Methotrexate Polyglutamates Research Articles

OA26 Erythrocyte mean corpuscular volume as a surrogate marker for methotrexate polyglutamation during early treatment in rheumatoid arthritis

Abstract Background/Aims The long-term outcomes for patients with rheumatoid arthritis (RA) depend on early and effective disease control. Methotrexate remains the key first-line disease-modifying therapy for the majority of patients. The gradual onset of action of methotrexate has been proposed to be linked to the build-up of intracellular stores of the drug, achieved through the process of polyglutamation. This is a complex and expensive assay. Recent work has shown an association between the increase in mean corpuscular volume (MCV) of erythrocytes and improvements in disease activity. This study seeks to relate intracellular methotrexate and 5-methyl-tetrahydrofolate polyglutamate concentrations with changes in MCV and their association with early ‘steroid free’ remission in RA patients. Methods 68 drug-naïve, newly diagnosed RA patients were recruited form the Newcastle Early Arthritis Clinic (NEAC). These patients were followed up for 6 months with serial blood sampling and quantification of the erythrocyte methotrexate and 5-methyl-tetrahydrofolate polyglutamate levels by liquid chromatography-mass spectrometry (LC-MS). This was then related to the contemporaneous erythrocyte MCV values from the clinical monitoring bloods on those occasions and the treatment outcome at 6 months. The outcome measure used was 4 component DAS28CRP remission (<2.4) with only the baseline intramuscular steroid permitted for the definition of remission. Results The majority of patients displayed a gradual increase in MCV over the first 6 months of treatment (MCV change from patient baseline used to normalise the data). When grouped by the ‘steroid free’ remission outcome, there was a divergence in MCV change with greater MCV increases observed in those achieving remission from 4 months of treatment onward (p < 0.05). In the same patient group, the accumulation of longer chain methotrexate polyglutamates (chain length 3 to 5) was observed to follow a similar trend to MCV with higher polyglutamate concentrations in those achieving the remission outcome from 2 months of treatment (P < 0.05). When the contemporaneous MCV and MTX polyglutamate samples were compared, an association was observed between the longer chain MTX polyglutamates and the change in MCV values (R2 = 0.39, p < 0.001), but not between intracellular 5-methyl-tetrahydrofolate and MCV (R2 = 0.01, p = NS). Conclusion Change in MCV shows an association with early remission induction with MTX therapy in treatment-naïve RA patients. This change in MCV appears to be associated with the accumulation of intracellular MTX metabolites, rather than via depletion of intracellular folates. MCV may therefore represent a tractable proxy measure of methotrexate polyglutamation for application in larger studies or for the purpose of drug monitoring. Disclosure P.M. Brown: None. A.G. Pratt: None. A.E. Anderson: None. A.W. Morgan: None. J.D. Isaacs: None.

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 12.39L/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.

Correlation between serum methotrexate-polyglutamate 3 (MTX-PG3) level and disease activity in rheumatoid arthritis patients: A prospective cohort study

Background: Rheumatoid arthritis (RA) is one of the most common autoimmune diseases, characterized by systemic inflammation, joint destruction and disability. Methotrexate (MTX) is used as the primary treatment for RA patients. However, the response to MTX therapy is highly varied and difficult to predict. This study sought to determine the role of MTX by measuring the MTX polyglutamate 3 (MTX-PG3) levels and the disease activity score 28 based on C-reactive protein (DAS28-CRP) of RA patients. Method: A prospective cohort study was conducted at the Rheumatology Polyclinic of Dr. Cipto Mangunkusumo General Hospital. Thirty-four patients with RA were included and followed up to 12 weeks. The RA patients were treated with MTX 10 mg per week and an increased dose of 5 mg per week every month. DAS28-CRP and MTX-PG3 level were assessed at week 8 and 12. Multivariate logistic regression analysis was used to determine the correlation between MTX-PG3 and DAS28-CRP. Result: A total of 34 RA patients were followed and the MTX was well tolerated in which no increase of serum glutamic oxaloacetic transaminase (SGOT), serum glutamic pyruvic transaminase (SGPT) and glomerular filtration rate (GFR) were observed. The mean scores of DAS28-CRP decreased following the MTX-treatment: 3.93, 3.22 and 2.82 at week 0, 8 and 12, respectively. In contrast, the median concentration of MTX-PG3 increased from week 8 to week 12 followed by increasing the dose of MTX. Our analysis suggested there was a moderate positive correlation between MTX-PG3 levels and DAS28-CRP score at week 8 and week 12 post-MTX treatment. Conclusion: The level of MTX-PG3 is correlated with DAS28-CRP score suggesting that MTX-PG3 could be used as an indicator to assess the disease activity in RA patients. Nevertheless, a prospective study with a higher number of patients is needed to confirm this finding.

