Bosphorus

3rd BBBB
International Conference
on Pharmaceutical Sciences
October 26-28, 2009 - Antalya –TURKEY
With the Participation of Representatives of
University - Industry - Regulatory Scientists

Bosphorus
Bled
Balaton
Baltic
with the collaboration of the
Phage Societies (UK, USA, GE) and European Association of Faculties of Pharmacy (EAFP)

organized by
Turkish Pharmaceutical Technology Scientists’ Association (TÜFTAD)

www.bbbb-eufeps.org
 PREFACE
W
ithin the last decade, very rapid changes have
taken place in pharmaceutical sciences, phar-
maceutical industry activities, regulatory ap-
proaches and economical platforms. Among these, the im-
pact of developments in computer sciences, technology and
medical sciences have also added tremendous challenges to
pharmaceutical practices in industry, regulatory body and
academia. We all know what we are facing new challenges
everyday in our mutual applications. What we have seen and
experienced within this last decade explained us that we
should transfer our experience, knowledge and guidance to
young scientists, and new generations.
The BBBB International Conferences have been initi-
ated under the auspices of EUFEPS by the joint proposal of
four founder countries’ associations and have been decided
to initiate organizing international conferences biennially on
the attractive, historical and well-known shore of a lake or on
the seacoast, e.g. in the region of Lake Balaton, the Baltic sea,
Bled, and the Bosphorus in order to support young, bright
and future promising scientists of these countries and other
participant countries as well.  Another important reason to
emphasize was to open the doors in order to create close sci-
entific contacts between founding and participant countries’
scientists. Under the light of above mentioned approaches,
the 1st BBBB Balaton International Conference in Siofok,
Hungary was jointly organized by Hungary and Slovenia in
2005. 2nd BBBB Baltic International Conference in Tartu
was jointly organized by Estonia and Finland in 2007.
The 3rd BBBB-Bosphorus International Conference
on Pharmaceutical Sciences was organized from October
26 to 28, 2009 in Antalya-Turkey by Turkish Pharmaceutical
Technology Scientists’ Association (TÜFTAD).
Main target of the 3rd BBBB-Bosphorus International
Conference on Pharmaceutical Sciences was to create a
pharmaceutical sciences platform in between the university,
industry and regulatory scientists for discussing the main
topics of this symposium in a friendly atmosphere in histori-
cally and naturally attractive part of Turkey called “Turkish
Riviera”. BBBB Bosphorus (Antalya-Turkey, 2009) Internation-
al Conference consisted of six main themes:
• Regulatory Issues
• Towards Innovative Novel Drug Delivery Systems
• Biotechnology, Delivery of Biologics and Vaccination
• Progresses in Medicinal Chemistry and Analytical Tech-
niques of Drug Development
• Clinical Pharmacy - Pharmaceutical Care
• New Progresses in Pharmacy Education, Training and
Pharmacy Progression
However, under these six main themes, BBBB Bosphorus
Conference brought the attendees a lot of hot topics and very
new area of interests for pharmaceutical sciences, such as:
• Modeling and simulation in modern pharmacy
• HTS and solubility testing
• New approaches and techniques for drug delivery sys-
tems
October 26 - 28, 2009 / Antalya - TURKEY
• Regulatory issues related  to
- disharmonization of BE of highly variable drugs,
- herbal medicinal products,
- medical devices, and
- pharmaceuticals
• Recent strategies for the oral delivery of poorly soluble
drugs
• Utilisation of bacteriophages  as  medicines in drug 
delivery systems
• Bacterophages and lytic enzymes to combat new and
emerging bacteria
• Self assembling nanoparticles as vaccine delivery ve-
hicles for bacteriophages
• Bacteriophages in targeted medications
• Biotechnology, biological systems for delivery of drugs
• New analytical techniques for enantiomeric purity of
chiral drugs
• Microchips in drug analysis
• SCCE in pharmaceutical and bioanalysis
• QSAR and prediction of  drug properties
• Alicylic beta aminoacids in drug research
• Further topics on medicinal chemistry and analytical
techniques of drug development
• Disease-drug interactions
• Cost effectiveness of drug therapy using pharmacoge-
netic testings
• Clinical pharmacy services in hospitals
• Clinical pharmacy and pharmaceutical care
3
• PHARMINE or pharmacy education in Europe
BBBB Bosphorus (Antalya-Turkey, 2009) International
Conference hosted around 200 participants from university,
industry, and regulatory agencies from all over the world.
The contributions as oral and poster presentations were
published in this special issue. We hope these excellent pre-
sentations serve as a guide to readers for new areas of phar-
maceutical sciences.
As the guest editors of this issue, we wish to thank all the
authors for their scientific contributions and also to Elsevi-
er giving us the chance of publishing this special issue. We
would like to extend our thanks to the sponsoring compa-
nies from the Turkish Pharmaceutical Industry.
September 26, 2009
Guest Editors
Prof. Dr. A. Atilla Hıncal
İDE Information-Consultancy-Education, Inc., Ankara, Turkey
Prof. Dr. Nevin Çelebi
Gazi University, Faculty of Pharmacy, Ankara, Turkey
Assoc. Prof. Dr. Nilüfer Yüksel
Ankara University, Faculty of Pharmacy, Ankara, Turkey
“Spelling mistakes and any other errors are under the responsibility
of the authors.”
ORAL PRESENTATIONS
 ORAL PRESENTATIONS

O01
In vitro blood brain barrier (BBB) models
and their application in drug research
Christian R. Noe
University of Vienna, Austria

D
evelopment of new drugs acting on the central ner-
vous system (CNS) is one of the major tasks in pharma-
ceutical sciences. In this context, CNS targeted drugs
have to cross the BBB. The BBB maintains the homeostasis
of the brain microenvironment, which is crucial for neuronal
activity and function. Unlike peripheral endothelium, brain
microvascular endothelial cells are characterised by the
presence of tight intercellular junctions, minimal pinocytotic
activity and the absence of fenestrations.
The lecture will report about the side-by-side comparison
of several BBB cell culture in vitro models. Transwell models
and dynamic hollow-fiber models based on different cell
lines were established and characterised with regard to their
BBB specific properties, such as tight junction formation and
functionality. A comparative analysis of the models will be
presented. In order to assess their reliability to predict in vivo
permeability of drugs, transport studies with sets of drugs of
different classes were performed. The broad applicability of
BBB models beyond transport studies will be presented as
well. The models may be used to study the impact of drugs
on the BBB system itself. There is significant evidence that
dysfunction of the BBB might play a crucial role in neurode-
generative and other CNS diseases. BBB in vitro models are
an excellent toll to study options for specific therapeutic in-
tervention by design of drugs exerting their effects directly
at the BBB.

October 26 - 28, 2009 / Antalya - TURKEY

 ORAL PRESENTATIONS
O02
AN ILLUSTRATION OF REGULATORY DISHARMONIZATION:
BIOEQUIVALENCE OF HIGHLY VARIABLE DRUGS
Laszlo Endrenyi and Laszlo Tothfalusi
University of Toronto and Semmelweis University of Medicine, Canada
H
armonization of regulations is a praiseworthy princi-
ple. In practice, however, various regulatory authori-
ties develop their expectations and procedures with
almost complete disregard of each other. As a reminder of
this divergence, the issue of individual bioequivalence (BE)
was strongly promoted for a decade in the United States but
nowhere else.
Another, currently timely topic is the determination of BE
for highly variable (HV) drugs and drug products which has
been, for a long time, a substantial problem. The usual reg-
ulatory requirement for satisfying BE is that the 90% confi-
dence interval around the ratio of geometric means of phar-
macokinetic parameters (AUC and Cmax) for the contrasted
drug products be between 0.80 and 1.25. It is very difficult to
satisfy this expectation, by utilizing the procedure of average
8 BE, when the within-subject variation of a drug is high unless
very many subjects are enrolled in a study.
Various other approaches have been devised in order to re-
duce this burden. Steady-state investigations often lower the
intrasubject variation. However, some regulatory authorities
have expressed reluctance to apply this approach.
In the past, regulatory disharmonization proceeded to an al-
most ironic extent. For instance, in Europe it was possible to
extend substantially, for a long time, the regulatory criterion
for Cmax provided that a clinical justification was presented
in the protocol. FDA in the United States did not permit at
the time such exceptions. In the late 1990`s the roles re-
versed. In Europe, the tolerance was constricted whereas
FDA became, for a while, more relaxed.
Currently, FDA appears to prefer the procedure of scaled
average BE. Accordingly, the usual criterion for average BE
is scaled by the within-subject standard deviation. FDA ex-
pects that the procedure be implemented in replicate-de-
sign investigations.
Computer-simulated studies demonstrate that by apply-
ing this approach, comparatively few subjects are needed
to satisfy regulatory expectations. For instance, a few more
than 36 subjects could be sufficient in a 3-period investiga-
tion, whereas only about 24 individuals could be needed in
a 4-period study. The producer risk is independent of the
within-subject variation.
3rd BBBB International Conference on Pharmaceutical Sciences
Scaled average BE can be interpreted as an equivalence test
for clinical effects sizes. It is also a special case of therapeutic
switchability.
FDA imposes also a secondary regulatory criterion. This
expects that the point estimates of the ratio of geometric
means of the pharmacokinetic parameters should be be-
tween 0.80 and 1.25. This criterion has only political but no
scientific background. It substantially complicates the eval-
uation of BE for HV drugs.
The FDA procedure, as stated currently, has some techni-
cal difficulties. Notably, the BE limits are discontinuous at a
within-subject variation of 30%. As a consequence, the con-
sumer risk can be much higher than 5%, and the regulatory
uncertainty for making a decision about acceptance or rejec-
tion is enhanced.
EMEA in the European Union issued a Concept Paper in 2006
on the problem of HV drugs. This was later withdrawn, and
EMEA resisted, until very recently, further consideration of
the topic. Currently, EMEA ponders the application of an ap-
proach which parallels that of scaled average BE. Unscaled
average BE would be utilized in which the BE limits would ex-
pand in proportion to the within-subject standard deviation.
However, the expansion would be limited to a range when
the intrasubject coefficient of variation is between 30% and
50%. There is no scientific justification for this limitation.
Health Canada contemplates also unscaled average BE with
expanding limits. However, it is concerned that various sub-
missions for the same drug could report differing within-
subject variations which could result in differing regulatory
procedures. Therefore the BE limits would be determined on
the basis of all available information. It is contemplated that
the BE limits would expand stepwise by decades of within-
subject variation. The problem with this approach is that the
BE limits would be discontinuous again with the consequent
difficulties.
The Medicines Control Council of South Africa considers accept-
able the use of alternative methods of evaluation, e.g. of scaled
average BE, provided that this is stated a priori in the protocol.
The possible presence of outliers is an issue related to the de-
termination of BE for HV drugs and drug products. BE trials
 ORAL PRESENTATIONS

often fail not because the drug is highly variable but because
outliers could inflate the variability. Regulatory authorities
propose different approaches to address this problem. FDA
suggests a re-dosing strategy. In contrast, this approach is
missing from a recent CHMP draft. Both guidelines are very
negative on removing outliers merely for statistical reasons
whereas a recent Canadian proposal is more permissive. At
least, the application of sequential designs is a common
feature of the recent proposals on which consensus could
be reached. However, it is not clear at present, how scaling
and sequential designs will be combined, for example, how
a study will be assessed if a drug product is HV in one stage
of the investigation but not in the other.
Thus, the various regulatory authorities make slow progress
but separately from each other and without taking into ac-
count considerations and procedures developed by the
other agencies. It is difficult to think of this as a harmonized
process.

October 26 - 28, 2009 / Antalya - TURKEY

 ORAL PRESENTATIONS

O03
REGULATORY ISSUES for HERBAL MEDICINAL PRODUCTS
Bilge Şener
Gazi University Faculty of Pharmacy Department of Pharmacognosy 06330 Ankara-Turkey

H
erbal medicinal products have been playing an im-
portant role in the primary health care of the people
around the world, specially in the developing coun-
tries. In order to keep pace with the modern medicines, the
production of safe and effective herbal medicinal products
in a standardized way is essential.
Assessment of the quality, safety and efficacy of herbal me-
dicinal products are an important issue. Standardization of raw
materials, intermediates and final product of herbal medicines
are the main issue for the quality control of herbal medicinal
products. All of the supporting evidence behind the use of phy-
tomedicines has been on use of standardized extracts of the
plant material to ensure reproducibility in the clinical setting.
With the growing interest for alternative approaches in trea-
ting diseases, herbal medicinal products have also an impor-
tant role for the development of new therapeutic agents.
Herbal medicinal products named as “Phytomedicines” ex-
hibit a variety of biological activities on human health. These
range from the control of regulatory processes by Health Au-
thorities is essential for human life. Therefore, herbal medici-
nal products are also subject to the same legislative controls
as other medicines.
The overview of the herbal medicinal products in the world-
wide along with current registration guidelines and criteria
for the control and market situation of herbal medicinal
products will be highlighted.

10

3rd BBBB International Conference on Pharmaceutical Sciences

 ORAL PRESENTATIONS

O04
Development of paediatric medication – opportunity or hurdle?
Tom Sam
Director Global Regulatory Affairs CMC, Schering-Plough, The Netherlands

INTRODUCTION Children are a diverse patient population and their needs

The continued widespread practice of unverified extempora-
neous preparation and “off-label” use of adult medicines for
children has led to the introduction of legislation in the EU
and USA to stimulate the industrial development of paediatric
medicines. In Europe it has become a regulation both for new
and existing patented products to include paediatric data in
the registration dossier in accordance with an agreed investi-
gation plan and/or a waiver and/or a deferral. This hurdle of
extra work beyond developing an adult medicine is meant to
be counterbalanced by an incentive for industry of 6 months
extension of the Supplementary Patent Certificate.
should be addressed both in terms of delivering the dose
of drug appropriate for their age and size and providing
the drug in a dosage form that will make it easy to admin-
ister and facilitate compliance. In order to create a situation
where substantially more children have access to safe and ef-
fective medication, an environment is needed that provides
the right balance between predictable and consistent regu-
latory assessment and industrial feasibility.

11

October 26 - 28, 2009 / Antalya - TURKEY

 ORAL PRESENTATIONS

O05
Medical Devices and Pharmaceuticals:
Differences and Regulatory Approach
Sema Çalış
Hacettepe University, Faculty of Pharmacy, Department of Pharmaceutical Technology, Sıhhiye, Ankara

M
edical devices have become an increasingly impor-
tant health care area in relation to their impact on
health and health care expenditure. This sector cov-
ers numerous types of products ranging from simple ban-
dages to hemodialysis machines, through life maintaining
implantable devices, equipment to screen and diagnose dis-
ease and health conditions to the most sophisticated diag-
nostic imaging and minimal invasive surgery equipment.
According to the definition of The Global Harmonization
Task Force, a “Medical device” means any instrument, ap-
paratus, implement, machine, appliance, implant, in vitro
reagent or calibrator, software, material or other similar or
related article, intended by the manufacturer to be used,
alone or in combination, for human beings for one or more
of the spesific purposes of: diagnosis, prevention, monitor-
12 ing, treatment or alleviation of disease; diagnosis, monitor-
ing, treatment, alleviation of or compensation for an injury;
investigation, replacement, modification, or support of the
anatomy or of a physiological process; supporting or sustain-
ing life; control of conception; disinfection of medical devic-
es; providing information for medical purposes by means of
in vitro examination.
Medical devices are distinguished from drugs and biologi-
cal products on the basis of their intended modes of action,
in which, they are not metabolic, pharmacologic or immu-
nologic, whereas drugs primary mode of action are meta-
bolic, pharmacologic or immunologic. For stating the main
differences between the drugs and medical devices; drugs
are pure molecules, have toxicologic concerns and drug-
interactions might be present, clinically studied and have a
long market life. Medical devices are complex components,
have biocompability concerns, device mulfunctions might
be possible. For production of drugs, Good Manufacturing
Practices are a requirement whereas Quality Managements
Systems are important for Medical devices. In devices ad-
verse effects are most often local in nature, they are intend-
ed for professional use.
According to the US FDA’s definition, “a combination device
comprises two or more regulated components, i.e., drug/
device, biologic/device, or drug/device/biologic, that are
physically, chemically, or otherwise combined or mixed and
produced as a single entity: or two or more separate prod-
ucts packaged together in a single package or as a unit and
comprised of drug and device products, device and biolgi-
cal products, or biological and drug products”. Using locally
controlled drug delivery, combination products have already
found applications in various areas of cardiovascular disease,
diabetes, orthopedic, and cancer.
The regulatory aspects of medical devices differ from coun-
try to country. Manufacturers are obliged to establish the
safety of their products before they are marketed. Medical
devices were addressed by three medical device directives,
which were intended to ensure the safety and performance
of medical device in the EU:
1. Active implantable medical devices directive –

Table 1. European medical devices classification

EU Class Device Type Example
Class I Low risk to the patient and, except for sterile products or measuring Stethoscopes, EEG and ECG electrodes, body liquid collection devices,
devices, such devices generally do not enter into contact or interact with wheel chair
the body.
Class IIA Medium risk, and are invasive in their interaction with the human body, Syringes and tubing intended for use with infusion pumps, blood
but that interact with the body only through natural body orifices. Also transfusion devices, non-medicated gauze dressing, urinary catheters
include therapeutic devices used in diagnosis or in wound management.
Class IIB Medium risk, and are either partially or totally implantable within the Dialysis apparatus, medicated gauze, dressing
human body, and may modify the biological or chemical composition of
body fluids
Class III High risk, and generally affect the functioning of vital organs and/or life Cardiac pacemarkers and defibrillators, drug-eluting stents, breast
support system. implants, knee impart

3rd BBBB International Conference on Pharmaceutical Sciences

 ORAL PRESENTATIONS

90/385/EEC 4: covers all powered implantable medical
devices.
2. Medical device directive – 93/42/EEC 5: regulates
most medical devices, e.g. bandages, condoms and di-
agnostic X-ray machines.
3. The in vitro diagnostic medical devices directive –
98/79/EC: this directive regulates products used to ex-
amine substance derived from human body.
Medical devices should be classified according to their in-
tended use. There are 18 rules for classification of medical
devices. They helped the manufacturer to determine wheth-
er the device is Class I (low risk), Class II ( medium risk), or
Class IIb or III (high risk).(Below please see European medi-
cal devices classification up to the device type and related
example)
REFERENCES
1. R. Swami, J. Singh, D. Kaushik, H.Dureja, Medical Devices: A
Regulatory Overview, Latest Reviews, 2009 Vol. 7 Issue 1.
2. J. Bright, European medical device regulatory law and
product liability, Journal of Hospital Infection, 1999, Vol.
43, Suppl. 1, S169-S173.
3. www.fda.gov
4. www.who.int

13

October 26 - 28, 2009 / Antalya - TURKEY

 ORAL PRESENTATIONS

O06
Cellular automata model for drug release sımulatıons
Timo Laaksonen1,Hannu Laaksonen2, Lasse Mutomäki3,4, Jouni Hirvonen1
1Division of the Pharmaceutical Technology, University of Helsinki, Finland
2Brain Research Unit, Low Tempreture Laboratory, Helsinki University of Technology, Finland
3Centre for Drug Research, University of Helsinki, Finland
4Laboratory of Physical Chemistry and Electrochemistry, Helsinki University of Technology, Finland

C
ontrolled release of drugs from polymeric tablets or abilities for various events such as moving to a neighboring cell
implants is a technologically and therapeutically im- or being converted into another type of a cell. The probability
portant field. In order to create and plan new dosage for its movement is set to 1 and all other probabilities are rela-
forms efficiently, it is important to be able to design the re- tive to this. Swelling and permeation have lower probabilities.
lease profile of the drug. These characteristics are influenced
by several factors, such as diffusion, geometry and solubility Table 1. Cell states considered in the model and the cellular
of the polymer used. There are four major release mecha- automata rule applied for each cell state in the simulations.
nisms which affect the release profile: diffusion, erosion, per- Adapted from ref. 2.
meation and swelling. Normally they are considered by solv-
Cell state & Cellular automata rule θ and γ values
ing differential equations, with relevant boundary and initial
symbol
conditions specific to each system. This can be quite time
consuming and must be done separately for every system. Water (W) No rules. θ = 0, γ = 0
Solid drug (D) When in contact with a W, p or O θ = θ0.
Here, a new approach for drug release modeling based on will convert into a mobile drug
a cellular automata model for virtually any geometry is pro- cell. Drug cells are not allowed to
posed1-2. It takes into account all four major drug release diffuse except through water or
14 mechanisms. Similar models have been used to model drug wet polymer.
release from eroding polymeric matrices3-7. In this model a
2D representation of the release device is divided into a grid Mobile drug Will randomly move to any If a transfer takes place,
space, where each cell represents a small part of the whole (dissolved) (d) neighboring water cell and con- the θ value of this cell is
system. Each cell is connected to 4 neighboring cells and vert it into a d cell. Can move into decreased by one and θ
can interact with them according to a simple set of rules de- a wet polymer cell with a prob- of the receiving cell is
signed to model the physics and chemistry of drug release. ability pp and form an O cell. increased by one.
For example, diffusion is modeled by allowing drug cells to Will be removed from the simu- Converts into a W cell
randomly move into adjacent water cells but not into other lation if it reaches the boundary when the value of θ is
drug cells. Diffusion inside permeable membranes is modeled of the simulation area. reduced to 0.
in the same manner, but with a lower diffusion coefficient. The
Polymer (P) Will become a p cell when in con- γ = γ0.
idea behind the model is to consider microscopic domains of
tact with water with a probabil-
the release device and treat them statistically instead of con-
ity pw. Polymer cells are assumed
sidering molecular interactions directly. For each physical or
to be passive unless they come
chemical reaction possible in the system, a probability for that
into contact with water.
occurrence is given. As this is the basis behind diffusion coeffi-
cients and kinetic rate constants, it is relatively straightforward Wet polymer Can swell with a probability ps if If swelling takes place,
to relate the probabilities to these constants. (p) γ > 1. the cell will increase
Has a probability pe to be eroded. the γ of a nearby cell
The model divides an assumed spherical drug release device The probability is higher if there by one and lower its
or tablet into a matrix of cells. The state of each cell represents are more water cells adjacent to own γ by one. Erosion
the physical contents of that part of the tablet. The states can it. decreases γ by one.
be either water, polymer, drug or a combination of the three. Converts into a W cell
Each cell represents a domain of the release device, not single when γ is reduced to 0.
molecules or even groups of molecules. During the simulations,
cells are allowed to interact with the 4 adjacent cells and change Wet polymer Obeys the rules of both d and p If θ falls to zero, this
their state according to a simple set of rules , shown in Table 1. with drug (O) cells. cell is converted into a
A dissolved drug filled domain is always assumed to be at the p cell. If γ falls to zero,
saturation concentration and concentration is represented by this cell is converted
the density of these cells. Each cell has a predefined set of prob- into a d cell.

3rd BBBB International Conference on Pharmaceutical Sciences

 ORAL PRESENTATIONS
As an example, swelling-controlled release is considered. In
this case, water as well as drug diffusion and polymer swell-
ing has to be taken into account. Adding these rules to the
cellular automata model is quite straightforward as can be
seen from Table 1. An example of a swelling-controlled re-
lease simulation is shown in Fig 1. The typical near-zero or-
der release profile is seen for most of the release time as the
thickness of the hydrogel layer stays constant throughout
the dissolution2.
Figure. 1: Illustration of a single simulation run for a swell-
ing-controlled drug release system. The status of the drug
release matrix in different time points during the simulation
is shown above. The release curve and the location of the dif-
ferent fronts during the simulation are shown below. Black
spots represent polymer, dark gray spots represent solid
drug, gray spots represent wet polymer and light gray spots
represent wet polymer with drug cells. Adapted from ref. 2.
In conclusion, a cellular, extremely flexible model for con-
trolled drug release simulations was presented. The model
can take into account swelling and erosion of the polymer,
drug and water permeations and solubility of the drug. The
model was simple to implement and gave realistic results
October 26 - 28, 2009 / Antalya - TURKEY
with limited calculation power required. It is therefore sug-
gested that it could prove to be useful for the design of swell-
ing drug formulations and other controlled release systems.
REFERENCES
1. Laaksonen, T., Laaksonen, H., Hirvonen, J., Murtomäki,
L., Cellular automata model for drug release from binary
matrix and reservoir polymeric devices, Biomaterials,
2009, 30: 1978-1987.
2. Laaksonen, H., Hirvonen, J., Laaksonen, T., Cellular
automata model for swelling-controlled drug re-
lease, Int. J. Pharm., 2009, In press, doi:10.1016/j.ijp-
harm.2009.06.023
3. Göpferich, A., Langer, R., Modeling monomer release from
bioerodible polymers. J. Controlled Release, 1995, 33: 55-69.
4. Barat, A., Ruskin, H.J., Crane, M., Probabilistic methods
for drug dissolution. Part 2. Modelling a soluble binary
drug delivery system dissolving in vitro, Simul. Model.
Pract. Theory, 2006, 14: 857-873.
5. Barat, A., Ruskin, H.J. & Crane, M., Probabilistic models
for drug dissolution. Part 1. Review of Monte Carlo and
stochastic cellular automata approaches, Simul. Model.
Pract. Theory, 2006, 14: 843-856.
6. Zygourakis, K., Development and temporal evolution of
erosion fronts in bioerodible controlled release devices,
Chem. Eng. Sci., 1990: 45: 2359-2366.
7. Zygourakis, K., Markenscoff, P.A., Computer-aided de-
sign of bioerodible devices with optimal release char-
acteristics: a cellular automata approach, Biomaterials,
1996, 17: 125-135.
8. Colombo, P., Santi, P., Bettini, R., Brazel, C.S., Peppas, N.A., 15
Drug Release from Swelling-Controlled Systems. In:
Wise, D.L. (ed), Handbook of Pharmaceutical Controlled
Release Technology, Marcel Dekker, New York, 2000, pp.
183-209.
 ORAL PRESENTATIONS

O07
Modelling and simulation in modern pharmacy –bridging
regulatory bodies, academia and industry
Sebastian Polak
Unit of Pharmacoepidemiology and Pharmacoeconomics, Faculty of Pharmacy, Jagiellonian University Medical
College, Medyczna 9 Str., 30-688 Krakow, Poland
Simcyp Ltd, Blades Enterprise Centre, John Street,  S24SU Sheffield, UK

T
he main reasons for growing role of modelling and sim-
ulation tools in pharmacy are widely known. Among
them dramatic cost increase of the drug discovery &
development process, ethical reasons forcing scientist to re-
duce number of animals sacrificed during drug development
process could be listed, but the most important reason is a
high reliability of currently used in silico methods. It is worth
mention that modelling and simulation procedures are not
only in common use at the universities during scientific re-
search studies and by pharmaceutical industry but also more
and more widely accepted and even requested by the main
regulatory bodies like FDA.  drug behaviour with use of a virtual population (Simcyp)
M&S methods and tools are in common use beginning from
the very early discovery (physico-chemical parameters pre-
diction), thorough qualitative forecasting and quantitative
16 assessment of toxicity, pharmacokinetic characterization
(including in vitro –  in vivo extrapolation and clinical trials
simulation), pharmacodynamic activity, biopharmacy, com-
puter aided drug formulation and Phase IV clinical trial con-
ducting (effect prediction for pharmacoeconomic studies).  croemulsions composition) systems.
Prediction of the basic physico-chemical parameters based
on substance structure plays important role at the initial
stage of the drug discovery. Having possibility to use and
compare both free (i.e. vcclab.org) and commercially avail-
able (i.e. Marvin, ACDlabs) software every substance can be
characterized according to the BCS classification. Specialized
chemical software (MOE, Dragon) offers calculation of the
theoretical descriptors which can be further used for estima-
tion of potential pharmacological and toxicological activity
(i.e. hERG channel inhibition, skin irritation, hepatotoxicity).
At the development level using in vitro information obtained
in the research laboratories it is possible to simulate in vivo
what enables considering inter-individual variability. Such a
facility can be directly used to plan Phase I clinical trials and
reduce costs of unnecessary studies. It becomes extraordi-
narily important for the paediatric population which can be
simulated with use of Simcyp Paediatric module. Concomi-
tantly carried formulation studies can be optimized by drug
absorption prediction platforms (Simcyp) for various routes
of administration and specific, formulation focused (i.e. mi-

3rd BBBB International Conference on Pharmaceutical Sciences

 ORAL PRESENTATIONS

O08
Microtensiometry - novel surface tension based HTS method for
solubility testing of drug materials
Tiina Heikkilä, Leena Peltonen, Saila Taskinen, Timo Laaksonen and Jouni Hirvonen
Division of Pharmaceutical Technology, University of Helsinki, P.O. Box 56 (Viikinkaari 5E), 00014 University of
Helsinki, Finland

INTRODUCTION
High throughput screening (HTS) techniques are mostly based
on the determination of kinetic solubility. Active pharmaceuti-
cal ingredient (API) is dissolved in dimethyl sulfoxide (DMSO)
and small volumes of the aqueous media are added until the
solubility limit is reached at the point where the API precipitates.
In the existing 96-well plate methods, the kinetic solubility can
be determined by turbidity, nephelometry or UV absorption1-2.
Still, these methods have some weaknesses. For example, with
UV absorption, problems can arise from the low absorption of
poorly soluble material. Also possible absorption by residues
or other materials, like buffering salts, in the same wavelength
area as the studied compound can cause problems2.
drug and diluting the next sample to half the concentration of the
previous one. Last samples were blank. The presolvent solutions
were then added to the medium in the 96-well plate where the
single well volume was 50 µl. The samples were let to stand for 10
minutes prior to the measurements without shaking5. Measure-
ments were repeated at least 4 times at room temperature (appr.
24 oC). Determined solubilities of ibuprofen and indomethacin var-
ied between 0.0005 – 10 mg/ml and 0.004 – 5 mg/ml, respectively.
The surface tension measurements were done with a fully automat-
ed Delta-8 multichannel microtensiometer (Kibron inc., Finland).
The method is based on Du Noüy maximum pull force technique
and the measurement of one 96-well plate lasted appr. 5 min.

In this study a microtensiometric method based on surface
tension measurements is introduced for drug solubility re-
search purposes. Amphiphilic molecules decrease the surface
tension (γ) within a concentration range limited by air/water
partitioning at the lower concentration end and solubility/
critical micelle concentration (CMC) at the higher end, which
is described by the classical Gibbs adsorption isotherm:
, ibuprofen in water. The solubility of ibuprofen can be seen from
where Γ is surface excess, R is gas constant, T is temperature
and c is concentration.
If the compound is more likely to precipitate than to form mi-
celles, the value for solubility can be determined from these
isotherms3 (Figure 1). In practice, almost all drug compounds
have some surface activity and the measurement is possible.
The shake-flask solubility tests were done in duplicate un-
der the same conditions as the microtensiometric measure-
ments according to a standard procedure. Samples were 17
analyzed after filtration by UV-spectrophotometry (λ= 264
nm for ibuprofen and 254 nm for indomethacin, Pharmacia
LKB, Ultrospec, Sweden).
RESULTS AND DISCUSSION
Figure 1 shows an example of a measured Gibbs isotherm for
the focal point between the slope and the level part of the iso-
therm. In this point, there is an equilibrium state between the
solid and solvated phases. At higher concentrations, all material
exceeding the solubility limit will be in the solid phase, which
has no effect on the surface tension. Also the effect of changing
pH is seen in Figure 1; lowering of the pH decreased the solubil-
ity, which was seen as a shift in the adsorption curve.

The aim of this study was to utilize interfacial tension measure-

ments at the air/water interface for the kinetic solubility deter-
minations. Poorly water-soluble BCS class II drugs ibuprofen
and indomethacin were used as the model drugs. Different sol-
ubility areas were tested by changing the medium. Traditional
shake-flask measurements were used as a reference method.

MATERIALS AND METHODS

Solubilities of ibuprofen and indomethacin were studied in
buffer solutions pH 1.2 and pH 6.8 (4) and in water. Either
DMSO (10% v/v) or methanol (10 % v/v) was used as presol-
vent. Also the effects of NaCl and CaCl2 were studied. Figure 1: Gibbs isotherm for ibuprofen in water in two differ-
ent pHs. Surface tension is decreased with increasing drug
The studied drugs were first dissolved in presolvent. 12-22 samples concentration until a solubility limit is reached. Methanol
were prepared in each medium starting from ca. 100 mg/ml of the was used as a presolvent (n=12).

October 26 - 28, 2009 / Antalya - TURKEY

 ORAL PRESENTATIONS
The correlation between shake-flask and microtensionmeter
methods was good (Figures 2 and 3). As expected for weak
carboxylic acid, the solubility of ibuprofen decreased in the
shake-flask method from 0.09 mg/ml in pure water to 0.05
mg/ml at pH 1.2, and increased to 3.1 mg/ml at pH 6.8. The
similar trends were obtained for indomethacin. NaCl and
CaCl2 concentrations of 0.9% and 2%, had only limited ef-
fects on the solubility of ibuprofen (Figure 3). Still, a linear
dependence exists in all the media tested between the two
methods for both the model substances.
Figure 2: Solubilities of ibuprofen from the surface tension
measurements (y-axis) as compared to the shake-flask mea-
surements (x-axis).
18
3rd BBBB International Conference on Pharmaceutical Sciences
Figure 3: Solubilities of ibuprofen and indomethacin (marked
in the figure) by the surface tension measurements (black
pillars, n=8-12) and shake-flask measurements (grey pillars,
n=2).
The developed surface tension based solubility testing meth-
od is fast, can be easily repeated and gives a large amount of
qualitative data. As compared to other screening methods
for solubility testing, microtensiometric analysis does have
some clear benefits. For example, in UV-based methods
problems arise if the absorption areas of different materi-
als overlap or the impurities with absorbance potential may
cause inaccuracy to the results or the material is poorly ab-
sorbing. One limitation for the technique is that the CMC and
solubility limits may not be differentiated. Though this is not
likely to cause problems for most of the drugs, very insoluble
drugs can, in some cases, not be analyzed since they might
not partition sufficiently to the water/air interface.
CONCLUSION
This study shows that the microtensiometric determination
of drug solubility is a promising new HTS method. The mea-
surements are fast to perform, can be easily repeated and
large amount of qualitative data with a minimum amount of
analyses required are achieved.
REFERENCES
1. Bevan and Lloyd, Anal. Chem., 72(2000), 1781.
2. Pan et al., J. Pharm. Sci., 90(2001), 521.
3. Johans, www.kibron.com, 2004.
4. USP XXXI, 2007.
5. Heikkilä et al., Letters Drug Design Deliv., 5(2008), 471.
 ORAL PRESENTATIONS

O09
THE POTENTIAL OF MAGNETIC RESONANCE IMAGING AS ANALYTICAL TOOL
FOR THE INVESTIGATION OF CONTROLLED RELEASE MATRIX TABLETS
S. Baumgartner1, J. Kristl1, U. Mikac2, A. Sepe2
1
University of Ljubljana, Faculty of Pharmacy, Aškerčeva 7, 1000 Ljubljana/Slovenia
2
Jožef Stefan Institute, Jamova 39, 1000 Ljubljana/Slovenia

INTRODUCTION
Various hydrophilic polymers have been investigated as the
basis for gel-forming, controlled release tablets, of which
HPMC currently appears to be among the most useful1. How-
ever, the polymers of the natural origin, like xanthan (XAN),
are becoming more and more important in the pharmaceu-
tical research. The drug release from such tablets is a very
complex process and is to a great deal influenced by the gel
layer structures forming around the dry tablet core2.
The polymer swelling process has been studied by variety of
analytical techniques. Among them, magnetic resonance im-
aging (MRI) has been proven to be a powerful method for fol-
lowing the formation of hydrogel because it is non-invasive,
spatial selective and can provide quantitative information.
Various approaches have been taken in using MRI for quanti-
tative characterization of the swollen tablets3,4. In one of the
latest, benchtop MRI was introduced into this research5.
The aim of this study was to investigate the influence of pH
and ionic strength of medium on dynamics of hydrogel for-
mation of the XAN matrix tablets. For resolving specific XAN
structural changes influencing drug release, measuring and
modeling tools of MRI were used.
The NMR spin-lattice (T1) and spin-spin relaxation (T2) times
were measured at 1H NMR frequency of 100MHz for hydro-
gels with known XAN concentrations (Bruker, G, with a su-
perconducting magnet Oxford Instruments Ltd., UK). The T1
was measured by standard inversion recovery sequence and
T2 by CPMG pulse sequence.
MRI of XAN tablets during swelling
For T2-mapping multi-echo pulse sequence was used: TE =
6.2 ms, number of echoes N = 50, TR = 200 ms. The field of
view was 50 mm with in-plane resolution of 200 µm and slice
thickness of 3 mm. The concentration profiles were calculat-
ed from the signal intensity of the image with N = 1, i.e. TE =
6.2 ms. Sequential MR-images of matrix tablets during swell-
ing in different media were taken and as a reference medium
was used.
19
RESULTS AND DISCUSSION
Drug release studies
Release of PF from XAN tablets was prolonged by more than
24 h in water and in HCl pH 3.0 medium. The slowing of re-
lease was accompanied by evident swelling of the matrix
tablet (Fig 1)

MATERIALS AND METHODS

Materials
As polymer matrix xanthan (XAN, Sigma-Aldrich Chemie, G)
was used. As water soluble drug pentoxifylline (PF) was sup-
plied by Krka, d.d., SI. For drug release and MRI studies 6 dif-
ferent media were prepared: purified water pH 5.5, HCl pH
1.2 and pH 3.0. To increase ionic strength for 0.2M, NaCl was
added to each media.

Preparation of matrix tablets

For MRI, XAN was directly compressed (SP 300, Kilian&co.,
Cologne, G) to form tablets of hardness 100N±10 (VanKel
VK200, USA), m=500mg, 2r=12mm. For drug release 100 mg
of PF was incorporated into 400mg tablets.
Drug release from XAN matrix tablets
Dissolution studies were performed in all 6 media on a dis-
solution apparatus, paddle method (Apparatus II, VK7000,
USA), 900ml, 50rpm, 37°C±0.5. Samples were UV analyzed at
274 nm (HP diode array UV spectrophotometer, 8453, G).
Measurements of NMR relaxation times
Figure 1: The release profile of pentoxifylline from XAN tab-
lets in different media
When tablets were placed in medium with increased ionic
strength or pH 1.2 the drug release was much faster. Since
the dynamics of swelling and arrangement of water mole-
cules within the formed gel structure is crucial for drug re-
lease, XAN tablets during swelling process were investigated
by MRI.

October 26 - 28, 2009 / Antalya - TURKEY

 ORAL PRESENTATIONS
MR imaging and T2-mapping
To be able to determine the polymer concentration pro-
file during swelling, the relaxation times T1 and T2 of gels at
known XAN concentrations were measured. From these re-
sults theoretical signal intensities (S) of gels were calculated
according to eq.
where k is the constant independent of polymer, ρ is the pro-
ton density, TE and TR are experimental parameters.
The images at different times after hydration of tablets in all
6 media were taken (Fig 2). The dry tablet core is black and
the brightness of the newly formed gel
layer depends on its concentration.
Figure 2: MR images of XAN during swelling in HCl pH 1.2
+ NaCl medium. Square on the 1st image denotes the area
20 that was chosen for evaluating the polymer concentration
profile. White arrows indicate different domains within gel.
The polymer concentration profiles across swollen tablet are
much steeper at low pH and in presence of NaCl, proving
denser, but thinner gel network. Unusual gel formation at
pH 1.2 was observed from MRI. For certain conditions during
swelling, T2 can be quite long even at high XAN concentra-
tion (Fig 3).
3rd BBBB International Conference on Pharmaceutical Sciences
Figure 3: XAN polymer concentration profiles across the tab-
let and T2 profiles in HCl pH 1.2 + NaCl medium
Differently hydrated domains were proved as the conse-
quence of XAN conformational changes during swelling.
Furthermore, the drug release was faster, due to the pres-
ence of larger fraction of free water molecules and thinner
gel layer.
REFERENCES
1. Colombo P, Bettini R, et al. Swellable matrices for con-
trolled drug delivery: gel-layer behaviour, mechanisms
and optimal performance. Pharm. Sci. Technol. Today
2000; 3: 198-204.
2. Baumgartner S, Pavli M, Kristl J. Effect of calcium ions on
the gelling and drug release characteristics of xanthan
matrix tablets Eur. J. Pharm. Biopharm. 2008; 69: 698 –
707.
3. Fyfe CA, Blazek-Welsh AI. Quantitative NMR imaging
study of the mechanism of drug release from swelling
HPMC tablets. J. Control. Release 2000; 68:313-333.
4. Baumgartner S, Lahajnar G, Sepe A, Kristl J. Quantita-
tive evaluation of polymer concentration profile during
swelling of hydrophilic matrix tablets using 1H NMR and
MRI methods. Eur. J. Pharm. Biopharm. 2005; 59: 299-
306.
5. Metz H, Mäder K. Benchtop-NMR and MRI–a new ana-
lytical tool in drug delivery research, Int. J. Pharm. 2008;
364 170-175.
 ORAL PRESENTATIONS

O10
USING MESOPOROUS INORGANIC MATERIALS FOR
DELIVERING POORLY SOLUBLE DRUGS
Vesa-Pekka Lehto
Department of Physics, University of Kuopio, FI-70211 Kuopio, Finland

INTRODUCTION
Loading of therapeutic compounds into the confined space
of mesoporous inorganic materials can radically change their
properties and fate when delivered in human body via various
administration routes. Not only the modified physico-chemical
properties obtained with the nanostructuring of the payloads,
but also the protection of the payloads against the harsh con-
ditions of the gastrointestinal tract, together with the possibil-
ity to cross biological barriers make inorganic porous nano/
microparticles attractive carriers for drug delivery purposes.
However, safety and biocompatibility are the basic require-
ments for any drug carrier. Biodegradability is also preferential
feature when concerning parenteral administration.
It is estimated that over 95% of the new drug candidates suf-
fer from poor pharmaco-kinetic/ADME-properties when deliv-
ered orally. The main reasons for the inadequate oral bioavail-
ability are poor thermodynamic solubility and/or dissolution
rate of the compound in the intestinal lumen, inadequate
permeability properties of the drug across the gastrointesti-
nal (GI) wall, as well as high intestinal and hepatic first pass
metabolism. Thus, due to these problems in drug discovery,
delivery systems addressing issues regarding low solubility,
enzymatic degradation and means to modulate drug perme-
ability (absorption) have been emphasized as key methodolo-
gies.
Silicon based mesoporous materials have been reported to
have potential to overcome problems encountered in the de-
velopment of effective drug delivery systems1,2. Mesoporous
silicon (PSi) is a top-down material produced typically from
silicon wafers via electrochemical etching3. PSi is a nano-
structured material with closely spaced 2 – 50 nm wide pores
throughout its volume. Silicon materials are biodegradable,
and they possess some beneficial properties regarding drug
delivery applications. The small size of the pores confines the
space of a drug and emphasizes the effects of surface inter-
actions of the drug molecules and the pore wall (Figure 1).
The size of the pores and the surface chemistry of the pore
walls may be easily controlled. Depending on the size and
the surface chemistry of the pores, increased or sustained re-
lease of the loaded drug can be obtained. Drug loading from
a solution at room temperature (or below) enables the use of
PSi also with sensitive therapeutic compounds like peptides
and proteins.
Figure 1. A schematic drawing of drug loaded PSi particle
with functionalized surfaces.
When aiming at oral delivery, microparticles are typically uti-
lized; when aiming at systems for parenteral administration, 21
nanoparticles are employed. However, utilizing nanoparticles
also in oral drug delivery may be advantageous as the residence
time in gastrointestinal (GI) tract can be prolonged by mucoad-
hesion or the particles can even cross the GI epithelium.
The presentation focuses on the results obtained in a Finnish
research consortium to manifest the feasibility of mesoporous
silicon as a drug carrier4-6. The encouraging results concern
high payloads, stabilization of the payloads, improved disso-
lution, reduced pH dependence of dissolution, improved per-
meation, biocompatibility/non-toxicity, bio-degradation and
sustained release. The aim has been in the development of PSi
as a safe and effective platform for delivery of therapeutics.
MATERIALS AND METHODS
The PSi micro/nanoparticles were prepared by electrochemi-
cal etching as described previously3. The pore structure and
pore diameters were varied by changing the current density
in the etching or by a post-treatment of annealing. Particles
with different size fractions were produced by milling of the
PSi films and by a subsequent wet sieving. The surfaces of
the particles were modified according to the intended appli-
cation as hydrogen terminated, oxidized, hydrocarbonized
or carbonized. The surfaces can also be grafted with carboxyl
or amino groups to facilitate a further bioconjugation. Typi-
cally the loading was performed by immersing the particles
in an appropriate drug solution. Dry powders of drug loaded
PSi microparticles were obtained after filtration and drying.
Regarding nanoparticles the material was used as suspen-

October 26 - 28, 2009 / Antalya - TURKEY

 ORAL PRESENTATIONS

sion. The unloaded and loaded particles were studied with

various physical methods including FTIR, DSC and TG to ana-
lyze, e.g., the stability and the loading degree of the drug.
The drug loaded particles were characterized in vitro con-
cerning dissolution behaviour, permeation and cytotoxic-
ity (flow cytometric analysis, CellTiter-Glo Luminescent Cell
Viability assay and CellTiter-Fluor Cell Viability assay) with
Caco-2 cells. In vivo experiments were performed to charac-
terize the toxicity (cytokine release tests after subcutaneous
administration), biodegradation and biocompatibility of PSi
after s.c. administration in mice.

RESULTS AND DISCUSSION

The typical loading degrees (mdrug/mtotal) for small molecules
were ca. 30% ranging from 20% to 60%. The latter case con-
cerns material with high porosity and optimal loading. With
peptides the obtained loading degrees were around 20%. By
loading poorly water soluble drugs in PSi particles their dissolu-
tion behaviors was significantly improved. For example, with in-
domethacin a 28-fold improvement in the dissolution rate was
achieved (Figure 2). For furosemide, which dissolution behavior
is very pH dependent, the dependence was minimized when
loaded in PSi. The permeability across Caco-2 cell monolayer
was improved 1.5-4 fold without any adverse effects on the
monolayer integrity. The large PSi particles were well tolerated
even with high concentrations by Caco-2 cells in vitro, but small
particles (<25 µm) seemed to reduce the viability of the cells
(Figure 3). However, cytokine release tests showed normal cy-
tokine activity in mice in vivo after s.c. administration suggest-
ing the particles are not causing any inflammatory effects (Ta-
Table 1: Plasma concentrations of various cytokines after s.c. ad-
22 ble I). Microscopic examinations of the injection sites supported
ministration of thermally hydrocarbonised PSi microparticles.
this finding. Importantly, the particles were biodegradable.
CONCLUSION
Based on the recent results obtained in our consortium and
elsewhere mesoporous silicon seems to be a potential platform
for delivery of therapeutics. However, various administration
routes demand specific concern regarding the safety of the
mesoporous silicon material with different surface chemistries.

Figure 2: Release kinetics of indomethacin as free powder
and loaded in thermally carbonized PSi microparticles.
ACKNOWLEDGEMENTS
Jouni Hirvonen, Kristiina Järvinen, Karl-Heinz Herzig, Marjat-
ta Louhi-Kultanen, Ale Närvänen, Anu Airaksinen and Jarno
Salonen are thanked for fruitful collaboration. The financial
support from the Academy of Finland is acknowledged.

Figure 3: Number of living cells (%) determined by flow cytom-
etry when Caco-2 cells were treated and incubated for 3 hours
with PSi microparticles with various surface chemistries.
REFERENCES
1. Prestidge C, Barnes T, Lau CH, Barnett C, Loni A, Canham
L, Expert Opin. Drug Deliv. 2007; 4: 101-110.
2. Salonen J, Kaukonen AM, Hirvonen J, Lehto VP, J. Pharm.
Sci. 2008; 97, 632-653.
3. Salonen J, Lehto VP, Chem. Eng. J. 2008; 137: 162-172.
4. Salonen J, Laitinen L, Kaukonen AM, Tuura J, Björkqvist
M, Heikkilä T, Vähä-Heikkilä K, Hirvonen J, Lehto V-P, J.
Control. Release 2005; 108: 362-374.
5. Kaukonen AM, Laitinen L, Salonen J, Tuura J, Heikkilä T,
Limnell T, Hirvonen J, Lehto VP, Eur. J. Pharm. Biopharm.
2007; 66: 348-356.
6. Kilpeläinen M, Riikonen J, Vlasova MA, Huotari A, Lehto
VP, Salonen J, Herzig KH, Järvinen K, J. Control. Release
2009; 137: 166-170.

3rd BBBB International Conference on Pharmaceutical Sciences

 ORAL PRESENTATIONS
O11
Carbon nanotubes as drug delivery systems
İsmail Tuncer Değim
Gazi University, Faculty of Pharmacy, Department of Pharmaceutical Technology, 06330, Etiler, Ankara, Turkey
C
arbon is the most common and versatile element in
the world. The type, strength, and number of bonds
of carbon in the molecule can form many different
elements with variety of properties. The diversity of bonds
and their corresponding geometries enable the existence
of structural isomers, geometric isomers, and enantiomers.
These are found in large, complex, and diverse structures
and allow for an endless variety of organic and inorganic
molecules. The properties of carbon are a direct conse-
quence of the arrangement of electrons around the nucleus
of the atom. Carbon in the solid phase can exist in three allo-
tropic forms: graphite, diamond, and buckminsterfullerene.
Diamond has a crystalline structure where the different ge-
ometry of the chemical bonds makes graphite soft, slippery,
opaque, and electrically conductive. Buckminsterfullerenes,
or fullerenes, are the third allotrope of carbon and consist
of a family of spheroidal or cylindrical molecules. The tubu-
lar form of the fullerenes called nanotubes (CNTs), will be
the subjected to this presentation and their usage as drug
carrier system will be discussed. Although CNTs were first
discovered in 1952 by Radushkevich and Lukyanovich in
Russia1 it was not announced through worldwide, therefore
it is generally accepted that at a carbon-carbon composites
workshop, Rick Smalley proposed the existence of a tubular
fullerene in 19902. Experimental evidence of the existence of
CNTs came in 1991 when Iijima imaged multiwalled carbon
nanotubes (MWCNTs) and Iijima was first to recognize that
nanotubes were concentrically rolled graphene sheets with
a large number of potential helicities and chiralities rather
than a graphene sheet rolled up like a scroll as originally pro-
posed by Bacon. Iijima initially observed only MWCNTs with
between two and twenty layers3, but in a subsequent publi-
cation in 1993, he confirmed the existence of single-walled
carbon nanotubes (SWCNTs) and elucidated their structure4.
Since the discovery of CNTs, numerous ideas for applications
have arose in a wide variety of scientific disciplines, such as
electronics and opto-electronics, sensors, batteries/fuel cells,
medicine/biology (fluorescent markers, labeling and drug
delivery carriers), catalysis and gas storage5. Because of
their intrinsic optical properties, nanotubes have been con-
sidered potential candidates for drug delivery carriers. The
capped ends of nanotubes may be opened up by oxidation,
allowing for the insertion of molecules of interest inside the
nanotube. Smith et al. observed peapods6, SWNTs filled with
C60, via high-resolution transmission electron microscopy,
These peapods can form suggests that nanotubes may serve
October 26 - 28, 2009 / Antalya - TURKEY
as carriers for other encapsulated molecules such as drugs or
imaging reagents. Fullerene adducts or organic derivatives
of fullerene molecules7 are the ideal candidates for applica-
tion in peapod syntheses to create advanced materials. For
example, a C60 molecule could serve as a vector to enable a
second, covalently conjugated molecule to be encapsulated
inside a SWNT. This approach could facilitate the assembly of
molecular “packages” wrapped in a protective SWNT for the
purpose of drug delivery. Unfortunately, fullerene adducts
typically decompose at relatively low temperatures, necessi-
tating mild filling conditions using solutions8 or supercritical
fluids. Although there are some reports and successful re-
sults, drug delivery attempts with CNTs rather concentrated
to use of CNTs9.
An ideal cancer treatment would deliver a high dose of drugs
to tumor sites while minimizing side effects. Unfortunately, as 23
anticancer drugs are quickly cleared from the bloodstream,
large doses are usually needed to ensure that enough drugs
reach the tumor. And since these drugs can be absorbed by
normal cells, high doses cause unpleasant side effects. Now
research from Stanford University10 has shown that carbon
nanotubes loaded with anticancer drugs can target tumor
cells while steering clear of healthy tissue. The CNTs on av-
erage 100 nanometers long and a few nanometers wide
pass easily through the leaky walls of tumor blood vessels
but do not get into healthy blood vessels. So the researchers
realized that drugs attached to the nanotubes could be car-
ried inside tumors without harming normal tissue. To make
working nano-drug transporters, the researchers coated the
nanotubes with a molecule called polyethylene glycol (PEG),
which has three branches on one end, then attached mol-
ecules of the anticancer drug paclitaxel to each branch10. The
drug-delivery technique was tested in breast cancer model
in mice and they found that the tumors treated by nanotube
delivery were less than half the size of the tumors treated by
the second most effective available conventional treatment
with Taxol10. However, despite such a dramatic improve-
ment, lingering concern over the potential toxicity of carbon
nanotubes means that they may be a long way from clinical
use as a drug-delivery mechanism. That safety concerns will
probably delay the approval of carbon nanotubes for medi-
cal purposes.
It has been also shown that single-walled carbon nanotubes
(SWNTs) and multiwalled carbon nanotubes (MWNTs) can
 ORAL PRESENTATIONS
be internalized by living cells and pass across the biological
membranes in cell culture studies11. The internalization of
carbon nanotubes by corneo-cytes has been shown12 in the
literature but their drug carrying properties through the skin
have not been fully evaluated. But in our previous study it
has been first shown that single-walled carbon nanotubes
(SWNTs) and multiwalled carbon nanotubes (MWNTs) can
be used to deliver drug molecules through deeper skin lay-
ers. The application of iontophoresis using carbon nanotube
electrode having adsorb drug molecules on their surface has
been shown and molecules successfully transferred through
deeper skin layers13. Indomethacin was selected as a hydro-
phobic drug (Log Ko/w = 4.5) and it penetrates through full
thickness skin6. The penetration of indomethacin through
fullthickness of rat skin was enhanced when indomethacin
adsorbed CNTs were used (Flux values were 0.119±0.037
μg/h for indomethacin alone; 0.330±0.052 μg/h for indo-
methacin with MWCNTs and 0.347±0.106 indomethacin with
double walled carbon nanotubes (DWCNTs, n=3,±SD) (14).
The penetration enhancement was higher with DWCNTs
and MWCNTs, however the mechanism was still unknown.
The CNTs may act to facilitate presentation of the drug to
the lipophillic membrane and/or they facilitate penetration
through the skin accompanying the drug into the dermal
tissue. Similar results were obtained when pig ear skin was
used. Penetration of indomethacin was found to be much
higher when indometahcin molecules were introduced to
the skin surface with CNTs14.
All previous studies have shown that carbon nanotubes
24 can be used to deliver drug molecules to the active site.
Therefore it was aimed to determine adsorption of pacli-
taxel, oxaliplatin and other non-steroidal anti-inflammatory
drug naproxen. Recent studies indicated and showed that
cyclooxygenase (COX) can have a role in development of
cancer and tumors. Nonsteroidal anti-inflammatory drugs
(NSAIDs) are therefore have noticeable effects on reducing
tumor mass and its development15-18. Naproxen sodium was
selected as a model NSAID. The drug adsorption capacity of
DWCNTs and MWCNTs were found to be 14.25 and 14.70%
(weightCNTs/weightDrug) respectively19.
The adsorption properties of DWCNTs and MWCNTs were
also investigated for oxaliplatin and MWCNTs were found
to be adsorbed oxaliplatin higher19. Oxaliplatin and pacli-
taxel were also adsorbed to the surface of fullerene and it
was found that in cell culture studies (MDCK cell lines) they
can actively stop cell proliferation more than their free forms
(MTT tests)19.
As a conclusion CNTs were found to be suitable material
for drug transport having higher surface area and being an
adsorptive material, CNTs can be useful materials for deliv-
ery of drug molecules by simply adsorption and desorption
mechanism.
REFERENCES:
1. http://en.wikipedia.org/wiki/Timeline_of_carbon_nan-
otubes
2. Smalley RE, Formation and properties of C60 and the
fullerenes, National Institute of Standards and Technol-
3rd BBBB International Conference on Pharmaceutical Sciences
ogy,1990, 6–7.
3. Iijima S, Helical microtubules of graphitic carbon, Na-
ture, 1991, 354: 56–58.
4. S. Iijima and T. Ichihashi. Single-shell carbon nanotubes
of 1-nm diameter, Nature, 1993, 363: 603–605.
5. O’Connell, MJ, Carbon Nanotubes Properties and Appli-
cations, CRC Press, Taylor & Francis Group, FL, US, 2006.
6. Smith BW, Monthioux M, and Luzzi DE, Encapuslated
C60 in carbon nanotubes, Nature, 1998, 396: 323–324.
7. Kadish, K.M. and Ruoff, R.S., Eds., Fullerenes: Chemistry,
Physics and Technology, Wiley, New York, 2000.
8. Yudasaka, M. et al., Nano-extraction and nano-conden-
sation for C60 incorporation into single-wall carbon
nanotubes in liquid phases, Chem. Phys. Lett., 2003, 380:
42.
9. Britz DA. et al, Selective host–guest interaction of single-
walled carbon nanotubes with functionalised fullerenes,
Chem. Commun., 2004, 176.
10. h t t p : / / w w w . t e c h n o l o g y r e v i e w . c o m /
Nanotech/21316/?a=f
11. Lacerda L, Raffa V, Prato M, Bianco A, Kostarelos K, Cell-
penetrating carbon nanotubes in the delivery of thera-
peutics, Nano Today, 2007, 2: 38–43.
12. Monteiro-Riviere NA, Inman AO, Wang YY, Nemanich RJ,
Surfactant effects on carbon nanotube interactions with
human epidermal keratinocytes, Nanomedicine: Nano-
technology, Biology and Medicine, 2005.1: 293-299.
13. Martínez-Pla JJ, Martín-Biosca Y, Sagrado S, Villanueva-
Camañas RM, Medina-Hernández MJ, Evaluation of the
pH effect of formulations on the skin permeability of
drugs by biopartitioning micellar chromatography, J.
Chromatogr. A., 2004,1047: 255-262.
14. Degim IT, Verma S, Papadimitrakopoulos F, Burgess D,
Carbon nanotubes (CNTs) for enhancing drug transport
through skin, Controlled Release Society Annual Meet-
ing & Exposition, July 12-16 2008, New York, U.S.A. (Oral
presentation).
15. İlbasmış Tamer S, Yılmaz Ş, Degim IT, Nanoscience Nano-
technology, Nanobiotechnology, Nanomedicine Con-
ference, June 11-14-2007. Bilkent, Ankara, Turkey (Oral
presentation).
16. Xin B, Yokoyama Y, Shigeto T, Mizunuma H, Anti-tumor ef-
fect of non-steroidal anti-inflammatory drugs on human
ovarian cancers, Pathology Oncology Research,2007,13:
365-369.
17. Piffara PM et al,, Naproxen, clenbuterol and insulin ad-
ministration ameliorates cancer cachexia and reduce
tumor growth in Walker 256 tumor-bearing rats, Cancer
Letters, 2003, 201: 139–148.
18. Chen JH, Liu TY, Wu CW,. Chi C, Nonsteroidal anti-inflam-
matory drugs for treatment of advanced gastric cancer:
cyclooxygenase-2 is involved in hepatocyte growth
factor mediated tumor development and progression,
Medical Hypotheses, 2001, 57: 503-505.
19. Ilbasmıs-Tamer S, DEGIM IT, Carbon nanotubes to de-
liver drug molecules, NanoTR5, , 5th National Congress
on NanoScience and Nanotechnology, June 8-12-2009.
Bilkent, Anadolu University, Eskişehir, Turkey (Poster pre-
sentation).
 ORAL PRESENTATIONS

O12
Colloidal Dispersions for Targeted Brain Drug Delivery
Paolo Blasi1, Aurélie Schoubben1, Stefano Giovagnoli1, C. Puglia2, L. Barberini3, C. Cirotto3, Carlo Rossi1,
Maurizio Ricci1
1
Dipartimento di Chimica e Tecnologia del Farmaco; Università degli Studi di Perugia, Italy
2
Dipartimento di Scienze Farmaceutiche, Università degli Studi di Catania, Italy
3
Dipartimento di Biologia Cellulare e Ambientale, Università degli Studi di Perugia, Italy

INTRODUCTION gies was employed to optimize the fabrication procedure and

The concentration and clearance of endogenous and ex-
ogenous molecules in the brain are strictly regulated by its
anatomic and physiologic features, such as the blood-brain
barrier (BBB). Furthermore, the enormous efforts made for
the individuation of new specific and powerful drug candi-
dates are often vain. For these reasons, different technologi-
cal strategies have been proposed and evaluated to solve
the BBB issue. Among them, the use of nanoparticles (NPs)
has been regarded as having great potential for the delivery
of drugs into the CNS1. In particular, polysorbate-coated NPs
have shown the capability to deliver active molecules within
the brain parenchyma, probably thanks to a preferential apo-
lipoprotein adsorption and successive BBB transcytosis2,3.
to individuate the most favourable working conditions. The
obtained NPs were characterized using photon correlation
spectroscopy (PCS), transmission electron microscopy (TEM),
and differential scanning calorimetry (DSC). Lipid colloids
were loaded with a fluorescent dye, nile red, and loperamide,
a model pharmacologically active compound used to investi-
gate the transport across the BBB. Method reproducibility and
long term stability were investigated.
In vivo biocompatibility studies were performed on chick
embryos using the chorioallantoic membrane (CAM) assay.
Fertilized eggs, windowed at day 4 and treated at day 7, were
monitored for 1, 2 and 5 days after treatment.

In the scaring scenario of a continuous growing incidence of RESULTS AND DISCUSSION

CNS pathologies (in Europe ~35% of all illnesses is represented
by CNS diseases), a fast translation from the benchside to the
bedside is highly desirable. Unfortunately, most of the nano-
technologies developed in the last two decades to deliver
drugs within the brain suffer from serious drawbacks, such as
low biocompatibility of the employed material, limited carrier
stability and difficult, if not impossible, industrial scale-up1.
Therefore, the aim of this study is to develop a highly stable
and biocompatible colloidal dispersion using an easily scal-
able method.
MATERIALS AND METHODS
Materials
Cetyl palmitate (CP) (m.p. ~ 55°C) was a kind gift of Gattefos-
sé (Milan, Italy); polysorbate 80 (HX)™ of injectable grade was
a gift of NOF corporation (Tokyo, Japan). All other materials
were of analytical grade and used as provided.
Colloidal dispersion preparation
CP (5g) was heated above its melting point and added to a
hot aqueous solution of surfactant (Polysorbate 80, P80) un-
der mixing with a high-speed stirrer (Ultra Turrax T25, Jahnke
and Kunkel, Germany) for 1 minute at 8000 rpm to form a pre-
emulsion. Homogenization was carried out by using an Emul-
siFlex-C5 homogenizer (Avestin, Ottawa, Canada) at the same
temperatures used to prepare the pre-emulsion and at a pres-
sure of 1500 Bar. Ten homogenization cycles were performed.
The hot dispersion was immediately cooled in an ice bath. A
statistical approach exploiting response surface methodolo-
Hot high pressure homogenization was chosen as produc- 25
tion methodology due to avoidance of toxic solvents, high
reproducibility and easy scalability.
Even though both surfactant concentration and number
of homogenization cycles were seen to affect colloid mean
diameter (MD) and polydispersity, expressed as Gaussian
distribution width (GDW), a preponderant effect of P80 con-
centration on nanoparticle dimensions was found. ANOVA
validation showed a high adequacy (P<0.0001) and non-
significant lack-of-fit for both models (MD and GDW), con-
firmed also by the values of the variance (1.76; 5.70) and the
correlation coefficients (r2 0.9622; 0.7773). Check-point anal-
ysis confirmed the good agreement between predicted and
experimental values. Seven homogenization cycles and 2%
of P80 were individuated as optimal fabrication conditions.
NPs were prepared in replicate of four to assess method repro-
ducibility. The prepared formulations showed a mean diameter
of 185 ± 8 nm, a method yield of ∼92 % and a good physical
stability at 4°C for 18 months. The encapsulation of the model
drug, loperamide, and of the fluorescent die, nile red, did not in-
fluence significantly particle size and distribution. Loperamide
was entrapped with an encapsulation efficiency of about 70%,
corresponding to 2.3% of drug content. TEM analysis evidenced
a similar morphology for blank and loaded NPs.
CAM assay showed high biocompatibility. In fact, neither in-
flammatory reaction nor angiogenesis was stimulated by

October 26 - 28, 2009 / Antalya - TURKEY

 ORAL PRESENTATIONS

NPs, 2% P80 solution, and the raw material at 1, 2 and 5 days af-
ter treatment. On the contrary, carragenin and sodium dodecyl
sulphate, used as positive controls, showed a high level of in-
flammation and angiogenesis around the application site.
CP NPs possessed the characteristics needed for brain drug
delivery and, for this reason, their capability to deliver drugs
to the brain is under evaluation.
REFERENCES
1. P. Blasi, S. Giovagnoli, A. Schoubben, M. Ricci, C. Rossi,
Solid lipid nanoparticles for targeted brain drug deliv-
ery. Adv. Drug Deliver. Rev. 59 (2007) 454-477.
2. J. Kreuter, Application of nanoparticles for the delivery
of drugs to the brain. Int. Congr. Ser. 1277 (2005) 85-94.
3. A. Zensi, D. Begley, C. Pontikis, C. Legros, L. Mihoreanu,
S. Wagner, C. Büchel, H. von Briesen, J. Kreuter, Albu-
min nanoparticles targeted with ApoE enter the CNS by
transcytosis and are delivered to neurones. J. Control.
Release 137 (2009) 78-86.

26

3rd BBBB International Conference on Pharmaceutical Sciences

 ORAL PRESENTATIONS
O13
Novel Lab-on-chip devices towards nanomedicine study at single
cell level
Giorgia Pastorin1 and Vincent Lee2
1 Department of Pharmacy, National University of Singapore
2 Department of Electrical and Computer Engineering, National University of Singapore
ABSTRACT
Single-cell detection is a crucial aspect in several branches
of medicine and biology,allowing the study of immune sys-
tem function, disease diagnosis and natural cell death. With-
regards to biology, an in-depth evaluation of the activities
associated with a particular cell, interms of its molecular up-
take or production of unique features at its surface. Similarly,
in the caseof medicine, the clear understanding of cells’ be-
haviour, on the basis of their exposure toparticular stimuli
and conditions, represents a fundamental requirement in
the development ofbioactive agents aimed to target a spe-
cific cell subtype without unwanted side effects.
Several approaches have been recently developed to iden-
tify a limited number of cellswith good accuracy, and they
have being divided into two main classes, namely Poly-
meraseChain Reaction(PCR)-based methods and cytometric
procedures. Both of them show advantagesand drawbacks,
so that no ideal technique is available. PCR in general shows
more sensitivity,since it is usually based on the design of spe-
cific sequences for the gene(s) of interest, but on theother
hand it might fail in distinguishing between intact units and
genetic material shed from dyingcells. On the other hand,
flow cytometry represents a valuable technique, dealing
with theintroduction of cells into an artificial flow and a “real-
time” laser counting each cell during itspassage through a
single-file flow. Unfortunately, some cell loss is inevitable,
and cell entrapmentis not feasible with this technique. In
other words, the development of techniques able not only
toidentify cells, but also to isolate them from an entire cell
population in order to carry out theirmorphological and mo-
lecular characterization, is extremely lacking and defective.
Therefore, in this project we present a novel cytometric sys-
tem consisting of an advanceddevice that is able to isolate a
few cells among a heterogeneous mixture, and to enhance
theentrapment yields, which still represent the limiting as-
pect of this methodology. To that purpose,we will combine a
micromechanical fluidic approach with a detection mecha-
nism within a uniquecell-based lab-on-chip (LOC) device, in
order to provide a fast and effective tool able to entrapcells
mainly on the basis of their dimensions. The realization of
the LOC device required an initialphase of platform design,
in which we chose samples of blood as it is made up of het-
erogeneouscells and platelets, with dimensions ranging
from a few micrometers (e.g. erythrocytes, of 6-8 μm)to huge
October 26 - 28, 2009 / Antalya - TURKEY
macrophages (of about 21 μm). The basic separation princi-
ple, analyzed with manysimulation designs, makes use of the
inherent characteristic size differences between thedifferent
cell populations, to provide a quick and simple separation
method. Coupled withmicromechanical filters and microflu-
idics designs, some cell prototypes (e.g. cancer cells) have-
demonstrated to be effectively segregated from the majority
of other cells without the need foradditional active control
or external forces. It has been shown, through fluidic simula-
tion, that cellsof larger radius (our target, of about 30-40 μm)
are readily exposed to the central flow and carriedaway from
the blood stream. A row of pillars with certain separation
gap (of about 8 μm) islocated further in the downstream and
arranged either parallel or oblique to the flow. This allowsthe
smaller cells type to be efficiently removed from the main
channels. Hence, only the cells ofinterest are obtained at the
end of the main channel. If confirmed with real samples, this 27
resultcould overcome the drawbacks of current diagnostic
approaches, since it enables an efficientidentification and
isolation of a few abnormal cells circulating into the blood-
stream, thus providingan early prognosis of more problem-
atic diseases.
So far, research studies aimed to detect a few cell subtypes
have often shownremarkable limitations in terms of rapid-
ity of sample processing, entrapment yields andinformation
regarding the mechanisms involved in cellular response.
Taking into account all theseinadequacies, we have elabo-
rated a promising device that not only is able to surmount
thecurrent problems, but it also paves the way for interest-
ing advances in the field ofNanomedicine. In fact our fluidic
simulations, together with preliminary estimations, seem
tosupport ambitious goals: first, our platform provides tools
for rapid screening of cell populations inorder to identify a
few cell subtypes at very low concentrations. The main ad-
vantage relies on theuse of a disposable chip that leads the
evaluation of several cells at the same time, whilesignificant-
ly raise up the flow rate, such that micro-litre sample will be
examined in a range of fewtens of minutes to one hour. This
will be achieved through a high-pressure flow into the LOC
witha concomitant increase of the flow rate. Second, it in-
corporates advanced technologies to entrapthese cells and
allow further investigations “in real-time”. Results obtained
from additional studiesat individual cell level are expected to
revolutionize cell diagnostics in vitro. Finally, it offersunique
possibilities to enhance week signals through a read-out sys-
 ORAL PRESENTATIONS
tem. As concerns this lastaspect, integrated signal enhancer
will enable us to characterize individual cells simultaneous-
lywhen treated with nanomedicines. This additional strategy
should enable us to overcome the lackof sensitivity usually
attributed to conventional detection approaches.
A further logical development of this project is represented
by the combination of our LOCwith a drug delivery system
(DDS), in order to study the effects of the administration of
bioactiveagents at the single cell level. Amidst the myriad
of systems currently employed for drug delivery,carbon
nanotubes (CNTs) represent intriguing devices, due to their
ability to carry bioactivederivatives and, at the same time,
molecules that can increase cell penetration. Previous in vit-
rostudies on uptake of functionalized CNTs by cell lines, while
providing useful insights on thedynamics of penetration, yet
gave limited information regarding the mechanism and the
distinctphysiological effects of various types of single cells or
tissues to CNTs. Due to the multiple cell-tocell interactions,
reactions of groups of cells often impair understanding the
intracellularmechanisms underlying the observed respons-
es. Therefore, the evaluation of single cellsexposed to CNTs
would be extremely beneficial in disclosing the real process
of interactionbetween the delivery of bioactive agents from
28
3rd BBBB International Conference on Pharmaceutical Sciences
suitable DDS and their direct cellular target. Thepresent
project is aimed to develop a novel, improved platform for
CNTs-based drug delivery andfor real-time monitoring of
responses deriving from different living cells. It is worth
mentioningthat, although CNTs represent suitable DDS, the
platform provided by the LOC device showshigh versatility:
after the identification of the cancer cell subtype, CNTs are
introduced in thedevice through a branched channel, hence
it theoretically enables other systems (such asliposomes or
polymeric structures) to be investigated as well. Using this
platform, the effects ofDDS and DDS-drug conjugates on
individual or assembled living cells could be analyzed in-
depth.
On the whole, this project offers a high degree of innova-
tion, in which state-of-the-artnanotechnology materials,
novel biosensing techniques and micromechanical systems
basedLOC are to be developed in order to elaborate a novel
detecting system for important biomedicalapplications. Due
to the advantageous aspects presented in our platform, to-
gether with the urgentneed and the precious role of these
devices, it is reasonably envisaged that the system willde-
termine a remarkable impact and a significant commercial
value in relatively short time.
 ORAL PRESENTATIONS

O14
NANOBIOTECHNOLOGY and NANOTOXICITY
Filiz Hıncal
Hacettepe University Faculty of Pharmacy Deparment of Toxicology, Ankara, 06100, TURKEY

N
anotechnology is defined as the design and manipu-
lation of materials at the atomic and molecular scale.
Exciting applications of nanotechnology in diverse
medical fields have recently provided significant health ben-
efits through the introduction of many intelligent nanomate-
rials such as biomarkers, molecular diagnostics, nanodevices,
new drug discoveries and drug delivery systems. Primary
goals of nanotechnology in drug delivery are more specific
and efficacious drug targeting and delivery, while minimiz-
ing side effects to provide greater safety and biocompatibil-
ity and leading to better patient compliance. However, there
is a concern that certain nanomaterials could have negative
impacts on human health or the environment as a result of
their new properties. The diversity of nanomaterial is vast,
and their physical, chemical and biological properties differ
from the bulk material. Results from the limited toxicological
database have fostered the perception that nanoparticles are
likely to be more toxic than fine-sized particulates, and sur-
face properties and free radical generation resulting from the
interactions of particles with cells may play important roles
in nanoparticle toxicity. Emphasizing that there exist at pres-
ent little understanding of the basics of the interactions of
nanoparticles with living cells, organs and organisms, there
are strong arguments that even if a certain nanoparticle does
not appear toxic by itself, its interaction with other common
components in human body may cause problems to cell func-
tions. It is certain that further research and developments and
improved knowledge of physiological effects and kinetics of
nanoparticles in the body, their long term effects, and persis-
tence and fate in the environment are needed as the integral
part of the technological development processes.
29

October 26 - 28, 2009 / Antalya - TURKEY

 ORAL PRESENTATIONS

O15
Poly(anhydride) nanoparticles as adjuvants for oral vaccination
Juan M. Irache
Department of Pharmaceutics and Pharmaceutical Technology, University of Navarra, 31008-Pamplona, Spain

I
n the last years, nanoparticles have been proposed as ad-
juvants for mucosal vaccination. However, conventional
nanoparticles usually display a low capability to target in
larger extent specific sites within the gut (i.e. Peyer’s patch-
es). Therefore, the immune response elicited with these
antigen carriers is usually not as high as necessary to offer
the adequate degree of protection to the host. In order to
render nanoparticles more efficient as adjuvants for vacci-
nation, one possible strategy can be their association with
compounds or molecules involved in the colonization pro-
cess of microorganisms. This biomimetic approach may be of
interest to put in place more effective and safer vaccines.
Systemic and mucosal immune responses, reported after ad-
ministration of a single dose, indicated that both OVA-M-NP
and OVA-F-NP elicited higher and balanced systemic specific
antibody responses [IgG1 (Th2-response) and IgG2a (Th1-
response)] compared to non-coated ones. In addition, oral
immunization using OVA-M-NP or OVAF-NP was able to elicit
higher levels of intestinal secretory IgA compared to subcu-
taneous administration. All of these findings may be directly
related with the high capability and tropism of both flagel-
lin- and mannosylated-nanoparticles to interact with the
Peyer’s patches and other intestinal regions rich in antigen
presenting cells.

Thus poly(anhydride) nanoparticles (NP) were either coated In summary, oral immunization by biomimetic nanoparticles
with flagellin from Salmonella enteritidis (F) or mannosamine demonstrated strong long lasting systemic and mucosal im-
(M). The mucosal affinity of the resulting nanoparticles (F-NP mune responses than the respective non-conjugated vec-
30 and M-NP) was investigated by bioadhesion and fluores- tors.
cence microscopy studies. The immunoadjuvant proper-
ties when administered by the oral route were evaluated in
Balb/C mice using ovalbumin (OVA) as an antigen model.

3rd BBBB International Conference on Pharmaceutical Sciences

 ORAL PRESENTATIONS

O16
Different medicinal forms of bacteriophage preparations for
treatment of infectious diseases
Dr. Mzia Kutateladze
G. Eliava Institute of Bacteriophages, Microbiology and Virology, Tbilisi, Georgia

D
uring several years, phage therapy was included as a
powerful tool against infectious diseases of bacterial
etiology. Unfortunately, phage treatment was forgot-
ten in the Western World shortly after antibiotics became
widely available. Although, nowadays rapid increase of multi-
drag resistant bacterial strains develop renewed interest to-
wards phage therapy.
Phage therapy remained as a standard part of healthcare
system in Eastern Europe and USSR. Phage preparations
were used for therapy, prophylaxis and diagnostic of various
bacterial infections. Such preparations have been success-
fully used against intestinal problems (dysentery, diarrhea,
typhoid) and purulent-septic infections (wounds, infectious
complications of burns, inflammation of organs, etc).
Bacteriophages have number of advantages in comparison
to antibiotics; they are ecologically safe - harmless to human,
plant and animal; they are very specific and do not affect
normal microflora; phage preparations are readily available
and easy to apply. There is no resistance appears to multi-
component phage preparation. Today, the uncontrolled use
of antibiotics (among other factors) has limited the efficacy
of their treatment and has contributed to the wide-spread
evolution of multi-drug-resistant bacterial pathogens. Phage
therapy as an alternative approach for treatment of infec-
tions has become an evident and promising remedy.
G. Eliava Institute of Bacteriophages, Microbiology and Vi-
rology, Tbilisi, Georgia is the most famous institutions in the
world focused on bacteriophage study, particularly isola-
tion and selection of phage components, active to various
pathogenic and conditionally pathogenic bacterial strains.
The preparations developed in the Eliava Institute have been
studied through extensive preclinical and clinical trials. How-
ever, little of this information has ever been published and
even when details are available, the trial reports do not meet
internationally approved regulations and standards.
Depending of the source of infections to be cured, the phage
preparations have been produced at the Eliava Institute in
different forms: liquid (ampoules, vials), ointments, sup-
positories, tablets, preparation for intravenous application.
The method for construction of phage-based product –
phagebioderm has been developed at the Institute together 31
with the scientists from the Tbilisi Technical University. This
preparation includes original biodegradable polymer that
is impregnated with phages against several pathogens. The
original technology for preparing bioactive composite act-
ing in bacteriophages’ sustained/controlled release fashion
is developed at the Institute. The preparation is successfully
used in infected wounds, burns, tropical ulcers etc.

October 26 - 28, 2009 / Antalya - TURKEY

 ORAL PRESENTATIONS

O17
Utilization of Bacteriophages and lytic enzymes in Treatment and
Prevention of Staphylococcus aureus MRSA and other Infections
David Trudil1, Michał Bartoszcze2, Romuald Gryko2, Mzia Kutateladze3.
1Battelle/NHDe, 12616 Mt. Laurel Ct, Reisterstown, MD 21136 USA davidt@nhdetect.com
2Biological Threats Identification and Countermeasure Center of MIHIE, Pulawy, Poland
3G. Eliava Institute of Bacteriophages, Tbilisi, Georgia

B
acteriophages (phage), one of the most common or-
ganisms on earth, are viruses which infect bacteria.
The discovery of bacteriophages in the early 1900s had
primarily pointed to the possibilities of using them as natural
antibacterial agents; and led to numerous attempts to use
them to treat bacterial infections in humans and animals.
The discovery and subsequent practical application of anti-
biotics (1940) halted both research work and clinical studies
in this area (particularly in the West) for many years; and it
was only the growing number of bacterial strains resistant
to antibiotics that brought phage therapy into the limelight
again. These studies concerning the possibilities offered by
phage therapy, however, were centered mainly in the former
Soviet Union and Poland.
Recent efforts focused on phages lytically active to a number
32 of pathogens, including antibiotic-resistant strains, are gen-
erating the most interest. Worth noting is the lytic activity
to MRSA (Methicillin-resistant Staphylococcus aureus),
VRE (Vancomycin-resistant enterococcus), multi-resistant
strains of Pseudomonas aeruginosa, or Escherichia coli which
produce lactamase of extended substrate spectrum (ESBL).
These harbour in purulent infections (wounds, dermatologi-
cal complications) as well as respiratory system and urinary
tract infections.
The G. Eliava Institute of Bacteriophages, Microbiology and
Virology in Tbilisi has developed a phage cocktail which has
been proven to be effective against MRSA. The results of a
limited, one person challenge study of a chronically ill, MRSA
infected patient treated with the Eliava phage cocktail will
be provided. Additionally, the status of phage therapies, as
well as phage derived treatments and preventatives will also
be discussed.

3rd BBBB International Conference on Pharmaceutical Sciences

 ORAL PRESENTATIONS

O18
Bacteriophages as vaccine delivery vehicles
Jason Clark and John March
BigDNA Ltd., Wallace Building, Roslin Biocentre, Midlothian, EH25 9PP,UK.

B
acteriophages (or phages) are viruses which infect bac- We are currently designing an optimised lambda vector in
teria. Most phages are relatively simple and a number which the genetic material will be customised and produced
of systems are available where the genetic material or entirely synthetically. It will include several safety features
protein coat can be easily modified. This versatility makes and optimisations to improve yield. We are also developing
them ideal tools for nanotechnology and for biotechnology the means to grow these phages in fermentors at high yield
in general. In recent years phages have been used as alter- and the procedures to purify them to a level suitable for in-
natives to antibiotics, as biosensors, for targeting specific cell jection into humans.
types in anti-cancer therapies, as molecular scaffolds in bat-
tery production or as drug or vaccine delivery vehicles1,2. In May 2007, BigDNA ltd. was set up to commercialise the
phage vaccine technology. It has several patents including
One of the most well characterised phages is phage lambda, those for genetic and chimeric phage vaccines. We are cur-
which has a 50 nm head, is 200nm long and has a 50 kilobase rently planning and performing pre-clinical trials and aim to
genome (figure 1). We have used bacteriophage lambda as a start phase I clinical trials in 2011.
DNA vaccine delivery vehicle. In standard DNA vaccination,
genetic material containing a vaccine gene in a form that will
be expressed in mammalian cells is used as an immunogen.
Host cells near the site of injection take up the vaccine DNA, 33
the vaccine protein is expressed and an immune response is
raised. In bacteriophage vaccination, a DNA vaccine cassette
is cloned into the genome of the phage and whole phage
particles are used to inoculate the host. To the immune sys-
tem, phages structurally resemble a pathogenic virus and
they are rapidly cleared by the host’s immune system to
sites of antigen presentation (i.e. where the immune system Figure 1: Schematic representation of a bacteriophage lamb-
works most efficiently), where the vaccine component is ex-
da particle.
pressed. In effect, the host’s immune cells are turned into
vaccine producing factories. We have tested several vac-
cines in a number of animal species (mice3, rabbits4, sheep5
and fish). Example results using a vaccine against Hepatitis B
are shown in figure 2.

Phage vaccines have several advantages over traditional vac-

cines. They are relatively cheap to produce and purify, being
produced on E. coli in simple culture media. It is also therefore
very rapid to produce large quantities of vaccine. The phage
coat protects the DNA making the vaccines very stable, so
they are easy to store and transport6. No antibiotic resistance
is required on the vector and large inserts (up to 20kb) can be
used, so in theory multiple vaccines can be delivered in the
same vector. The versatility of the phage technology confers
a great deal of flexibility onto the platform. For example, it is Figure 2: Comparison of antibody responses in rabbits (5 per
possible to modify the phage coat to display fusion proteins, group) given a commercially available protein vaccine (A), a
so that the phages themselves can be targeted to specific cell bacteriophage vaccine based on the same antigen (B) and a
types. Alternatively a vaccine protein can be displayed on the control phage (C). Vaccinations were at weeks 0, 4 and 8. Re-
phage surface, so that protein and DNA vaccines are delivered sponses were measured by ELISA against the recombinant
on the same construct (a chimeric vaccine - figure 3). protein version of the antigen

October 26 - 28, 2009 / Antalya - TURKEY

 ORAL PRESENTATIONS

2. Clark JR and March JB. (2006). Bacteriophages and Bio-

Figure 3: Modifying bacteriophage lambda to display vac-
cine proteins, contain DNA vaccines or a combination of
both. The whole phage particle is used as the vaccine.
REFERENCES
1. Clark JR, March JB. (2004). Bacterial viruses as human
vaccines? Expert Review of Vaccines 3, 463-474.
technology: Vaccines, Gene therapy and Antibacterials.
Trends Biotechnol. 24, 212-218.
3. March, J.B. et al. (2004) Genetic immunisation against
hepatitis B using whole bacteriophage lambda particles.
Vaccine 22, 1666-1671.
4. Clark, J.R. and March, J.B. (2004) Bacteriophage-medi-
ated nucleic acid immunisation. FEMS Immunol. Med.
Microbiol. 40, 21-26.
5. Rocchi RM, Jepson CD, Clark JR, March JB, and McKeever
DJ. (2003). Efferent lymphatic cannulation for the evalu-
ation of immunogenicity of DNA vaccines. Immunology
110 (Supplement 1), 121.
6. Jepson, C.D. and March, J.B. (2004). Bacteriophage lamb-
da is a highly stable DNA vaccine delivery vehicle. Vac-
cine 22, 2413-2419.

34

3rd BBBB International Conference on Pharmaceutical Sciences

 ORAL PRESENTATIONS

O19
PHAGE-TARGETED MEDICATIONS
Valery A. Petrenko
Department of Pathobiology, Auburn University, AL 36849, U.S.A.,
Professor, office:334-844-2897; fax: 334-844-2652;

T
he more than 20-year evolution of phage display has
dramatically affected the potential of this technique
amid other bioengineering methods. The marriage of
combinatorial chemistry and biological selection has
been very powerful changing the methodology of biochemi-
cal research by allowing selection simultaneously among bil-
lions of genetic species in one test tube. Genetically driven
phage biotechnology has allowed development of libraries
of diverse nanostructures expressed on the phage surface
providing a rich resource of diagnostic, detection and phar-
maceutical probes. Phage engineering, which is based on
natural mechanisms of selection, amplification and self-as-
sembly, allows directed fabrication of bioselective materials
with possible applications in gene/drug-delivery, biosensors,
biosorbents, and other areas of medicine and technology. In
particular, landscape phage expressing tumor-specific pep-
tides fused to all copies of the major coat protein pVIII can be
converted easily into gene-encapsulating particles or drug-
loaded vesicles that acquire the ability to recognize the same
receptors, cells, tissues and organs that have been used for
selection of the precisely targeted phage. The merge of
phage display technology with nanotechnology during the
last several years is very promising and has already shown its
vitality and productivity contributing vigorously to different
areas of medicine and technology, such as medical diagnos-
tics and monitoring, molecular imaging, targeted drug and
gene delivery, vaccine development, as well as bone and tis-
sue repair. This presentation focuses on the progress made
in the development of these new materials and discusses
the prospects of using phage as a bioselectable molecular
recognition interface in medical and technical devices based
on the experience of the author in this area. 35

October 26 - 28, 2009 / Antalya - TURKEY

 ORAL PRESENTATIONS

O20
Novel glutathion analogues and their effects in biological systems
Ursel Soomets
Department of Biochemistry, Faculty of Medicine, University of Tartu, The Centre of Excellence of Translational
Medicine, Ravila Str. 19, 50411 Tartu, Estonia

G
lutathione (GSH, γ-Glu-Cys-Gly) is the major intracel-
lular low molecular weight antioxidant. Its intracellu-
lar concentration is up to 10 mM, whereas extracel-
lular concentrations are relatively low (5-50 µmol/l). Most of
the cellular reduced glutathione (GSH; 85-90%) is located in
cytosol where it serves as non-enzymatic water-soluble an-
tioxidant. Additionally, GSH carries several other functions in
the human body concerning the metabolism of xenobiotics
and eicosanoids, cellular signalling and thiol disulphide ex-
change reaction. High grade oxidative stress and decrease of
GSH level is related to the development of several pathologi-
cal states including cardiovascular diseases, neurodegenera-
tive diseases, cancer, HIV, inflammation, psoriasis, ischemia/
reperfusion injury, chronic obstructive pulmonary disease
(COPD) etc. Compensatory administration of GSH is not ef-
fective because of its rapid degradation, while more stable
36 and nontoxic glutathione analogues could be an effective
alternative.
The aim of our study was to create more stable and effective
antioxidants than GSH, investigate their structure-activity re-
lationships and describe their biological activity in psoriasis
and COPD patient blood cells.
By adding a fourth amino acid to the GSH molecule, we de-
signed and synthesized library of tetrapeptidic glutathione
analogues called UPF peptides. We showed that UPF pep-
tides were far more active hydroxyl radical scavengers than
glutathione (EC50 of GSH: 1231.0 mM; EC50 of UPF peptides:
from 0.03 to 35 μM. GSH had slightly lower EC50 of DPPH radi-
cal scavenging than UPF peptides but comparing antiradi-
cal efficiencies (reflects the speed of DPPH scavenging), UPF
peptides exceeded GSH by antioxidative potential. In both
scavenging reactions the most effective structural modifica-
tion was the substitution of the γ-Glu with the α-Glu moiety
in GSH/UPF backbone. UPF peptides did not affect the viabil-
ity and membrane integrity of human erythroleucemia K562
cells. The influences of selected UPF peptides (UPF1, UPF6,
UPF17, UPF19) on the activities of antioxidant enzymes (glu-
tathione peroxidase (GPx), -reductase (GR) etc.) and poten-
tially pro-oxidative NAD(P)H oxidase were investigated. UPF
peptides inhibited GPx activity only at high concentrations
and this effect was more expressed for α-glutamyl moiety
containing UPF17; did not affect the activity of GR but inhib-
ited the human neutrophil NAD(P)H oxidase.
Psoriasis is a chronic inflammatory T-cell mediated auto-
immune disease. Many different factors such as genetic
features, immunological disorders, biochemical changes,
environmental effects, trauma and stress contribute to the
pathogenesis of psoriasis. Recent studies have suggested
that increased ROS production and deficient function of an-
tioxidant systems activities play a role in the pathogenesis of
psoriasis. We found that the expression levels of GSH-linked
enzymes in the peripheral blood mononuclear cells (PBMC)
of psoriasis patients were lower compared to healthy sub-
jects. Statistically significant differences in “glutathione sys-
tem” enzymes expressions were established between the
study groups. Influence of antioxidant peptides (UPF1 and
UPF17) to the expression of enzymes involved in glutathione
metabolism was examined in the PBMCs. UPF peptides had
different efficacy on control and patient cells, but in general,
they were more effective in patient samples and normalized
the expression of studied enzymes to the same level as con-
trols.
Chronic obstructive pulmonary disease (COPD) is an increas-
ing global health problem that is characterized by a specific
pattern of chronic inflammation in small airways and lung pa-
renchyma. Cigarette smoking is the major etiological factor
in this condition. COPD kills one person in every 10 seconds
worldwide. The effects of UPF analogues (UPF1 and UPF17)
on the antioxidative and proinflammatory enzymes expres-
sion levels in the blood mononuclear cells were studied. We
found that these glutathione analogues affect expressions
of studied enzymes by unknown mechanism.
Our study confirmed that UPF peptides are promising leads
for design of powerful non-peptidic antioxidants that can be
used in treatment of different diseases involving oxidative
stress.

3rd BBBB International Conference on Pharmaceutical Sciences

 ORAL PRESENTATIONS

O21
THE ROLE OF ABC MULTIDRUG TRANSPORTERS IN NORMAL
AND TUMOR STEM CELLS
András Váradi1, Gergely Szakács1, László Homolya2, Csilla Özvegy-Laczka2, and Balázs Sarkadi2
Institute of Enzymology, Hungarian Academy of Sciences, H-1113 Budapest, Hungary,
1

2
Membrane Research Group of the Hungarian Academy of Sciences, H-1113 Budapest, Hungary,

F
orty-eight genes coding ABC-transporters have been
annotated in the human genome. Most of these pro-
teins facilitate outward transport of small molecules
from the cytosol, thus functioning as export pumps, while
some of them act as ion channels or ATP/ADP-sensors/recep-
tors regulating ion channels.
The human ABC drug transporters play an important role in
cancer drug resistance, protection against xenobiotics, and
in general in the passage of drugs through cellular and tis-
sue barriers. This review explores how human ATP-Binding
Cassette (ABC) transporters modulate the pharmacological
effects of various drugs, and how this predictable modula-
tion can be used during the process of drug discovery and
development. We provide a description of the relevant hu-
man ABC drug transporters, and review the models and as-
say systems that can be applied for the analysis of their ex-
pected drug interactions. The use of the in vitro, in vivo, in
silico models, their combination, and the emerging clinical
information are evaluated with respect to their potential
application in early drug screening. We also discuss the de-
tails of the chemoimmunity network hypothesis explaining
how a very few ABC transporters can be involved in a very
wide substrate recognition thus providing a defense system
against toxic xenobiotic and metabolites.

37

October 26 - 28, 2009 / Antalya - TURKEY

 ORAL PRESENTATIONS

O22
Characterization of novel polimeric materıals from the point of
theır physical ageıng
Romána Zelkó and Virág Szente
University Pharmacy Department of Pharmacy Administration Semmelsweis UniversirtH-1092 Budapest, 7-9 Högyes
E. Street, Hungary

D
ifferent polymers are widely used in pharmaceutical
technology for a diversity of purposes. They can be ap-
plied as fillers, binders, matrix-forming and film-form-
ing excipients in solid dosage forms, and most of the gelling
agents of semisolid preparations and solutions are polymers,
as well. Furthermore, a wide variety of packaging materials is
based on macromolecules. Although the chemical structure
of the above mentioned polymers is rather heterogeneous,
their physical state is usually similar, as most of them are
amorphous or partly amorphous. A well-known property of
such materials is that they undergo physical ageing, which is
accompanied by volume and enthalpy relaxation and thus
might result in severe structural changes in the polymer. The
enhanced molecular mobility, caused by the plasticization
effect of absorbed water, has been proposed to be the ma-
jor underlying factor in chemical and physical instability of
38 amorphous pharmaceutical materials. This so called physi-
cal ageing can be initiated by several factors (e.g. humidity)
and is manifested in macrostructural (enthalpy and volume)
and microstructural (free volume distribution) alterations.
These changes also occur during the processing and storage
of pharmaceutical dosage forms containing such polymers,
thus influencing their stability and bioavailability. Tracking
the ageing process of polymeric excipients is possible using
an array of techniques. Macrostructural changes can be fol-
lowed by dilatometry and calorimetry, while microstructural
alterations can be monitored by means of positron annihila-
tion lifetime spectroscopy, electron spin resonance spectros-
copy, fluorescence spectroscopy, small angle X-ray scattering
and scanning electron microscopy. Such investigations are of
high importance because of the consequences considering
the stability of the excipients themselves and dosage forms
prepared using these materials.

3rd BBBB International Conference on Pharmaceutical Sciences

 ORAL PRESENTATIONS
O23
New analytical technologies and materials for determination of
enantiomeric purity of chiral drugs
Bezhan Chankvetadze
Department of Chemistry, School of Exact and Natural Sciences and Molecular Recognition and Separation Science
Laboratory, Tbilisi State University, Chavchavadze Ave 1, 0179 Tbilisi, Georgia
C
hirality represents increasingly important issue in drug
development and use. The world-wide market for chi-
ral fine chemicals reached US dollar 6.63 billion in 2000
and is expected to grow at 13.2% annually1. The drug indus-
try is considered to be the engine that is driving this strong
growth, accounting for 81.2% of the total, or US dollar 5.38
billion worth. These numbers become even more impressive
when transferred to drug formulations. Thus, the world-wide
market for dosage forms of single-enantiomer drugs was US
dollar 123 billion in 2000, up 7.2% from US dollar 115 billion
in 19991. The major current trend in enantioseparations is to
leave the classical column size and separation scale in two
opposite directions: On the one hand chromatography is
becoming a valuable tool for obtaining of enantiomerically
pure drugs on preparative and production scale. On the oth-
er hand, the techniques for analytical-scale enantiosepara-
tions are continuously miniaturized and microtechnologies,
such as capillary liquid chromatography (CLC), capillary elec-
trophoresis (CE) and capillary electrochromatography (CEC)
are gaining importance in this field. In addition, lab-on-chip
technologies are establishing very rapidly. This presentation
summarizes new developments for preparation of effective
chiral stationary phases for liquid phase enantioseparations
as well as the innovations from the viewpoint of applied sep-
aration technologies.
Enantiomeric impurities, as well as other impurities in active
pharmaceutical ingredients and drug formulations shall be
determined at the level below 0.1%. This applies very strict
requirements to the analytical technology used for determi-
nation of enantiomeric purity of chioral drugs.Together with
separation techniques some other instrumental techniques
such as polarimetry and nuclear magnetic resonance (NMR)
spectroscopy can be used for determination of enantiomeric
purity of chiral drugs. However, chromatographic separa-
tion techniques offer important conceptual advantages
compared to the “one step” processes such as polarimetry
and NMR spectroscopy. In chromatography as in a “multi-
step” separation method the overall resolution of the solute
originates from a large number of stereoselective “one-step”
adsorption-desorption cycles. The cumulative nature of the
chromatographic separation is the reason that a free energy
difference in interactions of enantiomers as small as 0.025 kJ
mol-1 may in principle be sufficient for baseline chromato-
graphic enantioseparations, whereas the free energy dif-
ference required in “one-step” techniques is several orders
October 26 - 28, 2009 / Antalya - TURKEY
higher. In addition, the less strict requirement to the value
of enantioselectivity allows to employ effectively a large
number of chiral selectors with relatively low chiral recogni-
tion ability. In chromatography it is possible to obtain both
enantiomers in the optically pure from, even when the chiral
selector has an optical purity less than 100 %. Moreover, the
absence of irreversible chemical reactions or phase transi-
tion during direct enantioseparations further favours the
application of this technique for analytical and preparative
scale enantioseparations.
Recent developments in chiral chromatography (more uni-
versal, easily available, stable, tailor-designed chiral station-
ary phasess) and technology (recycling, displacement and
especially, simulated moving bed (SMB)) makes chromatog-
raphy a valuable alternative to the classical techniques for
the analysis and preparation of pure enantiomers. 39
At present, high performance liquid chromatography (HPLC)
with common size columns (250x4.6 mm) still dominates
chromatographic enantiomeric analysis in industrial labo-
ratories. However, microanalytical techniques such as CLC,
CE and CEC are progressing very rapidly. It must be noted
that the acceptance of aforementioned microanalytical tech-
niques is not always the same by the conservative communi-
ty of separation scientists. Thus, despite the early remark by
Jorgenson that “The high theoretical plate counts make CE
particularly amenable to separating chiral molecules” [2] this
technique could not be established as the major technique
for enantiomeric purity determination of chiral pharmaceu-
ticals. CLC relies certainly on the same separation principle as
HPLC but offers the advantages of miniaturisation compared
to the latter one. In particular, CLC requires less amount of
the packing material, mobile phases and samples. It is cost-
effective and environmentally friendly technique and offers
also the advantage of higher sensitivity when coupled with
mass spectrometer.
CE offers higher peak efficiency compared to HPLC. It is clear
that even only at the expense of higher peak efficiency CE
may allow to observe enantioseparations for certain chiral
analyte-selector pairs where the separation power of HPLC
is insufficient for achieving this goal. In addition, chiral CE
offers almost unlimited possibility from the viewpoint of
adjustment of separation factor and the criticism “The effi-
ciency of CZE is high but, the flexibility of chromatography
 ORAL PRESENTATIONS
in adjusting separation factor is lost” 3 does not apply at all to
chiral CE. Together with aforementioned conceptual advan-
tages CE offers some favorable technical characteristics for
achieving high separation selectivity. Thus, chiral stationary
phases (CSPs) in HPLC contain commonly limited and pre-
defined amounts of a chiral selector, whereas the concentra-
tion of a chiral selector is easily variable and just limited by
the solubility (for charged selectors also with Joule heating)
of a chiral selector in a CE buffer. In addition, the combination
of two or more (chiral) selectors is technically much easier
and not associated with instrumental difficulties in CE com-
pared to column-coupling in HPLC. Again, two columns are
coupled with given amounts of the chiral selectors in HPLC
whereas the ratio of chiral selectors in a combination can be
easily optimized in CE. Thus, chiral CE offers really enormous
flexibility from the viewpoint of the adjustment of the sepa-
ration selectivity. This in combination with the inherently
high separation efficiency makes chiral CE a very powerful
technique for enantioseparations4.
Another electromigration technique, in particular CEC most
likely does not offer a different separation mechanism for
enantiomers compared to HPLC but higher peak efficiency.
Basically there are three different effects which may be har-
nessed in order to obtain higher peak efficiency in CEC com-
pared to CLC: a) The plug-like flow profile of the electrically-
driven flow; b) the potential of the application of smaller
particle size materials and longer separation beds and c) the
40
3rd BBBB International Conference on Pharmaceutical Sciences
perfussive flow through the pores of the particles. As recent
comparative studies on CLC and CEC indicated, the high
plate numbers of CEC may allow observing the enantiosepa-
rations in this technique under the conditions in which CLC
hardly provides any indication of chiral recognition5.
In this presentation newly developed particulate and mono-
lithic chiral stationary phases will be described for liquid-
phase enantioseparations 6-8.
Thus, in the coming years separation science may turn from
the workhorse HPLC with standard size chromatographic
columns to microanalytical techniques (CLC, CE, CEC) for
enantiomeric purity determination of chiral drugs.
REFERENCES:
1. S.C. Stinson, Chem. Eng. News, 2001, 79 (20), 45.
2. A. M. Rouhi, Chem. Eng. News, 2003, 81 (18) 56.
3. G. Guiochon, Int. Lab., 1999, January, 13C.
4. B. Chankvetadze, Capillary Electrophoresis in Chiral Anal-
ysis, 1997, Wiley&Sons, Chichester, UK.
5. B. Chankvetadze, J.Sep. Sci., 2001, 24, 691.
6. S. Fanali, G. D’Orazio, K. Lomsadze, B. Chankvetadze, J.
Chromatogr. B, 2008, 875, 296-303.
7. K. Lomsadze, A. B. Martinez-Giron, M. Castro-Puyana, L.
Chankvetadze, A. L. Crego, A. Salgado, M. L. Marina, B.
Chankvetadze, Electrophoresis, 2009, 30, 2803-2811.
8. B. Chankvetadze, Electrophoresis, 2009, 30, S211-S221.
 ORAL PRESENTATIONS

o024
Microchips in drug analysis
Risto Kostiainen
Division of Pharmaceutical Chemistry, P.O. Box 56, FI-00014 University of Helsinki, Finland

M
iniaturization of analytical instruments utilizing mi-
cro-fabrication technology has been one of the hot-
test research topics in analytical chemistry over the
past decade. Driving force to this is an increasing demand
for low-cost instruments capable of rapidly analyzing very
small amounts of samples with a high level of automation.
A concept termed both “Miniaturized total analysis systems
(µ-TAS)” and “Lab-on-a-chip” aims to develop integrated mi-
cro-analytical systems to perform complete analysis cycles
(e.g. sample pre-treatment, chemical reactions, analytical
separation, detection and data handling steps) on a single
micro-device.
In the most micro-fluidic applications so far, on-chip detec-
tion has relied on optical detection, and for sensitivity rea-
sons, fluorescence (FL) detection has been the most com-
monly utilized. Among the detection techniques alternative
to optical detection, mass spectrometry (MS) has gained rap-
idly enhanced interest in chip-based analysis, and during the
last few years great amount of reports have been published
in the field. At present, the main focus is in integrating ion-
ization methods to micro separation systems with MS.
Miniaturization of atmospheric pressure ionization tech-
niques has gained rapidly enhanced interest in chip-based
analysis. Electrospray ionization (ESI) is currently the method
of choice to connect a microchip with mass spectrometry
(MS). The flow rates used with microfluidic devices (nl-µl/
min scale) are ideal for optimal sensitivity in ESI-MS. Differ-
ent materials, such as silicon, glass, polymers have been used
in fabrication of microchips. Recently SU-8 polymer has been
shown to be highly suitable material for microfluidic separa-
tions and electrospray ionization.
Even though ESI is an excellent method for polar and ionic
compounds, its sensitivity for neutral and non-polar com-
pounds may be poor. Atmospheric pressure chemical ioniza-
tion (APCI) and especially atmospheric pressure photoion-
ization (APPI) offer alternative ionization techniques that is
capable to ionize with high efficiency non-polar compounds. 41
Recently we presented microchip APCI and APPI, which allow
flow rates down to 50 nl/min making it directly compatible
with microseparation systems. The chips provide excellent
sensitivity, robust analysis, good reproducibility and cost ef-
ficient manufacturing. The feasibility of the APCI and APPI
microchips in coupling of micro liquid chromatography (LC),
gas chromatography and microchip LC to mass spectrom-
etry is presented.

