Radiosensitization of solid tumors by nitroimidazoles

Im. J. Rodiatwn

Oncology

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Phys.. 1978. Vol. 4, pp. 143-151.

Pergamon

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??Radiation Sensitizers for Hypoxic Cells RADIOSENSITIZATION OF SOLID TUMORS BY NITROIMIDAZOLES J. DENEKAMP, Gray Laboratory

Ph.D., B.Sc. and J. F. FOWLER, of the Cancer

D.Sc., Ph.D., F.1nst.P.

Research Campaign, Mount Vernon Hospital, Northwood, Middlesex HA6 2RN, England

Recently a new cla.ss of drugs has been identified by Adams et al. at the Gray Laboratory, which act as specific radiosensitizers of hypoxic cells. This sensitization results from their high electron affinity, and their presence at the time of irra’diation abolishes the protection afforded by hypoxia. Amongst the various nitro-heterocyclic compounds that have been tested the nitroimidazoles seem to give the greatest sensitization in uiuo. The 5nitroimidazole metronidazole, and its 2-nitro derivative Ro-07-0582 have both been shown to be potent radiosensitizers, and to have a low toxicity in uioo. Tumor studies with Ro-07-0582 have been performed on at least 12 different experimental animal tumors, using a variety of endpoints, e.g. tumor regrowth delay, local control, and cell survival assays in uitro or in viva after in uiuo irradiation. The degree of sensitization achieved depends upon the drug dose administered, over the range 0.1-1.5 mg/g body weight. All tumors that contain hypoxic cells have been sensitized to single doses of radiation, often showing an enhancement ratio of 2.0. Normal tissues that are well oxygenated are not sensitized. Studies with fractionated doses of drug and radiation have also been performed on five tumor lines. Of these, three tumors have continued to show a considerable therapeutic advantage for treatments with the drug. The use of Ro-07-0582 with X-rays has been compared with the fast neutron beam from the MRC Cyclotron at Hammersmith Hospital on two mouse tumor lines. The X-ray plus sensitizer treatments were as effective as the fast neutron treatments. Fast neutrons used together with Ro-07-0582 were more effective than any of the other treatments. The time of administration of the drug relative to irradiation is critical. It must be present at its maximum concentration at the time of irradiation. This occurs between 15 and 60 min in mice, where the biological half-life is short ‘(l-1.5 hr). In man there is a plateau in serum concentration between 1 and 5 hr after administration. Because of this and the longer half-life (10-18 hr), the timing is less critical. In addition to cthedirect radiosensitization there is a specific cytotoxicity for hypoxic cells if the exposure to the drug is long enough. This effect is small compared to the radiosensitization, but it will be of more importance in ma.n than in the mouse, because of the difference in half-lives of drug in the serum. This cytotoxicity can be greatly enhanced by moderate hyperthermia. It would represent systemic chemotherapy for hypoxic (probably non-proliferating) cells throughout the body. Electron affinic, Badiosensitizers,

Tumors,

Hypoxic cells.

The local control of tumors by radiotherapy is limited because the dose that can be tolerated by the surrounding normal tissue must not be exceeded. Physical methods of improving the depth dose distribution have produced dramatic improvements in some sites, but local failure is still the cause of death in many cancer patients.25 It seems likely that the radioresistance of some of these tumors may be a result of low oxygen concentrations in regions where the intercapillary distances exceed 300 Fm.26 Hyperbaric oxygen, high-LET radiation (such as neutrons or ne-

gative pi-mesons) and electron-affinic chemicals are the three most commonly suggested ways of overcoming the radioresistance of hypoxic cells, without a corresponding increase in the damage to normal tissue. Some other types of radiosensitizer depend upon their preferential uptake into tumor cells because of their more rapid proliferation rate. The electron affinic drugs, however, will work without preferential uptake, by means of the difference between the micro-environmental oxygen concentration in tumors and in normal tissues.

Acknowledgements-The authors have pleasure in thanking Drs. G. E. Adams, S. Dische, R. H. Thomlinson, I. R. Flockhart, I. J. Stratford, R. Urtasun, A. M. Rauth, Miss S.

A. Hill and Mr. P. W. Sheldon for stimulating discussions and permission to quote their data, both published and unpublished.

