James C. Drake

Neurotoxicity and Elevated Cerebrospinal-Fluid Methotrexate Concentration in Meningeal Leukemia

Abstract Cerebrospinal-fluid methotrexate concentration was measured in 25 patients receiving intrathecal therapy for prophylaxis or treatment of meningeal leukemia. In 20 patients with no manifestations of neurotoxicity, the mean antifolate value in the cerebrospinal fluid was 1.7 X 10–7 M two days after administration of 12 to 15 mg per square meter of intrathecal methotrexate, and declined thereafter with a half-life of 12 to 18 hours. Five patients with severe neurotoxicity had cerebrospinal-fluid methotrexate concentrations averaging 13.8 times higher than the mean, and these concentrations were consistently higher than the range of antifolate values in the asymptomatic patients. One patient with values 20 to 100 times greater than the mean in asymptomatic patients sustained a fatal myelopathy, and in another, with an apparent antifolate half-life of 48 hours, irreversible neurologic sequelae developed. These observations suggest that the neurotoxicity associated with intrathecal methotrexate may be ...

Fluorouracil and high-dose leucovorin in previously treated patients with metastatic breast cancer.

The efficacy and toxicity of leucovorin 500 mg/m2 administered intravenously (IV) over 30 minutes daily for five days followed in one hour by fluorouracil (5-FU) 375 mg/m2 administered IV daily for five days, each given every 3 weeks, was assessed in 54 previously treated patients with metastatic breast cancer. An overall objective response rate of 24% was achieved (95% confidence interval, 13% to 38%), with an additional 56% of patients maintaining stable disease. Eleven of 12 patients who responded had received previous 5-FU therapy. Toxicity of this regimen included grade 3 diarrhea in 13%, grade 3 or 4 mucositis in 33%, grade 3 or 4 granulocytopenia in 65%, and grade 3 or 4 thrombocytopenia in 19%. Delay of treatment was required for hematologic toxicity in 44 patients. Thirty-eight patients required dose reductions due to toxicity. Biochemical evaluation of tumor biopsy specimens obtained from 17 patients used as their own controls with and without leucovorin was performed. These studies reveal an increased stabilization of the 5-fluorodeoxyuridylate (FdUMP)-thymidylate synthase (TS) folate ternary complex with the addition of leucovorin. There was a 71% +/- 14% occupancy or inhibition of the enzyme with the use of both 5-FU and leucovorin, v 30% +/- 13% for 5-FU alone (P2 less than .037). The percent TS bound in responding patients was substantially higher than in those patients with progressive disease. Finally, the mean total tumor TS pre-therapy in seven patients was 31 fmol/mg compared with a mean of 81 fmol/mg in these same seven patients 24 hours after therapy. This 2.6-fold increase suggests that there is an induction of the enzyme, TS, with 5-FU treatment.

Trimetrexate for the Treatment of Pneumocystis carinii Pneumonia in Patients with the Acquired Immunodeficiency Syndrome

Preclinical studies have demonstrated that trimetrexate is a potent inhibitor of dihydrofolate reductase from Pneumocystis carinii. On the basis of this evidence, this lipid-soluble antifolate was used as an antipneumocystis agent in 49 patients with the acquired immunodeficiency syndrome (AIDS) and pneumocystis pneumonia. Simultaneous treatment with the reduced folate leucovorin was used as a specific antidote to protect host tissues from the toxic effects of the antifolate without affecting the antipneumocystis action of trimetrexate. Patients were assigned to three groups and treated for 21 days: in Group I, trimetrexate with leucovorin was used as salvage therapy in patients in whom standard treatments (both pentamidine isethionate and trimethoprim-sulfamethoxazole) could not be tolerated or had failed (16 patients); in Group II, trimetrexate with leucovorin was used as initial therapy in patients with a history of sulfonamide inefficacy or intolerance (16 patients); and in Group III, trimetrexate with leucovorin plus sulfadiazine was used as initial therapy (17 patients). The response and survival rates were, respectively, 69 percent and 69 percent in Group I; 63 percent and 88 percent in Group II; and 71 percent and 77 percent in Group III. Trimetrexate therapy had minimal toxicity; transient neutropenia or thrombocytopenia occurred in 12 patients and mild elevation of serum aminotransferases in 4. We conclude that the combination of trimetrexate and leucovorin is safe and effective for the initial treatment of pneumocystis pneumonia in patients with AIDS and for the treatment of patients with intolerance or lack of response to standard therapies.

