Metabolite 7-hydroxymethotrexate Research Articles

Population pharmacokinetics of methotrexate and 7-hydroxymethotrexate and delayed excretion in infants and young children with brain tumors

PurposeThe objectives of this study were to develop a population pharmacokinetic model of methotrexate (MTX) and its primary metabolite 7-hydroxymethotrexate (7OHMTX) in children with brain tumors, to identify the sources of pharmacokinetic variability, and to assess whether MTX and 7OHMTX systemic exposures were related to toxicity. MethodsPatients received 2.5 or 5 g/m2 MTX as a 24-hour infusion and serial samples were analyzed for MTX and 7OHMTX by an LC-MS/MS method. Pharmacokinetic parameters were estimated using nonlinear mixed-effects modeling. Demographics, laboratory values, and genetic polymorphisms were considered as potential covariates to explain the pharmacokinetic variability. Association between MTX and 7OHMTX systemic exposures and MTX-related toxicities were explored using random intercept logistic regression models. ResultsThe population pharmacokinetics of MTX and 7OHMTX were adequately characterized using two-compartment models in 142 patients (median 1.91 y; age range 0.09 to 4.94 y) in 513 courses. The MTX and 7OHMTX population clearance values were 4.6 and 3.0 l/h/m2, respectively. Baseline body surface area and estimated glomerular filtration rate were significant covariates on both MTX and 7OHMTX plasma disposition. Pharmacogenetic genotypes were associated with MTX pharmacokinetic parameters but had only modest influence. No significant association was observed between MTX or 7OHMTX exposure and MTX-related toxicity. ConclusionsMTX and 7OHMTX plasma disposition were characterized for the first time in young children with brain tumors. No exposure-toxicity relationship was identified in this study, presumably due to aggressive clinical management which led to a low MTX-related toxicity rate.

CNS penetration of methotrexate and its metabolite 7-hydroxymethotrexate in mice bearing orthotopic Group 3 medulloblastoma tumors and model-based simulations for children

The brain penetration of methotrexate (MTX) and its metabolite 7-hydroxymethotrexate (7OHMTX) was characterized in non-tumor bearing mice and mice bearing orthotopic Group 3 medulloblastoma. Plasma pharmacokinetic studies and cerebral and ventricular microdialysis studies were performed in animals dosed with 200 or 1000 mg/kg MTX by IV bolus. Plasma, brain/tumor extracellular fluid (ECF) and lateral ventricle cerebrospinal fluid (CSF) MTX and 7OHMTX concentration-time data were analyzed by validated LC-MS/MS methods and modeled using a population-based pharmacokinetic approach and a hybrid physiologically-based model structure for the brain compartments. Brain penetration was similar for MTX and 7OHMTX and was not significantly different between non-tumor and tumor bearing mice. Overall, mean (±SD) model-derived unbound plasma to ECF partition coefficient Kp,uu were 0.17 (0.09) and 0.17 (0.12) for MTX and 7OHMTX, respectively. Unbound plasma to CSF Kp,uu were 0.11 (0.06) and 0.18 (0.09) for MTX and 7OHMTX, respectively. The plasma and brain model were scaled to children using allometric principles and pediatric physiological parameters. Model-based simulations were adequately overlaid with digitized plasma and CSF lumbar data collected in children receiving different MTX systemic infusions. This model can be used to further explore and optimize methotrexate dosing regimens in children with brain tumors.

Comparison of four immunoassays to an HPLC method for the therapeutic drug monitoring of methotrexate: Influence of the hydroxylated metabolite levels and impact on clinical threshold.

