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PubMed1998

Accumulation of methotrexate in human tissues following high-dose methotrexate therapy.

Methotrexate concentration was analyzed in a number of tissues of a patient of osteogenic sarcoma who had been on high-dose methotrexate therapy for nearly 6 months. Gall bladder and kidney contained the highest concentration of the drug, followed by testis, small intestine, skeletal muscle, bone marrow, lung, spleen, heart and liver. Although, compared to kidney the liver contained relatively small amount of the drug, yet nearly 1/5th of the total drug in liver was in bound form. This bound form of methotrexate is most likely associated with multiple forms of dihydrofolate reductase. The total concentration of methotrexate in kidney is 80 fold higher than the concentration of the drug in liver and 28 fold higher than the concentration in bone marrow. This suggests that in high-dose methotrexate therapy, nephrotoxicity is the more immediate threat to the patient than hepatotoxicity and bone marrow suppression.

Adolescent↗

PubMed1992

Characterization of transport-mediated methotrexate resistance in human tumor cells with antibodies to the membrane carrier for methotrexate and tetrahydrofolate cofactors.

An earlier report (Matherly, L. H., Czajkowski, C. A., and Angeles, S. M. (1991) Cancer Res. 51, 3420-3426) described a K562 human erythroleukemia line (K562.4CF), characterized by an elevated uptake capacity for methotrexate (MTX) and 5-formyltetrahydrofolate, and the identification of a highly glycosylated membrane transporter (GP-MTX) by radioaffinity labeling with N-hydroxysuccinimide [3H] methotrexate. In the present study, radioaffinity-labeled GP-MTX from K562.4CF cells was isolated by Ricinus communis agglutinin I-agarose affinity chromatography, coupled with gel filtration and preparative electrophoresis. Antiserum to the purified, radio-labeled protein was raised in a rabbit and screened by immunoblot analysis of K562.4CF plasma membrane proteins or purified GP-MTX. The antiserum detected a broad GP-MTX band centered at 92 kDa on 7.5% gels. On 4-10% gels, the apparent molecular mass for GP-MTX shifted to 99 kDa. Antiserum specificity was established by quantitatively converting the immunoreactive glycoprotein in plasma membrane homogenates to its N- and O-deglycosylated forms with N- and O-glycanases, respectively. Whereas the methotrexate uptake capacity of K562.4CF cells was elevated 6.1-fold over parental cells, the GP-MTX content on immunoblots was increased approximately 3-fold. For two methotrexate-resistant K562 lines (33- and 70-fold), decreased drug uptake (28 and 18% of parental levels) closely correlated with their reduced GP-MTX contents. A GP-MTX isoform was also detected on immunoblots of membrane proteins from CCRF-CEM human lymphoblastic leukemia cells. With a transport-impaired CCRF-CEM line (13% of wild type uptake), an aberrant electrophoretic migration for GP-MTX was observed, establishing the presence of structural modifications in the transport protein. These structural differences were independent of carrier glycosylation since they were detected following the glycosidase treatments. These findings implicate important roles for distinct carrier-specific alterations in the expression of diminished drug transport in methotrexate-resistant human tumor cells.

ATP Binding Cassette Transporter, Subfamily G, Mem↗

PubMed1988

Methotrexate suppresses passive adjuvant arthritis: studies on the metabolism of methotrexate in mononuclear cells derived from normal and adjuvant arthritic rats.

Passive transfer of adjuvant arthritis by spleen cells is suppressed by methotrexate. Mononuclear cells derived from spleens of normal and adjuvant arthritic Lewis rats were incubated with [3H]-methotrexate and harvested at various periods of time. The amount of methotrexate and its various polyglutamates were quantitated. The results of these studies indicate that the kinetics of uptake of methotrexate by mononuclear cells from normal and adjuvant arthritic rats are similar. However, the amount of methotrexate polyglutamates accumulating in the mononuclear cells of adjuvant arthritic rats was significantly lower than that observed in mononuclear cells derived from normal rats.

Animals↗

PubMed1982

[Experimental study on pharmacokinetics of methotrexate and its metabolite (7-hydroxy-methotrexate) in tissue].

