Insights into oxazaphosphorine resistance and possible approaches to its circumvention
Introduction
Anticancer oxazaphosphorines are alkylating agents and include the commonly used cyclophosphamide (CPA, Cytoxan, Neosar) and ifosfamide (IFO, Ifex) as well as the less commonly used trofosfamide (Ixoten) (Fig. 1). CPA, IFO and trofosfamide were introduced into clinical practice 50, 40, and 30 years ago, respectively. CPA and IFO have been widely used in the treatment of haematological malignancies and solid tumours including breast, lung and prostate cancer, ovarian cancer, lymphomas and multiple myeloma. The development of trofosfamide, however, has been hindered by the lack of intravenous (i.v.) formulation. It is orally administered for palliative therapy and as maintenance therapy in a broad spectrum of cancers (Latz et al., 2004), but there are no Phase III trials for this drug to date. CPA is also used for the mobilization of hematopoietic progenitor cells from the bone marrow into peripheral blood (Szumilas et al., 2005).
More recently, additional oxazaphosphorine derivatives have been synthesized and evaluated for improved activity and selectivity. These include mafosfamide (NSC 345842), glufosfamide (D19575, β-d-glucosylisophosphoramide mustard), NSC 612567 (aldophosphamide perhydrothiazine) and NSC 613060 (aldophosphamide thiazolidine) (Fig. 1). Phase III trials of glufosfamide are ongoing for the treatment of pancreatic cancer, non-small cell lung cancer (NCSLC), and recurrent glioblastoma multiforme, while Phase I studies of mafosfamide have been recently completed for the treatment of meningeal malignancy secondary to leukaemia, lymphoma, or solid tumours (Blaney et al., 2005a).
Conventional oxazaphosphorine anticancer agents, such as CPA and IFO, are prodrugs that are activated through 4-hydroxylation by hepatic cytochrome P450s such as CYP2B6, CYP2C9 and CYP3A4 (Chen et al., 2004), yielding nitrogen mustards which react with DNA ultimately leading to cell death. They also produce cytotoxic metabolites such as chloroacetaldehyde (CAA) that causes neurotoxicity and nephrotoxicity and acrolein that causes urotoxicity. The toxic by-products are detoxified by various aldehyde dehydrogenases (ALDHs) and, through conjugation with glutathione (GSH), by GSH S-transferases (GSTs).
Newer oxazaphosphorines are activated through other enzymatic and/or non-enzymatic pathways. For example, both NSC 612567 and NSC 613060 can be activated by common phosphodiesterases (PDEs) in plasma and other tissues, or by high-affinity nuclear 3′–5′ exonucleases associated with DNA polymerases such as DNA polymerase δ and ɛ (Voelcker et al., 1997, Voelcker and Hohorst, 1998). Glufosfamide does not require metabolic activation in the liver; the active moiety, isophosphoramide mustard, is released upon entry of glufosfamide into tumour cells by spontaneous or intracellular glucosidase-catalysed hydrolysis.
Metabolic activation of CPA and IFO generates bifunctional alkylating nitrogen mustards which are converted to chemically reactive carbonium ions at neutral pH and react with the 7-nitrogen atom of purine bases in DNA, especially when they are flanked by adjacent guanines. The second arm in phosphoramide mustard can react with a second guanine moiety in an opposite DNA strand or in the same strand to form crosslinks. The O6 atom of guanine may also be a target for oxazaphosphorines. The different intramolecular distance between the chloroethyl groups in CPA or IFO results in a different range of cross-linked DNA. Tumour cells have differential ability to repair these DNA injuries through several pathways.
A sensitive, standard method for measuring DNA strand breaks induced by DNA-damaging agents in vitro and in clinical samples is the single cell gel electrophoresis Comet assay. By using this assay, DNA cross linking was observed in the lymphocytes of patients treated with ifosfamide at 3.0 g/m2/day by continuous intravenous (i.v.) infusion over 3–5 days or as a 3-h infusion daily for 3 days (Hartley et al., 1999). Cross-links of DNA molecules were detected in all cancer patients within 3 h following IFO infusion, whereas DNA single strand breaks were not observed. In patients receiving continuous infusion, a plateau of DNA cross-linking was achieved by 24 h, while a marked decrease in the peak level of cross-linking was observed in the patients receiving IFO infusion over 3 h (Hartley et al., 1999). The clinical study demonstrated the utility of the Comet assay for monitoring DNA cross-links as a clinical response marker in oxazaphosphorine chemotherapy. The cross-link formation is dependent on dosage and dosing regimen.
CPA also kills circulating endothelial progenitors and augments the efficacy of antitumour immune response by depleting CD4+/CD25+ regulatory T cells and increasing T lymphocyte proliferation and T memory cells. The immunostimulatory effect of CPA is associated with marked inhibition of the inducible nitric oxide synthase (Loeffler et al., 2005). Low-dose CPA has gained increased application as an antiangiogenic/immunostimulatory agent in combination with immunotherapies in the treatment of cancer.
