Methotrexate accumulation in target intestinal mucosa and white blood cells differs from non-target red blood cells of patients with Crohn's disease

Background: Intracellular methotrexate polyglutamates (MTX-PGs) concentrations are measurable in red blood cells (RBCs) during MTX treatment. MTX-PG₃ concentrations correlate with efficacy in patients with Crohn's disease (CD). Since RBCs are not involved in pathogenesis of CD and lack extended MTX metabolism, we determined MTX-PGs accumulation in peripheral blood mononuclear cells (PBMCs: effector cells) and intestinal mucosa (target cells) and compared those with RBCs as a potential more precise biomarker. Methods: In a multicentre prospective cohort study, blood samples of patients with CD were collected during the first year of MTX therapy. Mucosal biopsies were obtained from non-inflamed rectum and/or inflamed intestine. MTX-PGs concentrations in mucosa, PBMCs and RBCs were measured by liquid chromatography–tandem mass spectrometry. Results: From 80 patients with CD, a total of 27 mucosal biopsies, 9 PBMC and 212 RBC samples were collected. From 12 weeks of MTX therapy onwards, MTX-PG₃ was the most predominant species (33%) in RBCs. In PBMCs, the distribution was skewed towards MTX-PG₁ (48%), which accounted for an 18 times higher concentration than in RBCs. Long-chain MTX-PGs were highly present in mucosa: 21% of MTX-PG_total was MTX-PG₅. MTX-PG₆ was measurable in all biopsies. Conclusions: MTX-PG patterns differ between mucosa, PBMCs and RBCs of patients with CD.