Br. J. Cancer (1978) 37, Suppl. III, 255
THE EFFECT OF MISONIDAZOLE IN COMBINATION WITH RADIATION DOSE FRACTIONATION R. P. HILL AND R. S. BUSH From the Ontario Cancer Institute incorporating The Princess Margaret Hospital, 500 Sherbourne Street, Toronto, Ontario, Canada M4X 1K9
Summary.-Single doses or multiple fractions of 2, 3, 5 or 10 Gy were given daily to KHT Sarcomas, growing in C3H mice, in combination with a misonidazole dose of 05 mg/g body weight administered 30-40 min before each radiation dose. Cell survival assays were performed on groups of tumours after different total doses to determine tumour cell survival curves for each fractionation schedule. The results indicate that misonidazole is effective in sensitizing the tumours to single doses and to large dose fractions, but that the degree of sensitization declines with fraction size such that there is no difference between the survival curves obtained for 2 Gy fractions given with or without prior drug treatment. Comparison of iso-effect curves, derived from the data, with those for normal skin suggests that, even though misonidazole increases the effect of the radiation on the tumour when large dose fractions are used, the small dose fractions probably still give a better "therapeutic ratio".
MISONIDAZOLE has been shown experimentally to be an effective sensitizer of hypoxic cells both in vitro and in vivo, and a number of trials are presently being planned or are under way investigating its clinical usefulness. Most of the in vivo animal tumour studies evaluating the radiation sensitizing ability of this drug have been done using single doses of radiation. Information concerning its value in combination with multiple dose fractions is limited (Fowler, Adams & Denekamp, 1976). If this drug is to make a contribution to the tumour control rate achieved by radiation therapy then it is almost certain that it will have to be combined with present therapy regimes, since most clinicians will be unwilling to alter a treatment already successful in a proportion of patients. The present study was undertaken, therefore, to investigate the effect, in an animal tumour model system, of combining misonidazole with multiple daily doses of X-irradiation. The study is still in progress and the results presently available are given in this paper.
MATERIALS AND METHODS
KHT Sarcomas growing in the left hind leg of male C3H mice were used throughout the experiments. Treatments were started when the tumours reached a size of 035-0 45 g. The tumour response was determined in terms of tumour cell survival using either an in vivo lung colony assay (Hill and Bush, 1969) or an in vitro agar colony assay (Thomson and Rauth, 1974), which give equivalent results. Groups of mice (2-4 per group) with tumours of equal size were selected on Day zero. One group was kept as a control while the others were given their first radiation dose fraction (with or without drug) on Day zero, with subsequent treatments being given daily. After different numbers of dose fractions and, therefore, different total doses, the irradiated groups were sacrificed and the number of surviving cells per tumour assayed. The unirradiated control group was assayed on Day zero and the number of colony-forming cells per tumour determined. The total number of colony-forming cells per tumour for each treated group was then divided by the number of colony-forming cells per tumour in the control group to give a tumour surviving fraction. This fraction should be
256
R. P. HILL AND R. S. BUSH
unaffected by any loss of radiation killed cells which may have occurred during the fractionated treatment. All irradiations were performed using a double headed 100 kVp X-ray unit giving a dose rate of 11-4 Gy/min. The tumour-bearing mice were unanaesthetized and confined by a specially constructed jig (Hill and Bush, 1977) during the radiation treatment. The mice breathed warm (24°C), humidified air (flow-rate 3-4 1/min) for 5 min before and during each radiation dose. Misonidazole (donated byDr C. E. Smithen, Roche Products Ltd, Welwyn Garden City, Herts., England) was dissolved in phosphate buffered saline, 1-2 h before use, at a concentration which allowed 0 05 ml to be injected per g body weight. The drug was injected i.p. 30-40 min before each radiation treatment. Mice receiving radiation but no drug treatment were not injected with saline only.
