Comparison of cardioprotective efficacy of two thromboxane A2 receptor antagonists

Article abstract of DOI:10.1097/00005344-200303000-00018

The purpose of the current study was to compare the efficacy of two structurally unrelated thromboxane A₂ (TXA₂) receptor antagonists, KT2-962 and daltroban (BM 13.505), in a dog model of myocardial ischemia/reperfusion injury. Pentobarbital-anesthetized dogs were subjected to left circumflex coronary artery occlusion for 90 minutes followed by 5 hours of reperfusion. Vehicle, KT2-962 (10 mg/kg), or daltroban (10 mg/kg) were administered as intravenous boluses 10 minutes before coronary occlusion. Systemic hemodynamics were measured throughout the experiments and regional myocardial blood flow was measured by the radioactive microsphere technique. At the end of the reperfusion period, myocardial infarct size was quantified by staining with triphenyltetrazolium chloride. Neither KT2-962 nor daltroban significantly altered heart rate, mean arterial blood pressure, or regional myocardial blood flow. The content of myeloperoxidase activity in the ischemic/reperfused tissue, an index of neutrophil infiltration, was not significantly different among the three treatment groups. However, administration of KT2-962, but not daltroban, significantly reduced the incidence of ventricular fibrillation during the ischemic period and significantly reduced myocardial infarct size expressed as a percentage of the risk region (approximately 40%). Subsequent in-vitro assays using electron spin resonance spectroscopy demonstrated that KT2-962 inhibited the formation of hydroxyl radicals, whereas daltroban had no effect. These results suggest that the beneficial effects of KT2-962 may be due to its direct free radical scavenging properties rather than its ability to block TXA₂ receptors.

Full text of DOI:10.1097/00005344-200303000-00018

ORIGINAL ARTICLE
Comparison of Cardioprotective Efficacy of Two
Thromboxane A2 Receptor Antagonists
*Zhi-Dong Ge, *‡John A. Auchampach, †‡Galen M. Piper, and *Garrett J. Gross
n experimental animals and humans, myocardial ische-
Abstract: The purpose of the current study was to compare the
Imia and reperfusion results in increased production and
efficacy of two structurally unrelated thromboxane A2 (TXA2)
release of thromboxane A2 (TXA2) (1–6), a metabolite of
receptor antagonists, KT2–962 and daltroban (BM 13.505), in a
arachidonic acid generated by its sequential metabolism
dog model of myocardial ischemia/reperfusion injury. Pentobar-
by cyclooxygenases and thromboxane synthase. Little is
known, however, regarding the roles of TXA2 in the patho-
genesis of ischemia/reperfusion injury. Exogenous appli-
cation of TXA2 or its analogues has been shown to be po-
tent inducers of both platelet aggregation and arterial
vasoconstriction (7,8). Accordingly, the endogenous pro-
duction of TXA2 could theoretically promote reperfusion
injury by reducing nutritional blood flow to the ischemic
myocardium due to regional vasoconstriction and neutro-
phil–endothelial adherence. Indeed, exogenous adminis-
tration of TXA2 has been shown to worsen ischemia/reper-
fusion injury in experimental animal models (9,10).
The actions of TXA2 are mediated by thromboxane–
prostanoid (TP) receptors expressed in many different tis-
sues including the myocardium (11). Some TP receptor an-
tagonists have been reported to be cardioprotective during
ischemia/reperfusion injury (12–18), but an absence of
protection with TP antagonists has also been reported in
other studies (19). The cardioprotective efficacy of TP re-
ceptor antagonists appears to vary with the drugs used for
reasons that remain unclear.
A large number of studies exist in the literature dem-
onstrating the development of oxidative stress due to in-
creased formation of reactive oxygen species (ROS) and
decreased antioxidant reserve in the ischemic/reperfused
myocardium (20,21). The reactive ROS include hydroxyl
and peroxy radicals, superoxide anions, and oxidants such
as H2O2 (20). Increasing evidence suggests that hydroxyl
radicals are important contributors to myocardial damage
(22–24) and that scavenging hydroxyl radicals by thera-
peutic agents provides protection against myocardial
ischemia/reperfusion injury (21,25–28).
bital-anesthetized dogs were subjected to left circumflex coronary
artery occlusion for 90 minutes followed by 5 hours of reperfu-
sion. Vehicle, KT2–962 (10 mg/kg), or daltroban (10 mg/kg)
were administered as intravenous boluses 10 minutes before
coronary occlusion. Systemic hemodynamics were measured
throughout the experiments and regional myocardial blood flow
was measured by the radioactive microsphere technique. At the
end of the reperfusion period, myocardial infarct size was quan-
tified by staining with triphenyltetrazolium chloride. Neither
KT2–962 nor daltroban significantly altered heart rate, mean ar-
terial blood pressure, or regional myocardial blood flow. The
content of myeloperoxidase activity in the ischemic/reperfused
tissue, an index of neutrophil infiltration, was not significantly
different among the three treatment groups. However, adminis-
tration of KT2–962, but not daltroban, significantly reduced the
incidence of ventricular fibrillation during the ischemic period
and significantly reduced myocardial infarct size expressed as a
percentage of the risk region (approximately 40%). Subsequent
in-vitro assays using electron spin resonance spectroscopy dem-
onstrated that KT2–962 inhibited the formation of hydroxyl radi-
cals, whereas daltroban had no effect. These results suggest that
the beneficial effects of KT2–962 may be due to its direct free
radical scavenging properties rather than its ability to block
TXA2 receptors.
