Population Pharmacokinetics For Low-dose Methotrexate And SLCO1B1 Genetic Polymorphism In Patients With Rheumatoid Arthritis

OBJECTIVE1.To establish a method for determining the concentration of methotrexate polyglutamates(MTXPGs)in erythrocytes of patients with rheumatoid arthritis(RA)who are taking low-dose of methotrexate(MTX).2.To analyze the relationship between the solute carrier organic anion transporter family member 1B1(SLCO1B1)388A>G and 521T>C gene polymorphism and MTXPGs concentration in erythrocytes,clinical efficacy and hepatotoxicity in RA patients.3.To study the correlation of MTXPGs concentration with efficacy and hepatotoxicity in RA patients,and explore the factors that affect efficacy and steady-state concentration of MTXPGs.4.To establish a population pharmacokinetics(PPK)model of MTXPGs,quantitatively examine the factors affecting pharmacokinetic parameters,and provide a reference for RA individualized medication.METHODS1.High performance liquid chromatography-mass spectrometry(HPLC-MS/MS)was used to measure the free MTX and total MTX in whole blood,respectively,so as to calculate the concentration of MTXPGs in red blood cells.2.Prospectively collected RA patients receiving low-dose MTX treatment in a hospital from April 2018 to October 2019.The gene chip method was used to detect the SLCO1B1 gene.SPSS software was used to analyze the relationship between gene polymorphism and MTXPGs concentration,efficacy and hepatotoxicity.3.Correlation of MTXPGs concentration with efficacy and hepatotoxicity was analyzed by non-parametric tests,and receiver operating characteristic curve(ROC)was drawn to find the treatment window of MTXPGs;Logistic regression and multivariate linear regression were used to explore the influence on patients’ efficacy and the steady-state concentration of MTXPGs,respectively.4.Nonlinear mixed effect model(NONMEM)was used to establish a PPK model of MTXPGs,and the effects of SLCO1B1 genotypes,physiological parameters,and combined medications on the pharmacokinetic parameters of MTXPGs were investigated.RESULTS1.The calibration curve of MTX was linear over the range of 1～300 ng·mL-1(r=0.999 4),and the limit of detection(LOD)was 0.5 ng·mL-1.The accuracy(RE)was within±5%and the intra-day and inter-day precision(RSD)were less than 7.96%and 10.88%,respectively.The extraction recovery rate was higher than 70%.2.A total of 137 RA patients were collected.The frequency of allele G was 73.7%and the frequency of allele C was 11.7%.The distribution of SLCO1B1 genotypes was consistent with Hardy-Weinberg equilibrium test.There were no differences in MTXPGs concentration,efficacy and hepatotoxicity between the 388A>G and 521T>C mutant groups and the wild group.3.The effective rate of treatment for 128 RA patients was 73.4%,and there were large individual differences in MTXPGs concentrations among patients,and the MTXPGs concentration was related to the effect and hepatotoxicity.When the MTXPGs concentration was 25～38 ng·mL-1,the patients had good effect and low hepatotoxicity.MTXPGs concentration,age,hemoglobin,platelets,and alkaline phosphatase were factors that affect the efficacy.MTXPGs steady state concentration was related to age,dose and red blood cells.4.Based on 269 MTXPGs concentration data and related clinical information of 128 RA patients,the final model of MTXPGs was established as:CL=2.08×(22/ALT)0.153×(RBC/4)1.17×e0.346(L·h-1),V=673(L).The model diagnostic plot,bootstrap method and normalized predictive distribution error showed that the model had good validity,stability and predictive ability.CONCLUSION1.The HPLC-MS/MS method for indirect detection of MTXPGs concentration established in this study is fast,sensitive,highly specialized and reproducible,and is suitable for the clinical monitoring of MTX in patients with RA.2.There is no relationship between SLCO1B1 388A>G and 521T>C gene polymorphism and MTXPGs concentration,efficacy and hepatotoxicity.3.MTXPGs concentration is related to effecacy and hepatotoxicity.The therapeutic window is 25～38 ng·mL-1.MTXPGs concentration,age,hemoglobin,platelets,and alkaline phosphatase are factors that affect the efficacy,while age,dose,and red blood cells are factors that affect the steady-state concentration of MTXPGs.4.The PPK model of MTXPGs for RA patients in China was established for the first time.This model is effective and stable,and has good predictive ability,which can provide a basis for the formulation of individualized medication regimens for MTX.