Synthesis and biological evaluation of novel selective androgen receptor modulators (SARMs). Part II: Optimization of 4-(pyrrolidin-1-yl)benzonitrile derivatives.

We recently reported a class of novel tissue-selective androgen receptor modulators (SARMs), represented by a naphthalene derivative A. However, their pharmacokinetic (PK) profiles were poor due to low metabolic stability. To improve the PK profiles, we modified the hydroxypyrrolidine and benzonitrile substituents of 4-(pyrrolidin-1-yl)benzonitrile derivative B, which had a comparable potency as that of compound A. This optimization led us to further modifications, which improved metabolic stability while maintaining potent androgen agonistic activity. Among the synthesized compounds, (2S,3S)-2,3-dimethyl-3-hydroxylpyrrolidine derivative 1c exhibited a suitable PK profile and improved metabolic stability. Compound 1c demonstrated significant efficacy in levator ani muscle without increasing the weight of the prostate in an in vivo study. In addition, compound 1c showed agonistic activity in the CNS, which was detected using sexual behavior induction assay.

Synthesis and biological evaluation of novel selective androgen receptor modulators (SARMs) Part III: Discovery of 4-(5-oxopyrrolidine-1-yl)benzonitrile derivative 2f as a clinical candidate.

We previously reported that 4-(pyrrolidin-1-yl)benzonitrile derivative 1b was a selective androgen receptor modulator (SARM) that exhibited anabolic effects on organs such as muscles and the central nervous system (CNS), but neutral effects on the prostate. From further modification, we identified that 4-(5-oxopyrrolidine-1-yl)benzonitrile derivative 2a showed strong AR binding affinity with improved metabolic stabilities. Based on these results, we tried to enhance the AR agonistic activities by modifying the substituents of the 5-oxopyrrolidine ring. As a consequence, we found that 4-[(2S,3S)-2-ethyl-3-hydroxy-5-oxopyrrolidin-1-yl]-2-(trifluoromethyl)benzonitrile (2f) had ideal SARM profiles in Hershberger assay and sexual behavior induction assay. Furthermore, 2f showed good pharmacokinetic profiles in rats, dogs, monkeys, excellent nuclear selectivity and acceptable toxicological profiles. We also determined its binding mode by obtaining the co-crystal structures with AR.

Synthesis and biological evaluation of novel selective androgen receptor modulators (SARMs). Part I.

To develop effective drugs for hypogonadism, sarcopenia, and cachexia, we designed, synthesized, and evaluated selective androgen receptor modulators (SARMs) that exhibit not only anabolic effects on organs such as muscles and the central nervous system (CNS) but also neutral or antagonistic effects on the prostate. Based on the information obtained from a docking model with androgen receptor (AR), we modified a hit compound A identified through high-throughput screening. Among the prepared compounds, 1-(4-cyano-1-naphthyl)-2,3-disubstituted pyrrolidine derivatives 17h, 17m, and 17j had highly potent AR agonistic activities in vitro and good tissue selectivity in vivo. These derivatives increased the weight of the levator ani muscle without influencing the prostate and seminal vesicle. In addition, these compounds induced sexual behavior in castrated rats, indicating that the compounds could also act as agonists on the CNS.

Design, synthesis, and biological evaluation of 3-aryl-3-hydroxy-1-phenylpyrrolidine derivatives as novel androgen receptor antagonists.

We designed and synthesized a series of 3-aryl-3-hydroxy-1-phenylpyrrolidine derivatives D and evaluated their potential as novel androgen receptor (AR) antagonists therapeutically effective against castration-resistant prostate cancer (CRPC). Introduction of a methyl group at the 2-position (R(2)) of the pyrrolidine ring increased the AR binding affinity. The (2S,3R) configuration of the pyrrolidine ring was favorable for the AR antagonistic activity. It was found that introduction of an amide substituent (R(1)) and a pyridin-3-yl group (Q) was effective for reducing the AR agonistic activity which appeared during the optimization of lead compound 6. Compound 54 showed potent antitumor effects against a CRPC model of LNCaP-hr cell line in a mouse xenograft, in which bicalutamide exhibited only partial suppression of tumor growth. Thus, the pyrrolidine derivatives such as 54 are novel AR antagonists, and their properties having efficacy against CRPC are distinct from those of a representative first-generation antagonist, bicalutamide.

