Endocrine Journal 1994, 41(4), 445-452 Antiandroenic~Activity and Endocrinological Profile of a Novel Antiandrogen, 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 steroidal antiandrogen. Antiandrogenic activity and endocrinological profile of TZP-4238 were investigated in rats, except that progestational activity was determined in rabbits. TZP-4238 suppressed the testosterone propionate-induced increases in the weights of the ventral prostate, 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 luteinizing hormone and testosterone. TZP-4238 did not have such an inhibitory effect on the weight of the adrenal gland as seen in other steroidal antiandrogens. 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 drug for the treat- ment of androgen-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 cyproterone acetate, megestrol acetate (MS) and action is the competition with androgen for the an- chlormadinone acetate (Prostal®) do not induce drogen receptor in the target organ. Non-steroidal any compensatory increase in the serum concen- antiandrogens such as flutamide and nilutamide 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 androgen receptor. 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- androgens 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 steroid 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-pregnadiene-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). Female 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 cyproterone acetate (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 Testosterone propionate (TP) and estradiol 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- Norethisterone (NE) and medroxyprogesterone 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 ligand 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]. Estrogen 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 estradiol benzoate 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 Glucocorticoid 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 liver 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