Antimetabolites are structural analogs of naturally occurring compounds. Antimetabolites interfere with the production of nucleic acids. They work through a variety of mechaninsms including competition for binding sites on enzymes and incorporation into into nucleic acids. Antimetabolites inhibit the growth of the most rapidly proliferating cells in the body (e.g., bone marrow, G.I. tract, etc.). Their are three categories of antimetabolites: antifolates, purine analogs and pyrimidine antimetabolites.

Antifolates - Methotrexate (Folex, Mexate) The importance of folates in tumor cell growth was demonstrated in 1948 by Farber and colleagues where they (aminopterin) were shown to produce remissions in leukemia. Antifolates produced both the first striking remissions in leukemia and the first cure of a solid tumor, choriocarcinama. Although aminopterin was the first clinically useful folate methotrexate was soon introduced in therapy and it has become the major folate used in cancer therapy.

Folic acid is an essential growth factor from which is derived a series of tetrahydrofolate cofactors that provide single carbon groups for the synthesis of RNA and DNA precursors such as thymidylate and purines. Folic acid must be reduced in two successive steps by dihydrofolate reductase before it can functiion as a coenzyme. The fully reduced form in the one that picks up and delivers single carbon units in various metabolic processes. The enzyme dihydrofolate reductase (DHFR)is the primary site of action of most folate analogs such as methotrexate. Inhibition of this enzyme leads to toxicity through partial depletion of cofactors required for the synthesis of purines and thymidylate. Methotrexate is a strong inhibitor of DHFR. It has a high affinity for the tumor cell enzyme blocking dihydrofolate reductase (DHFR) and formation of tetrahydrofolate needed for thymidylate and purine synthesis. Cell death probably results from inhibition of DNA synthesis. As with most antimetabolites methotrexate is only partially selective for tumor cells and is toxic to all rapidly dividing normal cells such as those of the gastrointestinal epithelium and and bone marrow.

Several factors influence the effectiveness of methotrexate treatment. Included among them are factors that concentrate the drug intracellularly such as transport and conversion to the higher molecular weight polyglutamates that are preferentially retained within the cell. Also important are changes in the amount and structure of dihydrofolate reductase. Changes in the latter will influence methotrexate binding and determine how well the drug is able to inhibit nucleotide synthesis.

Resistance>Several biochemical mechanisms of resistance have been demonstrated. The major mechanisms are decreased drug uptake, amplification of the dihydrofolate reductase gene and thus an increase in the target enzyme, mutations in the DHF gene, and decreased ability to form methotrexate polyglutamate inside cells. In the clinic there are now several documented cases where there is a lack of response to methotrexate and a correlation to amplified DHFR genes in tumor cells.

Pharmacology Methotrexate can be administered by several different routes including chronic oral, intermittent oral or i.v.high-dose intravenous and intrathecal. Its absorption from the gastrointestinal tract depends on the dose, with large doses being only partially absorbed while low doses are efficiently absorbed. These differences are most likely due to dependence on a saturable folate carrier. At high doses methotrexate is most likely taken up by passive diffusion. Only a small percentage crosses the into the CSF thus necessitatating intrathecal administration for the treatment of tumor cells in the CNS. Approximately 50% of the methotrexate is bound to plasma proteins thus there is the distinct possibility of drug interactions with drugs that also are significantly bound to plasma proteins. Metabolism is usually minimal although after administration in high dose therapy 7-hydroxymethotrexate is formed which is potentially nephrotoxic. Elimination is primarily renal and there is a clear relationship between serum levels of MTX and toxicity.

Therapeutic Uses Methotrexate is useful for the treatment of several different tumors. It is the drug of choice for gestational choriocarcinoma and related trophoblastic tumors of women where cures are obtained in a substantial number of women. In this cancer, it is usually used in combination with dactinomycin. Methotrexate is very useful for the treatment of acute lymphocytic leukemia in children where it is used for the maintenance of remissions. It is however of limited value for adult leukemias. It has also been used for the treatment and prevention of leukemic meningitis where it is given by intrethecal administration. Finally, high dose methotrexate along with leukovorin rescue is used for the treatment of osteogenic sarcoma and leukemias and lymphomas. Methotrexate is also used to treat several non-neoplastic diseases such as psoriasis and several immunological diseases such as rheumatoid arthritis, Wegener's granulomatosis and systemic lupus erythematosis.

