Screening of antihypertensive agents using in vivo models (rat model, dog model, monkey model and transgenic model) and in vitro models

1. Screening Methods for the
Evaluation of Anti-hypertensive
Activity of a Compound
Dr. Suchi Jain
Junior Resident
Dept. of Pharmacology & Therapeutics,
K.G.M.U, Lucknow

2. Introduction
 Hypertension is a complex multifactorial disease
 Is one of the leading causes of mortality & morbidity due
to stroke, heart attack & kidney failure
 Use of experimental animal models may provide valuable
information regarding many aspects of the disease , that
includes :
– Etiology
– Pathophysiology
– Complications
– Treatment

3. In Vitro Models
 Endothelin Receptor Antagonism in Porcine Isolated
Hearts
 Monocrotaline Induced Pulmonary Hypertension

4. In Vivo Models
 Rat Models of Hypertension
• Renovascular Hypertension
• Neurogenic Hypertension
• Dietary Hypertension
• Endocrine Hypertension
• Psychogenic Hypertension
• Genetic Hypertension
 Dog Models of Hypertension
• Chronic renal hypertension
• Neurogenic hypertension
 Monkey Model of Hypertension
• Renin inhibition in monkeys
 Trangenic Models

5. Renovascular hypertension
Experimentally, renal hypertension can be produced
by constriction of the renal artery
Activates Renin angiotensin aldosterone system (RAAS)
and sympathetic nervous system
Renal hypertension

6. Decreased blood flow
Renin secreted by kidney
Renin converts angiotensinogen to
angiotensin-I angiotensin-II by
angiotensin converting enzyme (ACE)
Angiotensin –II = potent vasoconstrictor and
also causes release of aldosterone →to salt
and water retention
Increased blood volume &
hypertension

7. In Vivo Models

8. Rat Models of
Hypertension
Renovascular
Hypertension
Neurogenic
Hypertension
Dietary
Hypertension
Endocrine
Hypertension
Psychogenic
Hypertension
Genetic
Hypertension

9. Renovascular Hypertension
 A. Two-kidney one clip (Goldblatt hypertension, 2K1C)
 B. Chronic renal hypertension in rats
• 1-kidney-1-clip method
• Two kidney two clip (2K2C) method

10. Renovascular Hypertension
 A. Two-kidney one clip (Goldblatt hypertension, 2K1C)
• Constriction of only one renal artery while the
contralateral kidney is left intact
• 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

11. Two-kidney one clip (Goldblatt hypertension, 2K1C)
Sprague Dawley rats (300 g) are anesthetized with hexobarbital
sodium (100mg/kg, intraperitoneally)
Trachea cannulated → facilitate spontaneous respiration
Through a pressure transducer connected to carotid artery, blood
pressure is measured. Jugular vein is cannulated for administration
of test compound

12. A poly vinyl chloride (PVC) coated clip is placed into the left hilum
of the kidney and fixed to the back muscles. The renal artery is
occluded for 3.5 – 4 hours
Ganglionic blockade is performed with pentolinium and after
obtaining stable reduced blood pressure values, the ‘renal arterial
clip’ is removed
Due to elevated plasma renin level, blood pressure rises
Test compound is administered by intravenous route
Increase in BP after re-opening of renal artery & reduction after
administration of test compound is determined

13. Renovascular Hypertension
 B. Chronic renal hypertension in rats (1-kidney-1-clip
method)
• Constriction of the renal artery is done on one side and
on the other side the contralateral kidney is removed

14. Chronic renal hypertension in rats (1-kidney-1-clip method)
Sprague Dawley rats (200 to 250g)- anesthetized with
pentobarbitone sodium (50mg/kg, intraperitoneally)
In the left lumbar area, a flank incision is made parallel to the
long axis of the rat. The renal artery is dissected, cleaned and a U
shaped silver clip is slipped around it near the aorta
The size of the clip is adjusted so that the internal gap ranges from
0.25 to 0.38
The right kidney is removed after tying off the renal pedicle

15. Four to five weeks after clipping, blood pressure is measured and
rats are divided into different groups of different doses. For
individual dose each animal is used as its own control
Test compounds are administered for 3 days. Pre- drugs and 2 hours
post- drug blood pressure reading are taken
Antihypertensive activity of test drug is determined by comparing
treatment blood pressure value with day 1, pre- drug BP
Comparisons are made using the paired t-test for evaluation of
statistical significance

16. Renovascular Hypertension
 C. Chronic renal hypertension in rats [Two kidney two
clip (2K2C) method]
• Constriction of aorta or both renal arteries is undertaken
When aorta or both renal arteries are constricted,
there is severe renal ischemia caused by renal clipping,
occasioning the activation of renin-angiotensin and the
sympathetic nervous system
the elevation of serum vasopressin, leading to
increased BP

