David P. Ryan
Bruce A. Chabner
Nucleoside analogs compete with their physiologic counterparts for incorporation into nucleic acids and have earned an important place in the treatment of acute leukemia. The most important of these are the arabinose nucleosides, a unique class of antimetabolites originally isolated from the sponge Cryptothethya crypta1 but now produced synthetically.2 They differ from the physiologic deoxyribonucleosides by the presence of a 2′-OH group in the cis configuration relative to the N-glycosyl bond between cytosine and the arabinose sugar (Fig. 8.1). Several arabinose nucleosides have useful antitumor and antiviral effects. The most active cytotoxic agent of this class is cytosine arabinoside (ara-C, cytarabine). A related nucleoside, adenine arabinoside, has antitumor and antiviral action,3 and its analog, 2-fluoro-ara-adenosine monophosphate, has strong activity in lymphomas and in chronic lymphocytic leukemia.4 Another member of the group is arabinosyl-5-azacytidine, a synthetic analog that failed in the clinic.5
Ara-C is one of the most effective agents in the treatment of acute myelogenous leukemia6 and is incorporated into virtually all standard induction regimens for this disease, generally in combination with an anthracycline (daunorubicin hydrochloride or idarubicin hydrochloride). Ara-C is also a component of consolidation and maintenance regimens in acute myelogenous leukemia after remission is attained. Clear clinical evidence now exists that a dose-response effect is present for ara-C, both as induction7 and consolidation8 therapy in acute myelogenous leukemia. High-dose ara-C confers particular benefit in patients with certain cytogenetic abnormalities related to the core binding factor that regulates hematopoiesis (t8:21, inv 16, del 16, t16:16)9 (Table 8.1). Ara-C is also active against other hematologic malignancies, including non- Hodgkin's lymphoma,10 acute lymphoblastic leukemia,11 and chronic myelogenous leukemia,12 but has little activity as a single agent against solid tumors. This limited spectrum of activity has been attributed to the lack of metabolic activation of this agent in solid tumors and its selective action against rapidly dividing cells. The essential features of ara-C pharmacology are described in Table 8.2.
Mechanism of Action
Ara-C acts as an analog of deoxycytidine and has multiple effects on DNA synthesis. Ara-C undergoes phosphorylation to form arabinosylcytosine triphosphate (ara-CTP), which competitively inhibits DNA polymerase α in opposition to the normal substrate deoxycytidine 5′-triphosphate (dCTP).13 This competitive inhibition has been demonstrated with crude DNA polymerase from calf thymus13 and with purified enzyme from human leukemic cells,14 as well as with enzyme from a variety of murine tumors.15, 16 Ara-CTP has an affinity for human leukemia cell DNA polymerase α in the range of 1 × 10-6 mol/L, and the inhibition is reversible in cell-free systems by the addition of dCTP or in intact cells by the addition of deoxycytidine, the precursor of dCTP.17 When present at high intracellular concentrations, ara-CTP also inhibits DNA polymerase β.18 The effects of ara-C on DNA polymerase activity extend not only to semiconservative DNA replication but also to DNA repair. Repair of ultraviolet light damage to DNA, a function that depends on polymerase α, is blocked more potently than the repair of photon-induced or γ radiation-induced strand breaks,19, 20 the repair of which is accomplished by a different polymerase. In addition to having an effect on eukaryotic DNA polymerases, ara-CTP is a potent inhibitor of viral RNA-directed DNA polymerase (Ki [inhibition constant] = 0.1 µmol/L).
Figure 8.1 Structure of cytidine analogs.
More important than the effects of ara-C on DNA synthesis, however, is its incorporation into DNA, a feature that correlates closely with cytotoxicity21, 22 (Fig. 8.2). In fact, a preponderance of evidence suggests that this is the major cytotoxic lesion in ara-C–treated cells. Drugs that prevent ara-C incorporation into DNA, such as aphidicolin, also block its cytotoxicity.23 A given level of ara-C incorporation can be achieved by various combinations of concentrations (C) and times (T) of exposure that yield a specific C × T product. A linear relationship exists between picomoles of ara-C incorporated and the log of cell survival for a wide range of drug concentrations and durations of exposure. Thus, drug toxicity is a direct function of incorporation into DNA, and the latter varies directly with the C × T product.24 Once it is incorporated into DNA, tumor cells excise ara-C slowly,25 and the incorporated ara-C inhibits template function and chain elongation.23, 26, 27 In experiments with purified enzyme and calf thymus DNA, the consecutive incorporation of two ara-C or two arabinosyl-5-azacytidine (ara-5–aza-C) residues effectively stopped chain elongation by DNA polymerase α.14 At high concentrations of ara-C, one finds a greater than expected proportion of ara-C residues at the 3′-terminus, which confirms a direct effect on chain termination.25 These observations support the hypothesis that ara-C incorporation into DNA is a prerequisite for drug action and is responsible for cytotoxicity.
TABLE 8.1 COMPLETE REMISSION (CR) DURATION BY CYTOGENETIC GROUP ACCORDING TO CYTOSINE ARABINOSIDE (ARA-C) DOSE RANDOMIZATION
Ara-C also causes an unusual reiteration of DNA segments.28 Human lymphocytes exposed to ara-C in culture synthesize small reduplicated segments of DNA, which results in multiple copies of limited portions of DNA. These reduplicated segments increase the possibility of recombination, crossover, and gene amplification; gaps and breaks are observed in karyotype preparations after ara-C treatment. The same mechanism, reiteration of DNA synthesis after its inhibition by an antimetabolite, may explain the high frequency of gene reduplication induced by methotrexate sodium, 5-fluorouracil, and hydroxyurea (see Chapters 6, 7, and 10). In summary, although ara-C has multiple effects on DNA synthesis, the most important effect seems to be its incorporation into DNA.
TABLE 8.2 KEY FEATURES OF CYTOSINE ARABINOSIDE (ARA-C) PHARMACOLOGY
Figure 8.2 Relationship between acute myelogenous leukemia blast clonogenic survival and incorporation of tritium-labeled cytosine arabinoside (ara-C) in DNA at ara-C concentrations of 10-7 mol/L (▲), 10-6 mol/L (●), 10-5 mol/L (▪), and 10-4 mol/L (○) during periods of 1, 3, 6, 12, and 24 hours. (From Kufe DW, Spriggs DR. Biochemical and cellular pharmacology of cytosine arabinoside. Semin Oncol 1985;12:34.)
Other biochemical actions of ara-C have been described, including inhibition of ribonucleotide reductase29 and formation of ara-CDP-choline, an analog of cytidine 5′- diphosphocholine (CDP-choline) that inhibits synthesis of membrane glycoproteins and glycolipids.30 Ara-C also has the interesting property of promoting differentiation of leukemic cells in tissue culture, an effect that is accompanied by decreased c-myc oncogene expression.31, 32 These changes in morphology and oncogene expression occur at concentrations above the threshold for cytotoxicity and may simply represent terminal injury of cells. Molecular analysis of clinical bone marrow samples from patients in remission has revealed persistence of leukemic markers,33 which suggests that differentiation may have occurred in response to ara-C in clinical use.
The molecular mechanism of cell death after ara-C exposure is unclear. Both normal and malignant cells undergo apoptosis in experimental models.34, 35 A complex system of interacting transduction signals ultimately determines whether a cell exposed to a cytotoxic agent is destined to die. Exposure of leukemic cells to ara-C stimulates the formation of ceramide, a potent inducer of apoptosis.36 On the other hand, an increase in protein kinase C (PKC) activity is observed in leukemic cells in response to ara-C in vitro.37 This is thought to be the result of ara-C induction of diacylglycerol, which in turn induces PKC activity. Because PKC activation is known to oppose apoptosis in hematopoietic cells, the lethal actions of ara-C may depend, at least partially, on its relative effects on the PKC and sphingomyelin pathways. Transcriptional regulation of gene expression is another key mechanism through which the growth and differentiation of mammalian cells are controlled. The induction of some transcription factors, such as AP-1 (a dimer of jun-fos or jun-jun proteins) and NF-kB, has been temporally associated with ara-C–induced apoptosis.38, 39 Whether increased expression of these transcription factors plays a direct role in the molecular signaling that leads to anticancer drug-induced programmed cell death is not clear. The ability of PKC inhibitors to promote ara-C–induced apoptosis despite antagonizing c-jun up-regulation illustrates the fact that apoptosis can occur by a mechanism that does not involve the induction of c-jun expression.40 Some have also reported that induction of pRb phosphatase activity by DNA-damaging drugs, including ara-C, is at least one of the mechanisms responsible for p53-independent, Rb-mediated G1 arrest and apoptosis.41 The resulting hypophosphorylated pRb binds to and inactivates the E2F transcription factor, which inhibits the transcription of numerous genes involved in cell-cycle progression.42
Figure 8.3 Correlation between accumulation of arabinosylcytosine triphosphate (ara-CTP) and nucleoside transport capacity measured by the maximal number of nitrobenzylthioinosine (NBMPR) binding sites on leukemic cells (r = 0.87; P <.0001). Ara-CTP accumulation was measured after incubation of cells with 1 µmol/L of tritium-labeled cytosine arabinoside for 60 minutes. (● acute myelogenous leukemia; ○ non-T-cell acute lymphoblastic leukemia; ▲, T-cell leukemia/lymphoma, lymphoblastic leukemia;▪, acute undifferentiated leukemia; □, chronic lymphocytic leukemia. (From Wiley JS, Taupin J, Jamieson GP, et al. Cytosine arabinoside transport and metabolism in acute leukemias and T-cell lymphoblastic lymphoma. J Clin Invest 1985;75:632.)
