EXPRESS: A rapid LC-MS/MS assay for the measurement of serum methotrexate in patients who have received high doses for chemotherapy

Background Methotrexate is used in high doses to treat a number of cancers, particularly certain haematological malignancies. Monitoring of serum methotrexate concentration is important due to the potential toxicity of methotrexate and the variation in methotrexate pharmacokinetics in different patients on the same treatment regimen. Objective To develop a rapid liquid chromatography-tandem mass spectrometry method for monitoring serum methotrexate in patients on high-dose chemotherapy. Method Isotopically labelled internal standard was added to sample prior to protein precipitation with methanol. Diluted supernatant was injected into a Waters Acquity UPLC system linked to a TQS-Micro mass spectrometer. Separation by chromatography was achieved with a Waters Phenyl Vanguard with a retention time of approximately 0.5 minutes. The quantifier and qualifier transitions for methotrexate were 455.2>134.1 and 455.2>175.2 respectively. Results Mean recovery was 111% for 3 different concentrations of methotrexate spiked into 7 different patient samples, with ion suppression <1%. Between-batch and within-batch CVs were <5% at three different concentrations of methotrexate in fresh frozen plasma. The lower limit of quantification was 0.02 µmol/L and the assay was shown to be linear to approximately 25 µmol/L. The LC-MS/MS assay showed a mean bias of -8.6% compared to an immunoassay, whilst mean bias compared to weighed in targets in EQA samples was 1.6 %. Discussion A rapid LC-MS/MS assay for methotrexate has been developed and validated. The LC-MS/MS method is likely to offer superior accuracy and specificity to more widely available immunoassays.

Urine Methotrexate Concentration at 46–48 Hours Post-Treatment Reflects Methotrexate-Induced Acute Kidney Injury

Early identification of methotrexate-induced acute kidney injury (AKI) and delayed elimination of methotrexate are critical to limiting toxicity of the drug. The current monitoring strategy consists of serial serum methotrexate concentrations at 24, 36, 42, and 48 hours. Appropriate serum concentration monitoring and intervention does not always prevent AKI. Therefore, ongoing study of biomarkers and improved methods of screening for methotrexate-induced AKI is critical to reduce toxicity. This case series reports urine methotrexate values of 4 patients undergoing treatment with high-dose methotrexate. Urine methotrexate concentration was measured 46 to 48 hours after methotrexate infusion. Urine methotrexate concentration was compared with the duration of drug clearance from the serum. Only 1 patient (case 3) developed AKI. Serum concentration of methotrexate were &lt; 0.3 μmol/L at 42, 48, and 48 hours in patients 1, 2, and 4, respectively, and at 168 hours in patient 3 (p &lt; 0.01). Urine methotrexate concentrations were 2.77, 6.45, and 7.8 (μmol/L), in patients 1, 2, and 4, respectively, and 113.69 (μmol/L) in patient 3 (p &lt; 0.001). This case series provides preliminary data that urine methotrexate concentration at hours 46 to 48 may reflect AKI. Future studies should investigate the ability of serial urine methotrexate concentrations to predict delayed drug clearance and the development of AKI.

A second administration of glucarpidase in a different cycle of high-dose methotrexate: Is it safe and effective in adults?

Introduction Methotrexate intoxication following high-dose methotrexate-induced acute kidney injury is a life-threatening complication. Glucarpidase can quickly reduce extracellular methotrexate to safe levels, but the effectiveness and safety of its use in different episodes of nephrotoxicity remain an unknown area. Case Report A 30-year-old male diagnosed with acute lymphoblastic T-cell lymphoma received methotrexate 5 g/m2 intravenous (IV) as part of the first consolidation cycle. On Consolidation 3, he restarted methotrexate at a dose of 3 g/m2 IV showing slow methotrexate elimination, associated myelosuppression, and hepatic toxicity. Glucarpidase was administered (total dose of 2000 International Units (IU)). No adverse events were observed, and his renal function returned to normal. One hundred and six days later, he was diagnosed with leptomeningeal and cerebellar relapse and treatment with methotrexate 3,5 g/m2 IV day 1 and cytosine arabinoside (Ara-C) 2 g/m2 IV twice per day days 1, 3, and 5 was started. At 36 h from methotrexate infusion, serum creatinine increased up to 1.89 mg/dL and methotrexate concentration was 100 µmol/L. Management and Outcome: Ara-C was suspended, and a second administration of glucarpidase (2000 IU) was dispensed. No adverse events were noticed, methotrexate levels decreased and renal function progressively improved, recovering completely three weeks later. Discussion The effectiveness and safety of the use of glucarpidase in different episodes of nephrotoxicity remain an unknown area, and the rate and consequences of antiglucarpidase antibody formation remain poorly understood. This case report is, to our knowledge, the first case of a second administration of glucarpidase in a different cycle of high-dose methotrexate in an adult patient.

