In Vitro Metabolic Stability of Exendin-4: Pharmacokinetics and Identification of Cleavage Products

Abstract

The aim of this study was to investigate the metabolic stability and cleavage sites of exendin-4 in rat tissue homogenates, as well as to identify the types of proteases involved in exendin-4 degradation. The stability of exendin-4 in kidney and liver homogenates from rats was evaluated using liquid chromatography-electrospray ionization mass spectrometry (LC-ESI-MS) with gradient elution. Furthermore, we used a combination of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and LC-ESI-MS/MS to identify the structures of the major degradation products of exendin-4, and peptidase inhibitors were used to characterize exendin-4 degradation in rat liver and kidney homogenates and to identify the proteases involved in exendin-4 metabolism. Exendin-4 had a half-life of 7.8 and 100.9 min in the kidney and liver homogenate, respectively. The enzymes most likely to be involved in the degradation of exendin-4 were aminopeptidases, serineproteases, and metalloproteases. Exendin-4(15-39) and exendin-4(16-39) were the predominant direct exendin-4 metabolites in the kidney, and the main product of exendin-4 metabolism in the liver was exendin-4(12-39). Our results indicated that the metabolism of exendin-4 involved an initial endoproteolytic cleavage and subsequent exoproteolytic digestion. The degradation of exendin-4 in the kidney and liver homogenates followed distinct patterns, and the primary cleavage sites of exendin-4 degradation in rat kidney homogenates were located after AA-14, and -15, whereas those in rat liver homogenates were located after AA-11.

Ureidopeptide GLP-1 analogues with prolonged activity in vivo via signal bias and altered receptor trafficking

“…In light of these interesting results, we sought to understand the underlying causes of such improvements of exenatide and lixisenatide by the substitution of one amino acid over 39 and 45, respectively. Our hypothesis was that the cleavage between positions 1 and 2 of exenatide (9) and lixisenatide (12) was a determining factor for their in vivo half-life 17 and, as we managed to improve the proteolytic stability of the peptides in vitro, as demonstrated by the increased mouse plasma stability, this would be reected in improved in vivo half-life proles. To validate this hypothesis, we conducted pharmacokinetic studies on healthy mice with Ex4 L (10) and Ex4 L [Ala u ] 2 (11).…”

“…10,11 However, despite having a glycine in position 2, exenatide, an FDA approved once-weekly treatment for T2DM, is still susceptible to degradation at its Nterminus. 17 We therefore hypothesize that modifying the backbone of exenatide might improve its proteolytic stability and prolong its activity in vivo. However, backbone modications at the N-terminal part of incretins and other ligands of class B GPCRs have scarcely been reported 15,[18][19][20] which reects the difficulty to mimic the complex network of interactions in the binding pocket of the receptor transmembrane domain.…”

Exendin‐4 from Heloderma suspectum venom: From discovery to its latest application as type II diabetes combatant

“…The enzymes responsible for the proteolytic actions of exendin‐4 are metalloproteases, aminopeptidases and serine proteases . The in vitro data also suggest that the breakdown of exendin‐4 in the liver first occurs through the actions of endopeptidases before being further excised by exopeptidases . The main peptide fragments produced from the proteolytic degradation of exendin‐4 are inactive metabolites indicates that they neither act as agonists nor antagonists of the GLP‐1 receptor …”

“…administration of exendin‐4 exhibits a better bioavailability compared to oral ingestion (Table ). This is because when exendin‐4 is ingested orally, it is absorbed via intestinal epithelial cells and enters the hepatic portal system which can be metabolised by the enzyme peptidases in liver . The first‐pass effects of oral ingestion reduce the bioavailability of exendin‐4.…”

“…Passive glomerular filtration is the primary route of renal elimination for exendin‐4 . The enzymes responsible for the proteolytic actions of exendin‐4 are metalloproteases, aminopeptidases and serine proteases . The in vitro data also suggest that the breakdown of exendin‐4 in the liver first occurs through the actions of endopeptidases before being further excised by exopeptidases .…”