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Experimental study of dexrazoxane–anthracycline combinations using the model of isolated perfused rat heart
Toxicology Letters 161 (2006) 37–42
Experimental study of dexrazoxane–anthracycline combinations using the model of isolated perfused rat heart Jo¨elle Pland´e a,b , Denis Platel a,b , Liliane Tariosse c , Jacques Robert a,b,∗ a
Laboratoire de Pharmacologie des Agents Anticanc´ereux, Institut Bergoni´e, 229 Cours de l’Argonne, 33076 Bordeaux-cedex, France b Universit´ e Victor Segalen Bordeaux 2, 146 rue L´eo-Saignat, 33076 Bordeaux-cedex, France c INSERM U441, Avenue du Haut-L´ evˆeque, 33600 Pessac, France Received 29 June 2005; received in revised form 26 July 2005; accepted 28 July 2005 Available online 29 August 2005
Abstract We have studied the protective effect of dexrazoxane on the cardiac toxicity induced by the anthracyclines currently used in clinics, doxorubicin, epirubicin, daunorubicin and idarubicin, with special emphasis on determining the optimal dose of dexrazoxane. This was performed using the model of isolated perfused rat heart after 12-day combination treatment of anthracyclines used at equicardiotoxic doses, and dexrazoxane used at 10-fold or 20-fold the anthracycline dose. We have shown in this study that dexrazoxane by itself was not cardiotoxic, and was able to significantly reduce anthracycline cardiac toxicity without increasing the general toxicity induced by these drugs. Using dexrazoxane at 20 times the anthracycline dose provided a better cardioprotection than using it at 10 times the anthracycline dose; at the higher dexrazoxane dose, the functional cardiac parameters (developed pressure, contractility and relaxation) were not different from those recorded in control animals. © 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Anthracyclines; Dexrazoxane; Cardiac toxicity models
1. Introduction The common use of anthracyclines in various malignancies is hindered by their cardiac toxicity, which remains a major problem 50 years after the discovery of daunorubicin and doxorubicin and their introduction in clinics (Minotti et al., 2004). This toxicity has elicited a large number of studies aimed both at understanding the mechanisms involved in the cardiac toxicity of these drugs and at circumventing it. Several approaches have
∗
Corresponding author. Tel.: +33 556 33 33 27; fax: +33 556 33 33 89. E-mail address: [email protected] (J. Robert).
been considered in this respect: (1) the development of anthracycline analogues devoid of cardiac toxicity; (2) the use of alternative schedules of administration such as protracted infusions; (3) the encapsulation of the anticancer drug in liposomal or other particulate formulations; (4) the combination of anthracycline with a cardioprotector. In this last area, a large number of potential drugs have been developed in preclinical models, such as free radical scavengers and anti-oxidants. However, only one drug has proven its efficacy in the clinical setting: dexrazoxane (ICRF187), a prodrug of an intracellular iron chelator (ADR925). It is admitted that this drug allows, in heart tissue, a decrease rate of the Fenton reaction, a pathway leading doxorubicin-induced free radical formation (Hasinoff et al., 1998). The poten-
0378-4274/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.toxlet.2005.07.013
38
J. Pland´e et al. / Toxicology Letters 161 (2006) 37–42
tial efficacy of dexrazoxane on the cardiac toxicity of anthracyclines other than the classical drugs, doxorubicin and daunorubicin, has never been studied in detail; also, the optimal dose of dexrazoxane in the prevention of anthracycline cardiac toxicity has never been assessed with accuracy and different schedules are currently used in different countries. We have already shown that that the isolated perfused rat heart may represent a useful model for the preclinical evaluation of anthracycline cardiotoxicity and its circumvention (Pouna et al., 1996). We have shown, for instance, the lower cardiac toxicity of idarubicin as compared to doxorubicin (Platel et al., 1999b), the protective role of doxorubicin conjugation to glucuronic acid (Platel et al., 1999a), or the potentiation of doxorubicin cardiac toxicity by paclitaxel (Platel et al., 2000). These preclinical evidences were all in agreement with the clinical observations, thus justifying the use of this short-term test to predict the cardiac side effects of anthracyclines in various conditions. We wanted in this study evaluate the role of the dose of dexrazoxane administered in combination with several anthracyclines currently used in the clinical setting. In the United States, dexrazoxane is administered at a dose of equal to 10-fold the doxorubicin dose (10:1 ratio), whereas in Europe a 20:1 ratio between dexrazoxane and the anthracycline is currently prescribed. Our results show that a higher dose of dexrazoxane may enhance the cardioprotective effect of dexrazoxane. 2. Materials and methods 2.1. Drugs and chemicals Doxorubicin, epirubicin, daunorubicin and idarubicin were provided as clinical formulations by the Hospital Pharmacy of Institut Bergoni´e. They were diluted with sterile water to a final concentration of 5 mg/ml and stored at +4 ◦ C for a maximum of 4 days. Dexrazoxane was provided as clinical formulation by Chiron France, diluted with sterile water at a final concentration of 20 mg/ml and kept at +4 ◦ C for a maximum of 4 days. 2.2. Experimental animals All experiments reported here were done in accordance with the guidelines of the Institut National de la Sant´e et de la Recherche M´edicale. Treatments were administered i.p. to male Sprague Dawley rats aged 10–12 weeks every other day for 12 days. The rats were weighed every 2 days and assessed for possible abnor-
