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Experimental Therapeutics, Preclinical Pharmacology |
Department of Medical Oncology, University Hospital Vrije Universiteit, 1081 HV Amsterdam [F. A. A. v. A., E. B., W. J. F. v. d. V.]; Department of Pharmacology and Toxicology, University Maastricht, 6200 MD Maastricht [F. A. A. v. A., G. R. M. M. H., A. B.]; and Department of Safety and Environmental Affairs, Vrije Universiteit, 1081 HV Amsterdam [K. K.], the Netherlands
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Recently, we have shown in mice that the semisynthetic flavonoid monoHER2
(Fig. 1)
protects against doxorubicin-induced cardiotoxicity (5
, 6)
without compromising the antitumor activity (7)
. This strongly supports the free radical hypothesis, because this flavonoid is an excellent antioxidant. Apart from vitamin C and the fat-soluble tocopherols, flavonoids are the most abundant and active antioxidant compounds naturally occurring in food (8)
.
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To select the most promising candidate from this series, we screened these new flavonoids for their antioxidant activity, their cardioprotective properties in vitro, and their possible cytotoxic effects.3
Frederine (Fig. 1B)
was selected from the series because this compound is a potent antioxidant, protects against doxorubicin-induced cardiotoxicity in vitro, and is nontoxic to hepatocytes (at 250 µM).
The purpose of the present study was to investigate Frederine for its toxicity in mice and for its cardioprotective properties in mice treated with doxorubicin using telemetry. Frederine was also investigated for its possible influence on the antitumor activity of doxorubicin in tumor cell lines in vitro as well as in human ovarian cancer xenografts grown in nude mice.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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pH 7.8–8). Frederine (3',4'-dihydroxy-3-glucosylflavone; MW 432.4) was synthesized as described earlier (9)
. For the toxicity study in mice, Frederine was dissolved in 18 mM NaOH in sterile water, giving a final concentration of 20 mg/ml. For the cardiotoxicity and antitumor activity studies in mice, Frederine was dissolved in 5 mM NaOH in sterile water, giving a final concentration of 10 mg/ml (pH 7.8–8). Formulated doxorubicin (doxorubicin hydrochloride; 2 mg/ml) was obtained from Pharmachemie B.V. (Haarlem, the Netherlands). Before injection, the contents of the vial were dissolved in 0.9% NaCl solution (sterile).
Animals
All of the protocols were approved by the Ethics Committee for Animal Experiments of the Vrije Universiteit in Amsterdam. Thirty-six male BALB/c mice (20–25 g) obtained from Harlan Nederland B.V. (Horst, the Netherlands) were kept in a light- and temperature-controlled room (21–22°C; humidity, 60–65%). The animals were fed a standard diet (Hope Farms B.V., Woerden, the Netherlands). Tap water and food pellets were provided ad libitum. The animals were allowed to adapt to the laboratory housing conditions for at least 1 week before starting the experiment. Nude female mice (Hsd:athymic-nu) were obtained from Harlan Nederland B.V. (Horst, the Netherlands) at the age of 6 weeks. The animals were maintained in isolation under controlled atmospheric conditions (temperature, 23–25°C; humidity, 50–60%). Animal handling was carried out under sterile conditions.
Toxicity Study
In the toxicity study, a dose of 170 mg/kg Frederine was used to compensate for the shorter period of the experiment (2 weeks) compared with the cardiotoxicity study (68 mg/kg for 6 weeks). BALB/c mice were treated 5 times/week with 170 mg/kg Frederine (n = 5), which is equimolar to 250 mg/kg monoHER, or 0.9% NaCl solution (n = 5) i.p. for 2 weeks. During treatment, body weight was determined daily as a measure of general toxicity. After 2 weeks, mice were killed by decapitation, and heart, liver, and kidneys were removed quickly after visual inspection. Organ weights were determined as a measure for organ toxicity. Organ weight was expressed as relative organ weight, i.e., the weight of an organ relative to the body weight of the mouse (g/g body weight x 100%).
Cardiotoxicity Study
Telemetry System
The telemetry system, which consisted of implantable transmitters (TA10ETA-F20) and telemetry receivers (RPC-1), was obtained from DATA Sciences International (St. Paul, MN). The frequency output (Hz) from the transmitter was monitored by the telemetry receivers placed underneath the animal’s cage. Samples were taken every 10 min using the Data Sciences data acquisition system (Dataquest A.R.T. 1.01).
