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Discovery and development of clofarabine: a nucleoside analogue for treating cancer

Key Points

  • Acute leukaemia is the most common paediatric cancer, with acute lymphoblastic leukaemia (ALL) and acute myelogenous leukaemia (AML) being the two most common types.

  • Despite the progress that has been made in the treatment of paediatric leukaemias and the high overall survival rate, a major need still exists for alternative therapies that are not cross-resistant to other cytotoxic agents and do not have overlapping toxicities with currently available drugs.

  • Clofarabine was discovered through a programme searching for new nucleoside anticancer drugs in which novel analogues of existing agents cladribine and fludarabine were iteratively designed, synthesis and screened with a view to addressing known deficiencies of the prior agents.

  • Following entry into cells, clofarabine is phosphorylated in a stepwise manner by cytosolic kinases to the nucleotide analogues, clofarabine 5′-mono-, di-, and triphosphate, with clofarabine triphosphate being the active form.

  • Once the triphosphate moiety is formed, the anticancer activity of clofarabine is believed to be due to three mechanisms: potent competition with dATP for DNA polymerase α and ε, which then incorporate clofarabine-monophosphate into internal and terminal DNA sites, impairing DNA elongation and/or repair; potent inhibition of ribonucleotide reductase, limiting DNA synthesis; and induction of apoptosis.

  • Preclinical models showed that clofarabine has a broad spectrum of antitumour activity in preclinical models of leukaemia and solid tumour malignancies and that its mechanism of activity was different from that of other nucleoside analogues, such as fludarabine and cladribine.

  • Based on the promising data seen in Phase I studies in paediatric patients with hematologic malignancies, interest by investigators and physicians, and positive feedback by the FDA, development for paediatric patients with ALL and AML was prioritized.

  • On the basis of Phase II studies, clofarabine became the first new drug approved by the FDA for the treatment of paediatric acute leukaemia in over a decade and was recently approved in the European Union for the same indication.

Abstract

The treatment of acute leukaemias, which are the most common paediatric cancers, has improved considerably in recent decades, with complete response rates approaching 90% in some cases. However, there remains a major need for treatments for patients who do not achieve or maintain complete remission, for whom the prognosis is very poor. In this article, we describe the challenges involved in the discovery and development of clofarabine, a second-generation nucleoside analogue that received accelerated approval from the US FDA at the end of 2004 for the treatment of paediatric patients 1–21 years old with relapsed or refractory acute lymphoblastic leukaemia after at least two prior regimens. It is the first such drug to be approved for paediatric leukaemia in more than a decade, and the first to receive approval for paediatric use before adult use.

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Figure 1: Structure of clofarabine and related nucleoside analogues.
Figure 2: Schematic of clofarabine mechanism of action.

References

  1. Pui, C. H. Childhood leukemias. N. Engl. J. Med. 332, 1618–1630 (1995).

    Article  CAS  Google Scholar 

  2. Nachman, J. B. et al. Augmented post-induction therapy for children with high-risk acute lymphoblastic leukemia and a slow response to initial therapy. N. Engl. J. Med. 338, 1663–1671 (1998).

    Article  CAS  Google Scholar 

  3. Nachman, J. et al. Augmented Berlin–Frankfurt–Munster therapy abrogates the adverse prognostic significance of slow early response to induction chemotherapy for children and adolescents with acute lymphoblastic leukemia and unfavorable presenting features: a report from the Children's Cancer Group. J. Clin. Oncol. 15, 2222–2230 (1997).

    Article  CAS  Google Scholar 

  4. Kersey, J. H. Fifty years of studies of the biology and therapy of childhood leukemia. Blood 90, 4243–4251 (1997).

    CAS  Google Scholar 

  5. Sadowitz, P. D. et al. Treatment of late bone marrow relapse in children with acute lymphoblastic leukemia: a Pediatric Oncology Group study. Blood 81, 602–609 (1993).

