Monohydroxyethylrutoside, a Dose-dependent ......Vol. 3, 1747-1754. October 1997 Clinical Cancer Research 1747 Monohydroxyethylrutoside, a Dose-dependent Cardioprotective Agent, Does

Vol. 3, 1747-1754. October 1997 Clinical Cancer Research 1747

Monohydroxyethylrutoside, a Dose-dependent Cardioprotective

Agent, Does Not Affect the Antitumor Activity

of Doxorubicin’

Saskia A. B. E. van Acker, Epie Boven,

Karin Kuiper, Dirk-Jan van den Berg,

Joop A. Grimbergen, Klaas Kramer, Aalt Bast,

and Wim J. F. van der Vijgh2

Leiden Amsterdam Center for Drug Research. Division of Molecular

Pharmacology. Department of Pharmacochemistry. Faculty of

Chemistry. Vrije Universiteit (S. A. B. E. v. A.. D-J. v. d. B.. K. Kr..

A. B.l: Department of Medical Oncology. University Hospital Vrije

Universiteit IS. A. B. E. V. A., E. B., K. Ku., D-J. v. d. B..

w. j. F. v. d. VI; and Clinical Animal Laboratory. Faculty of

Medicine, Vrije Universiteit lJ. A. G.l, 1081 HV Amsterdam. the

Netherlands

ABSTRACT

The cumulative dose-related cardiotoxicity of doxoru-

bicin is believed to be caused by the production of oxygen-

free radicals. 7-Monohydroxyethylrutoside (monoHER), a

semisynthetic flavonoid and powerful antioxidant, was in-

vestigated with respect to the prevention of doxorubicin-

induced cardiotoxicity in mice and to its influence on theantitumor activity of doxorubicin in vitro and in vivo. Non-

tumor-bearing mice were equipped with a telemeter in the

peritoneal cavity. They were given six weekly doses of 4

mg/kg doxorubicin i.v., alone or in combination with either100 or 250 mg/kg monoHER i.p., 1 h prior to doxorubicin

administration and for the following 4 days. Cardiotoxic

effects were measured from electrocardiogram changes up

to 2 weeks after treatment. Protection against cardiotoxicity

was found to be dose dependent, with 53 and 75% protec-

tion, respectively, as calculated from the reduction in theincrease in the ST interval. MonoHER and several other

flavonoids with good antioxidant properties were tested fortheir antiproliferative effects in the absence or the presenceof doxorubicin in A2780 and OVCAR-3 human ovarian

cancer cells and MCF-7 human breast cancer cells in vitro.

Some flavonoids were directly toxic at 50 and 100 �isi,

whereas others, including monoHER, did not influence the

antiproliferative effects of doxorubicin at these concentra-

tions. The influence of monoHER was further tested on the

Received 12/13/96: revised 7/1/97: accepted 7/1/97.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby marked

advertisement in accordance with 18 U.S.C. Section 1734 solely to

indicate this fact.

I This work was supported by Dutch Cancer Society Grant IKA 92-127.

2 To whom requests for reprints should be addressed, at Department of

Medical Oncology. University Hospital Vrije Universiteit, Dc Boelelaan

I I I 7, 108 1 HV Amsterdam, the Netherlands. Phone: 31-20-4442631:Fax: 31-20-4443844.

growth-inhibitory effect of 8 mg/kg doxorubicin i.v., given

twice with an interval of 1 week in A2780 and OVCAR-3

cells that were grown as s.c. xenografts in nude mice.

MonoHER, administered 1 h before doxorubicin in a doseschedule of 500 mg/kg i.p. 2 or 5 days per week, was not toxic

and did not decrease the antitumor activity of doxorubicin.

It can be concluded that monoHER showed a dose-depen-dent protection against chronic cardiotoxicity and did not

influence the antitumor activity of doxorubicin in vitro or in

vivo.

INTRODUCTION

Doxorubicin, an anthracycline with potent antitumor

activity against a wide range of human malignancies, has the

major acute toxicity of bone marrow suppression. The long-

term clinical usefulness is limited by a cumulative dose-

related cardiotoxicity. The chronic toxicity to the heart is

believed to be caused by the formation of oxygen-free radi-

cals ( 1 ), although other mechanisms of toxicity have been

suggested. With the current use of hematopoietic growth

factors, which allow for high-dose chemotherapies, including

anthracycline therapy, cardiotoxicity will become an even

more frequent treatment-limiting factor. This is because of

higher peak plasma levels, which are more cardiotoxic than

low levels and, in turn, lead to the maximum cumulative dose

in an earlier stage of treatment.

