Endocrine Journal 1994, 41(4), 445-452

Antiandroenic~Activity and Endocrinological Profile of a Novel , TZP-4238, in the Rat

MAMORUMIEDA, YOSHIHIROOHTA, T0M0YUKI SAITO, HIROO TAKAHASHI, EIICHIROSHIMAZAWA ANDKATSUHIKO MIYASAKA PharmacologicalResearch Department, Teikoku Hormone Mfg. Co., Ltd., Kawasaki213, Japan

Abstract. TZP-4238 is a new potent, orally active . Antiandrogenic activity and endocrinological profile of TZP-4238 were investigated in rats, except that progestational activity was determined in rabbits. TZP-4238 suppressed the propionate-induced increases in the weights of the ventral , seminal vesicle and levator ani in castrated immature male rats. TZP- 4238 also decreased the weights of the ventral prostate, seminal vesicle and levator ani in intact adult male rats, but did not affect the weight of the testis or the serum concentrations of and testosterone. TZP-4238 did not have such an inhibitory effect on the weight of the as seen in other steroidal . It exhibited potent progestational activity. Although TZP-4238 did not exert androgenic or estrogenic activity, it had weak antiestrogenic activity. These results suggest that TZP-4238 exerts an antiandrogenic effect on the prostate without any compensatory change in the serum concentration of luteinizing hormone or testosterone in rats, and it is a useful for the treat- ment of -dependent diseases such as prostatic hypertrophy and prostatic cancer.

Key words: TZP-4238, Antiandrogenic activity, Endocrinological profile. (Endocrine Journal 41: 445-452,1994)

ONE OF THE main mechanisms of antiandrogenic acetate, (MS) and action is the competition with androgen for the an- (Prostal®) do not induce drogen in the target organ. Non-steroidal any compensatory increase in the serum concen- antiandrogens such as and tration of LH or testosterone by a centrally medi- (Anandron®) exert their action exclusively through ated antigonadotropic action. However, it is pos- competition for the . They, how- sible for them to decrease the blood testosterone ever, induce a compensatory increase in serum concentration by their anti-gonadotropic action [2, concentrations of luteinizing hormone (LH) and 4, 5], resulting in inhibition of the reproductive testosterone [1-3] by blocking the negative feed- function. Furthermore, these steroidal anti- back at the hypothalamic-pituitary level. There- are considered to suppress the function fore, non-steroidal antiandrogens have a potent of the adrenal gland by their high affinity with glu- antiandrogenic action under the orchiectomized cocorticoid receptor [5-7]. It is very desirable to condition, but the activity is reduced under the find an orally active antiandrogen whose undesir- nonorchiectomized condition. able actions such as compensatory increase in LH In contrast, steroidal antiandrogens such as and testosterone concentrations, inhibition of re- productive function and suppression of the adre- Received: December 17, 1993 nal function are minimized. On the basis of this Accepted: April 7, 1994 idea, we have synthesized a series of de- Correspondence to: Dr. Hiroo TAKAHASHI, Pharmaco- rivatives and discovered 17a-acetoxy-6-chloro-2- logical Research Department, Teikoku Hormone Mfg. Co., Ltd., 1604 Shimosakunobe, Takatsu-ku, Kawasaki 213, oxa-4,6--3, 20-dione (TZP-4238) [8] Japan (Fig. 1). 446 MIEDA et al.

