PPT

1. EVALUATION SEMINAR ON
SCREENING OF ANTIHYPERTENSIVE
AGENTS
By
Mallappa. Shalavadi,
Lecturer,
Department of Pharmacology,
HSK College of Pharmacy,
Bagalkot.

2. CONTENTS
• Definition
• Types
• Pathophysiology
• Screening models
In vitro models
In vivo models
CONTENTS
• Definition
• Types
• Pathophysiology
• Screening models
In vitro models
In vivo models

3. WHAT IS HYPERTENSION ?
• Hypertension is the most common cardiovascular
disease.
• Hypertension is defined conventionally as a
sustained increase in blood pressure ≥ 140/90 mm
Hg.
2- TYPES
a) Primary or essential hypertension:
 Cause for rise in the blood pressure is unknown, several
factors implicated in its genesis:
• High salt intake
• Cigarette smoking
• Hypersensitivity of sympathetic system
WHAT IS HYPERTENSION ?
• Hypertension is the most common cardiovascular
disease.
• Hypertension is defined conventionally as a
sustained increase in blood pressure ≥ 140/90 mm
Hg.
2- TYPES
a) Primary or essential hypertension:
 Cause for rise in the blood pressure is unknown, several
factors implicated in its genesis:
• High salt intake
• Cigarette smoking
• Hypersensitivity of sympathetic system

4. b) Secondary hypertension:
 Common disorders causing hypertension are:-
• Cushing syndrome
• Acute or chronic renal disease
• Renal artery stenosis
• Drugs like oral contraceptives, estrogen, steroids.
b) Secondary hypertension:
 Common disorders causing hypertension are:-
• Cushing syndrome
• Acute or chronic renal disease
• Renal artery stenosis
• Drugs like oral contraceptives, estrogen, steroids.

5. PATHOPHYSIOLOGY

6. SCREENING MODELS OF ANTIHYPERTENSION
AGENTS
IN VITRO MODELS
1. α2-adrenoreceptor binding
2. Electrically stimulated release of [3H]norepinephrine from
brain slices
3. Inhibition of angiotensin converting enzyme in vitro
4. Quantitative autoradiographic localization of angiotensin
converting enzyme
5. Angiotensin II receptor binding
6. Angiotensin II induced contraction in isolated rabbit aorta
7. Renin-inhibitory activity using human kidney renin and a
synthetic substrate .
SCREENING MODELS OF ANTIHYPERTENSION
AGENTS
IN VITRO MODELS
1. α2-adrenoreceptor binding
2. Electrically stimulated release of [3H]norepinephrine from
brain slices
3. Inhibition of angiotensin converting enzyme in vitro
4. Quantitative autoradiographic localization of angiotensin
converting enzyme
5. Angiotensin II receptor binding
6. Angiotensin II induced contraction in isolated rabbit aorta
7. Renin-inhibitory activity using human kidney renin and a
synthetic substrate .

7. 8. Inhibition o endothelin converting enzyme
IN VIVO MODELS
1. Acute renal hypertension
2. Chronic renal hypertension in rats
3. Chronic renal hypertension in dogs
4. Neurogenic hypertension in dogs
5. DOCA-salt induced hypertension in rats
6. Fructose induced hypertension in rats
7. Genetic hypertension in rats
8. Pulmonary hypertension induced by monocrotaline
9. Blood pressure in conscious rats (tail cuff method)
8. Inhibition o endothelin converting enzyme
IN VIVO MODELS
1. Acute renal hypertension
2. Chronic renal hypertension in rats
3. Chronic renal hypertension in dogs
4. Neurogenic hypertension in dogs
5. DOCA-salt induced hypertension in rats
6. Fructose induced hypertension in rats
7. Genetic hypertension in rats
8. Pulmonary hypertension induced by monocrotaline
9. Blood pressure in conscious rats (tail cuff method)