Design, synthesis, and biological evaluation of 4-arylmethyl-1-phenylpyrazole and 4-aryloxy-1-phenylpyrazole derivatives as novel androgen receptor antagonists.

A series of 4-arylmethyl-1-phenylpyrazole and 4-aryloxy-1-phenylpyrazole compounds B were designed, synthesized, and evaluated for their potential as new-generation androgen receptor (AR) antagonists therapeutically effective against castration-resistant prostate cancer (CRPC). Introduction of a bulky amide substituent (R(2)) to the terminal aryl ring of the 4-arylmethyl group favored the reduction of agonistic activity and improved the pharmacokinetic (PK) properties. Similarly, introduction of a bulky substituent in the 4-aryloxy derivatives also resulted in improved PK properties. Compounds 28 h and 44b exhibited potent antitumor effects against a CRPC model of LNCaP-hr cell line in a mouse xenograft model. On the contrary, bicalutamide showed only partial suppression of tumor growth. These results suggest that the novel pyrazole derivatives are new-generation AR antagonists, different from the 'first-generation' antagonists such as bicalutamide in a CRPC treatment model.

Design, synthesis, and biological evaluation of 4-phenylpyrrole derivatives as novel androgen receptor antagonists.

A series of 4-phenylpyrrole derivatives D were designed, synthesized, and evaluated for their potential as novel orally available androgen receptor antagonists therapeutically effective against castration-resistant prostate cancers. 4-Phenylpyrrole compound 1 exhibited androgen receptor (AR) antagonistic activity against T877A and W741C mutant-type ARs as well as wild-type AR. An arylmethyl group incorporated into compound 1 contributed to enhancement of antagonistic activity. Compound 4n, 1-{[6-chloro-5-(hydroxymethyl)pyridin-3-yl]methyl}-4-(4-cyanophenyl)-2,5-dimethyl-1H-pyrrole-3-carbonitrile exhibited inhibitory effects on tumor cell growth against the bicalutamide-resistant LNCaP-cxD2 cell line as well as the androgen receptor-dependent JDCaP cell line in a mouse xenograft model. These results demonstrate that this series of pyrrole compounds are novel androgen receptor antagonists with efficacy against prostate cancer cells, including castration-resistant prostate cancers such as bicalutamide-resistant prostate cancer.

17,20-lyase inhibitors. Part 4: design, synthesis and structure-activity relationships of naphthylmethylimidazole derivatives as novel 17,20-lyase inhibitors.

A novel series of naphthylmethylimidazole derivatives and related compounds have been investigated as selective 17,20-lyase inhibitors. Optimization of the substituent at the 6-position on the naphthalene ring was performed to yield a methylcarbamoyl derivative, which exhibited potent inhibitory activity against human 17,20-lyase and promising selectivity (>200-fold) for 17,20-lyase over CYP3A4. Further modifications of the methylcarbamoyl derivative led to the discovery of the corresponding tricyclic compound, which showed highly potent activity against human 17,20-lyase (IC(50) 19 nM) and good selectivity (>1000-fold) for inhibition of 17,20-lyase over CYP3A4. Additional biological evaluation revealed that the tricyclic compound had potent in vivo efficacy in monkeys and favorable pharmacokinetic profiles when administered in rats. Asymmetric synthesis of the selective tricyclic inhibitor was also achieved using a chiral α-hydroxy ketone.

17,20-Lyase inhibitors. Part 3: Design, synthesis, and structure-activity relationships of biphenylylmethylimidazole derivatives as novel 17,20-lyase inhibitors.

A novel series of biphenylylmethylimidazole derivatives and related compounds were synthesized as inhibitors of 17,20-lyase, a key enzyme in the production of steroid hormones, and their biological activities were evaluated. In an attempt to identify potent and selective inhibitors of 17,20-lyase over the related CYP3A4 enzyme, a homology model for human 17,20-lyase was developed using the X-ray crystallographic structure of the mammalian CYP2C5 enzyme. With the aid of molecular modeling, optimization of the biphenyl moiety was performed to give an acetamide derivative, which was resolved by HPLC to give the active (-)-enantiomer. The obtained active enantiomer showed not only potent inhibition of both rat and human 17,20-lyase,with IC(50) values of 14 and 26 nM, respectively, but also excellent selectivity (>300-fold) for inhibition of 17,20-lyase over CYP3A4. Moreover, the active enantiomer significantly reduced both serum testosterone and DHEA concentrations in a monkey model after single oral administration. Asymmetric synthesis of the active enantiomer was also developed via a chiral intermediate using a diastereoselective Grignard reaction.

