A number of anticancer drugs, particularly those developed during the era of cytotoxic chemotherapy, exert their antitumor effects on DNA synthesis. Cells are thus most vulnerable during periods of active DNA synthesis (S-phase), and least affected during quiescent (G0) stages of their life cycle. Thus tumors that have a high proliferative rate, such as leukemias and aggressive lymphomas, are most vulnerable to these agents.
Farber and associates showed that the folate antagonist aminopterin induced a complete remission in children with ALL, thereby launching the modern era of chemotherapy. Unfortunately, these remissions were short-lived, and the leukemia invariably became resistant to further treatment. Subsequently, methotrexate, a 4-amino, N-10 methyl analogue of folic acid, supplanted aminopterin because it had more predictable side effects. Methotrexate continues to be a key drug in the induction and maintenance therapy of ALL, in the intrathecal prophylaxis and treatment of CNS leukemia, in the primary treatment of CNS lymphomas, and in combination therapy of high-grade lymphomas.
Methotrexate enters cells through an active uptake process mediated in most tumor cells by the reduced folate transporter20 and is actively effluxed from cells by the MRP class of exporters.21 A second uptake transporter, the membrane folate-binding protein (FBP), has lower affinity for methotrexate, but may contribute to uptake of other antifolates, such as pemetrexed. The FBP is found on many solid malignancies, and is an active target for folate analogue- and antibody-mediated drug development. A third, low pH transporter may also participate in methotrexate influx, particularly in the intestine, but its role in tumor uptake is uncertain.22 By virtue of its 4-amino substitution, methotrexate potently inhibits the enzyme DHFR, which recycles oxidized dihydrofolate to its active tetrahydrofolate state. Inhibition of DHFR leads to rapid depletion of the intracellular tetrahydrofolate coenzymes required for thymidylate and purine biosynthesis. As a result, DNA synthesis is blocked and cell replication stops. Methotrexate is retained intracellularly as a consequence of an enzymatic process that adds up to six glutamate moieties in an unusual peptide linkage to the γ-carboxyl group of the drug (Fig. 22–1). Polyglutamation is an important determinant of leukemic cell sensitivity to methotrexate. Methotrexate polyglutamates, in addition to their long persistence in cells and their potent inhibition of DHFR, have greatly increased inhibitory effects on other folate-dependent enzymes, including thymidylate synthase and enzymes that synthesize purines (Fig. 22–2). Cells that convert the drug to polyglutamates efficiently, such as leukemic myeloblasts and lymphoblasts, are more susceptible to the drug than are normal myeloid precursors, which have limited capability for polyglutamation.23 Accumulation of polyglutamates correlates with increased cytotoxicity and treatment response in childhood lymphoblastic leukemia.24 Hyperdiploid ALLs are particularly efficient in transporting methotrexate and in producing polyglutamated species, factors that may contribute to their favorable prognosis.25 Polyglutamates are slowly degraded to their readily effluxed monoglutamate form by γ-glutamyl hydrolase, and a polymorphism (T127I) that deceases γ-glutamyl hydrolase activity is associated with enhanced polyglutamate accumulation in leukemic cells.26 Acquired resistance to methotrexate in patients with leukemia is associated with several different alterations: increased levels of DHFR as a consequence of gene amplification,13 defective polyglutamation,27 impaired drug uptake,28 or increased efflux by the MRP class of transporters.29
Structures of folate, tetrahydrofolate, and its analogue methotrexate. The vitamin is absorbed as a monoglutamate and converted intracellularly to a polyglutamate, in which form it is both physiologically active and is stored in cells. Methotrexate, the 2,4-diamino analogue of folic acid, is shown in the bottom panel and is also converted to a polyglutamate intracellularly. (Reproduced with permission from Brunton L, Chabner B, and Knollman B: Goodman & Gilman's The Pharmacological Basis of Therapeutics, 12th ed. New York, NY: McGraw-Hill; 2011.)
Mechanism of methotrexate action. Sites of enzyme inhibition by methotrexate and its polyglutamates (PG). AICAR, aminoimidazole-carboxamide ribonucleotide; dUMP, deoxyuridine monophosphate; FH2, dihydrofolate; FH4, tetrahydrofolate; GAR, glycine amide ribonucleotide.
Methotrexate is well absorbed when administered orally at low doses (5 to 10 mg/m2), but when doses exceed 30 mg/m2, absorption is variable. Consequently, doses greater than 25 mg/m2 should be administered parenterally.
