Microdialysis Technology-based Pre-clinical Pharmacokinetic Study Of Innovative Drugs Pentapeptide

Pentapeptide (Met-Gln-Cys-Asn-Ser), as a new anti-brain stem drug, has been synthesized by modern technology. Dipeptide (Asn-Ser) is the major metabolite in vivo.Our study is based on the preclinical pharmacokinetics study of Pentapeptide (Met-Gln-Cys-Asn-Ser) and its metabolite (Dipeptide) in SD rats and Beagle dogs. The results provide a reference for the further conduct of clinical research.1. Preparation of laboratory-made microdialysis probe and study on the probe in-vivo recoveryMicrodialysis probes were self-designed and completed, the in vitro recovery of the probes were investigated. The experimental results show that in vivo recoveries can be determined by retrodialysis method. The microdialysis recoveries of TMP were inversely proportional to the perfusing rates while independent of TMP concentrations, the performance of the micordialysis system was stable over a120-min study. The microdialysis technology could be used for the pharmacokinetic study of pentapeptide (Met-Gln-Cys-Asn-Ser).2. The establishment of analytical method of Pentapeptide and its major metabolite (Dipeptide) in dialysate matrixA rapid and sensitive method based on microdialysis combined with liquid chromatography-tandem mass spectrometry was developed for the determination of Pentapeptide and its major metabolite (Dipeptide) in dialysate matrix. The analytes were separated on a Hanbon Hedera CN column (5μm,1004.6mm) with the mobile phase composed of methanol-water containing0.1%formic acid (60:40, v/v) at a flow rate of0.5mL/min. The method had a chromatographic total run time of3min and the injection volume was lOul. Detection was performed in electrospray positive mode and quantification was executed in multiple reaction monitoring mode. All the validation data, such as specificity, accuracy, precision, linearity, matrix effect and stability were within the required limits. The method has been successfully applied to pharmacokinetic study of the Pentapeptide and its major metabolite (Dipeptide) in dialysate matrix.3. The pharmacokinetics study of Pentapeptide and its major metabolite (Dipeptide) in SD rats blood dialysateSprague-Dawley rats were divided into three groups and received0.5,1and2mg/kg injected doses of PEP-801solutions (calculated by Pentapeptide) using Intravenous push.The validated LC-MS/MS methd was used to determine the concentration of Pentapeptide and its major metabolite (Dipeptide) in rat blood dialysate. Pharmacokinetic parameters were determined by probes calibration and non-compartment analysis method. The elimination half-life (t12) of Pentapeptide were1.92±0.23min、2.27±0.35min、2.09±0.30min, the mean residence time (MRT) of Pentapeptide were6.62±0.17min、6.84±0.18min、6.68±0.08min, the max blood drug level (Cmax) of Pentapeptide were176.85±9.62ng/mL、282.45±29.07ng/mL、556.27±26.41ng/mL, the clearance of Pentapeptide(CL) were0.27±0.02L/min/kg、0.33±0.02L/min/kg、0.33±0.01L/min/kg, the apparent volume of distribution(VD) of Pentapeptide were1.76±0.13L/kg、2.23±0.17L/kg、2.21±0.06L/kg, the AUC0～τ and AUC0～∞values of Pentapeptide were1831.11±132.68ng·min/mL,3008.2±202.14ng·min/mL、6005.73±192.46ng·min/mL and1888.21±148.32ng·min/mL、3080.88±191.99ng·min/mL、6058.71±182.61ng·min/mL, respectively. The t1/2of Dipeptide