Simultaneous determination of plasma methotrexate and 7-hydroxy methotrexate by UHPLC-MS/MS in patients receiving high-dose methotrexate therapy.

The plasma concentrations of methotrexate (MTX) and its major metabolite 7-hydroxy methotrexate (7-OH-MTX) are highly correlated with the toxicities in patients with high-dose MTX therapy. Routine monitoring of MTX and 7-OH-MTX plasma levels is useful for dose adjustment of rescue drugs and toxicity prevention. A UHPLC-MS/MS method for simultaneous determination of plasma MTX and 7-OH-MTX was developed, validated, and applied in 181 plasma samples. The ion transition was m/z 455.2 → 308.2 for MTX and m/z 471.2 → 324.1 for 7-OH-MTX. The flow rate was 0.4 mL/min with a run time of 2.6 min. The calibration range was 0.002-2 µM for MTX, and 0.01-10 µM for 7-OH-MTX. The intra-day and inter-day inaccuracy and imprecision were -5.50% to 10.93% and less than 9.20% for both analytes. The internal standard (MTX-D3) normalized recovery and matrix factor were consistent at four quality control levels. 14 h, 38 h, and 62 h after dosing, MTX and 7-OH-MTX plasma levels were significantly higher in patients with impaired renal function compared to those with normal renal function. 7-OH-MTX plasma levels were significantly higher in patients with impaired liver function compared to those with normal liver function.

Methotrexate-induced epidermal necrosis: A case series of 24 patients.

BACKGROUND: Methotrexate-induced epidermal necrosis (MEN) is a rare but life-threatening cutaneous reaction that mimics Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN). OBJECTIVES: To investigate the clinicopathology, risk factors, and prognostic factors of MEN. METHODS: We enrolled 24 patients with MEN and 150 controls and analyzed the demographics, pathology, and plasma concentrations of methotrexate (MTX). RESULTS: Patients with MEN showed extensive skin necrosis (mean, 33.2% total body surface area) but no target lesions. The histopathology displayed keratinocyte dystrophy. Early signs of MEN included painful skin erosions, oral ulcers, and leukopenia/thrombocytopenia. Although 79.2% patients received leucovorin treatment, there was 16.7% mortality. Risk factors for MEN included older age (>60 years), chronic kidney disease, and high initial dosage of MTX without folic acid supplementation. Renal insufficiency delayed MTX clearance. Severe renal disease and leukopenia predicted poor prognosis in MEN, but none of the SCORe of Toxic Epidermal Necrosis criteria were associated with mortality of MEN. LIMITATIONS: The study was limited by the small sample size. CONCLUSION: MEN exhibited distinct clinicopathologic features from SJS/TEN. Recognition of the early signs and prognostic factors is important, because the rapid institution of leucovorin may be helpful. To reduce the risk of MEN, physicians should avoid prescribing MTX to high-risk patients and titrate the dosage slowly upward with folic acid supplementation.

High-dose leucovorin as sole therapy for methotrexate toxicity.

PURPOSE: Hemodialysis, hemoperfusion, thymidine, and carboxypeptidase have been recommended together with high-dose (HD) leucovorin (LV) to treat patients at risk for methotrexate (MTX) toxicity. To elucidate the efficacy of high LV rescue as the sole salvage modality for severe MTX intoxication, we studied 13 patients who were treated in this fashion at Memorial Sloan-Kettering Cancer Center (New York, NY). PATIENTS AND METHODS: To identify patients at high risk for severe MTX toxicity, we performed a retrospective review of all patients with MTX levels greater than 100 micromol/L at 24 hours and greater than 10 micromol/L at 48 hours after HD MTX. RESULTS: A total of 13 patients were identified. The median MTX concentration was 164 micromol/L at 24 hours (range, 102 to 940 micromol/L), 16.3 micromol/L at 48 hours (range, 10.5 to 190 micromol/L), and 6.2 micromol/L at 72 hours (range, 1.35 to 39 micromol/L). MTX levels remained greater than 0.1 micromol/L for an average of 11 +/- 3 days (mean +/- SD) (range, 7 to 17 days). In addition to supportive treatment with hydration and sodium bicarbonate administration, all patients were treated solely with HD LV, which was started within the first 24 hours in nine patients, 48 hours in three patients, and 72 hours in one patient in doses that varied from 0.24 to 8 g/d. Significant neutropenia (neutrophil count < 1,000/ microL) occurred in eight patients and lasted for 1 to 5 days. Thrombocytopenia (platelet count < 100,000/microL) occurred in seven patients and lasted for 5 to 10 days. Other toxic manifestations included mucositis of varying degrees, diarrhea, and neutropenic fever, but all patients recovered. CONCLUSION: In the range of MTX levels observed, HD LV can be used as a sole therapy for MTX toxicity without the need for extracorporeal removal and with tolerable morbidity.