October 26 - 28, 2009 / Antalya - TURKEY

 ORAL PRESENTATIONS
O25
Sensitive chiral capillary electrophoresis:
Application in bioanalysis
Éva Szökő, Tamás Tábi
Department of Pharmacodynamics, Semmelweis University, Budapest, Hungary
C
hirality has a great impact on the pharmacological
properties of drug enantiomers, thus studying the
stereochemistry of the pharmacodynamic and phar-
macokinetic properties of a drug candidate in early phase
of development has gained importance. Capillary electromi-
gration methods are especially suitable for the simultaneous
enantiomer separation of several compounds with similar
structures, such those formed during biotransformation pro-
cesses. In contrast to chiral HPLC, capillary electrophoresis
(CE) methods have high efficiency and chemical selectivity
needed for the resolution of plethora of analytes in a sam-
ple. The low reagent and sample requirement, as well as the
quick and flexible method development provide a rapid and
inexpensive way of analyzing enantiomers. These properties
are especial advantages, when screening is to be performed
or e.g. in in vitro metabolism studies. Drug quality control
42 (chiral impurity testing), pharmacodynamic and pharma-
cokinetic studies, or forensic toxicological analysis are other
major fields of application of enantioselective CE.
In capillary electrophoresis, the chiral selector is dissolved in
the background electrolyte and acts as a pseudo-stationary
phase that can interact with one of the enantiomers prefer-
ably. The enantioselectivity derives from the different sta-
bility of diastereomer complexes the enantiomers form with
the chiral selector. This way, in chiral CE, the separation of the
enantiomers is based on their distinct distribution between
the separation buffer and the pseudo-stationary phase (chro-
matographic principle), as well as on the different mobility
of the enantiomers under the separation conditions (electro-
migration principle). The combination of the two separation
measures provides the high peak capacity of the chiral CE
3rd BBBB International Conference on Pharmaceutical Sciences
methods. However, when simultaneous enantioseparation
of several sample components is required, even in case of
the suitable enantioselectivity, achieving the needed chemi-
cal (achiral) selectivity can be problematic. Separation pH,
applied electric field, capillary length and temperature are
the main parameters to be optimized. Besides these, there
are several other ways for influencing chemical selectivity
mainly through the improvement of separation efficiency
and/or widening the separation time window. We have used
buffer additives, such as organic modifier and chiral poly-
mer, or dual cyclodextrin system to improve achiral selectiv-
ity when simultaneous enantiomeric separation of the chiral
drug deprenyl, and several of its metabolites were to be per-
formed in metabolism studies.
Major drawback of using CE techniques in bioanalysis is the
poor concentration detection sensitivity by UV due to the
small sample volume and short light path. The complex ma-
trix of biological samples further impairs separation efficien-
cy and the detection limits. However, during sample clean-
up the composition of the sample matrix can be modified to
allow on-capillary sample stacking, and remove disturbing
sample components. On-capillary sample stacking can be
reached by decreasing the conductivity of the sample ma-
trix, allowing higher sample injection volume without the
loss of separation efficiency. Stacking provides about ten
times increase in detection sensitivity. Liquid-liquid or solid
phase extractions are common sample clean-up methods
for biological specimens. Their carefully designed use can
provide a sample matrix allowing the on-capillary sample-
stacking and quantification limits in the submicromolar con-
centration range.
 ORAL PRESENTATIONS
O26
QSAR IN PREDICTION OF TARGET AND ANTITARGET DRUG PROPERTIES
Milena Jadrijević - Mladar Takač
Department of Medicinal Chemistry, Faculty of Pharmacy and Biochemistry, University of Zagreb, Zagreb/Croatia
INTRODUCTION
Drug discovery has entered a new century with a wealth
of sophisticated technologies and information generation
platforms that theoretically will allow the more rapid devel-
opment of medicines with improved selectivity and safety
profiles. The most used, the quantitative structure activity
relationship (QSAR) provides a great deal of information re-
garding the nature of investigated NCEs and so far many ap-
proaches were developed. In an extension of our earlier stud-
ies in attempting the role of the use of topological indices
(TIs) for QSAR studies in lead compound search among 1,4- (I)
and 3,1-benzoxazinones (II) ((n=62) we have undertaken the
present investigation in that we have used Wiener (W) and
molecular connectivity (χ1, χMOD, CID) indices, molecular
descriptors (MDs), the lipophilicity parameter, log P (AlogP
and miLogP) and van der Waals volume (Sv) in correlations
with antimicrobial activity (MICx10-3moldm-3) obtained in
tests with various microorganisms, including bacteria and
fungi. The Lipinski’s rule-of-five study was also undertaken
with the aim to explore, the potential oral bioavailability
and druglikeness properties of these compounds, in order
to lead identification with the lowest potential toxophoric
features. For this purpose the ‘n toxophoric scores’ were cal-
culated and used in correlations with data computed using
Molinspiration software for druglikeness (GPCR ligand, ion
channel modulator, kinase inhibitor, nuclear receptor li-
gand), lipophylicity parameter, miLogP and molecular polar
surface area (TPSA).
R1
R O R1
R
O The 2D correlations with the same TIs (W, χ1, χMOD and CID)
N O N O
CH2N R2R3 CH2 NR2R3
I II
F
F
F
F F
Cl O
Cl
O
F Lipinski’s rule-of-five analysis
NH O NH O
Efavirenz 1,4- isomer of efavirenz
Computed data for efavirenz and its 1,4- isomer were also
included in this study.
MATERIAL AND METHODS
All compounds I and II were previously synthesized and bio-
logically tested. The TIs (W, χ1, χMOD and CID) and MDs (Sv,
Mv and AlogP) were computed using Dragon for Windows,
October 26 - 28, 2009 / Antalya - TURKEY
2007. The Lipinski’s rule-of-five analysis and parameters
(miLogP, MW, TPSA, n atoms, nON, nOH NH, nrotab, volume)
as well as druglikeness scores were computed using Mo-
linspiration molecular processing engine, 2007.10. The
‘n toxophoric score’ for each compound was calculated on
the basis of presence one or more main toxophoric groups
in molecule. All analyses were performed and data were
graphed using OriginPro 7.5 software (Origin Laboratories,
Northampton, MA).
RESULTS AND DISCUSSION
QSAR study
In present QSAR study W, χ1, χMOD and CID were correlated
with AlogP, Sv and MICs (x10-3 moldm-3) for Staphilococcus
aureus ATCCC 56511, Klebsiella pneumoniae ATCC 10031
and Candida monosa. The 2D correlations with similar sig- 43
nificancy between TIs (W, χ1, χMOD) and Sv versus MICx10-
3moldm-3 against S. aureus, were obtained and they are as
follows: i. MIC x 10-3 (mol dm-3) = 4.6115 – 0.00125 W (n =
20, r = - 0.8627); ii. MIC x 10-3 (mol dm-3) = 7.35675 – 0.39864
χ1 (n = 20, r = - 0.8952); iii. MIC x 10-3 (mol dm-3) = 7.35268
– 0.06072 χMOD (n = 20, r = - 0.88495); and iv. MIC x 10-3
(mol dm-3) = 6.87874 – 0.16685 Sv (n = 20, r = - 0.86517). The
lipophilicity parameter, AlogP were used in 2D and 3D cor-
relations. The 3D multiparameter correlations offered an ad-
ditional insight in relationships among TIs, lipophilicity and
MIC. QSAR results revealed that with an increasing of W, χ1
and AlogP, the MIC is decreasing for this set of compounds.
and Sv versus MIC against K. pneumoniae and C. monosa,
showed comparatively less colinearities between investigat-
ed parameters.
Lipinski’s rule-of-five predict poor absorption or permeation
of orally administered compound if it meets the following
criteria: molecular mass greater than 500 Da, high lipophi-
licity (expressed as cLogP greater than 5), more than 5 hy-
drogen bond donors and more than 10 hydrogen bond ac-
ceptors. Lipinski’s rule-of-five analysis was applied on a set
(n = 64) of I and II derivatives, including efavirenz and its 1,4-
isomer. Eight compounds out of 64 did not meet Lipinski’s
rule-of-five requirements (18, 19, 23, 27, 28, 30, 35 and 56),
mostly because of TPSA and number of H-bond acceptors in
molecule.
 ORAL PRESENTATIONS

In addition to druglikeness data estimated using various mo- CONCLUSION

lecular properties, the ‘n toxophoric score’ were calculated on The results of QSAR study could be used in predicting more
the basis of presence one or more main toxophoric groups in active analogues of this series of compounds. The Lipinski’s
molecule. In correlations calculated n toxophoric scores with rules-of-five analysis revealed valuable information relevant
estimated druglikeness scores, the miLogP and TPSA, the 6 to potential drug candidate properties. The ‘n toxophoric
subgroups with relative and potential toxicity from 2 to 7 score’ have to be more sofisticated for the prediction of po-
were obtained. The ‘n toxophoric score’ have to be consid- tential toxicity of NCEs.
ered as relative value of indication for possible toxicity. By
this correlations only relative position of each compound in REFERENCES
particular subgroup and in relation to efavirenz for the par- 1. Höltje HD, Sippl W, Rognan D, Folkers G, Moelcular mod-
ticular druglikeness score, estimated miLogP and TPSA could eling: Basic principles and applications, Wiley-VCH Wein-
be evaluated. heim, DE, 2008.
6
Staphylococcus aureus ATCC 56511
2. Vaz RJ, Klabunde T, Antitargets: Prediction and preven-
Y = 4.6115 - 0.00125 X
r = -0.8627 tion of drug side effects, Vol. 38, Wiley-VCH, Weinheim,
n = 20

4
DE, 2008.
3. Lipinski CA, Lombardo F, Dominy BW, Feeney PJ, Experi-
-3
MIC/10 mol dm

mental and computational approaches to estimate solu-

-3

2
bility and permeability in drug discovery and develop-
ment settings, Adv.Drug.Delivery Rev. 1997; 23: 4-25.
0
0 500 1000 1500 2000 2500 4. Jadrijević-Mladar Takač M, Kos I, Takač V, QSAR studies
W
on antimicrobial activity of benzoxazindione analogues,
68th International Congress of FIP. Abstracts Book. Basel,
CH, September 2008.

4
-3
MIC/10 mol dm
-3

0 3,0
500
2,5
2,0
1000 1,5
1500 1,0
W 2000 0,5 AlogP
2500 0,0

44
Figure 1: 2D and 3D correlations of W versus AlogP and MIC
(Staphylococcus aureus ATCC 56511)

3rd BBBB International Conference on Pharmaceutical Sciences

 ORAL PRESENTATIONS

O27
Alicyclic β-amino acids: new, valuable scaffolds in drug research
Ferenc Fülöp
Institute of Pharmaceutical Chemistry, University of Szeged, H-6720 Szeged, Eötvös utca 6, Hungary

A
lthough the drug discovery process seems to be an au- EtOOC COOEt EtOOC COOEt
tomatic and routine protocol in most pharmaceutical N N 1R
4R
COOEt + N N 1R
4S COOEt
N
companies, one of the most important key elements 2R 3R
N
2R 3R
for success is the availability of good hit molecules. In the past O
HO NHBoc HO NHBoc

10 years, fragment-based drug discovery (FBDD) become one NH

of the most accepted efficient and promising protocols.1 EtOOC COOEt EtOOC COOEt

N N 1R 4S COOEt N N 1R 4R COOEt
+ N
N
In our university project, we work on small, sometimes exotic, 2R 3S 2R 3S
HO NHBoc
well-designed molecules possessiny a great diversity of func- HO NHBoc

tionalities. The present lecture will demonstrate that cyclic
β-amino acids meet the above criteria. The alicyclic β-amino
acids have acquired great interest in recent years in view of
their pharmacological potential.2 Cispentacin, an antifungal
antibiotic with a cyclopentane skeleton, is one of the most
important derivatives. (1R,2S)-2-Amino-4-methylenecyclo­
pentane­carboxylic acid (Icofungipen) is a known antifungal
agent.2 Cyclic, conformationally rigid β-amino acids such
as cis and trans-2-aminocyclo­pentane­carboxylic acid have
been used as building blocks in the synthesis of peptides.2
The present lecture will point out different strategies that re-
sult in functionalized β-amino acids from 2-aminocycloalk-
enecarboxylic acids, and will also focus on different proto-
cols for the preparation of enantiomeric substances.3
The Scheme illustrates an example of the preparation of four
1,2,3-triazole-substituted ethyl 2-amino-3-hydroxycyclopen-
tanecarboxylate diastereomers (3,4-disubstituted cispent-
acins) with a cyclopentane skeleton in enantiomerically pure
form from racemic β-lactam via enzymatic ring opening, ep-
oxidation and selective ring opening of the oxirane ring with
sodium azide. The formation of the 1,2,3-triazole ring system
involved click chemistry: 1,3-dipolar cycloaddition of the 45
corresponding 4-substituted azidocarboxylates with diethyl
acetylenedicarboxylate
REFERENCES
1. Keserű, M.G., Makara, G.M. Nature Rev. Drug Discovery
2009, 8, 203.Congreve, M., Chessari, G., Tisi, D., Wood-
head, A.J. J. Med. Chem. 2008, 51, 3661.
2. Fülöp, F. Chem. Rev. 2001, 101, 2181. Fülöp, F., Martinek,
T.A., Tóth, G.K. Chem. Soc. Rev., 2006, 35, 323. Mándity,
I.M., Wéber, E., Martinek, T.A. Olajos G., Tóth, G.K., Vass, E.,
Fülöp, F., Angew. Chem. Int. Ed., 2009, 48, 2171.
3. Kiss, L., Forró, E., Sillanpää, R., Fülöp, F. Tetrahedron:
Asymmetry, 2008, 19, 2856. Kiss, L., Nonn, M., Forró, E.,
Sillanpää, R. Fülöp, F. Tetrahedron Lett., 2009, 50, 2605.

October 26 - 28, 2009 / Antalya - TURKEY

 ORAL PRESENTATIONS
O28
HYDROXAMIC AND AMIDE DERIVATIVES OF NONSTEROIDAL
ANTIINFLAMMATORY DRUGS – SYNTHESIS AND BIOLOGICAL ACTIVITY
Zrinca Rajić, Branka Zorc
Department of Medicinal Chemistry, Faculty of Pharmacy and Biochemistry, University of Zagreb, 10000,
Zagreb/Croatia
NTRODUCTION
Nonsteroidal antiinflammatory drugs (NSAIDs) are well-
known class of drugs with anagesic, antipyretic and antiin-
flamatory activity based on their inhibition of cyclooxyge-
nase. Their numerous derivatives have been synthesized in
order to modify the pharmacokinetics, improve the selectiv-
ity towards the enzymes or increase the variety of their ac-
tion1.
Hydroxamic acids are well known to form strong complexes
with transition metals, and thus inhibit enzymes of human
and bacterial origin2. One of them is lipoxygenase, the cru-
cial enzyme in the synthesis of leukotrienes. Many NSAIDs
have been reported to act either as inhibitors of free radical
production or as radical scavengers.
46 On the other hand, they are potential anticancer drugs
and effective chemopreventive agents3. In our previous
papers, potency of NSAID amides as cytostatic agents was
screened4,5. It was shown that ketoprofen amidation signifi-
cantly enhanced antiproliferative activity of the parent com-
pound and that lipophilicity of the compounds had a signifi-
cant influence on their activity.
Here we report the synthesis of a new series of lipophilic
ketoprofen amides and NSAID hydroxamic acids (deriva-
tives of ibuprofen, fenoprofen, ketoprofen, diclofenac and
indomethacin) and their O-alkyl and O-benzyl derivatives.
Compounds were evaluated for their cytostatic, antioxidant
and antimicrobial activity, as well as their ability to inhibit
soybean lipoxygenase (LO).
RESULTS AND DISCUSSION
Chemistry
Ketoprofen was the starting compound in the synthetic
pathway leading to reduced analogues 2a,b and their novel
amides 4a-i, 5a-f and 6a-f, while various NSAIDs (ibuprofen,
fenoprofen, diclofenac, indomethacin, ketoprofen, 2a,b)
started the reaction path leading to the derivatives of NSAID
hydroxamic acids 7a-x. Compounds 2a,b were synthesized
by the catalytic hydrogenation of ketoprofen under differ-
ent reaction conditions. Further on, NSAIDs reacted with 1
equivalent of 1-benzotriazole carboxylic acid chloride (1) af-
fording benzotriazolides 3a-g (6). In the reaction of benzot-
riazolides 3g,e with 1.1 equivalent of the appropriate amine
in the presence of 5 equivalents of triethylamine in toluene
3rd BBBB International Conference on Pharmaceutical Sciences
amides 4a-i and 5a-f were obtained. Derivatives 6a-f were
synthesized by the catalytic hydrogenation of compounds
5a-f, by using heterogeneous Pd-ethylenediamine complex
catalyst. Benzotriazolides 3a-e,g were converted to NSAID
hydroxamic acids 7a-x in the reaction with the correspond-
ing hydroxylamine (hydroxylamine, N-methylhydroxylam-
ine, O-methylhydroxylamine, O-ethylhydroxylamine or O-
benzylhydroxylamine) or by the catalytic hydrogenation.
Biological Evaluation
Compounds 4b,c,e,f, 5c,e,f and 6c,e,f were screened in vitro
for their potential antiproliferative effects on a panel of 5 tu-
mor cell lines: HCT 116, SW620, MCF-7, MOLT-4, and H 460.
The tested compounds showed very similar, moderate anti-
proliferative effect on the presented panel cell lines. NSAID
hydroxamic acid derivatives 7 were evaluated in vitro for the-
ir cytostatic effects against the following malignant tumor
cell lines: L1210, FM3A, Molt4/C8, MOLT-4, CEM, HeLa, MCF-
7, MiaPaCa-2, NCI H727, SW620, HCT 116, H 460 and SK-BR-3
and compared with their effects on the growth of human
normal fibroblasts (WI 38). Indomethacin hydroxamic acid
(7f) showed the strongest, but nonselective, antiproliferative
activity on the tested tumor cell panel. In contrast, selective
and differential activity was exerted on MiaPaCa-2 by 7b (IC50
= 2.2 μmol/L) and on HeLa by 7c (IC50 = 6.5 μmol/L).
Antioxidant activity, soybean lipoxygenase inhibition and
inhibition of linoleic acid lipid peroxidation was tested for
compounds 4−7.
Antioxidant activity was tested by the reaction with the sta-
ble free radical 1,1-diphenyl-2-picrylhydrazyl (DPPH) and the
results have shown that derivatives of the NSAID hydroxamic
acids 7 showed better activity than amide derivatives 4−6.
The highest activity was exerted by compounds 7c and 7n.
Inhibition of LO was tested by the UV absorbance based en-
zyme assay. Ketoprofen derivatives 4−6 were better LO inhi-
bitors than NSAID hydroxamic acids derivatives 7. Perusal of
IC50 values showed that compound 6f was the most active
by far (IC50 = 20.5 µmol/L).
Inhibition of linoleic acid lipid peroxidation (LP) was studied
by using 2,2’-azobis(2-amidinopropane) dihydrochloride
(AAPH) as a free radical initiator to follow oxidative chan-
ges of linoleic acid to conjugated diene hydroperoxide.
The best LP inhibitors were 4d,e,f, 5c,d,e,f, and 7s,x,v,q,n
(92.2−99.9%).
 ORAL PRESENTATIONS

Antimicrobial activity was tested for NSAID hydroxamic acid
derivatives 7. The results of the hole-plate diffusion method
showed that only NSAID hydroxamic acids with free and O-
ethyl substituted hydroxy groups possessed noticeable anti-
microbial activity at concentration 29.6 mg/mL. The minimal
inhibitory (MIC) and the minimal microbicidal concentrations
(MMcC) were determined by the microdilution broth meth-
od. Compounds 7a and 7c showed the lowest MIC/MMcC
against B. subtilis and S. enterica subsp. enterica species.
REFERENCES
1. Bonina F, Puglia C, Santagati NA, Saija A, Tomaino A, Tita
B. Oligoethylene ester derivatives of ketoprofen, naprox-
en and diclofenac as oral prodrugs: a pharmacological
evaluation, Pharmazie. 2002; 57: 552−555.
2. Muri EMF, Nieto MJ, Sindelar RD, Williamson, JS. Hy-
droxamic acids as pharmacological agents, Curr Med
Chem. 2002; 9: 1631−1653.
3. Thun MJ, Henley SJ, Patrono C. Nonsteroidal anti-inflam-
matory drugs as anticancer agents: mechanistic, phar-
macologic, and clinical issues, J Nat Cancer Inst. 2002;
94: 252−266.
4. Wittine K, Benci K, Rajic Z, Zorc B, Kralj M et al. The novel
phosphoramidate derivatives of NSAID 3-hydroxypro-
pylamides: Synthesis, cytostatic and antiviral activity
evaluations, Eur J Med Chem. 2009; 44: 143−151.
5. Marjanovic M, Zorc B, Pejnovic L, Zovko M, Kralj M. Fe-
noprofen and ketoprofen amides as potential antitumor
agents. Chem Biol Drug Des. 2007; 69: 222−226.
6. Zorc B, Antolic S, Butula I. Macromolecular prodrugs. I.
Synthesis of some non-steroidal anti-inflammatory drug
esters. Acta Pharm. 1993; 43: 127–133.

47

October 26 - 28, 2009 / Antalya - TURKEY

 ORAL PRESENTATIONS
O29
Improving the effectiveness and cost effectiveness of drug therapy
using pharmacogenetic testing and comprehensive screening of
drug interactions
Lajos Botz
Pharmaceutical Institute, Faculty of Medicine, University of Pécs, Pécs, Szigeti u. 12, Hungary H-7624
R
ecently more and more controversial opinions surfaced
regarding the effectiveness of traditional pharmacot-
herapy. These critics refer to the inadequate effective-
ness of drugs, the high prevalence of adverse reactions and
the excessive use of medication. In many cases drug interac-
tions may be the root of the problem. The aims of the current
study were (1) to identify the most common drug interacti-
ons in clinical practice; (2) to characterise of drug interactions
with computer analysis (3); to develop a software application
which utilises a drug interaction database based on the CYP
450 metabolism; (4) to assess the influence of polymorphic
drug metabolism on the utilization of antidepressant drugs.
Many studies have demonstrated marked ethnogeographic
variance in the distribution of CYP2D6 and CYP2C19 genoty-
pes throughout the world. Many antidepressants are metab-
olized by the polymorphic cytochrome P450 enzymes. The
48 activities of CYP 2D6 and 2C19 enzymes in the metabolism
of antidepressants are very different. The pharmacogenetic
information may be beneficial in evaluating the drug-drug
interactions. The combination of these two fields for pre-
screening the drug interactions is useful for increasing the
efficiency of drug-drug interaction control software. We
would also like to map and visualise the complex network
of interactions. This combined methodology may be used
for optimising the drug therapy for patients suffering from
depression. Two drug interaction software applications have
been developed to detect drug interactions. The first soft-
ware application was developed by the Pharmacy Institute
of University of Pécs for community pharmacies. The interac-
tion analysis was based on three data sources: (1) the official
summary of product characteristics for drugs, (2) relevant
drug information databases and literature and (3) the effect
of the interactions on the cytochrome P450 enzyme system.
After having analysed the official Hungarian drug specifica-
tions and drug interactions, we developed the first software
application which filters out undesirable drug interactions.
We only targeted drug interactions which are relevant in the
clinical practice. The novel software application is based on
the drug interaction database. The second software was an
internationally used professional tool: Micromedex® Health-
care series drug interaction system (Thomson Reuters). The
first data collection was 1997, when 709,000 prescriptions
were analysed. The second systematic data collection was
between 2001 -2004. This analysed 1,227,000 prescriptions
by a computer program. Our third survey included 401,000
prescriptions in 2007. We determined the genotype of indi-
3rd BBBB International Conference on Pharmaceutical Sciences
viduals in the selected patients groups with increasing ex-
posure to drug-drug interactions. The alleles selected were
CYP2D6 and CYP2C19. In Caucasians, the most common
non-coding alleles are CYP2D6*4, CYP2D6*3, CYP2D6*5 and
CYP2D6*6. CYP2C19 has about 15 allele variants of which two
(CYP2C19*2 and CYP2C19*3) play the most important role in
the altered metabolism of antidepressant drugs. CYP2C19*3
is extremely rare in Caucasians, but accounts for approxi-
mately 85% of all poor metabolisers. We used two methods
to detect the patients’ genotype. The first method is based
on DNA melting point analysis. This method is less expensive
but only validated for experimental use. The second method,
which is based on a DNA chip analysis, is validated for clini-
cal use and capable to detect 29 polymorphisms (33 alleles)
of CYP2D6 and 2 polymorphisms (2 alleles) of CYP2C19. This
method is very expensive and not approved by National
Health Insurance System and health insurance companies.
The patients for the experiment were selected from the Clin-
ics of Psychiatry and Psychotherapeutics at the University of
Pécs in Hungary. Non-responsiveness was defined as ineffec-
tive therapeutic response evaluated by the physician after a
four-week treatment period with an adequate dosage. The
type and number of adverse effects and therapeutic conse-
quences were recorded and reported at the end of the treat-
ment with a particular antidepressant. The aims of the study
were to learn the drug interactions that occur in practice
and to compare various aspects of the analysis. The results
show that the most frequent drug-drug interactions were
the acenocoumarol - acetylsalicylic acid and the naproxen -
enalapril. The overall rate of clinically significant interactions
were 3.64% and 2.65%. The software application identifies
and prevents the prescription of clinically dangerous drug
interactions. The analysis of the drug interactions in patients
with depression also revealed startling relationships. Almost
8% of all drug administrations and more than 35 % of patients
were using alprazolam in a combination. The Micromedex®
drug interaction software application revealed 240 drug in-
teractions during the study period. Within 38% of all drug
administration at least one drug interaction occurred. The
most important drugs were antidepressants and antiplate-
let agents. The relationships between the frequency of ADRs
and genotype, the interactions in CYP enzyme-mediated
drug metabolism and ADRs were investigated. However, we
could not find correlation between phenotype and the oc-
currence of ADRs prior to this. We also analysed relationships
between drug interactions of CYP enzyme mediated drug
 ORAL PRESENTATIONS

metabolism and the presence of ADRs. Such analysis of drugs
may lead to a personalised medical therapy in the future.
Drug interactions via the inhibition or induction of a CYP en-
zyme have been also analysed. These results show that there
are correlations between genetic background and therapy
outcome. Drug therapy decisions based on pharmacogenet-
ics are very complex and clear link cannot be confirmed yet.
Our software application contains three screening methods
regarding an active agent pair. To improve our software ap-
plication bioinformatics network matrix and a bioinformatics
software environment were introduced. A new database was
added to the existing one to characterize the relationship
between active agent pairs. The possibility of analysis of the
complex network of interactions of the active ingredients of
drugs helps to predict future interactions. This analysis can
be facilitated by forming clusters of all used active ingredi-
ent pairs. The relationship function resembles the independ-
ent network models (logplot). This network analysis shows
a map of all drug-drug interactions, and highlights every in-
teraction of a selected drug.. The analysis shows that severe
drug interactions can occur in 3-4% of patients and this may
cause 6-10% increase in medical costs.

49

October 26 - 28, 2009 / Antalya - TURKEY

 ORAL PRESENTATIONS
O30
IMPLEMENTATION OF CLINICAL PHARMACY SERVICES IN HOSPITALS: WHERE TO
START?
Kutay Demirkan
Hacettepe University, School of Pharmacy, Ankara, Turkey
I
mplementation of clinical pharmacy requires an exten-
sive knowledge of therapeutics and medical terminology,
a good understanding of disease and drug monitoring
processes, therapeutic planning skills, ability to assess and
interpret physical and laboratory findings and provision of
information about medicines.
By means of its definition, responsibilities and duties of a
clinical pharmacist include selection of the correct drug,
proper dosage form, appropriate route of administration;
choice and timing of specimens for therapeutic drug moni-
toring; interpretation of pharmacokinetics data, detection
and prevention of drug interactions, adverse drug effects
and toxicity.
It is clear that clinical pharmacists have the potential to con-
50 tribute in reduction of drug expenditures and improving
medication and patient safety. To achieve these outcomes,
clinical pharmacists should obtain medication histories,
perform medication reviews, attend ward rounds, provide
recommendations on drug selection and follow-up, and
provide counseling to patients and healthcare providers.
The clinical pharmacists should be willing to improve them-
selves through ongoing learning activities about their dis-
cipline and should be able to keep up with current medical
literature.
Clinical pharmacy services are well developed in some coun-
tries (such as USA, Canada and the UK) and clinical pharma-
cists are integral members of the health care team. However,
clinical pharmacy is a new arising issue in many countries
and therefore education and provision of clinical pharmacy
are not available yet.
Implementing a new clinical pharmacy service in a hospital
is challenging. Before the implementation of any service, the
barriers such as lack of time, lack of communication skills, al-
location of new resources, lack of training and lack of clinical
pharmacist, lack of reimbursement need to be identified and
the plans to overcome for each barrier need to be consid-
ered.
Schools of Pharmacy in most countries do not satisfy minimal
requirements for an appropriate education in clinical phar-
3rd BBBB International Conference on Pharmaceutical Sciences
macy. Clinical pharmacy education and practice have been
developed by contribution from both pharmacy schools and
university-based teaching hospitals.
Since only few clinical pharmacists are available and clinical
pharmacy practices are not routinely performed in hospitals
in Turkey, most of the physicians are not familiar with clini-
cal pharmacy practices. Clinical pharmacy practices have
been first implemented in nine-bed medical intensive care
unit (ICU) at the Hacettepe University Hospital in Türkiye.
The practicing clinical pharmacist was a part-time provider;
therefore clinical pharmacy service was limited to morning
visits of ICU throughout almost all week-days. Also, the clini-
cal pharmacist was available on call when needed. The rea-
son of “why ICU to start with” is very clear and simple: the
clinical pharmacist had a specialty in the ICU and the attend-
ing physician who also had an experience working with clini-
cal pharmacists in USA. Implementation of clinical pharmacy
practice was also started in nutrition unit and neuro-ICU unit
upon requests from physicians after the ICU activities at the
Hacettepe University Hospital. Other requests for implemen-
tation of clinical pharmacy services from other units such
as oncology, infectious diseases are not fulfilled yet due to
a lack of clinical pharmacist at the moment. Moreover, bed-
side teaching for pharmacy students at the hospital was ap-
proved by the hospital administration office and chief phy-
sicians, where a clinical pharmacist act as a mentor during
medication review process for undergraduate education.
The implementation of clinical pharmacy practice from a
pilot unit and thereafter an establishment of a clinical phar-
macy service at the hospital might be easier to prove the
efficacy and necessity of the service. It is better to choose
a pilot unit that patients are more complex and need more
attention, so pharmacists can have more active role and in-
terventions in short time period.
Although implementing a new clinical pharmacy service in
a hospital seems to be challenging; having qualified clinical
pharmacists, highly cooperative and enthusiastic healthcare
professionals along with supportive hospital administrative
board, would be an initiative force to establish clinical phar-
macy services.
 ORAL PRESENTATIONS

O31
THE ROLE OF THERAPEUTIC DRUG MONITORING IN OPTIMAZING
PHARMACOTHERAPY OF SELECTED ANTIEPILEPTICS
T. Vovk1, I. Grabnar1, M. Kerec Kos1, M.B. Jakovljević2, K. Vučičević3, A. Mrhar1
1
The Chair of Biopharmaceutics and Pharmacokinetics, Faculty of Pharmacy, University of Ljubljana, Ljubljana,
Slovenia
2
Pharmacology Department, Medical Faculty, University of Kragujevac, Kragujevac, Serbia
3
Department of Pharmacokinetics, Faculty of Pharmacy, University of Belgrade, Belgrade, Serbia

INTRODUCTION the TPR study volume of distribution (V/F) were estimated,

Clinical experience has demonstrated that individualized
dose adjustment by the aid of therapeutic drug monitoring
(TDM) can significantly improve treatment with antiepileptic
drugs (AED). Development of a population pharmacokinetic
(PK) model is a logical extension of TDM, because it allows
estimation of individual patients’ PK parameters based on
sparse concentration measurements and ultimately effec-
tive control of dosing. In addition to being a valuable tool in
designing a safe and effective dosing regimen for patients
with epilepsy, population PK models permit evaluation of
various factors that can influence PK characteristics1.
This paper presents the summary of carbamazepine2, valpro-
ic acid and topiramate studies aiming to develop population
PK models to evaluate the influences of demographic factors
and comedications on drugs PK.
METHODS
Patients
For development of carbamazepine (CBZ) and valproic acid
(VPA) population pharmacokinetic models patients’ char-
acteristics and plasma drug concentrations were collected
retrospectively from patients’ charts as part of routine TDM.
Topiramate (TPR) study was prospective, and was not part of
routine TDM.
while the absorption rate constant was fixed. Effects of con-
tinuous and categorical covariates were tested against the
base model. Alternative models were compared by the likeli-
hood ratio test (α = 0.05). Significant covariates were rank-
ordered and introduced into the full model. The final model
was determined by backward elimination of covariates one
by one from the full model to see if they should remain in
the model using the likelihood ratio test. Additional criterion
for retention of a covariate in the model was reduction in the
unexplained interindividual variability. The model adequacy
was evaluated by standard diagnostic plots, convergence of
minimization, number of significant digits more than 3, suc-
cessful covariance step and gradients in the final iteration in
the range between 10-3 and 102.
RESULTS AND DISCUSSION 51
Carbamazepine
Patient weight (WT), daily carbamazepine dose (DCBZ), daily
dose of phenobarbitone (DPB) and valproic acid (VPA), when
its daily dose exceeded 750 mg, significantly influenced CL/F,
and were included in the final model:

Blood sampling
Blood samples were drawn at the end of the dose interval,
before the morning dose, while in all cases of TPR study and
same cases in CBZ and VPR study a second blood sample was
drawn after the dosing approximately at the time of the peak
concentration. , where VPA is 1 if DVPA>750 mg or 0 otherwise. The rela-
tionship between CBZ CL/F and DCBZ is nonlinear, while that
Bioanalitical assay with DPB is linear. The effects of CBZ and PB on CZB CL/F are
In CBZ and VPR studies drugs were measured using homog- attributed to induction of CBZ metabolism. Moreover, VPA
enous enzyme immunoassay technique (EMIT; Cobas Mira, increases the CBZ CL/F probably by displacement of CBZ
Roche Diagnostics, Basel, Switzerland), while the TPR was de- from plasma proteins.
termined with HPLC fluorescent labelling method adapted
from literature. Valproic acid
The VPA CL/F was significantly influenced by patient’s WT,
Population modelling daily dose of VPA, and co-therapy with topiramate (TPR).
PK analysis was performed by a population PK modelling These covariates were included in the final pharmacokinetic
approach using NONMEM software. The structural model model which is described with the following equation:
used was a one-compartment PK model with first-order ab-
sorption and elimination. Apparent clearance (CL/F) and in

October 26 - 28, 2009 / Antalya - TURKEY

 ORAL PRESENTATIONS

, where VPA is 1, if VPA dose is greater than 1000 mg/day, or 0
otherwise, and TPR equals 1, if patient is co-treated with TPR,
or 0, if not. This study confirms the interaction of VPA with
TPR, which is presumably dependent on VPA dose.
Topiramate
The final model of TPR CL/F was significantly influenced by
co-treatment with CBZ and patient’s age. While the TPR V/F
was only dependent on patient’s weight. The final models
are presented by the following equations:
CONCLUSIONS
The proposed PK models can be used for Bayesian estima-
tion of drugs CL/F in individual patients based on sparse
concentration measurements and for selection of optimum
dosing regimen in routine patient care.
REFERENCES
1. Johannessen SI, Landmark CJ. Value of therapeutic drug
monitoring in epilepsy, Expert Rev Neurother. 2008; 8:
929-939.
2. Vučićević K, Miljković B, Veličković R, Pokrajac M, Mrhar
A, et al. Population pharmacokinetic model of carbam-
azepine derived from routine therapeutic drug monitor-
ing data, Ther Drug Monit. 2007; 29: 781-788

, where CBZ is 1 in patients co-treated with carbamazepine,

or 0 otherwise. CBZ most probably induces TPR metabolism
and therefore its clearance.

52

3rd BBBB International Conference on Pharmaceutical Sciences

 ORAL PRESENTATIONS
O32
IDENTIFYING TARGETS FOR THE TREATMENT OF DYSTONIA AND
PRECLINICALTESTING OF THE LEAD COMPOUNDS
Aygül Balcıoğlu
Department of Neurology, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA
A
common early-onset dystonia is a disease that is char-
acterized by an abnormal rigidity of muscle, and pro-
longed, repetitive muscle contractions that may cause
twisting or jerking movements of the body or abody part.
Most cases of early onset torsin dystonia (DYT1) are caused
by a ΔGAG mutation in TOR1Agene resulting in the loss of a
glutamic acid in the carboxy terminal region of torsinA pro-
tein in theheterozygous state1.
There are 300,000 DYT1 patients in USA and over 3 mil-
lion dystonia patients in the worldwide waitingfor a treat-
ment2. Therapy is primarily limited to either neurosurgery
or chemodenervation3. Asmutant torsinA (ΔE) expression
results in decreased torsinA function, therapeutic strategies
directedtoward enhancement of wildtype (WT) torsinA ac-
tivity in patients, who are heterozygous for mutanttorsinA,
may restore normal cellular functionality. One of many such
efforts includes screening of thePrestwick C. elegans chemi-
cal library purchased from Prestwick Chemical Inc. (Illkirch,
France). Thesecompounds are evaluated in nematodes (C.
elegans) to enhance WT torsinA activity in two separateas-
says.
Transgenic nematode strains were utilized to quantitative-
ly measure changes in protein misfolding as afunctional
readout in a screen for small molecule effectors of torsinA
activity. We screened thePrestwick C. elegans library, a col-
lection of 240 chemically and therapeutically diverse drugs
(prescreenedfor maximal dosage and toxicity to worms),
for compounds that either inhibited the dominanteffect of
mutant torsinA (DE) or enhanced the normal activity of WT
torsinA. This screen wasperformed in worms that presum-
ably best reflect the human DYT1 condition in that they ex-
pressed boththe WT and DE gene products (WT/DE). At the
third larval stage of C. elegans, a 60% increase inaggregates
was reproducibly observed in worms expressing WT/DE,
when compared to animalsexpressing only the WT human
protein. This difference represented an ideal developmental
stage forreadily distinguishing molecules that significantly
altered torsinA chaperone function.
Work in our collaborative team has also focused on mouse
models of DYT1 dystonia, with the goal ofunderstanding
the underlying mechanisms, which may be shared with
other forms of dystonia. In atransgenic DYT1 model in which
mice express human mutant torsinA, we have identified
October 26 - 28, 2009 / Antalya - TURKEY
abnormalitiesin striatal DA release4 as well as function of
dopamine transporter (DAT) and amphetamine-induced-
locomotion. We also described a deficit in motor learning5,
which resembles that observed in humans.These animals do
not have overt dystonia, but they do not learn to stay on the
rotarod and they travelless distance under basal condition
as well as following amphetamine administration as a condi-
tion ofDYT1 dystonia.
Using in vivo microdialysis in awake animals, we found the
response to amphetamine to be markedlyattenuated in the
human mutant torsinA (hMT1) expressing mice compared
to their non-transgeniclittermates (253±71% vs. 561±132%,
p<0.05, two-way ANOVA). Extracellular striatal tissue DA lev-
elsmeasured using HPLC revealed no marked difference be-
tween hMT1 mice and their non-transgeniclittermates (48±9
vs. 34±6 nmol/mg, tissue respectively, n=6), suggesting no 53
degeneration in striatum. Two targets involved in amphet-
amine’s action are DAT and vesicular monoamine trans-
porter (VMAT2)are very important components of uptake,
storage and release of DA. Using the specific DAT inhibitor-
nomifensine (NOM), we demonstrated that the efficiency of
the inhibition was different at the sameconcentration (20
μM), with means of 50% for transgenics vs. 91% for non-
transgenics, but this did notreach statistical significance. This
indicated to us that DAT is involved in amphetamine-induced
DArelease in transgenic mice, and the reduced DA release
would be consistent with fewer or improperlyprocessed DAT
on the cell surface of neurons in hMT1 mice. Consequently,
we studied the dynamics ofDAT in vivo using no-net flux
microdialysis6,7. The extraction fraction (indicates function
of DAT)for non-transgenic animals was 0.88±0.04 and for
hMT1 transgenic mice 0.73±0.05 (p<0.05). Thisdifference in-
dicates that DAT in hMT1 mice work less efficiently than in
their controls, which couldresult from fewer or functionaly
compromised transporters on the cell surface. Additionally,
basal (datanot shown) and amphetamine-induced locomo-
tion was much less in hMT1 transgenic mice compared to-
control littermates. The distance traveled following amphet-
amine administration was 48.5±6.7 metersfor transgenics vs.
73.7±9.8 meters for non-transgenics (p<0.05). These results
support altered dynamicsof DAT in hMT1 transgenic mice
consistent with behavioral consequences. These functional
analyses ofDAT support potential defects in processing of
this protein by mutant torsinA, which is responsible from-
DYT1 dystonia.
 ORAL PRESENTATIONS
We are currently working on the promising leading com-
pounds from Prestwick C. elegans library onimpaired am-
phetamine-induced DA release as well as basal and am-
phetamine-induced locomotion in ananimal model of DYT1
dsytonia. In a transgenic DYT1 model in which mice express
human mutanttorsinA, we have identified abnormalities in
A. Striatal amphetamine-induced DA release, B. Functionof
dopamine transporter (DAT), and C. Amphetamine-induced
locomotion. These studies provide therationale that a com-
promised DA system might be an important phenotype in
animal models of DYT1dystonia. Some compounds have
emerged as a promising candidate for DYT1 dystonia to test
itsefficacy in vivo before it (and/or similar derivatives) could
be given to DYT1 patients. Our workaccelerates determining
the efficacy and relevance to behavioral outcome of prom-
ising compounds bytesting them on in vivo phenotype for
DYT1 dsytonia. It is critical to perform these translationalpre-
clinical tests to validate potential therapies under disease-
specific conditions in animal models beforethey can enter
the development pipeline on their way to the bedside.
REFERENCES
1. Ozelius LJ, Hewett J, Page C, Bressman S, Kramer P, Shal-
54
3rd BBBB International Conference on Pharmaceutical Sciences
ish C, de Leon D, Brin M, Raymond D, CoreyDP, Fahn S,
Risch N, Buckler A, Gusella JF, Breakefield XO (1997) The
early-onset torsion dystoniagene (DYT1) encodes an
ATP-binding protein. Nat Genet 17:40-48.
2. Defazio G (2007) in Handbook of Dystonia, ed Stacey ME
(Informa Healthcare USA,Inc. New York), pp 11-20.
3. Jankovic J (2006) Treatment of dystonia. Lancet Neurol
5: 864-872.
4. Balcioglu A, Kim MO, Sharma N, Cha JH, Breakefield XO,
Standaert DG. (2007) Dopamine release isimpaired in a
mouse model of DYT1 dystonia. J Chem. 102: 783-788.
5. Sharma N, Baxter MG, Petravicz J, Bragg CD, Schienda
A, Standaert DG, Breakefield XO (2005)Impaired motor
learning in mice expressing torsinA with the DYT1 dys-
tonia mutation. J Neurosci25: 5351-5355
6. Jones SR, Gainetdinov RR, Jaber M, Giros B, Wightman
RM, Caron MG (1998) Profound neuronalplasticity in
response to inactivation of the dopamine transporter.
Proc Natl Acad Sci 95: 4029-4034
7. Chefer VI, Czyzyk T, Bolan EA, Moron J, Pintar JE, Ship-
penberg TS (2005) Endogenous kappa-opioidreceptor
systems regulate mesoaccumbal dopamine dynamics
and vulnerability to cocaine. J Neurosci25: 5029-5037.v
 ORAL PRESENTATIONS
O33
A EUROPEAN PERSPECTIVE ON THE FUTURE OF THE PHARMACY PROFESSION
Daisy Volmer
Department of Pharmacy, University of Tartu, 50411 Tartu, Estonia
INTRODUCTION
Historically the preparation of medicines has been considered
as one the core competencies of the pharmacy profession.
During the past fifty years described professional activity has
been shifted from community and hospital setting to drug
industry and changed from preparation to manufacture of
medicinal products. The pharmacy profession, having a mo-
nopoly concerning preparation of medicines in the past, has
had to review its´ core values, practice roles and educational
methods. With a decrease in the preparation of medicines in
community and hospital pharmacies, provision of drug infor-
mation has become an important service. To ensure the qua-
lity treatment of patients, new approaches including clinical
pharmacy and pharmaceutical care were initiated.
Assurance of the professional competency in pharmacy
Nowadays the pharmacy profession has been considered
as dynamic and multifaceted discipline. In the EU Directive
2005/36/EC on the recognition of professional qualifications
pharmacists should be able to provide the following activi-
ties and services:
• preparation and manufacturing of medicinal products;
• testing the quality of medicinal products;
• storage, preservation and distribution of medicinal
products at the wholesale stage;
• preparation, testing, storage and supply of medicinal
products at the retail sale stage;
• preparation, testing, storage and dispensing of medici-
nal products at hospitals;
• provision of information and advice on medicinal prod-
ucts1).
The question is how to provide the education to cover all afore-
mentioned aspects of the pharmaceutical activities. Directive
2005/36/EC presents general info concerning length and con-
tent of the pharmacy studies at higher education institutions.
During the last two decades there have been several discus-
sions and attempts to develop unified pharmacy education
programme for Europe. In 2008 the PHARMINE programme has
been introduced by European Association of Faculties of Phar-
macy to map the pharmacy education in Europe and based on
the received information to develop core curriculum for the
pharmacy studies in Europe. In addition the extended curricula
in three main disciplines of pharmacy - community pharmacy,
clinical pharmacy and pharmaceutical industry will be devel-
oped. The new curricula could serve as a standard for Europe to
October 26 - 28, 2009 / Antalya - TURKEY
assure unified professional competency of the pharmacists.
Implementation of the professional competencies into practice
Community pharmacy setting
Within 15-20 years community pharmacy services have be-
come more accessible and patient oriented. Patients are re-
ceiving more detailed drug information and pharmacists are
regarded as professionals in the use of medicines2.
Community pharmacies provide new services as patient
medication review, taking blood pressure and other analysis,
monitoring the quality of therapy of patients with chronic
conditions. Pharmacists are considered as advisors on
healthcare issues and prescribers of medicines.
New initiatives within the pharmacy profession relate more
to providing effective drug therapy and resolving drug re- 55
lated problems. This is welcome, because a systematic ap-
proach about how to avoid the problems connected with
use of medicines is missing.
However, there are several factors serving as obstacles to
the development of the community pharmacy practice. The
liberal pharmacy system with mostly private ownership of
community pharmacies not restricted by the pharmacy pro-
fession and operation of pharmacy chains has introduced
not healthcare but business oriented pharmacies. Shortage
of professional staff and burn out syndrome of the pharma-
cists decrease interest towards professional continuing edu-
cation initiatives.
It would be impossible to give one universal recipe for the
development of community pharmacy practice. For the ac-
tual steps, it is important to analyse existing situation, learn
the local health care traditions and develop guidelines, work
out quality assurance instruments for provided or planned
services, cooperate with different institutions, apply for the
financial assistance from state and private organisations and
from EU funds.
Hospital pharmacy setting
In 1983 European Society of Clinical Pharmacy first pre-
sented document with requirements and standards for the
expertise and skills of clinical pharmacists. Following 10-15
years several different organizations have been contributed
to modernization and presentation of the professional roles
 ORAL PRESENTATIONS
of the clinical pharmacist3. However, nowadays there is con-
siderable variation in hospital pharmacy practice in Europe.
The development of clinical pharmacy education and prac-
tice has not been as fast as was expected.
In the countries where the clinical pharmacy is novel disci-
pline and/or the number of hospital pharmacies is small it
would be problematic to compose separate curriculum for
clinical pharmacy. Nevertheless, it is possible to introduce
some of the basic knowledge during the undergraduate
pharmacy studies followed by continuing educational cours-
es abroad.
Possible challenges in implementation of the professional
competencies
- Development of IT technologies has introduced new
powerful way to buy medicines – e-pharmacies. Online-
pharmacies obtain popularity due to ready availability and
cost benefits of medicines. Currently each country in EU can
establish rules for opening of e-pharmacy (26). Despite on
national rules regulating the operation of Internet pharma-
cies, the nonexistent contact with the pharmacist, question-
able quality of the drug counselling and unclear conditions
to order prescription medicines may serve as the potential
threat to the effective and safe medical treatment of the pa-
tients. However, according to the recent developments the
e-pharmacies do not decrease the role of traditional com-
munity pharmacies. It would be important to educate public
to use this source of medicines properly.
- Deregulation of the medicines distribution system at the
end of 1990`s and on the beginning of 2000´s has ended
56 pharmacy monopoly of the sale of OTC medicines in many
European countries. Does the better accessibility to OTC-
medicines outweigh missing consultation of the pharma-
cist?
3rd BBBB International Conference on Pharmaceutical Sciences
- There could be mistrust towards new professional roles of
the pharmacists by the patients and other health care profes-
sionals. Physicians may feel threatened by the pharmacists
who perform the medical review or prescribe medicines.
- Trend towards business orientation may turn community
pharmacies to the shops of medicinal products, where the
pharmacist is only a seller with no obligation to give any
counselling to the patient.
CONCLUSION
Regardless of the reorientation of the core values, practice
and education of the pharmacy profession within the past
20-30 years, the new initiatives and activities serve promis-
ing opportunity for the future developments. Pharmacist
can consider themselves as a part of the system that discov-
ers, develops, produces, distributes and provides informa-
tion on medicines.
REFERENCES
1. EU Directive 2005/36/EC. Available at: http://eur-lex.eu-
ropa.eu/LexUriServ/LexUriServ.do?uri=OJ:L:2005:255:0
022:0142:EN:PDF, visited July, 2009.
2. Leufkens H, Hekster Y, & Hudson S, (1997). Scenario anal-
ysis of the future of clinical pharmacy. Pharm World and
Sci, 1997; 19 182–198.
3. Volmer D, Bell JS, Janno R, Raal A, Hamilton DD, Airak-
sinen MS, Change in public satisfaction with community
pharmacy services in Tartu, Estonia, between 1993 and
2005. Research in Social and Administrative Pharmacy,
in press.
 ORAL PRESENTATIONS

O34
PHARMINE OR PHARMACEUTICAL EDUCATION IN EUROPE,
A CHALLENGE TO ALL PHARMACISTS
Bart Rombaut
(EAFP) Dept. Pharmaceutical Biotechnology and Molecular Biology, Vrije Universiteit Brussel,
Laarbeeklaan 103, 1090 Brussels, Belgium

I
n the XXI century EU pharmacists will play an increasingly
important role as partners in the efficient use of the health
care resources of the EU (community and hospital phar-
macists). They will also be major players in the development
of the EU pharmaceutical industry (industrial pharmacists).
Whilst abiding by the recommendations for the duration
and course content for EU pharmacy education and train-
ing given in the directive 2005/36/EC on the recognition of
professional qualifications, PHARMINE will examine the op-
portunities for the introduction of the principles of the Bolo-
gna declaration into pharmacy education and training with
the aim of tuning the latter to the future needs in the three
areas of pharmaceutical expertise: community, hospital and
industrial pharmacy.
The PHARMINE consortium consists of universities which
are members of the European Association of Faculties of
Pharmacy (EAFP) and EU partner associations representing
community, hospital of industrial pharmacy, together with
the European Pharmacy Students’ Association and other in-
terested bodies. It will develop a bachelor/master/doctorate
system for pharmacy education and training taking into ac-
count the need for basic pharmaceutical competences (and
mutual recognition of pharmacy qualifications) and the op-
tion needed for pharmaceutical expertise in, for example,
industry.
To do this, the consortium will survey existing EU pharmacy cur-
ricula and attempts to adapt these to the Bologna process.

57

October 26 - 28, 2009 / Antalya - TURKEY

 ORAL PRESENTATIONS

O35
The PHARMINE survey of pharmacy education and training in Europe:
a databank of EU HEIs delivering pharmacy education and training
and a task force for the survey of competency curricula (work
programme 7 (WP7))
Jeffrey Atkinson and Bart Rombaut
*EAFP, c/o Dept. Pharmaceutical Biotechnology and Molecular Biology, Vrije Universiteit Brussel, Laarbeeklaan 103,
1090 Brussels, Belgium

Aims and objectives Performance indicators

The aims and objectives of WP7 are to produce a databank of
pharmacy education and training leading to basic pharma-
cist qualifications and to qualifications required for optional
activities, in pharmacy higher education institutes (HEIs) of
the EU.
WP7 aims to survey to what extent the “Bologna” and the
“Sectoral profession” (based on 2005/36/EC) models for
pharmacy education and training are compatible (if at all:
see figure).
Organisation
The organisation of WP7 will be by electronic means and
telephone conference. WP7 will collect data directly from
pharmacy HEIs in the EU, from the EAFP archives (especially
58 the previous TEMPUS programme, see abstract of Rombaut
and Atkinson, this meeting), from PGEU, EAHP, EIPG and EPSA
and from all other web and other sources available.
Milestones
The principal milestone will be the delivery of the data re-
quired for the production of the white papers in WP2.
The main performance indicator is the retrieval of full data
from at least one HEI in each member state that delivers a
pharmacy diploma qualification. In the larger member states
an objective of two HEIs would be useful. As far as relation-
ships with other WPs and overall project it is obvious that the
success of the other WPs depends upon the quality of the
data obtained by this WP.
Relationships with other WPs and overall project.
This WP will work together with other WPs regarding what
type of information is to be gathered, in which form it is to
be handed over to the other WPs.
Although the main activity of WP7 will be over by month 12,
its activities will continue (and be slowly turned over to the
European Working Group on Pharmacy Education) with an
aim to ensuing that the information available in the data-
bank is kept up-to-date.
Anyone wishing to join WP7 should apply via the PHARMINE
website:
www.pharmine.orgv

3rd BBBB International Conference on Pharmaceutical Sciences

POSTER PRESENTATIONS
 POSTER PRESENTATIONS
P001-Characterization and In-
Vitro Dissolution Studies of Solid
Dispersion Systems of Gliclazide with
Macrogols
Ibrahim Khattab1, Abel-Azim Zaghloul1, Aly Nada1
Department of Pharmaceutics, faculty of Pharmacy, Kuwait
University, Kuwait1
INTRODUCTION
Solid dispersion techniques are very useful in pharmaceuti-
cal sciences because of the increasing number of novel drug
candidates that are poorly soluble, but there is still limited
information available on their ability to be processed into
the final dosage forms 1,2. Many substances can be used as
carriers to prepare solid dispersions among which, one of
the popular carriers used are polyethylene glycols (PEG)3,4 .
The objective of the present investigation was to improve
the aqueous solubility and dissolution rate of Gliclazide
(GLC), a hydrophobic anti-diabetic drug by solid dispersion
(SD) technique, using a water soluble carrier i.e. Macrogol
(Polyethylene glycol).
EXPERIMENTAL METHODS
Preliminary screening of various grades of PG’s (4000, 10000
and 20000) at different concentrations (0.5, 1, 2, 5 and 10%)
was done by phase solubility studies in order to analyze their
62 influence on solubility of GLC. SD’s of GLC and PG in differ-
ent ratios (1:1, 1:2, 1:5 and 1:10) were prepared by the fusion
technique. The SD’s were compared with the original drug
as well as the physical mixtures (PM) in the same ratios for
in-vitro dissolution in distilled water and phosphate buffer
(pH 7.4) by the USP Type II method. DSC studies were per-
formed to identify the physicochemical interaction between
the drug and the carrier. SD with satisfactory characteristics
was used for the formulation of tablets by wet granulation
method and compared with a commercial brand.
RESULTS AND DISCUSSION
It was evident from the phase solubility studies that the drug
solubility increased linearly with increasing PEG concentra-
tions indicative of the AL type of phase diagram. Intrinsic
solubility of GLC in water was found to be only 6.144 µg/
ml. Dissolution of GLC improved significantly in solid dis-
persions as compared with the original drug and physical
mixtures. SD’s of GLC with various grades of PEG in 1:2 w/w
ratio showed almost 100% release in phosphate buffer (pH
7.4) in 30 mins, as compared to 23% dissolution of plain GLC.
Results of DSC studies demonstrated that enhanced dissolu-
tion of GLC from SD might be due to a decrease in crystallin-
ity of GLC in the molten state while its formulation by fusion
method. SD tablets of GLC and PEG 20,000 in 1:2 w/w ratio
exhibited a better dissolution profile (54.21%) than the com-
mercial brand (30.26%).
CONCLUSION
The use of GLC:PEG 20,000 solid dispersion gave satisfy-
ing and reproducible drug dissolution. Also, the SD based
3rd BBBB International Conference on Pharmaceutical Sciences
tablets exhibited good technological properties. Thus, this
study reveals the possibility of preparing SD tablets of GLC
with improved aqueous solubility and dissolution rate.
ACKNOWLEDGEMENT
The authors would like to acknowledge Kuwait University for
funding this research project (grant No. PP01/07). Thanks is
extended to Dr Farzana Bandarkar for excellent technical as-
sistance.
REFERENCES
1. L. Chengsheng, G. Kashappa. Characteristics of Rofecox-
ib-Polyethylene glycol 4000 Solid Dispersions and Tab-
lets based on Solid Dispersions. Pharmaceutical devel-
opment and Technology. 10; 467-477 (2005).
2. D. Leonardi, M. G. Barrera, M. C. Lamas, and C. J. Salomón.
Development of Prednisone:Polyethylene Glycol 6000
Fast-Release Tablets From Solid Dispersions: Solid-State
Characterization, Dissolution Behavior, and Formula-
tion Parameters. AAPS PharmSciTech. 8 (4); Article 108
(2007).
3. Y. Özkan, N. Doğanay, N. Dikmen, A. Işimer. Enhanced
release of solid dispersions of etodolac in polyethylene
glycol. Farmaco. 55; 433-438 (2000).
4. M. Franco, G. Trapani, A. Latrofa, C. Tullio, M.R. Proven-
zano, M. Serra, M. Muggironi, G. Biggio, G. Liso. Dissolu-
tion properties and anticonvulsant activity of phenytoin
polyethylene glycol 6000 and -polyvinylpyrrolidone
K-30 solid dispersions. Int. J. Pharm. 225; 63-73 (2001).
P002-PREPARATION AND EVALUATION
OF KETOPROFEN SOLID DISPERSIONS
Seda Rençber1, Mine Özyazıcı1, Tamer Güneri1, Gökhan
Ertan1
Ege University Faculty of Pharmacy Department of
Pharmaceutical Technology1 , İzmir, Turkey
INTRODUCTION
Ketoprofen [2-(3-benzoylphenyl)propionic acid] (KP) is an
analgesic and non-steroidal anti-inflammatory (NSAI) drug,
widely used in order to reduce pain, inflammation and stiff-
ness caused by several condition such as osteorarthritis,
rheumatoid arthritis, ankylosing spondylitis or abdominal
cramps associated with menstruation. The mechanism of ac-
tion of KP is mainly associated to the inhibition of the body’s
ability to synthesise prostaglandins (1, 2). KP has poor solu-
bility in water and acidic conditions and has short half-life,
low bioavailability3.
Solid dispersions (SDs) of many poorly water soluble drugs
with hydrophilic carrier matrix have been formulated for im-
proving drug dissolution rate, which can be further improved
if such polymers have surface active properties. Moreover,
SDs may improve the bioavailability of poorly soluble drugs
by increasing the drug dissolution rate4,5.
Poloxamers are polyoxyethylene-polypropylene block co-
polymer nonionic surfactants that have been widely used
 POSTER PRESENTATIONS

as wetting and solubilizing agents, and surface adsorption

excipients. They have been employed to enhance the solu-
bility, dissolution and bioavailability of many poorly water
soluble drugs. Poloxamer 188 (P188) is selected to prepare
SDs because of its low melting point (about 56-57oC), sur-
factant properties and oral safety4.

The aim of the this study was to prepare KP–P188 solid dis-
persions in different ratios as drug-carrier system using melt-
ing method and to compare the in-vitro release of formula-
tion with KP and commercial product.

MATERIALS and METHODS Figure 2: The release rates of KP, SDs and CP in pH 4.5
Materials: Ketoprofen was a gift from Eczacibasi (Eczacıbaşı
İlaç S.T.A.Ş. Turkey). P188 was a gift from Basf.

Preparation of Formulation: KP and P188 1:1 (A1), 1:2 (A2),

1:3 (A3), 1:4 (A4), 2:1 (A5), 3:1 (A6) and 4:1 (A7) weight ratios
(w/w) were prepared by melting method. KP and P188 were
mixed at 1:1, 1:2, 1:3, 1:4, 2:1, 3:1 and 4:1 weight ratio and ob-
tained mixture was melted on water bath with stirring. After
10 -15 minutes, the mixture was cooled at fridge for 24 h. The
solidified solid dispersions were sieved and stored in a vial
at room temperature for further use. The formulations were
used containing equivalent to 150 mg KP.

In Vitro Release Studies: In vitro release studies of KP, SDs and

Figure 3: The release rates of KP, SDs and CP in pH 6.8
commercial product were performed using United Stetes
CONCLUSION
Pharmacopeia (USP) model digital tablet dissolution test ap-
Dissolution rate of KP were enhanced by preparing KP-P188
paratus at the paddle rotation speed of 50 rpm in 900 ml pH
(1:3) SDs in reletively easy, simple, quick, inexpensive and re-
1.2±0.1 (simulated gastric fluid), pH 4.5±0.1, pH 6.8±0.1 and
producible manner using low temperature melting method. 63
pH 7.5±0.1 (simulated intestinal fluid) at 37oC. The studies
Immediate release of free KP from A3 obtained rapid absop-
were tested and estimated at different time intervals spec-
tion and improved bioavailability compared to KP and CP.
trophotometrically at 259, 260, 261 and 260 nm, respectively
The solubility of KP was slightly increased from A3 formula-
(n=3).
tion in the pH 1.2, 4.5, 6.8 and 7.5.
RESULTS and DISCUSSION
REFERENCES
The in vitro release of KP, SDs and commercial product (CP)
1. Reynolds, J.E.F. (Ed.) Martindale The Extra Pharmacopeia,
in pH 1.2, 4.5, 6.8 and 7.5 were shown in Figure 1, Figure 2,
Vol.29, The Pharmaceutical Press, London, 1989; pp.25.
Figure 3 and Figure 4, respectively.
2. Bonina, F.P., Puglia, C., Barbuzzi, T., Caprariis, P., Palagiano,
F., Rimoli, M.G., Saija, A. In Vitro and In Vivo Evaluation
As seen in the Figure 1, 2, 3 and 4 the proporties of dissolu-
of Polyoxyethylene Esters as Dermal Prodrugs of Keto-
tion profiles of KP, SDs and CP were significantly different. Re-
profen, Naproxen and Diclofenac, Eur J Pharm Sci. 2001;
lease profile of A3 in pH 7.5 were obtained as 100% release,
14:123-134.
an the end of the 15 min.
3. Roda, A., Sabatini, L., Mirasoli, M., Baraldini, M., Roda,
E. Bioavailability of a New Ketoprofen Formulation
for Once-Daily Oral administration, Int J Pharm. 2002;
241:165-172.
4. Newa, M., Bhandari, K. H., Li, D. X., Kwon, T. H., Kim, J. A.,
Yoo, B. K., Woo, J. S., Lyoo, W. S., Yong, C. S., Choi, H. G.
Preparation, Characterization and In Vivo Evaluation of
Ibuprofen Binary Solid Dispersions with Poloxamer 188,
Int J Pharm. 2007; 343: 228-237.
5. Dordunoo, S. K., Ford, J. L., Rubinstein, M. H. Preformula-
tion Studies on Solid Dispersions Containing Triamterene
or Temazepam in Polyethylene Glycols or Gelucire 44/14
for Liquid Filling of Hard Gelatin Capsules, Drug Dev Ind
Pharm. 1991;17(12):1685-17.
Figure 1: The release rates of KP, SDs and CP in pH 1.2

October 26 - 28, 2009 / Antalya - TURKEY

 POSTER PRESENTATIONS

Figure 4: The release rates of KP, SDs and CP in pH 7.5

Figure 1: Gelling profile of 10% P1670/water dispersion
P003-Study Of Gel-Forming
Properties Of Sucrose Esters For Contact angle measurements
Thermosensitive Drug Delivery The contact angle (Θ) of a solid was determined by the ses-
sile drop technique, using the OCA 20 Optical Contact Angle
Systems Measuring System (Dataphysics, Filderstadt, Germany), and
the method of Wu. The liquids used for contact angle mea-
Angela Szuts 1, Maria Budai Szucs1, Istvan Eros1, Naoya surement were bidistilled water and diiodomethane. The
Otomo2, Piroska Szabo Revesz1 polarity percentage was calculated from the γp and γ values:
(γp/γ )*100.
University of Szeged, Department of Pharmaceutical
Technology, Hungary 1 Dissolution studies
Mitsubishi Kagaku Foods Corporation, Tokyo, Japan2 For the dissolution tests, PAR-SE physical mixtures were filled
into hard gelatine capsules. The capsules contained 50 mg of
INTRODUCTION PAR and 50 mg of SE. The release of the drug was studied by
Sucrose esters (SEs) are biodegradable, non-toxic, non-ionic using Pharmatest equipment (Hainburg, Germany) at a pad-
64 surfactants. The aims of the present work were to study the dle speed of 100 rpm. 900 ml artificial gastric juice with a pH
gelling behaviour of SEs and the effects of this property on of 1.2 (±0.05) at 37 °C (±0.5 °C) was used. The drug contents
drug release. of the samples were measured spectrophotometrically (λ =
244 nm) (Unicam UV/Vis spectrophotometer).
MATERIALS AND METHODS
Materials Drug release kinetics
Sucrose palmitate P1670 (HLB=16), and sucrose stearate In order to describe the kinetics of the drug release from
S970 (HLB=9) were kindly provided by Syntapharm GmbH the formulations, the zero-order, first-order, Hixson-Crowell
(Germany). The model drug paracetamol (PAR) was supplied cube root law, Higuchi model and Korsmeyer–Peppas equa-
by Hungaropharma Ltd. (Hungary). tions were used.

Rheological measurements RESULTS AND DISCUSSION

The rheological properties were studied with a Physica MCR101 Rheological measurements
rheometer (Anton Paar, Austria). The measuring system was of The concentration dependences of the gelling temperatures
plate and plate type (diameter 50 mm, gap 0.1 mm). of the SE dispersions can be seen in Figure 2.

Dynamic frequency sweep tests were carried out at 37 °C,

at 1.0 Pa in the limit of the linear viscoelastic region. Stor-
age modulus (G’) and loss modulus (G”) were determined for
frequencies between 0.1 and 100 Hz. The gelling character-
istics were measured at a constant frequency of 1.0 Hz at a
constant strain of 1.5% (this strain was within the linear vis-
coelastic range of the SE gels). The heating rate was 2 °C/min.
The gelling temperature of an SE was defined as the tem-
perature where the storage modulus was half way between
G’ for the SE dispersion and G’ for the SE gel (Figure 1).

Figure 2: Concentration dependence of gelling tempera-

tures of SE dispersions

3rd BBBB International Conference on Pharmaceutical Sciences

 POSTER PRESENTATIONS

According to the frequency sweep tests, both dispersions Table 2 shows the correlation coefficients (r²) of the formula-
display gel characteristics, and S970 dispersions have more tions.
elastic behaviour (higher G’ values) and better gel character-
istics (lower slopes of the curves) (Figure 3). Table 2: Correlation coefficients of the linearization of drug
release plots
Sample Zero- First- Higuchi Hixson- Korsmeyer-
order order Crowell Peppas
PAR-P1670 0.8760 0.9703 0.9740 0.9444 0.9816
PAR-S970 0.9497 0.9604 0.9953 0.9570 0.9944

CONCLUSION
The results revealed that the gelling of the SEs is temperature-
and concentration-dependent. The examined sucrose stear-
ate (S970) has a stronger gel structure than that of sucrose
palmitate (P1670) and this behaviour has a great effect on the
drug release. The analysis of the dissolution kinetic data in this
study revealed that the dissolution follows the Korsmeyer-
Peppas (PAR-P1670) or Higuchi (PAR-S970) equations.
Figure 3: Frequency dependence of 5% aqueous dispersions
of P1670 and S970
P004-Preparation Of Famotidine Orally
Contact angle measurements Disintegrating Tablets
The results of contact angle measurements, which provide
information on the surface free energies and polarities of the Senem Sevtap Ölmez1, İmran Vural1
samples, are presented in Table 1.
Department of Pharmaceutical Technology, Faculty of
Table 1: Contact angles, surface free energies and polarities Pharmacy, Hacettepe University, 06100 Ankara/ Türkiye1
of the materials
Materials Θwater[°] Θdiiodomethane[°] Υ[mNm-1] Polarity INTRODUCTION
[%] The aim of this study was to formulate famotidine orally disinte- 65
P1670 18.49±0.85 58.76±0.72 70.10 60.96 grating tablets (ODTs) with sufficient mechanical integrity, and
to prepare ODTs by direct compression which has simple and
S970 46.79±1.76 62.99±1.10 55.25 53.85
low-cost manufacturing processes using materials with proven
PAR 39.32±1.77 28.72±2.42 67.96 40.52 safety as excipients. ODTs are solid dosage forms containing me-
PAR-P1670 23.11±1.83 47.13±1.46 70.55 53.61 dicinal substances that disintegrate rapidly in the mouth and re-
PAR-S970 37.19±1.13 54.74±1.81 62.37 53.05 quiring no chewing or additional water to facilitate swallowing.
ODTs are easy to use and convenient for the patient who cannot
swallow, such as the elderly, stroke victims, bedridden patients,
Dissolution studies patients affected by renal failure or dysphagia and patients who
Due to the swelling properties of the SEs, they sustained the refuse to swallow such as pediatric, geriatric and psychiatric pa-
drug release (Figure 4). The stronger gel structure (Figure 3) tients1. ODTs have convenience of administration and accurate
and lower polarity (Table 1) of S970 resulted in a slower drug dosing as compared to liquid dosage forms as well.
release.
MATERIALS AND METHODS
Materials
Famotidine was kindly provided by Mustafa Nevzat İlaç Sanayi,
İstanbul, Türkiye. Sodium stearyl fumarate (Pruv®) was obtained
as gift sample from JRS Pharma, Germany. Avicel PH102 (MCC),
low hydroxypropyl cellulose-11 (LHPC) and aspartame were
kindly provided by Abdi İbrahim İlaç Sanayi, İstanbul, Türkiye.

Figure 4: Dissolution curves of PAR and PAR-SE samples
Drug release kinetics
Preparation OF ODTs
MCC and LHPC were selected as the basic excipients due to
their proven safety and excellent compactibility. LHPC was
also used as a disintegrant. Pruv® was used as lubricant and
aspartame was used as sweetener. ODTs were prepared ac-
cording to the following procedure. Firstly, MCC and L-HPC
at 4:1 or 9:1 ratios were mixed for 5 min. Famotidine was
mixed well with the mixture of MCC and L-HPC and then, as-

October 26 - 28, 2009 / Antalya - TURKEY

 POSTER PRESENTATIONS
partame (1%) was added and mixed. Lastly, sodium stearyl
fumarate (2%) was added and mixed for 5 min. To prepare
200 mg tablets, the above-mentioned mixtures were com-
pressed by Erweka AR 400 Korsch/Germany.
EVALUATION OF TABLET CHARACTERISTICS
Weight Variation: Twenty tablets were randomly selected from
each formulation and weighed using a Shimadzu digital bal-
ance. The mean ± standard deviation (SD) were recorded.
Measurement of Diameter and Thickness: Ten tablets from
each formulation were taken randomly and their diameter
and thickness were measured using a micrometer. The mean
± SD values were calculated.
Hardness: Tablet hardness, which is defined as the force required
to break a tablet by radial compression, was measured with a
tablet hardness tester (n=10) (Schleuniger 2E/Sweetzerland).
Measurement of Friability: Friability of the tablets was deter-
mined using a friabilator (Smiths/England) at 25 rpm/min for 4
min. Pre-weighed tablets were placed in a plastic chambered
friabilator then, the tablets were de-dusted, reweighed and
percentage weight loss (friability) was calculated (n=20).
In vitro disintegration time: In vitro disintegration time of the
ODTs was determined using USP disintegration test appara-
tus without disk(Manesty tablet disintegration test unit MK4)
for six tablets. The 1000 mL of distilled water at 37 ± 1 °C was
used as test fluid at the rate of 30 ± 2 cycles / min.
66
In vitro dissolution test: It was performed according to USP
XXX using the paddle method at 50 rpm in pH 4.5, 0.1 M
phosphate buffer utilizing a dissolution system (Sotax CH
4123). Samples (5 mL) were collected at predetermined time
intervals (2, 5, 10, 15, 20 and 30 min) and replaced with equal
volume of fresh medium and filtered. The detection of famo-
tidine was performed spectrophotometrically at 266 nm.
RESULTS AND DISCUSSION
The results of tablet characteristics of prepared ODT formu-
lations containing MCC and L-HPC at 4:1 or 9:1 ratios were
given in Table 1.
Table 1: Results of the tablet characteristics
Formulation
Tablet
MCC: LHPC (4:1) MCC: LHPC (9:1)
Characteristics
Mean ± SD Mean ± SD
Mean mass (mg) 200.89 ± 0.002 200.97 ± 0.003
Diameter (cm) 1.220 ± 0 1.220 ± 0
Thickness (cm) 0.15 ± 0 0.16 ± 0
Hardness (kp) 8.50 ± 0.67 8.57 ± 0.38
Friability (%) 0.33 0.38
Disintegration 14.6 ± 4.21 10.0 ± 2.13
time (s)
% dissolved after 94.62 ± 4.40 96.67 ± 10.05
30 min.
3rd BBBB International Conference on Pharmaceutical Sciences
Friability was observed between % 0.33-0.38 which were
below 1% indicating the sufficient mechanical integrity and
strength of the prepared tablets. According to EP, orodis-
persible tablets disintegrate within 3 min using conventional
disintegration apparatus. The prepared tablets were disinte-
grated in 14.55 ± 4.21 and 10.03 ± 2.13 s for MCC: LHPC (4:1)
and MCC: LHPC (9:1), respectively. The tablets containing
lower concentration of L-HPC showed rapid disintegration
time and high crushing tolerance. The high concentration of
L-HPC in tablets results decreasing in the degree of wetting,
so increasing disintegration time. For in vitro dissolution test,
the prepared tablets were complied the USP requirement for
famotidine tablets (dissolved more than 75 % of the labeled
amount of famotidine in 30 min) (Table 1). The dissolution
data is shown in Figure 1.
Figure 1: Dissolution profiles of prepared famotidine ODTs
(: MCC: LHPC (4:1), º: MCC: LHPC (9:1)) (mean±SD, n=6)
CONCLUSION
In conclusion, famotidine tablets with sufficient crushing tol-
erance and short disintegration time can be prepared using
Avicel PH102 and LHPC-11.
REFERENCES
1- Sastry SV, Nyshadham JR, Fix JA. Recent technological ad-
vances in oral drug delivery - A review, Pharm Sci Technolo
Today. 2000; 3: 138-145.
P005-Effect Of Curing And Tableting
On Thermomechanical And Release
Properties Of Eudragit-Based
Diclofenac Sodium Pellets
Mohammadreza Abbaspour1, Masoud Alimorad2, Iman
Beygi1
School of pharmacy,Ahwaz Jundishapur University of Medical
Sciences, Ahwaz, Iran1
SOHA pharmaceutical co., Tehran, Iran2
INTRODUCTION
One of the most popular oral drug delivery systems is the
multiparticulates such as pellets which their benefits over
single unit dosage forms have been widely studied. Such
pellets can be either filled in hard gelatin capsules or com-
pacted into multiple unit tablets. One challenge in the pro-
 POSTER PRESENTATIONS

duction of such tablets is maintaining the desired drug re-

lease after compaction, as the applic-ation of compaction
force can damage the pellets and alter the drug release. It
has been shown that curing of eudragit based ibupr-ofen
pellets leads to decrease in drug release rate and improves
the mechanical strength of pellets to ensure maintaining pel-
lets integ-rity and their drug release rate after compact-tion1.
The aim of this study was to invest-igate the effect of curing
and tableting on thermo-mechanical and release properties
of diclofenac eudragit-based pellets and to design multipar-
ticulate tablets which upon oral ingestion rapidly disinte-
grate into comp-rising pellets and preserve primary release.

MATERIALS AND METHODS

Materials: Diclofenac Na (Hakim Pharmac-euticals, Iran), Avi-
cel PH101 (Razak Pharm-aceuticals, Iran), Eudragit RL PO and
RS PO (Rohm Pharma , Germany), PVP K30 and cross-linked
povidone (PVP xl) (Fluka, Switzerland), PEG 4000 and Mg-
stearate (Merck, Germany).

METHODS
Preparation of pellets: 9 formulations containing different
levels of diclofenac (10, 20, and 30%), different amounts of
Eudragit RS:RL (1:1) (20, 40 and 60%), 10% Avicel and 3% PVP
K30 were manufactured and studied based on 32 full facto-
rial design using extrusion spheronization. The obtained pel-
lets were dried at 40˚C for 10h. The pellets were cured at 60˚C
for 24h and then were allowed to cool at room temperature.
Mechanical tests: The crushing strength and elastic modu-
lus of 30 uncured and cured pellets were determined using a
material testing machine (Hounsfield, UK). The speed of the
upper mobile platen fitted with a 1 kN load cell was set at 1
mm/min.
Tableting: Tablets containing 60% pellet and 40% filler blend
(59.5% Avicel, 10% PVP xl, 30% PEG 4000, 0.5% Mg-stearate)
were prepared by manual filling 1000 mg of above mixture
in a 12 mm die and compressed using a single punch tablet-
ing machine (Erweka, Germany)
Dissolution Test: The tests were carried out on pellets or tab-
lets containing 48 mg of drug (n=6) in USP apparatus I, at
100 rpm, in phosphate buffer (pH 7.4), at 37˚C. The samples
were taken at determined intervals and assayed by spectro-
photometer at 274 nm and converted to mean dissolution
time (MDT)2.
DSC: DSC analysis was performed on Eudragit RS or RL, di-
clofenac and their physical mixtures using a DSC (Mettler
Toledo DSC 822e, Switzerland).
Figure 1: Influence of amount of diclofenac and Eudragit on
MDT of: a) uncured and b) cured pellets
In this study all pellets had brittle behavior under the me- 67
chanical tests and the curing process had no effect on their
strength and mechanical properties (data were not shown).
DSC of diclofenac and eudragit physical mixtures showed
that diclofenac had no effect on Tg of polymers (Figure 2),
but the peak showing melting point of drug at 300°C were
disappear probably due to electrostatic interactions be-
tween drug and polymers3.
Cured pellets containing 10% drug and 60% Eudragit which
had the maximum MDT was chose to study the effect of
tableting on pellet properties. Tablets containing 60% pel-
lets and 40% filler blend with acceptable hardness (60±8 N)
and disintegration time (5min) have been produced.
In vitro release profiles for compacted and un-compacted
pellets showed that no apparent damage to the pellets has
occurred as a result of the compaction process (Figure 3).
Intact pellets were rapidly released from single unit dosage
form upon contact with dissolution medium.

RESULTS
The results showed that increase in amount of drug in both
uncured and cured pellets led to decrease in MDT, whereas
increasing amount of eudragit in uncured pellets had no
considerable effect on drug release but in cured pellets, spe-
cially at lower amounts of drug, led to increase in MDT or
decrease in drug release rate (Figure 1).

October 26 - 28, 2009 / Antalya - TURKEY

 POSTER PRESENTATIONS
, ing attention has been paid on this drug since it has the
Figure 2: DSC of physical mixture of diclofenac and eudragit RL
Figure 3: Dissolution profile for cured pellets and compacted
68 pellets
CONCLUSION
Curing has no effect on pellets hardness but decrease drug
release rate. Multiparticulate tablets can be prepared with a
release profile similar to original pellets
REFERENCES
1. Abbaspour MR. et al., Design and study of ibuprofen dis-
integrating sustained-release tablets comprising coated
pellets, Eur. J. Pharm. Biopharm. 2008; 68: 747-759.
2. Costa FA. et al., Comparison of dissolution profiles of
ibuprofen pellets, J. Control. Release 2003; 89: 199-212.
3. Sipos P. et al., An assessment of the interactions be-
tween diclofenac Na and ammonio methacrylate copo-
lymer using thermal analysis and Raman spectroscopy,
J. Pharm. Biomed. Anal. 2008; 46: 288-294.
3rd BBBB International Conference on Pharmaceutical Sciences
P006-Formulation Of Sustained Release
Oral Dosage Forms Of Hydralazine
Hydrochloride For The Treatment Of
Hypertension And Cancer
Melike Üner1, Benek Çelebi1
Istanbul University, Faculty of Pharmacy, Department of
Pharmaceutical Technology, 34116 Beyazit, Istanbul Turkey1
INTRODUCTION
Hydralazine hydrochloride (HH) has been used as a hypoten-
sive drug for years since it lowers the blood pressure by af-
fecting plain muscles of vessels1. In the last years, an increas-
demethylating effect on various suppressor genes. There-
fore, it can be used for various types of cancer in order to
support chemotherapy2.
Plasma half-life of HH is 3-4 hours. It is administered up to
200 mg daily as 3-4 divided doses by oral route1,4. The aim
of this study is to design oral dosage forms of HH release in
order to maintain drug in the systemic circulation for a lon-
ger time period. Therefore, its side effects are minimized by
lower dose administration, drug bioavailability is increased
and patient compliance is improved. For this aim, sutained
release (SR) tablets of HH were prepared by using various
polymers. Compression characterization of SR tablets and in
vitro HH release were studied. This study is continued. It is
already detailed according to the data obtained.
MATERIALS AND METHODS
Materials
Hydralazine hydrochloride (Novartis, Turkey), Methocel® K15
Premium EP (Colorcon, England), Carbopol® 971 PNF (No-
veon Inc., U.S.A.), Compritol® 888 ATO (Gattefossé, France),
cetyl alcohol (Cognis, Turkey), Avicel® PH-101 (Select-chemie
AG, Switzerland), magnesium stearate (Prever, Italy), Aerosil®
200 (Degussa, Germany).
Preparation of SR Tablets
Tablets were prepared by direct compression method (Table).
For this aim, a hydrolic press equipped with 10 mm diameter
flat-punch (Yeniyurt, Turkey) was used under 1500 psi force.
Physical characterization of tablets was tested in order to
determine weight variation, tablet diameter and thickness,
disintegration time, friability % and hardness5.
In Vitro Dissolution Studies
Release of HH from SR tablets was studied according to the USP
32 Method II (paddle) at 37°C ± 0.5 in 900 mL distilled water as
a dissolution medium at 50 rpm rotation speed. At predeter-
mined time intervals, 0.8 ml samples were taken, diluted to 10
ml and filtered through S&S5893 type blue ribbon filter paper.
The solution was assayed by UV spectrometry at 260 nm (Shi-
madzu UV-1700 Spectro- photometer, Japan) in water.
 POSTER PRESENTATIONS

Table 1: Composition (%) of tablets REFERENCES

Formula 1. Martindale. 31st Edition. Royal Pharmaceutical Society,
Tablet ingredients London, 1996.
A B C D
2. Song Y, Zhang C. Hydralazine inhibits human cervical
HH 28.57 28.57 28.57 28.57
cancer cell growth in vitro in association with APC dem-
Avicel 28.43 28.43 28.43 28.43 ethylation and re-expression. Cancer Chemo.Pharmacol.
Methocel 40 - - - 2009; 63: 605-613.
Carbopol - 40 - - 3. Estrada Flores L, Duenas Gonzalez A, Borbolla Garcia A,
Serna Martinez R, Rivera Hernandez A, Mohar Betan-
Compritol - - 40 -
court A. Pharmaceutical composition for the sustained
Cetyl alcohol - - - 40 release hydralazine and use thereof as a asupport for
Magnesium sterate 2 2 2 2 cancer treatment. 2007; EP 1 803 447 A1.
Aerosil 1 1 1 1 4. Kirsten R, Nelson K, Kirsten D, Heintz B. Clinical pharma-
cokinetics of vasodilators, Part II. Clin.Pharmacokinet.
RESULTS AND DISCUSSION 1998; 35: 9-36.
SR tablets were found to meet compendial requirements 5. Üner M, Altınkurt T. Evaluation of honey locust (Gleditsia
related to compression quality (weight variation, diameter, triacanthos Linn.) gum as sustaining material in tablet
thickness, friability % and hardness). dosage forms. Il Farmaco. 2004; 59: 567-573.

The highest release was observed with Formula D containing

cetyl alcohol (Figure). This formula released 100 % of drug
after 7 hours. Whereas, Formulas A, B and C released 79.05
%, 61.24 % and 89.32 % of drugs from tablets after 8 hours,
respectively. When a system composed of hydrophylic poly-
mers gets in contact with aqueous medium, medium passes
into the system and polymer at surface turns into a gel at
first. When medium transfer into the system continues, gel
layer is improved to be thicker and the system swells. There-
fore, drug release occurs by passive diffusion. The system was
waxy matrix because of containing cetyl alcohol in Formula
D. The release was highest since cetyl alcohol concentration
was low in the formulation.
Drug release from all formulations were kinetically evaluated
and they were found to fit to Higuchi kinetic model.
P007-IMMOBILIZATION OF PANCREATIN
USING ALGINATE BEADS
Nefise Özlen Şahin1, S. Ektiren2, S. Soylu3, A. Yüksel2
Mersin University, Faculty of Pharmacy, 1Department
of Pharmaceutical Biotechnology, 2Department of
Pharmaceutical Technology, Yenisehir Campus, Mersin 33169,
Turkey.
3
Mustafa Kemal University, Research Center, Hatay, Turkey.
69
INTRODUCTION
Impeded flow of pancreatic juice due to mechanical obstruc-
tion of the pancreatic duct in patients with cancer of the
pancreatic head region results in exocrine pancreatic insuf-
ficiency with steatorrhoea and creatorrhoea. This may cause
profound weight loss that often occurs in these patients.
Weight loss in patients with pancreatic cancer may be cured
by pancreatic enzyme replacement therapy which will lead
to significant improvement in fat and protein absorption.
Various dosage forms of pancreatin including enteric-coated
tablets, pellets and microspheres are in the market.

Among the various drug delivery systems investigated to

Figure 1: Release profiles of HH from Formulas A, B, C and D
in water.
CONCLUSION
Methocel, Carbopol and Compritol were determined to be
suitable polymers for preparation of SR tablets of HH in order
to reduce dose intensity in the treatment of hipertension.
Additionally, they can be administrated as a supportive drug
in the cancer treatment since demethylating effect of HH will
also occure in a long time period.
achieve efficient and selective delivery of therapeutic agents
to the site of action, beads have gained more attention. Re-
cently, beads were prepared with natural polymers such as
alginate, chitosan, pectin become the focus of research in
this field. With all these in mind, we set our goal to prepare
pancreatin bearing beads for treatment of weight loss in the
patients with pancreatic cancer. Alginate was used as bead
forming polymer to explore the temporary protection of
pancreatin against acidic and enzymatic degradation during
gastric passage. In vitro dissolution results were compared
to that of drug itself to ensure such protective effect.
MATERIALS AND METHODS
Materials:Both sodium alginate (medium viscosity) and pan-
creatin were bought from Sigma Chem. Co, MO, USA. All
other reagents were analytical grade.

October 26 - 28, 2009 / Antalya - TURKEY

 POSTER PRESENTATIONS

METHODS RESULTS & DISCUSSIONS

Preparation of gel beads: Alginate gel beads were prepared
by a slightly modified method of Yotsuyanagi et al. Pancre-
atin was added to 6 ml of 1.5-3.0 % (w/v) sodium alginate so-
lution and dissolved completely. This solution was dropped
into an 85 ml 1-2% Barium chloride solution with mild agi-
tation within a period of 17 mins. The beads were formed
instantaneously. Subsequently, they were transferred into
falcon tubes and centrifuged at 900 rpm for 2 min. The su-
pernatant was removed by means of aspiration via vacuum.
Centrifugation process was repeated several times until no
barium chloride residue left on beads. Then, beads were
dried at 50ºC for 3 h using an incubator.
Determination of the size of the beads: The size distribution
of the beads was determined by using sieve analysis (Reich,
Germany) (n =3).
Assessment of drug loading capacity: 100 mg of beads were
digested in 100 ml of pH 7.4 phosphate buffer. The solution
was filtered and the absorbance of pancreatin was measured
spectrophotometrically at 660 nm.
Erosion studies: A certain amount of beads was placed in
a 10 ml of screw-capped bottle containing 5 ml phosphate
buffer (pH 7.4) and agitated at 37ºC in a shaker bath. At vari-
ous time intervals, beads were removed, washed and dried.
Weight lose was determined gravimetrically (n=8).
Using the method described above and electronic beads
generator, spherical and smooth surfaced beads with high
drug content (59.7 % entrapment efficiency) could be pro-
duced. SEM photographs revealed that the surface of the
beads is porous. The bead size varied between 0.86-1.15 mm.
The drug loading capacity of the beads prepared with 1.5 %
alginate solution was %59.7. Drug loading capacity showed
an increasing trend as the polymer (3.0%) and initial drug
amount (%1) increased upto 76.4%.
In vitro drug release profiles of the beads are given in Fig-
ure 1. Release was slower in a controlled manner over 6 h
for both formulations. The data indicated that in vitro drug
release was affected by alginate, drug and barium chloride
concentrations. An increase in alginate concentration from
1.5% to 3.00 % reduced the release of pancreatin from the
beads. Figure 2 indicates the effect of drug concentration on
the release profile. The release of the drug showed delayed
characteristics as the drug concentration was increased.
Lowering barium chloride concentration from 2% to 1% de-
creased the drug release (Figure 3). Comparison of the re-
lease profiles of encapsulated drug and that of plain drug
indicated that bead formation provides controlled release of
pancreatin.

Microscopic investigations: Shape and surface morphology

70 of the beads were investigated by both polarized and scan-
ning electron microscopes.

In vitro dissolution studies: Pancreatin bearing beads were

suspended in phosphate buffer (pH 7.4) contained in wide-
mouthed bottles, and stirred at 100 rpm in a shaker bath
at 37ºC. Samples were withdrawn at certain time intervals
(15, 30, 45, 60, 90, 120, 180, 240, 300, 360 min) and analyzed
spectrophotometrically at 660 nm.

Determination of swelling rate: Adequate amount of beads Figure 1: Effect of alginate concentration on the release pro-
were weighed and suspended in distilled water. 3 hours later, file of pancreatin (n=3).
they were filtered an the diameters were measured under the
dissection microscope. Diameters of the beads before and after
swelling process were compared for each bead. The differences
were calculated and converted into percentage swelling rate.

Table 1: Variables used for preparation of different alginate

bead formulations.
Variables Values Codes
Alginate concentration (% w/w) 1.5 F1A15*

3.0 F1A3
Drug concentration (% w/w) 0.5 F2P05*

1.0 F2P1
Barium chloride concentration (% w/w) 1.0 F3B1* Figure 2: Effect of drug concentration on the release profile
of pancreatin (n=3).
2.0 F3B2
*F1A15, F2P05 and F3B1 are the same formulations.

3rd BBBB International Conference on Pharmaceutical Sciences

 POSTER PRESENTATIONS
Figure 3: Effect of Barium chloride concentration on the re-
lease profile of pancreatin (n=3)
Erosion profiles of alginate beads are plotted against time in
Figure 4. It indicates that a considerable erosion occurs dur-
ing the 180 min.
Figure 4: Erosion profiles of beads.
Swelling rate of pancreatin bearing alginate beads were
found as 35.2%.
CONCLUSIONS
Based on the data obtained in this study, we can suggest
that alginate beads can serve as a convenient carrier for the
controlled release of pancreatin to be used in the treatment
of weight loss associated with pancreatic cancer.
REFERENCES
1. Edelman ER, Mathiowitz E, Langer R, Klagsbrun M. Con-
trolled and modulated release of basic fibroblast growth
factor, Biomaterials 1991; 12: 619-625.
2. Hermes RS, Narayani R. Polymeric alginate films and al-
ginate beads for the controlled delivery of macromole-
cules, Trends Biomater. Artif. Organs. 2002; 15(2), 54-56.
3. Arnesjö B, Ihse I, Odeberg H, Stahl E. Deficiency of lipoly-
tic pancreatic enzymes in an adult man. Digestion, 1974;
11: 147-151.
4. Bhopatkar D, Anal AK, Stevens WF. Ionotropic alginate
beads for controlled intestinal protein delivery: effect of
chitosan and barium counter-ions on entrapment and
release. J Microencapsul. 2005; 22(1): 91-100.
October 26 - 28, 2009 / Antalya - TURKEY
P008-Evaluation Of Physical Stability
Of Piroxicam Using Gelucire 44/14,
Labrasol And Polyethylene Glycol
4000
Ayşegül Karataş1, Şerife Bekmezci2
Department of Pharmaceutical Technology, Faculty of
Pharmacy,Ankara University, 06100-Tandoğan, Ankara, Turkey1
Department of Pharmaceutical Technology, Faculty of
Pharmacy, Ankara University, 06100-Tandoğan, Ankara, Turkey2
INTRODUCTION
Piroxicam is non-steroidal anti-inflammatory drug, clasified
in the Biopharmaceutic Drug Clasification System (BCS) as a
Class II drug with low solubility and high permeability1. In our
previous study2, semi-solid dispersion containing the mix-
ture of 20 % of Gelucire 44/14 and 80 % of labrasol brought
about the dissolution not less than 85 % of piroxicam within
30 min in each of the dissolution media (namely, acidic me-
dium, pH 1.2 SGF; pH 4.5 buffer; pH 6.8 buffer; and water). It
was probable that this formulation filled into the hard gelatin
capsules was thermosoftening at room temperature might
have tendency to turn into liquid form during storage. In this
study, Polyethylene glycol 4000 (PEG 4000) was added to Ge-
lucire and labrasol combination to solidify the formulation.
The modified semi-solid formulation containing gelucire
44/14, Labrasol and PEG 4000 were prepared in following ra-
tios: 20 %, 78 % and 2 %, respectively. Ratio of drug-excipient
was 1:20. Formulation was studied for in vitro dissolution be- 71
haviour and 12-month physical stability test at temperature
of 25oC and relative humidity (RH) of 60%. Characterization
of the drug/carrier combination was conducted using Fou-
rier –transformation- IR spectroscopy (FT-IR) and differential
scanning calorimetry (DSC).
MATERIALS AND METHODS
Materials: Piroxicam was obtained from dipharma (Turkey),
Gelucire 44/14 and labrasol were supplied by Gattefosse
(Saint-Priest cedex, France). PEG 4000 was purchased from
Merck (USA). Hard gelarine capsules were provided by Shion-
ogi (Qualicaps SA, Spain).
Preparation of semi-solid dispersion: Necessary amounts of
excipients heated to abouth 5 oC above the melting point
of Gelucire 44/14 on the water bath. The calculated amount
of drug was added to the molten vehicle with continuous
stirring. Then, mixture was poured into a plastic injector and
volumetrically filled into hard gelatin capsules at the tem-
perature close to the solidification point of the material.
Dissolution testing: Dissolution studies were conducted us-
ing USP Apparatus 1 (rotating basket method) (Sotax AT 7
Smart, Swithzerland) with three replicates, according to the
USP monograph of the drug3. In all experiments, 5 mL of dis-
solution sample was withdrawn at 5, 10, 20, 30, 45, 60 min
and replaced with an equal volume of the fresh medium.
Samples were assayed by UV spectrophotometry.
 POSTER PRESENTATIONS

FT-IR Spectroscopy: Drug–carrier interactions during storage

conditions were determined based on IR spectra measured
with a FT-IR spectroscope (Jasco, FT/IR 420) by the KBr meth-
od in the 4000-400 cm-1 region.

DSC: DSC analysis was carried out on a differential scanning

calorimeter (DSC 60 Shimatzu). The themograms were ob-
tained by heating the samples (5 mg) at a rate of 5 oC/min
from 25 oC to 250 oC under nitrogen purge.

Storage stability studies: Formulation was stored in closed glass Figure 1c

vials for a period of 12 months at 25°C ± 2°C/60% RH ± 5% RH. Dis-
solution testing was conducted at 3-6- and 12-month intervals to
assess changes in drug release characteristics. The dissolution pro-
cedure used was the same as described above. Changes in physical
state of the samples were also performed on the drug, excipients
and at 0-3-6- and 12-month intervals of formulation by FT-IR and
DSC analyses.

RESULTS AND DISCUSSION

Dissolution studies: It was show that the addition of PEG
4000 to the formulation afforded a solidification effect on
the formulation. In addition, the presence of the PEG poly-
mer in combination with Gelucire and labrasol showed simi-
lar results in dissolution when compared to the original for-
mulation. Namely, this formulation (initial) also provided at
least 85 % piroxicam dissolution within 30 min in each of the
media (Fig 1).
³²°°°°µµ°°°
72
Figure 1d
Figure 1.Dissolution profiles of formulation monitored initial
sample and samples stored for 3,6 and 12-month at 25°C ±
2°C/60% RH ± 5% RH at different buffers (a) pH 1.2 (b) pH 4.5
(c) pH 6.8 (d)water
Storage stability studies
When dissolution of the stored samples (3-6- and 12-month
intervals) were compared with that of initial formulation,
samples at 6- and 12- month intervals showed a significant
decrease in dissolution (Fig 1).

FT-IR Spectroscopy
The presence of an interaction in formulation during storage
period could not be confirmed by FT-IR spectroscopy (Fig 2).

Figure 1a

Figure 1b Figure 2: FT-IR spectrograms

3rd BBBB International Conference on Pharmaceutical Sciences

 POSTER PRESENTATIONS
DSC
The melting peak of formulation (Fig 3 b) was observed at
around 38 oC showing amourphous state of the drug in
the dispersion. Melting peaks of stored samples were ob-
served at 59.57 oC (at 6-month) and 113.1 and 120.67 oC (at
12-month) (Fig 3 d,g).
Figure 3: DSC thermograms
CONCLUSION
In terms of storage stability, formulation exhibited short-
term stability of at least 3 months but beyond that seemed
to decrease in dissolution at all pH’s
DSC results indicated the present of a chemical interaction
between the drug and excipients during storage period that
results in a decrease in dissolution rates.
REFERENCES
1. Amidon GL, Lennernas H, Shah VP, Crison JR. A theoreti-
cal basis for a biopharmaceutics drug clasification: The
correlation of in vitro drug product dissolution and in
vivo bioavailability. Pharm Res. 1995; 12:423-420.
2. Karataş A,Yüksel N, Baykara T. Improved solubility and
dissolution rate of piroxicam using Gelucire 44/14 and
Labrasol. Il Farmaco. 2005; 60:777-782.
3. US Pharmacopoeia 24, US pharmacopeial Convention
Rockville, MD, 2000, pp.1342
P009-Effects Of A Sucrose Ester On
Co-Micronized Mannitol-Based Drug
Formulation
Rita Ambrus1, Anita Pomazi1, Peter Sipos1, Naoya Otomo2,
Piroska Szabo Revesz1
University of Szeged Department of Pharmaceutical
Technology1
Market Development Department, Mitsubishi Kagaku Foods
Corporation2
INTRODUCTION
The application of carrier-based drug formulation is strongly
dependent on the preparation factors and the physicochem-
ical properties of the carrier systems. Mannitol is a carrier
that is frequently used in the field of pharmaceutical appli-
cations, especially in dry powder inhalation (DPI) systems. In
this case, the surfactant Tween 80 is generally used, as a sta-
bilizer. The sucrose esters, as wetting and solubilizing agents,
and liberation and absorption-modifying agents, offer new
October 26 - 28, 2009 / Antalya - TURKEY
possibilities in this field of pharmaceutical technology. Co-
micronization involving the use of spray-drying as a one-
step and non-conventional processing procedure presents
new possibilities for the development of different carrier
systems and for investigations of how the physicochemical
properties of the raw carrier can be modified. Moreover, the
addition of adequate stabilizers may prevent the agglomera-
tion of the resulting microparticles of the carrier. This study
demonstrates the effects of a sucrose ester (SE) on the habit
and polymorph modification of mannitol as compared with
Tween 80.
MATERIALS AND METHODS
Materials
Sucrose myristate M1695 (HLB=16) (SE) was kindly provided
by Syntapharm GmbH (Germany). β-D-Mannitol (M) and
Tween 80 (polysorbate 80) (HLB=15) (T) were from Hungaro
Pharma Rt.
Preparation of co-micronized carrier systems
In the sample preparation, SE or T was chosen as surfactant
to modify and stabilize the M crystals.
The micronized carrier systems were prepared (Table 1) from
1 g of M and three different concentrations of SE or T and
ad 20 g of water, using an Ultraturrax (T-25 IKA-WERKE Ger-
many) and for spray-drying (SD) a Büchi Mini Dryer B-191
(Switzerland) was applied. A spray-dried mannitol (M-SD)
was also prepared, as a control sample without additives.
Investigations 73
The particle size was determined with a LEICA Q500MC Im-
age Processing and Analysis System (LEICA Cambridge Ltd.,
UK). The OCA Contact Angle System (Dataphysics OCA 20,
Dataphysics Inc., GmbH, Germany) was used for studies of
the wettability. The morphology of the particles was exam-
ined with a scanning electron microscope (SEM) (Hitachi
S4700, Hitachi Scientific Ltd., Japan). The DSC measurements
were achieved with a Mettler Toledo DSC 821e thermal anal-
ysis system (Mettler Inc., Schwerzenbach, Switzerland). The
crystallinity index for mannitol (CIM) was calculated from the
heats of fusion. The physical state of M was evaluated by X-
ray powder diffraction (XRPD), using an X-ray Diffractometer
Miniflex II (Rigaku Co. Tokyo, Japan).
RESULTS AND DISCUSSION
The SEM pictures (Figure 1) and Table 2 demonstrate the dif-
ferences in size and morphology of crystals produced.
M has an irregular shape with a rough surface. After spray-
drying, we got significantly smaller particles were obtained
in both cases, but the resulting crystals were a spherical only
when SE was used, which is suitable for the 1-5 μm range of
DPI.
The wettability study (Table 2) indicated that significantly
lower contact angles were measured with lower concentra-
tions of additives. When T was applied, the polarity values
were nearly the same as for M. With SE however, especially at
lower concentration, the polarity increased.
 POSTER PRESENTATIONS

Table 3. lists the changes in the melting point and CIM of M. It Table 3. DSC analysis of products
may be seen that little change occured in the melting point. Samples Melting Point [°] CIM(%)
After spray-drying, the crystallinity of the products was near-
ly the same as that of M-SD, but at the highest concentration M Pure 166.62 100
of SE the recrystallization was blocked. M-SD 166.01 94.31
MP-SE1 166.01 94.45
The three crystal forms of M can easily be distinguished via MP-SE2 165.95 95.97
their XRPD patterns, as shown in Figure 2. The lower concen- MP-SE3 165.46 54.57
tration of M resulted in a turn from β to α, and the highest to MP-T1 166.58 91.15
δ form. But T presented only the α form of the carrier. MP-T2 165.58 85.32
MP-T3 166.30 96.46
CONCLUSION
Our study has demonstrated that the crystal habit and struc-
ture of mannitol can be modified by the use of additives. We
suggest a sucrose ester for co-micronized carrier-based drug
formulation, and especially DPI formulation.
/e-mail: arita@pharm.u-szeged.hu/

Table 1: Concentrations of the additives in micronized prod-

ucts (MPs)
SE-M1695 Tween 80
0.05 g (0.25%) MP-SE1 MP-T1
0.1 g (0.5%) MP-SE2 MP-T2
0.1 g (2.5%) MP-SE3 MP-T3
Figure 2: XRPD patterns of MP-SE1-3

P010-TESTING OF THE GASTRO-RESISTANCE

74 PROPERTIES OF OMEPRAZOLE CAPSULES AT
DIFFERENT PH VALUES

E. Vranić1, A. Uzunović2, O. Rahić1, A. Atilla Hıncal3

1
Department of Pharmaceutical Technology Faculty of
Pharmacy, University of Sarajevo, Čekaluša 90, 71000
Sarajevo, Bosnia and Herzegovina
2
Institute for Quality Control of Medicines, M. Tita 9, 71 000
Sarajevo, Bosnia and Herzegovina
3
Department of Pharmaceutical Technology, Faculty of
Figure 1: SEM pictures of pure carrier (A), product MP-T3 (B) Pharmacy, Hacettepe University, 06100 Sıhhiye-Ankara, Turkey
and product MP-SE3 (C)
INTRODUCTION
Table 2. Particle size and wettability of the samples Omeprazol, a substitued benzimidazole compound and
Main prototype antisecretory agent, is a potent non-reversible
Θwater Polarity inhibitor of the gastric proton pump H+/K+-ATPase which is
Materials particle Θdiiodomethane [°]
[°] (%) reversible for gastric acid secretion. It is weak base, so it is
size [μ]°
acid labile???, and could be rapidly degraded unless it may
M Pure 86.74 21.34 17.55 43.72
be protected against acid conditions1. Moreover, it decom-
M-SD 1.61 20.13 24.87 45.71
posing rapidly at pH< 5.0 and is sensitive to heat, moisture,
MP-SE1 2.33 8.40 62.15 64.35 organic solvents and to some degree, light. The degradation
MP-SE2 2.40 13.20 65.55 65.87 of omeprazole manifests itself in a loss of drug content and
MP-SE3 3.81 36.95 64.10 58.90 increasing amounts of degradation products2.
MP-T1 6.90 9.20 9.85 44.64
MP-T2 2.30 14.80 18.73 45.27 Currently the USP pharmacopoeial requirements and test
MP-T3 not measurable method for gastro-resistant capsules are that not more than
10% of the active ingredient is released within two hours in
0.1 mol/dm3 hydrochloric acid3.