Correlation between serum methotrexate-polyglutamate 3 (MTX-PG3) level and disease activity in rheumatoid arthritis patients: A prospective cohort study.

Background: Rheumatoid arthritis (RA) is one of the most common autoimmune diseases, characterized by systemic inflammation, joint destruction and disability. Methotrexate (MTX) is used as the primary treatment for RA patients. However, the response to MTX therapy is highly varied and difficult to predict. This study sought to determine the role of MTX by measuring the MTX polyglutamate 3 (MTX-PG3) levels and the disease activity score 28 based on C-reactive protein (DAS28-CRP) of RA patients. Method: A prospective cohort study was conducted at the Rheumatology Polyclinic of Dr. Cipto Mangunkusumo General Hospital. Thirty-four patients with RA were included and followed up to 12 weeks. The RA patients were treated with MTX 10 mg per week and an increased dose of 5 mg per week every month. DAS28-CRP and MTX-PG3 level were assessed at week 8 and 12. Multivariate logistic regression analysis was used to determine the correlation between MTX-PG3 and DAS28-CRP. Result: A total of 34 RA patients were followed and the MTX was well tolerated in which no increase of serum glutamic oxaloacetic transaminase (SGOT), serum glutamic pyruvic transaminase (SGPT) and glomerular filtration rate(GFR) were observed. The mean scores of DAS28-CRP decreased following the MTX-treatment: 3.93, 3.22 and 2.82 at week 0, 8 and 12, respectively. In contrast, the median concentration of MTX-PG3 increased from week 8 to week 12 followed by increasing the dose of MTX. Our analysis suggested there was a moderate positive correlation between MTX-PG3 levels and DAS28-CRP score at week 8 and week 12 post-MTX treatment. Conclusion: The level of MTX-PG3 is correlated with DAS28-CRP score suggesting that MTX-PG3 could be used as an indicator to assess the disease activity in RA patients. Nevertheless, a prospective study with a higher number of patients is needed to confirm this finding.

Changes in methotrexate polyglutamate concentration in patients with rheumatoid arthritis during treatment and after discontinuation (review of two cases)

Introduction. The dynamics of changes in the concentration of polyglutamates of methotrexate (MTPG) over the past 20 years has been studied by several scientifc groups using various methods. For a number of reasons, the results of these studies cannot be called uniform and cannot be confdently projected onto the Russian population of patients with rheumatoid arthritis (RA). At the same time, therapeutic drug monitoring of methotrexate (MT) with clearly defned target values of metabolites could be an extremely useful tool in routine clinical practice.Purpose of the study. To characterize the concentration of MTPG in dynamics during treatment and 12 weeks after discontinuation of MT.Materials and methods. Two patients with early RA were traced 4, 12, 24 weeks after MT appointment, and also 12 weeks after its cancellation due to nausea that appeared during treatment. At each visit, an analysis was made for MTPG content by tandem gas chromatography-mass spectrometry.Results. Against the background of treatment, the signifcantly predominant metabolite was MTPG with three and gour residues of glutamic acid (the so-called long-chain), while 12 weeks after discontinuation, MTPG 2 was the predominant fraction.Conclusions. Low values of MTPG 3 and MTPG 4 with high values of MTPG 2 may indicate a recent initiation of treatment or MTB cancellation within the next 3 months. In the event of subjective adverse reactions (ADRs), it is advisable to consider the possibility of switching to a drug analogue of another manufacturer.

P312 Erythrocyte methotrexate polyglutamate concentrations in patients with Crohn’s Disease: towards a new therapeutic drug monitoring tool

