Arlene R. Cashmore

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.

Separation of substituted pteroyl monoglutamates and pteroyl oligo-γ-l-glutamates by high pressure liquid chromatography

Abstract Rapid analysis of substituted and unsubstituted pteroyl-oligo-γ- l -glutamates at the nanomole level is carried out by high performance liquid chromatography. The use of a siliceous microparticulate anion-exchanger column and gradient elution at pH 6.5 with increasing salt concentration facilitates the separation of the species containing up to seven glutamyl residues without decomposition in 30 min. The column effluent is monitored with a uv detector at 254 nm, and the peaks are conveniently identified by their retention.

Retention behavior of pteroyl-oligo-γ-l-glutamates in reversed-phase chromatography

The effect of eluent pH on the retention of pteroyl-oligo-γ-l-glutamates containing up to eight glutamyl residues is investigated in reversed-phase chromatography with octadecyl-silica column. When the carboxylic groups of the solutes are largely undissociated, at pH 2, the retention of oligoglutamates increases with the number of glutamyl residues and the elution order parallels that in anion-exchange chromatography. At sufficiently high eluent pH the carboxylic groups are dissociated and the elution order is reversed so that solute molecules having smaller number of charges. i.e. less glutamyl residues, are retained stronger. The logarithm of capacity factor, with the exception of folic acid, is linearly dependent on the number of glutamyl residues over a wide range of eluent pH. The dependence of the capacity factor on pH for oligoglutamates is quantitatively interpreted considering the different dissociation constants for the α- and γ-carboxyls. The results suggest that in reversed-phase chromatography the selectivity of separation for polyionogenic compounds can be drastically modulated by changing the pH of the eluent.

Improved purification of tetrahydrofolate dehydrogenase from L1210 leukemia by affinity chromatography

Abstract Tetrahydrofolate dehydrogenase (5,6,7,8-tetrahydrofolate:NADP + oxidoreductase, EC 1.5.1.3; formerly known as dihydrofolate reductase) from a high enzyme mutant of L1210 mouse leukemia was purified to homogeneity by a simple two step procedure involving pH 5.1 precipitation of inert protein, and affinity chromatography employing a methotrexate-agarose column. By raising the pH and ionic strength of the eluting buffer from 0.05 M citrate (pH 6.0) to 0.05 M Tris-HCl (pH 8.5) containing 0.1 M KCl, a peak was eluted containing pure enzyme (spec. act. 2800 μmoles/h per mg). Purity was confirmed by methotrexate titration and polyacrylamide disc electrophoresis. A second small peak containing tetrahydrofolate dehydrogenase activity was eluted by further increasing the pH of the 0.05 M Tris-HCl buffer to 9.0, and increasing the KCl concentration to 0.4 M. The material in this peak showed an absorbance at 258 nm, suggesting that a nucleotide was bound to the enzyme. The identify of this material has yet not been established.

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.