hf. .I. Radration Oncology Bid. Ph?s. Vol. 21, Prmted I” the U.S.A All nghts reserved
pp.1231-1234 Copyright
0360.3016191 $3.00 + .OO Q 1991 Pergamon Press plc
??Biology Original Contribution
IN VZVO RADIOSENSITIZING ACTIVITY OF A NEW FLUORINATED HYPOXIC CELL RADIOSENSITIZER, KU-2285 IN COMBINATION WITH RADIATION DOSE FRACTIONATION KEISUKE SASAI, M.D.,’
MASATO FUSHIKI, M.D.
HIROYUKI IWAI, M.
SC.,~
,2 YUTAKA YUKAWA,
YUTA SHIBAMOTO, M.D.,’
MASAJI TAKAHASHI,
M.D.’
R. T. ,l SUMIO SUYAMA, B. SC. ,2
SEI-ICHI NISHIMOTO, PH.D.,~
AND MITSUYUKI ABE, M.D.’
‘Department of Radiology, Faculty of Medicine, 4Department of Hydrocarbon Chemistry, Faculty of Engineering, 5Chest Disease Research Institute, Kyoto University, Kyoto 606; ‘Department of Radiology, Shiga University of Medical Science, Otsu 520-21; and 3Chemical Division,
Daikin Industries,
Ltd., Settsu 566, Japan
Since most clinical radiotherapy is given as multiple small irradiation fractions, the present study was undertaken to test the in vivo radiosensitizing activity of a new hypoxic cell radiosensitizer, KU-2285 in combination with radiation dose fractionation. Radiosensitizing activity was measured by a growth delay assay using a transplanted mammary tumor in C3H/He mice, and by an in vivo-in vitro assay using the SCC VII tumor. KU-2285 was injected intraperitoneally 30 min before irradiation in all experiments. The in vivo-in vitro assay using SCC VII tumors showed that 12.5 kg/g of KU-2285 sensitized the tumors to irradiation (5 Gylfr x 5 fr/48 hr or 6 Gy/fr x 3 fr/48 hr). KU-2285 also sensitized the transplanted mammary tumors to fractionated irradiation. We concluded that KU-2285 was able to sensitize two different murine tumors when given in combination with radiation dose fractionation. KU-2285, Hypoxic cell radiosensitizer,
Fractionated
irradiation.
macokinetic properties of KU-2285 are summarized in Table 1 in comparison with etanidazole. The reduction potential of KU-2285 is -0.96 V, and its octanol/water partition coefficient is 0.25. The acute toxicity measured as the LD,,,, (drug dose necessary to kill 50% of the tested mice within seven days) in 8-week-old female C3H/HeJ mice was found to be 2.4 g/kg (i.v.), 2.1 g/kg (i.p.), and 4.25 g/kg (p.0.) for KU-2285, whereas it was 4.75 g/kg (i.v.) for etanidazole (11). KU-2285 was dissolved in physiological saline and given intraperitoneally 30 min before irradiation in all experiments in this study on the basis of the results of previous sensitization experiments (11).
INTRODUCTION
The in vivo radiosensitizing activity with single-dose irradiation, acute toxicity, and pharmacokinetic properties of a new fluorinated hypoxic cell radiosensitizer, KU-2285, were described previously in comparison with etanidazole (11). Since most clinical radiation treatment is given as multiple small doses of irradiation, the present study was undertaken to test the in vivo radiosensitizing activity of KU-2285 in combination with radiation dose fractionation. The radiosensitizing activity was measured by a growth delay assay using a transplanted mammary tumor in C3H/He mice, and by an in Co-in vitro assay using SCC VII tumor.
MATERIALS
AND
Animals In all experiments, we used 8- or 9-week-old female C3H/He mice obtained from Japan SLC (Hamamatsu, Japan) or Clea Japan Inc. (Tokyo, Japan).
