High-dose trimetrexate and minimal-dose leucovorin: a case for selective protection?

Our objective was to find the minimum dose of leucovorin (LV; 5-formyltetrahydrofolate) needed to potentially provide selective protection of normal tissue in patients with tumors resistant to methotrexate (MTX) by virtue of transport during prolonged therapy with high-dose trimetrexate (TMTX). Based upon the known daily requirement for folate, that tumors are often resistant to methotrexate via a transport-based mechanism, and that large doses of trimetrexate can be given with large doses of leucovorin for the treatment of patients with Pneumocystis carinii, a protocol was designed to find the minimum LV dose required to allow the administration of large doses of TMTX. Patients were treated in 28-day cycles consisting of 14 consecutive days of oral TMTX (45 mg/m2 every 12 h), followed by 14 days of rest. The dose of concurrent LV was started at 5 mg/m2 twice daily. Cohorts of patients received successive half doses of LV so long as three consecutive patients had less than or equal to grade 3 toxicity. Ten patients received 29 courses of therapy. The most common toxicities encountered were thrombocytopenia (38%), mucositis (14%), and neutropenia (10%). At a LV dose of 2.5 mg/m2, toxicities were consistently limited to less than or equal to grade 3 and only one episode of grade 4 hematological toxicity. Although there was marked interpatient variability, the minimally effective LV dose for selective protection seems to be 2.5 mg/m2. If tumors are resistant to methotrexate because of decreased transport of drug (and also folate), then the same pharmacological principle used to develop TMTX/LV for the treatment of P. carinii may be applied to treatment of some patients with cancer.

The murine-reduced folate carrier gene can act as a selectable marker and a suicide gene in hematopoietic cells in vivo.

Increased expression of the reduced folate carrier confers sensitivity to the antifolate drug methotrexate because it results in increased cellular uptake of this drug, and increased resistance to trimetrexate, a lipid-soluble antifolate drug, because it enables cells to take up exogenous folates that rescue cells from antifolate cytotoxicity. We therefore hypothesized that the reduced folate carrier could act as a suicide gene after treatment with methotrexate and as a selectable marker after exposure to trimetrexate. To test this hypothesis, we constructed replication-defective retroviruses containing the murine-reduced folate carrier (mRFC). Murine bone marrow cells transduced with the mRFC-containing retrovirus showed increased sensitivity to methotrexate and increased resistance to trimetrexate compared to empty vector-transduced controls in colony forming assays. Furthermore, colonies surviving trimetrexate and methotrexate treatment showed an enrichment of the mRFC gene after exposure to trimetrexate and a decrease after exposure to methotrexate. Lethally irradiated mice transplanted with bone marrow cells transduced with the mRFC-retrovirus and treated with the antifolate drugs after hematopoietic recovery demonstrated a relative increase in the number of cells containing the mRFC transgene after trimetrexate treatment and a decrease after methotrexate treatment. Therefore, these studies demonstrate the potential of the reduced folate carrier gene to play a dual role in gene therapy applications.

Clinical pharmacokinetics and pharmacology of trimetrexate.

Trimetrexate represents one of a number of new antimetabolites that have been studied in malignant, rheumatological and infectious disease. Methotrexate, the classical antifolate agent, is active in a broad spectrum of clinical settings, but its use is limited ny pre-existing or acquired cellular resistance. Trimetrexate is an agent that does not require uptake by the folate carrier transport system, a major mechanism of cellular resistance both in vitro and in vivo. Both dihydrofolate reductase inhibition and high performance liquid chromatography (HPLC) assays can be used to determine drug concentrations. Clearance of trimetrexate has been reported to follow biphasic or triphasic patterns. Elimination is primarily by biotransformation with less than 5% of the drug excreted renally in an unchanged form. Both active and inactive metabolites have been found, but the precise metabolic pathways have yet to be defined. The role of trimetrexate in the treatment of Pneumocystis carinii pneumonia is limited to compassionate use, as clinical studies have shown cotrimoxazole (trimethoprim-sulfamethoxazole) to be superior to trimetrexate. However, in a wide spectrum of malignant processes, trimetrexate appears to have a role either as a high-dose single agent, with calcium folinate (leucovorin calcium) rescue, or in combination with other antineoplastic agents. However, further trials are needed to fully establish the efficacy of trimetrexate in these settings. Increased knowledge of the pattern of resistance for individual tumours and tumour types may result in trimetrexate becoming more widely used clinically.

