lometrexol and raltitrexed

Antifolate drugs, as a class, have broad-spectrum activity against both hematologic and solid human malignancies. The pharmacokinetics of the classical antifolate methotrexate have been well-defined and pharmacokinetic data can be exploited to reduce the toxicity and enhance the activity of the drug. Methotrexate remains the only anticancer drug for which plasma drug level monitoring is used in routine clinical practice. Recently, novel classical and nonclassical antifolates have been developed that target either specific folate-dependent enzymes (e.g., thymidylate synthase [CB3717, raltitrexed, ZD9331, 1843U89, nolatrexed, AG331], glycinamide ribonucleotide transformylase [lometrexol, LY309887, AG2034] or multiple folate-dependent enzymes (e.g., MTA/LY231514). In the early clinical trials of these agents, a number of pharmacokinetic-pharmacodynamic relationships were identified and it is highly likely that the full therapeutic potential of these new drugs will also require the exploitation of pharmacokinetic data.

Topics: Antimetabolites, Antineoplastic; Enzyme Inhibitors; Folic Acid; Folic Acid Antagonists; Humans; Indoles; Isoindoles; Methotrexate; Quinazolines; Tetrahydrofolates; Thiophenes; Thymidylate Synthase

Folic acid is a water-soluble vitamin associated with the other B vitamins. In its fully reduced form (tetrahydrofolate), folate serves as a 1-carbon donor for synthesis of purines and thymidine as well as in the remethylation cycle of homocysteine to methionine. Folate is essential for normal cell growth and replication. It therefore is not surprising that folate analogues have served and continue to serve well as antibiotics and cytotoxic drugs in the treatment of cancer, autoimmune diseases, psoriasis, and bacterial and protozoal infections. During the past 50 years, many of the enzymes requiring folate as a co-factor (ie, thymidylate synthase), and molecules critical in folate homeostasis (ie, the reduced folate carrier, folylpolyglutamate synthase), have been purified and even crystallized. The genes have been cloned, sequenced, and mapped, providing detailed knowledge of their regulation and three-dimensional structure. This has, in part, led to the rational synthesis of a large number of folate analogues that differ from methotrexate, the "classical antifolate," in transport, metabolism, and intracellular targets. Currently, several new folate analogues with unique biochemical properties and clinical applications are being tested. The goals of this brief review are to review folate homeostasis, to highlight the similarities and differences between natural folate and antifolates with respect to biochemistry and metabolism, and to present the pharmacology of methotrexate and several next-generation folate analogues, such as trimetrexate and raltritrexed, with an emphasis on mechanisms of drug resistance.

Topics: Antimetabolites, Antineoplastic; Enzyme Inhibitors; Folic Acid; Folic Acid Antagonists; Humans; Methotrexate; Quinazolines; Tetrahydrofolates; Thiophenes; Thymidylate Synthase; Trimetrexate

Many novel antifolate compounds with unique pharmacologic properties are currently in clinical development. These newer antifolates differ from methotrexate, the most widely used and studied drug in this class, in terms of their lipid solubility and cellular transport affinity, their level of polyglutamation, and their specificity for inhibiting folate-dependent enzymes, such as dihydrofolate reductase, thymidylate synthase, or glycinamide ribonucleotide formyltransferase. The current status (ie, mechanism of action, clinical response rates, and toxicity) of some of the newer antifolate compounds presently in clinical testing, including edatrexate, piritrexim, raltritrexed, LY 231514, AG337, AG331, 1843U89, ZD 9331, and lometrexol, is reviewed.

Topics: Aminopterin; Animals; Antimetabolites, Antineoplastic; Clinical Trials as Topic; Drug Design; Enzyme Inhibitors; Folic Acid Antagonists; Glutamates; Guanine; Humans; Indoles; Isoindoles; Pemetrexed; Pyrimidines; Quinazolines; Tetrahydrofolates; Thiophenes; Thymidylate Synthase

A number of promising new antifolates have been entered in clinical trials in recent years. These agents have been rationally designed based on the current understanding of folate transport and metabolism and of the mechanisms by which cells become resistant to methotrexate. Methotrexate-resistant cell lines are generally sensitive to one or more of the newer antifolates, which differ from methotrexate by being either more lipid soluble, more extensively polyglutamated, or by inhibiting folate-requiring enzymes other than dihydrofolate reductase. Five of the agents furthest along in clinical testing, trimetrexate, piritrexim, edatrexate, lometrexol, and D1694, are discussed. These drugs offer exciting opportunities to expand the role of antifolates in cancer chemotherapy, as well as in antimicrobial and antirheumatic therapy.

