Anticancer Antifolates: Current Status and Future Directions

Abstract

Antifolates are the oldest of the antimetabolite class of anticancer agents and were one of the first modern anticancer drugs. The first clinically useful antifolate, described in 1947, was 2,4-diamino-pteroylglutamate (4-amino-folic acid; aminopterin; AMT) which yielded the first-ever remissions in childhood leukemia. AMT was soon superseded by its 10-methyl congener, methotrexate (MTX), based on toxicity considerations; MTX remains, with one limited exception, the only antifolate anticancer agent in clinical use to this date. Because of the safety and utility of MTX, considerable effort has been invested in attempting to design more therapeutically selective antifolates or antifolates with a wider tumor spectrum. Initially, the design was based on the burgeoning knowledge of folate-dependent pathways and the determinants of the mechanism of action of MTX. These determinants include transport, the tight-binding inhibition of its target (the folate-dependent enzyme dihydrofolate reductase (DHFR)), and metabolism of MTX to poly-gamma-glutamate (Glu(n)) metabolites. These early studies led to the development of other antifolate DHFR inhibitors of two types: (1). "classical" analogs that use the same cellular transport systems as MTX and are also metabolized to Glu(n); and (2). "nonclassical" (i.e., lipophilic) analogs that do not require transport systems and that are not metabolized to Glu(n). Although several of these analogs have undergone clinical trial, none is proved superior to MTX. Detailed examination of the mechanisms of cytotoxicity and selectivity of MTX showed that inhibition of both dTMP synthesis and de novo purine synthesis, secondary to DHFR inhibition, led to DNA synthesis inhibition and subsequent cell death; inhibition of other folate-dependent pathways did not appear necessary for cell death. Further studies showed that the contribution of inhibition of dTMP or purine synthesis to cell death varied in different cell types. These data suggested that inhibition of one of these pathways individually might (at least in some cases) be therapeutically superior to the dual inhibition induced by MTX. Thus in rational design and in structure-based design studies, two new classes of antifolate enzyme inhibitors were elaborated-direct inhibitors of thymidylate synthase (TMPS) and direct inhibitors of one or both of the two folate-dependent enzymes of de novo purine synthesis. Members of each class included both classical and nonclassical types. After preclinical evaluation, several of these have moved into clinical trials. To date only one new TMPS inhibitor has successfully completed clinical trials and been approved for routine use; this drug, Tomudex (D1694, raltitrexed) is currently approved only in Europe and only for the treatment of colon cancer. This still represents a step forward for antifolates, however, since MTX is well-known to be ineffective in colon cancer; thus Tomudex extends the tumor range of antifolates. Antifolate development continues. Based on the immense body of knowledge now exta...

Raltitrexed increases tumorigenesis as a single agent yet exhibits anti-tumor synergy with 5-fluorouracil in ApcMin/+ Mice1

“…To determine the impact of a "pure" TS inhibitor, we examined the effects of RTX, a folate analog whose sole known target is TS. 2 Apc Min/+ mice were weaned to a folic aciddeficient diet, 25 and treated with RTX between 4 and 10 weeks of age using one of several doses and schedules described in Table 1. Mice that were treated according to protocol I (3 mg/kg, twice a week; see Table 1) exhibited a 4-fold increase in average tumor number in the small intestine as compared to control, PBS-treated animals (114 ± 60 tumors in drug-treated mice vs. 27 ± 5 tumors in controls; Fig.…”

“…RTX is a folate analog that has been approved for treatment of advanced CRC in Europe, Australia, Canada and Japan. 2 The inhibitor is taken up by cells via the reduced folate carrier and modified through addition of glutamate moieties by the enzyme folylpolyglutamate synthetase. Polyglutamylated forms of RTX have enhanced intracellular retention and bind to TS with higher affinity compared to unmodified forms of the drug.…”

Dihydrofolate Reductase and Thymidylate Synthase Transgenes Resistant to Methotrexate Interact to Permit Novel Transgene Regulation

“…Inhibition of these proteins adversely affects the de novo synthesis of purine and thymidine nucleotides, which is vital for survival of rapidly replicating cells. MTX has proven valuable for treating rapidly replicating cancers such as leukemia, but the impact of MTX on healthy, replicating tissue leads to dose-limiting toxicities such as bone marrow suppression (2,3). This disadvantage of MTX has led to intense study into mechanisms of resistance (4) and alternative antifolates for the treatment of cancer (3).…”

A role for Drosophila in understanding drug-induced cytotoxicity and teratogenesis

“…MTX, in addition to the C4-amino group, has a methyl group at N10. These changes to the structure are sufficient to make MTX a potent inhibitor of DHFR, and thus a valuable pharmaceutical (McGuire 2003). Indeed, MTX is used for the treatment of a wide variety of cancers (Huennekens 1994) as well as for the treatment of ectopic pregnancy (Fernandez et al 1998), inflammatory skin disease (Goujon et al 2006), Crohn's disease (Sun and Das 2005), rheumatoid arthritis (Nakazawa et al 2001), and systemic lupus (Wise et al 1996).…”

“…DNA synthesis is compromised when levels of 5,10-CH 2 -THF are reduced as the thymidylate synthase cycle requires this THF derivative to donate a methyl group to dUMP for synthesis of de novo dTMP. It is this DNA inhibition that is thought to cause most of MTX's cytotoxicity (McGuire 2003). Purine synthesis by glycinamide ribonucleotide and 5-aminoimidazole-4-carboxamide ribonucleotide is also affected by a deficiency of reduced folates, further disrupting DNA synthesis as well as RNA synthesis.…”