Pre-clinical Pharmacodynamics And Pharmacokinetics Study Of Domestic Rh-anti-CD20ximAb

Compared with foreign-made drug (Rituximab), whether domestic recombinant anti-CD20 monoclonal antibody (rh-anti-CD20ximAb) have similar or better binding with the tumor cell surface CD20 molecule,pharmacodynamic action and pharmacokinetics in vivo. According to the requirements of new biological products'effectiveness study, we provide pre-clinical pharmacodynamics and pharmacokinetics study data.The paper include two parts:Part 1: The pre-clinical Pharmacodynamic study of rh-anti-CD20ximAb1,We study rh-anti-CD20ximAb's complement-dependent cytotoxicity action (CDC) in vitro using Lactate dehydrogenase (LDH) cytotoxicity test method. In the experiment compared with Rituximab ,we evaluate rh-anti-CD20ximAb's CDC role in vitro.Rh-anti-CD20ximAb have a significant CDC effect on positive cells (Raji and Daudi cells) in vitro,and is very similar to the reference standard Rituximab :for Raji cells the average value of half inhibition concentration (IC₅₀) respectively were 740±76 ng / mL and 809±39 ng / mL, the average of EC₅₀ ratio was 91.31±5.27%. And for Daudi cells the average value of half inhibition concentration (IC₅₀) respectively were 729±57 ng/mL and 846±145 ng/mL, the average of EC₅₀ ratio was 87.42±7.88%.2,In the experiment we select positive cells (Raji and Daudi cells) and negative cells (MX-1 cell) as target cells and human peripheral blood mononuclear cells (PMBC) as effector cells.We study rh-anti-CD20ximAb's antibody-dependent cell-mediated cytotoxicity action (ADCC) in vitro using Lactate dehydrogenase (LDH) cytotoxicity test method in different E/T ratios. Compared with Rituximab,we evaluate rh-anti-CD20ximAb's ADCC role in vitro.Rh-anti-CD20ximAb have a significant ADCC effect on positive cells (Raji and Daudi cells) in vitro,and is very similar to the reference standard Rituximab.For positive cells , the killing rate of rh-anti-CD20ximAb and Rituximab on positive cells were all decreased with lower E/T ratios. But the killing rate was similar with same E/T ratios. For negative cells , the killing rate was below 10%.3,Rh-anti-CD20ximAb and Raji cell's high affinity binding test include saturation binding assay and competitive inhibition test. The conclusion of saturation binding assay explain that rh-anti-CD20ximAb and Raji cell have very high affinity and reversible. The conclusion of competitive inhibition test illustrate rh-anti-CD20ximAb and Rituximab have similar inhibition on 125I-rh-anti-CD20ximAb and Raji cell's binding. The IC₅₀ were 3.13±0.38μg/mL and 3.45±0.59μg/mL separately and have no difference statistically.4,Cynomolgus monkeys B-lymphocyte levels in peripheral blood was observed after intravenous drip rh-anti-CD20ximAb and Ritximab. And we determine whether rh-anti-CD20ximAb and Ritximab have the same CDC and ADCC action in vivo. Flow cytometry examination showed that the amount of C20 positive B lymphocytes before infusion was in 14.37-47.91% and each group showed no statistical difference. The same dose of rh-anti-CD20ximAb and Ritximab (30mg·kg-1) have no statistical difference in 2h-20d after infusion(P>0.05).Part 2: The pre-clinical pharmacokinetic study of rh-anti-CD20ximAb1,We determine monkey serum concentrations of rh-anti-CD20ximAb with a double antibody sandwich ELISA assay. Through methodology validation, standard curve,specificity,precisio,accuracy,LLOQ,recovery,stability and dilution all meet the requirements of rh-anti-CD20ximAb pharmacokinetic study.2,The results of a pharmacokinetics of rh-anti-CD20ximAb after single intravenous infusion to cynomolgus monkeys were submitted, groups of animals received 10,30 and 100 mg·kg-1 for 30 miutes, the drug concentration in serum reached peak after 0.5h in most monkeys. And the concentration was parallel to dosage of injected drug. The terminal phase half lives have no significantly extending with the increase of dosages. The drug concentration in serum of low, medium and high dosage groups decreased to 1/10～1/20 of the peak concentration 480h after drug injection. Comparing the pharmacokinetic parameters of the different groups, the average residence time have no significantly extending with the increase of dosages. The dosages rate was 1:3:10, the growth of AUC was 1:3.0:9.2, increase parallel to the dosage. The clearance have no significantly change with the increase of dosages, showing typical linear kinetic characters.Domestic rh-anti-CD20ximab were intravenous adminstration to cynomolgus monkeys weekly after four times, the drug concentrations in serum and the pharmacokinetic parameters were dissimilar between the first time administration and the last time. The rate of AUC_0-ι(AUC_{0-ι(第4次)}/AUC_{0-ι(第1次)})of three cynomolgus monkeys were 1.72,1.87 and 1.84 after the first time administration and the last time, 1.81±0.08 in average, indicating that there was accumulation administration weekly 4 times.The experiment result of intravenous infusion of comparable product Rituximab and domestic rh-anti-CD20ximab to cynomolgus monkeys indicated that, generally, the shapes of C-T curve of Rituximab and domestic rh-anti-CD20ximab in every monkey were similar, and the pharmacokinetic parameters as well. The Student's coupled t-examination of the concentrations of Rituximab and domestic rh-anti-CD20ximab in three cynomolgus monkeys showed that no statistical variances were found (P>0.05) according to the concentrations at all time points after treatment of Rituximab and domestic rh-anti-CD20ximab. On the other hand , the result of pharmacokinetics indicated that, all the pharmacokinetic parameters (AUC_0-480h,MRT,CL_S,V_SS,C_max) of the two drugs did not have statistical variances (P>0.05), indicating that the pharmacokinetic manners of domestic rh-anti-CD20ximab and Roche Company Rituximab were similar.