The Prostate 37:10–18 (1998) Novel Nonsteroidal Inhibitor of Cytochrome P45017␣ (17␣-Hydroxylase/C17–20 Lyase), YM116, Decreased Prostatic Weights by Reducing Serum Concentrations of Testosterone and Adrenal Androgens in Rats Yukitaka Ideyama,1 Masafumi Kudoh,1 Kyoko Tanimoto,1 Yoko Susaki,1 Taiki Nanya,1 Takahito Nakahara,1 Hiroko Ishikawa,1 Toru Yoden,1 Minoru Okada,1 Takashi Fujikura,1 Hideyuki Akaza,2 and Hisataka Shikama1* 1 Metabolic Diseases Research, Pharmacology Laboratories, Institute for Drug Discovery Research, Yamanouchi Pharmaceutical Co., Ltd., Tsukuba, Ibaraki, Japan 2 Department of Urology, Institute of Clinical Medicine, University of Tsukuba, Tsukuba, Ibaraki, Japan BACKGROUND. The purpose of this study was to determine the effects of a nonsteroidal C17–20 lyase inhibitor, 2-(1H-imidazol-4-ylmethyl)-9H-carbazole (YM116), on serum concentrations of androgens and ventral prostatic weight in rats. METHODS. Serum concentrations of testosterone and of dehydroepiandrosterone sulfate and prostatic weights were measured in rats treated with YM116. RESULTS. YM116 inhibited testicular C17–20 lyase competitively (Ki, 0.38 nM), and decreased the serum testosterone concentration in gonadotropin-releasing hormone-treated rats (ED50, 0.7 mg/kg), indicating that YM116 was about 21–24 times more potent than other C17–20 lyase inhibitors such as ketoconazole and liarozole, and was twice as potent as CB7630. YM116 also reduced dehydroepiandrosterone sulfate levels in ACTH-treated castrated rats (ED50, 11 mg/kg). YM116 (40 mg/kg, p.o., for 2 weeks) was almost comparable to bilateral orchiectomy with respect to the time course and magnitude of the reduction in prostatic weight. Each of these two treatments decreased the prostatic weight 3 days following the treatment. Contrarily, leuprolide transiently increased the prostatic weight and then decreased it. YM116 (100 mg/kg) had no effect on the serum cortisol level in guinea pigs, and slightly decreased the serum aldosterone level in rats. CONCLUSIONS. YM116 is a selective C17–20 lyase inhibitor which decreases rat prostatic weight by reducing androgen production in the testes and adrenal glands. Prostate 37:10–18, 1998. © 1998 Wiley-Liss, Inc. KEY WORDS: adrenal gland; orchiectomy; ketoconazole; leuprolide; CB7630; liarozole INTRODUCTION Since most prostate cancers show androgendependent growth, several antiandrogenic therapies have been attempted against this disease. Gonadotropin-releasing hormone (GnRH) agonists are widely recognized as a medical alternative to orchiectomy in © 1998 Wiley-Liss, Inc. the treatment of prostate cancer because of the potent inhibition of testicular testosterone synthesis by GnRH *Correspondence to: Hisataka Shikama, Ph.D., Metabolic Diseases Research, Pharmacology Laboratories, Institute for Drug Discovery Research, Yamanouchi Pharmaceutical Co., Ltd., 21 Miyukigaoka, Tsukuba, Ibaraki 305, Japan. Received 23 September 1997; Accepted 20 March 1998 Inhibition of P45017␣ and Prostatic Weight agonists . However, neither GnRH agonists nor orchiectomy can modify the production of adrenal androgens such as dehydroepiandrosterone (DHEA), its sulfate, and androstenedione. Adrenal androgens are converted to testosterone or dihydrotestosterone in the peripheral tissues. The prostatic concentration of testosterone or dihydrotestosterone was reported to be 25% or 10% of the pretreatment level, even after treatment with a GnRH agonist for 3 months [2,3]. The combined usage of a GnRH agonist or orchiectomy with an antiandrogen (flutamide or nilutamide) improved the median time to progression and survival in patients compared with those given the GnRH agonist or orchiectomy alone [4,5]. Recently, it was also reported that adrenal androgens activated mutant androgen receptors expressed in androgen-dependent human prostate cancer cells [6,7]. The exchange of a single valine into methionine in the androgen receptor promoted transactivation not only by testicular but also by adrenal androgens. This pattern of liganddependent transactivation may have significance in the process controlling the progression of prostatic carcinoma. These findings strongly suggest a significant