Red Blood Cell Methotrexate Polyglutamate Research Articles

P384 A higher red blood cell methotrexate polyglutamate 3 concentration is associated with methotrexate drug-survival in patients with Crohn’s Disease: first results of a prospective cohort

Abstract Background Prediction and monitoring of drug response of methotrexate (MTX) in patients with Crohn’s disease (CD) is an unmet need. MTX-polyglutamates (MTX-PGs) in red blood cells (RBC) are potential markers for response in other immune-mediated inflammatory diseases. Our objectives were investigating the relation between MTX-PGs and efficacy and defining predictors of response in CD patients treated with MTX. Methods In a multicenter prospective cohort study, CD patients starting subcutaneous (s.c.) MTX without biologics were included and followed for 12 months or until MTX s.c. was discontinued or a concomitant step-up therapy was started. At baseline clinical and biochemical predictors were recorded. At 2, 3, 6 and 12 months after start of therapy or when dropping out, blood samples were collected and individual MTX-PGs (MTX-PG1 - MTX-PG5) were analyzed in RBC using a validated UHPLC-MS/MS method. The outcome was either MTX s.c. discontinuation or initiation concomitant step-up therapy, due to disease activity or toxicity. Predictors were analysed in an univariate Cox regression model and MTX-PGs in an extended Cox model, corrected for prednisone (at start) and budesonide. Results Eighty CD patients were included (age mean±SD 55±13y, 35% male). The median Harvey Bradshaw Index (HBI) was 4 (IQR 2-7). After 12 months 21 patients were still on MTX s.c. monotherapy, 21 patients stopped because of disease activity, 29 because of toxicity, 4 because of a combination of both and 5 patients were censored (4 ended study participation, 1 MTX was stopped on patient’s own initiative) [Figure 1]. A higher HBI at baseline was associated with an increased rate of MTX s.c. monotherapy discontinuation (HR 1.08, 95% CI 1.02-1.16). Predictors of discontinuation because of disease activity (cause specific hazards) were male sex (3.83, 1.62-9.05), baseline eGFR (1.06, 1.02-1.09), baseline HBI (1.12, 1.02-1.23) and baseline plasma folate (0.94, 0.88-0.99). Sex and plasma folate were not correlated with HBI. No cause specific hazards for stopping MTX because of toxicity were identified. MTX-PG3 was the most abundant MTX-PG subspecie with a median concentration of 51 nmol/L RBC (IQR 37-62) at month 3 and was associated with better MTX survival (HR 0.98, 95% CI 0.971-0.999). For every ten points increase in the MTX-PG3 concentration, the rate of MTX s.c. monotherapy discontinuation decreases with 14%. Conclusion Higher eGFR, higher HBI, lower plasma folate at baseline and male sex are predictors for MTX failure in the first year in CD patients. The last two parameters are likely predictors of MTX response specifically rather than prognostic disease factors. The measurement of MTX-PG3 in packed RBC holds potential as a tool for therapeutic drug monitoring in CD.

Methotrexate Polyglutamation in a Myasthenia Gravis Clinical Trial

Methotrexate (MTX) is an immunosuppressive and anti-inflammatory drug used to treat rheumatoid arthritis (RA) and other autoimmune conditions. MTX is transported into cells, where glutamate moieties are added and is retained as methotrexate polyglutamates (MTXPGs). In the RA literature, it has been reported that the degree of polyglutamation correlates with the anti-inflammatory effect of MTX in RA. There are no prior studies evaluating the relationship between MTXPGs and myasthenia gravis (MG) outcome measures. The objective of this study was to assess the correlation between methotrexate (MTX) polyglutamates (MTXPGs) with Myasthenia Gravis (MG) outcome measures. An analysis was done of blood drawn from patients enrolled in the 12-month randomized, placebo-controlled study of MTX in MG study. Red blood cell MTXPGs were measured via ultra-performance liquid chromatography and tandem mass spectrometry. MTXPG was correlated to MG outcome measures using Spearman Correlation Coefficient. A two-group t-test was used to determine the difference in MTXPG based on clinical outcome responder definitions. Twenty-one polyglutamate samples were analyzed of subjects on MTX while eight samples were analyzed from subjects on placebo. Pentaglutamate had the strongest correlation with the MG-ADL (0.99), while tetraglutamate had the strongest correlation with the QMG (0.54). Triglutamate had the strongest correlation with MGC (0.76). There were variable correlations between MTXPG1-5 and MG outcomes (rho range: 0.08 to 0.99). There are strong correlations between MTXPG and the MG-ADL, QMG, and MGC. Long chain methotrexate polyglutamates correlate better with MG outcomes.

