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Aims: To develop a non-linear mixed-effects population pharmacokinetic and pharmacodynamic (PK-PD) model describing the change in the concentration of methotrexate polyglutamates in erythrocytes (ery-MTX-PG n with "n" number of glutamate, representing PK component) and how this relates to modified 28-joint Disease Activity Score incorporating erythrocyte sedimentation rate (DAS-28-3) for rheumatoid arthritis (RA), representing PD component., Methods: An existing PK model was fitted to data from a study consisting of 117 RA patients. The estimation of population PK-PD parameters was performed using stochastic approximation expectation maximisation algorithm in Monolix 2021R2. The model was used to perform Monte Carlo simulations of a loading dose regimen (50mg subcutaneous methotrexate as loading doses, then 20mg weekly oral methotrexate) compared to a standard dosing regimen (10mg weekly oral methotrexate for 2 weeks, then 20mg weekly oral methotrexate)., Results: Every 40 nmol/L increase in ery-MTX-PG 3-5 total concentration correlated with 1-unit reduction in DAS-28-3. Significant covariate effects on the therapeutic response of methotrexate included the use of prednisolone in the first 4 weeks (positive use correlated with 25% reduction in DAS-28-3 when other variables were constant) and patient age (every 10-year increase in age correlated with 3.4% increase in DAS-28-3 when other variables were constant). 4 methotrexate loading doses led to a higher percentage of patients achieving a good/moderate response compared to the standard regimen (Week 4: 87.6% vs. 39.8%; Week 10: 64.7% vs. 57.0%)., Conclusions: A loading dose regimen was more likely to achieve higher ery-MTX-PG concentration and better therapeutic response after 4 weeks of methotrexate treatment., (© 2024 The Author(s). British Journal of Clinical Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)

The aim of this study was to investigate the impact of genetic polymorphisms on methotrexate (MTX) metabolism in Korean children and young adults with acute lymphoblastic leukemia, specifically focusing on MTX polyglutamates (MTX-PGs) in erythrocytes, which have been rarely studied. Korean children and young adult patients undergoing maintenance therapy for acute lymphoblastic leukemia, who were receiving weekly oral MTX doses of 20 mg/m²/week, were prospectively included. We investigated erythrocyte MTX-PG (PG1 to PG5) levels, MTX-PG/MTX dose ratios, and 222 genetic polymorphisms spanning 78 genes and three intergenic areas related to MTX transport, folate cycle metabolism, purine/pyrimidine pathways, and non-pathway genes (including TPMT and NUDT15 genotypes) to explore their association with MTX metabolism. MTX-PG levels were associated with MTX dose (p < 0.05), and MTX-PG3 comprised the majority of the total MTX-PGs, with a median of 39.3 %. Various polymorphisms within the same gene demonstrated differing associations with each type of MTX-PG, underscoring the complexity of MTX pharmacogenetics. Among the polymorphisms examined, 14 across 13 genes showed significant associations with MTX-PG2-5 levels, even after adjusting for the false discovery rate (ABCC5, ATG16L1, CEP72, FSTL5, GMPS, HTR3A, IMPDH1, NT5C2, SLC28A3, SLCO1B3, SUCLA2, TPMT, and TYMS). This study enhances our understanding of genetic polymorphisms in MTX metabolism and therapeutic monitoring for MTX maintenance, promoting personalized medicine in acute lymphoblastic leukemia patients., Competing Interests: Declarations of competing interest The authors have declared no conflicts of interest., (Copyright © 2024. Published by Elsevier B.V.)

