Bristol-Myers Squibb Pharmaceutical Research Institute,
Departments of Pharmacology (A.W.G., R.A.R., R.E.S., A.J.B., C.S.S.,
G.J.G.) and
Chemistry (R.W.B., H.J.M.), Princeton, New Jersey
The effect of the timing of treatment with the ATP-regulated potassium
channel agonist BMS-180448 was evaluated in isolated rat heart and
ferret models of ischemia and reperfusion. In rat hearts, 10 µM
BMS-180448, given before and after global ischemia as well as only
during reflow, improved reperfusion contractile function and attenuated
lactic dehydrogenase release, although reperfusion-only treatment was
less effective. Cromakalim (10 µM) and bimakalim (10 µM) treatment
before and after global ischemia afforded a degree of protection
similar to that of BMS-180448, although they were not cardioprotective
when given only during reperfusion. Pre- and post-treatment
cardioprotection were abolished by glyburide. Ischemia/reperfusion
significantly increased cytosolic calcium concentration
([Ca++]i) and BMS-180448 given only during
reperfusion attenuated this change. In anesthetized ferrets, BMS-180448
(2 mg/kg) or vehicle was infused i.v. during a 40-min interval
beginning 1) 10 min before coronary occlusion, 2) at the 45th min of
ischemia or 3) at the 5th min of reperfusion. Preocclusion
administration of BMS-180448 was associated with a 35% reduction in
infarct damage from that recorded in vehicle-treated control ferrets.
Drug administered at the midpoint of ischemia reduced infarct size
~44%, whereas delaying BMS-180448 infusion until the 5th min of
reperfusion reduced, but still provided a significant (17%) level of
salvage. The favorable effects of BMS-180448 in the ferret were not
associated with changes in either collateral blood flow or peripheral
hemodynamics. Thus BMS-180448 shows some protective effects when given
only during reperfusion. Cromakalim and bimakalim did not exert similar actions and the difference may be secondary to the faster penetration of BMS-180448.
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Introduction |
Several members of a structurally
diverse group of KATP openers have been shown to promote
myocardial salvage and enhance function recovery in vivo
(Auchampach et al., 1991
; Endo et al., 1988;
Grover et al., 1990c) and in vitro (McCullough
et al., 1991; Mitani et al., 1991; Ohta et
al., 1991). Recently, pyranyl cyanoguanidine analogs have been
found to be relatively devoid of vasorelaxant activity although
retaining the glyburide-reversible cardioprotective activity of other
KATP openers. BMS-180448, a member of this chemical class
(Atwal et al., 1993, 1995), has been shown to reduce
ischemic/reperfusion injury in vitro in isolated rats
(Grover et al., 1995b) and in vivo in
anesthetized dogs (Grover et al., 1996) hearts, whereas it
is significantly less hypotensive than agents such as cromakalim in
these species as well as the ferret (Weselcouch et al.,
1994). In addition to minimal vasodilator effects, BMS-180448 has a
reduced propensity to reduce action potential duration (Grover et
al., 1995a), which suggests an intracellular site of action,
perhaps on mitochondrial KATP (Inoue et al.,
1991).
Some of the protective effects of KATP openers are exerted
during the ischemic event per se as the time to the onset of
contracture is increased in rat hearts and this is accompanied by
conservation of ATP (McPherson et al., 1993). ATP levels are
restored significantly better during reperfusion in KATP
opener-treated hearts (Baird et al., 1996), although it is
not clear whether this is secondary to protection during ischemia or to
a direct effect on reperfusion injury. At the present time, a
protective effect of KATP openers on reperfusion injury
cannot be completely ruled out. Presentation of the KATP
openers aprikalim and cromakalim only during reperfusion did not result
in cardioprotective effects (Auchampach et al., 1991; Grover
et al., 1990a,b). It is possible that insufficient time is
allowed for adequate drug penetration when the KATP openers are given only during reperfusion. A recently published paper from
Gross's laboratory (Mizumura et al., 1995) showed that
bimakalim reduced infarct size in dogs when it was given 10 min before
the onset of reperfusion, although the protection was not as good as
observed for pretreatment. This suggests that KATP openers may not penetrate rapidly enough when given only during reperfusion. Recent data have shown that BMS-180448 may penetrate ischemic myocardium more rapidly than other KATP openers such as
cromakalim (Grover and Sleph, 1995) and therefore may have a better
chance for working when given only during reperfusion. In the present investigation, the cardioprotective efficacy of BMS-180448 when administered either before myocardial ischemia, during the occlusive interval, or after the initiation of reflow was investigated. This was
done both in vitro in an isolated rat heart model of ischemia and reperfusion, and in vivo in an anesthetized
ferret model of coronary artery occlusion and reperfusion.
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Methods |
Isolated Rat Heart Studies
General procedures.
