levoleucovorin and 7-hydroxymethotrexate

Topics: Animals; Anions; Binding, Competitive; Biological Transport, Active; Carrier Proteins; Cell Line; Cell Membrane; Computers; Dose-Response Relationship, Drug; Drug Interactions; Drug Resistance; Folic Acid Antagonists; Humans; In Vitro Techniques; Leucovorin; Membrane Proteins; Methotrexate; Models, Biological; Neoplasms; Polyglutamic Acid; Structure-Activity Relationship; Tetrahydrofolates

The pharmacokinetics of methotrexate and 7-hydroxy-methotrexate were studied in patients undergoing very high-dose methotrexate monotherapy. The patients received, first, two methotrexate intravenous bolus test doses (50 mg/m2) one with and one without concomitant administration of folinic acid (15 mg every 6 h) in a random sequence, and, second, an 8 h infusion, individualized to achieve a peak plasma concentration of 5 X 10(-4) M methotrexate (infusion rates greater than 1000 mg/h). Methotrexate and 7-hydroxy-methotrexate concentrations were measured by specific radioimmunoassays and the data were analysed simultaneously by an integrated pharmacokinetic model. Following test dose administration, methotrexate and 7-hydroxy-methotrexate plasma concentration kinetics were best described by assuming that methotrexate elimination (and 7-hydroxy-methotrexate formation) occurred from a peripheral compartment reaching rapid equilibrium with the plasma. Folinic acid administration did not influence the disposition of either compound. Following the infusion, a significant (P less than 0.01) decrease of methotrexate total plasmatic clearance occurred without modification of 7-hydroxy-methotrexate formation and elimination.

Topics: Adolescent; Adult; Aged; Female; Humans; Infusions, Intravenous; Injections, Intravenous; Leucovorin; Male; Methotrexate; Middle Aged; Models, Biological; Time Factors

Methotrexate (MTX), an antifolate, is the anchor drug for the treatment of rheumatoid arthritis (RA). It is inexpensive, effective, and generally safe. When clinical response is inadequate, biological therapies are commonly used in combination with MTX. However, biological agents have safety concerns (i.e. infections, malignancy) and the addition of a biologic agent is expensive, making strategies to improve MTX efficacy important. Inhibition of pathways of folate metabolism involving purine metabolism by MTX, have been traditionally emphasized as important in MTX efficacy. However, inhibition MTX catabolism may also be important. MTX is irreversibly hydroxylated to form 7-hydroxy methotrexate (7-OH-MTX) by aldehyde oxidase (EC 1.2.3.1) (AOX). Catabolism of MTX to 7-OH-MTX is the first metabolic process imposed on an oral dose of MTX and will alter subsequent interactions of MTX with other enzymes. 7-OH-MTX is less potent than MTX in the treatment of rat adjuvant arthritis. RA patients with a low capacity to catabolize MTX to 7-OH-MTX do better clinically than individuals who are rapid formers of 7-OH-MTX. Therefore, altering the catabolism of MTX may be an innovative way to improve MTX efficacy. Raloxifene is a FDA-approved therapy for postmenopausal osteoporosis and for the reduction of invasive breast cancers but has no known activity in RA. Raloxifene is a potent inhibitor of human liver AOX. Postmenopausal women with RA frequently have low bone mineral density and would be candidates for raloxifene and MTX combination therapy. The effect of raloxifene on MTX metabolism has never been studied. Our hypothesis is that in postmenopausal women with RA and osteoporosis treated with MTX and raloxifene, the inhibition of AOX with resultant decreased formation of 7-OH MTX; will increase MTX levels and improve MTX efficacy. This hypothesis could be studied in an open-label, proof of concept clinical study in individuals before and after the addition of raloxifene. Red blood cell MTX and 7-OH-MTX levels and RA disease activity (DAS28) would be measured. In possible future studies, there are dietary substances, as supplements, (e.g. epigallocatechin gallate in green tea and resveratrol) which inhibit human liver AOX which could be evaluated.