role of adrenal androgens in stimulating cancer growth. C17–20 lyase is one of the key enzymes responsible for the biosynthesis of androgenic hormones . This enzyme is responsible for production of testosterone and adrenal androgens in the testes and adrenal glands. The inhibition of this enzyme may therefore result in a decrease in the production of androgenic hormones in both the testes and adrenal cortices, suggesting that an inhibitor of this enzyme may be useful for the treatment of androgen-dependent diseases . It thus seems likely that a C17–20 lyase inhibitor would be more effective than agents which only inhibit testicular androgen production, such as GnRH agonists. Ketoconazole, an antifungal agent, inhibits C17–20 lyase activity and has been used clinically in the treatment of advanced prostate cancers [9,10]. These clinical studies showed that ketoconazole could produce prolonged responses in previously hormonerefractory prostate cancers. Although this agent received favorable evaluation in some studies, it proved less promising in others [11,12]. Ketoconazole was withdrawn from clinical use because it caused gastrointestinal intolerance (nausea and vomiting) and/or hepatic lesions. These combined findings prompted a search for a more specific and safe inhibitor of C17–20 lyase that could block adrenal androgens [13,14]. In this study we examined the effects of a novel nonsteroidal compound, YM116 (Fig. 1), on C17–20 lyase activity, on serum concentrations of testosterone and DHEA sulfate, and on ventral prostatic weight in rats. In addition, we examined the specific inhibition 11 Fig. 1. Chemical structure of YM116. of androgen synthesis by YM116, estimated by the changes in serum concentrations of cortisol in guinea pigs and aldosterone in rats. MATERIALS AND METHODS Materials The materials used in this study were obtained from the following sources: gonadotropin-releasing hormone (GnRH), Peptide Institute, Inc., Osaka, Japan; Leuplin威 (sustained-release microspheres of leuprolide acetate), Takeda Chem. Ind. Co., Ltd., Osaka, Japan; pregnant mare’s serum gonadotropin (PMSG), Seikagaku Co., Tokyo, Japan; ACTH (synacthen depot), Ciba-Geigy, Basel, Switzerland; ketoconazole, Paesel + Lorei GMBH & Co., Frankfurt, Germany; flutamide, Sigma Chemical Co., St. Louis, MO; and 17␣[1,2-3H]-hydroxyprogesterone (1480 GBq/mmol), DuPont NEN, Boston, MA. The radioimmunoassays (RIA) used in this study were obtained from the following sources: estradiol, Daiichi Radioisotope Lab., Tokyo, Japan; aldosterone, Dainabot, Tokyo, Japan; cortisol, Incstar Co., Stillwater, MN; testosterone, Diagnostic Products Co., Los Angeles, CA; and dehydroepiandrosterone sulfate, ICN Biomedicals, Inc., Costa Mesa, CA. 2-(1H-imidazol-4-ylmethyl)-9H-carbazole monohydrochloride monohydrate (YM116) and other drugs such as CB7630 , liarozole , and bicalutamide  were synthesized at Yamanouchi Pharmaceutical Co., Ltd. (Tokyo, Japan). All other reagents were of analytical grade commercially available. Preparation of Rat Testicular Microsomes Testes were obtained from anesthetized male 10week-old Wistar rats (Japan SLC, Shizuoka, Japan). Testicular microsomes were prepared according to the method described by Schatzman et al. . Briefly, the testes were minced with scissors and homogenized in 4 volumes of 0.25 M sucrose with a polytron homogenizer (Kinematica GMBH, Lucerne, Switzerland). The homogenate was centrifuged at 12,000g for 10 12 Ideyama et al. min, and the resulting supernatant was centrifuged at 105,000g for 60 min. After decantation of the supernatant, the microsomal pellets were washed and resuspended in the solution containing 50 mM phosphate buffer (pH7.4) and glycerol (3:1). All of the above procedures were performed at 4°C. The final protein concentration was 0.5 mg/ml. The protein concentration was determined with a Bio-Rad protein assay kit (BioRad Laboratories, Inc., Hercules, CA). Measurement of C17–20 Lyase Activity C17–20 lyase activity was determined by incubating 50 g of rat testicular microsomes in an incubation mixture containing, in a final volume of 200 l, 50 mM phosphate buffer (pH 7.4), 1 M 17␣-[1,2- 3 H]hydroxyprogesterone (9.25 kBq), the