Abstract Background Methotrexate (MTX) is an immunomodulatory drug for patients with Crohn’s Disease (CD), used as first-line therapy, as a second-line in case of failure to thiopurine, and concomitantly with anti-TNFα agents to decrease production of anti-drug antibodies. Nevertheless, MTX is underutilised in the treatment of CD, despite its proven efficacy and good safety profile. This is for a large part due to the lack of therapeutic drug monitoring (TDM) of MTX because no stable plasma MTX levels are reached. Intracellular MTX-polyglutamates (MTX-PGs), formed after folylpolyglutamate synthetase attaches glutamate residues to MTX, could be used as a TDM tool as MTX-PG is thought to mediate MTX’s efficacy. We present the results of our cross-sectional study in CD patients, aiming to gain insight into erythrocyte MTX-PG levels. Methods CD adults on MTX treatment who visited the outpatient clinic of Amsterdam UMC between May 2019 and February 2020 were included consecutively. An established LC-ESI-MS/MS method1 was used to measure erythrocyte MTX-PGs. Results Nineteen patients were included. Mean disease duration was 17 years (SD±13.7). Montreal disease location and behaviour were as follows (n=): L1 = 2, L2 = 4, L3 = 13; B1 = 11, B2 = 5, B3 = 3. Only 4 patients had a flare according to Physician Global Assessment. Twelve patients received MTX monotherapy, whereas 7 patients were on concomitant anti-TNFα agents. The mean dose of MTX was 15.5 mg (SD±2.8) and 12 (63%) patients had subcutaneous (sc.) MTX. We successfully measured MTX-PG2-5 in 18 patients, showing substantial variability in measured concentrations of total MTX-PG(tot) and the individual species. The median MTX-PGtot was 117.1 nmol/L [min:46.4-max:358.7] with preferential accumulation of MTX-PG3 (43.1 [15.3-96.1]); the least predominant species being MTX-PG5 (9.4 [1.1-24.1]). Patients on sc. compared to oral MTX had higher MTX-PGtot levels (177.8 [58.8-358.7] vs. 93.2 [46.4-120.8] nmol/L, p=0.067) and significantly higher long-chain MTX-PG4-5 levels (55 [3.7-84.3] vs. 8.9 [2.4-15.0] nmol/L, p=0.010); see figure. Conclusion We showed that erythrocyte MTX-PGs can be measured in CD patients by tandem MS. Large variability in concentrations was demonstrated, similar to our previously published results in rheumatoid and juvenile arthritis2,3 which is a pre-requisite for MTX-PG use as a TDM tool. We showed for the first time that MTX-PG accumulation was higher in sc. MTX vs. oral MTX treatment. This work provides the first step towards establishing TDM for MTX in CD, a goal we aim to realize in our upcoming longitudinal study. References

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.

AB0277 ASSOCIATION BETWEEN INTRA-ERYTHROCYTE METHOTREXATE POLYGLUTAMATE CONCENTRATION AND CLINICAL RESPONSE IN RHEUMATOID ARTHRITIS IN PATIENTS TREATED WITH METHOTREXATE INJECTABLE SUBCUTANEOUSLY

Background:Methotrexate (MTX) is the first-line treatment for rheumatoid arthritis (RA). Due to its short half-life, blood MTX dosage is not performed in current practice. Erythrocyte MTX-polyglutamate (MTX-PG), which penetrates into the red blood cells, would be correlated with the area under the MTX curve and would be more accessible for dosing. When treatment is initiated, its concentration correlates with efficacy and therapeutic adherence.Objectives:To determine the interest of erythrocyte MTX-PG dosage in case of failure of 1st line MTX treatment.Methods:In this single-centre cross-sectional study, RA patients presenting for consultation at the Saint-Etienne University Hospital, with a stable dose of MTX for more than 3 months at least 15 mg/week subcutaneously with either clinical remission (DAS28<2.6) or active disease (DAS28>3.2) were included between July 2nd 2018 and May 28th 2020. In order to assess therapeutic compliance, the patient completed the compliance questionnaire of Rheumatology (CQR) questionnaire. The determination of erythrocytic MTX-PG was performed on a 5 mL blood sample by liquid chromatography method for the determination of the different PG forms.Results:Sixty patients were included, 34 in the active RA group and 26 in the RA group in remission. One patient withdrew his consent. Only 16% of patients were observed with a CQR score > 80%. Patients in remission were leaner with a longer duration of disease and MTX treatment. The sex ratio, RA status, creatinine clearance and MTX dose was not different in both groups. The CQR was better in the remission group than in the active RA group. However, total MTX-PG was not different in the two groups. The same results were observed for the different forms of MTX-PG 1, 2, 3 or 4. In contrast, in the remission PR group, total MTX-PG or MTX-PG2, 3, 4 and 5 correlated inversely with BMI while MTX-PG3 correlated positively with BMI in the active PR group. In the active PR group, MTX-PG5 correlated with MTX dose.Conclusion:MTX-PG dosage is not a biomarker of good response to MTX in our study. However, compliance is a key factor to be considered in RA with active disease in adapting patient management.

Potential Role of Methotrexate Polyglutamates in Therapeutic Drug Monitoring for Pediatric Inflammatory Bowel Disease.

