Potent Metalloporphyrin Peroxynitrite Decomposition Catalyst
Protects Against the Development of Doxorubicin-Induced
Pál Pacher, MD, PhD; Lucas Liaudet, MD; Péter Bai, MSc; Jon G. Mabley, PhD;
Pawel M. Kaminski, MD, PhD; László Virág, MD, PhD; Amitabha Deb, PhD; Éva Szabó, MD;
Zoltán Ungvári, MD, PhD; Michael S. Wolin, PhD; John T. Groves, PhD; Csaba Szabó, MD, PhD, DSc
Background—Increased oxidative stress and dysregulation of nitric oxide have been implicated in the cardiotoxicity of
doxorubicin (DOX), a commonly used antitumor agent. Peroxynitrite is a reactive oxidant produced from nitric oxide
and superoxide in various forms of cardiac injury. Using a novel metalloporphyrinic peroxynitrite decomposition
catalyst, FP15, and nitric oxide synthase inhibitors or knockout mice, we now delineate the pathogenetic role of
peroxynitrite in rodent models of DOX-induced cardiac dysfunction.
Methods and Results— Mice received a single injection of DOX (25 mg/kg IP). Five days after DOX administration, left
ventricular performance was significantly depressed, and high mortality was noted. Treatment with FP15 and an
inducible nitric oxide synthase inhibitor, aminoguanidine, reduced DOX-induced mortality and improved cardiac
function. Genetic deletion of the inducible nitric oxide synthase gene was also accompanied by better preservation of
cardiac performance. In contrast, inhibition of the endothelial isoform of nitric oxide synthase with N-nitro-L-arginine
methyl ester increased DOX-induced mortality. FP15 reduced the DOX-induced increase in serum LDH and creatine
kinase activities. Furthermore, FP15 prevented the DOX-induced increase in lipid peroxidation, nitrotyrosine formation,
and metalloproteinase activation in the heart but not NAD(P)H-driven superoxide generation. Peroxynitrite neutraliza-
tion did not interfere with the antitumor effect of DOX. FP15 also decreased ischemic injury in rats and improved
cardiac function and survival of mice in a chronic model of DOX-induced cardiotoxicity.
Conclusions—Thus, peroxynitrite plays a key role in the pathogenesis of DOX-induced cardiac failure. Targeting
peroxynitrite formation may represent a new cardioprotective strategy after DOX exposure or in other conditions
associated with peroxynitrite formation, including myocardial ischemia/reperfusion injury. (Circulation. 2003;107:896-
Key Words: cardiac function ? doxorubicin ? oxidative stress ? nitric oxide ? heart failure
used to treat a variety of cancers, including severe leukemias,
lymphomas, and solid tumors.1–4The clinical use of DOX is
limited because of its severe cardiotoxic side effects: irrevers-
ible degenerative cardiomyopathy and chronic heart failure.3,4
The cardiotoxicity of DOX involves increased oxidative
stress in cardiomyocytes, alteration of cardiac energetics, and
a direct effect on the DNA.5–11The production of peroxyni-
trite, a reactive oxidant formed from the rapid reaction of
nitric oxide (NO) and superoxide, was recently demonstrated
oxorubicin (DOX; Adriamycin) is a broad-spectrum
antitumor anthracycline antibiotic that is commonly
in rodent models of heart failure.10–13Using a novel metal-
loporphyrinic peroxynitrite decomposition catalyst molecule,
we have now directly tested the potential pathogenetic role of
peroxynitrite in a DOX-induced acute and chronic cardiac
dysfunction and heart failure in acute and chronic murine
The investigation conformed to the Guide for the Care and Use of
Laboratory Animals published by the US National Institutes of
Health (NIH Publication No. 85-23, revised 1985) and was per-
Received September 26, 2002; accepted October 28, 2002.
From Inotek Pharmaceuticals Corp, Beverly, Mass (P.P., P.B., J.G.M., L.V., A.D., E.S., C.S.); the Critical Care Division, Department of Internal
Medicine, University Hospital, Lausanne, Switzerland (L.L.); the Department of Physiology, New York Medical College, Valhalla (P.M.K., Z.U.,
M.S.W.); the Department of Chemistry, Princeton University, Princeton, NJ (J.T.G.); and the Experimental Research Department and Institute of Human
Physiology, Semmelweis University Medical School, Budapest, Hungary (C.S.).
P. Pacher, P. Bai, J.G. Mabley, L. Virág, A. Deb, E. Szabó, and C. Szabó are full-time employees of Inotek Corp, a for-profit organization involved
in the commercial development of poly(ADP-ribose) polymerase inhibitors and peroxynitrite decomposition catalysts for various therapeutic indications.