    CAS  PubMed  Google Scholar 

  6. Henze, G. et al. Six-year experience with a comprehensive approach to the treatment of recurrent childhood acute lymphoblastic leukemia (ALL-REZ BFM 85). A relapse study of the BFM group. Blood 78, 1166–1172 (1991).

    CAS  PubMed  Google Scholar 

  7. Stahnke, K. et al. Duration of first remission predicts remission rates and long-term survival in children with relapsed acute myelogenous leukemia. Leukemia 12, 1534–1538 (1998).

    Article  CAS  Google Scholar 

  8. Bosi, A. et al. Second allogeneic bone marrow transplantation in acute leukemia: a multicenter study from the Gruppo Italiano Trapianto Di Midollo Osseo (GITMO). Leukemia 11, 420–424 (1997).

    Article  CAS  Google Scholar 

  9. Henze, G. et al. BFM group treatment results in relapsed childhood acute lymphoblastic leukemia. Haematol. Blood Transfus. 33, 619–626 (1990).

    CAS  PubMed  Google Scholar 

  10. Barrett, A. J. et al. Bone marrow transplants from HLA-identical siblings as compared with chemotherapy for children with acute lymphoblastic leukemia in a second remission. N. Engl. J. Med. 331, 1253–1258 (1994).

    Article  CAS  Google Scholar 

  11. Sierra, J. et al. Transplantation of marrow cells from unrelated donors for treatment of high-risk acute leukemia: the effect of leukemic burden, donor HLA-matching, and marrow cell dose. Blood 89, 4226–4235 (1997).

    CAS  PubMed  Google Scholar 

  12. Lindemalm, S., Liliemark, J., Juliusson, J., Larsson, R. & Albertioni, F. Cytotoxicity and pharmacokinetics of cladribine metabolite, 2-chloroadenine, in patients with leukemia. Cancer Lett. 210, 171–177 (2004).

    Article  CAS  Google Scholar 

  13. Avramis, V. I. & Plunkett, W. 2-Fluoro-ATP: a toxic metabolite of 9-β-D-arabinoxyl-2-fluroadenine. Biochem. Biophys. Res. Commun. 113, 35–43 (1983).

    Article  CAS  Google Scholar 

  14. Montgomery, J. A. & Secrist, J. A. III. 2-Fluoro-9-(2-fluoro-β-D-arabinofuranosyl)adenine nucleosides. US Patent 5,034,581 (1991).

  15. Montgomery, J. A. & Secrist, J. A. III. 2-Fluoro-2-haloarabinoadenosines and their pharmaceutical compositions. US Patent 5,384,310 (1995).

  16. Montgomery, J. A. & Secrist, J. A. III. US Patent 5,661,136 (1997).

  17. Montgomery, J. A., Shortnacy-Fowler, A. T., Clayton, S. D., Riordan, J. M. & Secrist, J. A. III. Synthesis and biologic activity of 2′-fluoro-2-halo derivatives of 9-β-D-arabinofuranosyladenine. J. Med. Chem. 35, 397–401 (1992).

    Article  CAS  Google Scholar 

  18. King, K. M. et al. A comparison of the transportability, and its role in cytotoxicity, of clofarabine, cladribine, and fludarabine by recombinant human nucleoside transporters produced in three model expression systems. Mol. Pharmacol. 69, 346–353 (2006). This report shows that the efficiency of cellular uptake of clofarabine by nucleoside transporters was hCNT3>hENT2>hENT1>hCNT2. No uptake was observed with hCNT1.

    CAS  PubMed  Google Scholar 

  19. King, K. M. et al. Nucleoside transporter proteins are determinants of cytotoxicity of clofarabine (Clofarex) in cultured leukemic cell lines. Blood 100, A1247 (2002).

    Google Scholar 

  20. Parker, W. B. et al. Effects of 2-chloro-9-(2-deoxy-2-fluoro-β-D-arabinofuranosyl)adenine on K562 cellular metabolism and the inhibition of human ribonucleotide reductase and DNA polymerases by its 5′-triphosphate. Cancer Res. 51, 2386–2394 (1991).

