Pharmacokinetics Of A Novel Peptide AWRK6 In SD Rats

AWRK6 is a novel Synthetic Cationic polypeptide composed of 18 amino acid?SWVGKHGKKFGLKKHKKH,2130.5 Da?,which was modified from antibacterial peptide dybowskin2-CDYa?SAVGRHSRRFGLRKHRKH,GenBank number:ACF08009.1?by altering its amino acid sequence through substituting Lys for Arg,aiming to improve its biological activity and stability.Previous laboratory studies have shown that as a new GLP-1 receptor agonist,AWRK6 can promote the secretion of insulin from MIN6 cells in T2DM mice and relieve insulin resistance;treat the inflammatory response induced by endotoxin lipopolysaccharide?LPS?and alleviate liver damage;have broad-spectrum killing effects for both Gram-positive and Gram-negative bacteria.Based on the results of early pharmacology and pharmacodynamics,the pharmacokinetics of AWRK6 in SD rats was studied in this study.First,in this study,the stability of AWRK6 in vitro were determined.It was examined that AWRK6 was placed for the short-term storage stability and three freezes-melting cycle.Additionally,the stability of AWRK6 in organic solvents was investigated,as well as the stability AWRK6 in acidic and alkaline environments.A quantitative method of AWRK6 in SD rat plasma samples was developed and validated using liquid chromatography in tandem triple quadrupole mass spectrometry.The plasma sample pretreatment method was also developed.The established quantitative analysis method has been systematically validated.Methodological validation results showed that the method was in accordance with the requirements of the FDA biological sample analysis method principles.SD rats were selected and received intraperitoneal injections and intravenous injection.Rats blood samples were quickly collected from the orbital vein after administrations.The confirmed LC-MS/MS method was successfully applied to quantitatively analyze the drug concentration of AWRK6 in SD rat plasma samples.The drug concentration-time curve was drawn,the absolute bioavailability of AWRK6 intraperitoneal injection was calculated,and the main pharmacokinetic parameters and pharmacokinetic behavior were analyzed and evaluated using pharmacokinetic analysis software combined with statistical methods.The results showed that after a short-term storage and three freeze-thaw cycles,the stability of AWRK6 was good.But significant degradation occurred after freezing at-20°C for 30 d,and the degradation of AWRK6 was more intense at higher concentrations than at lower concentrations.Acetonitrile has a bad effect on the structural stability of AWRK6,while methanol is relatively mild.As a cationic antibacterial peptide,AWRK6 undergoes severe degradation in alkaline environments,and has better stability in acidic and neutral environments.Methodological validation results showed that the established quantitative method of AWRK6 in SD rat plasma has a linear range between 0.05?g/mL and 10?g/mL,and the lower limit of quantification reached 50 ng/mL.The extraction recovery rate of AWRK6 were at82.178%-86.368%,the matrix effect was from 99.523%to 104.772%.Extraction recovery rate of internal standard was about 82.805%,and the matrix effect of that was approximately 97.787%.Plasma endogenous impurities and organic solvents did not interfere with the chromatographic peaks of the components to be measured,nor did they inhibit the ionization and mass spectral response of the components to be measured.The intra-day precision of high,medium and low concentration QC samples was between 1.245 and 3.132%,and the inter-day precision was between 2.970%and3.490%.The relative error?RE?were between-7.940%and-13.456%,the precision and accuracy of the AWRK6 quantitative method were within±15%.Plasma samples had good short-term storage stability under a variety of storage conditions,while significant degradation occurred after storage at-20°C for more than 30 days?RE in the low-concentration group reached-34.737%?.After validation,the quantification method has the characteristics of high sensitivity,strong specificity,good reproducibility,simple and convenient sample processing methods with short analysis time,which meets the requirements of the FDA in vivo drug analysis method principle.After SD rats had received intraperitoneal injections,AWRK6 absorbed faster(T_maxax between 1.167 and 1.25 h)and cleared slowly.The half-life(T_1/2)was between 2.781 h and 2.946 h,with CL of 2.879?3.263 L/h/kg.MRT was about 2.256?2.525 h,and the absolute bioavailability?F?of the intraperitoneal injection is higher?between48.807%?52.54%?.As for Intravenous injection,t_1/2 of AWRK6 was 1.983±0.583 h,CL was 1.699±0.219 L/h/kg,with Vd of 4.884±1.736 L/kg.The linear dynamics related parameters C_max and AUC_0-t increase proportionally with the increase of the approximate dose,and linear relationship is good,as a result of showing linear pharmacokinetic characteristics.After fitting the compartmental model,the pharmacokinetic behavior of AWRK6 was in accordance with the two-compartment model with a weight coefficient of 1.The present study evaluated the plasma pharmacokinetics of the novel peptide AWRK6 in rats.This research will provide significant reference for the subsequent clinical development and safe use of AWRK6,the design of new dosage forms,the further modification of peptide sequences,and the in-depth development of new functions of AWRK6.