The role of the oxygen effect in promoting tumor cell inactivation by ionizing radiation has been well demonstrated in vitro and in animal tumor models for several decades. Mounting evidence of this influence in tumor control and patient survival has become available.²^–⁷ Clearly, the microenvironment, the nutritional state of the tumor, and the presence of hypoxia are only some of the many factors that contribute to tumor radioresistance, and it is likely that this resistance is multifactorial, that is, caused by tumor hypoxia, proliferation rates, and inherent cell radioresistance, as well as other biologic microenvironmental factors such as the presence of cytokines. Nonetheless, the clinical data that hypoxia, by conferring radiation resistance, is a prognostic indicator of poor response to standard radiotherapy, at least for some tumors, is reasonably clear. Hypoxia, however, has other consequences beyond conferring radiation resistance. First, hypoxia causes cells to slow their rate of proliferation and to come out of cycle. Because most anticancer drugs are more effective against rapidly proliferating than slowly or nonproliferating cells, this slowing of cell proliferation leads to decreased cell killing in the hypoxic cells. In addition, because the concentration of anticancer drugs is higher closer to blood vessels than further away, both as a consequence of geometry and the reactivity of the drugs, there is less killing of the hypoxic cells, which are invariably the farthest from the blood vessels.

In summary, there are a number of well-established phenomena that cause a gradient of reduced cell killing by most anticancer agents as a function of distance from the vasculature (Fig. 4-1). Such a gradient has been shown in experimental tumors and in spheroid systems.⁸^,⁹

FIGURE 4-1 • Gradient of reduced cell killing as a function of distance from the vasculature as seen in experimental tumors and spheroids.

Recent studies have also shown that hypoxia in solid tumors has an important consequence in addition to conferring a direct resistance to radiation and chemotherapy. Graeber and colleagues ¹⁰ have demonstrated that low oxygen levels cause apoptosis in minimally transformed mouse embryo fibroblasts, and by selecting for mutant p53, might predispose tumors to a more malignant phenotype. Clinical data support this conclusion: Studies both with soft tissue sarcomas⁵ and with carcinoma of the cervix¹¹^,¹² have shown that hypoxia is an independent and highly significant prognostic factor predisposing tumors to metastatic spread. Thus a more hypoxic tumor, in addition to being more difficult to control locally, is also more likely to have spread to distant sites and hence be more difficult to cure. In addition, hypoxia stabilizes the transcription factor HIF-1α, which increases levels of various survival factors such as vascular endothelial growth factor, which can protect against radiation damage to the tumor vasculature and hence, at least theoretically, protect the tumor from radiation damage.¹³^,¹⁴

The rationale for developing hypoxic cell sensitizers has been based on the assumption that sensitizing hypoxic cells to radiation killing would improve the outcome of radiotherapy. The possibility of doing this was based on pioneering studies by Adams and Chapman and colleagues on the use of electron-affinic drugs to sensitize hypoxic bacteria and mammalian cells in vitro.¹⁵^–¹⁷ The first drug of this class to show significant activity in sensitizing mouse tumors was the 5-nitroimidazole metronidazole, a drug that was already in clinical use.¹⁸^,¹⁹ Data on this and other such compounds are discussed in the following text.

Nitroimidazole Compounds

Under hypoxic tissue conditions, electron-affinic nitroimidazoles oxidize the radiation-induced free radicals on deoxyribonucleic acid (DNA), thereby mimicking oxygen for the fixation of DNA damage. However, unlike oxygen, nitroimidazoles are not rapidly metabolized by the cells through which they penetrate and are thus able to reach areas beyond the oxygen-diffusion distance. They were shown by various groups to be effective in preclinical studies with transplanted mouse tumors in radiosensitizing the tumors to large single radiation doses without sensitizing normal tissues.²⁰^,²¹

The first compound to be investigated clinically in terms of oral and intravenous pharmacokinetics, toxicity, and efficacy in patients with solid tumors was metronidazole (a 5-nitroimidazole). The plasma β-half-life of the drug was 9.8 hours and the absolute oral availability was estimated to approximate 100%.¹⁹ The dose-limiting toxicity was manifested in gastrointestinal and peripheral neuropathic effects; therefore, this compound reached an estimated SER of only 1.2. Given this limitation, the first randomized control clinical trial of a hypoxic radiosensitizer showing efficacy was performed in patients with glioblastoma multiforme. This study demonstrated the relevance of tumor hypoxia in terms of patient survival, showing that the results with a less-than-optimal radiation fractionation regimen approached the level of the results obtained with conventional fractionation.²²

Of interest, a major spin-off of these early investigations with the pharmacokinetics of metronidazole, particularly the use of a high-dose intravenous route, was not in the field of oncology but in the practice of abdominal surgery and infectious diseases, in which the compound was used as a parenteral agent for anaerobic bacteria. These initial investigations prompted a level of high activity in the investigation of nitroimidazole compounds in human solid tumors and the search for new and better nitroimidazole compounds with less toxicity and higher SER. The first clinical studies on the second generation of these drugs, misonidazole, a 2-nitroimidazole, were initiated in both Europe and North America during the early 1980s (Table 4-1). The structures of the hypoxic sensitizers discussed are shown in Fig. 4-2.

Table 4-1 The Evaluation from First- to Third-Generation Nitroimidazole Compounds During the Past 25 Years

FIGURE 4-2 • The structures of the hypoxic sensitizers tested in clinical studies.

Misonidazole and Nimorazole

Misonidazole, a 2-nitroimidazole, was developed as a more efficient radiosensitizer because of its known increased electron affinity. In the oral form, the dose-limiting toxicity was again manifested in gastrointestinal effects (nausea, vomiting) and peripheral neuropathy, which limits the effective SER.²³^,²⁴ Not surprisingly, almost all of the clinical trials of radiotherapy combined with misonidazole turned out to be negative,²⁵ an outcome consistent with the small degree of radiosensitization expected with the clinically used low doses.²⁶ Once more the inability to deliver a sufficient dose may have been one of the reasons that a significant proportion of large international clinical trials showed no benefit to misonidazole (Table 4-2). However, it has been shown that selected populations of patients with specific tumors did benefit by using this compound.²⁷ This observation was made in patients with head and neck cancers. Overgaard and colleagues²⁸ reported that not only with misonidazole but also with nimorazole (a 5-nitroimidazole), there was a significant benefit in a cohort of patients with stage T₁ and T₄ pharynx carcinoma in terms of local regional control (Fig. 4-3). Patients with hemoglobin levels lower than 9 mmol/L showed particular improvement. These two studies also showed that the compounds had a similar benefit, despite the fact that fewer fractions of radiation were “sensitized” in the misonidazole groups than in the nimorazole groups, in which the drug was administrated with every fraction of radiation (first 30 fractions). The significant improvement of the effect of the radiotherapeutic management of supraglottic and pharynx tumors was again demonstrated in a randomized double-blind phase III study of 414 patients receiving nimorazole or placebo in association with conventional primary radiotherapy (62-68 Gy, 2 Gy per fraction, 5 fractions per week).²⁸ Of interest is that in this study the nimorazole could be given without major side effects. Nimorazole is currently standard treatment for patients in Denmark receiving radiation therapy for head and neck cancer.²⁹ The benefit of misonidazole was not observed in another large clinical trial conducted in North America under the Radiation Therapy Oncology Group (RTOG) in patients with stage III and IV head and neck tumors, in which misonidazole was used with two of the five radiation fractions per week. In this study, the administration of the radiosensitizer was limited by neurologic complications, and the use of efficient doses was prevented.³⁰ A prospective randomized trial was initiated afterward in the same cooperative group with a newer third-generation compound, etanidazole. A significantly greater dose of etanidazole than misonidazole could be administered for a sensitizer effect with acceptable gastrointestinal and neurologic toxicity.³¹

