Phase Ⅰ Clinical Trial Of Recombinant Human Thrombopoietin In Healthy Volunteers

AIM Recently, thrombopoietin, an important hematopoietic factorhas been cloned. Recombinant human thrombopoietin (rhTPO) is a full-length glycosylated molecule produced in a genetically modified mammalian cell line and purified by standard technique by our Chinese boffin, National First new drug. Our Phase I clinical trial was performed to assess clinical tolerance and pharmacokinetics after a single subcutaneous injection of domestic rhTPO (a product of Zhongxinguojian Pharmaceutical Co Ltd) in healthy volunteers, and provide safety and reasonable administration for Phase II clinical trial. METHODS The study was designed in 3 parts. The first part was to set up the analytic methodology; and the second part was mainly study the clinical tolerance after a single subcutaneous injection of rhTPO(0.5².0 ug-Kg^-1) in healthy volunteers; the third part was study the pharmacokinetics after a single subcutaneous injection of rhTPO (0.5².0 ug.Kg^-1) in healthy volunteers. 1. Set up the analytic methodology:An ELISA method with high precision, high particularity andsensibility was validated for rhTPO assay in serum sample. Method recovery rate and drawn recovery rate, RSD of inter-plate precision and intra-plate precision, selective and ruggedness to proved whether it could be study the pharmacokinetics of rhTPO. 2. Study on clinical tolerance of rhTPO:27 subjectes voluntarily signed an informed consent concent agreement that was in compliance with Chinese Food and Drug Administration regulation and approved by the Ethic Committee of Xijing Hospital of the fourth military medical university, Xian, Shaanxi. Volunteers were judged to be in good health based on the results of a medical history, physical examination, laboratory profile and electrocardiogram. All were not possibly sensitive to some medication. The exclusion criteria included participation in any investigational drug trial within 3 month prior to the current study; receipt of any medications on a longterm basis. Volunteers avoided using other drugs for at least one week prior to the study and untilafter its completion.27 healthy volunteers were randomized to 3 groups and fasted for 12 hours before received a single subcutaneous injection of rhTPO in a dosage of 0.5 μg.Kg^-1. 1.0 μg.Kg^-1.2.0μg.Kg^-1 respectively. They were instructed to remain in a sitting or standing position for the first 4h after the dose, and abstained from food and beverages until 4h after dose administration. Water intake was allowed after 2h of dose; water, lunch and dinner were provided to all volunteers according to a time schedule. They were also refained from alcohol, tea, coffee, alcoholic beverages, and smoking.The symptoms, side-effects were monitored, and laboratory teste, inculding a complete blood cell count, platelet form, thrombin time,serum chemistry and urinalysis were followed serially. PRACS Institute staff throughout the confinement period of study continually monitored the volunteers.Results are expressed as mean ±standard deviation, and evaluated statistically using a paired t-test, ANOVA or x2 test by statistical software Excel or SPSS. 3. Study on pharmacokinetics of rhTPO:Approximately 6 ml of blood samples for rhTPO assay were drawn before (Oh) and at 0.5,1,2,4,6,8,10,12,24,36,48, 72,96,120,144, 168 and 216 h after dose administration. The blood samples were stored for 30 min before centrifuged at 1000g for 5 min at room temperature; serum was separated and stored at -20°C until* analysis instantly.RhTPO was extracted from serum samples and quantified by ELISA, The Tmax and Cmax were directly read from the individual serum concentration-time plots, and calculated the AUC (0-216h) by linear trapezoidal method, and Calculated Ke, Ka, t1/2 by pharmacokinetics statistical software 3P97. RESULTS1. 27 healthy volunteers, 17 males and 10 females. Analysis of the volunteers' mean age, weight, and height of 3 groups does not indicateany significant differences. All the volunteers completed the study.2. A final tolerance assessment took place within 21 days of trial completion. Out of the 27 healthy volunteers receiving single subcutaneous injection of 0.5 μg.Kg-1.1.0μg.Kg-1,2.0μg.Kg-1 domestic rhTPO, only one showed tetters on limbs 36 hours after the injection, one showed tetters on the part of injection, one showed transient elevation of HGB, and one showed transient elevation ofWBC. The others had no adverse drug reaction (ADR) or any side-effectes. No any organ, system toxicity was observed.3. All volunteers were associated with some increase of platelet count, peaking at the day 14 and returning to the baseline level by day 21. No abnormality was observed in form and function when the platelet count peaked.4. Serum chemistry, thrombin time and urinalysis were tested beforeand at 14d, no abnormal results were observed.5. In this experiment, rhTPO was extracted from serum samples and quantified by ELISA, and the limit of quantification (LOQ) for rhTPO was 50pg-ml-1 in serum; regression equation of rhTPO in serum was OD=-3.10264+1.0459XC (r=0.99645, n=6); and drawn recovery rate were 99.2%, 100.2%, 100.8%; inter- plate precision were 0.5%, 1.3%, 10.8%; intra-plate precision were 1.95%, 0.647% , 1.49% respectively.6.The main pharmacokinetics parameters were: T1/2ke: 46.74±6.36 h, 48.53±2.29 h and 51.88±3.34 h; T1/2ka: 2.17±0.53 h,2.64±0.53 h and 2.84±0.62 h; TmaX: 10.00±1.51 h,10.22±1.20 h and 10.00±1.00h; Cmax: 312.29±61.65pg.ml₁,465.14±46.94pg.ml-1 and 811.34±106.73 pg.ml-1; AUC (0-216h) :17269.92±4470.23 pg.ml.h-1,29710.56±3890.28 pg.ml.h-1 and 3358.41±5608.04 pg.ml.h-1 respectively. CONCLUSION1. When administrated subcutaneously in healthy volunteers within dosage of 0.5-2.0 ug.Kg-1, rhTPO was well tolerated by human beings and without significance adverse effectes. RhTPO increases peripheral platelet count and has no effect on its form and aggregation function.2. This analysis method has high sensitivity, accuracy and wonderful...