The concentration of methotrexate in plasma declines in a polyexponential manner. A very rapid initial disposition phase persists for only a few minutes after intravenous administration. The intermediate disposition phase has a 2- to 4-hour half-life and continues for 12 to 24 hours after dosing. The terminal phase of drug decay is considerably slower, with an 8- to 10-hour half-life, and this phase becomes important in determining drug toxicity and the effectiveness of leucovorin rescue in patients treated with high-dose methotrexate. Methotrexate is primarily excreted unchanged by the kidney, while a minor fraction of the drug (7 to 30 percent) is inactivated by hepatic hydroxylation at the 7 position. Thus, doses should be reduced in proportion to the decrease in creatinine clearance (CrCl) in patients with renal impairment (CrCl <60 mL/min), because the prolonged exposure to high blood levels may result in life-threatening hematologic and gastrointestinal toxicity.30 High-dose methotrexate (>0.5 g/m2) followed by leucovorin rescue is used to treat patients with high-grade lymphoma, osteosarcoma, and ALL.
In ALL, dose adjustment of methotrexate to maintain a specific area under the concentration × time (C × T) curve improves treatment outcome.31 Patients receiving high-dose methotrexate can be rescued from drug toxicity by administering small doses of N-10-formyltetrahydrofolate (leucovorin), which replenishes the intracellular pool of reduced folates. Leucovorin is administered intravenously or orally in doses of 10 to 15 mg/m2 at 6-hour intervals, starting 6 to 24 hours after the infusion of methotrexate, and continuing until plasma concentrations of the drug fall below 1 μM. In patients receiving high-dose methotrexate, drug levels are routinely assayed 24 to 48 hours after dosing to determine the rate of drug elimination and the safety of discontinuing leucovorin. Both methotrexate and its hydroxylated metabolite are organic acids, which, like uric acid, are much more soluble in alkaline urine. In patients receiving such therapy, renal toxicity may result from intrarenal precipitation of the parent drug or its 7-OH metabolite, and is generally the primary cause of decreased drug clearance and overwhelming toxicity. Renal dysfunction can be prevented by alkalinizing the urine to pH 7 with intravenous sodium bicarbonate prior to and during therapy. Patients should be given intensive hydration, as well. If drug concentrations in plasma exceed 1 μM at 48 hours after high-dose therapy, leucovorin should be continued at higher doses of 50 to 100 mg/m2 every 6 hours until methotrexate concentrations fall below 0.1 μM. The higher doses of leucovorin are necessary to compete with methotrexate for transport and polyglutamation. In cases of extreme renal failure, with stable drug levels in the 10 μM range, leucovorin will not be effective. In this setting, continuous flow hemodialysis may provide a sustained reduction in drug levels.32 An alternative effective measure in this circumstance is the administration of glucarpidase, a commercially available bacterial enzyme that instantly degrades antifolates33 and prevents further toxicity.
The dose-limiting toxicities of methotrexate are myelosuppression and gastrointestinal toxicity. Toxic doses of methotrexate can induce thrombocytopenia and/or leukopenia, although leukopenia is more common. An early indication of methotrexate toxicity to the gastrointestinal tract is oral mucositis, whereas more severe toxicity may be manifested as diarrhea and gastrointestinal bleeding. Less-common toxic effects of methotrexate are skin rash (10 percent), pneumonitis, and chemical hepatitis. Transaminase elevations are frequently seen after high-dose methotrexate but rapidly return to normal in most patients, and without sequelae, but low-dose chronic administration, as employed to treat psoriasis or rheumatoid arthritis, may lead to portal fibrosis and cirrhosis.
Methotrexate, given intrathecally in doses of 12 mg every 4 days for children older than age 3 years and for adults, is used to prevent or treat meningeal leukemia and lymphoma. Dose adjustment is required for children younger than age 3 years, and should be made according to established protocols. Because the drug distributes poorly into the ventricular system after spinal injection, patients with active meningeal leukemia are frequently treated through an indwelling ventricular reservoir. Toxicities caused by intrathecal administration of methotrexate include acute arachnoiditis with nuchal rigidity and headache, as well as more chronic CNS toxicities, such as dementia, motor deficits, seizures, and coma.34 Rarely, these neurotoxicities develop hours after intrathecal drug administration, but more commonly they occur in the days or weeks after initiation of intrathecal treatment, and are most often seen in patients with active meningeal leukemia. Leucovorin is ineffective in reversing or preventing these toxicities. Patients with such signs should undergo evaluation to rule out progressive CNS tumor, and if malignancy is not found, intrathecal cytarabine should be used for further therapy.