Surgery
BALB/c mice were anesthetized with 0.07 ml/10 g i.p. of a mixture of Hypnorm (0.315 mg/ml fentanyl and 10 mg/ml fluanisone), Dormicum (5 mg/ml midazolam), and sterile water in the ratio 1:1:2. Surgery was performed as described in detail by Kramer et al. (15)
. In short, the transmitter was implanted in the peritoneal cavity of each mouse 2 weeks before the start of the treatment. The leads of the transmitter were sutured s.c. in lead II position [the (-) lead at the right shoulder and the (+) lead toward the lower left chest].
Treatment
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 1, (n = 7) 0.9% NaCl solution i.v.; group 2, (n = 7) 0.9% NaCl solution i.p., followed by 4 mg/kg doxorubicin i.v. after 1 h; group 3, (n = 5) 100 mg/kg monoHER i.p., followed by 4 mg/kg doxorubicin i.v. after 1 h and 100 mg/kg monoHER i.p. every 24 h for 4 days (7)
; group 4, (n = 6) 68 mg/kg Frederine i.p., followed by 4 mg/kg doxorubicin i.v. after 1 h and 68 mg/kg Frederine i.p. every 24 h for 4 days; and group 5, (n = 5) 68 mg/kg Frederine i.p. and 68 mg/kg Frederine i.p. every 24 h for 4 days.
To compare the potency of Frederine with monoHER, we administered 68 mg/kg Frederine, which is an equimolar dose of 100 mg/kg monoHER. Using this concentration, a decreased or increased potency would be identifiable, because 100 mg/kg monoHER provided approximately 50% protection in the present dose schedule. For the monoHER group (group 3), data were used, which were presented earlier (7) .
i.v. injections were administered in the tail vein. After the treatments, the animals were observed for 2 additional weeks. During treatment and the observation period, body weight was determined once/week as a measure of general toxicity. ECG and heart rate were registered telemetrically in the freely moving animals once/week until the end of the study.
Parameters
Changes in weight were taken as an indication of general toxicity of the treatments, as were behavior of the animal and general impression of the condition. For interpretation of the ECG, five complexes were analyzed in detail. The ST interval was determined as mean ± SD of these five complexes. At the end of the study, the animals were sacrificed by decapitation.
Antitumor Activity Studies
Cell Culture
Three cell lines were used, i.e., the human ovarian cancer cell lines, A2780 (16)
and OVCAR-3 (17)
, and the human breast cancer cell line, MCF-7 (18)
. All of the cell lines were cultured in DMEM (Life Technologies, Inc.) supplemented with 10% heat-inactivated FCS (Sebak, Aidenbach, Germany), 50 IU/ml penicillin, and 50 µg/ml streptomycin (Flow, Irvine, United Kingdom) in an incubator with a humidified atmosphere containing 5% CO2 at 37°C.
A stock solution of Frederine was prepared in DMSO. Doxorubicin was dissolved in sterile water. Drugs were diluted in culture medium immediately before their addition to the culture plates. The DMSO concentration never exceeded 0.55%, which did not 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 quadruplicate at a density of 5000 cells/well for MCF-7 and OVCAR-3 and 3000 cells/well for A2780. After 24 h (day 1), Frederine was added in a final concentration of 100 µM in the presence or absence of doxorubicin (final concentrations ranging from 10-14 to 10-5 M; dosing interval, 5 min before doxorubicin and, for OVCAR-3 cells, 1 h before doxorubicin). The total exposure time was 96 h. At the end of the drug exposure period (day 5), growth inhibitory effects were evaluated with the standard tetrazolium test (19) . Briefly, after removal of the supernatant, 50 µl of tetrazolium-solution (0.4 mg/ml) was added to each well, and the cells were incubated at 37°C and 5% CO2 for at least 1.5 h. Subsequently, 200 µl of DMSO with 0.5% FCS was added to the cells, and the absorption of the formed formazan product was measured using a multiscan bichromatic platereader (A540 nm-A690 nm; Reader Microelisa System; Organon Teknika, Turnhout, Belgium). Data were collected and analyzed using Excel. The results were expressed as the IC50, which is the concentration of the drug giving a 50% inhibition of cell growth of treated cells when compared with the growth of control cells.
In Vivo Antitumor Activity
Experimental Design.
The drug tolerance and antitumor activity studies were performed with the same dose as that for the cardiotoxicity study. For drug tolerance studies, nontumor-bearing nude mice (8 weeks of age) were used. Each group contained three mice. After treatment, which was the same as for the antitumor studies, the mice were weighed daily to determine gross toxicity.