Key Words: Hydroxyl radicals—Ischemia—KT2–962-TXA2 re-
ceptor antagonist—Reperfusion.
( J Cardiovasc Pharmacol ™ 2003;41: 481–488)
*Department of Pharmacology and Toxicology, †Division of Transplant
Surgery, and ‡Cardiovascular Research Center, Medical College of
Wisconsin, Milwaukee, Wisconsin.
Received June 18, 2001; accepted July 31, 2002.
Address correspondence to Dr. Garrett J. Gross at Department of Phar-
macology and Toxicology, 8701 Watertown Plank Road, Medical
College of Wisconsin, Milwaukee, WI 53226. E-mail: ggross@mcw.edu
Supported in part by grants HL 08311 and HL 60051 from the National
Institutes of Health (Bethesda, MD, U.S.A.), by grant 9630083N from
the National American Heart Association (Dallas, TX, U.S.A.), and by
funds from Kotobuki-Seiyko (Osaka, Japan). Kotobuki-Seiyko pro-
vided the sample of KT2–962.
In the current study, we investigated the efficacy of
two structurally unrelated TP receptor antagonists, KT2–
962 [6-isopropyl-3-[4-(p-chlorobenzene-sulfonylamino)-
butyl]-azulene-1-sulfonic acid] and daltroban ([4-[2-(4-
chlorobenzene-sulfonylamino) ethyl]-benzene acetic acid];
BM 13.505), on infarct size in a clinically relevant dog
J Cardiovasc Pharmacolீ • Volume 41, Number 3, March 2003
481

Z.-D. Ge et al.
J Cardiovasc Pharmacolீ • Volume 41, Number 3, March 2003
model of myocardial ischemia/reperfusion injury. Interest-
ingly, we found that the azulene derivative KT2–962, but
not the sulfonamide derivative daltroban, decreased the in-
cidence of ventricular fibrillation and significantly reduced
myocardial infarct size. Subsequent in-vitro assays using
electron spin resonance (ESR) spectroscopy demonstrated
that KT2–962 is a potent hydroxyl radical scavenger.
These results suggest that KT2–962 is effective at protect-
ing against ischemia/reperfusion injury by scavenging free
radicals rather than by blocking TP receptors.
Study Protocol
The experimental model of infarction, assessment of
infarct size, and assessment of neutrophil accumulation by
measuring myeloperoxidase activity are as described pre-
viously in detail (29–31). All dogs were subjected to 90
minutes of LCX coronary artery occlusion followed by 5
hours of reperfusion. During reperfusion, the occluder was
used to maintain blood flow at preischemic levels for the
first 30 minutes of reperfusion to attenuate the hyperemic
response. Hemodynamics were measured continuously
throughout the protocol and regional myocardial blood
flow was assessed using radioactive microspheres (29–31).
Doses of vehicle (0.9% NaCl), KT2–962 (10 mg/kg), or dal-
troban (10 mg/kg) were administered as intravenous bo-
luses 15 minutes before the coronary occlusion. These
doses of the antagonists have been used previously to ef-
fectively block TP receptors (18,32–35). We also deter-
mined in preliminary studies that these doses of the antago-
nists abolished completely the hypertensive effects of
intravenously administered U46619 (10 µg) for at least 5
hours following administration. Dogs that developed ven-
tricular fibrillation were defibrillated using 15–20 J of cur-
rent applied directly to the heart. Strict criteria were used to
ensure that the animals used in the data analysis were
healthy and exposed to similar degrees of ischemia. Dogs
were excluded if heartworms were discovered, subendo-
cardial blood flow was greater than 0.15 ml/min/g, or
more than three consecutive attempts to defibrillate were
required. At the end of the experiments, infarct size was
expressed as a percentage of the risk region following dual
staining with triphenyltetrazolium chloride and patent
blue dye (29–31). Following infarct size assessment, my-
eloperoxidase (MPO) activity was determined in myocar-
dial samples obtained from two areas within the area at risk
(AAR): the area directly adjacent to the infarcted area (bor-
der zone) and the distant noninfarcted area (29).
METHODS
Anesthetized Dog Preparation
All the procedures in the current study conformed to
the principles outlined in the Guide for the Care and Use of
Laboratory Animals published by the National Institutes of
Health (NIH Publication No. 85–23, revised 1996). The
experimental preparation is the same as that described pre-
viously (29–31). Briefly, adult mongrel dogs were anesthe-
tized with a mixture of sodium pentobarbital (15 mg/kg
intravenously) and barbital sodium (200 mg/kg intrave-
nously) and connected to a respirator. Aortic and left ven-
tricular pressures were measured by inserting a double-
pressure-transducer–tipped catheter (PC 771; Millar
Instruments, Houston, TX, U.S.A.) into the aorta and left
ventricle via the left carotid artery. Left ventricular dP/dt
was recorded by electronic differentiation of the left ven-
tricular pulse pressure and heart rate was determined by a
tachometer (Grass Instruments, Quincy, MA, U.S.A.). The
right femoral vein and artery were cannulated for drug
administration and for withdrawal of reference blood
flow for the measurement of myocardial tissue blood flow,
respectively.