METHODS
Radiosensitizer KU-2285 has the structure shown in Figure 1 and was developed by our group (4, 9, 11, 13). This compound has a very similar side chain to etanidazole, but it has a CF, structure in its side chain. The physiochemical and phar-
Sensitizer Tumor assay (7, mice, and
Presented at the 32nd Annual Meeting of the American Society for Therapeutic Radiology and Oncology, Miami Beach, FL, 15-20 October 1990. Reprint requests to: Keisuke Sasai, M.D., Department of Ra-
testing system response was measured by an in vivo-in vitro 8, 11, 12) using the SCC VII tumor in C3H/He by a growth delay assay (5-7,11) using a trans-
diology, Faculty of Medicine, Kyoto University, oto 606, Japan. Accepted for publication 26 April 1991.
1231
Sakyo-ku,
Ky-
1232
I. J. Radiation Oncology 0 Biology 0 Physics
CHZCONHCH~
October 1991, Volume 21, Number 5
cH?cF> CONHCHICH20H
CH? OH
KU2285
Etanidazole
IL
Is) la3 .-c
Fig. 1. Structural formula of KU-2285.
planted mammary tumor in C3H/He mice. SCC VII tumor cells were subcutaneously inoculated into both hind legs of each mouse. In the growth delay assay, the transplanted mammary tumor in its third transplant generation was subcutaneously inoculated into the right hind legs of the C3H/He mice. Radiation treatment was started when the SCC VII tumors reached a size of 500 mm3 and the transplanted mammary tumor reached a size of 300 mm3. In the growth delay assay, tumor volume was estimated by caliper measurements on every day or alternate day of the three perpendicular diameters assuming an ellipsoid shape as described previously (7, 8, 10, 11). Irradiation Irradiation was carried out using 10 MV X rays generated by a linear accelerator at a dose rate of 5.6 Gy/min, as described previously (7, 8, 10, 11). For the in vivo-in vitro assay, the mice received whole body irradiation, whereas in the growth delay assay, only the tumor-bearing leg was irradiated. Mice were not restrained for whole body irradiation, but they were fixed with adhesive tape with their limbs extended without anesthesia for local irradiation. Irradiation of 5 fractions of 5 Gy each (every 12 hr) or 3 fractions of 6 Gy each (every 24 hr) was given for the SCC VII tumors, and 5 fractions of 6, 8, 10 or 12 Gy each (every 24 hr) were given for the transplanted mammary tumors. Data analysis Statistical differences in growth delay or tumor cell survival in the in vivo-in vitro assay were assessed using the
Table 1. Physiochemical
and pharmacokinetic
data for KU-2285
and etanidazole Comoound P* Ey;t AUC in mice (kg/g Brain Sciatic nerve
Etanidazole
KU-2285
0.040 - 1.05
0.25 -0.96
280 6200
690 5700
x min)$
*P: Partition coefficient in octanol/water. tERED: Reduction potential vs. Ag/Ag+ in N,N-dimethylformaidey* $Sasai, K. et al. Int. J. Radiat. Biol. 1990. AUC: Area under the concentration-time curve when 200 )*g/g of each compound was injected.
i
1p-i rftr 0
12.5 Drug
-1
25
50
Dose
(w
100
200
/ 9)
Fig. 2. SCC VII tumor cell survival following 3 fractions of 6 Gy each (every 24 hr) plotted as a function of the dose of KU-2285 given to the tumor-bearing animals prior to each radiation treatment. Error bars are 1 S. E. from more than four experiments.
Student’s t test. All statistical analysis of tumor cell survival was carried out using the logarithm values of the survival data.
