Folate Antagonists

Folates exist predominantly as polyglutamates within cells to facilitate intracellular retention in excess of the freely trans portable monoglutamate form. They must first be reduced to tetrahydrofolate (FH4) by the enzyme dihydrofolate reductase (DHFR) to become active coenzymes in one-carbon transfer reactions, among which are those required for the de novo purine synthesis mediated by glycineamide ribonucleotide formyltransferase (GARFT) and aminoimidazole carboxamide ribonucleotide transformylase (AICART), as well as the methylation of 2-deoxyuridylate for de novo synthesis of thymidylate through thymidylate synthetase (TS). Antifo-lates inhibit these reactions (Figure 2.5), which lead to formation of DNA strand breaks upon depletion of thymidylate and purine nucleotides, accumulation of deoxyuridine monophosphate (dUMP), and incorporation of deoxyuridine triphosphate (dUTP) into DNA. They are transported into the cell primarily via the reduced folate carrier (RFC) and to a smaller extent by the folate receptor protein (FRP). The primary toxicities seen with antifolates are myelosuppression and mucositis.

Methotrexate

Methotrexate (MTX) is the most commonly used antifolate agent in cancer chemotherapy. Cellular uptake of MTX is faster in rapidly dividing cells, with a concomitant decreased rate of efflux as opposed to slowly growing cells. It is then subsequently polyglutamated by the enzyme folylpolyglu-tamyl synthetase (FPGS), although less avidly and at slower rates compared to, in order of decreasing affinity, FH2, FH4, or

5fu Mucositis Mechanism

figure 2.5. Mechanism of action of fluoropyrimidines and antifolates. AICART, aminoimidazole carboxamide ribonucleotide transformylase; dTMP, deoxythymidine monophosphate; dUMP, deoxyuridine monophosphate; DHFR, dihydrofolate reductase; DHFU, dihydrofluorouracil; CH2-FH4, 5,10-methylenetetrahydrofo-late; 10-CHO-FH2, 10-CHO-FH4, 10-formyl-, -dihydro-, -tetrahydro-, -folate, respectively; DPD, dihydropyrimidine dehydrogenase; FH2, FH4, dihydro-, tetrahydro-, -folate, respectively; 5-FU, 5-fluorouracil;

5'-dFUrd, 5'-deoxy-5-fluorouridine; FUrd, 5-fluorouridine; FdUMP, FdUDP, FdUTP, fluorodeoxyuridine-, -mono-, -di-, -tri-, -phosphate, respectively; FUMP, FUDP, FUTP, fluorouridine-, -mono-, -di-, -tri-, -phosphate, respectively; GARFT, glycineamide ribonucleotide formyltransferase; MTX (Glu)n, polyglutamated methotrexate; RR, ribonucleotide reductase; dTMP, deoxythymidine monophosphate; dUMP, deoxyuridine monophosphate; TK, thymidine kinase; TP, thymidine phosphorylase; TS, thymidylate synthase.

figure 2.5. Mechanism of action of fluoropyrimidines and antifolates. AICART, aminoimidazole carboxamide ribonucleotide transformylase; dTMP, deoxythymidine monophosphate; dUMP, deoxyuridine monophosphate; DHFR, dihydrofolate reductase; DHFU, dihydrofluorouracil; CH2-FH4, 5,10-methylenetetrahydrofo-late; 10-CHO-FH2, 10-CHO-FH4, 10-formyl-, -dihydro-, -tetrahydro-, -folate, respectively; DPD, dihydropyrimidine dehydrogenase; FH2, FH4, dihydro-, tetrahydro-, -folate, respectively; 5-FU, 5-fluorouracil;

5'-dFUrd, 5'-deoxy-5-fluorouridine; FUrd, 5-fluorouridine; FdUMP, FdUDP, FdUTP, fluorodeoxyuridine-, -mono-, -di-, -tri-, -phosphate, respectively; FUMP, FUDP, FUTP, fluorouridine-, -mono-, -di-, -tri-, -phosphate, respectively; GARFT, glycineamide ribonucleotide formyltransferase; MTX (Glu)n, polyglutamated methotrexate; RR, ribonucleotide reductase; dTMP, deoxythymidine monophosphate; dUMP, deoxyuridine monophosphate; TK, thymidine kinase; TP, thymidine phosphorylase; TS, thymidylate synthase.

leucovorin. Polyglutamated MTX is a more potent and avid reversible inhibitor of DHFR than monoglutamated MTX. The ability to generate MTX polyglutamates seem to correlate with cytotoxicity in both murine and human tumor cells,70,71 whereas increasing concentration of reduced folates can competitively reverse MTX toxicity.