3rd BBBB International Conference on Pharmaceutical Sciences

 POSTER PRESENTATIONS

In this study, we have evaluated differences in the dissolu- Table 1.1: Fraction of dissolved omeprazole at pH value 1.1
tion characteristics of four omeprazole products (purchased pH = 1.1
from market) at different pH values.Our primary purpose in
F1 F2 F3 F4
this study was to determine amount of the active ingredient
released under dissolution test conditions at pH 4.5. 0.00 0.22 0.96 0.31

% dissolved
0.08 0.00 1.12 0.31
MATERIALS AND METHODS 0.12 0.26 2.11 0.34
Materials
The following reagents were used and provided by different 0.67 0.49 0.91 0.38
producers: omeprazole working standard (Ulkar Kimya), ac- 0.32 0.32 0.28 0.35
etonitrile-HPLC grade (Sigma Aldrich), phosphoric acid 85% 0.45 0.22 1.05 0.39
(Carlo Erba), sodium hydroxyde, hydrochloric acid (Merck),
X 0.27 0.25 1.07 0.35
sodium phosphate, disodium hydrogen-phosphate, potas-
sium hydrogen phosphate (Fluka). S.D. 0.25547 0.15930 0.590098 0.03386
R.S.D. 93.47 63.30 55.15 9.77
Dissolution Test Conditions
The dissolution tests of omeprazole gastro-resistant cap- Table 1.2: Fraction of dissolved omeprazole at pH value 6.8
sules (n=6) were performed using Apparatus 2 (n=6), Van
pH = 6.8
Kel VK 7010 dissolution tester, at stirring speed of 100 rpm.
The dissolution test was carried under the following condi- F1 F2 F3 F4
tions: -first stage: dissolution testing was performed for two 98.67 95.89 85.96 92.88

% dissolved
hours in 1000 ml of 0.1 mol/dm3 hydrochloric acid at pH=1.1; 101.78 90.74 81.77 93.74
-second stage: the same capsules were carried to dissolution
vessel, and 750 ml of phosphate buffer solution, pH=4.5, was 100.89 91.89 80.34 98.21
added. Samples in the amount of 10 ml were withdrawn after 95.43 89.06 86.98 95.44
45 minutes. Dissolution samples were collected for analysis 95.87 90.45 82.19 97.34
and replaced with an equal volume of fresh dissolution me-
92.57 94.39 8.04 98.51
dium to maintain a constant total volume; -final stage: within
the next five minutes 250 ml of phosphate buffer solution, X 3.53324 2.58691 2.62660 2.37205
pH=6.8, was added. The rotation speed was maintained at S.D. 97.54 92.07 83.21 96.02
100 rpm and continued to operate for next 45 minutes. R.S.D. 3.62 2.81 3.16 2.47 75
-the apparatus was maintained at 37oC throughout the
The results obtained in this study indicated problems in dis-
whole experiment
solution rate of F4 omeprazole formulation at pH 4.5 and
-all dissolution tests were performed in triplicate.
6.8.
HPLC Method
Additionally, excessive release at pH 4.5 is in contradiction
The following experiment examined the amount of the ac-
with insufficient dissolution rate in phosphate buffer at pH
tive ingredient released was carried out by high-performance
6.8. So, the results represent a sufficient reason for further
liquid chromatography (HPLC) method for omeprazole cap-
testing of the preparation in order to determine the reasons
sules in accordance with British Pharmacopoeia 2009 (4).
that would explain the results.
The system consisted of a pump, injection valve, autosam-
pler and variable wavelength detector (Shimadzu, Kyoto, Ja-
Table 2.1: Fraction of dissolved omeprazole at pH value 4.5
pan). The mobile phase consisted of phosphate buffer pH
7.6: water: acetonitrile = 250:350:400 v/v/v). The flow rate pH = 4.5
was 1.3 ml/min, the injection volume 50 µl, the column tem- F1 F2 F3 F4
perature 30oC and the detection wavelengths 302 nm. Zor- 0.00 0.08 1.06 27.73
% dissolved

bax Stable Bond C-18 column (250mm×4.6mm×5μm) was

used throughout the experiments. Optimization of original 0.04 0.05 1.4 28.02
isocratic HPLC method was performed using excel program 0.09 0.08 1.64 25.97
called ‘‘HPLC calculator v2.1’’5. 0.06 0.09 2.49 27.42
0.10 0.07 1.74 28.57
RESULTS AND DISCUSSION
The results of dissolution studies for Formulation (F) 1, 2, 0.00 0.06 1.14 27.60
3 and 4 are summarised in Table 1.1, 1.2, 2.1 and 2.2 which X 0.05 0.07 1.58 27.55
show fraction of the dissolved drug at different pH values, S.D. 0.04309 0.01472 0.52032 0.87287
1.1, 4.5 and 6.8., respectively.
R.S.D. 89.00 21.00 33.00 3.00

October 26 - 28, 2009 / Antalya - TURKEY

 POSTER PRESENTATIONS

Table 2.2: Fraction of dissolved omeprazole at pH value 6.8 INTRODUCTION

pH = 6.8 Verapamil hydrochloride (VH) is a calcium-channel blocker
and is classified as a class IV anti-arrhythmic agent. It is used
F1 F2 F3 F4
in the control of supra ventricular tachyarrhythmia, and in
94.19 93.75 81.30 37.08 the management of classical and variant angina pectoris. It is
% dissolved

97.58 95.01 84.32 34.43 also used in the treatment of hypertension. These drugs are
91.87 97.42 80.63 32.49 available in sustained release and immediate release prepa-
rations. Overdose is associated with a high incidence of mor-
92.61 95.50 82.09 33.48 bidity and mortality. Calcium channel blockers accounted
96.62 98.01 81.82 33.81 for 16% of all cardiovascular drug exposures reported to the
98.29 98.50 85.12 37.39 American Association of Poison Control Centers, but they ac-
counted for 38% to 48% of deaths caused by cardiovascular
X 95.19 96.37 82.55 34.78
drug overdosage1.
S.D. 2.68499 1.88678 1.77376 2.00533
R.S.D. 3.00 2.00 2.00 6.00 Drug–excipient interactions have the potential to affect
many physiological processes and factors, such as: the pH of
CONCLUSION the microenvironment, protein binding, GI transit time, sta-
The results obtained in this study indicate that the existing bility in the GI tract, effects on gut flora, and so on. The po-
USP pharmacopeia request for gastro-resistance testing (2 tential outcome of all of these interactions might be to alter
hours in 0.1 mol/dm3 HCl (≤ 10% of declared content) is not the bioavailability of the drug2,3.
sufficient and that the additional criteria should be estab-
lished (such as gastro-resistance testing in buffer pH 4.5). Our primary purpose in this study was to investigate degree
of permeation of three different tablet formulations of VH
REFERENCES (purchased from the market) under various test conditions.
1. Espinosa Bosch M., Ruiz Sánchez A.J., Sánchez Rojas F.,
Bosch Ojeda C. Analytical methodologies for the deter- MATERIALS AND METHODS
mination of omeprazole: An overview. J. Pharm. Biomed. The used reagents were of analytical grade, unless otherwise
Anal. 2007; 44: 831–844 stated. Verapamil hydrochloride working standard was pro-
2. Murakami F. S., Lang K.L., Mendes C., Cruz A.P., Marco vided by Merck (Darmstadt, Germany). Phosphatidylcholine,
A.S., Filho C., Silva M.A.S. Physico-chemical solid-state cholesterol, chloroform, methanol were provided by Sigma
76 characterization of omeprazole sodium: Thermal, spec- Aldrich (Buchs, Switzerland).
troscopic and crystallinity studies. J. Pharm. Biomed.
Anal. 2009; 49: 72-80 Assessment of permeation of VH was performed using appro-
3. U.S. Pharmacopeia, 31st rev.; U. S. Pharmacopeial Con- priate in house prepared in vitro passive absorption model.
vention, Rockville, MD, 2007 In this model, four HPLC vials (acceptor compartment) were
4. British Pharmacopoea 2009, British Pharmacopoeia adjusted on paddle of dissolution tester and positioned ver-
Commission, Stationery Office, London, UK tically with our artificial membrane downside oriented into
5. Guillarme D., Nguyen D.T.T., Rudaz S., Veuthey J.L. Met- dissolution vessel (donor compartment).
hod transfer for fast liquid chromatography in pharma-
ceutical analysis.: Application to short columns packed Polytetrafluoroethylene (PTFE) filters, pore size 0.45μm (Sar-
wit small particle. Part I: Isocratic separation, Eur. J. torious GmbH, Goettingen, Germany), were impregnated
Pharm. Sci. 2007; 66: 475–482 with mixture of phosphatidylcholine and cholesterol (22:78
w/w). Chloroform/ methanol solution (70/30 v/ v) was used
as a solvent to prepare impregnation solution. Impregnation
procedure was completed after PTFE filters were submerged
p011-ASSESSMENT OF PERMEATION OF into phosphatidylcholine/ cholesterol solution during 30
THREE DIFFERENT TABLET FORMULATIONS minutes, on Sartorius Certomat MO II shaker (Sartorious
OF VERAPAMIL HYDROCHLORIDE BY IN VITRO GmbH, Goettingen, Germany), rotation speed 200 rpm. Un-
coated PTFE filters were used as blank during testing proce-
PASSIVE ABSORPTION MODEL
dure.
A. Uzunović1, E. Vranić2, O. Rahić2, A. Atilla Hıncal3
In vitro tests on VH (80 mg) tablets were performed on dis-
solution tester ERWEKA DT 800; USP apparatus 2 (n= 6); ro-
1
Institute for Quality Control of Medicines, M. Tita 9, 71 000
tating speed 50 rpm at 370C±0.50C; volume of dissolution
Sarajevo, Bosnia and Herzegovina
medium 900 ml.
2
Department of Pharmaceutical Technology, Faculty of Phar-
macy, University of Sarajevo, Čekaluša 90, 71000 Sarajevo,
In our experiments, three different media were used in both
Bosnia and Herzegovina
compartments: simulated gastric fluid at pH 1.2, simulated
3
Department of Pharmaceutical Technology, Faculty of Phar-
gastric fluid at pH 4.5 and phosphate buffer pH 7.4. Four
macy, Hacettepe University, 06100 Sıhhiye-Ankara,Turkey
HPLC vials were adjusted on each paddle of dissolution
tester. Three of the four vials were impregnated with phos-

3rd BBBB International Conference on Pharmaceutical Sciences

 POSTER PRESENTATIONS
pholipids; the fourth was blank and was used as a control to
confirm that there is no significant permeation through non-
impregnated membrane. Samples were withdrawn from the
vials, one by one, at the following intervals: 1, 2 and 4 hours.
From the fourth vial (blank, non-impregnated PTFE filters)
the sample was withdrawn after 4 hours.
Quantification of VH was performed by UV/VIS spectropho-
tometric method at the absorption maximum at 277 nm (cal-
ibration curves: y = 11.69605x-0.00398, R2=0.99995 simulat-
ed gastric fluid at pH 1.2; y= 12.04581x+0.01054, R2=0.99977
simulated gastric fluid at pH 4.5 and y= 11.95440x-0.00106,
R2=1.00000 in phosphate buffer pH 7.4, respectively. The
precision of method was expressed by relative standard de-
viation (RSD) from 10 replications of samples, concentration
range from 0.00125 mg/cm3 to 0.005 mg/ cm3. RSD was in
the range of 0.14 %-0.60 %
RESULTS AND DISCUSSION
In donor cells, concentration of VH was identified by both
weighing and spectrophotometric determination (not pre-
sented).Permeation was calculated as apparent permeability
coefficient measured in cm min−1 (P) and results were pro-
vided in Figure 1. and Table 2.4,5
Figure 1: Permeation profile of VH at different pH values of
media and test duration
The pH of the GI tract varies greatly, from pH 1.0 –3.5 in the
stomach to pH 7.0 –8.5 in the large intestines. Some drugs
can be preferentially absorbed from a particular section of
the GI tract, although both weakly acidic and basic drugs are
October 26 - 28, 2009 / Antalya - TURKEY
absorbed from the small intestine. Drugs that are weak acids
are un-ionized in the stomach and hence might be absorbed
well from there, whereas drugs that are weak bases might
not be well absorbed from the stomach because they are
highly ionized. Also, most of the excipients are weak acids
or weak bases and possess various degree of ionisation in
different part of GI tract.
A substance that increases the gastric pH would therefore
be expected to increase the absorption of weak bases and to
decrease the absorption of weak acids in the stomach. Also,
decrease or increase of pH of the GI tract change degree of
ionisation of excipients (weak acids/ weak bases) and change
quality of drug-excipient interactions. A drug–excipient in-
teraction can be actively used to increase the bioavailabil-
ity of the drug (e.g. complexation with cyclodextrins). There
are also many cases in which an interaction might not be
expected and might adversely affect the bioavailability of a
drug (e.g. where the mechanism of the interaction is surface
adsorption).
Table 2: Influence of different pH values of media and test
duration on VH in vitro permeation
P x 104 [cm min−1] of VH
Test Tablet Tablet Tablet
pH of
duration formulation I formulation II formulation
media
(minutes) ± 2xSD ± 2xSD III ± 2xSD
60 315.64 ±41.3 213.69 ±35.1 125.64 ±12.6
1.2 ± 0.5 120 58.33 ±10.1 81.82 ±10.5 105.47 ±15.2 77
240 41.65 ±7.8 43.86 ±11.1 40.71 ±5.9
60 303.78 ±38.4 242.07 ±31.7 240.61 ±44.3
4.5 ± 0.5 120 137.16 ±9.9 102.40 ±16.3 72.40 ±21.3
240 51.99 ±10.0 37.50 ±8.4 33.89 ±6.8
60 248.47 ±42.3 156.36 ±24.1 89.95 ±19.4
7.4 ± 0.5 120 86.70 ±7.2 76.19 ±8.9 60.07 ±10.1
240 57.70 ±12.4 52.66 ±11.8 35.98 ±8.4
Apparent permeability coefficient achieved the highest val-
ue after the first hour of testing. This was caused by concen-
tration gradient between the acceptor/donor compartment
which value was decreased during further testing. In addi-
tion, as a result of decreased concentration gradient, two-
way permeation process equilibrium from one to another
compartment was established.
CONCLUSION
Traditionally, excipients have been regarded as inert. How-
ever, there are many instances in which excipients have been
shown to have a significant effect on the biological availa-
bility of the drug. Many of the drug–excipient interactions
affected the process of dissolution/ membrane permeation.
In fact, an interaction between a drug and an excipient that
alters the dissolution or the membrane permeation of some
drugs has been shown to have a marked impact on the ab-
sorption and bioavailability of that drug.
 POSTER PRESENTATIONS

Used in vitro model of passive absorption is discriminatory ting machine (Korsh-Erweka, Germany). The hardness of the
and suitable for testing the level of permeation for various tablets was kept constant (approximately 35 N) and was mea-
technological formulations (different preparation proce- sured with a hardness tester (Korsh-Erweka GmbH, Germany).
dures and different excipients). Future tests could be used
for the evaluation the nature of interactions with individual Table 1: Compositions of the tablets
excipients and active substances. T1 T2 T3 T4
Pioglitazone 13.6 13.6 13.6 13.6
REFERENCES
1. Verbrugge L.B., Harry B. van Wezel H.B. Pathophysiology HPMC K15M 25 - - -
of verapamil overdose: new insights in the role of insu- HPMC K4M - 25 - -
lin. J. Cardiothor. Vasc. An. 2007; 21(3):406-409. HPMC K100LV - - 25 -
2. Pifferi G., Restani P. The safety of pharmaceutical excipi-
ents. Il Farmaco. 2003; 58(8):541-550. HPMC E4M - - - 25
3. Jivraj M., Martini L.G., Thomson C.M. An overview of the Foam Powder 10 10 10 10
different excipients useful for the direct compression of Avicel pH200 50.9 50.9 50.9 50.9
tablets. Pharm. Sci. Technol. Today. 2000; 3(2):58-63.
Mg stearate 0.5 0.5 0.5 0.5
4. Corti G., Maestrelli F., Cirri M., et al. Development and
evaluation of an in vitro method for prediction of human
drug absorption I. Assessment of artificial membrane Table 2: Compositions of the tablets
composition. Eur. J. Pharm. Sci. 2006; 27(4):346-353. T5 T6 T7
5. Avdeef A. Absorption and drug development - Solubil- Pioglitazone 13.6 13.6 13.6
ity, permeability, and charge state. First edition, John
HPMC K15M 15 25 35
Wiley & Sons Inc., 2003.
Foam Powder 10 10 10
Avicel pH200 60.9 50.9 40.9
P012-Floating Matrix Tablets Based On Mg stearate 0.5 0.5 0.5
Low Density Foam Powder
Floating behaviour of the tablets
Sevgi Takka1, Didem Kuzu2 The in vitro floating behaviour of the tablets was studied by
placing them in 900 ml glass containers filled 500 ml pre-
78 heated 0.1 N HCl (pH 1.2, 37°C ± 2 ) for 8 hours. The float-
University of Gazi, Faculty of Pharmacy, Pharmaceutical
Technology Department, Etiler Ankara/Turkey1 ing lag times (time period between placing the tablet in the
Republic of Turkey Social Security Institution, Ataturk medium and tablet floating) and floating durations of the
Boulevard, Kavaklıdere Ankara/Turkey2 tablets were determined by visual.

INTRODUCTION Dissolution test

Several difficulties are faced in designing controlled release sys-
tems for better absorption and enhanced bioavailability. One of
such difficulties is the inability to confine the dosage form in the
desired area of the gastrointestinal tract1,2. The aim of this study
was to develop single unit, floating controlling drug delivery
systems and to study the effect of formulation parameters on
the floating and drug release behaviour of these systems.
MATERIALS AND METHODS
Materials
Floating tablets were prepared by using low density foam
powder (Aeorosil) (Evonik Degussa GmbH, Germany). Pio-
glitazone HCl and HPMC were a generous gift from Jubilan
Organosys, India and Colorcon, Orpington, UK, respectively.
Dissolution profiles of pioglitazone tablets were determined
using the USP 26 method II (Van Kel USA) with a paddle at 50
rpm. Dissolution was performed in 500 mL 0.1 N HCl at 37°C
± 2 temperature. The amount of drug released was detected
UV-spectrophotometrically at 269 nm for 8 hours.
RESULTS AND DISCUSSIONS
The effect of the type and concentration of matrix polymer
(HPMC K4M, HPMC K100LV, HPMC E5 or HPMC K15M) used
for the preparation of floating, low density tablets on the
resulting drug release is shown in Figs.1 and 2. The in vitro
floating behaviour of the tablets was shown in Table 3.

Tablet preparation
Tablets were prepared by direct compression. All excipients
were screened through an 80-mesh sieve before mixing. The
respective powders (drug, foam powder and polymer) and the
additive (Avicel pH 200) were blended for 10 minutes. Then the
mixture was lubricated with magnesium stearate for another 3
minutes. The magnesium stearate level was fixed at 0.5 % for all
formulations (Tables 1 and 2). The mixture was compressed into Figure 1: Effect of type of matrix-forming polymer on piogli-
flat-faced tablets 10 mm in diameter using single punch tablet- tazone release in 0.1N HCl from floating matrix tablet

3rd BBBB International Conference on Pharmaceutical Sciences

 POSTER PRESENTATIONS

P013-In Vivo Investigations On Liquid

Crystal Coated Naproxen

Altan Yüksel1, Tadeusz Librowski2, Nefise Özlen Şahin1

Mersin University, Faculty of Pharmacy, Mersin, Turkey1

Jagellonian University, Faculty of Pharmacy, Krakow, Poland2

INTRODUCTION
Figure 2: Effect of concentration of matrix-forming polymer on Naproxen (NPX), a nonsteroidal anti-inflammatory drug, has
pioglitazone release in 0.1N HCl from floating matrix tablet been widely used for analgesic effect. It has low aqueous
solubility and hence, its absorption is low. The peak plasma
Table 3: Floating time specifications of formulations concentration is reached late. As NPX stays in the gastroin-
testinal track for a long time, it results in gastric irritation.
Formulation Floating Time Lag Time
T1 8 hours **** Preparing salts of water insoluble drug substances, forming
T2 8 hours **** water soluble complexes with various vehicles such as cy-
clodextrins have been used to improve the solubility of sub-
T3 6 hours 1.5 hours
stances with low aqueous solubility1. Coating insoluble drug
T4 **** **** powders with liquid crystals is another way of enhancing the
T5 **** **** aqueous solubility.
T6 8 hours 3 minutes
Liquid crystals are stable structures with various solvents
T7 8 hours **** (mostly water) of amphiphilic substances. However, they are
homogenous-looking structures behaving as if single phase
CONCLUSION unlike emulsions2.
A single unit, floating drug delivery system has been developed.
Incorporation of the highly porous foam powder in the matrix In this study it was aimed to improve the aqueous solubil-
tablets provides densities that are optional lower than the den- ity of naproxen upon coating with liquid crystal. In the first
sity of the release medium. The three HPMC types/grades dif- part of our study which was presented elsewhere, it was de-
fer in the type of substitution and/or molecular weight which termined that coating naproxen with liquid crystal formula-
79
can be correlated with the polymer viscosity. The viscosities of tions improved the solubility efficiently under in vitro condi-
2 % aqueous solutions of HPMC E5, K 100LV, K4M and K15M tions. In this part of the study, the analgesic effect of liquid
at 20°C are 5, 100, 4000 and 15000 cps, respectively. The drug crystal coated naproxen formulations was investigated to
release rate decreased in the rank order HPMC E5>HPMC test whether liquid crystal coating influenced the pharma-
K100LV>HPMC K4M>HPMC K15M. With increasing macromo- cological activity of the drug substance.
lecular weight, the degree of entanglement of the polymer
chains increases and this leads to decreased drug release rates. MATERIALS AND METHODS
With decreasing amounts of HPMC, the density of the swollen Materials
hydrogel network decreases, presenting less hindrance for drug Sodium lauryl sulfate and vaseline were purchased from
diffusion and consequently, the drug release increase. Merck, Co., Germany. Distilled water was obtained by Mili-
pore water purifying system. Naproxen was obtained from
REFERENCES Sigma (St. Louis, USA). All other chemicals were of analytical
1. Streubel A, Siepmann J, Bodmeier R. Gastroretentive quality.
drug delivery systems, Expert Opin. Drug Deliv. 2006;
3(2):217-233. Preparation of liquid crystal formulations for coating
2. Li S, L, Lin S, Daggy BP, Mirchandani HL and Chien YW. Formulations consisting of sodium lauryl sulfate-vaseline-
Effect of HPMC and Carbopol on the release and floating water were prepared at room temperature as described else-
properties of gastric floating drug delivery system using where3. Adequately weighed substances at various ratios
factorial design, Int J Pharm. 2003; 253: 13-22. were mixed in a scintillation vial using a simple agitation ap-
plied with a glass rod for 5 minutes. Liquid crystal formations
were confirmed upon examination under polarized light mi-
croscope.

Amount of naproxen was kept constant as 250 mg in each

formulation. Coating procedure for naproxen powder by
liquid crystal was carried out in a mortar. Liquid crystal was
added at various ratios (12.5; 25 and 37.5% w/w). It was mixed
with naproxen for 2 minutes without applying any pressure
on the material.

October 26 - 28, 2009 / Antalya - TURKEY

 POSTER PRESENTATIONS

Analgesic activity of liquid crystal coated naproxen formulations
Albino male mice weighing approximately 25 g were used
in our study. Prior to the experiments, approval of ethical
committee at Jagellonian University was obtained. Analge-
sic activity was determined with “Writhing Test”4. Eight mice
(n= 8) were used in each group. Group I (control group): mice
without any drug treatment; Group II: mice treated with
naproxen powder; Group III: mice treated with 12.5% liquid
crystal coated naproxen formulation; Group IV: mice treated
with 25% liquid crystal coated naproxen formulation; Group
V: mice treated with 37.5% liquid crystal coated naproxen
formulation
Initially, phenylquinone was applied intraperitonealy to in-
duce pain. Each formulation was dispersed in distilled water
and then, administered orally by means of a gavage at the
doses of 7.5; 15; and 30 mg/kg to the groups II to V. One and
a half hour later, wriggling movements made in 10 minutes
were counted. Data obtained were evaluated statistically.
RESULTS AND DISCUSSION
Table 1: Analgesic activity and ED50 values of pure naproxen
Analgesic
Dose (mg/kg) ED50 (mg/kg)
Activity (%)
30 90,51
15 86,75 6,49
7,5 53,61
increases with the amount of liquid crystal used in coating.
As expected, ED50 values decrease with increasing coating
ratio. This is in good agreement with our hypothesis.
CONCLUSION
Liquid crystal coated naproxen formulations may be used to
obtain high analgesic activity at low doses of application.
REFERENCES
1. Milic-Askrabic J, Rajic DS, Tasic L, Djuric S, Kasa P,
Pintye-Hodi K. Etodolac and solid dispersion with
β-cyclodextrin, Drug development and industrial phar-
macy, 1997, 23(11): 1123-1129.
2. Friberg SE, Mandel L, Larsson M. Mesomorphous phases,
a factor of importance for the properties of emulsions, J.
Coll. Interface Sci., 1969; 29: 155-156.
3. Uslu S, Ermis D, Yuksel A, Baykara T. Increasing of disso-
lution rates tablets containing naproxen using isotropic
liquid crystals, 5th International Congress of Pharma-
ceutical Sciences, 1997, June 24-27, Ankara, pp.6.
4. Singh PP, Junnarkar AY, Rao CS, Varma RK, Shridhar DR.
Acetic acid and phenylquinone writhing test: a critical
study in mice, Methods Find Exp Clin Pharmacol., 1983,
5(9):601-606.
P014-EVALUATION OF SOLUBILITY CLASS OF
WEAK ACIDIC DRUG: ETODOLAC

Table 2: Analgesic activity and ED50 values of liquid crystal Huriye Demir, Betül Arıca Yeğin, Levent Öner
80 coated naproxen formulations (12.5% of liquid crystal)
Analgesic Department of Pharmaceutical Technology, Faculty of
Dose (mg/kg) ED50 (mg/kg) Pharmacy, Hacettepe University, 06100, Ankara, Turkey
Activity (%)
30 86,16
INTRODUCTION
15 79,50 4,84 The BCS is a scientific framework for classifying drug sub-
7,5 59,04 stances based on their aqueous solubility and intestinal
permeability1. Etodolac was selected as a model drug which
Table 3: Analgesic activity and ED50 values of liquid crystal belongs to BCS Class II. BCS Class II drugs exhibit low solubil-
coated naproxen formulations (25% of liquid crystal) ity and high permeability. In this study, the total solubility
of a insoluble and ionizable weak acid etodolac (pKa=4.65)2
Analgesic
Dose (mg/kg) ED50 (mg/kg) in surfactant containing buffers was investigated. The aim of
Activity (%)
this study was determining pH-solubility profile and solubil-
30 88,57 ity class of etodolac with the presence of surfactant within
15 83,13 4,84 the intestinal pH range.
7,5 63,47 MATERIALS AND METHODS
Materials
Table 4: Analgesic activity and ED50 values of liquid crystal Etodolac was supplied by Dr. Reddy’s Laboratory. Sodium
coated naproxen formulations (37.5% of liquid crystal) Lauryl Sulfate (SLS) and disodium hydrogen phosphate were
Analgesic purchased from Merck Co., and citric acid was purchased
Dose (mg/kg) ED50 (mg/kg)
Activity (%) from Carlo Erba. All the other chemicals used were analytical
30 93,38 grade and used without further purification.
15 71,07 3,64 Preparation of media
7,5 73,47 Mcllvaine buffers at pH 4.4, 5.6 and 6.8 were prepared for
solubility studies.
Data indicated that analgesic activity of coated naproxen
formulation is independent of amount of coating at high Solubility Studies
doses (30 mg/kg). Unlikely, at low doses, analgesic activity For determining solubility of etodolac at pH 4.4, 5.6 and 6.8,

3rd BBBB International Conference on Pharmaceutical Sciences

 POSTER PRESENTATIONS

each medium containing 0.5% and 1% (w/v) surfactant or Solubility results were used to determine the solubility class
without surfactant, etodolac powder was weighed as 200 mg, of etodolac. The solubility class was determined by calculat-
400 mg and 600 mg, respectively. Etodolac powder were shak- ing the volume of an aqueous medium sufficient to dissolve
en in rubber-capped vials containing 10 mL buffer solutions at the highest dose strength of etodolac (400 mg) in the pH
37ºC in a horizontal shaker water bath (Memmert, Germany) range 4.4-6.8.
with 80 rpm. At the end of first hour and twenty fourth hours,
samples were collected and drug content were determined at Dose/solubility values for etodolac are presented in Table 2.
273 nm with UV spectrometer (Shimadzu, Japan). A drug substance is considered highly soluble when the
highest dose strength is soluble in 250 mL or less of aque-
RESULTS AND DISCUSSION ous media over the pH range of l-7.51. According to this, sol-
The equilibrium solubility of etodolac in different buffer so- ubility class evaluation for etodolac is shown at Table 3.
lutions containing 0.5 - 1% SLS is shown in Table 1.
Table 2: Dose/solubility values of etodolac at various buffer
Table 1: Equilibrium solubility of etodolac at various buffer solutions
solutions (n=6, x±SD). SLS Concentration Dose/solubility (mL)
SLS Solubility (mg.mL-1) (%) pH 4.4 pH 5.6 pH 6.8
Concentration 0 3216 521 62
pH 4.4 pH 5.6 pH 6.8
(%) 0.5 432 206 53
0 0.124±0.003 0.767±0.020 6..371±0.231 1 248 124 41
0.5 0.925±0.010 1.933 ±0.066 7.460 ±0.075
1 1.608±0.020 3.213±0.047 9.717±0.189 Table 3: Solubility classification of etodolac in various buffer
solutions.
The solubility results indicate that the total solubility of et-
odolac increases with pH and SLS concentration. At pH<pKa, SLS Concentration Solubility Classification (250 mL)
total solubility was low however when pH>pKa, solubility in- (%)
pH 4.4 pH 5.6 pH 6.8
creased. Solubility enhancement was found to be 5-50 folds 0 Low Low High
with pH and 1.5-12 folds with SLS concentration changes.
0.5 Low High High
Figures 1 and 2 illustrate surface plot and contour graphics
1 High High High
of solubility with Equation 1 as a function of pH and SLS con-
centration.
Stotal=38,940-16,127x1-2,696x2+1,664x12+0,776x1x2-0,7762x22
Figures 3 and 4 illustrate surface plot and contour graphics 81
1 of dose/Solubility values with Equation 2 as a function of pH
and SLS concentration.
D/S=15879,0x1- 4178,6x1-9889,3x2+271,9x12+1227,5x1x2+1886,2x2 2

Figure 1: Total solubility as function of pH and SLS concentration.

Figure 3: Dose/solubility of etodolac as function of pH and
SLS concentration.

Figure 2: Total solubility as function of pH and SLS concentra-

tion.

October 26 - 28, 2009 / Antalya - TURKEY

 POSTER PRESENTATIONS
Figure 4. Dose/solubility of etodolac as function of pH and
SLS concentration.
CONCLUSION
Studies for combined effect of pH and surfactant on the
solubility showed that etodolac solubility is pH-dependent
and enhancement of pH and SLS concentration increased
its solubility. Dose/solubility values indicated at pH 6.8 and
pH 5.6 with SLS that etodolac is highly soluble. In summary,
because of high solubility at pH range of absorption region
in the gastrointestinal tract, etodolac exhibits BCS class I be-
havior.
REFERENCE
1. CDER/FDA. (2000). Waiver of In Vivo Bioavailability and
Bioequivalence Studies For Immediate-Release Solid
Oral Dosage Forms Based on a Biopharmaceutics Clas-
82 sification System.
2. Yazdanian, M., Briggs, K., Jankovsky, C.,Hawi, A. (2004)
The “High Solubility” Definition of the Current FDA Guid-
ance on Biopharmaceutical Classification System May
Be Too Strict for Acidic Drugs. Pharmaceutical Research,
21 (2), 293-299.
P015-CHITOSAN COATED LIPOSOMES
LOADED WITH FUROSEMIDE:
CHARACTERIZATION AND CELLULAR
INTERACTION
Can Sarısözen1, Senem Sevtap Ölmez2, İmran Vural2
Department of Pharmaceutical Technology, Faculty of
Pharmacy, Hacettepe University, Ankara, Turkey1
Department of Pharmaceutical Technology, Faculty of
Pharmacy, Hacettepe University , Ankara, Turkey 2
INTRODUCTION
The aim of this study was to develop chitosan coated lipo-
somal carrier systems for poorly soluble and permeable
drug Furosemide, which is a BCS Class IV compound and was
used as a model drug1. Chitosan is a natural cationic poly-
saccharide derived by deacetylation of chitin, which is, after
cellulose, the most abundant polymer found in nature2. Chi-
tosan was used as a coating material for liposomes due to
its bioadhesive and permeability enhancer effects. The effect
3rd BBBB International Conference on Pharmaceutical Sciences
of chitosan coating and liposomal encapsulation on the per-
meability, cellular toxicity, and characterization parameters
of the drug delivery system was investigated.
MATERIALS AND METHODS
Materials
L-α-lecithin (PC, soybean phospholipid, 95.6% phosphatidyl-
choline) and cholesterol (Col) was obtained by Calbiochem
(USA) and Acros Organics (USA), respectively. Protosan UP
CL-113 (MW: 110 kDa, DD: 87%) was purchased from FMC
Biopolymers, Norway. Caco-2 cells were obtained from ATCC
(Rockville, MD, USA). All the other chemicals used were ana-
lytical grade and used without further purification.
Preparation of the Liposomes
Liposomes were prepared by the evaporation-sonication
method and characterized in terms of particle size, zeta po-
tential, encapsulation efficiency (EE) and cell viability and
permeability on Caco-2 cells. PC and Col (10:1 molar ratio)
were dissolved in chloroform. The lipid mixture purged with
nitrogen and organic solvent was removed by rotary evapo-
rator for forming the lipid film. The formed lipid film was de-
hydrated with phosphate buffer (pH 7.4) and vortexed. Then
it was ultrasonicated for 15 min (SONOPULS Ultrasonic ho-
mogenizer HD 2200, Bandelin, Germany). For chitosan coat-
ing, 0.5 mL of the liposome solution was added drop wise
to 0.05%, 0.1%, 0.5% and 1% chitosan solutions. They incu-
bated at room temperature under continuous stirring for 2
hours. Furosemide encapsulated liposomes were prepared
as described above but this time furosemide was dissolved
in methanol and added to the lipid mixture before evaporat-
ing step. Prepared liposomes evaluated by the particle size
and zeta potential measurements (n=3)
Cytotoxicity of the Formulations
Cytotoxicity of formulations was evaluated by using Caco-2
cells and cell viability was assessed by using MTT assay.
Permeability Studies
Furosemide permeation studies were carried out with
Caco-2 cells. The cells were incubated in the flasks and they
trypsinized when near to confluency. 1.5 mL of medium was
transferred to the basolateral side of the wells. Cell suspen-
sions from 25 passages in 0.5 mL of medium were introduced
into the inserts’ apical side at 6.0x104 cells/well (THINCERTS,
pore diameter 1µm, surface area 1.13cm2, Greiner Bio-one,
NC, USA). Medium was changed on every 2nd day. The cell
monolayer had reached confluence after 20 days of seeding.
Wells with TEER value of >400 ohm.cm2 were selected for
furosemide transport studies. Culture medium was replaced
from each well by 0.5 mL and 1.5 mL HBSS in the apical and
basolateral side of the well. Free furosemide solution, loaded
uncoated liposomes and chitosan coated loaded liposomes
were added to the apical side of the monolayer. The wells
were then placed on a shaker at 30 rpm and 37°C for 2 hours.
After that the samples from the basolateral side was analyzed
by validated HPLC method (r2 > 0.999). Permeability coeffi-
cients (Papp, cm/s) were calculated using Eq. 1: Papp=(dQ/
dt)(1/AC0) where dQ/dt is the flux across the monolayer, A
is the surface area of the cell monolayer and C0 is the initial
concentration on the apical side of the membrane.
 POSTER PRESENTATIONS
RESULTS AND DISCUSSION
Liposomes coated with 0.5% chitosan was used due to its
zeta potential and particle size values. The results are given
below in Table 1.
Table 1: Characterization parameters of the liposomes (n=3,
mean ± SD)
Particle Size Zeta Potential EE
Formulation
(nm) (mV) (%)
UL 50.04 ± 0.78 2.00 ± 0.55 - meation enhancer effect observed for the chitosan coated
UFL 49.84 ± 0.85 -13.58 ± 0.56 42.6
CL 68.38 ± 1.10 22.05 ± 2.66 - administration.
CFL 115.40 ± 2.86 32.42 ± 2.26 71.1
Uncoated empty liposomes (UL) and uncoated furosemide
loaded liposomes (UFL) showed similar particle size. Chitosan
coating increased the average particle size for both unload-
ed (CL) and loaded liposomes (CFL). For unloaded liposomes
the zeta potential was nearly neutral, thus the mechanism
of coating involved hydrogen bonding and hydrophobic
interactions. On the other hand when furosemide encapsu-
lated into the liposomes the zeta potential became negative
and the main interaction was electrostatic attraction. So the
drug loaded liposomes adsorbed more chitosan, and have
increased particle sizes. Since chitosan carries high positive
charge, the adsorption of chitosan increased the density of
positive charge made the zeta potentials positive.
The encapsulation efficiencies of the formulations were giv-
en in Table 1. The results showed that the chitosan coating
increased the encapsulation of furosemide.
The cell viability results show that the chitosan coating in-
creased the Caco-2 cell viability both for furosemide loaded
and unloaded liposomes. This observation supports the idea
that cell viability may be increased by chitosan coating.
The results of permeation studies were given in Figure 1. The
Papp values of the formulations were also indicated above
the bars in the figure.
Figure 1: Permeability study results of the formulations (n=3,
mean±SD)
October 26 - 28, 2009 / Antalya - TURKEY
An 8 fold increase on Furosemide permeability was obtained
for chitosan coated liposomes when compared other formu-
lations. The UFL and free drug showed similar permeation
values of 1.96% and 1.77% respectively.
CONCLUSION
Furosemide which is a BCS Class IV drug was successfully en-
capsulated in the liposome formulations. According to the
cytotoxicity studies, chitosan coated liposome formulations
showed less cytotoxicity than the uncoated ones. The per-
liposomes make them interesting candidates that could be
effective to improve Class IV drugs bioavailability after oral
REFERENCES
1. Wu CY, Benet LZ. Predicting Drug Disposition via Ap-
plication of BCS: Transport/Absorption/Elimination In-
terplay and Development of a Biopharmaceutics Drug
Disposition Classification System, Pharm Res. 2005; 22:
11-23.
2. Kaş HS. Chitosan: Properties, preparations and applica-
tion to microparticulate systems, J Microencapsulation.
1997; 14: 689-711.
P016-PEG-PE BASED MIXED MICELLES
LOADED WITH PACLITAXEL FOR
ANTITUMOR ACTIVITY: ENHANCED DRUG
SOLUBILIZATION 83
Can Sarısözen1, İmran Vural1, Vladimir P. Torchilin2, A. Atilla
Hıncal1
Department of Pharmaceutical Technology, Faculty of
Pharmacy, Hacettepe University, 06100, Ankara, Turkey1
Department of Pharmaceutical Sciences and Center
for Pharmaceutical Biotechnology and Nanomedicine,
Northeastern University, Boston, MA, USA2
INTRODUCTION
Paclitaxel is an anticancer drug derived from Taxus brevifo-
lia. It possesses its activity by stabilizing the microtubules.
In the recent years the researches showed that paclitaxel
has also induces apoptosis via the mitochondrial or intrin-
sic pathway. Even though paclitaxel is effective against vari-
ous cancer types like breast and ovarian cancers, it has very
low solubility. In the literature there are values for paclitaxel
solubility of 0.7 µg/ml1 to 30 µg/ml2. Due to its hydrophobic
properties, currently paclitaxel is formulated with alcohol
and Cremophor EL, which has severe side effects like neph-
rotoxicity, neurotoxicity and hypersensitivity. Also paclitaxel
itself represents relatively high toxicity. To overcome these
issues encapsulation of paclitaxel in different carriers sys-
tems have been developed extensively.
In the present work, the preparation and in vitro character-
ization of paclitaxel loaded PEG-PE/Vitamin E mixed micelles
have been described. Different molar rations of PEG2000-PE:
 POSTER PRESENTATIONS

vitamin E and different paclitaxel amounts were used. The celles is formed by the hydrophobic diacyllipid part and the the
micellar drug solubilization effect and increased drug encap- corona is made of hydrophilic PEG chains. Thus the hydrophobic
sulation was evaluated. paclitaxel will be solubilized within in micelle core. Addition of
vitamin E is believed to increase the micelle size due to the hy-
MATERIALS AND METHODS drophobicity of the molecule. When vitamin E is incorporated
Materials into the micelle structures it increases the hydrophobic core vol-
1,2–distearoyl–sn–glycerol–3–phosphoethanolamine–N- ume of the micelle, thus increase the micelle size. This effect of
[methoxy(polyethylene glycol)-2000] (PEG2000-PE) was ob- the vitamin E can be clearly seen from the particle size data. The
tained from Avanti Plar Lipids (Alabaster, AL, USA). Vitamin increased vitamin E molar ratio resulted larger micelles (p<0.05).
E was purchased from Sigma (St. Louis, MO, USA). Paclitaxel
was from Applichem (Darmstadt, Germany). All the other As can be seen in the table, increased vitamin E ratio in-
chemicals used were analytical grade and used without fur- creased the encapsulation of paclitaxel. As explained be-
ther purification. fore, when vitamin E ratio was increased, core volume of the
mixed micelles becomes larger. The percent of paclitaxel in
Preparation of the paclitaxel loaded mixed micelles the micelles were found to be higher than 3% for both for-
The mixed micelles loaded with paclitaxel were prepared mulations. Also the amounts of the drug in 1 mL of micelle
with the following method. PEG2000-PE and vitamin E stock dispersion were about 1 mg. These results are consequent
solutions were prepared in chloroform. 85:15 and 90:10 mo- with literature3.
lar ratios of PEG2000-PE: vitamin E mixtures were prepared
and different amounts of paclitaxel solution (from the stock Table 1: Characterization parameters of the micelles (n=3,
solution of 1 mg/ml in methanol) was added to these mix- mean ± SD)
tures. Organic solvents were removed by rotavapor and mi- Formulation Particle Size Zeta Potential
celle material-paclitaxel film was formed. This film was freeze (PEG-PE: Vit. E) (mm) (mV)
dried to remove organic solvent residues. To form drug load- 85:15 (Empty) 23.9±2 -7.7±1.2
ed micelles, film was hydrated with PBS pH 7.4. The final con- 90:10 (Empty) 20.1±0.3 -9.0±0.4
centration of the micelle materials was 10 mM. 85:15 23.1±0.3 -24.6±0.2
90.19 21.4±0.2 -34.4±3
Particle size and zeta potential measurements
Particle size distribution and the zeta potentials of the micelles
Table 2: Incorporated drug values of the micelle formulations
were determined by laser diffractometry using a Malvern Na-
(mean ± SD)
84 noZS (Malvern Instruments Ltd., UK). The micelle samples were
diluted with MilliQ ultra pure water prior to measurements. All Drug Drug Drug amount
measurements have been made triplicate. Formulation incorporated weight in per mL of
(%) micelle (%) micelle (μg)
Drug solubilization and encapsulation efficiency 85:15 77.0±4.7 3.62±0.11 1095.7
The drug encapsulated in the micellar phase was measured 90:10 66.7±3.9 3.14±0.71 948.5
by a validated HPLC method. Mobile phase was methanol:
water (68:32, v/v), column was Develosil ODS-UG-5 C18 RP CONCLUSION
(4.6x150 mm, 5µm, Nomura Chemical, Seto city, Japan). The The results obtained showed that the PEG2000-PE/vitamin
injection volume was 10 µl and the flow rate was 1 ml/min. E mixed micelles can efficiently solubilized the paclitaxel, a
Paclitaxel was detected by UV detector at 230 nm. The ana- poorly soluble anticancer drug. The solubilization effect is
lytical method was validated by terms of specificity, linearity, significantly higher and 1 mg of paclitaxel can be incorpo-
accuracy, and precision. rated in 1 mL of mixed micelles at 10 mM concentration. The
sizes are around 20 nm, which is also very suitable for passive
The drug loaded micelle solutions was filtered through 0.22 targeting due to the EPR effect.
µm filter to remove non-incorporated and precipitated pa-
clitaxel crystals. The clear micelle suspensions then diluted REFERENCES
with methanol prior to injection. Micelle structure was dis- 1. Mathew AE, Mejillano MR, Nath JP, Himes RH, Stella VJ,
turbed by methanol and incorporated paclitaxel can be de- Synthesis and evaluation of some water-soluble prod-
termined. rugs and derivatives of taxol with antitumor activity,
Journal of Medicinal Chemistry, 1992; 35: 145-151
RESULTS AND DISCUSSION 2. Swindell CS, Krauss NE, Horwitz SB, Ringel I, Biologically
In our study the particle sizes and the zeta potentials of the active taxol analogs with deleted A-ring side chain sub-
micelle formulations are given in Table 1. The initial paclitaxel stituents and variable C-2’ configurations, Journal of Me-
amounts were added as the different percents of PEG-PE by dicinal Chemistry 1991; 34: 1176-1184
weight. The highest encapsulation was achieved by using 3. Sawant RR, Sawant RM, Torchilin VP, Mixed PEG-PE/vi-
the 5% paclitaxel of the PEG-PE weight. The results also indi- tamin E tumor targeted immunomicelles as carriers for
cate that micellar structure was intact. poorly soluble anti-cancer drugs: Improved drug solu-
bilization and enhanced in vitro cytotoxicity, European
The average size of both empty and loaded PEG2000-PE/Vitamin Journal of Pharmaceutics and Biopharmaceutics, 2008;
E mixed micelles was about 22 nm. The core of the PEG-PE mi- 70: 51-57

3rd BBBB International Conference on Pharmaceutical Sciences

 POSTER PRESENTATIONS
P017-FORMULATION AND IN VITRO
EVALUATION OF EUDRAGIT-L100/PLGA
NANOPARTICLES CONTAINING SALMON
CALCITONIN
Meltem Çetin1, İmran Vural2, Alptuğ Atila3, Can Sarısözen4,
Yücel Kadıoğlu5
Ataturk University, Faculty of Pharmacy, Dept. of
Pharmaceutical Technology1
Hacettepe University, Faculty of Pharmacy, Dept. of
Pharmaceutical Technology2
Ataturk University, Faculty of Pharmacy, Dept. of Analytical
Chemistry3
Hacettepe University, Faculty of Pharmacy, Dept. of
Pharmaceutical Technology, Ankara4
Ataturk University, Faculty of Pharmacy, Dept. of Analytical
Chemistry, Erzurum, Turkey5
INTRODUCTION
Salmon calcitonin (sCT) is an endogenous polypeptide hor-
mone composed of 32 amino acids (3,432 Da)1. sCT is current-
ly formulated as either a sterile solution for intramuscular or
subcutaneous injection or as a nasal spray2,3. This drug plays
a crucial role in both calcium homeostasis and the treatment
of bone disease such as osteoporosis1. Therefore, it is very
important to develop appropriate oral dosage forms of sCT
to increase patient compliance.
In the present study, we developed salmon calcitonin-load-
ed Eduragit®/PLGA nanoparticles by solvent evaporation
method. The formulations were characterized in vitro with
regard to encapsulation efficiency, surface morphology, size
and surface potential.
MATERIALS AND METHODS
Materials
Salmon calcitonin was purchased from PolyPeptide Lab.
(LimHamn, Sweden). Eudragit® L100 was supplied by Rohm
GmbH&Co. KG (Darmstadt, Germany). Acetone, polyvinyl
alcohol (Mw 30 000-70 000) were purchased from Sigma
(USA), poly (D, L-lactide-co-glycolide) (PLGA (50:50) Resomer
RG 502H) from Boehringer Ingelheim (Germany).
Preparation of Eud/PLGA-salmon calcitonin nanoparticles
The mixture of Eudragit® L100/PLGA polymers (Eud:PLGA)
was used for preparation of nanoparticles. Eudragit®/ L100
and PLGA were dissolved in anhydrous ethanol and acetone,
respectively. Then, the Eudragit® solution in ethanol was
added slowly to the PLGA solution in acetone (1:2 v/v) with
a constant stirring rate of 500 rpm on magnetic stirrer at am-
bient temperature. The organic phase was added into 6 ml
of PVA solutions (3 % w/v) containing salmon calcitonin in
the amber glass vial. The mixture was emulsified using 40
% power of ultrasonic probe (Sonoplus, HD 2070; Bandelin.
Electronics, Berlin, Germany) for 1,30 min. Finally, organic
phases were evaporated under reduced pressure in a rota-
ry evaporator at 40 °C. After evaporation of the solvent, NP
were recovered by centrifugation at 17,000 rpm for 30 min.
October 26 - 28, 2009 / Antalya - TURKEY
and washed with distilled water. The washing step was re-
peated once before NP were resuspended in distilled water
and lyophilized overnight.
Characterization of Nanoparticles
The morphological examination of the nanoparticles was
performed using AFM (NanoMagnetics Instruments Ltd., UK).
The size (Z-average mean) and zeta potential of the nanopar-
ticles were analyzed by photon correlation spectroscopy and
laser doppler anemometry, respectively, in triplicate using a
Zetasizer 3000HS (Malvern Instruments, UK). Size and zeta
potential measurements were performed in triplicate fol-
lowing a dilution of the nanoparticles suspension in distilled
water at 25 °C. Each measurement was done in triplicate. The
entrapment efficiencies determined with high performance
liquid chromatogram (HPLC).
RESULTS AND DISCUSSION
The nanoparticles were spherical and smooth on surface
as observed via AFM (Figure 1). Zeta potential of Eud/PLGA
and salmon calcitonin-loaded Eud/PLGA nanoparticles were
found to be -10,56 and -10,97 mV, respectively. There was no
significant differences were observed at the zeta potential
values (p> 0.05). Sizes of Eud/PLGA and salmon calcitonin-
loaded Eud/PLGA nanoparticles were 218,5 and 234,1 nm
with a narrow polydispersity index of 0.068 and 0.051, re-
spectively. The entrapment efficiency of salmon calcitonin
was 68 %.
85
Figure 1: AFM images of drug-loaded Eud/PLGA nanoparticles.
CONCLUSION
This study reports the preparation of salmon calcitonin-
loaded Eudragit® L 100/PLGA nanoparticles and their in vitro
characteristics. Eudragit® L 100/PLGA nanoparticles might
be well suited delivery systems for the oral delivery of salm-
on calcitonin.
ACKNOWLEDGEMENTS
This project was supported by the TUBITAK (Project no.
SBAG-108S275).
 POSTER PRESENTATIONS

REFERENCES Table 1:Contents of tablet formulations

1. Na DH, Youn YS, Lee SD, Son MW, Kim WB et al. Moni-
toring of peptide acylation inside degrading PLGA mi- Magnesium Clopidogrel
Avicel HPMC
crospheres by capillary electrophoresis and MALDI-TOF Formulation Stearate Bisulphate
(mg) (mg
mass spectrometry, J Control Release. 2003; 92: 291– (mg) (mg)
299.
2. Azria M, The calcitonins, Physiology and pharmacology, 1 0 0 0.1 75
Karger, Basel, 1989. 2 0 25 0.1 75
3. Lee KC, Park MO, Na DH, Youn YS, Lee SD et al. Intranasal 3 0 50 0.1 75
delivery of PEGylated salmon calcitonins: hypocalcemic 4 25 0 0.1 75
effects in rats, Calcif Tissue
5 25 25 0.1 75
6 25 50 0.1 75
P018-DEVELOPMENT OF SUSTAINED RELEASE 7 50 0 0.1 75
8 50 25 0.1 75
FORMULATION OF ANTITHROMBOTIC DRUG
9 50 50 0.1 75
10 0 75 0.1 75
İsmail Tuncer Değim1, Celil Tan1
11 0 100 0.1 75
Gazi University, Faculty of Pharmacy, Department of 12 0 150 0.1 75
Pharmaceutical Technology, Ankara, Turkey1
Calculation of theoretical plasma profiles
The theoretical plasma profiles after virtually taking imme-
INTRODUCTION diate and sustained release formulations containing clopi-
A novel polymeric salt form of clopidogrel (clopidogrel resi- dogrel were calculated using known pharmacokinetic pa-
nate) has been also developed to for sustained release of ac- rameters and computer program (Stella v.6.0, Hanover, US).
tive compound1 and a patent application has been made to
prepare more effective and controlled release dosage forms In-vivo experiments
of clopidogrel2. The best commercially available tablets (fastest release), for-
mulation 3 and formulation 12 were subjected to animal ex-
Serious adverse drug reactions have been reported to be as- periments. Number of platelets (thrombocytes), prothrom-
86 sociated with clopidogrel therapy3. A controlled release dos- bine time, fibrinogen and thrombine time, active partial
age form of clopidogrel was developed to enhance the ben- thromboplastine time (APTT), collagen/ADP closer times
efit of the therapy and to lower incidence of side/adverse were determined.
effects. The design of the study was performed using Fuzzy
logic technique. Dissolution and release characteristics of RESULTS AND DISCUSSION
prepared tablets were determined. Commercially available Dissolution profiles of prepared tablets in 0.1 N HCl for first
tablets in Turkish market were also subjected to drug release two hours and then pH 6.8 were performed. Dissolution
study. The theoretical plasma profiles after virtually taking profiles of all formulations in 0.1 N HCl were also performed
immediate and sustained release formulations containing (Figure.1).
clopidogrel were calculated using known pharmacokinetic
parameters and computer program. Selected formulation
was given to rats and rabbits and blood samples were ana-
lyzed in terms of coagulation parameters.

MATERIALS AND METHODS

Clopidogrel bisulphate was obtained from Dr. Reddy’s, India.
Commercially available tablets were purchased from Turkish
market.

Methods
Preparation of tablet formulations
Formulations were designed using Fuzzy Tech 5.70b (IN-
FORM GmbH, Germany) computer program.

Dissolution and determination of drug release from tablets

Pharma Test-PTW S3C (Hainburg, Germany) dissolution tes-
ter was used. Drug release tests were performed using pro-
cedure described in USP 28 as paddle method A.
Figure 1: Dissolution profiles of all formulations in 0.1 N HCl
The theoretical blood profiles were calculated using a com-
puter simulation program. Blood drug concentrations were
calculated after virtually given commercial tablets and after
virtually administration of formulation3 and.

3rd BBBB International Conference on Pharmaceutical Sciences

 POSTER PRESENTATIONS
One commercially available formulation was seleceted as
a reference considering best release. All formulations were
given rabbits orally and their blood samples were analyzed.
The number of platelets in their blood were found to be sig-
nificantly increased after 4 hours for commercial tablets and
formulation 12 (p<0.01).A significantly different results were
obtained after 4 hours with formulation 12 and commercial
tablet (p<0.5) for prothrombin times.Percentage of fibrinojen
coagulation results show that after first and 6 hours of tablets
administration, statistically significant results were obtained
for commercial tablets and formulation 3 (p<0.05) and sig-
nificant difference between commercial tablets and formu-
lation 12 and formulation 3 after 6 (p<0,01). When collagen/
ADP closer times were measured a significant difference were
found between control and formulation 3 and 12 after 6 hours
(p<0.01). Dade Behring has performed a clinical study and the
reference value for collagen/ADP closer time was given with
the value of 71-118 seconds. It is accepted with the general
agreement that if this value is higher than 133 seconds it can
be said that this parameter is prolonged4. In our experiments
this parameter was found to be 255 (210-300) and 180 (78-
274) seconds after 6 hours for formulation 3 and 12 respec-
tively. This indicates that our sustained release formulations
were found to be potentially active after 6 hours and they can
be used as an alternative to conventional tablets.
CONCLUSION
In conclusion, two sustained clopidogrel releasing formula-
tions were developed and showed to be effective.
REFERENCES
1. M.H. Ki, M.H. Choi, K.B. Ahn, B.S. Kim, D.S. Im, S.K. Ahn,
and H.J. Shin. The Efficacy and safety of clopidogrel resi-
nate as a novel polymeric salt form of clopidogrel. Arch
Pharm Res Vol. 31: 250-258 (2008).
2. USPTO Patent Application #: 20070003628: Nanopar-
ticulate clopidogrel formulations
3. http://en.wikipedia.org/wiki/Clopidogrel
4. A.J. Grau, S. Reiners, C. Lincy, F. Buggle, and A. Ruf. Plate-
let function under aspirin, clopidogrel, and both after
ischemic stroke. Stroke. April: 849-855 (2003).
P019-STUDIES ON THE IMPROVEMENT
OF ENCAPSULATION EFFICIENCY OF A
HYDROPHILIC DRUG
Göksu Yağ1, Betül Arıca Yeğin1, Sema Çalış1
Department of Pharmaceutical Technology, Faculty of
Pharmacy, Hacettepe University, Ankara, Turkey1
INTRODUCTION
Generally, the O/W method is one of the simplest techniques
to prepare microspheres and it provides many advantages
for efficient encapsulation of lipophilic drugs, such as high
drug loading and the ability of preparing in a wide range of
particle sizes. Although these advantages, hydrophilic drugs
are poorly encapsulated by O/W method, which is due to
October 26 - 28, 2009 / Antalya - TURKEY
dissolving of the drug in the external aqueous phase during
emulsification and hardening process, resulting with a low
drug loading or encapsulation efficiency2.
In recent years, emulsion solvent diffusion method is pro-
posed and began to be used widely for microencapsula-
tion of various kinds of drugs. When the emulsion solvent
diffusion method is compared with the solvent evaporation
method, it is observed that the solidification of the liquid
droplets realized much more faster in the former. Further-
more, in the solvent evaporation process, it is known that, the
solvents used widely for preparation is classified in the “risky”
groups whereas the heating stage to evaporate the solvents
are avoided in emulsion solvent diffusion method1,2.
Tolmetin sodium (TS) is an antiinflammatory drug which is
freely soluble in water. Therefore, in this study it is regarded
as a model drug for the realization of the microsphere for-
mulation in the present study using solvent diffusion tech-
nique with a high encapsulation efficiency.
In the light of the above stated facts, in the present study,
the objective can be summarized as, to encapsulate the
model hydrophilic drug tolmetin sodium within Eudragit®
S100 polymer, so to increase the encapsulation efficiency of
the drug in microspheres with the addition of different salts
(sodium chloride, potassium dihydrogen phosphate and
sodium bromide) into aqueous continuous phase to avoid
dissolving of drug, that will end up with a low encapsulation
efficiency, also succeed the formulation of a nonsteroidal an-
tiinflammatory drug for oral application discarding the gas- 87
trointestinal side effects3.
MATERIALS AND METHODS
Materials
Microsphere formulations were prepared by using Eudragit® S100 as
polymer. Eudragit® S100(ES 100) was purchased from Röhm Pharma
Polymers (Germany). Poly vinyl alcohol (PVA) was provided from Sig-
ma (USA). Dichloromethane (DCM) and methanol were purchased
from Merck (Germany). Potassium dihydrogen phosphate (KH2PO4)
and sodium chloride (NaCl) were provided from Carlo Erba, (Italy),
sodium bromide (NaBr) was purchased from Merck (Germany) and
tolmetin sodium (TS) was provided from Santa Farma (Turkey).
METHODS
Microspheres were prepared by emulsification diffusion
technique by using 0,5 % PVA solution as the continuous
phase. The continuous phase contained NaBr, NaCl and
KH2PO4 in different concentrations (see Table 1). For prepa-
ration process, first polymer was dissolved in methanol and
DCM mixture, then the drug was added in polymer solution
and stirred until it was dissolved. Following this step, this so-
lution was added into the continuous phase and emulsified
by stirring at 300 rpm for 1 hour with a propeller agitator.
Afterwards, the microspheres were collected by filtration.
Tolmetin sodium, a hydrophilic model drug loaded micro-
sphere formulations were characterized for their particle
size, drug loading and in vitro release properties for evalu-
ation. For preparing the formulations, all of the formulation
parameters kept same except for the continuous phase. The
codes of formulations are summarized Table 1 and the codes
 POSTER PRESENTATIONS

were given according to the variability of the salts in the salts in the external phase were increased, the drug loading
outer phase. was increased. Also it was observed that the pH dependent
polymer provided a controlled release of the drug from mi-
Table 1: Formulation Codes crospheres. As a result, Eudragit® S100 was determined to be
suitable polymer for formulation of an oral microsphere for-
Codes Concentration of Salts
mulation of TS, thus emulsion diffusion technique in which
ES1 0,1 M NaCl various salts were included in the outer phase for satura-
tion, so reducing the dissolving of the drug appeared to be a
ES2 1 M NaCl proper technique for encapsulation of hydrophilic drugs.
ES3 0,1 M NaBr REFERENCES
1. Laleci, F., Çalış, S., Hıncal, L, A.A. Preparation and In Vit-
ES4 1 M NaBr ro Characterization of Tolmetin Sodium Loaded Oral
ES5 0,1 M KH2PO4 Eudragit Microspheres, Eur. J. Pharm. Sci. (Suppl. 1),
2005, 25:146-148.
ES6 1 M KH2PO4 2. Guinebretiere S., Briançon S., Lieto J. Study of the Emul-
sion Diffusion of Solvent Preparation and Characteriza-
RESULTS AND DISCUSSION tion of nanocapsules , Drug Dev. Research, 2002, 57:18-
The average production yields of microspheres and standard de- 33
viations were calculated, the results are as shown Table 2. 3. Maaieh Al A., R., Flanagan R., D. Salt and Cosalvent Ef-
The average encapsulation efficiency (% E.E.) and standard devia- fects on Ionic Drug Loading into Microspheres Using
tions were calculated, the results are as summarized in Table 3. an O/W Method, Journal of Controlled Release, 2001,
70:169-181
Table 2: Production yield (%) of microsphere formulations
ES1 ES2 ES3 ES4 ES5 ES6
P020-DEVELOPMENT OF LIPOSOME
Yield 62.3 69.7 70.1 72.1 55.8 60.7 FORMULATION OF RIVASTIGMINE AND
S.D. 0,54 0,34 0,78 0,87 1,12 0,67 INVESTIGATION OF TRANSPORT PROPERTIES
THROUGH MDCK AND CACO-2 CELL LINES
Table 3: Encapsulation efficiency of TS
88 Zelihagül Değim1, N. Başaran Mutlu1, Şükran Yılmaz2, Dinç
ES1 ES2 ES3 ES4 ES5 ES6 Eşsiz3
% E.E. 69.8 93.1 70.8 82.0 73.1 95.7
Gazi University,Department Of Pharmaceutical Technology,
S.D. 0.41 0.34 0.65 0.76 1.09 0.55 Faculty Of Pharmacy, Ankara, 063301
Food And Mouth Diseases Institute, Ankara, 065202
The mean particle size of TS loaded microspheres ranged Kafkas University, Department Of Pharmacology And
from 50μm - 81μm. The in vitro release profiles are shown Toxicology, Faculty Of Veterinary, Turkey3
in Figure 1.
INTRODUCTION
Alzheimer disease (AD) is one of the most common cause of
demantia which is characterised by decreased level of AChE
in brain. Rivastigmine is a carbamate derivative pseudo-irre-
versible cholinesterase inhibitor which can both inhibit AChE
and BuChE1, These drugs have been used for symptomatic
treatment of AD. MDCK (Madin Darby Canine Kidney) and
Caco-2 (Human Colon Carsinoma) cells are epithelial origi-
nated cells, which are widely used to test or determine oral
drug absorption. They are also increasingly used as an in
vitro blood-brain barrier (BBB) model in passive permeability
and membrane transport studies. This cell lines have similar
protein structures with BBB tight junctions and possess high
Figure 1. Release profiles of TS from microspheres transendothelial electrical resistance (TEER) values just like
brain cells2,3.
CONCLUSION
Finally, it was concluded that formulation parameters were In this study, liposome formulations of rivastigmine were
very important in achieving the high encapsulation efficien- developed. Cytotoxicity (MTT) tests were performed with
cy values espacially for hydrophilic drugs. When preparing different concentrations of the materials that were used in
microsphere formulations, addition of different salts into ex- the formulations. Transport studies were performed with
ternal phase, improve encapsulation efficiency of hydrophilic rivastigmine containing liposomes through Caco-2, MDCK
drugs. Moreover, it was observed that when the amount of cell cultures and also dialysis membrane. All results were

3rd BBBB International Conference on Pharmaceutical Sciences

 POSTER PRESENTATIONS

compared with rivastigmine solution. AChE inhibition effect
of rivastigmine was also studied on mice and in vitro/in vivo
correlation studies were performed.
MATERIALS AND METHODS
Materials
Rivastigmine tartrate was obtained from Dr. Reddy’s (India)
and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium
bromide (MTT) were obtained from Sigma (Germany). Re-
agents which used in cell culture studies were obtained from
Gibco Life Technologies (England).
Preperation of cell culture
MDCK cells were seeded on semipermiable polycarbonate
filter inserts for 7 days (1.2 cm diameter, 0.4 µm pore size)
with 60000 cells/mL density. Caco-2 cells were cultured on
polycarbonate membrane filter inserts for 21 days. Cells
were seeded on polycarbonate cell culture inserts (1.2 cm
diameter, 0.4 µm pore size) with a density of 80000 cells/ml.
was 35.4%. 1.5 mg/ml rivastigmine used for both Caco-2 and
MDCK transport studies according to MTT study results. Cu-
mulative amount of rivastigmine penetrated through Caco-
2 and MDCK cells from apical to basolateral compartment
were found to be 60.9% and 54% respectively. Cumulative
amount of rivastigmine released from liposomes and passed
through Caco-2 and MDCK cells were found to be 3.6% and
15.2%(Figure 1). The apparent permeability coefficients (log.
values) of rivastigmine solution and rivastigmine liposomes
were calculated as -1.96±0.04 cm/hour, -1.80±0.03 cm/hour,
-1.03±0.29 cm/hour and -1.26±0.12 cm/hour for Caco-2 and
MDCK cell respectively. AChE inhibition values of rivastig-
mine solution were calculated for both blood and brain in
mice treated with rivastigmine solution and liposomes (oral
and intraperitonally), and more effective inhibition was ob-
served with rivastigmine liposomes.

MTT test
MTT test was performed for studying rivastigmine effect on
the viability of MDCK and Caco-2 cells. The serum effect on
cell viability was also investigated by using serum contain-
ing and serum free DMEM for 24 hours time period. Various
rivastigmine concentrations (1, 1.5, 3, 4.5, 6 µg/mL), were
studied in DMEM.

Transport Experiments
According to the MTT test results, the cell viability was
seemed to be higher with DMEM containing serum and it
was used for transport studies. Rivastigmin concentration
was not exceeded to 1.5 mg/mL for 24 hours of transport
period. MDCK and Caco-2 monolayer containing membrane
was placed between donor and receptor compartments of
vertical diffusion cells. 95% O2 and 5% CO2 were delivered
to the system within the experiment period at 37ºC to main-
tain cell viability. Transport experiments were performed
from apical to basolateral compartment. Dialysis membrane
transport studies were also performed at 37ºC using pH 7.4
phosphate buffer Samples were taken out at specific time
periods. Removed sample volume was always replenished
with fresh DMEM. The rivastigmine content of samples were
analysed by HPLC.
AChE inhibiton experiments
Ellman method was used for the determination AChE % in-
hibition4. Rivastigmine solutions were applied to the mice
both intraperitonally and orally. Blood samples were taken
at specific time periods. 20 µL of whole blood and 25 µL of
DTNB were added to the 3 mL pH 8 0.1 M phosphate buffer
containing spectrophotometer cuvettes and autozeroed at
the wavelength of 412 nm. Then 20 µL of AChI was added
to the sample only and absorbans changes were recorded
during 6 minutes. AChE % inhibition values were then calcu-
lated considering the control group.
Figure 1: Cumulative amount of rivastigmine transported 89
through MDCK, Caco-2 cells and dialyse membrane.
In conclusion more efficient and prolonged theraphy can be
possible for AD with rivastigmine liposomes after oral deliv-
ery and high in vivo/invitro correlation coefficients showed
that Caco-2 and MDCK cell lines are good tools for predicting
brain permeability of drugs.
ACKNOWLEDGMENTS
This study was supported by grants from Gazi University
(02/2006-17).
REFERENCES
1. Williams BR, Nazarians A, Gill M. Clin.Ther. 2003;
25(6):1651-1663.
2. Waterbeemd HV, Gifford E. Drug Discovery, 2003;2:192-
204.
3. Mahar Doan KM, et al. J Pharmcol. Exp. Ther.
2002,303(3),1029-1037
4. Ellman GL, et al. Biochem.Pharmacol. 1961; 7:88-95.

RESULTS AND DISCUSSION

Positive effect of serum on cell viability was shown by MTT
test results. Therefore rivastigmine concentration should
be below 1.5 mg/mL. Encapsulation efficiency of liposomes

October 26 - 28, 2009 / Antalya - TURKEY

 POSTER PRESENTATIONS

P021-CNS DELIVERY OF THERAPEUTIC
PROTEINS IN LIVING CELLS
Elena Batrakova1, Anna Brynskikh1, Yuling Zhao1, Shu Li1, R.
Lee Mosley2, Alexander Kabanov1, Howard Gendelman3
anticipated that these studies will lead to the developing a
new technology based on cell-mediated active delivery of
therapeutic polypeptides that attenuate neuroinflammation
and produce neuroprotection in patients with PD.

Center for Drug Delivery and Nanomedicine, Department

of Pharmaceutical Sciences, University of Nebraska Medical
Center1
Center for Neurovirology and Neurodegenerative Disorders,
University of Nebraska Medical Center2
Center for Drug Delivery and Nanomedicine, Center for
Neurovirology and Neurodegenerative Disorders, Department
of Pharmacology and Experimental Neuroscience, University
of Nebraska Medical Center3

INTRODUCTION
The need for delivery of therapeutic polypeptides to restore
brain tissues in PD cannot be overstated. Selective delivery
of antioxidants to the substantia nigra pars compacta (the
regions most affected in human disease) can potentially at-
tenuate oxidative stress and increase survival of dopaminer-
gic neurons in patients with PD. To this end, we developed
a bone-marrow derived monocyte (BMM) system to deliver
redox enzymes to PD affected brain regions in an animal
model (Scheme 1). To preclude BMM-mediated enzyme deg-
radation, catalase was packaged into a block ionomer com-
plex producing particles of nanoscale size, “nanozymes”.
MATERIALS AND METHODS
90 Enzyme molecules were coupled with a cationic block co-
polymer, polyethyleneimine-poly(ethylene glycol) (PEI-PEG),
a synthetic polyelectrolyte of opposite charge, and cross-
linked to obtain stable polyion complexes. Loading and re-
lease of nanozyme from cell carriers were studied in vitro. A
neuro-immunological approach for the nanozyme delivery
was employed in MPTP mouse model of PD.
RESULTS AND DISCUSSION
To build a protective nanocontainer for catalase, we synthe-
sized a block copolymer by conjugation of PEI (2,000 Da) and
PEO (10,000 Da)1. The self-assembled catalase/PEI-PEG com-
plexes, “nanozymes”, were rapidly taken up by BMM, retained
catalytic activity, and released in active form for greater than
24 hours2. Significant amount of nanozyme was found in
MPTP-intoxicated brain in mice.
Treatment of MPTP-intoxicated mice with catalase-loaded
BMM prevented neuroinflammation (microglial activation
and astrocytosis) at the level in healthy animals. Thus, MPTP
injections caused significant increase in GFAP staining par-
ticularly within the nigrostrial system. In contrast, treatment
of MPTP-injected mice with catalase-loaded monocytes pre-
vented neuroinflammation at the level in healthy animals.
Similar results were found for microglial activation. As a re-
sult, the neuroprotective effect of catalase-loaded mono-
cytes in murine PD model was recorded.
The obtained data suggest that “nanozyme”-loaded BMM mi-
grate across the BBB toward the site of pathology and release
the nanoparticles maintaining therapeutic concentrations of
the drug and providing prolonged antioxidative effect. It is
ACKNOWLEDGMENTS
We appreciate the support by the United States National In-
stitute of Health grant RO1NS057748.
REFERENCES
1. Vinogradov S, Batrakova E, Li S, Kabanov A. Polyion com-
plex micelles with protein-modified corona for receptor-
mediated delivery of oligonucleotides into cells. Biocon-
jug Chem.1999; 10: 851-860.
2. Batrakova EV, Li S, Reynolds A, Thomas M, Bronich TK,
Kabanov AV, Gendelman HE. A macrophage-nanozyme
delivery system for Parkinson’s Disease. Bioconjugate
Chem. 2007; 18: 1498-1506.
P022-DRUG RELEASE FROM PACLITAXEL
LOADED NIOSOMES AND EVALUATION
OF THEIR STABILITY AGAINST
GASTROINTESTINAL ENZYMES
Zerrin Sezgin Bayındır1, Nilüfer Yüksel1
Ankara Üniversitesi School of Pharmacy Department of
Pharmaceutical Technology, Ankara. Turkey1
INTRODUCTION
Paclitaxel (PCT) is an antineoplastic agent isolated from
the bark of Taxus brevifolia. Development of oral delivery
systems of PCT has attracted considerable attention1,2. In
our previous study we developed several niosome formu-
lations loaded with PCT. In this study in vitro drug release
and release mechanisms of PCT from niosome formulations
were investigated. The efficiency of niosomes to protect PCT
against gastrointestinal enzymes was researched for PCT
oral delivery.

3rd BBBB International Conference on Pharmaceutical Sciences

 POSTER PRESENTATIONS

MATERIALS AND METHODS Table 2: PCT encapsulation efficiency of niosomes and % PCT
Film hydration method was used to incorporate PCT in nio- released at 24 hours.
somes. Briefly a thin film of surfactant (Table 1), dicetyl phos- Formula Code Entrapment efficiency % PCT released at 24 hours
phate, cholesterol and PCT was formed. After hydration of F1 96.6±0.48 23.3±1.96
the film with ultrapure water, unloaded drug was removed
by ultracentrifuge. F2 91.5±1.53 28.6±1.96
F3 86.4±2.04 19.9±0.87
The dialysis method was used to investigate PCT release from F4 20.3±0.62 32.1±1.39
niosomal formulations. Niosomal suspensions containing 60
F5 42.5±0.35 33.1±0.28
μg of PCT was placed into dialysis membrane bag and sunk
in the 50 mL of release medium, phosphate buffered saline F6 79.5±1.32 19.7±0.36
(PBS), pH 7.4 containing %0.1 (v/w) Tween 80 to maintain the F7 12.1±1.36 27.6±1.52
sink condition. The samples were placed in a water bath and
F8 81.1±0.12 23.8±0.88
shaken at 100 rpm 37 ºC. 1 mL of samples were taken from
the release medium at predetermined time intervals up to
24 hours and fresh medium was added to the dissolution Release data have been applied to various mathematical
medium (n=3). The collected samples were directly analyzed models and Higuchi model fits best to the release data. It is
by HPLC. concluded that drug is released from the niosomes by a dif-
fusion controlled mechanism.
The stability of formulations in gastrointestinal (GI) enzymes
trypsin, α-chymotrypsin and pepsin was investigated3. 500
μl of niosome containing 30 μg/mL PCT or free PCT in PBS
pH 7.4 was mixed with 500 μl of 5 IU trypsin solution in
phosphate buffer pH 7.8 or 16 IU α-chymotrypsin solution
in phosphate buffer pH 7.8 or pepsin solution in simulated
gastric fluid (SGF) pH 1.2. The mixtures were incubated at 37
ºC, 3 hours for trypsin and α-chymotrypsin and 1 hour for
pepsin. At the end of the incubation time 100 μl trifluoroa-
cetic acid (TFA) was added to 100 μl of sample to stop the en-
zymatic reaction. Samples were diluted with methanol and
PCT content was measured by HPLC (n=3). 91
Figure 1: PCT release from the niosome formulations made
Table 1: Chemical names and HLB values of the used non-
with different non-ionic surfactants.
ionic surfactants in the niosome formulations.
Code Surfactant Chemical Name HLB
Pure drug preserved 96.1 % of its structure without degrada-
Value
tion in SGF containing pepsin, while this percent decreased
F1 Span 40 Sorbitan monopalmitate 6.70 to 75.8 % and 27.8 % in trypsin and chymotrypsin containing
F2 Span 20 Sorbitan monolaruate 8.60 phosphate buffer pH 7.8 respectively. In general niosomes
F3 Span 60 Sorbitan monostearate 4.70 seem to protect PCT against chymotrypsin degradation.
F4 Tween 20 Polyoxyethylene (20) sorbitan monoaruate 16.7 F1 formulation showed the best protection against all en-
F5 Tween 60 Polyoxyethylene (20) sorbitan monostearate 15.0 zymes.
F6 Brij 76 Polyoxyethylene (10) stearyl ether 12.4
F7 Brij 78 Polyoxyethylene (20) stearyl ether 15.3
F8 Brij 72 Polyoxyethylene (2) stearyl ether 4.90

RESULTS AND DISCUSSION

A negative correlation was observed between encapsulation
efficiency and drug release (Table 2). The release of PCT from
Span niosomes (F1-F3) is increased while the HLB (see Table
1) of the surfactants increased.

When we consider all formulations, drug release from Tween

niosomes seems faster (Figure 1). This result can be contrib-
uted to structural differences on niosome assembly. The bi-
layer structure upon hydration of the film would be looser
and more flexible due to high aqueous solubility of Tweens,
leading an increased permeability to solutes. It has been
stated that more hydrophobic Span surfactants form more Figure 2: Stability of pure PCT and PCT loaded niosomes in
compact niosomes when hydrated in presence of choles- GI enzymes.
terol.

October 26 - 28, 2009 / Antalya - TURKEY

 POSTER PRESENTATIONS
In vitro studies showed that F1 formulation might be more
beneficial for oral drug delivery among other formulations.
F1 will be further evaluated for their in vivo behaviors.
ACKNOWLEDGMENTS
This study has been supported by TUBITAK (The Scientific
and Technological Research Council of Turkey) under grant
SBAG-3742.
REFERENCES
1. Peltier, S.; et al. Enhanced oral paclitaxel bioavailability
after administration of paclitaxel-loaded lipid nanocap-
sules. Pharm. Res. 2006; 23(6): 1243-1250.
2. Kruijtzer, C. M. F. et al. Weekly oral paclitaxel as first-line
treatment in patients with advanced gastric cancer. An-
nals of Oncology 2003; 14: 197–204.
3. Pardakhty, A.; Varshosaz, J.; Rouholamini, A. In vitro study
of polyoxyethylene alkyl ether niosomes for delivery of
insulin. Int. J. Pharm. 2007; 328: 130-41.
P023-Optimization of flow and
compressibility characteristics
of starch and lactose using quasi-
emulsion solvent diffusion method
aimed for using in direct compression
Abbas Akhgari1, Mohammad Ali Dabbagh1, Hasti Sadeghi1
92 School of Pharmacy- Ahwaz Jundishapur University of Medical
Sciences- Ahwaz- Iran1
INTRODUCTION
Direct compression is one of the methods for tablet prepara-
tion. Despite its advantages over granulation methods it has
not been completely generalized as the latter approaches
which could be due to the limitation of compressibility and
flow of the usual excipients and drug powders. There are
some approaches to modify the excipient characteristics
such as flow and compressibility aimed for using in direct
compression. Quasi-emulsion solvent diffusion (QESD) is a
crystal modifying method by which the characteristics of ex-
cipients and drug substances could be changed1. Starch and
lactose are two excipients generally used for tabletting but
with poor flow. The aim of present study was to modify the
flow and compressibility characteristics of these excipients
using quasi-emulsion solvent diffusion (QESD) method.
MATERIALS AND METHODS
Materials
Corn starch (Darusazi Hakim, Iran), lactose (Merck, Germa-
ny), Flowlack® (Aburayhan, Iran), naproxen (Zambon, Spain),
ethanol (Simintak, Iran), and hydrochloric acid (HCl) (Merck,
Germany) were prepared from indicated sources.
Crystal modification of starch using QESD method
Firstly, 200 mL HCl 0.1 M was heated until 80°C and 5 g starch
was gradually added to this solution. The solution was then
cooled and reached to 40°C. 200 mL ethanol was gradually and
3rd BBBB International Conference on Pharmaceutical Sciences
with stirring added, the solution was stirred for 15 min and was
left to cool down the room temperature. The resulted sediment
was collected and kept in an oven at 40°C for 24 h. Finally, the
powder was kept in a desiccator at room temperature.
Crystal modification of lactose using QESD method
30 g lactose was dissolved in 50 mL of 80°C distilled water.
The solution was then cooled and reached to 40°C. 100 mL
ethanol was gradually and with stirring added to the slou-
tion and was left to cool down the room temperature. The
resulted sediment was collected and kept in an oven at 40°C
for 24h. Finally, the powder was kept in a desiccator at room
temperature.
Powder flow
Flow of 20 g of powders (untreated and modified) was re-
corded by use of Erweka flow meter (n=6).
Angle of repose
The repose angle of 20 g of powders (untreated and modi-
fied) was measured using the static method (n=6).
Densities and compressibility indices
Bulk and tapped density of powders were measured using
the tapping method by a 100 mL cylinder (n=6). Hausner ra-
tio and Carr’s index were calculated for each powder.
Tablet preparation and evaluation
200 mg of each powder (untreated or modified) was fully
mixed with 200 mg naproxen and the resulted powder was
compressed in a single punch tabletting machine (Korsch,
Germany) under the same pressure. The hardness of tablets
was measured using Erweka hardness tester (n=10).
Data analysis
One-way analysis of variance was used to assess the sig-
nificance of the differences among different groups. Tukey–
Kramer post-test was used to compare the means of differ-
ent treatment groups. Results with P < 0.05 were considered
to be statistically significant.
Results and discussion
According to Fig. 1 untreated starch did not have a measur-
able flow. But modified starch had flow equal to 37.78±3.44
g/sec and it was even higher than free flow excipient, Flow-
lack®. Increase in flow due to crystal modification could be
due to adhering of the particles to each other and producing
the large and relatively round crystals2. Scanning electron
micrographs of the resulted powder (Fig. 2) confirmed this
theory and showed that the modification method produced
the larger crystal particles compared to untreated starch. In
accordance with starch, modified lactose showed more flow
ability (7.55±0.6 g/sec) than untreated powder and round-
ness of modified lactose particles (Fig. 2) was the related
mechanism.
Bulk and tapped density of lactose and starch increased
with the crystal modification (Table 1) which could be due to
change in morphology of particles, more uniformity in par-
ticle shape and the resulted better particle packing3. Also,
Carr’s index and Hausner ratio of modified powders were de-
creased which confirms the above phenomenon.
 POSTER PRESENTATIONS

As shown in Fig. 3, tablets made of untreated starch had no

significant hardness. However, tablets containing modified
starch had suitable crushing strength. This phenomenon
could be explained by adhering smaller particles to each
other in the modified starch and more primary consolidation
of particles which could enhance by compression force and
conclude harder tablets. However, the increase in hardness
of lactose tablets was not very significant by crystal modifi-
cation (p>0.05).

Figure 3: Hardness of tablets made of different excipients

CONCLUSION
The results of this study demonstrated that quasi-emulsion
solvent diffusion (QESD) is a suitable method for modifying
tablet excipients aimed for use in direct compression. Change
in crystal morphology of starch and lactose enhanced their
flow characteristics. Also, by use of QESD method compress-
ibility of starch powder was increased and the resulted tab-
lets had good hardness and strength.

ACKNOWLEDGMENTS
The authors acknowledge for supporting this project by a
grant from Vice Chancellor for Research, Ahwaz University of
Medical Sciences.

REFERENCES
Figure 1: Flow of untreated and modified excipients
1. Katta J, Rasmuson A. Spherical crystallization of benzoic
acid. Int J Pharm. 2008; 348: 61-69.
2. Liu L, Marziano I, Bentham A, Litster J. Effect of particle
properties on the flowability of ibuprofen powders. Int J 93
Pharm. 2008; 362: 109-117.
3. Yadav V. Preparation and evaluation of polymeric car-
bamazepin spherical crystals by solvent diffusion tech-
nique. Asian J Pharm.2009; 3: 18-25.

P024-FORMULATION OF CHITOSAN COATED

SODIUM ALGINATE MICROCAPSULES
CONTAINING MELOXICAM

Hakan Eroğlu1, Levent Öner2

Figure 2: SEM of (a) untreated starch, (b) modified starch, (c)
untreated lactose and (d) modified lactose
Table 1: The results of factors related to flow and compress-
ibility of powders (mean ± SD)
HAcettepe University Faculty of Pharmacy Department of Basic
Pharmaceutical Sciences1
Hacettepe University Faculty of Pharmacy Department of
Pharmaceutical Technology2

Bulk Tapped Carr’s Hauser INTRODUCTION

Samples
density density index ratio Meloxicam (MLX) is a well known member of non-steroidal
anti-inflammatory drugs (NSAIDs) with its analgesic and
untreated com starch 0.49±0.02 0.61±0.03 20±1 1.24±0.02 antipyretic effects. It is generally used for the treatment of
rheumatoid arthritis osteoarthritis in elderly patients. In this
modified com starch 0.71±0.01 0.85±0.00 17±1 1.20±0.02 study, we have formulated microcapsule formulations pre-
pared by the combination of two natural polymers chitosan
untreated lactose 0.37±0.01 0.49±0.02 25±1 1.34±0.02 and sodium alginate.

modified lactose 0.70±0.01 0.90±0.04 23±2 1.30±0.04 MATERIALS and METHODS

Materials
Flowlach 0.62±0.01 0.71±0.01 13±1 1.22±0.10
The model drug MLX was from Dr. Reddy’s Laboratories. The

October 26 - 28, 2009 / Antalya - TURKEY

 POSTER PRESENTATIONS
polymers used in the preparation of microcapsule formula-
tions were chitosan and sodium alginate (Sigma Aldrich,
USA). For the hardening agent, calcium chloride anhidr (Car-
lo Erba, Italy) was used in the preparation of microcapsule
formulations.
Preparation of Microcapsule Formulations
Microcapsule formulations were prepared by using the ori-
fice-ionic gelation method1. As the matrix material, chitosan
(Medium M.Wt.) and sodium alginate (Medium M.Wt.) were
used. MLX was homogeneously dispersed in the solution of
sodium alginate at concentrations varying from 1% up to
2% (w/v) in water with Ultra Turrax T-25 homogenizer. Sepa-
rately, the hardening agent CaCl2 was dissolved in 1000 mL
of water and mixed with 25 mL of previously prepared chi-
tosan solution in diluted acetic acid. MLX dispersion in sodi-
um alginate solution was added drop wise into the solution
containing CaCl2 and chitosan. The resulting droplets were
mixed for 12 hours over a magnetic stirrer and filtered. They
were washed with deionized water and dried at 30oC until
they reach a constant weight.
Preparation Efficiency
The preparation efficiencies of the microcapsule formula-
tions were calculated with the formula: Yield=A/B * 100
where A is the total weight of the microcapsules and B is the
sum of the total weights of polymer and MLX used for prepa-
ration of microcapsules.
Surface Morphology
94 The surface morphologies of the microcapsule formulations
were investigated with Scanning Electron Microscopy (SEM).
The SEM investigations were further evaluated with JSM
5600 device in 20 kV.
Particle Size Analysis
The particle size distribution of the microcapsule formula-
tions containing MLX has been evaluated by using Ende-
cotts standard sieves having mesh aperture size in a range
of 125-810 µm.
Assay of MLX
The in-vitro quantification of MLX was carried out by using
a HPLC equipped with an UV detector after the modification
of previously used method in literature2. The mobile phase
was composed of 50 mM phosphate buffer –acetonitrile-
methanol mixture at the ratio of 50:25:25 (v:v:v) The injec-
tion volume and flow rate of the mobile phase was set as
15 µL and 1 mL/min, respectively. The UV detection of the
samples was recorded at wavelength (λ) of 363.4 nm. ACE
C18 reverse phase column was used for separation (150 x 4.6
mm). For the validation of the in-vitro HPLC method, linear-
ity, accuracy, precision, specificity and sensitivity parameters
have been investigated.
Determination of the MLX Content
Microcapsules containing exactly 15 mg of MLX have been
accurately weighed and added into 25 mL of HCl solution
(0.1N). They were mixed for 2 hours and afterwards 8 mL
of tribasic sodium phosphate was added. This solution was
mixed for 4 hours and at the end of this period, by the ad-
3rd BBBB International Conference on Pharmaceutical Sciences
dition of methanol, the total volume was completed up to
50 mL. The final solution was again mixed for 2 hours over a
magnetic stirrer. The concentration of MLX has been deter-
mined by the HPLC method.
In-Vitro Release Studies
The in-vitro release tests were carried out with USP- Apparatus
1. 15 mg of MLX were placed in the basket. The test was initially
performed in 750 mL of 0.1 N HCl solution (pH=1.2) for 2 hours
at 75 rpm. After this period, 250 mL of 0.2 M tribasic sodium
phosphate solution that has been previously conditioned at
37±0.5 oC was added in order to maintain the pH of the me-
dium at 6.8. At certain time intervals starting from the begin-
ning of the experiment, samples of 5 mL were withdrawn and
immediately replaced with fresh medium. The amount of MLX
was quantified with the HPLC method. The release profiles of
two different microcapsule formulations were compared with
each other by using model independent approach
RESULTS AND DISCUSSION
Preparation Efficiency
The preparation efficiencies of the microcapsule formula-
tions were calculated as 92.64 ± 2.57% and 95.12 ± 1.92 % for
the formulations prepared by using 1% (w/v) and 2% (w/v)
sodium alginate, respectively.
Surface Morphology
The microcapsules prepared by using sodium alginate at
different concentrations have similar surface properties.
The particles neither had perfect circular shape nor smooth
regular surface.
Particle Size Analysis
The particle size distributions for microcapsule formulations
prepared with 1% (w/v) sodium alginate and 2% (w/v) sodi-
um alginate polymer solutions were calculated as 505 ± 1.38
µm and 740 ± 1.39µm, respectively.
Assay of MLX
The linearity of the calibration curve has been established
within the concentration range of 1-40 µg/mL for MLX
(r2=0.9999) and the retention time for MLX was recorded as
2.62 min. The method was found to be accurate, precise, spe-
cific and sensitive for MLX.
Determination of the MLX Content
MLX contents of the microcapsule formulations were deter-
mines as 99.51 ± 1.38 % and 100.83 ± 2.46% for the formula-
tions prepared with sodium alginate polymer solution at the
concentrations of 1% (w/v) and 2% (w/v), respectively.
In-Vitro Release Studies
The release profiles of the formulations clearly indicate that
negligible release of MLX from microcapsule formulations
was determined within the first 2 hours of the experiment
where the pH of the release medium was 1.2. On the other
hand, as the pH was shifted to 6.8, release of MLX from micro-
capsule formulations starts depending on the increase in the
solubility of chitosan coating. The increase in sodium alginate
polymer concentrations from 1% (w/v) to 2% (w/v), governs
the release pattern of MLX from microcapsule formulations
 POSTER PRESENTATIONS
as soon as the pH of the release medium is increased.
CONCLUSION
The results clearly indicate that release of MLX may be modi-
fied by using sodium alginate at varying concentrations. Also
chitosan coating successfully prevented MLX release in gas-
tric conditions. These formulations should further be used in
in-vitro/in-vivo correlation studies.
REFERENCES
1. Gonzales-Rodriguez M.L., Holgado M.A., Sanchez-Lafu-
ente C., et al. Alginate/chitosan particulate system for
sodium diclofenac release. Int. J. Pharm. 2002, 232: 225-
234.
2. Nemutlu E. and Kır S. Method development and valida-
tion for the analysis of meloxicam in tablets by CZE. Jour-
nal of Pharmaceutical and Biomedical Analysis 2003, 31:
393-400.
P025-MUCO/BIOADHESIVE PROPERTIES AND
IN VIVO BIODISTRIBUTION OF CHITOSAN-CA-
ALGINATE MICROPARTICLES LOADED WITH 5-FU
Marija Glavas Dodov1, Sema Çalış2, Sonja Kuzmanovska3,
Maja Simonoska Crcarevska1, Vesna Petrovska1, Nikola
Geskovski1, Katerina Goracinova1
Department of Pharmaceutical Technology, Faculty of
Pharmacy, University Ss Cyril & Methodius, Skopje, Macedonia1
Pharmaceutical Technology Division, Faculty of Pharmacy,
Hacettepe University, Ankara, Turkey2
Department of Patophysiology, Medical faculty, University of
Ss Cyril & methodius, Skopje, Macedonia3
INTRODUCTION
The delivery of chemotherapeutic agents for the treatment of
colon cancer using polymeric microparticles (MPs) has become
one of the most popular areas of research because of the possi-
bilities of reducing toxicity, enhancing controlled release activity
and also localizing the drug delivery. Working on this rationale,
chitosan-calcium-alginate MPs loaded with 5-FU were previously
prepared and characterized1. The aim of this contribution was to
investigate the binding efficiency of the prepared particles on rat
intestinal mucosa and to test the biodistribution of this new mi-
croparticulated system after peroral administration in rats.
MATERIALS AND METHODS
Materials
5-FU was supplied as a gift sample from EBEWE Pharma, Ger-
many. Chitosan (CTS) was obtained from Fluka, Switzerland and
sodium alginate (ALG) was kindly donated by FMC BioPolymer,
Norway. Hydroxypropyl methylcellulose phthalate (HP-55) was
purchased from Shin Etsu Chemical Co. Ltd., Japan. Calcium
chloride (CaCl2) was obtained from Alkaloid, Macedonia.
Preparation procedure
Using one-step spray-drying process (Buchi 290, Mini Spray
Dryer, Swiss) cross-linked hydrogel MPs composed of acid-
resistant (HP-55 coated) particles of 5-FU in ALG core coated
October 26 - 28, 2009 / Antalya - TURKEY
with CTS containing different concentrations of CaCl2 (0.65,
1.25 and 2.5; samples 1, 2 and 3) were prepared and evalu-
ated.
In vitro release studies
To compare the drug release from MPs under different pH
conditions, the experiment were performed in buffer solu-
tions at pH 2.0, 4.5, 6.8 and 7.4. The released 5-FU in different
time intervals was quantified spectrophotometrically (266
nm; Lambda 16, Perkin Elmer, USA).
Ex vivo mucoadhesion studies and in vivo biodistribution
The muco/bioadhesive potential of the prepared MPs was de-
termined by modified everted sac method2. Unfasted Wistar
rats, weighed ~400g were sacrificed, the colons were removed
and flushed with phosphate buffer saline pH 7.4 with 200 mg/
dl glucose (PBSG). Six cm segments were everted, filled with
2ml of PBSG and introduced in conical tubes containing 40
mg of MPs, previously dispersed in 5ml PBSG. The sacs were
incubated at 37 °C on horizontal shaker (50 rpm, 30 min; Shak-
er Unitronic OR, Selecta, Spain). Afterwards, the sacs were re-
moved and the remaining MPs were freeze-dried (- 40 °C, 0.75
mBa, period 24h; Labconco, FreeZone 2.5L Feeze Dry System,
USA). Colon segments in PBSG (blank sample) were used as
control. The muco/bioadhesive potential expressed as binding
efficiency (%) was calculated using the following equation:
binding efficiency (%) = MPs - (fdS-fdPBSG)/MPs x 100
MPs - mass of the microparticles
fdS - mass of the sample after freeze-drying 95
fdPBSG - mass of blank sample after freeze-drying
In order to determine the fate of prepared particles in vivo
upon oral administration, optimized formulation (sample 3)
was labeled with Technetium-99 (99mc-Tc). A dose of 400 µCi
of 99mTc-labeled MPs dispersed in purified water was admin-
istered orally to fasted Wistar rats. Imaging was performed at
different time intervals using an E-Cam Single Head gamma
camera (Siemens, Germany) in an order to determine the dis-
tribution of activity in GI tract.
RESULTS AND DISCUSSION
Using one-step spray-drying process spherical particles with
mean size of ~7 µm, narrow, unimodal particle size distribution,
high 5-FU loading efficiency, positive surface charge and pH
dependent swelling were prepared. The results from in vitro dis-
solution studies pointed that the rate and extent of drug release
was a function of i). incorporation of acid-resistant particles of
5-FU into CTS-Ca-ALG matrix and ii). the degree of cross-linking
between both polymers; drug release decreased with increasing
CaCl2 concentration and. Formulation prepared with 2.5% CaCl2
(sample 3) has properties to minimize drug absorption in the
upper part of GIT and at the same time could provide high drug
concentration over a prolonged period of time in colon region.
All prepared formulation showed excessive adhesion to rat
colon mucosa probably due to the design of the matrix and
preserved flexibility of the polymer chains as well as posi-
tive surface charge and significantswelling of the particles in
phosphate buffer at pH 7.4
 POSTER PRESENTATIONS
Figure 1: Interaction of MPs with rat colon segments
Also, the amount of interacted MPs with colon mucosa sur-
face decreased with increasing the concentration of CaCl2.
Our biodistribution data pointed that the MPs are traveling slow-
ly through GIT with higher concentration in the small intestine
after 5 hours and more than 65% of administered quantity pres-
ent in the colon after 8 hours. 24 hours post administration ~55%
of the administered radioactivity was still present in the colon.
Several reasons might be involved in the pattern of MPs distribu-
tion, among them the particle size and surface properties of the
carrier as well as pH dependent swelling could probably lead to
increased interaction with biological surfaces and muco/bioad-
hesion, thus providing the prolonged residence time of the car-
rier at the site of action and better therapeutic efficiency.
CONCLUSION
Using an innovative microencapsulation technique, particles
with optimal physicochemical and biopharmaceutical prop-
96 erties were prepared. Also, presented studies confirmed the
potential of CTS-Ca-ALG MPs for local colon delivery of 5-FU
and efficient treatment of colon cancer.
REFERENCES
1. Glavas Dodov, M., et al., WGA-conjugated chitosan–Ca–
alginate microparticles for local colon delivery of 5-FU:
Development and in vitro characterization. Int. J. Pharm.
(2009), doi:10.1016/j.ijpharm.2009.06.037.
2. Santos, C. A., et al., Correlation of two bioadhesion as-
says: the everted sac technique and the CAHN microbal-
ance. J. Control. Release (1999), 61(1-2), 113-122.
p026-DEVELOPMENT AND
CHARACTERIZATION OF THEOPHYLLINE
LOADED PLGA MICROSPHERES
Dania Shamil1, Can Sarısözen2, Sibel Bozdağ Pehlivan2,
Abdul Basit1, Selma Şahin2, İmran Vural2
University of London, The School of Pharmacy, 29-39
Brunswick Square, London WC1N 1AX, United Kingdom1
Hacettepe University, Faculty of Pharmacy, Pharmaceutical
Technology Department 06100 Ankara Türkiye2
INTRODUCTION
PLGA microspheres are biocompatible and biodegradable
which show reproducible and slow-release characteristics in
vivo. Therefore, they are widely used in research and clini-
3rd BBBB International Conference on Pharmaceutical Sciences
cally. Theophylline is a hydrophobic drug and has a narrow
therapeutic index. Therefore, the dose must be monitored
and sustained release dosage forms have to be prepared. The
aim of this study was to load theophylline into PLGA micro-
spheres and to characterize them in vitro for the oral delivery
of the drug. In order to optimise the preparation process, two
formulation variables were investigated: 1) Concentration of
polymer solution by variation in the weight of PLGA polymer
and 2) The rate and duration of agitation during emulsifica-
tion. The prepared microspheres were tested for the particle
size, zeta potential, encapsulation efficiency, in vitro release
studies and cell culture studies for cytotoxicity.
MATERIALS AND METHODS
Materials
Theophylline was kindly provided by Nobel İlaç Sanayi,
İstanbul, Türkiye. Polyvinyl Alcohol (PVA) (Mw 30,000-70,000)
was purchased from Sigma (USA). 50:50 Poly(D,L-lactide-co-
glycolide)–PLGA (Resomer RG503) was obtained from Boeh-
ringer Ingelheim (Germany).
Preparation of PLGA Microspheres
PLGA microspheres were prepared using the oil-in-water
(o/w) emulsion/solvent evaporation method. PLGA was dis-
solved in dichloromethane and then injected using a syringe
into PVA solution. The prepared microspheres were then fil-
tered under vacuum filtration using 0.45µm filter paper to
collect the microspheres and they were left to dry at room
temperature for 48 hours.
Particle Size Distribution and Zeta Potential
Particle size distribution of microspheres and zeta potential
were determined using a Malvern Mastersizer (Malvern In-
struments Ltd, UK). The samples were analysed and average
particle size was expressed as the mean diameter and mean
zeta potential.
Encapsulation Efficiency
Drug loaded microspheres was dissolved in 1M NaOH, then
vortexed for 1hour and kept overnight in dry, dark environ-
ment. The following day, the solution was vortexed and 1M
HCL was added. The solution was vortexed and the absor-
bance was measured using the UV spectrophotometer to
determine the concentration of theophylline in solution. The
blank microspheres were used as control.
In Vitro Release Study
The in vitro release of theophylline from PLGA microspheres was
determined in distilled water. Microspheres were suspended
in distilled water in centrifuge tube and placed into a shaking
water bath at 37.5oC at 50cpm. At predetermined time inter-
vals, samples were centrifuged for 5 minutes and supernatant
removed for analysis in UV spectrophotometer. Fresh replace-
ment media was added to re-suspend the microspheres.
Cytotoxicity Studies
Cytotoxicity of formulations was evaluated by using Caco-
2 cells (ATCC, Rockville, MD, USA). The cells were grown in
DMEM. Cell viability was assessed by using MTT assay. Caco-
2 cells in culture medium were seeded in 96-well plates and
following 24 h incubation (for adherence) at 37oC in 5%
CO2, 20 µL of microsphere suspensions for each formulation
were added into each well. After incubation, 25 mL of MTT
 POSTER PRESENTATIONS

solution (5 mg/mL) was added to each well and the plates

were incubated for further 4 h. The produced formazan was
solubilized and kept overnight at 37oC, the optical densities
at wavelength of 570 nm were measured using microplate
reader (Spectramax Plus, Molecular Devices).

RESULTS AND DISCUSSION

There was a general pattern of increase in particle size when
increasing the PLGA polymer ratio (weight of polymer to
drug) from 3:1 to 15:1. However, there was no apparent rela-
tionship between the stirring speed and particle size. When
increasing PLGA polymer amount, the encapsulation effi-
ciency also increased. When increasing the speed of stirring/
mixing from 3000rpm to 5000rpm, the encapsulation effi-
ciency decreased consecutively for all three concentrations
of PLGA polymer
(Table 1).
Figure 1. Cell viability values of formulations (mean ± SD,
n=3)
For all three concentration of PLGA, there was a similar trend
seen in the cumulative release profiles. Initial burst release
was observed followed by a constant release of theophylline.
For cytotoxicity studies, it was shown that, the PLGA micro- p027-ENHANCED ORAL BIOAVAILABILITY
spheres loaded with theophylline presented no cytotoxicity OF LORATADINE VIA A PH-INDEPENDENT
during the MTT assay (Figure 1). INCLUSION COMPLEX
CONCLUSION Agnes Nacsa1, Zoltan Aigner1, Peter Sipos1, Tamas Martinek2,
In conclusion, theophylline loaded PLGA microspheres have Gabor Blazso3, Agnes Balogh3, Piroska Szabo Revesz1
been prepared using an o/w single-emulsion solvent evapo-
ration method and characterized in vitro. This work can be
University of Szeged Department of Pharmaceutical
considered for further studies on evaluation of the in vivo
Technology1
efficacy of theophylline PLGA microspheres. 97
University of Szeged Institute of Pharmaceutical Chemistry2
University of Szeged Department of Pharmacodynamics and
Table 1: Particle characteristics of formulations.
Biopharmacy3
Polymer: Drug Ratio at
3000 rpm INTRODUCTION
One of the prerequisites for successful oral drug therapy is
adequate intestinal absorption. Loratadine (LOR), a second-
3:1 10:1 15:1
generation antihistamine, chosen for the current studies, is
a BCS II drug1, i.e. it has dissolution- or solubility-limited ab-
Mean Particle Size ┴ SD 3.57 4.93 7.44 sorption. In this work we studied the stoichiometry of inclu-
(μm) ┴0.90 ┴1.24 ┴0.74 sion complexes of LOR with dimethyl-β-cyclodextrin (DIMEB)
and their bioavailability.
-22.8 -21.28 -23.13
Zeta Potential ± SD MATERIALS AND METHODS
±0.69 ±0.57 ±0.56
Materials
Encapsulation Efficiency % 13.1% 21.50% 81.76% LOR was kindly provided by TEVA Ltd., Hungary. DIMEB was
purchased from Cyclolab Ltd., Hungary. Other chemical re-
Polymer: Drug Ratio at agents were of analytical grade purity.
3000 rpm
Preparation of products
3:1 10:1 15:1 The products were prepared in two molar ratios (LOR:DIMEB
= 1:1 and 1:2) by the kneading method (KP). The physical mix-
tures were suspended with the same mass of 50% ethanol,
Mean Particle Size ± SD 4.54 6.313 8.57
and the solvent was evaporated off at room temperature. Af-
(μm) ±0.75 ±2.61 ±0.97
ter drying, the products were ground and sieved (100 µm).

-18.6 -20.8 -21.9 Diffusion ordered spectroscopy (DOSY)

Zeta Potential ┴ SD
┴0.56 ┴0.42 ┴0.57 NMR measurements were performed on a Bruker Avance III
400 MHz spectrometer with a triple resonance 2.5-mm capil-
Encapsulation Efficiency % 2.02% 14.01% 25.25% lary probe with a z-gradient coil in D2O solution at 298.1 K.

October 26 - 28, 2009 / Antalya - TURKEY

 POSTER PRESENTATIONS

The DOSY measurements were performed by using the stim- ESI-MS

ulated echo and longitudinal eddy current delay sequence.
Parameters: dephasing gradient pulse length: 2 ms; diffusion
delay: 250 ms; gradient strength: 5 to 95% of the maximum;
number of steps: 32. Each measurement was run with 256
scans and 2K time domain points.

Electron spray ionization mass spectrometry studies (ESI-MS)

The ESI-MSD-ion trap (AGILENT 1100 LC-MSD TRAP SL ion-
trap MS) was operated under positive ion and auto MS-MS
mode with previously described parameters2.

In vitro dissolution
The modified paddle method with the USP dissolution ap- Figure 2: The ESI of the products revealed that the complex stoi-
paratus (Pharmatest, Germany) was used to examine 200 mg chiometry is 1:1.
samples of pure LOR or products containing 200 mg of LOR In vitro dissolution
in 900 ml of simulated intestinal medium (pH = 7.0 ± 0.1, T =
37 ± 0.1). The rotation rate was 100 rpm. The LOR contents of
the samples were determined spectrophotometrically (λSIM
= 248 nm).

Study of the effect of pH on the solubility

Seven buffer solutions were prepared with different pH values
between 1.2 and 7.51. Products containing 10 mg of LOR were ex-
amined in 900 ml of dissolution media at 37 °C. The paddle was
rotated at 100 rpm. After 2 h, removed samples were filtered and
the LOR concentrations were measured spectrophotometrically.

In vivo experiments
In male rats of the Wistar strain weighing 150 ± 5 g, oedema Figure 3: The results of the in vitro dissolution.
98 was induced in the hind paw by the subplantar injection of
10 μg/0.1 ml compound 48/80. The extent of the oedema was Study of the effect of pH on the solubility
determined with a plethysmometer (Ugo Basile, Harvard Ap-
paratus, Germany) 30 min after the subplantar injection. The
animals were divided into five groups with 6 rats per group.
Group I served as a control and received the vehicle. The fol-
lowing materials were tested in this study: LOR, DIMEB, KP
1:1, KP 1:2. The materials were given orally in suspension in
0.25% methylcellulose (MC) at a LOR concentration of 10 mg/
kg 1 h before the subplantar injection of compound 48/80.
Evaluation was performed by one-way analysis of variance.

RESULTS AND DISCUSSION

DOSY

Figure 4: illustrates the pH-dependence of the solubility of

LOR and the products.

