Bioreductive Drugs: from Concept to Clinic

Clinical Oncology (2007) 19: 427e442 doi:10.1016/j.clon.2007.03.006

Overview

Bioreductive Drugs: from Concept to Clinic S. R. McKeown*, R. L. Coweny, K. J. Williamsy *Institute of Biomedical Sciences, University of Ulster, Coleraine, Northern Ireland BT52 1SA, UK; yExperimental Oncology Group, School of Pharmacy and Pharmaceutical Sciences, University of Manchester, Oxford Road, Manchester M13 9PT, UK

ABSTRACT: One of the key issues for radiobiologists is the importance of hypoxia to the radiotherapy response. This review addresses the reasons for this and primarily focuses on one aspect, the development of bioreductive drugs that are specifically designed to target hypoxic tumour cells. Four classes of compound have been developed since this concept was first proposed: quinones, nitroaromatics, aliphatic and heteroaromatic N-oxides. All share two characteristics: (1) they require hypoxia for activation and (2) this activation is dependent on the presence of specific reductases. The most effective compounds have shown the ability to enhance the anti-tumour efficacy of agents that kill betteroxygenated cells, i.e. radiation and standard cytotoxic chemotherapy agents such as cisplatin and cyclophosphamide. Tirapazamine (TPZ) is the most widely studied of the lead compounds. After successful pre-clinical in vivo combination studies it entered clinical trial; over 20 trials have now been reported. Although TPZ has enhanced some standard regimens, the results are variable and in some combinations toxicity was enhanced. Banoxantrone (AQ4N) is another agent that is showing promise in early phase I/II clinical trials; the drug is well tolerated, is known to locate in the tumour and can be given in high doses without major toxicities. Mitomycin C (MMC), which shows some bioreductive activation in vitro, has been tested in combination trials. However, it is difficult to assign the enhancement of its effects to targeting of the hypoxic cells because of the significant level of its hypoxia-independent toxicity. More specific analogues of MMC, e.g. porfiromycin and apaziquone (EO9), have had variable success in the clinic. Other new drugs that have good pre-clinical profiles are PR 104 and NLCQ-1; data on their clinical safety/efficacy are not yet available. This paper reviews the pre-clinical data and discusses the clinical studies that have been reported. McKeown, S. R. et al. (2007). Clinical Oncology 19, 427e442 ª 2007 The Royal College of Radiologists. Published by Elsevier Ltd. All rights reserved. Key words: Bioreductive drugs, clinical studies, pre-clinical studies, review, tumour hypoxia

Introduction One aspect of radiobiology that has had a central role in cancer therapy is the importance of hypoxia to the radiotherapy response. In the early 1950s, Gray and colleagues [1,2] stressed the importance of hypoxia in reducing the effectiveness of radiation treatments. It soon became clear that at low oxygen levels tumour cells were up to three times more resistant to radiation than those containing oxygen [3]. Indeed, fractionation schedules were designed to optimise the re-oxygenation of tumour cells. In the early 1970s, Sartorelli’s group [4] proposed that the presence of hypoxia could be used to an advantage if a prodrug could be designed that was metabolised to a cytotoxic compound only in hypoxic cells; this would provide a method of killing these treatment-resistant cells while having no (or very little) systemic toxicity. The concept of bioreduction had been born. Over the last 35 years, many radiobiology groups have searched for ‘bioreductive’ drugs that meet this specification and it is only in the last decade that a few have reached 0936-6555/07/190427þ16 $35.00/0

phase II/III clinical trials. A major advantage of this approach is that a bioreductive drug should have limited, if any, systemic toxicity, as it requires hypoxia for activation, a feature that is found rarely in normal tissues [5,6]. Thus, a bioreductive drug will have specificity for tumour cells. However, as they predominantly target the hypoxic cell compartment, the remaining well oxygenated cells will be largely unaffected and consequently will require treatment with cytotoxic agents that work better in the presence of oxygen. Radiation is the prime example of this, as it predominantly targets the better-oxygenated cells. In addition, most standard chemotherapy drugs are more effective at killing aerobic cells (as they are more likely to be dividing) and, therefore, bioreductive drugs should also enhance their effects. The importance of tumour hypoxia to the wider cancer research community has become more apparent in the last 20 years, as it has become clear that hypoxia is a major determinant of the response to therapy whatever the treatment. This was made possible partly by the availability of sensitive oxygen electrodes that allow, before treatment,

ª 2007 The Royal College of Radiologists. Published by Elsevier Ltd. All rights reserved.

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the measurement of tumour oxygenation in situ. The results have clearly shown that hypoxia is common in a wide range of human tumours and that it signals a poor prognosis, irrespective of the treatment. Indeed, even when tumours were surgically excised, patients with hypoxic tumours did worse, suggesting that a hypoxic phenotype is bad news [7,8]. These studies have now been carried out in many tumour types, confirming the ubiquitous nature of tumour hypoxia and the associated poor prognosis. Not surprisingly, this applies to both primary and metastatic tumours [9e14].

The Problem of Tumour Hypoxia It could be argued that hypoxic cells are not really a problem as they are quiescent and, hence, not contributing to tumour growth. There are five reasons why this is not true. First, any treatment that kills better-oxygenated cells will allow hypoxic tumour cells to acquire increased levels of oxygen and, hence, start to regrow, making a significant contribution to repopulation of the tumour [3]. Second, it should be realised that hypoxia is not a static state and both chronic (diffusion limited) and acute forms of hypoxia exist [15]. The latter arises through the occlusion of blood vessels, which is transient in nature. The period of hypoxia is normally short lived and, hence, cellular proliferation will not be inhibited. The existence of these proliferating hypoxic cells has been confirmed in preclinical tumours [16]. It should be noted that the transiently hypoxic cells will be associated with treatment resistance and once re-oxygenated they will be particularly well placed to contribute to tumour repopulation. Third, it has become clear that tumour cells that survive in the hypoxic environment are more hypoxia tolerant/ stress resistant. Therefore, treatments that allow the reoxygenation of hypoxic cells will result in repopulation of the tumour with more malignant cells. Direct evidence for this selection was provided by a study of a mixed population of p53 wild type and p53 null immortalised mouse embryonic fibroblasts that were exposed to repeated rounds of hypoxia in vitro. The null cells were resistant to hypoxia-induced apoptosis and quickly became the dominant population in the co-culture [17]. Fourth, it has been reported that hypoxic exposure is associated with genetic instability. In a number of preclinical studies, hypoxia has been shown to induce gene amplification, chromosomal rearrangements, DNA overreplication and DNA strand breaks, showing that the tumour microenvironment is a potent endogenous stimulus of genetic change [18e20]. One marked consequence of these events is the acquisition of drug resistance [21]. Moreover, recent observations have suggested that instability can also arise through modifications in DNA repair pathways after hypoxic exposure [22,23]. As genetic instability is a required determinant of malignant progression, this highlights a further mechanism linking hypoxia with an aggressive disease phenotype. Finally, hypoxia would be less of a problem if tumour cells did not mount an adaptive response to promote survival