For the antitumor studies, 106 cells from OVCAR-3 were inoculated s.c. into both flanks of 8-week-old mice. The solid tumors arising were used for transplantation of small fragments in subsequent recipients. Treatment was started when the tumor size was approximately 100 mm3; the first treatment day was designated as day 0. Treatment and control groups consisted of five to six mice each. Mice were weighed, and tumors were measured twice/week.
Treatment.
Treatments are as follows: group 1, no treatment; group 2, 8 mg/kg doxorubicin i.v. on days 0 and 7; group 3, 8 mg/kg doxorubicin i.v. on days 0 and 7, 68 mg/kg Frederine i.p. on days 0 and 7 (5 min before doxorubicin), and in 24-h intervals on days 1–4 and days 8–11; group 4, 8 mg/kg doxorubicin i.v. on days 0 and 7, 68 mg/kg Frederine i.p. on days 0 and 7 (1 h before doxorubicin), and in 24-h intervals on days 1–4 and days 8–11; and group 5, 68 mg/kg Frederine i.p. on days 0–4 and in 24-h intervals on days 7–11.
Previously, we established that the maximum tolerated dose for one i.v. injection of doxorubicin/week during 2 weeks was 8 mg/kg. At this schedule, tumor-bearing nude mice showed a reversible weight loss of approximately 10% of the initial weight within 2 weeks after the first injection (20) .
Tumor volumes were calculated according to the formula 0.5 x length x width x height (21) . The tumor volume from the start of the treatment (V0) until the value at any given time (Vt) was calculated for each tumor and expressed as the relative tumor volume (Vt/V0) on the day of measurement. The mean of these values was used to calculate the efficacy of the treatment as a ratio of the relative tumor values between treated (T) and control (C) tumors (T/C x 100%). Growth inhibition was expressed as 100% - (T/C x 100%).
Statistical Analysis
All of the parameters were expressed as mean ± SE unless stated otherwise. Parameters were evaluated using Student’s t test in Excel or ANOVA with Fisher’s LSD test for multiple comparisons when ANOVA indicated significant differences between groups. The program used for this ANOVA analysis was "NCSS" (by Dr. J. L. Hintze, Kaysville, Utah). For weekly comparisons between groups (multiple comparisons), the level of significance chosen was 99% (P < 0.01).
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RESULTS |
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No visible abnormalities of the organs were detected. The organ weights of the heart, liver, and kidneys were not significantly different between the two groups. For the saline-treated group, relative weights were 4.07 ± 0.16%, 1.46 ± 0.04%, and 0.44 ± 0.02% for liver, kidneys, and heart, respectively. In case of the Frederine-treated group, relative organ weights were 4.22 ± 0.05%, 1.53 ± 0.03%, and 0.45 ± 0.02% for liver, kidneys, and heart, respectively.
Cardiotoxicity Study
General Toxicity.
After surgery, recovery of the animals was indicated by an increase in weight after an initial decrease (15)
and by normal behavior such as building a nest of the available paper towels. Animals appeared lively throughout the study, and no behavioral changes were observed compared with mice without transmitters. There were no signs of decreased activity, which would indicate general toxicity.
Weight Gain.
A trend was observed that weight gain in the doxorubicin-treated group was somewhat less than that in the doxorubicin/monoHER and saline groups [percentage weight gain at the end of the study, mean ± SE, in groups 1 to 5 was 10.3 ± 4.1% (saline), 3.5 ± 1.9 (doxorubicin), 13.4 ± 1.9 (monoHER; Ref. 7
), 7.7 ± 1.6 (doxorubicin + Frederine), and 9.8 ± 2.1 (Frederine), respectively].
Pathology.
Three deaths occurred: one in the control group (group 1) during week 8, one in the doxorubicin group (group 2) during week 4, and one in the doxorubicin + Frederine group (group 4) during week 5. No explanation could be found for these three deaths after necropsy. The abdominal organs, such as kidney, liver, and intestine, appeared normal in all of the mice, including the three deaths. This indicates that both the transmitter and the cardioprotectors did not cause any visible abnormalities.
ECG Measurements.
