This presentation gives you a detailed information about the screening methods used to evaluate the antihypertensive action of the investigational product. It delineate the animal models used for testing of the antihypertensive agents. This presentation will provide you insight to the methods using in-vivo animal model for preclinical testing of antihypertensive agents.

1. Dr. Rajendra Gode Institute of Pharmacy,
University - Mardi Road, Amravati - 444602
Presented by
SHRADDHA S. RAUT
B. Pharm
Guided by
DR. SACHIN PADOLE
M. Pharm, Phd (Pharmacology)
Pharmacy Council of India, New Delhi
PHARMACOLOGICAL AND TOXICOLOGICAL SCREENING
(MPL 103T)
“ Preclinical Screening of Antihypertensive Agent ”
1

2.  Contents :
2
Introduction
Screening of Antihypertensive agent
Animals used
Methods using In-vivo animal model

3. 3
 Introduction
Preclinical Screening: It is the process by which potential drugs are identified and
optimized before undergoing in the process of clinical trial.
Aim: To collect data in support of the safety of the new treatment.

4. 4
Hypertension: Increase in the arterial blood pressure above normal.
Conti……
Blood Pressure
Normal B.P. Chronic B.P.
Pre-hypertension B.P.
120/80
mmHg
STAGE 1: 150/100
mmHg
STAGE 2: 180/120
mmHg
> 200/150
mmHg

5. 5

6. 6

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8. 8
 Screening of Antihypertensive Agent
Antihypertensive agent : Drugs used to treat high blood pressure.
Animal models are developed
By utilizing etiological factors responsible for human hypertension
Excessive salt
intake
Hyperactivity of
RAAS
Genetic factors

9.  Animals Used :
9
Dog
b
Rat
SHR
b

10. 10
Acute renal hypertension in rats
Chronic renal hypertension in rats
Chronic renal hypertension in dogs
Neurogenic hypertension in dogs
Fructose induced hypertension in rats
Genetic hypertension in rats
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2
3
4
5
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 Methods using in-vivo animal model

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• Purpose and Rationale
1. Goldblatt proved that ischemia of kidney causes increase in BP by activating RAAS.
2. Hypertension is induced by clamping the left renal artery (2-Kidney 1-clip method)
• Procedure
Male Sprague Dawley rats weighing 300 g
anesthetized by i.p. injection of hexobarbital sodium (100mg/kg)
Dieffenbach clip is placed onto the left hilum of the kidney
1) Acute renal hypertension in rats

12. 12
Conti……
The renal artery is occluded for 3.5 – 4 h
3.5 hr following the surgery, animals are anesthetized by
pentobarbital sodium (30-40 mg/kg, i.p.)
Trachea is cannulated to facilitate spontaneous respiration
To measure systolic and diastolic BP, the cannula in carotid artery is
connected to a pressure transducer
Jugular vein is cannulated to administer test compound

13. 13
Conti……
After obtaining stable reduced BP values, the renal arterial
clip is removed
Leads to rise in BP due to increased plasma renin level
Within 15 min stable hypertension is achieved
Then test compound is administered by i.v. injection at
doses of 10 & 100 ug/kg
BP is monitored continuously

14. 14
Conti….
• Evaluation
1. Increase in BP after reopening renal artery and decrease in BP after administration of
test compound is determined.
2. % inhibition is calculated by comparing pre-treatment and post-treatment BP values.
• Modification
Berthold et al. (1989) – BP associated with reflex bradycardia can be elicited by
injection of 5-HT into coronary artery.
James et al. (1975) – proved 5-HT as most powerful agent to induce hypertension.

15. 15
• Purpose and Rationale
1. Based on findings of Goldblatt, various modifications are described.
2. This is one of the most effective modified method (1 Kidney-1 Clip method)
• Procedure
Male Sprague Dawley rats weighing 200-250 g
anesthetized by i.p. injection of pentobarbital (50 mg/kg)
Fur on back is shaved and skin is disinfected
2) Chronic renal hypertension in rats

16. 16
Conti……
In left lumbar area, flank incision is made. Renal pedicel is exposed
Renal artery is dissected clean & U-shaped silver clip is slipped around it
near aorta
Right kidney is removed through incision and incision is closed by
wound clips
4-5 weeks after clipping, BP is measured
(Rat with >150 mmHg BP are selected)
BP readings are taken 3 days prior to drug treatment

17. 17
Conti……
Drug is administered (10 ml/kg) for 3 days
Predrug and 2 hrs Postdrug BP readings are taken
• Evaluation
1. Activity is determined by comparing treatment BP values with control BP values
(Predrug BP of Day 1)
2. Comparison is done using paired t-test for evaluation of statistical significance.
• Modification
Duan et al. (1996) – induced renal hypertension in male Hartley Guinea Pigs by
ligation of left caudal renal artery & right nephrectomy

