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Keywords:

  • clofarabine;
  • acute leukemias;
  • solid tumors;
  • nucleoside analogs

Abstract

  1. Top of page
  2. Abstract
  3. Pharmacology, Mechanisms of Action, and Metabolism
  4. Preclinical Development
  5. Clinical Development
  6. Conclusions
  7. Note Added in Proof
  8. REFERENCES

Clofarabine is a new-generation nucleoside analog that has been synthesized to combine the most favorable pharmacokinetic properties of its congeners fludarabine and cladribine. In addition to inhibition of DNA polymerases and DNA synthesis, clofarabine acts as a strong inhibitor of ribonucleotide reductase (RnR), an enzyme involved in regulating intracellular deoxynucleotide pools, and has a high affinity to the enzyme deoxycytidine kinase (dCyd), the rate-limiting step in nucleoside phosphorylation.A review of the English literature was performed that included original articles and related reviews from the MEDLINE (PubMed) data base and from abstracts based on the publication of meeting materials.Although it was synthesized early in the 1980s, the development of clofarabine was stalled until 1993, when, through efforts at The University of Texas M. D. Anderson Cancer Center, animal toxicology studies were conducted, and the first Phase I study was initiated in patients with hematologic and solid malignancies. Since then, clofarabine has demonstrated single-agent antitumor activity in pediatric and adult acute leukemias. By way of its unique metabolic properties, clofarabine also has lent itself to biochemical modulation strategies with other nucleoside analogs, such as cytarabine. Combinations of clofarabine with cytarabine have been studied in acute leukemia and currently are being evaluated in untreated elderly patients with acute myeloid leukemia. Novel schedules are being explored in lymphoproliferative disorders and solid tumors.Clofarabine is a new nucleoside analog with considerable activity and an acceptable safety profile in acute leukemias. Cancer 2005. © 2005 American Cancer Society.

The biology of tumor cells is comprised of a network of complicated processes that disrupt multiple layers of a cell's regulatory and survival pathways, eventually altering transcription of a variety of target genes. However intricate these processes, the capacity to synthesize DNA remains a vital feature of tumor cells. Proliferating cells, therefore, require a constant pool of purine and pyrimidine nucleotides to replicate; interference with their metabolism will lead to cell death.

Since their introduction in the 1960s, nucleoside analogs have become some of the most prevalent and active components of antitumor therapy.1, 2 Nucleoside analogs disrupt nucleotide metabolism in one of two major ways: 1) incorporation into DNA and 2) inhibition of ribonucleotide reductase (RnR), an enzyme involved in the recycling of the cellular nucleotide pool. The combined effect of the analogs leads to termination of DNA chain elongation, inhibition of DNA synthesis, interference with DNA repair mechanisms, and apoptosis as the most likely final pathway of activity.3–6 An intriguing aspect of nucleoside analogs is how apparently minor alterations in structure can result in major differences with respect to pharmacology, metabolism, and spectrum of activity.7 The activity of cytarabine (1-β-D-arabinosylcytosine [ara-C]), a deoxycytidine analog, is restricted almost exclusively to acute myeloid leukemias (AML); whereas gemcitabine (2′,2′-difluorodeoxycytidine), another carbohydrate-modified analog of deoxycytidine, has a therapeutic spectrum that has been expanded into solid tumor malignancies.8, 9 Fludarabine (9-β-D-arabinofuranosyl-2-fluoroadenine 5′-monophosphate) and cladribine (2-chlorodeoxyadenosine [2-CdA]) have been among the first clinically useful purine nucleoside analogs.10 Both are highly active in indolent lymphoproliferative disorders. Cladribine is inactive in adult AML but has shown efficacy in children with AML. Fludarabine is active in adult AML, but only at high doses, resulting in severe neurotoxicity.11, 12

Clofarabine (2-chloro-2′-fluoro-2′-deoxy-9-β-D-arabinofuranosyladenine) has provided an impressive example of how new nucleoside analogs can contribute to our understanding of alternative metabolic pathways and mechanisms of action that may clarify as yet unexplained features of nucleosides with regard to the relation between structure, pharmacology, and activity. It is a next-generation deoxyadenosine analog that was synthesized as a rational extension of the experience with fludarabine and cladribine. Minor modifications in structure, such as halogenation at the 2-adenine and 2′ positions of the carbohydrate, resulted in clofarabine retaining the mechanistically favorable properties of both previous agents: potent inhibition of DNA polymerases and of RnR. Clofarabine has shown antitumor activity in preclinical models and in clinical trials. The current emphasis of clofarabine lies in the acute leukemias, but its activity in other disease groups is being investigated actively.

Pharmacology, Mechanisms of Action, and Metabolism

  1. Top of page
  2. Abstract
  3. Pharmacology, Mechanisms of Action, and Metabolism
  4. Preclinical Development
  5. Clinical Development
  6. Conclusions
  7. Note Added in Proof
  8. REFERENCES