In vivo experiments

Figure 5: depicts the volume of the oedema in the control

and treated groups.
Figure 1: The DOSY spectra of KP 1:1

3rd BBBB International Conference on Pharmaceutical Sciences

 POSTER PRESENTATIONS
CONCLUSION
The stoichiometry of the complex is 1:1, but this amount of
DIMEB is not sufficient for pH-independent dissolution to
be achieved. Thus, there is a need for excess DIMEB, i.e. the
1:2 product is required. The KP 1:2 decreased the oedema by
more than 50% relative to LOR alone, in consequence of the
higher blood concentration, through better and smoother
bioavailability.
ACKNOWLEDGEMENTS
This work was supported by a Sanofi-Aventis Fellowship.
REFERENCES
1. Khan MZ et al. Classification of loratadine based on the
Biopharmaceutics Drug Classification concept and pos-
sible in Vitro - in Vivo Correlation, Biol. Pharm. Bull. 2004;
27(10):1630–1635
2. alázsik K et al. A new rigid cinchona modified (α-IQ) plat-
inum catalyst for the enantioselective hydrogenation of
activated ketones: Data to the origin of enantioselection,
J. Molecular Catalysis A: Chemical 2007; 272: 265-274
p028-PRELIMINARY STUDIES ON
PRONIOSOME FORMULATIONS
Nilüfer Yüksel1, Murat Oral1
Ankara University School of Pharmacy Department of
Pharmaceutical Technology Ankara-TURKEY1
INTRODUCTION
Proniosomes are in the form of water soluble carrier par-
ticles coated by a film of non-ionic surfactant and choles-
terol. Proniosomes can be hydrated easily prior to use and
converted to niosomal dispersions. In addition, they can be
prepared as per-oral dosage forms like tablets, capsules and
beads. Because of being dry powders, proniosomes improve
physical and chemical stability of niosomes. In this study, we
aimed to evaluate two methods by which proniosomes are
formed, and to investigate the effects of the formulation and
process variables on the characteristics of proniosomes.
MATERIALS AND METHODS
Materials
Sorbitol (Merck, Germany) was used as the water soluble
carrier. A non-ionic surfactant, Span 60 (Sigma Aldrich, USA)
and cholesterol (Sigma Aldrich,USA) were used to form nio-
somes. Dicetyl phosphate (Sigma Aldrich,USA) is used as the
negative charged agent for the stabilization of niosomal dis-
persion. All other chemicals were of analytical grade.
Methods
Spray Method
A 100 mL round bottomed flask containing required amount
of sorbitol powder was attached to a rotary evaporator (Ro-
tavapor, Buchi R-200,Germany). Span 60, cholesterol and
dicethylphosphate were dissolved in chloroform. This stock
solution was intermittently sprayed onto the surface of sor-
bitol powder. During spraying period the rate of application
October 26 - 28, 2009 / Antalya - TURKEY
was controlled in order to prevent extremely wetting of the
powder bed of sorbitol. The flask was rotated in the water
bath at 65-70°C under vacuum until sorbitol appeared to be
dry, and then, another portion of stock solution was sprayed.
This process was repeated until all the stock solution had
been applied. After addition of the final aliquot, evapora-
tion was continued until the powder was completely dry.
The material was further dried in an oven (Selecta Vaciotem,
Australia) under vacuum at room temperature overnight to
remove the residual chloroform (Formulation K1-spray, Table
1). Proniosomes were obtained as dry powder.
Slurry Method
Span 60, cholesterol and dicethylphosphate were dissolved
in chloroform. The whole of this solution was introduced into
the round-bottom flask containing required amount of sor-
bitol powder. After this process, a slurry was formed in the
round-bottom flask. The flask was attached to a rotary evap-
orator to evaporate chloroform at a temperature of 45 + 2 °C.
Evaporation was continued until the powder was completely
dry. The material was further dried in owen under vacuum
at room temperature overnight (Formulation K1-Slurry,
Table 1). Proniosomes were obtained as dry powder. In this
method, different formulations were prepared (i)changing
the amount of chloroform (K2 formulation), (ii)using differ-
ent particle size range of sorbitol (K3 formulation), and (iii)
changing the ratio of surfactant mixture:sorbitol (K3-Medi-
um, K4 and K5 formulations).
Table 1 :Composition of the proniosome formulations.
Ingredients K1 K2 K3 K4 K5 99
Span 60 49.28 49.28 49.28 49.28 49.28
Cholesterol 44.23 44.23 44.23 44.23 44.23
DCP 6.490 6.490 6.490 6.490 6.490
Chloroform 10 5 10 10 10
Sorbitol 1000 1000 1000 2000 3000
Particle size range of sorbitol used in the formulations (mm):
K1: 0.25-0.75, K2: 0.25-0.75, K3-Large: 0.75-1.00, K3-Medium: 0.25-0.75,
K3-Small: 0.25-0.75, K4: 0.25-0.75, and K5: 0.25-0.75
Determination of Niosome Size, Zeta Potential and Shape
The proniosomal powder were hydrated at 80°C and vor-
texed (Vortex mixer Heidolph D-91126, Germany) for 2 min.
The effect of different vortexing times were evaluated on K4
formulation. The size and zeta potential of the formed nio-
somes was measured using dynamic light scattering (DLS)
and phase analysis light scattering (PALS) with a zetasizer
(Malvern Zetasizer Nano ZS, UK). Leica Optical Microscope
(DM4000B, USA) was used to image the niosomes.
RESULTS AND DISCUSSION
Spray and slurry methods were compared for particle size
and shape of niosomes obtained by hydrating the pronio-
somes (K1-spray and K1-slurry).
The niosomes formed by the spray method are smaller than
those formed by the slurry method although the greater
amount of aggregated vesicles are seen in micrograph (Fig.
1). Slurry method was chosen as the preparation method of
 POSTER PRESENTATIONS

another proniosome formulations. The amount of chloroform
in K2 formulation was lowered (Table 1). Chloroform affected
the consistency of the slurry during preparation resulting in
bigger niosomes with a more heterogenous particle size dis-
tribution (Table 2). Optimum particle size of sorbitol used
as carrier for niosomes was also evaluated (K3 formulations,
Table 2). The niosomes formed from proniosomes prepared
with medium particle size of sorbitol had monomodal par-
ticle size distribution and average particle size was 580.6nm.
In the formulations of K3-Medium, K4, and K5, the ratio of
surfactant mixture:sorbitol was changed. Increasing amount
of sorbitol caused to increase of particle size of proniosome-
derived niosomes (K4 and K5 formulations, Table 2).
Zeta potentials of all the proniosome-derived niosomes were
negative and between -32,2 to -68,8mV due to dicetylphos-
phate (Table 2 and 3). These values indicates thermodynami-
cally stabilized preparations.
CONCLUSION
According to the experimental findings, slurry method was
found to be more convenient method for producing pronio-
somes by using lower amount of chloroform and sorbitol at
the medium size. Niosomes could be formed without vortex
mixing indicating the convenience of the proniosomes for
per-oral administration as a solid dosage form (tablets and
capsules) or a solution. In light of this observations, drug
loaded proniosomes will be prepared with the different type
of surfactants in an attempt to enhance drug solubility.

REFERENCES
1. Chengjiu H, Rhodes D, Proniosomes: A novel drug car-
rier preparation, Int. Journal of Pharm. 185 (1999) 23-35.
2. Solanki B, Parikh R, Formulation and optimization of
proxicam proniosomes by 3-factor 3-level Box Behnken
design, AAPS PharmSciTech 2007, 8 (4) Article 86.
3. Varshosaz et al, Sorbitan monopalmitate based pronio-
Figure 1: Optic micrographs of the niosomes obtained from somes for transdermal delivery of clorpheniramine
K1 formulations prepared by two different methods. maleate, Drug Delivery 12:2, 75-82

Table 2 : Zeta potentials and particle sizes of the niosomes

obtained from proniosomes. P029-OPTIMISATION STUDIES FOR MAKING
Peaks (nm) (% area of peak
Zeta potential FREE-FLOWING COMPACTABLE POWDER AND
indicating the volume of particle GRANULE CONTAINING RAMIPRIL
100 Formulation (mV±S.E.)
population)
1st peak 2nd peak Buket Aksu1, Ayça Yıldız2, Özgen Özer3, Tamer Güneri3
K1-spray 117.9 (100) - -32.2±0.482
K1-slurry 1156 (97.7) 125.3 (2.3) -35.7±0.99 Ege University, Pharmacy Faculty, Department of
Pharmaceutical Technology, Bornova-İzmir-Turkey Santafarma
K2 1326 (94.3) 314.9 (5.7) -46.7±0.698
Pharmaceuticals, 34382, Istanbul-Turkey1
K3-Large 1771 (89.6) 154.9 (10.4) -44.6±1.12 Istanbul University, Pharmacy Faculty, Department of
K3-Medium 580.6 (100) - -44.9±1.13 Pharmaceutical Technology, 34116, Istanbul-Turkey2
K3-Small 882.2 (62.9) 171.8 (14.6) -39.9±1.55 Ege University, Pharmacy Faculty, Department of
Pharmaceutical Technology, Bornova-İzmir-Turkey3
K4 1189 (100) - -67.1±3.04
K5 1178 (98.5) 158 (1.5) -57.0±1.68 INTRODUCTION
Granulation is a process by which fine powders are agglom-
The time of vortex mixing was changed to see its effect on erated into larger particles using a liquid binder. Flow prop-
particle size of niosomes after hydrating proniosomes. Par- erties of granules are of critical importance in fabrication of
ticle size of the niosomes was not considerably changed de- reproducible tablet formulations1. An optimum flow of gran-
pending on the vortex process indicating that niosomes can ules should be achieved to ensure uniform feed from funnel
also be formed without vortex mixing (Table 3). into dies to obtain tablet formulations with acceptable con-
tent uniformity, weight variation and physical consistency2.
Tablo 3 : The effect of vortex mixing time on particle size of In our study, therefore, with an eye to developing optimum
niosomes derived from proniosomes (K4). tablet formulations of ramipril which is an angiotensin-con-
Formulation Peaks (nm) (% area Zeta potential verting enzyme inhibitor, granule formulations were fabri-
of peak) (mV±S.E.) cated by wet granulation technique and the effect of some
parameters such as granule size, type and amount of lubri-
K4-without vortex 1046 (100) -68.8 ± 50
cants on their flow characteristics were investigated.
K4-2 min. vortex 1189 (100) -67.1 ± 3.01
K4-10 min. vortex 1227 (100) -33.3 ± 0.946 MATERIAL AND METHODS
Materials
Ramipril was purchased from Santa Farma Drug Company.

3rd BBBB International Conference on Pharmaceutical Sciences

 POSTER PRESENTATIONS

Hydroxypropyl methylcellulose (HPMC, Collorcon), Magne- ing amounts of both lubricants. Granules which were sieved
sium stearate (Faci/Italy), Sodium stearyl fumarate (JRS Phar- through the 1.20 mm screen and lubricated with sodium
ma). All the other excipients were of pharmaceutical grade. stearyl fumarate showed less caking characteristics than
others due to their lowest caking strength (Figure 2).
Fabrication Granule Formulations
Ramipril (5 mg) and other additives (Lactose monohydrate,
pregelatinised starch, Ac-Di-Sol, sodium hydrogen carbon-
ate, ferrum oxide) were granulated in “High shear granulator”
(ProCept, Belgium) HPMC solution and passed through an
oscillating granulator fitted with 0,250 mm screen, and dried
in a hot-air oven at 60ºC. After re-sieving using 0.80 mm and
1.25 mm screens, the granules were blended with different
amount of two distinct lubricants (magnesium stearate and
sodium stearyl fumarate) for 5 min (Table 1).

Granule Flow Characterisation Figure 1: Effect of granule size and different amount of (a)
Granule characterization was performed on unlubricated Magnesium stearate, (b) Sodium stearyl fumarate on cohe-
and lubricated granules using Powder reometer (TA-XTPlus, siveness of granule formulations
Stable Micro Systems, UK). The instrument was calibrated for
force and distance measurements. A cylindrical vessel was
partly filled (140 mL) with each granule formulation and
set on the platform of the apparatus. A helical blade which
moves into the granule bed was used to probe and to mea-
sure a) the cohesion (Cohesion test), b) the caking (Caking
test) and c) the changes on granule flow properties due to
the increasing and decreasing flow speeds (Powder flow
speed dependence test).

Table 1. Formulation parameters of ramipril granule formula-

tions
Amount of Lubricant (%) 101
Formulations Sleve mesh size Magnesium Sodium stearyl
starate fumerate
F1 0.8 - -
Figure 2: Effect of lubricant type and amount on cake strength
F2 0.8 0.75 - of granule formulations
F3 0.8 1.00 -
F4 0.8 - 0.60 Granule flow properties may change with increasing and de-
creasing flow speeds. Powders/granules may become more
F5 0.8 - 1.20 resistant to flow as it is forced to flow faster or indeed it may
F6 1.2 - - become more free flowing as the flow speed increases. While
F7 1.2 0.75 - marginal or no change of the compaction coefficient with
flow speed would show that the granule is flow speed in-
F8 1.2 1.00 -
dependent, an increase or decrease in the compaction coef-
F9 1.2 - 0.60 ficient as the test speed dependence may result inadequate
F10 1.2 - 1.20 feed from bulk container into dies during tablet preparation.
Flow stability also gives important information about the
RESULTS AND DISCUSSION flow resistance of the granules. Compaction coefficients of
Cohesion is tendency for particles of granules to cling to- the lubricated granules were not significantly change with
gether. Cohesion test results showed that the addition of the increasing of tip speed (Figure 3) and their flow stability
increasing amounts of both lubricants decreased the co- values were found to be very close to 1.00 (Figure 4). On the
hesiveness of granule formulations. Whereas the screen other hand, granules which were sieved through the 1.2 mm
size (0.8 mm and 1.2 mm) did not significantly change the screen and lubricated with 0.6 % sodium stearyl fumarate
cohesiveness of the granules, sodium stearyl fumarate as a had lowest compaction coefficient.
lubricant was found to be more effective than magnesium
stearate (Figure 1).

Caking is the tendency of granules to form larger agglom-

erates during storage and transportation. Caking test indi-
cated that the caking strength decreased with the increas-

October 26 - 28, 2009 / Antalya - TURKEY

 POSTER PRESENTATIONS
Figure 3: Powder flow speed dependence test results. Effect
of tip speed on compaction coefficent of granules
102
Figure 4: Flow stability values of unlubricated and lubricated
granule formulations.
CONCLUSION
As a conclusion, sodium stearyl fumarate was found to be
the most effective lubricant for ramipril granule formulations
due to its suitable properties.
REFERENCES
1. Kani T, Suzuki T, Tsukada M, Kamiya H. Influence of sur-
face-adhered nanoparticles and nanoporous structure
on bulk flowability of silica Powder Technology, 176,
108-113, 2007.
2. Navaneethan CV, Missaghi S, Fassihi R. Application of
Powder Rheometer to Determine Powder Flow Proper-
ties and Lubrication Efficiency of Pharmaceutical Par-
ticulate Systems. AAPS PharmSciTech, 6(3), Article 49,
2006.
3rd BBBB International Conference on Pharmaceutical Sciences
P030-ENHANCEMENT OF THE RELEASE OF
IBUPROFEN FROM SOLID DOSAGE FORM
Andres Lust1, Urve Paaver1
University of Tartu, Department of Pharmacy, Tartu, Estonia 1
INTRODUCTION
Release of poorly soluble drugs from oral solid dosage forms
in organism is guaranteed by endogenous surface-active
agents. For checking in vitro quality of pharmaceutical prep-
arations, bile acids are used for imitating in vivo conditions.
Bile acids are expensive and their usage for in vitro investiga-
tions is hardly reproducible because these are poorly soluble
and inadequately stable in various buffer solutions. Bile acids
are substituted with non-ionic surfactants that improve the
wettability of poorly soluble active pharmaceutical ingredi-
ent (API). The results of in vitro release should be in corre-
lation with pharmacokinetic characteristics and as a result
make possible to assess based on in vitro results the quality
of conventional solid oral pharmaceutical preparations con-
taining poorly soluble API.
Aims of the study
The aim of the study was
- to determine the effect of non-ionic surfactants with differ-
ent HLB value to the in vitro release of ibuprofen at different
pH conditions
MATERIALS AND METHODS
Formulation studied: Ibumetin® 200 (Nycomed) (ibuprofen -
II class by BCS, D0 = 80, solubility 0.01 mg/ml).
Surfactants: Polysorbate 60 – HLB 14.9, Polysorbate 80 – HLB
15.0, Polysorbate 40 – HLB 15.6, Polysorbate 20 – HLB 16.7,
Triton X-100 HLB 13.4, Triton X-102 – HLB 14.4, Triton X-165 –
HLB 15.5, Triton X-305 – HLB 17.3 (Sigma-AldrichTM)
The dissolution apparatus: Thermostat – Sotax AT7; Peristal-
tic pump – Watson-Marlow 202 U/AA; Flow-through spec-
trophotometer – Ultrospec III (LKB Biochrom Ltd.); Software
– TDS (LKB Biochrom Ltd.).
Testing conditions: Rotating paddle method. Speed of rota-
tion (r.p.m.) – 50. Dissolution medium – pH 1.2 and pH 4.5.
Volume of dissolution medium – 900 ml Wavelength – 221
nm. Temperature of dissolution medium - 37±0.5° C.
RESULTS AND DISCUSSION
Ibuprofen is used mainly for relief of acute pain. Ibuprofen
is rapidly and completely absorbed after oral administration
through-out the gastrointestinal tract. In studies with solu-
tions for oral administration, peak plasma concentrations
were achieved 0.5 to 0.7 hours after administration [1]. For
conventional tablets tmax values of 1.3 to 2.7 h have been
reported2.
In the present study, in vitro release of ibuprofen at pH 1.2 and
4.5 was very low, 10.3±0.7% and 19.8±2.1% respectively.
 POSTER PRESENTATIONS
Figure 1: Ibuprofen release at pH 1.2 and 4.5 during 1 hour in
the presence of various concentrations of Tritons
Extent of dissolution increased in the presence of surfac-
tants. Tritons increased the release of ibuprofen more at pH
1.2 than at pH 4.5. Except for Triton X-100, that acted contrary
(Figure 1). At pH 4.5 low concentrations (0.1%) of Polysor-
bates increase the release of ibuprofen on the average from
50% to 57.7% but not differentiate by HLB value.
It can be seen from Figure 2 and 3 that at pH 1.2 that to ob-
tain the same release of active ingredient (AI), the amount of
Tritons is 5 to 10 times smaller than in case of Polysorbates.
Also the presumption that a surfactant with higher HLB val-
ue facilitates the release of API was not true.
Figure 2: Ibuprofen release at pH 1.2 during 1 h in the pres-
ence of various concentrations of Tritons
The surfactants have mainly a wetting activity. The experi-
ments performed at pH 1.2 revealed that the increase of the
hydrophilicity of the amphiphilic surfactive polysorbates
does not correlate with the amounts of AI released. Addi-
tion of the 0.5% and 1% Polysorbate 60 (HLB 14.9) into the
dissolution medium increased the ibuprofen release during
one hour from 67.5±2.9% up to 91.2±1.1%. For Polysorbate
80 (HLB 15.0) was concentration 0.5% enough for releasing
87.2±4.2% of drug. However, the higher HLB value Polysor-
bate 40 (HLB 15.6) and Polysorbate 20 (HLB 16.7) addition
increased the release of the ibuprofen only by 65.6±1.1%
and 84.8±1.7 %.
October 26 - 28, 2009 / Antalya - TURKEY
Figure 3: Ibuprofen release at pH 1.2 during 1 h in the pres-
ence of various concentrations of Polysorbates.
In case of Polysorbates, PS 80 (HLB 15.0) should be preferred.
In case of Tritons, the best results at low concentrations were
obtained with Triton X-100 (HLB 13.4 – 0.035%) and Triton
X-165 (HLB 15.5 – 0.05%).
CONCLUSION
For enhancement the release of ibuprofen from solid dosage
forms at pH 1.2 Triton X-100, Triton -165, Polysorbate 80 and
Polysorbate 60 were at most suitable.
At pH 4.5 among the surfactants studied Triton X-100 in low
concentrations (below 0.1%) guaranteed the best result.
ACKNOWLEDGMENT
This study was supported by the research fund of Estonian 103
Academical Society of Pharmacy.
REFERENCES
1. W.R. Gillespie, A.R. DiSanto, R.E. Monovich and K.S. Albert, Rel-
ative bioavailability of commercially available ibuprofen oral
dosage forms in humans, J. Pharm. Sci. 1982; 71: 1034–1038.
2. Bramlage P, Goldis A, Bioequivalence study of three ibu-
profen formulations after single dose administration in
healthy volunteers, BMC Pharmacology 2008; 8:18.
p031-VITAMIN E TPGS COATED
BIODEGRADABLE POLYMERIC
NANOPARTICLES OF MELOXICAM FOR
COLON-SPECIFIC DELIVERY
Ceyda Tuba Şengel Türk1, Canan Hasçiçek1, Nurşin Gönül1
Ankara University Faculty of Pharmacy Department of
Pharmaceutical Technology, Ankara, Turkey1
INTRODUCTION
Colon-specific drug delivery has been widely investigated
for the treatment of local colonic diseases such as Crohn’s
diseases, ulcerative colitis, inflammatory bowel disease and
colorectal polyps and cancer. The effect of non-steroidal anti-
inflammatory drugs (NSAIDs) on colorectal polyps and can-
cer arises interests in recent years1. Meloxicam, an enolic ac-
 POSTER PRESENTATIONS

id-type NSAID, is used in the treatment of arthritis and other Table 2: Characteristics of the NPs.
joint diseases. Recent investigations about relationship be- Code EE (%) Sign (nm) PDI Zeta potential (mV)
tween meloxicam and colorectal cancer have reported that F1 80.17 178.30 0.146 -21.23
meloxicam has been regarded as a potential drug for the V1 80.63 178.60 0.149 -18.20
prevention and treatment of colorectal polyps and cancer2. V2 80.08 205.20 0.133 -23.30
Therefore, the development of a particulate carrier system
63.41
such as nanoparticles (NPs) which are designed to localize V3 79.96 0.279 -23.50
231.80
drugs at the target site for the therapy of colonic cancer is
valuable. The aim of this research is to develop a polymeric
The diameter of the NPs coated with higher than 2.5% coat-
nanoparticulate drug delivery system of Meloxicam for colon
ing ratio was found to be bigger than uncoated nanoparti-
cancer and investigate the effects of the coating process on
cles (p<0.05). This incerase in size was expected and attrib-
the physicochemical properties of the NPs. NP formulations
uted to the thickness of the coating. The size distribution of
which based on poly(D,L-lactide-co-glycolide) (PLGA) as a
the NP formulations was narrow (PDI: 0.133-0.149) except V3
biodegradable polymer were prepared by a combined tech-
coded NPs (PDI: 0.279). Zeta potential value is an important
nique that is based on salting-out and emulsion-evaporation
particle characteristics for the stability of the NP suspension.
steps and they were coated with Vitamin E TPGS which is a
All NPs were found to be stable in dispersion state, having
new water soluble derivative of natural Vitamin E by using
negative surface charges.
surface adsorpsion technique3.
The morphology of the prepared NPs was investigated by
MATERIALS AND METHODS
using transmission electron microscopy (TEM). As shown
Materials
in Figure 1, NPs have exhibited spherical shapes within the
Meloxicam (Ulkar Chem. Co., Turkey), PLGA (Mw: 40-75 kDa,
nanometer size.
copolimer ratio 50:50) and PVA (Mw: 9-10 kDa) (Sigma-Al-
drich, Germany), Vitamin E TPGS (Eastman Chemical, USA)
Dissolution rate studies on NPs were performed with static
were supplied and used as received.
method and resultant drug release profiles shown in Figure
Preparation of Nanoparticles
2, were found to be significantly different depending on the
PLGA and Meloxicam were dissolved in an organic mixture of
variation of the coating percentage for the formulation in-
acetone and methylene chloride. This solution was poured
vestigated (p<0.05). With an increase in the % coating ratio,
into a 25 ml aqueous phase containing PVA with magnesium
the drug release rate significantly decreased as a result of the
chloride and emulsified by ultrasonication in an ice bath for 3
thickness of the coating.
104 min at 80% output energy. Additional aqueous PVA was then
added under gentle stirring and stirring was continued for 24
h to insure complete organic solvents evaporation. NPs were
purified using ultracentrifugation and then freeze-dried for
48 h. For NPs coating, various ratios of Vitamin E TPGS was
dissolved in water and an adequate amount of lyophilized
NPs were suspended in this solution by ultrasonication for 30
sec. The suspension of NPs were then freeze-dried for 48 h3.

Characterization of Nanoparticles
Uncoated and Vitamin E TPGS coated PLGA NPs were evalu-
ated for their encapsulation efficiency (EE), zeta potential, Figure 1: TEM images of NPs coded F1.
particle size and polydispersity index, morphology and in-
vitro dissolution rate. Formulations, codes and the coating
percentages are given in Table 1.

Table 1: Formulation codes and the coating percentages.

Formulations Codes Coating percentage (%, w/w)
Uncoated NPs F1 0
Coated NPs V1 2.5
Coated NPs V2 5.0
Coated NPs V3 10.0

RESULTS AND DISCUSSION

The physicochemical NPs are summarized in Table 2. EE of
meloxicam within coated and uncoated NPs was between
79.96-80.63% and was not affected significantly by coating
process and percentages (p>0.5). Figure 2. In-vitro drug release profiles of the NPs.

3rd BBBB International Conference on Pharmaceutical Sciences

 POSTER PRESENTATIONS

CONCLUSION MATERIALS AND METHODS

In this present research, it is evident that PLGA NPs were suc- Materials
cessfully coated with Vitamin E TPGS for the colonic delivery Triamcinolone acetonide (TA) was kindly provided by I.E.
of meloxicam. The physicochemical properties and the re- Ulagay Drug Company (Turkey). Carbopol 934® (C934) (No-
lease behaviour of the uncoated NPs were modified by coat- veon-USA), high molecular weight chitosan (HMW - viscosity
ing with various of Vitamin E TPGS at different percentages. > 400mPa), medium molecular weight chitosan (MMW - vis-
cosity 200mPa–400mPa) (Fluka Biochemika-Japan), HPMC-
ACKNOWLEDGMENTS Methocel E 4M (Colorcon LTD-USA) were used as bioadhesive
This research has been supported by Management of Scien- polymers. Poloxamer 407® (P407) (BASF-Germany) were used
tific Research Projects of Ankara University (project number for it’s good gelling property. Transcutol (TC) (Fluka Bioche-
is 07B3336003). mika, Japan) and propylene glycol (PG) (Sigma-Aldrich, USA)
REFERENCES were selected as penetration enhancer. As the commercial
1. Makhlof A, Tozuka Y, Takeuchi, pH-sensitive nanospheres product contained 0.1% TA (Kenacort–A orabase®) was used.
for colon-specific drug delivery in experimentally in- All other reagents were analytical grade.
duced colitis rat model, Eur. J. Pharm. and Biopharm.
2009; 72: 1-8. Preparation of the gel systems
2. Goldman AP, Williams CS, Sheng H, Lamps LW, Williams Bioadhesive gels containing P407 – C934 were prepared by
VP, et al. Meloxicam inhibits the growth of colorectal cold method2. Other mucoadhesive formulations were pre-
cancer cells, Carcinogenesis 1998; 19: 2195-2199. pared by dissolving chitosan (MMW or HMW) and HPMC
3. Şengel-Türk CT, Hasçiçek C, Gönül N, Development in diluted lactic acid solution. Compositions of the gels are
of PLGA nanoparticles surface modified with didode- seen in Table1.
cyldimethyl ammonium bromide: preparation and in-
vitro characterization, 36th Annual Metting and Exposi- Table 1: Compositions of the formulations
tion of the Controlled Release Society 2009; PO 898. P407 C934 TC PG TA
(w/w) (w/w) (w/w) (w/w) (w/w)
F3 15 2 - 12 0.1
p032-A COMPARISION OF MECHANICAL F4 20 1.5 - 12 0.1
PROPERTIES OF BUCCAL GEL FORMULATIONS HPMC chitosan TC PG TA
BASED ON DIFFERENT BIOADHESIVE (w/w) (w/w) (w/w) (w/w) (w/w)
POLYMERS CM-3 - 3(MMV) 10 12 0.1
105
CH-3 - 3(HMV) 10 12 0.1
Gülin Amasya1, Sinem Y. Karavana2, Tangül Şen1, Esra X1 1 3(MMV) 10 12 0.1
Baloğlu2, Nilüfer Tarımcı1 X2 2 3 (MMV)
10 12 0.1

Ankara University, Faculty of Pharmacy, Department of Viscosity measurements

Pharmaceutical Technology, 06100 Ankara, TURKEY1 The viscosity measurements were carried on using by digital
Ege University, Faculty of Pharmacy, Department of viscometer (Brookfield DV II) spindle no: T96 at 20 rpm and
Pharmaceutical Technology, 35100 İzmir, TURKEY2 at 25±1°C.

INTRODUCTION Texture profile analysis of TA gels

Bioadhesive drug delivery formulations are very beneficial,
since they provide controlled drug release overtime and
localize the drug to a specific site of the body. They should
exhibit suitable mechanical properties including acceptable
viscosity, easy of expression from the container, easy of ap-
plication, good spreadability, appropriate hardness and pro-
longed residence time in the oral cavity. These mechanical
properties may affect the performance of the formulations
and their acceptance by patients1.
The aim of this study was to investigate the mechanical and
adhesive properties of buccal gel formulations containing
0.1% triamcinolone acetonide prepared with different poly-
mers. The results were also compared with the commercial
product contained the same amount of the triamcinolone
acetonide.
The mechanical properties of the gels were determined us-
ing software-controlled penetrometer, TA-XT Plus texture
analyzer (Stable Micro System, UK). From the resultant force-
time plot, mechanical parameters such as compressibility,
hardness, cohesiveness and elasticity were defined3.
Mucoadhesion testing
The mucoadhesive properties of formulations were evaluated
with using TA-XT Plus texture analyzer (Stable Micro Systems,
UK). Freshly excised bovine buccal tissue was employed and
three replicates were performed for each sample.
RESULTS AND DISCUSSION
pH and viscosity results are shown in Table 2. For chitosan
based formulations, the pH values are lower than commer-
cial product because of used lactic acid. The pH values of
P407 - C934 combinations were more applicable to the buc-
cal mucosa than chitosan based formulations and the com-
mercial product. Enhanced viscosity could suitable for easily
spread on the lesions as a thin film layer, the viscosity values

October 26 - 28, 2009 / Antalya - TURKEY

 POSTER PRESENTATIONS

of developed formulations except CM-3 were also more fa- The cohesiveness parameter increases the performance of
vorable for topical application than commercial product. the product at the application site. The high value of cohe-
siveness provides full structural recovery following gel appli-
Table 2: pH, viscosity and bioadhesion work values of the for- cation. Elasticity which defines the rate of deformed sample
mulations and commercial product returns to its undeformed condition should be high. Accord-
Work of ing to the TPA results elasticity decreased when bioadhesive
Viscosity (Pa.s) polymer concentration increased for C934 based formula-
pH mucoadhesion
25 ± 1⁰C tions. Also negative correlation was obtained between the
(mJ/cm2)
F3 6.17 ± 0.01 175.56 ± 3.88 0.102 ± 0.02 molecular weight of the chitosan and elasticity values.
F4 6.43 ± 0.02 231.78 ± 2.05 0.082 ± 0.00
CM-3 4.53 ± 0.00 6.28 ± 0.36 0.004 ± 0.03 CONCLUSION
The results suggest that chitosan, Carbopol 934® and HPMC
CH-3 4.44 ± 0.01 29.00 ± 0.83 0.029 ± 0.06
can be used as vehicle for active substance to the oral cavity
X1 4.72 ± 0.01 10.06 ± 0.68 0.107 ± 0.00 because of their good textural properties. Also patients’ ac-
X2 4.17 ± 0.01 26.89 ± 1.24 0.115 ± 0.01 ceptance can be increased when compare with the commer-
Kenacort-A Orabase® 5.13 ± 0.01 200.00 ± 1.63 0.010 ± 0.03 cial product. In addition, different amounts of HPMC (1%-
2%) were improved bioadhesiveness of the chitosan based
Decreasing concentration of C934 from 2% to 1.5% (w/w), formulations without affecting the mechanical properties.
decreased work of mucoadhesion as seen Table 2. Adhe-
siveness of chitosan based gels was affected by polymer REFERENCES
molecular weight. According to these studies, the adhesive 1. Jones D S, Woolfson A D, Brown A F. Textural, viscoelas-
properties of the formulations increased as a function of tic and mucoadhesive properties of pharmaceutical gels
bioadhesive polymer concentration and molecular weight composed of cellulose polymers, Int J Pharm. 1997; 151:
of chitosan. The highest work of mucoadhesion was deter- 223-233.
mined for the formulation coded X2 (0.115±0.01 mJ/cm²). It 2. Scmolka I V. Artificial skin I. Preparation and properties
was also noted that, the mucoadhesive performance of the of Pluronic F-127 gels for treatment of burns, J Biomed.
chitosan based formulations was improved with HPMC. The Mater. Res. 6: 571-582.
textural profiles results are given in Table 3. Hardness and 3. Cevher E, Taha M A, Orlu M, Araman A. Evaluation of me-
compressibility values should be low for taking the formula- chanical and mucoadhesive properties of clomiphene
tions from the container and applying easily to the mucosa. citrate gel formulations containing carbomers and their
106 Increasing the hardness and compressibility values of the thiolated derivatives, Drug Deliv. 2008; 15: 57–67.
formulations depended on the viscosity. Commercial prod-
uct had the highest hardness and compressibility values
when compared with all the developed formulations. Also
HPMC (1% - 2%) were improved the viscosity without affect-
P033-PREPARATION OF A CHITOSAN-BASED
ing the hardness and compressibility. FORMULATION FOR THE TREATMENT OF
ORAL MUCOSAL DISORDERS
Table 3: Mechanical properties of gel formulations
Compressibility Ayşe Özdemir1, Gülçin Akça2, Z. Gülşen Öner3, Merve
Codes Hardness (N) ± SD Günbeyaz3, Sevda Şenel3
(N.mm) ± SD
F3 0.26 ± 0.03 0.90 ± 0.11
University of London, The School of Pharmacy, London, United
F4 0.59 ± 0.06 1.84 ± 0.14 Kingdom1
CM-3 0.02 ± 0.00 0.09 ± 0.00 Gazi University, Faculty of Dentistry, Department of Basic
Medical Sciences, Microbiology Laboratory, Ankara, Turkey2
CH-3 0.04 ± 0.00 0.12 ± 0.01
Hacettepe University, Faculty of Pharmacy, Department of
X1 0.02 ± 0.00 0.09 ± 0.01 Pharmaceutical Technology, 06100- Ankara, Turkey3
X2 0.03 ± 0.00 0.10 ± 0.00
INTRODUCTION
Kenakort-A Orabase® 0.87 ± 0.03 1.92 ± 0.13 The aim of this study was to develop a chitosan based for-
Cohesiveness ± SD Elasticity ± SD mulation for the treatment of oral mucosal infections. In ad-
F3 0.86 ± 0.03 0.91 ± 0.01 dition to its favorable properties such as biodegradability,
biocompatibility, and bioadhesivity, chitosan itself exhibits
F4 0.93 ± 0.03 0.96 ± 0.01 antimicrobial activity as well1. Gel formulations were pre-
CM-3 0.94 ± 0.02 1.05 ± 0.10 pared using different types of chitosan. An antimicrobial
agent, chlorhexidine gluconate (Chx) was incorporated into
CH-3 0.95 ± 0.01 0.98 ± 0.01
the formulations at 0.1 and 0.2% w/v concentrations. For-
X1 0.93 ± 0.03 1.02 ± 0.02 mulations were investigated in vitro for rheological, release
X2 0.96 ± 0.01 1.00 ± 0.02 properties, and antimicrobial activities.
Kenakort-A Orabase® 0.63 ± 0.12 0.96 ±0.06

3rd BBBB International Conference on Pharmaceutical Sciences

 POSTER PRESENTATIONS

MATERIALS AND METHODS

Preparation of chitosan gels
Various types of chitosan with different molecular weight and
solubility were used to prepare gel formulations (Table 1).
Table 1: Gel formulations used in the study
Formualation Chitosan Concn of
Chx DD** chitosan Solvent
Code Type MV* (kDa)
(%w/w) (%) (%w/w)
S - Chitopharm 1% (v/v)
S-01 0.1 S (Cognis, 50 - 1000 >70 3 aqueous
S-02 0.2 Germany) lactic acid
M - Chitopharm 1% (v/v) Figure 1: Flow curves of chitosan gels
M-01 0.1 M (Cognis, 300 - 2000 >70 2 aqueous
M-02 0.2 Germany) lactic acid
L - Chitopharm 1% (v/v)
L-01 0.1 L (Cognis, 500 -5000 >70 1 aqueous
L-02 0.2 Germany) lactic acid
Sig - Chitosan
1% (v/v)
Sig-01 0.1 medium
190 - 310 75 - 85 2 aqueous
(Sigma
Sig-02 0.2 lactic acid
adrich, USA)
Pro - Protasan
Pro-01 0.1 UP CI 213 Distilled
150 - 400 75 - 76 3
(Novomatrix, water
Pro-02 0.2 Figure 2: Release profiles of Chx from gels in distilled water
Norway)
at 37°C
Viscosity measurements
Antimicrobial activity
Viscosity measurements of gels were performed on a Brook-
Inhibition zone diameters obtained for chitosan gels are
field digital viscometer (Model DV-II+) at 25°C.
given in Fig. 3a-c. 107
In vitro release studies
Chitosan gels alone were shown to exert antimicrobial activity.
Release from gels was studied using Franz diffusion cells. The
Water soluble chitosan was found to be less effective against
samples were assayed for Chx at 253 nm using UV 1800 Shi-
all the strains investigated when compared to basic chitosans.
madzu Spectrophotometer.
Highest antimicrobial activity was obtained with the high mo-
lecular weight chitosan in absence of Chx whereas in presence
Antimicrobial activity studies
of Chx the activity seemed to be influenced by the viscosity.
In vitro antimicrobial activity and growth inhibition of for-
Increasing the concentration of Chx in the gels did not signifi-
mulations against various microorganisms were evaluated
cantly enhance the antimicrobial activity except for the Sig-02
using well diffusion method on their specific agar media
formulation. Chitosan has a synergic effect which offers an ad-
according to the measurement of the inhibition zones. Can-
vantage in delivery of antimicrobial agents at lower concen-
dida albicans (ATCC#90028) and an oral isolate of Candida al-
trations avoiding undesired side effects.
bicans strains were used as fungal pathogens, and standard
strains of Agregatibacter actinomycetemcomitans (A.a) and
Porphyromonas gingivalis (ATCC#33277) and were used as
bacterial pathogens which can be found in oral cavity.

RESULTS AND DISCUSSION

Viscosity measurements
Viscosity was found to increase significantly with the increas-
ing chitosan concentration as well as increasing molecular
weight (Fig. 1). Chitosan gels exhibited pseudoplastic behav-
ior which indicates a gel structure.

In vitro release studies

The release of Chx was found to be higher from the gels
prepared with low molecular weight chitosans. Release was
found to be drug concentration dependent(Fig. 2).

October 26 - 28, 2009 / Antalya - TURKEY

 POSTER PRESENTATIONS
Figure 3: Inhibition zone diameters for gels a) C. albicans, b)
P. gingivalis and c) A.a.
108
CONCLUSION
Chitosan gels can be suggested as promising systems not
only for delivery but also for treatment of the oral mucosal
infections due to its antibacterial/ antifungal activity and
also due to its bioadhesive properties which would provide
occlusion on the applied site.
REFERENCES
1. İkinci G, Şenel S, Akıncıbay H, Kas S, Ercis S, Wilson CG,
Hıncal AA. Effect of chitosan on a periodontal pathogen P.
gingivalis, Int J. Pharm. 2002; 235: 121– 127.
p034-The degradation of artesunate in
aqueous solutions
Muder Al Haydar1, Bruce Sunderland2
Pharmacy School , Curtin Unniversity, WA Australia1
Pharmacy School, Curtin University, WA Australia2
INTRODUCTION
Artesunate, dihydroarteminsinin -10α – hemisuccinate, is a
semi-synthetic derivative of artemisinin which is used for the
treatment of both uncomplicated and severe malaria1. It is for-
mulated for oral, parenteral (intramuscular and intravenous)
and rectal administration and used clinically worldwide2. Al-
though it is frequently employed as a water soluble drug, it is
not sufficiently stable to allow for long term storage in solution.
3rd BBBB International Conference on Pharmaceutical Sciences
The main factor, which contributes in its instability, is hydrolysis
of the ester linkage3. This study demonstrates the rate degra-
dation of artesunate in different buffer solutions at a range of
pH values (2-10.5) and with different IV fluids. The temperature
dependency was also investigated at 5, 25 and 37 °C.
MATERIALS AND METHODS
Materials
Artesunate powder was provided by Apin chemical LTD
Company. All other solvents used were HPLC grade and wa-
ter was purified through a Milli-Q apparatus
Methods
The high performance liquid chromatography instrument
consisted of a high pressure pump (Waters 501, HPLC pump,
Millipore USA), a Apollo C18 (5 µm) column of 150 mm length
and ID of 4.6 mm, a Rheodyne Model 7125 syringe loading
sample injector with 20 µL sample loop, a ultraviolet detector
(Waters 484, Tunable Absorbance Detector, Millipore, USA).
Mobile phase used was phosphate buffer- acetonitrile (70:30)
50 mM at pH 7. The monitoring wavelength was 210 nm.
Artesunate powder was dissolved in buffer solutions sepa-
rately to make a concentration of 0.6 mg ml-1. Buffer solu-
tions (phosphate at pH 2.00, 2.50, 5.50, 6.50 and 7.50, citrate
at pH 3.50, acetate at pH 4.00 and 5.00, Bis Tris at pH 8.50 and
carbonate at pH 9.50 and 10.50) were employed to evalu-
ate the degradation of artesunate. These buffer solutions of
varying concentrations were prepared to evaluate the rate of
degradation of artesunate at a constant pH, temperature (37
°C), and ionic strength of 0.5 mol.L-1 (NaCl). Concentrations
of residual drug were determined by HPLC. Degradation
rate of artesunate in 0.6 mg mL-1 in Hartman, glucose and
normal saline fluids was investigated at 37 °C. Temperature
dependence was investigated by preparing a concentration
of 0.6 mg mL-1 artesunate in buffer solutions of pH 1.20, 6.00
and 10.50. Each selected pH of artesunate was separately in-
vestigated at three temperatures (278, 295 and 303K).
A plot of log percent drug concentration versus time was
plotted to determine the rate of degradation and to find the
rate constant of each reaction. Rate constant were reproduc-
ible to within ± 4%
RESULTS AND DISCUSSION
A plot of log percent remaining concentration of artesunate
in different type of buffer solution versus time showed a lin-
ear relationship over than ≥ 2 t1/2 of reaction which indi-
cated first order of degradation of artesunate.
The catalytic effect of the buffer systems used in the kinetic stud-
ies was determined by experiments at constant pH, temperature,
ionic strength and drug concentration, the only variation being
was in the overall buffer concentration at a given pH. This was ex-
amined at 0.6 mg mL-1 of artesunate, 37˚C, µ of 0.5 mol L-1 and
at selected pH values. The plots of log residual artesunate con-
centrations versus time indicated linear relationship which also
indicated a first-order rate for artesunate degradation.The rate
constant values did not show significantly increased catalysis
with increased of buffer concentration as shown in Table (1). This
indicates that the buffer system does not have a marked catalytic
effect on the degradation rate of artesunate.
 POSTER PRESENTATIONS

Table 1: The observed rate values of artesunate in different

pH values.
Rate constant (hr-1) at the following buffer
concentration
pH 0.1 mol L-1 0.15 mol L-1 0.2 mol L-1
2 1.57 1.6 1.64
2.5 0.297 0.331 0.337
3.5 0.226 0.243 0.268
4 0.15 0.162 0.165
5 0.086 0.091 0.094
5.5 0.083 0.882 0.893
6.5 0.083 0.088 0.089
7.5 0.06 0.073 0.074 Figure 2: log k’- pH profile of artesunate degradation in aque-
8.5 0.07 0.071 0.072 ous solution at 37˚C and µ of 0.5 mol L-1
9.5 0.138 0.179 0.19
10.5 0.34 0.34 0.345 CONCLUSION
The degradation rate of artesunate in the buffer systems ki-
The rate constant values at zero buffer concentration, k΄, were ob- netically obeyed a pseudo-first order reaction and with gen-
tained from the plots of pseudo first-order rate constants versus eral acid-base catalysis, however; it was not a major factor in
the total buffer concentration Figure (1). At zero buffer concentra- artesunate degradation. Maximum stability of artesunate in
tion only specific acid-base catalysis (H+ / OH¯) or a spontaneous aqueous solution was between pH 5.50 and 7.50. Stability
water reaction occurs. The logarithms of these rate constants are was markedly temperature dependent.
plotted versus pH in the pH range of 2.00 – 10.50. The pH-rate
profile Figure (2) has a U-shape which is characteristic of reaction ACKNOWLEDGMENT
susceptible to specific acid-base catalysis. The pH of maximum I would sincerely thank Prof. Bruce Sunderland for his contin-
stability for artesunate in buffer free conditions is over the pH ual support and encouragement in his roles as supervisor.
range of 5.50 to 7.50. Hence below pH 5.50 the rate of reaction
increases with decrease in pH whereas above 7.50 the rate of re- REFERENCES
action again increases with increased pH. There were three im- 1. Lalloo DG, Shingadia D, Pasvol G, Chiodini PL, Whitty
portant pH regions. One where the acid catalysed reaction domi- CJ, Beeching NJ, et al. UK malaria treatment guidelines. 109
nated, which was below pH 5.50, the pH-independent region Journal of Infection. 2007; 54(2):111-121.
over the pH range of 5.50 to 7.50 and the third one where the 2. Dondorp AM, Day NPJ. The treatment of severe malaria.
hydroxide ion catalysed reaction took place, which was above pH Transactions of the Royal Society of Tropical Medicine
7.50. The shelf lives of artesunate at pH 5.5, 6.50 and 7.50 and 37 and Hygiene. 2007; 101(7):633-634.
°C were estimated to be 1.3, 2.2 and 1.5 hr respectively. 3. Batty KT, Ilett KF, Davis T, Davis ME. Chemical stability
of artesunate injection and proposal for its administra-
The temperature dependent studies on artesunate indicated tion by intravenous infusion. J Pharm Pharmacol. 1996;
that the degradation of artesunate was increased with in- 48(1):22-6.
creased temperature. The Arrhenius plots were obtained and
the apparent activation energy values were calculated at pH p035-VALIDATION OF AN HPLC METHOD
1.2, 6.5 and 10.5 to be 100.38, 100.96 and 95 kJ mol-1 respec-
FOR THE DETERMINATION OF NAPROXEN
tively. These indicate a significant temperature dependence.
SODIUM IN POLY(LACTIDE-CO-GLYCOLIDE)
CORNEAL SCAFFOLDS

Seren Kayıran1, Sibel Bozdağ Pehlivan1, Mustafa Çelebier2,

Nurşen Ünlü1

Hacettepe University Faculty of Pharmacy Department of

Pharmaceutical Technology1
Hacettepe University Faculty of Pharmacy Department of
Analytical Chemistry2

Figure 1: Catalytic effect of the buffer solutions on 0.6mg
mL-1 artesunate at 37 °C and µ of 0.5 mol L-1
INTRODUCTION
The aim of this study was to develop a new specific HPLC
method for the determination of released naproxen sodium
(NS) from poly-(lactide-co-glycolide) (PLGA) (50:50) scaffolds
and to assess the related validation parameters for the ana-
lytical method. We intend to formulate PLGA (50:50) scaffolds

October 26 - 28, 2009 / Antalya - TURKEY

 POSTER PRESENTATIONS
containing corneal epithelial cells and NS to treat severe cor-
neal injuries by the replacement of epithelial cells with NS
into the damaged, inflammatory area1. The total amount of
NS in scaffolds and the released amount of NS from the for-
mulations will be determined by developed HPLC method.
MATERIALS AND METHODS
Materials
The HPLC system consisted of Waters 2690 Separations
Module equipped with a Waters 2695 Fluorescence Detec-
tor (Waters, USA) was used for the validation of the method.
NS was supplied from Abdi İbrahim (Turkey). PLGA (50:50)
was commercially available as Resomer RG 503 and was pur-
chased from Boehringer-Ingelheim (Germany). HPLC grade
methanol and acetic acid were purchased from Sigma Al-
drich (Germany). High purity water was prepared by using
Millipore Milli-Q plus water purification system.
HPLC Method
An HPLC method with fluorescence detection was selected
for the method of analyses. The reversed-phase procedure
utilized a Waters Spherisorb S10 ODS2 column (C18; 200 x
4.6mm). The mobile phase comprised of methanol and ac-
etate buffer (pH 5.1) (55:45 v/v). Analyses were run at a flow-
rate of 1 mL.min-1 at an ambient temperature. The fluoro-
metric detector was set at 254 nm ex/ 352 nm em. At this
wavelength, peak areas were measured and used for the
quantitative evaluations.
Standard solution of NS was prepared in simulated lacrimal
110 fluid (SLF). SLF was selected as the solvent because of its
property as a release medium for ophthalmic formulations.
Analytical Method Validation
Specificity
Specificity was examined by analyzing release medium and
the blank PLGA (50:50) scaffold in the release medium.
Linearity Range
The linearity of an analytical procedure is its ability to ob-
tain test results which are directly proportional to the con-
centration of analyte in the sample2. Seven concentrations
between 0.08-75 μg.mL-1 of NS solutions were prepared to
show the linearity of the method.
Sensitivity
The sensitivity of the analytical method was evaluated by de-
termining limits of detection (LOD) and quantitation (LOQ).
The signal-to-noise ratios of 3:1 and 10:1 were taken as LOD
and LOQ, respectively2.
Precision and Accuracy
Three different concentrations of standard NS were analyzed
three consecutive days (inter-day precision) and within the
same day (intra-day precision). The relative standard devia-
tion (RSD) an the Bias of intra- and inter-day studies were
calculated.
3rd BBBB International Conference on Pharmaceutical Sciences
Stability
The standard solutions of NS were stored at +4 °C for 24 hour
and protected from daylight. During this period, it was ana-
lyzed periodically at 6, 12, 24 h.
RESULTS AND DISCUSSION
Specificity
The chromatogram obtained from NS containing PLGA(50:50)
scaffold was identical with that obtained from the blank scaf-
fold containing PLGA(50:50). The representative chromato-
grams (Figure 1) show no other peaks on the retention time
of NS. Accordingly, the proposed method can be considered
selective.
Linearity Range
The peak areas of NS were plotted against the correspond-
ing nominal concentration to obtain the calibration graph.
Thus, the method was evaluated linear in the range of 0.08
to 75 μg.mL-1 for NS.
The r2 for the regression line is 0.9996 with a slope of
24239118.360 and a y-intercept of 230293899.733. These re-
sults were considered acceptable.
Sensitivity
The values of LOD and LOQ for NS were 0.03 μg.mL-1 and
0.08 μg.mL-1.
Precision and Accuracy
The relative standard deviation (RSD) and the Bias of intra-
and inter-day studies were within acceptable range indi-
cating that the precision and the accuracy of the method
were satisfactory. The intra- and inter-day precision relative
standard deviation was 3.71 % or less, and the accuracy was
within 2.96 % deviation of the nominal concentration.
Stability
During 24 h, no unexpected peak appeared and the peak
areas did not changed significantly (p > 0.05) for the com-
pound when it was compared with freshly prepared one.
Figure 1: The chromatogram obtained in optimum condi-
tions. a: blank solution; b: PLGA(50:50) standart solution (1
μg.mL1); c: NS standard solution
 POSTER PRESENTATIONS

HPMC were evaluated for gelling capacity in order to identify

the compositions suitable for use as in-situ gelling systems. In-
situ gel formulations were prepared in a citrophosphate buffer
(pH 6). Buffer salts were dissolved in purified water, HPMC was
added and allowed to hydrate. Carbopol was sprinkled over
this solution and allowed to hydrate. CPH or inclusion com-
plex was dissolved in NaOH solution and benzalkonium chlo-
ride was added. The drug solution was added to the carbopol/
HPMC solution under constant stirring. Then the volume was
made up to 100 ml with purified water (Table 1).

Figure 2: The regression line for NS (0.08-75 μg.mL-1). Table 1: Ingredients of the developed formulations
Code
C1 C2 C3 C4 C5 C6 C7 C8 C9 C10
CONCLUSION w/v %
In this study, a simple, very specific, and reliable HPLC meth- CPH 0.35 0.35 0.35 0.35 0.35 0.35 0.35 - - -
od using fluorometric detection has been developed and Inclusion
- - - - - - - 1.675 1.675 1.675
validated for the assay of NS from PLGA (50:50) scaffolds. complex
C940 0.5 0.5 0.3 0.5 - - - - - -
REFERENCES C934 - - - - 0.5 0.3 0.5 0.5 0.3 0.5
1. Nishida K. Tissue engineering of the cornea, Cornea HPMC 1.5 1.5 1.5 1 1.5 1.5 1 1.5 1.5 1
2003; 22(7):28-34. Tween 20 - 1 - - - - - - - -
2. ICH Guideline for Industry, Text on Validation of Analyti-
Benzalkonium
cal Procedures Q2A ,1995. 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02 0.02
Chloride

Drug Content Uniformity and pH

P036-PH INDUCED IN SITU OPHTHALMIC
GELLING SYSTEM FOR CIPROFLOXACIN /
HP-Β-CD COMPLEX
Berrin Başaran1, Asuman Bozkır1
Ankara University Faculty of Pharmacy Department of
Pharmaceutical Technology, Ankara, Turkey1
INTRODUCTION
In this study, a pH induced in-situ gel for Ciprofloxacin HCl (CPH)
was developed and different carbopol types were studied as
the gelling agent in combination with hydroxypropylmethyl-
cellulose (HPMC) which acted as a viscosity enhancing agent.
CPH was complexed with hydroxypropyl-beta-cyclodextrin
(HP-β-CD) and this complex was also used in the formulations.
Viscosities of formulations were measured. In vitro release and
antimicrobial efficacy of selected formulations were evaluated.
MATERIALS AND METHODS
Materials
Ciprofloxacin HCl (Bayer, Germany), Carbopol 934
(C934),Carbopol 940 (C940) (BF Goodrich,USA), Carbopol
980 (C980) (Noveon,USA), HPMC (Fluka,Switzerland) All oth-
er reagents were of analytical grade.
The CPH concentration of formulations was determined at
271 nm. pH of the formulations were measured with SenTix
82 pH electrode.
Viscosity Measurements
111
Viscosity of formulations were measured with a Brookfield
RVTDV-II viscometer at 25°C, pH 6 and at 34°C, pH 7,4.
In Vitro Release Studies
The in vitro release of CPH from the formulations was stud-
ied using a dialysis membrane in a circulating water bath at
80 rpm and 34±1°C. The diffusion medium was 50 ml of pH
7,4 phosphate buffer.
Antimicrobial Efficacy Studies
Antimicrobial efficacy of formulations were determined by
agar diffusion test employing the cup plate technique2.
RESULTS AND DISCUSSION
Gelling Capacity
C980 containing formulations showed the weakest gelation.
Formulations prepared with 0,5% C934 or C940 with 1 or
1,5% HPMC and 0,3% C934 or C940 with 1,5% HPMC were
shown to have the best gelling capacities.

Preparation and Characterization of CPH: HP-β-CD Inclusion Drug Content and pH

Complex
The inclusion complex of CPH with HP-β-CD in a 1:1 molar
ratio was prepared by freeze-drying technique. Formation of
the complex was confirmed using FTIR Spectrophotometry,
DSC analysis and 1H NMR spectroscopy (Data not shown)1.
Preparation of In Situ Gelling System
Solutions of varying concentrations of carbopol types and
The drug content and pH of the formulations were satisfac-
tory. Standard deviations were found to be below 2 %.
Rheological Studies
Decrease in viscosity was observed with increase in angular
velocity (pseudoplastic rheology)3. C934 containing formula-
tions exhibited lower viscosity than formulations containing
C940 at 25°C and pH 6. C7 and C10 formulations containing

October 26 - 28, 2009 / Antalya - TURKEY

 POSTER PRESENTATIONS

0,5% C934 and 1% HPMC exhibited lower viscosity values.
After gelation of C7 and C10 formulations, viscosity values
increased 3 fold and 7 fold, respectively (Table 2). So, C7 and
C10 formulations were selected in terms of both instilling
convenience and viscosity increase after gelation.
Antimicrobial Efficacy Studies
The zone of inhibition values against P. aeruginosa were
higher than that against S. aureus (Table 3). This study indi-
cates that CPH retained its antimicrobial efficacy when incor-
porated in an in situ gel system.

Table 2: Viscosity of formulations (10 rpm) (n=3) Table 3. Antimicrobial efficacy of formulations
Viscosity (cp) Zone of inhibition (mm) (% efficiency)
Formulations Viscosity (cp) (250C)
(340C, pH 7.4) Concentration
C1 14333 ± 1443 13000 ± 0 Std C7 C10
(μg/ml)
C3 11000 ± 2500 13500 ± 1250
100 42 28 (66.67) 28 (66.67)
C4 7333 ± 1443 15000 ± 2500 S.aureus
500 45 32 (71.11) 33 (73.33)
C5 10000 ± 0 13333 ± 1443
C6 9500 ± 1250 11670 ± 1443 P.aeruginosa 500 37 33 (89.19) 35 (94.60)
C7 3500 ± 1250 9670 ± 1443
C8 9500 ± 1250 21000 ± 2500 CONCLUSION
C9 8500 ±1250 11333 ± 1443 C7 and C10 formulations are an alternative to eye drops
because of instillation easiness and decreased frequency
C10 3000 ± 0 20333 ± 1443
of administration for better patient acceptance. Inclusion
complex was used for increasing the stability of CPH. These
In Vitro Release Studies
formulations seem promising opthalmic delivery system for
Increasing carbopol concentration decreased the drug re-
CPH.
lease for C5, C6, C7 formulations. While carbopol concentra-
tion was pegged, increase in HPMC concentration decreased
REFERENCES
the drug release. The most slowly drug release was observed
1. Basaran B, Bozkır A. Thermosensitive, prolonged release
with C5 and C8 formulations, but these formulations were
ophthalmic gel for ciprofloxacin/HP-β-CD complex. 2nd
found to have higher viscosity before gelation and instilla-
PharmSciFair, Nice-France, 2009.
tion wasn’t easy. C7 and C10 formulations showed the slower
2. Jain SP, Shah SP, Rajadhyaksha NS. In situ ophthalmic gel
drug release than C6 and C9 formulations and their viscosity
of ciprofloxacin hydrochloride for once a day sustained
values were convenience for instillation, so C7 and C10 for-
delivery. Drug Dev. Ind. Pharm. 2008; 34: 445-452.
112 mulations were selected (Figures 1 and 2).
3. Basaran B, Bozkır A. Preparation and evaluation of chito-
san-based in-situ gelling ophthalmic formulations con-
taining ciprofloxacin. Advances in Chitin Science 2007;
X: 343-348.

P037-SURFACE ANALYSIS AND STRUCTURE

CHARACTERIZATION OF A DRY POWDER
CONTAINING CHITOSAN-HYALURONIC ACID
NANOPARTICLES

S. Al-Qadi1, C. Remuñán-López2

Figure 1: In vitro release of C5, C6 and C7 formulations Department of Pharmaceutical technology, Faculty of
Pharmacy, University Santiago de Compostela,Spain1
Department of Pharmaceutical technology, Faculty of
Pharmacy, University Santiago de Compostela,Spain2

INTRODUCTION
Recently, we proposed the micro-encapsulation of nano-
particles in dry powders for pulmonary administration of
therapeutic macromolecules such as proteins and DNA1. It is
known that particle surface chemistry interfaces with biolog-
ical environments, and this governs its biological behaviour2.
The aim of this study was to characterize the structure and
surface composition of chitosan/hyaluronic acid/ tripoly-
phosphate nanoparticles (CS NPs) encapsulated in mannitol
Figure 2: In vitro release of C8, C9 and C10 formulations
microspheres (M-NPs). Confocal laser scanning microscopy
(CLSM) was used to investigate the spatial distribution of NPs

3rd BBBB International Conference on Pharmaceutical Sciences

 POSTER PRESENTATIONS

in the microcarrier. Besides, the surface-sensitive analytic

techniques X-ray spectroscopy (XPS) and time of flight sec-
ondary ion mass spectrometry (TOF-SIMS) were employed
for further characterization.

MATERIALS AND METHODS

Materials
Ultrapure chitosan (CS) HCl salt (Protasan UP CL 113) was
purchased from FMC Biopolymers [Norway]; hyaluronic acid
was provided by Bioiberica; fluorescein sodium salt, penta-
sodium tripolyphosphate (TPP) and D-mannitol were ob-
tained from Sigma-Aldrich [Spain]; Bodipy® 630/650-X was
provided by Molecular Probes [Netherlands].
Figure1b: SEM micrograph of M-NPs
Preparation and characterization of microencapsulated
The CLSM micrographs depicted in Fig. 2 show the labelled
nanoparticles
mannitol forming a continuous matrix in which NPs are ho-
CS was labelled by fluorescein (FITC) following the method
mogeneously distributed.
described by De Campos et al3. Morphology visualization
of NPs was conducted by transmission electron microscopy
(TEM) [CM 12 Philips, Eindhoven, Netherlands]. To prepare
the NPs-loaded M microspheres (M-NPs), mannitol was la-
belled with the flourophore Bodipy® and then co-spray dried
with the NPs suspension using a laboratory-scale spray-dryer
[Büchi mini spray dryer, B-290, Switzerland].The resultant mi-
crospheres were observed using a scanning electron micro-
scope [SEM, Leo 435VP, U. K.] and their particle size charac-
terized with an Aerosizer®-Aerodisperser® [Amherst Process
Instruments, Inc., Amherst, MA, USA].

Structural Characterization using CLSM

Fig.2. CLSM images of microspheres: (a) mannitol stained by
The internal structure of the microspheres was observed by 113
CLSM, using a TCS-SP2 vertical microscope [Leica GmbH, Ger-
many]. Laser excitation λ of 488 nm and 633 nm were used to
scan the powder; and fluorescent emissions from FITC (emis-
sion λ) (492-550 nm) and Bodipy® (emission λ) (650-725 nm)
were collected using separate channels.
Analysis Using XPS and TOF-SIMS
Surface analysis of microencapsulated NPs was performed
using XPS [VG Escalab 250 iXL ESCA, VG Scientific, U. K.] and
TOF-SIMS [TOF-SIMS IV, Ion-TOF GmbH, Germany]. Mannitol
microspheres and isolated NPs were used as controls.

RESULTS AND DISCUSSION (b) NPs labelled by FITC (green channel)

NPs displayed a regular shape and M-NPs showed a spherical
morphology (Fig. 1).

and (c) overlapping of both channelsBodipy® (red channels)

Analysis using XPS and TOF-SIMS

As shown in Table 1, the ratio C/O and the absence of the
Figure 1a: TEM micrograph of CS/HA/TPP NPs signal of P and Na in the case of mannitol and M-NPs indicate
that NPs are embedded inside the mannitol microspheres.

October 26 - 28, 2009 / Antalya - TURKEY

 POSTER PRESENTATIONS

Table 1: Surface composition (atomic percentage) following P038-SURFACE-MODIFIED PLGA

XPS analysis of CS/HA/TPP NPs, mannitol microspheres (M), MICROPARTICLES AS PULMONARY DELIVERY
and microencapsulated nanoparticles (M-NPs). C: carbon,O:
oxygen,N:nitrogen,P:phosphor,Na:sodium.
VEHICLES FOR RHIL-2

Element CS/HA/TPP NPs M M-NPs Burcu Devrim1, Asuman Bozkır1, Kandemir Canefe1
C 51,7 50.6 43.3
Department of Pharmaceutical Technology, Faculty of
O 37.1 43.0 37.0
Pharmacy, Ankara University, 06100- Tandoğan, Ankara/
N 3.4 0.5 1.0 Turkey1
P 0.6 0 0
Na 1.7 0 0 INTRODUCTION
C/O 1.39 1.176 1.170 Interleukin-2 (IL-2), a lymphokine produced by activated T
cells, has a wide variety of actions and plays a central role
The mass spectra obtained by TOF-SIMS proves that the char- in immune regulation. Because of this biological function,
acteristic mass of mannitol m/z 183 (molecular ion + H+) is recombinant human IL-2 (rhIL-2) has been tested in clinical
present in the case of mannitol and M-NPs and absent in the trials for treatment of tumors1. Since the protein has a short
case of the isolated NPs (Fig.3). Also, the mass of phosphate half-life, frequent administration of rhIL-2 is required to ob-
fragment (PO2, PO3), attributed to TPP, appears in the case tain a therapeutic effect, which can result in severe toxic side
of NPs but not in the spectrum of mannitol microspheres (M) effects. Controlled-release technology lends itself naturally
and M-NPs, suggesting that the NPs are totally covered with to this type of medical problem. Systems can be designed
mannitol (data not shown). that provide release of molecules over days to weeks, in such
a manner that a systemic effect is achieved without toxic-
ity. In addition to this, modifying of microparticles with mu-
coadhesive polymers may help to increase the residence
time and increase efficacy when administered by the nasal
or pulmonary routes2.

In this study, surface-modified PLGA microparticles was

prepared by using w/o/w double emulsion solvent extrac-
114 tion method. And the physicochemical properties of rhIL-2
loaded PLGA microparticles was determined. Chitosan (CS)
chloride and Carbopol 971P was used as surface modifying
agents.

MATERIALS AND METHODS

Figure 3: TOF-SIMS mass spectrum of NPs, mannitol and M-
NPs.
CONCLUSION
CLSM, XPS and TOF-SIMS analysis prove that NPs are efficiently
encapsulated and homogeneously distributed within M-NPs.
Materials
Microparticle formulations containing rhIL-2 were prepared
by PLGA with a lactide/glycolide ratio of 50:50 (Resomer RG
502) (Bohringer Ilgelheim, Germany). CS chloride (Protosan
UP CL 113) was obtained from Novomatrix (ABD). Carbopol
971P was obtained from Noveon (ABD). rhIL-2 was a gift from
Novartis (Novartis, Turkey) and all the other chemicals used
were analytical grade.

ACKNOWLEDGEMENTS Preparation of Surface-Modified PLGA Microparticles

MAEC-AECID fellowship of the Spanish Agency to S. Al-Qadi
and the Spanish Government (CICYT, SAF2002-03314) are
highly appreciated. C. Serra (CACTI, Vigo) is acknowledged
for her assistance.
REFERENCES
1. Grenha A, Seijo B, Remuñán-López C. Micro-encapsulat-
ed chitosan nanoparticles for lung protein delivery, Eur.
J. Pharm. Sci. 2005; 25:427-437.
2. Mc Arthur S. Applications of XPS in bioengineering, Surf.
Interface Anal. 2006; 38: 1380-1385.
3. De Campos A M, Diebold Y, Carvalho ELS et al. Chitosan
nanoparticles as new ocular drug delivery systems: in
vitro stability, in vivo fate, and cellular toxicity, Pharm.
Res. 2004; 21: 803–810.
PLGA microparticles containing rhIL-2 were prepared by a
w/o/w double emulsion solvent extraction method3. In brief,
aqueous solution of rhIL-2 was poured into a PLGA solution
in dicholoromethane. The primary emulsion was generated
by a high-speed homogenizer (Ultraturrax T-25 Homogeniz-
er, Germany). This water-in-oil emulsion, was added to exter-
nal aqueous solution of polyvinyl alcohol (PVA) and stirred
to produce the secondary emulsion. To modify the micropar-
ticle surfaces, 0.5% (w/v) CS chloride or Carbopol 971P was
added to PVA solution. The w/o/w emulsion was poured into
additional aqueous PVA solution and stirred with a mechani-
cal stirrer for 3 h. Finally, the microparticles were collected by
centrifugation, washed with bidistilled water and lyophilized
to obtain a dry powder.

3rd BBBB International Conference on Pharmaceutical Sciences

 POSTER PRESENTATIONS
Characterization of Microparticles
To determining morphology of the microparticles, samples
were coated with a thin layer of gold and viewed using a
JEOL-6400 scanning electron microscopy(SEM)(JEOL, Ja-
pan).
The particle size distributions of the microparticles were deter-
mined by laser diffraction (Sympatec laser diffraction particle
sizer, Germany) after suspending the microparticles in water.
Three determinations were carried out for each formulation.
Estimation of rhIL-2 Content in PLGA Microparticles
The encapsulation efficiency for biodegradable PLGA micropar-
ticles containing rhIL-2 was determined by dissolving micropar-
ticles in 2% (w/v) SDS in 0.1 M sodium hydroxide solution. After
the solution was neutralized with 0.1 M HCl, the amount of rhIL-2
was determined by micro bicinchoninic acid (microBCA) assay.
Placebo microparticles (without protein) were used as control.
Measurement of Zeta Potantial
Surface charge of freeze-dried microparticles was deter-
mined by zeta potential measurement on a Malvern Zeta-
sizer (Malvern, UK).
In vitro Release of rhIL-2 from Microparticles
The release of rhIL-2 from the PLGA microparticles was de-
termined by suspending microparticles in pH 7.2 phosphate
buffer (PB) containing 0.1% SDS and 0.02% sodium azide.
The samples were incubated at 37 °C in a shaking water bath.
A vial was withdrawn at each time-point, the contents of vial
were centrifuged and the supernatant containing released
rhIL-2 was collected. After each sample collection, the sam-
ple PB was replaced with fresh buffer. The measurement of
protein concentration was performed by microBCA.
RESULTS AND DISCUSSION
The surface-modified PLGA microparticles prepared by dou-
ble emulsion method were spherical in shape with smooth
surface (Figure 1).
Figure 1: SEM micrographs of PLGA microparticles. a)Unmod-
ified microparticles (F1) b)chitoasan hydrochloride coated
microparticles (F2) c)Carbopol 971
The particle size of microparticles was affected on surface
October 26 - 28, 2009 / Antalya - TURKEY
modification with mucoadhesive polymers. CS chloride
coating led to an increase in the particle size of micropar-
ticles. In contrast to the CS chloride, surface modification
with Carbopol 971P was resulted decrease in particle size of
microparticles (Table 1).
Table 1: Characterization of unmodified and surface modi-
fied microparticles
Zeta
Formulation Coating Particle size E.E
potantial
Code material (μm) ± S.D. (%) ± S.D.
(mV) ± SD.
F1 - 4.03 ± 0.0058 99.22 ± 1.20 -8.31 ± 1.24
F2 Chitosan HCl 5.68 ± 0.026 97.57 ± 2.36 +19.30 ± 0.30
Carbopol
F3 1.37 ± 0.010 98.12 ± 1.53 -28.60 ± 0.44
971P
The rhIL-2 encapsulation efficiency of PLGA microparticles
was found in range of 97.54-99.20% determined by microB-
CA assay.
The modification of the surfaces of PLGA microparticles with
CS chloride or Carbopol 971P was confirmed by resultant
changes in zeta potential. The surface charge of the unmodi-
fied PLGA microparticles was negative, whereas the zeta po-
tantial was recorded to be positive when the microparticles
were modified with CS chloride. Conversely, the zeta poten-
tial of Carbopol 971P coated microparticles became more
negatively charged compared with un-coated microparticles
(Table 1).
115
In vitro release results indicate that coating the micropar-
ticles with mucoadhesive polymers reduced the initial burst
of drug release.
Figure 2: Cumulative release of rhIL-2 from unmodified and
surface modified PLGA microparticles.
CONCLUSION
PLGA microparticles coated with mucoadhesive polymers
were successfully prepared by double emulsion solvent ex-
traction method. The surface coating of microparticles with
polymer was confirmed by the change in zeta potential.
Additionally, surface modification with mucoadhesive poly-
mers reduced the initial burst of drug release.
REFERENCES
1. Skubitz KM, Anderson PM. Inhalational interleukin-2 li-
 POSTER PRESENTATIONS

posomes for pulmonary metastases: a phase I clinical
trial, Anti-Cancer Drugs 2000, 11:555-563.
2. Alpar HO, Somavarapu S, Atuah KN, Bramwell VW. Biode-
gradable mucoadhesive particulates for nasal and pul-
monary antigen and DNA delivery, Adv. Drug Del. Rev.
2005, 57:411-430.
3. Devrim B, Bozkır A, Canefe K. Biodegradable PLGA mi-
croparticles as a delivery system for protein: effect of
molecular weight of PLGA on the microparticle charac-
teristics, Pharm. J. Slovenia 2008; 59:107-108.
p039-FORMULATION AND CHARACTERIZATION
OF SURFACTIN-CONTAINING SELF-
EMULSIFYING DRUG DELIVERY SYSTEMS
F. Hande Kural1, R. Neslihan Gürsoy1
Hacettepe University, Faculty of Pharmacy, Department of
Pharmaceutical Technology, Ankara, Turkey1
INTRODUCTION
Surfactin, one of the most powerful biosurfactants, is a cyclic
lipopeptide that is produced by Bacillus subtilis. It has been
shown to exhibit antiviral, antifungal, antimicrobial and anti-
tumor effects1. In this study, we have designed and character-
ized surfactin-containing self-emulsifying drug delivery sys-
tems (SEDDS) for future therapeutic applications of surfactin.
samples were obtained at a scanning rate of 10 °C/min with
a temperature range of 25–140 °C. Melting point of surfactin
was also determined by a capillary melting point apparatus
(Thomas Hoover, USA).
HPLC Analysis
An HPLC system (Waters) equipped with a photodiode array
detector (DAD) was utilized for the quantitative analysis of
surfactin. The mobile phase A was composed of 0.5% trifluo-
roacetic acid in water; B was 100% acetonitrile at a flow rate
of 0.7 ml/min. The injection volume was 40 μl. A reversed-
phase C18 column (4.6x250 mm, 5μm) kept at 20°C was
used. The detection wavelength was 205 nm.
RESULTS AND DISCUSSION
Preparation of Formulations
Several compositions of oils and surfactants were prepared
using ternary phase diagrams as part of preformulation
studies. Of the excipients tested, the composition of Gelucire
44/14, Labrasol, PEG 3000 and Vitamin E gave the most sta-
ble SEDDS (Figure 1). Stable microemulsions were obtained
as shown in region A. In region B, unstable microemulsions
which became cloudy as a function of time, were obtained.
In region C, coarse emulsions were obtained.

116 MATERIALS AND METHODS

Materials
Surfactin (≥ 98% purity) was purchased from Sigma. Gelucire
44/14 and Labrasol were purchased from Gattefossé. Vitamin
E was a kind gift from Roche Pharmaceuticals (İstanbul, Tur-
key). Poly ethylene glycol 3000 (PEG 3000) was purchased
from Fluka. Nanopure water was used throughout the ex-
periments.

Preparation of Formulations
In the preformulation studies, optimum surfactant, co-sur-
factant and oil combinations were determined with ternary
phase diagrams. After mixing the excipients on a water bath
at 40°C, the formulations were diluted to 10 ml with phos-
phate buffer (pH 7).

Microscopic Examination
The formulations were examined utilizing a polarizing light
microscope (Leica DM EP, Germany) to observe the appear-
ance of possible liquid crystal structures due to the presence
of high amounts of surfactants in the formulations.

Droplet Size and Zeta Potential

Droplet size and zeta potential measurements were carried
out by Malvern Nanosizer ZS 2000 (United Kingdom). Each
sample was analyzed in triplicate.

DSC Analysis
The thermal analyses of the excipients, surfactin and formu-
lations were performed with a differential scanning calorim-
eter (TA Instruments Q100, USA). The thermograms of the

3rd BBBB International Conference on Pharmaceutical Sciences

 POSTER PRESENTATIONS

Figure 1: Ternary phase diagram of SEDDS formulations

The SEDDS formulations in region A exhibited Newtonian
type rhelogical behavior upon aqueous dilution.

Microscopic Examination
Due to the nano size range of the SEDDS formulations, no
image was observed under the polarizing light microscope,
Figure 2: Chromatogram of surfactin solution (100 μg/ml)
as expected. Moreover, no liquid crystal structures were ob-
served, which are known to exhibit characteristic images un-
CONCLUSION
der the polarizing light microscope.
Stable SEDDS formulations were designed and character-
ized, and the bioactive surfactin compound was successfully
Droplet Size and Zeta Potential
incorporated into these formulations. The characterization
A monomodal droplet size distribution with a narrow range
studies of surfactin-containing SEDDS formulations indicat-
was obtained with stable SEDDS formulations. The average
ed that the addition of surfactin caused no changes in the
particle size of the SEDDS formulations were in the 15–21
stability and physicochemical properties of the formulations.
nm range. The average droplet size of the SEDDS formula-
To the best of our knowledge, this is the first study showing
tion containing surfactin was found to be 19.41±0.42 nm.
the successful incorporation of surfactin into a drug delivery
The average zeta potential values were in the range of -9.5–
system such as SEDDS.
5.0 mV. Since only nonionic surfactants were used in these
formulations, zeta potential values close to neutrality were
ACKNOWLEDGEMENT
obtained. The average zeta potential of the surfactin-con-
This project was supported by State Planning Office (DPT) of
taining formulation was -1.53±0.88 mV.
the Republic of Turkey.
DSC Analysis
REFERENCES
One of the most stable formulations from region A was test-
1. Seydlova G, Svobodova J, Review of Surfactin chemical
ed for DSC experiments.
properties and the potential biomedical applications,
Cent Eur J Med, 2008;3(2):123-133.
Table 1: DSC data for the excipients and the SEDDS formula-
2. Oka K, et al., Satisfactory separation and MS-MS spec-
tions (n=3)
trometry of six surfactins isolated from Bacillus subtilis
Component Tm(0C) ∆H (J/g) 117
natto,1993;41(5):1000-1002.
Gelucire 44/14 47.88 ± 2.09 60 ± 7.12 3. Abdel-Mawgoud AM et al., Characterization of surfactin
PEG3000 61.97 ± 0.85 194.68 ± 2.04 produced by Bacillus Subtilis isolate BS5, appl Biochem
SEDDS before dilution 39.48 ± 0.79 12.81 ± 4.31 Biotechnol, 2008;150:289-303.
SEDDS after dilution 70.50 ± 3.51 205.94 ± 149.13t

The melting temperature of surfactin was found to be 131.54 p040-THE EFFECTS OF PULMONARY
°C. This data is in accordance with the results obtained by the ADMINISTRATION OF INTERLEUKIN-2
capillary melting point method (132 °C) and the literature2. LIPOSOMES ON IMMUNE SYSTEM IN RATS
Table 1 illustrates the melting temperature (Tm) and the en-
thalpy of transition (ΔH) for Gelucire 44/14, PEG 3000 and the
Nevin Çelebi1, Ayşe Çilek1, Ayşegül Atak2, Gonca Akbulut3
formulations.

It can be inferred from the DSC data that excipients forming
the SEDDS behave differently before and after dilution. It ap-
pears that following aqueous dilution, the molecules exhib-
ited structural rearrangements so only the Gelucire melting
endotherm appeared before dilution (39.48 °C) and only the
PEG 3000 melting endotherm (70.50 °C) following dilution,
both of them exhibiting a shift from their actual values. This
may be due to the hydrophilicity of PEG 3000 which prob-
ably leads to more interaction with the water molecules. The
melting endotherm of surfactin was not observed in the ther-
mogram of the surfactin-containing SEDDS formulation.
HPLC Analysis
Figure 2 shows that surfactin isoforms eluted in 20 minutes.
The chromatographic profile of surfactin was comparable to
those obtained by other researchers utilizing different meth-
ods3.
Gazi University Faculty of Pharmacy Department of
Pharmaceutical Technology, Ankara, Turkey1
Gazi University Faculty of Medicine Department of
Immunology, Ankara, Turkey2
Gazi University Faculty of Medicine Department of Physiology,
Ankara, Turkey3
INTRODUCTION
Interleukin-2 (IL-2) is a cytokine which is produced by acti-
vated hepler T lymphocytes and is an important mediator of
the immune response. Because of this biological function,
recombinant human IL-2 (rhIL-2) has been tested in clinical
trials for treatment of tumors1. rhIL-2 is a relatively hydro-
phobic protein with limited aqueous solubility.Since it has a
short-half life, frequent administration of rhIL-2 is required to
obtain a therapeutic effect, which to can result in toxic side
effects. In order to overcome these problems, drug delivery

October 26 - 28, 2009 / Antalya - TURKEY

 POSTER PRESENTATIONS

systems (e.g liposomes and polymatrices) have been investi-

gated to increase the therapeutic efficacy and minimize the
side effects of rhIL-22. Recently, pulmonary administration of
IL-2 have been gained importance. In the previous study, we
have reported that the effects of pulmonary administration
of IL-2 liposome formulation on pharmacokinetics param-
eters in rats3. The objective of in this part of our study was to
investigate the effects of pulmonary administration of IL-2
liposomes on immunological parameters in serum and lung
homogenisate and to compare with pulmonary administra-
tion of IL-2 solution and intravenous (i.v) administration.

MATERIALS AND METHODS

Preparation of Liposomes
Liposomes were prepared by using the film method as re-
ported previously3. Liposomes were characterized by par-
ticle size and zeta potantial measurements. Encapsulation
efficiency of IL-2 in the liposomes were determined3.

In vivo Studies
Wistar male rats were used throughout the study. In vivo
experiments were conducted in accordance with the eth-
ics guidelines upon obtaining the approval Gazi University
laboratory animal care committee. The rats were divided into
three major groups including 8 rats each. Pulmonary admin-
istration of IL-2 formulations (liposomes and solution) to rats
were performed using PennCentury device. IL-2 was admin-
istered as 107IU/mL and 2.106 dose levels via pulmonary
and intravenous, respectively. IL-2 containing formulations
was administered at the beginnig and on the seventh day in
118 order to generate secondary immunity. The immunological
parameters (IgA, IgM, IgG, TNFα, IFNγ and subgroups IgG1,
IgG2a) in serum and lung homogenizate were measured by
ELISA kits (Biosource).The same immunolgical parameters
were once again measured on the fourteenth day. On the
other hand, the blood samples were taken from hearts at
the end of the seventh and on the fourteenth days. Then, the
animals were sacrificed. The lung homogenisate was pre-
pared as the following procedures:The lung was removed
and washed with saline solution. Traceal parts of lung have
been cleaned and was divided into small parts. After then, Figure 1: The IgM Levels in Serum
the parts have been homogenized with saline solution and
centrifuged at 4000 rpm for 20 minutes. The supernatant
was separated and the above-mentioned immunological
parameters were measured.

RESULTS AND DISCUSSION

The IgM levels were significantly reduced with pulmonary ad-
ministration of IL-2 liposome on the fourteenth day (p<0.05)
(Figure 1). When IgG levels in lung were evaluated, the best
result was obtained with liposome formulation since sup-
pressed the immune system at most ( Figure 2).

According to the experimental findings, pulmonary IL-2 lipo-

some formulation caused long-time reduction in serum IgM
and IgA levels. It was shown that intravenous IL-2 solution
increased the TNFα and IFNγ levels more than the other ad- Figure 2: The IgM Levels in Lung Homogenisate
ministration methods did. Reduction of IgG, IgG2 and IgG2a
levels by the pulmonary IL-2 liposome was shown on the CONCLUSION
fourteenth day. The results indicate that the prepared IL-2 liposome for pul-
monary administration can be effective on immune system.

3rd BBBB International Conference on Pharmaceutical Sciences

 POSTER PRESENTATIONS

ACKNOWLEDGEMENTS
This study was supported by grants from Turkish Scientific
and Technical Research Council (TUBITAK, 105S084).
REFERENCES
1. Cadee JA, Gnoot CJ,Jiskoot W, et al.Release of recombi-
nant human interleukin-2 from dextran-based hydro-
gels, J Control Rel. 2002; 78:1-13.
2. Kanaoka E,Takahashi K, Yoshikawa T, et al. A significant
enhancement of therapeutic effect against hepatic me-
tastases of M5076 in mice by a liposomal interleukin-2
(mixture),J Control Rel. 2002; 82:183-187.
3. Çelebi N, Çilek A, Evaluation of pharmacokinetics of in-
terleukin- liposomes for pulmonary delivery, 2nd Pharm-
SciFair, June 8-12 2009.
Methods
SLN were produced by modified microemulsion dilution
technique using stearic acid as matrix lipid, Tween 80 as sur-
factant and ethanol as co-surfactant, distilled water as dis-
pertant and either EQ1 (N,N-di-(b-steaorylethyl)-N,N-dime-
thylammonium chloride) or stearylamine as charge carrier.
The lipids were melted with a mixture of water, co-surfactant
and the surfactant at 80ºC. A transparent, thermodynami-
cally stable system was formed when the compounds were
mixed at certain ratios as seen in Table 1. This microemulsion
was then dispersed in a cold aqueous medium (+2ºC ±1) un-
der mild mechanical stirring at a ratio of 1:10 microemulsion/
aqueous medium3.
Table 1: SLN formulations

Distilled water (% w/w)

Stearylamine (% w/w)
Stearic Acid (% w/w)

Esterquat1 (% w/w)

Tween 80 (% w/w)

Ethanol (% w/w)
p041-PREPARATION AND

*Formulations
CHARACTERISATION OF CATIONIC SOLID
LIPID NANOPARTICLES AS A POTENTIAL DNA
DELIVERY SYSTEM
Gülten Kantarcı1, Devrim Demir Dora1, Hasan Akbaba1
1 2 2.5 2.5 15 12 66
2 2 2 2 15 12 67
Ege University Faculty Of Pharmacy- Pharmaceutical
3 2 2 2 15 12 68
Biotechnology Department, İzmir, Turkey 1
4 2 1 1 15 11 70
INTRODUCTION 5 2 3 3 15 12 65
In recent years, solid lipid nanoparticles (SLN) have been *In all formulations, 1ml microemulsion was dispersed in 10
developed as potential carriers for a number of drugs. SLN ml of dispertion medium. 119
usually consists of physiologically well-tolerated ingredients
already approved for pharmaceutical applications in hu-
mans. SLN can readily be produced in large scale, have good Droplet Size and Zeta Potential Determinations
storage capabilities including freeze-drying, can be sterilised The particle size distribution and average droplet size of
and show low cytotoxicity, when injected intravenously. In microemulsions were determined using Zetasizer Nano ZS
addition, the advantage of SLN is that the charge of the par- (Malvern Instruments Ltd., UK).
ticles can be modulated via the composition, thus allowing
binding of oppositely charged molecules via electrostatic in- pH Measurements
teractions1. SLN can be produced in nano-scale size (10–200 The observed pH values of the samples were measured by
nm), wherefore the particles are sufficiently small to traverse pH meter (Inolab Level 1, WTW Wissenschaftlich-Techniche
the microvascular system and prevent macrophage uptake Werkstätten GmbH, Weilheim, Germany), at 20±2ºC.
and are therefore particularly suitable for systemic delivery2.
Conductivity Measurements
The purpose of this study is to develop a novel cationic solid The electrical conductivity was measured using a conducto-
lipid nanoparticle formulations for targeting plasmid DNA meter (Jenway 4071, UK), at 20±2ºC.
for non-viral gene therapy.
Stability of SLN Formulations
MATERIALS AND METHODS The particle size and the polydispersity index of cationic SLN
Materials formulations were measured after two weeks storage in dis-
Tween 80....................... Merck-Co. (Hohenbrunn, Germany), persion medium at 4-8 ºC.
Stearic Acid................... Merck-Co. (Hohenbrunn, Germany),
Ethanol........................... Merck-Co. (Hohenbrunn, Germany), Plazmid DNA Purification
Stearylamine................ Sigma-Aldrich Co. (St. Louis, MO, USA). The Green Fluorescent Protein (GFP) expression plasmid
Esterquat 1(EQ1.......... Gerbu Biochemicals GmbH (Gaiberg, (pcDNA3.1/NT-GFP, 6160 bp) purchased from Invitrogen
Germany) (USA) was transformed into Escherichia coli JM109 cells. Plas-
Distilled Water............. pH 7.4 (adjusted with sodium hydrox- mid DNA purification was done by MiniPrep Plasmid DNA
ide) Isolation Kit (Fermentas, Germany), from the overnight liquid
50 µS (adjusted with sodium chloride) culture of transformed E. coli cells in Luria-Bertani medium
at an Ampicillin concentration of 80 µg/mL. The DNA con-
centration was determined by using Qubit fluorometer with

October 26 - 28, 2009 / Antalya - TURKEY

 POSTER PRESENTATIONS
Quant-iT™ ds DNA BR Assay Kit (Invitrogen). The purity of the
isolated plasmid DNA was checked by 0.8 % (w/v) agarose
gel electrophoresis and contamination with RNA or genomic
DNA was not detected.
Preparation of SLN-pDNA Complexes
SLN-pDNA complexes were prepared by incubating 0.1 µg/
mL pDNA solution with a cationic SLN suspension for 30 min-
utes at 25ºC, at a DNA/EQ1 ratio of 1:0.5, 1:1, 1:2, 1:4, 1:8, 1:12,
1:16 and 1:20 w/w. The complex formation was then checked
by agarose gel electrophoresis (Figure 1) (4).
Agarose Gel Electrophoresis
SLN-pDNA complexes were diluted in Ultra-Pure distilled wa-
ter to a final concentration of 0.1 µg DNA/µL and subjected
to electrophoresis on an 0.8 % (w/v) agarose gel (0.5 % (w/w)
ethidium bromide included for visualization) for 40 min at
120 V. The bands were visualized by a UV transilluminator
(Vilber-Lourmat) and images were captured using a digital
camera.
RESULTS AND DISCUSSION
Table 2 summarizes particle size, zeta potential and polydis-
persity index of SLN composed by different amounts of EQ1
and Stearylamine. Formulation 2 was selected for further
studies because of its appropriate zeta potential (41,3±3,11
mV) for DNA binding.
Table 2: Particle Size, PDI and Zeta Potential of the formula-
tions
120 Particle Size Zeta Potential
Formulations PDI
(nm) (mV)
1 34,93 (±0,6431 0,235 (±0,006) 51,7 (±0,681)
2 33,14 (±0,4954) 0,237 (±0,007) 41,3 (±3,11
3 30,85 (±0,3848) 0,285 (±0,006) 30,9 (±3,88)
4 42,83 (±0,5327) 0,271 (±0,001) 29,7 (±2,44)
5 35,18 (±0,2552) 0,217 (±0,005) 27,1 (±1,61)
We observed that the particle size and the polydispersity
index of cationic SLN formulations were increased after two
weeks storage in dispersion medium at 4-8 ºC. The results of
the measurements showed that the particle size of all formu-
lations increased after 15 days of storage in distilled water
(50µS, pH 7.4). Therefore, samples lyophilized at -45ºC and
0,07 mbar for 48h (Christ Alpha 1-2 LD Plus freze-dryer, İzmir)
for the stability. Lyophilized samples were reconstituted in
distilled water (adjusted to pH 7,4 and 50 µS) by mixing with
vortex then the resulting formulations filtered at 0,22 µm
millipore filter5.
Binding of the cationic SLN to pDNA was checked by the
analysis of the electrophoretic mobility of the pDNA within
an agarose gel. As shown in Figure 1, the cationic SLN formu-
lation 2 was immobilised detectable amounts of DNA at 1:8
DNA/EQ1 (w/w) ratios (Lane 6).
3rd BBBB International Conference on Pharmaceutical Sciences
Figure 1. Agarose gel electrophoresis photograph of SLN-
pDNA complexes at different DNA/EQ1 ratios. Lanes from
left: moleculer weight marker (GeneRuler™ 1kb DNA Ladder,
Fermentas), naked pDNA, DNA/EQ1 mixtures w/w ratios of
1:0,5, 1:1, 1:2, 1:4, 1:8, 1:16. 1:20, 0:16 respectively.
The cationic SLN was developed as DNA delivery system and
it was planned to determine its transfection efficiency in hu-
man cancer cell lines.
ACKNOWLEDGMENTS
We would like to thank to Sevil Zencir for her kind help.
REFERENCES
1. Müller, R.H., Mader, K. and Gohla, S. Solid lipid nanopar-
ticles (SLN) for controlled drug delivery - a review of the
state of the art, European Journal of Pharmaceutics and
Biopharmaceutics, 2000; 50: 161-177.
2. Pedersen, N., Hansen, S., Heydenreich, A. V., Kristensen,
H.G. and Poulsen, H.S. Solid lipid nanoparticles can effec-
tively bind DNA, streptavidin and biotinylated ligands,
European Journal of Pharmaceutics and Biopharmaceu-
tics, 2006; 62: 155-162.
3. M.R. Gasco., Method for producing solid lipid micro-
spheres having a narrow size distribution, US Patent
5250236, 1993.
4. Heydenreich, A.V., Westmeier, R., Pedersen, N., Poulsen,
H.S., Kristensen, H.G. Preparation and purification of
cationic solid lipid nanospheres-effects on particle size,
physical stability and cell toxicity, International Journal
of Pharmaceutics, 2003; 254: 83–87.
5. Vighi, E., Rouzi, B., Montanari, M., Batini R. and Leo, E.
Re-dispersible cationic solid lipid nanoparticles (SLNs)
freeze-dried without cryoprotectors: Characterization
and ability to bind the pEGFP-plasmid, European Jour-
nal of Pharmaceutics and Biopharmaceutics, 2007; 67:
320–328.
p042-Long-term biocompatibility of a
new liquid polyurethane embolization
material
Zsuzsa Tóvölgyi1, István Hudák2, Tamás Dóczi2, Lajos Botz1
Pharmaceutical Institute, Faculty of Medicine, University of
Pécs, Pécs, Szigeti u. 12, Hungary H-76241
Department of Neurosurgery, Faculty of Medicine, University of
Pécs, Pécs, Rét u. 2, Hungary H-76232
 POSTER PRESENTATIONS
INTRODUCTION
Endovascular embolization offers a novel and minimally in-
vasive treatment to several vascular pathologies in the pe-
riphery and central nervous system - including occlusion of
arteriovenous malformations (AVMs), arteriovenous fistulas,
saccular aneurysms and even hypervascularized tumours.
The most commonly used liquid embolic materials are the
tissue adhesive acrylics, such as isobutyl cyanoacrylate
(IBCA, Bucrylate) and n-butyl cyanoacrylate (nBCA, TRUFILL®
by Cordis Neurovascular Inc. US) and non-adhesives, such
as ethylene vinyl alcohol copolymer (EVOH, ONYX® by ev3
Inc. US) and cellulose acetate. However, these devices do
not meet all requirements of the endovascular surgeon and
therapeutical problems often arise after embolisation mainly
due to cellular reaction in the vessel wall and in surrounding
tissue. Mutagenic and carcinogenic potential of IBCA was re-
ported in animal experiments, therefore, the use of IBCA was
ceased very early1. The cyanoacrylate glues induce a signifi-
cant acute and chronic inflammatory response due to toxic
reaction products and the exothermic polymerization2. After
successful embolization of aneurysms and AVMs by means
of cellulose acetate polymer solution only mild inflamma-
tory reactions with no chronic granulomatous reaction was
reported, however, recanalisation was also seen3. Natarajan
et al. 4 observed inflammation in equal frequency (90-91%),
with both EVOH and nBCA embolization groups. Angione-
crosis occurred in higher rate in patients treated with EVOH
(59%) than with nBCA (40%). Chronic foreign body giant
cells and recanalisation was observed after EVOH but not
after nBCA embolization. The maximum exposure time of
specimens embolized with nBCA was less than 24 days in this
study, this time span had been probably not long enough for
the development of foreign body giant cells and recanalisa-
tion (4). Studies on technical feasibility and histotoxicity of
EVOH pointed out that the carrier solvent, DMSO (0,5 to 1,0
ml) caused severe acute vasospasm and toxic effects, such as
angionecrosis, endothelial denudation, subarachnoid hae-
morrhage, cerebral infarction and death, but angitoxicity of
DMSO may be dose-dependent5.
An ideal transcatheter embolization device must have low
toxicity, should cause minimal inflammation and produce
reliable and permanent occlusion without extravasation and
recanalisation.
The authors examined the properties of a potential emboli-
sation polyurethane material. The purpose of this study is to
determine the long-term biocompatibility of polyurethane
a new embolisation material in a preclinical animal studies.
Gross and microscopic changes were studied in the embo-
lised artery and the surrounding tissue, presence of angion-
ecrosis, arterial revascularization, intra- and perivascular in-
flammatory reactions were searched for in rats in a 6 months
experiment.
MATERIALS AND METHODS
Ingredients of the polyurethane are 4,4’-methylene-bisphe-
nyl-isocyanate (MDI), 1,4- butandiol (BD) and polytetrahy-
drofurane (PTHF). The chemical structure of polyurethane is
linear. This property offers numerous advantages compared
to the other monomers or polymers like nBCA, EVOH and
October 26 - 28, 2009 / Antalya - TURKEY
cellulose acetate. . The polyurethane was dissolved in a ster-
ile solution of DMSO and ethanol. As soon as an aqueous so-
lution, e.g. blood, or a water-containing surface comes into
contact with the liquid polyurethane, solvents rapidly diffuse
out from the mixture, precipitation process starts and is fin-
ished within minutes. A solid, elastic intravascular “implant”
without adhesion to the vascular wall is the result. Sterile, mi-
cronized tantalum powder (BALT Extrusion, France) was sus-
pended in the solution to obtain appropriate radioopacity
during endovascular occlusion of vessels. Forty-eight adult
male and female CD rats weighing 250-350 g from Charles
River (Hungary) were employed. Under general anaesthesia
and aseptic conditions the femoral arteries of the animals
were disclosed carefully. The right femoral arteries of 24 rats
were embolized with the liquid polyurethane embolization
material. In the control group (n=24) animals received physi-
ologic NaCl injection into the right femoral artery. Animals
were sacrificed at 1 day (n=6), 1 week (n=6), 8 weeks (n=6),
24 weeks (n=6) after vessel occlusion to explant vessels and
surrounding tissue. The removed arterial segments were
immediately immersed and fixed in 4% (w/w) neutral buff-
ered (pH=7.4) formalin at 4°C. The embedded tissue sample
blocks were frozen cut 10 μm thick sections, stained with
haematoxylin and eosin (HE) and periodic acid-Schiff (PAS)
to evaluate cellular and collagen states in the artery and the
surrounding tissue.The sections were also immunostained
with an antibody (CD31) to identify the presence of endothe-
lium in vessel walls within the embolizing material.
RESULTS AND DISCUSSION
All sections were examined under light microscope for pres- 121
ence of occlusion, recanalisation and inflammation and also
to evaluate vessel wall and tissue integrity and regeneration.
No inflammation in the perivascular tissues and no evidence
of necrosis was observed. Only mild intravascular inflam-
mation response with scattered foreign body-type reaction
with giant cells containing embolisation material were de-
tected and minor changes to the vessel wall were seen. As a
prove to good biocompatibility of the agent examined, it has
not induced severe inflammation or toxic destruction of ves-
sel wall, accordingly no contraindication has been found to
its application for endovascular procedures. As vessel occlu-
sions have remained stable for up to 6 months the suitability
for embolization vascular disorders, such as arteriovenous
malformations and aneurysms has also been proved.
REFERENCES
1. Vinters HV, Lundie MJ, Kaufmann JC: Long-term patho-
logical follow-up of cerebral arteriovenous malforma-
tions treated by embolization with bucrylate. N Engl J
Med 1986;314:477-483.
2. Vinters HV, Galil KA, Lundie MJ, Kaufmann JCE: The histo-
toxicity of cyanoacrylates. Neuroradiology 1985;27:279–
291.
3. Sugiu K, Kinugasa K, Mandai S, Tokunaga K, Ohmoto T.:
Direct thrombosis of experimental aneurysms with cel-
lulose acetate polymer (CAP): technical aspects, angio-
graphic follow up, and histological study. J Neurosurg.
1995;83(3):531-538.
4. Natarajan SK, Born D, Ghodke B, Britz GW, Sekhar LN.:
Histopathological changes in brain arteriovenous mal-
 POSTER PRESENTATIONS

formations after embolization using Onyx or N-butyl cy-
anoacrylate. J Neurosurg. 2009;111(1):105-13.
5. Chaloupka JC, Viñuela F, Vinters HV, Robert J: Technical
feasibility and histopathologic studies of ethylene vinyl
copolymer (EVAL) using a swine endovascular emboliza-
tion model. Am J Neuroradiol 1994;15:1107-1115.
p043-EVALUATION OF MICROEMULSION
FORMULATIONS FOR PARENTERAL DRUG
DELIVERY OF APROTININ: FORMULATION,
DESIGN AND STABILITY STUDIES
H. Yeşim Karasulu1, Neslihan Üstündağ1, Zeynep Ay
Şenyiğit1, Tamer Güneri1
Ege University, Faculty of Pharmacy, Department of
Pharmaceutical Technology, İzmir, Turkey1
INTRODUCTION
Microemulsions are thermodynamically- stable, isotropic,
single-phase dispersions of oil and water stabilized by one or
more surface active agents Microemulsion formulations have
distinct advantages over macroemulsion systems when de-
livered parenterally; because of the fine particle microemul-
sion is cleared more slowly than the coarse particle emulsion
and therefore, has longer residence time in the body1.
Aprotinin is a monomeric globular polypeptide derived from
122 bovine lung tissue; it has a molecular weight of 6512 and
consists of 16 different amino acids arranged in a chain of 58
amino acid residues. It is a Kunitz protease inhibitor and has
a wide action with particular against trypsin, chymotrypsin
and kallikrein, making it theorically attractive in ameliorating
the effects of acute pancreatitis2.
The aim of this study is to design and evaluate novel micro-
emulsion formulations for parenteral aprotinin administra-
tion.
MATERIALS AND METHODS
Materials
Aprotinin (Sigma, Germany), oleic acid (Merck, Germany),
isopropyl myristat (Sigma, Germany), Labrasol (Gattefosse,
France), Cremophor EL (Sigma, Germany), ethanol (J.T.Baker,
Holland) and isopropyl alcohol (Merck, Germany) were used
for microemulsion formulation. All other chemicals were
used as analytical grade.
aprotinin into pre-weighted water phase, and then adding
into the oil, surfactants and co-surfactants mixture.
Determination of phase inversion temperatures (P.I.T.) of the
microemulsions :
Blank microemulsions were placed in a beaker and the bea-
ker was heated in a water bath. The temperature of the bath
was increased at 1°C/min steadily. The microemulsions were
agitated with a stirrer and the change in the conductivity
was recorded. The measurements were taken with a conduc-
tivity meter (Conductivity meter 4071, Jenway) (4).
Stability of microemulsions
Stability of the different microemulsion formulations was
tested at 5±3°C, 25±2°C and 40±2°C. Microemulsions were
evaluated with their pH, viscosity, droplet size, electrical
conductivity, density and refractive index changes during 6
months.
Determination of the osmolality of water phase of the mi-
croemulsions
Measurement of the osmalality of the water phase with or
without aprotinin was based on the freezing-point method.
After calibration of the osmometer (Knauer Semi-Micro Os-
mometer) with reference standarts (distilled water and 400
miliosmol NaCl solution), the osmolality was measured with
0.15 mL of sample5.
Characterization of aprotinin-loaded microemulsion
formulations
The characteristic properties of drug-loaded microemulsions
such as pH, viscosity, refractive index, electrical conductivity,
droplet size and zeta potential were evaluated.
Determination of zeta potential
Zeta potential measurements were performed at 25 oC us-
ing a zetasizer (Malvern Zetasizer Nano ZS, UK).
RESULTS AND DISCUSSION
The optimum formulations according to phase diagrams are
shown in Table 1.
Table 1: Composition of the microemulsion formulations
M1 M2 M3
Isopropyl
Oil phase Oleic acid 33.92% Oleic acid 9.34%
myristate 10.18%
Cremophor EL /
Surfactants/co- Cremophor EL/ Labrasol/Isopropyl
Isopropyl alcohol
surfactants Ethanol 51.62% alcohol 66.58%
54%

Preparation of microemulsion formulations 0.9% NaCl 0.9% NaCl 0.9% NaCl

Water phase
The microemulsion systems were formulated with various solution 14.46% solution 36.66% solution 23.25%
compositions of oil, surfactants and co-surfactants. The HLB value 12.5 12.5 14
pseudo-ternary phase diagram was constructed by titration The phase inversion temperatures of M1, M2 and M3 were
of homogenous liquid mixtures of oil, surfactant and co-sur- found 48.00±0, 50.666±0.577, 47.666±0.015 respectively.
factant with 0.9% NaCl solution as water phase at ambient Also, the results were shown in Fig. 1, Fig. 2 and Fig. 3.
temperature. After the identification of microemulsion area
in the phase diagrams, optimum formulations were selected Microemulsion formulations didn’t show significant changes
according to the gravity centers of these diagrams3. Drug- and were determined stabil at 5±3°C, 25±2°C and 40±2°C
loaded microemulsions were prepared by dissolving 2 mg of during 6 mounts.

3rd BBBB International Conference on Pharmaceutical Sciences

 POSTER PRESENTATIONS

The water phase of the parenteral microemulsions should be Table 2: Characterization of the aprotinin-loaded microemul-
augmented by the incorporation of ionic or osmotic agents sion formulations
because emulsified oil exerts no osmotic effect. Therefore,
M1 M2 M3
the osmolaltity of water phase (0.9% NaCl solution) and 2
mg aprotinin incorporarted water phases of the formulatons pH 4.073±0.012 4.5±0.010 4.693±0.030
were tested. The osmolality these water phases were found
in range of 300-320 milliosmol/kg.
Viscosity (cP) 19.71±0.043 17.00±0.017 11.223±0.025
Characterization of the aprotinin-loaded microemulsion for-
mulations were listed in Table 2.

Zeta potential of the blank M1, M2 and M3 microemulsion Electrical 118.7±0.100 0.43±0.020 mS/ 0.155±0.007
formulations were found +0.113±0.153, +0.0197±0.0834, Conductivity µS/cm cm mS/cm
-0.0472±0.228, respectively. In addition, zeta potential
of aprotinin loaded M1, M2 and M3 formulations were
14.066±1.250, -0.038±0.033, 0.199±0.021, respectively.
Refractive index
1.4325±0.0006 1.3942±0.0059 1.4149±0.0016
(nm)

Droplet size (nm) 1.023±0.074 6.411±0.509 2.902±0.044

Polydispersity
0.288±0.042 0.254±0.078 0.493±0.161
Index

Figure 1: Change of conductivity of M1 with various tem-
perature.
CONCLUSION
The results of characterization and stability studies, physi-
cochemical property tests showed that all microemulsion
formulations studied may be appropriate vehicles for paren-
teral uses. In conclusion, microemulsion formulations con- 123
taining aprotinin may be appropriate vehicles for aprotinin
parenteral application.

ACKNOWLEDGEMENTS
This study was supported by The Scientific and Technologi-
cal Research Council of Turkey (Tubitak-108 S 083).