Recently, we reported that the semisynthetic flavonoid

antioxidant monoHER3 (Fig. 1 ) provides excellent protection

against chronic doxorubicin-induced cardiotoxicity in a

mouse model at a dose of 500 mg/kg i.p.. given I h before

doxorubicin and for the next consecutive 4 days. At that dose

level, it showed full protection, and no toxic side effects were

observed (2). This is in contrast with the clinically successful

compound Cardioxane (ICRF-187), which has the disadvan-

tage of being toxic to bone marrow (3). We found that the

protection offered by monoHER against doxorubicin-induced

cardiotoxicity was comparable to that of ICRF-l87, but the

toxicity, expressed by weight loss, was more severe in ICRF-

187-treated mice than it was in monoHER-comedicated mice

(2).

MonoHER is the best antioxidant in the registered fla-

vonoid mixture Venoruton (4, 5), which possesses protective

activity against doxorubicin-induced cardiotoxicity (6). To eval-

uate its possible clinical application, we investigated the pro-

tecting capacity of monoHER in two lower doses, 250 and 100

, The abbreviations used are: monoHER. 7-monohydroxyethylrutoside:ECG. electrocardiogram: T/C%, treated versus control, multiplied by

l(XYk.

Research. on February 25, 2020. © 1997 American Association for Cancerclincancerres.aacrjournals.org Downloaded from

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MonoHER

OH

Cyanidanol

�OH

Taxifolin

OH 0

1748 monoHER in Doxorubicin Cytotoxicity

OH

� -#4, OH

OGlu-Rha

OH 0

OH

Fig. 1 Molecular structures ofmonoHER, cyanidanol, taxifo-lin, rutin, kaempferol, and quer-

cetin.

HO.

,,, OH

OH

OH 0

Kaempferol

OH

Rutin � OH

HO�O��O’

OH

OH 0

Quercetin

mg/kg, which were compared with the initial 500 mg/kg dose, to

obtain insight in the presence of a dose dependency with respect

to cardioprotection. Furthermore, we investigated the influence

of monoHER on the in vitro and in vivo antitumor activity of

doxorubicin.

MATERIALS AND METHODS

Chemicals

MonoHER (MW 654.6) was kindly donated by Zyma S.A.

(Nyon, Switzerland). Doxorubicin (Adriblastina; 2 mg/ml) was

obtained from Farmitalia Carlo Erba (Milan, Italy). ICRF-187

(Cardioxane; 20 mg/mi) was kindly provided by Chiron (Am-

sterdam, the Netherlands). Doxorubicin and ICRF- 187 were

dissolved in 0.9% NaC1 solution and stored at -20#{176}Cuntil use.

monoHER was dissolved in 36 mrvi NaOH in sterile water (final

concentration, 33 mg/ml; pH approximately 7.8-8) and stored

at 4#{176}Cfor a maximum of 3 days.

Cardiotoxicity Study

Telemetry System

As described earlier (7), the telemetry system, which con-

sisted of implantable transmitters (model TA1OETA-F20), a

telemetry receiver (model RA1OIO), and an analogue ECG

adapter (option R08), was obtained from DATA Sciences (St.

Paul, MN). The data acquisition system consisted of a MacLab

(MLO2O MacLab/8; ADlnstruments Ltd. London, England),

which was connected to an Apple Macintosh LCII 4/80 com-

puter with the program Chart from MacLab. The transmitter was

activated by a magnet, after which the output of the transmitter

was received by an antenna that was mounted in a receiver

board, which was placed under the animal cage. This board was

connected to the data acquisition system. The sampling rate was

400 samples/s.

Surgery

For the chronic cardiotoxicity study, male BALB/c mice

(20-25 g) obtained from Harlan CPB (Zeist, The Netherlands)

were kept in a light- and temperature-controlled room (21-

22#{176}C;humidity 60-65%). The animals were fed a standard diet

(Hope Farms, Woerden, the Netherlands) and were allowed tap

water ad libitum. Animals were kept in quarantine for at least 1

week before surgery.