Male Sprague-Dawley rats (400-500 g)
were anesthetized with 100 mg/kg sodium pentobarbital (i.p.). The
trachea was intubated and then the jugular vein was injected with
heparin (1000 U/kg). While being mechanically ventilated, their hearts
were perfused in situ via retrograde cannulation of the
aorta. The hearts were then excised and quickly moved to a Langendorff
apparatus where they were perfused with oxygenated Krebs-Henseleit
solution containing (in mM): NaCl, 112; NaHCO3, 25; KCl, 5;
MgSO4, 1.2; KH2PO4, 1; CaCl2, 1.2; glucose, 11.5 and pyruvate, 2, at a constant
perfusion pressure (85 mm Hg). A water-filled latex balloon attached to a metal cannula was inserted into the left ventricle and connected to a
Statham pressure transducer for measurement of left ventricular pressure. The hearts were allowed to equilibrate for 15 min, at which
time EDP was adjusted to 5 mm Hg; this balloon volume was maintained
for the duration of the experiment. Preischemia or predrug function,
heart rate and coronary flow (extracorporeal electromagnetic flow
probe, Carolina Medical Electronics, King, NC) were measured.
Contractile function was calculated by subtracting EDP from left
ventricular peak systolic pressure, resulting in LVDP. Cardiac
temperature was maintained throughout the experiment by submerging the
hearts in 37°C buffer, which was allowed to accumulate in a
stoppered, heated chamber.
Treatment protocols.
After equilibration, the hearts were
subjected to one of several treatments: 1) vehicle (0.04% DMSO) given
before ischemia and during reperfusion (n = 8) (fig.
1A); 2) BMS-180448 at 1 to 30 µM given only during
reperfusion (n = 4-8 per group) (fig. 1B); 3) 10 µM
BMS-180448 given before ischemia and during reperfusion (n = 8) (fig. 1A); 4) 1 µM glyburide given only
during reperfusion (n = 5) (fig. 1C); 5) 10 µM
BMS-180448 and 1 µM glyburide given only during reperfusion
(n = 5) (fig. 1D); or 6) 1 µM glyburide and 10 µM
BMS-180448 given before and after ischemia (n = 8)
(fig. 1E). All hearts were subjected to 25 min of global ischemia and 30 min of reperfusion. Ischemia was initiated by completely shutting off perfusate flow. The respective drug treatments were included in the
perfusate and were given either 10 min before ischemia and during the
30 min of reperfusion, or only during reperfusion. In all cases when
glyburide and BMS-18044 were given together, they were started
simultaneously. At the end of the reperfusion period, contractile
function, coronary flow and LDH release were measured. Indices of
severity of ischemia included time to contracture, recovery of
contractile function at 30 min into reperfusion and LDH release into
the reperfusate.
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Fig. 1.
Diagrams of experimental treatment designs used in
the isolated rat heart model of ischemia and reperfusion. BMS,
BMS-180448; Cro, cromakalim; BK, bimakalim; Veh, vehicle.
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A second study was undertaken to determine whether cromakalim could
protect when given only during reperfusion. Hearts were given: 1)
vehicle (0.04% DMSO) (n = 6) (fig. 1B); 2) 10 µM
cromakalim only during reperfusion (n = 6) (fig. 1B);
3) 10 µM cromakalim before and after reperfusion (n = 6) (fig. 1A); 4) 10 µM cromakalim and 1 µM glyburide before and
after ischemia (n = 6) (fig. 1E); or 5) 10 µM
cromakalim before and after ischemia plus 1 µM glyburide only during
reperfusion (n = 6) (fig. 1F). An additional study was
performed to assess the effects of bimakalim in isolated rat hearts
when given only during reperfusion or when given before ischemia. The
hearts were treated with: 1) vehicle (0.04% DMSO) before and after
ischemia (n = 6) (fig. 1A); 2) 10 µM bimakalim only
during reperfusion (n = 4) (fig. 1B); or 3) bimakalim
before and after ischemia (n = 6) (fig. 1A). All hearts
were subjected to 25 min of global ischemia and 30 min reperfusion as
described above. The recovery of postischemic contractile function and
LDH release during reperfusion were measured. The concentrations of bimakalim and cromakalim were selected because of equivalence in terms
of cardioprotection with BMS-180448. These compounds have been
previously shown to be equally potent cardioprotectants (Grover
et al., 1995a; Grover and Sleph, 1995)
Measurement of intracellular calcium.
