Effect of Azone on the iontophoretic transdermal delivery of metoprolol tartrate through human epidermis in vitro

journal o f

ELSEVIER

Journal of Controlled Release 42 (1996) 57-64

controlled release

Effect of Azone on the iontophoretic transdermal delivery of metoprolol tartrate through human epidermis in vitro a~

S. Ganga " , R Ramarao b, J. S i n g h c aProduct Development Cell, National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110067, India bNational Institute of Pharmaceutical Education and Research, Sector 67, Phase X, SAS Nagar, Mohali, 160 062, India CDepartment of Pharmaceutical Sciences, College of Pharmacy, North Dakota State University, Fargo, ND 58105, USA

Received 7 July 1995; revised 9 November 1995; accepted 5 February 1996

Abstract

This study was attempted to investigate the effect of Azone on the transdermal iontophoretic transport of metoprolol tartrate (MPL) through human epidermis in vitro. Investigations were done to probe the mechanism of action of this enhancer, and to see whether there is any synergistic effect of this enhancer in conjunction with iontophoresis. Along with comparison of iontophoretic and passive transport of the drug in presence of the enhancer, parameters like steady state flux (Jss), permeability coefficient (Kp) and enhancement factors (E) were evaluated. It was found that both during passive diffusion and iontophoresis, Azone caused increased transport of the drug through human epidermis and the transport was increased 130-fold during iontophoresis compared to passive flux. These results were supported by scanning electron microscopy studies of the epidermis after experimentation. Keywords: Transdermal; Iontophoresis; Metoprolol tartrate; Azone; Human epidermis

I. I n t r o d u c t i o n

Iontophoresis (G.K. iontos = ion, phoresis = to bear) is the process of moving charged or ionized species across a membrane under a potential gradient. Iontophoretic transdermal delivery uses DC electric current between two electrodes to enhance the movement of ionized drugs through intact skin. Several investigators have shown the feasibility of delivering drugs iontophoretically both in-vitro [1-5] and by in-vivo methods [6-9]. Penetration enhancers may moderate the iontophoretic regimen required to achieve the target flux, thus improving the tolerability of skin to iontophoretic regimen. Few workers Corresponding author.

have examined the effect of iontophoresis in combination with permeation enhancers as a potential means to enhance and control the transdermal delivery of drugs [10-15]. Since not much work has been done in this direction, there is need to explore the possibility of a synergistic effect of a chemical penetration enhancer and iontophoresis on the transdermal transport of the drugs. This would enable the drug to reach the therapeutic levels at much lower current density than would be necessary with iontophoresis alone. The mechanism by which most enhancers act is largely unknown and under thorough investigations. Hence the visualization of the changes in the surface of human skin by scanning electron microscopy (SEM) due to application of permeation enhancers either alone or in combination

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S. Ganga et al. / Journal of Controlled Release 42 (1996) 5 7 - 6 4

with iontophoresis may help in predicting the possible mechanism of action of permeation enhancers. Metoprolol tartrate (MPL) chosen for the present studies is a selective beta-l-adrenoreceptor blocking agent routinely used in oral antihypertensive therapy. It lowers mean arterial blood pressure through a reduction in cardiac output produced primarily by slowing the heart rate [16]. Orally administered metoprolol undergoes extensive hepatic first pass elimination with the result of which only 50% of the initial dose reaches the systemic circulation. This drug is basic in nature with a pKa value of 9.5, a t 1/2 of 3.5 h and molecular weight of about 685, hence a potential candidate for the present work on transdermal studies [17]. In this work, we attempted to investigate the effect of Azone on the passive and iontophoretic in vitro transport of metoprolol tartrate from human epidermis, followed by scanning electron microscopy of skin samples, to probe the mechanism of action of Azone.

2. Materials and methods Metoprolol tartrate was a gift sample from Cipla Pvt. Ltd., Bombay. Trisodium citrate, glycine, and sodium chloride was obtained from Glaxo Lab (India) Ltd., Bombay. Sodium cacodylate, osmium tetraoxide were obtained from Central Drug House (Pvt. Ltd) Bombay and Loba-Chemie Bombay. Azone was obtained from Nelson Research, Irvine, California, USA.

