M Pharmacology Pharmacological Toxicological Studies Screening Models of Antianginal drugs (Invitro and In vivo) methods

1. SUBMITTED
BY- Ananaya
Pandey
M.Pharmacy(Ph
armacology)
1st year
SCREENING MODEL OF
ANTIANGINAL
NOIDA INSTITUTE OF
ENGINEERING&TECHN
OLOGY(PHARMACY
INSTITUTE)

2.  Angina Pectoris : It is a pain syndrome due to induction of an
adverse oxygen supply/demand situation in a portion of the
myocardium .It occurs when oxygen needs of heart is not
meet.
 Classical angina : Instable angina, episodes of chest
discomfort are usually predictable. They can occur during
exertion (such as running to catch a bus) or during mental or
emotional stress. Normally, the chest discomfort is relieved
with rest, use of nitroglycerin
 Unstable Angina
 In unstable angina, chest pain can occur at any
often while a person is resting. The discomfort may
more severe and last longer than in typical angina.

3.  The most common cause is reduced blood
flow to the heart muscle because the
coronary arteries are narrowed by fatty
buildups.
 Variant Angina Pectoris
 Variant angina pectoris can happen at any
time. Unlike typical angina, it nearly
always occurs when a person is resting. .
Variant angina is caused by spasms in the
coronary arteries. About two-thirds of
people with variant angina have severe
coronary blockages in at least one major
vessel.

5.  IN VITRO MODELS
 1)Isolated heart Langendorf technique
 Isolated heart lung preparation
 Relaxation of Bovine Coronary artery
 Coronary artery ligation in Isolated heart
 Plaster cast from coronary vasculature in
dose
 IN VIVO MODELS
Coronary artery oculsion
Isopretenol Induced myocardial necrosis
Stenosis- Induced coronary thrombosis
Myocardial Ischemia precondition model
Estimation induced coronary thrombosis

6. IN VITRO
MODELS
This Photo by Unknown author is licensed under CC BY.

7.  This allows you to record and analyze
multiple cardiac parameters such as left
ventricular developed pressure, perfusion
pressure, cardiac electrical activity, heart
rate and temperature all in real time.
 The primary goal of the Langendorff
method is to provide an isolated heart with
oxygen and metabolites via a cannula
inserted into the aorta. The Langendorff
heart is a complex in vitro technique used
primarily in pharmacological and
physiological research that allows the
evaluation of multiple cardiac
hemodynamic parameters including, but
not limited to, contractility and heart rate.

8.  Established in 1897 by Oswald lagandroff
 The heart is first surgically removed from the rat body
 The isolated mouse heart preparation is more complicated and challenging because of its rapid heart
rate and small size Before anesthesia, the mouse is injected with heparin to reduce the risk of
thrombus formation. There are several protocols for anesthesia based on different
institutional guidelines. Following the induction of anesthesia, the mouse is placed in a
supine position.
 Anesthesia can be induced either by the inhalation of volatile agents or injection . Efforts should be
made to keep the animal free from stressful stimuli. In some protocols, heparin is intravenously
injected prior to excision to prevent the formation of thrombi in the excised heart
 A thoracotomy is performed to expose the heart. Subsequently, the aorta is suspended in the
Langendorff apparatus and the heart is perfused via the aorta, usually with a nutrient rich
oxygenated solution [Krebs and Henseleit solution (KHB)]. The delivery of nutrients and oxygen
allows the heart to continue beating and also allows the evaluation of the effects of different drugs
on the heart

9.  Temporarily stop the heart from beating, and to protect the heart from ischemic injury. The
time of ischemia during this period should be minimized and kept constant, since a longer
temporary ischemia period can induce ischemic preconditioning of the heart
 It is important to inspect the cannula and tubes connecting the aortic cannula prior to
cannulation to ensure that they are free of air bubbles. With any technique, laceration of the
aorta should be avoided.
 The heart is then connected to the Langendorff apparatus with a clip, and the contractile
function of the heart returns within seconds. It should be noted that there is a strong
relationship between cardiac function and temperature.
 The temperature of the heart may be influenced by the temperature of the air surrounding the
heart as well as the temperature of the perfusate . Considering the importance of temperature
effects, efforts should be made to maintain a correct and constant temperature.
 In some protocols, the heart is immersed in a solution of physiological saline at 37 °C to better
control its temperature during the ischemic period. Suspending the heart from its aortic cannula
in room air is a less desirable method for ischemia studies

10.  Since longer ischemic periods can lead to ischemic preconditioning of the heart, the time of
ischemia during the set-up should be minimized and kept constant. A new technique has been
devised that is cost effective, faster, cardioprotective, and does not require the use of a
magnifying instrument. In the traditional technique, the mouse heart is exposed to the open air
without protective solution during the challenging hanging process or is under the magnifying
microscope.
 A simple modification of the Langendorff apparatus allows the mouse heart to be protected
inside the perfusate and allows the heart to be hung within a few seconds. In this modification,
the hanging needle is positioned inside the perfusate, allowing the heart to be kept under the
surface of the perfusate while the needle is located close to the aorta in the perfusate.
 The aortic orifice is completely distended by matching the diameter of the needle with that of
the aorta. The needle and aorta are clipped together, and the needle is separated from the
syringe and reconnected to the Langendorff apparatus. This short-length needle can be either
included in the main apparatus or kept as a readily available separate instrument.