The heart was exposed through a midline ster-
notomy and a left thoracotomy was performed in the fifth
intercostal space. A segment (1.0–1.5 cm) of the left cir-
cumflex (LCX) coronary artery was dissected free from
surrounding tissue at the border of the left atrial append-
age, and a calibrated electromagnetic flow probe (Statham
5P 7517; Gould-Statham, Cleveland, OH, U.S.A.) was
placed around the vessel to measure LCX coronary flow. A
micrometer-driven mechanical occluder was placed distal
to the flow probe and the occluder. The occluder was used
to zero the flow probe and to later occlude the artery. A
catheter was placed into the left atrium via the atrial ap-
pendage for the injection of radioactive microspheres. The
heart was paced at 150 beats/min (2.5 Hz) with rectangular
pulses of 4-millisecond duration and a voltage of twice
threshold via bipolar electrodes clipped to the atrium. The
electrocardiogram was monitored using limb lead II. All
measurements were recorded throughout the experiment
on a Grass model 7 polygraph (Grass Instruments).
Analysis of Hydroxy Radical Scavenging
Properties of KT2–962 and Daltroban
We examined the direct hydroxyl radical scavenging
properties of the two TP receptor antagonists using an in-
vitro ESR spectroscopy assay. For these studies, hydroxyl
radicals were generated by a Fenton reaction consisting of
1.2% H2O2 and 50 µM FeSO4 (prepared in nitrogen-
aerated 0.1 N HCl) in 50 mM phosphate buffer (pH 7.4
containing 20 µM EDTA) in the presence or absence of
increasing concentrations of KT2–962 or daltroban. Hy-
droxyl radicals were trapped by using 5,5-dimethypyr-
roline-N-oxide (DMPO; 3 mM). In preliminary studies, we
have determined that this assay is specific for trapping hy-
droxyl radicals as the DMPO-OH adduct is prevented in
the presence of 2% ethanol, a known hydroxyl radical scav-
enger. Samples were placed in the loop gap resonator of a
482
© 2003 Lippincott Williams & Wilkins

J Cardiovasc Pharmacolீ • Volume 41, Number 3, March 2003
TXA2 Receptor Antagonists and Cardioprotection
Varian E-109 spectrometer (Varian Medical Systems, Palo
Alto, CA, U.S.A.) at room temperature. Spectra were ob-
tained using 9.1 GHz of microwave frequency, 2 mW of
microwave power, 100 kHz of modulation frequency, 2.5
G of modulation amplitude, and a field set of 3,280 G with
a scan range of 100 G. The samples were scanned for 4
minutes with a time constant of 0.5 seconds.
Statistical Analysis
The results are expressed as means SEM. Statistical
differences among the groups in hemodynamic variables
and collateral blood flow were calculated by a two-way
analysis of variance (ANOVA) for repeated measures and
Fisher least significant difference test. A one-way ANOVA
and Fisher least significant difference test was used to de-
termine differences among groups with respect to infarct
size and MPO activity. Linear regression analysis was used
to determine the correlation between transmural collateral
blood flow in the ischemic area and myocardial infarct size
expressed as a percentage of the AAR. Analysis of covari-
ance, with collateral blood flow as the covariate, was used
to determine if differences in this relationship were ob-
served among the treatment groups. Differences in the in-
cidence of dogs fibrillating were determined by the Fisher
Exact test. A P value < 0.05 was considered statistically
significant.
FIG. 1. Percentage of dogs fibrillating in the control group
(n = 8) and the KT2–962–treated (n = 8) and daltroban-treated
(n = 8) groups during the 90-minute occlusion period, the
5-hour reperfusion period, and the combined total. *P < 0.05
vs. the control group by Fisher Exact test.
or LCX coronary artery blood flow among the three
groups at any of the time points measured throughout the
experiments.
Regional Myocardial Blood Flow
Regional myocardial blood flow data in the nonis-
chemic left anterior descending (LAD) coronary artery and
ischemic LCX coronary artery regions are summarized in
Table 2. In the nonischemic region, there were no signifi-
cant differences among the groups in any layer of the myo-
cardium at any time point measured. Similarly, there were
no differences in any layer of the myocardium in the
ischemic/reperfused region, indicating that both groups
were subjected to equivalent degrees of ischemia and that
neither of the TP receptor antagonists influenced collateral
blood flow.
RESULTS
Exclusions and Incidence of
Ventricular Fibrillation
A total of 30 dogs were initially included in the study.
One dog was excluded from the control group (during oc-
clusion) and one dog was excluded from the KT2–962–
treated group (during reperfusion) because of intractable
ventricular fibrillation. Four additional dogs were ex-
cluded from the study (one each from the control and dal-
troban-treated groups and two from the KT2–962–treated
group) because subendocardial blood flow exceeded the
preset limit of 0.15 ml/min/g. Thus, a total of 24 dogs were
included in data analysis (n = 8 per group).