malities (ascites, bleeding, diarrhea, etc.). Rats were killed on the 12th day after the first injection; hearts were removed and perfused, and cardiac functional parameters were monitored as described below. Treatment includes an anthracycline at a selected dose providing, whenever possible, equi-cardiotoxicity, combined, 30 min earlier, with dexrazoxane at either 10-fold or 20-fold the anthracycline dose. Doxorubicin was used at 3 mg/kg per day (18 mg/kg total dose), epirubicin at 3.5 mg/kg per day (21 mg/kg total dose), daunorubicin at 4 mg/kg per day (24 mg/kg total dose) and idarubicin at 0.6 mg/kg per day (3.6 mg/kg total dose). These doses were chosen after multiple trials and corresponded to those giving an acceptable general toxicity together with major cardiac functional symptoms. The purpose of the study was not to compare the various anthracyclines, but to evaluate the improvement of cardiac symptoms when combined with dexrazoxane. For each anthracycline studied, groups of rats were treated (1) with the anthracycline alone at the selected dose, (2) with dexrazoxane alone at 20-fold the theoretical anthracycline dose, (3) with the anthracycline combined with dexrazoxane at either 10-fold or 20-fold the anthracycline dose, or (4) with shadow injections of saline. 2.3. Perfusion of isolated rat hearts Rats were heparinized i.p. (5000 U/kg) and anesthetized with diethylether. The heart was quickly excised and soaked in Krebs-Henseleit solution at +4 ◦ C. Coronary perfusion was initiated through a short cannula in the aortic root and maintained at a constant pressure of 90 mm Hg in a non-recirculating way by the Langendorff technique as described by Lorell et al. (Lorell et al., 1986). Perfusion pressure was measured by a P23Db transducer (Bentley Trantec) connected to the aortic infusion cannula. The heart was electrically paced at a rate of 300 beats/min (5 Hz) through stimulatoractivated stainless steel electrodes placed on the heart. A latex balloon attached to one end of a polyethylene catheter was placed in the left ventricle through the mitral valve. The catheter was filled with water and the other end was linked to an electronic amplifier (Thomson Medical) via a second P23Db transducer. The coronary perfusion pressure and the left ventricular pressure were recorded on a computer that allowed continuous monitoring of heart rate, left ventricular systolic pressure (LVSP), left ventricular end-diastolic pressure (LVEDP), left ventricular developed pressure (LVDP = LVSP − LVEDP) and the maximal and minimal first derivatives of LVDP as a function of time
J. Pland´e et al. / Toxicology Letters 161 (2006) 37–42
[LV(dp/dt)max and LV(dp/dt)min ], respectively. The perfusate consisted in a modified Krebs-Henseleit buffer, pH 7.4, containing NaCl (118 mM), KCl (4.7 mM), MgSO4 (1.2 mM), KH2 PO4 (1.2 mM), NaHCO3 (25 mM), glucose (11 mM), CaCl2 (0.95 mM) and insulin (10 i.u./l). It was continuously bubbled with a mixture of 95% O2 /5% CO2 and maintained at 37 ◦ C. The latex balloon inserted in the left ventricle was periodically dilated with distilled water in order to produce a LVEDP of 5–6 mm Hg. After 30–45 min stabilization, necessary to reach the maximal functional cardiac values, the above parameters were recorded. 2.4. Statistical analysis of the data Statistical comparisons between untreated and treated groups were made by Student’s t-test after ANOVA assumption of the validity of t-test; all data are expressed as mean value ± S.D. Statistical significance was determined as a p-value below 0.05.
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3. Results 3.1. General toxicity of the treatments All anthracyclines exerted a marked general toxicity at the doses used in these experiments. Diarrhea remained relatively infrequent but spontaneous bleeding occurred in 25–50% of the animals. A weight loss of 10–20% of initial weight was apparent for all anthracyclines on the 12th day, to be compared to the 20–30% weight gain recorded in control animals during the same period (Table 1). At the high doses that were used with most anthracyclines (60–80 mg/kg per day × 6), dexrazoxane appeared to exert a significant general toxicity to the experimental animals. No special symptoms were recorded, but the weight increase during the 12-day period was reduced in animals dosed with dexrazoxane alone. At the dose of 12 mg/kg per day which was used with idarubicin, no such sign of general toxicity was evidenced.
Table 1 General toxicity of the treatments administered Early death
Diarrhea
Bleeding
Weight variation (%)
p-Value vs. control
p-Value vs. dex alone
p-Value vs. ant alone
p-Value vs. D+A
Doxorubicin experiments Control Dexrazoxane 60 mg/kg Doxorubicin 3 mg/kg Dox 3 mg/kg + dex 30 mg/kg Dox 3 mg/kg + dex 60 mg/kg
0/12 0/7 1/18 6/19 5/15
0/12 0/7 0/18 0/19 0/15
0/12 0/7 9/18 9/19 11/15
+21.0 +12.4 −13.8 −22.2 −4.1
± ± ± ± ±
4.6 4.7 8.5 9.5 9.4
0.005 2 × 10−10 2 × 10−10 5 × 10−8
5 × 10−7 2 × 10−7 2 × 10−5
0.04 n.s.
0.05
Epirubicin experiments Control Dexrazoxane 70 mg/kg Epirubicin 3.5 mg/kg Epi 3.5 mg/kg + dex 35 mg/kg Epi 3.5 mg/kg + dex 70 mg/kg
0/5 0/3 0/8 2/8 2/7
0/5 0/3 0/8 0/8 2/7
0/5 0/3 2/8 1/8 5/7
+25.1 +10.9 −11.4 −15.1 −18.0
± ± ± ± ±
2.2 2.4 7.1 6.3 8.9
2 × 10−4 6 × 10−7 6 × 10−7 1 × 10−5
0.001 5 × 10−4 0.003
n.s. n.s.
n.s.
Daunorubicin experiments Control Dexrazoxane 80 mg/kg Daunorubicin 4 mg/kg Dau 4 mg/kg + dex 40 mg/kg Dau 4 mg/kg + dex 80 mg/kg
0/10 0/7 3/21 10/24 9/22
0/10 0/7 7/21 1/24 4/22
0/10 0/7 8/21 1/24 2/22
+20.1 +4.4 −20.9 −7.8 −9.6
± ± ± ± ±
5.4 11.8 9.7 7.8 6.1
0.003 4 × 10−12 3 × 10−9 2 × 10−10
2 × 10−5 0.015 0.005
0.0004 0.02
n.s.