Background and Objective: High-dose methotrexate (HD-MTX) is the mainstream therapy of current acute lymphoblastic leukemia (ALL) regimens, but frequent intra- and interindividual differences in the clinical response to HD-MTX lead to chemotherapeutic interruption or discontinuation. The exact mechanism of transport across the cell membrane and the disposition of active methotrexate metabolites-methotrexate polyglutamates (MTXPGs)-are not well described in the literature. The aim of this study was to gain more insight into the plasma distribution of methotrexate and MTXPGs in pediatric patients with ALL and to clarify the obscure pathways of MTXPGs., Methods: We prospectively measured the concentrations of MTXPG 1-7 in plasma samples from three male pediatric patients treated with HD-MTX and leucovorin rescue according to the IC-BFM 2009 protocol using liquid chromatography-mass spectrometry (LC-MS). Blood samples were obtained at 24, 36, 42, and 48 h after the start of HD-MTX treatment., Results: Noticeable plasma concentrations of MTXPGs with a 2.2-fold interpatient variability were detected. The highest interindividual variability in total plasma MTXPG concentration was observed at 36 h, and ranged from 13.78 to 30.82 μmol/L. Among all patients, the predominant polyglutamate types in relation to the total plasma MTXPG concentration at each time point were MTXPG 3 (16.71-30.02%) and MTXPG 5 (26.23-38.60%), while MTXPG 7 was the least abundant MTXPG (3.22-5.02%)., Conclusion: The presence of MTXPGs in plasma of patients with ALL could be related to the action of ABC efflux transporters on blood cells and hepatocytes resulting from the administration of high doses of methotrexate. This study may not draw definitive conclusions, but it does reduce uncertainty about the dynamics of methotrexate and its active metabolites, which may be of vital importance for achieving a clinical response., (© 2021. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Purpose: Osteonecrosis is a burdensome treatment-related toxicity that is mostly diagnosed during or soon after 6-mercaptopurine (6MP)/methotrexate (MTX) maintenance therapy for acute lymphoblastic leukemia (ALL), possibly indicating a pathogenic role of these drugs., Methods: We prospectively registered symptomatic osteonecrosis during treatment of 1234 patients aged 1.0-45.9 years treated according to the Nordic Society of Hematology and Oncology (NOPHO) ALL2008 protocol. MTX/6MP metabolites were measured as part of the NOPHO ALL2008 maintenance therapy study., Results: After a median follow-up of 5.6 years [interquartile range (IQR) 3.6-7.5], 68 patients had been diagnosed with symptomatic osteonecrosis. The cumulative incidence was 2.7% [95% confidence interval (CI) 1.6-3.8%] for patients aged < 10 years, 14.9% (95% CI 9.7-20.2%) for patients aged 10.0-17.9 years, and 14.4% (95% CI 8.0-20.8%) for patients aged ≥ 18 years. The median time from diagnosis of ALL to diagnosis of osteonecrosis in these age groups was 1.0 year (IQR 0.7-2.0), 2.0 years (IQR 1.1-2.4), and 2.2 years (IQR 1.8-2.8), respectively (p = 0.001). With 17,854 blood samples available for MTX and 6MP metabolite analysis, neither erythrocyte levels of 6-thioguanine (TG) nucleotides (p > 0.99), methylated 6MP metabolites (p = 0.37), MTX polyglutamates (p = 0.98) nor DNA-TG (p = 0.53) were significantly associated with the hazard of osteonecrosis in Cox models stratified by the three age groups and adjusted for sex., Conclusion: Maintenance therapy intensity determined by 6MP and MTX metabolites was not associated with the risk of developing osteonecrosis in the NOPHO ALL2008 cohort., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Methotrexate is the gold standard treatment in rheumatoid arthritis. Once absorbed, it is internalized in cells, where glutamate residues are added to produce polyglutamated forms, which are responsible for the effect of methotrexate. The aim of the current study is to determine the relationship between methotrexate triglutamate concentrations and the clinical evolution in rheumatoid arthritis patients, as well as to characterize the variability in both features to propose strategies for low-dose methotrexate optimization. The quantification of methotrexate triglutamate concentration in red blood cells was performed through ultra-performance liquid chromatography coupled with mass spectrometry. Polymorphisms of genes involved in the formation of polyglutamates were determined by real-time polymerase chain reaction. A multivariate regression was performed to determine the covariates involved in the variability of methotrexate triglutamate concentrations and a population pharmacokinetics model was developed through nonlinear mixed-effects modeling. Disease activity score changed according to methotrexate triglutamate concentrations; patients with good response to treatment had higher concentrations than moderate or nonresponding patients. The methotrexate triglutamate concentrations were related to time under treatment, dose, red blood cells, and body mass index. A 1-compartment open model was selected to estimate the pharmacokinetic parameters; the typical total clearance (L/day) was determined as 1.45 * (body mass index/28 kg/m 2 ) * (red blood cells/4.6 × 10 6 cells/μL) and the volume of distribution was 52.4 L, with an absorption rate of 0.0346/day and a fraction metabolized of 1.03%. Through the application of the model, the initial dose of methotrexate is proposed on the basis of stochastic simulations and considering methotrexate triglutamate concentrations found in responders patients., (© 2021, The American College of Clinical Pharmacology.)

Objectives: The aim of the study was to explore the effect of total glucosides of paeony (TGP) and Tripterygium wilfordii polyglycosides (TWP) on erythrocyte methotrexate polyglutamates (MTXPGs), the metabolites of methotrexate (MTX)., Methods: An ultra-high-performance liquid chromatography (UPLC)-tandem mass spectrometry (MS/MS) method was developed to determine MTXPGs. The effects of MTXPGs were analysed using 24 male Sprague-Dawley rats that were randomly divided into the MTX alone, MTX-TGP combined, and MTX-TWP combined groups. Rats were administered MTX at a dose of 0.9 mg/kg once a week, TGP at 0.054 g/kg and TWP at 1.8 mg/kg three times a day. Venous blood (1.0 ml) was collected at weeks 2, 4, 6, 9, 12 and 15 and then analysed using the developed UPLC-MS/MS method., Key Findings: Specificity, linear range, inter-and intra-day precision, recovery, matrix effect and stability of MTXPGs met the standard regulations. This method was successfully used for the detection of MTXPGs. After administration of MTX alone, erythrocyte MTXPGs increased and accumulated in a time- and dose-dependent manner. Compared to MTX alone, the combination with TGP significantly decreased the content of total MTXPGs and short-chain MTXPGs (Methotrexate [MTX/MTXPG1] and 4-amino-10-methylpteroyldiglutamic acid [MTXPG2], P < 0.05), but had no significant effect on long-chain MTXPGs (4-amino-10-methylpteroyltriglutamic acid [MTXPG3], P > 0.05) and very long-chain MTXPGs (4-amino-10-methylpteroyltetraglutamic acid [MTXPG4] and 4-amino-10-methylpteroylpentaglutamic acid [MTXPG5], P > 0.05) at week 15. The combination of MTX with TWP had no significant effect on the content of total MTXPGs, short-chain MTXPGs and long-chain MTXPGs (P > 0.05), but it significantly decreased the content of very long-chain MTXPGs (P < 0.05) at week 15., Conclusions: The UPLC-MS/MS method was successfully used to determine MTXPGs in rat erythrocytes. TGP and TWP in combination with MTX affected the production of MTXPGs of different chain lengths in erythrocytes., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Royal Pharmaceutical Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

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).

