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A comparison of radiosensitization by etanidazole and pimonidazole in mouse tumors
Inr _I Rndrarmn Oncolu~~ Emi f’h’hrs Vol. Printed in the U.S.A. All rights reserved.
20, pp.
0360.3016/91 $3.00 + .I0 Copyright 0 I99 Pergamon Press plc
987-995
I
0 Original Contribution A COMPARISON OF RADIOSENSITIZATION AND PIMONIDAZOLE IN MOUSE HELEN
B. STONE, YUNG
PH.D., H. Luu,
l V. KATE
HIRST,
B.S.PHARM.’
B.SC.,~
BY ETANIDAZOLE TUMORS
RANDOLPH
AND J. MARTIN
BROWN,
CRIBBS,
B.S.,
B.A.,’
D.PHIL.~
‘Radiation Oncology Research Laboratory, MCB-200, Department of Radiation Oncology, University of California, San Francisco, CA 94143; and ‘Department of Radiation Oncology, Stanford University, Stanford, CA Radiosensitization by pimonidazole (Ro 03-8799) was tested in three murine tumors, EMT6/SF using the excision assay, SCC-VII/SF using the excision and growth delay assays, and MDAH-MCa-4 using TCDSO assays with both single doses and 6 fractions of radiation with a 24-hr interfraction interval. Results were compared with those using etanidazole (SR-2508), both at equitoxic doses and at doses giving tumor concentrations similar to those achievable in the clinic. In excision assays with EMT6/SF and SCC-VII/SF tumors, pimonidazole and etanidazole gave similar radiosensitization at similar concentrations in the tumors. Pimonidazole, however, did not demonstrate radiosensitization in SCC-VII/SF tumors in the growth delay assay, despite tumor concentrations that gave maximum sensitization in the excision assay. Furthermore, pimonidazole gave less than expected sensitization in single dose and 6-fraction TCDSO assays with MDAH-MCa-4 tumors, and less sensitization than comparable levels of etanidazole in this tumor line. When the concentration of pimonidazole in the tumors was approximately 0.36 pmoles/ g the dose modification factor (DMF = dose without sensitizer/dose with sensitizer to give an isoeffect) was 1.56 (1.40-l-74, 95% c.1.) in single dose TCDSO assays. Etanidazole, however, gave a DMF of 1.92 (1.59-2.32) with a tumor concentration of approximately 0.32 pmoles/g and 1.69 (1.46-1.96) with a tumor concentration of approximately 0.21 pmoles/g. Thus, etanidazole gave more consistent sensitization for different tumors and different endpoints than did pimonidazole. The results appear to confirm the disappointing performance of pimonidazole in the clinic. Hypoxic cell radiosensitizers,
Tumors, Etanidazole (SR-2508), Pimonidazole (Ro 03-8799).
There is convincing clinical evidence that, at least for some types of malignancies, hypoxic tumor cells limit the cure rates that can be achieved by conventional radiotherapy (4, 11). Hypoxic cell radiosensitizers have been developed to address this problem, but the first of these to receive extensive clinical testing, misonidazole, has not achieved unequivocal success due, at least in part, to inadequate tumor concentrations because of the limitations imposed by the neurotoxic side effects of the drug (2). However, new radiosensitizers such as etanidazole (SR-2508) and pimonidazole (Ro 03-8799) have been developed that show less neurotoxicity or other types of toxicity than misonidazole. Higher doses of these new drugs can be obtained in humans compared to those with misonidazole.
Nevertheless, further reduction of toxicity is a desirable goal in trying to achieve maximal radiosensitization for clinical trials of hypoxic cell radiosensitizers. One appreach that has been suggested is to use two or more sensitizers together or in alternation: etanidazole, which induces peripheral neuropathy with chronic administration (5), and pimonidazole, which induces acute central neuropathy (22). We previously reported that when these drugs were given together in single doses to mice, there was overlapping toxicity, but that there was a slight enhancement of sensitizing activity compared to that with each agent alone at equitoxic doses (25). In that study, we used an excision assay with EMT6/SF tumors in BALB/c mice. Here, we report additional studies using these two sensitizers alone and together in excision assays with EMT6/SF tumors, excision and growth delay assays
Reprint requests to: Dr. Helen B. Stone, Radiation Oncology Research Laboratory, MCB-200, Department of Radiation Oncology, Box 0806, University of California, San Francisco, CA 94143. Acknowledgments-We gratefully acknowledge the excellent technical assistance of Kitty Lam, David Hyun, Clare PetersLibeu, Hiep Nguyen, and James Evans. We thank Dr. Michael Tracy of SRI International for analysis of pimonidazole. This
project has been funded with federal funds from the Department of Health and Human Services under Contracts CM 37578 and CM 37868 and research Grant CA25990. The contents of this publication do not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government. Accepted for publication 9 November 1990.
INTRODUCTION
987
I. J. Radiation
988
Oncology
0 Biology 0 Physics
with SCC-VII/SF tumors, and TCDSO assays with MDAH-MCa-4 tumors. Although the two sensitizers appeared to be equally effective at equimolar concentrations in EMT6/SF and SCC-VII/SF tumors using excision assays, pimonidazole was less effective than etanidazole in growth delay assays and TCDSO assays with SCC-VII/SF and EMT6/SF tumors, respectively. We conclude that sensitization by pimonidazole in vivo varies with tumor line and with assay and is not predicted by tumor concentrations alone, whereas radiosensitization by etanidazole was independent of tumor line and assay, was predictable by tumor concentrations, and therefore appears to be more likely to succeed in clinical trials.
METHODS
AND
MATERIALS
Tumors and mice All tumors were stored in a liquid nitrogen refrigerator. This stock was used, according to a strict protocol, to supply tumor cells for culture in vitro or in viva. EMT6/ SF and SCC-VII/SF were passaged alternately in cell culture and in the subcutaneous tissue of the flanks of male BALB/cAnNCr and C3Hf/HeNCRMTV mice, respectively. EMT6/SF and SCC-VII/SF tumors implanted subcutaneously in the flanks of mice were used for excision assays. SCC-VII/SF tumors were implanted subcutaneously in the right hind leg for growth delay assays. TCD.50 assays were performed with fifth and sixth generation implants of MDAH-MCa-4 growing in the right hind legs of male C3Hf/HeNCRMTV mice. Pathogen-free mice were supplied by the Biological Testing Branch of the National Cancer Institute. Irradiation Mice were irradiated without anesthesia in one of two cesium irradiators, with dose rates of 2.28 to 2.17 Gy/ min and 3.33 Gy/min, as measured by lithium fluoride dosimetry. For excision assays, mice were irradiated whole-body, restrained in a modified 50 cc plastic centrifuge tube. For growth delay and TCDSO assays, mice were restrained in a jig with the leg extended across the 3 cm wide field, and the foot and remainder of the body shielded. Sensitizers Etanidazole (SR 2508, NSC 30 1467) pimonidazole (Ro 03-8799, NSC 3 18502, as the hydrochloride salt), and misonidazole (NSC 261037) were provided by the Drug Synthesis and Chemistry Branch of the National Cancer Institute. They were prepared fresh daily and dissolved in Ringer’s solution. Etanidazole and pimonidazole were given intravenously, and misonidazole was given intraperitoneally. Measurement At various pimanidazole,
of plasma and tumor levels of sensitizers times after injection of either etanidazole or mice were sacrificed and samples of blood
May
1. 1991, Volume 20, Number 5
and tumor collected as described in the following section. At the time of analysis, samples were thawed and blood samples were centrifuged at 3000 g for 10 min. Blood and tumor homogenates (20% w/v in distilled water) were analyzed by reverse phase high performance liquid chromatography (HPLC) as described previously (37) except that the concentration of methanol in the mobile phase was 15% for etanidazole, and for the analysis of pimonidazole, the mobile phase was 40% methanol in 50 mM NH4H2P04 and 5 mM heptane sulphonic acid adjusted to pH 6.0. The flow rate for both sensitizers was 1.5 ml/ min and samples of etanidazole were read at a wavelength of 324 nm; for pimonidazole the wavelength was 326 nm. The column was a Waters C 18 uBondapak. Concentrations of pimonidazole are reported as the free base. Assays of’tumor response For excision assays, mice bearing tumors of 50 to 150 mg were injected with sensitizers, then irradiated with 20 Gy. The aorta was severed immediately after cervical dislocation, and a sample of blood was collected in heparinized capillary tubes. The blood sample and half of each tumor were frozen immediately in liquid nitrogen for later analysis of sensitizer concentration by HPLC. A wedge was removed from the other half of the tumor and processed for microscopic examination, and the remainder was assayed in vitro for cell survival. The tissue was minced and incubated with a mixture of pronase, DNAse, and collagenase at 37°C for 30 min (25). The suspensions were filtered through a 200 mesh polyester screen, centrifuged, and the pellets resuspended. Viable cells were counted in a hemacytometer using trypan blue exclusion and phase contrast microscopy. Serial dilutions were made and plated in triplicate. After incubation at 37°C in a humidified atmosphere of 5% CO2 + 95% air for 13 days, colonies were stained with methylene blue and those of >50 cells were counted. For the growth delay assay, tumors were measured daily with a vernier caliper as they approached treatment size of 8 mm diameter, and after treatment until they reached 14 mm. There were 8 to 9 mice/group. Geometric means of the number of days for individual tumors to reach 14 mm diameter were calculated for each treatment group. The experiment was performed three times and the data were pooled. TCDSO assays were performed as described previously (26). Tumors were measured daily and treated when 8 mm in diameter. There were 5 to 8 dose groups of 8 to 12 mice each. All animals were given 50 mg diazepam/l drinking water during the period of acute skin reactions, approximately 20 to 35 days after treatment. Starting at about 30 days and weekly thereafter, animals were checked for recurrent tumors and were killed when the tumors reached 11 mm in diameter, or at 120 days after treatment if the tumor did not recur. Experiments were performed one to four times, and data were pooled for analysis. TCDSO values and their 95% confidence limits
Etanidazole
% $ 42 5 E
and pimonidazole
4
989
The doses of etanidazole and pimonidazole were 60% of the LD50 for the sensitizers administered together in equitoxic proportions determined from single dose experiments (25). The interval between injection of sensitizer and irradiation was 30 min.
ETANIDAZOLE, PIMONIDAZOLE
36
z
0 H. B. STONE eta/.
30 Local tumor control, single doses
I E 24 E g 18
3 a
E e
12 6
t
co
2
r
0’ 0
5
10
20
15
25
DOSE, GY Fig. I. Growth delay in XC-VII/SF tumors irradiated at 8 mm. in diameter. The doses of sensitizers were: misonidazole (MISO), 0.78 mg/g body weight; etanidazole (ETAN), 1.76 mg/g body weight; and pimonidazole (PIM), 0.27 mg/g body weight. These doses were equitoxic (0.6 LD50). For etanidazole and pimonidazole, the doses were based on the toxicity of the combined drugs. The interval between injection of sensitizers and irradiation was 30 min.
were computed, using pooled data, using the logit method of analysis, with a correction for censored data as described by Walker and Suit (32).
