John J. McGuire

Lipophilic methotrexate conjugates with antitumor activity

Lipophilic methotrexate (MTX)-lipoamino acid conjugates coupled with amide or ester linkages (1a-1r) were synthesised. The inhibitory activity of the conjugates was evaluated on bovine liver DHFR. The in vitro growth inhibitory effect against MTX-sensitive human lymphoblastoid CCRF-CEM cells and an MTX-resistant sub-line (CEM/MTX), which displays defective intracellular transport of MTX, was determined under short-term and continuous (120-h incubation) exposure conditions. The alpha, gamma, or alpha,gamma amide conjugates showed different activity in inhibiting the growth of parent cells. CEM/MTX cells were much less susceptible than CCRF-CEM cells to inhibition by alpha or alpha,gamma-substituted lipoamino acid conjugates, whereas both cell lines were almost equally sensitive to the MTX-gamma conjugates. Although less potent than MTX, they could partially circumvent the impaired transport system. These findings confirm that lipophilic MTX conjugates may be good lead compounds on the drug development for the treatment of some MTX-resistant tumors. Ester-type conjugates displayed an interesting activity against parent CCRF-CEM cells, although they were less potent against the transport-resistant sub-line. Stability studies on these molecules indicated that they are not degraded into MTX in the culture medium, thus suggesting that they are not able to over-cross cell resistance despite of their lipophilicity.

Mechanisms of resistance to N-[5-[N-(3, 4-dihydro-2-methyl-4-oxoquinazolin-6-ylmethyl)-N-methylamino]-2-thenoyl]-L-glutamic acid (ZD1694), a folate-based thymidylate synthase inhibitor, in the HCT-8 human ileocecal adenocarcinoma cell line

N-[5-[N-(3,4-Dihydro-2-methyl-4-oxoquinazolin-6-ylmethyl)-N- methylamino]-2-thenoyl]-L-glutamic acid (ZD1694) is a folate-based thymidylate synthase (TS; EC 2.1.1.45) inhibitor. Metabolism to higher chain length polyglutamates is essential for its optimal cytotoxic effect. A ZD1694-resistant (300-fold) human ileocecal carcinoma cell line (HCT-8/DW2) was developed, and its mechanism of resistance was evaluated. TS activities in situ and TS protein levels in the HCT-8 parental line and HCT-8/DW2 were similar (168 +/- 47 vs 137 +/- 25 pmol/hr/10(6) cells and 2.05 +/- 0.28 vs 2.07 +/- 0.19 pmol/mg protein, respectively). The IC50 values of ZD1694 for TS inhibition in cell-free extracts were similar in both lines, but the IC50 of ZD1694 for TS inhibition in situ in HCT-8/DW2 cells was 27- and 268-fold higher than that in HCT-8 cells at 0 and 24 hr, respectively, after a 2-hr drug exposure. Folylpolyglutamate synthetase (FPGS; EC6.3.2.17) activity was significantly lower in resistant HCT-8/DW2 cells as compared with parental HCT-8 cells (88 +/- 40 vs 1065 +/- 438 pmol/hr/mg protein when ZD1694 was used as substrate). The combined endogenous pool of methylenetetrahydrofolate and tetrahydrofolate in HCT-8/DW2 cells was also decreased. In addition, HCT-8/DW2 cells accumulated lower levels of methotrexate (MTX) in a 2-hr period, although the initial velocity of MTX transport was similar to that in parental HCT-8 cells. The lower level of FPGS activity and the lower level of (anti)folate accumulation in HCT-8/DW2 correlated with drug resistance and with the higher IC50 of ZD1694 for in situ TS inhibition. In addition, drug resistance was also correlated with the rapid recovery of in situ TS activity after drug treatment. In brief, in this highly ZD1694-resistant HCT-8 cell line, resistance is associated with decreased FPGS activity, which, in turn, affects the metabolism of ZD1694 and consequently the extent and duration of in situ TS inhibition by the drug.

5-Amino-4-Imidazolecarboxamide Riboside Potentiates Both Transport of Reduced Folates and Antifolates by the Human Reduced Folate Carrier and Their Subsequent Metabolism

