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.

Enantiomer ratio of MK-0767 in humans and nonclinical species.

MK-0767, (+/-)-5-[(2,4-dioxothiazolidin-5-yl)methyl]-2-methoxy-N-[[(4-trifluoromethyl)phenyl]methyl]benzamide, is a thiazolidinedione-containing dual peroxisome proliferator-activated receptor (PPAR) alpha/gamma agonist that has been studied as a potential treatment for patients with type 2 diabetes. MK-0767 contains a chiral center at the C-5 position of the thiazolidinedione ring and was being developed as the racemate, due to the rapid interconversion of its enantiomers in biological samples. In the present work the in vitro and in vivo concentration ratios of the (+)-(R) to (-)-(S) enantiomers of MK-0767 were determined in plasma from humans (in vitro only) and nonclinical species used in the toxicological evaluation of rac-MK-0767, namely CD-1 mice, Sprague-Dawley rats, beagle dogs, New Zealand white rabbits, and rhesus monkeys. The R/S ratio was determined by chiral liquid chromatography/tandem mass spectrometry. Species differences were observed in the in vitro and in vivo enantiomeric ratios, as well as differences between in vitro and in vivo in some species. The in vitro R/S ratio was similar in dogs and humans (approximately 1.5-1.7). In rats and monkeys, the ratio was approximately unity, both in vitro and in vivo. In mice, the ratio was higher in vitro (approximately 1) than in vivo (approximately 0.6), while in rabbits it was higher in vivo (approximately 1) than in vitro (approximately 0.5). These results suggested that differential binding of the MK-0767 enantiomers to plasma and tissue proteins and other macromolecules may be affecting the R/S ratio both in vitro and in vivo, since in protein-free systems MK-0767 exists as the racemate.