Table 4-2 Summary of Efficacy of Clinical Trials With Nitroimidazoles 1974-2008

FIGURE 4-3 • Results from the Danish Head and Neck Cancer Study Group 5 study. Patients with carcinoma of the pharynx and supraglottic larynx were randomly assigned to receive nimorazole or placebo in conjunction with radiotherapy.

(From Overgaard J, Hansen SH, Overgaard M, et al: The Danish Head and Neck Cancer Study Group [DAHANCA] randomized trials with hypoxic radiosensitizers in carcinoma of the larynx and pharynx. In Dewey WC, Edington M, Fry MRG, et al [eds]: Radiation Research: A 20th century perspective, vol 2, Toronto, 1992, Academic Press, p 576.)

Etanidazole

Etanidazole (originally known as SR2508) was developed by a team led by Brown and Lee with the aim of reducing the neurotoxicity seen with misonidazole.³¹ It was postulated that because etanidazole is less lipophilic, it would be associated with a lower incidence of neuropathies. This was confirmed in a phase I pharmacokinetic and toxicity study in which doses three times higher than with misonidazole were delivered and fewer peripheral neuropathies were observed.³¹

Based on the encouraging results of the Danish Group in pharynx carcinoma with misonidazole and nimorazole, the RTOG initiated a two-part study in advanced head and neck cancer. The first part, a toxicity and logistic study in which etanidazole was used three times a week for 17 doses in combination with standard radiation, was completed with acceptable toxicity (although the 22% incidence of peripheral neuropathies seen in that study still presented a problem). Subsequently, a phase III study in a similar patient population and with the same frequency of sensitizer administration was completed in 1992. The results showed that adding etanidazole to conventional radiotherapy produced no benefit for patients with advanced head and neck carcinomas, except for a suggestion of benefit in a subset of patients with early stages (N₀-N₁ disease).³² A randomized study of 374 patients from 27 European centers, conducted between 1987 and 1990, adding etanidazole to conventional radiotherapy did not afford any benefit for patients with head and neck carcinoma. Furthermore the study failed to confirm the hypothesis of benefit for patients with early disease.³³

A single large dose of etanidazole (12 g/m²) administered with intraoperative radiation was considered an ideal setting to assess hypoxic cell sensitizers in a phase I study, which was also completed under the RTOG. The serum and tissue concentrations of etanidazole observed in the trial were 5 to 10 times higher than the levels seen when this compound was given with fractionated radiation. The estimated SER was 2.5 to 3. In 1993, a phase III trial involving intraoperative radiation and single-dose etanidazole was initiated in patients with locally recurrent rectosigmoid carcinoma. In addition, an RTOG study was initiated to test the use of etanidazole in combination with stereostatic radiosurgery in recurrent malignant gliomas or central nervous system metastases. None of these studies demonstrated an improvement in patient outcome, but were limited in power by small numbers of patients.

Evidence exists that prolonged exposure to severe hypoxia can lead to increased sensitization beyond the oxygen effect, owing to the formation of reactive reduced metabolic species. Based on this evidence, an evaluation was made of etanidazole administered in 48-hour and 96-hour continuous intravenous infusions to patients undergoing brachytherapy.³⁵ The use of etanidazole under these particular conditions is considered worth pursing.

Hypoxia marker studies (see the following text) that used ³H-misonidazole or ¹²³I-iodoazomycin arabinoside showed evidence of tumor hypoxia in more than 50% of patients with small cell carcinoma (SCC) of the lung and indicated that tumor hypoxia could be one of the causes of chemoresistance and radioresistance in these patients. Therefore, a phase I and II clinical prospective study was initiated in patients with limited stage of small cell lung cancer, in which etanidazole was given in doses of 1.7 g/m² three times a week with concomitant chemotherapy and thoracic irradiation. In patients with limited-stage disease, the median and the crude rates of survival at 5 years with no evidence of disease were superior to the best results reported in the literature from similar radiotherapy and chemotherapy regimens in which etanidazole was not used.³⁶

Pimonidazole (a 2-nitroimidazole) was developed in Europe at the same time that etanidazole was developed as a third-generation sensitizer, and was considered to be more potent than misonidazole because of its potential to be concentrated in tumors as a result of its pH-dependent sidechain charge. The maximum tolerated dose, when administered with a daily 20-fraction course of radiotherapy, was established at 750 mg/m². The dose-limiting toxicity has been in the central nervous system, manifesting as disorientation and malaise. A randomized clinical trial in advanced carcinoma of the cervix was conducted by 16 centers in Western Europe under the guidance of the Medical Research Council of the UK. Patient accrual was completed in May 1989. Overall and disease-free survival rates were found to be poor among the patients who received pimonidazole in combination with external radiation.³⁷

Conclusions

The current status after decades of clinical investigations with hypoxic cell sensitizers can be summarized by stating that these compounds have not become part of the standard practice of radiotherapy, at least in North America. In Denmark, however all head and neck cancer patients routinely are given nimorazole based on the positive phase III randomized trial of this drug with conventional radiotherapy.²⁸ The only patients who appear to have benefited significantly from this approach are those with advanced pharyngeal and supraglottic carcinomas treated with radiation and either misonidazole or nimorazole. In the immediate future and with the advent of noninvasive markers of tumor hypoxia, it should be possible to select patients for the use of these agents. In addition, the increased use of stereotactic body radiotherapy is likely to make these compounds more attractive in the future because hypoxia is a bigger problem for large single or a limited number of large radiation doses and the individual doses of the particular sensitizers that can be delivered will be higher.

Possible Explanation for the Inconclusive Therapeutic Benefits Seen With Nitroimidazoles in Most Solid Tumors

The failure to demonstrate a major improvement of the therapeutic ratio with nitroimidazoles could be related to the following:

1 It has been postulated that the response of tumors to multiple doses of radiation similar to those used in therapy is governed not by the most hypoxic cells in the tumor, but by the more abundant cells at intermediate oxygenation.³⁸ Such cells require much higher concentrations of nitroimidazoles to give adequate radiosensitization, levels that are higher than clinically tolerated doses.³⁹

2 Some of the tumor sites chosen did not have a steep radiation-dose response when a 10% dose increment could have made a difference in the results.

