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PubMed1983

Effects of folinic acid on hepatoma cells containing methotrexate polyglutamates.

The effects of folinic acid on a toxic pulse exposure of cultured hepatoma cells to methotrexate (4-amino-10-methylpteroylglutamic acid) is reported. Inclusion of folinic acid (5-formyl-5,6,7,8-tetrahydropteroylglutamic acid) (10 micro M) with the 2-hr pulse of methotrexate (10 micro M) nearly completely prevents the uptake and gamma-glutamylation of methotrexate and prevents toxicity. Addition of folinic acid after methotrexate results in a partial rescue that is time and concentration dependent. Restoration of cell growth in the presence of increasing amounts of folinic acid is accompanied by a concentration-dependent elevation in tritium release from [5-3H]deoxyuridine. In the absence of folinic acid, the release of tritium from [5-3H]deoxyuridine remains inhibited for three days after exposure to methotrexate, which can be related to the cellular formation and retention of methotrexate polyglutamates. Following the 2-hr pulse of methotrexate, the cellular pool consists of 70% polyglutamates of which the predominant species has three glutamate residues (4-NH2-10-CH3PteGlu3). When methotrexate is removed from medium, following the pulse, unmetabolized methotrexate rapidly leaves the cells, and 4-NH2-10-CH3PteGlu3 is converted to methotrexate polyglutamates containing four to six glutamate residues. Addition of folinic acid after the methotrexate pulse prevents the conversion of 4-NH2-10-CH3PteGlu3 to the higher-chain-length derivatives and causes a reduction in the total methotrexate cell pools over the next 48 hr. These results suggest that the effects of folinic acid on methotrexate polyglutamates may play a role in the rescue of cells containing these derivatives.

Animals↗
PubMed1983

In vitro formation of polyglutamyl derivatives of methotrexate and 7-hydroxymethotrexate in human lymphoblastic leukemia cells.

The intracellular synthesis of polyglutamyl derivatives of both methotrexate (4-amino-N-10-methylpteroylglutamic acid) and 7-hydroxymethotrexate, the primary plasma metabolite of methotrexate in humans, was evaluated in a methotrexate-sensitive, acute lymphoblastic leukemia cell line, MOLT 4. These studies were performed using a highly specific ion-pairing high-pressure liquid chromatography method which permits the simultaneous determination of methotrexate, 7-hydroxymethotrexate, and their corresponding polyglutamyl derivatives. When MOLT 4 cells were exposed to 1 microM methotrexate, the monoglutamate attained a steady state after 30 min, and polyglutamyl derivatives having from one to 4 additional glutamyl residues were observed over 4 hr. Four additional metabolites were also detected upon incubation with 1 microM 7-hydroxymethotrexate. On the basis of the retention times for these compounds relative to methotrexate polyglutamyl standards and since these metabolites reverted to 7-hydroxymethotrexate upon treatment with a preparation of hog kidney conjugase, they were identified as polyglutamyl derivatives of 7-hydroxymethotrexate. The identification of 7-hydroxymethotrexate polyglutamyl derivatives in vitro raises the possibility of an important new dimension in the pharmacological action of methotrexate. We investigated the effect of extracellular 7-hydroxymethotrexate on net methotrexate uptake and metabolism when cells were exposed simultaneously to 1 microM [3H]-methotrexate and unlabeled 7-hydroxymethotrexate. A decrease in the levels of both intracellular methotrexate and the corresponding polyglutamyl derivatives was noted for cells treated with 1 or 10 microM 7-hydroxymethotrexate. However, no appreciable effect of 7-hydroxymethotrexate on the amount of polyglutamyl derivatives formed relative to the total intracellular antifolate was noted. These studies show that in MOLT 4 cells (a) both methotrexate and 7-hydroxymethotrexate are rapidly converted to polyglutamyl derivatives, and (b) 7-hydroxymethotrexate interferes with net methotrexate accumulation and metabolism when present simultaneously in the extracellular medium. These results, moreover, suggest a potential role for 7-hydroxymethotrexate in modulating the biochemical effects of methotrexate in vivo.

Cell Line↗
PubMed1984

Interaction of probenecid with methotrexate transport and release in the isolated rat hepatocyte in suspension.

