Methotrexate Accumulation In Patient Guts: Target Intestinal Mucosa Variation By Disease State
Overview
This study investigates the accumulation of intracellular methotrexate polyglutamates (MTX-PGs) in different cell types to identify a more accurate biomarker for methotrexate (MTX) therapy in Crohn’s disease (CD) patients. MTX-PG3 concentrations in red blood cells (RBCs) are known to correlate with treatment efficacy in CD, but RBCs are not involved in CD pathology and have limited MTX metabolism. Therefore, the study assessed MTX-PG levels in peripheral blood mononuclear cells (PBMCs), which are involved in the disease process, and intestinal mucosa, the primary target tissue.
In a multicenter prospective cohort study, blood samples and mucosal biopsies were collected from CD patients during their first year of MTX treatment. The study included 80 patients, with 27 mucosal biopsies, 9 PBMC samples, and 212 RBC samples analyzed. MTX-PGs were quantified using liquid chromatography-tandem mass spectrometry.
Results showed that MTX-PG3 was the predominant form (33%) in RBCs after 12 weeks of treatment. In contrast, PBMCs predominantly contained MTX-PG1, with concentrations 18 times higher than in RBCs. Long-chain MTX-PGs were more prevalent in the mucosa, where MTX-PG5 constituted 21% of the total MTX-PGs, and MTX-PG6 was detected in all biopsies.
The study concludes that MTX-PG distribution varies significantly between mucosal tissue, PBMCs, and RBCs in CD patients, suggesting that using RBC-based measurements alone may not fully represent MTX metabolism and therapeutic response in CD.
Introduction
Methotrexate (MTX) is a low-dose anti-folate medication used to treat various immune-mediated inflammatory diseases (IMIDs), such as rheumatoid arthritis (RA), juvenile idiopathic arthritis (JIA), and Crohn’s disease (CD). In CD, MTX is often used either alone to reduce reliance on steroids or in combination with infliximab. While MTX monotherapy can decrease disease activity in about 65% of patients, over 40% experience side effects, leading to only 26% of patients continuing treatment after one year.
To optimize MTX therapy and minimize toxicity, therapeutic drug monitoring (TDM) has been proposed. Although stable plasma levels are not typically achieved with low-dose MTX, measuring MTX-polyglutamates (MTX-PGs) in red blood cells (RBCs) is common for TDM, particularly in RA patients. Studies have shown that higher concentrations of MTX-PG in RBCs correlate with reduced disease activity. Recent research suggests that RBC MTX-PG3 levels might also be a useful marker for TDM in CD patients, correlating with therapeutic efficacy and longer drug survival.
MTX-PGs are intracellular metabolites formed when MTX is polyglutamylated by the enzyme folylpolyglutamate synthetase (FPGS) after being taken up by cells. These metabolites are retained intracellularly and inhibit enzymes involved in folate metabolism, contributing to MTX’s therapeutic effects. MTX-PGs can be broken down by γ-glutamyl hydrolase, and their levels are influenced by cellular processes and the lifespan of the cell.
Traditionally, MTX-PGs have been measured in RBCs due to their abundance and ease of analysis. These levels reflect MTX-PG accumulation from earlier stages of erythrocyte development, as mature RBCs have limited FPGS activity. However, RBCs are not directly involved in the pathophysiology of CD nor are they the primary target of MTX therapy in these patients. Thus, understanding MTX-PG dynamics in immune effector cells, such as peripheral blood mononuclear cells (PBMCs), and in the intestinal mucosa could provide additional insights into MTX pharmacology and therapy optimization.
This study explores alternative TDM strategies by evaluating MTX-PG concentrations and distribution in mucosal tissue and PBMCs compared to RBCs in CD patients during the first year of MTX treatment. It also examines the impact of MTX dosage and treatment duration on MTX-PG accumulation, aiming to refine and improve monitoring approaches for better therapeutic outcomes.