The effect of
BMS-180448 on reperfusion [Ca++]i was
measured in isolated Langendorff perfused rat hearts by
19F-NMR. The experimental procedure and data analysis has
been described in detail elsewhere (Behling and Malone, 1995). After
isolation the hearts were perfused at constant flow (12 ml/min) with
pyruvate-deficient Krebs-Henseleit solution containing 300 ml of 5 µM
acetoxymethyl ester of 5F-BAPTA. Subsequent to loading with 5F-BAPTA,
hearts were perfused with vehicle (5 µM DMSO), placed into the NMR
probe and then inserted into the magnet (Bruker 360 WB). The NMR data acquisition began after an equilibration period lasting ~15 min. Each
NMR spectrum took 6.25 min. Cardiac temperature was 30.0 ± 0.2°C during the experiments to give equivalent times to contracture as observed in hearts without 5F-BAPTA (Behling and Malone, 1995). In
this study, each experiment was divided into three periods: preischemia
(19 min), global ischemia (25 min) and reperfusion (25 min). The hearts
were randomly divided into four treatment groups. All hearts were
treated only with vehicle during preischemia and ischemia. Hearts were
reperfused with: 1) vehicle (n = 7); 2) 20 µM
BMS-180448 (n = 9); 3) 20 µM BMS-180448 plus 0.3 µM
glyburide (n = 7); or 4) 0.3 µM BMS-180448
(n = 7). Hearts were paced at 4 Hz during the
experiments, and NMR data acquisition was gated to the diastolic phase
of each heart cycle (repetition time for NMR data acquisition 
TR = 250 msec). Hearts were not paced during ischemia, but
TR remained constant. The treatment groups do not include
hearts rejected because of failure to pace properly or poor
signal-to-noise ratio in the NMR experiment.
[Ca++]i was determined from the ratio of
bound to free 5F-BAPTA measured from the NMR spectra with a
KD of 285 nM at 30°C (Marban et
al., 1987). Results were averages of the time periods required to
acquire the NMR spectra, unless otherwise indicated. Data points are
plotted at the midpoint of each period
Ferret Model of Infarction
General procedures.
Experiments were conducted in castrated
male ferrets,1.1 to 1.8 kg b.wt., anesthetized i.p. with pentobarbital
Na (40-45 mg/kg). Supplemental doses of pentobarbital were
administered i.p. or i.v. as needed to maintain a stable plane of
surgical anesthesia during the course of an experiment. Body
temperature was monitored with an esophageal thermistor probe and held
constant at 38 ± 1°C by maintaining the animal on a warmed
circulating water heating pad. A patent airway was established by
placement of a balloon-cuffed endotracheal tube (Magill type, internal
diameter 3.0 mm, Mallincrodt Critical Cars, Glen Falls, NY), and the
animals were respired (Harvard Apparatus, Model 655, South Natick, MA)
with room air at a volume and rate to maintain eucapnia. The latter was
verified by periodic blood gas measurements (Radiometer ABL500,
Deerfield, IL).
The heart was exposed via a thoracotomy at the fifth
intercostal space and supported in a pericardial cradle. A needle
affixed to a 5-0 Prolene ligature was passed under the LAD coronary
artery and incorporated into a soft rubber closure snare. A lead II ECG was monitored via needle electrodes inserted subdermally.
Catheters (PE50) were placed into a jugular vein, the left atrium and a carotid artery, respectively, for infusion of drug (or anesthetic), blood sampling and measurement of blood pressure by a Statham (P23ID)
transducer. Monitored variables were recorded on a Gould polygraph
(Model TA4000 or TA5000, Valley View, OH).
After completion of surgery and stabilization, ferrets were subjected
to 90-min occlusion of the LAD coronary artery followed by a 5-hr
interval of reflow. At the conclusion of the reperfusion interval, the
hearts were removed from the fully anesthetized animals and prepared
for determination of infarct size (see below).
Drug treatment.
To establish an optimally effective dose,
BMS-180448 (0.5, 1, 2, 4 mg/kg) or equal volume of vehicle (2 ml) was
injected over 20-min beginning at the 30th min of a 60-min ischemic
interval. BMS-180448 was dissolved in PEG400 and diluted with
PEG400/distilled water (1:1). The volume-infusion rate was 0.1 ml/min.
Vehicle-treated control ferrets received matched volumes of PEG400
diluted with 50% PEG-distilled water.
In the timing of treatment assessments, ferrets were given BMS-180448
(2 mg/kg) or vehicle (0.1 ml/min) i.v. over a 40-min interval
beginning: 1) 10 min before LAD coronary artery occlusion and
continuing until the 30th min of occlusion (fig. 2A); 2)
beginning at the 45th min of ischemia and continuing until the 85th min (fig. 2B); or 3) beginning at the 5th min and terminating at 45th min
of reperfusion (fig. 2C).
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Fig. 2.
Diagrams of treatment protocols used in the
anesthetized ferret coronary artery occlusion and reperfusion model.
Pre-Occ, preocclusion; Occ, occlusion; ReP, reperfusion.
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For observations on KATP blockade, glyburide (2 mg/kg) or
vehicle (0.1 ml/min) was infused i.v. over a 10-min interval beginning at either the 10th min before LAD occlusion (fig. 2D) or the 45th min
of coronary artery occlusion (fig. 2E). In interaction studies, glyburide (2 mg/kg) was given i.v. during a 10-min interval starting 10 min before occlusion and BMS-180448 (2 mg/kg) was administered between
the 45th and 85th min of ischemia (fig. 2F). Glyburide was administered
in PEG400/distilled water solution as described previously for
BMS-180448.
Assessment of infarct size.
Ferret hearts removed at the
completion of reperfusion were trimmed of adipose tissue, and whole
heart as well as left ventricular size were determined gravimetrically.
