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At least 19 recordsLinked to original sources
PubMed1979

Evaluation of ribonucleoside and deoxyribonucleoside triphosphate pools in cultured leukemia cells during exposure to methotrexate or methotrexate plus thymidine.

Continuous exposure to inhibitory concentrations of methotrexate produces distinct rates of steady-state growth of murine leukemia L1210 and human leukemia CCRF-CEM cells in culture. Addition of thymidine to the medium produces reversal (6 to 40%) of this steady-state growth rate inhibition. This study utilized combinations of methotrexate and thymidine for an evaluation of the accompanying relationship between steady-state growth rate and changes in the ribo- and deoxyribonucleoside triphosphate pools. In L1210 cells exposed to methotrexate alone, the deoxythymidine 5'-phosphate (dTTP) pools decreased, whereas deoxyadenosine 5'-triphosphate, deoxyguanosine 5'-triphosphate, and deoxycytidine 5'-triphosphate (dCTP) remained relatively constant up to 70% inhibition of growth rate, with dCTP at a constant 112% of controls. The corresponding ribonucleoside triphosphates decreased only slightly. With the combination of methotrexate and thymidine resulting in up to 40% inhibition of growth rate, there was also a decrease in the dTTP pool while the other deoxyribonucleoside triphosphates remained relatively constant, and the corresponding ribonucleoside triphosphates again decreased only slightly. The dCTP pool was reduced to a constant 42% of control comparable to that produced by thymidine alone. With greater than 40% (with thymidine) or 70% (without thymidine) inhibition of growth rate, all pools decreased, but only dTTP was substantially reduced in proportion to the growth rate inhibition caused by methotrexate. The dTTP pool became depleted in spite of the presence of exogenous thymidine. Evaluation of CCRF-CEM cells indicated that inhibition of growth rate and nucleotide pool perturbations by methotrexate were similar to those observed in L1210 cells. However, in the presence of thymidine, inhibition of growth rate appeared related to decreased pools of dCTP, deoxyadenosine 5'-triphosphate, and deoxyguanosine 5'-triphosphate, rather than dTTP as was observed for L1210 cells. Hence, mammalian cells were capable of responding in a differential fashion to pharmacological perturbations, and this capacity may play a role in determining therapeutic selectivity. Since the ribonucleoside triphosphate decreases were slight and relatively uniform during methotrexate-induced perturbations, the deoxyribonucleoside triphosphate pools appear to be more directly related to inhibition of growth rate. The results are consistent with the concept that slight imbalances in the deoxyribonucleoside triphosphate pools dramatically inhibit DNA synthesis, as mediated through their interaction with DNA polymerase.

Animals↗
PubMed1977

[The carrier potential of liposomes for methotrexate. Changing of the tissue levels of methotrexate in the organs of mice (author's transl)].

Drugs entrapped in liposomes (artificial lipid vesicles) exhibit different pharmacokinetics after intravenous application than drugs injected in a free form. The folidacidantagonist methotrexate can be entrapped in liposomes in a therapeutically useful concentration (0.5 mg MTX/ml) and can be stored with high stability of entrappment. After intravenous injection into the tail vein of mice liposomes entrapped methotrexate is found more enriched in cell systems with high rate of endocytosis and not eliminated by the kidneys within 3 h like free methotrexate. It can be shown, that for the organs liver, spleen, kidney, gut, lung, and blood over a 6 h period liposomes entrapped methotrexate is enriched in the tissues and that for example after 6 h the methotrexate level in the liver is 20 fold higher in comparison to free injected methotrexate.

Animals↗
PubMed1978

Radioimmunoassay of methotrexate: use of 75Se-labelled methotrexate.

75Se-labelled methotrexate has been used as the radiolabel in a radioimmunoassay for methotrexate and its use compared with that of 3H-methotrexate. The gamma-labelled drug has proved to be extremely reliable and easier to use than 3H-methotrexate. Results obtained with it compare well with those obtained with tritiated drug. There are practical advantages in using a gamma emitter, and it is suggested that the modified radioimmunoassay is invaluable for routine monitoring of methotrexate in patients undergoing chemotherapy with the drug.