2.1. Electrodes, cell design and instruments Platinum electrodes and side-by-side diffusion cells were selected for the present studies. The excised human epidermis was sandwiched between the cells with the dermal side towards receiver compartment. It was supported in this position by a wire mesh. A thin film of silicone grease was spread on lapped glass surfaces of the cell to provide a water-tight seal. For passive diffusion studies the cells were clamped and immersed in a water bath at 37 _+ 0.5°C. On the other hand, iontophoretic experiments using anodal iontophoresis were carried out by inserting platinum electrodes (platinum wires,

99.9% purity, 2 cm x 0.5mm) in the diffusion cells at a current density of 0.53 m A / c m 2. The constant current required in the iontophoretic studies was generated from a D.C. constant current source (fabricated at the University Science and Instrumentation Center, BHU, Varanasi, India). Glycine buffer at pH 7.4 was used as the donor buffer while phosphate buffer saline (PBS) at pH 7.4 was used as the receptor buffer. Changes in the pH were monitored and corrected by the addition o f / z l amounts of 1 M HC1 or 1 M NaOH solutions. By this technique, the pH was kept within _+0.2 units of the desired pH as determined at the end of the experiment. Three and a half ml of donor and receiver volumes was used. Samples were withdrawn at regular intervals from the receiver compartment (0.1 ml) for drug analysis and the same volume was replaced each time by fresh fluid. Also, at the start and end of the experiment, 0.1 ml of the donor solution was sampled out for analysis. The samples were suitably diluted with PBS, pH 7.4, and the absorbance was measured at 221 nm on a Beckman Model-24 spectrophotometer. Azone was added to the donor compartment at 5% w/v concentration. The drug solution was emulsified with the aid of 0.11% w/v Tween 20 [18]. The Beckman Model-24 spectrophotometer was used for analyzing the amount of drug transported through skin during different studies. The absorbance was read at 221 nm A max.

2.2. Collection and preparation of human skin samples Samples of skin of 21-50-year-old Caucasian cadavers of either sex, including the subcutaneous fat, approximately 25 cm x 6 cm were removed from the mid-abdominal region within 24 h of death and stored at -20°C. The subcutaneous fat was trimmed and the method of Kligman and Christophers was adopted to remove the epidermis [19]. The full thickness skin was immersed in water maintained at 60 - 0.5°C for 3 min. The skin was removed from the water, blotted dry and pinned dermal side down to a dissecting tray. The epidermis was peeled off with the help of ear buds, washed several times with water, dried overnight at room temperature and stored at - 2 0 ° C before use. The epidermis was allowed to thaw overnight at room

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S. Ganga et al. / Journal of Controlled Release 42 (1996) 5 7 - 6 4

temperature and rehydrated by immersing in water for 1 h before experimentation [20]. Scanning electron microscopy of skin samples after experimentation was done on a Phillips 515 Scanning Electron Microscope at varying magnifications and the selected areas were photographed using Indu 125 ASA Black and White 120 mm roll film.

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Fig. 1 shows the effect of Azone on the permeation profile of MPL during passive diffusion. As can be seen, Azone increased the transport of the drug through human epidermis, compared to the control where no enhancer has been used. When iontophoresis was used in conjunction with Azone, it produced greater transport of the drug compared to iontophoresis alone (Fig. 2). Azone increased the permeability coefficient of the drug during passive diffusion; however, it was greater during iontophoresis (Fig. 3). Table 1 gives the steady state flux values (Jss) for the transport of the drug. Jss was obtained from the slope of the linear portion of the plot of time vs. cumulative amount of the drug released per cm 2 area of the skin. The highest steady state flux was obtained when Azone and ion2000

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tophoresis are used in conjunction. Table 2 gives the enhancement factors for MPL with Azone. As evident iontophoresis coupled with Azone enhanced the flux of MPL by 130 folds compared to the passive flux. The scanning electron microscopy (SEM) of some samples of human epidermis was carried out to observe the changes in the surface characteristics of

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(PD = PASSIVE DIFFUSION ) Fig. 1. Effect of Azone on the transport of MPL through human epidermis during passive diffusion. (Data are shown as mean _+ SD, n = 3).

