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• History

  An alkylating anticancer agent with another strategy to lower the toxicities on normal cells and increase the specifi city for cancer cells was attempted by O.M. Friedman and A.M. Seligman at Harvard University in 1949. Based on the previous fi nding that cancer cells have higher phosphamidase activity, they predicted that if the activity of this enzyme were used to develop a drug that existed as an innocuous prodrug that becomes activated by phosphoamidases inside the cancer cells, this could specifi cally eliminate cancer cells [12]. However, it was later determined that this active form is generated by a different drug metabolic pathway, contrary to the initial assumption that phosphamidases would be involved. In other words, cyclophosphamide is mostly activated to 4-hydroxycyclophosphamide by cytochrome P450 enzymes in the liver microsomes, secreted to the bloodstream, and absorbed by cancer cells.With the therapeutic effect of cyclophosphamide verifi ed in clinical tests on patients with malignant lymphoma, it was registered as an FDA-approved lymphoma anticancer agent in 1959. Later, cyclophosphamide has been widely used in anticancer treatment for not only lymphocytic leukemia such as Hodgkin’s lymphoma, Burkitt lymphoma, childhood acute lymphoblastic leukemia, chronic granulocytic leukemia, acute myeloid leukemia, and multiple myeloma, but also solid cancers such as breast cancer, ovarian cancer, and sarcoma. ;

• Overview

  Cyclophosphamide is one of the most successful anticancer agents ever synthesized. Even today, 50 years after its synthesis, cyclophosphamide is still widely used as a chemotherapeutic agent and in the mobilization and conditioning regimens for blood and marrow trans plantation (BMT). Among 1,000 selected compounds and antibiotics tested against 33 tumors, cyclophosphamide was the most effective molecule. The initial clinical trials^{[1, 2]} of cyclophosphamide for the treatment of cancer were performed in 1958, and in 1959 it became the eighth cytotoxic anticancer agent approved by the FDA. It is also approved for minimal change disease of the kidney in children (a disease that causes nephrotic syndrome), but despite its widespread use in other autoimmune disorders and BMT, it has never been approved for these indications.
  
  Figure 1 the chemical structure of cyclophosphamide;;

• Mode of action and resistance

  The chemical design of cyclophosphamide—substitution of an oxazaphosphorine ring for the methyl group of nitrogen mustard—was based on the rationale that some cancer cells express high levels of phosphamidase, which is capable of cleaving the phosphorus–nitrogen (P–N) bond, releasing nitrogen mustard.^[3] Thus, cyclophosphamide was one of the first agents rationally designed to selectively target cancer cells. Although cyclophosphamide is in fact a prodrug that requires metabolic activation, the original hypothesis that it would function as targeted anticancer therapy via phosphamidase activation proved to be inaccurate.
  The cytotoxic action of nitrogen mustard is closely related to the reactivity of the 2chloroethyl groups attached to the central nitrogen atom. Under physiological conditions, nitrogen mustards undergo intramolecular cyclizations through elimination of chloride to form a cyclic aziridinium (ethyleneiminium) cation.
  This highly unstable cation is readily attacked on one of the carbon atoms of the three-member aziridine ring by several nucleophiles, such as DNA guanine residues (Figure 1). ^[5] This reaction releases the nitrogen of the alkylating agent and makes it available to react with the second 2chloroethyl side chain, forming a second covalent linkage with another nucleophile, thus interfering with DNA replication by forming intrastrand and interstrand DNA crosslinks. In contrast to aliphatic (or open chain) nitrogen mustards, cyclophosphamide is an inactive prodrug that requires enzymatic and chemical activation to release active phosphoramide mustard. Hydroxylation on the oxazaphosphorine ring by the hepatic cytochrome P450 system generates 4-hydroxycyclophosphamide, which coexists with its tautomer, aldophosphamide.
  The major mechanism of cyclophosphamide detoxification is oxidation of aldophosphamide to carboxyphosphamide by cellular aldehyde dehydrogenase (AlDH). ^[7] withdrawal of the hydrogen adjacent to the aldehyde, by a base, is a necessary step for decomposition of aldophosphamide. Conversion of the aldehyde to carboxylic acid by AlDH makes this hydrogen less acidic for removal,^[8] subsequently releasing the active phosphoramide mustard. Thus, cellular concentrations of AlDH are serendipitously responsible for many of the differential activities of cyclophosphamide in cells. ^[9] Carboxyphosphamide and its degradation products are the major metabolites found in urine. ^[10]
  Although AlDH1A1 expression is the major determinant of normal cellular sensitivity to cyclophosphamide and is associated with resistance to cyclophosphamide in tumor cell lines, ^[11] it has a minor role in the clinical response of cancer cells to cyclophosphamide^{[9, 12]}. In particular, leukemia and lymphoma specimens from newly diagnosed patients rarely express high levels of AlDH isozymes^[9] Increased cellular levels of glutathione and glutathione S-transferases have been shown to cause cyclophosphamide resistance in tumor cell lines, but the lack of a similar correlation in vivo suggests that this is not the major mechanism contributing to cyclophosphamide resistance^[12]. The inherent sensitivity of cancer cells to undergo apoptosis following DNA damage is the most important determinant of the clinical sensitivity of cancer cells to cyclophosphamide^{[13, 14]}. ;