Toxicity The primary toxic effects are against the rapidly dividing cells of the bone marrow and gastrointestinal epithelium. The severity of the clinical effects depends largely on the duration of exposure to inhibitory levels of the drug. All of the stem-cell types of the marrow can be affected to produce leukopenia, thrombocytopenia and with long-term administration, anemia. Methotrexate therapy must therefore be modified according to the patients hematological status and leukocyte and platelet counts must be carefully monitored. Mucositis is one of the earliest signs of toxicity and its appearance indicates that the dose must be reduced or other serious toxicities will occur. If diarrhea and ulcerative stomatitis occur therapy with the drug must be stopped. Methotrexate causes kidney damage which is a frequent complication of high dose therapy. It is manifested by elevated serum creatinine and decreased creatinine clearance. Crystalline deposits of methotrexate and methotrexate derived material have been found in the renal tubules which seems to account for most of the nephrotoxicity. Alkalinizing the urine to increase the solubility and ensuring good urine flow minimizes most of the nephrotoxicity due to high-dose methotrexate. Both low and high dose therapy can cause hepatotoxicity. High dose therapy results in elevated liver enzymes and low dose therapy produces a different type of hepatotoxicity which includes cirrhosis. Methtrexate can cause a reversible pulmonary syndrome which has been observed primarily in children undergoing maintenance therapy. Intrathecal and high-dose administration is accompanied by several types of neurotoxicity. These range from acute manifestations to long-term delayed toxicity in the form of encephalopathy. Nausea and anorexia frequently occur as acute side effects of methotrexate therapy.

High dose MTX-rescue therapyAdministration of high doses of methotrexate plus folinic acid (leucovorin) can be very beneficial in certain types of cancer. This therapy involves administration of methotrexate at high doses along with leucovorin to rescue host tissues from the effects of the intense methotrexate therapy. The leucovorin provides the normal tissues with the reduced folate leucovorin which circumvents the inhibition of DHFR. The protection seems to be selective in that it does not alter the antitumor effect of the methotrexate. Apparently only the host cells are able to utilize the leucovorin. A more recent explanation is that of differential reactivation of DHFR in host and tumor cells. Beneficial effects have been observed in patients with osteosarcoma as well acute leukemia.


6-mercaptopurine (6-MP, Purinethol) and 6- thioguanine (6-TG). The two major anticancer drugs in this category are 6-mercaptopurine and 6-thioguanine. These drugs are analogs of hypoxanthine and guanine, respectively. Several other purine analogs are also now commercially available. Among these compounds are not only anticancer drugs but immunosuppressives (azathioprine) and antiviral compounds (acyclovir, ganciclovir etc.). The antipurines can both inhibit nucleotide and nucleic acid synthesis and be incorporated into nucleic acid and sometimes they can do both.

Mechanism of action Most studies indicate that the thiopurines work at multiple sites and that their mechanism of action is a result of combined effects at these different sites. The thiopurines must first be converted into the nucleotide form in order to be active. This conversion is catalyzed by phosphoribosyltransferase enzymes. The nucleotide forms inhibit the first committed step in the de novo purine synthesis pathway (PRPP amidotransferase) and the key step in guanine nucleotide biosynthesis, IMP dehydrogenase. This latter site is the branch point where IMP is channeled towards either guanine nucleotide synthesis or adenine nucleotide synthesis. The mononucleotide derivatives are ultimately converted to triphosphates which can be incorporated into RNA and DNA.

6-MP is an analog of hypoxanthine and thus can't be incorporated directly into DNA but must first be converted into a TG analog (thio-IMP to thio-GMP). Both drugs are thus incorporated as the same form. Although RNA incorporation can be significant most studies indicate that the biological effects of the drugs are due to incorporation into DNA. Exactly why these compounds are toxic to tumor cells is not known but it may result from multiple effects buy it is well known that these compounds exhibit significant antitumor activity.