The left ventricle was subsequently sectioned into rings measuring
approximately 4 to 5 mm from apex to base, parallel to the
atrioventricular groove. The sections were incubated in 1%
2,3,5-triphenyltetrazolium in 20 mM phosphate buffer (pH 7.4) at 37°C
for 5 to 10 min. 2,3,5-triphenyltetrazolium is an agent that turns into
a bright red formazan precipitate when it undergoes reduction in the
presence of dehydrogenase enzymes present in viable myocardial tissue.
The extent of ischemic damage was thus demarcated by its negative
staining characteristics, and infarct size was quantified
gravimetrically.
Measurements of regional myocardial blood flow.
Radiolabeled
15 ± 3 µm diameter microspheres, 57Co,
113Sn, 85Sr or 46Sc (DuPont-NEN,
North Billerica, MA) injected in random order were used to measure
regional myocardial blood flow by the reference withdrawal method
(Flaim et al., 1984; Heymann et al., 1977) as described previously (Gomoll et al., 1994). Blood flow
determinations were performed just before occlusion, at the 85th min of
occlusion, and at the 120th min of reperfusion in ferrets given
BMS-180448 or vehicle over a 40-min interval beginning at the 45th min
of ischemia. Reference arterial blood samples were obtained from the
abdominal aorta via a femoral catheter at a constant rate of
0.4 ml/min with a calibrated pump (Ranin Rabbit Peristaltic Pump,
Woburn, MA). The withdrawal of reference blood was initiated 15 sec
before 0.2 to 0.4 ml of a well-mixed microsphere suspension diluted
with ~0.5 ml warmed normal saline was injected via a
cannula placed in the left atrium during a 15- to 20-sec interval
followed by an additional 2-ml rinse with warmed saline. Reference
blood withdrawal was discontinued 90 sec after completion of
microsphere injection. Between 150,000 and 450,000 microspheres were
given at each time point. After sacrifice and tissue zone
identification, myocardial samples from the subepicardial and
subendocardial halves of the ischemic and nonischemic regions of the
left ventricle were taken for blood flow analyses. Radioactivity in
each tissue and reference blood sample was determined in a 
-counter
(MICRAD, Inc. Automated Measurement System, Knoxville, TN or Beckman
Autogamma 8000, Irvine, CA).
Statistical Analysis
All data are presented as means ± S.E.M. Rat ischemia data
were analyzed by repeat measures ANOVA with post hoc
Neuman-Keuls test. Statistical differences in intracellular calcium at
each time period were determined by ANOVA with the Scheffé
post hoc test to interpret differences among all groups.
Ferret tissue weight data were compared by a one-way ANOVA. Hemodynamic
data collected at multiple time points were subjected to ANOVA with repeat measures; contrasts were used to detect mean within- and between-group treatment differences. In two-group comparisons, a
t-test was used. In all analyses, a P value of <.05 was
considered statistically significant.
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Results |
Isolated rat heart studies.
The effect of BMS-180448, when
given only during reperfusion, on coronary flow and cardiac function
are shown in table 1. Baseline function and coronary
flow were similar for all groups. After ischemia, contractile
functional recovery was poor in vehicle-treated hearts, which indicates
some degree of damage. In addition, significant bradycardia was
observed, probably because of a conduction disturbance. Reperfusion
coronary flow also did not return to baseline values. When given only
during reperfusion, BMS-180448 improved reperfusion contractile
function (LVDP) starting at the 10 µM concentration. This protective
effect was not clearly concentration dependent, and the degree of
protection was not great compared with pretreatment (table 1).
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TABLE 1
Effect of BMS-180448 given at the time of reperfusion on postischemic
cardiac function and coronary flow in isolated rat hearts
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The most marked effect of 10 µM BMS-180448 when given only during
reperfusion was on the bradycardia such that the double product of
heart rate (HR) × LVDP/1000 was significantly enhanced (fig.
3). Some of the protective effect on functional recovery was lost for BMS-180448 at the 30 µM concentration, although it was
still protective. Most of this reduced efficacy was caused by a loss of
protection against bradycardia. LDH release during reperfusion was
significantly attenuated, but not concentration dependently, by
BMS-180448 when given only during reperfusion at the 10 and 30 µM
concentrations (fig. 3). BMS-180448 at 100 µM was not used because it
had negative inotropic effects at this concentration.
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Fig. 3.
Effect of increasing concentrations of BMS-180448
on reperfusion LDH release and double product at 30 min after 25 min of global ischemia in rat hearts. BMS-180448 was given only during reperfusion in this study. At 10 and 30 µM, cumulative LDH release was significantly (*P < .05, > .01) reduced by BMS-180448,
although this effect was not clearly concentration dependent.
Reperfusion function was also significantly improved at these
concentrations, and the best protective effect was observed at 10 µM.