Cellular Pharmacology and Metabolism
Ara-C penetrates cells by a carrier-mediated process shared by physiologic nucleosides.43, 44 Several different classes of transporters for nucleosides have been identified in mammalian cells45; the most extensively characterized in human tumors is hENT1, the equilibrative transporter, identified by its binding to nitrobenzylthioinosine (NBMPR). The number of transport sites on the cell membrane is greater in acute myelocytic leukemia than in acute lymphocytic leukemia cells and can be enumerated by incubation of cells with NBMPR. The hENT1 transporter is highly up-regulated in biphenotypic leukemia associated with the 11q23 MLL gene (4:11) translocation.45a A steady-state level of intracellular drug is achieved within 90 seconds at 37°C. Studies of Wiley et al.44, 46and others45, 47, 48 suggest that the NBMPR transporter plays a limiting role in the action of this agent in that the formation of the ultimate toxic metabolite ara-CTP is strongly correlated with the number of transporter sites on leukemic cells46 (Fig. 8.3). At drug concentrations above 10 µmol/L, the transport process becomes saturated, and further entry takes place by passive diffusion.49 hENT1 is strongly inhibited by various receptor tyrosine kinase inhibitors, an interaction that could limit ara-C use with targeted drugs.49a
As shown in Figure 8.4, ara-C must be converted to its active form, ara-CTP, through the sequential action of three enzymes: (a) deoxycytidine (CdR) kinase, (b) deoxycytidine monophosphate (dCMP) kinase, and (c) nucleoside diphosphate (NDP) kinase. Ara-C is subject to degradation by cytidine deaminase, forming the inactive product uracil arabinoside (ara-U); arabinosylcytosine monophosphate (ara-CMP) is likewise degraded by a second enzyme, dCMP deaminase, to the inactive arabinosyluracil monophosphate (ara-UMP). Each of these enzymes, with the exception of NDP kinase, has been examined in detail because of its possible relevance to ara-C resistance.
The first activating enzyme, CdR kinase, is found in lowest concentration (Table 8.3) and is believed to be rate-limiting in the process of ara-CTP formation. The enzyme is a 30.5-kd protein that phosphorylates deoxycytidine, deoxyguanosine, deoxyadenosine, ara-C, dideoxycytidine, fludarabine, gemcitabine, and other cytidine and purine analogs. The complementary DNA (cDNA) coding for CdR kinase has been cloned as well as cDNAs with specific mutations that lead to ara-C resistance in experimental cells.50, 51 The rate-limiting role of CdR kinase in ara-C activation is illustrated by the fact that transfection of malignant cell lines with retroviral vectors containing CdR kinase cDNA substantially increases their susceptibility to ara-C, 2-chloro-2′-deoxyadenosine, 2-fluoro-9-β-D-arabinofuranosyladenine, and less potently to gemcitabine.52 Moreover, some investigators have demonstrated higher ara-C cytotoxicity in intradermal and intracerebral gliomas transduced with CdR kinase in rat models than in the same tumor models with no CdR kinase transduction.53 This transduction of genes that sensitize tumor cells to prodrugs in vivo represents a potential strategy for cancer gene therapy.
Figure 8.4 Metabolism of cytosine arabinoside (ara-C) by tumor cells. The conversion of arabinosyluracil monophosphate (ara-UMP) to a triphosphate has not been demonstrated in mammalian cells. ara-CMP, arabinosylcytosine monophosphate; ara-CDP, arabinosylcytosine diphosphate; ara-CTP, arabinosylcytosine triphosphate; ara-U, uracil arabinoside; dCMP, deoxycytidine monophosphate; NDP, nucleoside diphosphate.
CdR kinase activity is highest during the S phase of the cell cycle.54 The Km, or affinity constant, for ara-C is 20 µmol/L, compared with the higher affinity or 7.8 µmol/L for the physiologic substrate CdR.55 This enzyme is strongly inhibited by dCTP but weakly inhibited by ara-CTP. This lack of “feedback” inhibition allows accumulation of the ara-C nucleotide to higher concentrations. Protein kinase C-α, the activity of which is increased after ara-C exposure, has been implicated in phosphorylation of deoxycytidine kinase, increasing its overall activity at concentrations of substrate greater than the Km. This observation raises the possibility that ara-C at high doses may potentiate its own metabolism by induction of the PKC activator diacylglycerol.56
The second activating enzyme, dCMP kinase,57 is found in several hundred-fold higher concentration than CdR kinase. Its affinity for ara-CMP is low (Km = 680 µmol/L) but greater than the affinity for the competitive physiologic substrate dCMP. Because of its relatively poor affinity for ara-CMP, this enzyme could become rate-limiting at low ara-C concentrations. The third activating enzyme, the diphosphate kinase, appears not to be rate-limiting because the intracellular pool of arabinosylcytosine diphosphate (ara-CDP) is only a fraction of the ara-CTP pool.58
TABLE 8.3 KINETIC PARAMETERS OF CYTOSINE ARABINOSIDE (ARA-C) METABOLIZING ENZYMES
Opposing the activation pathway are two deaminases found in high concentration in some tumor cells as well as normal tissues. Cytidine deaminase is widely distributed in mammalian tissues, including intestinal mucosa, liver, and granulocytes.59, 60, 61, 62 It is found in granulocyte precursors and in leukemic myeloblasts in lower concentrations than in mature granulocytes, but even in these immature cells the deaminase level exceeds the activity of CdR kinase, the initial activating enzyme.55, 61 The second degradative enzyme, dCMP deaminase (Fig. 8.4), regulates the flow of physiologic nucleotides from the dCMP pool into the deoxyuridine monophosphate pool that is ultimately converted to deoxythymidine 5′-phosphate (dTMP) by thymidylate synthase.63 The enzyme dCMP deaminase is strongly activated by intracellular dCTP (Km = 0.2 µmol/L) and strongly inhibited by deoxythymidine triphosphate in concentrations of 0.2 µmol/L or greater. Ara-CTP weakly activates this enzyme (Km = 40 µmol/L)64 and thus would not promote degradation of its own precursor nucleotide, ara-CMP. The affinity of dCMP deaminase for ara-CMP is somewhat higher than that of dCMP kinase for the same substrate, but the activity of these competitive enzymes depends greatly on their degree of activation or inhibition by regulatory triphosphates (dCTP), and dCMP deaminase concentration in leukemic myeloblasts is slightly less than that of dCMP kinase (Table 8.3).
The balance between activating and degrading enzymes thus is crucial in determining the quantity of drug converted to the active intermediate, ara-CTP. This enzymatic balance varies greatly among cell types.55 Kinase activity is higher and deaminase activity lower in lymphoid leukemia than in acute myeloblastic leukemia. Enzyme activities vary also with cell maturity; deaminase increases dramatically with maturation of granulocyte precursors, whereas kinase activity decreases correspondingly.61 Thus admixture of normal granulocyte precursors with leukemic cells in human bone marrow samples complicates the interpretation of enzyme measurements unless normal and leukemic cells are separated. In general, cytidine deaminase (D) activity greatly exceeds kinase (K) (the kinase:deaminase ratio averages 0.03) in human acute myeloblastic leukemia, whereas the enzyme activities are approximately equal in acute lymphoblastic leukemia and Burkitt's lymphoma. Thus, the biochemical setting seems to favor drug activation by lymphoblastic leukemia cells if these initial enzymes play a rate-limiting role.
In fact, this may not be the case. Chou et al.58 found that human acute myeloblastic leukemia cells formed 12.8 ng of ara-CTP per 106 cells after 45 minutes of incubation with 1 × 10-5 mol/L ara-C. Acute lymphoblastic leukemia cells formed less ara-CTP, 6.3 ng/106 cells, and as expected, the more mature chronic myelocytic and chronic lymphocytic leukemia cells formed lesser amounts of ara-CTP (4.7 to 5.2 ng/106 cells). From this study and others,46, 48 the likelihood is that other factors, such as transport across the cell membrane, may limit ara-CTP formation.
In addition to its activation to ara-CTP, ara-C is converted intracellularly to ara-CDP-choline,65 an analog of the physiologic CDP-choline lipid precursor. However, ara-C does not inhibit incorporation of choline into phospholipids of normal or transformed hamster embryo fibroblasts.30 Ara-CMP does inhibit the transfer of galactose, N-acetylglucosamine, and sialic acid to cell surface glycoproteins. Further, ara-CTP inhibits the synthesis of cytidine monophosphate–acetylneuraminic acid, an essential substrate in sialylation of glycoproteins, although high ara-CTP concentrations (0.1 to 1 mmol/L) are needed to produce this effect.66 Thus, ara-C treatment could alter membrane structure, antigenicity, and function.
Biochemical Determinants of Cytosine Arabinoside Resistance
The foregoing consideration of ara-C metabolism and transport makes it clear that a number of factors could affect ara-C response. Not surprisingly, many of these factors have been implicated in various preclinical models of ara-C resistance. The most frequent abnormality found in resistant leukemic cells recovered from mice treated with ara-C has been decreased activity of CdR kinase.67, 68 In cultured cells exposed to a mutagen and then to low concentrations of ara-C, some single-step mutants developed high-level resistance to ara-C through loss of activity of CdR kinase, whereas other resistant clones exhibited markedly expanded dCTP pools, presumably through increased cytidine-5′-triphosphate (CTP) synthetase activity or through deficiency of dCMP deaminase.69, 70, 71, 72As mentioned previously, specific mutations and deletions in the CdR kinase coding cDNAs derived from resistant cells have been described by Owens et al.51
The role of cytidine deaminase in experimental models of resistance is less clear. Retrovirus-mediated transfer of the cytidine deaminase cDNA into 3T3 murine fibroblast cells significantly increases drug resistance to ara-C and other nucleoside analogs such as 5-aza-2′-deoxycytidine and gemcitabine. This phenotype of increased cytidine deaminase activity and drug resistance is reversed by the cytidine deaminase inhibitor tetrahydrouridine.73 Other genes, including proto-oncogenes, may affect ara-C response. Transfection of rodent fibroblasts and human mammary HBL 100 cells with c-H-ras conferred resistance to ara-C, an event attributed to decreased activity of CdR kinase.74 On the other hand, N-ras or K-ras mutations strongly correlated with increased ara-C sensitivity in the screening of human tumor cell lines from the National Cancer Institute's in vitro drug screen.75
Although various metabolic lesions have been implicated as causing ara-C resistance in animals, their relevance to resistance in human leukemia is less certain. Studies have described specific biochemical changes in drug-resistant cells from patients with leukemia, including deletion of CdR kinase,76 increased cytidine deaminase,77 a decreased number of nucleoside transport sites,46 and increased dCTP pools.78 Other clinical investigators have not been able to correlate resistance with either CdR kinase or cytidine deaminase,79, 80 but with the exception of Wiley et al.,46 who correlated clinical response with in vitro transport, few have examined transport. All studies have shown extreme variability in enzyme levels among patients with acute myelocytic or lymphocytic leukemia (Fig. 8.5). Thus, no agreement exists as to the specific changes responsible for resistance in human leukemia.