Methotrexate requires therapeutic drug monitoring in oncology because of narrow therapeutic index, especially the metabolite 7-hydroxymethotrexate exhibits nephrotoxicity. The goal of this study was to evaluate different assays and their impact on clinical decisions. Following routine measurement with Abbott TDxFLx® assay (MTX-TDX), 62 samples were analysed on Architect®i1000 (MTX-ARCHI), Xpand® (ARK/XPND), Indiko® (ARK/INDI), and HPLC (MTX-HPLC) as the reference method. The influence of 7-hydroxymethotrexate was explored on ARK reagent to document the cause of the observed bias. ROC curves were built to study the impact of the method on the discharge thresholds for the patients at three levels. Total imprecision was below 2.60% for the methotrexate-ARCHI and close to 10% for both ARK assays for plasma pools. The correlation coefficients were 0.93, 0.93, 0.89 and 0.95, the Bland-Altman difference plot revealed a bias of 0.075, 0.037, 0.049 and -0.002, and the number of results exceeding the TE criteria of 0.1 µM was 17 (27%), 13 (21%), 15 (24%) and 15 (24%) for MTX-TDX, ARK/INDI, ARK/XPND and MTX-ARCHI, respectively. Cross reactivity with 7-hydroxymethotrexate was between 1 and 9%. Overestimation of methotrexate concentration was between -4% and +32%. The most robust clinical level was found to be the highest level (0.2 µM) with ROC curves. The authors found the best results for imprecision with chemiluminescent microparticle immunoassay method on methotrexate-ARCHI, with bias below to the RICOS recommendations and best correlation to the reference method. Impact on the threshold values for clinical decision need to be clearly exposed to clinicians.

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.

Validation Of LC-Ms/Ms Method for Monitoring of Methotrexate and its Metabolite 7-Hydroxymethotrexate Levels in Human Serum

Validation Of LC-Ms/Ms Method for Monitoring of Methotrexate and its Metabolite 7-Hydroxymethotrexate Levels in Human Serum

Impact of Abcc2 [Multidrug Resistance-Associated Protein (Mrp) 2], Abcc3 (Mrp3), and Abcg2 (Breast Cancer Resistance Protein) on the Oral Pharmacokinetics of Methotrexate and Its Main Metabolite 7-Hydroxymethotrexate

The ATP-binding cassette (ABC) transporters ABCC2 [multidrug resistance-associated protein (MRP) 2], ABCC3 (MRP3), and ABCG2 (breast cancer resistance protein) are involved in the efflux of potentially toxic compounds from the body. We have shown before that ABCC2, ABCC3, and ABCG2 together influence the pharmacokinetics of the anticancer and antirheumatic drug methotrexate (MTX) and its toxic metabolite 7-hydroxymethotrexate (7OH-MTX) after intravenous MTX administration. We now have used Abcc2;Abcc3;Abcg2(-/-) and corresponding single and double knockout mice to investigate the relative impact of these transporters on MTX and 7OH-MTX pharmacokinetics after oral MTX administration (50 mg/kg). The plasma areas under the curve (AUC(plasma)) in Abcg2(-/-) and Abcc2;Abcg2(-/-) mice were 1.7- and 3.0-fold higher than those in wild-type mice, respectively, suggesting additive effects of Abcc2 and Abcg2 on oral MTX pharmacokinetics. However, the AUC(plasma) in Abcc2;Abcc3;Abcg2(-/-) mice was not different from that in wild-type mice, indicating that Abcc3 protein is necessary for increased MTX plasma concentrations in the absence of Abcc2 and/or Abcg2. Furthermore, 2 h after administration, MTX liver levels were increased in Abcg2-deficient strains and MTX kidney levels were 2.2-fold increased in Abcc2;Abcg2(-/-) mice compared with those in wild-type mice. The absence of Abcc2 and/or Abcg2 also led to significantly increased liver and kidney levels of 7OH-MTX. Our results suggest that inhibition of ABCG2 and/or ABCC2, genetic polymorphisms or mutations reducing expression or activity of these proteins may increase the oral availability of MTX. Such conditions may also present risk factors for increased MTX-related toxicity in patients treated with oral MTX.