Methotrexate and 6-mercaptopurine (6-MP) are synergistic in their inhibition of purine biosynthesis. L-Asparaginase, an inhibitor of protein synthesis, blocks cells from entering DNA synthesis and antagonizes the effects of methotrexate, when used before the antifolate. The two drugs are not used concurrently.
Nonsteroidal antiinflammatory drugs, which diminish renal blood flow, may reduce methotrexate clearance, as may nephrotoxic antibiotics and platinum derivatives, and these or other renal toxins should be avoided in patients during high-dose methotrexate.
CYTARABINE (CYTOSINE ARABINOSIDE, ARABINOSYL CYTOSINE, ARA-C)
Ara-C is an antimetabolite analogue of cytidine, differing in the configuration at the substituent on C2′ position of the sugar, in which the C2′-hydroxyl group is cis-oriented relative to the C1′-N-glycosyl bond, in contrast to the trans configuration of the ribose nucleoside. Ara-C is a mainstay in the induction of remission in patients with acute myelogenous leukemia (AML).
High doses (1 to 3 g/m2) of intravenous ara-C given at 12-hour intervals for 6 to 12 doses are more effective alone or in a combination with anthracyclines than conventional doses (100 to 150 mg/m2 q12h) in consolidation therapy of AML, and they confer particular benefit in patients with cytogenetic abnormalities (t[8:21], inv, t[9:16], and del) related to the core binding factor that regulates hematopoiesis.35 Other subsets of leukemia may have increased sensitivity to ara-C. ALL patients with mixed lineage leukemia (MLL) gene translocations have upregulation of the human equilibrative nucleoside transporter (hENT) and have a greater sensitivity to ara-C.36 AML patients with K-RAS gene mutations seem to derive greater benefit from high-dose ara-C than do patients with wild-type K-RAS in their tumors.37
Ara-C is converted to the nucleoside triphosphate, cytarabine triphosphate (ara-CTP) intracellularly. The first step is catalyzed by deoxycytidine kinase (dCK); polymorphisms of the dCK gene may affect the rate of activation, and ultimately response.38 Ara-CTP is an inhibitor of DNA polymerase and is also incorporated into DNA, where it terminates strand elongation.39 If repair is unsuccessful, apoptosis is initiated. Ara-C and its mononucleotide are deaminated and inactivated by two intracellular enzymes, cytidine deaminase and deoxycytidylate deaminase, respectively.
Acquired ara-C resistance in experimental leukemias consistently results from the loss of dCK.40 Other changes implicated in experimental tumors include decreased drug uptake because of decreased expression of the equilibrative nucleoside transporter, increased deamination, increased pool size of competitive deoxycytidine triphosphate, and inhibition of the apoptotic pathway. Some of these changes, particularly loss of dCK activity, have been reported in studies of human leukemia, but these results have not been confirmed in definitive trials.41
Ara-C is administered intravenously either as a bolus injection or, more commonly, as a continuous infusion. It is not orally bioavailable because of its degradation by cytidine deaminase, which is present in the gastrointestinal epithelium and liver. Two standard schedules of administration are used: (1) rapid infusion of 100 mg/m2 every 12 hours for 7 days; or (2) continuous infusion of 100 to 200 mg/m2 per day for up to 7 days. Ara-C distributes rapidly throughout total-body water and is eliminated from plasma with a biologic half-life of 7 to 20 minutes. Most of the dose is excreted as arabinosyluracil (ara-U), an inactive metabolite, which is formed in plasma, the liver, granulocytes, and other tissues. Inhibition of ara-C deamination by ara-U may be responsible for the prolongation of the biologic half-life of the drug as larger doses are administered.42 Single-bolus injections and short infusions (30 minutes to 1 hour duration) at doses as high as 5 g/m2 produce little myelotoxicity because of the drug’s rapid clearance, whereas continuous intravenous infusion of only 1 g/m2 over 48 hours produces severe marrow toxicity. High-dose ara-C (3 g/m2 q12h for 3 days on days 1, 3, and 5), is routinely used for consolidation therapy of AML, but lower doses of 1 gm/m2 or less should be used in patients older than 60 years to avoid CNS toxicity. Unlike most drugs, a relatively high concentration of ara-C is achieved in the cerebrospinal fluid after intravenous administration, and may approach 50 percent of the corresponding plasma concentration.