• Toxicity effect

  Bone marrow suppression is the most common toxic effect of cyclophosphamide. Neutropenia is dose dependent. Patients treated with low-dose cyclophosphamide should be monitored closely, although they rarely develop significant neutropenia. Leukopenia, thrombocytopenia and anemia are common after high dose cyclophosphamide administration. Rapid hematologic recovery invariably occurs within 2–3 weeks in patients with normal bone marrow reserve, regardless of the dose.
  Cardiotoxicity is the dose limiting toxic effect of cyclophosphamide, and is observed only after administration of high doses. The cardiac manifestations that result from high dose cyclophosphamide are heterogeneous and range from innocuous to fatal. The most severe form is hemorrhagic necrotic perimyocarditis, with a reported incidence of <1–9% after the most commonly used high doses of cyclophosphamide (60 mg/kg daily × 2 days or 50 mg/kg daily × 4 days) ^{[15, 16]}. However, in most transplant centers the rate of hemorrhagic myocarditis is less than 0.1%. This clinical syndrome occurs abruptly within days of drug infusion and is fatal. Perimyocarditis is manifested by severe congestive heart failure accompanied by electro cardiographic findings of diffuse voltage loss, cardio megaly, pleural and pericardial effusions. Postmortem findings reveal hemorrhagic cardiac necrosis.
  Gonadal failure is a major complication of cyclophosphamide administration, especially in females. The patient’s age at treatment, the cumulative dose, and the administration schedule are major determinants for this adverse effect. The risk for sustained amenorrhea in patients with lupus receiving monthly intermediate dose cyclophosphamide is 12% for women under 25 years of age, and greater than 50% for women over 30 years of age. The risk for ovarian failure following high dose cyclophosphamide administration seems to be less than that of intermediate dose.
  Hemorrhagic cystitis is the most common form of cyclophosphamide bladder toxicity,^[17] but bladder fibrosis and transitional or squamous cell carcinoma can also occur. Hemorrhagic cystitis can occur early or late after cyclophosphamide administration. Early onset disease, in the first few days after cyclophosphamide administration, seems to be caused by acrolein^[18]. Vigorous hydration, forced diuresis and MESNA, which interacts with acrolein to form nontoxic adducts, can prevent acute hemorrhagic cystitis by limiting uroepithelial exposure to acrolein^[19]. Hemorrhagic cystitis can develop weeks to months after treatment in 20–25% of patients who receive high dose cyclophosphamide.
  Cyclophosphamide is carcinogenic. In addition to bladder cancer, secondary acute leukemia (often preceded by myelodysplastic syndrome) and skin cancer are the most common malignancies after cyclophosphamide therapy. The probability of acquiring a therapy-related malignancy is proportional to the length of drug exposure and cumulative dose. Therapy-related leukemia occurs in roughly 2% of patients treated with chronic cyclophosphamide, primarily in patients who have received the drug for more than 1 year^[20]. ;

