Method for simulating heart failure

A method of chronically instrumenting an animal enabling one to simulate congestive heart failure. A method for assessing the effects of a test compound on cardiac function and systemic vascular dynamics.

BACKGROUND OF THE INVENTION 
The preclinical assessment of agents for the treatment of heart failure has 
been hampered by the lack of appropriate animal models. Previous models 
have either utilized non-clinically relevant insults to induce the disease 
state, or have failed to produce controllable, stable and predictable 
damage. Pressure overloading to induce ventricular hypertrophy and 
failure, produced by a variety of techniques including corticosteroid 
administration, renal artery occlusion, unilateral nephrectomy with 
contralateral occlusion of the renal artery, and most extensively banding 
of major outflow tracts such as the aorta, has been used in a variety of 
species including rat, cat and dog (Smith and Nutall, Cardiovascular 
Research 19: 181-186, 1985). 
However, acute severe fixed afterload augmentation in animal models 
probably differs significantly from the gradual events that occur with 
pressure overload failure in man. The major limitation of animal pressure 
overload models include the propensity for the development of hypertrophy 
but not failure and/or a protracted time frame for the development of 
failure. Volume overloading produced by arteriovenous fistulae and 
valvular incompetence has been used to induce heart failure in dogs; 
however, this method has been limited by difficulty in controlling the 
degree of cardiac damage (Smith and Nutall, Cardiovascular Research 19: 
181-186, 1985). 
Cardiotoxic agents, including doxorubicin, have been used in several 
species including rat and dog to induce heart failure. This approach is 
limited by difficulty in controlling dose of cardiotoxic agent to induce 
sufficient but not excessive damage, extracardiac toxicity and the 
production of calcium overload-injury that may render the model unsuitable 
for the assessment of positive inotropic agents (Czarnecki, Comparative 
Biochemistry and Physiology 79C: 9-14, 1984; Smith and Nutall, 
Cardiovascular Research 19: 181-186, 1985). Several experimental 
procedures have been utilized to effect coronary artery occlusion, 
myocardial ischemia and resultant heart failure primarily in rats and 
dogs. These procedures include direct coronary ligation, embolism with 
liquid mercury, injection of preformed thrombus, wedged catheters, and 
sequential coronary microembolization with microspheres (Khomaziuk et al, 
Kardiologiya 5: 19-23, 1965; Rees and Redding, Cardiovascular Research 2: 
43-53, 1968; Lumicao et al, American Journal of Medical Science 261: 
27-40, 1971; Millner et al, Annals of Thoracic Surgery 52: 78-83, 1991; 
Sabbah et al, American Journal of Physiology 260: H1379-H1384, 1991). 
Problems associated with coronary artery ligation/ischemia models of heart 
failure include the inability of ischemic rodent models to develop 
myocardial dysfunction which meets the hemodynamic criteria of heart 
failure, as well as a high degree of malignant arrhythmia and mortality 
associated with myocardial ischemia. Damage to the heart from repeated DC 
shocks has been shown to induce heart failure in dogs (McDonald et al, 
Journal of the American College of Cardiology 19: 460-467, 1992); however 
the clinical relevance of this method of damage is uncertain. 
Recently, several laboratories have adopted the method of rapid ventricular 
pacing-induced heart failure in dogs (Riegger and Liebau, Clinical Science 
62: 465-469, 1982). One prominent criticism of the pacing-induced dog 
failure model is that while it does induce a predictable, controllable 
degree of myocardial failure, this condition is reversible with the 
termination of pacing. Also, the underlying mechanism for the development 
of failure in the pacing model is not understood at this time. 
SUMMARY OF THE INVENTION 
This invention relates to a method for determining whether a test compound 
is therapeutically useful for the treatment of heart failure, including 
but not limited to congestive heart failure. The assay employs a 
chronically instrumented conscious pig with heart failure wherein the 
heart failure is induced by myocardial ischemia and intermittent rapid 
cardiac pacing and provides a method for assessing the effects of the test 
compound on cardiac function and systemic vascular function.

DETAILED DESCRIPTION OF THE INVENTION 
The invention relates to a method for simulating heart failure in a 
conscious, instrumented animal comprising the steps of: 
(a) producing myocardial ischemia in the instrumented animal; and 
(b) introducing rapid cardiac pacing in the instrumented animal, until the 
hemodynamic measurements, wherein hemodynamic measurements include: left 
atrial pressures; arterial pressure; aortic blood flow; total peripheral 
resistance; LV end-diastolic dimension; LV end systolic dimension; 
fractional shortening, mean velocity of circumferential fibre shortening 
and regional blood flows by radiolabeled microspheres; are indicative of 
heart failure. 
An embodiment of this invention relates to the method for simulating heart 
failure in a conscious, instrumented animal as recited above, wherein 
production of myocardial ischemia comprises the steps of: 
(a) inflating the distally implanted hydraulic occluder to occlude the 
distal left circumflex coronary artery about 7 to 10 days after the 
surgery to instrument the animal; 
(b) waiting about 6 hours to 30 days after the distal left circumflex 
coronary artery occlusion; and 
(c) inflating the proximally implanted hydraulic occluder to occlude the 
proximal left circumflex coronary artery. 
A further embodiment of this invention relates to the method for simulating 
heart failure in a conscious, instrumented animal as recited above, 
wherein the rapid cardiac pacing comprises the steps of: 
(a) using a cardiac pacemaker to introduce rapid cardiac pacing at a rate 
of about 190-210 beats/minute for about 1 to 7 days; 
(b) waiting for about 0 to 72 hours after the first pacing; and 
(c) repeating steps (a) and (b) at least until the hemodynamic 
measurements, wherein hemodynamic measurements include: left atrial 
pressures; arterial pressure; aortic blood flow; total peripheral 
resistance; LV end-diastolic dimension; LV end systolic dimension; 
fractional shortening, mean velocity of circumferential fibre shortening 
and regional blood flows by radiolabeled microspheres; are indicative of 
heart failure. 
