Design and synthesis of novel spirocyclic carboxylic acids as potent and orally bioavailable DGAT1 inhibitors and their biological evaluation.

A series of novel spirocyclic DGAT1 inhibitors containing the oxadiazole motif were designed and synthesized for biological evaluation. Several compounds exhibited potent diacylglycerol acyltransferase 1 (DGAT1) inhibitory activity. Optimization of the series led to the identification of five lead compounds 8, 9, 10, 11 and 12 that showed excellent in-vitro activity with IC50 values ranging from 7 to 20 nM against human DGAT1. All compounds demonstrated good druggability as well as microsomal stability and safety profiles such as hERG and CYP. Compound 12 significantly reduced plasma triglyceride levels in-vivo in the mouse model of acute lipid challenge. Significant reduction in plasma TG excursion was observed, thus indicating DGAT1 inhibition in-vivo.

Effect of parthenolide, an NLRP3 inflammasome inhibitor, on insulin resistance in high-fat diet-obese mice.

The activation of Nod-like receptor proteins (NLRP3) containing the pyrin domain inflammasome is a hallmark of the pathogenesis of metabolic disorders. Inhibition of the NLRP3 inflammasome by phytoconstituents has been attempted as a strategy to mitigate these disorders. Therefore, the present study aimed to evaluate the efficacy of an NLRP3 inflammasome inhibitor, parthenolide (PN; 5 mg/kg i.p.) against inflammation and insulin resistance in high-fat diet (HFD) - obese mice. Treatment with PN and pioglitazone (PIO; 30 mg/kg p.o.) attenuated lipopolysaccharide (LPS; 1 ng/ml) - induced elevation of tumor necrosis factor-α and interleukin-1ß in mouse peritoneal macrophages in a dose-dependent manner. Sixty days of PN and PIO treatment marginally reduced obesity-induced insulin resistance in HFD-obese mice. PN treatment also decreased blood glucose from 14th to 60th day, supporting the hypothesis of simultaneous attenuation of inflammation and insulin resistance in obese mice. Thus, PN treatment was also evident with significant improvement in glucose tolerance and peripheral insulin resistance validated through the respective tolerance tests. Therefore, the present study suggests that PN, an NLRP3 inflammasome inhibitor, could be a possible therapeutic agent for attenuating obesity-induced insulin resistance.

Discovery and pharmacological evaluation of indole derivatives as potent and selective RORγt inverse agonist for multiple autoimmune conditions.

Targeting nuclear receptor RORγ is recognized to be beneficial in multiple autoimmune disorders. We disclosed new indole analogues as potent RORγ inverse agonists. RO-2 as one of the potent and orally bioavailable compounds was evaluated in various models of autoimmune disorder. It showed potent suppression of downstream markers of RORγt activity in murine and human primary cells, ex vivo PD assay and in multiple animal models of autoimmune diseases. The results indicate the potential of these indole analogues as orally bioavailable small molecule inverse agonists of RORγt, efficacious in various Th17 driven models of autoimmune disorders.

Discovery and evaluation of 1H-pyrrolo[2,3-b]pyridine based selective and reversible small molecule BTK inhibitors for the treatment of rheumatoid arthritis.

In a pursuit to identify reversible and selective BTK inhibitors, two series based on 7H-pyrrolo[2,3-d]pyrimidine and 1H-pyrrolo[2,3-b]pyridine as the hinge binding core, have been identified. Structure activity relationship (SAR) exploration led to identification of two advanced lead molecules, 11 and 13, which demonstrated desired BTK inhibitory potency in different cellular assays, excellent selectivity in a panel of 50 diverse kinases, favorable in vivo PK properties in mice and anti-arthritic effect in a mouse model of CIA.

Discovery of a potent and selective small molecule hGPR91 antagonist.

