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PubMed1988

Evidence for a functional defect in the translocation of the methotrexate transport carrier in a methotrexate-resistant murine L1210 leukemia cell line.

Properties of the methotrexate (MTX) transport carrier were examined in a stable single-step 16-fold MTX-resistant L1210 murine leukemia cell line with unchanged dihydrofolate reductase gene copy and thymidylate synthase and dihydrofolate reductase levels and activities. MTX influx was markedly depressed due to a decrease in Vmax without a change in Km. From this cell line a clonal variant with greater resistance to MTX was identified due solely to a further decrease in influx Vmax. Trans-stimulation of MTX influx by 5-formyltetrahydrofolate was induced in parental but not resistant cells. Analysis of specific MTX surface binding demonstrated a small increase in the number of carriers in the first- and second-step resistant lines. Affinity labeling of cells with an N-hydroxysuccinimide ester derivative of [3H]MTX demonstrated carriers with comparable molecular weights in the parent and second-step transport defective lines. In two partial revertants with increased MTX sensitivity isolated from the second-step resistant lines, MTX influx was increased but surface membrane-binding sites were unchanged suggesting that recovery of transport was due to normalization of carrier function rather than an increase in the number of carriers. These studies suggest that impaired MTX transport in these lines is not due to an alteration in the association of the transport carrier with its substrate at the cell surface. Rather, resistance may be due to an alteration in the mobility of the carrier possibly associated with a protein change in the carrier itself or the cell membrane that surrounds it.

Animals↗
PubMed1985

Clinical controlled trial in advanced breast cancer: CMF (cyclophosphamide, methotrexate, 5-fluorouracil) versus CMF + T (cyclophosphamide, methotrexate, 5-fluorouracil, tamoxifen). CMEA Cooperative Treatment Group.

A controlled study with two arms was performed in 153 evaluable patients with advanced breast cancer. All patients were postmenopausal and untreated. The first combination included cyclophosphamide, methotrexate, 5-fluorouracil (CMF, 82 patients) while the second one contained the same cytostatic combination and tamoxifen (zitazonium) (CMF + T, 71 patients). Patients were prospectively stratified according to the dominant sites of metastases and randomized to the respective treatment schedule. In CMF combination the response rate (CR + PR) was 33/82 (40%) and complete response (CR) 8/82 (10%), respectively. In the CMF + T arm the corresponding values were 29/71 (41%) and 14/71 (20%), respectively. From the analysis of the data the superiority of the CMF + T arm could be established concerning CR in soft tissue and CR + PR in bone localization.

Adult↗
PubMed1987

Comparison of methotrexate and sequential methotrexate-5-fluorouracil for patients with recurrent squamous cell carcinoma of the oral cavity.

Forty-eight fully assessable previously treated patients with biopsy-proven recurrent squamous cell carcinoma of the oral cavity were randomized to receive either methotrexate (MTX), 40 mg/m2 iv push weekly, or sequential MTX and 5-fluorouracil (5-FU) (MTX 150 mg/m2 iv for 1 h; 1 h after the end of MTX, 5-FU 600 mg/m2 iv for 2 h; 24 h later, leucovorin rescue 10 mg/m2 iv and the same dose was given orally every 6 h 4 times; the treatment was repeated every 10 days). There were 1 complete response (CR) and 5 partial responses (PR) in the MTX group; median remission duration = 84 days. There were 3 CR and 11 PR in the MTX-5-FU group (overall response 14/24, 58.3%--p less than 0.05); median remission duration = 125 days. Median survival was 6.2 months in the MTX group and 8.1 months in the MTX-5-FU group. There was no difference in mucositis between the two groups, and a prevalence of leukopenia and moderate gastro-intestinal toxicity in the MTX-5-FU group.

Adult↗
PubMed1987

Reductions in methotrexate and folate influx in methotrexate-resistant lines of Leishmania major are independent of R or H region amplification.