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Tumors often have an inadequate vascular supply, which makes it difficult to deliver drug to all the tumor cells. But it is this same vascular inadequacy that provides the hypoxic micro-environment within the tumor. A lack of oxygen protects cells from X-ray damage by a factor of 2.5-3.0, and this resistance must be overcome before tumor eradication can be complete. During conventional fractionated radiotherapy an improvement in local oxygenation within a tumor may occur between fractions and the tumor radiosensitivity may increase throughout the course of treatment because of this “reoxygenation”. When this process is inadequate, a therapeutic alternative is afforded by chemicals which, like oxygen, sensitize hypoxic cells, but unlike oxygen, are not metabolised before diffusing to the target cells. In 1963 Adams and Dewey’ proposed that a correlation existed between the ability of chemical compounds to sensitize hypoxic bacteria and their electron affinity. Subsequent work on bacterial and mammalian cells has verified this relationship3 and a strong correlation has now been demonstrated between sensitization of mammalian cells and the electron affinity of various nitro-imidazoles.’ Another group of electron affinic drugs, the nitrofurans, have also been studied in detail,6.‘9 but their usefulness in uivo has been limited by their short biological halflife. ANIMAL

EXPERIMENTS

A number of the drugs shown to be effective in vitro have been tested in a range of animal tumors, and are now being tried with terminal cancer patients. The drugs were first tested in a normal tissue made artificially hypoxic, because it was a rapid and accurate in viuo test. The first demonstration of in uivo sensitization with one of these compounds involved the drug NDPP, a soluble derivative of paranitroacetophenone.’ Epidermal cell survival was assayed using the in situ cloning technique of Withers.3’ Mouse skin was made artificially hypoxic by giving the animals nitrogen to breathe for 35 set, during the last few seconds of which the skin was irradiated with electrons.’ The dose to reduce clone survival to a particular level was determined. A maximum enhancement ratio of 1.5 was obtained for a dose of NDPP that was toxic enough to kill half the animals. This was soon followed by tests of other compounds and the data in Fig. 1 show the sensitization achieved with different concentrations of Flagyl and Ro-070582,’ rising to an enhancement ratio of 2.0. No sensitization was observed in mice breathing oxygen or given the drug just after irradiation. An increasing sensitization was observed with increasing concentrations of both drugs if they were present at the time of irradiation. Ro-07-0582 was more efficient as a sensitizer than Flagyl.

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The experiments on artificially hypoxic skin demonstrated that substantial sensitization could be achieved in uiuo with doses that were well tolerated, and that the drug could diffuse from the peritoneum, through the blood stream and out to the target cells. In the early clinical testing of these two drugs at Mount Vernon Hospital, a similar approach was used to ensure that adequate serum levels could be achieved in patients to sensitize any hypoxic cells that were present.13 Patient volunteers had small areas of skin irradiated with low single doses of P-rays, so that pigmentation could be scored between 1 and 3 months later. The limb was made artificially hypoxic for some irradiations by applying a tourniquet and irradiating in a local atmosphere of nitrogen. In this way dose response curves were obtained for skin under well oxygenated or hypoxic conditions, and hypoxic in the presence of the sensitizer.” The drug enhancement ratio could then be determined, as could the oxygen enhancement ratio. The ratio of these two can be expressed as the Relative Sensitizing Efficiency (RSE). Figure 2 summarises this data on human skin, and relates it to the mouse skin data described above. The RSE is plotted as a function of the serum concentration at the time of irradiation. Figure 2 shows that the same degree of sensitization can be achieved in human subjects as in mice for any particular serum level of either Flagyl or Ro-07-0582. It should be emphasized that no enhancement was expected or seen in skin irradiated under normal aerobic conditions. When sensitization had been demonstrated in artificially hypoxic mouse skin, tumor studies in mice were undertaken using a variety of tumor assay techniques. These included regrowth delay, local control and cell survival measurements assayed in vitro or in uivo after irradiation in uiuo. An example of tumor regrowth data is shown in Fig. 3. The growth of a transplanted mouse carcinoma, “Ca NT”, was delayed for about 20 days by 2000 rad of X-rays. If the animals received Flagyl or Ro-07-0582 before irradiation this delay was increased, showing that more cell killing had been achieved in the presence of the sensitizer.’ A similar effect is shown in Fig. 3(b) for 1000 rad of X-rays, of neutrons, or of neutrons plus Ro-07-0582. Neutrons were more effective than X-rays, and the sensitizer made them more effective still.” From a series of curves such as those in Fig. 3, dose effect curves can be derived and the sensitizing efficiency of any treatment can be determined from the radiation dose necessary to achieve a given level of delay in the absence of the drug, divided by the X-ray dose necessary to achieve the same delay in the presence of the drug. This ratio of X-ray doses is termed the enhancement ratio. Figure 4 shows that if tumors were made uniformly hypoxic by clamping

