Background/Purpose: Optimal dosing of low-dose methotrexate (MTX) is a challenge in rheumatoid arthritis (RA) because of substantial interpatient variation of response and because no stable plasma MTX levels are reached. MTX-polyglutamates (PG) in red blood cells (RBC) have been suggested as a potential marker of response, however contradictory results have been reported.

We aimed to evaluate the association between MTX-PG_3-5 exposure and response in rheumatoid arthritis patients starting MTX and to identify clinically relevant covariates that could explain the inter-patient variability in this association.

Methods: Data of three prospective cohorts (MTX-R, tREACH and MeMo, The Netherlands, 395 patients) were available. RBC MTX-PG concentrations were determined by LC-MS/MS. Individual MTX-PG concentrations were summed in two categories: MTX-PG_1-2 and MTX-PG_3-5. The relationship between exposure and DAS28 was analysed using a semi-mechanistic pharmacokinetic-pharmacodynamic (PK-PD) model. In the population model relevant covariates were tested using full covariate modelling, followed by backward elimination. Reference values were adopted from Pan et al. 2014. Forest plots were used to define clinically significant covariates on MTX exposure and response. Covariates were considered clinically relevant if the 95% confidence interval (CI) of this covariate effect at the 1^st and 9^th quantile of the full dataset fell outside -25 or +25% deviation from the typical value. Only statistical significant covariates (p< 0.001) are displayed.

Results: We developed a PK-PD model (Fig 1) with 3387 individual MTX-PG concentrations and 1337 DAS28 outcome measurements between 0-300 days after the start of MTX treatment. The developed model adequately described the time course of MTX-PG_3-5 and DAS28. The median MTX-PG_3-5 level at month 1 was 30.9 nM (IQR 17.4) and at month 3: 69.3nM (IQR 56.9). Clearance of MTX-PG_3-5 from RBCs was significantly lower (14%, 95%CI 8% – 17%) in females and older patients (log age=140% (CI 119%-164%, e.g. -38% (CI 32%-44%) for patients of 85 years compared with patients of 65 years). Patients with FPGS rs4451422 exhibited a reduced formation of MTX-PG₃₄₅ (K_FPGS2, 85%, CI 78%-93%). MTX-PG_3-5 levels were associated with DAS28: E_max was 2.0 (95%CI 1.9-2.3) and an EC₅₀ was 11.6 nM (CI 6.8nM-19.7nM), which is below most observed MTX-PG_3-5 concentrations. Patients with a high baseline DAS28 showed a higher chance to improve (log DAS on E_MAX: 153%, (CI 141%-166%), e.g. a baseline DAS28 of 5.0 vs 3.5 resulted in 55% (CI 50%-59%) higher E_MAX). Co-administration of DMARDs and prednisolone lowered the EC₅₀; 69% (CI 61%-79%) and 46% (CI 33%-66%), respectively. Smoking and BMI negatively influenced E_MAX (not clinically relevant). None of the genetic variables influenced MTX-response.

Conclusion: With this PKPD model in RA patients starting methotrexate, exposure of methotrexate polyglutamates 3-5 in RBCs was associated with response. Baseline DAS28 and comedication affected the response clinically relevant. Low MTX-PG_3-5 EC₅₀ in RBCs was predictive for response, suggesting that the early measurements of long chain methotrexate polyglumates, before steady state is reached, are useful for dose individualisation.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/methotrexate-polyglutamates-exposure-response-modelling-in-a-large-cohort-of-rheumatoid-arthritis-patients-starting-methotrexate/