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PubMed1988

Proliferation-dependent cytotoxicity of methotrexate in murine L5178Y leukemia.

The basis for the proliferation-dependent cytotoxicity of methotrexate has been investigated in mice bearing the L5178Y ascites leukemia. Methotrexate at 60 mg/kg i.p. reduced the viability of logarithmically growing ascites cells (55% active S phase cells) to 28% of control, whereas the viability of the slowly growing cells (18% active S phase) was decreased to only 59% of control. Log phase tumor cells accumulated 8-fold higher levels of methotrexate polyglutamates compared to cells that had approached the stationary phase. However, no differences between log phase and slowly growing tumor cells were observed in the cellular levels of unmetabolized methotrexate. Intestinal mucosa and bone marrow from non-tumor-bearing mice resembled slowly growing tumor cells and had markedly lower levels of methotrexate polyglutamates than logarithmically growing cells. The greater accumulation of methotrexate polyglutamates in the logarithmically growing tumor cells was consistent with an increased synthesis of methotrexate polyglutamates in these cells. The enhanced methotrexate polyglutamylation in log phase versus slowly growing cells was not related to changes in the rates of either cellular methotrexate transport, transmembrane efflux of methotrexate, or hydrolysis of methotrexate polyglutamates. Thymidylate synthase activity measured in situ and in extracts from log phase cells was 4- and 2-fold higher, respectively, than in the more slowly growing cells. Methotrexate produced a 2.4-fold greater depletion of poly-gamma-glutamyl derivatives of 5,10-methylenetetrahydropteroylglutamate in log phase cells compared to slowly growing cells, and this was a function of both the increased methotrexate polyglutamate accumulation and thymidylate synthase activity in the rapidly proliferating cells. These results provide further evidence that the selectivity of methotrexate for tumors with a high growth fraction is a consequence of the rapid rates of both cellular methotrexate polyglutamate synthesis and oxidation of 5,10-methylenetetrahydropteroyl polyglutamates by thymidylate synthase.

Animals↗

PubMed1980

Plasma methotrexate as determined by liquid chromatography, enzyme-inhibition assay, and radioimmunoassay after high-dose infusion.

Three techniques for measuring methotrexate show various cross reactivities with methotrexate-related compounds: "high-pressure" liquid chromatography, by principle, is virtually specific for methotrexate; the enzyme-inhibition assay quantitates methotrexate, methotrexate diglutamate, and methotrexate triglutamate equally well, but has a 10% cross reactivity with 4-amino-4-deoxy-N10-methylpteroic acid and 1% with 7-hydroxymethotrexate; radioimmunoassay shows an equal cross reactivity with methotrexate, 4-amino-4-deoxy-N10-methylpteroic acid, methotrexate diglutamate and triglutamate, and a 5 to 10% cross reactivity with 7-hydroxymethotrexate. Radioimmunoassay almost always yielded the highest values for methotrexate, followed by enzyme-inhibition assay then liquid chromatography. The presence of two methotrexate-related compounds, 7-hydroxymethotrexate and 4-amino-4-deoxy-N10-methylpteroic acid, was confirmed in human urine samples and quantitated in patients' plasma by liquid chromatography, the respective maximum plasma concentrations being 250 and 16 mumol/L. Materials cross reacting with methotrexate in radioimmunoassay of chromatographic fractions from plasma were also noted in fractions corresponding to methotrexate diglutamate and triglutamate peaks, in quantities estimated to be 47 and 30 nmol/L methotrexate equivalents, respectively. 7-Hydroxymethotrexate is eliminated more slowly than methotrexate and its production increases with dosages of methotrexate.

Adolescent↗

PubMed1980

Irreversible inactivation of the methotrexate transport system of L1210 cells by carbodiimide-activated substrates.

Methotrexate and 1-ethyl-3(3-dimethylaminopropyl)carbodiimide react to form a relatively unstable product (activated methotrexate) that irreversibly inhibits methotrexate transport in L1210 mouse leukemia cells. The rate of methotrexate transport was reduced 50% by pretreatment of cells with activated methotrexate at a concentration of 1 microM and by 98% upon exposure to an excess (40 microM) of this agent. Specificity was demonstrated by the fact that activated methotrexate had no effect on other transport systems (i.e. leucine and phosphate) and that complete protection against inactivation was afforded by either the unlabeled substrate (methotrexate) or a competitive inhibitor (phosphate). The carbodiimide-generated derivatives of aminopterin, folate, and p-aminobenzoylglutamate were also irreversible inhibitors of methotrexate transport, and their relative effectiveness paralleled the ability of the parent compounds (aminopterin greater than methotrexate greater than folate greater than p-aminobenzoylglutamate) to act as reversible transport inhibitors. Measurements at 4 degrees C showed that activated methotrexate (Ki = 0.8 microM) was a competitive inhibitor for binding of methotrexate (KD = 0.4 microM) to the transport protein. Treatment of the cells with sufficient activated methotrexate to inhibit transport increased the KD for methotrexate to 0.9 microM but had no effect on total methotrexate-binding activity. Methotrexate efflux was also reduced but was not blocked by an excess of this reagent.

