| PEP801(Met-Gln-Cys-Asn-Ser), a synthetic pentapeptide produced by Entamoebahistolytic in axenic culture, was found to be an anti-inflammatory functional peptide.In vivostudies have suggested that it can inhibit the locomotion of human monocytes. Latestresearches demonstrated PEP801can protect the brain from ischemic injury. Up to now,PEP801is in the stage of preclinical-study. The pre-clinical pharmacokinetic research data ofPEP801has not been reported in recent years. This study was designed to evaluate the pre-clinical pharmacokinetics of PEP801in animals to support the pharmacokinetics study inhuman, clinical dosage and dosage form development.1. Preliminary investigation on the stability and metabolism of PEP801in animalsPEP801(Met-Gln-Cys-Asn-Ser) has Cys and Met which easily undergo oxidation,especially for Cys which contains thiol. In addition, in biological matrix, peptides oftenundergo rapid degradation in presence of different metabolic enzymes to obtain extreme shorthalf-life time. Therefore, a preliminary investigation on stabiliity and metabolism of PEP801(Met-Gln-Cys-Asn-Ser) using LC-MS/MS was performed.Through basic acid/base/organicsolvent stability test, PEP801was found to be more stable in acid enviroment and organicsolvent. However, remarkable degradation was found in blood. Low temperature, antioxidantand enzyme inhibitors could not prevent its rapid degradation in blood. However, directmethonal addition could stabilize PEP801in blood, which was selected as the samplepretreatment. The main degradation site might be blood and liver vial rat blood and tissuehomogenate incubaton stability tests. In addition, the metabolic stability of PEP801in bloodand liver microsome of different species such as SD rat, beagle dog and human demonstratedthe labile of PEP801: SD rat> beagle dog> human.Thus, in vitro metabolites in rat blood and liver microsome have been indentified usingQ-Trap and Q-TOF. The main metabolism pathway was the cleavage of amide acid bone andpotential dehydrogenation after amide acid bone cleavage. Four main metabolits, tetrapeptide(Gln-Cys-Asn-Ser), dipeptide (Met-Gln, Gln-Cys, Asn-Ser) have been indentified andsynthesized. Meanwhile from preliminary determination of in vivo metabolites study,dipeptide (Asn-Ser) was relatively dominant and stable, which was selected as acharacterization of main metabolites and monitored with PEP801(Met-Gln-Cys-Asn-Ser), inorder to acknowledge the comprehensive pharmacokinetic and toxicokinetics of PEP801(Met- 2. Methods establishment of PEP801and metabolite, dipeptide (Asn-Ser) in biologicalmatrixMethods of determination of PEP801(Met-Gln-Cys-Asn-Ser) and metabolite, dipeptide(Asn-Ser) in rat and beagle dog blood by LC-MS/MS were developed. PEP801(Met-Gln-Cys-Asn-Ser) was very unstable in blood. Therefore, direct deactivate enzyme by methanolcontaining0.2%FA was used, which was followed by simple protein precipitation.Considering different chemical behavior of PEP801and metabolite dipeptide (Asn-Ser),different LC conditions were used to determine PEP801and metabolite dipeptide (Asn-Ser) inrat and dog blood. After methods validation, the intra-and inter-day precision and accuracy,sensitivity, specificity and linear range met the acceptance criteria, which can be applied forthe pre-clinical pharmacokinetic and toxicokinetics studies of PEP801in rat and dog.3. Pharmacokinetic study of PEP801in animalsThe validated methods were successfully applied in measuring levels of PEP801(Met-Gln-Cys-Asn-Ser) and metabolite, dipeptide (Asn-Ser) in blood following intravenousinfusion of low, middle and high doses of pentapeptide in rats and dogs, respectively.Pharmacokinetic parameters were calculated using non-compartment alanalysis method.The blood concentration-time profile demonstrated that the concentrations of PEP801(Met-Gln-Cys-Asn-Ser) and dipeptide (Asn-Ser) in rat blood were reduced quickly followingintravenous injection administration of PEP801at0.5,1,2mg/kg. The estimated eliminationhalf-life (T1/2) of PEP801and dipeptide (Asn-Ser) was0.24±0.02min,0.24±0.01min,0.28±0.03min and1.94±0.04min,2.93±0.23min,2.81±0.06min, respectively.Thearea under curve (AUC0-τ) of PEP801and dipeptide (Asn-Ser) was65.46±3.17ng min/ml,154.51±6.90ng min/ml,355.19±15.43ng min/ml and1133.28±32.70ng min/ml,2385.88±83.26ng min/ml,4263.99±74.85ng min/ml, erspectively. AUC0-τofPEP801and metabolite dipeptide (Asn-Ser) were positively correlated with the doses and thecorrelation coefficients which were r2=0.9936and r2=0.9921, respectively. Considering theshort half-life time, intravenous drip was recommended for clinical usage.It was also shown by the blood concentration-time data that the concentrations ofPEP801and dipeptide (Asn-Ser) in dog blood were reduced quickly following intravenousinjection administration of PEP801at0.2,0.4,0.8mg/kg by syringe pumps. The estimatedelimination half-life (T1/2) of PEP801and dipeptide (Asn-Ser) was0.47±0.05min,0.40±0.05 min,0.56±0.06min;8.55±0.47min,12.50±1.42min,13.06±1.20min, respectively.The areaunder curve (AUC0-τ) of PEP801and dipeptide (Asn-Ser) was1546.09±65.50ng min/ml,2675.17±54.4ng min/ml,6666.53±151.43ng min/ml and1258.04±102.95ng min/ml,2771.37±103.71ng min/ml,5117.49±352.76ng min/ml, erspectively. AUC0-τofPEP801and metabolite, dipeptide (Asn-Ser) were positively correlated with the doses and thecorrelation coefficients which were r2=0.9763and r2=0.9833, respectively. Considering theshort half-life time, intravenous drip was recommended for clinical usage.4. Toxicokinetics study of PEP801in animalsPEP801(Met-Gln-Cys-Asn-Ser) and metabolite, dipeptide (Asn-Ser) levels weredetermined in rat and dog blood following28days of repeat-dose intravenous infusion of15mg/kg,30mg/kg, and60mg/kg doses of PEP801. Toxicokinetics parameters were calculatedusing non-compartment alanalysis method.Comparing the blood concentration-time data in rat and blood of the first day and the28thday, no distinctish differences of the maximum concentration (Cmax), clearance (Cl) andAUC0～τwere found as admister time was prolonged, which indicated no significantmetabolism damage was observed under28days of repeating intravenous infusion amongthree levels for rat and dog species. |
PDF Full Text Request