0360-3016/92 $5.00 + .oO Copyright 0 1992 Pergamon Press Ltd.

Inf J. Radmtron Onco/o~y Bml. Phys Vol. 24. PP. 669-673 Printed in the U.S.A. All rights reserved.

??Hyperthermia

Original Contribution

HYPERTHERMIA ENHANCES MITOXANTRONE CYTOTOXICITY ON HUMAN BREAST CARCINOMA AND SARCOMA XENOGRAF’TS IN NUDE MICE G.

M.D.,’ 0. MELLA, M.D., PH.D.,* S. ROSZINSKI, M.D.,3 C. WEISS, M.D., PH.D. (Lond.)3 AND TH. WAGNER, M.D.’

WIEDEMANN,

‘Dept. of Internal Medicine, ‘Dept. of Physiology, Medical University of Liibeck, Ratzeburger Allee 160, D-2400 Liibeck, Germany; and 2Dept. of Oncology, Haukeland University Hospital, N-502 I Bergen, Norway In this preclinical in viva study, we measured antitumor response, local side effects and systemic toxicity of locally applied water-bath hyperthermia given alone or simultaneously with mitoxantrone (3 mg/kg b.w. i.v.; LD 10) on a human derived breast carcinoma (MX 1) or a human sarcoma (S 117) transplanted to female athymic (nude) mice. A single hyperthermia treatment at a tumor temperature up to 43°C for 1 hr caused in both tumor lines only minor tumor regressions and transient tumor growth delay. However, the antitumor effect of mitoxantrone was significantly enhanced by local hyperthermia at 42’C and particularly at 43°C. In both tumor lines complete tumor regressions were achieved only if mitoxantrone was combined with hyperthermia. Undesired side effects and drug toxicity were not enhanced by hyperthermia. According to in vitro data and the results of the present in vim study mitoxantrone seems to be a good candidate for clinical trials in combination with locoregional hyperthermia. Mitoxantrone,

Hyperthermia,

Human tumor xenografts.

Female athymic (nude) NMRI mice (Zentralinstitut ftir Versuchstierzucht, DW-3000 Hannover, Germany) were

maintained in laminar flow installations with free access to pelleted food* and drinking water (HCl added up to pH 2.5, 1.35 g potassium sorbate/l). Mice were kept up to 6 per Makrolon size-2 cages at room temperature 24 ? 1°C and relative humidity of 70%. Tumor pieces for implantation were obtained from donor mice carrying subcutaneously in the neck region a human derived breast carcinoma (MX 1, obtained from Deutsches Krebsforschungszentrum, DKFZ, DW-6900 Heidelberg, Germany; a human tumor line that has been included in the drug screening panel of the NC1 since 1972), and a humanderived sarcoma (S 117, DKFZ, a human tumor line with primary drug resistance noted in the patient from which this line was derived). The recipient mice were anesthesized with i.p. pentobarbital (0.4 to 0.5 ml of 5 mg/ml solution). The tumors were excised from the donor mice and cut into pieces of l-2 mm diameter, which were implanted subcutaneously to the dorsum of the hind paw of recipient mice aged 6-7 weeks. After 5-6 weeks, at tumor volumes of approximately 180 mm3 (MX 1: 190 + 15 mm3, S 117: 170 + 26 mm3), mice with solitary tumors were stratified according to tumor size and assigned to one of the treatment or control groups, each comprising

Presented in part at the 1 lth Conference of the European Society for Hyperthermic Oncology, Latina, Italy, 17-20 September 1990. Reprint requests to: G. Wiedemann, M.D. Acknowledgements-The authors are grateful to D. J. Honess, Cambridge, U.K. for valuable suggestions on this manuscript.

Financial support was provided by the “Werner und Klara Kreitz-Stiftung Kiel”, Miinkeberg, Germany and by the “Gesellschaft der Freunde und FGrderer der Medizinischen Universit& zu Liibeck e.V. Accepted for publication 8 May 1992. * Special nude mouse diet Atromin 14 14 tpf.

