CA2150710A1 - Alleviation of cardiotoxic effects of anthracycline glycosides - Google Patents

Abstract

Anthracycline glycosides such as doxorubicin (adria-mycin) and daunorubicin have valuable anti-tumorous properties but also display cardiotoxic side effects that limit their usefulness. It has now been discovered that the cardiotoxic side effects in a patient can be reduced or alleviated if the patient is also treated with a lipid-lowering drug having antioxidant property, for example probucol.

Description

ALLEVIATION OF CARDIOTOXIC EFFECTS OF ANTHRACYCLINE GLYCOSIDES
The present invention relates to a method of treat-ing a patient who is receiving, or about to receive, treatment with an anthracycline compound to protect the patient against adverse side effects induced by the anthracycline compound.
Backqround of the Invention and Discussion of the Prior Art The anthracycline glycoside compounds are a well known class of compounds in the antineoplastic group of agents, wherein doxorubicin is a typical, and the most widely used, representative: Doxorubicin. Anticancer Antibiotics, Federico Arcamone, 1981, Publ.: Academic Press, New York, N.Y.; Adriamycin Review, EROTC International Symposium, Brussels, May, 1974, edited by M. Staquet, Publ.: Eur. Press Medikon, Ghent, Belg.; Results of Adriamycin Therapy, Adria-mycin Symposium at Frankfurt/Main 1974 edited by M. Ghione, J. Fetzer and H. Maier, publ.: Springer, New York; N.Y.
Doxorubicin, also called adriamycin, is an effective antitumour drug used against a variety of carcinomas. How-ever, the potential usefulness of doxorubicin is restricted because of its cardiotoxic side effects and the development of congestive heart failure that is refractory to all known therapeutic procedures. Doxorubicin-induced myocardial dys-function has been suggested to involve inhibition of nucleic acid as well as protein synthesis, release of vasoactive amines, changes in adrenergic functions, abnormalities in the mitochondria, lysosomal alterations, alterations in sarco-lemmal Ca2+ transport, membrane-bound enzymes, imbalance of myocardial electrolytes, free radical formation, and lipid peroxidation.
Although a close ex~m;n~tion of this list indicates that doxorubicin-induced injury may be multifactorial and complex, one mechanism common to most of these suggestions is the increased oxidative stress, see Singal P.K. et al, Sub-cellular effects of adriamycin in the heart: a concise review. J.Mol. Cell Cardiol. 1987; 19:817-828. Because of the presence of semiquinone in the tetracyclic aglycone molecule of doxorubicin, the drug is reported to increase the oxygen radical activity as well as peroxidation of polyunsaturated fatty acids within the membrane phase. This may also explain doxorubicin-induced defects in membrane function.
An acute study involving a single injection of adriamycin (15 mg/kg) in mice showed that a single pretreat-ment with 85 IU of vitamin E provided protection with respect to cardiac cell damage that was also accompanied by a reduc-tion in lipid peroxidation, Myers et al, Adriamycin: the role of lipid peroxidation in cardiac toxicity and tumour response.
Science, 1977; 19:165-167.
Summary of the Invention I have now discovered that many of the adverse side effects of the anthracycline glycosides such as doxorubicin can be prevented or alleviated if there is administered to the patient an effective amount of a lipid-lowering drug with strong antioxidant property. The lipid-lowering drug is administered over the same period of time that the patient is receiving the anthracycline glycoside or, preferably, the lipid-lowering drug is administered both prior to and during the period of time that the patient is receiving the anthra-cycline glycoside.
Accordingly, in one aspect the present invention provides a method of treating a patient receiving, or about to receive, treatment with an anthracycline glycoside or a pharm-aceutically acceptable salt thereof, to protect against adverse anthracycline-induced side effects, which method com-prises administering to the patient an effective amount of a lipid-lowering drug having antioxidant property.
Description of the Preferred Embodiments Anthracycline glycosides used in conjunction with the invention include, for example, those disclosed in UK
Patents Nos. 1,161,278; 1,217,133; 1,457,632; 1,500,421 and 1,511,559, the disclosures of which are incorporated herein by reference. Particular mention is made of doxorubicin, 4'-epi-doxorubicin (i.e. epirubicin), 4'-desoxy-doxorubicin (i.e.
esorubicin), daunorubicin (also known as daunomycin) and 4-demethoxy-daunorubicin (i.e. idarubicin). Preferred anthra-cycline glycosides are daunorubicin and, especially, doxo-rubicin. The compounds can be administered in the form of their pharmaceutically acceptable salts. Examples of suitable salts include the salts of mineral acids such as hydrochloric, hydrobromic, sulfuric, phosphoric and the like, and also salts with organic acids such as succinic, tartaric, ascorbic, citric, methanesulphonic, ethanesulphonic and the like. The hydrochloride salt is preferred, particularly with doxo-rubicin.