Animals↗

PubMed1989

Role of the membrane-associated folate binding protein (folate receptor) in methotrexate transport by human KB cells.

The uptake of methotrexate by KB cells was observed to be dependent on time, temperature, and concentration of extracellular methotrexate. The Kd for methotrexate surface binding to KB cells was approximately 200 nM. Following exposure of KB cells to trace quantities of [3H]methotrexate for periods ranging from 6 min to 24 h, the cellular methotrexate was progressively formed into methotrexate polyglutamates and was bound to dihydrofolate reductase as well as to a particulate folate binding protein. To further study the mechanism of methotrexate uptake in KB cells, the N-hydroxysuccinimide ester of methotrexate was used to covalently label the surface of KB cells and to inhibit transport of methotrexate. The N-hydroxysuccinimide ester of methotrexate was bound to a species of protein with an apparent molecular weight of 160,000 in 1% (v/v) Triton X-100 that bound folic acid and was specifically precipitated by antiserum raised against the previously purified high-affinity folate binding protein (the folate receptor) from human KB cells. In addition, trypsin was utilized to remove surface-accessible covalently bound methotrexate. The amount of covalently bound methotrexate that could be released by trypsin initially decreased on incubation at 37 degrees C, suggesting that the methotrexate and binding protein were internalized. However, with time, trypsin could again release the covalently bound methotrexate, suggesting that the binding protein cycles from the external cell surface to the inside of the cell and out again.

Binding, Competitive↗

PubMed1986

Liposome-mediated delivery of pteridine antifolates to cells in vitro: potency of methotrexate, and its alpha and gamma substituents.

We have examined the growth-inhibitory potency of several pteridines encapsulated in negatively charged liposomes, including methotrexate, methotrexate-gamma-methylamide, methotrexate-gamma-dimethylamide, methotrexate-alpha-aspartate, and a lipophilic methotrexate-phosphatidylethanolamine conjugate. The potency of encapsulated methotrexate is greater than the potency of the free drug for CV1-P cells, but not for other cell lines. The potency of methotrexate-gamma-methylamide and methotrexate-gamma-dimethylamide is only minimally improved by encapsulation. The potency of methotrexate-alpha-aspartate is increased by encapsulation. In addition, the lipophilic methotrexate derivative has demonstrable potency when incorporated in liposomes. We have also examined the potency of several pteridines under conditions where the cells are exposed to the drug for periods shorter than the entire growth assay. Reduction of the exposure time decreases the potency of both encapsulated and free drugs. However, the difference in potency between the encapsulated and free drug is increased, because the potency of the encapsulated drug is affected less. Consequently, encapsulated methotrexate-gamma-aspartate is 300-fold more potent than free drug, if CV1-P cells are exposed to drug for 4 h. Moreover, encapsulated methotrexate is more potent than free methotrexate for growth inhibition of L929 fibroblasts, if the term of exposure is less than 8 h. Potency is least affected by reduction of exposure length for the lipophilic methotrexate derivative.

Cell Line↗

PubMed1984

Protection of cells from methotrexate toxicity by 7-hydroxymethotrexate.

Cell growth survival studies have revealed that 7-OH methotrexate is two orders of magnitude less cytotoxic to human melanoma and human acute lymphoblastic leukaemia (ALL) cells in vitro than methotrexate. The influence of 7-OH methotrexate on methotrexate toxicity was investigated by studying cell growth in the presence of methotrexate and its 7-OH metabolite and by studying [3H]-methotrexate movement across the plasma membrane of isolated human cells. Transport was followed for net entry of the drug into drug-free cells, net exit of drug into drug-free medium and for unidirectional exit fluxes with drug and/or metabolite in the extracellular medium (exchange exit). Results indicate that 7-OH methotrexate (10(-6) M) interacts with melanoma cells to reduce the initial cellular uptake rate of [3H]-methotrexate but that no such interaction occurs with ALL cells. Efflux measurements revealed that a stimulatory effect of extracellular methotrexate on [3H]-methotrexate exit was apparent and that extracellular 7-OH methotrexate had a less stimulatory effect. Overall, loss of intracellular drug was greater from melanoma cells than from ALL cells. The results suggest that the drug resistance encountered following high dose therapy may be due to reduced cellular uptake and/or increased efflux of methotrexate from cells, both events being enhanced by 7-OH methotrexate. In addition, there is an apparently endogenous resistance of the melanomas to methotrexate as regards time of exposure to this agent which could also contribute to the lack of clinical response when compared to ALL.