Probenecid has been shown to delay the plasma clearance of methotrexate in the rat and to reduce both hepatic and renal excretion of methotrexate in this animal model. In order to probe the mechanism by which probenecid alters hepatic excretion of the antifolate, studies assessed the effects of probenecid on transport, accumulation, distribution, and release of methotrexate in the rat hepatocyte in suspension. Probenecid was found to effectively inhibit methotrexate influx with a Ki of approximately 100 microM. Inhibition of methotrexate influx was accompanied by a reduction in methotrexate accumulation; with 200 microM probenecid, the levels of exchangeable and nonexchangeable intracellular methotrexate were reduced by 43.4 +/- 2.4 (S.E.) and 41.8 +/- 7.7%, respectively. As a consequence of reduced accumulation of the methotrexate substrate, the formation of cellular polyglutamate derivatives of methotrexate was likewise reduced. Concentrations of probenecid which inhibited methotrexate influx and accumulation by 70 to 80% did not markedly alter methotrexate efflux under conditions where efflux was effected by a washout procedure or by the presence of inducing agents, such as N6,O2'-dibutyryl cyclic adenosine 3':5'-monophosphate or alpha-agonists. These studies suggest that the inhibition of hepatic methotrexate secretion by probenecid in vivo is likely to be a consequence of interference with hepatic uptake of the antifolate rather than an interaction of probenecid and methotrexate at a hepatic "secretory" site.

Animals↗
PubMed1993

Accelerated nodulosis and systemic manifestations during methotrexate therapy for rheumatoid arthritis.

OBJECTIVES: Methotrexate is successfully used in the treatment of arthritis but little is known about its effects on extra-articular manifestations of rheumatoid arthritis. We focused this work on the incidence and clinical course of extra-articular manifestations during long-term treatment with methotrexate. METHODS: The effect of methotrexate on extra-articular manifestations was investigated in 176 patients with rheumatoid arthritis who had obtained, in a prospective study, a good clinical response to methotrexate (10 mg/week) taken for 33 months (range 4-68). RESULTS: Before taking methotrexate, 44 patients (25.1%) had extra-articular manifestations: nodules (n = 40) and vasculitis (n = 9). With methotrexate, nodulosis and vasculitis were stable in 31 cases, improved in 3 and worsened in 10 (23%). Among the 132 patients without extra-articular manifestations before methotrexate therapy, 15 (11%) developed accelerated nodulosis preferentially located on the fingers, 7 had a vasculitis and 3 a pericarditis during methotrexate therapy. Extra-articular manifestations occurred between 1 and 24 months of initiating methotrexate therapy. Rheumatoid factor was positive in 88% of the patients with extra-articular manifestations. No relationship was noted between extra-articular manifestations and HLA type or antinuclear antibodies. In 3 out of 4 patients who developed accelerated nodulosis while taking methotrexate, the addition of hydroxychloroquine (400 mg/day) resulted in a significant reduction in the number and size of the nodules within 3 to 10 months after starting combined therapy. CONCLUSION: These data suggest that methotrexate is not effective in the treatment of extra-articular manifestations in rheumatoid arthritis and that nodulosis may occur in about 11% of patients taking methotrexate therapy for rheumatoid arthritis. The combination of hydroxychloroquine and methotrexate may have a beneficial effect on nodulosis that needs to be evaluated.

Adolescent↗
PubMed1983

Studies of formation and efflux of methotrexate polyglutamates with cultured hepatic cells.

Methotrexate polyglutamates are extensively synthesized when cultured hepatocytes and H35 hepatoma cells are exposed to micromolar concentrations of methotrexate. The predominant species found within the cell have from two to four additional gamma-linked glutamate residues. When either cell type containing a mixture of methotrexate and its polyglutamate derivatives is exposed to medium lacking methotrexate, there is a rapid release of methotrexate. This release has a T1/2 of 2 to 4 min and is apparently complete within 30 to 60 min. Methotrexate polyglutamates leave the cells much more slowly and appear to do so by two mechanisms. Although cleavage to methotrexate and subsequent efflux appears to be quantitatively the more important pathway, there is also a slow, finite loss of intact methotrexate polyglutamates from cells which exclude trypan blue. The T1/2 for the loss of methotrexate polyglutamates by both cell types, when placed in medium lacking methotrexate, is approximately 6 to 8 hr. These results, together with those of an earlier study (Galivan, J. (1980) Mol. Pharmacol. 17:105-110), suggest that the polyglutamate derivatives are forms of methotrexate which are as cytotoxic as methotrexate but which offer a potentially greater capacity for cellular destruction because they are retained longer in the tissue.