Discovery of orteronel (TAK-700), a naphthylmethylimidazole derivative, as a highly selective 17,20-lyase inhibitor with potential utility in the treatment of prostate cancer.

A novel naphthylmethylimidazole derivative 1 and its related compounds were identified as 17,20-lyase inhibitors. Based on the structure-activity relationship around the naphthalene scaffold and the results of a docking study of 1a in the homology model of 17,20-lyase, the 6,7-dihydro-5H-pyrrolo[1,2-c]imidazole derivative (+)-3c was synthesized and identified as a potent and highly selective 17,20-lyase inhibitor. Biological evaluation of (+)-3c at a dose of 1mg/kg in a male monkey model revealed marked reductions in both serum testosterone and dehydroepiandrosterone concentrations. Therefore, (+)-3c (termed orteronel [TAK-700]) was selected as a candidate for clinical evaluation and is currently in phase III clinical trials for the treatment of castration-resistant prostate cancer.

A mutation in beta-tubulin and a sustained dependence on androgen receptor signalling in a newly established docetaxel-resistant prostate cancer cell line.

The mechanisms of docetaxel resistance in PC (prostate cancer) are unclear because of the lack of suitable experimental models, and no effective treatment exists for docetaxel-resistant PC. We established a docetaxel-resistant cell line, LNDCr, from an androgen-refractory PC cell line, LNCaP-hr, by intermittent exposure to docetaxel in vitro. The LNDCr cells harboured an F270I mutation in class I beta-tubulin, and demonstrated impaired tubulin polymerization by docetaxel. AR signalling was sustained in LNDCr cells, and AR knockdown suppressed the growth of LNDCr cells. These results suggest that an acquired mutation in beta-tubulin is associated with docetaxel resistance in PC and that a novel AR-targeted therapy is effective for docetaxel-resistant PC.

A selective androgen receptor modulator SARM-2f activates androgen receptor, increases lean body mass, and suppresses blood lipid levels in cynomolgus monkeys.

SARM-2f a selective androgen receptor (AR) modulator, increases skeletal muscle mass and locomotor activity in rats. This study aimed to clarify its pharmacological effects in monkeys. In reporter assays, the EC50 values of SARM-2f for rat, monkey, and human AR were 2.5, 3, and 3.6 nmol/L, respectively; those of testosterone were 12, 3.2, and 11 nmol/L, respectively. A single oral administration (10 mg/kg SARM-2f) produced a maximal plasma concentration of 3011 ng/mL, with an area under the 24 hours concentration-time curve of 8152 ng·h/mL in monkeys. Body weight (BW), lean body mass (LBM), and plasma levels of total cholesterol, triglyceride, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, lipoprotein (a), alanine aminotransferase, and asparagine aminotransferase were measured after 4 weeks of treatment with SARM-2f (1, 3, and 10 mg/kg/day, QD, p.o.) or testosterone enanthate (TE; 2 mg/kg/2 weeks, s.c.) in monkeys. BW and LBM were significantly increased by 12% each by SARM-2f at 10 mg/kg, and by 5% and 8%, respectively, by TE, but these effects were not statistically significant. Plasma levels of all lipids were either decreased or showed a tendency to be decreased by SARM-2f. TE decreased the triglyceride level and increased the low-density lipoprotein cholesterol level. Liver marker levels were not changed by either SARM-2f or TE. Our data demonstrated that SARM-2f exerted anabolic effects and produced a lipid profile that differed from that produced by testosterone in monkeys, suggesting that SARM-2f might be useful for diseases such as sarcopenia.

Prevention of body weight loss and sarcopenia by a novel selective androgen receptor modulator in cancer cachexia models.

