Phase I Clinical Pharmacokinetic Study Of Anti-recombinant Human Cytotoxic T Lymphocyte Related Antigen-4 In Rheumatoid Arthritis Patients

【Background】Recombinant human Cytotoxic T Lymphocyte associate Antigen-4 Fusion Protein (rhCTLA4-Ig), a selective costimulation modulator, inhibits T cell (T lymphocyte ) activation by binding to CD80 and CD86, thereby blocking interaction with CD28. This interaction provides a costimulatory signal necessary for full activation of T lymphocytes found in the synovium of parients with RA. It is indicated for reducing signs and symptoms, slowing the progression of strural damage, and improving physical function in adult patients with moderately to senerely active rheumatoid arthritis who have had an inadequate response to one or more DMARDs. RhCTLA4-Ig is produced by recombinant DNA technology, and a fusion protein combining 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). According to the regulations of drug registration issued from State Food and Drug Administration(SFDA),rhCTLA4-Ig belongs to the national therapy new drug,class seven, namely, has been on market on aboard but not yet in China. Our Phase I clinical trial of this new drug is to assess the clinical tolerance and pharmacokinetics in healthy volunteers, and the in Chinese rheumatoid arthritis patients.【Objectives】The aim of present study is to establish an Enzyme-linked immunoassay assay ( ELISA ) method to monitor the concentration of rhCTLA4-Ig in rheumatoid arthritis patients blood serum samples, to assess clinical pharmacokinetics after successive intravenous injection of rhCTLA4-Ig in rheumatoid arthritis patients, and provide safety and reasonable administration for 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 detection in rheumatoid arthritis patients'blood serum. An ELISA blood serum with high precision,high specifity and sensibility was validated for rhCTLA4-Ig determination in blood samples. RSD of inter-plate precision and intra-plate precision,selective and ruggedness to proved whether it could study the pharmacokinetics of rhCTLA4-Ig. Procedure: CTLA4 monoclonal mouse anti-human antibody was diluted with a proportional buffer and added to presidium on the enzyme scale plate. The enzyme scale plate was then incubated at 4℃and closed by buffer 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-secondary antibody was then immediately added. Enzyme scale plate was placed, shocked and incubated for 2 hours at room temperature. After incubation with the HRP-labeled enzyme linked avidin, enzyme scale plate placed and shocked, incubated at room temperature, kept from light. Upoun termination of reaction absorbance (A) values was read at 450 nm by enzyme scale equipment, results were recorded. Duplicate experiments were carried out, and a set standard curve for every kit was used to measure concentration of unknown samples.The second part was to study the pharmacokinetics of recombinant human Cytotoxic T Lymphocyte associate Antigen-4 Fusion Protein injection after multiple-dose administration to Chinese rheumatoid arthritis patients. 9 subjects voluntarily signed an informed consent 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.According to the experimental design,9 subjects were food forbidden for 12 hours and blood sampled just before drug administration and then at 0,2,4,12,24h,2,3,4,7,14,28,42,56,70,84 days after drug administration. Plasma samples were immediately centrifuged at 3,600 rpm for 10 min at 4℃, and then stored at -80℃until analysis. RhCTLA4-Ig was extracted from blood samples and quantified by ELISA. The T max and C max were actual data,and the AUC calculated by statistical moment methods and other pharmacokinetic parameters by software Origin. Resuls are expressed as mean±standard deviation,Analysis of Variance (ANOVA) and two-one side Student's t test were adopted to analyze the data.The third part was the pharmacokinetics of rhCTLA4-Ig in healthy adult subjecta after a single 10 mg·kg ^-1 intravenous injection and in RA patients after multiple 10 mg·kg ^-1 intravenous infusions. Pharmacokinetics parameters was compared to those of RA patients or healthy adult. Analysis of Variance (ANOVA) and two-one side Student's t test were adopted to statistically evaluate. 