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.

Comparison of efficacies of a dipeptidyl peptidase IV inhibitor and alpha-glucosidase inhibitors in oral carbohydrate and meal tolerance tests and the effects of their combination in mice.

E3024 (3-but-2-ynyl-5-methyl-2-piperazin-1-yl-3,5-dihydro-4H-imidazo[4,5-d]pyridazin-4-one tosylate) is a dipeptidyl peptidase IV (DPP-IV) inhibitor. Since the target of both DPP-IV inhibitors and alpha-glucosidase inhibitors is the lowering of postprandial hyperglycemia, we compared antihyperglycemic effects for E3024 and alpha-glucosidase inhibitors in various oral carbohydrate and meal tolerance tests using normal mice. In addition, we investigated the combination effects of E3024 and voglibose on blood glucose levels in a meal tolerance test using mice fed a high-fat diet. ER-235516-15 (the trifluoroacetate salt form of E3024, 1 mg/kg) lowered glucose excursions consistently, regardless of the kind of carbohydrate loaded. However, the efficacy of acarbose (10 mg/kg) and of voglibose (0.1 mg/kg) varied with the type of carbohydrate administered. The combination of E3024 (3 mg/kg) and voglibose (0.3 mg/kg) improved glucose tolerance additively, with the highest plasma active glucagon-like peptide-1 levels. This study shows that compared to alpha-glucosidase inhibitors, DPP-IV inhibitors may have more consistent efficacy to reduce postprandial hyperglycemia, independent of the types of carbohydrate contained in a meal, and that the combination of a DPP-IV inhibitor and an alpha-glucosidase inhibitor is expected to be a promising option for lowering postprandial hyperglycemia.

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.

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 IC(50) value of 0.089 microM, whereas its IC(50) values toward human DPP8 and DPP9 were >100 microM. 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.

E3024, 3-but-2-ynyl-5-methyl-2-piperazin-1-yl-3,5-dihydro-4H-imidazo[4,5-d]pyridazin-4-one tosylate, is a novel, selective and competitive dipeptidyl peptidase-IV inhibitor.

Dipeptidyl peptidase IV (DPP-IV) inhibitors are expected to become a useful new class of anti-diabetic agent. The aim of the present study is to characterize the in vitro and in vivo profile of E3024, 3-but-2-ynyl-5-methyl-2-piperazin-1-yl-3,5-dihydro-4H-imidazo[4,5-d]pyridazin-4-one tosylate, which is a novel imidazopyridazinone-derived DPP-IV inhibitor. E3024 inhibited recombinant human and mouse DPP-IV with IC50 values of approximately 100 nM. E3024 inhibited DPP-IV in human, mouse, rat and canine plasma with IC50 values of 140 to 400 nM. In contrast, E3024 did not inhibit DPP-8 or DPP-9 activity. Kinetic analysis indicated that E3024 is a competitive DPP-IV inhibitor. In Zucker fa/fa rats, E3024 (1 mg/kg) reduced glucose excursion after glucose load, with increases in plasma insulin and active glucagon-like peptide-1 levels. In fasted rats, this compound did not cause hypoglycemia. In a rat 4-week toxicological study, no notable changes were found at doses up to 750 mg/kg. The present preclinical studies indicate that E3024 is a novel selective DPP-IV inhibitor with anti-diabetic effects and a good safety profile.

The combination of metformin and a dipeptidyl peptidase IV inhibitor prevents 5-fluorouracil-induced reduction of small intestine weight.