C. Szabó is also a board member and shareholder of the same organization.
Correspondence to Csaba Szabó, MD, PhD, DSc, Inotek Pharmaceuticals Corporation, Suite 419E, 100 Cummings Center, Beverly, MA 01915. E-mail
© 2003 American Heart Association, Inc.
Circulation is available at http://www.circulationaha.orgDOI: 10.1161/01.CIR.0000048192.52098.DD
formed with the approval of the local Institutional Animal Care and
Male BALB/c, C57BL6 (inducible nitric oxide synthase [iNOS]?/?),
or C57BL6-NOSII (iNOS?/?) mice (Jackson Laboratories, Bar Har-
bor, Me) weighing 25 to 35 g were given a single dose of DOX HCl
(Sigma/Aldrich) at 25 mg/kg IP and used for biochemical measure-
ments at 2 days and for functional measurements at 5 days as
described previously.10,14In a separate set of experiments, DOX was
injected in 3 equal doses of 9 mg · kg?1· d?1on days 1, 10, and 20,
and hemodynamics was measured on day 25. Treatment with FeCl
tetrakis-2-(triethylene glycol monomethyl ether) pyridyl porphyrin
(FP15) (0.03 to 1 mg · kg?1· d?1PO), aminoguanidine (AG; 50 and
100 mg · kg?1· d?1IP), or N-nitro-L-arginine methyl ester (L-NAME,
10 and 20 mg · kg?1· d?1IP) started 2, 24, and 24 hours before DOX
injection and continued until hemodynamic measurements were
completed or survival studies were terminated.
Hemodynamic Measurements in Mice
Five days after DOX administration in the acute model or on day 25
in the chronic model, left ventricular (LV) performance was ana-
lyzed in mice anesthetized with injections of ketamine (80 mg/kg IP)
and xylazine (10 mg/kg IP). A microtip pressure-volume catheter
(SPR-839; Millar Instruments) was inserted into the right carotid
artery and advanced into the LV under pressure control as de-
scribed.14,15After stabilization for 20 minutes, the signals were
recorded continuously with an ARIA pressure-volume conductance
system (Millar Instruments) coupled with a Powerlab/4SP A/D
converter (AD Instruments), stored, and displayed on a personal
computer. The heart rate, maximal LV systolic (LVSP) and end-di-
astolic (LVEDP) pressures, maximal slope of systolic pressure
increment (?dP/dt) and diastolic pressure decrement (?dP/dt),
stroke volume (SV), stroke work (SW), ejection fraction, and cardiac
output (CaO) were calculated and corrected according to in vitro and
in vivo volume calibrations with a cardiac pressure-volume analysis
program (PVAN2.9; Millar Instruments). After these measurements,
the catheter was pulled back into the aorta for the measurement of
mean arterial blood pressure (mean BP).
Malondialdehyde (MDA) formation was used to quantify the lipid
peroxidation in tissues and was measured as thiobarbituric acid–
reactive material from heart homogenates as described.16
Xanthine Oxidase Assay and NAD(P)H
NAD(P)H oxidase and xanthine oxidase activities in heart homoge-
nates were measured by the lucigenin chemiluminescence method of
Mohazzab et al,17modified to the use of 10 ?mol/L lucigenin.
Protein content was measured in an aliquot of the homogenate by the
Paraffin-embedded 3-?m sections were processed for immunohisto-
chemical determination of 3-nitrotyrosine (3-NT) as described.13,18
Serum LDH and Creatine Kinase Measurement
Forty-eight hours after DOX treatment, serum LDH and creatine
kinase (CK) activities were determined by end-point activity assay
kits (Sigma Diagnostics).14
Forty-eight hours after DOX treatment, hearts were homogenized
and used for matrix metalloproteinase (MMP) zymography as
Animals exposed to an acute dose of DOX (aDOX; 25 mg/kg IP,
n?180) received either FP15 (0.3, 0.1, 0.03 mg · kg?1· d?1PO), AG
(50 and 100 mg · kg?1· d?1IP), L-NAME (10 and 20 mg · kg?1· d?1
IP), or vehicle (isotonic saline, 0.2 mL PO or IP) starting 2, 24, and
24 hours before DOX injection, respectively. Mortality was moni-
tored and recorded twice daily for 7 days. In a separate set of chronic
experiments, DOX (cDOX, n?120) was injected in 3 equal doses of
9 mg · kg?1· d?1every 10 days, and survival was followed for 30
days in the presence of FP15 (1, 0.1, 0.01, 0.03 mg · kg?1· d?1PO)
or vehicle treatment.
Mouse Breast Carcinoma Model
The effect of FP15 on tumor growth and the antitumor effect of DOX
in a mouse model of breast cancer were investigated in 4T1
mammary adenocarcinoma cells.19Cells (n?106) were injected into
the mammary fat pad of female BALB/c mice. Fifteen days later,
mice were randomized into 4 groups (n?10 per group) and received
FP15 (1 mg · kg?1· d?1PO), DOX (4 mg · kg?1· d?1IP twice a
week), DOX?FP15, or vehicle. Tumor diameters (x, y, and z) were
recorded twice a week, and tumor size was estimated in mm3.
