Genetic polymorphisms and their association with methotrexate polyglutamates during maintenance treatment in Korean children and young adults with acute lymphoblastic leukemia.

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.

Pharmacokinetic-pharmacodynamic modelling and simulation of methotrexate dosing in patients with rheumatoid arthritis.

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-PGn 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-PG3-5 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.

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-PGtotal was MTX-PG5. MTX-PG6 was measurable in all biopsies. CONCLUSIONS: MTX-PG patterns differ between mucosa, PBMCs and RBCs of patients with CD.

Cellular pharmacokinetic of methotrexate and its modulation by folylpolyglutamate synthetase and γ-glutamyl hydrolase in tumor cells.

BACKGROUND AND PURPOSE: Clinical studies showed that prolonged infusion of methotrexate (MTX) leads to more severe adverse reactions than short infusion of MTX at the same dose. We hypothesized that it is the saturation of folate polyglutamate synthetase (FPGS) at high MTX concentration that limits the intracellular synthesis rate of methotrexate polyglutamate (MTX-PG). Due to a similar accumulation rate, a longer infusion duration may increase the concentration of MTX-PG and, result in more serious adverse reactions. In this study, we validated this hypothesis. EXPERIMENTAL APPROACH: A549, BEL-7402 and MHCC97H cell lines were treated with MTX at gradient concentrations. Liquid chromatograph-mass spectrometer (UPLC-MS/MS) was used to quantify the intracellular concentration of MTX-PG and the abundance of FPGS and γ-glutamyl hydrolase (GGH). High quality data were used to fit the cell pharmacokinetic model. KEY RESULTS: Both cell growth inhibition rate and intracellular MTX-PG concentration showed a nonlinear relationship with MTX concentration. The parameter Vmax in the model, which represents the synthesis rate of MTX-PG, showed a strong correlation with the abundance of intracellular FPGS. CONCLUSION AND IMPLICATIONS: According to the model fitting results, it was confirmed that the abundance of FPGS is a decisive factor limiting the synthesis rate of MTX-PG. The proposed hypothesis was verified in this study. In addition, based on the intracellular metabolism, a reasonable explanation was provided for the correlation between the severity of adverse reactions of MTX and infusion time. This study provides a new strategy for the individualized treatment and prediction of efficacy/side effects of MTX.

Quantitation of methotrexate polyglutamates in human whole blood, erythrocytes and leukocytes collected via venepuncture and volumetric absorptive micro-sampling: a green LC-MS/MS-based method.

Low-dose methotrexate (MTX) plays a key role in treatment of rheumatoid arthritis. However, not all patients respond satisfactorily, and no therapeutic drug monitoring has been implemented in clinical practice, despite the fact that MTX therapy has now been available for decades. Analysis of individual intracellular MTX metabolites among rheumatoid arthritis (RA) patients is hampered by the low intracellular concentrations of MTX-PGs which require a highly sensitive method to quantify. Here, we present a rapid and highly sensitive LC (HILIC) MS/MS method with LLOQ 0.1 nM, 0.8 nmol/L for each metabolite of MTX-PG1-5 and MTX-PG6-7 respectively. Over a linear range of 0.1-100 nM, 0.8-100 nmol/L for each metabolite of MTX-PG1-5 and MTX-PG6-7, respectively, the inter- and intra- accuracy and precision were within 15% of the nominal value for all MTX metabolites. The presented assay was used to assess and compare MTX metabolite concentrations extracted from four different matrices: red blood cells, plasma, peripheral blood mononuclear cells, and whole blood that have been collected either using traditional venepuncture or volumetric absorptive micro-sampling (VAMS) sampling techniques. The presented method not only improves analyte coverage and sensitivity as compared to other published methods; it also improves the greenness.

Factors influencing methotrexate and methotrexate polyglutamate in patients with rheumatoid arthritis: a systematic review of population pharmacokinetics.