The incidence of dogs fibrillating during the occlu-
sion period was reduced significantly (P < 0.05 by
Fisher Exact test; Fig. 1) in the dogs pretreated with KT2–
962. In this group, none of the dogs fibrillated during the
occlusion period, whereas 30% and 50% of the dogs fibril-
lated at least once during the occlusion period in the con-
trol and daltroban-treated groups, respectively.
Myocardial Infarct Size
AAR size and myocardial infarct size data are pre-
sented in Figure 2. The anatomic AAR was not signifi-
cantly different among the three experimental groups (con-
trol, 40 3%; KT2–962, 48 3%; daltroban-treated, 44
2%). However, myocardial infarct size expressed as a per-
centage of the AAR was significantly smaller in the KT2–
962–treated group (control, 41 6%; KT2–962, 26 3%;
daltroban, 47 6%).
Figure 3 shows the relationship between myocardial
infarct size expressed as a percentage of the AAR and
transmural collateral blood flow measured at 30 minutes of
the occlusion period. In all of the groups, there is an inverse
relationship between these two parameters, and the data
can be fitted by linear regression analysis. In the KT2–962–
treated group, this relationship was shifted downward
compared with both the control and daltroban-treated
groups (P < 0.05 by analysis of covariance). Analysis
of these data demonstrates that infarct size was reduced in
Hemodynamics
Systemic hemodynamic data are summarized in
Table 1. There were no significant differences in heart
rate, mean arterial blood pressure, left ventricular dP/dt,
© 2003 Lippincott Williams & Wilkins
483

Z.-D. Ge et al.
J Cardiovasc Pharmacolீ • Volume 41, Number 3, March 2003
TABLE 1. Hemodynamics variables
HR
(beats/min)
MBP
(mm Hg)
LV dP/dt
(mm Hg/sec)
CBF
(ml/min)
Preocclusion
Control (n = 8)
KT2–962 (n = 8)
BM 13.505 (n = 8)
Occlusion (30 min)
Control
156
152
154
3
3
3
102
106
102
3
5
7
2200 250
2460 260
1950 180
51
57
46
5
6
8
155
148
154
2
3
3
101
106
95
6
6
8
2150 170
2440 250
1720 130
—
—
—
KT2–962
BM 13.505
Occlusion (90 min)
Control
KT2–962
157
149
153
2
2
2
97
111
101
5
5
6
2090 203
2600 240
1800 160
—
—
—
BM 13.505
Reperfusion (3 hr)
Control
KT2–962
153
148
156
3
3
7
98
93
90 10
4
4
1750 70
1840 170
1480 180
55
41
36
8
3
5
BM 13.505
All values are mean SEM.
CBF, coronary blood flow; HR, heart rate; LV dP/dt, left ventricular dP/dt; MBP, mean arterial blood
pressure.
the KT2–962–treated group at any given degree of collat-
eral blood flow, demonstrating that the reduction in infarct
size produced by KT2–962 was not related to differences in
collateral blood flow.
among the treatment groups in either the border zone or in
the distal noninfarcted tissue.
In-vitro Analysis of Hydroxyl Radical
Scavenging Properties
In-vitro assays were performed to determine the po-
tential hydroxyl radical scavenging properties of KT2–962
and daltroban. Hydroxyl radicals were generated by H2O2
Myocardial MPO Measurements
The MPO data are summarized in Table 3. There
were no significant differences in tissue MPO activity
TABLE 2. Regional myocardial blood flow data (ml/min/gm)
Non-ischemic region (LAD)
Ischemic-reperfused region (LCX)
Epicardium
Midmyocardium
Endocardium
Epicardium
Midmyocardium
Endocardium
Preocclusion
Control (n = 8)
KT2–962 (n = 8)
BM 13.505 (n = 8)
Occlusion
1.18 0.12
1.30 0.16
1.39 0.25
1.23 0.10
1.44 0.19
1.51 0.23
1.26 0.12
1.94 0.27
1.61 0.24
0.96 0.07
1.18 0.16
1.23 0.17
1.01 0.08
1.18 0.17
1.30 0.18
1.17 0.09
1.36 0.17
1.43 0.16
Control
KT2–962
BM 13.505
1.32 0.13
1.15 0.15
1.25 0.14
1.37 0.13
1.42 0.16
1.50 0.18
1.48 0.15
1.52 0.18
1.68 0.20
0.19 0.06
0.22 0.07
0.19 0.06
0.10 0.04
0.09 0.02
0.06 0.02
0.05 0.01
0.06 0.01
0.03 0.01
Reperfusion (3 hr)
Control
KT2–962
1.01 0.12
1.06 0.07
1.16 0.24
1.13 0.12
1.24 0.10
1.48 0.28
1.23 0.15
1.24 0.13
1.54 0.27
0.90 0.09
0.90 0.09
0.84 0.21
0.86 0.17
0.68 0.17
0.66 0.17
0.70 0.15
0.70 0.14
0.75 0.26
BM 13.505
All values are mean SEM.