Twenty male Wistar rats (Charles River) weighing 300 to 330 g were
anesthetized with thiopentone sodium (60 mg/kg IP), tracheos-
tomized, and mechanically ventilated. Myocardial infarction was
induced by a 1-hour ligation of the left anterior descending coronary
artery, and infarct size and area at risk were quantified by use of the
phthalo blue/triphenyl tetrazolium chloride technique as described
previously.20Ten minutes before reperfusion, the rats received an
intravenous injection of either FP15 (0.3 mg/kg, n?8) or vehicle
(isotonic saline, 0.5 mL; n?12).
Results are reported as mean?SEM. Statistical significance between
2 measurements was determined by the 2-tailed unpaired Student’s t
test, and among groups it was determined by ANOVA with Bonfer-
roni’s correction. Survival curves were compared by the log-rank
test. Probability values of P?0.05 were considered significant.
Reagents were obtained from Sigma/Aldrich unless indicated other-
wise. The peroxynitrite decomposition catalyst FP15 was synthe-
sized as described.21
Both acute and chronic administration of DOX induced a
significant decrease in heart rate, mean BP, LVSP, ?dP/dt,
?dP/dt, SV, SW, ejection fraction, and CaO and an increase
in LVEDP, in mice (Figures 1 and 2). Treatment with FP15
(1 mg · kg?1· d?1PO) or AG (100 mg · kg?1· d?1IP) significantly
attenuated the DOX-induced changes in ventricular function. FP15
or AG alone exerted no significant effects on hemodynamic
parameters (Figures 1 and 2). Better cardiac function was also seen
in iNOS?/?mice treated with DOX than in iNOS?/?littermates
FP15 significantly attenuated the DOX-induced increase in
MDA formation in hearts (Figure 3A), indicative of an
overall reduction in oxidative stress in the presence of the
peroxynitrite decomposition catalyst compound.
Pacher et al Role of Peroxynitrite in Doxorubicin Cardiotoxicity
Figure 1. Effects of FP15 on DOX-induced acute and chronic cardiac dysfunction. Effect of DOX and FP15 on LVSP, LVEDP, LV ?dP/
dt, LV ?dP/dt, mean BP, heart rate, SV, SW, ejection fraction, and CaO in BALB/c mice. CO, control; aDOX, DOX-treated (single dose
of 25 mg/kg IP); CO?FP15, control treated with FP15 (1 mg · kg?1· d?1PO for 5 days); aDOX?FP15, treated with DOX (single dose of
25 mg/kg IP) and FP15 (1 mg · kg?1· d?1PO for 5 days); cDOX, DOX-treated (3 doses of 9 mg/kg IP every 10th day for 25 days),
cDOX?FP15, treated with DOX (3 doses of 9 mg/kg IP every 10th day for 25 days) and FP15 (1 mg · kg?1· d?1PO for 25 days). He-
modynamic parameters were measured 5 (aDOX) or 25 (cDOX) days after DOX administration. Results are mean?SEM of 10 to 14
experiments in each group. *P?0.05 vs CO; #P?0.05 vs aDOX or cDOX.
February 18, 2003
Figure 2. Effects of pharmacological inhibition or genetic deletion of iNOS gene on DOX-induced acute cardiac dysfunction. Effect of
DOX, AG, and genetic deletion of iNOS on LVSP, LVEDP, LV ?dP/dt, LV ?dP/dt, mean BP, heart rate, SV, SW, ejection fraction, and
CaO in mice. iNOS?/?, control; iNOS?/?, control; iNOS?/??AG, control treated with AG (100 mg · kg?1· d?1IP); iNOS?/??DOX, iNOS?/?
treated with DOX (single dose of 25 mg/kg IP); iNOS?/??DOX?AG, iNOS?/?treated with DOX (single dose of 25 mg/kg IP) and AG
(100 mg · kg?1· d?1IP); iNOS?/??DOX, iNOS?/?treated with DOX (single dose of 25 mg/kg IP). Hemodynamic parameters were mea-
sured 5 days after DOX administration. Results are mean?SEM of 8 to 11 experiments in each group. *P?0.05 vs iNOS?/?or iNOS?/?;
#P?0.05 vs iNOS?/?DOX.