Radiosensitization of solid tumors by nitroimidazoles

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Fig. 1. The response of mouse epidermal cells to radiation, assayed by island regrowth.’ Skin irradiated in mice breathing nitrogen for 35 set is about 2.5 times less sensitive to radiation than in oxygen breathing mice. This sensitivity is partly restored if the mice have received Flagyl or Ro-07-0582 before irradiation. The degree of sensitization increases with drug dose. No effect is seen in oxygen, or if the drug is administered 5 min post-irradiation (redrawn from Denekamp et d.“j. them 15 min after administering the drug Ro-07-0582, sensitization by a factor of 2.1 was observed at all

radiation dose levels.’ However, if the tumors were irradiated under normal aerobic conditions a biphasic curve was obtained (Fig. 4), with a sensitive response at low doses, and a resistant response at higher doses where the hypoxic cells; became important. At these high doses the same enhancement ratio was obtained (2.1), but at lower doses a much smaller effect was observed.’ Two sets of clinical data have been obtained with electron-affinic drugs that are comparable to these regrowth delay experiments. Thomlinson et ~1.~’ measured regrowth delay after palliative treatment with single doses in subcutaneous and lung metastases in 7 patients. Some of the nodules were treated

before administration of Ro-07-0582, and some after. Three patients died before tumor regrowth would be assessed, and of the remaining 4 sensitization was clearly demonstrated in 3 with subcutaneous nodules, but not in the patient with lung metastases. The regrowth curves from 1 patient are reproduced in Fig. 5, and show that 8OOrad of X-rays used with Ro-070582 was more efficient than 960 rad alone, but less than 1120 rad of X-rays. This yields an enhancement ratio of between 1.2 and 1.4 even though a very low radiation dose was used (cf. mouse data in Fig. 4b). The other clinical study is by Urtasun et aLZB on survival time of patients with glioblastoma multiforme. In this series 9 fractions of 330rad of X-rays were used either alone or after administration of 9-11 g of Flagyl before each fraction. The patients

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Fig. 2. The sensitizing effect of Flagyl and Ro-07-0582, on artificially hypoxic skin, as a function of serum concentration for mice and men. The sensitization achieved with the drug is expressed as a percentage of that which can be achieved by 100% oxygen. The human dataI fall close to the curves obtained from mice.*

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Fig. 4. Dose response curves for carcinoma NT, i.e. the delay in regrowth as a function of dose. (!Ilm) Animals treated with their tumors clamped to make all the cells hypoxic. 1 mg/g Ro-07-0582 sensitizes by a factor of 2.1 at all dose levels. (00) Tumors in air breathing mice. The enhancement ratio is 2.1 at high doses, but is much lower below 1200rad, where well oxygenated cells are deterand (redrawn from Denekamp mining the response Harris’). Open symbols, with Ro-07-0582. Closed symbols, without this drug.

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Fig. 3. Growth curves for carcinoma NT; each curve represents the average of 10 mice or more. (a) Untreated animals, or tumors treated with 2000 rad of X-rays alone, or after administration of 1 mg/g Flagyl or Ro-07-0582. The drug treated animals show more tumor delay.’ (b) Tumors treated with 1OOOrad of X-rays, of neutrons, or of neutrons plus Ro-07-0582. The drug increases the sensitivity to neutrons, which themselves are more effective than X-rays because of their greater efficiency in killing hypoxic cells.”

Radiosensitization

of solid tumors by nitroimidazoles 0 J.

