Tadashi Nagakura

Enhanced secretion of glucagon-like peptide 1 by biguanide compounds

Metformin was reported to increase plasma active glucagon-like peptide-1 (GLP-1) in humans. There are two possible mechanisms for this effect: (1) metformin inhibits dipeptidyl peptidase IV (DPPIV), an enzyme degrading GLP-1, and (2) metformin enhances GLP-1 secretion. To elucidate the mechanism(s), we examined (1) IC(50) of metformin for DPPIV inhibition, (2) plasma active GLP-1 changes after oral biguanide (metformin, phenformin, and buformin) treatment in fasting DPPIV-deficient F344/DuCrj rats, and (3) plasma intact GLP-1 excursions after oral administration of metformin and/or valine-pyrrolidide, a DPPIV inhibitor, in fasting DPPIV-positive F344/Jcl rats. Our in vitro assay showed that metformin at up to 30mM has no inhibitory activity towards porcine or rat DPPIV. Metformin treatment (30, 100, and 300mg/kg) increased plasma active GLP-1 levels dose-dependently in DPPIV-deficient F344/DuCrj rats (approximately 1.6-fold at 3 and 5h after administration of 300mg/kg). This treatment had no effect on blood glucose levels. Similarly, phenformin and buformin (30 and 100mg/kg) elevated plasma intact GLP-1 levels in F344/DuCrj rats. In DPPIV-positive F344/Jcl rats, coadministration of metformin (300mg/kg) and valine-pyrrolidide (30mg/kg) resulted in elevation of plasma active GLP-1, but neither metformin nor valine-pyrrolidide treatment alone had any effect. These findings suggest that metformin has no direct inhibitory effect on DPPIV activity and that metformin and the other biguanides enhance GLP-1 secretion, without altering glucose metabolism. Combination therapy with metformin and a DPPIV inhibitor should be useful for the treatment of diabetes.

Improvement of high fat-diet-induced insulin resistance in dipeptidyl peptidase IV-deficient Fischer rats

F344/DuCrj rats are genetically deficient in dipeptidyl peptidase IV (DPPIV). This enzyme degrades glucagon-like peptide-1 (GLP-1), which induces glucose-dependent insulin secretion. Glucose tolerance of F344/DuCrj rats is improved as a result of enhanced insulin release induced by high levels of plasma GLP-1. In this study, we fed F344/DuCrj rats and DPPIV-positive F344/Jcl rats, aged five weeks, on a high-fat (HF) diet to examine the effect of DPPIV deficiency on food intake and insulin resistance. F344/Jcl rats gained significantly more body weight and consumed significantly more food than F344/DuCrj rats from Week 4 on either control or HF diet. Glucose excursion in the oral glucose tolerance test (OGTT) was improved in F344/DuCrj rats fed on the control or HF diet at all times examined, compared with F344/Jcl rats. Homeostasis model assessment (HOMA) insulin resistance values of F344/DuCrj and F344/Jcl rats fed on HF diet were higher than those of animals fed on control diet up to Week 6. However, HOMA insulin resistance values of F344/DuCrj rats fed on HF diet became significantly lower than those of F344/Jcl rats on HF diet during Weeks 8-10. The area under the insulin curve in the OGTT at Week 10 showed that the insulin resistance of HF-diet-fed F344/DuCrj rats was greatly ameliorated. Plasma active GLP-1 concentrations of F344/DuCrj rats in the fed state were significantly higher than those of F344/Jcl rats. These observations suggest that DPPIV deficiency results in improved glucose tolerance and ameliorated insulin resistance owing to enhanced insulin release and inhibition of food intake as a result of high active GLP-1 levels.

7-But-2-ynyl-9-(6-methoxy-pyridin-3-yl)-6-piperazin-1-yl-7,9-dihydro-purin-8-one Is a Novel Competitive and Selective Inhibitor of Dipeptidyl Peptidase IV with an Antihyperglycemic Activity

7-But-2-ynyl-9-(6-methoxy-pyridin-3-yl)-6-piperazin-1-yl-7,9-dihydro-purin-8-one (ER-319711) is a novel dipeptidyl peptidase (DPP)-IV inhibitor discovered in our laboratories. In this study, we have characterized this DPP-IV inhibitor in vitro and in vivo as an antidiabetic agent. The trifluoroacetate salt form of ER-319711, ER-319711-15, inhibited human DPP-IV with an IC50 value of 0.089 μM, whereas its IC50 values toward human DPP8 and DPP9 were >100 μM. Inhibition kinetic pattern analysis indicated that ER-319711-15 inhibited DPP-IV in a competitive manner. ER-319711-15 (1 mg/kg) reduced glucose excursion in an oral glucose tolerance test (OGTT) using Zucker fa/fa rats, with significant increases in plasma insulin and active glucagon-like peptide-1 levels. In an OGTT using mice fed a high-fat diet in which ER-319711-15 (0.1–10 mg/kg) was orally administered at 0 h, and glucose was loaded at 0 and 5 h, this compound improved glucose tolerance dose dependently at both 0- and 5-h glucose loading. Next, we compared efficacy of ER-319711-15, E3024, a competitive DPP-IV inhibitor having an imidazopyridazinone structure, or vildagliptin, a slow-binding and long-acting DPP-IV inhibitor, at the same dose, 10 mg/kg, in the same procedures. At the first glucose challenge, all compounds lowered area under the curve (AUC) values of delta blood glucose between 0 and 2 h significantly to the same degree. At the second glucose load, the AUC values between 5 and 7 h were significantly decreased by ER-319711-15 and vildagliptin, but not by E3024. Therefore, ER-319711 might be a potent, competitive, and selective DPP-IV inhibitor with an antihyperglycemic activity.

