A single assessment of methotrexate levels at 42 hours permits safe administration and early discharge in children with lymphoblastic lymphoma and leukemia receiving high-dose methotrexate.

High-dose methotrexate (HDMTX) is an important component of treatment in pediatric acute lymphoblastic leukemia (ALL) and lymphoblastic lymphoma (LL). Optimal rescue therapy is essential for the safe administration of HDMTX. A cost-effective strategy that does not compromise safety is necessary for low- and middle-income countries. Consecutive admissions for HDMTX in children with ALL and LL over 12 months were analyzed. The dose of HDMTX was 3 g/m2 in B-ALL and B-LL and 5 g/m2 in T-ALL and T-LL. A methotrexate level was measured at 42 hours of starting HDMTX infusion (T42-MTX). Three doses of folinic acid at T42, T48, and T54 and alkalinized hydration till T54 were administered if T42-MTX <1 µM. A total of 282 cycles of HDMTX that were administered in 71 patients were analyzed. T42-MTX was <1 µM in 266 (94.3%) cycles. T42-MTX was ≥1 µM in 12% and 3% of cycles of HDMTX administered at a dose of 5 g/m2 and 3 g/m2, respectively (p = .074). The median duration of hospitalization for HDM was three days and did not differ with the dose of HDMTX administered (p = .427). Mucositis, delayed recovery of blood counts, and hospitalization for reversible toxicity occurred after 21 (7.4%), 28 (9.9%), and 19 (6.7%) cycles of HDMTX, respectively. Mucositis was greater following the administration of 5 g/m2 of HDMTX. A single T42-MTX measurement permits the safe administration of HDMTX and an expedited discharge from the hospital within three days in more than 90% of children with ALL/LL.

Translating research into clinical practice: quality improvement to halve non-adherence to methotrexate.

OBJECTIVE: MTX remains the cornerstone for therapy for RA, yet research shows that non-adherence is significant and correlates with response to therapy. This study aimed to halve self-reported non-adherence to MTX at the Kellgren Centre for Rheumatology. METHODS: An anonymous self-report adherence questionnaire was developed and data collected for 3 months prior to the introduction of interventions, and then regularly for the subsequent 2.5 years. A series of interventions were implemented, including motivational interviewing training, consistent information about MTX and development of a summary bookmark. Information on clinic times was collected for consultations with and without motivational interviewing. Surveys were conducted to ascertain consistency of messages about MTX. A biochemical assay was used to test MTX serum levels in patients at two time points: before and 2.8 years following introduction of the changes. Remission rates at 6 and 12 months post-MTX initiation were retrieved from patient notes and cost savings estimated by comparing actual numbers of new biologic starters compared with expected numbers based on the numbers of consultants employed at the two time points. RESULTS: Between June and August 2016, self-reported non-adherence to MTX was 24.7%. Following introduction of the interventions, self-reported non-adherence rates reduced to an average of 7.4% between April 2018 and August 2019. Clinic times were not significantly increased when motivational interviewing was employed. Consistency of messages by staff across three key areas (benefits of MTX, alcohol guidance and importance of adherence) improved from 64% in September 2016 to 94% in January 2018. Biochemical non-adherence reduced from 56% (September 2016) to 17% (June 2019), whilst remission rates 6 months post-initiation of MTX improved from 13% in 2014/15 to 37% in 2017/18, resulting is estimated cost savings of £30 000 per year. CONCLUSION: Non-adherence to MTX can be improved using simple measures including focussing on the adherence and the benefits of treatment, and providing consistent information across departments.

Solitary serum methotrexate level 36 hours post high-dose methotrexate: A safe, efficacious, and cost-effective strategy to monitor methotrexate toxicities in childhood leukemia in resource-limited centers.

BACKGROUND: The standard practice during high-dose methotrexate (HD-MTX) in acute lymphoblastic leukemia (ALL) to mitigate toxicity is to serially monitor levels till serum MTX < 0.01 µmol/L. Most resource-limited centers lack in-house access to MTX levels, and therefore repeated monitoring is costly and cumbersome. We studied the efficacy and safety of "solitary 36 hours post HD-MTX levels (MTX36 )." PROCEDURE: This prospective observational study consecutively enrolled children with ALL receiving HD-MTX. Cycles with unavailable MTX36 and MTX36  > 10 µmol/L were excluded. HD-MTX was administered over 24 hours (BFM-2009 protocol) with 12 hours of prehydration. MTX36 were performed at other centers. Leucovorin was given in six hourly doses 36 hours post HD-MTX. Hydration was continued until the last dose of leucovorin. MTX toxicities, including change of creatinine from baseline at 36 hours (∆Cr36 ), were noted. Two groups depending on MTX36 (≤1 µmol/L vs > 1 µmol/L) received six versus eight doses of leucovorin, and toxicities were compared. RESULTS: Twenty-nine children with median age five years (1-11) who received 100 HD-MTX cycles with a median MTX dose of 3 g/m2 (2-5) were analyzed. The median MTX36 level was 1.165 µmol/L (0.1-7.32). Toxicities of HD-MTX (CTCAE-4.0): transaminitis-22%; creatinine elevation ≥ 1.25 times baseline-24%; cytopenias-16%; mucositis-17%; acute kidney injury (AKI)-6%. All toxicities were ≤CTCAE grade 3. Creatinine elevation, AKI, and mucositis were significantly higher in the group with higher MTX36 . There was no correlation (r = 0.3) between ∆Cr36 and MTX36 . MTX36 was thrice more economical than the standard protocol. CONCLUSION: MTX36 is a potential cost-effective, efficacious, and safe limited sample strategy to monitor HD-MTX, particularly in centers where in-house MTX levels are unavailable.