NADPH generating system (1 mM NADPH, 40 mM G6P, and 12 IU/ml G6P dehydrogenase), and 2 l of a test compound and/or solvent at 37°C for 60 min. Following quenching of the incubates by 400 l tetrahydrofuranmethanol (3:2), the mixture was centrifuged at 2,000g for 5 min and the upper layer was filtered (UltrafreeMC, 0.45 m, Millipore Co., Bedford, MA). Androstenedione and testosterone were separated and quantified from their peak areas relative to the peak of the internal standard, using high-performance liquid chromatography (HPLC), a Shimadzu LC-10A system equipped with an autosampler (Shimadzu, Kyoto, Japan), and a flow-scintillation analyzer (Series A-500, Flow/bata, Packard Instrument Co., Meriden, CT), according to the method described by Schatzman et al. . The sum of the radioactivity in the fractions of androstenedione and testosterone was defined as the C17–20 lyase activity. Ki and the mode of enzyme inhibition were estimated by double-reciprocal plots. Serum Concentration of Testosterone in Male Rats Treated With or Without GnRH GnRH (60 ng) was administered intramuscularly (i.m.) to rats which had been pretreated with several doses of YM116 (0.4–1.5 mg/kg), ketoconazole (5–30 mg/kg), CB7630 (0.75–2 mg/kg), or liarozole (5–40 mg/kg) 1 hr earlier . Blood specimens were obtained 1 hr after GnRH administration in the GnRHtreated rats or 4 hr after the single oral administration of each test compound in normal rats. The serum concentration of testosterone was measured by a specific RIA. The experimental protocol was approved by the local ethics committee for animal studies. Serum Concentration of Adrenal Androgen in Castrated Rats Pretreated With ACTH ACTH (synacthen depot, 1 mg/kg) was administered subcutaneously (s.c.) to the rats which had been castrated 6 days before. YM116, ketoconazole, or CB7630 was orally administered 13 hr after ACTH treatment, and blood specimens were taken 2 hr after each drug administration. The serum concentration of DHEA sulfate was measured by RIA. To demonstrate the accuracy of the assay, the known amount (3 ng/ ml) of DHEA sulfate was added to the assay medium, including the standard ranging from 0.5–25 ng DHEA sulfate/ml; the hormone recovered was 96.7 ± 8.2%. When 3 ng/ml DHEA sulfate were added to five previously evaluated serum samples (6.9–9.1 ng/ml), the DHEA sulfate recovered was 167.7 ± 4.9%. The results of the recovery tests indicated that the assay was interfered with serum to a certain extent. Further dilution samples did not improve assay values (data not shown). We did not know the reasons for this, but one possible reason may be the high cross-reactivity of the antiserum used in this study to other adrenal androgens (cross-reaction percentage at 50% displacement compared to DHEA sulfate standard curve: 58.5% for DHEA, and 30.5% for androstenedione, according to the manufacturer’s instructions). Estrogen Content in Rat Ovaries Treated by PMSG The in vivo inhibition of estradiol synthesis was evaluated by methods described elsewhere . Briefly, 100 IU of PMSG were administered s.c. to female Wistar rats. Three days later, the ovaries were removed 3 hr after YM116 or CB7630 treatment. Estradiol was extracted with diethyl ether from the ovarian homogenate and then measured by RIA. Weights of Ventral Prostate and Seminal Vesicles Male adult Wistar rats were treated with several inhibitors for C17–20 lyase, antiandrogens, or a vehicle for 2 weeks. The ventral prostates were removed and weighed 16–20 hr after the last dose. Male Wistar rats at age 6 weeks were treated with leuprolide (3 mg/kg, s.c., once on day 0) [21,22] or YM116 (40 mg/kg, p.o., for 20 days). Some of the rats were castrated on day 0. The animals were sacrificed serially, and the weights of the ventral prostate and seminal vesicles were measured 2 hr after the last treatment. Serum Concentrations of Aldosterone in Rats and of Cortisol in Guinea Pigs The serum concentrations of aldosterone in male Wistar rats and of cortisol in guinea pigs were measured by RIA. The rats were pretreated with ACTH (synacthen depot, 1 mg/kg). All animals were treated Inhibition of P45017␣ and Prostatic Weight 13 TABLE I. Inhibitory Effects of