Idarubicin experiments Control Dexrazoxane 12 mg/kg Idarubicin 0.6 mg/kg Ida 0.6 mg/kg + dex 6 mg/kg Ida 0.6 mg/kg + dex 12 mg/kg
0/12 0/6 16/23 5/17 7/20
0/12 0/6 0/23 0/17 0/20
0/12 0/6 13/23 10/17 6/20
+27.8 +26.5 −16.6 −13.6 −5.8
± ± ± ± ±
4.8 4.3 5.7 10.8 9.9
n.s. 2 × 10-11 2 × 10-10 8 × 10-10
10−7 7 × 10−7 2 × 10−6
n.s. 0.03
0.1
Abbreviations as follows — dex: dexrazoxane; dox: doxorubicin; epi: epirubicin; dau: daunorubicin; ida: idarubicin; ant: anthracycline; D + A: dexrazoxane + anthracyclines; n.s.: not significant. The doses indicated in the first column are daily doses repeated six times over 12 days. Results are given as means ± S.D. p-Value have been calculated using Student’s t-test; values were compared vs. control, vs. dexrazoxane only, vs. the anthracycline only and vs. the combination of the anthracycline with dexrazoxane at 10-fold the anthracycline dose.
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Table 2 Cardiac toxicity of the treatments administered LVDP (mmHg) p-Value vs.
Doxorubicin experiments Control Dexrazoxane 60 mg/kg Doxorubicin 3 mg/kg Dox 3 mg/kg + dex 30 mg/kg Dox 3 mg/kg + dex 60 mg/kg
12 7 17 13 10
103.2 105.2 84.6 94.8 102.6
± ± ± ± ±
17.3 11.8 14.2 17.2 18.9
n.s. 0.04 n.s. n.s.
Epirubicin experiments Control Dexrazoxane 70 mg/kg Epirubicin 3.5 mg/kg Epi 3.5 mg/kg + dex 35 mg/kg Epi 3.5 mg/kg + dex 70 mg/kg
5 3 8 6 5
108.0 128.0 92.6 95.5 121.0
± ± ± ± ±
13.1 9.0 15.7 14.3 13.8
n.s. n.s. n.s. n.s.
Daunorubicin experiments Control Dexrazoxane 80 mg/kg Daunorubicin 4 mg/kg Dau 4 mg/kg + dex 40 mg/kg Dau 4 mg/kg + dex 80 mg/kg
10 7 18 14 13
108.6 108.6 92.8 98.8 113.8
± ± ± ± ±
19.7 16.4 17.0 14.3 21.3
n.s. n.s. n.s. n.s.
Idarubicin experiments Control Dexrazoxane 12 mg/kg Idarubicin 0.6 mg/kg Ida 0.6 mg/kg + dex 6 mg/kg Ida 0.6 mg/kg + dex 12 mg/kg
12 6 7 12 13
109.3 121.7 67.2 92.7 98.3
± ± ± ± ±
9.3 10.0 24.3 21.9 23.7
Same legend as Table 1.
cont
dex
ant
D+A
LV(dP/dt)max (mmHg/s)
p-Value vs. cont
dex
ant
D+A
dex
ant
n.s. 0.02 n.s.
−2122 ± 417 −2082 ± 191 −1585 ± 360 −1646 ± 393 −2003 ± 397
n.s. 0.02 0.04 n.s.
0.01 0.02 n.s.
n.s. 0.04 0.05
n.s. 0.02 0.05
−2127 ± 320 −2374 ± 94 −1450 ± 279 −1525 ± 495 −1992 ± 312
n.s. 0.04 n.s. n.s.
0.003 n.s. n.s. n.s. 0.01 n.s.
n.s. 0.02 0.02
−2005 ± 404 −1975 ± 219 −1494 ± 428 −1591 ± 268 −1902 ± 246
n.s. 0.01 0.01 n.s.
0.01 0.01 n.s.
−1968 ± 182 −2277 ± 138 −1112 ± 470 −1592 ± 376 −1721 ± 428
0.003 0.0001 0.003 0.007 0.001 0.05 n.s. 0.01 0.02 n.s.
n.s. 0.04 n.s.
n.s. 0.05 n.s. n.s.
n.s. 0.01 n.s.
3521 ± 458 3780 ± 290 2574 ± 564 2662 ± 755 3516 ± 469
n.s. 0.02 n.s. n.s.
n.s. 0.03 n.s.
3094 ± 484 2985 ± 525 2480 ± 621 2493 ± 466 3063 ± 516
n.s. 0.02 0.01 n.s.
0.02 0.0001 0.0001 0.05 0.001 0.05 n.s. 0.04 0.03 n.s.
2719 ± 379 3618 ± 377 1722 ± 782 2376 ± 553 2638 ± 612
0.0003 0.003 0.002 n.s. 0.0003 0.05 n.s. 0.004 0.02 n.s.
0.03 n.s. n.s.
n.s. n.s. n.s.
0.01 0.03 n.s.
0.05 n.s. n.s.
0.05 0.05 n.s.
p-Value vs. cont
2819 ± 636 3040 ± 409 2262 ± 538 2438 ± 549 3015 ± 685
0.01 n.s. n.s.