This study investigated the impact of seven polymorphisms in genes of folate transport and (de)glutamation pathway on methotrexate polyglutamate levels and response in patients with rheumatoid arthritis. This prospective study included patients with rheumatoid arthritis. They were treated with methotrexate (up to 25 mg per week) for 24 weeks and categorized by EULAR response criteria into responders (good and moderate) and non-responders. Using real-time Taqman discrimination assay, SNPs were genotyped-rs1045642 (ABCB1 3435C>T), rs1128503 (ABCB1 1236C>T), rs10106 (FPGS 1994A>G), rs1544105 (FPGS G>A), rs11545078 (GGH 452C>T), rs3758149 (GGH -401C>T), and rs1051266 (RFC1 80G>A). RBC methotrexate polyglutamate 1-5 (MTX-glu 1-5 ) levels were determined at 4, 8, 16, and 24 weeks using by reverse phase HPLC using C-18 column followed by post column photo-oxidation. This study included 117 patients with rheumatoid arthritis (M:F = 14:103). The mean dose of methotrexate at 24 weeks was 22.0 ± 4.0 mg, with data on DAS28(3) at 24 weeks available in 96 patients-61 responders and 35 non-responders. Minor allele of GGH 452C>T had an association with non-response (odds ratio 2.9, 95% CI 1.4-5.6) and assuming the dominance of C, the recessive genetic model found GGH 452C>T CC genotype (odds ratio 9.5, 95% CI 1.2 to 76.0) was significantly associated with response. However, there was no difference in MTX-glu 1-5 levels among the various genotypes of this SNP (p = 0.9). Other SNPs were neither associated with response nor with alteration in methotrexate polyglutamate levels. On logistic regression, GGH 452C>T CC genotype and DAS28(3) at baseline were independent predictors of response. GGH 452C>T CC genotype was associated with response to methotrexate. None of the SNPs affected MTX-glu 1-5 levels.

Purpose: A pharmacokinetic (PK) model is available for describing the time course of the concentrations of methotrexate (MTX or MTXGlu1) and its active polyglutamated metabolites (MTXGlu2-5) in red blood cells (RBCs). In this study, we aimed to simplify the MTX PK model and to optimise the blood sampling schedules for use in future studies., Methods: A proper lumping technique was used to simplify the original MTX RBC PK model. The sum of predicted RBC MTXGlu3-5 concentrations in both the simplified and original models was compared. The sampling schedules for MTXGlu3-5 or all MTX polyglutamates in RBCs were optimised using the Population OPTimal design (POPT) software., Results: The MTX RBC PK model was simplified into a three-state model. The maximum of the absolute value of relative difference in the sum of predicted RBC MTXGlu3-5 concentrations over time was 6.3 %. A five blood sample design was identified for estimating parameters of the simplified model., Conclusions: This study illustrates the application of model simplification processes to an existing model for MTX RBC PK. The same techniques illustrated in our study may be adopted by other studies with similar interest.

Background and Objectives: Therapeutic drug monitoring in patients with rheumatoid arthritis (RA) receiving methotrexate (MTX, MTXGlu1) has not been established. In this study, we aim to explore the relationship between red blood cell (RBC) concentrations of MTX and its polyglutamate metabolites (MTXGlu(n); n = 2, 3, 4, 5) and clinical response in RA patients commencing MTX., Methods: The binding activity of MTXGlu(n) to three putative enzymes involved in the MTX mechanism of action—dihydrofolate reductase, thymidylate synthase, and 5-aminoimidazole-4-carboxamide ribonucleotide transformylase—was simulated. RBC MTXGlu(n) concentrations that gave the highest inhibition activity across all three enzymes were linked with the disease activity score DAS28-3v (C-reactive protein [CRP]). A population pharmacokinetic-pharmacodynamic model was developed to describe the relationship between RBC MTX polyglutamate concentrations and clinical response in 12 RA patients commencing MTX., Results: The highest inhibition activity was with RBC MTXGlu(3-5). These polyglutamates were further evaluated for their relationship with DAS28-3v (CRP). Three of the 12 patients had a high DAS28-3v (CRP) at baseline (mean = 6.1) and showed a delayed response to MTX treatment. The remaining nine patients with a lower DAS28-3v (CRP) baseline (mean = 3.6) showed an immediate response. The developed MTX pharmacokinetic-pharmacodynamic model provided an acceptable description of the observed DAS28-3v (CRP) across all patients., Conclusions: The developed model describes a longitudinal relationship between RBC MTXGlu(3-5) concentrations and DAS28-3v (CRP) in patients with RA commencing MTX. Further work is required to determine whether measurement of RBC MTX polyglutamates might be useful for dose individualisation in patients with RA.

