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.

Determinants of the sensitivity of human small-cell lung cancer cell lines to methotrexate.

We have characterized the determinants of methotrexate (MTX) responsiveness in eight patient-derived cell lines of small-cell lung cancer (SCLC). Clonogenic survival was correlated with factors known to affect sensitivity to drug. NCI-H209 and NCI-H128 were most drug sensitive, with drug concentrations required to inhibit clonogenic survival by 50% with less than 0.1 microM MTX. Six cell lines (NCI-H187, NCI-H345, NCI-H60, NCI-H524, NCI-H146, and NCI-N417D) were relatively drug resistant. In all cell lines studied, higher molecular weight MTX-polyglutamates (MTX-PGs) with 3-5 glutamyl moieties (MTX-Glu3 through MTX-Glu5) were selectively retained. Relative resistance to low (1.0 microM) drug concentrations appeared to be largely due to decreased intracellular metabolism of MTX. Five of the six resistant lines were able to synthesize polyglutamates at higher (10 microM) drug concentrations, although one resistant cell line (NCI-N417D) did not synthesize higher molecular weight MTX-PGs, even after exposure to 10 microM drug. Two cell lines with resistance to 10 microM MTX (NCI-H146 and NCI-H524) synthesized and retained higher molecular weight MTX-PGs in excess of binding capacity after exposure to 10 microM drug. However, the specific activity of thymidylate synthase in these cell lines was low. MTX sensitivity in patient-derived cell lines of SCLC requires the ability of cells to accumulate and retain intracellular drug in the form of polyglutamate metabolites in excess of dihydrofolate reductase, as well as a high basal level of consumption of reduced folates in the synthesis of thymidylate.

Intravenous administration of the glycoprotein IIb-IIIa receptor antagonist 7E3 induces reperfusion of an acute thrombotic occlusion of the canine coronary artery.

The ability of the F(ab')2 fragment of the murine monoclonal antibody 7E3 directed against the platelet glycoprotein IIb-IIIa receptor complex, to cause reperfusion of a totally occluding coronary artery thrombus was examined alone and in combination with aspirin and heparin in a canine model of coronary artery thrombosis. A localized thrombus was produced in the left circumflex coronary artery in open-chest dogs by electrolytic injury of the endothelium. Intravenous administration of a single injection of 5.0 mg/kg aspirin and heparin (80 U/kg bolus plus 30 U/kg/hr x 2 hr) maintained vessel patency for approximately 101 +/- 15 minutes. After vessels had been completely occluded for 5 minutes (in the presence of aspirin + heparin), a single intravenous injection of saline (10 ml) or 0.8 mg/kg 7E3 was administered. Reperfusion was observed in all dogs (6 of 6) receiving 7E3; 4 of 6 dogs maintained vessel patency throughout the course of the 2 hour observation period. Activated partial thromboplastin and thrombin times were elevated 1.4 and 9 fold, respectively, in groups that received heparin. Template bleeding times were significantly elevated in the groups receiving 7E3. In the control group, 2 of 5 dogs reperfused briefly, however neither were patent at the end of the observation period. A third group of 4 dogs which did not receive the aspirin + heparin regimen was allowed to occlude and 5 minutes later received a single intravenous injection of 0.8 mg/kg 7E3. None of the 4 dogs in this group reperfused at any time during the study. There were no significant differences between groups in regards to hematological or hemodynamic measurements during the experiment. We concluded from these findings that the monoclonal antibody, 7E3 can promote the dissolution of friable coronary artery thrombi that evolve during standard anticoagulant and antiplatelet therapy.

Synthesis, binding and intracellular retention of methotrexate polyglutamates by cultured human breast cancer cells.