Modified enzyme multiplied immunoassay technique of methotrexate assay to improve sensitivity and reduce cost.

BACKGROUND: Methotrexate is an important component in many chemotherapy protocols. The blood concentration of Methotrexate is used to determine the regimen of folinic acid. However, the lower limit of Siemens assay kit is 0.30 µmol/L in China. This study extended the limit from 0.3 to 0.05 µmol/L and reduced the test cost by optimizing the parameters of Enzyme Multiplied Immunoassay Technique assay. METHODS: Parameters of Enzyme Multiplied Immunoassay Technique assay were modified to decrease the volume of reagents A and B. Then a standard curve with a new custom set of calibrators was prepared to detect low concentration. Intra-day and inter-day imprecision were assessed by control material and samples. The linearity of the modified assay was verified by analyzing a range of quality controls with known concentration from 0.05 to 1.00 µmol/L. At last, the same samples were tested by modified Enzyme Multiplied Immunoassay Technique assay and Liquid Chromatography-tandem Mass Spectrometry assay respectively. A simple linear regression was performed to verify the validity of the modified Enzyme Multiplied Immunoassay Technique assay. RESULTS: Intra-day and inter-day imprecision show good reproducibility at all levels (0.05, 0.12, 0.43, 0.82 µmol/L). The linearity equation of modified assay was y = 0.9913x + 0.0046, in which y was the mean of measured concentration and x was the target concentration (R2 = 0.9994). In the range of 0.05-10.00 µmol/L, correlation between the Modified assay and Liquid Chromatography-tandem Mass Spectrometry assay was significant (r = 0.9968). In the range of 0.30-10.00 µmol/L, the correlation between modified and commercial assays was significant (r = 0.9987) as well. CONCLUSIONS: The modified assay enhanced the sensitivity of Siemens VIVA-E to 0.05 µmol/L. In addition, the test number of a reagent Kit increased from 140 to 210. This means the cost of detection was reduced about 30%.

A simple, rapid and reliable liquid chromatography-mass spectrometry method for determination of methotrexate in human plasma and its application to therapeutic drug monitoring.

A simple, rapid and reliable liquid chromatography-electrospray ionization tandem mass spectrometry method was established and validated for the determination of methotrexate in human plasma. After a straightforward protein precipitation by acetonitrile-water (70:30, v/v), methotrexate (MTX) and p-aminoacetophenone (used as internal standard, IS) were separated on a Column C18 column (50 × 2.1 mm, 3 µm; Column Technology, Fremont, CA, USA) using a gradient elution with mobile phase of acetonitrile and 0.03% acetic acid aqueous solution at a flow rate of 0.5 mL/min. The total chromatographic runtime was 5 min for each injection. Quantification detection was performed in a triple-quadruple tandem mass spectrometer under positive mode monitoring the following mass transitions: m/z 455.3 → 308.3 for MTX and m/z 136.1 → 94.4 for IS. The calibration curve was linear over the range of 0.05-25.0 µmol/L with a lower limit of quantification of 0.05 µmol/L. The intra- and interday precisions were <5.2%, the accuracy varied from -4.1 to 4.5%. The recovery was >94%. The LC-MS/MS method showed an excellent agreement with the existing HPLC-UV method using Passing-Bablok regression and Bland-Altman difference plot analysis. The validated LC-MS/MS can be successfully applied to the routine therapeutic drug monitoring of MTX in clinical laboratories.

Role of methotrexate polyglutamation and reduced folate carrier 1 (RFC1) gene polymorphisms in clinical assessment indexes.

