Inf. J Radialion Oncology Bid Php.. Vol. 23, pp. 593-598 Printed in the U S.A. All rights reserved.
0360.3016/92 $5.00 + .oO Copyright Q 1992 Per&mm Press Lid.
??Hyperthermia Original Contribution
ENHANCEMENT
Department
OF MISONIDAZOLE CHEMOSENSITIZATION BY MILD LOCAL HYPERTHERMIA
KA-Ho
WONG,
PH.D.
of Radiation
Medicine,
University
AND MUNEYASU of Kentucky
URANO, M.D.,
Medical Center,
EFFECT
PH.D.
800 Rose St., Lexington,
KY 40536
In an attempt to increase the chemosensitization effect of the alkylating agents 1,3 bis(2-chloroethyl)-1-nitrosourea (BCNU) and cyclophosphamide (CY), by misonidazole (MISO) at the tumor site, mild hyperthermic treatment (41S”C, 1 hr) was applied at various administration sequences. BHf/Sed mice bearing subcutaneous FSa-II tumors in the foot were used for a tumor growth time assay. Local hyperthermic treatment increased the antitumor activities of BCNU and CY by 1.4 and 2.4 fold, respectively. MIS0 at 2.5 mmole/kg potentiated the antitumor activities of BCNU, but not CY, at normal body temperature. There were no significant improvement of MIS0 chemosensitization when heat was given before the administration of BCNU and CY. However, a significant enhancement of chemosensitization was observed when heat was given after the administration of MIS0 and the alkylating agents. Enhancement ratios of about 2.4 and 4.7 were observed with BCNLJ and CY, respectively. There may be two mechanisms responsible for this thermal enhancement. First, the MIS0 pre-incubation time that was required for the expression of chemosensitization effect decreased substantially at elevated temperatures. This hypothesis was supported by the pharmacokinetic studies that MIS0 was rapidly eliminated from tumors in the first 10 min during the local heat treatment and remained at a plateau with a concentration of about S-fold less than the peak MIS0 concentration in the control tumors. This rapid elimination might result from the increase in the rate of hypoxic metabolism of MIS0 in heated tumors. Second, heat may increase the MISO-alkylating agent interactions, which are independent of pre-incubation time. This effect was especially pronounced in CY because pre-incubation-induced chemosensitization of CY in unheated tumor was insignificant in this study. The significant improvement of MIS0 chemosensitization at moderately elevated temperatures can be useful clinically in combined hyperthermia and chemotherapy treatment. Chemosensitization, metabolism.
Hyperthermia,
Misonidazole,
Alkylating agents, Pharmacokinetics,
Pre-incubation,
Hypoxic
lective sensitization of cytotoxicity in the tumor over the normal tissues ( 11, 18). Recent studies using the rat SC9L tumor system (24, 25) indicated that the hypoxic metabolism of 2-NI in tumors was a prerequisite for chemosensitization in viva In addition, there was no observable correlation between a MISO-induced increase in the peak concentration of alkylating agent in tumor and that in the cytotoxicity (25). If hypoxic metabolism is a necessary condition for chemosensitization of MISO, increasing hypoxic metabolism of MIS0 in tumors through altering the tumor environment should enhance the chemosensitization effect. Recently, Siemann (16) demonstrated that hydralazine enhanced the chemosensitization of 2-NIs by decreasing the tumor O2 tension. In our present study, local hyperthermia was used to increase the efficacy and selectivity of both
INTRODUCTION
Numerous reports have demonstrated that 2-nitroimidazole (2-NI) such as misonidazole (MISO), when used in nontoxic doses, could sensitize the anti-tumor activity of a variety of alkylating agents in vivo with a minimal norma1 tissue toxicity ( 14, 18). This potentiation of cytotoxicity has been termed chemosensitization. The major mechanism(s) of chemosensitization in vivo is not clearly understood at this time. It was suggested that 2-NI, as a potent inhibitor of the hepatic P-450 enzyme system, could interfere with the metabolic inactivation process of some alkylating agents leading to an increase in the effective concentration of the alkylating agents in the body (8, 26). However, this alteration of alkylating agents’ pharmacokinetics hypothesis failed to account for the seReprint requests to: Ka-Ho Wong, Ph.D., Department of Radiation Medicine, University of Kentucky Medical Center, 800 Rose St., Lexington, KY 40536. Acknowledgements-The authors wish to thank N. R. Lomax of the Drug Synthesis and Chemistry Branch, Division of Cancer Treatment, National Cancer Institute for supplying the miso-
nidazole, 1,3 bis (2-chloroethyl)-1-nitrosourea and cyclophosphamide. We would also like to thank R. Reynolds for technical assistance and C. Bowen for preparation of this manuscript. Supported by NC1 grant CA 26350 awarded to M. Urano. Accepted for publication 2 January 1992.
593
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I. J. Radiation Oncology 0 Biology 0 Physics
MIS0 and some alkylating agents by providing a favorable tumor environment. The clinical benefits of local hyperthermia as an adjuvant to radiation therapy has been well-documented and summarized by Overgaard ( 15). Our proposal to use local hyperthermia for enhancing the chemosensitizing effectiveness of MIS0 is based on several experimental observations. Urano and Kahn (22) demonstrated that the hypoxic tumor cell fractions increased in both mouse mammary carcinoma and FSa-II tumors after local hyperthermic treatments. Song et al. (19) reported similar observations. Furthermore, hyperthermia has been shown to increase the bioreductive activation of some 2-NIs in vivo (7, 23). In addition, it has been shown that the antitumor effect of some alkylating agents that are sensitized by MIS0 is also enhanced by hyperthermic treatment (25, 12). The localized heat treatment can confine the enhanced drug interaction at a heated tumor site. Finally, we were encouraged by an early report by Mulcahy et al. (13) that a marked enhancement of CCNU cytotoxicity by MIS0 was achieved at 4 1“C whole body hyperthermia treatment. The two alkylating agents, BCNU and cyclophosphamide (CY), which have demonstrated chemosensitization effect with MIS0 in this study were used (14). To determine whether an increase in chemosensitization is caused by a hyperthermic enhancement of the hypoxic metabolism of MIS0 or an enhancement of the MISO-alkylating agent interactions, two administration schedules were tested: heat given before or after the alkylating agents. The pharmacokinetics of MIS0 undergoing local hyperthermia were also studied to determine the mechanism of action. METHODS
AND MATERIALS
Drugs
MISO, BCNU, and CY were obtained from the Drug Synthesis and Chemistry Branch, Division of Cancer Treatment, National Cancer Institute, Bethesda, MD. BCNU was dissolved in absolute ethanol and mixed with saline. All other drugs were dissolved in saline. Tumor growth (TG) time assay
C3Hf/Sed mice derived from our microorganism-free mouse colony were used as hosts for FSa-II tumors. Procedures for tumor inoculation into the recipient mouse foot are described elsewhere (21). Briefly, single tumor cell suspensions were prepared by trypsinization from the 4th generation tumors and approximately 2 X lo5 cells in 5 ~1 were injected subcutaneously into the foot. The TG time assay measures the time required for a tumor to reach a specific volume ( 1000 mm3 or equivalent to 5 tumor doubling times in this study) from the day of treatment. Tumors at an average diameter of 4 mm (1% hyp* Lauda, Germany.
