In Vitro Evaluation of a Series of N-Dodecanoyl-l -Amino Acid Methyl Esters as Dermal Penetration Enhancers

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In Vitro Evaluation of a Series of N-Dodecanoyl-L-amino Acid Methyl Esters as Dermal Penetration Enhancers TIMOTHY K. FINCHER, SUN D. YOOX, 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, 1996X. * 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 (Q24), and 24 h full-thickness skin steroid content. Controls received no enhancer pretreatment of the skin. N-Dodecanoyl-L-proline (10) showed the highest Q24 value for total steroid (ER 13.7) while N-dodecanoyl-Lphenylalanine (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 intercellular 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, intercellular route, and transappendageal route. The intercellular pathway is believed to be primarily responsible for the steadystate 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 21acetate 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 Ndodecanoyl-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 enhancement.16,17 Compound 11 was reported previously and was included in this study.18 X

Abstract published in Advance ACS Abstracts, August 1, 1996.

920 / Journal of Pharmaceutical Sciences Vol. 85, No. 9, September 1996

Figure 1sChemical structures of compounds 1−11.

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-F254, and visualization was accomplished with iodine vapor. The melting points were determined on an electrothermal apparatus and were uncorrected. 1H-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 EsterssLAmino 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 procedure 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 CaCl2 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

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© 1996, American Chemical Society and American Pharmaceutical Association

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Table 1sMelting Points, Yields, and Empirical Formulas of Compounds 1−11 Compound

Mp (°C)

Yield (%)