The mice were anesthetized i.p. with 0.07 ml per 10 g of a

mixture of 0.315 mg/mI fentanyl and 10 mg/mi fluanisone

(Hypnorm), midazolam (Dormicum, 5 mg/mi), and sterilized

water in the ratio 1 :1:2. Surgery was performed as described in

detail by Kramer et al. (7). In short, the transmitter was im-



Clinical Cancer Research 1749

planted in the peritoneal cavity of each mouse at least 2 weeks

before the start of the treatment. The leads of the transmitter

were sutured s.c. in the lead II position [the (-) lead at the right

shoulder and the (+) lead toward the lower left chest].

Experimental Design

After surgery, the mice were allowed to recover for 2

weeks, after which they were submitted to one of the following

weekly dose schedules for 6 weeks: group I (ii = 6), 0.05 ml of

a 0.9% NaCl solution iv.; group 2 (ii = 5), 4 mg/kg doxorubicin

iv.; group 3 (n 6), 500 mg/kg monoHER i.p., 4 mg/kg

doxorubicin iv. after 1 h, followed by 500 mg/kg monoHER i.p.

every 24 h for 4 days (monoHER 500); group 4 (n = 6), 250

mg/kg monoHER i.p., 4 mg/kg doxorubicin iv. after I h,

followed by 250 mg/kg monoHER i.p. every 24 h for 4 days

(monoHER 250); or group S (n = 5), 100 mg/kg monoHER i.p.,

4 mg/kg doxorubicin iv. after 1 h, followed by 100 mg/kg

monoHER i.p. every 24 h for 4 days (monoHER 100).

iv. injections were administered in the tail vein. After 6

weeks of treatment, the animals were observed for another 2

weeks. During treatment and the observation period, the body

weights were determined once a week as a measure of general

toxicity. ECG was registered in the freely moving animal once

a week until the end of the study.

Parameters

Telemetry Parameters. For interpretation of the ECG,

four consecutive complexes were analyzed in detail. The PR

segment, QRS complex, QT interval, and ST interval were

calculated as the mean ± SD of these four complexes. In case

of an unacceptable variation in the complexes due to noise

caused by movement of the animal (coefficient of variation

> 10%), four other consecutive complexes were selected for

evaluation.

Other Parameters. Changes in weight were taken as a

measure of general toxicity, as were behavior of the animal and

general impression of the condition. At the end of the study, the

animals were sacrificed by decapitation. The organs were sub-

mitted to pathological examination and investigated for any

abnormalities, either due to doxorubicin or to the transmitter.

Cell Culture

Three cell lines were used, the human ovarian cancer cell

lines A2780 (8) and OVCAR-3 (9) and the human breast cancer

cell line MCF-7 (10). The cells were routinely grown at 37#{176}C

and 5% CO2 in DMEM (Flow Laboratories, Irvine, Scotland)

supplemented with 7.5% heat-inactivated FCS (Life Technolo-

gies, Inc., Gaithersburg, MD).

Stock solutions of the flavonoids tested were freshly pre-

pared in DMSO. Doxorubicin was dissolved in sterile 0.9%

NaCI solution. Drugs were diluted in culture medium immedi-

ately prior to their addition to the culture plates. The DMSO

concentration never exceeded 0.55%, which was found not to

influence cell growth.

Exponentially growing cells were harvested and plated as

single-cell suspensions in 96-well flat-bottomed microtiter

plates (Greiner, Solingen, Germany). Cells were seeded in trip-

licate in 100 p.1 of medium at a density of 5000 cells/well for

A2780 and MCF-7 and 8000 cells/well for OVCAR-3. After

24 h (day I), 100 p.1 of medium containing the test compound

were added, together with 25 p.1 of doxorubicin or medium. The

total exposure time to the drugs was 72 h. At the end of the drug

exposure period (day 4), growth-inhibitory effects were evalu-

ated with the standard sulforhodamine B test ( I 1). Absorbance

was read at 540 nm using an automated spectrophotometric

microplate reader (Argus 400; Canberra Packard, Tilburg, The

Netherlands). Data were collected and analyzed using Kineti-

Calc (Bio-Tek EIA Application Software, Bio-Tek Instruments,

Winooski, VT). The results were expressed as the IC5�, which is

the concentration of the drug giving a 50% inhibition of cell

growth of treated cells when compared to the growth of control

cells. Doxorubicin growth inhibition curves were plotted, and

IC��s were calculated in the presence and absence of the fia-

vonoids.