Glucagon-like peptide 2 (GLP-2), which has intestinotrophic effects, is secreted from L-cells in the intestine in response to nutrient ingestion and is degraded by dipeptidyl peptidase IV (DPPIV). In this report, we show that biguanides promote GLP-2 release. Plasma GLP-2 levels were significantly increased by 1.4- to 1.6-fold in fasted F344 rats 1 h after oral meformin (300 mg/kg), phenformin (30 and 100 mg/kg) and buformin (100 mg/kg) treatment. In addition, metformin administration (300 mg/kg, p.o.) significantly elevated plasma GLP-2 in fasted CD-1 mice by about 2.0-fold 1 and 3 h after the treatment. Metformin and/or valine-pyrrolidide, a DPPIV inhibitor, was orally given (300 and 30 mg/kg, respectively, p.o., b.i.d., 3 days) to BALB/c mice treated with 5-fluorouracil (5-FU; 60 mg/kg, s.i.d.), which induces gastrointestinal damage leading to a reduction of small intestine wet weight. Metformin and valine-pyrrolidide co-administration prevented the 5-FU-induced reduction of wet weight of the small intestine, whereas metformin or valine-pyrrolidide alone had no effect. These results suggest that GLP-2 is co-secreted with GLP-1 flollowing biguanide stimulation, and that the combination of metformin with a DPPIV inhibitor might a useful oral treatment for gastrointestinal damage, based on GLP-2 actions.

Metformin causes reduction of food intake and body weight gain and improvement of glucose intolerance in combination with dipeptidyl peptidase IV inhibitor in Zucker fa/fa rats.

An incretin hormone, glucagon-like peptide-1 (GLP-1), has been shown to lower plasma glucose via glucose-dependent insulin secretion and to reduce appetite. We previously found that the biguanide metformin, an antidiabetic agent, causes a significant increase of plasma active GLP-1 level in the presence of dipeptidyl peptidase IV (DPPIV) inhibitor in normal rats. This finding suggested that the combination treatment might produce a greater antidiabetic and anorectic effect, based on enhanced GLP-1 action. In this study, we assessed the effects of subchronic treatment with metformin and a DPPIV inhibitor, valine-pyrrolidide (val-pyr), on glycemic control, food intake, and weight gain using Zucker fa/fa rats, a model of obesity and impaired glucose tolerance. The combination treatment caused a significant increase of GLP-1 level in Zucker fa/fa rats. In a subchronic study, val-pyr, metformin, or both compounds were administered orally b.i.d. for 14 days. The combination treatment significantly decreased food intake and body weight gain, although neither metformin nor val-pyr treatment alone had any effect. In an oral glucose tolerance test on day 1, the coadministration caused a greater improvement of glucose tolerance and a prominent increase of plasma active GLP-1 without marked insulin secretion. The 14-day combination treatment produced a potent reduction of fasting blood glucose and plasma insulin levels. These results demonstrate that the combination therapy of metformin with DPPIV inhibitor leads to reduced food intake and body weight gain, most likely through the significant increase of plasma GLP-1 level. The combination therapy seems to be a good candidate for treatment of type 2 diabetes with obesity.

Enteroinsular axis of db/db mice and efficacy of dipeptidyl peptidase IV inhibition.

In type 2 diabetic patients, the administration of glucagon-like peptide-1 (GLP-1), known as an incretin, exerts antidiabetic effects. However, GLP-1 is rapidly degraded by dipeptidyl peptidase IV (DPPIV) after its release. DPPIV inhibition is thought to be a rational strategy to treat type 2 diabetes. In this study, using C57BLKS/J-db/db (db/db) mice as a model of type 2 diabetes, we examined the effect of acute DPPIV inhibition on glucose tolerance at the early and later stages of diabetes, determining plasma active GLP-1 and insulin levels. In addition, we investigated changes of plasma DPPIV activity. Compared with normal C57BL6/J (B6) and db/+ mice, significantly increased plasma DPPIV activities were observed in db/db mice. Expression of the proglucagon gene encoding GLP-1 was significantly upregulated in the colon of db/db mice. The administration of valine-pyrrolidide, a DPPIV inhibitor, resulted in potentiated insulin secretion mediated by increased endogenous GLP-1 action, leading to improved glucose tolerance in db/db mice at 6 weeks of age. However, although acute DPPIV inhibition with valine-pyrrolidide resulted in higher plasma active GLP-1 and insulin levels in db/db mice at 23 weeks of age, it did not improve glucose tolerance. The function of the enteroinsular axis is preserved in both stage of diabetes and the DPPIV inhibitor potentiated it, but the progression of insulin resistance appeared to block the improvement of glucose tolerance through DPPIV inhibition. Our results suggest that DPPIV inhibition is a suitable approach for treatment of impaired glucose tolerance (IGT), and type 2 diabetes in the early stage.

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.