High-dose 7-hydromethotrexate: acute toxicity and lethality in a rat model.

To elucidate mechanisms for methotrexate (MTX)-induced renal and hepatic toxicity, we investigated the acute effects of bolus plus continuous infusion of up to 0.4 g/kg 7-hydroxymethotrexate (7-OH-MTX) in the rat. We demonstrate for the first time in any species the occurrence of acute lethal toxicity within a few hours after 7-OH-MTX administration. Serum concentrations of 7-OH-MTX measured at the time of death were 1.4 mM (mean), about one-half of those achieved in some patients after infusion of high-dose MTX (HD-MTX) in the clinic. The data suggest an approximate LD50 (the dose lethal to 50% of the study population) of 0.3 g/kg and a steep dose/lethality curve for 7-OH-MTX. Moreover, acute renal and hepatic toxicity occurred as evidenced by severe morphological findings and increased serum levels of creatinine and liver transaminases. In all rats subjected to continuous infusion of 7-OH-MTX, yellow microscopic precipitations were apparent in the kidney tubules. Crystallization was also seen in bile ducts of the liver in some of the rats. These results further support that the formation of 7-OH-MTX is disadvantageous and that reported attempts to prevent its formation during MTX treatment are warranted.

High-performance liquid chromatographic determination of methotrexate, 7-hydroxymethotrexate, 5-methyltetrahydrofolic acid and folinic acid in serum and cerebrospinal fluid.

A method for the simultaneous determination of the antifolates methotrexate and 7-hydroxymethotrexate as well as the folates 5-methyltetrahydrofolic acid and folinic acid (5-formyltetrahydrofolic acid) in serum and cerebrospinal fluid (CSF) is described. High-performance liquid chromatography with gradient elution and dual detection (ultraviolet absorption and fluorescence) was used to separate and quantitate the analytes. Serum samples containing high levels of the substances of interest and CSF samples were injected directly onto the HPLC column. For determination of low concentrations, serum samples were subjected to a solid-phase extraction method for clean-up and concentration purposes. The determination limits were 10 ng/ml for both antifolates, 100 ng/ml for folinic acid, and 0.1 ng/ml for the physiologically occurring methylated folate which is about 1/100 the serum concentration in healthy children. The suitability of the method for pharmacokinetic monitoring of high-dose methotrexate therapy combined with leucovorin rescue administered to children with acute lymphoblastic leukemia was demonstrated. Minimum values of the serum folate during treatment ranged from 0.2 to 3.1 ng/ml. Even those very low concentrations could be reliably measured.

7-Hydroxy-methotrexate and clinical toxicity following high-dose methotrexate therapy.

7-Hydroxy-MTX production after consecutive high-dose MTX therapy (12 g/m2) was measured in 7 patients with osteosarcoma by HPLC. 7-Hydroxy-MTX serum values in the last cycle were found to be significantly lower compared with the first high-dose MTX treatment of the adjuvant chemotherapy protocol (COSS 80). Moreover, in another patient highly reduced 7-hydroxy-MTX production was correlated with severe clinical toxicity. As 7-hydroxy-MTX is a 200 fold less potent dihydrofolic acid reductase inhibitor compared with MTX decreased production of the metabolite may lead to enhanced clinical toxicity which may not be predictable monitoring MTX serum levels alone.

A simple radiometric assay for methotrexate and other folate antagonists.

A rapid radiometric assay for the folate antagonist MTX, based on the inhibition of chicken liver dihydrofolate reductase activity, has been developed. In the system the inhibition by MTX of the reduction of tritiated FH2 is standardized and used to quantitate this antagonist in plasma of subjects receiving high-dose MTX therapy. The assay permits the detection of plasma MTX levels to 5 X 10(-9)M with a coefficient of variation of 15%. The predominating circulating folate, 5 methyltetrahydrofolate, or the rescue agent leucovorin (5 formyltetrahydrofolate) do not significantly interfere with the assay at concentrations attainable with current rescue programs. Analyses of multiple plasma samples with the present assay show close agreement with the competitive protein-binding assay.