Animals↗
PubMed1999

Characterization of the bile acid sensitive methotrexate carrier of rat liver cells.

The chemotherapeutic agent methotrexate is widely used in tumor therapy for different forms of leukemia and for the therapy of arthritis. Methotrexate is eliminated from systemic blood circulation by the liver and its transport into hepatocytes is therefore described in detail in this paper. Methotrexate uptake is energy- and sodium-dependent. The Km and the Vmax are 23 microM and 36 pmol/mg protein min, respectively. The apparent activation energy (E(app)) of methotrexate uptake (5 microM [3H]methotrexate) is 53.73 kJ/mol, which indicates an energy-dependent carrier-mediated process. Although methotrexate is a folate derivative, folate itself does not inhibit methotrexate uptake, whereas the reduced folates, dihydrofolate and tetrahydrofolate are weak uncompetitive inhibitors. In contrast, the bile acids taurocholate and cholate are effective competitive inhibitors of methotrexate uptake into hepatocytes. Further strong inhibitors are the loop diuretic bumetanide, the mycotoxin ochratoxin A and bromosulfophthalein. Because tumor patients develop drug resistance during methotrexate therapy, the uptake of methotrexate was tested in different hepatoma cell lines. In HepG2-cells and Reuber hepatoma Fao-cells the transport was non-existent or very small. However, the hepatocytoma fusion cell line HPCT-1E3, a hybrid cell line between primary rat hepatocytes and rat Reuber Fao-cells, shows an intermediate transport activity with a threefold increase of the methotrexate uptake. These results indicate the presence of a bile acid sensitive methotrexate carrier in hepatocytes which is absent in dedifferentiated hepatoma cells. The carrier differs from previously described transporters for the uptake of organic anions.

Animals↗
PubMed1990

Unexpectedly high serum methotrexate levels in cystectomized bladder cancer patients with an ileal conduit treated with intermediate doses of the drug.

The pharmacokinetics of serum methotrexate were studied in 45 bladder cancer patients receiving 250 mg. per m.2 as part of the initial cycle of combination chemotherapy. Serum methotrexate was determined routinely 43 to 49 hours after administration. If the methotrexate levels remained at more than 80 nmol. per l. measurements were repeated daily until the serum levels decreased below this point. The patients were classified into group 1-23 with a bladder in situ and no ureteral obstruction, group 2-11 with a bladder in situ and unilateral hydronephrosis, and group 3-11 who had had cystectomy and ileal conduit diversion before chemotherapy. Of the patients in groups 1 and 2, 5 and 6, respectively, had serum methotrexate levels of 80 nmol. per l. or more 43 to 49 hours after administration, which decreased to below this level on the next day. Of the 11 patients in group 3, 8 had elevated methotrexate levels at the initial determination. Daily methotrexate analyses showed a delayed elimination in 4 of 7 patients and levels of more than 80 nmol. per l. for 3 to 9 days. Low creatinine clearance but, in particular, the previous performance of an ileal conduit predicted high methotrexate levels on day 2 after treatment. The most likely explanation for this observation is the resorption of methotrexate by the small bowel mucosa in the ileal conduit. Patients with an ileal conduit performed 2 years or less before chemotherapy and/or those with a long ileal segment seem to have a particularly high risk for delayed methotrexate elimination. Bladder cancer patients with an ileal conduit who receive methotrexate-containing chemotherapy have a high risk of delayed methotrexate elimination and increased clinical methotrexate toxicity. Leukovorin rescue should be used liberally in these patients together with other prophylactic means (intensive hydration and alkalization of the urine).

Adult↗
PubMed1998

Resolution of hormonal markers of ectopic gestation: a randomized trial comparing single-dose intramuscular methotrexate with salpingostomy.