Bioanalytical method development for a generation 5 polyamidoamine folic acid methotrexate conjugated nanoparticle

Generation 5 poly(amidoamine) dendrimer nanoparticles conjugated with folic acid and methotrexate (G5-MTX-FA) for targeted treatment of cancer are of recent interest. The increased efficacy of these nanodevices over the free methotrexate has been shown in vitro and in vivo. The heterogeneous nature of this nanoparticle together with possible release of active compounds complicated the method development. This work presents a bioanalytical assay for the detection of nanoparticle-conjugated methotrexate, released methotrexate, and its main plasma metabolite 7-hydroxymethotrexate in rat plasma. Determination of G5-MTX-FA-associated methotrexate occurred by a reductive cleavage of the C9-N10 bond in methotrexate, resulting in a highly fluorescent 2,4-diamino-6-methylpteridine reporter molecule that could be measured by reversed-phase chromatography and fluorescence detection. It was found that reduction should occur directly in the plasma matrix to avoid irreversible adsorption of the nanodevice during sample preparation. The method was linear over a range from 50 to 10,000 nM G5-MTX-FA utilizing 100 microL of plasma. Nanoparticle-released methotrexate and its metabolite 7-hydroxymethotrexate were determined by reversed-phase chromatography followed by online post-column photochemical derivatization and fluorescence detection. The method was specific for these analytes irrespective of nanoparticle concentration. Sample preparation consisted of perchloric acid protein precipitation followed by a strong anion exchange solid-phase extraction. Limits of quantification were about 50 nM for methotrexate and 10 nM for 7-hydroxymethotrexate. Preliminary pharmacokinetic profiles of intravenous and subcutaneous administered G5-MTX-FA in rats were obtained. These data indicated that less than 0.1% of the methotrexate mass is released from the nanoparticle in plasma.

Abcc2 (Mrp2), Abcc3 (Mrp3), and Abcg2 (Bcrp1) are the main determinants for rapid elimination of methotrexate and its toxic metabolite 7-hydroxymethotrexate in vivo

The multidrug transporters ABCC2, ABCC3, and ABCG2 can eliminate potentially toxic compounds from the body and have overlapping substrate specificities. To investigate the overlapping functions of Abcc2, Abcc3, and Abcg2 in vivo, we generated and characterized Abcc3;Abcg2-/- and Abcc2;Abcc3;Abcg2-/- mice. We subsequently analyzed the relative impact of these transport proteins on the pharmacokinetics of the anticancer drug methotrexate (MTX) and its main, toxic, metabolite 7-hydroxymethotrexate (7OH-MTX) after i.v. administration of MTX (50 mg/kg). Whereas in single and double knockout mice, the plasma and liver concentrations of MTX and 7OH-MTX decreased rapidly after MTX administration, in the Abcc2;Abcc3;Abcg2-/- mice, they remained very high. One hour after administration, 67% of the MTX dose was still present in livers of Abcc2;Abcc3;Abcg2-/- mice as MTX or 7OH-MTX versus 7% in wild-type, showing dramatic liver accumulation of these toxic compounds when Abcc2, Abcc3, and Abcg2 were all absent. Furthermore, the urinary and fecal excretion of the nephrotoxic metabolite 7OH-MTX were increased 27- and 7-fold, respectively, in Abcc2;Abcc3;Abcg2-/- mice. Thus, Abcc2, Abcc3, and Abcg2 together mediate the rapid elimination of MTX and 7OH-MTX after i.v. administration and can to a large extent compensate for each other's absence. This may explain why it is still comparatively safe to use a toxic drug such as MTX in the clinic, as the risk of highly increased toxicity due to dysfunctioning of ABCC2, ABCC3, or ABCG2 alone is limited. Nevertheless, cotreatment with possible inhibitors of ABCC2, ABCC3, and ABCG2 should be done with utmost caution when treating patients with methotrexate.