Clofarabine is a rationally designed, second-generation, purine nucleoside analog (Fig. 1). Purine nucleoside analogs are made up of a purine base (e.g., adenine), which is linked to a deoxyribose sugar through a glycosidic bond. Deactivation of nucleosides occurs partly through deamination by adenosine deaminase and partly by cleavage of the glycosidic bond by the activity of the bacterial enzyme purine nucleoside phosphorylase (PNP). To create compounds with higher resistance to these deactivation mechanisms, a series of analogs was synthesized that incorporated a halogen group at the 2-position of the adenine.13, 14 Both fludarabine (2-fluoro-adenine aglycone) and cladribine (2-chloro-adenine aglycone) have been active against a number of indolent lymphoproliferative disorders (chronic lymphocytic leukemia [CLL], hairy cell leukemia, Waldenstrom macroglobulinemia). However, both deoxyadenosine analogs proved ineffective at clinically safe doses in adult acute leukemias. The functional consequences of minor modifications in the structure of these compounds led to the synthesis of a series of 2-halo-2′ halo-2′-deoxyarabinofuranosyladenine analogs, in which further substitution of fluorine at the arabinosyl configuration at the critical 2′-position of the carbohydrate (and retaining the 2-chloro adenine aglycone of cladribine) was more cytotoxic in cell cultures than substitution with either chlorine or bromine.15–17 Of 3 candidate molecules, clofarabine proved to be the most cytotoxic against P388 tumors in mice and was chosen for further evaluation in preclinical models. Similar to fludarabine and cladribine, clofarabine is highly resistant to deamination by adenosine deaminase. In addition, clofarabine is a poor substrate for the enzyme PNP, resulting in a high resistance to cleavage of the glycosidic bond. The 2′-flourine also makes clofarabine more acid stable, and these attributes (increased acid stability and poor substrate for PNP) increase its oral bioavailability.17–21

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Figure 1. The structure of clofarabine.

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Like its congeners fludarabine and cladribine, clofarabine requires intracellular phosphorylation by deoxycytidine (dCyd) kinase to its active triphosphate form for cytotoxic and therapeutic activity.18, 22 However, clofarabine appears to be a more efficient substrate for dCyd kinase, exceeding the affinity of dCyd kinase for other nucleoside analogs, such as cladribine.23 Retention of the triphosphate form of clofarabine in cells is longer than the retention of fludarabine or cladribine. Activity of clofarabine triphosphate is based on several mechanisms of action: 1) inhibition of DNA polymerases, 2) inhibition of RnR, and 3) induction of apoptosis through induction of DNA strand breaks and disruption of mitochondrial integrity, resulting in the release of proapoptotic proteins (cytochrome c, apoptosis-inducing factor).5, 24–27 The latter activity may be a factor in the cytotoxic effects of clofarabine toward nondividing lymphocytes. Whereas these mechanisms are shared with fludarabine or cladribine, significant differences exist: 1) The triphosphate of fludarabine primarily inhibits DNA polymerases after incorporation into DNA; 2) cladribine acts mainly by inhibition of RnR; 3) although antiapoptotic effects of all three purine nucleoside analogs occur by induction of DNA strand breaks, only clofarabine and cladribine have a direct impact on mitochondrial integrity and activity. Therefore, clofarabine represents not only a structural hybrid between fludarabine and cladribine but also a functional hybrid combining the mechanistically favorable properties of both congener compounds with regard to inhibition of DNA polymerases and RnR.28

Preclinical Development

  1. Top of page
  2. Abstract
  3. Pharmacology, Mechanisms of Action, and Metabolism
  4. Preclinical Development
  5. Clinical Development
  6. Conclusions
  7. Note Added in Proof
  8. REFERENCES

Clofarabine has shown cytotoxicity to a variety of human tumor cell lines derived from hematologic and solid tumor malignancies (Table 1).29–32 Clofarabine is a potent inhibitor of the L1210 mouse leukemia and K562 chronic myeloid leukemia (CML) blast cell lines. Exposure to 5 μM of clofarabine for 72 hours inhibited K562 cell growth by 50%. Incubation with 50 μM of clofarabine for 4 hours inhibited incorporation of thymidine into DNA by 50%. Incubation with 0.1 μM, 1.0 μM, or 10 μM of clofarabine for 4 hours depressed deoxyadenosine triphosphate (TP), deoxycytidine TP, and deoxyguanine TP (but not deoxythymidine TP pools).5, 13, 18, 29 Whereas the sensitivity of solid tumor cells was greater to clofarabine than to fludarabine, the sensitivity of leukemic cell lines to the cytotoxic effects of the nucleoside analogs was comparable between fludarabine and clofarabine but was more favorable for clofarabine compared with cladribine. Marked activity also was shown in murine and human tumor xenograft models (P388 leukemia, colon 36, and mammary 16/c).29 Curative activity was noted in both advanced and early colon carcinoma in the tumor model colon.36 Good activity also was noted in renal and breast tumor models but not in nonsmall cell tumors.22, 29 The compound also has shown cytotoxicity at nanomolar concentrations to normal lymphocytes and macrophages, making it potentially useful in the treatment of autoimmune diseases, indolent lymphoproliferative disorders, and allograft conditioning.19, 22

Table 1. Cytotoxicity of Clofarabine in Human Cell Linesa
NucleosideIC50 (μm)
ACHNCAKI-1SNB-7NCI-H23DLD-1SK-MEL-28K562CEMWI-38
  • IC50: 50% inhibitory concentration; ACHN and CAKI-1: renal carcinoma cell lines; SNB-7: central nervous system tumor; NCI-H23: nonsmall cell lung adenocarcinoma; DLD-1: colon adenocarcinoma; SK-MEL-28: melanoma; K562: chronic myelogenous leukemia in blast crisis; CEM: T-cell acute lymphoblastic leukemia; WI-38: normal fibroblasts.