OBJECTIVE: To evaluate resolution of serum hCG and progesterone in patients with ectopic pregnancy receiving single-dose intramuscular (IM) methotrexate as compared with those undergoing laparoscopic salpingostomy. METHODS: In this prospective randomized clinical trial, 75 hemodynamically stable women with a diagnosis of ectopic pregnancy were randomized to treatment with single-dose IM methotrexate (1 mg/kg) or laparoscopic salpingostomy. All women had initial, day 4, and weekly serum hCG and progesterone measurements taken until hCG levels were less than 15 mIU/mL. Methotrexate therapy was repeated if posttreatment day 7 hCG levels did not decrease by 15%, as compared with day 4 levels. Success rate was defined as ectopic resolution without the need for the alternate mode of therapy. RESULTS: Thirty-eight women were randomized to treatment with methotrexate and 37 to laparoscopic salpingostomy. The mean (+/-standard deviation) time required for serum progesterone concentrations to decrease to less than 1.5 ng/mL was significantly less for laparoscopic salpingostomy than for treatment with methotrexate: 7.8+/-1.7 and 17.6+/-2.2 days, respectively (P < .01). Within each treatment group, serum progesterone levels resolved (less than 1.5 ng/mL) more rapidly than did hCG levels (less than 15 mIU/mL) (P < .01). No further treatment was required once serum progesterone levels had decreased to less than 1.5 ng/mL. Success rates were similar in both groups: 94.7% (36 of 38) for methotrexate and 91.4% (33 of 36) for laparoscopic salpingostomy. Mean time required for hCG concentrations to decrease to less than 15 mIU/mL was significantly less for laparoscopic salpingostomy than for methotrexate therapy: 20.2+/-2.7 and 27.2+/-2.3 days, respectively (P < .05). Additional methotrexate injections were required in 15.8% (6 of 38) of women randomized to methotrexate therapy. Initial serum hCG levels for patients receiving additional methotrexate doses were 4830+/-1588 mIU/mL as compared with 2133+/-393 mIU/mL for women receiving only one dose (P = .07). CONCLUSION: Serum progesterone levels of less than 1.5 ng/mL are a good predictor of ectopic pregnancy resolution regardless of treatment, and because its return to normal values occurs more rapidly than that of hCG levels, serum progesterone may be a better marker for predicting successful treatment. Although laparoscopic salpingostomy leads to faster resolution of hormonal markers of ectopic gestation, methotrexate is equally successful for treating small unruptured ectopic pregnancies. Initial hCG levels may be a marker for women requiring additional doses of methotrexate.

Abortifacient Agents, Nonsteroidal↗
PubMed2001

Role of kidney-specific organic anion transporters in the urinary excretion of methotrexate.

BACKGROUND: High-dose folinic acid is used to accelerate methotrexate elimination to avoid renal toxicity of the drug. The present study was carried out to examine the role of the renal organic anion transporters OAT-K1 and OAT-K2 in the urinary excretion of methotrexate, especially in the methotrexate-folinic acid rescue therapy. METHODS: Madin-Darby canine kidney cells stably expressing OAT-K1 and OAT-K2 were used for the in vitro transport study; 5/6 nephrectomized rats were used to detect changes in mRNA expression levels of OAT-K1 and OAT-K2 and to evaluate methotrexate pharmacokinetics under conditions of renal insufficiency. RESULTS: Methotrexate efflux mediated by these transporters in stable transfectants was stimulated in the presence of extracellular folic acid and folinic acid, suggesting that they could serve as anion exchangers to enhance the apical efflux of methotrexate. The mRNA expression levels of OAT-K1 and OAT-K2 were markedly diminished after 5/6 nephrectomy, but those of multidrug resistance associated protein 2, which could transport methotrexate, were maintained. Renal clearance of methotrexate was markedly decreased in 5/6 nephrectomized rats compared with that in sham-operated rats. Additional folinic acid treatment resulted in a significant increase in methotrexate renal clearance in sham-operated rats but not in 5/6 nephrectomized rats. CONCLUSIONS: The decreased expressions of OAT-K1 and OAT-K2 may be attributable to the longer exposure to methotrexate and ineffective folinic acid rescue. In terms of contributing to patient safety, renal clearance of methotrexate, especially folinic acid-stimulated tubular secretion of the drug via these transporters, would be a key factor in methotrexate therapy.

Animals↗
PubMed1996

Methotrexate alters carbon flow through the hepatic folate-dependent one-carbon pool in rats.

