A novel series of indazole-/indole-based glucagon receptor antagonists.

A novel, potent series of glucagon receptor antagonists (GRAs) was discovered. These indazole- and indole-based compounds were designed on an earlier pyrazole-based GRA lead MK-0893. Structure-activity relationship (SAR) studies were focused on the C3 and C6 positions of the indazole core, as well as the benzylic position on the N-1 of indazole. Multiple potent GRAs were identified with excellent in vitro profiles and good pharmacokinetics in rat. Among them, GRA 16d was found to be orally active in blunting glucagon induced glucose excursion in an acute glucagon challenge model in glucagon receptor humanized (hGCGR) mice at 1, 3 and 10mg/kg (mpk), and significantly lowered acute glucose levels in hGCGR ob/ob mice at 3 mpk dose.

Discovery of novel, potent, selective, and orally active human glucagon receptor antagonists containing a pyrazole core.

A novel class of 1,3,5-pyrazoles has been discovered as potent human glucagon receptor antagonists. Notably, compound 26 is orally bioavailable in several preclinical species and shows selectivity towards cardiac ion channels, other family B receptors such hGIP and hGLP1, and a large panel of enzymes and additional receptors. When dosed orally, compound 26 is efficacious in suppressing glucagon induced plasma glucose excursion in rhesus monkey and transgenic murine pharmacodynamic models at 1 and 10 mpk, respectively.

Cloning and expression of canine glucagon receptor and its use to evaluate glucagon receptor antagonists in vitro and in vivo.

Glucose homeostasis is maintained by the combined actions of insulin and glucagon. Hyperglucagonemia and/or elevation of glucagon/insulin ratio have been reported in diabetic patients and in animal models of diabetes. Therefore, antagonizing glucagon receptor function has long been considered a useful approach to lower hyperglycemia. Dogs serve as an excellent model for studying glycemic control and various aspects of glucagon biology in vivo; however, the amino acid sequence of the dog glucagon receptor has not been reported. To better understand the pharmacology of the dog glucagon receptor and to characterize glucagon receptor antagonists, we cloned a cDNA corresponding to the glucagon receptor from dog liver RNA. The dog glucagon receptor shares a significant (>75%) homology at both nucleotide and amino acid levels with the glucagon receptor from human, monkey, mouse, and rat. The protein is highly conserved among all species in areas corresponding to the 7 trans-membrane domains. However, it shows significant divergence at the carboxy terminus such that the receptor from dog has the longest cytoplasmic tail among all species examined. When expressed in chinese hamster ovary cells, the dog glucagon receptor bound [125I]Glucagon with a K(d) of 477+/-106 pM. Glucagon stimulated the rise of intracellular cAMP levels in these cells with an EC(50) of 9.6+/-1.7 nM and such effects could be blocked by known peptidyl and non-peptidyl small molecule antagonists. In addition we show that a small molecule glucagon receptor antagonist with significant activity in cell based assays also blocked the ability of glucagon to induce elevation in blood glucose in beagle dogs. These data demonstrate that the cloned cDNA encodes a functional dog glucagon receptor. The availability of the dog cDNA will facilitate the understanding of glucagon pharmacology and aid in the characterization of novel glucagon antagonists that may serve as anti-hyperglycemic treatment for type 2 diabetes mellitus.

A novel glucagon receptor antagonist inhibits glucagon-mediated biological effects.

Glucagon maintains glucose homeostasis during the fasting state by promoting hepatic gluconeogenesis and glycogenolysis. Hyperglucagonemia and/or an elevated glucagon-to-insulin ratio have been reported in diabetic patients and animals. Antagonizing the glucagon receptor is expected to result in reduced hepatic glucose overproduction, leading to overall glycemic control. Here we report the discovery and characterization of compound 1 (Cpd 1), a compound that inhibits binding of 125I-labeled glucagon to the human glucagon receptor with a half-maximal inhibitory concentration value of 181 +/- 10 nmol/l. In CHO cells overexpressing the human glucagon receptor, Cpd 1 increased the half-maximal effect for glucagon stimulation of adenylyl cyclase with a KDB of 81 +/- 11 nmol/l. In addition, Cpd 1 blocked glucagon-mediated glycogenolysis in primary human hepatocytes. In contrast, a structurally related analog (Cpd 2) was not effective in blocking glucagon-mediated biological effects. Real-time measurement of glycogen synthesis and breakdown in perfused mouse liver showed that Cpd 1 is capable of blocking glucagon-induced glycogenolysis in a dosage-dependent manner. Finally, when dosed in humanized mice, Cpd 1 blocked the rise of glucose levels observed after intraperitoneal administration of exogenous glucagon. Taken together, these data suggest that Cpd 1 is a potent glucagon receptor antagonist that has the capability to block the effects of glucagon in vivo.