A Pilot Randomized Controlled Double-Blind Trial of High- Versus Low-Dose Weekly Folic Acid in People With Rheumatoid Arthritis Receiving Methotrexate.

The aim of this study was to determine whether reducing the dose of supplemental folic acid used in conjunction with methotrexate (MTX) therapy in people with active rheumatoid arthritis (RA) improved disease control and/or increased MTX-related adverse effects. A randomized double-blind randomized controlled trial comparing 5 mg/wk and 0.8 mg/wk folic acid was undertaken. Rheumatoid arthritis patients on MTX for 3 months or more at a stable dose for 1 month or more were recruited. All participants had DAS28 of 3.2 or greater or required a change in therapy determined by the treating clinician. Disease activity, full blood count, liver function tests, red blood cell (RBC) folate, and RBC MTX polyglutamates were assessed at weeks 0, 4, 8, 16, and 24 along with reports of adverse events. Forty participants were recruited. The mean (SD) change in RBC folate between week 0 and 24 was +87.9 (57.4) nmol/L in the high-dose group and -113.3 (65.7) nmol/L in the low-dose group (p < 0.05). There was no significant difference in the change in DAS28 between the high- and low-dose groups at 24 weeks (-0.13 [95% confidence interval, -0.69 to 0.43] vs -0.25 [-0.87 to 0.37], respectively [p = 0.78]). There was no significant difference in MTX-related adverse effects between the 2 groups. A reduction in RBC folate secondary to reduction in folic acid dose was not associated with a change in RA disease activity or MTX-related adverse effects. The prevention of MTX-related adverse effects remains the primary reason for coprescribing folic acid with MTX. Australian New Zealand Clinical Trials Registry (ANZCTR12610000739011).

The role and utility of measuring red blood cell methotrexate polyglutamate concentrations in inflammatory arthropathies—a systematic review

Evidence regarding the relationship between red blood cell methotrexate polyglutamate concentration and response to treatment and adverse drug reactions in patients using methotrexate for inflammatory arthropathies is complex and in some respects appears conflicting. Accordingly, we undertook a systematic analysis of available evidence to determine the clinical utility of dosing methotrexate to a target red blood cell methotrexate polyglutamate concentration. A systematic literature review was conducted to identify all studies that had reported an association between red blood cell methotrexate polyglutamate concentration and disease activity or adverse drug reactions in users of methotrexate for the treatment of rheumatoid arthritis, juvenile idiopathic arthritis or psoriatic arthritis. No randomised controlled trials were identified. Thirteen studies (ten in patients with rheumatoid arthritis and three in patients with juvenile idiopathic arthritis) were identified. All studies evaluated an association between red blood cell methotrexate polyglutamate concentration and response to treatment, and eight evaluated an association with toxicity. Eight studies identified lower disease activity with at least one higher red blood cell methotrexate polyglutamate concentration, although there was at least moderate potential for bias in all of these studies. Relatively large increases in concentration appeared to be required to produce a meaningful reduction in disease activity. Only one study identified an association between red blood cell methotrexate polyglutamate concentration and methotrexate-induced side effects, although studies were likely underpowered to detect this type of association. The manner in which data were presented in the included studies had many limitations that hampered its conclusive assessment, but red blood cell methotrexate polyglutamate concentrations appear to be a potentially useful guide to treatment in patients with inflammatory arthropathies, but the specific polyglutamate that should be monitored and how monitoring could be integrated into treat-to-target approaches should be clarified before it can be routinely implemented.

