Ara-C acts as an analogue of deoxycytidine (CdR) 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).⁹ This competitive inhibition has been demonstrated with crude DNA polymerase from calf thymus⁹ and with purified enzyme from human leukemic cells¹⁰ and from murine tumors.¹¹ Ara-CTP has an affinity for human leukemia cell DNA polymerase α in the range of 1 × 10⁻⁶ mol/L and competes with dCTP. In intact cells, its effects are antagonized by the addition of CdR, the precursor of dCTP.¹² Ara-CTP inhibits DNA polymerase β with lesser potency.¹³ 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 of polymerase α, is blocked more potently than the repair of photon-induced or γ radiation-induced strand breaks,¹⁴ which are repaired by a different polymerase. In addition to its inhibition of DNA synthesis, however, it becomes incorporated into DNA, a feature that correlates closely with cytotoxicity¹⁵,¹⁶ (Fig. 10-2). 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, block its cytotoxicity.¹⁷ In cell culture experiments, a linear relationship exists between picomoles of ara-C incorporated into DNA 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 drug concentration and duration of exposure.¹⁸ Once incorporated into DNA, ara-C is excised slowly,¹⁹ and the incorporated ara-C inhibits template function and chain elongation.¹⁷,²⁰,²¹ 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 stops chain elongation.¹⁰ At high concentrations of ara-C, one finds a greater than expected proportion of ara-C residues at the 3′-terminus, a finding that implies potent chain termination.¹⁹ These observations support the hypothesis that ara-C incorporation into DNA is a prerequisite for drug action and is responsible for cytotoxicity.

Ara-C also causes an unusual reiteration of DNA segments.²² 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, 5-fluorouracil, and hydroxyurea (see relevant chapters). In summary, although ara-C has multiple effects on DNA synthesis, the most important antitumor mechanism seems to be its incorporation into DNA.

Other biochemical actions of ara-C have been described, including a relatively weak inhibition of ribonucleotide reductase²³ and formation of arabinosylcytosine diphosphate (ara-CDP)-choline. The latter functions as an analog of cytidine 5′-diphosphocholine (CDP-choline) and inhibits synthesis of membrane glycoproteins and glycolipids.²⁴ 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.²⁵ These changes in morphology and oncogene expression occur at concentrations above the threshold for cytotoxicity and may simply represent terminal injury of cells. After exposure to ara-C, both normal and malignant cells undergo apoptosis in experimental models.²⁶

The mechanism by which ara-C induces apoptosis is uncertain. It may be triggered by p53 in response to DNA breaks (see above) and by futile attempts to excise incorporated ara-C nucleotides. Ara-C stimulates the formation of ceramide, a potent inducer of apoptosis.²⁷ Ara-C induces production of diacylglycerol, which activates protein kinase C (PKC), a response that opposes apoptosis in hematopoietic cells. The lethal actions of ara-C may depend, at least partially, on its relative effects on the PKC and ceramide pathways.

Cells exposed to ara-C display marked alterations in transcription factors. Ara-C induces AP-1 (a dimer of jun-fos or jun-jun proteins) and NF-kB, and induction has been temporally associated with apoptosis.²⁸,²⁹ PKC inhibitors promote ara-C-induced apoptosis despite their antagonizing c-jun up-regulation, calling into question the involvement of c-jun expression in apoptosis secondary to ara-C.³⁰ Ara-C induces pRb phosphatase activity, another possible mechanism responsible for p53-independent G₁ arrest and apoptosis.³¹ The resulting hypophosphorylated pRb binds to and inactivates the E2F transcription factor, which inhibits the transcription of genes responsible for cell-cycle progression.³²

Ara-C penetrates cells by a carrier-mediated process shared by physiologic nucleosides.³³ Several different classes of transporters for nucleosides have been identified in mammalian cells; 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 AML cells than in acute lymphocytic leukemia cells and can be enumerated by incubation of cells with NBMPR.³⁴ The hENT1 transporter is highly upregulated in biphenotypic leukemia associated with the 11q23 MLL gene (4:11) translocation.³⁵ Uptake occurs rapidly. A steady-state level of intracellular drug is achieved within 90 seconds at 37°C.³⁶,³⁷ Studies of Wiley et al.³³ and others³⁴,³⁷ 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 cells (Fig. 10-3). A single point
mutation in the hENT1 carrier confers resistance in leukemic cell lines in vitro.³⁶ At drug concentrations above 10 μmol/L, the transport process becomes saturated, and further entry takes place by passive diffusion.³⁷ hENT1 is strongly inhibited by various receptor tyrosine kinase inhibitors, an interaction that could limit ara-C use with targeted drugs.³⁸

