Inr. I. Radialion

0

Oncology

Rio/. Phys., 1978, Vol. 4. PP. 153-156.

Pergamon

Press.

Printed

in the U.S.A.

Radiation Sensitizers for Hypoxic Cells

DIFFERENTIAL CYTOTOXIC EFFECTS OF METRONIDAZOLE AND OTHER NITRO-HETEROCYCLIC DRUGS AGAINST HYPOXIC TUMOUR CELLS J. L. FOSTER, B.Sc., M.Sc. Research Institute, Churchill Hospital, Headington, Oxford, England Drugs which have been used as radiosensitizers of hypoxic tumour cells also work directly as cytotoxic agents. Whilst aerated mammalian cells are killed by these drugs, in oifro experiments have shown that hypoxic cells are sensitive to mu’ch lower drug concentrations. The nitroimidan;ole drugs metronidazole (Flagyl@) and Ro-07-0582 and the nitrofuran, nitrofurazone, show activity in this respect. This phenomenon is affected by drug concentration, temperature and oxygen tension. In oiuo experiments have shown that metronidazole and Ro-07-0582 can kill hypoxic cells in murine solid tumours, though the latter drug has only been used at very high dosages. Some of the difficulties encountered during the in uiuo investigation of the specific toxicity of these drugs for hypoxic cells are discussed. Cytotoxics,

Hypoxic tumour

cells, Metronidazole,

Oxygen tension.

INTRODUCTION

plating efficiencyi3*” or the in viuo assay of cell viability after in vitro treatment as criteria of drug toxicity. Several different drugs have been shown to produce a differential cell killing effect when used in vitro, these include a 5-nitroimidazole, metronidazole;6*‘33’6a 2-nitroimidazole, R0-07-0582~~.“~‘~and the nitrofuran, nitrofurazone.‘3 Drug concentrations required for the production of cytotoxic effects against hypoxic cells range from 50 JLMdmm3 for nitrofurazone to 10 mM dme3 for metronidazole. Increasing the temperature during the drug incubation period both promoted the cell killing effects of all the drugs tested and enhanced the specificity of the drugs for hypoxic cells. Thus, although these drugs are also toxic to aerated cells at higher concentrations an increased temperature during the period of drug exposure provides the possibility of a larger therapeutic gain.15 Somewhat similar effects due to hyperthermia have been described using higher concentrations of the drug Ro-07-0582.” In vitro work relating the cytotoxicity of a given drug to the oxygen tension in the medium surrounding the cellsI has important repercussions. It has

Evidence indicating that nitroimidazole and nitrofuran radiosensitizing drugs also kill hypoxic cells directly has been reviewed.6 Noncycling and presumably hypoxic tumour cells might also be susceptible to treatment with this kind of drug.16 In more thorough investigations lmetronidazole was found to kill hypoxic cells in oitro and in viuo.6a’6There is now a considerable body of evidence obtained from in vitro systems showing that many nitro-heterocyclic drugs are much more toxic to hypoxic cells than to aerated cells.‘“~“~‘3~‘Further s in viuo work demonstrating the activity of nitroimidazole drugs against hypoxic tumour cells has now been reported.2’3’6’8As a result it is proposed that a cancer patient would receive a course of metronidazole therapy, lasting perhaps 1 week, to remove the hypoxic tumour cells followed by a standard course of radiotherapy to kill the aerated tumour cells. FACTORS AFFECTING THE CYTOTOXIC ACTION OF DRUGS IN VITRO The cytotoxic effects of nitro-heterocyclic drugs have been described using reduced growth rate,

is gratefully acknowledged. I also wish to thank my colleague Dr. Robin Willson for many helpful and stimulating discussions and Dr. J. Hopewell for critically reading the manuscript. Roche Products Ltd. kindly supplied the drug Ro-07-0582.

