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PubMed1990

The effect of corticosteroid or methotrexate therapy on lung lymphocytes and macrophages in sarcoidosis.

Methotrexate appears to be an effective alternative to corticosteroid therapy for some patients with sarcoidosis. The mechanism of action of methotrexate as an immunosuppressive is unknown. Patients with symptomatic pulmonary sarcoidosis underwent pulmonary function tests and bronchoscopy with bronchoalveolar lavage. Patients were treated with 10 mg methotrexate or prednisone weekly for at least 6 months and repeat studies were performed. A comparison was made between those patients receiving methotrexate (12 patients) and those receiving prednisone (12 patients). For both groups, there was a significant improvement in the vital capacity with therapy (Prednisone: Pre = 2.5 +/- 0.14 L (Mean +/- SEM); Post = 3.1 +/- 0.18 L, p less than 0.01; Methotrexate: Pre = 2.4 +/- 0.14 L; Post = 2.8 +/- 0.18 L, p less than 0.01). In addition, the percentage of lymphocytes in the lavage fell significantly for both the prednisone (Pre: 30 +/- 3.5%; Post: 16 +/- 2.7%, p less than 0.001) and methotrexate (Pre: 37 +/- 3.4%; Post: 13 +/- 2.9%, p less than 0.001) groups. Alveolar macrophages from the symptomatic sarcoid patients were found to be spontaneously releasing hydrogen peroxide and tumor necrosis factor. After treatment with either prednisone or methotrexate, alveolar macrophages retrieved by lavage spontaneously released less of either macrophage product. We found that effective doses of methotrexate for sarcoidosis led to significant changes in lymphocyte and macrophages retrieved by lavage.

Adult↗

PubMed1973

Threshold methotrexate concentration for in vivo inhibition of DNA synthesis in normal and tumorous target tissues.

The suppression of DNA synthesis in host and tumor tissues by methotrexate has been monitored in mice by determining the in vivo incorporation of tritium-labeled deoxyuridine ([(3)H]UdR) into DNA. The duration of inhibition of [(3)H]UdR incorporation in normal tissues was related to the dose of methotrexate and was a direct function of plasma drug concentration. [(3)H]UdR incorporation recovered to 50% of pretreatment levels in bone marrow when plasma methotrexate concentration was 10(-8) M or less, irrespective of the dose administered, while 50% recovery of DNA synthesis in intestinal epithelium was not observed until plasma methotrexate levels were 5 x 10(-9) M or less. Ascitic L1210 leukemia cells did not fully return to pretreatment levels of [(3)H]UdR incorporation at any time, although a partial recovery of incorporation was noted at methotrexate ascitic fluid concentrations of approximately 10(-8) M. Methotrexate did not suppress the incorporation of tritium-labeled thymidine ([(3)H]TdR) into bone marrow and duodenal mucosa, confirming the specificity of its action in inhibiting thymidylate synthesis in host tissues. In the ascites tumor a gradual decline in [(3)H]TdR incorporation was seen after methotrexate, indicating that the tumor tissue depression of [(3)H]UdR incorporation is not solely due to inhibition of thymidylate synthesis. These studies indicate that host tissues are inhibited by extremely low concentrations of methotrexate, and indicate the importance of the slow final phase (t((1/2))=12 h) of drug elimination from plasma in producing a prolonged exposure of sensitive host tissues to inhibitory drug concentrations.

Animals↗

PubMed2004

Unaltered etanercept pharmacokinetics with concurrent methotrexate in patients with rheumatoid arthritis.