21~0710 The anthracycline drugs are normally administered by injection, in known manner and dosages. Injectable composi-tions of these drugs are described, for example, in Canadian Patents Nos. 1,248,453 and 1,291,307, which also discuss dosages. The disclosures of these two Canadian patents are incorporated herein by reference.
The preferred lipid-lowering drug with strong anti-oxidant property is probucol. This drug is normally admini-stered orally, and is already approved for and used as an antihyperlipoproteinemic. Hence methods of, and formulations for, administration are already known to the medical pro-fession. Probucol does have a high margin of safety against overdose, and is often given to adults in an amount of 500 mg twice daily.
Probucol contains two phenol moieties in the mole-cule and it is possible that these contribute to the antioxi-dant properties of probucol. Other lipid-lowering drugs whose molecule contains one or more phenol moieties can be used as replacement, or partial replacement, for probucol.
By the expression "strong antioxidant property" is meant that in an esc vivo system the drug is effective in mitigating the process of oxidative stress injury equal to or better than vitamin E. In a standard test known lipids are subjected to oxidative stress and lipid peroxidation, for example with hydrogen peroxide, and MDA is assayed.
It is of course clearly desirable that the lipid-lowering antioxidant drug shall not interfere in the effect-iveness of the anthracycline glycoside on the patient. In the 215071~

best case, of course, there should be no interference but in a less desirable case some interference may be tolerated if the overall effect is beneficial. Tests are available to deter-mine whether the effectiveness of the anthracycline glycoside is impeded, and it is demonstrated below that probucol does not adversely affect the anti-tumor effectiveness of adriamy-cin.
In the description of the invention there are refer-ences to the "concurrent" or "simultaneous" administration of the anthracycline glycoside and the lipid-lowering drug. This does not mean that once the one drug has been administered the other drug should immediately be administered. There can elapse several hours between the administration of the anthra-cycline glycoside and the lipid-lowering drug. What is meant is that a patient who is on a regime of treatment with an anthracycline glycoside should also be put on a regime of treatment with a lipid-lowering drug, so that the two treat-ment regimes are concurrent or simultaneous. In fact it is preferred that the regime of administration of the lipid-lowering drug should precede the regime of administration ofthe anthracycline glycoside, as well as be concurrent with that regime. Administration of the lipid-lowering drug can also continue after administration of the anthracycline glycoside has ceased.
In another aspect, the invention extends to the use of a lipid-lowering drug having antioxidant property to pro-tect against cardiotoxic effects of anthracycline glycosides.
It also extends to the concurrent use of an anthracycline gly-coside and a lipid-lowering drug as an antitumor treatment.
It also extends to a commercial package containing, as active pharmaceutical ingredient, a lipid-lowering drug with anti-oxidant property, together with instructions for its use with an anthracycline glycoside to protect against cardiotoxic side effects in an antitumor treatment. It also extends to a com-mercial package containing both an anthracycline glycoside and a lipid-lowering drug with antioxidant property, together with instructions for their use in an antitumor treatment.
Experiments reported below show the development of cardiomyopathy and congestive heart failure in rats, due to adriamycin. The rat model is considered to be a good repro-ducible and cost effective system for testing beneficial effects of different drugs; see Mattler, F.P. et al, Adria-mycin-induced cardiotoxicity (cardiomyopathy and congestive heart failure) in rats. Cancer Res. 1977; 37:2705-2714.
Cardiomyopathy and congestive heart failure were established by myocardial cell damage, depressed systolic pressures, increase in left ventricular end-diastolic pressure (LVEDP), ascites and congestive changes in liver. The first series of experiments reported below demonstrates that concurrent treat-ment with probucol and adriamycin delayed or decreased devel-opment of cardiomyopathy, but complete protection was not achieved. The second series of experiments, in which probucol was administered both prior to and concurrent with administra-tion of adriamycin, did demonstrate complete prevention of adriamycin cardiomyopathy, as indicated in the zero mortality in the group of rats given probucol and adriamycin, and the 21~0710 maintenance of the hemodynamic function and the myocardial structure of the animals.
Description of the Drawinqs Figures lA and lB are photomicrographs showing myocardial cell damage in rats exposed to doxorubicin.
Figures 2A and 2B are photomicrographs showing portions of a myocardial cell from a rat that had been treated with doxorubicin and probucol, in accordance with the invention.
Figure 3A and 3B is also a photomicrograph showing doxorubicin-induced cell damage. Figure 3B shows absence of such damage in a doxorubicin-probucol treated group.
Figure 4 is a plot showing the effects of adriamycin alone, probucol alone and adriamycin plus probucol on the regression in tumor size in lymphoma-bearing DBA/2 mice.
In Figures lA, lB, 2A, 2B, 3A and 3B, the bar indicates l~m.
The invention is further illustrated, by way of example, in the following experiments. The beneficial effects of repeated treatment with probucol in a chronic model of adriamycin-induced congestive heart failure in rats were examined. Hemodynamic function, myocardial ultrastructure, lipid peroxidation and various antioxidant enzyme activities were also studied.
Methods Animal Model Male Sprague-Dawley rats, body weight 250+10g, were maintained on a normal rat chow diet. Rats were divided into - 21~0~10 four groups: CONT (control), ADR (adriamycin treated), PROB
(probucol treated), and PROB+ADR (probucol+adriamycin treat-ed). Adriamycin (doxorubicin hydrochloride) was administered intraperitoneally in six equal injections (each containing 2.5 mg/kg adriamycin) to animals in the ADR and PROB+ADR
groups over a period of 2 weeks for a total cumulative dose of 15 mg/kg body weight. Probucol (cumulative dose, 60 mg/kg body weight) was also administered intraperitoneally to PROB
and PROB+ADR groups in six equal injections (each treatment containing 10 mg/kg) over a period of 2 weeks, alternating with adriamycin injections. CONT animals were injected with the vehicle alone (lactose, 75 mg/kg in saline) in the same regimen as ADR. Treated as well as CONT animals were observed for up to 3 weeks after the last injection for their body weight, general appearance, behaviour, and mortality. At the end of the 3-week posttreatment period, animals were hemody-namically assessed. Hearts were used for the study of myo-cardial antioxidants, lipid peroxidation, and ultrastructure.
Hemodynamic Studies Animals were anaesthetized with sodium pentobarbital (50 mg/kg IP). A miniature pressure transducer (Millar Micro-Tip) was inserted into the left ventricle via the right caro-tid artery. Left ventricular systolic (LVSP), left ventricu-lar end-diastolic (LVEDP), aortic systolic (ASP), and aortic diastolic (ADP) pressures were recorded on a Beckman Dyno-graph.

