INT. J. HYPERTHERMIA,
1992,
VOL.
8,
NO.
5, 645-658
Comparative study of thermoradiosensitization by misonidazole and metronidazole in vivo: antitumour effect and pharmacokinetics KA-HO WONGt, HIROSHI MAEZAWA and MUNEYASU URANO University of Kentucky Medical Center, Department of Radiation Medicine, 800 Rose Street, Lexington, KY 405364084. USA Int J Hyperthermia Downloaded from informahealthcare.com by University of Otago on 01/01/15 For personal use only.
(Received 18 November 1991; revised 3 Februury 1992; accepted 7 February 1992)
Tumour control by local hyperthermia (43-5"C, 30 min) and radiation (20 Gy) given in combination with misonidazole (MISO) or metronidazole (METRO) was studied using FSa-I1 murine fibrosarcoma. When MISO or METRO (5 mmollkg) was given 30 min before heat and subsequently treated with radiation, tumour regression was observed for both agents. Radiation dose-response curves for MISO and METRO with heating at 43.5"C for 30 min were identical. Mouse foot reaction was used to evaluate local toxicity following combined heat, a nitroimidazole and radiation treatment. MISO enhanced the magnitude of foot reaction and prolonged the recovery time compared with heat plus radiation controls. There were no observable differences of foot reaction between animals treated with heat plus radiation and those animals treated with heat, radiation and METRO. Pharmacokinetics of the nitroimidazoles heated at 43.5"C for 30 min in FSa-I1 tumours were investigated as a possible mechanism of thermal sensitization. Local hyperthermia did not alter the pharmacokinetics of METRO. Tumour concentration and tumour/plasma ratio of MISO were slightly decreased during heating. Since the hypoxic metabolism of the nitroimidazoles did not increase significantly during the heat treatment, the thermal enhancement of MISO or METRO radiosensitization cannot be explained by the increase in hypoxic cytotoxicity of the nitroimidazoles at elevated temperature alone. The two nitroimidazoles also were not accumulated in the tumour after heating. Therefore, alternation of pharmacokinetics is not the major mechanism for the thermal enhancement of nitroimidazole radiosensitization. The METRO radiosensitization effect became identical to that of MISO at elevated temperatures is of particular importance in clinical radiosensitization. The very low local and systemic toxicity together with the high efficacy of METRO at elevated temperatures will make it an attractive candidate as a future clinical radiosensitizer. Key words: Misonidazole, metronidazole, thermoradiosensitization, skin reaction, pharmacokinetics
1. Introduction It has long been recognized that the hypoxic cell population in tumours is the most probable cause of radiation resistance (Gray 1953). Nitroimidazoles (NIs) have been designed as radiosensitizers which mimic the effect of oxygen. Similar to oxygen, NIs are believed to 'fix' the damage induced by free radicals generated from ionizing radiation in tumour tissues (Kagiya 1984). NIs have an advantage over oxygen as a radiosensitizer: they can diffuse to a greater distance into the hypoxic cell foci without being extensively metabolized by the surrounding aerobic cells. Moreover, NIs may be metabolized at the hypoxic tumour sites and release cytotoxic metabolites (Hall and Roizin-Towle 1975; Heimbooke and Sartorelli 1985, Varghese e? al. 1976). Metronidazole (METRO) is a 5-nitroimidazole that has been studied in the clinic as a potential radiosensitizer. The early phase I and I1 clinical trials demonstrated that, although tTo whom correspondence should be addressed. 0265-6736/92 $3.00 01992 Taylor & Francis Lid
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the drug was relatively non-toxic, the radiosensitizing efficacy to human tumours was minimal (Urtasun et al. 1975, 1976). Thus a more efficacious hypoxic cell sensitizer was sought and misonidazole (MISO) was synthesized (Adams et al. 1979). MISO was one of the few compounds that demonstrated effective radiosensitization with tolerable normal tissue toxicity in animals (Denekemp 1977, Sheldon and Hill 1977). However, subsequent clinical trials were disappointing. MISO-induced neurotoxicity in patients limited the maximum cumulated dose to 12 g/m2 (Wasserman et al. 1981). Radiosensitization was minimal at this dose when it was used with conventional fractionated radiotherapy (Coleman 1985). Since then, radiosensitizers improved through chemical modifications (Narayana and Lee 1982) have been introduced, but clinically useful NI that has a higher efficacy than MISO has yet to be demonstrated. Besides using a chemical modifier in radiosensitization, local hyperthermia has been used to enhance the radiosensitivity of tumours (Dewey et al. 1977). Various mechanisms of thermo-radiosensitization have been proposed, e.g. independent cell killing of heat and radiation (Song 1984), and potentiation of radiation damage by inhibiting the repair mechanisms (Li er al. 1976, Sapareto et al. 1979). Clinical results for combined hyperthermia and radiation treatment of superficial tumours are encouraging (Overgaard 1989). However, the effectiveness of local hyperthermia is limited by the location and size of the tumours (e.g. tumours < 4 cm size were best heated). Furthermore, it has been reported that the hypoxic cell fraction in rodent tumours actually increases after hyperthermic treatment in certain treatment schedules, leading to a subsequent decrease in radiation sensitivity (Song et al. 1982, Urano and Kahn 1983). It was thought that NIs would increase the synergistic interactions between heat and radiation. The NIs may overcome some problems of local hyperthermia (e.g. tumour size limitation and hypoxia). In addition, the hypoxic cytotoxicity of NI may favour treating large bulky tumours that contain significant hypoxic cell fractions. On the other hand, local hyperthermia may increase the ‘activation energy’ of the NI radiosensitization and lead to increased cellular damage in a confined area. Althouth the mechanism for thermal enhancement of NI radiosensitization is unknown, almost all studies have demonstrated that MISO radiosensitization was significantly enhanced by heat (Goldfeder et al. 1979, Hofer et al. 1981, Wondergem et af. 1986). A pilot clinical trial which included combining heat and fractionated radiotherapy with MISO at its maximum tolerable dose demonstrated that this combination resulted in a more superior tumour clearance rate than that of heat plus radiation or radiation alone (Arcangeli et al. 1980). Although the efficacy of MISO has been increased at elevated temperatures, the drug-induced permanent neurotoxicity still limits the clinical application. Therefore, using NI analogues that are less systemically toxic in thermo-radiosensitization appears to be a logical approach, since local hyperthermia may specificially increase the efficacy of these NIs at the tumour sites without increasing the overall systemic toxicity. The investigation to compare the thermo-radiosensitizationof METRO and MISO was initiated because METRO is far less toxic than MISO (Urtasun et al. 1975, 1976), and because there is evidence in vifro that the thermal enhancement of hypoxic cytotoxicity for an NI of less electron affinity is larger in comparison with an NI of higher electron affinity (Rajaratnam et al. 1982). Radiosensitization of MISO and METRO at elevated temperatures has been studied by Hofer et al. (1977). They found enhancement of radiosensitization in hypoxic L1210 tumour cells by both agents. MISO was slightly more effective than METRO. The antitumour activity of MISO and METRO will be further compared using a solid tumour model, FSa-11. Local damage to normal tissues was also investigated as were the roles of pharmacokinetics of these agents in combined heat, NI and radiation treatment.
