Enhancement of transdermal permeation of aspirin/Asian Journal of Pharmaceutical Sciences 2007, 2 (3): 96-105

Evaluation of chemical penetration enhancers for transdermal delivery of aspirin

H. O. Ammara, *, M. Ghorabb, S. A. El-Nahhasa, R. Kamela
a

Department of Pharmaceutical Sciences, National Research Center, Dokki, Cairo, Egypt b Faculty of Pharmacy, Cairo University, Cairo, Egypt
Received 13 February 2007; Revised 28 May 2007; Accepted 14 June 2007

_____________________________________________________________________________________________________________

Abstract
Purpose: Aspirin, the most widely drug used in the world, has been and will remain the standard reference for long-term treatment of platelet hyperactivity. Aspirin induces a long-lasting functional defect in platelets by permanently inactivating the COX enzyme system. Orally administered aspirin requires high and frequent dosing because it undergoes extensive presystemic metabolism. Also, chronic oral aspirin is associated with serious gastrointestinal side-effects. Transdermal delivery offers an alternative, more convenient and safer route for aspirin administration. Methods: This study involved formulation of aspirin in CMC hydro-alcoholic gel and oleaginous ointments bases, modication of the oleaginous base to improve aspirin permeation, and evaluation of the effect of some different classes of chemical penetration enhancers (fatty acids and their esters, terpenes, sulphoxides, urea and cyclodextrins) on the in-vitro permeation of aspirin from the investigated bases. Results: CMC gel showed a high permeation, and 5-10% oleic acid and 20% urea provide the greatest enhancing effects. In the case of ointments, a combination of vaseline and liquid parafn allowed the highest permeation and 10 % oleic acid had the greatest enhancing effect and offered a 7-fold enhancement of permeation compared with the control and, accordingly this formula was selected for in-vivo investigation using aggregometry to measure its effect on platelet aggregation in male Wistar rats. Conclusion: The results obtained suggest the feasibility of designing a successful transdermal delivery system providing continuous, controlled and constant aspirin input and overcoming the disadvantages of oral administration. Keywords: Aspirin; Transdermal; Permeation; Penetration enhancer _____________________________________________________________________________________________________________

1. Introduction
Aspirin is the most widely used antiplatelet drug for the primary and secondary prevention of cardiovascular disease. Aspirin produces its primary antithrombotic effects through the inhibition of PGH-synthase/COX by the irreversible acetylation of a specic serine moiety (serine 530 of COX-1 and serine 516 of COX-2) [1] and is about 170-fold more potent in inhibiting COX-1 than COX-2 [2]. The resulting reduced production of prostaglandins and TXA2 likely accounts for its therapeutics effects, as well as its toxic effects. Regular aspirin is rapidly absorbed from the acid environment of the stomach but enteric coating of aspirin results in its release in the alkaline environment of the small bowel, where it is hydrolyzed. As a result, enteric-coated aspirin has
__________ *Corresponding author. Address: Department of Pharmaceutical Sciences, National Research Center, Dokki, Cairo, Egypt. Tel.: +20124472851; Fax: +2023370931. E-mail: drhusseinammar@yahoo.com

lower bioavailability than regular aspirin. The plasma half-life of aspirin is only 20 min; however, because platelets are anucleated and cannot generate new COX [3], the effects of aspirin last for the duration of the life of the platelets (5-10 days). After a single dose of aspirin, platelet COX activity recovers by 10% per day as a function of platelet turnover [4]. Recent data have supported the use of aspirin in reducing the risk of ischemic heart disease, ischemic stroke as well as rheumatoid arthritis [5], digestive tract cancer [6], Alzheimer disease and other forms of dementia [7]. Unfortunately, chronic administration of oral aspirin is associated with an increased risk of gastrointestinal (GI) mucosal damage, which can offset its clinical benet by predisposing patients to GI haemorrhage. Aspirin is subjected to extensive presystemic metabolism in the GIT and liver, converting it into salicylic acid which is devoid of an antiplatelet effect and, therefore, orally administered aspirin requires high and frequent dosing [8] which is associated with an increased risk of GIT side-effects [9]. Although aspirin