Objective: Low-dose methotrexate (MTX) is the anchor drug in the treatment for rheumatoid arthritis (RA). Response to MTX is related to the intracellular MTX-polyglutamate (MTX-PG) levels and little is known about its determinants. We aimed to define the determinants of erythrocyte MTX-PG concentrations in 2 prospective cohorts of patients with RA., Methods: Patients with RA treated with MTX from 2 longitudinal cohorts were included: 93 from the MTX-R study (Rotterdam, the Netherlands derivation cohort), and 247 from the treatment in Rotterdam Early Arthritis Cohort study (validation cohort). MTX-PG concentrations were measured at 3 months of treatment using liquid chromatography/mass spectrometry. The MTX-PG were used as outcome measure. Various sociodemographic, clinical, biochemical, and genetic factors were assessed at baseline. Associations with MTX-PG levels were analyzed using multivariate regression analysis., Results: Age was positively associated with MTX-PG1 (stβ 0.23, p=0.033) and total MTX-PG (stβ 0.23, p=0.018) in the derivation cohort, and with all MTX-PG in the validation cohort (MTX-PG1: stβ 0.13, p=0.04; MTX-PG2: stβ 0.21, p=0.001; MTX-PG3: stβ 0.22, p<0.001; MTX-PG4+5: stβ 0.25, p<0.001; and total MTX-PG: stβ 0.32, p<0.001). Erythrocyte folate levels were positively associated with MTX-PG3 (stβ 0.3, p=0.021) and total MTX-PG levels (stβ 0.32, p=0.022) in the derivation cohort, which was replicated for MTX-PG3 (stβ 0.15, p=0.04) in the validation cohort. Patients with the folylpolyglutamate synthase (FPGS) rs4451422 wild-type genotype had higher concentrations of MTX-PG3 (p<0.05), MTX-PG4+5 (p<0.05), and total MTX-PG (p<0.05) in both cohorts. In the combined cohort, MTX dose was positively associated with levels of MTX-PG3 (stβ 0.23, p<0.001), MTX-PG4+5 (stβ 0.30, p<0.001), and total MTX-PG (stβ 0.20, p=0.002), but negatively associated with MTX-PG2 levels (stβ -0.22, p<0.001)., Conclusion: Our prospective study shows that higher age, higher MTX dose, higher erythrocyte folate status, and the FPGS rs4451422 wild-type genotype are associated with higher MTX-PG concentrations. While only up to 21% of interpatient variability can be explained by these determinants, this knowledge may aid in the development of personalized treatment in RA.

Objective: The combination of methotrexate (MTX) with infliximab can modify infliximab pharmacokinetics and lower the incidence of antibodies against infliximab (ATIs). We hypothesised that the pharmacokinetic interaction between MTX and infliximab is related to activation of MTX to immunosuppressive MTX polyglutamates (MTXPGs)., Methods: Adult patients with rheumatoid arthritis receiving weekly MTX with infliximab for more than 3 months were enrolled in a cross-sectional study. Blood was collected at trough before the infusion of infliximab. Red blood cell (RBC) MTXPGs were measured using liquid chromatography, and circulating levels of infliximab were measured using a cell-based assay. ATIs were measured using enzyme immunoassays. Statistical analyses consisted of multiple regression and Wilcoxon tests., Results: In the 61 patients enrolled in the study, ATIs were detected in 11 (18%). Regression analyses revealed that lower infliximab levels (median 3.3 µg/ml) were associated with the presence of ATIs and lower RBC MTXPG levels (median 28 nmol/l) (p<0.05). Logistic regression revealed that RBC MTXPG levels above 25 nmol/l were associated with a 4.7-fold lower likelihood of having ATIs (OR=4.7; 95% CI 1.1 to 20.8; p=0.02). None of the 12 patients with RBC MTXPG levels above 50 nmol/l tested positive for ATIs., Conclusions: These hypothesis-generating data indicate that MTXPGs are associated with infliximab pharmacokinetics and ATI formation.

Background: Measurement of intracellular concentrations of methotrexate (MTX) and its polyglutamated metabolites (MTXGlu(2-5)) in red blood cells (RBCs) has been suggested as a potential means of monitoring low-dose MTX treatment of rheumatoid arthritis (RA). However, a possible correlation between RBC MTX and MTXGlu2-5 concentrations and clinical outcomes of MTX treatment in RA is debated. A better understanding of the dose-concentration-time relationship of MTX and MTXGlu(2-5) in RBCs by population pharmacokinetic modelling is desirable and will facilitate assessing a potential RBC concentration-effect relationship in the future., Aim: The purpose of this analysis was to describe the pharmacokinetics of MTX and MTXGlu(2-5) in RBCs. Secondary objectives included investigation of deglutamation reactions and the loss of MTX and MTXGlu(2-5) from the RBC., Methods: A model was developed using NONMEM(®) version 7.2 based on RBC data obtained from 48 patients with RA receiving once-weekly low-dose MTX treatment. This model was linked to a fixed two-compartment model that was used to describe the pharmacokinetics of MTX in the plasma. A series of five compartments were used to describe the intracellular pharmacokinetics of MTX and MTXGlu(2-5) in RBCs. Biologically plausible covariates were tested for a significant effect on MTX plasma clearance and the intracellular volume of distribution of all MTX species in RBCs ([Formula: see text]). The developed model was used to test hypotheses related to the enzymatic deglutamation of MTXGlu(2-5) and potential loss of MTXGlu(2-5) from RBCs., Results: The final RBC pharmacokinetic model required the intracellular volumes of distribution for the parent and metabolites to be set to the value estimated for the parent drug MTX alone, and the rate constants describing the polyglutamation steps were fixed at literature values. Significant covariates included effect of body surface area-adjusted estimated glomerular filtration rate on renal plasma clearance and effect of allometrically scaled total body weight with a fixed exponent of 0.75 on non-renal plasma clearance of MTX. The only significant covariate with an effect on [Formula: see text] was mean corpuscular volume (MCV). The model supported single deglutamation steps and a single mechanism of MTX and MTXGlu(2-5) loss from RBCs., Conclusions: The developed model enabled acceptable description of the intracellular kinetics of MTX and MTXGlu(2-5) in RBCs. In the future it can form the basis of a full pharmacokinetic-pharmacodynamic model to assess the time-RBC concentration-effect relationship of low-dose MTX treatment in RA.