Figure 2: Change of conductivity of M2 with various tem-
perature.
REFERENCES
1. Jadhav KR, Shaikh IM, Ambade KW, Kadam VJ. Applica-
tions of Microemulsion Based Drug Delivery System,
Curr Drug Deliv. 2006; 3:267-273.
2. Smith M, Kocher H, Hunt BJ. Aprotinin in severe acute
pancreatitits, The Int J Clin Pract. 2009; 1-9
3. Karasulu HY, Üstündağ N, Ay Şenyiğit Z, Güneri T. A
Novel Aprotinin Microemulsion For Pancreatit: Design,
Optimization And Physichemical Characterization, Drug
Reserach and Development Abstract Book, Ankara, Tur-
key, 4-7 May 2009.
4. Yue Y, San-ming L, Li-min Y, Pan D, Da-fang Z. Physico-
chemical properties and evaluation of microemulsion
systems for transdermal delivery of meloxicam, Chem
Res Chinese U. 2007; 23(1):81-86.
5. Ganta S, Paxton JW, Baguley BC, Garg S. Pharmacokinet-
ics and pharmacodynamics of chlorambucil delivered in
parenteral emulsion, Int J Pharm. 2008; 360:115–121.

Figure 3: Change of conductivity of M3 with various tem-

perature.

October 26 - 28, 2009 / Antalya - TURKEY

 POSTER PRESENTATIONS
p044-DEVELOPMENT OF A PARENTERAL
SOLVENT SYSTEM FOR THE SOLUBILIZATION
OF AZOLE TYPE ANTIFUNGALS
Kristóf Kovács1, György Stampf1, István Antal1, Imre
Klebovich1, Krisztina Ludányi1
Semmelweis University, Department of Pharmaceutics, Hogyes
Endre street 7, H-1092 Budapest, Hungary1
INTRODUCTION
Poor water solubility of active pharmaceutical ingredients is
a great issue in pharmaceutical development. Unfortunately
a wide range of chemicals exhibit inadequate solubility1,2.
Generally systemic mycoses are chronic and develop slowly.
A group of active pharmaceutical ingredients, the azoles
have been shown to be effective agents against various
fungi and can be used in the treatment of systemic fungal in-
fections. Although these compounds are potent molecules,
most of them show poor water solubility, thus formulation
of a composition, which comprises therapeutically effective
quantities of active agent, is difficult3. It has been shown that
the solubility of azoles can be advantageously altered by the
use of cyclodextrins and other solubilizers, but all of them
have certain disadvantages.
In this study we developed a solvent system for azoles, which
is a novel combination of well established excipients show-
124 ing synergetic solubilizing effect.
MATERIALS AND METHODS
Materials
Ketoconazole (MW: 531.44) and miconazole (MW: 416.1) of
minimum 98% purity were obtained from Bosche Scien-
tific, New Brunswick, NJ, USA. Acetic acid, ammonium ac-
etate, ethanol 96%, calcium gluconate, glycerol, macrogol
400, phosphoric acid, polysorbate 20, 60 and 80, potassium
dihydrogen phosphate, propylene glycol and sodium tau-
rocholeate were used as solubilizers and were all pharma-
copoeial grade (Molar Chemicals Ltd., Budapest, Hungary).
Ammonium acetate, acetic acid, methanol and water were
all of HPLC gradient grade (Sigma-Aldrich Ltd., Budapest,
Hungary).
Methods
Solubility studies
Solubility tests were conducted using pH adjusters, co-sol-
vents, surfactants and their combinations. Solvent systems
were prepared and an excess of drug was added to the sol-
vents. After reaching equilibrium the excess active ingre-
dient was removed and an HPLC method was used for the
quantitative determination of miconazole.
Optimization of the solvent system
After ensuring adequate solubility of miconazole, the com-
position of the ternary solvent system was optimized by
reducing the amount of polysorbate and ethanol based on
solubility and dilution tests. Dilution tests were performed
using 5% dextrose and 0.9% sodium chloride infusions.
3rd BBBB International Conference on Pharmaceutical Sciences
Sterility tests
The stability of the solvent system was examined on auto-
claving and on filtration. Stability of the solvent system was
confirmed on autoclaving on 120°C for 20 minutes.
Static filter tests were conducted on 0.2µm pore size cellu-
lose acetate, cellulose nitrate and polyamide membrane fil-
ters. Integrity of the membrane was examined prior to and
following testing and the ability of the membrane to bond
miconazole was also examined.
Stability test
Accelerated stability test of the solvent system was per-
formed according to Guideline on stability testing: stability
testing of existing active substances and related finished
products.
RESULTS AND DISCUSSION
Solubility studies
The solubility tests were systematically built up in order to achieve
adequate solubility of miconazole. First aqueous solutions of the
solubilizing excipients were examined, then binary and ternary
solvent systems were tested. The solubilizing effect of one of the
solvent systems from each group is shown in figure 1.
Figure 1: Solubilizing effect of various solvent systems
As seen in the table the a ternary solvent system compris-
ing 5% polysorbate 80, 25% ethanol and 0.05M ammonium
acetate (pH 3.1) is able of solubilizing more than 40 mg/mL
of miconazole. Furthermore, the same composition is able of
dissolving more than 150 mg/mL of ketoconazole.
Optimization of the solvent system
Optimization of the solvent system was performed and re-
sulted in a solvent system containing less excipients, but
with a similarly substantial solubilizing capacity.
Sterility tests
The final composition was autoclaved and was found to be
stable upon heat sterilization.
The tested membrane filters did not lose their integrity
during the static filter tests, but the cellulose nitrate mem-
brane filter absorbed substantial miconazole during the test.
 POSTER PRESENTATIONS
Therefore it was concluded that aside filtration with cellulose
nitrate membrane filters, all of the tested methods of steril-
ization may be applied for the novel solvent system.
Stability test
Accelerated stability tests performed over a period of 12
months prove the long term stability of the solvent system.
CONCLUSION
Based on the results it can be concluded that a novel solvent
system was worked out, which showed a very substantial solu-
bilizing power. After optimizing the composition of the solvent
system it was shown that the solvent system is readily capable
of being sterilized by both heat sterilization and filtration. The
accelerated stability tests show long term stability of the sol-
vent system. Based on these results a concentrate for infusion
containing miconazole or ketoconazole was formulated.
REFERENCES
1. Strickley RG. Solubilizing excipients in oral and inject-
able formulations, Pharm Res. 2004; 21(2): 201-230.
2. Yalkowsky SH, Solubility and Solubilization in Aqueous
Media, New York, 1999.
3. Richardson MD, Jones BL, Therapeutic Guidelines in Sys-
temic Fungal Infections, 3rd Ed., England, 2004.
P045-PREPARATION OF W/O
MCIROEMULSIONS WITH DIFFERENT
VEGETABLE OILS FOR DRUG DELIVERY
Gülten Kantarcı1, Mustafa Kotmakchiev1, Buket Aksu2, Tamer
Güneri3
Ege University, Faculty of Pharmacy, Department of
Pharmaceutical Biotechnology1, İzmir, Turkey
Ege Universiry, Faculty of Pharmacy, Department of
Pharmaceutikal Technology2, İzmir, Turkey
Ege Universiry, Faculty of Pharmacy, Department of
Pharmaceutical Technology3 , İzmir, Turkey
INTRODUCTION
Microemulsions are thermodynamically stable, isotropic so-
lutions of water and oil stabilized by appropriate surfactants
and co-surfactants. Several plant oils, in combination with
different surfactants or surfactant/co-surfactant mixtures
have been studied in order to construct biologically suitable
microemulsions1,2,3.
The purpose of this study was to investigate the effects of
different vegetable oils on microemulsion formulation.
MATERIALS AND METHODS
Materials
Soybean oil and Olive oil (as oil phase) from Küçükbay Yağ
Sanayi A.Ş. (Izmir, Turkey), Tween® 80 (T80) (as surfactant)
from Merck-Co. (Hohenbrunn, Germany), Span® 85 (S85)
(as surfactant) from Sigma-Aldrich Co. (St. Louis, MO, USA).
Distilled water was used in all experiments as the aqueous
phase.
October 26 - 28, 2009 / Antalya - TURKEY
Construction of phase diagrams
Phase diagrams were constructed at 5,57 and 8,40 HLB values
with S85 and T80. Oil phase was mixed with the surfactant
phase at the ratios of 10:90, 20:80, 30:70, 40:60, 50:50, 60:40,
70:30, 80:20, 90:10. These mixtures were slowly titrated with
distilled water until the turbidity was observed. Microemulsion
areas (MA) were detected with the aid of phase diagrams drawn
using a computer program developed in the computer center,
Faculty of Pharmacy, University of Ege [4]. Four microemulsion
formulations were chosen for further studies from the gravity
centers of microemulsion formation areas (Table 1).
Table 1: The contents of the microemulsion formulations.
% of ingredients
Oils Formations HLB
Oil Surf Water
OM1 5.57 31.0 66.5 2.5
Olive
OM2 8.40 31.6 64.4 4.0
SM1 5.57 32.1 65.4 2.5
Soybean
SM2 8.40 21.0 75.0 4.0
Physichochemical characterization
pH values were measured using pH meter (Inolab Level 1,
WTW Wissenschaftlich-Techniche Werkstätten GmbH, Weil-
heim, Germany), at 25±2°C. Viscosity and flow characeris-
tics were determined by Programmable DV-III+ Rheometer
(Brookfield Engineering Labs. Inc., Middleboro, USA) (Spin-
dle: SC4-21) at 25.0±0.1°C. Electrical conductivity was mea-
sured using a conductometer (Jenway 4071, UK), at 25±2°C.
Droplet size distribution and PDI were determined by Zeta-
sizer Nano ZS (Malvern Instruments Ltd., UK) at 25±0,1°C. 125
Stability of microemulsion formulations
The centrifuge method (5000 rpm, 15 min), and particle size
measurements are used for determining the physical stability
of microemulsions. The storage stability of microemulsions
were checked after 6 months storage at room temparature.
RESULTS
As shown in Table 1, W/O microemulsion formulations were de-
veloped with soybean oil and olive oil at different HLB values.
The clear transparent W/O microemulsion areas are plotted
on pseudoternary phase diagram for the systems oil/T80:S85/
water. At 8,40 HLB, w/o microemulsion areas were larger than
those at lower HLB with both oils. The MA’s with 5.57 HLB were
so similar for SM1 (166.90) and for OM1(166.31). At 8.40 HLB,
the area of OM2 (267.49) was larger than area of SM2 (231.69).
This may be related to the difference between the triglyceride
compositions of olive oil and soybean oil1,5. Olive oil contains
78 percent monounsaturated, 8 percent poly unsaturated and
12 percent saturated fatty acids, while soybean oil contains 25,
62 and 11.5 percent, respectively. The two oils showed similar
microemulsion forming areas which are narrow. These results
agree with the results of Parris et al. who concluded that trig-
lyceride oils could hardly be microemulsified5.
The viscosity was decreased after storage for all formula-
tions. As seen in table 2, the change of viscosity was so small
for OM2. The formulations showed newtonian flow before
and after the storage (Figure2, only before storage). On pH
values, little changes were observed.
 POSTER PRESENTATIONS

Table 2: Physichochemical properties of microemulsions. DS- bility after OM2. They also exhibited good stability in their
droplet size; PDI- polydispersity index. droplet size after 6 months storage, and may be appropriate
Before storage vehicles for drug delivery.
Formulations Viscosity
pH DS (mm) PDI REFERENCES
(cP)
OM1 214.3 6.84 58.21±2.480 0.096±0.017 1. Do LD., Withayyapayanon A., Harwell JH. and Sabatini
OM2 220.0 7.11 60.48±2.151 0.266±0.038 DA. Environmentally Friendly Vegetable Oil Microemul-
sions Using Extended surfactants as Linkers, J Surfact
SM1 177.3 6.80 73.08±0.850 0.086±0.050
Deterg. 2009; 12: 91-99.
SM2 328.7 7.24 74.09±12.457 0.181±0.007 2. Gupta S.; Sanyal S.K.; Datta S.; Moulik S.P. Preparation of
After storage prospective plant oil derived micro-emulsion vehicles
OM1 134.4 6.90 57.10±1.412 0.243±0.006 for drug delivery, Indian J Biochem Biophys. 2006; 43:
OM2 21..2 7.24 58.83±1.403 0.320±0.026 254-257.
SM1 118.9 7.05 72.53±1.105 0.090±0.042 3. Kantarci G.; Özgüney I.; Karasulu H.Y.; Arzık S.; Güneri
SM2 242.7 7.12 66.10±0.746 0.228±0.023 T. Comparison of Different Water/Oil Microemulsions
Containing Diclofenac Sodium: Preparation, Character-
The conductivity results are not included in Table 2, since all ization, Release Rate, and Skin Irritation Studies, AAPS
formulations exhibited non-conducting behavior at prepa- PharmSciTech, 2007; 8(4): Article 91
ration and after the storage period. 4. Ege MA, Karasulu HY, Guneri T. Triangle phase diyagram
analysis software, The 4th International Postgraduate
All formulations were proven stable by the centrifuge meth- Research Symposium on Pharmaceutics, Istanbul ,2004.
od. Droplet sizes of the formulations showed a little bit de- 5. Parris N., Joubran RF. and Lu DP. Triglyceride microemul-
crease after storage, except SM2. sions: Effect of nonionic surfactants and the nature of
the oil, J Agric Food Chem. 1994; 42: 1295-1299.

p046-NANOEMULSION FORMULATION OF A
NEW Candida albicans CYP51 INHIBITOR

Aurélie Schoubben1, Elio Cenci2, Fausto Schiaffella1, Paolo

126 Blasi1, Lara Milanese1, Carlo Rossi1, Anna Vecchiarelli2,
Maurizio Ricci1, Renata Fringuelli1

Department of Drug Chemistry and Technology, University of

Figure 1: Pseudoternary phase diagrams of W/O type micro-
emulsions.
Perugia1
Department of Experimental Medicine and Biochemical
Sciences, University of Perugia2
INTRODUCTION
In a program aimed at the design and synthesis of novel azole
inhibitors of C. albicans CYP51 (CA-CYP51), a series of azole
1,4-benzothiazines and 1,4-benzoxazines were recently syn-
thesized. Among them a novel derivative, 2-butyl-6-{1-[(4-
chlorobenzyl)oxy]-2-(1H-1-imidazoyl)ethyl}-4-methyl-3,4-di-
hydro-2H-1,4-benzothiazin-3-one (Fig.1), characterized by a
very good MIC against C. albicans CA-6, showed a low water
solubility that probably limits its in vivo activity1.

The water solubility, determined at 25°C, was of about 4 µg/

mL that, according to the European Pharmacopoeia, corre-
sponds to a “practically insoluble” compound2.

Therefore, the aim of the study was to assess the feasibility

Figure 2: Flow behavior of w/o type microemulsions before
storage.
CONCLUSION
Among all tested formulations OM2 showed minimal chang-
es on physicochemical properties. SM1 has also good sta-
of using nanoemulsion technology to overcome the com-
pound solubility issues.
MATERIALS AND METHODS
Materials and animals
PEG 400 was purchased from Sigma (Milan, Italy). Flucon-
azole was obtained from Pfizer (Latina, Italy).

3rd BBBB International Conference on Pharmaceutical Sciences

 POSTER PRESENTATIONS
For experimental systemic candidiasis, mice were inoculated
with the highly virulent C. albicans strain CA-6 isolated from clini-
cal specimens in the laboratories of the Microbiology Section,
University of Perugia3. Female, 8-10 weeks old, outbred CD1 mice
were obtained from Harlan Nossan Laboratories (Milan, Italy) and
housed at the Animal Facilities of the University of Perugia, Italy.
Methods
To prepare the nanoemulsion, the compound was first solu-
bilized in PEG 400 and then added dropwise to deionised
water under stirring. The obtained emulsion was character-
ized in terms of dimensions and stability using photocorrela-
tion spectroscopy.
In vitro anti-Candida activity was determined according to
the NCCLS guidelines4.
For systemic infection, mice were infected intravenously on
day 0 with 2.5x105 blastospores of the highly virulent C. al-
bicans CA-6 strain and untreated or treated 2 h before chal-
lenge and once a day for 6 consecutive days after challenge
with the synthetic compound, nanoemulsion, fluconazole
(all at 10 mg/kg body weight, i.p.). PEG 400 (0.2 mL, i.p.) was
used as a negative control. Infected animals were monitored
for survival and organ clearance.
RESULTS AND DISCUSSION
The nanoemulsion was characterized by dimensions of
189±55 nm and the formulation showed to be stable for at
least 7 months even when diluted (Fig. 2).
The nanometric compound droplets should accelerate
its in vivo solubilization with respect to the drug solution
(DMSO:H2O, 1:4). In fact, it can be hypothesized that once
injected, DMSO dilution or absorption may lead to drug
phase separation and low effective surface available for dis-
solution. In addition to the improved solubility properties,
this formulation allowed to eliminate the use of DMSO that
should be preferably avoided in vivo.
The preliminary results of in vivo experiments showed that
the use nanoemulsion turned out to be a valuable strategy
to overcome solubility problems encountered during early
discovery and at the formulation stage as well.
Figure 1: Chemical structure of 2-butyl-6-{1-[(4-chloroben-
zyl)oxy]-2-(1H-1-imidazoyl)ethyl}-4-methyl-3,4-dihydro-2H-
1,4-benzothiazin-3-one
October 26 - 28, 2009 / Antalya - TURKEY
Figure 2: Volume diameter distribution of the nanoemul-
sion.
REFERENCES
1. Schiaffella F, Macchiarulo A, Milanese L, Vecchiarelli A,
Costantino G, Pietrella D, Fringuelli R. Design, Synthesis,
and Microbiological Evaluation of New Candida albicans
CYP51 Inhibitors. J Med Chem. 2005; 48: 7658-7666.
2. European Pharmacopoeia. 6th Edition; 1: 5.
3. Bistoni F, Vecchiarelli A, Cenci E, Puccetti P, Marconi P,
Cassone A. Infect Immun. 1986; 51: 668.
4. Reference Method for Broth Dilution Antifungal Suscep-
tibility Testing of Yeast; Approved Standard NCCLS Doc-
ument M27-A; National Committee for Clinical Labora-
tory Standards:Waye, PA, 1997.
p047-EFFECTS OF STERILIZATION 127
TECHNIQUES ON INJECTABLE STEALTH BONE
TARGETED NANOPARTICLES
İpek Özcan1, Freimar Segura Sanchez2, Kawthar Bouchemal2,
Özlem Abacı3, Özgen Özer1, Tamer Güneri1, Gilles Ponchel2
Ege University, Faculty of Pharmacy, Department of
Pharmaceutical Technology, 35100 Izmir, Turkey1
Paris-South University, UMR CNRS 8612, Faculty of Pharmacy,
Physicochimie Pharmacotechnie Biopharmacie, 92290
Chatenay-Malabry, France2
Ege University, Faculty of Science, Department of Biology, Basic
& Industrial Microbiology, 35100 Izmir, Turkey3
INTRODUCTION
Synthetic biodegradable polymers are well-known materi-
als for the preparation of nanoparticles. Poly(γ-benzyl-L-
glutamate) PBLG, a synthetic degradable polypeptide and
various chemical moieties could be quite easily introduced in
the structure of PBLG to form various copolymers1. Nanopar-
ticles exhibit different properties such as targeting, stealth
properties, efficient drug loading and controlled drug re-
lease.
Sterilization of injectable nanoparticles is a major challenge
in the designing of appropriate drug delivery systems. Mem-
brane filtration is a safe technique based on physical removal
of present microorganisms that does not require excessive
heat or radiation causing irreversible effects on the nano-
 POSTER PRESENTATIONS

particles. However, it is very much limited to the size of the
particles. Heat sterilization by autoclaving is a highly effec-
tive technique involving high temperatures, which may in-
fluence decomposition or degradation of active ingredient
as well as the nanoparticle material2,3.
RESULTS AND DISCUSSION
Characterizations of nanoparticles were evaluated on sterile
and non-sterile samples. Table 1 and 2 summarize the mean
diameter, polydispersity index and zeta potential values of
non-sterile and sterile nanoparticles, respectively.

In the present work, effect of sterilization technique on pre- Table 1: Physicochemical characteristics of non-steril nano-
viously designed stealth bone targeted nanoparticles prop- particles
erties such as particle size, zeta potential and polydispersity
index were investigated. Formulation Mean diameter Zeta potential Polydispersity
code (nm) (mV) index
MATERIALS AND METHODS
Preparation of Nanoparticles
The surfaces of the nanoparticles were modified with alen- F1 50±14 -25.6±0.77 0.137±0.03
dronate (ALD) (Chemos GmbH) as targeting agent, polyeth-
ylene glycol (PEG) (Shearwater Corporation) as hydrophilic
F2 49±16 -25.5±0.81 0.118±0.05
agent and fluorescein isothiocyanate (FITC) (Sigma Aldrich)
as imaging agent. F1 and F2 coded nanoparticles were pre-
pared by nanoprecipitation technique using composite Table 2: Physicochemical characteristics of nanoparticles af-
polymers (PBLG-ALD/PBLG-PEG/PBLG-FITC and PBLG-PEG- ter different sterilization techniques
ALD/PBLG-PEG/PBLG-FITC) at a ratio of 2:2:1, respectively).
Mean diameter Zeta potential Polydispersity
Sterilization of Nanoparticles Formulation code (nm) (mV) index
Membrane Filtration
The formulations were filtered through a Milipore Express™ Mean brane filtration
membrane filter system (Millipore®, Volketswil, Switzerland). Af-
terwards, sterile suspension was poured into sterile glass vials. F1 52±10 27.4±0.85 0.132±0.05

F2 48±14 -23.9±0.48 0.122±0.04

Autoclaving (Heat Sterilization)
Samples were divided into two groups of equal volume af-
Autoclaving
128 ter preparation. They were put into glass vials, which were
sealed with rubber stoppers and aluminum caps. One group 100±11 -20.2±0.55 0.355±0.15
F1
was sterilized at 121 °C for 20 min while the other group (ref-
erence) was kept at 8 °C for comparative evaluation of physi- F2 115±16 -22.9±0.32 0.272±0.26
cochemical properties with their sterilized analogue.

Characterizations of Nanoparticles As seen in tables, membrane filtration does not seem to have
The mean diameter and particle size distribution was deter- any effect on particle size and polydispersity index. Also,
mined before and after sterilization by dynamic laser light slight changes was seen in zeta potential of nanoparticles.
scattering (Nanosizer Coulter N4 Plus®) and also their obser- The clogging doesn’t observed because of the particle size
vation in transmission electron microscopy (TEM-Philips EM is significantly smaller than the membrane pore size. How-
208). Zeta potential measurements were carried out using a ever, alternative technique, autoclaving, has a direct impact
Zetasizer 4, Malvern Instrument (n=6). on nanoparticle size. Nanoparticles tend to aggregate at
high temperature employed in autoclaving which leads to
Microbial Sterility Testing of Formulations increased particle size and polydispersity index.
The sterility testing was performed on the nanoparticles,
following USP XXIII guidelines. Thioglycollate resazurine From TEM results it could be seen that nanoparticles exhib-
broth (BioMerieux®, Marcy, France) was used as anaerobic ited elipsoidal geometries. After the sterile filtration, there
medium for the detection of bacteria and tripcase soy broth was no change in particle shape (Figure 1a,b).
was used as medium for the detection of yeasts and fungi.
Mediums were sterilized at 121 °C/ 1.1 atm for 15 min. Then,
steril nanoparticles were immersed in tubes containing ap-
propriate media. The tubes were incubated for 14 days at 37
°C (thioglycollate resazurine medium) or at 25 °C (tripcase
medium). Non-sterile nanoparticles were used as positive
controls. The turbidity of the media was then observed over
a basic period of 14 days in comparison to positive controls.
The experiment was done three times.

Figure 1: TEM micrographs of (a) non-steril nanoparticles; (b)

steril nanoparticles

3rd BBBB International Conference on Pharmaceutical Sciences

 POSTER PRESENTATIONS
In order to ensure the sterility of the final nanoparticles (i.e.
free of all forms of viable microorganisms), the sterility test-
ing was performed according pharmacopoeial requirements.
Sterility was assessed by the observation of the media during
the incubation period. All tested steril nanoparticles showed
no detectable visible growth of microorganisms at the end
of the 14 days for three different medium (Figure 2a,b). Con-
trarily to positive controls for which a substantial increase of
turbidity was systematically observed.
Figure 2: Photos of incubation medium (a) 1. day; (b) 14.day
CONCLUSION
According to the experimental findings, the sterilization of
nanoparticle formulations by autoclaving is not advisable. It
was shown that membrane filtration was a suitable method
for the sterilization of nanoparticles without measurable
physical properties loss.
REFERENCES
1. Barbosa EM, Montembault V, Cammas-Marion S, Ponchel
G, Fontaine L. Synthesis and characterization of novel
poly(γ -benzyl-L-glutamate) derivatives tailored for the
preparation of nanoparticles of pharmaceutical interest,
Polym Int. 2007; 56: 317-324.
2. Memisoglu-Bilensoy E, Hincal AA. Sterile, injectable cy-
clodextrin nanoparticles: Effects of gamma irradiation
and autoclaving, Int J Pharm. 2006; 311: 203-208.
3. Konan YN, Gurny R, Alleman E. Preparation and charac-
terization of sterile and freeze-dried sub-200 nm nano-
particles, Int J Pharm. 2002; 233: 239-252.
4. Ozcan I, Segura-Sánchez F, Bouchemal K, Ozer O, Guneri
T, Ponchel G. Design of nanoparticles composed of poly
(benzyl glutamate) derivatives for targeted drug deliv-
ery to bone, The 7th Central European Symposium on
Pharmaceutical Technology and Biodelivery Systems,
Ljubljana, Slovenia, September 2008.
October 26 - 28, 2009 / Antalya - TURKEY
p048-TUMOR TARGETED NANOSIZED
LIPOSOME-BASED DIAGNOSTIC AGENTS FOR
HYBRID IMAGING SYSTEMS
Mine Silindir1, Suna Erdoğan1, A. Yekta Özer1, A. Lale Doğan2,
Murat Tuncel 3, Vladimir P. Torchilin4
Department of Radiopharmacy, Faculty of Pharmacy,
Hacettepe University, 06100, Ankara, Turkey1
Department of Oncology, Faculty of Medicine, Hacettepe
University, 06100, Ankara, Turkey 2
Department of Nuclear Medicine, Faculty of Medicine,
Hacettepe University, 06100, Ankara, Turkey3
Department of Pharmaceutical Sciences, Northeastern
University, 02129, Boston, M A, U S A4
INTRODUCTION
Among the currently used cancer imaging methods; Nuclear
Medicine modalities provide metabolic wheras the radiolog-
ical modalities provide anotomical information. In the evalu-
ation of the patients, the data obtained from both of these
modalities were used collaboratively. However the different
acquisition times, on separate machines, decrease the speci-
ficity and accuracy of both modalities. To solve this problem
hybrid imaging modalities like SPECT/CT which opened a
new era in the field of cancer imaging were developed.
After the wide spread usage of the hybrid imaging modali-
ties there is a need for a single contrast agent which can be
used for both imaging modality. Nanoparticle carrier sys- 129
tems which can carry multiple contrast group are very prom-
ising. Among these systems; liposomes are the most popular
ones.
Herein, we studied the preparation, characterization and
in vitro cell binding of plain and tumor targeted PEGylated
liposomes contains iopromide as a contrast agent in liquid
compartment and radionuclide (Tc-99m)on the surface.
MATERIAL AND METHODS
Synthesis of DTPA-PE
DTPA-phosphatidylethanolanime was synthesized as de-
scribed before1.
Synthesis of pNP-PEG-PE
pNP-PEG-PE was synthesized as described before2.
Preparation of plain liposomes
Lipid mixtures (200 mmol/L) of DPPC, PEG2000-PE, choles-
terol and DTPA-PE were dissolved in ethanol at 70°C. The
lipid- ethanol solution was then hydrated with Iopromide
(185 mg/mL of iodine). The initial ethanol content was 10%
vol. The resulting multilamellar vesicles were then extruded
5 times with 2 stacked polycarbonate membranes of 0.2 µm
and 0.1 µm pore size at 70°C. Then, liposomes were dialyzed
overnight against 20 mM Tris buffer (pH 7.4) (3). For liposome
formulation consist of PL 90G as phospholipid, all procedures
were carried out at 35°C.
Preparation of immunoliposomes
To prepare antibody (mAb 2C5) conjugates with PEG3400-
 POSTER PRESENTATIONS

PE, solution of pNP-PEG-PE in citrat buffer saline (pH 5.0)

was added to an equal volume of a 1 mg/mL solution of a
antibody in Tris-buffered saline (pH 8.5). The mixtures were
incubated overnight at 4°C.

Then, 2C5(PEG-PE) conjugate were co-incubated with Iopro-

mide-loaded plain liposomes for 1 day at 4°C. Following the
incubation, free mAb 2C5 were removed by dialysis using
cellulose ester dialysis tubes with a cutoff size of 350 kDa.

Mean particle size Figure 1: In vitro binding of Tc-99m labeled Iopromide con-
The mean particle size of liposomes was measured by dynam- taining plain and immunoliposomes to different tumor cell
ic light scattering (DLS) at 25°C using Malvern Zeta Sizer. lines

Entrapment efficiency Table 1: Compositions of liposomes

After removing free iopromide by dialysis, vesicles were ly- Formulations Codes
sed by 20 mM Tris (pH 7.4) buffer containing 10% (w/v) Triton
X-100. Encapsulated iopromide amount was determined by DPPC:PEG2000:Chol:DTPA-PE A
spectrophotometrically at 297nm.
PL90G:PEG2000:Chol:DTPA-PE B
Radiolabeling of liposomes with Tc-99m mAb 2C5- DPPC:PEG2000:Chol:DTPA-PE C
Liposomes were labeled with Tc-99m by the tin reduction
method. Briefly, 0.5 mL SnCl2.2H2O (1 mg.ml-1) and 1.5 mCi mAb 2C5- PL90G:PEG2000:Chol:DTPA-PE D
Tc-99m were mixed with liposome dispersions and were left
to react for 30 min. After incubation samples were dialyzed Table 2: Characterization results of liposome formulations
against to 20mM Tris Buffer (pH 7.4) to remove free Tc-99m.
Formulations Particle Size (nm) Entrapment Efficiency (%)
In vitro cell culture experiments A 94 (0,13*) 10,3±0,02
The cells (MCF-7 and EL4) were seeded at (800.000 cells/
well) into six-well tissue culture plates and allowed to reat- B 99 (0,15*) 8,59±0,01
130 tach overnight at 37°C, 5% CO2 in a humidified atmosphere.
After 24h incubation, the cells were treated with 1% BSA in C 110 (0.14*) 16,1±0,02
a serum free media for 1h to prevent a nonspecific binding. D 115 (0,16*) 13,59±0,01
Then, the medium was replaced with plain and mAb 2C5
modified Iopromide-loaded liposomes for 2h at 37°C. The * PI: Polydispersity index
cells were washed with serum free media, trypsinised, and
resuspended in 1 mL serum-free media. Their radioactivities ACKNOWLEDGEMENTS
were measured by gamma counter. This work is supported by TUBITAK-SBAG (Project No:
108S058).
RESULTS AND DISCUSSION The authors would like to thank to Schering-Turkey for gen-
The objective of this study was to formulate liposomes contain- erous gift of Iopromide.
ing iopromide in their aqueous phase and DTPA-PE on their sur-
face to evaluate the feasibility of these vesicles for hybrid imag- REFERENCES
ing systems. All formulations are described in Table 1. 1. Grant CWM, Karlık S, Florio E. Magnetic Resonance in
Table 2 illustrated the results of mean particle size and en- Medicine, A liposomal MRI contrast agent: Phosphoti-
trapment efficiency for the plain and antibody modified li- dylethanolamine-DTPA, 1989; 11: 236-243.
posomes. It was seen that tumor specific antibody modified 2. Torchilin VP, Levchenko TS, Whiteman KR et al. p-nitro-
liposomes have bigger particle size and higher entrapment phenylcarbonyl-PEG-PE-liposomes: fast and simple
efficiency than plain liposomes. attachment of speci¢c ligands, including monoclonal
antibodies, to distal ends of PEG chains via p-nitrophe-
The results of in vitro cell binding experiments were shown in nylcarbonyl groupsBiomaterials 2001; 22: 3035-44.
Figure 1. The data presented in Fig. 1 clearly demonstrate that 3. Zheng J, Perkins G, Kirilova A et al. Multimodal contrast
Iopromide containing PEGylated 2C5-immunoliposomes ca- agent for combined computed tomography and mag-
pable of specific binding to various cancer cells than control netic resonance imaging applications, Investigative Ra-
non-specific liposomes of the same composition diology, 2006; 41: 339-348.

3rd BBBB International Conference on Pharmaceutical Sciences

 POSTER PRESENTATIONS
p049-EFFECT OF SOD AND KET RELEASE
FROM BIODEGRADABLE MICROSPHERES ON
THE IN VIVO RESPONSE TO IMPLANTED ALG-
PLO-ALG MICROCAPSULES
Stefano Giovagnoli1, Paolo Blasi1, Giovanni Luca2, Francesca
Fallarino3, Mario Calvitti3, Francesca Mancuso3, Aurélie
Schoubben1, Maurizio Ricci1, Giuseppe Basta4, Ennio
Becchetti5, Carlo Rossi1, Riccardo Calafiore4
Dipartimento di Chimica e Tecnologia del Farmaco, Faculty of
Pharmacy, University of Perugia1
Dipartimento di Medicina Interna (Di.M.I.), Sezione di Medicina
Interna e Scienze Metaboliche ed Endocrine, University of
Perugia, School of Medicine, 2
Dipartimento di Medicina Sperimentale e Scienze Biochimiche,
University of Perugia,3
Dipartimento di Medicina Interna (Di.M.I.), Sezione di Medicina
Interna e Scienze Metaboliche ed Endocrine, University of
Perugia, School of Medicine4
Dipartimento di Medicina Sperimentale e Scienze Biochimiche,
University of Perugia5
INTRODUCTION
Two major obstacles affect Pancreatic islet transplantation:
the restricted availability of human donors and the need for
life-long recipient’s general immunosuppression1.
These drawbacks could be solved by using neonatal por-
cine islets for human substitutes2 and by entrapping cells
into multicompartmental alginate-poly L-ornithine- alginate
(ALG-PLO-ALG) microcapsules (MC) supplied with systems
enabling delivery of vitamins and enzymes, and growth fac-
tors [3]. Sustained release of these actives could be used to
treat post-transplant adverse events as well4.
Therefore, this work was aimed to determine the in vivo ef-
fect of SOD and KET controlled release from poly(D,L-lac-
tide-co-glycolide) (PLGA) and poly(D,L-lactide) (PLA) micro-
spheres (MS) on unexplained random in vivo host’s response
to intraperitoneal grafting of ALG-PLO-ALG MC prepared
with purified ALG.
MATERIALS AND METHODS
MS Preparation and characterization
SOD loaded PLGA (Bidachem, Italy) MS and KET loaded PLA
(Sigma, Milan, Italy) MS were prepared by a W/O/W double
emulsion and an O/W emulsion solvent extraction/evaporation
methods respectively. The protein content was determined by
Micro-BCA protein assay (Sigma, Milan, Italy) by using a 8453
Agilent UV/VIS spectrophotometer (Agilent Technologies,
Germany). KET content was determined by UV/VIS after dis-
solving the MS in methylene chloride. MS size distribution was
determined by an Accusizer TM 770 Optical Particle Sizer (PSS
Inc., Santa Barbara, CA, USA). Morphology was investigated by
scanning electron microscopy (SEM) using a Philips XL30 mi-
croscope (Philips, the Netherlands). The entrapped SOD activ-
ity retention was evaluated in vitro according to the pyrogallol
(Sigma, Milan, Italy) autoxidation inhibition assay.
October 26 - 28, 2009 / Antalya - TURKEY
The in vitro SOD and KET release was determined in 0.1M
pH 7.4 phosphate buffer, at 37°C. Samples were analyzed by
Micro-BCA protein assay and UV.
Alginate analysis and MC fabrication and characterization
The purified ALG was assayed by the Bradford protein assay,
the LAL (limulus amebocyte lysate) test at both the Universi-
ty of Perugia (Italy) and Living Cell Technologies laboratories
(SOP Q209, Belgium) and for metals at Sereco Biotest s.p.a.
(Perugia, Italy). MS inclusion into ALG/PLO/ALG MC was car-
ried out according to a previously optimized method. SOD
and KET release from ALG/PLO/ALG MC was performed in
0.1M pH 7.4 phosphate buffer at 37°C. PLO leakage from the
MC was also investigated by Micro-BCA analysis.
MC transplant and cell overgrowth analysis
The experiment was performed according to a protocol pre-
viously approved by the Ethic Committee of the University
of Perugia (p.n. 19382 PRE 805/2003). CD1 mice (average
weight 25 g, Charles River Laboratories, Wilmington, MA),
divided into (n=9) one control with conventional MC, one
multicompartmental MC with SOD and one with KET, were
terminated at day 10, 20, and 30.
The recovered MC were examined by optical microscopy (Le-
ica, Italy) for cell overgrowth and cells were detached from
the MC and treated with rat anti-CD16/32 (2.4G2) and as-
sayed by EPICS flow cytometer (Beckman Coulter). Rat anti-
F4/80 and anti-CD8 or anti-CD11b, anti-CD25 and anti-CD69
were applied.
131
RESULTS AND DISCUSSION
Protein loading afforded 90-92% efficiency and 9% drug con-
tent. The mean volume diameter ranged between 13 and 18
µm. Span was 2.46. SOD activity within MS was successfully
retained. KET loaded PLA MS showed the same characteris-
tics previously recorded, with mean size around 5 µm and
99% of the population < 20 μm [5]. Drug content and encap-
sulation efficiency were 17% (w/w) and 75%, respectively.
SEM microphotographs highlighted a rather smooth surface
and fairly regular shape for both MS formulations (Fig. 1).
Optical microscopy showed MC of rather regular and uni-
form shape and size (Fig. 2). Both conventional (Panel 2A)
and multicompartmental (Panel 2B, 2C) had the same fea-
tures. All MC showed similar size around 450 µm. The MS dis-
tribution within the MC was homogeneous and no adhesion
to the double coated ALG membrane was observed.
SOD had a sustained release from PLGA MS with just 25% ini-
tial burst and the release reached 90% after 64 days in an al-
most linear manner (r2=0.993) (Fig. 3). SOD release from multi-
compartmental MC barely approached 40% at 30 days with a
slow progression over time and stopped afterwards (Fig. 3).
KET release reached 90% after 7 days from PLA MS, while it
dropped down to 70% from inside the MC. PLO was not re-
leased from the MC as no significant amounts (2-3% w/w)
were found in the release medium up to 10-12 days.
 POSTER PRESENTATIONS

Although the purified ALG was considered biocompatible,

the conventional MC collected at day 10, 20 and 30 were
completely coated by cells (Fig. 4A). In turn, the MC contain-
ing KET became completely covered by cells only at day 20
(Fig. 4B), while the MC with SOD did not show significant cell
growth over the entire post-transplant follow-up (Fig. 4C).

The profile of cell phenotypes recovered from the collected

MC is summarized in Table 1. The control group showed, at
day 10, 50% of F4/80+ peritoneal macrophages, which de-
creased to about 40% at day 30. 30% of the macrophages
expressed a constant percentage of CD11b+ cells over time.
The KET group presented 92% CD11b+ cells since day 10,
which at day 30 decreased down to 51%, while the F4/80+
cells were 61%. The SOD group displayed, at day 20, 61% of
F4/80+ cells, among which 48% expressed also for CD11b+,
while, at day 30, a bare 8% of F4/80+ cells and no CD11b+
phenotype.

T-cell expression at day 30 was about 19% CD8+ in the con-

trol, 6% in the KET and barely 4% in the SOD group. In the
control group, 50% of CD8+ expressed an activated pheno-
type.

132

Figure 2 - Optical observations of the obtained MC: A) conven-

tional and multicompartmental MC with B) SOD and C) KET.

Figure 1 - SEM microphotographs of A) SOD loaded PLGA MS

and B) KET loaded PLA MS.

Figure 3 - In vitro release profiles of SOD loaded PLGA MS

alone and from inside the MC.

3rd BBBB International Conference on Pharmaceutical Sciences

 POSTER PRESENTATIONS

REFERENCES
1. Shapiro AM, Lakey JR, Ryan EA, Korbutt GS, Toth E, et al.
Islet transplantation in seven patients with type 1 diabe-
tes mellitus using glucocorticoid-free immunosuppres-
sive regimen, N. Engl. J. Med. 2000; 343: 230-238.
2. Korbutt GS, Elliott JF, Ao Z, Smith DK, Warnock GL, et al.
Large scale isolation, growth, and function of porcine
eonatal islet cells, J. Clin. Invest. 1996; 97: 2119-2129.
3. Calafiore R Alginate microcapsules for pancreatic islet
cell graft immunoprotection: struggle and progress to-
wards the final cure for type1 diabetes mellitus, Expert.
Opin. Biol. Ther. 2003; 3: 201–205.
4. Ricci M, Blasi P, Giovagnoli S, Rossi C, Macchiarulo G, et
al. Ketoprofen controlled release from composite mi-
crocapsules for cell encapsulation: effect on post-trans-
plant acute inflammation, J. Control. Release 2005; 107:
Figure 4 - Microscopy observations of MC overgrowth at 395–407.
each time point for A) conventional MC and multicompart- 5. de Vos P, Faas MM, Strand B, Calafiore R Alginate-based
mental MC with B) KET and C) SOD. microcapsules for immunoisolation of pancreatic islets,
Biomaterials 2006; 27: 5603–5617.
Table 1: Cell expression on the recovered MC
*F4/80+ (CD11b+) CD8+
Timepoints (days)
Control P050-EVALUATION OF VAGINAL TABLETS
10 50% (30%) - CONTAINING POLYCARBOPHIL AND
20 *** - HYDROXYPROPYLMETHYLCELLULOSE AS
30 40% (30%) 19% MUCOADHESIVE POLYMERS
KET
Aslıhan Hilal Kurtoğlu, Tamer Baykara
10 100% (92%) -
20 *** - 133
Department of Pharmaceutical Technology, Faculty of
30 61% (51%) 6% Pharmacy, Ankara University, 06100 Tandoğan, Ankara/Turkey
SOD
10 n.a.** - INTRODUCTION
Mucoadhesive vaginal drug delivery systems extend the res-
20 61% (48%)" -
idence time in the vagina, so that reduce dosing frequency
30 8% n.d.† 4% and quantity of drug administered. Thus the therapeutic ef-
*Total activated cells = F4/80+, CD11b is a fraction of the ficacy of locally acting drugs may be improved due to the
F4/80+ phenotype. The number of the recovered cell was increased availability of the vaginal epitelium through mu-
qualitatively estimated being in the order: Control group > coadhesion1. The purpose of this work was to develop mu-
KET group >> 500 group >>> SOD group of all timepoints. coadhesive tablets able to prolong the permanence of the
**not applicable. The mass of cells collected at day 10 for the drug in the vaginal cavity. As a model drug we chose a board
SOD group was not sufficient to allow flow cytometry analy- spectrum antifungal agent Miconazole nitrate which is used
sis. As alternative data at day 20 were used. to treat topical fungal and yeast infections. The matrix tablets
***Day 20 for the control and KET groups were not used as a were designed using a combination of bioadhesive polymers
slight contamination was observed. polycarbophil (Noveon® AA1) and hydroxypropyl methylcel-
†
not detected lulose (Methocel K15M) in different ratios. The swelling, in
vitro drug release and ex vivo mucoadhesion properties of
CONCLUSIONS the tablets were evaluated.
In spite of ALG of tested clinical-grade, host’s reactions to the mate-
rial employed cannot be excluded. Therefore, the use of a combina- MATERIALS AND METHODS
tion of KET and SOD releasing MS is recommended to preserve the Materials
efficiency of grafts in the immediate and longer post-transplant fol- Miconazole nitrate (MCZ) (Selectchemie AG, Switzerland),
low-up and to warrant success of cell transplantation and survival. Polycarbophil (PCP) (Noveon® AA1), hydroxypropyl methyl-
cellulose (Methocel K15M) (Dow Chemical Company), Man-
ACKNOWLEDGMENTS nitol (Partect® M100). All other chemicals used were analyti-
This work was supported by the “Consorzio Interuniversitario cal grade.
per i Trapianti d’Organo”, Rome, Italy. The authors wish to thank
Dr Luca Poletti from Sereco Biotest s.p.a. for his support to ALG Preparation of mucoadhesive tablets
metal content analysis. Mucoadhesive tablets were basically formulated PCP alone

October 26 - 28, 2009 / Antalya - TURKEY

 POSTER PRESENTATIONS

and in combination with HPMC. Each tablet contained a RESULTS AND DISCUSSION
constant amount of miconazole nitrate (MCZ), mannitol and Swelling studies of tablets
magnesium stearate (Table 1). The extension in swelling time is favorable for mucoadhe-
sion3 and this time is prolonged by, through increasing
Table 1: The composition of tablet formulations (mg) HPMC concentration in our tablets (Figure 1).
HPMC Magnesium
Formulations MCZ PCP Mannitol
(K15M) stearate
F1 100 100 - 300 5
F2 100 80 20 300 5
F3 100 60 40 300 5
F4 100 50 50 300 5

Figure 1: Swelling index (%) of the mucoadhesive tablets

Tablets were prepared by direct compression of powder
mixture using a hydraulic press (Ayaşlı, Üçler, Ankara, Turkey) In vitro drug release studies
equipped with flat-faced punches of 16 mm in diameter for Increasing HPMC concentration in the tablets forms a viscous
swelling and in vitro drug release studies and 8 mm in diam- gel layer and slows down the release rate of drug by provid-
eter for ex vivo mucoadhesion studies. ing a control4. HPMC also modifies the release profiles of the
drug (F2-F4) as well as regulates the hydration (Figure 2).
Swelling studies of tablets
Swelling properties of the tablets were evaluated by gravi-
metric method. Each tablet was accurately weighed (W1)
and immersed in a petri dish containing pH 4.5 phosphate
buffer solution at 37°C. The swollen tablets were withdrawn
from the medium at the end of predetermined time intervals
and weighed (W2) after removal of excess surface water with
a filter paper (n=3). The swelling index (%) was determined
by Eq.1
134
Figure 2: In vitro release profiles of MCZ from the mucoad-
SI (%) = (W2-W1) / (W1) x 100 Eq.1
hesive tablets
In vitro drug release studies
Ex vivo mucoadhesion studies
Phosphate buffer (pH 4.5) with 35% of 1,4-dioxane was used
Mucoadhesive performance was gradually decreased with
as release medium because of the low solubility of micon-
the increase in HPMC amount in the tablets (Table 2).
azole nitrate. Drug release test from mucoadhesive tablets
was carried out in a shaking water bath at 37°C. Each tablet
Table 2: The work of adhesion of the 8 mm tablets
was put separately in bakers containing 150 ml of release
Formulations Work of adhesion N.mm(mJ)±SE
medium at 37°C. The bakers were shaken horizontally at 50
rpm in the water bath maintaining 37±1°C. Samples of 1 ml F1 0.263±0.017
were withdrawn for 12 hours and replaced with equal vol- F2 0.141±0.017
ume of release medium equilibrated 37°C (n=3). F3 0.090±0.014
Ex vivo mucoadhesion studies F4 0.065±0.023
Mucoadhesion testing of the tablets was carried out using
a texture analyzer2 (TA.XT plus, Stable Micro Systems, UK). Even so PCP has a better mucoadhesion alone, it was pre-
8 mm tablet was attached to the cylindrical probe with dicted from the retardation of the hydration for F2-F4, HPMC
double-sided adhesive tape. Cow vaginal tissue (20x20 mm, tablets could stay on mucosa for an adequate time during
stored at -30°C) was equilibrated for 15 min at 37oC before the treatment.
placing onto the holder stage of mucoadhesive holder and
maintained at 37°C during the test in 700 ml of the medium CONCLUSION
(pH 4.5). A volume of 0.1 ml buffer (pH 4.5) was slowly added Our overall results indicate that, HPMC retards drug release
using Hamilton syringe onto the tablet for pre-hydration and appropriately and has an adequate mucoadhesion prop-
waited for five minutes before the probe was placed. The in- erty when it is combined with PCP. Consequently, mixtures
strumental parameters and test conditions were constant of PCP/HPMC polymers could be preferred for the develop-
during the study. The total amount of the forces involved in ment of mucoadhesive vaginal tablet formulations.
during the probe withdrawal from the tissue (work of adhe-
sion, Wad) was calculated (n=3). ACKNOWLEDGEMENTS
We wish to thank to Ege University, Faculty of Pharmacy,
Department of Pharmaceutical Technology for the permis-

3rd BBBB International Conference on Pharmaceutical Sciences

 POSTER PRESENTATIONS
sion of using the texture analyzer TA.XT plus, Stable Micro
Systems.
REFERENCES
1. Ndesendo VMK, Pillay V, Choonara YE, Khan RA, Meyer L,
Buchmann E, Rosin U. In vitro and ex vivo bioadhesivity
analysis of polymeric intravaginal caplets using physico-
mechanics and computational structural modeling Int J
Pharm. 2009; 370:151-159
2. Thirawong N, Nunthanid J, Puttipipatkhachorn,S, Sri-
amornsak, P. Mucoadhesive properties of various pec-
tins on gastrointestinal mucosa: An in vitro evaluation
using texture analyzer, Eur J of Pharm Biopharm. 2007;
67:132–140
3. Baloğlu E, Özyazıcı M., Hızarcıoğlu SY, Şenyiğit T, Özturt
D, Pekçetin Ç. Bioadhesive controlled release systems of
ornidazole of vaginal delivery. Pharm Dev Technology,
2006;11: 477-484
4. Ceschel GC, Bergamante V, Calabrese V, Biserni S. Design
and evaluation in vitro of controlled release mucoad-
hesive tablets containing chlorhexidine, Drug Dev Ind
Pharm. 2006; 32:53-61
p051-STUDIES ON MUCOADHESIVE
VAGINAL TABLETS OF ECONAZOLE NITRATE
FORMULATED WITH THIOMERS
Esra Baloğlu1, Zeynep Şenyiğit1, Anna Vetter2, Sinem Yaprak
Karavana1, Tamer Güneri1, Andreas Bernkop- Schnuerch2
University of Ege, Faculty of Pharmacy, İzmir, Turkey1
University of Innsbruck, Institute of Pharmacy, Inssbruck, Austria2
INTRODUCTION
The increasing incidence of Candida vaginitis has highlighted
the importance of establishing therapeutic strategies aimed
at ensuring successful eradication of the infectious agent,
short-term treatment, achievement of high drug amount at
the target site and safety. From such a perspective, a ratio-
nal choice for management of vaginitis is topical treatment.
Traditional vaginal delivery systems might not ensure suffi-
cient therapeutic efficacy, most likely short residence time
at the site of administration1. Therefore, topical treatment of
vaginal infections is focused on developing suitable drug de-
livery systems to enable a prolonged intravaginal residence
time for administered drug.
Mucoadhesive vaginal tablets are able to adhere on the mucus
layer of vagina and prolong the residence time of the dosage
form and allows a sustained-release in order to maximize the
therapeutic effect2. A new generation of mucoadhesive poly-
mers, called ‘thiolated polymers’ are promising hydrophilic
polymers derivatised with thiol groups on their side chains.
Due to the formation of inter- and/or intrachain disulphide
bonds, these conjugates show strongly improved cohesive
and excellent mucoadhesive properties3. Econazole nitrate is
an imidazole derivative which plays an important role in the
treatment of diseases caused by different fungal infections 4.
October 26 - 28, 2009 / Antalya - TURKEY
The aim of this study is to prepare the mucoadhesive vagi-
nal tablet formulations of econazole nitrate with thiomers
for the treatment of vaginal candidiasis and to evaluate their
properties in vitro conditions.
Keywords: Thiomers, econazole nitrate, PAA-Cys, vaginal
drug delivery, mucoadhesion
MATERIALS AND METHODS
Materials
Econazole nitrate (EN) and poly(acrylic acid) (PAA) (450 kDa)
were purchased from Sigma-Aldrich (Germany). All other
chemicals were of analytical grade.
Synthesis of poly(acrylic acid)–cysteine conjugates (PAA–Cys)
The PAA–Cys were synthesized according to a method de-
scribed previously5.
Determination of the thiol group content
The amount of free thiol groups immobilized on polymer
backbones was determined photometrically with Ellman’s
reagent. In addition, the oxidized thiol moieties available in
form of disulphide bonds were quantified after reduction
with NaBH4 and addition of Ellman’s reagent.
Particle Size Determination of EN
Particle size of EN was determined using a laser particle size
analyzer (FRITSCH, Germany). The size of the particles was
determined in water as a non-dissolving medium.
Preparation of Tablets
For each tablet, 40 mg of the PAA-Cys conjugate and PAA 135
were hydrated in dematerialized water. 5 mg of EN was add-
ed for each tablet, homogenized and lyophilized. The 45 mg
of polymer-drug lyophilisates were compressed into 5.0 mm
diameter flat faced tablets. The compaction pressure was
kept constant during the preparation of all tablets.
After the preparation of the tablets, the quality control stud-
ies such as weight variation, friability, hardness and diam-
eter/thickness ratio were performed according to USP XXX.
In vitro disintegration studies
Disintegration behavior of the tablets examined using a dis-
integration test apparatus according to European Pharma-
copeia. Tablets were exposured in simulated vaginal fluid at
37 °C and the oscillation frequency was set to 0.5 s−1.
Evaluation of the swelling behavior
The swelling capacity of the tablets was determined by a gravi-
metric method. Tablets were fixed to a needle and immersed in
a beaker containing simulated vaginal fluid at 37°C. At sched-
uled time intervals, the swollen tablets were taken out of the in-
cubation medium, excess water was removed, and the amount
of water uptake was determined gravimetrically. The swelling
ratio was then calculated according to the following equation:
Swelling ratio =Wut/Wo
Wut: weight of uptaken water at time t; Wo: initial weight of
the dry tablet
Mucoadhesion studies via rotating cylinder
Time of adhesion of compressed discs to the bovine vaginal
 POSTER PRESENTATIONS

mucosa was established by the rotating cylinder as follows. The results of tablet quality control studies are shown in
Tablets were attached to freshly excised mucosa which was pro- Table 2.
vided by the local slaughterhouse and fixed on a stainless-steel
cylinder using a cyanoacrylate adhesive. Thereafter the cylinder Table 2: Results of tablet quality control studies
was placed into the dissolution apparatus and completely im- Weight
mersed into the simulated vaginal fluid at 37°C. The cylinder Friability Hardness Diameter/
Formulations variation
was rotated at a speed of 100 rpm. Every 30 min the changes in (%) (kg) Thickness (mm)
(mg)
the test system were observed visually and registered until all
4.963±0.016/
of the discs were either disintegrated or detached from the mu- EN+PAA 48.744±2.362 0.0676 > 16
1.855±0.052
cosa. This test was performed at least five times for each tablet.
4.987±0.008/
EN+PAA-Cys 46.850±2.076 0.426 > 16
Release studies 1.834±0.0533
The release rate of EN from tablets was determined in vitro us-
ing a perfusor as it was shown in Figure 1. Tablets were placed Tablets prepared with unmodified PAA showed a complete
into tubes and kept at 37±0.5°C with a heater. A perfusor (Per- disintegration and erosion within 5.4±1.140 hours. On the
fusor Fm, Braun) was used to give 6 mL of vaginal fluid to tab- other hand, thiolated tablets showed no disintegration or
lets in 24 hours because the daily production of vaginal fluid erosion within 72 hours.
is approximately 6 mL/day and 0.5-0.75 mL continually present
in the vagina6. The same amount of samples was collected con- The swelling ratio of the tablets in simulated vaginal fluid at
currently. The samples were assayed for EN content by HPLC. 37°C is shown in Figure 3.

136
Figure 1: Schematic drawing of the in vitro release studies

RESULTS AND DISCUSSION Figure 3: Swelling behaviour of EN+PAA tablets () and
The amount of free thiol groups and disulfide bonds of the EN+PAA-Cys tablets () simulated vaginal fluid at 37°C. Indi-
polymer are listed in Table 1. cated values are the me

Table 1: The amount of thiol groups and disulfide bonds im- The swelling state of the polymer contributes to its mucoadhe-
mobilized on PAA sive behavior. The formulation prepared with PAA-Cys showed
longer mucoadhesion time than PAA formulation (Figure 4).
-SH [µmol/g polymer ±S. D.] -S-S [µmol/g polymer ±S. D.] It was thought that the presence of thiol groups allowed the
formation of covalent bonds with cysteine- rich sub domains
PAA-Cys 172.80 ± 30.33 377.93 ± 85.10
of the mucus gel layer, leading to increased residence time.
The mean size of econazole nitrate in distilled water was
determined to be 34.245 µm. Size distribution results are
shown in Figure 2.

Figure 4: Comparison of the mucoadhesion time of tablets

on the rotating cylinder. Indicated values are means ±SD of
at least three experiments.
Figure 2: Size distribution of econazole nitrate

3rd BBBB International Conference on Pharmaceutical Sciences

 POSTER PRESENTATIONS
The presence of disulphide bonds may significantly alter the
mechanism of drug release from the delivery system due to
increased rigidity and cross linking7. Figure 5 showed the sig-
nificant difference between the release profiles of EN from
the formulation containing thiomer or not.
Figure 5: Release rate of EN+PAA tablets () and EN+PAA-
Cys tablets () simulated vaginal fluid at 37°C. Indicated val-
ues are the means of
CONCLUSION
The overall results obtained during this study suggested that
thiolated EN tablet formulation can be inferred as a prom-
ising candidate for topical treatment of Candida vaginitis.
It was planned that the formulation would be evaluated in
vivo.
ACKNOWLEDGEMENTS
This work was financed by The Scientific and Technological
Research Council of Turkey (106/S/196).
REFERENCES
1. Knuth K, Amiji M, Robinso JR. Hydrogel delivery system
for vaginal and oral applications, Adv Drug Delivery Rev.
1993; 11:137-167.
2. Vermani K, Garg S. The scope and potential of vaginal
delivery, Pharm Sci Tech Today. 2000; 3:1-9.
3. Leitner VM, Walker GF, Bernkop-Schnürch A. Thiolated
polymers: evidence for the formation of disulphide
bonds with mucus glycoproteins, Eur J Pharm Biopharm.
2003; 56:207-214.
4. Czeizel AE, Vargha P. A population-based case–control
teratological study of vaginal econazole treatment dur-
ing pregnancy, Eur J Obstet Gynecol Reprod Biol. 2003;
111:135–140.
5. Bernkop-Schnürch A, Schwarz V, Steininger S. Polymers
with thiol groups: a new generation of mucoadhesive
polymers?, Pharm Res. 1999; 16:876–881.
6. Bernkop-Schnürch A, Steininger S. Synthesis and Char-
acterisation of mucoadhesive thiolated polymers, Int J
Pharm. 2000; 22;1861-1867.
7. Andrews GP, Laverty TP, Jones DS. Mucoadhesive poly-
meric platforms for controlled drug delivery. Eur J Pharm
Biopharm. 2009; 71:505–518.
October 26 - 28, 2009 / Antalya - TURKEY
P052-Liposomal Gels for Vaginal Drug
Delivery of Cidofovir
Fatmanur Tuğcu Demiröz1, Fusun Acartürk1
Department of Pharmaceutical Technology, Faculty of
Pharmacy, Gazi University, Ankara, Turkey1
INTRODUCTION
Vaginal delivery of local and systemically acting drugs has in-
creased over the past decade presumably reflecting greater
attention to women’s health care1. Liposomes can provide
a controlled release of an incorporated drug, phospholipid
vesicles have a potential as vaginal delivery system. The hy-
drogels prepared from Carbopol resins as a vehicle for incor-
poration of liposomes destined for vaginal delivery2.
Cervical cancer which is mainly associated with Human Papil-
loma Virus (HPV) infection is the second most common cause
of cancer death among women worldwide affecting approx-
imately 1% of all female population3. The therapy regimens
for HPV-related genital diseases include the administration
of antiviral agents such as cidofovir (CV). CV is a prototype of
antiviral compounds, which is active against HPV, as well as
against a large number of other DNA viruses4.
Here, we report the development of a liposomal delivery
system for vaginal administration of CV, able to provide con-
trolled release of drug. CV was entrapped in liposomes pre-
pared by the polyol dilution method (PD), whereby different
phospholipid compositions were used.
137
MATERIALS AND METHODS
Materials
L-alpha-Dipalmitoyl phosphatidylcholine (DPPC) and 1-2 Dipalm-
itoyl-sn-glycero-3- phosphatidylcholine (DGPC) were purchased
from Acros Organics, USA and Carbopol 974P NF from Noveon,
USA. Stearylamine (SA) was also provided from Sigma, USA. Cido-
fovir was generous gift from Gilead Sciences, Inc., USA.
Preparation of Liposomes
Liposomes of CV were prepared according to the PD method5.
After preparation, the liposome suspension was extruded
through polycarbonate filter (pore size, 400 nm) by using a Mini
Extruder (Avanti Polar Lipids). PS2 formulation was prepared by
the freeze-thawing (FT) method5. For this purpose, preparations
were rapidly frozen in liquid nitrogen and then brought to 37°C
to thaw completely. The procedure was performed five times.
The composition of the liposomes are given in Table 1.
Particle Size and Zeta Potential of the Liposomes
The particle size and ζ-potential of liposomes were deter-
mined using Zetasizer Nano Series (Nono-ZS, Malvern Inst.,
UK). The analysis was performed at a temperature of 25°C,
using samples appropriately diluted with distilled water.
Encapsulation Efficiency Determination
The encapsulation efficiency (EE) was calculated indirectly
by determining the remaining concentration of CV in the
supernatant after centrifugation. Samples were ultracentri-
 POSTER PRESENTATIONS

fuged at 44,803 g for 1 h. The supernatant containing the dis- leased. The release of CV from the P2 formulation prepared with
solved free CV was used to determine the concentration. The DPPC and DGPC was observed to be high. Drug release varied
EE was calculated using the following equation: with the lipid content in the formulations. The PS2 formulation
exhibited a drug release of 88.1±2.1 % at the end of 48 hours.
EE = [(CVtot−CVsup)/ CVtot]x100 Thus, considering the release of liposomes during vaginal ad-
ministration, as well as controlled release of CV, hydrogels made
where, CVtot is the total amount of CV and CVsup is the of Carbopol 974P would be the proper choice of vehicle.
amount of CV in supernatant after centrifugation.
Table 1: Composition and preparation method of the lipo-
Preparation of Carbopol Gel Formulations some formulations
Carbopol 974P NF (1 g) was dispersed in demineralised water Liquid Preparation
(88 g) by stirring at 800 rpm for 60 min. Then propylene glycol Code CV (Molar Ratio) Molar Ratio
Composition Method
(10 g) was added and the mixture was neutralised by drop-wise P1 - DPPC: DPPC 9:1 PD
addition of 10% NaOH. Mixing was continued until a transpar- P2 0.5 DPPC: DPPC 9:1 PD
ent gel appeared, whereas the amount of base was adjusted to PS1 0.5 DPPC:S4 9:3 PD
achieve a hydrogel with pH 4.5. Carbopol 974P gel was kept at
PS2 DPPC:S4 9:3 PD-FT
+4°C overnight before application, in order to remove air bub-
bles. The PS2 liposomes added to the gel in the ratio of 10:90 to
Table 2: Characterization of liposomes
prepare the liposomal gel formulation PS2KR.
Code of liposomal Mean Zeta Potential Encapsulation
Drug Diffusion Studies of the Liposomes and Liposomal Gels gel formulation Diameter (nm) (mV) efficiency (EE)(%)
All glass Franz type diffusion cells were used. The diffusional P1 286.2±0.8 -0.522±0.171 -
cross sectional area was 1 cm2. Dialysis membrane (Sigma®, P2 298.7±0.9 -0.488±0.354 26.4
USA) having a pore size of 12,000 Da was used. The stirred recep-
PS1 326±9 2.54±0.405 15.53
tor phase (2.5 mL) containing citrate phosphate buffer (pH 4.5)
was thermostatted at 37°C. The donor compartment contained PS2 315±5 30.83±2.79 41.09
liposomes or liposomal gel formulation. Samples were taken
periodically from the receptor phase. Permeant concentrations
were determined by spectrophotometrically at 278.4 nm.

138 RESULTS AND DISCUSSION

Characterization of liposomes
Particle size of empty liposome formulation P1 was found
to be 286.2±0.8 nm. As shown in Table 2, liposome formu-
lations P1 and P2 had statistically significant difference be-
tween mean particle diameters (p>0.05). The modification of
liposomes with SA increased the particle size, depending on
its molar ratio (PS1 and PS2 formulations).
Figure 1: Diffusion profiles of CV from liposomes and lipo-
The zeta potential values of P1 and P2 formulations were somal gel formulations through dialysis membrane (n=6;
negative. The addition of SA caused a higher positive surface bars represent SD)
charge. Incorporation of DPPC or SA into liposome mem-
brane resulted negative zeta potential values for DPPC/DGPC CONCLUSION
liposomes or positive values for DPPC/SA liposomes respec- The polyol dilution method has been proven to be simple, repro-
tively. Regarding the encapsulation of CV, P2 liposomes en- ducible and appropriate for encapsulation of CV. Negatively and
capsulated 26.4% of the amount of drug. However, positively positively charged liposomes yielded different encapsulation ef-
charged PS2 liposomes prepared by the FT method encap- ficiencies. Incorporation of liposomes in to bioadhesive Carbopol
sulated a higher amount of the drug (41%). This results have 974P gel (PS2KR formulation) further confirmed for the applicabil-
shown that preparation method can affect the encapsula- ity of liposomes as a novel vaginal delivery system for controlled
tion of CV and the zeta potential of liposomes. release of encapsulated CV for the local treatment of genital HPV.

Drug Diffusion the Liposomes and Liposomal Gels ACKNOWLEDGMENTS

The drug diffusion profiles of CV from liposome formula-
tions are shown in Figure 1. P2 formulation released 99.5±2
% of the drug at the end of 48 h. PS1 and PS2 formulations
containing SA released 96.4±1.8 and 88.1±2.1 % of the drug
throughout the diffusion studies, respectively (Fig. 1).
This study was supported by a research grant (SBAG 2807-
104S324) from the Scientific and Technical Research Council
of TURKEY (TÜBİTAK) and Research Foundation of Gazi Uni-
versity (02/2006-08). We would like to thank Gilead Sciences,
Inc., USA for the gift sample of Cidofovir.

It was observed that, the liposomal gel formulations with Car- REFERENCES
bopol 974P released about 40.4 % of CV at 48 h. This showed 1. Acartürk F, Robinson JR, Pharm. Res. 1996; 13: 950-951.
that, liposomal gels almost prevented the drug from being re-

3rd BBBB International Conference on Pharmaceutical Sciences

 POSTER PRESENTATIONS

2. Pavelić Ž, Škalko N, Jalšenjak I, Eur. J. Pharm. Sci. 1999; 8: 345–351.
3. Bilensoy E, Çırpanlı Y, Şen M, Doğan L, Çalış S, J Incl Phe-
nom Macrocycl Chem. 2007; 57: 363-370.
4. Gross G, Int. J. Antimic. Agents. 1999; 12: 1-3.
5. Pavelić Ž, Škalko-Basnet N, Schubert R, J. Control. Rel.
2001; 219: 139-149.
p053-THE INFLUENCE OF CHEMICAL
ENHANCERS ON SKIN ACCUMULATION OF
TOPICAL CORTICOSTEROIDS FROM GEL
FORMULATIONS
Taner Şenyiğit1, Cristina Padula2, Özgen Özer1, Patrizia Santi2
Ege University, İzmir, Turkey1
Universita degli Studi di Parma, Dipartimento Farmaceutico,
Parma, Italy2
INTRODUCTION
Topical glucocorticoids (TG) are the most frequently prescribed
drugs by dermatologists. Despite their benefit in the therapy of
inflammatory diseases, TG are associated number of side effects
that limit their use1. In this study, two TG were selected as model
active agents: clobetasol-17-propionate (CP) and mometasone
furoate (MF). One of the approaches to reduce the systemic
adverse effects of TG is to enhance their permeability so as to
reduce the topically applied dose2. The use of chemical penetra-
tion enhancers is the most widely used approach to increase
topical and transdermal delivery3. Two terpene derivatives (D-
limonene and nerolidol) and Transcutol® P were evaluated as
chemical penetration enhancers in this study. The aim of this
work was to investigate the effect of chemical enhancers on the
skin accumulation and permeation of CP and MF from chitosan
gels. The goal was to increase skin retention without increasing
penetration, to reduce the possible side effects of these drugs.
ethyl alcohol-PBS (3:7) mixture thermostatted at 37°C, was about
4 ml. Infinite dose regimen was applied in all experiments.
At the end of the 6 h permeation experiments, the excess amount
of the formulation was removed and SC and epidermis were sep-
arated from dermis with heat application. The drug was extracted
using acetonitrile:water (60:40) mixture. The amount of CP or MF
in the permeation and accumulation samples was determined by
HPLC using Luna C18 (2) 150 mm x 3 mm column (Phenomenex,
USA) and a mobile phase composed of acetonitrile/water (55:45)
at 1 ml/min. UV detection at 240 nm was employed.
Statistical Analysis
All experiments were replicated six times. Statistical differ-
ences were determined using ANOVA followed by Dunnet
multiple comparison test.
RESULTS AND DISCUSSION
In vitro permeation and accumulation of CP and MF in pig
ear skin layers after passive diffusion was studied using chi-
tosan gel containing different penetration enhancers as do-
nor reservoir. During the permeation studies, CP or MF was
never found in the receptor medium at the end of 6 h.
CP or MF skin concentration was calculated by normalizing the
amount of drug recovered in the SC + epidermis and dermis by the
weight of tissues and expressed as μg of drug per mg of tissue.
Figure 1 and Figure 2 present the effect of chemical enhanc-
ers on the accumulation of CP and MF in the epidermis and
dermis, respectively. 139

MATERIALS AND METHODS

Materials
CP and MF were kind gifts from GlaxoSmithKline (Turkey)
and Orva Drug Company (Turkey), respectively. Medium
molecular weight chitosan from Sigma (Germany). All other
chemicals were of analytical grade.
Figure 1: Effect of enhancers on the accumulation of CP (dark
Preparation of Chitosan Gels bars) and MF (light bars) in the epidermis. *significantly dif-
For the preparation of chitosan gel, 2 % chitosan was dis- ferent from chitosan gel.
solved in 1.5 % (w/v) acetic acid solution. 0.05 % CP or 0.1 %
MF was dissolved in 10 % PEG-400 and added to the chitosan
solution with continuous stirring until uniformity.

The concentration of terpenes and Transcutol P in chitosan

gels was 2 and 20 %, respectively.

In Vitro Permeation Experiments

The in vitro permeation experiments were performed using
Franz-type diffusion cells across pig ear skin. Previous studies
demonstrated that pig skin is a reasonable model for the human
barrier in passive condition4. The available diffusion area was 0.6
cm2 and the volume of the receptor compartment, containing

October 26 - 28, 2009 / Antalya - TURKEY

 POSTER PRESENTATIONS
Figure 2: Effect of enhancers on the accumulation of CP (dark
bars) and MF (light bars) in the dermis. *significantly differ-
ent from commercial cream
In the case of CP, nerolidol provided a significant enhance-
ment of both epidermis and dermis retention (p < 0.01 and
p < 0.05, respectively). Besides, D-limonene improved only
dermis accumulation (p < 0.01) while, surprisingly, Transcu-
tol had only a modest, non significant, effect on epidermis
retention.
When the effect of enhancers on MF accumulation data is
considered, only nerolidol produced a statistically signifi-
cant increase at the epidermis level (p < 0.05). No significant
increment was observed in the dermis accumulation of MF
with three penetration enhancers tested.
CONCLUSION
According to the experimental findings of this study, it was
showed that Nerolidol resulted to be the best chemical pen-
etration enhancer among the three enhancers tested, with
significantly higher accumulation data especially in the epi-
dermis of both active agents. D-Limonene and Transcutol
did not increase the skin accumulation of active agents.
REFERENCES
1. Wiedersberg S, Leopold CS, Guy RH. Bioavailability and
bioequivalence of topical glucocorticoids, Eur J Pharm
Biopharm. 2008; 68:453–466.
2. Fang JY, Fang CL, Sung KC, Chen HY. Effect of low fre-
140 quency ultrasound on the in vitro percutaneous ab-
sorption of clobetasol 17-propionate, Int J Pharm. 1999;
191:33-42.
3. Moster K, Kriwet K, Naik A, Kalia YN, Guy RH. Passive skin
penetration enhancement and its quantification in vitro,
Eur J Pharm Biopharm. 2001; 52: 103-112.
4. Sekkat N, Kalia YN, Guy RH. Biophysical study of porcine
ear skin in vitro and its comparison to human skin in
vivo, J Pharm Sci. 2002; 91(11): 2376-2381.
p054-Evaluation of transdermal
absorption and anti-inflammatory
and analgesic activities of naproxen
through rat skin: ex vivo and in vivo
H. Yeşim Karasulu1, Neslihan Üstündağ1, N.Ülkü Karabay
Yavaşoğlu2, Şebnem Apaydın3
Department of Pharmaceutical Technology, Faculty of
Pharmacy, University of Ege1
Department of Biology, Faculty of Science, Ege University2
Center for Drug R&D and Pharmacokinetic Applications, Ege
University3
INTRODUCTION
Naproxen is a non-steroidal anti-inflammatory drug (NSAID)
compound with analgesic and antipyretic effects, used for
treatment of rheumatoid arthritis, osteoarthritis and trau-
matic contusions. Oral therapy of NSAIDs is very effective,
3rd BBBB International Conference on Pharmaceutical Sciences
but the clinical use is often limited because of their potential
to cause adverse effects such as irritation and ulceration of
the gastro-intestinal mucosa. Administration of these agents
via dermal route can bypass these disadvantages of the oral
route and may maintain relatively consistent plasma levels
for long term therapy from a single dose1. Microemulsions
have several advantages such as enhanced drug solubility,
good thermodynamic stability, enhancing effect on trans-
dermal ability over conventional formulations2. The aim of
this study is to develop new naproxen microemulsion formu-
lations as drug carrier for topical application and to compare
the ex vivo and in vivo permeation rate of naproxen from
these formulations with the commercial formulation (C).
MATERIALS AND METHODS
Materials
Naproxen (Deva Holding, Turkey (gift)), Isopropyl myristate
(IPM) (Sigma, Germany), Span 80 (Merck, Germany),Labrafil-
M (Gattefosse, France), Labrasol (Gattefosse, France), Ethanol
(J.T.Baker, Holland), Cremophor-EL (Sigma, Germany), Isopro-
pyl alcohol (IPA) (Merck, Germany), Methanol (Merck, Hohen-
brunn, Germany), Acetonitrile (Sigma, Aldrich, Germany). All
the other chemicals and reagents used were of HPLC or ana-
lytical grade samples.
Preparation of w/o and o/w microemulsion formulations
Microemulsions were prepared using isopropyl myristate
(IPM) as oil phase, Span 80, Labrafil-M, Labrasol, Cremophor-
EL as surfactants, ethanol, isopropyl alcohol as co-surfac-
tants and 0.5 N NaOH solution as aqueous phase. M1 and M2
formulations were loaded with 10% naproxen, M3 and M4
formulations are blank.
Characterization of microemulsions
The physicochemical properties of microemulsions were
measured.
Ex-Vivo permeation studies
Diffusion cell was used in the permeability studies of naprox-
en. The apparatus consisted of clamped preconditioned male
rat abdominal skin on to glass diffusion cell between donor
and receptor compartments. Phosphate buffer pH 7.4 (10
mL) (600 rpm, 37oC) was used in the receptor compartment.
The samples were withdrawn at predetermined time inter-
vals then, immediately analyzed HPLC system consisting of a
UV spectrometric detector and C18 column (270 nm), direct-
ly. The mobil phase contained methanol/acetonitrile/ puri-
fied water (20/28/52 v/v/v) and 0.4mL triethylamine . Three
replicates of each experiment were performed.
Assessment of anti-inflammatory activity
The anti-inflammatory activity was evaluated by the carra-
geenan-induced paw edema test in the rat. Male Wistar rats
were deprived of food overnight and treated by dermal route
with M1, M2, M3, M4, C and serum physiologic (SP), 60 min-
ute before 0.1 mL 1% carrageenan in isotonic saline was in-
jected subplantarly into left hind paw. The contralateral paw
was injected with 0.1 mL saline and used as a control. Paw
volume was measured by water plethysmometer (n = 7).
Assessment of Analgesic activity
 POSTER PRESENTATIONS

Tail flick test Permeation rate from microemulsion formulations and com-
The tail flick test was evoked by a score of radiant heat, which mercial formulation were given Figure 1.
was focused on the dorsal surface of the tail. Rats were ex-
amined for latency (seconds) to withdraw their tails from a
noxious thermal stimulus using a tail-flick meter (MAY-TF
0703, Turkey). Each rat was then tested before and 30, 45, 60,
75, 90, 105, 120 and 180 minute after the topical administra-
tion of formulations (n = 7) (4).

Hot plate test

Rats were placed on aluminium hot plate (MAY-AHP 0603,
Turkey) kept at a temperature of 62±0.5 oC for a maximum
Figure 1: Permeation rate from microemulsion formulations
time of 30 s. Reaction time was determined (when animals
and commercial formulation (n=3)
licked their fore and hind paws and jumped) before and 15,
30, 45, 60, 90, 120 and 180 min after the topical application
Statistical comparison of flux values obtained from ex vivo
of formulations (n = 7) (4).
studies throughout 24 h showed that most of the microe-
mulsions provided fluxes (P<0.05) higher than the commer-
Statistical data analysis
cial formulation.
The statistical analysis of values, were performed using a one
way anaysis of variance (ANOVA) to test the difference be-
The intraplantar injection of carrageenan caused a time-
tween the means of microemulsion formulations and com-
dependent paw edema in the rat, although saline injection
mercial formulation permeation experiments. Data with P <
caused no swelling. Dermal application of formulations (M1,
0.05 are considered statistically significant.
M2, C) inhibited paw swelling. However, M3, M4 and SF for-
mulations did not inhibit paw swelling (Figure 2).
RESULTS AND DISCUSSION
Composition of the microemulsion formulations were given
in Table 1. M1, M2 formulations contain naproxen, however
M3, M4 are blank microemulsion formulations.
Table 1: Composition of the Microemulsion Formulations
Formulation(%) M1(w/o) M2(o/w) M3(w/o) M4 (o/w)
IPM 26.22 8.53 26.22 8.53 141
Labrafil-M - 3.964 - 3.964
Labrasol 1.38 - 1.38 -
Span 80 6.91 - 6.91 -
Cremophor EL - 11.893 - 11.893
Figure 2: Volume increase % during 6 hours (n=7) for M1,M2,C,
Ethanol 58.10 - 58.10 - 1,2,3 h after the injection, p<0.05; according to SP. For M1,
IPA - 47.572 - 47.572 M2, 4,5,6 h after the inje
Distilled water 7.38 28.04 7.38 28.04
Naproxen 10 10 - - C, M1 and M2 administration increased the tail-flick and hot
HLB value 5.916 10.375 5.916 10.375 plate latencies when compared with control rats (SP, M3 and
The physicochemical parameters of the microemulsions M4 administration on rats) (Figure 3 and Table 3).
were listed in Table 2. In the skin irritation study, no formulation was found to be
irritant.
Table 2: Characterization of microemulsion formulations
Formulation /
M1 M2 M3 M4
Characterization
pH 6.39 ±0.025 6.62± 0.020 7.12 ± 0.01 8.3± 0.025
15.33± 10.33± 15.33± 10.33±
Viscosity (cP)
0.577 0.577 0.577 0.577
1.405 ± 5.617 ±
Droplet size (nm) 1.007±0.011 4.583±0.560
0.040 0.199
141.5±0.1 0.51±0.01 149.5 ± 0.1 0.59±0.02
Conductivity
(µs/cm) (ms/cm) (µs/cm) (ms/cm)
0.922 ± 0.875 ± 0.925 ±
Density (g/mL) 0.881±0.001
0.025 0.010 0.025
1.405 1.413 ± 1.452 ± Figure 3: Tail flick results, *p<0.05 according to SP (n=7)
Refractive index 1.461±0.002
±0.002 0.001 0.002
PIT (oC) 41 ± 0.006 61 ± 0.020 41 ± 0.006 61 ± 0.020

October 26 - 28, 2009 / Antalya - TURKEY

 POSTER PRESENTATIONS

Table 3: Hot plate results (n=7) results can be found on the effectiveness of antifungal treat-
0 15 30 45 60 90 120 180 ment. Only three of the papers were based on double-blind,
Group
(min.) (min.) (min.) (min.) (min.) (min.) (min.) (min.) placebo-controlled studies and of these three only one was
4.85± 4.75± 4.90± 4.85± 4.70± 4.40± 4.57± 4.34± multicentric, making the interpretation of the results rather
SP (sn)
0.15 0.22 0.24 0.18 0.22 0.15 0.14 0.14 difficult. The treatment period ranged between 4 and 80
4.71± 5.08± 8.41± 9.28± 8.87± 7.41± 6.52± 5.34± weeks, and the form of drug/placebo application also varied.
M1 (sn)
0.28 0.23 0.72 *# 0.44 *# 0.43 *# 0.35 *# 0.28 *# 0.22 * The concentration and the daily amount of amphotericin B
M2 (sn)
4.55± 5.02± 8.31± 9.55± 9.54± 8.28± 6.91± 5.81± recommended also differed. The conditions of drug storage
0.31 0.29 0.70 *x 0.45 *x 0.42 * 0.37 *x 0.32 *x 0.39 *x and checking the stability of the amphotericin B solutions
4.41± 8.35± 9.38± 8.88± 9.02± 8.54± 5.62± 4.87± was completely ignored in most cases. Due to the fact that
C (sn)
0.24 0.85 * 0.38 * 0.70 * 0.59 * 0.73 * 0.25 * 0.19 the patient compliance was examined in only one study it
4.85± 4.71± 4.68± 4.75± 4.51± 4.51± 4.48± 4.52± is extremely difficult to draw conclusions concerning the ef-
M3 (sn)
0.42 0.32 0.40 0.37 0.37 0.41 0.39 0.42 fectiveness or ineffectiveness of amphotericin B treatment.
4.54± 4.34± 4.28± 4.34± 4.35± 3.94± 3.94± 3.92± Thus during the preparation phase of the pilot study at Pécs
M4 (sn)
0.25 0.21 0.29 0.17 0.22 0.22 0.24 0.20 we had to face several pharmaceutically important questions
* SP; p<0.05, # M3; p<0.05, x M4; p<0.05
such as the preparation of study samples; optimal dosage
form; proper concentration of the active ingredient; optimal
CONCLUSION storage conditions, stability and analysis of samples and the
According to the ex vivo and in vivo studies, it can be con- compliance of the included patients. The significance and
cluded that when naproxen was incorporated into the brief summary of these topics is introduced and the outcome
microemulsion, its permeation rate from the formulation of the pilot clinical study is presented by the authors.
increases. Furthermore, microemulsion formulation contain-
ing naproxen may be an appropriate vehicle for the topical MATERIALS AND METHODS
application of naproxen. As the current work is a summary of different projects in di-
verse pharmaceutical sciences, the detailed description of
REFERENCES the materials and methods can be found in recently pub-
1. Beetge E, Plessis E, Müler J, Goosen DG, Rensburg FJ. The lished papers.1, 2, 3, 4
influence of the physicochemical characteristics and
pharmacokinetic properties of selected NSAID’s on their RESULTS AND DISCUSSION
transdermal absorption, Int. J. Pharm. 2000; 193: 261- In a double-blind, randomized, placebo-controlled study,
264.
142 33 patients received amphotericin B or placebo nasal sprays
2. Chen H, Chang X, Yang X. Microemulsion-based hydro- for 12 months after endoscopic polypectomy. Our aim was
gel formulation of ibuprofen for topical delivery. Int. J. to determine whether any difference could be observed
Pharm. 2006; 315: 52-58. between the two groups in the rates of recurrence of nasal
3. Guneli E., Yavaşoğlu-Karabay N.Ü., Apaydın, Ş., Uyar M., polyposis, in the symptoms, in the quality of life or in the end-
Uyar M. Analysis of the Antinociceptive Effect of System- socopic findings. The protocol of the study was approved by
ic Administration of Tramadol and Dexmedetomidine the Regional Ethics Committee of the University of Pécs. Our
Combination on Rats Models of Acute and Neuropatic patients displayed marked improvements in the sinus com-
Pain, Pharmacology, Biochemistry and Behavior. 2007; plaints by the end of the 1-year course of treatment, though
88: 9-17. without a significant difference in the extent of change be-
tween the two groups in the primary (computer tomogra-
phy evaluation) and secondary (sinonasal symphtoms and
p055-CLINICAL AND ANALYTICAL ASPECTS quality of life) outcome measures. Our clinical study does
OF THE IMPROVEMENT OF A LOCAL not justify the need for the administration of amphoteri-
ANTIFUNGAL PREPARATION FOR THE cin B nasal spray for 12 months following endoscopic nasal
TREATMENT OF NASAL POLYPOSIS polypectomy.

Andras Fittler1, Lajos Botz1
Department of Pharmaceutics and Central Clinical Pharmacy1
Pharmaceutical Institute, Faculty of Medicine, University of
Pécs, Pécs, Szigeti u. 12, Hungary H-76242
INTRODUCTION
In 2006 a double blind, randomized pilot clinical study was
launched to assess the efficacy of long term intranasal anti-
fungal treatment in patients suffering from chronic rhinosi-
nusitis with nasal polyposis, because it has been hypnotized
that fungal cells in the sinuses exacerbate adverse immune
response that result in the formation of nasal polyps. Several
studies have been published on the issue and controversial
The knowledge of patient adherence (the extent to which
the patient’s actual history of drug administration corre-
sponds to the prescribed regimen) is important both in
medical research and in clinical practice. The results of clini-
cal trials cannot be interpreted realistically without adher-
ence information. Two indirect methods were used for the
measurement of adherence: (1) recording of the amount of
medication self-administered and (2) self-reporting by the
patient via the Hungarian translated version of the standard-
ized BMQ revised in 2003. Assessments were made of how
the adherence changed during the 12-month period, which
factors influenced the attitudes of the patients and what
adverse events were reported. It can be stated that patient
adherence was relatively good; only a moderate decline was

3rd BBBB International Conference on Pharmaceutical Sciences

 POSTER PRESENTATIONS
seen during the 12 months. At the end of the study, the over-
all adherence was 94,7 ± 11,5 %. During the last month of
treatment, 55,6 % of the patients were totally adherent, in
37 % sporadic non-adherence was measured, and only 2 pa-
tients (7,4 %) were categorized as non-adherent.
Stability testing requires the measurement of the active
ingredient. Analysis and quantitaive determination of poly-
enes can be carried out with different methods such as
chemical and microbiological analysis. The two types of
analytical methods differ fundamentally from each other.
With the aid of chemical methods, those parameters (polar-
ity, light absorption) are studied which are associated to the
molecular structure of the molecule. On the other hand with
the aid of biological assays directly the biologic effect can
be measured. In our three month stability test we investi-
gated the stability of 5mg/ml amphotericin B solutions (Fun-
gizone®) with chemical (spectrophotometry) and biological
(bioassay) detection. The effect of storage temperature and
the addition of 5% glucose was evaluated on the stability of
the solutions for three months. The two detection methods
showed strikingly different results. According to the chemi-
cal analysis the samples are considered relatively stable un-
der all observed conditions. As opposed to chemical analysis,
bioassay showed complete loss of antifungal activity after 35
days of storage, at room temperature and notable decrease
was observed at 4°C. These results drew our attention to
the major differences that can be detected with the two dif-
ferent methods. Further analytical studies were performed
with high performance liquid chromatography (HPLC) and
agar diffusion (bioassay) to get better understanding of the
changes in the amphotericin B solutions during storage.
We have optimized previously used HPLC eluent systems
and with the aid of a gradient program, four heptaene com-
ponents could be detected besides AmB main component
at 407 nm. The amount of these constituents is about one
hundredth of the main AmB. Only traces of tetraenes were
detected in the samples at the wavelength of 305 nm.
In our bioassay study we evaluated five commonly used as-
say media and two test microorganisms for the quantitative
determination of amphotericin B in a water base solutions.
Our result showed, that Mueller Hinton agar supplemented
with 2 % glucose and 0,5 µg/ml methylene blue, inoculated
with C. albicans (ATCC 90028) proved to be the most suitable
bioassay parameters for the measurement of amphotericin
B during a stability test, where a relatively wide concentra-
tion change is to be measured. The linear segment of the log
dose-response curve was between 1,54-60,0 μg/ml and the
inhibition zones could be read easily and accurately.
CONCLUSION
Our poster illustrates the complex view of hospital pharma-
cists which can be highly beneficial during the preparations
for and execution of clinical studies.
REFERENCES
1. Gerlinger I, Fittler A, Fónai F, Patzkó A, Mayer A, Botz L,
Postoperative application of amphotericin B nasal spray
in chronic rhinosinusitis with nasal polyposis, with a re-
October 26 - 28, 2009 / Antalya - TURKEY
view of the antifungal therapy, Eur Arch Otorhinolaryn-
gol. 2009; 266: 847-55.
2. Gerlinger I, Fittler A, In reference to The Effect of Topi-
cal Amphotericin B on Inflammatory Markers in Patients
with Chronic Rhinosinusitis: A Multicenter Randomized
Controlled Study, Laryngoscope. 2009; 119:401-408
3. Fittler A, Mayer A, Kocsis B, Gerlinger I, Fónay F, Botz L,
Stability testing of amphotericin B nasal spray solutions
with chemical and biological analysis, Acta Pharm Hung.
2007; 77: 159-64.
4. Fittler A, Matus Z, Kocsis B, Botz L, Chemical and micro-
biological aspects of the quantitative analysis of ampho-
tericin B, Acta Pharm Hung. 2008; 78:95-102.
P056-CO-ENZYME Q10 LOADED SOLID LIPID
NANOPARTICLES INCORPORATED INTO HPMC
GELS: IN VITRO ANTIOXIDANT ACTIVITY
Evren Homan Gökçe1, Emrah Korkmaz1, Sibel Konyalıoğlu2,
Özgen Özer1
Department of Pharmaceutical Technology, Faculty of
Pharmacy, University of Ege, 35100, Izmir, Turkey 1
Department of Biochemistry, Faculty of Pharmacy, University
of Ege, 35100, Izmir, Turkey 2
INTRODUCTION
Solid Lipid Nanoparticles (SLNs) are lipidic colloidal systems
with an inner structure based on pure solid lipids. Advantag-
143
es of SLNs for dermal and transdermal applications include
skin occlusion, modulation of drug/cosmetic release, incre-
ment in skin hydration and elasticity, UV blocking effects,
drug targeting and enhancement of stability1. Coenzyme
Q10 (Q10) is known as an endogenous cellular antioxidant.
In terms of dermal/cosmetic applications, this active has
shown the ability to reduce photoaging in vivo with a cor-
responding decrease in wrinkle depth. Due to its structure,
the aqueous solubility is very low, causing difficulties in for-
mulation steps2.
HPMC hydrogels possess good rheological properties result-
ing in long residue times at the site of administration3.
The aims of the present work were to prepare Q10 loaded
SLNs incorporated into HPMC hydrogels and to investigate
their antioxidant properties in vitro.
EXPERIMENTAL METHODS
Materials
Compritol 888 ATO (C) was a kind gift from Gattefossèe.
Poloxamer 188 (Plu) (Pluronic® F 68, Basf ) and Tween 80
(T80) (Fluka) were used as surfactants. Q10 was purchased
from Fluka. HPMC (Methocel K100M) were obtained from
Colorcon.
MATERIALS AND METHODS
SLN Preparation
SLNs were prepared using high shear homogenization and
ultrasound method (4). C and Q10 (24%) as the lipid phase
 POSTER PRESENTATIONS

(100 mg) was melt. The aqueous phase was based on Plu (50
mg) and T80 (25 mg) dissolved in 12.5 ml of bidistilled water
and heated to 80°C. Aqueous phase was poured into the lip-
id phase under homogenization at 24000 rpm for 5 min with
Ultra-Turrax (T25). Subsequently particles were dispersed in
12.5 ml bidistilled water at 4°C and kept at 4°C for one day.
Hardness and compressibility values of gels should be low to
take the prepared gel from the container. The highest hard-
ness value was obtained with gels containing Q10 loaded SLNs.
There was a significant difference between HPMC gels and Q10
and SLN incorporated gel formulations (p<0.05). Nothing incor-
porated HPMC gel showed the highest compressibility.

SLN Characterization Table 2: The mechanical properties of gel formulations.

SLNs were characterized for particle size (PS), polydispersity Hardness Compressibility
index (PI) and zeta potential (ZP) by means of PCS (Malvern Elasticity ± SD
(N) ± SD (Nnum)± SD
Zetasizer Nano ZS). HPMC2.5 0.145 ± 0.01 0.991 ± 0.001 17.804 ± 1.36

Preparation of SLN HPMC Hydrogels HPMC2.5Q10 7.631 ± 0.12 0.994 ± 0.001 10.267 ± 0.43
HPMC gels were prepared at a concentration of 2.5% un- HPMC2.5SLN 6.608 ±0.19 0.993 ± 0.001 8.768 ± 0.24
der magnetic stirring with (HPMC2.5Q10) and without Q10 HPMC2.5Q10SLN 10.84 ±0.36 0.991 ± 0.001 14.131 ± 0.32
(HPMC2.5). HPMC were added to the nano-suspension after
the preparation of blank (HPMC2.5SLN) and loaded SLNs The high value of cohesiveness provides full structural recov-
(HPMC2.5SLNQ10). The resulting mixture was stirred at room ery following gel application. When we compared our formula-
temperature with a magnetic stirrer. The formulations were tions, the cohesiveness was significantly different from HPMC
put in an ultrasonicator (Transsonic 660/h) to avoid air bub- gels (Table 3). Incorporated gels showed similar cohesiveness.
bles before TPA analysis. The most adhesive formulation was HPMC gel (Fig.1).

Texture Profile Analysis (TPA) Table 3: The mechanical properties of gel formulations.
Evaluation of the mechanical properties of the preparations
Adhesiveness ± SD Coheiveness ± SD
was performed using an STS Stable Micro Systems texture
analyzer (Model TA-XT. plus) in the TPA mode. Gel formula- HPMC2.5 22.576 ± 1.78 0.975 ± 0.01
tions were transferred into a 10 mL beaker and packed to a HPMC2.5Q10 14.118 ± 0.35 1.017 ± 0.02
fixed height, taking care to avoid the introduction of air into HPMC2.5SLN 10.632 ±0.29 1.014 ± 0.02
the samples. The analytical probe (10 mm diameter) was
HPMC2.5Q10SLN 17.540 ±0.48 1.011 ± 0.01
compressed twice into each sample to a depth of 15 mm at
144 a rate of 8.0 mm s-I. A delay period of 15 s was allowed be-
tween the end of the first and the beginning of the second
compression. All tests were performed at least in quadrupli-
cate on samples at ambient temperature.

In vitro Antioxidant Capacity

The trolox equivalent antioxidant capacity (TEAC) assay was
employed for the assessment of antioxidant capacity. 7mM
of ABTS [2,2’-azino-bis(3-ethylbenzthiazoline-6-sulphonic
acid)] was converted to its radical cation by the addition of
potasium persulfate and keeping at 30°C for 12-16 h in a dark
incubator.
The formulations were heated to 80°C in 5mM phosphate
Figure 1: Texture profile analysis graphs of the gel formula-
buffer and 100 μl of these mixtures were added to the ABTS
tions
radical cation. The reaction was monitored spectrophoto-
metrically at 746 nm for 6 min. The difference between the
TEAC assay showed that the antioxidant capacity of Q10
absorbance was evaluated5.
loaded SLN incorporated HPMC gels was the highest in com-
parison to other formulations. The percentage of inhibition
RESULTS AND DISCUSSION
was detected as 8.7% which is equivalent to a trolox concen-
The determined PS of formulations was approximately 220
tration as 1.76 mmol/L.
nm and PI indicated a uniform system. ZP was found nega-
tive for both blank and loaded formulations. The charge of
CONCLUSION
the particles seemed to decrease after the incorporation of
It can be stated that Q10 loaded SLN incorporated HPMC
Q10 in the system (Table 1).
(2.5% w/v) gel formulations performed suitable mechanic
Table 1: PS, PI and Ç of SLNs (n=6)
properties with a significant antioxidant capacity. These sys-
PS (nm)±SD PI ±SD ZP ±SD tems might be evaluated as promising delivery systems for
Blank 143.8 ±23.60 0.317 ±0.03 -23.8 ±3.23 dermal/cosmetic applications of Q10.
Loaded 136.5 ±11.14 0.253 ±0.01 -18.7 ±2.96
Blank SLNs without Q10 REFERENCES
Loaded SLNs loaded with Q10 1. V. Teeranachaideekul et al. Eur. J. Pharm. Biopharm.

3rd BBBB International Conference on Pharmaceutical Sciences

 POSTER PRESENTATIONS

2007;67:141-148. RESULTS AND DISCUSSION

2. C. Hsu et al. AAPS PharmSciTech 2003 ;4(3) Article 32.
3. S.Y. Karavana et al. Pharm. Dev. Technol. (2009)
doi:10.1080/10837450902882351
4. E.H. Gokce et al. Int. J. Pharm. 2008;364:76-86.
5. C.C. Wang et al. Clinical Chem. 2004;50:952-954.
p057-FURTHER SCREENING FOR NEW
ANTIOXIDATIVE COMPOUNDS FOR TOPICAL
ADMINISTRATION USING LIPID MODEL
SYSTEMS
Figure 1 shows the results of the thiobarbituric acid assay of
the simple stratum corneum lipid model system after the ad-
dition of different drugs as test substances. The figure shows
surprising effects for ascorbic acid and amantadine. Whereas
the latter drug acted antioxidatively, for the samples includ-
ing ascorbic acid the secondary lipid peroxidation products
were increased significantly.

Trommer Hagen1, Neubert Reinhard1

Martin Luther University Halle Wittenberg, School of

Pharmacy, Institute of Pharmaceutics and Biopharmaceutics,
Halle, Germany1

INTRODUCTION
Ultraviolet radiation is able to damage all important biomol-
ecules, such as lipids, DNA, carbohydrates and proteins se-
verely via the initiation of free radical reactions1. The human
skin is of particular interest, because it is constantly exposed
to UV radiation, oxygen and other noxious environmental Figure 1: Concentration of the TBA reaction products (TBA-
influences2. RP Conc.) and influence of UV irradiation and different test
drugs (100 µM each) in the LLA
In this study, lipid model systems of different complexity
were used as in vitro counterparts of the intercellular lipid Among the four flavonoids tested in this study only the St.
matrix of the stratum corneum. The effects of forty seven Mary`s thistle ingredient silibinin and the flavone quercetin 145
different substances (drugs, plant extracts, plant ingredients showed a lipid protecting potency when added to the sam-
and polysaccharides) on UV irradiation induced lipid peroxi- ples before UV irradiation treatment (Figure 2). Hesperetin
dation were investigated3. and rutin did not act antioxidatively in this setting. All the
plant extracts used in the screening showed protective ef-
MATERIALS AND METHODS fects by decreasing the amount of UV irradiation induced
Sample Preparation lipid peroxidation. Figure 2 illustrates this for St. John`s Wort,
Two lipid systems were used as in vitro screening models. The melissa and sage.
simple system was an oil in water dispersion of alpha-Linolenic
acid. The complex system consists of liposomes prepared by use
of the thin layer method. The lipids used for liposome prepara-
tion were: alpha-Linolenic acid (LLA), L-alpha-Dipalmitoylphos-
phatidylcholine (DPPC) and Cholesterol (CHOL). Ferrous sulfate
(10 µM) was added to the samples as an electron donor to initi-
ate oxygen radical generation via a Fenton type reaction4.

Ultraviolet Irradiation
UV-B irradiation experiments were carried out using a UV ir-
radiation chamber (Dr. Gröbel UV-Elektronik, Ettlingen, Ger-
many) enabling a selective exposure to UV-B. The samples
were treated with an UV-B dose of 0.25 J/cm².

Thiobarbituric Acid Assay

The thiobarbituric acid (TBA) test was used to determine ma-
londialdehyde (MDA) as a classic lipid peroxidation second-
ary product. Two millilitres of a stock TBA reagent were add-
ed to 1.0 ml of the UV-B treated sample. During the reaction, Figure 2: Concentration of the TBA reaction products (TBA-
a red TBA:MDA complex (2:1) is formed which is detected by RP Conc.) and influence of UV irradiation, flavonoids (100 µM
fluorescence measurement. HPLC was used to quantify the each) and plant extracts (0.2%)
pigment.

October 26 - 28, 2009 / Antalya - TURKEY

 POSTER PRESENTATIONS
The buckwheat extract effects on the lipids is demonstrated
by showing the results of both simple and complex screen-
ing system (Figure 3). The flavonoid fraction of Fagopyrum
esculentum contains several flavonoids which showed an
antioxidative effect already when tested as a single com-
pound such as the aglycon quercetin.
Figure 3: Concentration of the TBA reaction products (TBA-RP
Conc.) and influence of UV irradiation and buckwheat extract (0.1
%, 0.2 %, 0.5 %) in the simple and complex screening system
The screening for antioxidative compounds for topical ad-
ministration resulted in new promising findings. In the drug
testings amantadine was found to act antioxidatively where-
as for ascorbic acid prooxidative effects were determined.
146 Buckwheat extract significantly reduced the level of irradia-
tion induced lipid peroxidation as well as the extracts of St.
John`s Wort, melissa and sage.
CONCLUSIONS
Human skin has constant exposure to UV light and oxygen.
Therefore, the administration of protectors in cosmetic formu-
lations or sunscreens, as found in this study, may be helpful for
skin protection against UV induced damage. Our results show
new ways of skin protection by topical applicable substances
with antioxidative potency. In vivo experiments with the pro-
tector substances should follow as well as preformulation stud-
ies to test the stability of the potential antioxidants in several
formulations and examinations of the penetration behaviour.
REFERENCES
1. Krutmann J. New developments in photoprotection of
human skin, Skin Pharmacol Appl Skin Physiol. 2001;
14(6):401-407.
2. Thiele JJ. Oxidative targets in the stratum corneum. A
new basis for antioxidative strategies, Skin Pharmacol
Appl Skin Physiol. 2001; 14(Suppl 1):87-91.
3. Trommer H, Neubert RHH. Screening for new antioxida-
tive compounds for topical administration using skin
lipid model systems, J Pharm Pharm Sci. 2005; 8(3):494-
506.
4. Trommer H, Wagner J, Graener H, Neubert RHH. The ex-
amination of skin lipid model systems stressed by ultra-
violet irradiation in the presence of transition metal ions,
Eur J Pharm Biopharm. 2001; 51(3):207-214.
3rd BBBB International Conference on Pharmaceutical Sciences
P058-THE EFFECT OF HOMOGENIZATION
SPEED ON THE IN VITRO CHARACTERISTICS
OF THE PLGA MICROPARTICLES PREPARED
USING O/W EMULSION – SOLVENT
EVAPORATION METHOD
F. Ulya (Numanoğlu) Badıllı1, Tangül Şen1, Nilüfer Tarımcı1
Ankara University Faculty Of Pharmacy Department Of
Pharmaceutical Technology, Ankara, Turkey 1
INTRODUCTION
Psoriasis is a chronic, genetically influenced and immuno-
logically based inflammatory skin disease1. Clobetasol pro-
pionate (CP), which is a superpotent topical corticosteroid,
is commonly used for the topical treatment of psoriasis.
However, the local and systemic side effects of topical cor-
ticosteroids have limited their use2. In this study, clobetasol
propionate loaded PLGA microparticles were prepared as a
topical drug delivery system for psoriasis treatment. By this
way, a reduction in the side effects of the drug was intended.
The effect of the homogenization speed on the character-
istics of the microparticles were investigated. Also, in vitro
drug release studies of the particles prepared with two dif-
ferent drug : polymer ratios were carried out.
MATERIALS AND METHODS
Materials
Clobetasol propionate (CP) was kindly provided by Sandoz
(Gebze-Kocaeli, Turkey). Poly(D,L-lactic-co-glycolic acid)
copolymer (PLGA 50:50, Mw=40000-75000) and Poly(vinyl
alcohol) (PVA) (Mw=30000-70000) were purchased from
Sigma-Aldrich (Steinheim, Germany). All of the solvents and
reagents were analytical grade.
Preparation of PLGA Microparticles
PLGA microparticles containing CP were prepared by oil-in-
water (O/W) emulsion – solvent evaporation technique as de-
scribed in our previous studies (3,4). Briefly, CP was dissolved
in the solution of polymer in DCM. The organic phase obtained
was added to aqueous PVA solution (0.5%) and homogenized
using UltraTurrax T25 Homogenizer®. This mixture was stirred
at 25 °C to evaporate the organic solvent. Microparticles were
collected by centrifugation, washed three times with ultra
pure water and liyophilized for 48 hours. The codes of the mi-
croparticle formulations are shown in Table 1.
Table 1: The codes of the microparticle formulations
Formul Code Drug: Ploymer Ratio Homogenization speed (rpm)
F1 1:10 8000
F2 1:10 9500
F3 1:5 8000
F4 1:5 9500
In vitro Characterization of Microparticles
The production yield, encapsulation efficiency and particle
size of the microparticle formulations were determined. The
shape and surface morphology of the F1 and F3 were ex-
 POSTER PRESENTATIONS

amined using scanning electron microscope. DSC and XRD

analysis of CP, polymer, F1, F3 and blank microparticles were
performed. In vitro drug release studies of the microparticles
were carried out using modified Franz diffusion cells during
24 hours at 37 °C. The CP content was analyzed by HPLC at a
wavelength of 242 nm.

RESULTS AND DISCUSSION

Encapsulation efficiencies for all microparticle formulations
were found to be higher than 90% (Table 2).