484
© 2003 Lippincott Williams & Wilkins

J Cardiovasc Pharmacolீ • Volume 41, Number 3, March 2003
TXA2 Receptor Antagonists and Cardioprotection
TABLE 3. Myeloperoxidase activity data
Non-infarcted area
1.1 0.4
Border zone
Control (n = 8)
KT2-962 (n = 8)
BM 13.505 (n = 8)
6.1 1.2
5.0 1.0
8.5 2.7
1.4 0.4
1.3 0.3
All values are mean SEM.
tude compared with the positive control of the hydroxyl
radical scavenger mannitol. In contrast, daltroban had
little effect on hydroxyl radical formation. At a concentra-
tion of 500 µM, the ESR signal was decreased by only 11
5% compared with the control (Fig. 4).
FIG. 2. Myocardial infarct size data in control (n = 8), KT2–
962–treated (n = 8), and daltroban-treated (n = 8) dogs. AAR,
area at risk; LV, left ventricle; IF, infarcted area. *P < 0.05 versus
the control group. Values are shown as mean ע SEM.
DISCUSSION
The present study compared the efficacy of two TP
receptor antagonists, KT2–962 and daltroban, on myocar-
in the presence of FeSO4 and the spin trap agent DMPO.
Using ESR spectroscopy, an ESR signal was observed con-
sisting of a quartet (1:2:2:1 intensity ratio) and a hyperfine
splitting constant of aN = aH = 14.9 Gauss, which is char-
acteristic of the trapped hydroxyl radical adduct of DMPO
(DMPO-OH; Fig. 4). The production of this radical was
reduced by the hydroxyl radical scavenger mannitol. In
addition, KT2–962 produced a concentration-dependent
reduction in the ESR signal, which was initially observed at
the threshold concentration of 25 µM and elicited a 50%
reduction at approximately 500 µM (Fig. 4). The inhibition
curve of hydroxyl radical formation by KT2–962 was
shifted to the left by approximately 1.5 orders of magni-
FIG. 3. The relationship between transmural collateral blood
flow in the ischemic area at 30 minutes of occlusion and myo-
cardial infarct size expressed as a percentage of AAR. In all
groups, there is an inverse relationship between collateral
blood flow and infarct size. Regression analysis was performed
for each group (control, y = 50 מ 74x [r = 0.44]; KT2–962,
y = 37 מ 58x [r = .73]; daltroban, 65 מ 178x [r = .78]), and
the KT2–962-treated group was found to be significantly dif-
ferent from the control group by analysis of covariance.
FIG. 4. Inhibition of DMPO-OH adduct formation by KT2–
962. A. Example showing DMPO-OH adduct formation in the
absence (control) or presence of equimolar concentrations
(500 µM) of the thromboxane receptor antagonists. B. Con-
centration–response curves of KT2–962 and mannitol to in-
hibit DMPO-OH adduct formation.
© 2003 Lippincott Williams & Wilkins
485

Z.-D. Ge et al.
J Cardiovasc Pharmacolீ • Volume 41, Number 3, March 2003
dial infarct size in an open-chest dog model of myocardial
ischemia/reperfusion injury. We found that: (i) both KT2–
962 and daltroban completely blocked the hypertensive
responses to U46619; (ii) blockade of TP receptors by both
KT2–962 and daltroban did not alter heart rate, arterial
blood pressure, regional myocardial blood flow, or MPO
activity; (iii) KT2–962, but not daltroban, decreased the
incidence of ventricular fibrillation during coronary occlu-
sion; (iv) KT2–962, but not daltroban, significantly re-
duced myocardial infarct size; and (v) KT2–962 potently
inhibited the formation of hydroxyl radicals, whereas dal-
troban had no significant effect. These results indicate that
KT2–962, but not daltroban, is an effective cardioprotec-
tive agent against irreversible ischemia/reperfusion injury.
The reason for the discrepancy in the observations be-
tween the two TP receptor antagonists on infarct size ap-
pear to be related to the fact that, in addition to blockade of
TP receptors, KT2–962 inhibits the formation of hydroxyl
radicals.
and regional myocardial blood flow, although potent and
direct actions of TXA2 on the blood vessels are well
known.
It has been reported that daltroban provided benefi-
cial effects in myocardial ischemia/reperfusion injury in
rats and cats (14,18). In the current study, however, daltro-
ban was found not to reduce the incidence of ventricular
fibrillation or reduce myocardial infarct size in dogs. This
result is consistent with a previous study (19) demonstrat-
ing that blockade of TP receptors alone does not alter myo-
cardial ischemia/reperfusion injury. Furthermore, these re-
sults are consistent with the recent observation that infarct
size is not reduced in mice lacking TP receptors (TP recep-
tor knockout mice) (19,39), suggesting that TXA2 has a
small role, if any, in the pathogenesis of cardiac
ischemia/reperfusion injury. An alternative explanation
for this unusual observation, however, is that daltroban
may not have been effective because it is a partial agonist of
TP receptors (40).
