Daisuke Nakata

Pharmacologic profiles of investigational kisspeptin/metastin analogues, TAK-448 and TAK-683, in adult male rats in comparison to the GnRH analogue leuprolide.

Kisspeptin/metastin, a hypothalamic peptide, plays a pivotal role in controlling gonadotropin-releasing hormone (GnRH) neurons, and we have shown that continuous subcutaneous administration of kisspeptin analogues suppresses plasma testosterone in male rats. This study examined pharmacologic profiles of investigational kisspeptin analogues, TAK-448 and TAK-683, in male rats. Both analogues showed high receptor-binding affinity and potent and full agonistic activity for rat KISS1R, which were comparable to natural peptide Kp-10. A daily subcutaneous injection of TAK-448 and TAK-683 (0.008-8μmol/kg) for consecutive 7 days initially induced an increase in plasma luteinizing hormone and testosterone levels; however, after day 7, plasma hormone levels and genital organ weights were reduced. Continuous subcutaneous administrations of TAK-448 (≥10pmol/h, ca. 0.7nmol/kg/day) and TAK-683 (≥30pmol/h, ca. 2.1nmol/kg/day) induced a transient increase in plasma testosterone, followed by abrupt reduction of plasma testosterone to castrate levels within 3-7 days. This profound testosterone-lowering effect was sustained throughout 4-week dosing periods. At those dose levels, the weights of the prostate and seminal vesicles were reduced to castrate levels. These suppressive effects of kisspeptin analogues were more rapid and profound than those induced by the GnRH agonist analogue leuprolide treatment. In addition, TAK-683 reduced plasma prostate specific antigen (PSA) in the JDCaP androgen-dependent prostate cancer rat model. Thus, chronic administration of kisspeptin analogues may hold promise as a novel therapeutic approach for suppressing reproductive functions and hormone-related diseases such as prostate cancer. Further studies are warranted to elucidate clinical significance of TAK-448 and TAK-683.

Discovery of 1-{4-[1-(2,6-Difluorobenzyl)-5-[(dimethylamino)methyl]-3-(6-methoxypyridazin-3-yl)-2,4-dioxo-1,2,3,4-tetrahydrothieno[2,3-d]pyrimidin-6-yl]phenyl}-3-methoxyurea (TAK-385) as a Potent, Orally Active, Non-Peptide Antagonist of the Human Gonadotropin-Releasing Hormone Receptor

We previously discovered an orally active human gonadotropin-releasing hormone (GnRH) receptor antagonist, thieno[2,3-d]pyrimidine-2,4-dione derivative 1 (sufugolix). To reduce the cytochrome P450 (CYP) inhibitory activity and improve in vivo GnRH antagonistic activity, further optimization of this scaffold was carried out. We focused our synthetic efforts on chemical modification at the 5 and 3 positions of the thieno[2,3-d]pyrimidine-2,4-dione ring based on computational modeling, which resulted in the discovery of 1-{4-[1-(2,6-difluorobenzyl)-5-[(dimethylamino)methyl]-3-(6-methoxypyridazin-3-yl)-2,4-dioxo-1,2,3,4-tetrahydrothieno[2,3-d]pyrimidin-6-yl]phenyl}-3-methoxyurea (16b) as a highly potent and orally active GnRH antagonist. Compound 16b showed potent in vitro GnRH antagonistic activity in the presence of fetal bovine serum (FBS) without CYP inhibition. Oral administration of 16b maintained the suppressive effect of the plasma luteinizing hormone levels in castrated cynomolgus monkeys at a 3 mg/kg dose for more than 24 h. Compound 16b is currently under clinical development with the code name of TAK-385.

A novel androgen-dependent prostate cancer xenograft model derived from skin metastasis of a Japanese patient

The incidence of, and mortality from, prostate cancer (PCa) has increased in Asian countries over the past decades, partly due to a change in dietary habits. Recent reports have revealed differences in the molecular basis of PCa among people of differing racial or ethnic backgrounds. PCa xenograft models established from Asian patients would be useful for understanding the basis of PCa in Asian populations; we therefore established and characterized a novel PCa xenograft model, JDCaP, from a metastatic skin lesion of a Japanese hormone‐refractory prostate cancer (HRPC) patient.

Suppression of the hypothalamic–pituitary–gonadal axis by TAK-385 (relugolix), a novel, investigational, orally active, small molecule gonadotropin-releasing hormone (GnRH) antagonist: Studies in human GnRH receptor knock-in mice