Acknowledgements-The author wishes to thank Dr. J. F. Fowler and the Misses S. Fyairman and S. Hill for help with the in uioo experiments re:ported herein. These were done at the C.R.C. Gray Laboratory, Mount Vernon Hospital, Northwood before the author moved to Oxford. Financial support for this work from the Cancer Research Campaign 153

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been found that the highly electron affinic drug nitrofurazone is highly toxic at oxygen concentrations well above those required for the less electron affinic drug metronidazole. This is important for two reasons. First it has been reported that many nitrobenzene compounds and Ro-07-0582 affect the rate of oxygen utilization of well aerated cells.5.‘0 Metronidazole has also been shown to have effects on the rate of oxygen utilization-a slight depression being reported at 1 mM dme3 drug concentration,’ a result not confirmed by more recent findings.” Indeed many of the drugs investigated increase the rate of oxygen utilization. If these effects occur in viuo then the oxygen concentration in normal tissues may fall to produce conditions which render the drug toxic to these tissues. If one can generalise from the work involving nitrofurazone and metronidazole then it would appear that the more active highly electron affinic drugs may be expected to be cytotoxic at relatively high oxygen tensions. Only after further work, covering a wider range of drugs, will a proper assessment of the relationship between cytotoxicity and oxygen concentration be possible. The second point is that a tissue culture plate containing cells in equilibrium with air is not a suitable model for normal tissues in the intact mammal. The actual oxygen tension of normal tissue cell populations will always be below that of a tissue culture plate in equilibrium with air and will vary from time to time accordmg to the physiological state of the animal involved. This effect of oxygen tension will make it very difficult to predict the outcome of in viuo experiments involving hyperthermia plus a drug cytotoxic for hypoxic cells, particularly with regard to unwanted side effects on normal tissues. THE MECHANISM OF ACTION OF METRONIDAZOLE AND OTHER NITROHETEROCYCLIC DRUGS AGAINST HYPOXIC CELLS The basis of the differential action of metronidazole and other similar drugs against hypoxic mammalian cells is still a matter of speculation.20 A favoured theory is that a toxic reduction product is produced by the cell particularly under hypoxic conditions. This product, or a more reactive intermediate, then reacts with vital biomolecules in the cells. It is also possible that reduction of the drug may in itself cause sufficient perturbation of cellular metabolism to cause directly, or indirectly, many of the toxic effects reported for this type of drug. It would be necessary for the relevant cellular processes to be more sensitive to interference by the drug under hypoxic conditions, and for the effect to be reversible by exposure to oxygen or drug removal. It has, for instance, been suggested that lipoic acid and pyruvate

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dehydrogenase may be interfered with by metronidazole. Is this mere speculation? Perhaps not as it was shown 20 years agoI that the nitrofuran compound furacin reversibly inhibits the formation of acetyl coenzyme A from pyruvate. Using a testis microsome preparation it was found that this effect was greatly increased in the absence of oxygen and it was reversible. The immediate effect of this inhibition of coenzyme A production must be to severely reduce the manufacture of adenosine triphosphate (ATP) by the Krebs’ cycle and to interfere with fatty acid metabolism. It is interesting to note that two of the most sensitive tissues to nitro-heterocyclic drugs are testis and brain: both these tissues are particularly dependent upon the Krebs’ cycle for the production of ATP, other tissues having a greater capacity for anaerobic glycolysis. It has been reported that hypoxic mammalian cells treated with the drug Ro-07-0582 also show some powers of recovery from damage if oxygen is admitted.ls Whether or not recovery may occur from sequelae such as peripheral neuropathy which is a side effect produced by this type of drug’2,‘8 will be dependent on how far the damage to the neuronal cells has advanced. This tacitly assumes that peripheral neuropathy results from disordered carbohydrate metabolism in the neuronal or supporting cells and that it is not a direct effect of the drug.12 Many other enzyme systems have been shown to be affected by metronidazole or similar compounds. Which, of these if any leads to cell death under aerated and hypoxic conditions is unknown at present. IN VW0 EXPERIMENTS INVESTIGATING HYPOXIC CELL CYTOXICITY When summarising the in vitro data, it is clear that the property of differential cytotoxicity for hypoxic cells is a widespread property of nitroaromatic and nitro-heterocyclic drugs. Important factors affecting the efficiency of such drugs in vitro are drug concentration, temperature and oxygen tension. The latter factor emphasizes the importance of carrying out in viuo experiments for assessing the possible clinical usefulness of this mode of attack on hypoxic tumour cells. The use of a murine solid tumour system to investigate the cytotoxic effects of drugs against hypoxic tumour cells is subject to three major difficulties. The half-life of metronidazole and Ro-07-0582 in mice is short (i.e. 2 hr or less). Thus it is difficult to maintain a steady drug concentration in the tumour over a period of days without frequent drug administration. The rate at which fresh hypoxic cells are generated in these fast growing murine tumour systems is unknown but likely to be rapid, thus many tumour cells