Acute Disease↗

PubMed1991

Methotrexate inhibits neutrophil function by stimulating adenosine release from connective tissue cells.

Although commonly used to control a variety of inflammatory diseases, the mechanism of action of a low dose of methotrexate remains a mystery. Methotrexate accumulates intracellularly where it may interfere with purine metabolism. Therefore, we determined whether a 48-hr pretreatment with methotrexate affected adenosine release from [14C]adenine-labeled human fibroblasts and umbilical vein endothelial cells. Methotrexate significantly increased adenosine release by fibroblasts from 4 +/- 1% to 31 +/- 6% of total purine released (EC50, 1 nM) and by endothelial cells from 24 +/- 4% to 42 +/- 7%. Methotrexate-enhanced adenosine release from fibroblasts was further increased to 51 +/- 4% (EC50, 6 nM) and from endothelial cells was increased to 58 +/- 5% of total purine released by exposure to stimulated (fMet-Leu-Phe at 0.1 microM) neutrophils. The effect of methotrexate on adenosine release was not due to cytotoxicity since cells treated with maximal concentrations of methotrexate took up [14C]adenine and released 14C-labeled purine (a measure of cell injury) in a manner identical to control cells. Methotrexate treatment of fibroblasts dramatically inhibited adherence to fibroblasts by both unstimulated neutrophils (IC50, 9 nM) and stimulated neutrophils (IC50, 13 nM). Methotrexate treatment inhibited neutrophil adherence by enhancing adenosine release from fibroblasts since digestion of extracellular adenosine by added adenosine deaminase completely abrogated the effect of methotrexate on neutrophil adherence without, itself, affecting adherence. One hypothesis that explains the effect of methotrexate on adenosine release is that, by inhibition of 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) transformylase, methotrexate induces the accumulation of AICAR, the nucleoside precursor of which (5-aminoimidazole-4-carboxamide ribonucleoside referred to hereafter as acadesine) has previously been shown to cause adenosine release from ischemic cardiac tissue. We found that acadesine also promotes adenosine release from and inhibits neutrophil adherence to connective tissue cells. The observation that the antiinflammatory actions of methotrexate are due to the capacity of methotrexate to induce adenosine release may form the basis for the development of an additional class of antiinflammatory drugs.

Adenine↗

PubMed1997

Carboxypeptidase-G2, thymidine, and leucovorin rescue in cancer patients with methotrexate-induced renal dysfunction.

PURPOSE: Methotrexate nephrotoxicity can lead to delayed methotrexate elimination and the development of life-threatening toxicity, which may not be preventable with the standard rescue agent leucovorin. In preclinical studies, we previously demonstrated that carboxypetidase-G2 (CPDG2) rapidly hydrolyzes methotrexate to nontoxic metabolites. A protocol for the compassionate use of CPDG2 in patients who develop nephrotoxicity while receiving high-dose methotrexate was therefore developed. The pharmacologic and clinical outcome of CPDG2 rescue administered with thymidine and leucovorin in 20 patients is presented here. METHODS: Patients with high-dose methotrexate-induced renal dysfunction received one to three doses of CPDG2, 50 U/kg body weight intravenously (i.v.), thymidine 8 g/m2/d by continuous i.v. infusion, and standard pharmacokinetically guided leucovorin rescue. Plasma concentrations of methotrexate and its inactive metabolite 4-deoxy-4-amino-N10-methylpteroic acid (DAMPA) were measured before and after CPDG2 using high-pressure liquid chromatography (HPLC). Tolerance of CPDG2 and thymidine, development of methotrexate toxicities, and recovery of renal function were monitored. RESULTS: Twenty patients who received high-dose methotrexate for osteosarcoma (n = 11), lymphoid cancers (n = 8), and gastric cancer (n = 1) developed nephrotoxicity (median serum creatinine, 3.2 mg/dL) and elevated plasma methotrexate concentrations (median, 201 mumol/L at hour 46). CPDG2 and thymidine rescue was well tolerated and resulted in a rapid 95.6% to 99.6% reduction in the plasma methotrexate concentration. Methotrexate-related toxicity was mild to moderate. Serum creatinine returned to normal values at a median of 22 days. CONCLUSION: CPDG2, thymidine, and leucovorin rescue was highly effective in 20 patients at high risk for developing life-threatening methotrexate toxicity after the onset of methotrexate-induced nephrotoxicity and delayed methotrexate excretion.

