Evaluation of the radiosensitizing effects of RK28 intravenous, intraarterial, and intratumoral injections on the rabbit VX2 tumor system

Int. 3. kxliation

Oncology

Biol.

Phys.,

Vol. 36, No. 5, pp. 1107-

1116,

1996

Copyright 0 1996 Elsevier ScienceInc. Printed in the USA. All rights reserved 0360-3016196$15.00 + .OO

PII: SO360-3016(96)00407-S

ELSEVIER

l

Biology Original Contribution EVALUATION INTRAVENOUS,

OF THE RADIOSENSITIZING EFFECTS INTRAARTERIAL, AND INTRATUMORAL ON THE RABBIT VX2 TUMOR SYSTEM

OF RK28 INJECTIONS

KAZUYU EBE, M.D.,* HIROKI SUDA, M.D.,* TATSUYA KURAMITSU, M.D.,* YUTAKA HONMA, M.D.,* TAKASHI NAKANISHI, M.D.,* YOSHIYUKI MIYATA, B.Sc.,+ MASAKAZU SAKAGUCHI, PH.D.? AND NAOFUMI MATSUNAGA, M.D.* *Departmentof Radiology, Yamaguchi University School of Medicine, 1144Kogushi, Ube, Yamaguchi 755, Japan; and +POLA PharmaceuticalR and D Laboratory, 27-l Takashimadai,Yokohama, Kanagawa221, Japan Purpose: To evaluate thk differencesin the radiosensitizingeffects of intravenous 0.v.) injection, intraarterial -injection, and intratumoral (i.t.) injections of the hypoxic cell radiosensitizerRK28 ([l-(4’~hydroxy-2’-butenoxy)methyl-2-nitroimidazole],a 2-nitroimidazolewith an acyclicsugaranaloguesubstitutedat the N-l position of the hnidazolering) usingan animal experimentalsystem. Methods and Materials: Rabbit VX2 tumors, which were implantedin the muscleof left hind legsand grown to 3 cm in diameter,weretreated with RK28 (80 mg/kg*b.wt.) before 15Gy of localx-ray irradiation. The auricular vein and the left saphenousartery were usedfor systemicinjection and regionalinjection, respectively.For i.t. injection, a 21-gaugeneedlewith three lateral holeswas positionedin the central area of the tumor. Tumor regressionwaspreciselyevaluatedby computedtomograpy (CT), and survival time wasalsostudied.Usinghighperformanceliquid chromatography(HPLC), pharmacokineticstudiesfor RK28 and its sevenmajor metabolites were performedin tumor and serumat 0, 10,20,30, and 60 min after drug injection wascompleted. Results:RadiosensitZngeffectsof RK28 wereconsideredpresentafter ia injection(p < 0.05)and i.t. injection(p < 0.05)after analyzingtumor volumeson day 21 after treatment.Increasedsurvival wasnot observedin any group with RK28 injectioncomparedwith survival in the group treatedby x-ray irradiation alone.Pharmacokinetic studies showedthe averageconcentrationof RK28 in the tumor during x-ray irradiationwas13 thneshigherafter i.a. injection and 3.5 timeshigherafter i.t. h+ction than that after i.v. injection.The time modifyingfactor, (TMFs: ratio of time for tumor to decrease by 50%, radiationalonevs. radiationplusdrug) wascalculatedto be 1.5after i.v. injection,1.7 after i.a injection,and 2.3atIer i.t. we&on. The valuesof TMFsocorrelatedto the averageconcentrationsof RK28 iu the tumor. As to metabolib of RK28,&$umronated compoundandcysteineconjugatewerehighly detected.The concentrationsof cystetie coeugatewerehigherin the tumor than in serumvia i.v. injection. Conclusions:Radiosensitizingeffects of RK28 were observedon the rabbit VX-2 tumor systemafter i.a. or i.t. injection. Pharmacokinetic studiesproved that radiosensitizingeffects dependedon the concentration in the tumor, though the administration routes were different. Combinedforms with nonprotein thiols were detected. However, survival benefitswere not obtained by RK28. For clinical applicationsof RK28, i.a. or i.t. injection could facilitate better local control of cancer. Copyright 0 1996by ElsevierScienceInc. Radiosensitizers,Hypoxic cells,RK28,2-Nitrohnidazole nucleoside,VX2 tumor, Pbarmacokineticand metabolic studies.