Topics: Aminopterin; Animals; Antineoplastic Agents; Chemistry, Pharmaceutical; Clinical Trials as Topic; Drug Evaluation; Folic Acid Antagonists; Forecasting; Humans; Neoplasms; Pyrimidines; Quinazolines; Tetrahydrofolates; Thiophenes; Trimetrexate

Metabolic reprogramming of tumor cells toward serine catabolism is now recognized as a hallmark of cancer. Serine hydroxymethyltransferase (SHMT), the enzyme providing one-carbon units by converting serine and tetrahydrofolate (H4 PteGlu) to glycine and 5,10-CH2 -H4 PteGlu, therefore represents a target of interest in developing new chemotherapeutic drugs. In this study, 13 folate analogues under clinical evaluation or in therapeutic use were in silico screened against SHMT, ultimately identifying four antifolate agents worthy of closer evaluation. The interaction mode of SHMT with these four antifolate drugs (lometrexol, nolatrexed, raltitrexed, and methotrexate) was assessed. The mechanism of SHMT inhibition by the selected antifolate agents was investigated in vitro using the human cytosolic isozyme. The results of this study showed that lometrexol competitively inhibits SHMT with inhibition constant (Ki ) values in the low micromolar. The binding mode of lometrexol to SHMT was further investigated by molecular docking. These results thus provide insights into the mechanism of action of antifolate drugs and constitute the basis for the rational design of novel and more potent inhibitors of SHMT.

Topics: Folic Acid Antagonists; Glycine Hydroxymethyltransferase; Humans; Methotrexate; Molecular Docking Simulation; Quinazolines; Tetrahydrofolates; Thiophenes

We determined the mechanisms of resistance of human CCRF-CEM leukemia cells to methotrexate (MTX) vs. those to six novel antifolates: the polyglutamatable thymidylate synthase (TS) inhibitors ZD1694, multitargeted antifolate, pemetrexed, ALIMTA (MTA) and GW1843U89, the non-polyglutamatable inhibitors of TS, ZD9331, and dihydrofolate reductase, PT523, as well as DDATHF, a polyglutamatable glycinamide ribonucleotide transformylase inhibitor. CEM cells were made resistant to these drugs by clinically relevant intermittent 24 hr exposures to 5-10 microM of MTX, ZD1694, GW1843U89, MTA and DDATHF, by intermittent 72 hr exposures to 5 microM of ZD9331 and by continuous exposure to stepwise increasing concentrations of ZD9331, GW1843U89 and PT523. Development of resistance required only 3 cycles of intermittent drug exposure to ZD1694 and MTA, but 5 cycles for MTX, DDATHF and GW1843U89 and 8 cycles for ZD9331. The predominant mechanism of resistance to ZD1694, MTA, MTX and DDATHF was impaired polyglutamylation due to approximately 10-fold decreased folylpolyglutamate synthetase activity. Resistance to intermittent exposures to GW1843U89 and ZD9331 was associated with a 2-fold decreased transport via the reduced folate carrier (RFC). The CEM cell lines resistant to intermittent exposures to MTX, ZD1694, MTA, DDATHF, GW1843U89 and ZD9331 displayed a depletion (up to 4-fold) of total intracellular reduced folate pools. Resistance to continuous exposure to ZD9331 was caused by a 14-fold increase in TS activity. CEM/GW70, selected by continuous exposure to GW1843U89 was 50-fold resistant to GW1843U89, whereas continuous exposure to PT523 generated CEM/PT523 cells that were highly resistant (1550-fold) to PT523. Both CEM/GW70 and CEM/PT523 displayed cross-resistance to several antifolates that depend on the RFC for cellular uptake, including MTX (95- and 530-fold). CEM/GW70 cells were characterized by a 12-fold decreased transport of [3H]MTX. Interestingly, however, CEM/GW70 cells displayed an enhanced transport of folic acid, consistent with the expression of a structurally altered RFC resulting in a 2.6-fold increase of intracellular folate pools. CEM/PT523 cells displayed a markedly impaired (100-fold) transport of [3H]MTX along with 12-fold decreased total folate pools. In conclusion, multifunctional mechanisms of resistance in CEM cells have a differential impact on cellular folate homeostasis: decreased polyglutamylation and transport defects lead to folate deple