Chemotherapy

of hypoxic tumour cells 0 J. L. FOSTER

may escape, becoming hypoxic towards the end of a period of drug treatment. The effect of the drug is only on the hypoxic cells and it is desirable, therefore, to be able to measure the viability of this tumour cell sub-population in isolation. To some extent this latter problem has been solved by measuring the tumour response to a large single dose of X-rays given shortly after prolonged drug treatment of tumour bearing mice.6 The radiosensitivity of such tumours is largely governed by their hypoxic cell fraction. Suc.h an experiment has already been reported after repeated doses of metronidazole, (six 6-hr doses of 0.3 mg,‘g body weight) followed by graded doses of X-rays given 6-8 hr after the last drug dose.6 An enhance:d response to X-rays was shown by the dose required to cure 50% of the mice (TCDso) being reduced f:rom 6081? 135 rad for controls to 4643 f 97 for drug treated mice (ER 1.3). A similar experiment is reported here using the same tumour system and experimental procedure to examine the effects of thle drug Ro-07-0582. Six 6-hr intraperitoneal doses of 0.15 mg/g or 0.3 mg/g body weight of Ro-07-0582 were given to tumour bearing mice and followed by graded X-ray doses 6-8 hr after the last drug dose. The results are shown in Fig. 1.

\o 50

x

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8

)25

used in the metronidazole experiment were selected on the basis that the serum concentration produced by these doses in mice would be near to that known to be tolerated in man.4*pS’pAt the time the experiment with Ro-07-0582 was started some uncertainty existed regarding the human tolerance of this drug. However, it is now clear that the lower dose used in the above experiment produces serum concentrations

(120 pg/ml) above that tolerated by man in repeated doses.‘* Other reports exist of in viva experiments on tumour bearing mice investigating the effect of Ro-070582 either alone3 or in combination with hypertherrnia’.’ on hypoxic tumour cells. Although positive effects were demonstrated the experiments were in all cases using very high drug doses (1 mg/g body weight or more) and in two cases2’3 the tumour system used was complicated by transplantation immunity problems. For these reasons and those outlined at the beginning of this section it would at present be premature to draw firm conclusions from the in uivo data as to the relative drug efficiency or the possible clinical usefulness of such a treatment modality for cancer patients. Fortunately some of the difficulties met with in these animal experiments will be less of a problem in man. First, it is known that the half-life of metronidazole and Ro-07-0582 in man is of the order of 10 hr.4*7Thus it is reasonably easy to maintain steady drug concentrations over an extended period of time. Second, most human tumours have volume doubling times of the order of weeks rather than days, so it is to be expected that the rate at which hypoxic cells are generated will be less than in the murine tumours used in the work referred to above. Consequently the risk that hypoxic cells will be generated at the end of a course of drug treatment will be less. In any case this could be guarded against by continuing the drug treatment into the early part of a course of fractionated radiotherapy. The use of “immunity free” slowly growing murine tumour involving the prolonged adsystems, ministration of clinically realistic drug dosages in future work is necessary before an assessment of the possible clinical values of this new treatment modality can be made. This must also include an assessment of normal tissue damage as a vigorous course of radiotherapy will still be required to eliminate the aerated population of tumour cells. Finally it should be recognised that many of the phenomena described above have only been investigated using one particular drug. It should not be assumed that such effects are universal for the nitroheterocyclic drugs as a whole because they have been juxtaposed to form the basis of a discussion.

l-._ld? x

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O40

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Fig. 1. The percentage of tumour controlled as a function of X-ray dose for transplanted anaplastic carcinomata in CBA/Ht mice. 0, TCDso for mice treated with metronidazole, 0.3 mg/g; 0, mice given 0.3 mg/g Ro-07-0582; A,

mice given 0.15 mg/g Ro-07-0582; III, control mice treated concurrently with metronidazole treated mice; X, controls.