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2. Materials and methods
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2.1. Drugs MISO and METRO were obtained from the Drug Synthesis and Chemistry Branch, Division of Cancer Treatment, National Cancer Institute. Drugs were dissolved in saline solution before administration. 2.2. Tumour response study C3Hf/Sed mice derived from our microorganism-free mouse colony were used as hosts for FSa-II tumours. Tumours were early generation isotransplants of a spontaneously arising fibrosarcoma from a C3Hf/Sed mouse. Sterilized mouse diets and acidified water were provided ad libitum. Procedures for tumour inoculation into the mouse foot have been described elsewhere (Urano et ul. 1980). Tumours with an average diameter of 4 mm (35 mm' and containing approximately 1 % hypoxic cell fraction) were used (Urano and Kahn 1983). The animals were killed when tumour size reached 1000 mm3 (five tumour doubling times) or the tumour was locally controlled for more than 30 days after treatment. The three diameters of the tumour (a, b and c) were measured using a caliper every 2 days after treatment and the volumes were calculated using the formula: nabd6. Since tumour growth time is distributed log-normally (Urano et al. 1980), the tumour growth (TG) time or the time for 50% of the treated tumours to reach lo00 mm3 was determined by the logit method. Local hyperthermia was performed without anaesthesia using a specially designed holder (Urano et al. 1980). The tumour-bearing foot was exposed and submerged in a water bath equipped with a constant temperature immersion circulator (Lauda model MS. Lauda, Germany). Heating at 43.5"C for 30 rnin was used. Irradiation was performed using a 4 kCi cesium-137 irradiator (J. L. Shepherd Co., San Fernando, CA, USA). Mice were anaesthetized by 60 mg/kg pentobarbital administered i .p. immediately before irradiation. Mice were fitted into a lucite box and a constant air flow was maintained within. Each animal was appropriately shielded by a lead and tungsten collimator and the tumours were irradiated through a 30 x 20 cm field. Dose distribution was uniform ( f 2 %) in the central 6 cm where tumours were located. Each animal was turned over in the middle of irradiation to obtain a uniform dose distribution in the tumour. Dose rate was approximately 5 . 6 Gy/min. Radiation dose to the mouse foot was 20 Gy. Equal doses of MISO or METRO at 5 mmol/kg were given i.p. Four treatment schemes were tested: (i) heat was given 20 min before irradiation; (ii) NI (MISO or METRO) was given 30 min before heat; (iii) MISO or METRO was given 30 rnin before irradiation; and (iv) MISO or METRO was given 30 rnin before heat, then followed by irradiation 20 rnin after the completion of heating. Further studies were performed to compare the efficacy of MISO and METRO at an elevated temperature as a function of radiation dose. A constant dose of MISO or METRO at 2.5 mmol/kg was used. Heat was given 15 rnin before irradiation and irradiation was given 15 min after the completion of heating. 2.3. Normal tissue damage Since skin reaction is the major toxicity in combined local hyperthermia and radiation treatment, the effects of MISO and METRO on the thermo-radiosensitization of the nontumour-bearing mouse foot were studied. Treatment schemes for local hyperthermia and irradiation were the same as in the radiation dose-response study. Skin reaction was scored every 2 days using the reaction score according to our numerical score system (Urano et al. 1988). Treatments were radiation (10 Gy) alone, heat (43.5"C for 30 min) and
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radiation, MISO ( 2 - 5 mmol/kg) or METRO (2-5 mmol/kg) followed by heat and radiation. Ten animals were used in each treatment group, and their daily scores averaged.
2.4. Pharmacokinetics Drug and hyperthermia treatments were identical to those for the tumour and normal tissue assays. MISO or METRO was given at 2 . 5 mmol/kg, i.p. Tumour-bearing mice were killed at various times after treatment and the plasma and tumour samples were collected. Procedures for sample preparation have been described in detail elsewhere (Wong et ul. 1989). A high-performance liquid chromatography (HPLC) method was used to measure MISO and METRO concentrations. The HPLC system (Shimadzu, Columbia, MD, USA) was equipped with a system controller, two solvent delivery units, a solvent mixer and a fixed ultraviolet wavelength detector. A C-18 (5 pm) reverse phase column (Alltech Assoc. Inc., Deerfield, IL, USA) was used. Solvents were 30% acetonitrile and 70% water, isocratic. Flow rate was I ml/min. MISO and METRO concentration was obtained by integrating the peak area at 320 nm and calculated from a standard calibration curve using Dynamax data management software (Rainin, Wobum, MA, USA). Retention times for MISO and METRO were 3.9 and 4.2 min, respectively. The initial portion of the pharmacokinetic curve was fitted by eye since the data were insufficient for a statistical fit. The exponential portion (elimination phase) was fitted by using a computer least-square fitting software. Curves were compared using a r-test for the quality of slopes.
3. Results 3.1 . Tumour response The volume doubling time of the subcutaneous FSa-I1 foot tumour is normally about 2 days. It takes approximtely 10 days for a 4-mm diameter tumour to reach loo0 mm3. As shown in Figure lA, local heat treatment of 43 -5°C for 30 min did not affect the overall TG time (10.6* 1 a2 days). A single dose of 20 Gy irradiation significantly prolonged the TG time for approximately 8 days from the control value to 1 8 . 6 & 2 . 1 days. Heat given 20 min before radiation prolonged the TG time to 2 2 - 7 & 3 3 2 days, an additional 4 days or a total of 12 days' prolongation from the saline-treated control. Figure 1 B illustrates the effect of local hyperthermia on MISO radiosensitization. When MISO alone (5 mmol/kg, i.p.) was administered 30 min before local hyperthermia, a slight increase in TG time of 2 days (12.5 f 1 - 2 days) was observed. MISO also sensitized the FSa-I1 tumour cells to radiation. TG time was increased by 15 days to 25 - 2 i 1 -9 days, an additional 7 days compared with that with 20 Gy irradiation alone. The trimodality combined treatment (MISO plus heat plus radiation) produced a significant potentiation effect. Average tumour size shrank after 20 days and no regrowth was observed. Figure 1C shows the effect of local hyperthermia on METRO radiosensitization. METRO (5 mmol/kg, i.p.) plus hyperthermia did not change the tumour growth. METRO given at room temperature produced a slight radiosensitization; it increased the TG time by about 2 . 6 days to 21.2 f 2.9 days compared with that with radiation alone. However, when METRO was given with heat and radiation, a significant potentiation of tumour response was observed. Average tumour size was maintained at about 250 mm3 for more than 25 days. Radiosensitization by MISO and METRO at 43.5"C was also compared as a function of radiation dose (Figure 2). Since a MISO or METRO dose of 2 . 5 mmol/kg or higher did not further increase thermo-radiosensitization (data not shown), 2 - 5 mmol/kg NI was therefore used for these and subsequent experiments. TG time increased exponentially with radiation doses for both MISO and METRO (some tumour cures occurred when MISO
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or METRO were given with heat and 20 Gy radiation). Their radiation dose-response curves were identical (Figure 2). 3.2. Normal tissue damage Non-tumour bearing mouse feet were exposed to 43.5"C heat for 30 rnin and 10 Gy radiation with or without NI treatment (Figure 3). A 10 Gy irradiation alone did not inflict any normal tissue damage for 48 days (data not shown). Heat plus radiation treatment induced a maximum skin damage (desquamation) between days 15-20 post-treatment and recovered at about 35 days. MISO pretreatment enhanced the magnitude of skin damage. Skin reaction reached a maximum average score of 2 . 5 between days 11-20 post-treatment and, after a slight recovery, the skin reaction score remained at 1 - 5 thereafter. In contrast, METRO pretreatment did not further enhance the skin damage induced by heat plus radiation.
3.3. Pharmacokinetics Pharmacokinetics of MISO in plasma showed that the drug reached a peak concentration of about 600 pg/ml in 20 min and eliminated monoexponentially with a T,,, of 44.7* 2.5 rnin (Figure 4A). The TI,, for MISO elimination in plasma was 54.1 + 8 . 9 rnin during and after the local hyperthermia. No significant difference @>0.5) was found between these two treatments, indicating that local heating at 43 -5°C did not affect MISO TII* * Pharmacokinetics of MISO in the tumour with or without local hyperthermia are shown in Figure 4B. MISO reached a peak concentration of about 100 pg/g in tumours at 30-45 rnin after drug administration. Elimination followed an apparent monoexponential kinetic with a T,,, of 70.9* 16.8 min at natural body temperature. Local hyperthermia slightly decreased the peak tumour concentration ( = 60 pg/g) and slightly increased the elimination TI,, to 109.1 *31.8 min, but the values were not significantly different from that at the natural body temperature (p > 0.5). Some mice in the control group were shamtreated (i.e. they were held in the holders without heat treatment). They showed no difference from the control (data not shown). METRO achieved a peak plasma concentration of approximately 350 pg/ml about 30 rnin after drug administration (Figure 5A). Elimination was also monoexponential with a TI,? of 70.9 f I I - 9 min. Local hyperthermia did not alter the pharmacokinetics of METRO in plasma: the elimination TI/, at 4 3 ~ 5 ° Cwas 70.1 lt6.3 rnin ( p > O - 5 ) . METRO reached a peak tumour concentration in 30 rnin of about 150 pg/g (Figure 5B). Elimination was fitted to a monoexponential function with a TI,, of 169.0*86.2 min. Local hyperthermia slightly reduced the elimination TI,, to 99.6 *23.7 min, which was not significantly different from the control (p > 0.2). Figure 6A shows the tumour/plasma ratio of MISO with or without hyperthermia as a function of time after drug administration. The ratio at natural body temperature was 0-3-0-4, but it was reduced to approximately 0 . 2 during heat treatment. The ratio returned to the unheated level after the heating was terminated (ratio of METRO is shown in Figure 6B). The ratio reached approximately 0 . 4 after 30 rnin at normal body temperature but local hyperthermia did not alter the ratio of METRO. 4. Discussion Alteration in pharmacokinetics has been implicated as a mechanism of thermoradiosensitization for many NIs. Increased release of reactive metabolites of NIs at elevated temperatures may partially account for the cause of the thermal enhancement of NI radiosensitization. Honess et al. (1980) studied the pharmacokinetics of MISO in EMT6 tumourbearing mice. Tumours were heated locally at 44°C for 1 h after anaesthesia and they