The invention relates to the method for simulating heart failure in a 
conscious, instrumented animal as recited above, wherein the rapid cardiac 
pacing is introduced in the left or right ventricle. 
The invention relates to the method for simulating heart failure in a 
conscious, instrumented animal as recited above, wherein the rapid cardiac 
pacing is introduced in the left or right atrium. 
The invention relates to the method for simulating heart failure in a 
conscious, instrumented animal as recited above, wherein the rapid cardiac 
pacing is introduced in the right ventricle. 
The invention relates to the method for simulating heart failure in a 
conscious, instrumented animal as recited above, wherein the instrumented 
animal is selected from the group consisting of: pig, primate, including 
monkey and baboon, rabbit, cat, dog, sheep, goat, horse and cow. 
The invention relates to the method for simulating heart failure in a 
conscious, instrumented animal as recited above, wherein the instrumented 
animal is mobile. 
The invention relates to the method for simulating heart failure in a 
conscious, instrumented animal as recited above, wherein the instrumented 
animal is immobile. 
The invention relates to the method for simulating heart failure in a 
conscious, instrumented animal as recited above, wherein the instrumented 
animal is a pig. 
The invention relates to a method for simulating heart failure in an 
immobilized, conscious, instrumented pig comprising the steps of: 
(a) inflating the distally implanted hydraulic occluder to occlude the 
distal left circumflex coronary artery about 7 to 10 days after the 
surgery to instrument the animal; 
(b) waiting about 6 hours to 30 days after the distal left circumflex 
coronary artery occlusion; and 
(c) inflating the proximally implanted hydraulic occluder to occlude the 
proximal left circumflex coronary artery. 
(d) waiting about 24 to about 48 hours after the proximal left circumflex 
coronary artery occlusion; 
(e) using a cardiac pacemaker to introduce rapid cardiac pacing at a rate 
of about 190-210 beats/minute for about 1 to 7 days; 
(f) waiting for about 0 to 72 hours after the first pacing; and 
(g) repeating steps (e) and (f) at least until the hemodynamic 
measurements, wherein hemodynamic measurements include: left atrial 
pressures; arterial pressure; aortic blood flow; total peripheral 
resistance; LV end-diastolic dimension; LV end systolic dimension; 
fractional shortening, mean velocity of circumferential fibre shortening 
and regional blood flows by radiolabeled microspheres; are indicative of 
heart failure. 
The invention relates to the method for simulating heart failure in an 
immobilized, conscious, instrumented pig as recited above, which comprises 
introducing rapid cardiac pacing in the left or right ventricle. 
The invention relates to the method for simulating heart failure in an 
immobilized, conscious, instrumented pig as recited above, which comprises 
introducing rapid cardiac pacing in the left or right atrium. 
The invention relates to the method for simulating heart failure in an 
immobilized, conscious, instrumented pig as recited above, which comprises 
introducing rapid cardiac pacing in the right ventricle. 
The invention relates to a method for assessing whether a test compound is 
therapeutically useful for the treatment of heart failure in an animal 
with simulated heart failure comprising the steps of: 
(a) recording hemodynamic measurements, wherein hemodynamic measurements 
include: left atrial pressures; arterial pressure; aortic blood flow; 
total peripheral resistance; LV end-diastolic dimension; LV end systolic 
dimension; fractional shortening, mean velocity of circumferential fibre 
shortening and regional blood flows by radiolabeled microspheres; 
continuously before injection of the test compound in the animal with 
simulated heart failure; 
(b) administering a dose of the test compound in the animal with simulated 
heart failure; and 
(c) recording hemodynamic measurements continuously for about 30 minutes to 
6 weeks; 
(d) repeating steps (a)-(c), administering a placebo dose in the animal 
with simulated heart failure as a control measurement; and 
(e) comparing the hemodynamic measurements of the test compound with the 
placebo control to determine if the hemodynamic burden on the heart and 
any associated congestion has been relieved by administration of the test 
compound. 
The invention relates to the method for assessing whether a test compound 
is therapeutically useful for the treatment of heart failure in an animal 
with simulated heart failure as recited above, wherein the animal with 
simulated heart failure is selected from the group consisting of pig, 
primate, including monkey, baboon, rabbit, cat, dog, sheep, goat, horse 
and cow. 
The invention relates to the method for assessing whether a test compound 
is therapeutically useful for the treatment of heart failure in an animal 
with simulated heart failure as recited above, wherein the method of 
administration of the test compound is selected from the group consisting 
of: oral, intravenous, intramuscular or subcutaneous, single and multiple 
doses. 
The invention relates to the method for assessing whether a test compound 
is therapeutically useful for the treatment of heart failure in an animal 
with simulated heart failure as recited above, wherein the animal with 
simulated heart failure is mobile. 
The invention relates to the method for assessing whether a test compound 
is therapeutically useful for the treatment of heart failure in an animal 
with simulated heart failure as recited above, wherein the hemodynamic 
measurements are recorded using instrumentation with telemetry. 
The invention relates to the method for assessing whether a test compound 
is therapeutically useful for the treatment of heart failure in an animal 
with simulated heart failure as recited above, wherein the animal with 
simulated heart failure is immobile. 
The invention relates to the method for assessing whether a test compound 
is therapeutically useful for the treatment of heart failure in an animal 
with simulated heart failure as recited above, wherein the method of 
administration of the test compound is oral or intravenous, single and 
multiple doses. 
The invention relates to the method for assessing whether a test compound 
is therapeutically useful for the treatment of heart failure in an animal 
with simulated heart failure as recited above, wherein the method of 
administration of the test compound is oral, single and multiple doses. 
