j. Soc. Cosmet. Chem., 45, 119-134 (May/June 1994) Influence of liposomal encapsulation on the penetration of retinoic acid through human skin in vitro DELPHINE IMBERT, GERALD B. KASTING, and R. RANDALL WICKETT, The University of Cincinnati College of Pharmacy, 3223 Eden Avenue, Cincinnati, OH 45267-0004 (D.I., R.R.W.), and The Procter & Gamble Company, Miami Valley Laboratories, Cincinnati, OH 45239-8707 (G.B.K. ). Accepted May 21, 1994. Synopsis The penetration of liposome-encapsulated trans-retinoic acid (t-RA) through the different strata of the skin was investigated using in vitro diffusion experiments on human cadaver skin. Simple phospholipid liposomes and a more complex pH-sensitive preparation containing 0.05% t-RA were applied to isolated human stratum corneum, isolated human epidermis, and dermatomed human skin, along with one of two non- liposomal controls. The control solutions were either 0.05% t-RA in ethanol (for small-dose experiments) or saturated solutions of t-RA in Transcutol©/water mixtures (for large-dose experiments and some small- dose experiments). Skin-specific interactions were differentiated from solution thermodynamic effects by repeating some experiments using a silicone rubber membrane. The influence of dose volume and occlusion was investigated. The results showed no evidence of enhanced epidermal penetration, nor of decreased percutaneous absorption from liposomal t-RA vs unencapsulated controls under realistic dose conditions. However, under one set of exaggerated dose conditions (consisting of a large, non-occluded dose of t-RA in PC liposomes), there was a suggestion that diffusion of t-RA across the lower skin layers may have been retarded by the liposomal components. INTRODUCTION Over the past fifteen years liposomes have been claimed to provide topical delivery benefits for both systemically active and locally active drugs. For systemic drugs, workers have reported increased percutaneous absorption from either liposomal disper- sions (1-2) or other related phospholipid formulations (3-5), leading to higher systemic levels for a given topical dose. For topical drugs, a commonly cited benefit is increased levels of active in the skin (including, in many cases, the lower epidermis and dermis), combined with decreased percutaneous absorption (6-13). This implies reduced drug clearance by the capillary bed. Other investigators have found no effects of liposomes on topical delivery (14). These findings have been reviewed by Egbaria and Weiner (15). Trans-retinoic acid (t-RA, also called tretinoin or vitamin A acid), a widely used anti-acne medication, is one of the drugs for which the skin localization benefit has been claimed (6,7, 16). Given the side effects associated with systemic retinoid therapy (17), 119

120 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS such a change in delivery pattern would be expected to widen the safety margin asso- ciated with topical use. Somewhat awkward for liposome proponents is the lack of a coherent explanation of how liposomes might increase drug concentrations in skin while reducing systemic absorp- tion. The phenomenon implies that liposome components penetrate through the stra- tum corneum and affect drug clearance from the underlying tissues. Yet, careful inves- tigations have failed to show appreciable concentrations of either intact liposomes or of liposome components (phosphatidylcholine or cholesterol) in lower skin layers following topical applications (6,14,18-20), and to our knowledge, no effects of liposomes on cutaneous blood flow have been reported. Thus, the mechanism for the liposome effect remains a mystery, and one is led to examine the methods by which the unexpected results were derived. Most of the evidence for increased drug concentrations in skin following liposomal application has been obtained from skin stripping experiments. This technique, while useful for estimating drug levels in the stratum corneum, does not clearly differentiate between the stratum corneum and the underlying skin layers. We reasoned that a properly controlled technique that measured delivery rates through increasingly thick layers of skin should be able to identify formulations leading to drug accumulation in the lower skin layers. Either the input rate (flux through the stratum corneum) must be higher or the output rate (flux through the whole tissue) must be lower in order for accumulation to occur. With this in mind, we studied the flux of liposomally encap- sulated t-RA vs unencapsulated controls through human stratum corneum, heat- separated human epidermis, and dermatomed human cadaver skin. We studied two liposome systems--a simple soy phosphatidylcholine system and a more complex, four- component system claimed to deliver enzymes through skin (21,22)--and compared their performance against an ethanolic vehicle and a saturated t-RA solution in transcutol/water. We also tested the influence of dosing conditions (dose volume, oc- clusion) on the penetration results and used an inert membrane to identify purely thermodynamic effects. This allowed us to determine whether or not a specific skin/ liposome interaction was taking place. MATERIALS AND METHODS MATERIALS Soya phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were obtained from Avanti Polar Lipids (Alabaster, AL). Cholesteryl hemisuccinate (CHEMS), oleic acid (OA), ot-tocopherol, all trans-retinoic acid (t-RA), [11, 12-3H(N)]-retinoic acid ([3H]t- RA, specific activity 49.30 Ci/mmol), bovine pancreas trypsin, type II-S soybean trypsin inhibitor, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), Dul- becco's phosphate-buffered saline (PBS), and EDTA (disodium salt) were purchased from Sigma (St. Louis, MO). Transcutol (diethylene glycol monoethylether) was obtained from Gattefosse (Elmsford, NY). Tritiated water (specific activity 1.6 ptCi/ml) was obtained from Dupont/NEN (Boston, MA). All other chemicals were of analytical grade.