Kumar V.S. Nemmani

Acute administration of GPR40 receptor agonist potentiates glucose-stimulated insulin secretion in vivo in the rat

Recently, several in vitro studies have shown that GPR40 receptor activation by free fatty acids (FFAs) results in glucose-dependent insulin secretion. However, whether GPR40 receptor activation results in glucose-dependent insulin secretion in vivo in rats is not known. Therefore, we evaluated the effect of synthetic GPR40 receptor agonist (compound 1) on glucose tolerance test (GTT) in fed, fasted, and insulin-resistant rats. In oral GTT, intraperitoneal GTT, and intravenous GTT, GPR40 receptor agonist improved glucose tolerance, which was associated with increase in plasma insulin level. Interestingly, in GTTs, the rise in insulin levels in agonist-treated group was directly proportional to the rate of rise and peak levels of glucose in control group. Although glibenclamide, a widely used insulin secretagogue, improved glucose tolerance in all GTTs, it did not display insulin release in intraperitoneal GTT or intravenous GTT. In the absence of glucose load, GPR40 receptor agonist did not significantly change the plasma insulin concentration, but did decrease the plasma glucose concentration. Fasted rats exhibited impaired glucose-stimulated insulin secretion (GSIS) as compared with fed rats. Compound 1 potentiated GSIS in fasted state but failed to do so in fed state. Suspecting differential pharmacokinetics, a detailed pharmacokinetic evaluation was performed, which revealed the low plasma concentration of compound 1 in fed state. Consequently, we examined the absorption profile of compound 1 at higher doses in fed state; and at a dose at which its absorption was comparable with that in fasted state, we observed significant potentiation of GSIS. Chronic high-fructose (60%) diet feeding resulted in impaired glucose tolerance, which was improved by GPR40 receptor agonist. Therefore, our results demonstrate for the first time that acute GPR40 receptor activation leads to potentiation of GSIS in vivo and improves glucose tolerance even in insulin-resistant condition in rats. Taken together, these results suggest that GPR40 receptor agonists could be potential therapeutic alternatives to sulfonylureas.

Isocytosine-based inhibitors of xanthine oxidase: Design, synthesis, SAR, PK and in vivo efficacy in rat model of hyperuricemia

Structure-activity relationship studies were carried out for lead generation following structure-guided design approach from an isocytosine scaffold identified earlier for xanthine oxidase inhibition. A 470-fold improvement in in vitro IC(50) was obtained in the process. Five most potent compounds with nanomolar IC(50) values were selected for pharmacokinetics and in vivo experiments. The best compound showed good in vivo activity when administered intraperitoneally but was not active by oral route. The results suggest that improvement in oral exposure could improve the in vivo efficacy of this series.

Synthesis and biological evaluation of isoxazole, oxazole, and oxadiazole containing heteroaryl analogs of biaryl ureas as DGAT1 inhibitors

Diacylglycerol acyltransferase, DGAT1, is a promising target enzyme for obesity due to its involvement in the committed step of triglyceride biosynthesis. Amino biphenyl carboxylic acids, exemplified by compound 4, are known potent inhibitors of hDGAT1. However the high cLogP and poor solubility of these biphenyl analogs might tend to limit their development. We have synthesized and evaluated compounds containing 3-phenylisoxazole, 5-phenyloxazole, and 3-phenyl-1,2,4-oxadiazole biaryl units for their hDGAT1 inhibition. Our aim in synthesizing such heterocyclic analogs was to improve the cLogP and solubility of these molecules while retaining hDGAT1 potency. Several compounds within the 3-phenylisoxazole series exhibited potent hDGAT1 inhibition when evaluated using an in vitro enzymatic assay. Certain promising compounds were studied for their potential to reduce triglyceride levels using an in vivo fat tolerance test in mice and were also evaluated for any possible improvement to their solubility. Compound 40a (IC(50) = 64 nM) with an in vivo plasma triglyceride reduction of 90 percent, and a solubility of 0.43 mg/ml at pH 7.4 may serve as a new lead for developing newer anti-obesity agents.

Identification of novel isocytosine derivatives as xanthine oxidase inhibitors from a set of virtual screening hits.