  • a

    Data adapted from Waud WR, Schmid SM, Montgomery JA, Secrist JA III. Preclinical antitumor activity of 2--chloro-9-(2-deoxy-2-fluoro-b-D-arabinofuranosyl) adenine (C1-F-Ara-A). Nucleosides Nucleotides Nucleic Acids. 2000;19:447–460;31 and Plunkett W, Gandhi V. Purine and pyrimidine nucleoside analogs. In: Giaccone G, Schilsky R, Sondel P, editors. Cancer chemotherapy and biologic response modifiers, annual 19. Amsterdam: Elsevier Science, BV, 2001:21–45.32

Clofarabine0.110.290.290.2911.00.670.0280.156.8
Fludarabine6.441.054.045.070.039.00.580.295.1

In 1992, the group at The University of Texas M. D. Anderson Cancer Center became interested in developing clofarabine further and conducted the animal toxicology studies to define the starting dose in human trials (unpublished data). In mice, the use of intraperitoneal clofarabine showed that doses of 25–100 mg/kg (75–300 mg/m2) daily for 7 days were safe. In the first 2 studies in mice, all animals survived single daily doses of 100 mg/kg per day (300 mg/m2 per day) for 7 days. Thus, a dose of 75 mg/kg per day (or 225 mg/m2 per day) for 7 days was considered safe and is the presumed lethal dose to 10% (LD10) in rodents. This predicted a safe dose of 22 mg/m2 per day for 7 days in human patients (one-tenth the LD10 in rodents). In dogs, a clofarabine dose of 7.5 mg/kg per day for 5 days, equivalent to 150 mg/m2 intravenously daily for 5 days, was used. Severe myelosuppression and gastrointestinal toxicities (nausea and emesis) were observed in all dogs, which died on Days 5–8 of therapy. At autopsy, necrosis of the bowels was observed, which, in dogs, generally is indicative of septic shock. In a second dog study, 3 dogs were treated at 0.75 mg/kg clorfarabine intravenous daily for 5 days, equivalent to 15 mg/m2 per dose daily for 5 days: No toxicities occurred. Autopsies did not show any adverse events or organ damage. Therefore, toxicology studies predicted a safe dose of 15 mg/m2 per dose daily for 5 days for human Phase I clinical trials.

Clinical Development

  1. Top of page
  2. Abstract
  3. Pharmacology, Mechanisms of Action, and Metabolism
  4. Preclinical Development
  5. Clinical Development
  6. Conclusions
  7. Note Added in Proof
  8. REFERENCES

Phase I studies in adult and pediatric malignancies

Phase I studies were pioneered at The University of Texas M. D. Anderson Cancer Center in 1998 and were conducted to determine dose-limiting toxicities (DLT) and the maximum tolerated doses (MTD) of clofarabine for acute leukemias, chronic lymphoproliferative disorders (LPDs), and solid tumors in both adult and pediatric patients.24, 33 In both adult and pediatric studies, clofarabine was given as a 1-hour infusion daily for 5 days. However, the starting doses differed (15 mg/m2 per dose in adults and 11.25 mg/m2 per dose in children), partly because the adult study preceded the pediatric trial and served as a reservoir experience for subsequent clinical studies.

The first part of the adult study (n = 51 patients) defined the MTD and DLT in solid tumors (n = 13 patients) and LPDs (n = 6 patients).29 Accrual was then opened for acute leukemias (n = 32 patients) assuming myelosuppression as the most significant DLT during the initial part. Indeed, myelosuppression necessitated multiple dose deescalations for patients with solid tumors and LPDs. Eventually, clofarabine doses of 2 mg/m2 or 4 mg/m2 daily × 5 were suggested for Phase II studies in patients with solid tumors and patients with LPDs. Dose escalation in patients with leukemia was then started at 7.5 mg/m2 daily × 5 and proceeded through several dose levels up to a maximum of 55 mg/m2 daily × 5. At a dose of 55 mg/m2, Grade 3 hepatic toxicity was observed in 2 of 4 treated patients, so that the number of patients was expanded at the lower dose schedule of 40 mg/m2 daily for 5 days. Although none of the first 3 patients at that level had Grade 3 hepatic toxicity, 1 patient in the expanded cohort experienced Grade 3 hepatic toxicity, and 1 patient experienced Grade 2 hepatic toxicity. The schedule of clofarabine of 40 mg/m2 daily for 5 days was recommended for Phase II studies in adult patients with acute leukemias. This is significant, because the clofarabine dose for acute leukemias (using a daily × 5 schedule) was 20 times higher than the dose for solid tumors. The usual difference between the dose schedules of drugs in solid tumors (limited by myelosuppression) and in acute leukemias (limited by extramedullary toxicities) is usually three to eight times higher. The Phase I toxicity profile is summarized in Table 2.

Table 2. Toxicities ≥ Grade 3 in 25 Children and 32 Adults with Acute Leukemia: Phase I Studies by Dose Levela
ToxicityNo. of children/adults with toxicity at each dose level (mg/m2/dose)
7.511.2515.022.530.040.052.055.070.0
  • a

    Data adapted from Kantarjian HM, Gandhi V, Kozuch P, et al. Phase I clinical and pharmacology study of clofarabine in patients with solid and hematologic cancers. J Clin Oncol. 2003;21:1167–117329 and Jeha S, Gandhi V, Chan KW, et al. Clofarabine, a novel nucleoside analog, is active in pediatric patients with advanced leukemia. Blood. 2004;103:784–789.33

  • b

    Other includes fatigue, anorexia, edema, sinus congestion (in children), chest pain (in children), and tachycardia (in children).

