5 Combinations of Hypoxia-Targeting Compounds and Radiation-Activated Prodrugs with Ionizing Radiation

Combinations of Hypoxia-Targeting Compounds and Radiation-Activated Prodrugs with Ionizing Radiation 67 5 Combinations of Hypoxia-Targeting Compounds and Radiation-Activated Prodrugs with Ionizing Radiation

G-One Ahn and J. Martin Brown

CONTENTS 5.1 Targeting Tumor Hypoxia 5.1 Targeting Tumor Hypoxia 67 5.2 Oxygen-Level Enhancers 68 5.3 Hypoxia-Selective Radiosensitizers 69 Tumor hypoxia was first postulated from histo- 5.3.1 Nitroimidazoles 69 logical studies of human lung adenocarcinomas by 5.3.2 Mixed-Function Radiosensitizers 69 Thomlinson and Gray (1955). They reasoned that, 5.3.3 DNA-Affinic Radiosensitizers 71 because of unrestrained growth, tumor cells are 5.3.4 Limitations of HSR 71 forced away from blood vessels beyond the effec- 5.4 Hypoxic Cytotoxins 72 5.4.1 Introduction 72 tive diffusion distance of oxygen (O2) in respiring 5.4.2 Combination of Hypoxic Cytotoxins with Ionizing tissues, hence becoming hypoxic and eventually Radiation 72 necrotic (Fig. 5.1a). Given the typical values for 5.4.3 Nitroimidazoles 73 intracapillary O2 tensions and consumption rates, 5.4.4 Other Nitroaromatics 73 they calculated that O diffusion distances would 5.4.4.1 CB 1954 73 2 be approximately 150 Pm and this was consistent 5.4.4.2 SN 23862 and PR-104 74 Thomlinson 5.4.4.3 RSU 1069 and RB 6145 74 with their histological observations ( 5.4.5 Quinones 75 and Gray 1955). This type of hypoxia has come to be 5.4.5.1 Mitomycin C 75 termed “chronic,” or “diffusion-limited,” hypoxia. 5.4.5.2 Porfiromycin 75 Acute hypoxia also develops in tumors through 5.4.5.3 EO9 75 temporal (reversible) cessation or reduction of 5.4.6 Benzotriene di-N-Oxides 76 5.4.7 Tertiary Amine N-Oxides 78 tumor blood flow resulting from highly disorga- 5.4.7.1 Nitracrine N-Oxides 78 nized tumor vasculature (Fig. 5.1b; Brown 1979). 5.4.7.2 AQ4N 79 Definitive evidence for acute hypoxia and fluctu- 5.5 Combination of Radiation-Activated Prodrugs ating blood flow has been demonstrated in trans- with Ionizing Radiation 79 planted tumors in mice injected at some time apart 5.5.1 Concepts of RAP 79 5.5.2 Nitro(Hetero)Cyclic Methylquarternary with two different diffusion limited fluorescent Ammonium Salts 79 dyes showing mismatch of labeled cells (Chaplin et 5.5.3 5-Fluorouracil (5-FU)-Releasing Prodrugs 81 al. 1986; Trotter et al. 1989); however, acute and 5.5.4 Transition Metal Complexes 81 chronic hypoxia are in fact the two ends of a con- 5.6 Other Hypoxia-Targeting Strategies 82 tinuum with fluctuations in blood flow without total 5.6.1 GDEPT Targeting Tumor Hypoxia 82 5.6.2 Clostridia-Directed Enzyme Prodrug Therapy 82 occlusion, which are common in both experimental 5.6.3 Targeting HIF-1 83 (Kimura et al. 1996) and human tumors (Hill et 5.7 Conclusion 83 al. 1996), producing a dynamic situation with fluc- References 83 tuating oxygen diffusion distances in many parts of tumors. Tumor hypoxia is a major factor contributing to the failure of radiotherapy (Fig. 5.2). This is largely because DNA damage produced by ionizing radiation, which would otherwise become fixed and lethal G. Ahn, PhD to cells by reacting with O under well oxygenated J. M. Brown, P D 2 h conditions, can be restored to its undamaged form Division of Radiation and Cancer Biology, Department of Brown Wilson Radiation Oncology, Stanford School of Medicine, 269 Campus under hypoxic conditions ( and Drive, Center for Clinical Science and Research, Rm 1255, 2004). Clinically hypoxia predicts poor local control Stanford, CA 94305-5152, USA and survival of patients undergoing radiotherapy 68 G. Ahn and J. M. Brown

a b

Fig. 5.1. a A diagram of a tumor capillary and surrounding tumor cells at decreasing oxygen concentrations (in the direction of arrows). Cells become hypoxic (green) and eventually necrotic (blue); chronic hypoxia. Cellular proliferation and chemo- therapeutic drug concentration are also decreasing in the same direction, as a function of distance from the capillary. (From Brown 1999). b A diagram of normal (left) and tumor (right) blood vasculature. The tumor vasculature is highly disorganized resulting in acutely hypoxic regions in the tumor. (From Brown and Giaccia 1998)

teins, such as vascular endothelial growth factor (VEGF), are increased under hypoxic conditions, potentially resulting in increased tumor angiogenesis (Shweiki et al. 1992; Fang et al. 2001). Thus, there is substantial evidence that hypoxia both interferes with the effective therapy of solid tumors and con- tributes to a more malignant phenotype; however, hypoxia may also prove to be a therapeutic advantage: because it is virtually unique to tumor cells, therapies that target hypoxic regions may have the potential to kill malignant cells while leaving non- malignant cells relatively untouched. This chapter discusses some examples of hypoxia targeting com- Fig. 5.2. A Kaplan-Meier plot of overall survival of patients pounds and approaches for combination with ioniz- with head and neck carcinoma undergoing radiotherapy. ing radiation in experimental or clinical settings. Well-oxygenated tumors (pO2 >10 mmHg; dotted line) showed better prognosis than poorly oxygenated (pO2 <10 mmHg; solid line) tumors. (From Brizel et al. 1997) 5.2 Oxygen-Level Enhancers for carcinoma of the head and neck (Nordsmark et al. 1996; Brizel et al. 1997), and cancer of the cervix One of the earliest attempts to overcome the prob- (Hockel et al. 1993; Fyles et al. 1998). lem of the resistance of hypoxic cells in tumors to Hypoxia further complicates cancer manage- radiotherapy was to increase O2 levels in the blood ment by limiting the access of conventional chemo- stream, thereby increasing the diffusion distance rown Wilson therapeutic drugs (Fig. 5.1a; B and of O2. A number of trials were performed with 2004). Hypoxia also increases genomic instability patients breathing 100% O2 at a pressure of 3 atmo- by increasing mutation frequency (Reynolds et al. spheres, but the results were mixed (Watson et al. 1996) or selecting for cells expressing an anti-apop- 1978; Dische et al. 1983; Henk 1986). One potential totic phenotype such as mutated p53 (Graeber et al. reason for such failures is that increasing the diffu- 1996). This leads to a more metastatic phenotype as sion distance of O2 would not be expected to reduce has been observed clinically (reviewed by Rofstad the levels of acute hypoxia. In some systems, the 2000). In addition, expression of proangiogenic pro- use of carbogen (95% O2/5% CO2) appears to have Combinations of Hypoxia-Targeting Compounds and Radiation-Activated Prodrugs with Ionizing Radiation 69