Cancer cachexia is a syndrome that impairs the quality of life and overall survival of patients, and thus the effectiveness of anticancer agents. There are no effective therapies for cancer cachexia due to the complexity of the syndrome, and insufficient knowledge of its pathogenesis results in difficulty establishing appropriate animal models. Previously, promising results have been obtained in clinical trials using novel agents including the ghrelin receptor agonist anamorelin, and the selective androgen receptor modulator (SARM) enobosarm to treat cachexia in patients with cancer. The present study examined the pharmacological effects of SARM-2f, a novel non-steroidal small molecule SARM, in animal models. SARM-2f increased body and skeletal muscle weight without significantly increasing the weight of the seminal vesicles or prostates of the castrated male rats. In the mice with tumor necrosis factor α-induced cachexia, SARM-2f and TP restored body weight, carcass weight, and food consumption rate. In the C26 and G361 cancer cachexia animal models, body and carcass weight, lean body mass, and the weight of the levator ani muscle were increased by SARM-2f and TP treatments. Tissue selectivity of SARM-2f was also observed in these animal models. The results demonstrate the anabolic effects of SARM-2f in a cytokine-induced cachexia model and other cancer cachexia models, and suggest that SARM-2f may be a novel therapeutic option for cachexia in patients with cancer.

Amelioration of sexual behavior and motor activity deficits in a castrated rodent model with a selective androgen receptor modulator SARM-2f.

Sarcopenia and cachexia present characteristic features of a decrease in skeletal muscle mass and strength, anorexia, and lack of motivation. Treatments for these diseases have not yet been established, although selective androgen receptor modulators (SARMs) are considered as therapeutic targets. We previously reported that a novel SARM compound, SARM-2f, exhibits anabolic effect on muscles, with less stimulatory effect on prostate weight compared with testosterone, in rat Hershberger assays and cancer cachexia models. In this study, we studied the mechanism of action for SARM-2f selectivity and also assessed whether the muscle increase by this compound might lead to improvement of muscle function and physical activity. First, we examined the tissue distribution of SARM-2f. Tissue concentration was 1.2-, 1.6-, and 1.9-fold as high as the plasma concentration in the levator ani muscle, brain, and prostate, respectively. This result showed that the tissue-selective pharmacological effect did not depend on SARM-2f concentration in the tissues. The ability of SARM-2f to influence androgen receptor (AR)-mediated transcriptional activation was examined by reporter assays using human normal prostate epithelial cells (PrEC) and skeletal muscle cells (SKMC). SARM-2f exerted higher activity against AR in SKMC than in PrEC. Mammalian two hybrid assays showed different co-factor recruitment patterns between SARM-2f and dihydrotestosterone. Next, we studied the effect of SARM-2f on motivation and physical functions such as sexual behavior and motor activities in castrated rat or mouse models. SARM-2f restored the sexual behavior that was lost by castration in male rats. SARM-2f also increased voluntary running distance and locomotor activities. These results suggest that tissue-specific AR regulation by SARM-2f, but not tissue distribution, might account for its tissue specific androgenic effect, and that the muscle mass increase by SARM-2f leads to improvement of physical function. Together, these findings suggest that SARM-2f might represent an effective treatment for sarcopenia and cachexia.

Enhanced androgen receptor signaling correlates with the androgen-refractory growth in a newly established MDA PCa 2b-hr human prostate cancer cell subline.

Bone metastasis is commonly found in prostate cancer (PC) patients. Although the mechanisms for the recurrence of bone metastasis-derived PC during medical or surgical castration therapy are still unclear because of the lack of suitable experimental models, one hypothesis is that enhanced androgen receptor (AR) signaling causes androgen-refractory PC growth. To test this hypothesis, we first established a novel androgen-refractory MDA PCa 2b cell subline, MDA PCa 2b-hr, which was generated in vitro from bone metastasis-derived, androgen-dependent MDA PCa 2b human PC cells after approximately 35 weeks of growth suppression by androgen-depletion treatment to mimic the clinical PC recurrence during androgen-ablation therapy. The changes of the androgen responsiveness of growth and the AR expression levels during the transition from an androgen-dependent to androgen-refractory proliferative phase through a temporal growth-suppressed phase precisely paralleled that of the basal growth rate. Furthermore, the androgen-refractory growth of MDA PCa 2b-hr cells in androgen-depleted medium was suppressed by an antiandrogen, bicalutamide. Next, we established nude mouse xenograft models to clarify whether AR signaling in MDA PCa 2b-hr cells is also enhanced in vivo. Both the MDA PCa 2b and MDA PCa 2b-hr tumors grew in gonadally intact mice, but only the MDA PCa 2b-hr tumors grew in castrated mice. The growth rate of MDA PCa 2b-hr tumors was significantly higher in gonadally intact mice than in castrated mice. Treatment with dehydroepiandrosterone pellets, which produced clinical castration levels of serum testosterone, accelerated the MDA PCa 2b-hr but not MDA PCa 2b tumor growth in castrated mice and increased blood prostate-specific antigen levels in castrated mice bearing MDA PCa 2b-hr tumors but not in mice bearing MDA PCa 2b tumors. Our data suggest that the enhanced AR signaling should be closely correlated with the androgen-refractory growth of human bone metastasis-derived PC, which might come to use adrenal androgens remaining in the blood even after castration therapy and warrant the continuation of hormone therapy for the recurrent PC.