Objectives: Methotrexate (MTX) is a cornerstone in the treatment of rheumatoid arthritis (RA). Although in general MTX is very effective, the major drawback is the large inter-patient variability in clinical response. The circulating levels of MTX polyglutamates (MTXPGs) are supposed to correlate with clinical efficacy, therefore having a potential role in drug monitoring. However, there is a controversial discussion about the importance of methotrexate polyglutamates as outcome parameters in the therapy of rheumatoid arthritis. The aim of the present study was to investigate the formation and pharmacokinetics of MTXPGs and to correlate their concentration with clinical response in MTX-naïve patients., Methods: The pharmacokinetics of erythrocyte MTXPGs was determined in samples of nineteen MTX-naïve patients by high pressure liquid chromatography (HPLC) using post-column photo-oxidation and fluorimetric detection. The relationship between erythrocyte concentrations of MTXPGs and the primary outcome parameter DAS-28 was assessed using the Spearman's correlation coefficient., Results: The short-chain polyglutamate MTXPG2 revealed to be a potential marker for clinical outcome in rheumatoid arthritis with a statistically significant positive correlation of MTXPG2 Cmax levels and improvement in DAS-28 (+0.518, p=0.023) over 16 weeks. Furthermore, Cmax levels of MTXPG2 negatively correlated with basophils (-0.478, p=0.038) and eosinophils (-0.531, p=0.019), both pro-inflammatory cells involved in the disease., Conclusions: MTXPG2 seems to be a potential indicator for clinical response and may serve as a marker for drug monitoring.

Methotrexate (MTX) is the first-line treatment for rheumatoid arthritis and is frequently used in the management of other forms of inflammatory arthritis. It is currently challenging to predict which patients will achieve adequate disease control and which patients will develop adverse effects while taking MTX. As an analog of dihydrofolic acid, MTX enters cells through the reduced folate carrier-1 protein, and is polyglutamated. MTX polyglutamates inhibit key enzymes in the folate pathway to produce an anti-inflammatory effect. It has been suggested that genetic polymorphisms in the folate pathway may influence intracellular folate and MTX polyglutamates pools, and thus MTX response. However, studies to identify genetic predictors have yielded inconclusive results. Nonreplication across studies has been attributed to insufficient statistical power as well as pharmacological and clinical confounders. Prospective studies, standardizing the definitions of response and toxicity, and application of genome-wide approaches may advance the search for genetic predictors of MTX response.

Purpose: To determine whether shortening the infusion duration of high-dose methotrexate (HDMTX; 1 g/m(2)) affects the in vivo accumulation of active methotrexate polyglutamates (MTXPG(1-7)) in leukemia cells and whether this differs among major acute lymphoblastic leukemia (ALL) subtypes., Methods: From June 2000 through October 2007, 356 children with ALL were randomly assigned to receive initial single-agent treatment with HDMTX (1 g/m(2)) as either a 24-hour infusion or a 4-hour infusion at two pediatric hospitals in the United States. The primary outcome measures were the accumulation of MTXPG(1-7) in leukemia cells and the antileukemic effects (eg, inhibition of de novo purine synthesis in bone marrow ALL cells, and decrease in circulating ALL cells)., Results: The 24-hour infusion resulted in significantly higher amounts of MTXPG(1-7) in bone marrow leukemia cells (median: 1,695 v 1,150 pmol/10(9) cells, P = .0059), and better antileukemic effects. The 24-hour infusion had the greatest effect on MTXPG(1-7) accumulation in hyperdiploid ALL (median: 3,919 v 2,417 pmol/10(9) cells, P = .0038); T-cell ALL exhibited smaller differences in MTXPG(1-7) but greater antileukemic effects with the longer infusion (median decrease in leukemia cells: 88.4% v 51.8%, P = .0075). In contrast, infusion duration had no significant impact on MTXPG(1-7) accumulation or antileukemic effects in ALL with the t(12;21)/(ETV6-RUNX1) chromosomal translocation., Conclusion: Shortening the infusion time of HDMTX reduces accumulation of active methotrexate in leukemia cells and decreases antileukemic effects, with differing consequences among major ALL subtypes.

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 one of the most commonly prescribed and most effective drugs for the treatment of rheumatoid arthritis (RA). Given the partial response of many patients and the side effect profile of the drug, there is considerable interest in identifying biomarkers to guide MTX therapy in RA. Upon entering cells, MTX is polyglutamated. Measuring MTX polyglutamate (MTX PG) levels in circulating red blood cells has been proposed as an objective measure to help optimize MTX therapy in RA. Data are conflicting with regard to the clinical utility of MTX PG measurements as a predictor of the efficacy or toxicity of low-dose MTX effects in RA. Should large, randomized clinical trials of this assay show consistent, reproducible, long-term correlations between MTX PG levels and efficacy or toxicity, this test could become a prominent tool for clinicians to optimize the use of MTX in treating RA.

