Targeted Delivery of

Antimicrobials
Project Proposal

Written and Submitted by

Deelha Ambreen
Submission date:

MSC IN PHARMACEUTICAL SCIENCES ,FACULTY OF HEALTH, LIFE AND SOCIAL

SCIENCES, EDINBURGH NAPIER UNIVERSITY

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Table of Contents
1.0 Introduction...........................................................................................3
Figure 1.1: Structure of TPP conjugate prodrug of metronidazole.........4
1.2 Hypothesis.............................................................................................4
2.0 Metronidazole........................................................................................4
Figure 2.1: The mechanism action of Metronidazole.............................5
2.1 The resistance to Metronidazole.........................................................5
3.0 Mitochondria..........................................................................................6
4.0 Triphenylphosphonium Cation (TPP)......................................................7
Fig 2.2: Structure of the Triphenylphosphonium Cation.......................7
5.0 Crisis of resistance to current antibiotics..............................................8
6.0 Methodology..........................................................................................9
6.1 Product Characterization....................................................................9
6.1.1 NMR (nuclear magnetic resonance).............................................9
6.1.2 Mass spectrometry.......................................................................9
6.2 Distribution Coefficient assay...........................................................10
6.3 Antibacterial Testing.........................................................................10
6.3.1 MIC testing....................................................................................10
6.3.2 Disk Diffusion testing.....................................................................10
7.0 Timeline...............................................................................................11
References.................................................................................................12

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1.0 Introduction
In this project a series of new triphenylphosphonium (TPP) conjugate
prodrugs of the antibiotic metronidazole will be synthesised. These
compounds will then be characterised, their physiochemical properties
e.g. distribution coefficients will be studied and some antibacterial testing
will be carried out.
Prodrugs of metronidazole with novel TPP carrier linked are synthesised in
order to target resistant bacteria in an attempt to improve the efficacy of
the drug, by potentially bypassing the bacterial membrane barrier and
associated efflux pumps. Prodrug delivery systems with improved uptake
of drug into the cytoplasm of bacteria may increase efficacy. The new
carrier which will be used to prepare new is Triphenylphosphonium cation
(TPP) conjugate therefore prodrugs will be ester linked to HO group of the
metronidazole to give TPP-O-metronidazole derivatives (fig 1.1). This is
introduced by reaction of TPP bromide with the primary alcohol group of
metronidazole.

N
P

N
C O
O

O
N
O

Figure 1.1: Structure of TPP conjugate prodrug of metronidazole

1.1 Aims
To synthesise a series of novel TPP- conjugate prodrugs of metronidazole
as potential improved, targeted antibacterial agents.
To determine the antibacterial activity of new conjugates against the
panel of pathogenic Gram-negative bacteria.

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1.2 Hypothesis
It

is

hypothesised

that

ester-linked,

TPP-labelled

conjugates

of

metronidazole will be transported through the periplasmic space in

bacteria into the cytoplasm, where esterase enzymes can cleave
(activate) the antibacterial prodrug.
It is hypothesised that a greater concentration of active agent can be
achieved by the prodrug approach in comparison to the free drug.

2.0 Metronidazole
Metronidazole

(2-(2-methyl-5-nitro-1H-imidazole-1-yl)

ethanol)

is

an

antibacterial agent which was discovered in the 1950s, it is an effective

agent against anaerobic bacteria and protozoan diseases. It is one of the
most potent antimicrobials but the emerging resistance of this antibiotic is
limiting its long term use (Miljkovic et al., 2014). Metronidazole is very
effective against microbes with 70-99% of progress rate which vary with
individuals infections (Lofmark et al., 2010).
Metronidazole from the nitroimidazole class shows no activity in vivo prior
to reaching microbial cells, therefore it is often considered as a prodrug as
it uses sensitive organisms to activate metabolically (Lau et al., 1992).
Metronidazole activates by reduction but in only harsh reducing
environment (Koch et al., 1997). The mechanism of action comprise of 4
steps as shown in figure 1.
1. Entry into the bacterial cell
2. Reduction of the nitro group
3. Cytotoxic effect of the reduced product
4. Finally release of end products that are
inactive

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Figure 2.1: The mechanism action of Metronidazole reproduced from

Muller et al., 1983.