Cross Reactions↗
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↗
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↗
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↗
PubMed1977

Purification and properties of dihydrofolate reductase from methotrexate-sensitive and methotrexate-resistant Chinese hamster ovary cells.

We have previously described methotrexate-resistant Chinese hamster ovary cells which appear to contain normal levls of a structurally altered dihydrofolate reductase (EC 1.5.1.3) (Flintoff, W.F., Davidson, S.V., and Siminovitch, L. (1976) Somatic Cell Genet.2,245-261). By selecting for increased resistance form these class I cells, class III resistant cells were isolated which appeared to possess an increased activity of the altered enzyme. In the report, we describe the purification and several properties of the reductase from wild-type cells, two independently selected class I cells, and class III resistant cell. The reductases from wild-type and resistant cells had similar specific activities using folate and dihydrofolate as substrates, and similar molecular weights as determined by sodium dodecyl sulfate gel electrophoresis. The mutant enzymes, however, were about six- to eight-fold more resistant to inhibition by methotrexate than the wild-type enzyme, suggesting a decreased affinity of the mutant reductases to methotrexate-binding. Small differences between various enzymes were also seen in other physicochemical properties such as pH optima and Km values for folate, and in their heat stabilities, which suggest that different structural alterations may lead to the same mutant phenotype. As expected from earlier studies with crude extracts, class III cells did produce a higher (about 10-fold) yield of the reductase than the class I or wild-type cells.

Cell Line↗
PubMed1976

Methotrexate pneumonitis induced by intrathecal methotrexate therapy: a case report with pharmacokinetic data.

A patient with adenocarcinoma of the breast metastatic to the leptomeninges was treated with 10 doses of intrathecal methotrexate (MTX) administered at intervals of 2 days. Following these treatments she developed fever, hypoxemia, and bilateral pulmonary infiltrates without documented pulmonary infection. Autopsy findings were consistent with the pneumonitis that has been associated with intermittent oral, intramuscular, and intravenous MTX therapy. It is suggested that this patient's pulmonary process represented MTX pneumonitis following intrathecal MTX. Cerebrospinal fluid and serum MTX concentrations determined retrospectively on frozen samples reflect an atypically rapid transport of MTX from this patient's cerebrospinal fluid to a slowly decaying systemic pool. Because of this, serum MTX levels probably exceeded 10-8M during the entire 20-day course of therapy, thus exposing the pulmonary parenchyma to significant drug concentrations for a prolonged interval. It is suggested that these unfavorable pharmacokinetics may have contributed to this patient's susceptibility to MTX pneumonitis.

Adenocarcinoma↗
PubMed1979

Low dose chemotherapy of metastatic breast cancer with cyclophosphamide, adriamycin, methotrexate, 5-fluorouracil (CAMF) versus sequential cyclophosphamide, methotrexate, 5-fluorouracil (CMF) and adriamycin.

Seventy-eight advanced breast cancer patients with hormone-resistant disease or visceral metastases were randomized to receive either of two low dose regimens consisting of cyclophosphamide (C), methotrexate (M), 5-fluorouracil (F), and Adriamycin (A) as their initial chemotherapy. One group was treated with CAMF, and the other with CMF until progression, followed by A (CMF leads to A). C was given at 50 mg/m2, po, days 1-14; M at 20 mg/m2, F at 300 mg/m2, and A at 20 mg/m2, iv, days 1 and 8 of each 28-day cycle. The response rates for CAMF vs. CMF did not differ significantly (complete and partial responses-62% vs. 49%; stabilizations-23% vs. 31%). Responses by site of metasis, median times to progression and median survivals were similar for both groups. Poor and good risk partial responders had similar survivals. Twelve percent of CMF patients treated with Adriamycin at the time of progression had partial responses with an associated improved survival. Since CMF is as effective as CAMF, but has less toxicity, low dose therapy with CMF is more acceptable than CAMF as an initial chemotherapy regimen for metastatic breast cancer. Adriamycin may be reserved for subsequent regression induction.

