Preclinical screening models for antihypertensive drugs

1. Preclinical screening models for
Antihypertensives
Presented To : Presented by:
Dr . M Rupesh Kumar Sharanappa
Head of department Reg no: 22MPY113
Department of pharmacology M Pharma I semester
SACCP, B. G. Nagara Pharmacology department
SACCP, B. G. Nagara

2. contents
 Introduction
Pathophysiology
Antihypertensive drugs
Screening models
References
2

3. Hypertension:
Hypertension is an elevation of systolic and / or diastolic BP above 140/90 mm Hg.
It is common cardiovascular disease affecting worldwide population.
Primary or essential hypertension
Secondary hypertension
As per Joint National Committee (JNC), Blood pressure is graded as:
3
Systolic BP (mmHg) Diastolic BP (mmHg)
Normal 120 80
Prehypertension 120-139 80-89
Stage 1 HTN 140-159 90-99
Stage 2 HTN ≥160 ≥100

4. Pathophysiology:
The normal blood pressure is maintained by four mechanisms:
Sympathetic nervous system activities
Activities of vascular endothelium
Activities of renal system
Activities of endocrine system
4

5. SYMPATHETIC NERVOUS SYSTEM ACTIVITIES
When the BP is decreasing the activation of SNS will occur. The increased SNS
activity increases the heart rate and cardiac contraction.
The increased the heart rate and cardiac contraction produce vasoconstriction in
the peripheral arterioles and promotes the release of renin from kidney.
The net effect of SNS activation is to increase the arterial blood pressure by
increasing cardiac output (CO) and systemic vascular resistance.(SVR)
BP=CO X SVR
5

6. ACTIVITIES OF VASCULAR ENDOTHELIUM
The vascular endothelium is a single cell layer that lines the blood vessel.
It will produce vasoactive substances and growth factors like nitric acid,
endothelin etc..
These substances are potent vasoconstrictors and causes increases blood pressure
level.
6

7. ACTIVITIES OF ENDOCRINE SYSTEM:
 When the angiotensin-II is stimulated in the adrenal cortex, it will secrete
aldosterone.
The aldosterone will stimulate the kidneys to retain sodium and water. Thus the
BP and cardiac output will get increased
7

8. Pathophysiology:
8
Fig no: 1 Pathophysiology of hypertension

9. Antihypertensive drugs:
1. Diuretics
Thiazides: Hydrochlorothiazide, Chlorthalidone, Indapamide
High ceiling: Furosemide, etc.
K+ Sparing: Spironolactone, Amiloride
2. ACE inhibitors: Captopril, Enalapril, Lisinopril, Perindopril, Ramipril,
Fosinopril etc.
3. Angiotensin (AT receptor) blockers: Losartan, Candesartan, Irbesartan,
Valsartan, Telmisartan
4. Direct renin inhibitor: Aliskiren
5. Calcium channel blockers: Verapamil, Diltiazem, Nifedipine, Felodipine,
Amlodipine, Nitrendipine, Lacidipine etc.
9

10. 6. β Adrenergic blockers: Propranolol, Metoprolol, Atenolol, etc.
7. β+α Adrenergic blockers* Labetalol, Carvedilol
8. α Adrenergic blockers: Prazosin, Terazosin, Doxazosin,
Phentolamine, Phenoxybenzamine
9. Central sympatholytic: Clonidine, Methyldopa
10.Vasodilators:
Arteriolar: Hydralazine, Minoxidil, Diazoxide
• Arteriolar + venous: Sodium nitroprusside
10

11. Mechanism of actions of antihypertensive drugs:
11

12. Screening methods for antihypertensive agents:
The animal models of hypertension share many features which are common to
human hypertension.
 Many of these models have been developed by utilizing the etiological factors
that are presumed to be responsible for human hypertension.
 Excessive salt intake.
 Hyperactivity of renin-angiotensin- aldosterone system (RAAS)
 Genetic factors.
12

13. Animals Used:
 Earlier, most studies on experimental hypertension were carried out on Dogs.
 Currently, Rat is the preferred species.
 Spontaneous Hypertensive Rat(SHR), the genetic strain of hypertensive rat, is the
animal of choice.
13

14. Dissected view of experimental animal:
14
Fig no: 2 Dissected view of experimental animal and heart

15. In vitro screening models:
Pulmonary Hypertension Induced by Monocrotaline
 Endothelin Receptor Antagonism in Porcine Isolated Hearts
15

16. In vivo screening models :
1. Renovascular hypertension
2. Dietary hypertension
3. Endocrine hypertension
4. Neurogenic hypertension
5. Psychogenic hypertension
6. Genetic hypertension
7. Other models
16

17. In vivo screening models:
1.Renovascular hypertension
Experimentally renal hypertension can be produced by constriction of renal artery
Activates RAAS and sympathetic nervous system
Renal hypertension
17

18. 1. Renovascular hypertension (Continuation…)
It can be performed in following methods:
a. Goldblatt Method: Goldblatt et.al (1934)
Three variants of hypertension are produced by this method.
 Two kidney one clip method(2K1C)
 One kidney one clip method(1K1C)
 Two kidney two clip method(2K2C)
18

19. Two kidney one clip method(2K1C)
The renal artery is constricted on only one side with the other artery (or kidney) left
untouched.
In rats, clamping renal artery for 4 hours can induce acute renal hypertension by activation
of the RAAS.
After re-Opening of the vessel, accumulated renin is released into circulation leading to
acute hypertension.
This test is used to evaluate antihypertensive activities of drugs.
19