Fig. 3. Permeability coefficient of MPL from different formulations through human epidermis.

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S. Ganga et al. / Journal of Controlled Release 42 (1996) 57-64

Table 1 Effect of Azone on the steady state flux (Jss) of MPL through human epidermis at pH 7.4 during passive diffusion and iontophoresis

the skin due to application of Azone either alone or in combination with iontophoresis. With scanning electron microscopy, the horizontal plane of the epidermis can be satisfactorily inspected with the final image having a three dimensional appearance

[21]. The microphotograph of the untreated epidermis at x 2 0 0 magnification (Fig. 4) shows closely united assembly of squamous cells, with a ridge and furrow pattern [22]. The cells show irregular folds, convolutions and occasionally a villous appearance. Cell margins are difficult to observe as adjacent cells appear to merge because of their close apposition. The central bulge or depression present on few cells m a y be attributed to the position of nucleus before 'cell death' [21]. These cells enclose keratin filaments embedded in amorphous matrix of mainly lipid and non-fibrous protein. All the cells show irregular folds and convolutions. SEM reveals the polygonal shape of desquamative cells [23]. SEM of the skin samples after passive diffusion with Azone shows loosening of the closely compact structure (Fig. 5). Under high magnification, it can be seen that epidermal cells are not swollen and they seem to be disengaged from each other (Fig. 6). The intercellular space is slightly distended. Iontophoresis alone produced considerable effect on the epidermal surface of human skin as observed in Fig. 7. It has been reported that the presence of an electric field may change the permeability of the skin by increasing the fluidity of lipids by polarizing skin proteins [24]. As can be seen, the dense compact structure has become loose and the cells are completely flattened. When Azone was used for ion-

Fig. 4. Scanning electron microphotograph showing epidermal surface of untreated human skin. (x 200).

Fig. 5. Scanning electron microphotograph showing epidermal surface of human skin after passive diffusion with Azone. (x 200).

Experimental

Jss (/~g/cm2/h)

conditions

Passive diffusion

Iontophoresis

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10.39 -+ 3.17 83.97 +_ 10.2

478 +- 23.11 1369.18 +_ 17.96

Table 2 Enhancement factors for MPL Enhancement types

Enhancement factors

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"E~ = Jpe/Jp; b E 2 = Jie/Jpe; C E 3 = Jie/Jp. Jpe = Jss during passive diffusion using Azone. Jie = Jss during iontophoresis using Azone. Jp = Jss during passive diffusion without enhancer.

S. Ganga et al. I Journal of Controlled Release 42 (1996) 57-64

61

4

Y S Fig. 6. Scanning electron microphotograph showing epidermal surface of human skin after passive diffusion with Azone.

Fig. 8. Scanning electron microphotograph showing epidermal surface of human skin after iontophoresis using Azone. (x200).

(x 1000).

tophoretic studies, the scanning electron microphotograph shows dramatic change at the intercellular regions (Fig. 8). It can be observed that the junctions between the cells have become extremely loose and the cell separation has increased considerably resulting in very large intercellular spaces. The addition of Azone to the donor solution has

Fig. 7. Scanning electron microphotograph showing epidermal surface of human skin after iontophoresis. (X200).

increased the flux and permeability of MPL through human epidermis as observed from the results, suggesting a decrease in the barrier resistance of stratum corneum, by the enhancer. Most of the penetration enhancers act by altering the fluid properties of the stratum corneum lipids, or altering the conformation, or denaturing skin proteins [25]. There is strong evidence that penetration enhancers act on different permeation pathways to varying degrees [26]. Morphological and biochemical studies support the physical and chemical evidence of separate hydrophilic and lipophilic domains in the barrier area and the penetrants with both hydrophilic and lipophilic properties probably penetrate the stratum corneum more readily [27,28]. Barry observed in some detail the effects of Azone on the stratum corneum, with DSC thermograms and permeability studies [29]. It was found that Azone dramatically affected the lipid structure. Azone did not enter cells in significant amounts. The DSC data and Azone's non-polar nature suggested that it partitions directly into the lipid bilayer structure, hence increasing the drug permeation through this less rigid environment. A number of other workers have also tried to elucidate the mechanism of action by Azone and have reported that Azone exerts a considerable effect on the fluidity of lipids of stratum corneum [14,18,30-37].