RESULTS
Growth delay The mean number of days required for SCC-VII/SF tumors to reach a mean diameter of 14 mm are shown as a function of radiation dose in Figure 1. At an isoeffect of 18 days to reach 14 mm, the DMF for misonidazole alone (0.8 mg/g.), etanidazole alone (1.76 mg/g.), and etanidazole (1.76 mg/g.) and pimonidazole (0.27 mg/g.) together was 1.4, and for pimonidazole alone was 1.0.
Local control of MDAH-MCa-4 tumors was determined in mice given etanidazole, pimonidazole, or both, or misonidazole, or saline given 30 min before irradiation (25, 27). The doses were 10% of the LD50 for the sensitizers administered together in equitoxic proportions (25). There was no significant reduction in the TCDSO by pimonidazole alone at a dose of 0.04 mg/g (DMF = 1.05) compared to saline controls (Table 1). Although pimonidazole gave an additional reduction in TCDSO when given with etanidazole, compared to etanidazole alone at a dose of 0.29 mg/g, the DMF values were not significantly different (DMF = 1.69 for etanidazole alone, and 1.84 for etanidazole with pimonidazole).
Local tumor control, 6 fractions Local control of MDAH-MCa-4 tumors was determined for treatment with 6 fractions with a l-day interval between each fraction. Because etanidazole and pimonidazole were to be given on alternate days, the equitoxic doses were 10% of the LD50 values for the drugs given separately in a single dose (25), namely, 0.07 mg pimonidazole/g and 0.44 mg etanidazole/g. A 60-min interval was used between injection and irradiation. The results are shown in Table 2. During the course of these experiments, information became available on pimonidazole toxicity in humans ( 1E), and additional groups were tested using a higher dose that would be equivalent to the daily dose of 0.75 g/m* given to patients. This was 0.25 mg/g, or 36% of the acute LD50. Neither dose of pimonidazole gave a significant reduction in TCDSO, compared to controls, when given with 3 of the 6 fractions in alternation with Ringer’s solution, or when given in alternation with etanidazole, compared to the mice treated with etanidazole alone. The DMFs for etanidazole given with 3 of the
Table 1. Single dose TCDSO of MDAH-MCa-4
Group
MG/G
RTE
Control Misonidazole Pimonidazole Etanidazole Pimonidazole + etanidazole
0.13 0.04 0.29 0.04 + 0.29
ip ip iv iv iv iv
TCDSO (f95% c.1.) 80.5 49.7 76.6 47.5 43.7
(68.8-94.1) (46.9-52.7) (59.4-98.7) (41.6-54.4) (36.5-52.4)
DMF (k95% c.1.) 1.62 1.05 1.69 1.84
(1.40-1.87) (0.82-1.34) ( 1.46- 1.96) (1.57-2.16)
Note: Doses were 10% of the LD50. For pimonidazole and etanidazole, this was based on the LD50 when the drugs were given together in equitoxic proportions. All sensitizers were given 30 min before irradiation. Controls were given an equivalent volume of Ringer’s solution. RTE = Route of administration: intraperitoneal (ip) or intravenous (iv); MIN = Minutes between drug administration and irradiation; DMF = Dose modification factor = TCDSO (control)/TCDSO (sensitizer).
990
I. J. Radiation Oncology 0 Biology 0 Physics Table 2. 6 fraction
May 1, 1991, Volume 20, Number 5
TCDSO in MDAH-MCa-4
Group
FX
MG/G
TCDSO (f95% c.1.)
Control Sensitizer with 3 of 6 fractions Pimonidazole Pimonidazole Etanidazole Sensitizer with 6 of 6 fractions Pimonidazole + etanidazole Pimonidazole + etanidazole Etanidazole
-
-
102.0 (98.0-106.2)
l, 3, 5 l, 3, 5 l, 3, 5
0.07 0.25 0.44
95.9 (87.7-104.7) >93 79.9 (76.8-83.2)
1.07 (0.99-l. 16)
l, 3, + 1, 3, + l-6
0.07 + 0.44 0.25 + 0.44 0.44
8 1.1 (74.6-88.2)
1.26 (1.17-1.37)
85.0 (82.4-87.6)
1.21 (1.15-1.27)
66.6 (62.0-7 1.7)
1.54 (1.43-l .65)
5 2, 4, 6 5 2, 4, 6
DMF (k95% C.I.) -
Note: The lower dose of pimonidazole and the dose of etanidazole are 10% of the LD50 for the drugs given separately, and the higher dose of pimonidazole is equivalent, on a mg/m2 basis, to the dose proposed for clinical use. The sensitizers were given iv 60 min before irradiation. Tumor: MDAH-MCa-4, 8mm diameter. FX = Fraction numbers with which sensitizer was given; Ringer’s solution was given on days when sensitizer was not given; MG/ G = Sensitizer dose given with each designated fraction number.
6 fractions were 1.2 1- 1.26, with or without pimonidazole. When etanidazole was given with all 6 fractions, the TCDSO was significantly lower than when it was given with only 3 fractions: the DMF was 1.54. The finding of little or no sensitization by pimonidazole in the tumor control experiments prompted a series of experiments to determine whether our drug samples had decomposed, whether pharmacokinetics were different in C3H mice than in BALB/c mice, or whether tumor levels of pimonidazole were lower in MDAH-MCa-4 tumors than in EMT6/SF tumors. During the course of these experiments, pharmacokinetic data became available from clinical trials (20, 21) and an attempt was made to use doses of pimonidazole equivalent to those used for patients, and later to mimic the tumor concentrations achieved clinically.
mined by correcting the concentrations at the time of sacrifice, within 7 min after irradiation, assuming a halflife of 23 min. Concentrations of etanidazole were similarly corrected, assuming a half-life of 48.6 min (3). Excision assays with pimonidazole The fraction of tumor cells surviving a dose of 20 Gy is shown in Figure 3 as a function of time between intravenous injection of 0.25 mg pimonidazole/g body weight and irradiation of EMT6/SF and SCC-VII/SF tumors. The surviving fractions after an equitoxic dose of misonidazole, 0.8 mg/g, injected intraperitoneally 30 min be-
PIMONIDAZOLE
IN BLOOD AND TUMOR
Drug purity Samples of pimonidazole were analyzed for chemical purity by reverse phase HPLC, ‘H-NMR, and by melting point determinations, and were found to be >97.8% pure. Half-life in tumors and blood Concentrations of pimonidazole in tumor tissue and blood are shown in Figure 2 as a function of time between iv injection of 0.25 mg pimonidazole/g body weight and sacrifice of the mice. Elimination half-lives of pimonidazole in the blood of tumor-bearing BALB/c or C3H mice were not significantly different statistically, nor were half-lives in EMT6/SF, SCC-VII/SF, or MDAH-MCa-4 tumors (Table 3). The tumor/blood concentration ratios for pimonidazole were 2.08 + 0.20 (s.e.) for EMT6/SF, 2.02 + 0.17 for SCC-VII/SF, and 2.72 & 0.22 for MDAHMCa-4 tumors. The ratios were significantly higher (p < 0.05) for MDAH-MCa-4 than for the other two tumor lines, based on Student’s t-test. Concentrations in individual tumors at the midpoint of irradiation were deter-
0
MIN”% A6% of 0.25mg/g
90 120 150 IV INJECTION body weight
Fig. 2. Concentration of pimonidazole in blood (open symbols) and tumor tissue (solid symbols) as a function of time after intravenous injection of 0.25 mg/g body weight. Each symbol represents a single tumor or blood sample from a single mouse.
Etanidazole and pimonidazole 0 H. B. STONEet al.
991
Table 3. Half-life of pimonidazole
Tumor
Mice
EMT6/SF SCC-VII/SF MDAH-MCa-4
BALB/c C3H C3H Pooled data
T 1,2, tumor, min. (?95% cl.)
T,,z, blood, min. (k95% c.1.) 23 30 23 25
(19-30) (25-38) (20-30) (23-30)
(I 7-23) (23-30) (22-30) (22-27)
20 25 25 23
dose: 0.25 mg/g body weight, injected intravenously.
Pimonidazole
fore irradiation, is shown for comparison. In both tumors, the maximum sensitization by pimonidazole was observed within 30 min, although there is clearly a broad nadir in surviving fraction. Little or no sensitization remained at 90 min. The surviving fractions were replotted as a function of the concentrations of pimonidazole in each tumor at the midpoint of irradiation (Fig. 4). For both tumor lines, maximum sensitization was seen with concentra-
tions of pimonidazole of about 0.2 pmoles/g or greater. The lower concentrations were obtained at the longer intervals after injection.
Excision assays with etanidazole The fractions of SCC-VII/SF and EMT6/SF tumor cells surviving a dose of 20 Gy are shown in Figure 5 as a function of the concentration of etanidazole in each tumor at the midpoint of irradiation. The doses of etanidazole
PlMONlDAZOLE
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10’
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IO0
h’
F c.J 10-l
PIMONIDAZOLE CONT NOT IFIR-
a. EMT6
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2
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z 5 5
1o-5
5 (0
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5 5
1o-3
PIM IRF
t;
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T 5 1o-3 lo-’
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_ CONT
B (3 1o-z
4 a
10'
30 60 90 120 MIN. BEMlEEN INJ. & IRR.
Survival of cells from EMT6/SF (a) and SCC-VII/SF (b) treated in vivo and assayed in vitro. The radiation dose Gy (IRR). The dose of pimonidazole was 0.25 mg/g body and of misonidazole, 0.78 mg/g body weight. Controls: Each symbol represents a single tumor.
E
1 I
1
1
I
I
I
1
I
I
I
0.0 0.1 0.2 0.3 0.4 IPIMONIDAZOLEI
IN TUMOR,
I
I
0.5 pmoles/g
Fig. 4. Survival of cells from EMT6/SF (a) and SCC-VII/SF (b) tumors as a function of pimonidazole concentration at the midpoint of irradiation. Tumors were irradiated 15 to 90 min after a dose of 0.25 mg pimonidazole/g body weight. Each symbol represents a single tumor.
