Nonadherence to disease modifying antirheumatic drugs in the first year after diagnosis: comparing three adherence measures in early arthritis patients.

OBJECTIVES: The aim of this study was to compare three measurement methods for non-adherence to DMARDs in early arthritis patients: the Compliance Questionnaire Rheumatology (CQR), the intracellular uptake of MTX in the form of MTX-polyglutamates (MTX-PGs) and electronic measurement with Medication Event Monitoring Systems (MEMS). METHODS: DMARD naïve early arthritis patients were included in an ongoing cohort study. MEMS were used to measure adherence continuously, while every 3 months MTX-PGs were collected together with the CQR. The associations between the measures were estimated with Spearman rank correlations. Sensitivity and specificity of the CQR against a MEMS cut-off was compared at 3, 6, 9 and 12 months. The same applied to MTX-PGs against a MEMS cut-off and MTX-PGs against a CQR cut-off. For the association between MEMS, the CQR and MTX-PGs, a multilevel linear regression model was performed with age, gender, weeks of treatment and MTX dosage as covariates. RESULTS: We included 206 patients. Non-adherence measured with MEMS varied over time and between DMARDs. The CQR score was not associated with MEMS non-adherence at 3, 9 and 12 months. At 9 months, MTX-PGs was associated with MEMS non-adherence, as well as with the CQR. All correlations were below 0.30. CONCLUSION: Associations between the three measures are weak. Explanations are individual differences in the uptake of MTX, and little variance in adherence between patients. Moreover, the measurement domains differ: perceptions (CQR), behaviour (MEMS) and pharmacokinetics (MTX).

Methotrexate polyglutamates in erythrocytes are associated with lower disease activity in juvenile idiopathic arthritis patients.

OBJECTIVE: To determine association of erythrocyte methotrexate polyglutamates (MTX-PG) with disease activity and adverse effects in a prospective juvenile idiopathic arthritis (JIA) cohort. METHODS: One hundred and thirteen JIA patients were followed from MTX start until 12 months. Erythrocyte MTX-PGs with 1-5 glutamate residues were measured at 3 months with tandem mass spectrometry. The outcomes were Juvenile Arthritis Disease Activity Score (JADAS)-27 and adverse effects. To determine associations of MTX-PGs with JADAS-27 at 3 months and during 1 year of MTX treatment, linear regression and linear mixed-model analyses were used. To determine associations of MTX-PGs with adverse effects during 1 year of MTX treatment, logistic regression was used. Analyses were corrected for JADAS-27 at baseline and co-medication. RESULTS: Median JADAS-27 decreased from 12.7 (IQR: 7.8-18.2) at baseline to 2.9 (IQR: 0.1-6.5) at 12 months. Higher concentrations of MTX-PG3 (ß: -0.006, p=0.005), MTX-PG4 (ß: -0.015, p=0.004), MTX-PG5 (ß: -0.051, p=0.011) and MTX-PG3-5 (ß: -0.004, p=0.003) were associated with lower disease activity at 3 months. Higher concentrations of MTX-PG3 (ß: -0.005, p=0.028), MTX-PG4 (ß: -0.014, p=0.014), MTX-PG5 (ß: -0.049, p=0.023) and MTX-PG3-5 (ß: -0.004, p=0.018) were associated with lower disease activity over 1 year. None of the MTX-PGs was associated with adverse effects. CONCLUSIONS: In the first prospective study in JIA, long-chain MTX-PGs were associated with lower JADAS-27 at 3 months and during 1 year of MTX treatment. Erythrocyte MTX-PG could be a plausible candidate for therapeutic drug monitoring of MTX in JIA.

Methotrexate polyglutamates in erythrocytes are associated with lower disease activity in patients with rheumatoid arthritis.

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

Using fluorescence polarization immunoassay for determination of erythrocyte methotrexate polyglutamates, a quick and easy test?