Nausea/emesis—/00/01/0—/00/01/01/——/01/—
Diarrhea—/00/00/0—/00/01/00/——/00/—
Mucositis—/00/00/0—/00/00/00/——/00/—
Hepatic—/10/10/0—/00/04/42/——/21/—
Skin—/10/00/0—/00/00/00/——/01/—
Myalgias—/00/00/0—/00/00/00/——/00/—
Cardiac—/00/00/0—/00/00/10/——/10/—
Drug fever—/00/00/0—/00/00/00/——/00/—
Neurologic—/00/00/0—/00/00/00/——/00/—
Otherb—/00/00/0—/00/00/10/——/00/—
Total treated—/31/31/3—/32/36/1213/——/42/—

Objective responses were seen in 5 adults (16%) with acute leukemias (6% complete response [CR] rate; 9% rate of CR without platelet recovery to > 100 × 109/L [CRp]) (Table 3). Both patients who had CRs (1 patient with AML and 1 patient with acute lymphoblastic leukemia [ALL]) achieved their response at a clofarabine dose of 40 mg/m2, whereas the CRp responses occurred at doses of 11.25 mg/m2, 22.5 mg/m2, and 55 mg/m2. Of the 2 patients with CLL, 1 patient achieved a 50% decrease in the size of palpable lymphadenopathy, and the other patient achieved a 75% decrease in peripheral lymphocytosis, both at the 15 mg/m2 dose level. No objective responses occurred among any of the 13 patients with solid tumors.

Table 3. Phase I Response Rates to Clofarabine in 29 Adult Patients with Acute Leukemiaa
DiseaseNo. of patientsResponse: no. of patients (%)b
CRCRp (HI)PRTotal
  • CR: complete response; CRp: complete response without platelet recovery; HI: hematologic improvement; PR: partial response; AML: acute myeloid leukemia; ALL: acute lymphoblastic leukemia.

  • a

    Data adapted from Kantarjian HM, Gandhi V, Kozuch P, et al. Phase I clinical and pharmacology study of clofarabine in patients with solid and hematologic cancers. J Clin Oncol. 2003;21:1167–1173.29 Three patients with chronic myeloid leukemia in blast phase were not included in this table.

  • b

    A CR was defined according to established criteria. A CRp (HI) in adults was defined as a CR without platelet recovery ≥ 100 × 109/L. A PR was defined the same as a CR but with 6–25% bone marrow blasts.

AML161 (6)1 (6)0 (0)2 (12)
ALL131 (8)1 (8)0 (0)2 (16)
Total292 (7)2 (7)0 (0)4 (14)

The pediatric Phase I study was restricted to children with advanced acute leukemias.33 Six dose levels, starting at 11.25 mg/m2 and escalating up to a dose of 70 mg/m2, were evaluated in 25 patients (8 patients with AML and 17 patients with ALL). The treatment schedule was identical to that used in the adult study. The children were pretreated heavily: Five of the 8 patients with AML and 4 of 17 patients with ALL developed disease recurrences after undergoing prior stem cell transplantation. Reversible DLTs at the 70 mg/m2 dose level occurred in 2 patients (≥ Grade 3 hyperbilirubinemia/transaminitis, skin rash), so that the number of patients was expanded at the next lower dose level of 52 mg/m2. Among 13 patients who received that dose, only 2 patients experienced Grade 3 elevation in transaminases, and another 3 patients experienced Grade 2 hyperbilirubinemia. In all of these episodes, hepatotoxicity was reversible and resolved by Day 14 of therapy. Toxicities are summarized in Table 2. Unlike the experience in the adult studies, some of the children experienced irritability when clofarabine was given in the established 1-hour infusion time, a phenomenon that disappeared with longer infusion times of 2 hours. This led to the recommendation of a clofarabine dose of 52 mg/m2 intravenously over 2 hours daily for 5 days for subsequent Phase II studies in children.

Responses occurred in 8 patients (32%) (Table 4). Five patients achieved a CR (4 patients with ALL and 1 patient with AML), and there were 3 partial responses (defined as 6–25% bone marrow blasts with an absolute neutrophil count [ANC] > 0.5 × 109/L and platelet counts > 25 × 109/L; 1 patient with ALL and 2 patients with AML). One of the patients who achieved a CR had a diagnosis of Philadelphia chromosome-positive ALL received clofarabine as fourth salvage therapy (including prior transplantation) at a dose of 30 mg/m2 and persisted in CR for 70 weeks. The remaining CRs occurred at the 40 mg/m2 and 52 mg/m2 dose levels.

Table 4. Phase I Response Rates to Clofarabine in 25 Children with Acute Leukemiaa
DiseaseNo. of patientsResponse: no. of patients (%)b
CRHIPRTotal
  • CR: complete response; HI: hematologic improvement; PR: partial response; AML: acute myeloid leukemia; ALL: acute lymphoblastic leukemia.

  • a

    Data adapted from Jeha S, Gandhi V, Chan K W, et al. Clofarabine, a novel nucleoside analog, is active in pediatric patients with advanced leukemias. Blood. 2004;103:784–789.33

  • b

    A CR was defined according to established criteria. HI included patients with bone marrow CR or PR without peripheral blood recovery (including neutrophils). A PR was defined as 6–25% bone marrow blasts, an absolute neutrophil count > 0.5 × 109/L, and a platelet count > 25 × 109/L.