Table 2: Production yield, encapsulation efficiency and par- Figure 2. In vitro release profiles of F3 and F4
ticle size of the microparticles
Encapsultaion CONCLUSION
Production PArticle Size PLGA microparticles containing CP were succesfully pre-
Formul code Efficiency (%)±
Yield (%) (μ+SD) pared using O/W emulsion – solvent evaporation method
SD
and the effect of the homogenization speed was investi-
F1 59.66 93.73 ± 0.22 14.61 ±1.45
gated. In conclusion, the particle size decreased when the
F2 77.56 95.09 ± 0.88 6.39 ±1.63 homogenization speed was increased.
F3 73.25 97.06+ 0.63 8.54 ±1.66
F4 77.285 08.34 ±0ç45 4.57 ±2.47 ACKNOWLEDGMENTS
This study is supported by Management of Scientific Re-
The particle size of the microparticles prepared with 1:10
search Projects of Ankara University (Project number:
drug : polymer ratio at 9500 rpm (F2) was significantly lower
07B3336002).
than the microparticles prepared at 8000 rpm (F1) (p<0.05).
Similar results were obtained for the microparticle formu-
REFERENCES
lations prepared with 1:5 drug : polymer ratio at different
1. Linden KG, Weinstein GD. Psoriasis: current perspectives with
speed values. The results of the particle analysis showed that
an emphasis on treatment, American Journal of Medicine.
the particle size significantly decreased when the homog-
1999; 107: 595-605.
enization speed was increased from 8000 rpm to 9500 rpm.
2. Lebwohl M, Ali S. Treatment of psoriasis. part 1. topical ther- 147
apy and phototherapy, J Am Acad Dermatol. 2001; 45: 487-
Scanning electron micrographs showed that the microparticles
498.
were spherical in shape and no drug crystals were observed
3. Numanoglu U, Şen T, Tarimci N. Preparation and in vitro eval-
on the surface of the microparticles. It was also observed that,
uation of clobetasol propionate loaded PLGA microparticles
F3 coded microparticles had highly porous surfaces whereas
as topical drug delivery system for treatment of psoriasis vul-
the surface of the F1 coded microparticles was non-porous.
garis, FIP World Congress. 2008, Basel-Switzerland; 182.
4. Badilli (Numanoğlu) FU, Şen T, Tarimci N. Clobetasol propi-
DSC and XRD results showed that CP was totally entrapped
onate loaded PLGA microparticles for topical drug delivery:
in the polymer and the drug was present in an amorphous
the effect of the emulsifier ratio, 36th CRS. 2009, Cophenha-
phase in the microparticles.
gen-Denmark; Poster No: 1024.
The results of the in vitro release studies showed that, the release
rate of the drug from the F2 coded microparticles was significant-
ly higher than the F1 microparticles (Figure 1). This was attributed P059-ATR-FTIR SPECTROSCOPIC ANALYSIS
to the smaller particle size of the F2 coded microparticles. Unex- OF THE EFFECT OF TERPENE/CO-SOLVENT
pectedly, the amount of the drug released from F4 microparticles MIXTURES ON PIG SKIN
was lower than that of F3 coded microparticles (Figure 2). It was
thought that, the release of the drug increased due to the highly M. Sedef Erdal1, Deniz Özdin1, Sevgi Güngör1
porous surface of the F3 coded microparticles.
Istanbul University Faculty of Pharmacy Department of
Pharmaceutical Technology, Istanbul, Turkey1

INTRODUCTION
Terpene penetration enhancers are widely used in combina-
tion with ethanol and propylene glycol (PG). It has been pro-
posed that the interaction of terpenes with SC in presence of
different solvents may not be similar1.

Fourier Transformed Infrared (FTIR) spectroscopic imaging in

Figure 1: In vitro release profiles of F1 and F2 Attenuated Total Reflection (ATR) mode is a useful tool for

October 26 - 28, 2009 / Antalya - TURKEY

 POSTER PRESENTATIONS

studying biomedical samples. It provides considerable in- at 2851.63±0.10 and 2920.39±0.14 cm-1, respectively. In
formation about the interactions of penetration enhancers the presence of EU and LM, SSV shifted by ~1.9 cm-1 to
with the lipids and proteins of the intercellular matrix of SC2. 2853.53±0.29 cm-1 which indicates the introduction of
gauche conformers in the carbon skeleton of lipid alkyl
The aim of our study was to investigate the interaction of chains.
three different terpenes: nerolidol (amphiphilic sesquiter-
pene), dl-limonene (cyclic terpene) and eucalyptol (oxygen As both SSV and ASSV collapse in the presence of NR, we can
containing monoterpene) with intact pig SC in two co-sol- suggest that the inter-chain interactions of epidermal lipids
vent systems, propylene glycol (PG) or Transcutol (TC) with are affected (Fig 1).
the goal of comparing these two co-solvents and the effect
of TC for the first time. Treatment of skin with pure PG or pure TC revealed no signif-
icant shift in lipid peak frequency, however, the dramatically
ATR-FTIR spectroscopy was employed to understand the ef- decrease in area of C-H stretching peaks is indicative of lipid
fect of terpene/co-solvent systems on molecular organiza- extraction from SC (Table 2).
tion of intact pig SC.
Table 1: Composition of terpene/co-solvent systems
MATERIALS AND METHODS Formulation Code Treatment (terpene 3%)
Materials LM dl-im onene
Nerolidol, Dl-limonene, Eucalyptol and Propylene Glycol LMPG dl-lim onene +PG
were purchased from Sigma. Transcutol® was kindly provid- LMTC dl-lim onene + TC
ed by Gattefossè. NR Nerolidod
NRTG Nerolidod +PG
Preparation of terpene/co-solvent systems NRTC Nerolidod + TC
Terpenes were added in PG or TC at 3% concentration (Table EU Eucalyptol
1). After 3 min vortexing, mixtures were equilibrated for 30 EUPG Eucalyptol + PG
min without stirring. EUTC Eucalyptol TC
PG Propylene Gylcol
Skin samples
The subcutaneous fat tissue of excised pig skin was removed TC Transcutol
with a scalpel and the hairs were clipped with hair clippers.
148 The skin was stored frozen at -25 oC (wrapped in Parafilm
and packed in ZipLock bags) for not longer than 2 months
prior to use.
Before collection of ATR-FTIR spectra, samples were de-
frosted 8 h at 4 oC. After defrosting, skin surface was wiped
2 times with wet cotton swab and excess water was gently
dried with paper tissue. 100 µl of terpene, co-solvent or ter-
pene/co-solvent system was applied on the surface and kept
for 3h at 25 oC. After the treatment period, excess of formu-
lation was gently blotted away with paper tissue from the
surface of the skin.
Figure 1: The IR spectra of intact pig SC treated with NR, EU
IR Spectroscopy and LM.
All spectra were collected with a spectral resolution of 4cm-1
in the 4000-650 cm-1 range using a Perkin Elmer Spectrum Table 2: Peak areas of SSV and ASSV of intact pig SC treated
100 FT-IR spectrometer (UK) equipped with a ZnSe ATR crys- with terpenes, co-solvents and terpene/co-solvent systems
tal. Each spectrum was an average of 4 scans. Skin sample,
Treatment Peak Area ASSV Peak Area of (SSV)
cutted to dimensions 2x2 cm, was placed SC side down onto
the ATR crystal. To ensure reproducible contact between the CNT 4.49 ± 0.22 2.25 ± 0.11
sample and the crystal, always the same pressure on top of PG 0.32 ± 0.25 n.a*
samples was applied (force gauge 100 N). TC 0.36 ± 0.32 n.a*
LM 3.60 ±0.11 1.56 ± ±0.17
For each skin sample peak area was measured after enhancer NR n.a* n.a*
treatment. Attention was focused on characterizing the oc- EU 2.01 ±1.21 1.42 ±0.39
currence of peaks near 2850 and 2920 cm-1 which were due LMPG 1.23 ±0.26 0.06 ±0.034
to the symmetric (SSV) and asymmetric (ASSV) C-H stretching LMTC 1.23 ±0.26 n.a*
vibrations, respectively. A non treated skin sample served as NRPG 1.81 ±0.05 0.05 ±0.04
control (CNT). Perkin Elmer Spectrum Version 6.0.2 software
NRTC 0.17 ±0.01 n.a*
was used for calculations.
RESULTS AND DISCUSSION EUPG 1.12 ±0.17 0.3 ±0.30
The SSV and ASSV of control skin were observed to be EUTC 1.17 ±0.25 0.26 ±0..26

3rd BBBB International Conference on Pharmaceutical Sciences

 POSTER PRESENTATIONS
CONCLUSION
The synergistic effect of TC in this study can be explained
by improved partitioning of terpenes into the SC in the
presence of TC. Our investigation will provide a basis of us-
ing a suitable terpene/co-solvent system in a transdermal
formulation. Ex vivo permeation studies will be carried out
using hydrophilic and lipophilic model drugs to investigate
the relationship between the observed flux enhancements
and the effect of terpene/co-solvent systems on skin barrier
property.
REFERENCES
1. Babita K., Kumar V., Rana V., Jain S, Tiwary A.K. Thermo-
tropic and spectroscopic behavior of skin : relationship
with precutaneous permeation enhancement, Curr Drug
Del. 2006, 3 : 95-113
2. Kazarian S.G., Chan K.L.A. Applications of ATR-FTIR spec-
troscopic imaging to biomedical samples, Biochim Bio-
phys Acta. 2006,1758 : 858-867.
p060-DETERMINATION OF SPF AND
MOISTURIZING EFFECTS OF LIPOSOMAL AND
CONVENTIONAL FORMULATIONS OF OMC AS
A SUNSCREEN
Shiva Golmohammadzadeh1, Mahmoud Reza Jaafari2,
Noman Khalili3
Pharmaceutical Research Center and School of Pharmacy,
Mashhad University of Medical Sciences, Mashhad, Iran1
Pharmaceutical Research Center and Biotechnology Research
Center, School of Pharmacy, Mashhad University of Medical
Sciences2
School of Pharmacy, Mashhad University of Medical Sciences,
Mashhad, Iran3
INTRODUCTION
It has been known for decades that sunscreens are capable
of protecting man from harmful effects of solar radiation1, 2
Ramon et al. showed that liposomes could be regarded as al-
ternatives to conventional oil/water emulsions in the formu-
lations of lipidic sun filters. When liposomes with a composi-
tion and structural organization similar to that of the stratum
corneum lipids are used, the skin penetration is retarded1.
The objectives of this study were to determine and compare
the SPF (Sun Protection Factor) and moisturizing effects of the
liposomal and conventional formulations containing octyl
methoxycinnamte (OMC) as a sunscreen by in vivo methods.
MATERIALS AND METHODS
Materials
OMC, cholesterol and vitamin E were purchased from Merck
(Darmstadt, Germany). Lanolin, white petrolatum, stearic
acid, propylparaben, methylparaben disodium EDTA, pro-
pylene glycol and triethanolamine were purchased from
Sigma (USA). Soya Phosphatidylcholine was obtained from
the Avanti Polar Lipids (Alabaster, Alabama, USA). Phosphate
October 26 - 28, 2009 / Antalya - TURKEY
buffered saline (PBS) ingredients were supplied from Sigma
(USA). All solvents used in this study were High Performance
Liquid Chromatography (HPLC) grade. All chemicals were of
the purest grade available.
Methods
Preparation of Liposomes Containing OMC
The multilamellar liposomes (MLVs) containing OMC were
prepared by fusion method and o/w emulsion was prepared
as FDA standard sunscreen method (3, 4).
Morphology and Size Analysis of Liposomes
Optical microscope (OLYMPUS, Germany) was used for study-
ing the morphological features of MLV liposomes containing
OMC. The mean diameter and particle size distribution of li-
posomes were determined by a particle size analyzer (PSA;
Klotz, Germany)1,3.
SPF determination of Homosalate reference, COLIPA stan-
dard, o/w emulsion and liposomes containing OMC by in
vivo method
The SPFs of the formulations were determined by in vivo
method according to Australian standard. The exposure area
was the back of 10 volunteers. Subsites of the backs were
exposed to solar simulator. The Minimum erythemal dose for
unprotected skin MED (US) was observed in the next day. (Fig
1) The sunscreen was spread (2mg/cm2) over the area. Each
test subsite in a series was exposed to controlled amounts of
simulated sunlight by a constant ratio. In the third day the
MEDs of the formulations were observed. The SPF was deter- 149
mined by the ratio between the MED while using sunscreen
and MED (US)4.
Moisture Content of the Skin Following Application of o/w
Emulsion, Liposomes Containing OMC and NaCl 3% in Euc-
erin Using Corneometer
The moisture content of the skin was determined before and
after 30 min, 2, 3, 6 and 10 h post application of the formu-
lations using Corneometer by measuring electrical capaci-
tance5.
RESULTS AND DISCUSSION
The liposomes made by fusion method were morphologi-
cally homogenous multilamellar vesicles, as observed under
optical microscope. Mean diameters of MLVs liposomes con-
taining OMC determined by PSA were 2.21 ± 0.06 μm (n = 3)
(Fig 2). Small unilamellar vesicles (SUV) were with a size of
62.41 ± 21.2 μm (n = 300) (Fig 2).
The SPF of the liposomes containing OMC was greater than
lotion at the same concentration of OMC. However this was
considered not quite significant by unpaired t test (Table 1).
It was observed that in each formulation there is significant
increase in moisture content after 30 minutes. After 2 hours
the moisture contents were decreased in all of the formu-
lations. At 30 minutes, the highest water content was ob-
served for OMC lotion. However, there were no significant
differences between NaCl 3% in eucerin as control and OMC
liposomes.
 POSTER PRESENTATIONS

P061-EVALUATION OF LIPOSOMAL AND

CONVENTIONAL FORMULATIONS ON THE
HUMAN PERCUTANEOUS ABSORPTION OF
OMC AS A SUNSCREEN
Figure 1; The MED observed 20 hours after UV
Shiva Golmohammadzadeh1, Mahmoud Reza Jaafari2,
Table 1:The SPF values of COLIPA, OMC lotion and OMC-MLV Noman Khalili3
liposomes by in vivo method in ten subjects (S) with differ-
ent skin types and MEDs. Pharmaceutical Research Center and School of Pharmacy,
Mashhad University of Medical Sciences, Mashhad, Iran1
SPS Pharmaceutical Research Center and Biotechnology Research
Sunscreen S1 S2 S3 S4 S5 S6 S7 S8 S9 S10
(Men±SD) Center, School of Pharmacy, Mashhad University of Medical
Sciences, Mashhad, Iran2
Homosalate
reference
4 4.46 4.56 4.90 4 4.02 3.62 4.44 5.63 4.05 4.35±0.64 School of Pharmacy, Mashhad University of Medical Sciences,
Mashhad, Iran3
COLIPA 12.4 1.5 19.4 14.5 17.1 12.4 15.5 15.5 12.4 19.4 15.2±3.4
INTRODUCTION
OMC lotion 14.2 15.5 12.4 12.3 1.5 17.1 12.3 12.4 15.5 12.3 13.95±1.83
It has been known for decades that sunscreens are capable of
OMC liposomes 17.1 19.3 19.4 12.4 15.5 14.5 19.4 17.1 14.5 15.5 16.85±1.40 protecting man from harmful effects of solar radiation. Many
factors manage the delivery of the drugs and cosmetics into
the skin from topically applied formulations1,2. The objective
of this study was to determine the influence of vehicle on
the penetration of octyl methoxycinnamate (OMC), as a UV
absorber, to the skin layers by stripping method. The formu-
lations were a conventional o/w emulsion, multilamellar and
small unilamellar liposomes (MLVs and SUVs).
Figure 2: Optical microscopic picture of liposomes prepared
by fusion method containing OMC (magnification of image MATERIALS AND METHODS
is × 1000). Materials
150 OMC, cholesterol and vitamin E were purchased from Merck
CONCLUSION (Darmstadt, Germany). Lanolin, white petrolatum, stearic
The results of this study indicate that the SPFs obtained from acid, propylparaben, methylparaben disodium EDTA, pro-
our in vivo results is almost the same as published SPFs for pylene glycol and triethanolamine were purchased from
Homosalate reference and MLVs prepared by fusion method Sigma (USA). Soya Phosphatidylcholine was obtained from
is a good vehicle for OMC as a sunscreen since it provides the Avanti Polar Lipids (Alabaster, Alabama, USA). Phosphate
proper SPF and increase the moisture content of the skin. buffered saline (PBS) ingredients were supplied from Sigma
(USA). All solvents used in this study were High Performance
ACKNOWLEDGMENTS Liquid Chromatography (HPLC) grade. All chemicals were of
This study was supported financially by the Pharmaceuti- the purest grade available.
cal Research Center and Biotechnology Research Center,
Shool of Pharmacy, Mashhad University of Medical Sciences Methods
(MUMS). Preparation of liposomes containing OMC
MLVs containing OMC were prepared by fusion method and
REFERENCES then converted to SUVs by probe sonication3.
1. Ramon E, Alonso C, Coderch L et al. Liposomes as alter-
native vehicles for sun filter formulations. Drug Deliv. Determination of percutaneus absorption of OMC using
2005; 12: 2, 83-88. stripping method
2. Chatelain E, Gabard B, Surber C. Skin penetration and Formulations were applied at the level of the forearm of six
sun protection factor of five uv filters: effect of the ve- volunteers in a dose of 2 mg/cm2. Afterwards stripping was
hicle. Skin Pharmacol Appl Skin Phys. 2003: 16, 28-35. performed by 22 stripping and divided to different groups.
3. Foldvari M. 1998, Biphasic Liposomes. In US Patent. No. The sunscreen agent was assessed by HPLC4,5.
5,853,755.
4. Australian/New Zealand Standard. Sunscreen products. Morphology and Size Analysis of Liposomes
Evaluation and classification. AS/NZS 2604:1998, 1-32. Optical microscope (OLYMPUS, Germany) was used for study-
5. Alanen E, Nuutinen J, Niclen K et al. Measurement of hy- ing the morphological features of MLV liposomes containing
dration in the stratum corneum with the MoistureMeter OMC. The mean diameter and particle size distribution of li-
and comparison with the Corneometer. Skin Res Tech- posomes were determined by a particle size analyzer (PSA;
nol. 2004; 10: 32-37. Klotz, Germany)2.

3rd BBBB International Conference on Pharmaceutical Sciences

 POSTER PRESENTATIONS
RESULTS AND DISCUSSION
The liposomes made by fusion method were morphologi-
cally homogenous multilamellar vesicles, as observed under
optical microscope. Mean diameters of MLVs liposomes con-
taining OMC determined by PSA were 2.21 ± 0.06 μm (n = 3)
(Fig 1). Small unilamellar vesicles (SUV) were with a size of
62.41 ± 21.2 μm (n = 300) (Fig 2).
Figure 1: Optical microscopic picture of liposomes prepared
by fusion method containing OMC (magnification of image
is × 1000).
Figure 2: Transmission electron micrographs of SUV lipo-
somes containing OMC by negative staining using phospho-
tungstate and viewed in an electron microsc
The results of this study indicate that skin accumulation of
OMC in MLVs was significantly greater than o/w emulsion
and SUVs. The penetration of SUVs into the deeper skin lay-
er was significantly more than MLVs and conventional o/w
emulsion. Higher amounts of OMC were recovered from
the upper layers of stratum corneum than deeper layers in
all formulations. The conventional lotion may remain on the
surface without penetration to the skin compared with lipo-
somes (Fig 3, 4).
Figure 3: Amount of OMC in the group of strips, after 3 h of
application, (mean ± SD; ns p >0.05, **p <0.01, ***p <0.001
vs. MLV), Group 1: strips 1-2; G
Figure 4: Amount of OMC in the group of strips after 7 h of
application, (mean ± SD; ns p >0.05, **p <0.01, ***p <0.001
vs. MLV), Group 1: strips 1-2; Gr
CONCLUSION
The result of this study indicate that MLVs prepared by fusion
method are better vehicle for OMC as a sun screen since it
remains more in the stratum corneum and its penetration to
the deeper layers is less.
ACKNOWLEDGMENT
This study was supported financially by the Pharmaceutical
October 26 - 28, 2009 / Antalya - TURKEY
Research Center and Biotechnology Research Center, Mash-
had University of Medical Sciences (MUMS).
REFERENCES
1. Sarveiya V, Risk S, Benson HAE. Liquid Chromatographic
assay for common sunscreen agents: application to in
vivo assessment of skin penetration and systemic ab-
sorption in human volunteers. J Chromatogr B 2004;
803: 225-231.
2. Verma D, Fahr A. Particle size of liposomes influences
dermal delivery of substances into skin, Int J Pharm.
2003; 258: 141-151.
3. Foldvari M. 1998, Biphasic Liposomes. In US Patent. No.
5,853,755.
4. Chatelain E, Gabard B, Surber C. Skin penetration and
sun protection factor of five uv filters: effect of the ve-
hicle. Skin Pharmacol Appl Skin Phys. 2003: 16, 28-35.
5. Periolia L, Ambrogi V, Bertini B, Ricci M, Nocchetti M et al.
Anionic clays for sunscreen agent safe use: Photoprotec-
tion, photostability and prevention of their skin penetra-
tion. Eur J Pharm Biopharm. 2006; 62: 185–193.
P062-THE KNOWLEDGE OF PHARMACISTS
IN REGARDE TO THE APPLICATION OF
SUNSCREENS AND MOISTURIZERS
Shiva Golmohammadzadeh1, Jebraeel Movaffagh2, Yousef
Setayesh3
151
Pharmaceutical Research Center and School of Pharmacy,
Mashhad University of Medical Sciences, Mashhad, Iran1
School of Pharmacy, Mashhad University of Medical Sciences,
Mashhad, Iran2
School of Health, Mashhad University of Medical Sciences,
Mashhad, Iran 3
INTRODUCTION
Sunscreen usage is a widely accepted method of primary
prevention against deleterious effects of ultraviolet radiation
(UVR); skin cancer, sunburn, freckles and photoaging. How-
ever, studies have shown that rates of regular sunscreen use
are very low, despite evidence of the efficacy of sunscreen
application1. Also it has been known for decades that mois-
turizers are useful for skin health care. Pharmacists are piv-
otal to prescribe the drugs and cosmetics. A pharmacist has
different functions; the pharmacist as a dispenser of drugs;
the pharmacist as a drug consultant and the pharmacist as a
‘substitute doctor’2.
The aim of this study was to assess the knowledge of phar-
macists in regard to the application of sunscreens and mois-
turizers in Mashad, Iran.
MATERIAL AND METHODS
In May to July 2007, 81 pharmacists completed surveys by
filling the questionnaire. In this descriptive study, 25 ques-
tions addressed to determine the demographic character-
istics plus the knowledge of the pharmacists towards sun-
 POSTER PRESENTATIONS
screens and moisturizers application. The factors influencing
the knowledge regarding sunscreens and moisturizers appli-
cation were evaluated3.
RESULTS AND DISCUSSION
This is the first report which compares the knowledge and beliefs of
pharmacists on sunscreens and moisturizers use in Iran and other
countries. Our study includes a sample of 81 Pharmacists from the
pharmacies in Mashhad including 41 (50.6%) men with 31.6±14.4
age and 39 (48.1%) women with 36.6±12.3 age. with 67% between
the ages 26 and 35. The median period of practicing as a pharma-
cist since graduation was 6 (5–14) years (Table 1). From the overall
respondents 56.8% had used sunscreens and 12.3% had used the
sunscreens in the correct time. 45.7% knew the correct applica-
tion of sunscreens, 34.6% didn’t know and 19.8% gave no answer.
In the case of the moisturizers, 65.4% had used moisturizers. The
respondents in this study generally performed very poorly on the
knowledge test. The mean score received, 39.33±16.77 out of 100
points, which is less than 50%. Sex was not significantly associated
with the knowledge. In the overall sample the results showed that
higher knowledge scores were associated with younger age. Also
higher knowledge scores were associated with less work history.
The mean score of the knowledge in formulations received was
9.25±7.59 out of 100 points (figure 1).
Table 1: Percentage distribution of selected characteristics,
in pharmacists in Mashad, Iran, 2007(n=81)
Characteristics Percentage
Use of sunscreens 56.8
Use of sunscreens in correct time 12.3
152 Use of sunscreens in all seasons 34.6
SBF definiton correctly answered 46.9
Accurate application of sunscreens 45.7
Use of moisturizers 65.4
Knowledge regarding to sunscreens and moisturizers 39.33±16.77
Knowledge regarding to conscistuence ofsunscreens and 9.25±7.59
moisturizers
Figure 1: Histogram curve of knowledge distribution in phar-
macists regarding to sunscreen sand moisturizers in Mashad
city in 2007.
CONCLUSION
This study showed that higher knowledge scores in formula-
tions were associated with more knowledge. In this study a
significant proportion of pharmacists do not have adequate
knowledge concerning the sunscreens and moisturizers. These
results indicate that more education in cosmetic and toiletries
field is necessary for pharmacists, specially the older ones.
3rd BBBB International Conference on Pharmaceutical Sciences
ACKNOWLEDGMENTS
Grant support was provided by Mashad University of Medi-
cal Science (No. 86054). Mohammad Naser Shafiee, Faculty
member of MUMS is gratefully acknowledged.
REFERENCES
1. Ling TC, Faulkner C, Rhodes LE. A questionnaire survey
of attitudes to and usage of sunscreens in northwest
England. Photodermatol Photoimmunol Photomed
2003;19(2):98-101.
2. van Grootheest K, Olsson S, Couper M, de Jong-van den
Berg L. Pharmacists’ role in reporting adverse drug reac-
tions in an international perspective. , Pharmacoepide-
miol Drug Saf 2004; 13(7): 457–464.
3. Ling TC, Faulkner C, Rhodes LE. A questionnaire survey
of attitudes to and usage of sunscreens in northwest
England. Photodermatol Photoimmunol Photomed
2003; 19(2):98-101.
P063-THE CONTENT OF TOTAL
POLYPHENOLS, TANNINS AND FLAVONOIDS
IN DIFFERENT PARTS OF EPILOBIUM
ANGUSTIFOLIUM L.
Ain Raal1, Siiri Jürgenson1
University of Tartu, Tartu, Estonia1
INTRODUCTION
Epilobium species (Onagraceae) are commonly used herbal
remedies in traditional, adjuvant therapy of benignus pros-
tate hyperplasia. Willow herb (Epilobium anguistifolium L.)
products usually consist of mixtures from various species,
with different phenoloid content, often only partially identi-
fied. The main constituent, oenothein B belong to the group
of tannins, but also lot of tannins, flavonoids and other vari-
ous polyphenols were detected in the willow herb1,2,3.
The aim of the study was to investigate the dynamics of total
polyphenols, tannins and flavonoids in different parts of wil-
low herb growing in Estonia.
MATERIALS AND METHODS
The plant material - 15 plants of willage herb were collected
from May to November 2006 in Unipiha village (Tartu county,
Estonia). The plants were cut at the height of 20 cm to avoid
stems without leaves. As soon as possible, the plants were
separated into different organs (roots, stems, leaves, flowers
and fruits), which were dried during a week at the room tem-
perature. For receiving the herb, plant material was collected
from the same site in July 2007.
The herb cut at 20 cm is henceforth called whole herb. Herb
without main stems was obtained with separating main stalk
from the whole herb. The dried and cut herb was sieved (diam-
eter 1 mm) before extraction of biologically active substances.
Total polyphenols
The plant material was extracted with 40% ethanol during
30 minutes at 60 degrees in thermostat. Folin-Denise re-
 POSTER PRESENTATIONS

agent and sodium carbonate saturated solution were added CONCLUSION

to the obtained extract. After an hour the optical density of
the solutions was detected spectrophotometrically at the
730 nm. Results were calculated according to the absorption
coefficient of 1% chlorogenic acid (123, 5).
Total tannins
Plant material was extracted while boiling in distilled water
for 30 minutes on electric cooker. 1% solution of potassium-
ferricyane and 1% solution of ferri(III)chloride were added to
the filtered solution. After shaking and after 5 minutes, opti-
cal density at the 720 nm was detected spectrophotometri-
cally. The results were calculated based on the optical den-
sity of tannin solutions. The standard chart was composed
based on the optical density of tannin solutions with differ-
ent concentration and the two solutions mentioned above
were added in advance.
Based on the content of flavonoids and tannins, it seems to
be optimum to use herb without main stalk collected while
flowering.
REFERENCES
1. Kosalec I, Zovko M, Sankovic K, Kremer D, Pepeljnjak S.
Antioxidant and antimicrobial activity of willow herb
(Epilobium angustifolium L.). Planta Med. 2008; 74(9):
948-948.
2. Shikov AN, Pozharitskaya ON, Ivanova SA, Makarov VG,
Tikhonov VP, Galambosi B. Variation in concentration of
oenothein B in different samples of cultivated Epilobium
angustifolium L. Planta Med. 2008; 74(9): 1120-1120.
3. Hevesi Tóth B, Blazics B, Kéry A. Polyphenol composition
and antioxidant capacity of Epilobium species, J Pharm
Biomed Anal. 2009; 49(1): 26-31.

Total flavonoids
Plant material was extracted while boiling with 70% ethanol p064-EFFECT OF CATECHIN DERIVATIVES
for 30 minutes under cooled reflux condenser. 10% sulphuric ON SERUM ALBUMIN CONFORMATION
acid was added to the extract and it was boiled under cooled
reflux condenser for 30 minutes for the hydrolysis of gluco- AND ITS GLYCATION BY REACTIVE ALPHA-
sides. Alcohol was steamed on the water bath and the ex- DICARBONYL COMPOUNDS
tract was filtered. Sediment on the filter paper was dissolved
in 95% ethanol warmed up to 50 degrees. The solution of Jaroslav Dršata1, Lucie Trnková2, Iva Bousová1
aglycones was diluted and the optical density was detected
at the 370 nm spectrophotometrically. Charles University Faculty of Pharmacy, Czech Republic1
University of Hradec Králové, Czech Republic 2
RESULTS AND DISCUSSION
During the vegetation period, the most polyphenols can be 153
INTRODUCTION
found in the herb with main stalks collected during full flow-
Flavonoids are natural polyphenolic compounds that oc-
ering. It can also be found in significant amounts in roots and
cur ubiquitously in plants. Multiple biological effects of fla-
stems, but not so much in leaves, fruits and flowers. In roots,
vonoids including antioxidant, antiviral, antibacterial, anti-
stems and leaves the tannins can be found the most in May
inflammatory, and vasodilatory activity have been reported.
(14.9-41.4%). In herbs collected in July the content is about
Besides positive acting, flavonoids exert also pro oxidative
50% lower (11.9-18.7%). The least flavonoids can be found
effects in vitro and are able to bind with proteins via hydro-
in stems and the most in leaves. Significant amounts of fla-
phobic interactions and hydrogen bonds. Such interactions
vonoids can be also found in flowers and fruits (Table 1).
with proteins may on one side influence relative antioxidant
potency of these compounds as well as physiological role of
Table 1: The content of polyphenols, tannins and flavonoids
affected protein. Catechins (flavan 3 ols), a group of naturally
in different parts of Epilobium angustifolium L. from May to
occurring flavonoids, can be found predominantly in green
November 2006
tea. Trapping of reactive alpha-dicarbonyl species (e.g. meth-
Total of ylglyoxal) by epigallocatechin, epicatechin 3 gallate (ECG),
Total of Total of and epigallocatechin 3-gallate in vitro has been described.
Organs flavonoids,
polyphenols, % tannins, %
mg% MATERIALS AND METHODS
Roots 4.0-8.5 7.7-16.2 4.7-19.3 The aim of this work was to evaluate both direct impact of
eight catechin derivatives on conformation of human serum
Stems 5.5-8.3 2.7-14.9 5.7-20.8 albumin (HSA) and their possible anti-glycation activity in
Leaves 2.4-6.3 16.0-41.4 113.1-235.7 the model containing HSA, methylglyoxal (MGO), and cat-
echin derivative (0-50 µM) using fluorescence spectroscopy.
Flowers 3.4* 36.1* 154.7*
Fruits 3.5-4.6** 17.5-20.2** 68.1-121.7** RESULTS AND DISCUSSION
Whole herb 10.2*** 11.9*** 30.4*** For catechin-albumin interaction, catechins proved to be
reactive compounds, which were able to cause changes in
Herb without protein conformation. Their binding affinity decreased in fol-
6.5*** 18.7*** 345.1*** lowing order: ECG > catechin gallate > epigallocatechin gal-
main stalk
*Collected in July 2006, ** collected in July, August and Sep- late > gallocatechin gallate > epicatechin > catechin > gallo-
tember 2006, *** collected in July 2007 catechin ≥ epigallocatechin. Furthermore, these compounds

October 26 - 28, 2009 / Antalya - TURKEY

 POSTER PRESENTATIONS
were able to decrease catalytical activity of glutathione
S-transferase in vitro. For anti-glycation study, fluorescence
of advanced glycation end products (AGEs) was measured
(370/440 nm) and % of inhibition of AGEs for individual cat-
echin derivatives were determined. The inhibitory activity of
these compounds was compared to effect of aminoguani-
dine (1 mM) and Trolox (2.5 mM). The most effective anti-gly-
cation agent, even better than aminoguanidine, appeared to
be ECG. Its inhibitory effect as well as effects of other cat-
echin derivatives was concentration-dependent. This study
could help to elucidate role of catechin derivatives in the
process of glycation and also contribute to assessment of
their interaction with protein molecules.
ACKNOWLEDGMENTS
Supported by the Development program of the Czech Minis-
try of Education No. 14/87/2009.
p065-EFFICACY OF CHLOROFORM, ETHANOL
AND WATER EXTRACTS OF MEDICINAL
PLANTS, MALVA SYLVESTRIS AND MALVA
NEGLECTA ON SOME BACTERIAL AND
FUNGAL CONTAMINANTS OF WOUND
INFECTIONS
Payman Zare1, AmirBabak Sioofi 1, Saeed Kolahian 2
Department of Pathobiology, Faculty of Veterinary Medicine,
154
University of Tabriz, Tabriz, Iran.1
Department of Basic Sciences, Faculty of Veterinary Medicine,
University of Tabriz, Tabriz, Iran.2
INTRODUCTION
Valuable gains have been documented on the bactericidal,
bacteriostatic, antifungal and immuno-modulatory proper-
ties of Malva sylvestris and Malva neglecta extracts. The pres-
ent study investigates the efficacy of chloroform, ethanol
and water extracts of these medicinal plants on some bacte-
rial and fungal contaminants of wound infections.
MATERIALS AND METHODS
Whole plants of wild Malva sylvestris and Malva neglecta
were collected freshly from free pastures in mountain areas
of East Azerbaijan, Iran. Dried ground powder from various
parts of plants were extracted with water (aqueous extracts),
ethanol, chloroform (Soxhlet extracts). All of the extracts
were filtered through 0.45 mm filters under sterile condi-
tions. Different concentrations of these extracts were added
to fresh cultures of test microorganisms in MHB broth media
and as previously described by NCCLS/ NCLI Documents4,5.
Test microorganisms included Staphylococcus aureus,
Pseudomonas aeruginosa, Streptococcus pyogenes, Proteus
vulgaris, Aspergillus niger, Aspergillus fumigatus and Can-
dida albicans. Broth dilution assay was performed to evalu-
ate antimicrobial activity of the extracts according to NCCLS/
NCLI guidelines4,5. After shaker incubation at 35°C for 24 and
48 h, test cultures as well as control media were evaluated
spectrophotometrically at 570 nm3,5, and Minimal inhibitory
3rd BBBB International Conference on Pharmaceutical Sciences
concentration (MIC) and minimal bactericidal concentration
(MBC) values of extracts were recorded4.
RESULTS AND DISCUSSION
Among all obtained extracts, ethanol extracts of both spe-
cies especially that of Malva sylvestris, showed the best ac-
tivity against bacteria, followed by aqueous extracts. The
best of antibacterial MIC values was 0.4 g/ml obtained with
S. pyogenes cultures. All extracts were active against S. au-
reus, P. aeruginosa, P. vulgaris. Aqueous and chloroform ex-
tracts had better antifungal activity. The best of antifungal
MIC values was 0.6 g/ml for Malva sylvestris aqueous extract
against A. niger cultures. All extracts had activity against A.
niger, A. fumigatus and C. albicans.
The prevention and treatment of wound infections espe-
cially after contaminated burn and surgical events is a criti-
cal issue. The proper interpretation of cultures taken from
wounds is uncertain. Multiple organisms are invariably iso-
lated from non-sterile wounds including indigenous aerobic
and anaerobic flora, aerobic gram-negative rods, and fungi.
Antibiotic trials with routine antimicrobial agents are losing
efficacy due to continual development of antibiotic resis-
tances in the causative microorganisms. On the other hand,
the antibacterial spectrum of an agent used for burn and
surgical would treatment and prophylaxis should include
coverage for a wide range of microorganisms including
pathogenic agents and normal flora, especially strains of an-
tibiotic multi-resistant bacteria, S. aureus and P. aeruginosa
with different and varying sensitivity patterns. Presence of
fungal agents including C. albicans and A. niger adds to this
coverage problem. The inflammation and healing process of
wounds is an additional issue. An ideal treatment may also
accelerate the healing process. Natural medicinal plant ex-
tracts have an ancient background in this aspect and mod-
ern medicine gains valuable benefits with them. The present
study shows the acceptable efficacy of chloroform, water
and ethanol extracts of medicinal plants Malva sylvestris and
Malva neglecta on some bacterial and fungal contaminants
of wound infections. Previous studies have got to valuable
gains on the bactericidal, antifungal and immune-modulato-
ry properties of these plant extracts (1, 2, 3). Our results show
that all extracts are active against S. aureus, P. aeruginosa, P.
vulgaris which have been reported to be troublesome bacte-
ria in wound infections, especially in the aspect of antibiotic
resistance as multi-resistant microorganisms. The ethanol
extracts had the highest antibacterial activity than all other
solvents. The results suggest that essential oils as non-polar
organic compounds could be the main active compounds in
these plants. Anthocyanin of Malva sylvestris has approved
bacteriostatic activity1. This water soluble pigment can be
responsible for acceptable antibacterial effects of aqueous
extracts of both plants. aqueous extracts of Malva sylvestris
have shown great antifungal activity against stored products
fungi including A. niger3. A. niger is also one of the most im-
portant fungal contaminants of wound infections. Efficacy of
all extracts especially aqueous extracts against A. niger, A. fu-
migatus and C. albicans can be a promising result of the pres-
ent study. The polysaccharide ingredients of aqueous extract
of Malva sylvestris has approved immunomodulatory effects
in in vitro studies; it has shown to switch off IL-4 transcription
 POSTER PRESENTATIONS
and to activate macrophages and T helper-1 lymphocytes2.
Ongoing studies of the authors of the present study on the
in vivo application of a combination of extracts especially
aqueous and ethanol extracts on infectious and non infec-
tious wounds will be complementary to these results.
CONCLUSION
Our results add more reasons to the clinical application of
these extracts in the prophylaxis and treatment of wound in-
fections. The wide range of efficacy on different bacterial and
fungal agents and possible healing acceleration obtained by
these extracts makes them acceptable candidates for the
promotion of healing in wound infections.
REFERENCES
1. Cheng Cui-lin and Wang Zhen-yu. Bacteriostatic activ-
ity of anthocyanin of Malva sylvestris J. of Forestry Res.
2006; 17(1):83-85.
2. Ghaoui WB, Ghanem EB, Chedid LA and Abdelnoor AM.
The effects of Alcea rosea L., Malva sylvestris L. and Sal-
via libanotica L. water extracts on the production of An-
ti-egg albumin antibodies, interleukin-4, gamma inter-
feron and interleukin-12 in BALB/c mice. Phytother Res.
2008 Dec; 22(12):1599-604.
3. Magro A, Carolino M, Bastos M and Mexia L. Efficacy
of plant extracts against stored products fungi. Rev
Iberoam micol 2006; 3:176-178.
4. NCCLS. 2002. Reference method for broth dilution an-
tifungal susceptibility testing of filamentous fungi. Ap-
proved standard M38-A. NCCLS, Wayne, Pa.
5. NCCLS. 2003. Methods for dilution antimicrobial sus-
ceptibility testing of bacteria that grow aerobically; ap-
proved standard – sixth edition. Document M7-A6. NC-
CLS. Wayne PA.
p066-Volatile constituents and anti
candida activity of the aerial parts
essential oil of Dittrichia graveolens
(L.) Greuter grown in Iran
Nasrin Aghel1, Ali Zarei Mahmoudabadi1
Joundishapour Medical Sciences University, Ahvaz, Iran1
INTRODUCTION
Because of the antimicrobial properties showed by essential
oils, the aromatherapy has been used for treatment of seri-
ous skin diseases, in special, superficial mycoses1.
The aim of present study was to identify the chemical com-
positions of the aerial parts essential oil of Dittrichia gravol-
ens grown in Khuzestan, south-west of Iran. Also, in order to
estimate the possibility of using it as an antifungal agent for
skin diseases (cutaneous candidiasis and dermatomycosis),
the antifungal activity of this oil against Candida albicans
was studied.
Plant Material
Aerial parts of Dittrichia graveolens were collected during
October 26 - 28, 2009 / Antalya - TURKEY
the flowering stage from Shush (latitude 32°11’21’’ N, lon-
gitude 48°15’28’’ E, Khuzestan Province, Iran) in September
2006.
Air-dried aerial parts were subjected to hydrodistillation us-
ing a Clevenger-type apparatus for 3 h to yield 0.08% v/w of
yellowish oil. The obtained oil with a distinct odor was col-
lected in pentane (1 mL), dried over anhydrous sodium sul-
phate and was stored under N2 atmosphere in amber vials
at 4°C until analysis. The oil was analyzed within 24 h from its
production.The identification of the chemical constituents
was based on comparisons of their retention times and mass
spectra with those obtained from authentic samples and/or
the NIST/NBS, Wiley libraries’ spectra as well with literature
data2.
Susceptibility tests
Teen strains of C. albicans (isolated from different patients)
were studied. Anti fungal activity of D. graveolens essential
oil was investigated by agar well diffusion method3.
RESULTS AND DISCUSSION
A total of twenty two components added up to 98.69% of
the oil was identified. The main constituents were 1,8 Cineol
(54.89%), P-Cymen (16.2%), [beta]-pinene (6.94%) and Bor-
neol (5.44%).
The oil components can be classified into monoterpene hy-
drocarbons (28%), oxygenated monoterpenes (66.70%) and
sesquiterpene hydrocarbons (3.99%).
155
In the present study the anti Candida activities of the essen-
tial oil of D. graveolens were evaluated against teen isolates
of C. albicans by agar well diffusion method. Several concen-
trations of the essential oil of D. graveolens were applied on
isolates. The results showed remarkable activities against
tested isolates. As shown the lowest MIC for teen isolates of
C. albicans was 28.84 mg/ml. Totally the MIC for 10 isolates of
C. albicans was 30.675mg/ml. In the present study the MIC of
hyphal forms of C. albicans was 15-16 mg/ml.
Numerous essential oils have been tested for in vivo and in
vitro antimycotic activity and some demonstrated to be po-
tential antifungal agents. Their mechanism of action appears
to be predominantly on the fungal cell membrane, disrupt-
ing its structure causing leakage and cell death; blocking the
membrane synthesis; inhibition of the spore germination,
fungal proliferation and cellular respiration1. Because of high
volatility and lipophilicity of the essential oils, they are read-
ily attached to penetrate into the cell membrane to exert
their biological effect4.
REFERENCES
1. Harris R. Progress with superficial mycoses using essen-
tial oils. International Journal of Aromatherapy, 2002; 12:
83-91.
2. Adams RP. Identification of Essential Oil Components by
Gas Chromatography/Quadropole Mass Spectroscopy.
3rd ed. Illinois: Allured Publlishing Corporation; 2001.
3. Shahidi Bonjar GH, Aghighi S, Karimi Nik A. Antibacte-
 POSTER PRESENTATIONS
rial and Anti fungal Survey in Plants used in Indigenous
Herbal-Medicine of South East Regions of Iran. J Biol Sci.
2005; 4: 405-412
4. Inouye S. Laboratory evaluation of gaseous essential oils
(part 1). International Journal of Aromatherapy, 2003;
13: 95-107.
P067-HISTON DEACETYLASE INHIBITION
ACTIVITIES OF ROSMARINIC ACID AND
QUERCETIN
Deniz Ulutürk1, G. Bora Tatar2, D. Dayangaç Erden2, A. S.
Demir3, S. Dalkara4, H. Erdem Yurter2
Hacettepe University1
Department of Medical Biology, Faculty of Medicine, Hacettepe
University, Ankara, Turkey2
Department of Chemistry, Faculty of Arts and Science, Middle
East Technical University, Ankara, Turkey3
Department of Pharmaceutical Chemistry, Faculty of
Pharmacy, Hacettepe University, Ankara, Turkey4
INTRODUCTION
The acetylation and deacetylation of histones have been shown
to play an important role in transcription regulation and are
regulated by histone deacetylases (HDACs) and histone acetyl-
transferases (HATs). HATs add acetyl groups to lysine residues
of amino terminal of histone proteins, whereas HDACs remove
156 the acetyl groups. HDACs can act as transcription repressors
due to histone deacetylation that causes chromatin condensa-
tion. HDACs have emerged as attractive drug targets and sev-
eral HDAC inhibitors have been developed as drug candidates.
According to the accumulated preclinical data, HDAC inhibitors
may be used for the treatment of numerous neurodegenerative
disorders such as spinal muscular atrophy1,2.
During the past 15 years, a number of structurally diverse HDAC
inhibitors have been identified including carboxylic acids3. In
our previous studies we found that cinnamic acid-like pheno-
lic compounds (caffeic acid derivatives) are HDAC inhibitors
with at least equal or better activity compared to conventional
carboxylates such as sodium butyrate (NaBA) and valproic acid
(VPA). Based on these data, two of the well-known natural caf-
feic acid derivatives, chlorogenic acid and curcumin showed
high activities5. These results prompted us to investigate HDAC
inhibitory activity of rosmarinic acid and quercetin, which are
dimer and cyclic ester analogs of caffeic acid, respectively:
Formulas
3rd BBBB International Conference on Pharmaceutical Sciences
MATERIALS AND METHODS
In vitro HDAC Inhibition Activity Screening
Among pan-HDAC enzymes, HDAC8 was the first human
HDAC whose three-dimensional and X-ray crystal structure
was described. Therefore pan-HDAC and HDAC8 inhibition
screening studies are important to develop new inhibitor
candidates.
Pan-HDAC and HDAC8 inhibition activity screening of the
compounds were determined in vitro by using a fluoromet-
ric HDAC inhibitor drug screening kit (BioVision/Biomol) ac-
cording to the manufacturer’s protocol. Briefly, HeLa nuclear
extract/HDAC8 enzyme and a HDAC fluorometric substrate,
which comprises an acetylated lysine side chain, were incu-
bated with/without one of the compounds at 50 and 500 µM
concentrations for 37°C at 30 min. Deacetylation sensitized
the substrate and treatment the with lysine developer (37°C
at 30 min) produced the fluorophore. Fluorescence was
measured with fluorescence plate reader (Molecular Devices
Spectramax M2) at excitation 350 nm and emmision 440 nm.
TSA was used as negative control according to the protocol
and NaBA was used as reference compound in all tests as
well. Compounds were screened in triplicate.
Data Analysis
HDAC inhibition activity was analyzed according to the con-
centration against arbitrary fluorescence unit (AFU). HDAC
inhibition activity was assessed according to the decrease in
fluorescence signal. Decreasing fluorescence signal showed
HDAC activity.
RESULTS AND DISCUSSION
In both pan-HDAC and HDAC8 assays, HDAC inhibition ac-
tivities of rosmarinic acid and quercetin were analyzed (Figs
1. and 2). We found that both of them have more potent
HDAC inhibition activities than NaBA which is a well-known
carboxylate inhibitor.
Figure 1: Inhibition of pan-HDAC activity by rosmarinic acid
and quercetin
 POSTER PRESENTATIONS
Figure 2: Inhibition of HDAC8 activity by rosmarinic acid and
quercetin
CONCLUSION
Rosmarinic acid and quercetin showed a higher HDAC inhi-
bition activity than NaBA. This result is in accordance with
our previous data and support our proposal that caffeic acid
is a promising lead structure as HDAC inhibitor (4,5). There-
fore, cell culture studies of these two caffeic acid derivatives
is considered for future to conclude that these compounds
may be potential candidate molecules for treatment of can-
cer, neurodegenerative, hereditary and inflammatory dis-
eases.
ACKNOWLEDGEMENT
This project was supported by The Scientific and Technologi-
cal Council of Turkey (TÜBİTAK, Project number: 105G014)
REFERENCES
1. Hahnen E, Hauke J, Tränkle C, Eyüpoglu IY, Wirth B,
Blümcke I. Histone deacetylase inhibitors: possible im-
plications for neurodegenerative disorders, Expert Opin
Investig Drugs. 2008; 17(2): 169-84.
2. Balasubramanian S, Verner E, Buggy JJ. Isoform-specific
histone deacetylase inhibitors: the next step?, Cancer
Lett. 2009; 8;280(2): 211-21.
3. Miller TA, Witter DJ, Belvedere, S. Histon Deacetylase In-
hibitors, J Med Chem. 2003; 46 (24): 5097-5116.
4. Bora-Tatar G, Dayangaç-Erden D, Demir A.S, Dalkara S,
Yelekçi K, , Erdem-Yurter H. Molecular modifications on
carboxylic acid derivatives as potent histone deacety-
lase inhibitors: Activity and docking studies. Bioorg Med
Chem. 2009 Jul 15;17(14):5219-28.
5. Dayangaç-Erden D, Bora G, Ayhan P, Kocaefe C, Dalkara
S, Yelekçi K, Demir AS, Erdem-Yurter H. Histone deacety-
lase inhibition activity and molecular docking of (e)-
resveratrol: its therapeutic potential in spinal muscular
atrophy, Chem Biol Drug Des. 2009; 73(3): 355-64.
October 26 - 28, 2009 / Antalya - TURKEY
P068-IDENTIFICATION OF
PHARMACOPHORES IN ANTIOXIDATIVE UPF
PEPTIDES.
Kalle Kilk1, Riina Mahlapuu1, Säde Viirlaid2, Jaak Järv2, Ursel
Soomets1
Institute of Biochemistry, Medical Faculty, University of Tartu,
The Centre of Excellence for Translational Medicine, 19 Ravila
St, 50411 Tartu, Estonia1
Institute of Chemistry, University of Tartu, 2 Jakobi St, 51014
Tartu, Estonia2
INTRODUCTION
Oxidative stress and its control by antioxidants is an issue
related to many pathological conditions. Peptides UPF1 and
UPF17 are analogues of one of the most important natural
antioxidant glutathione (GSH). In addition to glycyl, cysteinyl
and glutamyl residues they contain an extra N-terminal O-
methyl-tyrosine (Fig 1). These peptides have demonstrated
excellent free radical scavenging properties exceeding GSH1.
We have aimed to determine the main pharmacophores in
these novel antioxidants.
157
Figure 1: Structures of UPF1 (A) and UPF17 (B)
MATERIALS AND METHODS
Liquid chromatography – mass spectrometry
For peptide oxidation studies 10µM CuCl2 in 200 µl PBS and
10mM H2O2 were mixed in order to generate OH• in a Fen-
ton like reaction. Peptide was added to final concentration
100µM.
LC-MS was carried out on a Shimadzu Prominence - Applied
Biosystems QTrap 3200 tandem system. C18 column was
used to separate the buffer salts, peptides and oxidation
products from each other. Flow rate was 0.2 ml/min. Step-
wise gradient started with 10 min isocratic flow of 99.9%
H2O 0.01% HCOOH, after which acetonitrile concentration
rose to 50% within 10 min. Finally, 5 min elution at 50% ac-
etonitrile was done. Mass spectrometry detection range was
50-1500 Da. Ions were generated in negative mode, -4500
volts, 150 ºC.
Determination of pKa
The principe of thiol and thiolate ratio measurement was
their different absorbance at 240nm. 1 ml 50 μM solution of
peptide in PBS was titrated with 10 or 5 μl volumes of 1 M
NaOH. pH and absorbance changes were determined after
each addition. Absorbance was recorded on PerkinElmer
Lambda 25 spectrometer.
 POSTER PRESENTATIONS
Kinetic measurements
Scavenging of 2,2-diphenyl-1-picrylhydrazyl radical (DPPH)
was monitored spectrophotometrically. Briefly, 0.25 ml of
peptide solution in citric acid–sodium citrate buffer (20 mM,
pH 3.9) was added to 0.25 ml of DPPH solution in 95% etha-
nol. Changes in the absorbance at 517 nm, occurring due to
scavenging the free radical, were monitored in thermostated
quartz cuvette at 25 °C (UV–VIS spectrophotometer Unicam
UV300, ThermoSpectronic, USA). Data analysis was done by
Graphpad Prism 4.0 software.
RESULTS AND DISCUSSION
By liquid chromatography – mass spectrometry we deter-
mined that under used conditions aromaticity containing
UPF peptides oxidize further than non-aromatic controls
(Fig. 2). Oxidation products beyond expected disulphide
were identified as sulfinic (-SO2H) and sulfonic (-SO3H) acids.
No alteration in the aromatic ring was found and analogues
without the thiol group remained unreactive. Thus, the cen-
tre of the oxidative defense is the thiol group.
158
Figure 2: Mass spectra of oxidation of (A) GSH and (B) UPF17
by OH• at the beginning of the reaction and in 5 h.
By titration assays it was found that additional N-terminal
amino acids do not lower the pKa of the thiol, but rather
increase it slightly (8.97±0.3 vs 9.22±0.1). Hence the reason
why aromatic UPF peptides oxidize more readily than non-
aromatic analogues must lie in pi-electron cloud – free radi-
cal interaction. This interaction could prolong the time a free
radical spends near the thiol and thereby “catalyze” the reac-
tion.
3rd BBBB International Conference on Pharmaceutical Sciences
Figure 3: Kinetic differences in DPPH radical scavenging by
alpha and gamma glutamyl containing peptides.
The linker between the thiol and aromatic ring, glutamyl
residue, was found to have an impact as well. The glutamyl
residue can be attached to cysteine either via its alpha or
gamma carboxylic group. Compounds with alpha glutamyl
residue showed significatly faster reaction rates compared
to respective gamma-glutamyl analogues (Fig. 3). Thus, ei-
ther the length of this linker or position of the free carbocylic
group is a determinant radical scavenging kinetics.
CONCLUSION
In summary, we have identified the major structural features
that determine the biological activity of antioxidative UPF
peptides (Fig. 4).
Figure 4: Main pharmacophores of UPF peptides.
ACKNOWLEDGEMENT:
This study was financed by Estonian Science Foundation
grants 6503, 7494, 7856, Estonian Ministry of Education and
Research (Grant SF0180064s08), and by the European Re-
gional Development Fund.
REFERENCES
1. Ehrlich K, Viirlaid S, Mahlapuu R, Saar K, Kullisaar T,
Zilmer M, Langel U, Soomets U. Design, synthesis and
properties of novel powerful antioxidants, glutathione
analogues. Free Radic Res. 2007 Jul;41(7):779-87
p069-Wfs1 KO AND WILD TYPE MICE DIFFER
IN THEIR STRIATAL DOPAMINE OUTPUT
IN RESPONSE TO K+ BUT NOT MORPHINE
CHALLANGE
Vallo Matto1, Anton Terasmaa2, Sulev Kõks2
Department of Pharmacy, University of Tartu, Estonia1
Department of Physiology and Centre of Excellence for
Translational Medicine, University of Tartu , Estonia 2
INTRODUCTION
Wolfram syndrome is a very rare inherited disorder caused
by the loss-of-function mutations of in the Wfs1 gene and
 POSTER PRESENTATIONS
characterized by a complex of endocrinological symptoms
and/or neuropsychological problems. In mutant mice mod-
els with Wfs1 gene knocked out (Wfs1 KO) evidence has been
collected that the wfs1 protein is an important link between
the endocrinological and CNS systems. The Wfs1 KO mice
are physically retarded, they have serious carbohydrate me-
tabolism dysregulation, and they are also emotionally devi-
ated from wild type conspecifics1. Dopamine (DA) is a major
neuromediator involved in the mediation of the emotional,
motivational, and locomotor components of behavior. The
present set of experiments was intended to explore the pos-
sible differences in extracellular DA concentrations in wild
type and Wfs1 KO mice in response to acute morphine treat-
ment and high concentration K+ challenge.
MATERIALS AND METHODS
Animals and microdialysis surgery
The mice (male 3–5 months old F2 hybrids) were anesthe-
tized with Fentanyl-Fluanizone-Midazolam 30 min before
the surgery. The mice were attached from outer ear canals to
the stereotaxic apparatus equipped with mouse nose hold-
er. The coordinates for the guide cannulae (CMA/7, CMA/Mi-
crodialysis) were aimed at the point above the striatum (AP =
+0.6 mm, L = +2.0 mm, DV = -2.3 mm, relative to bregma).
Microdialysis experiments
The microdialysis experiments were carried out using the
technique described by Airavaara et al., 2, with some modi-
fications. A microdialysis probe was inserted into the guide
cannula, and the probe was infused with a modified Ringer
solution (147 mM NaCl, 1.2 mM CaCl2, 2.7 mM KCl, 1.0 mM
MgCl2, and 0.04 mM ascorbic acid) at the flow rate of 1.3 mi-
crol/min. The collection of the actual microdialysis samples
(every 20 min, 26 microl/sample) was started after two hours
of stabilization period. The DA concentrations were deter-
mined by an Agilent (HP 1100 Series) high performance liq-
uid chromatography (HPLC) system additionally equipped
with an electrochemical detector (Coulochem II, ESA), a
model 5011 analytical cell, an Agilent 35900E interface, and a
Rheodyne 7725i manual injector. The column (Zobirax SB-C-
18, 4×140 mm, 3.5 micron ODS) was kept at room tempera-
ture (20±1 °C). The mobile phase used (flow rate 1.0 ml/min)
consisted of 0.1 M NaH2PO4 buffer, pH 4.2 (adjusted with 1.0
mM citric acid), 0.6–0.8 mM octanesulphonic acid, 12–15%
methanol, and 1.2 mM EDTA.
20 microliters of dialysate per sample was injected into the
column. DA was reduced with an amperometric detector
(-100 mV) after being oxidized with a coulometric detector
(+600 mV). The DA concentrations are presented as relative
to the baseline (± SEM, the average concentration of three
consecutive stable samples were defined as 100%). The ani-
mals received after one hour an i.p. injection of 10 mg/kg
morphine, one hour before the completion of the experi-
ment the modified Ringer solution was replaced with the
[K+]-rich modified Ringer solution (corresponds to 100 mM
[K+]).
October 26 - 28, 2009 / Antalya - TURKEY
RESULTS AND DISCUSSION
The concentrations of DA of the baseline samples were 0.8
nM to 1.4 nm for the wild type animals and 0.6 to 1.3 nM
for the Wfs1 KO mice. Repeated measures ANOVA revealed
a significant time (F(1,11)= 18.9, p<0.001) and KO animal
(F(1,11)= 14.6, p<0.01) effect, as well as an interaction be-
tween these factors (F(1,11)= 10.6, p<0.01). 10 mg/kg mor-
phine injection increased the striatal DA output in all tested
wild type animals (up to 160% from baseline) while in the
KO animals only a minor increase (up to 105% from baseline,
with a large in-group variation) was found. Time-point-by-
time-point post hoc analysis (20 – 120 min) considered this
difference as statistically insignificant. Expectedly, the appli-
cation of the [K+]-rich modified Ringer solution dramatically
increased the striatal DA output in wild type animals (up to
3000% for the last time point measured) but the increase of
the striatal DA output of Wfs1 KO mice was less pronounced
yielding only up to 450% from the baseline values (Figure
1). There was a statistically significant difference between
these two mice groups (p<0.01 and p<0.001 for the time
points 160 and 180 min, respectively). Our results show that
the regulation of the DA output in striatum of the Wfs1 KO
mice is impaired. This can be due to the impaired regulatory
pathways or alternatively, the Wfs1 KO mice have limited DA
intraneuronal storage capacity. Further studies are needed
to assess the exact mechanisms of these findings and the
possible causal relationship between the deviated behavior
and impaired striatal DA output of Wfs1 KO mice.
159
Figure 1:Striatal DA output in wild type and Wfs1 KO mice.
ACKNOWLEDGEMENTS
This study was financed by a University of Tartu grant No
PARFA 08902.
REFERENCES
1. Kõks S, Vasar E (Eds.), Wfs1 protein (Wolframin): Emerg-
ing link between the emotional brain and endocrine
pancreas, Research Signpost, Kerala, India, 2008.
2. Airavaara M, Mijatovic J, Vihavainen T, Piepponen TP,
Saarma M, Ahtee L. In heterozygous GDNF knockout
mice the response of striatal dopaminergic system to
acute morphine is altered, Synapse. 2006; 59: 321-329.
 POSTER PRESENTATIONS

p070-WFS1- DEFICIENT MICE AS A POSSIBLE

ANIMAL MODEL OF PATHOLOGICAL ANXIETY

Sirli Raud1

Department of Physiology, University of Tartu, 19 Ravila Street,

50411 Tartu, Estonia; Centre of Excellence for Translational
Medicine, 19 Ravila Street, 50411 Tartu, Estonia1

INTRODUCTION
Mutations in coding region of Wolframin (WFS1) gene cause
Wolfram syndrome, a rare autosomal recessive disorder char-
acterized by diabetes insipidus, juvenile-onset non-autoim-
mune diabetes mellitus, optic atrophy and deafness1,2. Evi- Figure 1: Lower exploratory activity in Wfs1-deficient mice
dence exists that around 60 % of Wolfram syndrome patients at high illumination levels.White bars - wild-type mice, black
have psychiatric symptoms related to depression, psychosis, bars - Wfs1-deficient mice.
impulsivity, and aggression3. The exposure of rats to cat
odour induces the expression of Wfs1 gene in the amygdala Anxiety-like behaviour
and CA1 region of hippocampus.4,5. The major goal of pres- Short-term isolation for 15-20 minutes before the experiment
ent study was to extend our understanding about the func- had a significant anxiogenic-like effect on the behaviour of
tional role of Wfs1 in the regulation of stress and anxiety us- Wfs1-deficient mice in dark/light exploration test (Figure 2)
ing the genetically modified mice.

METHODS AND MATERIALS

According to our preliminary studies, the severity of outcome
of Wfs1 gene ablation is sex-dependent and progresses with
age. Therefore adult female mice of F2 hybrids (129SvEvTac
and C57Bl/6 mixed background), lacking Wfs1 gene was
used. Figure 2: Anxiety-like behaviour in Wfs1-deficient mice in
160 light-dark box test. White bars - wild-type mice, black bars -
Behavioural testing included: models of anxiety (elevated Wfs1-deficient mice. (*) p <
plus-maze, dark-light exploration, fear conditioning and
hyponeophagia tests), tests measuring motor performance Lower exploratory activity of Wfs1-deficient mice in the plus-
(rota-rod test) and locomotor activity (motility box). Ad- maze was antagonised by pre-treatment with diazepam (1
ditionally, mice were tested for sensitivity to diazepam, an mg/kg). In Wfs1-deficient mice, the administration of diaz-
anxiolytic GABAA receptor agonist. Quantitative real-time epam caused a robust anxiolytic-like effect as evidenced by
PCR was used to determine the expression of alpha1- and increased number of open arm entries and longer time spent
alpha2-subunits of GABAA receptors, the major targets in in exploring of open arms (Figure 3).
the action of diazepam in the frontal cortex, temporal lobe,
hippocampus and mesolimbic area.

RESULTS AND DISCUSSION

The most peculiar aspect of the overt phenotype of Wfs1-de-
ficient population was the presence of individuals producing
spontaneous audible vocalizations. The proportion of such
individuals varied across different batches from around 15
to 50 percent, and was comparable in males and females. It
was noted that the number of mice producing audible vocal-
izations was higher under stressful conditions (bright light,
handling, injection stress). In most individuals, calling was
blocked by the administration of diazepam (1 mg/ml).

Motor performance and locomotor activity

Wfs1-deficient mice did not display obvious shortcomings in mo-
tor functioning as exemplified by intact responses in rota-rod test
(data not shown). Locomotor activity of Wfs1-deficient mice was
significantly lower only in brightly lit environment (Figure 1).

3rd BBBB International Conference on Pharmaceutical Sciences

 POSTER PRESENTATIONS
Figure 3: Anxiolytic effect of diazepam (1 mg/kg) on explor-
atory behaviour of Wfs1-deficient mice in the elevated plus-
maze test. White bars - wild-ty
Wfs1-deficient mice displayed also increased anxiety-like be-
haviour in hyponeophagia test. Latency to start eating was
significantly longer in Wfs1-deficient mice (162±8 sec) when
compared to wild-type littermates (135±10 sec). There were
no differences between Wfs1-deficient mice and wild-types
in conditioned fear responses.
Gene expression studies
Wfs1-deficient mice displayed reduced expression of al-
pha1- and alpha2-subunits of GABAA receptor in the tempo-
ral lobe and frontal cortex. The exposure of wild-type mice
to the elevated plus-maze also reduced the expression level
of these genes in the temporal lobe and frontal cortex. The
exposure of Wfs1-deficient mice to the elevated plus-maze
did not cause a further reduction in the expression of the
alpha1- and alpha2 subunit genes compared to the experi-
mentally naïve mutant mice (Figure 4).
Figure 4: Effect of Wfs1 gene invalidation and plus-maze ex-
posure on the expression of the alpha1- and alpha2 subunit
genes in the forebrain structure
CONCLUSION
Altogether the present study demonstrates impaired behav-
ioural adaptation of Wfs1-deficient mice in stress-inducing
situations. It is possible that the genetic invalidation of Wfs1
compromises the function of GABAA receptors in the limbic
structures. Therefore, one could suggest that Wfs1 protein
plays a major role in the mechanisms of behavioural adapta-
tion to stressful environment and it may be possible target
for developing new effective compounds against anxiety.
ACKNOWLEDGEMENTS
This study was supported by Estonian Science Foundation
grant 7651 and European Union via European Regional De-
velopment Fund (MARFR08059R).
REFERENCES
1. Strom TM, Hortnagel K, Hofmann S, Gekeler F, Scharfe C,
Rabl W, et al. Diabetes insipidus, diabetes mellitus, optic at-
rophy and deafness (DIDMOAD) caused by mutations in a
novel gene (wolframin) coding for a predicted transmem-
brane protein. Hum Mol Genet, 1998; 7(13): 2021-2028.
October 26 - 28, 2009 / Antalya - TURKEY
2. Wolfram D, Wagener H. Diabetes mellitus and simple op-
tic atrophy among siblings: report of four cases. Mayo
Clin Proc, 1938; 9: 715–718.
3. M. Swift, R.G. Swift, Wolframin mutations and hospital-
ization for psychiatric illness, Mol Psychiatry, 2005; 10(8):
799-803.
4. Kõks, S., Luuk, H., Nelovkov, A., Areda, T., and Vasar, E. A
screen for genes induced in the amygdaloid area during
cat odor exposure. Genes Brain Behav, 2004; 3(2): 80-89.
5. Kesner, Y., Zohar, J., Merenlender, A., Gispan, I., Shalit,
F., and Yadid, G. WFS1 gene as a putative biomarker for
development of post-traumatic syndrome in an animal
model. Mol Psychiatry, 2009; 14(1): 86-94.
p071-ACUTE VALPROIC ACID IMPROVES
GLUCOSE TOLERANCE IN Wfs1 KO MICE
Anton Terasmaa1, Ursel Soomets2, Mats Hansen3, Vallo
Matto4, Eero Vasar5, Sulev Kõks5
Dept of Physiology, University of Tartu, Tartu, Estonia1
Dept of Biochemistry and Centre of Excellence for Translational
Medicine, University of Tartu, Tartu, Estonia 2
Dept of Biochemistry, University of Tartu, Tartu, Estonia 3
Dept of Physiology, University of Tartu, Tartu, Estonia4
Dept of Physiology and Centre of Excellence for Translational
Medicine, University of Tartu, Tartu, Estonia 5
INTRODUCTION 161
Wolfram syndrome is a very rare inherited disorder caused
by the loss-of-function mutations of in the WFS1 gene and is
characterized by a complex of endocrinological and neurop-
sychological problems1.
Wfs1 protein is a 890 amino acids long transmembrane pro-
tein located in endoplasmic reticulum (ER), lack of its func-
tion results in impaired ER stress response and apoptosis2-5.
Wfs1 is involved in the activation and secretion of the bioac-
tive peptides, including insulin. Diminished function of Wfs1
leads to apoptosis of pancreatic beta-cells.
Wfs1 KO mice exhibit impaired glucose tolerance, are sig-
nificantly smaller than their wild-type littermates despite
elevated growth hormone (GH) and insulin-like growth fac-
tor (IGF-1) levels6. Thus, Wfs1 deficiency seems to modulate
signaling cascade downstream of insulin and IGF receptors.
Expression of PI3K is reduced in temporal lobe of Wfs1 KO
mice as revealed with Affymetrix gene chip array6. Therefore,
we hypothesize that signaling cascade linking insulin ecep-
tor to glycogen synthase (PI3K/Akt/GSK3 pathway) is com-
promised as a result of inactivation of Wfs1 gene.
Glycogen synthase kinase 3 (GSK3) is a key component of
large number of cellular processes including glucose homeo-
stasis and apoptosis. Reduced inhibitory control of GSK3 ac-
tivity is one mechanism for development of type 2 diabetes
and insulin resistance, thus GSK3 activity is above normal in
diabetic rodents and in skeletal muscle of patients with type
2 diabetes7,8. Moreover, mood stabilizers are direct inhibitors
 POSTER PRESENTATIONS
of GSK3, thus pointing to importance of GSK3 in mechanism
of psychiatric disorders.
Lithium and valproic acid (VPA), widely used mood stabilis-
ers are inhibitors of GSK3 activity in vivo. The mood stabilizer
VPA induces Wfs1 protein expression and facilitates the ER
stress response9, thus linking GSK3 activity with ER stress re-
sponse10.
The aim of this study is to characterize effect of valproic acid
on the glucose homeostasis in the Wfs1 KO mice.
MATERIALS AND METHODS
Animals and glucose tolerance test: generation of Wfs1
knockout mice are described previously6, male 3 to 5 months
old mice were used in this study. On the day of experiment,
tail vein glucose was measured using hand held glucometer,
drugs were administered IP, and 15 minutes later blood glu-
cose was measured and glucose solution (2g/kg) was admin-
istered IP. Thereafter, blood glucose levels were monitored
for 3 hours.
Animals were non-fasted and had no access to food during
the experiment. Water was available ad libidum.
Drug doses were: valproic acid 300 mg/kg, lithium 200 mg/
kg, Diazepam 3 mg/kg.
Statistical analysis: repeated-measures ANOVA (genotype x
treatment - time) with Tukey post-hoc test was used.
162
RESULTS AND DISCUSSION
Wfs1 KO mice had lower body weight as compared with
their WT littermates (22.5+0.5 g vs 32.0+0.5g, mean+S.E.M.,
p<0.0001, student t-test), which confirms previous findings
(ref 41).
Blood glucose levels were similar between genotypes at
resting conditions. Glucose administration induced tran-
sient increase in blood glucose levels in WT animals which
were back to baseline after 60 min. In contrast, glucose ad-
ministration led to much larger and longer lasting increase
of blood glucose level in Wfs1 KO animals (genotype x treat-
ment, F(6,264)=22.0, p<0.001)
Acute valproate (300 mg/kg) prevented blood glucose rise
in Wfs1 KO mice, but had no effect on blood glucose levels
in WT mice (genotype x treatment, F(6,264)=24.2, p<0.001,
Fig1).
Lithium, another GSK3 inhibitor, failed to mimic the effect
of valproate on blood glucose intolerance in Wfs1 KO mice
(p=0.99, data not shown).
Diazepam, an allosteric modulator of GABA(A) receptor en-
hanced glucose intolerance of Wfs1 Ko mice (genotype x
treatment, F(6,264)=4.8, p<0.001, Fig1).
Acute treatment with valproic acid restores glucose tolerance of
Wfs1 KO mice to normal levels. As lithium did not mimick the ef-
fect of valproate, it is hard to say whether effect of valproate is in
3rd BBBB International Conference on Pharmaceutical Sciences
fact mediated via GSK3 inhibition. Valproate has multiple sites of
action, including GSK3, GABA receptors and several transcription
factors. Potentiating of GABA signaling with diazepam results in
opposite action compared to valproate. Valproate itself is an indi-
rect agonist at GABA receptors, thus effect of valproate is unlikely
to be mediated via activation of GABA receptors. The molecular
mechanism of action, by which valproate restores glucose utiliza-
tion in Wfs1 KO mice remains to be investigated at this time.
ACKNOWLEDGEMENTS
This study was financed by a University of Tartu grant No
PARFS 07904, grant from Estonian Science Foundation No
GARFS 0062J and by Centre of Excellence for Translational
Medicine, University of Tartu.
Figure 1: Glucose tolerance test in male Wfs1 KO mice.
Drugs or vehicle were administered 15 minutes before (left
arrow) administration of glucose (right arrow). WT mice
(filled circles) did not exhibit glucose intolerance, which was
not modified by valproate (300 mg/kg, filled triangles) or di-
azepam (3 mg/kg, filled squares). Wfs1 KO mice (open circles)
show impaired glucose tolerance, valproate (open triangles)
restores glucose tolerance to the level of WT mice, while di-
azepam (open squares) impairs glucose tolerance in Wfs1 KO
mice. (Data are presented as mean ± S.E.M, n=8 to 16).
REFERENCES
1. Kõks S., and Vasar E., (Eds.), Wfs1 protein (Wolframin): Emerg-
ing link between the emotional brain and endocrine pan-
creas, Research Signpost, Kerala, India, 2008.
2. Kakiuchi C, Ishiwata M, Hayashi A, Kato T. XBP1 induces WFS1
through an endoplasmic reticulum stress response element-
like motif in SH-SY5Y cells. J Neurochem 2006; 97: 545-555.
3. Yamada T, Ishihara H, Tamura A, Takahashi R, Yamaguchi S et
al. WFS1-deficiency increases endoplasmic reticulum stress,
impairs cell cycle progression and triggers the apoptotic
pathway specifically in pancreatic beta-cells. Hum Mol Gen-
et 2006; 15: 1600-1609.
4. Ueda K, Kawano J, Takeda K, Yujiri T, Tanabe K et al. Endo-
plasmic reticulum stress induces Wfs1 gene expression in
pancreatic beta-cells via transcriptional activation. Eur J En-
docrinol 2005; 153: 167-176.
5. Fonseca Sg, Fukuma M, Lipson Kl, Nguyen Lx, Allen Jr et al.
WFS1 is a novel component of the unfolded protein response
and maintains homeostasis of the endoplasmic reticulum in
 POSTER PRESENTATIONS
pancreatic beta-cells. J Biol Chem 2005; 280: 39609-39615.
6. Koks S, Soomets U, Paya-Cano Jl, Fernandes C, Luuk H et al.
Wfs1 gene deletion causes growth retardation in mice and
interferes with the growth hormone pathway. Physiol Ge-
nomics 2009; 37: 249-259.
7. Eldar-Finkelman H. Glycogen synthase kinase 3: an emerg-
ing therapeutic target. Trends Mol Med 2002; 8: 126-132.
8. Nikoulina Se, Ciaraldi Tp, Mudaliar S, Mohideen P, Carter L et
al. Potential role of glycogen synthase kinase-3 in skeletal
muscle insulin resistance of type 2 diabetes. Diabetes 2000;
49: 263-271.
9. Kakiuchi C, Ishigaki S, Oslowski Cm, Fonseca Sg, Kato T et al.
Valproate, a mood stabilizer, induces WFS1 expression and
modulates its interaction with ER stress protein GRP94. PLoS
ONE 2009; 4: e4134.
10. Chuang Dm. The antiapoptotic actions of mood stabilizers:
molecular mechanisms and therapeutic potentials. Ann N Y
Acad Sci 2005; 1053: 195-204.
P072-NAPROXEN SODIUM IN ACUTE
MIGRAINE TREATMENT: A META-ANALYSIS OF
RANDOMIZED CONTROLLED TRIALS
Naeti Suksomboon1, Chuthamanee Suthisisang2,
Nalinee Poolsup3, Vorachart Lertpipopmetha1, Bhakanit
Tepwitukgid1
Department of Pharmacy, Faculty of Pharmacy, Mahidol
University, Bangkok, Thailand1
Department of Pharmacology, Faculty of Pharmacy, Mahidol
University, Bangkok, Thailand2
Department of Pharmacy, Faculty of Pharmacy, Silpakorn
University, Nakhon-Pathom, Thailand3
INTRODUCTION
Migraine is a recurring headache of moderate to severe
intensity. It is typically described as an intense pulsing or
throbbing pain in one area of the head and is often char-
acterised by neurological, gastrointestinal, and autonomic
symptoms. Treatment strategies include non-pharmacologic
and pharmacologic treatments for acute migraine and for
prophylaxis1. Naproxen is an effective inhibitor of cyclo-ox-
ygenase, resulting in the inhibition of prostaglandin synthe-
sis. Naproxen sodium improved the solubility of naproxen,
achieving fast absorption and rapid onset of action. Several
studies have investigated the efficacy of naproxen sodium
in the treatment of migraine headache2, 3. We undertook a
meta-analysis to evaluate the efficacy of naproxen sodium in
an attempt to determine its therapeutic benefit in the treat-
ment of acute migraine attacks.
MATERIALS AND METHODS
Identification of studies
Reports of randomized controlled trials of naproxen sodium
were identified through a systematic search on MEDLINE, EM-
BASE, and the Cochrane Library. The bibliographic databases
were searched from their respective inception to June 2009.
The MeSH search terms used were “migraine disorders”, “head-
October 26 - 28, 2009 / Antalya - TURKEY
ache”, “vascular headache”, “naproxen”, “adult”, and “clinical tri-
al”. This was followed by a key word search using “migraine”,
“cephalalgia”, and “cephalgia” as key words. The reference lists
of relevant articles were also scanned to identify possible pub-
lished trials. No language restriction was imposed.
Study selection and data extraction
The studies were included in the meta-analysis if they were 1)
double-blind, randomized, placebo-controlled trials that evalu-
ated naproxen sodium tablet in moderate or severe migraine
attacks in adult patients, and 2) reporting the efficacy in terms
of headache relief or pain-free at 2 hours. Data extraction was
performed independently by 2 investigators using a standard-
ized form. Disagreements were resolved by a third investigator.
Statistical analysis
Treatment effect was expressed as risk ratio (RR) and num-
ber needed to treat (NNT) as well as 95% confidence inter-
val. The inverse variance-weighted method was used for the
pooling of RR and the estimation of 95% confidence interval.
A random effects model was used when the Q-statistic for
heterogeneity was significant at the level of 0.1, otherwise
the fixed effects model was used. The degree of heteroge-
neity was quantified using I-squared statistic. The statistical
analysis was undertaken with RevMan version 5.0 (Cochrane
collaboration). The significant level was set at p < 0.05.
RESULTS AND DISCUSSION
We identified 18 published randomized controlled trials of
naproxen sodium in the acute migraine treatment. All were
published in English. Three trials met our inclusion criteria 163
and were included2-4. Of these, one3 reported the results
from two trials and, therefore, was treated as two separated
studies. Dose of naproxen was 500 mg2,3 and 825 mg4. Char-
acteristics of these studies are presented in Table 1.
Table 1: Characteristics of randomized controlled trials of
naproxen sodium for the treatment of acute migraine attack
Study
Intervention N
(ref.)
Smith naproxen sodium 500 mg 972
20052 sumatriptan 50 mg. N=350
sumatriptan 50 mg plus naproxen sodium 500mg S=229
or placebo for a single migraine attack N+S=251
P=242
Brandes naproxen sodium 500 mg 1461
20073 sumatriptan 85 mg. N=361
sumatriptan 85 mg plus naproxen sodium 500mg S=365
or placebo for a single migraine attack N+S=370
P=365
Brandes naproxen sodium 500 mg 1495
20073 sumatriptan 85 mg. N=371
sumatriptan 85 mg plus naproxen sodium 500mg S=370
or placebo for a single migraine attack N+S=367
P=387
Wentz naproxen sodium 825 mg 284
20084 gw406381 70 mg N=93
or placebo for a single migraine attack GW406381=92
P=99
N=naproxen sodium; S=sumatripan; P=placebo
 POSTER PRESENTATIONS
As expected, naproxen sodium was more effective than
placebo in terms of both headache relief and pain-free at
2 hours. The pooled RRs were 1.58 (95%CI 1.41-1.77, p <
0.00001), and 2.22 (95%CI 1.46-3.37, p = 0.0002), respectively
(Figure 1). The corresponding numbers needed to treat were
7 (95%CI 5-9) and 10 (95%CI 7-25), respectively, for headache
relief and pain-free.
Based on the mean NNT for headache relief at 2 hours, naprox-
en sodium seems to be inferior to ibuprofen 400 mg (7 vs 4)5.
This may be explained by the rapid onset of action and shorter
time to peak plasma concentration of ibuprofen. The mean
NNT of 10 for pain-free at 2 hours for naproxen sodium was
comparable to that of ibuprofen 400 mg (NNT 9)5. However, its
effectiveness relative to other active comparators needs to be
better defined by appropriate head to head trials.
Figure 1: Efficacy of naproxen sodium vs. placebo
164 CONCLUSIONS
The current evidence indicates that naproxen sodium is an
effective treatment for moderate to severe migraine attacks.
It is consistently effective in reducing moderate or severe
pain to mild or no pain and rendering patients pain-free
within 2 hours.
REFERENCES
1. Silberstein SD, Migraine, Lancet 2004; 363: 381-391.
2. Brandes JL, Kudrow D, Stark S, O’Carroll CP, Adelman JU,
et al. Sumatriptan-naproxen for acute treatment of mi-
graine: a randomized trial, JAMA 2007; 297: 1443–1454.
3. Smith TR, Sunshine A, Stark SR, Littlefield DE, Spruill SE
et al. Sumatriptan and naproxen sodium for the acute
treatment of migraine, Headache 2005; 45: 983–991.
4. Wentz AL, Jimenez TB, Dixon RM, Aurora SK, Gold M on
behalf of the CXA20008 Study Investigators. A double-
blind, randomized, placebo-controlled, single-dose
study of the cyclooxygenase-2 inhibitor, GW406381, as
a treatment for acute migraine, Eur J Neurol. 2008; 15:
420-427.
5. Suthisisang C, Poolsup N, Kittikulsuth W, Pudchakan P,
Wiwatpanich P. Efficacy of low-dose ibuprofen in acute
migraine treatment: systematic review and meta-analy-
sis, Ann Pharmacother. 2007; 41: 1782-1791.
3rd BBBB International Conference on Pharmaceutical Sciences
p073-SYSTEMATIC REVIEW OF THE EFFECT OF
CINNAMON ON GLYCEMIC CONTROL IN TYPE
2 DIABETES
Sukamai Boonkaew1, Naeti Suksomboon1, Nalinee Poolsup2
Department of Pharmacy, Faculty of Pharmacy, Mahidol
University, Bangkok, Thailand1
Department of Pharmacy, Faculty of Pharmacy, Silpakorn
University, Nakhon-Pathom, Thailand2
INTRODUCTION
Type 2 diabetes is a chronic metabolic disease resulting from
impaired insulin action, impaired insulin secretion or both.
Management of type 2 diabetes comprises of diet and ex-
ercise, insulin replacement therapy and oral antidiabetic
drugs. Natural medicines with antihyperglycemic activities
have also been used in folk medicine and traditional heal-
ing systems around the world. Cinnamon, one of herbal
medicines, has been shown to enhance glucose uptake and
glycogen synthesis resulting in improved insulin sensitivity.
There was a systematic review published in 2008 that evalu-
ated the impact of cinnamon on glucose and lipids control in
diabetes1. Cinnamon failed to show significantly changes in
glycosylated hemoglobin (HbA1c), fasting blood glucose or
lipid parameters in diabetes patients. Since their review, we
identified one additional study. We therefore performed sys-
tematic review and meta-analysis of the effect of cinnamon
on glucose control in type 2 diabetes to confirm its effect.
MATERIALS AND METHODS
Identification of studies
Reports of randomized controlled trials of cinnamon
were identified through a systematic search on MEDLINE
(Pubmed), EMBASE, and the Cochrane Library. The reference
lists of related reviews and original articles were reviewed.
The bibliographic databases were searched from respec-
tive inceptions to June 2009. The MeSH search terms used
were “cinnamon”, “cinnamomum cassia”, “cinnamomum aro-
maticum”, “clinical trial” each crossed with the term type 2
diabetes mellitus. Hand searching and personal contact with
experts in the area were also undertaken. There was no lan-
guage restriction.
Study selection and data extraction
To be included in this meta-analysis, a study had to 1) be
randomized placebo-controlled trial of cinnamon aimed to
assess glycemic control in type 2 diabetes and 2) report out-
come on HbA1c. Data extraction was performed indepen-
dently by 2 reviewers using a standardized form. Disagree-
ments were resolved by a third investigator.
Statistical analysis
Treatment effect was estimated with mean difference in the
final value of HbA1c between the cinnamon and the placebo
group. The inverse variance-weighted method was used for
the pooling of mean difference and the estimation of 95%
confidence interval. A random effects model was used when
the Q-statistic for heterogeneity was significant at the level of
0.1, otherwise the fixed effects model was used. The degree
 POSTER PRESENTATIONS

of heterogeneity was quantified using the I-squared statistic. REFERENCES

The statistical analysis was undertaken with RevMan version
5.0 (Cochrane Collaboration). The significant level was set at
P < 0.05.
RESULTS AND DISCUSSION
We identified 7 published randomized controlled trials of
cinnamon in diabetes. All were published in English. Four
trials met our inclusion criteria and were included2-5. Char-
acteristics of these studies are presented in Table 1. These
involved a total of 208 patients. There was no significant dif-
ference between cinnamon and placebo. The pooled mean
difference in HbA1c was 0.13% (95% CI -0.11% to 0.36%) (Fig.
1). We found that most of enrolled patients had been pre-
scribed oral antidiabetic drugs therefore we could not com-
pletely evaluate the sole effect of cinnamon on HbA1c.
1. Baker WL, Gutierrez-Williams G, White MC, Kluger J, Cole-
man CI. Effect of cinnamon on glucose control and lipid
parameters, Diabetes Care. 2008; 31: 41-43.
2. Suppapitiporn S, Kanpaksi N, Suppapitiporn S. The effect
of cinnamon cassia powder in type 2 diabetes mellitus, J
Med Assoc Thai. 2006; 89: s200-s205.
3. Mang B, Wolters M, Schmitt B, Kelb K, Lichtinghagen R
et al. Effect of a cinnamon extract on plasma glucose,
HbA1c , and serum lipids in diabetes mellitus type 2, Eur
J Clin Invest. 2006; 36: 340-344.
4. Vanschoonbeek K, Thomassen BJ, Senden JM, Wodzig
WK, van Loon LJ. Cinnamon supplementation does not
improve glycemic control in postmenopausal type 2
diabetes patients, J Nutr. 2006; 136: 977-980.
5. Blevins SM, Leyva MJ, Brown J, Wright J, Scofield RH et
al. Effect of cinnamon on glucose and lipid levels in non-
insulin-dependent type 2 diabetes, Diabetes Care. 2007;
30: 2236-2237.

P074-SYSTEMATIC REVIEW OF THE EFFECT

OF TELMISARTAN ON ADIPONECTIN IN
Figure 1: Effect of cinnamon on HbA1c in type 2 diabetes HYPERTENSIVE PATIENTS WITH INSULIN
RESISTANCE OR DIABETES

Tananya Prasit1, Naeti Suksomboon1, Nalinee Poolsup2

Table 1: Characteristics of the studies included in the meta-
analysis
Department of Pharmacy, Faculty of Pharmacy, Mahidol
Study (ref.) N Study population Intervention Duration 165
University, Bangkok, Thailand1
Suppapitiporn 60 Type 2 diabetes Cinnamon capsule 12 weeks Department of Pharmacy, Faculty of Pharmacy, Silpakorn
20062 treated with either (500mg) 1 capsule University, Nakhon-Pathom, Thailand2
oral antidiabetic three times a day
drugs or diet only with meal or placebo
INTRODUCTION
three times a day
Thiazolidinediones (TZDs), namely rosiglitazone and pioglita-
Mang 20063 65 Type 2 diabetes Cinnamon capsule 4 months zone, stimulate peroxisome proliferator-activated receptor-γ
treated with either (1000mg) 1 capsule (PPAR-γ). It has a role in regulating carbohydrate and lipid
oral antidiabetic three times a day metabolism. TZDs inhibit the expression of tumor necrosis
drugs or diet only with meal or placebo factor-α and resistin which promote insulin resistance. Be-
three times a day sides, TZDs may stimulate the production of adiponectin
Vanschoonbeek 25 Postmenopausal Cinnamon capsule 6 weeks which promote free fatty acid oxidation and insulin sensi-
20064 women with type (500mg) 1 capsule tivity in muscle and liver. A recently evidence shows struc-
2 diabetes treated three times a day tural resemblance between telmisartan and pioglitazone. In
with either oral with meal or placebo vitro study, telmisartan activates approximately 25-30% of
antidiabetic drugs three times a day PPAR-γ compared to TZDs. Telmisartan has ability to stimu-
or diet only late adiponectin protein expression in obese Zucker rats. We
therefore undertook a meta-analysis to evaluate the efficacy
Blevius 20075 58 Type 2 diabetes Cinnamon capsule 3 months
of telmisartan on adiponectin in hypertensive patients with
treated with either (500mg) 1 capsule
insulin resistance or diabetes.
oral antidiabetic with breakfast and 1
drugs or diet only capsule with dinner
MATERIALS AND METHODS
or placebo 1 capsule
Identification of studies
with breakfast and 1
Reports of randomized controlled trials of telmisartan were iden-
capsule with dinner
tified through a systemic search on MEDLINE, EMBASE, and the
Cochrane Library. The bibliographic databases were searched
CONCLUSION
from their respective inception to June 2009. The MeSH terms,
The current evidence indicates that the daily use of cinna-
“telmisartan”, “insulin resistance”, “diabetes mellitus”, “adiponec-
mon 1-3 g for 1.5-4 months does not improve glycemic con-
tin”, “metabolic syndrome X”, and “clinical trial” were used. This
trol in type 2 diabetes.
was followed by a keyword search using “metabolic syndrome”

October 26 - 28, 2009 / Antalya - TURKEY

 POSTER PRESENTATIONS

as keywords. The reference lists of relevant articles were also increase adiponectin level implying an improvement in insu-
scanned to identify possible published trials. lin resistance, there should be further large randomized con-
trolled trials before its clinical benefit can be established.
Study selection and data extraction
The studies were included in the meta-analysis if they were Table 1: Characteristic of the studies included in the meta-
1) randomized controlled trials that evaluated telmisartan analysis
on metabolic parameters in hypertensive patients who have Study (ref.) N Study population Intervention Duration
either insulin resistance or diabetes, 2) adding or switching Negro 20061 43 • BMI≥30.0 Telmisartan 80 6 months
to fixed dose of telmisartan for hypertension, and 3) report- • FPG<105 mg/dl mg., or Irbesartan
ing adiponectin level as an outcome measure. Data extrac- • not known to 150 mg
tion was performed independently by 2 investigators using be hypertensive
a standardized form. Disagreements were resolved by a third and did not take
investigator. anti-hypertensive
medications
Statistical analysis • SSPG>190 mg/dl
Efficacy was assessed based on the final value of adiponectin
level. Treatment effect was estimated with mean difference Negro 20061 38 • treated with Telmisartan 80 mg., 4 months
in the final values of adiponectin level between the telmis- metmorfin plus Rosiglitazone
artan and the control groups. The inverse variance-weighted • HbAlc<7.0% 4mg, or Amlodipine
method was used for the pooling of mean difference and • never been 10 mg plus
the estimation of 95% confidence interval. A random ef- treated with anti- Rosiglitazone 4mg
fects model was used when the Q-statistic for heterogeneity hypertensive drugs
was significant at the level of 0.1, otherwise the fixed effect and mean blood
model was used. The degree of heterogeneity was quanti- pressure > 115
fied using I-squared statistics. The statistical analysis was un- mmHG
dertaken with RevMan version 5.0 (Cochrane Collaboration). Derosa 20073 118 • Caucasian patients Telmisartan 80 mg., 12 months
The significant level was set at p < 0.05. • aged ≥ 18 years plus Rosiglitazone
• Type 2 DM 4mg, or Irbesartan
RESULTS AND DISCUSSION • HbAlc<7.0%, 10 mg plus
Five trials met the inclusion criteria and were included in the diagnosed with Rosiglitazone 4mg
166 meta-analysis1-5. Three trials were open study1,2,5, one was metabolic syndrome
double-blind3, and the other was open-label cross-over de- and hypertension
sign4. Characteristics of these trials are presented in Table 1. • fasting C-peptide
Telmisartan was compared against other ARBs1,3-5 and amlo- • BMI 26.5-28.9 kg/m2
dipine4. In two studies, rosiglitazone 4 mg was also added Naklayama 56 • Type 2 diabetes Telmisartan 40 3 months
in both the treatment and the control groups2,3. A total of 20084 mellitius and mg., or Olmesartan
353 patients were involved in these five trials. Adiponectin hypertension 150 mg
level significantly increased with telmisartan both 40 and 80 • treated with
mg compared with the control group (mean difference 0.78, valsartan 80
95%CI 0.30-1.26, p = 0.001) (Figure 1). mg OD at least
8 weeks during
runin period
Naklayama 40 • Type 2 diabetes Telmisartan 40 mg., 24 weeks
20085 • essential or Candesartan 8 mg
hypertension

CONCLUSION
The available evidence shows that telmisartan may increase
Figure 1: Effect of telmisartan on adiponectin in hyperten- adiponectin level in hypertensive with either insulin resis-
sive patients with insulin resistance or diabetes tance or diabetic states.

It was noted that a trial was undertaken in obese patients REFERENCES

who tended to be insulin resistant with lower adiponectin
level1. Besides, two out of the five trials combined telmisar-
tan with rosiglitazone2,3 which may exert synergistic effect
in increasing adiponectin level resulted from enhanced act-
ing on PPAR-γ. Some studies instructed patients to perform
life-style modification resulting in improved insulin sensitiv-
ity3-5. These may cause heterogeneity in the study result, yet
it was not statistically significant. Although telmisartan may
1. Negro R, Formoso G, Hassan H. The effect of irbesartan
and telmisartan on metabolic parameters and blood
pressure in obese, insulin resistant, hypertensive pa-
tients, J Endocrinol Invest. 2006; 29: 957-961.
2. Negro R, Hassan H. The effect of telmisartan and amlo-
dipine on metabolic parameters and blood pressure in
type 2 diabetic, hypertensive patients, J Renin Angio-
tensin Aldosterone Syst. 2006; 7: 243-246.

3rd BBBB International Conference on Pharmaceutical Sciences

 POSTER PRESENTATIONS
3. Derosa G, Fogari E, D’Angelo A, Cicero AF, Salvadeo SA
et al. Metabolic effects of telmisartan and irbesartan
in type 2 diabetic patients with metabolic syndrome
treated with rosiglitazone, J Clin Pharm Ther. 2007; 32:
261-268.
4. Nakayama S, Watada H, Mita T, Ikeda F, Shimizu T et al.
Comparison of effects of olmesartan and telmisartan on
blood pressure and metabolic parameters in Japanese
early-stage type-2 diabetics with hypertension, Hyper-
tens Res. 2008; 31: 7-13.
5. Yamada S, Ano N, Toda K, Kitaoka A, Shiono K et al. Telmis-
artan but not candesartan affects adiponectin expression
in vivo and in vitro, Hypertens Res. 2008; 31: 601-606.
p075-CARDIOPROTECTIVE AND ANTIOXIDAN
ACTIVITY OF BLUEBERRY ANTHOCYANINE
AND POLYPHENOLS IN RATS
Cemşit Karakurt1, Şule Gürsoy2, Mehmet Abdullah Alagöz3,
Arzu Karakurt3, Göknur Aktay4, Gülendam Koçak1
İnönü University, Faculty of Medicine, Department of Pediatric
Cardiology, 44280 Malatya, Turkey1
İnönü University, Faculty of Medicine, Department of
Biochemistry, 44280 Malatya, Turkey2
İnönü University, Faculty of Pharmacy, Department of
Pharmaceutical Chemistry, 44280 Malatya, Turkey3
İnönü University, Faculty of Pharmacy, Department of
Pharmacology, 44280 Malatya, Turkey4
INTRODUCTION
Anthracyclines, such as adriamycine and daunorbicine are
highly effective chemoterapeuticals used in the various
types of cancer. Their usage has been limited primarily by
cardiotoxic side effects. Anthracyclines are toxic to tumor
cells DNA. Anthracycline induced cardiotoxicity is believed
to be related to generation of highly reactive free radicals, to
cause membrane lipid peroxidation and damage to the car-
diac myocyte membrane. Another important factor may be
poor antioxidant defense system of the heart. Antioxidant
compounds have shown some promise in animals models of
anthracycline induced cardiototoxicity. Recently, antioxidant
effect of blueberry anthocyanine and invitro antileukemic
activity of bluberry exxtract have been showed.
The aim of our study was to determine the blueberry anthocya-
nine and polyphenols’ antioxidant activity and cardioprotective
effects in anthracycline induced cardiotoxicity in rats. The levels
of thiobarbituric acid reactive substances (TBARS), non-protein
thiol groups (NP-SH; GSH) and biochemical parameters such as
ALT, AST, CK-MB and Tro-T were investigated to evaluate the car-
diotoxicity, oxidative stress or antioxidant status in tissues
MATERIALS AND METHODS
After ethical committee approve, Wistar Albino male rats
(250-300 g) were divided into 5 groups. Blueberry antho-
cyanine and polyphenols was obtained from Medox, Med-
Palett AS, and Norway. One capsule of Medox containes 80
mg anthocyanine and 115 mg polyphenol. The dosage was
October 26 - 28, 2009 / Antalya - TURKEY
calculated according to the 100 mg/kg (body weight of rats)
anthocyanine.
Group 1: Saline (i.p.)
Group 2: Saline (oral)
Group 3: Antocyanine (oral) 100 mg/kg
Group 4: Antocyanidine (oral) 100 mg/kg/ Adriamicin (i.p.) 15
mg/kg
Group 5: Adriamicin (i.p.) 15 mg/kg
Anthocyanine was maintaned daily for 7 days, adriamycine
was given for last 3 days via ip. The experiment was determi-
nated at the end of 7 days and all animals were sacrificed. 3
ml bloods were obtained and stored at – 80 oC for biochemi-
cal analysis. AST, ALT enzyme activities and Troponin T levels
were determined in this blood samples. Hepatic and heart
tissue samples were also obtained from all rats and stored
tissue samples at – 80 oC. Tissues were immediately removed
and washed with ice-cold (4 oC) saline, labeled and stored at
– 80 oC until the homogenization.
TBARS levels in tissues
The methode of Jamall and Smith was used to determine
TBARS in tissues samples. The method is bared on the forma-
tion of red chromofor which absorbs at 532 nm, following
reaction of thiobarbituric acid with malondialdehyde and
other break down products of peroxidized lipids. The results
were expressed as nmol / g wt weight of tissue.
GSH levels in tissues
The methode of Sedlac and Linsay was used to determine
GSH in tissues samples. The method is based on the quantita- 167
tion at 532 nm of the 2-nitro-5-mercapto benzoc acid which
appeared following reaction of tiol group with 5, 5’-dithiobis
(2-nitrobenzoic acid) (DTNB). The results were expressed as
μmol/g wet weight of tissue.
Statistical analysis
Statistical calculations were done by using INSTAT. Results
were expressed as means±standard error (SEM). Differences
between groups were examined using one-way analysis of
variance (ANOVA) followed by Tukey’s post-hoc test.
RESULTS AND DISCUSSION
It is generally believed that oxidative stress is associated with
depletion antioxidant system parameters with increased
lipid peroxidation. Adriamicine can disrupt the mitocho-
drial respiratory chain leading to the increased generation
of superoxide anions and hydrogen peroxide . It has been
shown that antioxidant supplementation can be used in
combination with adriamycin to protect against oxidative in-
juries without attenuating the clinical efficacy of adriamycin,
avoiding the need to take other medications, and improving
the patients’ quality.
The results of the our study suggest that treatment of adri-
amicine increased lipid peroxidation as TBARS levels in liver
and heart significantly. However, the little increased level of
GSH in the treatment groups may related the GSH synthesis
feedback system. On the other hand, it is clearly seen that
pre-treatament of anthociyanine can inhibit the adriamicine-
induced oxidative stres.
 POSTER PRESENTATIONS

Table 1: Biochemical parameters P076-BIOLOGICAL EFFECT OF SALMON

Control 1 Control 2 Anthociyanine
Anthociyanine + Adriamicine FIBRIN GLUE ON RATS
Adriamicine c a, b
532.8±86.4 710.0±68.5
AST (U/L) 127.7±4.9 156.6±15.6 107.2±7.2 Ivo Laidmäe1, Raivo Uibo2
(% 24.9) ***, ***
165.2±16.0 266.7±27.8
ALT(U/L) 51.0±2.2 69.5±9.1 50.5±2.5
*** (8) ***, *** University of Tartu, Department of Pharmacy; University of
Troponin-T
0.76±0.2 0.84±0.2 0.82±0.2
1.57±0.3 * 3.01±0.5 Tartu, Department of Immunology, Tartu, Estonia1
(ng/mL) (% 47.8) ***, *** University of Tartu, Department of Immunology, Tartu, Estonia2
a:compare to control group 1 ; b:compare to control group
2, c:compare to adriamicin group * p<0.05, ** p<0.01, *** INTRODUCTION
p<0.001 Fibrin glues (FG) of human or bovine origin are widely used
for haemostasis and wound healing. Our previous studies
Table 2: TBARS levels of liver and heart tissues showed that Atlantic salmon represents a potentially safer,
Anthociyanine + equally effective, and less costly alternative to human or
Control 1 Control 2 Anthociyanine Adriamicine a, b
Adriamicine c other mammalian blood proteins. A concern, especially with
Liver 250.4±8.3 243.4±9.5 269.1±5.8 215.5±12.1*** 289.9±11.3 *, * non-autologous proteins, is the potential that antibodies de-
Heart 134.1±7.7 144.3±6.8 152.2±8.9 130.8±2.8 ** 177.9±11.9 **, * veloped in response to the foreign protein may crossreact
a:compare to control group 1; b:compare to control grouıp with endogenous proteins leading to coagulation abnor-
2, c:compare to adriamicin group * p<0.05, ** p<0.01, *** malities. Immune response to Factor V used in sealants fre-
p<0.001 quently accompanies use of FG.1 The cross-reaction of anti-
bodies to endogenous coagulation proteins which has been
Table 3: GSH levels of liver and heart tissues. reported in several cases can lead to minor coagulation dis-
Anthociyanine + Adriamicine
turbances and in more rare instances severe hemorrhage.2,3
Control 1 Control 2 Anthociyanine Therefore, although antibodies to the foreign protein appear
Adriamicine c a, b
Liver 16.6±1.6 18.1±1.4 7.6±0.9 22.1±1.0 20.5±1.2 to be harmless, the risk of cross-reactivity with human pro-
Heart 5.7±0.8 4.1±0.4 3.2±0.3 @ 2.9±0.3 5.4±0.8 teins is a significant concern.
a:compare to control 1; b:compare to control 2, c:compare to
adriamicin group * p<0.05, ** p<0.01, *** p<0.001 @ p<0.05 In this study we examined immunological status of 46 rats
(compare to control 1) treated with salmon-derived FG. We studied possible cross-
168 reactivity of antibodies against FG components to human
CONCLUSION Factor Va (FVa).
- Oxidative stress may be an important mechanism in the
adriamicin-induced cardiotoxicity.. MATERIALS AND METHODS
- Antioxidants like anthociyanine may provide protection de- Coagulation proteins
pending on the concentration. Salmon fibrinogen and thrombin obtained from Professor
- This finding might be rational to understand the beneficial Paul Janmey (Institute for Medicine and Engineering, Uni-
effects of anthociyanine in cardioprotection against adriami- versity of Pennsylvania) were used for administration to ex-
cin induced myocardial injury, in which oxidative stress was perimental animals. Salmon fibrinogen was treated with 10
long known to contribute to the pathogenesis. kGy gamma irradiation. Human FVa was obtained from US
-Further studies necessary to affirmer the antioxidant power Biologicals (Massachusetts, MA, USA).
of anthociyanine in adriamicine- induced toxicity.
Secondary antibodies, enzyme substrates and other materials
ACKNOWLEDGEMENT Rabbit alkaline phosphatase-conjugated anti-rat IgG (Sig-
This study was supported by Inonu University Scientific Re- ma, St. Louis, USA) was used in combination with 5-bromo-
search Unit (No:2006/69). 4-chloro-3-indolyl phosphate/nitroblue tetrazolium (BCIP/
NBT) (Sigma-Aldrich, St. Louis, USA) in immunoblot assay.
REFERENCES Trans-Blot® Transfer Medium Pure Nitrocellulose Membrane
1. Elly V. Barry, Steven E. Lipshultz, and Stephen E. Sallan. Anthra- (NCM) at 0.2 um (Bio-Rad Laboratories, CA, USA) was used in
cycline-induced Cardiotoxicity: Natural History, Risk Factors, immunoblotting throughout the studies. Swine serum was
and Prevention. ASCO Educational Book.2008;2008:449-453. separated from clotted swine blood obtained from a local
2. Richard S Vander Heide, Thomas J. L Ecuyer. Molecular basis of slaughterhouse by centrifugation.
anthracycline induced cardiotoxicity. Heart Metab. 2007;35:1–
4. Experimental animals
3. Jamall, I.S., Smith, J.C.: Effects of cadmium on glutathione per- Forty-six Wistar rats (24 males, 22 females) were bred and
oxidase, superoxide dismutase and lipid peroxidation in the kept at the local animal care facility (Biomedicum’s Animal
rat heart: A possible mechanism of cadmium cardiotoxicity, House, University of Tartu, Estonia). Females and males
Toxicol. Appl. Pharmacol 80, 33-42, 1985. were separately randomized in two groups, an experimen-
4. Sedlak, J., Lindsay, RH: Estimation of total, protein-bound and tal group that was treated with salmon fibrin and a control
nonprotein sulfhydryl groups in tissue with Ellman’s reagent. group treated identically with 0.9% sodium chloride in place
Analytical Biochemistry, 25, 192-205, 1968. of fibrin.