were4.31±0.42min、4.38±0.67min,6.33±0.45min,, the MRT of Dipeptide were8.29±0.27min、8.14±0.08min、8.04±0.10min, the Cmax of Dipeptide were241.44±8.74ng/mL、349.61±18.31ng/mL、705.7±30.59ng/mL, the CL of Dipeptide were0.19±0.01L/min/kg、0.22±0.01L/min/kg、0.23±0.01L/min/kg, the VD of Dipeptide were1.54±0.12L/kg、1.83±0.05L/kg、1.85±0.08L/kg, The AUC0～τ and AUC0～∞, values of Dipeptide were2613.94±149.23ng·min/mL,4397.25±141.28ng·min/mL8596.52±278.4ng·min/mL and2705.15±156.71ng-min/mL、4449.54±149.96ng·min/mL、8693.46±279.48ng·min/mL, respectively. the result shown that the AUC0～∞values increased as the dose increased in a linear manner, meeting the characteristics of the linear pharmacokinetics.Sprague-Dawley rats were divided into three groups and received0.5,1and2mg/kg injected doses of PEP-801solutions (calculated by Pentapeptide) using Intravenous push. The validated LC-MS/MS methd was used to determine the concentration of Pentapeptide and its major metabolite (Dipeptide) in rat blood dialysate. Pharmacokinetic parameters were determined by probes calibration and non-compartment analysis method. The mean residence time (MRT) of Pentapeptide was20.53±4.40min, the max blood drug level (Cmax) of Pentapeptide was54.81±3.11ng/mL, the clearance of Pentapeptide(CL) was1.84±0.25L/min/kg, the apparent volume of distribution(VD) of Pentapeptide was36.96±1.69L/kg, The AUCO-24values of Pentapeptide was467.48±12.58ng·min/mL4. The pharmacokinetics study of Pentapeptide and its major metabolite (Dipeptide) in Beagle dogs blood dialysateBeagle dogs were divided into three groups and received0.2,0.4and0.8mg/kg injected doses of PEP-801solutions (calculated by Pentapeptide) using intravenous push.The validated LC-MS/MS methd was used to determine the concentration of Pentapeptide and its major metabolite (Dipeptide) in beagle dogs blood dialysate. Pharmacokinetic parameters were determined by probes calibration and non-compartment analysis method. The elimination half-life (t1/2) of Pentapeptide were2.76±0.17min、2.18±0.21min、2.17±0.18min, the mean residence time (MRT) of Pentapeptide were17.43±0.19min、17.22±0.17min、17.57±0.00min, the max blood drug level (Cmax) of Pentapeptide were81.08±6.88ng/mL、142.44±4.65ng/mL、252.00±7.13ng/mL, the clearance of Pentapeptide(CL) were0.09±0.01L/min/kg、0.1±0.00L/min/kg、0.12±0.00L/min/kg, the apparent volume of distribution(VD) of Pentapeptide were1.54±0.11L/kg、1.73±0.05L/kg、2.17±0.07L/kg, the AUC0～τ and AUC0～∞values of Pentapeptide were2223.7±147.02ng·min/mL、3941.57±124.1ng·min/mL、6452.32±166.76ng·min/mL and2274.22±141.33ng·min/mL、3974.67±132.72ng·min/mL、6484.11±168.43ng·min/mL, respectively. The t1/2of Dipeptide were3.56±0.17min、5.62±0.38min、7.32±0.77min, the MRT of Dipeptide were20.59±0.61min、19.97±0.2min、20.18±0.43min, the Cmax of Dipeptide were124.78±9.82ng/mL、179.68±15.12ng/mL、276.85±2.76ng/mL, the CL of Dipeptide were0.06±0.01L/min/kg、0.08±0.00L/min/kg、0.1±0.00L/min/kg, the VD of Dipeptide were1.18±0.16L/kg、1.56±0.06L/kg、2.03±0.08L/kg, The AUC0～τ and AUC0～∞values of Dipeptide were3444.4±333.91ng·min/mL、5033.76±227.54ng·min/mL、7848.42±200.36ng·min/mL and3512.9±337.41ng·min/mL、5117.61±230.66ng·min/mL、7945.58±188.33ng·min/mL, respectively. The result shown that the AUC0～∞values increased as the dose increased in a linear manner, meeting the characteristics of the linear pharmacokinetics.