8. IN VITRO MODELS
α2- ADRENORECEPTOR BINDING
PURPOSE AND RATIONALE
• α2-adrenoceptors are widely distributed and are activated by
norepinephrine released from sympathetic nerve terminals
• Prejunctionally mediated inhibition of the release of
neurotransmitters from many peripheral and central neurons.
• α2-adrenoceptors are also present at postjunctional sites,
where they mediate actions such as smooth muscle
contraction, platelet aggregation and inhibition of insulin
secretion.
• Clonidine is a centrally-acting antihypertensive agent, which
lowers blood pressure mostly through reducing sympathetic
tone by acting at the nucleus tractus solitarius in the brain
stem
α2- ADRENORECEPTOR BINDING
PURPOSE AND RATIONALE
• α2-adrenoceptors are widely distributed and are activated by
norepinephrine released from sympathetic nerve terminals
• Prejunctionally mediated inhibition of the release of
neurotransmitters from many peripheral and central neurons.
• α2-adrenoceptors are also present at postjunctional sites,
where they mediate actions such as smooth muscle
contraction, platelet aggregation and inhibition of insulin
secretion.
• Clonidine is a centrally-acting antihypertensive agent, which
lowers blood pressure mostly through reducing sympathetic
tone by acting at the nucleus tractus solitarius in the brain
stem

9. • Alpha-adrenergic agonists most potently displace 3H
clonidine.
• The purpose of this assay is to assess the interaction of
hypotensive agents with central α2-receptors and determine
possible clonidine-like mechanisms of action.
PROCEDURE
• Reagents
1. Tris buffer pH 7.7
2. [4-3H]-Clonidine hydrochloride
3. Clonidine-HCl
4. Test compounds:
1 mM stock solution is made up in a suitable solvent and
serially diluted, so that the final concentrations in the assay
range from 10–5 to 10–8 M.
• Alpha-adrenergic agonists most potently displace 3H
clonidine.
• The purpose of this assay is to assess the interaction of
hypotensive agents with central α2-receptors and determine
possible clonidine-like mechanisms of action.
PROCEDURE
• Reagents
1. Tris buffer pH 7.7
2. [4-3H]-Clonidine hydrochloride
3. Clonidine-HCl
4. Test compounds:
1 mM stock solution is made up in a suitable solvent and
serially diluted, so that the final concentrations in the assay
range from 10–5 to 10–8 M.

10. Tissue preparation ASSAYTissue preparation ASSAY

11. EVALUATION
• IC50 calculations are performed using log-probit
analysis.
• The percent inhibition at each drug concentration is
the mean of triplicate determinations.
MODIFICATIONS OF THE METHOD
• Perry and U’Prichard (1981) described
[3H]rauwolscine (α-yohimbine) as a specific
radioligand for brain α2-adrenergic receptors.
• Goldberg and Robertson (1983) reviewed yohimbine
as a pharmacological probe for the study of the α2-
adrenoreceptor.
EVALUATION
• IC50 calculations are performed using log-probit
analysis.
• The percent inhibition at each drug concentration is
the mean of triplicate determinations.
MODIFICATIONS OF THE METHOD
• Perry and U’Prichard (1981) described
[3H]rauwolscine (α-yohimbine) as a specific
radioligand for brain α2-adrenergic receptors.
• Goldberg and Robertson (1983) reviewed yohimbine
as a pharmacological probe for the study of the α2-
adrenoreceptor.

12. Inhibition of angiotensin converting enzyme in
vitro
PURPOSE AND RATIONALE
• An in vitro system can be used to screen potential
angiotensin-converting-enzyme inhibitors.
• Fluorescence generated by an artificial substrate in
presence or absence of the inhibitor is measured to
detect inhibitory activity.
PROCEDURE
• Reagents
1. 50 mM Tris-HCl buffer, pH 8.0 + 100 mM NaCl
2. 10 mM potassium phosphate buffer, pH 8.3
Inhibition of angiotensin converting enzyme in
vitro
PURPOSE AND RATIONALE
• An in vitro system can be used to screen potential
angiotensin-converting-enzyme inhibitors.
• Fluorescence generated by an artificial substrate in
presence or absence of the inhibitor is measured to
detect inhibitory activity.
PROCEDURE
• Reagents
1. 50 mM Tris-HCl buffer, pH 8.0 + 100 mM NaCl
2. 10 mM potassium phosphate buffer, pH 8.3