• Clinical efficacy

  Cyclophosphamide is one of the few drugs with a broad indication for cancer. Although it is effective as a single agent in malignancies, it is usually used in combination with other antineoplastic agents. Even though it has been substituted by newer agents (such as platinums, taxanes and targeted therapies) for the treatment of many solid tumors, it is quite active for many of these indications, and there often remains limited evidence for the superiority of the newer approaches.
  Cyclophosphamide based therapy is used extensively for lymphomas and is often curative for aggressive non-Hodgkin lymphoma, with Burkitt lymphoma being particularly sensitive. Although modern therapeutic regimens employ intensive cyclophosphamide based combination chemotherapy^[21], in the 1960s durable complete remissions were reported following a single course of cyclophosphamide.^[22] RCHoP (rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone) remains the most commonly employed regimen for aggressive non-Hodgkin lymphoma, with cure rates of 30–40%. Many newer cyclophosphamide based multidrug combinations have been developed for aggressive non-Hodgkin lymphoma, but none have proven to be superior to CHoP.^[23] Myeloablative therapy, usually including high dose cyclophosphamide, followed by BMT is the most effective treatment for relapsed aggressive non-Hodgkin lymphoma.
  Cyclophosphamide, in combination with other agents, has been the mainstay of adjuvant and metastatic breast cancer chemotherapy regimens such as CMF (cyclophosphamide, methotrexate, 5-fluorouracil) and FEC (5-fluorouracil, epirubicin, cyclophosphamide) for decades. AC (doxorubicin, cyclophosphamide) was demonstrated to be equivalent to 6 months of classic CMF in two separate National surgical Adjuvant Breast and Bowel Project (NsABP) studies^{[24, 25]}. The addition of a sequential taxane to this adjuvant regimen further improved outcomes and has since become the standard of care for human epidermal growth factor receptor 2 (HER2) negative early-stage breast cancer.
  Cyclophosphamide is the cornerstone of curative chemotherapy regimens for numerous newly diagnosed and recurrent pediatric malignancies. Combinations of carboplatin and etoposide, and vincristine, cyclophosphamide and doxorubicin (CAdo), showed 5 year overall and event free survival rates of over 90% in infants with initial localized and unresectable neuroblastoma as well as allowance for subsequent surgical excision.^[27] Cyclophosphamide in combination chemotherapy regimens as an adjunct to surgery and radiation has also been used in a variety of rare cancers such as retinoblastoma, wilms tumor, and rhabdomyosarcoma as well as Ewing sarcoma. ;

