Screening of antihypertensive agents

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Screening of antihypertensive agents

  1. 1. <ul><li>ANTIHYPERTENSIVE & </li></ul><ul><li>VASODILATIVE </li></ul>SCREENING OF AGENTS S.K.LAL Ist M.Pharm Dept. PHARMACOLOGY JKKMMRF KPM
  2. 2. What is Hypertension <ul><li>High blood pressure: blood forced through the arteries at an increased rate. </li></ul><ul><li>It is measured as two numbers, e.g. 120/80 mmHg. Normal blood pressure is below this value. </li></ul><ul><ul><li>The first number is the systolic blood pressure. This is the maximum pressure in the arteries when heart contracts/beats. </li></ul></ul><ul><ul><li>The second number is the diastolic blood pressure. This is the minimum pressure in the arteries when heart is at rest between beats. </li></ul></ul><ul><li>Until it becomes extreme, there are no symptoms. Hence, it is called the “Silent Killer.” </li></ul><ul><li>Severe hypertension may cause headache, sleepiness, confusion or coma. </li></ul>
  3. 4. <ul><li>IN VIVO MODELS </li></ul><ul><li>ACE Inhibition in rat </li></ul><ul><li>Goldbalt Hypertension </li></ul><ul><li>Chronic renal hypertension in rats </li></ul><ul><li>Tail cuff method </li></ul><ul><li>Salt- sensitive Dahl Rats </li></ul><ul><li>Renin inhibition in Monkeys </li></ul><ul><li>Chronic renal hypertension in Dogs . </li></ul><ul><li>Antihypertensive and vasodilator action in rats. </li></ul>
  4. 5. <ul><li>IN VITRO & EX-vivo models </li></ul><ul><li>Monocrotaline Induced Pulmonary Hyperyension </li></ul><ul><li>ACE Inhibition in GUENIA PIG illeum </li></ul><ul><li>β1 Sympatholytic Activity in GUENIA PIG Atria </li></ul>
  5. 6. ACE INHIBITION IN RAS <ul><li>HYPERTENSION </li></ul>Inactive Angiotensin -I Active Angiotensin -II ACE
  6. 7. <ul><li>Male Sprague- Dawley rats (200-225gm) are selected.. </li></ul><ul><li>They are anaesthetised with 60mg/kg of phenobarbitone sodium i.v. </li></ul><ul><li>The intubated trachea is artificially respirated with 30strokes/min and a stroke volume of 6-8ml. </li></ul><ul><li>The right artery is cannulated for recording the pressure. </li></ul><ul><li>The jujular vein is cannulated for i.v. test injection. </li></ul><ul><li>The B.P is diminished by the administration of 5mg/kg of pentolinium i.p. </li></ul><ul><li>Atropine(40µg/kg) is also injected i.m to inhibit the mucous secretion. </li></ul><ul><li>Now 310ng/kg of Ang I is injected i.v. in 0.1ml saline. </li></ul><ul><li>The injection is repeated in 5min interval until an identical pressure is attained. </li></ul>
  7. 8. <ul><li>The test drug is administered at a dose of 10mg/kg intravenously or 25mg/kg intradeudenally. </li></ul><ul><li>Again Ang I is injected as the similar dose above. </li></ul><ul><li>The diminution of the pressure after the administration of potent ACE inhibitors is compared . </li></ul>
  8. 9. In vivo models GOLDBALT HYPERTENSION (2 Kidney 1 Clip Method) <ul><li>Ischemia of kidney Blood pressure </li></ul><ul><li>METHOD: Renin- Angiotensin Mechanism </li></ul><ul><li>Clamping of renal artery for 4hr and reopening </li></ul><ul><li>Accumulated renin is released </li></ul>ACUTE HYPERTENSION