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Figure 1. Growth curves of FSa-I1 tumours treated with various combinations of nitroimidazoles. heat and radiation. Treatment doses were fixed: radiation dose was 20 Gy. local hyperthermia was at 43.5"C for 30 min, and the MISO or METRO dose was 5 mmol/kg. (A) Effect of local heat and radiation treatment on tumour growth; El, saline-treated controls; hyperthermia; A, single dose of radiation at 20 Gy; U, hyperthermia given 20 rnin after radiation. (B) Effect of MISO given with heat andlor radiation: heat given 30 rnin after MISO; A, radiation given 30 min after MISO; MISO given 30 rnin before heat, and radiation 20 min after heat. (---) Saline treated control from A. (C) Effect of METRO given
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found a marked decrease both in tumour MISO concentration and tumour/plasma ratio. They also found that the tumour location and size affected the alteration of pharmacokinetics by heat. For other NIs, Walton et al. (1989a,b) showed a decrease in the peak tumour concentration of Ro-03-8799 and benznidazole in KHT sarcoma following local hyperthermia at 4 3 ~ 5 ° Cfor 30 min. They also found a corresponding increase in the concentration of reduced metabolites. Increase in hypoxic metabolism of NI could be responsible for the altered pharmacokinetics observed in these NIs at elevated temperatures. However, the thermal enhancement in radiosensitization was not investigated in most of these pharmacokinetic studies. In the present study, local hyperthermia did not significantly alter the pharmacokinetics of MISO in plasma (Figure 4A), but the MISO concentration was slightly decreased (Figure 4B) in FSa-I1 tumours during the heating period. Decrease was also reflected in the tumour/plasma ratio. These findings are consistent with the results obtained in other tumour systems, although the magnitude of reduction in MISO concentration was much smaller in the FSa-I1 tumours. The present study showed no alteration of METRO pharmacokinetics in either plasma or tumour (Figure 5A,B). Reasons for the different pharmacokinetic alterations observed between MISO and METRO at 43.5"C are unknown. Different enzymes may be involved in the metabolic activation of MISO and METRO, and they may be enhanced differently in a FSa-I1 tumour. The potentiation of the radiation response by local hyperthermia has been shown in numerous animal tumour models (Goldfeder et al. 1979, Hofer et al. 1981, Wondergem et al. 1986). We have shown a significant enhancement of the radiation response in FSaI1 tumours when heated at 43.5"C for 30 min and then irradiated at 20 Gy 20 mm later (Figure IA). MISO is an effective radiation sensitizer; a significant tumour growth delay
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Radiatlon Dose (Gy) Figure 2. Comparison of the effect of MISO and METRO on thermo-radiosensitizationas a function of radiation dose. MISO or METRO ( 2 - 5 mmol/kg) were given 15 min before local hyperthermia (43.5"C. 30 min) and then followed by radiation 15 rnin later. 0, MISO plus heat plus radiation; A, METRO plus heat plus radiation. Each datum is the average from 12-16 mice from two independent experiments. Each error bar is the 95% confidence limit.
was observed when it was given 30 min before radiation (Figure 1B).The pharmacokinetic studies (Figure 4B) showed that MISO concentration was highest in the tumours at this time. When heat was given at 30 min after MISO administration (without radiation), a slight but significant prolongation of tumour growth time was observed. This is probably due to the enhancement of the hypoxic cytotoxicity of MISO at elevated temperatures, which has been demonstrated in various studies in virro (Rajaratnam er al. 1982, Stratford and Adams 1977). The slight increase in hypoxic metabolism of MISO in tumours was supported by our pharmacokinetic study. The MISO concentration and the tumour/plasma ratio were slightly decreased during heating (Figures 4B, 6A). However, a substantial enhancement in MISO radiosensitization was observed when local hyperthermia was given prior to radiation (Figure 1B). This thermal enhancement of MISO radiosensitization cannot be solely explained by a simple additive effect of MISO hypoxic cytotoxicity at a high temperature and radiation sensitization. METRO sensitized the response of FSa-I1 tumour to radiation at normal body temperature (Figure lC), although this sensitization was less than that of MISO. The lower radiosensitization of METRO at normal body temperature is predictable because of lower electron affinity than MISO (Adams et al. 1979). METRO given with local hyperthermia alone did not prolong tumour growth. This appeared to be contradictory to the prediction from studies in vitro that the hypoxic cytotoxicity of an NI of lower electron affinity should have a larger thermal enhancement effect than an NI of higher electron affinity (Rajaratnam et al. 1982). METRO pharmacokinetics in the tumour showed neither a decrease in concentration nor in the tumour/plasma ratio at the elevated temperature, also indicating that the hypoxic metabolism of METRO did not increase in the tumour at this temperature.
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Day8 After Treatment Figure 3. Commparison of the effect of MISO and METRO given in combination with local hyperthermia (43.5"C. 30 min) and radiation (10 Gy) on the skin reaction in non-tumourbearing mice. MISO and METRO doses were 2 . 5 mmol/kg given i.p. El, Local hyperthermia and radiation treatment alone; 4, MISO plus 30 min heat plus 15 min radiation; W, METRO plus 30 min heat plus 15 min radiation. Radiation alone (10 Gy) did not produce observable skin damage. There were 10 animals per group. Each datum is the average &S.E.M.
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Figure 4. Pharmacokinetics of MISO ( 2 . 5 mmollkg) with or without heat (43.5"C, 30 min) treatment. (A) MISO concentration in plasma; (B) MISO concentration in tumours. Curves are for animals treated at room temperature. Exponential portions of the curves were fitted by linear regression. 0, MISO alone; 0, mice treated with local hyperthermia 30 min after administration of MISO. Thick bar on the x-axis represents the heating period. Data were from five to six independent determinations and each is an average from at least three individual mice. Error bars indicate S.E.M.
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Figure 5. Pharmacokinetics of METRO (2.5 mmol/kg) with or without heat (43.5"C. 30 min) treatment. (A) METRO concentration in plasma; (J3) METRO concentration in turnours. Curves are for animals treated at room temperature. Exponential portions of the curves were fitted by linear regression. 0,METRO alone; 0,mice treated with local hyperthermia 30 min after administration of METRO. Thick bar on the x-axis represents the heating period. Data were from five to six independent determinations and each is an average from at least three individual mice. Error bars indicate S.E.M.
However METRO showed the same magnitude of thermal enhancement of radiosensitization as MIS0 when it was combined with heat and radiation (Figures 1C and 2). This potentiation effect cannot be explained by the additive effect of the METRO hypoxic cytotoxicity at a high temperature and radiosensitization. The NI potentiation of the radiation effect by heat cannot be accounted for by the observed pharmacokinetic mechanism. It is especially true for METRO that the enhancement of thermoradiosensitization was substantial without pharmacokinetic alteration at
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Figure 6. Effect of local hyperthermia on the tumour/plasma ratio of the two nitroimidazoles. (A) Tumour/plasma ratio of MISO; (B) tumour/plasma ratio of METRO. Each value was calculated from the data shown in Figures 4 and 5 . Both lines were fitted by eye. 0, Nitroimidazole treatment alone; 0, nitroimidazole with heat treatment. Thick bars on the x-axis represents the heating period. Error bars indicate the 95% cdnfidence limit.
43.5"C.Other mechanisms involved in the thermal potentiation in MISO or METRO radiosensitization must be taken into account. For example, MISO or METRO may alter the repair process of heat and radiation damage, or heat may increase the fixation of combined NI and radiation-induced damages. Although this study showed that the thermal enhancement of NI hypoxic cytotoxicity may not play an important role in vivo, it is intriguing that the thermal enhancement of NI radiosensitization in vivo showed a similar relationship between electron affinity and hypoxic cytotoxicity, i.e. an NI with less electron affinity produces a larger thermal enhancement than one with higher electron affinity. METRO, with less electron-affinity, was able to sensitize the tumour response to radiation at 43-5°C with the same magnitude as MISO, with higher electron affinity. In order to study this thermal enhancement phenomenon further, the hypoxic metabolism (cytotoxicity) of NI has to be dissected out from the NI radiosensitization (oxygen-mimicking) process. We
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are currently investigating this thermal enhancement effect using a cytoplasmic-free (therefore, removing the nitroreductase enzymes) FSa-I1 tumour cell system. Effect of MISO and METRO given in combination with heat and radiation on localized skin reaction was also compared. It has been demonstrated that MISO increased the localized skin reaction to heat plus radiation (Hofer ef al. 1981, Overgaard 1980). However, the enhancement ratio for skin reaction was less than that for tumours, resulting in a therapeutic gain. The increased skin damage may be related to the reactive oxidative species generated during the reductive metabolism of NIs under aerobic conditions (Perez-Reyes et al. 1980). Hyperthermia may enhance this redox process. In contrast, METRO which has lower electron affinity than MISO, may not generate enough oxidative species to enhance the skin reaction. This may explain the less severe skin reaction to METRO given with heat and radiation than that with MISO. In conclusion, this study may have important implications for the clinical use of NIs, heat and radiation: (1) a substantial NI thermoradiosensitization effect was observed with a small (4 mm) tumour containing a small hypoxic cell fraction of about 1 % (Urano and Kahn 1983). One would therefore expect a larger effect in tumours that contained a larger hypoxic cell fraction. (2) METRO with heat produced the same amount of radiosensitization as MISO at the elevated temperature. It should be noted that METRO is a less toxic NI than MISO systemically. The toxic side-effect (GI disturbance) developed in patients had been minimum following daily METRO doses as high as 6 g/m’ (Urtasun et al. 1975, 1976), and the toxicity can be controlled by antiemetics. In addition, this study showed that METRO is also less toxic than MISO to localized skin reaction. Therefore, being less toxic and more effective than most radiosensitizers, METRO plus local hyperthermia would be an attractive treatment regimen in future radiosensitizer clinical trials.
