[CANÅ’R RESEARCH 58. 3826-3832. September I. I998| Effects of Some Novel Inhibitors of C17v20-Lyaseand Sa-Reductase in Vitro and in Vivo and Their Potential Role in the Treatment of Prostate Cancer1

Ivo P. Nnane, Katsuya Kalo," Yang Liu, Qing Lu, Xin Wang, Yang-zhi Ling,3 and Angela Brodie4 l)e¡'i¿rtrnt'ntttfPhtirtniU'tilucy unii Experimental Therapeutics, University of Marylund at Baltimore. Baltimore. Marykind 21201

ABSTRACT prostatic cancer patients initially respond to androgen ablation therapy but eventually relapse. Furthermore, orchidectomy results in reduced The effects of some novel steroidal compounds were evaluated against both human <',- ,,,-l\;isr and 5a-reductase in vitro and also against an- androgen production by the testis but fails to alter androgen produc drogen synthesis in normal male rats. L-2, L-36, I,-.17. and 1-41 showed tion by the adrenals, which may contribute androgen precursors to the potent inhibition of human testicular C,7 2(>-lyase,with IC'50sof 43, 39, 42, prostate (10). Thus, total androgen blockade may be a therapeutically and 58 MM,respectively. In contrast, ketoconazolc, a competitive inhibitor more effective option than conventional androgen ablation therapy of C17 2,,-lyase, had an 1C,,, of 76 n\i. L-36 also showed potent inhibitory (10, 11). activity against 5Ketoconazole has been used clinically asteride, a potent inhibitor of 5a-reductase, had an ICSOof 33 n\i. When to reduce testosterone biosynthesis in patients with advanced prostate normal male rats were treated with these novel compounds (50 or 100 carcinoma. However, ketoconazole is only a moderate inhibitor of this mg/kg/day) for 14 consecutive days, the wet weight of the prostate was significantly reduced by I,-36, L-37, and 1-41, compared to the control enzyme and is not selective. This agent inhibits other steroidogenic group. Testosterone levels in rat serum were also reduced by L-36 (55%), enzymes and may cause adverse effects (18, 19). Several inhibitors of L-37 (86%), and 1-41 (53%). The concentrations of testosterone in rat 5a-reductase have been described, and in fact, finasteride (Prosear) testes were reduced by these novel compounds by 13-74%. The com has recently been approved for treatment of BPH (20). Another pounds also reduced the concentration of testosterone in rat prostates by azasteroid, MK-434. with similar activity to finasteride has also been 35-75%. Similarly, dihydrotestosterone (DHT) concentration in rat serum described, although its efficacy in man has not yet been evaluated was reduced 30-89% by these compounds, compared to the control (21). These compounds are effective in reducing DHT levels in group. Prostatic DHT levels were also lower in rats treated with L-36 patients with prostate cancer, but unfortunately, they also increase (48%), L-37 (54%), or 1-41 (26%). In contrast, L-2 enhanced serum serum testosterone levels in these patients (22). Although DHT binds testosterone and prostatic 1)111 concentrations by >50%. These findings to the androgen receptor with higher affinity than testosterone, in the suggest that the dual activities of several of these novel inhibitors of C i7.2o-l.vase and 5a-reductase accounts for the diminished levels of circu absence of DHT, testosterone may also bind to the androgen receptor lating androgens in vivo. and stimulate prostatic growth (23). It is, therefore, apparent that compounds that strongly inhibit C)7 -.„-lyaseand5a-reductase may be more suitable for development as potential treatments for BPH and INTRODUCTION prostate cancer. Prostate cancer is the most prevalent cancer in men in the United Recently, we have identified compounds with dual actions that States (1, 2). Development of therapeutic strategies is, therefore, an reduce testosterone and DHT synthesis by inhibiting both C,7 -,,,-lyase important objective for reducing the incidence of this disease. Andro and 5a-reductase (24-27). In this study, we evaluated the effects of gens play an important role in the growth of the normal prostate and some of these novel steroidal compounds (L-2, L-36, L-37, and 1-41; in promoting BPHS and prostatic cancer (3). C172()-lyase is a key see Fig. 1 for structures and chemical names) on human testicular enzyme in the androgen synthesis pathway that converts the steroids C1720-lyase and prostatic 5a-reductase in microsomal fractions and (pregnenolone and progesterone) to the C,,( androgens: dehydroepi- compared them to those of ketoconazole and finasteride. We also androsterone, 5-androstenediol, and testosterone in the testis and examined the effects of these compounds on the wet weights of the adrenals (4). In the prostate, testosterone, the major circulating an prostate and other organs and on tissue testosterone and DHT con drogen in males is converted to the more potent androgen, DHT, by centrations in normal male rats compared to surgical removal of the 5a-reductase (5. 6). Both testosterone and DHT stimulate prostatic testes. growth, although DHT plays a much more important role than does testosterone in the organogénesisand homeostasis of the prostate (7, MATERIALS AND METHODS 8). It has also been observed that men with prostatic carcinoma show Chemical Inhibitors and Reagents. The inhibitors (L-2, L-36, and L-37) an increased rate of conversion of testosterone to DHT (9). Most were synthesized in our laboratory according to procedures described by Ling el al. (27). 1-41 was synthesized as described by Li et al. (24. 26, 28). Received 3/3/98; accepted 7/2W8. Finasteride was a gift from Merck Research Laboratories (Rahway. NJ). and The costs of publication of this article were defrayed in part by the payment of page ketoconazole was purchased from Sigma Chemical Co. (St. Louis. MO). charges. This article must therefore be hereby marked advertisement in accordance with [21-'H]17a-hydroxypregnenolone was prepared in our laboratory as described 18 U.S.C. Section 1734 solely lo indicale Ihis fact. 1This work was supported by NIH Grant CA-27440 and a grani from Paramount previously (29). [7-'H]Testosterone (20 Ci/mmol) and [UC]DHT (50 mCi/ Capilal Inc.. New York. : Present address: Department Chemistry. National Industrial Research Institute of mmol) were obtained from DuPont (Boston. MA). Silica gel OF TLC plates (20 X 20 cm, w/uv 254, 500 /um) were obtained from Analtech (Newark, DE). Nagoya. Nagoya 462. Japan. ' Present address: School of Pharmaceutical Sciences, Beijing Medical University. The active testosterone-coated tube RIA kits and the DHT-coated tube RIA Beijing 100.083. People's Republic of China. kits for quantitative measurement of testosterone and DHT, respectively, were 4 To whom requests for reprints should he addressed, at Department of Pharmacology purchased from DSL Inc. (Webster. TX). All other reagents were purchased and Experimental Therapeutics. University of Maryland at Baltimore. 655 West Baltimore from Sigma. Street, Baltimore. MD 2I20I. * The abbreviations used are: BPH. benign prostatic hyperplasia; DHT, dihydroles- Preparation of Microsomes. Human testes and prostate tissue (from pa loslerone. tients with BPH I were obtained from Dr. James Mohler (University of North 3826

