Dipam Patel

Discovery of potent, selective and orally bioavailable triaryl-sulfonamide based PTP1B inhibitors

A novel series of pTyr mimetics containing triaryl-sulfonamide derivatives (5a-r) are reported as potent and selective PTP1B inhibitors. Some of the test compounds (5o and 5p) showed excellent selectivity towards PTP1B over various PTPs, including TCPTP (in vitro). The lead compound 5o showed potent antidiabetic activity (in vivo), along with improved pharmacokinetic profile. These preliminary results confirm discovery of highly potent and selective PTP1B inhibitors for the treatment of T2DM.

Discovery of Orally Active, Potent, and Selective Benzotriazole-Based PTP1B Inhibitors**

The worldwide incidence of metabolic syndromes such as obesity and diabetes are increasing at an alarming rate. Patients that suffer from obesity-induced type 2 diabetes (informally known as diabesity) are at increased risk of cardiovascular disease; their numbers pose a significant economic burden on health services. Type 2 diabetes mellitus (T2DM) is clinically characterized by increased blood glucose levels, either due to defects in insulin secretion, insulin resistance, or both. Current treatments for diabetic patients include various oral antihyperglycemic agents; however, over a period of time nearly half of T2DM sufferers lose their response to these agents and thereby require insulin therapy. Except incretin therapies, most of the available anti-hyperglycemic agents including insulin promote weight gain, which further aggravates obesity-associated cardiovascular risk and insulin resistance. Thus, there is an urgent need to develop novel agents for glycemic control that can complement existing therapies and prevent the progression of secondary complications associated with diabesity. In recent years, development of protein tyrosine phosphatase 1B (PTP1B) inhibitors has been considered as one of the best validated biological targets for the treatment of T2DM. PTP1B acts as a negative regulator in insulin signaling pathways; it dephosphorylates key tyrosine residues within the regulatory domain of the b-subunit of the insulin receptor. Thus, the inhibition of PTP1B activity has the potential for enhancing insulin action by prolonging the phosphorylated state of the insulin receptor. Gene knockout studies in animals have also demonstrated that PTP1B / mice show increased insulin sensitivity and are resistant to diet-induced obesity. Over the past two decades, several structurally diverse small-molecule-based PTP1B inhibitors have been developed, including Ertiprotafib, which was discontinued in phase II clinical trials owing to lack of efficacy and dose-dependent side effects. Most of the initial PTP1B inhibitors, such as phosphonates, carboxylic acids, and difluoromethylphosphonates (DFMPs), were designed to bind to the active site (site 1/A) by mimicking the phosphotyrosine (pTyr) substrate. However, achieving PTP1B selectivity over closely associated PTPs (PTPa, LAR, CD45, VHR, SHP-1, SHP-2, and T-cell protein tyrosine phosphatase (TCPTP)) is one of the major challenges, as most of the closely associated PTPs, particularly TCPTP, share a high degree of primary sequence identity (92%) in the active site (pTyr binding pocket). Lack of oral bioavailability is another important issue in the development of potent and selective PTP1B inhibitors, as the majority of the active-site-directed PTP1B inhibitors exhibit limited cell permeability due to the presence of negatively charged polar groups. To address this problem, Zhang and colleagues identified an additional noncatalytic aryl phosphate binding site (site 2/B) proximal to the catalytic phosphate binding site. Site B of PTP1B differs from that of TCPTP by a few amino acids (F52Y and A27S) and thus offers an opportunity to improve selectivity over TCPTP. Consequently, dual-site inhibitors were designed to bind across both sites A and B, to achieve additive effects and thereby improve potency and selectivity toward PTP1B over closely associated PTPs. Based on this dual binding site concept, various DFMP-based PTP1B inhibitors such as arylketone 1, benzotriazoles 2a and 2b, and naphthyl derivative 3 were developed (Figure 1). The X-ray crystal structure of PTP1B in complex with compound 2b reveals that sites A and B each have a DFMP moiety anchored into it. The benzotriazole ring system also functions as an anchor and is located under the YRD loop, thereby rigidly locking the molecule into the active site and providing good selectivity for PTP1B over other PTPs. The fourth substituent (benzene ring) occupies a hydrophobic pocket. Altogether, this signifies that the presence of all four substituents oriented rigidly by the molecule’s stereocenter is essential for high potency and selectivity. Although results of oral bioavailability and in vivo antidiabetic activity assays for compound 2a have yet to be published, in vitro results show improved PTP1B inhibitory activity (IC50= 5 nm) and moderate selectivity (sevenfold) over TCPTP (IC50= 36 nm). The X-ray crystal structure of PTP1B in complex with compound 2a illustrates that a methoxy group aligns very closely (3.7 ) to the side chain of F52 (site B). Oral administration of compounds 1 and 3 demonstrated good antidiabetic activity (compound 3 : ED50=0.8 mgkg , p.o.) and oral bioavailability (compounds 1 and 3: F=13 and 24%, respectively) in different animal species, despite their moderate in vitro PTP1B inhibitory activity (IC50=120 nm) and poor selectivity [a] D. Patel, Dr. M. Jain, Dr. R. Bahekar, P. Jadav, B. Darji, Y. Siriki, Dr. D. Bandyopadhyay, Dr. A. Joharapurkar, S. Kshirsagar, H. Patel, M. Shaikh, Dr. K. V. V. M. Sairam, P. Patel Department of Medicinal Chemistry, New Drug Discovery Division Zydus Research Centre, Sarkhej-Bavla N.H. 8A Moraiya, Ahmedabad 382210 (India) Fax: (+91)2717-665-355 E-mail : rajeshbahekar@zyduscadila.com [b] D. Patel, Prof. S. R. Shah Department of Chemistry, Faculty of Science M.S. University of Baroda, Vadodara 390002 (India) Fax: (+91)0265-79-3693 E-mail : Shailesh-chem@msubaroda.ac.in [**] ZRC communication No. 378 (part of PhD thesis work of D.P.) Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/cmdc.201100077.