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appears at rst sight not to be a suitable drug for transdermal delivery, the transdermal route actually offers an alternative way for administering aspirin that bypasses the gut and may be a more convenient, safer and noninvasive means of aspirin delivery, especially for longterm use. The low bioavailability of transdermal aspirin and the avoidance of direct contact with COX-1 expressed on gastric mucosal cells may provide a safer means of inhibiting platelet function [10]. Transdermal drug delivery systems (TDDS) have many advantages compared with the corresponding classical oral, injectable and inhaler systems. The most important advantages are improved systemic drug bioavailability, because the rst-pass metabolism by the liver and digestive system are avoided, leading to a controlled constant drug delivery prole (that is, controlled zeroorder absorption). Other benets include a longer duration of therapeutic action from a single application, and a reversible action [11]. Despite the obvious advantages of TDD, this route presents unique challenges. Considerable research efforts have focused on discovering methods for overcoming the impermeability of the human stratum corneum, and the most popular technique is the use of chemical penetration enhancers, which reversibly disrupt the permeability barrier of the stratum corneum [12]. The present study is an extrapolation of our previous study concerning the transdermal delivery of aspirin and the feasibility of successfully inuencing platelet function [13]. The low skin permeability together with aspirin instability impede the development of a successful transdermal formula for aspirin. This study involves the evaluation of the in vitro percutanoeus absorption of aspirin from gel and ointment bases, and optimization
Table 1 Composition of CMC gel. Component CMC Ethanol Glycerin Propylene glycol Distilled water w/w (%) 5 8 20 20 47

of the chosen topical formulations to allow high drug permeation as well as assuring its stability. Furthermore, we assessed the ability of some penetration enhancers such as fatty acids and their esters, terpenes, sulphoxides, urea and cyclodextrins to improve the permeation of aspirin. Then we selected an optimized formula for invivo investigation by measuring its anti-platelet effect.

2. Materials and methods

2.1. Materials Aspirin, -cyclodextrin (-CyD) and limonene were purchased from Sigma Chemical Company (St.Louis, USA). Hydroxypropyl--cyclodextrin (HP--CyD) was purchased from Aldrich Chemical Company (USA). Oleic acid was purchased from Fluka (Switzerland). Propylene glycol (PG) was purchased from BDH Chemicals Ltd. (Poole, England). Carboxymethyl cellulose sodium (CMC) was purchased from Adwic (Cairo, Egypt). Urea was purchased from Merck (Schuchardt, Germany). Adenosine diphosphate (ADP) was purchased from DiaMed AG (Switzerland). All other materials were of analytical grade. 2.2. Preparation of different bases for transdermal delivery 2.2.1. Gel preparation The CMC gel composition is shown in Table 1 [14]. Briey, CMC was dispersed in ethanol and, then, glycerin, propylene glycol and distilled water were added with stirring. The mixture was stirred thoroughly till gelation occured. 2.2.2. Ointment preparation (oleaginous bases) The compositions of the ointments are shown in Table 2. Vaseline was melted on a water bath maintained at 60C and the other component was warmed to the same temperature. The mixture was then removed from the water bath and stirred together until it congealed [15, 16].

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2.3. In-vitro permeation studies of aspirin through full thickness rat abdominal skin The abdominal hair of male Wistar rats (200 to 250 g) was removed carefully using an electric razor. After the animals were sacriced, the abdominal skin was excised and the adhering fat removed. Prepared skin obtained from each rat was divided into two equal parts, one part was used for the medicated preparation and the other one was used for the unmedicated base (as a blank) so that any interference due to the skin or the plain base can be eliminated. This membrane was mounted on a vertical Franz-type diffusion cell (Vangard International Inc., New Jersey, USA) with the dermis facing the receptor compartment. The donor side was loaded with 187.5 mg of the investigated base containing 20% aspirin. The membrane surface area available for diffusion was 5.3066 cm2. The donor chamber was covered with paralm. The receptor compartment was completely lled with 100 ml phosphate buffer (pH 7.4). The temperature was maintained at 37C 0.5C and the receptor compartment was constantly stirred at 300 r/min. Samples of the receptor uid (1 ml) were withdrawn at various times up to 24 h and replaced immediately with an equal volume of fresh buffer solution to maintain a constant volume and then the samples were assayed spectrophotometrically at 302 nm [17] using a Shimadzu UV Spectrophotometer (2401/PC), Japan. Several chemical penetration enhancers belonging to different groups were incorporated into the bases to investigate their effect on permeation. The tested enhancers were: oleic acid, methyl myristate, limonene, dimethyl
Table 2 Composition of ointments. Constituents Vaseline Liquid parafn PG Almond oil