Low dose methotrexate (MTX) is commonly used in the treatment of rheumatoid arthritis. The clinical effect is mediated by its metabolite, methotrexate polyglutamate (MTX-PGn). The drug exhibits high interindividual pharmacokinetic variability and the optimal MTX dose is different among individuals. Thus, several MTX population pharmacokinetic (PopPK) models were developed to characterize factors affecting MTX pharmacokinetic variability. This review summarizes significant predictors for MTX pharmacokinetics and identifies knowledge gaps to be further examined. A total of 359 articles were identified from a systematic search of four databases: PubMed, Science Direct, and CINAHL Complete. Of these eight studies were included. Most studies investigated influential factors on MTX pharmacokinetics, but information on MTX-PGn is limited, with only one study performing a parent-metabolite (MTX-PG3) model. MTX pharmacokinetics was described using a two-compartment model with first-order elimination in most studies, with the MTX clearance ranging from 6.94 to 12.39 L/h. Significant predictors influencing MTX clearance included weight, creatinine clearance, sex, OATP1B3 polymorphism, and MTX multiple dosing. While body mass index and red blood cell counts were significant predictors for MTX-PG3 clearance. Providing that MTX-PGn plays a crucial role in clinical effect, further studies should determine other factors affecting MTX-PGn as well as its relationship with clinical response.

Comparison of two dose escalation strategies of methotrexate in active rheumatoid arthritis: a multicentre, parallel group, randomised controlled trial.

OBJECTIVES: There 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. METHODS: This 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. RESULTS: 178 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. CONCLUSION: A 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 NUMBER: CTRI/2018/12/016549.

Maintenance therapy and risk of osteonecrosis in children and young adults with acute lymphoblastic leukemia: a NOPHO ALL2008 sub-study.

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.

Adverse effects related to methotrexate polyglutamate levels: adjudicated results from the cardiovascular inflammation reduction trial.

OBJECTIVES: Methotrexate is widely used at low dosages (LD-MTX) for non-oncologic indications and is associated with a variety of adverse effects (AEs). We sought to determine whether concentrations of the active metabolite, MTX polyglutamates (MTX-PGs) 1-5, correlate with AEs. METHOD: We examined data from the LD-MTX arm of the randomized double-blind Cardiovascular Inflammation Reduction Trial (CIRT). All AEs were blindly adjudicated and monitoring laboratories were tested centrally. The MTX-PGs 1-5 were assessed in one reference laboratory using liquid chromatography-tandem mass spectrometry. Based on prior literature, MTX-PGs 3-5 were chosen as the exposure of interest and quartiles of MTX-PGs 3-5 were assessed for their relationship with all AEs and each pre-specified category of AE using adjusted Cox proportional hazards regression. RESULTS: Of the 2391 subjects randomized to LD-MTX, MTX-PG levels were available for 1319 subjects (median dosage 16.1 mg/week) from the 8 month visit. We followed these subjects for a median of 2.2 years [interquartile range (IQR) 1.5-2.9]. Higher MTX-PG3-5 levels were related to an increased risk of anaemia [compared with quartile 1 (Q1); hazard ratio (HR) for Q4 1.27 (95% CI 0.98, 1.65), P for trend = 0.05] and a decreased risk of thrombocytopenia [HR for Q4 0.52 (95% CI 0.32, 0.84), P for trend = 0.05]. MTX-PG3-5 levels >134 nmol/l were associated with an increased risk of liver abnormalities [HR 1.36 (95% CI 1.08, 1.72)]. CONCLUSIONS: Higher MTX- PG3-5 levels were modestly associated with LD-MTX AEs, including anaemia and liver function abnormalities, but a reduced risk of thrombocytopenia and haemorrhage. CLINICAL TRIAL REGISTRATION: NCT01594333.

Methotrexate Polyglutamates Analysis by Chromatography Methods in Biological Matrices: A Review.