The invention relates to the method for assessing whether a test compound 
is therapeutically useful for chronic treatment of heart failure in an 
animal with simulated heart failure as recited above, wherein the oral 
dose is about 0.1 .mu.g/kg to 100 mg/kg, single and multiple doses. 
The invention relates to the method for assessing whether a test compound 
is therapeutically useful for the treatment of heart failure in an animal 
with simulated heart failure as recited above, wherein the method of 
administration of the test compound is intravenous, single and multiple 
doses. 
The invention relates to the method for assessing whether a test compound 
is therapeutically useful for the treatment of heart failure in an animal 
with simulated heart failure as recited above, wherein the intravenous 
dose is about 0.1 .mu.g/kg to 10 mg/kg, single and multiple doses. 
The invention relates to the method for assessing whether a test compound 
is therapeutically useful for the treatment of heart failure in an animal 
with simulated heart failure as recited above, wherein the animal with 
simulated heart failure is a pig. 
The invention relates to the method for assessing whether a test compound 
is therapeutically useful for the treatment of heart failure in an animal 
with simulated heart failure as recited above, wherein the hemodynamic 
measurements are recorded continuously for about 30 minutes to 24 hours. 
The invention relates to the method for assessing whether a test compound 
is therapeutically useful for the treatment of heart failure in an animal 
with simulated heart failure as recited above, wherein the hemodynamic 
measurements are recorded continuously for about 30 minutes to 8 hours. 
The invention relates to the method for assessing whether a test compound 
is therapeutically useful for the treatment of heart failure in an animal 
with simulated heart failure as recited above, wherein the hemodynamic 
measurements are recorded continuously for about 90 minutes to 180 
minutes. 
The invention relates to a method for assessing whether a test compound is 
therapeutically useful for chronic treatment of heart failure in a 
conscious, instrumented animal comprising the steps of: 
(a) administering the test compound to a conscious, instrumented animal 
prior to or during: (1) the production of myocardial ischemic injury 
and/or (2) the introduction of rapid cardiac pacing until the hemodynamic 
measurements are indicative of heart failure, wherein hemodynamic 
measurements include: left atrial pressures; arterial pressure; aortic 
blood flow; total peripheral resistance; LV end-diastolic dimension; LV 
end systolic dimension; fractional shortening, mean velocity of 
circumferential fibre shortening and regional blood flows by radiolabeled 
microspheres; 
(b) recording the hemodynamic measurements continuously for about 30 
minutes to 6 weeks of the conscious, instrumented animal; 
(d) repeating steps (a)-(b), administering a placebo dose to a conscious, 
instrumented animal as a control measurement; and 
(e) comparing the hemodynamic measurements of the test compound with the 
placebo control to determine if the hemodynamic burden on the heart and 
any associated congestion has been relieved by administration of the test 
compound. 
The invention relates to the method for assessing whether a test compound 
is therapeutically useful for chronic treatment of heart failure in a 
conscious, instrumented animal as recited above, wherein the animal is 
selected from the group consisting of pig, primate, including monkey and 
baboon, rabbit, cat, dog, sheep, goat, horse and cow. 
The invention relates to the method for assessing whether a test compound 
is therapeutically useful for chronic treatment of heart failure in a 
conscious, instrumented animal as recited above, wherein the method of 
administration of the test compound is selected from the group consisting 
of: oral, intravenous, intramuscular or subcutaneous, single or multiple 
doses. 
The invention relates to the method for assessing whether a test compound 
is therapeutically useful for chronic treatment of heart failure in a 
conscious, instrumented animal as recited above, wherein the animal with 
simulated heart failure is mobile. 
The invention relates to the method for assessing whether a test compound 
is therapeutically useful for chronic treatment of heart failure in a 
conscious, instrumented animal as recited above, wherein the hemodynamic 
measurements are recorded using instrumentation with telemetry. 
The invention relates to the method for assessing whether a test compound 
is therapeutically useful for chronic treatment of heart failure in a 
conscious, instrumented animal as recited above, wherein the animal with 
simulated heart failure is immobile. 
The invention relates to the method for assessing whether a test compound 
is therapeutically useful for chronic treatment of heart failure in a 
conscious, instrumented animal as recited above, wherein the method of 
administration of the test compound is oral or intravenous, single or 
multiple doses. 
The invention relates to the method for assessing whether a test compound 
is therapeutically useful for chronic treatment of heart failure in a 
conscious, instrumented animal as recited above, wherein the method of 
administration of the test compound is oral, single or multiple doses. 
The invention relates to the method for assessing whether a test compound 
is therapeutically useful for chronic treatment of heart failure in a 
conscious, instrumented animal as recited above, wherein the oral dose is 
about 0.1 .mu.g/kg to 100 mg/kg, single or multiple doses. 
The invention relates to the method for assessing whether a test compound 
is therapeutically useful for chronic treatment of heart failure in a 
conscious, instrumented animal as recited above, wherein the method of 
administration of the test compound is intravenous, single or multiple 
doses. 
The invention relates to the method for assessing whether a test compound 
is therapeutically useful for chronic treatment of heart failure in a 
conscious, instrumented animal as recited above, wherein the intravenous 
dose is about 0.1 .mu.g/kg to 10 mg/kg, single or multiple doses. 
The invention relates to the method for assessing whether a test compound 
is therapeutically useful for chronic treatment of heart failure in a 
conscious, instrumented animal as recited above, wherein the animal is a 
pig. 
An instrumented animal is defined as an animal that has undergone surgery 
to implant the hardware necessary to induce heart failure and to obtain 
the hemodynamic measurements. The hemodynamic measurements include: left 
atrial pressures; arterial pressure; aortic blood flow; total peripheral 
resistance; LV end-diastolic dimension; LV end systolic dimension; 
fractional shortening and mean velocity of circumferential fibre 
shortening. Additionally, an echocardiogram can be utilized to measure the 
hemodynamic effects on the heart. The procedure described in Example 1 
represents a method of instrumenting an animal, specifically a pig. FIG. 8 
is a drawing of the instrumentation utilized in Example 1. This method of 
simulating heart failure is understood to include all known ways of 
producing myocardial ischemia including the coronary hydraulic occulsion 
procedure described in Example 1 and a microsphere microembolization 
procedure, preferably the coronary hydraulic occulsion procedure. 
