INT. J. HYPERTHERMIA, 1999, VOL. 15, NO. 1, 1± 6

Reduction of tumour blood ¯ ow with KB-R8498 potentiates the response

of tumours to hyperthermia

A. SHAKIL, A. OGAWA, R. J. GRIFFIN and C. W. SONG*

Department of Therapeutic Radiology± Radiation Oncology, Radiation BiologySection, University of Minnesota Medical School, 420 Delaware Street S.E.,Box 494, Minneapolis, MN 55455, USA

(Received 29 April 1998; accepted 10 August 1998)

The anti-tumour activity and the e� ect on tumour and normal tissue perfusion ofa newly discovered anticancer agent, KB-R8498 (Kanebo Ltd., Osaka, Japan),were investigated in FSa II tumours of C3H mice. The tumour perfusion, asmeasured by the 86Rb-uptake method, markedly decreased with relatively littlechange in the normal tissue perfusion after an i.v. injection of KB-R8498.Furthermore, the drug potentiated the e� ect of hyperthermia at 42.5ë C for60min to suppress the tumour growth. The results suggest that the preferentialreduction in tumour blood ¯ ow relative to normal tissue blood ¯ ow by KB-R8498may be exploited to enhance the anti-tumour e� ect of hyperthermia.

Key words: Tumour blood ¯ ow, KB-R8498, hyperthermia.

1. Introduction

It is expected that destruction of tumour vascular beds or reduction of tumourblood ¯ ow would potentiate the e� ect of hyperthermia by increasing heating e� -ciency and increasing the acidity of the intratumour environment (Rhee et al. 1984,Song 1984, Vaupel et al. 1989, Lin and Song 1990, Hasegawa and Song 1991).Unfortunately, the methods used in the past to reduce tumour blood ¯ ow, such asinjection of hydralazine or large doses of glucose, have been found to be impracticalin clinical situations for various reasons. It has recently been reported that KB-R8498 (4-[t-3, t-4-dihydroxycyclopentan-r-1-yl)oxy]-2-(1-piperazinyl)quinazolinehydro chloride) causes vasoconstriction and reduces tumour blood ¯ ow and thatthe e� ect of KB-R8498 was antagonized by ketanserin, which binds to type-2 ser-otonin (5-HT2) receptors (Sekida et al. 1997b). Daily injection of this drug totumour-bearing mice signi® cantly suppressed tumour growth, probably by deprivingthe nutritional supply to the tumour cells (Sekida et al. 1997a). The present paperreports on the e� ect of KB-R8498 on blood ¯ ow in a mouse tumour model, aswell as the anti-tumour activity of the drug alone and in combination withhyperthermia.

2. Materials and methods

2.1. Drug treatment and determination of blood perfusionFSa II tumours transplanted s.c. in the right rear thigh of C3H mice were used

when they grew to a size of 200± 250mm3. KB-R8498 (Kanebo Ltd, Osaka, Japan)was dissolved in 0.9% saline at a concentration corresponding to 5, 15 or 30mg/kg in

0265± 6736/99 $12.00 Ñ 1999 Taylor & Francis Ltd.

* To whom correspondence should be addressed.

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0.1ml on the day of each experiment. The drug was injected i.v. through the tail vein,while the control animals received an i.v. injection of 0.1ml saline. Tumour bloodperfusion was measured by the radioactive Rubidium (86Rb) uptake method(Sapirstein 1958, Song et al. 1989). Anaesthetics (mixture of ketamine 100mg/kgand xylazine 10mg/kg) were administered i.p. 3min before the injection of 86Rb.Therefore, for the determination of perfusion immediately after the treatment withKB-R8498, anaesthetics were administered 3min before the injection of the drug.The mice were sacri® ced by cervical dislocation 60 s after the radioisotope injectionand tumour-bearing legs were immediately removed and the tumours dissected.Several normal tissues were also removed and the activity of 86Rb in tumours andeach normal tissue was counted and the percentage of the total injected activity of86Rb per g tissue was calculated, which is equivalent to the per cent cardiac outputper g of tissue (Sapirstein 1958).

2.2. Tumour growthTumour growth was measured following a single i.v. treatment of 5± 30mg/kg of

the drug alone, or in combination with local hyperthermia for 60 min at 42.5ë C,applied immediately or 1h after the drug treatment (Song et al. 1989). The mice wereanaesthetized just prior to locally heating the tumour-bearing leg by extending it intoa preheated water bath. Tumour size was measured on the day of treatment andevery alternate day thereafter.