greater effect than 100% O2 in increasing O2 level in 5.3.1 the blood stream (Rockwell 1997) possibly by pre- Nitroimidazoles venting the vasoconstriction caused by high partial pressures of O2. Metromidazole and misonidazole (Fig. 5.3) are the Other potential approaches to overcome hypoxia prototype members of this class. They were admin- include the use of nicotinamide (Horsman et al. istered to patients in the 1970s. Disappointingly, they 1987; Chaplin et al. 1991; Kjellen et al. 1991) in showed very little efficacy sensitizing tumors but a conjunction with carbogen (Corry and Rischin high incidence of peripheral neuropathy (Urtasun 2004), agents to increase tumor blood flow such as et al. 1976; Dische et al. 1977). With the limited flunarizine (Jirtle 1988), artificial blood substi- dose of misonidazole that can be administered to Teicher tutes carrying increased levels of O2 ( and patients, almost all of the clinical trials of radio- Rose 1984; Rockwell et al. 1986), drugs to reduce therapy combined with misonidazole were negative Hirst Wood Dische the affinity of hemoglobin for O2 ( and ( 1985); however, a meta-analysis of 50 ran- 1989), blood transfusion (Bush et al. 1978), and domized trials later showed a small but significant hyperthermia (Song et al. 2001). Recently, RSR13, benefit of misonidazole and other hypoxic radio- a drug reducing hemoglobin O2-binding affinity, sensitizers when added to radiotherapy in head and has been claim to benefit non-small cell lung cancer neck cancers (Overgaard 1994). patients receiving radiotherapy in phase-II trials Attempts to produce superior drugs to misoni- (Choy et al. 2005). dazole resulted in the development of etanidazole (Fig. 5.3; Brown et al. 1981), pimonidazole (Fig. 5.3; Smithen et al. 1980), and nimorazole (Fig. 5.3; Dische 1985); of these, nimorazole showed a signifi- 5.3 cant benefit of loco-regional control when given in Hypoxia-Selective Radiosensitizers conjunction with radiotherapy to patients with invasive carcinoma of larynx and pharynx (Overgaard In the 1960s Adams and Cooke (1969) proposed that et al. 1998; Overgaard et al. 2005) and is now given electron-affinic drugs might act like O2, a potent as part of the standard of care for radiotherapy of head electron-affinic molecule, to sensitize hypoxic and neck cancer patients in Denmark (Table 5.1). tumor cells. These agents (hypoxia-selective radiosensitizers (HSR) mimic O2 by reacting with the short-lived DNA free radicals generated by ionizing 5.3.2 radiation; however, unlike O2, HSR are not rapidly Mixed-Function Radiosensitizers metabolized by the cells through which they pen- etrate and are thus able to reach areas beyond the The neurotoxicity of misonidazole stimulated the O2 diffusion distance. Some examples of HSR are search for drugs not only with less toxicity, but also discussed below. with increased efficiency as radiosensitizers on a

Metronidazole Misonidazole Etanidazole

Fig. 5.3. Examples of nitroimidazole compounds as hypoxia-selective Pimonidazole Nimorazole radiosensitizers (HSR) 70 G. Ahn and J. M. Brown et al. et al. et al. et al. ogaert B et al. (1998) et al. et al. (1989) et al. et al. (1996) et al. et al. (1993) et al. en et al. (2003) et al. et al. (2004) et al. et al. (1995); (1995); et al. Reference Henk Urtasun et al. (1994); (1994); et al. Beard Lawton Lee et al. (1997) et al. Eschwege Dische et al. (1998) et al. Overgaard (1995) et al. et al. Overgaard (1989b) Van D et al. et al. Overgaard Grigsby (1989a); (1999) Chan Simpson t t t t; unusually unusually t; t t t t t cant benefi cant cant benefi cant cant benefi cant cant benefi cant cant benefi cant cant benefi cant cant benefi cant benefi cant cant benefi cant No signifi No previous CHART study CHART previous good response in the control arm in the control good response regional control rate and cancer- rate control regional death) related Outcome 73 signifi No 61 Positive effect compared with 523 signifi No patients No. of No. y 422 effect (better loco- Positive ractionated ractionated accelerated radiation therapy therapy radiation accelerated (CHART) External beam radiation 39 58, signifi No Conventional radiotherapyConventional 374 521, signifi No Conventional radiotherapyConventional 183 signifi No Split-course radiotherapySplit-course 626 fractionation Conventional radiotherapy signifi No Radical radiotherapy Conventional radiotherapyConventional 120 331, signifi No Conventional radiotherapyConventional 239 signifi No sults from the clinical trials from sults Small-cell lung cancerSmall-cell lung radiotherapy Conventional 30 prostate cancer carcinoma uterine cervix Invasive carcinoma of carcinoma Invasive larynx and pharynx head and Advanced neck cancer Carcinoma of the Carcinoma cervix Carcinoma of the Carcinoma uterine cervix small cell lung cancer lung small cell Type of cancerType of radiotherapy Type Phase II and neck Head hyperf Continuous Phase III of the Carcinoma Phase III non- Locally advanced Phase II Locally advanced Phase III and neck Head radiotherap Conventional Phase III and neck Head Type of trial HSR Drug evalua- category tion Pimonidazole HSR Nimorazole HSR Etanidazole HSR Misonidazole HSR Name of Name drug , hypoxia-selective radiosensitizers hypoxia-selective , HSR Examples of radiosensitizers 5.1. Examples and re nitroimidazole Table Combinations of Hypoxia-Targeting Compounds and Radiation-Activated Prodrugs with Ionizing Radiation 71 dosage basis. The latter strategy has been achieved by cellular metabolism occurring at 37qC (Roberts by incorporating other functional moieties in the et al. 1987) and poor extravascular penetration drug molecule. through tumor cell layers (Wilson et al. 1986). CB 1954 (2,4-dinitro-5-aziridinylbenzamide; 2-NLP-3 (5-[3-(2-nitro-1-imidazolyl)-propyl]-phen- Fig. 5.4) is an HSR-bearing DNA alkylating moiety anthridinium bromide; Figure 5.5) and 2-NLP-4 (5-[3-(2-nitro-1-imidazolyl)-butyl]-phenanthri- dinium bromide; Fig. 5.5) are 2-nitroimidazoles attached to the DNA intercalator phenanthridine. They were shown to be 10–100 times more efficient as HSR than misonidazole both in vitro and in vivo (Cowan et al. 1991). The structurally similar compound, NLA-1 (9-[3-(2-nitro-1-imidazolyl) propylamino]acridine hydrochloride; Fig. 5.5) is CB 1954 RSU 1069 a DNA-targeted acridine-linked 2-nitroimidazole ( Papadopoulou et al. 1992). Although NLA-1 is a Fig. 5.4. Examples of mixed-function compounds as HSR potent HSR in vitro, it lacked in vivo activity (Denny et al. 1992). of aziridine, which showed more efficient radiosensitization than misonidazole (Chapman et al. 1979). 5.3.4 Similarly, RSU 1069 (1(2-nitro-1-imidazolyl)3- Limitations of HSR aziridinyl-2-propanol; Fig. 5.4), the second generation of CB 1954, showed in vitro radiosensitization The major drawback of HSR is that the radiosensi- potency tenfold greater than misonidazole at equi- tization efficacy of HSR to tumors decreases at the molar doses (Stratford 1982; Adams et al. 1984); radiation doses used in clinically relevant frac- however, it appears that the more effective sensitiz- tionated irradiation. Experimental results showed ing ability of RSU 1069 is due to a greater degree of that 2–3 Gy fractionated irradiation resulted in hypoxic cell cytotoxicity rather than to enhanced lower enhancement ratios than single large doses radiosensitizing ability (Hill et al. 1986). Both CB for misonidazole (Denekamp and Stewart 1954 and RSU 1069 are discussed further as hypoxic 1978; Hill and Bush 1978; Sheldon and Fowler cytotoxins in section 5.4.4. (1978) or etanidazole (Brown and Yu 1984). One explanation for the lack of radiosensitization at fractionated low doses could be reoxygenation 5.3.3 occurring between each fraction (Brown and Yu DNA-Affinic Radiosensitizers 1984); however, an equally likely reason is that it is not the cells at maximum radiation resis- This class of HSR incorporates a DNA-affinic moiety tance, but those at intermediate oxygenation and thereby attracting the drug molecule closer to DNA intermediate radioresistance that dominate the where they exert their radiosensitizing effect. Nitra- response to fractionated irradiation (Wouters crine, 1-nitroacridine derivative (1-nitro-9-(dime and Brown 1997). Though hypoxic cell radiosen- thylaminopropylamino)acridine; Fig. 5.5) showed sitizers at clinically realistic doses can sensitize 1700 times more efficient in vitro radiosensitization the maximally hypoxic cells to radiation killing, than misonidazole (Roberts et al. 1987); however they have little effect on the radiosensitivity of the development of nitracrines as HSR was limited the cells at intermediate hypoxia and radiosen-

