THE EFFECT OF ANTI-ANDROGENS ON THE RESPONSE OF RAT PREPUTIAL GLANDS TO TESTOSTERONE* E. LINN JONES, M.D.t AND LINDA WOODBURY, M.D. In recent years, a number of new compounds have been discovered which have anti-androgenic effects on animals. In experimental animals, these compounds antagonize the effects of testosterone, yet have little or no estrogenic effect. Such compounds are of interest because of their potential usefulness in the management of certain diseases which are believed to be aggravated by androgens, such as carcinoma of the prostate, acne vulgaris, hirsutism, and patterned male baldness. Estrogens have been widely used as antagonists in the treatment of prostatic carcinoma, in spite of major undesirable side-effects such as breast enlargement. In the treatment of acne vulgaris, the unwanted side-effects of estrogens have tended to restrict the use of these substances topically as well as orally. If a compound could be found which was capable of reducing the effect of androgenic hormones on the pilosebaceous apparatus, but was free from other anti-androgenie or undesirable side-effects, such a compound might be very useful in the management of acne vulgaris. A search of the literature and inquiries to drug manufacturers revealed fifteen compounds to which a.nti-androgenic activity has been attributed. Five of these were made available to the authors for animal experimentation. Their chemical structures are shown in Figure 1. Compound SC-9420, which is currently marketed as a diuretic under the trade name "Aldactone," has been studied intensively because of its ability to block the action of aldosterone. Ro2-239 has been demonstrated to have a very weak androgenic action when tested on the chick comb (1), yet it is able to interfere with the action of the highly potent androgen, testosterone * From the Section of Dermatology, Department of Medicine, University of Chicago, Chicago 3, Illinois. This research was supported by the U. S. Army Research and Development Command, Office of the Surgeon General, under Contract No. DA-49-193-MD-2341 and by United States Public Health Service Training Grant No. 2A-523(C2). t Present address: Medical Research Division, Eli Lilly and Company, Indianapolis, Indiana. Received for publication November 20, 193. 15 propionate, on the seminal vesicles, prostate, and levator ani muscle of the rat (2). Lauppi studied the action of Ro2-230 on rat epidermis and found that it lowered the rate of mitosis, reduced the thickness of the epidermis, and inhibited the induction of epidermal proliferation and acanthosis by testosterone (35). Since no reports of the effect of these compounds on the pilosebaceous apparatus could be found, it was decided to undertake such studies. Three possible mechanisms by which antiandrogenic compounds might act are as follows: 1. Reducing the ability of target organs to respond to androgens. 2. Reducing the release of androgenic substances by the gonads and adrenal glands. 3. Reducing the release of pituitary hormones which stimulate the gonads and adrenals to secrete androgenic hormones. The studies presented here are concerned with the direct effects of compounds on the target organs. By using castrated animals receiving exogenous androgens, it is possible to eliminate most of the effect mediated through the second and third mechanisms. In a recent review of anti-androgenic substances by Dorfman (), two animal tests for anti-androgenic activity were described. One of these uses the comb of the chick as a target organ, and the other uses the seminal vesicles, prostate, and levator au muscle of the rat. The comb of the chick is of interest to the dermatologist because it is a dermal structure which is highly responsive to both systemic and topical androgens. Androgens cause the comb to enlarge, apparently as the result of both vascular engorgement and an increase in the amount of extra-cellular metachromatic polysaccharides. The weight of the chick comb is a useful index for the bio-assay of androgenic activity. An increased rate of incorporation of sulfur into sulfated polysaccharide in the androgen-treated chick comb has been demonstrated by radioisotopic methods (). The chick comb is not useful for the study of the effects of hormones on dermal appendages, however, since it is devoid of these.
1 THE JOURNAL OF INVESTIGATIVE DERMATOLOGY 0J SC9li20 so-b5 OH3 0000520H211(0520192 50 33 ao2239 15 1280 0000113 0 I N 3 0113 0 053 FIG. 1. Structural formulas of five compounds with anti-androgenie activity. The structure of testosterone is shown for comparison. FIG. 2. A midline incision in the pubic area of a male rat revealing the bilateral subcutaneous preputial glands. The sebaceous glands of the rat have been used for experimental study of the effect of hormones on the size of sebaceous structures. The volume of the sebaceous glands can be calculated from planimetrie measurements of projections of serial sections of the glands (5, 9). This method, while useful, has the disadvantage of being quite timeconsuming. The male rat has two bilateral modified sebaceous glands, the preputial glands, which arise from the hair-bearing skin around the penis (Fig. 2 and 3). In the female rat, homologous glands are present. The preputial glands appear to be subject to the same hormonal influences as the sebaceous glands (, 11), but because of their greater size the preputial glands can be readily dissected and weighed. The preputial glands have been shown to enlarge in response to testosterone, androsterone, and androstenediol (12). For this reason, they were consideredlikely to be suitable organs for the study of the effects of anti-androgenie compoullds on sebaceous structures.