3. Results

No noticeable change in activity was observed in any animal after the injection ofthe drug. The mean per cent 86Rb-uptake of 56 control tumours was 3.0 0.1%/gand a single i.v. injection of 5, 15 or 30mg/kg KB-R8498 markedly reduced the 86Rbuptake in FSa II tumours, indicating that tumour perfusion was markedly reduced.Figure 1 shows the e� ect of KB-R8498 on the 86Rb uptake in tumours, taking thecontrol tumour 86Rb uptake as 100%. The 86Rb uptake or perfusion decreased toless than 25% of the control immediately after the treatment. Tumour perfusiongradually recovered thereafter, but it was still 75% or less than the control tumourperfusion at 1h post-treatment for all three drug doses tested (p < 0.05).

The changes in normal tissue perfusion are shown in table 1. Immediately afterthe treatment with 5± 30mg/kg, the liver perfusion was slightly increased (non-sig-ni® cant) while the spleen, muscle and skin perfusion were all signi® cantly (p < 0.05)decreased. The kidney perfusion decreased slightly immediately after treatment with15 or 30mg/kg (p < 0.05), but not with 5mg/kg. The perfusion in the liver, spleenand kidney returned to control level by 30min after injection with 5± 30mg/kg ofKB-R8498. The perfusion in the skin and muscle had not fully returned to controllevel by 60min after 15 or 30mg/kg of KB-R8498 (table 1).

Figure 2 shows a growth delay plot for the 15mg/kg conditions. An i.v. injectionof 15mg/kg KB-R8498 had no e� ect on the tumour growth, while heating thetumour at 42.5ë C for 60min slightly suppressed the tumour growth. The tumourgrowth was markedly suppressed when KB-R8498 was given to the tumour-bearingmice and the tumours were heated immediately or 1h after the drug administration.Hyperthermic treatment of tumours immediately after drug administration appearedto be slightly more e� ective than hyperthermia given 1h after drug treatment.

Detailed information on the e� ect of di� erent amounts of KB-R8498, alone or incombination with hyperthermia, on the tumour growth time is shown in table 2. A

2 A. Shakil et al.

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single i.v. treatment of 30mg/kg of KB-R8498 alone caused only about a 1 day delayin the growth of the tumours to four times the starting volume. Treatment with 5 or15mg/kg KB-R8498 caused no tumour growth delay (data not shown).Hyperthermia alone (following saline injection) caused a tumour growth delay of2 days. When hyperthermia was applied to the tumours immediately or 1h aftertreatment with 5, 15, or 30mg/kg KB-R8498, a 6± 8 day delay in the growth time was

Reduction of blood ¯ ow by KB-R8498 3

0

25

50

75

100

Rb-

86 u

ptak

e (%

of c

ontro

l)

0 30 60

min after treatment

30 mg/kg

15 mg/kg

5 mg/kg

Control

FSa II

Figure 1. Tumour perfusion plotted as the percentage of the control tumour value(3.0 0.1% 86Rb uptake/g) measured at di� erent intervals after an i.v. injection of KB-R8498 (mean SE).

Table 1. Percent 86Rb uptake (of total injected activity) per gram of tissue in normal tissuesat di� erent times after an i.v. injection of 5, 15 or 30mg/kg KB-R8498 (mean SEshown).

Skin Muscle Liver Spleen Kidney

Control 1.4 0.04 3.0 0.2 3.9 1.6 9.0 0.3 41.0 1.45mg/kg 0 min 0.7 0.1* 2.0 0.2* 5.7 0.5 4.9 0.4* 41.4 2.3

30 min 1.1 0.1* 3.1 0.3 3.8 0.4 7.7 0.3 39.5 2.160 min 1.3 0.1 3.4 0.3 4.1 0.3 9.0 0.4 43.9 2.6

15mg/kg 0 min 0.5 0.1* 2.0 0.2* 5.2 0.6 4.3 0.5* 34.8 2.5*30 min 0.9 0.1* 2.4 0.3 3.1 0.2 8.9 0.8 40.8 1.860 min 1.2 0.1* 2.6 0.4 3.8 0.3 9.9 0.7 39.8 1.4

30mg/kg 0 min 0.3 0.1* 1.9 0.2* 5.5 0.5 3.5 0.3* 32.2 2.7*30 min 0.9 0.1* 2.2 0.2* 4.2 0.3 8.8 0.4 38.7 0.860 min 1.2 0.1 2.3 0.2* 4.3 0.4 9.3 0.6 37.1 3.0

*Indicates that the 86Rb uptake in the drug treated group and that in the control groupsigni® cantly di� ered at p < 0.05 using Welch’s alternate t-test.

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encountered, compared to the untreated control (p < 0.05 for all drug and hyper-thermia treatments vs. control). Another ongoing study found that a 5-day treatment(i.p.) with 5± 30mg/kg/day of the drug alone caused a 6 day delay in the tumourgrowth (data not shown).