Inside cells, the immunomodulator methotrexate (MTX) undergoes the addition of glutamates to form methotrexate polyglutamates (MTX-Glu)—promising biomarkers of systemic exposure and treatment response to MTX in rheumatology. MTX-Glu are underexplored in Inflammatory Bowel Disease (IBD), with no data in pediatrics. In this cross-sectional secondary analysis, we assessed the relationships between MTX-Glu and MTX dose and treatment response in pediatric IBD. Twenty-one children with IBD, receiving maintenance therapy with infliximab (IFX) and MTX, had MTX-Glu1–6 concentrations and IFX troughs/antibodies measured and disease activity assessed for comparison in remission vs. active IBD using non-parametric tests, with associations explored using Spearman’s correlation (ρ) and regression analyses; SASv9.4 (α = 0.05). Total and long-chain MTX-Glu correlated with MTX dose (ρ = 0.51 and 0.56, respectively; p ≤ 0.02). In children with Crohn’s disease (n = 19), short-chain MTX-Glu1–2 were 2.5-fold higher in remission vs. active disease, approaching statistical significance (p = 0.066), with no statistical differences in IFX trough (p = 0.549) between groups. Our study highlights a potential role for long-chain MTX-Glu in the therapeutic drug monitoring of MTX in IBD. It is the first study in pediatric IBD and, although statistical significance was not reached, our findings also suggest that higher short-chain MTX-Glu levels may be associated with IBD treatment response to MTX in children.

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.

Front Cover: Simultaneous determination of erythrocyte methotrexate polyglutamates by a novel and simple HPLC‐MS/MS method with stable isotope‐labeled internal standards

DOI: 10.1002/jssc.202001081 The cover picture shows a new HPLC strategy for quantitative analysis of multiple components via a single marker (QAMS) to determine nine nucleosides (cytidine, uridine, 2'‒deoxyuridine, inosine, guanosine, 2'‒deoxyguanosine, thymidine, adenosine and 2'‒deoxyadenosine) in Rhizoma Paridis for the first time. The results of methodology validation showed that there was no significant difference between QAMS method and the external standard method, indicating that QAMS method can applied to solve the problem of a shortage of reference materials and high cost in multicomponent analysis. Our work provide an efficient and feasible quality assessment method for other natural products containing nucleosides.

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.

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). 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. 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. 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 10nM 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 500.0nmol/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 0.100ng/ml, and the run time was only 3min. 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).

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

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. 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. 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)]. 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. NCT01594333.

Therapeutic drug monitoring of methotrexate and its metabolites in the red blood cells and mononuclear cells of patients with rheumatoid arthritis

Objective: to assess the time course of changes in the concentration of methotrexate (MTX) and its main metabolites in the red blood cells (RBC) and mononuclear cells (MNC) of patients with rheumatoid arthritis (RA), by taking into account individual characteristics (age, statin therapy, and smoking).Patients and methods. The investigation enrolled 33 MTX-treated patients (mean age 53.2±11.7 years) with RA, who underwent therapeutic drug monitoring to measure the RBC and MNC concentrations of free MTX and MTX polyglutamates (MTXPGs) with 2, 3, and 4 glutamate residues (MTXPG 2–4) in using tandem chromatomass spectrometry after 4, 12, and 24 weeks of therapy.Results and discussion. Following 12 weeks, the concentration of MTXPG4 in the MNC was higher in patients taking statins, while that of MTX and MTXPG2 in the RBC were significantly lower than in smokers. At 24 weeks, older patients were observed to have a higher MTX level and a lower MTXPG4 concentration in the RBC.Conclusion. After 24 weeks of therapy, the RBC concentration of MTPG4 was lower and that of MTX was higher in older patients than in others, which confirms data on a slower MTX metabolism in the elderly. The use of statins is likely to have a positive impact on the accumulation of MTXPG. There is a statistically significantly lower RBC concentration of MTXPG in at 12 weeks of therapy.

Weekly split-dose regimen for oral methotrexate reduced polyglutamation in red blood cells in patients with rheumatoid arthritis compared with single-dose regimen: Results from a multicentered randomized control trial.

We compared the incidence of adverse events between single and divided-dose regimens of methotrexate (MTX) by using a multicenter randomized controlled trial. Eighty-nine patients with insufficient control on MTX 8mg/wk were randomly assigned into single-dose (39 patients) or triple dose (39 patients) groups. The MTX dose for all patients was gradually increased to 16mg/wk. The primary endpoint was the occurrence of liver dysfunction during the observation period (20weeks). There were no differences in baseline data and MTX dose at Week 20 between groups. There was no significant difference in the incidence of liver dysfunction between groups (single dose, 3 [7.7%] patients vs. triple dose, 5 [13.2%] patients; P=.455). The incidence of adverse event increased in triple dose (single dose, 12 [30.8%] patients vs. triple dose, 20 [51.3%]), but the difference was not significant (P=.066). There was no significant difference in disease activity between groups, although MTX-triglutamate (PG3), MTX-PG4, and MTX-PG5 were significantly higher in the single dose group. Weekly split dosing reduced the polyglutamation of MTX. There was no significant difference in efficacy and safety between the 2 groups.