20. Procedure:
Male Sprague Dawley rats (=300g) are anaesthetized by hexobarbital sodium (100
mg/kg) IP
A PVC-coated clip of 0.2 mm internal diameter is placed onto the left hilum of the
kidney & fixed to the back muscles. Constriction of renal artery should be > 50%.
Renal artery is occluded for 3.5-4 hours.
Ganglionic blockade is performed with pentolinium & after obtaining a stable
reduced BP values, the renal arterial clip is removed.
Subsequently the animals are anaesthetized with pentobarbitone sodium (30-40
mg/kg)IP
20

21. 21
The trachea is cannulated to facilitate spontaneous respiration.
Through a pressure transducer connected to carotid artery, BP is measured.
Jugular vein is cannulated for administration of test compound (iv)
As a consequence of elevated plasma renin level ,BP rises . BP is monitored
continuously.
Evaluation :
Increase in blood pressure after reopening of the renal artery and reduction in
blood pressure after administration of the test drug are determined.
Percent inhibition of hypertensive B.P. values under drug treatment are calculated
as compared to pretreatment hypertension values
Duration of effect is also determined.
Statistical significance is assessed by paired t-test.

22. 2. Dietary hypertension
a. Fructose induced hypertension in rats.
b. Increased salt induced hypertension in rats
22

23. a. Fructose induced hypertension in rats:
23
Fructose Induced Hypertension in Rats The increased intake of either sucrose or
glucose was shown to enhance the development of either spontaneous
hypertension or salt hypertension in rats (Hall and Hall 1966).
 Hwang et al. (1987) first reported that hypertension could be induced in normal
rats by feeding a high-fructose diet.

24. 24
PROCEDURE:
Groups of 8 male Wistar rats weighing 210–250 g are used.
The are housed two per cage on a 12-h light 12- h dark cycle and are allowed free access
to standard laboratory diet (Purina rat chow) and drinking fluid
Drinking fluid consists either of tap water or 10%- fructose solution
Body weight, food intake and fluid intake of each rat are measured every week during
treatment.

25. Evaluation:
Since maximum effects on the chosen parameters are achieved after 6 weeks, the
duration of treatment can be limited to this time.
Statistical analysis is performed using a one-way or two-way analysis of variance,
followed by the Newman-Keuls test.
25
Using the tail-cuff method, systolic blood pressure and pulse rate is measured
before and every week during treatment.
Blood samples are collected before and every second week during treatment
for determination of plasma glucose, insulin, and triglycerides

26. Invitro method:
Endothelin Receptor Antagonism in Porcine Isolated Hearts:
Potent long lasting contractions of isolated blood vessel strips and increase blood
pressure in vivo is elicited by endothelin peptides.
Endothelin have been implicated in the pathophysiology of cardiovascular disorders.
In this model, isolated porcine coronary artery is used since the smooth musculature
of artery is considered to contain the ET receptors.
26

27. Balanced coronary dominance ;Posterior view of the porcine
isolated heart:
27
RCA: Right Coronary Artery. PIA: Posterior Interventricular
Artery. CXA: Circumflex Artery. (*): Cordis Crux. RV: Right
Ventricle. LA: Left Atrium. LV: Left Ventricle
Fig no: 3 Dissected view of experimental porcine heart

28. Procedure:
From porcine hearts left anterior descending coronary arteries are isolated. The
endothelium-denuded arteries are cut into spiral strips about 10 mm long and 1
mm wide.
The intimal surface of the spiral rings is then rubbed gently with filter paper to
remove the vascular endothelium.
Each strip is suspended in an organ bath containing Krebs-Henseleit solution
bubbled with 95% CO, at 37°C. Once the isolated preparation is stabilized
reference contraction is isometrically obtained
28

29. Evaluation:
Concentration-response curves for ET-1 are obtained by cumulative addition of ET-1.
Twenty minutes before the addition of ET-1 the endothelin receptor antagonist test
drug is added to the organ bath and concentration response curve is recorded.
The pA2 values and slopes are obtained by analysis of Schild plots.
29

30. METHODS TO MEASURE BP IN RATS:
a) Direct method:
It is an invasive method .
Femoral/carotid cannula is inserted into an
anaesthetized animal.
On day of screening, cannula is connected to
a pressure transducer then to the pre-amplifier.
BP is recorded on the polygraph.
30
Fig no: 4 shows photo of the carotid artery
cannulation with suture of the needle cannula in
place.

31. 31
b) Indirect /Non invasive method:
 Tail cuff method:
 It is a common and convenient method.
 Tail cuff is inflated and deflated
 Pulsations disappear when cuff is inflated.
 Pulsations start appearing when pressure in the cuff equals systolic pressure while
deflating.
 The cuff is attached to a tail cuff sphygmomanometer or pressure transducer and BP is
recorded on a chart..

32. 32
Fig no: 5 Overview of Tail cuff system method

33. References:
• Vogel & Vogel, drug discovery and evaluation ,chapter A2, methods to induce
experimental Hypertension,
• Jian, Ziyan & Chen, Yi-Je (Jay) & Shimkunas, R. & Jian, Y. & Jaradeh, Mark &
Chavez, Karen & Chiamvimonvat, N. & Tardiff, Jil & Izu, L.T. & Ross, R.S. &
Chen-Izu, Ye. (2016). In vivo cannulation methods for cardiomyocytes isolation
from heart disease models. 11. 10.1371/journal.pone.3474065.
• Pharmacological screening methods and Toxicology by A S Rao
33

34. 34