GPR91, a 7TM G-Protein-Coupled Receptor, has been recently deorphanized with succinic acid as its endogenous ligand. Current literature indicates that GPR91 plays role in various pathophysiology including renal hypertension, autoimmune disease and retinal angiogenesis. Starting from a small molecule high-throughput screening hit 1 (hGPR91 IC(50): 0.8 µM)-originally synthesized in Merck for Bradykinin B(1) Receptor (BK(1)R) program, systematic structure-activity relationship study led us to discover potent and selective hGPR91 antagonists e.g. 2c, 4c, and 5 g (IC(50): 7-35 nM; >1000 fold selective against hGPR99, a closest related GPCR; >100 fold selective in Drug Matrix screening). This initial work also led to identification of two structurally distinct and orally bio-available lead compounds: 5g (%F: 26) and 7e (IC(50): 180 nM; >100 fold selective against hGPR99; %F: 87). A rat pharmacodynamic assay was developed to characterize the antagonists in vivo using succinate induced increase in blood pressure. Using two representative antagonists, 2c and 4c, the GPR91 target engagement was subsequently demonstrated using the designed pharmacodynamic assay.

First-in-Human, Healthy Volunteers Integrated Protocol of ETC-206, an Oral Mnk 1/2 Kinase Inhibitor Oncology Drug.

In the last decade, drug development has tackled substantial challenges to improve efficiency and facilitate access to innovative medicines. Integrated clinical protocols and the investigation of targeted oncology drugs in healthy volunteers (HVs) have emerged as modalities with an increase in scope and complexity of early clinical studies and first-in-human (FIH) studies in particular. However, limited work has been done to explore the impact of these two modalities, alone or in combination, on the scientific value and on the implementation of such articulated studies. We conducted an FIH study in HVs with an oncology targeted drug, an Mnk 1/2 small molecule inhibitor. In this article, we describe results, advantages, and limitations of an integrated clinical protocol with an oncology drug. We further discuss and indicate points to consider when designing and conducting similar scientifically and operationally demanding FIH studies.

Discovery of liver-directed glucokinase activator having anti-hyperglycemic effect without hypoglycemia.

Glucokinase activators (GKAs) are among the emerging drug candidates for the treatment of type 2 diabetes (T2D). Despite effective blood glucose lowering in clinical trials, many pan-GKAs "acting both in pancreas and liver" have been discontinued from clinical development mainly because of their potential to cause hypoglycemia. Pan-GKAs over sensitize pancreatic GK, resulting in insulin secretion even at sub-normoglycemic level which might be a possible explanation for hypoglycemia. An alternative approach to minimize the risk of hypoglycemia is to use liver-directed GKAs, which are reported to be advancing well in clinical development. Here, we report the discovery and structure-activity relationship (SAR) studies on a novel 2-phenoxy-acetamide series with the aim of identifying a liver-directed GKA. Incorporation of a carboxylic acid moiety as an active hepatocyte uptake recognizing element at appropriate position of 2-phenoxy-acetamide core led to the identification of 26, a potent GKA with predominant liver-directed pharmacokinetics in mice. Compound 26 on oral administration significantly reduced blood glucose levels during an oral glucose tolerance test (oGTT) performed in diet-induced obese (DIO) mice, while showing no sign of hypoglycemia in normal C57 mice over a 10-fold dose range, even when dosed at fasted condition. Together, these data demonstrate a liver-directed GKA has beneficial effect on glucose homeostasis with reduced risk of hypoglycemia.

Beneficial effects of fenoldopam treatment on renal function in streptozotocin-induced diabetic rats.

We have studied the effect of chronic treatment with dopamine D1 receptor agonist fenoldopam (1 mg/kg, i.p. daily for 6 weeks) on renal function and metabolic parameters in streptozotocin (STZ)-diabetic rats. Diabetes was induced by a single tail vein injection of STZ (45 mg/kg). STZ produced severe hyperglycemia, hypoinsulinemia, hypercholesterolemia, hypertriglyceridemia, hypertension and bradycardia. Fenoldopam treatment significantly reduced fasting but not fed blood glucose levels and lowered the blood pressure in diabetic animals. Significant change was not observed in insulin, cholesterol, triglyceride levels. Diabetic animals showed increase in AUCglucose and decrease in AUCinsulin during oral glucose tolerance test. Fenoldopam treatment did not significantly change these values in diabetic animals. STZ produced increase in serum urea, creatinine and blood urea nitrogen. Diuresis and urinary sodium retention was observed in diabetic animals. Renal hypertrophy was observed as seen from increased kidney weight/body weight ratio and increased total RNA content as well as decreased total DNA content. Fenoldopam treatment significantly lowered serum urea, creatinine and blood urea nitrogen. Urinary sodium retention was significantly reduced and renal hypertrophy was prevented with fenoldopam treatment as seen from the improved kidney weight/body weight ratio. Fenoldopam treatment significantly prevented reduction in total DNA content and increase in total RNA content further substantiating reduced renal hypertrophy. Our data suggest that STZ induced diabetes is associated with renal dysfunctions and fenoldopam treatment could be beneficial in a condition where diabetes mellitus co-exists with hypertension and compromised renal function.