DENEKAMP

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Fig. 5. Changes in volume relative to that at the time of treatment of subcutaneous metastases of human carcinoma of the cervix uterus. Single dose treatments to each group of 7 nodules (after Thomlinson et al.*‘). treated with Flagyl (wh.ich is a much less efficient sensitizer than Ro-07-0582) showed a definitely prolonged survival time, consistent with more cell killing and a longer delay before regrowth of the residual tumor (Fig. 6). Although the X-ray treatment schedule was probably :suboptimal because the total dose used was rather low, this result is an unequivocal demonstration that hypoxic cells exist in human tumors, that they limit radiocurability, and that they are accessible to a blood-borne drug. Figure 7 shows results using the local control of an anaplastic tumor in mice as a function of X-ray dose in the presence or absence of different concentrations of the sensitizers Flagyl and Ro-07-0582. This data is part of a series on six different compounds tested by Sheldon and Hill.‘* Significant sensitization is seen

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Fig. 6. Kaplan-Meier survival plots for glioblastoma patients treated with 9 fractions of X-rays alone, group I or 9 fractions of X-rays used with doses of 9-11 g Flagyl before each treatment group II. A significantly increased survival time was observed in the Flagyl treated group (from Urtasun et ~1.~).

with both drugs; an increasing drug dose yields an increasing enhancement ratio, which can be read off at any level of tumor control probability. The enhancement ratio of 1.5 corresponds to only twothirds of the X-ray dose being necessary to achieve 50% local control rates when the drug is present. This was achieved with 1 mg/g of Flagyl or with 0.1 mglg Ro-07-0582, again showing that the 2-nitroimidazole is a more efficient sensitizer than the 5-nitro compound. Figure 7 shows that with an enhancement ratio of this magnitude, an X-ray treatment that would yield no local controls if used alone (e.g. 6000 rad) would give more than 75% local controls if used with the sensitizer. The hatched area in Fig. 7(b) corresponds to serum concentrations that can be attained in man, although approximately 30% less drug (mglkg) is necessary in man relative to the mouse.15 In addition, the concentration of Ro-07-0582 in human tumors reaches 80-95% of the serum concentration at 14 hr, whereas the tumor concentration in mice does not exceed 30-50%, probably because of the shorter biological half-life.15Z” Therefore human tumor responses may be even better than would be anticipated from mouse data. A large degree of sensitization has also been demonstrated in other tumors, by these techniques and also by cell survival assays in vitro or in vivo after excision of a tumor irradiated in vivo. These data are summarised in Fig. 8 where Enhancement Ratio is plotted as a function of drug dose administered for mouse tumors that were tested at several drug doses. The heavy line represents the data from artificially hypoxic skin.’ The data from tumors are represented by different symbols, depending on the method of assay used. The hatched area

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Fig. 7. Tumor control at 80 days as a function of X-ray dose for the anaplastic response curves for animals treated with X-rays only or with 0.1 and 1.0 mglg Increasing sensitization is seen with increasing drug dose, the maximum being Ro-07-0582. This drug is more effective, with a maximum enhancement ratio of represents the clinically attainable serum concentrations. In this range one tumor shows no sensitization because it was used with a small dose of radiation, but most show an enhancement ratio between 1.2 and 1.8. If such an effect represented a real therapeutic gain it could increase the rate of local control from 0 to almost 100% in animal tumors, such as those used by Sheldon and Hill (Fig. 7) and should also give a big gain clinically, based on tumor response curves such as those of Shukovsky,” or Morrison,” if it is maintained during a fractionated irradiation. Since no sensitization of well oxygenated normal tissues occurs, an enhancement ratio is equal to a therapeutic gain, unless hypoxic cells occur in the normal tissue included in the beam. For the experiments described above the time interval between intraperitoneal administration of the

and NO. 2

mice

SHELDON

3

I

tumor “MT”. (a) Dose body weight of Flagyl. 1.5. (b) Treatment with 2.1 (Sheldon and Hi1122).

drug and irradiation varied between 15 and 45 min. It is known that high serum levels are achieved within 10min in mice and that the half-life for removal is about 90 min.15 The time of peak tumor concentration is somewhat later than 10 min. A number of mouse experiments have been performed to determine the degree of sensitization at different times after administration. Brown4 found that 30 min was optimal for the EMT6 carcinoma whereas McNally (unpublished) and Sheldon (unpublished) have each found that 45-60 min is optimal in the fibrosarcoma Fib/T and the anaplastic tumor “MT”. In tumor regrowth experiments on two different tumors, 1530min seem to give the maximal effect (Denekamp and Stewart, unpublished). For the interpretation of mouse experiments timing is very important, but for clinical application it will be much less critical be-