Synthesis, Retention, and Biological Activity of Methotrexate Polyglutamates in Cultured Human Breast Cancer Cells

To determine the pharmacologic importance of methotrexate (MTX) polyglutamates, we examined the formation, retention, and effect of these metabolites in cultured human breast cancer cells. Two cell lines (MCF-7 and ZR-75-B) converted the drug to gamma-polyglutamate derivatives in a dose- and time-dependent reaction. After 24-h incubations with 2 muM MTX, polyglutamates of two to five amino acids in length accounted for 55.4% (51.9 nmol/g) of intracellular drug in the MCF-7 cells and 87.6% (62.4 nmol/g) of drug in ZR-75-B cells. In contrast, MDA-231 cells showed lesser accumulation of MTX, and only 32% (4.06 nmol/g) of the intracellular drug was in the form of polyglutamates, a difference that could only partially be explained by decreased ability of these cells to take up free drug from the medium. When MCF-7 and ZR-75-B cells containing polyglutamates were transferred to drug-free medium for 24 h, 22 and 51% of the total intracellular drug were, respectively, retained in each cell line. The loss of intracellular drug was primarily accounted for by disappearance of parent compound and polyglutamates containing 1-3 additional glutamyl residues. The rates of disappearance from cells decreased with increasing glutamyl chain length. All of the 4-NH(2)-10-CH(3)-PteGlu(5) and 47 and 38% of the 4-NH(2)-10-CH(3)-PteGlu(4) remained in the MCF-7 and ZR-75-B cells, respectively, and could be identified in the cytosol after 24 h in drug-free medium. The retention of MTX polyglutamates in these two cell lines in excess of dihydrofolate reductase binding capacity led to prolonged inhibition of thymidylate synthesis and loss of cell viability after removal of extracellular MTX. After 24-h incubation with 2 muM MTX and an additional 24 h in drug-free medium, [(3)H]deoxyuridine incorporation was still inhibited to 30% of control in the MCF-7 cells and 34.7% of control in ZR-75-B cells; this persistent inhibition was associated with a 30% reduction in cell numbers in each cell line during the 24-h period in drug-free medium. In contrast, [(3)H]deoxyuridine incorporation and cell growth quickly recovered to normal in the MDA-231 cells following removal of 2 muM MTX from the medium after a 24-h incubation. Prolonged inhibition of both thymidylate synthesis and cell growth was observed in this cell line in drug-free medium only after a 24-h incubation with 10 muM MTX, a condition that leads to the synthesis of 11.3 nmol/g of MTX polyglutamates. These studies demonstrate that polyglutamate formation allows a prolonged retention of drug in a noneffluxable form and prolonged inhibition of both thymidylate synthesis and cell growth following removal of extracellular drug.

Presence of 2,4-diamino-N10-methylpteroic acid after high-dose methotrexate

Assay of plasma methotrexate has been established as important to its safe use. We have investigated the specificity of 2 assay procedures for methotrexate: the competitive dihydrofolate reductase binding assay (CRBA) and the radioimmunoassay (RIA). The RIA of plasma methotrexate resulted in consistently higher values than the CRRA, with greater differences at later measurement times. A compound that strongly cross‐reacts in the RIA, but not the CRBA, has been identified in plasma and urine of patients on high‐dose methotrexate therapy, and appears to be the carboxypeptidase cleavage product (2,4‐diamino‐N10‐methylpteroic acid) on the basis of chromatographic and ultraviolet spectral properties. Although this compound is present as a minor contaminant in commercial methotrexate preparations, quantitative assessment of urinary excretion suggests that in man a major portion of the compound is derived from methotrexate metabolism.

Potent antipneumocystis and antitoxoplasma activities of piritrexim, a lipid-soluble antifolate.

Piritrexim, a lipid-soluble antifolate, was evaluated for its activity against Pneumocystis carinii and Toxoplasma gondii. The concentration of piritrexim needed to inhibit 50% of the catalytic activity of P. carinii dihydrofolate reductase (DHFR) was 19.3 nM, and that for T. gondii DHFR was 17.0 nM, concentrations that were 40- to over 1,000-fold less than those needed for the inhibition of activity by trimethoprim and pyrimethamine, the antifolates conventionally used in treating these organisms. Piritrexim was able to inhibit replication of T. gondii in a mouse peritoneal macrophage model at concentrations of 0.1 to 1.0 microM. Leucovorin, a reduced folate that can bypass the inhibition of DHFR by antifols in mammalian cells but not in protozoa, did not affect the ability of piritrexim to inhibit T. gondii replication. The addition of sulfadiazine, which alone was ineffective, to piritrexim allowed inhibition of T. gondii replication at lower concentrations of piritrexim than when piritrexim was used alone. These results suggest that piritrexim, alone or combined with a sulfonamide, may be a highly potent antitoxoplasma and antipneumocystis agent that could provide major pharmacologic and clinical advantages over available agents.

Potent in vitro and in vivo antitoxoplasma activity of the lipid-soluble antifolate trimetrexate.

