PERMEATION AND METABOLISM OF tx-T and tx-TAc 37 reported in the literature. Also, the kinetics and rate and extent of permeation and metabolism of tx-TAc has not been very clearly elucidated thus far. The permeability of pig and miniature pig skin has been widely studied, and they have proven to be good animal models for human skin. Pig and human skin have similar surface lipids, barrier thickness, and morphology, making excised pig skin a useful model to estimate i, vitro human skin permeation behavior. The animal model chosen for this study, excised viable micro-Yucatan pig skin, permitted us to evaluate the permeation and bioconversion of (x-TAc to its major metabolite. Also, the formulation vehicle may modify properties of the stratum corneum (e.g., increased hydradon), which could influence the penetration of active ingredients (16). It is believed that skin is capable of many of the same types of metabolic processes that are present in the liver and other organs (9). The overall metabolizing capacity of the skin is less than that of the liver by nearly two orders of magnitude. However, Shiratori (17) suggested that the skin may be an important storage site for tx-T and play a major role in the distribution and metabolism of (x-T. Studies have shown that mouse skin (18) and human skin (19) contain esterases capable of converting prodrug ester compounds to bioactive drugs. Meyer and Neurand (20) found non-specific esterases to be located in the epidermis, subcuds, hair follicles, dermis, and sebaceous and apocrine glands of pig skin. The present work was carried out with the intent to (i) find out if metabolism of (x-TAc occurs in pig skin, (ii) identify the nature of the metabolite, and (ii) elucidate the kinetics in terms of rate and extent of such metabolism. The influence of two prototypic formulations on permeation and metabolism, viz., a solution of (x-TAc and (x-T and an (x-TAc emulsion, was determined. MATERIALS AND METHOD CHEMICALS AND INSTRUMENTS D-(x-Tocopheryl acetate, Covitol © 1360, was obtained as a gift from Henkel Nutrition and Health Group (Illinois). D-tx-Tocopherol was obtained as a gifk from Archer Daniels Midland Company (Illinois). The following chemicals were obtained directly from the manufacturer and used without purification: SD alcohol from Eastman (Tennessee) isopropyl myristate and mineral oil from Sigma Chemical Company (New Jersey) diisopropyl adipate and isocetyl alcohol (Ceraphyl © 230 and Ceraphyl ©, respectively) from ISP Vandyk (New Jersey) carbomer (Carbopol ©) from BF Goodrich (Ohio) Bis (2-hydroxyethyl)-ammonium hexadecyl hydrogen phosphate, DEA-cetyl phosphate (Amphisol ©) from Roche Vitamins and Fine Chemicals (New Jersey) diazolidinyl urea (Germall ©) from Sutton Laboratories (New Jersey) Ethomeen C/25 from Akzonobel (Illinois) and hydroxypropyl cellulose (Klucel ©) from Hercule (Germany). Special 30-ml centrifuge tubes were obtained from Corning Inc. The solvents acetonitrile, chloroform, and n-hexane of HPLC grade were obtained from Fisher Scientific (Springfield, N J). Water refers to freshly deionized water. The centrifuge used was from Fisher Centrific (Fisher Scientific). Organic solvents were evaporated using a Savant Speed Vac ©, SC 110. The wrist-action shaker used was from Burrell (Ohio). Vortexing was done with a Vortex-Genie (New York).

38 JOURNAL OF COSMETIC SCIENCE TOPICAL FORMULATIONS All formulations were prepared on a weight/weight basis. There were three formulations used in this study, a simple solution of ot-TAc (5%) in isopropyl myristate (IPM), an emulsion formulation of ot-TAc (5%), and a solution of ot-T (1%) in IPM. The com- position of the emulsion formulation is given in Table I. RECEPTOR FLUID In the metabolism study the receptor media must serve two important functions, viz., maintaining the viability of skin tissue and ensuring the solubility of the prodrug and its metabolite. Dulbecco's modified phosphate buffered saline (DMPBS) was used to maintain the tissue viability (21,22). As compounds that are essentially insoluble in water (such as the ot-T derivatives) may not partition freely from excised skin into an aqueous receptor fluid, bovine serum albumin (3%) was added to DMPBS (21,23). Fresh deionized water was used to prepare the buffer, and the solutions were adjusted to pH 7.4 with 10% w/v NaOH prior to use. ANIMAL TISSUE AND PREPARATION Fresh viable micro-Yucatan pig skin was obtained from Charles River Laboratories (Wilmington, MA). The pig skin was cut into squares of 10 x 10-cm 2 pieces and placed in Tupperware © containers filled with DMPBS. The skin was then set on cool packs and used for the metabolism experiments within one day of its arrival. Upon receipt the fresh skin was gently washed with a 1% (w/w) mild soap and deionized water. A 250-300- pro-thick layer of the skin was cut from the surface with a Padgett Electrodermatome TM instrument (Padgett Instrument Co., Kansas City, MO). The dermatomed skin was used the same day and was cut into 10-mm circular pieces with a brass punch and placed epidermis-side-up in Bronaugh diffusion cells. DOSING Finite dosing was used to simulate actual use conditions in all the ], vitro permeation and metabolism experiments. The smallest volume of the formulation required to obtain complete and uniform coverage of the diffusion cell surface area (0.636 cm 2) was de- Table I Composition of •-TAc (5 %) Emulsion Formulation Concentration (% w/w) Ingredient ot-TAc •-TAc 5 Diisopropyl adipate 7.5 Mineral oil 7.5 DEA-cetyl phosphate 2 Carbomer 0.3 Diazolidinyl urea 0.3 Water q.s. 100