BACKGROUND: The folate antagonist methotrexate (MTX) is the anchor drug in the treatment of rheumatoid arthritis. The therapeutic effects of MTX are attributed to the intracellular levels of MTX, present in the cell as polyglutamates (MTX-PGs). We aimed to validate an immunoassay for the measurement of MTX-PG in erythrocytes. METHODS: Samples were analyzed by an adapted fluorescence polarization immune assay (FPIA) method on the FLx analyzer (Abbott). Cross-reactivity was determined in both plasma and erythrocyte pellet. In erythrocyte pellet, the imprecision, linearity, and lower limit of quantitation were determined. The method was compared with our in-house liquid chromatography tandem mass spectrometry (LC-MS/MS) method for total MTX-PG. RESULTS: For the adapted FPIA method, a linear range of 25-1000 nmol/L (R = 0.993) was obtained for total MTX-PG in erythrocytes. A coefficient of variation of <17% for interday and <8% for intraday imprecision was found and average recovery was 91%. Lower limit of quantitation was determined at 50 nmol/L total MTX-PG with a coefficient of variation of 15%. There was no significant proportional bias of the FPIA assay compared with our in-house LC-MS/MS method, but a (nonsignificant) constant positive bias was present [FPIA = 1.00 (95% confidence interval: 0.60-1.95) × LC-MS/MS + 31.00 nmol/L (95% confidence interval: -11.83 to 61.00)]. Results could be very different for individual patients as reflected in the poor R of 0.419. CONCLUSIONS: The FPIA method can be used to measure total MTX-PG in erythrocytes. Although there was no significant bias detected compared with the LC-MS/MS method, the FPIA method showed constant positive bias, probably because of interference from folates and MTX metabolites 2,4-diamino-N10-methylpteroic acid and 7-hydroxy-MTX. The correlation between both methods was average and resulted in large differences in individual patients, most likely because of problems during sample preparation.

A U-HPLC-ESI-MS/MS-based stable isotope dilution method for the detection and quantitation of methotrexate in plasma.

INTRODUCTION: High-dose methotrexate (MTX) is used in the treatment of proliferative diseases such as acute lymphoblastic leukemia. Therapeutic drug monitoring of plasma MTX is important to monitor efficacy and adverse events. The authors aimed to develop a liquid chromatography, electrospray ionization, tandem mass spectrometry (LC-ESI-MS/MS)-based method to determine MTX in plasma for therapeutic drug monitoring and pharmacokinetic studies. METHODS: Samples were analyzed using a Waters Acquity UPLC and Quattro Premier XE. A Waters Acquity UPLC BEH C18 column (2.1 mm × 100 mm, 1.7 µm) was used running an isocratic mobile phase of 21% methanol and 10 mM ammonium bicarbonate. The electrospray was operated in the positive ionization mode monitoring the following mass transitions: m/z 455.2 > 308.2 for MTX and m/z 458.2 > 311.2 for MTXd3. The analysis combined straightforward sample preparation, consisting of dilution and protein precipitation, with a 3-minute run time. RESULTS: The method was linear up to 50 µM (r > 0.99), and the coefficient of variation was <6% for intraday and <10% for interday precision. Average recovery was 99%. There were no significant matrix effects. The lower limit of quantitation, defined as the lowest concentration at which the coefficient of variation <20% and S/N > 1:10, was 5 nM. Method comparison with the Abbott TDx fluorescent polarization immunoassay (FPIA) showed excellent agreement, and a small but significant negative constant bias was detected (LC-MS/MS = 0.98 × FPIA - 7.3). CONCLUSIONS: [corrected] The authors developed a specific and sensitive stable isotope dilution LC-ESI-MS/MS method to monitor MTX concentrations in plasma within the clinically relevant range. The method can be easily applied in clinical laboratories because it combines straightforward sample pretreatment with LC-MS/MS.

Assessment of intracellular methotrexate and methotrexate-polyglutamate metabolite concentrations in erythrocytes by ultrafast matrix-assisted laser desorption/ionization triple quadrupole tandem mass spectrometry.

