Design and synthesis of potent amido- and benzyl-substituted cis-3-amino-4-(2-cyanopyrrolidide)pyrrolidinyl DPP-IV inhibitors.

The cis-3-amino-4-(2-cyanopyrrolidide)-pyrrolidine template has been shown to afford low nanomolar inhibitors of human DPP-IV that exhibit a robust PK/PD profile. An X-ray co-crystal structure of 5 confirmed the proposed mode of binding. The potent single digit DPP-IV inhibitor 53 exhibited a preferred PK/PD profile in preclinical animal models and was selected for additional profiling.

Plasma glucose levels are reduced in rats and mice treated with an inhibitor of glucose-6-phosphate translocase.

The activity of glucose-6-phosphatase (G-6-Pase) in isolated rat microsomes was inhibited by a new selective inhibitor of the multi-subunit G-6-Pase system, 1-[2-(4-chloro-phenyl)-cyclopropylmethoxy]-3,4-dihydroxy-5-(3-imid azo[4,5-b]pyridin-1-yl-3-phenyl-acryloyloxy)-cyclohexanecarboxylic acid (compound A) with a 50% inhibitory concentration (IC50) of approximately 10 nmol/l. Compound A (500 nmol/l) inhibited the uptake of [14C]glucose-6-phosphate (G-6-P) into intact isolated rat microsomes, confirming that this agent blocks G-6-P translocation, as suggested by previous studies using intact and permeabilized microsomes. The inhibition of microsomal G-6-P transport by compound A was associated with inhibition of the rate of glucose output from rat hepatocytes incubated in the presence of 25 nmol/l glucagon (IC50 approximately 320 nmol/l.) Compound A (1 micromol/l) also inhibited the basal rate of glucose production by rat hepatocytes by 47%. Intraperitoneal administration of compound A to fasted mice lowered circulating plasma glucose concentrations dose-dependently at doses as low as 1 mg/kg. This effect was comparatively short-lived; glucose lowering was maximal at 30 min after dosing with 100 mg/kg compound A (-71%) and declined thereafter, being reversed within 3 h. A similar time course of glycemic response was observed in fasted rats; glucose lowering was maximal 30 min after dosing with 100 mg/kg compound A (-36%) and declined until the effect was fully reversed by 3 h postdose. In rats subjected to compound A treatment, liver glycogen content was increased. G-6-P and lactate levels were maximally elevated 30 min after dosing and declined thereafter. Cumulatively, these results suggest that the mechanism of glucose lowering by compound A was via inhibition of G-6-Pase activity, mediated through inhibition of the T1 subunit of the microsomal G-6-Pase enzyme system. Drug levels measured over the same time course as that used to assess in vivo efficacy peaked within 30 min of administration, then declined, which is consistent with the transient changes in plasma glucose and liver metabolites.

Binding site on the murine IFN-gamma receptor for IFN-gamma has been identified using the synthetic peptide approach.

We have studied the structural parameters involved in the binding of murine IFN-gamma (MuIFN-gamma) to its receptor. Ten synthetic overlapping peptides corresponding to the extracellular domain of the MuIFN-gamma receptor (MuIFN-gamma R) were synthesized. In direct binding studies, biotinylated MuIFN-gamma bound specifically to receptor peptide (95-120). Further, the NH2-terminal IFN-gamma peptide, MuIFN-gamma (1-39), also specifically bound to receptor peptide (95-120). Binding of both labeled MuIFN-gamma and MuIFN-gamma (1-39) to MuIFN-gamma R peptide (95-120) was inhibited by either unlabeled molecule. The COOH-terminal receptor binding peptide, MuIFN-gamma (95-133), neither bound to any receptor peptides nor blocked the binding of intact MuIFN-gamma or MuIFN-gamma (1-39) to receptor peptide (95-120). Polyclonal antibodies to each of the peptides were then produced. Each of the anti-peptide antisera recognized its corresponding peptide and bound denatured cloned soluble receptor by Western blotting. Furthermore, the antisera to peptides representing the inner region of the extracellular domain of the receptor bound to nondenatured soluble MuIFN-gamma R. Specifically, antisera to the receptor peptides (73-97), (95-120), (118-143), (142-163), and (161-182) bound to soluble MuIFN-gamma R, whereas antisera to peptides (1-21), (20-49), (46-74), (178-203), and (202-227) did not bind. Most important, antisera to peptides (95-120) and (118-143) competed with [125I]MuIFN-gamma for binding to soluble receptor. These results show that the region of the MuIFN-gamma R encompassing amino acid residues (95-120) is a binding site on the receptor for the NH2-terminal of MuIFN-gamma by direct binding, and that the larger region (95-143) on the receptor may play a role in binding of intact MuIFN-gamma based on blocking of binding by site-specific antibodies.

Modulation of insulin secretion and glycemia by selective inhibition of cyclic AMP phosphodiesterase III.

The effects of selective inhibition of cyclic AMP phosphodiesterase type III on insulin and glucose levels during an oral glucose challenge were evaluated in obese, diabetic ob/ob mice and in lean, non-diabetic littermates using the selective inhibitor, milrinone. Oral administration of milrinone increased plasma insulin levels both in ob/ob and in lean mice. Glucose tolerance was improved in lean, but not in ob/ob mice, where glucose levels were increased by milrinone treatment. In isolated hepatocytes from normal rats incubation with 200 microM milrinone caused a 30% increase in glucose release with a corresponding depletion of glycogen stores. Stimulation of isolated rat adipocytes with 200 microM milrinone increased glycerol release 7-fold. We conclude that selective inhibitors of cyclic AMP phosphodiesterase III are effective insulin secretagogues, but their therapeutic utility may be limited by their concurrent stimulation of lipolysis and hepatic glucose output.

Cyclic AMP phosphodiesterases of human and rat islets of Langerhans: contributions of types III and IV to the modulation of insulin secretion.

This study evaluated the contribution of isozymes of cAMP phosphodiesterase (PDE) to total PDE activity in human and rat islets using type-selective inhibitors. The effects of selected PDE inhibitors on insulin secretion from human and rat islets were also measured in order to assess the contribution of the various PDE isozymes to the modulation of insulin secretion. The data suggest that PDE III is quantitatively the most important PDE isozyme present in islets, accounting for up to 70% of the total activity. Lower, but measurable, levels of PDE IV activity were present. Approximately 20% of islet PDE is not inhibitable by agents selective either for PDE III or IV. Selective inhibition of PDE III stimulated insulin secretion, but inhibition of PDE IV had no effect. The effects of type-selective inhibitors on PDE activity and insulin secretion were similar in human and rat islets.