RESULTS In vivo-in vitro assay The radiosensitizing ability of the drug was studied as a function of the dose given to the tumor-bearing mice. The results for irradiation with 6 Gy fractions (every 24 hours) are shown in Figure 2. The surviving fraction of the tumor cells in the mice pretreated with even 12.5 pg/g of KU2285 was significantly lower than that in mice pretreated with physiological saline (p < 0.05). A similar series of experiments was performed using 5 fractions of 5 Gy each (every 12 hr), and the results are shown in Figure 3. Pretreatment with 12.5 p,g/g of KU-2285 also sensitized the tumor to irradiation in this experiment @ < 0.05). Transplantable mammary tumor The growth curves of tumors after 5 fractions of 6 Gy each are shown in Figure 4. Each group consisted of at least eight mice. The growth curves for tumors in mice without irradiation are included for comparison. Time zero represents the day on which the irradiation was started. When the irradiated and regrowing tumors returned to their initial volume (growth delay time, GDT) (5, 6), the difference in tumor growth between the groups was compared. Tumor growth in the group treated with 200 Fg/g of KU2285 plus irradiation was significantly delayed compared to that in the group treated with physiological saline plus irradiation. Tumor growth in the group given 200 Fg/g of KU-2285 without irradiation was not different from that in mice treated with physiological saline without irradiation. Figure 5 shows GDT as a function of the dose fractionated
Fractionated irradiation plus KU-2285
??K.
1233
SASAIef al
5Gyx5fr (q 12hrs)
+
*
* +* ;
L
rid0
12.5 25 Drug
50
Dose
*
*
100 200
(c1g / 9)
Fig. 3. SCC VII tumor cell survival following 5 fractions of 5 Gy each (every 12 hr) plotted as a function of the KU-2285 dose given to the tumor-bearing animals prior to each radiation treatment. Error bars are 1 S. E. from more than four experiments.
d
0
I
I
I
I
I
I
I
2
4
6
8
10
12
14
Gy/Fr
of irradiation. Pretreatment with 200 kg/g of KU-2285 significantly sensitized the tumors to fractionated irradiation. DISCUSSION
To develop new hypoxic cell radiosensitizers, we have incorporated fluorine atoms into the side chain of 2-nitro-
Fig. 5. Dose-response curves for transplanted mammary tumors, that is, growth delay time as a function of the radiation dose. Error bars are 1 S. E. from more than eight mice. Open circles: 200 kg/g of KU-2285 was administered intraperitoneally 30 min before irradiation. Closed circles: physiological saline was given intraperitoneally 30 min before irradiation. Sr: Four of the 8 mice had no tumor regrowth. *: p < 0.05 **: p < 0.01
6 5
I
I
I
20
25
30
1.1111
Days Fig. 4. Tumor growth curves for transplanted mammary tumors as a function of time after 5 fractions of 6 Gy each (every 24 hr). Error bars are 1 S. E. from more than eight mice. Open circles: 200 pg/g of KU-2285 was given intraperitoneally 30 min before each dose of radiation. Closed circles: physiological saline was given intraperitoneally 30 min before each dose of irradiation. Open diamonds: 200 pg/g of KU-2285 was given intraperitoneally every day for 5 days without irradiation. Closed diamonds: physiological saline was given intraperitoneally every day for 5 days without irradiation. Arrowheads indicate the time of irradiation.