Resistance to the thiopurines For these antipurines to work efficient generation and maintenance of the nucleotide forms is necessary. In experimental tumors, lack of an altered phosphoribosyltransferase enzyme is the most commonly encountered mechanism of resistance. This enzyme is primarily responsible for forming the nucleotide. A different pattern is seen in humans receiving thiopurine therapy where increased alkaline phosphatase activity seems to be a major cause of resistance. This enzyme catalyzes the breakdown of the nucleotide form and could protect tumor cells by antagoning the accumulation of thiopurine nucleotides. As would be expected there is cross resistance between these two purine analogs.


6- Mercaptopurine (Purinethol) After oral ingestion, absorption is incomplete and variable, but it is still routinely given this way. 6-MP is widely distributed in the body with the exception of the CNS where little drug is found. As far as metabolism 6-mercaptopurine is methylated on the sulfhydryl group with subsequent oxidation of the methylated derivatives. In addition 6-mercaptopurine is oxidized to thiouric acid by xanthine oxidase. This pathway is blocked by allopurinol. Therefore, if it used along with allopurinol the dosage of 6-MP must be reduced otherwise toxicity will be increased. 6-Thioguanine is also given orally but it is slowly absorbed after oral administration. Unlike mercaptopurine it is metabolized mainly to inorganic sulfate. There is no formation of thiouric acid with 6-thioguanin thus unlike 6-MP it can be used in combination with allopurinol without the dosage being reduced.

Therapeutic Uses Both drugs are used primarily in the treatment of leukemias. Response rates are higher in children than adults. 6-MP is used in the maintenance therapy of acute lymphocytic leukemia and 6-TG in the treatment of acute nonlymphocytic leukemia.

Toxicity Bone marrow depression is dose limiting with both drugs. The maximum effect on the blood count may be delayed and it is important to discontinue these drugs temporarily if there is an abnormally large fall in the leukocyte count or abnormal depression in the bone marrow. Other major toxicities include nausea and vomiting and stomatitis. Hepatotoxicity is seen as jaundice in ]about 33% of the patients treated with 6-MP. As mentioned above 6-MP is a substrate for xanthine oxidase which converts it to 6-thiouric acid by oxidation. This appears to be an important route for inactivation of the drug. Allopurinol, a drug used to prevent the hyperuricemia and uricosuria that often follow marked cell kill consequent to leukemia therapy, inhibits this conversion. When 6-MP and allopurinol are used together, the dose of 6-MP is usually lowered. 6-TG is not extensively deaminated and only a small amount is converted to 6-thiouric acid. Therefore, no reduction in dosage is necessary with allopurinol.

Chlorodeoxyadenosine and Pentostatin These are two newly introduced purine analogs which have good activity against certain types of leukemia. The presence of a halogen in the adenine ring of several purine analogs led to a group of compounds which resisted metabolic degradation by deamination. 2-chlorodeoxyadenosine (CdA) showed the greatest activity of these compounds. It was found to be an inhibitor of ribonucleotide reductase and consequently DNA synthesis. It also inhibited DNA strand elongation after incorporation into the DNA. This drug was found to cause extensive DNA damage. It has been found to be very useful in the treatment of indolent B-cell lymphocytic leukemias such as hairy cell leukemia and chronic lymphocytic leukemia. Many of these have been resistant to other therapies. Additional studies have shown that this drug may be promising in lymphomas, and acute and chronic granulocytic leukemias also. The dose limiting toxicity is mild bone marrow depression.

Pentostatin (2'-deoxycoformycin)is a natural product isolated from bacteria. It binds tightly to and inhibits the enzyme adenosine deaminase. Lack of this enzyme has been implicated in some immunodeficiency syndromes. It has significant activity in patients with B-cell chronic lymphocytic leukemias. Toxicities are varied and somewhat unpredictable. Lymphopenia is often encountered leading to infectiions. Acute renal failure and neurological toxicities are less common but have been seen to be life threatening.


There are several different compounds in this group but the principal members used in the treatment of cancer include the fluoropyrimidines and cytosine arabinoside. Pyrimidine analogs have also been used in the treatment of diseases as diverse as cancer, psoriasis, fungal infections and viral infections. The best characterized agents in this class are the halogenated pyrimidines.


