Evaluation of Anti-Anginal Agents Polished

Evaluation of Anti-Anginal
Agents
Dr. Jayesh U Patil
Dept. of Pharmacology
T.N.M.C. Mumbai
Angina Pectoris
The chest discomfort due to
myocardial ischemia

It is a visceral discomfort that is
usually described as a heaviness,
pressure, or squeezing type of
pain.

It is due to adverse oxygen
supply/demand situation in a
proportion of myocardium.

Angina Pectoris
Ischemic Heart Disease
Classical Angina (Stable Angina
Pectoris)
Variant /Prinzmetal/Vasospastic
Angina
Unstable Angina
Myocardial Infarction
Silent Ischemia

Stable Angina Pectoris
Predictably provoked by exercise , emotions, eating etc. & relieved by
rest
Underlying pathology involves fixed obstruction due to stable
atherosclerotic plaque.
Diagnosis : Based on clinical picture , confirmed by stress
Electrocardiography(Reproducible ST segment elevation), or
Anatomical localization of obstruction on PCI.

Managed with lifestyle modifications, Drug therapy ( Nitrates, Beta
blockers, CCBs, Pot.ch. Openers etc.) or with Percutaneous Coronary
Interventions (PCI), / Coronary Artery Bypass Graft (CABG)
Variant/Prinzmetal/Vasospastic angina
Unpredictable- may occur at rest/ during sleep.
Usually due to recurrent localized coronary vasospasm ,which
may be superimposed on a atherosclerotic segment on coronary
artery.

Diagnosis: Observation of ECG ( Ambulatory Holter Monitor), By
Coronary Angiography (Vasospasm provoked by Ach)

Factors precipitating coronary vasospasm like smoking, Cocaine
should be avoided .
Medical management involves use of Nitrates, CCBs
Beta blockers should be avoided.

Unstable Angina
Rapid increase in the duration and severity of attacks mostly
due to rupture of an atheromatous plaque.

Managed as an Acute Coronary Syndrome with anti platelet
agents (Aspirin, Clopidogrel etc) Nitrates, Beta Blockers,
Morphine Sulphate , & Anticoagulants (Heparin, Enoxaparin
etc) .

Thrombolytics should be avoided.
Early PCI is warranted.

Drug Therapy for Angina
Nitrates : Glyceryl Trinitrate (GTN), Isosorbide Mononitrate,
Isosorbide Dinitrate, Erythityl Tetranitrate, Pentaerythitol Tetranitrate.
Beta Blockers: Propranolol, Metoprolol, Atenolol etc.
Calcium Channel Blockers: Verapamil, Diltiazem, Nifedipine,
Felodipine, Amlodipine, Nitredipine, Nimodipine, Lacidipine,
Lercanidipine, Clevidipine, Benidipine.
Potassium Channel Openers: Nicorandil
Other Drugs : Trimetazidine, Ranolazine, Ivabradine
Evaluation
IN-Vitro Models
In Vivo Models
Clinical Evaluations
IN-Vitro Models
IN-Vitro Models
Langendorff Heart Preparation
Coronary Artery Ligation in Isolated working Rat Heart.
Relaxation of Bovine Coronary Artery
Isolated Rabbit Aorta Preparation
Heart Lung Preparations
Plastic Casts from Coronary Vascular Bed.

Described by Langendorff
(1895).

Highly reproducible model
for evaluating effect of
drugs on Coronary blood
flow.

It is based upon the
principle of Retrograde
Perfusion.

Retrograde Perfusion

Retrograde perfusion closes the aortic valves
during diastole. The perfusate is displaced
through the coronary arteries flowing off the
coronary sinus and the opened right atrium.

In this original set-up the ventricles do not
fill with perfusate and therefore do not
perform pressure-volume work.

Canula
Aorta
Coronary Artery
Coronary Sinus
Rt. Atrium
Coronary Effluent
Procedure
Guinea pigs of either sex weighing 300500 g are sacrificed
by stunning. The heart is removed as quickly as possible
and placed in a dish containing Ringers solution at 37C.
The aorta is located and cut just below the point of its
division.