Adolescent↗

PubMed1989

Methotrexate and dipyridamole combination chemotherapy based upon inhibition of nucleoside salvage in humans.

We have carried out a clinical trial in 23 patients to determine whether dipyridamole modulates the clinical effect of methotrexate. This trial was based upon in vitro studies which indicate that dipyridamole potentiates the cytotoxic action of methotrexate through inhibition of thymidine salvage. Methotrexate was given as a bolus injection 24 h after initiation of a high dose dipyridamole infusion. The trial was designed so that methotrexate was escalated in individuals until toxicity occurred and then the methotrexate dose resulting in toxicity was repeated without dipyridamole. During the course of this study the methotrexate dose was escalated from 10 to 130 mg/m2. While individual patient tolerance varied, moderate to severe myelosuppression and/or mucositis occurred frequently in patients receiving the combination with methotrexate doses greater than or equal to 60 mg/m2. Ten of 10 patients who experienced moderate or severe toxicity with the combination had significantly less toxicity when treated with methotrexate alone. Dipyridamole did not increase toxicity by an alteration in methotrexate elimination. The potentiation of methotrexate by dipyridamole in these patients suggests that physiological thymidine levels are sufficient to perturb the clinical effects of methotrexate and that thymidine salvage may represent a mechanism for clinical resistance to methotrexate. These results also suggest that a high dose dipyridamole regimen can be used as a pharmacological approach to test the role of nucleoside membrane flux on the clinical action of other standard chemotherapeutic drugs. Phase II studies testing the clinical efficacy of this combination should use a methotrexate dose of 60 mg/m2 with a provision for methotrexate dose escalation based upon individual patient tolerance.

Antineoplastic Combined Chemotherapy Protocols↗

PubMed1980

Relationship between membrane transport of methotrexate and endogenous cyclic adenosine 3':5'-monophosphate in the Ehrlich ascites tumor.

Transport of methotrexate in mammalian cells is a complex process. Although the drug is pumped uphill into cells, metabolic poisons enhance influx and transmembrane gradients for this agent. Structurally unrelated inorganic and organic anions nonspecifically depress these parameters in a variety of tumor cells. It has been suggested that cell cyclic adenosine 3':5'-Monophosphate (cyclic AMP) plays a regulatory role in the transport of this agent. To evaluate this further, a number of different experimental approaches were used to analyze the relationship between methotrexate transport and cell cyclic AMP in the Ehrlich ascites tumor. The following results indicate that for the Ehrlich ascites tumor, at least, there is no evidence for a regulatory role for cyclic AMP in the transport of methotrexate. (a) A marked increase in cell cyclic AMP by cholera-toxin or reduction in cyclic AMP by ascorbate was unaccompanied by changes in methotrexate influx. (b) Instantaneous and constant inhibition of methotrexate influx by isobutylmethylxanthine was temporally dissociated from the slower rise in cell cyclic AMP that was induced by this agent. (c) Although isobutylmethylxanthine and dibutyryl cyclic AMP augment cell cyclic AMP and decrease methotrexate influx, they have different effects on efflux and net transport of methotrexate. Isobutylmethylxanthine stimulates net methotrexate uptake and slows methotrexate efflux but dibutyryl cyclic AMP depresses net methotrexate uptake without an effect on methotrexate efflux. (d) Azide markedly stimulates methotrexate influx and net transport without a change in cell cyclic AMP. (d) Dinitrophenol inhibits methotrexate influx and augments net methotrexate accumulation but does not alter cell cyclic AMP.

1-Methyl-3-isobutylxanthine↗

PubMed1981

The influence of methotrexate pretreatment on 5-fluorouracil metabolism in L1210 cells.