  9. 10. <ul><li>Sprague – Dawley Rats (300gm) are anesthetized ĉ Hexobarbital sodium (100mg/kg) Intra peritonially. </li></ul><ul><li>Cannulate the trachea for respiration and the Jugular vein for test compound administration. </li></ul><ul><li>A transducer is connected to the carotid artery for recording the pressure. </li></ul><ul><li>A PVC coated clip is placed in the left hilum of the kidney by fixing with the back muscle for 3.5 - 4hr. </li></ul><ul><li>Pentolinium is administered for ganglionic blockache. </li></ul><ul><li>Relaese the clip and record the rise in B.P. </li></ul><ul><li>Administer the test drug througfh I.V. and moniter the pressure continuously. </li></ul><ul><li>Increase in B.P after releasing the clip and reduction after the drug administration is determined. </li></ul><ul><li>Compare using percentage values </li></ul>
  10. 11. CHRONIC RENAL HYPERTENSION (1 Kidney- I -Clip Method) <ul><li>Sprague – Dawley Rats (200 - 300gm) are anesthetized ĉ Phenobarbitone sodium (100mg/kg) Intra peritonially. </li></ul><ul><li>A flank parallel incision is made in the left lumbar area. </li></ul><ul><li>Renal artery is dissected, cleaned and ‘U’ shaped silver clip is slipped around near the aorta. </li></ul><ul><li>The internal gap b/w the clip is adjusted to 0.25 – 0.38 nm. </li></ul><ul><li>The right kidney is removed after tying off the renal pedicel. </li></ul><ul><li>After 4-5 weeks the B.P is measured and the animals are divided into groups of different doses. </li></ul><ul><li>Test drug is administered for 3 days </li></ul><ul><li>Pressure before and after drug administration(3min) are recorded. </li></ul><ul><li>Percent reduction in pressure is calculated and compare to the Std. </li></ul>
  11. 12. TAIL CUFF METHOD (Blood Pressure in Conscious rats) <ul><li>Method without any surgical procedure, Similar to sphygomanometry in humans (systolic pressure). </li></ul><ul><li>Widely used to evaluate the antihypertensive drugs in experimently induced animals. </li></ul><ul><li>Charles River male rats(300-350g) are anaesthetized using I.P injection of 0.8ml of 4% of chloral hydrate. </li></ul><ul><li>Both the kidneys are exposed, hypertension is induced by placing a silver clip on both renal arteries.(0.2mm dm & 4mm l) </li></ul><ul><li>After 5 to 6 weeks operated animals attain renal hypertension with systolic B.P of 170 to 200mmHg. </li></ul><ul><li>A tubular inflatable cuff is placed around the base of tail and a Pizo- electric pulse detector is positioned distal to the cuff. </li></ul>
  12. 13. <ul><li>The cuff is inflated to approximately 300mmHg. </li></ul><ul><li>As the pressure in the cuff is released slowly, the systolic pressure is detected and recorded in the poly graph. </li></ul><ul><li>Test substance is administered intra peritonially for alternative days in three times. </li></ul><ul><li>Decrease in the systolic pressure is determined by the following steps. </li></ul><ul><ul><li>Day 1: Predose & 2hr postdrug </li></ul></ul><ul><ul><li>Day 3: Predose & 2hr postdrug </li></ul></ul><ul><ul><li>Day5: Predose , 2hr post drug & 4hr postdrug </li></ul></ul>
  13. 14. SALT- SENSITIVE DAHL RATS <ul><li>These rats Develop severe fatal hypertension when fed high – salt </li></ul><ul><li>diets, whereas salt resistance rats cant. </li></ul><ul><li>Sprague – Dawley Rats (250 - 300gm) are used in this study. </li></ul><ul><li>Drinking water is replaced with 8% NaCl soln. high salt diet is prepared by mixing salt with regular diet. </li></ul><ul><li>The animals are fed with the above diet and saline. </li></ul><ul><li>The test group rats are administered the drug orally for 1 month. </li></ul><ul><li>Blood pressure changes are recorded. </li></ul><ul><li>After the experiment the animals (both cntrl & test) are sacrificed. </li></ul><ul><li>The hearts are isolated, the mass, weight of LV & RV is compared and measured. </li></ul><ul><li>Upon salt feeding animals B.P rises steeply (up to 36%). </li></ul><ul><li>The ability of the drug to reverse these changes is studied. </li></ul><ul><li>Cardiac failure occurs at 4 to 5 months of age in thse kind of rats. </li></ul>
