A novel ryanodine receptor 2 inhibitor, M201-A, enhances natriuresis, renal function and lusi-inotropic actions: Preclinical and phase I study.

BACKGROUND AND PURPOSE: The ryanodine receptor 2 (RyR2) is present in both the heart and kidneys, and plays a crucial role in maintaining intracellular Ca2+ homeostasis in cells in these organs. This study aimed to investigate the impact of M201-A on RyR2, as well as studying its effects on cardiac and renal functions in preclinical and clinical studies. EXPERIMENTAL APPROACH: Following the administration of M201-A (1,4-benzothiazepine-1-oxide derivative), we monitored diastolic Ca2+ leak via RyR2 and intracellular Ca2+ concentration in isolated rat cardiomyocytes and in cardiac and renal function in animals. In a clinical study, M201-A was administered intravenously at doses of 0.2 and 0.4 mg·kg-1 once daily for 20 min for four consecutive days in healthy males, with the assessment of haemodynamic responses. KEY RESULTS: In rat heart cells, M201-A effectively inhibited spontaneous diastolic Ca2+ leakage through RyR2 and exhibited positive lusi-inotropic effects on the rat heart. Additionally, it enhanced natriuresis and improved renal function in dogs. In human clinical studies, when administered intravenously, M201-A demonstrated an increase in natriuresis, glomerular filtration rate and creatinine clearance, while maintaining acceptable levels of drug safety and tolerability. CONCLUSIONS AND IMPLICATIONS: The novel drug M201-A inhibited diastolic Ca2+ leak via RyR2, improved cardiac lusi-inotropic effects in rats, and enhanced natriuresis and renal function in humans. These findings suggest that this drug may offer a potential new treatment option for chronic kidney disease and heart failure.

Contributions of hepatic gluconeogenesis suppression and compensative glycogenolysis on the glucose-lowering effect of CS-917, a fructose 1,6-bisphosphatase inhibitor, in non-obese type 2 diabetes Goto-Kakizaki rats.

Contributions of gluconeogenesis suppression in liver, kidney, and intestine as major gluconeogenic organs to the glucose-lowering effect of CS-917, a fructose 1,6-bisphosphatase inhibitor, was evaluated in overnight-fasted Goto-Kakizaki (GK) rats. CS-917 decreased plasma glucose by suppressing glucose release and lactate uptake from liver but not from kidney and intestine. These results suggest that hepatic gluconeogenesis suppression predominantly contributes to the glucose-lowering effect of CS-917 in GK rats. Moreover, the mechanism by which CS-917 decreased plasma glucose more in overnight-fasted GK rats than in non-fasted ones was investigated. Lactate uptake from liver was suppressed by 15 mg/kg of CS-917 in both states, but glucose release from liver and plasma glucose were decreased only in the overnight-fasted state. CS-917 at 30 mg/kg decreased hepatic glycogen content in both states and depleted it in the overnight-fasted state. In the non-fasted GK rats, co-administration of CS-917 with CP-91149, a glycogen phosphorylase inhibitor, suppressed hepatic glycogen reduction by CS-917 and decreased plasma glucose more than single administration of CS-917. These results suggest that gluconeogenesis suppression by CS-917 was counteracted by hepatic glycogenolysis especially in the non-fasted state and that combination therapy with CS-917 and CP-91149 is efficacious to decrease plasma glucose in GK rats.

Metformin primarily decreases plasma glucose not by gluconeogenesis suppression but by activating glucose utilization in a non-obese type 2 diabetes Goto-Kakizaki rats.

Metformin is an anti-diabetic agent that has been reported to decrease plasma glucose by multiple mechanisms, such as decreasing hepatic glucose production and activating peripheral glucose utilization. In order to elucidate the primary glucose-lowering mechanism of metformin, the present study focused on a comparison of the acute effect between metformin and CS-917 as a direct gluconeogenesis inhibitor. We examined the effect of metformin and CS-917 on glucose turnover in intravenous glucose-loaded Goto-Kakizaki (GK) rats, and on gluconeogenesis and glucose utilization in rat hepatocytes. Moreover, the glucose-lowering effect of metformin and CS-917 was compared in a fed and a fasted state in GK rats. In intravenous glucose-loaded GK rats, metformin and CS-917 lowered plasma glucose by increasing the glucose disappearance rate and by decreasing the glucose appearance rate, respectively. In rat hepatocytes, CS-917 but not metformin suppressed gluconeogenesis (IC(50)=0.136microM). Instead, metformin dose-dependently increased glucose uptake and the following lactate production at 30 to 100microM. Metformin decreased plasma glucose more in a fed state than in a fasted state in GK rats. CS-917, however, decreased plasma glucose more in a fasted state. These results confirm that metformin primarily decreases plasma glucose not by gluconeogenesis inhibition but by activating glucose utilization in GK rats. Moreover, metformin and CS-917 have different glucose-lowering effects depending on the nutrient state, which may be related to differences in their mechanisms of action. Such differences in action may have implications for metformin and CS-917 in the treatment of type 2 diabetes patients.