Pretreatment of L1210 cells with methotrexate in concentrations which produced free intracellular methotrexate and near maximal inhibition of dihydrofolate reductase resulted in an enhancement of intracellular 5-fluorouracil (FUra) accumulation. This enhancement of FUra accumulation was maximum (5-fold increase) after a 6-h exposure to 100 microM methotrexate. The nucleotide derivatives of FUra, including a 5-fluoro-2'-deoxyuridylate, and 5-fluorouridine-5'-triphosphate were also increased nearly 5-fold following methotrexate treatment. In cells pretreated with methotrexate, there was an increase in intracellular 5-phosphoribosyl-1-pyrophosphate pools which ranged from 2 to 8 times control values following concentrations of methotrexate between 0.1 microM and 10 microM. Both the increase in 5-phosphoribosyl-1-pyrophosphate and FUra accumulation could be prevented by the addition of Leucovorin (N5-formyltetrahydrofolate) at concentrations which rescued cells from the inhibitory effects of methotrexate. Pretreatment with 6-methylmercaptopurine riboside, which inhibits amidophosphoribosyltransferase, the first committed step in de novo purine synthesis, also resulted in a similar elevation in 5-phosphoribosyl-1-pyrophosphate pools and enhancement of FUra accumulation. If the 5-phosphoribosyl-1-pyrophosphate pools were reduced following methotrexate pretreatment by the addition to the cultures of hypoxanthine, which utilizes 5-phosphoribosyl-1-pyrophosphate for the conversion to IMP, the intracellular accumulation of FUra was not enhanced. Also, if the inhibitor of 5-phosphoribosyl-1-pyrophosphate synthetase, 7-deazaadenosine, was given to cultures with methotrexate, there was no increase in 5-phosphoribosyl-1-pyrophosphate pools, nor enhancement of FUra accumulation. In addition, when 5-fluoro-2'-deoxyuridine was added with the methotrexate to cell cultures, there was no increase in 5-phosphoribosyl-1-pyrophosphate pools, nor enhancement of intracellular FUra accumulation. These results indicate that the ability of methotrexate to enhance FUra accumulation was probably the consequence of the antipurine effect of methotrexate which resulted in a reduction of the complex feedback inhibition on 5-phosphoribosyl-1-pyrophosphate synthesis and utilization. The resultant increased 5-phosphoribosyl-1-pyrophosphate pools were then capable of being utilized for the conversion of FUra to 5-fluorouridylate, the possible rate-limiting step in FUra intracellular metabolism and the major determinant of the rate of intracellular FUra accumulation. When methotrexate preceded FUra, there was synergistic cell killing as determined by soft agar cloning. The exact mechanism of this sequential synergistic antitumor activity may be the result of the enhanced incorporation of FUra into RNA, since the increased 5-fluoro-2'-deoxyuridylate which is formed is unlikely to increase substantially the inhibition of dTMP synthesis induced by methotrexate pretreatment.

Animals↗

PubMed1995

Treatment of recalcitrant cardiac allograft rejection with methotrexate. Cardiac Transplant Team.

Acute rejection continues to be a major cause of mortality and morbidity among cardiac allograft recipients. In this retrospective analysis, we evaluated the efficacy and safety of methorexate in the treatment of recalcitrant rejection in 16 heart transplant patients. Methotrexate was initiated in these patients for rejection refractory to conventional therapy or for multiple, recurrent rejection episodes. Before methotrexate therapy, these patients had experienced 3.2 +/- 1.1 (mean +/- SD) episodes of allograft rejection. Methotrexate was administered at 5.9 +/- 5.3 months postransplant, at a starting oral dose of 7.8 +/- 2.7 mg/week. The methotrexate dose was increased as tolerated by white blood cell counts to 10-25 mg/week. These patients had been followed for 26 +/- 12 months after initiation of methotrexate. All ongoing rejection episodes were reversed with methotrexate. Rejection resolution was typically delayed and was observed at 19 +/- 15 days after methotrexate initiation. Compared to the 6 months before methotrexate therapy, there was significant reduction in the linearized rejection rate (0.44 +/- 0.14 vs 0.06 +/- 0.09 episodes/patient/month), and the time spent in rejection (29.8 +/- 14.0 vs 5.8 +/- 8.7 days) in the 6 months after methotrexate initiation. Nadir white blood cell counts were observed at 4.0 +/- 1.8 weeks after methotrexate initiation, but were above 2000/mm3 in all patients. Multiple infections occurred in 2 patients who received repeat courses of methotrexate and the highest cumulative doses of methotrexate. These findings suggest that methotrexate may be effective in the management of recalcitrant cardiac allograft rejection. Methotrexate therapy appears to be well tolerated by most patients.(ABSTRACT TRUNCATED AT 250 WORDS)

Administration, Oral↗

PubMed1987

Alteration of methotrexate metabolism in rats by administration of an elemental liquid diet. I. Changes in drug enterohepatic circulation.