YM116, Ketoconazole, CB7630, and Liarozole on Rat Testicular C17–20 Lyase Activities* Inhibition of C17–20 lyase activities, IC50 (nM) YM116 Ketoconazole CB7630 Liarozole 5.4 587 8.2 356 *Results are expressed as the concentration required to inhibit the enzyme activity by 50% (IC50). with either a test compound or a vehicle. The animals were sacrificed 2 hr after drug treatment. Analytical Procedures In order to determine the ED50 value, which was the dose required to decrease the serum concentration of each hormone observed in the control group by 50%, the logarithm of the dose of a test compound vs. the serum concentration of each hormone was linearized by least-squares fitting (SAS Software, SAS Institute Japan, Tokyo, Japan). The regression equation was used to determine the ED50 value. Statistical Analyses Comparisons between experimental groups were made using the one-way analysis of variance (ANOVA) test, followed by Dunnett’s multiple range test. Differences were accepted as significant at the P < 0.05. RESULTS Inhibition of C17–20 Lyase Activity in Rat Testicular Microsomes YM116 inhibited C17–20 lyase activity in rat testicular microsomes with an IC50 value of 5.4 nM, a value similar to that for CB7630 (Table I). YM116 was a much more potent inhibitor of this enzyme than either ketoconazole or liarozole. The kinetic analysis showed that YM116 was a competitive inhibitor of C17–20 lyase, with a Ki of 0.38 nM (Fig. 2). Decreases in Serum Concentrations of Testosterone in Male Rats Treated With or Without GnRH GnRH increased the serum testosterone concentration from a basal value of 0.82 ± 0.14 ng/ml to Fig. 2. Inhibition of rat testicular C17–20 lyase by YM116; Lineweaver-Burk plots of enzyme activities at varying concentrations of 17␣-hydroxyprogesterone and YM116. Inset: Replot of the slope of each reciprocal plot vs. YM116. 9.82 ± 1.01 ng/ml. YM116 dose-dependently reduced the concentration of serum testosterone with an ED50 value of 0.7 mg/kg in the GnRH-treated rats (Fig. 3). YM116 was about 21–24 times more potent than ketoconazole and liarozole, and was twice as potent as CB7630 in decreasing the serum testosterone concentration. In untreated normal rats, YM116 as well as CB7630 decreased serum testosterone concentrations in a dose-dependent manner (Table II). Decrease in Estradiol Content in Rat Ovaries Stimulated by PMSG YM116 decreased the estradiol content in the ovaries pretreated with PMSG, with an ED50 of 1.1 mg/kg (Fig. 4), indicating that YM116 blocked both estrogen and androgen synthesis; its potency was about twice that of CB7630. Decrease in Serum Concentration of DHEA Sulfate in Castrated Rats Pretreated With ACTH YM116 reduced the serum concentration of DHEA sulfate (one of the adrenal androgens) in castrated rats, with an ED50 value of 11 mg/kg. YM116 was about 5 times more potent than ketoconazole. CB7630, at a dose of up to 40 mg/kg, induced no significant reduction in the DHEA sulfate level (Fig. 5). Reduction in Weight of the Rat Ventral Prostate and Seminal Vesicles Treatment with YM116 for 2 weeks to rats decreased their prostatic weights in a dose-dependent 14 Ideyama et al. Fig. 3. Dose-dependent decreases in the serum concentrations of testosterone by YM116, ketoconazole, CB7630, and liarozole in GnRH-treated male rats. GnRH (60 ng) was administered i.m. to rats which were pretreated with YM116 (䊉), ketoconazole (䉱), CB7630 (䊐), or liarozole (䊊) 1 hr earlier. Blood specimens were obtained 1 hr after GnRH administration from GnRH-treated rats. Serum concentrations of testosterone were measured by a specific RIA. Each point with a vertical line shows the mean ± SEM of 5–10 rats. The serum testosterone concentration in GnRHtreated rats was 9.82 ± 1.01 ng/ml. ED50 was the dose required to decrease the serum concentration of testosterone by 50%. Fig. 4. Dose-dependent inhibition by YM116 of estradiol production stimulated by PMSG in rat ovaries. PMSG (100 IU) was administered s.c. to female Wistar rats. Three days later, the ovaries were removed 3 hr after YM116 or CB7630 treatment. Estradiol was measured by RIA after the extraction. Each column represents the mean ± SEM of 4–5 rats. Statistical significance