ICANCERRESEARCH52, 194-201,January 1, 19921 Comparative Study of Doxorubicin, Mitoxantrone, and Epirubicin in Combination with ICRF-187 (ADR-529) in a Chronic Cardiotoxicity Animal Model' Patricia M. Alderton, Janet Gross, and Michael D. Green2 Department ofMedical Oncology, Royal Melbourne Hospital, Parkville, Victoria, 3050 [P. M. A., M. D. G.J, and Department ofPathology, University of Melbourne, Parkville, Victoria, 3052 Ii. G.J,Australia ABSTRACF superoxide dismutase, catalase, glutathione peroxidase, and reduced glutathione, are intrinsically very low, rendering the In this study doxorubicin,epirubicin,and mitoxantronewerecompared heart very susceptible to free radical attack (10, 11). By com for their cardiotoxic potential in a chronic mouse model in an effort to identify and compare their mechanism(s) of toxicity. In addition, the parison with doxorubicin, mitoxantrone has been shown to be cardioprotective ability of ICRF-187 t(±)-1,2-bis(3,5-dioxopiperazinyl-1- less susceptible to enzymatic activation to its free radical form yl)propanejwith each anticancer drug was evaluated in this model.The and inactive in stimulating oxygen free radical production or antioxidant capacity (superoxide dismutase, reduced glutathione, cata lipid peroxidation (12—15).This study is designed to determine lase, and glutathione peroxidase) was assessed following drug treatment. whether similar mechanisms of action apply to anthracene Five-week-old BALB/c mice received weekly i.p. injections of each diones and anthracyclines. drug or the drug and ICRF-187 over a 3-month period. ICRF-187 was Another approach to abrogating doxorubicin cardiotoxicity administered 30 mm prior to the anticancer drug. The hearts were has been the use of a cardioprotective agent, ICRF-187 (ADR examined by electron and light microscopy to assess subcellular changes, 529),@ administered just prior to doxorubicin (16, 17). ICRF and the cardiac and hepatic antioxidant levels were measured concur 187 may protect against cardiac damage by preventing the rently. Chronic treatment with these drugs or each combinedwith ICRF 187 did not change the antioxidant levels relative to the control values. induction of oxygen free radicals by doxorubicin (18, 19). In However,all three drugs causedcardiacdamageduringchronicexposure. contrast to its cardioprotective role ICRF-187 acts synergisti Both epirubicin and mitoxantrone caused less severe damage than doxo cally with respect to the cytotoxicity of doxorubicin, actually rubicin, and epirubicin was the least cardiotoxic of the three. ICRF-187 enhancing its antitumor activity (20, 21). These observations was cardioprotectivefor epirubicinand doxorubicinbut not for mitoxant lead to the proposal that the antitumor activity of doxorubicin rone. These results suggest epirubicin acts by a mechanism similar to can be separated from its cardiotoxic action, and these two that of doxorubicin that is probably mediated by oxygen-free radicals, actions may occur by different mechanisms. ICRF-187 is able whilemitoxantroneacts bya differentmechanismto cause cardiotoxicity. to block one mechanism and enhance the other. This work was undertaken to extend previous observations INTRODUCTION (18) about the effect of the combination of cardiotoxic drugs and ICRF-187 on the cardiac antioxidant capacity. In this Doxorubicin is one of the most effective chemotherapeutic particular study three anticancer drugs (doxorubicin, epirubicin, drugs used for cancer treatment, but its usefulness is compro and mitoxantrone) are compared with respect to their effect on mised by the development of chronic dose-dependent cardi cardiac morphology and antioxidant capacity in a chronic otoxicity. One approach to overcoming doxorubicin cardiotox mouse model of cardiotoxicity. The potential cardioprotective icity is to develop structural analogues which retain the anti ability of ICRF-187 when coadministered with each drug is cancer activity but are less or not cardiotoxic. Two of the most evaluated. promising drugs that have been developed include epirubicin, an epimer of doxorubicin, and mitoxantrone, an anthracenedione. MATERIALS AND METHODS Epirubicin has been reported to be less cardiotoxic than its Doxorubicin hydrochloride, epirubicin hydrochloride, and ICRF-l87 parent drug, doxorubicin, and has comparable antitumor activ (ADR-529) were obtained from Farmitalia-Carlo (Milan, Italy). Mi ity (1—3).Mitoxantrone was initially described as noncardi toxantrone hydrochloride (Novantrone) was purchased from Lederle otoxic in animal models (4, 5) with an antitumor activity similar Laboratories Division, Cynamid Australia (New South Wales, Aus to that ofdoxorubicin (6),but morerecentstudieshavereported tralia). All other