Formula

1 2 3 4 5 6 7 8 9 10 11

61−61.5 56−57 50−50.5 26−26.5 54−54.5 83−83.5 59−59.5 55−56 70.5−71 Oil Oil

39 61 41 59 43 47 86 87 67 23

C15H29NO3 C16H31NO3 C18H35NO3 C19H37NO3 C22H35NO3 C22H35NO4 C18H35NO3S C16H31NO4 C24H36N2O3 C18H33NO3 C12H21NO3

reaction mixture was cooled to 0 °C and suction filtered. The crude material was recrystallized from methanol/water (9:1, v/v) to yield white needles (1.22 g, 39%): mp 61-61.5 °C; 1H-NMR (CDCl3) δ 0.86 (t, 3H, terminal CH3), 1.23 (m, 16H, (CH2)8), 1.63 (m, 2H, CH2 β to CO), 2.22 (t, 2H, CH2 R to CO), 3.74 (s, 3H, OCH3), 4.03 (d, 2H, CONHCH2CO), 5.92 (br s, 1H, NH) ppm. Anal. (C15H29NO3) Calcd: C, 66.38; H, 10.77; N, 5.16. Found: C, 66.48; H, 10.82; N, 5.21. N-Dodecanoyl-L-alanine Methyl Ester (2)sThe crude material was recrystallized from methanol/water (9:1, v/v) to yield a white solid (3.69 g, 61%): mp 56-57 °C; 1H-NMR (CDCl3) δ 0.82 (t, 3H, terminal CH3,), 1.22 (m, 16H, (CH2)8), 1.30 (d, 3H, CHCH3), 1.60 (m, 2H, CH2 β to CO), 2.22 (t, 2H, CH2 R to CO), 3.74 (s, 3H, OCH3), 4.55 (m, 1H, CHCH3), 6.10 (br d, 1H, NH) ppm. Anal. (C16H31NO3) Calcd: C, 67.33; H, 10.95; N, 4.91. Found: C, 67.46; H, 11.00; N, 4.68. N-Dodecanoyl-L-valine Methyl Ester (3)sThe crude material was recrystallized from methanol/water (9:1, v/v) to yield a white solid (1.53 g, 41%): mp 50-50.5 °C; 1H-NMR (CDCl3) δ 0.83-0.92 (complex m, 9H, terminal CH3, gem-dimethyls), 1.24 (m, 16H, (CH2)8), 1.58 (m, 2H, CH2 β to CO), 2.07-2.24 (complex m, 3H, CH2 R to CO, methine H of isopropyl group), 3.70 (s, 3H, OCH3), 4.55 (m, 1H, CHCH3), 6.10 (br d, 1H, NH) ppm. Anal. (C18H35NO3) Calcd: C, 68.97; H, 11.25; N, 4.47. Found: C, 68.69; H, 11.23; N, 4.48. N-Dodecanoyl-L-leucine Methyl Ester (4)sThe crude material was recrystallized from methanol/water (9:1, v/v) to yield a white solid (3.16 g, 59%): mp 26-26.5 °C; 1H-NMR (CDCl3) δ 0.85 (t, 3H, terminal CH3), 0.95 (m, 6H, CH(CH3)2), 1.22 (m, 16H, (CH2)8), 1.40-1.61 (complex m, 5H, CH2 β to CO, CH2CH(CH3)2), 2.22 (t, 2H, CH2 R to CO), 3.70 (s, 3H, OCH3), 4.55 (m, 1H, CHCH3), 5.90 (br d, 1H, NH) ppm. Anal. (C19H37NO3) Calcd: C, 69.68; H, 11.39; N, 4.28. Found: C, 69.44; H, 11.36; N, 4.22. N-Dodecanoyl-L-phenylalanine Methyl Ester (5)sThe crude material was recrystallized from methanol/water (9:1, v/v) to yield a white solid (1.98 g, 43%): mp 54-54.5 °C; 1H-NMR (CDCl3) δ 0.86 (t, 3H, terminal CH3), 1.22 (m, 16H, (CH2)8), 1.50 (m, 2H, CH2 β to CO), 2.19 (t, 2H, CH2 R to CO), 3.15 (m, 2H, CH2 Ar) 3.70 (s, 3H, OCH3), 4.90 (m, 1H, CHCH2), 5.90 (br d, 1H, NH), 7.0-7.15 (m, 5H, Ar H) ppm. Anal. (C22H35NO3) Calcd: C, 73.09; H, 9.76; N, 3.87. Found: C, 73.03; H, 9.82; N, 3.83. N-Dodecanoyl-L-tyrosine Methyl Ester (6)sThe crude material was recrystallized from methanol/water (9:1, v/v) to yield a white solid (2.30 g, 47%): mp 83-83.5 °C; 1H-NMR (CDCl3) δ 0.86 (t, 3H, terminal CH3), 1.22 (m, 16H, (CH2)8), 1.50 (m, 2H, CH2 β to CO), 2.22 (t, 2H, CH2 R to CO), 3.05 (m, 2H, CH2 Ar), 3.72 (s, 3H, OCH3), 4.90 (m, 1H, CHCH2), 5.90 (br d, 1H, NH), 6.85-6.95 (m, 4H, Ar H) ppm. Anal. (C22H35NO4) Calcd: C, 69.99; H, 9.34; N, 3.71. Found: C, 70.05; H, 9.34; N, 3.62. N-Dodecanoyl-L-methionine Methyl Ester (7)sThe crude material was recrystallized from methanol/water (9:1, v/v) to yield a white solid (4.30 g, 86%): mp 59-59.5 °C; 1H-NMR (CDCl3) δ 0.86 (t, 3H, terminal CH3), 1.25 (m, 16H, (CH2)8), 1.58 (m, 2H, CH2 β to CO), 1.92 (m, 2H, CHCH2CH2), 2.00 (s, 3H, SCH3), 2.22 (t, 2H, CH2 R to CO), 2.40 (m, 2H, CH2S), 3.70 (s, 3H, OCH3), 4.70 (m, 1H, NHCH), 6.10 (br d, 1H, NH) ppm.

