| This article uses recombinant anti-RANKL humanized monoclonal antibodies as analyte to establish different detection methods to study their pharmacokinetics,tissue distribution and excretion,drug efficacy and characteristics of anti-drug antibodies in cynomolgus monkeys.To understand the dynamic changes of the analyte in vivo,provide a basis for further in-depth clarification of the mechanism of drug action,drug efficacy and toxicology research,and the design of clinical drug delivery trials.A sensitive and specific enzyme-linked immunosorbent assay(ELISA)was established to detect the concentration of the analytes in the serum,and the methodological verification results meet the requirements of the quantitative analysis method verification guidelines for biological samples.Pharmacokinetic studies on cynomolgus monkeys as test animals.The experiment was divided into 4 groups,with a single subcutaneous injection of low(0.5 mg·kg-1),medium(3 mg·kg-1),high(18 mg·kg-1)group and single intravenous injection(3 mg·kg-1)group.Used the validated ELISA method to detect the concentration of analytes in the serum,used noncompartmental model to fit the drug time curve and calculated the pharmacokinetic parameters.Results showed that the peak concentration(Cmax)of cynomolgus monkeys after single subcutaneous injection of 0.5,3,18 mg·kg-1 analytes were(7.57±0.872),(37.9±6.72),(250±35.5)μg·mL-1,exposure level(AUC(0-t))were(1630±496),(8810±3800),(94700±34900)hr.·ig·mL-1.It shows that the exposure level of the drug body increases with the dose.After intravenous injection of 3 mg·kg1 analytes,Cmax was(87.2±13.8)μg·mL-1;AUC(0-t)was(12000±4370)hr·μg·mL-1.2、Established radionuclide tracing method combined with trichloroacetic acid(TCA)precipitation and molecular exclusion high performance liquid chromatography to study the tissue distribution and excretion characteristics of the analyte in cynomolgus monkeys.It has been verified that the activity of the analyte has not changed after being labeled with the radionuclide tracer,which can be used to detect the tissue distribution and excretion of the analyte in the cynomolgus monkey body.The results of the tissue distribution and excretion experiment showed that:after the subcutaneous injection of 125I-T,the cynomolgus monkey was mainly distributed in the lung,liver,spleen,heart and kidney;28 days after administration,79.5%±14.6%of metabolites were excreted through urine and 6.83%±2.18%of metabolites were excreted through feces,indicating that the analyte was mainly excreted through urine and feces.3、Used sensitive and specific ELISA kit to detect the levels of bone turnover markers tartrate resistant acid phosphatase 5b(TRACP 5b),bone alkaline phosphatase(BAP)and collagen type I cross-linked C-telopeptide(CTX-I)in serum,to study the pharmacodynamic effects of the analyte in cynomolgus monkeys.The data showed that after administration,the concentrations of TRAcP 5b,BAP and CTX-I in the serum began to decrease at 1d or 7d.It shows that the above biomarkers can be used as indicators for the evaluation of drug efficacy.4、Established Bridging-ELISA to detect anti-drug antibodies in serum,and its verification results meet the requirements of the 2019 US Food and Drug Administration(FDA)immunogenicity methodology.Biological samples collected from a single subcutaneous injection of vehicle control group(0 mg·kg-1),low-dose group(20 mg·kg-1)and high-dose group(100 mg·kg-1).The results showed that no anti-drug antibody was detected in the vehicle control group and low-dose administration group,one in the high-dose group individuals have detected anti-drug antibodies;suggesting that the analyte is immunogenic in cynomolgus monkeys,and the production of anti-drug antibodies may have an effect on drug efficacy and allergic reactions. |
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