INTRODUCTION:

Transdermal drug delivery systems are self contained discrete dosage forms which when applied to the intact skin deliver the drug through skin at a controlled rate in systemic circulation¹. Unfortunately, only very few drugs can overcome the formidable barrier of the stratum corneum (SC), which possesses a multilamellar lipid structure interposed by proteinaceous corneocytes². However, drugs proposed for transdermal delivery should possess several physico-chemical prerequisites such as shorter half-life, molecular size below 500 Da, low dose, low melting point, to be a suitable candidate for transdermal drug delivery system (TDDS) due to formidable barrier action of keratinized cells present in SC of skin³. Since, LP possesses all the above mentioned characteristics; we decided to explore it for transdermal delivery. However, LP is hydrophilic molecule and for ionisable and hydrophilic molecules, the SC prevents the attainment of a therapeutic systemic level. Therefore, several strategies can be employed to enhance the transdermal transport of such drugs through the stratum corneum like chemical enhancers, iontophoresis, sonophoresis and electroporation^4-7.

Chemical enhancers are the substances which temporarily diminish the barrier of the skin and can be used for enhancement of drug flux. Such enhancers with different proposed mechanisms of action were tested by several authors for their effects at various concentrations. The more commonly studied chemical enhancers can be broken down into three broad categories. The first class is lipid disrupting agents (LDAs), usually consisting of a long hydrocarbon chain with a cis-unsaturated carbon carbon double bond⁸. These molecules have been shown to increase the fluidity of the SC lipids, thereby increasing drug transport. In our study, oleic acid (OA) was investigated as lipid disrupting agent. A second class of permeation enhancers relies on improving drug solubility and partitioning into the skin^9-10. The lipophillic vehicle isopropylmyristate (IPM) menthol as well as the organic solvent ethanol were examined in present study.

The third class of enhancers studied were surfactants. These molecules have affinity to both hydrophilic and hydrophobic groups, which might facilitate in traversing the complex regions of the SC¹¹. An anionic surfactant sodium lauryl sulphate (SLS) and a nonionic surfactant polysorbate 80 (tween 80)were tested for their effect on LP delivery. Hence, all the chemical enhancers chosen for present studies are generally regarded as safe (GRAS) status.

LP is the first angiotensin receptor antagonist which is extensively used for treatment of hypertension. The drug is readily absorbed from the GI tract, following oral administration. The drug undergoes extensive first pass metabolism, hence its bioavailability is only 32%. The drug has a low elimination half life (2h). The normal daily dose of LP is 50 mg and its molecular weight is 461.01 Da¹². All these properties make it suitable candidate for research in transdermal delivery system. As the drug belongs to BCS class III, a suitable enhancement approach is required to deliver the therapeutic effective dose in body.

Therefore, aim of the present study was to evaluate the different concentrations of vehicles and potential of chemical enhancers like menthol, OA, IPM, SLS, tween 80 in transdermal delivery of LP across full thickness albino rat skin.

MATERIALS AND METHODS:

Materials:

Losartan potassium was received as a gift sample from Jubiliant Organosys (Noida, India). Oleic acid (OA), menthol, isopropyl myristate (IPM) (Qualikems Fine Chemicals Pvt. Ltd, Mumbai, India), sodium lauryl sulphate (SLS), tween 80 (Thomas Baker Pvt. Ltd, Mumbai, India) and n-octanol (Loba Chemie Pvt. Ltd, Mumbai, India) were procured commercially. All other chemicals and reagents used were of analytical grade.

Methods:

Apparent Partition Coefficient Determinations:

The Log K_o/w value of LP was determined using LP, n-octanol as the oil phase and PBS (7.4) as the aqueous phase. The phase volume ratio was 1:1. After equilibrating for 24 h in water bath at 37⁰ C, the organic and aqueous phase were separated, centrifuged (1000 rpm for 5 min) and filtered through 0.45 µm membrane. The filtrate was assayed spectrophotometrically.

The oil-water partition coefficient was calculated as follows:

Log K_o/w= Log (C_o/C_w)

Where, C_ois the concentration of LP in oil phase and C_wis the concentration of LP in the aqueous phase^13-14.