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N OH tube. To remove residual steroids that may remain after the chloroform extraction. 0.75 ml of charcoal solution (2.5 g of activated charcoal per 1(X)ml L-2 20-Hydroximino-4,16-pregnadien-3-one of distilled water) was added to each tube and vortexed vigorously. After standing for 30 min. the charcoal was pelleted by centrifugation at 2000 X g for 20 min. Finally. 0.75 ml of the supernatant was analyzed for tritium by liquid scintillation spectrometry. The reaction conditions were optimi/ed with 0.1-6.0 mM of [2l-'H|17a-hydroxypregnenolone and the Km and Vlmxs were determined at the optimum conditions. IC50s for inhibitors were calculated from the linear regression line in the plot of logit of en/.yme activity against log L-36 17-(3'-PyrazolyI)androsta-4,16-dien-3[l-one of inhibitor concentration. Kf were also determined at the same reaction conditions with addition of appropriate concentrations of inhibitors. The ex periments were performed in duplicate on at least two separate occasions. 5a-Reductase Assay. The effects of novel compounds on human prostatic 5a-reductase activity were evaluated as described previously (24. 25) with some modifications. Ethanolic solutions of [7-'H]testosterone (600.000 dpm). cold testosterone (4.8 ng). indicated inhibitors (0-200 nM). and propylene L-37 17-(3'-Pyrazolyl)androsta-5,16-dien-3|i-ol glycol ( 10 /al) were added to sample tubes in duplicate. The control incubations were prepared without the addition of the indicated inhibitors. The elhanol was evaporated to dryness under a gentle stream of air. The samples were recon stituted in phosphate buffer (0.1 M. pH 7.4. 400 fil) containing DTT (78 fi.M). and the NADPH-generating system (6.5 mM NADP. 71 mM glucose-6-phos- phate. and 2.5 units of glucose-6-phosphate dehydrogenase in 100 ftl of phosphate buffer) was added to each tube. The tubes were preincubated at 37°Cfor 15 min. The enzymatic reactions were initiated by addition of human 1-41 17ß-(4'-lmidazolyt)androst-4-en-3-one BPH microsomes (~180 fig of microsomal protein in 500 fil of phosphate buffer) in a total volume of I ml, and the incubations were performed for 10 min under oxygen in a shaking water bath at 37°C.The incubations were terminated by placing the sample tubes on ice. and | I4C|DHT (3(XX)dpm) and Fig. 1. Chemical structures of steroidal inhibitors of androgen synthesis evaluated in vitro and in vivo. cold DHT (50 fig) were added to each tube as an internal standard and visualization marker, respectively. These additions were immediately followed by ether (1 ml). The steroids were extracted with ether (3 X 1 ml), separated Carolina, Chapel Hill, NC) and stored at -70°C prior to use. Testicular and by TLC (chloroform-ether, 80:20). and visualized by exposure to iodine vapor. prostatic microsomes were prepared as described previously (24). The micro- The extracts were analyzed for 'H and 14C using a liquid scintillation counter. somal protein content was determined by the Lowry method (30). and the The percentage conversion of [7-'H]testosterone to |7-'H]DHT was calculated microsomal fractions were stored at -70°C until required for assay. and used to determine 5a-reductase activity. The reactions conditions were Ci7,2o-Lyase Activity. The measurement of the activity of the human optimized with testosterone (0-60 nM) and the Km and Vm.,xwere estimated at Ci72o"'yase in testicular microsomes in the absence and presence of inhibitors the optimum conditions. The lC<,(,swere determined from plots of 5a-reduc- was performed as described previously (24, 25). Briefly, the C,720-lyase activity was determined by measuring the release of [^HJacetic acid during the tase activity against four different concentrations of the inhibitor. Ai,swere also conversion of (21-'H]17a-hydroxypregnenolone to dehydroepiandrosterone. determined using the optimum reaction conditions with the addition of appro priate concentrations of testosterone (0-60 nM) and inhibitors (0-40 nM). The The incubations were carried out in a total volume of 1.01 ml. Sample tubes were supplied with 10 /nl of propylene glycol, 300,000 dpm of [21-'*H]17or- experiments were performed in duplicate and were repeated at least once. Animal Studies. Adult Male Sprague Dawley rats (240 ± 10 g) were hydroxypregnenolone (13.61 /uCi/fimol ), and the indicated inhibitors. The supplied by Charles River Breeding Laboratories (Wilmington, MA). The control incubations were prepared without the addition of the indicated inhib animals were maintained in a controlled environment of ~25°C, 50* relative itors. After evaporation of the ethanolic solution, in each tube. 750 /al of 0. l M sodium phosphate buffer (pH 7.4, with 78 jiM DTT) and 50 fil of an NADPH- humidity, and 12 h of light/12 h of dark cycles and were allowed free access generating system (phosphate buffer containing 6.5 min of NADP+, 71 mM to food and water. About six to eight rats were assigned to the different glucose-6-phosphate, and 1.25 units of glucose-6-phosphate dehydrogenase) treatment groups. The compounds were suspended in 0.3*2- hydroxypropylcel- were added. The tubes were preincubated for 15 min at 37°C.and the reaction lulose and administered s.c. at a dose level of 50 mg/kg for 14 consecutive was started by the addition of 200 ¿dof human microsomes (300 jug protein days. The control group was injected with the vehicle alone. Another group of per 200 /xl of phosphate buffer). The reaction tubes were incubated at 37°C rats (six to eight) was castrated and injected with the vehicle alone for 14 days. under oxygen. After 60 min, the tubes were placed in an ice bath, and the In a further experiment. L-37 was administered at a higher dose level of 1(X) reaction mixture was extracted twice with chloroform ( 1 ml). The tubes were mg/kg daily for 14 days. The rats were sacrificed at the end of the treatment allowed to stand at 4°Cfor 20 min, centrifuged at 4°Cfor 15 min at 2000 X g, period (1-2 h after the last administered dose), and testes, prostate, epididymis. and then 0.75 ml of the aqueous phase of each tube was placed into a fresh and seminal vesicles were removed. The organs were weighed and stored at