The methotrexate (MTX) and folate transport properties of five MTX-resistant lines of Leishmania major have been examined. These resistant lines all show a decreased Vmax for MTX influx, with no change in apparent affinity (Kt). The Vmax of folate influx is also proportionately decreased without alteration in Kt, supporting our proposal that there is a single carrier mediating influx of both ligands. Amplifications of two regions of DNA, the R region (encoding dihydrofolate reductase-thymidylate synthase) and the H region (Beverley, S.M., Coderre, J.A., Santi, D.V., and Schimke, R.T. (1984) Cell 38, 431-439), were also observed. In a given line, the amplifications occurred singly, in combination, or not at all. No other regions of amplification were detected. The phenotype of reduced MTX transport was moderately stable in the highly resistant R1000 line, being retained for more than 200 generations in the absence of MTX in vitro and during one passage through an infected mouse; in contrast, R- and H-amplified DNA were less stable. The lack of correlation of R and H amplification with reduced MTX transport suggests that alterations in transport are not causally mediated by gene amplification in Leishmania, but are a separate mode of MTX resistance. The onset of decreased MTX transport was also examined; wild-type Leishmania developed a reduced Vmax of MTX influx within 24 h following exposure to 1 microM MTX, which is extremely unstable in the absence of drug pressure. A comparable decrease in the Vmax of influx is seen in cells exposed to MTX in media in which cytotoxicity is eliminated. As the folate/MTX transporter is regulated by exogenous folate, these data suggest that the initial rapid decrease in MTX transport may be a cellular regulatory response, in contrast to that found within the R1000 line which resembles a more stable genetic mutation.

Animals↗
PubMedDate not supplied

High-performance liquid chromatographic and enzyme-inhibition assays of methotrexate concentrations in serum from patients receiving high-dose therapy: tight binding of some methotrexate to a protein that could be dihydrofolate reductase.

In the serum of patients receiving high-dose methotrexate (MTX), concentrations of the drug were monitored using both high-performance liquid chromatographic (HPLC) and enzyme-inhibition assays. In samples obtained greater than or equal to 24 hours after drug application, the HPLC assay measured higher MTX concentrations than the enzyme-inhibition assay. In 72-hour samples, the increase was usually greater than 200%. This difference was not observed in serum spiked with MTX. While the HPLC assay needs sample cleanup, ie, protein precipitation, the enzyme-inhibition assay routinely employs native serum. When MTX was measured in the supernatant with the enzyme inhibition assay, the results equaled those obtained with the HPLC procedure. In patient serum, MTX eluted from a Sephadex G-75 column in two fractions. One had the same retention volume as free MTX and could be assayed directly. The other had a retention volume like dihydrofolate reductase. In this fraction, MTX could only be determined after denaturing proteins. Only small amounts of tightly bound MTX were found in the serum of patients on intermediate-dose therapy (500-600 mg/m2). The observation that after high-dose MTX therapy part of the MTX in patient serum is strongly bound to a protein which could be DHFR raises the question as to the source and fate of this complex. Furthermore, the present finding has clinical relevance to the extent that the accepted limits for risk of MTX toxicity are based on methods using native serum. However, any procedure employing protein precipitation for sample cleanup may grossly overestimate active MTX serum concentrations, especially in the 72-hour serum samples.

Adult↗
PubMed1977

Liver ultrastructure in psoriatics related to methotrexate therapy. 1. A prospective study of findings in hepatocytes from 24 patients before and after methotrexate treatment.

To show what damage occurs in the hepatocytes of psoriatics receiving Methotrexate (MTX) therapy liver biopsies from 24 psoriatics with severe psoriasis before and after MTX therapy were studied blind by light and electron microscopy. We also aimed to determine the severity of lesions and find possible correlations, and to seek a relationship of these observations with light microscopical and clinical findings. The present study has shown that MTX probably caused damage to the hepatocytes reflected in the membrane whorls (p less than 0.05) and the accumulation of lipid droplets (p less than 0.05). There was an increase (p less than 0.05) in autophagic vacuoles, which mostly contained glycogen and cell sap with residual bodies. These residual bodies were then found in an increased number in the nearby Kupffer cells. Crystals were found in megamitochondria in most patients before and after MTX therapy. Some of these crystals were found free in the cytoplasm. Mitochondria containing crystals were shown in autophagic vacoules, representing possible pathways of their breakdown. Bile canaliculi commonly contained debris but only one patient had evidence of cholestasis. There was no significant change in nuclei, Golgi apparatus, or the endoplasmic reticulum and no statistically significant correlation between the shown changes and the total dose of MTX given.

Cell Membrane↗
PubMed1977

Liver ultrastructure in psoriatics related to methotrexate therapy. 2. Findings in bile ducts from 11 methotrexate treated psoriatics and 2 controls.