Long-acting peptidomimetics based DPP-IV inhibitors

Pyrrolidine based peptidomimetics are reported as potent and selective DPP-IV inhibitors for the treatment of T2DM. Compounds 16c and 16d showed excellent in vitro potency and selectivity towards DPP-IV and the lead compound 16c showed sustained antihyperglycemic effects, along with improved pharmacokinetic profile.

Design, synthesis and biological evaluation of novel aminomethyl-piperidones based DPP-IV inhibitors

A series of novel aminomethyl-piperidones were designed and evaluated as potential DPP-IV inhibitors. Optimized analogue 12v ((4S,5S)-5-(aminomethyl)-1-(2-(benzo[d][1,3]dioxol-5-yl)ethyl)-4-(2,5-difluorophenyl)piperidin-2-one) showed excellent in vitro potency and selectivity for DPP-IV over other serine proteases. The lead compound 12v showed potent and long acting antihyperglycemic effects (in vivo), along with improved pharmacokinetic profile.

Central and Peripheral Glucagon Reduces Hyperlipidemia in Rats and Hamsters

Increased lipid levels in blood contribute to increasing the risk of diabetic complications. Glucagon exerts lipid lowering effects in diabetic state. However, the mechanism behind the lipid reduction by glucagon independent of glucose homeostasis is not well understood. We assessed the actions of glucagon on lipid modulation in blood and markers in liver in hyperlipidemic hamsters and rats. Male Sprague Dawley rats and Golden Syrian hamsters on a hyperlipidemic diet for 2 weeks were administered a single dose of glucagon by subcutaneous (SC, 150 and 300 µg/kg) or intracerebroventricular (ICV, 15 and 30 µg/animal) route. Effect of acute treatment was observed on tyloxapol-induced hypertriglyceridemia, corn oil-induced post-prandial lipemia, and bile flow. A repeated dose treatment by subcutaneous (300 µg/kg) or intracerebroventricular (30 µg/animal) route was done for 2 weeks, following which circulating and hepatic lipids, hepatic markers of lipid metabolism and bile flow were assessed. Acute administration of glucagon (SC and ICV) decreased triglyceride absorption, hepatic triglyceride secretion rate and increased excretion of cholesterol in bile fluid in dose related manner. Repeated dose treatment reduced circulating and hepatic lipids and mainly LDL, and enhanced cholesterol excretion in bile. In liver, expression of HMG-CoA reductase was reduced while that of ABCA1 was increased after repeated treatment, whereas pair fed group did not show significant changes when compared to the control group. These findings demonstrate that central as well as peripheral glucagon effectively reduces hyperlipidemia in rat and hamster model, by modulating hepatic lipid metabolism.