5-fluorouracil(Adrucil), 5-fluorodeoxyuridine (FdUrd) (Floxuridine) The most important members of this group are 5-fluorouracil(5-FU) and 5-fluorodeoxyuridine (FdUrd).

Mechanism of action Members of this group are direct inhibitors of thymidylate synthetase the key enzyme in thymidylate synthesis. Methotrexate in contrast is an indirect inhibitor of this enzyme through inhibition of dihydrofolate reductase. These agents produce multiple biochemical lesions as a result any one of which has the potential to be cytotoxic. 5-FU must first be converted to the nucleotide form to be active as a cytotoxic agent. The nucleotide (5'-FUMP) can be formed by several different pathways. FUMP can be incorporated into RNA and also can be converted to the deoxynucleotide(F-dUMP). This latter reaction is crucial to the cytotoxic effects of 5-FU. FdUMP may also be formed by the direct conversion of FdURD by thymidine kinase. FdUMP inhibits the enzyme thymidylate synthetase which leads to deletion of TTP, a necessary constituent of DNA. Ordinarily, thymidylate synthetase catalyzed the methylation of dUMP in a multistep process. When FdUMP is utilized the process becomes inhibited at an intermediate step. A complex between the enzyme, the pseudosubstrate and the folate cofactor is formed. The complex dissociates very slowly and is sufficiently stable so that the enzyme no longer can catalyze the reaction. DNA synthesis is inhibited until the drug is removed and new enzyme synthesis occurs. Since FdUrd is converted to FdUMP directly in one step it is a more potent inhibitor of TMP synthetase than is 5-FU. It is often effective in the low nanomolar concentration range. On the other hand 5-FU will require micromolar concentrations to be effective and at such concentrations other active metabolites are formed such as FUTP which can become incorporated into RNA in place of UTP. Incorporation into RNA has resulted in observed effects on the function of both rRNA and mRNA. Although incorporation into RNA is much higher than incorporation into DNA recent studies have shown this to occur also. The biological consequences of this are not known, however. An important question that is always asked is whether the effects on RNA or on those on DNA are important for the cytotoxic action of these compounds. There are many conflicting studies on this point. It is most probable that the cytotoxic action of these drugs is not the same in all cell lines and that this heterogeneity is the basis for the wide variability in response to FU.

Resistance A number of biochemical mechanisms have been identified that are associated with resistance to 5- FU and FudR. The major ones include decreased conversion to the nucleotide form and increased breakdown of the nucleotide. For each of these mechanisms changes in several different enzymes might account for resistance.

Pharmacology The oral absorption of fluorouracil is erratic, so it is usually given parenterally either by bolus (intravenous) or by continuous infusion (intravenous or arterial). Administration by continuous infusion overcomes the problem of rapid disappearance of FU from the circulation. Since these drugs work best if they are present during the S phase of the cell cycle their short time in the circulation would ordinarily present a problem as the plasma levels would greatly fluctuate. Protocols using continuous infusion were developed to overcome this potential problem. Metabolic degradation occurs particularly in the liver. 5- FU readily enters the CSF.

Therapeutic Uses Fluorouracil is used to treat several common solid tumors. It produces partial responses in up to about 1/3 of patients with metastatic carcinomas of the breast and the gastrointestinal tract. Topically it has been used for the treatment of basal cell carcinomas and premalignant skin keratoses. FU is used in many different combinations in order to enhance its activity. Some involve combination with other cytotoxic agents like methotrexate while others involve combination with agents that by themselves lack toxicity but modulate the cytotoxic effects of FU. Combination with leucovorin has been very successful as the leucovorin enhances formation of the ternary complex. FudR is primarily used by continuous infusion into the hepatic artery for treatment of metastatic colon cancer.