Antitumor Activity Study

Experimental Design

Nude female mice (Hsd:Athymic-nu) were obtained from

Harlan CPB at the age of 6 weeks. The animals were maintained

in isolation under controlled atmospheric conditions (tempera-

ture, 23-25#{176}C; humidity, 50-60%). Animal handling was car-

ned out under sterile conditions. For drug tolerance studies,

non-tumor-bearing mice (8 weeks old) were used. Each group

contained three mice. After treatment, the mice were weighed

daily to determine toxicity.

For the antitumor studies, lO� cells from A2780 and

OVCAR-3 were inoculated s.c. into both flanks of 8-week-old

mice. The solid tumors arising were used for the transplantation

of small fragments in subsequent recipients. As xenografts,

A2780 shows an undifferentiated pattern and has a volume

doubling time of approximately 3.5 days. OVCAR-3 shows a

pattern of a poorly differentiated serous adenocarcinoma and

has a volume doubling time of approximately S days. Treatment

was started when the tumor size was approximately 600 mm3

for A2780 and 150 mm3 for OVCAR-3; the first treatment day

was designated as day 0. Treatment and control groups consisted

of five or six mice each. Mice were weighed and tumors were

measured twice a week for A2780 and once a week for

OVCAR-3.

Treatment Evaluation

Treatments were as follows: group I , no treatment; group

2, 8 mg/kg doxorubicin iv., days 0 and 7; group 3, 8 mg/kg

doxorubicin iv., days 0 and 7, 500 mg/kg monoHER i.p., days

0 and 7 (1 h before doxorubicin), 1-4, and 8-11; group 4: 8

mg/kg doxorubicin iv., days 0 and 7, 500 mg/kg monoHER i.p.,

days 0 and 7 (1 h before doxorubicin) and days I and 8; group

5, 8 mg/kg doxorubicin iv., days 0 and 7, 100 mg/kg ICRF-l87

i.p., days 0 and 7 (1 h before doxorubicin); and group 6, 500

mg/kg monoHER i.p., days 0-4 and 7-I I.

Earlier, we established the maximum tolerated dose of 8

mg/kg i.v. for doxorubicin for two weekly injections. At this

schedule, tumor-bearing nude mice will show a reversible

weight loss of approximately 10% of the initial weight within 2

weeks after the first injection.

Tumor volumes were calculated according to the formula

0.5 X length x width X height (12, 13). The tumor volumes

from the start of treatment (V0) until any given time (V,) was

calculated for each tumor and expressed as the relative tumor



0

.544

.0444’

Fig. 2 Weight gain per treatment group in the cardiotoxicity study.

Data points, mean percentage of weight gain; bars, SE. Control, 0.9%NaCI solution iv. once weekly for 6 weeks; doxorubicin, 4 mg/kg iv.

once weekly for 6 weeks. MonoHER 500, 250, and 100 indicate 500,250, and 100 mg/kg, respectively, i.p., I h prior to doxorubicin and

every 24 h for 4 days.

Weeks

2 4 6 8

Weeks

10

Fig. 3 Increase in ST interval per treatment group in the cardiotoxicity

study. Data points, mean increase in ST interval; bars, SE. For doses,see legend to Fig. 2.


vi

E

4’

0

ri�

0

4’viCO4’

0

Table I Changes in ST interva 1 per treatment group in the cardiotoxicity study

Week 0.9% NaCl Doxorubicin monoHER 100 monoHER 250 monoHER 500

1

23

4

5

6

7

8

ND”

D”D

D

D

D

D

D

ND

I�I

I

I

I

I

I

ND

NSD

D

D,I

D,I

D,I

D,I

ND

DD,I

D,I

D,I

D,I

D,I

D,I

ND

DD

D

D

D,I

D

D

ANOVA at P < 0.01 with Fisher’s least significant difference test (multiple comparisons).“ ND, no difference indicated by ANOVA; NS, not significant.b D, significant decrease in ST interval when compared to mice treated with doxorubicin.

