Analysis of the effect of canagliflozin on renal glucose reabsorption and progression of hyperglycemia in zucker diabetic Fatty rats.

Sodium-glucose cotransporter 2 (SGLT2) plays a major role in renal glucose reabsorption. To analyze the potential of insulin-independent blood glucose control, the effects of the novel SGLT2 inhibitor canagliflozin on renal glucose reabsorption and the progression of hyperglycemia were analyzed in Zucker diabetic fatty (ZDF) rats. The transporter activity of recombinant human and rat SGLT2 was inhibited by canagliflozin, with 150- to 12,000-fold selectivity over other glucose transporters. Moreover, in vivo treatment with canagliflozin induced glucosuria in mice, rats, and dogs in a dose-dependent manner. It inhibited apparent glucose reabsorption by 55% in normoglycemic rats and by 94% in hyperglycemic rats. The inhibition of glucose reabsorption markedly reduced hyperglycemia in ZDF rats but did not induce hypoglycemia in normoglycemic animals. The change in urinary glucose excretion should not be used as a marker to predict the glycemic effects of this SGLT2 inhibitor. In ZDF rats, plasma glucose and HbA1c levels progressively increased with age, and pancreatic ß-cell failure developed at 13 weeks of age. Treatment with canagliflozin for 8 weeks from the prediabetic stage suppressed the progression of hyperglycemia, prevented the decrease in plasma insulin levels, increased pancreatic insulin contents, and minimized the deterioration of islet structure. These results indicate that selective inhibition of SGLT2 with canagliflozin controls the progression of hyperglycemia by inhibiting renal glucose reabsorption in ZDF rats. In addition, the preservation of ß-cell function suggests that canagliflozin treatment reduces glucose toxicity via an insulin-independent mechanism.

Novel Indole-N-glucoside, TA-1887 As a Sodium Glucose Cotransporter 2 Inhibitor for Treatment of Type 2 Diabetes.

Inhibition of the renal sodium glucose cotransporter (SGLT) increases urinary glucose excretion (UGE) and thus reduces blood glucose levels during hyperglycemia. To explore the potential of new antihyperglycemic agents, we synthesized and determined the human SGLT2 (hSGLT2) inhibitory potential of novel substituted 3-benzylindole-N-glucosides 6. Optimization of 6 resulted in the discovery of 3-(4-cyclopropylbenzyl)-4-fluoroindole-N-glucoside 6a-4 (TA-1887), a highly potent and selective hSGLT2 inhibitor, with pronounced antihyperglycemic effects in high-fat diet-fed KK (HF-KK) mice. Our results suggest the potential of indole-N-glucosides as novel antihyperglycemic agents through inhibition of renal SGLT2.

C-Glucosides with heteroaryl thiophene as novel sodium-dependent glucose cotransporter 2 inhibitors.

Canagliflozin (1), a novel inhibitor for sodium-dependent glucose cotransporter 2 (SGLT2), has been developed for the treatment of type 2 diabetes. To investigate the effect of replacement of the phenyl ring in 1 with heteroaromatics, C-glucosides 2 were designed, synthesized, and evaluated for their inhibitory activities against SGLT2. Of these, 3-pyridyl, 2-pyrimidyl or 5-membered heteroaryl substituted derivatives showed highly potent inhibitory activity against SGLT2, while 5-pyrimidyl substitution was associated with slightly reduced activity. In particular, 2g (TA-3404) had remarkable anti-hyperglycemic effects in high-fat diet fed KK (HF-KK) mice.

Automated recognition and quantification of pancreatic islets in Zucker diabetic fatty rats treated with exendin-4.

Type 2 diabetes is characterized by impaired insulin secretion from pancreatic ß-cells. Quantification of the islet area in addition to the insulin-positive area is important for detailed understanding of pancreatic islet histopathology. Here we show computerized automatic recognition of the islets of Langerhans as a novel high-throughput method to quantify islet histopathology. We utilized state-of-the-art tissue pattern recognition software to enable automatic recognition of islets, eliminating the need to laboriously trace islet borders by hand. After training by a histologist, the software successfully recognized even irregularly shaped islets with depleted insulin immunostaining, which were quite difficult to automatically recognize. The results from automated image analysis were highly correlated with those from manual image analysis. To establish whether this automated, rapid, and objective determination of islet area will facilitate studies of islet histopathology, we showed the beneficial effect of chronic exendin-4, a glucagon-like peptide-1 analog, treatment on islet histopathology in Zucker diabetic fatty (ZDF) rats. Automated image analysis provided qualitative and quantitative evidence that exendin-4 treatment ameliorated the loss of pancreatic insulin content and gave rise to islet hypertrophy. We also showed that glucagon-positive α-cell area was decreased significantly in ZDF rat islets with disorganized structure. This study is the first to demonstrate the utility of automatic quantification of digital images to study pancreatic islet histopathology. The proposed method will facilitate evaluations in preclinical drug efficacy studies as well as elucidation of the pathophysiology of diabetes.