Trimetrexate. A review of its pharmacodynamic and pharmacokinetic properties and therapeutic potential in the treatment of Pneumocystis carinii pneumonia.

Trimetrexate is a folinic acid analogue structurally related to methotrexate, whose primary mechanism of action is believed to be inhibition of dihydrofolate reductase. This reduces the production of DNA and RNA precursors and leads to cell death. Trimetrexate is lipophilic and can passively diffuse across cell membranes including those of Pneumocystis carinii and its mammalian host. To minimise toxicity, trimetrexate must be coadministered with calcium folinate (leucovorin calcium), a reduced folate coenzyme, which is transported into, and protects, mammalian host cells but not P. carinii cells. In noncomparative trials trimetrexate was effective in the treatment of P. carinii pneumonia (PCP) in patients with AIDS who were intolerant of or refractory to cotrimoxazole (trimethoprim/sulfamethoxazole) and pentamidine treatment. In these patients, 2- to 4-week survival rates of 48 to 69% were reported. In a comparative trial in the initial therapy of PCP, trimetrexate was less effective than cotrimoxazole in moderate to severe disease as evidenced by a significantly higher failure rate. Trimetrexate was better tolerated than cotrimoxazole when used in this setting, however. Significantly fewer patients receiving trimetrexate plus calcium folinate discontinued treatment because of adverse events than did patients receiving cotrimoxazole. The most common adverse effect associated with trimetrexate is myelosuppression (neutropenia and thrombocytopenia); this is mitigated by coadministration of calcium folinate and is generally reversible upon dosage reduction or discontinuation. Other adverse effects include increases in serum aminotransferase levels, anaemia, fever, rash/pruritus, and increased alkaline phosphatase or serum creatinine levels. Further research into the use of trimetrexate, including its efficacy as prophylaxis, in combination with other agents and as an oral formulation, is needed to clearly define its role in the treatment of PCP and to identify patients most likely to benefit. Currently, trimetrexate should be considered as an alternative treatment option in immunocompromised patients with moderate to severe PCP who have not responded to or are intolerant of first-line therapy.

Pharmacokinetics of trimetrexate and dapsone in AIDS patients with Pneumocystis carinii pneumonia.

The objective of this study was to determine the pharmacokinetics of trimetrexate and dapsone in AIDS patients with moderate to severe pneumocystis pneumonia. Trimetrexate, leucovorin, and dapsone were administered for 21 +/- 3 days in the following doses: trimetrexate glucuronate, 45 mg/m2; leucovorin, 20 mg/m2; and dapsone, 100 mg daily. The pharmacokinetics of trimetrexate, dapsone, and dapsone's metabolite, monoacetyldapsone, were determined at three separate periods over the course of treatment. Serial blood samples were obtained over 24 hours after dosing and analyzed for trimetrexate, dapsone, and monoacetyldapsone, and pharmacokinetic parameters were determined. The mean parameters obtained for the early, mid-, and late collection periods were the following: trimetrexate: t1/2 = 8.29, 9.15, 10.00 hr; AUC = 16.85, 22.38, 24.49 mg.hr/l; CI = 5.58, 4.14, 3.96 l/hr, respectively. DDS: t1/2 = 14.99, 16.59, 15.13 hr; AUC = 30.60, 35.29, 36.08 mg.hr/l; CI = 3.82, 3.49, 3.01 l/hr, respectively. Monoacetyldapsone: t1/2 = 20.25, 18.66, 16.32 hr; AUC = 24.05, 24.06, 23.86 mg.hr/l, respectively. No statistically significant changes in pharmacokinetics for trimetrexate or dapsone were observed over the 21 +/- 3 day course of treatment. The results suggest that there are no major interactions between trimetrexate and dapsone when administered together in acutely ill patients.

N10-propargyl-5,8-dideazafolic acid (CB3717): inhibitory effects on human leukemia cell lines resistant to methotrexate or trimetrexate.