INTRODUCIION

anticancer agents presently used clinically have demonstrated significantly increased cytotoxicity at elevated temperatures (3, 7,9, 11; for review: 2,5,6). So far, in vitro studies have shown that the cytotoxicity of mitoxantrone (MITOX), a thermostable (20) synthetic amino anthraquinone, is considerably enhanced by hyperthermia (8, 12). Although it is known that MITOX may be effective as monotherapy in certain human malignancies such as breast cancer (for review: 16) there are to our knowledge no in vivo or clinical data published where the therapeutic efficacy of thermochemotherapy with MITOX has been evaluated. To determine quantitatively the antitumor response of thermochemotherapy with MITOX, a human derived carcinoma sensitive to MITOX and a human sarcoma primarily drug resistant were exposed to either MITOX or heat alone or in combination. Many

METHODS

AND

MATERIALS

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1. J. Radiation Oncology 0 Biology 0 Physics

8-10 mice. The MX 1 contained no significant estrogen (< 2 fmol/mg) or progesterone (6 fmol/mg) hormone receptor protein and, therefore no supplementary estrogen was used. Hyperthermia Prior to treatment, all mice were anesthetized with pentobarbital (0.4 to 0.5 ml of a 5 mg/ml solution) by i.p. injection. The anesthetized animals were fixed on a flat perspex holder. The extremity bearing the tumor was immersed in a thermostatically controlled waterbath. A chromel-alumel microthermocouple+ with a diameter of 0.25 was inserted into the tumor and the signals fed into a digital thermometer circuit.’ During hyperthermia, steady state temperature in the tumor center was held at 43”C, 42”C, 41 “C, 40°C, 39°C or 37°C for 60 min. In order to attain these temperatures in the tumor center the temperature of the waterbath had to be about 0S”C above that of the tumor center. When placing the thermoprobe tip at different depths from the periphery to the tumor center, no temperature differences exceeding 0.5”C occurred. Rectal temperatures were continuously measured with similar thermocouples and maintained at 37 (& 0.5)“C by adjusting the distance of an infrared lamp. Cytotoxic drug Solutions of commercially available MITOX” were freshly made at a concentration of 2 mg/ml and administered at 3 mg/kg b.w. (equal to the LD 10) by tail vein injection. A corresponding volume of 0.9% NaCl solution was given i.v. to mice assigned to hyperthermia alone. To obtain maximal cytotoxicity in combined treatment groups (4), the drug was given i.v. 6-4 min before hyperthermia. Tumor evaluation Tumors were measured with vernier calipers 3X/week, and individual tumor volumes were calculated by the modified ellipsoid formula (?r/6ABZ; 3,4), where A is the longest and B the shorter perpendicular axis of an assumed ellipsoid. Complete tumor regression (CR) was defined as no visible or palpable tumor found at two subsequent measurements. Local tumor control was defined as no visible or palpable tumor observed during a period of 50 days. Tumor growth time was defined as the time required for individual tumors to double their initia! volume. Individual tumor growth was determined and the results for each treatment group pooled at each measurement. Systemic toxicity and local side eflects Blood samples were taken 3X/week with heparin-coated 10 ~1 capillary tubes from the retrobulbar venous plexus

+ K 2/2, Philips, Germany. * Keithley, USA, type 871 A.

Volume 24, Number 4. I992 MX l/3 turnour volume

1.4

O,

“8“--__*_.___ II -- 15

18

22

25

29

32

36 .

days following treatment

Fig. I. Growth curves of human derived MX 1 breast carcinoma xenografts in female nude mice given mitoxantrone 3 mg/kg b.w. i.v., local hyperthermia, or combined modality treatment. Bars: f SEM.

