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Adjustment of mercaptopurine and methotrexate maintenance therapy of acute lymphoblastic leukaemia by leucocyte count is confounded by natural variations. Cytotoxicity is primarily mediated by DNA-incorporated thioguanine nucleotides (DNA-TGN). The aim of this study was to establish whether DNA-TGN concentrations in blood leucocytes during maintenance therapy are associated with relapse-free survival. In this substudy of the NOPHO ALL2008 phase 3 trial done in 23 hospitals in seven European countries (Denmark, Estonia, Finland, Iceland, Lithuania, Norway, and Sweden), we analysed data from centralised and blinded analyses of 6-mercaptopurine and methotrexate metabolites in blood samples from patients with non-high-risk childhood acute lymphoblastic leukaemia. Eligible patients were aged 1·0–17·9 years; had been diagnosed with non-high-risk precursor B-cell or T-cell leukaemia; had been treated according to the Nordic Society of Pediatric Hematology and Oncology ALL2008 protocol; and had reached maintenance therapy in first remission. Maintenance therapy was (mercaptopurine 75 mg/m2 once per day and methotrexate 20 mg/m2 once per week, targeted to a leucocyte count of 1·5–3·0 × 109 cells per L). We measured DNA-TGN and erythrocyte concentrations of TGN nucleotides, methylated mercaptopurine metabolites, and methotrexate polyglutamates. The primary objective was the association of DNA-TGN concentrations and 6-mercaptopurine and methotrexate metabolites with relapse-free survival. The secondary endpoint was the assessment of DNA-TGN concentration and 6-mercaptopurine and methotrexate metabolites during maintenance therapy phase 2. Between Nov 26, 2008 and June 14, 2016, 1509 patients from the NOPHO ALL2008 study were assessed for eligibility in the DNA-TGN substudy, of which 918 (89%) of 1026 eligible patients had at least one DNA-TGN measurement and were included in the analyses. Median follow-up was 4·6 years (IQR 3·1–6·1). Relapse-free survival was significantly associated with DNA-TGN concentration (adjusted hazard ratio 0·81 per 100 fmol/μg DNA increase, 95% CI 0·67–0·98; p=0·029). In patients with at least five blood samples, erythrocyte concentrations of TGN, methylated mercaptopurine metabolites, and methotrexate polyglutamates were associated with DNA-TGN concentration (all p<0·0001). Our results suggest the need for intervention trials to identify clinically applicable strategies for individualised drug dosing to increase DNA-TGN concentration, and randomised studies to investigate whether such strategies improve cure rates compared with current dose adjustments based on white blood cell counts. Danish Cancer Society, Childhood Cancer Foundation (Denmark), Childhood Cancer Foundation (Sweden), Nordic Cancer Union, Otto Christensen Foundation, University Hospital Rigshospitalet, and Novo Nordic Foundation.

Objective To investigate if erythrocyte-methotrexate-polyglutamate (MTX-PG) concentrations in patients with rheumatoid arthritis (RA) are associated with disease activity or adverse events. Methods We used a longitudinal study design with two cohorts. The derivation cohort included 102 and the validation cohort included 285 patients with RA on MTX. We measured erythrocyte-MTX-PG with 1–5 glutamate residues at 3 months, 6 months and 9 months after MTX start with a liquid chromatography (LC)-mass spectrometry (MS)/MS assay. Outcomes were disease activity score in 28 joints (DAS28) and adverse events. Longitudinal associations of MTX-PG concentrations after 3 months, 6 months and 9 months with DAS28 were tested with a linear mixed model adjusted for age, gender, baseline DAS28, MTX dose and comedication. Results In the derivation cohort, mean DAS28 decreased from 4.26 (SE=0.14) at baseline to 2.72 (SE=0.13) after 9 months. Thirty per cent of patients in the derivation cohort experienced more than three adverse events after 3 months, which decreased to 18% after 9 months. In the validation cohort, DAS28 and adverse events were comparable with the derivation cohort. In the derivation cohort, MTX-PG1 (ß=−0.005), MTX-PG2 (ß=−0.022), MTX-PG3 (β=−0.007) and total MTX-PG (ß=−0.004) were associated (p<0.05) with lower DAS28 over 9 months. In the validation cohort, MTX-PG2 (ß=−0.015), MTX-PG3 (ß=−0.010), MTX-PG4 (ß=−0.008) and total MTX-PG (ß=−0.003) were associated with lower DAS28 over 9 months. None of the MTX-PGs was associated with adverse events. Conclusions In this first longitudinal study, we showed that an increase in erythrocyte-MTX-PG concentration was associated with a decreased DAS28 over 9 months in two cohorts, and is therefore a potential tool for therapeutic drug monitoring of MTX in RA.