Balanced Coagonist of GLP-1 and Glucagon Receptors Corrects Dyslipidemia by Improving FGF21 Sensitivity in Hamster Model

Hyperlipidemia is often associated with obesity and diabetes, and can lead to serious complications like atherosclerosis and fatty liver disease. Coagonist of GLP-1 and glucagon receptors is a therapy under clinical investigation for treatment of obesity and diabetes. In this study, we have characterized the mechanism of hypolipidemic effect of a balanced coagonist using high cholesterol-fed hamsters. Tyloxapol-induced hypertriglyceridemia, lipolysis in adipose tissue, and bile homeostasis were assessed after repeated dose treatment of the coagonist of GLP-1 and glucagon receptors (Aib2 C24 chimera 2, SC). Antagonists of GLP-1, glucagon, and FGF21 receptors were coadministered, and FGF21 sensitivity was determined in liver and adipose tissue. Repeated dose treatment of coagonist reduced cholesterol and increased FGF21 in blood and liver. Coagonist treatment reduced hepatic triglyceride secretion, increased lipolysis and reduced body weight. Antagonism of GLP-1 and glucagon receptors partially blocked the effect of the coagonist on lipid metabolism in circulation and liver, while FGF21 receptor antagonist completely abolished it. Glucagon and GLP-1 receptors antagonists blocked the action of coagonist on cholesterol excretion and bile flow in liver, but FGF21 antagonist was not effective. Treatment with the coagonist increased expression of FGF21, FGF21R and cofactor ßKlotho in liver and adipose. In conclusion, coagonist of GLP-1 and glucagon receptors improved lipid metabolism in liver of dyslipidemic hamsters. This effect is partially mediated by GLP-1 and glucagon receptors, and the improved FGF21 sensitivity could be the mechanism of hypolipidemic action of the coagonist of GLP-1/glucagon receptors.

Discovery of liver selective non-steroidal glucocorticoid receptor antagonist as novel antidiabetic agents

Series of benzyl-phenoxybenzyl amino-phenyl acid derivatives (8a-q) are reported as non-steroidal GR antagonist. Compound 8g showed excellent h-GR binding and potent antagonistic activity (in vitro). The lead compound 8g exhibited significant oral antidiabetic and antihyperlipidemic effects (in vivo), along with liver selectivity. These preliminary results confirm discovery of potent and liver selective passive GR antagonist for the treatment of T2DM.

Coagonist of GLP-1 and glucagon receptors ameliorates non-alcoholic fatty liver disease

Nonalcoholic fatty liver disease (NAFLD) is often associated with obesity and type 2 diabetes. Coagonists of glucagon-like peptide-1 receptor (GLP-1R) and glucagon receptor (GCGR) are under clinical investigation for the treatment of obesity and type 2 diabetes. In this study, we have demonstrated the effect of a balanced coagonist in the treatment of NAFLD using mouse models. GLP-1R agonist exendin-4, glucagon, and coagonist (Aib2 C24 chimera2) were administered to C57BL6/J mice, in which NAFLD was induced by carbon tetrachloride (CCl4) treatment after high-fat diet (HFD) feeding, and choline-deficient, L-amino-acid-defined HFD (CDAHFD) feeding. Repeated dose administration of coagonist significantly attenuated liver inflammation and steatosis induced by acute and long-term treatment with CCl4 in HFD-fed mice. Coagonist markedly attenuated the CDAHFD-induced expression of TIMP-1, MMP-9, TNF-α, MCP-1, COL1A1, and α-SMA. It also inhibited progression of hepatic steatosis and fibrosis in mice. Exendin-4 was better than glucagon, but coagonist was most effective in reduction of hepatic inflammation as well as steatosis. Coagonist of GLP-1R and GCGR improved NAFLD in C57BL6/J mice. This effect is mediated by reduction in lipotoxicity and inflammation in liver.