AML81 (13)1 (13)2 (25)4 (50)
ALL174 (24)3 (18)1 (6)8 (48)
Total255 (20)4 (16)3 (12)12 (48)

Plasma and cellular pharmacology studies accompanied the Phase I studies and are summarized in part below.29, 30, 33, 34 Although heterogeneity among individuals was common, in both trials, dose-dependent increases of clofarabine in plasma and accumulation of clofarabine triphosphate in leukemic blasts could be demonstrated. Comparison of the triphosphate concentration in leukemic blasts of each patient at the end of the infusion relative to that present at 24 hours indicated retention of > 50% of the initial concentration of clofarabine triphosphate at 24 hours, irrespective of lymphoid or myeloid lineage. This slow elimination of clofarabine triphosphate from blast cells is in contrast to what has been observed with other nucleoside analogs, such as fludarabine, cladribine, and even cytarabine, in AML cells. The ability of the cells to retain the active clofarabine compound has been related to its inhibitory activity on DNA synthesis. This influenced decisions about scheduling and combination strategies with other agents, which will become obvious from subsequent clinical studies.

Keeping heterogeneity among individuals as limitations in mind, a few key points have emerged with regard to clofarabine pharmacology in humans29, 30, 34: 1) Peak levels of clofarabine in plasma occur at the end of the infusion. 2) There is a dose-proportional increase in the plasma clofarabine concentration with median plasma clofarabine levels of 1.5 μM administering clofarabine at the acute leukemia MTD of 40 mg/m.2 3) A dose-proportional accumulation has been shown for intracellular clofarabine triphosphate, although, at clofarabine doses > 30 mg/m2, this proportionality was lost with no further increases; the saturation in the rates of triphosphate accumulation in cells has been explained by the dCyd kinase activity as the rate-limiting step, although evidence suggests that clofarabine triphosphate accumulation is limited by the rate of phosphorylation of the monophosphate to the diphosphate compound.25, 27 4) Whereas no difference has been observed between myeloid and lymphoid leukemic blasts, responding patients accumulated higher levels of the triphosphate metabolite; leukemia blasts retained > 50% of the initial concentration of clofarabine triphosphate at 24 hours after the first infusion. This observation also is significant in that subsequent infusions resulted in incremental increases in the levels of the active metabolite in the blasts of responding patients. 5) Finally, dose-responsive duration of inhibition of DNA synthesis in acute leukemia blasts resulted in a decrease of circulating leukemia cells.

Development of clofarabine in acute leukemias and myelodysplastic syndromes

Single-agent Phase II studies in adult acute leukemias and myelodysplastic syndromes.

Following the lead of the Phase I studies, we conducted a Phase II study of clofarabine in 62 patients with recurrent and refractory AML (n = 31 patients), myelodysplastic syndrome (MDS) (n = 8 patients), ALL (n = 12 patients), and CML in myeloid blast phase (n = 11 patients).34 All patients received clofarabine at an intravenous dose of 40 mg/m2 daily for 5 days every 3–6 weeks, depending on blood recovery and leukemia response. Overall, 48% of the patients responded: There were 20 CRs (32%), 1 partial response, and 9 patients who had hematologic improvements (15%) (Table 5). Responses differed by diagnosis, salvage status, and duration of first remission. Of the 31 patients with AML, 13 patients (42%) achieved a CR, and 4 patients (13%) achieved a CRp, for an overall response rate of 55%. Response rates were higher in patients who had preceding CR durations ≥ 12 months and in patients who received clofarabine in first salvage. Among the 6 patients with MDS, 2 patients achieved a CR, and 2 patients achieved a CRp, for an overall response rate of 50%. Overall response rates were 58% in patients with CML in myeloid blast phase (including 4 patients who achieved a hematologic CR) and 16% in patients with ALL (including 1 patient who achieved a CR).

Table 5. Phase II Response Rates to Single-Agent Clofarabine in Adults with Acute Leukemiaa
DiseaseNo. of patientsResponse: no. of patients (%)
CRCRp (HI)/PROR
  • CR: complete response; CRp: complete response without platelet recovery; HI: hematologic improvement; PR: partial response; OR: overall response; AML: acute myeloid leukemia; MDS: myelodysplastic syndrome; ALL: acute lymphoblastic leukemia; CML BP: chronic myeloid leukemia in blast phase.

  • a

    Data adapted from Kantarjian H, Gandhi V, Cortes J, et al. Phase 2 clinical and pharmacologic study of clofarabine in patients with refractory or relapsed acute leukemia. Blood. 2003;102:2379–2386.34

AML3113 (42)4 (13)19 (55)
MDS82 (25)2 (25)4 (50)
ALL121 (8)1 (8)2 (17)
CML BP114 (36)3 (27)7 (64)

Common adverse events followed the pattern observed during the Phase I study. Most frequently observed were transient liver dysfunction, skin rashes, palmoplantar ertythrodysesthesia, and mucositis. Liver dysfunctions included both hyperbilirubinemia and/or elevation of transaminases. Typically, they started around Day 5 and resolved by Day 10–15 of therapy.