The purpose of this study was to evaluate the potential impact of concurrent weekly oral methotrexate administration on the pharmacokinetics of etanercept in patients with rheumatoid arthritis (RA) in a phase 3B trial. As part of a double-blind randomized trial of 682 patients with rheumatoid arthritis who received etanercept (25 mg subcutaneously twice weekly), methotrexate (weekly oral dose, median weekly dose: 20 mg), or etanercept (25 mg subcutaneously twice weekly) plus methotrexate (weekly oral dose, median weekly dose: 20 mg), serum etanercept concentrations were measured in a subset of patients. Serum samples for 98 randomly selected patients (48 receiving etanercept-alone treatment, 50 receiving etanercept plus methotrexate combination treatment) were analyzed to assess the pharmacokinetics of etanercept. A single blood sample was drawn from each patient at baseline and at the week 24 visit. Given the variable sampling time for patients in both groups, a population pharmacokinetic analysis using NONMEM was conducted for etanercept. A final covariate population pharmacokinetic model was constructed based on previously obtained etanercept data from both healthy subjects (n = 53) and patients with RA (n = 212) in 10 prior clinical trials. The predictive performance of the final model was assessed by both bootstrap and data-splitting validation approaches. The final model was then used to estimate Bayesian pharmacokinetic parameters for the patients in both treatments in the current trial. The potential effect of the concurrent administration of methotrexate on the pharmacokinetics of etanercept was examined by comparing the clearance values between 2 treatments using statistical criteria. A population 2-compartment model with first-order elimination from the central compartment and with either zero-order (intravenous administration) or first-order (subcutaneous administration) input was selected based on the data from the prior 10 etanercept clinical studies. The following pharmacokinetic parameters (typical value +/- standard error) were estimated: clearance (CL: 0.072 +/- 0.005 L/h), volume of distribution in the central compartment (V(c): 5.97 +/- 0.45 L), volume of distribution in the peripheral compartment (V(p): 2.05 +/- 0.32 L), intercompartment clearance (Q: 0.0645 +/- 0.0093 L/h), first-order absorption rate constant (k(a): 0.0282 +/- 0.0039 1/h), and absolute bioavailability for subcutaneous administration (F: 0.626 +/- 0.056). Interindividual variability of the pharmacokinetic parameters was quantified for CL (25.1%), V(c) (41.7%), k(a) (53.1%), and F (24.2%). Residual variability consisted of combined additive (11.4 ng/mL) and proportional error (49.9%). Both age (< 17 years) and body weight (< 60 kg) were found to be important covariates on CL. The results of both validation tests indicated the adequate predictive performance of the population model. Based on the bioequivalence criteria, the Bayesian-estimated clearance for patients receiving etanercept alone (mean: 0.070 L/h) was comparable to that for patients receiving a combination of etanercept and methotrexate (mean = 0.066 L/h). The pharmacokinetics of etanercept were not altered by the concurrent administration of methotrexate in patients with rheumatoid arthritis. Thus, no etanercept dose adjustment is needed for patients taking concurrent methotrexate.

Antirheumatic Agents↗

PubMed2002

Central nervous system prophylaxis with high-dose methotrexate does not give rise to significant electroencephalographic changes in children with acute lymphoblastic leukemia.

Acute, subacute, and chronic neurologic complications have been reported in children treated with high-dose methotrexate for various malignant diseases. It was the aim of this study to monitor central nervous system treatment with high-dose methotrexate in children with acute lymphoblastic leukemia by serial electroencephalographic (EEG) examinations. Electroencephalographic examinations with quantitative computed analysis were performed in 21 children before and on the third day after each of four high-dose methotrexate infusions with leucovorin rescue according to protocol M of trial ALL-BFM 90 of the German Society for Pediatric Haematology and Oncology. Six patients with a medium risk of relapse also received L-asparaginase. In the cohort treated with methotrexate solely, no statistically significant changes of the quantitative EEG parameters could be demonstrated. Only two children with delayed serum methotrexate clearance showed reversible diffuse EEG slowing of a slight to moderate degree. In the group with additional L-asparaginase treatment, slight transient EEG slowing also occurred. Our findings indicate that in patients with a normal methotrexate clearance during central nervous system treatment with high-dose methotrexate according to trial BFM-ALL 90, usually no subacute or cumulative EEG changes have to be expected. If neurologic or psychiatric symptoms or EEG slowing occur, delayed methotrexate clearance must be suspected.