2.1 The resistance to Metronidazole

Antimicrobial resistance can take place mainly in three ways they are
intrinsic, mutational and acquired. Bacteria use three main ways to
become resistant to antibiotics, first by inhibiting the drug from reaching
its target, second by modification of the target and third by inactivating
the antibiotic (Miljkovic et al., 2014).
In the mechanism of resistance metronidazole interferes with the
synthesis of nucleic acid. In H.pylori treatment resistance has emerged by
use of the metronidazole, but the main mechanism of resistance is
observed to be the slower uptake and decreased intracellular reduction of
the drug (Soares et al., 2011). The resistance of metronidazole is
becoming very common and targets approximately all types of infections
resulting in the failure of treatment. It has been reported by Upcroft et al.,
2001 that clinical resistance of 10-20% has been observed for patients
with giardiasis, and 2-4% of clinical resistance with varying degrees has
been shown for T.vaginalis (Krashin et al., 2010). The major resistance of
50% to metronidazole is acquired in H. pylori cases in most developing
countries (Secka et al., 2013).
Metronidazole is an essential antibiotic recorded by the World Health
Organisation as it is very effective against numerous essential anaerobic
pathogens. Activity of the metronidazole against various important
microbes can be improved by making changes to the basic drug structure
and target many forms of resistance (Miyamoto et al., 2013). Another way
to overcome the resistance of metronidazole may be by using the
alternative pathways such as mitochondrial delivery strategies in order to
bypass detoxification or efflux pathways. Mitochondria are not present in
bacteria, however evolutionary similarities exist between them. The
mitochondria targeted strategy of the antimicrobials has been applied to

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treat bacterial infections and has shown to increase the therapeutic

efficacy of a drug (Pereira et al., 2011).

3.0 Mitochondria
Mitochondria are present in almost all eukaryotic cells except in
erythrocytes, mitochondria ranges from one to several thousands in cells
(Desler et al., 2011). A mitochondrion is known as the powerhouse of cell
who generates energy for cell through oxidative phosphorylation and
plays a major role in the cell apoptosis (Schumacker et al., 2014).
Generating energy and assisting in cell death pathways are just the main
roles of mitochondria, But mitochondria is also involved in many other
processes of cells such as the production of ATP, thermal regulation,
reactive

oxygen

species

(ROS)

containment,

movement

of

cell,

inflammation, provides response to cell during stress and danger. There

are many different kinds of mitochondria in cell, which differ in their
functions,

size,

and

shape

are

known

as

heteroplasmy,

whereas

mitochondria which varies in numbers and copies of mitochondrial DNA

(mtDNA) are known as polyplasmy (Milane et al., 2015).
Mitochondria are the only organelle in a cell with its own mitochondrial
DNA (mtDNA), which contains 37 genes and it is not part of a nuclear DNA
(Penta et al., 2001). Mutations in the mtDNA are the main reason of many
mitochondrial

diseases

such

as

age-related

diseases,

cancers,

neurological disorders and premature ageing (Desler et al., 2011). As

mitochondria is involved in many human diseases it is being explored as a
potential drug target to overcome some problems such as host toxicity,
resistance to the therapeutic agent and the production of undesirable
secondary effects (Jean et al., 2014).
Mitochondrial drug targeting strategy is being used for antimicrobials due
to the similarities shared between mitochondria and bacteria (Jean et al.,
2014). A mitochondrion structure is composed of two membranes- a
permeable outer mitochondrial membrane (OMM) and enclosed inner
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mitochondrial

membrane

(IMM).

IMM

is

packed

with

saturated

phospholipids and contains high ratio of protein to lipid than plasma

membrane (Smith et al., 2012). Gram-negative negative bacterial
surrounds the periplasm with two lipid bilayers- inner membrane (IM) and
outer membrane (OM). The IM is made up of lipopolysaccharide (LPS) and
phospholipid bilayer (Jiang et al., 2012). There are many similarities
shared between mitochondria and bacteria are therefore, same strategy
of mitochondrial drug delivery can be used for bacteria which are
resistance to antibiotics.
In this research project same strategy will be applied to a new carrier
linked prodrug metronidazole to overcome the resistance, by improved
uptake of drug into the cytoplasm of bacteria.