Anal. (C18H35NO3S) Calcd: C, 64.62; H, 10.54; N, 4.19. Found: C, 62.76; H, 10.41; N, 4.03. N-Dodecanoyl-L-serine Methyl Ester (8)sA solution of L-serine methyl ester hydrochloride (10 mmol), (dimethylamino)pyridine (20 mmol), and acetonitrile (20 mL) was stirred at -10 °C under a CaCl2 drying tube while lauroyl chloride (2.19 g, 10 mmol) was slowly added. The reaction mixture was stirred for 15 min at room temperature. The reaction was quenched with distilled water (50 mL), and the reaction mixture was suction filtered. The crude material was recrystallized from ethyl acetate/hexane (1:1, v/v) to yield a white solid (2.72 g, 87%): mp 55-56 °C; 1H-NMR (CDCl3) δ 0.92 (t, 3H, terminal CH3), 1.15 (m, 16H, (CH2)8), 1.65(m, 2H, CH2 β to CO), 1.91-2.1 (br s, 1H, OH), 2.25 (t, 2H, CH2 R to CO), 3.70 (s, 3H, OCH3), 3.95 (m, 2H, HOCH2), 4.65 (m, 1H, HOCH2CH), 6.38 (br d, 1H, NH) ppm. Anal. (C16H31NO4) Calcd: C, 63.74; H, 10.36; N, 4.66. Found: C, 63.88; H, 10.47; N, 4.67. N-Dodecanoyl-L-tryptophan Methyl Ester (9)sThe crude material was recrystallized from methanol/water (9:1, v/v) to yield a white solid (3.24 g, 67%): mp 70.5-71 °C; 1H-NMR (CDCl3) δ 0.86 (t, 3H, terminal CH3,), 1.22 (m, 16H, (CH2)8), 1.60 (m, 2H, CH2 β to CO), 2.15 (t, 2H, CH2 R to CO), 3.30 (m, 2H, CH2 Ar), 3.74 (s, 3H, OCH3), 4.70 (m, 1H, NHCH), 5.90 (br d, 1H, NH), 6.95-7.60 (m, 5H, Ar H), 8.05 (s, 1H, Ar NH) ppm. Anal. (C24H36N2O3) Calcd: C, 71.96; H, 9.06; N, 6.99. Found: C, 71.69; H, 9.10; N, 6.95. N-Dodecanoyl-L-proline Methyl Ester (10)sThe crude material was recrystallized from methanol/water (9:1, v/v) to yield an oil (0.65 g, 23%): 1H-NMR (CDCl3) δ 0.86 (t, 3H, terminal CH3,), 1.22 (m, 16H, (CH2)8), 1.60 (m, 2H, CH2 β to CO), 1.80-2.40 (complex m, 6H, NCH2CH2CH2CH, CH2 R to CO), 3.40-3.60 (m, 2H, NCH2), 3.71 (s, 3H, OCH3), 4.25 (m, 1H, NCH) ppm. Anal. (C18H33NO3) Calcd: C, 69.41; H, 10.68; N, 4.50. Found: C, 69.18; H, 10.55; N, 4.36. Synthesis of n-Pentyl N-Acetylprolinatesn-Pentyl N-acetylprolinate (compound 11) was synthesized by a method described previously.18 Diffusion Cell ExperimentssMale hairless mice [SKH1 (hr/hr)], 6-8 weeks old, were obtained from Charles River Laboratories, Inc. (Wilmington, MA). The mice were euthanized with carbon dioxide, and dorsal portions of full thickness skin were removed. Skins were placed in Franz diffusion cells with a diffusional area of 3.14 cm2 and a receptor volume of 12 mL and were allowed to hydrate for 1 h. Diffusion cells were maintained at 37 ( 0.5 °C throughout the experiment. Following hydration, 8 µL of enhancer solution was applied to each skin using methanol as an intermediate solvent. The methanol facilitated uniform spreading of the enhancer solution and was immediately evaporated off each skin. Enhancers 1-9 were applied as saturated suspensions in propylene glycol, and compound 2 was applied at 0.4 M. Following a 1 h pretreatment period, 80 µL of a saturated suspension of hydrocortisone in PG was applied per cell (hydrocortisone solubility in PG at 32 ( 0.5 °C was 0.03 M). The donor cells were then occluded with Parafilm. The receptor phase consisted of phosphate buffer (isotonic, pH 7.2) with 0.1% v/v 36% formaldehyde as a preservative19 and 0.5% w/v polyethylene 20 cetyl ether as a solubilizer.20 The receptor phase was constantly stirred at 600 rpm throughout the experiment. Receptor samples (300 µL) were taken at 0.5, 1, 1.5, 2, 3, 4, 6, 10, 12, and 24 h and were replaced with fresh buffer. Analysis of each subsequent sample was adjusted for removed volume to represent cumulative amounts of drug for each sampling time. Control samples were treated as described above with the exception that no enhancer solution (enhancer, PG or methanol) was used to pretreat the skin. Samples were stored at -80 °C until assayed by HPLC. Skin Retention ExperimentssSkins were removed from the diffusion cells at 24 h, washed twice in 100 mL of methanol for 5 s, blotted dry, and weighed (mean skin weight ) 0.107 ( 0.095 g). These were then homogenized using a Kinematica GmbH tissue homogenizer, filtered through a C18 Sep pak filter, and frozen at -80 °C until HPLC analysis. Extraction ProceduresA 50 µL aliquot of sample was combined with 25 µL of dexamethasone (internal standard, 10 µg/mL), 200 µL of methanol, and 2 mL of methylene chloride. The mixture was vortexed for 30 s and centrifuged for 3 min at 5000 rpm, and the aqueous layer was removed. The organic layer was then dried under nitrogen and reconstituted with 50 µL of mobile phase. An aliquot (20 µL) was injected into the HPLC.