Although specific biochemical lesions associated with resistance in humans are unclear, the current understanding of ara-C action suggests that the ultimate formation of ara-CTP and the duration of its persistence in leukemic cells determine response.58, 81 Chou et al.58 found greater ara-CTP formation in leukemic cells of responders when these cells were incubated in vitro with ara-C, but in other series of patients no correlation was seen between remission induction or duration of complete remission and ara-CTP formation.82, 83, 84
Preisler et al.85 found a strong correlation between duration of remission and the ability of cells to retain ara-CTP in vitro after removal of ara-C from the medium (Table 8.4). Attempts to monitor ara-CTP formation in leukemic cells taken from patients during therapy have yielded useful information on rates of nucleotide formation and disappearance (the intracellular ara-CTP half-life is approximately 3 hours) but have not disclosed useful correlations of ara-CTP levels with response.84, 86 Again, considerable variability has been observed in the rates of formation of ara-CTP, and this rate does not correlate well with plasma ara-C concentrations in individual patients (Fig. 8.6).
Figure 8.5 Response as a function of deoxycytidine kinase (K) and cytidine deaminase (D) activities and their ratio in patients with acute myelogenous leukemia. ara-C, cytosine arabinoside; K/D, kinase/deaminase ratio. (From Chang P, Wiernik PH, Reich SD, et al. Prediction of response to cytosine arabinoside and daunorubicin in acute nonlymphocytic leukemia. In: Mandelli F, ed. Therapy of Acute Leukemias: Proceedings of the Second International Symposium, Rome, 1977. Rome: Lombardo Editore, 1979:148.)
Although specific steps in ara-C activation and degradation exert a strong influence on its ultimate action, the cellular response to ara-C–mediated DNA damage also governs whether the genotoxic insult results in cell death. In this sense, overexpression of Bcl-2 and Bcl-XL in leukemic blasts have been associated with in vitro resistance to ara-C–mediated apoptosis.87 The intracellular metabolism of ara-C and its initial effects on DNA are not modified by Bcl-2 expression, which suggests that Bcl-2 primarily regulates the more distal steps in the ara-C–induced cell death pathway. Although the precise mechanism by which these proteins prevent ara-C–induced cytotoxicity remains to be elucidated, Bcl-2 and Bcl-XL have been shown to antagonize ara-C–mediated cell death by a mechanism that prevents the activation of Caenorhabditis elegans deathlike proteases, such as Yama/CPP32 protease, which are involved in the execution of apoptosis.87 The fact that antisense oligonucleotides directed against Bcl-2 increase the susceptibility of leukemic blasts to ara-C–induced apoptosis in vitro,88 and that patients whose blasts express high levels of Bcl-2 respond poorly to ara-C–containing regimens,89 further illustrates the potential role of Bcl-2 in ara-C resistance. Exceptions are seen, however, in which even high levels of Bcl-2 expression apparently fail to prevent cell death.
TABLE 8.4 CORRELATION OF IN VITRO ARA-CTP POOLS AND RETENTION OF ARA-CTP 4 HOURS AFTER DRUG REMOVAL WITH DURATION OF COMPLETE RESPONSE OF PREVIOUSLY UNTREATED PATIENTS WITH ACUTE NONLYMPHOCYTIC LEUKEMIAa
Phosphorylation of apoptotic or DNA damage response factors may also determine the outcome of ara-C exposure. Studies have shown that phosphorylation of Bcl-2 is required for its antiapoptotic function, and a functional role for PKC-α in Bcl-2 phosphorylation and suppression of apoptosis has been postulated,90although this observation has not been confirmed by others.91 Altered phosphorylation of transcription factors also influences the cellular response to ara-C toxic insult. Ara-C–induced activation of PKC and mitogen-activated protein kinase (MAPK) has been reported to increase c-jun expression and phosphorylation,37, 92 and hyperphosphorylation of the AP-1 transcription factor has been associated with ara-C resistance in human myeloid leukemic cell lines in vitro.93
Clinical studies of determinants of ara-C response are complicated by the fact that ara-C is almost always given in combination with an anthracycline or an anthraquinone. Thus, a complete response or long remission duration does not necessarily imply sensitivity to ara-C. A lack of response does imply resistance to both agents in the combination, except for the not-infrequent cases in which failure can be attributed to infection or inability to administer full dosages of drug. With these limitations, the duration of complete response is probably the most appropriate and most important single yardstick of drug sensitivity because it reflects the fractional cell kill during induction therapy.
Figure 8.6 Pharmacokinetics of arabinosylcytosine triphosphate (ara-CTP) in leukemia cells and of cytosine arabinoside (ara-C) in plasma. Blood samples were drawn at the indicated times during and after infusion of ara-C, 3 g/m2, to patients with acute leukemia in relapse. Symbols for each analysis are the same for individual patients. (From Plunkett W, Liliemark JO, Estey E, et al. Saturation of ara-CTP accumulation during high-dose ara-C therapy: pharmacologic rationale for intermediate- dose ara-C. Semin Oncol 1987;14[2(Suppl 1)]:159.)
Cell Kinetics and Cytosine Arabinoside Cytotoxicity
In addition to biochemical factors that determine response, cell kinetic properties exert an important influence on the results of ara-C treatment. As an inhibitor of DNA synthesis, ara-C has its greatest cytotoxic effects during the S phase of the cell cycle,94 perhaps because of the requirement for its incorporation into DNA and the greater activity of anabolic enzymes during S phase. The duration of exposure of cells to ara-C is directly correlated with cell kill because the longer exposure period allows ara-C to be incorporated into the DNA of a greater percentage of cells as they pass through S phase (Fig. 8.7). The cytotoxic action of ara-C is not only cell-cycle phase–dependent but also affects the rate of DNA synthesis. That is, cell kill in tissue culture is greatest if cells are exposed during periods of maximal rates of DNA synthesis, as in the recovery period after exposure to a cytotoxic agent. In experimental situations it has been possible to schedule sequential doses of ara-C to coincide with the peak in recovery of DNA synthesis and thus to improve the therapeutic results.95,96, 97
Burke and colleagues96 and Vaughan and colleagues98 have attempted to exploit kinetic patterns of leukemic cell recovery after ara-C by optimizing sequential doses of drug. Thus, retreatment 8 to 10 days after an initial dose of ara-C has yielded a promising improvement in the duration of unmaintained remission in adult patients with leukemia in uncontrolled studies.98
In humans, the influence of tumor cell kinetics on response is unclear. Although earlier studies showed that the complete remission rate seems to be higher in patients who have a high percentage of cells in S phase,99 remissions are longer in patients with leukemias that have long cell-cycle time.100
Figure 8.7 Thymidine incorporation into DNA of M19 human melanoma cells as a function of drug concentration and duration of exposure to cytosine arabinoside (ara-C). The exposure duration in hours is indicated by the numbers adjacent to the individual curves. The data indicate a near-linear relationship between inhibition of thymidine incorporation and drug concentration but a lesser dependence on time for intervals longer than 12 hours, perhaps because of the cell-cycle dependence of the drug. Thus, most replicating cells are exposed to ara-C during their period of DNA synthesis if the exposure time is 12 hours or longer.
Clinical Pharmacology—Assay Methods
A number of assay methods have been used to measure ara-C concentration in plasma.101, 102, 103, 104, 105 The preferred method for assay of ara-C and its primary metabolite ara-U is high-pressure liquid chromatography, which has the requisite specificity and adequate (0.1 µmol/L) sensitivity.106, 107 An alternative method using gas chromatography–mass spectrometry combines high specificity with greater sensitivity (4 nmol/L) but requires derivatization of samples and thus prolonged performance time.108 Because of the presence of cytidine deaminase in plasma, the deaminase inhibitor tetrahydrouridine must be added to plasma samples immediately after blood samples are obtained.
The important factors that determine ara-C pharmacokinetics are its high aqueous solubility and its susceptibility to deamination in liver, plasma, granulocytes, and gastrointestinal tract. Ara-C is amenable to use by multiple schedules and routes of administration and has shown clinical activity in dosages ranging from 3 mg/m2 twice weekly to 3 g/m2 every 12 hours for 6 days. Remarkably, over this wide dosage range, its pharmacokinetics remains quite constant and predictable.
As a nucleoside, ara-C is transported across cell membranes by a nucleoside transporter and distributes rapidly into total-body water.109, 110 It crosses into the central nervous system (CNS) with surprising facility for a water-soluble compound and reaches steady-state levels at 20 to 40% of those found simultaneously in plasma during constant intravenous infusion.102 At conventional doses of ara-C (100 mg/m2 by 24-hour infusion), spinal fluid levels reach 0.2 µmol/L, which is probably above the cytotoxic threshold for leukemic cells. High doses of ara-C yield proportionately higher ara-C levels in the spinal fluid.111, 112, 113
The pharmacokinetics of ara-C are characterized by rapid disappearance from plasma owing to deamination, with some variability seen among individual patients.102, 104, 108, 110 Peak plasma concentrations reach 10 µmol/L after bolus doses of 100 mg/m2 and are proportionately higher (up to 150 µmol/L) for doses up to 3 g/m2 given over a 1- or 2-hour infusion111, 114 (Fig. 8.8). Thereafter, the plasma concentration of ara-C declines, with a half-life of 7 to 20 minutes. A second phase of drug disappearance has been detected after high-dose ara-C infusion, with a terminal half-life of 30 to 150 minutes, but the drug concentration during this second phase has cytotoxic potential only in patients treated with high-dose ara-C.113, 115 Seventy to eighty percent of a given dose is excreted as ara-U,102 which, within minutes of drug injection, becomes the predominant compound found in plasma. Ara-U has a longer half-life in plasma (3.2 to 5.8 hours) than does ara-C and may enhance the activation of ara-C through feedback inhibition of ara-C deamination in leukemic cells.115
Figure 8.8 Cytosine arabinoside (ara-C) pharmacokinetics in plasma after doses of 3 g/m2 given over 2 hours, 100 mg/m2 per hour by continuous infusion for 24 hours, 4 mg/m2 per hour (a conventional antileukemic dose) by continuous intravenous infusion, and 10 mg/m2 subcutaneously or intravenously as a bolus.
The steady-state level of ara-C in plasma achieved by constant intravenous infusion remains proportional to dose for dose rates up to 2 g/m2 per day. At this dosage, steady-state plasma levels approximate 5 µmol/L. Above this rate of infusion, the deamination reaction is saturated and ara-C plasma levels rise unpredictably, which leads to severe toxicity in some patients.116 To accelerate the achievement of a steady-state concentration, one may give a loading dose of three times the hourly infusion rate before infusion.110 Equivalent drug exposure (area under the curve) is achieved by subcutaneous or intravenous infusion of ara-C,117 although one study has reported higher ara-CTP concentrations in leukemia cells after subcutaneous administration.118
Owing to the presence of high concentrations of cytidine deaminase in the gastrointestinal mucosa and liver, orally administered ara-C provides much lower plasma levels than does direct intravenous administration. Threefold to 10-fold higher doses must be given in animals to achieve an equal biologic effect. The oral route, therefore, is not routinely used in humans.