as previously described (2 1, 24). White blood cells and platelets were analyzed by standard laboratory techniques. The animals were weighed 3X/week following treatment and the time of minimum weight was noted. The foot reactions were noted 48 hr following hyperthermia and at every tumor measurement. They were numerically scored using an established scoring system ( 19) where 1.O was a red foot, 1.5 slight edema, 2.0 severe edema, which was the maximal acute reaction seen. Statistical evaluation For comparison of the tumor growth time of different treatment groups, the Mann-Whitney test was used. RESULTS Both untreated MX 1 and S 117 tumors showed initial rapid exponential growth. In the MX 1 tumors hyperthermia at 42°C and 43°C for 1 hr resulted in some minor tumor regressions and transient growth delay before regrowth began at approximately the same rate as in untreated controls (Fig. 1). Though not causing tumor regressions, MITOX alone (3 mg/kg b.w. = LD 10) led to reduced growth rates (Fig. 1). MITOX given prior to hyperthermia at 42’C for 1 hr resulted in a growth delay greater than either modality alone, but no CR was seen (Fig. 1 and 2). When MITOX and 43°C for 1 hr were combined, 8 of 10 mice had complete tumor regressions (Table 1) lasting at least 36 days. 7 mice of 10 were recurrence-free at day 50 (Table 1). The effects of low grade hypertheimia were extensively investigated in the S 117 tumor (Fig. 3). Only 43°C

9Novantrone,

Lederle.

Thermochemotherapy

MX

l/3

human

breast

with mitoxantrone

0 G. WIEDEMANN

671

et al

S 117 HYPERTHERMIA ALONE

carcinama

tumor volume (mm31 .-.9-.. .a+-

Mitox n = IO Mi,oz + 43-C Ih n = l0 -t Mlox+42% Ih n = 8 . ..+..Mtox + 4l’C Ih n : 0 .-0-Ylox t 40’CIh n = w) .-A-,yifox + 39X Ih n = IO -.uito~ + 37’C Ih n = l0 _c cMllrds

1000

s

’

4

01,I 8

4

8

Tage

II

days

Fig. 2. Semilogarithmic presentation of the growth curves of human derived MX 1 breast carcinoma xenografts in female nude mice given mitoxantrone 3 mg/kg b.w. i.v. alone or combined with local hyperthermia at various temperatures (for I hr). Bars: * SEM.

showed an effect on the tumors, although no complete regressions were seen. The S 117 tumor was not sensitive to MITOX alone at the given dose (Fig. 4). Higher concentrations of MITOX were used to see if the S 117 sarcoma was truly drug resistant. and demonstrated only higher systemic toxicities, but no significant tumor regressions (data not shown). In contrast, MITOX alone seems to accelerate the growth of S 117 sarcomas. This is very likely because of a MITOX-induced suppression of the remaining immune response of the host. There was a significant interaction between MITOX and hyperthermia at 42°C for 1 hr (Fig. 4 and 5), even though hyperthermia at that level and MITOX applied separately had no antitumor effect (Fig. 4). The enhancement of the efficiency by combination was even more pronounced at 43°C for 1 hr, where all mice had CR. By day 22, in 2 mice of 8 tumors had recurred (Fig. 5). Com-

II

15 18 22 25 days followinq treatment

29

32

36

Fig. 3. Growth curves of human derived S 117 sarcoma given local hyperthermia at different temperatures for I hr. Bars: +- SEM.

bined 43°C hyperthermia and MITOX was significantly more effective than both hyperthermia alone and MITOX alone in both tumors 0, < 0.00 1). The peak foot reactions were slight, typically 1 to 2, and were not significantly increased by MITOX as compared to the effect of hyperthermia alone (data not shown). The systemic toxicity of MITOX and hyperthermia is shown in Table 2. There was no indication of increased weight loss or increased myelodepression as compared to the effect of MITOX alone when the drug was combined with local hyperthermia (Table 2). DISCUSSION Clinical evidence is accumulating showing that hyperthermia is effective when given together with radiation in recurrent tumors. Therefore, hyperthermia is currently evaluated in Phase III studies as first-line therapy given together with radiation (for review, see 14). In vitro and s II7

Table 1. Antitumor efficacy of mitoxantrone (MITOX) 3 mg/ kg b.w. i.v. given alone or in combination with local hyperthermia at various temperatures (for 1 hr)

Treatment MITOX MITOX MITOX MITOX MITOX MITOX MITOX

+ + + + + +

37”C/l 39”C/I 40°C/l 4l”C/l 42’C/l 43”C/l

hr hr hr hr hr hr

MX l/3: human breast carcinoma

S 117: human sarcoma

CR

LTC

CR

LTC

o/10 o/10 o/10 O/IO o/10 O/8 8/10

o/10 o/10 o/10 o/10 O/IO O/8 7110

O/9 Of10 O/9 o/10 8/8

O/9 oil0 o/9 o/10 618

Note: CR = complete tumor regression; LTC = local tumor control.

iumour volume+ ron,rob (mm31 *- ‘lx Ihr

n

___ _________.__.._ 29

32

36

days falowinq lreatmeni

Fig. 4. Growth curves of human derived S 117 sarcoma given mitoxantrone 3 mg/kg, local hyperthermia, or combined drug and hyperthermia. Bars: + SEM.