Methotrexate (MTX) is used as an immunosuppressant and antineoplastic drug in clinical practice. MTX is a parent drug and converts to MTX polyglutamates (MTXPGs) to exhibit its biological activity. Clinical studies found that MTXPG levels were associated with MTX response and toxicities, especially at low doses. Due to huge variance of MTX response and toxicities between individuals, therapeutic drug monitoring is necessary for its use in individualized therapy. Various chromatography methods coupled with ultraviolet-visible detector, fluorescence detector and mass spectrometry have been reported for MTXPG analysis in various biological matrices. The aim of this paper is to review the chromatographic based methods for the measurement of total and/or individual MTXPGs. We searched Embase, Science Direct and PubMed databases using "methotrexate polyglutamate" and "chromatography" as search terms, and found 745 articles. Of those, 14 articles were extracted for this study. The key steps for method development (sample pretreatment, parameter optimization of liquid chromatography and mass spectrometry, selection of internal standard) and validation (lower limit of quantitation, accuracy, precision, recovery, matrix effect and stability) were analyzed and summarized, which might be helpful for researchers to develop their own methods.

Methotrexate polyglutamate quantification for clinical application in patients with pediatric acute lymphoblastic leukemia in association with genetic polymorphisms.

We developed and validated a quantification method for methotrexate (MTX) polyglutamates (MTX-PGs, MTX-PG1 to MTX-PG5) by liquid chromatography-tandem mass spectrometry using stable isotope-labeled internal standards and applied to 196 clinical samples collected from pediatric acute lymphoblastic leukemia patients treated with MTX. MTX-PGs levels and their proportions (%) in sum of all MTX-PGs (MTXSum) were evaluated in relation to TPMT, NUDT15, and MTHFR genotypes. For the developed method, linearity ranges 1-500 nmol/L, bias for accuracy 0.3-13.5 %, coefficient of variation for within- and between-run imprecision of 3.2-9.5% and 1.5-12.0%, respectively. Recoveries achieved were 74.2-105.8 %. There was no significant carryover. The median level of the MTXSum for 196 clinical samples was 129.4 nmol/L (interquartile range 28.1-241.2). MTX dose and MTX-PGs were associated (P < 0.05) and among five MTX-PGs, MTX-PG3 was the predominant form (median 41.7 %). The MTX-PG3 level was significantly higher in patients with TPMT *1/*3C than in patients with wild type and MTX-PG3% was significantly higher and MTX-PG5% was significantly lower in NUDT15 intermediate metabolizers than normal or indeterminate phenotypes (P < 0.05). This validated MTX-PGs quantification method can facilitate a better understanding of MTX metabolism and therapeutic drug monitoring for MTX treatment.

Effect of total glucosides of paeony and Tripterygium wilfordii polyglycosides on erythrocyte methotrexate polyglutamates in rats, analysed using ultra-high-performance liquid chromatography-tandem mass spectrometry.

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.

Simultaneous determination of erythrocyte methotrexate polyglutamates by a novel and simple HPLC-MS/MS method with stable isotope-labeled internal standards.

Low-dose methotrexate is the first-line therapy for juvenile idiopathic arthritis. In vivo, methotrexate is converted into a series of methotrexate polyglutamates whose intracellular levels contribute significantly to its efficacy and toxicity. In this study, a novel high-performance liquid chromatography-tandem mass spectrometry method was developed and validated to simultaneously determine erythrocyte methotrexate polyglutamates using stable isotope-labeled internal standards. Erythrocyte samples were precipitated by perchloric acid and then determined on an XBridge BEH C18 column with an XP vanguard precolumn in 12 min. The mobile phase consisted of 10 nM ammonium acetate (pH 10) and methanol under gradient elution. The detection was carried out in multiple reaction monitoring mode via an electrospray ionization source in positive ionization mode. The calibration curve for each metabolite was linear from 2.0 to 500.0 nmol/L (r2  > 0.99). The intraday and interday accuracies were between 93.0 and 107.0%, and the corresponding precisions were between 0.8 and 5.2%. The relative recovery ranged from 82.7 to 105.1%, and the relative matrix effect varied from 96.5 to 104.4%. The erythrocyte metabolites were stable for 30 days at -80°C. This simple and accurate method is applicable to routine monitoring of the concentration of erythrocyte methotrexate polyglutamates in patients to achieve individualized treatment.