Adolescent↗

PubMed1997

Low-dose methotrexate spares steroid usage in steroid-dependent asthmatic patients: a meta-analysis.

STUDY OBJECTIVES: To determine if treatment with low-dose methotrexate spares oral steroids in adult, steroid-dependent, asthmatic patients. DESIGN: Articles identified by computer search were excluded if they (1) did not contain original data relating to the primary question, (2) had no controls, or (3) described subjects younger than 18 years of age. MEASUREMENTS: From each article, the following was abstracted: reference citation; type of control; whether study incorporated a run-in period in which the baseline level of prednisone was reduced to the lowest possible dose; dosage and length of methotrexate therapy; baseline dosage; and type of steroid. Also, the following was determined during both placebo and methotrexate arms of each study: steroid dosage, FEV1, serious side effects, and alteration in level of serum aspartate aminotransferase. RESULTS: Eleven eligible studies were identified. Methotrexate treatment resulted in a decrease in prednisone or prednisolone usage by an average of 4.37 mg/d or 23.7% of the initial dosage. The summary effect size in standard deviations (SDs) was -0.53 (95% confidence interval=-0.29 to -0.77). Subgroup analysis showed that patients treated with prolonged (6-month) therapy with methotrexate, those with low long-term usage of steroids (< or = 20 mg/d), and those whose study design incorporated a run-in period, tended to have the greatest steroid-sparing effects with methotrexate. CONCLUSIONS: Low-dose methotrexate has a significant steroid-sparing effect in steroid-dependent asthmatic patients. The greatest effect was evident in patients in whom an effort was made to reduce baseline steroid dosage and in whom methotrexate was used for 24 weeks.

Adult↗

PubMed1990

The practical use of methotrexate in psoriasis.

Psoriasis is an inflammatory disease of the skin associated with increased epidermal proliferation. The aetiology of the disease is unknown, but there seems to be a genetic predisposition. The goal of therapy in the treatment of psoriasis is to decrease the rate of epidermal proliferation and the underlying inflammation. Topical application of steroids and coal tar are the therapies of choice; however, for those patients with severe recalcitrant psoriasis who have failed conventional topical therapy methotrexate is an established alternative. The use of methotrexate in psoriasis is limited by its toxicity, and proper patient selection and close monitoring are essential in achieving good clinical response. The dosage used should be the lowest that will maintain the patient in comfort, not necessarily that which produces total resolution. Caution should be exercised when other agents are used concurrently with methotrexate, and possible drug interactions should be identified as these may influence the effectiveness and toxicity of methotrexate therapy. The common side effects associated with the use of methotrexate in psoriasis include bone marrow suppression, gastrointestinal symptoms and hepatotoxicity. Liver damage is a major concern in long term methotrexate therapy and thus liver biopsies are warranted to monitor any pathological changes. The drug is a known teratogen and should be avoided in pregnant patients. Women of childbearing age should use reliable contraception during therapy. Patients should be made aware of the signs and symptoms of methotrexate toxicity and inform their physicians promptly as most adverse effects can be ameliorated with appropriate dosage adjustment. Methotrexate will continue to play a major role in the treatment of psoriasis and it is thus important that it be used safely.

Humans↗

PubMed1990

Accumulation, intracellular metabolism, and antitumor activity of high- and low-dose methotrexate in human osteosarcoma xenografts.