Topics: Animals; Antirheumatic Agents; Arthritis, Rheumatoid; Biological Products; Drug Therapy, Combination; Female; Folic Acid; Folic Acid Antagonists; Humans; Leucovorin; Male; Methotrexate; Osteoporosis; Purines; Raloxifene Hydrochloride; Rats; Rats, Inbred Lew; Treatment Outcome

To assess the catabolism of methotrexate (MTX) to 7-hydroxy-MTX (7-OH-MTX) in patients with rheumatoid arthritis as well as the effect of folic acid and folinic acid on this catabolism.. Urinary excretion of MTX and its catabolite, 7-OH-MTX, was measured in 2 24-hour urine specimens collected after MTX therapy. Urine samples were collected from patients after the sixth and seventh weekly doses of MTX. MTX and 7-OH-MTX concentrations were determined by high-performance liquid chromatography mass spectrometry. Swelling and pain/tenderness indices were used to measure symptoms before and at 6 and 7 weeks of therapy. Patients received either folic acid or folinic acid supplements (1 mg/day) from week 6 to week 7.. Folic acid inhibited aldehyde oxidase (AO), the enzyme that produces 7-OH-MTX, but folinic acid did not. Excretion of 7-OH-MTX (determined as a percentage of the dose of MTX or as mg 7-OH-MTX/gm creatinine) was not normally distributed (n=39). Patients with marked improvement in swelling and pain/tenderness indices had a lower mean 7-OH-MTX excretion level (P<0.05). Patients who received folic acid supplements had decreased 7-OH-MTX excretion (P=0.03). Relatively high 7-OH-MTX excretion was correlated with relatively high MTX excretion and with relatively low MTX retention in vivo (P<0.05) (n=35).. Our findings of a non-normal distribution of 7-OH-MTX excretion suggest that there are at least 2 phenotypes for this catabolism. Decreased 7-OH-MTX formation suggests folic acid inhibition of AO and a better clinical response, while increased 7-OH-MTX formation may interfere with MTX polyglutamylation and binding to enzymes and, therefore, may increase MTX excretion and decrease MTX retention and efficacy in vivo.

Topics: Aldehyde Oxidase; Arthritis, Rheumatoid; Chromatography, High Pressure Liquid; Folic Acid; Health Status; Humans; Joints; Leucovorin; Methotrexate; Pain; Tandem Mass Spectrometry; Treatment Outcome

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.

Topics: Calibration; Child; Chromatography, High Pressure Liquid; Folic Acid Antagonists; Humans; Leucovorin; Methotrexate; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Specimen Handling; Tetrahydrofolates

The pharmacokinetics of methotrexate (MTX), 7-hydroxymethotrexate (7-OHMTX), 2,4-diaminomethylpteroic acid (APA), folinic acid, and 5-methyltetrahydrofolate (5-MTHF) have been studied during 21 high-dose MTX (HDMTX) infusions (5 g.m-2 in 24 h) with leucovorin (LCV) rescue, a component of the therapy of 5 children with acute lymphoblastic leukemia (ALL). The median steady-state concentration of MTX was 66 mumol.l-1. Three elimination half-lifes were determined for MTX: 1.8 h, 6.4 h and a terminal 15 h. The median systemic MTX clearance was 110 mg.m-2.min-1. The 7-OHMTX level increased during each infusion and a Cmax of 19 mumol.l-1 was achieved at the end. Its initial half-life was 5 h and the terminal half-life was 12 h. Thus, the peak serum concentration ratio of 7-OHMTX to MTX was reached 24 h after the end of the infusion at a median ratio of 8. The MTX metabolite APA was detected in concentrations less than 0.06 mumol.l-1. The median folinic acid level during rescue, 48 h after starting the infusion, was 7.0 mumol.l-1 and 18 h following the last dose of LCV it was 0.44 mumol.l-1, leading to ratios of folinic acid to MTX of 31 and 6, respectively. The median 5-MTHF level during rescue was 0.44 mumol.l-1 with a median ratio of 5-MTHF to MTX of 2. Twenty infusions with 48 h MTX levels of less than 0.5 mumol.l-1 were without marked toxicity. Only one patient with a 48 h MTX concentration of 5.5 mumol.l-1 and a ratio of 5-MTHF to MTX of 0.08 suffered from ulcerating mucositis and septicaemia despite increased and prolonged LCV rescue.