Journal of Pharmaceutical Sciences / 921 Vol. 85, No. 9, September 1996

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Figure 2sCumulative amount (µM) of steroid penetrated through hairless mouse skin over 24 h for enhancers 10 (b) and 7 (O) and control (4). HPLC AnalysissThis was conducted using an HPLC consisting of a Model CR501 integrator, SPD 10AV UV-vis detector, LC-10AS pump (Shimadzu) and Microsorb reversed-phase C18 column (15 × 0.46 cm, 5 µm, Rainin). The mobile phase consisted of methanol/ water (60:40) with a flow rate of 1.0 mL/min. HC, cortisone and dexamethasone (internal standard) were detected at 242 nm. Data AnalysissPermeation parameters were defined as permeability coefficient (P), 24 h receptor concentrations (Q24), and skin content. Data obtained for compounds 1-11 were derived from a plot of the cumulative concentration of drug (µM) in the receptor versus time. The slope of the linear portion was used to calculate P, and extrapolation of that line yielded the lag time. Sink conditions were assumed to exist during the times in which P was measured. Skin content was expressed as amount of total steroid per gram of hydrated skin at 24 h. Enhancement ratios (ER) were calculated as the permeation parameter after enhancer treatment divided by the permeation parameter for control. All statistical analyses were performed using Student’s t-test. All data were shown as mean ( SD. Statistical significance was set at p < 0.05.

Results and Discussion Plots of the cumulative steroid concentration vs time data are shown for enhancers 10 and 7 and control in Figure 2. Profiles of other enhancers not shown in Figure 2 fell between those of enhancer 10 and control. The permeation parameters for enhancers 1-11 are shown in Table 2. Values for permeability coefficient were calculated for HC only, with the exception of compound 11, which was presented as the sum