2-NLP-3 (n=3) Nitracrine or 2-NLP-4 (n=4) NLA-1

Fig. 5.5. Examples of DNA afﬁ nic compounds as HSR 72 G. Ahn and J. M. Brown sitivity. This is a consequence of the logarithmic Selectivity for hypoxia is usually a result of a futile nature of the curve of radiosensitization vs dose: redox cycling in which the presence of O2 reoxidizes as cell sensitivity increases it takes geometrically the one-electron reduced intermediate thereby more drug to increase their radiosensitivity still regenerating the parent drug (Fig. 5.6a; Mason and oltzman x- further; thus, a hypoxic cell partially sensitized H 1975). Superoxide anion (O2 ), a major by some oxygen takes much more drug to increase by-product of this reaction, is capable of producing its sensitivity by a given amount compared with a DNA strand breaks and other oxidative damage, but fully hypoxic cell. can be detoxified by superoxide dismutase and cata- It thus seems unlikely that hypoxic cell radio- lase (Biaglow et al. 1982). Hypoxia selective cyto- sensitizers – even newer ones with greater potency toxicity therefore occurs when the reduction of the – have a major influence on the outcome of frac- hypoxic cytotoxin produces a more toxic interme- x- Wardman tionated radiotherapy. They may, however, play diate than the O2 radical ( et al. 1995; a role – or probably should play a role – in those Wardman 2001). situations where large, single doses of radiation are given such as with stereotactic radiotherapy of brain tumors. 5.4.2 Combination of Hypoxic Cytotoxins with Ionizing Radiation

5.4 Hypoxic cytotoxins are the ideal drug to combine Hypoxic Cytotoxins with ionizing radiation because they produce a profile of cytotoxicity as a function of distance from 5.4.1 blood vessels in tumors that are the opposite to that Introduction produced by ionizing radiation (Fig. 5.6b; Brown 1999). In other words, hypoxic cytotoxins kill the An alternative strategy to overcome the problem of tumor cells that are resistant to ionizing radiation. hypoxic tumor cells is to selectively kill them by In addition, hypoxic cytotoxins may release stable using hypoxic cytotoxins. Typically these are pro- and diffusible cytotoxins capable of killing the sur- drugs of very low cytotoxicity that are reduced by rounding tumor cells at relatively higher O2 con- enzyme(s) in hypoxic tumor cells. This results in centrations, producing a so-called bystander effect conversion to potent cytotoxins killing the activat- (Denny and Wilson 1993). Unlike HSR, the efficacy ing cell and, in some cases, the surrounding cells. of hypoxic cytotoxins is independent of the dose of The development of hypoxic cytotoxins was origi- radiation because there is no interaction between the nally stimulated by the findings that nitroimid- two agents; hence, the benefit of adding a hypoxic azoles were more toxic to hypoxic than oxic tumor cytotoxin to fractionated irradiation increases with cells even without irradiation (Sutherland 1974; the number of times the drug is administered and Hall and Roizon-Towle 1975). can produce greater benefit than if all of the tumor

Fig. 5.6. a The concept of hypoxic cytotoxins (indicated as D). Hypoxia selectivity is achieved by the futile cycle by oxygen converting the reduced intermediate (D•-) back to its non- toxic prodrug form (D). b The prediction of tumor cell killing by a hypoxic cytotoxin or radiation and most anticancer drugs as a function of the distance from the capillary. Note the opposite proﬁ le in cytotoxicity towards tumor cells; the combination of the two should yield complementary cell killing and is shown as a dashed line. (From Brown 1999) b Combinations of Hypoxia-Targeting Compounds and Radiation-Activated Prodrugs with Ionizing Radiation 73 were fully oxygenated as shown in the theoretical Moselen et al. 1995), and NLCQ-1 (Papadopoulou study by Brown and Koong (1991). et al. 2000). NLCQ-1 is a nitroimidazole-linked chloroquinoline (4-[3-(2-nitro-1-imidazolyl)-propylamino]-7-chloroquinone hydrochloride; Fig. 5.8), a 5.4.3 member of NLP-1/NLA-1/THNLA-1 series, which is Nitroimidazoles also an efficient HSR (Papadopoulou et al. 1994; Papadopoulou et al. 1996). It shows time-dependent A general scheme (Fig. 5.7) for nitroheterocycle increase in hypoxic cytotoxicity in vitro and is cur- reduction is that the addition of an odd number rently under preclinical evaluation (Papadopoulou of electrons (1, 3, 5) leads to radical intermediates, et al. 2000; Squillace et al. 2000). while an even number of electrons (2, 4, 6) leads to In mammalian cells, aldehyde oxidase, DT- the nitroso (R-NO), hydroxylamine (R-NHOH), and diaphorase, xanthine oxidase, NADPH:cytochrome Mason amine reductants (R-NH2), respectively ( and P450 reductase, cytochrome b5 reductase, NADH- Holtzman 1975; Perez-Reyez et al. 1980). Oxygen dehydrogenase, and succinate dehydrogenase is able to reverse or inhibit reduction at the one-elec- (Heimbrook and Sartorelli 1986; Walton and tron radical anion, although it could in principle act Workman 1987; Hodgkiss 1998) have been reported at various stages (Wardman and Clarke 1976). In as nitroreductases. the absence of O2, further reduction occurs primar- x- ily via disproportionation reactions of R-NO2 , ulti- mately leading to the fragmentation of the imidazole 5.4.4 ring (Varghese et al. 1976; Flockhart et al. 1978). Other Nitroaromatics Two- and four-electron reduced derivatives may have different stabilities and reactivities depending 5.4.4.1 upon the nature of the aromatic ring and its sub- CB 1954 stituents (McClelland et al. 1984). Some examples of this class of hypoxic cytotoxins Though not strictly a hypoxic selective cytotoxin, are metronidazole, misonidazole (Fig. 5.8; Rauth et CB1954 has been an important lead for the develop- al. 1984), bis-nitroimidazole (Fig. 5.8; Hay et al. 1994; ment of such agents. CB 1954 (Fig. 5.9) is a mono-