17. Renovascular Hypertension
 C. Chronic renal hypertension in rats [Two kidney two clip (2K2C)
method]
• The 2K2C, with a high incidence of spontaneous stroke, can be used
as independent of a genetic deficiency
• The lesioned small artery or arteriole with thrombotic occlusion is
the main cause of cerebral infarction in 2K2C
• This may be similar to lacunar infarction in the human brain
• One of the most common causes of renal hypertension in human
beings is a patchy ischaemic kidney disease

18. Chronic renal hypertension in rats 2K2C method
Sprague Dawley rats (300 g) anesthetized; trachea, carotid artery
and jugular vein are cannulated. The renal arteries are located, U
shaped silver clip is slipped around them or near the aorta
Either the renal arteries or the aorta is occluded using renal arterial
clips
Test compound are administered by intravenous route via the
jugular vein. Blood pressure is monitored continuously
Percent reduction of blood pressure values under drug treatment is
calculated as compared to pre-treatment values

19. Neurogenic Hypertension
 Is defined as a permanent increase in BP resulting from
a primarily neural change
 Negative feedback in the control of BP originates from
baroreceptors located in the carotid sinus & aortic arch
 Denervation of sinoaortic baroreceptors (SAD) is the
neurogenic model of hypertension most often used
 Blood pressure in pithed rats

20. Neurogenic Hypertension
 Blood pressure in pithed rats
• The pithed rat model is devoid of neurogenic reflex
control that may modulate the primary drug effect
• It is frequently used to evaluate drug action on the CVS

21. Blood pressure in pithed rats
Male wistar rats (250 to 350 g) are anaesthetized with halothane.
The carotid artery is cannulated for monitoring blood presssure &
blood sampling
The trachea is cannulated & the animal is maintained on artificial
respiration using a ventilation pump (60 cycles/min). The jugular
vein is also cannulated for the administration of test drug
Pithing is done by inserting a steel rod ,2.2 mm in diameter & 11
cm in length, through the orbit & foramen magnum down the
whole length of spinal canal

22. Inspired air is oxygen enriched by providing a flow of oxygen
across a T piece attached to the air inlet of the ventilation pump
Thirty minutes after pithing , a 0.3 ml blood sample is withdrawn
from the carotid canula & analyzed for pO2, pCO2, pH &
Bicarbonate concentration using blood gas analyzer. Through the
carotid artery blood pressure & cardiac frequency is recorded

23. To measure a1 & a2 antagonism first dose response curves are
registered with phenylephrine, a selective a1 agonist (0.1 – 30
mg/kg, i.v) & BHT 920, a selective a2 agonist ( 1 – 1000 mg/kg, i.v)
Test drug is administered i.v & agonist dose response curves are
repeated 15 min
The curve of BP response to agonist is obtained. Dose response
curves are plotted on a logarithmic probit scale. Potency ratios
are calculated from the dose response curves

24. Dietary Hypertension
Long term exposure to a special diet (high salt, fat, sugar) results in
dietary hypertension in some rats
Oxidative stress &
inactivation of Nitric
Oxide (NO) in rats
maintained on the
high fat or high sugar
diet
Enhanced generation
of ROS
Development of
hypertension
The reduction in NO
availability was
associated with
marked salt
sensitivity
Evidenced by a
significant rise in BP
on the high salt diet
Dietary intake of fats,
carbohydrates
(simple sugars)
Resultant effects of
plasma insulin,
adipokine, lipid
concentrations
May affect
cardiomyocyte size &
function

25. Dietary Hypertension
 A. Fructose induced hypertension in rats
 B. Increased salt induced hypertension in rats

26. Fructose induced hypertension in rats
Wistar rats (200
– 250g) are
housed per cage
on a 12 hr light &
dark cycle, fed
water & chow
diet ad libitum
Drinking water
consists of 10%
fructose solution
Fluid intake, food
intake, & body
weight of each
rat are measured
every week
Systolic BP &
pulse rate is
measured using
the tail cuff
method
Blood samples
are collected
before & every
second week
during treatment
plasma glucose,
insulin,
triglycerides are
measured

27. Increased Salt induced hypertension in rats
Wistar rats (200
– 250g) are fed
chow diet ad
libitum
Drinking water is
replaced with
1 – 2% Sodium
chloride solution
Fluid intake, food
intake & body
weight of each
rat are measured
every week
Systolic BP &
pulse rate is
measured using
the tail cuff
method
Blood samples
are collected
before & every
second week
during treatment
High salt intake hypertension in rats produced by replacing
drinking water with 1 – 2% sodium chloride for 9 – 12 months