imidazole, 3-nitro-1,2,4_triazole, and 4-nitro-1,2,3-triazole derivatives to modify their tumor affinity and thereby their radiosensitizing activity (4, 13). Of the resulting compounds that were produced, KU-2285 is considered to be the most useful as a hypoxic cell radiosensitizer (13). Although its side chain is similar to that of etanidazole, KU-2285 has a much higher octanol/water partition coefficient than etanidazole, and is similar in this respect to misonidazole. Thus, incorporation of a -CF,group into the side chain structure of -CH,CONHCH,CH,OH belonging to etanidazole has made the compound more lipophilic (11). The in vivo radiosensitizing activity of KU-2285 was previously found to be similar to or a little higher than that of etanidazole at the same dose when tested by an in vivo-in vitro assay, a growth delay assay, and a tumor control assay using SCC VII or transplanted mammary tumors in C3H/He mice (11). Although the radiosensitizing activity of etanidazole was reduced when it was administered orally, there was no significant difference in its activity when given intravenously, intraperitoneally, or orally. In this study, KU-2285 was found to be able to sensitize both SCC VII and transplanted mammary tumors to fractionated irradiation in C3HkIe mice. Although the acute toxicity of KU-2285 is higher than that of etanidazole, it is lower than that of misonidazole (11). Etanidazole was given as a single dose of 2 g/m2 (1, 2) and
1234 misonidazole
I. J. Radiation Oncology was
administered
??Biology
as a single
than 0.4 g/m2 (14) in their respective suggests that the administration doses
dose
0 Physics
October 1991, Volume 21, Number 5
of more
that for KU-2285
clinical trials. This of over 0.4 g/m2 of
KU-2285 should be easily tolerated in clinical use. Even as little as 12.5 kg/g of KU-2285 could significantly sensitize SCC VII tumors to fractionated irradiation in this study. As 12.5 kg/g is almost equal to a dose of 0.4 g/m2 in humans, there seems to be a good possibility that KU-2285 could be clinically useful. Although Hill (3) reported that doses above 500 kg/g of misonidazole or etanidazole could not further increase tumor sensitization with dose fractionation, he did not test lower doses of the hypoxic sensitizers. This study showed
maximal
sensitization
occurred
at a dose
of 25 p.r/g, with tumor survival tending to plateau at higher dose. Since most radiotherapy is given as multiple small fractions, KU-2285 appears to be a hypoxic cell radiosensitizer that could be useful in such regimens. These results have encouraged us to continue our preclinical studies of this compound.
CONCLUSION KU-2285 sensitized two different murine tumors when it was combined with fractionated irradiation, and it was considered to be an effective hypoxic cell radiosensitizer.
REFERENCES 1 Coleman, C. N.; Urtasun, R. C.; Wasserman, T. H.; Hancock, S.; Harris, J. W.; Halsey, J.; Hirst, V. K. Initial report of the Phase I trial of the hypoxic cell radiosensitizer SR-2508. Int. J. Radiat. Oncol. Biol. Phys. 10: 1749-1753; 1984. 2. Coleman, C. N.; Wasserman, T. H.; Urtasun, R. C.; Halsey, J.; Hirst, V. K.; Hancock, S.; Phillips, T. L. Phase I trial of the hypoxic cell radiosensitizer SR-2508: the results of the five to six week drug schedule. Int. J. Radiat. Oncol. Biol. Phys. 12:1105-l 108; 1986. 3. Hill, R. P. Radiation dose fractionation studies with hypoxic cell radiosensitizers using a murine tumor. Int. J. Radiat. Oncol. Biol. Phys. 8:483485; 1982. 4. Kagiya, T.; Nishimoto, S.; Shibamoto, Y.; Wang, J.; Zhou, L.; He, Y.; Sasai, K.; Takahashi, M.; Abe, M. Importance of tumor affinity of nitroazoles in hypoxic radiosensitization. Int. J. Radiat. Oncol. Biol. Phys. 16:1033-1037; 1989. 5. Nishidai, T.; Abe, M.; Revesz, L. Time dependence of response of transplanted mouse mammary tumors to single or split radiation doses. Int. J. Radiat. Oncol. Biol. Phys. 9:1351-1356; 1983. 6. Ono, K.; Komuro, C.; Nishidai, T.; Shibamoto, Y.; Dodo, Y.; Takahashi, M.; Abe, M.; Shrieve D. C. The radiosensitizing effects of misonidazole (MISO) in combination with diethyl maleate (DEM) in mouse mammary tumors. Int. J. Radiat. Oncol. Biol. Phys. 12:1843-1847; 1986. 7. Sasai, K.; Ono, K.; Nishidai, T.; Tsutsui, K.; Shibamoto, Y.; Takahashi, M.; Abe, M. Variation in tumor response to Fluosol-DA. Int. J. Radiat. Oncol. Biol. Phys. 16:11491152; 1989.