The rapid cardiac pacing is introduced using a cardiac pacemaker. The 
pacing can be done continuously or in cycles of 1 to 7 days with breaks of 
up to 72 hours at least until heart failure is evident. Heart failure is 
typically evident after 2 to 3 cycles of 7 days of rapid cardiac pacing 
with breaks of about 72 hours. Continous rapid cardiac pacing after heart 
failure is evident, is also within the scope of this invention. 
In smaller "immobilized" animals (e.g. rabbit or cat in an activity box) in 
the absence of telemetry, one can record hemodynamics for 0.5-24 hours 
after dosing. With larger animals "immobilized" animals (e.g. dog, pig, 
primate, cow or horse) in a sling or chair in the absence of telemetry, 
one can record hemodynamics for 0.5-8 hours after dosing. The hemodynamic 
measurements can be recorded continuously using instrumentation with 
telemetry, for experiments set up to evaluate the effects of chronic 
treatment with a test compound on heart failure or the prevention of heart 
failure. 
The animals can be instrumented such that they are mobile during the 
simulation of heart failure and during the adminstration of the test 
compound and hemodynamic measurements are recorded using instrumentation 
with telemetry. Alternatively, the animals are immobilized and hemodynamic 
measurements are recorded directly from the wire probes leading from the 
heart. The experiments carried out on immobilized animals are generally 
carried out over about 3 to 8 hours with hemodynamic measurements taken 
for upwards of 3 hours. The hemodynamic measurements can be recorded 
continuously for upwards of about 6 weeks with animals that are mobile 
using intrumentation with telemetry, for experiments set up to evaluate 
the effects of chronic treatment with a test compound on heart failure or 
the prevention of heart failure. 
The test compound is administered in a pharmacologically effect dose range, 
and will depend on the potency of the individual test compound, the weight 
of the animals and the route of adminstration. The method of 
administration of the test compound includes: oral (p.o.), intravenous 
(i.v.), intramuscular (i.m.) or subcutaneous (s.c.). The preferred dose 
range would encompass about 0.1 .mu.g/kg to 10 mg/kg i.v. or i.m. and 
about 0.1 .mu.g/kg to 100 mg/kg p.o. or s.c. Test compounds could be 
administered as acute single doses (i.v., i.m., s.c. or p.o.) after the 
establishment of heart failure, or chronically with multiple doses (i.v., 
i.m., s.c. or p.o.) either after the establishment of heart failure or 
before the establishment of heart failure (even preceding surgical 
preparation, myocardial ischemia or rapid ventricular pacing) to assess 
the potential of the test compound to prevent heart failure. For purposes 
of comparision the instrumented animals are administered a placebo dose. 
The placebo dose comprising in the case of intravenous administration the 
vehicles used to solubilize the test compound or in the case of oral 
administration the binding agents used to formulate the test compound for 
oral administration. 
The instant invention can be understood further by the following examples, 
which do not constitute a limitation of the invention. 
EXAMPLE 1 
Step A: Implantation of Instrumentation 
Five farm pigs of either of sex and weighing 34.5.+-.2.5 kg were sedated 
with ketamine hydrochloride (25 mg/kg, i.m.) and xylazine (6 mg/kg, i.m.). 
After tracheal intubation, general anesthesia was maintained with 
isoflurane (1.5-2.0 vol % in oxygen). Using sterile surgical technique, a 
left thoracotomy was performed at the fifth intercostal space. Catheters 
made of Tygon tubing (Norton Performance Plastics Co., Akron, Ohio) were 
implanted in the descending aorta, left and right atria for measurement of 
pressures. A solid-state miniature pressure gauge (Konigsberg Instruments 
Inc., Pasadena, Calif.) was implanted in the left ventricular (LV) chamber 
to obtain LV pressure and the rate of change of LV pressure (LV dP/dt). A 
flow probe (Transonic System Inc., Ithaca, N.Y.) was placed around the 
main pulmonary artery for measurement of blood flow. One pair of 
piezoelectric ultrasonic dimension crystals were implanted on opposing 
anterior and posterior endocardial regions of the LV to measure the 
short-axis internal diameter. Proper alignment of the endocardial crystals 
was achieved during surgical implantation by positioning the crystals so 
as to obtain a signal with the greatest amplitude and shortest transit 
time. A pacing lead (model 5069, Medtronic Inc., Minneapolis, Minn.) was 
attached to the right ventricular free wall, and stainless steel pacing 
leads were attached to the left atrial appendage. The left circumflex 
coronary artery was isolated and two hydraulic occluders, made of Tygon 
tubing, were implanted proximally and distally to the first obtuse 
marginal branch. The wires and catheters were externalized between the 
scapulae, the incision was closed in layers, and air was evacuated from 
the chest cavity. See FIG. 8. 