Ara-C may also be administered by intraperitoneal infusion for treatment of ovarian cancer.119 After instillation of 100 µmol/L of drug, ara-C levels fall in the peritoneal cavity with a half-life of approximately 2 hours. Simultaneous plasma levels are 100- to 1,000-fold lower, presumably because of deamination of ara-C in liver before it reaches the systemic circulation. In 21-day continuous infusion, patients tolerated up to 100 µmol/L intraperitoneal concentrations but developed peritonitis at higher concentrations.120
Cerebrospinal Fluid Pharmacokinetics
After intravenous administration of 100 mg/m2 of ara-C, parent drug levels reach 0.1 to 0.3 µmol/L in the cerebrospinal fluid (CSF), with a decline in levels thereafter characterized by a half-life of 2 hours. Proportionately higher CSF levels are reached by intravenous high-dose ara-C regimens; for example, a 3 g/m2 infusion intravenously over 1 hour yields peak CSF concentrations of 4 µmol/L,114 whereas the same dose over 24 hours yields peak CSF ara-C concentrations of 1 µmol/L.116
Ara-C is effective when administered intrathecally for the treatment of metastatic neoplasms. A number of dosing schedules for giving intrathecal ara-C have been recommended, but twice weekly or weekly schedules of administration are the most widely used. The dose of ara-C ranges from 30 to 50 mg/m2. The dose is generally adjusted in pediatric patients according to age (15 mg for children below 1 year of age, 20 mg for children between 1 and 2 years, 30 mg for children between 2 and 3 years, and 40 mg for children older than 3 years). The clinical pharmacology of ara-C in the CSF following intrathecal administration differs considerably from that seen in the plasma following a parenteral dose. Systematically administered ara-C is rapidly eliminated by biotransformation to the inactive metabolite ara-U. In contrast, little conversion of ara-C to ara-U takes place in the CSF following an intrathecal injection. The ratio of ara-U to ara-C is only 0.08, a finding that is consistent with the very low levels of cytidine deaminase present in the brain and CSF. Following an intraventricular administration of 30 mg of ara-C, peak levels exceed 2 mmol/L, and levels decline slowly, with the terminal half-life being approximately 3.4 hours.102Concentrations above the threshold for cytotoxicity (0.1 µg/mL, or 0.4 µmol/L) are maintained in the CSF for 24 hours. The CSF clearance is 0.42 mL/minute, which is similar to the CSF bulk flow rate. This finding suggests that drug elimination occurs primarily by this route. Plasma levels following intrathecal administration of 30 mg/m2 of ara-C are less than 1 µmol/L, which illustrates again the advantage of intracavitary therapy with a drug that is rapidly cleared in the systemic circulation.
Depocytarabine (DTC 101) is a depot formulation in which ara-C is encapsulated in microscopic Gelfoam particles (DepoFoam) for sustained release into the CSF so that the need for repeated lumbar punctures is avoided. The encapsulation of ara-C in DepoFoam results in a 55-fold increase in CSF half-life after intraventricular administration in rats, from 2.7 hours to 148 hours. Cytotoxic concentrations of free ara-C (>0.4 µmol/L) in CSF were maintained for more than 1 month following a single intrathecal dose administration of 2 mg of DTC 101 in rhesus monkeys. A phase I trial of DTC 101 given intraventricularly has been performed in patients with leptomeningeal metastasis. Free ara-C CSF concentration decreased biexponentially. After a dose of 50 mg of DTC, ara-C concentration was maintained above the threshold for cytotoxicity for an average of 12 ± 3 days. The maximum tolerated dosage was 75 mg administered every 3 weeks, and the dose-limiting toxicity was headache and arachnoiditis.121 Preliminary results of a randomized study involving patients with lymphomatous meningitis demonstrate a possible prolongation of time to neurologic progression in patients treated with 50 mg of DTC 101 every 2 weeks compared with patients treated with standard intrathecal ara-C.122 DTC appears to give equivalent results to standard intrathecal methotrexate, given every 4 days, for treatment of carcinomatous meningitis.122a
Alternate Schedules of Administration
Although ara-C is used most commonly in regimens of 100 to 200 mg/m2 per day for 7 days, other high-dose and low-dose schedules have been used in treating leukemia. The more effective of these newer regimens have been high-dose schemes, usually 2 to 3 g/m2 every 12 hours for six doses.123 High-dose ara-C is used primarily in the consolidation phase for acute myelocytic leukemia.8 The rationale for the higher-dose regimen initially rested on the assumption that ara-C phosphorylation is the rate-limiting intracellular step in the drug's activation and could be promoted by raising intracellular concentrations to the Km of deoxycytidine kinase for ara-C, or approximately 20 µmol/L. Above this level, further increases in ara-C do not lead to increased ara-CTP because the phosphorylation pathways become saturated.48, 124
Others have examined the clinical activity of low-dose ara-C, particularly in older patients with myelodysplastic syndromes.125 These regimens have used dosages in the range of 3 to 20 mg/m2 per day for up to 3 weeks. The rationale for low-dose regimens has been based primarily on the expectation that they would produce less toxicity; low concentrations of ara-C were also thought to promote leukemic cell differentiation (or apoptosis) in tissue culture. In isolated cases, the persistence of chromosomal markers for the leukemic cell line in remission granulocytes has been documented, findings that support differentiation.126, 127 In general, although the low-dose regimens produce less toxicity, particularly at the lower end of the dose spectrum, the therapeutic results have been disappointing in that less than 20% of patients achieve a clinical remission.128 Continuous exposure of normal myeloid precursor cells to drug concentrations as low as 10 nmol/L inhibits proliferation, a further problem in MDS treatment.129 After intravenous doses as low as 3 mg/m2, peak plasma levels reach 100 nmol/L and remain above the inhibitory concentration (10 nmol/L) for 30 to 60 minutes. Thus, low-dose ara-C regimens have not avoided the myelosuppressive effects of standard schedules.
The primary determinants of ara-C toxicity are drug concentration and duration of exposure. Because ara-C is cell cycle phase-specific, the duration of cell exposure to the drug is critical in determining the fraction of cells killed.130 In humans, single-bolus doses of ara-C as large as 4.2 g/m2 are well tolerated because of the rapid inactivation of the parent compound and the brief period of exposure, whereas constant infusion of drug for 48 hours using total doses of 1 g/m2 produces severe myelosuppression.131
Myelosuppression and gastrointestinal epithelial injury are the primary toxic side effects of ara-C. With the conventional 5- to 7-day courses of treatment, the period of maximal toxicity begins during the first week of treatment and lasts 14 to 21 days. The primary targets of ara-C are platelet production and granulopoiesis, although anemia also occurs. Little acute effect is seen on the lymphocyte count, although a depression of cell-mediated immunity is found in patients receiving ara-C.132 Megaloblastic changes consistent with suppression of DNA synthesis are observed in both the white and red cell precursors.133
Gastrointestinal symptoms, including nausea, vomiting, and diarrhea, are frequent during the period of drug administration but subside quickly after treatment. Severe gastrointestinal lesions occur in patients treated with ara-C as part of complex chemotherapy regimens, and the specific contribution of ara-C is difficult to ascertain in these cases. All parts of the gastrointestinal tract are affected. Oral mucositis also occurs and may be severe and prolonged in patients receiving more than 5 days of continuous treatment. Clinical symptoms of diarrhea, ileus, and abdominal pain may be accompanied by gastrointestinal bleeding, electrolyte abnormalities, and protein-losing enteropathy. Radiologic evidence of dilatation of the terminal ileum, termed typhlitis, may be associated with progressive abdominal pain and bowel perforation. Pathologic findings include denudation of the epithelial surface and loss of crypt cell mitotic activity. Reversible intrahepatic cholestasis occurs frequently in patients receiving ara-C for induction therapy but requires cessation of therapy in fewer than 25% of patients.134, 135 It is manifested primarily as an increase in hepatic enzymes in the serum, together with mild jaundice, and rapidly reverses with discontinuation of treatment. Ara-C has been implicated as the cause of pancreatitis in a small number of patients.136
Toxicity of High-Dose Cytosine Arabinoside
High-dose ara-C significantly increases the incidence and severity of bone marrow and gastrointestinal toxic effects.7 Hospitalization for fever and neutropenia is required in 71% of the treatment courses in patients receiving 3 g per m2 per 12 hours given on alternative days for six doses, and platelet transfusions are required in 86%.8 Treatment-related deaths, primarily the result of infection, occurred in 5% of the patients treated with this schedule.8 In addition, high-dose ara-C produces pulmonary toxicity, including noncardiogenic pulmonary edema, in approximately 10% of patients, and a surprisingly high incidence of Streptococcus viridans pneumonia is seen, especially in pediatric populations.137, 138, 139 The pulmonary edema syndrome is frequently irreversible.
Cholestatic jaundice and elevation of serum glutamic-oxaloacetic transaminase, serum glutamic-pyruvic transaminase, and alkaline phosphatase, with underlying cholestasis and passive congestion on liver biopsy, are also frequently observed with the high-dose regimen.140 These changes, however, are generally clinically unimportant and reversible. A more dangerous toxicity involving cerebral and cerebellar dysfunction occurs in 10% of patients receiving 3 g/m2 for 6 doses8 and in two-thirds of patients receiving 4.5 g/m2 for 12 doses.141 Age over 40 years, abnormal alkaline phosphatase activity in serum, and compromised renal function142 are risk factors associated with an increased susceptibility to CNS toxicity, which is manifested as slurred speech, unsteady gait, dementia, and coma.141 Patients with two or more of these risk factors treated with high-dose ara-C develop CNS toxicity in 37% of the cases, whereas the incidence is less than 1% when fewer than two of these criteria are present.142 Symptoms of neurologic toxicity resolve within several days in approximately 20% of patients and gradually recede in approximately 40%; however, a permanent disability is present in the remaining 40%, and occasionally patients have died of CNS toxicity.8 Progressive brainstem dysfunction143 and an ascending peripheral neuropathy144 also have been reported after high-dose ara-C.
