Stability and degradation patterns of chemically modified analogs of apelin‐13 in plasma and cerebrospinal fluid

Abstract

Apelin is the endogenous ligand of APJ, which belongs to the superfamily of G protein-coupled receptors. In recent years, the apelin/APJ system has been detected in many tissues and emerges as a promising target for the treatment of various pathophysiological conditions. Pyr1-apelin-13 is the major isoform of apelin in human plasma; however its stability and proteolytic degradation pattern remain poorly understood. The aim of the present study was first to identify the cleavage sites of Pyr1-apelin-13 in mouse, rat and human plasma and rat cerebrospinal fluid, then to determine its stability to proteolytic degradation following intravenous administration in rats. Secondly, key residues were substituted by natural and unnatural amino acids in order to examine the impact on in vitro stability and degradation pattern. The kinetics of degradation revealed that the Leu5-Ser6 peptide bond of Pyr1-apelin-13 is the first cleavage observed in plasma, independently of the species. Replacement of Phe13 by unnatural amino acids showed a 10-fold increase in plasma stability although the hydrolysis of Pro12-Phe13 bond, previously described as a site of cleavage by ACE-2, was not observed. In vivo, this Pro12-Phe13 bond was cleaved yet appears as a minor product compared to hydrolysis of the Pro10-Met11 bond. This study pinpoints the most critical amino acids targeted by proteases and will be instrumental for the design of Pyr1-apelin-13 analogs possessing increased stability.

Role of the Vasopressin/Apelin Balance and Potential Use of Metabolically Stable Apelin Analogs in Water Metabolism Disorders

“…In addition, Gerbier et al (33) showed that K17F and pE13F have a half-life in mouse plasma of 4.6 and 7.2 min, respectively, and Murza et al (42) showed that pE13F has a 14 min half-life in rat plasma. Regarding apelin-36, Japp et al (43) suggested from experiments conducted in healthy human subjects that the half-life of apelin 36 is lower than 5 min.…”

“…Numerous technologies, such as PEGylation (44, 45), palmytoylation (46) conjugation to albumin, N-terminal acetylation (33), C-terminal amidation (47), use of unnatural amino acids (15, 33, 42, 48), or main chain modifications (cyclization) (49–51) have now been set up to increase the in vivo plasma half-life of peptides (52). Table 1 summarizes the pharmacological characteristics of the main metabolically stable apelin analogs described in this section.…”

“…Most studies that aim to develop apelin analogs have focused on pE13F (15, 42, 46, 48, 50, 51, 53) or apelin-36 (44, 45); however, K17F, which had an affinity 10 times higher than that of pE13F for human ApelinR, was 10 times more efficient at inducing internalization of rat ApelinR and also decreased arterial BP more strongly than did pE13F (20). …”

Regulation of the Apelinergic System and Its Potential in Cardiovascular Disease: Peptides and Small Molecules as Tools for Discovery

“…ACE2 cleavage occurred only in vivo. 59 Reproduced with permission from Biopolymers (Murza, A.; Belleville, K.; Longpre, J. M.; Sarret, P.; Marsault, E. Stability and degradation patterns of chemically modified analogs of apelin-13 in plasma and cerebrospinal fluid. 2014 , 102 , 297–303).…”

[Pyr1]-Apelin-13 delivery via nano-liposomal encapsulation attenuates pressure overload-induced cardiac dysfunction

“…The plasma instability of apelin is due to the rapid degradation of the peptide [22,25,26,45] which results in significantly short plasma half-life (<8 min) [46]. Nanocarrier drug formulations have been found beneficial for the sustained delivery of therapeutic compounds in various cardiac disease [27–29].…”