I. J. Radiation Oncology 0 Biology 0 Physics
992
ETANIDAZOLE
ETAN & PIM IN EMTG/SF & SSVWSF
t CONT NOT IRR
May I, I99 I, Volume 20, Number 5
a. EMT6
10’ CPNT NOT IRR
-
0.0
0.1
PM&/E IRR
E
P
0.2
0.3
0.4
0.5
0.8
CONC. IN TUMOR, pmoles/g Fig. 6. Data from Figures 4 and 5 plotted together. ETAN: etanidazole; PIM: pimonidazole; /E: data from EMT6/SF tumors; /S: data from SCC-VII/SF tumors. The bars indicate concentrations achievable in human tumors (20).
lo-’
11
I
I
I
t
1%
I*
0.0 0.2 0.4 0.6 0.8 1.0 [ETANIDAZOLEI IN TUMOR, pmoles/g Fig. 5. Survival of cells from EMT6/SF (a) and SCC-VII/SF (b) tumors as a function of etanidazole concentration in the tumors at the midpoint of irradiation. Measured concentrations were individually corrected assuming a half-life of 48.6 min (3). Tumors were irradiated 30 min after injection of pimonidazole doses of 0.11 to 0.35 mg/g body weight (a) or 40 min after doses of 0.05 to 1.O mg/g body weight (b). Each symbol represents a single tumor.
were 0.11 to 0.35 mg/g body weight for mice with EMT6/ SF tumors, and 0.05 to 1.0 mg/g body weight for mice with SCC-VII/SF tumors. The intervals between injection and irradiation were 30 and 40 min, respectively. Maximum sensitization was observed with concentrations of about 0.2-0.3 pmoles/g or greater. To facilitate comparison of the sensitizing efficiency of pimonidazole and etanidazole, the data from Figures 4 and 5 were replotted together in Figure 6 for tumor concentrations < 0.6 pmoles/g. There was no significant difference between the sensitizers or between the two tumor lines: a common line could be fitted through all the data.
Local tumor control, single doses, high doses of pimonidazole The studies of tumor and plasma half-life suggested that sensitization by pimonidazole might be observed in
TCDSO assays in MDAH-MCa-4 if both a high pimonidazole dose and short interval between injection and irradiation were used, when tumor concentrations of pimonidazole would be higher. We therefore performed an additional TCDSO assay using single doses of radiation with the higher dose of pimonidazole at a shorter interval. Mice were given pimonidazole (0.25 mg/g) 20 min before the midpoint of irradiation, or etanidazole (0.44 mg/g) 40 min before the midpoint of irradiation (25). The DMF for pimonidazole was 1.56 (1.40-l .74, 95% conf. lim.) and for etanidazole, 1.92 (1.59-2.32) (Table 4). The TCDSO value for the group treated with etanidazole was significantly lower, 4 1.6 Gy, than that for the group treated with pimonidazole, 5 1.3 Gy (p < 0.05).
DISCUSSION
Sensitization in EA4T6/SF and SCC- VII/SF tumors The present data raise questions about the reasons for the observed differences in sensitization by pimonidazole in the different tumors and assays. The KC-VII/SF tumor, the only tumor we evaluated with two different assays, showed sensitization by pimonidazole in the excision assay, but not the growth delay assay. The failure to sensitize in the growth delay assay cannot be explained by the time interval between injection and irradiation, because an administered dose of 0.27 mg/g would be expected to give a tumor concentration of 0.25 pmoles/g at the midpoint of irradiation (Fig. 2). This should have given nearly the maximum sensitization possible with pimonidazole (Fig. 6). Although little or no sensitization at ra-
Etanidazole and pimonidazole 0 H. B. STONE et al. Table 4. Single dose TCDSO of MDAH-MCa-4
using a shorter interval between
Group
MG/G
RTE
Min
Control Etanidazole Pimonidazole
0.44 0.25
iv iv iv
30 40 20
Min: Minutes
between
injection
and the midpoint
of the radiation
diation doses below 10 to 15 Gy is a common observation in growth delay studies (35, 36) both misonidazole and
etanidazole demonstrated sensitization at radiation doses where pimonidazole did not (Fig. 1). Previous studies have demonstrated sensitization of EMT6 tumors by pimonidazole and etanidazole, alone and together, in excision assays ( 12, 25). The tumor concentrations of 0.24 pmole of etanidazole/g and 0.14 pmole pimonidazole/g reported by Honess et al. (12) gave additional sensitization when the two sensitizers were given together or when the dose of each drug was doubled.
Sensitization in MDAH-MCa-4
tumors
In the case of the MDAH-MCa-4 tumor, part of the lack of efficacy of pimonidazole in the first single dose TCDSO experiments and in the 6-fraction TCDSO experiments could be explained by low concentrations of pimonidazole in the tumors at the time of irradiation. However, similar pharmacokinetics of pimonidazole in the two strains of mice and in the three tumor lines would lead us to expect some radiosensitization in MDAH-MCa4 tumors based on the findings in the excision assays. However, the measured tumor concentrations do not discriminate between intra- and extracellular pimonidazole, concentrations in clonogenic or nonclonogenic or necrotic cells, or concentrations in vascular and supportive tissues within the tumor. They also do not identify regional heterogeneity within the tumor (36). Thus, there may have been clonogenic hypoxic cells in the tumor that contained lower concentrations of pimonidazole. Such subpopulations govern the TCD.50 even when present in small numbers (28). Although it would have been desirable to test pimonidazole sensitization in an excision assay with MDAH-MCa-4, this tumor cannot be dissociated enzymatically into a single cell suspension and has a very low plating efficiency in vitro. The presence of large areas of necrosis in MDAH-MCa4 could indicate a low pH in this tumor line, which could limit uptake of pimonidazole into the cells (6, 33). This has been demonstrated for cells in culture, and presumably as a result of the higher intracellular than extracellular pH. Saunders et al., however, found that correcting for the amount of necrosis in the tumor samples had little effect on the range of concentrations observed in tumors (23). Vaupel et al. (3 1) have reported pH values of 5.8 to 7.2 (mean, 6.73) in MDAH-MCa-4 tumors in anesthetized
sensitizer
TCDSO (k95% c.1.) 80.0 (73.6-87.0) 41.6 (35.2-49.2) 5 1.3 (47.4-55.5) treatment;
iv: intravenous;
993 injection
and irradiation DMF (k95% c.1.) 1.92 (1.59-2.32) 1.56 (1.40-1.74)
ip: intraperitoneal.
mice. The extensive necrosis we observed in these tumors probably signified a low pH in our MDAH-MCa-4 tumors as well. We do not have information on the pH levels in EMT6/SF or SCC-VII/SF tumors, but little necrosis was observed in the histological samples from these tumors. Measurements of pH in human tumors indicate that they are slightly more acidic than subcutaneous tissues, with reported averages in tumors of 6.81 + 0.09 (SEM) (29), 6.8 1 -t 0.25 (SD) (13), 7.04 f 0.19 (SD) (13), 7.29 f 0.05 (SEM) (30), and 7.25 -I 0.29 (SD) (34), and in subcutaneous tissues of 7.63 + 0.03 (SEM) (30) and 7.54 -t 0.09 (SD) (34). By using a shorter interval between injection of pimonidazole and irradiation, we were able to show radiosensitization of MDAH-MCa-4 tumors, but pimonidazole was still less effective than etanidazole at comparable tumor concentrations. In human tumors, the half-life in plasma for both pimonidazole and etanidazole is 5 to 6 hr (1, 5, 22, 23, 24) compared to less than 1 hr in mice (3) so that timing of sensitizer administration and irradiation should be less critical in patients. Etanidazole, however, gave more consistent radiosensitization in the three tumor lines and assays that we tested.
Concentrations of etanidazole and pimonidazole achievable in human tumors Concentrations of both etanidazole and pimonidazole achieved in human tumors appear to vary widely (9, 10, 17-24). This may relate to the difficulty of obtaining adequate samples of tissue from needle biopsies (17, 18), metabolism of the drug during and after removal of the biopsy and prior to analysis ( 17), variability in distribution of the drug within the tumor (17), as well as variability between tumors. In a comparison of concentrations in murine tumors, Minchinton and Stratford (16) found little variability in small, fast-growing SaFa tumors, but greater variability in large, fast-growing tumors. Large, slowgrowing CaRH tumors, however, showed little variability. et al. (20) in Concentrations measured by Newman human tumors other than gliomas averaged 34 + 19 (S.D.) pg pimonidazole/g 15 min after intravenous administration of 0.75 g/m2, and 34 f 24 pg/g at 30 min. Etanidazole concentrations, corrected to an administered dose of 1.5 g/m*, averaged 58 + 19 pg/g at both 15 and 30 min. These concentrations are about 0.12 -t 0.06 pmoles of pimonidazole/g and 0.27 + 0.09 pmoles etanidazole/g, and are
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indicated by bars on Figure 6. Although this figure shows that there was no apparent difference in radiosensitizing efficacy between the two drugs at equivalent tumor concentrations, the lower concentrations of pimonidazole achieved in human tumors is on a region of the curve where sensitization is more dependent on the concentration of the sensitizer in the tumor. Lower and more variable radiosensitization would therefore be expected of pimonidazole in the clinic. Note that the concentration of pimonidazole in the growth delay assay, 0.25 pmoles/g, where no sensitization was observed, and that in the only TCDSO assay in which significant but moderate sensitization was observed, 0.36 pmoles/g, were greater than the upper limit of the range achievable in patients (Fig. 6). The findings appear to confirm the disappointing results in the Multicentre Trial conducted in Europe and coordinated by the British Medical Research Council, of pimonidazole in the treatment of carcinoma of the cervix (S. Dische, written communication, October, 1990). Although we found similar tumor/plasma ratios of about 2.5 for pimonidazole in all three tumor lines, some pigmented tumors appear to concentrate this sensitizer to a greater extent than nonpigmented tumors (7, 8, 14, 15). Whether greater radiosensitization is achieved in such tumors has not been determined. Etanidazole, in contrast, does not concentrate in tumors and is excluded from the brain because of its low lipophilicity (3, 15, 18). Concentrations of etanidazole achieved in gliomas are lower than in other tumors, averaging 0.17 pmoles/g for a dose of 1.5 g/m2; pimonidazole concentrations are even lower, averaging 0.09 pmoles/g for a dose of 0.75 g/m2 (20), suggesting that both sensitizers could be less effective or ineffective in brain tumors.
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The concept of achieving additional sensitization by giving two sensitizers together at doses that each give partial sensitization has not been disproved by the present work, because ineffective doses of pimonidazole were used in combination with etanidazole in the growth delay and TCDSO experiments with single radiation doses. However, the greater effectiveness of etanidazole when given with all treatments in a 6-fraction irradiation schedule than when given with every other fraction suggests that using two effective sensitizers in alternation could be a feasible approach, if the two had different mechanisms of toxicity.
CONCLUSIONS Etanidazole, although it does not concentrate in tumors to the extent that pimonidazole does, appears to be the better sensitizer. Etanidazole sensitized EMT6/SF, SCCVII/SF, and MDAH-MCa-4 tumors in mice, as tested by excision, growth delay, and TCDSO assays at concentrations achievable in human tumors. In 6-fraction TCDSO assays, significant sensitization was seen when it was given with only three of the fractions, and greater sensitization when it was given with all six fractions. Pimonidazole, in contrast, showed sensitization in EMTB/SF and SCC-VII/ SF tumors in excision assays, but not in SCC-VII/SF tumors in the growth delay assay. The TCDSO of MDAHMCa-4 tumors was reduced by a factor of 1.56 when tumor concentrations of pimonidazole were approximately 0.36 pmoles/g, but similar concentrations of etanidazole reduced the TCDSO by a factor of 1.92. These findings appear to confirm the disappointing results of clinical trials of pimonidazole.