Other bothersome toxicities complicate high-dose AraC. Conjunctivitis, responsive to topical steroids, also has been a frequent side effect of high-dose ara-C.145 Rarely, skin rash146 and even anaphylaxis have been noted.147 Neutrophilic eccrine hydradenitis, an unusual febrile cutaneous reaction manifested as plaques or nodules during the second week after chemotherapy, is being reported with increasing frequency after high-dose ara-C.148 Finally, reports have appeared sporadically in the literature of cardiac toxicity associated with ara-C, generally at high dosages. Findings have included arrhythmias, pericarditis, and congestive heart failure. None of these reports provide conclusive evidence for a cause-and-effect relationship.149
Toxicity of Intrathecal Cytosine Arabinoside
Ara-C given intrathecally is infrequently associated with fever and seizures occurring within 24 hours of administration, and arachnoiditis occurring within 4 to 7 days.150 Rarely, it causes a progressive brainstem toxicity that may be fatal.151 Intrathecal ara-C should be used with caution in patients who have previously experienced methotrexate neurotoxicity.
Although ara-C causes chromosomal breaks in cultured cells and in the bone marrow of patients receiving therapy,152 it is not an established carcinogen in humans. The drug is teratogenic in animals.153
Ara-C has synergistic antitumor activity with a number of other antitumor agents in animal tumor models. These other agents include alkylating agents (cyclophosphamide154 and carmustine [BCNU]155), cisplatin,156 purine analogs,157, 158 methotrexate,159, 160 and etoposide.161 In the past, ara-C and 6-thioguanine (6-TG) were frequently combined in the treatment of acute leukemia. This interaction seems to be highly schedule-dependent. Ara-C, an inhibitor of DNA synthesis, blocks the incorporation of 6- TG into DNA; however, if ara-C is given 12 hours before 6-TG, enhanced incorporation of the purine analog is observed.162 On the other hand, evidence exists that 6-TG, given before or with ara-C, enhances ara-C incorporation into DNA by blocking exonuclease activity.163
The basis for ara-C potentiation of alkylating agents and cisplatin is thought to be inhibition of repair of DNA-alkylator adducts. The hypothesis is consistent with the finding that ara-C exposure preceding cisplatin is synergistic—perhaps allowing for inhibition of repair164—whereas ara-C after cisplatin is not.156
Tetrahydrouridine (THU), a potent inhibitor of cytidine deaminase (Ki = 3–10-8 mol/L),62 also enhances ara-CTP formation in acute myelocytic leukemia cells in vitro but not in chronic lymphocytic leukemia cells, which lack deaminase activity.165 THU enhances the growth- inhibitory effects of sublethal concentrations of ara-C in experiments with the sarcoma 180 cell line, which contains high amounts of cytidine deaminase.166 Initial clinical evaluation of the combination indicates that THU in intravenous doses of 50 mg/m2 markedly prolongs the plasma half-life of ara-C from 10 to 120 minutes and causes a corresponding enhancement of toxicity to bone marrow.167, 168 In combination with THU, the tolerable dosage of ara-C is reduced 30-fold to 0.1 mg/kg per day for 5 days. Whether the combination has greater therapeutic effects and a better therapeutic ratio than ara-C alone is unclear.
Figure 8.9 Interactions of thymidine and cytosine arabinoside (ara-C). ara-CMP, arabinosylcytosine monophosphate; ara-CTP, arabinosylcytosine triphosphate; ara-UMP, arabinosyluracil monophosphate; CDP, cytidine diphosphate; CdR, deoxycytidine; dCDP, deoxycytidine diphosphate; dCMP, deoxycytidine monophosphate; dCTP, deoxycytidine triphosphate; TTP, thymidine triphosphate.
Inhibitors of ribonucleotide reductase—such as hydroxyurea,169 2,3-dihydro-1 H-imidazolo(1,2-b)pyrazole,170 and thymidine triphosphate171—enhance ara-C toxicity by decreasing dCTP pools (Fig. 8.9). A decrease in dCTP should have several beneficial effects on ara-C activity. CdR kinase, the enzyme that converts ara-C to ara-CMP (Fig. 8.4), is inhibited by dCTP, whereas dCMP deaminase, which would convert ara-CMP to the inactive ara-UMP, is activated by dCTP; a decrease in dCTP pools should thus increase ara-CTP formation. Second, because ara-CTP and dCTP compete for the same active site on DNA polymerase, a decrease in dCTP pools should lead to a relative increase in the amount of ara-C incorporated into DNA.
Experimental studies have confirmed that synergy between ara-C and thymidine occurs in some but not all tumor cell lines171, 172 and experimental chemotherapy settings.173, 174 The combination of ara-C and thymidine has received limited clinical evaluation in patients with refractory leukemia and lymphoma, and the initial results have not been favorable as only 7 of 26 patients in the largest study achieved remission.174, 175, 176 Thymidine (75 g/m2 per day) is extremely cumbersome to administer because of the massive fluid load required.174, 175 Tumor cells may develop resistance to both agents by a single-step mutation related to expansion of the dCTP pool as a result of increased de novo synthesis of pyrimidines.69
The conversion of ara-C to its active form, ara-CTP, is also augmented by pretreatment with methotrexate, according to studies of the murine lymphoma cell lines L1210 and L5178Y.159, 160 Simultaneous administration of ara-C and methotrexate is associated with greater retention of ara-CTP in tumor cells and better therapeutic results than achieved with schedules using ara-C alone or ara-C and methotrexate administered 24 hours apart.177 The combination has not been evaluated in definitive clinical trials. Ara-C is commonly used in combination with daunorubicin or etoposide for the treatment of acute myelocytic leukemia. In experimental systems, minute (0.01 µmol/L) concentrations of ara-C cause an increase in levels of topoisomerase II, enhance the rate of protein-associated DNA strand breaks induced by etoposide,161 and increase their cytotoxicity. Ara-C has no apparent direct effect on topoisomerase II activity.178
Ara-CTP formation, a requisite step for cytotoxicity, is markedly augmented by prior exposure of leukemic cells to fludarabine (fluoro-ara-adenine) phosphate, but this combination decreases the intracellular levels of fluoro-arabynosyl-adenine-triphosphate (F-ara-ATP).157, 179 Increased ara-CTP results from the inhibition of ribonucleotide reductase by fludarabine triphosphate. Approximately a 50% increase in leukemic cell ara-CTP is associated with pretreatment of chronic lymphocytic leukemia patients with fludarabine. Ara-C also may shorten the plasma half-life of fludarabine.158 Clinical studies performed during treatment of patients with acute myelogenous leukemia demonstrated that the accumulation of ara-CTP in circulating leukemia blasts was increased by a median of twofold when fludarabine was infused 4 hours before ara-C. The augmentation depended on the cellular concentration of fludarabine triphosphate. Fludarabine at 15 mg/m2 infused over 30 minutes consistently produced cellular fludarabine triphosphate levels that maximized ara-CTP accumulation in acute myelocytic leukemia blasts.180
Considerable interest has focused on the use of ara-C in combination with hematopoietic growth factors (HGFs). The theoretical gain of this combination would be that administration of HGFs before the administration of a cell-cycle–specific drug, such as ara-C, would recruit leukemia cells into the susceptible S phase of the cell cycle, which would thereby enhance cytotoxicity. In fact, several in vitro studies have shown that cytokines, particularly interleukin 3 and granulocyte-macrophage colony-stimulating factor, stimulate myeloid leukemia proliferation181 and increase leukemic blast susceptibility to ara-C–induced apoptosis.182 Growth regulatory molecules might also affect the therapeutic index by increasing the ara-CTP to dCTP ratio183 and the ara-C incorporation into DNA.184 Conflicting results have been observed in in vivo studies, however, and several randomized clinical trials have shown no advantage in response rate or survival in patients with acute myelocytic leukemia treated with HGFs in combination with ara- C compared with patients treated with ara-C alone.185
As resistance to a broad range of chemotherapeutic agents, including ara-C, may arise from defects in damage recognition and apoptosis pathways, a major field of investigations has been the modulation of signal transduction-apoptotic pathways. Staurosporine, a highly potent but nonspecific inhibitor of PKC (20 to 50 nmol/L), significantly potentiated ara-C–mediated apoptosis in human myeloid leukemia cell lines HL-60 and U937, but was ineffective when given alone at these concentrations.186 In contrast, coadministration of another nonspecific PKC inhibitor, H7, and two highly selective PKC inhibitors, calphostin C and chelerythrine, also increased the extent of DNA fragmentation observed in ara-C–treated cells, but only at concentrations that were themselves sufficient to induce DNA damage.186 Sustained exposure to bryostatin 1, a macrocyclic lactone PKC activator, also enhanced ara-C–mediated apoptosis. These apparently conflicting observations may be explained by the down-regulation of PKC expression that follows a period of sustained activation, or by effects of PKC activation on Bcl-2 phosphorylation.91, 187
OTHER CYTIDINE ANALOGS
One objective of analog development in the general area of cytidine antimetabolites has been to find compounds that preserve the inhibitory activity of ara-C but are resistant to deamination. This goal is based primarily on the assumptions that the rapid metabolism of ara-C and its short half- life in plasma constitute an inconvenience because they require continuous infusion of drug rather than intermittent bolus administration, and that deamination may play a role in tumor cell resistance. As reviewed previously in this chapter, the evidence that nucleoside deamination is responsible for resistance is limited to the study of Steuart and Burke77 and has not been confirmed by subsequent work. Nonetheless, a number of deaminase-resistant analogs have been developed, and several, including cyclocytidine (O2, 2′-cyclocytidine)188 and N4-behenoyl ara-C,189 showed antileukemic activity in limited clinical trials, but had undesirable side effects.190, 191, 192 Representative compounds are listed in Table 8.5.
