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Antifolates or antifols are compounds commonly used to treat various forms of cancer. They act as antitumor agents by suppressing the effects of folic acid and its derivatives on cellular processes. Antifolate drugs produce an intracellular state of folic acid deficiency in order to inhibit folate-dependant enzymes along the folate metabolic pathway. DNA synthesis and cell division, processes involved in malignant tumor growth, are hindered by folic acid deficiency. Antifolates are used to treat: breast cancer, head and neck cancer, bladder cancer, acute lymphocytic leukemia, non-Hodgkin’s lymphoma, choriocarcinoma, and osteogenic sarcoma. Antifolates are also being used in the treatment of non-cancerous diseases such as malaria, bacterial infections, psoriasis, and rheumatoid arthritis.

When the effects of folate on cellular metabolic processes were discovered in the 1940s, doctors began a clinical investigation began that would lead to the development of the first folate antagonists. The chief investigators found that the antifolate called aminopterin (AMT) could cause pediatric patients with acute leukemia to go into remission. Shortly thereafter, methotrexate (MTX) – a derivative of aminopterin – was found effective in mice with the L1210 form of leukemia. Because of its success and higher efficacy with decreased toxicity compared to aminopterin, it became the primary antifolate.

With its multitude of uses, MTX is still the most widely used folate antagonist in medical oncology today. For some neoplastic diseases such as choriocarcinoma, MTX is highly effective, apparently providing a cure for about half of the patients with this disease who use it. While MTX is effective when used by itself, it is commonly used in combination with other antineoplastic drugs. The anticancer activity of antifolates can be enhanced if they are given with drugs that inhibit nucleoside transport by preventing neoplastic cells from recovering nucleoside precursors. Currently, it serves as the principle biochemical prototype for the clinical research of all new antifolates. In addition to MTX and AMT, original antifolates still under clinical investigation and being used in medicine include: thymidylate synthase (TS), serine hydroxymethyltransferase (SHMT), folyilpolyglutamyl synthetase (FPGS), g-glutamyl hydrolase (g-GH), glycinamide-ribonucleotide transformylase (GARTfase), leucovorin (LV), amino-imidazole-carboxamide-ribonucleotide transformylase (AICARTfase), 5-fluorouracil (5-FU), and folate transporters.

Folate metabolism is a fundamental intracellular process, without which cells cannot survive. Folates are essential to single-carbon metabolism within cells. This transfer of single-carbon groups with nonfolate groups is the underlying process responsible for folate’s primary function in the body: the production and maintenance of new cells. The molecules with which folate interact are also responsible for cell growth and survival. Antifolates are useful in demonstrating the importance of folate metabolism to cell survival.

At some level antifolates hinder the folate metabolic pathway. At the molecular level, the substrates along this pathway are forced to transform into tight-binding inhibitors of the enzyme DHFR. This occurs because of the structural difference of the antifolate. Instead of having a hydroxyl at the 4-position of the pteridine ring, antifolates have an amino group at this location, changing the way upon which the substrate binds to the enzyme’s active site. The enzyme involved in this reaction, DHFR, is the one that maintains the production of tetrahydrofolates, which are the reduced forms of folate within the cell. These reduced folates become depleted by the presence of antifolates, which in fact, use the reduced folate carrier route into the cell to take up the intracellular folates. Tetrahydrofolates play a crucial part in the formation of the DNA molecule since they are the cofactors that donate a carbon atom in the enzymatic formation of thymidylate and purine nucleotides, essential precursors for the synthesis of DNA. Therefore, the interference of the production of tetrahydrofolates by antifolates inhibits the biosynthesis of essential nucleotides for DNA synthesis. Examples of tetrahydrofolates include 10-formyltetrahydrofolate and 5, 10-methylenetetrahydrofolate, both of which donate single carbon groups in the biosynthetic reaction that forms nucleotides. They become converted out of their biologically active, reduced form into dihydrofolate. To be converted back into the reduced form as a tetrahydrofolate again, they require the presence of the enzyme, DHFR. Therefore, when DHFR is reduced by the interference of an antifolate, the amount of intracellular folates is also reduced, which contribute significantly to the prevention of the formation of nucleotide precursors needed for the synthesis of DNA. The impediment of DNA production eventually leads to cell death, hence causing the antitumor effect of the antifol.