3 Patients with proven tumor hypoxic fractions were not preselected with hypoxia markers, and therefore most trials were not large enough to detect significant statistical difference at the 10% level.⁴⁰

4 Resistance contributed by high tumor thiol concentrations was not accounted for.

Of interest is an updated review of all randomized clinical trials on approximately 10,108 patients combining tumor hypoxia modifiers (hyperbaric and normobaric oxygen plus carbogen and hypoxic radiosensitizers) with curative radiation, showing an improved effect of radiotherapy on local-regional control, without evidence of increase of radiation side effects in normal tissues.⁴⁰

What Has Been Learned?

It has been learned to predict (1) which patients could be at risk for drug-related neurotoxicity according to the initial pharmacokinetics of the drug in the individual patient, (2) that intravenous administration is the preferable route, and (3) that it is necessary to assess the characteristics of each tumor prior to the course of radiation by developing practical techniques to measure tumor hypoxia. It has also been recognized that sensitizers might be best tested in those tumors in which the dose response is sharp. Thus, to see a 20% to 30% improvement in local tumor control, there is only a need to increase the dose by 5%.

The resistance of tumor hypoxic cells to conventional fractionated radiation in humans is a more complex problem than it appeared to be 25 years ago, when the first clinical investigations were initiated with these agents. We now know that in addition to the classically described state of chronic tumor hypoxia, there is intermediate acute hypoxia, and that both states could be influenced by manipulation of the tumor microenvironment. We also know that drugs that kill, rather than sensitize, hypoxic cells, might be a preferable way of dealing with, and indeed of exploiting tumor hypoxia, than the classic nitroimidazole radiosensitizers.

Current Status and Future Directions in Tumor Hypoxia

Microinvasive Methods of Measuring Tumor Hypoxia

One possible interpretation of the clinical sensitizer trials is that hypoxia is less important for the therapy of human tumors than for murine tumors and other model systems. Arguing against this is a series of studies using a newly developed semiautomatic needle electrode (Eppendorf Histograph).⁴¹^–⁴⁴ These studies not only confirm the radiation resistance of hypoxic human tumors, but demonstrate their more “aggressive” phenotype for other types of treatment failure (chemotherapy–resistance, metastasis, surgical). The overall “aggressiveness” of hypoxic tumors determined by this pioneering work is now much better understood since the discovery and subsequent elucidation of the central role played by hypoxia-inducible factor-1 (HIF-1) in many aspects of tumor behavior.⁴⁵ The resolution of the Eppendorf system is roughly 0.7 mm and several assays capable of much finer resolution have been developed for immunohistochemical analysis.

Some of these microassays involve endogenous molecular changes (often HIF-mediated ⁴⁶) or inherent radiation resistance.⁴⁷ The most extensively developed microscopic assay of hypoxia, also central to the development of noninvasive assays (see the following text), was based on the finding that 2-nitroimidazole metabolism led to the hypoxia-dependent formation of adducts between the metabolized drug (activated by nitro-reduction) and cellular macromolecules, later shown to be primarily thiol-containing proteins.⁴⁸^,⁴⁹ Chapman first proposed that this metabolism could be used for the practical measurement of tissue hypoxia, and one resulting technique (autoradiography after excision of the labeled tumor) progressed to a clinical trial.⁵⁰ The measurement of tritiated misonidazole metabolism by autoradiography was problematic for a general-use assay, but this problem was solved through the development of antibody-based assays for the detection of 2-nitroimidazole adducts, particularly using pimonidazole or EF5 as the hypoxia-detecting agent.⁵¹^,⁵²

As described previously for the sensitizer trials, most former drug development has emphasized the use of polar drugs to reduce access to the central nervous system (thus avoiding neurotoxicity) while promoting rapid excretion. In contrast, EF5 is highly lipophilic, with the recognition that neurotoxicity is unlikely at the low drug concentrations used for hypoxia detection.

One endogenous hypoxia marker currently capable of predicting outcome is carbonic anhydrase IX (CA-IX). CA-IX is one of several carbonic anhydrase enzymes, and is strongly upregulated under hypoxic conditions through the HIF-1α mechanism. CA-IX is expressed on the surface of cells and therefore can be detected by antibodies. However, its correlation with gold-standard measures of hypoxia is suboptimal.⁵³

Noninvasive Methods of Measuring Tumor Hypoxia

Determination of therapy-relevant tumor hypoxia using nuclear medicine techniques holds many potential benefits, including timely stratification of treatment, prediction of outcome, and even the spatial optimization of radiation therapies using image-guided radiation therapy (IGRT), intensity-modulated radiation therapy (IMRT), and protons.⁵⁴ It has become clear that the first of these is much more important than previously considered—that is, heterogeneity of the extent and degree of hypoxia between tumors or patients with otherwise similar characteristics can ultimately stifle the development and interpretation of hypoxia-specific therapies, resulting in the waste of clinical trial resources on patient cohorts for which the hypoxia-specific therapy is inappropriate. With the exception of Copper(II)-diacetyl-bis(N⁴-methylthiosemicarbazone) (Cu-ATSM) and the antibody developed against CA-IX, most agents being considered for the noninvasive detection of hypoxia are also 2-nitroimidazoles. Thus, it is fitting that the first agent developed for noninvasive imaging of hypoxia [¹⁸F]fluoromisonidazole (¹⁸F-FMISO) was first used to demonstrate the predictive value of the first clinically developed hypoxic cell cytotoxin (tirapazamine [TPZ]).⁵⁵^,⁵⁶ Nevertheless, this delay in the successful application of ¹⁸F-FMISO imaging has resulted in the extensive development of other possible noninvasive hypoxia markers.

Several different principles have formed the basis for potential improvements (compared with FMISO) in marker properties.

Half-Life of Isotope versus Drug

Chapman and colleagues¹⁷ suggested that the relatively short isotope half-life of ¹⁸F (≈110 minutes) was suboptimal in detecting tumor hypoxia because, first, this does not allow adequate clearance (pharmacologic half-life of drug) of nonmetabolized drug (which forms the image background), and second, binding to hypoxic cells should increase with time, so that inherent limitations exist in generating optimal tumor versus normal tissue contrast during the relatively short imaging times suitable for ¹⁸F.⁵⁷ Use of iodine as the detecting isotope was considered superior because of its variety of half-lives and decay types. However, directly iodinating the imidazole ring of misonidazole did not lead to suitably stable compounds, so a variety of nucleoside derivatives were devised, with the iodine conjugated to the sugar moiety rather than the 2-nitroimidazole. These compounds (exemplified by iodoazomycin arabinoside [IAZA] and iodoazomycin galactopyranose [IAZGP]) were also more polar than FMISO (see next paragraph). Both single photon emission computed tomography (SPECT) (¹³⁸I) and positron emission tomography (PET) (¹³⁹I) isotopes have been used to evaluate the utility of these drugs. Clinical trials of IAZA successfully imaged hypoxia but because of deiodination this agent was not considered optimal.⁵⁷ IAZGP is under current investigation clinically at the Memorial Sloan Kettering Institute. Although the positive aspects of longer-lived isotopes seem clear, there are also negative aspects that must be mentioned. First, to provide a suitable image quality, the number of counts and imaging time must be specified. For these to be equivalent for long- versus short-lived isotopes, the former must necessarily cause a substantially higher total radiation dose burden to the patient. This is exaggerated for many PET isotopes (e.g., Cu and I), because the fraction of positron emissions per decay can be much less than one. This problem cannot be resolved by use of SPECT isotopes, because, other factors being equal, SPECT imaging has an inherently lower resolution than does PET imaging. Ultimate resolution of PET depends on the energy spectrum of the positron, and ¹⁸F is optimal in this respect.