Table I Kinetic constants (IC^ tinti K¿)ofsome inhibitors agititi*! human C17 ¿y/Vil-vcamiSa-iTtliiclast' lC50s and /C,s of these steroidal compounds against human testicular C¡7-»0-lyaseand prostatic 5a-reduclase from human BPH microsomes were determined as described in "Materials and Methods." The control incubations were prepared without the indicated inhibitors. Values are the means ±SEs from three experiments (n = 3).

Ci7.2o-Lyase 5a-Reducíase

CompoundL-2L-36L-371-41FinasterideKetoconazoleIC50(nM)43 (nM)15 (nM)75311277151331.32.0394.1I.INIK, (nM)ND"20 ±1.239 ±1.18 ±1.042 ±0.64 ±1.1NDND36 ±1.958 ±0.530 ±2.8NI76 ±2.5NI38 ±1.7NI ±2.0K, ±1.0IC50 ' ND. not determined; Nl, no inhibition.

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the activities of ketoconazole and finasteride. The Km and Vmaxfor (A) C|720-lyase were 0.48 JIM and 40 pmol/mg/min, respectively. L-2, L-36, L-37, and 1-41 showed potent inhibition of Cl720-lyase with IC50s of 43, 39, 42. and 58 nM, respectively (Table 1). L-36 showed competitive inhibition and L-37 exhibited mixed-type competitive inhibition against C,7 20-lyase with corresponding K¡sof8 and 4 nM, respectively (Figs. 2 and 3). On the other hand, L-2 and 1-41 showed noncompetitive inhibition against C17 2()-lyase with corresponding Kts of 15 and 30 nM, respectively (Figs. 4 and 5). Thus, these compounds are more potent than ketoconazole (IC50 = 76 nM, K{ = 38 nM), the competitive inhibitor for this enzyme that is used clinically. L-36 also showed potent and mixed-type competitive inhibition against 5a- reductase in human prostatic microsomes with an IC50 of ~31 nM and K, of 20 nM (Fig. 6). The Km and Vmaxof 5a-reductase were 40 nM and 2 pmol/mg protein/min, respectively. In comparison, finasteride, a -3-2-10123456 potent and competitive inhibitor of 5a-reductase used clinically, had an IC50 of 33 nM and a K-, of 36 nM against 5a-reductase. The [OH-P]-1(uM)-1 inhibitory activity of L-2 on 5a-reductase was moderate with an IC50 of 75 nM. 1-41 (IC50, —¿151nM)was —¿5-foldlesspotent than finas teride, whereas L-37 (IC50, —¿1277nM)showed little activity against this enzyme (Table 1). Male Sprague-Dawley rats were assigned to different treatment