21.30 710 Bioassays Catalase Assay Ventricles (1 g) were homogenized in 10 mL 0.05 potassium phosphate buffer (pH 7.4) and centrifuged at 40 OOOg for 30 minutes. Supernatant, 50 ~L, was added to the cuvette containing 2.95 mL of 19 mmol/L H2O2 solution prepared in potassium phosphate buffer, see Clairborne A. Catalase Activ-ity. In: Greenwald RA, ed. Handbook of Methods for Oxygen Radical Research. Boca Raton, Fla: CRC Press; 1985:283-284.
The color was read at 240 nm on a Zeiss spectrophotometer every minute for 5 minutes. Commercially available catalase was used as a standard. Specific activity of the enzyme was expressed as units per milligram tissue protein.
Glutathione Peroxidase (GSHPx) Assay GSHPx activity was expressed as nanomoles reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidized to nicotinamide adenine dinucleotide phosphate (NADP) per minute per milligram protein, with a molar extinction coeffi-cient for NADPH at 340 nm of 6.22x106, see Paglia DE et al.
Studies on the quantitative and qualitative characterization of erythrocyte glutathione peroxidase. ~ Lab Clin Med. 1967;
70:158-169. Cytosolic GSHPx was assayed in a 3-mL cuvette containing 2.0 mL of 75 mmol/L phosphate buffer, pH 7Ø The following solutions were then added: 50 ~L of 60 mmol/L
glutathione, 100 ~L glutathione reductase solution (30 U/mL), 50 ~L of 0.12 mol/L NaN3, 0.10 of 15 mmol/L Na2EDTA, 100 ~L of 3.0 mmol/L NADPH, and 100 ~L of cytosolic fraction obtained after centrifugation at 20 OOOg for 25 minutes. Water was 21~3 0710 added to make a total volume of 2.9 mL. The reaction was started by the addition of 100 ~L of 7.5 mmol/L H2O2, and the conversion of NADPH to NADP was monitored by a continuous recording of the change of absorbance at 340 nm at l-minute intervals for 5 minutes. Enzyme activity of GSHPx was ex-pressed in terms of milligrams of protein.
Superoxide Dismutase Assay Supernatant (20 000g for 20 minutes) was assayed for superoxide dismutase (SOD) activity by following the inhibi-tion of pyrogallol auto-oxidation, see Marklund S.L. Pyro-gallol autooxidation. In: Greenwald RA, ed. Handbook of Methods for Oxygen Radical Research. Boca Raton, Fla: CRC
Press; 1985:243-247. Pyrogallol (24 mmol/L) was prepared in 10 mmol/L HCl and kept at 4C before use. Catalase (30 ~mol/L
stock solution) was prepared in an alkaline buffer (pH 9.0).
Aliquots of supernatant (150 ~g protein) were added to Tris HCl buffer containing 25 ~L pyrogallol and 10 ~L catalase.
The final volume of 3 mL was made up of the same buffer.
Changes in absorbance at 420 nm were recorded at l-minute intervals for 5 minutes. SOD activity was determined from a standard curve of percentage inhibition of pyrogallol auto-oxidation with a known SOD activity. This assay was highly reproducible, and the standard curve was linear up to 250 ~g protein with a correlation coefficient of 0.998. Data are expressed as SOD units per milligram protein compared with the standard.