A glass or plastic cannula is introduced into the aorta,
perfusion is started with oxygenated Ringers solution

The heart is transferred to a double walled plexiglass
apparatus . (Heart Chamber)

Procedure
Contractile force is measured isometrically by a force
transducer with a preload of 2.5 g and recorded on a
polygraph.
Coronary flow is measured by a drop counteror by a
mechanic-electronic flow meter.
Heart rate is measured through a chronometer coupled to
the polygraph.

Drugs are injected into the perfusion medium just above the
aortic cannula.

Oxygenated Ringers
Solution
Advantages
Highly reproducible, low cost preparation.
Broad spectrum of parameters can be measured (Coronary
Blood flow, Heart rate, Force of contration etc.)
Due to denervation Absence of Neurohumoral
confounding factors.
We can ligate LCA or any of its branches, thus it Allows
induction of regional or global ischemia.
It is also useful for the studying effects of Reperfusion
Injury and Anti-arrhythmic effects of drugs.

Disadvantages

Does not represent in vivo settings. Endogenous factors
(reflex tachycardia , changes in end diastolic pressure)are
not considered.
Constantly deteriorating preparation. (after 3 hrs)

Coronary Artery Ligation in Isolated Working
Rat Heart
Modification of Langendorff Heart Preparation.
In working heart preparations of rats, ischemia can be induced by
clamping the left coronary artery close to its origin.
After removal of the clip, changes in the reperfusion period can be
observed.
Prevention of changes due to Ischemia can be an indicator of the
efficacy of coronary drugs.

Rat Heart
Procedure : Wistar rats of either sex weighing 280300 g are
sacrificed by decapitation. Heart is isolated in the heart chamber
as described by langendorff. In this preparation number
additional instruments are used to measure various parameters
Including

Teflon Balloon : In the left ventricle a balloon closely fitting the
ventricular cavity measures Stroke volume
Statham pressure transducer Measures LVP (left ventricular
pressure),LV dp/dtmax and HR (heart rate).
pH/blood gas monitor, Electromagnetic flow probes (measure
coronary flow ,Cardiac Output).
coronary effluent samples taken for biochemical analysis

Isolated heart is perfused for a period of 20 min (pre-ischemic
period) with modified Krebs-Henseleit buffer at a constant pressure
of 65mm Hg

Thereafter, acute myocardial ischemia is produced by clamping
the left coronary artery close to is origin for 15min (ischemic
period).

The clip is then reopened, and changes during reperfusion are
monitored for 30 min (reperfusion period).

The test drugs are given into the perfusion medium either before
occlusion or 5 min before reperfusion.

Rat Heart
Rat Heart
Evaluation :
The incidence and duration of ventricular fibrillation after
treatment with coronary drugs is compared with controls.

Number of other parameters can also be compared:
Left ventricular pressure, coronary flow
Enzyme activities in the effluent (LDH, CK)
Myocardial content of glycogen, ATP and creatine
phosphate etc.

Spiral strips from bovine coronary
artery can be used for assaying
relaxing activity of test
compounds.

This activity is compared to
relaxation caused by PGI2.

Procedure:

The left descending coronary artery (obtained from freshly
slaughtered beef heart) and several of its primary branches are cut
into spiral strips (about 20mm long and 23mm wide).

The strips are suspended in a organ bath under an initial tension of
2 g and immersed in a Krebs bicarbonate solution at 37C gassed
with oxygen containing 5%CO2

The strips are superfused with a solution of the test compounds in
concentrations of 0.01, 0.1, 1.0 g/ml at a rate of 1020 ml/min with
oxygenated Krebs solution


Isometric contractions are recorded with Grass force-
displacement transducers (type FT 03C) on a Grass
polygraph.

Standard compounds are 100 ng/ml PGE2 inducing
contraction and 100 ng/ml PGI2 inducing pronounced
relaxation.

Evaluation: The relaxation induced by the test compound is
expressed as percentage of maximal response to 100 ng/ml
PGI2.

Principle:
Contraction of aortic rings is
induced by adding potassium
chloride or norepinephrine.