Topics: Biological Transport; Drug Resistance, Multiple; Drug Screening Assays, Antitumor; Folic Acid; Folic Acid Antagonists; gamma-Glutamyl Hydrolase; Glutamates; Guanine; Homeostasis; Humans; Leukemia; Methotrexate; Ornithine; Pemetrexed; Peptide Synthases; Polyglutamic Acid; Pterins; Quinazolines; Tetrahydrofolates; Thiophenes; Thymidylate Synthase; Tumor Cells, Cultured

The impact of p53 status on cellular sensitivity to antifolate drugs has been examined in seven human cell lines (A549, MCF7, T-47D, CCRF-CEM, COR-L23, A2780, and HCT-116) and p53 nonfunctional counterparts of two of the cell lines (HCT-116/N7 and A2780/CP70). p53 status was determined by sequencing and functional assays. The sensitivities of the cell lines to growth inhibition (sulphorhodamine B assay) produced by four antifolate drugs (Alimta, methotrexate, raltitrexed, and lometrexol) were studied. There was no clear relationship between functional p53 status and sensitivity to methotrexate or lometrexol, whereas a functional p53 status was possibly associated with resistance to Alimta- and raltitrexed-induced growth inhibition. In contrast, in the two pairs of related human tumor cell lines (HCT-116 and HCT-116/N7 and A2780 and A2780/CP70) cells with functional p53 were more sensitive to Alimta- and raltitrexed-induced growth inhibition (P = 0.002). Detailed studies were performed with the A2780 cell lines, and in the parental cells sensitivity to Alimta- and raltitrexed-induced cytotoxicity (clonogenic assay) was similar to the sensitivity determined in the sulphorhodamine B assay. However, in A2780/CP70 cells, 1 microM of drug resulted in only 40-60% growth inhibition yet > or = 85% cytotoxicity. After Alimta and raltitrexed exposure for < or = 72 h, there were no differences between the A2780 and A278/CP70 cell lines in cell cycle phase distribution, absolute cell number, or the induction of apoptosis. However, the cellular protein content of the A2780/CP70 cells was 3-6-fold higher than in A2780 cells after Alimta and raltitrexed treatment, suggesting that cells without functional p53 can maintain protein synthesis in the absence of cell division (unbalanced cell growth). In conclusion, the apparent impact of functional p53 status on sensitivity to antifolate drugs may depend upon the phenotypic/genotypic background as well as the assay used to measure cellular sensitivity.

Topics: Apoptosis; Blotting, Western; Cell Cycle; Cell Division; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; DNA Mutational Analysis; DNA, Neoplasm; Dose-Response Relationship, Drug; Folic Acid Antagonists; Gene Expression Regulation, Neoplastic; Genotype; Glutamates; Guanine; Humans; Methotrexate; Mutation; Pemetrexed; Quinazolines; Sensitivity and Specificity; Tetrahydrofolates; Thiophenes; Tumor Cells, Cultured; Tumor Suppressor Protein p53