sulphoxide (DMSO), urea, -CyD and HP--CyD. The cumulative amounts of drug permeated in mg/cm2 and the percentage of the cumulative amount permeated after 24 h (Q24%) were calculated. The effectiveness of penetration enhancers can be determined by the enhancement ratio which is expressed as ER = Q24% with enhancer/Q24% without enhancer (control). The data were analyzed statistically by the one-way ANOVA test followed by the least signicant difference procedure. This statistical analysis was carried out using SPSSsoftware. 2.4. In-vivo study The inhibition of platelet aggregation was used as a pharmacodynamic parameter for evaluating the efcacy of the selected formulation [9]. Male Wistar rats weighing 200250 g were kept on a standard diet and maintained under controlled conditions of humidity (30% 70%) and temperature (24C 2C) and fasted overnight before blood collection [18]. Aspirin (30 mg) in an equal weight of vaseline-liquid parafn (7-3) ointment, containing 10% oleic acid as a penetration enhancer, was spread as a lm of surface area 5.3066 cm2 on aluminum foil and applied using an adhesive tape to the naked back of the animals of the test groups (n = 6). The non-medicated base was applied to the control groups (n = 6). The adhesive tapes were removed 48 h after application; no signs of irritation, edema nor erythema were observed. Blood samples were withdrawn from the orbital sinus of the animals 72 h after application. The absorbance of platelet-rich plasma falls as the platelet aggregate. The amount and the rate of this fall

w/w (%) Ointment A 100 Ointment B 70 30 Ointment C 70 30 Ointment D 70 30

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are dependent on the platelet reaction to the added agonist if other variables such as temperature, platelet count and mixing speed are controlled. [18] Then, 4.5 ml blood samples were collected in plastic tubes containing 0.5 ml sodium citrate (0.9%). Platelet-rich plasma (PRP) was obtained from citrated blood by centrifugation at 200 g for 15 min. Platelet-poor plasma (PPP) was prepared from the remaining volume of blood by centrifugation at 1600 g for 15 min. PRP (450 l) was added to an aggregation cuvette containing a stirring bar and 50 l aggregation-inducing agent (Adenosine diphosphate, 10-3 mol/l) was added. Then, 500 l PPP was placed in the other cuvette. Platelet aggregation was then measured turbidometrically until the response reached a plateau or for a period of 3 min using a whole Blood Aggregometer (Chrono-Log Corporation, Havertown, PA, USA). The main indicator of platelet function was the maximal aggregation intensity induced by ADP and inhibited by the administration of aspirin [18]. The anti-platelet aggregation effect of aspirin was evaluated as a percentage inhibition of platelet aggregation which was calculated as follows [19]: The in-vivo data were analyzed statistically by the one-way ANOVA test followed by the least signicant difference procedure. This statistical analysis was carried out using SPSS software.

