In Vitro Evaluation of a Series of N-Dodecanoyl-L-amino Acid Methyl Esters as Dermal Penetration Enhancers TIMOTHY K. FINCHER,SUN D. YOO X ,MARK R. PLAYER*, J. WALTER SOWELL,SR., AND BOZENA B. MICHNIAK Received February 13, 1996, from the College of Pharmacy, University of South Carolina, Columbia, SC 29208. Accepted for publication June 17, 1996 X . * Present address: Section on Biomedical Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892. Abstract 0 A series of N-dodecanoyl-L-amino acid methyl esters (1- 10) and n-pentyl N-acetylprolinate (11) were evaluated for dermal enhancement properties using an in vitro diffusion cell technique. Methods of synthesis of these compounds were described. Enhancers were applied 1 h prior to drug treatment. Hydrocortisone was used as the model drug and was applied to excised hairless mouse skin as a saturated suspension in propylene glycol. Enhancement ratios (ER) were determined for permeability coefficient, 24 h diffusion cell receptor concentration (Q 24 ), and 24 h full-thickness skin steroid content. Controls received no enhancer pretreatment of the skin. N-Dodecanoyl-L-proline (10) showed the highest Q 24 value for total steroid (ER 13.7) while N-dodecanoyl-L- phenylalanine (5) showed the highest total steroid skin retention (ER 16.5). The stratum corneum serves as the primary barrier to absorption of substances contacting the skin. This layer consists of enucleated cells evolving from keratinocytes in the basal epidermal layer. Each cell is surrounded by a thick envelope of intermediate filament proteins, and the intercel- lular spaces are filled with strongly hydrophobic lamellar lipids. It has been described as hydrophilic protein “bricks” in a hydrophobic lipid “mortar” and is an efficient barrier to both hydrophilic and hydrophobic substances. 1 Diffusion of drugs through the stratum corneum is considered to take place by three possible mechanisms: intracellular route, intercel- lular route, and transappendageal route. The intercellular pathway is believed to be primarily responsible for the steady- state transport of drugs through the stratum corneum. 2 In recent years, various skin penetration enhancers have been evaluated for their ability to reversibly reduce the barrier to drug permeation in the intercellular regions. 2-7 Of these, Azone has been the most widely studied. Azone is thought to disrupt the organization of lipids and to increase the water content of proteins in the stratum corneum. It consists of a seven-membered ring with a hydrocarbon side chain. This imparts both hydrophilic and lipophilic character to the compound, allowing it to partition between the lipophilic and hydrophilic components of the stratum corneum. Previously, we have evaluated multiple cyclic and acyclic Azone and pyrrolidinone analogs in hairless mouse and hairless rat skin using hydrocortisone and hydrocortisone 21- acetate as model drugs. 8-15 In the present study, we have synthesized and tested 11 amino acid derivatives for their ability to enhance the penetration of hydrocortisone (HC) through hairless mouse skin. Compounds 1-10 are N- dodecanoyl-L-amino acid derivatives. An 11-carbon side chain was utilized since it has been reported that side chain lengths of 10-12 carbons are most effective for penetration enhance- ment. 16,17 Compound 11 was reported previously and was included in this study. 18 Experimental Section All chemicals were purchased from Aldrich Chemical Co. except hydrocortisone, cortisone, dexamethasone, polyoxyethylene 20 cetyl ether, and propylene glycol (PG), which were obtained from Sigma Chemical Co. Baxter Diagnostics, Inc., supplied reagent grade solvents, except for methanol and acetonitrile, which were HPLC grade. Dimethylformamide (DMF) was redistilled and stored over 3A molecular sieves prior to use. Thin layer chromatography (TLC) was performed with Merck precoated silica gel plates, type 60-F 254, and visualization was accomplished with iodine vapor. The melting points were determined on an electrothermal apparatus and were uncorrected. 1 H-NMR spectra were obtained on a Bru ¨ ker AM 300 NMR spectrometer. UV spectra were recorded on a Beckman DU-6 spectrometer. Elemental analyses were performed by Atlantic Microlabs (Atlanta, GA) and were within (0.4% of theoretical values for all compounds. Synthesis of N-Dodecanoyl-L-amino Acid Methyl EsterssL- Amino acid methyl ester hydrochlorides were converted to their N-dodecanoyl amides by treatment with lauroyl chloride in the presence of pyridine with the exception of compound 8. The reaction solvent was optimized, and it was observed that DMF afforded the highest yield of acylated product. The use of DMF as solvent also simplified product isolation (vide infra). Chemical structures of the synthesized permeation enhancers are shown in Figure 1. Empirical formulas, melting points (mp), and synthetic yields are listed in Table 1. Of the compounds listed in Table 1, only compounds 10 and 11 were liquids and the remaining compounds were solids. The proce- dure given for the synthesis of compound 1 was utilized in the preparation of compounds 2-7, 9, and 10. Procedures for Synthesis of the N-Dodecanoyl-L-amino Acid Methyl EsterssN-Dodecanoylglycine Methyl Ester (1)sA solution of glycine methyl ester hydrochloride (1.26 g, 10 mmol) and pyridine (0.79 g, 10 mmol) in dry DMF (30 mL) was stirred at 0 °C under a CaCl 2 drying tube while lauroyl chloride (2.19 g, 10 mmol) was slowly added. The reaction mixture was heated to 50 °C and stirred for 2 h and then cooled to room temperature and stirred for an additional 3 h. The reaction was quenched with distilled water (50 mL), and the X Abstract published in Advance ACS Abstracts, August 1, 1996. Figure 1sChemical structures of compounds 1-11. S0022-3549(96)00078-0 CCC: $12.00 920 / Journal of Pharmaceutical Sciences © 1996, American Chemical Society and Vol. 85, No. 9, September 1996 American Pharmaceutical Association + +