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PubMed1992

Folinic acid rescue used routinely in psoriatic patients with known methotrexate "sensitivity".

Many combinations of methotrexate and folic or folinic acid have been used to limit the side effects of methotrexate therapy in psoriasis or psoriatic arthropathy. Methotrexate inhibits the enzyme dihydrofolate reductase and prevents the formation of DNA and RNA. Folinic acid, the 5-formyl derivative of tetrahydrofolic acid, is the active form of folic acid. We have confirmed in 5 patients that continuous administration of folinic acid with weekly oral methotrexate prevents improvement of psoriasis. When folinic acid was ceased on the day of methotrexate in these patients their psoriasis improved. Five other patients with previous sensitivity to methotrexate, forcing cessation of therapy, were given weekly oral methotrexate and folinic acid every day except the day of methotrexate. Marked improvement of psoriasis or arthropathy occurred in each case without side effects. This method precisely limits the exposure to methotrexate, allowing a therapeutic effect without complication even in those patients who exhibit methotrexate sensitivity.

Adult↗

PubMed2000

Methotrexate is steroid sparing in acute sarcoidosis: results of a double blind, randomized trial.

BACKGROUND AND AIM OF THE WORK: Methotrexate has been steroid sparing for some patients with chronic sarcoidosis. We wished to determine whether methotrexate can be steroid sparing in the first year of corticosteroid therapy in sarcoidosis. METHODS: Patients with new onset, symptomatic disease within four weeks of starting on prednisone were randomized to receive either methotrexate or placebo for the next year. They were seen monthly and prednisone dosage was tapered following a pre-determined schedule. RESULTS: Of 24 patients enrolled, only 15 received at least six months of therapy. Since methotrexate appears to take six months to be effective, only patients who completed six or more months of therapy were evaluated. The amount of prednisone per day decreased for both groups: methotrexate (First 6 months: Median 26 (Range 15-37) mg/day); Second 6 months 8 (1-22) mg/day, p < 0.01) and placebo (First 6 Months 28 (24-33) mg/day; Second 6 months 16 (11-22) mg/day, p < 0.02), with less prednisone used for the methotrexate patients versus placebo in the last six months (p < 0.01). There was also less weight gain for those patients receiving methotrexate. There was no difference in toxicity between methotrexate and placebo. The difference between methotrexate versus placebo was not seen when all patients (including the dropouts) were analyzed. CONCLUSIONS: Methotrexate can be a steroid sparing agent in acute sarcoidosis.

Adult↗

PubMed1975

Interaction of chemotherapeutic agents with methotrexate and 5-fluorouracil and its effect on de novo DNA synthesis.

The transport of methotrexate is known to be affected by corticosteroids and vincristine in L1210 leukemia cells. The deoxyuridine suppression test was used to measure the metabolic consequences of using these drugs with the antimetabolites, methotrexate and 5-fluorouracil, in both L1210 leukemia cells and normal human marrow cells. The deoxyuridine suppression test can be utilized as a sensitive measure of methotrexate and 5-fluorouracil biological activity in producing defective de novo DNA synthesis. The deoxyuridine suppression test was found to detect changes in biological activity equal to 20 ng (0.044 nmole) of methotrexate and 200 ng (1.94 nmoles) of 5-fluorouracil. Hydrocortisone and prednisone, but not dexamethasone or prednisolone, decreased the methotrexate effect to one-half in both L1210 and human cells as measured by the deoxyuridine suppression test. 5-Fluorouracil biological activity was not affected by any steroid studied. Vincristine produced variable results, but on the average it decreased the methotrexate effect in human marrow. Vincristine consistently decreased the methotrexate effect in L1210 systems. Cephalosporin, 75 mug/ml (0.214 mumole), had no effect. In parallel studies, hydrocortisone decreased the uptake of methotrexate, but no folic acid, in human and L1210 cells. The deoxyuridine suppression test warants further investigation as a method of screening drugs for interaction with antagonists of de novo DNA synthesis. This tudy extends earlier evidence of drug interaction with methotrexate in a murine system to human cells and demonstrates that there is a metabolic consequence, reduced potency of methotrexate, as a result of reduced transport produced by certain corticosteroids.

Animals↗

PubMed2003

[Treatment of patients with juvenile rheumatoid arthritis with combination of leflunomide and methotrexate].