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Fig. 8. Summary of the enhancement ratio with increasing drug concentration for anoxic skin and for a range of experimental tumors assayed by means of local control (C), by tumor regrowth (R)I, or by excision and dilution in vitro or in vivo (D). The name of the experimenter is shown against each curve. Skin data from Ref. 8. The hatched area represents the drug doses that can be tolerated clinically.

cause of the longer half-life in man. In man the peak serum level is usually achieved within 1 hr, it remains high for 4-5 hr, and then decreases with a half-life of 10-18 hr.17 Thus, providing the drug has been ingested between 3 and 5 hr before irradiation it should be present in high concentration. Whilst significant sensitization has been observed with single doses in all these tumors (even at low drug doses), single dose experiments do not seem relevant to the clinical situation since most clinical treatments involve many small fractions, and reoxygenation may occur within the treatment period. For this reason fractionated studies, us.ing multiple doses of X-rays together with multiple doses of drug are necessary. These have been performed on only a few tumors. In two of these the effect of X-rays plus drug has been compared with the effect of cyclotron produced fast neutrons, which are also more effective on hypoxic cells. Figure 9 shows the data obtained from regrowth delay experiments on Carcinoma NT. In order to assess the therapeutic gain for the different treatments the tumor delay was assessed for each treatment for doses that would produce a constant level of normal tissue damage.“.” For the chosen level of skin reaction, (0.8 = erythema and slight desquamation), about 16 days’ delay in tumor growth could be achieved with a single dose of X-rays. This could be increased to almost 40 days by using 5 fractions of X-rays given over 9 days. 40 days’ delay could also be achieved from a single dose treatment, either of neutrons, or of X-rays after administering 0.67 mglg body weight of Ro-07-0582. These single dose treatments

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Fig. 9. The response of carcinoma NT to 1, 2 or 5 fractions of X-rays alone (X), X-rays plus Ro-07-0582 (O), and fast neutrons (N). The sensitizer plus X-rays and the neutrons give a therapeutic advantage (i.e. more tumor delay for a constant skin reaction) for all three schemes. 0 represents a single dose of neutrons plus Ro-07-0582, which was one of the most effective treatments tested.“‘.”

could be improved again by fractionation (Fig. 9). The effect of conventional X-rays plus drug was very similar to the effect of the high-LET radiation alone. This has obvious economic implications, although the drug doses were rather high. The most effective single dose treatment was irradiation by neutrons in the presence of sensitizer, i.e. by use of two methods of overcoming hypoxia together. A further gain has recently been demonstrated for fractionated treatments using both 2 and 5 fractions of neutrons plus the sensitizer.‘* Thus the most effective treatment seems to be high-LET radiation plus an electron-affinic sensitizer, and they should be considered as complementary to, not competitive with, each other. Another set of data from fractionation experiments is shown in Fig. 10. Again a standard level of skin reaction was chosen, but in this case it was much more severe, since higher doses were needed for local control than for studying tumor regrowth. The skin reaction of 2.0 corresponds to severe ulceration, with subsequent healing within 30 days. The hatched area represents the range of local control values achieved by a series of different X-ray only fractionation schedules extending from single doses to 15 fractions in 18 days.“j Four of these schedules were tested with X-rays plus sensitizers and with fast neutrons.‘6’2’ The sensitizer results are shown in Fig. 10 for single doses,

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Fig. 10. The probability of local control for a constant skin damage (level 2.0) for a variety of fractionation schemes in C3H mammary tumors. The hatched area represents the range observed in X-ray-only experiments. Four schedules were tested with 0.7-l mg/g Ro-07-0582 and the improvement relative to concurrent controls is shown by the arrows. A

similar gain was observed with fast neutrons (Fowler et al.“).