Pacher et al Role of Peroxynitrite in Doxorubicin Cardiotoxicity
Xanthine Oxidase Assay and NAD(P)H
In heart samples from DOX-treated mice, NADH- and
NADPH-driven increases in lucigenin chemiluminescence
were significantly greater than in samples from control mice
(Figure 3B). FP15 treatment did not significantly decrease
NADH- and NADPH-driven signal, consistently with the
concept that it primarily intercepts the reactions of peroxyni-
trite, which is downstream from the formation of superoxide
(Figure 3B). Xanthine oxidase seemed to be a minor source of
superoxide in each group (Figure 3B).
Five days after DOX injection, there was a significant
increase in cardiac nitrotyrosine formation (a marker of
peroxynitrite formation or, more generally, of nitrosative
stress). As expected, nitrotyrosine immunoreactivity was
attenuated by FP15 (Figure 4).
Serum LDH and CK
Serum LDH and CK activities were significantly elevated 48
hours after DOX injection compared with the activities
measured in the control mice (Figure 5, A and B). FP15
significantly attenuated the DOX-induced elevations in serum
LDH and CK activities, indicative of reduced myocardial
necrosis (Figure 5, A and B).
On the gelatin zymography gels, only 1 band was detected with
increase of 327?37% (P?0.01, n?3) in MMP activity in hearts
165?37% of control) (Figure 5C).
Figure 6 shows the results of acute (A–C) and chronic (D)
survival experiments. At 0.1 and 0.3 or 1 mg/kg FP15, a
significant protection was noted against DOX-induced mortality
in both acute (Figure 6A) and chronic (Figure 6D) models.
To determine the sources of NO that contribute to per-
oxynitrite formation and associated cytotoxicity, we used a
combined approach (pharmacological inhibition and geneti-
cally deficient mice). Significant protection against mortality
was seen with the iNOS inhibitor AG (100 mg · kg?1· d?1IP;
Figure 6B). In contrast, the primarily constitutive NOS
inhibitor L-NAME (10 and 20 mg · kg?1· d?1IP; Figure 6B)
significantly increased mortality. There was no difference in
the survival of iNOS?/?and iNOS?/?mice treated with DOX;
L-NAME (20 mg · kg?1· d?1IP) further aggravated DOX-
induced mortality in iNOS?/?mice (Figure 6C).
Figure 3. Effects of FP15 on DOX-
induced MDA formation (A) and
NAD(P)H- or xanthine oxidase–driven
superoxide generation in heart (B). A,
MDA was measured from heart homoge-
nates 48 hours after treatment with DOX
(25 mg/kg IP) or DOX (25 mg/kg
IP)?FP15 (1 mg · kg?1· d?1PO). Hearts
of untreated mice were used as controls
(CO). Results are mean?SEM of 6
experiments in each group. *P?0.05 vs
CO; #P?0.05 vs DOX. B, Lucigenin
chemiluminescence was measured after
addition of NADH (10?4mol/L), NADPH
(10?4mol/L), or xanthine (10?4mol/L) to
heart homogenates 48 hours after treatment with DOX (25 mg/kg IP) or DOX (25 mg/kg IP)?FP15 (1 mg · kg?1· d?1PO). Hearts of
untreated mice were used as controls (CO). Results are mean?SEM of 6 to 10 experiments in each group. *P?0.05 vs CO.
Figure 4. Evidence of increased cardiac
NT formation 5 days after DOX injection;
effect of FP15. Immunohistochemical
staining for NT, an indicator of peroxyni-
trite formation, in control (A), DOX-
treated (B) and FP15?DOX-treated (C)
mouse hearts. B, Widespread NT forma-
tion in myocytes in mice 5 days after
DOX injection (25 mg/kg IP). Treatment
with FP15 for 5 days (10 mg · kg?1· d?1
PO.) C, Reduced NT formation in mouse
hearts treated with DOX. Similar immu-
nohistochemical profiles were seen in
n?5 to 6 hearts per group.
February 18, 2003
Effect of FP15 on Tumor Growth and
Antineoplastic Effect of DOX
FP15, at the highest dose used (1 mg · kg?1· d?1PO), was
tested on tumor growth and on the antineoplastic activity of
DOX, and it failed to affect these parameters, indicating that
peroxynitrite formation does not represent an important
mechanism for DOX-induced antitumor effects in the present
experimental models (Figure 7, A and B).
Effects of FP 15 on Myocardial Damage Produced
by Transient Coronary Artery Ligation
To test whether the cardioprotective effect of FP15 extends to
other forms of myocardial injury associated with peroxyni-
trite generation, a rat model of acute myocardial infarction
was used. The area at risk was comparable in vehicle and
FP15 groups (45.9?1.7% versus 46.5?4.3%, respectively).
