Free fatty acid receptor 1 agonist, MR1704, lowers blood glucose levels in rats unresponsive to the sulfonylurea, glibenclamide.

Preclinical Research & Development MR1704 is a selective G protein-coupled receptor 40/free fatty acid receptor 1 agonist, which exhibited favorable pharmacokinetic profiles and glucose-lowering effects in animal models. We studied the effects of MR1704 in a sulfonylurea-desensitized Sprague-Dawley rat model and evaluated the risk of pancreatic ß-cell exhaustion compared to that of glibenclamide in Zucker fatty rats. Rats fed ad libitum a diet containing 0.03% glibenclamide exhibited lower non-fasting blood glucose levels compared to those in rats fed a control diet during the first 6 days. However, the response to glibenclamide disappeared on day 9. In a rat oral glucose tolerance test (OGTT), MR1704 reduced the plasma glucose excursion, whereas glibenclamide did not show this effect. In Zucker fatty rats, oral administration of MR1704 reduced glucose excursion during the OGTT, and the effects of MR1704 were maintained after 2-week treatment. In contrast, the glucose-lowering effects of glibenclamide were diminished, and glucose tolerance was aggravated after 2-week treatment. These results indicated that MR1704 provided more sustainable effects compared to those of the sulfonylurea, glibenclamide suggesting that MR1704 may be an attractive therapeutic option for diabetic patients who are unresponsive to sulfonylurea treatment.

A novel free fatty acid receptor 1 (GPR40/FFAR1) agonist, MR1704, enhances glucose-dependent insulin secretion and improves glucose homeostasis in rats.

Activation of G protein-coupled receptor 40/Free fatty acid receptor 1 (GPR40/FFAR1), which is highly expressed in pancreatic ß cells, is considered an important pharmacologic target for the treatment of type 2 diabetes mellitus. The aim of this study was to determine the effect of MR1704, a novel GPR40/FFAR1 agonist, on glucose homeostasis in rats. MR1704 is a highly potent and selective, orally bioavailable agonist with similar in vitro potencies among humans, mice, and rats. Treatment of rat islets with MR1704 increased glucose-dependent insulin secretion. Augmentation of glucose-dependent insulin secretion was abolished by adding a GPR40/FFAR1 antagonist. In mouse, insulinoma MIN6 cells, palmitic acid induced the activity of caspase 3/7 after a 72-h exposure, while pharmacologically active concentrations of MR1704 did not. In an oral glucose tolerance test in normal Sprague-Dawley rats, orally administered MR1704 (1-10 mg·kg-1 ) reduced plasma glucose excursion and enhanced insulin secretion, but MR1704 did not induce hypoglycemia, even at 300 mg·kg-1 , in fasted Sprague-Dawley rats. In addition, orally administered MR1704 reduced plasma glucose excursion and enhanced insulin secretion in diabetic Goto-Kakizaki rats. Oral administration of MR1704 once daily to Goto-Kakizaki rats reduced their blood glucose levels during a 5-week treatment period without reducing pancreatic insulin content; as a result, hemoglobin A1C levels significantly decreased. These results suggest that MR1704 improves glucose homeostasis through glucose-dependent insulin secretion with a low risk of hypoglycemia and pancreatic toxicity. MR1704 shows promise as a new, glucose-lowering drug to treat type 2 diabetes mellitus.

Discovery of novel aminobenzisoxazole derivatives as orally available factor IXa inhibitors.

Using structure based drug design, novel aminobenzisoxazoles as coagulation factor IXa inhibitors were designed and synthesized. Highly selective inhibition of FIXa over FXa was demonstrated. Anticoagulation profile of selected compounds was evaluated by aPTT and PT tests. In vitro ADMET and pharmacokinetic (PK) profiles were also evaluated.

Simultaneous targeting of CD14 and factor XIa by a fusion protein consisting of an anti-CD14 antibody and the modified second domain of bikunin improves survival in rabbit sepsis models.

