ANTIHYPERTENSIVE &ANTIHYPERTENSIVE &

VASODILATIVEVASODILATIVE

SCREENING SCREENING

OF OF AGENTSAGENTS

S.K.LALIst M.Pharm

Dept. PHARMACOLOGYJKKMMRF KPM

What is Hypertension• High blood pressure: blood forced through the arteries at an

increased rate.• It is measured as two numbers, e.g. 120/80 mmHg. Normal

blood pressure is below this value.

– The first number is the systolic blood pressure. This is the maximum pressure in the arteries when heart contracts/beats.

– The second number is the diastolic blood pressure. This is the minimum pressure in the arteries when heart is at rest between beats.

• Until it becomes extreme, there are no symptoms. Hence, it is called the “Silent Killer.”

• Severe hypertension may cause headache, sleepiness, confusion or coma.

IN VIVO MODELSIN VIVO MODELS ACE Inhibition in rat Goldbalt Hypertension Chronic renal hypertension in rats Tail cuff method Salt- sensitive Dahl Rats Renin inhibition in Monkeys

Chronic renal hypertension in Dogs. Antihypertensive and vasodilator action in rats.

IN VITRO & EX-vivo models IN VITRO & EX-vivo models

Monocrotaline Induced Pulmonary Hyperyension ACE Inhibition in GUENIA PIG illeum β1 Sympatholytic Activity in GUENIA PIG Atria

ACE INHIBITION IN RASACE INHIBITION IN RAS

HYPERTENSIONHYPERTENSION

Inactive

Angiotensin -I

Active

Angiotensin -II

ACE

Male Sprague- Dawley rats (200-225gm) are selected..Male Sprague- Dawley rats (200-225gm) are selected.. They are anaesthetised with 60mg/kg of phenobarbitone They are anaesthetised with 60mg/kg of phenobarbitone

sodium i.v.sodium i.v. The intubated trachea is artificially respirated with The intubated trachea is artificially respirated with

30strokes/min and a stroke volume of 6-8ml.30strokes/min and a stroke volume of 6-8ml. The right artery is cannulated for recording the pressure.The right artery is cannulated for recording the pressure. The jujular vein is cannulated for i.v. test injection.The jujular vein is cannulated for i.v. test injection. The B.P is diminished by the administration of 5mg/kg of The B.P is diminished by the administration of 5mg/kg of

pentolinium i.p.pentolinium i.p. Atropine(40µg/kg) is also injected i.m to inhibit the mucous Atropine(40µg/kg) is also injected i.m to inhibit the mucous

secretion.secretion. Now 310ng/kg of Ang I is injected i.v. in 0.1ml saline.Now 310ng/kg of Ang I is injected i.v. in 0.1ml saline. The injection is repeated in 5min interval until an identical The injection is repeated in 5min interval until an identical

pressure is attained.pressure is attained.

The test drug is administered at a dose of 10mg/kg The test drug is administered at a dose of 10mg/kg intravenously or 25mg/kg intradeudenally.intravenously or 25mg/kg intradeudenally.

Again Ang I is injected as the similar dose above.Again Ang I is injected as the similar dose above. The diminution of the pressure after the administration of The diminution of the pressure after the administration of

potent ACE inhibitors is compared .potent ACE inhibitors is compared .

In vivo modelsIn vivo modelsGOLDBALT HYPERTENSIONGOLDBALT HYPERTENSION

(2 Kidney 1 Clip Method)(2 Kidney 1 Clip Method)

Ischemia of kidney Blood pressure Ischemia of kidney Blood pressure

METHOD:METHOD: Renin- Angiotensin Mechanism Renin- Angiotensin Mechanism

Clamping of renal artery for 4hr and reopening Clamping of renal artery for 4hr and reopening

Accumulated renin is released Accumulated renin is released

Sprague – Dawley Rats (300gm) are anesthetized Sprague – Dawley Rats (300gm) are anesthetized ĉ ĉ Hexobarbital sodium (100mg/kg) Intra peritonially.Hexobarbital sodium (100mg/kg) Intra peritonially.

Cannulate the trachea for respiration and the Jugular Cannulate the trachea for respiration and the Jugular vein for test compound administration.vein for test compound administration.

A transducer is connected to the carotid artery for A transducer is connected to the carotid artery for recording the pressure.recording the pressure.