Infarct size was significantly reduced by FP15 (vehicle,
54.2?2.9%; FP15, 40.9?4.3% of area at risk; P?0.018)
DOX continues to be an effective and widely used broad-
spectrum chemotherapeutic agent. However, its clinical use is
limited because of its serious dose-dependent cardiotoxic-
ity.1–4Clinical and experimental investigations suggested that
increased oxidative stress associated with an impaired anti-
oxidant defense status plays a critical role in subcellular
remodeling, calcium-handling abnormalities, alteration of
cardiac energetics, and subsequent cardiomyopathy and heart
failure associated with DOX treatment.5,6,8–11,22Increased
iNOS expression and nitrotyrosine formation have been
shown in cardiomyocytes of mice 5 days after a single dose
Our results indicate that peroxynitrite is formed in the heart
after DOX exposure and plays a pathogenetic role in the
development of acute and chronic DOX-induced heart failure.
Treatment with the peroxynitrite decomposition catalyst
FP15 attenuated the development of cardiac dysfunction,
increased survival, and reduced the DOX-induced increase in
serum LDH and CK activities, consistent with protection
against peroxynitrite-mediated myocyte necrosis. FP15 also
abolished tyrosine nitration in the hearts of DOX-treated
animals. Nitrotyrosine was initially considered a specific
marker of peroxynitrite generation. Now it is clear that other
pathways can sometimes also induce tyrosine nitration.23
Thus, nitrotyrosine is now generally considered a collective
index of reactive nitrogen species.23,24Nevertheless, the
increase in nitrotyrosine in myocytes of DOX-treated mice
and its abolishment by FP15 suggest that a causative link
exists between oxidative and nitrosative stress and cardiotox-
icity of DOX. FP15 also prevented DOX-induced cardiac
lipid peroxidation and MMP activation. MMP contributes
importantly to the development of various pathophysiological
conditions, including dilated cardiomyopathy, congestive
heart failure, and reperfusion injury.25–28Oxidative stress
causes tissue injury through activation of the precursors of
MMPs (proMMPs). The activation of proMMPs is triggered
by peroxynitrite generation via an extensive S-glutathiolation
reaction.29By inhibiting this reaction, peroxynitrite decom-
position catalysts may reduce MMP activation. In addition to
direct oxidation, peroxidation, and nitration reactions and
MMP activation, likely additional downstream cytotoxic
mechanisms elicited by peroxynitrite during DOX-induced
cardiac injury include DNA injury and activation of the
nuclear enzyme poly(ADP-ribose) polymerase, as well as the
inhibition of myofibrillar CK.10–14,30
Peroxynitrite is formed from the reaction of superoxide
anion and NO. Our results indicate that NAD(P)H oxidase–
dependent superoxide generation but not xanthine oxidase
upregulation contributes to the DOX-induced increased oxi-
dative stress in the myocardium. The cardiac mitochondria
may represent additional sources of superoxide and other
oxygen free radicals.3,4,6With regard to the source of NO, low
levels of constitutively produced NO are present in the heart
under all conditions. Upregulation of iNOS may represent an
additional source of NO during DOX cardiotoxicity.10,11An
inhibitor of iNOS, AG, as well as genetic deletion of the
Figure 5. Effects of FP15 on DOX-induced
increase in serum LDH (A) and CK (B)
activities and MMP activation (C) in hearts.
A and B, CO, control; DOX, DOX-treated
(single dose of 25 mg/kg IP); DOX?FP15,
treated with DOX (single dose of 25 mg/kg
IP) and FP15 (10 mg · kg?1· d?1PO for 5
days). LDH and CK activities, indirect
indexes of myocardial tissue damage,
were measured 48 hours after DOX admin-
istration. Results are mean?SEM of 6
experiments. *P?0.05 vs CO; #P?0.05 vs
DOX. C, On gelatin zymography gels, only
1 band was detected with a molecular
weight of 34 kDa. Densitometric analysis
of these bands showed significant
increase of MMP activity in hearts from
DOX-treated mice compared with controls
(n?3 in each groups). FP15 treatment
resulted in significant reduction in MMP
activity (n?3). Hearts were assayed after
48 hours of DOX or DOX?FP15 treatment.
Hearts of untreated mice were used as
Pacher et al Role of Peroxynitrite in Doxorubicin Cardiotoxicity
iNOS gene, preserved cardiac performance in DOX-treated
animals and in the case of AG also improved survival in this
very severe model. In agreement with our results, Mostafa et
al31demonstrated in a chronic rat model that AG given
concurrently with DOX normalized LDH and lipid peroxida-
tion. Furthermore, AG reduced the mortality and improved
the histopathology of the DOX-treated heart. In sharp con-
trast, an inhibitor of constitutive NOS, L-NAME, aggravated
DOX-induced mortality in both BALB/c and iNOS?/?mice.