    CAS  PubMed  Google Scholar 

  21. Eriksson, S. et al. Properties and levels of deoxynucleoside kinases in normal and tumor cells; implications for chemotherapy. Adv. Enzyme Regul. 34, 13–25 (1994).

    Article  CAS  Google Scholar 

  22. Arner, E. S. & Eriksson, S. Mammalian deoxyribonucleoside kinases. Pharmacol. Ther. 67, 155–186 (1995). A thorough review of the biochemisty and molecular biology of a clofarabine-activating enzyme, deoxycytidine kinase.

    Article  CAS  Google Scholar 

  23. Lotfi, K. et al. Biochemical pharmacology and resistance to 2-chloro-2′-arabino-fluoro-2′-deoxyadenosine, a novel analogue of cladribine in human leukemic cells. Clin. Cancer Res. 5, 2438–2444 (1999).

    CAS  PubMed  Google Scholar 

  24. Parker, W. B. et al. Comparison of the mechanism of cytotoxicity of 2-chloro-9-(2-deoxy-2-fluoro-β-D-arabinofuranosyl)adenine, 2-chloro-9-(2-deoxy-2-fluoro-β-D-ribofuranosyl)adenine, and 2-chloro-9-(2-deoxy-2,2-difluoro-β-D-ribofuranosyl)adenine in CEM cells. Mol. Pharmacol. 55, 515–520 (1999).

    CAS  PubMed  Google Scholar 

  25. Spasokoukotskaja, T. et al. Activation of deoxycytidine kinase by various nucleoside analogues. Adv. Exp. Med. Biol. 431, 641–645 (1998).

    Article  CAS  Google Scholar 

  26. Ruiz van Haperen, V. W. & Peters, G. J. New targets for pyrimidine antimetabolites for the treatment of solid tumours. 2: Deoxycytidine kinase. Pharm. World Sci. 16, 104–112 (1994).

    Article  CAS  Google Scholar 

  27. Xie, C. & Plunkett, W. Metabolism and actions of 2-chloro-9-(2-deoxy-2-fluoro-β-D-arabinofuranosyl)-adenine in human lymphoblastoid cells. Cancer Res. 55, 2847–2852 (1995). This report shows clofarabine is inserted into internal and terminal sites of DNA and the accumulation in nucleotide metabolites is independent of cell cycle.

    CAS  PubMed  Google Scholar 

  28. Xie, K. C. & Plunkett, W. Deoxynucleotide pool depletion and sustained inhibition of ribonucleotide reductase and DNA synthesis after treatment of human lymphoblastoid cells with 2-chloro-9-(2-deoxy-2-fluoro-β-D-arabinofuranosyl) adenine. Cancer Res. 56, 3030–3037 (1996). This report demonstrates that clofarabine is a potent inhibitor of ribonucleotide reductase which produces a prolonged decrease in dNTP pools and inhibition of DNA synthesis.

    CAS  PubMed  Google Scholar 

  29. Genini, D. et al. Deoxyadenosine analogs induce programmed cell death in chronic lymphocytic leukemia cells by damaging the DNA and by directly affecting the mitochondria. Blood 96, 3537–3543 (2000). This report shows clofarabine directly interferes with mitochondrial integrity and possesses three modes of cytotoxic action in non-proliferating CLL cells.

    CAS  PubMed  Google Scholar 

  30. Carson, D. A. et al. Oral antilymphocyte activity and induction of apoptosis by 2-chloro-2′-arabino-fluoro-2′-deoxyadenosine. Proc. Natl Acad. Sci. USA 89, 2970–2974 (1992).

    Article  CAS  Google Scholar 

  31. Waud, W. R., Schmid, S. M., Montgomery, J. A. & Secrist, J. A., III. Preclinical antitumor activity of 2-chloro-9-(2-deoxy-2-fluoro-β-D- arabinofuranosyl)adenine (Cl-F-ara-A). Nucleosides Nucleotides Nucleic Acids 19, 447–460 (2000). This report shows clofarabine has significant activity against a wide spectrum of preclinical tumor models.