A hybrid of ara-C and 5-azacytidine, 5-aza-cytosine arabinoside193 (Fig. 8.1), is activated by the same pathway as ara-C and is incorporated into DNA, where it inhibits DNA synthesis. It is not deaminated and has broad activity against human xenografts in nude mice and against murine solid tumors, but it failed to demonstrate clinical activity. A related analog, 5-aza-2′-deoxycytidine (decitabine), is incorporated into DNA and, as does 5-azacytidine, inhibits DNA methylation and promotes differentiation.194 In human K562 cells, decitabine was a more effective inducer of erythroid differentiation than its related analog 5-azacytidine, with less acute cell toxicity.195 In addition, decitabine showed a greater antileukemic activity than ara-C when the two drugs were compared in vitro on a panel of human leukemia cell lines of different phenotypes,196 and also in some animal tumor models.197Decitabine has entered human clinical trials. Encouraging antileukemic activity has been observed in patients with untreated and heavily pretreated acute myelocytic leukemia and acute lymphoblastic leukemia, and the drug has been shown to induce trilineage responses in patients with advanced myelodysplastic syndromes.198, 199 Its most frequent side effects are myelosuppression and moderate emesis, with no other major extrahematologic toxicities.198, 199
TABLE 8.5 ALTERNATIVE FORMS OF CYTIDINE ANTIMETABOLITE CHEMOTHERAPY
The success of ara-C as an antileukemic agent has encouraged the search for other cytidine analogs, particularly those that would not require activation by deoxycytidine kinase (the enzyme deleted in many ara-C–resistant tumors). Considering ribonucleosides with structural changes in the basic pyrimidine ring was logical because these would be activated in all likelihood by uridine- cytidine kinase, an entirely separate enzyme. Considerable enthusiasm greeted the introduction of 5-azacytidine, an analog of cytidine synthesized by Sorm and colleagues in 1963200 and later isolated as a product of fungal cultures.201 The compound was found to be toxic to both bacterial and mammalian cells. In clinical trials, however, its most important cytostatic action was exerted against myeloid leukemias and myelodysplasia (MDS), 202, 203, 204, 205 Other actions of 5-azacytidine have awakened interest among biologists and clinicians, however, particularly its ability to inhibit DNA cytosine methylation and, as a consequence, to promote expression of “suppressed” genes. For example, the drug is able to promote the synthesis of fetal hemoglobin, an effect believed to be mediated by hypomethylation of the γ-globin gene in erythroid precursor cells.206, 207The use of 5-azacytidine for gene demethylation in inherited diseases, a subject of considerable interest in molecular genetics, has been limited by its bone marrow toxicity and by concerns about carcinogenesis. The important features of the pharmacokinetics and clinical effects of 5-azacytidine are summarized in Table 8.6.
Structure and Mechanism of Action
The biochemistry and pharmacology of 5-azacytidine have been reviewed in depth by Glover and Leyland-Jones.208 The analog 5-azacytidine differs from cytidine in the presence of a nitrogen at the 5 position of the heterocyclic ring (Fig. 8.10). This substitution renders the ring chemically unstable and leads to spontaneous decomposition of the compound in neutral or alkaline solution, with a half-life of approximately 4 hours. The product of this ring opening, N-formylamidinoribofuranosylguanylurea, may recyclyze to form the parent compound but is also susceptible to further spontaneous decomposition to ribofuranosylurea.209 This spontaneous chemical instability is important in the drug's use in two ways: (a) the ultimate antitumor activity of the drug has been attributed to its incorporation into nucleic acids and subsequent spontaneous decomposition, and (b) the preparation formulated for clinical application must be administered within several hours of its dissolution in dextrose and water or saline.210 In buffered solutions such as Ringer's lactate and at acidic pH, the agent is considerably more stable, with a half-life of 65 hours at 25°C and 94 hours at 20°C.211
TABLE 8.6 KEY FEATURES OF 5-AZACYTIDINE PHARMACOLOGY
The mechanism of 5-azacytidine action has not been firmly established, although the balance of evidence suggests that, as a triphosphate, it competes with CTP for incorporation into RNA,212 the primary event that leads to a number of different effects on RNA processing and function.213 These effects include an inhibition of the formation of ribosomal 28 S and 18 S RNA from higher molecular-weight species,214 defective methylation215 and acceptor function of transfer RNA,216 disassembly of polyribosomes,217 and a marked inhibition of protein synthesis.218
Figure 8.10 Metabolic activation and degradation of 5-azacytidine.
Other effects of 5-azacytidine, however, may be more relevant to its antitumor activity. This analog is also incorporated into DNA,219, 220 although to a lesser extent than into RNA. The consequences of 5-azacytidine incorporation into DNA are not fully understood, but one important effect is the inhibition of DNA methylation. The methylation of cytosine residues in DNA inactivates specific genes, whereas treatment of cells with 5-azacytidine inhibits the function of DNA methyl transferase and leads to enhanced expression of a broad variety of genes, depending on the cell type studied.221, 222 Transferase inhibition occurs through formation of a covalent bond between the azacytidine base and a prolylcysteine dipeptide group on the enzyme223 (Fig. 8.11).
The analog 5-azacytidine readily enters mammalian cells by a facilitated nucleoside transport mechanism shared with the physiologic nucleosides uridine and cytidine.220 The initial step in its activation consists of conversion to a monophosphate by uridine-cytidine kinase (Fig. 8.10), which is found in low concentration in human acute myelocytic leukemia cells,209 has low affinity for 5-azacytidine (Km = 0.2 to 11 mmol/L),224, 225 and probably represents the rate-limiting step in 5-azacytidine activation. Either uridine226 or cytidine is capable of preventing 5-azacytidine toxicity in the whole animal and in tissue culture227 by competitively inhibiting its phosphorylation. Deletion of uridine-cytidine kinase has been observed in mutant Novikoff hepatoma cells resistant to 5-azacytidine,221 as well as in other resistant cell types.228 Cytidine deaminase, found in 10-fold to 30-fold higher concentration than uridine-cytidine kinase in leukemic cells, degrades 5-azacytidine to 5-azauridine. The role of this enzyme in resistance to 5-azacytidine has not been defined.
Figure 8.11 Formation of a 5,6-dihydropyrimidine intermediate during methylation of a target DNA containing (a) Cyt, (b) deoxy-5 flurocytidine; and (c) 5-azacytidine.
Further activation of 5-azacytidine monophosphate (5-aza-CMP) to a triphosphate probably occurs by the enzyme dCMP kinase and nucleoside diphosphate kinase. One hour after exposure of cells to the drug, 60 to 70% of acid-soluble radioactivity was identified as 5-azacytidine triphosphate.221
Both drug concentration and duration of exposure are important determinants of 5-azacytidine cytotoxicity in tissue culture, a finding consistent with a preferential action on rapidly dividing cells. In tissue culture experiments it has greatest lethality for cells in the S phase of the cell cycle and relatively little effect against nondividing cells.229, 230 Dose-survival curves in vivo for L1210 and normal hematopoietic cells are both biphasic, however, which indicates perhaps the presence of more than a single site or mechanism of cytotoxic action.231 A closely related analog, 5-aza-2′-deoxycytidine, causes an induction of p21WAF1 and cell cycle arrest in G1 at very low concentrations (2–4 × 10-8m) while at levels of 10-7m it induced phosphorylation of MAP kinase and G2 arrest as well. At these higher doses, cells become apoptotic.232
In addition to its cytotoxic effects, 5-azacytidine has other biologic actions of possible importance in its clinical use. Through its inhibition of DNA methylation, it has been found to induce the synthesis of various proteins, including hepatic enzymes (tyrosine aminotransferase),233 metallothionein,234 β- and γ-globin,206 histocompatibility proteins,235, 236 and T-cell surface markers.237 It can reactivate repressed genes coding for thymidine kinase,238 hypoxanthine-guanine phosphoribosyl transferase,239 or DNA repair240 and in doing so may convert drug-resistant cells to drug-sensitive, or vice versa. Probably through its effects on DNA methylation, 5-azacytidine is able to increase the immunogenicity of tumor cells,235 induce senescence in cell lines,241 and increase the phenotypic diversity of tumor cell lines in mice.238 The drug has mutagenic and teratogenic effects,229, 242, 243 but it is not known to be carcinogenic in humans.
At present, no assay method specific for 5-azacytidine has been developed for clinical use. Future attempts to develop such methods will undoubtedly be complicated by the chemical instability of the drug, its very limited lipid solubility (which will complicate attempts at extraction and concentration from plasma), and the presence in serum of cytidine deaminase, an enzyme that hydrolyzes 5-azacytidine.
Clinical Pharmacology and Pharmacokinetics
The limited information available on 5-azacytidine pharmacokinetics in animals and humans is based on studies using drug labeled with radioactive carbon (14C) 210, 244, 245, 246 and provides an incomplete understanding of drug disposition because of the drug's extensive metabolism and chemical decomposition. After subcutaneous injection [14C]5-azacytidine is well absorbed, as judged by radioactivity levels in plasma.237 Radioactivity distributes into a volume approximately equal to or greater than total-body water (0.58 to 1.15 L/kg) with little plasma protein binding. Peak plasma levels of 0.1 to 1.0 mmol/L are reached by drug infusion at a rate of 2 to 6 mg/hour in adult patients. The primary half-life of radioactivity in plasma is approximately 3.5 hours after bolus intravenous injection but after 30 minutes, less than 2% of radioactivity is associated with intact drug.210 Isolated measurements of radioactivity in the CSF indicate poor penetration of drug, with a CSF:plasma ratio of less than 0.1.
The identity of metabolites is unclear in humans. 5-Azacytidine is known to be susceptible to deamination by cytidine deaminase,247, 248 an enzyme found in high concentrations in liver, granulocytes, and intestinal epithelium and in lower concentration in plasma. A number of metabolic products have been identified in the urine of beagle dogs, including 5-azacytosine, 5-azauracil, and ring cleavage products.244 The last-named product may result from decomposition of the parent compound or of its deamination product, 5-azauridine.
In patients with acute myelogenous leukemia, a number of schedules of administration have been used for 5-azacytidine, 202, 203, 204, 205 including single weekly intravenous doses of up to 750 mg/m2, daily doses of 150 to 200 mg/m2 for 5 to 10 consecutive days, and continuous infusion of similar daily doses for up to 5 days (Table 8.7). With each of these schedules, the primary toxicity has been leukopenia, although nausea and vomiting have been extremely bothersome for patients receiving the drug in bolus doses, which has led some investigators to favor continuous intravenous infusion.204 The latter schedule is also supported by cell kinetic considerations, in view of the drug's greater activity in the S phase of the cell cycle and its very rapid metabolism in humans. The continuous infusion of 5-azacytidine requires fresh preparation of drug at frequent intervals, usually every 3 to 4 hours, because of the chemical instability of the agent. The response rate to 5-azacytidine in previously treated patients with acute myelocytic leukemia has varied from 17 to 36% and seems to be approximately equivalent for the bolus and continuous-infusion schedules.