A PVC coated clip is placed in the left hilum of the A PVC coated clip is placed in the left hilum of the kidney by fixing with the back muscle for 3.5 - 4hr.kidney by fixing with the back muscle for 3.5 - 4hr.

Pentolinium is administered for ganglionic blockache.Pentolinium is administered for ganglionic blockache. Relaese the clip and record the rise in B.P.Relaese the clip and record the rise in B.P. Administer the test drug througfh I.V. and moniter the Administer the test drug througfh I.V. and moniter the

pressure continuously.pressure continuously. Increase in B.P after releasing the clip and reduction Increase in B.P after releasing the clip and reduction

after the drug administration is determined.after the drug administration is determined. Compare using percentage valuesCompare using percentage values

CHRONIC RENAL HYPERTENSIONCHRONIC RENAL HYPERTENSION(1 Kidney- I -Clip Method)(1 Kidney- I -Clip Method)

Sprague – Dawley Rats (200 - 300gm) are anesthetized Sprague – Dawley Rats (200 - 300gm) are anesthetized ĉ ĉ Phenobarbitone sodium (100mg/kg) Intra peritonially.Phenobarbitone sodium (100mg/kg) Intra peritonially.

A flank parallel incision is made in the left lumbar area.A flank parallel incision is made in the left lumbar area. Renal artery is dissected, cleaned and ‘U’ shaped silver clip is Renal artery is dissected, cleaned and ‘U’ shaped silver clip is

slipped around near the aorta.slipped around near the aorta. The internal gap b/w the clip is adjusted to 0.25 – 0.38 nm.The internal gap b/w the clip is adjusted to 0.25 – 0.38 nm. The right kidney is removed after tying off the renal pedicel.The right kidney is removed after tying off the renal pedicel. After 4-5 weeks the B.P is measured and the animals are divided After 4-5 weeks the B.P is measured and the animals are divided

into groups of different doses.into groups of different doses. Test drug is administered for 3 daysTest drug is administered for 3 days Pressure before and after drug administration(3min) are recorded.Pressure before and after drug administration(3min) are recorded. Percent reduction in pressure is calculated and compare to the Std.Percent reduction in pressure is calculated and compare to the Std.

TAIL CUFF METHODTAIL CUFF METHOD(Blood Pressure in Conscious rats)(Blood Pressure in Conscious rats)

Method without any surgical procedure, Similar to Method without any surgical procedure, Similar to sphygomanometry in humans (systolic pressure).sphygomanometry in humans (systolic pressure).

Widely used to evaluate the antihypertensive drugs in experimently Widely used to evaluate the antihypertensive drugs in experimently induced animals.induced animals.

Charles River male rats(300-350g) are anaesthetized using I.P Charles River male rats(300-350g) are anaesthetized using I.P injection of 0.8ml of 4% of chloral hydrate.injection of 0.8ml of 4% of chloral hydrate.

Both the kidneys are exposed, hypertension is induced by placing a Both the kidneys are exposed, hypertension is induced by placing a silver clip on both renal arteries.(0.2mm dm & 4mm l)silver clip on both renal arteries.(0.2mm dm & 4mm l)

After 5 to 6 weeks operated animals attain renal hypertension with After 5 to 6 weeks operated animals attain renal hypertension with systolic B.P of 170 to 200mmHg.systolic B.P of 170 to 200mmHg.

A tubular inflatable cuff is placed around the base of tail and a Pizo- A tubular inflatable cuff is placed around the base of tail and a Pizo- electric pulse detector is positioned distal to the cuff.electric pulse detector is positioned distal to the cuff.

The cuff is inflated to approximately 300mmHg.The cuff is inflated to approximately 300mmHg. As the pressure in the cuff is released slowly, the systolic As the pressure in the cuff is released slowly, the systolic

pressure is detected and recorded in the poly graph.pressure is detected and recorded in the poly graph. Test substance is administered intra peritonially for alternative Test substance is administered intra peritonially for alternative

days in three times.days in three times. Decrease in the systolic pressure is determined by the Decrease in the systolic pressure is determined by the

following steps.following steps.