Step B: Experimental Measurements 
Hemodynamic recordings were made using a data tape recorder (RD-130TE, 
TEAC, Montebello, Calif.) and a multiple-channel oscillograph (MT95K2, 
Astro-Med, West Warwick, R.I.). Aortic and left atrial pressures were 
measured using strain gauge manometers (Statham Instruments, Oxnard, 
Calif.), which were calibrated in vitro using a mercury manometer, 
connected to the fluid-filled catheters. The solid-state LV pressure gauge 
was cross-calibrated with aortic and left atrial pressure measurements. LV 
dP/dt was obtained by electronically differentiating the LV pressure 
signal. Blood flow was measured using a volume flow meter (T208, Transonic 
System Inc., Ithaca, N.Y.). Mean arterial pressure, left atrial pressures, 
and pulmonary blood flow (cardiac output) were measured using an amplifier 
filter. Stroke volume was calculated as the quotient of cardiac output and 
heart rate. Cardiac output was normalized by body weight to yield cardiac 
index. LV dimension was measured with an ultrasonic transit-time dimension 
gauge (Model 203, Triton Technology Inc., San Diego, Cailf.). Total 
peripheral resistance was calculated as the quotient of mean arterial 
pressure and cardiac output. LV short-axis end-diastolic dimension (EDD) 
was measured at the beginning of the upstroke of the LV dP/dt signal. LV 
end-systolic dimension (ESD) was measured at the time of maximum negative 
dP/dt. The percent shortening of LV internal diameter was calculated as 
(EDD-ESD)/EDD*100. LV mean velocity of circumferential fibre shortening 
(Vcf) was calculated from the dimension measurements using the following 
formula:(EDD-ESD)/EDD/Ejection time (sec.sup.-1). Ejection time was 
measured as the interval between maximum and minimum LV dP/dt. A 
cardiotachometer triggered by the LV pressure pulse provided instantaneous 
and continuous records of heart rate. 
Step C: Heart Failure Model 
Experiments were initiated 10-14 days after surgery, when the pigs were 
fully recovered from surgery. During the post-operative period, the pigs 
were introduced to a sling for training. Heart failure was produced by 
progressive myocardial ischemia induced by two coronary artery occlusions 
followed by intermittent ventricular pacing. Briefly, after post-surgical 
hemodynamic control monitoring was performed, the left circumflex coronary 
artery was occluded by inflating the distally implanted hydraulic 
occluder. Approximately 48 hrs after the first occlusion, the proximal 
coronary artery occluder was inflated. One to two days following the 
second myocardial infarction, the right ventricle was paced at a rate of 
190-210 beats/min using a programmable external cardiac pacemaker (model 
EV4543, Pace Medical, Waltham, Mass.). Pacing was continued for 1 week and 
then terminated for 3 days. This procedure was repeated another 1-2 
cycles, until heart failure was evident and the hemodynamic parameters 
were stable. 
Step D: Experimental Protocols 
Hemodynamic experiments were performed after 2 cycles of tachycardic pacing 
in the presence of myocardial ischemia injury, after the animal had 
achieved a stable state of heart failure. During the experiments, the pigs 
were conscious and quietly restrained in a sling. The test compound is 
dissolved in saturated NaHCO.sub.3 (10% by Vol) and 0.9% saline (90% by 
Vol) at a concentration of 2 mg/ml, was studied in four conscious pigs 
with heart failure. A dose of 0.5 mg/kg was injected intravenously over a 
period of 2 minutes, and hemodynamics were continuously recorded before 
and 90 minutes following injection of the test compound in all 4 pigs. In 
two of these pigs, recording continued until 3 hours following injection 
of the test compound. The vehicle was tested on separate days. The effects 
of 0.1 to 0.5 .mu.g/kg cumulative bolus doses in 0.1 .mu.g/kg steps of 
endothelin-1 (Peptide Institute, Inc., Osaka, Japan) were also examined on 
separate days before and 90 minutes after injection of the test compound 
in three pigs during development of heart failure to characterize the 
extent of endothelin block by the test compound. ET-1 was dissolved in 0.1 
N NaHCO.sub.3 (95% by Vol) and 0.9% saline (95% by Vol) at a concentration 
of 20 .mu.g/ml. 
Step E: Data Analysis 
All data were stored on an AST 4/d computer. Data before and after 
development of heart failure were compared using Student's t-test for 
paired data. The data at baseline and after injection of the test compound 
also were compared using Student's t-test for paired data with a 
Bonferroni correction. All values are expressed as the mean.+-.S.E. 
Statistical significance was accepted at the p&lt;0.05 level. 
EXAMPLE 2 
Experimental Protocol Using an Endothelin Antagonist: Compound 1 
Step A: Hemodynamic Study with Compound 1 
Hemodynamic experiments were performed after 2 cycles of tachycardic pacing 
in the presence of myocardial ischemia injury, after the animal had 
achieved a stable state of heart failure. During the experiments, the pigs 
were conscious and quietly restrained in a sling. Compound 1, ((+)-(5S*, 
6R*, 
7R*)-2-butyl-6-carboxy-7-2-(2-carboxypropyl)-4-methoxyphenyl!-5-(3,4-meth 
ylenedioxyphenyl)-cyclopenteno1,2-b!pyridine), dissolved in saturated 
NaHCO.sub.3 (10% by Vol) and 0.9% saline (90% by Vol) at a concentration 
of 2 mg/ml, was studied in four conscious pigs with heart failure. A dose 
of 0.5 mg/kg was injected intravenously over a period of 2 minutes, and 
hemodynamics were continuously recorded before and 90 minutes following 
injection of Compound 1 in all 4 pigs. In two of these pigs, recording 
continued until 3 hours following injection of Compound 1. The vehicle was 
tested on separate days. The effects of 0.1 to 0.5 .mu.g/kg cumulative 
bolus doses in 0.1 .mu.g/kg steps of endothelin-1 (Peptide Institute, 
Inc., Osaka, Japan) were also examined on separate days before and 90 
minutes after injection of Compound 1 in three pigs during development of 
heart failure to characterize the extent of ET block by Compound 1. ET-1 
was dissolved in 0.1 N NaHCO.sub.3 (95% by Vol) and 0.9% saline (95% by 
Vol) at a concentration of 20 .mu.g/ml. For comparative purpose, the 
effects of intravenous injection of Compound 2(enalaprilat), at doses of 1 
mg/kg and 4 mg/kg, were studied on different days in three of the pigs 
that were used to study Compound 2 and in one additional pig. Compound 2 
was dissolved in 0.9% saline. 