Fig. 5.7. The reduction of nitroimidazole hypoxic cytotoxins. R generalized nitroimidazole ring

Metronidazole Misonidazole

Fig. 5.8. Examples of nitroimidazole Bis-nitroimidazole NLCQ-1 compounds as hypoxic cytotoxins 74 G. Ahn and J. M. Brown

RSU 1069

CB 1954 SN 23862

RB 6145

Fig. 5.9. Examples of nitroaromatic hypoxic cytotoxins functional alkylating agent that can be enzymatically et al. 2003). Good bystander effect in both in vitro activated to a potent difunctional alkylating agent and in vivo systems together with substrate speci- which crosslinks DNA (Knox et al. 1988a; Knox et ficity to Escherichia coli B nitroreductase warrants al. 1988b; Brown and Wilson 2004). Reduction of development of SN 23862 for GDEPT (Anlezark et CB 1954 produces the potent cytotoxic metabolites, al. 1995; Wilson et al. 2002). the 4-hydroxylamine, and 2-amine (Helsby et al. PR-104 is a phosphate pre-prodrug of the dini- 2004). The activation of CB 1954 also occurs through trobenzamide mustard PR-104A that is activated a second activation step by a non-enzymatic reac- in hypoxia to become a potent bifunctional alkyl- tion with a thioester (such as acetyl CoA) to form ating agent producing DNA interstrand crosslinks the final DNA reactive species, which is presumably (Douglas et al. 2005). In addition, PR-104 has dem- 4-(N-acetoxy)-5-(aziridin-1-yl)-2-nitrobenzamide onstrated substantial bystander effect killing aero- (Knox et al. 1991). The reduction of CB 1954 has bic as well as hypoxic cells in solid tumors (Wilson been shown to proceed at about equal rates under et al. 2005). Because of this ability to kill both aero- aerobic and hypoxic conditions, presumably because bic and hypoxic cells in tumors PR-104 shows sig- the two-electron reduction by DT-diaphorase is not nificant antitumor activity as a single agent alone. inhibited by O2. Currently, there is interest in CB It is also superior to tirapazamine in combination 1954 in gene-directed enzyme-prodrug therapy with fractionated irradiation in preclinical models (GDEPT) using Escherichia coli B. nitroreductase (Dorie et al., unpublished data). PR-104 entered (Bridgewater et al. 1995; Green et al. 1997), and clinical phase-1 trials in December 2005. is in currently phase-I clinical trials (Chung-Faye et al. 2001; Dachs et al. 2005). 5.4.4.3 RSU 1069 and RB 6145 5.4.4.2 SN 23862 and PR-104 RSU 1069 (Fig. 5.9) is bioreductively activated by NADPH-cytochrome P450 reductase forming a Structurally similar to CB 1954, a nitrogen bifunctional crosslinking agent (Stratford et al. mustard compound, SN 23862 (5-[N,N-bis(2- 1986; Whitmore and Gulyas 1986). In air, RSU 1069 chloroethyl)amino]-2,4-dinitrobenzamide; Fig. 5.9) functions as a monofunctional alkylating agent due exploits the nitro-group as an electronic switch in to the presence of the aziridine group ( Stratford et that nitro-to-amine conversion shifts electron den- al. 1986). It shows in vivo antitumor activity against sity modifying the reactivity of a drug molecule KHT sarcoma and RIF-1 tumor when given before (Siim et al. 1997; Helsby et al. 2003). The chemi- or after irradiation (Hill et al. 1986). cal reduction of each of the 2-nitro and the 4-nitro The bromoethylamine derivative RB 6145 group of SN 23862 showed an increase in cytotoxic- (Fig. 5.9) was developed as a prodrug of RSU 1069 ity by 160- and 9-fold in AA8 cell line, and by 4400- because of irreversible gastrotoxicity observed in and 83-fold in UV 4 cell line, respectively, and that early phase-I trials of RSU 1069 (Horwich et al. the reduction of both nitro groups led to further 1986). RB 6145 showed slightly less hypoxia selec- increase in cytotoxicity (Palmer et al. 1995; Helsby tive cytotoxicity than RSU 1069 in vitro (Jenkins Combinations of Hypoxia-Targeting Compounds and Radiation-Activated Prodrugs with Ionizing Radiation 75 et al. 1990) but was active against hypoxic cells in 1986). Mitomycin C can also be reductively activated vivo with lowered toxicity compared with RSU 1069 via two-electron reducing DT-diaphorase generat- Jenkins Cole Iyanagi ( et al. 1990; et al. 1990); however, fur- ing an O2-insensitive hydroquinone ( and ther development of this compound was stopped Yamazaki 1970; Keyes et al. 1984). because preclinical studies showed irreversible Despite variable results in hypoxia selective cyto- cytotoxicity toward retinal cells in mice (Parker et toxicity observed in preclinical studies ( Rockwell al. 1996; Breider et al. 1998). and Kennedy 1979; Rockwell et al. 1982; Keyes et al. 1984), clinical trials have reported that mitomycin C in combination with radiation shows a sig- 5.4.5 nificant benefit in local regional control rates for Quinones patients with head and neck cancer (Weissberg et al. 1989; Haffty et al. 1993; Haffty et al. 1997), 5.4.5.1 squamous cell carcinoma of the cervix (Roberts et Mitomycin C al. 2000), and laryngeal and hypopharyngeal cancer (Saarilahti et al. 2004) (Table 5.2). Mitomycin C (Fig. 5.10), isolated from Streptomyces caespitosus, was introduced into the clinic in 1958, 5.4.5.2 and was subsequently shown to have a moderate in Porfiromycin vitro hypoxia selective cytotoxicity [hypoxic cytotoxicity ratio (concentration of drug in air divided Porfiromycin (Fig. 5.10), a second-generation version by concentration of drug in hypoxia to produce the of mitomycin C, showed superior in vitro hypoxic same level of cell killing; Brown 1993) of about 1–5; selectivity over mitomycin C as a result of lowered Rockwell et al. 1982; Fracasso and Sartorelli aerobic cytotoxicity (Fracasso and Sartorelli 1986]. The cytotoxicity of mitomycin C is associ- 1986; Rockwell et al. 1988) and an improved in ated with formation of monofunctional alkylation vivo therapeutic index as a result of higher LD50 (the and more potently with intra- and inter-strand dosage required to kill 50% of the treated popula- crosslinks of DNA, all of which require bioreduc- tion) in mice (Keyes et al. 1985). Although a phase-I tive activation (Iyer and Szybalski 1963; Tomasz trial showed an acceptable toxicity profile, a recent et al. 1987; Volpato et al. 2005). phase-III randomized trial showed that porfiromy- The one-electron reduction of mitomycin C results cin was inferior to mitomycin C as an adjunct to in a semiquinone, which under hypoxic conditions radiotherapy in squamous cell cancer of the head activates the aziridine ring and results in binding and neck (Haffty et al. 2005). For this and other of the drug to DNA (Pan et al. 1984). Following the reasons (Brown (1999), it therefore seems unlikely initial covalent attachment of mitomycin C to DNA, that the clinical activity of mitomycin C is the result the drug can undergo further reductive activation to of its (modest) selectivity as a hypoxic cytotoxin. form a second alkylating site (Pan et al. 1984). The one-electron reduction pathway can be catalysed 5.4.5.3 by any of several enzymes including NADPH:cyto- EO9 chrome P450 reductase (Pan et al. 1984; Keyes et al. 1984) and xanthine oxidase (Pan et al. 1984), in EO9 ([3-hydroxymethyl-5-aziridinyl-2-methyl-2- Bachur a process that can be reversed by O2 ( et al. (H-indole-4,7-indione)-propenol]; Fig. 5.10) was 1978; Bachur et al. 1979; Pritsos and Sartorelli originally developed as a synthetic analog of mito-