TAK-385 (relugolix) is a novel, non-peptide, orally active gonadotropin-releasing hormone (GnRH) antagonist, which builds on previous work with non-peptide GnRH antagonist TAK-013. TAK-385 possesses higher affinity and more potent antagonistic activity for human and monkey GnRH receptors compared with TAK-013. Both TAK-385 and TAK-013 have low affinity for the rat GnRH receptor, making them difficult to evaluate in rodent models. Here we report the human GnRH receptor knock-in mouse as a humanized model to investigate pharmacological properties of these compounds on gonadal function. Twice-daily oral administration of TAK-013 (10mg/kg) for 4 weeks decreased the weights of testes and ventral prostate in male knock-in mice but not in male wild-type mice, demonstrating the validity of this model to evaluate antagonists for the human GnRH receptor. The same dose of TAK-385 also reduced the prostate weight to castrate levels in male knock-in mice. In female knock-in mice, twice-daily oral administration of TAK-385 (100mg/kg) induced constant diestrous phases within the first week, decreased the uterus weight to ovariectomized levels and downregulated GnRH receptor mRNA in the pituitary after 4 weeks. Gonadal function of TAK-385-treated knock-in mice began to recover after 5 days and almost completely recovered within 14 days after drug withdrawal in both sexes. Our findings demonstrate that TAK-385 acts as an antagonist for human GnRH receptor in vivo and daily oral administration potently, continuously and reversibly suppresses the hypothalamic-pituitary-gonadal axis. TAK-385 may provide useful therapeutic interventions in hormone-dependent diseases including endometriosis, uterine fibroids and prostate cancer.

Growth Inhibition by Testosterone in an Androgen Receptor Splice Variant‐Driven Prostate Cancer Model

Castration resistance creates a significant problem in the treatment of prostate cancer. Constitutively active splice variants of androgen receptor (AR) have emerged as drivers for resistance to androgen deprivation therapy, including the next‐generation androgen‐AR axis inhibitors abiraterone and enzalutamide. In this study, we describe the characteristics of a novel castration‐resistant prostate cancer (CRPC) model, designated JDCaP‐hr (hormone refractory).

Anti‐tumor efficacy of a novel CLK inhibitor via targeting RNA splicing and MYC‐dependent vulnerability

The modulation of pre‐mRNA splicing is proposed as an attractive anti‐neoplastic strategy, especially for the cancers that exhibit aberrant pre‐mRNA splicing. Here, we discovered that T‐025 functions as an orally available and potent inhibitor of Cdc2‐like kinases (CLKs), evolutionally conserved kinases that facilitate exon recognition in the splicing machinery. Treatment with T‐025 reduced CLK‐dependent phosphorylation, resulting in the induction of skipped exons, cell death, and growth suppression in vitro and in vivo. Further, through growth inhibitory characterization, we identified high CLK2 expression or MYC amplification as a sensitive‐associated biomarker of T‐025. Mechanistically, the level of CLK2 expression correlated with the magnitude of global skipped exons in response to T‐025 treatment. MYC activation, which altered pre‐mRNA splicing without the transcriptional regulation of CLKs, rendered cancer cells vulnerable to CLK inhibitors with synergistic cell death. Finally, we demonstrated in vivo anti‐tumor efficacy of T‐025 in an allograft model of spontaneous, MYC‐driven breast cancer, at well‐tolerated dosage. Collectively, our results suggest that the novel CLK inhibitor could have therapeutic benefits, especially for MYC‐driven cancer patients.

The RNA helicase DDX39B and its paralog DDX39A regulate androgen receptor splice variant AR-V7 generation

Mounting evidence suggests that constitutively active androgen receptor (AR) splice variants, typified by AR-V7, are associated with poor prognosis and resistance to androgen deprivation therapy in prostate cancer patients. However, mechanisms governing the generation of AR splice variants are not fully understood. In this study, we aimed to investigate the dynamics of AR splice variant generation using the JDCaP prostate cancer model that expresses AR splice variants under androgen depletion. Microarray analysis of JDCaP xenografts before and after expression of AR splice variants suggested that dysregulation of RNA processing pathways is likely involved in AR splice variant generation. To explore factors contributing to generation of AR-V7 mRNA, we conducted a focused RNA interference screen in AR-V7-positive JDCaP-hr cells using an shRNA library targeting spliceosome-related genes. This screen identified DDX39B as a regulator of AR-V7 mRNA expression. Simultaneous knockdown of DDX39B and its paralog DDX39A drastically and selectively downregulated AR-V7 mRNA expression in multiple AR-V7-positive prostate cancer cell lines. DDX39B was upregulated in relapsed JDCaP xenografts expressing AR splice variants, suggesting its role in expression of AR splice variants. Taken together, our findings offer insight into the mechanisms of AR splice variant generation and identify DDX39 as a potential drug target for the treatment of AR splice variant-positive prostate cancer.

