Yasuharu Urano

Discovery of Novel Forkhead Box O1 Inhibitors for Treating Type 2 Diabetes: Improvement of Fasting Glycemia in Diabetic db/db Mice

Excessive hepatic glucose production through the gluconeogenesis pathway is partially responsible for the elevated glucose levels observed in patients with type 2 diabetes mellitus (T2DM). The forkhead transcription factor forkhead box O1 (Foxo1) plays a crucial role in mediating the effect of insulin on hepatic gluconeogenesis. Here, using a db/db mouse model, we demonstrate the effectiveness of Foxo1 inhibitor, an orally active small-molecule compound, as a therapeutic drug for treating T2DM. Using mass spectrometric affinity screening, we discovered a series of compounds that bind to Foxo1, identifying among them the compound, 5-amino-7-(cyclohexylamino)-1-ethyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (AS1842856), which potently inhibits human Foxo1 transactivation and reduces glucose production through the inhibition of glucose-6 phosphatase and phosphoenolpyruvate carboxykinase mRNA levels in a rat hepatic cell line. Oral administration of AS1842856 to diabetic db/db mice led to a drastic decrease in fasting plasma glucose level via the inhibition of hepatic gluconeogenic genes, whereas administration to normal mice had no effect on the fasting plasma glucose level. Treatment with AS1842856 also suppressed an increase in plasma glucose level caused by pyruvate injection in both normal and db/db mice. Taken together, these findings indicate that the Foxo1 inhibitor represents a new class of drugs for use in treating T2DM.

Prevention of renal interstitial fibrosis via histone deacetylase inhibition in rats with unilateral ureteral obstruction.

Acute rejection following renal transplantation has become manageable with the introduction of calcineurin inhibitors, FK506 and cyclosporine A. However, chronic allograft dysfunction accompanied by renal interstitial fibrosis, which induces graft loss, remains unresolved. Here, we evaluated the effect of FR276457, a pan-histone deacetylase (HDAC) inhibitor, on interstitial fibrosis in the injured kidneys of a rat model of unilateral ureteral obstruction. The injured kidneys, harvested on Day 14 following the operation, showed progression of interstitial fibrosis, increases of hydroxyproline contents, and mRNA expression of collagen type Ialpha1 and monocyte chemotactic protein 1 (MCP-1). However, these changes were found to be prevented with daily oral administration of FR276457. In addition, given that MCP-1 is believed to contribute to progressive fibrosis, we investigated the direct effect of FR276457 on MCP-1 production by activated THP-1 cells in vitro. Results showed that FR276457 administration decreased MCP-1 production in these cells in a concentration-dependent manner. Findings from the present study suggested that a pan-HDAC inhibitor may exert a prophylactic effect against renal interstitial fibrosis by inhibiting MCP-1 production.

Effects of the novel foxo1 inhibitor as1708727 on plasma glucose and triglyceride levels in diabetic db/db mice

Recent evidence suggests that the forkhead transcription factor Foxo1 plays an important role in the regulation of glucose and triglyceride metabolism at the gene transcription level for glucose-6 phosphatase (G6Pase), phosphoenolpyruvate carboxykinase (PEPCK), and apolipoprotein C-III (apoC-III). Here, we report on the pharmacological effects of the novel Foxo1 inhibitor AS1708727, which we identified by compound screening. Chronic treatment of diabetic db/db mice with AS1708727 for four days significantly reduced blood glucose and triglyceride levels with decrease of gene expression levels of hepatic G6Pase, PEPCK, and apoC-III. No reports have yet examined the influence of Foxo1 inhibitors on these pharmacological effects. In this study, we newly identified a Foxo1 inhibitor compound capable of exerting both an anti-hypertriglyceridemic and anti-hyperglycemic effect. These effects were dependent on maintaining a stable blood concentration of AS1708727 and achieving a high rate of compound transition to the liver. We also investigated the action mechanism of AS1708727 on gluconeogenesis in vitro and in vivo. The compound inhibited gene expression of key gluconeogenic molecules and suppressed gluconeogenesis in Fao hepatocyte cells in vitro. Further, in the pyruvate challenge study using db/db mice in vivo, AS1708727 suppressed increases in blood glucose level by inhibiting gluconeogenic gene expression. These results indicate that the novel Foxo1 inhibitor AS1708727 may exert anti-diabetic and anti-hypertriglyceridemic effects by improving blood glucose and triglyceride metabolism at the gene expression level, and may represent a new class of drugs useful for treating type 2 diabetes mellitus and hypertriglyceridemia.