To determinate what damage occurred in the bile ducts of psoriatics receiving Methotrexate (MTX) therapy liver biopsies from 11 patients were studied with the light and electron microscope and compared with normal material. Thick sections (1 micrometer) showed light and dark cells in biliary epithelium and lipofuscin granules. At the ultrastructural level these were confirmed. The lumen of the bile ducts contained debris. The microvilli were decreased in number and damaged forms appeared. Damage to the biliary epithelial mitochondria was widespread and there were foci of intracellular oedema. The Golgi apparatus was hypertrophied and dilated. Atrophic cells were seen. The lateral intercellular spaces were dilated and contained debris and the basement membrane showed zones of duplication. Similar changes were found in the ducts of Hering.

Adult↗
PubMed1981

The role of methotrexate and dihydrofolate polyglutamates in the enhancement of fluorouracil action by methotrexate.

The effect of the methotrexate (MTX)-5-fluorouracil (FUra) combination on L1210 cell viability was highly sequence dependent in that a 1.4 log (25-fold) increase in cytotoxicity was observed when the MTX preceded FUra, as compared to the reverse sequence. The formation of ternary complexes of thymidylate synthetase has been examined as a basis for the interaction of FUra and MTX in L1210 cells. L1210 cells converted 39% of the total intracellular MTX into MTX-poly-gamma-glutamates within 4 hours of a 1 microM MTX exposure. MTX-diglutamate (2,4-diamino,N 10-methylpteroyl-diglutamate) and MTX-triglutamate were the predominant metabolites. In contrast to the ternary complexes formed with MTX and MTX-diglutamate, the 5-fluorodeoxy-uridylate (FdUMP)-7,8-dihydropteroylpentaglutamate-enzyme and the FdUMP-5,10-methylenetetrahydropteroylpentaglutamate-enzyme complexes were stable to polyacrylamide gel electrophoresis under nondenaturing conditions. MTX-diglutamate enhanced the extent of tight-binding inhibition of thymidylate synthetase activity by FdUMP in the presence of saturating 5,10-methylenetetrahydropteroylpentaglutamate, suggesting that MTX-diglutamate did not antagonize the formation of FdUMP-5,10-methylenetetrahydropteroylpentaglutamate enzyme complex. We propose that the sequence-dependent effect of MTX plus FUra on L1210 cell viability results from MTX and MTX polyglutamate inhibition of dihydrofolate reductase, and consequently a trapping of intracellular folates as dihydrofolate polyglutamates that could increase the extent of FdUMP binding to thymidylate synthetase.

Animals↗
PubMed1984

A methotrexate-resistant human breast cancer cell line with multiple defects, including diminished formation of methotrexate polyglutamates.

Methotrexate (MTX)-resistant human breast cancer cells (MTXR ZR-75) were obtained following serial passage of the wild-type ZR-75-1 cells (wild-type ZR-75) in MTX. The resistant cell line contains neither quantitative nor qualitative changes in dihydrofolate reductase compared to the parental line. Resistance is associated with a 3-fold decrease in MTX transport into MTXR ZR-75 cells as well as a 3-fold decrease in the activity of thymidylate synthetase in the resistant subline. Moreover, marked differences were observed between the wild-type and MTXR ZR-75 cells in their ability to convert MTX to its polyglutamate derivatives. Wild-type ZR-75 cells accumulate significant intracellular levels of antifolates during prolonged (24 h) exposure to 2 microM MTX, due to the formation of MTX polyglutamates. In contrast, essentially no polyglutamates are formed in the MTXR cells even during conditions which result in a vast excess of free intracellular drug in these cells. This defect is not associated with any apparent change in the activity of the enzyme folylpolyglutamyl synthetase, nor is there any alteration in the apparent Km of this enzyme for MTX in the resistant cells. Further studies demonstrate that the MTXR ZR-75 cells are cross-resistant to antifolate analogues which can be converted to polyglutamate derivatives (aminopterin and dichloromethotrexate), yet they are relatively sensitive to antifolate analogues such as 2,4-diamino-5-(3',4'-dichlorophenyl)-6-methylpyrimidine, triazinate, and trimetrexate, which cannot be converted to polyglutamate forms. These studies identify a new mechanism (diminished accumulation of MTX polyglutamates) associated with resistance to MTX and lend additional support to the hypothesis that the formation of these derivatives is an important determinant of MTX cytotoxicity.

Breast Neoplasms↗
PubMed1981

Activity of free and carrier-bound methotrexate against transport-deficient and high dihydrofolate dehydrogenase-containing methotrexate-resistant L1210 cells.