Acknowledgements The authors are grateful to Ms R. Reynolds for her technical and editing assistance. This work is supported by National Cancer Institute grant CA-26350, DHHS. References ADAMS,G. E., CLARKE, E. D., FLOCKHART, I. R., JACOBS,R. S.,SEHMI,D. S., STRATFORD, I. J.. WARDMAN, P., WATTS,M. E., PARRICK, J., WALLACE, R. G. and SMITHEN, C. E.. 1979, Structure-activity relationships in the development of hypoxic cell radiosensitizers. I. Sensitization efficiency. International Journal of Radiation Biology, 35, 133- 150. ARCANGELI, G., BAROLAS, A., MAURO,F., NERRI,C., SPARO,M. and TABOCCHINI, A.. 1980, Multiple daily fractionation radiotherapy in association with hyperthermia and/or misonidazole: experimental and clinical results. Cancer, 45, 2707-27 1 1. COLEMAN, C. N., 1985, Hypoxic cell radiosensitizers: expectations and progress in drug development. International Journal of Radiation Oncology, Biology und Physics, 11, 323-329. DENEKEMP, J., 1977, Tumor regression as a guide to prognosis: a study with experimental animals. British Journal of Radiology, 50, 27 1-279. L. E., SAPRETO,S. A. and GERWECK, L. E., 1977, Cellular responses DEWEY,W. C., HOPEWOOD, to combination of hyperthermia and radiation. Radiobiology, 123, 463-474. GOLDFEDER, A., BROWN,D. M. and BERGER, A., 1979, Enhancement of radioresponse of a mouse mammary carcinoma to combine treatments with hyperthermia and radiosensitizer misonidazole. Cancer Research, 39, 2966-2970. GRAY,L. H., GORGER.A. 0..EBERT,M., HORNSEY, S. andscorn, 0. C. A., 1953, Theconcentration of oxygen dissolved in tissues at the time of irradiation as a factor in radiotherpy. British Journal of Radiology, 26, 638-648. HALL,E. J. and ROIZIN-TOWLE, L., 1975, Hypoxic sensitizers: radiobiological studies at the cellular level. Radiology, 117, 453-457. HEIMBOOKE, D. C. and SARTORELLI, A. C., 1985, Biochemistry of misonidazole reduction by NADPH-cytochrome (P-450)reductase. Molecular Pharmacology, 29, 168- 172.
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HOFER,K. G., HOFER,M. G., IERACITANO, J. and MCLANGHLIN, W. H., 1977,Radiosensitization of hypoxic tumor cells by simultaneous administration of hyperthermia and nitroimidazoles. Radiation Research, 70, 362-377. HOFER,K. G., MACKINNON, A. R.. SCHUBERT, A. L., LEHR,J. E. and GRIMMETT, E. V., 1981, Radiosensitization of tumors and normal tissues by combined treatment with misonidazole and heat. Radiology, 141, 801-809. HONESS,D. J., WORKMAN, P., MORGAN,J. E. and BLEEHEN, N. M., 1980,Effects of local hyperthermia on the pharmacokinetics of misonidazole in the anesthetized mouse. British Journal of Cancer, 41, 529-540. KAGIYA.T., WADE,T. and NISHIMOTO, S. C., 1984,Molecular mechanism of radiosensitization by nitro compounds. In: Modification of Radiosensitivity in Cancer Treatment, edited by T. Sugahara (Japan: Academia Press), pp. 85-106. LI. G. C . , EVANS,R. G. and HAHN,G. M., 1976,Modification and inhibition of potentially lethal X-ray damage by hyperthermia. Radiation Research, 67, 491-501. NARAYANA.V. L. and LEE, W. W., 1982.Development of radiosensitizers: a medicinal chemistry perspective. Advances in Pharmacology and Chemotherapy, 19, 155-204. OVERGAARD, J. ,1980,Effect of misonidazole and hyperthermia on the radiosensitivity of a C3H mouse mammary carcinoma and its surrounding normal tissue. British Journal of Cancer, 41, 10-21. OVERGAARD, J., 1989.The current and potential role of hyperthermia in radiotherapy. International Journal of Radiation Oncology, Biology and Physics, 16, 535-549. PEREZ-REYES, E.. KALYANARAMAN. B. and MASON,R. P., 1980,The reductive metabolism of metronidazole and ronidazole by aerobic liver microsomes. Molecular Pharmacology, 17,
239-244. RAJARATNAM, S.. ADAMS,G. E., STRATFORD, 1. J. and CLARKE, C., 1982,Enhancement of the cytotoxicity of radiosensitizers by modest hyperthermia: the electron-affinity relationship. British Journal of Cancer, 46, 912-917. SARARETO, S. A., RAAPHORST. G. P. and DEWEY,W. C.. 1979,Cell killing and the sequencing of hyperthermia and radiation. International Journal of Radiation Oncology, Biology and Physics, 5 , 343-348. SHELDON, P. W. and HILL. S. A.. 1977,Hypoxic cell radiosensitizers and local control by X-ray of a transplanted tumor in mice. British Journal of Cancer, 35, 795-808. SONG,C. W.. 1984,Effects of local hyperthermia on blood flow and microenvironment: a review. Cancer Research. (Supplement), 44, 472I s-4730s. SONG,C. W., RHEE.J. G. and LEVITT,S. H., 1982,Effect of hyperthermia on hypoxic cell fraction in tumor. International Journal of Radiation Oncology, Biology and Physics, 8, 85 1-856. STRATFORD. I. J. and ADAMS,G. E., 1977,Effect of hyperthermia on differential cytotoxicity of a hypoxic cell radiosensitizer, Ro-07-0582,on mammalian cells in vitro. British Journal of Cancer, 35, 307-313. URANO,M., GERWECK, L. E., EPSTEIN, R., GUNNINGHAM, M. and SUIT,H. D., 1980,Response of a spontaneous murine tumor to hyperthermia: factors which modify the thermal response in vitro. Radiation Research, 83, 3 12-322. URANO, J. and KAHN,J., 1983,The change in hypoxic and chronically hypoxic cell fraction in murine tumor treated with hyperthermia. Radiation Research, 96, 549-559. URANO,M.,KENTON,L. and KAHN,J., 1988,The effect of hyperthermia on the early- and lateappearing mouse foot reactions and on the radiation carcinogensis. I. Effect on the earlyand late-appearing reactions. International Journal ($Radiation Oncology, Biology nnd Physics,
15, 159-166. URTASUN. R., BAND,P.. CHAPMAN, D. J., FELDSTEIN, M. L., MIELKE,B. and FRYER,C., 1976, Radiation and high-dose metronidazole in supratentorial glioblastomas. New England Journal of Medicine, 294, 1364-1367. URTASUN, R., CHAPMAN, J. D., BAND,P., RABIN.H. R., FRYER, C. G. and STURMWIND, J., 1975, Phase I study of high-dose metronidazole: a specific in vivo and in vitro radiosensitizer of hypoxic cells. Radiology, 117, 129-133. VARGHESE, A. J., GALYAS,S. and MOHINDRA, J. K., 1976, Hypoxia-dependent reduction of by Chinese hamster ovary cells and KHT tumor I-(2-nitro- 1-imidazolyl)-3-methoxy-2-propanol cells in vitro and in vivo. Cancer Research, 36, 3761-3765. WALTON,M. I.. BLEEHEN, N. M. and WORKMAN, P., 1989a, Effects of localized tumor hyperthermia on pimonidazole (Ro 03-8799)pharmacokinetics in mice. Brirish Journal of Cancer. 59, 667-673.