Transport is required before reduced folates and anticancer antifolates [e.g., methotrexate (MTX)] exert their physiologic functions or cytotoxic effects. The folate/antifolate transporter with the widest tissue distribution and greatest activity is the reduced folate carrier (RFC). There is little evidence that RFC-mediated influx is posttranscriptionally regulated. We show that [(3)H]MTX influx in CCRF-CEM human childhood T-leukemia cells is potentiated up to 6-fold by exogenous 5-amino-4-imidazolecarboxamide riboside (AICAr) in a AICAr and MTX concentration-dependent manner. Metabolism to more biologically active polyglutamate forms is also potentiated for MTX and other antifolates. That potentiation of influx by AICAr is mediated by effects on the RFC is supported by analyses +/-AICAr showing (a) similarity and magnitude of kinetic constants for [(3)H]MTX influx; (b) similarity of inhibitory potency of known RFC substrates; (c) lack of potentiation in a CCRF-CEM subline that does not express the RFC; and (d) similarity of time and temperature dependence. Potentiation occurs rapidly and does not require new protein synthesis. Effects of specific inhibitors of folate metabolism and the time and sequence of AICAr incubation with cells suggest that both dihydrofolate reductase inhibition and metabolism of AICAr are essential for potentiation. Acute folate deficiency or incubation of CCRF-CEM with AICAr-related metabolites (e.g., adenosine) does not initiate potentiation. AICAr increases growth inhibitory potency of MTX and aminopterin against CCRF-CEM cells when both AICAr and antifolate are present for the first 24 hours of a 120-hour growth period. AICAr is the first small molecule that regulates RFC activity.

The expanding role of folates and fluoropyrimidines in cancer chemotherapy

Determinants of Response to Fluopopyrimidines in Combination with CF.- Overview: Rational Basis for Development of Fluoropyrimidine/5-Formyltetrahydrofolate Combination Chemotherapy.- The Natural and Unnatural Diastereomers of Leucovorin: Aspects of Their Cellular Pharmacology.- Distribution and Metabolism of Calcium Leucovorin in Normal and Tumor Tissue.- Pole of Dose, Schedule and Route of Administration of 5-Formyltetrahydrofolate: Preclinical and Clinical Investigations.- Pharmacokinetic Analysis of (6S)-5-Formyltetrahydrofolate (1-CF), (6R)-5-Formyltetrahydrofolate (d-CF) and 5-Methyltetrahydrofolate (5-CH3-THF) in Patients Receiving Constant i. v. Infusion of High-Dose (6R,S)-5-Formyltetrahydrofolate (Leucovorin).- Evidence for Thymidylate Synthase as the Critical Site of Action of Fluoropyrimidines in Combination with CF.- The Role of Thymidylate Synthase in the Response to Fluoropyrimidine-Folinic Acid Combinations.- Tumor Cell Responses to Inhibition of Thymidylate Synthase.- Selectivity of CF and 5-Fluorouracil: Critical Role of Polyglutamylation.- Methods for Thymidylate Synthase Pharmacodynamics: Serial Biopsy, Free and Total TS, FdUMP and dUMP, and H4PteGlu and CH2-H4PteGlu Assays.- The Treatment of Metastatic Breast Cancer with 5-Fluorouracil and Leucovorin.- Thymidylate Synthase and Fluorouracil.- Mechanisms for Cisplatin-FUra Synergism and Cisplatin Resistance in Human Ovarian Carcinoma Cells both In Vitro and In Vivo.- Summation of Sessions 1 and.- Summation of Session 1.- Summation of Session 2.- Therapeutic Efficacy and Toxicity of Fluoropypimidines in Combination with CF.- The Roswell Park Memorial Institute and Gastrointestinal Tumor Study Group Phase III Experience with the Modulation of 5-Fluorouracil by Leucovorin in Metastatic Colorectal Adenocarcinoma.- Mount Sinai Clinical Experience with Leucovorin and 5-Fluorouracil.- High-Dose Weekly Oral Leucovorin and 5-Fluorouracil in Previously Untreated Patients with Advanced Colorectal Carcinoma: A Phase I Study.- A Controlled Clinical Trial Including Folinic Acid at Two Distinct Dose Levels in Combination with 5-Fluorouracil (5FU) for the Treatment of Advanced Colorectal Cancer: Experience of the Mayo Clinic and North Central Cancer Treatment Group.- 5-Fluorouracil (FUra) and Folinic Acid (FA) Therapy in Patients with Colorectal Cancer.- A Northern California Oncology Group Randomized Trial of Single Agent 5-FU vs. High-Dose Folinic Acid + 5-FU vs. Methotrexate + 5-FU + Folinic Acid in Patients with Disseminated Measurable Large Bowel Cancer.- Clinical Experience with CF-FUra.- A Randomized Trial of 5-Fluorouracil Alone versus 5-Fluorouracil and High Dose Leucovorin in Untreated Advanced Colorectal Cancer Patients.- Pharmacodynamics of 5-Fluorouracil and Leucovorin.- 5-Fluorouracil and 5-Formyltetrahydrofolate in Advanced Malignancies.- Folinic Acid (CF)/5-Fluorouracil (FUra) Combinations in Advanced Gastrointestinal Carcinomas.- Fffective Salvage Therapy for Refractory Disseminated Breast Cancer with 5-Fluorouracil and High-Dose Continuous Infusion Folinic Acid.- Progress Report on a Phase II Trial of 5-Fluorouracil Plus Citrovorum Factor in Women with Metastatic Breast Cancer.- Adjuvant Therapy for Colorectal Cancer: The NSABP Clinical Trials.- Chemotherapy of Advanced and Relapsed Squamous Cell Cancer of the Head and Neck with Split-Dose Cisplatinum (DDP), 5-Fluorouracil (Fura) and Leucovorin (CF).- 5-Fluorouracil/Folinic Acid/Cisplatin-Combination and Simultaneous Accelerated Split-Course Radiotherapy in Advanced Head and Neck Cancer.- Progress Report on Studies of FAM-CF for Gastric Cancer and Intra-peritoneal Administration of FUra-CF Followed by Cisplatin (DDP).- Clinical Experience with 5-FU/DDP +- OHDW Combination Chemotherapy in Patients with Advanced Colorectal Carcinoma.- 5-Fluorouracil and Folinic Acid: Summary of Clinical Experience.- Panel Discussion of Future Directions.- Abbreviations.- Author Index.