The metabolism of methotrexate was investigated in rabbits with six hours infusion of (3'5'7-3H) methotrexate (50mg/kg). Methotrexate and its metabolites were analyzed by reverse-phase high-pressure liquid chromatography and by counting of radioactivity. 7-Hydroxymethotrexate was found to be the major metabolite in the plasma, urine, bile and tissues of rabbits. Cumulative production of 7-Hydroxymethotrexate during the first six hours was 31.8% of the total dose. The 7-Hydroxymethotrexate concentration in plasma was reached to peak level in six hours after the initiation of the infusion, and was declined slower than that of methotrexate. The highest total radioactivity was found in the kidney after six hours, which was 7.24 times higher than that in plasma. Ratio of 7-Hydroxymethotrexate/methotrexate increased in the liver, small intestine, lung, kidney and testis. This data suggested that conversion of methotrexate to 7-hydroxymethotrexate took place in lung and kidney as well as in liver.

Animals↗

PubMed1999

Comparing abortion induced with methotrexate and misoprostol to methotrexate alone.

This was a dual-cohort study performed to determine if, by using methotrexate alone, abortions could be induced with fewer side effects, without sacrificing effectiveness and patient satisfaction. The subjects were 358 consecutive patients who requested elective medical termination of pregnancy at < 7 weeks gestation. Women were given information and allowed to choose between methotrexate alone (50 mg/m2 intramuscularly) or methotrexate followed 4 days later by 800 micrograms misoprostol vaginally. The two groups were compared with respect to the number of surgical aspirations required, the number of days until the abortion was completed, side effects, and the number of women who would choose the same method again. The surgery rate for methotrexate was 17.2%, whereas for the combination it was 10.9%. The mean numbers of days until the abortion were complete was 23 and 12, respectively. Side effects, mean pain scores, total days of bleeding, and satisfaction rates were not significantly different in the two groups. The high failure rate with methotreaxate alone suggests that it should not be offered despite the high level of satisfaction.

Abortifacient Agents, Nonsteroidal↗

PubMed1993

Plasma methotrexate levels in patients receiving etoposide, methotrexate, actinomycin, cyclophosphamide, and vincristine (EMA-CO) chemotherapy for trophoblastic neoplasia.

Methotrexate serum levels were measured in eight patients receiving etoposide, methotrexate, actinomycin, cyclophosphamide, and oncovin (EMA-CO) chemotherapy for high-risk and actinomycin-resistant gestational trophoblastic neoplasia. During 41 courses of EMA there were only 3 instances where the level of methotrexate was greater than 9 x 10(-7) M/liter at 24 hr postinfusion. A level higher than this may be associated with subsequent toxicity. Use of postmethotrexate leucovorin rescue should continue, but routine measurement of methotrexate with EMA-CO is unnecessary unless there is compromise of renal or hepatic function.

Antineoplastic Combined Chemotherapy Protocols↗

PubMed1988

Methotrexate and its polyglutamate derivatives in erythrocytes during and after weekly low-dose oral methotrexate therapy of children with acute lymphoblastic leukemia.

Methotrexate and methotrexate polyglutamates were quantitatively determined in red blood cells from 12 children with acute lymphoblastic leukemia who were treated with MTX (15-20 mg/m2 per week) and daily 6-mercaptopurine orally during the steady-state period of erythrocyte MTX concentration (ery-MTX). The terminal decline of the ery-MTX and its polyglutamate metabolites were determined for up to 15 weeks after cessation of MTX treatment. Methotrexate polyglutamates with 2-4 extra glutamyl derivatives (MTX-glu2-5) constituted 75% of the MTX in the entire red blood cell population. MTX-glu3 was the principal metabolite; no MTX-glu6-7 was identified. After discontinuation of MTX therapy, the ery-MTX declined in a non-linear manner because of different half-lives for the individual polyglutamates. From about 5 weeks until 13-15 weeks after the last MTX dose, the erythrocyte MTX elimination curve was linear. The approximate half-life of MTX-glu1 was 2-3 days; for MTX-glu2 it was 4-15 days. The concentration of MTX-glu3-5 remained constant in the erythrocyte throughout its life span and declined only with age-dependent destruction of the red blood cell. It was calculated that 80%-90% of MTX in newly formed reticulocytes was MTX-glu1+2, the remainder being MTX-glu3-5. Mature red blood cells did not form methotrexate polyglutamates to any significant degree. There was a significant correlation between the amount of MTX-glu3-5 and the steady -state ery-MTX, which to some extent explained the interindividual variation of the ery-MTX in children with ALL in maintenance therapy.