CS-917, a fructose 1,6-bisphosphatase inhibitor, improves postprandial hyperglycemia after meal loading in non-obese type 2 diabetic Goto-Kakizaki rats.

Postprandial hyperglycemia is one of the features of type 2 diabetes. Increased hepatic gluconeogenesis is a predominant cause of postprandial hyperglycemia in type 2 diabetes. In this study, we evaluated the effect of gluconeogenesis inhibition on postprandial hyperglycemia using CS-917, a novel inhibitor of fructose 1,6-bisphphosphatase (FBPase) which is one of the rate-limiting enzymes of gluconeogenesis. The suppressive effect of CS-917 on postprandial hyperglycemia was evaluated in a meal loading test in Goto-Kakizaki (GK) rats, non-obese type 2 diabetic animal model characterized by impaired insulin secretion. In addition, we describe acute effect of CS-917 on fasting hyperglycemia in overnight-fasted GK rats and chronic effect of CS-917 in multiple dosing GK rats.CS-917 suppressed plasma glucose elevation after meal loading in a dose-dependent manner at doses ranging from 10 to 40 mg/kg. In an overnight-fasted state, CS-917 decreased the plasma glucose levels dose-dependently at doses ranging from 2.5 to 40 mg/kg. Consistent with the inhibition of FBPase, glucose-lowering was associated with an accumulation of hepatic d-fructose 1,6-bisphosphate and a reduction in hepatic d-fructose 6-phosphate. Chronic treatment of CS-917 decreased plasma glucose significantly, and no significant increase in plasma lactate and no profound elevation in plasma triglycerides were observed by both acute and chronic treatment of CS-917 in GK rats.These findings suggest that enhanced gluconeogenesis contributes to hyperglycemia in postprandial conditions as well as in fasting conditions, and that CS-917 as an FBPase inhibitor corrects postprandial hyperglycemia as well as fasting hyperglycemia.

Identification of molecular target of AMP-activated protein kinase activator by affinity purification and mass spectrometry.

We show an efficient method to identify molecular targets of small molecular compounds by affinity purification and mass spectrometry. Binding proteins were isolated from target cell lysate using affinity columns, which immobilized the active and inactive compounds. All proteins bound to these affinity columns were eluted by digestion using trypsin and then were identified by mass spectrometry. The specific binding proteins to the active compound, a candidate for molecular targets, were determined by subtracting the identified proteins in an inactive compound-immobilized affinity column from that in an active compound-immobilized affinity column. This method was applied to identification of molecular targets of D942, a furancarboxylic acid derivative, which increases glucose uptake in L6 myocytes through AMP-activated protein kinase (AMPK) activation. To elucidate the mechanism of AMPK activation by D942, affinity columns that immobilized D942 and its inactive derivative, D768, were prepared, and the binding proteins were purified from L6 cell lysate. NAD(P)H dehydrogenase [quinone] 1 (complex I), which was shown as one of the specific binding proteins to D942 by subtracting the binding proteins to D768, was partially inhibited by D942, not D768. Because inhibition of complex I activity led to a decrease in the ATP/AMP ratio, and the change in the ATP/AMP ratio triggered AMPK activation, we identified complex I as a potential protein target of AMPK activation by D942. This result shows our approach can provide crucial information about the molecular targets of small molecular compounds, especially target proteins not yet identified.

Liver X receptor agonists inhibit tissue factor expression in macrophages.

Exposure of blood to tissue factor (TF) rapidly initiates the coagulation serine protease cascades. TF is expressed by macrophages and other types of cell within atherosclerotic lesions and plays an important role in thrombus formation after plaque rupture. Macrophage TF expression is induced by pro-inflammatory stimuli including lipopolysaccharide (LPS), interleukin-1beta and tumor necrosis factor-alpha. Here we demonstrate that activation of liver X receptors (LXRs) LXRalpha and LXRbeta suppresses TF expression. Treatment of mouse peritoneal macrophages with synthetic LXR agonist T0901317 or GW3965 reduced TF expression induced by pro-inflammatory stimuli. LXR agonists also suppressed TF expression and its activity in human monocytes. Human and mouse TF promoters contain binding sites for the transcription factors AP-1, NFkappaB, Egr-1 and Sp1, but no LXR-binding sites could be found. Cotransfection assays with LXR and TF promoter constructs in RAW 264.7 cells revealed that LXR agonists suppressed LPS-induced TF promoter activity. Analysis of TF promoter also showed that inhibition of TF promoter activity by LXR was at least in part through inhibition of the NFkappaB signaling pathway. In addition, in vivo, LXR agonists reduced TF expression within aortic lesions in an atherosclerosis mouse model as well as in kidney and lung in mice stimulated with LPS. These findings indicate that activation of LXR results in reduction of TF expression, which may influence atherothrombosis in patients with vascular disease.