Sa1255 Higher Red Blood Cell Methotrexate Polyglutamates Correlate With Increased Disease Activity, and Are Useful in Assessing Adherence

Introduction Methotrexate (MTX) is commonly used in patients with inflammatory bowel disease (IBD). Within red blood cells (RBC), MTX is activated by sequential addition of glutamic acid residues to form polyglutamates (MTXPG 1–5 ). In rheumatoid arthritis, low [MTXPG] has been associated with active disease, whereas other studies have demonstrated an inverse relationship, including the only published data in IBD. The aim of this study was to determine if RBC [MTXPG] reflect clinical response in IBD patients and whether they are useful in assessing adherence. Methods This was a single-centre, retrospective pilot study of 21 IBD patients treated with weekly MTX. RBC MTXPG 1–5 was measured using high-performance liquid chromatography. Clinical status (active disease or remission) was assessed by 2 IBD physicians blinded to [MTXPG], using a combination of prospectively recorded clinical activity indices (Simple Colitis Activity Index, Harvey Bradshaw Index), endoscopy, faecal calprotectin and C reactive protein (CRP). Pearson correlation coefficient, r was calculated to assess the relationship between MTX dose and [MTXPG]. Association between [MTXPG] and clinical response was analysed with unpaired t-test. Results 4/21(22%) patients (3 of whom admitted non-adherence) had undetectable MTXPGs and were excluded from further analysis. MTXPG 2–4 were detected in all adherent patients. PG 3 was the predominant polyglutamate accounting for a mean of 43% of total MTXPG. A linear relationship between dose of MTX and PG 1–5 was observed. 12/21(57%) patients were assessed as having active disease. No significant difference in mean [MTXPG n ] was observed between those with active disease and remission. For each MTXPG n , a non-significant trend towards a higher concentration was observed in patients with active disease. Conclusion In this study, the largest to date in IBD, measuring RBC MTXPG was useful in assessing adherence to MTX. A trend towards higher PG concentrations was associated with active disease confirming the findings in the only other study in IBD. Whether this is confounded by higher doses being used in patients with more active disease warrants further study in larger, prospective trials. Reference Disclosure of Interest None Declared.

P469 Higher red blood cell methotrexate polyglutamates correlate with increased disease activity, and are useful in assessing adherence

P469 Higher red blood cell methotrexate polyglutamates correlate with increased disease activity, and are useful in assessing adherence

AB0593 Subcutaneous methotrexate is more efficacious and better tolerated than oral methotrexate: The experience of a large group of patients in the rheumatology department of a district general hospital

BackgroundWith the emphasis in the management of inflammatory arthritis on early disease modification it is vital that prescribed treatments are tolerated and adhered to. Methotrexate can prove difficult for patients...

AB0183 Methotrexate induced apoptosis in synoviocyte of rheumatoid arthritis

BackgroundMethotrexate (MTX) was the corner stone treatment of rheumatoid arhtirits. So for long time, MTX was used in rheumatoid arhtirits treatment with other Immunosuppressive drug. There were many hypothesis in...

Methotrexate polyglutamation in relation to infliximab pharmacokinetics in rheumatoid arthritis

ObjectiveThe 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...

Effects of Changing from Oral to Subcutaneous Methotrexate on Red Blood Cell Methotrexate Polyglutamate Concentrations and Disease Activity in Patients with Rheumatoid Arthritis

To determine the effects of changing from oral to subcutaneous (SC) methotrexate (MTX) in patients with rheumatoid arthritis (RA) on red blood cell MTX polyglutamate (RBC MTXGlu(n)) concentrations, disease activity, and adverse effects. Thirty patients were changed from oral to SC MTX. Trough RBC MTXGlu(n) concentrations were measured for 24 weeks and concentrations fitted to a first-order accumulation model. Disease activity was assessed by 28-joint Disease Activity Score (DAS28). MTXGlu(3), MTXGlu(4), and MTXGlu(5) concentrations, but not MTXGlu(1) and MTXGlu(2), increased significantly over 24 weeks, reaching 90% of new steady-state concentrations by about 40 weeks. A decrease in DAS28 was associated with increased RBC MTXGlu(5) (p = 0.035) and RBC MTXGlu(3-5) (p = 0.032). No change in adverse effect frequency occurred. Changing to SC MTX results in increased long-chain MTXGlu(n). However, it takes at least 6 months for RBC steady-state concentrations to be achieved. Increased long-chain MTXGlu(n) concentrations were significantly associated with reduced disease activity.