REFERENCES Bleehen, N. M.; Newman, H. F. V.; Maughan, T. S.; Workman, P. A multiple dose study of the combined radiosensitizers Ro 03-8799 (pimonidazole) and SR 2508 (etanidazole). lnt. J. Radiat. Oncol. Biol. Phys. 16:1093-1096; 1989. 2. Brown, J. M. Clinical trials of radiosensitizers: what should 1.
we expect? lnt. J. Radiat. Oncol. Biol. Phys. 10:425-9; 1984. considerations in radiosensitizer development. In: Brady, L. W., ed. Radiation sensitizers. Their use in the clinical management of cancer. New York: Masson Publishing USA, Inc.; 1980:213. 4. Bush, R. S.; Jenkin, R. D.; Alit, W. E.; Beale, F. A.; Bean, H.; Dembo, A. J.; Pringle, J. F. Definitive evidence for hypoxic cells influencing cure in cancer therapy. Brit. J. Cancer 37(Suppl. 111):302-6; 1978. T. H.; Han5. Coleman, C. N.; Urtasun, R. C.; Wasserman, cock, S.; Harris, J. W.; Halsey, J.; Hirst, V. K. Initial report of the Phase 1 trial of the hypoxic cell radiosensitizer SR2508. lnt. J. Radiat. Oncol. Biol. Phys. 10:1749-1753; 1984. M. F.; Stratford, M. R.; Wardman, P.; Watts, 6. Dennis, M. E. Cellular uptake of misonidazole and analogues with acidic or basic functions. lnt. J. Radiat. Biol. 47:629-43; 1985.
3. Brown, J. M.; Lee, W. W. Pharmacokinetic
using the hypoxic cell sensitizer 7. Dische, S. Radiotherapy Ro 03-8799 in malignant melanoma. Radiother. Oncol. 10: 11 l-6; 1987. 8. Dische, S.; Bennett, M. H.; Orchard, R.; Stratford, M. R.; Wardman, P. The uptake of the radiosensitizing compound Ro 03-8799 (pimonidazole) in human tumors. lnt. J. Radiat. Oncol. Biol. Phys. 16: 1089-92; 1989. 9. Dische, S.; Saunders, M. I.; Bennett, M. H.; Dunphy, E. P.; Des, R. C.; Stratford, M. R.; Minchinton, A. I.; Wardman, P. A comparison of the tumour concentrations obtainable with misonidazole and Ro 03-8799. Brit. J. Radiol. 59:9 1 l7; 1986. 10. Dische, S.; Saunders, M. I.; Dunphy, E. P.; Bennett, M. H.; Des, R. C.; Stratford, M. R.; Minchinton, A. I.; Orchard, R. A. Concentrations achieved in human tumors after administration of misonidazole, SR-2508 and Ro 03-8799. lnt. J. Radiat. Oncol. Biol. Phys. 12:1109-l 1; 1986. J. S.; Coia, 11. Gatenby, R. A.; Kessler, H. B.; Rosenblum, L. R.; Moldofsky, P. J.; Hartz, W. H.; Broder, G. J. Oxygen distribution in squamous cell carcinoma metastases and its relationship to outcome of radiation therapy. lnt. J. Radiat. Oncol. Biol. Phys. 14:831-8; 1988. 12. Honess, D. J.; Wasserman, T. H.; Workman, P.; Ward, R.; Bleehen, N. M. Additivity of radiosensitization by the com-
Etanidazole and pimonidazole 0 H. B. STONEet al.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
bination of SR 2508 (etanidazole) and Ro 03-8799 (pimonidazole) in a murine tumor system. Int. J. Radiat. Oncol. Biol. Phys. 15:671-675; 1988. Kallinowski, F.; Vaupel, P.; Runkel, S.; Berg, G.; Fortmeyer, H. P.; Baessler, K. H.; Wagner, K.; Mueller-Klieser, W.; Walenta, S. Glucose uptake, lactate release, ketone body turnover, metabolic micromilieu, and pH distributions in human breast cancer xenografts in nude rats. Cancer Res. 48~7264-7272; 1988. Laurent, F.; Canal, P.; Soula, G. Pharmacokinetics of Ro 03-8799 in mice bearing melanosarcoma: comparison with tumors without melanin. Int. J. Radiat. Oncol. Biol. Phys. 16:1101-l 103; 1989. Lespinasse, F.; Thomas, C.; Bonnay, M.; Malaise, E. P.; Guichard, M. Ro 03-8799: Preferential relative uptake in human tumor xenografts compared to a murine tumor: comparison with SR-2508. Int. J. Radiat. Oncol. Biol. Phys. 16:1105-l 109; 1989. Minchinton, A. I.; Stratford, M. R. L. A comparison of tumor and normal tissue levels of acidic, basic and neutral 2-nitroimidazole radiosensitizers in mice. Int. J. Radiat. Oncol. Biol. Phys. 12: 1 117- 1120; 1986. Newman, H. F. V.; Bleehen, N. M.; Ward, R.; Workman, P. Hypoxic cell radiosensitizers in the treatment of high grade gliomas: a new direction using combined Ro 03-8799 (pimonidazole) and SR 2508 (etanidazole). Int. J. Radiat. Oncol. Biol. Phys. 15:677-684; 1988. Newman, H. F. V.; Bleehen, N. M.; Workman, P. A phase I study of the combination of two hypoxic cell radiosensitizers, Ro 03-8799 and SR-2508: toxicity and pharmacokinetics. Int. J. Radiat. Oncol. Biol. Phys. 12:1113-l 116; 1986. Newman, H. F. V.; Bleehen, N. M.; Workman, P. A phase I study of the combined hypoxic cell radiosensitizers, Ro 03-8799 and SR 2508: a preliminary report of single-dose toxicity, pharmacokinetics and tumour concentrations. Brit. J. Radiol. 59:423-425; 1986. Newman, H. F. V.; Ward, R.; Workman, P.; Bleehen, N. M. The multi-dose clinical tolerance and pharmacokinetics of the combined radiosensitizers, Ro 03-8799 (pimonidazole) and SR 2508 (etanidazole). Int. J. Radiat. Oncol. Biol. Phys. 15:1073-1083; 1988. Roberts, J. T.; Bleehen, N. M.; Walton, M. I.; Workman, P. A clinical phase I toxicity study of Ro 03-8799: plasma, urine, tumour and normal brain pharmacokinetics. Brit. J. Radiol. 59:107-l 16; 1986. Roberts, J. T.; Bleehen, N. M.; Workman, P.; Walton, M. I. A phase I study of the hypoxic cell radiosensitizer Ro03-8799. Int. J. Radiat. Oncol. Biol. Phys. 10:1755-1758; 1984. Saunders, M. I.; Anderson, P. J.; Bennett, M. H.; Dische, S.; Minchinton, A.; Stratford, M. R.; Tothill, M. The clinical
24.
25.
26.
27.
28.
29.
30.
3 1.
32.
33.
34.
35.
36.
37.
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testing of Ro 03-8799-pharmacokinetics, toxicology, tissue and tumor concentrations. Int. J. Radiat. Oncol. Biol. Phys. 10:1759-63; 1984. Saunders, M. I.; Dische, S.; Ferment, D.; Bishop, A.; LenoxSmith, I.; Allen, J. G.; Malcolm, S. L. The radiosensitizer Ro 03-8799 and the concentrations which may be achieved in human turnours: a preliminary study. Brit. J. Cancer 46: 706-10; 1982. Stone, H. B.; Luu, Y. H.; Lam, K. N. Sensitization by SR2508 plus Ro 03-8799. Int. J. Radiat. Oncol. Biol. Phys. 12: 1097-100; 1986. Stone, H. B.; Withers, H. R. Tumor and normal tissue response to metronidazole and irradiation in mice. Radiology 113:441-4; 1974. Stone, H. B.; Withers, H. R. Enhancement of the radioresponse of a murine tumour by a nitroimidazole. Brit. J. Radiol. 48:411-2; 1975. Stone, H. B.; Withers, H. R. Metronidazole: Effect on radiosensitivity of tumor and normal tissues in mice. JNCI 55: 1189-94; 1975. Thistlethwaite, A. J.; Leeper, D. B.; Moylan, D. J., III; Nerlinger, R. E. pH distribution in human tumors. Int. J. Radiat. Oncol. Biol. Phys. 11:1647-1652; 1985. Van den Berg, A. P.; Wike-Hooley, J. L.; Van den BergBlock, A. E.; Van der Zee, J.; Reinhold, H. S. Tumour pH in human mammary carcinoma. Eur. J. Cancer Clin. Oncol. 18:457-462; 1982. Vaupel, P. W.; Frinak, S.; Bicher, H. I. Heterogeneous oxygen partial pressure and pH distribution in C3H mouse mammary adenocarcinoma. Cancer Res. 4 1:2008- 13; 198 1. Walker, A. M.; Suit, H. D. Assessment oflocal tumor control using censored tumor response data. Int. J. Radiat. Oncol. Biol. Phys. 9:383-6; 1983. Watts, M. E.; Dennis, M. F.; Woodcock, M. Uptake and additivity of the radiosensitizing effects of Ro 03-8799 and SR-2508 in mammalian cells in vitro. Brit. J. Radiol. 60: 1233-5; 1987. Wike-Hooley, J. L.; Van den Berg, A. P.; Van der Zee, J.; Reinhold, H. S. Human tumour pH and its variation. Eur. J. Cancer Clin. Oncol. 21:785-791; 1985. Williams, M. V.; Denekamp, J.; Minchinton, A. I.; Stratford, M. R. In vivo assessment of basic 2-nitroimidazole radiosensitizers. Brit. J. Cancer 46: 127-37; 1982. Williams, M. V.; Denekamp, J.; Minchinton, A. I.; Stratford, M. R. In vivo testing of a 2-nitroimidazole radiosensitizer (Ro 03-8799) using repeated administration. Int. J. Radiat. Oncol. Biol. Phys. 8:477-8 1; 1982. Workman, P.; Little, C. J.; Marten, T. R.; Dale, A. D.; Ruane, R. J.; Flockhart, I. R.; Bleehen, N. M. Estimation of the hypoxic cell sensitizer misonidazole and its O-demethylated metabolite in biological chromatography. J. Chromatogr. 145:507-512; 1978.