Severe sepsis is a complex, multifactorial, and rapidly progressing disease characterized by excessive inflammation and coagulation following bacterial infection. To simultaneously suppress pro-inflammatory and pro-coagulant responses, we genetically engineered a novel fusion protein (MR1007) consisting of an anti-CD14 antibody and the modified second domain of bikunin, and evaluated the potential of MR1007 as an anti-sepsis agent. Suppressive effects of MR1007 on lipopolysaccharide (LPS)-induced inflammatory responses were assessed using peripheral blood mononuclear cells or endothelial cells. Its inhibitory activity against the coagulation factor XIa was assessed using a purified enzyme and a chromogenic substrate. Anticoagulant activity was assessed using human or rabbit plasma. Anti-inflammatory and anti-coagulant effects and/or survival benefits were evaluated in an endotoxemia model and a cecal ligation and puncture model. MR1007 inhibited LPS-induced cytokine production in peripheral blood mononuclear cells and endothelial cells, inhibited factor XIa, and exhibited anticoagulant activity. In an endotoxemia model, 0.3-3mg/kg MR1007 suppressed pro-inflammatory and pro-coagulant responses in a dose-dependent manner; at a dose of 3mg/kg, the protein improved survival even when administered 8h after the LPS injection. In addition, 10mg/kg MR1007 administered 2h post cecal ligation and puncture improved survival. However, MR1007 administered at doses up to 30mg/kg did not increase ear bleeding time or bacterial counts in the cecal ligation and puncture model. Thus, simultaneous targeting of CD14 and factor XIa improves survival in the rabbit endotoxemia and sepsis models and represents a promising approach for the treatment of severe sepsis.

Establishment of immunoassay for platelet-derived soluble glycoprotein VI, a novel platelet marker.

Soluble Glycoprotein VI (GPVI) is an attractive marker for disorders marked by platelet activation, such as thrombotic microangiopathy, myocardial infarction, and stroke. Several groups have already developed an immunoassay for soluble GPVI; however, there are several discrepancies between the groups' assays. In this study, we prepared the two types of recombinant soluble GPVI, the monomeric form GPVI (GPVI-His) and the dimeric form of GPVI (GPVI-Fc), moreover, we generated four anti-GPVI antibodies, F1232-7-1 (7S1), F1232-10-2 (10S2), F1232-19-1 (19D1), and F1232-21-1 (21D1). The former 2 antibodies (7S1 and 10S2) had a high affinity for both GPVI-His and GPVI-Fc, while the latter 2 antibodies (19D1 and 21D1) showed a high affinity for GPVI-Fc but low affinity for GPVI-His. All of the antibodies comparably recognized surface GPVI on resting platelets. Furthermore, we established two immunoassays for soluble GPVI, 7S1/10S2-HRP and 19D1/21D1-HRP (capture antibody/detection antibody). 7S1/10S2-HRP showed equivalent reactivity with GPVI-His and GPVI-Fc, whereas 19D1/21D1-HRP had high affinity for GPVI-Fc but low reactivity with GPVI-His. In terms of reactivity with platelet-derived soluble GPVI, 7S1/10S2-HRP demonstrated sensitive detection whereas 19D1/21D1-HRP was nonreactive. Taken together, 7S1/10S2-HRP is a suitable candidate for a reliable soluble GPVI immunoassay as it has a high affinity for monomeric GPVI.

Properties of soluble glycoprotein VI, a potential platelet activation biomarker.

Glycoprotein VI (GPVI) plays a critical role in the platelet response to collagen. Clinical studies suggest that the plasma level of soluble GPVI (sGPVI) is a highly specific and useful platelet activation marker. However, many properties of sGPVI have not been fully characterized, such as its sensitivity in detecting platelet activation and its elimination rate from the blood. In this study we established a sandwich enzyme-linked immunosorbent assay for human sGPVI, which cross-reacts to cynomolgus monkey sGPVI, and evaluated the time course of sGPVI production in a cynomolgus monkey model of lipopolysaccharide (LPS)-induced thrombocytopenia. The sGPVI levels in this model were dramatically elevated and returned to baseline by 24 hours after LPS injection, the change was more pronounced than the existing platelet activation biomarker, soluble P-selectin (sP-selectin) levels. The elimination half-life of recombinant human sGPVI was about 2.5 hours following intravenous administration to monkeys. These results suggest that plasma sGPVI closely reflects platelet activation in the bloodstream and has a short half-life. sGPVI would be a useful biomarker for disorders marked by platelet activation and for monitoring anti-platelet therapy.

Elevated plasma levels of soluble platelet glycoprotein VI (GPVI) in patients with thrombotic microangiopathy.