4.0 Triphenylphosphonium Cation (TPP)

Triphenylphosphonium (TPP) is a lipophilic cation which is used as a
mitochondria targeting vector. TPP is a nano targeting vector with a
positive charge which is resonance stabilized on 3 phenyl groups. TPP also
contains a hydrophobic surface area which gives efficient interaction with
the inner mitochondrial membrane (IMM) (Murphy et al., 2008). This
interaction allows the TPP inside the mitochondrial matrix to freely
solubilize or adsorb to the inner leaflet of IMM, based on the lipophilicity of
a cargo (Murphy et al., 2007). In this project TPP will be linked to
metronidazole to bypass the bacterial membrane barrier in an attempt to
overcome the resistance of this antibacterial agent.

O
C

Fig 2.2: Structure of the Triphenylphosphonium Cation

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This carrier is being used because of its known unique properties which
deliver the drug to mitochondria to treat mitochondrial dysfunction
disease. TPP is a non-toxic nanocarrier which provides an effective
therapeutic cargo delivery through surface conjugation or electrostatic
interaction (Biswas et al., 2012).
TPP has also been effective in delivering anti-cancer drugs to the
mitochondria. For example doxorubicin (DOX) an anti-cancer agent has
been administered with polyethyleneimine (PEI) and TPP to humans with
prostate carcinoma cells, and it has been observed that PEI-TPP-DOX
target cancer cells very efficiently and selectively (Theodossiou et al.,
2013).
TPP is limited to only low molecular compounds for its cargo, TPP is the
only small carrier which can be constructed to add a cleavable tag that
can cleave chemically unaltered cargo only by specific mitochondrial
enzymes (Ripcke et al., 2009). Ripcke et al., 2009 demonstrates a lipoic
acid derivative reversibly tagged to Triphenylphosphonium which is
endogenously cleaved by the mitochondrial aldehyde dehydrogenase
(ALDH-2) after mitochondrial accumulation.

5.0 Crisis of resistance to current antibiotics

Antibiotics resistance is becoming a worldwide threat to health
organisations, because antibiotics are no longer effective for bacterial
pathogens. Resistance to antibiotics is the capacity of bacteria to remain
effective in antibiotic environment that inhibits the growth or finish
bacteria of the same species (Alos et al., 2014). Antibiotics are a vital
modern medicine which has been very essential for important medical
practices such as anti-cancer chemotherapy, implantation of medical
devices, surgery of contaminated districts, stem cell and organ
transplantations. However most of the antibiotics have lost their efficacy
with time because of development and spread of antibiotic resistance
between bacterial pathogens (Rossolini et al., 2014). Resistance to
antibiotics has spread among Gram-positive and Gram-negative species
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which also includes Staphylococcus aureus, Enterococcus spp,

Pseudomonas aeruginosa, Acinetobacter spp. Enterobacteriaceae and
Neisseria gonorrhoeae. However Gram-negative bacteria has been
affected most because it has become resistance to all the existing
antimicrobials, and the development of extremely drug resistance and
totally drug resistance phenotypes has been regularly reported for Gramnegative diseases (Livermore et al., 2012). Therefore new strategies need
to be developed to bypass the membrane barrier in resistant isolates to
diffuse antibiotics through bacterial membranes (Bolla et al., 2011).
In this project Gram-negative bacteria will be targeted using prodrug
metronidazole along with carrier TPP, in order to cross the inner
membrane bilayer by potentially bypassing drug efflux mechanisms.

6.0 Methodology
An established literature methods will be followed for the synthesis of
metronidazole TPP conjugate prodrug in order to form an ester bond
between metronidazole and the TPP carrier.
6.1 Product Characterization
Synthesised products will then be characterised using NMR (nuclear
magnetic resonance) and mass spectrometry. Chromatographic
purification will also be carried out on the synthesised products.
6.1.1 NMR (nuclear magnetic resonance)

NMR spectroscopy was developed by physical chemists in the late 1940s

to study the properties of atomic nuclei. In 1951, chemists realised that
NMR spectroscopy could also be used to determine the structure of
organic compounds. Because hydrogen nuclei (proton) were the first
nuclei studied by nuclear magnetic resonance, the acronym NMR is
generally assumed to mean proton NMR (proton magnetic resonance).
Proton NMR is most useful because in it's spectrum series of signals
occurring at slightly different frequencies can spot different types of H's
get signals at characteristic frequencies. Proton NMR not only gives
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information on the number of hydrogens on a carbon but also the

amounts of hydrogens present on adjacent carbons (Bruice et al).
6.1.2 Mass spectrometry

Mass spectrometry is used to measure the molecular mass of a product,

mass spectrometer converts the molecules into ions, so they can be easily
moved around by electric and magnetic fields, and this technique is
usually 0.01% accurate (Shariatgorji et al., 2014).