OBJECTIVE: To evaluate the efficacy and safety of the combined therapy with leflunomide and methotrexate in the patients with juvenile rheumatoid arthritis (JRA). METHODS: Forty patients with active polyarthritis JRA were divided into 2 groups. Group 1 (n = 21) received leflunomide tablet (1 mg/(kg x day) on days 1 - 3; then [(0.2 - 0.4) mg/kg per day] plus methotrexate (0.3 mg/kg i.v. every two weeks till clinical remission, then oral tablet 0.2 mg/kg weekly). Group 2 received the same doses of methotrexate in the same way. Permitted concomitant drugs included stable doses of NSAIDs and a low dose of prednisone during the course of treatments. The clinical assessments included the number of tender and swollen joints, tender articular index, swollen articular index, general articular function score, parents and physician's evaluation score, erythrocyte sedimentation rate, serum C-reactive protein and rheumatoid factor. Drug safety was assessed by observing the reaction of mucous membrane, skin, gastrointestinal tract, nervous system, hematologic changes, liver and renal function. Statistical comparison between two groups was performed by using analysis of variance, t test and chi(2) test. RESULTS: Efficacy and safety was assessed at 12th and 26th week. Average improvement rate of leflunomide plus methotrexate group at 12th week and 26th week was respectively 39.6% and 71.9%; while that of control group was 27.5% and 49.5%, i.e., there was significant difference between the two groups (P < 0.01). Average remission rate of leflunomide plus methotrexate group at 12th week and 26th week was respectively 4.76% and 38.10%; while that of control group (methotrexate only) was respectively 0, 0. The clinical improvement in the group treated with leflunomide plus methotrexate was significantly greater than control group (P < 0.01). There was no significant difference (9.5% v 5.3%) in occurrence rate of side effects between the two groups. Side effects included leucocytopenia and raised aminotransferase. They were mostly mild and tolerable. CONCLUSION: The effect of the leflunomide and methotrexate therapy in patients with active JRA was better than methotrexate alone. The combination therapy with leflunomide and methotrexate was safe and well tolerated.

Adolescent↗

PubMed1990

Interaction of methotrexate polyglutamates and dihydrofolate during leucovorin rescue in a human breast cancer cell line (MCF-7).

Previous investigations have suggested that high-dose methotrexate with leucovorin rescue is a potentially useful strategy for overcoming antifolate resistance. Interactions between methotrexate (MTX) and leucovorin and their respective metabolites appear to occur at multiple intracellular sites, including dihydrofolate reductase (MTX/MTX polyglutamates versus dihydrofolate) and other folate-dependent enzymes (MTX polyglutamates versus reduced folate substrates). The present studies were designed to test the ability of dihydrofolate to compete with methotrexate and methotrexate polyglutamates for dihydrofolate reductase activity using an intact human breast carcinoma cell line (MCF-7) as the model system. Exposure of the breast cells to methotrexate for 24 h resulted in a concentration-dependent formation of methotrexate polyglutamates that markedly exceeded the dihydrofolate reductase-binding capacity for up to 24 h after the removal of drug from the growth media. Under these conditions of dihydrofolate reductase inhibition, we found that tritium-labeled dihydrofolate was capable of competing with methotrexate and its metabolites for dihydrofolate reductase activity as evidenced by the appearance of tritium-labeled reduced folates in the treated cells. We found the interaction between dihydrofolate and methotrexate to be dependent on the exposure concentrations of both methotrexate and dihydrofolate. These studies provide direct evidence that competition during leucovorin rescue occurs at the level of dihydrofolate reductase between methotrexate polyglutamates and dihydrofolate polyglutamates in intact human cells.

Binding, Competitive↗

PubMed1991

Herpes zoster in patients with rheumatoid arthritis treated with weekly, low-dose methotrexate.

