Pharmacological characterization of ZYDPLA1, a novel long-acting dipeptidyl peptidase-4 inhibitor.

OBJECTIVE: Dipeptidyl peptidase-4 (DPP-4) is responsible for degradation of glucagon-like peptide 1 (GLP-1) and gastric inhibitory polypeptide (GIP), the endogenous incretins that stimulate glucose-dependent insulin secretion. The objective was to evaluate preclinical profile of a novel DPP-4 inhibitor ZYDPLA1. METHODS: In vitro inhibition potency and selectivity were assessed using recombinant enzymes and/or plasma. In vivo efficacy was determined in oral glucose tolerance test or mixed meal tolerance test in C57BL/6J mice, db/db mice and Zucker fatty rats. Pharmacokinetics/pharmacodynamics was studied in mice, rats, dogs, and non-human primates. RESULTS: ZYDPLA1 is a potent, competitive and long acting inhibitor of DPP-4 (Ki 0.0027 µM; Koff 2.3 × 10(-4 ) s(-1) ). ZYDPLA1 was more than 7000-fold selective for recombinant DPP-4 relative to DPP-8 and DPP-9, and more than 60 000-fold selective relative to fibroblast activation protein (FAP) in vitro. DPP-4 inhibition was comparable across species. In vivo, oral ZYDPLA1 elevated circulating GLP-1 and insulin levels in mice and rats and showed dose-dependent anti-hyperglycemic effect. Anti-hyperglycemic effect was also observed in db/db mice and Zucker fatty rats. ZYDPLA1 showed low clearance, large volume of distribution, and a long half-life with excellent oral bioavailability in all species. It significantly inhibited plasma DPP-4 activity in mice and rats for more than 48 h, and for up to 168 h in dogs and non-human primates. Allometric scaling predicted a half-life in humans of 53 to 166 h. CONCLUSION: ZYDPLA1 is a potent, selective, long-acting oral DPP-4 inhibitor with potential to become once-a-week therapy for treatment of type 2 diabetes mellitus.

Validation of 96-well equilibrium dialysis with non-radiolabeled drug for definitive measurement of protein binding and application to clinical development of highly-bound drugs.

Definitive plasma protein binding (PB) studies in drug development are routinely conducted with radiolabeled material, where the radiochemical purity limits quantitative PB measurement. Recent and emerging regulatory guidances increasingly expect quantitative determination of the fraction unbound (Fu) for key decision making. In the present study, PB of 11 structurally- and therapeutically-diverse drugs spanning the full range of plasma binding was determined by equilibrium dialysis of non-radiolabeled compound and was validated against the respective definitive values obtained by accepted radiolabeled protocols. The extent of plasma binding was in agreement with the radiolabeled studies; however, the methodology reported herein enables reliable quantification of Fu values for highly-bound drugs and is not limited by the radiochemical purity. In order to meet the rigor of a development study, equilibrium dialysis of unlabeled drug must be supported by an appropriately validated bioanalytical method along with studies to determine compound solubility and stability in matrix and dialysis buffer, nonspecific binding to the dialysis device, and ability to achieve equilibrium in the absence of protein. The presented methodology establishes an experimental protocol for definitive PB measurement, which enables quantitative determination of low Fu values, necessary for navigation of new regulatory guidances in clinical drug development.