Discovery of canagliflozin, a novel C-glucoside with thiophene ring, as sodium-dependent glucose cotransporter 2 inhibitor for the treatment of type 2 diabetes mellitus.

We discovered that C-glucosides 4 bearing a heteroaromatic ring formed metabolically more stable inhibitors for sodium-dependent glucose cotransporter 2 (SGLT2) than the O-glucoside, 2 (T-1095). A novel thiophene derivative 4b-3 (canagliflozin) was a highly potent and selective SGLT2 inhibitor and showed pronounced anti-hyperglycemic effects in high-fat diet fed KK (HF-KK) mice.

Xanthine oxidase inhibition improves left ventricular dysfunction in dilated cardiomyopathic hamsters.

BACKGROUND: Oxidative stress is implicated in cardiac remodeling and failure. We tested whether xanthine oxidase (XO) inhibition could decrease myocardial oxidative stress and attenuate left ventricular (LV) remodeling and dysfunction in the TO-2 hamster model of dilated cardiomyopathy. METHODS AND RESULTS: TO-2 hamsters were randomized to treatment with the XO inhibitor, allopurinol, or vehicle from 6 to 12 weeks of age. F1B hamsters served as controls. TO-2 hamsters treated with vehicle progressively developed severe LV systolic dysfunction and dilation between 6 and 12 weeks. Marked cardiac fibrosis was apparent in these hamsters at 12 weeks in comparison with F1B controls. The ratio of reduced to oxidized glutathione (GSH/GSSG) was decreased and malondialdehyde levels were increased in the hearts of vehicle-treated TO-2 hamsters. Treatment with allopurinol from 6 to 12 weeks attenuated LV dysfunction and dilation as well as myocardial fibrosis and the upregulation of a fetal-type cardiac gene. Allopurinol also inhibited both the decrease in GSH/GSSG ratio and the increase in malondialdehyde levels in the heart. CONCLUSIONS: These results indicate that chronic XO inhibition with allopurinol attenuates LV remodeling and dysfunction as well as myocardial oxidative stress in this model of heart failure. Allopurinol may prove beneficial for the treatment of heart failure.

Differential cardiovascular effects of endotoxin derived from Escherichia coli or Pseudomonas aeruginosa.

Sepsis is characterized by various symptoms, signs and underlying pathophysiology. To investigate possible mechanisms underlying this diversity, we compared the cardiovascular effects of lipopolysaccharide (LPS) derived from Escherichia coli (E-LPS) with those of LPS from Pseudomonas aeruginosa (P-LPS) in rats. We also examined the possible roles of tumor necrosis factor-alpha (TNF-alpha) and oxidative stress in LPS-induced cardiovascular damage. E-LPS (10 mg/kg body weight) or P-LPS (2 mg/kg body weight) was administered intravenously to Wistar rats. Echocardiography was serially performed. E-LPS induced an increase in left ventricular fractional shortening that persisted for at least 6 h, whereas P-LPS elicited an initial increase and a subsequent decrease in this parameter. Histological analysis revealed that P-LPS induced interstitial edema, congestion, intramyocardial bleeding, myocardial necrosis, infiltration of inflammatory cells, and formation of fibrin thrombi in the heart, whereas no pathological changes were apparent in the hearts of rats treated with E-LPS. Furthermore, the plasma concentration of TNF-alpha in rats treated with P-LPS was greater than that in rats treated with E-LPS, but the glutathione redox ratio in the heart was not affected by either type of LPS. In conclusion, E-LPS and P-LPS induced distinct patterns of functional and structural responses in the cardiovascular systems of rats. These differential responses may be attributable in part to the difference in the associated increases in the plasma concentration of TNF-alpha. The cardiovascular effects of LPS thus depend on the causative organisms.

Attenuation of ventricular hypertrophy and fibrosis in rats by pitavastatin: potential role of the RhoA-extracellular signal-regulated kinase-serum response factor signalling pathway.

1. Inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A reductase (statins) manifest pleiotropic effects that may contribute to their therapeutic efficacy. However, the mechanism of the beneficial action of statins on cardiac hypertrophy and fibrosis remains unclear. We have now investigated this action of pitavastatin in Dahl salt-sensitive (DS) rats. 2. The DS rats progressively develop marked hypertension when fed a diet containing 8% NaCl from 7 weeks of age. These animals exhibited pronounced cardiac hypertrophy and fibrosis, as well as upregulation of fetal-type cardiac gene expression at 12 weeks of age, compared with DS rats fed a diet containing 0.3% NaCl. The abundance of mRNAs for collagen types I and III, angiotensin-converting enzyme, transforming growth factor-beta1 and connective tissue growth factor was also increased in the heart of rats on the high-salt diet. 3. Treatment of rats on the high-salt diet with a non-antihypertensive dose of pitavastatin (0.3 or 1 mg/kg per day) from 7 to 12 weeks of age attenuated the development of cardiac hypertrophy and fibrosis, as well as inhibiting the upregulation of cardiac gene expression. Pitavastatin also blocked the translocation of RhoA to the membrane fraction of the left ventricle and RhoA activation, as well as the phosphorylation of the mitogen-activated protein kinases extracellular signal-regulated kinase (ERK)-1 and ERK-2 and an increase in the DNA binding activity of serum response factor (SRF) in the heart induced by the high-salt diet. 4. These findings suggest that the effects of pitavastatin on load-induced cardiac hypertrophy and fibrosis are independent of its cholesterol-lowering action and may be mediated, at least in part, through inhibition of RhoA-ERK-SRF signalling.

Pitavastatin improves cardiac function and survival in association with suppression of the myocardial endothelin system in a rat model of hypertensive heart failure.

Statin therapy may be associated with lower mortality in patients with heart failure, but the underlying mechanism of such an association is unknown. We have evaluated the effects of pitavastatin on cardiac function and survival in a rat model of hypertensive heart failure and investigated the molecular mechanism of the observed effects. Dahl salt-sensitive rats fed with high-salt diet from 7 weeks of age developed compensatory left ventricular hypertrophy at 12 weeks and heart failure at 19 weeks. Dahl salt-sensitive rats were treated with either vehicle or pitavastatin (0.3 mg/kg per day) from 7 or 12 weeks. Both early-onset and late-onset pitavastatin treatment reduced left ventricular fibrosis, improved cardiac function, and increased the survival rate apparent at 19 weeks. The increases in the expression levels of hypertrophic, profibrotic, and metalloproteinase genes as well as in gelatinase activities in the heart induced by the high-salt diet were suppressed by pitavastatin treatment. Furthermore, the level of cardiac endothelin-1 was increased in association with the development of heart failure in a manner sensitive to treatment with pitavastatin. Both early and late pitavastatin treatment thus improved cardiac function and survival, with modulation of extracellular matrix remodeling and endothelin-1 signaling possibly contributing to these beneficial effects.

Mineralocorticoid receptor antagonism attenuates cardiac hypertrophy and failure in low-aldosterone hypertensive rats.

Chronic elevation of plasma aldosterone contributes to heart failure. Mineralocorticoid receptor (MR) antagonism is cardioprotective in such a setting, but whether such protection occurs in the presence of low-aldosterone concentrations remains unclear. We investigated whether MR blockade attenuates cardiac hypertrophy and failure in rats with salt-sensitive hypertension. Dahl salt-sensitive (DS) rats fed a high-salt diet from 7 weeks develop concentric left ventricular (LV) hypertrophy secondary to hypertension at 12 weeks followed by heart failure at 19 weeks (DS-CHF). DS rats on such a diet were treated with a non-antihypertensive dose of the selective MR antagonist eplerenone from 12 to 19 weeks. Renin activity and aldosterone concentration in plasma were decreased in DS-CHF rats compared with controls. LV hypertrophy and fibrosis, as well as macrophage infiltration around coronary vessels, were apparent in DS-CHF rats. The amounts of mRNAs for 11beta-hydroxysteroid dehydrogenase type 1, MR, monocyte chemoattractant protein 1, and osteopontin were increased in these hearts. Treatment of DS-CHF rats with eplerenone inhibited these changes in gene expression, as well as coronary vascular inflammation and heart failure. Eplerenone attenuated both the decrease in the ratio of reduced to oxidized glutathione and the increase in NADPH oxidase activity apparent in DS-CHF rat hearts. MR blockade with eplerenone thus resulted in attenuation of LV hypertrophy and failure, without an antihypertensive effect, in rats with low-aldosterone hypertension. The beneficial cardiac effects of eplerenone are likely attributable, at least in part, to attenuation of myocardial oxidative stress and coronary vascular inflammation induced by glucocorticoid-activated MRs.