The clinical relevance of the concept of bioavailability rests on two main principles. First, that measurement of the active component at the site of action is generally not possible and, secondly, that a relationship exists between on the one hand efficacy and/or safety and on the other hand concentration of the active compound or its active metabolite(s) in the systemic circulation. Applying these principles to the current knowledge on methotrexate (MTX), it is clear that bioavailability of MTX is an important parameter for optimal dosing. In this manuscript the current knowledge on MTX bioavailability is reviewed. This review reveals that bioavailability of MTX in higher oral doses is decreased, most probably by limitation of absorption from the gastro-intestinal tract. It is suggested that higher doses can be given either by splitting the oral dose or by parenteral administration. Both will result in improved bioavailability as compared with one higher oral dose. However, larger, prospective studies directly comparing the efficacy and safety of the splitted oral dose strategy and the switch to parenteral MTX are needed.

Methotrexate is used widely in the pharmacotherapy of juvenile idiopathic arthritis. Polyglutamates of methotrexate are active metabolites which accumulate in cells including erythrocytes. Their intracellular concentration may reflect methotrexate bioavailability and, at the same time, may serve as a bioindicator for optimization of methotrexate therapy and drug monitoring. Therefore, a simple and selective isocratic reversed phase chromatographic method with fluorescence detection (excitation/emission wavelengths of 370/463 nm) was developed which quantifies the sum of all methotrexate polyglutamates in erythrocytes as methotrexate after their enzymatic conversion with gamma-glutamylhydrolase. Separation was carried out on a Phenomenex GEMINI C18 column using a mobile phase flowing at a rate of 0.6 ml/min and consisting of a mixture (110:890:0.25 v/v) of acetonitrile, ammonium acetate buffer (0.05 M, pH=5.5) and hydrogen peroxide 30% (w/w). The method was found linear over the concentration range of 25-400 nmol/l. Its intra- and inter-day precision and accuracy were characterized by coefficients of variation and relative errors less than 20%. The limits of detection and quantification achieved 10.9 and 32.9 nmol/l, respectively. The method was proved suitable for monitoring the concentration of methotrexate polyglutamates in erythrocytes of patients with juvenile idiopathic arthritis.

Objective: There is evidence supporting a therapeutic range for methotrexate polyglutamate (MTXGlu) concentrations in the treatment of rheumatoid arthritis (RA). Knowledge of the pharmacokinetics of MTXGlu1-5 is required for optimal timing of blood sampling. The aim of this study was to determine the time to steady state and the half-life of accumulation of red blood cell (RBC) MTXGlu1-5 in patients with RA commencing oral MTX, and the time for RBC MTXGlu1-5 to become undetectable and the half-life of elimination of RBC MTXGlu1-5 in patients ceasing treatment with oral MTX., Methods: Ten patients beginning treatment and 10 patients stopping treatment with low-dose oral MTX were recruited. Blood samples were initially collected weekly, with gradual extension to monthly collection over the study period. RBC MTXGlu1-5 concentrations were assayed by high-performance liquid chromatography. Results were analyzed using a first-order exponential method., Results: The median times to reach steady state in RBCs (defined as 90% of the maximum concentration) were 6.2, 10.6, 41.2, 149, and 139.8 weeks, respectively, for MTXGlu1, MTXGlu2, MTXGlu3, MTXGlu4, and MTXGlu5. The median half-life of accumulation for RBC MTXGlu1-5 ranged from 1.9 weeks to 45.2 weeks. The median times for MTXGlus to become undetectable in RBCs were 4.5, 5.5, 10, 6, and 4 weeks, respectively, for MTXGlu1, MTXGlu2, MTXGlu3, MTXGlu4, and MTXGlu5. The median half-life of elimination for RBC MTXGlu1-5 ranged from 1.2 weeks to 4.3 weeks., Conclusion: There is wide interpatient variability of RBC MTXGlu1-5 accumulation and elimination in adults with RA. These data also suggest that after a dose change, >6 months are required for RBC MTXGlu1-5 to reach steady state. Such delays in achieving steady state suggest that more rapid dose escalation or subcutaneous administration from the outset should be considered.

Methotrexate (MTX) use in inflammatory bowel disease (IBD) is complicated by unpredictable efficacy and toxicity. In rheumatoid arthritis, total and individual red blood cell MTX polyglutamates (RBC MTXGlu1-5) correlate with disease activity and possibly toxicity, and therefore may be useful in guiding treatment. It is unclear if this applies in IBD. The aim of this pilot study was to measure total and individual RBC MTXGlu1-5 concentrations in patients with IBD to see if these relate to efficacy and adverse effects. RBC MTXGlu1--5 concentrations were measured on three occasions in 18 patients with IBD receiving MTX at a constant dose for 3 or more months. The intrapatient variability, expressed as the coefficient of variation, of RBC MTXGlu1-5 concentrations at steady state was determined, and disease activity and adverse effects were assessed against concentrations. The intrapatient coefficients of variation of individual MTXGlu1-5 varied from 12% to 27%. In Crohn's disease, higher RBC MTXGlu4&5 concentrations correlated with worse disease activity (r = 0.42 and 0.53, respectively, P