The chemotherapeutic value of methotrexate resides in its ability to perturb folate-dependent one-carbon metabolism and subsequently inhibit DNA synthesis. To assess the functional effect of methotrexate on hepatic one-carbon metabolism, we have developed and applied tracer kinetic techniques in vivo to quantify the carbon flux through the folate-dependent one-carbon pool in rats. Following a 7-day treatment period with methotrexate (0.2 mg/kg body weight), the tracers L-[ring-2-14C] histidine and L-[methyl-3H] methionine were simultaneously infused into control and methotrexate-treated rats. Methotrexate treatment decreased hepatic tetrahydrofolate, methyltetrahydrofolate, and formyltetrahydrofolate, concentrations by 63, 83, and 58%, respectively. Furthermore, the enzymatic activity of 10-formyltetrahydrofolate dehydrogenase, the terminal enzyme in the catabolism of the ring-2-carbon of histidine to CO2, was diminished by 32% in methotrexate-treated animals. These changes in enzyme activity and folate coenzyme concentrations did not result in a significant decrease in the oxidative flow of carbon from histidine to CO2 in methotrexate-treated rats compared to control animals (2.40 and 3.22 micromol/h/kg3/4, respectively). Oxidative carbon flow was reflective of tetrahydrofolate and formyltetrahydrofolate pools when expressed as a percent of total folate: neither coenzyme pool was diminished as a result of methotrexate treatment. In contrast, the reductive carbon flux through the one-carbon pool from histidine to methionine was significantly decreased 59% in methotrexate-treated (7.63 micromol/h/kg3/4) versus control rats (18.73 micromol/h/kg3/4)). Likewise, methyltetrahydrofolate, as a percent of total folate, was reduced 51% in methotrexate-treated rats. Consequently, total measured carbon flow (oxidative+reductive) was 54% lower in rats subjected to subchronic methotrexate treatment. These tracer kinetic experiments quantitatively demonstrate the extent to which methotrexate alters the actual carbon flow through the hepatic folate-dependent one-carbon pool, primarily directed at diminishing the reductive carbon flow towards methyltetrahydrofolate and methionine synthesis.

Animals↗
PubMed1984

Persistence of antifolate activity in skin of rats following systemic administration of methotrexate.

Studies of the time course of methotrexate-derived antifolate activity (methotrexate activity) in plasma and skin of rats have been carried out to evaluate the hypothesis that the formation of therapeutically active poly-gamma-glutamyl conjugates of methotrexate in skin after systemic administration of the drug will result in the prolongation of methotrexate effects in skin. Such a finding may eventually bear on the use of the drug in the treatment of psoriasis. Three groups of 4 rats received a single i.v. dose of methotrexate, 5, 20, or 40 mg/kg, respectively. Serial skin and plasma samples were obtained over 48 h from each rat and analyzed for methotrexate activity by a dihydrofolate reductase enzyme inhibition assay. The values of pharmacokinetic parameters describing the disposition of the drug were calculated from plasma data using standard methods. Skin to plasma ratio of methotrexate activity was also calculated each time a skin sample was obtained. Pharmacokinetic parameter values and skin to plasma ratios were not significantly different between doses. Methotrexate activity declined biexponentially in plasma and skin. The terminal half-life in plasma (mean +/- SD) was 20.3 +/- 9.2 h. At early times, methotrexate activity in skin was less than plasma, but at later times activity in skin was an order of magnitude greater than that in plasma. In a preliminary study to determine whether the persistent activity in skin is associated with the presence of poly-gamma-glutamyl conjugates of methotrexate, a rat was dosed with tritiated methotrexate, 0.18 mg/kg, i.v., and skin was obtained 24 h later. Skin homogenate was treated with papain and applied to an anion-exchange column for cleanup prior to injection on a reversed-phase high-pressure liquid chromatography system. Methotrexate poly-gamma-glutamates were identified by elution with authentic standards and specific hydrolysis with carboxypeptidase G1.

Animals↗
PubMed2004

Lumiracoxib does not affect methotrexate pharmacokinetics in rheumatoid arthritis patients.