Step B: Baseline Hemodynamics Before and After Development of Heart Failure 
Tables 1 and 2 summarize the baseline LV function and systemic vascular 
dynamics before (i.e., post surgical control) and after heart failure 
induced by serial myocardial infarctions in combination with intermittent 
tachycardic stress in conscious pigs. Heart failure resulting from at 
least 2 cycles of tachycardic pacing in the presence of myocardial injury 
was manifested by significant increases in LV end-diastolic (+10.7.+-.0.4 
mm from 40.2.+-.3.6 mm) and end-systolic diameters (+14.6.+-.1.1 mm from 
31.2.+-.2.7 mm) and in mean left atrial pressure (+19.+-.3 mmHg from 
4.+-.2 mmHg). LV dP/dt, LV fractional shortening, Vcf, and cardiac index 
significantly decreased by 45.+-.4, 54.+-.6, 46.+-.1 and 29.+-.8%, 
respectively. Also, total peripheral resistance increased significantly 
(+46.+-.14%), while mean arterial pressure and heart rate were unchanged. 
In addition to these hemodynamic changes, which are shown in FIGS. 1 and 
2, heart failure, particularly at the advanced stages, was characterized 
by anorexia, peripheral and pulmonary edema, and reduced activity. 
TABLE 1 
______________________________________ 
Baseline Left Ventricular Function in Controls and 
Conscious Pigs with Heart Failure. 
Control Heart Failure 
______________________________________ 
LV End-Diastolic Diameter (mm) 
40.2 .+-. 3.6 
50.9 .+-. 3.5* 
LV End-Systolic Diameter (mm) 
31.2 .+-. 2.7 
45.8 .+-. 3.6* 
LV Fractional Shortening (%) 
22.3 .+-. 1.5 
10.3 .+-. 1.3* 
Vcf (sec.sup.-1) 1.1 .+-. 0.1 
0.6 .+-. 0.0* 
LV dP/dt (mmHg/sec) 
3012 .+-. 153 
1681 .+-. 184* 
______________________________________ 
*Significantly different from control, p&lt;0.05. 
Data are mean .+-. SE with n = 3, except that LV dP/dt n = 4. 
TABLE 2 
______________________________________ 
Baseline Cardiac and Systemic Hemodynamics in Controls 
and Conscious Pigs with Heart Failure. 
Control Heart Failure 
______________________________________ 
Mean Arterial Pressure (mmHg) 
90 .+-. 3 88 .+-. 5 
Mean Left Atrial Pressure (mmHg) 
4 .+-. 2 23 .+-. 3* 
Cardiac Index (ml/min/kg) 
123 .+-. 11 86 .+-. 9* 
Total Peripheral Resistance 
0.73 .+-. 0.06 
1.04 .+-. 0.09* 
(mmHg/ml/min/kg) 
Heart Rate (beat/min) 
139 .+-. 4 143 .+-. 15 
______________________________________ 
*Significantly different from control, p&lt;0.05. 
Data are mean .+-. SE with n = 4. 
Step C: Effects of Compound 1 on Hemodynamics in Heart Failure 
The time course of hemodynamic changes following intravenous administration 
of Compound 1 (0.5 mg/kg) are shown in FIGS. 3 and 4 Tables 3 and 4 
summarize LV function and systemic hemodynamic responses to Compound 1 at 
15 minutes and 60 minutes after administration of Compound 1. FIGS. 1 and 
2 illustrate the cardiac and systemic dynamic measurements made before and 
after development of heart failure, as well as 60 minutes after 
administration of Compound 1 during the heart failure stage. 
Compound 1 mainly induced a sustained increase in cardiac index, and 
prolonged decreases in mean arterial pressure and total peripheral 
resistance. For example, at 60 minutes after administration of Compound 1, 
mean arterial pressure was significantly decreased by 10.+-.2% and cardiac 
index was increased by 17.+-.4%. Thus, total peripheral resistance was 
significantly decreased by 22.+-.3% with Compound 1, which basically 
constitutes a complete restoration of the elevated vascular resistance of 
heart failure back to pre-heart failure control values (FIG. 1). Although 
Compound 1 also decreased left atrial pressure and increased heart rate, 
these changes were not statistically significant. Vcf was increased by 
12.+-.2%, while LV dP/dt, LV end-diastolic and systolic diameter, and LV 
fraction shortening were not affected by Compound 1. The vehicle did not 
induce any significant changes throughout 180 minutes of observation 
(FIGS. 3 and 4). 
The salutary effects of acute administration of Compound 1 in this heart 
failure model were predominantly due to the reversal of elevated vascular 
resistance. 
TABLE 3 
______________________________________ 
Effects of Intravenous Injection of Compound 1 (0.5 mg/kg) 
on LV Function in Conscious Pigs with Heart Failure. 
Change from Baseline 
Baseline 
15 min 60 min 
______________________________________ 
LV End-Diastolic 
50.9 .+-. 3.5 
+0.1 .+-. 0.2 
+0.2 .+-. 0.3 
Diameter (mm) 
LV End-Systolic 
45.8 .+-. 3.6 
-0.3 .+-. 0.3 
-0.2 .+-. 0.3 
Diameter (mm) 
LV Fractional 
10.3 .+-. 1.3 
+0.6 .+-. 0.2 
+0.7 .+-. 0.2 
Shortening (%) 
Vcf (sec.sup.-1) 
0.60 .+-. 0.04 
0.05 .+-. 0.01* 
+0.07 .+-. 0.02* 
LV dP/dt (mmHg/sec) 
1681 .+-. 184 
+25 .+-. 40 +56 .+-. 65 
______________________________________ 
*Significantly different from baseline, p&lt;0.025. 