Red blood cell methotrexate polyglutamates emerge as a function of dosage intensity and route of administration during pulse methotrexate therapy in rheumatoid arthritis

MTX is a prodrug producing anti-arthritic effects through a folylpolyglutamate synthase-mediated activation to MTX polyglutamates (MTXPGs). Our objective was to characterize the pharmacokinetics of intracellular MTXPGs and the factors associated with their accumulation in adult RA patients treated with MTX weekly. MTX pharmacokinetics were evaluated in 47 MTX-naïve patients enrolled in an MTX dose-escalation study for an average of 20 weeks and 223 patients enrolled in a cross-sectional study under long-term MTX therapy. Short-chain (MTXPG1-2), long-chain (MTXPG3) and very long-chain (MTXPG4-5) concentrations were measured in circulating red blood cells using liquid chromatography. Statistical analyses consisted of non-linear mixed models, multivariate regression analyses and Wilcoxon signed-rank test. The accumulation of MTXPG1-5 was sigmoidal and steady-state concentrations were achieved after 7 weeks of therapy. However, additional exposure and MTX dosage escalation produced a selective redistribution towards longer chain MTXPGs at the expense of shorter chain MTXPGs. Age, glomerular filtration rate and route of MTX administration were the most important predictors of MTXPG accumulation. In 10 patients, a switch from oral to parenteral MTX was associated with a 37% increase in long-chain MTXPGs, a 132% increase in very long-chain MTXPGs and a concomitant 31% reduction in disease activity (P<0.02). The selective emergence of long-chain MTXPGs is function of dose, time of exposure and hence dosage intensity. Switching from oral to parenteral MTX produces a selective accumulation of longer chain MTXPGs that are known to be more potent inhibitors of de novo purine biosynthesis than shorter chain MTXPGs.

Polymorphisms within the folate pathway predict folate concentrations but are not associated with disease activity in rheumatoid arthritis patients on methotrexate

To determine whether genetic polymorphisms within the folate pathway are associated with red blood cell (RBC) methotrexate (MTX) polyglutamate concentrations, RBC folate concentrations and MTX efficacy/toxicity. Disease activity in 200 rheumatoid arthritis patients on MTX was assessed by swollen and tender joint counts and Disease Activity Score 28. Genetic polymorphisms shown to have an effect on gene expression or protein function were examined. RBC folate concentrations were significantly associated with MTHFR 677C>T (P=0.002), MTRR 66A>G (P<0.0001), MTHFD1 1958G>A (P=0.001) and SHMT 1420C>T (P=0.012), whereas no association of these polymorphisms with disease activity was observed. None of the polymorphisms tested predicted RBC MTX polyglutamate concentrations. There were weak associations between central nervous system adverse effects and AMPD1 34C>T (P=0.04) and between gastrointestinal adverse effects and MTHFD1 1958G>A (P=0.03) and ABCC2 IVS23+56T>C (P=0.045). There was a stronger association between any adverse effect and ABCG2 914C>A (P=0.004). Although RBC folate concentrations are associated with genetic polymorphisms within the folate pathway, these variants do not predict disease activity. To accurately evaluate whether any polymorphisms are reliable predictors of MTX efficacy or toxicity, large prospective clinical trials are required. Furthermore, application of a genome-wide association strategy is likely to uncover novel predictors of MTX response.

Red Blood Cell Methotrexate Polyglutamate Concentrations in Inflammatory Bowel Disease

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 </= 0.03). RBC MTXGlu5 concentrations were higher in patients experiencing at least one adverse effect as a result of MTX compared with those without adverse effects (mean, 26.7 versus 11.1 nmol/8 x 10 RBCs; mean difference, 15.6; 95% confidence interval, 3.5-27.7; P = 0.04). Similar trends were observed for RBC MTXGlu4. Furthermore, patients who specifically experienced gastrointestinal adverse effects had higher RBC MTXGlu4&5 concentrations than patients without (P = 0.03 and 0.04, respectively). In summary, RBC MTXGlu1-5 concentrations can be measured accurately with low intrapatient variation. Unexpectedly, RBC MTXGlu4&5 concentrations correlated inversely with efficacy in Crohn's disease. RBC MTXGlu4&5 concentrations were higher in patients experiencing adverse effects. These findings suggest that RBC MTXGlu1-5 concentration monitoring may be of value in IBD and could assist with planning of larger studies.