Antineoplastic Agents↗
PubMed1979

Methotrexate-induced oral mucositis and salivary methotrexate concentrations.

We examined the plasma and saliva levels of methotrexate (MTX) achieved during the treatment and rescue periods of ten patients receiving 42-h MTX infusions followed by citrovorum rescue. Saliva MTX levels were generally 1%--2% of the simultaneous plasma levels. Four patients developed severe oral mucositis; three patients developed mild to moderate oral toxicity, and three others had no evidence of mucositis. MTX levels in the patients with severe mucositis were not higher and did not persist longer than the levels achieved in patients with mild or absent toxicity. Attempts at reducing the severity of oral mucositis with topical citrovorum mouthwashes or with atropine to suppress salivation were unsuccessful. MTX-induced oral mucositis is not related to salivary MTX concentrations, and the use of topical citrovorum therapy or the suppression of salivation does not appear to ameliorate this toxicity.

Humans↗
PubMedDate not supplied

Microcalorimetric evaluation of the effects of methotrexate and 6-thioguanine on sensitive T-lymphoma cells and on a methotrexate-resistant subline.

Isothermal microcalorimetry was used in order to continuously monitor and quantitatively assess the action of two antineoplastic drugs, methotrexate (MTX) and 6-thioguanine (6-TG), on a human T-lymphoma cell line, CCRF-CEM. The results with MTX were compared with data from experiments with a MTX-resistant subline, CEM/MTX. The slope of the power-time curve after drug injection relative to that obtained during unperturbed growth, was used to construct dose-response curves. The normal cell line was characterized by a D50 value (i.e., the dose producing half the maximal response) of 0.05 microM for MTX and 0.38 microM for 6-TG. For the MTX-resistant subline the D50 value was 8 microM of MTX. Comparisons of the continuous power-time curves showed the inhibitory effect of 6-TG to be faster than that of MTX.

Calorimetry↗
PubMed1977

Liver biopsies from psoriatics related to methotrexate therapy. 3. Findings in post-methotrexate liver biopsies from 160 psoriatics.

The purpose of this paper is to report findings in post-MTX liver biopsies from 160 psoriatics treated with Methotrexate (MTX) in single biopsy and B. 68 patients with serial biopsies. At the time of liver biopsy the 92 patients had received a mehosis and six patients had fibrosis. Comparing these 7 patients with patients having normal liver histology (13 patients) revealed no statistically significant difference in cumulative doses of MTX, but a statistically significant higher admitted alcohol intake during MTX therapy (p less than 0.002) and an older age (p less than 0.01) in the patients with cirrhosis or fibrosis. in the 68 patients MTX had accumulated to a mean dose of 3940 mg (range 32k-8355 mg) at the time the latest liver biopsies were taken. Among the latest liver biopsies were 14 cirrhosis (21 per cent, 95 per cent confidence limits: 12-32 per cent) and 16 fibrosis (24 per cent, 95 per cent confidence limits: 14-35 per cent). The 14 patients with cirrhosis when compared to patients with normal histology (9 patients), had taken an equal total dose of MTX at the latest liver biopsy, but had consumed a statistically significant higher amount of alcohol (p less than 0.05) during MTX therapy and also tended to be older (p less than 0.006). Comparison of a material A and B indicates that the prevalence of cirrhosis and fibrosis among MTX treated psoriatics increases rapidly beyond a cumultative dose of two to four grams of MTX. No MTX treated psoriatics should thus be allowed to pass this dosage range without having a liver biopsy performed.

Adult↗
PubMed1978

Direct administration of methotrexate into the central nervous system of primates. Part 1: Distribution and degradation of methotrexate in nervous and systemic tissue after intraventricular injection.