28. Endocrine Hypertension
Deoxycorticosterone acetate (DOCA) produces hypertension in rats
DOCA salt rats
Increased DOCA
induced reabsorption
of salt & water
Increased blood
volume & increased
BP
Increased secretion
of vasopressin
Water retention &
vasoconstriction
Altered activity of
RAAS
Increased
sympathetic activity

29. DOCA salt rats
Male Sprague
Dawley rats (250
– 300g) are
anaesthetized
with ether
The left kidney is
removed through
a flank incision
DOCA (20 mg/kg)
is dissolved in
olive oil &
injected s.c. to
rats, twice
weekly for four
weeks
Drinking water is
replaced with 1%
NaCl solution
Test drug is
administered
orally for one
month
BP is measured
before & after
the
administration of
the test drug &
their values are
compared
Mineralocorticoid induces hypertension by in plasma volume
Salt loading & unilateral nephrectomy in rats further increases the
hypertensive effect

30. Psychogenic Hypertension
Elevation of BP resulting from repeated exposure to stressful situation
Lead to a state of persistent hypertension
 Stress : Emotional stimuli, psychosocial stress, immobilization
stress, electric stimuli
 Hypertension in these animals is not renin dependent
 Stress plays an important role in development of hypertension in
humans this model is frequently used to study
pathophysiology of hypertension
 Air - jet stimulation induced hypertension

31. Air - jet stimulation induced hypertension
BHRs are
exposed to daily
sessions of either
short (20 min) or
long (120 min)
duration air –jet
stimulation
BP is monitored
at regular
intervals using
tail cuff method
Animals exposed
to 120 min stress
sessions have
significantly
higher systolic
BP relative to the
20 min group
Deleterious
effect of stress
depends-
Critical period of
exposure,
duration, type
All these factors
may alter
functions of the
basic regulatory
stress response
- HPA axis
- Sympatho
adrenal
medullar
system
- RAAS
- Sympathetic
Nervous System
Borderline hypertensive rats (BHR) are useful for psychogenic model

32. Genetic Hypertension
 A. Salt sensitive Dahl rats
 B. Spontaneously hypertensive rats

33. Salt sensitive Dahl rats
Upon salt loading
Salt sensitive Dahl rats
develop severe & fatal
hypertension
Salt resistance Dahl rats do
not develop such severe
hypertension
When fed normal salt diets, the salt sensitive rats become
hypertensive
Demonstrates that this is a model of genetic hypertension
With the extra feature of salt sensitivity

34. Salt sensitive Dahl rats
Animals are fed
the prepared
diet, 8 % NaCl
solution ad
libitum
Test group rats
are administered
the drug orally
for 1 month
BP changes
recorded
After the
experiment is
complete
animals of test &
control group
sacrificed
Their hearts are
removed, total
cardiac mass,
weight of Lt & Rt
ventricle is
measured &
Upon salt feeding
BP rises to levels
higher than
found in SHR
The ability of the
test drug to
reverse these
changes is
studied
Sprague Dawley rats are used (250-300g).The drinking water is
replaced with 8 % NaCl saline solution.High Dahl salt diet is prepared
by mixing salt with regular diet

35. Spontaneously hypertensive rats (SHR)
Breeding a strain of Spontaneously hypertensive wistar
rats with a female having slightly raised BP
A strain of rats with spontaneous hypertension obtained
 BP rises around 5 to 6 weeks of age & steadily increases
to reach SBP of 180 to 200 mm Hg
 They develop features of end organ damage -
Cardiac hypertrophy, cardiac failure, renal dysfunction

36. Dog Models of Hypertension
 A. Chronic renal hypertension
 B. Neurogenic hypertension

37. Chronic renal hypertension
Dogs (8 to 12 kg) are anaesthetized i.v. with 15 mg/kg
thiopental
Midline abdominal incision is made, one kidney is exposed &
wrapped in cellophane & then replaced
The contralateral kidney is exposed, artery, vein, ureter
ligated & kidney is removed
Abdomen is closed & sutured back

38. After 6 weeks of surgery, BP is measured
BP is recorded either by tail cuff method or by direct
measurement through the carotid artery
Test drugs are administered for 5 days
On day 1 readings are taken every 2h, just before & 2 and 4h
after oral treatment
On day 3 & 5 BP is recorded before, 2 and 4h after drug
treatment and fall of bp is assessed at various recording times

39. Neurogenic Hypertension
 Baroreceptors in carotid sinus & aortic arch
Regulation of BP
 Stimulation of afferent buffer fibres exerts an inhibitory
influence on the vasomotor centre
 On sectioning the baroreceptors , a persistent rise in BP is
observed this procedure is used to induce
neurogenic hypertension in dogs

