In vitro metabolic stability of exendin-4: pharmacokinetics and identification of cleavage products.
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.
Beijing Institute of Pharmacology and Toxicology, 27 Taiping Road, Haidian District, Beijing, 100850, PR China.
Sha
S
Liao
Beijing Institute of Pharmacology and Toxicology, 27 Taiping Road, Haidian District, Beijing, 100850, PR China.
Yuanjun
Y
Liang
Beijing Institute of Pharmacology and Toxicology, 27 Taiping Road, Haidian District, Beijing, 100850, PR China.
Zhiwei
Z
Zhang
Beijing Institute of Pharmacology and Toxicology, 27 Taiping Road, Haidian District, Beijing, 100850, PR China.
Jinglai
J
Li
Beijing Institute of Pharmacology and Toxicology, 27 Taiping Road, Haidian District, Beijing, 100850, PR China.
Juan
J
Wang
Beijing Institute of Pharmacology and Toxicology, 27 Taiping Road, Haidian District, Beijing, 100850, PR China.
Xiaoying
X
Wang
Beijing Institute of Transfusion Medicine, 27 Taiping Road, Haidian District, Beijing, 100850, PR China.
Guifang
G
Dou
Beijing Institute of Pharmacology and Toxicology, 27 Taiping Road, Haidian District, Beijing, 100850, PR China.
Zhenqing
Z
Zhang
Beijing Institute of Pharmacology and Toxicology, 27 Taiping Road, Haidian District, Beijing, 100850, PR China.
Keliang
K
Liu
D000595:Amino Acid Sequence; D000818:Animals; D002853:Chromatography, Liquid; D000077270:Exenatide; D007668:Kidney / Q000378:metabolism; D008099:Liver / Q000378:metabolism; D010455:Peptides / Q000737:chemistry / Q000378:metabolism* / Q000493:pharmacokinetics; D011480:Protease Inhibitors / Q000494:pharmacology; D055550:Protein Stability / Q000187:drug effects; D059748:Proteolysis; D051381:Rats; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014688:Venoms / Q000737:chemistry / Q000378:metabolism* / Q000493:pharmacokinetics
D010455:Peptides; D011480:Protease Inhibitors; D014688:Venoms; D000077270:Exenatide