Novel mutations of androgen receptor: a possible mechanism of bicalutamide withdrawal syndrome.

Most prostate cancers (PCs) become resistant to combined androgen blockade therapy with surgical or medical castration and antiandrogens after several years. Some of these refractory PCs regress after discontinuation of antiandrogen administration [antiandrogen withdrawal syndrome (AWS)]. Although the molecular mechanisms of the AWS are not fully understood because of the lack of suitable experimental models, one hypothesis of the mechanism is mutation of androgen receptor (AR). However, bicalutamide, which has become the most prevalent pure antiandrogen, does not work as an agonist for any mutant AR detected thus far in PC. To elucidate the mechanisms of the AWS, we established and characterized novel LNCaP cell sublines, LNCaP-cxDs, which were generated in vitro by culturing androgen-dependent LNCaP-FGC human PC cells in androgen-depleted medium with bicalutamide to mimic the combined androgen blockade therapy. LNCaP-FGC cells did not grow at first, but they started to grow after 6-13 weeks of culture. Bicalutamide stimulated LNCaP-cxD cell growth and increased prostate-specific antigen secretion from LNCaP-cxD cells both in vitro and in vivo. Sequencing of AR transcripts revealed that the AR in LNCaP-cxD cells harbors a novel mutation in codon 741, TGG (tryptophan) to TGT (cysteine; W741C), or in codon 741, TGG to TTG (leucine; W741L), in the ligand-binding domain. Transactivation assays showed that bicalutamide worked as an agonist for both W741C and W741L mutant ARs. Importantly, another antiandrogen, hydroxyflutamide, worked as an antagonist for these mutant ARs. In summary, we demonstrate for the first time that within only 6-13 weeks of in vitro exposure to bicalutamide, LNCaP-FGC cells, whose growth had initially been suppressed, came to use bicalutamide as an AR agonist via W741 AR mutation to survive. Our data strongly support the hypothesis that AR mutation is one possible mechanism of the AWS and suggest that flutamide might be effective as a second-line therapy for refractory PC previously treated with bicalutamide.

Selective androgen receptor modulator activity of a steroidal antiandrogen TSAA-291 and its cofactor recruitment profile.

Selective androgen receptor modulators (SARMs) specifically bind to the androgen receptor and exert agonistic or antagonistic effects on target organs. In this study, we investigated the SARM activity of TSAA-291, previously known as a steroidal antiandrogen, in mice because TSAA-291 was found to possess partial androgen receptor agonist activity in reporter assays. In addition, to clarify the mechanism underlying its tissue selectivity, we performed comprehensive cofactor recruitment analysis of androgen receptor using TSAA-291 and dihydrotestosterone (DHT), an endogenous androgen. The androgen receptor agonistic activity of TSAA-291 was more obvious in reporter assays using skeletal muscle cells than in those using prostate cells. In castrated mice, TSAA-291 increased the weight of the levator ani muscle without increasing the weight of the prostate and seminal vesicle. Comprehensive cofactor recruitment analysis via mammalian two-hybrid methods revealed that among a total of 112 cofactors, 12 cofactors including the protein inhibitor of activated STAT 1 (PIAS1) were differently recruited to androgen receptor in the presence of TSAA-291 and DHT. Prostate displayed higher PIAS1 expression than skeletal muscle. Forced expression of the PIAS1 augmented the transcriptional activity of the androgen receptor, and silencing of PIAS1 by siRNAs suppressed the secretion of prostate-specific antigen, an androgen responsive marker. Our results demonstrate that TSAA-291 has SARM activity and suggest that TSAA-291 may induce different conformational changes of the androgen receptor and recruitment profiles of cofactors such as PIAS1, compared with DHT, to exert tissue-specific activity.