11.  Disadvantages of the traditional technique
 There are several disadvantages associated with the traditional Langendorff technique. The longer
hanging time with this technique in the mouse heart leads to ischemic damage to the heart.
Additionally, exposure of the heart to the air without a protective solution contributes to ischemia,
tissue temperature changes, and inconsistent results. Because of the small size of the mouse heart,
there is increased damage to the aorta and surrounding tissues, resulting in an increased failure rate
and higher costs. Finally, the traditional technique requires an assistant or the use of a magnifying
instrument.
 Advantageous of the new technique
 This new technique provides improved visualization of the aortic orifice because of the absence of
air pressure within the solution. There is decreased hanging time, and the heart is kept in a
protective solution instead of being exposed to the air, Because the procedure is performed within
the solution, the introduction of air bubbles is prevented. Additionally, both hands are free to work
and no assistant or magnification is needed. Ultimately, this new technique causes less damage to
the surrounding tissues with lower failure rates and costs. Most importantly, there is improved heart
quality with less ischemic damage.

12.  Testing of coronary vasodilator drugs,
electrophysiological evaluations
 Recording positive inotropic effects, negative
inotropic effects, calcium antagonism, effect
on potassium outflow induced by glycosides
and determination of hypoxic damage.
 Metabolic studies- arrhythmogenic,
antiarrhythmic and Anti fibrillatory effects.
 To study EDRF release from coronary
vascular bed

14.  For coronary artery occlusion experiment (Scholz et al. 1992, 1993), the isolated working
hearts are perfused for a period of 20 min (pre-ischemic period) with modified Krebs- Henseleit
buffer at a constant pressure of 65 mm Hg.
 Thereafter, acute myocardial ischemia is produced by clamping the left coronary artery close
to is origin for 15 min (ischemic period).The clip is then reopened, and changes during
reperfusion are monitored for 30 min (reperfusion period). After coronary artery ligation and
reperfusion the hearts develop ventricular fibrillation
 From the coronary effluent samples are taken for lactate, lactate dehydrogenase (LDH), and
creatine kinase (CK) determinations. After the experiment, glycogen, lactate, ATP, and creatine
phosphate in myocardial tissue are measured. The test drugs are given into the perfusion
medium either before occlusion or 5 min before reperfusion. For ex vivo studies, the rats are
treated orally with the test drug 1 h before sacrifice and preparation of the isolated working
heart.

15.  Coronary artery ligation to induce myocardial infarction (MI) in mice is typically performed by an
invasive and time-consuming approach that requires ventilation and chest opening (classic method),
often resulting in extensive tissue damage and high mortality. We developed a novel and rapid
surgical method to induce MI that does not require ventilation.
 Male C57/B6 mice were grouped into 4 groups: new method MI (MI-N) or sham (S-N) and classic
method MI (MI-C) or sham (S-C). In the new method, heart was manually exposed without
intubation through a small incision and MI was induced. In the classic method, MI was induced
through a ventilated thoracotomy. Similar groups were used in an ischemia/reperfusion injury
model. This novel MI procedure is rapid, with an average procedure time of 1.22 ± 0.05 minutes,
whereas the classic method requires 23.2 ± 0.6 minutes per procedure. Surgical mortality was 3% in
MI-N and 15.9% in MI-C. The rate of arrhythmia was significantly lower in MI-N. The postsurgical
levels of tumor necrosis factor-α and myeloperoxidase were lower in new method, indicating less
inflammation. Overall, 28-day post-MI survival rate was 68% with MI-N and 48% with MI-C.
Importantly, there was no difference in infarct size or post-MI cardiac function between the
methods.
 Conclusions: this new rapid method of MI in mice represents a more efficient and less damaging
model of myocardial ischemic injury compared with the classic method.

16. IN VIVO
METHODS

17. 1)CORON
ARY
ARTERY
OCULSIO
N
 PURPOSE AND RATIONALE
 The size of infarcts is studied after proximal occlusion of the
left anterior descending coronary artery in open chest dogs.
 Compounds potentially reducing infarct size are tested. To
delineate the post-mortem area at risk, coronary arteriograms
are made after injection of a BaSO4-gelatin mass into the left
coronary ostium.
 The infarct’s area is visualized with nitro-blue tetrazolium
chloride in myocardial sections.

18.  Dogs of either sex weighing approximately 30 kg are used. The animals are anesthetized by
intravenous injection of pentobarbital sodium (bolus of 35 mg/kg followed by continuous
infusion of 4 mg/kg/h).
 The animals are placed in the right lateral position. Respiration is maintained through a
tracheal tube using a positive pressure respirator.
 Arterial blood gases are checked, and the ventilation rate and/or oxygen flow rate are adjusted
to achieve physiological blood gas values (PO2: 100–140 mm Hg, PCO2: 32–40 mm Hg, and
pH 7.47).
 A peripheral vein (saphenous vein) is cannulated for the administration of test compound. The
ECG is recorded continuously from lead II.