3. Results and discussion

3.1. Permeation of aspirin from CMC gel base The cumulative amount of aspirin permeated per unit area (mg/cm2), the percentage of the cumulative amount permeated after 24 h (Q24%) and the enhancement ratio were calculated and listed in Table 3. CMC hydro-alcoholic gel base, containing alcohol together with PG, was shown to inhibit in-vitro deacetylation [20] and, therefore, may increase aspirin stability [14]. CMC gel showed high and rapid permeation. Initially, rapid permeation was observed, gradually reaching a constant value for the rest of the experiment (Table 3). The initial quick permeation (burst effect) would be benecial as it would help achieving a therapeutic plasma concentration of the drug in a minimum time. The burst effect may be

due to the initial migration of drug molecules throughout the three-dimensional network of the gel [15]. Also, the presence of PG which is a cosolvent, not only solubilizes aspirin, but also alter the skin structure and, therefore, modies the percutaneous absorption [21, 22, 23]. Ethanol and PG can exert a permeation enhancing activity via various mechanisms [23, 24, 25]. The use of propylene glycol in combination with alcohol may offer synergistic enhancement [13, 26]. Since transdermal delivery offers the greatest opportunity for controlled release of drugs, overcoming the problem of low skin permeability represents a crucial advance that allows transdermal delivery to realize its great potential. The use of penetration enhancers offers a cheap, simple and convenient method of improving transdermal bioavailability [13, 27]. Fatty acids are currently receiving much attention as penetration enhancers [28]. Percutaneous drug absorption has been increased by a wide variety of long chain fatty acids, the most popular of which is oleic acid. Table 3 shows the effect of different concentrations of oleic acid on the permeation of aspirin from CMC gel base. The data showed that oleic acid enhanced aspirin permeation from this base. Oleic acid, at a concentration of 5% or 10% enhanced permeation (P < 0.001), and the ER was found to be 1.2 and 1.197 respectively. However, a concentration of 20% enhanced the permeation when compared with the control (P < 0.01) but didn't lead to a further increase in permeation (P < 0.01), and the ER was found to be 1.166. This may be attributed to an increase in the lipophilicity of the vehicle [13]. Oleic acid is a mono-unsaturated fatty acid with 18 carbon atoms, which interacts with stratum corneum lipids and disrupts their structures increasing their uidity and consequently increasing the ux [12, 23, 29]. Also the major mechanism of enhancement of oleic acid is an increase in permeation through the non-polar route, as it increases both diffusivity and partitioning. However, it also increases the partitioning parameter in the polar route by increasing hydration of the stratum corneum [21]. Another example of this class monitored for its permeation enhancing activity, was methyl myristate, an ester of saturated fatty acid with 14 carbon atoms. Incorporation of this ester in CMC gel base at a concentration

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of 5 % led to an increase in drug permeation (P < 0.05), and the ER was =1.103 (Table 3); however, increasing the ester concentration up to 20% only slightly increased the permeation (P > 0.05) from CMC gel, but did not result in a further increase, and this may be attributed to an increase in the lipophilicity of the vehicle [13]. The ER was found to be 1.052 and 1.046 for a concentration of 10% and 20%, respectively, Beside being used as penetration enhancers, both oleic acid and methyl myristate have stabilizing and solubilizing effects on the drug, according to the results

of Mizobuchi et al. [16] who reported the stabilizing and solubilizing effects of organic acids having 2 to 20 carbon atoms and their esters on aspirin. D-limonene, the non-polar group containing cyclic terpene, was found to increase aspirin permeation from CMC gel base when used at a concentration of 5% (P < 0.01); increasing the limonene concentration to 10% slightly increased drug permeation from this base (P > 0.05) (Table 3). The ER was found to be 1.159 and 1.029, respectively. Terpenes improve drug partitioning into the stratum corneum by modifying its solvent nature,

Table 3 Effect of penetration enhancers on permeation of aspirin from CMC gel. Penetration enhancer Cumulative amount permeated (mg/cm2) SE 0.5 h 1h 2h 3h 4h 5h 24 h Q24% SE 74.011.84 1.200 1.197 1.166 1.103 1.052 1.046 1.159 1.029 0.658 1.076 0.893 1.029 1.155 1.195 0.461 1.113 0.245 1.111 1.103 ER

Control (no enhancer) 0.230.02 0.570.04 1.710.16 2.650.14 3.580.16 4.340.20 5.230.13 5% oleic acid 10% oleic acid 20% oleic acid 5% methyl myristate