Severe enteritis and death consistently occur within 150 hours of a single intraperitoneal dose of 25 mg/kg methotrexate given to rats fed an elemental diet. Rats fed a regular chow diet show no clinical evidence of gastrointestinal morbidity or mortality at this dose. The etiology of this enhanced toxicity is not clear, however. It this study rats were randomized to receive an elemental diet (n = 45) or a regular chow diet (n = 45) for 7 days. They were then given a bolus dose of 3H methotrexate (25 mg/kg) intraperitoneally. At 20 and 40 minutes and at 1, 2, 12, 24, 48, 72, and 96 hours after injection, five animals from each group were anesthetized, and mean methotrexate levels in bile and serum, and tissue methotrexate concentrations in proximal, mid, and distal small intestine and in liver were obtained. Mean methotrexate levels in bile were significantly elevated in rats fed an elemental, chemically defined diet at 12, 24, 48, and 72 hours compared with regular diet fed rats. Mean serum methotrexate levels were elevated in both the inferior vena cava (IVC) and portal vein (PV) at 12 (IVC), 24 (PV), and 48 hours (IVC) in elemental, liquid diet fed rats. There were no statistical differences between methotrexate levels in rats fed an elemental diet or a regular chow diet in proximal or mid small intestine at any of the time points measured. In the distal small intestine, methotrexate levels were significantly elevated at 24 hours in rats fed an elemental, chemically defined liquid diet (P less than 0.03) compared with regular diet fed rats. Methotrexate levels in liver of rats fed an elemental, chemically defined liquid diet were significantly elevated at 24 and 48 (P less than 0.05) hours. Administration of an elemental, chemically defined liquid diet prolongs the enterohepatic circulation of methotrexate in rats and delays clearance of the drug from the systemic circulation. Thus, prolonged exposure of methotrexate to the cells of the intestinal mucosa explains the increased gastrointestinal side effects of the drug in rats fed an elemental, chemically defined liquid diet. If these results are applicable to man, elemental, chemically defined liquid diets are contraindicated as the sole nutritional source in patients receiving methotrexate. Such qualitative alteration of dietary intake may be a factor in the unpredictability of gastrointestinal side effects associated with high dose methotrexate administration.

Animals↗

PubMed2003

Comparison between methotrexate and azathioprine in the treatment of chronic active Crohn's disease: a randomised, investigator-blind study.

BACKGROUND AND AIMS: The efficacy of azathioprine in the treatment of chronic active Crohn's disease is well established. However, this drug has a long onset of action. Methotrexate has also been shown to be effective in chronic active Crohn's disease. The aim of this study was to evaluate the efficacy and safety of methotrexate in comparison with azathioprine, and to establish whether methotrexate has a shorter onset of action in this setting. METHODS: Patients with chronic active Crohn's disease were admitted to this investigator-blind study. Chronicity was defined as the need for steroid therapy of > or = 10 mg/day for at least 4 months during the preceding 12 months, with at least one attempt to discontinue treatment. The disease had to be clinically active at entry, with a Crohn's Disease Activity Index of > or = 200. Six patients treated with azathioprine and methotrexate, respectively, were found to have enterocutaneous and perianal fistulas. At entry, all patients received prednisolone (40 mg once a day) which was tapered over a period of 12 weeks unless their clinical condition deteriorated. All patients were randomised to receive i.v. methotrexate 25 mg/week, or oral azathioprine 2 mg/kg per day, for a 6-month follow-up period. After the first 3 months, methotrexate was switched to oral administration maintaining the same dose. The primary efficacy outcome considered was the proportion of patients entering first remission after 3 and 6 months of therapy. Clinical remission was defined as the lack of need for steroid treatment and a Crohn's Disease Activity Index score of < or = 150 points at each scheduled visit. RESULTS: In the 54 patients (26 F, 28 M, mean age 34 years, range 18-60) randomly assigned to methotrexate (n=27) or azathioprine (n=27), no statistically significant difference was found between the two treatment regimens with respect to remission rate after 3 (methotrexate 44%, azathioprine 33%, p=0.28, (95% CI, 0.369-0.147), and 6 months (methotrexate 56%, azathioprine 63%, p=0.39, 95% CI, 0.187-0.335), respectively. Six patients withdrew from therapy due to adverse events: 3/27 (11%) in methotrexate and 3/27 (11%) in azathioprine. Drug-related adverse events (asthenia, nausea and vomiting) that did not require withdrawal from therapy were more frequent in the methotrexate group (azathioprine: 2/27 (7%); methotrexate: 12/27 (44%), p=0.00009). The frequency of these adverse events was comparable during the intravenous or oral administration of the drug. CONCLUSIONS: This study confirms that methotrexate is effective in inducing remission in patients with chronic active Crohn's disease, therapeutic efficacy being comparable, but not faster, than that of azathioprine.