Studies on the intracellular accumulation process of methotrexate and its correlation with the key protein using an LC-MS/MS method: a novel way to realize prospective individualized medication.

High-dose methotrexate (HDMTX) combined with leucovorin (LV) is the first-line drug therapy for many kinds of malignant tumors. However, the specific treatment plans, such as dosage and duration of administration, are usually formulated according to the clinician's experience and therapeutic drug monitoring (TDM) of methotrexate in patients' plasma, which are responsible for strong individual differences of drug usage. A large number of studies have shown that methotrexate targets the inside of the cell. The key cytotoxic component is the methotrexate polyglutamates (MTXPGs) in the cell. The concentration of methotrexate in plasma does not reflect the efficacy and side effects well. Based on mass spectrometry technology, we developed and validated an accurate, sensitive, and stable method to quantify the intracellular MTX (MTXPG1) and its metabolites MTXPG2-7 simultaneously. The lower limit of quantification was 0.100 ng/ml, and the run time was only 3 min. Moreover, our team has already developed two LC-MS/MS-based methods to respectively quantify methotrexate in plasma samples and two key proteins (γ-glutamyl hydrolase [GGH] and folylpolyglutamate synthetase [FPGS]) in peripheral blood mononuclear cells (PBMC). Through these highly sensitive and accurate approaches, we have gained a deep understanding of the whole pharmacokinetic process of MTX and explored the key factors affecting the accumulation process of intracellular active components (MTXPGs). Based on this research, it is possible to find a more effective way to provide an accurate reference for clinical drug use than traditional therapeutic drug monitoring (TDM).

Methotrexate Triglutamate as a Determinant of Clinical Response in Mexican Patients With Rheumatoid Arthritis: Pharmacokinetics and Dose Recommendation.

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/m2 ) * (red blood cells/4.6 × 106 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.

Interaction of nobiletin with methotrexate ameliorates 7-OH methotrexate-induced nephrotoxicity through endoplasmic reticulum stress-dependent PERK/CHOP signaling pathway.

Drug-induced nephrotoxicity is a frequent adverse event that contributes to acute kidney injury with tubular and/or glomerular lesions. Methotrexate (MTX) is a folate analog used against a myriad of malignancies and autoimmune diseases. Unfortunately, ambiguous renal toxicology limits its safe clinical usage. Based on our previous studies, 7-OH MTX as an overlooked oxidative metabolite of MTX was proposed to be the main culprit responsible for nephrotoxicity, while nobiletin, a naturally occurring polymethoxylated flavonoid screened from our prepared total phenolic extracts of Citrus aurantium L. (TPE-CA), was employed as a therapeutic agent for drug-drug interactions. According to the present study, nobiletin can ameliorate the renal accumulation of 7-OH MTX through the interaction with aldehyde oxidase. RNA-seq analysis revealed that 7-OH MTX was mainly related to protein processing in endoplasmic reticulum (ER) stress, with the PERK/CHOP pathway selected as the most significant for metabolic nephrotoxicity. Meanwhile, the cross-linked proteins and conducted signals were investigated by western blotting and further verified by GSK inhibition analyses. These results indicated that nobiletin protected renal function from MTX-induced nephrotoxicity by modulating metabolism and ameliorated the metabolic toxicity of 7-OH MTX on ER stress-induced PERK/CHOP conduction by maintaining Ca2+ homeostasis and reducing the production of reactive oxygen species.

Pharmacogenomics of intracellular methotrexate polyglutamates in patients' leukemia cells in vivo.

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).

Simultaneous Determination of Urine Methotrexate, 7-Hydroxy Methotrexate, Deoxyaminopteroic Acid, and 7-Hydroxy Deoxyaminopteroic Acid by UHPLC-MS/MS in Patients Receiving High-dose Methotrexate Therapy.