On the basis of these findings, we hypothesize that much of
the NO that contributes to peroxynitrite formation is derived
from iNOS. The detrimental effects of L-NAME are probably
related to the fact that endothelial NOS–derived NO is a
maintainer of basal myocardial blood flow and its inhibition
leads to severe cardiac ischemia.32A multitude of compounds
that modulate endogenous antioxidant systems or exert anti-
Figure 6. Effects of FP15, AG, L-NAME,
or genetic deletion of iNOS on survival in
a DOX-induced acute (A–C) or chronic
(D) heart failure models in mice. A, Effect
of various doses of FP15 (0.03, 0.1, and
0.3 mg · kg?1· d?1PO) on DOX-induced
mortality (25 mg/kg IP) in mice. B, Effect
of various doses of AG (50 or 100 mg ·
kg?1· d?1IP) or L-NAME (10 or 20 mg ·
kg?1· d?1IP) on DOX-induced mortality
(25 mg/kg IP) in mice. C, Effect of
genetic deletion of iNOS?L-NAME (20
mg · kg?1· d?1IP) on DOX-induced mor-
tality (25 mg/kg IP) in mice. D, Effect of
various doses of FP15 (0.03, 0.1, and 1
mg · kg?1· d?1PO) on DOX-induced
chronic mortality (3 doses of 9 mg · kg?1
· d?1IP every 10 days) in mice.
Figure 7. Effects of FP15 on mouse
breast carcinoma growth and antineo-
plastic effect of DOX. A, CO, controls;
CO?FP15, control treated with FP15 (1
mg · kg?1· d?1PO); DOX, DOX-treated
(twice 4 mg · kg?1· wk?1IP);
DOX?FP15, treated with DOX (twice 4
mg · kg?1· wk?1IP) and FP15 (1 mg ·
kg?1· d?1PO). Tumor growth was indi-
vidually followed in all mice, and tumor
diameters (x, y, and z) were measured
twice a week after initiation of treat-
ments. Tumor size was calculated in
cubic millimeters and expressed as per-
centage of increase over time compared
with initial size at start of treatment (day
0). Results are mean?SEM of 10 mice in
each group. *P?0.05 vs CO. B, Repre-
sentative pictures show primary solid
breast carcinomas in mice 2 weeks after
initial treatment. Scale is in millimeters.
February 18, 2003
oxidant properties have been proposed for the prevention of
DOX-induced cardiotoxicity.4,8,9,33,34Nevertheless, the pre-
vention and treatment of DOX-induced cardiomyopathy re-
mains an unresolved clinical problem. Preclinical experimen-
tal and clinical studies have shown that the iron-chelating
agent dexrazoxane is protective against anthracycline cardio-
toxicity in various animal models33and humans.4The thiol
compound amifostine is also in clinical use.34However,
because thiols react very slowly with peroxynitrite,35it is
unlikely that thiol compounds (or traditional antioxidants)
could be applied at sufficiently high doses to interfere with
the rapid reactivity of peroxynitrite. Although we did not
directly compare the efficacy of dexrazoxane or amifostine in
the present experimental models, overall, the efficacy of
FP15 in our model seems to be comparable to or better than
the efficacy of many previously published approaches.33,34,36
The fact that FP15 does not interfere with the antitumor
actions of DOX provides an additional indication that potent
peroxynitrite decomposition catalysts should be tested in
additional preclinical and clinical models of DOX toxicity.
FP15 is an N-PEGylated-2-pyridyl iron porphyrin that has
shown superior performance as a peroxynitrite decomposition
catalyst.21Endogenous reducing agents such as ascorbate and
glutathione react too slowly with peroxynitrite to complete
with trans-membrane diffusion and reactions with metal
centers.37,38Because peroxynitrite reacts very efficiently with
synthetic metalloporphyrins,39compounds in this class have
been investigated as peroxynitrite decomposition catalysis.40
Several water-soluble iron41and manganese38porphyrins
have shown very high rates of reaction with peroxynitrite.
One such porphyrin, FeTMPS, has been shown to reduce
carrageenan-induced paw edema and cause reductions in
inflammatory mediator production.42-44
Many pathophysiological conditions of the heart are asso-
ciated with peroxynitrite formation, including acute myocar-
dial infarction, chronic ischemic heart failure, and diabetic
cardiomyopathy.13,15,21,30It seems that peroxynitrite decom-
position catalysts improve cardiac function and overall out-
come in these models. For instance, FP15 reduced myocardial
necrosis in our present rat model of acute myocardial infarc-
tion (present study) as well as in a recent porcine study.45
Furthermore, FP15 significantly improved cardiac function in
a diabetic cardiomyopathy model.21These observations, cou-
pled with the protective effect of FP 15 against DOX-induced
cardiotoxicity reported here, support the concept that per-
oxynitrite is a major mediator of myocardial injury in various
pathophysiological conditions, and its effective neutralization
can be of significant therapeutic benefit.