Adult↗

PubMed1999

A trial of etanercept, a recombinant tumor necrosis factor receptor:Fc fusion protein, in patients with rheumatoid arthritis receiving methotrexate.

BACKGROUND: Patients treated with methotrexate for rheumatoid arthritis often improve but continue to have active disease. This study was undertaken to determine whether the addition of etanercept, a soluble tumor necrosis factor receptor (p75):Fc fusion protein (TNFR:Fc), to methotrexate therapy would provide additional benefit to patients who had persistent rheumatoid arthritis despite receiving methotrexate. METHODS: In a 24-week, double-blind trial, we randomly assigned 89 patients with persistently active rheumatoid arthritis despite at least 6 months of methotrexate therapy at a stable dose of 15 to 25 mg per week (or as low as 10 mg per week for patients unable to tolerate higher doses) to receive either etanercept (25 mg) or placebo subcutaneously twice weekly while continuing to receive methotrexate. The primary measure of clinical response was the American College of Rheumatology criteria for a 20 percent improvement in measures of disease activity (ACR 20) at 24 weeks. RESULTS: The addition of etanercept to methotrexate therapy resulted in rapid and sustained improvement. At 24 weeks, 71 percent of the patients receiving etanercept plus methotrexate and 27 percent of those receiving placebo plus methotrexate met the ACR 20 criteria (P<0.001); 39 percent of the patients receiving etanercept plus methotrexate and 3 percent of those receiving placebo plus methotrexate met the ACR 50 criteria (for a 50 percent improvement) (P<0.001). Patients receiving etanercept plus methotrexate had significantly better outcomes according to all measures of disease activity. The only adverse events associated with etanercept were mild injection-site reactions, and no patient withdrew from the study because of adverse events associated with etanercept. CONCLUSIONS: In patients with persistently active rheumatoid arthritis, the combination of etanercept and methotrexate was safe and well tolerated and provided significantly greater clinical benefit than methotrexate alone.

Adult↗

PubMedDate not supplied

[Methotrexate, liver and rheumatoid arthritis in tropical areas].

Rheumatoid arthritis (RA) is a common disease in tropical areas. Methotrexate which has become the main first-line treatment in western countries is increasingly used in tropics. Well documented liver toxicity of methotrexate led the American College of Rheumatology to provide guidelines about monitoring patients. In endemic areas of hepatitis B and C methotrexate may interfere with the natural history of these infections and exarcerbate liver damage, on the other hand, RA causes extra-articular manifestations which are rare and exceptionnally serious in the liver. The most important hepatic disorders associated with RA are: intrahepatic portal hypertension without cirrhosis, amyloidosis, drug hepato-toxicity and viral interferences. - Intrahepatic portal hypertension Several cases of portal hypertension without cirrhosis have been reported. Most cases were related to Nodural Regenerative Hyperplasia (NRH) which is made of diffuse nodules of hepatocytes without fibrosis. The pathogenesis of this entity is unknown. Distal vascular changes and abnormal perfusion of liver are mentioned. Presentation is cholestasis in one third of cases. Portal hypertension has no particularity and may cause esophageal variceal bleeding. NRH is closely associateed with Felty's syndrome. - Amyloidosis Hepatic amyloidosis is a classical complication of RA even rare. It is a secondary amyloidosis of AA type. Hepatic injury is generally silent and renal symptoms are dominant. - Drug hepatotoxicity Several medications used in the management of RA are potentially hepatotoxic : salicylates, nonsteroidal antiinflammatory drugs (NSAID), corticosteroids, gold, sulfasalazine and methotrexate above all. Methotrexate hepatotoxicity is well documented in carcinology with high doses but also in psoriasis patients treated with low doses. Hepatic damage related to methotrexate includes elevation of aminotransferases, portal fibrosis and cirrhosis. But data on methotrexate toxicity show small risk of serious liver disease in RA patients. Long duration of therapy and age (> 60 years) have been found to be independent risk factors for the development of hepatic disease. Other identified risk factors are alcohol intake, diabetes, obesity and prior history of hepatitis B or C. The American College of Rheumatology has published guidelines about monitoring patients for liver toxicity. Hepatic tests and Hepatitis B and C serologic studies are recommended before starting treatment with methotrexate. Liver biopsy is only recommended in case of alcoholism, prolonged abnormalities of aminotransferases and chronic hepatitis B or C infection. - Methotrexate and hepatitis B or C infection These infections are endemic in tropical areas. Chronic hepatitis B or C is a contra indication for methotrexate therapy, due to the immuno-suppressive effect of the drug. Positive ELISA tests to C virus must be confirmed with RIBA tests to avoïd false positive tests which have been reported in Africa. The "healthy" HBs Ag carrier state is not in theory a contra indication for methotrexate therapy but the risk of hepatitis B reactivation needs a close monitoring. The biological tests required for are sophisticated and quite impossible in routine practice in tropical areas. So HBs Ag carrier state is usually incompatible with methotrexate treatment. Studies would be useful to prove that in endemic areas of viral hepatitis B and C as in western countries, methotrexate is enough safe to become the main first-line treatment of rheumatoid arthritis.