13. 3. Substrate: O-aminobenzoylglycyl-p-nitro-L-
phenylalanyl-L-proline
4. Test compounds
• Compounds are made up to a concentration of 1
mM in 50 mM Tris-HCl buffer, pH 8.0 + 100 mM
NaCl or 10% methanol in Tris/NaCl if insoluble in
aqueous buffer alone. This will give a final
concentration in the assay of 0.1 Mm.
3. Substrate: O-aminobenzoylglycyl-p-nitro-L-
phenylalanyl-L-proline
4. Test compounds
• Compounds are made up to a concentration of 1
mM in 50 mM Tris-HCl buffer, pH 8.0 + 100 mM
NaCl or 10% methanol in Tris/NaCl if insoluble in
aqueous buffer alone. This will give a final
concentration in the assay of 0.1 Mm.

14. Enzyme preparationEnzyme preparation

15. Enzyme inhibition studies
1. Enzyme activity is measured with a Perkin Elmer LS-5
Fluorescence Spectrophotometer or equivalent at an
excitation wavelength of 357 nm and an emission wavelength
of 424 nm.
2. Enzyme assay
• 50 μl vehicle or inhibitor solution and 40 μl enzyme are
preincubated for 5 min, then 410 μl substrate working
solution is added.
• Samples are mixed by drawing fluid back up into the pipette
and by pipetting into the cuvette.
• For the initial control run of the day, the auto zero is pushed
immediately after placing the sample in the cuvette.
Enzyme inhibition studies
1. Enzyme activity is measured with a Perkin Elmer LS-5
Fluorescence Spectrophotometer or equivalent at an
excitation wavelength of 357 nm and an emission wavelength
of 424 nm.
2. Enzyme assay
• 50 μl vehicle or inhibitor solution and 40 μl enzyme are
preincubated for 5 min, then 410 μl substrate working
solution is added.
• Samples are mixed by drawing fluid back up into the pipette
and by pipetting into the cuvette.
• For the initial control run of the day, the auto zero is pushed
immediately after placing the sample in the cuvette.

16. EVALUATION
• The individual fluorescence slope is measured and %
inhibition is calculated as follows:
• Inhibitor concentrations on either side of the IC50 should be
tested to generate a dose-response curve.
• The IC50 is calculated using Litchfield-Wilcoxon log probit
analysis.
EVALUATION
• The individual fluorescence slope is measured and %
inhibition is calculated as follows:
• Inhibitor concentrations on either side of the IC50 should be
tested to generate a dose-response curve.
• The IC50 is calculated using Litchfield-Wilcoxon log probit
analysis.

17. IN VIVO MODELSIN VIVO MODELS
1. Acute renal hypertension in rats
PURPOSE AND RATIONALE
• Ischemia of the kidneys causes elevation of blood pressure
by activation of the renin-angiotensin system.
• In rats acute renal hypertension is induced by clamping the
left renal artery for 4 h.
• After reopening of the vessel, accumulated renin is released
into circulation.
• The protease renin catalyzes the first and rate-limiting step
in the formation of angiotensin II leading to acute
hypertension.
• The test is used to evaluate antihypertensive activities of
drugs.

19. PROCEDUREPROCEDURE

20. • EVALUATION
• Increase in blood pressure after reopening of the
renal artery and reduction in blood pressure after
administration of the test drug are determined [mm
Hg].
• Percent inhibition of hypertensive blood pressure
values under drug treatment are calculated as
compared to pretreatment hypertension values.
• Duration of the effect is determined [min].
• Statistical significance is assessed by the paired t-
test.
• EVALUATION
• Increase in blood pressure after reopening of the
renal artery and reduction in blood pressure after
administration of the test drug are determined [mm
Hg].
• Percent inhibition of hypertensive blood pressure
values under drug treatment are calculated as
compared to pretreatment hypertension values.
• Duration of the effect is determined [min].
• Statistical significance is assessed by the paired t-
test.