【Results】1. In this experiment,rhCTLA4-Ig was extracted from blood samples and quantified by ELISA. The limit of quantification for rhCTLA4-Ig was 6.25μg·L^-1;the linear range is 100～6.25μg·L^-1.The double-pipe was repeatable in this linear range,CV% were 100.4% to 112.1%,accuracy rating were during 0.73% to 4.6%. All indicatives to achieve pharmacokinetics test requirement.2. Ten rheumatoid arthritis patients,3 males and 7 females,other 1 rheumatoid arthritis patient was to reject because right bundle branch block. One male was enrollede experiment. During the study, no one experienced visible adverse effects at all doses of rhCTLA4-Ig. No unexpected severe adverse effect was observed, all subjects showed normal respiration,heart rate,blood pressure,pulse,body temperature and other vital things,and normal ECG. The injection part did not showed any flares,ecchymosed,rashes or any other irrigative responses. In general,there has been no abnormal physical signs in all subjects. As to the blood biochemical indicators or metaphase and urine routines indicators dielectric before or metaphase or after the test,we found no statistics significant deviations in all index between two groups by t-test in all 9 subjects and with no abnormal changes.3. The main pharmacokinetic at steady state were as follows: Cssmax were 247.1±50.3mg·L^-1, C ssmin were 19.7±9.5 mg·L^-1,C ssav were 65.4±25.3 mg·L^-1, DF were 3.48±1.61. At once every four weeks 10 mg·kg^-1 intravenous drip infusion of rhCTLA4-Ig,patients do not accumulate phenomenon?. At eighth weeks, the average drug concentrations were 27.1±8.1 mg·L^-1,and at sixteenth weeks 19.1±9.0 mg·L^-1. No difference was found between these two time points ( P<0.05 ) . Back the main pharmacokinetic at stead state were as follows:After the 0,2,4,8,12,16 weeks mult-dose 10 mg·kg ^-1 intravenous drip infusion of rhCTLA4-Ig,drug blood level peaked fast but eliminated torpidly. Tmax was 0.063±0.039d,C max was 247.1±50.3 mg·L^-1, and T1/2 was 4.7±12.6d .After serial 10 mg·kg ^-1 intravenous drip infusion of rhCTLA4-Ig,AUC(0-Z) was 2347±901mg·d^-1·L^-1 , AUC(0-∞) was 2362±906 mg·d^-1·L^-1,CL was 0.0050±0.0023 L·d·Kg^-1,VSS was 0.074±0.031 L·kg^-1.4. Clinical assessments was done according to the American College of Rheumatology criteria(ACR). As compared with the control group, the etanercept treatement group had a more rapid improvement in ACR20 and ACR50 in disease activity during the first three(78%,11%)treatment?.At the end of sixth treatment, the etanercept treatement group had a more rapid improvement in ACR20 and ACR50 in disease activity during the first three(100%,78%).As to the immunological blood sedimentation,C reactive protein and rheumatoid factor before or metaphase or after the test,we found no statistics significant difference in all index between two groups by t-test in all 9 subjects and with abnormal changes:blood sedimentation was valued at 16.67±19.83 mm·L^-1,C reactive protein 204.41±300.91 IU·mL^-1and rheumatoid factor 1.55±2.30 mg·L^-1.【Conclusions】1. This analysis method has high sensitivity,accuracy and wonderful specificity. The lineal concentrations correlate well the lineal area under curve;the linearity was in the scope of 6.25～100μg·L^-1 in plasma. The limit of quantization for rhCTLA4-Ig was all below 10%,the limit of quantization for rhCTLA4-Ig was 6.25μg·L^-1 in plasma;CV% of inter-case precision and intra-case precision were all below 10%.There was no significant difference. All the profiles meet the criterion of the SFDA. The results showed that the present method is suitable for the study of pharmacokinetics of plasma rhCTLA4-Ig.2. The ELISA was used to test concentration of rhCTLA4-Ig in plasma. Blood drug level increases dose dependently,concentrations of which of some point has statistics differences in groups of dose of homo-middle,time to steady state concentration for eighth weeks. At once four weeks 10 mg·kg ^-1 intravenous infusion of rhCTLA4-Ig,patients do not show accumulated phenomenon. At eighth weeks the average drug density were 27.1±8.1 mg·L^-1,at sixteenth weeks the average drug density were 19.1±9.0 mg·L^-1. No difference was found between these two groups(p<0.05). Variations of concentration at each point has no statistical differences,as well as the blood drug levels at the same points are between dosage at first time and at the sixth by using matched t-test. The result of test is similar to the clinical report for foreign new drugs of Orencia and may provide safe and reasonable administration for Phase II clinical trial.3. When administrated subcutaneously in rheumatoid arthritis patients with the dose of 10 mg·kg^-1,rhCTLA4-Ig was well rated by patients and without any adverse effect's but not drug therapy. RhCTLA4-Ig multiple administration of 10 mg·kg ^-1at the 0,2,4,8,12,16 weeks. During the study no adverse effect was observed.