Pharmacokinetic studies confirmed individual heterogeneity with regard to the accumulation of plasma clofarabine and intracellular clofarabine triphosphate in all disease groups.34 When measured at the end of the infusion and then again at 24 hours just prior to the second infusion, clofarabine triphosphate levels were maintained in the leukemia cells, indicating an elimination half-life ≥24 hours. A corollary of this observation is that there is incremental accumulation of clofarabine triphosphate when clofarabine continues to be given every 24 hours for up to 5 days. Furthermore, early data indicate a difference between responding and nonresponding patients with regard to the accumulation of clofarabine triphosphate. Responding patients displayed higher levels of clofarabine triphosphate at the end of the first 24-hour infusion and at the end of the first 24-hour period just prior to the next infusion of clofarabine. In essence, responding patients showed greater accumulation and higher incremental increases in clofarabine triphosphate during the 5-day treatment course.

A second Phase II multicenter study was conducted in the U.S. in adult patients with refractory and recurrent AML.35 The dose and schedule were identical to the Phase II dose and schedule established by The University of Texas M. D. Anderson Cancer Center study. Among 15 patients who were treated, 1 CR was reported, 5 patients were discontinued from the study for lack of response, and the response status in the remaining patients was not known at the time the data were presented. Differences in responses and outcomes between the Phase II studies await further confirmation but may be attributed to different eligibility criteria and patient selection.

Developing mechanism-based combination therapies.

Phase I and II studies of single-agent clofarabine established its dose schedule and confirmed its activity in leukemias. An important extension of these trials is the combination of clofarabine with other active antileukemia agents based on the metabolic and pharmacokinetic properties of clofarabine. Emphasis in AML shifted to combinations with cytarabine and anthracyclines.

Cytarabine is one of the most active antileukemic agents and is the backbone of many combination regimens in patients with AML. Biochemical modulation strategies that aim at increasing intracellular nucleoside concentrations, such as cytarabine triphosphate (ara-CTP), have been tested and validated clinically in combinations of cytarabine with fludarabine in adults with AML and with cladribine in children with AML.36–39 Synergy between cytarabine and clofarabine has been demonstrated in vitro.40 K562 cells were incubated with cytarabine alone, with cytarabine plus clofarabine at the same time, and with clofarabine followed 3 hours later by cytarabine. Whereas concomitant incubation resulted in an actual decrease in the amount of intracellular ara-CTP, clofarabine followed by cytarabine 3 hours later caused an up to 2-fold increase of intracellular ara-CTP levels relative to cells that were incubated with cytarabine alone. The possible mechanism by which biochemical modulation may occur is summarized in Figure 2. In short, the rate-limiting step in the synthesis of ara-CTP is catalyzed by dCyd kinase. The activity of dCyd kinase, in turn, is regulated by intracellular levels of deoxynucleotide triphosphates (dNTPs). By combining clofarabine with ara-C, inhibition of RnR by clofarabine triphosphate will result in a decrease in the levels of dNTPs, causing a subsequent decrease in the feedback inhibition of dCyd kinase. Therefore, in addition to having antileukemic activity by itself, the activity of clofarabine and cytarabine may be enhanced in leukemic cells by a biochemical synergy between these two agents.

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Figure 2. Overview of the biochemical synergy between clofarabine and cytarabine. Cytarabine is phosphorylated intracellularly. The rate-limiting step occurs between cytarabine and its conversion to the monophosphate form by the enzyme deoxycytidine kinase (dCyd). The activity of dCyd in turn is regulated by the intracellularly available pool of deoxynucleotide phosphates (dNTP). This pool is regulated by the key enzyme ribonucleotide reductase (RnR). High levels of dCTP inhibit the activity of dCyd. Clofarabine is a strong inhibitor of RnR, leading to depletion of dCTP and removal of its feedback inhibition of dCyd, resulting in increased dCyd activity and increasing levels of cytarabine triphosphate (ara-CTP). Ara-C: cytosine arabinoside.

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Clofarabine plus cytarabine combination studies.

The first study of clofarabine plus cytarabine was designed as a combined Phase I and II study in adults with acute leukemias and high-risk MDS who either were in first recurrence or who were primary refractory to the respective induction regimen.41 The cytarabine dose of 1 g/m2 per day intravenously on Days 2–5 remained constant; clofarabine was dose escalated during the Phase I part starting at 15 mg/m2 intravenously daily on Days 2–6 and proceeding through dose levels of 22.5 mg/m2, 30.0 mg/m2, and 40.0 mg/m2. Because no DLTs occurred during the Phase I component, an ara-C dose of 1 g/m2 and a clofarabine dose of 40 mg/m2 were recommended for Phase II studies. Among 32 patients in the study, 25 patients had AML, 4 patients had MDS, 2 patients had ALL, and 1 patient had CML in myeloid blast phase. Their median age was 60 years (range, 18–84 years). Of the 29 patients who had AML and MDS, 7 patients (24%) achieved a CR, and 5 patients (17%) achieved a CRp, for an overall response rate of 41%. With a median follow-up of 14 months (range, 5.5–15.0 months), the median remission duration (CR plus CRp) was 3.2 months (range, 0.5–14.0 months), the median survival was 5.5 months (range, 0.2–15.0 + months) overall and 8 months (range, 2–8 + months) in responders. The combination regimen was safe without additional or unexpected adverse events, and there was a low early death rate (i.e., within 4 weeks of therapy) of 3%. Liver function abnormalities of any grade were observed in up to 90% of patients but usually were transient and were < Grade 3 according to National Cancer Institute Common Toxicity Criteria. Plasma and cellular pharmacokinetic studies showed that 1) plasma clofarabine levels increased with the clofarabine dose without evidence of nonlinearity, 2) the infusion of cytarabine did not affect plasma clofarabine levels significantly, and 3) five of eight patients studied demonstrated an increase in ara-CTP levels after the clofarabine infusion.