This article describes the endocrinological pro- 4238, CMA and CPA, 3-week-old male rats were files of TZP-4238 and its main metabolite in man castrated, and one week later TP (1 mg/kg) was and rats, 17a-acetoxy-6-chloro-15-hydroxy-2-oxa-4, administered subcutaneously (s.c.) to the animals 6-pregnadiene-3, 20-dione(150H-TZP) (Fig. 1), with concurrently with oral administration of each anti- special reference to their antiandrogenic action. androgen or vehicle (5% arabic gum physiological saline solution) for 7 successive days. Each animal was weighed on the day following the final admin- Materials and Methods istration, and after the animals were sacrificed by ether anesthesia, the ventral prostate, seminal Animals vesicle and levator ani were excised and weighed. To compare of the antiandrogenic activity of TZP- Wistar rats were supplied by SLC (Hamamatu, 4238 and 150H-TZP, TP was administered s.c. at a Japan). immature rabbits were obtained dose of 0.05 mg/rat to the animals concurrently from Kanamaru Experimental Animals Co. with oral administration of each compound or ve- (Tokyo, Japan). The animals were maintained un- hicle for 5 successive days. til the end of the experiment with commercially available pellets (CE-2, CLEA Japan Inc.) and tap Antiandrogenic activity in intact mature male rats water given ad libitum in rooms controlled at a room temperature of 22 ± 2°C and 55 ± 15% hu- Fifteen-week-old male rats were orally given midity and illuminated from 0800 to 2000 h. TZP-4238, CMA or vehicle for 14 successive days. Each animal was weighed on the day following the Steroidsand referencecompounds final administration. After blood sampling from the abdominal aorta under ether anesthesia, the TZP-4238, 150H-TZP, chlormadinone acetate testis, ventral prostate, seminal vesicle and levator (CMA) and (CPA) were syn- ani were excised and weighed. After centrifuga- thesized in the chemical section of our laboratory. tion of blood samples, the serum was collected and (TP) and ben- frozen at -20°C until used to determine the serum zoate were obtained from Roussel UCLAF (Paris). concentrations of LH and testosterone. Estradiol was obtained from Schering A G (Berlin). The serum testosterone concentration was deter- (NE) and ac- mined by gas chromatography-mass spectrometry etate (MPA) were obtained from Sigma Chemical after conversion of testosterone to a Co. (St. Louis, MO). trifluoroacetate derivative, in which 2H3-testoster- one was used as an internal standard. The serum Antiandrogenic activity in castrated immature male LH concentration was determined by Amersham's rats rat luteinizing hormone (yLH) [125I] assay (Amersham, Buckinghamshire, England). To assess the antiandrogenic activity of TZP-

Fig. 1. The chemical structures of TZP-4238 and 150H-TZP. ANTIANDROGENIC ACTIVITY OF TZP-4238 447

mM ethylenediaminetetraacetic acid (EDTA), 0.5 And rogenic activity mM dithiothreitol (DTT), 10 mM Na2Mo04, 0.1 mM phenylmethanesulfonyl fluoride (PMSF),10% Three-week-old male rats were castrated, and 2 (v/v) glycerol, pH 7.4] under cooling with ice. The weeks later TZP-4238was administered orally and homogenates were centrifuged at 105,000 g for 60 testosterone was injected s.c. The animals were min, and the supernatant was used as the proges- sacrificed by ether anesthesia 72 h after a single terone receptor source. The was 3H-R5020 injection, and the ventral prostate was then excised (3215.3 GBq/mmol, the final concentration of 2.88 and weighed. nM/l; NEN Research Products, Boston, MA) and the binding assay followed the method of Leavitt Progestational activity (Clauberg's test) et al. [10]. receptor assay: Three- to 4-week-old fe- Immature female rabbits were primed by subcu- male rats were sacrificed by ether anesthesia, and taneous injection of at 2 µg/rab- the uterus was excised. The uterus samples were bit for 7 successive days. For 5 successive days homogenized with 5 times the quantity of TED after that, TZP-4238,150H-TZP and reference com- buffer (10 mM Tris-HCI, 1.5 mM EDTA, 0.5 mM pounds (CMA, MPA and NE) were orally adminis- DTT, pH 7.4) under cooling with ice. The tered. Each animal was weighed on the day fol- homogenates were centrifuged at 105,000 g for 60 lowing the final administration, and sacrificed by min, and the supernatant was used as the estrogen injecting a fatal dose of pentobarbital-Na. The receptor source. The ligand was 3H-estradiol (6253 uterus was excised from each rabbit, and weighed. GBq/mmol, the final concentration of 1.67 nM/I The uterine horn was fixed in 10% formalin buff- NEN Research Products), and the binding assay ered solution. After fixation, the uterus was sec- followed the method of Lieberman et al. [11]. tioned so that the cut surfaces would be at right receptor assay: Six-week-old angles to the long axis, and hematoxylin-eosin- male rats whose adrenal glands had been excised stained sections were prepared. Progestational ac- 3-7 days before the experiment were sacrificed by tivity was measured by taking the growth of the ether anesthesia. The was perfused with ice- endometrium as a parameter (the McPhail index) cooled physiological saline solution to remove the [9]. The minimum effective dose was represented blood. The liver was homogenized with twice the as the minimum dose required to induce endome- quantity of TEND buffer (pH 7.4) under ice cool- trial hyperplasia in all the animals in the group. ing. The homogenates were centrifuged twice at 105,000 g for 60 min, and the supernatant was used Estrogenic and antiestrogenic activity as the glucocorticoid receptor source. The ligand was 3H-triamcinolone acetonide (740 GBq/mmol, Either TZP-4238 or 150H-TZP was orally admin- the final concentration of 1.67 nM/l; Amersham, istered, and estradiol was concurrently injected s.c. Buckinghamshire, England), and the binding assay into 3-week-old female rats for 3 successive days. followed the method of Agarwal et al. [12]. On the day following the final administration, each animal was weighed. After the animals were sacri- Statistic analysis ficed by ether anesthesia, the uterus was excised and weighed. Statistical comparisons were made by analysis of variance followed by a Dunnett's test [13]. The receptor assay relative of TZP-4238 and each of the other antiandrogens was calculated by four point assay. receptor assay: Nine-week-old fe- male rats to which estradiol (10 jig/O.1 ml of corn oil/rat/day) had been administered for 4 succes- Results sive days before the experiment, were sacrificed by ether anesthesia, and the uterus was excised. The Table 1 shows the antiandrogenic activity of uterus samples were homogenized with 3 times TZP-4238 against the exogenous androgen. In the the quantity of TEND buffer [10 mM Tris-HCI, 1 castrated immature rats, oral treatment with TZP- 448 MIEDA et al.