EFFECT OF ANTI-ANDROGENS ON PREPUTIAL GLANDS 1. I p V ' J\ Fm. 3. A histologic section of a preputial gland from a castrated male rat treated with testosterone showing the sebaceous nature of the gland. METROnS AND MATERIALS All rats used were castrated males of the Sprague-Dawley strain fed an ad lib diet of Purina Laboratory Chow and water. Room temperature and lighting were not closely controlled. The rats were castrated two weeks or more before the beginning of the experiments. All animals were weighed at the beginning and conclusion of the experiments, and were distributed into the various test groups in a random manner and identified by notching of the ears. Each test group was housed in a separate cage. The animals were injected daily with a or 1.0 cc volume of a sesame oil solution of the compounds to be tested. Injections were made subcutaneously in the scapular area for to days, and the animals were sacrificed on the day following the last injection. After sacrificing, the rats were tagged with code numbers, and the preputial glands. seminal vesicles, ventral prostate and levator ani muscles were dissected by one operator who was not aware of the coding. The moist weights of the organs were recorded immediately after dissection, The levator ani muscle was dissected using the method described by Eisenberg and Gordan (13). Compounds SC-9420 (Aldactone), SC-33, and SC-495, were obtained from G. D. Searle & Co.t Compound Ro2-239 was obtained from t Courtesy of Francis J. Saunders, Ph.D., Division of Biological Research Hoffman-LaRoche Inc. Compound BS-1280 was obtained from Syntex Laboratories, Inc.f In some experiments, the antiandrogen and testosterone were injected separately at different sites. In other experiments they were dissolved in the same vial and injected together. Benzyl alcohol, 3 percent or percent was added to maintain sterility and to achieve complete solution of the dissolved compounds. All solutions were sterilized by heat after preparation. For each animal, determinations were made of body weight, initial and final, and organ weights of preputial glands, seminal vesieles, ventral prostate, and levator ani muscle. RESULTS The results of four experiments are shown in Table 1. In the first experiment, Compound SC- 9420 given in a to 1 ratio with testosterone caused a marked reduction of the response of the preputial gland to testosterone. The response of the seminal vesicles and prostate did not appear to have been reduced. SC-495 increased the growth of the seminal vesieles, but the inhibition of the preputial glands was not statistically significant. Courtesy of Mr. W. E. Scott Courtesy of Kenneth Dumas, M.D.
Compound (s) mg Days + SC-9420 + SC-495 Sesame Oil + SC-33 + Ro2-239 + Ro2-239 0.1 20.0 0.1 20.0 + RS-1280 12 12 5 5 TABLE Mean (Gin.) SE. Mean Organ Weight (mg) ifz S.E. Initial Weight Final Weight Preputial Gland Seminal Vesicle Ventral Prostate Levator ani Muscle 42 8 483 11 193.4 29.5 4.3 43.4 190.4 2.2 421 15 483 18 4.3 1.9 438.2 35.2 193.3 1.1 441 11 500 15 139.5 25.8 594.2 23.0 283.4 3.0 228 + 3 230 3 25 302 5.2 9. 113.5 1.2 91.1 12.8 28.0 3 53. 3. 9.8.1 222±2 25±3 9.1 12.0 352.4± 11.1 14.±23.0 221 220 5 293 5 290 8 122. 1.0 9.9 15.3 820.0 29. 331.4 31.1 203.0. 1.8 13.0 221 5 28.2 1 584.2 43.1 19..2 21 4 288 8.5.8 15.8 1.9 1 11.5 308 8 30 8.2 +.1 24.2 3.1 222.1. 318 12 344 1.1 8.3 11.0 22.0 0.1.9 C
EFFECT OF ANTI-ANDROGENS ON PREPUTIAL GLANDS 19 In the second experiment, Compound SC-33 in a to 1 ratio with testosterone inhibited the response of the preputial glands, but did not inhibit the response of the seminal vesicles and prostate. In the third experiment, 20 mg of Ro2-239 was given in conjunction with nnd 0.1 mg of testosterone. The response of the seminal vesicles appeared to have been inhibited; however, the differences in the other organs were too small to be significant. In the fourth experiment, RS-1280 was given in a to 1 ratio. The seminal vesicles and levator ani demonstrated an anti-androgenic effect, but the inhibition of the preputial glands was probably not significant. There was definite asymmetry between the right and left preputial glands of the rats, with the left gland usually being larger than the right (mean 22 per cent greater). DISCUSSION The results of weighing the seminal vesicles, ventral prostates, and levator ani muscles