LV(dP/dt)min (mmHg/s)
D+A
n.s. 0.01 0.01
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When combined to dexrazoxane at 10 times the anthracycline dose, the general toxicity symptoms (especially weight loss) were not improved as compared to those observed with the anthracycline alone, except in the case of daunorubicin. When using a dose ratio dexrazoxane:anthracycline of 20:1, no significant change in general toxicity was noticed as compared to the 10:1 dose ratio. 3.2. Cardiac toxicity of the treatments Three parameters were used to evaluate the cardiac toxicity of anthracyclines and dexrazoxane– anthracycline combinations: the left ventricular pressure developed under a constant perfusion pressure (LVDP), the rate of variation of this parameter during systole (contractility) and during diastole (relaxation). High dose dexrazoxane (60–80 mg/kg per day × 6) did not induce a significant modification of any of these parameters, but at low dose (12 mg/kg per day × 6), there was a significant inotropic effect of dexrazoxane alone (Table 2). All anthracyclines exerted a significant cardiac toxicity at the doses and schedules used, which was generally more pronounced on relaxation than on the other parameters. When combined with dexrazoxane at 10 times the anthracycline dose, there was some reduction in cardiac toxicity, which remained, however, below the level of significance in most cases when compared to the anthracycline administered alone. When used at 20 times the anthracycline dose, the effect of dexrazoxane became significant for all anthracyclines (p = 0.01–0.05, see Table 2), and the parameters recorded were no longer significantly different from those obtained in control animals. 4. Discussion The model of isolated perfused rat heart appears as useful for a rapid preclinical evaluation of anthracycline cardiotoxicity. As a model, it does not pretend to mimic the delayed cardiac toxicity of anthracyclines in humans, which generally occurs 1–2 years after treatment completion in adults, and even much later in children (Shan et al., 1996). In clinics, the myocardic cellular lesions induced by anthracyclines occur rapidly after each administration, as evidenced from the early elevation of serum troponin levels (Herman et al., 1999), but there is, to a certain degree, a physiological compensation to the loss of cardiac fibers, explaining the delay between anthracycline administration and the occurrence of heart failure. However, it appears possible to detect functional alterations shortly after administration
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and to compare, in a short-term model as the one we have implemented, the ability of different drugs and drug combinations to induce heart fibers damage. We have already shown how this model could be useful to predict anthracycline cardiotoxicity in various situations (Pouna et al., 1996; Platel et al., 1999a,b, 2000). We show in this study that dexrazoxane exerts a significant protective effect on the cardiac toxicity induced by the four anthracyclines currently used in the clinical setting. This effect had already been observed in the model of isolated perfused rat heart in the case of doxorubicin (Pouna et al., 1996). We confirm here that it also occurs for the other anthracyclines when used at equi-cardiotoxic doses. The optimal dose of dexrazoxane to be prescribed has not been determined with accuracy in the clinical setting. It was admitted from the early studies that dexrazoxane should be prescribed as a function of the anthracycline dose, but the ratio was set at 10:1 in the United States and at 20:1 in Europe. The 10:1 ratio had been chosen because of the risk that dexrazoxane could potentiate the myelosuppression effects induced by doxorubicin. Was also questioned the possibility that this combination could decrease the response rate to doxorubicin chemotherapy. The prescription of dexrazoxane in combination with doxorubicin is currently limited to the patients having already received a minimal dose of 300 mg/m2 of doxorubicin or equivalent. Dexrazoxane is a catalytic inhibitor of topoisomerase II, the target enzyme of doxorubicin and other drugs such as etoposide, which poison the enzyme by inhibiting the religation reaction without inhibiting the cleavage reaction (Ishida et al., 1991). No clinical study has shown an increase of doxorubicin myelotoxicity induced by dexrazoxane (see for review Wiseman and Spencer, 1998, or Cvetkovic and Scott, 2005), but an early study had shown a decrease in the response rate of breast cancer, without consequences on time to progression and overall survival (Swain et al., 1997). The interference between the two types of topoisomerase-targeting drugs has been carefully studied in several models; it has essentially been shown that dexrazoxane could exert a protection against topoisomerase II-induced DNA cleavage by daunorubicin or etoposide, but not by doxorubicin (Hofland et al., 2005). We observed in this study that the 20:1 ratio provided a better cardioprotection than a 10:1 ratio for all the anthracyclines used. This is in agreement with the results already obtained by Imondi et al. (1996) who studied the dose-effect relationships of dexrazoxane on doxorubicin cardiotoxicity in several animal models. It is remarkable that dexrazoxane protects against idarubicin
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cardiotoxicity at doses five times lower than those used to protect against doxorubicin cardiotoxicity: this confirms that the important parameter is the ratio between the anthracycline dose and that of dexrazoxane, and not the absolute dose of the cardioprotector. As a general rule, our study is rather in favor of the use of a 20:1 anthracycline:dexrazoxane ratio in the clinical setting. Acknowledgements This study was supported by a grant from Chiron France and was part of the Programme Hospitalier de Recherche Clinique. We are grateful to INSERM unit 441 for technical support of these experiments. References Cvetkovic, R.S., Scott, L.J., 2005. Dexrazoxane: a review of its use for cardioprotection during anthracycline chemotherapy. Drugs 65, 1005–1024. Hasinoff, B.B., Hellmann, K., Herman, E.H., Ferrans, V.J., 1998. Chemical, biological and clinical aspects of dexrazoxane and other bisdioxopiperazines. Curr. Med. Chem. 5, 1–28. Herman, E.H., Zhang, J., Lipshultz, S.E., Rifai, N., Chadwick, D., Takeda, K., Yu, Z.X., Ferrans, V.J., 1999. Correlation between serum levels of cardiac troponin-T and the severity of the chronic cardiomyopathy induced by doxorubicin. J. Clin. Oncol. 17, 2237–2243. Hofland, K.F., Thougaard, A.V., Sehested, M., Jensen, P.B., 2005. Dexrazoxane protects against myelosuppression from the DNA cleavage-enhancing drugs etoposide and daunorubicin but not doxorubicin. Clin. Cancer Res. 11, 3915–3924. Imondi, A.R., Della Torre, P., Mazue, G., Sullivan, T.M., Robbins, T.L., Hagerman, L.M., Podesta, A., Pinciroli, G., 1996. Dose-response relationship of dexrazoxane for prevention of doxorubicin-induced cardiotoxicity in mice, rats, and dogs. Cancer Res. 56, 4200–4204.