Fenoldopam treatment improves peripheral insulin sensitivity and renal function in STZ-induced type 2 diabetic rats.

Dopamine and diabetes mellitus are reported to have close link between them. We have studied the effect of six-week treatment with D1 receptor agonist fenoldopam (1 mg/kg, i.p., daily) on glucose, lipid, and renal profile in streptozotocin (STZ)-induced (non-insulin dependent) type 2 diabetic rats. Streptozotocin (90 mg/kg, i.p.) was injected to two day old Sprague-Dawley pups. Streptozotocin produced hyperglycemia, hyperinsulinemia, hyperlipidemia, hypertension, increase in serum urea and creatinine by the time animals were 10 week old. Treatment with fenoldopam significantly decreased serum glucose, insulin, cholesterol, triglyceride, urea, creatinine, and blood pressure. During oral glucose tolerance test (OGTT), diabetic rats showed increase in AUC(glucose) and AUC(insulin). Fenoldopam significantly decreased AUC(glucose) in diabetic rats. Diabetic rats showed lower insulin sensitivity index (K(TTT)) that was significantly increased by treatment with fenoldopam in diabetic rats. Diabetic rats showed decrease in urinary sodium. Fenoldopam treatment significantly increased urine output as well as urinary sodium indicating reduced sodium retention. Our data indicates fenoldopam treatment improves peripheral insulin sensitivity and renal function in STZ-induced type 2 diabetic rats.

Increased renal angiotensin II AT1 receptor function in obese Zucker rat.

Angiotensin II (Ang II) via the activation of AT1 receptors and subsequent stimulation of the tubular sodium transporters increases sodium and water reabsorption in the proximal tubule. An enhanced tubular action of Ang II is implicated in obesity related hypertension; however, the mechanism of such a phenomenon is unknown. Present study was designed to determine the AT1 receptor numbers and function in the proximal tubule of obese and lean Zucker rats. Obese Zucker rats were hypertensive and hyperinsulinemic. The plasma renin activity was similar in the lean and obese rats. Angiotensin II stimulated the Na,H-exchanger (NHE) activity in the proximal tubule, but the stimulatory response was markedly greater in obese than in lean rats. Similarly, Ang II caused greater inhibition in cAMP accumulation in the proximal tubule of obese compared to lean rats. The (125I]sar-Ang II binding revealed a 100% increase in the AT1 receptor number in the brush border membrane (BBM) of obese compared to lean rats. The Western blot analysis revealed a 36-51% increase in the Gi(alpha)1 and Gi(alpha)3 in the BBM of obese compared to lean rats. We conclude that increases in the AT1 receptor number and abundance of the Gi(alpha) on BBM may be responsible for the enhanced signaling and subsequent greater stimulation of NHE by Ang II in proximal tubules of obese rats. The greater stimulation of NHE by Ang II may contribute to the increased tubular sodium reabsorption and to the hypertension in obese Zucker rats.

Effect of sarpogrelate on altered STZ-diabetes induced cardiovascular responses to 5-hydroxytryptamine in rats.