.Iz
:Ez
0 RESULTS AND DISCUSSION
Initial experiments were done to determine the maximum level of drug which would be tolerated by the mice when given as a daily injection for up to 20 treatments. The results indicated that this level was 0-5 mg/g body weight and this concentration has been used throughout the radiation dose fractionation experiments although a small amount of data for 0-8 mg/g has also been obtained for small numbers of 5 Gy fractions. Single doses or multiple doses of 2, 3, 5 or 10 Gy were given to the tumours with or without misonidazole and the results are shown in Fig. 1, 2 and 3. For the 5 Gy fractions there appears to be little difference between the two drug doses used (0-5 mg/g and 0-8 mg/g) particularly at large total doses of radiation. The lines through the data have been drawn by eye and the results indicate that there is a D.M.F. of about 1-5 for single doses of radiation under these conditions. This value is slightly smaller than that found previously for this tumour (Rauth et al., 1978) and is on the low side of the range of values found for other animal tumours (Fowler et al., 1976). The D.M.F. is little changed when 10 Gy fractions are given
5
10
15 Dose (Gy)
20
25
30
FIG. 1.-Single dose survival curves for KHT Sarcomas irradiated in the presence (squares) or absence (circles) of misonidazole (0-5 mg/g, 30-40 min before irradiation). The points are mean values of at least 2 independent survival determinations with standard errors shown on points combining 3 or more survival determinations.
but as the fraction size becomes smaller the D.M.F. declines so that for 2 Gy fractions it is essentially 1-0. This decline in the D.M.F. with fraction size is not unexpected since the KHT Sarcoma is a tumour which is known to reoxygenate. Both Fowler et al. (1976) and van Putten and Smink (1976) have observed significant sensitization with large radiation dose fractions but there was only a small effect when fractions as small as 3 Gy were used. A number of similar results are reported in the present conference. All the dose fractionation results can be compared by determining an isosurvival (isoeffect) curve for the fractionated treatments. This has been done by determining the total dose required for each different fractionation regime to produce a survival level of 3 xlO-4 and the curves for drug and no drug treatment are shown in Fig. 4 as the solid lines. The points for
MISONIDAZOLE WITH RADIATION DOSE FRACTIONATION
r13
0
Total dose (Gy) FIG. 2.-Survival curves for KHT Sarcomas given multiple daily doses of 10 Gy (A) or 5 Gy (B) in the presence (squares 0 5 mg/g and triangles 0-8 mg/g) or absence (circles) of misonidazole, 30-40 min before each dose of irradiation. Each point represents an independent survival determination.
0
10
20
30
40
Total dose (Gy) FIG. 3.-Survival curves for KHT Sarcomas given multiple daily doses of 3 Gy (A) or 2 Gy (B) in the presence (squares) or absence (circles) of misonidazole (0-5 mg/g, 30-40 min before each dose of irradiation). Each point represents an independent survival determination. 18
257
258
R. P. HILL AND R. S. BUSH
100'
8060 -
'
'
Skin response
'
'
'
----
40
q,} 30
SD
G
B 20< 15
8 10 5 15 2030 3 No. of fractions FIG. 4.-Isoeffect curves for the KHT Sarcoma irradiated in the presence (squares) or absence (circles) of misonidazole (0-5 mg/g), derived at a survival level of 3 x 10 -4 from the results in Fig. 1, 2 and 3. The broken line representing skin response is taken from Fowler (1971).