of HC and cortisone. Skin content and 24 h receptor concentrations were presented as the sum of HC and cortisone (total steroid) since cortisone was detected during sample analysis. Both human and animal skin have been shown to metabolize hydrocortisone to cortisone.21-24 In sheep and nude mouse skin, 11β-hydroxy steroid dehydrogenase is responsible for this conversion with the percentage conversion as high as 49%.23,24 In the present study, compounds 1-10 yielded percentage conversions ranging from 1.1% (compound 10) to 7.7% (compound 7) based on Q24 values. Compound 11 had the highest percentage conversion of 51.4%. Enhancers 1-10 contained methyl esters, and 11 contained a pentyl ester linkage. These esters may have been hydrolyzed to some extent by esterases present in hairless mouse skin at relatively high concentrations.25 Whether hydrolysis products are partially responsible for enhancement activity was not determined in this study. However, we would expect that hydrolysis of the methyl esters in compounds 1-10 to the corresponding carboxylic acids would result in compounds with enhancement properties. Of the compounds tested, enhancement properties of compound 11 would likely be the most affected by cleavage of a longer hydrocarbon chain. Future work is warranted to evaluate the effects of compounds 1-11 on the extent of hydrocortisone metabolism and the effect of esterases present in the skin on these compounds. In addition, evaluation of possible hydrolysis products of compounds 1-11 for enhancement activity remains to be conducted. Differential scanning calorimetry has revealed that Azone affects the lipid structure of the stratum corneum by partitioning into the lipid bilayer and fluidizing this region.26,27 The structure of Azone is thought to be responsible for this effect.28 Azone consists of a seven-membered ring and a long hydrocarbon chain which imparts both hydrophilic and hydrophobic characteristics to the enhancer. By fluidizing the lipophilic domain of the stratum corneum, permeation of polar drugs such as HC is increased.15 Spectroscopic data, interaction with model structure lipids, and molecular graphics have revealed that molecular shape and charge distribution of Azone allows for this fluidization.28 Azone in combination with PG increases the permeation of HC through hairless mouse skin [P ) 215.2 ( 48.6 cm h-1 (×10-5), Q24 ) 218.9 ( 47.84 µM].8,9 Compounds 1-11 are amino acid derivatives with lipophilic N-dodecanoyl side chains similar to the Ndodecyl side chains of Azone and Azone analogs we have previously evaluated.8-15 Of compounds 1-11, compound 10 was structurally most similar to Azone and showed the highest values for P and Q24 (ERP ) 13.9, ERQ24 ) 13.7). Compound 11 was also similar to Azone and displayed high values for P and Q24, but differed from 10 and Azone in that it contained a shorter hydrocarbon side chain. Both com-

Table 2sEffect of Enhancers 1−11 on the Percutaneous Penetration and Skin Retention of Steroid in Hairless Mouse Skin Enhancer in PGa,b

Lag Time (h)

P (×10-5) (cm h-1)

ERPc

Q24d (µM)

ERQ24c

SCe (µg g-1)

ERSCc

None 1 2 3 4 5 6 7 8 9 10 11

8.8 ± 2.4 2.8 ± 1.1 1.8 ± 1.4 3.6 ± 2.3 2.4 ± 0.5 3.4 ± 1.7 3.8 ± 1.2 1.7 ± 0.3 1.3 ± 0.5 2.9 ± 0.8 1.5 ± 0.4 4.0 ± 1.0

8.8 ± 5.1 22.9 ± 9.2 101.6 ± 67.5 31.0 ± 17.4 47.9 ± 19.5 46.5 ± 23.3 42.3 ± 12.3 36.3 ± 12.3 52.4 ± 6.3 36.4 ± 8.4 122.5 ± 13.2 64.1 ± 11.8

1.0 2.6 11.6 3.5 5.5 5.3 4.8 4.1 6.3 4.1 13.9 3.6

9.1 ± 6.1 20.4 ± 5.1 98.6 ± 59.2 41.5 ± 10.4 65.8 ± 36.9 61.8 ± 37.5 64.3 ± 22.5 52.5 ± 12.8 92.3 ± 24.1 65.4 ± 7.1 125.1 ± 20.0 91.7 ± 29.8

1.0 2.2 10.8 4.6 7.2 6.8 7.1 5.8 10.1 7.2 13.7 10.0

23.9 ± 3.6 164.9 ± 55.8 280.6 ± 106.4 151.8 ± 73.5 238.3 ± 97.7 395.7 ± 128.5 92.4 ± 26.9 32.3 ± 7.3 64.5 ± 37.0 111.9 ± 26.2 122.4 ± 60.8 143.3 ± 82.2

1.0 6.9 11.7 6.3 10.0 16.5 3.9 1.4 2.7 4.7 5.2 6.0

a PG, propylene glycol. b n ) 5 for all. c ER: enhancement ratio calculated as permeation parameter after enhancer pretreatment divided by the corresponding parameter from control. d Q24; receptor concentration of total steroid at 24 h. e SC: total steroid skin content.