Levels of methotrexate (MTX) measured by both 3H radioactivity and dihydrofolate reductase assays were determined in cerebrospinal fluid (CSF), plasma, urine, and both neural and non-neural tissues at varying times after a single intraventricular injection into Cynomolgus monkeys (Macaca fascicularis). Clearance of the MTX from CSF was rapid after injection. A relatively constant level of 3HMTX was reached in plasma 2 1/2 hours after injection, and about 30% of the 3HMTX dose was excreted in the urine within 4 hours after injection. Maximum levels in CNS tissues were obtained by 4 hours after injection, and average concentrations of 10(-6) M MTX (moles/kg wet weight) were maintained in CSF for up to 12 hours and in brain for up to 24 hours after injection. Conversion of MTX to non-MTX products was detected in CSF between 4 and 12 hours, and in brain tissue between 12 and 24 hours after injection, and the amount of these products increased with time. Regional distribution studies in the cerebrum showed a U-shaped distribution curve for 3HMTX up to 12 hours after injection, which closely followed the 14C inulin distribution. Thus, the levels in deep cerebral tissue were less than the average level for brain, and this suggests that treatment of CNS tumors by intraventricular injection may have variable results, partly due to complex tissue distribution patterns.

Animals↗
PubMed1976

Exchangeable intracellular methotrexate levels in the presence and absence of vincristine at extracellular drug concentrations relevant to those achieved in high-dose methotrexate-folinic acid "rescue" protocols.

Studies were undertaken to (a) assess intracellular methotrexate (MTX) levels at extracellular drug concentrations comparable to those achieved in high-dose MTX-folinic acid rescue protocols and (b) establish whether there is a rationale for the use of vincristine in these regimens. The data indicate that only low levels of exchangeable MTX (intracellular MTX in excess of the tightly bound fraction) accumulated in Ehrlich ascites tumor cells at high extracellular MTX concentration. For instance, the exchangeable steady-state intracellular MTX level was approximately 6.5 muM when the extracellular drug concentration was 85 muM. Over the interval of these experiments, exchangeable intracellular MTX did not exceed approximately 10 muM even when extracellular MTX was raised to 250 muM. These exchangeable intracellular MTX concentrations are comparable to those levels required experimentally to suppress (a) tetrahydrofolate synthesis from dihydrofolate and (b) tetrahydrofolate-dependent purine, pyrimidine, and amino acid synthesis in these cells in vitro. Vincristine (10 muM) augmented net MTX accumulation when the extracellular MTX level was 10, 100, or 250 muM. The limited capacity of cells to accumulate exchangeable intracellular MTX and the apparent role for this intracellular MTX component in achieving the metabolic effects of this agent may account for the necessity for high MTX blood levels in the treatment of some tumors and may be the basis, in part, for the enhanced chemotherapeutic efficacy of high-dose MTX regimens. These studies provide a rationale for the combined use of vincristine and MTX in high-dose MTX protocols. The addition of vincristine may permit the achievement of the level of exchangeable intracellular MTX that is required to critically inhibit tetrahydrofolate synthesis without an increase in the extracellular MTX concentration. This may permit a reduced MTX dose, diminishing the excretory load on the kidney and minimizing nephrotoxicity due to deposition of MTX in the renal tubule and interstitium. While the data indicate that the ratio of the concentration of exchangeable intracellular MTX to the extracellular drug concentration may be very low under steady-state conditions at high extracellular drug levels, further studies are required to establish that these steady-state gradients for MTX represent nonequilibrium conditions.

Animals↗
PubMed1977

[Serum methotrexate determination by microbiological assay in patients receiving high-dosage methotrexate (author's transl)].

Microbiological assay according to Burchenal (modified by Mehta) is used for the determination of the serum methotrexate (MTX) level. Fifty cycles of high-dose MTX therapy were monitored in 11 patients with osteogenic sarcoma. The results showed close correlation with the clinical course of the therapy. The serum level of MTX was elevated in patients with symptoms of toxicity.

Bone Neoplasms↗
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↗