Ara-C is also used intrathecally to treat meningeal leukemia. Doses of 50 to 70 mg in adults are usually employed and afford cerebrospinal fluid levels of the drug near 1 mM, which decline with a half-life of 2 hours. Ara-C (50 mg given every 2 weeks) has been impregnated into a gel matrix, in a formulation called DepoCyt, for sustained release into the cerebrospinal fluid, thus avoiding the need for repeated spinal taps. Initial clinical results in spinal lymphomatous meningitis indicate that it has efficacy equal to that of methotrexate.43
The dose-limiting toxicity for conventional dosing regimens of intravenous ara-C, 100 to 150 mg/m2 per day for 5 to 10 days, is myelosuppression. Nausea and vomiting also occur at these doses, the severity of which increases markedly when higher doses are employed, although repeated administration of the drug results in some tolerance. The nadir of the white count and platelet count occurs at about days 7 to 10 after the last dose of drug. Cerebellar, gastrointestinal, and liver toxicity, as well as conjunctivitis have also been observed when high-dose regimens are used. Hepatotoxicity ranges from abnormalities in serum transaminase levels to frank jaundice. The severity of these effects increases as the duration of therapy is prolonged; however, toxic effects rapidly subside upon discontinuation of treatment. Pulmonary infiltrates as a result of noncardiogenic pulmonary edema, and occasionally associated with severe pulmonary dysfunction, occur in leukemic patients receiving ara-C, as do gastrointestinal ulcerations with bleeding and infrequently perforation. Ara-C treatment is also reported to predispose to Streptococcus viridans pneumonia.44
In patients older than 60 years of age, and in patients with renal dysfunction, intravenous high-dose ara-C (3 g/m2 every 12 hours, days 1, 3, and 5, for six doses) causes a high incidence of cerebellar toxicity, manifested as ataxia and slurred speech.45 Confusion and dementia may supervene, leading to a fatal outcome. Cerebellar toxicity is more frequent in patients with abnormal renal function because of slowed elimination of ara-U, with consequent inhibition of ara-C deamination. Intrathecal ara-C is usually well tolerated, but neurologic side effects have been reported (seizures, alterations in mental status).
Although primarily used for solid tumors, gemcitabine, a 2′-2′-difluoro analogue of deoxycytidine, has significant activity against Hodgkin lymphoma. Its mechanism of action is similar to ara-C, in that, as a triphosphate, it competes with deoxycytidine triphosphate for incorporation into the elongating DNA strand, where it terminates DNA synthesis. It is also self-potentiating in that at a second site of action, it inhibits ribonucleotide reductase and thereby reduces competitive pools of deoxycytidine triphosphate (dCTP). It achieves higher nucleotide levels in tumor cells than does ara-CTP, and has a longer intracellular half-life. Its clinical pharmacokinetics are determined primarily by its rapid deamination by cytidine deaminase, yielding a short plasma half-life (t1/2) of 15 to 30 minutes. Standard schedules use 1000 mg/m2 infused over 30 minutes, and produced peak drug concentrations of 20–60 μM in plasma. Longer infusion times may produce higher intracellular triphosphate concentrations, but the benefit is uncertain.46
Resistance in solid tumors arises from low expression of hENT, increased expression of ribonucleotide reductase, and low levels of the initial activating enzyme, dCK. Gemcitabine is an extremely potent radiosensitizer and should not be used concurrently with radiation therapy except in clinical trials.
Toxicities are acute myelosuppression, mild hepatic enzyme elevations, uncommonly a reversible pneumonitis, and with prolonged usage, a progressive hemolytic uremic syndrome with capillary leak, leading to pleural effusions, ascites, and renal failure.47
5-AZACYTIDINE AND 5-AZA-2′-DEOXYCYTIDINE
Both 5-azacytidine and decitabine (5-aza-2′-deoxycytidine), its closely related deoxy analogue, exhibit cytotoxic activity and also induce differentiation of malignant cells at low doses. The latter action results from their incorporation into DNA and their covalent inactivation of DNA methyltransferase. The resulting inhibition of methylation of cytosine bases in DNA leads to enhanced transcription of otherwise silent genes.48 The differentiating effects of 5-azacytidine are the basis for the induction of fetal hemoglobin synthesis in patients with sickle cell anemia and thalassemia53 and its approved use in low-dose therapy of myelodysplastic syndromes (MDS). The usual doses of 5-azacytidine are 75 mg/m2 subcutaneously or intravenously per day for 7 days, repeated every 28 days, whereas decitabine is used in doses of 20 mg intravenously every day for 5 days every 4 weeks. Responses become apparent in myelodysplasia after two to five courses.