Effects of germline DHFR and FPGS variants on methotrexate metabolism and relapse of leukemia

AbstractMethotrexate (MTX) during maintenance therapy is essential for curing acute lymphoblastic leukemia (ALL), but dosing strategies aiming at adequate treatment intensity are challenged by interindividual differences in drug disposition. To evaluate genetic factors associated with MTX metabolism, we performed a genome-wide association study in 447 ALL cases from the Nordic Society for Pediatric Haematology and Oncology ALL2008 study, validating results in an independent set of 196 patients. The intergenic single-nucleotide polymorphism rs1382539, located in a regulatory element of DHFR, was associated with increased levels of short-chain MTX polyglutamates (P = 1.1 × 10−8) related to suppression of enhancer activity, whereas rs35789560 in FPGS (p.R466C, P = 5.6 × 10−9) was associated with decreased levels of long-chain MTX polyglutamates through reduced catalytic activity. Furthermore, the FPGS variant was linked with increased relapse risk (P = .044). These findings show a genetic basis for interpatient variability in MTX response and could be used to improve future dosing algorithms.

FPGS relapse-specific mutations in relapsed childhood acute lymphoblastic leukemia

Although the cure rate for childhood acute lymphoblastic leukemia (ALL) has exceeded 80% with contemporary therapy, relapsed ALL remains a leading cause of cancer-related death in children. Relapse-specific mutations can be identified by comprehensive genome sequencing and might have clinical significance. Applying whole-exome sequencing to eight triplicate samples, we identified in one patient relapse-specific mutations in the folylpolyglutamate synthetase (FPGS) gene, whose product catalyzes the addition of multiple glutamate residues (polyglutamation) to methotrexate upon their entry into the cells. To determine the prevalence of mutations of the FPGS mutations, and those of two important genes in the thiopurine pathway, NT5C2 and PRPS1, we studied 299 diagnostic and 73 relapsed samples in 372 patients. Three more FPGS mutants were identified in two patients, NT5C2 mutations in six patients, and PRPS1 mutants in two patients. One patient had both NT5C2 and PRPS1 mutants. None of these alterations were detected at diagnosis with a sequencing depth of 1000X, suggesting that treatment pressure led to increased prevalence of mutations during therapy. Functional characterization of the FPGS mutants showed that they directly resulted in decreased enzymatic activity, leading to significant reduction in methotrexate polyglutamation, and therefore likely contributed to drug resistance and relapse in these cases. Thus, besides genomic alterations in thiopurine metabolizing enzymes, the relapse-specific mutations of FPGS represent another critical mechanism of acquired antimetabolite drug resistance in relapsed childhood ALL.

Association of NUDT15*3 and FPGS 2572C>T Variants with the Risk of Early Hematologic Toxicity During 6-MP and Low-Dose Methotrexate-Based Maintenance Therapy in Indian Patients with Acute Lymphoblastic Leukemia.

Genetic variants influencing the pharmacokinetics and/or pharmacodynamics of the chemotherapeutic drugs used in Acute Lymphoblastic Leukemia (ALL) therapy often contribute to the occurrence of treatment related toxicity (TRT). In this study, we explored the association of candidate genetic variants with early hematological TRT (grade 3–4) occurring within the first 100 days of low-dose methotrexate and 6-mercaptopurine based maintenance therapy (n = 73). Fourteen variants in the following candidate genes were genotyped using allele discrimination assay by real-time PCR: ABCB1, DHFR, GGH, FPGS, MTHFR, RFC1, SLCO1B1, TPMT, and NUDT15. Methotrexate polyglutamate (MTXPG3-5) levels in red blood cells were measured by LC-MS/MS. Early hematological TRT (grade 3–4) was seen in 54.9% of patients. The NUDT15c.415T allele was associated with early TRT occurrence [HR: 3.04 (95% CI: 1.5–6.1); p = 0.007]. Sensitivity of early TRT prediction improved (from 30.7% to 89.7%) by considering FPGS variant (rs1544105’T’) carrier status along with NUDT15c.415T allele [HR = 2.7 (1.5–4.7, p = 0.008)]. None of the considered genetic variants were associated with MTXPG3-5 levels, which in turn were not associated with early TRT. NUDT15c.415T allele carrier status could be used as a stratifying marker for Indian ALL patients to distinguish patients at high or low risk of developing early hematological TRT.