They indicate a reduction in the TCDso from 5953 * 93 rad for concurrent controls to 5722 ? 119 rad and 5379297 rad for the 0.15 mglg and 0.3 mg/g dose groups (ER 1.04 and 1.11) respectively. The doses of the drug Ro-07-0582 used in this experiment and that

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REFERENCES J.C., Foster, J.L., Willson, R.L.: Radiosensitization of hypoxic cells by metronidazole (Flagyl). (Abstract). Br. J. Radio!. 46: 648, 1973. Bleehen, N.M., Honess, D., Morgan, J.: The interaction of hyperthermia and the hypoxic cell sensitizer-Ro-070582 on the EMT6 mouse tumour. Br. J. Cancer 35: 299-306, 1976. Brown, J.M.: Select killing of hypoxic tumour cells by Ro-07-0582. Radiat. Res. in press. Deutsch, G., Foster, J.L., Ings, R.M.J., McFadzean, J.A.: Human studies with high dose metronidazole: a non-toxic radiosensitizer of hypoxic cells. Br. J. Cancer 31: 75-80, 1975. Durand, R.E., Biaglow, J.E., Sutherland, R.M.: Hypoxic radiosensitizers and cellular respiration. Br. J. Radio!. 49: 567-568, 1976. Foster, J.L., Conroy, P.J., Searle, A.J., Willson, R.L.: Metronidazole (Flagyl): Characterization as a cytotoxic drug specific for hypoxic tumour cells. Br. J. Cancer 33: 485490, 1976. Foster, J.L., Flockhart, I.R., Dische, S., Gray, A., Lenox-Smith, I., Smithen, C.E.: Serum concentration measurements in man of the radiosensitizer Ro-07-0582: Some preliminary results. Br. J. Cancer 31: 679-683, 1975. George, K.C., Hirst, D.G., McNally, N.J.: The effect of hyperthermia on the cytotoxicity of the radiosensitizer Ro-07-0582 on a solid mouse tumour. Br. J. Cancer 35: 372-375, 1976. Gray, A.J., Dische, S., Adams, G.E., Flockhart, I.R., Foster, J.L.: Clinical testing of the radiosensitizer Ro07-0582. I Dose tolerance, serum and tumour concentrations. Clin. Radio/. 27: 151-157, 1976. Hall, E.J., Biaglow, J.: Ro-07-0582 as a radiosensitizer and cytotoxic agent. ht. J. Radiat. Oncol. to be published. Hall, E.J. Roizin-Towle, L.: Hypoxic sensitizers:

1. Asquith,

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3. 4.

5.

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11.

Radiobiological studies at the cellular level. Radiology 117: 453457, 1975. 12. LeQuesne, P.M.: Peripheral neuropathy. In Recent Advances in Medicine 16th Edn, ed. by Baron, D.N., Compston, N., Dawson, A.M. Edinburgh, Churchill Livingstone, 1973, pp. 31-56. 13. Mohindra, J.E., Rauth, A.M.: Increased cell killing by metronidazole and nitro-furazone of hypoxic compared to aerobic mammalian cells. Cancer Res. 36: 930-936, 1976. 14. Paul, M.F., Paul, H.E., Kopko, F., Bryson, M.J., Harrington, C.: Inhibition by Furacin of citrate formation in testis preparations. J. Biol. Chem. 206: 491497, 1954. 15. 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, 1976. 16. Sutherland, R.M.: Selective chemotherapy of noncycling cells in an in vitro tumour model. Cancer Res. 34: 3501-3504, 1974. 17. Urtasun, R., Band, P., Chapman, J.D., Feldstein, M.L., Mielke, B., Fryer, C.: Radiation and high dose metronidazole (Flagyl) in supratentorial glioblastomas. N. Engl. J. Med. 294: 1364-1357, 1976. 18. Urtasun, R.C., Band, P., Chapman, J.D., Rabin, H.R., Wilson, A.P., Fryer, C.G.: Clinical Phase I study of the hypoxic cell radiosensitizer Ro-07-0582, a 2-nitroimidazole derivative. Radiology 122: 801-804, 1977. 19. Urtasun, R.C., Chapman, J.D., Band, P., Rabin, H., Fryer, C., Strumwind, J.: Phase I study of high dose metronidazole: A specific in vivo and in oitro radiosensitizer of hypoxic cells. Radiology 117: 129133, 1975. 20. Willson, R.L.: Metronidazole (Flagyl) in cancer radiotherapy. A historical introduction. Proc. Conf. Metronidazole Montreal, Canada, 1976, to be published.

Differential cytotoxic effects of metronidazole and other nitro-heterocyclic drugs against hypoxic tumour cells.

Inr. I. Radialion 0 Oncology Rio/. Phys., 1978, Vol. 4. PP. 153-156. Pergamon Press. Printed in the U.S.A. Radiation Sensitizers for Hypoxic C...
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