    Article  CAS  Google Scholar 

  32. Takahashi, T., Kanazawa, J., Akinaga, S., Tamaoki, T. & Okabe, M. Antitumor activity of 2-chloro-9-(2-deoxy-2-fluoro-β-D-arabinofuranosyl) adenine, a novel deoxyadenosine analog, against human colon tumor xenografts by oral administration. Cancer Chemother. Pharmacol. 43, 233–240 (1999).

    Article  CAS  Google Scholar 

  33. Stephenson, K. et al. Correlation between frequency of administration and efficacy of clofarabine in the H460 human non-small cell lung tumor xenograft model. Proc. Am. Assoc. Cancer Res. 44, A814 (2003).

    Google Scholar 

  34. Qian, M., Wang, X., Shanmuganathan, K., Chu, C. K. & Gallo, J. M. Pharmacokinetics of the anticancer agent 2-chloro-9-(2-deoxy-2-fluoro-β-D-arabinofuranosyl)adenine in rats. Cancer Chemother. Pharmacol. 33, 484–488 (1994).

    Article  CAS  Google Scholar 

  35. Spasokoukotskaja, T. et al. Treatment of normal and malignant cells with nucleoside analogues and etoposide enhances deoxycytidine kinase activity. Eur. J. Cancer 35, 1862–1867 (1999).

    Article  CAS  Google Scholar 

  36. Cooper, T., Ayres, M., Nowak, B. & Gandhi, V. Biochemical modulation of cytarabine triphosphate by clofarabine. Cancer Chemother. Pharmacol. 55, 361–368 (2005).

    Article  CAS  Google Scholar 

  37. Gandhi, V. et al. Clinical and pharmacokinetic study of clofarabine in chronic lymphocytic leukemia: strategy for treatment. Clin. Cancer Res. 12, 4011–4017 (2006).

    Article  CAS  Google Scholar 

  38. Gandhi, V. et al. Pharmacokinetics and pharmacodynamics of plasma clofarabine and cellular clofarabine triphosphate in patients with acute leukemias. Clin. Cancer Res. 9, 6335–6342 (2003).

    CAS  PubMed  Google Scholar 

  39. Bonate, P. L. et al. Population pharmacokinetics of clofarabine, a second-generation nucleoside analog, in pediatric patients with acute leukemia. J. Clin. Pharmacol. 44, 1309–1322 (2004). This report characterizes the pharmacokinetics of clofarabine in paediatric patients.

    Article  CAS  Google Scholar 

  40. Kantarjian, H. M. et al. Phase I clinical and pharmacology study of clofarabine in patients with solid and hematologic cancers. J. Clin. Oncol. 21, 1167–1173 (2003).

    Article  CAS  Google Scholar 

  41. Kantarjian, H. et al. Phase 2 clinical and pharmacologic study of clofarabine in patients with refractory or relapsed acute leukemia. Blood 102, 2379–2386 (2003).

    Article  CAS  Google Scholar 

  42. Foran, J. et al. A Phase II, open-label study of clofarabine in adult patients with refractory or relapsed acute myelogenous leukemia. Am. Soc. Clin. Oncol. Proc. 22, 587A (2003).

    Google Scholar 

  43. Jeha, S. et al. Clofarabine, a novel nucleoside analog, is active in pediatric patients with advanced leukemia. Blood 103, 784–789 (2004). This report is the first study to show clofarabine has activity in patients with paediatric leukaemia.

    Article  CAS  Google Scholar 

  44. Jeha, S. et al. Phase 2 study of clofarabine in pediatric patients with refractory or relapsed acute lymphoblastic leukemia. J. Clin. Oncol. 12, 1917–1923 (2006). This report confirms the activity of clofarabine in relapsed or refractory paediatric ALL.

    Article  Google Scholar 

  45. Johnson, J. Food and Drug Administration Medical Review for Clofarabine: NDA 21–673. 2005.

  46. Mathieu, M. New Drug Development: A Regulatory Overview (Parexel International, Waltham, 2005).

    Google Scholar 

  47. International Conference on Harmonisation. Good Clinical Practices: Consolidated Guideline (E6). (1996).

  48. Faderl, S. et al. Results of a Phase 1–2 study of clofarabine in combination with cytarabine (ara-C) in relapsed and refractory acute leukemias. Blood 105, 940–947 (2005).