Effect of an investigational CYP17A1 inhibitor, orteronel (TAK-700), on estrogen- and corticoid-synthesis pathways in hypophysectomized female rats and on the serum estradiol levels in female cynomolgus monkeys.

Orteronel (TAK-700) is an investigational, non-steroidal inhibitor of CYP17A1 with preferential inhibition of 17,20-lyase in NCI-H295 cells. Estrogen is synthesized from androgen by aromatase activity, and the effect of orteronel on estrogen synthesis was therefore evaluated. First, it was confirmed that orteronel does not directly inhibit aromatase activity. Second, the specific decline of serum estradiol and androgen levels in hypophysectomized female rats by orteronel in comparison with aromatase inhibitor anastrozole was evaluated; orteronel at doses ≥3mg/kg significantly suppressed serum estradiol, testosterone, androstenedione and 17-hydroxyprogesterone levels, and increased progesterone levels in the estrogen-synthesis pathway. Orteronel, at a dose of 300mg/kg, suppressed serum estradiol concentrations to a similar degree as 0.1mg/kg anastrozole. In contrast, in the corticoid-synthesis pathway, serum aldosterone, corticosterone, and progesterone levels did not change significantly following administration of 300mg/kg of orteronel. Third, the effect of multiple oral administration of orteronel on serum estradiol levels in regularly cycling female cynomolgus monkeys was evaluated. Orteronel at 15mg/kg/day (7.5mg/kg/treatment, twice daily [bid]) continued to suppress the estradiol surge prior to the start of luteal phase for 1.5-times the average duration of three consecutive, pre-treatment menstrual cycles, while serum progesterone was maintained at levels almost equal to those in the luteal phase although a certain portion of this increased level of progesterone could be of adrenal-origin. This suppressive effect on estradiol surge was thought to be reversible since serum estradiol levels started to rise immediately after the discontinuation of orteronel. Estradiol surge was not abrogated by treatment with anastrozole 0.2mg/kg/day (0.1mg/kg/treatment, bid). In summary, orteronel can suppress serum estradiol concentrations in hypophysectomized female rats and monkeys through selective inhibition of CYP17A1 activity, suggesting that orteronel might be effective for hormone-dependent breast cancers and estrogen-dependent diseases.

Effect of a novel 17,20-lyase inhibitor, orteronel (TAK-700), on androgen synthesis in male rats.

Endogenous androgens play a role in the development and progression of prostate cancer (PC), thus androgen suppression may offer an effective therapeutic strategy for this disease. Orteronel (TAK-700), 6-[(7S)-7-hydroxy-6,7-dihydro-5H-pyrrolo[1,2-c]imidazol-7-yl]-N-methyl-2-naphthamide, is a novel, non-steroidal, selective inhibitor of the 17,20-lyase activity of CYP17A--a key enzyme in the production of steroidal hormones--and is being developed as a therapy for PC. The purpose of this study was to elucidate the inhibitory activity of orteronel, in particular its specificity for androgen synthesis enzymes, in male rats--an androgen-synthesis model that largely reflects this pathway in humans. Orteronel inhibited 17,20-lyase activity in rats with an IC(50) of 1200 nM but did not inhibit 17α-hydroxylase or 11ß-hydroxylase (CYP11B1) activity in rats at concentrations up to 10 µM. In cellular steroidogenesis assays using rat testicular cells, orteronel suppressed testosterone and androstenedione production with an IC(50) of 640 nM and 210 nM, respectively, but did not suppress either corticosterone or aldosterone production in rat adrenal cells at concentrations up to 30 µM. In addition, serum testosterone and androstenedione levels in human chorionic gonadotropin-injected hypophysectomized rats were significantly reduced by single oral administration of orteronel at a dose of 30 mg/kg (both p ≤ 0.01); serum corticosterone and aldosterone levels in ACTH-injected hypophysectomized rats did not result in significant differences compared with controls, following orteronel administration at doses up to 300 mg/kg. Serum testosterone levels in intact male rats were significantly reduced by orteronel 4h after dosing at 100mg/kg (p ≤ 0.01); testosterone levels showed a tendency to recover afterward. In intact male rats, the weight of the prostate glands and seminal vesicles was decreased in a dose-dependent manner following multiple doses of orteronel at 37.5, 150, and 600 mg/kg, TID for 4 days. The reversibility of orteronel was further confirmed using a human adrenocortical tumor cell line. In summary, orteronel is a selective and reversible 17,20-lyase inhibitor, and decreases the weight of androgen-dependent organs in male rats. Our data suggests that orteronel would therefore be effective for androgen-dependent disorders such as PC.