4238 dose-dependently suppressed the increase Table 3 shows the antiandrogenic activities of induced with TP in the weights of the ventral TZP-4238 and CMA against endogenous andro- prostate, seminal vesicle and levator ani. The gens. In normal adult male rats, oral treatments minimum dose at which TZP-4238 significantly with TZP-4238 and CMA decreased the weights of decreased the weights of the organs was 4.5 mg/ the ventral prostate and seminal vesicle dose-de- kg for the ventral prostate and seminal vesicle, and pendently. The minimum dose at which the anti- 13.5 mg/kg for the levator ani. Thus, TZP-4238 androgens significantly decreased the weights of inhibited the trophic effect of TP on these target the ventral prostate and seminal vesicle was 2 and organs of the castrated immature rats. The relative 0.4 mg/kg for TZP-4238 and 10 and 2 mg/kg for potencies of the antiandrogenic activity of TZP- CMA, respectively. The potency of CMA relative 4238, CMA, CPA and 150H-TZP against the to that of TZP-4238 calculated from the inhibitory exogenous androgen were 1.00, 0.10, 0.72 and 1.60, effect on the weight of the ventral prostate was respectively in the ventral prostate (Table 1 and 2). 0.24. Neither TZP-4238nor CMA exerted a signifi-

Table 1. Effects of TZP-4238, CMA and cyproterone acetate on sex accessory glands and levator ani in castrated immature male rats

Table 2. Effects of TZP-4238 and 150H-TZP on sex accessory glands in castrated immature male rats ANTIANDROGENIC ACTIVITY OF TZP-4238 449

Table 3. Effects of TZP-4238 and CMA on accessory grands, testis, adrenal and serum hormone levels in mature male rats

Table 4. Progestational activity of TZP-4238, CMA, norethisterone and MPAb~ in the estrogen primed immature female rabbits

Fig. 2. Effects of testosterone and TZP-4238 on ventral prostate weight in castrated, immature male rats. Testosterone was injected s.c. at doses of 2, 8 and 32 mg/kg, and TZP-4238 was orally administered at 100, 400 and 1,600 mg/kg. Values represent the mean ± SEM obtained from 8 rats. **, P<0.01 compared to vehicle. cant effect on body weight (data not shown), the weight of the testis or serum concentrations of LH and testosterone. CMA decreased the weight of the adrenal gland dose-dependently, while TZP- 4238 exerted no effect on it. TZP-4238 did not induce a significant increase in the weight of the ventral prostate of castrated im- mature rats at oral doses of 100-1600 mg/kg, thus showing no androgenic activity (Fig. 2). The periment (Table 5). The estrogenic and progestational activities of TZP-4238 and reference antiestrogenic activities of TZP-4238 and 150H- compounds are shown in Table 4. The minimum TZP are shown in Table 6. There was no signifi- effective dose is 0.0025 mg/kg for TZP-4238, 0.02 cant change in the weight of the uterus of female mg/kg for CMA, 0.05 mg/kg for MPA and 0.1 immature rats after the oral administration for 3 mg/kg for NE. 150H-TZP showed no progesta- days of TZP-4238 and 150H-TZP at either 0.3 or 3 tional activity at the doses used in the present ex- mg/rat, thus showing no estrogenic activity. TZP- 450 MIEDA et al.