Since the clinical introduction of the antifolates aminopterin (AMT) and methotrexate (MTX) many promising analogs have been developed. A common feature of these compounds is their ability to induce bone marrow suppression. However, few studies have been undertaken on the effect of the folic acid analogs on the cells comprising the hematopoietic system.. In this paper we describe the effects of the novel thymidylate synthase (TS) inhibitors raltitrexed (Tomudex, ZD1694), AG337 (nolatrexed, Thymitaq), and the two closely related analogs 5,8-dideazaisofolic acid (IAHQ2a) and 2-desamino-2-methyl 5,8-dideazaisofolic acid (IAHQ2c), the glycinamide-ribonucleosyl (GAR) transformylase inhibitor lometrexol (DDATHF), and the dihydrofolate reductase (DHFR) inhibitors MTX, AMT, trimetrexate (TMTX), and edatrexate (EDX) on purified populations of early and late murine hematopoietic progenitor cells.. All the antifolates inhibited bone marrow proliferation in suspension cultures and all drugs except DDATHF inhibited colony formation by more mature progenitor cells (CFU-C) in clonogenic assays. The lipophilic agents TMTX and AG337 were most toxic, totally abolishing CFU-C colony formation at high concentrations. When IAHQ2c, raltitrexed, DDATHF, and MTX were investigated further for effects on the immature high proliferative potential colony-forming cells (HPP-CFCs) in semisolid and limiting dilution cultures, none of these agents were found to be toxic to the HPP-CFC, but induced a reversible developmental arrest in the progenitor cell population.

Topics: Aminopterin; Animals; Cell Division; Cells, Cultured; Enzyme Inhibitors; Female; Folic Acid; Folic Acid Antagonists; Hematopoietic Stem Cells; Methotrexate; Mice; Mice, Inbred BALB C; Quinazolines; Tetrahydrofolates; Thiophenes; Trimetrexate

N-[4-[2-(2-amino-3,4-dihydro-4-oxo-7H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl ]-benzoyl]-L-glutamic acid (LY231514) is a novel pyrrolo[2,3-d]pyrimidine-based antifolate currently undergoing extensive Phase II clinical trials. Previous studies have established that LY231514 and its synthetic gamma-polyglutamates (glu3 and glu5) exert potent inhibition against thymidylate synthase (TS). We now report that LY231514 and its polyglutamates also markedly inhibit other key folate-requiring enzymes, including dihydrofolate reductase (DHFR) and glycinamide ribonucleotide formyltransferase (GARFT). For example, the Ki values of the pentaglutamate of LY231514 are 1.3, 7.2, and 65 nM for inhibition against TS, DHFR, and GARFT, respectively. In contrast, although a similar high level of inhibitory potency was observed for the parent monoglutamate against DHFR (7.0 nM), the inhibition constants (Ki) for the parent monoglutamate are significantly weaker for TS (109 nM) and GARFT (9,300 nM). The effects of LY231514 and its polyglutamates on aminoimidazole carboxamide ribonucleotide formyltransferase, 5,10-methylenetetrahydrofolate dehydrogenase, and 10-formyltetrahydrofolate synthetase were also evaluated. The end product reversal studies conducted in human cell lines further support the concept that multiple enzyme-inhibitory mechanisms are involved in cytotoxicity. The reversal pattern of LY231514 suggests that although TS may be a major site of action for LY231514 at concentrations near the IC50, higher concentrations can lead to inhibition of DHFR and/or other enzymes along the purine de novo pathway. Studies with mutant cell lines demonstrated that LY231514 requires polyglutamation and transport via the reduced folate carrier for cytotoxic potency. Therefore, our data suggest that LY231514 is a novel classical antifolate, the antitumor activity of which may result from simultaneous and multiple inhibition of several key folate-requiring enzymes via its polyglutamated metabolites.

Topics: 5,10-Methylenetetrahydrofolate Reductase (FADH2); Acyltransferases; Aminohydrolases; Antimetabolites, Antineoplastic; Folic Acid Antagonists; Formate-Tetrahydrofolate Ligase; Glutamates; Guanine; Humans; Hydroxymethyl and Formyl Transferases; Methotrexate; Methylenetetrahydrofolate Dehydrogenase (NADP); Methylenetetrahydrofolate Reductase (NADPH2); Molecular Structure; Multienzyme Complexes; Oxidoreductases; Pemetrexed; Phosphoribosylaminoimidazolecarboxamide Formyltransferase; Phosphoribosylglycinamide Formyltransferase; Polyglutamic Acid; Quinazolines; Tetrahydrofolate Dehydrogenase; Tetrahydrofolates; Thiophenes; Thymidylate Synthase; Tumor Cells, Cultured