- 2~ 507~0 Malondialdehyde Assay Measurement of lipid peroxidation by determination of myocardial malondialdehyde (MDA) content was performed by a modified thiobarbituric acid (TBA) method, see Placer ZA.
et al. Estimation of product of lipid peroxidation (malondi-aldehyde) in biochemical systems. Anal Biochem. 1966:16:359-365. Hearts were quickly excised and washed in buffered 0.9%
KCl (pH 7.4). After the atria, extraneous fat, and connective tissue were removed, the ventricles were homogenized in the same buffer (10% wt/vol). The homogenate was incubated for 1 hour at 37C in a water bath. A 2-mL aliquot was withdrawn from the incubation mixture and pipetted into an 8-mL Pyrex tube. One milliliter of 40% trichloroacetic acid (TCA) and 1 mL of 0.2% TBA were promptly added. To ml n; ml ze peroxidation during the subsequent assay procedure, 2% butyl-ated hydroxytoluene was added to the TBA reagent mixture, see Aust SD. Lipid peroxidation. In: Greenwald RA, ed. Handbook of Methods for Oxygen Radical Research. Boca Raton, Fla: CRC
Press; 1985:203-207. Tube contents were vortexed briefly, boiled for 15 minutes, and cooled in a bucket of ice for 5 minutes. Two milliliters of 70% TCA was then added to all tubes, and contents were again vortexed briefly. The tubes were allowed to stand for 20 minutes. This was followed by a centrifugation of the tubes for 20 minutes at 3500 rpm. The color was read at 532 nm on a Zeiss spectrophotometer and compared with a known MDA standard.

21~0710 Ultrastructural Studies For ultrastructural studies, three to five hearts in each group were processed as described by Tong J. et al. Myo-cardial adrenergic changes at two stages of heart failure due to adriamycin treatment in rats. Am J Physiol. 1991; 260:
H909-H916, and Singal PK et al. Changes in lysosomal morphol-ogy and enzyme activities during the development of adria-mycin-induced cardiomyopathy. Can J Cardiol . 1985:1:139-147.
Hearts were washed in cold 0.1 mol/L sodium phosphate buffer (pH 7.4). Tissue samples 4 to 6 mm in size were taken from four different areas of the subendocardium as well as the subepicardium of the free left ventricular wall between the midregion and apex of the heart. The tissue pieces were immersed for 15 minutes in 0.1 mol/L phosphate buffer (pH 7.4) containing 3% glutaraldehyde. This briefly fixed tissue was further cut into cubes smaller than 1 mm. Aldehyde fixation was continued for a total duration of 2 hours. The tissues were washed for 1 hour in the above phosphate buffer contain-ing 0.05 mol/L sucrose. Postfixation was done in 2% OSO4 for 1.5 hours, after which the tissue pieces were dehydrated in graded alcohol series. Tissue embedding was done in epoxy.
Ultrathin sections were placed on Formvar-coated grids and stained with uranyl acetate and lead citrate. Electron micrographs of the subendocardial and subepicardial regions from the four groups were compared to establish ultrastruc-tural differences.

- 21S071~

Proteins and Statistical Analysis Proteins were determined by the method of Lowry and associates, see Lowry OH, et al. Protein measurements with the Folin phenol reagent. ~ Biol Chem. 1951;193:265-275.
Data were expressed as the mean+SEM. For a statistical analy-sis of the data, group means were compared by one-way ANOVA, and Bonferroni's test was used to identify differences between groups. Statistical significance was acceptable to a level of P<.05.
Results General Observations and Mortality The general appearance of all groups of animals was recorded during the time course of the study. After comple-tion of adriamycin treatment, the animals' fur became scruffy and developed a light yellow tinge, and there was red exudate around the eyes in both ADR and PROB+ADR groups, although more extensively in the ADR group. Animals in the ADR group also appeared to be sicker, weaker, and lethargic compared with the PROB+ADR group. The most predominant feature in the ADR group animals was the development of a grossly enlarged abdomen and ascites. This condition became apparent within a week after the completion of treatment with adriamycin. When they were killed, all ADR group animals had a significant amount of peritoneal fluid (Table 1 below). In addition, the liver was enlarged and congested in all ADR group animals. In the PROB+ADR group animals, the amount of peritoneal fluid was about one fifth that seen in animals in the ADR group (Table 1). During the posttreatment period, the mortality 2 1 ~ 0 7 1 0 rate was significantly higher in the ADR group than in the PROB+ADR group (Table 1). There were no deaths in the CONT
and PROB groups.
TABLE 1. Effects of Probucol on Adriamycin-Induced Changes In Heart Weight, Body Weight, Mortality Rate, and Ascites Heart Animal HeartWeight/Body Group Weight, g Weight Mortality, % Ascites, mL
RatioxlO3 CONT 1.25+0.05 2.84+0.12 0 0 ADR 0.75+0.03* 2.38+0.08* 30 92.2+13.2*
PROB 1.18+0.04 2.78+0.13 0 0 PROB+ADR 0.92_0.04t 2.73+0.14 10 19.9+6.4t_ CONT indicates control; ADR, adriamycin; and PROB, probucol. Data are mean+SEM of six to eight animals in all studies. Mortality data are mean+SEM of 50 animals each in the ADR and PROB+ADR groups and 25 animals each in the CONT
and PROB groups.
*and tP~.05 compared with all other groups.

