Endocrinol. Japon. 1980, 27 (1), 69-76 Action of a Novel Nonsteroidal Antiandrogen, AA560 KEIZO SHIDA1, HIDETOSHI YAMANAKA1, ATSUSHI KOYA1, KATSUMIWAKABAYASHI2, HIROSHIMORI3, KENYU SHIBATA3 ANDEIICHIRO SHIMAZAWA3 1D epartment of Urology School of Medicine, Gunma University, Maebashi, 'Hormone Assay Center, Institute of Endocrinology, Gunma University, Maebashi, 3Research Division, Teikoku Hormone Mfg. Co., Kawasaki Synopsis The antiandrogenic properties of a new nonsteroidal antiandrogens, AA560 (N- (2-chloromethyl-2-hydroxypropionyl)-3, 4, 5-trichloroaniline) were investigated. The ventral prostate, dorselateral prostate and coagulating gland weights in rats given AA560 at1-9mg/head were significantly less than those in the intact rats. The seminal vesicle weights in rats given3-9mg/head were significantly less than those of the intact group. In intact animals given daily3or9mg of AA560there were significantly increases of serum FSH, LH and testosterone concentrations. In the in vivo experiment, the pretreatment with AA560decreased the uptake of 3H-androgens in the nuclear fraction of the ventral prostate. On the other hand, a significant increase in the uptake of3H-radioactivity in the cytosol fraction was observed. It was proved by the in vitro displacement study that AA560inhibited the binding of5a-dihydrotestosterone with a receptor protein in the prostatic cytosol. An intensive effort has been directed the transfer of5a-DHT into nucleus (Belham toward the research of antiandrogens because and Neal, 1971; Mangan and Mainwaring, of usefulness of these druge in a variety of 1972). On the other hand, SK&F7690was endocrinological disorders including hirsu- reported to inhibit the cytosol receptor bin- tism, acne, male pattern baldness, benign ding to5a-DHT and the rate of 5a-DHT prostatic hypertrophy and prostatic cancer. formation (Tveter, 1971; Shimazaki et al., A number of antiandrogens have been re- 1972). The representative of nonsteroidal ported, most of which have steroidal stru- antiandrogens is SCH13521 (Flutamide). ctures. The representatives of steroidal an- SCH13521 has been reported to have potent tiandrogens are Cyproterone acetate, Chlor- madinone acetate, Megestrol acetate, SK& Abbreviations used are: F7690and BOMT. Cyproterone acetate and Cyproterone acetate; 6ƒ¿-chloro-17ƒ¿-hydroxy-1ƒ¿, 2a- BOMT compete with 5a-dinydrotestosterone methylene-4, 6-pregnadiene-3, 20-dione-17-acetate. Chlormadinone acetate; 6ƒ¿-chloro-17a-acetoxy-4, 6- (5a-DHT) for the specific, high affinity ben- pregnadiene-3, 20-dione. ding site in the cytosol of prostate and reduce Megestrol acetate; 17ƒ¿-hydroxy-6-methylpregna-4, 6- diene-3, 20-dione-acetate. Received June4, 1979. BOMT; 6ƒ¿-bromo-17/3-hydroxy-17a-methy1-4-oxa-5ƒ¿- * Department of Urology, School of Medicine, androstane-3-one. Gunma University, Maebashi. SK&F7690; 17ƒ¿-methyl-B-nortestosterone. ** Hormone Assay Center, Institute of Endocrino- SCH13521; N-butyryl-3-trifluoromethyl-4-nitroani- logy, Gunma University, Maebashi. line. *** Research Division, Teikoku Hormone Mfg. Co., AA560; N-(2-chloromethyl-2-hydroxypropionyl)-3, 4, Kawasaki. 5-trichloroaniline. Endocrinol. Japon. 70 SHIDA et al. February1980 antiandrogenic effects in the experimental animals (Neri et al., 1972; Neri and Mon- ahan, 1972; Liao et al., 1974; Varkarakis et al., 1975; Surfin and Coffey, 1976) and humans (Irwin and Prout, 1973; Stoliar and Albert, 1974; Prout et al., 1975). This re- -port deals with the antiandrogenic proper- ties of a new nonsteroidal compound, AA Fig.1. Chemical structure of AA560. 560 (N-(2-chloromethyl-2-hydroxypropionyl) -3, 4, 5-trichloroaniline)(Fig.1). concentration (8-12mg/ml). The condensed super- natant fluid was mixed with10ul/ of an ethanol solution of 3H-DHT and100ƒÊl of glycerol to give Materials and Methods final concentrations of2•~10-9m and10 (v/v), re- spectively, and was incubated for3.5hr at0•Ž by Chemicals gentle shaking in the absence or presence of non- radioactive competing compounds dissolved in 5ul (1, 2-3H) testosterone (59Ci/mmole) and (1, 2-3H) 5a-DHT (48Ci/mmol) were obtained from Radio- of ethanol. Then an equal volume of0.25% (w/v) chemical Centre, Amersham, England. Cyproterone dextran-2.5%(w/v) charcoal-10% (v/v) glycerol in -acetate was a gift from Schering AG, Berlin. Chlor- in TEM buffer was added and vortexed for5sec. madinone acetate, AA560and SCHI3521were ob- After centrifugation at8,000•~g for10min, clear tained from Teikoku Hormone Mfg. Co., Kawasaki. supernatant fluids were counted to estimate the pro- tein bound radioactivity. Animals Male Wistar strain rats were maintained on labo- Sucrose density gradient analysis ratory chow of Japan Clea Co. and water ad libitum. After dextran-charcoal treatment, 0.2ml of the supernatant