A difference in the mechanism of transmembrane transport was demonstrated for methotrexate (MTX) and MTX bound to the high molecular weight carrier bovine serum albumin (MTX-BSA) when the drug dose needed to reduce growth of cells to 50% of that of untreated cells (ID50) was compared in the sensitive L1210 leukemia and 3 L1210 sublines resistant to MTX by virtue of either deficient MTX transport or high levels of dihydrofolate dehydrogenase (DHFD). The loss of transport increased the ID50 for inhibition of growth rate by free MTX tenfold to twentyfold, whereas the elevation of DHFD levels increased the ID50 by tenfold. In contrast, deficiency of transport resulted in only a twofold increase in the ID50 for MTX-BSA, and elevation of DHFD caused a tenfold increase similar to that for free MTX. This difference was confirmed in studies of inhibition of DHFD activity by free and BSA-bound MTX. MTX-BSA but not MTX had antitumor activity against the transport-deficient L1210 line in (C57BL/6 x DBA/2)F1. These studies confirm a separate mode of cell entry for MTX-BSA and suggest a role for these complexes in overcoming resistance.

Animals↗
PubMed1982

Pharmacokinetics of methotrexate and 7-hydroxymethotrexate following infusions of high-dose methotrexate.

The pharmacokinetics of methotrexate (MXT) and 7-hydroxymethotrexate (7-OH-MTX) has been studied in seven patients treated for osteosarcoma with up to 27 cycles of MTX at doses of 140-350 mg/kg of body weight. The distribution volume of MTX was 0.186 +/- 0.062 liter/kg. Peak plasma levels ranged from 540 to 1700 microM for MTX and from 12 to 560 mu M for 7-OH-MTX. The MTX metabolite 2,4-diamino-N10-methylpteroic acid was occasionally detectable in plasma and urine at a level of 10(-7)M. Plasma disappearance of MTX was biphasic, with a terminal half-life of 2.1 +/- 0.6 hours (mean +/- SE). Plasma decay of 7-OH-MTX was mainly monoexponential, with a half-life of 23.8 +/- 15.2 hours. The renal clearance of MTX (0.0623 +/- 0.0232 liter/kg/hour) accounted for about 84% of the total clearance of MTX (0.0742 +/- 0.0288 liter/kg/hour). In urine, 70%-94% of the dose was recovered as MTX and 0.4%-11% as 7-OH-MTX. The renal clearance of 7-OH-MTX was in the range of 0.0173 +/- 0.0149 liter/kg/hour. The pharmacokinetics of MTX and 7-OH-MTX was influenced by orally coadministered activated charcoal, presumably by inhibition of enteral reabsorption of MTX and 7-OH-MTX.

Adolescent↗
PubMed1982

Effect of methotrexate on dihydrofolate reductase activity in methotrexate-resistant human KB cells.

A cloned subline of human cell variants derived from KB cells (KB/6b), 6500-fold resistant to methotrexate (MTX), exhibited a stable 40-fold elevation of the enzyme dihydrofolate reductase (EC 1.5.1.3., DHFR) in the absence of the drug. No differences were detected between the enzyme isolated from resistant and parental KB cells. After the culture medium was supplemented with MTX, the resistant cells were shown to have an additional 5-fold increase in DHFR levels. This increment was dependent upon the concentration of exogenous MTX and was freely reversible. This behavior differs from that of other DHFR-overproducing mammalian cell lines previously reported. The half-life of DHFR in these cells in the presence or absence of MTX was the same, thus eliminating stabilization of the enzyme by MTX as being responsible for the modulation or "induction" phenomenon. Furthermore, the effect was blocked by cycloheximide and was not affected by actinomycin D. These results suggest that modulation of DHFR specific activity by MTX occurs at either the translational level or by as yet undefined post-transcriptional mechanism(s).

Cells, Cultured↗
PubMed1993

Selective expression of methotrexate-resistant dihydrofolate reductase (DHFR) activity in mice transduced with DHFR retrovirus and administered methotrexate.