ObjectivesThere are no head-to-head trials of different dose escalation strategies of methotrexate (MTX) in RA. We compared the efficacy, safety and tolerability of ‘usual’ (5 mg every 4 weeks) versus ‘fast’ (5 mg every 2 weeks) escalation of oral MTX.MethodsThis multicentre, open-label (assessor blinded) RCT included patients 18-55 years of age having active RA with disease duration <5 years, and not on DMARDs. Patients were randomized 1:1 into usual or fast escalation groups, both groups starting MTX at 15 mg/week till a maximum of 25 mg/week. Primary outcome was EULAR good response at 16 weeks, secondary outcomes were ΔDAS28 and adverse effects (AE). Analyses were intention-to-treat.Results178 patients with mean DAS28-CRP of 5.4(1.1) were randomized to usual (n=89) or fast escalation groups (n=89). At 16 weeks, there was no difference in good EULAR response in the usual (28.1%) or fast escalation (22.5%) groups (p=0.8). There was no difference in mean ΔDAS28-CRP at 8 weeks (-0.9, -0.8, p=0.72) or 16 weeks (-1.3, -1.3, p=0.98). Even at 24 weeks (extended follow-up), responses were similar. There were no inter-group differences in ΔHAQ, or MTX-polyglutamates 1-3 levels at 8 or 16 weeks. Gastrointestinal AE were higher in the fast escalation group over initial 8 weeks (27%, 40%, p=0.048), but not over 16 weeks. There was no difference in cytopenias, transaminitis, or drug discontinuation/dose reduction between the groups. No serious AE were seen.ConclusionA faster MTX escalation strategy in RA was not more efficacious over 16-24 weeks, and did not significantly increase AE, except higher gastrointestinal AE initially.Trial registration numberCTRI/2018/12/016549

BACKGROUNDInterpatient differences in the accumulation of methotrexate's active polyglutamylated metabolites (MTXPGs) in leukemia cells influence its antileukemic effects.METHODSTo identify genomic and epigenomic and patient variables determining the intracellular accumulation of MTXPGs, we measured intracellular MTXPG levels in acute lymphoblastic leukemia (ALL) cells from 388 newly diagnosed patients after in vivo high-dose methotrexate (HDMTX) (1 g/m2) treatment, defined ALL subtypes, and assessed genomic and epigenomic variants influencing folate pathway genes (mRNA, miRNA, copy number alterations [CNAs], SNPs, single nucleotide variants [SNVs], CpG methylation).RESULTSWe documented greater than 100-fold differences in MTXPG levels, which influenced its antileukemic effects (P = 4 × 10-5). Three ALL subtypes had lower MTXPG levels (T cell ALL [T-ALL] and B cell ALL [B-ALL] with the TCF3-PBX1 or ETV6-RUNX1 fusions), and 2 subtypes had higher MTXPG levels (hyperdiploid and BCR-ABL like). The folate pathway genes SLC19A1, ABCC1, ABCC4, FPGS, and MTHFD1 significantly influenced intracellular MTXPG levels (P = 2.9 × 10-3 to 3.7 × 10-8). A multivariable model including the ALL subtype (P = 1.1 × 10-14), the SLC19A1/(ABCC1 + ABCC4) transporter ratio (P = 3.6 × 10-4), the MTX infusion time (P = 1.5 × 10-3), FPGS mRNA expression (P = 2.1 × 10-3), and MTX systemic clearance (P = 4.4 × 10-2) explained 42% of the variation in MTXPG accumulation (P = 1.1 × 10-38). Model simulations indicated that a longer infusion time (24 h vs. 4 h) was superior in achieving higher intracellular MTXPG levels across all subtypes if ALL.CONCLUSIONSThese findings provide insights into mechanisms underlying interpatient differences in intracellular accumulation of MTXPG in leukemia cells and its antileukemic effectsFUNDINGTHE National Cancer Institute (NCI) and the Institute of General Medical Sciences of the NIH, the Basque Government Programa Posdoctoral de Perfeccionamiento de Personal Investigador doctor, and the American Lebanese Syrian Associated Charities (ALSAC).