In mammalian cells, polyglutamylation occurs when the enzyme, folylpolyglutamate synthetase (FPGS), adds approximately seven additional glutamate molecules to both folate and antifolate molecules. It is important to take this process into account when evaluating the relative efficacy of the antifolate drug since it is partially responsible for the duration of the drug’s stay inside the cell. This is because the polyglutamylation of the antifolate adds additional negative charge to it, increasing its size, and ultimately reducing the efflux of the drug as it becomes trapped inside the cell, prolonging its overall effect. Also, antifolate polyglutamates are more effective as folate-dependant enzyme inhibitors than monoglutamated drugs. They are direct inhibitors of DHFR and are also able to effectively inhibit other folate-dependant enzymes such as glycinamide ribonucleotide (GAR), aminoimidazole carboxamide ribonucleotide (AICAR) transformylases, and thymidylate synthase (TS). Because they bind to the enzyme with greater strength, the glutamates are slower to become detached from it than the antifolate is in its original form.

When using antifolate drugs, the cell-cycle must be taken into consideration to ensure its maximum efficacy. Many of the same enzymes and proteins that are involved with folate metabolism fluctuate in accordance with the cell cycle. In fact, the folate-dependant enzymes, such as DHFR, increase during the S-phase of mitosis. Cells in the resting (G0) phase are less affected by the same amount of antifolate drug than are cells in other stages. Therefore, antifolates are most effective when there are relatively few cells, as in the G0 phase. Another important factor is that when using a folate antagonist, the synthesis of DNA in both normal and cancerous cells will be hindered. However, RNA and protein synthesis will still take place within the cell. If folates are not replenished, megaloblasts (giant cells), will form and cell death will then increase.

While extensive research efforts in the area of antimetabolic chemotherapy are taking place, the antifolates currently being administered to patients, like MTX, must be used with a degree of caution. Recent studies have found that high concentrations of MTX in the blood plasma are correlated with toxicity. Taking relatively low doses of antifolates is more beneficial because absorption is increased with the lower doses. Studies have shown that increasing oral doses of MTX in particular, decreases overall absorption. However, absorption can be enhanced when taken on an empty stomach with pure water. Also, increasing doses does have certain benefits, such as increased polyglutamate formation, which, in turn, leads to extended periods of DHFR inhibition.

Another precaution to take into consideration is the use of other drugs along with the antifolate as there are serious interactions that can occur. For example, the combination of an antifolate with an antibiotic produces a toxic effect. Even more serious, however, are the use of antifolates with the nonsteroidal, anti-inflammatory class of drugs (NSAIDs) as this combination can potentially lead to death. The NSAIDs, naproxen and ketoprofen, are especially to be used with caution. Other common NSAID drugs such as aspirin, phenylbutazone, salicylate, probenecid, and trimethoprim must also be carefully monitored when using antifolates. Alcohol should be completely avoided by patients taking antifolates as it increases the risk of developing hepatic fibrosis and cirrhosis. Notably, there are some drugs that can actually reduce antifolate toxicity, such as methylxanthines (e.g., caffeine and aminophylline).

Adverse effects of the prolonged administration of antifolates occasionally occur. Tissues that renew themselves, like bone marrow and epithelial cells, are the most subject to side effects by antifolates. Younger patients cope better than older ones, due to greater kidney efficiency, resulting in quicker elimination of the drug from the body. Sometimes it is necessary to halt the administration of the antifolate because of the severe side effects. These can include mucositis, a serious symptom of gastrointestinal toxicity and renal toxicity. Occasionally, the chronic use of antifolates causes hepatoxicity, neuro, pulmonary, and skin toxicity.

New antifolate compounds are currently in development. Medicinal chemists are trying to overcome biomechanisms that are immune to existing antifolates. The body’s resistance to antifolates is often natural, but can also be acquired. Known causes of resistance include: reduced influx of the drug into the cell by the reduced folate carrier or because of the decrease of polyglutamation; an increase in DHFR because of gene enhancement; or a mutation in the DHFR enzyme which leads to decreased binding to the antifolate. Newer antifolates will have greater solubility in lipids, improved cellular uptake, and/or enhanced polyglutamation ability. The newer antifolates have potential for improved therapeutic efficacy along with decreased toxicity.