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 by 16.9 ± 2.7 ms in week 8 (Fig. 2)
. When 100 mg/kg monoHER was given 5 days/week in combination with the weekly doxorubicin injections, the increase of the ST interval was 7.8 ± 1.7 ms after 8 weeks (P < 0.01, relative to doxorubicin and control; Ref. 7
). In contrast to monoHER, an equimolar dose of Frederine was able to reduce the increase in ST interval completely to 1.7 ± 0.3 ms (P < 0.01, relative to monoHER; P < 0.001, relative to doxorubicin; not significantly different from control). Frederine alone did not result in a change of the ST interval.
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. All of the three cell lines showed a similar sensitivity to doxorubicin. Frederine alone (100 µM) showed limited inhibition of cell growth, in particular in MCF-7 and OVCAR-3 cells (growth of 83.8% and 86.8% relative to control for MCF-7 and OVCAR-3, respectively). More importantly, Frederine given 5 min before doxorubicin did not reduce the antitumor activity of doxorubicin in the investigated cell lines. In the case of OVCAR-3 cells, no difference was found between the 5-min and 1-h dosing intervals.
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and Fig. 3
. Combination of Frederine with doxorubicin did not lead to a more pronounced weight loss than doxorubicin alone, whereas mice receiving Frederine alone did not show a significant weight loss. On day 15 of the experiment, both the doxorubicin-treated group and the Frederine-doxorubicin-treated groups had recovered from their weight loss. The Frederine-treated group showed a weight gain similar to the control animals. Doxorubicin treatment resulted in a significant growth delay of the OVCAR-3 xenografts, with a growth inhibition of 74% after 29 days (P < 0.05, relative to control). Treatment with 68 mg/kg Frederine alone did not influence tumor growth. Addition of Frederine to doxorubicin did not affect the antitumor activity of the latter when Frederine was administered 5 min before doxorubicin. However, when Frederine was administered 1 h before doxorubicin, Frederine significantly reduced the growth delay caused by doxorubicin alone (day 39, P < 0.05).
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Because a relatively high dose of monoHER is needed to provide complete protection (500 mg/kg, in mice), we have recently synthesized a series of new flavonoids, using monoHER as a lead compound. The combination of parameters such as antioxidant activity (lipid peroxidation), cytotoxicity, and cardioprotection against doxorubicin in vitro (isolated mouse left atrium model) is thought to be an efficient method for the screening of potential cardioprotectors. From this consecutive set of in vitro assays, the synthetic flavonoid Frederine has been selected.3 The antioxidant activity was established using LPO and trolox equivalent antioxidant capacity assays. Frederine is 5 times more potent in the LPO assay, provides similar protection against doxorubicin in the isolated mouse left atrium, and shows a slightly higher lactate dehydrogenase leakage and a slightly larger decrease in reduced glutathione concentration in hepatocytes, when compared with monoHER. The present study deals with possible toxic effects of Frederine in mice and its effect on the cardiotoxicity and antitumor activity of doxorubicin.
Frederine did not induce weight loss, significant changes in the relative organ weight, or visible changes to the organs when administered at the dose of 170 mg/kg, which is equimolar to 250 mg/kg monoHER. To the contrary, a significantly larger weight gain was observed with respect to controls, whereas heart, liver, and kidneys were not different from control nontreated organs. This weight gain could also be an indication for toxicity, because organ toxicity might lead to an increased organ weight and, thus, an increased body weight. Therefore, changes of relative organ weights are often used as an indication of organ toxicity (26 , 27) . As shown, the relative organ weights of heart, liver, and kidneys did not change. As a result, Frederine is considered to be safe at the dosing schedule used.
As a measure for cardiotoxicity, we have used the changes in ST interval, which correlate with the degree of cardiotoxicity in both rats (28) and mice (29) . We previously validated (30) this model by correlating the increase in ST interval with the histological score using Billingham’s grading scale. The histological scores/individual mouse were found to be in agreement with the increase in ST interval (31) . In a previous study, 500 mg/kg monoHER given as an i.p. injection 5 days a week was needed to completely prevent the cardiotoxicity in mice that was caused by 4 mg/kg doxorubicin given in six weekly i.v. injections. This treatment resulted in an increase in 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. 5 ). A combination of doxorubicin with 100 mg/kg monoHER gave approximately 53% protection against the toxicity of doxorubicin (increase in ST interval, 7.8 ± 1.7 ms).