8. Sasai, K.; Shibamoto, Y.; Takahashi, M.; Abe, M.; Wang, J.; Zhou, L.; Nishimoto, S.; Kagiya, T. A new, potent 2-nitroimidazole nucleoside hypoxic cell radiosensitizer, RP170. Jpn. J. Cancer Res. 80:1113-1118; 1989. 9. Sasai, K.; Shibamoto, Y.; Takahashi, M.; Ito, T.; Nishimoto, S.; Abe, M. Pharmacokinetics of 2-nitroimidazole hypoxic cell radiosensitizers in rodent peripheral nervous tissue. Int. J. Radiat. Biol. 57:971-980; 1990. 10. Sasai, K.; Shibamoto, Y.; Takahashi, M.; Zhou, L.; Hori, H.; Nagasawa, H.; Shibata, T.; Inayama, S.; Abe, M. KIH802, an acetohydroxamic acid derivative of 2nitroimidazole, as a new potent hypoxic cell radiosensitizer: radiosensitizing activity, acute toxicity and pharmacokinetics. Cancer Chemother. Pharmacol. 26:112-l 16; 1990. 11. Sasai, K.; Shibamoto, Y.; Zhou, L.; Nishimoto, S.; Takahashi, M.; Kagiya, T.; Abe, M. A fluorinate 2-nitroimidazole, KU-2285, as a new hypoxic cell radiosensitizer. Int. J. Radiat. Oncol. Biol. Phys. (In press). 12. Shibamoto, Y.; Sasai, K.; Abe, M. The radiation response of SCC VII tumor cells in C3H/He mice varies with the irradiation condition. Radiat. Res. 109:352-354; 1987. 13. Shibamoto, Y.; Nishimoto, S.; Shimokawa, K.; Hisanaga, Y.; Zhou, L.; Wang, J.; Sasai, K.; Takahashi, M.; Abe, M.; Kagiya, T. Characteristics of fluorinated nitroazoles as hypoxic cell radiosensitizers. Int. J. Radiat. Oncol. Biol. Phys. 16: 1045-1048; 1989. 14. Urtasun, R. C.; Coleman, C. N.; Wasserman, T. H.; Phillips, T. L. Clinical trials with hypoxic cell sensitizers: time to retrech or to push forward? Int. J. Radiat. Oncol. Biol. Phys. 10:1691-1696; 1984.
pp.1231-1234 Copyright
0360.3016191 $3.00 + .OO Q 1991 Pergamon Press plc
??Biology Original Contribution
IN VZVO RADIOSENSITIZING ACTIVITY OF A NEW FLUORINATED HYPOXIC CELL RADIOSENSITIZER, KU-2285 IN COMBINATION WITH RADIATION DOSE FRACTIONATION KEISUKE SASAI, M.D.,’
MASATO FUSHIKI, M.D.
HIROYUKI IWAI, M.
SC.,~
,2 YUTAKA YUKAWA,
YUTA SHIBAMOTO, M.D.,’
MASAJI TAKAHASHI,
M.D.’
R. T. ,l SUMIO SUYAMA, B. SC. ,2
SEI-ICHI NISHIMOTO, PH.D.,~
AND MITSUYUKI ABE, M.D.’
‘Department of Radiology, Faculty of Medicine, 4Department of Hydrocarbon Chemistry, Faculty of Engineering, 5Chest Disease Research Institute, Kyoto University, Kyoto 606; ‘Department of Radiology, Shiga University of Medical Science, Otsu 520-21; and 3Chemical Division,
Daikin Industries,
Ltd., Settsu 566, Japan
Since most clinical radiotherapy is given as multiple small irradiation fractions, the present study was undertaken to test the in vivo radiosensitizing activity of a new hypoxic cell radiosensitizer, KU-2285 in combination with radiation dose fractionation. Radiosensitizing activity was measured by a growth delay assay using a transplanted mammary tumor in C3H/He mice, and by an in vivo-in vitro assay using the SCC VII tumor. KU-2285 was injected intraperitoneally 30 min before irradiation in all experiments. The in vivo-in vitro assay using SCC VII tumors showed that 12.5 kg/g of KU-2285 sensitized the tumors to irradiation (5 Gylfr x 5 fr/48 hr or 6 Gy/fr x 3 fr/48 hr). KU-2285 also sensitized the transplanted mammary tumors to fractionated irradiation. We concluded that KU-2285 was able to sensitize two different murine tumors when given in combination with radiation dose fractionation. KU-2285, Hypoxic cell radiosensitizer,
Fractionated
irradiation.