5-Azacytidine and decitabine are rapidly deaminated to chemically unstable uridine metabolites that immediately degrade into inactive products. Pharmacologic activity results from phosphorylation of the parent compound by cytidine kinase (for 5-azacytidine) or dCK (for decitabine), with subsequent conversion to a triphosphate nucleotide that becomes incorporated into DNA. The primary clinical toxicities of both 5-azacytidine and decitabine49 include reversible myelosuppression, nausea and vomiting with higher doses, hepatic dysfunction, myalgia, and fever and rash. Resistance likely results from defects in drug activation or alternative mechanisms for gene silencing, such as histone methylation or acetylation.
Purine analogues (Fig. 22–3) occupy an important role in maintenance for childhood ALL, and in the past decade newer analogues have shown remarkable activity in chronic leukemias and small cell lymphomas. With methotrexate, 6-MP is a critical component in the maintenance phase of curative therapy of childhood ALL. Other purine analogues include azathioprine, a prodrug of 6-MP and potent immunosuppressive agent; allopurinol, an inhibitor of xanthine oxidase, useful in the prevention of uric acid nephropathy; 2-chlorodeoxyadenosine, effective in the treatment of hairy cell leukemia and other lymphoid malignancies; 6-thioguanine (6-TG), an infrequently used antileukemic agent; and fludarabine phosphate (2-fluoroara-adenosine monophosphate), an effective agent for chronic lymphocytic leukemia (CLL) and follicular lymphomas, and for suppression of graft-versus-host disease in transplantation. A new purine analogue, nelarabine, is an ara-guanine prodrug, with strong activity against T-cell diseases, including lymphoblastic leukemias and lymphomas.50 The basis for this T-cell sensitivity appears to be the resistance of arabinosylguanine (ara-G) to degradation by the catabolic enzyme, purine nucleoside phosphorylase. High levels of arabinosylguanine triphosphate (ara-GTP) accumulate in T-cell neoplasms, leading to Fas ligand-mediated apoptosis. The most recent addition, clofarabine, also an adenosine analogue, has notable activity against childhood ALL and adult AML. Deoxycoformycin, a potent inhibitor of adenosine deaminase, is also effective in the treatment of T-cell malignancies and hairy cell leukemia.
Mechanism of Action of 6-Thiopurines
Both 6-MP and 6-TG have a thiol group substituted for the 6-hydroxy group of hypoxanthine or guanine, respectively, and are converted to nucleotides by hypoxanthine guanine phosphoribosyltransferase. They block synthesis of purines. The nucleotides of both 6-MP and 6-TG are incorporated into DNA, where they become methylated and are recognized by the mismatch repair system. Attempts to correct miscoding lead to strand breaks and apoptosis.51 Cell death correlates with the extent of their incorporation into DNA. 6-MP has the added effect of inhibiting de novo purine synthesis through the action of its metabolite, methyl-thioinosine monophosphate.52
In experimental tumor cells, resistance to 6-MP is most commonly caused by decreased activity of hypoxanthine guanine phosphoribosyltransferase (HGPRT), by increased efflux by the transporter MRP-4, and by the absence of an effective mismatch repair process. Resistance in human leukemia is poorly understood, but is linked to HGPRT deficiency. Patients differ in their rates of metabolic clearance of 6-MP and in their ability to efflux 6-thiopurines from cells. Rapid systemic clearance of the drug, as mediated by methylation of the thiol group by 5-thiopurine-methyltransferase (TPMT),53 is associated with a high leukemia recurrence rate in ALL maintenance therapy. Low levels of red blood cell thiopurine nucleotides correlate with a high level of activity of TPMT (more often found in patients of African descent) and a high risk of clinical relapse in patients with ALL,54 whereas decreased expression of TPMT, resulting from an inherited polymorphism in the number of tandem repeats in the 5′ promoter region, is associated with increased drug toxicity. A commercial test for enzyme polymorphism, based on red cell enzyme activity or thioguanine nucleotide content, is available. A second polymorphism of significance involves the cellular efflux protein, MRP-4; an inactive variant is associated with high 6-TG nucleotide concentrations in cells, and may be responsible for great sensitivity of Japanese patients to 6-thiopurines, as the variant occurs in 18 percent of the Japanese population.55 A polymorphism affecting the inosine triphosphate pyrophosphorylase enzyme (rs41320251) responsible for degrading a thiopurine nucleotide intermediate is associated with increased methyl-mercaptopurine nucleotides and a high incidence of febrile neutropenia in children with ALL.56
Methotrexate and 6-MP are highly synergistic, possibly because methotrexate blocks the de novo synthesis of purines, elevates phosphoribosyl pyrophosphate (PRPP), and enhances the activation of 6-MP. 6-MP blocks warfarin anticoagulation in some patients, leading to a requirement for higher doses of warfarin in patients receiving chronic 6-MP therapy for immunosuppression.