under conditions of low oxygen tensions. The transcription factor hypoxia-inducible factor-1 (HIF-1) is pivotal to this response and has been termed a master regulator of hypoxia-mediated gene expression [24]. HIF-1 is a heterodimer that is comprised of a and b subunits. Both are constitutively expressed, but the a subunit is subject to enzymatic modification by prolyl hydroxylase enzymes and subsequently targeted for degradation via the proteosome in the presence of oxygen. Hypoxia inhibits the activity of the prolyl hydroxylase enzymes, leading to the rapid accumulation of the a subunit and formation of the active HIF-1 transcription factor [25]. Gene targets of HIF-1 share a common promoter motif, the hypoxia-responsive element, which contains the core recognition sequence 5’ RCGTG 3’. A vast array of HIF-1 target genes has been identified to date, including glucose transporters and glycolytic enzymes that facilitate the adaptive metabolic shift to anaerobic glycolysis and angiogenic growth factors and cytokines that enhance the potential for re-oxygenation through vessel recruitment. These could be seen as classical ‘pro-survival’ responses to the aberrant environment [24,25]. In addition, a number of HIF-1 targets have been identified that can contribute to the metastatic transformation of the hypoxia-exposed cell (e.g. matrix metalloproteinase 2; urokinase plasminogen activator receptor; fibronectin 1; keratins, vimentin [26] and lysyl oxidase [27]). Furthermore, a direct role for HIF-1 in the promotion of genetic instability through the transcriptional repression of DNA repair enzymes has recently been elucidated [23]. A number of in vitro and in vivo studies suggest that HIF-1 status can influence both radio- and chemotherapy responses such that targeting HIF-1 can yield an enhanced therapeutic benefit when combined with standard therapies [28e33]. As for hypoxia per se, clinical data also support correlations between HIF-1 and aggressive disease. HIF-1a expression is detectable in the vast majority of human cancers and over-expression is more prevalent in metastases compared with primary tumours [34,35]. Furthermore, HIF-1a over-expression can be predictive of a poor prognosis after radio- and/or chemotherapy, although this observation is not universal [36e39]. Interestingly, the prognostic significance of downstream targets of HIF-1 (e.g. carbonic anhydrase-9 and glucose transporter-1) is greater in determining disease-free survival or metastases-free survival rather than local control [40,41]. For all of the above reasons, it would seem far better to kill hypoxic cells rather than allow them to become the predominant phenotype. Bioreductive drugs, as an adjunct to any cancer treatment, could potentially fulfil this need.

Targeting Hypoxic Cells with Bioreductive Drugs: Pre-clinical Studies For any bioreductive drug to be effective it will, by definition, require hypoxia, which is primarily found in tumours, and an enzyme (or enzymes) capable of reducing the drug to a cytotoxic species. So far four groups of putative

BIOREDUCTIVE DRUGS

bioreductive drugs have been classified based on their chemistry (Table 1). The activation of these drugs and the pre-clinical evidence of their efficacy are discussed below.

Quinones The prototype bioreductive agent was the quinone-alkylating agent mitomycin C (MMC). Although MMC was in routine clinical use much earlier, it was not until the 1980s that it was first recognised that the hypoxic tumour environment could facilitate the bioreduction and activation of MMC [64]. When the in vitro cytotoxicity of MMC was measured in hypoxic as compared with normoxic cells (hypoxic cytotoxic ratio, HCR) the differential was generally less than five-fold [53,65,66]. The one-electron reductase NADPH/cytochrome P450 reductase (P450R) has been the predominant flavoenzyme implicated in the bioactivation of MMC (and other quinone agents) under hypoxic conditions [54]. One-electron reduction produces the semiquinone radical anion that covalently interacts with DNA causing cross-links as the major cytotoxic lesion. In the presence of oxygen the free radical is back-oxidised, thereby giving hypoxia selectivity to the generation of the cytotoxic species. However, P450R is not the only flavoenzyme associated with quinone reduction. MMC and other agents within this class (e.g. RH1 [55] and apaziquone [EO9] [56]) are substrates for the two-electron reducing enzyme, DT-diaphorase (DTD; NAD(P)H: quinone oxidoreductase, NQO1) [53,57,67]. DTD is over-expressed in a number of tumour types and has, therefore, been seen as an attractive target for bioreductive drug design [68e71]. However, as DTD is an oxygen-independent reductase, bioactivation is observed in aerobic conditions that can severely compromise hypoxic selectivity. Furthermore, the precise correlation between MMC sensitivity and DTD activity in vivo has been questioned [72,73].

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The identification of MMC as a potential bioreductive agent led to the search for analogues that showed a greater degree of preferential toxicity towards hypoxic cells. Porfiromycin, a methylated analogue of MMC, was found to have a much greater HCR than the parent compound [74]. Encouragingly, porfiromycin showed enhanced cytotoxicity against hypoxic cells, even in cell lines where the toxicity of MMC was independent of oxygen tension [75]. The enhanced selectivity of porfiromycin towards hypoxic cells was associated with reduced substrate specificity towards DTD, preferential activation via one-electron reductases and enhanced drug uptake in hypoxic conditions [76]. Pre-clinical studies combining porfiromycin with radiotherapy illustrated an additive effect in vitro and a supra-additive effect in vivo, consistent with the idea that porfiromycin successfully targeted the radioresistant hypoxic tumour compartment [77]. Further development within this area resulted in the synthesis of a series of indolequinones, of which EO9 showed the most promise in pre-clinical studies. The HCR of EO9 is about 30 and significantly superior to that of MMC [50,51]. P450R is implicated in the activation of EO9, particularly in hypoxia, and studies using cell-free systems have shown that P450R-mediated activation of EO9 results in the generation of a DNA-damaging radical [51,52]. However, EO9 is also an excellent substrate for oxygenindependent reduction by DTD that impacts on hypoxia selectivity of the drug in high DTD backgrounds [50,78,79]. Combination with radiotherapy revealed promising data in pre-clinical tumour models [80] and led to the initiation of clinical trials (see below).