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WALTON,M . I., BLEEHEN,N. M. and WORKMAN,P., 1989b, Stimulation of localized tumor hyperthermia of reductive bioactivation of 2-nitroimidazole benznidazole in mice. Cancer Research, 49, 2351-2355. WASSERMAN, T . H . , STETZ,J . and PHILLIPS,T. L., 1981, Clinical trials of misonidazole in the United States. Cancer Clinical Trials, 4, 7-16. WONDERGEM, J . , HAVEMAN, J . , VAN DER SCHUEREN, E., VAN DER HOEREN,H.and BEUR,K.,1986, Effect of hyperthermia and misonidazole on the radiosensitivity of a transplantable murine tumor: influence of factors modifying the fraction of hypoxic cells. International Journal of Radiation Oncology, Biology and Physics, 8, 1323-1331. WONG, K-H., WALLEN,C. A. and WHEELER,K. T., 1989, Biodistribution on misonidazole and I ,3-bis(2-chloroethyl)-l-nitrosourea(BCNU) in rats bearing unclamped and clamped 9L subcutaneous tumors. International Journal of Radiation Oncology, Biology and Physics, 17, 135- 143.
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Comparative study of thermoradiosensitization by misonidazole and metronidazole in vivo: antitumour effect and pharmacokinetics KA-HO WONGt, HIROSHI MAEZAWA and MUNEYASU URANO University of Kentucky Medical Center, Department of Radiation Medicine, 800 Rose Street, Lexington, KY 405364084. USA Int J Hyperthermia Downloaded from informahealthcare.com by University of Otago on 01/01/15 For personal use only.
(Received 18 November 1991; revised 3 Februury 1992; accepted 7 February 1992)
Tumour control by local hyperthermia (43-5"C, 30 min) and radiation (20 Gy) given in combination with misonidazole (MISO) or metronidazole (METRO) was studied using FSa-I1 murine fibrosarcoma. When MISO or METRO (5 mmollkg) was given 30 min before heat and subsequently treated with radiation, tumour regression was observed for both agents. Radiation dose-response curves for MISO and METRO with heating at 43.5"C for 30 min were identical. Mouse foot reaction was used to evaluate local toxicity following combined heat, a nitroimidazole and radiation treatment. MISO enhanced the magnitude of foot reaction and prolonged the recovery time compared with heat plus radiation controls. There were no observable differences of foot reaction between animals treated with heat plus radiation and those animals treated with heat, radiation and METRO. Pharmacokinetics of the nitroimidazoles heated at 43.5"C for 30 min in FSa-I1 tumours were investigated as a possible mechanism of thermal sensitization. Local hyperthermia did not alter the pharmacokinetics of METRO. Tumour concentration and tumour/plasma ratio of MISO were slightly decreased during heating. Since the hypoxic metabolism of the nitroimidazoles did not increase significantly during the heat treatment, the thermal enhancement of MISO or METRO radiosensitization cannot be explained by the increase in hypoxic cytotoxicity of the nitroimidazoles at elevated temperature alone. The two nitroimidazoles also were not accumulated in the tumour after heating. Therefore, alternation of pharmacokinetics is not the major mechanism for the thermal enhancement of nitroimidazole radiosensitization. The METRO radiosensitization effect became identical to that of MISO at elevated temperatures is of particular importance in clinical radiosensitization. The very low local and systemic toxicity together with the high efficacy of METRO at elevated temperatures will make it an attractive candidate as a future clinical radiosensitizer. Key words: Misonidazole, metronidazole, thermoradiosensitization, skin reaction, pharmacokinetics
1. Introduction It has long been recognized that the hypoxic cell population in tumours is the most probable cause of radiation resistance (Gray 1953). Nitroimidazoles (NIs) have been designed as radiosensitizers which mimic the effect of oxygen. Similar to oxygen, NIs are believed to 'fix' the damage induced by free radicals generated from ionizing radiation in tumour tissues (Kagiya 1984). NIs have an advantage over oxygen as a radiosensitizer: they can diffuse to a greater distance into the hypoxic cell foci without being extensively metabolized by the surrounding aerobic cells. Moreover, NIs may be metabolized at the hypoxic tumour sites and release cytotoxic metabolites (Hall and Roizin-Towle 1975; Heimbooke and Sartorelli 1985, Varghese e? al. 1976). Metronidazole (METRO) is a 5-nitroimidazole that has been studied in the clinic as a potential radiosensitizer. The early phase I and I1 clinical trials demonstrated that, although tTo whom correspondence should be addressed. 0265-6736/92 $3.00 01992 Taylor & Francis Lid
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the drug was relatively non-toxic, the radiosensitizing efficacy to human tumours was minimal (Urtasun et al. 1975, 1976). Thus a more efficacious hypoxic cell sensitizer was sought and misonidazole (MISO) was synthesized (Adams et al. 1979). MISO was one of the few compounds that demonstrated effective radiosensitization with tolerable normal tissue toxicity in animals (Denekemp 1977, Sheldon and Hill 1977). However, subsequent clinical trials were disappointing. MISO-induced neurotoxicity in patients limited the maximum cumulated dose to 12 g/m2 (Wasserman et al. 1981). Radiosensitization was minimal at this dose when it was used with conventional fractionated radiotherapy (Coleman 1985). Since then, radiosensitizers improved through chemical modifications (Narayana and Lee 1982) have been introduced, but clinically useful NI that has a higher efficacy than MISO has yet to be demonstrated. Besides using a chemical modifier in radiosensitization, local hyperthermia has been used to enhance the radiosensitivity of tumours (Dewey et al. 1977). Various mechanisms of thermo-radiosensitization have been proposed, e.g. independent cell killing of heat and radiation (Song 1984), and potentiation of radiation damage by inhibiting the repair mechanisms (Li er al. 1976, Sapareto et al. 1979). Clinical results for combined hyperthermia and radiation treatment of superficial tumours are encouraging (Overgaard 1989). However, the effectiveness of local hyperthermia is limited by the location and size of the tumours (e.g. tumours < 4 cm size were best heated). Furthermore, it has been reported that the hypoxic cell fraction in rodent tumours actually increases after hyperthermic treatment in certain treatment schedules, leading to a subsequent decrease in radiation sensitivity (Song et al. 1982, Urano and Kahn 1983). It was thought that NIs would increase the synergistic interactions between heat and radiation. The NIs may overcome some problems of local hyperthermia (e.g. tumour size limitation and hypoxia). In addition, the hypoxic cytotoxicity of NI may favour treating large bulky tumours that contain significant hypoxic cell fractions. On the other hand, local hyperthermia may increase the ‘activation energy’ of the NI radiosensitization and lead to increased cellular damage in a confined area. Althouth the mechanism for thermal enhancement of NI radiosensitization is unknown, almost all studies have demonstrated that MISO radiosensitization was significantly enhanced by heat (Goldfeder et al. 1979, Hofer et al. 1981, Wondergem et af. 1986). A pilot clinical trial which included combining heat and fractionated radiotherapy with MISO at its maximum tolerable dose demonstrated that this combination resulted in a more superior tumour clearance rate than that of heat plus radiation or radiation alone (Arcangeli et al. 1980). Although the efficacy of MISO has been increased at elevated temperatures, the drug-induced permanent neurotoxicity still limits the clinical application. Therefore, using NI analogues that are less systemically toxic in thermo-radiosensitization appears to be a logical approach, since local hyperthermia may specificially increase the efficacy of these NIs at the tumour sites without increasing the overall systemic toxicity. The investigation to compare the thermo-radiosensitizationof METRO and MISO was initiated because METRO is far less toxic than MISO (Urtasun et al. 1975, 1976), and because there is evidence in vifro that the thermal enhancement of hypoxic cytotoxicity for an NI of less electron affinity is larger in comparison with an NI of higher electron affinity (Rajaratnam et al. 1982). Radiosensitization of MISO and METRO at elevated temperatures has been studied by Hofer et al. (1977). They found enhancement of radiosensitization in hypoxic L1210 tumour cells by both agents. MISO was slightly more effective than METRO. The antitumour activity of MISO and METRO will be further compared using a solid tumour model, FSa-11. Local damage to normal tissues was also investigated as were the roles of pharmacokinetics of these agents in combined heat, NI and radiation treatment.