Nephrotoxicity, the most important toxicity in high-dose methotrexate (MTX) therapy, is partly caused by the formation of crystal deposits in the kidney due to poor water solubility of MTX and its metabolites 7-hydroxy methotrexate (7-OH MTX), deoxyaminopteroic acid (DAMPA) and 7-hydroxy deoxyaminopteroic acid (7-OH DAMPA). Plasma MTX level-guided urine alkalinization, leucovorin rescue and glucarpidase detoxification are common strategies to overcome MTX-related nephrotoxicity. However, overestimation is a problem for MTX analysis by immunoassays due to the cross-reactivity of MTX metabolites (7-OH MTX and DAMPA). An UHPLC-MS/MS method for the simultaneous determination of MTX, 7-OH MTX, DAMPA and 7-OH DAMPA in human urine was developed, validated and applied in clinical practice. Samples were treated by one-step protein precipitation and analyzed within 3 min. The calibration range was 0.02 to 4 µmol/L for MTX and DAMPA, and 0.1 to 20 µmol/L for 7-OH MTX and 7-OH DAMPA. For all analytes, the intra-day and inter-day bias and imprecision were -8.0 to 7.6 and <9.0%, the internal standard normalized recovery and matrix factor were 92.34 to 109.49 and <20.68%. The plasma MTX and 7-OH MTX levels increased with the urine drug levels, age, serum creatinine and alanine transaminase, but urine could not replace blood for MTX monitoring due to their poor correlation (R2, 0.16 to 0.51). Dose-normalized urine and plasma MTX and 7-OH MTX levels were similar between different patient groups (urine pH <7 or ≥7). Due to the large inter-individual variance of the analytes levels in both plasma and urine, these findings should be treated with caution.

In Silico Study Identified Methotrexate Analog as Potential Inhibitor of Drug Resistant Human Dihydrofolate Reductase for Cancer Therapeutics.

Drug resistance is a core issue in cancer chemotherapy. A known folate antagonist, methotrexate (MTX) inhibits human dihydrofolate reductase (hDHFR), the enzyme responsible for the catalysis of 7,8-dihydrofolate reduction to 5,6,7,8-tetrahydrofolate, in biosynthesis and cell proliferation. Structural change in the DHFR enzyme is a significant cause of resistance and the subsequent loss of MTX. In the current study, wild type hDHFR and double mutant (engineered variant) F31R/Q35E (PDB ID: 3EIG) were subject to computational study. Structure-based pharmacophore modeling was carried out for wild type (WT) and mutant (MT) (variant F31R/Q35E) hDHFR structures by generating ten models for each. Two pharmacophore models, WT-pharma and MT-pharma, were selected for further computations, and showed excellent ROC curve quality. Additionally, the selected pharmacophore models were validated by the Guner-Henry decoy test method, which yielded high goodness of fit for WT-hDHFR and MT-hDHFR. Using a SMILES string of MTX in ZINC15 with the selections of 'clean', in vitro and in vivo options, 32 MTX-analogs were obtained. Eight analogs were filtered out due to their drug-like properties by applying absorption, distribution, metabolism, excretion, and toxicity (ADMET) assessment tests and Lipinski's Rule of five. WT-pharma and MT-pharma were further employed as a 3D query in virtual screening with drug-like MTX analogs. Subsequently, seven screening hits along with a reference compound (MTX) were subjected to molecular docking in the active site of WT- and MT-hDHFR. Through a clustering analysis and examination of protein-ligand interactions, one compound was found with a ChemPLP fitness score greater than that of MTX (reference compound). Finally, a simulation of molecular dynamics (MD) identified an MTX analog which exhibited strong affinity for WT- and MT-hDHFR, with stable RMSD, hydrogen bonds (H-bonds) in the binding site and the lowest MM/PBSA binding free energy. In conclusion, we report on an MTX analog which is capable of inhibiting hDHFR in wild type form, as well as in cases where the enzyme acquires resistance to drugs during chemotherapy treatment.