Volume 23, Number 3, 1992
oxic cell fraction) were used (22). With the animals in individual holders, local hyperthermia was delivered without anesthesia by submerging the tumor-bearing foot into a water bath equipped with a Lauda model MS constant temperature immersion circulator* (kO.05 “C). The three diameters of each tumor (designated as a, b, and c) were measured using a caliper every 2 days after treatment and the tumor volume was calculated using the formula, aabc/6. Since the SCFSa-II tumor growth time is distributed log-normally (21.) the median TG time for 50% of the treated tumors to reach 1000 mm3 was determined by the logit method. All data were presented with a 95% confidence limit. There were eight animals per datum per experiment. The results were the average of two-three independent experiments. Pharmacokinetic
assay
Drugs and hyperthermia treatment were identical to the TG time assay. Tumor bearing mice were sacrificed at various time points after treatment and the plasma and tumor samples were collected. The procedure for sample preparation has been described in detail elsewhere (25). A high performance liquid chromatography (HPLC) method was used for the analysis of MIS0 concentration. MIS0 was separated through a C- 18 (5 CL)reverse phase column.+ The solvents (isocratic) were 30% acetonitrile and 70% water. The flow rate was 1 ml/min. The MIS0 concentration was obtained by integrating the peak area and calculated from a standard calibration curve. Each datum represents the mean f SEM of the natural log of the drug concentration. There were at least four animals (independent, not pool) per datum. Each pharmacokinetic curve was resulted from at least eight independent experiments. Administration schedule
MIS0 at 2.5 mmole/kg was given i.p. Local hyperthermia was given at 4 1.5 “C for 1 hr. Two administration schedules were used: (A) MIS0 + 15 min + alkylating agent + 15 min + heat, and (B) MIS0 + 30 min + heat + 15 min + alkylating agent. The controls were saline alone, MIS0 alone, alkylating agent alone, heat alone, MIS0 + heat, alkylating agent + heat and MIS0 + alkylating agent. In the last group, the administration intervals between MIS0 and alkylating agent were 15 min and 1 hr 45 min which corresponds to the above schedule (A) where MIS0 was given 15 min before alkylating agent then heat and schedule (B) where MIS0 was given followed by heat and alkylating agent, respectively. RESULTS TG time
The TG time was studied as a function of the dose of each alkylating agent, and the slope of the dose response +All&h
Assoc. Inc, Deerheld,
IL.
Chemosensitization and hyperthermia 0 K.-H.
curve was calculated for each treatment regimen by a linear regression method. The TG times of MIS0 alone, heat alone, and MIS0 + heat did not differ from the TG time of the saline alone control (data not shown). The enhancement ratio (ER) due to the chemosensitization effect is expressed as a ratio of the slope of the dose response curve following a test treatment to the slope of the dose response curve following an alkylating agent alone. MIS0 given 15 min before BCNU (ER = 1.24) or local hyperthermia given after BCNU slightly (ER = 1.35) increased the effect of BCNU (Fig. 1A). When heat was given after MIS0 and BCNU, the TG time was greatly
MIS0
+ BCNU + 41.5 C
13
16
14
12
10 0
10
20
30
40
BCNU
(ma/kg)
WONG
AND
M. URANO
595
prolonged (ER = 2.34) and the effect was greater than the summation of MIS0 + BCNU and BCNU + heat treatment. Figure 1-B illustrates the effect of heat on the MIS0 “preincubation” for BCNU chemosensitization. MIS0 given 1 hr 45 min before BCNU significantly increased the TG time (ER = 1.65) as compared to BCNU alone. This ER was larger than that of MIS0 given 15 min before BCNU without heat (ER = 1.24). However, MIS0 + heat followed by BCNU did not further increase the TG time following MIS0 + BCNU (Fig. 1-B). The ER of this treatment sequence (ER = 1.65, Fig. 1B) was significantly smaller than the ER of the MIS0 + BCNU + heat sequence (ER = 2.43, Fig. 1A). A similar sequence-dependent chemosensitization effect with heat was also observed for CY. As shown in Fig. 2A, MIS0 given 15 min before CY without heat did not increase CY activity significantly (ER = 1.07). The effect of CY alone was significantly increased by heat (ER = 2.38). If heat was given after MIS0 + CY treatment, the TG time was increased further to an ER of 4.68. This ER was larger than the summation effect of MIS0 and CY without heat (ER = 1.07) and CY alone given at 4 1.5”C (ER = 2.38). In Fig. 2B, MIS0 alone slightly enhanced the CY activity when it was given at 1 hr 45 min before CY administration (ER = 1.09). However, local hyperthermia showed a minimal effect on the chemosensitization in this treatment scheme (MIS0 + heat + CY, ER = 1.05), as shown by the nearly identical curves in Fig. 2B. The effects of the administration sequence on the chemosensitization of BCNU and CY were summarized as ER values in Table 1.
(A)
MIS0
+
41.5 C + BCNU
20 +
t
10 ’ 0
* 10
20
I 40
30 BCWU
(mglkg)
Fig. 1. The effect of local hyperthermia (41 S’C, 1 hr) on the chemosensitization of BCNU in SCFSa-II tumors. The TG time was expressed as a function of BCNU dose. MIS0 was given at 2.5 mmole/kg, ip. Heat was given after BCNU (A) and before BCNU (B). (Cl)-BCNU alone; (O)-BCNU + heat; (A)-MIS0 + 15 min + BCNU in (A) and MIS0 + 1 hr 45 min + BCNU in (B); (=)-MIS0 + BCNU + heat in (A) and MIS0 + heat + BCNU in (B). The dotted line in (B) was redrawn from the BCNU alone curve in (A).
Pharmacokinetics Figure 3-A shows the biodistribution of MIS0 in plasma. MIS0 reached a peak concentration of about 800 pg/ml and eliminated following an apparent monoexponential function in plasma with a T,,* of 37.3 + 3.3 min. Local hyperthermia applied to the foot tumor at 41.5”C did not change the pharmacokinetics of MIS0 in plasma. Fig. 3B shows the biodistribution of MIS0 in SC FSa-II tumors. MIS0 reached a peak concentration ( 100 pg/g) at about 30 min and eliminated with a TllZ of 45.6 f 14.0 min. Local hyperthermia, when applied 30 min after MIS0 treatment, rapidly decreased the MIS0 concentration in tumor (to about 20 pg/g) in the first 10 min and reached a plateau during the rest of the heating period. DISCUSSION
The effect of drug administration schedule on chemosensitization at normal body temperature has been studied by Siemann and Hill ( 17). They showed that the maximum chemosensitization of CCNU by MIS0 was achieved at least 1 hr after MIS0 treatment rather than when they were administered together. Our previous studies using in situ unclamped and clamped 9L tumors also demonstrated that a 1 hr preincubation of MIS0 was
596
I.