INTRODUCTION There have been a large number of animal experiments regarding the radiosensitizing effects of hypoxic cell radiosensitizers in viva. However, differences in the radiosensitizing effects of intravenous (i.v.) injection, intraarterial (ia.) injection, and intratumoral (i.t.) injection have not been reported, though studies have been done comparing intraperitoneal injection to i.t. injection in mice (18) and on i.t. injection in humans (1). RK28 [ 1-(4’-hydroxy-2’-butenoxy) methyl-2-nitroimidazole] , a 2-nitroimidazole with an acyclic sugaranalogue

substituted at the N-l position of the imidazole ring, is a potent hypoxic cell sensitizer, which was developed by Sakaguchi et aE. (21). Its main characteristics are lower toxicity than misonidazole or etanidazole with similar sensitizing effects in experimental systems(16,24). Cytotoxicity of RK28 has not been reported if the concentration is less than 1 mM and the incubation time is lessthan 1 h under both aerobic and hypoxic conditions in vitro (16). The early PhaseII clinical trials have been completed in Japan. The results will be published soon. Dose dependency of this compound in its radiosensitizing effects was

Reprint requeststo: Kazuyu Ebe, M.D., Departmentof Radiology Yamaguchi University School of Medicine, 1144

Kogushi, Ube, Yamaguchi755, Japan. Acceptedfor publication8 August 1996. 1107

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I. J. Radiation Oncology l Biology l Physics

shown in experimental studies, in vitro and in vivo (16, 23). One problem to be resolved is how to obtain a high drug concentration in the tumor while reducing systemic toxicity. It is considered that i.a. or i.t. injection of this radiosensitizer will achieve higher drug concentration in the tumor, providing greater radiosensitizing effects than i.v. injection. Furthermore, systemic toxicity will be reduced in comparison with that after i.v. injection. In the present study, we report the differences of radiosensitizing effects of RK28 via i.v., ia., and i.t. injections on tumor regression, survival terms, and pharmacokinetics using the rabbit VX2 tumor system, in which it is very easy to administer a drug by three different routes of injection.

METHODS

Co., Ltd., Tokyo.

‘Shionogi Pharmaceutical Co., Ltd., Osaka. 3Abbott Laboratories,

and i.t. injections. Rabbits were anesthetized with 20 mg/ kg. b.wt. of pentobarbital sodium3 during procedures. RK28 RK28 [ I-(4’-hydroxy-2’-butenoxymethyl-2-nitroimidazole) is yellowish powder with a molecular weight of 213.19 Da. Chemical structures of RK28 and its seven major metabolites are shown in Fig. 1 (22). Metabolites were designated as follows: LY-and ,&hydroxy carboxylic acid derivatives of RK28 as (a-l) and (a-2), tram- and ciscarboxylic acid derivatives of RK28 as (b) and (c), /?glucuronated RK28 as (d), cysteine and N-acetylcysteine conjugates of RK28 as (e) and (f). RK28 was dissolved at 50 mg/ml in saline and 80 mg/kg *b.wt. was administered via i.v., ia., or i.t. injection.

AND MATERIALS

Rabbit VX2 tumor Japanese white male rabbits (closed colony) each weighing 2.5-3.0 kg were purchased from Shizuoka Animal Center (Shizuoka, Japan). The animals were housed in institutional animal care facillities in our university. The VX2 tumor is a squamous cell carcinoma which originated in a Shope virus-induced papilloma (20). It is an allogeneic tumor. It was obtained from a donor rabbit maintained at Kobe University, then maintained by serial transplantation in vivo in our laboratory. Fragments of tissue taken fom the tumor in donor rabbits were minced and homogenized in Dulbecco’s Modified Eagle Medium (DMEM). ’ About 5 X lo7 of VX2 tumor cells in 2 ml of DMEM were implanted into the left hind limb muscle by a percutaneous injection. Thereafter, 12 mg/kg of body weight (b.wt.) of tobramycin’ as an antibiotic was intramuscularly injected to the contralateral hind limb. A day before implantation, 80 mg/kg . b.wt. of cyclophosphamide’ was intravenously administered to recipient rabbits to improve transplant efficiency (5, 9). Tumors that had grown to 3 cm in diameter 10 days after implantation were used for experiments. Experimental animals were divided into five groups, six rabbits in each group, as follows: (a) controls: natural growth with i.v. normal saline; (b) x-ray: 15 Gy of x-ray irradiation alone with i.v. normal saline; (c) i.v. + x-ray: 15 Gy of x-ray irradiation after i.v. injection of RK28; (d) i.a. + x-ray: 15 Gy of x-ray irradiation after i.a. injection of RK28; and (e) i.t. + x-ray: 15 Gy of x-ray irradiation after i.t. injection of RK28. If CT scans detected regional nodal metastases in tumor-bearing animals on treatment day, the animals were excluded from the experiments, because the tumors were too progressive to control locally. Furthermore, 18 other rabbits were divided into three groups, six rabbits in each group, for studies of pharmacokinetics of the RK28 and its metabolites after i.v., i.a., ’ Nissui Pharmaceutical

