Pharmacokinetics of methotrexate polyglutamates in peripheral blood mononuclear cells of ra patients is similar after subcutaneous or oral administration in the methotrexate monitoring (memo) trial

BackgroundPharmacokinetics of methotrexate (MTX) after oral and subcutaneous administration to RA patients differs: MTX levels in plasma and MTX-polyglutamate (MTX-PG) accumulation in erythrocytes are higher during equidosed subcutaneous compared to oral MTX treatment. (1,2) No data are available whether administration route of MTX differentially impacts the intracellular concentrations of MTX-PGs in peripheral blood mononuclear cells (PBMCs) during MTX therapy.ObjectivesTo investigate the pharmacokinetics of MTX-PGs in PBMCs of newly diagnosed RA patients receiving oral or subcutaneous MTX in the early phase (1, 2, 3 and 6 months) of MTX treatment.MethodsIn a clinical prospective cohort study (MeMo study (NTR7149)), RA patients wereadministered oral (n=24) or subcutaneous (n=22) MTX up to 25 mg MTX/week, as described before. (1) At 1, 2, 3 and 6 months after the start of therapy, PBMCs were isolated via Ficoll density gradient centrifugation. Individual MTX-PG forms (MTX-PG1-6) in PBMCs were analyzed by a UPLC-MS/MS method including custom-made stable isotopes of MTX-PG1-6 as internal standards (3). UPLC-MS/MS measurements of the PBMCs were performed with a Waters Acquity BEH C18 column coupled to an AB Sciex 6500+ with the ESI operating on the positive mode. Dosing, concomitant treatments and DAS28-ESR assessments were in conformity with clinical practice. (4)Results46 consecutive patients were included in this study; 76% female, mean age: 57.8 years, BMI: 25.8, smokers: 20%, mean baseline DAS28-ESR: 3.5, as described before. (1) MTX dose at baseline was 10.5 mg (SD: 1.5) for both groups, 15.4 mg (4.4) and 16.8 mg (1.8) at 1 month, 22.8 mg (3.9) and 22.4 mg (5.2) at 2 months, 20.1 mg (6.3) and 20.8 mg (5.6) at 3 months, and 19.7 mg (6.1) and 18.5 mg (6.7) at 6 months for oral and subcutaneous use, respectively. MTX-PG analyses in PBMCs for individual and total MTX-PGs revealed no significant differences between oral and subcutaneous administration groups at 1, 2, 3, and 6 months (Figure 1). Linear regression of LN transformed MTX-PG levels in PBMCs and administration route, corrected for age, baseline DAS28, smoking, BMI, eGFR and MTX dose, showed a trend towards higher MTX-PG levels in PBMCs after subcutaneous MTX administration compared to oral administration (data not shown). MTX-PG distribution in PBMCs was mainly composed of MTX-PG1 (58%), and to a lesser extent MTX-PG2 (27%) and MTX-PG3 (15%). Longer chain MTX-PGs beyond MTX-PG4 were detectable in PBMCs, but at levels lower than MTX-PG1-3 (mean: 4.0 – 6.7 fmol/106 cells). Total MTX-PG accumulation in PBMCs was approximately 10-20 fold higher than in erythrocytes. PBMC accumulation was rather stable, whereas RBC MTX-PG accumulation increased between 1 to 3 months to reach a plateau (Figure 1).Figure 1.Loess regression of MTX-PG concentrations in PBMCs (MTX-PG1-3) and RBCs (MTX-PG1-6) of RA patients during the first 6 months of oral or subcutaneous MTX administration. At 6 months, 18 patients using oral and 18 patients using subcutaneous MTX were still continuing MTX treatment. Means (lines) and SE (grey areas) are depicted.ConclusionThis study demonstrated that MTX-PG accumulation in PBMCs early on in the MTX treatment of RA patients was not significantly different between oral or subcutaneous MTX administration routes.References[1]RCF Hebing et al, Arthritis Rheum (2021); 60:339-348[2]M Hoekstra et al, J Rheumatol (2004); 31:645-8[3]E Den Boer et al, Anal Bioanal Chem (2013); 405: 1673-1681[4]J Smolen et al, Ann Rheum Dis (2020); 79:685-699AcknowledgementsWe would like to thank all participating patients and Pfizer (grant 53233663 / WI230458), NVKC (Noyons grant) and AmsterdamUMC (AI&II extension grant).Disclosure of InterestsRenske Hebing Grant/research support from: Pfizer, grant number 53233663 / WI230458, Amsterdam UMC (AI&II extension grant), NVKC (Netherlands Society for Clinical Chemistry, Noyons grant), Marry Lin: None declared, Eduard Struys: None declared, Sohaila Mahmoud: None declared, Ittai Muller: None declared, WIllem Lems: None declared, Bart van den Bemt Speakers bureau: Pfizer, UCB, Sanofi-Aventis, Galapagos, Amgen and Eli Lilly, Gerrit Jansen: None declared, Robert De Jonge Grant/research support from: NVKC (Netherlands Society for Clinical Chemistry, Noyons grant), Michael Nurmohamed Grant/research support from: Pfizer grant