Determination of the Peptide AWRK6 in Rat Plasma by Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) and Its Application to Pharmacokinetics.

Lili Jin,Hongchuan Xin,Haibo Ding,Qiuyu Wang,Volkan Degirmenci,Qifan Miao,Dianbao Zhang

doi:10.3390/molecules27010092

Abstract

AWRK6 was a synthesized peptide developed based on the natural occurring peptide dybowskin-2CDYa, which was discovered in frog skin in our previous study. Here, a quantitative determination method for AWRK6 analysis in rat plasma by using liquid chromatography-tandem mass spectrometry (LC-MS/MS) was established and validated following U.S. FDA guidelines. A combination of plasma precipitation and liquid–liquid extraction was applied for the extraction. For pharmacokinetics study, the rats were administrated with AWRK6 via intraperitoneal and intravenous injection. The prepared plasma samples were separated on an ODS column and analyzed by tandem MS using precursor-to-product ion pairs of m/z: 533.4→84.2 for AWRK6 and m/z: 401.9→101.1 for internal standard Polymyxin B sulfate in multiple reaction monitoring mode. AWRK6 concentrations in rat plasma peaked at about 1.2 h after intraperitoneal injections at 2.35, 4.7 and 9.4 mg/kg bodyweight. The terminal half-life was around 2.8 h. The absolute bioavailability of AWRK6 was 50% after 3 doses via injection, and the apparent volume of distribution was 4.884 ± 1.736 L. The obtained determination method and pharmacokinetics profiles of AWRK6 provides a basis for further development, and forms a benchmark reference for peptide quantification.

Highlights

Peptides are considered to be promising treatment options for microbial infections and metabolic disorders [1,2]
Developed peptides based on glucagon-like peptide-1 (GLP-1) or exendin-4; e.g., dulaglutide, exenatide, liraglutide, lixisenatide, and semaglutide have been approved for diabetes and obesity treatment in multiple markets across the world [4]
AWRK6 presented antibacterial and lipopolysaccharide-neutralizing activities, and it has been discovered that AWRK6 regulates glycolipid metabolism to improve type 2 diabetes mellitus and metabolic associated fatty liver disease (MAFLD) [8,9,10,11]

Summary

Introduction

Peptides are considered to be promising treatment options for microbial infections and metabolic disorders [1,2]. The gut-derived glucagon-like peptide-1 (GLP-1) presented glucose-dependent plasma glucose reduction effects. Developed peptides based on GLP-1 or exendin-4; e.g., dulaglutide, exenatide, liraglutide, lixisenatide, and semaglutide have been approved for diabetes and obesity treatment in multiple markets across the world [4]. A series of novel peptides were discovered in frog (Rana dybowskii) skin, and dybowskin-2CDYa was later modified to improve the stability, acquiring a novel peptide, namely AWRK6 [6,7]. AWRK6 presented antibacterial and lipopolysaccharide-neutralizing activities, and it has been discovered that AWRK6 regulates glycolipid metabolism to improve type 2 diabetes mellitus and metabolic associated fatty liver disease (MAFLD) [8,9,10,11]

Methods

Results

Conclusion