Drug Polarity (Octanol-to-Water Partition Coefficient)

Because of the potential high resolution and low patient radiation dose from ¹⁸F, several other fluorinated drugs have been developed. Some of these compounds have been significantly more polar than FMISO. The goal of minimizing neurotoxicity seems “off-target” for the use of noninvasive imaging agents, because their concentration is orders of magnitude lower than was used in the sensitizer trials. Additionally, there are data suggesting that highly polar nitroimidazoles do not achieve suitable access to brain and possibly other tissues.⁵⁸ Thus, it is possible that polar imaging agents will have to be evaluated much more thoroughly with respect to their biodistribution and ability to diffuse into actively metabolizing tissue.

Drug Biodistribution and Stability

EF5’s design was based on specific properties of oxygen dependence of bioreduction, drug stability, and biodistribution determined in former pharmacologic and biochemical studies. It was hypothesized that emphasis on the uniform biodistribution allowed by a lipophilic molecule, in effect minimizing excretion, might allow relatively low hypoxia-dependent bioreduction to be detected above the uniform background of the parent drug.⁵⁹ These properties were validated at the relatively high concentration used for immunohistochemistry (≈100 µm—using monoclonal antibodies against EF5 adducts) and appear to be maintained at approximately 1000-fold lower drug concentrations used for PET.⁶⁰ Recently, two other 18F-containing hypoxia markers have been tested in humans: [¹⁸F]fluoroazomycin arabinoside,⁶¹ the fluorinated equivalent of IAZA; and EF3,⁶² a tri-fluoro analog of EF5. These drugs are somewhat more lipophilic than FMISO, with octanol-to-water partition coefficients close to 1.

Several imaging agents have been found to concentrate in tumors, but their precise mechanism binding is not fully understood. The best characterized of these is Cu-ATSM. ATSM is a copper chelator related to the perfusion marker copper pyruvaldehyde bis (N⁴-methylthiosemicarbazone).⁶³ ATSM is very lipophilic but unexpectedly shows rapid biodistribution and binding. Like iodine, copper has a number of radioactive isotopes with selectable properties. Thus this hypoxia marker has many appealing properties.

What Is the “Optimal” Hypoxia Marker?

Because of the large number of methods and compounds under current study, the answer to this difficult question may finally be answered based on current and planned clinical trials. As indicated previously, the question is very complex, and, in fact, the range of oxygen levels important for tumor aggressiveness or therapeutic outcome is only minimally understood. Like many other aspects of tumor “heterogeneity” there may be no single answer. With the current ability to partition radiotherapy to specific volumes of larger tumors or likely metastatic sites (IGRT; protons), it may soon be possible to deliver more radiation dose to the more hypoxic tumor volumes (“dose-painting”).

Manipulation of the Tumor Microenvironment

Increasing the Oxygen-Carrying Capacity of Blood: Hyperbaric Oxygen and Fluosol

Clinical studies have been completed in which hyperbaric oxygen,⁶⁴ carbogen,⁶⁵ packed red cell transfusions,⁶⁶^,⁶⁷ and oxygen-carrier substances such as perfluorocarbons (Fluosol-DA) have being used.⁶⁸^,⁶⁹ Although the use of the hyperbaric oxygen chamber at three atmospheres presented technical difficulties, such as barotrauma and the limitations of its use to only a few high-dose fractions of radiation, 3 out of 10 clinical trials showed significant positive results. The beneficial effect was seen particularly in patients with advanced cancer of the head and neck and of the cervix.⁷⁰

A phase II study of Fluosol-DA and 100% oxygen in combination with radiotherapy in advanced head and neck tumors has shown promising results.⁶⁸^,⁶⁹ Investigators have also been assessing the compound 2-(4-[{3,5-dimethylanilino}carbonyl]methyl]phenoxy)-2-methylproprionic acid derivative, an allosteric hemoglobin modifier, and preliminary reports are encouraging.⁷¹

An alternative approach is to increase the level of hypoxia in tumor cells and to treat them with hypoxic cytotoxic agents. Attempts have been made in the past to reduce tumor perfusion by the use of agents like hydralazine. This has not proved to be of value because of the potential systemic side effects. Another approach to increase the level of tumor hypoxia by modifying the oxyhemoglobin disassociation curve with a specific chemical agent such as 5-(2-formyl-3-hydroxyphenoxy) pentanoic acid, combining this approach with the hypoxic cytotoxic agent, mitomycin C. This approach was investigated in patients with advanced gastrointestinal cancer.⁷²

Increasing Tumor Blood Flow: Nicotinamide and Carbogen/Arcon

Improvement in tumor PO₂ following carbogen breathing (95% O₂, 5% CO₂) has been shown in both animal and human tumors. Tumor tissue oxygenation was measured in humans with the polarographic electrode system (Eppendorf) PO₂ histograph. In one study, in 12 out of 17 patients with solid tumors there was a significant increase in median tumor PO₂ during the first 10 minutes of carbogen breathing. Measurements were taken in accessible superficial tumors; 15 were epithelial tumors (most of them breast and lung carcinomas) and 2 were soft tissue sarcomas.⁷³

It is hypothesized that nicotinamide decreases the presence of acute intermittent hypoxia and that carbogen breathing reoxygenates the chronic hypoxic cells.⁷⁴^,⁷⁵ The benefit of this combination could be further enhanced in tumors with rapidly proliferating stem cells if accelerated radiotherapy schedules are used. This approach, accelerated radiotherapy with carbogen, nicotinamide (ARCON), was initiated originally in a pilot study in the United Kingdom. A multiple-institution ARCON phase I trial was conducted under the European Organization for Research and Treatment of Cancer. In this three-step study, 115 patients with glioblastoma multiforme were registered. The overall survival was not different when compared with results of other series using radiotherapy alone.⁷⁶ Nicotinamide produced gastrointestinal toxicity, necessitating dose reduction. Two other phase I and II clinical trials using ARCON in non-small cell lung cancer (NSCLC)⁷⁷ and in 215 patients with advanced head and neck squamous cell carcinoma⁷⁸^,⁷⁹ were conducted recently with encouraging results in regard to tumor responses, but continued to require a reduction in the dose of nicotinamide because of the incidence of gastrointestinal acute toxicity. The use of ARCON in advanced bladder cancer has been studied to assess its potential gain, with encouraging results and no overt increase in normal tissue radiosensitivity.⁸⁰

The use of carbogen (without nicotinamide) has recently been explored in a randomized study comparing definitive hyperfractionated radiation therapy to the same radiation plus carbogen in T₂-T₄ head and neck tumors. This study did not appear to improve the local tumor control.⁸¹ However, another trial using carbogen breathing combined with radical radiotherapy also in advanced head and neck cancer patients suggested that carbogen breathing may be an alternative form for patients who are unfit to receive concomitant chemotherapy with the radiation treatments.⁸²