PURPOSE: Herpes zoster occurred in nine patients with methotrexate-treated rheumatoid arthritis. We compared these patients to a large group of methotrexate-treated rheumatoid arthritis patients in order to uncover potential factors explaining the occurrence of herpes zoster. PATIENTS AND METHODS: Data from 187 patients taking methotrexate were reviewed and compared with data from another nine patients who developed herpes zoster while taking the drug for rheumatoid arthritis, all from the same university-based arthritis clinic. Literature pertinent to infection in rheumatoid arthritis patients treated with methotrexate is reviewed. RESULTS: Herpes zoster occurred in 14.5 cases per 1,000 patient-years in our methotrexate-treated rheumatoid arthritis patients, as compared with the general population incidence of 1.3 to 4.8 cases per 1,000 patient-years. The infection was unrelated to duration of methotrexate usage, prednisone treatment, or the co-existence of diabetes mellitus, but appeared to occur in patients with high titers of rheumatoid factor and an overall longer duration of rheumatoid arthritis. There were no cases of systemic dissemination or recurrence of herpes zoster despite 27.4 years cumulative follow-up on continued methotrexate therapy. CONCLUSIONS: Herpes zoster may occur more frequently in patients with rheumatoid arthritis treated with low-dose methotrexate than in the general population. Herpes zoster in rheumatoid arthritis patients treated with methotrexate appears to be self-limited, benign, and statistically related to methotrexate use in the presence of longer-term rheumatoid disease.

Arthritis, Rheumatoid↗

PubMed1988

Long-term bone marrow culture as a model for host toxicity: the effect of methotrexate on hematopoiesis and adherent layer function.

Long-term bone marrow culture was used to examine the hematopoietic toxicity of the chemotherapeutic agent methotrexate. A dose-related suppression in myelopoiesis was seen when cultures were treated with 10(-3), 10(-4), or 10(-5) M methotrexate followed by folinic acid rescue. Differential counts revealed an initial decrease of postmitotic myeloid cells followed by mitotic precursors and myeloid colony-forming units (CFU-C). Myelopoiesis had disappeared by 7-10 days when 10(-3) M methotrexate was studied, by day 21 with 10(-4) M methotrexate, and by day 28 with 10(-5) M methotrexate. Although 10(-3) M methotrexate caused a predictable suppression in myelopoiesis, the effect of 10(-5) M methotrexate was more variable. In some studies this dose caused significant suppression of myelopoiesis, whereas in others less suppression occurred. This provides some evidence for varying host susceptibility to drug. Microenvironmental (adherent layer) cells were also affected by methotrexate. An increase in proliferation of these cells occurred in proportion to the dose of methotrexate added to culture. Methotrexate did not affect the ability of the adherent cells to produce colony-stimulating activity (CSA), but high doses did prevent the layer's ability to support the proliferation of adherent cell-free marrow. These results indicate that long-term bone marrow culture can be used to successfully predict and define chemotherapeutic host toxicity.

Bone Marrow↗

PubMed1975

Liver biopsy in methotrexate-treated psoriatics-a re-evalution.

Two-hundred and eighty-six liver biopsies were performed in 139 psoriatics on treatment or considered for treatment with methotrexate. In 56 psoriatics included in this study both pre- and post-methotrexate biopsies were performed, the average methotrexate dose being 936 mg. None of the data showed statistically significant differences between pre- and post-methotrexate biopsies, with the exception of an increase in fattly infiltration, found when comparing all pre-methotrexate biopsies with the total number of latest post-methotrexate samples. As expected, alcohol seemed to be significantly associated with liver fibrosis in pre-methotrexate biopsies. An patients, although potassium arsenite alone has not been proved to be the cause of liver damage among psoriatics included in this study. While only 1 of 22 psoriatics with a total normal biopsy had been on arsenite, 6 of 18 of the same group of psoriatics who had fibrosis had been on this drug earlier. Although no statistically significant differences related to fibrosis and cirrhosis could that in three cases liver cirrhosis did appear in a biopsy from a methotrexate-treated psoriatic who had signs of fibrosis of cirrhosis in a pre-methotrexate biopsy. This incidence is low in relation to the total number of patients treated. The relatively low incidence of cirrhosis found in the present study, as in earlier studies by our group is believed to be due to the use of an intermittent dosage schedule. The study showed that early fibrosis and cirrhosis seem to appear, with very minor abnormalities in laboratory results. This finding indicates the necessity of performing liver biopsies in the control of psoriatics on long-term methotrexate therapy. The difference between biopsies from psoriatics and liver biopsies from control patients may indicate that severity of disease may be a complicating factor in the pathogenesis of the liver damage.

Adult↗

PubMed1982

Optimal schedule of methotrexate and 5-fluorouracil in human breast cancer.