Nitroaromatics Coincidental with the development of quinones, a second class of bioreductive agents was identified. These studies

Table 1 e Classes of bioreductive drug and their metabolic profiles

Prodrug

Cytotoxic species

Final product

Enzymes thought to be responsible

Key references

Benzotriazines, e.g. tirapazamine (TPZ)

Free radical (SR4317)

Non-toxic (SR4330)

P450R; iNOS

[42e49]

Bi- (and mono-) functional alkylating agents, e.g. mitomycin C, porfiromycin, RB 6145, RSU 1069, EO9, RH1

DNA adduct

Non-functional reduced drug

P450R; DTD

[44,50e57]

Alkyl-amino-anthraquinones, e.g. banoxantrone (AQ4N)

AQ4

Stable persistent cytotoxin, topoisomerase II inhibitor

Cytochrome P450s d 1A1, 2B6, 3A4; iNOS

[58e60]

NLCQ-1

Nitro (1ee), nitroso (2ee) and hydroxylamine (4ee) metabolites

Unknown, shows weak DNA-intercalating activity

Cytochrome b5, other one-electron reductases

[61,62]

Phosphate esters of dinitrobenzamide mustards, e.g. PR-104 (Proacta)

Stable cytotoxin

Stable persistent cytotoxin

Phosphatases to generate alcohol then nitroreductases, including oneelectron reductases

[63]

DTD, DT-diaphorase; iNOS, inducible nitric oxide synthase; P450R, cytochrome P450 reductase; EO9, apaziquone.

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initially focused on the nitroaromatic hypoxic cell radiosensitiser misonidazole. Experiments using both monolayer cultures and multicellular spheroids (a three-dimensional in vitro model of chronic hypoxia) revealed that misonidazole possessed selective cytotoxicity towards hypoxic cells [81e84]. Nitroaromatic prodrugs are bioreduced via the stepwise addition of up to six electrons catalysed by various one-electron reductases. As with the quinones, the first reduction one-electron intermediate can be readily backoxidised in the presence of oxygen, although subsequent reductions are effectively irreversible. Misonidazole itself was found to have a HCR similar to, or greater than, that for MMC [83]. Furthermore, misonidazole could enhance radiotherapeutic outcome in vivo when given after radiotherapy [85]. As this cannot be attributable to radiosensitisation, which requires the agent to be present at the time of radiation, the enhancement was consistent with hypoxic cytotoxicity. A drug development programme around the nitroimidazole-based misonidazole ensued and the first agent to show a marked enhancement in hypoxia selectivity was RSU1069 [86,87]. This 2-nitroimidazole contains an aziridine group in the N1 side chain enabling RSU1069 to function as a bi-functional alkylating agent upon reduction. The one-electron reductase P450R has been implicated as an important bioactivator of this compound [44]. In vivo studies exemplified that RSU1069 could enhance radiation response irrespective of whether the drug was given before or after radiotherapy and that RSU1069 was also effective when used in combination with fractionated radiotherapy [66,80]. Clinical development of this compound was terminated, however, upon the discovery of marked gastrointestinal toxicity [88]. RB6145, a less toxic prodrug of RSU1069, was then developed and showed good efficacy in vivo. However, the R-enantiomer caused retinal toxicity in preclinical models, which blocked further development [89]. More recently, nitroaromatic bioreductive agents have been developed that exhibit weak DNA-intercalating ability. The lead agent, NLCQ-1 (NSC 709257) [62], exhibits up to 40-fold selectivity towards hypoxic tumour cells in vitro (Cowen and Williams, unpublished data, [90]). Furthermore, the hypoxic potency of the drug increases with increasing exposure time, a phenomenon that is associated with the ‘bis-bioreductive agents’ that contain two reducible centres. Analysis of the metabolism of NLCQ1 using cell-free systems suggests that the one-electron reductases P450R and cytochrome b5 reductase play a significant role in the bioactivation of NLCQ-1 [61]; the very poor reduction kinetics of NLCQ-1 by human recombinant DTD suggest that DTD is not important in the initial reduction steps of this drug. In a panel of transfected human tumour cell lines, P450R over-expression was consistently associated with enhanced NLCQ-1 toxicity, whereas DTD had little effect (Cowen and Williams, unpublished data). Combination studies have shown that NLCQ-1 is a potent radiosensitiser in vitro and can show a synergistic interaction with radiotherapy in vivo [62,91]. With chemotherapy, synergistic interactions are seen with the alkylating agents, cisplatin, cyclophosphamide or melphalan, the

anti-mitotic paclitaxel and the anti-metabolite 5-fluorouracil (5-FU). NLCQ-1 was found to be optimal when given before the alkylating agents; no schedule dependency was observed in the other combinations. Importantly, modifications in the efficacy of chemotherapy agents were not matched by any potentiation of bone marrow toxicity [62]. Furthermore, the efficacy studies undertaken so far suggest that NLCQ-1 compares favourably with the current lead bioreductive agent tirapazamine (TPZ) [90e96]. Currently, licensing opportunities are being negotiated for the clinical development of NLCQ-1. The bioreductive potential of dinitrobenzamide mustards has been investigated by the groups of Denny and Wilson [63]. These compounds are activated only under severe hypoxia and provide for a bystander effect due to the formation of relatively stable cytotoxic metabolites. They were initially developed as analogues of the weak monofunctional alkylating agent CB1954 [97]. This drug was found to have potent anti-tumour activity against the Walker rat carcinoma associated with its reduction by rat DTD, which is present at high levels in this tumour model [98]. CB1954 is reduced to the 4-hydroxylamino derivative, which undergoes further reaction with acetyl coenzyme A to produce a potent DNA inter-strand cross-linking agent. However, its therapeutic efficacy is limited in human tumours, as CB1954 is not as good a substrate for human DTD as it is for the rat isoform [99]. CB1954 was subsequently found to be a substrate for nitroreductase (NTR) [100] and has been clinically developed in gene therapy protocols (see below) [101]. The first generation of dinitrobenzamide mustards (e.g. SN 23862) had poor aqueous solubility and limited hypoxic selectivity, leading to the more recent development of phosphate ester analogues. These have excellent solubility and formulation characteristics, and act as ‘pre-prodrugs’; the systemic phosphatase activity generates the corresponding alcohols (prodrugs), which are subsequently activated by NTRs. A lead compound, PR-104 (Proacta), has now been identified and recently commenced its first phase I trial in solid tumours (NCT00349167) [102].