Test drugs with calcium channel
blocking activity have a relaxing
effect on isolated rabbit aorta
preparation
KCl
+
Rabbit
descending
aorta
Cut into 8 rings
of 4-5mm width
Mounted in tissue bath
containing Krebs HCO3 buffer

20 minutes later, test drug is added.
Percent relaxation reading- every 30 mins after adding test drug. 30 min
interval is kept before adding different Test drug

EVALUATION: Percentage relaxation by the test drug from the precontracted level
& ID
50
is calculated.
Heart Lung Preparation
Introduced by Knowlton and Starling (1912)
Principle:
Heart is isolated with lungs maintained on IPPV. Beating hearts
pumps the perfusate through aorta in to a pneumatic resistance
instead of systemic vascular resistance, which through a
reservoir returns the blood back to Inferior Vena Cava.

Thus this model mimics systemic circulation, lungs maintain
their ability to oxygenate & Preload & afterload may be adjusted
Useful to evaluate hemodynamic effects of drugs.

Wistar rats weighing 300320 g are anesthetized with 50
mg/kg pentobarbitone i.p.

Tracheostomy is performed and intermittent positive pressure
ventilation is instituted with air.

The chest is opened and flooded with ice-cold saline and the
heart arrested.

Cannulae from pneumatic resistance and blood reservoir are
inserted into the aorta and the superior (for measurement of
central venous pressure) and inferior venae cavae.

Perfusate:

The heart-lung preparation
is perfused with a solution
containing rat blood cells
from another rat and
Krebs-Ringer bicarbonate
buffer, with hematocrit 25
% and pH of 7.4.
perfusate pumped from the aorta
passes through a pneumatic resistance
and is collected in a reservoir
maintained at 37C and then returned
to the inferior vena cava.

Mean arterial pressure is regulated by the pneumatic
resistance.
Heart rate is recorded by a bioelectric amplifier and
cardiac output is measured with an electromagnetic
blood flow meter.
Arterial pressure and right atrial pressure are
measured with transducers and amplifiers.

Test drugs are administered into the perfusate 5min
after start of the experiment.

Evaluation:

Hemodynamic data within groups are
analyzed by two-way analysis of variance
(ANOVA) with repeated Measures.
Plastic Casts from Coronary Vascular Bed
Principle :

Prolonged administration of coronary
dilator drugs has been shown to increase
the number and size of interarterial
collaterals of dogs and pigs after
coronary occlusion

Filling the arterial coronary bed with
plastic cast provides the possibility to
make the collaterals visible and to
quantify them
Dogs weighing 1015 kg are anesthetized thorax is opened.
Ameroid cuffs are placed around major coronary branches.
Gradual swelling of the plastic material occludes the lumen.

The dogs are treated daily with the test drug or placebo. After 1
week recovery period they are submitted to exercise on a treadmill
ergometer.

After 6weeks treatment, the animals are sacrificed, the heart
removed and the coronary bed flushed with saline.

The liquid plastic Araldite is used to fill the whole coronary tree from
the bulbus aortae.

. Polymerization is complete after several hours. Then, the tissue is
digested with 35% potassium hydroxide.
Plastic Casts from Coronary Vascular Bed
Evaluation:

Plastic casts from drug treated
animals is compared with casts
from dogs submitted to the same
procedure without drug treatment
for Number and Size of Inter-
arterial Collaterals.
In-Vivo Models
In Vivo Methods
Isoproterenol Induced Myocardial Necrosis in Rats
Myocardial Infarction After Coronary Ligation in
Rodents
Occlusion of Coronary Artery in Anesthetized Dogs and
Pigs
Acute Ischemia by Injection of Microspheres in Dogs
Stenosis Induced Coronary Thrombosis Model
Electrical stimulation induced coronary thrombosis
Influence on Myocardial Preconditioning.
Isoproterenol Induced Myocardial Necrosis in
Rats
Principle
Cardiac necrosis can be produced by
injection of sympathomimetics in high
doses.
Infarct-like myocardial lesions in the rat
by isoproterenol have been described by
Rona et al. (1959).
These lesions can be totally or partially
prevented by several drugs such as
sympatholytics.