0.160.01 0.320.02 0.740.01 1.210.10 1.650.04 2.200.03 6.280.04 88.870.57a 0.260.02 0.670.05 1.930.04 2.960.05 4.010.11 4.800.12 6.260.09 88.581.27
a

0.220.01 0.530.05 1.590.07 2.520.13 3.370.20 4.110.15 6.100.17 86.322.41b 0.210.04 0.470.06 1.120.05 1.850.10 2.330.11 3.110.14 5.770.20 81.652.83
c

10% methyl myristate 0.440.10 0.920.09 1.830.11 2.670.12 3.430.05 4.200.21 5.500.15 77.832.12d 20% methyl myristate 0.770.09 1.430.11 2.100.14 3.000.15 3.700.06 4.400.20 5.470.17 77.412.41 5% limonene 10% limonene 5% DMSO 10% DMSO 20% DMSO 5% Urea 10% Urea 20% Urea 10% BCyD 20% BCyD 40% BCyD 10% HPBCyD 20% HPBCyD
d

0.180.02 0.480.07 1.570.11 2.860.06 3.880.21 4.730.11 6.060.15 85.752.12b 0.230.04 0.530.06 1.380.15 2.330.20 3.020.13 3.610.14 5.380.16 76.132.26 0.070.01 0.100.02 0.190.04 0.350.03 0.490.05 0.590.10 3.440.11 0.270.02 0.490.10 1.030.12 1.430.03 1.930.06 2.340.12 5.630.11
d

48.681.56a 79.671.56
c

0.090.03 0.210.05 0.590.03 0.910.11 1.200.12 1.500.11 4.670.13 66.081.84c 0.270.05 0.530.06 1.040.11 1.390.04 1.770.11 2.290.11 5.380.20 76.132.83
d

0.430.11 0.970.05 1.800.20 2.700.21 3.620.09 4.070.11 6.040.20 85.472.83b 0.150.02 0.430.04 0.900.09 1.480.11 2.050.20 2.500.16 6.250.22 88.443.11
a

0.150.04 0.170.11 0.260.08 0.350.11 0.470.12 0.590.07 2.410.05 34.100.71a 0.200.02 0.500.04 1.170.09 2.130.06 2.920.11 3.490.12 5.820.14 82.361.98 0.060.07 0.150.03 0.330.11 0.450.12 0.590.13 0.720.20 1.280.21
c

18.112.97a
c

0.200.01 0.400.03 1.360.06 2.340.07 3.120.11 3.890.04 5.810.14 82.221.98

0.300.04 0.640.06 1.590.05 2.720.11 3.630.14 4.260.06 5.770.14 81.651.98c

Each value represents the mean SE (n = 3). Q24%: percentage of the cumulative amount permeated after 24 h. ER: enhancement ratio = Q24% (with enhancer)/Q24% (control). aSignicant (P < 0.001) when compared with the control. bSignicant (P < 0.01) when compared with the control. cSignicant (P < 0.05) when compared with the control. dNon-signicant (P > 0.05) when compared with the control.

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and many terpenes exhibit high human skin permeability. With the loss of terpenes from a formulation, there could be an alteration in the thermodynamic activity of the permeant. Terpenes may also modify drug diffusivity through the membrane. Small angle X-ray diffraction studies have also indicated that d-limonene disrupts stratum corneum bilayer lipids, this interaction with the stratum corneum is reversible [23, 30]. Terpenes are not signicant skin irritants, possess good a toxicological prole, provide excellent enhancing ability and appear to be promising candidates for pharmaceutical formulations [31]. Dimethylsulphoxide (DMSO) is widely used to denature proteins and, on application to human skin, it has shown to change the intercellular keratin conformation, from helical to sheet [32]. It has been shown to interact with the intercellular lipid domains of human stratum corneum. Considering the small highly polar nature of this molecule, it is feasible that DMSO interacts with the head groups of some bilayer lipids to distort the packing geometry. Furthermore, DMSO within skin membranes may facilitate drug partitioning from a formulation into this universal solvent within the tissue [23]. Over a concentration range of 5%20%, only 10% DMSO increased the permeation of aspirin from CMC gel (P < 0.05), while 5% (P < 0.001) and 20% (P < 0.01) led to a reduction in permeation. The ER was found to be 0.658, 1.076 and 0.893 for 5%, 10% and 20% DMSO, respectively (Table 3). The incorporation of different concentrations of urea (5%20%) in CMC gel base produced a concentrationdependent increase in aspirin permeation (Table 3). While 5% urea caused only a slight increase in permeation (P > 0.05) (ER=1.029), 10 % produced a greater increase in permeation (P < 0.01) (ER=1.155) and 20% produced a further increase (P < 0.001) (ER=1.195). Urea is a hydrating agent and hydration of the stratum corneum increases the penetration rate of most substances; water opens up the compact structure of the horny layer. Urea also has keratolytic properties. The penetration enhancing activity of urea probably results from a combination of increasing the stratum corneum water content and its keratolytic activity [23]. Urea has also been shown to increase the permeation of aspirin from