U46619, a TXA2 mimetic, is known to be a potent
agonist of TP receptors. In dogs, it has previously been
reported that systemic administration of U46619 at doses
of 0.125–0.500 µg/kg increases blood pressure (36). To de-
termine whether KT2–962 and daltroban effectively
blocked TP receptors in our study, we tested the effects of
bolus administration of U46619 (10 µg/kg) and found that
pretreatment with either of the antagonists blocked the hy-
pertensive effect completely for at least 5 hours (U46619
increased mean arterial blood pressure 12 mm Hg 2 [n =
3] in control animals but had no effect in animals treated
with either antagonist). This observation demonstrates that
KT2–962 and daltroban at doses of 10 mg/kg completely
blocked vascular TP receptors in our studies. Furthermore,
these results indicate that the use of an insufficient dose of
daltroban was not responsible for its lack of a beneficial
effect in reducing infarct size.
TXA2 is known to be a potent coronary vasoconstric-
tor (37). In the blood, it is rapidly metabolized to the stable
metabolite thromboxane B2 (TXB2). Previous studies have
reported that baseline levels of TXB2 in the femoral arterial
blood of dogs were approximately 800 pg/ml (38). After
the release of a sustained coronary artery occlusion, TXB2
levels increased to over 2,500 pg/ml for at least 2 hours
(38). In the current study, we could not observe any signifi-
cant differences in heart rate, mean arterial blood pressure,
or regional myocardial blood flow between KT2–962- or
daltroban-treated and control dogs during 90 minutes of
ischemia or the subsequent 5 hours of reperfusion. These
results are consistent with several previous studies demon-
strating that TP receptor antagonists do not alter heart rate
and blood pressure in the context of myocardial ischemia
and reperfusion (13–18). Collectively, these results suggest
that TXA2 plays a minor role in regulating hemodynamics
In the current study, we observed no differences in
collateral blood flow measured by the use of radioactive
microspheres between the control and KT2–962–treated
groups during the occlusion period. We also observed no
differences in systemic hemodynamic parameters (heart
rate, blood pressure, left ventricular dP/dt) between the
two groups of dogs. Therefore, KT2–962 did not protect
against infarction by improving the oxygen
supply/demand balance. In addition, MPO activity in the
ischemic/reperfused myocardium was not significantly al-
tered in KT2–962–treated dogs, suggesting that the treat-
ment with KT2–962 did not reduce ischemia-induced neu-
trophil extravasation and capillary plugging. It is well
known that brief episodes of myocardial ischemia and
reperfusion are associated with generation of reactive oxy-
gen species (22,41,42) such as hydroxyl radicals, superox-
ide anions, and H2O2. Hydroxyl radicals and other cyto-
toxic oxidants are released in a burst immediately upon
reperfusion following periods of regional/global myocar-
dial ischemia (23,26,43). Considerable evidence suggests
that the production and release of hydroxyl radicals during
the initial ischemic challenge is important in the develop-
ment of myocardial necrosis (23,25–27,44). We therefore
examined the effects of KT2–962 on the formation of hy-
droxyl radicals. In the current study, it was found that the
formation of hydroxyl radicals was inhibited by KT2–962
but not by daltroban. KT2–962 was more potent and effi-
cacious than mannitol, a known hydroxyl radical scaven-
ger, in our in-vitro assay. Therefore, it is reasonable to sug-
gest that the reduction in infarct size produced by KT2–962
may be related largely to its ability to scavenge hydroxyl
radicals or perhaps remove other reactive oxygen species
produced during ischemia and reperfusion.
486
© 2003 Lippincott Williams & Wilkins

J Cardiovasc Pharmacolீ • Volume 41, Number 3, March 2003
TXA2 Receptor Antagonists and Cardioprotection
neutrophil accumulation and infarct size following thromboxane syn-
thetase inhibitor or receptor antagonist. Angiology. 1989;40:209–21.
14. Bhat AM, Sacks H, Osborne JA, et al. Protective effect of the specific
thromboxane receptor antagonist, BM-13505, in reperfusion injury
following acute myocardial ischemia in cats. Am Heart J. 1989;117:
799–803.
15. Brezinski ME, Yanagisawa A, Lefer AM. Cardioprotective actions of
specific thromboxane receptor antagonist in acute myocardial ische-
mia. J Cardiovasc Pharmacol. 1987;9:65–71.
16. Brezinski ME, Yanagisawa A, Darius H, et al. Anti-ischemic actions
of a new thromboxane receptor antagonist during acute myocardial
ischemia in cats. Am Heart J. 1985;110:1161–7.
17. Singh J, Seth SD, Manchanda SC, et al. Protective actions of a throm-
boxane receptor antagonist, SQ 29548 on the ischemic myocardium:
morphologic and hemodynamic effects. Prostaglandins Leukot Essent
Fatty Acids. 1997;56:105–10.
18. Smith EF III, Earl CQ, Egan JW. BM 13.505, a selective throm-
boxane receptor antagonist, reduces myocardial infarct size following
coronary artery reperfusion. Prostaglandins Leukot Essent Fatty Acids.
1989;38:15–23.
