Study On The Correlation Between Adalimumab Therapeutic Drug Monitoring And Theratpeutic Response In Patients With Ankylosing Spondylitis

Objectives:To establish immune assay methods to determine the concentrations of adalimumab?ADL?and anti-ADL antibody?AAA?in serum of ankylosing spondylitis?AS?patients,and to investigate the relationship between serum concentration of ADL,the level of AAA and therapeutic response and the predict ability of early drug serum concentration and AAA levels for therapeutic response.We hope to provide useful information for the application of therapeutic drug monitoring?TDM?in ADL.Methods:This study is a prospective cohort study.Active AS who met the inclusion criteria enrolled in this study after providing written informed consent and received 40 mg ADL subcutaneously every other week.Serum samples were prospectively collected at baseline and at week 2,4,8,12,18 and 24,prior to the following subcutaneous injection.The serun concentration of ADL was determined by enzyme-linked immunosorbent assay?ELISA?;In brief,AAA in serum was purified by pretreatment procedures of bead extraction and acid dissociation assay?BEAD?and the level of AAA was determined by direct ELISA.Clinical data of baseline characteristics and therapeutic efficacy of AS patients were collected at baseline and at week 2,4,8,12,18 and 24,respectively.Concentration-effect curve was used to investigate the relationship between serum concentration of ADL,the level of AAA and therapeutic response.And receiver operating characteristic curve?ROC?analysis was used to evaluate the ability of early drug serum concentration and AAA levels to predict primary non-responders.Results:?1?An ELISA method used to determine the concentration of ADL in human serum was established and validated.Our data showed a good concentration-response relationship of this method in the range of 20.00-0.63 ?g·mL.1.No hook effect was observed even though at high ADL concentration of 200.00 ?g·mL^-1.The intra-and inter-assay accuracies and precisions were always below 15%.Dilution linearity and parallelism of the method met the requirements of biological sample analysis.And the thaw-freeze and room temperature display showed good stability.?2?Establishment of a BEAD method used to determine the AAA levels in human serum:The quantification range of this method was 10.00⁰.31 ?g·mL^-1.The detection limit of the method was 0.16 ?g·mL^-1 determined by 38 ADL-naive samples?9 healthy subjects and 29 AS patients?.Full recoveries within 80%¹20%were obtained when various concentrations of ADL(0.00,1.00,10.00,and 50.00 ?gn·mL^-1)were spiked to 0.50,1.00 and 5.00 ?g·mL^-1 of AAA positive controls.Validation of the assay revealed that spiking of 50.00 ADL to both quality control samples demonstrated the total drug resistance of the BEAD assay.No significant interference was observed when 500.00 ng·mL^-1 of the tumor necrosis factor a?TNF-a?was spiked into 4.00 ?g·mL^-1 of AAA control sample,which was much higher than that in the physiological level.The precision and accuracy of the method was always below 15%.Results of dilution linearity and the research of hook effect test also met the need for determination in biological samples.?3?Relationship between drug serum concentrations and therapeutic response.32 AS patients enrolled in this study.There was a significant concentration-effect relationship between the concentration of ADL and AASDAS.We identified the therapeutic range of 8.00¹2.00 ?g·mL^-1 for maximal clinical effect.The serum concentration of ADL in primary responder group was significantly higher than that in primary non-responder group?p=0.002?.ROC curve analysis showed that primary non-responders would be distinguished accurately when the concentration of ADL was lower than 9.49 ?g·mL^-1?AUC=0.820,Sensitivity:76.9%,Specificity:78.9%,p=0.0001?.?4?Relationship between AAA levels,drug serum concentrations and therapeutic response:The positive frequencies of AAA increased following the time of ADL administration.At 4 weeks,the positive frequencies of AAA were 62%?18/29?while the positive frequencies of AAA increase to 72%?21/29?at 12 weeks.The serum concentration of AAA positive patients at each evaluating period was significantly lower than that of AAA negative patients?p=0.001?.Results of spearman rank correlation test showed that the AAA level was significantly inversely correlated with the drug serum concentration at 12 weeks.The AAA levels in primary responder group was much lower than that in primary non-responder group although there is no statistical difference?p?0.052?.The ROC analysis indicated that when the level of AAA was higher than 0.59?g·mL^-1-eq,primary non-responders may be accurately distinguished?AUC=0.710,Sensitivity:83.3%,Specificity:58.8%,p=0.036?.?5?Relationship between early serum drug concentrations,AAA levels and therapeutic response:The serum concentration of ADL at 4 weeks in primary responder group was significantly higher than that in primary non-responder group?p?0.0002?.ROC curve showed that the accuracy of distinguishing primary non-responders from primary responders is pretty high when the serum concentration of ADL at 4 weeks is lower than 3.66 ?g·mL^-1?AUC=0.900,Sensitivity:84.6%,Specificity:94.7%,p<0.0001?.The serum concentration of ADL in primary responder group at 2 weeks was also significantly higher than that in primary non-responder group?p=0.036?.ROC curve showed that prImary non-responders would be distinguished accurately when the concentration of ADL plasma at 2 weeks is lower than 3.34 ?g·m-L^-1?AUC=0.880,Sensitivity:100%,Specificity:70%,p?0.0002?.The AAA level at 4 weeks in primary non-responder group was significantly higher than that in primary responder group?p=0.011?.ROC curve showed that when the level of AAA at 4 weeks was higher than 2.18?g·mL^-1-eq,primary non-responders would be accurately distinguished?AUC=0.780,Sensitivity:75%,Specificity:82.4%,p=0.003?.Conclusions:?1?An ELISA method for determining the concentration of ADL and AAA levels of AS patients in serum was established and the method is with good stability and suitability.?2?There is a significant correlation in the serum concentration of ADL,the level of AAA and the therapeutic response.And this relationship can help adjust the therapeutic regimen when therapeutic drug monitor?TDM?was conducted.?3?Mohitoring early concentration of ADL and the level of AAA may help individualize the dose to patients who are:in sub-therapeutic drug level,and the incidence of primary non-response may be reduced.