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0360.3016/91 $3.00 + .I0 Copyright 0 I99 Pergamon Press plc
987-995
I
0 Original Contribution A COMPARISON OF RADIOSENSITIZATION AND PIMONIDAZOLE IN MOUSE HELEN
B. STONE, YUNG
PH.D., H. Luu,
l V. KATE
HIRST,
B.S.PHARM.’
B.SC.,~
BY ETANIDAZOLE TUMORS
RANDOLPH
AND J. MARTIN
BROWN,
CRIBBS,
B.S.,
B.A.,’
D.PHIL.~
‘Radiation Oncology Research Laboratory, MCB-200, Department of Radiation Oncology, University of California, San Francisco, CA 94143; and ‘Department of Radiation Oncology, Stanford University, Stanford, CA Radiosensitization by pimonidazole (Ro 03-8799) was tested in three murine tumors, EMT6/SF using the excision assay, SCC-VII/SF using the excision and growth delay assays, and MDAH-MCa-4 using TCDSO assays with both single doses and 6 fractions of radiation with a 24-hr interfraction interval. Results were compared with those using etanidazole (SR-2508), both at equitoxic doses and at doses giving tumor concentrations similar to those achievable in the clinic. In excision assays with EMT6/SF and SCC-VII/SF tumors, pimonidazole and etanidazole gave similar radiosensitization at similar concentrations in the tumors. Pimonidazole, however, did not demonstrate radiosensitization in SCC-VII/SF tumors in the growth delay assay, despite tumor concentrations that gave maximum sensitization in the excision assay. Furthermore, pimonidazole gave less than expected sensitization in single dose and 6-fraction TCDSO assays with MDAH-MCa-4 tumors, and less sensitization than comparable levels of etanidazole in this tumor line. When the concentration of pimonidazole in the tumors was approximately 0.36 pmoles/ g the dose modification factor (DMF = dose without sensitizer/dose with sensitizer to give an isoeffect) was 1.56 (1.40-l-74, 95% c.1.) in single dose TCDSO assays. Etanidazole, however, gave a DMF of 1.92 (1.59-2.32) with a tumor concentration of approximately 0.32 pmoles/g and 1.69 (1.46-1.96) with a tumor concentration of approximately 0.21 pmoles/g. Thus, etanidazole gave more consistent sensitization for different tumors and different endpoints than did pimonidazole. The results appear to confirm the disappointing performance of pimonidazole in the clinic. Hypoxic cell radiosensitizers,
Tumors, Etanidazole (SR-2508), Pimonidazole (Ro 03-8799).
There is convincing clinical evidence that, at least for some types of malignancies, hypoxic tumor cells limit the cure rates that can be achieved by conventional radiotherapy (4, 11). Hypoxic cell radiosensitizers have been developed to address this problem, but the first of these to receive extensive clinical testing, misonidazole, has not achieved unequivocal success due, at least in part, to inadequate tumor concentrations because of the limitations imposed by the neurotoxic side effects of the drug (2). However, new radiosensitizers such as etanidazole (SR-2508) and pimonidazole (Ro 03-8799) have been developed that show less neurotoxicity or other types of toxicity than misonidazole. Higher doses of these new drugs can be obtained in humans compared to those with misonidazole.
Nevertheless, further reduction of toxicity is a desirable goal in trying to achieve maximal radiosensitization for clinical trials of hypoxic cell radiosensitizers. One appreach that has been suggested is to use two or more sensitizers together or in alternation: etanidazole, which induces peripheral neuropathy with chronic administration (5), and pimonidazole, which induces acute central neuropathy (22). We previously reported that when these drugs were given together in single doses to mice, there was overlapping toxicity, but that there was a slight enhancement of sensitizing activity compared to that with each agent alone at equitoxic doses (25). In that study, we used an excision assay with EMT6/SF tumors in BALB/c mice. Here, we report additional studies using these two sensitizers alone and together in excision assays with EMT6/SF tumors, excision and growth delay assays
Reprint requests to: Dr. Helen B. Stone, Radiation Oncology Research Laboratory, MCB-200, Department of Radiation Oncology, Box 0806, University of California, San Francisco, CA 94143. Acknowledgments-We gratefully acknowledge the excellent technical assistance of Kitty Lam, David Hyun, Clare PetersLibeu, Hiep Nguyen, and James Evans. We thank Dr. Michael Tracy of SRI International for analysis of pimonidazole. This
project has been funded with federal funds from the Department of Health and Human Services under Contracts CM 37578 and CM 37868 and research Grant CA25990. The contents of this publication do not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government. Accepted for publication 9 November 1990.
INTRODUCTION
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with SCC-VII/SF tumors, and TCDSO assays with MDAH-MCa-4 tumors. Although the two sensitizers appeared to be equally effective at equimolar concentrations in EMT6/SF and SCC-VII/SF tumors using excision assays, pimonidazole was less effective than etanidazole in growth delay assays and TCDSO assays with SCC-VII/SF and EMT6/SF tumors, respectively. We conclude that sensitization by pimonidazole in vivo varies with tumor line and with assay and is not predicted by tumor concentrations alone, whereas radiosensitization by etanidazole was independent of tumor line and assay, was predictable by tumor concentrations, and therefore appears to be more likely to succeed in clinical trials.
METHODS
AND
MATERIALS
Tumors and mice All tumors were stored in a liquid nitrogen refrigerator. This stock was used, according to a strict protocol, to supply tumor cells for culture in vitro or in viva. EMT6/ SF and SCC-VII/SF were passaged alternately in cell culture and in the subcutaneous tissue of the flanks of male BALB/cAnNCr and C3Hf/HeNCRMTV mice, respectively. EMT6/SF and SCC-VII/SF tumors implanted subcutaneously in the flanks of mice were used for excision assays. SCC-VII/SF tumors were implanted subcutaneously in the right hind leg for growth delay assays. TCD.50 assays were performed with fifth and sixth generation implants of MDAH-MCa-4 growing in the right hind legs of male C3Hf/HeNCRMTV mice. Pathogen-free mice were supplied by the Biological Testing Branch of the National Cancer Institute. Irradiation Mice were irradiated without anesthesia in one of two cesium irradiators, with dose rates of 2.28 to 2.17 Gy/ min and 3.33 Gy/min, as measured by lithium fluoride dosimetry. For excision assays, mice were irradiated whole-body, restrained in a modified 50 cc plastic centrifuge tube. For growth delay and TCDSO assays, mice were restrained in a jig with the leg extended across the 3 cm wide field, and the foot and remainder of the body shielded. Sensitizers Etanidazole (SR 2508, NSC 30 1467) pimonidazole (Ro 03-8799, NSC 3 18502, as the hydrochloride salt), and misonidazole (NSC 261037) were provided by the Drug Synthesis and Chemistry Branch of the National Cancer Institute. They were prepared fresh daily and dissolved in Ringer’s solution. Etanidazole and pimonidazole were given intravenously, and misonidazole was given intraperitoneally. Measurement At various pimanidazole,
of plasma and tumor levels of sensitizers times after injection of either etanidazole or mice were sacrificed and samples of blood
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1. 1991, Volume 20, Number 5
and tumor collected as described in the following section. At the time of analysis, samples were thawed and blood samples were centrifuged at 3000 g for 10 min. Blood and tumor homogenates (20% w/v in distilled water) were analyzed by reverse phase high performance liquid chromatography (HPLC) as described previously (37) except that the concentration of methanol in the mobile phase was 15% for etanidazole, and for the analysis of pimonidazole, the mobile phase was 40% methanol in 50 mM NH4H2P04 and 5 mM heptane sulphonic acid adjusted to pH 6.0. The flow rate for both sensitizers was 1.5 ml/ min and samples of etanidazole were read at a wavelength of 324 nm; for pimonidazole the wavelength was 326 nm. The column was a Waters C 18 uBondapak. Concentrations of pimonidazole are reported as the free base. Assays of’tumor response For excision assays, mice bearing tumors of 50 to 150 mg were injected with sensitizers, then irradiated with 20 Gy. The aorta was severed immediately after cervical dislocation, and a sample of blood was collected in heparinized capillary tubes. The blood sample and half of each tumor were frozen immediately in liquid nitrogen for later analysis of sensitizer concentration by HPLC. A wedge was removed from the other half of the tumor and processed for microscopic examination, and the remainder was assayed in vitro for cell survival. The tissue was minced and incubated with a mixture of pronase, DNAse, and collagenase at 37°C for 30 min (25). The suspensions were filtered through a 200 mesh polyester screen, centrifuged, and the pellets resuspended. Viable cells were counted in a hemacytometer using trypan blue exclusion and phase contrast microscopy. Serial dilutions were made and plated in triplicate. After incubation at 37°C in a humidified atmosphere of 5% CO2 + 95% air for 13 days, colonies were stained with methylene blue and those of >50 cells were counted. For the growth delay assay, tumors were measured daily with a vernier caliper as they approached treatment size of 8 mm diameter, and after treatment until they reached 14 mm. There were 8 to 9 mice/group. Geometric means of the number of days for individual tumors to reach 14 mm diameter were calculated for each treatment group. The experiment was performed three times and the data were pooled. TCDSO assays were performed as described previously (26). Tumors were measured daily and treated when 8 mm in diameter. There were 5 to 8 dose groups of 8 to 12 mice each. All animals were given 50 mg diazepam/l drinking water during the period of acute skin reactions, approximately 20 to 35 days after treatment. Starting at about 30 days and weekly thereafter, animals were checked for recurrent tumors and were killed when the tumors reached 11 mm in diameter, or at 120 days after treatment if the tumor did not recur. Experiments were performed one to four times, and data were pooled for analysis. TCDSO values and their 95% confidence limits
Etanidazole
% $ 42 5 E
and pimonidazole
4
989
The doses of etanidazole and pimonidazole were 60% of the LD50 for the sensitizers administered together in equitoxic proportions determined from single dose experiments (25). The interval between injection of sensitizer and irradiation was 30 min.
ETANIDAZOLE, PIMONIDAZOLE
36
z
0 H. B. STONE eta/.
30 Local tumor control, single doses
I E 24 E g 18
3 a
E e
12 6
t
co
2
r
0’ 0
5
10
20
15
25
DOSE, GY Fig. I. Growth delay in XC-VII/SF tumors irradiated at 8 mm. in diameter. The doses of sensitizers were: misonidazole (MISO), 0.78 mg/g body weight; etanidazole (ETAN), 1.76 mg/g body weight; and pimonidazole (PIM), 0.27 mg/g body weight. These doses were equitoxic (0.6 LD50). For etanidazole and pimonidazole, the doses were based on the toxicity of the combined drugs. The interval between injection of sensitizers and irradiation was 30 min.
were computed, using pooled data, using the logit method of analysis, with a correction for censored data as described by Walker and Suit (32).