Members of the multidrug resistance protein family, notably MRP1-4/ABCC1-4, and the breast cancer resistance protein BCRP/ABCG2 have been recognized as cellular exporters for the folate antagonist methotrexate (MTX). Here we show that MRP5/ABCC5 is also an antifolate and folate exporter based on the following evidence: (a) Using membrane vesicles from HEK293 cells, we show that MRP5 transports both MTX (KM = 1.3 mmol/L and VMAX = 780 pmol per mg protein per minute) and folic acid (KM = 1.0 mmol/L and VMAX = 875 pmol per mg protein per minute). MRP5 also transports MTX-glu2 (KM = 0.7 mmol/L and VMAX = 450 pmol per mg protein per minute) but not MTX-glu3. (b) Both accumulation of total [3H]MTX and of MTX polyglutamates were significantly reduced in MRP5 overexpressing cells. (c) Cell growth inhibition studies with MRP5 transfected HEK293 cells showed that MRP5 conferred high-level resistance (>160-fold) against the antifolates MTX, GW1843, and ZD1694 (raltitrexed) in short-term (4 hours) incubations with high drug concentrations; this resistance was proportional to the MRP5 level. (d) MRP5-mediated resistance (8.5- and 2.1-fold) was also found in standard long-term incubations (72 hours) at low concentrations of ZD1694 and GW1843. These results show the potential of MRP5 to mediate transport of (anti)folates and contribute to resistance against antifolate drugs.

We reported that children with B-progenitor-cell acute lymphoblastic leukemia (BpALL) treated in the early 1980s whose lymphoblasts accumulated high levels of methotrexate (MTX) and of methotrexate polyglutamates (MTXPGs) in vitro had an improved 5-year event-free survival (EFS) (65% (standard error (s.e.) 12%) vs 22% (s.e. 9%)). We repeated this study in children with BpALL treated in the early 1990s. The major change in treatment was the addition of 12 24-h infusions of 1 g/M2 MTX with leucovorin rescue (IDMTX). In 87 children treated on Pediatric Oncology Group (POG) study 9005 and POG 9006, the 5-year EFS for those whose lymphoblasts accumulated high levels of MTX and MTXPGs (79.2%, s.e. 8.3%) was not significantly different from that of patients with lesser accumulation of MTX and MTXPGs (77.7%, s.e. 5.4%). These findings support the notion that higher dose MTX therapy has contributed to increased cure, particularly for patients whose lymphoblasts accumulate the drug less well.

The existence of an ATP-dependent methotrexate (MTX) efflux mechanism has long been postulated; however, until recently, the molecular components were largely unknown. We have previously demonstrated a role for the ATP-binding cassette transporter breast cancer resistance protein (BCRP) in MTX resistance (Volk et al., Cancer Res., 62: 5035-5040, 2002). Resistance to this antifolate directly correlated with BCRP expression, and was reversible by the BCRP inhibitors fumitremorgin C and GF120918. Here, we provide evidence for BCRP as a MTX-transporter using an in vitro membrane vesicle system. Inside-out membrane vesicles were generated from both drug-selected and stably transfected cell lines expressing either wild-type (Arg482) or mutant (Gly482) variants of BCRP. In the presence of the wild-type variant of BCRP, transport of MTX into vesicles was ATP-dependent, osmotically sensitive, and inhibited by fumitremorgin C. In contrast, no transport was observed in vesicles containing the mutant form of BCRP. Wild-type BCRP appeared to have low affinity, but high capacity, for the transport of MTX, with an estimated K(m) of 680 micro M and a V(max) of 2400 pmol/mg/min. MTX accumulation was greatly decreased by mitoxantrone, a known BCRP substrate, suggesting competition for transport. Furthermore, and in contrast to the multidrug resistance-associated proteins, BCRP also transported significant amounts of polyglutamylated MTX. Although transport gradually decreased as the polyglutamate chain length increased, both MTX-Glu(2) and MTX-Glu(3) were substrates for BCRP. Together, these data demonstrate that BCRP is a MTX and MTX-polyglutamate transporter and reveal a possible mechanism by which it confers resistance.

ABCG2 is a plasma membrane efflux pump that is able to confer resistance to several anticancer agents, including mitoxantrone, camptothecins, anthracyclines, and flavopiridol. The antimetabolite methotrexate (MTX) was inferred recently to be an additional substrate of the pump based on the analysis of ABCG2-overexpressing cell lines. However, the transport characteristics of the pump with regard to this agent have not been determined. In addition, physiological substrates of ABCG2 have not been identified. Here we examine the in vitro transport properties of the pump using membrane vesicles prepared from HEK293 cells transfected with ABCG2 expression vector. In so doing it is shown that MTX is a high capacity low affinity substrate of the pump, with K(m) and V(max) values of 1.34 +/- 0.18 mM and 687 +/- 87 pmol/mg/min, respectively. Unlike previously characterized multidrug resistance protein family members, ABCG2 is also able to transport MTX diglutamate and MTX triglutamate. However, addition of even one more glutamyl residue is sufficient to completely abrogate ABCG2-mediated transport. By contrast with the wild-type protein (ABCG2-R482), two ABCG2 variants that have been identified in drug selected cell lines, R482T and R482G, were unable to transport MTX to any extent. Similarly, folic acid was subject to efflux by the wild-type protein but not by the two mutants. However, transport of the reduced folate leucovorin was not detected for either the wild-type or the mutant proteins. Finally, it is shown that ABCG2 is capable of transporting E(2)17betaG with K(m) and V(max) values of 44.2 +/- 4.3 micro M and 103 +/- 17 pmol/mg/min, respectively. These results indicate that ABCG2 is a component of the energy-dependent efflux system for certain folates and antifolates, but that its transport characteristics with respect to polyglutamates and reduced folates are not identical to those of multidrug resistance protein family members. In addition, it is demonstrated that R482 mutations observed in drug-resistant cell lines have profound effects on the in vitro transport properties of the pump.