Trimetrexate, a highly lipid-soluble quinazoline antifolate now undergoing trials as an anticancer agent, was found to be a potent inhibitor of the dihydrofolate reductase (DHFR) isolated from Toxoplasma gondii. The concentration required for 50% inhibition of protozoal DHFR was 1.4 nM. As an inhibitor of this enzyme, trimetrexate was almost 600-fold (amount of antifolate required to inhibit catalytic reaction by 50%) and 750-fold (inhibition constant) more potent than pyrimethamine, the DHFR inhibitor currently used to treat toxoplasma infection. When the protozoan was incubated with 1 microM trimetrexate, the drug rapidly reached high intracellular concentrations. Since toxoplasma organisms lack a transmembrane transport system for physiologic folates, host toxicity can be prevented by co-administration of the reduced folate, leucovorin, without reversing the antiprotozoal effect. The effectiveness of trimetrexate against toxoplasma was demonstrated both in vitro and vivo. Proliferation of toxoplasma in murine macrophages in vitro was completely inhibited by exposure of these cells to 10(-7) M trimetrexate for 18 h. When used alone, trimetrexate was able to extend the survival of T. gondii-infected mice.

Resistance to Tomudex (ZD1694): Multifactorial in human breast and colon carcinoma cell lines

ZD1694 (Tomudex; TDX) is a quinazoline antifolate that, when polyglutamated, is a potent inhibitor of thymidylate synthase (TS), the enzyme that converts dUMP to dTMP. Continuous exposure of MCF-7 breast and NCI H630 colon cells to TDX, with stepwise increases in TDX up to 2.0 microM, resulted in stably resistant cell lines (MCFTDX and H630TDX) that were highly resistant to TDX. Initial studies revealed 34-fold increase in TS protein levels in MCFTDX and a 52-fold increase in TS levels in H630TDX cell lines. Despite continued exposure of these cells to 2.0 microM TDX, TS protein and TS mRNA expression decreased to parental levels in H630TDX cells, whereas in MCFTDX cells TS mRNA expression and TS protein levels remained elevated. Southern blot analysis revealed a 20-fold TS gene amplification in the MCFTDX cell line. TDX uptake was 2-fold higher in resistant MCFTDX cells than in parental MCF-7 cells, whereas in H630TDX cells TDX uptake was 50-fold less than that observed in parental H630 cells. In contrast, no change in the transport of either leucovorin or methotrexate into H630TDX cells was noted when compared with the H630 parental cells. In H630TDX cells, folylpolyglutamate synthetase (FPGS) activity was 48-fold less compared to parent H630 cells; however, FPGS mRNA expression was similar in both lines. H630TDX cells were also highly resistant to ZD9331, a novel quinazoline TS inhibitor that does not require polyglutamation, suggesting that defective transport by the reduced folate carrier was also an important mechanism of resistance in these cells. In MCFTDX and H630TDX resistant cells, several mechanisms of resistance are apparent: one increased TS expression; the others evolved over time from increased TS expression to decreased FPGS levels and decreased TDX transport.

Test dose for predicting high-dose methotrexate infusions

Eighteen evaluable patients were studied to determine whether individual methotrexate (MTX) kinetics, determined by test‐dose bolus injection, could be used to predict plasma drug concentrations during and after high‐dose infusion. Small nontoxic doses of MTX (10 mg/m2) was given to patients who were followed for 12 to 24 hr and the kinetic data were used to predict subsequent kinetic behavior of moderate‐ and high‐dose methotrexate infusions (150 to 1500 mg/m2 over 12 to 18 hr). After test‐dose injection, MTX clearance varied from 36 to 138 ml/min/m2and decreased with advancing age (r = −0.49, P < 0.05). MTX clearance varied from 24 to 100 m /min/m2 after high‐doses. Although there was a trend to decreasing clearance with advancing age, this was not as clear as with the test dose (r = −0.42, P > 0.05). There was no correlation between MTX clearance and Creatinine clearance in this group of patients in whom Creatinine clearance varied from 32 to 63 ml/min/m2. When the kinetic parameters derived from the test‐dose data were used, accurate predictions could be made of the infusion plateau (r = 0.89, P < 0.001) and 24‐hr (r = 0.92, P < 0.001) MTX concentrations after high‐dose infusions. Our results indicate that test‐dose MTX kinetics may serve as a guide to dose modification of MTX infusions in some high‐risk patients.

Anaphylactoid type reactions in two patients receiving high dose intravenous methotrexate

Two patients receiving intravenous high dose methotrexate (MTX) and intracutaneous BCG injections as adjuvant treatment for osteogenic sarcoma suffered sudden cardiovascular collapse within minutes of infusion of MTX. These cases demonstrate that anaphylactic or idiosyncratic reactions to MTX and/or contaminants in these preparations do occur and that careful patient monitoring is required.