Linking (Pyr)apelin-13 pharmacokinetics to efficacy: Stabilization and measurement of a high clearance peptide in rodents.

Apelin， the endogenous ligand for the APJ receptor， has generated interest due to its beneficial effects on the cardiovascular system. Synthesized as a 77 amino acid preproprotein， apelin is post-translationally cleaved to a series of shorter peptides. Though (Pyr)apelin-13 represents the major circulating form in plasma， it is highly susceptible to proteolytic degradation and has an extremely short half-life， making it challenging to quantify. Literature reports of apelin levels in rodents have historically been determined with commercial ELISA kits which suffer from a lack of selectivity， recognizing a range of active and inactive isoforms of apelin peptide. (Pyr)apelin-13 has demonstrated beneficial hemodynamic effects in humans， and we wished to evaluate if similar effects could be measured in pre-clinical models. Despite development of a highly selective LC/MS/MS method， in rodent studies where (Pyr)apelin-13 was administered exogenously the peptide was not detectable until a detailed stabilization protocol was implemented during blood collection. Further， the inherent high clearance of (Pyr)apelin-13 required an extended release delivery system to enable chronic dosing. The ability to deliver sustained doses and stabilize (Pyr)apelin-13 in plasma allowed us to demonstrate for the first time the link between systemic concentration of apelin and its pharmacological effects in animal models.