Synthesis, binding, and intracellular retention of methotrexate polyglutamates by cultured human breast cancer cells were investigated by gel filtration and high-pressure liquid chromatography to separate methotrexate from its metabolites. MCF-7, ZR-75-1, and MDA-231 human breast cancer cells were found to readily convert methotrexate to higher polyglutamates during a 24-hr incubation period, although at differing rates. Examination of that portion of intracellular methotrexate specifically bound to dihydrofolate reductase revealed that, with prolonged incubation, methotrexate polyglutamates become the predominant drug form bound to the enzyme. Similarly, methotrexate polyglutamates accumulated free in the cytosol and when cells were suspended in drug-free medium, were retained intracellularly both bound to dihydrofolate reductase and in the unbound fraction, indicating their slow passage through the cell membrane. Studies of methotrexate polyglutamate binding to purified bacterial dihydrofolate reductase revealed high affinity binding for compounds with up to 6 additional glutamyl residues. These studies demonstrate that methotrexate polyglutamates are readily formed in human breast cancer cells, bind intracellularly to dihydrofolate reductase, and are selectively retained both bound to the enzyme and free in the cell cytosol.

Methotrexate polyglutamate synthesis by cultured human breast cancer cells.

We studied the conversion of methotrexate to poly-gamma-glutamyl derivatives by cultured human breast cancer cells. After incubation with 2 micro M [3',5',9-3H]methotrexate, MCF-7 cells were washed free of extracellular drug and were boiled to lyse cells and to release drug bound to dihydrofolate reductase (tetrahydrofolate dehydrogenase; 5,6,7,8-tetrahydrofolate:NADP+ oxidoreductase, EC 1.5.1.3). The supernatant fraction was chromatographed on Sephadex G-15 to separate parent drug from polyglutamate forms. These cells rapidly and quantitatively converted methotrexate to polyglutamates, such that after 24 hr of incubation, 70 +/- (SEM) 3% of intracellular methotrexate existed as polyglutamates. Examination of that portion of intracellular methotrexate specifically bound to dihydrofolate reductase indicated that, with prolonged incubation, methotrexate polyglutamates become the predominant drug form bound to the enzyme. These studies demonstrate that methotrexate polyglutamates are readily formed in human tumor cells and bind to dihydrofolate reductase. Because these forms of the drug may be selectively retained within the cell, they may be important determinants of the duration of action and, ultimately, the cytotoxicity of methotrexate in human solid tumors.

Lack of stereospecificity at carbon 6 of methyltetrahydrofolate transport in Ehrlich ascites tumor cells. Carrier-mediated transport of both stereoisomers.

Nonlabeled and tritiated stereoisomers of 5-methyltetrahydrofolate were prepared and were both shown to be substrates for the high affinity H4 folate cofactor membrane transport carrier in Ehrlich ascites tumor cells. Both the enzymically active form and the isomer having the opposite configuration at carbon 6 inhibited the influx of enzymically synthesized (+)-5-methyltetrahydrofolate, methotrexate, and aminopterin. When added to the media of cells preloaded with methotrexate, both isomers stimulated a net efflux of the antifolate from the cell. Influx of the natural and unnatural isomers followed Michaelis-Menten kinetics with comparable Km values. Each isomer competitively inhibited influx of the other.

Rate-limiting steps in the interactions of fluoropyrimidines and methotrexate.

Rate-limiting steps are defined between methotrexate (MTX) and 5-fluorouracil (FU) or 5-fluorodeoxyuridine (FUdR) and [14C]-formate incorporation into RNA, DNA and protein as a function of the basal rate of dTMP synthesis. When Ehrlich cells are incubated with 0.1 microM FU dR, 1 microM FU and 50 microM MTX for 1-35 min. [3H]-deoxyuridine (UdR) incorporation into DNA is maximally inhibited within 1, 10 and 15 min respectively. The delay in suppression of [3H]-UdR incorporation into MTX-exposed cells compared to cells exposed to FU or FUdR is related to the slow transport of MTX and the increasing free intracellular MTX levels. Influx of MTX is 4 and 10 times slower than FU and FUdR respectively. At 2.5, 5, 10 and 15 min the free intracellular MTX levels (nmol/g dry wt) are 5.8, 7.4, 8.7 and 8.8 respectively. Free intracellular FdUMP is identified 1 min after exposure of cells to FU and FUdR. Antagonism to MTX-suppression of [14C]-formate incorporation into RNA, DNA and protein occurs when cells are simultaneously exposed to MTX and FU or FUdR. However, [14C]-formate incorporation into RNA, DNA and protein is maximally inhibited when Ehrlich tumor cells are incubated with 50 microM MTX for 10 min and then exposed to 1 microM FU for 1 min (a time in which free intracellular MTX is maximal and [3H]-UdR incorporation is maximally suppressed). Hence the sequence and time of administration of FU or FUdR and MTX inhibition of formate incorporation into RNA, DNA and protein is related to the rate of (a) FU, FUdR and MTX transport, (b) FU and FUdR metabolism to FdUMP and (c) generation of maximal free intracellular MTX.