Volume 36, Number 5, 1996

North Chicago.

Drug injection Intravenous injections were done via the auricular vein using a 24-gauge cannula
(C)

(d)

(e)

Fig. 1. Chemical structures of RK28 and its metabolites (a-f). 4PEIT Needle, Hakko Co., Ltd. Tokyo.

‘Orthovoltage unit, Shimazu Co., Ltd., Kyoto.

Radiosensitizing effects of RK28 injections

0

K.

EBE et al.

1109

Fig. 2. Retrograde arteriogram of a rabbit VX2 tumor via the left saphenous artery. 1: left femoral artery, 2: saphenous artery, 3: posterior femoral artery, 4: popliteal artery, 5: innominate artery, 6: tibial artery.)

distance was 37 cm. Irradiated dose over the tumor volume ranged from approximately 12.5 Gy to 17.0 Gy. The field size was 10 X 9 cm. Irradiation was performed immediately after drug injection. It took 21.4 min to complete irradiation. A dosimeter with an ion chamber6 was used to measure a dosage. Animals were anesthetized with 20 mg/kg . b.wt. of pentobarbital sodium during irradiation. X-ray irradiation was carried out at room temperature. Tumor growth Tumor volume was evaluated by computed tomograpy (CT) before and after treatments. CT scans were per610nex 2500/3, Nuclear Enterprises, ‘Siemens Co., Ltd., Erlangen.

Ltd., UK.

formed once every 5 -7 days with a Somatom DR3’ using intravenous administration of 612.4 mg/kg *b.wt. of contrast material. ’ When an animal died, CT scans were also done to evaluate the final tumor volume. Scanning times of 4 s were used with a slice thickness of 8 mm at 8 mm intervals. Tumor volume was calculated by integrating the tumor area of each slice. The equation was as follows:

V: tumor volume (mm3), ff: magnification factor, fl slice thickness (mm), a: long-axis distance of the tumor on a * Iopamidol,

Nihon Schering, Osaka.

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I. J. Radiation Oncology 0 Biology l Physics

Survival Survival curves in each group were determined by the Kaplan-Meier method. Survival was observed for a maximum of 200 days after treatment. Data were compared by the generalized Wilcoxon test.

5 days

Pharmacokinetic study in tumor tissue and serum Pharmacokinetic studies were performed using HPLC’ in tumor tissue and serum at 0, 10, 20, 30, and 60 rnin after i.v., i.a., or i.t. injection of RK28 was completed. Eighteen tumor-bearing rabbits 10 days after implantation were used, six rabbits in each group. One milliliter of nonheparinized blood sample was collected from the right femoral vein and 100 mg of tumor tissue was biopsied at each of the above times on the same animal. Therefore, one animal provided five blood samples and five tumor tissue samples. Tissue samples were not frozen and homogenated. Methanol extractions were done immediately after blood and tissue samplings. Animals were anesthetized during procedures. RK28 and its seven major metabolites were isolated and purified using affinity chromatography and preparative HPLC. Thereafter, these chemical structures were determined using fast atom bomberdment mass spectrometry (FAB-MS) lo and nuclear magnetic resonance (‘H-NMR and 13C-NMR). l1 Then, purified molecules were used as standards. Samples were examined by HPLC equipped with a TSK-gel ODS 80 TM column (4.6 X 150 mm), with a mobile phase of 10% acetonitril solution in 10 ml

10 days WY

Volume 36, Number 5, 1996

0)

20 days WY 10)

31 days (Day 21)

Fig. 3. CT findingsduring the naturalcourseof the VX2 tumor (*). Upper, 5 days after implantation;upper middle, 10 days after; lower middle, 20 days after; lower, 31 days after.