Aliphatic N-oxides Another group of compounds that has shown potential as bioreductive drugs are the aliphatic N-oxides. The lead compound is the bis-N-oxide banoxantrone (AQ4N), which is reduced under hypoxic conditions to yield the cytotoxic product AQ4 [103]. AQ4, an analogue of mitoxantrone, has high DNA binding affinity and also acts as a topoisomerase II inhibitor, characteristics that are absent in AQ4N. Bioreductive activation of AQ4N to AQ4 occurs through two 2electron additions via the intermediate AQ4M. The key enzymes implicated in this bioactivation are the cytochrome P450s (CYPs) [58,59], in particular CYP1A1, CYP2B6 and CYP3A4 [103]. Interestingly, P450R does not seem to play a substantial role [58], although the enzyme inducible nitric oxide synthase (iNOS), which exhibits considerable homology to P450R, seems to be a potent activator in vitro [60]. One of the key distinguishing features of AQ4N is the

BIOREDUCTIVE DRUGS

fact that the reduction product AQ4 is stable and persistent, thereby enhancing the potential for a bystander effect to be observed. Initial in vitro studies were hampered by the fact that a classic hypoxic cell sensitisation was not apparent. However, this is probably a consequence of the down-regulation of CYP proteins that is generally seen in cultured cells. Hypoxia selectivity was observed when cultured cells were incubated with NADPHsupplemented microsomes that are rich in CYPs [58]. In addition, evidence for AQ4N metabolism could be observed in cells prepared from murine tumours immediately after excision but not 24 h later [104]. When AQ4N was tested as a single agent in vivo, it had limited effect on tumour growth. However, in combination with methods to increase tumour hypoxia, a substantial growth delay could be observed, consistent with its action as a hypoxic selective cytotoxin [105,106]. Combination studies using murine tumour models have shown that AQ4N enhances the anti-tumour effects of radiation, cisplatin, cyclophosphamide and thiotepa [105e109]. These observations have been supported by human tumour xenograft studies with radiotherapy or chemo- (cisplatin) radiotherapy (Williams et al., unpublished data). Studies analysing DNA damage after radiotherapy found that the addition of AQ4N resulted in more persistent damage than radiation alone [104]. This may be taken as evidence of the long-lived nature of the AQ4 product and/or the fact that once formed, AQ4 may cause DNA damage in cells recruited to the cell cycle after radiotherapy. In all of these studies, AQ4N alone did not show any normal tissue toxicity at effective doses and could be scheduled appropriately with chemotherapy so as not to influence their inherent normal tissue effects. These data, combined with favourable pharmacokinetic parameters in murine studies [110], supported the promotion of AQ4N into clinical development (see below).

Heteroaromatic N-oxides The current lead bioreductive agent, TPZ, first emerged as a hypoxia-selective cytotoxin in the 1980s [42]. TPZ is a heteroaromatic N-oxide that exhibits impressive HCRs in the range 50e200. In vivo combinations with radiotherapy showed positive interactions, particularly with fractionated protocols [111e113]. TPZ is also beneficial when combined with a myriad of chemotherapeutic agents (cisplatin, cyclophosphamide, carmustine [BCNU], melphalan, etoposide, bleomycin, doxorubicin, taxol, carboplatin, paclitaxel and 5-FU), although there are some subtleties in relative scheduling to elicit the maximum benefit of the combination [96,114e120]. Encouragingly, reported enhancements in normal tissue toxicity were generally small, with the exception of 5-FU [118]. Initial studies suggested that the one-electron reduction product may be the cytotoxic moiety [42]. Subsequently, TPZ has been shown to be an excellent substrate for the classical one-electron reductases, cytochrome P450 and P450R [43e47] and more recently NOS [48,49]. One-electron reduction causes the generation of the nitroxide radical intermediate that can be readily back-oxidised in the presence of oxygen. In the

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absence of oxygen, the radical undergoes rearrangement by the loss of water to form an oxidising radical that can cause DNA damage through the abstraction of a hydrogen atom [97]. The short-lived nature of the one-electron product means that TPZ has no bystander effect. TPZ exhibits a markedly different cytotoxicity relationship with respect to oxygen availability than other bioreductive agents that generally require very low oxygen tensions to elicit bioactivation. In contrast, TPZ has been shown to become increasing cytotoxic as oxygen levels are diminished [121]. This means that TPZ can effectively target cells at intermediate oxygen tensions that are radioresistant but not sufficiently hypoxic for targeting with other bioreductive drugs. The importance of controlling this population in terms of both local control and the advent of secondary disease has been recently highlighted in a pre-clinical model [122]. Although a broadened oxygen selectivity is on the one hand beneficial when the reactive product is a very short-lived radical, there is a downside as it has been recognised for some time that the extent of metabolism of a bioreductive drug through an oxygen gradient is an important factor in determining drug distributions in vivo. A number of studies have suggested that the metabolism of TPZ at intermediate oxygen tensions could impede its delivery to chronically hypoxic sites within the tumour [123,124]. Although the effect of this on overall responsiveness has not been categorically addressed, it has been suggested that the development of TPZ analogues should focus upon molecules with better diffusion characteristics [124]. In the last 15 years, the potential of bioreductive drugs has been explored in the clinic with varying levels of success (see below). It is clear that the activation of bioreductive drugs is dependent both on the hypoxic tumour microenvironment and the presence of endogenous reductases to metabolise the drugs. Therefore, great efforts have been made to identify the key enzymes involved in bioactivation of the lead bioreductive agents. Enzyme profiling studies, in human tumours as compared with healthy tissues, have shown that several endogenous reductases have increased activity in tumours, most notably DTD, iNOS, carbonyl reductase and cytochrome P450s [70,125e128]. However, even for these enzymes the activity levels are heterogeneous between patients and between different tumour types. One approach, which is currently being developed, is the enhancement of bioreductive drug activation using gene-directed enzyme prodrug therapy (GDEPT) [129].