other bases [13]. Cyclodextrins have been used as unconventional skin penetration enhancers or novel drug delivery systems. One of the more extraordinary properties of cyclodextrins is that they are able to increase dermal and transdermal drug delivery without affecting the barrier function of the skin [33]. Table 3 shows the effect of different concentrations of -CyD and HP--CyD on aspirin permeation from CMC gel base. The results show that the use of 10% and 40% -CyD caused a pronounced reduction in the permeation of aspirin (P < 0.001) (the ER was found to be 0.461 and 0.245 respectively), While incorporation of 20% -CyD increased drug permeation (P < 0.05) (ER= 1.113). HP--CyD, at a concentration of 10% or 20% increased the permeation of aspirin (P < 0.05) (ER = 1.111 and 1.103 respectively), and a further increase in concentration to 40% did not signicantly affect aspirin permeation from CMC gel base (P > 0.05) (ER = 1.013). Cyclodextrins have been reported to be able to interact with some lipophilic components of the skin [34] and also can disrupt the skin barrier by extracting various constituents from it, but, under normal conditions, such extraction may be suppressed by drug molecules and other lipophilic molecules which are usually present in dermatologic preparations. These lipophilic molecules will compete with the membrane constituents for space in the cyclodextrin cavity and, in this way, will reduce the ability of the cyclodextrins to extract lipophilic compounds from the skin barrier [33]. Cyclodextrins solubilize lipophilic water-insoluble drug in aqueous vehicle systems and deliver the drug molecules to the barrier surface. At the surface, the drug molecules partition from CyD cavity into lipophilic barrier. Thus, drug delivery from aqueous CyD solutions is both diffusion-controlled and membrane-controlled. Only insignicant amounts of the hydrated cyclodextrin molecules and drug-cyclodextrin complexes are able to penetrate into lipophilic biological barriers, such as intact skin [35]. In addition, a number of studies using various biomembranes, under several sets of experimental conditions, have shown that excess CyD, i.e., more than needed to solubilize a given lipophilic drug in an aqueous vehicle, results in reduced drug penetration through the membrane [36]. Under linear regression analysis, the permeation pro-

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les of aspirin from CMC gel base as well as CMC gel base containing the investigated penetration enhancers, show a good correlation in the straight line portions between the cumulative amounts permeated and time (R2 > 0.99). Therefore, the penetration of aspirin from these formulations can be accurately described by zeroorder kinetics. 3.2. Permeation of aspirin from ointment (oleaginous) bases The composition of the investigated formulated ointments containing vaseline is shown in Table 2. Vaseline used as an ointment base provides a number of advantages: compatibility with a variety of medicaments, an emollient effect, being anhydrous it provides optimum stability for many drugs and, occlusive properties that increase skin hydration by reducing the rate of loss of surface water, which may increase drug permeation [15]. Vaseline is an ideal base for assuring aspirin stability [14, 16]. Unfortunately, being an anhydrous, lipophilic and highly viscous base, it exhibits very poor permeation (Table 4). Great efforts have been devoted to improve the permeation of aspirin from this base. Some substances having the ability to dissolve aspirin [16], such as glycerol fatty acid esters (almond oil), hydrocarbon oils (liquid parafn) and propylene glycol were considered for this purpose. The cumulative amount of aspirin permeated per unit area (mg/cm2), the percentage of the cumulative amount permeated after 24 h (Q24%) and the enhancement ratio were calculated and are listed in Table 4. The
Table 4 Permeation of aspirin from ointment (oleaginous) bases. Ointment A (control) B C D