slice in the CT film (mm), b: short-axis distance of the tumor on a slice in the CT film (mm), n: slice number. Tumor regression curves were depicted in the way of semi-logarithm. Tumor volumes were compared by the Mann-Whitney test 21 days after treatment, and the slopes of regression lines were compared by analysis of variance. The time for tumor volume to decrease by 50% after treatment (designated as T50) was measured on tumor regression curves. Then, a TMFsO (time modifying factor& was calculated as follows: TMFsO = T5,, in x-ray/T,, in each group that received the hypoxic cell radiosensitizer RK28. ‘Trirotar IV, JascoCo., Ltd., Tokyo. ‘“JMS-HXIOO,

Jeol, Tokyo.

0 Controls

X-ray

IV+X-ray IA+X-ray IT+X-ray

Fig. 4. Relative tumor volumeson day 21. Tumor volume is normalizedto 1 on day 0 for eachgroup. Significantdifferences were found betweencontrolsand x-ray (p < 0.05), betweenxray and La. + x-ray (p < 0.05), and betweenx-ray and i.t. + X-ray (p < 0.05). Each box showsthe meantumor volume + SE. The numberof animalswasthree in controls,five in x-ray, five in i.v. + x-ray, six in i.a. + x-ray, and six in i.t. + x-ray. I1JNM-GX400, Jeol, Tokyo.

Radiosensitizing

effects

of RK28

phosphate buffer (pH 7.0) with 10 mM tetrabutyl ammonium phosphate at a flow rate of 1.0 ml/mm. Ultraviolet (UV) absorption of the sample was measured at 320 nm on a spectrophotometer. ‘* Then the concentrations and identifications of RK28 and its seven major metabolites were determined by peak height and retention time. RESULTS Tumor growth

In controls, tumors grew to 35.7 -+ 5.3 cm3 (mean ? standard error) in tumor volume after 10 days, when treatments were given (designated as day 0), 117.0 2 19.9 cm3 after 20 days (day lo), and 182.2 2 54.0 cm3 after 31 days (day 21). As a sample, Figure 3 shows CT scans of one of the rabbit VX-2 tumors during the natural course. The tumor volume increased to 15.1 cm3 5 days after implan-

Day 0

injections

l

K.

EBE et al.

tation,, 40.2 cm3 10 days after, 109.3 cm3 20 days after, and 222.3 cm3 31 days after. Furthermore, CT scans revealed nodal metastasis 20 days later. Figure 4 shows comparisons of tumor volumes of the 25 surviving animals on day 21. Five of 30 animals died before day 21 (three animals in controls, one animal in x-ray, and one animal in i.v. + x-ray). Significant differences were observed in controls vs. x-ray (p < 0.05), in x-ray vs. i.a. + x-ray (p < O.OS), and x-ray vs. i.t. + x-ray (p < 0.05). This conclusively demonstrates radiosensitizing effects of RK28 (80 mg/kg *b.wt.) after i.a. or i.t. injection, but not after iv. injection. As a sample, Figure 5 shows CT scans of treated tumors on day 0 and day 21 in x-ray, i.v. + xray, La. + x-ray, and i.t. + x-ray. One of the tumors in xray slightly increased in volume from 33.8 cm3 to 35.6 cm’, one in i.v. + x-ray decreased from 20.5 cm3 to 15.8 cm3, one in i.a. + x-ray decreased from 60.8 cm3 to 17.0

Day 21

Fig. 5. CT findings in treated groups on day 0 (left side) and day 21 (right side). Upper, x-ray, upper middle, i.v. + x-ray; lower middle, i.a. + x-ray; lower, i.t. + x-ray. “UVI

DEC 100 VI, Jasco Co., Ltd., Tokyo.

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Volume 36, Number 5, 1996

O.OS), controls and i.a. + x-ray (p < O.Ol), and controls and i.t. + x-ray (p
0

10

20

30

40

50

60

70

Days Fig. 6. Tumor regression curves in controls (0), x-ray (A), i.v. x-ray (O), i.a. x-ray (U), and i.t. x-ray (A). T50representsthe time for the tumor volume to decreaseby 50%. Tumor volume is normalizedto 1 on day 0 for eachgroup.Each symbolshows the meantumor volume 2 SE. Significant difference wasobservedin x-ray vs. i.a. x-ray as to slopes(p < 0.01).