Method
The study focused on patients enrolled in the prospective longitudinal ‘MTX-PG//CD cohort’ study, which investigated the effects of subcutaneous methotrexate (MTX) treatment in Crohn’s disease (CD). A total of 80 adults beginning MTX therapy were included from ten medical centers in the Netherlands, comprising both university and general hospitals. Patients received an initial dose of 25 mg MTX subcutaneously once weekly, with a possible reduction to 15 mg weekly after eight weeks. All participants also received weekly folic acid supplementation. The study duration was 12 months or until an endpoint, such as MTX discontinuation or initiation of additional therapies, was reached. The research adhered to ethical guidelines, with all participants providing informed consent.
Data collection involved blood samples and mucosal biopsies at various intervals: baseline, and at weeks 8, 12, 24, and 52, or upon reaching the endpoint. Clinical data included self-reported drug adherence and MTX dosage. Blood samples were collected in EDTA tubes for red blood cell (RBC) and peripheral blood mononuclear cell (PBMC) analyses. RBCs were centrifuged, and the cell pellet was stored for later analysis. PBMCs were isolated using Ficoll gradient centrifugation and stored as dry pellets. Mucosal biopsies were obtained either during scheduled ileocolonoscopy or rectoscopy and were snap-frozen for analysis.
MTX polyglutamates (MTX-PGs) in RBCs were analyzed using ultra-high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UPLC-ESI-MS/MS). If RBC samples were unavailable, MTX-PGs in whole blood were measured and adjusted for hematocrit. PBMC MTX-PGs were also measured by UPLC-ESI-MS/MS. Mucosal biopsies were homogenized, and MTX-PGs were extracted and quantified.
Statistical analysis
Statistical analyses included descriptive statistics and comparisons between patients on MTX therapy and those who had discontinued it. Data were analyzed using linear mixed models to assess variations in MTX-PG accumulation and distribution over time, according to MTX dosage, biopsy site, and inflammation status. Results were reported as coefficients with standard errors, and variability was expressed as the coefficient of variation. All analyses were conducted using R software, with significance defined as a p-value less than 0.05.
Result
The MTX-PG//CD cohort initially included 80 patients, with 73 continuing through week 8, 64 through week 12, and 21 patients extending to a median of week 51. During follow-up, 213 red blood cell (RBC), 11 peripheral blood mononuclear cell (PBMC), and 28 mucosal samples were collected. Technical issues led to the exclusion of two PBMC samples and one mucosal sample due to abnormally high MTX-PG concentrations, likely from pre-analytical errors. The demographics and sample sizes for each tissue type are detailed in Table 1.
Throughout the study, the methotrexate (MTX) dose was reduced from 25 mg/week to 15 mg/week in 56 patients, with one patient reverting to the higher dose at week 26. Adherence to MTX was recorded at 100% for at least 75% of patients, while poorer adherence (VAS < 80%) was noted in one to three patients at various times.
Mucosal biopsies were taken from the ileum (n = 5, including one patient who discontinued MTX), colon (n = 6, all on MTX), and rectum (n = 16, including two patients who discontinued MTX). Ileal biopsies were from inflamed tissue, rectal biopsies from non-inflamed tissue, and colon biopsies included both inflamed (4) and non-inflamed (2) samples. The median duration of MTX use before biopsy was 28 weeks (range 5–54 weeks). Most patients were on 15 mg MTX/week, except for two on 25 mg/week, with one discontinuing before endoscopy.
MTX-PG profiles in RBCs of patients with Crohn’s disease (CD) on MTX therapy showed that MTX-PG 3 was the most prevalent polyglutamate form at week 12, followed by MTX-PG 1, MTX-PG 4, MTX-PG 2, and MTX-PG 5. RBC concentrations of MTX-PG 3 remained stable after 12 weeks, while MTX-PG 4 and 5 decreased over time, and MTX-PG 2 increased.
PBMCs accumulated higher concentrations of MTX-PGs compared to RBCs, with MTX-PG 1 being the most abundant in PBMCs. The levels of MTX-PGs in PBMCs were notably higher than in RBCs: 18-fold for MTX-PG 1, 12-fold for MTX-PG 2, 3-fold for MTX-PG 3, and 1.5-fold for MTX-PG 4.