macokinetic properties of KU-2285 are summarized in Table 1 in comparison with etanidazole. The reduction potential of KU-2285 is -0.96 V, and its octanol/water partition coefficient is 0.25. The acute toxicity measured as the LD,,,, (drug dose necessary to kill 50% of the tested mice within seven days) in 8-week-old female C3H/HeJ mice was found to be 2.4 g/kg (i.v.), 2.1 g/kg (i.p.), and 4.25 g/kg (p.0.) for KU-2285, whereas it was 4.75 g/kg (i.v.) for etanidazole (11). KU-2285 was dissolved in physiological saline and given intraperitoneally 30 min before irradiation in all experiments in this study on the basis of the results of previous sensitization experiments (11).
INTRODUCTION
The in vivo radiosensitizing activity with single-dose irradiation, acute toxicity, and pharmacokinetic properties of a new fluorinated hypoxic cell radiosensitizer, KU-2285, were described previously in comparison with etanidazole (11). Since most clinical radiation treatment is given as multiple small doses of irradiation, the present study was undertaken to test the in vivo radiosensitizing activity of KU-2285 in combination with radiation dose fractionation. The radiosensitizing activity was measured by a growth delay assay using a transplanted mammary tumor in C3H/He mice, and by an in Co-in vitro assay using SCC VII tumor.
MATERIALS
AND
Animals In all experiments, we used 8- or 9-week-old female C3H/He mice obtained from Japan SLC (Hamamatsu, Japan) or Clea Japan Inc. (Tokyo, Japan).
METHODS
Radiosensitizer KU-2285 has the structure shown in Figure 1 and was developed by our group (4, 9, 11, 13). This compound has a very similar side chain to etanidazole, but it has a CF, structure in its side chain. The physiochemical and phar-
Sensitizer Tumor assay (7, mice, and
Presented at the 32nd Annual Meeting of the American Society for Therapeutic Radiology and Oncology, Miami Beach, FL, 15-20 October 1990. Reprint requests to: Keisuke Sasai, M.D., Department of Ra-
testing system response was measured by an in vivo-in vitro 8, 11, 12) using the SCC VII tumor in C3H/He by a growth delay assay (5-7,11) using a trans-
diology, Faculty of Medicine, Kyoto University, oto 606, Japan. Accepted for publication 26 April 1991.
1231
Sakyo-ku,
Ky-
1232
I. J. Radiation Oncology 0 Biology 0 Physics
CHZCONHCH~
October 1991, Volume 21, Number 5
cH?cF> CONHCHICH20H
CH? OH
KU2285
Etanidazole
IL
Is) la3 .-c
Fig. 1. Structural formula of KU-2285.