Groups of 10 maleWistar rats weighing 150200 g are pretreated
with the test drug or the standard either s.c. or orally for 1
week.
Then, they receive 5.25 and 8.5mg/kg isoproterenol s.c. on two
consecutive days.
Hemodynamic parameters (HR, BP ) and mortality in each group
are recorded
48 hrs after the first isoproterenol administration, the rats are
sacrificed and autopsied.
The hearts are removed and weighed, and frontal sections are
embedded for histological examination.
Evaluation
Microscopic examination allows the following grading:

Grade 0: no change
Grade 1: focal interstitial response
Grade 2: focal lesions in many sections, consisting of mottled
staining and fragmentation of muscle fibres
Grade 3: confluent retrogressive lesions with hyaline necrosis and
fragmentation of muscle fibres and sequestrating mucoid edema
Grade 4: massive infarct with occasionally acute aneurysm and mural
thrombi

Rats
Evaluation

Changes of parameters (histological, biochemical and
hemodynamic ) of drug treated animals are compared to
Isoproterenol controls.

Rats
Microspheres-induced acute ischemia
Principle

Severe left ventricular failure is induced by repeated
injections of 50 m plastic microspheres into the left
main coronary artery of anesthetized dogs.

The test can be used to evaluate the influence of drugs
on myocardial performance during acute ischemic left
ventricular failure in dogs.

Dogs of either sex weighing approximately 30 kg are
anesthetized ( 40mg/kg iv) . Artificial respiration maintained.

Two peripheral veins are cannulated for the administration of
narcotic (brachial vein) and testcompounds (saphenous vein).

The heart is exposed through a left thoracotomy between
the fourth and fifth intercostal space, the pericard is opened
and the left circumflex coronary artery (LCX) is exposed.

The microspheres are injected through the angiogram catheter into the
left ostium.

The microsphere injections produce stepwise elevations of Left ventricular
end diastolic pressure (LVEDP).

Embolization is terminated when LVEDP has increased to 1618 mm Hg
and/or PAP has increased to 20 mm Hg and/or heart rate has reached 200
beats/min.

Hemodynamic variables are allowed to stabilize after coronary
embolization for at least 30 min.
Parameters measured: Systolic and Diastolic pressure, LVEDP, HR,
Pulmonary capillary pressure, Pulmonary Artery Pressure, Cardiac output
Dose of Microspheres, S O
2
, Aortic Pressure etc.


Image shows beneficial effects of Mild Hypothermia On
various Hemodynamic parameters in Microsphere induced
acute ischemia.
CME - coronary microembolisation, CME
NT- Normal Temperature (black)
MH Mild Hypothermia (mild Hypothermia )
Coronary artery occlusion
Principle
Compounds potentially reducing infarct-size are tested by studying the
size of the infarct after proximal occlusion of the left anterior
descending coronary artery dogs.
Area at risk of infarction is measured by coronary angiogram , injecting
BaSO4 gel into occluded artery.
Nitro-blue Tetrazolium Chloride stain is used to visualize the infarcted
area.
Dogs anaesthetized- pentobarbitone(35mg/kg i.p.)- Artificial
respiration started. Thorax is opened

LAD coronary artery is exposed and ligated for 360 min

Test substance is administered i.v. bolus

Hemodynamic parameters are measured

Animals are sacrificed
Coronary arteriograms are made after injection of BaSO
4

gelatin mass into LCA

LV cut into Transverse section

Area at the risk of infarction can be measured by Caronary
Arteriogram (injecting BsSO
4
Gel) as a Defect Opacity

Slices are then incubated with p-nitro-blue-tetrazolium to
visualize infarct tissue (histology)
Evaluation:

Mortality, Hemodynamic changes and Infarct size are
determined in drug treated animals are compared to
vehicle controls.
Principle
Clamping induces
thrombosis in coronary
artery .

Stenosis of the artery
alters the cyclical
variations in the
coronary blood flow.

Drugs with coronary
vasodilator action may
improve the flow pattern
in the stenosed artery

Dogs anaesthetized - pentobarbitone sodium( 40mg/kg ip)
and LCA exposed

Electromagnetic flowprobe placed on proximal part of LCA to
measure Blood Flow

Vessel is squeezed with haemostatic clamp for 5 seconds

Plastic constrictor placed around artery

Dogs with repeated cyclic flow variations of same intensity are
used for experimental purpose

Hemodynamic parameters are recorded

Test compound administered IV and cyclic flow variations
registered for 2 and 5 hrs. and compared to pre-treatment
values

Electrical Stimulation Induced Coronary
Thrombosis
Principle:

Electrical stimulation induces thrombosis in
coronary artery .
An alteration in coronary blood flow with
transient platelet aggregation at the site of
coronary constriction is assessed using this model.