Functionally Overlapping Roles of Abcg2 (Bcrp1) and Abcc2 (Mrp2) in the Elimination of Methotrexate and Its Main Toxic Metabolite 7-Hydroxymethotrexate In vivo

ABCC2 (MRP2) and ABCG2 (BCRP) transport various endogenous and exogenous compounds, including many anticancer drugs, into bile, feces, and urine. We investigated the possibly overlapping roles of Abcg2 and Abcc2 in the elimination of the anticancer drug methotrexate (MTX) and its toxic metabolite 7-hydroxymethotrexate (7OH-MTX). We generated and characterized Abcc2;Abcg2(-/-) mice, and used these to determine the overlapping roles of Abcc2 and Abcg2 in the elimination of MTX and 7OH-MTX after i.v. administration of 50 mg/kg MTX. Compared with wild-type, the plasma areas under the curve (AUC) for MTX were 1.6-fold and 2.0-fold higher in Abcg2(-/-) and Abcc2(-/-) mice, respectively, and 3.3-fold increased in Abcc2;Abcg2(-/-) mice. The biliary excretion of MTX was 23-fold reduced in Abcc2;Abcg2(-/-) mice, and the MTX levels in the small intestine were dramatically decreased. Plasma levels of 7OH-MTX were not significantly altered in Abcg2(-/-) mice, but the areas under the curve were 6.2-fold and even 12.4-fold increased in Abcc2(-/-) and Abcc2;Abcg2(-/-) mice, respectively. This indicates that Abcc2 compensates for Abcg2 deficiency but that Abcg2 can only partly compensate for Abcc2 absence. Furthermore, 21-fold decreased biliary 7OH-MTX excretion in Abcc2;Abcg2(-/-) mice and substantial 7OH-MTX accumulation in the liver and kidney were seen. We additionally found that in the absence of Abcc2, Abcg2 mediated substantial urinary excretion of MTX and 7OH-MTX. Abcc2 and Abcg2 together are major determinants of MTX and 7OH-MTX pharmacokinetics. Variations in ABCC2 and/or ABCG2 activity due to polymorphisms or coadministered inhibitors may therefore substantially affect the therapeutic efficacy and toxicity in patients treated with MTX.

Impact of Abcc2 (Mrp2) and Abcc3 (Mrp3) on the In vivo Elimination of Methotrexate and its Main Toxic Metabolite 7-hydroxymethotrexate

ATP-binding cassette sub-family C member 2 [ABCC2; multidrug resistance-associated protein 2 (MRP2)] and ABCC3 (MRP3) mediate the elimination of toxic compounds, such as drugs and carcinogens, and have a large overlap in substrate specificity. We investigated the roles of Abcc2 and Abcc3 in the elimination of the anticancer drug methotrexate (MTX) and its toxic metabolite 7-hydroxymethotrexate (7OH-MTX) in vivo. Abcc2;Abcc3(-/-) mice were generated, characterized, and used to investigate possibly overlapping or complementary roles of Abcc2 and Abcc3 in the elimination of MTX and 7OH-MTX after i.v. administration of 50 mg/kg MTX. Abcc2;Abcc3(-/-) mice were viable and fertile. In Abcc2(-/-) mice, the plasma area under the curve (AUCi.v.) for MTX was 2.0-fold increased compared with wild type, leading to 1.6-fold increased urinary excretion, which was not seen in Abcc2;Abcc3(-/-) mice. Biliary excretion of MTX was 3.7-fold reduced in Abcc2(-/-) but unchanged in Abcc2;Abcc3(-/-) mice. The plasma AUCi.v.s of 7OH-MTX were 6.0-fold and 4.3-fold increased in Abcc2(-/-) and Abcc2;Abcc3(-/-) mice, respectively, leading to increased urinary excretion. The biliary excretion of 7OH-MTX was 5.8-fold reduced in Abcc2(-/-) but unchanged in Abcc2;Abcc3(-/-) mice. 7OH-MTX accumulated substantially in the liver of Abcc2(-/-) and especially Abcc2;Abcc3(-/-) mice. Abcc2 is important for (biliary) excretion of MTX and its toxic metabolite 7OH-MTX. When Abcc2 is absent, Abcc3 transports MTX and 7OH-MTX back from the liver into the circulation, leading to increased plasma levels and urinary excretion. Variation in ABCC2 and/or ABCC3 activity may therefore have profound effects on the elimination and severity of toxicity of MTX and 7OH-MTX after MTX treatment of patients.