Data on heart weight and heart weight/body weight ratio are also given in Table 1. Despite the ascites, the body weight gain in the ADR group was significantly less.
Treatment with adriamycin resulted in a significant decrease in heart weight and ratio of heart to body weight in the ADR
group. In the PROB+ADR group, ratio of heart to body weight was not significantly different from the CONT and PROB groups.
Heart weight in the PROB+ADR group was significantly higher than in the ADR group but was still lower than in the CONT and PROB groups.

~1~0710 Hemodynamic Studies ASP, ADP, LVSP, and LVEDP were recorded in all groups; these data are shown in Table 2, below. There were significant changes in cardiac performance in the ADR group.
LVEDP in both ADR and PROB+ADR groups was higher than values in the CONT and PROB groups; however, this value was rela-tively more increased in the ADR than the PROB+ADR group.
LVSP was significantly decreased in the ADR group alone, whereas in the PROB+ADR group, it was no different from the CONT and PROB groups. There was no difference between CONT
and treated groups with respect to ADP. ASP values were significantly lower in the ADR group compared with all other groups.
Morpholoqical Studies Electron microscopic analysis of left ventricular free wall was conducted on heart tissue excised from all four groups of rats. The morphological appearance of different subcellular structures, including mitochondria, sarcoplasmic reticulum, sarcomeres, myofibrils, and intercalated disks, in hearts from control and probucol groups were typical of normal cells. Morphological changes due to adriamycin treatment alone included disruption of several subcellular elements including loss of myofibrils, swelling of mitochondria, vacuolization of the cytoplasm, formation of lysosomal bodies, and dilation of the sarcotubular system . Thus, Figure lA
shows swelling of mitochondria (M) as well as sarcoplasmic reticulum (arrow). Vacuolization (*) and dense bodies (double arrow) are also apparent. Mitochondrial injury in addition to - ~ 21S~710 the swelling of these organelles was also accompanied by dis-arrangement and disruption of cristae. Thus, Figure lB, at a higher magnification, shows loss of cristae from the mito-chondria and some membrane cisternae in the dense bodies are seen. Some of the electron-dense bodies showed lamellar inclusions (Fig. lB). These structural changes are typical for adriamycin cardiomyopathy. The ultrastructure of hearts from the PROB+ADR group showed regular myofibrillar structure, maintained sarcotubular reticulum, and preserved mitochondria.
Figure 2A shows a portion of a myocardial cell from a probu-col- and adriamycin-treated rat. Mitochondria (M), myofibrils (MF), sarcoplasmic reticulum (arrows) and other cellular details are better maintained. At higher magnification, intramitochondrial details were quite normal, but some intra-cellular edema was noticeable around the mitochondria (Fig. 2B).
Antioxidant Enzymes and Lipid Peroxidation Different antioxidant enzyme activities were examin-ed in all groups; these data are shown in Table 3. GSHPx activity in the ADR group was reduced by about 32% compared with the CONT group. In the PROB+ADR group, GSHPx activity was near control levels. Total SOD activity in the PROB and PROB+ADR groups was significantly higher than in the CONT and ADR groups. Catalase activity did not change in any group.
The amount of lipid peroxidation was determined by evaluating myocardial MDA content; these data are also shown in Table 3.
MDA levels were almost the same in the CONT, PROB, and 07ln PROB+ADR groups, whereas in the ADR group alone the MDA
content was significantly higher.