German Landrace pigs (40 kg)- anesthesized with Ketamine (2
mg/kg) Heart exposed- Electromagnetic flowmeter placed on
proximal part of LCA

Electrode is placed in the vessel on the intimal lining and connected
with the teflon coated wire of 9 volt battery, potentiometer and
amperometer

Intima is stimulated with 150 A for 6 hours- occluding thrombosis
occurs

Test drug - s.c. ,simultaneously or 30 mins following electrical
stimulation

Thrombosis
Evaluation:
Time interval until thrombotic occlusion
occurs, Thrombus size, hemodynamic
parameters are measured

Thrombosis
Myocardial Ischemic Preconditioning Model
Rationale:

Myocardial precondition (brief duration of ischemia &
reperfusion) can reduce the damage produced by
prolonged ischemia & reperfusion
.
Certain drugs like ACE inhibitors have protective on
cardiac myocytes by altering preconditioning response

This effect can be evaluated in this model.
Rabbits are anaesthetized with ketamine
Artificial respiration is established and vessels cannulated
4-0 suture is looped around marginal branch of LCA
Loop is tighten for 5 min, loosened for 10 min
Test drug administered
Tighten for 30 min followed by 120 min of reperfusion
Rabbits are sacrificed.

Evaluation:
Animals are sacrificed after the reperfusion duration.
Comparison between systemic hemodynamic data &
infarct size are analyzed by ANOVA using Statistical
Software.

Measurement of Coronary
Blood Flow
Measurement of Coronary Blood Flow
Measurement of Coronary Artery /Coronary sinus flow using
Electromagnetic Flowmeter:
Principle: magnetic field perpendicular to blood flow generates
voltage in the conductor (bloodstream) which is picked up by
electrode, amplified and recorded.

Inert gas technique:
A mixture of room air and inert gas (helium/nitrous oxide) is
inhaled.
A series of blood samples withdrawn from peripheral artery and
cardiac vein/coronary sinus.
Amount of substance taken up in unit time/A-V difference
This measures mean flow.

Coronary flow measurement using Radio-isotopes (I
121
, H
3

& Rubidium)
Radioactive Microsphere Technique : Useful to determine
regional flow. Batch of radioactive microspheres (9-15 m)
injected into left atrium. Number of spheres trapped /unit
of myocardium indicate proportion of blood flow received
by that part of myocardium.
Thermo-dilution Technique:
Coronary Arteriography:

Measurement of Coronary Blood Flow
Clinical Evaluation
Patient Selection
The patients included in the studies must suffer from stable angina
pectoris, preferably documented by a history of proven myocardial
infarction, previous coronary revascularisation and/or coronary
angiography.

With regard to dose-finding studies the documentation of unequivocal
coronary heart disease is mandatory.

Women and elderly patients should be sufficiently represented in the
study population.

The symptoms of angina pectoris and the nitrate consumption must
have been stable at least during the 2 weeks preceding the study. Any
concomitant medication should remain unchanged during the study.

Inclusion Criterias
Individuals (aged 1885 years)

They were eligible if they had angiographically documented
coronary artery disease, a positive exercise tolerance test (ETT),
and a history of symptoms of chronic, stable, effort-induced
angina for at least 2 months.

All concomitant anti-anginal drugs were allowed and
continued unchanged during the study.
Exclusion criterias
Inability of participant to do ETT because of back or leg
problems
Myocardial infarction or acute coronary syndrome for at least 2
months.
Coronary revascularisation (percutaneous or coronary artery
bypass graft) within the previous 6 months,
Left ventricular ejection fraction of less than 45%
Estimated glomerular filtration rate of less than 45 mL per min or
creatinine concentration greater than 180 mmol/mL,
Substantial valvular disease
Electrocardiogram (ECG) abnormalities interfering with ST-
segment interpretation.
Previous ventricular arrhythmias on ETT.
Efficacy Parameters
Exercise based
variables
Exercise capacity
Time to the onset of
angina
Time to ST segment
depression
Other exercise
variables
Measure of Anginal Pain
Canadian Clasification
Society (CCS) Angina
Grading Scale.
Concomitant use of short
acting nitrates

Health-Related Quality of
Life (HRQoL)

Morbidity and mortality
Exercise Testing
The most widely used test for both the diagnosis of
IHD and the estimation of risk and prognosis. It
involves recording the 12-lead ECG before, during,
and after exercise, usually on a treadmill.