Ishida, R., Miki, T., Narita, T., Yui, R., Sato, M., Utsumi, K.R., Tanabe, K., Andoh, T., 1991. Inhibition of intracellular topoisomerase II by antitumor bis(2,6-dioxopiperazine) derivatives: mode of cell growth inhibition distinct from that of cleavable complex-forming type inhibitors. Cancer Res. 51, 4909–4916. Lorell, B.H., Wexler, L.F., Momomura, S., Weinberg, E., Apstein, C.S., 1986. The influence of pressure overload left ventricular hypertrophy on diastolic properties during hypoxia in isovolumically contracting rat hearts. Circ. Res. 58, 653–663. Minotti, G., Menna, P., Salvatorelli, E., Cairo, G., Gianni, L., 2004. Anthracyclines: molecular advances and pharmacologic developments in antitumor activity and cardiotoxicity. Pharmacol. Rev. 56, 185–229. Platel, D., Bonoron-Ad`ele, S., Dix, R.K., Robert, J., 1999a. Preclinical evaluation of the cardiac toxicity of HMR-1826, a novel prodrug of doxorubicin. Br. J. Cancer 81, 24–27. Platel, D., Pouna, P., Bonoron-Ad`ele, S., Robert, J., 1999b. Comparative cardiotoxicity of idarubicin and doxorubicin using the isolated perfused rat heart model. Anticancer Drugs 10, 671– 676. Platel, D., Pouna, P., Bonoron-Ad`ele, S., Robert, J., 2000. Preclinical evaluation of the cardiotoxicity of taxane–anthracycline combinations using the model of isolated perfused rat heart. Toxicol. Appl. Pharmacol. 163, 135–140. Pouna, P., Bonoron-Ad`ele, S., Gouverneur, G., Tariosse, L., Besse, P., Robert, J., 1996. Development of the model of rat isolated perfused heart for the evaluation of anthracyline cardiotoxicity and its circumvention. Br. J. Pharmacol. 117, 1593–1599. Shan, K., Lincoff, A.M., Young, J.B., 1996. Anthracycline-induced cardiotoxicity. Ann. Intern. Med. 125, 47–58. Swain, S.M., Whaley, F.S., Gerber, M.C., Weisberg, S., York, M., Spicer, D., Jones, S.E., Wadler, S., Desai, A., Vogel, C., Speyer, J., Mittelman, A., Reddy, S., Pendergrass, K., Velez-Garcia, E., Ewer, M.S., Bianchine, J.R., Gams, R.A., 1997. Cardioprotection with dexrazoxane for doxorubicin-containing therapy in advanced breast cancer. J. Clin. Oncol. 15, 1318–1332. Wiseman, L.R., Spencer, C.M., 1998. Dexrazoxane. A review of its use as a cardioprotective agent in patients receiving anthracyclinebased chemotherapy. Drugs 56, 385–403.
Experimental study of dexrazoxane–anthracycline combinations using the model of isolated perfused rat heart Jo¨elle Pland´e a,b , Denis Platel a,b , Liliane Tariosse c , Jacques Robert a,b,∗ a
Laboratoire de Pharmacologie des Agents Anticanc´ereux, Institut Bergoni´e, 229 Cours de l’Argonne, 33076 Bordeaux-cedex, France b Universit´ e Victor Segalen Bordeaux 2, 146 rue L´eo-Saignat, 33076 Bordeaux-cedex, France c INSERM U441, Avenue du Haut-L´ evˆeque, 33600 Pessac, France Received 29 June 2005; received in revised form 26 July 2005; accepted 28 July 2005 Available online 29 August 2005
Abstract We have studied the protective effect of dexrazoxane on the cardiac toxicity induced by the anthracyclines currently used in clinics, doxorubicin, epirubicin, daunorubicin and idarubicin, with special emphasis on determining the optimal dose of dexrazoxane. This was performed using the model of isolated perfused rat heart after 12-day combination treatment of anthracyclines used at equicardiotoxic doses, and dexrazoxane used at 10-fold or 20-fold the anthracycline dose. We have shown in this study that dexrazoxane by itself was not cardiotoxic, and was able to significantly reduce anthracycline cardiac toxicity without increasing the general toxicity induced by these drugs. Using dexrazoxane at 20 times the anthracycline dose provided a better cardioprotection than using it at 10 times the anthracycline dose; at the higher dexrazoxane dose, the functional cardiac parameters (developed pressure, contractility and relaxation) were not different from those recorded in control animals. © 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Anthracyclines; Dexrazoxane; Cardiac toxicity models
1. Introduction The common use of anthracyclines in various malignancies is hindered by their cardiac toxicity, which remains a major problem 50 years after the discovery of daunorubicin and doxorubicin and their introduction in clinics (Minotti et al., 2004). This toxicity has elicited a large number of studies aimed both at understanding the mechanisms involved in the cardiac toxicity of these drugs and at circumventing it. Several approaches have
∗
Corresponding author. Tel.: +33 556 33 33 27; fax: +33 556 33 33 89. E-mail address: [email protected] (J. Robert).
been considered in this respect: (1) the development of anthracycline analogues devoid of cardiac toxicity; (2) the use of alternative schedules of administration such as protracted infusions; (3) the encapsulation of the anticancer drug in liposomal or other particulate formulations; (4) the combination of anthracycline with a cardioprotector. In this last area, a large number of potential drugs have been developed in preclinical models, such as free radical scavengers and anti-oxidants. However, only one drug has proven its efficacy in the clinical setting: dexrazoxane (ICRF187), a prodrug of an intracellular iron chelator (ADR925). It is admitted that this drug allows, in heart tissue, a decrease rate of the Fenton reaction, a pathway leading doxorubicin-induced free radical formation (Hasinoff et al., 1998). The poten-
0378-4274/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.toxlet.2005.07.013
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J. Pland´e et al. / Toxicology Letters 161 (2006) 37–42
tial efficacy of dexrazoxane on the cardiac toxicity of anthracyclines other than the classical drugs, doxorubicin and daunorubicin, has never been studied in detail; also, the optimal dose of dexrazoxane in the prevention of anthracycline cardiac toxicity has never been assessed with accuracy and different schedules are currently used in different countries. We have already shown that that the isolated perfused rat heart may represent a useful model for the preclinical evaluation of anthracycline cardiotoxicity and its circumvention (Pouna et al., 1996). We have shown, for instance, the lower cardiac toxicity of idarubicin as compared to doxorubicin (Platel et al., 1999b), the protective role of doxorubicin conjugation to glucuronic acid (Platel et al., 1999a), or the potentiation of doxorubicin cardiac toxicity by paclitaxel (Platel et al., 2000). These preclinical evidences were all in agreement with the clinical observations, thus justifying the use of this short-term test to predict the cardiac side effects of anthracyclines in various conditions. We wanted in this study evaluate the role of the dose of dexrazoxane administered in combination with several anthracyclines currently used in the clinical setting. In the United States, dexrazoxane is administered at a dose of equal to 10-fold the doxorubicin dose (10:1 ratio), whereas in Europe a 20:1 ratio between dexrazoxane and the anthracycline is currently prescribed. Our results show that a higher dose of dexrazoxane may enhance the cardioprotective effect of dexrazoxane. 