Day 1: Predose & 2hr postdrugDay 1: Predose & 2hr postdrug Day 3: Predose & 2hr postdrugDay 3: Predose & 2hr postdrug Day5: Predose , 2hr post drug & 4hr postdrugDay5: Predose , 2hr post drug & 4hr postdrug

SALT- SENSITIVE DAHL RATSSALT- SENSITIVE DAHL RATS These rats Develop severe fatal hypertension when fed high – saltThese rats Develop severe fatal hypertension when fed high – salt

diets, whereas salt resistance rats cant.diets, whereas salt resistance rats cant. Sprague – Dawley Rats (250 - 300gm) are used in this study.Sprague – Dawley Rats (250 - 300gm) are used in this study. Drinking water is replaced with 8% NaCl soln. high salt diet is prepared Drinking water is replaced with 8% NaCl soln. high salt diet is prepared

by mixing salt with regular diet.by mixing salt with regular diet. The animals are fed with the above diet and saline.The animals are fed with the above diet and saline. The test group rats are administered the drug orally for 1 month.The test group rats are administered the drug orally for 1 month. Blood pressure changes are recorded.Blood pressure changes are recorded. After the experiment the animals (both cntrl & test) are sacrificed.After the experiment the animals (both cntrl & test) are sacrificed. The hearts are isolated, the mass, weight of LV & RV is compared and The hearts are isolated, the mass, weight of LV & RV is compared and

measured.measured. Upon salt feeding animals B.P rises steeply (up to 36%).Upon salt feeding animals B.P rises steeply (up to 36%). The ability of the drug to reverse these changes is studied.The ability of the drug to reverse these changes is studied. Cardiac failure occurs at 4 to 5 months of age in thse kind of rats.Cardiac failure occurs at 4 to 5 months of age in thse kind of rats.

ANGIOTENSIN - II ANTAGONISMANGIOTENSIN - II ANTAGONISM

Male Sprague- Dawley rats (200-225gm) are selected..Male Sprague- Dawley rats (200-225gm) are selected.. They are anaesthetised with 60mg/kg of phenobarbitone They are anaesthetised with 60mg/kg of phenobarbitone

sodium i.v.sodium i.v. One carotid artery is cannulated & connected with a Statham One carotid artery is cannulated & connected with a Statham

transducer and the B.P is recorded in a polygraph.transducer and the B.P is recorded in a polygraph. Both the jujular veins are cannulated to administer the test Both the jujular veins are cannulated to administer the test

drug.drug. Pentolinium (10mg/kg) is injected to block the ganglionic Pentolinium (10mg/kg) is injected to block the ganglionic

activity.activity. Atleast 5 animals are used for the evaluation of test drug.Atleast 5 animals are used for the evaluation of test drug. In 10 min interval doses of 0.5,1.0, and 2µg/kg of angiotensin In 10 min interval doses of 0.5,1.0, and 2µg/kg of angiotensin

II are injected to establish the dose response curves.II are injected to establish the dose response curves. After 10 min a continuous infusion is started of the Ang II After 10 min a continuous infusion is started of the Ang II

blocker in a dose of 10µg/kg/ 0.1ml/ml.blocker in a dose of 10µg/kg/ 0.1ml/ml.

Again doses of 0.5,1.0, and 2µg/kg of angiotensin II are Again doses of 0.5,1.0, and 2µg/kg of angiotensin II are injected .injected .

Intensity and duration of fall in B.P is recorded.Intensity and duration of fall in B.P is recorded. The results are compared with the known std. drug.The results are compared with the known std. drug.

ATIHYPERTENSIVE & VASODILATIOR ACTIVITY IN ATIHYPERTENSIVE & VASODILATIOR ACTIVITY IN GANGLION BLOCKED ANG II SUPPORTED RATSGANGLION BLOCKED ANG II SUPPORTED RATS..

To demonstrate the direct vasodilator activity of To demonstrate the direct vasodilator activity of antihypertensive agent.antihypertensive agent.

This method is an anaesthetized ganglion blocked rat whose This method is an anaesthetized ganglion blocked rat whose B.P is maintained by an i.v. infusion of Ang II. B.P is maintained by an i.v. infusion of Ang II.

7-9 nos of Wister Rats (250-300g) are anaesthetized using a 7-9 nos of Wister Rats (250-300g) are anaesthetized using a combination of Urethrane & Chlorolase(60mg/kg).combination of Urethrane & Chlorolase(60mg/kg).