3 and 5 fractions given at 48 hr intervals and for 5 fractions given daily. In each case concurrent control X-ray only data are also shown. The sensitizer increased the probability of local control for all four treatments. X-ray treatments that were already quite effective because of reoxygenation, e.g. 3F/4 days, were improved only a little, but less effective treatments, e.g. 5F/4 days, showed a bigger benefit. Similar gains were seen with fast neutrons. The use of neutrons, or of X-rays plus Ro-07-0582, both removed the criticality due to reoxygenation from the choice of fractionated schedules and made them all of similar value, and amongst the best treatments tested. The effect was most marked for short fractionation schedules and the economic implications are obvious. Sheldon (personal communication) has shown that the enhancement ratio is reduced from 1.9 for a single dose to 1.5 for 5 fractions with 0.67 mglg Ro-07-0582 in the anaplastic tumor “MT,“. Regrowth delay experiments in a fibrosarcoma (Denekamp and Stewart, unpublished) have shown that no sensitization was observed with five daily fractions, although this

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tumor was not believed to reoxygenate very extensively. Van Putten3’ has also shown that the degree of sensitization is much reduced in a rat osteosarcoma if it is treated with 20 small daily fractions, but if the fraction size is increased more sensitization is observed. These results, together with the finding of peripheral neuropathy as a limitation in the clinical use of R0-07-0582,‘~*~~make it likely that the drug will be most useful in association with relatively few, large radiation doses. Another potential use of electron-affinic radiosensitizers is in combination with less conventional forms of therapy such as hyperthermia. Nitro-imidazoles have been shown to be specifically cytotoxic to hypoxic cells if the exposure is prolonged for several hours.‘*24 The effect is smaller than that of direct radiosensitization and appears to be significant in some types of mouse tumor but absent in others. This cytotoxicity has recently been shown to be greatly increased in vitro by heating the cells moderately, to 38”-41”C, during their exposure to the drug.23 The cytotoxicity is specific for hypoxic cells and the degree of cell kill can be increased by 2-4 orders of magnitude with heat treatments that are not themselves cytocidal. Whilst this effect seems to be small in mouse tumors relative to the direct radiosensitization, it will play a larger part in human tumors because the contact time is much longer and higher tumor drug levels are achieved. It could also act as a form of systemic chemotherapy for any metastatic deposits that contain hypoxic and presumably nonto most cycling cells, i.e. those that are resistant other forms of chemotherapy. Flagyl and Ro-07-0582 are amongst the best compounds that have been tested in uiuo, and because of

the large body of pharmacological data available on them they are already entering clinical trials. However, the large drug doses and the peripheral neuropathy encountered with them make it unlikely that they will be the ultimate drugs of choice. Therefore the search for an even better sensitizer continues at the basic level.

REFERENCES 4. Brown, J.M.: Selective radiosensitization of the Adams, G.E., Dewey, D.L.: Hydrated electrons and hypoxic cells of mouse tumors with the nitroimidazoles radiobiological sensitization. Biochem. Biophys. Res. metronidazole and Ro-07-0582. Radiat. Res. 64: 633, Comm. 12: 473-477, 1963. 1975. Adams, G.E., Flockhart, I.R., Smithen, C.E., Stratford, 5. Brown, J.M.: Cytotoxic effects of the hypoxic cell I.J., Wardman, P., Watts, M.E.: Electron affinic senradiosensitizer Ro-07-0582 to tumor cells in duo. sitization-VII. A correlation between structures, one Radiat. Res. in press. electron reduction potentials and efficiencies of nitroimidazoles as hypoxic cell radiosensitizers. Radiat. Res. 6. Chapman, J.D., Reuvers, A.P., Borsa, J., Petkau, A., 67: 9-20, 1976. McCalla, D.: Nitrofurans as radiosensitizers of hypoxic mammalian cells. Cancer Res. 32: 2616-2624, 1972. Asquith, J.C., Watts, M.E., Patel, K., Smithen, C.E., 7. Denekamp, J., Michael, B.D.: Preferential sensitization Adams, G.E.: Electron affinic sensitization-V. Radiosensitization of hypoxic bacteria and mammalian of hypoxic cells to radiation in vivo. Nature New Biol. cells in vitro by some nitroimidazoles and nitro239: 21-23, 1972. 8. Denekamp, J., Michael, B.D., Harris, S.R.: Hypoxic cell pyrazoles. Radiat. Res. 60: 108-118, 1974.

