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 radia- tion, 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 angiogene- sis (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 advan- tage: 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 inva- sive 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 radi- osensitizers (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) Table 5.1. Examples of nitroimidazole radiosensitizers and results from the clinical trials 70 Name of Drug evalua- Type of Type of cancer Type of radiotherapy No.