Biochemical and biological properties of methotrexate analogs containing D-glutamic acid or D-erythro, threo-4-fluoroglutamic acid.

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Potent inhibition of human folylpolyglutamate synthetase by a phosphinic acid mimic of the tetrahedral reaction intermediate

A phosphorous-containing pseudopeptide folate analog (Valiaeva et al., J Org Chem 2001;66:5146-54) was designed to mimic the tetrahedral intermediate formed in the ATP-dependent reaction catalyzed by folylpolyglutamate synthetase (FPGS). This analog, methotrexate-phosphinate (MTX-phosphinate; 4-amino-4-deoxy-10-methylpteroyl-L-Glu-gamma-[psi(P(O)(OH)-CH(2))]glutarate), is a highly potent (K(is), 3.1+/-0.5 nM), competitive inhibitor of recombinant human cytosolic FPGS. Within experimental limits, FPGS inhibition was not time-dependent, and preincubation of FPGS, inhibitor, and ATP did not potentiate the inhibition. These results suggest that slow phosphorylation to produce a more potent inhibitor form is not involved. MTX-phosphinate was not growth inhibitory to human CCRF-CEM leukemia cells at 1 microM (70-fold above the concentration of MTX giving 50% growth inhibition), probably because of poor transport. Because of its exceedingly high potency as an FPGS inhibitor, MTX-phosphinate represents a lead structure from which cell-permeable analogs may be developed to test the hypothesis that FPGS inhibition is therapeutically efficacious.

Structural specificity of inhibition of human folylpolyglutamate synthetase by ornithinecontaining folate analogs

A series of folate analogs containing ornithine instead of glutamate was synthesized and tested for inhibition of folylpolyglutamate synthetase (FPGS) and other folate-dependent enzymes of human leukemia cell lines. Reduced derivatives of 2-amino-4-oxo-10-methyl-pteroyl-ornithine had dramatically increased inhibitory potency against FPGS compared to the oxidized parent. The amino-pterin analog (2,4-diamino-pteroylornithine) was a potent inhibitor of both dihydrofolate reductase and FPGS. It was a much more potent linear competitive inhibitor of human FPGS than the corresponding methotrexate derivative previously described (Ki = 0.15-0.26 and 3 microM respectively). A quinazoline folate analog, 2-amino-4-oxo-5,8-dideazapteroyl-ornithine, was a relatively poor inhibitor of isolated dihydrofolate reductase and thymidylate synthase; however, it is the most potent human FPGS inhibitor identified to date (Ki = 100-150 nM). Because of the lack of appreciable interaction with other folate-dependent enzymes, structures incorporating the 2-amino-4-oxo-5,8-dideazapteroate nucleus may thus lead to selective inhibition of FPGS. Substitution of ornithine for glutamate caused a profound decrease in cytotoxic potency for these analogs; this was apparently the result of poor transport. Together with earlier studies, these data indicate that the potency of FPGS inhibition by an analog containing ornithine closely parallels the relative substrate activity of its glutamate-containing counterpart. The substitution of ornithine apparently does not perturb the pterin specificity of FPGS. The close parallel between substrate and inhibitor specificity may thus allow the use of currently available structure-activity studies on FPGS to design more potent and more selective inhibitors of FPGS.