(A) 60

2 -

40

-40 -30 -20 -10 0 10 20 30 40 50 60 70 [ L-36] nM C 1 - [L-36] nM 20

Fig. 2. Inhibition of human lesticular microsomal C,7 w-\yase by L-36. A, Lineweaver- Burk plot of enzyme activities at various substrate and inhibitor concentrations. B, slopes of each reciprocal plot against L-36 concentration. The Km and Vmax for C,7 ,,,-lyase. under the optimum incubation conditions, were 0.48 /AMand 40 pmol/mg protein/mm, respectively. Human testicular microsomes were prepared, and CI7^-lyase activity determined as described in "Materials and Methods." Dtita points, means of duplicate -6-5-4-3 -2 -1 determinations in at least two separate experiments. OH-P. 17a-hydroxypregnenolone. [OH-P]-1(uM)-1

-70°C until analysis. Blood samples were also collected, centrifuged to obtain serum, and stored at —¿70°Cuntil required. (B) 0.4 -, Testosterone RIA. Serum and testicular and prostatic tissues obtained from individual male rats were placed on ice. Portions (~ 100 mg) of testicular 0.3 and prostatic tissues were homogenized in phosphate buffer (pH 7.4, 0.1 M), and the homogenates were centrifuged at 2000 x g for 20 min. Serum (50 ¡u) and aliquots (50 /il) of the tissue supernatant were used for the determination 0.2 - of testosterone as described for the I25l-testosterone assay kit supplied by DSL Ine (Webster, TX). DHT RIA. Portions (100 mg) of prostatic tissues were homogenized in 0.1 Ki = 4 nM assay buffer provided with the DHT RIA kit obtained from DSL Inc. (Webster, TX), and the homogenates were centrifuged at 2000 x g for 20 min. Serum (0.4 ml) and aliquots (0.4 ml) of the prostatic supernatant were used for the 0.0 —¿â€”—¿i— determination of DHT as described in the I25I-DHT assay kit. -40 -30 -20 -10 O 10 20 30 40 50 60 70 Statistical Analysis. One-way ANOVA on SigmaStat for Windows ver [L-37] nM sion 1.0 was used to compare different treatment groups at the 95* confidence level. The Student's i test was also used to obtain Ps and to compare different [L-37] nM treatment groups. A P of S0.05 was considered statistically significant. Fig. 3. Inhibition of human testicular microsomal C17 2o-lyase by L-37. A. Lineweaver- Burk plot of enzyme activities at various substrate and inhibitor concentrations, ß,slope RESULTS of each reciprocal plot against L-37 concentration. The Km and Vmallfor C1720-lyase, under the optimum incubation conditions, were 0.48 |tM and 40 pmol/mg protein/min, respectively. Human testicular microsomes were prepared, and C,720-lyase activity The inhibitory potencies of L-2, L-36, L-37, and 1-41 were evalu determined as described in "Materials and Methods." Data points, means of duplicate ated against human Cl72(>-lyase and 5a-reductase and compared to determinations in at least two separate experiments. OH-P, 17a-hydroxypregnenolone. 3828

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[L-2] nM were significantly reduced by L-37 (74%, P = 0.0048). L-2, L-36, and (A) 1.5 - 1-41 also reduced the levels of testosterone in rat testis by —¿13,36, and 25% (P > 0.05), respectively (Table 3). L-37 significantly re duced the levels of testosterone in rat prostates by 75% (P = 0.015). E The levels of testosterone in the prostates were also lower in rats Ë treated with L-2, L-36, and 1-41 by 55, 58, and 35%, respectively. The 1 1-0 o effects of these compounds on the testosterone levels in rat prostates a were comparable to the effect achieved by surgical removal of the O) i o testes. Furthermore, DHT concentration in rat serum was reduced by L-36, L-37, and 1-41 by -69% (P = 0.022), 84% (P = 0.0206), and

I 0.5 89% (P = 0.016) respectively. L-2 also reduced the concentration of a DHT in serum by 30%, although this was not statistically significant. Although the compounds produced marked reduction in DHT con centrations, castration was more effective in lowering the concentra tion of DHT in rat serum than these novel compounds. The concen 0.0 tration of DHT in the prostates was also lower than controls in rats -3 -2 -1 2 treated with L-36, L-37, or 1-41 by 48% (P = 0.0063), 54% -1 (P = 0.0022), and 26% (P = 0.405), respectively, than it was in controls (Table 3). On the other hand, DHT concentration in rat 2.0 prostates was enhanced by L-2 (58%).

(A)

LO

0.0 o ÃŒ o 0.5 in —¿ Fig. 4. Inhibition of human lesticular microsoma] CI7 20-lyaseby L-2. A. Lineweaver- o Burk plol of enzyme activities at various substrate and inhibitor concentrations. B. slopes of each reciprocal plot against L-2 concentration. The K,ti and Vmaxfor C,7 ,,,-lyase, under I the optimum incubation conditions, were 0.48 /AMand 40 pmol/mg protein/min. Human testicutar microsomes were prepared, and C,7 2o-lyaseactivity determined as described in "Materials and Methods." Daui [mints, means of duplicate determinations in at least two separateexperiments. OH-P. I7or-hydroxypregnenolone. 0.0 -3 -2 [OH-P]-1(uM)-1 groups to receive one of these compounds (50 mg/kg daily for 14 days). The effects of L-37 were also examined at the higher dose of 100 mg/kg per day for 14 consecutive days. The animals were (B) 1.0 -i sacrificed at the end of the treatment period, and tissues were re moved, weighed, and examined for gross histopathological changes. 0.8 The effects of L-2, L-36, L-37, and 1-41 on the wet weight of tissue are shown in Table 2. L-36 reduced the weights of the prostate, seminal vesicle, and epididymis by 61, 56, and 24%, respectively. 0.6 - However, it did not alter testicular weight to any significant extent. ° L-37 showed a dose response effect and diminished the prostate co 0.4 weight by 26.1% at 50 mg/kg/day and by 50% at 100 mg/kg/day but had no effect on the weights of the other organs examined. 1-41 Ki = 30 nM 0.2 - reduced prostate weight (32%) and weight of the seminal vesicle (36%). L-2 did not alter the weights of any of the organs examined.