Gene Therapy to Sensitise Tumours to Bioreductive Drugs To achieve enhancement of bioreductive drug metabolism, a GDEPT strategy can be designed using genes encoding for reductases combined with specific bioreductive drugs. As P450R plays a major role in TPZ metabolism, a P450R/TPZ GDEPT strategy has been tested [130,131]. It should be noted that tumour cells endogenously over-expressing P450R are sensitised to TPZ [44,130]. However, when the

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P450R is over-expressed exogenously, TPZ toxicity increases in both hypoxic and well-oxygenated cells, reducing the HCR. A modified approach to achieve hypoxia-specific overexpression of P450R has been proposed [131,132]. This is achieved by the incorporation of hypoxia-responsive enhancer sequences within the promoter of the P450R expression cassette, which bind the transcription factor HIF-1 [133,134], resulting in HIF-dependent transcriptional activation of the P450R gene. A replication-defective adenoviral vector was used to deliver the hypoxia-responsive P450R cassette to both dividing and quiescent hypoxic tumour cells. When used in combination with radiotherapy, to control the well-oxygenated tumour, this strategy resulted in total tumour clearance [131]. AQ4N is an ideal prodrug for inclusion in a GDEPT strategy. Unlike TPZ, whose active metabolite is highly reactive and short lived, when AQ4N is reduced the product, AQ4, is very stable and can elicit a bystander effect (McKeown, unpublished data). One of the major limitations of gene therapy is the inability to deliver and express the gene in all tumour cells; this becomes less of a problem if the cytotoxic product can diffuse to neighbouring cells. Several CYP enzymes mediate the bioreduction of AQ4N and these have been tested in GDEPT strategies. When CYP2B6 was delivered by intra-tumoural injection into murine tumours, an enhanced response was observed with AQ4N in combination with radiation or cyclophosphamide. In the latter approach, the metabolism of both AQ4N and cyclophosphamide should be enhanced by CYP2B6 [130,135]. CYP3A4 and CYP1A1 injection into murine tumours can also increase tumour metabolism of AQ4N and enhance tumour control with radiation [136,137]. Although AQ4N is not a substrate for P450R, it has been shown that it is readily metabolised by iNOS [60]. The use of iNOS gene therapy to sensitise tumours to AQ4N is particularly attractive, as iNOS also catalyses the conversion of L-arginine to citrulline with the concomitant production of nitric oxide, which has been shown to be both directly cytotoxic and a potent radiosensitiser in tumours [138]. The discovery that CB1954 is efficiently reduced by Escherichia coli NTR, at a 60 times faster rate than by rat DTD [98e100] opened up the opportunity to develop CB1954/NTR GDEPT strategies. NTR is encoded by the nfsB/ nfnB gene, which has been delivered to human tumours in pre-clinical GDEPT studies by either retrovirus or replicationdefective adenovirus vector infection [139,140]. The toxic metabolite of CB1954 exhibits a beneficial bystander effect diffusing to, and killing, adjacent tumour cells that do not express NTR [139]. Systemic administration of CB1954 after viral-mediated NTR gene delivery resulted in a significant reduction in tumour growth compared with non-infected tumours and several complete tumour regressions were also observed. Consequently, several CB1954/NTR gene therapy clinical trials have been initiated using a replication-defective adenovirus vector (CTL102) to deliver the NTR gene to accessible tumours in patients with primary and secondary liver, head and neck, and prostate cancer [101,141]. In conclusion, there has been much pre-clinical evidence that, in the right setting, bioreductive drugs can enhance

the effectiveness of both radiotherapy and chemotherapy. For some situations it may be necessary to use GDEPT to enhance bioreduction. However, many of the compounds are effective without recourse to this more complex therapy. The more important clinical trials that have resulted from the pre-clinical studies of bioreductive drugs are now reviewed.

Clinical Trials Involving Bioreductive Drugs As discussed, there is much pre-clinical evidence that bioreductive drugs can effectively enhance the toxicity of standard cancer therapies. However, designing and carrying out clinical trials brings its own challenges. In particular, phase I trials of agents that are predicted to be unsuccessful when given alone are somewhat problematic, as it can be difficult to establish a maximum tolerated dose (MTD). However, several bioreductive drugs have now entered clinical trials with varying degrees of success. The most thoroughly investigated is TPZ and there are now several phase II and one phase III trial reported. MMC has also been widely tested, but its activity is complicated by its action on normoxic cells. Apart from TPZ, the most hopeful is AQ4N, which has been shown to be tolerated up to high doses. Details of some of the clinical trials are shown in Tables 2 and 3 and the more important aspects of these are discussed below.

Tirapazamine The first phase I trial of TPZ was reported in 1994 [161] and since then there have been nine phase I trials and 15 phase II/III trials that have resulted in published papers; most of the latter have been summarised in Table 2. In several phase I studies, the MTD was fixed at 330 mg/m2 [142,162e164]. In one phase I trial, the MTD of TPZ was 390 mg/m2 when combined with 100 mg/m2 cyclophosphamide. However, this combination had a dose-limiting toxicity of granulocytopenia and was not recommended unless growth factor support was available [165]. The most widely used range of TPZ doses has been 260e330 mg/m2, depending on the combination of radiation and/or chemotherapy schedules and the frequency of delivery. Neutropenia is commonly reported, but usually in a tolerable range; a few patients have developed a febrile neutropenia. The most frequent non-haematological toxicities reported are nausea, vomiting, diarrhoea and skin rash; with the highest TPZ doses some patients showed grade 3e4 toxicities. Two more unusual toxicities noted were reversible deafness and muscle cramping; the latter was frequently observed, but not found to be dose limiting [142,145,151,161,162]. The published phase II trials have investigated a range of tumour types in over 1100 patients. The results of these trials have generally been promising and have indicated progress to phase III investigations. Only a few randomised phase II or III studies have been completed and these have

Table 2 e Reported clinical trials using tirapazamine (TPZ) to target hypoxic tumour cells Reference

n

Tumour

Phase

Treatment

Outcome

39

Stage III/IV HNSCC

II

Radiotherapy (70 Gy in 7 weeks) with concurrent TPZ (159 mg/m2 3 times/week for 12 doses)

1- and 2-year local control rates: 64 and 59%, respectively

[143]

122

Stage III/IV HNSCC

II

3-year failure-free survival rates: 55% with TPZ/CIS and 44% with chemoboost arm. 3-year locoregional failure-free rates were 84% in the TPZ/CIS arm and 66% in the chemoboost arm

[144]

62

Stage IV HNSCC

II

Radiotherapy (70 Gy in 7 weeks) þ CIS (75 mg/m2) þ TPZ (290 mg/m2):day 2 of weeks 1, 4, and 7, þ TPZ alone (160 mg/m2) on days 1, 3, and 5, weeks 2 and 3 TPZ/CIS or CIS (50 mg/m2) on day 1 and infusional 5-FU (360 mg/m2) on days 1e5 of weeks 6 and 7 TPZ, CIS, 5-FU:induction chemotherapy (2), followed by simultaneous chemoradiotherapy (TPZ, CIS, and 5-FU) or the same regimen without TPZ. Radiotherapy (2 Gy fractions): 66e70 Gy to tumour. Dose to supra-clavicular region was 50 Gy (25  2 Gy) prescribed at depth of 3 cm.