J. Radiation Oncology 0 Biology 0 Physics MIS0
Volume 23, Number 3, 1992
+ CY + 41.5 c
BIODISTRIBUTION
OF MIS0
IN PLASMA
30
P f 3
28
E
26 24
IO.1
300 TIME
CY (WW
(A)
(A) MIS0
BlODlSTRlBUTlON
+ 41.5 c + CY
30
t P L
a-
2
26-
(mln)
OF MIS0
IN TUMOR
z loo0
t
. .. . . . . . .
24
A
m+cy
H
m+ht+y
B
E
y
x 2 5
OCONTRX 0 41.5 100
22 20
/ 10
l”olo
0
50
100
*
’
150
.
’
200
.
’
250 TIME
CY OWW
Fig. 2. The effect of local hyperthermia (41.S°C, 1 hr) on the chemosensitization of CY in SC FSa-II tumors. The TG time was expressed as a function of CY dose. MIS0 was given at 2.5 mmole/kg ip. Heat was given after CY (A) and before CY (B). (El)-CY alone; (O)-CY + heat; (A)-MIS0 + 15 min + CY in (A) and MIS0 + 1 hr 45 min + CY in (B); (w)-MIS0 + CY + heat in (A) and MIS0 + heat + CY in (B). The dotted line in (B) was redrawn from the CY alone curve in (A).
Table 1. The effect of the administration sequence of heat, MIS0 and alkvlatina aaent on chemosensitization
Treatments MIS0 + 15 min + Alk. A.* MIS0 + 1 hr 45 min + Alk. A. Alk. A. + heat MIS0 + Alk. A. + heat MIS0 + heat + Alk. A.
BCNU 1.2 1.7 1.4 2.4 1.7
+ + + + *
0.2 0.4 0.2 0.3 0.4
ratio for CY 1.1 + 1.1 + 2.4 f 4.7 * 1.1 f
’
300
(mln)
(B)
(6)
Enhancement
.
0.6 0.6 0.2 0.5 0.4
Note: Enhancement ratio (+S.D.) was the ratio of the slope of dose-response curve for the test treatment to the slope of dose-response curve for the alkylating agent alone. * Alk. A. = Alkylating agent.
Fig. 3. The biodistribution of MIS0 in plasma (A) and in SC FSa-II tumors (B) with or without local hyperthermia (41.5”C, 1 hr) treatment. MIS0 was given at 2.5 mmole/kg, ip. Open circles represent unheated control and close circles represent locally heated tumors. The dark bar represents the heating period.
for the maximum chemosensitization of BCNU at room temperature (K.-H. Wong, H. Zhang, C. A. Wallen, K. T. Wheeler, Int. J. Radiat. Oncol. Biol. Phys., in press). The present results were consistent with those previous observations. The sensitization of BCNU by MIS0 was larger when MIS0 and BCNU were administered with an interval of 1 hr 45 min (which allowed a sufficient preincubation time) as compared to when they were administered 15 min apart. The chemosensitization of CY was minimal for both 15 min and 1 hr 45 min MIS0 administration intervals (the ER values were 1.07 and 1.09, respectively). The reasons for the lack of MIS0 chemosensitization on CY in FSa-II tumor at normal body temperature are unknown. In the literature, there was a large variation in tumor response for the chemosensitization of CY ( 10). A range from no response to a dose modifying factor of 2 has been observed (1, 20). We are currently studying if the rate of hepatic activation of CY required
Chemosensitization and hyperthertnia 0 K.-H. WONG AND M. URANO
may account for the lack of chemosensitization. The administration schedule may have to adjust accordingly. This study indicated that the preincubation effect of MIS0 was not significantly increased by mild hyperthermic treatment for both BCNU and CY (Figure 1B and 2B). Heat did not further increase the MIS0 chemosensitization of BCNU and CY when it was given before the alkylating agent. These observations were in contrast to those obtained by Mulcahy et al. (13) who used whole body hyperthermia in their study. They observed an increase in chemosensitization effect of MIS0 at elevated temperatures. Since heat was given before CCNU in their experiments, they argued that the major mechanism for the thermal enhancement effect was the increased preincubation effect of MIS0 at elevated temperatures. These results can be reconciled: (A) MIS0 preincubation has reached the maximum in the tumors after 1 hr 45 min and heat added little to increase the preincubation effect (however, the chemosensitization effect of BCNU did increase by heat for the 15 min MIS0 preincubation schedule), and (B) the availability of nitroreductases and/or cofactors (e.g., /3-nicotinamide adenine dinucleotide) that were required for the hypoxic metabolism of MIS0 were limited in this tumor system, resulting in the failure of thermal increase in nitroreduction. The pharmacokinetic data seem to support these hypothesis. Local hyperthermia was given in approximately the peak drug concentration in the tumors, that is, 30 and 15 min following the administration of MIS0 and the alkylating agents, respectively. MIS0 was rapidly eliminated in the initial 10 min heating (probably because of an increase in the rate of MIS0 metabolism) and the MIS0 concentration reached a plateau thereafter. The rapid disappearance of MIS0 indicated that the time required for elimination of the same amount of MIS0 was shorter under local hyperthermia. Nitroreductases and cofactors might have limited the further reduction of MIS0 after IO min heating, resulting in a plateau of MIS0 concentration in the tumor. Taking the TG data into account, we speculated that mild local hyperthermic treatment increases the rate, but not the extent, of the hypoxic metabolism of MIS0 with a resultant decrease in the preincubation time of MIS0 which was required for chemosensitization. This can partly explain a substantial increase in chemosensitization when heat followed MIS0 and BCNU, in spite of the insufficient MIS0 preincubation time (i.e., 15 min) in this treatment schedule (Figure 1A), whereas heat did not enhance the chemosensitization when sufficient preincubation time of MIS0 was allowed (Fig. 1B). The shortening of MIS0 preincubation time is particularly critical for the BCNU treatment because BCNU eliminated very rapidly from the body (9).