2
the large dose fractions have been plotted as part fractions because each additional fraction results in such a large change in survival that to correct them to the nearest integral number of fractions would distort the isoeffect relationship. The broken line is an isoeffect curve for normal skin taken from the literature (Fowler, 1971). It is immediately apparent that the tumour curve for radiation alone is shallower than the curve for normal skin. Even for the tumours in radiation and drug treated animals the curve is less steep than that for the normal tissue. This suggests that, although misonidazole gives significant sensitization for single doses and large dose fractions, the small dose fractions still give a better "therapeutic ratio", and there is little benefit from using the drug. It should be noted, however, that we have so far only reached a total dose of about 30 Gy with such small
dose fractions and it is possible that a small sensitizing effect will be observed at higher total doses. For groups of patients, such as those with carcinoma of the cervix, in which significant cure-rates are already being obtained with conventional treatment, even a sensitizing effect equivalent to changing the surviving population of cells by a factor of 2 could lead to a significant change in the tumour control rate. Further studies are in progress to extend the data for 2 and 3 Gy fractions to lower levels of survival. The authors are pleased to acknowledge the financial support of the Ontario Cancer Treatment and Research Foundation and the National Cancer Institute of Canada, and the technical assistance of Mrs R. Sanders. REFERENCES FOWLER, J. F. (1971) Experimental Animal Results Relating to Time-dose Relationships in Radiotherapy and the "Ret" Concept. Br. J. Radiol., 44, 81. FOWLER, J. F., ADAMS, G. E. & DENEKAMP, J. (1976) Radiosensitizers of Hypoxic Cells in Solid Tumours. Cancer Preat. Rev., 3, 227. HILL, R. P. & BUSH, R. S. (1969) A Lung Colony Assay to Determine the Radiosensitivity of the Cells of a Solid Tumour. Int. J. Radiat. Biol., 15, 435. HiLL, R. P. & BUSH, R. S. (1977) Dose Fractionation Studies with a Murine Sarcoma under Conditions of Air or Carbogen (95%02+5%CO2) Breathing. Int. J. Radiat. Oncol. Biol. Phy8., 2, 913. RAUTH, A. M., CHIN, J., MARCHOW, L. & PACIGA, J. (1978) Testing of Hypoxic Cell Radiosensitizers In vivo. Br. J. Cancer, 37, Suppl. III, 202. THOMSON, J. E. & RAUTH, A. M. (1974) An In vitro Assay to Measure the Viability of KHT Tumor Cells not Previously Exposed to Culture Conditions. Radiat. Re8., 58, 262. VAN PIJTTEN, L. M. & S1IiNK, T. (1976) Effect of Ro-07-0582 and Radiation on a Poorly Reoxygenating Mouse Osteosarcoma. In Modification of Radiosensitivity of Biological System8. Vienna: IAEA, 179; and private communication.
THE EFFECT OF MISONIDAZOLE IN COMBINATION WITH RADIATION DOSE FRACTIONATION R. P. HILL AND R. S. BUSH From the Ontario Cancer Institute incorporating The Princess Margaret Hospital, 500 Sherbourne Street, Toronto, Ontario, Canada M4X 1K9
Summary.-Single doses or multiple fractions of 2, 3, 5 or 10 Gy were given daily to KHT Sarcomas, growing in C3H mice, in combination with a misonidazole dose of 05 mg/g body weight administered 30-40 min before each radiation dose. Cell survival assays were performed on groups of tumours after different total doses to determine tumour cell survival curves for each fractionation schedule. The results indicate that misonidazole is effective in sensitizing the tumours to single doses and to large dose fractions, but that the degree of sensitization declines with fraction size such that there is no difference between the survival curves obtained for 2 Gy fractions given with or without prior drug treatment. Comparison of iso-effect curves, derived from the data, with those for normal skin suggests that, even though misonidazole increases the effect of the radiation on the tumour when large dose fractions are used, the small dose fractions probably still give a better "therapeutic ratio".
MISONIDAZOLE has been shown experimentally to be an effective sensitizer of hypoxic cells both in vitro and in vivo, and a number of trials are presently being planned or are under way investigating its clinical usefulness. Most of the in vivo animal tumour studies evaluating the radiation sensitizing ability of this drug have been done using single doses of radiation. Information concerning its value in combination with multiple dose fractions is limited (Fowler, Adams & Denekamp, 1976). If this drug is to make a contribution to the tumour control rate achieved by radiation therapy then it is almost certain that it will have to be combined with present therapy regimes, since most clinicians will be unwilling to alter a treatment already successful in a proportion of patients. The present study was undertaken, therefore, to investigate the effect, in an animal tumour model system, of combining misonidazole with multiple daily doses of X-irradiation. The study is still in progress and the results presently available are given in this paper.