Clofarabine in elderly AML.

The encouraging results of clofarabine in acute leukemias led to its use alone or in combination in older patients with newly diagnosed AML and high-risk MDS. Choosing elderly patients with AML was justified for several reasons: 1) their poor prognosis and 2) the poor results (CR rates of 30–50%) and higher mortality rates (up to 50%) with standard intensive chemotherapy (e.g., “3 + 7.”)42–44 Worse outcomes in elderly patients with AML are due to 1) different AML biology (higher incidence of unfavorable karyotype, a higher rate of primary drug resistance associated with overexpression of P-glycoprotein, and a higher incidence of antecedent hematologic disorders, such as MDS) and 2) poor tolerance of traditional combination chemotherapy regimens. The development path of clofarabine, therefore, may have targeted a suitable group of patients for whom new approaches are needed and are investigated actively.

We first designed the clofarabine/cytarabine combination as frontline treatment for older patients with newly diagnosed AML.45 The dose and schedule were identical to those used in the prior salvage study. Up to 3 induction cycles were permitted followed by up to 6 maintenance courses with identical daily doses for 3 days every 4–6 weeks. Among 60 patients who have received treatment, 28 patients (47%) had either secondary AML with an antecedent hematologic disorder (MDS) or another prior malignancy; 28 patients (47%) had poor-prognosis karyotypes (trisomy 8, del[11], abnormalities of chromosomes 5 and/or 7). Two-thirds of patients were age ≥ 60 years. Overall, 36 patients (60%) responded (52% CR rate and 8% CRp rate). The response rate was higher in patients with diploid cytogenetics (60% CR rate vs. 43% CR rate). When considering the patients with diploid cytogenetics and Flt3 internal tandem duplications or mutations, 5 of 11 patients (45%) achieved a CR: The CR rate was 55% in patients with diploid karyotype and without detectable Flt3 abnormalities. Most responses occurred after one induction course. Among 11 patients who received a second induction course, 2 patients achieved a CR, and 2 patients achieved a CRp. Four patients died during induction (induction mortality rate, 7%). The most common adverse events included transient liver function abnormalities, rashes, and palmoplantar dysesthesias.

To validate the position of clofarabine and cytarabine combinations in AML therapy, further follow-up and comparison with standard AML induction regimens eventually will be needed.

Burnett et al. developed the use of single-agent clofarabine in elderly patients with AML using a clofarabine dose of 30 mg/m2 intravenously daily for 5 days every 28 days in patients who were not suitable to receive intensive chemotherapy and any patient age > 70 years.46 In their study, among 27 evaluable patients, 16 patients (59%) achieved a CR, making clofarabine probably 1 of the more active anti-AML agents. Five patients died before response could be assessed in that study, and another five patients did not enter remission, although a reduction in bone marrow blasts was observed in three of those patients.

Based on this experience, we initiated a randomized study of clofarabine at a dose of 30 mg/m2 daily × 5 with or without low-dose cytarabine (20 mg/m2 subcutaneously daily × 7–14). The rationale for using low-dose cytarabine was based on data from a randomized trial by the leukemia group in the U.K. that showed a superior CR rate (15% vs. 0%) and survival rates (30% at 1 year vs. < 5%) with low-dose cytarabine versus hydroxyurea (the proposed standard of care).

Other combination trials in adult acute leukemia.

Anthracyclines (mostly idarubicin) commonly are combined in AML therapy with nucleoside analogs, such as cytarabine, and are part of the standard induction regimens. A clear benefit has been demonstrated when cytarabine is combined with anthracyclines. This benefit can be based on 1) a direct cytotoxic activity of the anthracyclines, 2) anthracycline activity resulting in DNA strand breaks (the combination with nucleoside analogs synergizes its activity by inhibiting DNA repair damage), and 3) in vitro evidence that anthracyclines can potentiate the activity of cladribine in murine leukemia models. With cladribine and idarubicin combinations, a 60% increase of the life span of leukemic mice was observed, which was significantly better compared with the increase achieved with monotherapy schedules. Based on the activity of idarubicin in AML combination regimens and the preclinical evidence of possible synergism, a Phase I study of combined clofarabine and idarubicin versus combined clofarabine, idarubicin, and cytarabine is in progress in patients with recurrent and refractory AML and high-risk MDS.47

Pediatric Phase II studies in acute leukemias.

Phase II studies currently are under way separately in children with AML and ALL.48 In the pediatric Phase II studies, clofarabine was given at a dose of 52 mg/m2 daily for 5 days every 2–6 weeks, depending on toxicities and leukemia response. Thirty-five children with AML have been treated. Their median age was 11 years (range, 2–22 years); all patients were pretreated heavily (median, 3 prior regimens; range, 1–6 prior regimens). It is noteworthy that nearly half of all children had undergone and failed prior stem cell transplantation. Responses in the pediatric Phase II studies are summarized in Table 6. The overall response rate was 26% in children with AML (1 CRp and 8 partial responses). Among the 49 evaluable children with ALL, the median age was 12 years (range, 1–19 years). Similar to the AML study group, patients with ALL were pretreated heavily (median, 3 prior regimens; range, 1–6 prior regimens), and 20% had undergone prior stem cell transplantation. Six patients achieved a CR, 4 patients had a CRp, and 5 patients had a partial response, for an overall response rate of 31% (Table 6). The median survival was 42 weeks (range, 7.0–63.1 + weeks) for patients with ALL who responded and 39 weeks (range, 7.7–93.6 + weeks) for patients with AML who responded. Overall, 13 of 24 responding patients (54%) were able to undergo a subsequent stem cell transplantation. Toxicities were comparable in both disease groups and consisted mainly of nausea, emesis, fever, myelosuppression, skin rashes, hand-foot syndrome, and transient elevations of liver transaminases. Although preliminary, these data confirm the positive experience from the pediatric Phase I trial. Clofarabine combination trials in children are in development.