597
The greatest chemosensitization effect of BCNU will only be felt when it is administered at a point when a sufficient amount of MIS0 has been metabolized. An enhancement of chemosensitization effect was observed when heat was administered after MIS0 and the alkylating agent. This enhancement effect implies that heat may also increase the MIS0 interaction with BCNU or CY. This effect was especially pronounced on CY since there were very little chemosensitization at normal body temperature for the CY treatment and yet the effect was much larger than that for BCNU. The shortening of preincubation time probably will not play a major role in the CY treatments because MIS0 preincubation time is not a limiting factor in this case (since a 1 hr 45 min administration interval did not improve the chemosensitization of CY significantly). These additional interactions, which were independent of preincubation time, were also observed for BCNU. It was supported by the observation that MIS0 + 1 hr + BCNU + heat (therefore with sufficient preincubation time) gave a similar TG time as MIS0 + 15 min + BCNU + heat (data not shown). Thus, local hyperthermia given following administration of MIS0 and alkylating agents may produce two benefits, namely, the shortening of MIS0 preincubation time and the increase in MISO-alkylating agent chemosensitizing interactions. The relative contribution of these two effects will depend on the administration schedules and the type of alkylating agents used. Further investigations on these two effects that involve experiments of MIS0 metabolism and in vitro clonogenic assays by manipulating various variables (02, pH, co-factors, and temperatures) are currently underway. Finally, this study is of clinical relevance because it was conducted using only a moderately elevated temperature and yet the enhancement of chemosensitization of MIS0 for BCNU and CY was substantial. This mild temperature can be obtained by currently available clinical hyperthermic applicators (6). In addition, the shortening of MIS0 preincubation time implies that a prolonged presence of MIS0 in a tumor is not required before chemosensitization can be initiated at mildly elevated temperatures. The treatment may therefore encompass tumors that contain acute type of hypoxic cell populations. In conclusion, (a) mild local hyperthermia enhanced the MIS0 chemosensitization effect of BCNU and CY, (b) the extent of enhancement was dependent on the type of alkylating agents and administration sequence, (c) heat treatment may increase the rate (but not the extent) of hypoxic metabolism of MISO, leading to a shortening of preincubation time required for chemosensitization, and (d) heat treatment may also increase the MISO-alkylating agent interactions leading to a larger enhancement ratio.
REFERENCES 1. Clement, J. J.; Goman, M. S.; Wodinsky, I.; Catane, R.; Johnson, R. K. Enhancement of anti-tumor activity of alkylating agents by the radiosensitizer misonidazole. Cancer Res. 40: 4165-4172; 1980.
2. Dahl, 0.; Mella, 0. Effect of timing and sequence of hyperthermia and cyclophosphamide on a neurogenic rat tumor (BT4A) in vivo. Cancer 52: 983-987; 1983. 3. Dahl, 0.; Mella, 0. Enhanced effect of combined hyper-
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4. 5. 6. 7.
8.
9.
10. 11.
12.
13.
14.
15.
I. J. Radiation Oncology 0 Biology0 Physics thermia and chemotherapy (Bleomycin, BCNU) in a neurogenic rat tumor (BT4A) in vivo. Anticancer Res. 2: 359364; 1982. Engelhardt, R. Hyperthermia and drugs. Recent Results in Cancer Res. 104: 136-203; 1987. Hahn, G. M. Potential for therapy of drugs and hyperthermia. Cancer Res. 39: 2264-2268; 1979. Hand, J. W. Heat delivery and thermometry in clinical hyperthermia. Recent Results Cancer Res. 104: l-23; 1987. Honess, D. J.; Workman, P.; Morgan, J. E.; Bleehen, N. M. Effects of local hyperthermia on the pharmacokinetics of misonidazole in the anesthetized mouse. Br. J. Cancer 41: 529-540; 1980. Lee, F. Y. F.; Workman, P. Modification of CCNU pharmacokinetics by misonidazole-a major mechanism of chemosensitization in mice. Br. J. Cancer 47: 659-669; 1983. Levin, V. A.; Hoffman, W.; Weinkam, R. J. Pharmacokinetics of BCNU in man: a preliminary study of 20 patients. Cancer Treat. Rep. 62: 1305-1312; 1978. McNally, N. J. Enhancement of chemotherapy agents. Int. J. Radiat. Oncol. Biol. Phys. 8: 593-598; 1982. McNally, N. J.; Stephens, T. C.; Twentyman, P. R.; Hinchliffe, M.; Peacock, H.; Spooner, D. The effect of cytotoxic drugs with or without misonidazole on leucopenia in three strains of mice. Int. J. Radiat. Oncol. Biol. Phys. 8: 659662; 1982. Minor, D. R.; AlIen, R. E.; Alberts, D.; Peng, Y.-M.; Tardelli G. A clinical and pharmacokinetic study of isolated limb perfusion with heat and melphalan for melanoma. Cancer 55: 2638-2644; 1985. Mulcahy, R. T.; Gipp, J. J.; Tanner, M. A. Enhancement of misonidazole chemopotentiation by mild local hyperthermia (4 1“C) in vitro and selective enhancement in vivo. Int. J. Radiat. Biol. 52: 57-65; 1987. Mulcahy, R. T.; Trump, D. L. Clinical chemosensitization by misonidazole and related compounds: a critical evaluation. J. Clin. Oncol. 569-573; 1988. Overgaard, J. The current and potential role of hyperthermia in radiotherapy. Int. J. Radiat. Oncol. Biol. Phys. 16: 535549; 1989.
Volume 23, Number 3, 1992 16. Siemann, D. W. Enhancement of chemotherapy and nitroimidazole-induced chemopotentiation by vasoactive agent hydralazine. Br. J. Cancer 62: 348-353; 1990. 17. Siemann, D. W.; Hill, S. A. Enhanced tumor responses through therapies combining CCNU, MIS0 and radiation. Int. J. Radiat. Oncol. Biol. Phys. 10: 1623-1626; 1984. 18. Siemann, D. W.; Mulcahy, R. T. Sensitization of cancer chemotherapeutic agents by nitroheterocyclics. Biochem. Pharmacol. 35: 11 l-l 15; 1986. 19. Song, C. W.; Rhee, J. G.; Levitt, S. H. Effect of hyperthermia on hypoxic cell fraction in tumor. Int. J. Radiat. Oncol. Biol. Phys. 8: 851-856; 1982. 20. Stepthens, T. C.; Courtenay, V. D.; Mills, J.; Peacock, J. H.; Rose, C. M.; Spooner, D. Enhanced cell killing in Lewis lung carcinoma and a human pancreatic carcinoma xenograft by the combination of cytotoxic drugs and misonidazole. Br. J. Cancer 43: 451-457; 1981. 2 1. Urano, M.; Kahn, J. Some practical questions in the tumor regrowth assay. In: KaIlman, R. F., ed. Rodent tumor models in experimental cancer therapy. New York, NY: Pergamon Press; 1987: 122-127. 22. Urano, M.; Kahn, J. The change in hypoxic and chronically hypoxic cell fraction in murine tumor treated with hyperthermia. Radiat. Res. 96: 549-559; 1983. 23. Walton, M. I.; Bleehen, N. M.; Workman, P. Stimulation by localized tumor hyperthermia of reductive bioactivation of 2-nitroimidazole benznidazole in mice. Cancer Res. 49: 235 l-2355; 1989. 24. Wheeler, K. T.; Wallen, C. A.; Wolf, K. L.; Siemann, D. W. Hypoxic cells and in situ chemopotentiation of the nitrosoureas by misonidazole. Br. J. Cancer 49: 787-793; 1984. 25. Wong, K-H.; Wallen, C. A.; Wheeler, K. T. Chemosensitization of the nitrosoureas by 2nitroimidazoles in the subcutaneous 9L tumor model: pharmacokinetic and structureactivity considerations. Int. J. Radiat. Oncol. Biol. Phys. 18: 1043-1050; 1990. 26. Workman, P.; Twentyman, P. R.; Lee, F. Y. F.; Walton, M. Drug metabolism and chemosensitization: nitroimidazoles as inhibitors of drug metabolism. Biochem. Pharmacol. 32: 857-864; 1983.