further demonstrate that the effects of different antiandrogenic compounds on preputial glands in several cases were at variance with their effects on these other androgen-sensitive target organs. In screening compounds for anti-androgenic ability, measurement of the suppression of response of sebaceous structures to testosterone as determined by weighing the preputial glands would be a useful procedure. in testing compounds SC-9420 and SC-495, it would have been misleading to have considered only the seminal vesicles and prostate. The individual variation in preputial gland weight limits the ability to detect a small difference between test groups with confidence unless large groups of animals are used. Groups of 25 or 30 would be better than those used in this study, provided that enough test material was available. The finding of asymmetry of the preputial glands is in general agreement with that of Hess, who found left preputial glands to be 18 per cent heavier than the right (). The marked asymmetry of the preputial glands would seem to preclude using each animal as his own control by removing one gland at the beginning of the test period. Regarding the mechanism of anti-androgen action, Strickler has suggested that the net result of two antagonizing hormones can sometimes be satisfactorily explained by assuming a competitive adsorption on a common surface at the target cell (15). The dissociation constants of the surface-hormone complexes for each hormone and the ratio of the concentrations of the two hormones would then determine the net result of various mixtures. CONCLUSIONS 1. A method for determining the effect of antiandrogenic compounds on a specialized large sebaceous structure, the rat preputial gland, has been described. 2. Two of the five compounds tested, SC-9420 and SC-33, reduced the growth of the preputial gland in response to testosterone. 3. In screening compounds for anti-androgenic ability, measurement of the suppression of response of sebaceous structures to testosterone as detecting by weighing the preputial glands can be a useful procedure. REFERENCES 1. DOIIFMAN, RALPH I. AND DOEFMAN, ADELINE S.: A test for anti-androgens. Acta Endocr. (Kobenhavn), 33: 308, 190. 2. RANDALL, LOWELL 0. AND SELITTO, JOSEPH J.: Anti-androgenic activity of a synthetic phenanthrenc. Endocrinology, 2: 93, 1958. 3. LAuppi, E. AND Srunn, A.: Hemmung der experimentellen, testosteron-bedingten Epidermisproliferation an der weiblichcn Ratte Durch em synthetisches Phenanthrenderivat mit antiandrogener Wirkung. Experientia, 15: 24, 1959. 4. LAum, E. AND STUDER, A.: Additional studies on skin proliferation-inhibiting properties of a phenanthrene derivative (Ro2-239). IDermatologica (Basel), 120: 25, 190. 5. LAuppi, E.: The effect of Ro2-239 on nonspecific proliferation of the epidermis of the rat. Dermatologica, 122: 380, 191.. DOEFRAN, RALPH I.: Methods in hormone research. Acad. Press, 2: 35, and 31523, 192.. MANCINI, H. E., et al.: Autoradiographic and histochemical study of cock's comb in normal and hormonally-treated birds. Endocrinology, : 430, 1948. 8. EBLING, F. J.: Sebaceous glands. I. The effect of sex hormones on the sebaceous glands of the female albino rat. Endocrinology, 5: 29, 1948. 9. LASHER, N., et el: Hormonal effects on sebaceous glands in the wbite rat. J. Invest. Derm., 22: 25, 1954.. HUGGINS, C. H., ci ci.: Promotion of growth of preputial glands by steroids and the pituitary growth hormone. Endocrinology, 5: 25, 1955.
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY 11. LOJtJNCZ, A. L. AND LANCASTER, G.: Anterior pituitary preparation with tropic activity for sebaceous, preputial, and Harderian glands. Science, 12: 124, 195. 12. KOEENCHEvSKY, V., et al.: The prolonged treatment of male and female rats with androsterone, and its derivatives, alone or together with oestrone. Biochem. J., 29: 2534, 1935. 13. EISENEEI%G, E. AND GORDAN, G. S.: The levator ani muscle of the rat as an index of myotrophic activity of steroidal hormones. J. Pharmacol. Exp. Ther., 99: 38, 1950.. HEss, M., et at.: Response of preputial and adrenal glands of hypophysectomized rats to ACTH. Endocrinology, 52: 223, 1953. 15. STEICKLER, H. S., et at.: Application of physical concepts to dose response relations of mixed estrogens and mixed androgens. Proc. Soc. Exp. Biol. Med., : 309, 191.