RESULTS
Growth delay The mean number of days required for SCC-VII/SF tumors to reach a mean diameter of 14 mm are shown as a function of radiation dose in Figure 1. At an isoeffect of 18 days to reach 14 mm, the DMF for misonidazole alone (0.8 mg/g.), etanidazole alone (1.76 mg/g.), and etanidazole (1.76 mg/g.) and pimonidazole (0.27 mg/g.) together was 1.4, and for pimonidazole alone was 1.0.
Local control of MDAH-MCa-4 tumors was determined in mice given etanidazole, pimonidazole, or both, or misonidazole, or saline given 30 min before irradiation (25, 27). The doses were 10% of the LD50 for the sensitizers administered together in equitoxic proportions (25). There was no significant reduction in the TCDSO by pimonidazole alone at a dose of 0.04 mg/g (DMF = 1.05) compared to saline controls (Table 1). Although pimonidazole gave an additional reduction in TCDSO when given with etanidazole, compared to etanidazole alone at a dose of 0.29 mg/g, the DMF values were not significantly different (DMF = 1.69 for etanidazole alone, and 1.84 for etanidazole with pimonidazole).
Local tumor control, 6 fractions Local control of MDAH-MCa-4 tumors was determined for treatment with 6 fractions with a l-day interval between each fraction. Because etanidazole and pimonidazole were to be given on alternate days, the equitoxic doses were 10% of the LD50 values for the drugs given separately in a single dose (25), namely, 0.07 mg pimonidazole/g and 0.44 mg etanidazole/g. A 60-min interval was used between injection and irradiation. The results are shown in Table 2. During the course of these experiments, information became available on pimonidazole toxicity in humans ( 1E), and additional groups were tested using a higher dose that would be equivalent to the daily dose of 0.75 g/m* given to patients. This was 0.25 mg/g, or 36% of the acute LD50. Neither dose of pimonidazole gave a significant reduction in TCDSO, compared to controls, when given with 3 of the 6 fractions in alternation with Ringer’s solution, or when given in alternation with etanidazole, compared to the mice treated with etanidazole alone. The DMFs for etanidazole given with 3 of the
Table 1. Single dose TCDSO of MDAH-MCa-4
Group
MG/G
RTE
Control Misonidazole Pimonidazole Etanidazole Pimonidazole + etanidazole
0.13 0.04 0.29 0.04 + 0.29
ip ip iv iv iv iv
TCDSO (f95% c.1.) 80.5 49.7 76.6 47.5 43.7
(68.8-94.1) (46.9-52.7) (59.4-98.7) (41.6-54.4) (36.5-52.4)
DMF (k95% c.1.) 1.62 1.05 1.69 1.84
(1.40-1.87) (0.82-1.34) ( 1.46- 1.96) (1.57-2.16)
Note: Doses were 10% of the LD50. For pimonidazole and etanidazole, this was based on the LD50 when the drugs were given together in equitoxic proportions. All sensitizers were given 30 min before irradiation. Controls were given an equivalent volume of Ringer’s solution. RTE = Route of administration: intraperitoneal (ip) or intravenous (iv); MIN = Minutes between drug administration and irradiation; DMF = Dose modification factor = TCDSO (control)/TCDSO (sensitizer).
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I. J. Radiation Oncology 0 Biology 0 Physics Table 2. 6 fraction
May 1, 1991, Volume 20, Number 5
TCDSO in MDAH-MCa-4
Group
FX
MG/G
TCDSO (f95% c.1.)
Control Sensitizer with 3 of 6 fractions Pimonidazole Pimonidazole Etanidazole Sensitizer with 6 of 6 fractions Pimonidazole + etanidazole Pimonidazole + etanidazole Etanidazole
-
-
102.0 (98.0-106.2)
l, 3, 5 l, 3, 5 l, 3, 5
0.07 0.25 0.44
95.9 (87.7-104.7) >93 79.9 (76.8-83.2)
1.07 (0.99-l. 16)
l, 3, + 1, 3, + l-6
0.07 + 0.44 0.25 + 0.44 0.44
8 1.1 (74.6-88.2)
1.26 (1.17-1.37)
85.0 (82.4-87.6)
1.21 (1.15-1.27)
66.6 (62.0-7 1.7)
1.54 (1.43-l .65)
5 2, 4, 6 5 2, 4, 6
DMF (k95% C.I.) -
Note: The lower dose of pimonidazole and the dose of etanidazole are 10% of the LD50 for the drugs given separately, and the higher dose of pimonidazole is equivalent, on a mg/m2 basis, to the dose proposed for clinical use. The sensitizers were given iv 60 min before irradiation. Tumor: MDAH-MCa-4, 8mm diameter. FX = Fraction numbers with which sensitizer was given; Ringer’s solution was given on days when sensitizer was not given; MG/ G = Sensitizer dose given with each designated fraction number.
6 fractions were 1.2 1- 1.26, with or without pimonidazole. When etanidazole was given with all 6 fractions, the TCDSO was significantly lower than when it was given with only 3 fractions: the DMF was 1.54. The finding of little or no sensitization by pimonidazole in the tumor control experiments prompted a series of experiments to determine whether our drug samples had decomposed, whether pharmacokinetics were different in C3H mice than in BALB/c mice, or whether tumor levels of pimonidazole were lower in MDAH-MCa-4 tumors than in EMT6/SF tumors. During the course of these experiments, pharmacokinetic data became available from clinical trials (20, 21) and an attempt was made to use doses of pimonidazole equivalent to those used for patients, and later to mimic the tumor concentrations achieved clinically.
mined by correcting the concentrations at the time of sacrifice, within 7 min after irradiation, assuming a halflife of 23 min. Concentrations of etanidazole were similarly corrected, assuming a half-life of 48.6 min (3). Excision assays with pimonidazole The fraction of tumor cells surviving a dose of 20 Gy is shown in Figure 3 as a function of time between intravenous injection of 0.25 mg pimonidazole/g body weight and irradiation of EMT6/SF and SCC-VII/SF tumors. The surviving fractions after an equitoxic dose of misonidazole, 0.8 mg/g, injected intraperitoneally 30 min be-
PIMONIDAZOLE
IN BLOOD AND TUMOR
Drug purity Samples of pimonidazole were analyzed for chemical purity by reverse phase HPLC, ‘H-NMR, and by melting point determinations, and were found to be >97.8% pure. Half-life in tumors and blood Concentrations of pimonidazole in tumor tissue and blood are shown in Figure 2 as a function of time between iv injection of 0.25 mg pimonidazole/g body weight and sacrifice of the mice. Elimination half-lives of pimonidazole in the blood of tumor-bearing BALB/c or C3H mice were not significantly different statistically, nor were half-lives in EMT6/SF, SCC-VII/SF, or MDAH-MCa-4 tumors (Table 3). The tumor/blood concentration ratios for pimonidazole were 2.08 + 0.20 (s.e.) for EMT6/SF, 2.02 + 0.17 for SCC-VII/SF, and 2.72 & 0.22 for MDAHMCa-4 tumors. The ratios were significantly higher (p < 0.05) for MDAH-MCa-4 than for the other two tumor lines, based on Student’s t-test. Concentrations in individual tumors at the midpoint of irradiation were deter-
0
MIN”% A6% of 0.25mg/g
90 120 150 IV INJECTION body weight
Fig. 2. Concentration of pimonidazole in blood (open symbols) and tumor tissue (solid symbols) as a function of time after intravenous injection of 0.25 mg/g body weight. Each symbol represents a single tumor or blood sample from a single mouse.
Etanidazole and pimonidazole 0 H. B. STONEet al.
991
Table 3. Half-life of pimonidazole
Tumor
Mice
EMT6/SF SCC-VII/SF MDAH-MCa-4
BALB/c C3H C3H Pooled data
T 1,2, tumor, min. (?95% cl.)
T,,z, blood, min. (k95% c.1.) 23 30 23 25
(19-30) (25-38) (20-30) (23-30)
(I 7-23) (23-30) (22-30) (22-27)
20 25 25 23
dose: 0.25 mg/g body weight, injected intravenously.
Pimonidazole
fore irradiation, is shown for comparison. In both tumors, the maximum sensitization by pimonidazole was observed within 30 min, although there is clearly a broad nadir in surviving fraction. Little or no sensitization remained at 90 min. The surviving fractions were replotted as a function of the concentrations of pimonidazole in each tumor at the midpoint of irradiation (Fig. 4). For both tumor lines, maximum sensitization was seen with concentra-
tions of pimonidazole of about 0.2 pmoles/g or greater. The lower concentrations were obtained at the longer intervals after injection.
Excision assays with etanidazole The fractions of SCC-VII/SF and EMT6/SF tumor cells surviving a dose of 20 Gy are shown in Figure 5 as a function of the concentration of etanidazole in each tumor at the midpoint of irradiation. The doses of etanidazole
PlMONlDAZOLE
5
10’
E
IO0
h’
F c.J 10-l
PIMONIDAZOLE CONT NOT IFIR-
a. EMT6
??
2
loo
g q
lo-*
z 5 5
1o-5
5 (0
lo-’
1o-2
5 5
1o-3
PIM IRF
t;
CONT IRR -
8
2
loo
E 2
10-l
CONT
i
lRCQ n
fE (3 lo-*
1 MlSOf _ IRR
u
1o-5
5 5
lo-$
5 UJ 10“
1o-6
0 Fig. 3. tumors was 20 weight, CONT.
E 0
5 ul lo-'
10-l
e a
loo
g C-J 10"
T 5 1o-3 lo-’
2
_ CONT
B (3 1o-z
4 a
10'
30 60 90 120 MIN. BEMlEEN INJ. & IRR.
Survival of cells from EMT6/SF (a) and SCC-VII/SF (b) treated in vivo and assayed in vitro. The radiation dose Gy (IRR). The dose of pimonidazole was 0.25 mg/g body and of misonidazole, 0.78 mg/g body weight. Controls: Each symbol represents a single tumor.
E
1 I
1
1
I
I
I
1
I
I
I
0.0 0.1 0.2 0.3 0.4 IPIMONIDAZOLEI
IN TUMOR,
I
I
0.5 pmoles/g
Fig. 4. Survival of cells from EMT6/SF (a) and SCC-VII/SF (b) tumors as a function of pimonidazole concentration at the midpoint of irradiation. Tumors were irradiated 15 to 90 min after a dose of 0.25 mg pimonidazole/g body weight. Each symbol represents a single tumor.