3rd BBBB International Conference on Pharmaceutical Sciences

 POSTER PRESENTATIONS

Experiments were approved by the local Ethical Committee In these rats baseline and 11th day plasma samples were
for Animal Experimentation. studied side-by-side with 46th dayplasma samples. As seen
from Figure 3 both control (rat no. 36) and experimental (rat
Blood sampling no. 13) group rat plasma samples had antibody reactivity
Blood was taken from the tail vein in anaesthesized rats as against polypeptides at 75 and 79 kDa. These reactions were
described elsewhere.4 Plasma was stored at -20 °C. Blood also seen with plasma samples taken before the experiments
was taken at the start of experiment for baseline values and and on day 11, except for one experimental rat showing re-
on days 11, 20, 30, 33, 42 and 46 (Figure 1). activity to 110 kDa protein only on day 46.
Bleeding of rats
0 11 20 30 33 42 46

2 32
Administration of salmon fibrin glue or saline solution

Figure 1: Bood sampling and immunization of rats during the

study (numbers represent days after starting of experiment)

Administration of salmon thrombin/fibrinogen complex

preparation
Figure 2: Immunoblot with Human FVa in experimental
Salmon FG was prepared according to previous protocols.4
group (strips 2-24) and control group (strips 25-45) of 46 rat
Eleven mg of fibrinogen with 90 U of thrombin per kg body
study group using plasma samples ta
weight was injected intraperitoneally on the 2nd and 32nd
days to the experimental groups of rats. The same volume of
0.9% sodium chloride was administered to animals in control
groups (Figure 1).

SDS-gel electrophoresis, Western blot

SDS-gel electrophoresis and Western blot were done accord-
ing to previous protocols.5 Briefly, reduced samples of hu-
man FVa were applied on discontinuous SDS-polyacrylam-
ide gels. The gels were run in a MiniProtean II Electrophoresis 169
Cell apparatus from Bio-Rad. The proteins were transferred
onto NCM by semi-dry electroblotting. After the transfer,
molecular weight markers ranging from10 to 250 kDa (Pre-
cision Plus Protein (tm) standards of Bio-Rad) were stained
with Amido Black dye and the rest of the NCM was blocked
in a TBS-TM solution overnight at 5 °C. The membrane was
then incubated for 2 h with rat plasma diluted 1:500 in TBS-
TM. The rabbit alkaline phosphatase-conjugated anti-rat IgG
antibodies were diluted 1:5000 in TBS-TM. After 1.5 h of in-
cubation, bound antibodies were visualized with 5-bromo-
4- chloro-3-indolyl phosphate/nitroblue tetrazolium (BCIP/
NBT).

RESULTS AND DISCUSSION

Anti-human FVa antibodies as detected by immunoblotting
To test possible Factor V contamination in salmon FG and its
possible immunogenicity we used human FVa as an antigen
in immunoblots using rat plasma samples obtained at day
46. Staining with Amido Black for protein revealed reduced
human FVa subunits at 75, 79 and 110 kDa (Figure 2, lane 1).
After incubating strips with experimental group and control
group rat plasma samples we found potential reactions to
human FVa in both groups. One out of 23 rats from the ex-
perimental group showed antibody reactions against poly-
peptides at 75 and 79 kDa and one rat showed weak reaction
against a 110 kDa polypeptide. At the same time one out of
23 control group rats also displayed reactivity to polypep-
tides at 75 and 79 kDa.
Figure 3: Antibody reactivity towards human FVa prepara-
tion as revealed by immunoblot using plasma samples from
rats at baseline, 11th and 46th day of
CONCLUSION
Antibody reactivity to human FVa, revealed in three animals
out of 46 rats, was not associated with FG application. These
result together with our previous findings4,5 support the
suitability of salmon derived FG in the development of fibrin
sealants for medical use.

October 26 - 28, 2009 / Antalya - TURKEY

 POSTER PRESENTATIONS
ACKNOWLEDGEMENTS
This study was also supported by Sea Run Holdings Inc. and
Immunotron Ltd.
REFERENCES
1. Banninger H, Hardegger T, Tobler A, Barth A, Schupbach
P, Reinhart W, Lammle B, Furlan M. Fibrin glue in surgery:
frequent development of inhibitors of bovine thrombin
and human factor V. Br J Haematol 1993;85(3):528–32.
2. Zumberg MS, Waples JM, Kao KJ, Lottenberg R. Manage-
ment of a patient with a mechanical aortic valve and
antibodies to both thrombin and factor V after repeat
exposure to fibrin sealant. Am J Hematol 2000;64(1):59–
63.
3. Seifert J, Klause N, Stobbe J, Egbers HJ. Antibodies
formed against fibrin glue components and their circu-
latory relevance. J Invest Surg 1994;7(2):167–71.
4. Laidmae I, McCormick ME, Herod JL, Pastore JJ, Salum
T, Sawyer ES, Janmey PA, Uibo R. Stability, sterility, co-
agulation, and immunologic studies of salmon co-
agulation proteins with potential use for mammalian
wound healing and cell engineering. Biomaterials 2006;
27(34):5771–9.
5. Laidmae I, Salum T, Sawyer ES, Janmey PA, Uibo R. Char-
acterization of the biological effect of fish fibrin glue in
experiments on rats: Immunological and coagulation
studies. J Biomed Mater Res A. Published Online: May 29
2009
170 p077-SYSTEMATIC REVIEW OF THE EFFECT
OF VITAMIN K2 SUPPLEMENTATION ON BONE
MINERAL DENSITY IN POSTMENOPAUSAL
WOMEN
Nalinee Poolsup1, Naeti Suksomboon2, Poramed
Tangsakprasert1, Pinwadee Kungka1, Piyawan Sukjarern1,
Supanika Prateepjarussaeng1
Department of Pharmacy, Faculty of Pharmacy, Silpakorn
University, Nakhon-Pathom, Thailand1
Department of Pharmacy, Faculty of Pharmacy, Mahidol
University, Bangkok, Thailand2
INTRODUCTION
Postmenopausal women experience accelerated bone loss
and have an increased risk of fractures. Oral vitamin K (phy-
tonadione and menaquinone-4) supplementation has been
reported to prevent vertebral, hip, and nonvertebral frac-
tures. Vitamin K2, or menaquinone-4, both increases bone
formation and decreases bone resorption. There has been
no previously published systematic review to evaluate the
effect of vitamin K2 supplementation on bone mineral den-
sity in postmenopausal women. We therefore performed a
systematic review and meta-analysis of the available data.
MATERIALS AND METHODS
Identification of studies
Reports of randomized controlled trials of vitamin K2 were
3rd BBBB International Conference on Pharmaceutical Sciences
identified through a systematic search on MEDLINE, EMBASE,
EBM review, and the Cochrane Library. The bibliographic da-
tabases were searched from their respective inception to
May 2009. The MeSH terms, “vitamin K2”, “menatetrenone”,
“postmenopause”, “clinical trial”, and “bone density” were
used. This was followed by a keyword search using “me-
naquinone”, “bone mineral density”, and “bone mineral con-
tent” as keywords. The reference lists of relevant articles were
also scanned to identify possible published trials.
Study selection and data extraction
The studies were included in the meta-analysis if they were
1) randomized controlled trials that evaluated vitamin K2
monotherapy in postmenopausal women, 2) published in
English, 3) of at least 6-month duration, and 4) reporting
bone mineral density (BMD) in the lumbar spine (L2-4) as
an outcome measure. Data extraction was performed inde-
pendently by 2 investigators using a standardized form. Dis-
agreements were resolved by a third investigator.
Statistical analysis
Efficacy was assessed based on the percent change in BMD
from baseline to final assessment. Treatment effect was esti-
mated with mean difference in the percent change in BMD
between the vitamin K2 group and the control group. The
inverse variance-weighted method was used for the pooling
of mean difference and the estimation of 95% confidence in-
terval. A random effects model was used when the Q-statistic
for heterogeneity was significant at the level of 0.1, otherwise
the fixed effects model was used. The degree of heterogeneity
was quantified using I-squared statistics. The statistical analy-
sis was undertaken with RevMan version 5.0 (Cochrane col-
laboration). The significant level was set at p < 0.05.
RESULTS AND DISCUSSION
5 trials were included in the meta-analysis1-5. Four trial were
open studies1-4, one was single-blind design5. Characteristics
of these trials are presented in Table 1. Vitamin K2 was com-
pared against placebo or no treatment, vitamin D3, and the
combination of vitamin K2 and vitamin D3. These involved a
total of 605 patients. BMD significantly increased with vitamin
K2 compared with placebo or no treatment (mean difference
2.34%; 95%CI 0.51-4.17%, p = 0.01). No significant difference
was detected between vitamin K2 and vitamin D3. Although
vitamin K2 and vitamin D may exert synergistic effect in pro-
moting calcification in the bone, this was not evident in this
meta-analysis. BMD was not statistically different between
women receiving vitamin K2 alone and those treated with
vitamin K2 combined with vitamin D3. (Figure 1)
It was noted that four out of the five trials were undertaken
in Japanese women1-4, while one was conducted in Cauca-
sian women5. In addition, some studies recruited postmeno-
pausal women with osteoporosis and osteopenia2-4. These
may cause heterogeneity in the study results. Although vita-
min K2 may be effective in improving BMD in these women,
there should be further large randomized controlled trials
before its clinical benefit can be clearly established.
 POSTER PRESENTATIONS

Table 1: Characteristics of the studies included in the meta- CONCLUSION

analysis. The available evidence suggests that daily supplementation
Study (ref.) N Study population Intervention Duration with vitamin K2 45 mg for 1-3 years may protect against age-
(year) related decline in BMD. It was superior to placebo or no treat-
Iwamoto 75 Healthy, non- Vitamin K2 45 1 ment in increasing BMD. Vitamin K2 alone was as effective as
19991 hysterectomized mg/d, 1(OH)D3 vitamin D3 and the combination with vitamin D3. Vitamin K2
postmenapausal 1g/d, conjugated may offer an alternative treatment in preventing bone loss in
women equine estrogen postmenopausal women.
Had no medications 0.625 mg/d plus
and complications medroxyprogesterone REFERENCES
which affected bone acetate 2.5 mg/d , or 1. Iwamoto I, Kosha S, Noguchi S, et al. A longitudinal study
metabolism no treatment of the effect of vitamin K2 on bone mineral density in
Had no unbalanced postmenopausal woman a comparative study with vita-
diet for vitamin K min D3 and estrogen-progestin therapy, Maturitas 1999;
rich food 31: 161-164.
Non-smokers, non- 2. Iwamoto J, Takeda T, Ichimura S. Effect of combinated
drinkers, and did not administration of vitamin D3 and K2 on bone mineral
practice sports density of the lumbar spine in postmenopausal woman
with osteoporosis, J Orthop Sci. 2000; 5: 546-551.
Iwamoto 92 More than 5 years Vitamin K2 45 mg/d, 2 3. Ushiroyama T, Ikeda A, Ueki M. Effect of continuous com-
20002 Postmenapausal 1(OH)D3 0.75mcg/d, bined therapy with vitamin K(2) and vitamin D(3) on bone
women with vitamin K2 45 mineral density and coagulofibrinolysis function in post-
osteoporosis mg/d plus 1(OH) menopausal women, Maturitas. 2002; 41: 211-221.
D3 0.75mcg/d, or 4. Yasui T, Miyatani Y, Tomita J, et al. Effect of vitamin K2
calcium lactate treatment on carboxylation of osteocalcin in early post-
2 g/d menopausal woman, Gynecol Endocrionol. 2006; 22:
Ushiroyama 172 Postmenapausal Vitamin K2 45 mg/d, 3 455-459.
20023 women bone 1(OH)D3 0.75mcg/d, 5. Knapen M, Schurgers L, Vermeer C. Vitamin K2 supple-
mineral density of vitamin K2 45 mentation improves hip bone geometry and bone
the lumbar spine mg/d plus 1(OH)D3 strength indices in postmenopausal woman, Osteopo-
less than 0.98 g/ 1mcg/d, or dietary ros Int. 2007; 18: 963-972. 171
cm2 (osteopenia and therapy
osteoporosis)
Naklayama 34 Postmenapausal Vitamin K2 45 mg/d, 3 p078-EFFICACY OF AMITRIPTYLINE IN
20084 women bone or vitamin K2 45 IMPROVING SLEEP DISTURBANCE IN
mineral density of mg/d plus 1(OH)D3
the lumbar spine 0.75mcgg/d
FIBROMYALGIA: A META-ANALYSIS
less than 0.809 g/
cm2 (osteopenia and Somying Rattanasookchit1, Chuthamanee Suthisisang2,
osteoporosis) Nalinee Poolsup3, Naeti Suksomboon1
Naklayama 325 At least 2 years Vitamin K2 45 mg/d, 3
20085 postmenapausal or placebo Department of Pharmacy, Faculty of Pharmacy, Mahidol
Age 55-75 years University, Bangkok, Thailand1
Caucasian race Department of Pharmacology, Faculty of Pharmacy, Mahidol
Apparently healthy University, Bangkok, Thailand2
Department of Pharmacy, Faculty of Pharmacy, Silpakorn
University, Nakhon-Pathom, Thailand3

INTRODUCTION

Fibromyalgia (FM) is a distinctive chronic syndrome of unknown

Figure 1: Effect of vitamin K2 on bone mineral density in
postmenopausal women
etiology which is characterized by a widespread musculoskeletal
pain and multiple tender points, along with other related symp-
toms such as sleep disturbances, fatigue, headache, cognitive
disorder, irritable bowel, etc. Delayed onset of sleep, decreased
duration of sleep, frequent awakenings, non-restorative sleep,
and daytime fatigue are common complaints among patients
having problems with their sleep. To date, there is not yet a cure.
A multidisciplinary approach combining both pharmacologic
and non-pharmacologic therapy is strongly recommended. The

October 26 - 28, 2009 / Antalya - TURKEY

 POSTER PRESENTATIONS

treatment goal is geared toward reducing pain and improving ity and the depth of sleep by promoting deep restorative
patient’s quality of life. For many years amitriptyline, a conven- sleep - NREM stage ¾. Although a number of symptoms
tional tricyclic antidepressant, has been widely prescribed and are often identified at the same time and it remains unclear
recognized as the first-line agent for various types of chronic which happens first, patients could be expected of less fa-
pain including fibromyalgia. In the clinical trials of fibromyal- tigue, less painful, less depressed, and more pleasant with
gia, amitriptyline appears to have the greatest impact on sleep their lives when sleep problems are resolved.
compared to other core outcome measures. Our meta-analysis
evaluated the magnitude of its effect in improving sleep distur- Table 1: Characteristics of the studies comparing amitrip-
bance in patients with fibromyalgia. tyline versus placebo in improving sleep disturbance in fi-
bromyalgia.
MATERIALS AND METHODS
Identification of studies Study Design Intervention N
The computerized searches of Medline, EMBASE, Scopus,
and the Cochrane Library was performed up until May 2009. Goldenberg parallel 6 amitryptyline 25 mg, naproxen 500mg
The following MeSH terms and keywords: ‘amitriptyline’ and 62
19861 wk BID, amitryptyline+naproxen, or placebo
‘fibromyalgia’, with alternative search terms: ‘fibrositis’ and
‘fibromyositis’, were used without a language restriction.
References of review articles were screened for additional
relevant articles. Carette parallel 24 amitryptyline 10-50 mg, cyclobenzapine
92
19942 wk 30mg, or placebo
Study selection and data extraction
The studies were included in the meta-analysis if they met
these criteria: (1) double-blind, randomized placebo-con- Carette crossover
amitryptyline 25 mg, or placebo 172
trolled trials investigating the efficacy of amitriptyline (im- 19953 8 wk
mediate-released tablet) monotherapy in fibromyalgia, and
(2) assessed sleep disturbance using visual analog scale or
numeric rating scale. Study selection and data extraction Goldenberg crossover amitryptyline 25 mg, fluoxetine 20mg,
were undertaken by two investigators. Disagreements were 34
19964 6 wk amitryptyline+fluoxetine, or placebo
resolved by a third investigator.

172 Statistical analysis

Efficacy was assessed based on the change in sleep distur- Hannonen parallel 12 amitryptyline 25 mg, mocloblemide
325
bance scores from baseline to final assessment. The effect 19985 wk 450mg, or placebo
estimate was expressed as mean difference in the change
scores between the amitriptyline group and the placebo
group. Crossover and parallel-group trials were incorporated
into a single meta-analysis. The inverse variance-weighted
method was used for the pooling of mean difference and
the estimation of 95% confidence interval. The heterogene-
ity was tested by the Q-statistic, with calculated I-squared in-
dicating the percentage of total variation in effect estimates
across trials. A random effects model was applied when the
heterogeneity was significant at the level of 0.1, otherwise a
fixed effects model was used. The quantitative analysis was
undertaken with RevMan 5.0 (Cochrane collaboration). The
significant level was set at p < 0.05.
Figure 1: Efficacy of amitriptyline versus placebo in improv-
RESULTS AND DISCUSSION ing sleep disturbance
Twenty-two randomized controlled trials of amitriptyline
monotherapy in fibromyalgia were identified. Of these, 5 CONCLUSIONS
studies were eligible for the meta-analysis1-5. The characteris- Evidence from our meta-analysis indicates that a low-dose
tics of these studies are summarized in Table 1. amitriptyline is effective in improving sleep disturbance, and,
it should be considered for the treatment of fibromyalgia.
Mean baseline score of sleep disturbances was approximate-
ly 6.3 for a total of 298 patients. Treatment with amitriptyline REFERENCES
at the doses ranging from 10-50 mg once daily was undoubt- 1. Goldenberg DL, Felson DT, Dinerman H. A random-
edly more effective than placebo. The pooled estimate was ized, controlled trial of amitriptyline and naproxen in
-2.28 (95%CI -3.39,-1.17, p<.0001), as shown in Figure 1. the treatment of patients with fibromyalgia, Arthritis
Rheum. 1986; 29: 1371-1377.
Due to its sedating antihistaminergic and anticholinergic ef- 2. Carette S, Bell MJ, Reynolds WJ, Haraoui B, McCain GA, et al.
fects, a low-dose amitriptyline could improve both the qual-

3rd BBBB International Conference on Pharmaceutical Sciences

 POSTER PRESENTATIONS
Comparison of amitriptyline, cyclobenzaprine, and place-
bo in the treatment of fibromyalgia: a randomized, double-
blind clinical trial, Arthritis Rheum. 1994; 37: 32-40.
3. Carette S, Oakson G, Guimont C, Steriade M. Sleep elec-
troencephalography and the clinical response to ami-
triptyline in patients with fibromyalgia, Arthritis Rheum.
1995; 38: 1211-1217.
4. Goldenberg D, Mayskiy M, Mossey C, Ruthazer R, Schmid
C. A randomized, double-blind crossover trial of fluox-
etine and amitriptyline in the treatment of fibromyalgia,
Arthritis Rheum. 1996; 39: 1852-1859.
5. Hannonen P, Malminiemi K, Yli-Kerttula U, Isomeri R,
Roponen P. A randomized, double-blind, placebo-con-
trolled study of moclobemide and amitriptyline in the
treatment of fibromyalgia in females without psychiatric
disorder, Br J Rheumatol. 1998; 37: 1279-1286.
p079-EFFECT OF THIAZOLIDINEDIONES
VERSUS SULPHONYLUREAS AS ADD-ON
TREATMENTS TO METFORMIN IN TYPE 2
DIABETES MELLITUS:A META-ANALYSIS
Wanwaree Setwiwattanakul1, Nalinee Poolsup2, Naeti
Suksomboon1
Department of Pharmacy, Faculty of Pharmacy, Mahidol
University, Bangkok, Thailand1
Department of Pharmacy, Faculty of Pharmacy, Silpakorn
University, Nakhon-Pathom, Thailand2
INTRODUCTION
Metformin is recommended as the first choice monotherapy
of blood glucose-lowering agent in type 2 diabetes mellitus
(T2DM) patients. It is capable of maintaining a target glyce-
mic control only for a limited period of time. Combination
therapies of metformin with other antidiabetic agents are
therefore necessary. Thiazolidinediones and sulphonylu-
reas, with different mechanisms of action, have been recom-
mended as second line agents. We performed systematic
review and meta-analysis to assess the efficacy of thiazoli-
dinediones versus sulphonylureas as an add-on treatment to
metformin in T2DM patients who have not achieved thera-
peutic glycemic goal with metformin alone.
MATERIALS AND METHODS
Identification of studies
All reports of randomized controlled trials of thiazolidinediones
and sulphonylureas in T2DM patients who failed metformin
monotherapy were identified through a systematic literature
search of MEDLINE (Pubmed), EMBASE and The Cochrane Con-
trolled Trials Register. The bibliographic databases were searched
from respective inceptions to June 2009. The following MeSH
terms were used, “metformin”, “sulphonylureas”, “sulfonylureas”,
“thiazolidinediones”, “diabetes mellitus” and “clinical trial”. This was
followed by keyword search using as keywords “pioglitazone”,
“rosiglitazone”, “glibenclamide”, “glyburide”, “glipizide”, “gliclazide”
and “glimepiride”. Historical search of reference lists of relevant
October 26 - 28, 2009 / Antalya - TURKEY
randomized controlled trials, systematic and narrative reviews
were also undertaken. No language restriction was imposed.
Study selection and data extraction
The studies were included in the meta-analysis, if they a)
were randomized controlled trials that compared thiazoli-
dinediones versus sulphonylureas in T2DM patients who had
already been treated with metformin monotherapy, b) in-
cluded patients with baseline HbA1c ≥ 7%, c) lasted at least
12 weeks of treatment duration, and d) reported outcome
measure in terms of HbA1c. Data extraction was performed
independently by two investigators using a standardized
form. Disagreements were resolved by a third investigator.
Statistical analysis
Efficacy was reported as the change in HbA1c from baseline
to final assessment. Treatment effect was estimated using
mean difference in the change in HbA1c between the thi-
azolidinedione group and the sulphonylurea group. The in-
verse variance-weighted method was used for the pooling
of mean difference and the estimation of 95% confidence
interval. Subgroup analyses were undertaken with regard to
study duration. Random effects model was used to combine
the results of individual studies when Q-statistic test was sig-
nificant at the level of 0.1, otherwise the fixed effects model
was used. The degree of heterogeneity was quantified using
I-squared statistics. The statistical analysis was undertaken
with RevMan 5.0. (Cochrane Collaboration). The significance
was set at p < 0.05.
RESULTS AND DISCUSSION
Five trials met the inclusion criteria and were included in the 173
meta-analysis. All were published in English. Of these, three
were double-blind studies, two others were open studies.
Characteristics of these studies are presented in Table 1.
Thiazolidinediones was more effective than sulphonylureas in
decreasing HbA1c (pooled mean difference -0.12; 95%CI -0.19
to -0.05, p = 0.0008) in T2DM treated with metformin. Sub-
group analysis showed that for the treatment duration of ≤ 26
weeks, sulphonylureas showed a greater reduction of HbA1c
than did thiazolidinediones (pooled mean difference 0.18;
95%CI 0.01 to 0.35, p = 0.04). In the 52-104-week subgroup, no
significant difference was detected between the thiazolidine-
dione and the sulphonylurea groups (pooled mean difference
-0.01; 95%CI -0.13 to 0.12). In the study of 264-week duration,
the benefit of thiazolidinediones in reducing HbA1c was bet-
ter than that of sulphonylureas (mean difference -0.29; 95%CI
-0.39 to -0.19, p = 0.0008). It should be noted that in this study
13% of patients in the thiazolidinedione group and 17% in
the sulphonylurea group were treated with an additional an-
tidiabetic agent which may affect further reduction of HbA1c
level. The tendency towards the reduction in HbA1c in favor
of thiazolidinediones in the studies with longer duration sug-
gested that it may be more effective than sulphonylureas in
improving glycemic control in long-term use.
 POSTER PRESENTATIONS

Table 1: Characteristics of the studies included in the meta- 5. Home PD, Pocock S, Beck-Nielsen H, Curtis PS, Gomis R et
analysis. al. Rosiglitazone evaluated for cardiovascular outcomes
Duration in oral agent combination therapy for type 2 diabetes
Study (ref) N Intervention (RECORD): a multicentre, randomised, open-label trial,
(week)
Garber 158 (T) Rosiglitazone 4-8 mg vs. 24 Lancet. 2009; 373: 2125-2135.
20061 160 (S) Glibenclamide 5-10 mg
Umpierrez 109 (T) Pioglitazone 30-45 mg vs. Glimepiride 26
20062 101(S) 2-8 mg P080-THE ISOLATION OF DAIDZEIN AND
Roden 317 (T) Pioglitazone 15-45 mg vs. Gliclazide 104 GENISTEIN OF THE SOYBEAN COMPOUNDS
20093 313 (S) 80-320 mg BY ACCELERATED SOLVENT EXTRACTION
Hamann 294 (T) Rosiglitazone 4-8 mg vs. Glibenclamide 52 AND DEVELOPMENT OF A FULL VALIDATED
20084 302 (S) 5-20 mg or gliclazide 80-320 mg RP-HPLC METHOD
Home 1,117 (T) Rosiglitazone 4-8 mg vs. 264
20095 1,105 (S) Glibenclamide 2.5-15 mg or gliclazide Güler Yalçın1, Nuray Yüktaş2, Elif Sarayoğlu3, Levent Özgül4
80-240 mg or glimepiride 1-4 mg
Marmara University Faculty of Pharmacy, Analytical Chemistry
Dept., Haydarpaşa, İstanbul, Turkey1
M. Ü. Fac. of Phamacy, Center for Research Lab., Haydarpaşa,
İstanbul, Turkey2
M. Ü. Inst. of Sciences, Göztepe, İstanbul, Turkey 3
M. Ü. Institute of Health Sciences, Haydarpaşa, İstanbul,
Turkey4

INTRODUCTION
Soybean isoflavones daidzein (DZ) and genistein (GS) have
been considered as antioxidants in preventing tumour initia-
tion, promotion and oxidative damage1. Recently, genistein
has attracted a great deal of interest due to its in vitro in-
174 hibition of growth factor receptor tyrosine kinase activity2.
Figure 1: Effect of thiazolidinediones versus sulphonylureas in
Among the soybean isoflavones, genistein was found to be
T2DM inadequately controlled with metformin monotherapy
the most potent inhibitor of human breast carcinoma cell
lines, followed by daidzein and biochanin A3.
CONCLUSIONS
The current evidence suggests that thiazolidinediones may
The aim of this study is to improve a full validated RP-HPLC
improve glycemic control better than sulphonylureas in long
determination method for DZ and GS. Secondly, improving
term therapy in type 2 diabetes patients whose blood glucose
and standardizing of the extraction process of soy samples
levels are not adequately controlled with metformin alone.
by accelerated solvent extraction.
REFERENCES
MATERIALS AND METHODS
1. Garber A, Klein E, Bruce S, Sankoh S, Mohideen P. Me-
The registered Soybean PG/s 375 was kindly obtained from
formin-glibenclamide versus metformin plus rosigli-
Seed Progen Tarım Teknoloji ve Ltd. Şti. Daidzein (DZ) and
tazone in patients with type 2 diabetes inadequately
genistein (GS) were obtained from Fluka. Acetic acid (AcH),
controlled on metformin monotherapy, Diabetes Obes
dimethysulfoxide were from Merck and of reagent grade.
Metab. 2006; 8: 156-163.
Methanol (Merck) was of HPLC grade.
2. Umpierrez G, Issa M, Vlajnic A. Glimepiride versus pio-
glitazone combination therapy in subjects with type 2
Apparatus
diabetes inadequately controlled on metformin mono-
The HPLC (Agilent Technologies) diode array detector (DAD).
therapy: results of a randomized clinical trial, Curr Med
Data were processed through the Agilent ChemStation. Gen-
Res Opin. 2006; 22: 751-759.
ex beta 50 µL and Eppendorf 1000 µL automatic pipettes
3. Roden M, Mariz S, Brazzale AR, Pacini G. Free fatty acid
were used. Pressurised solvent extraction was performed by
kinetics during long-term treatment with pioglitazone
ASE100 (Dionex). Rotary vacuum evaparator was Heidolph
added to sulfonylurea or metformin in type 2 diabetes, J
vv 2000 with water bath Heidolph WB 2000.
Intern Med. 2009; 265: 476-487.
4. Hamann A, Garcia-Puig J, Paul G, Donaldson J, Stewart
Chromatographic Conditions
M. Comparison of fixed-dose rosiglitazone/metformin
The Synergi 4 µm, Hydro-RP 80A, 150mm×4.6 mm column
combination therapy with sulphonylureas plus met-
was used. Mobile Phase A: 0.1 % acetic acid, Mobile Phase
formin in overweight individuals with type 2 diabetes
B: 0.1 % acetic acid containing methanol. The gradient pro-
inadequately controlled on metformin alone, Exp Clin
gramme (FR1) was taken from Mitani K., et al4.
Endocrinol Diabetes. 2008; 116: 6-13.

3rd BBBB International Conference on Pharmaceutical Sciences

 POSTER PRESENTATIONS

Detection was performed with an Agilent 1100 Model DAD

at λ: 255 nm, Column temperature was 40 °C. Injection vol-
ume was 50 µL. Dimethylsulfoxide (DMSO) was used for void
volume ( 1.40 min).

EXTRACTION PROCEDURE
Soybean seeds were died at 100 °C for 1 hour. 90-50 % (v/v)
aqueous methanol was used as the extraction solvent. Sam-
ples (0.2g±5mg) of soybean powder was wrapped with a fil-
ter paper of Filter Dises (Quant) Grade 388. The mixture was Figure 1
transferred into a 10 mL stainless steel extraction cell (thim-
ble). The temperature was 100°C. Thimble volume was 10
mL. The static cycle was 1, flush volume was 30 %. The pres-
sure was 1500 1600 psi, The extracts were collected in 250
mL glass vials with Teflon-coated rubber caps. The superna-
tants were diluted to the volume with 50:50 (v/v) methonol:
distilled water to 50 mL. They were evaporated to dryness in
the rotary vacuum evaporator. The residue was dissolved in
Figure 2
the mobile phase of 50:50 (v/v) distilled water: methanol (0.1
% acetic acid) and filtered through 0.45µm teflon membrane
Table 1: Graident elution of the RP-HPLC system
filters minisort RC15 (sartoriusstedim) prior to injection into
the HPLC system. Time Flow Rate (FR1) Flow Rate (FR2)
B%
min mlmin-1 mlmin-1
Calibration Graph 0.00 40 1.0 1.2
Standard stock solutions: 10 mg GS and DZ dissolved in 0.5 6.00 70 1.5 1.2
ml DMSO and the volume diluted to 10 mL with MeOH. 9.00 70 1.5 1.2
11.00 40 1.0 1.2
Appropriate dilutions were made and the calibration graph
was constructed. Apigenin was used an internal Standard Table 2: The intercept (b) and slope (m) of daidzein (DZ) and
(IS). LOQ (S/N: 10/3) and LOD (S/N: 3/1) values for DZ and GS genistein (GS) for 2 different gradient elution programmes
200 ng/mL and 60 ng/mL were found. (S/N: 10/3, 3/1).The (FR1 and FR2) 175
concentration range was 0.03-0.60 µg/mL for GS; 0.10-3.20 Daidzein (DZ)
µg/mL for DZ. The RSD % was 0,2764 % for 12 injection. DZ (FR1)(%) DZ (FR2)
Genistein (GS)
intercept (b)
RESULTS AND DISCUSSION m b m b
slope (m)
A new HPLC-DAD method was developed in this study. How-
Mean DZ 2.236 0.042 2.101 0.012
ever, the method by Mitani et al5, has been applied in the first
RSD (%) DZ 6.7 177 8.8 120
place, some elution problems have been encountered. Mod-
erately apolar column has been used instead of C18 column. Mean GS 2.325 0.017 2.420 0.024
Another difficulty arised in the definition of the LOQ and LOD RSD (%) GS 6.6 212 5.9 8.3
values when using FR1 gradient elution. The founded values
were not repeatable. So, the elution profile chanced to FR2, ACKNOWLEDGMENTS
then we obtained reproducible LOQ and LOD values. There We would like to thank to Marmara University Research Fund,
was no problem for GS in these situation but, high intercept Istanbul, Turkey for its financial support.
RSD %’s obtained for DZ. The extraction yield was found
highest with 90 % aqueous methanol compared to 70, 60, REFERENCES
50 %. The correlation coefficients of the calibration graphes 1. Caragay, A. B., Cancer-preventive foods and ingredients,
for GS were 0,99578 (FR1) and 0,99914 (FR2) ; for DZ were Food technol. 1992; 46: 65-68.
0,99742 (FR1) and 0,99862 (FR2). 2. Akiyama T., Ishida J., Nakagawa S., Ogawara H., Wa-
tanabe S., Itoh N., Shibuya M. and Fukami Y., Genistein
Future Aims: 1. Because of the unstable and oxidazible na- a specific inhibitor of tyrosine-specific protein kinases, J.
ture of DZ, we suspect to some interferences. So, we plan Biol. Chem., 1987; 262: 5592-5595.
touse 0.05 % butylhydroxytoluen / methanol solution in the 3. Peterson G. And Barnes S., Genistein inhibition of the
calibration studies. 2. Checking the repeatability / reproduc- growth of human breast cancer cells: Independence from
ibility of the HPLC method and extraction procedure for the estrogen receptors and the multi-drug resistance gene,
full validation of the method. 3. Changing the extraction pa- Biochem. Biophy. Res. Comm., 1991; 179: 661-667.
rameters for obtaining the high efficiency. 4. Mitani K., Narimatsu S., Kataoka H., Determination of daid-
zein and genistein in soybean foods by automated on-line
in-tube solid-phase microextraction coupled to high-perfor-
mance liquid chromatography, Journal of Chromatography
A, 2003; 986: 169-177.

October 26 - 28, 2009 / Antalya - TURKEY

 POSTER PRESENTATIONS
p081-CHEMOMETRICAL APPROACH IN
CLARITHROMYCIN AND ITS RELATED
COMPOUNDS ANALYSIS BY RAPID
RESOLUTION RP-HPLC METHOD
Rumenka Petkovska1, Aneta Dimitrovska1, Claus Cornett2,
Liljana Bogdanovska1
Department of Applied Chemistry and Pharmaceutical
Analysis, Faculty of Pharmacy, Ss.Cyril and Methodiys
University, Vodnjanska 17, 1000 Skopje, R.Macedonia 1
Department of Pharmaceutics and Analytical Chemistry,
Faculty of Pharmaceutical sciences, University of Copenhagen,
Universitetsparken 2, 2100 Copenhagen, Denmark2
INTRODUCTION
Clarithromycin is a semisynthetic macrolide derived from
erythromycin A and consists of a 14-membered lactone ring
as well as cladinose desosamine residues at positions 3 and 5
of the ring, respectively. At very low pH, it degrades rapidly1.
For clarithromycin, five related compounds are specified as
impurities that can appear in the drug substance: KLN 1: 3-O-
decladinosyl-6-O-methyleritromycin A; KLN 3: eritromycin
A(E)-9-oxime; KLN 5:6,11-di-O-methyleritromycin A; KLN 7:
6-O-methyleritromycin A (E)-9-(O-methyloxime), KLN 8: 6-O-
methyleritromycinA(E)-9-oxime.
A literature search has shown HPLC method for the determi-
nation of macrolide antibiotic, including claritromycin,2 and
176 HPLC methods for determination of clarithromycin and its
related compound KLN53,4. This manuscript describes the de-
velopment and validation of a Rapid Resolution HPLC meth-
od for simultaneous determination of clarithromycin and its
impurities using Design of Experiments (DoE) approach.
MATERIAL AND METHODS
Investigation matrix was laboratory mixture of therapeutic
active substance clarithromycin and its five related com-
pounds in concentration ratio 1:5x10-4 respectively5. Experi-
mental design was used during method optimization (Full
factorial 23 design) and robustness testing (Central Com-
posite Circumscribed design). The MODDE 8.0 software was
used for generation and evaluation of the experimental de-
sign. Three factors: organic phase variation during gradient
elution, column temperature and gradient rise time were in-
dependent variables. For estimation of the system response
during optimization procedure and robustness testing, reso-
lution (Rs) and a Chromatographic Response Function (CRF)
were used as response factors.(Fig.1)
A Zorbax Eclipse XDB C18 Rapid Resolution HT 4.6 mm × 50
mm, 1.8 μm particle size column was used. UV detection was
performed at 205 nm.
Screening Experimental design
Full factorial design 23 (second order interaction model), was
used as a screening design, to identify influence of all factors
and its interactions on the conditions of separation. (Table 1)
y = bo + b1x1 + b2 x2 + b3 x3 + b12 x1x2 + b13 x1x3 + b23
3rd BBBB International Conference on Pharmaceutical Sciences
x2x3 + b123 x1x2x3
Rs values for all consecutive peak pairs were applied to esti-
mate coefficients of the linear model.
RESULTS AND DISCUSSION
Optimization
The screening experiments have shown that the separation of
the investigated substances as measured by the Rs values is
most affected by the organic phase variations during gradient
elution (factor x1) and column temperature (factor x2). In the
second stage these two most important factors were simul-
taneously varied. Resolution (Rs) of obtained peak pairs were
estimated for a combination of five values of column tempera-
ture (40, 45, 50, 55 and 60 0C) and six basic gradients, where
ACN content in the mobile phase was varied from 25 to 45%,
from 25 to 50%, from 25 to 55%, 25 to 60%, 25 to 65% and
from 25 to 67%, at gradient rise time of 7 min. CRF values were
calculated for these 25 experiments. RSM (Response surface
methodology) was applied for determination of an optimum
set of relevant experimental chromatographic factors for sep-
aration of impurities from drug substances. Experimental data
was fitted in a full quadratic 22 response surface model;
y = bo + b1x1 + b2 x2 + b11 x12 + b22 x22 + b12 x1x2
The separation quality of clarithromycin and related com-
pounds for achieving the maximum assay time was assessed
by calculating the value of CRF.
Minimum obtained value of individual Rs values of 1.5 as a
selection criterion was used. Contour diagram of the CRF
was constructed (Fig.2)
Separation was achieved using gradient elution (organic
phase fraction changed linearly from 25% to 55% over 7
min at 50 °C with a flow rate 0.9 ml min−1). The total time for
chromatographic separation was 6.0 min with a total analy-
sis time of 7.0 min. (Fig.3)
Figure 1: Chromatographic Response Function (CRF)
Table 1: Considered factors
Factor levels
Factors - + 0
x1
Organic phase variation during
25-45 25-75 25-60
gradient (v/v, %)
x2 Column temperature 0C 40 60 50
x3 Gradient rise time (min.) 5 12 7
 POSTER PRESENTATIONS
Figure 2: Contour diagrams of the CRF. The contour corre-
sponding to all Rs-values ≥1.5 is marked.
Figure 3: The chromatogram of test solution (organic phase
variation during gradient elution from 25 to 55 %, v/v, gradi-
ent rise time of 7
CONCLUSION
The proposed Rapid Resolution HPLC method permits si-
multaneous determination of clarithromycin and its related
compounds specified as impurities. The linear models ob-
tained demonstrate a large influence of organic phase varia-
tion during gradient elution and column temperature to the
resolution between investigated substances. Valuable infor-
mation about retention modelling was obtained by RSM,
using resolution as important component of CRF. The meth-
odology proposed represents an efficient and easily accom-
plishable approach in resolving the problem of searching for
optimum HPLC conditions.
REFERENCES
1. Omura S.(Ed), Macrolide antibiotics: chemistry, biology
and practice, Academic Press, Orlando, FL, 1984.
2. Leal C., Codony R., Compano R., Granados M., Prat M. D.,
J.Chromatogr. A 2001; 910: 285–290.
3. Erah P.O., Barrett D.A., Shaw P.N., J.Chromatogr. B 1996;
682: 73 -78.
4. Pappa-Louisi A., Agrafiotou P., Chromatographia 2002;
55:541-547.
5. ICH Guidelines. 2006. Topic Q3A (R2), Impurities in New
drug substances.
October 26 - 28, 2009 / Antalya - TURKEY
P082-A COMPARATIVE STUDY OF QUALITY
OF VITAMIN E PREPARATIONS PRODUCED IN
EUROPEAN UNION COUNTRIES AND RUSSIAN
FEDERATION
Kersti Teder1, Marko Paap2, Andres Meos1
Institute of Pharmacy, Univeristy of Tartu, Tartu, Estonia1
Interchemie Estonia Ltd, Tallinn, Estonia2
INTRODUCTION
To reduce the absence of vitamins and minerals, different
commercially produced oral nutritional supplements are
extensively used. Although the vitamin E (a well-known an-
tioxidant) deficiency is said to be rare and the real benefit of
these formulations is still not fully proven, its formulations
containing mainly α-tocopheryl acetate (TocAc) are one of
the most widely used supplements.
Since most oral supplements do not need mandatory regis-
tration in Estonian State Agency of Medicines it is difficult to
evaluate the quality of these formulations, since Veterinary
and Food Board’s registration form states the name and list
of the ingredients, the manufacturer information but no
quality or safety data.
Since the daily consumed amount of vitamin E seems to
have vital importance (in recent years, studies have shown
significantly increased mortality in case of high doses (>400
mg/day) of Vitamin E was used), the present experiment was 177
designed to evaluate the quantity as well as the quality of
the vitamin E formulations available on Estonian and Rus-
sian drug markets. The latter being important, since notable
amount of medications used in Estonia are self-imported
from Russian Federation.
MATERIALS AND METHODS
Materials
All used solvents were of HPLC-grade and purchased from
Sigma. The chemical reference substances (CRS) from Euro-
pean Pharmacopoeia (EP) (TocAc CRS, TocAc for peak identi-
fication CRS and Tocopheryl acid succinate CRS) were used.
The studied formulations (Table 1.) were obtained from Esto-
nian and Russian Federation community pharmacies, which
were stored in accordance to manufacturers’ information.
Different drug forms (Table 1.) were analysed, but all formu-
lations contained TocAc as an active pharmaceutical ingredi-
ent (API). Four out of seven had TocAc as the only API; the
others contained additional oil- and water-soluble vitamins
and minerals.
Methods
IR-spectrophotometric system
The IR spectra of vitamin E formulations’ content and sur-
faces were registered using Shimadzu FTIR-8400S IR-spec-
trophotometer coupled with Golden-Gate attenuated total
reflectance accessory. Whole process was controlled by soft-
ware IRSolution.
 POSTER PRESENTATIONS

HPLC system Table 1: The price of the vitamin E in Estonia and Russian Fed-
Contrary to other vitamins, the EP and USA Pharmacopoeia eration pharmacies
(USP) use gas chromatography (GC) to assay the vitamin E Price Price of
as the API. Since the HPLC is more common, the method Content of of the the 100
for qualitative and quantitative analysis of tochpherols was Formulationa Drug from
the API package mg of the
worked out (Picture 1.). (EUR) API (EUR)
FarmFab (RUS)
Rastvor alpha-
tocoferol atsetat (v
masle) 20 ml
oil-solution 10%, 20ml 0.66 0.03
OAO
Famatsetitseckaja
fabrika Lot:30907
Picture 1: The relative retention times for alpha tocopheryl Exp:10.2009
acid succinate, alpha tocopherol and alpha tocopheryl ac-
etate are about 0.6, 0.8, and 1.0 Ljumi (RUS)
alfatocoferola at
Accordingly to experimental data slightly modified USP method OOO Lujumi capsule 10 x 100mg 0.10 0.01
2 presented in monograph “Oil- and Water-Soluble Vitamins with Lot:140907
Minerals Tablets” was chosen for following analyses of Vitamin E Exp:10.2009
formulations: column Supelco’s Ascentis Express 100 x 4.6 mm, Queisser (EST)
2.7 µm packing C18, injection volume 20 µl, flow rate 1.0 ml/min, Vitamin E Forte N 60
UV detector at 280 nm, temperature 25°C, run time 18 min, elu- Queisser Pharma capsule 60 x 147mg 6.39 0.07
ent 240 ml methanol, 10 ml water and 0.3 ml 85% phosphoric Lot:4001017
acid diluted with acetonitrile to 1000 ml. HPLC system Shimadzu Exp:11.2009
Prominence with diode-array spectrophotometric detector SPD- Slovako (EST)
M20A was used for chromatography and the whole process was Vitamin E 400
directed, saved and analyzed by software LCsolution. Slovakofarma N 30 capsule 40 x 400mg 2.33 0.02
Lot:3390205
Sample preparation Exp:01.2008
178 All studied formulations contained different amounts of vi-
tamin E (Table 1.). During sample preparation, the aim was Rottendorf (EST)b
to prepare test solutions containing about 1 mg/ml of TocAc, Elevit prenatal N 30
so adequate portion of powdered formulation content was Rottendorf Pharma
tablet 30 x 15 IU 8.68
weighed. Since the content of TocAc was very low in multi- GmBH
vitamin formulations, 100% of the capsule content or tablet Lot:3656601
powder was dissolved. For dissolving, pure methanol or solu- Exp:04.2009
tion of methanol, heptane and isoprpanol (3:3:4) was used. Scherer (EST)c
Vitiron Suscaps N 30
RESULTS AND DISCUSSION R.P. Schrer GmbH&Co
Pure vitamin E capsules contained TocAc as the API that was capsule 30 x 2mg 8.11
KG
easily detectable on IR-spectra (Picture 2.). The formulations Lot:0651120
that contained different vitamins with minerals had very dif- Exp:02.2009
ferent IR-spectra and due to very low concentration it was
Wyeth (EST)d
not possible to see any specific absorbance of TocAc.
Centrum N 30
Wyeth Lederle S.p.A. tablet 30 x 30 UI 7.78
Lot:5GZ013B
Exp:03.2008
a
EST and RUS refer to the country of purchase, Estonia and Russian Federation respectively.
b
The prices are calculated on the basis of purchase date exchange rates
c
1 Vitamin E unit = 1 mg of dl-alpha-tocopherly acetate
d
formulation contain other vitamins and minerals

Although the look of the capsules was different – Ljumi and

Picture 2: The IR-spectra of the pure vitamin E capsules’ con-
tent: blue Slovako, yellow Queisser and pink Ljumi
Slovako were transparent red, Queisser transparent yellow
and Scherer opaque brown, the IR-spectra of all the surfaces
of capsules were almost coincidental (Picture 3.) proving that
probably the manufacturers were using the same polymer as
the base material of capsule cell. No considerable penetra-
tion of TocAc into cell wall was recorded.

3rd BBBB International Conference on Pharmaceutical Sciences

 POSTER PRESENTATIONS

CONCLUSION
As a result of this research an HPLC method for qualitative and
quantitative analysis of tocopherols producing comparable re-
sults to official pharmacopoeial GC method was worked out.

The experimental data showed that mono- and multi-ingre-

dient Vitamin E formulations produced in EU countries and
Russia contained TocAc as an API. In vitamin E formulations
containing it as the only ingredient, the presence of TocAc
Picture 3: The IR-spectra of the vitamin E capsules’ surface:
was clearly detectable by IR-spectrophotometry. The similar-
purple Scherer, blue Slovako, green Queisser and brown
ities of spectra also proved that all the manufacturers used
Ljumi
similar type of oil to dissolve the API.
All formulations, but Slovako and Scherer, contained TocAc
The low content and presence of other vitamins and miner-
within USP tolerance limits (USP monographs for Vitamin E
als made the observation of the vitamin E in multivitamin
mono- and multi-ingredient formulations, Table 2.)). Accord-
formulations using IR-spectra impossible. The presence of
ing to USP the tolerance limits for formulations containing
TocAc was proved only by chromatography.
vitamin E as the only API were 95 to 120% of labeled amount.
The Slovako contained 92.2 ± 1.7%. The multivitamins must
Parameters like purity of API and its quantity (of labeled
contain 90 to 165% of vitamin E stated on label, but the
amount) of these formulations were also rather similar. The
Scherer contained 85.6 ± 3.9%.
relative presence of impurities in preparations was rather
low, only the Scherer’s capsules contained impurity C more
Table 2: Quantitive Vitamin E Content in Estonian and Rus-
than EP tolerance limit for pure all-rac-α-TocAc. The latter fact
sian Federation Drug Market Formulations
was probably due to the very low (2 mg in capsule) content
Quantitive vitamin E Content of the TocAc in this formulation.
Formulationa
(%of loaded amount, means±SEM)
FarmFab (RUS) 103.2±4.1b The quantity of API in formulations was in accordance (ex-
Ljumi (RUS) 99.3±1.4 b cept Slovako and Scherer) with USP corresponding tolerance
limits for mono- and multi-ingredient vitamin preparations,
Queisser (EST) 98.7±1.4b
although the quantity of TocAc in both nonconforming
Slovako (EST) 92.2±1.7b preparations was close enough to the lower limit not to have 179
Rottendorf (EST) 125.2±10.5c any significant effect to the clinical efficiency.
Scherer (EST) 85.6±3.9c
Wyeth (EST) 118.6±4.5c
p083-HPLC METHOD DEVELOPMENT AND
a
EST and RUS refer to the country of purchase, Estonia and Russian Federation respectively
b
Vitamin E capsules and preparations contain one or more inert substances) must not contain not VALIDATION: SIMULTANEOUS DETERMINATION
less than 95.0 %and not more than120 %of the labeled amount of vitamin E. OF DICLOFENAC, METOPROLOL AND PHENOL
c
Oil-and Water-Soluble Vitamins with Minerals Tablets must contain vitamin E not less than 165.0
% of the labeled amount. RED IN BIOLOGICAL MATRIX FOR INTESTINAL
PERFUSION STUDIES
Three main impurities (A, B, C) were also identified and the
contents were measured (Picture 4.). According to the modi- Hatice Çağlar1, Mustafa Sinan Kaynak2, Selma Şahin3, Ebru
fied USP Test 2, all vitamin E formulations, but Scherer, con- Büyüktuncel1
tained API almost as pure as the EP all-rac-α-TocAc for peak
identification standard. The content of impurity B in Slovako
Department of Analytical Chemistry, Faculty of Pharmacy,
and impurity A in Queisser were more than stated limit, but
İnönü University, Malatya, Turkey1
since the content was higher less than 0.1% and the EP limit
Department of Pharmaceutical Technology, Faculty of
was for pure API, the latter was evaluated to be of high qual-
Pharmacy, İnönü University, Malatya, Turkey 2
ity. Scherer contained the impurity C 6.2%; while 0.5% being
Department of Pharmaceutical Technology, Faculty of
the EP tolerance limit.
Pharmacy, Hacettepe University, Ankara, Turkey3

INTRODUCTION
Diclofenac is a non-steroidal anti-inflammatory drug (NSAID)
with anelgesic, anti-inflammatory and antipyretic proper-
ties. Compared to other NSAIDs, diclofenac is well tolerated
and rarely produces gastrointestinal ulcerations or other se-
rious side-effects1. Phenol red which is not absorbed across
Picture 4: The chromatograms of vitamin E monoingredient the intestinal membrane, is widely used as a low permaebil-
formulations ity marker in the intestinal perfusion studies2. On the other
hand, metoprolol is a highly permeable drug that is used as

October 26 - 28, 2009 / Antalya - TURKEY

 POSTER PRESENTATIONS

a reference standard. Therefore, these two standard com-
pounds used for the determination of the permeability pro-
file of the test compound.
Several HPLC methods have been described in the literature
for determination of metoprolol and diclofenac in biologi-
cal fluids separately but not simultaneously3-5. In this study, a
reversed-phase liquid chromatographic method, optimised
for the simultaneous determination of diclofenac, meto-
prolol and phenol red in a biological matrix (blank perfusion
medium), was reported.
MATERIALS AND METHODS
Chemicals
Metoprolol tartarate was a generous gift by Novartis (No-
vartis Drug Co., Turkey) and diclofenac sodium salt by Deva
(Deva Drug Co., Turkey). Phenol red was purchased from
Merck and propyl paraben from Sigma Chemical Company.
Methanol was of HPLC grade, and all other reagents were of
analytical grade.
RESULTS AND DISCUSSION
Method Optimization
The effect of different concentrations of KH2PO4 in the mo-
bile phase (55% methanol, %45 KH2PO4) on the separation
of metoprolol, phenol red and diclofenac was investigated.
Since the retention time of diclofenac decreased with de-
creasing KH2PO4 concentration, the concentration of KH-
2PO4 in the mobile phase was chosen as 0.0125 M (Figure
1). When the effect of mobile phase pH on the separation
of metoprolol, phenol red and diclofenac was investigated,
the retention time of diclofenac increased with decreasing
pH (Figure 2). Therefore, the final composition of the mobile
phase was chosen as 55% methanol, 45% of 0.0125 M KH-
2PO4 adjusted to pH 7.0 with 0.3 % (v/v) triethylamine and
defined as the optimum conditions. The retention times for
metoprolol, phenol red, propyl paraben (IS) and diclofenac
using optimum conditions were 2.3, 2,7, 8.38 and 11.41 min-
utes, respectively

Instrumentation
A Hewlett-Packard 1110 series HPLC system with a ternary
solvent pump, on-line degasser, Rheodyne injection valve,
equipped with a 20µL loop, column heater and a diode-ar-
ray detector was used. The separation was performed using
a C18 analytical reversed phase column (5 µm, 4.6 mm x 150
mm).

Chromatographic Conditions
180 Isocratic chromatographic separation was accomplished.
The mobile phase was a mixture of 55% methanol, 45% of Figure 1: Effect of KH2PO4 concentration in the mobile phase
0.0125 M KH2PO4 aqueous solution adjusted to pH 7.0, to on the separation of metoprolol, phenol red and diclofenac.
which was added 0.3 % (v/v) triethylamine. Detection was
set at 205 and 227 nm. Analysis was run at a flow rate of 1
mL/min with a 12 min run time at ambient temperature.

Preparation of Perfusion Medium

The perfusion medium was consisted of 25 mM NaCl, 10 mM
KCl, 40 mM Na2SO4, 20 mM NaHCO3 and 80 mM mannitol.
The pH of buffer was adjusted to 7.0 by ortho-phosphoric
acid. Perfusion medium was prepared freshly and then fill-
tered through 0.45µm membrane filter.

Preparation of Standard Solutions

Primary stock solution was prepared by dissolving metoprolol
tartarate, diclofenac sodium, propyl paraben and phenol red
Figure 2: Effect of pH of the mobile phase on the separation
in methanol to produce concentration of 1mg/mL. Six stan-
of metoprolol, phenol red and diclofenac.
dard solutions of different concentrations were prepared by
spiking in blank perfusion solution. Propyl paraben (IS; 20 µg/
Method validation
mL) was used as the internal standard in methanol.
Selectivity
There was no interfering peak on the chromatogram follow-
Validation Procedure
ing injection of blank perfusion solution into HPLC column
A full validation of assay consisting of selectivity, linearity,
(Figure 3). The chromatogram obtained from standard solu-
lower limit of detection and quantitation (LOD and LOQ),
tion was identical with that obtained from spiked solution
intra- and inter-day accuracies and precision of the method
containing equivalent concentration of metoprolol tartarate,
was performed.
phenol red, propyl paraben (IS) and diclofenac (Figure 4).
Therefore, the proposed method was considered to be se-
lective.

3rd BBBB International Conference on Pharmaceutical Sciences

 POSTER PRESENTATIONS

control sample concentrations, and accuracy was expressed as

percentage recovery. Recoveries were calculated as peak area
ratio of reference standard/analyte (spiked blank perfusion
solution) at different concentration. Based on the results, the
method was judged to be accurate and precise for intended
purpose and minor variations did not affect the analysis.

Table 2: The intra-assay (intra-day) and between- assay (inter-

day) precision and accuracy results
Intra-day Inter-day
Figure 3: Chromatogram of blank perfusion solution.
Added Precision Accuracy Precision Accuracy
(μg/mL) RSD % % RSD % %
2.5 4.31 100.77 4.47 101.23
Metoprolol 10 1.29 99.61 1.60 99.43
40 3.4 98.75 3.75 98.58

2.5 4.49 98.32 4.74 97.93

Phenol Red 10 3.76 99.60 3.95 99.43
40 2.31 98.52 2.55 98.69

Figure 4: Chromatogram of standard solutions spiked in 2.5 3.98 104.84 3.43 102.87
blank perfusion solution (20 µg/mL). Diclofenac 10 2.32 101.34 3.54 103.55
40 1.95 101.60 4.16 102.11
Linearity
Quantifications of metoprolol, phenol red and diclofenac 2.5 3.93 100.14 3.43 98.54
were based on the calibration curves constructed under op- Propyl
20 3.24 100.58 2.97 99.12
timum conditions as the ratio of the peak areas of analysed paraben
40 1.34 99.66 2.64 102.2
subtance to internal standard (y) against the concentration
(x). Linearity of the method was determined by performing CONCLUSION
injections at eight different concentration levels in the linear 181
A simple, precise and accurate HPLC method using a C18
range over 6 different days. The peak shapes and symmetries column was developed and validated for the simultaneous
worsened after injection of 40 µg/mL concentration for di- determination of diclofenac, metoprolol and phenol red in
clofenac. The calibration curves were found to be linear in biological matrix (blank perfusion medium). This newly de-
the range of 0.1-80 µg/mL for metoprolol, 0.3-80 µg/mL for veloped and validated method can be readily used on a rou-
phenol red, 0.2-40 µg/mL for diclofenac (Table 1). tine basis for the standardization of in situ intestinal perme-
ability experiments.
Table 1: The linearity data.
Metoprolol Phenol Red Diclofenac REFERENCES
Regression 1. Roškar R., Kmetec V., Liquid chromatographic determina-
y=0.0371x+0.0064 y=0.0329+0.0034 y=0.0811x+0.0035
equation
tion of diclofenac in human synovial fluid, J. Chromatogr.
Standart error of B, 2003; 788, 57-64.
0.0003 0.0002 0.0005
intercept
2. Milani P.Z., Valizadeh H., Azarmi Y., Jalali M.B., Tajerzadeh H.,
Standart error of
0.0004 0.0005 0.0006 Simultaneous determination of metoprolol, propranolol
slope
and phenol red in samples from rat in situ intestinal perfu-
Correlation
coefficient (r)
0.9999 0.9995 0.9997 sion studies, DARU 2006; 14(2), 102-108.
3. Klimeš J., Sochor J., Doležal P., Körner J., HPLC evaluation of
Linearity range
0.1-80 0.3-80 0.2-40 diclofenac in transdermal therapeutic preparations, Int. J.
(μg/mL)
LOD (μg/mL) 0.05 0.1 0.075 Pharmaceut., 2001; 217, 153-160.
LOQ (μg/mL) 0.1 0.3 0.2
4. Mistry B., Leslie J., Eddington N.E., A sensitive assay of me-
toprolol and its major metabolite α-hydroxy metoprolol in
human plasma and determination of dextromethorphan
Precision and accuracy
and its metabolite dextrorphan in urine with high perfor-
The intra-assay (intra-day) and between- assay (inter-day) preci-
mance liquid chromatography and fluorometric detection,
sion and accuracy results for metoprolol, phenol red, diclofenac
J. Pharmaceut.Biomed. Analysis 1998; 16, 1041-1049.
and propyl paraben in blank perfusion solution at low, medium
5. Kaphalia L., Kaphalia B.S., Kumar S., Kanz M.F., Moslen M.T.,
and high concentrations were summarized in Table 2. Three dif-
Efficient high performance liquid chromatograph/ultra-
ferent concentrations of standard solutions were analyzed five
violet method for determination of diclofenac and 4’-hy-
consecutive days and five times within the same day. Precision
droxydiclofenac in rat serum, J. Chromatogr. B, 2006; 830,
was expressed as the relative standard deviation (RSD) of the
231-237.

October 26 - 28, 2009 / Antalya - TURKEY

 POSTER PRESENTATIONS
084-SIMULTANEOUS DETERMINATION OF
ETOPOSIDE AND IT’S CIS ISOMER IN HUMAN
PLASMA BY AN UV HIGH-PERFORMANCE
LIQUID CHROMATOGRAPHY USING
MONOLITHIC COLUMN
Roonak Saadati1, Simin Dadashzadeh1, Farhad Mostafavi
Shahab1
Department of Pharmaceutics, School of Pharmacy, Shaheed
Beheshti Medical University, Tehran, Iran1
INTRODUCTION
This paper describes the development, validation and stabil-
ity studies of an isocratic reversed phase HPLC method for
the determination of etoposide and its cis isomer in human
plasma using a monolithic C18 column with UV detection.
The most significant advantages of present method are its
simplicity and rapidness (one step extraction and 10 min run
time), use of very low volume of plasma (200 µl) as well as
extraction solvent (1ml) and high sensitivity (10 ng/ml).
We successfully applied the method to determine the pharmacoki-
netic characteristics of etoposide in patients treated with etoposide.
MATERIALS AND METHODS
Chemicals and reagents
Etoposide was kindly provided by Cipla (Cipla, India). Lamotrig-
182 ine the internal standard (IS) was supplied from Fluka Chemie
(Buchs, Switzerland). Cis-Etoposide was synthesized and char-
acterized according to the method previously described by
Strife and coworkers1.and all the other chemicals used were
analytical grade and used without further purification.
Chromatographic conditions
Analyses were carried out on a Chromolith Performance RP-
18e (100 mm × 4.6 mm i.d., Merck) coupled with a Chromo-
lith RP-18e guard cartridge (5.0 mm × 4.6 mm i.d., Merck).
The optimized mobile phase was a mixture of methanol– ac-
etonitrile–monopotassium phosphate (0.020 M) containing
0.007% triethylamine (TEA); (18:19:63, v/v) and pH adjusted
to 5.2 by phosphoric acid (5%, v/v). The mobile phase was
filtered by a millipore filter (0.45µM), degassed by vacuum
stirring for 30 min and delivered at a flow rate of 0.5 ml/min.
The detection wavelength was set at 240 nm and the injec-
tion volume was 100µL.
Calibration curves and quality control samples
Seven point calibration curves (final plasma etoposide con-
centrations of 20, 50, 100, 200, 400, 600, 800, and 1000 ng/
ml) and a fixed amount of the IS (30ng in each sample) were
constructed by plotting peak area ratio of the analyte to IS
versus the theoretical analyte concentration of the spiked
plasma standard.
Sample preparation
The sample preparation procedure involved simple, one step
liquid extraction of analytes and IS from plasma by 1 ml mix-
ture of chloroform, n-hexane
3rd BBBB International Conference on Pharmaceutical Sciences
Method Validation
A full validation (three analytical runs) and chemical stability
of etoposide in biomatrix in human plasma was carried out.
Pharmacokinetic analysis
Etoposide pharmacokinetic parameters in patients treated
with etoposide, were determined by non-compartmental
methods.
RESULTS AND DISCUSSION
Optimization of mobile phase composition
The optimized mobile phase composition provided an ac-
ceptable resolution between etoposide, cis-isomer, IS and
plasma interfering peaks in a total analysis time around 10
min and no need to post-run wash-out. The aqueous phase
(63% v/v) was composed of monopotassium phosphate
(0.020 M) containing 0.007% triethylamine (TEA) and the
organic phase was selected from methanol (18% v/v), ac-
etonitrile (19% v/v) and pH adjusted to 5.2.Retention times
were 5.5 min for the internal standard, 8.25 min for etopo-
side and 9.7 for cis isomer.
Sample preparation
We found that a combination of chloroform and nhexane
(0.8 and 0.2 ml respectively) can sifnificantly increase extrac-
tion capacity of the solvent and consequently reducing sol-
vent volume to one ml.
Method Validation
No interferes was observed in blank plasma samples using
six independent sources of the plasma.
The calibration curves were linear in the range of the assay
(20-1000ng/ml) and an excellent correlation coefficient (r2 >
0.996) was consistently found over different days.
The limit of detection of etoposide was around 10ng/ml and
determined as the concentration of drug corresponding to a
signal-to-noise ratio of 3:1. The limit of quantification was at
least 20ng/ml in plasma, at which the accuracy and precision
calculated were <20%.
The coefficients of variation for inter-day and intra-day as-
say were found to be less than 11 and 7% for etoposide and
less than 9 and 6.5% for cis isomer and accuracy ranged from
91.8 to 110.9% and 99.8 to 111% respectively and analytical
recovery was greater than 91%.
Stability
Etoposide was found to be stable in plasma for two freeze–
thaw cycles when stored at −200C.
Pharmacokinetic analysis
Etoposide pharmacokinetic parameters in patients were deter-
mined by non-compartmental methods. Blood samples were
collected predose and at 0, 10, 20, 40, 60, 180, 360, 420, 600 and
1440 minutes postdose. Samples were promptly centrifuged
and plasma was separated and stored at -200C until analyzed.
CONCLUSION
A simple and sensitive high-performance liquid chroma-
 POSTER PRESENTATIONS
tography (HPLC) assay was developed and validated for the
quantitation of the etoposide and its Cis isomer in human
plasma. The analysis was carried out on a monolithic silica
column, using a mixture of methanol, acetonitrile, phosphate
buffer (0.020 M) containing 0.007% triethylamine (TEA);
(18:19:63, v/v) adjusted to pH= 5.2 as mobile phase and UV
detection at 240 nm. The sample preparation procedure in-
volved simple, one step liquid extraction of analytes and IS
(lamotrigine) from plasma by 1 ml mixture of chloroform, n-
hexane and analytical recovery was greater than 91%. The
Retention times were 5.5 min for the internal standard, 8.25
min for etoposide and 9.7 for cis isomer. The assay enabled
the measurement of etoposide and its cis isomer with a mini-
mum quantification limit of 20 and 50ng ml-1. The calibration
curve was linear over the concentration range of 20-1000 ng
ml-1for etopodide and 50 - 500 ng ml-1 for cis isomer when
0.2 ml aliquot of plasma was used. The coefficients of varia-
tion for inter-day and intra-day assay were found to be less
than 11 and 7% for etoposide and less than 9 and 6.5% for cis
isomer and accuracy ranged from 91.8 to 110.9% and 99.8 to
111% respectively. The presented method was successfully
applied for pharmacokinetic studies in humans.
REFERENCES
1. Strife J, Jardine I, Colvin M, Analysis of the anticancer
drugs VP 16-213 and VM 26 and their metabolites by
high-performance liquid chromatography, J. Chro-
matogr.1980; 182: 211-220.
P085-ANALYTICAL METHOD DEVELOPMENT
AND VALIDATION OF TAMOXIFEN CITRATE
USING NORMAL PHASE HIGH PERFORMANCE
LIQUID CHROMATOGRAPHY
Fırat Yerlikaya1, Manon Launay2, Clement Gegu2, Thibaut
Leturgez3, Erem Bilensoy4, Sema Çalış4, Yılmaz Çapan4
Deparment of Pharmaceutical Technology, Faculty of
Pharmacy, Hacettepe University, 06100, Ankara, Turkey1
Faculty of Pharmacy, University of Angers, 49100 Angers,
France2
Faculty of Pharmacy, University of Lille, 59006 Lille, France3
Department of Pharmaceutical Technology, Faculty of
Pharmacy, Hacettepe University, 06100, Ankara, Turkey4
INTRODUCTION
Tamoxifen citrate (TAM) is an antagonist of the estrogen re-
ceptor in breast tissue and is therefore used in the treatment
of breast cancer1. Chromatographic systems are suitable to de-
termine TAM both in vitro and in vivo, including high perfor-
mance liquid chromatography (HPLC)2-4 . The aim of this study
is in vitro determination of TAM accurately and precisely.
MATERIALS AND METHODS
Materials
The HPLC system used was an Agilent 1200 series equipped
with a UV-Vis detector (Agilent, USA). 1-octanesulfonic acid
October 26 - 28, 2009 / Antalya - TURKEY
sodium salt (Merck, Germany), glacial acetic acid (Merck, Ger-
many) and methanol (Merck, Germany) were all HPLC grade.
The column used was a Clipeus cyano (5 µm particle size, 250
mm length, 4,6 mm internal diameter).
Method development
Since TAM is a polar compound, normal phase chromatogra-
phy has been preferred. According to the U.S. Pharmacopeia,
a mobile phase with the following composition was used: a
solution of 1,08 g of 1-octanesulfonic acid sodium salt and 2
ml of glacial acetic acid in water:methanol [322:678]. Stock
and standard solutions were prepared in the mobile phase.
The injection volume was 20 µl, the column oven tempera-
ture was 25°C, with a pump flow rate of 1 ml/min. The detec-
tor wavelength was set at 232 nm.
System suitability
System suitability parameters were performed and evalu-
ated due to the European Pharmacopoeia (5th Edition) rec-
ommendations.
Calibration
External standard method was used for the calibration of
TAM. 3 series of 7 concentrations of TAM solutions were pre-
pared (a dilution range from 500 ng/ml to 2000 ng/ml), each
sample was injected one time.
Method Validation
Analytical method validation parameters like accuracy, preci-
sion, linearity, range, specificity, detection and quantitation
limits were evaluated according to the ICH Q2R1 guideline. 183
Also, the stock solution stability was evaluated.
RESULTS AND DISCUSSION
System suitability
All of the system suitability parameters were evaluated sta-
tistically and data was given in Table 1.
Table 1: System suitability test results.
Acceptance criteria Experimental values
Peak area RSD<1% RSD % = 1,012 %
Peak height RSD<2% RSD % = 1,848 %
Retention time RSD<1% RSD % = 1,035 %
Capacity Factor k'>2 k'>2
Theoretical plates number N>2000 N>7000
Calibration
The equation of the regression line is: y = 0,037644x –
4,7457677 with a regression coefficient of 0,9996.
Method Validation
The results of accuracy study were given in Table 2. The intra-
day precision results show that there is no significant differ-
ence between samples prepared and analyzed in the same
day. And the interday precision assay shows that there is no
significant difference between samples prepared at the first
day and samples prepared in the second day. Also, the meth-
od is specific to TAM when comparing the TAM sample chro-
matogram and the blank sample chromatogram (Figure 1).
 POSTER PRESENTATIONS

TAM stock solution was stable for at least 2 days. The quanti- p086-SYNTHESIS, CHEMICAL
tation limit is 46 ng/ml and the detection limit is 13 ng/ml. CHARACTERIZATION AND BIOLOGICAL
Table 2: Evaluation of accuracy.
SCREENING OF NEW DIBENZO[B,E]THIEPINE
Theoratical Experimental Recovery DERIVATIVES
concentration (ng/ml) concentration (ng/ml) (%)
500 506.00 101.20 Camelia Elena Stecoza1, Miron Teodor Caproiu2, Corina Ilie3,
Olguta Dracea4, Ileana Cornelia Chirita1, Diana Camelia
4000 3977.67 99.44
Nuta1
20000 20136.00 100.68
Carol Davila University of Medicine and Pharmacy1
Table 3: Intraday precision data. C. D. Nenitescu Organic Chemistry Center of Romanian
True Obtained concentrations (ng/ml) Academy2
concentrations National Institute for Chemical Pharmaceutical Research and
I II III X SD RSD %
(ng/ml) Development3
500 512 495 511 506.00 9.54 1.89 Cantacuzino National Institute of Research and Development
4000 4095 3939 3949 3977.67 58.53 1.47 for Microbiology and Immunology4
20000 20430 19920 20058 20136.00 263.80 1.31
INTRODUCTION
Dibenzothiepine derivatives constitute an important class of
Table 4: Intraday precision data.
therapeutic agents in medicinal chemistry. A diversity of bio-
True Obtained concentrations (ng/ml) logical effects is associated with dibenzothiepine structure:
concentrations antidepressant, antihistaminic, antipsychotic, anxiolytic,
I II III X SD RSD %
(ng/ml) hypnotic, antimicrobial, anti-inflammatory, etc. In previous
500 514 499 512 508.33 8.14 1.60 studies we have reported the synthesis, structural character-
4000 4100 4058 3998 4052.00 51.26 1.27 ization and in vitro evaluation of the antimicrobial activity
20000 20122 20147 20569 20279.33 251.17 1.24 for some original O-acyloximino-dibenzo[b,e]thiepins1,2. As
part of our ongoing studies in developing new biological-
active compounds, in this study we report the synthesis,
chemical characterization and biological screening of new
184 dibenzo[b,e]thipine derivatives. Moreover, this study was
prompted by our previous data which made evident the fa-
vourable effects of replacement of S atom by SO2 group, for
the improvement of biological activity3.

MATERIALS AND METHODS

Synthesis of the compounds
The synthetic strategies adopted to obtain the target com-
pounds 2 are outlined in Fig. 1 and 2.

Figure 1: Chromatograms of TAM and blank samples.

REFERENCES
1. Memisoglu-Bilensoy, E., et al., Tamoxifen citrate loaded
amphiphilic beta-cyclodextrin nanoparticles: in vitro
characterization and cytotoxicity. J Control Release,
2005. 104(3): 489-496.
2. Kojima, T., et al., Effect of spectroscopic properties on
photostability of tamoxifen citrate polymorphs. Int J
Pharm, 2007. 336(2): 346-351. Figure 1: Synthesis of dibenzo[b,e]thiepins 2 (pathway A)
3. Gliesing, S., et al., HPLC-determination of tamoxifen di-
hydrogen citrate with the base selective column Ultra-
Sep ES Pharm RP8 in tablets. Pharmazie, 1997. 52(5):
399-401.
4. Jalonen, H.G., Simultaneous determination of tamoxifen
citrate and its E isomer impurity in bulk drug and tablets
by high-performance liquid chromatography. J Pharm
Sci, 1988. 77(9): 810-813.

3rd BBBB International Conference on Pharmaceutical Sciences

 POSTER PRESENTATIONS
Figure 2: Synthesis of dibenzo[b,e]thiepins 2 (pathway B)
The preparation of key intermediates ketones 1 (Fig. 3), was
accomplished by the synthetic sequences as previously re-
ported1,4,5.
Figure 3: Synthesis of the key intermediates 1
All the newly synthesized compounds were characterized
by elemental analyses, IR and NMR spectroscopic analyses.
The elemental analyses were performed on a Perkin Elmer
CHNS/O Analyser Series II 2400 apparatus. The IR spectra
were registered in potassium bromide pellets with a Fourier
transform infrared spectrophotometer FTS-135 BIORAD and
by ATR technique with an FT-IR Bruker instrument Vertex 70.
The NMR spectra were registered on a Varian Gemini 300BB
instrument at room temperature, operating at 300 MHz for
1H and 75 MHz for 13C and an Unity-Inova 400 operating at
400 MHz in proton and 100 MHz in carbon. Melting points
were determined in open capillary tubes on an Electrother-
mal 9100 apparatus and are uncorrected.
Antimicrobial activity evaluation
All the original synthesized compounds were screened for
their in vitro antimicrobial activity against bacterial strains,
Staphylococcus aureus, Bacillus subtilis, Escherichia coli,
Pseudomonas aeruginosa, Klebsiella pneumoniae and fun-
gal strains Candida albicans, Aspergillus niger, reference
ones (from Cantacuzino Institute Culture Collection Labo-
ratory) and recently isolated from clinical samples, respec-
tively. Quantitative assay of the antimicrobial activity was
performed by the binary microdilution method in 96 multi-
well plates, in order to establish the minimal inhibitory con-
centration (MIC) (µg/mL).
October 26 - 28, 2009 / Antalya - TURKEY
RESULTS AND DISCUSSION
Following the aforementioned synthesis procedures, we
obtained two series of new compounds O-acyloximino-
dibenzo[b,e] thiepins (X= S) and their sulfones (X= SO2).
Chemical structures of the new compounds are depicted in
Fig. 4.
Figure 4: Chemical structures of the new dibenzo[b,e]thi-
epine derivatives.
The new dibenzo[b,e]thipines derivatives are white or light
yellow crystals and they were characterized by their physical
properties (melting points, solubility). The compounds purity
was certified by elemental analyses, the results being within
±0.4% of the theoretical values, and their structures were
confirmed by spectral methods (IR, 1H-NMR, 13C-NMR).
From the biological evaluation results we conclude that the
new compounds exhibited a specific antimicrobial activity,
depending on the nature of the substituents and their posi- 185
tion. The MIC values ranged between 15.6 μg/mL and 500
μg/mL.
CONCLUSIONS
Novel dibenzo[b,e]thipine derivatives were synthesized and
their structures were elucidated by elemental analysis as
well as FTIR, 1H-NMR and 13C-NMR spectral data. The new
compounds, O-acyloximino-dibenzo[b,e]thiepins and their
sulfones were also evaluated for their antibacterial and an-
tifungal potential. All the tested compounds were found to
exhibit moderate to good antimicrobial activity.
ACKNOWLEDGEMENT
The authors acknowledge for the financial support offered
by Romanian Ministry of Education, Research and Youth
through the PN2 41-055/2007 grant.³¹¹¹>>
REFERENCES
1. Stecoza C. E., Căproiu M. T., Drăghici C., Chifiriuc M. C.,
Drăcea N. O., Synthesis, characterization and antimi-
crobial activity evaluation of some new derivatives of
6,11-dihydrodibenzo[b,e]thiepin-5,5-dioxide, Rev. Chim.
(Bucureşti), 2009, 60(2): 137-141
2. Ilie C., Stecoza C. E., Căproiu M. T. Hau Ramona, Guta R.,
Nanau-Andreescu D., Synthesis and characterization
of new dibenzo[b,e]thiepine derivatives II, Rev. Chim.
(Bucureşti), 2009, 60(6): 588-591
3. Tarko L. Stecoza C. E., Ilie C., Chifiriuc M. C., QSAR studies
on antibacterial activity of some substituted dihydrod-
 POSTER PRESENTATIONS

ibenzothiepins, Rev. Chim. (Bucureşti), 2009, 60(5): 476- Antioxidant activities

479 Then, antioxidant activities of compounds 2 were investigat-
4. Stecoza C., Missir Al., Draghici C, 6,11-Dihydrodibenzo[b,e] ed by different antioxidant tests, including 1,1-diphhenlyl-2-
thiepin derivatives, Annals of West University of Ti- picryl-hydrazyl free radical (DPPH.) scavenging, reducing ac-
misoara, Series Chemistry, 2001, 10(2): 485-492 tivity by Fe+3 –Fe+2 transformation and ferrous metal (Fe+2)
5. Stecoza C., Missir Al., Noi derivaţi ai chelating activities. BHT, BHA and α-tocopherol were used as
6,11-dihidrodibenzo[b,e]tiepinei. Nota III, Farmacia reference antioxidant compounds. For the measurement of
2000, 48(5), 51-59 the reductive ability, Fe3+-Fe2+ transformation was investi-
gated in the presence of compound using by the method of
Oyaizu1. The hydrogen atoms or electrons donation ability of
p087-ANTIOXIDANT ACTIVITIES OF the synthesized compound was measured by DPPH. using
SOME NOVEL 1-ACETYL-3-ALKYL-4-(4- the method of Blois2. The chelating effect of ferrous ions by
the compound was determined according to the method of
METHYLTHIOBENZYLIDENAMINO)-4,5- Dinis et al3.
DIHYDRO-1H-1,2,4-TRIAZOL-5-ONES
RESULTS AND DISCUSSION
Haydar Yüksek1, Faruk Kardaş2, Muzaffer Alkan3, Mustafa Reducing activitiy
Calapoğlu4 In the reducing activitiy measurement, an increase in ab-
sorbance is directly related to reducing power of the elec-
Dept. of Chemistry, Kafkas University, 36100 Kars/Turkey1
tron donating antioxidants present in the reaction mixture.
Kars MYO, Kafkas University, 36100-Kars, Turkey2
In this study, all the amounts of the compounds showed
Education Faculty, Kafkas University, Kars/Turkey3
lower absorbance than blank. Hence, no activities were ob-
Dept. of Biology, Süleyman Demirel University, Isparta/Turkey4
served to reduce metal ions complexes to their lower oxida-
tion state or to take part in any electron transfer reaction. In
INTRODUCTION other words, compounds did not show the ability of electron
In this study, five novel 1-acetyl-3-alkyl(aryl)-4-(4- donor to scavenge free radicals (Table 1)
methylthiobenzyliden-amino)-4,5-dihydro-1H-1,2,4-
triazol-5-one compounds2 were synthesized from the Table 1: Reducing power of the compounds 2a-e at different
reactions of 3-alkyl(aryl)-4-(4-methylthio-benzylidenamino)- concentrations (μg/mL, A700 nm)
4,5-dihydro-1H-1,2,4-triazol-5-ones1 with acetic anhydride Compounds 60 90 180
186 (Scheme-1). In addition, the newly synthesized compounds BHT 0.6631 0.7672 1.0809
were screened for their in-vitro antioxidant activities.
BHA 0.8898 1.1573 1.6034
a-tocopherol 0.353 0.4417 0.5734
2a Inactive Inactive Inactive
2b Inactive Inactive Inactive
2c Inactive Inactive Inactive
2d Inactive Inactive Inactive
2e Inactive Inactive Inactive
Scheme 1
Radical-scavenging activity
MATERIALS AND METHODS All of the compounds 2 tested with this method exhibited
Chemicals mild DPPH free radical scavenging activity in a concentra-
BHT was purchased from Merck. Ferrous chloride, tion-dependent manner. Table 2 illustrate a decrease in the
α-tocopherol, DPPH., ferrozine, butylated hydroxyanisole concentration of DPPH radical due to the scavenging ability
(BHA) and trichloracetic acid (TCA) were purchased from of these compounds. The interaction between an antioxi-
Sigma (Sigma –Aldrich GmbH, Sternheim,Germany). dant and ABTS+· is an indication of the ability of the carbonyl
functional group in the antioxidant to react with or scavenge
Synthesis of compounds 2 free radicals. The N–C bond in compounds can also react
The corresponding compound 1 (0.01 mol) was refluxed with with ABTS+·. These results indicate that the newly synthe-
acetic anhydride (15 mL) for 0.5 h. After addition of absolute sized compounds 2a-e showed weak activities as a radical
ethanol (50 mL), the mixture was refluxed for 1 h. more. scavenger (Table 2).
Evaporation of the resulting solution at 40-45 °C in vacuo
and several recrystallizations of the residue from EtOH gave
pure compounds 2a-e as colourless crystals (Scheme 1). The
newly synthesized compounds 2 were characterized by el-
emental analyses and IR, 1HNMR, 13C-NMR and UV spectral
data.

3rd BBBB International Conference on Pharmaceutical Sciences

 POSTER PRESENTATIONS

Table 2: DPPH scavenging activity of the compounds 2a-e at REFERENCES

different concentrations (μg/mL, A517 nm, % inhibition) 1. Oyaizu M. Studies on products of browning reaction
Compounds 40 80 160 prepared from glucosamine, Japan Nutri. 1986; 44: 307-
316.
BHT 31.6 50.9 59.9
2. Blois MS. Antioxidant determinations by the use of a
BHA 80.1 80.6 80.9 stable free radical, Nature, 1958; 26: 1199-1200.
a-tocopherol 80.6 81.7 82.1 3. Dinis TCP, Madeira VMC, Almeida LM. Action of phenolic
2a 9.4 10.6 13.1 derivatives (acetaminophen, salicylate, and 5-aminosali-
cylate) as inhibitors of membrane lipid peroxidation and
2b 3.6 8.9 6.5 as peroxyl radical scavengers, Arch Biochem Biophys.
2c 12.1 7.0 3.0 1994; 315: 161-169.
2d 7.8 9.0 21.4 4. Gordon MH. Food Antioxidants, Elsevier: London-New
York, 1990; pp. 1-18.
2e 6.2 7.1 7.2

Ferrous ions chelating activity

Ferrous ion chelating activities of the compounds, BHA, BHT
p088-UNEXPECTED RING TRANSFORMATIONS
and α-tocopherol are shown in Figure 1. In this study, metal OF AN AZOXYQUINOXALINE
chelating capacity was significant since it reduced the con-
Hermecz István1, Molnár Annamária1, Boros Sándor1, Simon
centrations of the catalyzing transition metal. It was report-
Kálmán1, Gönczi Csaba1
ed that chelating agents, that form σ-bonds with a metal, are
effective as secondary antioxidants because they reduce the
Chinoin Ltd, Budapest, Hungary1
redox potential thereby stabilizing the oxidized form of met-
al ion4. The data obtained from Figure 1 reveal that the com-
INTRODUCTION
pounds demonstrates a marked capacity for iron binding,
Treatment of azoxybenzene 1 and its derivatives with strong
suggesting that their action as peroxidation protector may
acids is known to result the corresponding hydroxyazo-
be related to its iron binding capacity. On the other hand,
benzenes 2 (Scheme 1), coined as Wallach rearrangement1.
free iron is known to have low solubility and a chelated iron
In kinetic studies much information about the mechanism
complex has greater solubility in solution, which can be con-
has been deduced from structural changes, but the exten-
tributed solely from the ligand. Furthermore, the compound-
sion into the heterocyclic azoxy compounds is not reported.
iron complex may also be active, since it can participate in 187
Only the phenylazoxypyridines and their N-oxides were in-
iron-catalyzed reactions.
vestigated by Buncel and his co-workers2.

Figure 1: Metal chelating effect of different concentrations of

the compounds 2a-e, BHA, BHT and α-tocopherol on ferrous
ions at different concentrations
CONCLUSION
The data here reported could be of the possible interest
because of their activities of hydrogen donating and metal
chelating could prevent redox cycling. Chelating agents,
which form σ-bonds with a metal, are effective as second-
ary antioxidants because they reduce the redox potential
thereby stabilizing the oxidized form of metal ion. Design
and synthesis of novel small molecules which can specifi-
cally protective role in biological systems are in perspective
in modern pharmacological chemistry. These results encour-
age further in vivo studies and explore their possible thera-
peutic applications.
Scheme 1: Wallach rearrangement
MATERIALS AND METHODS
We decided to extend the generic Wallach rearrangement
into the biologically interesting heterobicycles ring systems.
We started our investigations with azoxyquinoxaline 33which
have revealed some interesting and surprising reactions and
products depending on the reaction media.
RESULTS AND DISCUSSION
Firstly we applied the classic Wallach rearrangement conditions
treating azoxy compound 3 in conc. H2SO4 and expected to
obtain either 3- (4a) or 6- (4b) azo compound (Scheme 2).

October 26 - 28, 2009 / Antalya - TURKEY

 POSTER PRESENTATIONS

Scheme 2: Expected reaction products Scheme 4: Formation of compound 7

The isolated product was characterized by HRMS-assay. Sur-
prisingly the product did not contain O atom, instead of a
Wallach rearrangement formally a “HNO” eliminated from
3, which is consistent with observation of nitrous gas evo-
lution. For the structure, on the basis of 2D NMR measure-
ments, a new pentacyclic ring system, 5,7,8,13,13b-pentaa-
za-dibenzo[b,g]fluorene 5 is proposed (Scheme 3).
The question arose of possibility of a thermal reaction and ac-
tually the same product was prepared from other indifferent
organic solvents (Entry 7-8) and even in neat. Results of thermo-
analytical studies (DSC, TG, DTG) were on line these finding.
In solvents the N2 elimination proceeds even at lower tem-
peratures, this indicates a very strong solvent effect (Entry
6-8). To the best of our knowledge, this type of thermal trans-
formation is not well-described in literature; only one exam-
ple for thermolytical N2 loss of azoxy compounds is known5.

On the evidence of these studies we propose mechanistic

pathway depicted in Scheme 5. In the first step a nucleophilic
attack of oxygen of 3 on the positively charged C(3) carbon
of the farther quinoxaline ring furnished a tricyclic derivative
188 8a. In the next step disproportion of the tricyclic intermedi-
ate 8a gave a quinoxalinone anion 8b and 3-azoniumqui-
noxaline cation 7a. Consequently N2 elimination occurred
from 7a, and the recombination of the formed quinoxalinyl
cation 7b and 8b provided bisquinoxaline 7. To support the
proposed mechanism we could trap 7b cationic species by
Scheme 3: Formation of compound 5 morpholine to give 9 (Entry 9).

The structure was supported by an independent synthe- Table 1: The influence of the strength of the acid and tem-
sis starting from 2-amino- and 2,3-dichloroquinoxalines perature
(Scheme 3). 5 was obtained in a two-step synthesis, first Prod
Ent. Media pKa T [0C]
2-(3-chloroquinoxalylamino)quinoxaline was prepared in a (Yield %) [a]
nucleophilic substitution reaction, than the cyclization of 6 1 H2SO4 -3.0 140 5(63)
was carried out by a Buchwald-Hartwig protocol in 46% and
2 H2SO4 .-3.0 rt 5(67) [b]
43% yields, respectively.
3 CH3SO3H -2.6 140 5(56)
We investigated the influence of the strength of the acid 4 CF3CO2H -0.25 72 [c]
5(56)
and temperature on the reaction. Using strong mineral and 5 HCO2H 3.77 101 [c] 5(67)
organic acids results also pentacyclic product 5 (Entry 1-5),
lower temperature has no significant influence on the qual- 6 AcOH 4.76 118 [c]
7(85)
ity and quantity of the product but reaction time extremely 7 Ac2O - 140 [c]
7(87)
increases (Entry 2). 8 glycol - 140 7(80)
9 O NH - 129 [c] 9 [d](87)
When pKa was increased dramatic changes were observed
[a]
after 10 min; isolated, recrystallized yields; [b] after 48 h; [c] at boiling temperature;
above pKa 3.77. Reaction in MeCO2H gave a new compound [d]
1-morpholinoquinoxalin-(1H)-2-one9
7, instead of 5 (Entry 6, Scheme 4) The HRMS-assay interpreted
that N2 elimination occurred from 3 yielding bisquinoxaline 74.

3rd BBBB International Conference on Pharmaceutical Sciences

 POSTER PRESENTATIONS
Scheme 5: Proposed mechanism of thermal reaction of 3
CONCLUSION
In conclusion, the treatment of azoxy compound 3 with
strong acids and thermally led to two different reaction path-
ways to produce pentacyclic ring system 5 and bisquinox-
alinone 7, respectively. The structures of the products were
supported by detailed NMR analysis, and were confirmed by
independent synthesis (for 5) and X-ray crystallography (for
7). We have described herein a possible interpretation for the
formation of product 7.
REFERENCES
1. Cox RA, Buncel E, in: Patai S (Ed.) The Chemistry of Hy-
drazo, Azo and Azoxy Groups, Vol. 2, Wiley, New York,
1975, pp. 808-837.
2. Buncel E, Keum SR, Rajagopal S, Klepek E, Cox RA, Wal-
lach rearrangement of azoxypyridines and azoxypyri-
dine N-oxides – Charge distributions and dramatic reac-
tivity differences, Can J Chem 2008; 86:298-304.
3. Harsányi K, Gönczi Cs, Korbonits D, Synthese und Oxida-
tion von 2-(Hydroxyimino)-1,2-dihyd-ro-chinoxalin, Lie-
bigs Ann Chem. 1973: 190-194.
4. Iijima C, Quinoxalines XI. On Quinoxaline 1-oxide, Yaku-
gaku Zasshi 1967; 87:942-953.
5. Moskalenko GG, Sedova VF, Mamaev VP, Synthesis and
thermal transformation of azoxy-pyrimidines, Bull Acad
Sci USSR Div Chem Sci. (English Trans.) 1987; 36:642.
October 26 - 28, 2009 / Antalya - TURKEY
p089-SYNTHESIS AND CHARACTERIZATION
OF SOME NEW THIOSEMICARBAZONE
DERIVATIVES OF 1-BIPHENYL-4-YL-2-
IMIDAZOL-1-YL-ETHANONE
Ebubekir Septioğlu1, Ünsal Çalış1
Department of Pharmaceutical Chemistry, Faculty of
Pharmacy, Hacettepe University, 06100 Sihhiye-Ankara/
Turkey1
INTRODUCTION
New antiepileptic drug development which is more active and
has less toxicity is one of the current studies on development
of biological active drugs. Development of new drugs is over-
rating by researchers because of the drugs which have been
used for the symptomatic treatment of epilepsy are requiring
a long term and in some cases lifelong therapy, have more
adverse effects and failures on controlling particular kinds of
seizures1. Recently, the anticonvulsant activities of thiosemi-
carbazone derivatives were reported by various studies 2.
MATERIALS AND METHODS
All chemicals used in this study were purchased from Aldrich
Chemical and Riedel-de Haën. The purity of the compounds
was assessed by TLC on Kieselgel 60 F254. Three compounds
were synthesized according to the procedures given below:
General procedure for the preparation of 1-biphenyl-4-yl-2-
imidazol-1-yl-ethanone (1) 189
A mixture of 1-biphenyl-4-yl-2-bromo-ethanone (0.01 mol),
imidazole (0.03 mol), and 5 mL of DMF was stirred at 5-10
“C for 16 h. Then it was poured into ice, and the precipitate
was collected and washed with H2O. The crude product was
dissolved in benzene and filtered. The benzene was removed
and the precipitate was the compound 13.
General procedure for the preparation of substituted phe-
nylthiosemicarbazides (2a-c)
The appropriate isothiocyanate (10.0 mmol) was added to
a mixture of hydrazine hydrate (0.485 mL, 10.0 mmol) and
dichloromethane (DCM) (25 mL) at 5-10 °C. The bath was re-
moved and the suspension was stirred for 1 h at room tem-
perature, cooled in ice, and filtered. The crude was recrystal-
lized by ethanol4.
General procedure for the preparation of thiosemicarba-
zones derivatives (3a-c)
To a solution of 1-biphenyl-4-yl-2-imidazol-1-yl-ethanone
(1) (0.001 mol) in EtOH (15 mL) was added a solution of N-
substituted-thiosemicarbazides (2a-c) (0.001 mol). After ad-
dition of a drop of concd. H2SO4, the mixture was refluxed
on a water bath for 3 h (Scheme). The product formed after
cooling was filtered, purified by column chromatography,
where the solvent system is CHCl3 : CH3OH : NH3 (90:10:1).
 POSTER PRESENTATIONS
Scheme: Schematic representation and structures of synthe-
sized compounds.
RESULTS AND DISCUSSION
Some characteristics of the compounds are given below:
2-(1H-imi
dazol-1-yl)-1-(2-4-biphenyl)ethan-1-one N-phenylthiosemi-
carbazone (3a).
1H-NMR (DMSO-d6, δ, ppm, 400 MHz): 5,64 (s, 2H, -CH2 -),
6.86-8.09 (m, 17H, aromatic), 10.23 (s, 1H, phenyl-NH) and
11.46 (s, 1H, =N-NH-). 13C-NMR (DMSO-d6, δ, ppm, 400
MHz): 41, 120, 121, 125, 126, 127, 127, 127, 127, 128, 129, 129,
129, 129, 130, 130, 135, 138, 139, 140, 140, 142, 145 and 178.
ESI-MS (ESI+, m/z): 434 (M+23) and 412 (M+1). Melting point:
192 °C. Anal. C24H21N5S (M.W.: 411.52): C, H, N, S.
2-(1H-imidazol-1-yl)-1-(2-4-biphenyl)ethan-1-one N-(4-chlo-
190 rophenyl)thiosemicarbazone (3b).
1H-NMR (CHCl3-d3, δ, ppm, 400 MHz): 5,08 (s, 2H, -CH2 -), 6.92-
7.93 (m, 16H, aromatic), 8.79 (s, 1H, phenyl-NH) and 9.11 (s, 1H,
=N-NH-). 13C-NMR (CHCl3-d3, δ, ppm, 400 MHz): 53, 120, 125,
127, 127, 128, 128, 129, 129, 131, 136, 138, 139, 144, 145 and
176. ESI-MS (ESI+, m/z): 470 (M+25), 468 (M+23), 448 (M+3) and
446 (M+1). Melting point: 199 °C. Anal. C24H20ClN5S (M.W.:
445.97): C, H, N, S.
2-(1H-imidazol-1-yl)-1-(2-4-biphenyl)ethan-1-one N-(3-chlo-
rophenyl)thiosemicarbazone (3c).
1H-NMR (CHCl3-d3, δ, ppm, 400 MHz): 5,05 (s, 2H, -CH2 -),
6.99-7.86 (m, 16H, aromatic), 8.85 (s, 1H, phenyl-NH) and 9.12
(s, 1H, =N-NH-). 13C-NMR (CHCl3-d3, δ, ppm, 400 MHz): 52,
120, 122, 124, 126, 127, 128, 128, 129, 129, 129, 130, 130,
135, 138, 139, 140, 144, 146 and 176. ESI-MS (ESI+, m/z): 470
(M+25), 468 (M+23), 448 (M+3) and 446 (M+1). Melting point:
173-4 °C. Anal. C24H20ClN5S (M.W.: 445.97): C, H, N, S.
Thiosemicarbazone derivatives of 1-biphenyl-4-yl-2-imida-
zol-1-yl-ethanone were prepared to evaluate their anticon-
vulsant activities. All the targeted compounds (3a -c) were
prepared by the condensation of 1-biphenyl-4-yl-2-imidazol-
1-yl-ethanone and thiosemicarbazides in ethanol after add-
ing a drop of conc. H2SO4. The structures of the compounds
were elucidated by 1H and 13C-NMR, ESI-MS and elemental
analysis. The results of elemental analysis for C, H, N and S
were within ± 0.4 % of the theoretical values.
CONCLUSION
3rd BBBB International Conference on Pharmaceutical Sciences
Some new thiosemicarbazone derivatives of 1-biphenyl-4-
yl-2-imidazol-1-yl-ethanone were synthesized and the struc-
tures were identified. All the spectral data of the compounds
were in accordance with the assigned structures. On the other
hand, it is well known that the 1-biphenyl-4-yl-2-imidazol-1-
yl-ethanone and some thiosemicarbazone derivatives have
anticonvulsant activity1-3. In this study we aimed to gather
both structures and new compounds are expected to show
more effective activity. The compounds will be tested for their
anticonvulsant activities against MES and scMet induced sei-
zures and rotorod neurotoxicity test will be performed for
neurological toxicity according to the phase I tests of ADD
(Antiepileptic Drug Development) programme5.
ACKNOWLEDGEMENT
This project is supported by Hacettepe University Scientific
Research Unit. Project No: 07 01 301 002.
REFERENCES
1. Malawska B. New Anticonvulsant Agents, Curr Top Med
Chem. 2005; 5(1): 69-85.
2. Yogeeswari P, Sriram D, Mehta S, Nigam D, Kumar MM et
al. Anticonvulsant and neurotoxicity evaluation of some
6-substituted benzothiazolyl-2-thiosemicarbazones, Il
Farmaco. 2005; 60: 1-5.
3. Nardi D, Tajana A, Leonardi A, Pennini R, Potioli F et al. Syn-
thesis and Anticonvulsant Activity of N-(Benzoylalky1)
imidazoles and N-(ω-Phenyl-ω-hydroxyalkyl)imidazol, J
Med Chem. 1981; 24: 727-731.
4. Beukers MW, Wanner MJ, Künzel JKVFD, Klaasse EC, IJz-
erman AP et al. N6-Cyclopentyl-2-(3-phenylaminocar-
bonyltriazene-1-yl) adenosine (TCPA), a Very Selective
Agonist with High Affinity for the Human Adenosine A1
Receptor, J Med Chem. 2003; 46: 1492-1503.
5. Krall RL, Penry JK, White BG, Kupferberg HJ, Swinyard EA.
Antiepileptic drug development: II. Anticonvulsant drug
screening, Epilepsia, 1978; 9: 409-428.
p090-CYTOSTATIC AND ANTIVIRAL
ACTIVITY EVALUATIONS OF O-ALKYLATED
HYDROXAMIC DERIVATIVES OF SOME
NONSTEROIDAL ANTIINFLAMMATORY DRUGS
Zrinka Rajic1, Branka Zorc1, Miroslav Poznic2, Sandra
Kraljevic-Pavelic2, Karlo Hock2, Kresimir Pavelic2, Jan
Balzarini3, Mladen Mintas4
Department of Medicinal Chemistry, Faculty of Pharmacy and
Biochemistry, University of Zagreb, 10000, Zagreb/Croatia.1
Division of Molecular Medicine, Rudjer Boskovic Institute, 10000,
Zagreb/Croatia.2
Rega Institute for Medical Research, Katholieke Universiteit Leuven,
3000, Leuven/Belgium.3
Department of Organic Chemistry, Faculty of Chemical Engineering
and Technology, University of Zagreb, 10000, Zagreb/Croatia.4
INTRODUCTION
In our previous study we have shown that NSAID hydroxamic
 POSTER PRESENTATIONS
acids demonstrated a strong non-specific antiproliferative ef-
fect, higher than their NSAID parent compounds1. Among these
compounds diclofenac and indomethacin hydroxamic acids
showed the most pronounced and selective inhibitory activity,
while ibuprofen and fenoprofen hydroxamic acids demonstrat-
ed better activity if the hydroxy group was O-benzyl substitut-
ed. Here we report cytostatic and antiviral activity evaluation of
O-methyl and O-ethyl NSAID hydroxamic acids 1–9 (Figure).
Figure: NSAID hydroxamic acid derivatives 1–9
MATERIALS AND METHODS
Chemistry
NSAID hydroxamic acids, derivatives of ibuprofen, fenopro-
fen, ketoprofen, diclofenac and indomethacin were synthe-
sized according to our published procedure2.
Cytostatic Activity Assays
The cytostatic experiments were carried out on eleven hu-
man cell lines: murine leukemia (L1210), murine mammary
carcinoma (FM3A), human T-lymphocytes (Molt4/C8 and
CEM), cervical carcinoma (HeLa), breast carcinoma (MCF-7),
pancreatic carcinoma (MiaPaCa-2), non small cell lung can-
cer (NCI H727), colon carcinoma (SW620), breast adenocar-
cinoma-metastatic (SK-BR-3) and normal human fibroblasts
(WI 38). Proliferation assays, cell cycle analysis and Western
blot were performed according to the method described
previously1.
Antiviral Activity Assays
The antiviral assays, other than the anti-HIV assays, were
based on inhibition of virus-induced cytopathicity in HEL
[herpes simplex virus type 1 (HSV-1) (KOS), HSV-2 (G), vaccin-
ia virus and vesicular stomatitis virus], Vero (parainfluenza-3,
reovirus-1, Sindbis, Coxsackie B4, and Punta Toro virus), HeLa
(vesicular stomatitis virus, Coxsackie virus B4, and respiratory
syncytial virus) or MDCK (influenza A (H1N1; H3N2) and influ-
enza B) cell cultures3.
RESULTS AND DISCUSSION
Chemistry
NSAID hydroxamic acid derivatives 1-8 were prepared from
NSAID benzotriazolide and the corresponding hydroxylamine,
following our procedure2. Product 9 was obtained by catalytic
hydrogenation of ketoprofen O-benzylhydroxamic acid4.
Cytostatic Activity
NSAID hydroxamic acid derivatives 1–9 were evaluated in
vitro on their cytostatic effects against malignant tumor cells
October 26 - 28, 2009 / Antalya - TURKEY
L1210, FM3A, Molt4/C8, CEM, HeLa, MCF-7, MiaPaCa-2, NCI
H727, SW620 and SK-BR-3, and compared with their effects on
the growth of normal human fibroblasts (WI 38). Compounds
8 and 9 showed a rather pronounced antiproliferative activity
on the growth of breast cancer cell lines MCF-7 and SK-BR-3,
whereas compounds 4 and 7 markedly inhibited the growth
of colon carcinoma cells SW620. Strong and selective cyto-
static effects were observed for compounds 1 and 9 on Mi-
aPaCa-2 cells in comparison to normal fibroblasts WI 38. The
flow cytometric results for treatments of MiaPaCa-2 cells with
1 and 9 revealed a decrease of G0/G1 cell population. Accord-
ing to the Western blot results, procaspase 3 cleavage along
with the cleavage of procaspase 7 might play an important
role in MiaPaCa-2 apoptosis induced by compound 1 whereby
procaspase 3 cleavage seems important in apoptosis induc-
tion by compound 9. The possibility that Akt phosphorylation
occurs in dependence of caspase activity raised in this study,
should be tested in details by additional experiments.
Antiviral Activity
Compounds 1–9 were evaluated for their activity against a
broad spectrum of DNA and RNA viruses, in cell culture. Un-
fortunately, none of the compounds showed pronounced
antiviral activity at subtoxic concentrations. Only compound
9 showed marginal activity against parainfluenza virus and
Punta Toro virus (MIC = 20 µmol/L; MCC = 100 µmol/L).
References
1. Rajić Z, Butula I, Zorc B, Kraljević Pavelić S, Hock K, Pavelić
K, Naesens L, De Clercq E, Balzarini J, Przyborowska M,
Ossowski T, Mintas M. Cytostatic and antiviral activ- 191
ity evaluations of hydroxamic derivatives of some non-
steroidal anti-inflammatory drugs. Chem Biol Drug Des.
2009; 73: 328–338.
2. Rajić Z, Perković I, Butula I, Zorc B, Hadjipavlou-Litina D,
Pontiki E, Pepeljnjak S, Kosalec I. Synthesis and biological
evaluation of O-methyl and O-ethyl NSAID hydroxamic
acids. J Enz Inh Med Chem. 2009; in press.
3. Rajić Z, Zorc B, Poznić M, Kraljević-Pavelić S, Hock K et al.
Cytostatic and antiviral activity evaluation of O-alkylated
hydroxamic derivatives of some nonsteroidal antiinflam-
matory drugs. Chem Biol Drug Des. 2009; submitted.
P091-CONJUGATES OF PRIMAQUINE AND
NONSTEROIDAL ANTIINFLAMMATORY
DRUGS
Zrinka Rajic1, Branka Zorc1
Department of Medicinal Chemistry, Faculty of Pharmacy and
Biochemistry, University of Zagreb, 10000, Zagreb/Croatia.1
INTRODUCTION
Primaquine (PQ) is a well known antimalarial drug and inter-
esting molecule for derivatization in the search of potential
biologicaly active agents1. Recently we have reported the
synthesis of a series of primaquine derivatives bearing urea
substituent attached to the primaquine side-chain2. These
derivatives showed a modest antiproliferative activity on sev-
 POSTER PRESENTATIONS
eral tumor cell lines, whereby pyridine substituted derivative
of urea exhibited the highest cytostatic effect and some spe-
cific activity against human cytomegalovirus (HCMV). On the
other hand, nonsteroidal antiinflammatory drugs (NSAIDs)
are potential anticancer drugs and effective chemopreven-
tive agents3. The present work is aimed towards developing
amide PQ-NSAID conjugates with potential cytostatic and
antimalarial activity.
MATERIALS AND METHODS
Melting points were determined on a Stuart Melting Point
Apparatus SMP3 (Barworld Scientific, UK) and were uncor-
rected. IR spectra were recorded on a FTIR Perkin Elmer Para-
gon 500 spectrometer (Perkin Elmer, USA). 1H and 13C NMR
spectra were recorded on a Varian Gemini 300 spectrometer
(USA), operating at 300 and 75.5 MHz for the 1H and 13C
nuclei, respectively. Samples were measured in DMSO-d6
solutions at 20ºC in 5-mm NMR tubes. Chemical shifts (δ) in
ppm were referred to TMS. Coupling constants (J) are given
in Hz. The elemental composition of the compounds agreed
within ±0.4% (CHN-LECO-932, LECO Corporation, USA).
For thin-layer chromatography, precoated silica gel 60 F254
plates (Merck, Germany) and solvent systems cyclohexane/
ethyl acetate/methanol 3:1:0.5 and dichloromethane/
methanol 9:1 were used. Spots were visualized by short-
wave UV light and iodine vapor. Column chromatography
was performed with silica gel 0.063–0.200 mm (Merck,
Germany) and the following eluents: cyclohexane/ethyl ac-
etate/methanol 3:1:0.5, dichloromethane/methanol 9:1 and
192 dichloromethane→dichloromethane/methanol 99.5:0.5→di-
chloromethane/methanol 99:1.
Benzotriazole, triphosgene, triethylamine, ethylenediamine,
primaquine diphosphate and 10% Pd/C were purchased
from Sigma-Aldrich (USA). NSAIDs (diclofenak, ketoprofen,
fenoprofen, ibuprofen and indomethacin) were obtained
as a gift sample from Pliva and Belupo (Croatia) and Univer-
sity of Potchefstroom (Republic of South Africa). All solvents
were of analytical grade purity and were dried prior to use.
1-Benzotriazole carboxylic acid chloride (1), 2-(3-benzylphe-
nyl)propanoic acid (2a), 2-(3-(hydroxy(phenyl)methyl)phe-
nyl)propanoic acid (2b), NSAID benzotriazolides 3a-h and
10% Pd/C(en) were prepared according the published pro-
cedures4,5. PQ base was prepared from primaquine diphos-
phate prior to use. PQ solution was protected against light
during the whole procedure.
General Procedure for the Synthesis of 4a-h
A light-protected solution of PQ (0.156 g, 0.6 mmol), an ap-
propriate benzotriazolide 3 (0.5 mmol) and triethylamine
(0.209 mL, 1.5 mmol, except in the synthesis of 4g) in toluene
(5 mL) was stirred at room temperature for 0.5 h. The reac-
tion mixture was extracted with water at pH 9 (5×10 mL). The
organic layer was dryed over anhydrous sodium sulphate,
filtrated and evaporated. The obtained crude product was
purified by column chromatography.
3rd BBBB International Conference on Pharmaceutical Sciences
RESULTS AND DISCUSSION
The PQ-NSAID conjugates 4a-h were prepared by the re-
action of the PQ and an appropriate benzotriazolide 3a-h,
in toluene, at room temperature for 0.5 h (Figure). In gen-
eral, 3-fold excess of triethylamine was used. Triethylamine
formed a water soluble salt with benzotriazole, a by-product
of the reaction, which was readily extracted with water. The
synthesis of PQ-indomethacine derivative 4g was performed
with 1.3 equivalents of triethylamine because of the possibil-
ity of the indomethacine amide bond cleveage.
Figure: Synthesis of the PQ-NSAID conjugates 4a-h
Structures of the compounds 4a-h were deduced from the
analysis of their 1H and 13C NMR spectra and confirmed by
the elemental analysis. The chemical shifts were
consistent with the proposed structures of the novel com-
pounds.
Cytostatic and antimalarial activity evaluations are in prog-
ress.
REFERENCES
1. Vale N, Moreira R, Gomes P. Primaquine revisited six
decades after its discovery. Eur J Med Chem. 2009; 44:
937–953.
2. Džimbeg G, Zorc B, Kralj M, Ester K, Pavelić K, Balzarini J,
De Clercq E, Mintas M. The novel primaquine derivatives
of N-alkyl, cycloalkyl or aryl urea: synthesis, cytostatic
and antiviral activity evaluations. Eur J Med Chem. 2008;
43: 1180–1187.
3. Richter M, Weiss M, Weinberger I, Fürstenberger G, Mar-
ian B. Growth inhibition and induction of apoptosis in
colorectal tumor cells by cyclooxygenase inhibitors. Car-
cinogenesis. 2001; 22: 17–25.
4. Rajić Z, Hadjipavlou-Litina D, Pontiki E, Kralj M, Šuman L,
Zorc B. The novel ketoprofen amides – synthesis and bi-
ological evaluation as antioxidants, lipoxygenase inhibi-
tors and cytostatic agents. Chem Biol Drug Des. 2009;
submitted.
5. Zorc B, Antolić S, Butula I. Macromolecular prodrugs. I.
Synthesis of some non-steroidal anti-inflammatory drug
esters. Acta Pharm 1993; 43: 127–133.
 POSTER PRESENTATIONS
p092-THE NOVEL KETOPROFEN
AMIDOCARBAMATES: BIOLOGICAL ACTIVITY
EVALUATION
Zrinka Rajic1, Dimitra Hadjipavlou-Litina2, Eleni Pontiki2, Jan
Bazarinil3, Branka Zorc1
Department of Medicinal Chemistry, Faculty of Pharmacy and
Biochemistry, University of Zagreb, 10000, Zagreb/Croatia.1
Department of Pharmaceutical Chemistry, School of
Pharmacy, Aristotles University of Thessaloniki, 54124,
Thessaloniki/Greece.2
Rega Institute for Medical Research, Katholieke Universiteit
Leuven, 3000, Leuven/Belgium.3
INTRODUCTION
Ketoprofen is NSAID with pronounced analgesic and anti-
pyretic properties. In our previous research antioxidant and
cytostatic activity of various ketoprofen amides was tested1.
It was shown that lipophilic substances possessed better bi-
ological activity, therefore we have synthetized novel keto-
profen amidocarbamates 4a-j. Here we report their biological
screening as antioxidants, cytostatic and antiviral agents.
MATERIALS AND METHODS
Each experiment in vitro was performed at least in tripli-
cate and the standard deviation of absorbance was less
than 10% of the mean. 1,1-Diphenyl-picrylhydrazyl (DPPH),
2,2’-azobis(2-amidinopropane) dihydro-chloride (AAPH),
nordihydroguaiaretic acid (NDGA), sodium linoleate, soy-
bean lipoxygenase (LOX), caffeic acid and trolox were pur-
chased from Sigma-Aldrich (USA).
LOX Inhibition Activity2)
DMSO solution of the tested compound was incubated with
0.1 mmol/L sodium linoleate and 0.2 mL of LO solution (1/9
× 10-4 w/v in saline) at room temperature. The conversion
of sodium linoleate to 13-hydroperoxylinoleic acid was re-
corded at 234 nm and compared with the standard inhibitor
caffeic acid.
Interaction with DPPH Activity4
To a solution of 0.05 mmol/L DPPH in absolute ethanol an
equal volume of 0.05 or
0.1 mmol/L ethanolic solution of the tested compound was
added. After 20 and 60 min the absorbance was recorded at
517 nm and compared with the appropriate standard NDGA.
Ethanol was used as a control.
Inhibition of Linoleic Acid Lipid Peroxidation3
Ten microliters of 16 mmol/L linoleic acid dispersion was
added to the UV cuvette containing 0.93 mL of 0.05 mmol/L
phosphate buffer, pH 7.4 prethermostated at 37 °C. The oxi-
dation reaction was initiated at 37 °C under air by the ad-
dition of 50 μL of 40 mmol/L AAPH solution. Oxidation was
carried out in the presence of the tested compounds (10 μL,
final concentration 0.1 mmol/L). In the assay with no anti-
oxidant, lipid peroxidation was measured in the presence
of the same level of DMSO. The rate of oxidation was moni-
tored at 37 °C by recording the increase of absorption at 234
October 26 - 28, 2009 / Antalya - TURKEY
nm caused by conjugated diene hydroperoxides. The results
were compared to the standard inhibitor trolox.
Cytostatic Activity Assay
Murine leukemia L1210, murine mammary carcinoma FM3A
and human T-lymphocyte CEM cells were suspended at
300,000−500,000 cells/mL of culture medium, and 100 µL
of a cell suspension was added to 100 µL of an appropriate
dilution of the test compounds in wells of 96-well microti-
ter plates. After incubation at 37 °C for two (L1210, FM3A)
or three (CEM) days, the cell number was determined using
a Coulter counter. The IC50 was defined as the compound
concentration required to inhibit cell proliferation by 50%.
Antiviral Activity Assay
The antiviral assays were based on inhibition of virus-induced
cytopathicity in HEL [herpes simplex virus type 1 (HSV-1), HSV-2
(G), vaccinia virus and vesicular stomatitis virus], Vero (parainflu-
enza-3, reovirus-1, Sindbis, Coxsackie B4, and Punta Toro virus),
HeLa (vesicular stomatitis virus, Coxsackie virus B4, and respiratory
syncytial virus) or MDCK (influenza A (H1N1; H3N2); and influenza
B) cell cultures. Confluent cell cultures in microtiter 96-well plates
were inoculated with 100 CCID50 of virus (CCID50 being the virus
dose to infect 50% of the cell cultures). After a 1 h virus adsorption
period, residual virus was removed, and the cell cultures were in-
cubated in the presence of varying concentrations (200, 40, 8, ...
µmol/L) of the test compounds. Viral cytopathicity was recorded
as soon as it reached completion in the control virus-infected cell
cultures that were not treated with the test compounds.
RESULTS AND DISCUSSION 193
A series of novel ketoprofen derivatives 4a-j bearing both
amide and carbamate functionalities were prepared using the
benzotriazole method of carboxylic and hydroxy group acti-
vation. Selective reduction of ketoprofen produced hydroxy
derivative 2, which in the reaction with one or two moles of
1-benzotriazole carboxylic acid chloride (1) gave benzotriaz-
ole derivatives 3a and 3b, respectively. Compounds 3 with
various amines afforded amidocarbamates 4a-j.
Figure: The final step in the synthesis of ketoprofen amido-
carbamates 4a-j
Antioxidative screenings revealed that the prepared compounds
3b and 4a-j possess excellent lipid peroxidation inhibition at
0.1 mmol/L concentration, higher than 95% for the derivatives
bearing aromatic, cycloalkyl or heterocyclic substituents. Two of
the compounds, 3b and 4g, also show high soybean lipoxyge-
nase inhibition activity (95 and 83.5%, respectively) (Table).