MTX is rapidly absorbed orally, although incompletely at higher doses. It has a rather long terminal half-life, up to 27 hours.72 CSF penetration at conventional doses is poor. It is thus administered intrathecally in prophylactic CNS therapy for ALL. MTX slowly penetrates third-space fluid collections such as pleural effusion and ascites. The half-life of MTX sequestered in these situations is further prolonged because of slow reentry into the bloodstream. Enhanced clinical toxicity may be observed if fluid collections are not drained before methotrexate therapy. MTX is primarily cleared by the kidneys within the first 12 hours after administration, mostly as unchanged drug. It should therefore be used cautiously in patients with renal insufficiency. In these circumstances, the enterohepatic circulation may assume a more important role in drug excretion,73 and use of activated charcoal or cholestyramine may be tried to enhance plasma clearance through enhanced biliary excretion.74,75 Carboxypeptidase G, an enzyme that removes the terminal glutamate residue from MTX, leads to MTX inactivation and is another effective alternative.

Standard Intravenous Administration Major toxicities are myelosuppression and mucositis. Gastrointestinal epithelial cells are more sensitive to the effects of MTX, being inhibited at half the concentrations required to inhibit DNA synthesis in the bone marrow.76 Mucositis typically appears 3 to 5 days after treatment and precedes the fall in leukocytes or platelets by several days. Unless drug clearance is severely impaired, such as in renal failure, myelo-suppression and mucositis are usually reversed within 2 weeks.

High-Dose Therapy High-dose MTX yields therapeutic concentrations in the CSF. Nephrotoxicity, manifested as oliguria and azotemia, is the major adverse effect of high-dose MTX therapy. Although MTX may be a direct tubular toxin per se, nephrotoxicity arising from high-dose therapy is mainly attributed to intratubular precipitation of MTX and its less-soluble metabolites, 7-OH MTX and 2,4-diamino-N-10-methyl pteroic acid (DAMPA), in acidic urine. Vigorous hydration and urine alkalinization can diminish such complication. Some myelosuppression and mucositis may occur. Leucovorin rescue (described next) reduces the likelihood of systemic tox-icity. Acute transient transaminase elevations commonly occur after high-dose therapy, but late occurrence of liver failure or cirrhosis has not been reported. Repeated courses of high-dose MTX therapy can result in encephalopathy, dementia, paresis, and seizures.

Leucovorin Rescue Methotrexate blood levels should be assayed every 24 hours with high-dose therapy in patients with impaired renal function as well as patients who have had excessive toxicity with prior MTX therapy. Leucovorin rescue is initiated at 12 to 24 hours after the start of high-dose drug infusion and should continue until MTX level is less than 50 nM/L. Leucovorin is typically administered as 10mg/m2 intravenously followed by oral doses given every 6 hours for 10 to 12 more doses. If MTX levels are above 500nM/L, 1 |M/L, or 2 |mM/L at 48 hours, a general guideline is that leucovorin should be administered at 15mg/m2, 100mg/m2, or 200mg/m2, respectively, every 6 hours over 48 more hours. Patients should be vigorously hydrated using bicarbonate-enriched fluids (2.5-3.5 L/m2/day IV fluids + 45-50 mEq bicarbonate/L IV fluid) to maintain high urine output (more than 100mL/h) and alkaline urine (above pH 7.0) for 12 hours before and 48 hours after highdose therapy (more than 1 g/m2) to avoid renal failure. MTX is used clinically in leukemia, lymphoma, breast cancer, head and neck cancer, osteogenic sarcoma, and choriocarcinoma.