Hepatic glucagon receptor binding and glucose-lowering in vivo by peptidyl and non-peptidyl glucagon receptor antagonists.

Glucagon receptor antagonists have been actively pursued as potential therapeutics for the treatment of type 2 diabetes. Peptidyl and non-peptidyl glucagon receptor antagonists have been shown to block glucagon-induced blood glucose elevation in both animals and humans. How the antagonists and the glucagon receptor interact in vivo has not been reported and is the subject of the current study. Using (125)I-labeled glucagon as a radiotracer, we developed an in vivo glucagon receptor occupancy assay in mice expressing a human glucagon receptor in place of the endogenous mouse glucagon receptor (hGCGR mice). Using this assay, we first showed that the glucagon receptor is expressed predominantly in liver, to a much lesser extent in kidney, and is below detection in several other tissues/organs in the mice. We subsequently showed that, at 2 mg/kg body weight (mg/pk) dosed intraperitoneally (i.p.), peptidyl glucagon receptor antagonist des-His-glucagon binds to approximately 78% of the hepatic glucagon receptor and blocks an exogenous glucagon-induced blood glucose elevation in the mice. Finally, we also showed that, at 10 and 30 mg/kg dosed orally (p.o.), compound A, a non-peptidyl small molecule glucagon receptor antagonist, occupied 65-70% of the hepatic glucagon receptor, and significantly diminished exogenous glucagon-induced blood glucose elevation in the mice. At 3 mg/kg, however, compound A occupied only approximately 39% of the hepatic glucagon receptor and did not affect exogenous glucagon-induced blood glucose elevation in the mice. Taken together, the results confirmed previous reports that glucagon receptors are present predominantly in the liver, and provide the first direct evidence that peptidyl and non-peptidyl glucagon receptor antagonists bind to the hepatic glucagon receptor in vivo, and that at least 60% receptor occupancy correlates with the glucose lowering efficacy by the antagonists in vivo.

Anti-diabetic efficacy and impact on amino acid metabolism of GRA1, a novel small-molecule glucagon receptor antagonist.

Hyperglucagonemia is implicated in the pathophysiology of hyperglycemia. Antagonism of the glucagon receptor (GCGR) thus represents a potential approach to diabetes treatment. Herein we report the characterization of GRA1, a novel small-molecule GCGR antagonist that blocks glucagon binding to the human GCGR (hGCGR) and antagonizes glucagon-induced intracellular accumulation of cAMP with nanomolar potency. GRA1 inhibited glycogenolysis dose-dependently in primary human hepatocytes and in perfused liver from hGCGR mice, a transgenic line of mouse that expresses the hGCGR instead of the murine GCGR. When administered orally to hGCGR mice and rhesus monkeys, GRA1 blocked hyperglycemic responses to exogenous glucagon. In several murine models of diabetes, acute and chronic dosing with GRA1 significantly reduced blood glucose concentrations and moderately increased plasma glucagon and glucagon-like peptide-1. Combination of GRA1 with a dipeptidyl peptidase-4 inhibitor had an additive antihyperglycemic effect in diabetic mice. Hepatic gene-expression profiling in monkeys treated with GRA1 revealed down-regulation of numerous genes involved in amino acid catabolism, an effect that was paralleled by increased amino acid levels in the circulation. In summary, GRA1 is a potent glucagon receptor antagonist with strong antihyperglycemic efficacy in preclinical models and prominent effects on hepatic gene-expression related to amino acid metabolism.

Discovery of a novel glucagon receptor antagonist N-[(4-{(1S)-1-[3-(3, 5-dichlorophenyl)-5-(6-methoxynaphthalen-2-yl)-1H-pyrazol-1-yl]ethyl}phenyl)carbonyl]-ß-alanine (MK-0893) for the treatment of type II diabetes.