The inhibitory effects of N10-propargyl-5,8-dideazafolic acid (CB3717), a quinazoline antifolate and a potent thymidylate synthase inhibitor, were evaluated in human leukemia cell lines resistant to methotrexate (MTX) and trimetrexate (TMQ). MTX-resistant MOLT-3 cell lines, MOLT-3/MTX200 and MOLT-3/MTX10,000, were cross-resistant to CB3717; however, the degree of resistance was only tenfold for both cell lines, and increased dihydrofolate reductase activity in MOLT-3/MTX10,000 had little influence on the degree of CB3717 resistance. The MOLT-3 cell line made resistant to TMQ, MOLT-3/TMQ200, was as sensitive to CB3717 as the parent line. The cell growth inhibitory effect of CB3717 on MOLT-3 was reversed by the addition of thymidine. Leucovorin also partially reversed CB3717-induced growth inhibition. Cellular uptake of MTX and 5-methyl-tetrahydrofolate was hindered by the presence of a high concentration of CB3717, whereas TMQ uptake was not influenced by CB3717. CB3717 appears to enter the cells not only through reduced folate transport system, but by other route(s). CB3717 does not share the transport pathway with TMQ. Our observations that MTX-resistant cells with increased dihydrofolate reductase are not more resistant than cells without increased enzyme activity, and that TMQ-resistant cells are not cross-resistant to CB3717, may have clinical relevance.

Therapeutic strategies targeting proteins that regulate folate and reduced folate transport.

Folate is an essential vitamin which acts as a precursor for cofactors that regulate a variety of biochemical reactions. Cellular uptake of endogenous folates as well as antifolate agents such as methotrexate may be regulated by two independent transport proteins, the folate receptor and the reduced folate carrier. This paper reviews the molecular and functional characteristics of these transport systems and potential therapeutic approaches exploiting these targets in the treatment of cancer. Understanding of the molecular basis and functional characteristics of the transport of endogenous folates and folate analogs via the folate receptor and the reduced folate carrier has led to the development of novel antifolate agents through rational drug design and targeted therapeutic approaches for tumors that express or lack the presence of these transport proteins. With this knowledge, new and selective treatment will become available to more effectively treat patients with a variety of malignancies.

A phase I trial of trimetrexate (NSC352122) on a daily x 5 schedule in patients with refractory adult leukemia.

Seven adult patients with refractory acute leukemia were administered trimetrexate (TMTX), a non-classical folate antagonist, in a phase I trial. TMTX was administered as an intravenous bolus for five consecutive days at doses of 9-12 mg/m2 based on marrow response. The maximum tolerated dose was 12 mg/m2. Hepatotoxicity was the dose-limiting toxicity. Initial dosage reductions in patients with liver disease and/or low protein concentrations may be necessary since TMTX is significantly protein bound and cleared primarily by hepatic metabolism. The recommended phase II dose on this dosing schedule is 9 mg/m2.

Trimetrexate for Pneumocystis carinii pneumonia in patients with AIDS.

OBJECTIVE: The primary objective of this article is to introduce readers to the use of a new agent, trimetrexate (TMTX), in the treatment of Pneumocystis carinii pneumonia (PCP). The article also gives the readers an overview of PCP and discusses some of the controversies surrounding it. Pharmacokinetic data and clinical trials are reviewed, as well as adverse effects, drug interactions, and dosage guidelines. DATA SOURCES: A MEDLINE search was used to identify pertinent literature, including reviews. STUDY SELECTION: As both pharmacokinetic and clinical trials were few in number, all available trials were reviewed. DATA EXTRACTION: Pharmacokinetic data from trials involving patients with AIDS was sparse; therefore, those involving oncology patients, including a pediatric population, were included. Although more trials need to be done in AIDS patients, the results from the oncologic trials give us a baseline from which to extrapolate. All clinical trials available at the time of publication were reviewed as were all of the preliminary results from three ongoing trials, which were made available through a personal communication. DATA SYNTHESIS: TMTX has been found to be 1500 times more potent than trimethoprim as a dihydrofolate reductase inhibitor, and has the potential to provide an effective therapeutic option for PCP. TMTX is a lipid-soluble analog of methotrexate and is thus capable of greater penetration into Pneumocystis cells, which lack the folate membrane transport system necessary to take up classic folate structures like leucovorin and methotrexate, thereby negating any clinical effectiveness of methotrexate and allowing leucovorin to be used for host cell rescue. TMTX's pharmacokinetic parameters best fit a multicompartmental model with a terminal half-life of up to 12 hours. It is cleared both hepatically and renally with up to 41 percent excreted unchanged in the urine. Although TMTX's pharmacokinetic parameters are variable, the need for plasma concentration monitoring at present is unclear, as no dose-response relationship has been established.