Apparent Partition Coefficient (APC) of Drug in Rat Skin:

The APC of drug in full thickness skin/PBS systems was determined by placing the full thickness skin (100 mg) in conical flask containing 10 mL of drug solution in PBS. The conical flask was gently rotated for 24 h, after which the concentration of drug in full thickness albino rat skin was determined¹⁵ spectrophotometrically after passing through a 0.45 µm membrane (Pall Gelman, USA).

Preparation of Rat Skin for Permeation Studies:

Skin from male albino rats was used in present studies. The experiments were carried out under the approval of the Institutional Animal Ethics Committee, M.M.U., Mullana, India. The rats were anesthetized and the hairs were removed with help of electric clippers. Full thickness skin was separated from the connective tissues and fat with help of blades. Then, the skin was kept frozen at -20⁰ C and used within 2 weeks. Before starting the experiments, the skin was allowed to reach room temperature for at least 10 min^14.

Preparation of Vehicles:

LP was added to each vehicle in excess to give saturated solubility in order to produce an equivalent thermodynamic activity. These suspensions were used for skin permeation experiments. In order to investigate the effect of the binary vehicle system on skin permeation of LP, a skin permeation study was performed using various concentrations of ethanol and other chemical enhancers. LP was also added to each binary vehicle in excess amounts of saturated solubility^17.

In Vitro Skin Permeation Studies:

Modified Franz diffusion cell (FDC) was used to study the percutaneous absorption of LP. The volume of the donor and receptor chambers was 2 and 7 ml respectively and the effective surface area for permeation of the drug through skin was 0.784 cm². The skin was mounted on the modified FDC with dermis facing the receptor chamber¹⁸. Donor side was filled with 1 mL of the test solution and the receptor chamber was filled with 7 ml of PBS and temperature of cell was maintained 37±0.5⁰C¹⁹. At predetermined time intervals, 1ml sample was withdrawn from receiver chamber and replaced with a same amount of PBS to maintain a constant volume. The samples were assayed by UV spectrophotometer after proper dilution. The similar procedure was repeated for all vehicles and chemical enhancers. Triplicate studies were performed for each experiment.

Data Analysis:

The amount of drug permeating through the skin during sampling interval was calculated based on the receptor phase drug concentration and its volume²⁰. Permeation profiles of the drug were calculated by plotting time (h) against the cumulative amount of drug (µg/cm²) measured in the receptor solution²¹. Slope of linear portion of plot was estimated as steady state flux (J_ss). The permeability coefficient (P) was obtained by dividing J by the initial drug concentration in the donor phase (C_d).

P = Flux (J_ss)/C_d

To evaluate the promoting activity of each enhancer, the enhancement ratio (ER) was calculated as, ER = J_ss (with enhancer)/J (without enhancer)

Statistical analysis was performed using one way analysis of variance (P<0.05) using Graphpad (vesion 2.01, San Diego, CA).

RESULTS AND DISCUSSION:

Apparent Partition Coefficient Determinations:

The APC of drug in n-octanol has been reported to reflect the partitioning of drugs into the intercellular spaces²². A high value of APC was observed for LP in n-octanol/PBS (2.97) which reflects that partitions into the intercellular spaces and does not form a reservoir in the dead or viable cells of the epidermis^15.

The APC value of skin/PBS (1.81) indicates that LP has an affinity to the full thickness skin. Hence, LP is a good candidate for present transdermal delivery studies.

In Vitro Skin Permeation Studies:

As mentioned previously, the present study was carried out to investigate the effect of menthol, OA, IPM, SLS, tween 80 as chemical enhancers on permeability of LP across albino rat skin. For this purpose, experiments were done using penetration enhancers (3, 5 and 10%). The concentrations of enhancers were selected from previous reports in literature^23-24.

Effect of Drug Concentration on Flux:

In order to determine the role of donor phase composition on the permeation profile of drug, three separate permeation experiments were performed by using three different concentrations (10, 20 and 30% w/v) of LP. The flux of LP was found 0.039 ± 0.007, 0.043 ± 0.005,0.07 ± 0.015 for 10, 20 and 30 % w/v concentrations of drugs respectively. Fig 1 presents the cumulative release data for this study.