Determination of methotrexate and its major metabolite 7-hydroxymethotrexate in mouse plasma and brain tissue by liquid chromatography-tandem mass spectrometry

Methotrexate (MTX) is an anticancer agent that is widely used in a variety of human cancers including primary central nervous system lymphoma (PCNSL). Important pharmacological properties that directly bear on the use of MTX in PCNSL, such as mechanisms that govern its uptake into brain tumors, are poorly defined, but are amenable to investigation in mouse models. In order to pursue such preclinical pharmacological studies, a rapid and sensitive liquid chromatography-tandem mass spectrometry (LC/MS/MS) method for the determination of MTX and its metabolite, 7-hydroxymethotrexate (7-OH MTX) in plasma and microdialysate samples from brain tumors and cerebrospinal fluid (CSF) is needed. The plasma assay was based on 10 μl samples and following a protein precipitation procedure enabled direct injection onto a LC/MS/MS system using positive electrospray ionization. A column switching technique was employed for desalting and the clean-up of microdialysate samples from brain tissues. The methods were validated for MTX and 7-OH MTX in both plasma and microdialysate samples from brain tumor and CSF, and produced lower limits of quantification (LLOQ) in plasma of 3.7 ng/ml for MTX and 7.4 ng/ml for 7-OH MTX, and in microdialysate samples of 0.7 ng/ml for both MTX and 7-OH MTX. The utility of the method was demonstrated by estimation of pharmacokinetic (PK) and brain distribution properties of MTX and 7-OH MTX in conscious mice. The method has the advantages of low sample volume, rapid clean-up, and the simultaneous measurement of MTX and 7-OH MTX in plasma and brain tissues allowing detailed PK studies to be completed in individual mice.

Serum levels and pharmacodynamics of methotrexate and its metabolite 7-hydroxy methotrexate in Japanese patients with rheumatoid arthritis treated with 2-mg capsule of methotrexate three times per week

Methotrexate (MTX) is the first-choice drug for rheumatoid arthritis (RA); however, the pharmacodynamics of MTX in Japanese patients with RA treated legitimately according to the government recommended dosage, 6 mg per week, are unknown. Methotrexate and its metabolite, 7-hydroxy MTX (7-OH MTX), were measured in sera of 16 outpatients with active RA in the first week of MTX treatment and 4-12 weeks after the introduction at 0, 1, 2, 4, and 8 h after administration of the first and the third 2-mg capsule, followed by sampling at 48, 96, and 168 h. The mean maximal serum drug concentration (mean C(max)) of MTX attained at 1-2 h after ingestion of the first capsule was 0.215 and 0.252 microM, respectively, in the first and the follow-up week. The mean C(max) after ingestion of the third capsule was 0.223 microM and 0.357 microM. The mean C(max) of 7-OH MTX was 0.0334 and 0.0289 microM for the first capsule, and 0.0495 and 0.0672 microM for the third capsule. The results indicate that MTX does not accumulate or deposit in the body of Japanese patients with RA when treated with 6 mg per week, and pharmacodynamics of MTX are comparable to those in overseas patients treated with 7.5 mg per week.

