Synergistic ·Antiandrogenic Effects of Topical Combinations of Sa-Reductase and Androgen Receptor Inhibitors in the Hamster Sebaceous Glands Jonathan R. Matias, M.S., Virginia L. Malloy, M.S., and Norm.an Orentreich, M.D. Biomedical Research Station, Orcntreich Foundation for the Advancement of Science, Inc., Cold Spring-on-Hudson, New York ORM, VLM, NO), and Department of Dermatology, NYU School of Medicine, New York, New York U.S.A. (NO) The androgenic action of dihydrotestosterone (DHT) is an­ produced any effect on SGS. When combinations of these tagonized by agents that compete with testosterone for the two steroids were applied at low concentrations, SGS de­ Sa-reductase enzyme and by agents that block the binding of creased unilaterally to approximately SO%. This synergy oc­ DHT to its receptor. The topical synergistic effect of Sa-re­ curred best at a P : SL ratio of 1 : 2. The lower effective con­ ductase (SaRI) and androgen receptor inhibitors (ARI) was centrations of P may be explained by its greater percutaneous determined by measurement of the sebaceous gland size absorption. Synergy was also demonstrated at low concen­ (SGS) of the ventral ear skin of the intact, sexuall y mature trations with other antiandrogens: cyproterOlle acetate, can­ male Syrian hamsters. Progesterone (P), a SaRI, and spiro­ renone, hydroxyflutamide, and N -N-diethyl-4-methyl- nolac tone (SL), an ARI, produced a dose responsive decrease 3-oxo-4-aza-5a-androstane-17,B-carboxamide. The use of in SGS at topical concentrations of 0.01 % to 5.0%. At con­ anti-androgen combinations at low concentrations is of value centrations of 1,3, and 5%, P and SL combinations produced because of the decreased risk of systemic side effects while neither an additive nor synergistic inhibition ofSGS. At very maintaining potent topical efficacy. ] Jil l/est Dermatol low concentrations of up to 0.10%, neither P nor SL alone 91:429-433,1988. ntiandrogens are divided into two major classes. The each class. In this study the androgen-sensitive sebaceous glands of first is represented by Sa-reductase inhibitors (SaRI), the hamster ventral ear pinna were utilized as a target organ for which co mpete with testosterone for the binding site evaluating the effect of topically applied combinations of antian­ in the Sa-reducta se enzyme. The second group is drogens. W e report here that combinations of topically applied comprised of androgen receptor inhibitors (ARI), SaRI and ARI exert a potent synergistic effect at very low doses Aw hich interf ere with the binding of dihydrotestosterone (DHT) to without evidence of sys temic side effects. the androgen receptor protein . Details of the biochemical and phys­ iologic effects of antiandrogens can be found in numerous reviews MATERIALS AND METHODS [e.g. , Refs 1,2). Animals Male Syrian golden hamsters were purchased from Har­ Because both types of antiandrogens act through entirely differ­ lan-Sprag ue Dawley, Inc. (Farmersburg, IN), at 10-11 weeks of ent mechanisms, the overall effi cacy of antiandrogen therapy might age. The animals werc houscd at a density of 4 to 5 animals per cage be enhanced by using combinations of selected compounds from with a photoperiod of 14 h li ght and 10 h dark. Food and water were provided ad libitum. The hamsters were acclimated to the labora­ tory conditions for at leas t 1 week prior to the experiments. Manuscript received N ovember 3, 1987; accepted for publication April Chemicals The following were purchased commerciall y: spiron­ 2 1,1988. Reprint requ ests to: Jonathan R. Matias, Biomedical Research Station, olactone (SL) from Sigma C hemical Company (St. Louis, MO); Orentreich Foundation for the Advancement of Scicncc, Inc., RD 2, Box progesterone (P) from Upjohn Company (Kalamazoo, MI); 4-preg­ 375, Cold Spring-on-Hudso n, NY 10516. ncne-3-one (4P3) fro m SyntBiotech, Inc. (Ncw York, NY); desox­ Abbreviations: ycorticosterone acetate (DOCA) , medroxyproges terone acetate ARI: androgen receptor inhibitor (MPA) and 4-androsten-3-one-17p-ca rboxylic acid (1 7BCA) fro m CN: ca nrcnone Steral oids (Wilton, NH). The following compounds were received 17 PCA: 4-androstcn-3-onc-17P carboxylic acid as gifts: canrenone (C N) from G.D. Searle Company (Skokie, IL); CA: cy proterone acetate n,n dicthyl-4 methyl-3-oxo-4-aza-Sa-androstanc 17p-carboxa­ DHT: dihydrotcstostcronc lI1ide (4-MA) from Merck, Sharpe, and Dohme (Rahway, NJ); hy­ DOCA: desoxycorticosterone acetate HF: hydroxyfluta111id e droxyflutamide (HF) and cy proterone acctatc (CA) from Schering 4MA: 1l,Il-di ethyl-4 1l1cthyl- 3-oxo-4-aza-Sa-androstanc 17p-carboxa- AG (Berlin, West Germany). 111 ide Radiolabeled steroids were obtained from th e following sources: MPA: 111 edroxyprogcstcronc acetate [1,2-3H(N))-progesterone (S.A. 57.5 CI/l11mol) from New En­ P: Progesterone gland Nuclea r (B os ton, MA); [1 ,2-3H (N))-spironolac tone (S.A. 44 4P3: 4-prcgnene-3-one Ci/ mmol) fro m Amersham (Arlington Heights, IL). 