‘ I, significant increase in ST interval when compared to mice treated with 0.9% NaCl solution.

volume (V,/V0). The mean of these values for all evaluable

tumors in the control and treated groups were used to calculate

treatment efficacy. The ratio of the mean relative volume of

treated tumors and that of control tumors multiplied by 100%

(T/C%) was assessed at each day of measurement (12).

Animal deaths occurring within 2 weeks after the final drug

injection were considered toxic deaths and were excluded from

the evaluation.

Statistical Analysis

All measurements were expressed as mean ± SE unless

stated otherwise. All data were evaluated using ANOVA, with

Fisher’s least significant difference test for multiple compari-

sons when ANOVA indicated significant differences between

groups. The program used for this analysis was NCSS (devel-

oped by Dr. J. L. Hintze, Kaysviiie, UT). The level of signifi-

cance chosen was 99% (P < 0.01), to correct for comparisons

between groups per treatment week (multiple comparisons).

Power analysis of the antitumor activity study with a =

0.05 and 13= 0.1 revealed that a detection of �33% is possible

in the OVCAR-3 xenograft.

RESULTS

Cardiotoxicity Study

General Toxicity/Weight Loss. After surgery, recovery

of the animals was indicated by an increase in weight after an

initial decrease. Behavior appeared normal in all treatment

groups during the entire study as compared to control mice

without transmitters. There were no signs of decreased activity,

which indicated low general toxicity. This was confirmed by the

weight gain (Fig. 2), which revealed no significant differences

between groups. There was, however, a trend (P = 0.098)

toward somewhat less weight gain in the doxorubicin-treated

group than in the other groups (Fig. 2). Pathological examina-



Table 2 IC�0s of growth inhibition by doxorubicin in the presenceof flavonoid”

Cell line

“ Doxorubicin, 8 mg/kg iv.; monoHER, 500 mg/kg i.p.; ICRF-187,

100 mg/kg i.p.

I, Weight on day 0 is 100%. No significant differences were found

between groups.

a Mean ± SD of at least three independent experiments performed

in triplicate. None of the flavonoids had an influence on the control cell

growth, except for those labeled toxic and 50 p.M taxifolin, which had a

small influence.b Doxorubicin in the absence of flavonoid.


Doxorubicin +

flavonoid (p.M)

A2780

(l0_8 M)

MCF-7

(l0� M)

OVCAR-3

(l0� M)

None” 2.05 ± 0.66 2.09 ± 0.75 3.28 ± 1.70

Cyanidanol (50) 1.52 ± 0.16 1.50 ± 0.75 1.73 ± 1.57Cyanidanol (100) 0.85 ± 1.25 0.61 ± 0.08 3.64 ± 0.95

monoHER (50) 2.57 ± 1 .95 1 . 1 1 ± 0.04 4.29 ± I .95

monoHER (100) 2.00 ± 1.82 2.10 ± 0.22 5.67 ± 2.37

Taxifolin (50) 1 . 1 1 ± 2.82 4.7 1 ± 4.8 1 1.74

Taxifolin (100) Toxic Toxic Toxic

Rutin (50) 2.71 ± 1.36 4.78 ± 1.76 5.15 ± 4.07

Rutin (100) 1.83 ± 0.25 3.17 ± 0.49 6.42 ± 2.55

Kaempferol (50) Toxic Toxic ToxicQuercetin (50) Toxic Toxic Toxic

tion of the animals at the end of the study revealed no abnor-

malities caused by either the treatment or the transmitter.

ECG. As described earlier, the ECG signal in lead II

deflection of a mouse is somewhat different from that of man

(14), but it corresponds with the ECG measured in mice that are

under anesthesia or restrained (15, 16). The T wave immediately

follows the QRS complex; thus, there is no ST segment.

The ECGs of the control animals did not change during the

course of the study. Doxorubicin had a profound influence on

the shape of the ECG. The ST interval increased with time (Fig.

3 and Table I) by 16.7 ± 2.7 ms in week 8, whereas the PR

segment and the QRS complex remained constant. monoHER

was able to protect against this ECG change. When 500 mg/kg

monoHER was given S days a week in combination with the

weekly doxorubicin injections, the increase in the ST interval

was 1.7 ± 0.8 ms after 8 weeks (P < 0.001, relative to

doxorubicin, not significantly different from control; Ref. 2). In

a dose of 250 or 100 mg/kg, monoHER protected to a somewhat

lesser extent. The increase in ST interval after 8 weeks for 250

mg/kg monoHER was 4.6 ± 0.7 ms, and for 100 mg/kg mono-

HER, it was 7.8 ± 1.7 ms (both values had P < 0.01, relative

to doxorubicin). No arrhythmias were observed in animals of

any group.