19.  Mortality and the different hemodynamic
parameters are determined.
 Changes of parameters in drug-treated
animals are compared to vehicle controls. The
different characteristics are evaluated
separately.
 Mean values ± SEM of infarct area and of area
at risk are calculated. Statistical analyses
consist of regression and correlation analyses
and of the Student’s t-test. Results are
considered significant at p < 0.05.

20. 2)ISOPROTE
RENOL-
INDUCED
MYOCARDIA
L NECROSIS
PURPOSE AND RATIONALE
Cardiac necrosis can be produced by injection of natural and
synthetic 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 or calcium-antagonists.
EVALUATION
For each group the main grade is calculated with the standard
deviation to reveal significant differences

21.  Groups of 10 male Wistar rats (150-200g) are pretreated with test drugs orally or SC for at
least a week.
 Isoproterenol is injected SC on 2 consecutive days.
 Mortality as well as symptoms are recorded in each group and compared to group injected with
isoproterenol only.
 After 48 hrs. of 1st dose animals are sacrificed. Heart is removed , weighed and preserved for
various hemodynamic parameters.
 Degree of histopathological changes can be graded as follows:
 Grade 0: no change Grade 1: focal areas of necrosis Grade 2: focal areas of necrosis and
muscle fiber fragmentation Grade 3: confluent areas of necrosis, edema and inflammation and
muscle fiber fragmentation Grade 4: massive areas of necrosis, edema and inflammation and
mural thrombi

22. Preconditioning(brief duration of ischemia and reperfusion) can
reduce damage produced by prolonged ischemia and
reperfusion.
Preliminary preconditioning of the myocardium reduces : Infarct
size ,Leakage of cellular proteins indicative of myocyte
death. Improves post ischemic ventricular function Attenuates
cardiac arrythmia associated with frequent ischemia/reperfusion.

23.  Rabbits (3-4 kg) are anesthetized with ketamine xylazine. Trachea canulated and animal is set up
for artificial respiration
 Right femoral artery and vein are catheterized for measuring hemodynamic parameters. A 4-0
suture is looped loosely around the marginal branch of left coronary artery to facilitate
coronary occlusion.
 Ischemic preconditioning is induced by tightening the loop around the coronary artery for 5 min
and then loosening to reperfuse the myocardium for 10 min prior to a subsequent 30 min occlusion.
 After 30 min. ischemia, ligation is released for 120 min of reperfusion. Prior to 30 min of
occlusion rabbits are selected to receive ischemic preconditioning, no preconditioning or
preconditioning along with the administration of test compound. Animals are sacrificed after
reperfusion duration. Compared with the controlled groups.

24. In the study of cardiovascular biology, both in healthy and diseased conditions there is a vast
spectrum of measurable indices of function and injury.
The key feature of the In Vitro isolated animal hearts is that global or regional ischemia and
reperfusion can be imposed at will and the contractile biochemical, physiological and
morphological consequences can be easily assessed.
In Vivo preparations allow measurements, hemodynamic functions such as ECG, Ventricular
Wall Motion and Ejection Fraction that is of major diagnostic importance.
Data is analyzed by ANOVA using statistical software.

25. STENOSIS-
INDUCED
CORONAR
Y
THROMBO
SIS
 Dogs anaesthetized - pentobarbitone sodium( 40mg/kg ip)
and LCA exposed Electromagnetic flow probe placed on
proximal part of LCA to measure Blood Flow Vessel is
squeezed with hemostatic clamp for 5 seconds
 Principle: Clamping 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.
 Test compound is administered IV & the cyclic flow
variations are registered for 2-5 hrs. and compared with
pretreatment values Plastic constrictor placed around
artery at the site of damage- changed several times till
desired constriction is achieved.
 Dogs with repeated cyclic flow variations of same
intensity are used for experimental purpose

26. REFRENC
ES
Screening antianginal (1) (slideshare.net)
Rita Pavasini, Paolo G. Camici, Filippo Crea, Nicolas Danchin, Kim Fox,
Athanasios J. Manolis, Mario Marzilli, Giuseppe M.C. Rosano, José L.
Lopez-Sendon, Fausto Pinto, Cristina Balla, Roberto Ferrari
Corrigendum to “Anti-anginal drugs: Systematic review and clinical
implications” [Int. J. Cardiol. 283 (2019) 55–63]International Journal of
Cardiology, Volume 321, 15 December 2020, Pages 23
Screening methods for antianginal & antimalarial drugs (slideshare.net)
Erhe Gao 1, Yong Hong Lei, Xiying Shang, Z Maggie Huang, Lin
Zuo, Matthieu Boucher, Qian Fan, J Kurt Chuprun, Xin L Ma, Walter J
Koch (2010)A novel and efficient model of coronary artery ligation and
myocardial infarction in the mouse
Kazusa K, Nakamura Y, Watanabe Y et al (2014) Effects of pH on
nifekalant-induced electrophysiological change assessed in the
Langendorff heart model of Guinea pigs. J Pharmacol Sci 124:153–159

27. THANK YOU