I. J. Radiation Oncology 0 Biology 0 Physics
992
ETANIDAZOLE
ETAN & PIM IN EMTG/SF & SSVWSF
t CONT NOT IRR
May I, I99 I, Volume 20, Number 5
a. EMT6
10’ CPNT NOT IRR
-
0.0
0.1
PM&/E IRR
E
P
0.2
0.3
0.4
0.5
0.8
CONC. IN TUMOR, pmoles/g Fig. 6. Data from Figures 4 and 5 plotted together. ETAN: etanidazole; PIM: pimonidazole; /E: data from EMT6/SF tumors; /S: data from SCC-VII/SF tumors. The bars indicate concentrations achievable in human tumors (20).
lo-’
11
I
I
I
t
1%
I*
0.0 0.2 0.4 0.6 0.8 1.0 [ETANIDAZOLEI IN TUMOR, pmoles/g Fig. 5. Survival of cells from EMT6/SF (a) and SCC-VII/SF (b) tumors as a function of etanidazole concentration in the tumors at the midpoint of irradiation. Measured concentrations were individually corrected assuming a half-life of 48.6 min (3). Tumors were irradiated 30 min after injection of pimonidazole doses of 0.11 to 0.35 mg/g body weight (a) or 40 min after doses of 0.05 to 1.O mg/g body weight (b). Each symbol represents a single tumor.
were 0.11 to 0.35 mg/g body weight for mice with EMT6/ SF tumors, and 0.05 to 1.0 mg/g body weight for mice with SCC-VII/SF tumors. The intervals between injection and irradiation were 30 and 40 min, respectively. Maximum sensitization was observed with concentrations of about 0.2-0.3 pmoles/g or greater. To facilitate comparison of the sensitizing efficiency of pimonidazole and etanidazole, the data from Figures 4 and 5 were replotted together in Figure 6 for tumor concentrations < 0.6 pmoles/g. There was no significant difference between the sensitizers or between the two tumor lines: a common line could be fitted through all the data.
Local tumor control, single doses, high doses of pimonidazole The studies of tumor and plasma half-life suggested that sensitization by pimonidazole might be observed in
TCDSO assays in MDAH-MCa-4 if both a high pimonidazole dose and short interval between injection and irradiation were used, when tumor concentrations of pimonidazole would be higher. We therefore performed an additional TCDSO assay using single doses of radiation with the higher dose of pimonidazole at a shorter interval. Mice were given pimonidazole (0.25 mg/g) 20 min before the midpoint of irradiation, or etanidazole (0.44 mg/g) 40 min before the midpoint of irradiation (25). The DMF for pimonidazole was 1.56 (1.40-l .74, 95% conf. lim.) and for etanidazole, 1.92 (1.59-2.32) (Table 4). The TCDSO value for the group treated with etanidazole was significantly lower, 4 1.6 Gy, than that for the group treated with pimonidazole, 5 1.3 Gy (p < 0.05).
DISCUSSION
Sensitization in EA4T6/SF and SCC- VII/SF tumors The present data raise questions about the reasons for the observed differences in sensitization by pimonidazole in the different tumors and assays. The KC-VII/SF tumor, the only tumor we evaluated with two different assays, showed sensitization by pimonidazole in the excision assay, but not the growth delay assay. The failure to sensitize in the growth delay assay cannot be explained by the time interval between injection and irradiation, because an administered dose of 0.27 mg/g would be expected to give a tumor concentration of 0.25 pmoles/g at the midpoint of irradiation (Fig. 2). This should have given nearly the maximum sensitization possible with pimonidazole (Fig. 6). Although little or no sensitization at ra-
Etanidazole and pimonidazole 0 H. B. STONE et al. Table 4. Single dose TCDSO of MDAH-MCa-4
using a shorter interval between
Group
MG/G
RTE
Min
Control Etanidazole Pimonidazole
0.44 0.25
iv iv iv
30 40 20
Min: Minutes
between
injection
and the midpoint
of the radiation
diation doses below 10 to 15 Gy is a common observation in growth delay studies (35, 36) both misonidazole and
etanidazole demonstrated sensitization at radiation doses where pimonidazole did not (Fig. 1). Previous studies have demonstrated sensitization of EMT6 tumors by pimonidazole and etanidazole, alone and together, in excision assays ( 12, 25). The tumor concentrations of 0.24 pmole of etanidazole/g and 0.14 pmole pimonidazole/g reported by Honess et al. (12) gave additional sensitization when the two sensitizers were given together or when the dose of each drug was doubled.
Sensitization in MDAH-MCa-4
tumors
In the case of the MDAH-MCa-4 tumor, part of the lack of efficacy of pimonidazole in the first single dose TCDSO experiments and in the 6-fraction TCDSO experiments could be explained by low concentrations of pimonidazole in the tumors at the time of irradiation. However, similar pharmacokinetics of pimonidazole in the two strains of mice and in the three tumor lines would lead us to expect some radiosensitization in MDAH-MCa4 tumors based on the findings in the excision assays. However, the measured tumor concentrations do not discriminate between intra- and extracellular pimonidazole, concentrations in clonogenic or nonclonogenic or necrotic cells, or concentrations in vascular and supportive tissues within the tumor. They also do not identify regional heterogeneity within the tumor (36). Thus, there may have been clonogenic hypoxic cells in the tumor that contained lower concentrations of pimonidazole. Such subpopulations govern the TCD.50 even when present in small numbers (28). Although it would have been desirable to test pimonidazole sensitization in an excision assay with MDAH-MCa-4, this tumor cannot be dissociated enzymatically into a single cell suspension and has a very low plating efficiency in vitro. The presence of large areas of necrosis in MDAH-MCa4 could indicate a low pH in this tumor line, which could limit uptake of pimonidazole into the cells (6, 33). This has been demonstrated for cells in culture, and presumably as a result of the higher intracellular than extracellular pH. Saunders et al., however, found that correcting for the amount of necrosis in the tumor samples had little effect on the range of concentrations observed in tumors (23). Vaupel et al. (3 1) have reported pH values of 5.8 to 7.2 (mean, 6.73) in MDAH-MCa-4 tumors in anesthetized
sensitizer
TCDSO (k95% c.1.) 80.0 (73.6-87.0) 41.6 (35.2-49.2) 5 1.3 (47.4-55.5) treatment;
iv: intravenous;
993 injection
and irradiation DMF (k95% c.1.) 1.92 (1.59-2.32) 1.56 (1.40-1.74)
ip: intraperitoneal.
mice. The extensive necrosis we observed in these tumors probably signified a low pH in our MDAH-MCa-4 tumors as well. We do not have information on the pH levels in EMT6/SF or SCC-VII/SF tumors, but little necrosis was observed in the histological samples from these tumors. Measurements of pH in human tumors indicate that they are slightly more acidic than subcutaneous tissues, with reported averages in tumors of 6.81 + 0.09 (SEM) (29), 6.8 1 -t 0.25 (SD) (13), 7.04 f 0.19 (SD) (13), 7.29 f 0.05 (SEM) (30), and 7.25 -I 0.29 (SD) (34), and in subcutaneous tissues of 7.63 + 0.03 (SEM) (30) and 7.54 -t 0.09 (SD) (34). By using a shorter interval between injection of pimonidazole and irradiation, we were able to show radiosensitization of MDAH-MCa-4 tumors, but pimonidazole was still less effective than etanidazole at comparable tumor concentrations. In human tumors, the half-life in plasma for both pimonidazole and etanidazole is 5 to 6 hr (1, 5, 22, 23, 24) compared to less than 1 hr in mice (3) so that timing of sensitizer administration and irradiation should be less critical in patients. Etanidazole, however, gave more consistent radiosensitization in the three tumor lines and assays that we tested.
Concentrations of etanidazole and pimonidazole achievable in human tumors Concentrations of both etanidazole and pimonidazole achieved in human tumors appear to vary widely (9, 10, 17-24). This may relate to the difficulty of obtaining adequate samples of tissue from needle biopsies (17, 18), metabolism of the drug during and after removal of the biopsy and prior to analysis ( 17), variability in distribution of the drug within the tumor (17), as well as variability between tumors. In a comparison of concentrations in murine tumors, Minchinton and Stratford (16) found little variability in small, fast-growing SaFa tumors, but greater variability in large, fast-growing tumors. Large, slowgrowing CaRH tumors, however, showed little variability. et al. (20) in Concentrations measured by Newman human tumors other than gliomas averaged 34 + 19 (S.D.) pg pimonidazole/g 15 min after intravenous administration of 0.75 g/m2, and 34 f 24 pg/g at 30 min. Etanidazole concentrations, corrected to an administered dose of 1.5 g/m*, averaged 58 + 19 pg/g at both 15 and 30 min. These concentrations are about 0.12 -t 0.06 pmoles of pimonidazole/g and 0.27 + 0.09 pmoles etanidazole/g, and are
994
1. J. Radiation
Oncology
0 Biology 0 Physics
indicated by bars on Figure 6. Although this figure shows that there was no apparent difference in radiosensitizing efficacy between the two drugs at equivalent tumor concentrations, the lower concentrations of pimonidazole achieved in human tumors is on a region of the curve where sensitization is more dependent on the concentration of the sensitizer in the tumor. Lower and more variable radiosensitization would therefore be expected of pimonidazole in the clinic. Note that the concentration of pimonidazole in the growth delay assay, 0.25 pmoles/g, where no sensitization was observed, and that in the only TCDSO assay in which significant but moderate sensitization was observed, 0.36 pmoles/g, were greater than the upper limit of the range achievable in patients (Fig. 6). The findings appear to confirm the disappointing results in the Multicentre Trial conducted in Europe and coordinated by the British Medical Research Council, of pimonidazole in the treatment of carcinoma of the cervix (S. Dische, written communication, October, 1990). Although we found similar tumor/plasma ratios of about 2.5 for pimonidazole in all three tumor lines, some pigmented tumors appear to concentrate this sensitizer to a greater extent than nonpigmented tumors (7, 8, 14, 15). Whether greater radiosensitization is achieved in such tumors has not been determined. Etanidazole, in contrast, does not concentrate in tumors and is excluded from the brain because of its low lipophilicity (3, 15, 18). Concentrations of etanidazole achieved in gliomas are lower than in other tumors, averaging 0.17 pmoles/g for a dose of 1.5 g/m2; pimonidazole concentrations are even lower, averaging 0.09 pmoles/g for a dose of 0.75 g/m2 (20), suggesting that both sensitizers could be less effective or ineffective in brain tumors.
May 1, 1991, Volume
20, Number
5
The concept of achieving additional sensitization by giving two sensitizers together at doses that each give partial sensitization has not been disproved by the present work, because ineffective doses of pimonidazole were used in combination with etanidazole in the growth delay and TCDSO experiments with single radiation doses. However, the greater effectiveness of etanidazole when given with all treatments in a 6-fraction irradiation schedule than when given with every other fraction suggests that using two effective sensitizers in alternation could be a feasible approach, if the two had different mechanisms of toxicity.