Vasorelaxant effect of a phosphodiesterase 3 inhibitor, olprinone, on isolated human radial artery

The radial artery is currently used as a viable arterial conduit for myocardial revascularization. The aims of this study were to identify phosphodiesterase 3 isoenzyme in the human radial artery isolated for coronary artery bypass grafting, and to examine the vasorelaxant effect of a cardiotonic and vasodilating phosphodiesterase 3 inhibitor, 1, 2-dihydro-6-methyl-2-oxo-5-(imidazo[1,2-a]pyridin-6-yl)-3-pyridine carbonitrile hydrochloride monohydrate (olprinone). The phosphodiesterase 3 isoenzyme was separated from the radial artery by DEAE-Sepharose chromatography. Olprinone inhibited the phosphodiesterase 3 activity with an IC(50) value of 1.25 microM. Olprinone relaxed the phenylephrine-induced contractions of endothelium-denuded arterial strips with an EC(50) value of 0. 107+/-0.029 microM (n=5). These findings indicate that the human radial artery possesses phosphodiesterase 3 isoenzyme activity and olprinone causes potent relaxation of the arterial strip in vitro through inhibition of phosphodiesterase 3 isozyme activity.

Effects of the Combination of a Dipeptidyl Peptidase IV Inhibitor and an Insulin Secretagogue on Glucose and Insulin Levels in Mice and Rats

Several combination therapies have been tried for treating of type 2 diabetes to control more effectively fasting hyperglycemia and postprandial hyperglycemia. In this study, we have examined the effects of combining a novel, selective, and competitive dipeptidyl peptidase IV (DPP-IV) inhibitor, 3-but-2-ynyl-5-methyl-2-piperazin-1-yl-3,5-dihydro-4H-imidazo[4,5-d]pyridazin-4-one tosylate (E3024), with a representative of one of two types of insulin secretagogues, i.e., either glybenclamide (a sulfonylurea) or nateglinide (a rapid-onset/short-duration insulin secretagogue), on glucose and insulin levels in an oral glucose tolerance test (OGTT) using mice fed a high-fat diet. In addition, we have investigated the effects of these combinations on blood glucose levels in fasting rats. Two-way analysis of variance showed that the combination of E3024 and glybenclamide improved glucose tolerance additively and also caused a synergistic increase in insulin levels in the OGTT in mice fed a high-fat diet. In a similar way, the combination of E3024 and nateglinide ameliorated glucose tolerance additively and raised insulin levels additively. In fasting rats, coadministration of E3024 with glybenclamide or nateglinide treatment did not affect the glucose-lowering effects of the insulin secretagogues. Therefore, a DPP-IV inhibitor in combination with glybenclamide or nateglinide may be a promising option for the treatment of type 2 diabetes, and particularly, for controlling postprandial hyperglycemia in the clinic.

Selective tissue factor/factor VIIa Inhibitor, ER-410660, and its prodrug, E5539, have anti-venous and anti-arterial thrombotic effects with a low risk of bleeding.

INTRODUCTION Many anticoagulant drugs target factors common to both the intrinsic and extrinsic coagulation pathways, which may lead to bleeding complications. Since the tissue factor (TF)/factor VIIa complex is associated with thrombosis onset and specifically activates the extrinsic coagulation pathway, compounds that inhibit this complex may provide therapeutic and/or prophylactic benefits with a decreased risk of bleeding. MATERIALS AND METHODS The in vitro enzyme profile and anticoagulation selectivity of the TF/VIIa complex inhibitor, ER-410660, and its prodrug E5539 were assessed using enzyme inhibitory and plasma clotting assays. In vivo effects of ER-410660 and E5539 were determined using a TF-induced, thrombin generation rhesus monkey model; a stasis-induced, venous thrombosis rat model; a photochemically induced, arterial thrombosis rat model; and a rat tail-cut bleeding model. RESULTS ER-410660 selectively prolonged prothrombin time, but had a less potent anticoagulant effect on the intrinsic pathway. It also exhibited a dose-dependent inhibitory effect on thrombin generation caused by TF-injection in the rhesus monkey model. ER-410660 also reduced venous thrombus weights in the TF-administered, stasis-induced, venous thrombosis rat model and prolonged the occlusion time induced by arterial thrombus formation after vascular injury. The compound was capable of doubling the total bleeding time in the rat tail-cut model, albeit with a considerably higher dose compared to the effective dose in the venous and arterial thrombosis models. Moreover, E5539, an orally available ER-410660 prodrug, reduced the thrombin-anti-thrombin complex levels, induced by TF-injection, in a dose-dependent manner. CONCLUSION Selective TF/VIIa inhibitors have potential as novel anticoagulants with a lower propensity for enhancing bleeding.