Methotrexate (MTX) is widely used for the treatment of childhood acute lymphoblastic leukemia (ALL). The accumulation of MTX and its active metabolites, methotrexate polyglutamates (MTXPG), in ALL cells is an important determinant of its antileukemic effects. We studied 194 of 356 patients enrolled on St. Jude Total XV protocol for newly diagnosed ALL with the goal of characterizing the intracellular pharmacokinetics of MTXPG in leukemia cells; relating these pharmacokinetics to ALL lineage, ploidy and molecular subtype; and using a folate pathway model to simulate optimal treatment strategies. Serial MTX concentrations were measured in plasma and intracellular MTXPG concentrations were measured in circulating leukemia cells. A pharmacokinetic model was developed which accounted for the plasma disposition of MTX along with the transport and metabolism of MTXPG. In addition, a folate pathway model was adapted to simulate the effects of treatment strategies on the inhibition of de novo purine synthesis (DNPS). The intracellular MTXPG pharmacokinetic model parameters differed significantly by lineage, ploidy, and molecular subtypes of ALL. Folylpolyglutamate synthetase (FPGS) activity was higher in B vs T lineage ALL (p<0.005), MTX influx and FPGS activity were higher in hyperdiploid vs non-hyperdiploid ALL (p<0.03), MTX influx and FPGS activity were lower in the t(12;21) (ETV6-RUNX1) subtype (p<0.05), and the ratio of FPGS to γ-glutamyl hydrolase (GGH) activity was lower in the t(1;19) (TCF3-PBX1) subtype (p<0.03) than other genetic subtypes. In addition, the folate pathway model showed differential inhibition of DNPS relative to MTXPG accumulation, MTX dose, and schedule. This study has provided new insights into the intracellular disposition of MTX in leukemia cells and how it affects treatment efficacy.

Methotrexate (MTX) is an antifolic agent used in the first line of anti-inflammatory disease treatment and some oncologic issues. The metabolism of MTX is rapid, and the MTX concentration in the blood is not significant 24 h after administration. Unlike this, methotrexate polyglutamates (MTXPGs) can be informative biomarkers of drug exposure. It is widely concluded that MTXPG retention in red blood cells (RBCs) is related to appropriate efficacy, drug exposure, and toxicity during treatment. Therefore, the mentioned biomarker may be appropriately used for the PK/PD monitoring of low-dose MTX (LDMTX) treatment. The presented review study aimed to review published studies about MTX determination in clinical practice, including pre-analytical variability, bioanalytical considerations, and clinical applications of the methods for pharmacotherapy supporting target populations. In total, 14 papers from the field of bioanalytics have been included in the main review. For each phase of an analytical process, the best practises and main findings were defined as guidelines for proper analytical method optimisation, validation, and standard operation procedure implementation in clinical practice. The presented study is the first comprehensive review of MTX and its methods of metabolite determination to account for pre-analytical, analytical, and post-analytical phases concerning the TDM process.

Abstract Background and Aims Therapeutic drug monitoring (TDM) of methotrexate (MTX) is challenging due to its pharmacokinetics and short plasma half-life. Intracellular MTX–polyglutamates (PG1–5), which accumulate over time, have not been assessed in pediatric inflammatory bowel disease (IBD). This study aimed to evaluate erythrocyte MTX-PG as a potential TDM tool in pediatric IBD. Methods In this cross-sectional study, MTX-PG concentrations were measured in erythrocytes of children with IBD on stable low-dose MTX for at least 12 weeks using stable-isotope dilution liquid chromatography–tandem mass spectrometry. The influence of administration route, MTX dosage, and anthropometrics on MTX-PG concentrations was examined. Results Seventy-eight patients were included, showing MTX-PG3 as the predominant subspecies (median 27.0 nmol/L) with a median MTX-PGtotal of 74.8 nmol/L. A higher MTX dose correlated significantly with elevated levels of MTX-PG3, MTX-PG4, MTX-PG5, and MTX-PGtotal (P < .01). Adjusted for body surface area, MTX dose remained significantly associated with higher MTX-PG concentrations (P < .01). However, comparison by administration route was limited due to a few patients on subcutaneous MTX (n = 4). Conclusions We observed high interindividual variability in the reached erythrocyte MTX-PG concentrations. Body surface adjusted or unadjusted MTX dosage showed a positive linear correlation with erythrocyte MTX-PG concentrations in children with IBD. This is a prerequisite for TDM and provides a strong basis for further research into the relation between TDM of MTX, efficacy, and toxicity. Lay Summary This study investigated erythrocyte methotrexate–polyglutamate (MTX-PG) levels in pediatric inflammatory bowel disease patients, finding that higher MTX doses correlate with increased long-chain MTX-PG concentrations. Results showed substantial interindividual variability, supporting further research on MTX-PG’s potential as a therapeutic drug monitoring tool.