Mitomycin C Porﬁ romycin EO9

Fig. 5.10. Examples of quinones 76 G. Ahn and J. M. Brown mycin C (Oostveen and Speckamp 1987). The triazinyl radical, which can mediate the initial cyto- reductive bioactivation of EO9 occurs in a similar toxic process by abstracting a hydrogen atom from fashion to mitomycin C such that one- and two- the deoxyribose backbone of DNA (Anderson et al. electron reduction processes yield the correspond- 2003). The tirapazamine radical may also lead to the ing semiquinone and hydroquinone, respectively formation of the two-electron reduced product, SR (Maliepaard et al. 1995). The semiquinone is 4317, a major non-toxic metabolite detected in cul- believed to be more cytotoxic than the hydroqui- tured hypoxic cells (Baker et al. 1988; Hicks et al. none based on its ability to produce DNA interstrand 2003), in the mouse (Walton and Workman (1993), crosslinks and strand breaks (Bailey et al. 1993; and in humans (Grahams et al. 1997). Formation of Plumb et al. 1994; Maliepaard et al. 1995). SR 4317 can occur by a number of different routes, Although partial responses were observed including the direct two-electron reduction of tira- in a small number of patients in phase-I trials pazamine by DT-diaphorase (Riley and Workman (Schellens et al. 1994), no apparent antitumor 1992), by radical disproportionation reaction, or by activity by EO9 alone was demonstrated in phase- hydrogen abstraction from macromolecules other II studies in patients with advanced breast, gastric, than DNA (Brown and Wang 1998). Dirix pancreatic, and colorectal carcinoma ( et al. Tirapazamine has a unique O2 concentration 1996); however, some concerns about the design dependency such that its cytotoxicity does not level of the trials were raised in that enzymatic activ- off at high concentrations, but gradually decreases as Koch Hicks ity in patients’ tumors was not measured routinely the O2 concentration increases ( 1993; et (Phillips 1996) and that hypoxic cytotoxins, such al. 2004). Tirapazamine shows hypoxic cytotoxicity as EO9, should be combined with other treatment ratios of up to 200 in murine and 50 in human cell modalities, such as radiotherapy or chemother- lines (Zeman et al. 1986). apy, to demonstrate detectable clinical responses The hypoxic cytotoxicity of tirapazamine is due ( Workman and Stratford 1993). to the formation of DNA strand breaks resulting in chromosome aberrations (Beidermann et al. 1991; Wang et al. 1992; Siim et al. 1996). The chromosome 5.4.6 breaks produced by tirapazamine were shown to be Benzotriene di-N-Oxides less easily repaired than those produced by X-rays, and this has been suggested to be a result of prob- Tirapazamine (SR 4233; Fig. 5.11a) is the prototype able metabolism by reductases located close to DNA of this class of hypoxic cytotoxins. Under hypoxic (Wang et al. 1992). Although a large proportion of conditions, tirapazamine is reduced to an O2-sensi- tirapazamine is metabolized in the cytoplasm by tive tirapazamine radical (Lloyd et al. 1991). The enzymes, such as cytochrome P450 (Walton et al. tirapazamine radical then eliminates a water mol- 1992; Wang et al. 1993; Riley et al. 1993), NADPH ecule to form a nitrogen-centered oxidant, benzo- cytochrome P450 reductase (Cahill and White