    Article  CAS  Google Scholar 

  49. Burnett, A. K., Russell, N., Kell, J., Milligan, D. & Culligan, D. A Phase 2 evaluation of single agent clofarabine as first line treatment for older patients with AML who are not considered fit for intensive chemotherapy. Blood 104, 248A (2004).

    Google Scholar 

  50. Cunningham, C. C. et al. Oral administration of clofarabine daily x5 every 4 weeks in patients with advanced solid tumors in a Phase I and pharmacokinetic study. Eur. J. Cancer (Suppl. 2), A548 (2005).

  51. National Cancer Institute Dictionary of Cancer Terms. 2005.

  52. Bauta, W. et al. A new process for antineoplastic agent clofarabine. Org. Proc. Res. Dev. 8, 889–896 (2004).

    Article  CAS  Google Scholar 

  53. King, K. M. et al. Clofarabine and fludarabine cytotoxicity in cultured leukemic cell lines can be correlated with the activity of the human equilabrative nucleoside transport process hENT1. Proc. Am. Assoc. Cancer Res. 44, LB-115 (2003).

    Google Scholar 

  54. Gati, W. P. et al. Es nucleoside transporter content of acute leukemia cells: role in cell sensitivity to cytarabine (araC). Leuk. Lymphoma 32, 45–54 (1998).

    Article  CAS  Google Scholar 

  55. Baldwin, S. A., Mackey, J. R., Cass, C. E. & Young, J. D. Nucleoside transporters: molecular biology and implications for therapeutic development. Mol. Med. Today 5, 216–224 (1999).

    Article  CAS  Google Scholar 

  56. Bonate, P. L. et al. The distribution, metabolism, and elimination of clofarabine in rats. Drug Metab. Dispos. 33, 739–748 (2005).

    Article  CAS  Google Scholar 

  57. Lindemalm, S., Liliemark, J., Larsson, B. S. & Albertioni, F. Distribution of 2-chloro-2′-deoxyadenosine, 2-chloro-2′-arabino-fluoro-2′-deoxyadenosine, fludarabine and cytarabine in mice: a whole-body autoradiography study. Med. Oncol. 16, 239–244 (1999).

    Article  CAS  Google Scholar 

  58. Foss, F. M. Nucleoside analogs and antimetabolite therapies for myelodysplastic syndrome. Best. Pract. Res. Clin. Haematol. 17, 573–584 (2004).

    Article  CAS  Google Scholar 

  59. Reichelova, V., Liliemark, J. & Albertioni, F. Structure-activity relationships of 2-chloro-2′-arabino-fluoro-2′-deoxyadenosine and related analogues: protein binding, lipophilicty, and retention in reversed phase LC. J. Liq. Chrom. 18, 1123–1135 (1995).

    Article  CAS  Google Scholar 

  60. Cunningham, C. C. et al. Clofarabine administration weekly to adult patients with advanced solid tumors in a Phase I dose-finding study. Proc. Am. Soc. Clin. Oncol. 23, A7109 (2005).

    Article  Google Scholar 

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Acknowledgements

Drug development is a team effort. Clofarabine could not have been developed without the help of numerous unnamed, but dedicated, individuals. The authors would like to specifically thank B. Bauta, M. Bernstein, S. Schmid, B. Waud and B. Parker for their help in the preparation of this manuscript.

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Correspondence to Peter L. Bonate.

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Competing interests

P.L.B., L.A., W.J.C. and K.S. are employees of Genzyme, the manufacturer of clofarabine. S.W. is a previous employee of Genzyme and current consultant to Genzyme. J.A.S. is an employee of Southern Research Institute, an organization that has received funding from Genzyme.

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Glossary

Complete response

The disappearance of all signs of cancer in response to treatment, but not necessarily a sign that the patient has been cured. Also called a complete remission51.