planted mammary tumor in C3H/He mice. SCC VII tumor cells were subcutaneously inoculated into both hind legs of each mouse. In the growth delay assay, the transplanted mammary tumor in its third transplant generation was subcutaneously inoculated into the right hind legs of the C3H/He mice. Radiation treatment was started when the SCC VII tumors reached a size of 500 mm3 and the transplanted mammary tumor reached a size of 300 mm3. In the growth delay assay, tumor volume was estimated by caliper measurements on every day or alternate day of the three perpendicular diameters assuming an ellipsoid shape as described previously (7, 8, 10, 11). Irradiation Irradiation was carried out using 10 MV X rays generated by a linear accelerator at a dose rate of 5.6 Gy/min, as described previously (7, 8, 10, 11). For the in vivo-in vitro assay, the mice received whole body irradiation, whereas in the growth delay assay, only the tumor-bearing leg was irradiated. Mice were not restrained for whole body irradiation, but they were fixed with adhesive tape with their limbs extended without anesthesia for local irradiation. Irradiation of 5 fractions of 5 Gy each (every 12 hr) or 3 fractions of 6 Gy each (every 24 hr) was given for the SCC VII tumors, and 5 fractions of 6, 8, 10 or 12 Gy each (every 24 hr) were given for the transplanted mammary tumors. Data analysis Statistical differences in growth delay or tumor cell survival in the in vivo-in vitro assay were assessed using the
Table 1. Physiochemical
and pharmacokinetic
data for KU-2285
and etanidazole Comoound P* Ey;t AUC in mice (kg/g Brain Sciatic nerve
Etanidazole
KU-2285
0.040 - 1.05
0.25 -0.96
280 6200
690 5700
x min)$
*P: Partition coefficient in octanol/water. tERED: Reduction potential vs. Ag/Ag+ in N,N-dimethylformaidey* $Sasai, K. et al. Int. J. Radiat. Biol. 1990. AUC: Area under the concentration-time curve when 200 )*g/g of each compound was injected.
i
1p-i rftr 0
12.5 Drug
-1
25
50
Dose
(w
100
200
/ 9)
Fig. 2. SCC VII tumor cell survival following 3 fractions of 6 Gy each (every 24 hr) plotted as a function of the dose of KU-2285 given to the tumor-bearing animals prior to each radiation treatment. Error bars are 1 S. E. from more than four experiments.
Student’s t test. All statistical analysis of tumor cell survival was carried out using the logarithm values of the survival data.
RESULTS In vivo-in vitro assay The radiosensitizing ability of the drug was studied as a function of the dose given to the tumor-bearing mice. The results for irradiation with 6 Gy fractions (every 24 hours) are shown in Figure 2. The surviving fraction of the tumor cells in the mice pretreated with even 12.5 pg/g of KU2285 was significantly lower than that in mice pretreated with physiological saline (p < 0.05). A similar series of experiments was performed using 5 fractions of 5 Gy each (every 12 hr), and the results are shown in Figure 3. Pretreatment with 12.5 p,g/g of KU-2285 also sensitized the tumor to irradiation in this experiment @ < 0.05). Transplantable mammary tumor The growth curves of tumors after 5 fractions of 6 Gy each are shown in Figure 4. Each group consisted of at least eight mice. The growth curves for tumors in mice without irradiation are included for comparison. Time zero represents the day on which the irradiation was started. When the irradiated and regrowing tumors returned to their initial volume (growth delay time, GDT) (5, 6), the difference in tumor growth between the groups was compared. Tumor growth in the group treated with 200 Fg/g of KU2285 plus irradiation was significantly delayed compared to that in the group treated with physiological saline plus irradiation. Tumor growth in the group given 200 Fg/g of KU-2285 without irradiation was not different from that in mice treated with physiological saline without irradiation. Figure 5 shows GDT as a function of the dose fractionated
Fractionated irradiation plus KU-2285
??K.
1233
SASAIef al
5Gyx5fr (q 12hrs)
+
*
* +* ;
L
rid0
12.5 25 Drug
50
Dose
*
*
100 200
(c1g / 9)
Fig. 3. SCC VII tumor cell survival following 5 fractions of 5 Gy each (every 12 hr) plotted as a function of the KU-2285 dose given to the tumor-bearing animals prior to each radiation treatment. Error bars are 1 S. E. from more than four experiments.