TABLE 8.7 5-AZACYTIDINE IN THE TREATMENT OF ACUTE MYELOGENOUS LEUKEMIA
In patients with MDS, a lower dose of 75 mg/m2 per day for 7 days repeated every 28 days, yields a best response after the fifth cycle of therapy.205 Maximal dosages, as shown in Table 8.7, produce profound leukopenia and somewhat lesser thrombocytopenia. Hepatotoxicity also has been observed, particularly in patients with preexisting hepatic dysfunction.249 The lower doses in MDS cause an initial decrease in peripheral blood counts, with a subsequent rise with onset of response.
A syndrome of neuromuscular toxicity was observed by Levi and Wiernik250 in patients receiving 200 mg/m2 per day by intravenous bolus injection. Whether this peculiar reaction was related to the somewhat higher dosage of drug is unclear, but neurotoxicity has been reported only sporadically by other investigators using this agent.204 Several less worrisome acute toxic reactions have been associated with 5-azacytidine, including transient fever, a pruritic skin rash, and, rarely, hypotension during or immediately after bolus intravenous administration.203
Low-dose 5-azacytidine has been used in experimental trials to raise fetal hemoglobin levels in patients with sickle cell anemia and thalassemia,207 but concerns regarding carcinogenicity, as demonstrated in studies of rats exposed to the drug,251 have discouraged routine use to treat these diseases and it has been largely replaced by hydroxyurea for this indication. When given as a continuous infusion of 2 mg/kg per day for 5 days, one cycle per month, the drug regularly produces a reticulocytosis of fetal hemoglobin-containing cells and an increase in hemoglobin content in blood of 2 to 3 g/100 mL.252 On this schedule, little myelosuppression, nausea, or vomiting occur.
5-Azacytidine was approved for treatment of patients with MDS in 2004, based on the results of a randomized phase III trial comparing the drug to best supportive care. Thirty-five percent of patients achieved either a clear improvement in blood counts or decreased transfusion requirements, the transfusion benefits lasting a median of more than 330 days.205 In an overview of published trials on 5-azacytidine in MDS, 6% achieved a complete response in bone marrow and peripheral blood.253.
Decitabine, a deoxynucleoside analog of 5-azacytidine, has a similar spectrum of activity against myeloid leukemias and MDS, a similar spectrum of toxicity (myelosuppresion), but no evidence of carcinogenicity in preclinical tests.254 It inhibits DNA methyltransferase activity in a manner analogous to 5-azaC223becoming incorporated into DNA and forming a covalent link with the methyltransferase. It induces hemoglobin F synthesis in patients with sicle cell anemia. In seven patients, refractory to hydroxyurea, a schedule of 0.3 mg/kg per day for 5 days a week for 2 weeks, repeating every 6 weeks, raised Hb F levels from a baseline of 2% to a median of 14%, with a corresponding increase in total Hb of 1.5 g/dL.255 These promising results have led to larger trials aimed at establishing its beneficial effects on clinical end points, such as painful crisis and hospitalization.
Decitabine in higher doses (45 mg/m2 daily for 3 days every 6 weeks) produced a 49% response rate in patients with MDS, as judged by improvement in blood counts, but at the expense of severe myelosuppression, and a 7% mortality rate from infection.256 In significantly higher doses (50 to 100 mg/m2 twice daily for 5 days), it produced objective responses in 18 of 64 patients (28%) in blastic crisis of chronic myelogenous leukemia, including six complete hematologic remissions.257 This regimen produced severe myelosuppression in most patients, with platelet recovery above 30,000 per milliliter occurring at a median of 27 days at the lowest doses given. Other toxicities included drug-related fever in 21%, and drug-related infection in 34%. The role of this drug in leukemia treatment is still uncertain.
Gemcitabine (2,2-difluorodeoxycytidine, dFdC) is the most important cytidine analog to enter clinical trials since ara-C (Fig. 8.12). It has become incorporated into the standard first-line therapy for patients with pancreatic cancer, lung cancer, and transitional cell cancer of the bladder.258, 259, 260, 261 The drug was selected for development on the basis of its impressive activity against murine solid tumors and human xenografts in nude mice.262 In tissue culture it is generally more potent than ara-C; the 50% inhibition concentration values for human leukemic cells range from 3 to 10 nmol/L for 48-hour exposure compared with 26 to 52 nmol/L for ara-C.263 Although its metabolism to triphosphate status and its effects on DNA in general mimic those of ara-C, differences are found in kinetics of inhibition and additional sites of action of the newer compound, and clearly the spectrum of clinical activity is different.
Cellular Pharmacology, Metabolism, and Mechanism of Action
Gemcitabine retains many of the characteristics of ara-C. Its key features are shown in Table 8.2. Influx of gemcitabine through the cell membrane occurs via active nucleoside transporters,264 and deoxycytidine kinase phosphorylates gemcitabine intracellularly to produce difluorodeoxycytidine monophosphate (dFdCMP), from which point it is converted to its diphosphate and triphosphate difluorodeoxycytidine (dFdCDP, dFdCTP) (Fig. 8.12).265 Its affinity for deoxycytidine kinase is threefold lower than that of deoxycytidine itself, whereas it has a 50% lower affinity for cytidine deaminase than does deoxycytidine.266 Cytidine deaminase conversion of gemcitabine to difluorodeoxyuridine (dFdU) represents the main catabolic pathway.267 To a lesser extent, pyrimidine nucleoside phosphorylase clears gemcitabine by cleaving the pyrimidine base from the furanose ring.
Figure 8.12 Key steps in gemcitabine metabolism.
As with ara-C, in vitro studies of gemcitabine suggest potent inhibition of DNA synthesis as its mechanism of action,262, 265, 268 but kinetic studies indicate that the killing effects of gemcitabine are not confined to the S phase of the cell cycle, and the drug is as effective against confluent cells as it is against cells in log-phase growth.269 The cytotoxic activity may be a result of several actions on DNA synthesis: dFdCTP competes with dCTP as a weak inhibitor of DNA polymerase268; dFdCDP is a potent inhibitor of ribonucleotide reductase, which results in depletion of deoxyribonucleotide pools necessary for DNA synthesis270; and dFdCTP is a substrate for incorporation into DNA and, after the incorporation of one more nucleotide, leads to DNA strand termination.271This “extra” nucleotide may be important in hiding the dFdCTP from DNA repair enzymes because incorporation of gemcitabine into DNA appears to be resistant to the normal mechanisms of DNA repair.272 These effects on DNA synthesis represent the main action of gemcitabine, and evidence demonstrates that incorporation of dFdCTP into DNA is critical for gemcitabine-induced apoptosis.273, 274
Several important differences exist between ara-C and gemcitabine (Fig. 8.13). First, dFdCTP has a biphasic elimination from leukemic cells with α half-life (t1/2α) of 3.9 hours and β half-life (t1/2β) of 16 hours, whereas ara-CTP has a monophasic elimination with t1/2 = 0.7 hours.265 Also, dFdCDP is a stronger inhibitor of ribonucleotide reductase (50% inhibition concentration of 4 µmol/L), and exposure to the drug blocks incorporation of labeled cytidine into the cellular pool of dCTP.270 Further, dFdC causes a decrease in all intracellular deoxynucleotide triphosphates, consistent with inhibition of ribonucleotide reductase. The significance of ribonucleotide reductase inhibition is uncertain. Some cell lines selected for resistance to other inhibitors of this enzyme, such as hydroxyurea and deoxyadenosine, do not show cross-resistance to dFdC.275 On the other hand, resistance to gemcitabine has been demonstrated through overexpression of ribonucleotide reductase.276 Nevertheless, the significance of ribonucleotide reductase inhibition may be in the potentiating effects of deoxyribonucleotide depletion on other sites of gemcitabine action.277 For example, deamination of dFdCMP by dCMP deaminase requires activation by dCTP. As dCTP pools become depleted by the effect of gemcitabine on ribonucleotide reductase, less deamination of gemcitabine diphosphate occurs and intracellular accumulation of gemcitabine metabolites increases. Furthermore, high intracellular concentration of dFdCTP appears to inhibit dCMP deaminase directly.267
Figure 8.13 Accumulation of difluorodeoxycytidine triphosphate (dFdCTP) and arabinosylcytosine triphosphate (ara-CTP) as a function of time after incubation of cells with either dFdCTP or ara-C at drug concentrations of 1 µmol/L (A), 10 µmol/L (B), and 100 µmol/L (C). (Adapted from Heinemann V, Hertel LW, Grindey GB, et al. Comparison of the cellular pharmacokinetics and toxicity of 2′,2′-difluorodeoxycytidine and 1-beta-D-arabinofuranosylcytosine. Cancer Res 1988;48:4024–4031.)
The activity of dFdCTP on DNA repair mechanisms may allow for increased cytotoxicity of other chemotherapeutic agents, particularly platinum compounds. Cisplatin works by creating interstrand and intrastrand cross-links. A mechanism of resistance may be removal of these cross-links by nucleotide excision repair (NER). Preclinical studies of tumor cell lines show that cisplatin-DNA adducts are enhanced in the presence of gemcitabine.278 In cisplatin-resistant tumor cell lines, which have increased expression of NER, the addition of gemcitabine inhibited the repair of cisplatin-induced DNA lesions and correlated with cytotoxic synergism.274, 278 Combined gemcitabine and cisplatin are standard agents in the treatment of lung cancer and transitional cell cancer.
Mechanisms of Resistance
Resistance to gemcitabine is not fully understood. In vitro studies have suggested several possible mechanisms. Gemcitabine resistance has been correlated with tumor levels of deoxycytidine kinase.279 Induction of cytidine deaminase and high concentrations of heat-shock protein have also conferred gemcitabine resistance to cells.280, 281 Preclinical studies have also demonstrated that increased expression of ribonucleotide reductase may be associated with gemcitabine resistance.282 Lastly, inhibition of nucleoside transporters can prevent the influx of gemcitabine through the cell membrane and the absense of transporters has been associated with reduced survival in patients with pancreatic cancer.264, 283 Resistance to gemcitabine has not been associated with increased P-glycoprotein expression.284
In animals, gemcitabine pharmacokinetics is largely determined by deamination, which proceeds more rapidly in mice than in rats or dogs.285 Gemcitabine half-life in mice is 0.28 hours compared with 1.38 hours in dogs. In both species, the predominant elimination product is dFdU. Grunewald and colleagues286have found that, in both in vitro cell lines and cells taken from patients during treatment, maximal accumulation of dFdCTP occurs when plasma (or tissue culture) drug concentrations are in the range of 15 to 20 µmol/L, a level achieved during 3-hour infusions of 300 mg/m2.