Figure 1: Amount of different drug permeated for chosen concentrations of LP. Each point represents the mean ± S.E. of three experiments.

It is well known that maximum flux can be achieved from saturated solution in which thermodynamic activity is highest, however this may lead to drug crystallization in a patch or film^25-26. Hence maximum drug concentration was maintained at 30% w/v and donor concentration was not increased further. As highest permeation was recorded at 30% w/v drug concentration, subsequent studies were carried out at this concentration.

Effect of Vehicle Concentration on Flux:

The permeation parameters of LP from different vehicles across the excised hairless rat skin are listed in Table 1. The steady state flux of LP from PBS was found to be (0.065 mg/cm²/h).

Ethanol is one of the most commonly used skin permeation enhancers which is known to enhance the permeation of both hydrophillic and lipophillic drugs through rat and human skin^27-28. However, concentration of ethanol-water system greatly affected its activity. Volume fraction of 60-70% v/v or below improved drug permeation rate across the skin whereas permeation decreased with high concentration^29-31.

Therefore, a binary system of ethanol (25, 50, 75 and 100 percent) with PBS was chosen in present study. In this study, using ethanol-PBS cosolvent systems, the permeation rate of LP reached the maximum value at 50% v/v of ethanol (Fig 2 and Table 1). Results indicated that 50% v/v ethanol showed significantly (p<0.05) greater drug release as a vehicle and hence, selected for further studies using chemical enhancers.

Figure 2: Permeation profile of drug with different vehicle concentrations. Each point represents the mean ± S.E. of three experiments.

However, transdermal flux using 75% and absolute alcohol was reduced (Table 1). Different mechanisms were postulated regarding the effect of ethanol in transdermal permeation, which include: (i) ethanol enhances the rotational freedom of lipid alkyl chains, leading to an increase in the fluidity of the lipid bilayer and (ii) extraction of SC lipids, thereby altering the barrier property^32-33. However, the reduced transdermal flux by absolute alcohol was due to stabilization of the gel phase of the lipid bilayer, thereby leading to rigidization of the lipid bilayer, as reported in literature^34.

Table 1: The steady state flux data, P and ER value for different concentrations of vehicle and chemical enhancers.

Vehicles	Flux ± S.E. (mg/Cm²/h)	Permeability coefficient (P)×10^-3(cm/h)	Enhancement ratio
Control	0.07 ± 0.015	0.13 ± 0.02	1.0
25% ethanol	0.10 ± 0.003	0.19 ± 0.01	1.47
50% ethanol	0.14 ± 0.003*	0.29 ± 0.03	2.21
75% ethanol	0.09 ± 0.005	0.18 ± 0.04	1.40
Pure ethanol	0.08 ± 0.006	0.16 ± 0.07	1.20
3% Menthol	0.33 ± 0.008*	0.67 ± 0.13	5.08
5% Menthol	1.59 ± 0.026*	3.17 ± 0.23	24.19
10% Menthol	3.39 ± 0.085*	7.84 ± 0.08	59.89
3% Oleic acid	1.32 ± 0.013*	2.45 ± 0.20	20.04
5% Oleic acid	1.41 ± 0.054*	2.81 ± 0.11	21.47
10% Oleic acid	1.79 ± 0.053*	3.6 ± 0.08	27.26
3% IPM	0.72 ± 0.006*	1.44 ± 0.05	10.99
5% IPM	1.05 ± 0.036*	2.1 ±0.18	16.01
10% IPM	1.18 ±0.004*	2.36 ±0.14	18.07
3% SLS	1.23 ± 0.003*	2.62 ± 0.21	18.69
5% SLS	1.30 ± 0.026*	2.61 ± 0.17	19.90
10% SLS	2.25 ± 0.017*	4.5 ± 0.05	34.38
3% Tween 80	0.17 ± 0.003*	0.35 ± 0.03	2.66
5% Tween 80	0.20 ± 0.009	0.40 ± 0.09	3.06
10% Tween 80	0.89 ± 0.017*	1.78 ± 0.10	13.54

The flux values are presented as mean ± S.E. of three experiments. *p<0.05 compared with control.

