Marked suppression of the activity of some, but not all, antifolate compounds by augmentation of folate cofactor pools within tumor cells

Abstract

¹Folates have been co-administered with some antifolates to diminish host toxicity; however, the extent to which this will reduce antitumor activity is not known. To further clarify this issue, studies were undertaken to characterize and quantitate the impact of alterations in intracellular folate levels on the activities of a variety of antifolates in L1210 murine leukemia cells. Intracellular folate cofactor levels increased almost in proportion to the increase in extracellular 5-formyltetrahydrofolate (5-CHO-THF) over a concentration range that encompassed physiological levels of 5-methyltetrahydrofolate. This resulted in a spectrum of increases in the ic₅₀ values of antifolates upon continuous exposure to drugs [Lometrexol (DDATHF) (70x) > trimetrexate (TMQ) (30x), multitargeted antifolate, LY231514 (ALIMTA) (30x) > Raltitrexed, Tomudex (ZD1694) (10x), 6R-2′,5′-thienyl-5,10-dideazatetrahydrofolic acid (LY309887) (10x) > methotrexate (MTX) (6x) > (2S)-2-{o-fluoro-p-[N-(2,7-dimethyl-4-oxo-3,4-dihydroquinazolin-6-ylmethyl)-N-(prop-2-ynyl)amino]benzamido}-4-(tetrazol-5-yl) butyric acid (ZD9331) (3x), N^α-(4-amino-4-deoxypteroyl)-N^δ-hemiphthaloyl-l-ornithine (PT523) (3x)]. Upon a 4-hr pulse exposure to drug, the ic₅₀ values for DDATHF and ALIMTA were increased > 180- and 5-fold, respectively, with only a 2.5-fold increase in the extracellular 5-CHO-THF level within the physiological range. The reductions in drug sensitivities could be attributed to decreases in accumulation of polyglutamate derivatives of ALIMTA and DDATHF. Hence, in these studies, natural folates diminished the activity of agents that undergo polyglutamation by suppression of the formation of these active congeners at the level of folylpolyglutamate synthetase. For inhibitors of dihydrofolate reductase, the suppressive effect of endogenous folates appears to be due to competition between the antifolate and dihydrofolate at the level of the target enzyme. These data should be carefully considered in the design of regimens with antifolates, which incorporate co-administration of folates.

Introduction

Antifolates are competitive inhibitors of their target folate-requiring enzymes within cells. For the classical antifolates, MTX and aminopterin, dihydrofolate that builds up behind the block in DHFR is the basis for the competitive nature of the interaction among these agents, dihydrofolate, and their target enzyme [1]. For new generation antifolate inhibitors of thymidylate synthase and/or GAR transformylase that require formation of polyglutamate derivatives for activity, such as DDATHF, ALIMTA, and ZD1694 [2], [3], [4], the level of endogenous folate pools should modulate the rate and extent of formation of these congeners synthesized by FPGS within cells. Endogenous folate pools also have the potential for diminishing the activity of these agents due to competition at the level of their target enzymes. Low levels of endogenous folates in normal tissues are a determinant of the toxicity of antifolate inhibitors of GAR transformylase, and repleting these pools with added folate in animal models circumvents toxicity and may enhance therapeutic efficacy [5], [6]. Conversely, enhanced accumulation of folate pools within cells that occurs by perturbations of folate transporters can result in markedly diminished antifolate activity [7], [8]. Studies on drug resistance in vitro usually utilize media in which the folate growth substrate is folic acid. This folate has a low affinity for the major folate transporter, RFC1, and enters cells largely by other mechanisms [9]. Recent studies utilizing 5-CHO-THF as the growth substrate demonstrated that ALIMTA or DDATHF activities can be preserved in cell lines resistant to MTX due to impaired RFC1-mediated transport [10], [11], [12]. This was due to selective, partial preservation of transport of these antifolates along with the contraction of the endogenous folate pool that accompanies a decrease in 5-CHO-THF transport [12], [13].

This paper represents the first quantitative analysis of the relationship between levels of cellular folate cofactors and the activities of antifolates, using L1210 murine leukemia cells grown in 5-CHO-THF concentrations that include, and bracket, the physiological blood levels of 5-CH₃-THF. These data were then correlated with growth inhibitory activities upon continuous, or brief, exposure to antifolate agents with different enzyme targets, some of which do, while others do not, form polyglutamate derivatives. The data characterize the very critical role that the cellular folate pool can play as a determinant of antifolate activities, findings relevant to the design of clinical regimens in which the co-administration of folate supplements is considered to prevent toxicity.