Relationship between methotrexate in red and white blood cells with disease activity: towards optimal methotrexate dosing in rheumatoid arthritis.

Rationale

Low-dose methotrexate (MTX) is the anchor drug in the treatment of rheumatoid arthritis (RA), both as mono- and combination therapy with other chemical and biological disease-modifying anti rheumatic drugs (DMARDs). Moreover, its convenience, tolerability, safety and low costs contribute to the clinical and socio-economic benefits of MTX. In order to have a therapeutic effect, MTX should be converted into MTX-polyglutamates (MTX-PGn). MTX monoglutamate is poorly retained and extruded from cells and rapidly cleared from plasma within 24 hours. Thus, for assessment of clinically active levels of MTX, it is more relevant to measure MTX-PGn levels in blood cells rather than plasma MTX concentrations.

There is a highly variable intracellular MTX-PGn accumulation that is largely unexplained but some of the variation may be associated with intracellular folate levels, ABC-drug efflux transporter and folylpolyglutamate synthetase (FPGS) gene polymorphisms, MTX dosing and route of administration, BMI and age. Recently, it was also demonstrated that aberrant pre-mRNA splicing of FPGS could constitute a plausible basis for loss of FPGS activity and consequently decreased MTX-PGn levels.

Furthermore, it should be taken into account that red blood cells (RBCs) do not have nuclei and intracellular organelles to control folate and MTX homeostasis as immune-effector cells do. Since immune effector cells are the ultimate target of MTX, analysis of MTX-PGn levels in peripheral blood mononuclear cells (PBMCs) is more clinically relevant.

Together, to meet shortcomings of RBC MTX-PGn analysis as a tool to predict MTX response or toxicity, novel analytical tools to analyse MTX-PGn in PBMCs and novel molecular knowledge of the determinants of intracellular MTX-PGn accumulation (e.g. aberrant FPGS splicing) are now available to aid and improve MTX drug dosing and individualize MTX treatment to reach the optimally effective intracellular dose.



Study design and study population

In this pilot study, we will prospectively follow 40 consecutive RA patients in whom MTX therapy is initiated (20 treated with oral MTX and 20 treated with s.c. MTX). RBCs and PBMCs samples will be obtained at 0, 1, 2, 3 and 6 months. MTX-PGn and folate levels will be measured with Liquid chromatography tandem-mass spectrometry (LC-MS/MS). FPGS pre-mRNA splicing profiles in PBMCs will be determined in a multi polymerase chain reaction (PCR)-based approach.

Main study parameters/endpoints

1) MTX-PGn accumulation and MTX-PG distribution profiles in PBMCs and RBC from RA patients following 6 months MTX therapy.

2) Profiles of FPGS pre-mRNA splicing aberrations in PBMCs from RA patients following 6 months MTX therapy.

3) Clinical disease parameters, folate levels and impact of MTX on disease activity.

In this study, the primary objective is to compare MTX-PGn levels in PBMCs of RA patients with levels in RBCs and serum and to determine the correlations with clinical response.

The secondary objective is to investigate route of administration-related determinants of MTX-PGn accumulation in PBMCs.

Finally, a tertiary objective is to examine at a molecular level whether aberrant FPGS pre-mRNA splicing is a contributing factor to variability in MTX-PGn levels in PBMCs.

5 timepoints (baseline, 1 month, 2 months, 3, months and 6 months)

this pilot study, we will prospectively follow 40 consecutive RA patients in whom MTX therapy is initiated (20 treated with oral MTX and 20 treated with s.c. MTX). RBCs and PBMCs samples will be obtained at 0, 1, 2, 3 and 6 months. MTX-PGn and folate levels will be measured with Liquid chromatography tandem-mass spectrometry (LC-MS/MS). FPGS pre-mRNA splicing profiles in PBMCs will be determined in a multi polymerase chain reaction (PCR)-based approach.