Fig. 5.11. a Chemical structure of tirapazamine. b Synergistic activity of tirapazamine in killing SCCVII tumors in vivo when combined with fractionated irradiation (fi lled circles), deter- Relative clonogenic cells/tumor Relative mined by clonogenic cell survival. Tirapazamine alone and radiation alone (2.5 Gy per fraction) are shown as open circles and dots, respectively. From Brown and Lemmon (1990) Number of fractions b Combinations of Hypoxia-Targeting Compounds and Radiation-Activated Prodrugs with Ionizing Radiation 77 et al. (2004) et al. et al. (1989) et al. (2005) et al. (2000) et al. et al. (1991) et al. et al. (2004) et al. et al. (2001) et al. Saarilahti Widder Roberts Weissberg et al. (1993) Haffty et al. (1995) McKeown et al. Teicher (1990) Cole et al. Rischin Rischin et al. (1990) Cole et al. (2005) et al. Papadopoulou Reference t cant benefi cant ve effect ve Additive effect Additive Positive effect Positive effect Positive effect (4-year disease-free effect (4-year Positive survival) disease-free effect (5-year Positive survival free and local recurrence survival) Positive effect loco- 3-year free survival rates; rates) failure-free regional Outcome u3 Gy) Additive effect u3 Gy) Additive effect u1 Gy) No signifi nitive RT nitive failure- effect (3-year Positive 21 patients with accelerated with21 patients accelerated RT hyperfractionated 160 patients with160 patients radical RT with120 patients RT conventional 182 patients with182 patients RT conventional single-dose RT12-Gy effect Additive Fractionated RT (5 RT Fractionated Fractionated RT (5 RT Fractionated 122 patients, defi 122 patients, 5-Gy single-dose RT5-Gy with convention- 16 patients effect Additive RT fractionated ally Fractionated RT (4 RT Fractionated No. of patients and/or type of and/or patients No. of RT brosarcoma Advanced squa- Advanced FSaIIC murine FSaIIC murine KHT sarcoma single-dose RT 10-Gy Advanced laryngeal Advanced T50/80 murine T50/80 murine and hypopharyngeal cancer Advanced head and Advanced neck cancer mous-cell carcinoma carcinoma mous-cell of the cervix cell Squamous of the carcinoma head and neck mammary carcinoma fi KHT sarcoma single-dose RT 10-Gy Additi xenograft head and neck cancer Locally advanced advanced Locally U251 human glioma U251 human Type of cancer/ Type evaluated tumor ecent clinical/preclinical evaluation data evaluation clinical/preclinical ecent Phase III Phase-II trialrandomized evaluation system Clinical Phase II Clinical Phase I Preclinical vivo In Evaluation of trial/ Type BEP category , radiotherapy , AQ4N BEP RSU-1069 and BEP HSR Preclinical vivo In RB-6145 and BEP HSR Preclinical vivo In NLCQ-1 and BEP HSR Preclinical vivo In Tirapaza- mine Mitomycin C Mitomycin BEP RT Name of drugName Drug evaluation Potential candidates of hypoxic cytotoxins and their r of hypoxic candidates 5.2. Potential Table 78 G. Ahn and J. M. Brown

1990; Lloyd et al. 1991; Walton et al. 1992; Wang 5.4.7 et al. 1993; Patterson et al. 1995; Patterson et al. Tertiary Amine N-Oxides 1997), xanthine oxidase (Laderoute et al. 1988), and DT-diaphorase (Riley and Workman 1992; 5.4.7.1 Wang et al. 1993), it is the nuclear metabolism of Nitracrine N-Oxides tirapazamine (ca. 20% of the overall cellular metabolism) that accounts for essentially all of the tira- Nitracrine N-oxide (Fig. 5.12a) was developed as a pazamine-induced DNA damage under hypoxic prodrug of nitracrine, a hypoxia cytotoxin whose conditions (Evans et al. 1998; Delahoussaye et al. cytotoxicity is due to nitroreduction that is inhibited Wilson 2001). Recently, tirapazamine in hypoxic conditions by O2 ( et al. 1986). While nitracrine showed has also shown to markedly inhibit DNA replication the degree of hypoxic selectivity similar to misoni- (Peters et al. 2001) and to poison topoisomerase-II dazole (ca. tenfold) in vitro, its metabolism was too activity (Peters and Brown 2002) in vitro leading rapid to provide selective killing of hypoxic cells in to the suggestion that the DNA double-strand breaks vivo (Wilson et al. 1986). Derivatization with ter- produced by tirapazamine result from its poisoning tiary amine N-oxide lowered DNA binding (15-fold), topoisomerase II (Peters and Brown 2002). cell uptake, and aerobic cytotoxicity compared with Tirapazamine potentiates cell killing with frac- nitracrine, while its hypoxic selectivity was greatly tionated irradiation in mouse tumors (Fig. 5.11b; increased (1000- to 1500-fold; Wilson et al. 1992). Brown and Lemmon 1990), and with cisplatin in The very high hypoxia selectivity was suggested to a highly schedule-dependent manner (Dorie and be due to a requirement for both the nitro and N- Brown 1993). Tirapazamine is currently in phase- oxide moieties for full activation in an O2 inhibitable III evaluation and appears particularly effective in manner (Wilson et al. 1992). Despite some improve- combination with cisplatin (Rodriguez et al. 1996; ment of extravascular diffusion properties observed Miller 1997; Treat 1998; von Powel 2000) or in vitro (Wilson et al. 1992), in vivo activity against as an adjunct to cisplatin/radiotherapy treatment KHT tumors was only observed at doses lethal to the (Rischin et al. 2001; Rischin et al. 2005). host (Lee et al. 1996).

Nitracrine N-oxide AQ4N a

Fig. 5.12. a Examples of tertiary amine N-oxide compounds. b Tumor growth delay of T50/80 in BDF mice treated with AQ4N. Combination with a single dose radiation (ﬁ lled squares) produced a signiﬁ cantly greater antitumor activity compared with b AQ4N alone (open squares). (From McKeown et al. 1996) Combinations of Hypoxia-Targeting Compounds and Radiation-Activated Prodrugs with Ionizing Radiation 79

- 5.4.7.2 prodrugs (RAP) are reduced by eaq (aquated/ AQ4N hydrated electron) generated from the radiolysis of water, offering two distinct mechanisms strongly AQ4N (1,4-bis-{[2-(dimethylamino-N-oxide)ethyl] inhibited by O2. These mechanisms involve either amino}5,8-dihydroxyanthracene-9,10-dione; back-oxidization of the one-electron reduction x- - Fig. 5.12a) is a prodrug designed to be prevented intermediate of RAP (RAP ) or to scavenge eaq at from binding to DNA (Patterson 1993; Smith et diffusion controlled rate (Fig. 5.13; Other primary al. 1997b) until metabolized in hypoxic cells to give radicals formed by radiolysis of water (OHx and Hx) AQ4, a stable, oxygen insensitive metabolite (Smith are expected to be scavenged at very high rates by et al. 1997a). AQ4 is a DNA intercalator and potent other biomolecules. Some of the resulting organic inhibitor of DNA topoisomerase II ( Patterson radicals also have reducing properties so might 1993; Patterson et al. 1994; Smith et al. 1997b). make a further contribution to prodrug reduction. Early development of AQ4N has been confounded The RAP approach offers a number of potential by the lack of hypoxia selective activity in several advantages such as selective activation within the cell lines; however, a large (>100-fold) increase radiation field (tumor bearing volume); exploita- in cytotoxicity of AQ4N was observed when cells tion of necrotic hypoxic regions lacking reductase were incubated under hypoxic conditions with liver activity through release of a stable cytotoxin with microsomes (Patterson 1993). It was later shown a good bystander effect, no requirement for expres- that AQ4N is not activated by NAD(P)H-dependent sion of specific reductase(s); lack of two-electron cytochrome P450 reductase per se (Patterson et activation; and improved extravascular transport al. 1999), but by cytochrome P450 (CYP) isoforms. by allowing design of prodrugs that are not sub- Several studies have shown that the metabolism of strates for metabolism in tumors (Wilson et al. AQ4N correlates with levels of CYP1A1, 2B6, and 1998). Some of the main classes of the compounds 3A in humans (Patterson and McKeown 2000), that have been considered as candidates for RAP are 3A in mice (Patterson et al. 2000), and 2B and 2E discussed below. in rats (Raleigh et al. 1999). Reduction of the two N-oxide functionalities of AQ4N has been suggested to be involved in an oxygen atom transfer from the N-oxide side chains in a process that is O2 sensitive (Patterson and McKeown 2000). Moreover, heme- containing systems other than CYP, such as nitric oxide synthase, have also shown to mediate AQ4N reduction (Raleigh et al. 1998). AQ4N has shown in vivo antitumor activity when combined with radiation (Fig. 5.12b; McKeown et al. 1995), McKeown et Fig. 5.13. The concept of radiation-activated prodrugs (RAP). Hypoxia selectivity occurs by the futile cycling of oxygen al. 1996) or when combined with a range of methods reverting the reduced intermediate of RAP (RAP•-) or scav- inducing additional tumor hypoxia, e.g., hydralazine - enging hydrated electron (eaq ) generated from the radiolysis (Patterson et al. 2000), dimethylxanthenone acetic of water acid (Wilson et al. 1996), or clamping (Patterson et al. 2000). It is currently in phase-I clinical trials. 5.5.2 Nitro(Hetero)Cyclic Methylquarternary Ammonium Salts 5.5 Combination of Radiation-Activated NMQ ammonium salts such as 4-nitroimidazole Prodrugs with Ionizing Radiation (SN 25341; N,N-bis(2-chloroethyl)-N-methyl-N-[(1- methyl-4-nitro-5-imidazolyl)methyl]ammonium 5.5.1 chloride; 4-NIQ-HN2; Fig. 5.14a) and 5-nitropyrrole Concepts of RAP (N,N-bis(2-chloroethyl)-N-methyl-N-[(1-methyl- 5-nitro-1-pyrrolyl)methyl]ammonium chloride; 5- Another way to target hypoxic tumor cells is to use NPQ-HN2; Fig. 5.14a) that were originally developed ionizing radiation, rather than enzymes, to effect as hypoxic cytotoxins (Tercel et al. 2001), have the reduction of the prodrug. Radiation-activated also been reported as RAP that are reduced with 80 G. Ahn and J. M. Brown