Two methotrexate (MTX)-resistant human breast-cancer cell lines with impaired transport via the reduced folate carrier (RFC), one established in vitro (MTX(R)-ZR-75-1) and another inherently resistant (MDA-231), were adapted to grow in medium containing 2 nM folic acid. This induced the expression of previously undetectable membrane folate receptors (MFR) to levels of 8.2 and 2.3 pmol/10(7) cells, respectively. Polymerase chain reaction (PCR) quantitation revealed that MFR messenger-RNA levels of the isoform first described in human nasopharyngeal carcinoma KB cells (MFR-alpha) were increased in low-folate-adapted MTX(R)-ZR-75-1 cells, whereas placental transcripts (MFR-beta) coincided with MFR-alpha expression in low-folate (LF)-adapted MDA-231 cells. These cell lines were used to study the role of MFR in the uptake and growth-inhibitory effects of five different antifolates with varying affinities for MFR: N10-propargyl-5, 8-dideazafolic acid (CB3717) > 5,10-dideazatetra-hydrofolic acid (DDATHF) > N-5-[N-(3,4-dihydro-2-methyl-4-oxoquinazolin-6-methyl) -N-methyl-amino]-2-theonyl}-glutamic acid (ZD1694) >> MTX > edatrexate (EDX). Expression of MFR only slightly decreased the resistant phenotype for MTX, EDX, and ZD1694, suggesting that these drugs are not transported intracellularly to cytotoxic concentrations at these levels of MFR expression. On the other hand, both cell lines became from at least 180- to 400-fold more sensitive to growth inhibition by CB3717 and DDATHF, which may be correlated with their high affinity for MFR. These sensitivity/resistance profiles were largely similar following cell culture in medium containing 1 nM L-leucovorin, a folate with an affinity for MFR 10-fold lower than that of folic acid, the one exception being the increased sensitivity for ZD1694 seen in the LF-adapted cells with the highest level of MFR expression (MTX(R)-ZR-75-1). These results illustrate that the efficacy of MFR in mediating antifolate transport and cytotoxicity depends on their affinity for the folate antagonist, their degree of expression, and the levels of competing folates.

Topics: Aminopterin; Antimetabolites, Antineoplastic; Binding Sites; Breast Neoplasms; Carrier Proteins; Cell Membrane; DNA, Complementary; Female; Folate Receptors, GPI-Anchored; Folic Acid; Folic Acid Antagonists; Humans; Methotrexate; Polymerase Chain Reaction; Quinazolines; Receptors, Cell Surface; RNA, Messenger; Spectrometry, Fluorescence; Structure-Activity Relationship; Tetrahydrofolates; Thiophenes; Tumor Cells, Cultured

The role of a membrane-associated folate binding protein (mFBP) in transport of folate analogues was investigated in three epithelial cell lines that were grown in high folate medium and folate-conditioned medium and express different levels of mFBP: human nasopharyngeal KB cells, monkey kidney MA104 cells, and IGROV-I ovarian carcinoma cells. Folate analogues were selected for which mFBP exhibits a low affinity, i.e., methotrexate (MTX) and 10-ethyl-10-deazaaminopterin (10-EdAM) or a (moderately) high affinity as compared to folic acid, i.e., N-(5[N-(3,4-dihydro-2-methyl-4-oxoquinazolin-6-ylmethyl(-N-m ethylamino]-2-theonyl)-L-glutamic acid (ZD1694), N10-propargyl-5,8-dideazafolic acid (CB3717), and 5,10-dideazatetrahydrofolic acid. Regardless of the medium folate status, growth inhibition studies with IGROV-I and MA104 cells demonstrated a lack of correlation between the affinity of mFBP for the antifolate drugs and their sensitivity profile; both cell lines were highly sensitive to growth inhibition by MTX, 10-EdAM, ZD1694 and 5,10-dideazatetrahydrofolic acid, but were insensitive for CB3717. The same drug sensitivity profile was observed for KB cells, with the exception that these cells were also sensitive to growth inhibition by CB3717 but only in folate-conditioned medium. This overall drug sensitivity profile appeared to correlate with the differential efficiency of drug transport via the "classical" reduced folate/MTX carrier (RFC), rather than by mFBP. Characteristics that further supported functional RFC activity in KB, IGROV-I, and MA104 cells included: (a) the growth inhibitory effects of the drugs could be prevented by the reduced folate leucovorin rather than by folic acid; (b) rates for uptake of [3H]10-EdAM were 2-4-fold higher than for [3H]MTX at 1 microM extracellular concentrations and coincided with the affinity of the RFC for these drugs, rather than those of the mFBP; (c) uptake of [3H]10-EdAM and [3H]leucovorin was markedly inhibited by leucovorin and 10-EdAM, respectively, or by an N-hydroxysuccinimide ester of MTX (irreversibly labeling RFC) but only to a minor extent by folic acid or an N-hydroxysuccinimide ester of folic acid (irreversibly labeling mFBP); and, finally, (d) labeling with an N-hydroxysuccinimide ester of [3H]MTX identified a protein with a molecular weight within the range of that reported for the RFC in human leukemic cells. Altogether, these results indicate that both RFC and mFBP are coexpressed in three epithe