Surgical castration was considerably more effective at shrinking pros 0.0 tate size compared to these agents. -60 -40 -20 0 20 40 60 80 100 Testosterone and DHT levels in rat serum and testicular and pros- [1-41] nM tatic tissues were also assayed after treatment with L-2, L-36, L-37, and 1-41 (Table 3). Testosterone levels in rat serum were reduced by Fig. 5. Inhibition of human testicular microsomal C|72(>-lyaseby 1-41.A. Lineweaver- Burk plot of enzyme activities at various substrate and inhibitor concentrations. B, slope L-36 (55%), L-37 (86%), and 1-41 (53%), although these levels were of each reciprocal plot against 1-41 concentration. The A"mand V'II1J1XforCi7 20-lyase. not statistically significant. On the other hand, L-2 produced —¿2-fold under the optimum incubation conditions, were 0.48 /IM and 40 pmol/mg protein/min. respectively. Human testicular microsomes were prepared, and C|7 ,,,-lya.se activity increase in serum testosterone levels. Serum testosterone levels were determined as described in "Materials and Methods." Data points, means of duplicate undetectable after castration. Testosterone concentration in rat testis determinations in at least two separateexperiments. OH-P. I7a-hydroxypregnenolone. 3829

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(A) prostate cancer. Several previous inhibitors of C, 7-,,,-lyase and/or 5 -, 5a-reductase have been found to have a number of drawbacks. Ke- toconazole, an inhibitor of C17 2ll-lyase, is not selective and does not show any activity against 5a-reductase. Finasteride inhibits the con version of testosterone to the more potent androgen, DHT, and is used clinically to suppress prostate growth (22). However, finasteride and other 5a-reductase inhibitors fail to diminish and, in some cases, actually increase the circulating levels of testosterone (31, 32). In such a situation, testosterone would continue to stimulate prostatic growth, and therefore, a more complete ablation of circulating androgens is required for effective treatment of prostate cancer. Luteinizing hor mone-releasing hormone antagonists and agonists are also used to suppress testosterone, but they do not suppress adrenal androgens. A disadvantage of using these agents is that they are not p.o. active. The results of the current investigation indicate that these novel steroidal compounds exhibit dual-inhibitory actions against both -0.10 -0.05 0.00 0.05 0.10 C172()-lyase ar>d5a-reductase. In fact, when tested in the same assay, n 1 the inhibitory effects of L-2, L-36, L-37, and 1-41 on human testicular C,72()-lyase were more marked than that of ketoconazole, a clinically (B) available drug used to inhibit testosterone biosynthesis. L-36, for example, showed very potent inhibition of CI7 20-lyase with an IC5() of 39 nMand a K¡of8 nM (Table 1 and Fig. 2). In comparison, the IC50 for ketoconazole was 76 nM, and the AT¡was38 nM. L-36 showed competitive inhibition and binds with high affinity to the active site of C17 20-lyase. The results show that the interaction between L-2 or 1-41 and CI7 20-lyase is noncompetitive in nature, and this probably indi cates nonspecific binding of L-2 and 1-41 to the enzyme. L-36 was slightly more potent than finasteride, a potent inhibitor of 5a-reduc- tase. L-36 showed mixed-type competitive inhibition of 5a-reductase with an IC,() of 31 nM and a Kt of 20 nM (Table 1 and Fig. 6). This Kt for L-36 was lower than that for finasteride (K¡= 36 nM). Thus, lower -40 -20 concentrations of L-36 were required to inhibit 5a-reductase because

[L-36] nM Table 2 The effects ¡ifandrogen synthesis inhibitors on tissue weight of mirnuil acini/ Fig. 6. Inhibition of human prostatic licrosomal 5or-reductase by L-36. -4, inule rats Linewcuvcr-Burk plot of en/yme acl vities at arious substrate and inhibitor concentra lions, ß,slope of each reciprocal pio against lo concentration. The K,„andV'lll;lxfor Weights of tissues from animals in the same experiments shown in Table 3. Rats were 5tt-reductase, under the optimum in uhation onditions. were 40 nM and 2 pmol/mg injected s.c. with a 50 or 100 mg/kg/day (L-37) suspension of inhibitors in 0.5% protein/mill, respectively. Human pro tatie mi' osomes were prepared, and 5a-reductase hydroxypropylcellulose or vehicle alone (controls). Animals were autopsied after 2 weeks activity was determined as described n "Mater als and Methods." Dtiui ¡mints,means of and the following tissues were weighed: prostate, testes, seminal vesicles, epididymis. duplicate determinations in at least two separa •¿experiments.T. testosterone. adrenals, liver, and kidneys. There were no significant changes in the weights of adrenals, liver, kidneys, or total body weight between different treatment group. Values are the means ±SEs from six to eight rats.

