screening methods of antihypertensive agents
TRANSCRIPT
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Preclinical screening methods of Antihypertensive agents
(for MD Pharmacology)
- Dr. Advaitha M V
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Introduction • 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- aldosteronesystem (RAAS) and
Genetic factors.
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An ideal animal model of hypertension criteria
• It should be feasible in small animals.
• Simple to perform and uniformly reproducible.
• Should be able to predict the potential antihypertensive properties of an agent.
• Consume minimal quantities of compounds.
• It should be comparable to some form of human hypertension.
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Animals used
• In the past, most studies
on experimental hypertension were carried out on Dogs.
• Currently, rat is the preferred animal species.
• Spontaneous hypertensive rat (SHR), the genetic strain of hypertensive rat, is the animal of choice.
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Types of animal models of hypertension
1. Renovascular hypertension
2. Dietary hypertension
3. Endocrine hypertension
4. Neurogenic hypertension
5. Psychogenic hypertension
6. Genetic hypertension
7. Other models
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1. Renovascular Hypertension
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• This is a very commonly used model of hypertension.
• Experimentally, renal hypertension is produced by constriction of renal artery.
• This activates peripheral RAAS and sympathetic nervous system.
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Methods of inducing renovascularhypertension
Goldblatt method:
Goldblatt et al (1934)
• He reported that a partial constriction of renal arteries in dogs produced hypertension.
• This type of hypertension has also been induced in rabbits, rats and monkeys.
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• Rats weighing from 120 to 200 g are anaesthetized with hexobarbitone sodium (40 mg/kg body weight).
• A silver clip of 0.2 mm internal diameter is placed on the left renal artery close to the aorta.
• The renal artery in rats can also be ligatedwith 4-0 silk suture.
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• Constriction of renal artery should be more than 50%.
• The animal is considered hypertensive if systolic BP is more than 160 mm Hg for two consecutive days after 4 weeks of ligation/application of clip.
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• Three variants of hypertension produced by Goldblatt method-
• Two kidney one clip (2K1C) hypertension
• One kidney one clip (1K1C) hypertension
• Two kidney two clip (2K2C) hypertension
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Two kidney one clip (2K1C) hypertension
• The renal artery is constricted on only one side with the other artery (or kidney) left untouched.
• This results in a sustained increase in BP.
• Initially , there is no salt and water retention because of the other normal kidney being intact.
• So , the resultant hypertension at this stage is renin-angiotensin dependent.
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• After about 6 weeks, the increased angiotensin-II releases aldosterone from adrenal cortex leading to gradual retention of salt and water.
• Retention of salt and water leads to decreased renin production.
• From this stage onwards, hypertension is volume dependent.
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One kidney one clip (1K1C) hypertension
• Constriction of renal artery is done on one side and the contra lateral kidney is removed.
• There is an increase in BP within a few hours.
• Since there is no contra lateral kidney, there is no pressure diuresis and natriuresis.
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• So there is rapid salt and water retention.
• Plasma renin activity is usually normal.
• Hypertension soon becomes volume dependent
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Two kidney two clip (2K2C) hypertension
• Constriction of aorta or both renal arteries is done.
• There is a patchy ischemic kidney tissue, which secretes renin leading to increased BP.
• The remaining kidney tissue retains salt and water.
• one of the most common causes of renal hypertension in human beings is (such) a patchy ischaemic kidney disease.
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Hypertension induced by external compression of renal parenchyma
Grollman hypertension
• In this method, kidney tissue is compressed by securing a 'figure of 8' ligature.
• It is of two types:
• Two kidney one ligature (2K1L)
• One kidney one ligature (1K1L)
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Reduced renal mass
• Reducing renal tissue to five-sixth (5/6th) by renal mass ablation produces hypertension.
• Here , the right kidney is removed and 2 or 3 branches of left renal artery are ligated.
• This is to produce infarction of approximately 2/3rd of the left kidney.
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2. Dietary hypertension:
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I. Increased salt intake:
• Chronic ingestion of excess salt produces hypertension in rats, which mimics human hypertension morphologically.
• High salt intake hypertension has been produced in rats, rabbits and chicks by replacing drinking water with 1-2% sodium chloride for 9-12 months.
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• This method is also used to cause Accelerated high pressure in renal hypertension.
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3. Endocrine hypertension
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DOCA-Salt induced HTN
• Selye et al was the first to demonstrate that deoxycorticosterone acetate (DOCA) produces hypertension in rats.
• There is increased DOCA-induced reabsorption of salt and water leading to increased blood volume and hence increased BP.
• There is also increased secretion of vasopressin leading to water retention and vasoconstriction.
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• In addition, altered activity of RAAS leads to increased sympathetic activity
• Rats, especially female and young, are prone to DOCA-salt induced hypertension.
• DOCA induced hypertension is salt dependent
(neither administration of DOCA nor partial removal of renal mass is effective in increasing BP when applied without salt Administration).
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To produce hypertension,
• Rats weighing about 100 g are kept on a diet high in sodium chloride and drinking water is replaced by 2% sodium chloride solution.
• After they attain a weight of about 250 g, they are given DOCA dissolved in sesame seed oil at a dose of 10 mg/kg SC, twice weekly for 4 weeks.
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4. Neurogenic hypertension
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• One of the most important negative feedback in the control of BP originates from baroreceptorsin the carotid sinus and aortic arch.