Raltitrexed

Raltitrexed is a potent quinazoline analogue that selectively inhibits thymidylate synthase (TS). Similar to MTX, polyglu-tamation by folylpolyglutamyl synthetase (FPGS) is correlated with increasing cytotoxicity.77 It is poorly absorbed orally. Its excretion, mostly as unchanged drug, is similarly correlated with creatinine clearance. Patients with insufficient folate intake may be at increased risk for clinical toxic-ity. Transient elevation of liver enzymes may be seen with its convenient IV dosing once every 3 weeks. It is an effective alternative to 5-FU-based therapy in patients with metastatic colorectal cancer. Another advantage over 5-FU is lesser frequency of mucositis. It also exhibits antitumor activity in non-small cell lung and breast cancers. It is approved in several countries in Europe, Canada, Asia, and Australia.

Pemetrexed

Pemetrexed is a pyrrolo-pyrimidine-based antifolate analogue whose main activity is inhibition of TS. It is transported into cells via the RFC, with transport kinetics similar to that of methotrexate. It binds to folate receptor-a with a very high affinity, similar to that of folic acid. Furthermore, cellular influx is also facilitated by the presence of high-affinity and highly specific transport systems for pemetrexed in malignant mesothelioma cell lines.78 This transport system has a relatively low affinity for other inhibitors of DHFR or TS such as methotrexate and raltitrexed. Pemetrexed is likewise poly-glutamated by FPGS to enhance intracellular concentration, and consequently, cytotoxicity. The polyglutamation reaction occurs 90- to 195-fold more efficiently with pemetrexed than methotrexate.79 Polyglutamated pemetrexed is more than 60fold more potent in its inhibition of TS than the monoglutamate. Its prolonged intracellular retention allows for bolus intermittent dosing schedules. At high concentrations, peme-trexed causes an S-phase block and apoptosis. Under the same conditions, it also exhibits multitarget inhibition of several crucial folate-requiring enzymes such as DHFR, GARFT, and, to a lesser extent, AICART and C1-FH4 synthase.

Pemetrexed causes a rapid depletion of deoxythymidine-, deoxycytosine-, and deoxyguanosine triphosphates. Cells that rely on de novo purine synthesis and do not have purine salvage pathways are expected to be particularly sensitive to pemetrexed. It has been shown that pleural mesothelioma cells frequently (approximately 90%) exhibit codeletion of the gene coding for the enzyme methylthioadenosine phosphory-lase (MTAP) with the CDKN2A gene.80 Homozygous deletion of CDKN2A gene, which encodes for the cell-cycle regulatory proteins p16 and p14ARF, may be seen in 75% of pleural mesotheliomas.80 As MTAP catalyzes an important purine salvage pathway, the frequency of its codeletion with CKDN2A, as well as the presence of highly specific transport systems for pemetrexed in malignant mesothelioma, may in part account for its efficacy in this tumor type.

Folic acid has been shown to be 100- to 1,000-fold less effective than leucovorin in protecting tumor cells against the cytotoxic effects of pemetrexed.81 Nevertheless, folic acid reduces toxicity in mice while preserving the antitumor activity of pemetrexed. Myelosuppression and mucositis can be significantly ameliorated by folate and vitamin Bi2 supplementation without any demonstrable reduction in antitumor efficacy.

Pemetrexed has demonstrated broad antitumor activity in a wide variety of solid tumors, including mesothelioma, non-small cell lung, breast, cervical, colorectal, head and neck, and bladder cancers in a variety of Phase II trials. It exhibits synergistic antitumor activity with alkylating agents, irinote-can, and gemcitabine.82 Promising activity has been demonstrated when pemetrexed is combined with cisplatin and gemcitabine.82,83 A pivotal Phase III trial indicates the superiority of pemetrexed in combination with cisplatin versus cisplatin alone in malignant pleural mesothelioma.84 Pemetrexed demonstrated equivalent efficacy to docetaxel, but with significantly less toxicity, in second-line NSCLC.85

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