The aims of the present study were to define inter-individual differences in response to methotrexate (MTX) through MTX polyglutamate (MTX-PG) levels in red blood cells (RBC) and MTX-related gene polymorphisms. A total of 145 rheumatoid arthritis patients were recruited. MTX-PG1-5 concentrations in RBC were measured, and 11 single nucleotide polymorphisms, all in MTX-related genes involved in the folate pathway, were analyzed. Disease activity was also assessed. There was no direct relationship between any MTX-PG concentration and the patient's disease condition, but detectability of MTX-PG5 was extracted as a candidate marker for response to MTX. When disease activity was compared between patients in which MTX-PG5 was detectable and undetectable, all indexes except the visual analog scale (VAS) and C-reactive protein (CRP) were found to be significantly lower in the former patients. Reduced folate carrier 1 (RFC1) 80G>A was significantly associated with the detectability of MTX-PG5; detectability of MTX-PG5 was lower in patients with the A mutant allele. The present study suggests that detectability of MTX-PG5 in RBC is a possible biomarker for response to MTX, and the RFC1 80G>A mutation is associated with low detectability of MTX-PG5. Prospective studies with a sufficient number of patients are needed to confirm the present findings.

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.

High dose methotrexate population pharmacokinetics and Bayesian estimation in patients with lymphoid malignancy.

The purpose of present study was to develop a population pharmacokinetic model of high dose methotrexate (HD-MTX) infusion in patients with lymphoid malignancy, to investigate the biological and clinical covariates related to the drug distribution and elimination. It is also the purpose to propose a limited sampling strategy (LSS) for the estimation of the time above the threshold (0.2 micromol.L(-1)). A total 82 patients with lymphoid malignancy were involved in the study. A pharmacokinetic model was developed using nonlinear mixed-effect model. The influence of demographic characteristics, biological factors, and concurrent administration were investigated. The final predictive performance was validated by bootstrap and cross-validation. Bayesian estimation was evaluated. The pharmacokinetics of HD-MTX was described by a two-compartment model. The pharmacokinetic parameters and the inter-individual variability were as follows: the clearance CL, 7.45 L.h(-1) (inter-individual variability 50.6%), the volume of the central and peripheral compartment V(1), 25.9 L (22.5%), V(2), 9.23 L (97.8%), respectively, and the intercompartmental clearance Q, 0.333 L.h(-1) (70.4%). The influence of serum creatinine on CL and weight on V(1) was retained in the final model. The protocol involved one sampling time at 44 h after the start of the infusion, allowing one to predict the time at which the MTX concentration reached the expected threshold (0.2 micromol.L(-1)). Serum creatinine and weight showed significant influence on methotrexate CL and V(1), respectively. Furthermore, a Bayesian estimation based on the covariates and 44 h sample was developed, allowing prediction of the individual methotrexate pharmacokinetic parameters and the time to 0.2 micromol.L(-1).

Population pharmacokinetics of high-dose methotrexate in children with acute lymphoblastic leukaemia.

OBJECTIVE: To develop and a priori validate a methotrexate population pharmacokinetic model in children with acute lymphoblastic leukaemia (ALL), receiving high-dose methotrexate followed by folinic acid rescue, identifying the covariates that could explain part of the pharmacokinetic variability of methotrexate. METHODS: The study was carried out in 49 children (aged 6 months to 17 years) who received high-dose methotrexate (3 g/m(2) per course) in long-term treatment. In an index group (37 individuals; 1236 methotrexate plasma concentrations), a population pharmacokinetic model was developed using a nonlinear mixed-effects model. The remaining patients' data (12 individuals; 278 methotrexate plasma concentrations) were used for model validation. Age, sex, total bodyweight (TBW), height, body surface area, lowest urine pH during infusion, serum creatinine, ALT, AST, folinic acid dose and length of rescue were analysed as possible covariates. The final predictive performance of the pharmacokinetic model was tested using standardised mean prediction errors. RESULTS: The final population pharmacokinetic model (two-compartmental) included only age and total bodyweight as influencing clearance (CL) and volume of distribution of central compartment (V(1)). For children aged < or =10 years: CL (L/h) = 0.287 . TBW(0.876); V(1) (L) = 0.465 . TBW, and for children aged >10 years: CL (L/h) = 0.149 . TBW; V(1) (L) = 0.437 . TBW. From the base to the final model, the inter-individual variabilities for CL and V(1) were significantly reduced in both age groups (30-50%). The coefficients of variation of the pharmacokinetic parameters were <30%, while residual and inter-occasional coefficients maintained values close to 40%. Validation of the proposed model revealed the suitability of the model. CONCLUSION: A methotrexate population pharmacokinetic model has been developed for ALL children. The proposed model could be used in Bayesian algorithms with a limited sampling strategy to estimate the systemic exposure of individual patients to methotrexate and adapt both folinic acid rescue and methotrexate dosing accordingly.

External quality assessment of Syva Emit and Abbott TDx II assays for methotrexate in serum.