FR226807: a potent and selective phosphodiesterase type 5 inhibitor.

We describe the pharmacological characteristics of a novel phosphodiesterase type 5 inhibitor FR226807, N-(3,4-dimethoxybenzyl)-2-[[(1R)-2-hydroxy-1-methylethyl]amino]-5-nitrobenzamide. FR226807 inhibited phosphodiesterase type 5 isolated from human platelets with an IC(50) value of 1.1 nM. FR226807 also inhibited phosphodiesterase type 6 with an IC(50) of 20 nM; however, the IC(50) value for phosphodiesterase type 6 was 18-fold higher than that for phosphodiesterase type 5. The IC(50) values of FR226807 for other phosphodiesterases (phosphodiesterase type 1, phosphodiesterase type 2, phosphodiesterase type 3, and phosphodiesterase type 4) were 1000-fold higher than that for phosphodiesterase type 5. FR226807 increased the cyclic guanosine monophosphate (cGMP) content in corpus cavernosum isolated from rabbit, an effect associated with relaxation of the muscle. FR226807 enhanced the relaxation response induced by electrical field stimulation of corpus cavernosum isolated from the rabbit. In an anesthetized dog model for the evaluation of erectile function, intravenous administration of FR226807 prolonged the time to return to 75% of maximal intracavernosal pressure after cessation of electrical stimulation of the pelvic nerve. In summary, FR226807 is a potent and highly selective phosphodiesterase type 5 inhibitor with an augmentative effect on penile erection and will be useful for the treatment of erectile dysfunction.

A Histone Deacetylase Inhibitor, FR276457, Altered Characteristics of Infiltrating Cells into Allograft in a Rat Cardiac Transplant Model

Background: A recent study indicated that FR276457, a pan-histone deacetylase inhibitor, prevented allograft rejection in an ACI-to-Lewis rat heart transplant model, seemingly without affecting cellular infiltration into the transplanted heart. In this report, we investigated alterations in infiltrating cells in allografts by treatment of FR276457. Materials and Methods: Heterotopic cardiac allografts which were removed from August Copenhagen Irish rats were transplanted into the necks of Lewis rats. FR276457 40 mg/kg or vehicle was subsequently administered orally for 5 consecutive days beginning on the operation day after recovery from anesthesia (Day 0). The allograft recipients were sacrificed under anesthesia on Day 5 after transplantation. At first, we identified infiltrating cells into the allografts by the histopathological analysis. In addition, we isolated the infiltrating cells from allografts, sorted CD8 positive (CD8 + ) T cells, and then investigated cytotoxicity of CD8 + T cells against spleen cells from an August Copenhagen Irish rat. Results: Histopathological analysis of allografts on Day 5 after the heart transplant showed that infiltration of T cells was not suppressed in FR276457-treated allograft recipients, compared with those in vehicle-treated allograft recipients, although the infiltration of ED1 positive cells was tended to decrease. Ex vivo analysis revealed that alloantigen-specific cytotoxicity of CD8 + T cells, isolated from allografts in FR276457-treated allograft recipients, against ACI rat splenocytes was much lower than that in vehicle-treated allograft recipients. Conclusion: FR276457 treatment suppressed the alloantigen-specific cytotoxicity of CD8 + T cells infiltrated into