To determine the effect of in vivo pharmacological selective pressure on the insertion and expression of new gene sequences, retroviral-mediated transfer of a methotrexate-resistant dihydrofolate reductase (Mtxr-DHFR) gene in hematopoietic tissue was investigated using a murine syngeneic bone marrow transplant system. A series of recombinant retroviral vectors were constructed to contain long terminal repeat (LTR) regions from different murine retroviruses associated with various proliferative disorders of the lymphohematopoietic system, including Moloney leukemia virus, spleen focus-forming virus (anemia strain) and myeloproliferative sarcoma virus. High-titer DHFR virus (10(7) colony-forming units/ml) was generated by gene amplification adapting virus-producer cell lines to grow in medium containing increasing concentrations of Mtx. This high-titer DHFR virus was used to introduce the Mtxr-DHFR gene into murine hematopoietic tissue by injecting DHFR virus-exposed marrow into lethally irradiated syngeneic recipient mice that subsequently were administered Mtx. Southern blot analysis of spleen DNA demonstrated insertion of the DHFR provirus in all surviving mice transplanted with DHFR virus-exposed marrow. However, enzymatic assay of crude spleen extracts demonstrated the presence of Mtxr-DHFR activity only in mice that were administered Mtx; nonadministered animals or animals transplanted with control (neo) virus-infected marrow contained undetectable drug-resistant enzyme activity. These results suggest the selective outgrowth of hematopoietic tissue harboring and expressing a DHFR provirus in animals administered Mtx and have implications for the application of drug-resistance gene insertion in somatic tissues of animals and humans.

3T3 Cells↗
PubMed1996

Oral administration of an easily prepared solution of injectable methotrexate diluted in water: A comparison of serum concentrations vs methotrexate tablets and clinical utility.

OBJECTIVE: To investigate whether the injectable formulation of methotrexate (MTX) given as an easily prepared oral solution of MTX diluted in water results in serum concentrations similar to those obtained with MTX tablets; to describe an easy and safe method of dispensing the drug. METHODS: Six patients (5 women, 1 man) with rheumatoid arthritis were given 10 mg of liquid MTX orally. The liquid was prepared by diluting 0.4 ml of the injectable formulation of MTX (50 mg/2 ml) in 8 ounces of water. One to 2 weeks later these patients were given 10 mg of MTX in the tablet form. MTX serum concentrations were determined using a fluorescence polarization immunoassay. The area under the concentration vs time curve (AUC), maximum concentration (Cmax) and the time to reach maximum concentration (tmax) were determined from the resulting concentration vs time curves. RESULTS: There was no statistical difference in the variables measured (AUC, Cmax, tmax), demonstrating comparable concentrations with these 2 methods of MTX administration. Patients found the medication easy to administer, potential hazards with the use of needles were avoided, and the cost of the drug was greatly decreased. CONCLUSION: The administrator of this easily prepared MTX solution is an alternative to the conventional administration of MTX tables, and may be of particular benefit in patients with financial limitations.

Administration, Oral↗
PubMed2001

Methotrexate for treating juvenile idiopathic arthritis.

BACKGROUND: In both adult rheumatoid arthritis (RA) and juvenile arthritis, the focus has shifted from 'inflammation parameters' to more patient centered disability outcomes. In RA this resulted in the development of the Outcome Measures in Arthritis Clinical Trials (OMERACT), and in juvenile arthritis the Pediatric Rheumatology International Trials Organization (PRINTO) core set. This PRINTO-core set was established using a combination of statistical and consensus formation techniques. This core set contains a number of patient centered disability measures. This review systematically searched the available literature and reports the available evidence of efficacy of MTX, with special focus on patient centered disability measures in Juvenile Idiopathic Arthritis (JIA). OBJECTIVES: To perform a systematic review on the effects of MTX on functional ability, range of motion, quality of life, overall well-being and pain for patients with JIA. SEARCH STRATEGY: The Cochrane Controlled Trials Register (CCTR) and MEDLINE were searched up to March 2001, using the search strategy sensitive for randomised controlled trials, used by the Cochrane Collaboration. SELECTION CRITERIA: Randomized controlled trials and controlled clinical trials comparing MTX against placebo or standard care in patients with Juvenile Idiopathic Arthritis (JIA) were selected. DATA COLLECTION AND ANALYSIS: Two reviewers (TT, JN) determined the studies to be included in this review and extracted the data of patient centered disability measures. The data were pooled using standardized mean differences (SMD) for limited joint range score, number of joints with swelling, and number of joints with pain on motion. Physicians global assessment and parents global assessment were evaluated with pooled odds ratios (OR). MAIN RESULTS: Only two studies with a total 165 JIA patients under 18 years of age were included in this review. For JIA patients, MTX therapy had small to moderate effects on patients centered disability. The effect on joint range of motion, number of joints with pain and swelling and parent's assessment of disease activity showed a relative percentage improvement from 3 to 18% greater with MTX than with placebo. REVIEWER'S CONCLUSIONS: Current evidence suggests that MTX does not have clinically significant effects (>20%) on patient centered disability measures in JIA patients.

Adolescent↗