A new ultrafast quantitative and high-throughput mass spectrometric method using matrix-assisted laser desorption/ionization triple quadrupole tandem mass spectrometry has been developed and validated for determination of intracellular erythrocyte concentrations of the antifolate drug methotrexate (MTX) and its polyglutamate metabolites. The method consists of a solid-phase extraction of MTX and MTX-polyglutamate metabolites from deproteinized erythrocyte lysates spiked with aminopterin as internal standard. The newly developed method was validated according to the most recent FDA guidelines on linearity, recovery, within-run and between-run accuracy and precision and stability of the analytes. The low limit of quantification (LLOQ) was 10 nmol/L for all analytes while the limit of detection (LOD) determined at a signal-to-noise (S/N) ratio = 3:1 in drug- free erythrocyte lysate was on average 0.3 nmol/L. After validation, the new method was used in the measurement of intracellular erythrocyte concentrations of MTX and MTX-polyglutamate metabolites (MTXPG2 to MTXPG7) in packed human erythrocyte samples collected from patients with rheumatoid arthritis receiving low-dose oral methotrexate therapy. Mean (SD) intracellular erythrocyte concentrations observed in patient samples were 12.8 (12.6), 12.4 (9.4), 44.4 (30.0), 33.6 (35.9) and 9.4 (8.2) nmol/L for MTX to MTXPG5, respectively, in 10(6) erythrocytes. The highest observed glutamylation degree of MTX was MTXPG5, the very long chain MTX-polyglutamate metabolites MTXPG6 and MTXPG7 were not detected in the packed erythrocyte pellets collected from rheumatoid arthritis patients.

A new ultrafast and high-throughput mass spectrometric approach for the therapeutic drug monitoring of the multi-targeted anti-folate pemetrexed in plasma from lung cancer patients.

An analytical assay has been developed and validated for ultrafast and high-throughput mass spectrometric determination of pemetrexed concentrations in plasma using matrix assisted laser desorption/ionization-triple quadrupole-tandem mass spectrometry. Patient plasma samples spiked with the internal standard methotrexate were measured by multiple reaction monitoring. The detection limit was 0.4 fmol/µL, lower limit of quantification was 0.9 fmol/µL, and upper limit of quantification was 60 fmol/µL, respectively. Overall observed pemetrexed concentrations in patient samples ranged between 8.7 (1.4) and 142.7 (20.3) pmol/µL (SD). The newly developed mass spectrometric assay is applicable for (routine) therapeutic drug monitoring of pemetrexed concentrations in plasma from non-small cell lung cancer patients.

Determinants of erythrocyte methotrexate polyglutamate levels in rheumatoid arthritis.

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 (stß 0.23, p=0.033) and total MTX-PG (stß 0.23, p=0.018) in the derivation cohort, and with all MTX-PG in the validation cohort (MTX-PG1: stß 0.13, p=0.04; MTX-PG2: stß 0.21, p=0.001; MTX-PG3: stß 0.22, p<0.001; MTX-PG4+5: stß 0.25, p<0.001; and total MTX-PG: stß 0.32, p<0.001). Erythrocyte folate levels were positively associated with MTX-PG3 (stß 0.3, p=0.021) and total MTX-PG levels (stß 0.32, p=0.022) in the derivation cohort, which was replicated for MTX-PG3 (stß 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 (stß 0.23, p<0.001), MTX-PG4+5 (stß 0.30, p<0.001), and total MTX-PG (stß 0.20, p=0.002), but negatively associated with MTX-PG2 levels (stß -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.

Effect of methotrexate use and erythrocyte methotrexate polyglutamate on glycosylated hemoglobin in rheumatoid arthritis.