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• Purpose and Rationale
1. Goldblatt -1st described the process by clamping renal artery.
2. Abram and Sobin – modified it as wrapping technique
• Procedure
Dogs weighing 10-12 kg anesthetized by i.v. injection of Thiopental (15 mg/kg)
midline abdominal incision is made (aseptic condition)
1 Kidney is exposed & wrapped in cellophane & then replaced
The contralateral kidney is exposed
3) Chronic renal hypertension in dogs

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Conti……
The artery, vein and ureter are ligated and kidney is removed
The abdomen is closed by sutures and clips
BP is measured using tail-cuff method
(Dog with >150 mmHg BP are selected)
On day 1, readings are taken every 2 hr, just before treatment & 2-4 hr after treatment
with test compound
Drug administration is repeated for 5 days
On Day 3 & 4, BP readings are taken before and after treatment

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Conti……
• Evaluation
The starting value is the average of 2 readings before administration of drug
Readings obtained after administration of drug is subtracted from this value
And recorded as fall of BP at various recording times
• Modification
Duan et al. (1996) - achieved renal hypertension by removing the renal capsule with gentle
traction
Abram & Sobin (1947) - induced hypertension by encapsulating both kidneys with latex
rubber capsules

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• Purpose and Rationale
1. Vasomotor center is responsible for regulating BP.
2. By stimulating afferent buffer fibers, inhibitory influence is exerted on vasomotor
center, leads to acute neurogenic hypertension.
• Procedure
Adult Dogs weighing 10-15 kg anesthetized by i.v. injection of Thiopental (15 mg/kg)
femoral vein and artery is cannulated using polyethylene tubing to administer test
compound i.v.
Record arterial pressure and heart rate
4) Neurogenic hypertension in dogs

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Conti……
Left ventricular pressure is recorded - by Millar microtip pressure transducer
Pmax is recorded - by speeding up chart paper
Cardiac output is determined - by Swan Ganz Catheter
All recordings are made with polygraph
Both carotid arteries are cleared up to bifurcation of internal ad external carotid arteries
Carotid sinus nerve are isolated, ligated and sectioned & bilateral vagotomy is performed to
produce neurogenic hypertension
Dog - allowed to equilibrate - for 30 min

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Conti……
& bolus of test compound is administered through i.v.
H.R., arterial blood pressure, left ventricular pressure, Pmax are monitored for 90 min
• Evaluation
Changes of parameters are expressed as % of values before administration of drug.
• Modification
Angell James (1984) - described in rabbits
Krieger (1984) - described in rats

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• Purpose and Rationale
Hwang (1987) - First reported that hypertension could be induced in normal rat by
feeding high-fructose diet
• Procedure
Male Wistar rats weighing 210-250 g
Housed 2 per cage on 12-hr night & 12-hr dark cycle
& are allowed free access to standard laboratory diet (Purina rat chow) & drinking fluid
(10% fructose solution)
5) Fructose induced hypertension in rats

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Conti……
Body weight & Food and fluid intake is measured every week during
treatment
Using tail-cuff method, systolic BP and pulse rate is measured before
and every week during treatment
Blood sample is also taken to determine plasma glucose and insulin
• Evaluation
Statistical analysis is performed using one-way or two-way analysis of variance, followed
by Newman-Keuls test
• Modification
Hall et al. - reported effect of high-fat diet (for 6 weeks) on CV function in dogs.

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• Survey
1. Smik and Hall – described inherited hypertension in rats
2. Okamoto (1963) – Spontaneous Hypertensive Rats (SHR) / Wistar-Kyoto Rats (WKY)
He also separated several sub-strains from SHR and named them as stroke prone strains
3. Bianchi – Milan Hypertensive Strains (MHS)
6) Genetic hypertension in rats
n m n
Male
(High BP)
Female (Slightly
elevated BP)
SHR / WKY
> 200 mmHg

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Conti……
Dahl (1962) – developed inbreed strains named as DS (salt-hypertension-sensitive)
RD (salt-hypertension-resistant)
Deng and Rapp (1992) – increased BP of SHR is determined by multiple genetic loci
Peters (1993) – TGR27 as monogenetic model in hypertension research
• Assessment - Use of SHR to detect potential antihypertensive compound is well established.
• Modification
Pijel – described streptozotocin induced Diabetes Mellitus in SHR as a model for combined
effect of hypertension & diabetes
Linz et al. – proved that lifelong ACE inhibition doubled the lifespan in hypertensive rats

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 References
1) Drug Discovery and Evaluation by H. Gerhard Vogel, 2nd edition, Springer
Publication, Pg no. 172 – 179
2) Handbook of Experimental Pharmacology by S. K. Kulkarni, 4th edition, Vallabh
Publication.

29. T H A N K
Y O U
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Any question