Effect of Chemical Enhancers:

The objective of this set of studies was to assess the effects of chemical enhancers on LP penetration through rat skin. Five different chemical enhancers menthol, OA, IPM, SLS and tween 80 (3%, 5% and 10%) were employed for examining their enhancing effect of LP permeation using 50% v/v ethanol binary system as vehicle. Compared to 50% v/v ethanol binary system alone, the addition of chemical enhancers increased permeation flux regardless of the concentration of chemical enhancers.

OA, at a concentration of 3% enhanced permeation in relation to 50% v/v ethanol binary system (Fig 3). Among all these, using 3% chemical enhancers, OA showed highest flux value followed by SLS>IPM>menthol>tween 80. All the chemical enhancers showed significant flux when compared with control.

Figure 3: Cumulative amount of LP permeated hairless rat skin using 3% chemical enhancers. Each point represents the mean ± S.E. of three experiments.

The flux data, permeability coefficient and Enhancement Ratio are shown in Table 1. Data here indicated that there was a 20.04 fold increase in drug flux with 3% OA.

On further increasing the concentration from 3% to 5%, menthol showed significantly (p<0.05) highest LP flux among all 5% concentrations of different chemical enhancers (Table 1). However, at this level, the cumulative amount of drug permeated varied with the enhancers. In Fig 4, menthol (5%) showed greater cumulative amount permeated through rat skin than other chemical enhancers. With 5% concentrations, all chemical enhancers used showed significant (p<0.05) except tween 80 compared with control.

Figure 4: Cumulative amount of LP permeated hairless rat skin using 5% chemical enhancers. Each point represents the mean ± S.E. of three experiments.

Accordingly, it can also be said that penetration enhancer potencies appear to be drug specific, or at best may be predictive for a series of permeants with similar physicochemical properties. Menthol comes under the category of terpenes which was considered as a promising enhancer for transdermal use, considering the balance between efficacy and toxicity³⁵. In the present study, the increase in concentration of menthol from 5 to 10% showed a greater increase in LP flux. There was a 29.14 fold increase in flux using 5% menthol and 59.89 fold increase with 10% menthol concentration as shown in Table 1 and Fig 5. Using 10% concentrations of chemical enhancers, all flux values were found significant (p<0.05) as compared to control.

In Fig 6, the menthol at concentration 10% showed maximum flux followed by 10% SLS> 10% OA> 5% menthol> 5% OA> 3% OA> 5% SLS> 3% SLS> 10% IPM> 5% IPM> 10% tween 80> 3% IPM> 3% menthol> 5% tween 80> 3% tween 80.

Figure 5: Cumulative amount of LP permeated hairless rat skin using 10% chemical enhancers. Each point represents the mean ± S.E. of three experiments.

Figure 6: Effect of different concentrations of chemical enhancers on LP flux. Each column represents the mean ± S.E. of three experiments.

Terpenes isolated from natural essential oils are used as penetration enhancers for delivering materials across the skin membrane. Among terpenes, menthol is a monocyclic terpenoid alcohol used as promising enhancer for transdermal studies. It is also known that the polar group containing terpene (menthol) provides better enhancement for hydrophilic permeants. In addition, increased skin permeability of atenolol by menthol-ethanol system was also reported by Kobayashi et al³⁶. The mechanism by which this agent operates is by modifying the solvent nature of the SC and improving drug partitioning into the tissues. According to this hypothesis and in the case of LP (hydrophilic compound), the expected enhancement activity occurred and the Kp increased up to 11.9 fold when the concentration was increased from 3% to 10% w/v.

The surfactant like SLS played an important role in permeation enhancement of LP. In case of SLS, an increase in concentration of the surfactant resulted in an increase in permeation rate of LP and highest permeation was obtained from 10% SLS among all three concentrations (3, 5 and 10% w/v). It had been reported that anionic surfactants, like SLS, can penetrate and interact strongly with the skin, producing large alterations in the barrier properties^37-38. In particular, SLS is able to produce variations in the structural organization of lipids when it is used above the critical micellar concentration (CMC) ³⁹. An additional mechanism for the skin penetration enhancement by SLS could involve the hydrophobic interaction of the SLS alkyl chain with the skin structure which leaves the end sulfate group of the surfactant exposed, creating additional sites in the membrane which leads to permit an increase in skin hydration^{22, 40- 41.}

