Note: Descriptions are shown in the official language in which they were submitted.
2150710
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
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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
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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.
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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
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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-
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213 0710
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
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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
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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.
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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
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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.
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- 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.
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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.
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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
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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.
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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
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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
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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.
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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.
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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,
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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,
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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
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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).
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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.
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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
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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
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2150710
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.
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