重组抗RANKL人源化单克隆抗体在食蟹猴体内的药动学研究

Pharmacokinetics of Recombinant Anti-RANKL Humanized Monoclonal Antibody in Cynomolgus Monkey

目的建立并验证自主开发的酶联免疫吸附分析法(ELISA),检测食蟹猴血清中重组抗核因子κB受体活化因子配体(RANKL)人源化单克隆抗体(受试品,T)的浓度,以研究受试品在食蟹猴体内的药动学(PK)特征,为受试品潜在的生物类似药药效和毒理研究,以及临床给药方案的设计和优化提供依据。方法选取健康食蟹猴32只,分为4组,分别为0.5、3、18 mg·kg^(-1)皮下注射给药组和3 mg·kg^(-1)静脉注射给药组,对采集样品中的受试品浓度采用验证的ELISA进行检测,检测的血药浓度导入WinNonLin^(■)v6.4(Pharsight公司)对药动学参数进行计算。结果自主建立的ELISA法定量范围为31.3~2000 ng·mL^(-1),批内、批间精密度和准确度在3.3%~11.6%和-14.4%~14.8%内,该方法符合法规要求的准确度、精密度、特异性、灵敏度、稀释线性、钩状效应、选择性、平行性和稳定性。食蟹猴皮下注射0.5、3、18 mg·kg^(-1)和静脉注射3 mg·kg^(-1)受试品后,分别于24~72、10~72、24~168和0.0833~4 h达峰;达峰浓度(ρ_(max))分别为(7.57±0.872)、(37.9±6.72)、(250±35.5)和(87.2±13.8)μg·mL^(-1);暴露水平(AUC0-t)分别为(1630±496)、(8810±3800)、(94700±34900)和(12000±4370)μg·h·mL^(-1);皮下注射3 mg·kg^(-1)受试品的绝对生物利用度为73.4%。结论自主建立的ELISA法,可用于支持受试品的药动学研究;药动学研究结果表明,食蟹猴皮下注射0.5~18 mg·kg^(-1)受试品后,在给药剂量范围内呈线性药动学特征。

OBJECTIVE To develop and validate the in-house enzyme-linked immunosorbent assay(ELISA)to determine the concentration of recombinant anti-RANKL humanized monoclonal antibody(analyte,T)in cynomolgus serum to evaluate the pharmacokinetic behavior of T in cynomolgus monkey.METHODS A total of 32 healthy cynomolgus monkeys were selected and randomly divided into 4 dosing groups,which were subcutaneously injected 0.5,3 and 18 mg·kg^(-1)of T,and intravenously injected 3 mg·kg^(-1)of T,respectively.The concentrations of T in the serum samples was detected by the validated ELISA,and the measurements were imported into WinNonLin^(■)v6.4(Pharsight Inc.)to calculate the pharmacokinetic parameters.RESULTS The quantitative range of the in-hourse ELISA assay is 31.3 to 2000 ng·mL-1,and the intra-and inter-batch precision and accuracy are in the range of 3.3%to 11.6%and-14.4%to 14.8%,respectively.The method was validated with acceptable accuracy,precision,specificity,sensitivity,dilution linearity,hook effect,selectivity,parallelism,and stability required by regulations.After subcutaneous administration of 0.5,3 and 18 mg·kg^(-1)of T and intravenous injection of 3 mg·kg^(-1)T,concentrations in each group reach peak concentrations at 24-72,10-72,24-168 and 0.0833-4 h,respectively;the peak concentrations(ρ_(max))are(7.57±0.872),(37.9±6.72),(250±35.5)μg·mL-1 and(87.2±13.8)μg·mL-1;areas under concentration-time curve(AUC0-t)are(1630±496),(8810±3800),(94700±34900)and(12000±4370)μg·h·mL-1.The absolute bioavailability of the 3 mg·kg^(-1)subcutaneous injection group is 73.4%.CONCLUSION The in-house ELISA method can be used to support the pharmacokinetic study of T;the results of pharmacokinetic parameters indicate a linear pharmacokinetic behavior after subcutaneous injection of 0.5-18 mg·kg^(-1)of analyte in cynomolgus monkeys.