Clinical Pharmacology of 6-Thiopurines
Both 6-TG and 6-MP are given orally at doses of 50 to 100 mg/m2 per day. Oral absorption of 6-MP is erratic, as only 16 to 50 percent of an oral dose is systemically available.57 Food and antibiotics may decrease absorption. Both 6-MP and 6-TG are inactivated by metabolism, and have half-lives of approximately 1 to 1.5 hour in plasma. Peak plasma levels of 6-MP occur 2 hours after administration and reach 1 to 2 μM. During 6-TG treatment, 6-TG nucleotides accumulate to much higher levels in leukemic cells, as compared to 6-MP. Inactive 6-thiomethyl nucleotides are almost 30-fold higher after 6-MP, than after 6-TG.58 6-MP is inactivated by metabolism to 6-thiouric acid, a reaction catalyzed by xanthine oxidase. Allopurinol inhibits the metabolic inactivation of 6-MP, but not of 6-TG. Therefore, it is generally recommended that dosages of orally administered 6-MP be reduced by 75 percent in patients receiving allopurinol. 6-TG is inactivated primarily by S-methylation, followed by oxidation and desulfuration, but a second pathway, mediated by guanase and xanthine oxidase, contributes to clearance. Dose reduction is not necessary when 6-TG and allopurinol are administered together.
Adverse Effects of 6-Thiopurines
Both 6-TG and 6-MP are myelotoxic, producing nadirs of white blood cells and platelets at 7 to 10 days after treatment.59 Moderate nausea and vomiting may also be observed. Patients may experience mild but rapidly reversible hepatotoxicity after treatment with either compound. Cirrhosis has occurred in some children with leukemia who are receiving long-term therapy with 6-MP. TPMT, which inactivates 6-thiopurines, occurs in several polymorphic forms that fail to metabolize the analogues. Approximately one person in 10 of the white population is heterozygous for ineffective polymorphic forms of the enzyme and will have significantly greater myelosuppression, whereas one patient in 300 is homozygous for the inactive forms, accumulates high concentrations of thioguanine nucleotides in both tumor and normal cells, and is at risk for overwhelming toxicity, even with greatly reduced doses of 6-MP.60
Other toxicities may include hypersensitivity reactions (fever, rash), interstitial pneumonitis; pancreatitis; opportunistic infection, and an increased incidence of AML in patients receiving chronic immunosuppressive treatment with 6-MP.
Originally synthesized as a deamination-resistant analogue of adenosine, fludarabine phosphate contains two important substitutions: a fluorine attached to the purine ring, which renders the drug resistant to deamination, and an arabinose sugar in place of deoxyribose, which leads to its pharmacologic activity as an inhibitor of DNA synthesis and ribonucleotide reductase. It has outstanding activity in CLL.61 It is strongly immunosuppressive, like the other purine analogues, and is frequently used for this purpose in nonmyeloablative allogeneic marrow transplantation62 and in the treatment of autoimmune diseases.
Activation of fludarabine phosphate requires removal of the phosphate group in plasma to allow cellular uptake by nucleoside transporters, and then intracellular rephosphorylation. Fludarabine is activated to the monophosphate level by dCK. The triphosphate inhibits DNA polymerase and becomes incorporated into both DNA and RNA.63 Its mechanism of cytotoxicity results from DNA chain termination and induction of apoptosis, although it also inhibits ribonucleotide reductase (RNR), a self-potentiating activity that decreases intracellular deoxyadenosine triphosphate (dATP) and increases fludarabine incorporation into DNA.64 Its triphosphate has a long intracellular half-life of 15 hours in CLL cells. Resistance has been ascribed to decreased active uptake, a deficiency of dCK, increased efflux, or increased RNR.
