New Antifolates in Clinical Development
New Antifolates in Clinical Development
Numerous new antifolate drugs have been developed in an attempt to overcome the potential mechanisms of tumor cell resistance to methotrexate, which can include decreased drug transport into cells; decreased polyglutamation, leading to increased drug efflux from cells; decreased drug affinity for folate-dependent enzymes; mutations of dihydrofolate reductase (DHFR), a key enzyme required for the maintenance of adequate intracellular reduced folate levels that is inhibited by methotrexate; and increased expression of the DHFR protein. Promising antifolate compounds undergoing clinical testing as anticancer agents include trimetrexate (which was recently approved by the FDA for the treatment of Pneumocystis carinii pneumonia), edatrexate, piritrexim, Tomudex, and lometrexol. The mechnisms of action, dosage, pharmacokinetics, clinical toxicity, and antitumor activity of these drugs are profiled.
Antifolates, such as methotrexate, were one of the earliest classes of drugs developed for clinical use in cancer chemotherapy. Today, methotrexate is still used extensively in the treatment of human leukemia, breast cancer, head and neck cancer, choriocarcinoma, osteosarcoma, and lymphoma. Antifolate compounds also have important clinical utility outside the realm of oncology, in the treatment of such diverse diseases as rheumatoid arthritis, psoriasis, bacterial and plasmodial infections, and opportunistic infections associated with AIDS.
Currently, numerous promising antineoplastic antifolate drugs are in clinical development, and many of these agents have interesting and unique mechanisms of action. An understanding of the cellular pharmacology of these agents will help the practicing clinician effectively use these new drugs as they become available for the treatment of human malignancies.
Mechanism of Action
The most widely used and best understood antifolate in cancer therapy is methotrexate, which differs from the essential vitamin, folic acid, by having an amino group substituted for a hydroxyl at the 4-position on the pteridine ring (Figure 1). This change transforms the enzyme substrate into a tight-binding inhibitor of dihydrofolate reductase (DHFR), a key enzyme required to maintain adequate intracellular levels of reduced folates .
Dihydrofolate reductase is critically important because folate molecules are biochemically active only in their fully reduced form as tetrahydrofolates. The tetrahydrofolates are essential cofactors that donate one-carbon groups in the enzymatic biosynthesis of thymidylate and purine nucleotide precursors for DNA synthesis (Figure 2). One reduced folate, 5,10-methylenetetrahydrofolate, participates in the reaction catalyzed by the enzyme thymidylate synthase (TS), which converts deoxyuridylate (dUMP) into thymidylate (dTMP). As a consequence of this reaction, 5,10-methylenetetrahydrofolate undergoes oxidation to dihydrofolate, which must then be reduced by DHFR back to tetrahydrofolate in order to replenish the intracellular reduced-folate pools.
Another reduced folate, 10-formyltetrahydrofolate, serves as a one-carbon donor for the reactions catalyzed by glycinamide ribonucleotide (GAR) and aminoimidazole carboxamide ribonucleotide (AICAR) transformylases. These enzymes are involved in the de novo biosynthesis of purine nucleotides. Thus, inhibition of DHFR by antifolates can lead ultimately to the decreased production of several essential precursors for DNA synthesis.
Both methotrexate and naturally occurring folate compounds can undergo intracellular metabolism to polyglutamate derivatives. These reactions, catalyzed by the enzyme folylpolyglutamyl synthase (FPGS), attach up to six glutamate residues to the pteridine ring, which help trap these molecules within the cell by decreasing their efflux. Methotrexate polyglutamates are also potent direct inhibitors of DHFR, as well as other folate-dependent enzymes, such as TS and GAR and AICAR transformylases. Furthermore, DHFR inhibition leads to the accumulation of dihydrofolate polyglutamates within the cell, which can directly inhibit the folate-dependent enzymes involved in the synthesis of thymidylate and purine nucleotides [2,3]. Thus, inhibition of DNA synthesis by the antifolates is a multifactorial process, resulting from the partial depletion of the intracellular reduced folate pool and from the direct inhibition of folate-dependent enzymes.
Administration of exogenous reduced folates, such as leucovorin calcium, to methotrexate-treated nonmalignant cells efficiently replenishes the reduced folate pool and directly competes with the drug-induced inhibition of folate-dependent enzymes. This is the biochemical rationale for the clinical use of leucovorin rescue to prevent severe toxicity in high-dose methotrexate chemotherapy regimens.