19. Golino P, Ambrosio G, Villari B, et al. Endogenous prostaglandin
endoperoxides may alter infarct size in the presence of thromboxane
synthase inhibition: studies in a rabbit model of coronary artery oc-
clusion-reperfusion. J Am Coll Cardiol. 1993;21:493–501.
20. Engelman DT, Watanabe M, Engelman RM, et al. Hypoxic precon-
ditioning preserves antioxidant reserve in the working rat heart. Car-
diovasc Res. 1995;29:133–40.
In summary, KT2–962, but not daltroban, reduced
myocardial ischemia/reperfusion injury and the incidence
of ventricular fibrillation in an open-chest dog model of
regional ischemia and reperfusion. The cardioprotective
effect of KT2–962 may be due to its free radical scavenging
properties. Blockade of TP receptors by daltroban alone
did not provide protection against myocardial ische-
mia/reperfusion injury. Thus, our results suggest that
TXA2 produced endogenously during ischemia and/or
reperfusion plays a minor role in the pathogenesis of myo-
cardial ischemia/reperfusion injury. However, in situa-
tions in which TXA2 may play an important pathogenic
role in patients with heart disease, i.e., increased platelet
aggregation, the use of KT2–962 may be advantageous
because of its additional hydroxyl radical scavenging
property.
ACKNOWLEDGMENTS
The authors thank Jeannine Moore and Anna Hsu
for excellent technical assistance; and Dr. A. Tomiyama (of
Kotobuki-Seiyko) for providing the sample of KT2–962
and funds that partially supported this project.
21. Das DK, Maulik N, Moraru II. Gene expression in acute myocardial
stress. Induction by hypoxia, ischemia, reperfusion, hyperthermia
and oxidative stress. J Mol Cell Cardiol. 1995;27:181–93.
22. Bolli R, Jeroudi MO, Patel BS, et al. Marked reduction of free radical
generation and contractile dysfunction by antioxidant therapy begun
at the time of reperfusion: evidence that myocardial “stunning” is a
manifestation of reperfusion injury. Circ Res. 1989;65:607–22.
23. Powell SR, Hall D. Use of salicylate as a probe for .OH formation in
isolated ischemic rat hearts. Free Radic Biol Med. 1990;9:133–41.
24. Liu P, Hock CE, Nagele R, et al. Formation of nitric oxide, superox-
ide, and peroxynitrite in myocardial ischemia-reperfusion injury in
rats. Am J Physiol. 1997;272(5 part 2):H2327–36.
25. Merrill G, McConnell P, Vandyke K, et al. Coronary and myocardial
effects of acetaminophen: protection during ischemia-reperfusion.
Am J Physiol Heart Circ Physiol. 2001;280:H2631–8.
26. McHugh NA, Merrill GF, Powell SR. Estrogen diminishes postische-
mic hydroxyl radical production. Am J Physiol. 1998;274:H1950–4.
27. Merrill GF. Acetaminophen and low-flow myocardial ischemia: effi-
cacy and antioxidant mechanisms. Am J Physiol Heart Circ Physiol.
2002;282:H1341–9.
28. Yoshida T, Maulik N, Engelman RM, et al. Targeted disruption of the
mouse Sod I gene makes the hearts vulnerable to ischemic reperfu-
sion injury. Circ Res. 2000;86:264–9.
29. Auchampach JA, Pieper GM, Cavero I, et al. Effect of the platelet-
activating factor antagonist RP 59227 (Tulopafant) on myocardial
ischemia/reperfusion injury and neutrophil function. Basic Res Car-
diol. 1998;93:361–71.
REFERENCES
1. Kuzuya T, Hoshida S, Nishida M, et al. Increased production of ara-
chidonate metabolites in an occlusion–reperfusion model of canine
myocardial infarction. Cardiovasc Res. 1987;21:551–8.
2. Walinsky P, Smith JB, Lefer AM, et al. Thromboxane A2 in acute
myocardial infarction. Am Heart J. 1984;108:868–72.
3. Beitz A, Taube C, Beitz J, et al. Influence of drugs on the TXA2/PGI2
balance and on the atherogenic index in myocardial ischemia in
dogs. Biomed Biochim Acta. 1988;47:S149–52.
4. Tada M, Kuzuya T, Hoshida S, et al. Arachidonate metabolism in
myocardial ischemia and reperfusion. J Mol Cell Cardiol. 1988;20
(suppl 2):135–43.
5. Smith EF III, Lefer AM, Smith JB. Influence of thromboxane inhibi-
tion on the severity of myocardial ischemia in cats. Can J Physiol Phar-
macol. 1980;58:294–300.
6. Tanabe M, Terashita ZI, Fujiwara S, et al. Coronary circulatory fail-
ure and thromboxane A2 release during coronary occlusion and
reperfusion in anaesthetised dogs. Cardiovasc Res. 1982;16:99–106.
7. Johnson GJ, Leis LA, Francis GS. Disparate effects of the calcium-
channel blockers, nifedipine and verapamil, on ␣2-adrenergic recep-
tors and thromboxane A2-induced aggregation of human platelets.
Circulation. 1986;73:847–54.