cm3, and one in i.t. + x-ray markedly decreased from 44.7 cm3 to 4.3 cm3. Figure 6 shows the tumor regression curves. The slopes of regression lines were also compared (8). The difference was significant only in x-ray vs. i.a. + x-ray (p < 0.01). The p-values were 0.112 in x-ray vs. i.v. + x-ray and 0.5 11 in x-ray vs. Lt. + x-ray. Regrowth data after day 28 was omitted for analysis because it is considered that treatment effects disappeared when tumor regrowth began. On the tumor regression curves (Fig. 6), TJO was established as 27 days in x-ray, 18 days in iv. + x-ray, 16 days in La. + x-ray, and 12 days in i.t. + x-ray. Then the TMFsO was calculated as 1.0 (27/27) in x-ray as the control, 1.5 (27/18) in i.v. + x-ray, 1.7 (27/16) in i.a. + x-ray, and 2.3 (27/12) in i.t. + x-ray. Figure 7 shows the regression line when the values of TMFsO were plotted against the average concentrations of RK28 in the tumor during irradiation as derived by the pharmacokinetic studies (r’ = 1).

Figure 9 a and b shows the results of pharmacokinetic studies in the tumor and serum for i.v., i.a., and i.t. injections of RK28. The peak concentration of RK28 in tumor tissues (mean 2 SE) was 80 + 20 ,@g in i.v. injection, 159 + 17 ,ug/g in i.a. injection, and 260 + 154 &g in i.t. injection, respectively. The average concentrations of RK28 in the tumor (mean _+ SE) during irradiation were calculated as 60 + 13 pg/g (0.28 ? 0.06 n&l) after i.v. injection, 78 I? 7 pg/g (0.36 t 0.03 mM) after i.a. injection, and 210 2 65 pg/g (0.98 2 0.31 mM) after i.t. injection as derived from the time-concentration curves in Fig. 9a. The ratio of concentrations was 1 (60/60): 1.3 (78/ 60): 3.5 (210/60) for i.v. injection vs. i.a. injection vs. i.t. injection, respectively. The peak concentration of RK28 in serum (mean + SE) was 184 !I 58 pg/ml after i.v. injection, 158 2 22 pg/ml after i.a. injection, and 70 + 16 pg/ml after i.t. injection. The concentration of RK28 in serum after 60 min decreased to 7.1% (13/184) following i.v. injection, to 7.0% (1 l/158) following i.a. injection, and to 11.4% (8170) following i.t. injection. Excretion was very rapid as shown in Fig. 9b.

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Survival

Figure 8 shows survival curves by the Kaplan-Meier method. Median survival time was 17 days in controls, 33 days in x-ray, 63 days in i.v. + x-ray, 139 days in i.a. + x-ray, and 153 days in i.t. + x-ray. The lengths of survival periods were compared using the generalized Wilcoxon test. There was a significant difference between controls and x-ray (p < 0.05), controls and iv. + x-ray (p <

0

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.4 .6 .8 RK28 (mM)

1 1.2 1.4

Fig. 7. The regressionline showing the relationshipbetween TMFsOvaluesandaverageconcentrationsof RK28 (mean2 SE) in the tumor in x-ray (A), i.v. x-ray (a), i.a. x-ray (D), and i.t. x-ray (A) (2 = 1).

Radiosensitizing effects of RK28 injections

EBE

1113

et al.

shows tumor regression curves. Regression lines for i.v. injection and i.t. injection were parallel to the line of 15 Gy irradiation alone, but that of i.a. injection was steeper

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Fig. 8. Survival curves of controls (0), x-ray (A), iv. x-ray (o), i.a. x-ray (O), and i.t. x-ray (A). Significant differences were observed in controls vs. x-ray (p < O.OS), controls vs. i.v. xray (p < 0.05), controls vs. i.a. x-ray (p < O.Ol), and controls vs. i.t. x-ray (p < 0.01).

Figure 10 (a-c) shows time-concentration histograms of total metabolites after i.v., i.a., and i.t. injections. The sum of metabolite (a-l) and (a-2) was demonstrated as (a). Concentrations of total metabolites in tumor tissue were lower than those in serum after i.v. injection, i.a. injection, or i.t. injection at each interval except 60 min after i.v. injection. High concentrations of metabolites (d) and (e) were detected in the tumor and serum. The concentrations of metabolite (d) in the tumor were lower than those in serum at each interval in each group, but the concentrations of metabolite (e) in the tumor were higher than those in serum only in the i.v. injection group.