[145]

20

Advanced NSCLC

II

[146]

44

Advanced NSCLC

II

[147]

446

NSCLC (previously untreated)

II

[148]

45

NSCLC

II

[149]

21/49 NSCLC

I/II

Clinicopathologic response rate in lymph nodes: standard (90%) vs TPZ (74%) (P ¼ 0.08), response at the primary site and 89% and 90% (P ¼ 0.71). 5-year overall survival: 59%; cause-specific survival rate: 68%; loco-regional control rate: 77% for entire group TPZ (390 mg/m2) over 2 h; 1 h later CIS 25% had major objective responses; (75 mg/m2) over 1 h. Repeated every 21 days median duration 8 months; 1-year survival 40%. TPZ/CIS appears to be safe and active in the treatment of NSCLC without a substantial increase in toxicity compared to CIS alone 260 mg/m2. TPZ over 2 h; 1 h later CIS Median survival and response rate observed strongly suggest that TPZ 75 mg/m2 over 1 h; repeat every 21 days. with CIS is more active than CIS alone CATAPULT 1 study: TPZ: 390 mg/m2 over Median survival was longer (34.6 vs 2 h; 1 h later CIS 75 mg/m2 over 1 h, or 27.7 weeks; P ¼ 0.0078) and the response rate was greater 27.5% v 75 mg/m2 of CIS alone, every 3 weeks for a maximum of eight cycles 13.7%; P ! 001) for TPZ þ CIS than for CIS alone TPZ 330 mg/m2 (day 1), CIS 75 mg/m2 Response rate: 40%. Median (day 1) and Gemcitabine 1250 mg/m2 progression free survival: 6.7 months. (days 1 and 8) every 3 weeks Median survival: 8.1 months. One-year survival: 35% I: MTD not identified. Most toxicity I: day 1 TPZ (260, 330, or 390 mg/m2), grade 1/2 CIS (75 or 100 mg/m2), vinorelbine II: response rate: 47%; (25 or 30 mg/m2), maximum six cycles median survival: 50 weeks every 4 weeks. Vinorelbine repeated weekly. II: day 1 TPZ 390 mg/m2, CIS 100 mg/m2, and vinorelbine 30 mg/m2.

33% had grade 3 or 4 drug-related toxicities. No excessive radiotherapy–associated acute normal tissue reactions Both regimens feasible; significant but acceptable toxicity. Based on promising efficacy seen, TPZ/CIS is being evaluated in phase III trial

There was no difference with regard to any of the outcome parameters between the two treatment arms. Haematologic toxicity was greater with TPZ.

Toxicities: temporary hearing loss, muscle cramping, diarrhoea, skin rash, nausea, vomiting. No grade 3 or 4 haematologic or renal toxicity was observed

BIOREDUCTIVE DRUGS

[142]

Comments

Expected toxicities were treatable and no dose reductions were necessary. No incremental toxicity increases and no TPZ related deaths. TPZ enhanced the activity of CIS; confirms hypoxia is an exploitable therapeutic target Haematologic and non-haematologic toxicity were moderate Most frequently reported adverse event was neutropaenia. Asthenia, fever, anaemia, vomiting, weight decrease, nausea, and muscle cramps also noted 433

(continued on next page)

Treatment

Advanced NSCLC

III

Cycle 1: Paclitaxel 225 mg/m2 þ carboplatin (area under the curve ¼ 6) þ TPZ 260 mg/m2 (escalated, if tolerated, to 330 mg/m2 in cycle 2), or paclitaxel þ carboplatin, no TPZ. 21-day interval between cycles.

[151, 152] 48

Advanced malignant melanoma

II

TPZ (260 mg/m2) then CIS (75 mg/m2); every 21 days

[153]

124

Glioblastoma multiforme

II

[154]

64

Recurrent CIS-sensitive ovarian or peritoneal cancers

II

RT: 30  2-Gy. TPZ: 3 times/week for 12 treatments during radiotherapy. 55 received TPZ at 159 mg/m2; 69 received 260 mg/m2 TPZ was administered at a dose of 390 mg/m2 over 2 h; 1 h later CIS 60 mg/m2 every 3 weeks until disease progression or adverse effects prohibited further therapy

[155]

36

Carcinoma of the cervix (recurrent or stage 4)

II

TPZ (330 mg/m2) over 2 h; 1 h later CIS (75 mg/m2) over 1 h every 21 days to maximum of eight cycles

[156]

56

Carcinoma of the cervix

II

TPZ, 260 mg/m2, followed by CIS, 75 mg/m2, every 21 days for six cycles

367

Tumour

Outcome No differences in response rates, progression-free survival, or overall survival. TPZ arm caused more side effects (P ! 0.05). The trial was closed early as interim analysis showed projected improvement was unachievable Nine (mostly cutaneous melanoma) showed partial response; overall response rate: 19%; median duration of response: 6 months There was no significant survival advantage compared to controls (standard treatment population.) 26 patients received six or more cycles of therapy; 16 received one course of therapy. There were six complete responders, 28 partial responders; total response rate: 53%. Median progression-free and overall survival for all patients was 10.9 and 26.4 months, respectively Response rate was 27.8%; 11 patients had stable disease as best response. Response rate was greater in tumours outside the previous radiotherapy field (44.4 vs 11.1%). Median time to progression: 32.7 weeks TPZ þ CIS was active in patients who had not received CIS previously. Prior use of radio-sensitizing chemotherapy impacted response and survival significantly

Comments The addition of TPZ to paclitaxel and carboplatin does not improve survival in advanced NSCLC but substantially increases toxicity

All responders were chemotherapy naive

Grade 3/4 toxicities were more frequent in the higher dose regimen TPZ þ CIS has definite activity. Toxicity, mainly non-haematological, was substantial. Reducing the toxicity should be pursued in future trials

Most frequent adverse events were nausea, vomiting, and fatigue. Anaemia was most frequent grade 3/4 haematologic toxicity (8.3%) Systemic toxicity was acceptable but overall survival was thought too low to warrant further testing in this disease

MTD, maximum tolerated dose; n, number of patients enrolled; HNSCC, squamous cell carcinoma of the head and neck; NSCLC, non-small cell lung cancer; 5-FU, 5-fluorouracil; CIS, cisplatin.