BACKGROUND: Thrombotic microangiopathy (TMA) is caused by various conditions, such as decreased a ADAMTS13 level, activated or injured vascular endothelial cells or activated platelets. This study examined the soluble platelet glycoprotein VI (sGPVI) levels in patients with TMA to evaluate the activation of platelets in thrombotic states. MATERIALS AND METHODS: The plasma levels of sGPVI, ADAMTS13 activity, von Willebrand factor (VWF) and VWF propeptide (VWFpp) were measured in patients with TMA. RESULTS: The plasma levels of sGPVI were significantly higher in postoperative patients, patients with TMA and those with disseminated intravascular coagulation (DIC) than in those without thrombosis. The plasma levels of sGPVI were the highest in patients with TMA without markedly reduced ADAMTS13 and those were significantly reduced after plasma exchange. CONCLUSION: The measurement of sGPVI level is therefore considered to be important for the diagnosis and evaluation of TMA.

A novel antiplatelet antibody therapy that induces cAMP-dependent endocytosis of the GPVI/Fc receptor gamma-chain complex.

Platelet adhesion to vascular subendothelium, mediated in part by interactions between collagen and glycoprotein VI (GPVI) complexed with Fc receptor gamma-chain, is crucial for thrombus formation. Antiplatelet therapy benefits patients with various thrombotic and ischemic diseases, but the safety and efficacy of existing treatments are limited. Recent data suggest GPVI as a promising target for a novel antiplatelet therapy, for example, GPVI-specific Abs that deplete GPVI from the surface of platelets. Here, we characterized GPVI-specific auto-Abs (YA-Abs) from the first reported patient with ongoing platelet GPVI deficiency caused by the YA-Abs. To obtain experimentally useful human GPVI-specific mAbs with characteristics similar to YA-Abs, we generated human GPVI-specific mouse mAbs and selected 2 representative mAbs, mF1201 and mF1232, whose binding to GPVI was inhibited by YA-Abs. In vitro, mF1201, but not mF1232, induced human platelet activation and GPVI shedding, and mF1232 inhibited collagen-induced human platelet aggregation. Administration of mF1201 and mF1232 to monkeys caused GPVI immunodepletion with and without both significant thrombocytopenia and GPVI shedding, respectively. When a human/mouse chimeric form of mF1232 (cF1232) was labeled with a fluorescent endocytosis probe and administered to monkeys, fluorescence increased in circulating platelets and surface GPVI was lost. Loss of platelet surface GPVI mediated by cF1232 was successfully reproduced in vitro in the presence of a cAMP-elevating agent. Thus, we have characterized cAMP-dependent endocytosis of GPVI mediated by a human GPVI-specific mAb as what we believe to be a novel antiplatelet therapy.

Toll-like receptor 4 signal transduction inhibitor, M62812, suppresses endothelial cell and leukocyte activation and prevents lethal septic shock in mice.

Sepsis occurs when microbes activate toll-like receptors (TLRs) stimulating widespread inflammation and activating coagulation cascades. TLR4 signal transduction has been recognized as a key pathway for lipopolysaccharide (LPS)-induced activation of various cells and an attractive target for treatment of sepsis. We found a new benzisothiazole derivative, M62812 that inhibits TLR4 signal transduction. This compound suppressed LPS-induced upregulation of inflammatory cytokines, adhesion molecules and procoagulant activity in human vascular endothelial cells and peripheral mononuclear cells. The half maximal inhibitory concentrations in these assays ranged from 1 to 3 microg/ml. Single intravenous administration of M62812 (10-20 mg/kg) protected mice from lethality and reduced inflammatory and coagulatory parameters in a murine d-galactosamine-sensitized endotoxin shock model. M62812 (20 mg/kg) also prevented mice from lethality in a murine cecal ligation and puncture model. These results suggest that inhibition of TLR4 signal transduction can suppress coagulation as well as inflammation during sepsis and may be clinically beneficial in sepsis treatment.

Synthesis and evaluation of 1-arylsulfonyl-3-piperazinone derivatives as factor Xa inhibitors(1-6) VI. A series of new derivatives containing N,S- and N,SO2-spiro acetal scaffolds.

In the course of development of factor Xa (FXa) inhibitors, we have found unique compounds containing an N,O- and an N,N-spiro acetal structure. It appeared that the difference in overall conformation due to the N,X-spiro acetal structure might be important for FXa inhibitory activity. Therefore, other N,X-spiro acetal structures, an N,S- and an N,SO2-spiro acetal, were developed as analogues of the N,X-spiro acetal structure. Compound 7b (N,S-spiro acetal structure) was found to have the strongest activity in these series of N,X-spiro acetal compounds, which had ever been synthesized.(4,5)).

Synthesis and evaluation of 1-arylsulfonyl-3-piperazinone derivatives as factor Xa inhibitors V. A series of new derivatives containing a spiro[imidazo[1,2-a]pyrazine-2(3H),4'-piperidin]-5(1H)-one scaffold.