To compare the potency of Frederine with monoHER, we administered 68 mg/kg Frederine, which is equimolar to 100 mg/kg monoHER. At this dose, both a higher or lower potency of Frederine compared with monoHER would be identifiable. We found that 68 mg/kg Frederine given as an i.p. injection 5 times a week was able to completely protect against doxorubicin-induced cardiotoxicity. This treatment resulted in an increase in ST interval of only 1.7 ± 0.3 ms after 8 weeks (P < 0.001, relative to doxorubicin; not significantly different from control). Thus, 68 mg/kg Frederine provided at least the same protection as 500 mg/kg monoHER. This means that our new compound is at least 5 times more potent than monoHER. The increased potency for Frederine might be explained by the increased antioxidant potency compared with monoHER (IC50 values in the LPO assay for Frederine and monoHER were 2.8 ± 0.9 and 12.7 ± 3.7 µM, respectively), although Frederine did not show an increased protection on the isolated mouse left atrium. Other factors such as differences in pharmacokinetics, metabolism, or mechanisms of action may also play a role.
In the antitumor activity experiments with the human OVCAR-3 xenograft described here, 68 mg/kg Frederine administered 5 min before doxorubicin was found not to influence the growth inhibition of doxorubicin. In addition, Frederine did not increase weight loss caused by doxorubicin. However, if Frederine was administered 1 h before doxorubicin, it seemed to reduce the growth delay caused by doxorubicin alone. After 29 days, Frederine did not significantly influence the antitumor activity of doxorubicin, but after 39 days, a small but significant decrease in antitumor activity could be observed (P < 0.05). This is in contrast to the data obtained from the in vitro experiments, where Frederine did not affect the antitumor activity of doxorubicin in OVCAR-3 cells with both dosing intervals.
The effect on the growth delay of doxorubicin is not necessarily a drawback in the further development of Frederine as a cardioprotector. When Frederine was administered 5 min before doxorubicin, no significant effect on the antitumor activity was observed even after 39 days. This means that the dosing schedule could play an important role. Cardioprotective properties of Frederine could be investigated in mice using a 5-min dosing interval instead of the current interval of 1 h. Furthermore, it is possible that the lowest dose of Frederine that is needed to give complete protection is well below 68 mg/kg.
Doroshow (32 , 33) reported that the antitumor activity of doxorubicin is also free-radical mediated, meaning that the ratio of the protector and doxorubicin determines the effect on the antitumor activity. A reduction of the dose of Frederine might abolish its possible effect on the antitumor activity of doxorubicin.
In summary, we found that Frederine is an attractive compound, suggesting that the consecutive set of assays (LPO, cytotoxicity, and isolated mouse left atrium model) used for the screening of these compounds is an efficient method for the selection of potential cardioprotectors. The selected compound Frederine is not toxic to mice under the tested conditions and is at least 5 times more potent as a cardioprotector than monoHER in mice. Frederine did not reduce the antiproliferative effects of doxorubicin in A2780, OVCAR-3, and MCF-7 cells in vitro and in OVCAR-3 xenografts grown in nude mice when administered 5 min before doxorubicin. Therefore, it can be concluded that Frederine merits further investigation as a possible protector against doxorubicin-induced chronic cardiotoxicity in cancer patients.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 To whom requests for reprints should be addressed, at Department of Medical Oncology, BR-232, University Hospital Vrije Universiteit, De Boelelaan 1117, 1081 HV Amsterdam. Phone: 31204442773; Fax: 31204443844; E-mail: f.vanacker{at}azvu.nl 
2 The abbreviations used are: monoHER, 7-monohydroxyethylrutoside; ECG, electrocardiogram; LPO, lipid peroxidation.
3 F. A. A. Van Acker, J. W. Hulshof, G. R. M. M. Haenen, W. M. P. B. Menge, W. J. F. Van der Vijgh, and A. Bast. New synthetic flavonoids as potent protectors against doxorubicin-induced cardiotoxicity, submitted for publication.
Received 12/ 6/00; revised 2/27/01; accepted 2/27/01.
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REFERENCES |
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-phenyl-tert-butyl nitrone protects against myelotoxicity and cardiotoxicity of Adriamycin while preserving the cytotoxic activity. Anticancer Res., 13: 1607-1612, 1993.[Medline]
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); 100 mg/kg monoHER i.p. 1 h before doxorubicin and the next 4 days (significantly different from control from week 5 to week 8; #, P < 0.01; 
); 68 mg/kg Frederine i.p. 1 h before doxorubicin and the next 4 days (significantly different from 100 mg/kg monoHER; P < 0.01; 
); and 68 mg/kg Frederine i.p. 5 days/week (
).