In recent years, xanthine oxidase has emerged as an important target not only for gout but also for cardiovascular and metabolic disorders involving hyperuricemia. Contrary to popular belief, recent clinical trials with uricosurics have demonstrated that enhanced excretion of uric acid is, by itself, not adequate to treat hyperuricemia; simultaneous inhibition of production of uric acid by inhibition of xanthine oxidase is also important. Virtual screening of in-house synthetic library followed by in vitro and in vivo testing led to the identification of a novel scaffold for xanthine oxidase inhibition. In vitro activity results corroborated the results from molecular docking studies of the virtual screening hits. The isocytosine scaffold maintains key hydrogen bonding and pi-stacking interactions in the deep end of the xanthine-binding pocket, which anchors it in an appropriate pose to inhibit binding of xanthine and shows promise for further lead optimization using structure-based drug design approach.

Lead optimization of isocytosine-derived xanthine oxidase inhibitors

We report our attempts at improving the oral efficacy of low-nanomolar inhibitors of xanthine oxidase from isocytosine series through chemical modifications. Our lead compound had earlier shown good in vivo efficacy when administered intraperitoneally but not orally. Several modifications are reported here which achieved more than twofold improvement in exposure. A compound with significant improvement in oral efficacy was also obtained.

Evaluation of thiazole containing biaryl analogs as diacylglycerol acyltransferase 1 (DGAT1) inhibitors.

Biphenyl carboxylic acids, exemplified by compound 5, are known potent inhibitors of diacylglycerol acyltransferase, DGAT1, an enzyme involved in the final committed step of triglyceride biosynthesis. We have synthesized and evaluated 2-phenylthiazole, 4-phenylthiazole, and 5-phenylthiazole analogs as DGAT1 inhibitors. The 5-phenylthiazole series exhibited potent DGAT1 inhibition when evaluated using an in vitro enzymatic assay and an in vivo fat tolerance test in mice. Compound 33 (IC50 = 23 nM) exhibiting promising oral pharmacokinetic parameters (AUCinf = 7058 ng h/ml, T1/2 = 0.83 h) coupled with 87 percent reduction of plasma triglycerides in vivo may serve as a lead for developing newer anti-obesity agents.

Fermentation, Isolation, Structure, and antidiabetic activity of NFAT-133 produced by Streptomyces strain PM0324667

Type-2 diabetes is mediated by defects in either insulin secretion or insulin action. In an effort to identify extracts that may stimulate glucose uptake, similar to insulin, a high throughput-screening assay for measuring glucose uptake in skeletal muscle cells was established. During the screening studies to discover novel antidiabetic compounds from microbial resources a Streptomyces strain PM0324667 (MTCC 5543, the Strain accession number at Institute of Microbial Technology, Chandigarh, India), an isolate from arid soil was identified which expressed a secondary metabolite that induced glucose uptake in L6 skeletal muscle cells. By employing bioactivity guided fractionation techniques, a tri-substituted simple aromatic compound with anti-diabetic potential was isolated. It was characterized based on MS and 2D NMR spectral data and identified as NFAT-133 which is a known immunosuppressive agent that inhibits NFAT-dependent transcription in vitro. Our investigations revealed the antidiabetic potential of NFAT-133. The compound induced glucose uptake in differentiated L6 myotubes with an EC50 of 6.3 ± 1.8 μM without activating the peroxisome proliferator-activated receptor-γ. Further, NFAT-133 was also efficacious in vivo in diabetic animals and reduced systemic glucose levels. Thus it is a potential lead compound which can be considered for development as a therapeutic for the treatment of type-2 diabetes. We have reported herewith the isolation of the producer microbe, fermentation, purification, in vitro, and in vivo antidiabetic activity of the compound.

Oral bioavailability, efficacy and gastric tolerability of P2026, a novel nitric oxide-releasing diclofenac in rat.