Data are mean .+-. SE with n = 3, except that LV dP/dt n = 4. 
TABLE 4 
______________________________________ 
Effects of Intravenous Injection of Compound 1 (0.5 mg/kg) 
on Cardiac and Systemic Hemodynamics in Conscious Pigs 
with Heart Failure. 
Change from Baseline 
Baseline 
15 min 60 min 
______________________________________ 
Left Atrial Pressure 
23 .+-. 3 -4 .+-. 2 -2 .+-. 2 
(mmHg) 
Mean Arterial Pressure 
88 .+-. 5 -10 .+-. 1* 
-8 .+-. 1* 
(mmHg) 
Cardiac Index 
86 .+-. 9 +14 .+-. 2* 
+13 .+-. 2* 
(ml/min/kg) 
Total Peripheral Resist- 
1.04 .+-. 0.09 
-0.25 .+-. 0.03* 
-0.24 .+-. 0.05 
ance (mmHg/ml/min/kg) 
Heart Rate (beat/min) 
143 .+-. 15 
+5 .+-. 5 +7 .+-. 5 
______________________________________ 
*Significantly different from baseline, p&lt;0.025. 
Data are mean .+-. SE with n = 4. 
Step D: Effects of Compound 1 Compared with Compound 2 in Heart Failure 
FIG. 5 compares the effects of intravenous administration of Compound 1 
(0.5 mg/kg) and Compound 2 (enalaprilat) (1 mg/kg or 4 mg/kg) on total 
peripheral resistance in conscious pigs with heart failure. While the 
effects were not dose-dependent, suggesting maximal effect, both doses of 
Compound 2 did significantly reduce the total peripheral resistance to a 
similar level throughout 90-minute observation period. The reduction in 
total peripheral resistance by administration of Compound 1 (0.5 mg/kg) 
was greater than that induced by either dose of Compound 2. FIG. 6 
compares the changes in mean arterial pressure, LV dP/dt, cardiac output 
and total peripheral resistance at 60 minutes after administration of 
Compound 1 or Compound 2. 
Step E: Effects of ET-1 in the Absence and Presence of Compound 1 in Heart 
Failure 
FIG. 7 shows the effects of cumulative intravenous bolus injections of ET-1 
(total dose of 0.5 .mu.g/kg) on mean arterial pressure, mean left atrial 
pressure, total peripheral resistance and heart rate before and after 
intravenous administration of Compound 1 at a dose of 0.5 mg/kg. ET-1 
induced significant dose-dependent increases in mean arterial pressure, 
mean left atrial pressure and total peripheral resistance. Heart rate 
decreased, but not dose-dependently. The hemodynamic responses to ET-1 
were markedly attenuated after administration of Compound 1, suggesting 
the 0.5 mg/kg, i.v. dose of Compound 1 to be capable of significant 
blocking the effects of exogenous ET-1. 
Compound 1, at a dose of 0.5 mg/kg, i.v. reduced the elevated vascular 
resistance but did not affect myocardial contractility in conscious pigs 
with heart failure. This acute effect of Compound 1 was greater than that 
of 1 mg/kg or 4 mg/kg, i.v. Compound 2. The salutary effects of Compound 1 
in this heart failure model were attributed to ET receptor antagonism 
since the hemodynamic responses to an ET-1 challenge were markedly 
attenuated by the same dose of Compound 1. 
EXAMPLE 3 
Experimental Protocol Using an Angiotensin Converting Enzyme Inhibitor and 
an Angiotensin II Antagonist--Compound 2 and Compound 3 
Step A: Hemodynamic Study with ACEI-AII Combination 
Hemodynamic experiments were performed after 2 cycles of tachycardiac 
pacing in the presence of myocardial ischemic injury, after the animal had 
achieved a stable state of heart failure. During the experiments, the pigs 
were conscious and quietly restrained in a sling. The following eight 
treatment regimens (groups) were studied on different days: 
1) Compound 2 (enalaprilat) (1 mg/kg, n=3). 
2) Compound 3 
(3-(2'-tetrazol-5-yl)biphenyl-4-yl!methyl-5,7-dimethyl-2-ethyl-3H-imidazo 
4,5-b!pyridine) (1 mg/kg, n=4). 
3) Compound 2 (1 mg/kg) followed by a dose of Compound 3 (1 mg/kg, n=5). 
4) Compound 2 (1 mg/kg) followed by a second dose of Compound 2 (1 mg/kg, 
n=4). 
5) Compound 2 (1 mg/kg) and Compound 3 concomitantly (1 mg/kg, n=4). 
6) Compound 2 at high dose (4 mg/kg, n=4). 
7) Compound 3 at high dose (4 mg/kg, n=3). 
8) Vehicle (n=4). 
For groups 3 and 4 above, the additional doses of either Compound 3 or 
Compound 2 were administered 30 min after the first dose. Compound 2 was 
dissolved in 0.9% saline, and Compound 3 was dissolved in saturated 
NaHCO.sub.3 (10% by Vol) and 0.9% saline (90% by Vol) at a concentration 
of 2 mg/ml. Hemodynamic measurements were continuously recorded before and 
for 90 min after intravenously injecting each of the treatment or the 
vehicle over a 2-min period. 
Step B: Baseline Hemodynamics Before and After Development of Heart Failure 
Tables 1 and 2 summarize the baseline LV function and systemic vascular 
dynamics before (i.e., post-surgical control) and after heart failure 
induced by serial myocardial infarctions followed by intermittent 
tachycardiac stress in conscious pigs. Heart failure resulting from at 
least 2 cycles of tachycardiac pacing in the presence of myocardial injury 
was manifested by significant increases in LV end-diastolic (+13.5.+-.3.2 
mm from 39.4.+-.3.2 mm) and end-systolic diameters (+17.4.+-.3.6 mm from 
29.8.+-.2.6 mm) and in mean left atrial pressure (+18.+-.2 mmHg from 
4.+-.1 mmHg). LV dP/dt, LV fractional shortening, Vcf, and cardiac index 
significantly decreased by 43.+-.8, 55.+-.7, 51.+-.6 and 36.+-.5%, 
respectively. Also, total peripheral resistance increased significantly 
(+52.+-.12%), while mean arterial pressure and heart rate were unchanged. 