High doses of methotrexate with leucovorin rescue are routinely used in the treatment of patients with osteosarcoma; the rationale for this application is controversial. Using human osteosarcoma xenografts growing in mice as a clinically relevant model, we compared the accumulation, intracellular metabolism, and tumor response of methotrexate administered as either high-dose (2400 mg/kg) or low-dose (150 mg/kg) infusions. The high-dose regimen, which included i.v. hydration and leucovorin rescue, resulted in plasma methotrexate levels that approximated those in patients receiving the drug at 12 g/m2. The low-dose infusion produced essentially the same toxicity as the higher dose level, without use of leucovorin. The HxOs33 tumor line was moderately sensitive to the high-dose infusion (55-day delay in tumor volume doubling time), whereas the second line, HxOs2, did not respond. Neither xenograft had a measurable response to low-dose methotrexate. Methotrexate was present in both tumors for up to 72 hr post-infusion, regardless of the dosage regimen. Only shorter-chain polyglutamates (MTXglu2 and MTXglu3) were detected over this period in the high-dose trial, and levels of these derivatives were uniformly higher in the resistant HxOs2 xenograft. Low-dose infusions were associated with formation of longer-chain polyglutamate species, with more abundant production in the HxOs2 line. Methotrexate polyglutamates exceeded baseline [3H]MTX binding of dihydrofolate reductase, as measured in tumor homogenates, at all testing intervals through 72 hr in both tumor lines. Nonetheless, high-dose methotrexate-induced suppression of [14C]formate incorporation into DNA was greater in the drug-sensitive HxOs33 tumor than in HxOs2. These results suggest a therapeutic advantage for high-dose methotrexate regimens in the treatment of human osteosarcoma but show that formation of tumor MTX polyglutamates is not the sole determinant of response to this agent.

Animals↗

PubMed1991

Folate transport and the modulation of antifolate sensitivity in a methotrexate-resistant human breast cancer cell line.

The mechanism of acquired methotrexate-resistance in an estrogen-receptor positive human breast cancer cell line (MTX(R)ZR-75-1) was studied. MTX(R) ZR-75-1 cells are 250-fold resistant to methotrexate when grown in the presence of 1 microM folinic acid and 2,400-fold resistant in the presence of 1 microM folic acid. This drug resistant cell line also showed collateral sensitivity (10-fold) to trimetrexate (TMQ), when grown in the presence of folinic acid. Using fluoresceinated methotrexate (F-MTX), FACS analysis indicated that there is no intracellular accumulation of methotrexate into MTX(R) ZR-75-1 cells, as determined by competition of F-MTX and methotrexate binding to dihydrofolate reductase. These characteristics strongly indicate that the mechanism of resistance involved down regulation of the reduced-folate transporter. To investigate this further, the transport kinetics of parental and MTX(R) ZR-75-1 cells were examined. Although the V(max) for methotrexate transport in wild-type (WT) ZR-75-1 breast cancer cells was 1-2 orders of magnitude lower than that in the well characterized leukemia cell lines, such as L1210 and CCRF-CEM cells, kinetic analysis indicated that transport of methotrexate into WT ZR-75-1 cells involved a mechanism that was similar if not identical to the reduced folate transporter. In contrast, no specific uptake of methotrexate was detected in MTX(R) ZR-75-1cells. Furthermore, neither cell line expressed detectable levels of folate binding protein, a binding protein with high affinity for folic acid as well as for reduced folates and antifolates. These results indicate that the level of expression of the reduced-folate carrier may be an important factor in determining the sensitivity of breast cancer cells as well as leukemia cells to antifolate compounds.

Animals↗

PubMed1996

Methotrexate for inflammatory bowel disease: pharmacology and preliminary results.