Ultimately, the depletion of thymidylate and purine nucleotide cofactors for DNA synthesis leads to a cessation of DNA synthesis, but it is not clear whether this action alone is enough to induce cell death. Lethal DNA damage resulting from a drug-induced lack of essential nucleotides may occur because of ineffective DNA repair, or because of misincorporation of uracil deoxynucleotides into DNA. Additional studies on this important issue are clearly necessary. Furthermore, the relative importance of the inhibition of thymidylate or purine nucleotide synthesis in the generation of methotrexate-induced cytotoxicity has yet to be defined.
Influx into Cells--At least two distinct, energy-dependent, carrier-mediated transport systems are responsible for the uptake of methotrexate into mammalian cells . The classic reduced-folate carrier system, which transports reduced folates, such as 5-methyltetrahydrofolate, and antifolates, such as methotrexate, has affinity constants in the micromolar range, and it is a relatively less efficient transporter of folic acid. A second carrier system that utilizes the hydrophobic membrane-associated binding protein, the human folate receptor (hFR), has a higher affinity (nanomolar range) for folic acid and reduced folates than it does for methotrexate. Some tumors, such as human ovarian cancers, overexpress the hFR on their cell surface.
The exact contribution of these two transport pathways to the uptake of methotrexate in clinical cancer chemotherapy is an area of intensive research. However, methotrexate resistance resulting from the decreased activity of one or both of these transport systems has been demonstrated in vitro, suggesting that transport deficiencies may be clinically important . More lipophilic antifolates, such as trimetrexate and piritrexim, are not substrates for these carrier-mediated folate transport systems, and can enter cells by either passive diffusion or by other transport mechanisms. Cell lines that are resistant to methotrexate because of decreased transport generally retain their sensitivity to these more lipophilic antifolate agents .
Efflux from Cells--Efflux of methotrexate from the cell is also mediated by several different transport systems, some of which are clearly distinct from the influx systems. Methotrexate efflux is not associated with the P-glycoprotein, multidrug resistance (MDR) system that has been described for numerous other antineoplastic agents. However, drug efflux is heavily influenced by the degree of methotrexate polyglutamation. As mentioned previously, both normal and malignant cells contain the enzyme FPGS, which can add glutamyl groups in a gamma peptide linkage to the pteridine ring in naturally occurring folates and in some antifolate drugs as well. This reaction serves two important functions: First, it facilitates the accumulation of intracellular folates by converting them into large anions that are less readily transported out of the cell. Second, polyglutamation enhances the affinity of methotrexate for several folate-dependent enzymes, including TS and AICAR transformylase.
Polyglutamation of methotrexate occurs more slowly compared to naturally occurring folates; however, the resulting methotrexate polyglutamates have extremely long intracellular half-lives, and can be detected in some tissues several months following a single drug administration. The accumulation of methotrexate polyglutamates in normal tissues, such as the liver, reduces the natural polyglutamation of endogenous folates, and may account for the chronic hepatotoxicity associated with methotrexate therapy . In addition, the selective nature of methotrexate cytotoxicity may result, in part, from the increased polyglutamation of methotrexate in cancer cells compared to normal tissues. The decreased ability to polyglutamate antifolates also appears to be an important mechanism of clinical drug resistance .
Binding to DHFR--Methotrexate is a competitive inhibitor of DHFR that binds noncovalently to this enzyme at the same binding site as the normal substrate, dihydrofolate. This interaction also depends on the intracellular concentration of reduced nicotinamide dinucleotide phosphate (NADPH), which is a normal cofactor for DHFR. Point mutations both inside and outside of the enzyme's active site have been identified that decrease the binding affinity of DHFR for methotrexate . Thus, mutations in the DHFR enzyme are another potential mechanism of resistance to antifolates.
Sensitivity to methotrexate cytotoxicity is highly dependent on the absolute amount of DHFR enzyme within the cell. Both human tumors and cancer cell lines that have increased levels of DHFR due to gene amplification are relatively resistant methotrexate . More subtle mechanisms may also exist that allow cells to acutely increase DHFR expression in response to antifolate treatment.
The expression of DHFR appears to be controlled at least partially by the binding of the DHFR protein to its own mRNA (Figure 3) . This binding of DHFR prevents further synthesis of the DHFR protein, resulting in an autoregulatory negative feedback loop. Excess normal substrate, such as dihydrofolate, or inhibitors, such as methotrexate, interfere with the binding of the DHFR protein to its mRNA and block this negative feedback loop, thereby allowing the synthesis of DHFR protein to continue unimpeded. This permits normal cellular function to be maintained, even in the presence of inhibitors such as methotrexate or other antifolates. The importance of this mechanism in the development of clinical antifolate resistance must be defined.
For a detailed discussion of the pharmacokinetics of methotrexate, including specific dosage and scheduling information, readers are referred to reference 1.