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Endocrinol. Japon. 1980, 27 (1), 69-76

Action of a Novel , 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 , 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 concentrations. In the in vivo experiment, the pretreatment with AA560decreased the uptake of 3H- 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- 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 acetate, Chlor- madinone acetate, , SK& Abbreviations used are: F7690and BOMT. and Cyproterone acetate; 6ƒ¿-chloro-17ƒ¿-hydroxy-1ƒ¿, 2a- BOMT compete with 5a-dinydrotestosterone methylene-4, 6-pregnadiene-3, 20-dione-17-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, -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 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 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 rats cantly decreased on the3rd day after AA560 were decreased compared with those of the treatment. On the other hand, the weights control group. Throughout the period of this of testes in the AA569treated rats were not experiment no changes of testicular weights significantly different from those in the con- were observed in both AA560and chlor- trol rats in the35days'study (Table3). madinone acetate treated rats. Table4shows the changes of serum The effects of the oral administration of FSH, LH, testosterone and corticosterone AA560of chlormadinone acetate on serum levels in the rats in the same experiment. Endocrinol. Japon. 72 SHIDA et al. February1980

Table2. Effects of the oral administration of AA560or chlormadinone acetate on serum FSH, LH, testosterone and corticosterone levels in mature male rats.

Data are shown as mean•}standard error. Animals used in this experiment are the same as those in the experiment shown in Table1. S1: Immunoreactive FSH and LH values assayed with NIAMDD anti-rat NIH-FSH-Sl and NIH-LH-S1. * Significant difference from mean of the intact group, p<0.05. ** Significant difference from mean of the intact group, p<0.01.

Table3. Inhibition of endogeneous androgens in mature rats by the oral administration of3mg of AA560for1to35days

Data are shown as mean•}standard error. Mature male rats received3 mg of AA560daily by the oral administration for the indicated periods 24hr after the last administration, rats were killed and organs were removed and weighed. * Significant difference from mean of the intact group, p<0.05. ** Significant difference from mean of the intact group, p<0.01.

In animals given3mg of AA560daily for Effects of AA560on the castrated im- 3to35 days there was a slight increase of mature male rats given an injection of serum FSH concentration. In contrast, there testosterone. were significant increases of serum LH and Table5shows that AA560inhibited testosterone concentrations on the first day testosterone induced hypertrophy of accessory after the treatment of AA560compared with sex organs in castrated rats. Castration was the intact rats. The serum testosterone level performed4weeks after birth. The weights gradually increased and reached about seven of ventral prostates and seminal vesicles were fold higher than that of the intact rats on significantly less in those rats given125-1000 the35day after the treatment. There was no pg of AA560concomitantly with testosterone significant difference of corticosterone level propionate than in those given testosterone between AA560treated rats and the control propionate alone. On the other hand, there rats. were no significant changes in adrenal weights between these two groups. Vol.27, No.1 ACTION OF AA560 73

Table4. Changes of serum FSH, LH, testosterone and corticosterone levels in mature rats by the oral administration of3mg of AA560for1to35days.

Data are shown as mean•}standard error. Animals used in this experiment are the same as those in the experiment shown in Table3. * Significant difference from mean of the intact group, p<0.05. ** Significant difference from mean of the intact group, p<0.01.

Table5. Inhibition of exogeneous androged in castrated immature rats by the oral administration of AA560.

Data are shown as mean•}standard error. Drug treatment was begun on the8th day after castration. Both testosterone propionate and AA560 were administerd for7days. Testosterone propionate was injected subcutaneously at100g simul- taneously with varing doses of AA560 given orally for7days. 24hr after the last treatment, ventral prostate, seminal vesicle and adrenal gland were removed and weighed. * Significant difference from mean of testosterone propionate injected group, p<0.05. ** Significant difference from mean of testosterone propionate injected group, p<0.01.

Effects of AA560on the uptake of3H- nand, a significant increase was observed in androgens in vivo. the uptake of 3H-radioactivity in the cytosol The effect of the pretreatment of castrated fraction. rats with AA560on the uptake of3H-and- rogens in the whole homogenates, cytosol Effect of AA560on the3H-5a-DHT bin- fraction and nuclear fraction (2.4M sucrose) ding to8S macromolecules of castrated of the ventral prostate was investigated. As rat prostatic cytosol in vitro. shown in Table6., the pretreatment with 8S receptor is considered to be a specific, AA560decreased the amount of radioac- hign affinity but low capacity binding com- tivity in the nuclear fraction. On the other ponent according to the sucrose density Endocrinol. Japon. 74 SHIDA et al. February1980

Table6. The effect of AA560 on the uptake of 3H-testosterone in the ventral prostate, in vivo.

Two groups of three adult male Sprague-Dawley rats were castrated, and21hr later the first group Fig.2. 105,000•~g supernatant was prepared from received1mg of AA560by the intraperitoneal in- the ventral prostate of21hr castrated rats. Sam- jection. The second group (control) received aqueous ples were incubated with2•~10-9m 3H-5ƒ¿-dihydro- vehicle. Both groups were injected intraveneously testosterone alone or in the presence of an excess with0.5ƒÊg of3H-testosterone30min after the in- of non-radioactive competitors. After the treat- jection of AA560or aqueous vehicle. The ventral ment of dextrancoated charcoal, samples was prostates were removed30min after 3H-testosterone analyzed by the sucrose density gradient centrifuga- injection. Subcellular fractions were prepared as de- tion. The results are expressed as the percentage of scribed in text. The results are the mean of three the binding in control sample (without competitor) inde pendent determinations. and are plotted agaist the log of the concentration of competitor. C. M. A. and C. A. represent chlormadinone acetate and cyproterone acetate, respectively.