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2. Materials and methods
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2.1. Drugs MISO and METRO were obtained from the Drug Synthesis and Chemistry Branch, Division of Cancer Treatment, National Cancer Institute. Drugs were dissolved in saline solution before administration. 2.2. Tumour response study C3Hf/Sed mice derived from our microorganism-free mouse colony were used as hosts for FSa-II tumours. Tumours were early generation isotransplants of a spontaneously arising fibrosarcoma from a C3Hf/Sed mouse. Sterilized mouse diets and acidified water were provided ad libitum. Procedures for tumour inoculation into the mouse foot have been described elsewhere (Urano et ul. 1980). Tumours with an average diameter of 4 mm (35 mm' and containing approximately 1 % hypoxic cell fraction) were used (Urano and Kahn 1983). The animals were killed when tumour size reached 1000 mm3 (five tumour doubling times) or the tumour was locally controlled for more than 30 days after treatment. The three diameters of the tumour (a, b and c) were measured using a caliper every 2 days after treatment and the volumes were calculated using the formula: nabd6. Since tumour growth time is distributed log-normally (Urano et al. 1980), the tumour growth (TG) time or the time for 50% of the treated tumours to reach lo00 mm3 was determined by the logit method. Local hyperthermia was performed without anaesthesia using a specially designed holder (Urano et al. 1980). The tumour-bearing foot was exposed and submerged in a water bath equipped with a constant temperature immersion circulator (Lauda model MS. Lauda, Germany). Heating at 43.5"C for 30 rnin was used. Irradiation was performed using a 4 kCi cesium-137 irradiator (J. L. Shepherd Co., San Fernando, CA, USA). Mice were anaesthetized by 60 mg/kg pentobarbital administered i .p. immediately before irradiation. Mice were fitted into a lucite box and a constant air flow was maintained within. Each animal was appropriately shielded by a lead and tungsten collimator and the tumours were irradiated through a 30 x 20 cm field. Dose distribution was uniform ( f 2 %) in the central 6 cm where tumours were located. Each animal was turned over in the middle of irradiation to obtain a uniform dose distribution in the tumour. Dose rate was approximately 5 . 6 Gy/min. Radiation dose to the mouse foot was 20 Gy. Equal doses of MISO or METRO at 5 mmol/kg were given i.p. Four treatment schemes were tested: (i) heat was given 20 min before irradiation; (ii) NI (MISO or METRO) was given 30 min before heat; (iii) MISO or METRO was given 30 rnin before irradiation; and (iv) MISO or METRO was given 30 rnin before heat, then followed by irradiation 20 rnin after the completion of heating. Further studies were performed to compare the efficacy of MISO and METRO at an elevated temperature as a function of radiation dose. A constant dose of MISO or METRO at 2.5 mmol/kg was used. Heat was given 15 rnin before irradiation and irradiation was given 15 min after the completion of heating. 2.3. Normal tissue damage Since skin reaction is the major toxicity in combined local hyperthermia and radiation treatment, the effects of MISO and METRO on the thermo-radiosensitization of the nontumour-bearing mouse foot were studied. Treatment schemes for local hyperthermia and irradiation were the same as in the radiation dose-response study. Skin reaction was scored every 2 days using the reaction score according to our numerical score system (Urano et al. 1988). Treatments were radiation (10 Gy) alone, heat (43.5"C for 30 min) and
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radiation, MISO ( 2 - 5 mmol/kg) or METRO (2-5 mmol/kg) followed by heat and radiation. Ten animals were used in each treatment group, and their daily scores averaged.
2.4. Pharmacokinetics Drug and hyperthermia treatments were identical to those for the tumour and normal tissue assays. MISO or METRO was given at 2 . 5 mmol/kg, i.p. Tumour-bearing mice were killed at various times after treatment and the plasma and tumour samples were collected. Procedures for sample preparation have been described in detail elsewhere (Wong et ul. 1989). A high-performance liquid chromatography (HPLC) method was used to measure MISO and METRO concentrations. The HPLC system (Shimadzu, Columbia, MD, USA) was equipped with a system controller, two solvent delivery units, a solvent mixer and a fixed ultraviolet wavelength detector. A C-18 (5 pm) reverse phase column (Alltech Assoc. Inc., Deerfield, IL, USA) was used. Solvents were 30% acetonitrile and 70% water, isocratic. Flow rate was I ml/min. MISO and METRO concentration was obtained by integrating the peak area at 320 nm and calculated from a standard calibration curve using Dynamax data management software (Rainin, Wobum, MA, USA). Retention times for MISO and METRO were 3.9 and 4.2 min, respectively. The initial portion of the pharmacokinetic curve was fitted by eye since the data were insufficient for a statistical fit. The exponential portion (elimination phase) was fitted by using a computer least-square fitting software. Curves were compared using a r-test for the quality of slopes.
3. Results 3.1 . Tumour response The volume doubling time of the subcutaneous FSa-I1 foot tumour is normally about 2 days. It takes approximtely 10 days for a 4-mm diameter tumour to reach loo0 mm3. As shown in Figure lA, local heat treatment of 43 -5°C for 30 min did not affect the overall TG time (10.6* 1 a2 days). A single dose of 20 Gy irradiation significantly prolonged the TG time for approximately 8 days from the control value to 1 8 . 6 & 2 . 1 days. Heat given 20 min before radiation prolonged the TG time to 2 2 - 7 & 3 3 2 days, an additional 4 days or a total of 12 days' prolongation from the saline-treated control. Figure 1 B illustrates the effect of local hyperthermia on MISO radiosensitization. When MISO alone (5 mmol/kg, i.p.) was administered 30 min before local hyperthermia, a slight increase in TG time of 2 days (12.5 f 1 - 2 days) was observed. MISO also sensitized the FSa-I1 tumour cells to radiation. TG time was increased by 15 days to 25 - 2 i 1 -9 days, an additional 7 days compared with that with 20 Gy irradiation alone. The trimodality combined treatment (MISO plus heat plus radiation) produced a significant potentiation effect. Average tumour size shrank after 20 days and no regrowth was observed. Figure 1C shows the effect of local hyperthermia on METRO radiosensitization. METRO (5 mmol/kg, i.p.) plus hyperthermia did not change the tumour growth. METRO given at room temperature produced a slight radiosensitization; it increased the TG time by about 2 . 6 days to 21.2 f 2.9 days compared with that with radiation alone. However, when METRO was given with heat and radiation, a significant potentiation of tumour response was observed. Average tumour size was maintained at about 250 mm3 for more than 25 days. Radiosensitization by MISO and METRO at 43.5"C was also compared as a function of radiation dose (Figure 2). Since a MISO or METRO dose of 2 . 5 mmol/kg or higher did not further increase thermo-radiosensitization (data not shown), 2 - 5 mmol/kg NI was therefore used for these and subsequent experiments. TG time increased exponentially with radiation doses for both MISO and METRO (some tumour cures occurred when MISO
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or METRO were given with heat and 20 Gy radiation). Their radiation dose-response curves were identical (Figure 2). 3.2. Normal tissue damage Non-tumour bearing mouse feet were exposed to 43.5"C heat for 30 rnin and 10 Gy radiation with or without NI treatment (Figure 3). A 10 Gy irradiation alone did not inflict any normal tissue damage for 48 days (data not shown). Heat plus radiation treatment induced a maximum skin damage (desquamation) between days 15-20 post-treatment and recovered at about 35 days. MISO pretreatment enhanced the magnitude of skin damage. Skin reaction reached a maximum average score of 2 . 5 between days 11-20 post-treatment and, after a slight recovery, the skin reaction score remained at 1 - 5 thereafter. In contrast, METRO pretreatment did not further enhance the skin damage induced by heat plus radiation.