922 / Journal of Pharmaceutical Sciences Vol. 85, No. 9, September 1996

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pounds 10 and 11 are analogs of proline and contained a pyrrolidine ring (Figure 1). We previously reported that structurally similar compounds, N-dodecylpyrrolidine and N-dodecyl-2-pyrrolidinone, increased the skin permeation of hydrocortisone 21-acetate through hairless mouse skin. N-Dodecyl-2-pyrrolidinone was found to be significantly more effective at increasing permeability coefficient and Q24 values than N-dodecylpyrrolidine.13,15 Compounds 10 and 11, although pyrrolidine analogs, are similar to the pyrrolidinones in that there is a carbonyl group adjacent to the pyrrolidine nitrogen. This increased hydrophilicity made these compounds more effective than N-dodecylpyrrolidine. However, 10 and 11 were less effective than N-dodecyl-2-pyrrolidinone. Of the compounds evaluated, compounds 10 and 11 most closely resemble the “spoon” shape structure exhibited by Azone. However, further work will be necessary to determine the three-dimensional structure and charge distribution in the ring structures of these compounds. The remaining compounds are less similar to Azone but may act via similar mechanisms. Of compounds 1-9, 2 and 8 showed the highest ER values for P (11.6 and 6.3, respectively) and Q24 (10.8 and 10.1, respectively). The R group for compounds 1-9 appears to have some implications as to the effectiveness of these compounds. Compound 1 showed the lowest enhancement ratio with R ) H. The addition of a methyl group at this position (compound 2) resulted in a 4.5-fold increase in permeability coefficient. Compound 8 (R ) CH2OH) also showed increased permeability over compound 1. However, as chain length was increased (compounds 3, 4, and 7) or bulky substituents were added (compounds 5, 6, and 9) permeability was decreased compared to compound 2. Compound 2 is a derivative of alanine, and compound 8 is a derivative of serine. Both alanine and serine are hydrophilic in nature.29 Compounds 1, 3-7, and 9 are more hydrophobic in nature and appear to be less effective at increasing P and Q24. In addition, more hydrophilic model drugs may express different profiles. Azone has been reported to be more effective at increasing the permeation of hydrophilic drugs.15 This may also be true of the enhancers presented in this study. Penetration enhancers may have several different effects on drug delivery. They may increase penetration of drugs through the skin, increase the concentration of drug within the skin, or a combination of both.15 This was evident with respect to compounds 1-11. Compounds 4 and 5 showed high ER values for skin content (10.0 and 16.5, respectively) but low Q24 values, while compound 2 showed high ER values for both Q24 and skin content (10.8 and 11.7, respectively). High skin content values for 4 and 5 may be due in part to their relatively high lipophilicities.30 However, other compounds with relatively high lipophilicities such as compounds 7 and 9 did not display high skin contents compared to 4 and 5, indicating that other factors such as affinity of the enhancer for the stratum corneum were involved.30 In addition, the mechanism by which these enhancers increase skin retention could be model drug specific,31 since steroids have been shown to form epidermal reservoirs under both occluded and nonoccluded conditions.32,33 Thus, other model drugs may show a different skin content profile when evaluated with these enhancers. Compounds 1-10 may act via mechanisms similar to those of Azone and Azone analogs. Compounds 2, 8, 10, and 11

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possess both hydrophilic and lipophilic components similar to Azone which may have contributed to increased skin permeation. Structurally, compound 10 is the most similar to Azone and exhibits the best permeation profile. Compounds 2, 4, and 5 had high steroid skin content and may be useful for topical drug delivery. Future work is warranted to evaluate these compounds using different vehicles since changes in vehicle can affect permeation parameters.