Administration, Oral↗

PubMed1982

Effect of combination chemotherapy, duration of methotrexate administration, and patient's age on methotrexate pharmacokinetics.

The kinetics of methotrexate were studied in a group of 59 patients after infusion of high doses. From the triexponential analysis of the serum concentration curve, rate constants and relative concentrations in non-circulating pools were calculated, using a linear 'mammillary' three-compartment model. No significant variation as a function of dose (300-6,000 mg/m2) was observed. In circumstances the rapidly exchanging pool was a reflection of the circulating pool, suggesting that it is an extravascular, protein-bound pool of methotrexate. Considerable variations of the relative concentration and rate constants of the slowly exchanging pool were observed to be a function of the duration of methotrexate infusion, the presence of other drugs, and the patient's age. If this compartment is assumed to include the target cells for methotrexate, then the large variations observed could be responsible for individual differences in toxicity and/or effectiveness.

Acute Disease↗

PubMed1984

The use of a fluorescent methotrexate probe to monitor the effects of three vinca alkaloids on a mixed population of parental L1210 and gene-amplified methotrexate-resistant cells by flow cytometry.

Cells resistant to methotrexate (L1210/R7A) and possessing an increased level of dihydrofolate reductase due to gene amplification can be detected by the technique of flow cytofluorimetry using a new fluorescent derivative of methotrexate (F-MTX) based on a putrescine linker. Comparative studies of dihydrofolate reductase enzyme and cell growth inhibition following treatment with methotrexate and F-MTX suggest that the two agents possess similar modes of action. In an artificially mixed population of cells sensitive and resistant to methotrexate it is possible, using F-MTX, to recognise and separate distinct cell subpopulations showing differential fluorescence using a fluorescence-activated cell sorter (FACS IV). The selective removal of the resistant cells within a mixed population of sensitive and resistant cells has been demonstrated for 5 X 10(-8) M vinblastine by means of flow cytometry. The effectiveness of the vinca alkaloids decreases in the order vinblastine greater than vindesine greater than vincristine, which previously was shown to be the order of effectiveness in producing collateral sensitivity.

Animals↗

PubMed1983

Irreversible inhibitors of methotrexate transport in L1210 cells. Characteristics of inhibition by an N-hydroxysuccinimide ester of methotrexate.

Methotrexate, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide react to form an activated ester of methotrexate which is a potent irreversible inhibitor of methotrexate transport in L1210 cells. In cells treated with the reagent at 37 degrees C, inhibition was rapid (t1/2 less than 1 min), optimal at pH 6.8, half-maximal at an inhibitor concentration of 20 nM, and complete at high levels of the reagent. Specificity was indicated by the fact that excess methotrexate added during the pretreatment step protected the transport system against inactivation. Irreversible inhibition was also observed in cells exposed to the reagent at 4 degrees C. Inactivation in this case was qualitatively similar to the corresponding process at 37 degrees C; it appeared rapidly, was half-maximal at 20 nM, and could be prevented by the addition of high concentrations of the substrate. The extent of the inhibition, however, reached a maximum of only 75%, even in samples containing excess or multiple additions of reagent. The latter findings suggest that at 4 degrees C the transport protein exists in two forms, one (75% of the total) containing binding sites which are accessible to the active ester, and the other (25% of the total) with inaccessible sites. The identity of these sites is suggested to be transport proteins which have outward and inward orientations, respectively.

Animals↗

PubMed2005

Mucositis after allogeneic hematopoietic stem cell transplantation: a cohort study of methotrexate- and non-methotrexate-containing graft-versus-host disease prophylaxis regimens.