CLINICAL ONCOLOGY

Phase

[150]

n

434

Table 2 (continued ) Reference

Table 3 e Summary of current and previous trials of banoxantrone (AQ4N) Sponsoring body [reference]

n

Tumour

Phase

Treatment

22

Oesophageal tumours

I/II

Dose escalation study. Initial AQ4N dose was repeated 2 weeks later with same dose combined with palliative radiotherapy (5  4 Gy)

KuDOS Pharmaceuticals [158]

32

Bladder, breast, cervix, glioblastoma, head and neck

I

Single preoperative dose of AQ4N, 200 mg/m2, to surgical patients. Tumour and healthy tissue evaluated: HPLC (AQ4N and AQ4), immunohistochemistry (for hypoxia, using Glut-1), confocal microscopy (co-localisation of drug/hypoxia signals)

KuDOS Pharmaceuticals

(17)*

Advanced solid tumours

I

Safety and tolerability of AQ4N, administered in combination with cisplatin

KuDOS Pharmaceuticals

(20)*

Invasive bladder cancer

I

Safety and tolerability of AQ4N, given in combination with radiotherapy

NCT00109356y Novacea [159]

11

Hodgkin’s and Non-Hodgkin’s Lymphoma; chronic lymphocytic lymphoma

I/II

AQ4N once every 3 weeks for a maximum of 24 weeks: patients received the same dose for the entire study. Dose escalation 400 mg/m2 up to 1200 mg/m2

NCT00090727y Novacea [160]

16

Advanced Solid Tumours

I

NCT00394628y Novacea

(60)z

Glioblastoma multiforme

Ia and IIb

AQ4N administered over 30 min on days 1, 8, and 15 of a 28-day cycle. Aim to establish MTD and pharmaco-kinetic analysis for weekly AQ4N Safety and tolerability of AQ4N with radiotherapy and temozolomide. Dose levels selected from Phase I

The MTD with radiotherapy was not reached at the closing dose of 447 mg/m2. No DLT seen with or without radiotherapy. Pharmacokinetic and toxicity study Treatment was well-tolerated. All solid tumours metabolised and accumulated AQ4 (tumour levels exceeded those in healthy tissue and plasma). AQ4 co-localises with hypoxic signal. Levels achieved were potentially therapeutic (based on exposures achieved in animal models.) Recruiting. Current dose level: 400 mg/m2 AQ4N þ 80 mg/m2 cisplatin (both given day 1 of 21 day cycle; up to six cycles permissible) Recruiting. Current dose level: 400 mg/m2 AQ4N (days 1, 8) þ 30 Gy (3 Gy/day, days 1e5, 8e12). Single cycle only AQ4N is well-tolerated in repeat 21-day schedule at doses up to 1200 mg/m2. Further dose escalation was precluded since maximum solubility of AQ4N was reached Dosing was carried out up to 1200 mg/m2. MTD set at 786 mg/m2 due to DLT of respiratory distress (grade 5) and fatigue (grade 3) Recruiting. A range of outcomes will be measured

BIOREDUCTIVE DRUGS

Cancer Research UK/KuDOS Pharmaceuticals [157]

Comments

n, number of patients.DLT, dose-limiting toxicity; MTD, maximum tolerated dose. *Current number accrued. yhttp://www.clinicaltrial.gov/. zProjected number.

435

436

CLINICAL ONCOLOGY

so far shown a limited improvement in tumour control or no added effect for frequently an increased (if tolerated) toxicity [143,150]. This is disappointing after the encouraging results of some (but not all) of the phase II trials. One problem may be the statistical evaluation linked to the trials, as all cases are usually included in the analysis without trying to identify those with increased levels of hypoxia. A method to stratify the tumours into high and low hypoxic groups should improve the significance of the trials, as the patients with better-oxygenated tumours will have less likelihood of an improvement in outcome and, hence, may mask any bioreductive enhancement. This has been exemplified by a recent report comparing cisplatin/5-FU vs cisplatin/TPZ. Positron emission tomography, before treatment, was used to stratify the tumours into hypoxic and non-hypoxic. This study clearly showed that TPZ improved local control of hypoxic head and neck tumours. Cisplatin/ 5-FU showed 8/18 tumours recurring in the hypoxic group, whereas 0/26 recurred in patients treated with cisplatin/ TPZ. When the non-hypoxic tumours were analysed, TPZ did not provide an improvement in outcome: 2/27 (5-FU) vs 3/21(TPZ) [166,167]. This clearly shows that local control of hypoxic tumours is improved when a hypoxic cell cytotoxin is included in the treatment regimen, underlining the importance of treating these cells whenever they are present.

Banoxantrone The current and previous trials of AQ4N are summarised in Table 3. The first trial was carried out in oesophageal cancer to establish a MTD and evaluate its tolerability with a course of palliative radiotherapy. The design of this trial illustrates the problem of developing prodrugs that are potentially non-toxic. A two-step approach was used. Initially, AQ4N was given as a single agent with a 2 week follow-up. If no toxicity ensued, the patients received a second dose followed by a course of palliative radiotherapy; each patient was only treated at one dose level. The drug was well tolerated at all doses tested; a MTD was not established before the trial was closed. No dose-limiting toxicity was identified nor any enhancement of the radiotherapy reactions [157]. A second phase I trial has recently reported that AQ4N (200 mg/m2) was primarily localised (as the reduced product AQ4) in tumour tissues, which were excised 18 h after dosing. This tumour-specific deposition of AQ4 contributes to the safety profile of the product. Control tissues excised at the same time-point and plasma samples showed that most of the AQ4N had been eliminated. The high levels of intra-tumoural AQ4 achieved were in the range shown to be effective in animal models and, hence, of potential therapeutic benefit to man [158]. In a phase I trial to establish the MTD for AQ4N given every 3 weeks for patients with lymphoid neoplasms, a dose of 1200 mg/m2 was achieved without dose-limiting toxicity. The most common AQ4N-related adverse events in the 11 patients enrolled were the expected transient skin discoloration (due to the intense blue colour of the drug) (100%), transient chromaturia (36%), lymphopenia (27%), fatigue