Chlorisondamine is administered to block the ganglion ( both Chlorisondamine is administered to block the ganglion ( both sympathetic and para sympathetic nerve activity i.p)sympathetic and para sympathetic nerve activity i.p)

The right faemoral artery is cannulated to record the B.P.The right faemoral artery is cannulated to record the B.P. Artificial respiration is supported during the experiment.Artificial respiration is supported during the experiment. Ang II is infused at a rate of 3.5µg/min in a volume Ang II is infused at a rate of 3.5µg/min in a volume

equivalent to 0.05ml/min using equivalent to 0.05ml/min using Harvard Harvard infusion pump. infusion pump.

The steady state pressure is established with in 15-20min.The steady state pressure is established with in 15-20min. Test drugs are injected i.v. at an interval of 3min(2ml/kg).Test drugs are injected i.v. at an interval of 3min(2ml/kg). Arterial pressure is recorded on a polygraph at 5,10, 15, 20 & Arterial pressure is recorded on a polygraph at 5,10, 15, 20 &

30 min.30 min. Pressor response for graded dose of phenylephrine can be Pressor response for graded dose of phenylephrine can be

obtained 15min before test administration.obtained 15min before test administration. Data is obtained fron 5-6 animals.Data is obtained fron 5-6 animals. A fall in B.P indicates the vasodilative action in turn A fall in B.P indicates the vasodilative action in turn

antihypertensive action of the test drug.antihypertensive action of the test drug.

IN VITRO MODELSIN VITRO MODELS

MONOCROTALINE INDUCED MONOCROTALINE INDUCED HYPERTENSIONHYPERTENSION

MONOCROTALINE, a pyrrolizidine alkaloids derived from MONOCROTALINE, a pyrrolizidine alkaloids derived from Crotaloria spectabillis.Crotaloria spectabillis.

Sprague- Dawley rats (200-225gm) of either sex are selected.Sprague- Dawley rats (200-225gm) of either sex are selected. Animals are fed with test drug for one week prior to S.C Animals are fed with test drug for one week prior to S.C

injection of MONOCROTALINE 100mg/kg.injection of MONOCROTALINE 100mg/kg. Animals are sacrificed 7 or 14 days later and their hearts and Animals are sacrificed 7 or 14 days later and their hearts and

lungs are excised.lungs are excised. Left ventricle and lung are weighed, along with main, extra Left ventricle and lung are weighed, along with main, extra

and intrapulmonary, artery are isolated.and intrapulmonary, artery are isolated. After 1hr arteries are made to contract with KCl.After 1hr arteries are made to contract with KCl. Contractions are recorded using lever transducerContractions are recorded using lever transducer..

Maximum force generated by an artery is plotted as a function of Maximum force generated by an artery is plotted as a function of applied force and recorded on a polygraph.applied force and recorded on a polygraph.

Contraction and relaxation of agonist responses of artery are Contraction and relaxation of agonist responses of artery are assessed. assessed.

Cumulative Conc. responce of KCl, Angiotension II, and NOR-Cumulative Conc. responce of KCl, Angiotension II, and NOR-Epinephrine are plotted.Epinephrine are plotted.

Both the responses are plotted as a function of negative log of Both the responses are plotted as a function of negative log of agonist Conc.agonist Conc.

To compare the differences in mean responses, t-test is applied. To compare the differences in mean responses, t-test is applied.

ACE INHIBITION IN ISOLATED GUINEA PIG ILEUM

ANGIOTENSIN CONVERTING ENZYME

1. Inactive Angiotensin I Active Angiotensin II

2. Active Peptide Bradykinin Inactive products

Activity of Angiotensin II ACE Inhibition

Activity of Bradykinin

The Guinea Pig Ileum responds powerfully for both Angiotensin II, and Bradykinin.