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Because we observed a protective effect of BMS-180448, we determined
whether this effect is abolished by the KATP blocker glyburide (table 2; fig. 4). Also for the
purpose of comparison, the effect of BMS-180448 when given before and
after global ischemia was assessed. At 10 µM, BMS-180448
significantly protected hearts, particularly when given both before and
after global ischemia. Cardiac function during reperfusion was
significantly improved and reperfusion LDH release was reduced, with
pre- and post-treatment BMS-180448 being significantly more efficacious
than post-treatment alone. The protective effects of BMS-180448 when
given before and after ischemia, as well as reperfusion alone, was
abolished by glyburide. Glyburide alone (reperfusion only) had no
effect on severity of ischemia, and previous studies have shown that glyburide pretreatment is innocuous in this model. In an additional group (data not shown), pre- and postischemic treatment
cardioprotection with 10 µM BMS-180448 was not abolished by 1 µM
glyburide given only during reperfusion (LDH release = 11 ± 1.0 vs. 10 ± 1.0 U/g for BMS-180448 without and with
glyburide, respectively).
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TABLE 2
Effect of BMS-180448 with or without glyburide on postischemic cardiac
function and coronary flow in isolated rat hearts
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Fig. 4.
Effect of BMS-180448 (BMS, 10 µM) on reperfusion
LDH release and recovery of function after 25 min global ischemia in
rat hearts either with or without glyburide (GLY, 1 µM). BMS
significantly reduced LDH release and improved function (*P < .05, > .01) when given only during reperfusion (REPER) or when given
both before and after global ischemia (PRE). Glyburide abolished the
protective effects BMS.
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The effect of the KATP openers bimakalim and cromakalim
when given before and during ischemia or only during reperfusion are shown in tables 3 and 4 and figures
5 and 6. Both bimakalim (10 µM) and
cromakalim (10 µM) significantly protected hearts when given before
and after ischemia, and the degree of protection was similar to that
for 10 µM BMS-180448. Neither cromakalim nor bimakalim exerted
significant protective effects when given only during reperfusion.
Glyburide abolished the protective effects of cromakalim when given
before and after ischemia, but was without effect when given only
during reperfusion.
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TABLE 3
Effect of bimakalim given before or after ischemia on cardiac function
and coronary flow in isolated rat hearts
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TABLE 4
Effect of cromakalim with or without glyburide on postischemic cardiac
function and coronary flow in isolated rat hearts
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Fig. 5.
Effect of 10 µM bimakalim on reperfusion LDH
release and double product at 30 min after 25 min of global ischemia in
rat hearts when given either before and after ischemia or only during
reperfusion (REPER). Pretreatment with bimakalim significantly (*P < .05, > .01) protected these hearts, but reperfusion treatment had
no such protective activity.
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Fig. 6.
Effect of 10 µM cromakalim (CRO) given either
before and after (PRE) ischemia or only during reperfusion (REPER) on
postischemic LDH release and functional recovery. Pretreatment with
cromakalim significantly (*P < .05, > .01) reduced LDH release
and enhanced function, whereas reperfusion only treatment showed no
protection. Glyburide (GLY, 1 µM) when given before and after
ischemia completely abolished the protective effect of cromakalim
pretreatment. Glyburide given only during reperfusion did not alter the
protective effect of cromakalim pretreatment.
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The effect of BMS-180448, given only during reperfusion, on cytosolic
calcium concentration is shown in figure 7. Diastolic [Ca++]i during the preischemia period was
~200 to 300 nM in all groups. [Ca++]i
increased steadily during ischemia in all hearts, reaching levels of
600 to 800 nM after 25 min. During reperfusion,
[Ca++]i in hearts treated with vehicle,
glyburide or BMS-180448 plus glyburide continued to rise to 750 to 900 nM during the first 12 min of ischemia before decreasing during the
final 12 min of reperfusion. [Ca++]i in these
hearts was ~600 nM after 25 min of reperfusion. In contrast,
[Ca++]i decreased immediately in hearts
treated with BMS-180448 and was significantly lower after 6 min of
reperfusion. [Ca++]i in BMS-180448-treated
hearts returned to preischemia levels within the 25 min of reperfusion
without evidence of any calcium increase immediately upon reperfusion.
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Fig. 7.
The cardiac diastolic intracellular concentrations
of calcium [Ca++]i are shown for perfused rat
hearts divided into four treatment groups. Hearts in all groups were
treated with vehicle (5 µM DMSO) during the 18-min preischemia
period, followed by 25 min of global (no-flow) ischemia. Hearts were
then reperfused with perfusate containing one of the following
treatments: vehicle, 0.3 µM glyburide, 20 µM BMS-180448 or 20 µM
BMS-180448 plus 0.3 µM glyburide. Time-course changes of
[Ca++]i before and during ischemia, and
subsequent reperfusion are shown. Symbols indicate levels of
significant changes observed (*P < .05, > .01;  P < .005).
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Ischemic injury in ferrets.
In dose-response assessments,
significant levels of tissue salvage of 24.5 ± 9.2% (P < .05), 29.3 ± 18.8% (P < .05) and 29.3 ± 8.4%
(P < .01), respectively, were noted after 1, 2 and 4 mg/kg BMS-180448. In the ferret, a 0.5 mg/kg dose was inactive (1.7 ± 9.9%).