DISCUSSION % decrease in tissue weight CompoundL-2 vesiclesNC Testosterone and its metabolite, DHT, play a pivotal role in pro moting prostutic growth and cancer (3). Cn -,,,-lyase catalyzes the L-36L-37 61 ±1.8* 56 ±1.9* 24 ±0.8* early step in the biosynthesis of testosterone and other androgens in 50 ±1.3* NC NC 1-41CastrationProstateNC"32 ±0.9*90 36 ±1.1*87 NC99 both the testes and the adrenal glands, whereas 5a-reductase converts ±l.lrSeminal ±1.8'EpididymisNC ±1.0' testosterone to DHT in the prostate gland (6). Thus, dual inhibition of " NC, no significant change compared (o the control group. both cn/.ymes may result in complete ablation of androgens and, h P < 0.05 compared to controls. therefore, serve as a useful therapeutic strategy for the treatment of ' P < 0.01 compared to controls.

Table 3 Effects of L-2, L-36, L-37, und 1-41 on lesiosierone and DHT levels in serum und tissues of normal adult mule rats Normal adult male rats (240 kg body weight) were injected with the compounds listed (50 mg/kg/day); L-37 was given at a dose of 100 mg/kg tor 14consecutive days. Blood wa collected and serum testosterone and DHT concentrations were determined by RIA, as described in "Materials and Methods." Values are the means ±SEs from six to eight rats.

levelsTestis levelsProstate

TreatmentControl (ng/ml)2.60 (ng/g)179.13 (ng/g)3.91 (pg/ml)231.36± (ng/g)2.61 ±25.33156.31 ±0.61 25.58 ±0.38 C'astrated 0.05 ±0.04" 1.48 ±0.16* 5.39 ±1.84* 0.001 ±0.0001" L-2 4.93 ±0.90* ±18.19 1.74 ±0.98 161.67 ±23.57 4.12 ±0.14 L-36 1.18 ±0.24 114.64 ±15.11 1.62 ±0.38 72.66 ±9.31* 1.35 ±0.11" L-37 0.38 ±0.16* 45.96 ±9.53" 1.00 ±0.17* 36.0 ±8.30" 1.19 ±0.18* MlSerum 1.23±0.92±0.39Testosterone 134.79 ±14.46DHT 2.56 ±0.84Serum 25.75 ±7.98*Prostate 1.92 ±0.34 ' P < 0.01 compared to the control group. *P < 0.05 compared to the control group.