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Ogiso et al. have further elucidated the mechanism of action of Azone using shed snake skin and hairless rat skin using the techniques of scanning electron microscopy and ESR [38]. Observations of hairless rat skin treated with Azone indicated looseness and cell separation of stratum corneum probably caused by extensive extraction of intercellular lipids. ESR study demonstrated this fact, as increased fluidity of the corneum was observed which was probably caused by extensive extraction of intercellular lipids. This may be due to the effect of Azone on the stratum corneum lipids. Sugibayashi et al. have also studied the mechanism of skin penetration enhancing effect of Azone [39]. They studied the effects of Azone on skin components and compared with that of other penetration enhancers. Azone was found to markedly fluidize liposomal lipids compared to other enhancers. It also increased water content in the stratum corueum as was found by measuring skin conductance. They reported that Azone may act on a lipid rich route because it fluidizes the stratum corneum lipids more over the water rich route because it enriches the water content. Singh has also attributed the increased transport of methotrexate through human epidermis, in the presence of Azone, to the disruption of lipid bilayer [13]. Stoughton and McClure have found

2%-10% concentration of Azone to be appropriate for most of the formulations [40], The mechanism of action of Azone discussed earlier can be confirmed by SEM of skin samples treated with Azone. The cells seem to have loosened and, owing to disengagement, the intercellular space is distended. This may be attributed to the extraction of intercellular lipids by Azone. A second increase in flux after 22 h of study may be attributed to the phenomenon of hydration. Hydration aids drug mobility within the cell by creating more fluid character and competing for hydrogen bonding sites [29]. The above phenomenon is confirmed by SEM studies as very large intercellular spaces can be observed owing to disengagement of cells (Fig. 9).

4. Conclusions These results indicate that the effect of Azone and constant current iontophoresis is additive in enhancing the permeability of MPL. Iontophoresis alone produced considerable effect (Fig. 7) and Azone caused increased flux for MPL when coupled with iontophoresis compared to passive diffusion studies. SEM studies have confirmed the above observations as Azone has caused considerable loosening of cells with increased cell separation resulting in very large intercellular spaces. In addition, Azone not only fluidizes the skin lipids but also hydrates the skin, resulting in enhancement of water soluble drugs like MPL. Infact, Srinivasan et al., Singh, Gay et al., Hinsberg et al. and Lashmar and Manger have reported an additive effect of iontophoresis and chemical enhancers on the transdermal transport of drugs [10], [13], [4], [14,15]. Hence, from the present studies it can be concluded that despite the general observation that enhancers interact with the lipids and proteins, and iontophoresis drives ions across the skin through porous pathways, in combination the enhancement strategies do not necessarily operate in isolation and may be infact additive.

Acknowledgments Fig. 9. Scanning electron microphotograph showing epidermal surface of human skin after 22 h of iontophoretic studies depicting hydration effects.

We wish to thank M / s Cipla Ltd., Bombay for providing the gift sample of metoprolol tartrate, Dr.

S. Ganga et al. / Journal of Controlled Release 42 (1996) 57-64

S.C. Maitra, Assistant Director, Medicinal Chemistry Division, RSIC, Central Drug Research Institute, Lucknow, India for SEM studies and Dr. C.B. Tripathi, Head, Forensic Sciences, Institute of Medical Sciences, Banaras Hindu University, Varanasi, India.