The present study was designed to evaluate, P2026 [(2-((2-(nitrooxy)ethyl)disulfanyl)ethyl 2-(2-(2,6-dichlorophenylamino)phenyl)acetate)], a novel NO (nitric oxide) donor prodrug of diclofenac for its ability to release NO and diclofenac, and whether P2026 provides advantage of improved activity/gastric tolerability over diclofenac. Oral bioavailability of P2026 was estimated from plasma concentration of diclofenac and nitrate/nitrite (NOx). Anti-inflammatory activity was evaluated in three different models of inflammation: acute (carrageenan-induced paw oedema), chronic (adjuvant-induced arthritis), and systemic (lipopolysaccharide-induced endotoxic shock). Gastric tolerability was evaluated from compound’s propensity to cause gastric ulcers. P2026 exhibited dose-dependent diclofenac and NOx release. Similar to diclofenac, P2026 showed potent anti-inflammatory activity in acute and chronic model, whereas it improved activity in systemic model. Both diclofenac and P2026 inhibited gastric prostaglandin, but only diclofenac produced dose-dependent haemorrhagic ulcers. Thus, the results suggest that coupling of NO and diclofenac contribute to improved gastric tolerability while retaining the anti-inflammatory properties of diclofenac.

APD668, a G protein-coupled receptor 119 agonist improves fat tolerance and attenuates fatty liver in high-trans fat diet induced steatohepatitis model in C57BL/6 mice

ABSTRACT G‐protein coupled receptor 119 (GPR119) receptor is a rhodopsin‐like, class A G&agr;s‐coupled receptor, predominantly expressed in pancreatic islet cells and intestinal entero‐endocrine cells. GPR119 has been emerged as a novel therapeutic target for the treatment of dyslipidemia in type 2 diabetes. In this study, we investigated the effect of APD668, a GPR119 agonist alone and in combination with linagliptin, a DPPIV inhibitor on oral fat tolerance test. Our findings demonstrate that APD668, a GPR119 agonist inhibits the intestinal triglyceride absorption after acute fat load in mice. Single dose administration of APD668 increases incretin secretion and enhances total PYY levels in presence of fat load in mice. We found that, the anti‐dyslipidemic action of APD668 was reversed in presence of exendin‐3 in oral fat tolerance test. In addition, our results showed that exendin‐3 (9−39) failed to block the effect of APD668 on gastric emptying indicating that gastric emptying effects of APD668 are indeed mediated through GPR119 receptor dependent mechanism. Combined administration of APD668 and linagliptin significantly increased plasma active GLP‐1 levels in‐vivo and showed improvement in fat tolerance. However, APD668 failed to show anti‐dyslipidemic activity in tyloxapol‐induced hyperlipidemia in mice. Furthermore, we investigated the chronic effects of APD668 on hepatic steatosis in high trans‐fat diet fed steatohepatitis model in mice. Oral administration of APD668 in HTF diet fed mice ameliorated hepatic endpoints such as plasma ALT, AST, liver weight and steatosis. These findings suggest that GPR119 agonists may represent a promising therapeutic strategy for the treatment of dyslipidemia and non‐alcoholic steatohepatitis.

Effect of D-Ala2GIP, a stable GIP receptor agonist on MPTP-induced neuronal impairments in mice

Abstract The aim of the present study was to evaluate the ability of D‐Ala2GIP, a gastric inhibitory polypeptide (GIP) receptor agonist, to attenuate the behavioral phenotype of Parkinsons disease caused by 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) administration in mice. In the behavioral studies, MPTP administration led to spontaneous locomotor activity deficits, impaired rotarod performance, akinesia, muscular rigidity and increased tremor amplitude, which was attenuated by pretreatment with D‐Ala2GIP (50–100 nmol/kg, i.p.). This acute neuroprotective response by D‐Ala2GIP was found to be blocked by a selective GIP receptor antagonist, (Pro3)GIP (50 nmol/kg, i.p.), indicating that the observed effects are mediated through GIP receptor mediated signaling pathway. Biochemical studies revealed that D‐Ala2GIP reduced the brain malondialdehyde levels and enhanced the brain glutathione levels, thereby mitigating the MPTP‐induced oxidative stress. MPTP administration resulted in reduction of the striatal concentration of dopamine and its metabolites, homovanillic acid (HVA) and 3, 4‐Dihydroxyphenylacetic acid (DOPAC). Pretreatment with D‐Ala2GIP attenuated the loss of striatal dopamine levels without affecting the normal dopamine catabolism. Thus, the observed effects in the MPTP‐induced Parkinsonism model could be in part attributable to the antioxidant properties of D‐Ala2GIP and enhanced turnover of dopamine in the nigrostriatal pathways in mouse brain. These findings together suggest that GIP receptor could be a therapeutic target in the management of symptoms of Parkinsons disease.