0360.3016/92 $5.00 + .oO Copyright Q 1992 Per&mm Press Lid.
??Hyperthermia Original Contribution
ENHANCEMENT
Department
OF MISONIDAZOLE CHEMOSENSITIZATION BY MILD LOCAL HYPERTHERMIA
KA-Ho
WONG,
PH.D.
of Radiation
Medicine,
University
AND MUNEYASU of Kentucky
URANO, M.D.,
Medical Center,
EFFECT
PH.D.
800 Rose St., Lexington,
KY 40536
In an attempt to increase the chemosensitization effect of the alkylating agents 1,3 bis(2-chloroethyl)-1-nitrosourea (BCNU) and cyclophosphamide (CY), by misonidazole (MISO) at the tumor site, mild hyperthermic treatment (41S”C, 1 hr) was applied at various administration sequences. BHf/Sed mice bearing subcutaneous FSa-II tumors in the foot were used for a tumor growth time assay. Local hyperthermic treatment increased the antitumor activities of BCNU and CY by 1.4 and 2.4 fold, respectively. MIS0 at 2.5 mmole/kg potentiated the antitumor activities of BCNU, but not CY, at normal body temperature. There were no significant improvement of MIS0 chemosensitization when heat was given before the administration of BCNU and CY. However, a significant enhancement of chemosensitization was observed when heat was given after the administration of MIS0 and the alkylating agents. Enhancement ratios of about 2.4 and 4.7 were observed with BCNLJ and CY, respectively. There may be two mechanisms responsible for this thermal enhancement. First, the MIS0 pre-incubation time that was required for the expression of chemosensitization effect decreased substantially at elevated temperatures. This hypothesis was supported by the pharmacokinetic studies that MIS0 was rapidly eliminated from tumors in the first 10 min during the local heat treatment and remained at a plateau with a concentration of about S-fold less than the peak MIS0 concentration in the control tumors. This rapid elimination might result from the increase in the rate of hypoxic metabolism of MIS0 in heated tumors. Second, heat may increase the MISO-alkylating agent interactions, which are independent of pre-incubation time. This effect was especially pronounced in CY because pre-incubation-induced chemosensitization of CY in unheated tumor was insignificant in this study. The significant improvement of MIS0 chemosensitization at moderately elevated temperatures can be useful clinically in combined hyperthermia and chemotherapy treatment. Chemosensitization, metabolism.
Hyperthermia,
Misonidazole,
Alkylating agents, Pharmacokinetics,
Pre-incubation,
Hypoxic
lective sensitization of cytotoxicity in the tumor over the normal tissues ( 11, 18). Recent studies using the rat SC9L tumor system (24, 25) indicated that the hypoxic metabolism of 2-NI in tumors was a prerequisite for chemosensitization in viva In addition, there was no observable correlation between a MISO-induced increase in the peak concentration of alkylating agent in tumor and that in the cytotoxicity (25). If hypoxic metabolism is a necessary condition for chemosensitization of MISO, increasing hypoxic metabolism of MIS0 in tumors through altering the tumor environment should enhance the chemosensitization effect. Recently, Siemann (16) demonstrated that hydralazine enhanced the chemosensitization of 2-NIs by decreasing the tumor O2 tension. In our present study, local hyperthermia was used to increase the efficacy and selectivity of both
INTRODUCTION
Numerous reports have demonstrated that 2-nitroimidazole (2-NI) such as misonidazole (MISO), when used in nontoxic doses, could sensitize the anti-tumor activity of a variety of alkylating agents in vivo with a minimal norma1 tissue toxicity ( 14, 18). This potentiation of cytotoxicity has been termed chemosensitization. The major mechanism(s) of chemosensitization in vivo is not clearly understood at this time. It was suggested that 2-NI, as a potent inhibitor of the hepatic P-450 enzyme system, could interfere with the metabolic inactivation process of some alkylating agents leading to an increase in the effective concentration of the alkylating agents in the body (8, 26). However, this alteration of alkylating agents’ pharmacokinetics hypothesis failed to account for the seReprint requests to: Ka-Ho Wong, Ph.D., Department of Radiation Medicine, University of Kentucky Medical Center, 800 Rose St., Lexington, KY 40536. Acknowledgements-The authors wish to thank N. R. Lomax of the Drug Synthesis and Chemistry Branch, Division of Cancer Treatment, National Cancer Institute for supplying the miso-
nidazole, 1,3 bis (2-chloroethyl)-1-nitrosourea and cyclophosphamide. We would also like to thank R. Reynolds for technical assistance and C. Bowen for preparation of this manuscript. Supported by NC1 grant CA 26350 awarded to M. Urano. Accepted for publication 2 January 1992.
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MIS0 and some alkylating agents by providing a favorable tumor environment. The clinical benefits of local hyperthermia as an adjuvant to radiation therapy has been well-documented and summarized by Overgaard ( 15). Our proposal to use local hyperthermia for enhancing the chemosensitizing effectiveness of MIS0 is based on several experimental observations. Urano and Kahn (22) demonstrated that the hypoxic tumor cell fractions increased in both mouse mammary carcinoma and FSa-II tumors after local hyperthermic treatments. Song et al. (19) reported similar observations. Furthermore, hyperthermia has been shown to increase the bioreductive activation of some 2-NIs in vivo (7, 23). In addition, it has been shown that the antitumor effect of some alkylating agents that are sensitized by MIS0 is also enhanced by hyperthermic treatment (25, 12). The localized heat treatment can confine the enhanced drug interaction at a heated tumor site. Finally, we were encouraged by an early report by Mulcahy et al. (13) that a marked enhancement of CCNU cytotoxicity by MIS0 was achieved at 4 1“C whole body hyperthermia treatment. The two alkylating agents, BCNU and cyclophosphamide (CY), which have demonstrated chemosensitization effect with MIS0 in this study were used (14). To determine whether an increase in chemosensitization is caused by a hyperthermic enhancement of the hypoxic metabolism of MIS0 or an enhancement of the MISO-alkylating agent interactions, two administration schedules were tested: heat given before or after the alkylating agents. The pharmacokinetics of MIS0 undergoing local hyperthermia were also studied to determine the mechanism of action. METHODS
AND MATERIALS
Drugs
MISO, BCNU, and CY were obtained from the Drug Synthesis and Chemistry Branch, Division of Cancer Treatment, National Cancer Institute, Bethesda, MD. BCNU was dissolved in absolute ethanol and mixed with saline. All other drugs were dissolved in saline. Tumor growth (TG) time assay
C3Hf/Sed mice derived from our microorganism-free mouse colony were used as hosts for FSa-II tumors. Procedures for tumor inoculation into the recipient mouse foot are described elsewhere (21). Briefly, single tumor cell suspensions were prepared by trypsinization from the 4th generation tumors and approximately 2 X lo5 cells in 5 ~1 were injected subcutaneously into the foot. The TG time assay measures the time required for a tumor to reach a specific volume ( 1000 mm3 or equivalent to 5 tumor doubling times in this study) from the day of treatment. Tumors at an average diameter of 4 mm (1% hyp* Lauda, Germany.