4 A. Shakil et al.

0

200

400

600

800

1000

1200

1400

0 4 8 12 16Days after treatment

15mg/kg-1h-HT (13)

15mg/kg-0h-HT (13)

15mg/kg alone (8)

Saline+HT (24)

Control (14)

KB-R8498: 15 mg/kg i.v.HT: 42.5° C, 60 min

FSaII tumorM

ean

tum

or v

olum

e (m

m )3

Figure 2. Tumour growth plot for groups of FSaII tumours treated with 5± 30mg/kg KB-R8498 with or without hyperthermia at 42.5ë C for 60min, beginning immediately or 1hafter drug administration. Data points represent the mean SE of 8± 24 tumours.

Table 2. Time (mean number of days SE) required forthe tumour size to reach 4 the starting volume inFSa II tumours after various treatments.

Groups N Growth time(days)

Untreated control 14 8.5 0.5SalineÐ 0h, HT² 24 10.3 0.730 mg/kg alone³ 9 9.3 0.45 mg/kgÐ 0 h, HT 12 15.8 0.6*5 mg/kgÐ 1 h, HT 12 14.3 0.6*15 mg/kgÐ 0 h, HT 12 16.1 0.7*15 mg/kgÐ 1 h, HT 11 14.5 1.5*30 mg/kgÐ 0 h, HT 12 16.3 0.9*30 mg/kgÐ 1 h, HT 12 15.0 0.8*

² HT: local hyperthermia at 42.5ë C for 60 min.³ dose of KB-R8498/kg body weight.* Indicates that the increase in tumour growth time was

statistically signi® cant at p < 0.05.

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4. Discussion

A number of di� erent drugs have been reported to decrease tumour blood ¯ ow(Vaupel et al. 1989, Lin and Song 1990, Hasegawa and Song 1991, Hirst et al. 1991,Honess and Bleehen 1991, Peters et al. 1991). Unfortunately, these drugs were foundto signi® cantly a� ect not only the tumour blood ¯ ow but also the normal tissueblood ¯ ow. For example, hydralazine is a vasodilator for normal tissues and itdiverts blood ¯ ow from tumours to normal tissues. However, the decrease in tumourblood ¯ ow caused by hydralazine is very much dependent on the site of tumourgrowth (Lin and Song 1990, Hasegawa and Song 1991, Hirst et al. 1991). It wasfound that hydralazine e� ectively dilated blood vessels and increased blood ¯ ow inskin and muscles, but not in some other normal tissues (Hasegawa and Song 1991).In fact, hydralazine caused a decrease instead of an increase in blood ¯ ow in manyinternal organs, and the blood ¯ ow in tumours which grew in these organs did notdecrease by hydralazine (Hasegawa and Song 1991). Unlike hydralazine, KB-R8498causes vasoconstriction through a 5-HT2 receptor mediated pathway (Sekida et al.1997b). Type-1 and type-2 5-HT receptors have been shown to have increased reac-tivity in the vasculature feeding experimental tumours, and agonists directed towardseither one of these receptor types can cause vasoconstriction (StuÈ cker et al. 1997).Related to the present study, it has been shown that 5-HT receptors in the tumour-feeding vasculature are more responsive to agonists than in normal tissues (StuÈ ckeret al. 1991). This fact may explain the selective e� ect of KB-R8498 on the tumourblood ¯ ow when compared to other tissues. Apparently such a profound decrease intumour perfusion and resultant decline in heat dissipation during heating wasresponsible for the potentiation of the e� ect of hyperthermia to suppress the tumourgrowth, as shown in ® gure 2 and table 2.

It has also been observed that the tumour pO2 signi® cantly decreased from amedian value of 5.7 1.5mmHg before treatment to a median value of only1.6 0.1mmHg immediately after a dose of 15mg/kg of KB-R8498 (data notshown). The e� ect of the drug on tumour pH has not been determined. It wouldbe reasonable, however, to expect that tumour pH is decreased, due to the decreasein pO2 by the drug. Such a decrease in tumour pHand pO2 may also account, at leastin part, for the marked increase in response of tumours to hyperthermia. As shownin ® gure 2, hyperthermia applied 1h after KB-R8498 injection was almost equally ase� ective as application of hyperthermia immediately after the drug administration,although the tumour blood perfusion recovered to almost 70% of control 1h afterthe drug administration. It is probable that the tumour pO2 and/or pH were stilldecreased 1h after drug administration therefore enhancing the hyperthermic e� ect.It is hoped that KB-R8498 may also potentiate bioreductive drugs by decreasingtumour pO2. Further investigation on the potential usefulness of KB-R8498 as ananti-tumour drug used alone, or in combination with other modalities, is warranted.

Acknowledgements

This work was supported by NCI grant number CA44114. KB-R8498 was kindlyprovided by Kanebo, LTD, Osaka, Japan.

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Reduction of blood ¯ ow by KB-R8498 5

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