BACKGROUND: Methotrexate and nonsteroidal antiinflammatory drugs are frequently coadministered in the treatment of rheumatoid arthritis (RA). OBJECTIVE: To evaluate the effect of lumiracoxib, a novel cyclooxygenase-2 selective inhibitor, on methotrexate pharmacokinetics and short-term safety in patients with RA. METHODS: This multicenter, randomized, double-blind, placebo-controlled crossover study enrolled 18 patients (mean age 49.1 y) with stable RA. Patients were randomized to receive methotrexate 7.5-15 mg orally once weekly plus either lumiracoxib 400 mg/day or placebo for 7 days. Patients then received the other treatment combination for an additional 7 days. Serial blood and urine were collected for 24 hours after the methotrexate dose on day 1 (methotrexate alone) and days 8 and 15 (combination treatment). RESULTS: Plasma methotrexate pharmacokinetics (AUC(0-t), maximum concentration [C(max)], time to C(max)) and methotrexate protein binding were similar for methotrexate alone (108.0 ng.h/mL, 26.7 ng/mL, 1.5 h, and 57.1%, respectively), methotrexate/lumiracoxib (110.2 ng.h/mL, 27.5 ng/mL, 1.0 h, and 53.7%, respectively), and methotrexate/placebo (101.8 ng.h/mL, 22.6 ng/mL, 1.0 h, and 57.0%, respectively). Similarly, no clinically significant difference was found in the urinary excretion of methotrexate. Mean exposure to the 7-OH metabolite was lower when methotrexate was given with lumiracoxib compared with placebo, shown by a reduction in AUC and C(max), although similar amounts of the metabolite were recovered in urine following both lumiracoxib and placebo. Coadministration of methotrexate and lumiracoxib was well tolerated. CONCLUSIONS: Lumiracoxib had no significant effect on the pharmacokinetics, protein binding, or urinary excretion of coadministered methotrexate in patients with RA.

Adolescent↗
PubMedDate not supplied

Methotrexate pulse therapy in the treatment of recurrent acute heart rejection.

Despite cytolytic induction therapy and triple-drug immunosuppression, acute allograft rejection continues to cause important morbidity and occasional death after heart transplantation. Between November 1, 1988, and May 1, 1990, 24 patients received methotrexate pulse therapy for recurrent or persistent acute rejection despite methylprednisolone, OKT3, or antithymocyte globulin therapy. Methotrexate was administered as a daily oral dose of 2.5 to 15 mg on 1 day/week over 3 weeks (longer in 15 patients because of either severe leukopenia with temporary interruption of therapy or recurrent rejection during methotrexate therapy) with reduction or discontinuation of azathioprine. Rejection incidence was reduced from 1.1 episodes/patient month before methotrexate therapy to 0.2 episodes/patient month after completion of therapy (p = 0.0001). Two patients died within 3 months after treatment, one of cytomegalovirus pneumonia and one of lymphoma. Mean white blood count (WBC) fell from 6900 per ml before methotrexate therapy to 3700 during the first month of methotrexate therapy (p = 0.0005). The lowest WBC typically occurred about 3 weeks after starting methotrexate therapy, and a transient WBC of less than 1000/ml developed in seven patients. By multivariable analysis, the WBC 1 month after starting methotrexate therapy was significantly related to greater bone marrow suppression (lower WBC), immediately before methotrexate therapy, greater overall immunosuppression (more rejection episodes) during the 3 months before methotrexate therapy, and a higher total dose of methotrexate. The following conclusions can be drawn: (1) Methotrexate is a useful adjunct in the treatment of recurrent or persistent rejection.(ABSTRACT TRUNCATED AT 250 WORDS)

Acute Disease↗
PubMed1990

Methotrexate transport in variant human CCRF-CEM leukemia cells with elevated levels of the reduced folate carrier. Selective effect on carrier-mediated transport of physiological concentrations of reduced folates.