Cell Culture

A2780 cells were approximately 10-fold more sensitive to

doxorubicin than were MCF-7 and OVCAR-3 cells, as deter-

mined from the IC50s of 2.05 X lO_8, 2.09 X l0�, and 3.28 X

io� M, respectively. As can be seen from Table 2, the fla-

vonoids cyanidanoi, monoHER, and rutin were found not to be

cytotoxic at concentrations of 50 and 100 p.M. More importantly,

they did not significantly influence the IC50 of doxorubicin in

the investigated cell lines. Taxifolin showed some cytostatic

activity at 50 p.M. which was indicated by the fact that the

maximum cell growth could not be reached. This effect was the

largest in OVCAR-3 cells. The slight cytostatic activity of SO

p.M taxifolin had no influence on the IC,0 of doxorubicin, which

Table 3 Weight loss o f non-tumor-bearing nude mice

Days of Maximum weight Weight on day

Group” treatment loss” (%) 21b (%)

Doxorubicin 0, 7 9 ± 7 97 ± 5

+ monoHER 0-4, 7-1 1 9 ± 4 94 ± 8

+ ICRF-l87 0, 7 16 ± 8 94 ± 13

could still be calculated. At a concentration of 100 p.M. taxifolin

was toxic (no cell growth) in all cell lines. Kaempferol and

quercetin were toxic in all cell lines at SO p.si; no living cells

were found after the incubation period.

Antitumor Activity Study

First, we investigated the toxicity of monoHER and ICRF-

I 87 in combination with 8 mg/kg doxorubicin, which is the

maximum tolerated dose in a schedule of two weekly injections.

Three non-tumor-bearing nude mice were given 8 mg/kg doxo-

rubicin iv. on days 0 and 7. The toxicity of monoHER was

determined in three non-tumor-bearing nude mice receiving 8

mg/kg doxorubicin iv. on days 0 and 7 and 500 mg/kg mono-

HER i.p. on days 0 and 7 (1 h before doxorubicin) and days 1-4

and 8-11. The toxicity of ICRF-187 was also determined in

three non-tumor-bearing nude mice receiving doxorubicin as

described above and 100 mg/kg ICRF- 187 i.p. on days 0 and 7,

I h before doxorubicin. ICRF-l87 in combination with doxo-

rubicin appeared to cause more pronounced weight loss than

doxorubicin alone (Table 3). MonoHER did not affect the

weight loss caused by doxorubicin.

In the antitumor activity study, doxorubicin treatment re-

suited in a significant growth delay of A2780 xenografts, with a

T/C% of 30% (P < 0.05; Table 4). Treatment with 500 mg/kg

monoHER alone in a S-day schedule did not influence the tumor

growth; the T/C% was 61% (not significantly different from

doxorubicin-treated and control tumors) in A2780 xenografts.

The addition of monoHER or ICRF-187 to doxorubicin did not

affect the growth delay. No differences were observed between

the 2-day and the S-day schedules of monoHER (Table 4 and

Fig. 4A). Also, in OVCAR-3 xenografts, doxorubicin showed a

significant growth delay, with a T/C% of 45% (P < 0.001).

Treatment with 500 mg/kg monoHER alone did not influence

the tumor growth, as can be seen from the T/C% of 97% (not

significantly different from control tumors). Also, for

OVCAR-3 tumors, coadministration of doxorubicin and ICRF-

187 or monoHER by either schedule did not affect the growth

delay caused by doxorubicin (Table 4 and Fig. 4B).