CONCLUSIONS Etanidazole, although it does not concentrate in tumors to the extent that pimonidazole does, appears to be the better sensitizer. Etanidazole sensitized EMT6/SF, SCCVII/SF, and MDAH-MCa-4 tumors in mice, as tested by excision, growth delay, and TCDSO assays at concentrations achievable in human tumors. In 6-fraction TCDSO assays, significant sensitization was seen when it was given with only three of the fractions, and greater sensitization when it was given with all six fractions. Pimonidazole, in contrast, showed sensitization in EMTB/SF and SCC-VII/ SF tumors in excision assays, but not in SCC-VII/SF tumors in the growth delay assay. The TCDSO of MDAHMCa-4 tumors was reduced by a factor of 1.56 when tumor concentrations of pimonidazole were approximately 0.36 pmoles/g, but similar concentrations of etanidazole reduced the TCDSO by a factor of 1.92. These findings appear to confirm the disappointing results of clinical trials of pimonidazole.
REFERENCES Bleehen, N. M.; Newman, H. F. V.; Maughan, T. S.; Workman, P. A multiple dose study of the combined radiosensitizers Ro 03-8799 (pimonidazole) and SR 2508 (etanidazole). lnt. J. Radiat. Oncol. Biol. Phys. 16:1093-1096; 1989. 2. Brown, J. M. Clinical trials of radiosensitizers: what should 1.
we expect? lnt. J. Radiat. Oncol. Biol. Phys. 10:425-9; 1984. considerations in radiosensitizer development. In: Brady, L. W., ed. Radiation sensitizers. Their use in the clinical management of cancer. New York: Masson Publishing USA, Inc.; 1980:213. 4. Bush, R. S.; Jenkin, R. D.; Alit, W. E.; Beale, F. A.; Bean, H.; Dembo, A. J.; Pringle, J. F. Definitive evidence for hypoxic cells influencing cure in cancer therapy. Brit. J. Cancer 37(Suppl. 111):302-6; 1978. T. H.; Han5. Coleman, C. N.; Urtasun, R. C.; Wasserman, cock, S.; Harris, J. W.; Halsey, J.; Hirst, V. K. Initial report of the Phase 1 trial of the hypoxic cell radiosensitizer SR2508. lnt. J. Radiat. Oncol. Biol. Phys. 10:1749-1753; 1984. M. F.; Stratford, M. R.; Wardman, P.; Watts, 6. Dennis, M. E. Cellular uptake of misonidazole and analogues with acidic or basic functions. lnt. J. Radiat. Biol. 47:629-43; 1985.
3. Brown, J. M.; Lee, W. W. Pharmacokinetic
using the hypoxic cell sensitizer 7. Dische, S. Radiotherapy Ro 03-8799 in malignant melanoma. Radiother. Oncol. 10: 11 l-6; 1987. 8. Dische, S.; Bennett, M. H.; Orchard, R.; Stratford, M. R.; Wardman, P. The uptake of the radiosensitizing compound Ro 03-8799 (pimonidazole) in human tumors. lnt. J. Radiat. Oncol. Biol. Phys. 16: 1089-92; 1989. 9. Dische, S.; Saunders, M. I.; Bennett, M. H.; Dunphy, E. P.; Des, R. C.; Stratford, M. R.; Minchinton, A. I.; Wardman, P. A comparison of the tumour concentrations obtainable with misonidazole and Ro 03-8799. Brit. J. Radiol. 59:9 1 l7; 1986. 10. Dische, S.; Saunders, M. I.; Dunphy, E. P.; Bennett, M. H.; Des, R. C.; Stratford, M. R.; Minchinton, A. I.; Orchard, R. A. Concentrations achieved in human tumors after administration of misonidazole, SR-2508 and Ro 03-8799. lnt. J. Radiat. Oncol. Biol. Phys. 12:1109-l 1; 1986. J. S.; Coia, 11. Gatenby, R. A.; Kessler, H. B.; Rosenblum, L. R.; Moldofsky, P. J.; Hartz, W. H.; Broder, G. J. Oxygen distribution in squamous cell carcinoma metastases and its relationship to outcome of radiation therapy. lnt. J. Radiat. Oncol. Biol. Phys. 14:831-8; 1988. 12. Honess, D. J.; Wasserman, T. H.; Workman, P.; Ward, R.; Bleehen, N. M. Additivity of radiosensitization by the com-
Etanidazole and pimonidazole 0 H. B. STONEet al.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
bination of SR 2508 (etanidazole) and Ro 03-8799 (pimonidazole) in a murine tumor system. Int. J. Radiat. Oncol. Biol. Phys. 15:671-675; 1988. Kallinowski, F.; Vaupel, P.; Runkel, S.; Berg, G.; Fortmeyer, H. P.; Baessler, K. H.; Wagner, K.; Mueller-Klieser, W.; Walenta, S. Glucose uptake, lactate release, ketone body turnover, metabolic micromilieu, and pH distributions in human breast cancer xenografts in nude rats. Cancer Res. 48~7264-7272; 1988. Laurent, F.; Canal, P.; Soula, G. Pharmacokinetics of Ro 03-8799 in mice bearing melanosarcoma: comparison with tumors without melanin. Int. J. Radiat. Oncol. Biol. Phys. 16:1101-l 103; 1989. Lespinasse, F.; Thomas, C.; Bonnay, M.; Malaise, E. P.; Guichard, M. Ro 03-8799: Preferential relative uptake in human tumor xenografts compared to a murine tumor: comparison with SR-2508. Int. J. Radiat. Oncol. Biol. Phys. 16:1105-l 109; 1989. Minchinton, A. I.; Stratford, M. R. L. A comparison of tumor and normal tissue levels of acidic, basic and neutral 2-nitroimidazole radiosensitizers in mice. Int. J. Radiat. Oncol. Biol. Phys. 12: 1 117- 1120; 1986. Newman, H. F. V.; Bleehen, N. M.; Ward, R.; Workman, P. Hypoxic cell radiosensitizers in the treatment of high grade gliomas: a new direction using combined Ro 03-8799 (pimonidazole) and SR 2508 (etanidazole). Int. J. Radiat. Oncol. Biol. Phys. 15:677-684; 1988. Newman, H. F. V.; Bleehen, N. M.; Workman, P. A phase I study of the combination of two hypoxic cell radiosensitizers, Ro 03-8799 and SR-2508: toxicity and pharmacokinetics. Int. J. Radiat. Oncol. Biol. Phys. 12:1113-l 116; 1986. Newman, H. F. V.; Bleehen, N. M.; Workman, P. A phase I study of the combined hypoxic cell radiosensitizers, Ro 03-8799 and SR 2508: a preliminary report of single-dose toxicity, pharmacokinetics and tumour concentrations. Brit. J. Radiol. 59:423-425; 1986. Newman, H. F. V.; Ward, R.; Workman, P.; Bleehen, N. M. The multi-dose clinical tolerance and pharmacokinetics of the combined radiosensitizers, Ro 03-8799 (pimonidazole) and SR 2508 (etanidazole). Int. J. Radiat. Oncol. Biol. Phys. 15:1073-1083; 1988. Roberts, J. T.; Bleehen, N. M.; Walton, M. I.; Workman, P. A clinical phase I toxicity study of Ro 03-8799: plasma, urine, tumour and normal brain pharmacokinetics. Brit. J. Radiol. 59:107-l 16; 1986. Roberts, J. T.; Bleehen, N. M.; Workman, P.; Walton, M. I. A phase I study of the hypoxic cell radiosensitizer Ro03-8799. Int. J. Radiat. Oncol. Biol. Phys. 10:1755-1758; 1984. Saunders, M. I.; Anderson, P. J.; Bennett, M. H.; Dische, S.; Minchinton, A.; Stratford, M. R.; Tothill, M. The clinical
24.
25.
26.
27.
28.
29.
30.
3 1.
32.
33.
34.
35.
36.
37.
995
testing of Ro 03-8799-pharmacokinetics, toxicology, tissue and tumor concentrations. Int. J. Radiat. Oncol. Biol. Phys. 10:1759-63; 1984. Saunders, M. I.; Dische, S.; Ferment, D.; Bishop, A.; LenoxSmith, I.; Allen, J. G.; Malcolm, S. L. The radiosensitizer Ro 03-8799 and the concentrations which may be achieved in human turnours: a preliminary study. Brit. J. Cancer 46: 706-10; 1982. Stone, H. B.; Luu, Y. H.; Lam, K. N. Sensitization by SR2508 plus Ro 03-8799. Int. J. Radiat. Oncol. Biol. Phys. 12: 1097-100; 1986. Stone, H. B.; Withers, H. R. Tumor and normal tissue response to metronidazole and irradiation in mice. Radiology 113:441-4; 1974. Stone, H. B.; Withers, H. R. Enhancement of the radioresponse of a murine tumour by a nitroimidazole. Brit. J. Radiol. 48:411-2; 1975. Stone, H. B.; Withers, H. R. Metronidazole: Effect on radiosensitivity of tumor and normal tissues in mice. JNCI 55: 1189-94; 1975. Thistlethwaite, A. J.; Leeper, D. B.; Moylan, D. J., III; Nerlinger, R. E. pH distribution in human tumors. Int. J. Radiat. Oncol. Biol. Phys. 11:1647-1652; 1985. Van den Berg, A. P.; Wike-Hooley, J. L.; Van den BergBlock, A. E.; Van der Zee, J.; Reinhold, H. S. Tumour pH in human mammary carcinoma. Eur. J. Cancer Clin. Oncol. 18:457-462; 1982. Vaupel, P. W.; Frinak, S.; Bicher, H. I. Heterogeneous oxygen partial pressure and pH distribution in C3H mouse mammary adenocarcinoma. Cancer Res. 4 1:2008- 13; 198 1. Walker, A. M.; Suit, H. D. Assessment oflocal tumor control using censored tumor response data. Int. J. Radiat. Oncol. Biol. Phys. 9:383-6; 1983. Watts, M. E.; Dennis, M. F.; Woodcock, M. Uptake and additivity of the radiosensitizing effects of Ro 03-8799 and SR-2508 in mammalian cells in vitro. Brit. J. Radiol. 60: 1233-5; 1987. Wike-Hooley, J. L.; Van den Berg, A. P.; Van der Zee, J.; Reinhold, H. S. Human tumour pH and its variation. Eur. J. Cancer Clin. Oncol. 21:785-791; 1985. Williams, M. V.; Denekamp, J.; Minchinton, A. I.; Stratford, M. R. In vivo assessment of basic 2-nitroimidazole radiosensitizers. Brit. J. Cancer 46: 127-37; 1982. Williams, M. V.; Denekamp, J.; Minchinton, A. I.; Stratford, M. R. In vivo testing of a 2-nitroimidazole radiosensitizer (Ro 03-8799) using repeated administration. Int. J. Radiat. Oncol. Biol. Phys. 8:477-8 1; 1982. Workman, P.; Little, C. J.; Marten, T. R.; Dale, A. D.; Ruane, R. J.; Flockhart, I. R.; Bleehen, N. M. Estimation of the hypoxic cell sensitizer misonidazole and its O-demethylated metabolite in biological chromatography. J. Chromatogr. 145:507-512; 1978.