Table 6. Phase II Single-Agent Clofarabine Response Rates in Children with Acute Leukemiaa
DiseaseNo. of patientsNo. of prior regimens (range)Prior SCT (%)Response: no. of patients (%)
CRCRpPROR
  • SCT: stem cell transplantation; CR: complete response; CRp: complete response without platelet recovery; PR: partial response; OR: overall response; AML: acute myeloid leukemia; ALL: acute lymphoblastic leukemia.

  • a

    Data adapted from Jeha S, Razzouk B, Rytting ME, et al. Phase II trials of clofarabine in relapsed or refractory pediatric leukemia. Blood. 2004;104:196a.48

AML352 (1–6)531 (3)8 (23)9 (26)
ALL493 (1–6)206 (12)4 (8)5 (11)15 (31)

Clofarabine in LPDs and solid tumors

Experience with clofarabine in patients with LPDs and with solid tumors is very limited. Six patients with CLL or non-Hodgkin lymphomas were treated as part of the initial Phase I study at The University of Texas M. D. Anderson Cancer Center.29 Given at the acute leukemia schedule of clofarabine daily for 5 days with the starting dose of 15 mg/m2, 2 patients with CLL experienced Grade 4 myelosuppression. Significant myelosuppression continued to occur with further dose deescalations of 7.5 mg/m2 and 4 mg/m2. Eventually, at a dose of 2 mg/m2 daily × 5, myelosuppression was observed in 1 patient with a solid tumor and in 1 patient with follicular lymphoma. The MTD was 4 mg/m2 and 2 mg/m2 daily for 5 days for indolent leukemia and solid tumors, respectively, substantially lower than the MTD in patients with acute leukemias. Although no objective responses were observed, evidence of biologic activity was seen even at the lower doses. A 50% decrease in lymphadenopathy and a 75% decrease in peripheral lymphocytosis were observed in 2 patients with CLL who were on 15 mg/m2 of clofarabine. In both patients, CLL was refractory to multiple therapies, including fludarabine. Stable disease for 9 months was achieved in 1 patient with metastatic breast carcinoma who received clofarabine at a dose of 7.5 mg/m2.

Although the numbers were small, the daily × 5 schedule was not suitable for patients with LPDs and solid tumors to achieve high levels of clofarabine triphosphate. Changing the schedule from daily × 5 every 4 weeks to an administration of clofarabine once weekly may allow increased dose delivery of clofarabine, avoiding significant dose-limiting myelosuppression. Based on this expectation and on the comparatively long elimination half-life of clofarabine triphosphate in circulating cells, we recently started a new Phase I study of clofarabine given weekly for 3 weeks every 4 weeks for patients with CLL. In this respect, with clofarabine, we follow the lead from experience with earlier nucleoside analogs, such as gemcitabine, with regard to the importance of schedule, dose, and route of administration (e.g., bolus vs. continuous infusion). The starting dose chosen was 20 mg/m2 per week, below the dose that was been reached in another solid tumor Phase I study (> 100 mg/m2) using the same modified schedule. The role of clofarabine and its optimal dose schedule in patients with LPDs and solid tumors remains to be defined. An oral preparation of clofarabine is under development. Oral bioavailability of 50% has been reported. An oral agent would open further possibilities of testing improved schedules not only in patients with AML but also in patients with MDS, LPDs, and solid tumors.

Conclusions

  1. Top of page
  2. Abstract
  3. Pharmacology, Mechanisms of Action, and Metabolism
  4. Preclinical Development
  5. Clinical Development
  6. Conclusions
  7. Note Added in Proof
  8. REFERENCES

Research in nucleoside analogs has been among the most fruitful endeavors in the quest for antitumor agents. The last few years have seen the emergence of additional compounds with new metabolic properties and mechanisms of action. Clofarabine is a next-generation nucleoside analog that exemplifies how investigations of new compounds may help understand the still unexplained characteristics of nucleoside analogs with regard to correlations between structure, activity, and pharmacokinetic properties. Clofarabine is the first of the new generation with established single-agent antileukemic activity at tolerable doses. The development of clofarabine has been carried forward through adult and pediatric Phase II studies and in combination strategies based on biochemical modulation with cytarabine. Future studies hopefully will validate a role for clofarabine in hematologic and solid tumors.

Note Added in Proof

  1. Top of page
  2. Abstract
  3. Pharmacology, Mechanisms of Action, and Metabolism
  4. Preclinical Development
  5. Clinical Development
  6. Conclusions
  7. Note Added in Proof
  8. REFERENCES

Clofarabine has been approved by the U.S. Food and Drug Administration in December 2004 as treatment for children with relapsed ALL who have received at least two prior regimens.

REFERENCES

  1. Top of page
  2. Abstract
  3. Pharmacology, Mechanisms of Action, and Metabolism
  4. Preclinical Development
  5. Clinical Development
  6. Conclusions
  7. Note Added in Proof
  8. REFERENCES
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