In intestinal mucosal biopsies, all MTX-PG species were detectable with significant interpatient variability. MTX-PG 1 was the most common in mucosal samples, though long-chain MTX-PGs (MTX-PG 4–6) were also highly prevalent. The proportion of MTX-PG 3 was lower in the mucosa compared to RBCs, while MTX-PG 1 and MTX-PG 5 proportions were higher.
Following MTX discontinuation, significant drops in MTX-PG 1 concentrations were observed in all matrices, with MTX-PG 2 also showing decreased levels in mucosa and RBCs. Long-chain MTX-PGs (4–6) were retained longer. In PBMCs, MTX-PG concentrations decreased rapidly, with short-chain MTX-PGs dropping by 93–98% and MTX-PG 4 by 75% within the first month.
The effect of MTX dosing on MTX-PG accumulation showed that a higher dose (25 mg/week) resulted in significantly greater RBC concentrations of MTX-PG 4 and 5. However, the influence of dosing on mucosal and PBMC samples could not be assessed due to limited sample sizes. No significant relationship was found between biopsy location and mucosal MTX-PG accumulation, though MTX-PG 1 was higher in ileal biopsies compared to colon and rectal biopsies, with this effect diminishing after adjusting for inflammation status. Median concentrations of MTX-PG 2–6 were higher in inflamed biopsies, but these differences were not statistically significant.
Conclusion
The study revealed significant differences in methotrexate polyglutamates (MTX-PGs) accumulation across various tissues. In peripheral blood mononuclear cells (PBMCs), MTX-PG levels were about ten times higher compared to red blood cells (RBCs). Intestinal mucosa exhibited the highest accumulation of long-chain MTX-PGs. After discontinuing methotrexate (MTX), there was a rapid decline in short-chain MTX-PGs in PBMCs, highlighting the distinct dynamics of MTX-PG retention in different cell types and tissues. This variability could inform future therapeutic drug monitoring (TDM) strategies for Crohn’s disease (CD) patients.
Prior studies in rheumatoid arthritis (RA) and juvenile idiopathic arthritis (JIA), and preliminary research in CD patients, have observed dose-dependent kinetic profiles of MTX-PGs in RBCs, reaching a steady state after three months, with MTX-PG3 being predominant. Our study’s findings align with these observations but also reveal higher MTX-PG4 and MTX-PG5 concentrations in the current cohort, potentially due to the subcutaneous administration and higher MTX dosage compared to earlier studies. Notably, RBCs showed higher MTX-PG2 and lower MTX-PG4 and MTX-PG5 concentrations after one year of treatment, which may be due to decreased folylpolyglutamate synthetase (FPGS) activity over time.
This is the first study to examine MTX-PG accumulation in PBMCs of CD patients. The total MTX-PG levels in PBMCs were approximately ten times higher than in RBCs, likely due to the nucleated nature of PBMCs and their role in biosynthetic processes. The MTX-PG profile in PBMCs primarily consisted of short-chain MTX-PGs, similar to findings in RA patients. The lower levels of long-chain MTX-PGs in PBMCs could be due to their short lifespan and high γ-glutamyl hydrolase (GGH) activity, though this was not directly measured.
In contrast, the intestinal mucosa, the primary target tissue for CD treatment, displayed substantial long-chain MTX-PG concentrations, reflecting high FPGS activity. This finding aligns with previous reports of high FPGS activity in proliferative tissues. After MTX discontinuation, the most significant decrease in MTX-PGs occurred in PBMCs, especially for short-chain MTX-PGs. This rapid decline may be attributed to the low polyglutamylation rate in PBMCs, their short circulation lifespan, and the presence of efflux transporters that expel MTX-PGs.
The study underscores the importance of analyzing MTX-PG profiles in various tissues to better understand MTX efficacy and retention. Future research should focus on cell- and tissue-specific analyses to refine therapeutic strategies. While the study’s limitations include a lack of detailed cellular composition in mucosal biopsies and variability in MTX administration, it provides valuable insights into MTX-PG distribution and accumulation that could enhance treatment monitoring and optimization for CD patients.
Oncology Related Tools
Other
Latest Research