Volume 23, Number 3, 1992
oxic cell fraction) were used (22). With the animals in individual holders, local hyperthermia was delivered without anesthesia by submerging the tumor-bearing foot into a water bath equipped with a Lauda model MS constant temperature immersion circulator* (kO.05 “C). The three diameters of each tumor (designated as a, b, and c) were measured using a caliper every 2 days after treatment and the tumor volume was calculated using the formula, aabc/6. Since the SCFSa-II tumor growth time is distributed log-normally (21.) the median TG time for 50% of the treated tumors to reach 1000 mm3 was determined by the logit method. All data were presented with a 95% confidence limit. There were eight animals per datum per experiment. The results were the average of two-three independent experiments. Pharmacokinetic
assay
Drugs and hyperthermia treatment were identical to the TG time assay. Tumor bearing mice were sacrificed at various time points after treatment and the plasma and tumor samples were collected. The procedure for sample preparation has been described in detail elsewhere (25). A high performance liquid chromatography (HPLC) method was used for the analysis of MIS0 concentration. MIS0 was separated through a C- 18 (5 CL)reverse phase column.+ The solvents (isocratic) were 30% acetonitrile and 70% water. The flow rate was 1 ml/min. The MIS0 concentration was obtained by integrating the peak area and calculated from a standard calibration curve. Each datum represents the mean f SEM of the natural log of the drug concentration. There were at least four animals (independent, not pool) per datum. Each pharmacokinetic curve was resulted from at least eight independent experiments. Administration schedule
MIS0 at 2.5 mmole/kg was given i.p. Local hyperthermia was given at 4 1.5 “C for 1 hr. Two administration schedules were used: (A) MIS0 + 15 min + alkylating agent + 15 min + heat, and (B) MIS0 + 30 min + heat + 15 min + alkylating agent. The controls were saline alone, MIS0 alone, alkylating agent alone, heat alone, MIS0 + heat, alkylating agent + heat and MIS0 + alkylating agent. In the last group, the administration intervals between MIS0 and alkylating agent were 15 min and 1 hr 45 min which corresponds to the above schedule (A) where MIS0 was given 15 min before alkylating agent then heat and schedule (B) where MIS0 was given followed by heat and alkylating agent, respectively. RESULTS TG time
The TG time was studied as a function of the dose of each alkylating agent, and the slope of the dose response +All&h
Assoc. Inc, Deerheld,
IL.
Chemosensitization and hyperthermia 0 K.-H.
curve was calculated for each treatment regimen by a linear regression method. The TG times of MIS0 alone, heat alone, and MIS0 + heat did not differ from the TG time of the saline alone control (data not shown). The enhancement ratio (ER) due to the chemosensitization effect is expressed as a ratio of the slope of the dose response curve following a test treatment to the slope of the dose response curve following an alkylating agent alone. MIS0 given 15 min before BCNU (ER = 1.24) or local hyperthermia given after BCNU slightly (ER = 1.35) increased the effect of BCNU (Fig. 1A). When heat was given after MIS0 and BCNU, the TG time was greatly
MIS0
+ BCNU + 41.5 C
13
16
14
12
10 0
10
20
30
40
BCNU
(ma/kg)
WONG
AND
M. URANO
595
prolonged (ER = 2.34) and the effect was greater than the summation of MIS0 + BCNU and BCNU + heat treatment. Figure 1-B illustrates the effect of heat on the MIS0 “preincubation” for BCNU chemosensitization. MIS0 given 1 hr 45 min before BCNU significantly increased the TG time (ER = 1.65) as compared to BCNU alone. This ER was larger than that of MIS0 given 15 min before BCNU without heat (ER = 1.24). However, MIS0 + heat followed by BCNU did not further increase the TG time following MIS0 + BCNU (Fig. 1-B). The ER of this treatment sequence (ER = 1.65, Fig. 1B) was significantly smaller than the ER of the MIS0 + BCNU + heat sequence (ER = 2.43, Fig. 1A). A similar sequence-dependent chemosensitization effect with heat was also observed for CY. As shown in Fig. 2A, MIS0 given 15 min before CY without heat did not increase CY activity significantly (ER = 1.07). The effect of CY alone was significantly increased by heat (ER = 2.38). If heat was given after MIS0 + CY treatment, the TG time was increased further to an ER of 4.68. This ER was larger than the summation effect of MIS0 and CY without heat (ER = 1.07) and CY alone given at 4 1.5”C (ER = 2.38). In Fig. 2B, MIS0 alone slightly enhanced the CY activity when it was given at 1 hr 45 min before CY administration (ER = 1.09). However, local hyperthermia showed a minimal effect on the chemosensitization in this treatment scheme (MIS0 + heat + CY, ER = 1.05), as shown by the nearly identical curves in Fig. 2B. The effects of the administration sequence on the chemosensitization of BCNU and CY were summarized as ER values in Table 1.
(A)
MIS0
+
41.5 C + BCNU
20 +
t
10 ’ 0
* 10
20
I 40
30 BCWU
(mglkg)
Fig. 1. The effect of local hyperthermia (41 S’C, 1 hr) on the chemosensitization of BCNU in SCFSa-II tumors. The TG time was expressed as a function of BCNU dose. MIS0 was given at 2.5 mmole/kg, ip. Heat was given after BCNU (A) and before BCNU (B). (Cl)-BCNU alone; (O)-BCNU + heat; (A)-MIS0 + 15 min + BCNU in (A) and MIS0 + 1 hr 45 min + BCNU in (B); (=)-MIS0 + BCNU + heat in (A) and MIS0 + heat + BCNU in (B). The dotted line in (B) was redrawn from the BCNU alone curve in (A).
Pharmacokinetics Figure 3-A shows the biodistribution of MIS0 in plasma. MIS0 reached a peak concentration of about 800 pg/ml and eliminated following an apparent monoexponential function in plasma with a T,,* of 37.3 + 3.3 min. Local hyperthermia applied to the foot tumor at 41.5”C did not change the pharmacokinetics of MIS0 in plasma. Fig. 3B shows the biodistribution of MIS0 in SC FSa-II tumors. MIS0 reached a peak concentration ( 100 pg/g) at about 30 min and eliminated with a TllZ of 45.6 f 14.0 min. Local hyperthermia, when applied 30 min after MIS0 treatment, rapidly decreased the MIS0 concentration in tumor (to about 20 pg/g) in the first 10 min and reached a plateau during the rest of the heating period. DISCUSSION
The effect of drug administration schedule on chemosensitization at normal body temperature has been studied by Siemann and Hill ( 17). They showed that the maximum chemosensitization of CCNU by MIS0 was achieved at least 1 hr after MIS0 treatment rather than when they were administered together. Our previous studies using in situ unclamped and clamped 9L tumors also demonstrated that a 1 hr preincubation of MIS0 was
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+ CY + 41.5 c
BIODISTRIBUTION
OF MIS0
IN PLASMA
30
P f 3
28
E
26 24
IO.1
300 TIME
CY (WW
(A)
(A) MIS0
BlODlSTRlBUTlON
+ 41.5 c + CY
30
t P L
a-
2
26-
(mln)
OF MIS0
IN TUMOR
z loo0
t
. .. . . . . . .