This study reports the isolation and characterization of a variant of the human CCRF-CEM leukemia cell line that overproduces the carrier protein responsible for the uptake of reduced folates and the folate analogue methotrexate. The variant was obtained by adapting CCRF-CEM cells for prolonged times to stepwise decreasing concentrations of 5-formyltetrahydrofolate as the sole folate source in the cell culture medium. From cells that were grown on less than 1 nM 5-formyl-tetrahydrofolate, a variant (CEM-7A) was isolated exhibiting a 95-fold increased Vmax for [3H]methotrexate influx compared to parental CCRF-CEM cells. The values for influx Km, efflux t0.5, and Ki for inhibition by other folate (analogue) compounds were unchanged. Affinity labeling of the carrier with an N-hydroxysuccinimide ester of [3H]methotrexate demonstrate an approximately 30-fold increased incorporation of [3H] methotrexate in CEM-7A cells. This suggests that the up-regulation of [3H]methotrexate influx is not only due to an increased amount of carrier protein, but also to an increased rate of carrier translocation or an improved cooperativity between carrier protein molecules. Incubation for 1 h at 37 degrees C of CEM-7A cells with a concentration of 5-formyltetrahydrofolate or 5-methyltetrahydrofolate in the physiological range (25 nM) resulted in a 7-fold decline in [3H]methotrexate influx. This down-regulation during incubations with 5-formyltetrahydrofolate or 5-methyltetrahydrofolate could be prevented by either the addition of 10-25 nM of the lipophilic antifolate trimetrexate or by preincubating CEM-7A cells with 25 nM methotrexate. The down-regulatory effect was specifically induced by reduced folates since incubation of CEM-7A cells with 25 nM of either methotrexate, 10-ethyl-10-deazaaminopterin, aminopterin, or folic acid, or a mixture of purines and thymidine, had no effect on [3H]methotrexate influx. Similarly, these down-regulatory effects on [3H]methotrexate transport by 5-formyltetrahydrofolate, and its reversal by trimetrexate or methotrexate, were also observed, though to a lower extent, for parental CCRF-CEM cells grown in folate-depleted medium rather than in standard medium containing high folate concentrations. These results indicate that mediation of reduced folate/methotrexate transport can occur at reduced folate concentrations in the physiological range, and suggest that the intracellular folate content may be a critical determinant in the regulation of methotrexate transport.

Biological Transport↗
PubMed1986

Characterization of the individual transport routes that mediate the influx and efflux of methotrexate in CCRF-CEM human lymphoblastic cells.

The transport routes utilized by CCRF-CEM human lymphoblastic cells for the influx and efflux of methotrexate have been analyzed. Evidence was obtained for a single influx route for methotrexate: (a) Influx at 2 microM [3H]methotrexate was inhibited completely by high concentrations of unlabeled methotrexate, o-phthalate, and bromosulfophthalein, and the inhibition profile with each anion was monophasic; and (b) Pretreatment of the cells with an N-hydroxysuccinimide ester of methotrexate also blocked influx, and this inhibition was complete over a range of substrate concentrations from 2 to 50 microM. Influx was also saturable and proceeded with a maximum rate (Vmax) of 4.3 pmol/min/mg protein (at 37 degrees C) and with a Kt of 0.8 microM in an anion-deficient buffer and 4.6 microM in a 4-(2-hydroxyethyl)-1-piperazineethanesulfonate-buffered saline. The ratio of Vmax to the amount of carrier protein (0.3 pmol/mg protein) gave a turnover number for the transport system of 14.3/min. In contrast to influx, methotrexate efflux proceeded via three routes which could be separated by their sensitivity to specific inhibitors. The major portion of efflux occurred via the methotrexate influx carrier, the identity of which was established from its sensitivity to the N-hydroxysuccinimide ester of methotrexate and by its requirement for anions in the external medium. Methotrexate, adenosine monophosphate, and phosphate each stimulated efflux via this route and this stimulation was half-maximal at anion concentrations that approximated their Ki values for inhibition of methotrexate influx. A second efflux route was identified by its sensitivity to bromosulfophthalein. This route was relatively inactive and did not fluctuate significantly upon addition of various anions, glucose, or metabolic inhibitors. The third route was quantitated by its sensitivity to probenecid and its activity was increased in saline buffers and upon addition of glucose and was inhibited by oligomycin. Similar transport routes for methotrexate are present in L1210 mouse cells, although these two cell lines can be distinguished by the amount of transport protein and by the activity of the bromosulfophthalein-sensitive efflux route for methotrexate.

ATP Binding Cassette Transporter, Subfamily G, Mem↗
PubMedDate not supplied

Methotrexate therapy in pediatric heart transplantation as treatment of recurrent mild to moderate acute cellular rejection.