Table 2. Effects of Probucol on Adriamycin-Induced Pressure Changes Animal Group ASP ADP LVSP LVEDP
CONT 102.1+1.8 66.9+4.4 126.0+11.3 5.9+3.0 ADR 84.2+3.2* 57.8+9.4 89.5+6.7* 33.7+8.6*
PROB 91.0+9.8 62.6+13.5 113.9+6.5 12.4+6 PROB+ADR 110.5+8.2 72.1+6.3 123.3+7.2 20.1+5.3t ASP indicates aortic systolic pressure; ADP, aortic dia-stolic pressure; LVSP, left ventricular systolic pressure;
LVEDP, left ventricular end-diastolic pressure; CONT, control;
ADR, adriamycin; and PROB, probucol. Values are mm Hg, mean+
SEM of six to eight experiments.
*and tP<.05 significantly different from CONT and ADR
groups, respectively.
Table 3. Effects of Probucol on Adriamycin-Induced Changes in Antioxidant Enzyme Activities and Lipid Peroxidation Animal GSHPx, SOD, Catalase, MDA, Group nmol/mg U/mg U/mg nmol/g protein protein protein heart CONT 59.9+5.7 34.7+4.4 31.3+3.8 49.1+3.2 ADR 40.7+5.lt 41.0+2.7 32.7+2.0 82.1+3.1*
PROB 52.4+2.9 46.2+6.5t 36.7+3.2 54.3+3.2 PROB+ADR 54.6+5.3 64.1+4.2t 30.0+2.4 58.2+7.1 GSHPx indicates glutathione peroxidase; SOD, superoxide dismutase; MDA, malondialdehyde; CONT, control; ADR, adriamy-cin; and PROB, probucol. Data are mean+SEM from six to eight experiments.
*P<.05, tP<.02 different from all other groups, tP<.05 different from CONT and ADR groups.

- 21~710 Discussion Repeated administration of adriamycin beyond a certain dose has been shown to cause cardiomyopathic changes in patients as well as in a variety of animal species. The rat model is considered to be a good, reproducible, and cost-effective system for testing beneficial effects of different drugs. Both development of cardiomyopathy and congestive heart failure in rats in the present study were established by the myocardial cell damage, depressed systolic pressures, increase in LVEDP, ascites, and congestive changes in the liver. These failing hearts in vivo as well as in isolated myocardial preparations ex vivo have been shown to respond poorly to inotropic interventions. I have demonstrated for the first time that a simultaneous treatment with probucol mitigates adriamycin-induced cardiomyopathic changes as well as congestive heart failure, as indicated by the improved cardiac structure and function and a reduced mortality in the PROB+ADR group.
Probucol in the plasma is transported predominantly by low-density, very-low-density, and high-density lipopro-teins. Oral administration of probucol at 1 g/d increases its level in the blood as well as the adipose tissue. However, there seems to be no absolute correlation between the plasma levels of probucol and the extent of cholesterol lowering.
Although it is difficult to draw any parallel between the dosage used by us in rats (6 x 10 mg/kg IP) and therapeutic dosage (2x 500 mg/d for 3 to 6 months), the probucol treatment protocol used in my study was well tolerated.

- `~ 21~Q71Q

Probucol treatment in heterozygous familial hyper-cholesterolemia caused the regression of xanthomas, which did not correlate with the level of cholesterol reduction. Chole-styramine, another cholesterol-lowering drug, and probucol both sharply lowered the serum cholesterol levels in nonhuman primates, but only probucol caused regression of athero-sclerotic lesions. These observations clearly suggest that beneficial effects of probucol may be independent of cholest-erol lowering. Because of the two phenolic groups in its molecular structure, probucol has been reported to be a strong antioxidant, and it appears that the protection offered by probucol in this study may involve antioxidant mechanisms.
Adriamycin has been shown to promote the production of free radicals; these toxic species are known to cause myocardial dysfunction. Data on lipid peroxidation are also in concert with this suggestion inasmuch as probucol caused a significant attenuation in the adriamycin-induced increase in MDA levels.
The beneficial effect of probucol against restenosis after percutaneous transluminal coronary angioplasty has also been suggested to be due to its antioxidant properties.
In addition to its cholesterol-lowering and anti-oxidant properties, probucol may also have an effect on endo-genous antioxidant enzyme activities. Probucol not only prevented adriamycin-induced decreases in GSHPx activity but also increased SOD activity. It is important to note that in the ADR group, there was a small although statistically not significant increase in the SOD activity. Probucol alone caused a 33% increase in SOD; however, in the PROB+ADR group, - 21~710 there may have been some synergistic effect, since the increase in SOD activity was about 88%. Thus, probucol clearly improves "endogenous antioxidant reserve," and the latter has been suggested to improve myocardial structure and function. The mechanisms for an adriamycin-induced decrease in GSHPx and probucol-induced increase in antioxidants (GSHPx and SOD) are not clear. This study, however, clearly demon-strates that probucol may be providing protection by acting as an antioxidant as well as by promoting endogenous anti-oxidants.
In conclusion, it can be said that adriamycin cardi-omyopathy is associated with an antioxidant deficit and that probucol treatment improves the antioxidant status of the heart. Improved cardiac function due to treatment with probu-col may be related to the maintenance of the antioxidant status of the heart. Adriamycin, because of its histophilic nature, is cleared from the plasma and appears in the tissues within minutes. Since treatments with PROB and adriamycin were 24 hours apart, it is unlikely that PROB influenced adriamycin absorption.
A further series of experiments was carried out.
These experiments illustrate the preferred method of the invention in which probucol is administered both prior to and concurrent with administration of adriamycin. The Animal Model was as described above except that the cumulative dose of probucol given to the PROB and PROB+ADR groups was 120 mg/kg body weight. It was administered in 12 equal injections (each treatment containing 10 mg/kg) over a period of 4 weeks, `- ~ 915!~711) 2 weeks before adriamycin administration and 2 weeks concurrent with adriamycin administration.
Hemodynamic studies and bioassays, i.e., catalase assay, glutathione peroxidase (GSHPx) assay, superoxide dis-mutase assay and ultrastructural studies were as described above, as were protein and statistical studies. Malondialde-hyde assay was as follows.
Malondialdehyde Assay Measurement of lipid peroxidation by determining myocardial thiobarbituric acid reactive substance (TBARS) con-tent was performed using a modified thiobarbituric acid (TBA) method. Hearts were quickly excised and washed in buffered 0.9% KCl (pH 7.4). After the atria, extraneous fat, and con-nective tissue were removed, the ventricles were homogenized in the same buffer (10% w/v). The homogenate was incubated for 1 hour at 37C in a water bath. A 2-mL aliquot was with-drawn from the incubation mixture and pipetted into an 8-mL
Pyrex tube. One milliliter of 40% trichloroacetic acid (TCA) and 1 mL of 0.2% TBA were promptly added. To minimize per-oxidation during the subsequent assay procedure, 2% butylatedhydroxytoluene was added to the TBA reagent mixture. Tube contents were vortexed briefly, boiled for 15 minutes, and cooled in a bucket of ice for 5 minutes. Two milliliters of 70% TCA was then added to all tubes, and the contents were again vortexed briefly. The tubes were allowed to stand for 20 minutes. This was followed by centrifugation of the tubes for 20 min at 3500 rpm. The color was read at 532 nm on a Zeiss spectrophotometer and compared with a known TBARS