MATERIALS AND METHODS
KHT Sarcomas growing in the left hind leg of male C3H mice were used throughout the experiments. Treatments were started when the tumours reached a size of 035-0 45 g. The tumour response was determined in terms of tumour cell survival using either an in vivo lung colony assay (Hill and Bush, 1969) or an in vitro agar colony assay (Thomson and Rauth, 1974), which give equivalent results. Groups of mice (2-4 per group) with tumours of equal size were selected on Day zero. One group was kept as a control while the others were given their first radiation dose fraction (with or without drug) on Day zero, with subsequent treatments being given daily. After different numbers of dose fractions and, therefore, different total doses, the irradiated groups were sacrificed and the number of surviving cells per tumour assayed. The unirradiated control group was assayed on Day zero and the number of colony-forming cells per tumour determined. The total number of colony-forming cells per tumour for each treated group was then divided by the number of colony-forming cells per tumour in the control group to give a tumour surviving fraction. This fraction should be
256
R. P. HILL AND R. S. BUSH
unaffected by any loss of radiation killed cells which may have occurred during the fractionated treatment. All irradiations were performed using a double headed 100 kVp X-ray unit giving a dose rate of 11-4 Gy/min. The tumour-bearing mice were unanaesthetized and confined by a specially constructed jig (Hill and Bush, 1977) during the radiation treatment. The mice breathed warm (24°C), humidified air (flow-rate 3-4 1/min) for 5 min before and during each radiation dose. Misonidazole (donated byDr C. E. Smithen, Roche Products Ltd, Welwyn Garden City, Herts., England) was dissolved in phosphate buffered saline, 1-2 h before use, at a concentration which allowed 0 05 ml to be injected per g body weight. The drug was injected i.p. 30-40 min before each radiation treatment. Mice receiving radiation but no drug treatment were not injected with saline only.
.Iz
:Ez
0 RESULTS AND DISCUSSION
Initial experiments were done to determine the maximum level of drug which would be tolerated by the mice when given as a daily injection for up to 20 treatments. The results indicated that this level was 0-5 mg/g body weight and this concentration has been used throughout the radiation dose fractionation experiments although a small amount of data for 0-8 mg/g has also been obtained for small numbers of 5 Gy fractions. Single doses or multiple doses of 2, 3, 5 or 10 Gy were given to the tumours with or without misonidazole and the results are shown in Fig. 1, 2 and 3. For the 5 Gy fractions there appears to be little difference between the two drug doses used (0-5 mg/g and 0-8 mg/g) particularly at large total doses of radiation. The lines through the data have been drawn by eye and the results indicate that there is a D.M.F. of about 1-5 for single doses of radiation under these conditions. This value is slightly smaller than that found previously for this tumour (Rauth et al., 1978) and is on the low side of the range of values found for other animal tumours (Fowler et al., 1976). The D.M.F. is little changed when 10 Gy fractions are given
5
10
15 Dose (Gy)
20
25
30
FIG. 1.-Single dose survival curves for KHT Sarcomas irradiated in the presence (squares) or absence (circles) of misonidazole (0-5 mg/g, 30-40 min before irradiation). The points are mean values of at least 2 independent survival determinations with standard errors shown on points combining 3 or more survival determinations.
but as the fraction size becomes smaller the D.M.F. declines so that for 2 Gy fractions it is essentially 1-0. This decline in the D.M.F. with fraction size is not unexpected since the KHT Sarcoma is a tumour which is known to reoxygenate. Both Fowler et al. (1976) and van Putten and Smink (1976) have observed significant sensitization with large radiation dose fractions but there was only a small effect when fractions as small as 3 Gy were used. A number of similar results are reported in the present conference. All the dose fractionation results can be compared by determining an isosurvival (isoeffect) curve for the fractionated treatments. This has been done by determining the total dose required for each different fractionation regime to produce a survival level of 3 xlO-4 and the curves for drug and no drug treatment are shown in Fig. 4 as the solid lines. The points for
MISONIDAZOLE WITH RADIATION DOSE FRACTIONATION
r13
0
Total dose (Gy) FIG. 2.-Survival curves for KHT Sarcomas given multiple daily doses of 10 Gy (A) or 5 Gy (B) in the presence (squares 0 5 mg/g and triangles 0-8 mg/g) or absence (circles) of misonidazole, 30-40 min before each dose of irradiation. Each point represents an independent survival determination.