Methotrexate in saliva and risk in the development of chemotherapeutic mucositis in children

The aim of the study was to assess the possible direct effect of anticancer therapy with methotrexate (MTX) in bio liquids on the oral mucosa in the development of oral mucositis (OM). Twenty one children and adolescents participated in this study. Chemotherapy with MTX was administered in the following concentrations of 1; 2; 5 g/m2 of body surface area during 24 hours (including four episodes with OM). Twenty seven episodes of chemotherapy with high dose MTX were assessed in the samples of saliva on the 6th; 12; 24; 42; 48; 54 hour from the start of infusion and in the samples of blood on the 42; 48; 54 hour from the start of infusion. Сoncentration of methotrexate was measured by standard fluorescent polarization immunoassay using MTX reagent pac kit according to the manufacturer's instructions. Analysis of dynamics of methotrexate concentration in samples was performed using a linear model of mixed effects, on the basis of which the average values ​​(M) and confidence intervals for them were calculated (95 % CI). The analysis of the correlation of the levels of methotrexate in the blood and oral liquid was performed at individual time points (42; 48 and 54 hours) using the Spearman method (r). It was determined that excretion of MTX in the oral cavity repeated its clearance in blood. MTX concentration in saliva was less than 1/10 from its concentration in blood. During the first day, MTX concentration had decimicromol level and then until 54 hour it had santimicromol level. MTX concentration in saliva on the 6; 12; 24 hour in children with OM was lower by 2 times than in children without OM (P < 0,001). There was no significant difference in those parameters between two groups (with or without OM) in the following observed hours. This data does not support hypothesis concerning involvement of salivary MTX in OM pathogenesis.

DAMPAned Methotrexate: A Case Report and Review of the Management of Acute Methotrexate Toxicity

Rationale: Consensus guidelines on the management of methotrexate-induced nephrotoxicity using glucarpidase (Voraxaze) may be relatively unfamiliar to the nephrology community. Presenting concerns of the patient: A 61-year-old man with intravascular large B-cell lymphoma was admitted for cycle #1 of high-dose methotrexate (HDMTX) following 2 cycles of R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone) chemotherapy. On admission, he was clinically euvolemic and had a creatinine clearance of 98 mL/min. He received standard HDMTX toxicity prophylaxis with volume expansion, urinary alkalinization, and leucovorin rescue. Diagnoses: Despite prophylactic efforts, he developed a severe acute kidney injury, creatinine 63 to 226 µmol/L (2.56 mg/dL), following HDMTX, impaired methotrexate clearance, and neurotoxicity manifested by status epilepticus. Interventions: He was given glucarpidase to convert extracellular methotrexate into its inactive metabolites, glutamate and DAMPA (4-deoxy-4-amino- N10-methylpteroic acid) at 52 hours post-HDMTX. Cross-reactivity between commercial methotrexate immunoassays with DAMPA led to falsely elevated methotrexate concentrations for much longer than expected based on the current guideline (5 days instead of <48 hours). This required ongoing monitoring of methotrexate concentration by mass spectrometry. Outcomes: The patient remained nonoliguric and did not develop acute indications for dialysis. Serum creatinine peaked at 608 µmol/L (6.88 mg/dL) 6 days after HDMTX. He ultimately had a full renal and neurologic recovery. Lessons learned: Glucarpidase is an effective option for nonrenal elimination of methotrexate-induced nephrotoxicity. Timing of methotrexate concentration monitoring to assess for toxicity, how to access the drug, and the need for ongoing monitoring by mass spectrometry beyond the guideline recommendation are highlighted for centers where HDMTX therapy may be used.

Insulin potentiation of photodynamic activity in vitro

The present work reports on the investigation of the possible influence of insulin on the photodynamic effect of the photosensitizer radachlorin on four cell lines: HepG2, HT-29, BJ and BALB/3T3. For a quantitative comparison, the insulin effect on the standard chemotherapy drug methotrexate is also measured. The results show that the EC[Formula: see text] value for methotrexate is 40% higher than when combined with insulin; and for the lowest methotrexate concentration used, the difference exceeds 120%. For the case of the photodynamic effect, it is shown that insulin, when applied in combination with radachlorin, improves the cell permeability for the photosensitizer, thus increasing its concentration in the cells. The cytotoxic effect of photodynamic therapy with radachlorin as the photosensitizer has been quantitatively compared. The quantitative comparison of the photodynamic effect in the cases of using the photosensitizer radachlorin both alone and in combination with insulin, shows that the addition of insulin does not have an effect on the model of normal cell lines used; while for the HepG2 tumor cells, a two-fold increase in the EC[Formula: see text] value is measured.

Methotrexate Elimination When Coadministered With Levetiracetam

Background: Delayed elimination of methotrexate was previously reported in 2 patients receiving concomitant levetiracetam. Objective: To explore the potential interaction between methotrexate and levetiracetam in patients receiving high-dose methotrexate. Methods: This retrospective study reviewed the records of 81 adults receiving 280 cycles of methotrexate to determine the effects of levetiracetam on methotrexate elimination. Institutional review board approval was obtained. Results: Levetiracetam was administered in 33 (12%) cycles of methotrexate. Patients receiving levetiracetam had significantly lower 24-hour methotrexate concentrations compared with those not receiving levetiracetam (2.91 vs 7.37 µmol/L, P = 0.005). Despite this difference, concentrations at 48 and 72 hours were similar between groups. Times to nontoxic methotrexate concentration (<0.1 µmol/L) were the same regardless of the presence of levetiracetam. The frequency of delayed elimination at 24, 48, and 72 hours was similar in both groups as was the frequency of delayed elimination at any time point. Cox regression demonstrated that levetiracetam was not a significant predictor of time to nontoxic methotrexate concentration ( P = 0.796; HR = 1.058; 95% CI = 0.692-1.617), and logistic regression demonstrated that levetiracetam was not a significant predictor of delayed elimination at any time point. Levetiracetam use was similar between groups when comparing patients experiencing delayed elimination at any time point with those without delayed elimination (13% vs 10%, respectively, P = 0.527). Conclusion: This study does not support the previous reports of a significant interaction between levetiracetam and methotrexate. A clinically significant interaction is unlikely in those without additional risk factors for delayed elimination.