- 21~07i 0 standard. Proteins and statistical analysis were carried out as described above.
Results General Observations and Hemodynamics Within 1 week after the completion of treatment with adriamycin, animals in the ADR-only group had enlarged abdo-men, developed ascites, and appeared weaker and lethargic. At death, all ADR group animals had a significant amount of peri-toneal fluid (Table 4). In the PROB+ADR animals, the amount of peritoneal fluid was insignificant. Of 14 animals examined for ascites in the PROB+ADR group, 12 animals had no ascites, 1 animal had 6 mL of ascites, and 1 animal had 13 mL of ascites. During the posttreatment period, the mortality rate was approximately 32% in the ADR group. There were no deaths in the CONT, PROB, and PROB+ADR groups (Table 4).
ASP and LVSP were significantly depressed, whereas LVEDP was significantly elevated in the ADR group alone. In the PROB+ADR group, these parameters were no different from those of the CONT and PROB groups (Table 4).

Table 4. Effects of Probucol Pretreatment and Concurrent Treatment on Adriamycin-Induced Changes CONT ADR PROB PROBtADR
Heart weight, g 1.27+0.040.76+0.03* 1.20+0.06 1.04+0.04 Mortality, % 0 32 0 0 Ascites, mL0 105.2+19.3* 0 1.35+0.99 ASP103.2+2.7 83.3+4.5* 94.3+4.3 104.0+8.7 ADP 66.6+3.4 60.3+8.2 58.6+6.5 62.6+3.3 LVSP124.5+1.3 86.5+5.8* 117+2.7 112.22+9.5 LVEDP5.3_2.6 35.6+4.9* 7.4+3.6 9.1+1.3_ CONT indicates control; ADR, adriamycin; PROB, probucol;
PROB+ADR, probucol and adriamycin; ASP, aortic systolic pres-sure; ADP, aortic diastolic pressure; LVSP, left ventricular systolic pressure; and LVEDP, left ventricular end-diastolic pressure. Probucol treatment was started 2 weeks before and was continued for another 2 weeks concurrent with adriamycin treatment. Mortality data are expressed as percent of 25 animals each in the ADR and PROB+ADR groups and 12 animals each in the CONT and PROB groups. For ascites, 14 animals were studied. All other data are mean+SEM of 6 to 8 animals.
*P<.05 compared with all other groups.
Ultrastructure Morphological changes in the ADR group were typical for adriamycin-induced cardiomyopathy, as shown in Figure 3A, and included loss of myofibrils, swelling of mitochondria (M), and sarcoplasmic reticulum vacuolization (*) of the cytoplasm, formation of lysosomal bodies (double arrow), and dilation of the sarcotubular system (Fig. 3A). Ultrastructure of hearts from the PROB+ADR group, as shown in Figure 3B, was indisting-uishable from that of the CONT group and had regular myofi-brillar arrangement, maintained sarcotubular system, and pre-served mitochondria. Mitochondria (M), myofibrils (MF), sarcoplasmic reticulum (arrow) and other cellular details are normal.