Abbruzzese et al.287 performed a phase I study of gemcitabine given weekly as a 30-minute infusion on days 1, 8, and 15, followed by a 1-week rest in patients with refractory solid tumors. The maximum tolerated dose (MTD) was 1,000 mg/m2 per week. The dose-limiting toxicity was myelosuppression characterized by thrombocytopenia with relative sparing of granulocytes. Pharmacokinetic analysis showed a t1/2 of 8 minutes for the parent compound and a biphasic elimination of dFdU, with t1/2α = 27 minutes and t1/2β = 14 hours. No relationship was found between degree of myelosuppression and any of the pharmacokinetic parameters. The area under the curve of plasma dFdC was proportional to the dose over a range of 10 to 1,000 mg/m2 per week. Clearance was dose-independent but varied widely among individuals (39 to 1,239 L/hour per m2 at a dose of 1,000 mg/m2).
Although a higher gemcitabine dose of 2,200 mg/m2 administered over 30 minutes on days 1, 8, and 15 can be safely given to less heavily treated or chemonaïve patients, no improvement in efficacy has been demonstrated.288 This lack of dose responsiveness may be caused by the limited ability of cells to generate the active metabolite. In the case of ara-C, the ability of peripheral blood mononuclear cells to accumulate ara-CTP saturates at ara-C concentrations of greater than 10 µmol/L.289 A similar series of studies with gemcitabine have demonstrated that activation of gemcitabine by deoxycytidine kinase to dFdCTP is saturated at infusion rates of approximately 10 mg/m2 per minute.285, 290 This “dose-rate infusion” produced steady-state dFdC levels of 15 to 20 µmol/L in plasma. In leukemic patients, the maximum tolerated dose (MTD) for “dose-rate infusion” is 4,800 mg/m2 infused over 480 minutes.291, Biphasic elimination of gemcitabine was seen in the leukemic cells at this infusion rate, and inhibition of DNA synthesis was proportional to the intracellular level of dFdCTP. Based on these results, a phase I study using constant dose-rate infusion of gemcitabine on days 1, 8, and 15 every 28 days was carried out in patients with metastatic solid tumors.292 Although the first-cycle MTD was estimated to be 2,250 mg/m2 over 225 minutes, the recommended phase II dose of gemcitabine administered as a dose-rate infusion is 1,500 mg/m2 over 150 minutes because of the occurrence of cumulative neutropenia and thrombocytopenia at higher doses. The dose-rate infusion resulted in higher levels of dFdCTP in circulating leukemic cells than a fixed infusion duration of 30 minutes (Fig. 8.14).
In a proof-of-concept study, Tempero and colleagues293 performed a randomized phase II study of constant dose-rate infusion at 10 mg/m2 per minute versus dose-intense infusion over 30 minutes in patients with advanced pancreatic cancer. Patients were randomized to receive gemcitabine 1,500 mg/m2 over 150 minutes or 2,200 mg/m2 over 30 minutes. Constant dose-rate infusion resulted in a twofold increase in intracellular gemcitabine triphosphate in peripheral blood mononulear cells compared with the standard 30-minute infusion. Furthermore, activity appeared to be enhanced with the constant dose-rate infusion because 1-year survival increased from 9% to 28%. Further studies utilizing the dose rate infusion schedule are underway.
Figure 8.14 Relationship between dose of gemcitabine (difluorodeoxycytidine, dFdC) and area under the curve (AUC) of difluorodeoxycytidine triphosphate (dFdCTP) in circulating leukemia cells during gemcitabine infusion. Shown is the mean ± standard error of the mean of the AUC observed for patients at each dose during this phase I study of dose-rate infusion of gemcitabine in patients with leukemia. (Adapted from Grunewald R, Kantarjian H, Du M, et al. Gemcitabine in leukemia: a phase I clinical, plasma, and cellular pharmacology study. J Clin Oncol 1992;10:406–413.)
Gemcitabine has been studied in children and the maximum tolerated dose of gemcitabine given as a 30-minute infusion weekly for 3 of 4 weeks is 1,200 mg/m2. Myelosuppression is the dose-limiting toxicity, and pharmacokinetics in pediatric patients is similar to the adult population.294
The dose-limiting toxicity of gemcitabine is invariably hematologic, and the toxicity profile differs according to schedule. In general, the longer-duration infusions lead to greater myelosuppression. The MTD for a daily × 5 schedule every 21 days is 12 mg/m2 per day or 60 mg/m2 per cycle.295 The MTD for twice-weekly doses of gemcitabine administered for 3 weeks with a 1-week rest period depends on the time of infusion. When the drug is administered over 5 minutes, the MTD is 150 mg/m2, and when it is administered as a 30-minute infusion, the MTD is 75 mg/m2.296 For a 24-hour infusion given weekly in 3 of 4 weeks, the MTD is 180 mg/m2 per dose.297 The weekly dose schedule has gained popularity and is implemented as a 30-minute infusion in 3 of 4 weeks. The MTD for chemonaïve patients is 2,200 mg/m2 per week, and the MTD for pretreated patients is 800 to 1,000 mg/m2 per week.287, 288 A dose of 1,000 mg/m2per week, for 3 to 4 weeks, given over 30 minutes, is recommended for treatment of a variety of solid tumors.
The safety of gemcitabine has been evaluated in a database including 22 studies using the once-weekly treatment regimen.298 Nine hundred seventy-nine patients received at least one dose of gemcitabine and were evaluable for toxicity. World Health Organization (WHO) grade 3 and 4 neutropenia occurred in 19.3% and 6% of patients, respectively. WHO grade 3 and 4 thrombocytopenia occurred in 4.1% and 1.1% of patients, respectively. Clinically significant consequences of hematologic toxicity were uncommon: only 1.1% of patients experienced WHO grade 3 infection and 0.7% of patients required platelet transfusions. Among nonhematologic toxicities, flulike symptoms including fever, headache, back pain, and myalgias occur in approximately 45% of patients. The duration of these symptoms was short, and less than 1% of patients discontinued therapy because of flulike symptoms. Asthenia is also common, occurring in 42% of patients. A transient, mild elevation in liver function test results (WHO grade 1 or 2 elevations in alanine aminotransferase) was detected in 41% of cycles.
Although severe nonhematologic reactions are rare, several specific syndromes complicating gemcitabine therapy are emerging. Thrombotic microangiopathy as manifested by hemolytic-uremic syndrome or thrombotic thrombocytopenic purpura has been reported as a complication of gemcitabine therapy,299, 300, 301and a review of the manufacturer's database estimated an overall incidence rate of 0.015%.302 However, a large single institution review demonstrated 9 cases of gemcitabine-associated microangiopathy among a total of 2,586 cases of microangiopathy for an estimated incidence of 0.31%.303 Patients who are treated for prolonged periods (i.e., longer than 1 year) may be at higher risk for developing hemolytic-uremic syndrome or thrombotic microangiopathy.
Severe pulmonary toxicity as manifested by acute respiratory distress syndrome, capillary leak syndrome, or interstitial pneumonitis has been reported in patients treated with gemcitabine.304, 305 A review of the Lilly world-wide database identified 91 patients with serious pulmonary toxicity for an estimated incidence of less than 0.1%.306 Caution is warranted when combining gemcitabine with drugs known to cause pulmonary dysfunction, such as bleomycin. A study substituting gemcitabine for etoposide in the BEACOPP regimen in Hodgkin's lymphoma led to severe pulmonary toxicity, possibly as a result of interaction with bleomycin.307
A multicenter study evaluated the role of gemcitabine in patients with hepatic or renal dysfunction.308 Patients with elevated bilirubin experienced increased toxicity and should receive reduced doses; whereas, patients with elevated transaminases did not experience increased toxicity. Patients with elevated creatinine appeared to be more sensitive to gemcitabine but did not require dose reductions.
Because of its inhibition of ribonucleotide reductase and DNA polymerase, gemcitabine may have strong radiosensitization effects. Preclinical studies of gemcitabine have shown potent radiosensitization effects in human colon, pancreatic, head and neck, and cervical cancer cell lines.309, 310, 311, 312 These effects parallel the intracellular depletion of deoxyadenosine triphosphate and are most prominent when gemcitabine is administered before radiation therapy. Interestingly, the radiosensitization effect had no correlation with dFdCMP incorporation into DNA, which suggests that the inhibition of ribonucleotide reductase is the key mechanism of action.313 In vitro studies suggest that maximal enhancement of radiation sensitization occurs when gemcitabine is administered before radiation, and in vivo studies suggest that this effect is most pronounced when the time interval is 24 to 60 hours.309, 314, 315Gemcitabine radiosensitization may be best in mismatch repair-deficient cells when compared with mismatch repair-proficient cells 316
Despite the radiosensitization seen in preclinical studies, the initial phase I and II studies of gemcitabine and radiation therapy have not demonstrated markedly improved clinical activity, and are associated with increased toxicity. In a phase I trial of twice-weekly gemcitabine and concurrent radiation in patients with advanced pancreatic cancer, the MTD was 40 mg/m2 administered over 30 minutes on Monday and Thursday of each week.317 The dose-limiting toxicities were grade 3 neutropenia, thrombocytopenia, nausea, and vomiting. This regimen was subsequently evaluated in phase II study for patients with locally advanced pancreatic cancer, and the median survival was 7.9 months.318 When given once weekly with radiation at doses of 300 to 500 mg/m2 to patients with locally advanced pancreatic cancer, gemcitabine is associated with increased severe toxicity and similar survival when compared with historical conrols using flouropyrimidines with external beam radiation therapy.319 An attempt to combine weekly gemcitabine with fluorouracil and external beam radiation therapy in patients with locally advanced pancreatic cancer was stopped when five of the first seven patients experienced dose-limiting toxicities at gemcitabine doses of 100 and 50 mg/m2.320
The inability to deliver full-dose gemcitabine concurrent with radiation has been demonstrated in other tumor types as well. A phase II study of gemcitabine administered weekly with concurrent external beam radiation therapy in patients with unresectable head and neck cancer required dose de-escalation from 300 mg/m2 per week to 50 mg/m2 per week as the result of a high rate of mucosa-related toxicity.321 A phase I study of weekly gemcitabine with concurrent radiotherapy in patients with locally advanced lung cancer established a maximally tolerated dose of 300 mg/m2.322 Dose-limiting toxicities included grade 3 esophagitis and grade 3 pneumonitis. Ongoing trials are continuing to define the toxicity of efficacy of gemcitabine when combined with external beam radiation therapy.
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