Hypoxic Cytotoxins

Tirapazamine

The development of hypoxic radiosensitizers led to that of hypoxic cytotoxins, also known as bioreductive drugs. These are agents that can sensitize a solid tumor to radiotherapy or to chemotherapy by killing, rather than sensitizing, the resistant hypoxic cells. Hypoxic cells in tumors are not only resistant to radiation, they are also resistant to most anticancer drugs. This is because hypoxic cells, by definition, must be those farthest from functioning blood vessels, and also because cells at low oxygen levels divide much less rapidly than when fully oxygenated. These two factors lead to resistance to anticancer drugs, first because the majority of anticancer drugs are only effective against rapidly proliferating cells, and second because drugs have to reach all tumor cells regardless of their distance from blood vessels. Thus hypoxic cytotoxins are fundamentally different from conventional agents in that they target a different subpopulation of cells within the tumor. Typically, hypoxic cytotoxins have maximum cytotoxicity to the cells at maximum distance from tumor blood vessels, thereby complementing the pattern of cytotoxicity for both radiation and anticancer drugs, which is maximum for the cells immediately adjacent to the blood vessels (see Fig. 4-1). Thus these agents have the potential of overcoming a major cause of resistance of solid tumors to conventional therapies, namely that resulting from the inadequate oxygenation and drug delivery to tumor cells distant to blood vessels. The structures of the hypoxic cytotoxins discussed in this Chapter are shown in Fig. 4-4.

FIGURE 4-4 • Structures of the hypoxic cytotoxins that have been tested in the clinic.

Mitomycin C

Mitomycin C, a quinone antibiotic that requires reductive metabolism for activity, is the prototype bioreductive agent. Introduced into clinical use in 1958, mitomycin C has demonstrated activity toward a number of different tumors in combination with other chemotherapeutic drugs and radiation. Sartorelli and colleagues suggested that the lower oxidation reduction (redox) potential of tumor relative to normal tissue might be exploited to obtain greater activation of this compound to its cytotoxic species.⁸³ Although tumor redox potential did not turn out to be key for the activity of mitomycin C, Sartorelli and Rockwell were able to show that this drug preferentially kills hypoxic rather than aerobic cells in vitro.⁸⁴ However, the differential toxicity is modest: The ratio of drug concentrations under aerobic to hypoxic conditions for the same level of cell kill (hypoxic cytotoxicity ratio [HCR]) is in the range of 1 (no differential) to 5.⁸⁵ Nonetheless, this can be sufficient to overcome the resistance of hypoxic cells in animal tumors, and clinical trials have reported higher cure rates for head and neck cancers by adding mitomycin C to radiotherapy compared with radiotherapy alone,⁸⁶ although because mitomycin C is a chemotherapy drug with toxicity toward all cells, it is not clear whether the improved cure rates over radiotherapy alone were the result of selective killing of hypoxic cells.

RB 6145

A second class of hypoxic cytotoxins was developed by Adams and colleagues, who showed that nitroheterocyclic structures containing a bifunctional side chain with alkylating properties were not only more active as radiosensitizers of hypoxic cells, they were also potent and selective killers of hypoxic cells, both in vitro and in vivo. The lead compound of this group, RB 6145, showed considerable activity in vitro and in animal tumors, but proved too toxic to warrant clinical development.

Tirapazamine

A group led by Brown and Lee introduced a third class of bioreductive drugs in 1975. The compound introduced, SR 4233, now known as tirapazamine (TPZ), a benzotriazene di-N-oxide, had an HCR of 50 to 300 for different cell lines ⁸⁷ (Fig. 4-5), and (unlike the classic hypoxic radiosensitizers) is active when combined with fractionated radiation at doses comparable with those used clinically.⁸⁸ The mechanism for the selective toxicity of TPZ (and other members of this class) toward hypoxic cells is that the drug is reduced (an electron is added) by intracellular reductases to form a highly reactive radical that produces both single- and double-strand breaks in DNA that result in cell death, although the exact mechanism of this is quite complex.⁸⁹^,⁹⁰ However, under aerobic conditions, oxygen removes the electron from the TPZ radical, thereby back-oxidizing it to the nontoxic parent with a concomitant production of superoxide radical. Thus the differential hypoxic cytotoxicity results from the fact that the TPZ radical is much more cytotoxic than the superoxide radical. In addition to its toxicity to hypoxic cells, TPZ was shown to be remarkably efficient at enhancing the cytotoxicity of some chemotherapeutic agents, notably cisplatin (CIS), in experimental animal tumors.⁹¹ Following favorable results in phase I and II studies with the combination of CIS and TPZ, a phase III, multicenter, randomized clinical trial with TPZ combined with CIS in patients with advanced NSCLC showed a doubling of the overall response when TPZ was combined with CIS compared with CIS only and a significant increase in the median survival time of the patients.⁹² This increase of antitumor activity occurred without any evidence of increased systemic toxicity of the anticancer drug CIS, as was also seen in experimental animal systems. Promising results of phase I and II trials of TPZ combined with both CIS and fractionated irradiation have been reported for cervix and ovarian cancer⁹³^–⁹⁵ and for head and neck cancer.⁹⁶ Currently, there are phase III trials underway with TPZ combined either with chemotherapy in NSCLC or with radiotherapy and CIS in head and neck cancer, and the results are awaited.⁹⁶ However, the results of the large (861-patient) multicenter phase III trial of SCC were recently reported at the 2008 American Society of Clinical Oncology (ASCO) meeting.⁹⁷ Patients with previously untreated stage III or IV SCC of the oral cavity, oropharynx, hypopharynx, or larynx were randomized to receive definitive radiotherapy (70 Gy in 7 weeks) concurrently with either CIS (100 mg/m²) on day 1 of weeks 1, 4, and 7; or CIS (75 mg/m²) plus TPZ (290 mg/m²/day) on day 1 of weeks 1, 4, and 7, and TPZ alone (160 mg/m²/day) on days 1, 3, and 5 of weeks 2 and 3 (CIS/TPZ). There were no significant differences in failure-free survival or time to locoregional failure (LRF) between the two groups. Interestingly, however, 20% of patients were found to have major deviations in the radiotherapy plan, which was associated with an increased risk of death (hazard ratio [HR] = 1.56; p = <0.0001), and LRF (HR = 1.82; p = 0.0002), and there was a trend of a benefit with the addition of TPZ in patients without major radiotherapy deviations, HR for risk of LRF (CIS/TPZ:CIS) 0.74, 95% CI: 0.53 to 1.04. As well, there is an ongoing phase III study under the Gynecological Oncology Group (GOG) studying the role of TPZ in combination with CIS and radiation in the management of patients with advanced cervical cancer. One of the drawbacks to the use of TPZ is muscle and gastrointestinal toxicities,⁹⁵^,⁹⁶ although with the chemoradiotherapy studies, myelotoxicity is the dose-limiting toxicity. TPZ remains the most widely studied hypoxic cytotoxin at this time. One of the major conclusions from the TPZ trials is the importance of selection of those patients with hypoxic tumors for trials with hypoxic cytotoxins. This was not done and there is evidence that in patients with hypoxic tumors there is a substantial benefit of the addition of the drug.⁹⁸

FIGURE 4-5 • Effect of tirapazamine on tumor cells surviving fraction under air and hypoxic conditions.