Serum levels and pharmacodynamics of methotrexate and its metabolite 7-hydroxy methotrexate in Japanese patients with rheumatoid arthritis treated with 2-mg capsule of methotrexate three times per week

Methotrexate (MTX) is the first-choice drug for rheumatoid arthritis (RA); however, the pharmacodynamics of MTX in Japanese patients with RA treated legitimately according to the government recommended dosage, 6 mg per week, are unknown. Methotrexate and its metabolite, 7-hydroxy MTX (7-OH MTX), were measured in sera of 16 outpatients with active RA in the first week of MTX treatment and 4–12 weeks after the introduction at 0, 1, 2, 4, and 8 h after administration of the first and the third 2-mg capsule, followed by sampling at 48, 96, and 168 h. The mean maximal serum drug concentration (mean Cmax) of MTX attained at 1–2 h after ingestion of the first capsule was 0.215 and 0.252 µM, respectively, in the first and the follow-up week. The mean Cmax after ingestion of the third capsule was 0.223 µM and 0.357 µM. The mean Cmax of 7-OH MTX was 0.0334 and 0.0289 µM for the first capsule, and 0.0495 and 0.0672 µM for the third capsule. The results indicate that MTX does not accumulate or deposit in the body of Japanese patients with RA when treated with 6 mg per week, and pharmacodynamics of MTX are comparable to those in overseas patients treated with 7.5 mg per week.

Disparate mechanisms of antifolate resistance provoked by methotrexate and its metabolite 7-hydroxymethotrexate in leukemia cells: implications for efficacy of methotrexate therapy

AbstractMethotrexate (MTX) is one of the leading drugs in the treatment of leukemia, but extensive metabolism to 7-hydroxymethotrexate (7-OHMTX) can limit its therapeutic efficacy. In this study we investigated whether 7-OHMTX itself can provoke anti-folate resistance that may further disrupt MTX efficacy. For this purpose, we developed resistance to 7-OHMTX as well as MTX in 2 human leukemia cell lines (CCRF-CEM and MOLT-4) by stepwise exposure to increasing concentrations of 7-OHMTX and MTX. Consequently, both leukemia cell lines displayed marked levels of resistance to 7-OHMTX (> 10-fold) and MTX (> 75-fold). The underlying mechanism of resistance in the MTX-exposed cells was a marked decrease (> 10-fold) in reduced folate carrier (RFC)-mediated cellular uptake of MTX. This was associated with transcriptional silencing of the RFC gene in MTX-resistant CCRF-CEM cells. In contrast, the molecular basis for the resistance to 7-OHMTX was due solely to a marked decreased (> 95%) in folylpolyglutamate synthetase (FPGS) activity, which conferred more than 100-fold MTX resistance upon a short-term exposure to this drug. This is the first demonstration that 7-OHMTX can provoke distinct modalities of antifolate resistance compared with the parent drug MTX. The implications of this finding for MTX efficacy and strategies to circumvent MTX resistance are discussed.

Disparate Mechanisms of Antifolate Resistance Provoked by Methotrexate and Its Metabolite 7-Hydroxymethotrexate in Leukemia Cells: Implications for Efficacy of Methotrexate Therapy.

Methotrexate (MTX) is one of the leading drugs in the treatment of leukemia, but extensive metabolism to 7-hydroxymethotrexate (7-OHMTX) can limit its therapeutic efficacy. In this study we investigated whether 7-OHMTX itself can provoke antifolate resistance that may further disrupt MTX efficacy. For this purpose we developed resistance to 7-OHMTX as well as MTX in two human leukemia cell lines (CCRF-CEM and MOLT-4) by stepwise exposure to increasing concentrations of 7-OHMTX and MTX. Consequently, both leukemia cell lines displayed marked levels of resistance to 7-OHMTX (>10 fold) and MTX (>75 fold), respectively. The underlying mechanism of resistance in the MTX-exposed cells was a marked decrease (>10-fold) in reduced folate carrier (RFC)-mediated cellular uptake of MTX. This was associated with transcriptional silencing of the RFC gene in MTX-resistant CCRF-CEM cells. In contrast, the molecular basis for the resistance to 7-OHMTX was solely due to a marked decreased (> 95%) in folylpolyglutamate synthetase (FPGS) activity which conferred >100-fold MTX resistance upon a short term exposure to this drug. This is the first demonstration that 7-OHMTX can provoke distinct modalities of antifolate resistance as compared to the parent drug MTX.