21. 2. DOCA-salt induced hypertension in rats
PURPOSE AND RATIONALE
• Mineralocorticoid-induced hypertension is thought
to be due to the sodium retaining properties of the
steroid causing increases in plasma and
extracellular volume.
• The hypertensive effect is increased by salt loading
and unilateral nephrectomy in rats.
2. DOCA-salt induced hypertension in rats
PURPOSE AND RATIONALE
• Mineralocorticoid-induced hypertension is thought
to be due to the sodium retaining properties of the
steroid causing increases in plasma and
extracellular volume.
• The hypertensive effect is increased by salt loading
and unilateral nephrectomy in rats.

22. PROCEDURE
Male Sprague Dawley rats weighing 250–300 g are
anesthetized with ether.
Through a flank incision the left kidney is removed.
The rats are injected twice weekly with 20 mg/kg s.c.
desoxycorticosterone-acetate in olive oil for 4 weeks.
Drinking water is replaced with a 1% NaCl solution.
Blood pressure starts to rise after one week and reaches
systolic values between 160 and 180 mm Hg after 4 weeks.
PROCEDURE
Male Sprague Dawley rats weighing 250–300 g are
anesthetized with ether.
Through a flank incision the left kidney is removed.
The rats are injected twice weekly with 20 mg/kg s.c.
desoxycorticosterone-acetate in olive oil for 4 weeks.
Drinking water is replaced with a 1% NaCl solution.
Blood pressure starts to rise after one week and reaches
systolic values between 160 and 180 mm Hg after 4 weeks.

23. MODIFICATIONS OF THE METHOD
• DOCA-salt hypertension can also be achieved
without nephrectomy (Bockman et al. 1992).
• DOCA pellets or implants in silastic devices
(Ormsbee and Ryan 1973; King and Webb
1988) were used instead of repeated
injections.
MODIFICATIONS OF THE METHOD
• DOCA-salt hypertension can also be achieved
without nephrectomy (Bockman et al. 1992).
• DOCA pellets or implants in silastic devices
(Ormsbee and Ryan 1973; King and Webb
1988) were used instead of repeated
injections.

24. 3. Fructose induced hypertension in rats
PURPOSE AND RATIONALE
• Increases in dietary carbohydrate intake can raise blood
pressure in experimental animals.
• The increased intake of either sucrose or glucose was shown
to enhance the development of either spontaneous
hypertension or salt hypertension in rats
• Hypertension could be induced in normal rats by feeding a
high-fructose diet.
• Fructose feeding was also found to cause insulin resistance,
hyperinsulinemia, and hypertriglyceridemia in normal rats
• Dai and McNeill (1995) studied the concentration- and
duration-dependence of fructose-induced hypertension in
rats.
3. Fructose induced hypertension in rats
PURPOSE AND RATIONALE
• Increases in dietary carbohydrate intake can raise blood
pressure in experimental animals.
• The increased intake of either sucrose or glucose was shown
to enhance the development of either spontaneous
hypertension or salt hypertension in rats
• Hypertension could be induced in normal rats by feeding a
high-fructose diet.
• Fructose feeding was also found to cause insulin resistance,
hyperinsulinemia, and hypertriglyceridemia in normal rats
• Dai and McNeill (1995) studied the concentration- and
duration-dependence of fructose-induced hypertension in
rats.

25. PROCEDUREPROCEDURE

26. 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.
MODIFICATIONS OF THE METHOD
• Brands et al. (1991, 1992) found an increase of
arterial pressure during chronic hyperinsulinemia in
conscious rats.
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.
MODIFICATIONS OF THE METHOD
• Brands et al. (1991, 1992) found an increase of
arterial pressure during chronic hyperinsulinemia in
conscious rats.

27. • Hall et al. (1995) reported the effects of 6 weeks of a
high-fat diet on cardiovascular, renal, and endocrine
functions in chronically instrumented conscious
dogs. Body weight increased by approximately 16.9
kg, whereas MAP, cardiac output, and heart rate
increased by 28%, 77%, and 68%, respectively.
• Hall et al. (1995) reported the effects of 6 weeks of a
high-fat diet on cardiovascular, renal, and endocrine
functions in chronically instrumented conscious
dogs. Body weight increased by approximately 16.9
kg, whereas MAP, cardiac output, and heart rate
increased by 28%, 77%, and 68%, respectively.

