Tsuneo Masaki

Isolation of fatty acid amide as an angiogenic principle from bovine mesentery

Erucamide (13-docosenamide) was found to be the major bovine mesentery angiogenic lipid as assessed by chorioallantoic membrane (CAM) assay. Two micrograms of this lipid caused angiogenesis in the assay. Angiogenic activity of this naturally occurring lipid was also found by rat corneal micropocket and mouse dorsal air-sac assays. Specificity of the chemical structure which elicited activity was low, however. The mechanism of angiogenic activity is unknown and this lipid does not promote proliferation of endothelial cells or induce inflammatory effects.

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.

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.

Brain testosterone deficiency leads to down-regulation of mitochondrial gene expression in rat hippocampus accompanied by a decline in peroxisome proliferator-activated receptor-γ coactivator 1α expression.

Age-related decrease of testosterone levels in blood and brain is believed to be associated with neurodegenerative diseases such as Alzheimer’s disease. However, the effect of testosterone on brain function is not well understood. Therefore, we investigated the impact of testosterone deprivation on mitochondrial gene expression in the brain of male gonadectomized (GDX) rats. We found that peripheral castration led to testosterone deficiency in the brain and caused a significant reduction in protein and mRNA expression of genes encoded by mitochondrial DNA, namely NADPH dehydrogenase subunit 1, subunit 4, cytochrome b, and cytochrome c oxidase subunit 1 and subunit 3 in the hippocampus. In addition, gene expression of peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α), which is a master regulator of mitochondrial biogenesis, and its downstream transcriptional factors, nuclear respiratory factors 1 and 2 and mitochondrial transcription factors A and B2, were also decreased in the hippocampus of GDX rats. These reductions in the expression of mitochondrial gene and transcriptional coactivators and factors were recovered by androgen replacement. These findings indicate that androgen plays an important role in mitochondrial gene expression in the hippocampus.

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.

Acute effect of triiodothyronine on the dynamics of thyrotropin release from superfused anterior pituitary cells

The effects of triiodothyronine (T3) on the dynamics of thyrotropin (TSH) release induced by TSH-releasing hormone (TRH) were examined in the presence or absence of a protein synthesis inhibitor, cycloheximide (CX), in a superfusion system using primarily cultured cells of the rat anterior pituitary gland on microcarrier beads. When the cells were continuously stimulated with TRH (10 nM, 180 min), TSH release occurred in a biphasic manner and the profile of TSH release was characterized by an initial sharp peak (phase I), followed by a lower plateau form phase (phase II). Both phase-I and phase-II releases were significantly suppressed in the presence of T3 (1 ng/ml), which was added to the superfusion medium 1 h before initiation of TRH stimulation. The biphasic nature of the release profile was maintained in the presence of T3, suggesting that the site of the T3 action may be common between phase-I and phase-II release. We have already suggested that phase-I release is protein synthesis-independent and phase-II release protein synthesis-dependent using CX in TRH-stimulated cells. In the presence of CX, phase-I release was not suppressed by T3, while phase-II release was still suppressed by T3. The inability of CX to reverse the T3-induced suppression of phase-II release may be masked by the direct CX effect on phase-II release of TSH. The present study indicates that each component (phase I and phase II) of the biphasic release of TSH induced by TRH stimulation was acutely suppressed by T3 and suggests that the T3 action is mediated through protein synthesis.

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).

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.