gradient analysis. The ability of AA560to that of SCH31521 (Takahashi et al., 1977). compete with3H-5ƒ¿-DHT in the8S region Chlormadinone acetate is a representative was examined in comparison with SCH of steroidal antiandrogens and usefulness of 13521, cyproterone acetate, chlormadinone this drug in the treatment of human prostatic acetate, estradio1-17ƒÀ and5ƒ¿-DHT. As shown tumor has been reported (Shida et al., 1972; in Fig.2, 1000fold excess of SCH13521did Geller et al., 1969). Table 1 shows that not compete with 3H-5ƒ¿-DHT in8S region the magnitude of inhibition of endogeneous at all. On the other hand, 1000fold excess androgens in mature male rats by the oral

of AA560displaced 95% of 3H-radioactivity administration of AA560was about three in8S region. Fig.2also shows that both times as large as that of chlormadinone cyproterone acetate and chlormadinone ace- acetate. tate showed significantly inhibitory effects We reported in this paper that the pre- on the3H-5ƒ¿-DHT binding to8S macro- treatment with AA560in vivo decreased the molecules of the rat prostatic cytosol. uptake of 3H-androgens in the nuclear frac- tion and increased that in the cytosol frac- tion. Mainwaring et al.(1974) have reported Discussion similar results, namely, antiandrogens such as SCHI3521or BOMT increased the uptake

AA560 N-(2-chloromethl-2-hydroxypro- of 3H-androgens in the homogenates and

pionyl-3, 4, 5-trichloroanilin.e) is a derivative decreased the3H-uptake in the nuclear of anilide (Fig.1) and one of the potent fraction. They gave the most plausible ex- nonsteroidal antiandrogens. Its chemical planation about these phenomena that the structure resembles that of SCHI13521 compounds under evaluation affected the

(Flutamide). The antiandrogenic potency rate of entry of3H-testosterone into only of AA560was reported to be stronger than dependent cells such as those in Vol.27, No.1 ACTION OF AA560 75 the prostate. The mechanism of action of corticosterone level in male rats (Table1 AA560 is not completely obvious. and2). Further investigation will be needed The present in vivo and in vitro displa- in order to clarify the mechanism of action cement studies suggested that one mechanism of AA560on the hypothalamo-pituitary of action of this compound might be related adrenal axis. to an affinity of AA560itself and/or me- Our experiments showed that there were. tabolites of this compound for the androgen significant increases of serum LH, FSH and receptor, resulting in the interference of testosterone levels in intact male rats after transfer of5a-DHT-receptor complex into the oral administration of AA560. Prout et the nucleus. Therefore, the subcellular anta- al.(1975) have reported that the treatment of gonistic mechanism of AA560to testosterone male patients with stage D prostatic cancer seems to be similar to that of the antiand- with SCH13521increased serum LH levels. rogens such as SCH13521, BOMT and cypro- The absence of central inhibitory effects of terone acetate. According to Liao et al. SCHI3521was indicated in the failure to (1974), the molecular model building showed inhibit gonadotropin release in the perabiotic that the gross geometric structures of a rat (Neri et al., 1972). It has been reported nonsteroidal antiandrogens, SCH13521and (Kato and Onouchi, 1973a; Kato and Ono- 5a-DHT were more similar than their chemi- uchi, 1973b; Lieberburg et al., 1977) that cal formulas would suggest. Therefore, SCH there exist androgen receptors in the cytosol 13521 (or its metabolite) may act by com- fraction of male rat hypophysis and hypo- peting with5a-DHT for the androgen bin- physis and hypothalamus. We have already ding site on the receptor protein. shown (Yamanaka et al., 1979) that both It has been reported by Newmann and AA560and SCH13521had an ability to Steinbeck (1974) that a steroidal antiand- 5a-DHT binding to cytosol macro- rogen, cyproterone acetate, caused a decrease molecules from male rat hypophysis and of adrenal weight in male rats. Purvis et al. hypothalamus. (1977) reported that cyproterone acetate Thus, nonsteroidal antiandrogens such as reduced plasma corticosterone levels in im- AA560or SCHI3521might cause the in- mature male rats. This compound may not creased secretion of gonadotropins, since less. have a direct effect on the adrenal gland androgen was available for the negative since there is no suppression of adrenal feedback. Further studies for the detailed gland weight by this compound when injected mechanism of feedback control of gonado- into hypophysectomized rats substituted with tropin secretion in male sujects was ardently ACTH. Table1and2showed that chlor- awaited. madinone acetate had similar inhibitory effects on adrenal weight and serum corti- costerone levels in male rats. Acknowledgements It is unlikely that this inhibitory effect of cyproterone acetate or chiormadinone The authors are grateful for the expert technical assistance provided by Mrs. H. Yuasa and Miss Y. acetate on adrenal function is a consequence Sugiyama. This work was partly supported by a of their progestalional properties, because Grant from the Ministry of Education, Scienceand an antiandrogen, cyproterone, which is Culture, Japan and a Grant-in Aid for Cancer Re- devoid of progestational activity, has similar search from the Ministry of Health and Welfare of effects on plasma corticosterone level (Purvis Japan. et al., 1977). On the other hand, nonsteroidal antiandrogen, AA560, has the stimulatory effects on adrenal gland weight and serum Endocrinol. Japon. 76 SHIDA et al. February1980

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