PROCEDURE PROCEDURE

Guinea Pig of either sex (300 – 500gm) is selected.Guinea Pig of either sex (300 – 500gm) is selected. Animals are sacrificed by stunning, and abdomen is opened.Animals are sacrificed by stunning, and abdomen is opened. A chord is tied around the starting of intestine.A chord is tied around the starting of intestine. The intestine is gradually removed from the bottom and the The intestine is gradually removed from the bottom and the

mesentry is cut away.mesentry is cut away. When reached the colon, the intestine is cut halfway by passing When reached the colon, the intestine is cut halfway by passing

Tyrode solution to clean the surface.Tyrode solution to clean the surface. The distal pieces (more sensitive) are fixed in the tissue clamp The distal pieces (more sensitive) are fixed in the tissue clamp

and brought into organ bath of 37and brought into organ bath of 37OOC (oxygenated)C (oxygenated) Angiotensin I is added (10ng/ml) after 30mts of equilibrium in Angiotensin I is added (10ng/ml) after 30mts of equilibrium in

bath solution and contraction is recorded.bath solution and contraction is recorded. 5min after the addition of ACE Inhibitor (test drug), the 5min after the addition of ACE Inhibitor (test drug), the

diminished contraction is recorded.diminished contraction is recorded. The opposite response can be observed while using Bradykinin.The opposite response can be observed while using Bradykinin.

ββ11 SYMPATHOLYTIC ACTIVITY IN SYMPATHOLYTIC ACTIVITY IN

GUENIA PIG ATRIAGUENIA PIG ATRIA

β agonist (Isoprenaline)β agonist (Isoprenaline) the frequency of the the frequency of the force of contractionforce of contraction of right atria of right atria

.. (+ve ionotropic) (+ve ionotropic)

potentiatespotentiates the contraction of electrically the contraction of electrically stimulated left atria. stimulated left atria.

β1 sympatholytic β1 sympatholytic DrugsDrugs isoprenaline induced ionotropic action. isoprenaline induced ionotropic action.

β1 receptor blockingβ1 receptor blocking activity can be evaluated by this technique. activity can be evaluated by this technique.

Procedure:Procedure: Pirbright- Guineapig of either sex (250-300gm) is used.Pirbright- Guineapig of either sex (250-300gm) is used. The animal is sacrificed by stunning and exsanguination.The animal is sacrificed by stunning and exsanguination. Heart is removed and right/ left atrium is mounted in 50ml organ Heart is removed and right/ left atrium is mounted in 50ml organ

bath containing Krebs – Henseleit buffer with aeration (95%Obath containing Krebs – Henseleit buffer with aeration (95%O22, , 5% CO5% CO22) at 37) at 37ooC.C.

Contractions are recorded using lever transducer.Contractions are recorded using lever transducer.Right atrium Right atrium

Isoprenaline is administered in the organ bath after 30mins to Isoprenaline is administered in the organ bath after 30mins to induce ionotropy.induce ionotropy.

Cumulative dose is maintained starting from 0.5µgm/ml and Cumulative dose is maintained starting from 0.5µgm/ml and consecutive doses at 3 min intervals.consecutive doses at 3 min intervals.

The bath is flushed for 3-5 times after the stable max plateau is The bath is flushed for 3-5 times after the stable max plateau is achieved. achieved.

The test drug is added into the organ bath.The test drug is added into the organ bath. 5min later, again isoprenaline is added in above Concentrations 5min later, again isoprenaline is added in above Concentrations

& observe the response.& observe the response.

Higher conc. of ISP is necessary to attain same response

(potentation and ionotropy).

The increase in the frequency and ionotropy is reduced at

same ISP Conc. added before.

β1 receptor blocking activity

OBSERVATIONOBSERVATION

Left atrium (Electricaly stimulated)Left atrium (Electricaly stimulated)

LA is stimulated by square wave stimulator(2 impulses at 15V, LA is stimulated by square wave stimulator(2 impulses at 15V, dur- 1min)dur- 1min)

After equilibrium, repeat the same as above using Isoprenaline at After equilibrium, repeat the same as above using Isoprenaline at concentrations of 0.05-0.1 5µgm/ml.concentrations of 0.05-0.1 5µgm/ml.

The bath is flushed for 3-5 times after the stable max. plateau is The bath is flushed for 3-5 times after the stable max. plateau is achieved.achieved.

The test drug is added into the organ bath, 3min later again The test drug is added into the organ bath, 3min later again isoprenaline is added in above same doses.isoprenaline is added in above same doses.

If it has β1 receptor blocking activity, the ISP induced activity is If it has β1 receptor blocking activity, the ISP induced activity is inhibited.inhibited.

IC50 values are determined from the individual dose response.IC50 values are determined from the individual dose response.