Fig 6.1 Simplified schematic of a mass spectrometer adapted from

http://www.astbury.leeds.ac.uk/facil/MStut/mstutorial.htm (date accessed: 07-06-2015).

6.2 Distribution Coefficient assay

Distribution coefficient assay will be carried out to test the absorption
properties of synthesised metronidazole TPP conjugate prodrug. Measure
of lipophilicity is very important because it is involved in the transport of
drug through bacterial membrane. The assay will be carried out using
shake flask method, in the assay solute will be separated between 2 liquid
phases of the solvent system. Sample will then undergo equilibration and
centrifugation followed by separate determination of both layers using
TLC (thin layer chromatography) method (Berthod et al., 2004).

6.3 Antibacterial Testing

The antibacterial testing is used to determine which antibiotic is most

susceptible in treating a bacterial infection. In this project disk diffusion

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and minimal inhibitory concentration (MIC) test will be carried out, in order
to test synthesised products for their antibacterial properties.
6.3.1 MIC testing
In the MIC test an antimicrobial agent inhibits the growth of bacterial
isolate in its lowest possible concentration. It inhibits the bacteria from
multiplying and producing visible colonies. A broth media is normally
preferred for MIC test but agar media can also be used.
For reliable and accurate results to detect antimicrobial resistant bacteria
it is important to follow step by step precise instructions (Coyle et al.,
2005).
6.3.2 Disk Diffusion testing
Disk diffusion testing is being used since 1950s but until 1966 every
microbiologist used a different procedure. In 1966 including all the
variables (e.g. Temperature, media and depth of agar).A precise step by
step procedure was published by Drs.Bauer, Kirby Sherris, and Turck. It is
essential to follow step by step guide of experimental procedure for set of
accurate results for disk diffusion testing (Coyle et al., 2005).

7.0 Timeline
Week

Week Starting

Wed 20th May

Mon 25th May

Mon 1st June

Mon 8th June

Lab activities and targets

Writing activities and targets

Commence project
planning.
Project planning

Commencement of lab work+lab

inductions skills sessions.
Commencement of synthesis and
purification of metronidazole TPP
conjugate prodrug.

Submission of
project proposal
(Fri 5th June, 12
noon)
Continuation of
targeted literature
searching.

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Mon 15th June

Continuation of conjugate
prodrug synthesis and
chromatographic purification.

Submission of first 2
or 3 pages of
experimental.

Mon 22nd June

Submission of section
on rational design (for
R& D)
Submission of first 1
or 2 schemes and
discussion of
synthesis (for R& D).

Mon 29th June

Characterization by mass
spectrometry (EPSRC National
Mass Spectrometry Facility,
Swansea).
Characterization by nmr (Heriot
Watt University).
Commencement of antibacterial
testing against Pseudomonas
aeruginosa, E.coli and
Salmonella.
Continuation of antibacterial
testing.
Potential synthesis and
characterization of a second
metronidazole TPP conjugate
prodrug.

Mon 6th July

Continuation of antibacterial
testing

Mon 13th July

Continuation of antibacterial
Submission of
testing.
preliminary LOG D
Commencement of metronidazole results and methods.
TPP conjugate prodrug

10

Mon 20th July

Continuation of antibacterial
testing.
Completion of distribution
coefficient assay

11

Mon 27th July

12

Mon 3rd August

Completion of antibacterial
testing.
Completion of Lab activities
(Friday 7th August, latest)

13

Mon 10th
August
Mon 17th
August

14

Submission of all
chemical structures
and reaction schemes
and experimental for
antibacterial testing.
Submission of
preliminary results of
antibacterial testing.
Submission of 1st draft
of introduction
(including references).

Submission of draft of
complete project
report
Submission of final
thesis, Friday 21st
August, 12 noon.

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