was layered on the top of a sucrose Animals were housed in metabolic cages in a tem- perature-controlled room under a12hr light-12hr gradient (5to20% linear in Hitachi RPS 65T roter tube) containing1.5mm EDTA, 2mm2-mercaptoe- d ark lighting schedule. Orchiectomy was performed thanol, 10% (v/v) glycerol and50mm Tris-HC1, via the scrotal route under ether anaesthesia. The pH7.4. The tube was centrifuged at178,900•~g dose and schedule of the injection are given in detail for21hr at2•Ž. Fractions (0.1ml each) were col- in foot-notes to Table. Sprague-Dawley strain (JCL: lected using a density gradient fractionater (ISCO, SD) rats weighing250-300g were used for the in -vivo and in vitro displacement studies Model640), and the radioactivities were measured. Radioactivity measurement Preparation of subcellular fraction Aqueous samples containing tritium were dissolved The ventral prostates were removed and homo- in a mixture of1.0ml water, 1.0ml ethyl alcohol genized with a Potter-Elvehjem homogenizer in cold and10ml toluene containing0.4% PPO and0.01% v0.05M Tris-HC1 buffer, pH7.4containing0.25M POPOP, and then vortexed for30sec. to extract sucrose and1mM MgC12. The homogenate was cen- the labelled steroid from the aqueous phase. Radio- trifuged at600•~g for10min. and then at105,000•~g activity was counted in a Packard Tri Carb liquid for60min., to yield the prostatic cytosol fraction. scintillation spectrometer, Model 3380, with a count- The crude nuclear fraction (600•~g) was suspended ing efficiency of37-47%. in2.4M sucrose-1mM MgC12, and then centrifuged in a Hitachi RPS27roter at93700•~g for40min., at0•K-2•Ž, to yield the purified nuclear fraction. Protein assay Protein was determined by the method of Lowry et al. (1951). In vitro displacement studies The prostatic tissues were gently homogenized with a Potter-Elvehjem homogenizer in cold50mm Serum LH, FSH, testosterone and corticosterone Tris-HC1, pH7.4containing1.5mm EDTA disodium measurement salt and2mm2-mercapto-ethanol (TEM buffer). The Serum LH, FSH were measured by radioimmuno- homogenate was centrifuged at 105,000 xg for60min assay employing the RIA kits supplied by Dr. at2•Ž. The supernatant fluid was concentrated using A. F. Parlow of Rat Pituitary Hormone Program, Minicon B15 (Amicon) to get an appropriate protein NIAMDD, NIH., as described previously (Waka- Vol.27, No.1 ACTION OF AA560 71 Table1. Inhibition of endogeneous androgens in mature male rats by the oral administration of AA560or chlormadinone acetate for21days. Data are shown as mean•}standard error. Animals received daily the indicated dose of AA560or chlormadinone acetate suspended in Twin80 by the oral administration for21days and were killed24hr after21th administration. * Significant difference from mean of the intact group, p<0.05. ** Significant difference from mean of the intact group, p<0.01. bayashi, 1977). The results were expressed as the FSH, LH, testosterone and corticosterone equivalent of NIH-LH-S1and NIH-FSH-S1for LH levels in mature male rats are shown in and FSH. Serum testosterone was measured by radioimmunoassayaccording to the method of Ma- Table2. In animals given daily3or9mg kino (1973). Serum corticosterone was determined of AA560there was a significant increase also by radioimmunoassay(Kanbegawa et al., 1977). of serum FSH concentration. In contrast, in animals given27mg of chlormadinone acetate the opposite was observed. Table2 Results also shows there were significant increases of serum LH and testosterone levels in ani- Effects of AA560or chlormadinone ace- mals given AA560. Chlormadinone acetate tate administration to intact male rats. did not appear to affect the serum LH level Three different doses of AA560were in these experimental conditions. The daily given orally for21days and the changes in administration of9mg of AA560increased the weights of the accessory sex organs, adrenal weights and serum corticosterone adrenal gland and testis were compared with level, whereas27mg of chlormadinone those in rats given orally chlormadinone ace- acetate significantly decreased them. tate (Table1). The ventral prostate weight Effects of the duration of AA560treat- from rats given AA560at1-9mg/head were ment in intact mature male rats. significantly less than those in the intact The effects of the duration of AA560 control rats. The seminal vesicle weights treatment in intact male rats were examined. in rats given3-9mg/head were significantly Rats received3mg of AA560suspended in less than those of the control group. Adrenal Twin80daily by gavage for1to35days. weights were significantly increased in rats The weights of ventral prostates, dorsolateral receiving daily9mg of AA560, whereas prostates and seminal vesicles were signifi- those in chlormadinone acetate treated