2. Materials and methods 2.1. Drugs and chemicals Doxorubicin, epirubicin, daunorubicin and idarubicin were provided as clinical formulations by the Hospital Pharmacy of Institut Bergoni´e. They were diluted with sterile water to a final concentration of 5 mg/ml and stored at +4 ◦ C for a maximum of 4 days. Dexrazoxane was provided as clinical formulation by Chiron France, diluted with sterile water at a final concentration of 20 mg/ml and kept at +4 ◦ C for a maximum of 4 days. 2.2. Experimental animals All experiments reported here were done in accordance with the guidelines of the Institut National de la Sant´e et de la Recherche M´edicale. Treatments were administered i.p. to male Sprague Dawley rats aged 10–12 weeks every other day for 12 days. The rats were weighed every 2 days and assessed for possible abnor-
malities (ascites, bleeding, diarrhea, etc.). Rats were killed on the 12th day after the first injection; hearts were removed and perfused, and cardiac functional parameters were monitored as described below. Treatment includes an anthracycline at a selected dose providing, whenever possible, equi-cardiotoxicity, combined, 30 min earlier, with dexrazoxane at either 10-fold or 20-fold the anthracycline dose. Doxorubicin was used at 3 mg/kg per day (18 mg/kg total dose), epirubicin at 3.5 mg/kg per day (21 mg/kg total dose), daunorubicin at 4 mg/kg per day (24 mg/kg total dose) and idarubicin at 0.6 mg/kg per day (3.6 mg/kg total dose). These doses were chosen after multiple trials and corresponded to those giving an acceptable general toxicity together with major cardiac functional symptoms. The purpose of the study was not to compare the various anthracyclines, but to evaluate the improvement of cardiac symptoms when combined with dexrazoxane. For each anthracycline studied, groups of rats were treated (1) with the anthracycline alone at the selected dose, (2) with dexrazoxane alone at 20-fold the theoretical anthracycline dose, (3) with the anthracycline combined with dexrazoxane at either 10-fold or 20-fold the anthracycline dose, or (4) with shadow injections of saline. 2.3. Perfusion of isolated rat hearts Rats were heparinized i.p. (5000 U/kg) and anesthetized with diethylether. The heart was quickly excised and soaked in Krebs-Henseleit solution at +4 ◦ C. Coronary perfusion was initiated through a short cannula in the aortic root and maintained at a constant pressure of 90 mm Hg in a non-recirculating way by the Langendorff technique as described by Lorell et al. (Lorell et al., 1986). Perfusion pressure was measured by a P23Db transducer (Bentley Trantec) connected to the aortic infusion cannula. The heart was electrically paced at a rate of 300 beats/min (5 Hz) through stimulatoractivated stainless steel electrodes placed on the heart. A latex balloon attached to one end of a polyethylene catheter was placed in the left ventricle through the mitral valve. The catheter was filled with water and the other end was linked to an electronic amplifier (Thomson Medical) via a second P23Db transducer. The coronary perfusion pressure and the left ventricular pressure were recorded on a computer that allowed continuous monitoring of heart rate, left ventricular systolic pressure (LVSP), left ventricular end-diastolic pressure (LVEDP), left ventricular developed pressure (LVDP = LVSP − LVEDP) and the maximal and minimal first derivatives of LVDP as a function of time
J. Pland´e et al. / Toxicology Letters 161 (2006) 37–42
[LV(dp/dt)max and LV(dp/dt)min ], respectively. The perfusate consisted in a modified Krebs-Henseleit buffer, pH 7.4, containing NaCl (118 mM), KCl (4.7 mM), MgSO4 (1.2 mM), KH2 PO4 (1.2 mM), NaHCO3 (25 mM), glucose (11 mM), CaCl2 (0.95 mM) and insulin (10 i.u./l). It was continuously bubbled with a mixture of 95% O2 /5% CO2 and maintained at 37 ◦ C. The latex balloon inserted in the left ventricle was periodically dilated with distilled water in order to produce a LVEDP of 5–6 mm Hg. After 30–45 min stabilization, necessary to reach the maximal functional cardiac values, the above parameters were recorded. 2.4. Statistical analysis of the data Statistical comparisons between untreated and treated groups were made by Student’s t-test after ANOVA assumption of the validity of t-test; all data are expressed as mean value ± S.D. Statistical significance was determined as a p-value below 0.05.
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3. Results 3.1. General toxicity of the treatments All anthracyclines exerted a marked general toxicity at the doses used in these experiments. Diarrhea remained relatively infrequent but spontaneous bleeding occurred in 25–50% of the animals. A weight loss of 10–20% of initial weight was apparent for all anthracyclines on the 12th day, to be compared to the 20–30% weight gain recorded in control animals during the same period (Table 1). At the high doses that were used with most anthracyclines (60–80 mg/kg per day × 6), dexrazoxane appeared to exert a significant general toxicity to the experimental animals. No special symptoms were recorded, but the weight increase during the 12-day period was reduced in animals dosed with dexrazoxane alone. At the dose of 12 mg/kg per day which was used with idarubicin, no such sign of general toxicity was evidenced.
Table 1 General toxicity of the treatments administered Early death
Diarrhea
Bleeding
Weight variation (%)
p-Value vs. control
p-Value vs. dex alone
p-Value vs. ant alone
p-Value vs. D+A
Doxorubicin experiments Control Dexrazoxane 60 mg/kg Doxorubicin 3 mg/kg Dox 3 mg/kg + dex 30 mg/kg Dox 3 mg/kg + dex 60 mg/kg
0/12 0/7 1/18 6/19 5/15
0/12 0/7 0/18 0/19 0/15
0/12 0/7 9/18 9/19 11/15
+21.0 +12.4 −13.8 −22.2 −4.1
± ± ± ± ±
4.6 4.7 8.5 9.5 9.4
0.005 2 × 10−10 2 × 10−10 5 × 10−8
5 × 10−7 2 × 10−7 2 × 10−5
0.04 n.s.
0.05
Epirubicin experiments Control Dexrazoxane 70 mg/kg Epirubicin 3.5 mg/kg Epi 3.5 mg/kg + dex 35 mg/kg Epi 3.5 mg/kg + dex 70 mg/kg
0/5 0/3 0/8 2/8 2/7
0/5 0/3 0/8 0/8 2/7
0/5 0/3 2/8 1/8 5/7
+25.1 +10.9 −11.4 −15.1 −18.0
± ± ± ± ±
2.2 2.4 7.1 6.3 8.9
2 × 10−4 6 × 10−7 6 × 10−7 1 × 10−5
0.001 5 × 10−4 0.003
n.s. n.s.
n.s.