DISCUSSION

The formation of oxygen-free radicals is believed to be the

cause of doxorubicin-induced cardiotoxicity, although com-

pounds that reduce free radical formation, such as antioxidants

and iron chelators, have had limited success in its prevention

(17-20). Until now, only ICRF-187, which is hydrolyzed intra-

cellularly into an iron chelator, found its way to clinical practice



10 20 30

Days

A

4’Ea0

I.0

Ea

4’

CO4’

B

4’

Sa0

I..0

S

4’

CO4’

50

Days

Fig. 4 Antitumor activity of doxorubicin, with or without cardiopro-tector. Data points, mean relative tumor volume; bars, SE. A, A2780

xenografts; B, OVCAR-3 xenografts. Control, no treatment; doxorubi-

cm, 8 mg/kg iv., days 0 and 7; +monoHER 5-day, + 500 mg/kg i.p.,

days 0-4 and 7-I 1; +monoHER 2-day, + 500 mg/kg i.p., days 0 and

1 and 7 and 8; +ICRF-l87, + 100 mg/kg ICRF-187 i.p., days 0 and 7:

monoHER 5-day, 500 mg/kg i.p., days 0-4 and 7-1 1 without doxoru-

bicin. MonoHER and ICRF-l87 at days 0 and 7 were given 1 h prior to

doxorubicin.

-10 0 10 20 30 40


- Table4 Antitumor activity of doxorubicin with or without monoHER or ICRF-187 in nude mice bearing A2780 or OVCAR-3 xenografts

A2780 OVCAR-3Days of

treatmentGroup T/C% (day 17) Toxic death T/C% (day 27) Toxic death

Doxorubicin 0, 7 30 0/6 45 0/6

+ monoHER, 5 days 0-4, 7-1 1 29” 0/6 39” 0/6+ monoHER, 2 days 0. 1, 7, 8 34(1 0/6 37” 0/6

+ ICRF-l87 0, 7 41” 1/6 37” 0/6monoHER, 5 days 0-4. 7-1 1 61” 0/6 97” 0/5

“ Not significantly different from doxorubicin-treated tumors.

I) Not significantly different from control tumors.

(3, 2 1 ). Pretreatment with ICRF- I 87 allowed a 4-fold escalation

in the cumulative dose of doxorubicin, with attenuation of the

doxorubicin-induced cardiomyopathy in animals, and an esca-

lation of at least 2-fold has been reported in patients (21).

ICRF-1 87 did not prevent alopecia and other anthracycline-

induced toxicities. A clear disadvantage of ICRF-l87 is its

toxicity to mitotically active tissues, e.g. , bone marrow, lymph-

oid tissue, testes, and gastrointestinal mucosa (3). Because of

this toxicity and because there is concern about the drug’s

impact on antitumor efficacy, it has not become a popular

addition to doxorubicin treatment.

The success of ICRF-187 in preventing cardiotoxicity,

however, indicates that free radicals are likely to be involved

because iron-catalyzed reactions play an important role in the

formation of radicals in biological systems. In addition, it dem-

onstrates that antitumor activity and cardiotoxicity are probably

caused by different mechanisms or, at least, can be separated

because ICRF- 187 does not influence the antitumor activity of

doxorubicin. This has given researchers a new incentive to study

nontoxic iron chelators and other antioxidants.

Flavonoids are known to be excellent transition metal

chelators and radical scavengers. The combination of these

properties enables “site-specific” scavenging. If oxygen radicals

can still be formed around the iron, which is chelated by the

flavonoid, then these radicals are formed in the vicinity of the

flavonoid and can be scavenged immediately (5). MonoHER is

a powerful antioxidant, the most active compound of the hy-

droxyethylrutosides that are present in Venoruton (5, 22). Ve-

noruton itself was found earlier to partly protect against doxo-

rubicin-induced cardiotoxicity (4). The protection may,

therefore, be caused by the scavenging of oxygen-free radicals

that are generated by doxorubicin or by the chelation of iron,

which catalyzes the formation of more reactive radicals.

As a model for cardiotoxicity, we have used the changes in

ST interval, which have been found earlier to correlate with the

degree of cardiotoxicity in both rats (23) and mice (24, 25). In

a previous study, 500 mg/kg monoHER, given as an i.p. injec-

tion S days a week, was able to completely prevent the cardio-

toxicity in the mouse, which was caused by 4 mg/kg doxorubi-

cm given in six weekly iv. injections. This treatment resulted in

an increase in the ST interval of only 1 .7 ± 0.8 ms after 8 weeks

versus 16.7 ± 2.7 ms for doxorubicin (P < 0.001, relative to

doxorubicin, not significantly different from control; Ref. 2). In

the same study, monoHER alone was found not to exert any

effect on the ECG.