was analyzed by one-way ANOVA, followed by Dunnett’s multiple range test. *P < 0.05, significantly different from the controls treated with PMSG. TABLE II. Effects of YM116 and CB7630 on Serum Concentrations of Testosterone in Male Rats* Serum testosterone (ng/ml) Dose (mg/kg) 0.0 0.5 0.75 1.0 1.5 2.0 YM116 CB7630 0.82 ± 0.14 0.61 ± 0.11 0.60 ± 0.06 <0.2 <0.2 <0.2 0.82 ± 0.14 0.71 ± 0.13 0.44 ± 0.11 0.40 ± 0.17 <0.2 <0.2 *Results are mean ± SEM of 5 rats. <0.2, below the detection limit of the assay. The testosterone concentration was determined 4 hr after the single oral administration of YM116 or CB7630. manner (Fig. 6). The reduction in organ weight by YM116 at a dose of 40 mg/kg was almost the same as that induced by surgical castration. YM116 was about 2 times more potent than CB7630. Liarozole at a dose of 80 mg/kg produced no significant reduction in prostatic weight. Although flutamide and bicalutamide dose-dependently decreased prostatic weight, Fig. 5. Effects of YM116, ketoconazole, and CB7630 on the serum concentrations of DHEA sulfate in castrated rats pretreated with ACTH. ACTH (1 mg/kg) was administered to rats which had been castrated 6 days earlier. Thirteen hours later, the test compound was orally administered, and blood specimens were taken 2 hr later. Serum concentrations of DHEA sulfate were measured by RIA, and the mean concentration in the ACTHtreated rats was 6.2 ± 0.9 ng/ml. Each column represents the mean ± SEM of 4–5 rats. Statistical significance was analyzed by one-way ANOVA, followed by Dunnett’s multiple range test. **P < 0.01, *P < 0.05, significantly different from control rats treated with ACTH (100%). the maximum reduction at a dose of 80 mg/kg did not reach castration levels. In order to compare the differences in the time courses of the reduction of androgendependent organs induced by surgical castration and by medical castration, the effects of surgical castration, YM116, and a GnRH agonist were examined (Fig. 7). Inhibition of P45017␣ and Prostatic Weight Fig. 6. Effects of YM116, CB7630, liarozole, and antiandrogens on the weights of the ventral prostates in rats. Male adult Wistar rats were treated with YM116 (䊉), CB7630 (䊐), liarozole (䊊), flutamide (䉱), bicalutamide (䉭), or a vehicle for 2 weeks. The ventral prostates were removed and their weights were measured 16–20 hr after the last dosing. The weight of the ventral prostate in the control rats was 0.881 ± 0.048 mg/g body weight. Each point with a vertical line shows the mean ± SEM of 5–20 rats. The effects of YM116 or CB7630 at every dose used in this study were significantly different from the control value without treatment (data not shown). YM116 and surgical castration significantly reduced the weights of the prostate and seminal vesicles at 3 days after the start of treatment. There was no difference between these two groups with respect to the time course and magnitude of the reduction in the weights of both organs. In contrast, a single subcutaneous injection of leuprolide elicited a rapid increase in the weights of both organs on day 3, and subsequently decreased them. Effect on Serum Concentrations of Aldosterone in Rats and Cortisol in Guinea Pigs YM116 at a dose of 100 mg/kg had no significant effect on the serum cortisol concentration in the guinea pigs, but elicited a 37% decrease in the serum aldosterone level in the rats (Table III). CB7630 had no effect on cortisol or aldosterone levels. In contrast, ketoconazole and liarozole significantly decreased the aldosterone level, with ED50 values of 1.4 and 9.4 mg/ kg, respectively, although these drugs had no significant effect on cortisol levels. DISCUSSION Cytochrome P450 17␣ is known as 17␣hydroxylase/C17–20 lyase and plays a key role in the synthesis of androgens in the testes and of adrenal 15 androgens and cortisol in the adrenal glands in humans [8,13,23]. In rats and mice, however, it was reported that plasma concentrations of cortisol were near the detection limit of the assay, and that these low levels of cortisol amounted to less than 4% of the levels found for corticosterone. These findings could be attributable to the lack of 17␣-hydroxylase in rat adrenals [24,25]. In