270 JOURNAL OF COSMETIC SCIENCE discussed the effects of storage, heat separation, and freezing on human cadaver skin viability for in vitro percutaneous absorption. Another factor of potential importance is the dermal microvasculature in skin absorption, and therefore, appropriate use of full- thickness or split-thickness skin preparations in permeability studies also requires fur- ther attention. Use of full-thickness human or animal skin may result in an artificial reduction in permeation, particularly for compounds with high lipid solubility (e.g., c•-tocopherol). The absorption of hydrophobic compounds must be measured with an appropriate receptor fluid. Percutaneous absorption of vitamin E acetate into and through human skin has been studied by Wester and Maibach (35). Dermatomed human cadaver skin (500 pm) was mounted into glass cells with flow-through design, with a 1-cm 2 surface area. Two formulations, A and B, were used with labeled vitamin E acetate as the active. At 24 h the receptor fluid (buffered saline) was analyzed, and the skin surface was washed, digested, and analyzed. The majority of vitamin E acetate was recovered in the skin surface wash. There was no difference, however, in vitamin E acetate absorption between the two formulations, as both were able to deliver vitamin E acetate into and through human skin. Results are shown in Figure 12. About 1.5-1.7% of the applied dose was found in the skin, and minimal quantities, 0.63-0.78%, were found in the receptor fluid. This study showed that vitamin E acetate can be delivered into human skin and into the systemic circu- lation with a simple topical formulation. However, a limitation of this study is that it did not consider the effect of metabolism of the acetate into the active vitamin E or the skin enzymes involved in such a conversion. Also, the receptor medium used in the study, buffered saline at a flow rate of 3 ml/hour, did not ensure that sink conditions were maintained. In another in vitro study on rat skin 6 h after application of a 5% vitamin E alcoholic 120 100 • 8O 60 4o 20 Receptor Skin Fluid Content Surface Wash [] Fommla A [] Formula B Figure 12. In vitro percutaneous absorption of vitamin E acetate into and through human skin. Adapted from reference 35.

SKIN DELIVERY OF VITAMIN E 271 solution, 38.6% of the applied dose was recovered in the viable epidermis and dermis the amount detected in the horny layer was 7.12%, and the residual fraction persisting on the surface was 54.3% of the applied dose (109). I, vivo stzzdy. To establish the extent to which ot-tocopheryl acetate was bioconverted to ot-tocopherol, Beijersbergen van Henegouwen eta/. (96) studied the i, vivo permeation of vitamin E and its prodrug across rat skin membrane. Both 2.5% ot-tocopherol and 2.5% ot-tocopheryl acetate solutions in ethanol were applied to specified areas on the back of rats. At various time intervals the animals were sacrificed and the dorsal skin removed. The epidermis was separated from the dermis using 2 M KI for 1 h at room temperature. The epidermis was then removed with a scalpel. The authors found that both ot-tocopherol and its acetate behave similarly with regard to the penetration and horizontal migration through the epidermis. The pH of the stratum corneum is estimated to be about 5.0, and that of the viable tissue is about 7.4. The aromatic hydroxyl group in ot-tocopherol (pKa about 10, comparable to various phenol derivatives with similar molecular structures) is not dissociated. Hence the difference between ot-tocopheryl acetate and ot-tocopherol with regard to its physico- chemical parameters that determine skin transport was negligible. After a period of 5 h after a single application of ot-tocopheryl acetate, the amount of ot-tocopherol found did not differ significantly from that already present. Even after five days the amount of ot-tocopherol found in the epidermis did not differ significantly from that already found, indicating only minor amounts of hydrolysis. After correction for the ot-tocopherol already present without application of o•-tocopherol, hydrolysis amounted to less than 1% of the ot-tocopherol in the stratum corneum and about 5 % in the viable layer after five days. It was concluded that vitamin E acetate can be considered a prodrug that very slowly releases minute amounts of the protector vitamin E. A limitation of this study was that it did not take into consideration the whole skin, i.e., the epidermis and the dermis. The enzymatic activity in the dermis, with its system of capillary blood vessels, would be higher than the epidermis, and this may result in the higher production of ot-tocopherol from the acetate. Alberts et aL (110) conducted a Phase II cancer prevention study i, vivo to evaluate whether topically applied ot-tocopheryl acetate was absorbed in human skin and me- tabolized to the free or other forms. A cream containing ot-tocopheryl acetate (125 mg/g) was rubbed on the forearms of eleven subjects twice daily for three months. Punch biopsies were taken at the beginning and end of the time period. Results (Table IV) showed an elevated level of ot-tocopheryl acetate after three months of topical cream administration. However, the cleavage capacity of human skin was extremely low or absent, and free ot-tocopherol concentrations ranged from 38.9 + 17.9 lng/g skin at baseline to 36.3 + 20.9 lag/g skin after three months of topically applied ot-tocopheryl acetate cream. I, vivo experiments offer the advantage that skin viability is constantly maintained but can be frustrated by the difficulties of differentiating between skin metabolism and systemic metabolism. EFFECT OF FORMUL^T•O•S EmMsio,s. Emulsions are preferable to simple solutions because of their universal solu-