References [1] E Tyle, Iontophoretic devices for drug delivery. Pharm. Res. 3 (1986) 318-326. [2] M.S. Roberts, J. Singh, N. Yoshida and K.I. Currie, Iontophoretic transport of selected solutes through human epidermis, in: R.C. Scott, J. Hadgraft and R. Guy (Eds.), Prediction of Percutaneous Absorption, IBC Technical Service Ltd., London, 1989, pp. 231-241. [3] M.R. Lark and L.P. Gangarosa, Iontophoresis an effective modality for the treatment of inflammatory disorders of the temporomandibular joint and myofascial pain, J. Craniomandib. Pract. 8 (1990) 108-119. [4] C.L. Gay, P.G. Green, R.H. Guy and M.L. Francoeur, Iontophoretic delivery of piroxicam across the skin in-vitro, J. Control. Rel. 22 (1992) 57-68. [5] S.K. Gupta, S. Kumar, S. Bolton, C.R. Behl and A.W. Mallick, Optimization of iontophoretic transdermal delivery of a peptide and a nonpeptide drug delivery, J. Control. Rel. 30 (1994) 253-261. [6] R.V. Padmanabhan, J.B. Phipps, G.A. Lattin and R.J. Sawchuk, In vitro and in vivo evaluation of transdermal iontophoretic delivery of hydromorphone, J. Control. Rel. 11 (1990) 123-135. [7] T. Tsuji, Bleomycin: Iontophoretic therapy for verrucous carcinoma, Arch. Dermatol. 127 (1991) 973-975. [8] T.J. Petelenz, J.A. Buttke, C. Bonds, L.B. Lloyd, J.E. Beck, R.L. Stephen, S.C. Jacobsen and P. Rodriguez, Iontophoresis of dexamathasone: lab studies, J. Control. Rel. 20 (1992) 55-56. [9] Y. Sun, H. Xue, H. Gu and J. Zhang, Rapid drug absorption following transdermal iontophoresis, Proceed. Intern. Symp. Control. Rel. Bioact. Mater. 19 (1992) 479-480. [10] V. Srinivasan, M.H. Su, W.I. Higuchi and C.R. Behl, Iontophoresis of polypeptides: effect of ethanol pretreatment of human skin, J. Pharm. Sci. 79 (1990) 588-591. [11] L. Wearley and Y.W. Chien, Enhancement of the in-vitro skin permeability of Azidothymidine (AZT) via iontophoresis and chemical enhancers, Pharm. Res. 7 (1990) 34-40. [12] S. Singh, Ph.D Thesis, Passive and iontophoretic delivery of some model drugs through human skin. Banaras Hindu University, Varanasi, India, 1991. [13] W.H.M.C. Hinsberg, H.E. Bodde, C.J. Verhoef, L.J. Bax and H.E. Junginger, Effect of dodecyl Azone on skin resistance and iontophoretic peptide flux using two skin models: human and shed snake skin, Proceed. Intern. Symp. Control. Rel. Bioact. Mater. 19 (1992) 483-484.

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[14] J. Hirvonen, L, Murtomaki, K. Konturri, P. Paronen and A. Uttri, Transdermal permeation of model anions and cations: Effect of skin charge, iontophoresis and penetration enhancers, Proceed. Intern. Symp. Control. Rel. Bioact. Mater. 19 (1992) 452-453. [15] U.T. Lashmar and J. Manger, Investigations into the potential for iontophoresis facilitated transdermal delivery of acyclovir, Int. J. Pharm. 111 (1994) 73-82. [16] Martindale The Extra Pharmacopoeia (29th Eds.) London Pharmaceutical Press, 1989, pp. 791-792. [17] J.R. Luch, Metoprolol Tartrate, in: K. Florey (Eds.), Analytical Profiles of Drug Substances, Vol 12, Academic Press, USA, 1983, pp. 325-386. [18] Y. Morimoto, K. Sugibayashi, K. Hosoya and W.I. Higuchi, Penetration enhancing effect of Azone on the transport of 5-fluorouracil across hairless rat skin, Int. J. Pharm. 32 (1986) 31-38. [19] A.M. Kligman and E. Christophers, Preparation of isolated sheets of human statum comenm. Arch. Dermatol. 88 (1963) 702-710. [20] J. Swarbrick, G. Lee and J. Brom, Drug permeation through human skin. I. Effect of storage conditions of skin, J. Invest. Derm. 78 (1982) 63-66. [21] R.P.R. Dawber, R. Marks and J.A. Swift, Scanning electron microscopy of the stratum corneum, Br. J. Derm. 86 (1972) 272-281. [22] R. Marks and R.P.R. Dawber, Skin surface biopsy: An improved technique for the examination of the horny layer, Br. J. Derm. 84 (1971) 117-123. [23] J.P. Carteud, Scanning electron microscopy in skin pathology, in: Scanning Electron Microscopy: Proceedings of the workshop on scanning electron microscopy in pathology (IIT Research Institute, Chicago, Illinois, USA, 1973) 3 pp. 629-635. [24] L. Wearley, Jue-Chen Liu and Y.W. Chien, Iontophoresis facilitated transdermal delivery of verapamil. II. Factors affecting the reversibility of skin permeability, J. Control. Rel. 9 (1989) 231-281. [25] B.J. Aungst, N.J. Rogers and E. Shefter, Enhancement of naloxone penetration through human skin in-vitro using fatty acids, fatty alcohols, surfactants, sulfoxides and amides, Int. J. Pharm. 33 (1986) 225-234. [26] B.W. Barry and S.L. Bennet, Effect of penetration enhancers on the permeation of mannitol hydrocortisone and progesterone through human skin, J. Pharm. Pharmacol. 39 (1987) 535-546. [27] P.M. Elias, Epidermal lipids, barrier function and desquamation, J. Invest. Dermatol. 80 Suppl. (1983) 44-49. [28] A. Hoelgaard, B. Mollguard and E. Baker, Vehicle effect on topical drug delivery. IV. Effect of N-methylpyrrolidone and polar lipids on percutaneous drug transport, Int. J. Phann. 43 (1988) 233-240. [29] B.W. Barry, Penetration enhancers: Mode of action in human skin, in: B. Shroot and H. Schaefer (Eds.), Skin Pharmacokinetics., Karger, Basel, 6, 1987, pp. 121-137. [30] M. Goodman and B.W. Barry, Differential scanning calorimetry of human stratum corneum: effect of Azone, J. Pharm. Pharmacol. 37 Suppl. (1985) 80P.