We have already reported unique compounds containing a N,O-spiro acetal structure as an orally active factor Xa (FXa) inhibitor. This time, we described a N,N-spiro acetal structure as an analogue of the N,O-spiro acetal structure for an orally active FXa inhibitor. The synthesis of these analogues could be achieved in a similar fashion to the N,O-spiro acetal synthesis. Consequently, FXa inhibitory activity was increased and more active compounds could be found (M58163: IC50 = 0.61 nM, M58169: IC50 = 0.58 nM). Additionally, the absolute configuration could be determined by X-ray crystallography analysis (M58169: (R)-config.).

Pharmacological characterization of the active synthetic factor Xa inhibitors M55551 and M55165.

Factor Xa plays an important role in blood coagulation and is widely regarded as an attractive target for antithrombotic drug development. M55551 and M55165 (1-arylsulfonyl-3-piperazinone derivatives) are novel synthetic factor Xa inhibitors. In vitro, M55551 and M55165 competitively inhibited factor Xa with K(i) values of 3.2 nM and 2.3 nM, respectively, and prolonged clotting time in human and rat plasma. Pharmacokinetic analysis of these compounds revealed that M55551 was intravenously active with a short half-life (0.2 h) and that M55165 exhibited good bioavailability (31%) with a long half-life (3.9 h). Therefore, the antithrombotic effects of M55551 and M55165 were compared with those of the intravenous anticoagulant argatroban and the oral anticoagulant warfarin. Intravenous administration of M55551 and oral administration of M55165 inhibited thrombus formation at 0.3 mg/kg and 10 mg/kg, respectively, without significant prolongation of bleeding time. In contrast, although argatroban (0.3 mg/kg) and warfarin (1 mg/kg) also inhibited thrombus formation, significant prolongation of bleeding time was observed at dosages of 3 mg/kg and 1 mg/kg, respectively. These results suggest that M55551 and M55165 are potent factor Xa inhibitors that are active upon intravenous and oral administration, respectively, and that may prove clinically useful for the treatment of thrombosis while minimizing bleeding risks.

Synthesis and evaluation of 1-arylsulfonyl-3-piperazinone derivatives as factor Xa inhibitors IV. A series of new derivatives containing a spiro[5H-oxazolo[3,2-a]pyrazine-2(3H),4'-piperidin]-5-one skeleton.

In the course of development of factor Xa (FXa) inhibitor in an investigation involving the synthesis of 1-arylsulfonyl-3-piperazinone derivatives, we found new compounds containing a unique spiro skeleton. Among such compounds, (-)-7-[(6-chloro-2-naphthalenyl)sulfonyl]tetrahydro-8a-(methoxymethyl)-1'-(4-pyridinyl)-spiro[5H-oxazolo[3,2-a]pyrazine-2(3H),4'-piperidin]-5-one (28, M55529) had activity more favorable than those of previously reported compounds. The inhibitory activity of M55529 for FXa is IC(50)=2 nM, with high selectivity for FXa over thrombin and trypsin.

Synthesis and evaluation of 1-arylsulfonyl-3-piperazinone derivatives as factor Xa inhibitors III. Effect of ring opening of piperazinone moiety on inhibition.

Compounds containing an ethylenediamine structure in place of the piperazine ring of M55113 (1) and M55551 (2) were synthesized to investigate the effects of a piperazine moiety and evaluated for activity as factor Xa (FXa) inhibitors. Most such compounds, however, exhibited lower activity (1/10-1/100) than that of M55113 and M55551 as FXa inhibitors.

Synthesis and evaluation of 1-arylsulfonyl-3-piperazinone derivatives as a factor Xa inhibitor II. Substituent effect on biological activities.

Intravascular clot formation is an important event in a number of cardiovascular diseases. The prevention of blood coagulation has become a major target for new therapeutic agents. Factor Xa (FXa) is a trypsin-like serine protease that plays a key role in the blood coagulation cascade and represents an attractive target for anticoagulant drug development. We have investigated substituents in the central part of a lead compound (3: M55113), and discovered that compound M55551 (34: (R)-4-[(6-Chloro-2-naphthalenyl)sulfonyl]-6-oxo-1-[[1-(4-pyridinyl)-4-piperidinyl]methyl]-2-piperazinecarboxylic acid) is a potent inhibitor of FXa (IC(50)=0.006 microM), with high selectivity for FXa over trypsin and thrombin. The activity of this compound is ten times more powerful than the lead compound.