24
A
m+cy
H
m+ht+y
B
E
y
x 2 5
OCONTRX 0 41.5 100
22 20
/ 10
l”olo
0
50
100
*
’
150
.
’
200
.
’
250 TIME
CY OWW
Fig. 2. The effect of local hyperthermia (41.S°C, 1 hr) on the chemosensitization of CY in SC FSa-II tumors. The TG time was expressed as a function of CY dose. MIS0 was given at 2.5 mmole/kg ip. Heat was given after CY (A) and before CY (B). (El)-CY alone; (O)-CY + heat; (A)-MIS0 + 15 min + CY in (A) and MIS0 + 1 hr 45 min + CY in (B); (w)-MIS0 + CY + heat in (A) and MIS0 + heat + CY in (B). The dotted line in (B) was redrawn from the CY alone curve in (A).
Table 1. The effect of the administration sequence of heat, MIS0 and alkvlatina aaent on chemosensitization
Treatments MIS0 + 15 min + Alk. A.* MIS0 + 1 hr 45 min + Alk. A. Alk. A. + heat MIS0 + Alk. A. + heat MIS0 + heat + Alk. A.
BCNU 1.2 1.7 1.4 2.4 1.7
+ + + + *
0.2 0.4 0.2 0.3 0.4
ratio for CY 1.1 + 1.1 + 2.4 f 4.7 * 1.1 f
’
300
(mln)
(B)
(6)
Enhancement
.
0.6 0.6 0.2 0.5 0.4
Note: Enhancement ratio (+S.D.) was the ratio of the slope of dose-response curve for the test treatment to the slope of dose-response curve for the alkylating agent alone. * Alk. A. = Alkylating agent.
Fig. 3. The biodistribution of MIS0 in plasma (A) and in SC FSa-II tumors (B) with or without local hyperthermia (41.5”C, 1 hr) treatment. MIS0 was given at 2.5 mmole/kg, ip. Open circles represent unheated control and close circles represent locally heated tumors. The dark bar represents the heating period.
for the maximum chemosensitization of BCNU at room temperature (K.-H. Wong, H. Zhang, C. A. Wallen, K. T. Wheeler, Int. J. Radiat. Oncol. Biol. Phys., in press). The present results were consistent with those previous observations. The sensitization of BCNU by MIS0 was larger when MIS0 and BCNU were administered with an interval of 1 hr 45 min (which allowed a sufficient preincubation time) as compared to when they were administered 15 min apart. The chemosensitization of CY was minimal for both 15 min and 1 hr 45 min MIS0 administration intervals (the ER values were 1.07 and 1.09, respectively). The reasons for the lack of MIS0 chemosensitization on CY in FSa-II tumor at normal body temperature are unknown. In the literature, there was a large variation in tumor response for the chemosensitization of CY ( 10). A range from no response to a dose modifying factor of 2 has been observed (1, 20). We are currently studying if the rate of hepatic activation of CY required
Chemosensitization and hyperthertnia 0 K.-H. WONG AND M. URANO
may account for the lack of chemosensitization. The administration schedule may have to adjust accordingly. This study indicated that the preincubation effect of MIS0 was not significantly increased by mild hyperthermic treatment for both BCNU and CY (Figure 1B and 2B). Heat did not further increase the MIS0 chemosensitization of BCNU and CY when it was given before the alkylating agent. These observations were in contrast to those obtained by Mulcahy et al. (13) who used whole body hyperthermia in their study. They observed an increase in chemosensitization effect of MIS0 at elevated temperatures. Since heat was given before CCNU in their experiments, they argued that the major mechanism for the thermal enhancement effect was the increased preincubation effect of MIS0 at elevated temperatures. These results can be reconciled: (A) MIS0 preincubation has reached the maximum in the tumors after 1 hr 45 min and heat added little to increase the preincubation effect (however, the chemosensitization effect of BCNU did increase by heat for the 15 min MIS0 preincubation schedule), and (B) the availability of nitroreductases and/or cofactors (e.g., /3-nicotinamide adenine dinucleotide) that were required for the hypoxic metabolism of MIS0 were limited in this tumor system, resulting in the failure of thermal increase in nitroreduction. The pharmacokinetic data seem to support these hypothesis. Local hyperthermia was given in approximately the peak drug concentration in the tumors, that is, 30 and 15 min following the administration of MIS0 and the alkylating agents, respectively. MIS0 was rapidly eliminated in the initial 10 min heating (probably because of an increase in the rate of MIS0 metabolism) and the MIS0 concentration reached a plateau thereafter. The rapid disappearance of MIS0 indicated that the time required for elimination of the same amount of MIS0 was shorter under local hyperthermia. Nitroreductases and cofactors might have limited the further reduction of MIS0 after IO min heating, resulting in a plateau of MIS0 concentration in the tumor. Taking the TG data into account, we speculated that mild local hyperthermic treatment increases the rate, but not the extent, of the hypoxic metabolism of MIS0 with a resultant decrease in the preincubation time of MIS0 which was required for chemosensitization. This can partly explain a substantial increase in chemosensitization when heat followed MIS0 and BCNU, in spite of the insufficient MIS0 preincubation time (i.e., 15 min) in this treatment schedule (Figure 1A), whereas heat did not enhance the chemosensitization when sufficient preincubation time of MIS0 was allowed (Fig. 1B). The shortening of MIS0 preincubation time is particularly critical for the BCNU treatment because BCNU eliminated very rapidly from the body (9).