AQ4N (Banoxantrone)

The anthraquinone AQ4N was designed specifically as a hypoxia selective cytotoxin. It resembles TPZ in being a di-N-oxide, but has a distinct mechanism of activation and cytotoxicity. AQ4N is a prodrug of a potent DNA intercalator/topoisomerase poison, AQ4, which is formed by reduction of the two tertiary amine N-oxide groups that mask DNA binding in the prodrug form.⁹⁹ AQ4N has substantial activity against hypoxic cells in a variety of transplanted tumors,¹⁰⁰ and has recently completed phase I and II clinical trials with lymphomas and leukemias and a phase I and II trial in combination with radiotherapy and temozolomide, with glioblastoma multiforme in progress.

PR-104

PR-104 is a dinitrobenzamide mustard, developed by Wilson and colleagues at the University of Auckland, with considerable advantages over TPZ or AQ4N.¹⁰¹ The prototype for this class is the prodrug CB 1954, which first came to attention because of its dramatic curative activity against the Walker rat tumor.¹⁰² It was subsequently shown to be a bioreductive prodrug, activated within the tumors by rat DT-diaphorase, which reduces its 4-nitro group to the corresponding hydroxylamine, a potent DNA crosslinking agent.¹⁰³^,¹⁰⁴

PR-104 is a phosphate ester that is in effect a “pre-prodrug”; systemic or tumor phosphatases generate the corresponding alcohol (prodrugs), which is subsequently activated by reduction of one or more of the nitro groups by nitroreductases, including one-electron reductases, to produce hypoxia-selective cytotoxicity by virtue of its ability to form DNA interstrand crosslinks.¹⁰⁵

PR-104 has an interesting advantage over TPZ in that it is highly effective not only in killing the hypoxic cells in the tumor, but also better oxygenated cells, by virtue of the fact that the toxic metabolite formed in hypoxic cells can diffuse to kill nearby aerobic cells. PR-104 is therefore active against tumors alone, not requiring the addition of a second agent (such as radiation) to kill the aerobic cells of the tumor. The drug has completed a multicenter phase I trial and is progressing to phase II trials.

Modifiers of Hemoglobin Levels

Erythropoietin

Erythropoietin is a growth factor that has been synthesized in the laboratory. It has shown efficacy in the treatment of anemia related to systemic chemotherapy,¹⁰⁶ as well as in combined chemoradiation. A significant increase in hemoglobin levels compared with controls has been shown in patients receiving radiotherapy.¹⁰⁷^,¹⁰⁸ These studies did not address the question of whether the increase in hemoglobin levels seen when administering erythropoietin results also in improvement in local tumor control. A randomized phase III trial to assess the effect of erythropoietin on local-regional control in anemic patients treated with radiotherapy for advanced carcinoma of the head and neck was initiated by the RTOG. This study was terminated before completion of patient accrual because of negative results. Further, a review of all phase III studies have shown mixed results, with some studies reporting a decrease in patient survival despite an improvement in hemoglobin levels.¹⁰⁹^–¹¹¹ One phase III study evaluating erythropoietin in cervical cancer closed prematurely because of potential concerns with thromboembolic events with the use of this compound. The tumor recurrence status between treatment regimens were not statistically significant (GOG 191).¹¹² Also, similar results, with no improvement in tumor control, have been reported on head and neck cancer.¹¹³

Glutathione Depletion

Sulfhydryls are scavengers of free radicals, protecting chemical damage induced by either ionizing radiation or alkylating agents. It has been postulated and demonstrated in the laboratory that one approach to increasing the efficacy of the nitroimidazoles as sensitizers is to decrease the levels of the competing endogenous sulfhydryls. Glutathione is one of the major endogenous sulfhydryls. Buthionine sulfoximine was developed as a specific inhibitor of glutathione synthesis. It has been shown to deplete glutathione levels in both in vitro and in vivo systems, therefore making misonidazole a more effective sensitizer. Earlier studies in laboratory experiments showed little or no increase in the toxicity of misonidazole in normal tissue, but showed an increased sensitizing efficacy in the tumor tissue.¹¹⁴

The use of buthionine sulfoximine as a modulator for hypoxic cell sensitizers in combination with radiation and as a modulator of chemotherapeutic drugs such as alkylating agents and bioreductive hypoxic cytotoxics has been of interest in the laboratory.¹¹⁵^–¹¹⁸

An Alternative to Nitroimidazole Hypoxic Cell Radiosensitizers

Nitric Oxide

The hypoxic cell radiosensitization properties and vasodilator effects on tumor vasculature of nitric oxide gas have been described, and there is continued laboratory interest on the possible practical therapeutic use of nitric oxide–releasing compounds (NONOates) under specific physiologic conditions. Studies done in the laboratory under in vitro conditions has shown a marked radiosensitizer effect under hypoxic conditions.¹¹⁹ Further studies are being conducted in in vivo models. For the time being, this approach is still limited to the laboratory level.

Nonhypoxic Cell Sensitizers

The cancer chemotherapy agents hydroxyurea and 5-fluorouracil are not discussed in this chapter. The reader is referred to Chapter 6 for description of these agents as possible nonhypoxia-specific cell radiosensitizers.

Halogenated Pyrimidine Analogues

5-Bromodeoxyuridine and 5-Iodoeoxyuridine

The pyrimidine analogues 5-bromodeoxyuridine (BUDR) and 5-iodoeoxyuridine (IUDR) are considered cell-cycle–specific radiosensitizers and act independently of the oxygen effect. As previously discussed, the radioresistance of human solid tumors could be multifactorial, in which, in addition to the microenvironment (nutrition oxygenation and presence of endogenous sulfhydryl compounds), tumor cell kinetics play an important role. The presence of rapidly proliferating clonogens may substantially influence the control of tumors by irradiation. Because BUDR and IUDR sensitize only rapidly proliferating cells, in such tissues either normal or tumor cells could be effectively sensitized. Rapidly proliferating tumor cells surrounded by slowly proliferating and supporting normal tissue present the ideal scenario for an improved therapeutic ratio.

Unlike the hypoxic cell sensitizers, these agents require extended exposure of the cells for the necessary incorporation into DNA, while the cells are undergoing DNA synthesis.

Importance of Cell Labeling and DNA Incorporation

Clinical Investigations

The degree of incorporation and thymidine replacement and the SER are intimately related. Therefore, measurements of thymidine replacement in individual human tumors by flow cytometry to establish the potential doubling time, and assessment of thymidine replacement after short and long infusions are needed as part of the design of future clinical trials with these cell-cycle drugs.