In the timing of treatment studies, mean LV weights represented a
consistent 3.75 ± 0.22 to 4.09 ± 0.20 g (ns) or range
of 69.3 ± 0.63% to 70.3 ± 0.38% (ns) of the whole heart
in the three paired groups. Preocclusion drug administration
significantly (P < .01) decreased mean infarct weight from
0.92 ± 0.06 g (23.7 ± 1.8% of LV) in vehicle controls
to 0.58 ± 0.06 g (15.4 ± 0.9% of LV) after BMS-180448
(fig. 8). This represented a mean reduction in tissue
damage of 35 ± 4%.
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Fig. 8.
Comparisons of infarct size as a percent of the
left ventricle (%LV) in groups of ferrets subjected to 90 min
occlusion and 5 hr reperfusion of the LAD coronary artery. Different
treatment groups were given 40 min i.v. infusion of BMS-180448 (2 mg/kg) or vehicle (0.1 ml/min): 1) beginning 10 min before occlusion (Pre-Occ); 2) between the 45th and 85th min of occlusion (Post-Occ); or
3) starting at the 5th min of reflow (Post-ReP). Vertical lines represent S.E.M.; n = 6 in all Pre-Occ and Post-Occ
groups, whereas n = 5 and 7, respectively, in the
control and drug-treated Post-ReP groups. Asterisks (*) indicate
significant (*P < .05, > .01, **P < .01) group differences
from response in vehicle controls.
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Infusion of 2 mg/kg BMS-180448 during a 40-min interval between the
45th and 85th min of ischemia significantly (P < .01) reduced the
extent of myocardial tissue injury from a control level of 25.6 ± 1.4% (0.98 ± 0.07 g) to 14.4 ± 1.1% (0.58 ± 0.04g) of LV (fig. 8). This represented a mean decrease in infarct
damage of 44 ± 4%.
Initiation of BMS-180448 administration at the 5th min of reflow was
also significantly (P < .05) cardioprotective in reducing infarct
size 17 ± 4% from 20.9 ± 0.25% (0.78 ± 0.01 g)
to 17.4 ± 0.91% (0.66 ± 0.04 g) of the LV (fig. 8).
The hemodynamic effects associated with 10-min preocclusion i.v.
infusion of BMS-180448 or vehicle are shown in figure 9. Mean heart rates were significantly elevated from predrug control levels (P < .05) at the 90th min of occlusion and at all
intervals thereafter by BMS-180448. These increases in heart rate,
however, differed (P < .05) from those in vehicle controls only
at the 300th min of reperfusion. There were no significant within or between-treatment group differences in either mean blood pressure or
rate pressure product.
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Fig. 9.
Time-course hemodynamic effects of BMS-180448 (2 mg/kg) or vehicle (0.1 ml/kg) given i.v. in ferrets over 40 min
beginning at the 10th min preceding LAD coronary artery occlusion. Mean absolute responses and S.E.M. (vertical bars) are shown;
n = 6 for each treatment group. Asterisks (*)
designate significant within-group differences from baseline values
(*P < .05, > .01). Symbol ( ) indicates temporal response in
BMS-180448 group that is significantly different (P < .05, > .01) from that of vehicle control.
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The pattern of hemodynamic changes associated with midischemia
administration of BMS-180448 (table 5) were comparable
to those reported after preocclusion dosing (fig. 9). Heart rate was
significantly increased from basal levels only at the 180th min of
reperfusion after BMS-180448. Blood pressure remained unchanged throughout in both groups while alterations in the rate pressure product mirrored those observed in heart rate.
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TABLE 5
Effects of i.v. BMS-180448 (2 mg/kg) or vehicle (0.1 ml/min) on
hemodynamic variables during and after myocardial ischemia in
anesthetized ferrets
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Baseline regional myocardial blood flow was similar in all regions for
the two studied experimental groups (table 6). Flow into
the LAD region was significantly reduced by coronary artery occlusion,
and BMS-180448 had no significant effect on collateral blood flow.
Reflow into the formerly ischemic region was not affected by BMS-180448
administration. In nonischemic regions, blood flow during LAD occlusion
was significantly increased from basal values and from that in vehicle
controls by BMS-180448. A shift of flow toward the subepicardium was
noted, but this was not sustained during reflow.
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TABLE 6
Effect of BMS-180448 or vehicle on regional myocardial blood flow
before, during and subsequent to release of LAD occlusion in
anesthetized ferrets
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There were no between-group differences in mean LV weights (extreme
values, 3.29 ± 0.08 g and 3.54 ± 0.12 g; range,
65.2 ± 1.2% to 68.6 ± 1.4% of whole heart) of the vehicle
control and three groups of ferrets used for either the pre- or
postocclusion observations on glyburide and the interaction studies
between glyburide and BMS-180448. Glyburide administration alone either during or before ischemia was without effect on the extent of tissue
damage, namely, 0.69 ± 0.03 g or 0.71 ± 0.05 g
(19.6 ± 0.91% and 21.5 ± 1.34% of LV), respectively, from
0.73 ± 0.03 g (21.3 ± 1.0% of LV) recorded in vehicle
controls (fig. 10). These changes represented mean
decreases of only 7.9% and <1%. Pretreatment of ferrets with
glyburide abolished the previously observed cardioprotective effects of
2 mg/kg BMS-180448. Infarct size was reduced only 5.9% compared with
vehicle treatment in the presence of glyburide, i.e., to
0.70 ± 0.05 g or 20.0 ± 1.4% of LV (fig. 10).