chemicals were obtained from the sources detailed that mitoxantrone is cardiotoxic (7—9).Hence these drugs were previously (18). chosen for investigation to assess their cardiotoxic potential Experimental Animals. Female BALB/c mice were obtained from the and to contribute to an understanding of the biochemical mech Walter and Eliza Hall Institute's Breeding Laboratories (Melbourne, anisms involved in cardiotoxicity. Australia) at 5 weeks of age and then used in the experiments. Mice There is growing evidence in the literature that implicates were housed in plastic cages and fed a laboratory diet and water ad libitum. Mice wererandomlydividedinto eighttreatmentgroups,each oxygen free radicals as a primary mechanism in doxorubicin containing four mice which received (a) 0.9% NaCl solution, (b) ICRF cardiotoxicity. Doxorubicin can be reduced by cellular flavoen 187, (c) doxorubicin, (d) doxorubicin and ICRF-187, (e) epirubicin, zymes to a semiquinone free radical. This drug radical is pos (1) epirubicinandICRF-187,(g)mitoxantrone,and(h)mitoxantrone tulated to give rise to oxygen free radicals that can initiate and ICRF-187. The same treatment protocol utilized to develop a cellular damage if they overwhelm the antioxidant defense model for chronic doxorubicin cardiotoxicity was used (18). Briefly, mechanisms. The cardiac antioxidant defenses, which include each mouse, depending on the treatment group, received weekly i.p. injections over a 12-week period ofeither 0.9% NaC1 solution or ICRF Received 6/7/91; accepted 10/18/91. 187 (12.5 mg/kg) 30 mm prior to a 0.9% NaC1 solution, doxorubicin The costs of publication of this article were defrayed in part by the payment (1 mg/kg), epirubicin (1 mg/kg), or mitoxantrone (0.2 mg/kg) injection. of page charges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734solelyto indicatethis fact. The totalcumulative doses receivedwere 12mg/kg for both doxorubicin I This study was supported by a grant from the Anticancer Council of Victoria. 2 To whom requests for reprints should be addressed, at Department of Medical 3 The abbreviations used are: ICRF-l87 or ADR-529, (±)-l,2-bis(3,5-dioxo Oncology, c/o Post Office, Royal Melbourne Hospital, Victoria, 3050, Australia. piperazinyl-l-yl)propane. 194 Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1992 American Association for Cancer Research. CARDIOTOXICITYOF DOXORUBICIN, MITOXANTRONE, AND EPIRUBICIN and epirubicin, 2.4 mg/kg for mitoxantrone, and 150 mg/kg for ICRF the Z-bands. In comparison, doxorubicin induced more pro 187. A lower dose of mitoxantrone than of doxorubicin was used, since nounced disorientation of the cristae associated with clearing mitoxantrone has been reported to be 5—7timesmore potent and toxic out of the matrix in some mitochondria. These degenerative than doxorubicin in humans (22) and in animal models (4—6).One mitochondrial changes caused by doxorubicin were associated week after the final injection cycle the mice were sacrificed, the hearts with the appearance of myelin figures. Furthermore, there was and livers were removed for antioxidant assays, and a portion of the heart was retained for electron microscopy.This study consisted of two marked disruption of the organized myofibrillar array, includ experiments in which a total of6 mice/treatment group (3/experiment) ing misalignment of the Z-bands and myofibrillar lysis, leading were examined by electron and light microscopy, and the cardiac to focal loss of banding in the myocytes following chronic antioxidant capacity was assessed. doxorubicin exposure. Doxorubicin also caused dilation of sarc Antioxidant Assays. The mouse livers and hearts were homogenized otubular and t-tubular systems, separation ofthe adjacent mem and centrifuged to obtain a 100,000 xg supernatant fraction following branes of the intercalated disc, and swelling of the peri a modification of the procedure detailed by Doroshow et aL (10), as nuclear area, changes which were absent with epirubicin treat described previously (18). Aliquots of the tissue homogenate super ment. Neither epirubicin nor doxorubicin affected the mor natants were stored at —70Cuntil analysis. However, reduced gluta phology of the endothelial cells. The morphological changes thione is subject to oxidation during long-term storage, and therefore were scored as grade 1 for epirubicin and grade 3 for its concentration was determined within 3 days of sample preparation. The reducedglutathione, superoxide dismutase, glutathione peroxidase, doxorubicin. and catalase assays were performed as specified before (18) using Mitoxantrone was cardiotoxic in this chronic mouse model, previously published methods. with the degree and severity of the morphological changes Electron and