The means of achieving an optimal incorporation of these compounds in the cell in the clinical situation has been extensively explored over the years, particularly the route of administration and length of drug exposure. Early on, BUDR was used intra-arterially both to avoid dehalogenation by the liver and to increase the drug tumor concentration.¹²⁰ However, the necessary prolonged use of this route in patients over several weeks was laborious and had a high incidence of complications. Although there have been reports of rapid debromination of halopyrimidines occurring after intravenous therapy, Goffinet and Brown¹²¹ showed that following intravenous infusion, enough halopyrimidine apparently passes through the hepatic vessels to permit tumor radiosensitization, despite dilution of the drug by the systemic circulation. This has also been shown in human studies. There was a renewed interest in the 1980s and 1990s in the use of continuous intravenous infusion of halopyrimidines. It was observed that adequate steady-state arterial plasma levels could be maintained with this route of administration with acceptable systemic toxicities.¹²² A large study was reported by the Northern California Oncology Group in 160 patients with glioblastoma treated with 96-hour infusion of BrUdR at 800 mg/m² a day for a total of 6 weeks, in combination with 60 Gy irradiation directed to tumor plus a margin. The patients in this series received chemotherapy with procarbazine, lomustine (CCNU), and vincristine (PCV) for 1 year following radiotherapy. The median survival time was 12.8 months. Patients with anaplastic astrocytoma had a median survival time of almost 5 years, and the observation was made that the use of pyrimidine analogues in combination with radiation may be of greater benefit in this group of patients.¹²³ However a randomized study in anaplastic astrocytoma conducted by the RTOG using radiation and PCV chemotherapy compared with radiation and PCV plus BrUdR was terminated earlier because of the inferior time to tumor recurrence and survival observed in the arm using BrUdR.¹²⁴

In addition, because of the toxicities and limited tumor efficacy, the use of this drug with radiation was not warranted in patients with glioblastoma.¹²⁵

Conclusions on Radiosensitizers

After more than two decades of clinical investigations, the goal of using hypoxic cell or cell-cycle specific radiosensitizers in everyday standard radiotherapy remains elusive. There is, however, a better understanding of the multifactorial cause of radiation resistance and current efforts are concentrated on the selection of a specific tumor site in which one or two factors of resistance have been uncovered. Numerous studies indicate tumor hypoxia as one of the important factors in clinical radiotherapy. The application of radiosensitizers will not be universal in all solid tumors, but could be specifically directed to tumor sites according to their characteristics, including cell kinetics and presence or absence of hypoxia using noninvasive markers of hypoxia.

Chemical Radioprotectors

The first chemical radioprotector compounds intended for use on humans were developed for the purpose of protecting individuals from whole-body irradiation, such as in the event of nuclear warfare. The sulfhydryl compounds, including β-mercaptoethylamine and thiophosphates, were considered. An extensive drug developmental program was initiated by the U.S. Department of Defense (Walter Reed Army Research Institute [WR]). Out of 4000 screened compounds, the thiophosphate WR-2721 was the most promising. Clearly, these compounds were designed to protect all tissues, a very different requirement from their possible use in the field of oncology, in which protection of normal tissues to the exclusion of tumor tissues is essential to improve the therapeutic ratio. Initially, there were serious questions about whether these agents could also protect the tumor from the effects of radiation.¹²⁶^,¹²⁷

The assumption that the tumor tissues are not protected to the same degree as normal tissues is based on the probability that there is poor drug penetration in tumors because of their poor blood perfusion, the partition coefficient of the drug, and the probability that there is a higher concentration of the drug in normal tissues because of their higher pH. In addition, for WR-2721 to be active, the phosphate group must be cleaved by the enzyme alkaline phosphatase to form the dephosphorylated free thiol WR-1065. This enzyme is not as abundant in tumor tissues as in normal tissues; therefore, the levels of alkaline phosphatase and pH in tissues determine the uptake of WR-2721. There is also a final assumption that thiol compounds could have less protective effect on hypoxic cells. The mechanisms of radioprotection fit the competition-model, dual-action theory. Once inside the cell, the active free thiol WR-1065 can chemically reduce free radicals. However because protectors will have maximum effect at intermediate oxygen levels, the possibility of tumor protection can occur presumably at levels of intermediate hypoxia. It also takes part in the repair reaction of DNA damage. Of interest in the field of cancer chemotherapy is the fact the dephosphorylated WR-2721 can bind to the active species of alkylating agents, as well as prevent the formation of CIS-DNA adducts.¹²⁸

Biologic agents such as interleukin-1 have been shown to protect normal tissues in animal systems.¹²⁹

Amifostine WR-2721 (Ethyol)

The dose-modifying factor (DMF) of amifostine WR-2127 for both normal and tumor tissues was studied in animals carrying solid tumors.¹³⁰^,¹³¹ For normal tissues, the greatest protection is found in bone marrow, with a DMF of 2.7 to 3, and in the gastrointestinal tract, with a DMF of 1.6. The lowest DMF, at 1.2, is in lung tissue. However, this drug also protects tumor tissues, with DMFs ranging from 1.3 for cure of EMT-6 carcinoma to 2.2 for mean survival time of P-388 leukemia. Once more, the degree of protection to tumors appears to be related to the tumor blood perfusion, degree of hypoxia, the tissue pH, and the levels of alkaline phosphatase. A cautionary note is that most of these experiments were performed with single-radiation doses. It is possible that the differential protective effect between normal and tumor tissues would be less if multiple, daily, small doses in combination with radiation were used.

Experimental work has also been done in which amifostine is used as a chemoprotector of normal tissues. Three studies were performed with different animal tumor models and all three showed protection of bone marrow and intestine with no protection of the tumor when amifostine was used with melphalan ¹³² or in combined chemotherapy regimes with nitrogen mustard, cyclophosphamide, carmustine, CIS, and 5-fluorouracil.¹³³^,¹³⁴

It should be noted that amifostine does not protect the central nervous system tissues from radiation effects because of the blood-brain barrier.¹³⁵

Clinical Experience

Amifostine has been approved for clinical use and is available for intravenous route in a sterile lyophilized powder mixture with mannitol, requiring reconstitution for intravenous administration. It is administered over a 15-minute period prior to radiation or chemotherapy. Initial single-dose toxicity and pharmacokinetic studies were performed in 1983. The plasma initial-half-life is 9 minutes, and it is assumed that the protective concentrations are maintained in normal tissues for approximately 2 hours.¹³⁶^,¹³⁷

The maximum tolerated dose for multiple doses is 340 mg/m² given 4 days a week for 5 weeks, 15 minutes prior to external radiation. Toxicities at this dose level are manifested as nausea, vomiting, anorexia, malaise, transient moderate hypotension, and occasional hypocalcemia. It is recommended that the amifostine infusion be interrupted if there is a 25% decrease in systolic blood pressure. The maximum tolerated dose with single doses is 740 mg/m², although recently the dose was increased to 910 mg/m² given twice a week for at least five treatments, and this dose has been considered acceptable.¹³⁸

Since 2000, this compound has led most of the reported activity on clinical trials with chemical modifiers of radiation response. This interest has been triggered by the increasing use of chemoradiotherapy in solid tumors and in an effort to avoid the incidence of toxicities with this therapeutic combination on major sites such as NSCLC (protection for esophagitis and pneumonitis), head and neck tumors (protection from xerostomia and mucositis), as well as pelvic tumors (protection of rectal mucosa and the small bowel).