This work was supported by grants from the National Institutes of
Health: R01-HL-59266 and R43-CA-097559 (to Dr Szabó); R-43-
CA-95807 (to Dr Pacher); R01-GM-36928 (to Dr Groves) and
R01-HL-43023 (to Dr Wolin). Péter Bai was supported by Hungar-
ian Science and Technology (TET) Foundation grant 27/MO/01 and
Eötvös Fellowships, Dr Ungvári by a fellowship from the American
Heart Association, New York State Affiliate Inc, 0020144T, and Dr
Virág by the grant Hungarian Scientific Research Fund (OTKA)
T035182 and the Bolyai Scholarship of the Hungarian Academy of
Sciences. Dr Pacher is on leave from the Institute of Pharmacology
and Pharmacotherapy, Semmelweis University, Budapest, Hungary.
1. Blum RH, Carter SK. Adriamycin: a new anticancer drug with significant
clinical activity. Ann Intern Med. 1974;180:249–259.
2. Young RC, Ozols RF, Myers CE. The anthracycline antineoplastic drugs.
N Engl J Med. 1981;305:139–153.
3. Singal PK, Deally CM, Weinberg LE. Subcellular effects of adriamycin
in the heart: a concise review. J Mol Cell Cardiol. 1987;19:817–828.
4. Singal PK, Iliskovic N. Doxorubicin-induced cardiomyopathy. N Engl
J Med. 1998;339:900–905.
5. Myers CE, McGuire WP, Liss RH, et al. Adriamycin: the role of lipid
peroxidation in cardiac toxicity and tumor response. Science. 1977;197:
6. Doroshow JH, Davies KJ. Redox cycling of anthracyclines by cardiac
mitochondria, II: formation of superoxide anion, hydrogen peroxide, and
hydroxyl radical. J Biol Chem. 1986;261:3068–3074.
7. Liu LF. DNA topoisomerase poisons as antitumor drugs. Annu Rev
8. Siveski-Iliskovic N, Kaul N, Singal PK. Probucol promotes endogenous
antioxidants and provides protection against adriamycin-induced cardio-
myopathy in rats. Circulation. 1994;89:2829–2835.
9. Li T, Singal PK. Adriamycin-induced early changes in myocardial anti-
oxidant enzymes and their modulation by probucol. Circulation. 2000;
10. Weinstein DM, Mihm MJ, Bauer JA. Cardiac peroxynitrite formation and
left ventricular dysfunction following doxorubicin treatment in mice.
J Pharmacol Exp Ther. 2000;294:396–401.
11. Mihm MJ, Yu F, Weinstein DM, et al. Intracellular distribution of
peroxynitrite during doxorubicin cardiomyopathy: evidence for selective
impairment of myofibrillar creatine kinase. Br J Pharmacol. 2002;135:
12. Mihm MJ, Coyle CM, Schanbacher BL, et al. Peroxynitrite induced
nitration and inactivation of myofibrillar creatine kinase in experimental
heart failure. Cardiovasc Res. 2001;49:798–807.
13. Pacher P, Liaudet L, Mabley JG, et al. Pharmacological inhibition of
poly(ADP-ribose) polymerase may represent a novel therapeutic
approach in chronic heart failure. J Am Coll Cardiol. 2002;40:
14. Pacher P, Liaudet L, Bai P, et al. Activation of poly(ADP-ribose) poly-
merase contributes to development of doxorubicin-induced heart failure.
J Pharmacol Exp Ther. 2002;300:862–867.
15. Pacher P, Liaudet L, Soriano FG, et al. The role of poly(ADP-ribose)
polymerase in the development of cardiovascular dysfunction in diabetes
mellitus. Diabetes. 2002;51:514–521.
16. Liaudet L, Murthy KG, Mabley JG, et al. Comparison of inflammation,
organ damage, and oxidant stress induced by Salmonella enterica serovar
Muenchen flagellin and serovar Enteritidis lipopolysaccharide. Infect
17. Mohazzab KM, Kaminski PM, Wolin MS. Lactate and PO2modulate
superoxide anion production in bovine cardiac myocytes: potential role of
NADH oxidase. Circulation. 1997;96:614–620.
18. Szabó C, Zanchi A, Komjáti K, et al. Poly(ADP-ribose) polymerase is
activated in subjects at risk of developing type II diabetes and is asso-
ciated with impaired vascular reactivity. Circulation. 2002;106:
19. Connolly EM, Harmey JH, O’Grady T, et al. Cyclo-oxygenase inhibition
reduces tumour growth and metastasis in an orthotopic model of breast
cancer. Br J Cancer. 2002;87:231–237.
20. Liaudet L, Yang Z, Al-Affar EB, et al. Myocardial ischemic precondi-
tioning in rodents is dependent on poly (ADP-ribose) synthetase. Mol
21. Szabó C, Mabley JG, Moeller SM, et al. FP 15, a novel, potent per-
oxynitrite decomposition catalyst: in vitro cytoprotective actions and
protection against diabetes mellitus and diabetic cardiovascular compli-
cations. Mol Med. 2002;8:571–580.