Sarpogrelate, a specific 5-HT2A receptor antagonist is reported to produce a number of beneficial cardiovascular effects in diabetes mellitus. In the present investigation we have studied the effects of sarpogrelate on 5-HT receptors in heart and platelets in streptozotocin (STZ)-diabetic rats. Diabetes was induced by a single tail vein injection of STZ (45 mg/kg) and sarpogrelate (1 mg/kg, i.p.) was administered daily for 6 weeks. Injection of STZ produced significant loss of body weight, polyphagia, polydypsia, hyperglycemia, hypoinsulinemia, hypertension and bradycardia. Treatment with sarpogrelate significantly lowered fasting glucose levels with corresponding increase in insulin levels. It also significantly prevented STZ-induced polydypsia, hyperphagia, hypertension, and bradycardia but not the loss of body weight. 5-HT produced dose-dependent positive inotropic effect that was found to be decreased significantly in STZ-diabetic rats. Hearts obtained from sarpogrelate treated diabetic rats did not show any decrease in responsiveness to 5-HT. Relative platelet aggregation per se was found to be higher in STZ-diabetic rats as compared to control and this was significantly prevented by sarpogrelate treatment. 5-HT produced a dose-dependent increase in platelet aggregation in non-diabetic and sarpogrelate treated diabetic rats. However, 5-HT failed to produce any increase in platelet aggregation in untreated diabetic rats. Our data suggest that STZ-induced diabetes may produce down-regulation of cardiac 5-HT2A receptors and increased platelet aggregation. Treatment with sarpogrelate seems to prevent STZ-induced down-regulation of 5-HT receptors and increase in platelet activity in diabetic rats.

Effect of chronic treatment with losartan on streptozotocin-induced renal dysfunction.

The present investigation was undertaken to study the effect of chronic treatment with angiotensin (AT1) receptor antagonist losartan (2 mg/kg, p.o., 6 weeks) on streptozotocin (STZ) induced (45 mg/kg, i.v., single dose) renal dysfunctions in diabetic rats. Injection of streptozotocin produced not only the cardinal symptoms of diabetes mellitus like loss of body weight, hyperglycemia, and hypoinsulinemia but also the renal dysfunctions. Losartan treatment significantly prevented all these changes except STZ-induced hypoinsulinemia. There was a significant elevation of blood pressure in diabetic rats and treatment with losartan significantly brought it back to normal. Renal dysfunction in diabetic rats was characterized by a significant decrease in creatinine clearance, elevated levels of electrolytes and renal hypertrophy. Treatment with losartan prevented these changes. A good correlation was found between biochemical parameters and histopathological abnormalities. Our data suggests that, losartan may be considered as the drug of choice when there is a co-existence of diabetes mellitus and hypertension with compromised kidney function.

Rosiglitazone treatment restores renal dopamine receptor function in obese Zucker rats.

Earlier we have reported a defective dopamine D1-like receptor function, which was accompanied by a decrease in D1 receptor numbers and the inability of dopamine to inhibit Na,K-ATPase and Na,H-exchanger in proximal tubules of hyperinsulinemic obese Zucker rats. The present study was designed to test the hypothesis that the defect in dopamine receptor function is a result of hyperinsulinemia in obese rats. We designed experiments to study D1 receptor function in obese Zucker rats treated with rosiglitazone, as it lowers plasma insulin by improving insulin sensitivity. A group of untreated lean and obese rats served as controls. Rosiglitazone treatment (10 mg/kg orally, 4 weeks) caused significant decreases in plasma insulin, blood glucose, and blood pressure while causing an increase in renal sodium excretion compared with untreated obese rats. In the isolated proximal tubules obtained from untreated lean rats, dopamine caused concentration-dependent inhibition of the Na,K-ATPase activity, but this inhibitory effect was absent in untreated obese rats. In rosiglitazone-treated obese rats, the inhibitory effect of dopamine on Na,K-ATPase was significantly restored. This was accompanied by a complete restoration of D1 receptor numbers in proximal tubular membranes of treated obese rats. In another set of experiments, treatment of primary proximal tubule epithelial cells in culture medium with insulin caused a significant decrease in the D1 receptor abundance, suggesting a direct role of insulin on D1 receptor regulation. We conclude that hyperinsulinemia causes downregulation of D1 receptor function and lowering of plasma insulin levels leads to restoration of renal D1 receptor function.