SN 25341 5-NPQ-HN2

a 4-NBQ-AMAC 4-NIQ-AMAC 5-NPQ-AMAC

Fig. 5.14. a Examples of NMQ compounds as RAP. b Radiolytic activation of 4-NIQ-HN2 in human plasma determined as apparent IC50 against UV4 cells. Oxic (95% O2 and 5% CO2) and anoxic (95% N2 and 5% CO2) are shown in open and ﬁ lled circles, respectively. The IC50 for b authentic HN2 is also shown (ﬁ lled square) with an arrow

one-electron stoichiometry by pulse and steady- reduction potential of the prodrug (therefore sus- state radiolysis (Anderson et al. 1997; Wilson et ceptible for enzymatic reduction) and only modest al. 1998). Irradiating 4-NIQ-HN2 in anoxic human cytotoxicity of HN2 limited their utility to that of plasma, followed by exposure to UV4 cells showed only model compounds. that IC50 (concentration of a compound giving 50% In order to incorporate a more potent cyto- growth inhibition relative to the control cells) was toxin within the prodrug system, 4-NBQ-AMAC markedly decreased with radiation, approaching a (Fig. 5.14a), 4-NIQ-AMAC (Fig. 5.14a), and 5-NPQ- value equal to that of the cytotoxin, mechloretha- AMAC (Fig. 5.14a) were synthesized. These prodrugs mine (HN2), itself after ca. 20 Gy (Fig. 5.14b; Wilson were two orders of magnitude less cytotoxic than the et al. 1998); however, a relatively high one-electron released cytotoxin (AMAC) against a panel of tumor Combinations of Hypoxia-Targeting Compounds and Radiation-Activated Prodrugs with Ionizing Radiation 81 cell lines (Wilson et al. 1998). Irradiation in anoxic OFU001 (1-(2’-oxopropyl)-5-fluorouracil; Fig. 5.15) culture medium followed by exposure to UV4 cells and OFU101 (Fig. 5.15) have been reported to showed significant activation of 4-NIQ-AMAC and release 5-FU and 5-fluoro-2’deoxyuridine (FdUrd), 5-NPQ-AMAC at 2 Gy, although the yield of AMAC respectively, with higher efficiency (two-electron was lower than that for the HN2 analogs (Wilson stoichiometry) in anoxic buffer upon irradiation et al. 1998). The AMAC prodrugs showed appre- (Shibamoto et al. 2000; Shibamoto et al. 2004). - ciable enzymatic reduction in hypoxic cell cultures, The mechanism proposed is that attachment of eaq , and convulsion in mice by a mechanism unrelated generated by radiolysis of water, weakens the N(1)- to the cytotoxin release (Wilson et al. 1998), sug- C(1’) bond that links 5-FU or FdUrd to the oxoalkyl gesting that they are not likely to be useful as RAP side chain. Although these compounds showed cell prodrugs. killing after hypoxic irradiation, they failed to demonstrate any significant antitumor activity in vivo (Shibamoto et al. 2000, 2001, 2004). 5.5.3 5-Fluorouracil (5-FU)-Releasing Prodrugs 5.5.4 Nishimoto et al. (1992) first reported that the Transition Metal Complexes N(1)-C(5)-linked dimer of 5-FU (1-(5’-fluoro-6’- hydroxy-5’,6’-dihydrouracil-5’-yl)-5-fluorouracil) A number of transition metal complexes, such as releases 5-FU with ca. three-electron stoichiometry those of cobalt (III) (Co(III) (Fig. 5.16; Wilson et by irradiation in anoxic aqueous buffer and some al. 1994; Ahn et al. 2004b) and copper (II) (Cu(II) indication of antitumor effect in combination with (Fig. 5.16; Parker et al. 2004; Torre et al. 2005), radiation in mice bearing SCCVII tumors. Recently, have been reported as hypoxia-selective prodrugs. In particular, Co(III) complexes are kinetically inert when not reduced; however, when reduced radio- - 6 lytically by eaq , the inert d electron spin state of Co(III) becomes labile d7, which dramatically weakens the coordination bond hence releasing the cytotoxin (Fig. 5.17; Ware et al. 1993). A Co(III) complex bearing a very potent DNA minor groove alkylator cyclopropylindoline (Fig. 5.17) has demonstrated a number of attractive features as a RAP such as: a good masking of the cytotoxicity of cyclopropyl- OFU001 OFU001 indoline in the intact Co(III) complex; an efficient release of the cytotoxin with clinically relevant Fig. 5.15. Chemical structures of 5-FU prodrugs of RAP radiation dose of 2 Gy in anoxic human plasma

Co(III) complex Cu(II) complex

Fig. 5.16. Chemical structures of Co(III) and Cu(II) complexes of hypoxic prodrugs 82 G. Ahn and J. M. Brown