The Syva Emit and Abbott TDx II kits for determination of methotrexate in serum were compared using data from 14 samples distributed by the United Kingdom National External Quality Assessment Scheme to a mean of 38 European laboratories. For methotrexate concentrations above 0.2 mumol/L, there was no significant difference in the bias or coefficient of variation of measurements between the two techniques. A significantly greater number of measurements by Syva Emit (6.9%) were rejected as outliers > 3 SD from the sample mean compared with Abbott TDx (1.3%). The lower sensitivity of the Syva Emit assay was evident in data reported for samples containing methotrexate concentrations below 0.2 mumol/L.

Oral administration of an easily prepared solution of injectable methotrexate diluted in water: A comparison of serum concentrations vs methotrexate tablets and clinical utility.

OBJECTIVE: To investigate whether the injectable formulation of methotrexate (MTX) given as an easily prepared oral solution of MTX diluted in water results in serum concentrations similar to those obtained with MTX tablets; to describe an easy and safe method of dispensing the drug. METHODS: Six patients (5 women, 1 man) with rheumatoid arthritis were given 10 mg of liquid MTX orally. The liquid was prepared by diluting 0.4 ml of the injectable formulation of MTX (50 mg/2 ml) in 8 ounces of water. One to 2 weeks later these patients were given 10 mg of MTX in the tablet form. MTX serum concentrations were determined using a fluorescence polarization immunoassay. The area under the concentration vs time curve (AUC), maximum concentration (Cmax) and the time to reach maximum concentration (tmax) were determined from the resulting concentration vs time curves. RESULTS: There was no statistical difference in the variables measured (AUC, Cmax, tmax), demonstrating comparable concentrations with these 2 methods of MTX administration. Patients found the medication easy to administer, potential hazards with the use of needles were avoided, and the cost of the drug was greatly decreased. CONCLUSION: The administrator of this easily prepared MTX solution is an alternative to the conventional administration of MTX tables, and may be of particular benefit in patients with financial limitations.

Identification and quantitation of methotrexate and methotrexate metabolites in clinical high-dose therapy by high pressure liquid chromatography and field desorption mass spectrometry.

High-pressure liquid chromatography in combination with field desorption mass spectrometry as techniques of high specificity and sensitivity have been applied to the identification and quantitation of the anticancer drug methotrexate and its metabolites which occur in clinical high-dose therapy. Field desorption mass spectra of methotrexate and several methotrexate and folic acid derivatives, when investigated as free acids or ammonium salts, yield abundant protonated molecular ions and a consistent pattern of structurally significant fragments. High-pressure liquid chromatographic separation of methotrexate metabolites was performed on reverse-phase, C-18 columns using a volatile, ammonium bicarbonate/acetate containing mobile phase that was especially suited for the field desorption mass spectral analysis of isolated metabolites, and provided the definite identification of 7-hydroxymethotrexate and 4-[[2,4-diamino-6-pteridinyl]methyl]methylamino]-benzoic acid in serum and urine of patients treated with high-dose methotrexate. The high intensity and stability of the [MH]+ ions was found suitable for the quantitation of methotrexate and related folate analogues by field desorption mass spectrometry. A synthetic methotrexate derivative, methotrexate-gamma-(2-hydroxy)ethyl-amide was used as internal standard for the quantitative determination of methotrexate in serum and urine. In a study to comparatively assess the potential of specific quantitation methods, serum and urine levels of methotrexate and its major metabolite, 7-hydroxymethotrexate were determined by (i) an enzyme immunoassay, (ii) reverse phase high-pressure liquid chromatography and (iii) field desorption mass spectrometry. Results obtained from four patients with osteogenic sarcoma receiving high-dose methotrexate/leucovorin rescue therapy consistently show the sustained elimination of 7-hydroxymethotrexate over several days, thus indicating the utility of specifically monitoring this nephrotoxic metabolite, at massive methotrexate doses.

Determination of methotrexate in serum by a rapid, fully mechanized enzyme immunoassay (EMIT).

A homogeneous enzyme immunoassay for the determination of methotrexate in serum (EMIT, Syva Corp.) was fully mechanized by the use of an Eppendorf analyzer 5010. Coefficients of variation from day to day were in the range 2--16%. A comparison of the results obtained by EMIT and a radioimmunoassay (Methotrexate 125 I Radioimmunoassay Kit, Diagnostic Biochemistry Inc.) in a series of 50 samples from patients showed a good correlation between both methods. The specificity of the EMIT assay appeared to be adequate. The time required for the determination of about 20 patient samples by this procedure was only about 80 minutes. From our clinical experimence the EMIT assay appears to be well suited for routine monitoring of methotrexate serum concentrations above 0.1 mumol/1 during intrathecal and high dose methotrexate therapy.