40. Neurogenic hypertension
Adult dogs (10 to 15 kg) are anaesthetized using 15 mg/kg
thiopental, 200 mg/kg sodium barbital & 60 mg/kg sodium
pentobarbital
Femoral vein is cannulated for the administration of test
compounds
LVP & Dp/dt are recorded through common carotid artery
using Millar microtip pressure transducer
Carotid sinus nerves are isolated, ligated & sectioned
& a bilateral vagotomy is performed to induce neurogenic
hypertension

41. After 30 min period, a bolus of test compound is
administered by i.v. route
Heart rate, arterial pressure, LVP, dP/dt are monitored for 90
min
Changes in cardiovascular parameters are expressed as
percentage of the values before & after administration of the
drug

42.  Monkey Model of Hypertension
• Renin inhibition in monkeys
Transgenic Models
• Transgenic rats overexpressing the mouse
Ren-2-gene {TGR (m Ren 2)27}

43. In Vitro Methods

44. Endothelin receptor antagonism in
Porcine Isolated Hearts
 Potent long lasting contractions of isolated blood vessel
strips and increase BP in vivo is elicited by endothelin
peptides
 Endothelins have been implicated in the pathophysiology
of cardiovascular disorders
 In this model, isolated porcine coronary is used since
smooth muscles of artery→ contain the ET receptors

45. Endothelin receptor antagonism in Porcine Isolated Hearts
From porcine hearts→left anterior descending coronary
arteries are isolated
Endothelium-denuded arteries are cut into spiral strips about
10mm long and 1mm wide
Intimal surface of the spiral rings is then rubbed gently with
filter paper to remove vascular endothelium
Each strip is suspended in an organ bath containing Krebs –
Heinseleit solution bubbles with 95% O2 / 5% CO2 at 37◦C

46. Once isolated preparation is stabilized→ reference
contraction is isometrically obtained with 50 mM KCl
Twenty minutes before addition of ET1 the endothelin
receptor antagonist /test drug is added to the organ bath
Concentration response curve is recorded
The pA2 values and slopes are obtained by analysis of Schild
plots

47. Monocrotaline induced pulmonary
hypertension
 Monocrotaline - hepatotoxic and pneumotoxic agent→
used in rats to induce pulmonary hypertension
 Single injection→ progressive pulmonary hypertension →
right ventricular hypertrophy & cardiac failure
 Monocrotaline administration in rats may result in severe
right ventricular hypertrophy accompanied by ascites and
pleural effusion

48. Monocrotaline induced pulmonary hypertension
Sprague Dawley rats (200 -225g) are fed with test drug for 1
week prior to single subcutaneus injection of 100mg/kg
monocrotaline
4, 7 or 14 days later, the animals are sacrificed and their
hearts and lungs excised from the thoracic cavity
The left ventricle and left lung are weighed. Their pulmonary
artery segments, main pulmonary artery, right extra
pulmonary and an intra pulmonary artery are also isolated

49. Monocrotaline induced pulmonary hypertension
Each vessel is suspended between stainless steel hooks in
tissue baths containing Krebs- Hensleit buffer aerated with
95% O2 and 5 % CO2 at 37◦
At the end of the experiment vessel segments are blotted,
weighed and their dimensions are measured. After 1 hour
arteries are made to contract to KCl (6 * 1/100 M)
Maximum active force generated by an artery is plotted as a
function of applied force & changes in isometric force are
monitored using displacement transducers & recorded on a
polygraph. Contractile and relaxant agonist responses are
assessed in pulmonary arteries

50. Summary
In vitro models of hypertension
Endothelin receptor antagonism in porcine isolated hearts
Monocrotaline induced pulmonary hypertension

51. Summary
Renovasc
ular
Hypertens
ion
Neurogeni
c Induced
Diet
Induced
Endocrine
Induced
Psychogen
ic
Geneticall
y induced
Two
Kidney
One Clip
(2K1C)
Blood
pressure
in pithed
rats
Fructose
induced
DOCA salt
rats
Air-jet
stimulatio
n induced
hypertensi
on
Salt
sensitive
Dahl Rats
Chronic
Renal
Hypertens
ion
- 1K1C
- 2K2C
Increased
salt
induced
Spontaneo
usly
hypertensi
ve rats
(SHR)
In vivo models of hypertension : Rat Models

52. Summary
In vivo models of hypertension
Dog model of hypertension
• Chronic renal hypertension
• Neurogenic hypertension
Monkey model of hypertension
• Renin inhibition in monkeys
Transgenic model of hypertension
• Transgenic rats overexpressing the mouse Ren-2 gene { TGR
(mRen 2)27}

53. Thank You

54. References
• S K Gupta(ed.) Drug Screening Methods. 3rd ed. New Delhi: Jaypee
Brothers Medical Publishers (P) Ltd; 2016. p 266-277