• References

  1. Brock, N. & Wilmanns H. Effect of a cyclic nitrogen mustard-phosphamidester on experimentally induced tumors in rats; chemotherapeutic effect and pharmacological properties of B518 ASTA [German]. Dtsch. Med. Wochenschr. 83, 453–458 (1958).
  2. Gross, R. & Wulf, G. Klinische und experimentelle Erfahrungen mit zyk lischen und nichtzyklischen Phosphamidestern des N-Losl in der Chemotherapie von Tumoren [German]. Strahlentherapie 41, 361–367 (1959).
  3. Friedman, O. M. & Seligman, A. M. Preparation of N-phosphorylated derivatives of bis-P-chloroethylamine. J. Amer. Chem. Soc. 76, 655–658 (1954).
  4. Arnold, H., Bourseaux, F. & Brock, N. Chemotherapeutic action of a cyclic nitrogen mustard phosphamide ester (B 518-ASTA) in experimental tumours of the rat. Nature 181, 931 (1958).
  5. Dong, Q. et al. A structural basis for a phosphoramide mustard-induced DNA interstrand cross-link at 5'-d(GAC). Proc. Natl Acad. Sci. USA 92, 12170–12174 (1995).
  6. Boddy, A. v. & Yule, S. M. Metabolism and pharmacokinetics of oxazaphosphorines. Clin. Pharmacokinet. 38, 291–304 (2000).
  7. Chen, T. L. et al. Nonlinear pharmacokinetics of cyclophosphamide and 4-hydroxycyclophosphamide/aldophosphamide in patients with metastatic breast cancer receiving high-dose chemotherapy followed by autologous bone marrow transplantation. Drug Metab. Dispos. 25, 544–551 (1997).
  8. Silverman, R. B. The Organic Chemistry of Enzyme-catalyzed Reactions (Academic Press, 2002).
  9. Russo, J. E., Hilton, J. & Colvin, O. M. The role of aldehyde dehydrogenase isozymes in cellular resistance to the alkylating agent cyclophosphamide. Prog. Clin. Biol. Res. 290, 65–79 (1989).
  10. Joqueviel, C. et al. Urinary excretion of cyclophosphamide in humans, determined by phosphorus-31 nuclear magnetic resonance spectroscopy. Drug Metab. Dispos. 26, 418–428 (1998).
  11. Sladek, N. E. Leukemic cell insensitivity to cyclophosphamide and other oxazaphosphorines mediated by aldehyde dehydrogenase(s). Cancer Treat. Res. 112, 161–175 (2002).
  12. Tanner, B. et al. Glutathione, glutathione S-transferase alpha and pi, and aldehyde dehydrogenase content in relationship to drug resistance in ovarian cancer. Gynecol. Oncol. 65, 54–62 (1997).
  13. Banker, D. E., Groudine, M., Norwood, T. & Appelbaum, F. R. Measurement of spontaneous and therapeutic agent-induced apoptosis with BCL-2 protein expression in acute myeloid leukemia. Blood 89, 243–255 (1997).
  14. Zhang, J., Tian, Q., Chan, S. Y., Duan, W. & Zhou, S. Insights into oxazaphosphorine resistance and possible approaches to its circumvention. Drug Resist. Updat. 8, 271–297 (2005).
  15. Murdych, T. & Weisdorf, D. J. Serious cardiac complications during bone marrow transplantation at the University of Minnesota, 1977–1997. Bone Marrow Transplant. 28, 283–287 (2001).
  16. Cazin, B. et al. Cardiac complications after bone marrow transplantation. A report on a series of 63 consecutive transplantations. Cancer 57, 2061–2069 (1986).
  17. Stillwell, T. J. & Benson, R. C. Jr Cyclophosphamide-induced hemorrhagic cystitis. A review of 100 patients. Cancer 61, 451–457 (1988).
  18. Cox, P. J. Cyclophosphamide cystitis— identification of acrolein as the causative agent. Biochem. Pharmacol. 28, 2045–2049 (1979).
  19. Haselberger, M. B. & Schwinghammer, T. L. Efficacy of mesna for prevention of hemorrhagic cystitis after high-dose cyclophosphamide therapy. Ann. Pharmacother. 29, 918–921 (1995)
  20. Levine, E. G. & Bloomfield, C. D. Semin. Oncol. 19, 47–84 (1992).
  21. Magrath, I. et al. J. Clin. Oncol. 14, 925–934 (1996).
  22. Burkitt, D. Cancer 20, 756–759 (1967).
  23. Fisher, R. I. et al. N. Engl. J. Med. 328,1002–1006 (1993).
  24. Fisher, B. et al.. J. Clin. Oncol. 8, 1483–1496 (1990).
  25. Fisher, B. et al. J. Clin. Oncol. 19, 931–942 (2001)
  26. Rubie, H. et al. Med. Pediatr. Oncol. 36, 247–250 (2001).
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