The present results show a clear dose dependency in the




protective effect of monoHER. After 8 weeks, doxorubicin

combined with a dose of 250 mg/kg monoHER caused a small

but steady increase in ST interval (4.6 ± 0.7 ms, 73% protec-

tion, versus 16.7 ± 2.7 ms for doxorubicin). The combination of

doxorubicin with 100 mg/kg monoHER gave approximately

53% protection from the toxicity of doxorubicin (increase in ST

interval, 7.8 ± 1.7 ms). As already mentioned above, doxoru-

bicin combined with 500 mg/kg monoHER resulted in an in-

crease in the ST interval of I .7 ± 0.8 ms in week 8, which was

not significantly different from the ECG in control animals.

It appears that a relatively high dose of flavonoid is needed

for optimal protection against doxorubicin-induced cardiotoxic-

ity. This is probably caused by the high clearance of the fla-

vonoid (26), which, as a consequence, requires repeated dosing.

This in contrast to ICRF-l87, which is given only once, I h

before doxorubicin. As the hydrolyzed product (1CRF-198) is

charged, it cannot easily leave the cell and, therefore, remains at

its site of action. MonoHER is neutral at physiological pH and

can, therefore, probably cross the cellular membrane either way.

Venoruton, which contains about 10% monoHERs (2%

specifically 7-monoHER), was found not to have an influence

on the antitumor activity of doxorubicin in L12l0 and P388

cells in vitro and in vivo with a dose of 1 .5 g/kg ( 1 8, 27). In the

antitumor activity experiments in human tumor xenografts de-

scribed here, the higher dose of 500 mg/kg monoHER was also

found not to influence the growth inhibition of doxorubicin in

A2780 and OVCAR-3 xenografts in either the 2-day or the

S-day dose schedule. In addition, monoHER showed no increase

in weight loss caused by doxorubicin, whereas there was a trend

that weight loss was longer and more pronounced in mice

treated with ICRF-l 87 and doxorubicin than in mice given

doxorubicin alone (Table 3). It should be noted that cardiopro-

tection in the antitumor activity study (8 mg/kg doxorubicin in

combination with 500 mg/kg monoHER) is probably compara-

bie to the 250 mg/kg monoHER dose with 4 mg/kg doxorubicin

in the cardiotoxicity study, whereas ICRF-l87 was dosed in the

same concentration ratio versus doxorubicin in both this antitu-

mor study and the cardiotoxicity study (2, 25).

High doses of monoHER (up to 100 p.M) did not influence

the antiproliferative effects of doxorubicin in three cell lines

derived from human tumor types that are known to be sensitive

to anthracyclines in the clinic. Some other flavonoids tested in

vitro were cytotoxic at concentrations of SO p.M. which was the

lowest concentration tested. The cytotoxicity of flavonoids, in-

ciuding quercetin, has been observed earlier, and some authors

have studied the effect of quercetin as an antitumor agent in

patients (28). Our observations indicate that flavonoids such as

monoHER might be used in vito to modulate cardiotoxicity

because high doses do not affect the antitumor effect of doxo-

rubicin.

In our experiments, monoHER was shown to protect

against chronic doxorubicin-induced cardiotoxicity in a dose-

dependent way. At a dose of 500 mg/kg, it appeared to be at

least as effective as ICRF-187 (2), a compound that has shown

promising results in clinical practice. MonoHER is of interest,

despite the high dose needed for protection, because hydroxy-

ethylrutosides have little toxicity of their own. Importantly,

monoHER does not have a negative influence on the antiprolif-

erative effects of doxorubicin in A2780, OVCAR-3, and MCF-7

cells in vitro and in A2780 and OVCAR-3 xenografts grown in

nude mice. Therefore, it can be concluded that monoHER merits

further investigation as a possible protector against doxorubicin-

induced chronic cardiotoxicity in cancer patients.

ACKNOWLEDGMENTS

We thank Dr. G. Golden from Zyma (Nyon. Switzerland) for the

gift of monoHER. Caroline A. M. Erkelens is acknowledged for tech-

nical assistance.

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1997;3:1747-1754. Clin Cancer Res S A van Acker, E Boven, K Kuiper, et al. agent, does not affect the antitumor activity of doxorubicin.Monohydroxyethylrutoside, a dose-dependent cardioprotective

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