~ 21aO710 Antioxidants In addition to the study of different antioxidant enzyme activities, the amount of lipid peroxidation was deter-mined by evaluating myocardial TBARS content (Table 5). GSHPx activity was reduced and TBARS were increased significantly in the ADR group (Table 5). In the PROB+ADR group, GSHPx activ-ity as well as TBARS were near control levels. Total SOD
activity in the PROB and PROB+ADR groups was significantly higher, whereas catalase activity did not show change in any group.
Table 5. Effects of Probucol Pretreatment and Concurrent Treatment on Adriamycin-Induced Changes in Anti-oxidant Enzyme Activities and Lipid Peroxidation Animal GSHPx, SOD, Catalase, TBARS, Group nmol/mg U/mg U/mg nmol/g Protein Protein Protein Heart CONT 52.17+2.5 34.52+2.2 29.62+3.6 48.10+3.2 ADR 38.04+3.2* 36.1+4.3 33.33+2.6 98.62+4.5*
PROB 54.12+2.3 48.65+3.46t 31.12+4.8 52.16+4.1 PROB+ADR 65.15+2.4t 58.16+3.lt 30.06+2.8 52.19+2.3 CONT indicates control; ADR, adriamycin; and PROB, probu-col; and PROB+ADR, probucol and adriamycin. Data are mean+
SEM from 6 to 8 animals.
*P<.05 from all other groups.
tP<.05 different from CONT and ADR groups.
Antitumor Effect To assess the effects of probucol on the antitumor efficacy of adriamycin, subcutaneous tumor growth was studied in mice (Fig. 4). The L5178Y-F9 lymphoma model in mice was chosen because it was cloned directly from the L5178Y, one of - - * 2150710 the standard experimental tumors used to examine chemothera-peutic efficacy of different anticancer drugs, including adriamycin and its derivatives. A significant reduction in the tumor size was seen in the ADR group as well as the PROB+ADR group compared with the CONT and PROB groups. There was no significant difference in the tumor size between ADR
and PROB+ADR groups. The experimental procedure was as follows.
Male inbred DBA/2 mice (total number, 44) were inoculated with 106 L5178Y-F9 cells. A subcutaneous injection in 100-~L aliquot was made into the middle of a shave area on the back of each syngeneic DBA/2 mouse. Tumor size was assessed as surface area by multiplying the larger tumor dimension by that at a 90-degree angle from it measured with a vernier caliper on the days on which adriamycin or probucol was administered. Probucol and adriamycin administrations were initiated 10 days after tumor inoculation. In ADR (n=10) and PROB+ADR (n=12) groups, each animal received a total cumu-lative dose of 15 mg/kg of adriamycin in six equal IP injec-tions (i.e. six treatments) over 2 weeks. In PROB (n=12) and PROB+ADR groups, each animal received a total cumulative dose of 60 mg/kg of probucol in six equal IP injections (i.e. six treatments) over 2 weeks. The CONT group (n=10) received coconut oil (medium in which probucol was dissolved) in six IP
injections for a total cumulative dose of 6 mL/kg.
Figure 4 plots the effect of ADR, PROB and ADR+PROB
on the regression in tumor size in the lymphoma-bearing DBA/2 mice. Tumor size in the ADR group and the ADR+PROB group was significantly less compared with the CONT and PROB groups.
Data are mean+SEM of 10 to 12 animals. *P<.05 using ANOVA and tP<.05 using ANOVA and Bonferroni's post-hoc test indicate significant differences from the corresponding points in the CONT and PROB groups.

Claims (7)

1. A method of treating a patient receiving, or who is about to receive, treatment with an anthracycline glycoside, or a pharmaceutically acceptable salt thereof, to protect against adverse anthracycline-induced side effects, which method comprises administering to the patient an effective amount of a lipid-lowering drug having antioxidant property.

2. A method according to claim 1 wherein the anthra-cycline glycoside is doxorubicin or daunorubicin.

3. A method according to claim 1 wherein the lipid-lowering drug having antioxidant property is probucol.

4. A method according to claim 1 wherein the patient is subjected to a regime of treatments with an anthracycline glycoside and a concurrent regime of treatments with a lipid-lowering drug having antioxidant property.

5. A method according to claim 4 wherein the regime of administration of the lipid-lowering drug commences prior to the regime of administration of anthracycline glycoside.

6. A method according to claim 4 wherein the regime of administration of the lipid-lowering drug commences two weeks prior to the regime of administration of anthracycline glycoside.

7. A method according to claim 1 wherein the patient is treated with doxorubicin hydrochloride and probucol.