Oral mucositis occurs in up to 75% of recipients of high-dose chemoradiotherapy conditioning regimens used for allogeneic hematopoietic stem cell transplantation (HSCT). As a result of mucositis, narcotic analgesia and total parenteral nutrition (TPN) are commonly required after HSCT. Methotrexate, an antiproliferative graft-versus-host disease (GVHD) prophylaxis agent, impairs mucosal regeneration and worsens and prolongs mucositis. We assessed the effect of substituting sirolimus for methotrexate as GVHD prophylaxis on outcomes associated with mucositis. Two patient cohorts undergoing allogeneic HLA-matched related donor peripheral blood stem cell transplantation with cyclophosphamide/total body irradiation conditioning were prospectively analyzed for mucositis severity and retrospectively reviewed for correlative outcomes. GVHD prophylaxis consisted of sirolimus/tacrolimus (ST) in the study group and tacrolimus/methotrexate (TM) in the control group. Thirty patients received ST and 24 patients received TM as GVHD prophylaxis between October 2000 and May 2003. Mild, moderate, and severe mucositis was noted in 37%, 57%, and 7% of the ST group and 8%, 42%, and 50% of the TM group (P = .0002). Less TPN was used in the ST group than the TM group (17% versus 43% of posttransplantation hospital days; P = .02). The total number of narcotic days was lower in the ST group in comparison with the TM group (median, 13.5 versus 17 days; P = .08). The time to first hospital discharge was shorter in the ST group compared with the TM group (median, 18 versus 22 days; P = .07). The substitution of sirolimus for methotrexate as GVHD prophylaxis is associated with a reduction in mucositis severity. As a result, TPN and narcotic use are reduced, and hospitalization duration is shortened. Less toxic GVHD prophylaxis regimens without methotrexate may have a significant effect on patient quality of life, patient outcomes, and economic outcomes associated with allogeneic stem cell transplantation.

Adult↗

PubMed1995

Isolation of human cDNAs that restore methotrexate sensitivity and reduced folate carrier activity in methotrexate transport-defective Chinese hamster ovary cells.

This report describes the isolation, nucleotide sequencing, and functional expression of human cDNAs that restore reduced folate carrier activity in transport-defective cells. Based on homology to a partial murine cDNA probe, two functional cDNAs were isolated from a lambda gt11 library prepared from methotrexate transport upregulated K562 cells (K562.4CF). A 2.8-kilobase (kb) clone, KS43, contained a 1776-base pair open reading frame. The 2.5-kb clone, KS32, contained an internal deletion (626 base pairs) resulting a shortened open reading frame and 3'-untranslated region. KS43 and KS32 encoded proteins with multiple hydrophobic domains, one consensus N-glycosylation site, and predicted molecular masses of 65 and 58 kDa, respectively. The deduced amino acid sequence of KS43 is 79% and 80% homologous to the mouse and hamster sequences, respectively (Dixon, K. H., Lanpher, B. C., Chiu, J., Kelley, K., and Cowan, K. H. (1994) J. Biol. Chem. 269, 17-20; Williams, F. M. R., Murray R. C., Underhill, T. M., and Flintoff, W. F. (1994) J. Biol. Chem. 269, 5810-5816). Northern blots identified one primary transcript at 3.1 kb in parental K562, K562.4CF, and transport-impaired K500E cells; transcript levels varied by 7-fold. The expression of both KS43 and KS32 in methotrexate transport-defective Chinese hamster ovary cells restored methotrexate sensitivity and transport. Certain transport characteristics of the transfected cells resembled both the wild type human (K562) and hamster "classical" reduced folate carriers, suggesting the expression of a hybrid system. For instance, based on Ki values, up to a 4-fold increased affinity for 1843U89 over wild type hamster cells (typical of human cells), and a 19-fold increased affinity for methotrexate over K562 cells (typical of hamster cells) was observed. Further, a photoaffinity probe with high specificity for the reduced folate carrier labeled 94-kDa proteins in K562 cells and the transfectant containing the full-length KS43, and a 85-kDa protein in the transfectant containing the 3'-truncated KS32. No specifically labeled proteins were detected in wild type or mock-transfected hamster cells. Collectively, our results suggest that the KS43/KS32 cDNAs encode the human reduced folate carrier; however, additional modulatory/regulatory factors may be required to manifest the full spectrum of transport substrate activities typical of this system.