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Fig. 10.
Comparisons of infarct mass and infarct (Inf) as a
percent of the left ventricle (LV) in groups of ferrets subjected to 90 min occlusion and 5 hr reperfusion of the LAD coronary artery. Animals
were given a 10-min i.v. infusion of glyburide (2 mg/kg) beginning at
either 1) the 45th min during or 2) 10th min before ischemia, 3) a
combination of glyburide over 10 min starting 10 min before occlusion
plus BMS-180448 (2 mg/kg) between the 45th and 85th min of occlusion or
4) vehicle (0.1 ml/min). Vertical lines represent SEM;
n = 6 in each treatment group.
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The pattern of time-course changes in the measured hemodynamic
variables were similar in each of the treatment groups comprising these
studies. Analyses of the composite data using one (different timing of
treatment), as well as two (presence/absence of BMS-180448 with
glyburide) grouping factors revealed no significant between treatment
differences (table 7). Heart rate was progressively and
statistically increased above basal levels throughout the experimental
period in all groups. Mean arterial blood pressure remained unchanged,
whereas the rate pressure product was statistically elevated only at
isolated, but differing intervals either immediately before or during
the reperfusion phase.
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TABLE 7
Effects of glyburide (2 mg/kg i.v.) with and without BMS-180448 (2 mg/kg i.v.) on hemodynamic variables before and after myocardial ischemia in anesthetized ferrets
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Discussion |
As a pharmacologic class, KATP openers have been found
by several laboratories to protect the ischemic myocardium. Agents shown to improve reperfusion function and/or reduce infarct size in rat
hearts in vitro and anesthetized dogs or pigs in
vivo include pinacidil (Grover et al., 1990a),
nicorandil (Endo et al., 1988; Mitani et al.,
1991; Ohta et al., 1991; Woerkens et al., 1992), aprikalim (Auchampach et al., 1991; Grover et
al., 1990b), KRN2391 (Ohta et al., 1991) and bimakalim
(Mizumura et al., 1995; Woerkens et al., 1992),
as well as cromakalim (Grover et al., 1990a; McCullough et al., 1991). This effect appears to be caused by a direct
protective action on myocytes (Armstrong et al., 1995) and
associated with KATP activation (Grover et al.,
1995b; Rohmann et al., 1994).
BMS-180448 is a cromakalim analog which is efficacious as a
cardioprotective agent, but has weak vasorelaxant activity relative to
cromakalim (Grover et al., 1995b). The protective mechanism of action of KATP openers has previously been suggested to
be associated with action potential shortening or inhibition of
depolarization within the ischemic region (Cole et al.,
1991; D'Alonzo et al., 1992). Data from our laboratory have
shown that glyburide-reversible cardioprotective effects are not
dependent on action potential duration shortening (Grover et
al., 1995a). BMS-180448 is not only a weak vasodilator, but it
also neither shortens action potential duration within its
cardioprotective concentration range, nor readily opens single
KATP channels (Grover et al., 1995a,b). This suggests the possibility of an intracellular site of action which may be related to mitochondrial KATP (Paucek et
al., 1995).
Although some of the protective effects of KATP openers are
thought to be exerted during ischemia proper, their effects on reperfusion injury are not as clear. Previous studies from our laboratory (Grover et al., 1990a,b) have shown that
administration of KATP openers, cromakalim and aprikalim,
only during reperfusion in rat hearts after global ischemia did not
result in significant cardioprotection. This could indicate a lack of
direct effect on reperfusion injury, but could also suggest a slow
penetration of drug into its putative intracellular site of action.
Gross and colleagues (Mizumura et al., 1995) recently showed
that bimakalim exerted modest protective effects when given 10 min
before reperfusion in dogs; and, with this protocol, sufficient time
may have been allowed for adequate drug penetration.
