Studies On The Long-term Toxicity And Toxic Mechanismof Recombinant Human Anti-TNFα Monoclonal Antibody In Cynomolgus Monkeys

Part One： Long Term Toxicity Studies of Recombinantanti-TNF-α human monoclonal antibodyObjective: To learn about the toxicity reactions, the severity of the toxicity, the targetorgans and the reversibility of the harm in Cynomolgus monkeys with successiveadministration of recombinant human anti-TNFα monoclonal antibody, so as to providesupport and the setting of main observation indicators for its clinical use. Methods:30healthy Cynomolgus monkeys were divided into5groups according to the weight:0,10.0,33.0,200.0mg/kg dose groups and a positive control group (HUMIRA,33mg/kg), with6animals in each group to take subcutaneous injection. The drug was administrated once aweek for successive4weeks with4weeks of recovery period. During the first2hoursafter administration, we supervise body weight, respiration, rectal temperature and the foodintake were monitored in the experimental animals. The determined indexes were asfollows: NEUT, EOS, BASO, LUC, HCT, PLAT, TT, MCV, MCH, MONO, APTT, PT,LYMPH, RBC, WBC, MCHC, RETIC, HGB, FIB, GLU, TBIL, CREA, ALP, ALT, Tch,BU, ALB, TP, GGT, CPK, AST, LDH, UA, serum Ca, P, K, Na and Cl; urine Ph, bilirubin,glucose, specific gravity, protein, nitrite, leukocytes, urobilinogen, erythrocyte, ketones. At28thday and56thday after administration,15of the experimental animals were anatomizedto examine the histopathological and make bone marrow cell counting and classification.Results:(1) The antibody has no significant effection on respiration, rectal temperature,body weight, and pupil of monkeys.(2) The antibody caused significant changes inhematological indexes RBC, HGB, HCT and RETIC%, but had no effect on otherhematological indexes.(3) It had no obvious effect on blood biochemical parameters.(4)There was no significant effection on the urine.(5) The antibody induced hyperplasia ofbone marrow erythroid cells.(6) Pathological examination suggested the antibody inducedthymic and splenic atrophy and slight hyperplasia of bone marrow erythroid cells. Therewas no morphological evidence of injury in other tissues and organs, and no blood vesselsand muscle irritation was found at injection site. Conclusions: Recombinant humananti-TNFα monoclonal antibody exhibited significant immunotoxicity, it can cause thymic and splenic atrophy. In addation, the drug induced slight hyperplasia in bone marrowerythroid cells. Both of the immunotoxicity and hematologic toxicity were reversible. Part two: Studies on the immunotoxicity Mechanisms ofRecombiant human anti-TNFα monoclonal antibodyObjective: To investigate the possible mechanisms involved in thymus and spleenatrophy induced by recombinant human anti-TNFα monoclonal antibody in Cynomolgusmonkeys. Methods: Cynomolgus monkeys were administered with recombinant humananti-TNFα monoclonal antibody at dose of10.0、33.0and200.0mg/kg, the positive controlgroup was treated with33.0mg/kg HUMIRA, the vehicle control group was treated withthe vehicle in equal volume. The drug was administered once a week for successive4weeks with a recovery period of4weeks. At the end of the treatment and recovery period,the following indexs were detected.(1). Effect of the drugs on thymic and splenic cellapoptosis was examined. First, animals were sacrificed and thymus and spleen wereremoved to prepare single cell suspension. Thymic and splenic cells were then stained withHoechest Staining Kit. Secondly, thymus and spleen were removed to make paraffinsections, followed by the apoptosis detection with TUNEL method.(2). The cell cycle ofthe thymus cell were examined with the cell cycle analysis kit.(3). The influence of thedrugs on thymus cell proliferation were detected with the EdU staining kit.(4).Theinfluence of the drugs on T cell development was examined in Cynomolgus monkeys.Single cell suspension of thymus and spleen was made and the cell number was counted.Thereafter, the proportion of different stage of thymocytes, including double negative (DN,CD4–CD8–), double positive (DP, CD4+CD8+) and single positive (SP, CD4+or CD8+)thymocytes, was detected by flow cytometry.(5). Analysis on peripheral blood lymphocytephenotypes was performed. The proportion of CD3+、CD3+CD4+、CD3+CD8+Tlymphocytes and the ratio of CD3+CD4+T lymphocytes to CD3+CD8+T lymphocytes(CD4+/CD8+) were test by flow cytometry. Results: At the end of the treatment andrecovery period, the proportion of Hoechest positive cells in both thymus and spleen wasnot significantly different among each group (P＞0.05). In addition, TUNEL positive cellswere also found comparable in vehicle control and other treatment groups no matter for thymus or spleen. These results indicated that the test article did not induce cell apoptosisin thymus and spleen. So thymic and splenic atrophy observed in the study were notrelevant to cell apoptosis. Cell cycle analysis revealed that cell cycle of thymus cells wasarrested at G0/G1phase, and EdU staining indicated that thymus cell proliferation wasinhibited by recombinant human anti-TNFα monoclonal antibody. By cell counting, wefound a dramatic reduction in cell number of thymus and spleen in positive control andother treatment groups compared with vehicle control (P＜0.05). But the proportion ofthymocytes in different stages was not affected by drug treatment, indicating that theearlier stage, i.e. DN stage of T cell development may be disturbed, while the followingstages were not affected. The peripheral blood T lymphocyte phenotype was not influencedby drug treatment. Conclusions:(1). Recombinant human anti-TNFα monoclonal antibodymight inhibit cell proliferation to induce thymic and splenic atrophy in Cynomolgusmonkeys, and cell apoptosis was not involved in this effect;(2). The earlier stage (DNstage) of T lymphocyte development might be disturbed in thymus by recombinant humananti-TNFα monoclonal antibody, but the later stages were not affected.