OA is known to be an effective penetration enhancer for a wide variety of drugs. Among 3% chemical enhancers used, OA showed highest LP flux value followed by SLS> IPM> menthol> tween 80. As compared to other chemical enhancers, 10% OA in ethanolic solution gave LP flux in the order menthol> SLS> OA> IPM> tween 80 (Fig 4). It was reported that OA provides a pathway of diminished resistance for a drug absorption by disrupting the intercellular barrier in skin absorption²⁷. Moreover, the skin permeation enhancing effect of OA is quantitatively related to amount of OA incorporated into stratum corneum bilayer⁴². In our study, when applied together with ethanol, OA is also believed to cause lipid extraction.

Table 1 summarizes the effect of IPM on LP flux, permeability coefficient and enhancement ratio. Drug permeation in presence of IPM using concentration 3, 5 and 10% w/v was found to be 10.99, 16.01 and 18.07 fold greater respectively than that of control. IPM in combination with ethanol has been reported to increase the transdermal permeation of many drugs⁴³. It had been proposed that the ethanol/IPM system increases the drug concentration in the stratum corneum by increasing its partitioning. Moreover, these led to fluidization of stratum corneum lipids causing flux enhancement^44.

To determine the effect of tween 80 (a nonionic surfactant) on LP flux, different concentrations of tween 80 (3, 5 and 10%w/v) were used in the present study. The highest permeation rate was observed with 10% tween 80 among all the concentrations used.

There are two possible mechanisms by which drug permeation enhancement takes place using nonionic surfactants using nonionic surfactant^45-46. Initially, the surfactants may penetrate into the intercellular region of stratum corneum, increase fluidity and eventually solubilize and extract lipid components. Secondly, penetration of surfactants into intercellular matrix followed by interaction and binding with keratin filaments may results in disruption within corneocytes.

CONCLUSION:

Based on the present in vitro skin permeation, a number of enhancers were shown to increase the flux of LP across skin. The nature of enhancer seems to exert an important influence on cutaneous barrier impairment. The highest permeation is obtained with 10% menthol and the lowest permeation rate with 3% tween 80. This study also shows that different chemical enhancers acted on SC and made it easy for the drug to cross the main barrier. Hence, it is concluded that enhanced skin permeation of LP could help significantly to reduce systemic side effects of oral delivery.

REFERENCES:

1. Thakur R, Anwer MK, Shams MS, Ali A, Khar RK, Shakeel F, et al. Proniosomal transdermal therapeutic system of losartan potassium: development and pharmacokinetic evaluation. J Drug Targeting. 17; 2009: 442-449.

2. Nair A, Reddy C, Jacob S. Delivery of classical antihypertensive agent through the skin by chemical enhancers and iontophoresis. Skin Res Tech. 15; 2009: 87-194.

3. Budhathoki U, Thapa P. Effect of chemical enhancers on in vitro release of salbutamol sulphate from transdermal patches. Kathmandu University Journal of Science, Engineering and Technology.1; 2005:1-8.

4. Kang L, Poh AL, Fan SK, Ho PC, Chan YW, Chan SY. Reversible effects of permeation enhancers on human skin. Eur J Pharm Biopharm. 67; 2007: 149-155.

5. Dixit N, Bali V, Baboota S, Ahuja A, Ali A. Iontophoresis- An Approach for Controlled Drug Delivery: A Review. Curr Drug Del. 4; 2007: 1-10.

6. Rao R & Nanda S. Sonophoresis: recent advances & future trends. J Pharm Pharmacol. 61; 2009: 689-705.

7. Sharma A, Kara M, Smith FR, Krishnan TR. Transdermal drug delivery using electroporation. II. Factors influencing skin reversibility in electroporative delivery of terazosin hydrochloride in hairless rats. J Pharm Sci. 89(4); 2000: 536-544.

8. Kim DD, Chien YW. Transdermal delivery of dideoxynucleoside type anti- HIV drugs. 2. The effect of vehicle and enhancer on the skin permeation. J Pharm Sci. 85; 1996: 214-219.