Inactive IR

– eaq +

DNA alkylation Fig. 5.17. A Co(III) complex of cyclopropylindoline releasing its cytotoxin upon ionizing radiation (IR)

or buffer; cell exclusion property of the complex ing an HRE sequence into an appropriate expres- enhancing extravascular transport through multi- sion cassette (Dachs et al. 1997). With this system, cellular tumor layers; a HCR of up to 20 in human studies have shown that in transfected tumor cells, colorectal HT29 cells (Ahn et al. 2002; Ahn 2003); NADPH:cytochrome P450 reductase enhances the however, further development of this Co(III) com- antitumor efficacy of RSU 1069 or tirapazamine in plex as a RAP has failed due to the lack of efficacy combination with ionizing radiation (Patterson et in RIF-1 bearing mice when combined with ionizing al. 2002; Cowen et al. 2004). Another approach has radiation (Ahn 2003). shown antitumor activity with HRE driven nitroreductase enhancing the activity of CB1954 (Shibata et al. 2002). Dual responsive promoters have also been devel- 5.6 oped whose expression is driven not only by HRE Other Hypoxia-Targeting Strategies but also by radiation-responsive promoter sequence such as the early growth response-1 (Egr-1) to 5.6.1 enhance the reporter gene expression in response to GDEPT Targeting Tumor Hypoxia hypoxia in conjunction with low dose of radiation (Greco et al. 2002; Chadderton et al. 2005). As discussed in section 5.1, many genes supporting anaerobic metabolism and angiogenesis are upregulated under hypoxic conditions by the transcription 5.6.2 factor, hypoxia-inducible factor-1 (HIF-1). Specifi- Clostridia-Directed Enzyme Prodrug Therapy cally, HIF-1, once it heterodimerizes with D and E subunits, binds to a common hypoxia-responsive The above-mentioned GDEPT systems have the major element (HRE) found in the enhancer region of drawback of utilizing ex vivo manipulated (trans- all HIF-1 responsive genes (Semenza 2001). One fected) tumor cell lines implying that their clini- GDEPT approach targeting tumor hypoxia aims to cal applicability may be difficult. To overcome this express an exogenous therapeutic gene by introduc- limitation Brown and colleagues have exploited the Combinations of Hypoxia-Targeting Compounds and Radiation-Activated Prodrugs with Ionizing Radiation 83 necrotic regions as a target for cancer therapy using Interestingly, ionizing radiation has been recently a non-pathogenic strain of the bacterial genus Clos- shown to upregulate HIF-1 activity in tumors tridium, an obligate anaerobe (Lemmon et al. 1994; ( Moeller et al. 2004); however, the effect of HIF-1 Fox et al. 1996). This Gram-positive, spore-forming on the radiosensitivity of the tumor is controversial. bacteria becomes vegetative and grows only in the Moeller et al. (2005) showed that functional HIF-1 absence or at very low levels of oxygen (Brown and enhances tumor radiosensitivity by promoting ATP Wilson 2004). Clinical studies in 1970s already have metabolism, cell proliferation, and p53 activation, proven the safety of Clostridium and its tumor-spe- whereas others have reported that it is the defi- cific germination (Carey et al. 1967; Heppner and ciency in HIF-1 that increases tumor radiosensitiv- Mose 1978; Heppner et al. 1983). Recently, geneti- ity, independent of p53 function (Unruh et al. 2003; cally engineered Clostridium expressing E. coli Williams et al. 2005). The reason for this discrep- enzyme cytosine deaminase showed tumor-specific ancy is yet to be determined, but the difference in delivery of the enzyme and the consequent antitu- experiment systems (RNA interference vs cell lines mor activity upon 5-fluorocytosine (5-FC) prodrug deficient for either HIF-1D or -E) could have contrib- administration (Liu et al. 2002; Ahn (2004a). The uted. combination of CDEPT with ionizing radiation is expected to result in enhanced antitumor activity because of ionizing radiation killing well-oxygenated tumor cells and/or the radiosensitizing abilities 5.7 of 5-FC and 5-FU. Conclusion

The potential clinical benefit of exploiting tumor 5.6.3 hypoxia by combining a hypoxia activated drug with Targeting HIF-1 conventional cancer therapy has yet to be realized in routine clinical practice. Despite this, the positive In addition to its role in hypoxic conditions, HIF-1 clinical results with the combination of the hypoxic can be upregulated in the tumor in an O2-inde- cytotoxin tirapazamine with cisplatin in advanced pendent manner. Oncogenic mutations and loss- non-small cell lung cancer and with chemoradio- of-function mutations in tumor-suppressor genes therapy with advanced head and neck cancer demon- have been shown to be associated with the increased strate the potential of this approach. There is a good activity of HIF-1 (Semenza 2003; Li et al. 2005). reason to expect that future drugs or strategies will Evidence for HIF-1 serving as a potential target do better: indeed advances made in experimental for cancer therapy is substantial and extensively models identifying the determinants of the efficacy reviewed elsewhere (Semenza 2003; Giaccia et al. of these hypoxia-targeting compounds, together 2003). For example, HIF-1D (the O2 sensitive HIF-1 with other strategies to exploit tumor hypoxia, auger subunit) expression has been shown to be a poor well for the future of this field. prognostic factor in a number of cancers including bladder cancer (Nakanishi et al. 2005; Theodo- ropoulos et al. 2004), pancreatic cancer (Shibaji et al. 2003), and breast carcinoma (Bos et al. 2003). References HIF-1 is also reported to promote tumor growth in many studies (Ryan et al. 1998; Carmeliet et al. Adams GE, Cooke MS (1969) Electron-affinic sensitization. I. 1998; Chen et al. 2003; Stoeltzing et al. 2004). A structural basis for chemical radiosensitizers in bacteria. Int J Radiat Biol Relat Stud Phys Chem Med 15:457–471 Some recent approaches targeting HIF-1 include Adams GE, Ahmed I, Sheldon PW, Stratford IJ (1984) Radia- using small molecule inhibitors blocking transcription sensitization and chemopotentiation: RSU 1069, a tional activity of HIF-1 (Kung 2004; Kong et al. compound more efficient than misonidazole in vitro and 2005) or those blocking HIF-1D protein synthesis in vivo. Br J Cancer 49:571–577 (Tan et al. 2005), inhibitors of HIF-1 downstream Ahn G (2003) Investigation of an aza-chloromethylbenzindo- Majumder Zhong line cobalt(III) complex as a hypoxia-activated prodrug for target ( et al. 2004; et al. 2004; cancer therapy. Pathology Auckland, University of Auck- Fang et al. 2005), enhancement of HIF-1D protein land, New Zealand degradation (Li et al. 2004), and antisense (Sun et Ahn G, Ware DC, Botting KJ, Kriste AG, Denny WA, Wilson WR al. 2005) or RNA interference against HIF-1D (Li (2002) A novel Co(III) complex of the DNA minor-groove et al. 2005). binder aza-seco-CBI-TMI as a hypoxia-selective prodrug 84 G. Ahn and J. M. Brown

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