5 RI : Sa-rcductase inhibitor SL: spiro nolactone Procedures for the Topical Study The antiandrogens were dis­ SGS: sebaceous gland size solved in acctone either separately or in combinations. Twenty-five 0022-202X/88/S03.50 Copyright © 1988 by The Society for Investiga tive Dcrmatology, Inc. 429 430 MATIAS, MALLOY, AND ORENTREICH THE JOURNAL OF INVESTIGATIVE DERMATOLOGy microliters of each solution was applied with a micropipette on the ventral surface of the right ear pinna, once in the morning and once - \00 ~ in the afternoon of each day, Monday through Friday. The left ;§ 80 pinna of the same animal was not treated and served as an indicator '0 60 of any systemic effect of the antiandrogen treatment. Control ani­ ~ w 40 ~ N PROGES TERON E mals received topical applications of acetone alone to the right ;;;; 20 pinna. Unless otherwise specified, the treatments were terminated <=>z « after 4 weeks and the animals were killed by cervical dislocation. A -' '"" \00 -- ·-"O"-- -- --o--~ __o.., preliminary study indicated that chis was the minimum time for ' . :=>'" 0 80 b attaining an antiandrogenic response in intact mature males. ~ <D 60 The method used for evaluating the sebaceous gland response was W previously described in detail (3,4]. Briefly, the ear was cut at the cr'" 40 :5 SPIRONOLACTO N E proximal base with scissors, and the ventral ear skin, rich in seba­ 20 ceous glands, was manually separated from the cartilage. The skin was immediately stained for 3h with 0.1 % (w Iv) solution of Sudan 0 0. 1 I 10 Black B in propylene glycol. The ear skin was then fixed in 10% % CO NCE NTRATI ON buffered formalin after an overnight rinse in a solution of propylene Figure 1. Dose-dependent inhibitory effect of P and SL on the sebaceous glycol:water (85:15). A 2-mm punch biopsy of the medial zone (an glands of male Syrian hamsters (N=5-6 animals per data point). (e): area 5-8 mm from the apex of the pinna along the midline) was treated;(o): contralateral untreated. obtained. The whole-mount preparation of the ventral pinna was viewed from the dermal side at a magnification of 150X using an Ortholux low doses, i.e., doses of 0.1 % or lower. However, a marked syner, II microscope equipped with a drawing attachment (Leitz). The size gistic inhibition, particularly at the lowest dose (Exp. B, Table II), of each sebaceous gland unit was quantitated planimetrically using a was evident when P and SL were combined. When applied togethet computerized graphics calculator (Numonics Corp., Lansdale, PA). at a concentration of 0.025% for P and 0.05% for SL, the sebaceous Procedure for the Evaluation of Percutaneous Absorption gland size decreased to 58% of the control value. It is also important to note that the systemic side effects did not occur at such lo\\> The penetration rate of P and SL through the skin was determined concentrations (see Fig 1). This experiment was repeated two mor ~ by measuring the urinary excretion of each steroid according to the times and showed the same synergistic antiandrogenic effect. Fur, method of Feldmann and Maibach [5] . The radiolabeled steroid (0.5 therdilution of the 0.025% P and 0.05% SL combination resulted ill. .uCi) was dissolved in acetone with 4 .ug of unlabeled carrier, in a eventual loss of the antiandrogenic effect (Table III). total volume of 10.u1. After applying the solution on the ventral ear Different ratios of P and SL combinations at low concentrations pinna, the hamster was placed inside a plastic metabolic cage (Nal­ were also tested to determine the combination that would produce gene). Urine was collected daily for 5 d. An aliquot of urine was the optimal synergistic effect. As can be seen in Fig 2, synergisnl counted in a scintillation counter (Packard TriCarb) using 3H-tolu­ occurred best at a P:SL ratio of 1:2. To test whether this ratio was ene as the internal standard. The values were corrected for incom­ related to the penetration rates of these steroids through the skin, plete urinary excretion as described previously [5]. radiolabeled P and SL were applied to the ventral ear skin and the Calculation of the Index of Synergism or Antagonism To rate of percutaneous absorption was measured. The data (Table IV) determine whether or not a synergistic, antagonistic, or additive indicated that P was absorbed at twice the rate observed for SL. effect was present for each combination, an index was calculated as Synergistic effects were also demonstrated for other potent follows: lndex = (1 - D/c)/(1 - Alc) + (1 - Blc), where A is SaRI's, such as 4MA and 4P3, when used in combination with SL the mean value calculated for SaRI treated hamsters; B is the mean (Table V, experiments A and B).