Elsevier

Drug Resistance Updates

Volume 8, Issue 5, October 2005, Pages 271-297
Drug Resistance Updates

Insights into oxazaphosphorine resistance and possible approaches to its circumvention

https://doi.org/10.1016/j.drup.2005.08.003Get rights and content

Abstract

The oxazaphosphorines cyclophosphamide, ifosfamide and trofosfamide remain a clinically useful class of anticancer drugs with substantial antitumour activity against a variety of solid tumors and hematological malignancies. A major limitation to their use is tumour resistance, which is due to multiple mechanisms that include increased DNA repair, increased cellular thiol levels, glutathione S-transferase and aldehyde dehydrogenase activities, and altered cell-death response to DNA damage. These mechanisms have been recently re-examined with the aid of sensitive analytical techniques, high-throughput proteomic and genomic approaches, and powerful pharmacogenetic tools. Oxazaphosphorine resistance, together with dose-limiting toxicity (mainly neutropenia and neurotoxicity), significantly hinders chemotherapy in patients, and hence, there is compelling need to find ways to overcome it. Four major approaches are currently being explored in preclinical models, some also in patients: combination with agents that modulate cellular response and disposition of oxazaphosphorines; antisense oligonucleotides directed against specific target genes; introduction of an activating gene (CYP3A4) into tumor tissue; and modification of dosing regimens. Of these approaches, antisense oligonucleotides and gene therapy are perhaps more speculative, requiring detailed safety and efficacy studies in preclinical models and in patients. A fifth approach is the design of novel oxazaphosphorines that have favourable pharmacokinetic and pharmacodynamic properties and are less vulnerable to resistance. Oxazaphosphorines not requiring hepatic CYP-mediated activation (for example, NSC 613060 and mafosfamide) or having additional targets (for example, glufosfamide that also targets glucose transport) have been synthesized and are being evaluated for safety and efficacy. Characterization of the molecular targets associated with oxazaphosphorine resistance may lead to a deeper understanding of the factors critical to the optimal use of these agents in chemotherapy and may allow the development of strategies to overcome resistance.

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).

References (124)

  • F. Drablos et al.

    Alkylation damage in DNA and RNA—repair mechanisms and medical significance

    DNA Repair (Amst.)

    (2004)
  • M.E. Franks et al.

    New directions in cancer research: technical advances in biology, drug resistance, and molecular pharmacology

    Drug Resist. Update

    (2003)
  • G. Giaccone et al.

    Glufosfamide administered by 1-hour infusion as a second-line treatment for advanced non-small cell lung cancer; a phase II trial of the EORTC-New Drug Development Group

    Eur. J. Cancer

    (2004)
  • S.G. Lopez et al.

    Effects of cyclophosphamide and buthionine sulfoximine on ovarian glutathione and apoptosis

    Free Radic. Biol. Med.

    (2004)
  • C.S. Morrow et al.

    Combined expression of multidrug resistance protein (MRP) and glutathione S-transferase P1-1 (GSTP1-1) in MCF7 cells and high level resistance to the cytotoxicities of ethacrynic acid but not oxazaphosphorines or cisplatin

    Biochem. Pharmacol.

    (1998)
  • G. Muzio et al.

    Inhibition of cytosolic class 3 aldehyde dehydrogenase by antisense oligonucleotides in rat hepatoma cells

    Chem. Biol. Interact.

    (2001)
  • G. Muzio et al.

    Antisense oligonucleotides against aldehyde dehydrogenase 3 inhibit hepatoma cell proliferation by affecting MAP kinases

    Chem. Biol. Interact.

    (2003)
  • Y. Niitsu et al.

    A proof of glutathione S-transferase-pi-related multidrug resistance by transfer of antisense gene to cancer cells and sense gene to bone marrow stem cell

    Chem. Biol. Interact.

    (1998)
  • A.J. Noe et al.

    Characterization of the catecholamine extraneuronal uptake2 carrier in human glioma cell lines SK-MG-1 and SKI-1 in relation to (2-chloroethyl)-3-sarcosinamide-1-nitrosourea (SarCNU) selective cytotoxicity

    Biochem. Pharmacol.

    (1996)
  • G.K. Rekha et al.

    Inhibition of human class 3 aldehyde dehydrogenase, and sensitization of tumor cells that express significant amounts of this enzyme to oxazaphosphorines, by chlorpropamide analogues

    Biochem. Pharmacol.

    (1998)
  • A. Sparreboom et al.

    Pharmacogenomics of ABC transporters and its role in cancer chemotherpay

    Drug Resist. Update

    (2003)
  • L. Sreerama et al.

    Phenolic antioxidant-induced overexpression of class-3 aldehyde dehydrogenase and oxazaphosphorine-specific resistance

    Biochem. Pharmacol.

    (1995)
  • B. Tanner et al.

    Glutathione, glutathione S-transferase alpha and pi, and aldehyde dehydrogenase content in relationship to drug resistance in ovarian cancer

    Gynecol. Oncol.

    (1997)
  • F. Ali-Osman et al.

    Buthionine sulfoximine induction of gamma-l-glutamyl-l-cysteine synthetase gene expression, kinetics of glutathione depletion and resynthesis, and modulation of carmustine-induced DNA–DNA cross-linking and cytotoxicity in human glioma cells

    Mol. Pharmacol.