We have shown previously that methotrexate pretreatment of murine leukemia and human colon carcinoma cell cultures results in augmented intracellular accumulation of 5-fluorouracil metabolites. Both of these drugs are commonly used for the treatment of women with breast cancer; thus, sequencing of methotrexate before 5-fluorouracil was evaluated in vitro using a human mammary carcinoma cell line, 47-DN. Intracellular 5-fluorouracil accumulation was maximally increased 4-fold in cultures pretreated with 10 microM methotrexate for 24 hr. This enhancement of 5-fluorouracil metabolism was associated with increased intracellular levels of 5-phosphoribosyl 1-pyrophosphate, resulting from the antipurine effect of methotrexate. Brief exposure to exogenous hypoxanthine at physiological concentrations reversed the biochemical synergism between methotrexate and 5-fluorouracil. Other antimetabolites associated with elevations of 5-phosphoribosyl 1-pyrophosphate enhanced intracellular accumulation of 5-fluorouracil up to 2.5-fold. In cloning assays, 18 hr of methotrexate pretreatment followed by 5-fluorouracil resulted in optimal synergistic cytotoxicity, which could be prevented if high concentrations of leucovorin were given between methotrexate and 5-fluorouracil administration. Since these results indicated that optimal breast tumor toxicity in vitro was achieved by 18- to 24-hr sequencing of methotrexate and 5-fluorouracil, clinical toxicity study was carried out to assess whether this drug schedule could be tolerated. Seven patients with advanced cancer were treated with 21 courses of sequential therapy. No toxicity occurred with 38% of treatment courses; mild to moderate leukopenia and mucositis occurred with 29 and 38% of courses respectively. Toxicity was related to treatment interval and not cumulative drug dose or elevated serum methotrexate levels. These clinical results suggest that Phase II studies evaluating 24-hr-sequenced methotrexate and 5-fluorouracil in breast cancer are warranted.

Breast Neoplasms↗

PubMed1982

A new fluorescent dihydrofolate reductase probe for studies of methotrexate resistance.

A new fluorescent methotrexate analogue (PT430) was synthesized as a reported ligand for dihydrofolate reductase. The analogue was prepared by attachment of lysine in place of the glutamate side chain of methotrexate and conjugation to fluorescein isothiocyanate via the epsilon-amino group of lysine. Spectrophotometric enzyme inhibition assays showed PT430 to be about one-tenth as potent as methotrexate against either Lactobacillus casei or L1210 mouse leukemia enzyme; competitive radioligand binding assays using tritiated methotrexate gave similar results. In assays of L1210 cell proliferation in culture, on the other hand, PT430 was 100-fold less toxic than methotrexate. In dilute solution, the fluorescence intensity of PT430 was 5-fold lower than that of equimolar fluorescein and diminished with decreasing pH. On complexation with dihydrofolate reductase, however, fluorescence intensity was enhanced 3- to 5-fold depending on the pH. Measurement of fluorescence increase with added ligand provided data for the determination of the stoichiometric ratio, dissociation constant, and extent of fluorescence enhancement. Specificity of PT430 for methotrexate binding sites was indicated by the observation of decreased fluorescence uptake in PT430-treated L1210 cells in the presence of methotrexate. Fluorescence uptake occurred faster, and to a greater extent, in methotrexate-resistant dihydrofolate reductase overproducing L1210/R6 cells than in the methotrexate-sensitive parent line. Therefore, PT430 may be used as a flow cytometry probe to detect methotrexate resistance based on dihydrofolate reductase overproduction.

Animals↗

PubMedDate not supplied

Increased schedule-dependent synergism of vindesine versus vincristine in combination with methotrexate against L1210 leukemia.