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the affinity for the enzyme was higher. The inhibitory activity of L-2 Whittemore. A. S. Prostate Cancer. In: R. Doll, J. F. Fraumeni. and C. S. Muir (eds.). on 5a-reductase was moderate, and 1-41 was —¿5-foldlesspotent than Cancer Surveys: Trends in Cancer Incidence and Mortality. Vol. 19/20. pp. 309-322. Cold Spring Harbor. NY; Cold Spring Harbor Laboratory. 1994. finasteride. The dual actions of these novel inhibitors, particularly Cheng. E.. Lee, C.. and Grayhack. J. Endocrinology of the prostate, in: H. Lepor and L-36, make them superior to finasteride or ketoconazole as inhibitors R. K. Lawson (eds.). Prostate Diseases, pp. 57-71. Philadelphia: W. B. Saunders. 1993. of androgen synthesis. Nakajin. S.. Hall. P. F.. and Onada. M. Testicular microsomal P-450 for C2, steroid The effects of these novel compounds on tissue androgen levels and side chain cleavage: spectral and binding studies. J. Biol. Chem.. 256: 6134-6139. prostatic weights were evaluated in male rats (Tables 2 and 3). L-36, 1981. Bruchovsky, N.. and Wilson J. D. The conversion of testosterone to 5or-androsta- L-37, and 1-41 reduced the levels of both testosterone and DHT in rat 17o>ol-3-one by rat prostate in vivo and in vilro. J. Biol. Chem.. 243: 2012-2021, tissues. L-36 and L-37, for example, significantly reduced the levels 1968. of testosterone in rat prostate by >60% and diminished DHT levels by Saunders F. J. Some aspects of relation of structure of steroids to their prostate stimulating effects. In: E. P. Vollmer (ed.). Prostate and Related Tissues. National >40%. Their actions may be explained in terms of their dual inhibi Cancer Institute Monograph No. 12. pp. 139-159. Washington, DC: United States tion of Cl7,,,-lyase and 5a-reductase. the two critical enzymes in Government Printing Office. 1963. androgen biosynthesis. Some reports have shown that selective inhib Wilson, J. D. Role of dihydrolestosterone in androgen action. Prostate Suppt.. 6: 88-92. 1996. itors of 5a-reductase reduced tissue DHT levels but also produced an Peterson. R. E.. Imperanto-McGinley. J.. Gautier T., and Sturla. E. Male pseudohe- accumulation of testosterone (33-38), and consequently, the increased maphroditism due to steroid 5-a-reductase deficiency. Am. J. Med.. 62: 170-191, levels of testosterone would be expected to continue to promote 1977. prostatic growth. L36, L-37, the 17-(3'-pyrazolyl)-derivatives, and Meikle. A. W.. Smith. J. A., and Stringham J. D. Production, clearance and metab olism of testosterone in men with prostatic cancer. Prostate. 10: 25-31. 1987. 1-41 were effective in reducing both testosterone and DHT levels in Labrie, F.. Dupont. A.. Belanger. A.. Lacoursiere. Y.. Raynaud. J. P.. Husson. J. M.. rat tissues, but to different extents, and, therefore, offer promise as Gareau. J.. Fazekas. A. T. A.. Sandow. J.. Monfette. G.. Girard. J. G.. Emond. J.. and Houle. J. G. New approach in the treatment of prostate cancer: complete instead of selective inhibitors of 5a-reductase. This result may be due to phar- partial withdrawal of androgens. Prostate. 4: 579-594. 1983. macokinetic differences, because L-37, the 5-ene derivative, would be Crawford. E. D., Eisenberger. M. A., McLeod. D. G.. Spaulding. J. T.. Benson. R.. expected to be less active in the rat because progesterone is the Dorr, F. A.. Blumenslein. B. A.. Davies. M. A., and Goodman. P. J. A controlled (rial of leuprolide with and without flutamide in prostatic carcinoma. N. Engl. J. Med., preferred substrate of the rat CI7 2n-lyase. Due to insufficient material, 321: 419-424. 1989. we did not compare ketoconazole and finasteride with our compounds Angelastro. M. R.. Laughlin. M. E.. Schalzman. G. L.. Bey. P.. and Blohm. T. R. l7a-(cyclopropylamino)-androsta-5-en-3a-ol. a selective mechanism based inhibitor in vivo in these experiments. However, surgical castration has been of cytochrome P-45017„a(steroid l7a-hydroxylase/C1720lyase). Biochem. Biophys. the traditional approach to lower androgen levels in vivo (10). These Res. Commun.. 162: 1571-1577. 1989. novel steroids were effective in achieving similar reductions in tes 13. Ayub. M., and Levell. M. J. Inhibition of testicular 17a-hydroxylase and 17.20-lyase but not 3-hydroxysteroid dehydrogenase-isomerase or 17-hydroxysteroid oxidoreductase by tosterone concentration in the prostate compared to castration. In fact, ketoconazole and other imidazole drugs. J. Steroid Biochem.. 2K: 521-531. 1987. L-37 was slightly more effective in this regard. Nevertheless, DHT 14. Nakajin. S , Takahashi. K.. and Shinoda. M. Inhibitory effects and interaction of concentrations were reduced by only 40% of intact controls compared stanozolol with pig testicular cytochrome P-450 (l7a-hydroxylase/Cl7 2nlyase). Chem. Pharm. Bull. (Tokyo). 7: 1855-1858. 1989. to almost complete reduction by castration, although testosterone Nakajin, S.. Takahashi. K.. and Shinoda. M. Inhibitory effects and spectral changes levels in the prostate were similar (L-36) or lower (L-37) than cas on pig testicular cytochrome P-450 ( 17a-hydroxylasc/C, 7 2l)lyase) by 20a-hydroxy- tration. Prostatic growth and weight are regulated by both testosterone C,,-steroids. Yakugaku Zasshi. 108: 1188-1195. 1988. Jarman. M.. Barrie. S. E.. Deadman. J. J.. Houghton, J.. and McCague. R. Novel inhibitors and DHT, although to a greater extent by the latter. Therefore, the of enzymes of androgen biosynthesis. J. Med. Chem.. 33: 3050-3055. 1990. reduction in the levels of these hormones by these compounds may McCague. R.. Rowlands. M. G.. Barrie. S. E.. and Houghton. J. Inhibition of enzymes of estrogen and androgen biosynthesis by ester of 4-pyridylacetic acid. J. Med. explain the corresponding reduction in normal prostate size observed Chem., 33: 3050-3055, 1990. after treatment with L-36 and L-37. The fact that these novel steroidal Trachtenberg. J. Ketoconazole therapy in advanced prostalic cancer. J. Urol.. 132: compounds were not as effective as castration in lowering tissue DHT 61-63, 1984. concentrations or shrinking prostate size may also indicate that other Williams. G., Kerle. D. J.. Doble. A., Dunlop. H.. Smith. C., Allen. J., Yeo. T.. and Bloom, S. R. Objective responses to ketoconazole therapy in patients with relapsed mechanisms may be involved in prostate regulation. The effects of progressive prostatic cancer. Br. J. Urol.. 58: 45-51, 1986. these compounds on prostate tumors are in progress. L-2 increased Stoner, E. The clinical development of 5a-reductase inhibitor, finasteride. J. Steroid Biochem. Mol. Biol.. 37: 375-378. 1990. plasma testosterone levels and prostate DHT levels and failed to Cohen. S. M.. Werrman. G. H.. Rasmussen. G. H.. Tankaka. W. K.. Malalesta. P. F.. reduce prostate weight when administered to rats. These observations Prahalada. J. G.. Jacobs. J. G.. Harris. G.. and Nett. T. M. Comparison of the effects were unexpected because L-2 was a potent inhibitor of C172()-lyase of new azasteroids inhibitors of steroid 5a-reductase on canine hyperplastic prostate: and also a moderately active inhibitor of 5a-reductase in vitro. It is suppression of prostatic DHT correlated with prostate regression. Prostate. 26: 55-71, 1995. plausible that L-2 is biotransformed in vivo to some derivative that has Geller. J. Effects of finasteride. a 5a-reductase inhibitor of prostate tissue androgens androgenic activity or that it alters the metabolism of testosterone or and prostate specific antigen. J. Clin. Endocrino!. Metab.. 71: 1552-1555. 1990. Gormley, G. J. Role of 5t»-reductaseinhibitors in the treatment of advanced proslatic DHT. Further studies are necessary to verify this possibility. Signif carcinoma. Uro!. Clin. North Am. 18: 93-97, 1991. icant macroscopic changes in the liver and kidneys were also observed Li. J.. Li. Y. Son. C.. and Brodie. A. M. H. Inhibition of androgen synthesis by when L-2 was administered to rats. Thus, this particular compound 22-hydroximino-23.24-bisnor-4-cholen-3-one. Prostate. 26: 140-150. 1995. Klus, G. T.. Nakamura, J., Li, J. Ling, Y. Son, C.. Kemppainen. J. A.. Wilson. E. M.. will not be useful for further evaluation. L-36, L-37, and 1-41 are more and Brodie. A. M. H. Growth inhibition of human prostate cells in viiro by novel promising; L-36, in particular, was very effective in diminishing inhibitors of androgen synthesis. Cancer Res., 56: 4956-4964. 1996. Li, J., Li. Y.. Son. C.. Banks. P.. and Brodie, A. 4-Pregnen-3-one-20cr-carboxyaldc- androgen levels in rat tissues and reducing prostatic weight. hyde: a potent inhibitor of l7a-hydroxylase/C]72()lyase and of 5a-reductase. In conclusion, our studies indicate that inhibitors with dual activi J. Steroid Biochem. Mol. Biol.. 42: 313-321. 1992. ties against C172()-lyase and 5a-reductase can result in greater inhi Ling, Y.. Li. J.. Liu. Y.. Kato. K.. Klus. G. T.. and Brodie. A. 17-lmidazolyl. pyrazolyl and isoxa/.olyl androsteinc derivatives. Novel steroidal inhibitors of human bition of androgen synthesis in vivo. These novel compounds may cytochrome C,720-lyase (P-450l7Å“l) J. Med. Chem., 40: 3297-3304. 1997. provide potential new leads for research and development of drugs for 28. Li, J. S.. Li. Y. Son. C.. and Brodie. A. Synthesis and evaluation of pregnane androgen-dependent diseases. derivatives as inhibitors of human testicular 17a-hydroxylase/Cí720lyase. J. Med. Chem.. 39: 4335-4339, 1996. 29. Njar. V. C. O.. Kalo. K.. Nnane. I. P.. Grigoryev, D. N.. Long. B. J.. and Brodic. A. M. H. Novel azolyl steroids: potent inhibitors of human cytochrome I7a-hydrox- REFERENCES ylase/C|7 -,,,-lyase (P-450|7,,.,): potent inhibitors for the treatment of prostate cancer. J. Med. Chem.. 41: 902-912. 1998. I. Dijkman, G. A., and Debruyne, F. M. Epidemiology of prostate cancer. Eur. Urol.. 30. Lowry. O. H.. Roseborough. N. S., Fair. A. L.. and Randall. R. S. Protein measure 30: 281-295. 1996. ment with the folin phenol reagent. J. Biol. Chem.. 193: 265-275, 1951. 3831