Mechanism of the pharmacokinetic interaction between methotrexate and benzimidazoles: potential role for breast cancer resistance protein in clinical drug-drug interactions.

The antifolate drug methotrexate (MTX) is transported by breast cancer resistance protein (BCRP; ABCG2) and multidrug resistance-associated protein1-4 (MRP1-4; ABCC1-4). In cancer patients, coadministration of benzimidazoles and MTX can result in profound MTX-induced toxicity coinciding with an increase in the serum concentrations of MTX and its main metabolite 7-hydroxymethotrexate. We hypothesized that benzimidazoles interfere with the clearance of MTX and/or 7-hydroxymethotrexate by inhibition of the ATP-binding cassette drug transporters BCRP and/or MRP2, two transporters known to transport MTX and located in apical membranes of epithelia involved in drug disposition. First, we investigated the mechanism of interaction between benzimidazoles (pantoprazole and omeprazole) and MTX in vitro in membrane vesicles from Sf9 cells infected with a baculovirus containing human BCRP or human MRP2 cDNA. In Sf9-BCRP vesicles, pantoprazole and omeprazole inhibited MTX transport (IC50 13 microm and 36 microm, respectively). In Sf9-MRP2 vesicles, pantoprazole did not inhibit MTX transport and at high concentrations (1 mm), it even stimulated MTX transport 1.6-fold. Secondly, we studied the transport of pantoprazole in MDCKII monolayers transfected with mouse Bcrp1 or human MRP2. Pantoprazole was actively transported by Bcrp1 but not by MRP2. Finally, the mechanism of the interaction was studied in vivo using Bcrp1-/- mice and wild-type mice. Both in wild-type mice pretreated with pantoprazole to inhibit Bcrp1 and in Bcrp1-/- mice that lack Bcrp1, the clearance of i.v. MTX was decreased significantly 1.8- to 1.9-fold compared with the clearance of i.v. MTX in wild-type mice. The conclusion is as follows: benzimidazoles differentially affect transport of MTX mediated by BCRP and MRP2. Competition for BCRP may explain the clinical interaction between MTX and benzimidazoles.

Microvascular Perturbations in Rats Receiving the Maximum Tolerated Dose of Methotrexate or Its Major Metabolite 7-Hydroxymethotrexate

Methotrexate(MTX) is a clinically important cytostatic antifolate.The study describes the acute effects of maximum tolerated doses of MTX or its major metabolite 7-hydroxymethotrexate (7-OH-MTX) on the ultrastructure of rat liver and kidneys. The ultrastructural changes in rats receiving MTX or 7-OH-MTX were, in principle, indistinguishable and their severity and extension increased with time of survival or doses of medication. All lesions were focal, microvascular, or parenchymal. Microvascular changes were more severe in nature when blood cells were present. The endothelial cells were swollen with loss of pinocytotic vesicles, their luminal plasma membrane formed blebs or were disrupted. Partly detached endothelial cells or deendothelialized areas, various types of white blood cells, in particular, neutrophil granulocytes, were observed in the microcirculation. Single platelets or small platelet aggregates were found either in the lumen or adhering to deendothelialized areas of injured endothelial cells. Hepatocytesexhibited steatosis, edema, and manifest single cell necrosis. There were also nuclear changes, marked proliferation of smooth endoplasmatic reticulum, increased amounts of intracellular lipid vacuoles, and a decrease in glycogen particles in hepatocytes. The kidney presented the major changes in the tubules and in the interstitial part. MTX and 7-OH-MTX acute toxicity may primarily be related to microvascular perturbation.

Determination of methotrexate and its main metabolite 7-hydroxymethotrexate in human urine by high-performance liquid chromatography with normal solid-phase extraction.