Table: In vitro LOX and LP inhibition
Compd. 3b 4a 4b 4c 4d 4e 4f
LOX: inhibitiona
95.0b 26.9 22.3 40.8 69.6a 22.7 56.6b 83.8b 16.9 112.7 n.ac
(%)
LP inhibitiona
98.0 61.0 54.5 99.3 95.2 96.1 99.5
(%)
Concentration of the tested compounds a1 x 10-4mol/L
b
/c50 value was also determined:21 (3b), 86(4d), 95(4f ), 32(4g)
μmol/L, c not determined
On the other hand, the ketoprofen amidocarbamate deriva-
tives show only weak reducing activity against 1,1-diphenyl-
2-picrylhydrazyl radicals. No selective antiviral effects were
noted for the tested compounds against a broad variety of
DNA and RNA viruses. Most compounds were endowed with
a moderate (IC50: 10–25 µmol/L) cytostatic activity against
tested tumor cell lines.
REFERENCES
1. Rajić Z, Hadjipavlou-Litina D, Pontiki E, Kralj M, Šuman L,
Zorc B. The novel ketoprofen amides – synthesis and bi-
ological evaluation as antioxidants, lipoxygenase inhibi-
tors and cytostatic agents. Chem Biol Drug Des. 2009;
submitted.
2. Pontiki E, Hadjipavlou-Litina D, Synthesis and pharmaco-
chemical evaluation of novel aryl-acetic acid inhibitors
of lipoxygenase, antioxidants, and anti-inflammatory
agents, Bioorg Med Chem. 2007; 15: 5819-5827.
3. Re ER, Pellegrini N, Proteggente A, Pannala A, Yang M,
Rice-Evans C, Antioxidant activity applying an improved
194 ABTS radical cation decolorization assay, Free Rad Biol Med.
1999; 26: 1231-1237Table. In vitro LOX and LP inhibition
p093-ANTIOXIDANT ACTIVITIES OF SOME
4-(4-METOXYBENZOYLAMINO)-4,5-DIHYDRO-
1H-1,2,4-TRIAZOL-5-ONES
Sevilay Demirci1, Haydar Yüksek1, Muzaffer Alkan2, Şule
Bahçeci3, Mustafa Calapoğlu4
Dept. of Chemistry, Kafkas University, 36100 Kars/Turkey1
Education Faculty, Kafkas University, 36100 Kars/Turkey2
Fatih Education Faculty, KTÜ, Trabzon/Turkey3
Dept. of Chemistry, Süleyman Demirel University, Isparta/Turkey4
INTRODUCTION
In the last decade, a great deal of research has been devot-
ed to the study of different types of new antioxidant com-
pounds, either synthesized or obtained from natural sources
which may at least minimize the deleterious effects induced
by reactive oxygen species (ROS). Exogenous chemicals and
endogenous metabolic processes in human body or in food
system might produce highly reactive free radicals, especial-
ly oxygen derived radicals, which are capable of oxidizing
biomolecules by resulting in cell death and tissue damage.
A large number of heterocyclic compounds, containing the
1,2,4-triazole ring, are associated with wide ranges of biolog-
ical activities1. The identification of triazoles and determina-
tion of their antioxidant activities are of considerable interest
3rd BBBB International Conference on Pharmaceutical Sciences
POSTER PRESENTATIONS
because of the role they play in pharmacological actions. In
4g 4h 4i 4j the present investigation, radical scavenging and antioxi-
dative activity for the five recently reported2 3-alkyl-4-(p-
methoxybenzoylamino)-4,5-dihydro-1H-1,2,4-triazol-5-ones
(1) were evaluated using three antioxidant methodologies
98.4 99.0 77.2 97.4
Scheme 1
MATERIALS AND METHODS
Chemicals
BHT was purchased from Merck. Ferrous chloride,
α-tocopherol, DPPH.) ferrozine, butylated hydroxyanisole
(BHA) and trichloracetic acid (TCA) were purchased from
Sigma (Sigma –Aldrich GmbH, Sternheim,Germany).
Reducing power
The reducing power of the synthesized compounds was de-
termined according to the method of Oyaizu (3). Different
concentrations of the samples (60-180 µg/mL) in DMSO (1
mL) were mixed phosphate buffer (2.5 mL, 0.2 M, pH = 6.6)
and potassium ferricyanide (2.5 mL,1%). The mixture was in-
cubated at 50oC for 20 min. after incubation period; a por-
tion of trichloroacetic acid (2.5 mL, 10%) was added to the
mixture, which was then centrifuged for 10 min at 1000 x g.
The upper layer of solution (2.5 mL) was mixed with distilled
water (2.5 mL) and FeCI3 (0.5 mL, 0.1%), and then the ab-
sorbance was measured at 700 nm in a spectrophotometer.
Higher absorbance of the reaction mixture indicated greater
reducing power.
Free radical scavenging activity
Free radical scavenging activity of compounds was mea-
sured by DPPH., using the method of Blois (4). Briefly, a 0.1
mM solution of DPPH. in ethanol was prepared, and this so-
lution (1mL) was added to sample the solutions in DMSO (3
mL) at different concentrations (50-150 µg/mL). The mixture
was shaken vigorously and allowed to stand at room tem-
perature for 30 min. Then the absorbance was measured at
517 nm in a spectrophotometer. Lower absorbance of the
reaction mixture indicated higher free radical scavenging
activity. The DPPH. concentration (mM) in the reaction medi-
um was calculated from the following calibration curve and
determined by linear regression (R: 0.997):
Absorbance = 0.0003 x DPPH. – 0.0174
The capability to scavenge the DPPH radical was calculated
using the following equation:
DPPH. scavenging effect (%) = (A0 – A1/A0) x 100
where A0 is the absorbance of the control reaction and A1 is
the absorbance in the presence of the samples or standards.
 POSTER PRESENTATIONS

Metal chelating activity

The chelating ferrous ions by the synthesized compounds
and standards were estimated by the method of Dinis et al5.
Briefly, the compounds (50-150 µg/mL) were added to a 2
mM solution of FeCI2 (0.05 mL). The reaction was initiated by
the addition of 5 mM ferrozine (0.2 mL) and the mixture was
shaken vigorously and left standing at room temperature
for 10 min. After the mixture had reached equilibrium, the
absorbance of the solution was then measured spectropho-
tometrically at 562 nm in a spectrophotometer. All test and
analyses were run in triplicate and averaged. The percentage
of inhibition of ferrozine-Fe2+ complex formation was given
by the formula: % Inhibition = (A0 – A1/A0) x 100, where A0 Figure 1: Scavenging effect of compounds, BHA and a-tocopherol at dif-
is the absorbance of the control, and A1 is the absorbance ferent concentrations (50-100-150 μg/mL)
in the presence of the samples or standards. The control did
not contain compound or standard.

RESULTS AND DISCUSSION

Reducing activitiy
The reducing capacity of a compound may serve as a sig-
nificant indicator of its potential antioxidant activity. The
presence of reductants such as antioxidants substances in
the antioxidant samples causes the reduction of the Fe3+/
ferricyanide complex to the ferrous form. In this study, all the
amounts of the compounds showed lower absorbance than
blank. Hence, no activities were observed to reduce metal
ions complexes to their lower oxidation state or to take part Figure 2: Metal chelating effect of different amount of the compounds,
in any electron transfer reaction. BHT and a-tocopherol on ferrous ions

Radical-scavenging activity CONCLUSION

All the compounds tested with this method showed lower
absorbance than absorbance of the control reaction and
higher absorbance of the standard antioxidant reactions. The
decrease in absorbance of DPPH radical was caused by anti-
oxidants, because of reaction between antioxidant molecules
and radical, progresses, which result in the scavenging of the
radical by hydrogen donation. Figure 1 illustrates a decrease
the concentration of DPPH radical due to the scavenging abil-
ity of the compound and standards. The scavenging effects of
compounds and standards on the DPPH radical were found
as following order: α-tocopherol>BHA>1c>1d>1a>1e>1b at
the concentration of 150 µg/mL. These results indicate that
the newly synthesized compounds showed mild activities as
a radical scavenger, indicating that it has moderate activities
as hydrogen donors.
Ferrous ions chelating activity
Ferrous ion chelating activities of the compounds, BHT and
α-tocopherol are shown in Figure 2. In this study, metal
chelating capacity was significant since it reduced the con-
centrations of the catalyzing transition metal. It was report-
ed that chelating agents, that form σ-bonds with a metal,
are effective as secondary antioxidants because they reduce
the redox potential thereby stabilizing the oxidized form of
metal ion6. The percentage of metal chelating capacity of
compounds, BHT and α-tocopherol exhibited the following
order: 1b>1c>1a>BHT>1d>1e>α-tocopherol at the concen-
tration of 50-150 µg/mL. The data obtained from Figure 2
reveal that the compounds demonstrates a marked capacity
for iron binding, suggesting that their action as peroxidation
protector may be related to its iron binding capacity.
We are currently evaluating the antioxidant activity of the 195
3-alkyl-4-(p-methoxybenzoylamino)-4,5-dihydro-1H-1,2,4-
triazol-5-ones. The biochemical action of the compounds
could be due, in part, to their ability to scavenge oxygen free
radicals and chelating activity, which may be involved in pre-
venting redox cycling. Chelating agents, who form σ-bonds
with a metal, are effective as secondary antioxidants because
they reduce the redox potential thereby stabilizing the oxi-
dized form of metal ion. Design and synthesis of novel small
molecules which can specifically protective role in biologi-
cal systems are in perspective in modern pharmacological
chemistry. These results encourage further in vivo studies
and explore their possible therapeutic applications.
REFERENCES
1. Yüksek H, Demirbaş A, Ikizler A, Johansson CB, Çelik C,
Ikizler AA. Synthesis and antibacterial activities of some
4,5-dihydro-1H-1,2,4-triazol-5-ones, Arzneim-Forsch/
Drug Res.1997; 47: 405-409.
2. Yüksek H, Alkan M, Bahçeci Ş, Çakmak İ, Ocak Z, Baykara
H, Aktaş Ö, Ağyel E. Synthesis, determination of pKa val-
ues and GIAO NMR calculations of some new 3-alkyl-4-(p-
methoxybenzoylamino)-4,5-dihydro-1H-1,2,4-triazol-5-
ones, J Mol Struc. 2008; 873: 142-148.
3. Oyaizu M. Studies on products of browning reaction pre-
pared from glucosamine, Japan Nutri. 1986; 44: 307-316.
4. Blois MS. Antioxidant determinations by the use of a
stable free radical, Nature, 1958; 26: 1199-1200.
5. Dinis TCP, Madeira VMC, Almeida LM. Action of phenolic
derivatives (acetaminophen, salicylate, and 5-aminosali-
cylate) as inhibitors of membrane lipid peroxidation and

October 26 - 28, 2009 / Antalya - TURKEY

 POSTER PRESENTATIONS

as peroxyl radical scavengers, Arch Biochem Biophys. Computational analysis

1994; 315: 161-169. Lipophilicity log P parameters were calculated using differ-
6. Gordon MH. Food Antioxidants, Elsevier: London-New ent programmes available on the Internet (ALOGPs, CLOGP,
York, 1990; pp. 1-18. IA_logP, KowWin, MiLogP, MLOGP, XLOGP). Data was pro-
cessed in MS Office Excel 2003.

p094-APPLICABILITY OF RP-TLC METHOD RESULTS AND DISCUSSION

FOR THE ASSESSMENT OF LIPOPHILICITY OF Visualization of plates with standards of cephalosporines
BETA-LACTAMS USED IN TREATMENT OF CNS was done under UV light (254 nm), Figure 1.
INFECTIONS

Mirza Bojic1, Alemka Mitar1, Marica Medic-Saric1, Zrinka

Rajic1

Department of Medicinal Chemistry, Faculty of Pharmacy and

Biochemistry, University of Zagreb, 10000, Zagreb/Croatia.1

INTRODUCTION
80 years have passed since Fleming published discovery of
penicillin. Beta-lactams have been for years the safest and
most efficient antibiotics owing it to the specific action on
bacterial cell wall. Nowadays, beta-lactams are in their renais-
sance as multipotent agents against neurological diseases Figure 1: TLC plate of cephalosporines recorded under 254
(e.g. amylotrophic lateral sclerosis, Alzheimer, Parkinson and nm
prion diseases)1. Thus their lipophilicity, as major character-
istic determining passage through blood-brain barrier, is of As spectra of penicillins (Figure 2) have maximum at even
grate importance for their targeted delivery. lower UV, these plates were recorded at 200 nm.

As TLC is relatively inexpensive and time saving method

196 we decided to characterise each beta-lactam with chro-
matographic parameters RM0 and φ0 using linear solvent
strength model2.

MATERIALS AND METHODS

Materials
Standards of eight most commonly used beta-lactams for
treatment of CNS infections (amoxicillin, ampicillin, benzyl-
penicillin, cloxacillin, cefuroxime, ceftazidime, ceftrixaone,
cefotaxime) were obtained from Agency for Medicinal Prod-
ucts and Medical Devices (Zagreb, Croatia). RP-18F254s TLC
plates were purchased from Merck (Darmstadt, Germany).
Methanol and iodine were purchased from Kemika (Croatia,
Figure 2: Spectrum of ampicillin obtained with CAMAG TLC
Zagreb). MiliQ water was made in house.
Scanner 3
Sample preparation
Using linear solvent strength (LSS) equation:
Stock solutions were prepared by dissolving 1 mg of stan-
dard in 1 mL of methanol. These were diluted to 0.1 mg/mL
RM = RM0 - k φ
for standards of cephalosporines.
models could be constructed only for five beta-lactams.
TLC analysis
These are presented in Table 1.
Samples were applied in bands of 5 mm using CAMAG Lino-
mat 5 (Basel, Switzerland) in duplicate. Mobile phase was bi-
Table 1. LSS models of beta-lactams analysed
nary mixture of methanol and water. The amount of organic
Beta-lactam Equation Correlation
phase was varied from 10 to 90% with increment of 2.5, 5 or
10% to obtain linear solvent strength model for each beta- benzylpenicilin RM=1.664-0.028ϕ r2=0.9355
lactam individually. Plates were recorded using CAMAG TLC cloxacillin RM=1.112-0.019ϕ r2=0.9641
Scanner 3. Penicillins were additionally visualized using io- cefuroxime RM=0.405-0.223ϕ r2=0.9322
dine vapours.
ceftazidime RM=0.456-0.018ϕ r2=0.9718

3rd BBBB International Conference on Pharmaceutical Sciences

 POSTER PRESENTATIONS
Beta-lactam Equation Correlation
cefotaxime RM=0.279-0.019ϕ r2=0.9401
RM0 and φ0 as chromatographic measurements of lipophi-
licity were correlated to calculated log P values. The high-
est correlation was observed between RM0 and CLOGP
(0.7358).
LSS models could not be determined for ampicillin, amoxi-
cillin and ceftrixaone in wide range of organic modifier. Al-
though LSS models for other beta- lactams were suitable,
RP-TLC should be used as indicative rather than quantitative
measurement for characterisation of lipophilicity of beta-
lactams in methanol-water systems.
ACKNOWLEDGMENT
This work was supported by Ministry of Science, Educa-
tion and Sports of Republic of Croatia (grant number 006-
0061117-1237).
REFERENCES
1. Ji HF, Shen L, Zhang HY. Beta-lactam antibiotics are mul-
tipotent agents to combat neurological diseases, Bio-
chem Biophys Res Commun. 2005; 333(3):661-663.
2. Kaliszan R. QSRR: quantitative structure-
(chromatographic) retention relationships, Chem Rev.
2007; 107(7):3212-3246.
p095-ANTIOXIDANT ACTIVITIES OF SOME
3-ALKYL(ARYL)-4-ISOBUTYRYLAMINO-4,5-
DIHYDRO-1H-1,2,4-TRIAZOL-5-ONES
Mustafa Calapoğlu1, Haydar Yüksek2, Muzaffer Alkan3, Şule
Bahçeci4
Dept. of Chemistry, Süleyman Demirel University, Isparta/Turkey1
Dept. of Chemistry, Kafkas University, 36100 Kars/Turkey2
Education Faculty, Kafkas University, 36100 Kars/Turkey3
Fatih Education Faculty, KTÜ, Trabzon/Turkey4
INTRODUCTION
Antioxidants are extensively studied for their capacity to
protect organism and cell from damage that is induced by
the oxidative stress. A great deal of research has been de-
voted to the study of different types of natural and synthetic
antioxidant. A large number of heterocyclic compounds,
containing the 1,2,4-triazole ring, are associated with diverse
biological properties such as antioxidant, anti-inflammatory,
antimicrobial and antiviral activity1. Exogenous chemicals
and endogenous metabolic processes in human body or
in food system might produce highly reactive free radicals,
especially oxygen-derived radicals, which are capable of
oxidizing biomolecules by resulting in cell death and tissue
damage. Oxidative damages play a significantly pathological
role in human diseases. Herein, a series of 3-alkyl-4-isobutyr-
ylamino-4,5-dihydro-1H-1,2,4-triazol-5-ones, which were
synthesized according to literature2 (Scheme-1) with potent
antioxidant activities were reported.
October 26 - 28, 2009 / Antalya - TURKEY
Scheme 1
MATERIALS AND METHODS
Materials
Butylated hydroxyl toluene (BHT) was purchased from Mer-
ck. Ferrous chloride, α-tocopherol, 1,1-diphenyl-2-picrylhy-
drazyl (DPPH.), 3-(2-pyridyl)-5,6-bis(phenyl-sulfonic acid)-
1,2,4-triazine (ferrozine), butylated hydroxyanisole (BHA) and
trichloracetic acid (TCA) were purchased from Sigma (Sigma-
Aldrich GmbH, Sternheim, Germany). All other chemicals
used were analytical grade and obtained from either Sigma-
Aldrich or Merck.
Antioxidant activities
The antioxidant capacity of compound samples was exam-
ined by comparing to that of the known antioxidants, BHT,
BHA and α-tocopherol, by employing the following three
complementary in vitro assays: ferric reducing/antioxidant
power (FRAP) assay by the method of Oyaizu3, radical scav-
enging activity by the method of Blois(4 and The chelating
effect of ferrous ions according to the method of Dinis et al5. 197
RESULTS AND DISCUSSION
Reducing activitiy
The reducing power test, in which the capacity of breaking
radical chain reactions is reflected, is considered to be a good
indicator of antioxidant capacity Thus, this method was em-
ployed to determine the antioxidant capacity and, indirectly,
total reducing potentials of three different concentrations of
the compounds and reference standard.In this study, all the
amounts of the compounds showed lower absorbance than
blank. Hence, no activities were observed to reduce metal
ions complexes to their lower oxidation state or to take part
in any electron transfer reaction.
Radical-scavenging activity
All the compounds tested with this method showed lower
absorbance than absorbance of the control reaction and
higher absorbance of the standard antioxidant reactions.
The decrease in absorbance of DPPH radical was caused
by antioxidants, because of reaction between antioxidant
molecules and radical, progresses, which result in the scav-
enging of the radical by hydrogen donation. Figure 1 illus-
trates a decrease the concentration of DPPH radical due
to the scavenging ability of the compound and standards.
The scavenging effect of compounds and standards on the
DPPH radical decreased in the order of α-tocopherol>BHA
>1a>1b>1c>1e>1d at the concentration of 50 µg/mL, re-
spectively. These results indicate that the newly synthesized
compounds showed mild activities as a radical scavenger, in-
 POSTER PRESENTATIONS
dicating that it has moderate activities as hydrogen donors.
Figure 1: Scavenging effect of compounds, BHA and a-toco-
pherol at different concentrations (50-100-150 μg/mL)
Ferrous ions chelating activity
Ferrous ion chelating activities of the compounds, BHT and
α-tocopherol are shown in Figure 2. In this study, metal
chelating capacity was significant since it reduced the con-
centrations of the catalyzing transition metal. It was report-
ed that chelating agents, that form σ-bonds with a metal,
are effective as secondary antioxidants because they reduce
the redox potential thereby stabilizing the oxidized form of
metal ion6. The percentage of metal chelating capacity of
compounds, BHT and α-tocopherol exhibited the following
order: 1c>1a>BHT>1e>1b>1d>α-tocopherol at the concen-
tration of 12,5-37,5 µg/mL. The data obtained from Figure 2
reveal that the compounds demonstrates a marked capacity
for iron binding, suggesting that their action as peroxidation
198 protector may be related to its iron binding capacity.
Figure 2: Metal chelating effect of different amount of the
compounds, BHT and a-tocopherol on ferrous ions
CONCLUSION
Several antioxidant assays were utilized inorder to evalu-
ate the biological and chemical properties of the chemical
compounds.The data here reported could be of the possible
interest because of their activities of hydrogen donating
and metal chelating could prevent redox cycling. Chelating
agents, who form σ-bonds with a metal, are effective as sec-
ondary antioxidants because they reduce the redox poten-
tial thereby stabilizing the oxidized form of metal ion. In ad-
dition, the compound samples appeared to deserve further
investigation of to their individual biologically active com-
ponents, which may be an attractive source of nutraceuticals
and medicinal ingredients.
3rd BBBB International Conference on Pharmaceutical Sciences
REFERENCES
1. Yüksek H, Demirbaş A, Ikizler A, Johansson CB, Çelik C,
Ikizler AA. Synthesis andantibacterial activities of some
4,5-dihydro-1H-1,2,4-triazol-5-ones, Arzneim-Forsch/
Drug Res.1997; 47: 405-409.
2. Yüksek H, Alkan M, Ocak Z, Bahçeci Ş, Ocak M, Özdemir
M. Synthesis and acidic properties of some new po-
tential biologically active 4-acylamino-4,5-dihydro-1H-
1,2,4-triazol-5-one derivatives, Indian J Chem. 2004; 43B:
1527-1531.
3. Oyaizu M. Studies on products of browning reaction
prepared from glucosamine, Japan Nutri. 1986; 44: 307-
316.
4. Blois MS. Antioxidant determinations by the use of a
stable free radical, Nature, 1958; 26: 1199-1200.
5. Dinis TCP, Madeira VMC, Almeida LM. Action of phenolic
derivatives (acetaminophen, salicylate, and 5-aminosali-
cylate) as inhibitors of membrane lipid peroxidation and
as peroxyl radical scavengers, Arch Biochem Biophys.
1994; 315: 161-169.
6. Gordon MH. Food Antioxidants, Elsevier: London-New
York, 1990; pp. 1-18.
p096-SYNTHESIS AND ANTIOXIDANT
ACTIVITIES OF SOME 1-ACETYL-3-ALKYL-4-
(4-DIETHLYAMINOBENZYLIDENAMINO)-4,5-
DIHYDRO-1H-1,2,4-TRIAZOL-5-ONES
Mustafa Calapoğlu1, Haydar Yüksek2, Özlem Gürsoy Kol2,
Muzaffer Alkan3
Dept. of Chemistry, Süleyman Demirel University, Isparta/Turkey1
Dept. of Chemistry, Kafkas University, 36100 Kars/Turkey2
Education Faculty, Kafkas University, 36100 Kars/Turkey3
INTRODUCTION
1,2,4 Triazole and 4,5-dihydro-1H-1,2,4-triazol-5-one deriva-
tives are reported to possess a broad spectrum of biological
activities such as systemic pesticides, antifungal, microbial,
lukemic, parasitic, viral, inflammatory, antitumor, hyperten-
sive, hypocholesteremic, antioxidant and anti-human immu-
nodeficiency virus properties. The identification of triazoles
and determination of their antioxidant activities are of con-
siderable interest because of the role they play in pharma-
cological actions. In the present investigation, antioxidative
activity for the five newly synthesized 1-acetyl-3-alkyl(aryl)-
4-(4-diethylaminobenzylidenamino)-4,5-dihydro-1H-1,2,4-
triazol-5-ones were evaluated using three antioxidant meth-
odologies.
Scheme 1
MATERIALS AND METHODS
Chemicals
Butylated hydroxyl toluene (BHT) was purchased from
 POSTER PRESENTATIONS

Merck. Ferrous chloride, α-tocopherol, DPPH., ferrozine,
butylated hydroxyanisole (BHA) and trichloracetic acid
(TCA) were purchased from Sigma (Sigma –Aldrich GmbH,
Sternheim,Germany).
Synthesis of title compounds
The starting compounds 1a-e were synthesized according to
literature1. The corresponding compound 1 (0.01 mol) was
refluxed with acetic anhydride (15 mL) for 0.5 h. After addi-
tion of absolute ethanol (50 mL), the mixture was refluxed for
1 h more. Evaporation of the resulting solution at 40-45 °C in
vacuo and several recrystallizations of the residue from EtOH
gave pure compounds 2a-e as colourless crystals (Scheme
1). The newly synthesized compounds 2 were characterized
by IR, 1HNMR, 13C-NMR, UV and MS spectral data.
Ferrous ions chelating activity
Ferrous ion chelating activities of the compounds, BHT and
α-tocopherol are shown in Figure 3. In this study, metal chelat-
ing capacity was significant since it reduced the concentrations
of the catalyzing transition metal. It was reported that chelating
agents, that form σ-bonds with a metal, are effective as second-
ary antioxidants because they reduce the redox potential there-
by stabilizing the oxidized form of metal ion5. The data obtained
from Figure 2 reveal that the compounds demonstrates a marked
capacity for iron binding, suggesting that their action as peroxi-
dation protector may be related to its iron binding capacity.

Antioxidant activities
The antioxidant activity of tested compounds was determined
by the ferric-reducing antioxidant power (FRAP), 1,1-diphenyl-
2-picrylhydrazyl (DPPH) assays and Fe2+-metal chelating as-
say. Butylated hydroxytoluene (BHT) and α-tocopherol were
used as reference antioxidant compounds. The reducing pow-
er of the synthesized compounds was determined according
to a modified version of FRAP assay of Oyaizu2. The hydrogen
atoms or electrons donation ability of the synthesized com- Figure 1: Total reductive potential of different concentrations
pound was measured by DPPH. using the method of Blois3. (50-100-150 μg/mL) of 2a, 2b, BHA, BHT and α-tocopherol.
The chelating effect of ferrous ions by the compound was de- High absorbance at 700 nm indicates high reducing power
termined according to the method of Dinis et al4.

RESULTS AND DISCUSSION

Reducing activitiy 199
FRAP assay uses antioxidants as reuctants in a redox-linked
colorimetric method, employingan easily reduced oxidant
system present in stoichiometric excess. In this study, the re-
ducing ability of some synthesized compounds augmented
with increasing concentration of samples. As can be seen in
Figure 1, compound 2a and 2b showed a weak reducing activ-
ity for Fe3+ compared to the blank reaction. Compounds also
displayed. The other compounds showed lower absorbance
than the blank, hence, no reductive activities were observed.
Only compounds 2a and 2b may reduce metal ions complexes Figure 2. Scavenging effect of compounds 2a-e, BHA and
to their lower oxidation state or to take part in electron trans- α-tocopherol at different concentrations (12,5-25-37.5 μg/mL)
fer reaction. In other words, compounds 2a and 2b showed
the weak ability of electron donor to scavenge free radicals.
,
Radical-scavenging activity
All the compounds tested with this method showed lower ab-
sorbance than absorbance of the control reaction and higher
absorbance of the standard antioxidant reactions. The decrease
in absorbance of DPPH radical was caused by antioxidants, be-
cause of reaction between antioxidant molecules and radical,
progresses, which result in the scavenging of the radical by hy-
drogen donation. Figure 2 illustrates a decrease the concentra-
tion of DPPH radical due to the scavenging ability of the com-
pound and standards. The scavenging effect of compounds and
standards on the DPPH radical were found as following order: Figure 3. Metal chelating effect of different amount of the
α-tocopherol> BHA> 2a>2c>2b>2e>2d at the concentration of compounds 2a-e, BHT and α-tocopherol on ferrous ions
12,5 µg/mL. These results indicate that the newly synthesized
compounds showed mild activities as a radical scavenger, indi-
cating that it has moderate activities as hydrogen donors.

October 26 - 28, 2009 / Antalya - TURKEY

 POSTER PRESENTATIONS
CONCLUSION
Synthesis of a series novel 1-acetyl-3-alkyl(aryl)-4-(4-diethyl-
aminobenzyliden-amino)-4,5-dihydro-1H-1,2,4-triazol-5-ones
in high yields was accomplished. Some of them were found
to possess a weak reducing activity. All the compounds mild
radical scavenging activity and good metal chelating effect.
These points to the fact that any electron withdrawing and
stabilizing the oxidized form of metal ion substituent moiety
in 2a-e appears to prevent to some extent oxidative metabolic
pathways in the living systems. Design and synthesis of novel
small molecules which can specifically protective role in bio-
logical systems are in perspective in modern pharmacological
chemistry. These results encourage further in vivo studies and
explore their possible therapeutic applications.
REFERENCES
1. Alkan M, Yüksek H, Kol-Gürsoy Ö, Calapoğlu M. Synthe-
sis, Acidity and Antioxidant Properties of Some Novel
3,4-disubstituted-4,5-dihydro-1H-1,2,4-triazol-5-one
Derivatives, Molecules, 2008; 13: 107-121.
2. Oyaizu M. Studies on products of browning reaction
prepared from glucosamine, Japan Nutri. 1986; 44: 307-
316.
3. Blois MS. Antioxidant determinations by the use of a
stable free radical, Nature, 1958; 26: 1199-1200.
4. Dinis TCP, Madeira VMC, Almeida LM. Action of phenolic
derivatives (acetaminophen, salicylate, and 5-aminosali-
cylate) as inhibitors of membrane lipid peroxidation and
as peroxyl radical scavengers, Arch Biochem Biophys.
1994; 315: 161-169.
200 5. Gordon MH. Food Antioxidants, Elsevier: London-New
York, 1990; pp. 1-18.
P097-BIOLOGICAL SCREENING OF THE NOVEL
AMINO ACID UREIDOAMIDES
Ivana Perkovic1, Ivan Butula1, Zrinka Rajic1, Dimitra
Hadjipavlou-Litina2, Eleni Pontiki2, Branka Zorc1
Department of Medicinal Chemistry, Faculty of Pharmacy and
Biochemistry, University of Zagreb, 10000, Zagreb/Croatia.1
Department of Pharmaceutical Chemistry, School of
Pharmacy, Aristotles University of Thessaloniki, 54124,
Thessaloniki/Greece.2
INTRODUCTION
N-hydroxyureas previously synthesized by our group,
showed a significant antiviral and cytostatic activity1,2. There-
fore, we have prepared a series of analogue compounds, new
ureidoamides 4a-o, with hydroxy group detached from urea
nitrogen with two, three or five methylene spacers (Figure).
Here we report their biological screening.
3rd BBBB International Conference on Pharmaceutical Sciences
Figure: Ureidoamides 4a-o
MATERIALS AND METHODS
Each experiment in vitro was performed at least in tripli-
cate and the standard deviation of absorbance was less
than 10% of the mean. 1,1-Diphenyl-picrylhydrazyl (DPPH),
2,2’-azobis(2-amidinopropane) dihydro-chloride (AAPH),
nordihydroguaiaretic acid (NDGA), sodium linoleate, soy-
bean lipoxygenase (LO), caffeic acid and trolox were pur-
chased from Sigma-Aldrich (USA).
LO Inhibition Activity3
DMSO solution of the tested compound was incubated with
0.1 mmol/L sodium linoleate and 0.2 mL of LO solution (1/9
× 10-4 w/v in saline) at room temperature. The conversion
of sodium linoleate to 13-hydroperoxylinoleic acid was re-
corded at 234 nm and compared with the standard inhibitor
caffeic acid.
Interaction with DPPH Activity3
To a solution of 0.05 mmol/L DPPH in absolute ethanol an
equal volume of 0.05 or 0.1 mmol/L ethanolic solution of the
tested compound was added. After 20 and 60 min the ab-
sorbance was recorded at 517 nm and compared with the
appropriate standard NDGA. Ethanol was used as a control.
Inhibition of Linoleic Acid Lipid Peroxidation4
Ten microliters of 16 mmol/L linoleic acid dispersion was
added to the UV cuvette containing 0.93 mL of 0.05 mmol/L
phosphate buffer, pH 7.4 prethermostated at 37°C. The oxida-
tion reaction was initiated at 37°C under air by the addition
of 50 μL of 40 mmol/L AAPH solution. Oxidation was carried
out in the presence of the tested compounds (10 μL, final
concentration 0.1 mmol/L). In the assay with no antioxidant,
lipid peroxidation was measured in the presence of the same
level of DMSO. The rate of oxidation was monitored at 37°C
by recording the increase of absorption at 234 nm caused
by conjugated diene hydroperoxides. The results were com-
pared to the standard inhibitor trolox.
Cytostatic Activity Assays
Cytostatic activity against 5 human cell lines, which are de-
rived from 4 cancer types, was measured as described previ-
ously (1, 2). The following cell lines were used: MCF-7 (breast
carcinoma), SW 620 (colon carcinoma), HCT 116 (colon car-
cinoma), MOLT-4 (acute lymphoblastic leukemia) and H 460
(lung carcinoma).
 POSTER PRESENTATIONS
Antimicrobial Activity
Microbial species (Bacillus subtilis ATCC 6633, Staphylococcus
aureus ATCC 25923, Escherichia coli ATCC 10536, Pseudomo-
nas aeruginosa ATCC 27853, Candida albicans ATCC 10231
and Aspergilus niger ATCC 16404) used in the study were
obtained from the American Type Culture Collection (LGC
Promochem, UK). Trypticasa soy agar and Müller-Hinton
agar were purchased from Merck (Germany), Sabouraud 2%
(m/V)-glucose agar from BBL (Germany), amphotericin B
from Sigma (USA) and norfloxacin from Krka (Slovenia). An-
timicrobial activity was screened according to the method
previously described5.
RESULTS AND DISCUSSION
Ureidoamides 4a-o were screened for their antioxidative, an-
timicrobial and cytostatic activities. The results have shown
that biological activity of the tested compounds was rather
modest.
Inhibition of LO was determined by the UV absorbance
based enzyme assay, while antioxidant activity was studied
by monitoring the interaction of 4a-o with stable free radi-
cal DPPH and by inhibition of lipid peroxidation (oxidative
changes of linoleic acid to conjugated diene hydroperoxide).
Ureidoamides 4d, 4g and 4k exerted the strongest LO inhi-
bition (IC50 = 92.5, 88 and 98 μmol/L, respectively). On the
other hand, interaction of 4a-o with DPPH was very low. The
best inhibitors of lipid peroxidation were ureidoamides 4c
and 4m (94.6 and 87.4%).
Evaluation of cytostatic activities against malignant tumor
cell lines showed that ureidoamides 4a-o possess very weak
antiproliferative activity (data not shown), significantly lower
than the ureidoamides with hydroxy group directly attached
to urea nitrogen atom1,2.
Results of the microbiological screening using hole-plate
diffusion method5 revealed that ureidoamides 4a-o did
not show inhibition zones of growth, and were considered
inactive in concentration used (20 mg/mL)). The minimal
inhibitory (MIC) and the minimal microbicidal concentra-
tions (MMcC) were determined by the microdilution broth
method. MIC/MMcC values were high. The strongest activ-
ity was exerted towards Pseudomonas aeruginosa (1.25/2.5
mg/mL).
REFERENCES
1. Opačić N, Barbarić M, Zorc B, Cetina M, Nagl A et al. The
novel L- and D-amino acid derivatives of hydroxyurea
and hydantoins: synthesis, X-ray crystal structure study,
and cytostatic and antiviral activity evaluations, J Med
Chem. 2005; 48: 475-482.
2. Perković I, Butula I, Zorc B, Hock K, Kraljević Pavelić S et
al. Novel lipophilic hydroxyurea derivatives: synthesis,
cytostatic and antiviral activity evaluations, Chem Biol
Drug Des. 2008; 71: 546-553.
3. Pontiki E, Hadjipavlou-Litina D, Synthesis and pharmaco-
chemical evaluation of novel aryl-acetic acid inhibitors
of lipoxygenase, antioxidants, and anti-inflammatory
agents, Bioorg Med Chem. 2007; 15: 5819-5827.
4. Re ER, Pellegrini N, Proteggente A, Pannala A, Yang M,
October 26 - 28, 2009 / Antalya - TURKEY
Rice-Evans C, Antioxidant activity applying an improved
ABTS radical cation decolorization assay, Free Rad Biol
Med. 1999; 26: 1231-1237.
5. European Pharmacopoeia, ed. 5, Council of Europe,
Strasbourg, 2006.
p098-RP-HPLC DETERMINATION OF
LIPOPHILICITY IN SERIES OF COUMARIN
DERIVATIVES
A. Uzunović1, E. Vranić2, S. Muratović3, A. Atilla Hıncal 4
1
Institute for Quality Control of Medicines, M. Tita 9, 71 000
Sarajevo, Bosnia and Herzegovina
2
Department of Pharmaceutical Technology, Faculty of
Pharmacy, University of Sarajevo, Čekaluša 90, 71000 Sarajevo,
Bosnia and Herzegovina
3
Department of Pharmaceutical Chemistry, Faculty of
Pharmacy, University of Sarajevo, Čekaluša 90, 71000 Sarajevo,
Bosnia and Herzegovina
4
Department of Pharmaceutical Technology, Faculty of
Pharmacy, Hacettepe University, 06100 Sıhhiye-Ankara/Turkey
INTRODUCTION
4-hydroxycoumarin derivatives are of interest because of
their anticoagulant, spasmolytic, and rodenticidal activity.
Some coumarin derivatives also possess antifungal and anti-
HIV activity1,2.
201
Lipophilicity belongs to basic physico-chemical characteris-
tics of a novel chemical entity. In biological systems lipophi-
licity mostly determines the solubility of drugs in biological
fluids and penetration through the biological membranes.
Determination of lipophilicity at the early stage of develop-
ment can significantly limit the problems with poor ADME
properties of novel drug candidates. The basic experimental
method for determination of lipophilicity is performed us-
ing traditional shake-flask procedure with subsequent de-
termination of the concentrations of the compound in both
phases. Although different solvents were investigated for
this purpose, octanol–water system remains the most popu-
lar model. Using this approach lipophilicity of a compound is
expressed as a logarithm of partitioning coefficient3.
The aim of this study was to develop fast gradient HPLC
method to determine the lipophilicity of series of coumarins
(3,3’-(benzilidene) bis (4-hydroxycoumarin) and its deri-
vates).
In addition these parameters (retention times and logarithm
of capacity factors (log k) were compared with log P values
estimated using different software products.
MATERIALS and METHODS
Analyzed compounds, 3,3’-(benzilidene) bis (4-hydroxycou-
marin) and its derivates were synthesized in-house at the
Faculty of Pharmacy, University of Sarajevo (Table 1.).
 POSTER PRESENTATIONS
Table 1: Chemical structure of of 3,3’-(benzilidene) bis (4-hy-
droxycoumarin) and its derivates
Acetonitrile, methanol, potassium dihydrogen phosphate
and ortho-phosphoric acid 85% were purchased from Mer-
ck KGaA (Darmstadt, Germany). The water was prepared
by Milli-Q® Ultra-pure Water Purification System (Millipore).
In this study, we develop a gradient reversed-phase HPLC-
DAD method for studying series of 4-hydroxycoumarin de-
rivatives with similar chemical structures4. Optimization of
original isocratic HPLC method was performed using excel
program called ‘‘HPLC calculator v2.1’’5. A total of 22 differ-
ent samples were prepared by dissolving about 2 mg of each
202
sample in 10 ml of methanol. The analyses of all compounds
were carried out on Zorbax StableBond-C18 column (250 ×
4.6mm, 5 μm).
Prepared samples were analysed using a Shimadzu LC10-
Avp system, equipped with a DAD detector, an auto-injector
(10μl), at 350C and 1.5 ml min-1 flow rate. HPLC separation was
performed using mobile phase A (acetonitrile) and mobile
phase B (phosphate buffer, 10 mM, pH 3.0) in a gradient pro-
gram (A; 1.5-37 min: 40-80% A; 37-39 min: 80-40% 39-50 min:
hold at 40%). The samples were monitored at 280 nm.The
theoretical lipophilic parameters (log P) for all compounds
were calculated employing the chemical programs (Chem
Draw Ultra 8.03 (www.cambridgesoft.com),ClogP (www.bio-
byte.com) and ACD/logP (www.acdlabs.com)).
RESULTS AND DISCUSSION
Under the developed HPLC conditions, retention times
ranged from 4.28 min to 23.88 min and the peaks asymmetry
were in the range 0.93-1.28. Calculated log P values were in
the range 1.07-4.89.
Levels of correlation between retention times and log P val-
ues calculated by chemical softwares (Chem Draw Ultra 8.03,
ClogP and ACD/logP) are provided in Figure 1.
3rd BBBB International Conference on Pharmaceutical Sciences
Figure 1: Correlation of retention time values obtained by HPLC
and log P values calculated by various software products
Figure 2: Correlation of log k and log P values calculated by
various software products
Levels of correlation between log k and log P values calcu-
lated by chemical softwares (Chem Draw Ultra 8.03, ClogP
and ACD/logP) are provided in Figure 2.
According to the results obtained in this study, the measure-
ment of capacity may provide an insight whether retention
shifts are caused by the column (capacity factor is changing
with retention time changes) or by the system (capacity fac-
tor remains constant with retention time changes). In our
case, capacity factor is changing with retention time chang-
es, what is indicated in similarities of the obtained R2 values
(see Figure 1. and compare to Figure 2.)By the application of
C log P (www.biobyte.com) and ACD / log P (www.acdlabs.
com) programs similar results for log P of 4-3,3’-(benzilidene)
bis (4-hydroxycoumarin) derivatives were obtained (log P
values were in range from 2.43 to 4.89) in regard to Chem
Draw Ultra 8.03 (www.cambridgesoft.com) (log P values
were in range from 1.07 to 2.99).
CONCLUSION
Established HPLC method was suitable for investigation of
4-3,3’-(benzilidene) bis (4-hydroxycoumarin) derivatives
with similar chemical structures.
all three calculational methods showed higher lipophylicity val-
ues which were caused by the increasing of the retention time.
similar correlation results (based on the coefficient of deter-
mination - R2) were obtained comparing retention time and
 POSTER PRESENTATIONS
log k in regard to log P of the tested substances.
REFERENCES
1. Manolov I., Maichle-Moessmer C., et al. Synthesis, structure, toxi-
cological and pharmacological investigations of 4-hydroxy-
coumarin derivatives. Eur. J. Med. Chem. 2006; 41:882-890.
2. Mao P. C-M., Mouscadet J.-F., et al. Chemical modification
of coumarin dimer and HIV-1 integrase inhibitory activity.
Chem. Pharm. Bull. 2002; 50(12):1634-1637.
3. Valkó K. Application of high-performance liquid chroma-
tography based measurements of lipophilicity to model
biological distribution. J. Chromatogr. A. 2004; 1037 (1-
2):299–310.
4. Adamowicz P., Kała M. Simple HPLC method for the identifi-
cation of the most commonly used rodenticides in Poland.
Probl. Forens. Sci. 2005; 64:373-381.
5. Guillarme D., Nguyen D. T. T., Rudaz S., Veuthey J. L. Method
transfer for fast liquid chromatography in pharmaceutical
analysis: Application to short columns packed wit small
particle. Part I: Isocratic separation. Eur. J. Pharm. Sci.
2007; 66: 475–482.
p099-DESIGN, SYNTHESIS AND BIOLOGICAL
EVALUATION OF NEW ANALOGUES OF
PHENYL ETHYL AND PHENOXY METHYL
OXADIAZOL AS SELECTIVE INHIBITORS OF
CYCLOOXYGENASE-2 ENZYME
Hoda Abolhasani1, Afshin Zarghi2, Ahmad Abolhasani3
Student Research Committee, Tabriz University Of Medical
Sciences,Tabriz,Iran1
Shaheed Beheshti University of Medical Sciences,Tehran,Iran2
Tarbiat Modares university,Tehran,Iran3
INTRODUCTION
Non-steroidal anti-inflammatory drugs (NSAIDs) that use for
the treatment of inflammatory and pain and fever is often ac-
companied by adverse gastrointestinal and renal side effects.
Their anti-inflammatory activity results from inhibition of cy-
clooxygenases (COXs), which catalyzes the bioconversion of
arachidonic acid to prostaglandins1,2. Nowadays, it is well
established that there are at least two COX isozymes, COX-1
and COX-2. More recently, COX-2 over expression has been
demonstrated in several types of cancer, in angiogenesis and
in neurodegenerative diseases such as Alzheimer’s or Parkin-
son’s3. Cyclooxygenase inhibitors such as rofecoxib and cele-
coxib which selectively inhibit the inducible COX-2 isozyme
that causes inflammation, rather than the constitutive COX-1
isozyme that provides gastro protection and maintains vascu-
lar homeostasis, are clinically effective nonulcerogenic anti-in-
flammatory drugs, however, a precautionary concern regard-
ing the use of selective COX-2 inhibitors in patients at risk for
an adverse cardiovascular event such as myocardial infarction
has been raised4. The adverse cardiovascular effects of rofe-
coxib and valdecoxib appear to be due to their high COX-2
selectivity5. Accordingly there is still a need for the design of
COX-2 inhibitors with a greater safe profile for the treatment
October 26 - 28, 2009 / Antalya - TURKEY
of arthritis. In this study, we describe the design and synthesis
of a new series of 2-alkylsulfonyl-5-(phenyl ethyl or phenoxy
methyl)-1, 3, 4-oxadiazoles as selective COX-2 inhibitors.
MATERIALS AND METHODS
Chemistry
The synthesis of , 2-alkylsulfonyl-5-(phenyl ethyl)-1,3,4-oxadi-
azoles was started from phenyl propionic acid and the synthe-
sis of , 2-alkylsulfonyl-5-( phenoxy methyl)-1,3,4-oxadiazoles
was started from phenoxy acetic acid. Esterification of the acid
by methanol using acidic condition gave appropriate ester.
Reaction of the ester with hydrazine hydrate in absolute etha-
nol gave phenyl propionic acid hydrazide. The corresponding
hydrazide was converted to 2-mercapto-5-(phenyl ethyl or
phenoxy methyl)-1, 3, 4-oxadiazole after dissolved in metha-
nol and using carbon disulfide under alkaline condition. Alky-
lation of this compound using methyl and ethyl iodide and
propyl bromide in alkaline media and then oxidation by ox-
one gave 2-methyl or 2-ethyl or 2-propyl sulfone-5-(phenyl
ethyl or phenoxy methyl )-1,3,4-oxadiazole.
Molecular modeling study
Molecular modeling study shows that the SO2Me pharma-
cophore in selective COX-2 inhibitors induces COX-2 selectiv-
ity by insertion in to the secondary pocket of COX-2, which is
absent in the COX-1 isozyme and head of two aromatic rings
had an ability to match with COX-2 active site. Accordingly,
conformational analysis of the synthesized compounds and
rofecoxib were preliminarily performed by molecular me-
chanic methods implemented in Hyper hem software. The
conformers were optimized further by AM1 calculation and 203
global minimum were obtained by rigid rotation. Docking
experiments were performed using Auto dock software.
RESULTS & DISCUSSION
A series of new 2-alkylsulfonyl-5-(phenyl ethyl or phenoxy
methyl)-1, 3, 4 oxadiazoles was designed and synthesized
as selective cyclooxygenase-2 inhibitors. Molecular model-
ing and docking study of designed molecule that contained
methyl sulfone at 2 position of 1, 3, 4-oxadiazole ring, in the
active site of COX-2 enzyme revealed that these compounds
can bind to secondary pocket of enzyme. So we can expect
COX-2 inhibitory activity of these compounds. The purity of
synthesized compounds was tested by thin layer chroma-
tography using several solvents with different polarity. The
compounds were stable and crystalline powder and the end
products were oily. Several parameters such as media of re-
action, time and temperature of reaction and work up condi-
tion were important to increase the yield of reactions. The
yield of reactions ranged from 75% to 94% in several stages
of reaction. The structure of synthesized compounds was
confirmed by H nuclear magnetic resonance, infrared and
mass spectrometry. In vitro cyclooxygenase (COX) inhibition
assay, which was determined using an enzyme immunoas-
say (EIA) kit (Catalog No. 560101, Cayman Chemical, Ann Ar-
bor, MI, USA), exhibit selective COX-2 inhibitory activity for
most of the test compounds.
 POSTER PRESENTATIONS
Figure 1: The synthesis of designed compounds
204
Figure 2: Some representative selective COX-2 inhibitors
Figure 3: The test compounds were superimposed on corre-
sponding conformer of rofecoxib molecule which was con-
sidered as a reference COX-2 selective inhibitor
3rd BBBB International Conference on Pharmaceutical Sciences
Figure 4: Docking of designed molecule in active site of COX-
2 enzyme
Figure 5: COX-1 & COX-2 receptor
ACKNOWLEDGEMENTS
We are grateful to Iran Islamic Azad University of medical sci-
ence for providing laboratory facilities of this research.
REFERENCES
1. Zarghi A, Rao P, Knaus E. Sulfonamido, Azidosulfonyl and N-
acetylsulfonamido analogues of rofecoxib, Bioorganic Med
Chem Lett. 2004; 14: 1957-1960.
2. Zarghi A, Rao P, Knaus E. Synthesis and biological evaluation of
methanesulfonamide analogues of rofecoxib: Replacement
of methanesulfonyl by methanesulfonamido decreases cy-
clooxygenase-2 selectivity, Bioorganic & Medicinal Chemistry.
2007; 15: 1056–1061.
3. Zarghi A, Arfaee S, Rao P, Knaus E. Design, Synthesis and bio-
logical evaluation of 1,3-diarylprop-2-en-1-ones : A novel class
of cyclooxygenase-2 Inhibitors , Bioorganic Med Chem. 2006;
4: 2600-2605.
4. Zebardas T, Zarghi A, , Daraie B Hedayati M,G. Dadrass O. De-
sign and synthesis of 3-alkyl-2-aryl-1,3-thiazinan-4-one de-
rivatives as selective cyclooxygenase (COX-2) inhibitors, Bioor-
ganic & Medicinal Chemistry Letters. 2009; 19: 3162–3165.
5. Zarghi A, Najafnia L, Daraee B, G. Dadrassc O ,Hedayati M. Syn-
thesis of 2,3-diaryl-1,3-thiazolidine-4-one derivatives as selec-
tive cyclooxygenase (COX-2) inhibitors, Bioorganic & Medici-
nal Chemistry Letters. 2007; 17: 5634–5637.
 POSTER PRESENTATIONS

p100-SYNTHESIS OF CONFORMATIONALLY
CONSTRAINED TRICYCLIC Β-LACTAM
ENANTIOMERS THROUGH UGI-4C-3C
REACTIONS OF A MONOTERPENE-BASED
Β-AMINO ACID

Zsolt Szakonyi 1, Reijo Sillanpää 2, Ferenc Fülöp 1

Institute of Pharmaceutical Chemistry, University of Szeged,

H-6720 Szeged, Eötvös utca 6, Hungary1
Department of Chemistry, University of Jyväskylä, POB 35,
40351 Jyväskylä, Finland2
INTRODUCTION
Intramolecular Ugi reactions (U-4C-3CR and U-5C-4CR) in
which the amino acids used as starting materials contain
two functional groups on the same compound may furnish
a wide range of heterocyclic compounds1. This procedure is
an efficient tool for the preparation of β-lactams, which are
versatile building blocks for stereoselective syntheses. In the
recent years, both racemic and enantiopure β-amino acids
have proved to be excellent building blocks in the synthesis
of saturated or partially saturated heterocycles, β-peptide
oligomers and biologically active peptidomimetics2. Mono-
and bicyclic β-amino acids have recently been utilized in Ugi
reactions in organic solvents and in water, respectively3.
Our present aim was to prepare an apopinane-based
β-lactam library in methanol, water or under solvent free
condition in order to study the driving force of the confor-
mationally constrained pinane system on the yields, reaction
time and diastereoselectivity.
Scheme 1: Possible reaction mechanism for the synthesis of
β-lactams 2-6.
The configurations of the major diastereoisomers (2a-6a)
were determined by X-ray measurements.
The Ugi reaction was also attempted in water to compare
the effects of water with those of MeOH.
Under the conditions applied, the yields were comparable
only in the cases of pivalaldehyde and propionaldehyde,
while the reaction times were shorter and the diastereose-
lectivities of the reactions were similar in water relative to
MeOH. The results clearly demonstrate that the factor limit-
ing the application of water as solvent is the water solubility
of the Schiff base formed in the first step of the Ugi reaction.
These condesations were also carried out under solvent-free
conditions, but, similarly as in water, comparable yields were
observed only for pivalaldehyde and propionaldehyde. 205

MATERIALS AND METHODS

(1R,2R,3S,5R)-2-Amino-6,6-dimethylbi¬cyclo[3.1.1]heptane-
3-carboxylic acid 1 was prepared from (-)-(1S,5S)-apopinene
by a literature method4.

RESULTS AND DISCUSSION

(1R,2R,3S,4R)-Amino acid 1 was reacted with three alde-
hydes and two isocyanides in methanol, in water and under
solvent-free conditions to prepare enantiomeric β-lactams
via U-4C-3CRs. The building blocks of the Ugi reactions are
depicted in Figure 1. The yields in methanol were found to
be from acceptable to good. High diastereoselectivities of Figure 1: Building blocks of the U-4C-3CRs.
the reactions were observed, and the diastereoisomers were
successfully separated in most cases. Table 1: β-lactam derivatives 2-6 with diverse substituents
via Scheme 1.
A possible mechanism of β-lactam ring generation is presented Product R1 R2
in Scheme 1. The first step is the condensation of the aldehyde
2 tBu tBu
with the amino acid, resulting in the Schiff base, followed by the
formation of a seven-membered ring intermediate to give the 3 tBu CH2Ph
β-lactam derivative via a ring-contraction step (Scheme 1). 4 Ph rBu
5 Ph CH2Ph
6 Et tBu

October 26 - 28, 2009 / Antalya - TURKEY

 POSTER PRESENTATIONS
CONCLUSION
In conlusion, we found that the Ugi-4C-3CR is an efficient
method for the preparation of conformationally constrai¬ned
enantiomeric β-lactams. Relative to MeOH, water as solvent
has several advantages, such as its enviromentally benign
nature and the shorter reaction time, but it is additionally
noteworthy that the reaction also accured appropiately un-
der solvent-free conditions (with some restrictions).
ACKNOWLEDGEMENTS
We are grateful to the Hungarian Research Foundation (OTKA
No. NF69316 and T04407) for financial support.
REFERENCES
1. Dömling A. Recent developments in isocyanide based
multicomponent reactions in applied chemistry. Chem
Rev 2006; 106:17–89 and references cited therein.
2. Fülöp F, Martinek TA, Tóth GK. Application of alicyclic
beta-amino acids in peptide chemistry. Chem Soc Rev
2006; 35:323–334 and references cited therein.
3. Kanizsai I, Szakonyi Z, Sillanpää R, Fülöp F. A compara-
tive study of the multicomponent Ugi reactions of an
oxabicycloheptene-based β-amino acid in water and in
methanol. Tetrahedron Lett 2006; 47:9113–9116.
4. Szakonyi Z, Fülöp F. Chiral cyclic β-amino acids and
their derivatives, pharmaceutical compositions con-
taining them and the use of such compounds. PCT: WO
2008/059299 A1.
206 p101-SYNTHESIS OF SOME 1-(2-NAPHTHYL)-
2-(PYRAZOL-1-YL)ETHANONE OXIME ETHER
DERIVATIVES AND THEIR ANTICONVULSANT
ACTIVITIES
Zeynep Bulut1, Arzu Karakurt1, Ünsal Çalış2, Sevim Dalkara2
İnönü University, Faculty of Pharmacy, Department of
Pharmaceutical Chemistry, 44280, Malatya-Turkey1
Hacettepe University, Faculty of Pharmacy, Department of
Pharmaceutical Chemistry, 06100, Ankara-Turkey2
INTRODUCTION
There is continuing need to find more effective and safer
antiepileptic drugs mainly because of inadequate treatment
with existing drugs for almost one third of the patients and
significant toxic side effects. Nafimidone, 1-(2-naphthyl)-
2-(imidazol-1-yl)ethanone, is one of the representative of
(arylalkyl)azole anticonvulsant compounds1. In our previous
studies we found that oxime ether derivatives of nafimi-
done also showed anticonvulsant activity2. It is known that
some pyrazole derivatives in different chemical structures
show anticonvulsant activities3,4. Therefore in this project,
as a continuation of our previous studies on (arylalkyl)azole
derivatives we aimed to synthesize some new oxime ether
derivatives of pyrazole analog of nafimidone as potential an-
ticonvulsant compounds and then to evaluate their anticon-
vulsant activities.
3rd BBBB International Conference on Pharmaceutical Sciences
MATERIALS AND METHODS
Chemistry
The oxime ethers1-3 were synthesized by O-alkylation of
2-(pyrazol-1-yl)-1-(naphthalene-2-yl)ethanone oxime with
appropriate alkyl halides (Scheme 1). The oxime was pre-
pared by the reaction of hydroxylamine hydrochloride and
the ketone which was obtained by N-alkylation of pyrazole
with 2-bromo-1-(naphthalene-2-yl)ethanone according to
the literature procedures2.
Preparation of the compounds
0.01 mol 2-(pyrazol-1-yl)-1-(naphthalene-2-yl)ethanone oxi-
me and 0.011 mol Nao/EtOH (5 ml) were stirred and refluxed
for 30 minutes. Ethanol was evaporated in vacuo, the residue
was dissolved in DMF and 0.02 mol appropriate alkyl halide
was added. The mixture was stirred in room temperature for
4h. and then poured into ice. The precipitate was filtered and
crystallized from the appropriate solvents.
Anticonvulsant activity
Anticonvulsant activity of the compounds were determined
by maximal electroshock seizure (MES) and subcutaneous
metrazole seizure (ScM) tests in mice and neurotoxicity of the
compounds was evaluated by rotorod test according to the
Phase I tests of NIH-Anticonvulsant Screening Programme5.
The suspension of the compounds in methylcellulose was
administered to mice by intraperitoneal route 30 min or 4
h before application of anticonvulsant and neurotoxicity
screening tests for evaluation of their activity.
RESULTS AND DISCUSSION
Chemistry
The melting points and yields of the compounds synthesized
are given in Table 1. Their chemical structures were confirmed
by IR and H1-NMR spectral and elementary analysis data.
Table 1: The structures, melting points and yields of the com-
pounds.
M.p. Yield
Comp R
(0C) (%)
1 -CH3 52-4 72
2 -(CH2)2CH3 42-4 83
3 -CH2CH=CH2 45-7 52
Scheme 1: Synthesis of the compounds
 POSTER PRESENTATIONS

2-(Pyrazol-1-yl)-1-(naphthalene-2-yl)ethanone oxime methyl C, Unverferth K. 3-Amino- and 5-Aminopryazoles with

ether (1), IR (KBr, cm-1): (N-O) 906, (C-O) 1277. 1H-NMR(CDCl3- Anticonvulsant Activity. Arch. Pharm. Med. Chem. 1999;
d ppm): 4.15 (3H; s; -CH3), 5.56 (2H; s; CH2-N), 6.25 (1H; t; 332: 219-221.
pyrazole H4), 7.28-7.58 (4H; m; naphthalene H6-7 and pyrazole 5. Kupferberg HJ, Stables JP. In Challenge epilepsy-new an-
H3,5) 7.74-7.95 (4H; m; naphthalene H3-5,8), 8.18 (1H; s; naph- ticonvulsant drugs,: Stefan H, Kramer G, Mamoli B, Ed.;
thalene H1). Anal. Found: C,  71.02; H, 5.63; N, 15.74 Calc. for Blackwell Science Ltd.: Boston 1998; pp 7-29.
C16H15N3O.1/3 H2O (271.31): C, 70.83; H, 5.82; N, 15.49 %.

2-(Pyrazol-1-yl)-1-(naphthalene-2-yl) ethanone oxime propyl  p102-Factors Affecting Compliance

ether (2), IR (KBr, cm-1):(N-O) 916, (C-O) 1276. 1H-NMR (CDCl3-
d ppm): 0.97 (3H ;t; -CH3), 1.74 (2H; m; -CH2CH3), 4.24 (2H; t;
-O-CH2), 5.57 (2H; s; CH2-N), 6.20 (1H; t; pyrazole H4), 7.37-
7.57 (3H; m; naphthalene H6-7 and pyrazole H5), 7.77 (1H; d;
pyrazole H3), 7.82-7.95 (4H; m; naphthalene H3-5,8), 8.37 (1H; s;
naphthalene H1). Anal. Found: C, 72.24; H, 6.39; N, 14.10. Calc.
for C18H19N3O.1/3 H2O (299.15): C, 72.22; H, 6.62; N, 14.04 %.
2-(Pyrazol-1-yl)-1-(naphthalene-2-yl)ethanone oxime allyl
ether (3), IR (KBr, cm-1):(N-O) 914, (C-O) 1278. 1H-NMR (CDCl3-d
ppm): 4.80 (2H; d; -CH2-O), 5.24-5.40 (2H; dd; =CH2), 5.60 (2H;
s; CH2-N), 6.08 (1H; m; -CH=CH2), 6.196 (1H; t; pyrazole H4),
7.37-7.58 (3H; m; naphthalene H6-7 and pyrazole H5), 7.38 (1H;
d; pyrazole H3), 7.52-7.59 (2H; m; naphthalene H5,8), 7.78-7.94
(2H; m; naphthalene H3-4), 8.24 (1H; s; naphthalene H1). Anal.
Found: C, 72.97; H, 5.875; N, 14.26. Calc. for C18H17N3O.1/3 H2O
(297.14): C, 72.71; H, 5.99; N, 14.13 %.
Pharmacology
Compounds 1 and 3 showed anticonvulsant activity against
MES induced seizures in mice at 300 mg kg- 1 in 4h; but unfor-
tunately these compounds were found to be neurotoxic at
the same dose level in 30 min (Table II).
Table 2: Phase I anticonvulsant screening of the synthesized
compounds.
MES
½ hour 4 hour ½ hour
Comp
among Oral Hypoglycemic Agents
(OHA) Users at a Public Hospital in
Malaysia
Geok H. Yeo1, Mudassir Anwar1, Pei S. Wong1
1School of Pharmacy and Health Sciences, International
Medical University, Kuala Lumpur, Malaysia
INTRODUCTION
Diabetes mellitus is an important public health concern and
globally there is a rising trend in its prevalence. Nearly 1.2
millions of Malaysians are diagnosed with diabetes mellitus.
Type 2 Diabetes Mellitus (T2DM) accounts for 90% of all the
cases. Non-compliance is widespread with weighty con-
sequences1. Many factors have been reported toinfluence
compliance levels andovercoming these barriers will allow-
improvement of compliance.Compliance towards oral hypo-
glycaemicagents (OHA) in T2DM was reported tobe poor to
satisfactory in previousstudies. Little has been done oncom-
pliance to specifically OHA inT2DM patients2,3. The study 207
wasdesigned with the objectives ofinvestigating the compli-
ance and factorsaffecting compliance among patientstaking
oral hypoglycemic agents (OHA)treated in a public health-
care setting.
ScM Toxicity
4 hour ½ hour 4 hour MATERIALS AND METHODS

mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg The study was conducted in HospitalTuanku Ja’afar, Ser-
30 100 300 30 100 300 30 100 300 30 100 300 30 100 300 30 100 300
emban (HTJ) byusing a validated 30-item questionnairevia
1 0/1 0/1 0/1 0/1 0/1 1/1 0/1 0/1 0/1 0/1 0/1 0/1 0/4 0/4 4/4 0/2 0/2 0/2
2 0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/4 0/4 0/4 0/2 0/2 0/2
face-to-face interview. Conveniencesampling technique
3 0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/4 0/4 4/4 0/2 0/2 0/2
was used to selectthe subjects who were then givenIinfor-
mation sheets and written consentform to fulfill the ethical
requirements.A 5-point Lickert scale was used togather the
ACKNOWLEDGEMENTS
responses. Compliance wasassessed from the mean response
This project was supported by İnönü University Research
tocertain questions. The level of agreementfor certain state-
Fund (Project number: 2009/25 thesis).
ments regarding thefactors affecting compliance wasdeter-
REFERENCES
mined by set parameters. StatisticalPackage for Social Sci-
1. Walker KAM,Wallach BM, Hirschfeld RD. 1-(Naphthylalkyl)-
ences (SPSS) version 11.5 for Windows® was used toanalyze
1H-imidazole derivatives, a new class of anticonvulsant
the data obtained. Statisticaltests were used to evaluate the
agents. J Med Chem. 1981; 24: 67-74.
differencein mean among the groups and todetermine the
2. Karakurt A, Dalkara S, Özalp M, Özbey S, Kendi E, Stables
correlation betweenvariables.hical form.
JP. Synthesis of some 1-(2-naphthyl)-2-(imidazole-1-yl)
ethanone oxime and oxime ether derivatives and their
RESULTS AND DISCUSSION
anticonvulsant and antimicrobial activities. Eur. J. Med.
A total of 89 patients were approachedbut only 75 ful-
Chem. 2001; 36: 421–433.
filled the inclusion criteriaand/or agreed to participate in
3. Siddiqui N, Alam P, Ahsan W. Design, Synthesis and In-
the studyout of which 58.7% were males and therest were
vivo Pharmacological Screening of N,3-(Substituted
females. A majority of subjectsinterviewed were Indians
Diphenyl)-5-phenyl-1H-pyrazoline-1-carbothioamide
(48.0%),followed by Malays (26.7%), Chinese(20.0%), and
Derivatives. Arch. Pharm. Chem. Life Sci. 2009; 342: 173-
other races (5.3%). Thecompliance level as reported by thep-
181.
articipants was low as only 20% of thepatients were found
4. Lankau HJ, Menzer M, Rostock A, Arnold T, Rundfeldt
to be compliant(taking more than 90% of themedications

October 26 - 28, 2009 / Antalya - TURKEY

 POSTER PRESENTATIONS
prescribed). Patients’knowledge of their disease andmedi-
cation was generally poor, 2.924 ±0.722 and 2.907 ± 1.493
respectively.The perceived healthcare providerpatientrela-
tionship and knowledge aboutdiabetic medications were
shown topositively correlate with compliance inthis study.
Other influencing factors ashighlighted by the patients dur-
inginterview included side effectsassociated with the use of
prescribedOHAs, family support, preference of lifestyle mod-
ification over takingmedications, not having the symptoms
ofdiabetes and inadequate informationgiven by the health-
care professional.
CONCLUSION
The study suggested that patients’perceptions regarding
healthcareproviders and knowledge about diabeticmedica-
tion are the importantdeterminants of compliance.Interven-
tions to improve complianceamong T2DM patients should
includeimproving the knowledge of diabeticpatients about
their medication andstrengthening healthcare provider-
patientrelationship. Similar study can be doneover a large
population in order toidentify other influencing factors so as
tocome up with the suggestions tominimise the contribu-
tion of factors thatnegatively affect the compliance.
REFERENCES
1. Kardas P. The DIACOM study(effect of DosIng frequency
of oralAntidiabetic agents on theCOMpliance and bio-
chemicalcontrol of type 2 diabetes). Diabetes,Obesity
and Metabolism2005;7:722–728.
2. Bezie Y, Molina M, Hernandez N,et. al. Therapeutic com-
208 pliance: aprospective analysis of variousfactors involved
in the adherencerate in type 2 diabetes. DiabetesMetab-
olism 2006;32: 611 – 616.
3. Donnan P, MacDonald T, Morrist A.Adherence to pre-
scribed oralhypoglycaemic medication in apopulation
of patients with Type 2diabetes: a retrospective study.
Diabetes Medicine 2002;19:279–284.
p103-COUNTERFEIT MEDICINES – GLOBAL
PROBLEM AND CHALENGE
Arlinda Haxhiu1, Suzana Trajkovic - Jolevska2
Drug Quality Control Laboratory, Kosovo Medicines Agency,
Prishtine, Kosovo1
Department of Drug Quality Control, Faculty of Pharmacy,
Skopje, Macedonia2
INTRODUCTION
Counterfeit medicines are a global public health problem,
which is constantly growing and it present the serious threat
to human lives. Counterfeit medicines distinguish from oth-
er counterfeit products because they can harm patients and
sometimes it can lead to death as a result of incorrect dose
received, presence of toxic ingredients in the formulation,
lack of quality control and inability to monitor adverse reac-
tions.
3rd BBBB International Conference on Pharmaceutical Sciences
The aim of this study is to summarize relevant legislation,
case law and doctrine for counterfeit medicines and to com-
pare the national strategy and efforts of some countries on
combating counterfeit medicines.
Definition; Extent Of The Problem
The absence of common definition on counterfeit medicines
leads to the current difficulty in preventing medicines from
counterfeiting, inconsistencies in reporting cases of coun-
terfeit and difficulties in exchange of information between
countries.
WHO defines counterfeit medicines as :
“Deliberately and fraudulently mislabeled with respect to
identity and/or source. Counterfeiting can apply to both
branded and generic products. Counterfeit products may
include products with the correct ingredients or with the
wrong ingredients, without active ingredients, with insuffi-
cient active ingredients or with fake packaging”.
WHO identify the counterfeit as a problem in the early 1980’s
and since then it started to collect data, on voluntary ba-
sis, and to document them. In the period 1999-2000, WHO
received 46 confidential reports from 20 countries, 60% of
cases were in developing countries and 40% in developed
countries.
Assessment for the counterfeit occurrence is done by differ-
ent professional organization at European and international
level, pharmaceutical companies, regulatory authorities,
ad hoc studies on specific geographical areas or therapeu-
tic groups. According to the collected data on counterfeit
medicines (2002 until 2007) by the Pharmaceutical Security
Institute, (Fig.1), it is evident that the seizures of counterfeit
medicines in general are increased1.
Most of counterfeit are medicines with a high cost and for
treatment of serious chronic diseases which means that it is
a business of a large turn of money and a large profit. The
sale of fake medicines is estimated to increase up to 92%
between 2005 and 2010 and would reach $75 billion, glob-
ally2.
WHO analysis shows that industrialized countries with an
effective regulatory systems (EU, USA, Australia, Canada,
New Zealand and Japan) have a very low level of counterfeit
medicines (less than 1% of the market value), while many de-
veloping countries of Africa, parts of Latin America and Asia
have areas where counterfeit constitutes more than 30% of
the medicines on sale. Over 50% of the medicines purchased
over the Internet from illegal sites that camouflage their
physical address, are counterfeit.
Types of medicines affected by counterfeiting
The Counterfeit medicines survey report conducted by the
Council of Europe in 2003/2004, identify the following types
of medicines as a risk from counterfeiting: high level of pre-
scribing, high price, well known brand especially blockbust-
ers, lifestyle drugs, parenterals (in developing countries), all
generics3.
 POSTER PRESENTATIONS
Factors facilitating counterfeiting
There are several factors that facilitate counterfeiting: lack
of legislation; weak penal sanction; corruption and conflicts
of interest at Drug Regulatory Authorities (may affect the
handling of counterfeiting cases); inefficient cooperation
between stakeholders (DRA, custom services, police); lack of
political will (some authorities are not prepared to recognize
the existence of a counterfeit as a problem); lack of aware-
ness among health professionals and patients.
Combating counterfeit medicines
Combating counterfeit medicines depends mostly of the
legislation of a country. WHO and FIP (International Phar-
maceutical Federation) issued the guidelines on combating
and checklist for visual inspection of medicines in order to
recognize counterfeiting. MHRA (Medicines and Healthcare
products Regulatory Agency) prepared anti counterfeiting
strategy and guideline. General recommendation for buying
medicines over the Internet is to buy only from secure health
sites that have a certificate (in the USA it is VIPPS certifica-
tion, Verified Internet Pharmacy Practice Sites).
In order to increase the awareness and action against coun-
terfeit medicines, WHO has established the first global initia-
tive – IMPACT (International Medical Products Anti – Coun-
terfeiting Taskforce), approved with the Declaration of Rome
in 20064.
CONCLUSION
Counterfeit medicines are a global problem which is in the
expansion and threat patients’ health. Due to that it is nec-
essary to create and adopt the anti-counterfeiting strategy
in national and global level and prepare the guidance for
health professionals and the patients, as well. As a model of
national strategy, the MHRA-anti counterfeiting strategy and
guideline could be used.
REFERENCES
1. PSI-Pharmaceutical Security Institute http://www.psi-
inc.org/index.cfm
2. http://www.ocregister.com/articles/drug-counterfeit-
drugs-2089980-law-tracking
3. Counterfeit medicines-Survey report; Council of Europe
Publishing, 2006
4. Counterfeit drugs kill!-IMPACT/WHO brochure-updated
May 2008
Figure 1: Total number of incidents by year CY 2002 – CY
2007 source: PSI
October 26 - 28, 2009 / Antalya - TURKEY
p104-THE ROLE OF STUDENTS’ SCIENTIFIC
SOCIETY BY ORGANIZE OF STUDENT
RESEARCH WORK
Toivo Hinrikus1, Ain Raal1
University of Tartu, Tartu, Estonia1
INTRODUCTION
Many of current teachers and researchers at the University of
Tartu (UT) have participated as students or their supervisors
in the work of Students’ Scientific Society (SSS). The Society
was established 61 years ago, in 1948 and it organized stu-
dents’ scientific work until the Soviet Union collapsed and
Estonia re-gained independence at the beginning of 1990s.
MATERIAL AND METHODS
This research is based on the conference programs of the UT
SSS Pharmacy Unit, programs of meetings, several overviews
of SSS work written by students during different periods. We
also studied different materials and photos etc. at the Library
of the Department of Pharmacy and University archives.
RESULTS AND DISCUSSION
There was a special SSS unit at every department of the UT.
Each SSS unit had a scientific supervisor from among uni-
versity teachers and a leader chosen by students. The work
of SSS units was coordinated by Department’s SSS Council,
which was coordinated by the University SSS Council. Activ-
ists from different higher schools formed national SSS Coun- 209
cil, the representatives of that formed SSS Council of the
whole Soviet Union.
According to statutes, SSS was a voluntary scientific student
organization that acted in collaboration with the Rector’s
Office, the UT Council and public organizations. In yearly re-
ports of departments, there was a topic about department’s
work concerning SSS. In principle, that obligated depart-
ments to take systematic action in this field.
It is mentioned in the statutes introduced in 1979 that the
SSS contributes to the implementation the Communist Party
of the Soviet Union program and the acquisition Marxist-Le-
ninist methodology of scientific research. On the other hand,
the statutes emphasized the need to deepen and broaden
students’ professional knowledge and horizons, raise their
sense of duty and love to work, which in the reality turned
out to be the main content of the SSS.
There were associations similar to the SSS also in other soviet
countries, but for example in Hungary, its work was organized
by the Academy of Sciences. In the Soviet Union, the work on
the SSS was organized by the Ministry of Higher Education
and was subject to the Communist Party ideology. This can
be seen for example in the design of the certificates of honor
for the SSS competition works.
Those days scientific research was not in the curriculum and
the SSS was the only possibility for students to do research.
 POSTER PRESENTATIONS
The work of the UT SSS Pharmacy Unit was not only limited
to scientific research. The unit regularly organized expedi-
tions to collect Estonian medicinal plants and identify their
distribution areas. It was a tradition to have a field trip to the
Moscow All-union Research Institute of Medicinal Plants.
The number of research work was relatively small in the first
years of the SSS: during 1948-1953 there were averagely 3 con-
ference presentations per year. By the year 1963, the number
went up to 10. In following years the number of conference
presentations dropped again. The growth can be seen again
since 1976 (9 research works presented on the Pharmacy Unit
conference). In 30 years – during 1948–1977 – the Pharmacy
Unit produced 19 competition projects and 135 conference
presentations. The most successful were the 1980s, when the
number of presentations in the yearly SSS conferences was 12
to 16. During 1978-1987, there were 127 conference presenta-
tions and 19 competition projects. It has to be added that the
number of course participants was rather small. On each of
five courses there were about 25 students – in the whole Phar-
macy Department about 125 students altogether.
The research work was mainly supervised by the teaching
staff of the Department of Pharmacy. One research project
was often supervised by several teachers of different fields of
pharmacy. In reality the work of the SSS reflected the depart-
ments’ scientific work, based on teachers’ research work and
collaboration with other departments, institutes and also
practical institutions (such as control analysis laboratories,
pharmacy factory etc.). The SSS had the closest collaboration
210 with the Department of Microbiology. There was also some
research work supervised in collaboration with the teaching
staff of pharmacology and physiology. A good result of co-
operation between students and teachers is publishing the
research. Students with good experimenter qualities were
occupying them with contract works. Presentations by our
students can be found also in the conference programs of
other SSS units in the Faculty of Medicine.
In the field of pharmacy, a major achievement was introduc-
ing only experimental research work into the conference pro-
gram in 1980s. The Faculty of Medicine as a whole reached
this level little bit later – in the academic year 1989/90 the
SSS information declares that since then they do not deal
with only theoretical work anymore.
The programs of the SSS conferences were printed both in
Estonian and in Russian, which clearly indicated that we were
kept an eye on. In the 1980s several guest students from oth-
er soviet countries made presentations in our conference.
Also our students had the opportunity to participate similar
events in Leningrad, Riga, Minsk etc. Also pharmacists work-
ing in pharmacies and control analysis laboratories in Tartu
were often interested in the Pharmacy Unit conferences. In
addition to the conference that took place once a year, there
were also meetings with presentations.
The most successful research works were presented to na-
tional and all-union competitions. Gained experiences came
in handy and were worth the effort. Here students often got
the bug to do science for life.
3rd BBBB International Conference on Pharmaceutical Sciences
The end of the SSS organization did not of course mean the
end of student science. Student science is fostered in profes-
sional departments. It is also written in the statutes of several
student corporations and unions; there are national compe-
titions of student research work etc.
CONCLUSION
Analyzing the work of the SSS Pharmacy Unit, it became evi-
dent that all teaching staff from that period and the period
right after came from this organization. Their period of stu-
dent science was followed by independent research work
and supervising student research. That tendency was not
only significant to the active members of the Pharmacy Unit.
Contrary to many condemnable soviet traditions, the SSS
has to be highlighted for its positive role as the hotbed for
young teaching staff and scientists.
p105-THE STUDY OF STATINS CONSUMPTION
IN CROATIA
Milena Jadrijevic-Mladar Takac1, Tina Perisin2, Pero
Draganic3, Sinisa Tomic3, Vedran Takac4
Faculty of Pharmacy and Biochemistry, University of Zagreb1
Faculty of Pharmacy and Biochemistry, University of Zagreb,
Zagreb, Croatia2
Agency for Medicinal Products and Medical Devices of the
Republic of Croatia, Zagreb, Croatia3
PharmaSwiss Ltd. Croatia4
INTRODUCTION
The normalization of blood lipid profile has become an accept-
ed therapeutic measure of primary and secondary prevention
of vascular disease, with statins being the most popular group
of medicines prescribed to lower cholesterol and LDL choles-
terol levels. Statins are the HMC-CoA reductase inhibitors and
the most important group of lipid modifying agents (ATC C10).
In order to obtain the general insight of statins consump-
tion trend in Croatia, the study of consumption in urban
and rural surroundings in the period from 2004 to 2006 was
performed. Consumption data were compared with data for
these drugs in Norway, Finland and Denmark for the same
period (2004-2006).
MATERIAL AND METHODS
The statins consumption data were retrieved from the Croa-
tian Agency for Medicinal Products and Medical Devices
(ALMP) database. The data were extracted from annual re-
ports to ALMP for 2004, 2005 and 2006 year and were col-
lected from 44 pharmacies in total, out of which 22 were
from urban and 22 from rural surroundings. The statins con-
sumption is expressed in number of DDD/1000 inhabitants/
day (DDD – Defined daily dose). The estimation of DDD/1000
inhabitants/day was made on the basis of issued/sold pack-
ages of each statin using the following expression: DDD x
1000/population x 365. The consumption of simvastatin,
atorvastatin, fluvastatin, pravastatin and lovastatin were an-
alyzed using Farmakoekonomika – MaPIS software (Mapro
d.o.o. Croatia).
 POSTER PRESENTATIONS

RESULTS AND DISCUSSION

The results of the study showed an increase in overall con-
sumption of lipid modifying agents (ATC C10) in the period
from 2004 to 2006. In total issued drugs (DDD/1000 inhabit-
ants/day) in 2004, 2005 and 2006, the lipid modifying agents
(ATC C10) were represented with 28.13, 36.03 and 46.21
DDD/1000 inhabitants/day or 4.27, 5.26 and 6.64%, respec-
tively. (Table 1).

Table 1:The consumption of lipid modifying agents (ATC

C10), expressed in DDD/1000 inhab./day in total consump-
tion of medicines from 2004 to 2006
.
Figure 1: The percentage (%) of simvastatin and atorvastatin
Total issued C10 lipid modifying % of C10 in total consumption (DDD/1000 inhab./day) in total consumption
Year
drugs agents issued drugs of drugs (2004 to 2006)

2004 658.14 28.13 4.27 The comparative analysis of simvastatin and atorvastatin
consumption in Croatia (2004 to 2006) with consumptions
2005 684.96 36.03 5.26 in Norway, Denmark and Finland were performed. (Table 4,
Fig. 2 and Table 5, Fig. 3) The results of analysis showed that
2006 696.1 46.21 6.64 consumptions of both, simvastatin and atorvastatin, were
significantly lesser in Croatia than in European countries.

Of five statins (Table 2) issued in that period the most issued/ Table 4: The simvastatin consumption (DDD/1000 inhabit-
prescribed, in both urban and rural surroundings were sim- ants/day) in 2004, 2005 and 2006 year
vastatin and atorvastatin (more than 90%).
Year Norway Denmark Finland Croatia
Table 2: Estimated number of DDDs for statins in urban and 2004 40,6 49,6 30,86 17,81
rural surroundings (2004 to 2006)
2005 53,76 71,6 40,35 20,23 211
DDD in urban DDD in rural
Statins 2006 85,26 96 56,33 20,87
surroundings surroundings

Simvastatin 651 348.40 387 242.39

Table 5: The consumption of atorvastatin (DDD/1000 inhab-
Atorvastatin 614 110.01 404 475.00 itants/day) in 2004, 2005 and 2006 in Norway, Denmark, Fin-
land and Croatia.
Paravastatin 2 480.00 1 580.00 Year Norway Denmark Finland Croatia
Fluvastatin 195 214.42 109 647.10 2004 53,2 11 29,02 8,85
Lovastatin 3 307.08 1 360.17 2005 61,31 12,1 32,72 13,03
2006 51,04 14,5 29,22 20,73
The comparison of these two, the most prescribed statins,
atorvastatin and simvastatin (in DDD/1000 inhabitants/day)
showed a significant increase in atorvastatin consumption
from 2004 to 2006. (Table 3) The consumption of atorvasta-
tin significantly exceeded simvastatin consumption in 2006
(Fig. 1).

Table 3:The simvastatin and atorvastatin consumptions

(DDD/1000 inhabitants/day) and their percentages (%) in to-
tal drug consumption in 2004, 2005 and 2006 year

Year Simvastatin % Atorvastatin %

Figure 2: The simvastatin consumption (DDD/1000 inhabit-
2004 17.81 2.71 8.85 1.35 ants/day) in 2004, 2005 and 2006 in Norway, Denmark, Fin-
2005 20.23 2.95 13.03 1.9 land and Croatia

2006 20.87 3 20.73 2.98

October 26 - 28, 2009 / Antalya - TURKEY

 POSTER PRESENTATIONS

Figure 3: The consumption of atorvastatin (DDD/1000 inhab-

itants/day) from 2004 to 2006 in Norway, Denmark, Finland
and Croatia

CONCLUSION
The consumption trend of lipid modifying agents (ATC C10)
in Croatia is in permanent increase, and especially of statins.
The most significant consumption in the investigated period
(2004 to 2006) was obtained for simvastatin and atorvastatin.
The results of this study indicate on different degree of sta-
tins consumptions in different part of Croatia, and these dif-
ferences are especially significant between urban and rural
surroundings. The comparison of simvastatin and atorvasta-
tin consumptions data obtained for Croatia with consump-
tions in Norway, Denmark and Finland showed that statins
consumption in Croatia was significantly lesser than in other
countries, and that the consumption vary from country to
country. Additionally, more data are necessary for final con-
cludings of this research, including results of forthcoming
study for the same medicines in the period from 2007 to
2009.
212
REFERENCES
1. Reiner Z, Statins in primary and secondary prevention of
coronary diseases, Medicus. 2003; 12: 85-90.
2. Vaughan CJ, Gotto AM, Update on statins.2004; 110:886-
892.
3. Wertheimer AI, Santella TM, Problems using the defined
daily dose (DDD) as a statistical basis for drug pricing
and reimbursement, International Federation of Phar-
maceutical Manufacturers & Associations (IFPMA), 2007.

3rd BBBB International Conference on Pharmaceutical Sciences

INDEX
 INDEX

A Brynskikh, Anna  90 Erdoğan, Suna  129

Abacı, Özlem  127 Bulut, Zeynep  206 Eroğlu, Hakan  93
Abbaspour, Mohammadreza  66 Butula, Ivan  200 Eros, Istvan  64
Abolhasani, Ahmad  203 Büyüktuncel, Ebru  179 Ertan, Gökhan  62
Abolhasani, Hoda  203 Eşsiz, Dinç  88
Acartürk, Fusun  137 C
Aghel, Nasrin  155 Calafiore, Riccardo  131 F
Aigner, Zoltan  97 Calapoğlu, Mustafa  186, 194, Fallarino, Francesca  131
Akbaba, Hasan  119 197, 198 Fittler, Andras  142
Akbulut, Gonca  117 Calvitti, Mario  131 Fringuelli, Renata  126
Akça, Gülçin  106 Canefe, Kandemir  114 Fülöp, Ferenc  45
Akhgari, Abbas  92 Caproiu, Miron Teodor  184
Aksu, Buket  100, 125 Cenci, Elio  126 G
Aktay, Göknur  167 Chankvetadze, Bezhan  39 Gegu, Clement  183
Alagöz, Mehmet Abdullah 167 Chirita, Ileana Cornelia  184 Gendelman, Howard  90
Alimorad, Masoud  66 Cirotto, C.  25 Geskovski, Nikola  95
Alkan, Muzaffer  186, 194, 197, 198 Clark, Jason  33 Giovagnoli, Stefano  25, 131
Al-Qadi, S.  112 Cornett, Claus  176 Golmohammadzadeh, Shiva 149,
Amasya, Gülin  105 Crcarevska, Maja Simonoska  95 150, 151
Ambrus, Rita  73 Csaba, Gönczi  187 Goracinova, Katerina  95
Annamária, Molnár  187 Gökçe, Evren Homan  143
Antal, István  124 Ç Gönül, Nurşin  103
Anwar, Mudassir  207 Çağlar, Hatice  179 Grabnar, I.  51
Apaydın, Şebnem  140 Çalış, Sema  12, 87, 95, 183 Gryko, Romuald  32
Atak, Ayşegül  117 Çalış, Ünsal  189, 206 Günbeyaz, Merve  106
Atila, Alptuğ  85 Çapan, Yılmaz  183 Güneri, Tamer  62, 100, 122, 125,
Atkinson, Jeffrey  58 Çelebi, Benek  68 127, 135
Ay Şenyiğit, Zeynep  122 Çelebier, Mustafa  109 Güngör, Sevgi  147
Çelebi, Nevin  117 Gürsoy, R. Neslihan  116
B Çetin, Meltem  85 Gürsoy, Şule  167
216
Badıllı, F. Ulya (Numanoğlu) 146 Çilek, Ayşe  117
Bahçeci, Şule  194, 197 H
Balcıoğlu, Aygül  53 D Hadjipavlou-Litina, Dimitra 193
Balogh, Agnes  97 Dabbagh, Mohammad Ali  92 Hagen, Trommer  145
Baloğlu, Esra  105, 135 Dadashzadeh, Simin  182 Hasçiçek, Canan  103
Balzarini, Jan  190 Dalkara, S.  156 Haxhiu, Arlinda  208
Barberini, L.  25 Dalkara, Sevim  206 Haydar, Muder Al  108
Bartoszcze, Michał  32 Dayangaç Erden, D.  156 Heikkilä, Tiina  17
Basit, Abdul  96 Değim, İsmail Tuncer  23, 86 Hıncal, A. Atilla  74, 76, 83, 201
Basta, Giuseppe  131 Değim, Zelihagül  88 Hıncal, Filiz  29
Başaran, Berrin  111 Demir, A. S.  156 Hinrikus, Toivo  209
Batrakova, Elena  90 Demirci, Sevilay  194 Hirvonen, Jouni  14, 17
Baumgartner, S.  19 Demir, Huriye  80 Hock, Karlo  190
Bayındır, Zerrin Sezgin  90 Demirkan, Kutay  50 Homolya, László  37
Baykara, Tamer  133 Devrim, Burcu  114 Hudák, István  120
Bazarinil, Jan  193 Dimitrovska, Aneta  176
Becchetti, Ennio  131
Bekmezci, Şerife  71
Dóczi, Tamás  120 I
Dodov, Marija Glavas  95 Ilie, Corina  184
Beygi, Iman  66 Doğan, A. Lale  129
Bilensoy, Erem  183 Irache, Juan M.  30
Dora, Devrim Demir  119 István, Hermecz  187
Blasi, Paolo  25, 126, 131 Dracea, Olguta  184
Blazso, Gabor  97 Draganic, Pero  210
Bogdanovska, Liljana  176 Dršata, Jaroslav  153
J
Bojic, Mirza  196 Jaafari, Mahmoud Reza  149, 150
Boonkaew, Sukamai  164 Jadrijević, Milena  43
Bora Tatar, G.  156
E
Jakovljević, M.B.  51
Botz, Lajos  48, 120, 142 Ektiren, S.  69 Järv, Jaak  157
Bouchemal, Kawthar  127 Endrenyi, Laszlo  8 Jolevska, Suzana Trajkovic - 208
Bousová, Iva  153 Erdal, M. Sedef  147 Jürgenson, Siiri  152
Bozkır, Asuman  111, 114 Erdem Yurter, H.  156

3rd BBBB International Conference on Pharmaceutical Sciences

 INDEX

K Matto, Vallo  158 Puglia, C.  25

Kabanov, Alexander  90 Medic-Saric, Marica  196
Kadıoğlu, Yücel  85 Meos, Andres  177 R
Kálmán, Simon  187 Mikac, U.  19 Raal, Ain  152, 209
Kantarcı, Gülten  119, 125 Milanese, Lara  126 Rahić, O.  74, 76
Karabay Yavaşoğlu, N.Ülkü 140 Mintas, Mladen  190 Rajić, Zrinca  46
Karakurt, Arzu  167, 206 Mitar, Alemka  196 Rajic, Zrinka  190, 191, 193, 196,
Karakurt, Cemşit  167 Mosley, R. Lee  90 200
Karasulu, H. Yeşim  122, 140 Movaffagh, Jebraeel  151 Rattanasookchit, Somying 171
Karataş, Ayşegül  71 Mrhar, A.  51 Raud, Sirli  160
Karavana, Sinem Y.  105 Muratović, S.  201 Reinhard, Neubert  145
Karavana, Sinem Yaprak  135 Mutlu, N. Başaran  88 Remuñán-López, C.  112
Kardaş, Faruk  186 Mutomäki, Lasse  14 Rençber, Seda  62
Kayıran, Seren  109 Revesz, Piroska Szabo  64, 73, 97
Kaynak, Mustafa Sinan  179 N Ricci, Maurizio  25, 126, 131
Khalili, Noman  149, 150 Nacsa, Agnes  97 Rombaut, Bart  57, 58
Khattab, Ibrahim  62 Nada, Aly  62 Rossi, Carlo  25, 126, 131
Kilk, Kalle  157 Noe, Christian R.  7
Klebovich, Imre  124 Nuta, Diana Camelia  184 S
Koçak, Gülendam  167 Saadati, Roonak  182
Kõks, Sulev  158 O Sadeghi, Hasti  92
Kolahian, Saeed  154 Oral, Murat  99 Sam, Tom  11
Kol, Özlem Gürsoy  198 Otomo, Naoya  64, 73 Sanchez, Freimar Segura  127
Konyalıoğlu, Sibel  143 Sándor, Boros  187
Korkmaz, Emrah  143
Kos, M. Kerec  51
Ö Santi, Patrizia  139
Ölmez, Senem Sevtap  65, 82 Sarayoğlu, Elif  174
Kostiainen, Risto  41 Sarısözen, Can  82, 83, 85, 96
Kotmakchiev, Mustafa  125 Öner, Levent  80, 93
Öner, Z. Gülşen  106 Sarkadi, Balázs  37
Kovács, Kristóf  124 Schiaffella, Fausto  126
Kraljevic-Pavelic, Sandra  190 Özcan, İpek  127
Özdemir, Ayşe  106 Schnuerch, Andreas Bernkop- 135 217
Kristl, J.  19 Schoubben, Aurélie  25, 126, 131
Kungka, Pinwadee  170 Özdin, Deniz  147
Özer, A. Yekta  129 Sepe, A.  19
Kural, F. Hande  116 Septioğlu, Ebubekir  189
Kurtoğlu, Aslıhan Hilal  133 Özer, Özgen  100, 127, 139, 143
Özgül, Levent  174 Setayesh, Yousef  151
Kutateladze, Mzia  31, 32 Setwiwattanakul, Wanwaree 173
Kuzmanovska, Sonja  95 Özvegy-Laczka, Csilla  37
Özyazıcı, Mine  62 Shahab, Farhad Mostafavi  182
Kuzu, Didem  78 Shamil, Dania  96
Silindir, Mine  129
L P Sioofi, AmirBabak  154
Laaksonen, Hannu  14 Paap, Marko  177 Sipos, Peter  73, 97
Laaksonen, Timo  14, 17 Paaver, Urve  102 Soomets, Ursel  36, 157
Laidmäe, Ivo  168 Padula, Cristina  139 Soylu, S.  69
Launay, Manon  183 Pastorin, Giorgia  27 Stampf, György  124
Lee, Vincent  27 Pavelic, Kresimir  190 Stecoza, Camelia Elena  184
Lehto, Vesa-Pekka  21 Pehlivan, Sibel Bozdağ  96, 109 Sukjarern, Piyawan  170
Lertpipopmetha, Vorachart 163 Peltonen, Leena  17 Suksomboon, Naeti  163, 164,
Leturgez, Thibaut  183 Perisin, Tina  210 165, 170, 171, 173
Librowski, Tadeusz  79 Perkovic, Ivana  200 Sunderland, Bruce  108
Li, Shu  90 Petkovska, Rumenka  176 Suthisisang, Chuthamanee 163,
Luca, Giovanni  131 Petrenko, Valery A.  35 171
Ludányi, Krisztina  124 Petrovska, Vesna  95 Szakács, Gergely  37
Lust, Andres  102 Polak, Sebastian  16 Szente, Virág  38
Pomazi, Anita  73 Szökő, Éva  42
M Ponchel, Gilles  127 Szucs, Maria Budai  64
Pontiki, Eleni  193, 200 Szuts, Angela  64
Mahlapuu, Riina  157 Poolsup, Nalinee  163, 164, 165, 170,
Mahmoudabadi, Ali Zarei  155 171, 173
Mancuso, Francesca  131 Ş
Poznic, Miroslav  190
March, John  33 Prasit, Tananya  165 Şahin, Nefise Özlen  69, 79
Martinek, Tamas  97 Prateepjarussaeng, Supanika 170 Şahin, Selma  96, 179

October 26 - 28, 2009 / Antalya - TURKEY

 INDEX

Şenel, Sevda  106 Trudil, David  32 W

Şener, Bilge  10 Tuğcu Demiröz, Fatmanur  137 Wong, Pei S.  207
Şengel Türk, Ceyda Tuba  103 Tuncel, Murat  129
Şen, Tangül  105, 146
Y
Şenyiğit, Taner  139 U
Şenyiğit, Zeynep  135 Yağ, Göksu  87
Uibo, Raivo  168 Yalçın, Güler  174
Ulutürk, Deniz  156 Yeğin, Betül Arıca  80, 87
T Uzunović, A.  74, 76, 201 Yeo, Geok H.  207
Tábi, Tamás  42 Yerlikaya, Fırat  183
Takac, Milena Jadrijevic-Mladar 210 Ü Yıldız, Ayça  100
Takač, Mladar  43 Üner, Melike  68 Yılmaz, Şükran  88
Takac, Vedran  210 Ünlü, Nurşen  109 Yüksek, Haydar  186, 194, 197, 198
Takka, Sevgi  78 Üstündağ, Neslihan  122, 140 Yüksel, A.  69
Tan, Celil  86 Yüksel, Altan  79
Tangsakprasert, Poramed  170
Tarımcı, Nilüfer  105, 146
V Yüksel, Nilüfer  90, 99
Váradi, András  37 Yüktaş, Nuray  174
Taskinen, Saila  17
Teder, Kersti  177 Vecchiarelli, Anna  126
Tepwitukgid, Bhakanit  163 Vetter, Anna  135 Z
Terasmaa, Anton  158 Viirlaid, Säde  157 Zaghloul, Abel-Azim  62
Tomic, Sinisa  210 Volmer, Daisy  55 Zare, Payman  154
Torchilin, Vladimir P.  83, 129 Vovk, T.  51 Zarghi, Afshin  203
Tothfalusi, Laszlo  8 Vranić, E.  74, 76, 201 Zelkó, Romána  38
Tóvölgyi, Zsuzsa  120 Vučičević, K.  51 Zhao, Yuling  90
Trnková, Lucie  153 Vural, İmran  65, 82, 83, 85, 96 Zorc, Branka 46, 190, 191, 193, 200

218

3rd BBBB International Conference on Pharmaceutical Sciences