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S. Ganga et al. / Journal of Controlled Release 42 (1996) 5 7 - 6 4

[31] J.C. Beastall, J. Hadgraft and C. Washington, Mechanism of action of Azone as a percutaneous penetration enhancer: lipid bilayer fluidity and transition temperature effects, Int. J. Pharm. 43 (1988) 207-213. [32] J. Bouwstra, L. Peschier, J. Brusse and H. Bodde, Effect of n-alkyl azocycloheptanan-2-ones including Azone on the thermal behaviour of human stratum comeum, Int. J. Pharm. 52 (1989) 47-54. [33] P.P. Bhatt, J.H. Rytting and E.M. Topp, Influence of Azone and lauryl alcohol on the transport of acetaminophen and ibuprofen through shed snake skin, Int. J. Pharm. 72 (1991) 219-226. [34] J. Bouwstra, M. DeVries, G. Gooris, W. Bras, J. Brusse and M. Porrec, Thermodynamic and stuctural aspects of the skin barrier, J. Control. Rel. 15 (1991) 209-220. [35] F. Schuckler and G. Lee, Relating the concentration dependent effect of Azone and dodecyl-~-pyroglutamate to change in drug diffusivity, partition coeffient and flux, Int. J. Pharm. 80 (1992) 81-89.

[36] B.B. Michniak, M.R. Player, J.M. Chapman Jr. and J.W. Sowell Sr., In-vitro evaluation of a series of Azone analogs as dermal penetration enhancers, Int. J. Pharm. 91 (1993) 85 -93. [37] B.B. Michniak, M.R. Player, J.M. Chapman Jr. and LW. Sowell Sr., Azone analogues as penetration enhancers: effect of different vehicles on hydrocortisone acetate skin permeation and retention, J. Control. Rel. 32 (1994) 147-154. [38] T. Ogiso, M. Iwaki, K. Bechako and Y. Tsutsumi, Enhancement of percutaneous absorption by laurocapram, J. Pharm. Sci. 81 (1992) 762-767. [39] K. Sugibayashi, S. Nakayama, T. Seki, K. Hosoya and Y. Morimoto, Mechanism of skin penetration enhancing effect by laurocapram, J. Pharm. Sci. 81 (1992) 58-64. [40] R.B. Stoughton and W.O. McClure, Azone: A new nontoxic enhancer of cutaneous penetration. Drug. Dev. Ind. Pharm. 9 (1983) 725-744.