Monocytes and monocyte-derived macrophages play a key pathogenic role in inflammatory arthritis. Methotrexate (MTX) is the first-line disease-modifying antirheumatic drug (DMARD) for arthritis, yet the mechanisms and kinetics of its effects on monocyte/macrophages remain poorly understood. We have now investigated the temporal dynamics of the MTX's anti-inflammatory action by initially performing a phase I clinical trial (METOMAC) on healthy individuals following a single MTX dose, which revealed that MTX plasma levels peak at 1 h after MTX treatment, with maximal suppression of LPS-induced IL-1β and IL-6 in whole blood 5 days after MTX exposure. Building on these findings, we performed an observational clinical study (METOMAC-PAC) on 29 DMARD-naïve early arthritis patients receiving a weekly dose of MTX over a 3 month period. The METOMAC-PAC study revealed that MTX polyglutamates (MTX-PG) accumulates in peripheral blood monocytes, where MTX-PG is detected as early as 5 days post-treatment, and modulates the monocyte gene profile after 4 MTX doses, with specific enrichment of the aryl-hydrocarbon receptor (AhR)-molecular signature and genes coding for anti-inflammatory factors. Importantly, stratification of METOMAC-PAC patients according to their clinical response revealed that Good Responders exhibit increased expression of “non-classical monocyte”-specific genes ( MAF , FCGR3B , ICAM4 ) both before and after MTX treatment, while Partial Responders patients showed a higher baseline expression of genes preferentially expressed by “classical monocytes”. Our results elucidate the kinetics of the anti-inflammatory action of MTX, demonstrate that MTX modulates the monocyte transcriptional signature in vivo , and identify “non-classical monocyte”-associated genes as predictors for an effective MTX clinical response. Graphical abstract

The nutrient-sensing metabolite S-adenosylmethionine (SAM) controls one-carbon metabolism by donating methyl groups to biochemical building blocks, DNA, RNA, and protein. Our recent work uncovered a requirement for cytoplasmic arginine methylation during Wnt signaling through the activity of protein arginine methyltransferase 1 (PRMT1), which transfers one-carbon groups from SAM to many protein substrates. Here, we report that treatments that decrease levels of the universal methyl donor SAM were potent inhibitors of Wnt signaling and of Wnt-induced digestion of extracellular proteins in endolysosomes. Thus, arginine methylation provides the canonical Wnt pathway with metabolic sensing properties through SAM. The rapid accumulation of Wnt-induced endolysosomes within 30 minutes was inhibited by the depletion of methionine, an essential amino acid that serves as the direct substrate for SAM production. We also found that methionine is required for GSK3 sequestration into multivesicular bodies through microautophagy, an essential step in Wnt signaling activity. Methionine starvation greatly reduced Wnt-induced endolysosomal degradation of extracellular serum proteins. Similar results were observed by addition of nicotinamide (vitamin B3),which serves as a methyl group sink. Methotrexate, a pillar in the treatment of cancer since 1948, decreases SAM levels. We show here that methotrexate blocked Wnt-induced endocytic lysosomal activity and reduced canonical Wnt signaling. Importantly, the addition of SAM during methionine depletion or methotrexate treatment was sufficient to rescue endolysosomal function and Wnt signaling. Inhibiting the Wnt signaling pathway by decreasing one-carbon metabolism provides a platform for designing interventions in Wnt-driven disease.

Methotrexate (MTX) remains the first-choice treatment for rheumatoid arthritis (RA), but individual variability in response and adherence underscores the need for reliable biomarkers of long-term drug exposure. Intracellular methotrexate polyglutamates (MTXPGs), typically measured in red blood cells (RBCs), fulfill this role but require invasive venous sampling. This study aimed to develop and validate a multi-matrix LC–MS/MS method for measuring MTXPGs in capillary blood samples obtained via volumetric absorptive microsampling (VAMS) and dried blood spots (DBS), and to compare these methods with traditional matrices. The method was validated in accordance with ICH M10 guidelines across RBC, whole blood (WB), VAMS, and DBS samples. MTX and MTXPG2–5 and total MTXPG were measured in 40 matched clinical samples. MTXPG6–7 were not detected across the tested clinical samples. Validation using Passing–Bablok regression, Bland–Altman analysis, and Spearman correlation showed strong agreement between VAMS and DBS (slopes 0.95–1.07; bias −4.21% to 0.36%; SRCC ≥ 0.969), with up to 100% of samples within ±20% of the agreement limits for total MTXPG. Significant differences were observed between capillary matrices and RBCs, with higher MTXPG levels in erythrocytes (bias up to −28%). Whole blood showed closer agreement with microsampling methods. ISR pass rates ranged from 84% to 95%, and stability tests indicated matrix- and chain length-dependent degradation, particularly for long-chain MTXPGs. These findings show that VAMS and DBS yield comparable results and can be considered interchangeable within a capillary-sampling framework. However, interpretation must account for matrix-specific differences when relating measurements to RBC-based reference values. This validated method could support the analytical feasibility of decentralized MTXPG monitoring in RA. However, prospective studies linking matrix-specific thresholds with disease activity, adherence, and toxicity are required before implementation for therapeutic decision-making.