| Parathyroid hormone(PTH) is a polypeptide hormone, which can stimulate bone metabolism and reborn normal bone tissues. PTH can increase bone density, reinforce sclerotin and improve bone architecture, which make it be the main treatment for osteoporosis. Recombinant human parathyroid hormone(1-34) for injection is a high expressing engineering bacteria constructed of the nucleotide sequence of human PTH(1-34) N-telopeptide region. According to the management methods of drug registration issued from State Food and Drug Administration (SFDA), rhPTH(1-34) is classified to a new category seven(7) drug biologic product, which has already been sold in foreign market but not in the Chinese market.AIM:To establish an Immunoradiometric assay (IRMA) method to monitor concentrations of rhPTH(1-34) in human plasma samples, to assess clinical tolerance and pharmacokinetics after a single and successive subcutaneous injection of rhPTH(1-34) in healthy volunteers, and to provide safe and reasonable guidance for Phase II clinical trial.METHODS:The study includes three parts.The first part was to establish an IRMA method with high specificity, high precision and sensibility for rhPTH(1-34) assay in plasma samples. Procedure: 200uL of untested or standard samples with different concentrations was added into the labeled tubes, and then 100μL of mice PTH-antibody, 125I was added into the above tubes. The above tubes were vortexed and mixed, and a bead was added into every tube by forceps. Incline the test tube racket while adding the bead. Use sealing membrane to seal the tubes. Blot out the fluid in each tube after incubation under room temperature for 18 to 24 hours. 2mL of eluant was added into each tube to wash for three times, and blotted cleanly. Recorded the result of radioactive rates counted in a gamma counter. Double repeating survey for every sample, a set standard curve for every kit was used to measure concentrations of unknown samples.The second part was to investigate clinical tolerance of rhPTH(1-34) injection in healthy volunteers following single- and multiple-dose administration. Volunteers were judged to be in good health based on the results of a medical history, physical examination, laboratory tests (regular blood, liver and urea analysis, cardiac electronic graph) and electrocardiogram. No one was allowed to have any history of medication sensitive and some systematic diseases. The exclusion criteria included participaters in any investigational drug trial within 3 month prior to the current study. Volunteers avoided using other drugs for at least two week prior to the study and until after its completion. During the study, the symptoms, side-effects were monitored, and laboratory teste, inculding Ca,P and ALP in blood, plasma chemistry and urinalysis were tested regularly. The whole study period was monitored by doctors and nurses. Results are expressed as mean±standard deviation, and evaluated statistically by a paired t-test, ANOVA (statistical software Excel or SPSS 10.0).The third part was to study the pharmacokinetics of rhPTH(1-34) following a single- and multiple-dose administration in healthy volunteers. Forty healthy volunteers were randomized to three single-dose groups and one multiple-dose group. They were remained sitting or standing within the first 4h after administration, and abstained from food and beverages until 4h after administration. Water, lunch and dinner were provided to all volunteers according to a time schedule. They were also refrained from alcohol, coffeine beverages, and smoking. According to the experimental design, forty subjects were fasting for 12 hours and administered. Blood samples were collected according to the time schedule, which included a blank drug sample before adminstration and then at 5,10,15,20,30,45,60,90,120,150,180 and 240min after administration. Plasma samples were immediately centrifuged at 3,600 rpm for 10 min at 4℃, and then stored at -80℃until analysis. RhPTH(1-34) was extracted from plasma samples and quantified by IRMA. Tmax and Cmax were actual data, AUC was calculated by statistical moment methods and other pharmacokinetic parameters were calculated by software Origin. Analysis of Variance (ANOVA) and two-one side Student's t test were adopted to evaluate the results statistically, which were expressed as mean±standard deviation.RESULTS:1. Forty healthy volunteers, 20 males and 20 females. All the volunteers completed the study. Clinical tolerance was assessed within 7 days for single-dose group and 14 days for multiple-dose group. During the study period, no one experienced visible adverse events after different doses of rhPTH(1-34). Respiration rate, heart rate, blood pressure, pulse, body temperature and other vital signs were normal. ECG was observed normally too. No any flares, ecchymoses, rashes or any other irritative responses was observed in the injecting part. During the whole study period, there were no abnormal physical signs shown in all subjects.2. Observed single subcutaneous injection of rhPTH (1-34) for 7days at concentrations of 10,20 and 40μg, respectively, and obseved multiple subcutaneous injection of 20μg for 14 days. Detected the blood biochemical indicators before and after the trial. There were no statistically significant deviations in blood biochemical indicators between two tests by t-test. Detected the urine routine tests before and after the trial. There was no significant difference in urine routine test among subjects.3. In the study, rhPTH(1-34) was detected by IRMA. The limit of quantification (LOQ) for rhPTH(1-34) was 20pg?mL-1; the linear range was 25～2500pg?mL-1.The intra-plate double-pipe was reproducible in this linear range, CV% was within 10% and accuracy was during -1.7% to 2.8%. Inter-plate precision were within 10% and accuracy was during -3.0% to 4.4%. One standard curve was set for each plate to calculate unknown samples concentration.4. The main pharmacokinetic parameters of rhPTH(1-34) after single administration were as follows:T1/2ke were 47.7±12.1, 50.1±16.4 and 53.6±14.4min; MRT were 56.3±3.9, 57.8±11.9 and 84.7±9.3min; AUC(0-240) were 9786.9±1456.3, 15425.5±1661.1 and 32716.2±3158.0 pg·min·mL-1; Tmax were 32.5±9.5, 24.0±9.7 and 35.0±13.5min; Cmax were 139.8±36.3, 203.9±50.4 and 295.8±43.5pg·mL-1, repectively. The main pharmacokinetic parameters of rhPTH(1-34) after successive administration of 7 days were as follows:T1/2ke was 50.9±5.9min; AUC(0-240) was 96955.8±3076.3pg·min·mL-1; Tmax was 30.5±11.9min; Cmax was 16955.8±3076.3 pg·mL-1.CONCLUSIONS:1. This analysis method has high sensitivity, accuracy and wonderful specificity, reproducibility. RhPTH(1-34) was stable in plasma for at least one month when stored at–80℃at low and high concentrations samples. The method was applicated to determine rhPTH(1-34) in plasma.2. RhPTH(1-34) injection was well tolerated in Chinese healthy volunteers at doses of 10-40μg. No significance adverse effectes were observed during the study.3. The interruption of endogenous parathyroid hormone antigen is less than LOQ. Blood drug level increased with the dose of RhPTH(1-34). The pharmacokinetic parameters of multiple doses are similar to the single dose and there was no drug accumulation in human body. The result of trial is consistent with the character of pharmacokinetics in foreign human body.
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