Coagonist of GLP-1 and Glucagon Receptor Ameliorates Development of Non-Alcoholic Fatty Liver Disease

BACKGROUND Obesity, diabetes and dyslipidemica are the key pathogenic stimulus that enhances progression of Non-Alcoholic Fatty Liver Disease (NAFLD). Coagonist of Glucagon Like- Peptide-1 (GLP-1) Receptor (GLP-1R) and Glucagon Receptor (GCGR) are being evaluated for obesity and diabetes. GLP-1 analogs have shown to reverse diabetes and obesity. Glucagon treatment reduces lipids after acute and chronic treatment. OBJECTIVE In this study, we have investigated the effect of co-agonist on the prevention of NAFLD induced by long-term feeding of High Fat Diet (HFD). METHOD We have used HFD to induce NAFLD after chronic feeding in mice. Co-agonist treatment (150 µg.kg-1, s.c.) was initiated with induction of HFD, which was continued for 40 weeks. Body weight, food intake, glucose homeostasis, lipid profile, inflammatory and fibrotic markers were assessed at the end of treatment. RESULTS Co-agonist treatment prevented body weight gain, glucose intolerance and insulin resistance. Treatment with co-agonist reduced NEFA, increased FGF21 and adiponectin levels. Co-agonist increased glycerol release and energy expenditure, while decreased respiratory quotient. Co-agonist reduced lipids in circulation and liver. Expression of SREBP-1C, SCD-1, ACC and FAS were decreased, while ACOX1 and CPT1 were increased after co-agonist treatment. Inflammatory cytokine TNF-α and IL-6 in plasma and expression of MCP-1, TGF-ß, MMP-9, TNF-α, TIMP-1, α-SMA, and COL1A1 were decreased after co-agonist treatment. Plasma transaminases, hepatic TBARS, hepatic hydroxyproline and relative liver weight were suppressed after co-agonist treatment. Fat accumulation, inflammation and fibrosis were reduced in histological assessment of liver in co-agonist treated animals. CONCLUSION Co-agonist prevented development of HFD-induced NAFLD by ameliorating obesity, diabetes, inflammation and fibrosis.

Coagonist of GLP-1 and glucagon decreases liver inflammation and atherosclerosis in dyslipidemic condition

Dyslipidemia enhances progression of atherosclerosis. Coagonist of GLP-1 and glucagon are under clinical investigation for the treatment of obesity and diabetes. Earlier, we have observed that coagonist reduced circulating and hepatic lipids, independent of its anorexic effects. Here, we investigated the role of coagonist of GLP-1 and glucagon receptors in complications of diet-induced dyslipidemia in hamsters and humanized double transgenic mice. Hamsters fed on high fat high cholesterol diet were treated for 8 weeks with coagonist of GLP-1 and glucagon receptors (75 and 150 μg/kg). Pair-fed control was maintained. Cholesterol fed transgenic mice overexpressing hApoB100 and hCETP with coagonist (300 μg/kg) for 4 weeks. After the completion of treatment, biochemical estimations were done. Coagonist treatment reduced triglycerides in plasma, liver and aorta, plasma cholesterol and hepatic triglyceride secretion rate. Expressions of HMG-CoA reductase and SBREBP-1C were reduced and expressions of LDLR, CYP7A1, ABCA1 and ABCB11 were increased in liver, due to coagonist treatment. Coagonist treatment increased bile flow rate and biliary cholesterol excretion. IL-6 and TNF-α were reduced in plasma and expression of TNF-α, MCP-1, MMP-9 and TIMP-1 decreased in liver. Treatment with coagonist reduced oxidative stress in liver and aorta. Energy expenditure was increased and respiratory quotient was reduced by coagonist treatment. These changes were correlated with reduced hepatic inflammation and lipids in liver and aorta in coagonist treated hamsters. Coagonist treatment also reduced lipids in cholesterol-fed transgenic mice. These changes were independent of glycaemia and anorexia observed after coagonist treatment. Long term treatment with coagonist of GLP-1 and glucagon receptor ameliorated diet-induced dyslipidemia and atherosclerosis by regulating bile homeostasis, liver inflammation and energy expenditure.