Daunorubicin experiments Control Dexrazoxane 80 mg/kg Daunorubicin 4 mg/kg Dau 4 mg/kg + dex 40 mg/kg Dau 4 mg/kg + dex 80 mg/kg
0/10 0/7 3/21 10/24 9/22
0/10 0/7 7/21 1/24 4/22
0/10 0/7 8/21 1/24 2/22
+20.1 +4.4 −20.9 −7.8 −9.6
± ± ± ± ±
5.4 11.8 9.7 7.8 6.1
0.003 4 × 10−12 3 × 10−9 2 × 10−10
2 × 10−5 0.015 0.005
0.0004 0.02
n.s.
Idarubicin experiments Control Dexrazoxane 12 mg/kg Idarubicin 0.6 mg/kg Ida 0.6 mg/kg + dex 6 mg/kg Ida 0.6 mg/kg + dex 12 mg/kg
0/12 0/6 16/23 5/17 7/20
0/12 0/6 0/23 0/17 0/20
0/12 0/6 13/23 10/17 6/20
+27.8 +26.5 −16.6 −13.6 −5.8
± ± ± ± ±
4.8 4.3 5.7 10.8 9.9
n.s. 2 × 10-11 2 × 10-10 8 × 10-10
10−7 7 × 10−7 2 × 10−6
n.s. 0.03
0.1
Abbreviations as follows — dex: dexrazoxane; dox: doxorubicin; epi: epirubicin; dau: daunorubicin; ida: idarubicin; ant: anthracycline; D + A: dexrazoxane + anthracyclines; n.s.: not significant. The doses indicated in the first column are daily doses repeated six times over 12 days. Results are given as means ± S.D. p-Value have been calculated using Student’s t-test; values were compared vs. control, vs. dexrazoxane only, vs. the anthracycline only and vs. the combination of the anthracycline with dexrazoxane at 10-fold the anthracycline dose.
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Table 2 Cardiac toxicity of the treatments administered LVDP (mmHg) p-Value vs.
Doxorubicin experiments Control Dexrazoxane 60 mg/kg Doxorubicin 3 mg/kg Dox 3 mg/kg + dex 30 mg/kg Dox 3 mg/kg + dex 60 mg/kg
12 7 17 13 10
103.2 105.2 84.6 94.8 102.6
± ± ± ± ±
17.3 11.8 14.2 17.2 18.9
n.s. 0.04 n.s. n.s.
Epirubicin experiments Control Dexrazoxane 70 mg/kg Epirubicin 3.5 mg/kg Epi 3.5 mg/kg + dex 35 mg/kg Epi 3.5 mg/kg + dex 70 mg/kg
5 3 8 6 5
108.0 128.0 92.6 95.5 121.0
± ± ± ± ±
13.1 9.0 15.7 14.3 13.8
n.s. n.s. n.s. n.s.
Daunorubicin experiments Control Dexrazoxane 80 mg/kg Daunorubicin 4 mg/kg Dau 4 mg/kg + dex 40 mg/kg Dau 4 mg/kg + dex 80 mg/kg
10 7 18 14 13
108.6 108.6 92.8 98.8 113.8
± ± ± ± ±
19.7 16.4 17.0 14.3 21.3
n.s. n.s. n.s. n.s.
Idarubicin experiments Control Dexrazoxane 12 mg/kg Idarubicin 0.6 mg/kg Ida 0.6 mg/kg + dex 6 mg/kg Ida 0.6 mg/kg + dex 12 mg/kg
12 6 7 12 13
109.3 121.7 67.2 92.7 98.3
± ± ± ± ±
9.3 10.0 24.3 21.9 23.7
Same legend as Table 1.
cont
dex
ant
D+A
LV(dP/dt)max (mmHg/s)
p-Value vs. cont
dex
ant
D+A
dex
ant
n.s. 0.02 n.s.
−2122 ± 417 −2082 ± 191 −1585 ± 360 −1646 ± 393 −2003 ± 397
n.s. 0.02 0.04 n.s.
0.01 0.02 n.s.
n.s. 0.04 0.05
n.s. 0.02 0.05
−2127 ± 320 −2374 ± 94 −1450 ± 279 −1525 ± 495 −1992 ± 312
n.s. 0.04 n.s. n.s.
0.003 n.s. n.s. n.s. 0.01 n.s.
n.s. 0.02 0.02
−2005 ± 404 −1975 ± 219 −1494 ± 428 −1591 ± 268 −1902 ± 246
n.s. 0.01 0.01 n.s.
0.01 0.01 n.s.
−1968 ± 182 −2277 ± 138 −1112 ± 470 −1592 ± 376 −1721 ± 428
0.003 0.0001 0.003 0.007 0.001 0.05 n.s. 0.01 0.02 n.s.
n.s. 0.04 n.s.
n.s. 0.05 n.s. n.s.
n.s. 0.01 n.s.
3521 ± 458 3780 ± 290 2574 ± 564 2662 ± 755 3516 ± 469
n.s. 0.02 n.s. n.s.
n.s. 0.03 n.s.
3094 ± 484 2985 ± 525 2480 ± 621 2493 ± 466 3063 ± 516
n.s. 0.02 0.01 n.s.
0.02 0.0001 0.0001 0.05 0.001 0.05 n.s. 0.04 0.03 n.s.
2719 ± 379 3618 ± 377 1722 ± 782 2376 ± 553 2638 ± 612
0.0003 0.003 0.002 n.s. 0.0003 0.05 n.s. 0.004 0.02 n.s.
0.03 n.s. n.s.
n.s. n.s. n.s.
0.01 0.03 n.s.
0.05 n.s. n.s.
0.05 0.05 n.s.
p-Value vs. cont
2819 ± 636 3040 ± 409 2262 ± 538 2438 ± 549 3015 ± 685
0.01 n.s. n.s.