A potent, selective glucagon receptor antagonist 9m, N-[(4-{(1S)-1-[3-(3,5-dichlorophenyl)-5-(6-methoxynaphthalen-2-yl)-1H-pyrazol-1-yl]ethyl}phenyl)carbonyl]-ß-alanine, was discovered by optimization of a previously identified lead. Compound 9m is a reversible and competitive antagonist with high binding affinity (IC(50) of 6.6 nM) and functional cAMP activity (IC(50) of 15.7 nM). It is selective for glucagon receptor relative to other family B GPCRs, showing IC(50) values of 1020 nM for GIPR, 9200 nM for PAC1, and >10000 nM for GLP-1R, VPAC1, and VPAC2. Compound 9m blunted glucagon-induced glucose elevation in hGCGR mice and rhesus monkeys. It also lowered ambient glucose levels in both acute and chronic mouse models: in hGCGR ob/ob mice it reduced glucose (AUC 0-6 h) by 32% and 39% at 3 and 10 mpk single doses, respectively. In hGCGR mice on a high fat diet, compound 9m at 3, and 10 mpk po in feed lowered blood glucose levels by 89% and 94% at day 10, respectively, relative to the difference between the vehicle control and lean hGCGR mice. On the basis of its favorable biological and DMPK properties, compound 9m (MK-0893) was selected for further preclinical and clinical evaluations.

Discovery and investigation of a novel class of thiophene-derived antagonists of the human glucagon receptor.

A novel class of antagonists of the human glucagon receptor (hGCGR) has been discovered. Systematic modification of the lead compound identified substituents that were essential for activity and those that were amenable to further optimization. This SAR exploration resulted in the synthesis of 13, which exhibited good potency as an hGCGR functional antagonist (IC50 = 34 nM) and moderate bioavailability (36% in mice).

Discovery of novel, potent, and orally active spiro-urea human glucagon receptor antagonists.

A novel class of spiro-ureas has been discovered as potent human glucagon receptor antagonists in both binding and functional assays. Preliminary studies have revealed that compound 15 is an orally active human glucagon receptor antagonist in a transgenic murine pharmacodynamic model at 10 and 30 mpk. Compound 15 is orally bioavailable in several preclinical species and shows selectivity toward cardiac ion channels and other family B receptors, such as hGIP1 and hGLP.

Regulation of insulin signal transduction pathway by a small-molecule insulin receptor activator.

Insulin regulates cellular metabolism and growth through activation of insulin receptors (IRs). We recently identified a non-peptide small-molecule IR activator (compound 2), which induced human IR tyrosine kinase activity in Chinese-hamster ovary cells expressing human IR [Qureshi, Ding, Li, Szalkowski, Biazzo-Ashnault, Xie, Saperstein, Brady, Huskey, Shen et al. (2000) J. Biol. Chem. 275, 36590-36595]. Oral treatment with this compound resulted in correction of hyperglycaemia, hypertriacylglycerolaemia and hyperinsulinaemia in several rodent models of diabetes. In the present study, we have found that this compound increased tyrosine phosphorylation of the IR beta-subunit and IR substrate 1 in primary rat adipocytes as well as induced phosphorylation of Akt, the 70 kDa ribosomal protein S6 kinase and glycogen synthase-3 (deactivation) in Chinese-hamster ovary cells expressing human IR. Similar to insulin, compound 2 stimulated glucose uptake, glycogen synthesis and inhibited isoprenaline-stimulated lipolysis in adipocytes. A structurally related analogue (compound 3) was devoid of the above activities suggesting that the activity of compound 2 is specifically mediated by targeted IR activation. The effects of compound 2 on stimulation of glucose uptake, glycogen synthesis and inhibition of lipolysis were blocked by wortmannin, consistent with the involvement of a phosphoinositide 3-kinase-dependent pathway. In addition, compound 2, but not compound 3, exhibited additive or synergistic effects with sub-maximal concentrations of insulin in rat adipocytes. Thus the IR activator was capable of activating insulin-mediated signalling and metabolic pathways in primary adipocytes. These results demonstrate that IR activators have implications for the future development of new therapeutic approaches to Type I and Type II diabetes.