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The characteristics of rabbit VX-2 tumors are rapid growth and distant metastases to the lung and the liver as well as regional lymph nodes (15). Tumor-bearing animals died of cachexia within 36 days (day 26) after implantation in our studies. This tumor system is considered one of good in vivo models for human cancer treatment (11). The following three types of studies are available. (a) Analysis of different effects of the same compound . . of via i.v., i.a., and i.t. injections; (b) determination whether local control contributes to survival (3, 15); (c) imaging analysis by CT, angiography (3), NMR (7), scintigraphy (25), and ultrasonography (US) (19) because tumor size is large enough to detect by each imaging modality. In the present study, the hypoxic cell radiosensitizer RK28 was administered via iv., i.a., and it. injections. Radiosensitizing effects were observed after i.a. and i.t. injections by analysis on day 21 (Fig. 4). According to the pharmacokinetic studies, drug concentrations played a critical role in the radiosensitizing efficiency, although the administration routes were different (Fig. 7). Figure 6

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I. J. Radiation Oncology l Biology l Physics

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Volume 36, Number 5, 1996

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(c) of total metabolitesof RK28 in i.v. injection (a), La. injection (b), and i.t. injection (c). Tumor and serumwere abbreviatedas T and S, respectively.Metabolite levels are shownasbarsstackedon top of one another(seefigure for key). Fig. 10. Pharmacokinetics

than the control. This result suggests that i.a. injection induced additional effects on tumor regression.The mechanismshave not been clarified yet.

RK28 improved the tumor shrinkage of rabbit VX-2 tumors. However, the hypoxic cell fraction has not yet heen clarified becauseof difficulties in tissueculture in the in vivo-

Radiosensitizing effects of RK28 injections 0 K. EBE et al.

ipl vitro clonogenic assay. This should be studied in the near future using computerized pO2 histography (4,23). Hypoxic cells are considered present as correlation of magnetic resonance imaging (MRI) and histologically necrotic areas in VX2 carcinoma of rabbits has been reported (7). It is clear that local control of tumor contributed to survival benefits in the comparison between the untreated group receiving i.v. normal saline and the treated groups with or without RK28 as shown in Figs. 4 and 8. Furthermore, RK28 prolonged median survial times from 33 days in 15 Gy irradiation alone to 63 days after i.v. injection, to 139 days after i.a. injection, and to 153 days after i.t. injection. But these effects were not significant. Pharmacokinetic studies showed that RK28 was excreted very rapidly from blood. The concentrations of RK28 in serum were lower after i.a. and i.t. injections than those after i.v. injection. It is likely that systemic toxicity will be reduced after it. and i.a. injections compared with that after i.v. injection. Metabolites of RK28 consist of oxidized compounds indicated as (a-l), (a-2), (b), and (c), glucuronated compound shown as (d), and combined forms with nonprotein thiols shown as (e) and (f). It has been reported that an enhancement ratio (ER) of each metabolite (1 mM) against hypoxic EMT6 tumor cells in vitro was 1.29 in (a-l), 1.19 in (a-2), 1.23 in (b), 1.41 in (c), 1.07 in (d), 1.59 in (e), and 1.07 in (f) (22). The ER was

1115

calculated from the ratio of the radiation dose with and without the metabolites required to reduce the survival fraction to 1% (17). Radiosensitization by metabolites of misonidazole have been reported (14,26). Detection of metabolites (e) and (f) in our studies implies that RK28 deleted nonprotein thiols in vivo which may have acted as radioprotectors (2,24). It is worth noticing that metabolite (e) was highly detected in iv. injection and its concentrations were higher in the tumor than in serum as shown in Fig. 10a. If purified metabolite (e) alone were intravenously administered, and if its concentrations were higher in the tumor than in serum, metabolite (e) could possibly become a newly potent radiosensitizer and could also be available for tumor imaging once it has been radiolabeled. In our studies, CT findings provided three advantages: (a) It was easy to evaluate tumor volumes precisely since CT scans revealed VX-2 tumors clearly with contrast enhancement even if the tumors were almost nonpalpable; (b) it allowed exclusion of animals with nodal metastases from our experiments if these were demonstrated by CT on day 0; (c) the recorded images made it possible to review the results retrospectively. For clinical administration of RK28, i.a. or i.t. injection may facilitate better local control of cancer, especially for intraoperative radiation therapy and brachytherapy (6, 10, 13).

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