OBJECTIVE: To review the limited published experience with methotrexate treatment for inflammatory bowel disease, to examine the proposed anti-inflammatory and immune-modifying mechanism of action and pharmacologic properties of methotrexate, and to detail its limiting toxicities. DESIGN: A comprehensive synopsis of methotrexate is presented to aid physicians who treat patients with inflammatory bowel disease. RESULTS: Methotrexate and its polyglutamate metabolites are folic acid analogues with inhibitory activity against many enzymes in the metabolic pathway of folic acid. Long-term low-dose methotrexate therapy (25 mg or less once a week) inhibits production of thymidylate, purines, and methionine and leads to accumulation of adenosine, a potent anti-inflammatory substance. These actions inhibit cellular proliferation, decrease formation of antibodies, and decrease production of mediators of inflammation such as interleukins and eicosanoids. Three uncontrolled trials and two controlled trials have demonstrated efficacy of low-dose methotrexate therapy for induction of remission in Crohn's disease and have also suggested possible benefit for ulcerative colitis and for remission maintenance indications in both diseases. Although methotrexate is generally well tolerated for long-term use at a low dose, several serious toxicities potentially limit its use. CONCLUSION: Methotrexate is a promising new agent for the treatment of inflammatory bowel disease.

Anti-Inflammatory Agents↗

PubMed1987

Pulse methotrexate therapy in rheumatoid arthritis. A controlled prospective roentgenographic study.

STUDY OBJECTIVE: To assess whether weekly pulse methotrexate therapy alters radiographic progression of joint disease in patients with rheumatoid arthritis. DESIGN: Prospective, controlled study. Hand, wrist and foot roentgenograms obtained before, at the onset of, and during methotrexate treatment were scored for degree of joint-space narrowing and erosions by three rheumatologists using a standard method. PATIENTS: Sequential sample of 24 patients with active definite or classical rheumatoid arthritis and previous unsuccessful treatment; of these, 3 were excluded due to drug ineffectiveness; 2, due to side effects; and 1, due to refusal to take methotrexate. INTERVENTIONS: Treatment with nonsteroidal anti-inflammatory drugs and prednisone was continued. Methotrexate was given weekly to control clinical evidence of disease in patients. MEASUREMENTS AND MAIN RESULTS: After having had an average of 30 months of therapy, the 18 patients who continued to receive methotrexate therapy showed significant (p less than 0.05) clinical improvement, as evidenced by their decreased joint counts and joint scores, duration of morning stiffness, pain scales, and sedimentation rates. Despite patients' prolonged clinical improvement, the mean rate of development of erosions and joint-space narrowing during methotrexate therapy was not significantly different from the rate of radiographic progression before methotrexate therapy (0.043 compared with 0.041; p greater than 0.05). CONCLUSIONS: Weekly pulse methotrexate is effective for the long-term management of clinical disease activity in patients with refractory rheumatoid arthritis but may not be a disease-modifying agent by roentgenographic criteria.

Adult↗

PubMed1991

Influence of methotrexate and azathioprine on radiologic progression in rheumatoid arthritis. A randomized, double-blind study.

OBJECTIVE: To compare the effects of azathioprine and methotrexate on progression of radiologic damage in patients with rheumatoid arthritis. DESIGN: Double-blind, randomized 48-week trial. PATIENTS: Sixty-four patients with active rheumatoid arthritis who either have not responded to or who have reacted with side effects to at least parenteral gold and D-penicillamine. INTERVENTIONS: Either azathioprine, 100 mg daily, or methotrexate, 7.5 mg weekly, was administered orally. Depending on the clinical effect after 8 weeks, the dosage was increased to either azathioprine, 150 mg, or methotrexate, 15 mg. The dosages for nonsteroidal anti-inflammatory drugs and prednisone were held stable. MEASUREMENTS: Clinical and laboratory assessments were done by the same physician every 4 weeks for the first 24 weeks and every 8 weeks thereafter. Radiographs of hands, wrists, and feet obtained at baseline and after 24 and 48 weeks were scored by one rheumatologist blinded to medication and clinical findings. MAIN RESULTS: Initial radiologic scores were comparable in both groups and correlated with disease duration (r = 0.38). An intention-to-treat analysis after 24 and 48 weeks showed significantly fewer new erosions in the methotrexate group compared with the azathioprine group (difference, 2.0 [95% CI, 0.2 to 3.9] and 3.5 [CI, 1.3 to 5.8], respectively). The change in total joint score was also significantly less pronounced in the methotrexate group compared with the azathioprine group after 24 weeks (difference, 2.8 [CI, 0.2 to 5.2]) and after 48 weeks (difference, 3.9 [CI, 0.3 to 7.4]). Radiologic stabilization after 48 weeks was present in 10% of the azathioprine group compared with 29% of the methotrexate group. CONCLUSIONS: Patients with rheumatoid arthritis treated with low-dose methotrexate showed significantly less radiologic progression than patients treated with azathioprine. This result suggests that methotrexate therapy is clinically superior in these patients.