Topics: Aminopterin; Animals; Carrier Proteins; Cell Division; Female; Folate Receptors, GPI-Anchored; Folic Acid; Folic Acid Antagonists; Glutamates; Haplorhini; Humans; Kidney; Leucovorin; Methotrexate; Nasopharyngeal Neoplasms; Ovarian Neoplasms; Quinazolines; Receptors, Cell Surface; Tetrahydrofolates; Thiophenes; Tumor Cells, Cultured

5,10-Dideazatetrahydrofolic acid (DDATHF) reduces de nova purine biosynthesis by inhibiting glycinamide ribonucleotide transformylase. ICI D1964 and CB3717 are folate-based inhibitors of thymidylate synthase (TS). Fluorodeoxyuridine (FdUrd) following metabolism to FdUMP also inhibits TS. In cultured L1210 cells DDATHF reduced the toxicity of ICI D1694, CB3717 and FdUrd in a concentration-dependent manner. This protection correlated with a prevention of the increase in intracellular dATP pools seen in cells exposed to the TS inhibitors alone. The possibility that DDATHF protection might be due to competition for cell entry is not likely since CB3717 and FdURD but not ICI D1694 enter the cell by independent transport processes. Exogenous hypoxanthine (HX) had no effect on the toxicity of TS inhibitors. However, HX increased the protective effect of DDATHF from ICI D1694 toxicity, had no effect on the CB3717-DDATHF interaction, and reduced the protective effect of DDATHF on FdUrd toxicity. HX prevented the fall in dATP levels caused by DDATHF addition to cells treated with TS inhibitors. HX had different effects on dTTP levels in cells treated with DDATHF and quinazoline TS inhibitors compared to FdUrd. Together these results support the hypothesis that imbalance in dTTP and dATP pools is an important determinant of cytotoxicity in antifolate-treated cells. In addition, these findings suggests that intracellular reduced folates interconvert to catalyse reactions in metabolically perturbed cells.

Topics: Animals; Cell Survival; Deoxyadenine Nucleotides; Drug Interactions; Floxuridine; Folic Acid; Hypoxanthine; Hypoxanthines; Leukemia L1210; Quinazolines; Tetrahydrofolates; Thiophenes; Thymidylate Synthase; Tumor Cells, Cultured