Acute leukaemia

A rapidly progressing cancer that starts in the blood-forming tissue, such as the bone marrow, in which too many immature white blood cells (WBCs) are formed. When the WBCs are lymphocytes the disease is known as acute lymphocytic or lymphoblastic leukaemia (ALL) and when the WBCs do not refer to lymphocytes the disease is referred to as acute myelogenous or non-lymphocytic leukaemia (AML)51.

Blasts

Blast cells are immature precursors of lymphocytes (lymphoblasts) or granulocytes (myeloblasts) that do not normally appear in peripheral blood. Acute leukaemia is often indicated by the presence of peripheral blood blast cells.

Nucleoside

An organic compound consisting of a sugar, usually ribose or deoxyribose, linked to a heterocyclic nitrogenous base, particularly a purine or pyrimidine, especially a compound obtained by hydrolysis of a nucleic acid.

Nucleotide

The structural unit of nucleic acids, such as DNA or RNA, a nucleotide consists of a nucleoside and a (poly)phosphate group. A nucleotide that lacks a phosphate group is called a nucleoside.

V max

The maximum initial velocity of an enzyme-catalysed reaction.

IC50

The concentration of a substance that results in a 50% effect on some measure of biochemical function or substance–target binding interaction.

C max

The maximum concentration of drug in systemic circulation that is attained after dosing.

Palmoplantar erythrodysthesia

A cutaneous drug reaction manifesting in painful edematous erythema on the palms of the hands and soles of the feet.

Mucositis

Sore and inflamed mouth.

Grade

The grade of an adverse event ranges from 1–5, usually, and is an ordinal measure of the degree of severity. The higher the grade, the more severe the adverse event. Grades 1 and 2 are generally mild severity, whereas Grades 3 and 4 adverse events are usually thought to be dose-limiting. A grade of 5 relates to death. A common guideline for toxicity grading is the National Cancer Institute's Common Terminology Criteria for Adverse Events.

Serious adverse events

A serious adverse event, which has nothing to do with the severity of the adverse event (see Grade), is any adverse event that results in death, is life-threatening, requires or prolongs hospitalization, causes disability, results in a congenital abnormality, or requires intervention to prevent permanent impairment or damage.

Balanced clearance

Clearance is a proportionality constant between the concentration of drug in the body and its rate of removal from the body. A secondary definition is the volume of blood cleared by the drug per unit time. Balanced clearance refers to when there is balance between competing mechanisms of elimination, for example, the drug is cleared equally by the liver and kidneys.

Fast-track designation

The FDA assigns fast-track status to expedite review of new drugs used to treat serious or life-threatening conditions or which demonstrate the potential to address an unmet medical need.

Orphan drug designation

Orphan drug designation requires that the patient population affected is less than 200,000 per year and there is no reasonable expectation to recover the cost of developing the drug from sales in the United States. The advantages to obtaining such status are that sponsors of drugs granted orphan drug status do not have to pay FDA user fees for review of the application; the Internal Revenue Service grants up to a 50% tax credit to recoup expenses related to the development of the drug along with other tax-related incentives; and the FDA grants 7 years of marketing exclusivity for the drug.

Accelerated approval

Accelerated approval, also sometimes referred to as Subpart H, must be applied for after fast-track designation is approved. A drug can receive fast-track designation yet not gain accelerated approval. Traditional approval must demonstrate an effect on a clinically meaningful endpoint; in the case of cancer therapeutics, the 'gold standard' is survival. Under accelerated approval, a drug's approval might be based on a surrogate endpoint that is reasonably likely to be predictive of clinical benefit. When accelerated approval is granted, the sponsor is required to perform post-approval confirmatory studies.

Stable disease

Cancer that is neither decreasing nor increasing in extent or severity51.

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Bonate, P., Arthaud, L., Cantrell, W. et al. Discovery and development of clofarabine: a nucleoside analogue for treating cancer. Nat Rev Drug Discov 5, 855–863 (2006). https://doi.org/10.1038/nrd2055

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