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The greatest chemosensitization effect of BCNU will only be felt when it is administered at a point when a sufficient amount of MIS0 has been metabolized. An enhancement of chemosensitization effect was observed when heat was administered after MIS0 and the alkylating agent. This enhancement effect implies that heat may also increase the MIS0 interaction with BCNU or CY. This effect was especially pronounced on CY since there were very little chemosensitization at normal body temperature for the CY treatment and yet the effect was much larger than that for BCNU. The shortening of preincubation time probably will not play a major role in the CY treatments because MIS0 preincubation time is not a limiting factor in this case (since a 1 hr 45 min administration interval did not improve the chemosensitization of CY significantly). These additional interactions, which were independent of preincubation time, were also observed for BCNU. It was supported by the observation that MIS0 + 1 hr + BCNU + heat (therefore with sufficient preincubation time) gave a similar TG time as MIS0 + 15 min + BCNU + heat (data not shown). Thus, local hyperthermia given following administration of MIS0 and alkylating agents may produce two benefits, namely, the shortening of MIS0 preincubation time and the increase in MISO-alkylating agent chemosensitizing interactions. The relative contribution of these two effects will depend on the administration schedules and the type of alkylating agents used. Further investigations on these two effects that involve experiments of MIS0 metabolism and in vitro clonogenic assays by manipulating various variables (02, pH, co-factors, and temperatures) are currently underway. Finally, this study is of clinical relevance because it was conducted using only a moderately elevated temperature and yet the enhancement of chemosensitization of MIS0 for BCNU and CY was substantial. This mild temperature can be obtained by currently available clinical hyperthermic applicators (6). In addition, the shortening of MIS0 preincubation time implies that a prolonged presence of MIS0 in a tumor is not required before chemosensitization can be initiated at mildly elevated temperatures. The treatment may therefore encompass tumors that contain acute type of hypoxic cell populations. In conclusion, (a) mild local hyperthermia enhanced the MIS0 chemosensitization effect of BCNU and CY, (b) the extent of enhancement was dependent on the type of alkylating agents and administration sequence, (c) heat treatment may increase the rate (but not the extent) of hypoxic metabolism of MISO, leading to a shortening of preincubation time required for chemosensitization, and (d) heat treatment may also increase the MISO-alkylating agent interactions leading to a larger enhancement ratio.
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2. Dahl, 0.; Mella, 0. Effect of timing and sequence of hyperthermia and cyclophosphamide on a neurogenic rat tumor (BT4A) in vivo. Cancer 52: 983-987; 1983. 3. Dahl, 0.; Mella, 0. Enhanced effect of combined hyper-
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I. J. Radiation Oncology 0 Biology0 Physics thermia and chemotherapy (Bleomycin, BCNU) in a neurogenic rat tumor (BT4A) in vivo. Anticancer Res. 2: 359364; 1982. Engelhardt, R. Hyperthermia and drugs. Recent Results in Cancer Res. 104: 136-203; 1987. Hahn, G. M. Potential for therapy of drugs and hyperthermia. Cancer Res. 39: 2264-2268; 1979. Hand, J. W. Heat delivery and thermometry in clinical hyperthermia. Recent Results Cancer Res. 104: l-23; 1987. Honess, D. J.; Workman, P.; Morgan, J. E.; Bleehen, N. M. Effects of local hyperthermia on the pharmacokinetics of misonidazole in the anesthetized mouse. Br. J. Cancer 41: 529-540; 1980. Lee, F. Y. F.; Workman, P. Modification of CCNU pharmacokinetics by misonidazole-a major mechanism of chemosensitization in mice. Br. J. Cancer 47: 659-669; 1983. Levin, V. A.; Hoffman, W.; Weinkam, R. J. Pharmacokinetics of BCNU in man: a preliminary study of 20 patients. Cancer Treat. Rep. 62: 1305-1312; 1978. McNally, N. J. Enhancement of chemotherapy agents. Int. J. Radiat. Oncol. Biol. Phys. 8: 593-598; 1982. McNally, N. J.; Stephens, T. C.; Twentyman, P. R.; Hinchliffe, M.; Peacock, H.; Spooner, D. The effect of cytotoxic drugs with or without misonidazole on leucopenia in three strains of mice. Int. J. Radiat. Oncol. Biol. Phys. 8: 659662; 1982. Minor, D. R.; AlIen, R. E.; Alberts, D.; Peng, Y.-M.; Tardelli G. A clinical and pharmacokinetic study of isolated limb perfusion with heat and melphalan for melanoma. Cancer 55: 2638-2644; 1985. Mulcahy, R. T.; Gipp, J. J.; Tanner, M. A. Enhancement of misonidazole chemopotentiation by mild local hyperthermia (4 1“C) in vitro and selective enhancement in vivo. Int. J. Radiat. Biol. 52: 57-65; 1987. Mulcahy, R. T.; Trump, D. L. Clinical chemosensitization by misonidazole and related compounds: a critical evaluation. J. Clin. Oncol. 569-573; 1988. Overgaard, J. The current and potential role of hyperthermia in radiotherapy. Int. J. Radiat. Oncol. Biol. Phys. 16: 535549; 1989.
Volume 23, Number 3, 1992 16. Siemann, D. W. Enhancement of chemotherapy and nitroimidazole-induced chemopotentiation by vasoactive agent hydralazine. Br. J. Cancer 62: 348-353; 1990. 17. Siemann, D. W.; Hill, S. A. Enhanced tumor responses through therapies combining CCNU, MIS0 and radiation. Int. J. Radiat. Oncol. Biol. Phys. 10: 1623-1626; 1984. 18. Siemann, D. W.; Mulcahy, R. T. Sensitization of cancer chemotherapeutic agents by nitroheterocyclics. Biochem. Pharmacol. 35: 11 l-l 15; 1986. 19. Song, C. W.; Rhee, J. G.; Levitt, S. H. Effect of hyperthermia on hypoxic cell fraction in tumor. Int. J. Radiat. Oncol. Biol. Phys. 8: 851-856; 1982. 20. Stepthens, T. C.; Courtenay, V. D.; Mills, J.; Peacock, J. H.; Rose, C. M.; Spooner, D. Enhanced cell killing in Lewis lung carcinoma and a human pancreatic carcinoma xenograft by the combination of cytotoxic drugs and misonidazole. Br. J. Cancer 43: 451-457; 1981. 2 1. Urano, M.; Kahn, J. Some practical questions in the tumor regrowth assay. In: KaIlman, R. F., ed. Rodent tumor models in experimental cancer therapy. New York, NY: Pergamon Press; 1987: 122-127. 22. Urano, M.; Kahn, J. The change in hypoxic and chronically hypoxic cell fraction in murine tumor treated with hyperthermia. Radiat. Res. 96: 549-559; 1983. 23. Walton, M. I.; Bleehen, N. M.; Workman, P. Stimulation by localized tumor hyperthermia of reductive bioactivation of 2-nitroimidazole benznidazole in mice. Cancer Res. 49: 235 l-2355; 1989. 24. Wheeler, K. T.; Wallen, C. A.; Wolf, K. L.; Siemann, D. W. Hypoxic cells and in situ chemopotentiation of the nitrosoureas by misonidazole. Br. J. Cancer 49: 787-793; 1984. 25. Wong, K-H.; Wallen, C. A.; Wheeler, K. T. Chemosensitization of the nitrosoureas by 2nitroimidazoles in the subcutaneous 9L tumor model: pharmacokinetic and structureactivity considerations. Int. J. Radiat. Oncol. Biol. Phys. 18: 1043-1050; 1990. 26. Workman, P.; Twentyman, P. R.; Lee, F. Y. F.; Walton, M. Drug metabolism and chemosensitization: nitroimidazoles as inhibitors of drug metabolism. Biochem. Pharmacol. 32: 857-864; 1983.