permeation of aspirin from the investigated ointments is slow and constant over the time, thus resulting in a controlled and sustained release behavior. Table 4 shows shows that vaseline-liquid parafn (7: 3) base (oint. B) produces marked enhancement of aspirin permeation compared with that from vaseline base (oint. A) (P < 0.001). The ER was found to be 2.419. Liquid parafn not only stabilize aspirin but also dissolves it and, therefore, it has an enhancing effect on absorption of aspirin from skin [16]. Also, vaseline-propylene glycol (7: 3) base (oint. C) produces an increase in permeation of the drug compared with that from vaseline base (oint. A) itself (P < 0.01). The ER was found to be 1.391. Propylene glycol is a cosolvent which not only solubilizes aspirin, but also can alter the skin structure, thereby modifying the percutaneous absorption [22]. Almond oil is a plant oil which stabilizes aspirin and dissolves it leading to an enhancing effect on percutaneous absorption [16]. Hence vaseline- almond oil (7: 3) base (oint. D) allowed better permeation (P < 0.01) of the drug compared with vaseline (oint. A) itself. The ER was found to be 1.244. The permeation proles of aspirin from the previous ointment bases show a good correlation in the straight line portions in the graphic illustration of permeation as a function of time (R2 > 0.99), using linear regression analysis. Therefore, the penetraation of aspirin from these formulations can be accurately described by zero-order kinetics. Since ointment B afforded the highest permeation, it was chosen for further investigations concerning monitoring the effect of different chemical penetration

Cumulative amount permeated (mg/cm2) 0.5 h 0.170.04 0.120.03 0.130.05 0.070.01 1h 0.210.12 0.150.05 0.190.06 0.080.02 2h 0.290.11 0.210.04 0.260.07 0.120.02 3h 0.430.09 0.260.06 0.360.11 0.150.03 4h 0.570.06 0.310.05 0.460.12 0.180.05 5h 0.680.22 0.390.07 0.530.08 0.210.04 24 h 0.740.02 1.790.12 1.030.02 0.920.06

Q24% 10.470.48 25.331.70a 14.560.24

b

ER

2.419 1.391 1.244

13.020.89b

Each value represents the mean SE (n = 3). Q24%: percentage of the cumulative amount permeated after 24 h. ER: enhancement ratio = Q24% (oint.)/ Q24% (control). aSignicant (P < 0.001) when compared with the control. bSignicant (P < 0.01) when compared with the control.

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enhancers on aspirin permeation from ointments (Table 5). Oleic acid over a concentration range of 5%20% markedly enhances aspirin permeation (P < 0.001) (Table 5). Incorporation of oleic acid at a concentration of 10% in this base produced a maximum enhancing effect (ER = 2.899). The ER was found to be 1.475 and 1.994 for 5% and 20% oleic acid, respectively. Incorporation of 5% methyl myristate produced a reduction in permeation of the drug compared with its permeation from the corresponding base itself (P < 0.05), and the ER was found to be 0.872 (Table 5). This result agrees with the results previously reported by Ammar et al. [13]. The use of 5 % limonene as a penetration enhancer had an enhancing effect (P < 0.05) on the permeation of aspirin, and the ER was found to be 1.190 (Table 5). Incorporation of 10% DMSO as a penetration enhancer improved the permeation of aspirin (P < 0.001), and the ER was found to be 1.698 (Table 5). The use of 20% urea as a penetration enhancer produced a marked reduction in the permeation of aspirin from ointment B (P < 0.001) (Table 5), and the ER = 0.369. This may be due to the high viscosity and lipophilicity of the base, which mask the enhancing effect of urea.