Methotrexate (MTX) is one of the most widely used drugs for the treatment of childhood acute lymphoblastic leukemia (ALL). Interindividual differences in lymphoblast accumulation of MTX and its active metabolites, methotrexate polyglutamates (MTXPG), may contribute to the effectiveness of treatment among ALL subtypes. To better understand these differences in MTXPG accumulation, we developed a model to characterize the cellular influx and efflux of MTX, formation of MTXPG by the addition of glutamyl residues catalyzed by FPGS (folylpolyglutamate synthetase), and cleavage of glutamyl residues from MTXPG by GGH (gamma-glutamyl hydrolase). The model was fitted to in vivo intracellular MTXPG concentrations measured serially in leukemic blasts from 20 newly diagnosed patients with ALL treated with 24-h intravenous infusions of MTX. The observed median concentrations of total MTXPG at 44 h was higher in B-lineage than in T-cell ALL (1706 vs 518 pmol/10(9) cells, P<0.025), consistent with the higher estimated Vmax for FPGS activity in B-lineage vs T-lineage blasts (414 vs 93 pmol/10(9) cells/h, P<0.008). Simulations based on the model-estimated parameters indicated greater accumulation of MTX, MTXPGs (MTXPG(2-7)) and total MTX (MTXPG(1-7)) with longer MTX infusions and with higher MTX doses, with the highest concentrations in hyperdiploid B-lineage, intermediate in non-hyperdiploid B-lineage, and lowest in T-cell ALL. These differences provide mechanistic and treatment insights for lineage and ploidy differences in MTXPG accumulation in human leukemia cells in vivo.

Human MRP4 (ABCC4, MOAT-B) is a lipophilic anion transporter that is able to confer resistance to nucleotide analogues and methotrexate (MTX). We previously investigated the implications of the ability of MRP4 to confer resistance to nucleotide analogues and determined that the pump is competent in the MgATP-energized transport of cyclic nucleotides and estradiol 17beta-D-glucuronide. Here we examine the potential role of MRP4 in conferring resistance to MTX and related processes by determining the selectivity of the transporter for MTX, MTX polyglutamates, and physiological folates. In so doing, it is shown that MRP4 is active in the transport of MTX as well as the physiological folates folic acid (FA) and N(5)-formyltetrahydrofolic acid (leucovorin). MTX, FA, and leucovorin are subject to high capacity [V(max(MTX)), 0.24 +/- 0.05 nmol/mg/min; V(max (FA)), 0.68 +/- 0.14 nmol/mg/min; V(max(leucovorin)), 1.95 +/- 0.18 nmol/mg/min], low affinity [K(m(MTX)), 0.22 +/- 0.01 mM; K(m(FA)), 0.17 +/- 0.02 mM; K(m (leucovorin)), 0.64 +/- 0.23 mM] transport by MRP4. In addition, as would be expected were MRP4 a component of the MTX efflux system, its capacity to transport this agent is abrogated by the addition of a single glutamyl residue. It is also shown that glutamylation similarly affects the ability of MRP2 to transport MTX. On the basis of these transport properties, it is concluded that the efflux system for MTX includes MRP2 and MRP4, in addition to MRP1 and MRP3, and that MRP4 represents a common efflux system for both MTX and certain nucleotide analogues.

We have recently determined that human multidrug resistance protein (MRP) 3, which confers resistance to certain natural product agents and methotrexate (MTX), is competent in the MgATP-energized transport of MTX and the monoanionic bile constituent glycocholate as well as several glutathione and glucuronate conjugates. Of these capabilities, the facility of MRP3 in conferring resistance to and mediating the transport of MTX is of particular interest because it raises the possibility that this pump is a component of the previously described cellular efflux system for this antimetabolite. However, if this is to be the case, a critical property of cellular MTX efflux that must be addressed is its ability to mediate the export of MTX but not that of its intracellular polyglutamylated derivatives. Here we examine the role of MRP3 in these and related processes by determining the selectivity of this transporter for MTX, MTX polyglutamates, and physiological folates. In so doing, we show that MRP3 is not only active in the transport of MTX but is also active in the transport the physiological folates folic acid (FA) and N(5)-formyltetrahydrofolic acid (leucovorin) and that polyglutamylation of MTX abolishes transport. Both FA and leucovorin are subject to high-capacity (V(max(FA)), 1.71 +/- 0.05 nmol/mg/min; V(max(leucovorin)), 3.63 +/- 1.20 nmol/mg/min), low-affinity (K(m(FA)), 1.96 +/- 0.13 mM; K(m(leucovorin)), 1.74 +/- 0.65 mM) transport by MRP3. Addition of a single glutamyl residue to MTX is sufficient to diminish transport by >95%. We also show that polyglutamylation similarly affects the capacity of MRP1 to transport MTX and that physiological folates are also subject to MgATP-stimulated transport by MRP1. On the basis of the capacity to transport MTX but not MTX-Glu(2), it is concluded that MRP3 and MRP1 represent components of the previously described cellular efflux system for MTX. The capacity of MRP3 to transport folates indicates that it may reduce intracellular levels of these compounds and thereby indirectly influence antifolate cytotoxicity, and it also implies that this pump may play a role in the response to chemotherapeutic regimens in which leucovorin is a component.