22. Siveski-Iliskovic N, Hill M, Chow DA, et al. Probucol protects against
adriamycin cardiomyopathy without interfering with its antitumor effect.
23. Eiserich JP, Hristova M, Cross CE, et al. Formation of nitric oxide-
derived inflammatory oxidants by myeloperoxidase in neutrophils.
24. Halliwell B. What nitrates tyrosine? Is nitrotyrosine specific as a
biomarker of peroxynitrite formation in vivo? FEBS Lett. 1997;411:
Pacher et al Role of Peroxynitrite in Doxorubicin Cardiotoxicity
25. Mann DL, Spinale FG. Activation of matrix metalloproteinases in the Download full-text
failing human heart: breaking the tie that binds. Circulation. 1998;98:
26. Thomas CV, Coker ML, Zellner JL, et al. Increased matrix metallopro-
teinase activity and selective upregulation in LV myocardium from
patients with end-stage dilated cardiomyopathy. Circulation. 1998;97:
27. Cheung PY, Sawicki G, Wozniak M, et al. Matrix metalloproteinase-2
contributes to ischemia-reperfusion injury in the heart. Circulation. 2000;
28. Spinale FG. Matrix metalloproteinases: regulation and dysregulation in
the failing heart. Circ Res. 2002;90:520–530.
29. Okamoto T, Akaike T, Sawa T, et al. Activation of matrix metallopro-
teinases by peroxynitrite-induced protein S-glutathiolation via disulfide
S-oxide formation. J Biol Chem. 2001;276:29596–29602.
30. Ferdinandy P, Danial H, Ambrus I, et al. Peroxynitrite is a major con-
tributor to cytokine-induced myocardial contractile failure. Circ Res.
31. Mostafa AM, Nagi MN, Al Rikabi AC, et al. Protective effect of amino-
guanidine against cardiovascular toxicity of chronic doxorubicin
treatment in rats. Res Commun Mol Pathol Pharmacol. 1999;106:
32. Benyo Z, Kiss G, Szabo C, et al. Importance of basal nitric oxide
synthesis in regulation of myocardial blood flow. Cardiovasc Res. 1991;
33. Imondi AR. Preclinical models of cardiac protection and testing for
effects of dexrazoxane on doxorubicin antitumor effects. Semin Oncol.
34. Nelson MA, Frishman WH, Seiter K, et al. Cardiovascular considerations
with anthracycline use in patients with cancer. Heart Dis. 2001;3:
35. Beckman JS, Koppenol WH. Nitric oxide, superoxide, and peroxynitrite:
the good, the bad, and ugly. Am J Physiol 1996;271:C1424–1437.
36. Herman EH, Ferrans VJ. Preclinical animal models of cardiac pro-
tection from anthracycline-induced cardiotoxicity. Semin Oncol. 1998;
37. Lee J, Hunt JA, Groves JT. Rapid decomposition of peroxynitrite by
manganese porphyrin-antioxidant redox-couples. Bioorg Med Chem Lett.
38. Marla SS, Lee J, Groves JT. Perioxynitrite rapidly permeates phos-
pholipid membranes. Proc Natl Acad Sci U S A. 1997;94:14243.
39. Groves JT, Marla SS. Peroxynitrite-induced DNA strand scission
mediated by a manganese porphyrin. J Am Chem Soc. 1995;117:9578.
40. Groves JT. Peroxynitrite: reactive, invasive and enigmatic. Curr Op
Chem Biol. 1999;3:226–235.
41. Lee J, Hunt JA, Groves JT. Mechanisms of iron porphyrin reactions with
peroxynitrite. J Am Chem Soc. 1998;120:7493–7501.
42. Misko TP, Highkin MK, Veenhuizen AW, et al. Characterization of the
cytoprotective action of peroxynitrite decomposition catalysts. J Biol
43. Misko TP, Salvemini D, Wang Z-Q, et al. Peroxynitrite decomposition
catalysts: therapeutics for peroxynitrite-mediated pathology. Proc Nat
Acad Sci U S A. 1998;95:2659–2663.
44. Shimanovich R, Groves JT. Mechanisms of peroxynitrite decomposition
catalyzed by FeTMPS, a bioactive sulfonated iron porphyrin. Arch
Biochem Biophys. 2001;387:307–317.
45. Bianchi C, Wakiyama H, Faro R, et al. A novel peroxynitrite decompo-
sition catalyst (FP-15) reduces myocardial infarct size in an in vivo
peroxynitrite decomposer and acute ischemia-reperfusion in pigs. Ann
Thorac Surg. 2002;74:1201–1207.
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