Topics: Child; Child, Preschool; Chromatography, High Pressure Liquid; Half-Life; Humans; Leucovorin; Metabolic Clearance Rate; Methotrexate; Precursor Cell Lymphoblastic Leukemia-Lymphoma

This paper describes studies that further explore the pharmacologic activity of the 7-hydroxy catabolite of methotrexate (7-OH-MTX). A 3-hr exposure of L1210 leukemia cells to 100 microM 7-OH-MTX produced negligible suppression of cell growth despite the build-up of intracellular polyglutamyl congeners to levels 2.7 times greater than the dihydrofolate reductase (DHFR) binding capacity. There was no evidence for direct inhibition of DHFR under these conditions based upon measurements of cellular tetrahydrofolate cofactor and dihydrofolate levels, nor was there suppression of [3H]deoxyuridine incorporation into DNA or [14C]formate incorporation into purines. When the interval of exposure to 100 microM 7-OH-MTX was increased to 6 hr, cell growth was inhibited by 60% and there was mild (approximately 50%) inhibition of purine and thymidylate biosynthesis associated with a small increase in cellular dihydrofolate and a small decline in cellular tetrahydrofolates. Consistent with weak inhibition of DHFR was the absence of significant binding of 7-OH-MTX polyglutamates to DHFR as assessed by gel filtration of cell extracts. Mild direct inhibition of purine biosynthetics by 7-OH-MTX- or MTX-polyglutamyl congeners was demonstrated based upon inhibition of [14C]formate incorporation into purines in cells pretreated with fluorodeoxyuridine so as to prevent tetrahydrofolate cofactor depletion or dihydrofolate polyglutamate build-up. Effects of a 6-hr exposure of cells to 100 microM 7-OH MTX on cell growth were reversed completely by 10 microM leucovorin; effects on cells containing comparable levels of MTX polyglutamyl congeners were unaffected by leucovorin. These studies demonstrate very weak inhibition of L1210 leukemia cell growth and purine, pyrimidine and tetrahydrofolate synthesis by the polyglutamyl congeners of 7-OH-MTX. The data suggest that effects of 7-OH-MTX polyglutamates on folate-requiring enzymes are not likely to play an important role in moderate-dose MTX regimens. However, pharmacologic activity may be expressed in high-dose MTX protocols when high blood levels of 7-OH-MTX are sustained over long intervals to the extent to which polyglutamate congeners accumulate in tumor cells and add to the much more potent inhibitory effects of MTX polyglutamates already present. Pharmacologic activity, however, would be diminished, if not completely reversed, by the concurrent administration of leucovorin.

Topics: Animals; Deoxyuridine; Folic Acid; Folic Acid Antagonists; Formates; Leucovorin; Leukemia L1210; Methotrexate; Mice; Polyglutamic Acid; Tumor Cells, Cultured

A high-performance liquid chromatographic method for the determination of N5-methyltetrahydrofolic acid, leucovorin, methotrexate and 7-hydroxymethotrexate in plasma and liquor samples is presented. Gradient elution is used to increase the sensitivity. Four sample preparation methods were compared with respect to the stability of the injectable sample. Samples can be pretreated with a simple deproteinization method. For enhanced selectivity a solid-phase extraction procedure is described.

Topics: Chromatography, High Pressure Liquid; Humans; Leucovorin; Methotrexate; Spectrophotometry, Ultraviolet; Tetrahydrofolates

HPLC analysis of plasma samples obtained from patients included in a high-dose methotrexate-folinic acid Rescue (HD-MTX-CF) protocol, allowed the simultaneous determination of MTX, CF and their respective plasma metabolites, 7-hydroxymethotrexate (7-OH-MTX) and 5-methyltetrahydrofolate (5-CH3-FH4). These 4 compounds interact at the cellular level to ensure the selective effectiveness of the HD-MTX-CF rescue protocol. An in vitro study has been investigated in RAJI cells to better describe the interference of CF on uptake, accumulation and metabolism of [3H]7-OH-MTX. Results about uptake and accumulation of CF were also obtained using [3H]CF, in the absence or the presence of unlabeled 7-OH-MTX. The rate of [3H]7-OH-MTX influx in RAJI cells (Km = 25.30 +/- 7.75 microM, n = 3) was competitively inhibited by the presence of 10 microM CF with a Ki value of 6.00 +/- 1.94 microM (n = 2). Intracellular 7-OH-MTX accumulation was decreased by approximately 30% when extracellular CF concentration was twice as high as that of 7-OH-MTX, and 70% when CF extracellular concentration was 5 times higher. The metabolism of 7-OH-MTX to its intracellular polyglutamyl derivatives was depressed by 90% when 10 microM CF were incubated for 2 h with equimolar [3H]7-OH-MTX, and it was completely abolished in the presence of 100 microM CF.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Biological Transport; Cells, Cultured; Dose-Response Relationship, Drug; Drug Interactions; Humans; Kinetics; Leucovorin; Methotrexate

