Barbara A. Moroson

2,4-Diamino-5-methyl-6-[(3,4,5-trimethoxyanilino)methyl]quinazoline (TMQ), a potent non-classical folate antagonist inhibitor—I: Effect of dihydrofolate reductase and growth of rodent tumors in vitro and in vivo☆

Abstract Seventeen non-classical 2,4-diamino-6-[(anilino)methyl]quinazoline antifolates were tested as inhibitors of dihydrofolate reductase from L1210 leukemia cells and from human leukemia cells (acute lymphocytic leukemia). Several potent inhibitors of this enzyme were found, some with I50 values of 10−9 M, thus displaying activity comparable to that of methotrexate. In general, the potency of dihydrofolate reductase inhibition correlated with the inhibition of cell growth in vitro against L1210 cells. Two of these compounds, compound 14 (2,4-diamino-5-methyl-6-[(3,4,5-trimethoxyanilino)methyl]quinazoline; TMQ, JB-11, NSC 249008) and compound 3 (2,4-diamino-5-chloro-6-[(3,4-dichloroanilino)methyl]quinazoline; NSC 208652), were further evaluated against murine tumors in vivo and both showed a broad spectrum of antitumor effects. The results of these studies encourage further evaluation of these compounds, in particular compound 14, as possible anti-neoplastic agents in the treatment of human disease.

Cytotoxic effects of hyperthermia, 5-fluorouracil and their combination on a human leukemia T-Lymphoblast cell line, CCRF-CEM

The cytotoxic effects of 5-fluorouracil (FUra), hyperthermia, and the combination of these treatments were examined in a human T-lymphoblast cell line, CCRF-CEM. Simultaneous exposure of exponentially growing CCRF-CEM cells to hyperthermia (39 and 42 degrees C) and FUra (10, 50, and 100 microM) for 1 or 2 hr resulted in subadditive or additive cell kill. When CCRF-CEM cells were exposed to these agents in sequence (hyperthermia----FUra and FUra----hyperthermia) for 1 and 2 hr duration additive cell kill was also observed. Enhanced cytotoxic effects were observed when a longer exposure (4 and 8 hr) to FUra (100 microM) followed heat (42 degrees C for 1 and 2 hr). Heat exposure (42 degrees C, 1 and 2 hr) induced a rapid decrease in the synthesis of DNA of CCRF-CEM cells, followed by a rebound increase at 12 hr and a new decrease at 24 hr. Flow cytometry demonstrated an accumulation of cells in the S phase at 12 hr after heat exposure, followed by a marked increase of the G + M population (maximum at 24 hr). The exposure time, and the sequence of administration of hyperthermia and FUra were critical determinants of cytotoxicity in this in vitro system and might constitute important variables of treatment when these two agents are used clinically.

Biochemical modulation of fluoropyrimidines by antifolates and folates in an in vitro model of human leukemia.

Although 5-fluorouracil (FUra) is one of the most effective cytotoxic agents in the treatment of various solid tumors (carcinomas of the gastro-intestinal tract, breast, head and neck), remissions occur in only 20 to 30% of cases and usually are of short duration. Recently, preclinical studies have shown that the antitumor activity of FUra can be potentiated by modulating the metabolism of this drug by using other substances, in particular antifolates of folates. Pretreatment with antifolates may, by blocking de novo purine biosynthesis and consequently increasing phosphoribosyl pyrophosphate (PRPP) pools, enhance the conversion of FUra to active fluoronucleotide pools via orotate phosphoribosyltransferase. Methotrexate (MTX) pretreatment may also enhance binding of the fluoropyrimidine inhibitor, 5-fluodeoxyuridylate (FdUMP), to the target enzyme, thymidylate synthase (TS), indirectly by increasing dihydrofolate polyglutamates or directly, as MTX polyglutamates, by enhancing the formation of ternary complexes with FdUMP and TS. Exogenous folates, in particular 5-formyltetrahydrofolate (folinate, leucovorin, LV), can, by raising the intracellular levels of 5, 10-methylenetetrahydrofolate, lead to increased formation and stabilization of the ternary complex formed by TS, the folate coenzyme, and FdUMP. In vitro studies have also shown potentiation of FUra cytotoxicity by antifolates and folates against human lymphoblastic leukemia cell lines. Thus, while FUra may have little or no single agent activity in leukemias and lymphomas, it may be converted to an active drug in these neoplasms by appropriate modulation. Clinical studies of sequential MTX-FUra or combined LV-FUra based upon experimental tumor results reviewed herein, are warranted.