• Afferents of baroreceptors travel along 9th and 10th cranial nerves.
• Sectioning of the baroreceptor nerves leads to persistent rise in BP.
• Suitable in dogs, cats and rabbits
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5. Psychogenic hypertension
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• Repeated exposure to stressful situation may lead to a state of persistent hypertension.
• Borderline hypertensive rats (BHR) are useful for psychogenic hypertension.
• BHRs that were exposed to daily sessions of either short (20 min) or long (120 min) duration air-jet stimulation developed hypertension within 2 weeks.
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• Stress plays an important part in development of human hypertension.
• So, This model is also very frequently used to study the pathophysiology of hypertension.
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6. Genetic hypertension
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• The so called ‘spontaneous hypertensive rat (SHR)’ was developed by meticulous genetic inbreeding by Okamoto and Aoki.
• This inbreeding resulted in 100% of the progeny having naturally occurring hypertension
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How are SHR ?
• In SHRs, BP gradually increases until it is maintained at a markedly elevated level after approximately 12 weeks of age.
• In unrestrained male SHRs, mean arterial pressure is approximately 190- 200 mm Hg as compared to 115-130 mm Hg in normal rats.
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• During the early stable stages and developmental phase of hypertension-
elevated BP is maintained in large part by enhanced central sympathetic outflow.
• In the later stages
Increased total peripheral resistance.
normal cardiac output
decreased permeability of the glomerularmembranes
Forms the basis for the long term maintenance of the hypertension
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• SHRs also develop typical complications of hypertension.
• There are Stroke prone SHRs (SHRSP), which are selectively bred among SHRs.
• These develop cerebrovascular lesions spontaneously in over 80% of rats.
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• Because of apparent similarities of the SHR to essential hypertension-
SHR models are highly recommended for screening potential drug candidates for hypertension
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• Other models
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Obesity-related hypertension
• Wistar fatty rats (WFR) are derived from cross between obese Zucker and Wistar Kyoto rats.
• These show persistent hyperinsulinemia and hypertension after 16 weeks of age.
• A good model to elucidate the relationship between hyperinsulinemia and hypertension.
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Wistar Kyoto Rat
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Transgenic rat (TGR) models• Transgenic models of hypertension have
revolutionized the experimental work on hypertension.
• Here , an additional renin gene, the murineRen-2 gene, is introduced into the germ line of rats.
• This results in transgenic hypertensive rat strain, TGR (mREN2).
• It has an overexpression of renin.
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• Linkage has been described for the angiotensinogen gene in human hypertension.
• TGR model are also useful for studying the role of local RAAS system in hypertension.
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Others
• Angiotensin-II induced hypertension.
• Hypertension induced by cadmium
• Chronic nitric oxide inhibition-induced hypertension.
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How to measure BP ?
Direct Method :
• It is a invasive procedure.
• A week before the drug adminstration, The animals are anesthetized.
• Femoral/Carotid Cannula is inserted.
• On the day of screening, cannula is connected to a mercury manometer or a pressure transducer.
• BP is measured
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In rats
• A week before the experiment, each rat is anaesthetised with 40 mg/kg pentobarbital.
• Left or right carotid artery or femoral artery is cannulated under aseptic conditions with polyethylene cannula filled with 1% heparin in normal saline.
• Free end of the cannula is passed under the skin and allowed to protrude 3-4 cm from the skin behind the ears of the rat.
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• The skin incisions are sutured and a plastic skin dressing is applied.
• After recovery from anaesthesia (2-2.5 h) each rat is placed in an individual cage for 24 h habituation period
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• On the day of expt., Cannula is connected to a pressure transducer then to the pre-amplifier.
• BP is recorded on the polygraph or physiograph.
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Non-Invasive/ Indirect method :
In rats
Tail cuff Method :
• It is a common and convenient method.
• Tail cuff is inflated and then deflated.
• Pulsations disappear when cuff is inflated.
• When cuff is deflated, pulsations start appearing when pressure in the cuff equals systolic pressure.
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• The cuff is attached to a tail cuff sphygmomano-meter or pressure transducer and BP is recorded on a chart.
• Tail swelling method.
• Foot swelling method.
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Effects of antihypertensive agents
• Antihypertensive drugs, according to their mode of action, will affect the blood pressure in certain types of experimental hypertension, and not in all !
• Vasodilators like minoxidil, hydralazine and diazoxide are effective in renal hypertensive rats.
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• CCB, ACE inhibitors and AT-1 antagonists decrease BP in 5/6 nephrectomised SHR
• Diuretics, are active in mineralocorticoid or salt induced hypertension.
• Sympathomimetic Drugs decrease BP in both endocrine and neurogenic hypertension.
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An E.g. of data collection
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Invitro models
• Endocrine receptor antagonism in porcine isolated heart
• Monocrantaline-induced pulmonary hypertension.
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References • D.K. BADYAL, H. LATA , A.P. DADHICH. ANIMAL MODELS OF
HYPERTENSION AND EFFECT OF DRUGS. Indian Journal of Pharmacology 2003; 35: 349-362.
• Journal of Visualized Experiments videos
Video downloads from http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2794298/bin/jove-27-1291.flv
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3462562/bin/jove-59-3496.mov
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Thank You