Chemotherapeutic Antimetabolites

Antimetabolites: Methotrexate, 5-FU, & Others

Overview

Highlights

Methotrexate (MTX) inhibits dihydrofolate reductase. Methotrexate can lead to interstitial and alveolar infiltrates, as well as pleural effusions.
5-Fluorouracil (5-FU) inhibits thymidylate synthase.

Both of these chemotherapies inhibit pyrimidine synthesis and produce a so-called “thymineless death”.
Leucovorin (folinic acid) is used as a rescue to reverse the effects of MTX and enhance the effects of 5-FU.

6-Mercaptopurine (6-MP) inhibits the conversion of PRPP (phosphoribosyl pyrophosphate) to IMP (inosine monophosphate) to inhibit purine synthesis.
Cytarabine (Ara-C), a deoxycytidine analog, inhibits DNA polymerase to produce direct DNA damage.

Mechanisms

Pyrimidine Synthesis Inhibition: Thymineless Death

Some chemotherapies (and antibiotics) work by starving cells of thymine, called “thymineless death”.

Leflunomide

Through a series of reactions carbamoyl phosphate is converted to uridine diphosphate (UDP) and that this step is inhibited by leflunomide, which we discuss in further in our immunotherapy section. Leflunomide is classified as a disease-modifying antirheumatic drug (DMARD) that is used in the treatment of rheumatoid arthritis and other autoimmune conditions.

Hydroxyurea

Hydroxyurea is an antimetabolite myelosuppressive agent that blocks ribonucleotide reductase, which catalyzes the conversion of ribonucleotides into deoxyribonucleotides: here uridine diphosphate (UDP) to deoxyuridine diphosphate (dUDP). Hydroxyurea is used in the treatment of cancers, including leukemia (ie, CML), melanoma, and ovarian cancer, as well as other conditions, including, notably, sickle cell disease.

5-Fluorouracil (5-FU)

Thymidylate synthase converts the conversion of deoxyuridine monophosphate (dUMP) to thymidylate (dTMP): 5-fluorouracil (5-FU) inhibits thymidylate synthase, which prevents this step in pyrimidine synthesis. Cancers adapt to inhibit this prodrug activation as a key form a chemotherapy resistance.

Methotrexate, Trimethoprim

As part of the conversion from dUMP to dTMP, there is a folate recycling loop within which converts dihydrofolate (DHF) to tetrahydrofolate (THF) via the enzyme dihydrofolate reductase (DHFR). Methotrexate inhibits dihydrofolate reductase to inhibit this reduction and prevent the conversion of deoxyuridine monophosphate to deoxythymidine monophosphate. The antibiotic trimethoprim inhibits this enzyme in bacteria.
In response, as a resistance mechanism, cancers can develop point mutations in DHFR to reduce binding affinity of MTX, and thus reduce its efficacy or it can increase production of DHFR.

Leucovorin

It's an important counterpunch to folate depletion; leucovorin does not require dihydrofolate reductase in order to be utilized in pyrimidine synthesis, thus methotrexate’s inhibition of dihydrofolate reductase is rendered inconsequential (in simplistic terms). So, we give methotrexate, allow it time to work against cancer cells (typically 24 hours), and then circumvent its toxicity via leucovorin rescue to save the normal (healthy) cells from the toxicity of thymine deficiency.
It enhances 5-fluorouracil (5-FU) efficacy. 5-FU has improved efficacy when administered along with leucovorin; it allows 5-FU more opportunity to bind-up and inhibit thymidylate synthase and further the thymineless death.

Purine Synthesis Inhibition

Next, let’s focus on purine synthesis.

6-Mercaptopurine (6-MP)

Via de novo purine synthesis, 5-phosphoribosyl-1-pyrophosphate

(PRPP) undergoes several different steps to form the first purine product, inosine monophosphate (IMP). 6-mercaptopurine (6-MP, a thiopurine) inhibits the conversion of PRPP (phosphoribosyl pyrophosphate) to IMP (inosine monophosphate). In its parent form, the 6-MP thiopurine is inactive and it must undergo conversion by hypoxanthine-guanine phosphoribosyl transferase (HGPRT) to form thioinosinic acid (note that the detailed metabolites are: 6-thioguanosine-5'-phosphate (6-thioGMP) and 6-thioinosine monophosphate (T-IMP)).

Note that azathioprine, which we discuss along with immunotherapies is a prodrug of 6-MP; thus, it undergoes conversion to 6-MP and further conversion to thioinosinic acid.

6-MP Toxicity

Importantly, thiopurine S-methyltransferase (TPMT) deactivates 6-mercaptopurine; patients with an autosomal recessive gene that produces TPMT deficiency, thus develop toxic 6-MP toxicity. So, we test for adequate TPMT levels prior to administering 6-MP or azathioprine. It’s also important to be aware that 6-MP undergoes inactivation via a xanthine oxidase mediated oxidation reaction but allopurinol, which is commonly given in the acute leukemias to prevent hyperuricemia inhibits xanthine oxidase and, thus, can lead to 6-MP toxicity.

Note that, in contrast, a cousin drug, 6-thioguanine (6-TG), undergoes a deamination reaction (rather than xanthine oxidase oxidation) and, thus, is unaffected by allopurinol administration.

Direct DNA Toxicity

Cytarabine (Ara-C)

Cytarabine (Ara-C, Cytosine arabinoside) enters the cell and undergoes series of steps of phosphorylation to its active toxic form Ara-cytidine-5'-triphosphate

(Ara-CTP), which enters the nucleus and induces DNA damage. Along this pathway, cytidine deaminase (CDA) can catalyze the inactivation of Ara-C (cytosine arabinoside) to Ara-U (uracil arabinoside) and render the cytarabine ineffective. Thus, naturally, cancer cells can learn to upregulate the production of CDA to increase this inactivation and resist the cytotoxic capability of cytarabine. In fact, cytarabine is known for its rapid deamination to the inactive uracil metabolite.

For reference, cytarabine is used in the treatment of hematologic malignancies (acute myelogenous leukemia (AML) and non-Hodgkin lymphoma).

Chemotherapeutic Antimetabolites

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