Interaction of d, l-erythro- and d,l-threo-γ-fluoromethotrexate with human leukemia cell dihydrofolate reductase

Gamma-fluoromethotrexate (FMTX) is a poorly glutamylated mimic of the anti-cancer drug methotrexate (MTX) which is useful in studies of the roles of MTX poly-gamma-glutamates. A second chiral center occurs at C-4 of the 4-fluoroglutamate used to synthesize FMTX and, as a consequence, FMTX occurs as both D,L-erythro and D,L-threo diastereomers. The interaction of both diastereomers with intracellular dihydrofolate reductase has been examined in the human leukemia cell line CCRF-CEM, using a centrifugal column technique. Measurements of the rate at which radiolabel was displaced from [3H]MTX-saturated dihydrofolate reductase following suspension of the cells in unlabeled drug indicated that MTX and the erythro isomer of FMTX gave essentially the same rate of displacement; the rate of displacement by the threo isomer of FMTX was slower, but the interpretation of these data was ambiguous since the rate of transport of threo-FMTX may have been limiting. In reciprocal experiments in which dihydrofolate reductase was saturated with [3H]erythro-FMTX, the erythro isomer and MTX again behaved equivalently in terms of displacement. When dihydrofolate reductase was saturated with [3H]threo-FMTX, the radiolabel was clearly displaced at a much faster rate than either other radiolabel regardless of whether the displacing agent was MTX or the isomer. These results indicate a distinct stereospecificity for interaction of inhibitor with dihydrofolate reductase in which the threo isomer has a faster off-rate. Of the two FMTX diastereomers, the erythro isomer thus most closely mimics the properties of MTX.

Mechanisms of acquired resistance to modulation of 5-fluorouracil by leucovorin in HCT-8 human ileocecal carcinoma cells.

Repeated (10x) exposure of HCT-8 human ileocecal carcinoma cells to 5-fluorouracil (5-FU) for 2 or 72 hr, both incubations in the continuous presence of 20 microM leucovorin (LV), yielded two stable modulation-resistant sublines, FL2h and FL72h. Although LV potentiated growth inhibition by 5-FU 2-fold in parental HCT-8 cells, it did not potentiate the effect of 5-FU in the FL2h or FL72h sublines. LV modulation of 5-fluorodeoxyuridine (5-FdUrd) was also reduced (FL72h) or eliminated (FL2h). In the FL2h and FL72h sublines, the level of thymidylate synthase (TS) protein and TS activity in cell extracts, TS activity in situ, the rate of cellular uptake and metabolism of LV, and the level of 5-FU incorporation into total cellular RNA were similar to those in parental HCT-8 cells. However, LV significantly (P < 0.01) potentiated the inhibition of TS activity in situ in HCT-8 cells at 24 hr after a 2-hr treatment with either 5-FU or 5-FdUrd, but had no such activity in the FL2h and FL72h sublines (P > 0.1). Resistance to modulation of 5-FU by LV was associated with the inability of LV to increase the formation of intracellular TS-FdUMP-methylenetetrahydrofolate ternary complexes, and these complexes dissociated more rapidly (T1/2 > 1.5- to 3-fold faster) in the presence of different concentrations of 5,10-methylenetetrahydropteroylpentaglutamate. Thus, decreased stability of ternary complexes appears to be the mechanism of acquired resistance to the LV modulation of fluoropyrimidine cytotoxicity, possibly due to mutation(s) of TS in these two modulation-resistant HCT-8 sublines.

Antitumor Efficacy of Classical Non-Polyglutamylatable Antifolates that Inhibit Dihydrofolate Reductase

The non-polyglutamylatable dihydrofolate reductase (DHFR) inhibitors γ-methylene-l0 deazaaminopterin (MDAM) and 7-methylene-l0ethyl-10-deazaaminopterin (MEDAM)1,2 exhibited striking activity relative to methotrexate (MTX) against the growth of a number of human tumor cell lines in culture3,4. Since both MDAM and MEDAM are nonpolyglutamylatable and therefore could be cleared more efficiently from tissues than MTX, it was of interest to undertake a comparative study of the in vivo activity and toxicity of these compounds and MTX in normal and tumor bearing animals. These investigations were carried out with MEDAM using normal and ip implanted L1210 and P388 leukemic mice as the animal model using a number of experimental protocols. MDAM and MEDAM were previously shown to inhibit recombinant human DHFR at equivalent magnitude as MTX 1,2. They were neither substrates nor inhibitors of CCRF-CEW human leukemia cell folypolyglutamate synthetase (FPGS) and they competed more efficiently for folinic acid transport than MTX in H35 hepatoma cells. Both MDAM and MEDAM were excellent inhibitors of the growth of H35 hepatoma, CCRF-CEM human leukemia and Manca human lymphoma cells in culture. In NCI’s human tumor disease oriented in vitro screen, MDAM and MEDAM exhibited a wide spectrum of sensitivity and their activities were superior to MTX in a large number of tumor cells (Table I).