contrast, Touitou et al.  showed the in vitro syntheses of 17␣-hydroxyprogesterone and cortisol from progesterone in rodent adrenals, suggesting the presence of 17␣-hydroxylase activity in this tissue. There is thus a controversy regarding the role of 17␣-hydroxylase/C17–20 lyase in the synthesis of adrenal androgens and cortisol in rat adrenal glands. In this study, we examined the effects of YM116 on C17–20 lyase activity, on serum concentrations of testosterone and DHEA sulfate (one of the adrenal androgens), and on ventral prostatic weight in rats. In addition, we examined the specific inhibition of androgen production by YM116, as estimated by changes in the serum concentrations of aldosterone in rats and cortisol in guinea pigs. YM116 dose-dependently inhibited C17–20 lyase activity in rat testicular microsomes, with an IC50 value of 5.4 nM (Table I), and was a competitive inhibitor of this enzyme with a Ki of 0.38 nM, based on kinetic analysis (Fig. 2). The potency of this agent was comparable to or even greater than other known steroidal or nonsteroidal inhibitors of C17–20 lyase, i.e., ketoconazole , CB7630 , and liarozole . YM116 decreased serum concentrations of testosterone with an ED50 of 0.7 mg/kg in rats, whose testosterone level was increased about 12-fold by GnRH treatment (Fig. 3). YM116 was about 21–24 times more potent than ketoconazole and liarozole, and was twice as potent as CB7630 in decreasing the serum testosterone concentration. It should be pointed out that YM116 was one of the most potent inhibitors of C17– 20 lyase and serum testosterone production in rats, and that the relative potency of the four agents in inhibiting C17–20 lyase activity was very similar to that in decreasing serum testosterone in rats following oral administration. Consistent with these findings, ketoconazole and liarozole were reported to produce a drop in the level of circulating testosterone in humans [28–30]. YM116 and ketoconazole reduced serum concentrations of DHEA sulfate in castrated rats, with ED50 values of 11 and 60 mg/kg, respectively. CB7630 at a dose of up to 40 mg/kg produced no significant reduction in the DHEA sulfate level (Fig. 5). This last finding was unexpected, since the two other inhibitors significantly decreased DHEA sulfate in serum, and CB7630 significantly decreased the serum testosterone concentration. However, no information is available about 16 Ideyama et al. Fig. 7. Effects of YM116, leuprolide, and surgical castration on weights of the ventral prostate and seminal vesicles in rats. Male Wistar rats at age 6 weeks were divided into four groups. They received YM116 (䊉, 40 mg/kg, p.o., for 20 days), leuprolide (䊏, 3 mg/kg, s.c., once on day 0), surgical castration (䊊, on day 0), or a vehicle. The animals were sacrificed and the weights of both the ventral prostate and seminal vesicles were determined. The re- TABLE III. Effects of YM116, Ketoconazole, CB7630, and Liarozole on Serum Concentrations of Aldosterone in Rats and Cortisol in Guinea Pigs Aldosterone in rats, ED50 (mg/kg) YM116 Ketoconazole CB7630 Liarozole 37% decreasea 9.4b No effecta 1.4b Cortisol in guinea pigs, ED50 (mg/kg) No No No No effecta effecta effecta effecta a Rats or guinea pigs were orally given a test compound at a dose of up to 100 mg/kg. The number of animals used in each group was 5 rats and 7 guinea pigs. b ED50. the effect of CB7630 on adrenal androgen levels . An in vivo study of male volunteers reported by Bruynseels et al.  confirmed that ketoconazole (600 mg, b.i.d.) and liarozole (300 mg, b.i.d.) decreased plasma testosterone levels, and these levels were reduced close to or within castration levels. As to the suppression of adrenal androgen production, ketoco- sults are expressed as percentage of control value at the same time-point. During the 20-day treatment, prostate weight increased from a control value of 0.473 ± 0.024 mg/g to 0.662 ± 0.043 mg/g, and the weight of the seminal vesicles increased from 0.515 ± 0.071 mg/g to 2.095 ± 0.129 mg/g. Each point with a vertical line shows the mean ± SEM of 5–10 rats. *P < 0.05, significantly different from the control value at the same time-point. nazole was much more