Inhibition of methionine uptake by methotrexate in mouse leukemia L1210 cells

SummaryMethionine-auxotrophic L1210 cells were used to study the effect of methotrexate (MTX) on methionine utake and metabolism. MTX was shown to inhibit amino acid transport systems and cause a decrease of methionine uptake into L1210 cells. Conversely, a nonmetabolizable amino acid analogue reduced MTX uptake into L1210 cells. MTX also blocked the transfer of the beta carbon from serine into methionine. Therefore, methionine deprivation may be an additional mechanism of action for MTX in methionine-auxotrophic tumor cells.

Drug Resistance: New Approaches to Treatment

Mechanisms by which malignant cells may become resistant to chemotherapeutic agents are reviewed, with emphasis on methotrexate resistance. At least four mechanisms of resistance have been described in experimental systems, including human tumor cells propagated in vitro: impaired uptake of methotrexate, an altered target enzyme (dihydrofolate reductase), and an elevated level of dihydrofolate reductase, or decreased methotrexate polyglutamylation. Combinations of these changes have been noted to occur in cells acquiring resistance to methotrexate. In the clinic, examples of resistance due to alteration of dihydrofolate reductase or elevated levels of this enzyme due to gene amplification have been reported. A strategy for selectively eradicating these resistant cells with second generation antifolates that are cytotoxic to resistant cells is discussed.

The role of methionine in methotrexate-sensitive and methotrexate-resistant mouse leukemia L1210 cells

SummaryA mouse L1210 leukemia cell line was made 25-fold resistant to methotrexate (MTX) and had altered methionine transport and metabolism. L1210 cells resistant to methotrexate also had a 50-fold decrease in the exogenous methionine requirement for optimal cell growth compared to the parent cells. This change in methionine requirement was associated with differences in methionine metabolism between MTX-sensitive and MTX-resistant cell lines. Analysis of amino acid transport systems revealed different K1 and Vmax properties of methionine and nonmetabolizable amino acid analogues. There was a greater than twofold decrease in the initial sodium-dependent uptake of methionine in the resistant cells. Amino acid competition experiments revealed altered substrate specificities in the resistant cells. The cellular alterations occurring upon resistance may result from methotrexatemembrane interactions, and have been previously observed in cisplatinum-resistant cells. Thus modulation of methionine metabolism may provide the biochemical basis for MTX and cisplatinum collateral resistance.

Synthesis and evaluation of 2,4-diaminoquinazoline antifolates with activity against methotrexate-resistant human tumor cells

In an attempt to find potent antifolates with selectivity against tumor cells with intrinsic or acquired resistance to methotrexate, eleven nonclassical 2,4-diaminoquinazoline antifolates were synthesized and tested as inhibitors of dihydrofolate reductase from L5178Y cells. Several compounds appeared to be good enzyme inhibitors, with I50 values around 1 nM. Two of the compounds were also good inhibitors of cell growth in vitro. One of these (PKC-32, 9-(2,4-diamino-5-methylquinazoline-6-methylene)aminophenanthren e) appeared to be 100-fold more potent than methotrexate as an inhibitor of growth of a methotrexate-resistant cell line with impaired transport for methotrexate. PKC-32 and PKC-155 were also tested against mouse tumors in vivo. PKC-32 was modestly active in vivo as compared with methotrexate. This drug may be a useful agent in the treatment of methotrexate-resistant tumors.

Mechanisms of Drug Resistance in Human Leukemia

Drug resistance remains a major obstacle to eure of patients with acute leukemia. At the present time, most centers are reporting 90% complete remission rates in acute lymphatic leukemia (ALL) and 70%–80% complete remission rates in patients with acute nonlymphocytic leukemia (ANLL). However, 5-year disease-free survival rates are only 50% and 10%–15%, respectively, in these diseases. This is almost certainly due to the development of drug resistance even to the combination chemotherapy programs utilized to treat these diseases [1].