d
0
I
I
I
I
I
I
I
2
4
6
8
10
12
14
Gy/Fr
of irradiation. Pretreatment with 200 kg/g of KU-2285 significantly sensitized the tumors to fractionated irradiation. DISCUSSION
To develop new hypoxic cell radiosensitizers, we have incorporated fluorine atoms into the side chain of 2-nitro-
Fig. 5. Dose-response curves for transplanted mammary tumors, that is, growth delay time as a function of the radiation dose. Error bars are 1 S. E. from more than eight mice. Open circles: 200 kg/g of KU-2285 was administered intraperitoneally 30 min before irradiation. Closed circles: physiological saline was given intraperitoneally 30 min before irradiation. Sr: Four of the 8 mice had no tumor regrowth. *: p < 0.05 **: p < 0.01
6 5
I
I
I
20
25
30
1.1111
Days Fig. 4. Tumor growth curves for transplanted mammary tumors as a function of time after 5 fractions of 6 Gy each (every 24 hr). Error bars are 1 S. E. from more than eight mice. Open circles: 200 pg/g of KU-2285 was given intraperitoneally 30 min before each dose of radiation. Closed circles: physiological saline was given intraperitoneally 30 min before each dose of irradiation. Open diamonds: 200 pg/g of KU-2285 was given intraperitoneally every day for 5 days without irradiation. Closed diamonds: physiological saline was given intraperitoneally every day for 5 days without irradiation. Arrowheads indicate the time of irradiation.
imidazole, 3-nitro-1,2,4_triazole, and 4-nitro-1,2,3-triazole derivatives to modify their tumor affinity and thereby their radiosensitizing activity (4, 13). Of the resulting compounds that were produced, KU-2285 is considered to be the most useful as a hypoxic cell radiosensitizer (13). Although its side chain is similar to that of etanidazole, KU-2285 has a much higher octanol/water partition coefficient than etanidazole, and is similar in this respect to misonidazole. Thus, incorporation of a -CF,group into the side chain structure of -CH,CONHCH,CH,OH belonging to etanidazole has made the compound more lipophilic (11). The in vivo radiosensitizing activity of KU-2285 was previously found to be similar to or a little higher than that of etanidazole at the same dose when tested by an in vivo-in vitro assay, a growth delay assay, and a tumor control assay using SCC VII or transplanted mammary tumors in C3H/He mice (11). Although the radiosensitizing activity of etanidazole was reduced when it was administered orally, there was no significant difference in its activity when given intravenously, intraperitoneally, or orally. In this study, KU-2285 was found to be able to sensitize both SCC VII and transplanted mammary tumors to fractionated irradiation in C3HkIe mice. Although the acute toxicity of KU-2285 is higher than that of etanidazole, it is lower than that of misonidazole (11). Etanidazole was given as a single dose of 2 g/m2 (1, 2) and
1234 misonidazole
I. J. Radiation Oncology was
administered
??Biology
as a single
than 0.4 g/m2 (14) in their respective suggests that the administration doses
dose
0 Physics
October 1991, Volume 21, Number 5
of more
that for KU-2285
clinical trials. This of over 0.4 g/m2 of
KU-2285 should be easily tolerated in clinical use. Even as little as 12.5 kg/g of KU-2285 could significantly sensitize SCC VII tumors to fractionated irradiation in this study. As 12.5 kg/g is almost equal to a dose of 0.4 g/m2 in humans, there seems to be a good possibility that KU-2285 could be clinically useful. Although Hill (3) reported that doses above 500 kg/g of misonidazole or etanidazole could not further increase tumor sensitization with dose fractionation, he did not test lower doses of the hypoxic sensitizers. This study showed
maximal
sensitization
occurred
at a dose
of 25 p.r/g, with tumor survival tending to plateau at higher dose. Since most radiotherapy is given as multiple small fractions, KU-2285 appears to be a hypoxic cell radiosensitizer that could be useful in such regimens. These results have encouraged us to continue our preclinical studies of this compound.
CONCLUSION KU-2285 sensitized two different murine tumors when it was combined with fractionated irradiation, and it was considered to be an effective hypoxic cell radiosensitizer.
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