BD2F1 mice were inoculated ip with 10(6) L1210 leukemia cells and treated with vincristine or vindesine alone or in combination with methotrexate. Drug administration was begun on Day 1 and was continued every 4 days until a total of five doses were given or death occurred. Methotrexate (48 or 72 mg/kg ip) produced a 199% and a 222% increase in lifespan, respectively, as compared with untreated animals (6.9 +/- 0.5 days). When given as single agents, vincristine (0.5-1.0 mg/kg ip) or vindesine (0.5-1.5 mg/kg ip) produced between a 27% and an 88% increase in lifespan. The therapeutic benefit observed when either vinca alkaloid was used with methotrexate was schedule-dependent. With the exception of vindesine plus 72 mg/kg of methotrexate, the increase in lifespan produced by the simultaneous administration of methotrexate and either vinca alkaloid was additive. When vindesine was administered with 72 mg/kg of methotrexate, the increase in lifespan was greater than expected from an additive effect of the two agents. However, none of the trials employing single-agent therapy or simultaneous combination therapy produced long-term survivors (greater than or equal to 90 days after therapy). When either vinca alkaloid was given 24 hours after the folate analog, the increase in lifespan was almost 100% greater than that observed when the agents were given simultaneously; moreover, long-term survivors were produced. Vindesine in combination with 48 mg/kg of methotrexate produced 10%-25% long-term survivors, as compared to 5%-7% long-term survivors obtained with vincristine. In combination with 72 mg/kg of methotrexate, vindesine produced 27%-60% long-term survivors, as compared to 10%-20% long-term survivors obtained with vincristine. When either vinca alkaloid was administered 72 hours after methotrexate, the regimen was still synergistic, but the overall effect was less than with a 24-hour delay. When two doses of either vinca alkaloid were injected at 24 and 72 hours after the folate analog, the result was either highly therapeutic or very toxic. Two doses of 0.5 mg/kg of vindesine or vincristine with 48 mg/kg of methotrexate produced 35% and 20% long-term survivors, respectively. All other regimens were toxic.

Animals↗

PubMed1998

Comparative studies of quality and bioavailability of methotrexate in Thai patients with rheumatoid arthritis.

The bioavailability of the two generic methotrexate oral preparations (Emtrexate, Pharmachemie Company, Holland and Methotrexate Remedica, Remedica, Cyprus as the test preparations), were compared to the innovator (Methotrexate Lederle, Lederle, U.S.A. as the reference) in 10 patients with rheumatoid arthritis. A single 7.5 mg oral dose of each preparation was given to the subjects in a randomized, double-blind, three-period crossover design with a 1 week washout period. Serum methotrexate concentrations were determined by using Fluorescence Polarization Immunoassay (Abbott TDx). No significant differences in pharmacokinetic parameters (AUC, Cmax, and Tmax) were observed between the test and reference preparations. The mean and 90 per cent CI of the ratio Emtrexate/Methotrexate Lederle and Methotrexate Remedica/Methotrexate Lederle of the Cmax, AUC0-8, and AUC0-alpha were 0.93 (0.87-1.00), 0.9 (0.82-0.98), 0.88 (0.79-0.99) and 0.97 (0.93-1.02), 0.95 (0.90-0.99), 0.94 (0.86-1.02), respectively. These values were well within the acceptable bioequivalence range of 0.8-1.25. The mean and 90 per cent CI of Tmax difference between Emtrexate-Methotrexate Lederle and Methotrexate Remedica-Methotrexate Lederle also overlapped the stipulated bioequivalence range of the Tmax differences of +/- 0.25 hour. Thus, Emtrexate and Methotrexate Remedica were considered bioequivalent to the reference Methotrexate Lederle regarding the rate of absorption and the extent of absorption.

Adult↗

PubMed1999

Treatment of active rheumatoid arthritis with leflunomide compared with placebo and methotrexate. Leflunomide Rheumatoid Arthritis Investigators Group.