A reversed-phase HPLC method is described for the simultaneous determination of folinic acid, MTX, and their plasma metabolites 5-CH3-FH4 and 7-OH-MTX respectively. In addition, this technique allows the separation of FA another naturally occurring folate, and of AMT, used as internal standard. Separation of these compounds was achieved on a Waters Spherical C18 column at a flow rate of 0.8 ml.min-1. Elution was carried out with 0.1 M sodium acetate buffer (pH 5.5) as solvent A and 7.5% acetonitrile 92.5% bidistilled water as solvent B. UV detection was performed at 280 nm. This method was applied in a pharmacokinetic study of folinic acid and its plasma metabolite 5-CH3-FH4 following two different protocols: (1) i.v. bolus injection of 50 mg calcium folinate in six healthy volunteers and (2) simultaneous i.v. bolus injections of 50 mg/m2 MTX and 50 mg/m2 folinic acid in four cancer patients. Mean apparent half-life values for folinic acid and its metabolite were 7.02 +/- 1.81 h and 3.90 +/- 0.86 respectively in the first protocol, 4.80 +/- 1.48 h and 4.74 +/- 1.47 h in the second protocol. MTX and 7-OH-MTX were also quantified in the second protocol and were found not to affect the pharmacokinetics of folinic acid and 5-CH3-FH4. Since in vitro studies on metabolism of folinic acid might be of great interest in trying to assess the mechanism of action of the folates and the potential interaction of MTX and 7-OH-MTX in this mechanism via the metabolism, the chromatographic method we describe here has been adapted for the separation of all the potential intracellular monoglutamyl metabolites of folinic acid.

Topics: Adult; Aged; Chromatography, High Pressure Liquid; Female; Half-Life; Humans; Kinetics; Leucovorin; Male; Methotrexate; Middle Aged; Neoplasms; Tetrahydrofolates

Methotrexate was administered by IV infusion, 2g (1.19 +/- 0.05 g/m2) over 24 hours, to a homogeneous group of patients undergoing treatment for breast cancer. Three courses were given at three week intervals. Methotrexate and 7-hydroxy-methotrexate plasma and urine pharmacokinetics were investigated. The average terminal half-lives of methotrexate and 7-hydroxy-methotrexate in plasma were 15.02 and 15.19 hours respectively. The area under concentration-time curve was 723.8 +/- 196.4 microM x h for methotrexate and 598.1 +/- 212.5 microM x h for 7-hydroxy-methotrexate. The total average urinary excretions of methotrexate and 7-hydroxy-methotrexate over a 96 hour period were 52% and 5.4% respectively. Urinary clearance of methotrexate was 3.46 +/- 1.4 1/h. In contrast, urinary excretion of 7-hydroxy-methotrexate was not linear. These results confirm the protein binding of metabolite to serum albumin and may suggest that distribution of 7-hydroxy-methotrexate is different from unchanged drug or that the metabolite can be eliminated by another route, such as bile.

Topics: Aged; Biotransformation; Breast Neoplasms; Female; Humans; Infusions, Intravenous; Leucovorin; Methotrexate; Middle Aged

The cytotoxicity of 7-hydroxymethotrexate (7-OH-MTX), the primary plasma metabolite of methotrexate (MTX) in humans, was assessed by inhibition of colony formation in agar, using human bone marrow granulocyte-macrophage stem cells (CFU) from healthy volunteers and RAJI cells, a human Burkitt's lymphoma cell line. After a 2 hr exposure of cells to 7-OH-MTX, the concentrations necessary to produce a 50% inhibition of colony formation were 180 microM and 10 microM for bone marrow cells and for RAJI cells respectively. A continuous incubation with 20 microM folinic acid (CF) protected the RAJI cells from 7-OH-MTX cytotoxicity at concentrations below 5 microM but was not able to completely reverse 7-OH-MTX effects at higher doses. Continuous incubation of 7-OH-MTX-preloaded cells (2 hr, ID90) with the end products of folate-dependent reactions, adenosine (100 microM) and thymidine (10 microM), completely rescued RAJI cells from the 7-OH-MTX cytotoxic effects. Moreover, while thymidine alone had no effect on the 7-OH-MTX response curve, both adenosine alone or CF-adenosine combination produced 75% and 90% protection respectively. CF and adenosine concentrations necessary to achieve 90% protection were 20 and 100 microM respectively. This study demonstrates that 7-OH-MTX can exhibit a cytotoxic selectivity for this human Burkitt's lymphoma cell line as compared to human bone marrow stem cells and the cytotoxicity of 7-OH-MTX cannot be reversed by CF alone. These data suggest that 7-OH-MTX and/or its polyglutamylated derivatives may play an important role on different enzyme(s) involved in the interconversion of tetrahydrofolate cofactors necessary for the de novo purine biosynthesis.