Synthesis and biological activity of novel alpha-substituted beta-phenylpropionic acids having pyridin-2-ylphenyl moiety as antihyperglycemic agents.

We previously reported the identification of novel oximes having 5-benzyl-2,4-thiazolidinedione with antihyperglycemic activity. We now report the synthesis and biological activity of a novel series of oximes and amides having alpha-substituted-beta-phenylpropionic acids. In this series, we obtained potent PPAR alpha/gamma dual agonist (S)-9d, with which activation of PPAR alpha and PPAR gamma was considerably more potent than that of the reference compounds GW9578 22 and rosiglitazone 3, respectively. This means (S)-9d is of the strongest class of PPAR alpha/gamma dual agonists. In the course of this study, we also obtained 8h, which indicated potent plasma glucose lowering effect in spite of weak PPAR alpha/gamma agonistic activity.

High glucose potentiates palmitate-induced NO-mediated cytotoxicity through generation of superoxide in clonal beta-cell HIT-T15.

Prolonged exposure to free fatty acids induces beta-cell cytotoxicity. We investigated whether this fatty-acid-induced cytotoxicity is affected by high glucose levels. In clonal beta-cell HIT-T15, palmitate-induced cytotoxicity was potentiated depending on elevated glucose concentrations due to increased apoptosis without cytotoxic effects of high glucose per se. This palmitate cytotoxicity was blocked by NO synthase inhibitors, and palmitate actually increased cellular NO production. The potentiation of palmitate cytotoxicity under high glucose was reversed by decreasing superoxide production, suggesting that superoxide overproduction under high glucose enhances NO-mediated cytotoxicity in beta-cells, which may explain the mechanism of synergistic deterioration of pancreatic beta-cells by free fatty acids and high glucose.

Thiazolidinedione derivatives as novel therapeutic agents to prevent the development of chronic pancreatitis.

INTRODUCTION: Thiazolidinedione derivatives are known to be novel insulin-sensitizing agents and ligands of a nuclear hormone receptor peroxisome proliferator-activated receptor gamma (PPARgamma). Recently, ligands of PPARgamma have been shown to modulate proinflammatory cytokine production and NF-kappaB activation. AIM: To show that thiazolidinedione derivatives interfere with the development of chronic pancreatitis. METHODOLOGY: Rat chow containing 0.2% troglitazone was administered from 1 month to 7 months of age in WBN/Kob rats with spontaneous chronic pancreatitis. Morphologic evaluation of the pancreas was performed at 4 months and 7 months of age. Pancreas weight, protein, amylase, and insulin contents also were determined. Changes of cytokine levels were detected by enzyme-linked immunosorbent assay or semiquantitative reverse transcription-polymerase chain reaction. Localization and expression of PPARgamma in the pancreas and isolated peritoneal macrophages were examined by immunohistochemical study. RESULTS: Administration of troglitazone reduced the severity of morphologic pancreatic damage including inflammatory cell infiltration, and fibrosis markedly improved by the administration of troglitazone. Further, troglitazone was able to prevent the decrease in amylase content and pancreas atrophy that were observed in WBN/Kob rats. Serum IL-8 levels and TNF-alpha mRNA levels in the pancreas were significantly elevated in WBN/Kob rats, and these were dramatically attenuated by troglitazone. Peritoneal macrophages isolated from normal rats expressed PPARgamma at low levels, whereas those from WBN/Kob rat abundantly expressed PPARgamma. CONCLUSION: Troglitazone prevented the progression of pancreatic inflammatory process in an animal model of chronic pancreatitis. Macrophages may be one of the targets of the PPARgamma ligand to attenuate the severity of chronic pancreatitis, partially mediated by the inhibition of proinflammatory cytokine gene expression.

Angptl3 regulates lipid metabolism in mice.

The KK obese mouse is moderately obese and has abnormally high levels of plasma insulin (hyperinsulinemia), glucose (hyperglycemia) and lipids (hyperlipidemia). In one strain (KK/San), we observed abnormally low plasma lipid levels (hypolipidemia). This mutant phenotype is inherited recessively as a mendelian trait. Here we report the mapping of the hypolipidemia (hypl) locus to the middle of chromosome 4 and positional cloning of the autosomal recessive mutation responsible for the hypolipidemia. The hypl locus encodes a unique angiopoietin-like lipoprotein modulator, which we named Allm1. It is identical to angiopoietin-like protein 3, encoded by Angptl3, and has a highly conserved counterpart in humans. Overexpression of Angptl3 or intravenous injection of the purified protein in KK/San mice elicited an increase in circulating plasma lipid levels. This increase was also observed in C57BL/6J normal mice. Taken together, these data suggest that Angptl3 regulates lipid metabolism in animals.