0
10
20
30
40
Total dose (Gy) FIG. 3.-Survival curves for KHT Sarcomas given multiple daily doses of 3 Gy (A) or 2 Gy (B) in the presence (squares) or absence (circles) of misonidazole (0-5 mg/g, 30-40 min before each dose of irradiation). Each point represents an independent survival determination. 18
257
258
R. P. HILL AND R. S. BUSH
100'
8060 -
'
'
Skin response
'
'
'
----
40
q,} 30
SD
G
B 20< 15
8 10 5 15 2030 3 No. of fractions FIG. 4.-Isoeffect curves for the KHT Sarcoma irradiated in the presence (squares) or absence (circles) of misonidazole (0-5 mg/g), derived at a survival level of 3 x 10 -4 from the results in Fig. 1, 2 and 3. The broken line representing skin response is taken from Fowler (1971).
2
the large dose fractions have been plotted as part fractions because each additional fraction results in such a large change in survival that to correct them to the nearest integral number of fractions would distort the isoeffect relationship. The broken line is an isoeffect curve for normal skin taken from the literature (Fowler, 1971). It is immediately apparent that the tumour curve for radiation alone is shallower than the curve for normal skin. Even for the tumours in radiation and drug treated animals the curve is less steep than that for the normal tissue. This suggests that, although misonidazole gives significant sensitization for single doses and large dose fractions, the small dose fractions still give a better "therapeutic ratio", and there is little benefit from using the drug. It should be noted, however, that we have so far only reached a total dose of about 30 Gy with such small
dose fractions and it is possible that a small sensitizing effect will be observed at higher total doses. For groups of patients, such as those with carcinoma of the cervix, in which significant cure-rates are already being obtained with conventional treatment, even a sensitizing effect equivalent to changing the surviving population of cells by a factor of 2 could lead to a significant change in the tumour control rate. Further studies are in progress to extend the data for 2 and 3 Gy fractions to lower levels of survival. The authors are pleased to acknowledge the financial support of the Ontario Cancer Treatment and Research Foundation and the National Cancer Institute of Canada, and the technical assistance of Mrs R. Sanders. REFERENCES FOWLER, J. F. (1971) Experimental Animal Results Relating to Time-dose Relationships in Radiotherapy and the "Ret" Concept. Br. J. Radiol., 44, 81. FOWLER, J. F., ADAMS, G. E. & DENEKAMP, J. (1976) Radiosensitizers of Hypoxic Cells in Solid Tumours. Cancer Preat. Rev., 3, 227. HILL, R. P. & BUSH, R. S. (1969) A Lung Colony Assay to Determine the Radiosensitivity of the Cells of a Solid Tumour. Int. J. Radiat. Biol., 15, 435. HiLL, R. P. & BUSH, R. S. (1977) Dose Fractionation Studies with a Murine Sarcoma under Conditions of Air or Carbogen (95%02+5%CO2) Breathing. Int. J. Radiat. Oncol. Biol. Phy8., 2, 913. RAUTH, A. M., CHIN, J., MARCHOW, L. & PACIGA, J. (1978) Testing of Hypoxic Cell Radiosensitizers In vivo. Br. J. Cancer, 37, Suppl. III, 202. THOMSON, J. E. & RAUTH, A. M. (1974) An In vitro Assay to Measure the Viability of KHT Tumor Cells not Previously Exposed to Culture Conditions. Radiat. Re8., 58, 262. VAN PIJTTEN, L. M. & S1IiNK, T. (1976) Effect of Ro-07-0582 and Radiation on a Poorly Reoxygenating Mouse Osteosarcoma. In Modification of Radiosensitivity of Biological System8. Vienna: IAEA, 179; and private communication.