active in comparison with liarozole, indicating that there was a difference between these two agents in inhibiting adrenal androgen synthesis. In this study, a significant difference between the ED50 values for serum testosterone and adrenal androgen was also observed in the YM116- or ketoconazole-treated rats. An about 16-fold higher dose of YM116 was required to decrease the serum DHEA sulfate concentration compared to testosterone, and an about 4-fold higher dose of ketoconazole was required to do so. The reason for this difference is as yet unknown, but it is in part attributed to the difference in experimental conditions at which the ED50 values were determined. YM116 dose-dependently decreased rat prostatic weights, which were reduced to close to or within castration levels when YM116 was administered at a dose of 40 mg/kg for 2 weeks (Fig. 6). CB7630 at a dose of 80 mg/kg, but neither flutamide nor bicalutamide, decreased prostatic weights to castration levels. Liarozole produced no significant reduction in prostatic weight. YM116 significantly reduced the weights of the prostate and seminal vesicles as quickly Inhibition of P45017␣ and Prostatic Weight as did surgical castration (Fig. 7). There was no difference between YM116 and surgical castration with respect to the time course and magnitude of the reduction in the weights of the prostate and seminal vesicles. A single subcutaneous injection of leuprolide, however, elicited a rapid increase in the weights of the prostate and seminal vesicles and subsequently decreased the weights of these organs. YM116 at a dose of 100 mg/kg had no significant effect on serum cortisol concentration in guinea pigs, but elicited a 37% decrease in the serum aldosterone level in rats, suggesting that YM116 was a selective inhibitor of androgen production (Table III). Some limitations of ketoconazole when used clinically were high doses and frequent administration (400 mg, every 8 hr). Some patients experienced a moderate rise in serum testosterone levels 1 month after the start of administration, which might be due to a rise in LH . These observations may be caused by gradual escape from C17–20 lyase inhibition and a short elimination half-life. As shown in Figures 6 and 7, YM116 at a dose of 40 mg/kg decreased prostatic weight to castration levels until 2 or 3 weeks. Although we have not yet examined the long-term inhibitory effect of this agent on prostatic weight, our preliminary results demonstrated that such suppression was also observed after 1 month. In addition, our pharmacokinetics study showed that plasma concentration of YM116, which was administered orally to rats at a dose of 1 or 3 mg/kg, reached a maximum between 1–3 hr postdose and subsequently declined, with an elimination half-life of 2.5 or 10.9 hr (unpublished observations). These values were equal to or even greater than those (1.5–2.2 hr) of ketoconazole when administered orally at a dose of 5 mg/kg . Further study will be needed to clarify the long-term effect of YM116 on prostatic weight in rats and other species of the animals. It is well-established that the production of androgens in the testes must be inhibited to effectively treat hormone-dependent prostate cancer . Surgical castration or treatment with a GnRH agonist is an effective way to decrease the testicular production of androgens in prostate cancer patients. However, these treatments have no effect on the supply of androgens and their precursors in the adrenal glands. A GnRH agonist is used alone or in a combination with an antiandrogen for this purpose . The function of the antiandrogen in the combined treatment is to counteract the stimulatory action of androgens of adrenal origin on the androgen receptor in prostatic cancer cells. YM116 is a single agent which may inhibit both testicular and adrenal androgen production, and may have an advantage over the present combination therapy in clinical practice. 17 CONCLUSIONS YM116 is a potent inhibitor of C17–20 lyase, and produced a significant reduction in the weight of the rat prostate by decreasing the serum concentrations of testosterone and adrenal androgen. 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