We have used adjunctive therapy with methotrexate as treatment of recurrent mild-to-moderate acute cellular rejection and in an attempt to reduce rejection frequency and corticosteroid dosage. The purpose of this study was to review our experience with this treatment strategy. Eight patients, 13.1 +/- 1.1 years of age (mean +/- standard error of the mean) at the time of transplantation, were given methotrexate in addition to their standard triple therapy immunosuppression. Methotrexate was started at 6.2 +/- 2 months after transplantation after an average of 3.1 +/- 0.4 rejection episodes. Patients were given methotrexate weekly for 8 weeks at 2.5 or 5 mg orally every 12 hours for three doses (0.23 +/- 0.02 mg/kg/week). The time to resolution of rejection was 17.9 +/- 4 days after initiating methotrexate therapy. The number of rejections per month decreased significantly from the 2 months before methotrexate therapy (1.49 +/- 0.1) when compared with both the 2 months during methotrexate therapy (0.50 +/- 0.1) and the 2 months after methotrexate therapy was completed (0.44 +/- 0.3) (p < 0.005). Furthermore, when comparing total rejection frequency since transplantation and before methotrexate therapy to a follow-up period of 21.8 +/- 5 months after completion of methotrexate therapy, the rejection frequency was significantly less (0.81 +/- 0.2 versus 0.10 +/- 0.06 rejections/month) (p < 0.01). Prednisone dosage was also significantly less when comparing the time before methotrexate therapy to immediately after completion of methotrexate therapy (0.23 +/- 0.04 versus 0.15 +/- 0.03 mg/kg/day) (p < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Acute Disease↗
PubMed1996

[Methotrexate in gynecologic oncology].

Methotrexate produced the first remission in leukemia and the first cure of a solid tumor, choriocarcinoma. Methotrexate tightly binds to dihydrofolate reductase (DHFR), blocking the reduction of dihydrofolate to tetrahydrofolic acid, the active form of folic acid. Methotrexate also directly inhibits the folate-dependent enzymes of de novo purine and thymidylate synthesis. Resistance to methotrexate may develop as a result of elevated DHFR activity or defective transport of methotrexate into malignant cells. Increased DHFR enzyme levels may also result from amplification of the DHFR gene, which is now clinically significant in selected patients. Methotrexate is an active drug in the first-line treatment of gestational trophoblastic disease (GTD) and in metastatic squamous cell carcinoma of the cervix. Since the introduction of methotrexate chemotherapy for malignant GTD, most hospitals have reported almost 100% cure rates for patients with nonmetastatic disease using single-agent regimens. Patients with low-risk metastatic disease have been treated with methotrexate and folinic acid and over 50% complete remission rates have been reported. Patients with metastatic GTD who had one or more high-risk factors benefited from initial multiagent chemotherapy, rather than waiting for acquisition of drug-resistance to single-agent therapy to start multiagent treatment. Using multiagent combination chemotherapy such as MAC (methotrexate, actinomycin D, cyclophosphamide) or EMA-CO (etoposide, methotrexate, actinomycin D and cyclophosphamide, vincristine), most investigators have reported remission in approximately 60 to 80% of patients with high-risk metastatic GTD. Although the role of chemotherapy in carcinoma of the cervix has been limited for several reasons, trial of combination chemotherapy including methotrexate has been reported. However, it is still impossible to draw definite conclusions as to whether methotrexate combined with another clearly active drug may yield a superior response rate and survival.

Adult↗
PubMed1996

Rates of development of methotrexate resistance in heterogeneous mouse mammary tumor cell cultures.

Our previous studies have indicated that the expression by tumor cells of sensitivity to chemotherapeutic drugs such as methotrexate can be affected by the presence of other tumor cells; thus, otherwise methotrexate-resistant cells may respond to that drug in the presence of methotrexate-sensitive cells. In order to determine whether tumor heterogeneity also affects the emergence of drug resistance, we measured the rate of development of methotrexate resistance in mixed monolayer cultures of three mammary tumor subpopulation lines (66, 168TFAR, 4T07) that differ in degree of sensitivity to methotrexate. Cultures were treated weekly with 80 nM or 200 nM methotrexate. Each individual cell line was re-isolated from the mixture by passage in selective medium and then assayed for methotrexate sensitivity. Cultures of each of the three lines were treated and assayed in parallel. Few differences in the rate of development of methotrexate resistance were seen among cells from mixtures and cells cultured alone; line 4T07 appeared to become resistant somewhat more rapidly in mixtures. In untreated mixed cultures, line 66, the line least sensitive to methotrexate, gradually became dominant; this process was accelerated in treated cultures. One methotrexate-resistant subline from each parent cell line was tested to determine the mechanisms by which methotrexate resistance was increased. Two lines appeared to have increased levels of dihydrofolate reductase, and one exhibited decreased methotrexate transport as well. The third cell line had neither mechanism. Others have shown that tumor heterogeneity can act as a brake on the rate of development of new metastatic or immunogenic variants. Our data indicate that, at least in the model system we have tested, the rate of development of extrinsic drug resistance is substantially independent of pre-existing clonal diversity.

Animals↗