Orteronel (TAK-700), a novel non-steroidal 17,20-lyase inhibitor: effects on steroid synthesis in human and monkey adrenal cells and serum steroid levels in cynomolgus monkeys.

Surgical or pharmacologic methods to control gonadal androgen biosynthesis are effective approaches in the treatment of a variety of non-neoplastic and neoplastic diseases. For example, androgen ablation and its consequent reduction in circulating levels of testosterone is an effective therapy for advanced prostate cancers. Unfortunately, the therapeutic effectiveness of this approach is often temporary because of disease progression to the 'castration resistant' (CRPC) state, a situation for which there are limited treatment options. One mechanism thought to be responsible for the development of CRPC is extra-gonadal androgen synthesis and the resulting impact of these residual extra-gonadal androgens on prostate tumor cell proliferation. An important enzyme responsible for the synthesis of extra-gonadal androgens is CYP17A1 which possesses both 17,20-lyase and 17-hydroxylase catalytic activities with the 17,20-lyase activity being key in the androgen biosynthetic process. Orteronel (TAK-700), a novel, selective, and potent inhibitor of 17,20-lyase is under development as a drug to inhibit androgen synthesis. In this study, we quantified the inhibitory activity and specificity of orteronel for testicular and adrenal androgen production by evaluating its effects on CYP17A1 enzymatic activity, steroid production in monkey adrenal cells and human adrenal tumor cells, and serum levels of dehydroepiandrosterone (DHEA), cortisol, and testosterone after oral dosing in castrated and intact male cynomolgus monkeys. We report that orteronel potently suppresses androgen production in monkey adrenal cells but only weakly suppresses corticosterone and aldosterone production; the IC(50) value of orteronel for cortisol was ~3-fold higher than that for DHEA. After single oral dosing, serum levels of DHEA, cortisol, and testosterone were rapidly suppressed in intact cynomolgus monkeys. In castrated monkeys treated twice daily with orteronel, suppression of DHEA and testosterone persisted throughout the treatment period. In both in vivo models and in agreement with our in vitro data, suppression of serum cortisol levels following oral dosing was less than that seen for DHEA. In terms of human CYP17A1 and human adrenal tumor cells, orteronel inhibited 17,20-lyase activity 5.4 times more potently than 17-hydroxylase activity in cell-free enzyme assays and DHEA production 27 times more potently than cortisol production in human adrenal tumor cells, suggesting greater specificity of inhibition between 17,20-lyase and 17-hydroxylase activities in humans vs monkeys. In summary, orteronel potently inhibited the 17,20-lyase activity of monkey and human CYP17A1 and reduced serum androgen levels in vivo in monkeys. These findings suggest that orteronel may be an effective therapeutic option for diseases where androgen suppression is critical, such as androgen sensitive and CRPC.

Overcoming persistent dependency on androgen signaling after progression to castration-resistant prostate cancer.

Prostate cancer is the most common form of cancer in males in the United States, and the second leading cause of cancer death. Initially, most cases of prostate cancer respond well to hormone therapy; however, resistance often develops rapidly, leading to castration-resistant prostate cancer (CRPC). Several mechanisms for castration resistance have been proposed, of which the most significant seems to be the "intracrine" production of androgens from adrenal androgen or intratumorally via the de novo route. This mechanism stimulates disease progression through reactivation of androgen receptor signaling in patients who have previously undergone castration therapy. 17,20-lyase is essential for androgen synthesis in both the adrenal glands and CRPC tissue, and some 17,20-lyase inhibitors and second-generation anti-androgens that were developed to treat CRPC are currently under clinical investigation, with encouraging preliminary data reported so far. However, resistance to some of these therapies has already been noted. The study of circulating tumor cells will likely be important not only to identify patients likely to receive benefit from this therapeutic approach, but also to further understand the molecular mechanisms of resistance.