(27%) and nausea (27%). The adverse events were primarily mild (grade 1e2), with the exception of grade 3 lymphopenias (n ¼ 3) and grade 3 neutropenia (n ¼ 1) [159]. In a further phase I trial in solid tumours where AQ4N was given weekly in 16 patients, a dose-limiting toxicity at 1200 mg/m2 was found in 2/5 patients involving grade 5 respiratory distress and grade 3 fatigue. This has led to the MTD for weekly administration of AQ4N as a single agent to be set at 768 mg/m2 [160]. The most common AQ4N-related event profile was similar to the phase I trial in lymphoid neoplasms. Further phase I combination studies with cisplatin and phase II studies with radiotherapy are ongoing in Europe and a study of AQ4N, temozolomide and radiotherapy in glioblastoma is currently recruiting in the USA. In summary, the AQ4N trials have been promising and occasional patients have shown partial responses. Because AQ4N only targets hypoxic cells, it is not surprising that it shows limited efficacy as a single agent. The drug-related toxicities are generally mild and the MTD allows high levels of AQ4 to be sequestered in the tumour. The current combination trials will help to show how much this can enhance the effects of standard therapies. An additional advantage of the wide distribution of the prodrug is its ability to cross the bloodebrain barrier, which will allow AQ4N to be added to treatments aimed at controlling the growth of primary brain tumours and metastatic deposits from other sites.

Mitomycin C and Analogues Evaluation of the ability of MMC to act as a bioreductive drug in the clinical setting is confounded by the fact that this drug has significant activity in normoxic conditions. Many trials have been carried out and have been reviewed previously [168]. Currently, 20 trials are recruiting in the USA where MMC is part of a treatment regimen. However, none of the trial descriptions claims the bioreductive capability of MMC as a rationale for the study. Because the HCR in vitro is only about 5, this suggests that the bioreductive potential of MMC may be limited. This problem was further supported by a phase II study of 5-FU-resistant colorectal cancer, in which MMC was combined with BW12C, an agent that acts by left shifting the oxygen dissociation curve of haemoglobin, effectively reducing tumour oxygenation by about 50% [169]. No enhancement of the anti-tumour efficacy of MMC could be detected, suggesting that it may be difficult to show the bioreductive capabilities of MMC over its effect under normoxic conditions. An alternative explanation proposed for the trial failure was that drug resistance was responsible. More recently, intra-arterial administration of MMC has been investigated. These trials were designed to make use of the hypoxic enhancement of MMC combined with other treatments to reduce the growth of local tumours. For example, metastatic deposits in the liver have been treated with MMC combined with ischaemic damage caused by the embolisation of the vascular supply to the tumour [170]. The results were encouraging, showing that this approach

BIOREDUCTIVE DRUGS

was safe and feasible. A reasonable clinical response was seen in 9/13 patients. A less favourable outcome was obtained in a phase I/II study of primary pancreatic tumours, where 21 patients with advanced pancreatic carcinoma were treated with hypoxic abdominal perfusion (HAP) combined with MMC and melphalan followed by celiac axis infusion with the same agents 6 weeks later. In contrast to earlier reports of HAP, this approach did not show any benefit in terms of tumour response, median survival and pain reduction, compared with less invasive treatment options [171]. It is possible that this approach may have a more favourable outcome if a more active bioreductive drug, such as TPZ or AQ4N, was used. In pre-clinical studies, the MMC analogue, porfiromycin, showed an improved bioreductive profile and less systemic toxicity than MMC. This led to a prospective randomised trial comparing the effect of porfiromycin or MMC in combination with standard radiotherapy (64 Gy over 47 days) in 121 patients with squamous cell carcinoma of the head and neck. Unfortunately, the outcome showed porfiromycin to provide no advantage as compared with MMC and this has led to reduced interest in this compound [172]. Initial phase I studies of the synthetic MMC analogue EO9 showed no bone marrow toxicity and rapid clearance of EO9; the most important toxicity was proteinuria [173,174]. Phase II studies confirmed the toxicity findings, but the drug did not have any activity as a single agent in a range of tumour types [175,176]. This underlines the need to carry out combination studies when trialling a bioreductive drug. However, another problem, which may have had more influence on the results, was the short plasma half-life and poor diffusion properties of EO9. This may have resulted in poor drug delivery to the tumour and, hence, reduced the likelihood of efficacy [177]. More recently, EO9 has been used in superficial bladder cancer as an intravesical therapy where the drug is delivered directly to the tumour, reducing delivery problems. In two ‘marker-tumour’ studies, it was shown that EO9 had the ability to eradicate the markertumour as effectively as MMC and with less toxicity than bacillus Calmette-Guerin [178,179]. This shows that when tumour cells come into direct contact with EO9, effective anti-tumour cytotoxicity can be obtained.

Conclusion In conclusion, after considerable pre-clinical development of bioreductive drugs, some of the compounds have confirmed their effectiveness in human trials. In particular, TPZ has shown the ability to enhance other treatments, although disappointingly not in all combinations. AQ4N, which is significantly less toxic than TPZ, is well tolerated in reported phase I trials and it is hoped that phase II trials may show efficacy in combination with radiation or cisplatin, mirroring the pre-clinical data. Although there are some invasive methods to measure tumour hypoxia, the methods to non-invasively measure tumour hypoxia on a routine basis still await development of the technology. Currently, several imaging techniques

437

are at various stages of development (see recent reviews [180,181]). When these become more refined, stratification of tumours by oxygenation will hopefully become routine. This should allow both more accurate evaluation of the efficacy of bioreductive drugs and the pre-treatment selection of patients for bioreductive therapy. This would provide trials with a higher power and require fewer patients to show efficacy. There is one other major potential benefit of bioreductive drugs that is yet to be confirmed in the clinic. Because the hypoxic cell compartment contains the more malignant, stress-resistant cells, eradicating, rather than re-oxygenating, these cells may also reduce the incidence of metastasis, where this has not already occurred before treatment. Clearly, this field of radiobiology research has made many advances over the past 30 years, but there is still much to do. Acknowledgements. The authors would like to thank the following people who provided information to aid in the compiling of this review. Dr C. R. Dunk, KuDOS Pharmaceuticals, Cambridge, UK; Dr M. V. Papadopoulou, Evanston Northwestern Healthcare, IL, USA; Professor L. J. Peters, Peter MacCallum Cancer Centre, Melbourne, Australia; Professor W. R. Wilson, The University of Auckland, New Zealand; Dr A. Wong, Novacea Inc., San Francisco, CA, USA. Author for correspondence: S. McKeown, Institute of Biomedical Sciences, University of Ulster, Coleraine, Northern Ireland BT52 1SA, UK. E-mail: [email protected] Received 6 February 2007; received in revised form 20 February 2007; accepted 9 March 2007

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15 16

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27

28

29

30

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