CONTEXT: Leflunomide is a reversible inhibitor of de novo pyrimidine synthesis shown to be effective in a phase 2 trial in 402 patients with active rheumatoid arthritis (RA). OBJECTIVE: To compare the efficacy and safety of leflunomide treatment with placebo and methotrexate treatment in patients with active RA. DESIGN: Randomized, double-blind, placebo, and active-controlled 12-month study. SETTING: Forty-seven university and private rheumatology practices in the United States and Canada. PATIENTS: Diagnosis of RA by the American College of Rheumatology (ACR) criteria for duration of 6 months or longer and no previous methotrexate treatment. INTERVENTION: Leflunomide treatment (20 mg/d), placebo, or methotrexate treatment (7.5-15 mg/wk). MAIN OUTCOME MEASURES: American College of Rheumatology success rate (completed 52 weeks of treatment and met the ACR > or = 20% response criteria), disease progression as assessed by x-ray films, and improvement in function and health-related quality of life using the intent-to-treat population. RESULTS: The 482 patients studied were predominantly women (mean age, 54 years; mean disease duration, 6.7 years) for whom a mean of 0.8 disease-modifying antirheumatic drugs had failed. The ACR response and success rates for patients receiving leflunomide treatment (52% and 41%, respectively) and methotrexate treatment (46% and 35%, respectively) were significantly higher than those for patients receiving placebo (26% and 19%, respectively) (P<.001), and they were statistically equivalent, with mean time to initial response at 8.4 weeks for patients receiving leflunomide vs 9.5 weeks for patients receiving methotrexate therapy. X-ray analyses demonstrated less disease progression with leflunomide (P=.001) and methotrexate (P = .02) therapy than with placebo. Leflunomide and methotrexate treatment improved measures of physical function and health-related quality of life significantly more than placebo (P<.001 and P<.05, respectively). Common adverse events for patients receiving leflunomide treatment included gastrointestinal complaints, skin rash, and reversible alopecia. Asymptomatic transaminase elevations resulted in treatment discontinuations for 7.1% of patients receiving leflunomide therapy, 1.7% of patients receiving placebo, and 3.3% of patients receiving methotrexate therapy. CONCLUSIONS: Clinical responses following administration of leflunomide, a new therapeutic agent for the treatment of RA, were statistically superior to those with placebo and equivalent to those with methotrexate treatment. Both active treatments improved signs and symptoms of active RA, delayed disease progression as demonstrated by x-ray films, and improved function and health-related quality of life.

Adult↗

PubMed1976

Paraplegia following intrathecal methotrexate: report of a case and review of the literature.

A patient who developed paraplegia following the intrathecal instillation of methotrexate is discribed. The ten previously reported cases of this unusual complication are reviewed. The following factors appear to predispose to the development of this complication: abnormal cerebrospinal dynamics related to the presence of central nervous system leukemia, and epidural cerebrospinal leakage; elevated cerebrospinal fluid methothexate concentration related to abnormal cerebrospinal fluid dynamics and to inappropriately high methotrexate doses based on body surface area calculations in older children and adults; the presence of neurotoxic preservatives in commercially available methotrexate preparations and diluents; and the use of methotrexate diluents of unphysiologic pH, ionic content and osmolarity. The role of methotrexate contaminants, local folate deficiency, and cranial irradiation in the pathogenesis of intrathecal methotrexate toxicity is unclear. The incidence of neurotoxicity may be reduced by employing lower doses of methotrexate in the presence of central nervous system leukemia, in older children and adults, and in the presence of epidural leakage. Only preservative-free methotrexate in Elliott's B Solution at a concentration of not more than 1 mg/ml should be used for intrathecal administration. Periodic monitoring of cerebruspinal fluid methotrexate levels may be predictive of the development of serious neurotoxicity.

Adult↗

PubMed2001

Protective effect of a synthetic analog of prostaglandin E(1) on the small intestinal damage induced by the administration of methotrexate to rats.

Antitumor drugs like methotrexate cause damage to the small intestine, resulting in malabsorption. The present study evaluated this damage by determining the small intestinal absorption of 3-O-methyl-D-glucose (3-OMG) and a poorly absorbable marker, fluorescein isothiocyanate-labeled dextran (FD-4; average molecular mass, 4.4 KDa) using the in vitro everted intestine and in situ intestinal loop techniques. Methotrexate (15 mg/kg body weight) was orally administered to rats once daily for 5 days. A synthetic analog of prostaglandin E(1), OP-1206 (17S,20-dimethyl-trans-Delta(2)-prostaglandin E(1); 0.5 microg/kg body weight) was orally administered to rats twice a day for 5 days. The absorption clearance of FD-4 via the small intestine of the methotrexate-treated rats increased marked, but that of the methotrexate- and OP-1206-treated rats was significantly lower than that of the rats treated only with methotrexate. The absorption clearance of [(3)H]-3-OMG via the small intestine of the methotrexate-treated rats fell markedly, but that of the methotrexate- and OP-1206-treated rats was significantly greater than that of the rats treated only with methotrexate. The changes in AUC values of FD-4 and [(3)H]-3-OMG obtained from in situ intestinal loop experiment showed the same trends as those seen in the absorption clearance from the in vitro everted intestine experiment. These results show that OP-1206 alleviates the methotrexate-induced damage to the small intestine of rats.

3-O-Methylglucose↗

PubMed1982

The in vivo distribution of methotrexate between plasma and erythrocytes.

