Phase I Clinical Trial Of Recombinant Human Cytotoxic T Lymphocyte Associate Antigen-4 In Healthy Volunteers

Recombinant human CTLA4-antibody fusion protein is a fusion protein connected by human cytotoxic T lymphocyte-associated antigen 4 (CTLA4) and the extracellular region of human immunoglobulin G1 (IgG1) modified Fc fragment (hinge region, and CH2 zone CH3). It is a selective regulator for co-stimulation as the affinity to the molecular B7 on the antigen presenting cells is much higher than the affinity to CD28 on the surface of T cells. It can close B7 on the antigen presenting cells, and thus in combination with the CD28 pathway, can block the co-stimulation pathway and inhibit the activation of T cells (T-lymphocytes). The pathological process of Rheumatoid arthritis (RA) is related to T lymphocyte activation. We found copious activation of T lymphocytes in the intra-articular synovial of RA patients. According to the management methods of drug registration issued by the State Food and Drug Administration (SFDA), rhCTLA4-Ig is classified as a Category Seven (7) drug biologic product, which has already been sold in foreign markets but not in the Chinese market.Objective: To establish an Enzyme-linked immunosorbent assay (ELISA) method to monitor concentrations of rhCTLA4-Ig in human plasma samples, to assess clinical tolerance and pharmacokinetics after a single intravenous drip infusion of rhCTLA4-Ig in healthy volunteers, and to provide safe and reasonable guidance for a Phase II clinical trial.Methods: This study comprises the following three parts:The first part was to establish an ELISA method with high specificity, high precision and sensitivity for rhCTLA4-Ig assay in plasma samples. Procedure: CTLA4 monoclonal mouse anti-human was diluted with a proportional buffer and added to peridium on the enzyme scale plate. The enzyme scale plate was then incubated for 18 hours at 4℃and closed by buffer for 2 h at room temperature. The plate was washed with detergent solution. The standards of the different concentration and the diluted samples were loaded to the plate. The diluted biotin-labeled anti-second was then immediately added. Enzyme scale plate was placed, shocked and incubated for 2 hours at room temperature. Joined by dilution ratio of HRP-labeled enzyme linked avidin. Enzyme scale plate placed and shocked, incubation for half an hour at room temperature. Placed under the conditions of protection from light accession HRP liquid at room temperature reaction for 15 minutes. Termination of reaction, 30 min after termination of reaction read absorbance (A) values at 450 nm by enzyme scale equipment, record results. 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 rhCTLA4-Ig injection in healthy volunteers following single 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 sensitivity and some systematic diseases. The exclusion criteria included participants in any investigational drug trial within 3 month prior to the current study. Volunteers avoided using other drugs for at least two weeks prior to the study and until after its completion. During the study, the symptoms and side effects were monitored, and laboratory tests, inculding erythrocyte sedimentation rate in blood, plasma chemistry, and urinalysis were tested regularly. Comprehensive evaluation tolerance in healthy volunteers after used rhCTLA4-Ig was measured. The entire study period was monitored by doctors and nurses. Results are expressed as mean±standard deviation, and evaluated statistically by a paired t-test using ANOVA (statistical software Excel or SPSS 10.0).The third part was to study the pharmacokinetics of rhCTLA4-Ig following a single-administration in healthy volunteers. Twenty-seven healthy volunteers were randomized to three single-dose groups. They 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 also refrained from alcohol, caffeine beverages, and smoking. According to the experimental design, 27 subjects fasted for 12 hours and then administered. Blood samples were collected according to the time schedule, which included a blank drug sample before administration and then at 2, 4, 12, 24 hours, and 2, 3, 4, 7, 14, 28, 42, 56, 70 and 84 days after administration. Plasma samples were immediately centrifuged at 3,600 rpm, and then stored at -80℃until analysis. RhCTLA4-Ig was extracted from plasma samples and quantified by ELISA. DAS 2.1 software was used to calculated pharmacokinetic parameters, Tmax and Cmax were calculated from actual data, and AUC was calculated by statistical moment methods and other pharmacokinetic parameters were calculated by the Origin software. 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. All 27 healthy volunteers completed tests. Clinical tolerance was assessed within 84 days for single-dose group. During the study period, no one experienced visible adverse events after different doses of rhCTLA4-Ig. Respiration rate, heart rate, blood pressure, pulse, body temperature and other vital signs were normal. ECG was observed to be normal. No flares, ecchymoses, rashes or any other irritative responses were observed in the injecting part. During the study period, there were no abnormal physical signs in any subjects.2. We observed single intravenous infusion of rhCTLA4-Ig for 84 days at concentrations of 1, 10 and 20 mg, respectively, and detected the blood biochemical indicators before and after the trial. There were no statistically significant deviations in blood sedimentation rate between the two tests by t-test. We performed the urine routine tests before and after the trial. There was no significant difference in urine routine test among subjects.3. In the study, rhCTLA4-Ig was detected by ELISA. The limit of quantification (LOQ) for rhCTLA4-Ig was 0.4 ng?mL-1; the linear range was 1.95～500 ng·mL-1. The intra-plate double-pipe was reproducible in this linear range. CV % was less than 10 % and accuracy was in the range -5.3 to -1.1 %. Inter-plate precision was within 10 % and accuracy was in the range 0.6 to 6.2 %. One standard curve was set for each plate to calculate unknown sample concentration.4. After single intravenous infusions of 1, 10 and 20 mg rhCTLA4-Ig, the pharmacokinetic parameters were as follows: T1/2ke(15.13±2.62), (14.21±2.35) and (11.77±1.24) d;MRT (19.913±2.086), (19.630±1.832) and (18.795±0.832) d;AUC(0-84d) (170.64±27.75), (1490.26±231.23) and (2977.25±362.31)μg·d·mL-1;Tmax was 0.0416 d for all;Cmax (18.39±1.57), (186.91±24.70) and (416.84±34.40)μg·mL-1。CONCLUSIONS:1. Our analysis method showed high sensitivity, accuracy, specificity, and reproducibility. rhCTLA4-Ig was stable in plasma for at least 90 days when stored at–80℃at high, medium and low concentrations. The method was applied to determine rhCTLA4-Ig in plasma.2. rhCTLA4-Ig injection was well tolerated in healthy volunteers at doses of 1-20 mg. No significant adverse effects 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 rhCTLA4-Ig. The pharmacokinetic parameters of multiple doses are similar to the single dose and there was no drug accumulation in human body. The results of the trial are consistent with the character of pharmacokinetics in the human body as observed in other studies.