8. Ito BR, Roth DM, Engler RL. Thromboxane A2 and peptidoleuko-
trienes contribute to the myocardial ischemia and contractile dys-
function in response to intracoronary infusion of complement C5a in
pigs. Circ Res. 1990;66:596–607.
9. Morooka S, Kobayashi M, Takahashi T, et al. Experimental ischemic
heart disease–effects of synthetic thromboxane A2. Exp Mol Pathol.
1979;30:449–57.
30. Gumina RJ, Auchampach J, Wang R, et al. Na+/H+ exchange inhi-
bition-induced cardioprotection in dogs: effects on neutrophils versus
cardiomyocytes. Am J Physiol Heart Circ Physiol. 2000;279:H1563–70.
31. Gross GJ, Auchampach JA. Role of ATP dependent potassium chan-
nels in myocardial ischaemia. Cardiovasc Res. 1992;26:1011–6.
32. Kosakai K, Wakabayashi S, Sato T, et al. Pharmacologic properties of
KT2–962 (6-isopropyl-3-[4-(p- chlorobenzenesulfonylamino)-butyl]-
azulene-1-sulfonic acid sodium salt); a new TXA2/prostaglandin en-
doperoxide receptor antagonist. J Cardiovasc Pharmacol. 1993;21:
441–7.
10. Smith EF III, Lefer AM, Aharony D, et al. Carbocyclic thromboxane
A2: aggrevation of myocardial ischemia by a new synthetic throm-
boxane A2 analog. Prostaglandins. 1981;21:443–56.
11. Bowling N, Dube GP, Kurtz WL, et al. Characterization of throm-
boxane A2/prostaglandin H2 binding sites in guinea pig cardiac
membrane preparations. J Mol Cell Cardiol. 1994;26:915–23.
12. Schror K, Thiemermann C. Treatment of acute myocardial is-
chaemia with a selective antagonist of thromboxane receptors (BM
13.177). Br J Pharmacol. 1986;87:631–7.
33. Enzan K, Shouji K, Inaba H, et al. Inhibition of pulmonary hyper-
tensive response after antigen challenge. Shock. 1995;4:294–7.
34. Nagao T, Koseki J, Suzuki Y, et al. Thromboxane A2 causes retarded
clearance of aggregated protein in glomeruli of nephritic mice. Eur J
Pharmacol. 2001;413:271–9.
13. Nichols WW, Mehta J, Wargovich TJ, et al. Reduced myocardial
35. Yokota M, Uchibori S, Hayashi H, et al. Azulene derivatives as
© 2003 Lippincott Williams & Wilkins
487

Z.-D. Ge et al.
J Cardiovasc Pharmacolீ • Volume 41, Number 3, March 2003
TXA2/PGH2 receptor antagonists—II: synthesis and biological activ-
ity of 6-mono- and 6-dihydroxylated- isopropylazulenes. Bioorg Med
Chem. 1996;4:575–91.
40. Bertolino F, Valentin JP, Patoiseau JF, et al. Evidence for partial ago-
nist properties of daltroban (BM 13,505) at TP receptors in the anaes-
thetized open-chest rat. Naunyn Schmiedebergs Arch Pharmacol. 1997;
356:462–6.
41. Bolli R, Patel BS, Jeroudi MO, et al. Demonstration of free radical
generation in ‘stunned‘ myocardium of intact dogs with the use of the
spin trap alpha-phenyl N-tert-butyl nitrone. J Clin Invest. 1988;82:
476–85.
42. Zweier JL. Measurement of superoxide-derived free radicals in the
reperfused heart: evidence for a free radical mechanism of reperfu-
sion injury. J Biol Chem. 1988;263:1353–7.
43. Powell SR, Tortolani AJ. Recent advances in the role of reactive oxy-
gen intermediates in ischemic injury. I. Evidence demonstrating pres-
ence of reactive oxygen intermediates; II. Role of metals in site-
specific formation of radicals. J Surg Res. 1992;53:417–29.
44. Merrill GF, Goldberg E. Antioxidant properties of acetaminophen
and cardioprotection. Basic Res Cardiol. 2001;96:423–30.
36. Mehta J, Nichols WW, Goldman R. Prostacyclin release following
endoperoxide analogue infusion in the intact dog. Am J Physiol.
1984;246(2 part 2):R205–10.
37. Evers AS, Murphree S, Saffitz JE, et al. Effects of endogenously pro-
duced leukotrienes, thromboxane, and prostaglandins on coronary
vascular resistance in rabbit myocardial infarction. J Clin Invest. 1985;
75:992–9.
38. Tanabe M, Terashita Z, Nishikawa K, et al. Inhibition of coronary
circulatory failure and thromboxane A2 release during coronary oc-
clusion and reperfusion by 2-phenylaminoadenosine (CV- 1808) in
anesthetized dogs. J Cardiovasc Pharmacol. 1984;6:442–8.
39. Xiao CY, Hara A, Yuhki K, et al. Roles of prostaglandin I2 and
thromboxane A in cardiac ischemia-reperfusion injury: a study using
mice lacking their respective receptors. Circulation. 2001;104:2210–5.
488
© 2003 Lippincott Williams & Wilkins