In addition to these hemodynamic changes, which are shown in FIG. 9, heart 
failure, particularly at the advanced stages, was characterized by 
anorexia, peripheral and pulmonary edema, and reduced physical activity. 
TABLE 1 
______________________________________ 
Baseline Left Ventricular Function in Controls and 
Conscious Pigs with Heart Failure. 
Control Heart Failure 
______________________________________ 
LV End-Diastolic Diameter (mm) 
39.4 .+-. 3.2 
52.9 .+-. 2.5* 
LV End-Systolic Diameter (mm) 
29.8 .+-. 2.6 
47.2 .+-. 2.7* 
LV Fractional Shortening (%) 
24.7 .+-. 1.8 
10.9 .+-. 1.7* 
Vcf (sec.sup.-1) 1.21 .+-. 0.10 
0.58 .+-. 0.01* 
LV dP/dt (mmHg/sec) 
2890 .+-. 124 
1641 .+-. 208* 
______________________________________ 
*Significantly different from control, p&lt;0.05. 
Data are mean .+-. SE with n = 5 (n = 6 for LV dP/dt). 
TABLE 2 
______________________________________ 
Baseline Cardiac and Systemic Hemodynamics in Controls 
and Conscious Pigs with Heart Failure. 
Control Heart Failure 
______________________________________ 
Mean Arterial Pressure (mmHg) 
92 .+-. 3 86 .+-. 3 
Mean Left Atrial Pressure (mmHg) 
4 .+-. 1 22 .+-. 2* 
Cardiac Index (ml/min/kg) 
126 .+-. 9 80 .+-. 6* 
Total Peripheral Resistance 
0.72 .+-. 0.06 
1.08 .+-. 0.06* 
(mmHg/ml/min/kg) 
Heart Rate (beat/min) 
128 .+-. 7 143 .+-. 11 
______________________________________ 
*Significantly different from control, p&lt;0.05. 
Data are mean .+-. SE with n = 6 (n = 5 for cardiac index and total 
peripheral resistance). 
Step C: Effects of Compound 2 and/or Compound 3 on Hemodynamics in Heart 
Failure 
FIGS. 10 through 16 show the time course of mean arterial pressure, total 
peripheral resistance, LV dP/dt and LV mean velocity of circumferential 
fiber shortening (Vcf) changes during each of the treatment protocols 
mentioned in the Methods Section. The vehicle (FIG. 10) did not induce any 
significant changes throughout 90 min of observation. Compound 2 (FIG. 11) 
and Compound 3 (FIG. 12), administered individually at doses of 1 mg/kg, 
each induced a minor decrease in mean arterial pressure, while total 
peripheral resistance was reduced by approximately 15% from baseline 
levels. LV dP/dt was unchanged and Vcf was slightly increased by 
administration of these compounds. Compound 2 (FIG. 13) and Compound 3 
(FIG. 14), administered individually at high doses of 4 mg/kg, each caused 
similar changes in all indices compared to those observed at 1 mg/kg, 
indicating that the maximal effects of these agents had been achieved with 
the 1 mg/kg, i.v. doses. When Compound 3 (1 mg/kg) was injected either 
concomitantly or 30 min after Compound 2 (1 mg/kg) administered, the total 
peripheral resistance was reduced by more than 20% and Vcf was increased 
by approximately 10 to 15% from baseline levels (FIGS. 15 and 16). 
Step D: Combination Treatment Versus Mono-Treatment with Compound 2 and 
Compound 3 in Heart Failure 
FIG. 17 compares the effects of combination treatment with those of 
mono-treatment with Compound 2 and Compound 3 on total peripheral 
resistance 60 min after administration. The reduction in total peripheral 
resistance was greater following combination treatment than that following 
mono-treatment with either agent. FIG. 18 shows the time course of total 
peripheral resistance changes after separate administrations either of 
Compound 2 (1 mg/kg) and Compound 3 (1 mg/kg) or of two doses of Compound 
2 (each 1 mg/kg). Clearly, Compound 3 enhanced the reduction in total 
peripheral resistance in the presence of Compound 2, whereas a second dose 
of Compound 2 did not have the same effect. Also, Compound 2 and Compound 
3 administered together (each 1 mg/kg) decreased total peripheral 
resistance more than even a higher, single dose of Compound 2 (4 mg/kg) or 
Compound 3 (4 mg/kg) alone (FIG. 19). 
FIG. 20 compares the effects of combination treatment versus mono-treatment 
with Compound 2 and Compound 3 on LV dP/dt, mean arterial pressure, heart 
rate, fractional shortening, Vcf, cardiac output and total peripheral 
resistance 60 min after injection. The increases in fractional shortening, 
Vcf, and cardiac output were greater in response to the combination 
treatment than in response to the mono-treatment with either agent. The 
effects on mean left atrial pressure are shown in FIG. 21. The baseline 
left atrial pressure was similar in all groups, whereas left atrial 
pressure was reduced more in the combination treatment groups than in the 
mono-treatment groups. 
These preliminary data suggest that treatment with the ACE inhibitor, 
Compound 2 (enalaprilat), in combination with a selective AII antagonist, 
Compound 3 (Compound 3) reduced vascular resistance and left atrial 
pressure and increased cardiac output during heart failure more 
effectively than either compound alone. None of the treatments affected 
myocardial contractility as evidenced by the lack of significant changes 
in LV dP/dt. The increases in ejection phase indices therefore could be 
due to the decrease in afterload.