In Vivo Pharmacokinetics Of Polypeptide ZP-1 Specially Binding To Lung Cancer In Rats

The polypeptide ZP-1 specially binding to lung cancer(DDP-Arg-Gly-Asp), a small molecule tripeptide by produced, has targeted inhibition efficacy for Lung Cancer Cells. It can induce tumor cell apoptosis and the process associated with the activation of caspase-3. The characteristics of ZP-1 in latest researches were as following: smaller liver and kidney toxicity, higher tissue specificity, higher tumor inhibition rate and so on. Currently, ZP-1 is still in the stage of preclinical-study and the relevant pre-clinical pharmacokinetic parameter of ZP-1 has not been reported in recent years. The aim of this paper is to study its pre-clinical pharmacokinetics in rats to provides the reference for the clinical pharmacokinetic study. PartⅠ:Methods establishment of ZP-1 in plasma from SD ratsMethods of determination of ZP-1 in rat blood by HPLC were developed. By screening of the mobile phase, we choose 0.1% aqueous trifluoroacetic acid and 0.1% trifluoroacetic acid in acetonitrile(97: 3) as the mobile phase and using an isocratic elution mode; ZP-1 was very unstable in plasma. Therefore, direct deactivate enzyme by 50% aqueous trifluoroacetic acid was used, which not only was followed by simple protein precipitation, but also improve its recovery in plasma rate(> 80%). At the same time the analytical method was validated. The results show, the specificity, the standard curve, the precision, the accuracy, the recovery and quantitative limits met in the requirements of biological sample analysis methods, which can be applied for the pharmacokinetic of ZP-1 in the SD rats study. PartⅡ:Pharmacokinetic study of ZP-1 in SD ratsThe validated methods were successfully applied in measuring levels of ZP-1 in plasma following femoral vein intravenous infusion of high, middle and low doses(38.3 mg/kg, 25.5 mg/kg, 17 mg/kg of tripeptide in rats, respectively. The results showed, the half-life in vivo metabolism was faster, the apparent volume of distribution was relatively low, the dose was directly proportional to AUC and the pharmacokinetic behavior of ZP-1 in vivo was conformed to one compartment model. The average half-life(T1/2) of three dose ZP-1 was 15.05±1.69 min, 15.92±2.41 min, 17.94±2.23 min, respectively. The area under curve(AUC0-t) of ZP-1 was 1440.99±48.78 mg/L·min, 821.34±73.54 mg/L·min, 568.13±56.26 mg/L·min, respectively. The clearance(CL) of ZP-1 was 0.025±0.006 L/min/kg, 0.021±0.004 L/min/kg, 0.027±0.004 L/min/kg, respectively. The apparent volume of distribution(V) of ZP-1 was 0.55±0.062 L/kg, 0.61±0.075 L/kg, 0.70±0.12 L/kg, respectively. The mean residence time(MRTo-t) of ZP-1 was 13.22±3.41 min, 14.46±2.11 min, 16.56±2.31 min, respectively. Part Ⅲ:Preliminary investigation on the stability of ZP-1 in the tissue homogenatesThe polypeptide ZP-1 specially binding to lung cancer(DDP-Arg-Gly-Gln) has Cisplatin and Arg which easily oxidative hydrolysis. At the same time, in plasma, Peptide drugs often undergo rapid metabolism in presence of different proteolytic enzymes to obtain extreme short half-life time. To this point, the metabolic stability of ZP-1 in different tissue homogenates were performed. In the first chapter the results show that ZP-1 is stable in saline enviroment.T he study investigated the metabolism of ZP-1 in homogenates from rats liver, kidney, lung and intestinal in vitro incubation and this experiment found that the rank order of degradation rate of ZP-1 in the four biological matrices was lung> kidney> liver> intestine, with the half-life of 6.5, 12.1, 16.3, 19.7 min, respectively. The metabolism of ZP-1both happened in rat lungs, kidneys, liver and small intestine and the metabolic half-life of ZP-1both short. Part Ⅳ:The disposition of ZP-1 in the in situ vascularly perfused rat intestine-liver modelThis chapter we built the model of in suit perfused rat intestine-liver preparation to study the absorption and metabolism of ZP-1. A does of ZP-1 were delivered to the superior mesenteric artery of the one-through intestine-liver vascular perfusion preparation. The plasma perfusion samples were collected at different time points(0, 1, 3, 5, 10, 15, 30, 45, 60 min) at portal and hepatic vein. Analysis and Treatment of samples drawn that the steady-state intestinal extraction ratio of ZP-1 was smaller(0.48±0.61) compared to that for the liver(0.78±0.74), indicating that the metabolism of ZP-1 was faster in the liver cells than in the intestinal cell.With the later technique, an intraduodenal injection of a dose of ZP-1 was made close to the pyloric sphinctor, whereas the intestine preparation was recirculated with blank perfusate. Reservoir sampling times at 0, 1, 3, 5, 10, 15, 30, 45 and 60 min for each perfusion period. The partial results showed that ZP-1 was metabolism largely in the intestine, the absorption(0.81%) was very poor and the first-pass metabolism(including the intestines and liver metabolism) was serious. In summary, the ZP-1 of oral administration was inappropriate for clinical usage.