OBJECTIVE: To investigate whether methotrexate (MTX) use, as compared to other therapies, and erythrocyte methotrexate polyglutamate (MTXGlu) concentrations are associated with changes in glycosylated hemoglobin (HbA1c ) levels in rheumatoid arthritis (RA) patients. METHODS: The derivation cohort consisted of patients selected from the Treatment in the Rotterdam Early Arthritis Cohort who fulfilled the 2010 American College of Rheumatology/European League Against Rheumatism classification criteria for RA. Patients were randomized to 6 treatment arms: triple disease-modifying antirheumatic drug (DMARD) therapy (consisting of MTX, sulfasalazine, and hydroxychloroquine [HCQ]) + intramuscular (IM) glucocorticoids, triple DMARD therapy + oral glucocorticoids, MTX + oral glucocorticoid therapy, MTX therapy, oral glucocorticoid therapy, and HCQ therapy. HbA1c levels were determined at baseline and at 3 months. Concentrations of erythrocyte MTXGlu1-5 were measured after 3 months of treatment. Within treatment arms, changes in the level of HbA1c were compared by paired t-test. Associations of MTXGlu concentrations with changes in the level of HbA1c were tested using multiple linear regression analysis, adjusted for age, sex, body mass index, and comedication. Significant associations were validated using data on RA patients taking MTX who were enrolled in the Methotrexate in Rotterdam cohort. RESULTS: In the derivation cohort, the mean change in HbA1c level after 3 months of treatment was -1.9 mmoles/mole (-0.18%) (P = 0.001). Levels of HbA1c decreased in 4 of the individual treatment groups, as follows: for the triple DMARD therapy + IM glucocorticoids treatment arm, -5.5 mmoles/mole (-0.50%) (P < 0.001), for the triple DMARD therapy + oral glucocorticoids treatment arm, -3.7 mmoles/mole (-0.34%) (P < 0.001), for the MTX treatment arm, -0.8 mmoles/mole (-0.08%) (P = 0.018), and for the HCQ treatment arm, -2.0 mmoles/mole (-0.19%) (P = 0.175). Increased levels of MTXGlu2 (ß = -0.20, P = 0.005), MTXGlu3 (ß = -0.31, P < 0.001), MTXGlu4 (ß = -0.33, P < 0.001) after treatment, MTXGlu5 (ß = -0.39, P < 0.001), and total MTXGlu (ß = -0.29, P < 0.001) were associated with decreased levels of HBA1c . In the validation cohort, levels of HbA1c were decreased by 2.6 mmoles/mole (0.23%) (P < 0.001) after treatment, and MTXGlu3 was associated with decreased levels of HbA1c (ß = -0.26, P = 0.018). CONCLUSION: MTX use and higher concentrations of erythrocyte MTXGlu are associated with decreased levels of HbA1c in RA patients. Triple DMARD therapy and HCQ treatment resulted in reduced HbA1c levels, and glucocorticoid treatment resulted in increased levels of HbA1c .

Ultrafast selective quantification of methotrexate in human plasma by high-throughput MALDI-isotope dilution mass spectrometry.

BACKGROUND: A new analytical MS method using isotope dilution combined with MALDI-triple quadrupole MS/MS has been developed and validated for the determination of methotrexate and 7-hydroxymethotrexate in plasma. Methotrexate, methotrexate-d3, 7-hydroxymethotrexate and 7-hydroxymethotrexate-d3 were monitored by selected reaction monitoring using the transitions m/z 455.2→308.2, 458.2→311.2, 471.2→324.2 and 474.2→327.2 for methotrexate, methotrexate-d3, 7-hydroxymethotrexate and 7-hydroxymethotrexate-d3, respectively. RESULTS: The LLOQ was 1 nmol/l for methotrexate and 7-hydroxymethotrexate while the limit of detection was 0.3 nmol/l for both analytes. The new developed method was cross-validated by a fluorescence polarization immunoassay and tested for its clinical feasibility by measuring plasma samples from patients suffering from acute lymphoblastic leukemia. Plasma methotrexate concentrations ranged between 66.0 and 954 nmol/l and observed 7-hydroxymethotrexate/methotrexate ratios ranged between 0.1 and 32.4, respectively. CONCLUSION: The new method showed comparable analytical performances as the fluorescence polarization immunoassay, but analyte specificity and sensitivity of the newly developed method were significantly better.