28. 4. Genetic hypertension in rats
• Inherited hypertension in rats has been described
by Smik and Hall 1958; Phelan and Smirk 1960;
Laverty and Smirk 1961; Phelan 1968 as genetically
hypertensive (GH) rats.
• Okamoto et al. (1963, 1966) reported the
development of a strain of spontaneously
hypertensive rats from mating one Wistar male rat
with spontaneously occurring high blood pressure
with a female with slightly elevated blood pressure.
By inbreeding over several generations a high
incidence of hypertension with blood pressure
values of 200 mm Hg or more was achieved.
4. Genetic hypertension in rats
• Inherited hypertension in rats has been described
by Smik and Hall 1958; Phelan and Smirk 1960;
Laverty and Smirk 1961; Phelan 1968 as genetically
hypertensive (GH) rats.
• Okamoto et al. (1963, 1966) reported the
development of a strain of spontaneously
hypertensive rats from mating one Wistar male rat
with spontaneously occurring high blood pressure
with a female with slightly elevated blood pressure.
By inbreeding over several generations a high
incidence of hypertension with blood pressure
values of 200 mm Hg or more was achieved.

29. • These strains were called “Spontaneously
hypertensive rats (Akamoto-Aoki)” = SHR or
“Wistar-Kyoto rats” =WKY.
• Hypertension in these rats is clearly hereditary
and genetically determined, thus comparable
to primary hypertension in humans.
• Cardiac hypertrophy and cellular ionic
transport abnormalities have been observed
• These strains were called “Spontaneously
hypertensive rats (Akamoto-Aoki)” = SHR or
“Wistar-Kyoto rats” =WKY.
• Hypertension in these rats is clearly hereditary
and genetically determined, thus comparable
to primary hypertension in humans.
• Cardiac hypertrophy and cellular ionic
transport abnormalities have been observed

30. CRITICAL ASSESSMENT OF THE METHOD
• The use of spontaneously hypertensive rats to
detect potential antihypertensive compounds
is well established.
• On the basis of available data no preference
can be given to a particular strain.
• The most abundant experience has been
gained with the Wistar-Kyoto strain.
• Transgenic rats with well defined genomes are
gaining more importance.
CRITICAL ASSESSMENT OF THE METHOD
• The use of spontaneously hypertensive rats to
detect potential antihypertensive compounds
is well established.
• On the basis of available data no preference
can be given to a particular strain.
• The most abundant experience has been
gained with the Wistar-Kyoto strain.
• Transgenic rats with well defined genomes are
gaining more importance.

31. 5. Pulmonary hypertension induced by
monocrotaline
PURPOSE AND RATIONALE
• The pyrrolizidine alkaloid monocrotaline, derived
from Crotalaria spectabilis, is hepatotoxic and
pneumotoxic in the rat.
• A single injection of monocrotaline leads to
progressive pulmonary hypertension resulting in
right ventricular hypertrophy and cardiac failure
• Rats given monocrotaline develop severe right
ventricular hypertrophy often accompanied by
ascites and pleural efflusions.
5. Pulmonary hypertension induced by
monocrotaline
PURPOSE AND RATIONALE
• The pyrrolizidine alkaloid monocrotaline, derived
from Crotalaria spectabilis, is hepatotoxic and
pneumotoxic in the rat.
• A single injection of monocrotaline leads to
progressive pulmonary hypertension resulting in
right ventricular hypertrophy and cardiac failure
• Rats given monocrotaline develop severe right
ventricular hypertrophy often accompanied by
ascites and pleural efflusions.

32. • Amelioration by angiotensin-converting
enzyme inhibitors and by penicillamine has
been demonstrated.
• Amelioration by angiotensin-converting
enzyme inhibitors and by penicillamine has
been demonstrated.

33. REFERANCES
• H. Gerhard Vogel., Wolfgang H.Vogel., Bernward A.
Schölkens., Jürgen Sandow., Günter Müller., Wolfgang F.
Vogel, Drug discovery and evaluation, 2nd
ed. Springer-Verlag
Berlin Heidelberg, 2002;26-172.
• Harsh mohan, Text book of Pathology, 5th
ed., Jaypee,
2005;708-709.
• www. Google.com