Amino Acid Sequence↗

PubMed2004

Methotrexate and MX-68, a new derivative of methotrexate, limit infarct size via adenosine-dependent mechanisms in canine hearts.

Methotrexate, an anti-rheumatic agent, has recently been reported to show an anti-inflammatory action via ecto-5'-nucleotidase- and adenosine-dependent mechanisms. Because ecto-5'-nucleotidase contributes to the production of adenosine and adenosine has a potent cardioprotective effect against ischemia/reperfusion injury, we investigated whether methotrexate or MX-68 [N-1-((2,4-diamino-6-pteridinyl) methyl)-3,4-dihydro-2H-1,4-benzothiazine-7- carbonyl]-N-2- aminoadipic acid] could reduce infarct size via adenosine-dependent mechanisms. In beagle dogs, the left anterior descending coronary artery was perfused through a bypass tube, which was occluded for 90 minutes followed by 6 hours of reperfusion. The size of infarcts was assessed by TTC staining. MX-68 reduced infarct size compared with that in untreated dogs (13.7 +/- 1.9 versus 38.6 +/- 5.3%, P < 0.01). This effect was completely blunted by either the adenosine receptor antagonist 8-sulfophenyltheophylline (8-SPT) (45.0 +/- 4.6% and 46.8 +/- 5.8% in the 8-SPT and MX-68 + 8-SPT groups, respectively) or by the ecto-5'-nucleotidase inhibitoralpha,beta-methylenadenosine 5'-diphosphate (AMP-CP) (44.0 +/- 4.5% and 46.7 +/- 5.8% in the AMP-CP and MX-68 + AMP-CP groups, respectively). Methotrexate also reduced infarct size to a level comparable with that in the MX-68 group, and its effect was also blunted by 8-SPT. There were no significant differences of collateral blood flow or risk area between the groups. We conclude that methotrexate and its derivative (MX-68) both limit infarct size via adenosine-dependent mechanisms.

2-Aminoadipic Acid↗

PubMed1987

Comparative biodistribution of methotrexate and monoclonal antibody-methotrexate complexes in mice.

The biodistribution of radiolabelled methotrexate and immune complexes of methotrexate and a murine monoclonal anti-methotrexate antibody has been compared in mice. Complexes formed in-vitro with the antibody, but not with control immunoglobulin. The complexes were, characteristically, acid labile. In-vivo, blood levels, organ distribution and whole body catabolism of methotrexate in immune complexes were similar to those of free antibody, and markedly different from those of free drug. These findings suggest the feasibility of prolonging the survival of drugs and altering in-vivo distribution using complexes with monoclonal antibodies.

Animals↗

PubMed1978

Direct administration of methotrexate into the central nervous system of primates. Part 2: Distribution of 3H methotrexate after intrathecal lumbar injection.

The kinetics of distribution of 3H methotrexate (3HMTX) in the central nervous system, plasma, and urine after intraventricular, lumbar percutaneous puncture, and spinal catheter injections were compared. Levels of 3HMTX in whole brain after lumbar percutaneous injection were 40 times less than after intraventricular injection. Injection of 3HMTX via a spinal catheter increased the level of 3HMTX in whole brain but this was still tenfold less than after direct intraventricular instillation. Also, it was found that a disproportionately high amount of 3HMTX was in the brain-stem-cerebellum region which would further reduce the concentration of methotrexate in the cerebral hemispheres. Both intraventricular and lumbar spinal catheter administration of 3HMTX produced 3HMTX levels greater than 10(-6)M (moles/kg wet weight) in spinal cord tissue as measured by 3H specific activity between 2 to 8 hours after injection. Administration by lumbar percutaneous puncture, however, rarely resulted in this suggested therapeutic level of 10(-6)M. Initial 3HMTX levels in plasma after lumbar percutaneous instillation was 24 times greater than after intraventricular or lumbar spinal catheter injections. This indicated significant and unavoidable extradural leakage after lumbar percutaneous puncture, which may account for the substantially lower levels of 3HMTX in the brain and spinal cord tissue. It is concluded that intraventricular instillation of methotrexate is the best route of administering the drug to achieve therapeutic levels of methotrexate in both whole brain and throughout the spinal cord.