Table 5. Progestational activity of 150H-TZP in estrogen 4238 significantly suppressed the increase in the primed immature female rabbits weight of the uterus induced with estradiol, but this antiestrogenic activity was not dose-depen- dent. 150H-TZP had no antiestrogenic activity. Figure 3 shows the binding affinities of TZP- 4238, CMA and 150H-TZP for progesterone, estro- gen and glucocorticoid receptors. The binding af- finities of TZP-4238 and CMA for progesterone re- ceptor were about 1/120 and 1/20 that of R5020,

Table 6. Estrogenic, antiestrogenic activity of TZP-4238 and 150H-TZP in immature female rats

Fig. 3. Representative competition profile of antiandrogens to rat uterine progesterone and estrogen receptors and rat liver glucocorticoid receptor. Cytosol binding assays were performed with the 3H-ligands, R5020, estradiol and dexamethasone for the progesterone, estrogen and glucocorticoid receptor assays, respectively. After correction for non-specific binding, the results were expressed as a percent of maximal binding. ANTIANDROGENIC ACTIVITY OF TZP-4238 451

respectively. 150H-TZP showed no binding affin- antiestrogenic action is interesting but remains to ity for . TZP-4238 and CMA be elucidated. showed no binding affinity for . TZP-4238 showed a very potent, progestational The binding affinities of TZP-4238,150H-TZP and activity. Compared at the minimum effective CMA for glucocorticoid receptor were about 1/ dose, the progestational activity of TZP-4238 was 20,000, 1/50,000 and 1/1,000 that of 40 times that of NE, 20 times that of MPA and 8 dexamethasone, respectively. times that of CMA. On the other hand, 150H-TZP did not show any progestational activity. A simi- lar situation has been reported on CPA and its 15- Discussion hydroxy metabolite, that is, the progestational ac- tivity of the 15-hydroxy metabolite of CPA is re- The present study shows that TZP-4238 and duced to 1/10 to 1/100 while its antiandrogenic 150H-TZP exerted a stronger antiandrogenic action activity is approximately equal to that of the par- in castrated immature rats than CPA, the most po- ent compound [14]. These results are consistent tent steroidal antiandrogen so far known. Anti- with the fact that TZP-4238 had binding affinity androgens of the steroid type generally with progesterone receptor, but 150H-TZP did not. decrease the blood concentration of LH or test- TZP-4238 did not suppress the function of the osterone by the antigonadotropic action, resulting adrenal gland in castrated immature rats (data not in the inhibition of reproductive function. In fact, shown) or intact mature rats. This is consistent El Etreby et al. [5] have reported that CPA and MS with the fact that TZP-4238 and 150H-TZP have decreased serum the LH concentration at the sub- very low binding affinity with glucocorticoid re- cutaneous dose at which they exerted an ceptor. antiandrogenic action in intact adult rats. In con- From the abovementioned point of view, that is, trast, TZP-4238 had no influence on the serum con- hormonal activities, antihormonal activities and centrations of LH and testosterone at the oral dose binding affinities for hormonal receptors, TZP- to exert an antiandrogenic action in intact adult 4238 is evaluated as having favorable characteris- rats. The effect of TZP-4238 in decreasing the tics for treating androgen-dependent diseases such weight of the prostate is therefore considered to be as prostatic hypertrophy and prostatic cancer. attributable to the antiandrogenic effect on the prostate without any antigonadotropic action. The mechanism whereby TZP-4238 did not cause any Conclusions compensatory change in serum concentrations of LH and testosterone is not known with certainty, TZP-4238 is a new potent, orally active steroidal but may be an exquisite balance between its antiandrogen that exerts an antiandrogenic effect antiandrogenic and antigonadotropic actions. The on the prostate without any compensatory change antiandrogenic potency of TZP-4238 relative to in . TZP-4238 has no an- CMA was 4 times in intact rats and 10 times in drogenic activity and does not have the kind of castrated rats supplemented with TP. 150H-TZP inhibitory effect on the adrenal gland seen in other had an antiandrogenic action 1.6 times as potent as steroidal antiandrogens. These results suggest that that of TZP-4238 in castrated rats. TZP-4238 is a unique antiandrogen and an useful TZP-4238 did not induce an increase in the drug for the treatment of androgen-dependent dis- weight of the prostate in castrated immature rats, eases such as prostatic hypertrophy and prostatic suggesting no androgenic activity. TZP-4238 and cancer. 150H-TZP showed no sign of estrogenic activity in female immature rats. On the other hand, TZP- 4238 caused weak but significant suppression of Acknowledgments the increase in the weight of the uterus induced with estradiol, but this antiestrogenic action was We express our heartful thanks to Dr. Kenyu not dose-dependent. Furthermore, TZP-4238 did Shibata and Dr. Hajime Okumura of our company not show any binding affinity with estrogen recep- for valuable advice given during the work and for tor. The mechanism whereby TZP-4238 exerted an helpful criticism of the manuscript. 452 MIEDA et al.

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