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radiosensitizers: comparative tests of some electron affinic compounds using epidermal cell survival in oiuo. Radiat. Res. 60: 119-132., 1974. 9. Denekamp, J., Harris, S.-R.: Tests of two electron affinic radiosensitizers in vivo using regrowth of an experimental carcinoma. Radiat. Res. 61: 191-203, 1975. 10. Denekamp, J., Harris, S.R.: The response of a transplantable tumor to fractionated irradiation-I. X-rays and the hypoxic cell sen:sitizer Ro-07-0582. Radiat. Res. 66: 66-75, 1976. 11. Denekamp, J., Harris, S.R., Morris, C., Field, S.B.: The response of a transplantable tumor to fractionated irradiation-11. Fast neutrons. Radiat. Res. 68: 93-103, 1976. 12. Denekamp, J., Morris, C., Field, S.B.: The response of a transplantable tumor to fractionated irradiation-III. Neutrons plus Ro-07-0582. Radiat. Res. 70: 425-432, 1976. 13. Dische, S., Gray, A.J., Zanelli, G.D.: Clinical testing of the radiosensitizer Ro-07-0582-11. Radiosensitization of normal and hypoxic skin. Clin. Radiol. 27: 159-165, 1976. 14. Dische, S., Saunders, h4.1., Lee, M.E., Adams, G.E., Flockhart, I.R.: Clinical testing of the radiosensitizer Ro-07-0582: experience with multiple doses. Bit. J. Cancer 35: 567-579, 1977. 15. Flockhart, I.R.: personal communication, 1976. 16. Fowler, J.F., Sheldon, P.W., Denekamp, J., Field, S.B.: Optimum fractionation of the C3H mouse mammary carcinoma using X-rays, the hypoxic cell sensitizer Ro-07-0582 or fast neutrons. Int. J. Radiat. Oncol. Biol. Phys. 1: 579-592, 1976. 17. Gray, A.J., Dische, S., Adams, G.E., Flockhart, I.R., Foster, J.L.: Clinical te:jting of the radiosensitizer Ro07-0582-I. Dose, tolera.nce, serum and tumor concentrations. Clin. Radial. 2’7: 151-7, 1975. 18. Morrison, R.: The results of treatment of cancer of the bladder, a clinical contribution to radiobiology. Clin. Radiol. 26: 67-75, 1975. 19. Rauth, A.M., Kaufman, K.: In uiuo testing of hypoxic radiosensitizers using the KHT murine tumor assayed by a 2-nitroimidazole drug. Br. J. Radiol. 48: 209-220, 1975. 20. Shukovsky, L.J.: Dose, time volume relationships in

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squamous cell carcinoma of the supraglottic larynx. Am. J. Roentgenol. 108: 27-29, 1970. Sheldon, P.W., Hill, S.A., Foster, J.L., Fowler, J.F.: Radiosensitization of C3H mouse mammary tumours using fractionated doses of X-rays with the drug Ro-070582. Br. J. Radiol. 49: 76-80, 1975. Sheldon, P.W., Hill, S.A.: The effect of hypoxic cell radiosensitizing drugs on local control by single doses of X-rays of a transplanted anaplastic tumour in mice. Br. J. Cancer 35: 795-808, 1977. Stratford, I.J., Adams, G.E.: The effect of hyperthermia on the differential cytotoxicity of a hypoxic cell radiosensitizer, the 2-nitroimidazole Ro-07-0582, on mammalian cells in vitro. Br. J. Cancer 35: 307-313, 1977. Sutherland, R.M.: Selective chemotherapy of noncycling cells in an in vitro tumour model. Cancer Res. 34: 3501-3505, 1974. Suit, H.D.: Statement of the problem pertaining to the effect of dose fractionation and total treatment time on response of tissue to X-irradiation-VII-X. In Time and Dose Relationships in Radiation Biology as Applied to Radiotherapy. NCI-AEC Conf. Camel 1969. Brook-

haven Nat1 Lab. Publ. No. 50203 (C57). 26. Thomlinson, R.H., Gray, L.H.: The histological

structure of some human lung cancers and the possible implications for radiotherapy. Br. J. Cancer 9: 539-49,

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1%7.