We examined the antitumor effects of two antifolate inhibitors of thymidylate synthesis, N-(5-[N-(3, 4-dihydro-2-methyl-4-oxoquinazolin-6-ylmethyl)-N-methylamino ]-2-theno yl-L-glutamic acid (D1694; Tomudex) and 1843U89 as well as a folate-based inhibitor of purine synthesis, 5, 10-dideazatetrahydrofolic acid (DDATHF) on human soft tissue sarcoma cell lines having intrinsic resistance to methotrexate (MTX) due to impaired accumulation of polyglutamates of MTX (HS-16 and HS-42 cells) and to increased levels of dihydrofolate reductase and thymidylate synthase activity (HS-18 cells). Growth inhibition studies showed that ED50 values for D1694 and 1843U89 after a 24-h exposure were 11-19-fold and 22-222-fold lower, respectively, than those for MTX in HT-1080, a MTX-sensitive cell line, and the three MTX-resistant cell lines. In contrast, DDATHF was less cytotoxic than MTX in both the MTX-sensitive and the three resistant sarcoma cell lines. Uptake of D1694, 1843U89, or DDATHF was 2.5-4.5-fold higher than MTX in these sarcoma cell lines. However, D1694 and 1843U89, unlike MTX, accumulate in HS-16 and HS-42 cells as polyglutamate forms, reaching 70% of the total intracellular drug level after 24 h. DDATHF polyglutamates (9.4-24%) were less in the same cell lines. Much lower Km values for D1694 and 1843U89 as compared to MTX for folylpolyglutamate synthase were measured in the sarcoma cell lines, with Vmax values equal to or slightly higher than those obtained with MTX. D1694 and 1843U89 are significantly more cytotoxic than MTX in intrinsically MTX-resistant sarcoma cell lines as a result of extensive formation of polyglutamates. These two thymidylate synthase inhibitors should be evaluated in patients with soft tissue sarcomas.

Topics: Antimetabolites, Antineoplastic; Antineoplastic Agents; Biological Transport; Biotransformation; Cell Division; Cell Survival; Drug Resistance, Neoplasm; Folic Acid Antagonists; Humans; Indoles; Isoindoles; Kinetics; Methotrexate; Purines; Quinazolines; Sarcoma; Tetrahydrofolates; Thiophenes; Thymidine Monophosphate; Tumor Cells, Cultured

We have studied the cytotoxicity of 5,10-dideazatetrahydrofolate (DDATHF) and of D-1694 to human WiDr colonic carcinoma cells as a model system for the effects of pure inhibitors of either the de novo purine synthesis pathway or thymidylate synthesis. The growth of this cell line was inhibited by very low concentrations of either agent and the lethality of DDATHF and D-1694 was completely prevented by continuous exposure to either hypoxanthine or thymidine, respectively, indicating that these compounds were very potent metabolic inhibitors, each specific for one of these pathways. D-1694 was highly cytotoxic (> 3 logs of kill) after a 4-h exposure to 1 microM drug, or a 24-h exposure to very low concentrations (0.04 microM). On the other hand, the cytotoxicity of DDATHF was substantially lower, with 2 logs of cell kill requiring >> 100 microM with 4 h of exposure or approximately 40 microM for 72 h of exposure. Maximal cell kill induced by D-1694 was 5-6 logs, consistent with elimination of all viable cells except preexisting mutants. A maximum of 2-3 logs of cell kill was observed with DDATHF. Exposure of WiDr cells to either D-1694 or DDATHF caused striking cellular changes, but the morphologies of cells treated with the two drugs were remarkably different. D-1694-treated cells detached from the dish within 1-2 days after a megaloblastosis, whereas DDATHF-treated cells remained adherent to the dishes for at least 10 days after treatment. The addition of thymidine to D-1694-treated cultures or hypoxanthine to DDATHF-treated cells after up to 20 h of drug exposure completely prevented cytotoxicity of either drug. With longer exposures, cytotoxicity of both drugs progressively increased in spite of such rescue. Our results indicate that substantial (99-99.9%) tumor cell kill can be induced by a pure inhibitor of purine synthesis, but that the rate of commitment to cell death and the extent of cell kill is greater with a pure inhibitor of thymidylate synthesis.

Topics: Acyltransferases; Antineoplastic Agents; Cell Division; Cell Survival; Colonic Neoplasms; Folic Acid Antagonists; Humans; Hydroxymethyl and Formyl Transferases; Hypoxanthine; Hypoxanthines; Methotrexate; Phosphoribosylglycinamide Formyltransferase; Quinazolines; Tetrahydrofolates; Thiophenes; Thymidine; Thymidylate Synthase; Tumor Cells, Cultured