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Volume 24, Number 4, I992

Table 2. Systemic toxicity of mitoxantrone Minimum b.w. (mean + SD, g) 27 27 24 23

Controls 43“C/l hr MITOX MITOX + 43”C/l hr

f 3.0 f 3.18 + 3.28 & 2.79

and hyperthermia

Minimum WBC (mean + SD, G/L) 5.09 5.66 2.52 2.43

+ f + f

Minimum thrombocytes (mean f SD, counts/n]) 1363 1375 1109 1154

1.86 1.36 1.62 2.12

k f + f

278 631 154 178

Note: Pooled results from experiments with MX 1 breast carcinoma and S 117 sarcoma carrying nude mice. b.w. = body weight, WBC = white blood cells.

in vivo studies have identified drugs of potential value in clinical Phase II studies of combined hyperthermia and cytotoxic drugs (for review, see 2, 5, 6). The present in vivo study confirms previous in vitro data (8, 12). It demonstrates a significant interaction between MITOX and hyperthermia, leading to enhanced antitumor efficacy and tumor regrowth delay. There is experimental evidence for increased MITOX uptake and increased alkylating reaction rates with increased DNA interaction (8, 12). The present study shows that a tumor (S 117) that is insensitive to MITOX alone or hyperthermia alone at 42°C is significantly affected by the combined modalities; suggesting hyperthermia as a means to overcome primary drug resistance. However, a decreased tumor pH which may occur as a consequence of hyperthermia-induced vascular effects may partly inhibit the desired cytotoxicity of MITOX since low extracellular pH has been shown to protect from MITOX cytotoxicity (10). In some rapidly growing rodent tumors, pH is significantly lowered during hyperthermia due mainly to vascular effects ( 18, for review, see 17). In previous studies with both the present human-derived tumor lines grown in nude mice (24,25) and nude rats (15, see Fig. 6), there was no indication of a shutdown of tumor circulation with local hyperthermia at 43°C for 1 hr. In contrast, an initial increase in tumor oxygenation was measured and maintained during the whole period of S 117 human

heating (Fig. 6). The increased tumor pOz was probably caused by an increase in blood flow. The observed stable pH may be explained by an increased washout effect. An increase in overall tumor blood flow during hyperthermia is in accordance with patient derived data ( 1, 13, 22, 23).

The theoretical problem of reduced thermosensitization of MITOX at low pH may, therefore, not be important in the clinical setting, particularly not at the temperatures usually employed. Criticism has been raised concerning murine models used in experimental tumor therapy. Although human tumor xenografts have their blood vessels from the host, they still represent the animal model with the closest resemblance to human malignancies. The demonstration s 117 nude rots HT43’

s 117 nude mice HT43’

sarcoma

tumor volume (mm?

MX l/3 nude mice

II I

,

,

,

4

8

II

‘; I5 days

< 18

(

,

22

25

,

29

Fig. 5. Semilogarithmic presentation of the growth curves of human derived S 117 sarcoma given mitoxantrone 3 mg/kg b.w. i.v. alone or combined with local hyperthermia at various temperatures (for 1 hr). Bars: f SEM.

Fig. 6. Time course of the changes of mean tumor p02 and of mean tumor pH before, during, and after 1 hr of 43°C hyperthermia (HT) in female nude rats (data taken from 15) and female nude mice (data taken from 24).

Thermochemotherapy with mitoxantrone 0 G.

in this study of a temperature dependent MITOX enhancement is promising. It encourages further experi-

WIEDEMANN et a/.

673

mental studies and early clinical trials of the combination of MITOX and locoregional hyperthermia.

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