Animals↗

PubMed1987

A randomized trial of high-dose methotrexate versus standard-dose methotrexate following cyclophosphamide, doxorubicin (adriamycin), vincristine, and prednisone with or without bleomycin in the therapy of diffuse large cell lymphoma: preliminary report of Cancer and Leukemia Group B Study 7851.

Between 1979 and 1985, 166 patients with diffuse large cell (histiocytic) lymphoma were randomized to receive therapy with 3 courses of cyclophosphamide, doxorubicin (Adriamycin), vincristine, and prednisone (CAVP), with or without low-dose bleomycin, by continuous iv infusion. Responders were further randomized to 3 weeks of therapy with either high-dose methotrexate (3 g/m2 iv weekly with leukovorin rescue) or low-dose methotrexate (30 mg/m2 orally weekly without rescue). Therapy was concluded with 3 additional courses of CAVP. No significant differences among the 4 treatment programs were observed in complete response rates (ranging from 46% to 51%) or in failure-free survival. Of the 38 relapses that have occurred in patients treated with low-dose methotrexate, 5 included relapse in the central nervous system in conjunction with systemic relapse. However, none of 31 relapses observed in patients receiving high-dose methotrexate have occurred with involvement of the central nervous system. Patients entering this study with "B" symptoms had significantly poorer treatment results than those patients entering study without "B" symptoms.

Antineoplastic Combined Chemotherapy Protocols↗

PubMed1984

Sequential methotrexate and 5-FU in CMFP (cyclophosphamide, methotrexate, 5-FU, and prednisone) therapy for breast cancer.

Forty-one women with advanced breast cancer were treated with cyclophosphamide, methotrexate, 5-FU, and prednisone. Methotrexate (200 mg/m2) was given iv 1 hour before administering 5-FU (600 mg/m2). Leucovorin (10 mg orally every 6 hours for six doses) was begun 24 hours after the methotrexate. The response rate was 43%, with 19% of patients having a clinically complete remission. This overall objective response rate is lower than that achieved when this drug combination is given at standard doses of methotrexate and with no interval before 5-FU. Toxicity was primarily related to bone marrow suppression; eight women had wbc counts that fell to less than 1500/mm3.

Adult↗

PubMed1975

Superiority of conventional intrathecal methotrexate therapy with maintenance over intensive intrathecal methotrexate therapy, unmaintained, or radiotherapy (2000-2500 rads tumor dose) in treatment for meningeal leukemia.

In the treatment of patients with meningeal leukemia, conventional intrathecal methotrexate therapy followed by maintenance intrathecal methotrexate at intervals of 8 weeks has been shown superior to an intensive, unmaintained intrathecal methotrexate induction regimen, and to neuroaxis radiotherapy, tumor dose 2000-2500 rads. In each of the treatment regimens, all children completing proscribed therapy achieved complete CNS remission as judged by the return of CSF findings to normal. Median lengths of remission were: maintenance regimen 240 days; intensive induction regimen, 106 days; and radiotherapy, 216 days. Differences were of statistical significance between the maintenance regimen and the intensive regimen (p equal 0.001), and between the radiotherapy and intensive regimens (p equal 0.01). Maintenance intrathecal therapy appeared to affect favorably the duration of existing marrow remissions; the median time to marrow relapse for patients given maintenance therapy was significantly longer than for those given intensive induction therapy. Toxicity of the chemotherapy regimens was not prohibitive. Radiotherapy was associated with severe myelosuppression, interruptions of systemic therapy, and serious infections, which resulted in death in five children.

Adolescent↗