A practical and sensitive high-performance liquid chromatographic method using normal solid-phase extraction has been developed for the determination of methotrexate (MTX) and its main metabolite 7-hydroxymethotrexate (7-OH-MTX) in human urine. A urine specimen followed by the addition of pH 5.0 acetate buffer was purified by solid-phase extraction on a Sep-Pak silica cartridge. The analyte was chromatographed on a reversed-phase Inertsil ODS-2 column using phosphate buffer-acetonitrile at pH 5.3 as the mobile phase, and the effluent from the column was monitored at 303 nm. A good linear relationship between peak height and concentration was found for both of MTX and 7-OH-MTX in the range 5 to 1000 ng/ml of human urine. The inter-day coefficients of variation for the assay (n = 5) were 8.8% (5 ng/ml), 3.4% (50 ng/ml) and 2.0% (500 ng/ml) for MTX, and 7.2, 2.7 and 2.3% for 7-OH-MTX in urine, respectively. The present method should prove useful for the evaluation of urinary drug excretion in patients undergoing MTX low-dose therapy.

Determination of methotrexate and its metabolite 7-hydroxymethotrexate by direct injection of human plasma into a column-switching liquid chromatographic system using post-column photochemical reaction with fluorimetric detection

A simple, sensitive and fully automated column-switching system by direct injection of plasma samples for determination of methotrexate and its metabolite 7-hydroxymethotrexate was developed. The system utilized a C8 alkyl-diol silica precolumn coupled with a LiChrospher RP-18 analytical column, followed by a photoreactor and fluorimetric detection. The photo-oxidative irradiation was accomplished at UV 254 nm in the presence of 0.1% hydrogen peroxide in the eluent. Studies showed that the fluorimetric response was influenced by the reaction time, the degree of the reactor's transparency and the choice of the working wavelengths. By optimizing the content of acetonitrile in the eluent, methotrexate can be separated from 7-hydroxymethotrexate completely. The method validation revealed quantitative recoveries (> or = 94%) with coefficients of variation < or = 4.4%. The limits of detection and quantitation for determination of methotrexate were 0.20 and 0.36 ng, respectively, corresponding to 2.0 and 3.6 ng/ml for an injection volume of 100 microliters. It was possible to enhance the sensitivity further by injecting larger plasma volumes, up to 500 microliters.

Determination of methotrexate and 7-hydroxymethotrexate by liquid chromatography for routine monitoring of plasma levels

A high-performance liquid chromatographic (HPLC) method was designed to meet analytical and metrological requirements for routine blood level monitoring of methotrexate (MTX) and its main metabolite 7-hydroxymethotrexate (7OMTX). The metabolite, unavailable as a pure substance, was measured by reference to MTX calibration according to their respective ultraviolet absorbances. Acetonitrile deproteinization and chloroform clean-up provided plasma samples devoid of long-retained contaminants. The precision of the HPLC measurements, reproducibility of clean-up recovery, matrix effects and linearity were assessed by analysis of variance and linear regression in an appropriate experimental design, within a range from 0.205 to 16.7 mg/l of MTX and from 0.084 to 6.83 mg/l of 7OMTX. The clean-up recovery from plasma was 88% for MTX and 72% for 7OMTX, owing to retention on the protein precipitate. The assay was linear, the measurement precision was 3.3% for MTX and 6.2% for 7OMTX and the clean-up reproducibility was 4% for MTX and 3.6% for 7OMTX. By reference to automated fluorescence polarization immunoassay, the HPLC method resulted in plasma MTX values 10% lower, probably owing to the higher specificity of HPLC. Unsystematically sequenced plasma samples from 35 children following 24-h MTX infusions provided estimated half-decay times of 16 and 19 h for MTX and 7OMTX, respectively, and 7OMTX:MTX concentration ratios of 7 at 48 h and of 5 at 7 from starting infusions.