Topics: Burkitt Lymphoma; Cell Line; Colony-Forming Units Assay; Folic Acid Antagonists; Granulocytes; Hematopoietic Stem Cells; Humans; Leucovorin; Methotrexate

Kinetics of 7-hydroxy-methotrexate (7-OH-MTX) excretion after high-dose methotrexate (MTX) (12 g/m2) therapy were monitored in 93 consecutive drug cycles of 19 adolescent patients with osteosarcoma. A reversed-phase HPLC method was used. Serum elimination was found to be monophasic with a mean half-life of 5.5 h. Shortly after the 4-h MTX infusion period 7-OH-MTX levels exceeded those of the parent compound. By 12 h after MTX infusion 7-OH-MTX levels were 16.5 times higher than those of MTX itself. Autostimulation of MTX metabolism leading to enhanced 7-OH-MTX production after repeated drug cycles was not observed. The production of 7-OH-MTX decreased significantly from the first to the last high-dose MTX cycle of the adjuvant chemotherapy protocol.

Topics: Adolescent; Adult; Alanine Transaminase; Antineoplastic Combined Chemotherapy Protocols; Aspartate Aminotransferases; Child; Humans; Kinetics; Leucovorin; Methotrexate; Osteosarcoma; Retrospective Studies

9 patients with osteosarcoma were treated with a total of 122 infusions of high-dose methotrexate (MTX; 140-350 mg/kg) followed by leucovorin rescue. Plasma kinetics of MTX and 7-hydroxymethotrexate (7-OH-MTX) has been routinely monitored. Due to inadequate hydration and alkalinization, 1 of the 122 high-dose MTX infusions was followed by delayed disappearance of MTX and 7-OH-MTX from plasma with subsequent development of severe mucositis. Serious hepatotoxicity repeatedly developed in another patient with inconspicuous MTX kinetics. The benefit of monotherapy with high-dose MTX for adjuvant treatment of osteosarcoma remains questionable, since 6 of 8 patients with primary osteosarcoma developed pulmonary metastases within 4-12 months (median 5 months), 2 have been disease-free and alive for 25 and 53 months.

Topics: Adolescent; Adult; Bone Neoplasms; Chemical and Drug Induced Liver Injury; Female; Humans; Kinetics; Leucovorin; Male; Methotrexate; Middle Aged; Mucous Membrane; Osteosarcoma

Methotrexate (MTX) shows consistent cytotoxicity for melanoma cells in vitro but it is ineffective in clinical use at equivalent concentrations in vivo. This apparent paradox has been investigated by cell culture techniques and results quantified by cell number. In an in vitro model of high dose MTX therapy followed by leucovorin rescue (HD-MTX-LCR) there was survival of both melanoma and choriocarcinoma cell lines but not of an acute lymphocytic leukaemia cell line. The 70H metabolite of MTX was identified by HPLC in plasma samples of melanoma patients treated by HD-MTX-LCR, in which MTX concentrations approximately 10(-5) M were maintained for 24 h. However, metabolism per se is unlikely to account for the lack of response to MTX clinically. In vitro 70H MTX (10(-7) - 10(-6) M) was two orders of magnitude less cytotoxic for melanoma than MTX (10(-9) - 10(-8) M). The cellular accumulation of [3H]-MTX, using a rapid gradient centrifuge technique for separation of melanoma cells from medium, was reduced in the presence of 70H-MTX. The results suggest that reduced cellular uptake of MTX combined with biochemical rescue of tumour cells may partially explain the paradoxical lack of clinical response of melanoma to the drug.

Topics: Animals; Cell Count; Cell Line; Cell Survival; Choriocarcinoma; Humans; Leucovorin; Leukemia, Lymphoid; Male; Melanoma; Methotrexate; Mice

Topics: Child; Chromatography, High Pressure Liquid; Drug Therapy, Combination; Humans; Leucovorin; Leukemia, Lymphoid; Methotrexate