    (1996)
  • B.S. Andersson et al.

    Nucleotide excision repair genes as determinants of cellular sensitivity to cyclophosphamide analogs

    Cancer Chemother. Pharmacol.

    (1996)
  • V. Arora

    Antisense strategies for redirection of drug metabolism: using Paclitaxel as a model

    Methods Mol. Med.

    (2004)
  • ASTA Medica, 1999. Glufosfamide [investigator's brochure]. Frankfurt,...
  • S.M. Blaney et al.

    Intrathecal mafosfamide: a preclinical pharmacology and phase I trial

    J. Clin. Oncol.

    (2005)
  • S.M. Blaney et al.

    Phase I clinical trial of mafosfamide in infants and children aged 3 years or younger with newly diagnosed embryonal tumors: a pediatric brain tumor consortium study (PBTC-001)

    J. Clin. Oncol.

    (2005)
  • A.V. Boddy et al.

    Metabolism and pharmacokinetics of oxazaphosphorines

    Clin. Pharmacokinet.

    (2000)
  • A.G. Bosanquet et al.

    Bax expression correlates with cellular drug sensitivity to doxorubicin, cyclophosphamide and chlorambucil but not fludarabine, cladribine or corticosteroids in B cell chronic lymphocytic leukemia

    Leukemia

    (2002)
  • E.G. Brain et al.

    Modulation of P450-dependent ifosfamide pharmacokinetics: a better understanding of drug activation in vivo

    Br. J. Cancer

    (1998)
  • J.P. Braybrooke et al.

    Phase I study of MetXia-P450 gene therapy and oral cyclophosphamide for patients with advanced breast cancer or melanoma

    Clin. Cancer Res.

    (2005)
  • E. Briasoulis et al.

    Phase I trial of 6-hour infusion of glufosfamide, a new alkylating agent with potentially enhanced selectivity for tumors that overexpress transmembrane glucose transporters: a study of the European Organization for Research and Treatment of Cancer Early Clinical Studies Group

    J. Clin. Oncol.

    (2000)
  • H. Burger et al.

    RNA expression of breast cancer resistance protein, lung resistance-related protein, multidrug resistance-associated proteins 1 and 2, and multidrug resistance gene 1 in breast cancer: correlation with chemotherapeutic response

    Clin. Cancer Res.

    (2003)
  • J.E. Byfield et al.

    Carrier-dependent and carrier-independent transport of anti-cancer alkylating agents

    Nature

    (1981)
  • Y. Cai et al.

    Effect of O6-benzylguanine on alkylating agent-induced toxicity and mutagenicity. In Chinese hamster ovary cells expressing wild-type and mutant O6-alkylguanine-DNA alkyltransferases

    Cancer Res.

    (2000)
  • M. Carrio et al.

    Intratumoral activation of cyclophosphamide by retroviral transfer of the cytochrome P450 2B1 in a pancreatic tumor model. Combination with the HSVtk/GCV system

    J. Gene Med.

    (2002)
  • A. Cayre et al.

    O(6)-Methylguanine-DNA methyl transferase gene expression and prognosis in breast carcinoma

    Int. J. Oncol.

    (2002)
  • C.S. Chen et al.

    Activation of the anticancer prodrugs cyclophosphamide and ifosfamide: identification of cytochrome P450 2B enzymes and site-specific mutants with improved enzyme kinetics

    Mol. Pharmacol.

    (2004)
  • G. Chen et al.

    Biochemical characterization of in vivo alkylating agent resistance of a murine EMT-6 mammary carcinoma. Implication for systemic involvement in the resistance phenotype

    Cancer Biochem. Biophys.

    (1998)
  • L. Chen et al.

    Cytochrome P450 gene-directed enzyme prodrug therapy (GDEPT) for cancer

    Curr. Pharm. Des.

    (2002)
  • M.L. Citron

    Dose density in adjuvant chemotherapy for breast cancer

    Cancer Invest.

    (2004)
  • H.A. Dirven et al.

    Involvement of human glutathione S-transferase isoenzymes in the conjugation of cyclophosphamide metabolites with glutathione

    Cancer Res.

    (1994)
  • M.G. Dole et al.

    Bcl-xL is expressed in neuroblastoma cells and modulates chemotherapy-induced apoptosis

    Cancer Res.

    (1995)
  • R. Dollner et al.

    Ex vivo responsiveness of head and neck squamous cell carcinoma to glufosfamide, a novel alkylating agent

    Anticancer Res.

    (2004)
  • M.P. Ducharme et al.

    Phenytoin-induced alteration in the N-dechloroethylation of ifosfamide stereoisomers

    Cancer Chemother. Pharmacol.

    (1997)
  • R. Fahrig et al.

    Inhibition of induced chemoresistance by cotreatment with (E)-5-(2-bromovinyl)-2′-deoxyuridine (RP101)

    Cancer Res.

    (2003)
  • Ferguson, S.S., Chen, Y., Lecluyse, E.L., Negishi, M., Goldstein, J.A., in press. Human CYP2C8 is transcriptionally...
  • M. Filipits et al.

    Clinical role of multidrug resistance protein 1 expression in chemotherapy resistance in early-stage breast cancer: the Austrian Breast and Colorectal Cancer Study Group

    J. Clin. Oncol.

    (2005)
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