As shown in Figure 10-4, ara-C must be converted to its active form, ara-CTP, through the sequential action of three enzymes: (a) 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 10-2) and is believed to be rate limiting in the process of ara-CTP formation. The enzyme is a 30.5-kDa protein that phosphorylates ara-C and many pyrimidine and purine nucleosides and their analogs. The gene coding for CdR kinase and a mutated version found in ara-C resistant cells have been cloned.³⁹,⁴⁰ The rate-limiting role of CdR kinase in ara-C activation is illustrated by transfection of malignant cell lines with retroviral vectors containing CdR kinase cDNA, a maneuver that substantially increases the susceptibility of cells to ara-C, gemcitabine, and purine analogues.⁴¹ Ara-C cytotoxicity increases when intracerebral gliomas in rats are transduced with CdR kinase.⁴²

CdR kinase activity is highest during the S phase of the cell cycle.⁴³ The K_m, 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.⁴⁴ 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. PKC-α, the activity of which is increased after ara-C exposure, has been implicated in phosphorylation and activation of CdR kinase. This observation raises the possibility that ara-C at high doses may potentiate its own metabolism by induction of the PKC activator, diacylglycerol.⁴⁵

The second activating enzyme, dCMP kinase,⁴⁶ is found in several hundred-fold higher concentration than CdR kinase. Its affinity for ara-CMP is low (K_m = 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-CMP concentrations. The third activating enzyme, the diphosphate kinase, appears not to be rate limiting because it is present in very high concentration and the intracellular pool of ara-CDP is only a fraction of the ara-CTP pool.⁴⁷

Opposing the activation pathway are two deaminases found in high concentration in some tumor cells as well as in normal tissues. Cytidine deaminase is widely distributed in mammalian tissues, including intestinal mucosa, liver, and granulocytes.⁴⁸ 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.⁴⁴,⁴⁸ The second degradative enzyme, dCMP deaminase (Fig. 10-4), regulates the flow of physiologic nucleotides from the dCMP pool into the deoxyuridine monophosphate (dUMP) pool from which point dUMP is ultimately converted to deoxythymidine 5′-phosphate (dTMP) by thymidylate synthase.⁴⁹ The enzyme dCMP deaminase is strongly activated by intracellular dCTP (K_m = 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 (K_m = 40 μmol/L⁵⁰ 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 the affinity 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 10-2).

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.⁴⁴ CdR kinase activity is higher and cytidine 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.⁴⁸ 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 AML, 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.⁴⁷ found that human AML 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). The likelihood is that other factors, such as transport across the cell membrane and regulatory effects of intracellular nucleoside triphosphate concentrations, may limit ara-CTP formation.

In addition to its activation to ara-CTP, ara-C is converted intracellularly to ara-CDP-choline,⁵¹ 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.²⁴ Ara-CMP does inhibit the transfer of galactose, N-acetylglucosamine, and sialic acid to cell surface glycoproteins. Further, high concentrations (approaching 1 mM) of ara-CTP inhibit the synthesis of cytidine monophosphate (CMP)-acetylneuraminic acid, an essential substrate in sialylation of glycoproteins.⁵² Thus, ara-C treatment could alter membrane structure, antigenicity, and function.

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.⁸² 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.⁸³ Because of the presence of cytidine deaminase in plasma, the deaminase inhibitor THU 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.

Although ara-C is used most commonly in regimens of 100 to 200 mg/m²/d 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.⁹⁷ High-dose ara-C is used primarily in the consolidation phase for acute myelocytic leukemia.⁴ 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 K_m of CdR 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 enzymes become saturated.⁹⁸

Others have examined the clinical activity of low-dose ara-C, particularly in older patients with myelodysplastic syndromes.⁹⁹ These regimens have used dosages in the range of 3 to 20 mg/m²/d for up to 3 weeks, with the expectation that low doses would produce less toxicity and promote leukemic cell differentiation (or apoptosis). The persistence of chromosomal markers for the leukemic cell line in remission granulocytes has been documented, findings that support induction of differentiation.¹⁰⁰ In general, although the low-dose regimens produce less toxicity, myelosuppression often supervenes and less than 20% of patients achieve meaningful improvement in blood counts.

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.¹⁰¹ 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.¹⁰²

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.¹⁰³ Megaloblastic changes consistent with suppression of DNA synthesis are observed in both the white and red cell precursors.¹⁰⁴