9. Guy RH, Hadgraft J. The effect of penetration enhancers on the kinetics of percutaneous absorption. J Control Release. 5; 1987: 43-51.

10. Gorukanti SR, Li L, Kim KL. Transdermal delivery of antiparkinsonian agent, benztropine. I. Effect of vehicles on skin permeation. Int J Pharm. 192; 1999: 159-72.

11. Sarpotdar PP, Zatz JL. Evaluation of penetration enhancement of lidocaine by nonionic surfactants through hairless mouse skin in vitro. J Pharm Sci. 75; 1986: 176-181.

12. Goa KL, Wagstaff AJ. Losartan potassium: a review of its pharmacology, clinical efficacy and tolerability in the management of hypertension. Drugs. 51; 1996: 820-45.

13. Sales OD, Castellano AL, Lacer FJM, Dominguez MH. An in vitro percutaneous absorption study of non-ionic compounds across human skin. Pharmazie. 48; 1993: 684–686.

14. Wen Z, Fang L, He Z. Effect of chemical enhancers on percutaneous absorption of daphnetin in isopropyl myristate vehicle across rat skin in vitro. Drug Del. 16; 2009: 214-223.

15. Carelli V, Di Colo G, Nannipieri E, Serafini MF. Enhancement effects in the permeation of alprazolam through hairless mouse skin. Int J Pharm. 88; 1992: 89–97.

16. Scott RC, Walker M, Dugard PH. In vitro percutaneous absorption experiments: a technique for production of intact epidermal membranes from rat skin. J Soc Cosmet Chem. 37; 1986: 35-41.

17. Rhee YS, Huh JY, Park CW, Nam TY, Yoon KR, Chi SC, et al. Effects of vehicles and enhancers on transdermal delivery of celbopride. Arch Pharm Res. 30(9); 2007: 1155-1161.

18. Larrucea E, Arellano A, Santoyo S, Ygartua P. Combined effect of oleic acid and propylene glycol on the percutaneous penetration of tenoxicam and its retention in the skin. Eur J Pharm Biopharm. 52; 2001: 113-119.

19. Bounoure F, Skiba ML, Besnard M, Arnaud P, Mallet E, Skiba M. Effect of iontophoresis and penetration enhancers on transdermal absorption of metopimazine. J Derm Sci. 52; 2008: 170-177.

20. Fang L, Kobayashi Y, Numajiri S, Kobayashi D, Sugibayashi K, Morimoto Y. The enhancing effect of a triethanolamine- ethanol- isopropyl myristate mixed system on the skin permeation of acidic drugs. Biol Pharm Bull. 25(10); 2002: 1339-1344.

21. Paula DD, Oliveira DCR, Tedesco AC, Bentley MVLB. Enhancing effect of modified beta-cyclodextrins on in vitro skin permeation of estradiol. Rev Bras Cienc Farm. 43; 2007: 111-120.

22. Panchagnula R, Ritschel WA. Development and evaluation of an intracutaneous depot formulation of corticosteroids using Transcutol as a cosolvent: in-vitro, ex-vivo and in-vivo rat studies. J Pharm Pharmacol. 9; 1991: 609–614.

23. Zhao L, Fang L, Xu Y, Liu S, He Z, Zhao Y. Transdermal delivery of penetrants with differing lipophillicities using o- acylmenthol derivatives as penetration enhancers. Eur J Pharm Biopharm. 69; 2008: 199-213.

24. Shokri J, Nokhodehi A, Dashbolaghi A, Zadeh DH, Ghafourian T, Jalali MB. The effect of surfactants on the skin penetration of diazepam. Int J Pharm. 228; 2001: 99-107.

25. Hadgraft J. Passive enhancement strategies in topical and transdermal drug delivery. Int J Pharm. 184; 1999: 1-6.

26. Moser K, Kriwel K, Naik A,Kalia YN, Guy RH. Passive skin penetration enhancement and its quantification in vitro. Eur J Pharm Biopharm. 52; 2001: 103-112.