Cytotoxicity of trimetrexate against antifolate-resistant human T-cell leukemia cell lines developed in oxidized or reduced folate.

Cytotoxicity of trimetrexate (TMQ), a lipophilic dihydrofolate reductase inhibitor, was examined in antifolate-resistant human T-cell leukemia cell lines developed in oxidized or reduced folate. An approximately 60-fold methotrexate (MTX)-resistant subline was developed in oxidized folate (pteroylglutamic acid: PGA) (CCRF-CEM/MTX60-PGA) from human T-cell leukemia cell line CCRF-CEM; this line exhibited impaired membrane transport of the drug. Further enhancement of MTX resistance resulted in selection of an approximately 5000-fold MTX-resistant subline (CCRF-CEM/ MTX5000-PGA), which showed increased dihydrofolate reductase activity due to gene amplification in addition to further impairment of MTX transport. An approximately 140-fold MTX-resistant subline, and then a 1500-fold MTX-resistant subline were developed in reduced folate (10 nM leucovorin) (CCRF-CEM/MTX140-LV and CCRF-CEM/MTX1500-LV); they exhibited increased dihydrofolate reductase due to gene amplification accompanied by increased intracellular drug accumulation of MTX. While CCRF-CEM/MTX140-LV and CCRF-CEM/MTX1500-LV cells showed cross-resistance to TMQ, CCRF-CEM/MTX60-PGA and CCRF-CEM/MTX5000-PGA cells were at least as sensitive to TMQ as the parent cells. TMQ was more potent against approximately 200-fold N10-propargyl-5,8-dideazafolic-acid (CB3717)-resistant human T-cell leukemia MOLT-3 sublines developed in PGA (MOLT-3/CB3717(200)-PGA) or leucovorin (MOLT-3/CB3717(200)-LV), as compared to the parent cells; MOLT-3/CB3717(200)-PGA and MOLT-3/CB3717(200)-LV cells were resistant to CB3717 by virtue of impaired transport, only the former possessing gene amplification of thymidylate synthase. The cytotoxicity of TMQ in both MOLT-3/CB3717(200)-PGA and MOLT-3/CB3717(200)-LV cells was reduced by addition of leucovorin in a dose-dependent manner, suggesting intracellular folate deficiency as a cause of TMQ sensitivity. These results demonstrate that TMQ overcomes transport-impaired antifolate resistance, irrespective of gene amplification of dihydrofolate reductase or thymidylate synthase. Types of folate used during the development of antifolate resistance seem to be important in relation to the mechanism of TMQ responsiveness as well as that of antifolate resistance.

Defective transport as a mechanism of acquired resistance to methotrexate in patients with acute lymphocytic leukemia.

Although the mechanisms of resistance to methotrexate (MTX) are known in experimental tumors made resistant to this drug, little information is available regarding acquired resistance to MTX in patients. A competitive displacement assay using the fluorescent lysine analogue of MTX, N-(4-amino-4-deoxy-N10-methylpteroyl)-N epsilon-(4'-fluorescein-thiocarbamyl)-L-lysine (PT430), was developed as a sensitive method of detection of transport resistance to MTX in cell lines, as well as in blast cells from patients with leukemia. Rapid uptake of PT430 at high concentrations (20 mumol/L) in leukemic blasts resulted in achievement of steady-state levels within 2 hours. Subsequent incubation with the folate antagonists, MTX and trimetrexate (TMTX), which differ in the mode of carrier transport, produced characteristic patterns of PT430 displacement. Flow cytometric analysis of the mean fluorescence intensity in the human CCRF-CEM T-cell lymphoblastic leukemia cell line and its MTX-resistant subline clearly identified the presence of transport deficiency in the resistant subline. Analysis of blasts from 17 patients with leukemia, nine with no prior chemotherapy and eight previously treated with chemotherapy, found evidence of MTX transport resistance in two of the four patients who were treated with MTX and considered to be clinically resistant to the drug. The finding that blast cells of some patients with leukemia considered clinically resistant to MTX is due to decreased MTX transport has important implications for clinical use of this drug and for new drug development.