1. The concentration of methotrexate in whole blood, plasma and erythrocytes was measured in three patients receiving 250 mg methotrexate by continuous intravenous infusion over 12 h for different malignant diseases. 2. Methotrexate was measured using a double-antibody radioimmunoassay which facilitated drug monitoring for 1--2 weeks. 3. The concentration of methotrexate in plasma was much higher than that in whole blood and erythrocytes during the period of infusion, but this profile was reversed during the elimination phase. 4. The concentration in erythrocytes fell rapidly immediately after the infusion ended, but thereafter, in contrast to plasma levels, methotrexate concentrations in erythrocytes did not appear to decay during the elimination phase. In one patient the concentration/time profiles differed between treatment days. On the first occasion, at the initiation of chemotherapy, erythrocytes progressively accumulated methotrexate in the elimination phase against an apparent concentration gradient. On the second occasion this progressive increase was not observed, but as in the other two patients, methotrexate levels in red cells remained many times higher than drug levels in plasma throughout the period of observation. 5. Folinic acid administration did not appear to influence the distribution of methotrexate between red cells and plasma. 6. It was concluded that while the distribution between plasma and erythrocytes was probably mediated by complex mechanisms, the results were consistent with the erythrocyte mass behaving as a slowly exchanging kinetic compartment. Accumulation and persistence of a drug such as methotrexate in red cells might be expected to promote resistance and perhaps influence the expression of toxicity.

Erythrocytes↗

PubMed1992

Molecular cloning of a gene involved in methotrexate uptake by DNA-mediated gene transfer.

A methotrexate-resistant Chinese hamster ovary cell line deficient in methotrexate uptake has been complemented to methotrexate sensitivity by transfection with DNA isolated from a wild-type Chinese hamster ovary genomic cosmid library. Primary and secondary transfectants, which contain a limited number of cosmid sequences, have been shown to regain methotrexate sensitivity and to take up methotrexate. Furthermore, the DNA from three cosmid clones, isolated from a primary methotrexate-sensitive transfectant, after transfection rescued the methotrexate-resistant phenotype at a high frequency. Restriction endonuclease analysis of the DNA of these cosmid clones indicated that they overlapped extensively and shared two regions of Chinese hamster ovary DNA of 6.6 kb and 20.6 kb. These observations indicate that a gene involved in methotrexate uptake is contained in its entirety within one of these regions. This is the first report of the functional molecular cloning of a gene involved in methotrexate uptake. A general strategy is also described for screening large cosmid libraries from primary transfectants.

Animals↗

PubMed1979

Alterations of the carrier-mediated transport of an anionic solute, methotrexate, by charged liposomes in Ehrlich ascites tumor cells.

Interaction of positively charged liposomes with Ehrlich ascites tumor cells increases the bidirectional transmembrane fluxes of the anionic folic acid analog, methotrexate. Negative liposomes reduce methotrexate influx. Stimulation of methotrexate influx by positively charged liposomes is time and concentration dependent, requiring at least a 5-min incubation with 2.5 mM phosphatidylcholine containing 20% stearylamine for maximum effect. Stimulation is not appreciably reversed by washing the cells. Similar increases are observed for influx and efflux so that there is no change in the steady-state methotrexate electrochemical-potential difference across the cell membrane. The increase influx appears to be a stimulation of the carrier-mediated transport process for methotrexate since both control and stimulated influx are abolished by the competitive inhibitor, 5-formyltetrahydrofolate or the sulfhydryl group inhibitor, p-chloromercuriphenylsulfonic acid and the Q10 of the system remains unchanged. Influx of 5-methyltetrahydrofolate, which shares the same transport carrier as methotrexate, is also stimulated. However, the transport of folic acid, which is structurally similar to methotrexate but does not utilize the carrier, is unaffected. The kinetic change induced by positively charged liposomes is an increase in the Vma in, while the Kt in remains unchanged. Trans-stimulation of methotrexate influx by 5-formyltetrahydrofolate occurs to the same extent in the presence or absence of positively charged liposomes. The liposomes have no apparent effect on the intracellular water, the extracellular space, or the chloride distribution ratio. The data suggest that interaction of positively charged liposomes with Ehrlich ascites tumor cells accelerates the rate of transposition of the membrane carrier system for methotrexate, altering the kinetics of transport without a change in transport thermodynamics.

Animals↗