Aged↗

PubMedDate not supplied

Effects of PEGylated soluble tumor necrosis factor receptor type I (PEG sTNF-RI) alone and in combination with methotrexate in adjuvant arthritic rats.

OBJECTIVE: To determine the potential combination benefit of treatment with PEG sTNF-RI and methotrexate in adjuvant arthritic rats. METHODS: Lewis rats with adjuvant arthritis were treated by sc injections of either 3.0 or 0.3 mg/kg PEG sTNF-RI on days 9, 11, and 13 of adjuvant arthritis. The effects of PEG sTNF-RI treatment alone were compared to treatment with daily oral methotrexate (0.075, 0.06 or 0.045 mg/kg) or methotrexate in combination with PEG sTNF-RI. Efficacy was monitored by volume measurement of ankle joints, final paw weights and histologic evaluation with particular emphasis on bone lesions. RESULTS: Treatment with 3.0 or 0.3 mg/kg PEG sTNF-RI alone resulted in 52% or 28% inhibition, respectively, of paw swelling as assessed by final paw weight. Treatment with methotrexate at either 0.075, 0.06, or 0.045 mg/kg gave 84%, 51% or 18% inhibition and combination treatment resulted in additive inhibitory effects. Histologic evaluation of ankle joints demonstrated 68% or 25% inhibition of bone resorption with PEG sTNF-RI alone at 3.0 or 0.3 mg/kg. Treatment with 0.075, 0.06 or 0.045 mg/kg methotrexate resulted in 98%, 76% or 40% inhibition of bone resorption. Additive benefit was best seen with the lower doses of methotrexate. CONCLUSION: Combination therapy with PEG sTNF-RI and methotrexate results in additive benefit, with the final result being excellent inhibition of all arthritis parameters. Data from these studies supports the clinical investigation of the use of combination therapy of PEG sTNF-RI and methotrexate in rheumatoid arthritis patients.

Animals↗

PubMed1992

Intrinsic resistance to methotrexate in human soft tissue sarcoma cell lines.

A human fibrosarcoma cell line, HT-1080, and four new cell lines (HS-16, HS-28, HS-30, and HS-42) were established from untreated patients with mesenchymal chondrosarcoma, peripheral nerve sheath sarcoma, malignant hemangiopericytoma, and mixed mesodermal tumor, respectively, and were used for analysis of mechanisms of intrinsic resistance to methotrexate. All four new cell lines were resistant to methotrexate as determined by inhibition of thymidylate synthase in whole cells and by growth inhibition, as compared with HT-1080, a methotrexate sensitive cell line. Methotrexate uptake, level of dihydrofolate reductase, and inhibition of this enzyme by methotrexate in the four cell lines were comparable to HT-1080 cells. However, levels of long chain polyglutamates (glu3-5) of methotrexate achieved after a 24-h incubation with this drug were much lower in the four new cell lines as compared to the HT-1080 cell line (5- to 20-fold lower). The low levels of methotrexate polyglutamates formed is likely the major cause of intrinsic methotrexate resistance in these new sarcoma cell lines.

Drug Resistance↗

PubMedDate not supplied

Methotrexate for the treatment of patients with multiple episodes of acute cardiac allograft rejection.