27. Williams AC, Barry BW. Skin absorption enhancers. Crit Rev Ther Drug Carries Sys. 9; 1992: 305-353.

28. Kim DD and Chien YW. Transdermal delivery of Zalcitabine: in vitro skin permeation study. AIDS. 9; 1995: 1331-1336.

29. Chen GS, Kim DD, Chien YW. Dual- controlled transdermal delivery of levonorgestrol and estradiol: enhanced skin permeation and membrane modulatory delivery. J Control Release. 34; 1995: 129-143.

30. Kurihara- Bergstrom T, Knutson K, De Noble LJ, Goates CY. Percutaneous absorption enhancement of an ionic molecule by ethanol- water systems in human skin. Pharm Res. 7; 1990: 762-766.

31. Berner B, Mazzenga GC, Otte JH, SteffensRJ, Juang RH, Ebert CD. Ethanol: water mutually enhanced transdermal therapeutic system II: skin permeation of ethanol and nitroglycerin. J Pharm Sci. 78; 1989: 402-407.

32. Panchagnula R, Salve PS, Thomas NS, Jain AK, Ramarao P. Transdermal delivery of nalaxone: effect of water, propylene glycol, ethanol and their binary combinations on permeation through rat skin. Int J Pharm. 219; 2001: 95–105.

33. Bommannan D, Potts RO, Guy RH. Examination of the effect of ethanol on human stratum corneum in vivo using infrared spectroscopy. J Control Release. 16; 1991: 299-304.

34. Rowe ES. Lipid chain length and temperature-dependence of ethanol phosphatidyl choline interactions. Biochemistry. 22; 1983: 3299-3305.

35. Higaki K, Amnuaikit C, Kimura T. Strategies for overcoming the stratum corneum: chemical and physical approaches. J Drug Delivery. 1; 2003: 187–214.

36. Kobayashi D, Matsuzawa T, Sugibayashi K, Morimoto Y, Kobayashi M, Kimura M. Feasibility of use of several cardiovascular agents in transdermal therapeutic systems with l-menthol-ethanol system on hairless rat and human skin. Biol Pharm Bull. 16; 1993: 254–258.

37. Walters KA, Dugard PH, Florence AT. Non-ionic surfactants and gastric mucosal transport of paraquat. J Pharm Pharmacol. 33; 1981: 207–213.

38. Cheon Koo L, Takahiro U, Kazahosa K, Akira Y, Nak-Seo K, Shigeru GJ. Skin permeability of various drugs with different lipophilicity. J Pharm Sci. 8; 1994: 562–565.

39. Ribaud CH, Garson JC, Doucet J, Leveque JL. Organization of stratum corneum lipids in relation to permeability: influence of sodium lauryl sulphate and preheating. Pharm Res. 11; 1994: 1414-1418.

40. Rhein LD, Robbins CR, Fernee K, Cantore R. Surfactants structure effects on swelling of isolated human stratum corneum. J Soc Cosmet Chem. 37; 1986: 199-210.

41. Gibson KT, Teall MR. Interactions of C12 surfactants with the skin: changes in enzymes and visible and histological features of rat skin treated with sodium lauryl sulphate. Food Chem Toxicol. 21; 1983: 587-594.

42. Francoeur ML, Golden GM, Potts RO. Oleic acid: its effects on stratum corneum in relation to (trans) dermal drug delivery. Pharm Res. 7; 1990: 621-627.

43. Suwanpidokkul N, Thongnopnua P, Umprayn K. Transdermal delivery of zidovudine (AZT): the effects of vehicles, enhancers, and polymer membranes on permeation across cadaver pig skin. AAPS Pharm Sci Tech. 2004; 3, Article 48: E1–8.

44. Ogiso T, Hata T, Iwaki M, Tanino T. Transdermal absorption of bupranolol in rabbit skin in vitro and in vivo. Biol Pharm Bull. 5; 2001: 588–91.

45. Breuer MM. The interaction between surfactants and keratinous tissues. J Soc Cosmet. 30; 1979: 41–64.

46. Walters KA, Walker M, Olejnik O. Non-ionic surfactant effects on hairless mouse skin permeability characteristics. J Pharm Pharmacol. 40; 1987: 525-529.

Received on 12.01.2012 Modified on 30.01.2012

Research J. Pharm. and Tech. 5(3): Mar.2012; Page 346-352