The test consists of a standardized incremental
increase in external workload while symptoms, the
ECG, and arm blood pressure are monitored.

Exercise testing is usually carried out on using
Treadmill or Bicycle Ergometer

Treadmill Test

Treadmill Test
There are several standardized exercise protocols (Which
specify progression of exercise with respect to speed
workload Duration etc.) to perform Treadmill test
including Bruce protocol, Modified bruce protocol, Balke
protocol, Wilson protocol etc.

Most Commonly used among these is Bruce Protocol.
It increases treadmill speed & elevation every 3 minutes.

Treadmill Test
Exercise duration is usually symptom-limited,
and the test is discontinued upon evidence of
chest discomfort,
severe shortness of breath,
dizziness, severe fatigue,
ST-segment depression >0.2 mV (2 mm),
a fall in systolic blood pressure >10 mmHg,
development of a ventricular tachyarrhythmia.
Treadmill Test
Parameters Measured include
Total Exercise Capacity :
as per EMEA recommendations, Total Exercise Capacity
should be used as primary endpoint for clinical
evaluation of anti-anginal agents.
Measured by Total Exercise Time, Maximum Workload
Achieved, Maximum Heart rate.
Time to onset of Angina
Time to ST segment depression
Rate-pressure product, Magnitude of ST deviation, Exercise
induced Ventricular arrhythmias etc.
Bicycle Ergometer
Exercise testing can also be done using
Bicycle Ergometer
Anginal pain
The patient's experience of anginal pain should be recorded in a patient
diary.

Canadian Clasification Society (CCS) Angina Grading Scale. may be
used for assessing changes in symptoms or physical function.
Class I Angina only during strenuous or prolonged physical activity
Class II Slight limitation, with angina only during vigorous physical
activity
Class III Symptoms with everyday living activities, i.e., moderate
limitation of daily activities
Class IV Inability to perform any activity without angina or angina at
rest, i.e., severe limitation of daily activities.

concomitant use of short-acting nitrates is also used as a measure of
Anginal Pain.
Efficacy Parameters
Health-Related Quality of Life (HRQoL)
provides relevant supportive information on patients quality of life.
Many questionnaires are available to assess HRQoL , Most common is
SF-36 questionnaire .
SF-36 questionnaire ( measures vitality, physical functioning, bodily
pain, general health perceptions, physical role functioning, emotional
role functioning, social role functioning, mental health, Scaled 0-100 )

Morbidity and mortality
Should always be evaluated and assessed using the pooled data of the
(controlled) trials.

Study Design
At least in one study efforts should be made to enrol a study
population without concomitant antianginal drug therapy
where the test drug should be given as monotherapy.

Some of the trials should also include patients where the test
drug is given as add on therapy and compared to an
acceptable active control and /or placebo.

Rescue medication with short acting nitrate therapy should
always be allowed throughout the whole trial period.
Safety Aspects
Long-term data on adverse events must be presented as the
drug may be administered for long periods of time.

A sufficient number of patients should be treated for a minimum
of 6 months or for one year, preferably in controlled trials.

Specific effects should be studied, in particular:
Pro-anginal and pro-arrhythmic effects as well as other
cardiovascular effects.
Withdrawal phenomena and any effects related to rebound
phenomena
General effects influencing the function of other organ
systems, e.g. through alterations of regional blood flow

Thank You
Coronary Flow Measurement
Coronary Sinus /Coronary Artery Flow
measurement : using Electromagnetic
Flowmeter.
Inert Gas Technique: using
Helium/Nitrogen .Inert

Coronary Flow Measurement
Coronary flow measurement using Radio-
isotopes
Radioactive Microsphere Technique
Thermo-dilution Technique
Coronary Arteriography

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