The drug is available in the United States as an intravenous preparation, and for oral use. It has 60 to 80 percent bioavailability. Because it is resistant to adenosine deaminase, fludarabine is eliminated primarily by renal excretion (60 percent), with a terminal half-life of 10 hours. For patients treated with fludarabine, the standard intravenous dose is 25 mg/m2 daily for 5 days, whereas the approved oral dose is 40 mg/m2 daily for 5 days. In patients with renal impairment, a 20 percent dose reduction for a CrCl of 17 to 40 mL/min/m2, and a 40 percent dose reduction for a CrCl less than 17 mL/min/m2 yields an area under the curve approximately equal to that seen in patients with normal renal function receiving full doses of fludarabine.65,66
When administered at these doses, fludarabine causes only moderate myelosuppression. In CLL patients, its antileukemic effect will lead to a progressive improvement in marrow function over a period of two to three cycles of treatment, with a median time to disease progression of 31 months. However, the drug also exerts cytotoxic effects against both B and T lymphocytes, lowering CD4 T-cell counts to 150 to 200 cells/μL and predisposing patients to opportunistic infections. In patients with a large tumor burden, rapid tumor lysis may rarely lead to hyperuricemia, renal failure, and hypocalcemia (tumor lysis syndrome).67 Thus, patients should be well hydrated and their urine alkalinized prior to beginning therapy. The primary acute toxicity is reversible myelosuppression. Peripheral sensory and motor neuropathy may occur during standard-dose therapy; autoimmune phenomena, including prolonged hypothyroidism, neutropenia and hemolytic anemia with both warm and cold antibodies, have been reported.68 Approximately 10 percent of CLL patients receiving fludarabine may develop a hypersensitivity syndrome of pulmonary infiltrates, hypoxemia, and fever, responsive to glucocorticoids.69 Myelodysplasia and acute leukemias, with chromosome 7p deletions, have been reported as infrequent late complications.70
CLADRIBINE (2-CHLORODEOXYADENOSINE, 2-CDA)
The extreme sensitivity of normal and malignant lymphocytes to deamination-resistant purine analogues is further exemplified by the potent activity of cladribine in hairy cell leukemia, CLL, and low-grade lymphomas.71 A single course of cladribine, typically 0.09 mg/kg per day for 7 days by continuous intravenous infusion, induces complete response in 80 percent of patients with hairy cell leukemia. Administration by subcutaneous injection or by 2-hour intravenous infusion daily for 5 days to the same total dose achieves similar results. The drug has much the same intracellular fate as fludarabine, undergoing phosphorylation by dCK and further conversion to a triphosphate that becomes incorporated into DNA. The triphosphate of cladribine has a long intracellular half-life of 9.7 hours in CLL cells isolated from patients treated with the drug.72 The triphosphate has multiple metabolic effects, disrupting oxidative phosphorylation in mitochondria, inhibiting RNR and depleting nicotinamide adenine dinucleotide levels in tumor cells. All of these actions may explain the drug’s toxicity to slowly dividing lymphoid malignancies such as hairy cell leukemia and CLL. The actual mechanisms by which cladribine induces DNA strand breaks are not completely understood. However, similar to fludarabine, it inhibits DNA chain extension and daughter strand synthesis.73 Furthermore, the drug’s inhibition of RNR lowers levels of the competitive dATP. The cumulative effects of cladribine induce apoptosis (programmed cell death).
Cladribine is eliminated primarily (>50 percent) by renal excretion, with a terminal plasma half-life of 7 hours. In a patient with renal failure, continuous flow hemodialysis effectively cleared the drug and prevented serious myelosuppression.74 Cladribine retains effectiveness in at least a fraction of hairy cell leukemia patients resistant to deoxycoformycin or fludarabine, although clinical experience with sequential use of these drugs is limited. Toxicities of cladribine include transient myelosuppression, fever, tumor lysis syndrome, and occasional opportunistic infections possibly related to immunosuppression. The development of cumulative thrombocytopenia during treatment with repeated courses of the drug may limit its use. Resistance develops in experimental tumors through decreased uptake, loss of the activating enzyme dCK, increased RNR activity, increased efflux,75 or by induction of 5′-nucleotidase activity.
This analogue has halogen substitutions on both the purine ring and arabinose sugar, resulting in a ready uptake and activation, to a highly stable intracellular triphosphate (half-life of 24 hours), which terminates DNA synthesis, inhibits RNR, and induces apoptosis. The usual adult dose is 52 mg/m2 given as a 2-hour infusion daily for 5 days. Clofarabine has a plasma half-life of 6.5 hours. The primary route of clearance is through renal excretion, and dose adjustment according to CrCl is recommended for patients with abnormal renal function.