BMS-180448 appears to penetrate ischemic rat hearts somewhat faster
than agents such as cromakalim (Grover and Sleph, 1995). This was shown
by administering BMS-180448 or cromakalim for short durations of time
before ischemia. Under these conditions, BMS-180448 protected when
given as early as 1 min, whereas cromakalim did not. BMS-180448 is more
lipid soluble than cromakalim, although it is not clear whether this is
a complete explanation for faster penetration. We therefore determined
the effect of BMS-180448 on ischemic/reperfusion damage in rat hearts
when this agent was given only during reperfusion. BMS-180448
significantly protected isolated rat hearts when given only during
reperfusion, although the protective effect was modest compared with
pretreatment. This protective effect was abolished by glyburide, which
indicates a KATP-mediated mechanism. Bimakalim and
cromakalim were devoid of protective activity when given only during
reperfusion, although this does not rule out a protective effect for
these compounds on reperfusion injury. Interestingly, the protective
effect of cromakalim pretreatment was not affected by glyburide given
only during reperfusion, which does not suggest a protective effect of
cromakalim on reperfusion injury, although the degree of protection with pretreatment may be so good that it would be difficult for glyburide alone to overcome this. Glyburide given only during reperfusion also did not attenuate the protective effect of BMS-180448 pretreatment, whereas it abolished the protective effect of BMS-180448 given only during reperfusion. Another possibility is that
KATP opener pretreatment cannot be pharmacologically
overcome by subsequent blocker administration.
Data from the present investigation also demonstrate a potential role
of BMS-180448 in modifying intracellular calcium shifts in the
myocardial damage associated with ischemia/reperfusion. In the
"unprotected" rat heart, intracellular calcium increased during
early ischemia, an observation consistent with the calcium influx known
to be associated with reperfusion injury (Behling and Malone, 1995;
Poole-Wilson et al., 1984). BMS-180448 treatment during
reperfusion significantly prevented this continued influx. This
suggests that elimination/attenuation of calcium influx may reduce the
severity of reperfusion injury, and the extent of injury that is
observed may come from damage which arises during ischemia and thus is
not the result of further reperfusion injury.
In the ferret, preocclusion administration of BMS-180448 was associated
with a 35% reduction of myocardial tissue damage compared with that in
vehicle-treated controls. The 2 mg/kg drug dose used for this
assessment was the same as that previously reported to reduce infarct
damage by 50% in a canine model (Grover et al., 1995b). In
that investigation the timing of BMS-180448 infusion, as in the present
study, spanned a 40-min interval beginning 10 min before left
circumflex coronary artery occlusion.
When BMS-180448 administration was begun at the 45th min (and completed
at the 85th min) of occlusion, an associated 44% reduction in infarct
size was observed in the ferret. In dogs, BMS-180448 given 2 min before
reperfusion evoked a ~36% level of tissue salvage (Grover, in
press). Thus, reasonably comparable levels of salvage were observed
with the KATP opener BMS-180448 in both dogs and ferrets
when administered before and during the ischemic interval. When 40-min
BMS-180448 infusion was withheld in the ferret until the 5th min of
reperfusion, a marginal (17%), but significant cardioprotective effect
was obtained. Efficacy was therefore still demonstrable even when drug
administration was withheld until after the initiation of reperfusion.
This in vivo observation is thus consistent with that found
in vitro in the globally ischemic rat heart.
Evidence that the cardioprotective effect of BMS-180448 in the present
rat and ferret studies was caused by KATP channel
activation was provided by observations that it was abolished, both
in vitro and in vivo, by pretreatment with
glyburide, a known antagonist/inhibitor of this channel. Both glyburide
and sodium 5-hydroxydecanoate, another known inhibitor of
KATP, also reversed the protective effects of aprikalim
(Auchampach et al., 1991; Grover et al., 1990b)
and cromakalim (Grover et al., 1990c; McCullough et
al., 1991).
It is uncertain if BMS-180448 is attenuating reperfusion injury, or if
there is on-going ischemia, that it may be due to no-reflow even with
reperfusion. If underperfusion is observed during reflow then
BMS-180448 may be working by attenuating ischemic damage. BMS-180448
has been shown to enhance reperfusion ATP protection, which suggests
mitochondrial protection (Grover et al., in press). Pieper
and Gross (1992) claim that KATP openers reduce PMN
function and this could explain their reperfusion effects. In those
studies (Gross et al., 1992; Pieper and Gross, 1992),
nicorandil and bimakalim were shown to inhibit neutrophil superoxide
production. Our results are in crystalloid perfused hearts, and
PMN-related mechanisms may not be operative. Electron micrographs of
isolated rats hearts do not show neutrophils (Monticello et
al., 1996).
The present results also indicate that, in addition to a lack of effect
on hemodynamics, the reductions in tissue damage associated with
BMS-180448 administration were not caused either by alterations in
collateral blood flow during ischemia or by enhanced flow into the
ischemic region during reperfusion. The measured relative shifts in
myocardial blood flow toward the subepicardium are consistent with
drug-associated changes observed in normal ferrets and dogs (Weselcouch et al., 1994), as well as in canine
ischemia studies (Grover et al., 1995b).
The favorable cardioprotective effects and lack of hemodynamic flow
actions of BMS-180448 in the ferret are thus consistent with
observations made in canine models. The present data go one step
further in demonstrating the persistence of significant
cardioprotective actions when drug administration is initiated at not
only the very late stages of the occlusive interval, but also at the
early stages of reperfusion.
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Abstract
Introduction
-difluoro-1,2-bis(2-bis(2-aminophenoxy)ethane-N,N,N