Of 142 cardiac allograft recipients who underwent transplantation from December 1985 to January 1991, four women and seven men (mean age, 41 +/- 14 years) required multiple (10.5 +/- 3.3) courses of antirejection treatment over a total follow-up period of 30 +/- 15 months. The underlying heart disease was cardiomyopathy in six patients and coronary disease in five patients. These patients were treated with methotrexate (10 mg/wk for 6 weeks). Rejection treatment before methotrexate therapy included six courses of OKT3, one course of antithymocyte globulin, 33 courses of high-dose steroids, and 45 courses of low-dose steroids for the entire group. The average number of rejection treatments per patient before methotrexate therapy was 8.7 +/- 3.5 treatments or 0.90 +/- 0.51 treatments per month of follow-up. After methotrexate therapy the average number of rejection treatments fell to 1.7 +/- 1.1 treatments or 0.11 +/- 0.08 treatments per month of follow-up (p = 0.0002). Seven patients responded to a single course of methotrexate therapy; three patients required two courses (second course, 20 mg/wk for 6 weeks), and one patient required three courses of methotrexate therapy. The only complication associated with methotrexate therapy was one patient in whom cytomegalovirus interstitial pneumonitis developed while on therapy. Methotrexate was well tolerated and appeared to be effective in halting repeated episodes of rejection in this subset of patients who have had multiple episodes of acute rejection.

Adult↗

PubMed1988

Role of folylpolyglutamate synthetase in the regulation of methotrexate polyglutamate formation in H35 hepatoma cells.

The effect of culture conditions on the glutamylation of methotrexate by intact H35 hepatoma cells and folylpolyglutamate synthetase (FPGS) activity in the corresponding crude extracts has been examined. The rate of cellular glutamylation of methotrexate observed in rapidly dividing cultures was 4-fold higher than confluent cultures, and was accompanied by an increase in extract FPGS activity (2.2-fold). The depletion of cellular folates produced comparable increases in both cellular methotrexate glutamylation and extract FPGS activity (approximately 1.8-fold). Near-quantitative reductions in cellular methotrexate glutamylation were caused by media additions of reduced folates and methotrexate to confluent cultures of wild-type and folate-depleted H35 cells. However, these produced relatively modest reductions in FPGS activity in the corresponding crude extracts (approximately 50%). Methionine exclusion resulted in a greater than 50% decrease in FPGS activity in crude extracts of these cells compared to extracts of control cultures. The combination of methionine exclusion and folinic acid addition lowered the FPGS activity to less than 25% that of control. The data suggest that the changes in the glutamylation rate of methotrexate in whole cells due to culture conditions such as folate restriction, reduced folate addition, methionine exclusion, and growth state are at least in part a consequence of alterations in FPGS activity. This conclusion is consistent with the proposition that the metabolism of slow-acting substrates for FPGS (such as 4-amino antifolates and their corresponding polyglutamates) may be sensitive to changes in enzyme levels or activity (Cook et al., Biochemistry, 26: 530-539, 1987). Analysis of the products formed by FPGS from extracts using methotrexate as the substrate revealed no significant amounts of polyglutamate species higher than 4-NH2-10-CH3-PteGlu3. In contrast, when using the thymidylate synthase inhibitor N10-propargyl-5,8-dideazafolic acid as the starting substrate under identical assay conditions, FPGS from extracts catalyzed the formation of predominantly long chain polyglutamate derivatives (Glu4 and higher). These results reflect the relative efficacy of methotrexate and N10-propargyl-5,8-dideazafolic acid, as well as their polyglutamate derivatives, as substrates for FPGS.

Animals↗

PubMed1989

Enhancement of the frequency of methotrexate resistance by gamma-radiation in Chinese hamster ovary and mouse 3T6 cells.