Toxicity The toxicities of these two drugs are similar and somewhat dependent on the mode of administration. Anorexia and nausea are among the earliest toxicities seen. These are followed by stomatitis and diarrhea which are indicative of a sufficient dose being given. The frequency of effects varies with the treatment schedule employed. Stomatitis and diarrhea are the most common dose-limiting toxicities when continuous infusions are used. The major toxicity resulting from a bolus dose is bone marrow depression. This is manifested by leukopenia and somewhat less often by thrombocytopenia and anemia. The lowest blood counts occur at one to two weeks. Skin toxicity manifested by alopecia, thinning of the skin, nail changes, dermatitis and photosensitivity can also occur. FU also produces an acute cerebellar syndrome in less than 1% of patients. This includes ataxia, nystagmus, slurred speech and dizziness.

Cytosine Arabinoside (Cytosar; ara-C) This is an analog of 2'-deoxycytidine with the 2'-hydroxyl in a position trans to the 3'-hydroxyl of the sugar. The bases of polyarabinsides cannot stack normally as do the bases of polydeoxynucleotides.

Mechanism of action Ara-C is first converted to the monophosphate nucleotide (AraCMP) by deoxycytidine kinase. The monophosphate can then react with appropriate kinases to form the di and triphosphate nucleotides. AraCTP is believed to be the key active component. Its accumulation causes potent inhibition of DNA synthesis in many cells. This nucleotide is a competitive inhibitor of DNA polymerases. Also, the triphosphate is a substrate for DNA polymerases and it is incorporated into the DNA. It apparently causes inhibition of DNA chain elongation when ara-C in incorporated at the terminal position of a growing DNA chain. Unlike most of the antimetabolites the effects of ara-C are directed almost exclusively towards DNA and it has little or no effect on RNA synthesis or function. The relative biological impact of DNA synthesis inhibition versus incorporation into DNA has been studied for several years now. The evidence indicates that synthesis inhibition is secondary to analog incorporation. However, the precise cause of cellular death by ara-C is not known.

Resistance The overall balance between the anabolic(kinases) and catabolic enzymes (deaminases) involved in the metabolism of ara-C determine the amount of araCTP and thus the sensitivity or resistance of cells to araC. For example, in patients with refractory acute leukemia, who received high doses of ara-C upon relapse there was a significant correlation between the area under the curve and clinical response rate. The same relationship in newly diagnosed acute leukemias does not seem as well established.

Pharmacology Cytarabine is very poorly absorbed after oral administration in humans. It is routinely given i.v. by continuous infusion sometimes in high dose regimens. Intrathecal administration is also used for meningeal leukemia and lymphoma. Ara-C reaches the CNS reasonable well with CSF levels as high as 40% of the plasma levels. It has a very short plasma half-life due to cytidine deaminase activity in liver and elsewhere, and its metabolites are renally excreted. Metabolism accounts for 70-90% of the Ara-C eliminated with most of the drug being excreted as ara-U.

Therapeutic Uses AraC is an important antimetabolite used primarily for the treatment of acute myelocytic leukemia due to its potent myelosuppressive action. It is the single most effective agent for induction of remission in this disease and it is used primarily in combination with daunorubicin. It has occasionally been used to treat acute lymphocytic leukemia and in high doses it has been used for non-Hodgkins lymphoma and chronic myelocytic leukemia. It is not active against solid tumors.

Toxicity The principal toxicity is bone marrow depression manifested as granulocytopenia and thrombocytopenia. Other toxicities include oral ulceration, nausea, vomiting and diarrhea, and peripheral neurotoxicity with high doses. USE CAUTION WITH DECREASED HEPATIC FUNCTION since the liver is a major site of deamination.

Fludarabine A new nucleoside analog that has modifications in both the base and sugar moieties. Like ara-C it is metabolized to the nucleotide triphosphate by sequential action of several kinases. It then interferes with DNA synthesis by DNA polymerase inhibition and by incorporation into nascent DNA. Unlike ara-C this compound is much more likely to act as a chain terminator. It also apparently acts as an inhibitor of the proofreading exonuclease activity of DNA polymerase epsilon. Fludarabine has been shown to be very effective against chronic lymphocytic leukemia. It gives a response rate exceeding 80% in previously untreated patients. Moderate to severe myelosuppression occurs at therapeutic doses, while neurologic toxicity occurs especially at higher doses.