Evaluation of pharmacokinetics/pharmacodynamics and efficacy of one-month depots of TAK-448 and TAK-683, investigational kisspeptin analogs, in male rats and an androgen-dependent prostate cancer model

Abstract TAK‐448 and TAK‐683 are kisspeptin agonist analogs with improved in vivo stability and activity. Previous studies showed that continuous subcutaneous administration of TAK‐448 or TAK‐683 caused rapid and profound reductions in plasma testosterone levels in various species, including male healthy volunteers, suggesting their therapeutic potential as anti‐prostate cancer agents. For clinical drug development, one‐month sustained‐release depots of TAK‐448 and TAK‐683, TAK‐448‐SR(1M) and TAK‐683‐SR(1M), were designed to improve usability in clinical practice. In this study, the pharmacokinetics/pharmacodynamics (PK/PD) profiles of TAK‐448‐SR(1M) and TAK‐683‐SR(1M) were initially tested in male rats to ensure their eligibility as one‐month depots. The therapeutic advantages of TAK‐448‐SR(1M) and TAK‐683‐SR(1M) over TAP‐144‐SR(1M) were then investigated in a JDCaP xenograft rat model. TAK‐448‐SR(1M) and TAK‐683‐SR(1M) maintained certain levels of plasma TAK‐448 free form (TAK‐448F) and plasma TAK‐683 free form (TAK‐683F) for at least 4 weeks, before clearance from the circulation. Accompanying their desirable PK profiles, TAK‐448‐SR(1M) and TAK‐683‐SR(1M) showed favorable PD responses as one‐month depots and demonstrated better testosterone control than TAP‐144‐SR(1M). Both depots exerted rapid and profound suppression of plasma testosterone levels in male rats. These profound suppressive effects were maintained in dose‐dependent manners, before recovery toward normal levels. In the JDCaP xenograft model, TAK‐448‐SR(1M) and TAK‐683‐SR(1M) both showed better prostate‐specific antigen (PSA) control than TAP‐144‐SR(1M), although all treatment groups eventually experienced PSA recurrence and tumor regrowth. In conclusion, this study demonstrates that both TAK‐448‐SR(1M) and TAK‐683‐SR(1M) have desirable and better PK/PD profiles than TAP‐144‐SR(1M) in rats, which could potentially provide better clinical outcomes in androgen‐dependent prostate cancer.

Abstract 3247: Establishment of a castration-resistant prostate cancer model, JDCaP-hr: Implications for prostate cancer recurrence

There are few in vivo models of androgen-dependent prostate cancer (ADPC), and limited availability of the models makes it difficult to evaluate anticancer agents for early stage prostate cancer and to investigate the recurrence of ADPC. We previously reported JDCaP xenograft model, an ADPC model with wild type androgen receptor (AR) and TMPRSS2-ERG gene fusion, derived from skin metastatic lesions of a prostate cancer patient. JDCaP xenografts completely regressed after surgical or medical castration; however, some of them relapsed several months after castration. The relapsed JDCaP subline, designated JDCaP-hr (hormone refractory), was established by serial passage in castrated nude mice. The AR antagonist bicalutamide did not show any effect on tumor growth of JDCaP-hr, while testosterone treatment induced tumor regression of JDCaP-hr, indicating that ligand binding to AR is not necessary, rather cytotoxic, for the growth of JDCaP-hr. Direct sequencing analysis of full length AR revealed that JDCaP-hr retained wild type AR. AR mRNA expression in JDCaP-hr was approximately 7-fold higher than in JDCaP, and interestingly, truncated AR protein with molecular weight of approximately 75 kDa in addition to full length AR (110 kDa) was expressed in JDCaP-hr. Microarray analysis revealed that ubiquitin-dependent protein catabolism pathway was markedly upregulated in JDCaP-hr compared with JDCaP. JDCaP-hr cells proliferated in vitro in steroid-free medium under co-culture condition with mouse stromal cells, and secreted prostate-specific antigen into the conditioned medium. Taken together, JDCaP/JDCaP-hr is a valuable model which simulates the recurrence of ADPC to castration-resistant prostate cancer (CRPC) with common clinical features such as AR overexpression. This model provides not only useful in vivo and in vitro systems to evaluate anticancer agents for ADPC and CRPC but also intriguing implications for the recurrence of ADPC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3247.

CDK8/19 inhibition induces premature G1/S transition and ATR-dependent cell death in prostate cancer cells

The CDK8/19 kinase module comprises a subcomplex that interacts with the Mediator complex and regulates gene expression through phosphorylation of transcription factors and Mediator subunits. Mediator complex subunits have been increasingly implicated in cancer and other diseases. Although high expression of CDK8/19 has been demonstrated in prostate cancer, its function has not been thoroughly examined. Here we report that CDK8/19 modulates the gene expression of cell cycle regulators and thereby maintains the proper G1/S transition in prostate cancer cells. We show that highly selective CDK8/19 inhibitors exerted anti-proliferative activity in prostate cancer cells both in vitro and in vivo. In CDK8/19 inhibitor-sensitive prostate cancer cells, the compounds reduced the population of G1 phase cells and elevated that of S phase cells through the modulation of G1/S transition regulators at the level of mRNA expression. Furthermore, the premature G1/S transition induced a DNA damage response that was followed by ATR-dependent and caspase-independent cell death. These findings suggest a novel role of CDK8/19 in transcription-mediated cell cycle control, albeit with possible contribution of other proteins inhibited by the compounds. Our data provide a rationale for further investigation of CDK8/19 inhibitors as a new therapeutic approach to prostate cancer.