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1998 American Association for Cancer Research. EFFECTS OF NOVEL ANDROGEN SYNTHESIS INHIBITORS

31. Gormlcy, G. J.. Sloner. E.. Bruskewitz. R. C.. Impcranto-McGinlcy. J.. Walsh, P. C.. 35. Lamb. J. C., Levy, M. A.. Johnson, R. K.. and Isaacs. J. T. Response to ral and human McConnel. J. D., Andriole, G. L., Geller. J., Brachen, B. R.. Tenover, J. S., Vaughan, prosiate cancer to the novel 5o-reducta.se inhibitor. SK&F 105657. Prostate, 21: E. D., Pappas, F., Taylor. A.. Binkowitz. B.. and Ng, J. The effects of finasteride in 15-34, 1992. men with prostatic hyperplasia. N. Engl. J. Med., 327: 1185-1191. 1992. 36. Rittmaster. R. S.. Manning. A. P.. Wright. A. S. Thomas. L. N.. Whitefield, S., and 32. Peters. D. H.. and Sorkin. M. Finasteride: a review of its potential in the treatment of Norman, R. W. Evidence of atrophy and apoptosis in the ventral prostate of rats given benign prostalic hyperplasia. Drugs. 46: 177-208, 1993. the 5a-reductase inhibitor finasteride. Endocrinology, 136: 741-748. 1995. 33. Homma. Y.. Kaneko. M.. Kondo. Y.. Kawabe. K.. and Kakizoe. T. Inhibition of rat 37. Zaccheo, T.. Giudici. D.. and di Salle, E. Effects of turosteride. a 5a-reductase prostate carcinogenesis by a 5a-reductase inhibitor, FKI43. J. Nail. Cancer Inst. inhibitor, on the Dunning R3327 rat prostatic carcinoma. Prostate, 30: 85-91, iBethesda). 89: 803-807. 1997. 1997. 34. Kadohama. N., Karr. J. P., Murphy, G. P., and Sandberg. A. A. Selective inhibition 38. Nakayama. O.. Hirosomi. J.. Chida. N.. Takahashi. S., Sawada. K., Kojo, H.. and of prosialic tumour 5a-reductase by a 4-methyl-4-azasteroid. Cancer Res.. 44: 4947- Notsu, Y. FRI46687. A novel steroid 5a-reductase inhibitor: in vitro and in vivo 4954, 1984. effects on prostates. Prostate. 31: 241-249. 1997.

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Ivo P. Nnane, Katsuya Kato, Yang Liu, et al.

Cancer Res 1998;58:3826-3832.

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