References and Notes 1. Barry, B. W.; Bennett, S. L. J. Pharm. Pharmacol. 1987, 39, 535-546. 2. Idson, B. J. Pharm. Sci. 1975, 64, 901-924. 3. Kim, Y. H.; Ghanem, A. H.; Mahmoud, H.; Higuchi, W. I. Int. J. Pharm. 1992, 80, 17-31. 4. Williams, A. C.; Barry, B. W. Pharm. Res. 1991, 8, 17-24. 5. Wong, O.; Huntington, J.; Nishihata, T.; Rytting, J. H. Pharm. Res. 1989, 6, 286-295. 6. Michniak, B. B.; Player, M. R., Godwin, D. A., Sowell, J. W. Drug Delivery 1995, 2, 117-122. 7. Michniak, B. B.; Chapman, J. M.; Seyda, K. L. J. Pharm. Sci. 1993, 82, 214-219. 8. Michniak, B. B.; Godwin, D. A.; Phillips, C. A., Sowell, J. W. Proc. Pharm. Technol. Conf., 14th 1995, 2, 148-157. 9. Michniak, B. B.; Player, M. R.; Godwin, D. A.; Sowell, J. W. Pharm. Res. 1995, 12, S268. 10. Michniak, B. B.; Player, M. R.; Chapman, J. M.; Sowell, J. W. Int. J. Pharm. 1993, 91, 85-93. 11. Michniak, B. B.; Player, M. R.; Fuhrman, L. R.; Christensen, C. A.; Chapman, J.M.; Sowell, J. W. Int. J. Pharm. 1993, 94, 203-210. 12. Michniak, B. B.; Player, M. R.; Fuhrman, L. R.; Christensen, C. A.; Chapman, J.M.; Sowell, J. W. Int. J. Pharm. 1994, 110, 231-239. 13. Michniak, B. B.; Player, M. R.; Godwin, D. A.; Phillips, C. A.; Sowell, J. W. Int. J. Pharm. 1995, 116, 201-209. 14. Michniak, B. B.; Player, M. R.; Chapman, J. M.; Sowell, J. W. J. Controlled Release 1994, 32, 147-154. 15. Phillips, C. A.; Michniak, B. B. J. Pharm. Sci. 1995, 84, 14271433. 16. Aungst, B. J.; Rogers, N. J.; Shefter, E. Int. J. Pharm. 1986, 33, 225-234. 17. Cooper, E. R. J. Pharm. Sci. 1984, 73, 1153-1156. 18. Harris, W. T.; Tenjarla, S. N.; Holbrook, J. M.; Smith, J.; Mead, C.; Entrekin, J. J. Pharm. Sci. 1995, 84, 640-642. 19. Sloan, K. B.; Beall, H. D.; Weimar, W. R.; Villanueva, R. Int. J. Pharm. 1991, 73, 97-104. 20. Chien, Y. W. In Novel Drug Delivery Systems: Fundamentals, Developmental Concepts, Biomedical Assessments; Dekker: New York, 1982; pp 551-553. 21. Hsia, S. L.; Hao, Y. Biochemistry 1966, 5, 1469-1474. 22. Hsia, S. L.; Hao, Y. Steroids 1967, 10, 489-500. 23. Ward, K. A.; Kasmarik, S. E. Aust. J. Biol. Sci. 1979, 32, 197203. 24. Teelucksingh, S.; Mackie, A. D.; Burt, D.; McIntyre, M. A.; Brett, L.; Edwards, C. R. Lancet 1990, 335, 1060-1063. 25. Ghosh, M. K.; Mitra, A. K. Pharm. Res. 1990, 7, 251-255. 26. Barry, B. W. Int. J. Cosmet. Sci. 1988, 10, 281-293. 27. Barry, B. W. J. Controlled Release 1991, 15, 237-248. 28. Brain, K. R.; Walters, K. A. In Pharmaceutical Skin Penetration Enhancement; Walters, K. A., Hadgraft, J., Eds.; Dekker: New York, 1993; pp 389-416. 29. Stryer, L. Biochemistry; Freeman: New York, 1988; pp 17-21. 30. Sasaki, H.; Kojima, M.; Mori, Y.; Nakamura, J.; Shibasaki, J. J. Pharm. Sci. 1991, 80, 533-538. 31. Okamoto, H.; Hashida, M.; Sezaki, H. J. Pharm. Sci. 1991, 80, 39-45. 32. Woodford, R.; Barry, B. W. Curr. Ther. Res. 1977, 21, 877-886. 33. Woodford, R.; Barry, B. W. J. Pharm. Sci. 1977, 66, 99-103.

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Journal of Pharmaceutical Sciences / 923 Vol. 85, No. 9, September 1996