LV(dP/dt)min (mmHg/s)
D+A
n.s. 0.01 0.01
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Number evaluable
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When combined to dexrazoxane at 10 times the anthracycline dose, the general toxicity symptoms (especially weight loss) were not improved as compared to those observed with the anthracycline alone, except in the case of daunorubicin. When using a dose ratio dexrazoxane:anthracycline of 20:1, no significant change in general toxicity was noticed as compared to the 10:1 dose ratio. 3.2. Cardiac toxicity of the treatments Three parameters were used to evaluate the cardiac toxicity of anthracyclines and dexrazoxane– anthracycline combinations: the left ventricular pressure developed under a constant perfusion pressure (LVDP), the rate of variation of this parameter during systole (contractility) and during diastole (relaxation). High dose dexrazoxane (60–80 mg/kg per day × 6) did not induce a significant modification of any of these parameters, but at low dose (12 mg/kg per day × 6), there was a significant inotropic effect of dexrazoxane alone (Table 2). All anthracyclines exerted a significant cardiac toxicity at the doses and schedules used, which was generally more pronounced on relaxation than on the other parameters. When combined with dexrazoxane at 10 times the anthracycline dose, there was some reduction in cardiac toxicity, which remained, however, below the level of significance in most cases when compared to the anthracycline administered alone. When used at 20 times the anthracycline dose, the effect of dexrazoxane became significant for all anthracyclines (p = 0.01–0.05, see Table 2), and the parameters recorded were no longer significantly different from those obtained in control animals. 4. Discussion The model of isolated perfused rat heart appears as useful for a rapid preclinical evaluation of anthracycline cardiotoxicity. As a model, it does not pretend to mimic the delayed cardiac toxicity of anthracyclines in humans, which generally occurs 1–2 years after treatment completion in adults, and even much later in children (Shan et al., 1996). In clinics, the myocardic cellular lesions induced by anthracyclines occur rapidly after each administration, as evidenced from the early elevation of serum troponin levels (Herman et al., 1999), but there is, to a certain degree, a physiological compensation to the loss of cardiac fibers, explaining the delay between anthracycline administration and the occurrence of heart failure. However, it appears possible to detect functional alterations shortly after administration
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and to compare, in a short-term model as the one we have implemented, the ability of different drugs and drug combinations to induce heart fibers damage. We have already shown how this model could be useful to predict anthracycline cardiotoxicity in various situations (Pouna et al., 1996; Platel et al., 1999a,b, 2000). We show in this study that dexrazoxane exerts a significant protective effect on the cardiac toxicity induced by the four anthracyclines currently used in the clinical setting. This effect had already been observed in the model of isolated perfused rat heart in the case of doxorubicin (Pouna et al., 1996). We confirm here that it also occurs for the other anthracyclines when used at equi-cardiotoxic doses. The optimal dose of dexrazoxane to be prescribed has not been determined with accuracy in the clinical setting. It was admitted from the early studies that dexrazoxane should be prescribed as a function of the anthracycline dose, but the ratio was set at 10:1 in the United States and at 20:1 in Europe. The 10:1 ratio had been chosen because of the risk that dexrazoxane could potentiate the myelosuppression effects induced by doxorubicin. Was also questioned the possibility that this combination could decrease the response rate to doxorubicin chemotherapy. The prescription of dexrazoxane in combination with doxorubicin is currently limited to the patients having already received a minimal dose of 300 mg/m2 of doxorubicin or equivalent. Dexrazoxane is a catalytic inhibitor of topoisomerase II, the target enzyme of doxorubicin and other drugs such as etoposide, which poison the enzyme by inhibiting the religation reaction without inhibiting the cleavage reaction (Ishida et al., 1991). No clinical study has shown an increase of doxorubicin myelotoxicity induced by dexrazoxane (see for review Wiseman and Spencer, 1998, or Cvetkovic and Scott, 2005), but an early study had shown a decrease in the response rate of breast cancer, without consequences on time to progression and overall survival (Swain et al., 1997). The interference between the two types of topoisomerase-targeting drugs has been carefully studied in several models; it has essentially been shown that dexrazoxane could exert a protection against topoisomerase II-induced DNA cleavage by daunorubicin or etoposide, but not by doxorubicin (Hofland et al., 2005). We observed in this study that the 20:1 ratio provided a better cardioprotection than a 10:1 ratio for all the anthracyclines used. This is in agreement with the results already obtained by Imondi et al. (1996) who studied the dose-effect relationships of dexrazoxane on doxorubicin cardiotoxicity in several animal models. It is remarkable that dexrazoxane protects against idarubicin
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cardiotoxicity at doses five times lower than those used to protect against doxorubicin cardiotoxicity: this confirms that the important parameter is the ratio between the anthracycline dose and that of dexrazoxane, and not the absolute dose of the cardioprotector. As a general rule, our study is rather in favor of the use of a 20:1 anthracycline:dexrazoxane ratio in the clinical setting. Acknowledgements This study was supported by a grant from Chiron France and was part of the Programme Hospitalier de Recherche Clinique. We are grateful to INSERM unit 441 for technical support of these experiments. References Cvetkovic, R.S., Scott, L.J., 2005. Dexrazoxane: a review of its use for cardioprotection during anthracycline chemotherapy. Drugs 65, 1005–1024. Hasinoff, B.B., Hellmann, K., Herman, E.H., Ferrans, V.J., 1998. Chemical, biological and clinical aspects of dexrazoxane and other bisdioxopiperazines. Curr. Med. Chem. 5, 1–28. Herman, E.H., Zhang, J., Lipshultz, S.E., Rifai, N., Chadwick, D., Takeda, K., Yu, Z.X., Ferrans, V.J., 1999. Correlation between serum levels of cardiac troponin-T and the severity of the chronic cardiomyopathy induced by doxorubicin. J. Clin. Oncol. 17, 2237–2243. Hofland, K.F., Thougaard, A.V., Sehested, M., Jensen, P.B., 2005. Dexrazoxane protects against myelosuppression from the DNA cleavage-enhancing drugs etoposide and daunorubicin but not doxorubicin. Clin. Cancer Res. 11, 3915–3924. Imondi, A.R., Della Torre, P., Mazue, G., Sullivan, T.M., Robbins, T.L., Hagerman, L.M., Podesta, A., Pinciroli, G., 1996. Dose-response relationship of dexrazoxane for prevention of doxorubicin-induced cardiotoxicity in mice, rats, and dogs. Cancer Res. 56, 4200–4204.
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