J. Cosmet. Sci., 56, 91-103 (March/April 2005) Permeation study of five formulations of alpha-tocopherol acetate through human cadaver skin HANSA MAHAMONGKOL, ROBERT A. BELLANTONE, GRAZIA STAGNI, and FOTIOS M. PLAKOGIANNIS, Division of Pharmaceutical Sciences, Arnold and Marie Schwartz College of Pharmacy and Health Sciences, Long Island University, 75 DeKalb Avenue, Brooklyn, NY 11201. Accepted for publication February 16, 2005. Presented in part at the Annual Meeting of the American Association of Pharmaceutical Scientists, Baltimore1 November 7-111 2004. Synopsis Alpha-tocopherol (AT) is the vitamin E homologue with the highest in vivo biological activity. AT protects against the carcinogenic and mutagenic activity of ionizing radiation and chemical agents, and possibly against UV-induced cutaneous damage. For stability consideration, alpha-tocopherol is usually used as its prodrug ester, alpha-tocopherol acetate (ATA), which once absorbed into the skin is hydrolyzed to alpha­ tocopherol, the active form. The objective of this research was to characterize in vitro the permeation properties of ATA from various solutions and gel formulations. Permeation studies were conducted using modified Franz diffusion cells and human cadaver skin as the membrane. Specifically, 5% (w/w) alpha­ tocopherol acetate was formulated in the following vehicles: ethanol, isopropyl myriscate, light mineral oil, 1 % Klucel® gel in ethanol, and 3% Klucel® gel in ethanol (w/w). The receiver temperature was 37 ° C. Samples from the receiver were collected at 2, 4, 6, 8, 12, 24, 30, 36, and 48 hours and analyzed by HPLC for concentrations of alpha-tocopherol acetate and alpha-tocopherol. The permeabilities of ATA through human cadaver skin were 1.0 x 10-4, 1.1 x 10- 2 , 1.4 x 10-4, 2.1 x 10- 4 , and 4.7 x 10-4 cm/h for the ethanol solution, isopropyl myristate solution, light mineral oil solution, 1 % Klucel® gel, and 3% Klucel® gel, respectively. The results show that the formulation had relatively minor effects on the permeability coefficients of AT A through cadaver skin in all cases except for the isopropyl myristate solution. INTRODUCTION Alpha-tocopherol is the major lipophilic antioxidant in many biological systems (1). The main antioxidant function of AT is to prevent lipid peroxidation at the cell membrane site and therefore to promote the preservation of the structural integrity of the mem­ brane. AT is present in high concentration at the lower levels of the stratum corneum (2,3) where it represents the first line of defense of the skin from the oxidative stress of Address all correspondence to Fotios M. Plakogiannis. 91

92 JOURNAL OF COSMETIC SCIENCE sunlight and pollutants. Indeed, formation of free radicals and subsequent lipid peroxi­ dation is considered to be the major mechanism of UV irradiation-induced cutaneous damage (4). Exposure to UV light depletes the stratum corneum of AT, and regeneration of AT may be difficult in these conditions. Topical application of AT to the skin has been reported to protect animal skin from UV-induced damage (5) either by direct protection from free radicals or by indirect protection by means of increased epidermal thickness (6). In addition, topical application of AT may prevent mutations in critical genes (7) and effectively reduce cancer formation and immunosuppression induced by UV irradiation (8). Topical application of AT was far more effective at preventing the increase in lipid peroxidation than dietary supplementation, probably because of the higher tissue level attained (9). Other significant local actions of AT are improvement of skin microcirculation, inhibition of inflammation, promotion of hair growth, treat­ ment of alopecia and of various skin diseases (e.g., axillar bromidrosis, chilblains, acne vulgaris, mycosis in the nail) (10,11). In addition, AT is widely used in skin care products mainly as a natural moisturizer to relieve dry skin, and as an aid in the conceal­ ment of wrinkles and facial lines. Unfortunately, AT has a very short shelf life in topical formulations because it is sensitive to atmospheric oxygen, and it is therefore formulated as its prodrug ester, alpha-tocopherol acetate (ATA). ATA is biologically inactive because it lacks the free phenolic OH group. However, ATA is believed to hydrolyze to the active form, AT, in the skin. The skin is capable of many of the same types of metabolic processes that are present in the liver and other organs (12), but the overall metabolizing capacity of the skin is less than that of the liver by nearly two orders of magnitude. Therefore, the actual efficacy of ATA-containing products is still uncertain. The metabolic capability of the skin may differ among species. For instance, in rat skin, van Henegouwen et al. (13) found that after a period of five hours following a single application of AT A, the amount of AT in skin does not significantly differ from the amount already present in the skin. In pig skin, Rangarajan and Zatz (14) found that AT appeared as early as two hours after application, with the extent of metabolism reaching a peak at 6-12 hours after appli­ cation. No metabolism was detected in the stratum corneum, but it was detected in the viable skin ( 13, 15 ). They also demonstrated that the topical delivery and metabolism of ATA were dependent on formulation. Baschong et al. (15) did an ex vivo study in viable human skin. Their study confirms that also in humans bioconversion of AT A to AT is localized exclusively in the viable skin. Hydrolysis was absent on the skin surface as well as in the horny layer. Distribution of ATA in skin was dependent on the formulation. The in vitro study of ATA permeability is particularly challenging because ATA is very poorly soluble in water, and water is usually the major component of the receiver compartment of in vitro testing cells. The objectives of this study were to evaluate the permeation through human cadaver skin of ATA in vitro from various topical formula­ tions. The formulations tested were kept simple and ranged from solutions of increasing viscosity (ethanol, isopropyl myristate, and mineral oil) to gel formulations. A recently reported mathematical approach (16) for the determination of membrane permeability was used. The method has the advantage that accurate determinations of membrane permeabilities can be done using a common experimental technique and can be applied to systems in which the donor compartment is unstirred.