Amyloidosis↗

PubMed1980

Transport, binding, and polyglutamation of methotrexate in freshly isolated rat hepatocytes.

Influx of [3H]methotrexate into freshly isolated hepatocytes in suspension is mediated by two routes, one with a high affinity (Km = 5.9 microM) and another with a low affinity for methotrexate. Both transport routes are equally sensitive to the sulfhydryl group inhibitor, p-chloromercuriphenylsulfonic acid, alterations in temperature, substitution of extracellular Na+ with choline, and inhibition by ouabain or azide. The high-affinity pathway for methotrexate shows specificity for the 4-amino group of the pteridine moiety as methotrexate and aminopterin similarly inhibit influx of the labeled drug. On the other hand, 100 microM concentrations of the naturally occurring folates, folic acid, 5-methyltetrahydrofolate, and 5-formyltetrahydrofolate, are not inhibitory to influx of 1 microM methotrexate. Once in the cell, methotrexate rapidly reaches molar equivalence with dihydrofolate reductase following which both exchangeable and nonexchangeable intracellular methotrexate accumulates. The exchangeable component reaches steady state within 0.5 hr while the nonexchangeable component increases for at least 1 hr. The nonexchangeable component represents both bound methotrexate and methotrexate polyglutamates. Polyglutamates of methotrexate are a trivial component of total 3H within the cell until about 15 min, but thereafter, their rate of accumulation is constant so that by 1 hr they represent approximately 30% of total intracellular 3H. At steady state, there is a transmembrane chemical gradient for exchangeable methotrexate of 2.4:1; this is 24 times greater than the chemical gradient predicted for equilibrium when the transcellular membrane potential is considered. These results indicate that there are multiple routes for methotrexate transport in the rat hepatocyte that appear to be, at least in part, distinct from the routes for folic acid and the tetrahydrofolate cofactors. The data suggest that transport is energy and Na+ dependent and that the transport carrier requires intact sulfhydryl groups. Net association of methotrexate with the cells is a complex process determined by transport and binding to multiple sites within the cell and metabolism to polyglutamate derivatives that are retained within the cell.

4-Chloromercuribenzenesulfonate↗

PubMed1982

Augmentation of the intracellular levels of polyglutamyl derivatives of methotrexate by vincristine and probenecid in Ehrlich ascites tumor cells.

The intracellular accumulation of poly-gamma-glutamyl derivatives of methotrexate was evaluated in the presence of vincristine or probenecid (agents which raise the intracellular level of free methotrexate) in Ehrlich ascites tumor cells. The results show that both intracellular methotrexate and its metabolites are increased by these agents and that, in the presence of L-glutamine, polyglutamate derivatives are increased by a higher percentage than is methotrexate. From 1 to 50 microM vincristine increased the levels of polyglutamate derivatives from 25 to over 300%, whereas methotrexate was raised from 25 to 80%. Similarly, 50 to 200 microM probenecid increased methotrexate polyglutamate derivatives from 31 to 88%, whereas methotrexate was raised from 0 to 30%. A determination of the bound fraction of drug indicated that the proportion of dihydrofolate reductase bound with methotrexate polyglutamates increased in the presence of these agents. Efflux studies showed that over 90% of the large pools of intracellular methotrexate polyglutamates produced by these agents was retained for at least 1 hr in the absence of extracellular methotrexate, whereas the majority of intracellular methotrexate exited the cell. These studies (a) indicate that vincristine and probenecid may be potentially useful for selectively increasing methotrexate polyglutamates in tumor cells and (b) introduce another basis for synergism observed between alkaloids and methotrexate.

Animals↗