The use of 20% -CyD as a penetration enhancer with ointment B produced a reduction in aspirin permeation (P > 0.05) from this base, and the ER = 0.922 (Table 5). On the other hand, incorporation of 10% HP--CyD produced a pronounced reduction in the permeation of aspirin (P < 0.001), and the ER = 0.592. The straight line portions of the permeation proles of aspirin from ointment B containing different penetration enhancers show a good correlation between the cumulative amounts of drug permeated and time (R2 > 0.99), using linear regression analysis. Therefore, the penetration of aspirin from these formulations follows zeroorder kinetics. Although ointment B containing 10% oleic acid as a penetration enhancer is not the base that offers the best permeation for aspirin in this study, this base is promising. Modication of vaseline by the incorporation of 30% liquid parafn and then the addition of 10% oleic acid as a penetration enhancer increased the permeation about 7 fold and allowed high permeation of aspirin without altering the lipophilic nature of the original base which assures aspirin stability. Hence, it was selected for biological investigation.

Table 5 Effect of penetration enhancers on permeation of aspirin from vaseline-liquid parafn (7-3) ointment (ointment B) base. Enhancer Oint. B control (no enhancer) 5% oleic acid 10% oleic acid 20% oleic acid 5% methyl myristate 5% limonene 10% DMSO 20% urea 20% BCyD 10% HPBCyD Cumulative amount permeated (mg/cm2) 0.5 h 0.120.03 0.070.01 0.080.02 0.080.02 0.100.02 0.110.02 0.230.05 0.160.06 0.170.05 0.080.02 1h 0.150.05 0.090.02 0.130.06 0.120.06 0.170.05 0.170.11 0.410.06 0.180.04 0.210.08 0.100.04 2h 0.210.04 0.160.05 0.270.04 0.300.05 0.220.04 0.310.14 0.700.10 0.220.06 0.250.06 0.130.05 3h 0.260.06 0.250.06 0.480.10 0.510.11 0.270.09 0.540.12 0.900.11 0.260.06 0.270.04 0.160.04 4h 0.310.05 0.340.10 0.720.05 0.700.13 0.310.05 0.780.13 1.100.10 0.310.10 0.300.10 0.220.05 5h 0.390.07 0.500.07 0.980.11 0.800.16 0.380.06 0.940.21 1.340.11 0.340.11 0.350.12 0.260.09 24 h Q24% ER

1.790.12 25.331.70

2.640.11 37.361.55a 1.475 5.190.06 73.440.89a 2.899 3.570.15 50.522.12a 1.994 1.560.12 22.081.70c 0.872 2.130.12 30.141.70c 1.190 3.040.16 43.022.26a 1.698 0.660.02 9.340.48a 0.369

1.650.13 23.351.84d 0.922 1.060.07 15.000.99a 0.592

Each value represents the mean SE (n = 3). Q24%: percentage of the cumulative amount permeated after 24 h. ER: enhancement ratio = Q24% (with enhancer)/Q24% (control). aSignificant (P < 0.001) when compared with the control. cSignificant (P < 0.01) when compared with the control. dNonsignicant (P > 0.05) when compared with the control.

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3.3. In-vivo study The maximum intensity of platelet aggregation (Imax) was measured from platelet aggregation curves and was found to be 33.33 1.47 for the control group and 24.83 1.66 for the test group. Statistical analysis revealed a signicant difference (P < 0.05) between the test and the control groups. The percentage inhibition of platelet aggregation was found to be 25.50%.
[10]

[11]

[12] [13]

4. Conclusion
In conclusion, the results obtained suggest the feasibility of designing successful and effective transdermal delivery systems for aspirin overcoming the drawbacks of oral administration and offering additional advantages. Therefore, this study together with our previous one [13] concerning transdermal delivery of aspirin open up new ways to obtain a systemic anti-thrombotic effect by topical application of aspirin which, now, seems to be a suitable entity for transdermal delivery.

[14]

[15] [16]

[17]

[18]

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