Amifostine in combination with chemoradiation therapy for small cell lung cancer and NSCLC has been studied in several randomized phase III clinical trials, which have shown a statistically significant decrease in esophagitis and pneumonitis with no observed tumor protection.¹³⁹^–¹⁴² A meta-analysis of all published clinical trials (seven randomized involving 601 patients, with locally advanced NSCLC treated with radiotherapy with or without chemotherapy) revealed that amifostine has not protected the tumor from the therapeutic effect of either radiation or chemoradiation.¹⁴³ However, amifostine toxicity consisting of hypotension, nausea, and vomiting with the use of the intravenous route prevented a rather large proportion of patients from receiving the full dose according to protocol.¹⁴² To avoid the amifostine toxicity seen with intravenous use, studies have been initiated to explore the subcutaneous administration based on previous phase I and II trials demonstrating a reduction of amifostine toxicities when using this route.¹⁴⁴^–¹⁴⁷

Head and neck tumors treated with chemoradiotherapy is another area in which a large number of randomized phase III clinical trials were initiated over the past 7 years using amifostine as a radioprotector. Most of the reported clinical trials on this disease site have demonstrated a decreased incidence of xerostomia and mucositis, with no clear evidence of tumor protection as measured by local regional tumor control.¹⁴⁸ A significant reduction in the incidence of radiation-induced xerostomia at 12 months posttreatment was reported with the use of amifostine in a multinational phase III trial of 315 patients with head and neck cancer.¹⁴⁹ An update of this study at 18 and 24 months after initial treatment indicated that amifostine reduces the severity and duration of xerostomia 2 years after treatment and does not seem to compromise local-regional tumor control rates, progression-free survival, or overall survival.¹⁵⁰ Amifostine-related nausea, vomiting, and hypotension at a dose of 200 mg IV daily with each radiation fraction led to discontinuation of the drug in approximately 20% of patients with, in some cases, a delay of radiotherapy.¹⁵¹

Early phase II efficacy trials have been initiated using amifostine during chemoradiotherapy of pelvic tumors in an attempt to protect rectal mucosa, indicating significant protective effect in patients with cancers of the prostate, cervix, and rectum using either the intravenous or subcutaneous routes as well as with the intrarectal aqueous solution in a 48-ml enema.¹⁵²^,¹⁵³

Concerns about amifostine toxicity as well as the question of tumor tissue protection have been raised by some authors.¹⁵⁴ However, contradicting this view, a review of all clinical studies done up to 2003 have clearly indicated no evidence of amifostine tumor protection.¹⁵⁵

The practical application of amifostine in the standard practice of radiation oncology continues to be a controversial one. Up to 2008, the use of amifostine in the chemoradiotherapy of head and neck tumors to protect from xerostomia has been recommended by the ASCO and is approved by the U.S. Food and Drug Administration for (1) reduction of the incidence of moderate to severe xerostomia in patients undergoing postoperative radiation treatment for head and neck cancer, and (2) reduction in the cumulative renal toxicity associated with CIS in patients with advanced ovarian cancer.¹⁵⁶^,¹⁵⁷ Recent observations of the efficacy of IMRT in reducing xerostomia have raised questions about the expense of using amifostine for this purpose.

Chemoprotection

The clinical effectiveness of amifostine as a chemoprotector was first assessed with the use of bone marrow as the normal tissue endpoint and cyclophosphamide as the chemotherapeutic agent. Definitive evidence of bone marrow protection was observed. Tumor protection was not assessed.¹⁵⁸ In another study, renal damage and peripheral neuropathy were assessed as endpoints and CIS was used; normal tissue protection with no tumor protection was observed.¹⁵⁹ Another clinical study demonstrated the protective effect of amifostine in bone marrow, kidney, and peripheral nerves when cyclophosphamide is used in combination with CIS.¹⁶⁰ Protection of carboplatin myelotoxicity was observed in one study,¹⁶¹ but findings were inconclusive in another preliminary study.¹⁶² The use of amifostine in combination with chemotherapy and radiation is currently being used in clinical research protocols for both adult and pediatric patient populations.

Tempol

Nitroxide superoxide dismutase mimic (Tempol), a nonthio nitroxide, has been shown both in vitro and in vivo to be an effective radioprotector with the absences of tumor radioprotection. In addition, nitroxide compounds are known for their modest hypoxic cell radiosensitization. The potential of Tempol as an aerobic cell radioprotector has been described by Mitchell and colleagues.¹⁶³

Conclusions on Radioprotectors and Chemoprotectors

A spin-off of the work on radioprotectors is manifested in the increasing interest in the use of these compounds as chemoprotectors in the field of medical oncology.¹⁶⁴ The protective effects of amifostine have been seen in bone marrow during the use of alkylating and platinum compound agents. Phase III large clinical trials are ongoing to demonstrate conclusively normal tissue protection without tumor protection in esophageal mucosa, salivary glands, and bone marrow when these protectors are combined with radiation, and in kidney, peripheral nerves, and bone marrow when they are used with alkylating agents and platinum compounds.

An exciting evolving potential is in the combination of chemical protectors (amifostine or Tempol) with biologic stimulators (cytokines). Furthermore, the effect of thiol compounds might be even greater in the laboratory, based on their promising use as a probe in examining the mechanisms of radiation cell damage. It is likely that vital new information will be available in the near future on the molecular mechanisms of radiation protection.¹⁶⁴

References

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2 Gatenby RA, Kessler HB, Rosenblum JS, et al. Oxygen distribution of squamous cell carcinoma metastases and its relationship to outcome of radiation therapy. Int J Radiat Oncol Biol Phys. 1988;14(5):831.

3 Hockel M, Knoop C, Schlenger K, et al. Intratumoral Po₂ predicts the survival in advanced cancer of the uterine cervix: updated analysis of an open prospective clinical study. Radiother Oncol. 1992;26:45.

4 Bush RS, Jenkin RDT, Eltt WEC, et al. Definitive evidence for hypoxic cells influencing cure in cancer therapy. Br J Cancer. 1978;37(3):302.

5 Brizel DM, Scully SP, Harrelson JM, et al. Tumor oxygenation predicts for the likelihood of distant metastases in human soft tissue sarcoma. Cancer Res. 1996;56:941.

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7 Nordsmark M, Bentzen SM, Rudat V, et al. prognostic value of tumor oxygenation in 396 head and neck tumors after primary radiation therapy. An international multi-center study. Radiother Oncol. 2005;77(1):18-24.

8 Durand RE. Distribution and activity of antineoplastic drugs in a tumor model. J Natl Cancer Inst. 1989;81:146-152.

9 Sutherland RM. Cell and environment interaction in tumor microregions. The multicell spheroid model. Science. 1988;24:177.

10 Graeber TG, Osmanian C, Jacks T, et al. Hypoxia-mediated selection of cells with diminished apoptotic potential in solid tumours. Nature. 1996;379:88.