In addition, CPA has modulating effects on both humoral and cell-mediated immunity and has been widely used in combination with immunosuppressants such as prednisone, mycophenolic acid, or azathioprine to treat systemic lupus erythematosus (SLE), rheumatoid arthritis and other autoimmune diseases. In such cases, extended low-dose, pulse (e.g. a dose per month) or high-dose i.v. regimen for CPA is often used.
The pharmacokinetics of oxazaphosphorines, in particular CPA and IFO, have been extensively studied (Boddy and Yule, 2000, Yule et al., 2004). Both CPA and IFO are soluble in water and alcohol as monohydrates and can be readily administered orally. When given orally, CPA and IFO are well absorbed with high oral bioavailability (85–100%). A small fraction of the drug is probably metabolized in the liver and gut due to first-pass effect. The peak concentration appears 1–2 h following oral drug administration. CPA is orally administered at low doses (75–200 mg/day) when used as an immunosuppressive agent or as a component of the CMF (CPA, methotrexate and fluorouracil) regimen in the treatment of advanced breast cancer. IFO is less commonly used as an oral agent, as oral administration results in unacceptable neurotoxicity, probably due to increased formation of neurotoxic CAA compared to i.v. administration. After oral or i.v. administration, CPA is rapidly distributed throughout the body with ∼20% plasma protein binding, whereas the ability of protein binding is higher for its metabolite (<67%) (Boddy and Yule, 2000). The distribution of IFO is more extensive with lower plasma protein binding compared with CPA. Some studies in obese patients showed that a longer elimination half-life (t1/2β) for IFO due to the increased volume of distribution in obese patients. Like CPA, IFO and its active metabolite 4-hydroxy-IFO can pass the blood–brain barrier and reach cerebrospinal fluid (Boddy and Yule, 2000). Both CPA and IFO are primarily (70%) excreted in the urine and to a less extent in the faeces. The majority of CPA and IFO elimination is by metabolic transformation; only 10–20% remain unchanged in the urine and only 4% in the bile (Boddy and Yule, 2000). Considerable inter-patient variability in the pharmacokinetics of the two agents has been observed; they are markedly influenced by the route of administration and duration of treatment, age, co administered drugs, genetic factors, liver and renal function (Yule et al., 2004).
Resistance to oxazaphosphorine chemotherapy is a major reason for therapeutic failure. Low response rates and considerable variability in clinical response have been observed in a variety of tumours. Resistance to CPA in patients with autoimmune diseases has also been observed. For example, up to 40% of patients with lupus nephritis failed to achieve renal remission, and up to 20% of patients developed end-stage renal disease after a long course (30 months or longer) of pulse CPA therapy (Vyas et al., 2002). Based on in vitro and in vivo mechanistic studies, multiple factors associated with the pharmacokinetic and pharmacodynamic behaviour of oxazaphosphorines have been implicated in resistance. This review will discuss resistance mechanisms to oxazaphosphorines and potential approaches to circumvent it.
Section snippets
Resistance mechanisms to oxazaphosphorines
Intrinsic resistance to oxazaphosphorines is associated with multiple biochemical changes in tumour cells. In addition, exposing tumour cells to oxazaphosphorines can generate significant acquired resistance. Unlike natural product anticancer drugs, resistance to CPA and IFO is not associated with the multidrug resistance (MDR) phenotype or markedly elevated expression of P-glycoprotein (PgP), although cross-resistance with cisplatin and other alkylating agents has been observed (Frei et al.,
Potential approaches for circumventing resistance to oxazaphosphorines
Oxazaphosphorines are widely used in cancer chemotherapy and the management of autoimmune diseases. Thus, there is a compelling need to find potential and practical approaches to overcome oxazaphosphorine resistance. To date, several major approaches, including combination with other agents that modulate the disposition of, or cellular response to oxazaphosphorines, combined use with agents that reduce intracellular GSH level, antisense oligonucleotides directed against target genes, and
Concluding remarks and future directions
Drug resistance is an important reason for the failure of cancer chemotherapy. Multiple mechanisms of resistance to oxazaphosphorines have been identified (Fig. 4 and Table 1). These include decreased activation by CYP2B6, CYP2C9, CYP3A4, and CYP2C19; increased deactivation of oxazaphosphorines by deactivating enzymes such as ALDH and GSTs; increased cellular thiol level; altered efflux and/or influx of oxazaphosphorine and their metabolites in tumour cells; increased DNA repair capacity; and
Acknowledgements
The authors appreciate the support by the National University of Singapore Academic Research Funds (Grant No. R-148-000-054-112 and R-148-000-047-101).
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