Toxicities are myelosuppression; uncommonly, fever, hypotension, and pulmonary edema, suggestive of capillary leak caused by cytokine release; hepatic transaminitis; hypokalemia; and hypophosphatemia. As a single agent, the drug is well tolerated as second-line treatment for AML patients with remission rates of 30 percent.76
A guanine nucleoside analogue, nelarabine has useful activity as a secondary agent for T-cell lymphoblastic lymphoma and acute T-cell leukemias. Its mode of action is similar to the other purine analogues, in that it becomes incorporated into DNA and terminates DNA synthesis. Its selective action for T cells may relate to the ability of T cells to activate purine nucleosides and the lack of susceptibility of this drug to purine nucleoside phosphorylase, a degradative reaction.
Usual doses are an intravenous 2-hour infusion of 1500 mg/m2 for adults on days 1, 3, and 5, and a lower dose of 650 mg/m2 per day for 5 days for children. The drug is rapidly demethylated by adenosine deaminase after administration, yielding the ara-G, which is cleared by hydrolysis and has a longer plasma half-life of 3 hours. ara-G is converted intracellularly to its triphosphate77 which becomes incorporated into DNA. The primary toxicities are myelosuppression and abnormal liver function tests, but the drug may cause a spectrum of neurologic abnormalities, including seizures, delirium, somnolence, and the Guillain-Barré syndrome of ascending paralysis.
Pentostatin contains a unique seven-carbon primary ring system that closely resembles the transition-state intermediate of the adenosine deaminase reaction. As such, pentostatin is a potent inhibitor of the enzyme, leading to accumulation of intracellular adenosine and deoxyadenosine nucleotides. In addition, the triphosphate of pentostatin is incorporated into DNA. The imbalance in purine nucleotide pools produced by pentostatin probably accounts for its cytotoxicity.
Although initial trials of pentostatin demonstrated striking renal and neurologic toxicities at doses of 10 mg/m2 intravenously per day or greater, lower doses (4 mg/m2 biweekly) are extremely effective in inducing pathologically confirmed complete responses in hairy cell leukemia. At this lower dose, severe depletion of normal T cells occurs and may predispose to opportunistic infection.78 The optimal dose may be lower than 4 mg/m2 biweekly. The drug is eliminated entirely by renal excretion, necessitating proportional dose reduction in patients with reduced CrCl.
RIBONUCLEOTIDE REDUCTASE INHIBITOR: HYDROXYUREA
Hydroxyurea inhibits RNR, the enzyme that converts ribonucleotide diphosphates to deoxyribonucleotides. It chelates iron, an essential cofactor in the RNR reaction. In malignant disease, hydroxyurea is most commonly used for treating polycythemia vera, essential thrombocythemia, and the chronic phase of CML and to lower the myeloblast count in patients presenting with AML or blastic crisis of CML. It has also become the standard agent for preventing painful crisis and reducing hospitalization in patients with sickle cell disease and in thalassemia patients with hemoglobin (Hgb) C/SS. Its antisickling activity results from induction of Hgb F through its activation of a specific promoter for the γ-globin gene. It may also exert antisickling activity and decrease occlusion of small vessels through its generation of nitric oxide, a vasodilator, and through decreased expression of adhesion molecules such as L-selectin, on neutrophils.79 Resistance occurs in experimental tumors as a consequence of amplification of the catalytic subunit of RNR or through mutations in RNR that lower affinity for the enzyme.
Hydroxyurea is well absorbed orally, even when large doses such as 50 to 75 mg/kg orally are given for rapid lowering of the white blood cell count. In chronic therapy of myeloproliferative neoplasm, starting doses of 15 mg/kg orally are adjusted upward or downward based on neutrophil counts. In managing patients with sickle cell disease, neutrophils should be maintained above 2000 per mL.80 Hydroxyurea may also be given intravenously to rapidly lower the white blood cell count in patients with extreme leukemic leukocytosis or thrombocytosis. Peak plasma levels following oral administration are achieved at about 1 hour and decline with a half-life of 3 to 4 hours thereafter. Renal excretion is the major route of drug elimination, and doses should be decreased in proportion to the deficit in CrCl.
The major toxicities of hydroxyurea are leukopenia and the induction of megaloblastic changes. Nausea, drug fever, pneumonitis, maculopapular skin rash, and painful leg ulcers have been observed with this drug, although it is generally well tolerated. Hydroxyurea, like ara-C, is an S-phase–specific agent. Accordingly, single large doses cause little toxicity other than myelosuppression. The nadir of the leukocyte count occurs 3 to 5 days after a single dose of drug, and the leukocyte count recovers rapidly. It is a potent teratogen and should not be used in women of childbearing age. Its potential to cause leukemic transformation is uncertain, but small cases series suggest this may occur in patients with a myeloproliferative neoplasm.81