Pharmacological profile of recombinant, human activated protein C (LY203638) in a canine model of coronary artery thrombosis.

The antithrombotic activity of recombinant, human activated protein C (rh-APC, LY203638) was examined in a model of canine coronary artery thrombosis. Three doses of rh-APC (0.5, 1.0, and 2.0 mg/kg/h) were administered intravenously for 2 h. Whole blood clotting times (thrombin time, activated partial thromboplastin time), ex vivo platelet aggregation, and template bleeding times were determined. Activated partial thromboplastin time significantly increased 2- and 3.7-fold during the 2-h infusion of rh-APC (1.0 and 2.0 mg/kg/h, respectively); thrombin time did not change. Intravenous infusions of rh-APC (1.0 and 2.0 mg/kg/h) produced significant prolongations to occlusion, 186 +/- 21 and 190 +/- 22 min, respectively, compared with the vehicle and the 0.5 mg/kg/h group (86 +/- 12 and 93 +/- 17 min, respectively). Vessel patency was better at the end of the experiment in the intermediate- and high-dose groups (3 of 6 and 3 of 5 vessels, 1.0 and 2.0 mg/kg/h, respectively) compared with the vehicle and 0.5 mg/kg/h groups (0/5 and 0/6, respectively). Only the 1.0 mg/kg/h group was found to have significantly elevated template bleeding times, with peak increases seen 60 min into the drug infusion. All groups had returned to baseline values by the end of the study. There was no observed inhibition of platelet aggregation. These data demonstrate that recombinant, human activated protein C is an effective anticoagulant and antithrombotic agent in the dog.

Antithrombotic assessment of the effects of combination therapy with the anticoagulants efegatran and heparin and the glycoprotein IIb-IIIa platelet receptor antagonist 7E3 in a canine model of coronary artery thrombosis.

BACKGROUND: There is a paucity of data regarding the antithrombotic pharmacology of the drug-drug interactions between the newer anticoagulant and antiplatelet agents. In this investigation, we have studied the antithrombotic effects of combinations of minimum effective doses of the glycoprotein IIb-IIIa receptor antagonist 7E3 [murine F(ab')2] with both heparin and the novel tripeptide arginal antithrombin efegatran (LY294468) in a canine model of coronary artery thrombosis. METHODS AND RESULTS: Thrombogenesis was initiated by electrolytic injury of the intimal surface of the left circumflex coronary artery. The groups studied were efegatran (0.25 mg . kg-1. h-1), heparin (80 U/kg, single injection, plus 30 U . kg-1. h-1), 7E3 (0.4 mg/kg, single injection), 7E3+efegatran, and 7E3+heparin. The combination of 7E3+efegatran was found to maintain better vessel patency (P < .05) at the end of the experiment (4 of 5 vessels) than all other groups (0 of 5, 0 of 4, 1 of 6, 2 of 7, and 1 of 6 for the vehicle-, heparin-, 7E3-, efegatran-, and 7E3+heparin-treated groups, respectively). Bleeding times were increased (P < .05) in both the 7E3+heparin group (fourfold) and the 7E3+efegatran group (threefold). 7E3 alone and both combination treatments produced significant reductions in ADP, arachidonic acid, and thrombin-induced platelet aggregation, whereas efegatran and heparin abolished only thrombin-induced aggregation. CONCLUSIONS: The present investigation demonstrates that combination therapy with minimum effective doses of 7E3+efegatran provided enhanced antithrombotic efficacy compared with 7E3+heparin in this model of thrombosis.