3.3. Pharmacokinetics Pharmacokinetics of MISO in plasma showed that the drug reached a peak concentration of about 600 pg/ml in 20 min and eliminated monoexponentially with a T,,, of 44.7* 2.5 rnin (Figure 4A). The TI,, for MISO elimination in plasma was 54.1 + 8 . 9 rnin during and after the local hyperthermia. No significant difference @>0.5) was found between these two treatments, indicating that local heating at 43 -5°C did not affect MISO TII* * Pharmacokinetics of MISO in the tumour with or without local hyperthermia are shown in Figure 4B. MISO reached a peak concentration of about 100 pg/g in tumours at 30-45 rnin after drug administration. Elimination followed an apparent monoexponential kinetic with a T,,, of 70.9* 16.8 min at natural body temperature. Local hyperthermia slightly decreased the peak tumour concentration ( = 60 pg/g) and slightly increased the elimination TI,, to 109.1 *31.8 min, but the values were not significantly different from that at the natural body temperature (p > 0.5). Some mice in the control group were shamtreated (i.e. they were held in the holders without heat treatment). They showed no difference from the control (data not shown). METRO achieved a peak plasma concentration of approximately 350 pg/ml about 30 rnin after drug administration (Figure 5A). Elimination was also monoexponential with a TI,? of 70.9 f I I - 9 min. Local hyperthermia did not alter the pharmacokinetics of METRO in plasma: the elimination TI/, at 4 3 ~ 5 ° Cwas 70.1 lt6.3 rnin ( p > O - 5 ) . METRO reached a peak tumour concentration in 30 rnin of about 150 pg/g (Figure 5B). Elimination was fitted to a monoexponential function with a TI,, of 169.0*86.2 min. Local hyperthermia slightly reduced the elimination TI,, to 99.6 *23.7 min, which was not significantly different from the control (p > 0.2). Figure 6A shows the tumour/plasma ratio of MISO with or without hyperthermia as a function of time after drug administration. The ratio at natural body temperature was 0-3-0-4, but it was reduced to approximately 0 . 2 during heat treatment. The ratio returned to the unheated level after the heating was terminated (ratio of METRO is shown in Figure 6B). The ratio reached approximately 0 . 4 after 30 rnin at normal body temperature but local hyperthermia did not alter the ratio of METRO. 4. Discussion Alteration in pharmacokinetics has been implicated as a mechanism of thermoradiosensitization for many NIs. Increased release of reactive metabolites of NIs at elevated temperatures may partially account for the cause of the thermal enhancement of NI radiosensitization. Honess et al. (1980) studied the pharmacokinetics of MISO in EMT6 tumourbearing mice. Tumours were heated locally at 44°C for 1 h after anaesthesia and they
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Figure 1. Growth curves of FSa-I1 tumours treated with various combinations of nitroimidazoles. heat and radiation. Treatment doses were fixed: radiation dose was 20 Gy. local hyperthermia was at 43.5"C for 30 min, and the MISO or METRO dose was 5 mmol/kg. (A) Effect of local heat and radiation treatment on tumour growth; El, saline-treated controls; hyperthermia; A, single dose of radiation at 20 Gy; U, hyperthermia given 20 rnin after radiation. (B) Effect of MISO given with heat andlor radiation: heat given 30 rnin after MISO; A, radiation given 30 min after MISO; MISO given 30 rnin before heat, and radiation 20 min after heat. (---) Saline treated control from A. (C) Effect of METRO given
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found a marked decrease both in tumour MISO concentration and tumour/plasma ratio. They also found that the tumour location and size affected the alteration of pharmacokinetics by heat. For other NIs, Walton et al. (1989a,b) showed a decrease in the peak tumour concentration of Ro-03-8799 and benznidazole in KHT sarcoma following local hyperthermia at 4 3 ~ 5 ° Cfor 30 min. They also found a corresponding increase in the concentration of reduced metabolites. Increase in hypoxic metabolism of NI could be responsible for the altered pharmacokinetics observed in these NIs at elevated temperatures. However, the thermal enhancement in radiosensitization was not investigated in most of these pharmacokinetic studies. In the present study, local hyperthermia did not significantly alter the pharmacokinetics of MISO in plasma (Figure 4A), but the MISO concentration was slightly decreased (Figure 4B) in FSa-I1 tumours during the heating period. Decrease was also reflected in the tumour/plasma ratio. These findings are consistent with the results obtained in other tumour systems, although the magnitude of reduction in MISO concentration was much smaller in the FSa-I1 tumours. The present study showed no alteration of METRO pharmacokinetics in either plasma or tumour (Figure 5A,B). Reasons for the different pharmacokinetic alterations observed between MISO and METRO at 43.5"C are unknown. Different enzymes may be involved in the metabolic activation of MISO and METRO, and they may be enhanced differently in a FSa-I1 tumour. The potentiation of the radiation response by local hyperthermia has been shown in numerous animal tumour models (Goldfeder et al. 1979, Hofer et al. 1981, Wondergem et al. 1986). We have shown a significant enhancement of the radiation response in FSaI1 tumours when heated at 43.5"C for 30 min and then irradiated at 20 Gy 20 mm later (Figure IA). MISO is an effective radiation sensitizer; a significant tumour growth delay
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Radiatlon Dose (Gy) Figure 2. Comparison of the effect of MISO and METRO on thermo-radiosensitizationas a function of radiation dose. MISO or METRO ( 2 - 5 mmol/kg) were given 15 min before local hyperthermia (43.5"C. 30 min) and then followed by radiation 15 rnin later. 0, MISO plus heat plus radiation; A, METRO plus heat plus radiation. Each datum is the average from 12-16 mice from two independent experiments. Each error bar is the 95% confidence limit.
was observed when it was given 30 min before radiation (Figure 1B).The pharmacokinetic studies (Figure 4B) showed that MISO concentration was highest in the tumours at this time. When heat was given at 30 min after MISO administration (without radiation), a slight but significant prolongation of tumour growth time was observed. This is probably due to the enhancement of the hypoxic cytotoxicity of MISO at elevated temperatures, which has been demonstrated in various studies in virro (Rajaratnam er al. 1982, Stratford and Adams 1977). The slight increase in hypoxic metabolism of MISO in tumours was supported by our pharmacokinetic study. The MISO concentration and the tumour/plasma ratio were slightly decreased during heating (Figures 4B, 6A). However, a substantial enhancement in MISO radiosensitization was observed when local hyperthermia was given prior to radiation (Figure 1B). This thermal enhancement of MISO radiosensitization cannot be solely explained by a simple additive effect of MISO hypoxic cytotoxicity at a high temperature and radiation sensitization. METRO sensitized the response of FSa-I1 tumour to radiation at normal body temperature (Figure lC), although this sensitization was less than that of MISO. The lower radiosensitization of METRO at normal body temperature is predictable because of lower electron affinity than MISO (Adams et al. 1979). METRO given with local hyperthermia alone did not prolong tumour growth. This appeared to be contradictory to the prediction from studies in vitro that the hypoxic cytotoxicity of an NI of lower electron affinity should have a larger thermal enhancement effect than an NI of higher electron affinity (Rajaratnam et al. 1982). METRO pharmacokinetics in the tumour showed neither a decrease in concentration nor in the tumour/plasma ratio at the elevated temperature, also indicating that the hypoxic metabolism of METRO did not increase in the tumour at this temperature.
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Day8 After Treatment Figure 3. Commparison of the effect of MISO and METRO given in combination with local hyperthermia (43.5"C. 30 min) and radiation (10 Gy) on the skin reaction in non-tumourbearing mice. MISO and METRO doses were 2 . 5 mmol/kg given i.p. El, Local hyperthermia and radiation treatment alone; 4, MISO plus 30 min heat plus 15 min radiation; W, METRO plus 30 min heat plus 15 min radiation. Radiation alone (10 Gy) did not produce observable skin damage. There were 10 animals per group. Each datum is the average &S.E.M.
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Figure 4. Pharmacokinetics of MISO ( 2 . 5 mmollkg) with or without heat (43.5"C, 30 min) treatment. (A) MISO concentration in plasma; (B) MISO concentration in tumours. Curves are for animals treated at room temperature. Exponential portions of the curves were fitted by linear regression. 0, MISO alone; 0, mice treated with local hyperthermia 30 min after administration of MISO. Thick bar on the x-axis represents the heating period. Data were from five to six independent determinations and each is an average from at least three individual mice. Error bars indicate S.E.M.
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Figure 5. Pharmacokinetics of METRO (2.5 mmol/kg) with or without heat (43.5"C. 30 min) treatment. (A) METRO concentration in plasma; (J3) METRO concentration in turnours. Curves are for animals treated at room temperature. Exponential portions of the curves were fitted by linear regression. 0,METRO alone; 0,mice treated with local hyperthermia 30 min after administration of METRO. Thick bar on the x-axis represents the heating period. Data were from five to six independent determinations and each is an average from at least three individual mice. Error bars indicate S.E.M.
However METRO showed the same magnitude of thermal enhancement of radiosensitization as MIS0 when it was combined with heat and radiation (Figures 1C and 2). This potentiation effect cannot be explained by the additive effect of the METRO hypoxic cytotoxicity at a high temperature and radiosensitization. The NI potentiation of the radiation effect by heat cannot be accounted for by the observed pharmacokinetic mechanism. It is especially true for METRO that the enhancement of thermoradiosensitization was substantial without pharmacokinetic alteration at
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Figure 6. Effect of local hyperthermia on the tumour/plasma ratio of the two nitroimidazoles. (A) Tumour/plasma ratio of MISO; (B) tumour/plasma ratio of METRO. Each value was calculated from the data shown in Figures 4 and 5 . Both lines were fitted by eye. 0, Nitroimidazole treatment alone; 0, nitroimidazole with heat treatment. Thick bars on the x-axis represents the heating period. Error bars indicate the 95% cdnfidence limit.