We have studied the effects of gamma-radiation on the frequency of methotrexate resistance and dihydrofolate reductase gene amplification. gamma-Irradiation of Chinese hamster ovary cells resulted in enhancement of the frequency of methotrexate resistant colonies (maximum enhancement: 2000-fold after 1000 rads). The enhancement of methotrexate resistance was dependent on the dose of gamma-radiation and increased with time after irradiation; a maximum enhancement was observed when methotrexate was added 18 h after irradiation. Methotrexate resistant clones of Chinese hamster ovary cells showed no increase in dihydrofolate reductase gene copy number but were found to be defective in methotrexate transport. However, when these experiments were extended to 3T6 murine cells, 54% of nonirradiated and 44% of gamma-radiation induced methotrexate resistant clones showed an increase in dihydrofolate reductase gene copy number. These results suggest that the cells that survive irradiation have a very high probability of becoming methotrexate resistant and may explain why some malignant tumors (e.g., head and neck cancer) either do not respond or respond poorly to chemotherapy if the patient had prior radiotherapy.

Animals↗

PubMed1987

Blood-brain barrier disruption and methotrexate in the treatment of a readily transplantable intracerebral osteogenic sarcoma of rats.

An animal model of intracerebral osteogenic sarcoma has been developed to evaluate blood-brain barrier disruption as an adjunct to chemotherapy of intracerebral tumors. Adult Sprague-Dawley rats (n = 225) were inoculated intracerebrally with transplantable, methotrexate sensitive, osteogenic sarcoma cells and 3 days later randomized to receive either no treatment or methotrexate with or without blood-brain barrier disruption using intracarotid mannitol. Methotrexate was administered i.v., i.p., or directly into the carotid artery (i.c.) in doses of 2.5, 10, 20, 50, or 100 mg/kg. Survival was the study's end point. Surgery, anaesthesia, or blood-brain barrier disruption with mannitol did not affect survival. However, there was a significant effect of dose and route of administration of methotrexate on survival. The shortest survival was in rats receiving no treatment in which death occurred reproducibly at 7.6 +/- 0.2 days (n = 29) and the longest survival was 12.7 +/- 0.3 day (p less than 0.001) in those given methotrexate 50 mg/kg i.c. (n = 6). The i.c. route was most effective in prolonging survival followed by i.v. and the least effective was the i.p. route of methotrexate administration. Blood-brain barrier disruption followed by methotrexate (i.v. or i.c.) was deleterious to survival (two-way analysis of variance, p less than 0.003 and p less than 0.011, respectively) and the reduced survival was in part related to early complications such as intratumor hemorrhage or possibly a methotrexate induced encephalopathy. It is concluded that this is a useful model for the study of the chemotherapy of cerebral tumors, that blood-brain barrier disruption did not appear to improve the dose-response curve but resulted in reduced survival. We caution against the use of this procedure in the treatment of cerebral tumor in humans.

Animals↗

PubMed1988

Preclinical trials using an immunoconjugate of T101 and methotrexate in an athymic mouse/human T-cell tumor model.

We made an immunoconjugate (IC) using the anti-CD5 monoclonal antibody T101 and the chemotherapeutic drug methotrexate. Methotrexate was conjugated to T101 using an active ester intermediate, yielding a drug: antibody molar ratio of 12.4. Although T101 immunoreactivity was not significantly altered by conjugation, the IC did not demonstrate antigen-specific cytotoxicity in vitro. Methotrexate activity, assayed in vitro, decreased approximately 100-fold following conjugation. The IC was tested for in vivo efficacy in athymic mice bearing human T-cell (MOLT 4) xenografts. Experimental arms used in the study included i.p. injections of saline, T101, methotrexate, the IC, and a mixture of T101 and methotrexate. Doses ranged from 500 micrograms T101 (17.5 micrograms methotrexate) to 2 mg T101 (70 micrograms methotrexate). Injections were administered only after palpable tumors were established. In experiments at all doses, totaling 66 animals per arm, injection of the IC significantly inhibited tumor growth, and resulted in more tumor regressions and fewer animal deaths than the other four experimental arms. These data demonstrate that the IC promotes a potential advantage over the use of methotrexate through an increase in the therapeutic index.

Animals↗