43.5"C.Other mechanisms involved in the thermal potentiation in MISO or METRO radiosensitization must be taken into account. For example, MISO or METRO may alter the repair process of heat and radiation damage, or heat may increase the fixation of combined NI and radiation-induced damages. Although this study showed that the thermal enhancement of NI hypoxic cytotoxicity may not play an important role in vivo, it is intriguing that the thermal enhancement of NI radiosensitization in vivo showed a similar relationship between electron affinity and hypoxic cytotoxicity, i.e. an NI with less electron affinity produces a larger thermal enhancement than one with higher electron affinity. METRO, with less electron-affinity, was able to sensitize the tumour response to radiation at 43-5°C with the same magnitude as MISO, with higher electron affinity. In order to study this thermal enhancement phenomenon further, the hypoxic metabolism (cytotoxicity) of NI has to be dissected out from the NI radiosensitization (oxygen-mimicking) process. We
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are currently investigating this thermal enhancement effect using a cytoplasmic-free (therefore, removing the nitroreductase enzymes) FSa-I1 tumour cell system. Effect of MISO and METRO given in combination with heat and radiation on localized skin reaction was also compared. It has been demonstrated that MISO increased the localized skin reaction to heat plus radiation (Hofer ef al. 1981, Overgaard 1980). However, the enhancement ratio for skin reaction was less than that for tumours, resulting in a therapeutic gain. The increased skin damage may be related to the reactive oxidative species generated during the reductive metabolism of NIs under aerobic conditions (Perez-Reyes et al. 1980). Hyperthermia may enhance this redox process. In contrast, METRO which has lower electron affinity than MISO, may not generate enough oxidative species to enhance the skin reaction. This may explain the less severe skin reaction to METRO given with heat and radiation than that with MISO. In conclusion, this study may have important implications for the clinical use of NIs, heat and radiation: (1) a substantial NI thermoradiosensitization effect was observed with a small (4 mm) tumour containing a small hypoxic cell fraction of about 1 % (Urano and Kahn 1983). One would therefore expect a larger effect in tumours that contained a larger hypoxic cell fraction. (2) METRO with heat produced the same amount of radiosensitization as MISO at the elevated temperature. It should be noted that METRO is a less toxic NI than MISO systemically. The toxic side-effect (GI disturbance) developed in patients had been minimum following daily METRO doses as high as 6 g/m’ (Urtasun et al. 1975, 1976), and the toxicity can be controlled by antiemetics. In addition, this study showed that METRO is also less toxic than MISO to localized skin reaction. Therefore, being less toxic and more effective than most radiosensitizers, METRO plus local hyperthermia would be an attractive treatment regimen in future radiosensitizer clinical trials.
Acknowledgements The authors are grateful to Ms R. Reynolds for her technical and editing assistance. This work is supported by National Cancer Institute grant CA-26350, DHHS. References ADAMS,G. E., CLARKE, E. D., FLOCKHART, I. R., JACOBS,R. S.,SEHMI,D. S., STRATFORD, I. J.. WARDMAN, P., WATTS,M. E., PARRICK, J., WALLACE, R. G. and SMITHEN, C. E.. 1979, Structure-activity relationships in the development of hypoxic cell radiosensitizers. I. Sensitization efficiency. International Journal of Radiation Biology, 35, 133- 150. ARCANGELI, G., BAROLAS, A., MAURO,F., NERRI,C., SPARO,M. and TABOCCHINI, A.. 1980, Multiple daily fractionation radiotherapy in association with hyperthermia and/or misonidazole: experimental and clinical results. Cancer, 45, 2707-27 1 1. COLEMAN, C. N., 1985, Hypoxic cell radiosensitizers: expectations and progress in drug development. International Journal of Radiation Oncology, Biology und Physics, 11, 323-329. DENEKEMP, J., 1977, Tumor regression as a guide to prognosis: a study with experimental animals. British Journal of Radiology, 50, 27 1-279. L. E., SAPRETO,S. A. and GERWECK, L. E., 1977, Cellular responses DEWEY,W. C., HOPEWOOD, to combination of hyperthermia and radiation. Radiobiology, 123, 463-474. GOLDFEDER, A., BROWN,D. M. and BERGER, A., 1979, Enhancement of radioresponse of a mouse mammary carcinoma to combine treatments with hyperthermia and radiosensitizer misonidazole. Cancer Research, 39, 2966-2970. GRAY,L. H., GORGER.A. 0..EBERT,M., HORNSEY, S. andscorn, 0. C. A., 1953, Theconcentration of oxygen dissolved in tissues at the time of irradiation as a factor in radiotherpy. British Journal of Radiology, 26, 638-648. HALL,E. J. and ROIZIN-TOWLE, L., 1975, Hypoxic sensitizers: radiobiological studies at the cellular level. Radiology, 117, 453-457. HEIMBOOKE, D. C. and SARTORELLI, A. C., 1985, Biochemistry of misonidazole reduction by NADPH-cytochrome (P-450)reductase. Molecular Pharmacology, 29, 168- 172.
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239-244. RAJARATNAM, S.. ADAMS,G. E., STRATFORD, 1. J. and CLARKE, C., 1982,Enhancement of the cytotoxicity of radiosensitizers by modest hyperthermia: the electron-affinity relationship. British Journal of Cancer, 46, 912-917. SARARETO, S. A., RAAPHORST. G. P. and DEWEY,W. C.. 1979,Cell killing and the sequencing of hyperthermia and radiation. International Journal of Radiation Oncology, Biology and Physics, 5 , 343-348. SHELDON, P. W. and HILL. S. A.. 1977,Hypoxic cell radiosensitizers and local control by X-ray of a transplanted tumor in mice. British Journal of Cancer, 35, 795-808. SONG,C. W.. 1984,Effects of local hyperthermia on blood flow and microenvironment: a review. Cancer Research. (Supplement), 44, 472I s-4730s. SONG,C. W., RHEE.J. G. and LEVITT,S. H., 1982,Effect of hyperthermia on hypoxic cell fraction in tumor. International Journal of Radiation Oncology, Biology and Physics, 8, 85 1-856. STRATFORD. I. J. and ADAMS,G. E., 1977,Effect of hyperthermia on differential cytotoxicity of a hypoxic cell radiosensitizer, Ro-07-0582,on mammalian cells in vitro. British Journal of Cancer, 35, 307-313. URANO,M., GERWECK, L. E., EPSTEIN, R., GUNNINGHAM, M. and SUIT,H. D., 1980,Response of a spontaneous murine tumor to hyperthermia: factors which modify the thermal response in vitro. Radiation Research, 83, 3 12-322. URANO, J. and KAHN,J., 1983,The change in hypoxic and chronically hypoxic cell fraction in murine tumor treated with hyperthermia. Radiation Research, 96, 549-559. URANO,M.,KENTON,L. and KAHN,J., 1988,The effect of hyperthermia on the early- and lateappearing mouse foot reactions and on the radiation carcinogensis. I. Effect on the earlyand late-appearing reactions. International Journal ($Radiation Oncology, Biology nnd Physics,
15, 159-166. URTASUN. R., BAND,P.. CHAPMAN, D. J., FELDSTEIN, M. L., MIELKE,B. and FRYER,C., 1976, Radiation and high-dose metronidazole in supratentorial glioblastomas. New England Journal of Medicine, 294, 1364-1367. URTASUN, R., CHAPMAN, J. D., BAND,P., RABIN.H. R., FRYER, C. G. and STURMWIND, J., 1975, Phase I study of high-dose metronidazole: a specific in vivo and in vitro radiosensitizer of hypoxic cells. Radiology, 117, 129-133. VARGHESE, A. J., GALYAS,S. and MOHINDRA, J. K., 1976, Hypoxia-dependent reduction of by Chinese hamster ovary cells and KHT tumor I-(2-nitro- 1-imidazolyl)-3-methoxy-2-propanol cells in vitro and in vivo. Cancer Research, 36, 3761-3765. WALTON,M. I.. BLEEHEN, N. M. and WORKMAN, P., 1989a, Effects of localized tumor hyperthermia on pimonidazole (Ro 03-8799)pharmacokinetics in mice. Brirish Journal of Cancer. 59, 667-673.
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WALTON,M . I., BLEEHEN,N. M. and WORKMAN,P., 1989b, Stimulation of localized tumor hyperthermia of reductive bioactivation of 2-nitroimidazole benznidazole in mice. Cancer Research, 49, 2351-2355. WASSERMAN, T . H . , STETZ,J . and PHILLIPS,T. L., 1981, Clinical trials of misonidazole in the United States. Cancer Clinical Trials, 4, 7-16. WONDERGEM, J . , HAVEMAN, J . , VAN DER SCHUEREN, E., VAN DER HOEREN,H.and BEUR,K.,1986, Effect of hyperthermia and misonidazole on the radiosensitivity of a transplantable murine tumor: influence of factors modifying the fraction of hypoxic cells. International Journal of Radiation Oncology, Biology and Physics, 8, 1323-1331. WONG, K-H., WALLEN,C. A. and WHEELER,K. T., 1989, Biodistribution on misonidazole and I ,3-bis(2-chloroethyl)-l-nitrosourea(BCNU) in rats bearing unclamped and clamped 9L subcutaneous tumors. International Journal of Radiation Oncology, Biology and Physics, 17, 135- 143.