  14. 15. ANGIOTENSIN - II ANTAGONISM <ul><li>Male Sprague- Dawley rats (200-225gm) are selected.. </li></ul><ul><li>They are anaesthetised with 60mg/kg of phenobarbitone sodium i.v. </li></ul><ul><li>One carotid artery is cannulated & connected with a Statham transducer and the B.P is recorded in a polygraph. </li></ul><ul><li>Both the jujular veins are cannulated to administer the test drug. </li></ul><ul><li>Pentolinium (10mg/kg) is injected to block the ganglionic activity. </li></ul><ul><li>Atleast 5 animals are used for the evaluation of test drug. </li></ul><ul><li>In 10 min interval doses of 0.5,1.0, and 2µg/kg of angiotensin II are injected to establish the dose response curves. </li></ul><ul><li>After 10 min a continuous infusion is started of the Ang II blocker in a dose of 10µg/kg/ 0.1ml/ml. </li></ul>
  15. 16. <ul><li>Again doses of 0.5,1.0, and 2µg/kg of angiotensin II are injected . </li></ul><ul><li>Intensity and duration of fall in B.P is recorded. </li></ul><ul><li>The results are compared with the known std. drug. </li></ul>
  16. 17. ATIHYPERTENSIVE & VASODILATIOR ACTIVITY IN GANGLION BLOCKED ANG II SUPPORTED RATS . <ul><li>To demonstrate the direct vasodilator activity of antihypertensive agent. </li></ul><ul><li>This method is an anaesthetized ganglion blocked rat whose B.P is maintained by an i.v. infusion of Ang II. </li></ul><ul><li>7-9 nos of Wister Rats (250-300g) are anaesthetized using a combination of Urethrane & Chlorolase(60mg/kg). </li></ul><ul><li>Chlorisondamine is administered to block the ganglion ( both sympathetic and para sympathetic nerve activity i.p) </li></ul><ul><li>The right faemoral artery is cannulated to record the B.P. </li></ul><ul><li>Artificial respiration is supported during the experiment. </li></ul><ul><li>Ang II is infused at a rate of 3.5µg/min in a volume equivalent to 0.05ml/min using Harvard infusion pump. </li></ul>
  17. 18. <ul><li>The steady state pressure is established with in 15-20min. </li></ul><ul><li>Test drugs are injected i.v. at an interval of 3min(2ml/kg). </li></ul><ul><li>Arterial pressure is recorded on a polygraph at 5,10, 15, 20 & 30 min. </li></ul><ul><li>Pressor response for graded dose of phenylephrine can be obtained 15min before test administration. </li></ul><ul><li>Data is obtained fron 5-6 animals. </li></ul><ul><li>A fall in B.P indicates the vasodilative action in turn antihypertensive action of the test drug. </li></ul>
  18. 19. IN VITRO MODELS MONOCROTALINE INDUCED HYPERTENSION <ul><li>MONOCROTALINE, a pyrrolizidine alkaloids derived from Crotaloria spectabillis. </li></ul><ul><li>Sprague- Dawley rats (200-225gm) of either sex are selected. </li></ul><ul><li>Animals are fed with test drug for one week prior to S.C injection of MONOCROTALINE 100mg/kg. </li></ul><ul><li>Animals are sacrificed 7 or 14 days later and their hearts and lungs are excised. </li></ul><ul><li>Left ventricle and lung are weighed, along with main, extra and intrapulmonary, artery are isolated. </li></ul><ul><li>After 1hr arteries are made to contract with KCl. </li></ul><ul><li>Contractions are recorded using lever transducer . </li></ul>
  19. 20. <ul><li>Maximum force generated by an artery is plotted as a function of applied force and recorded on a polygraph. </li></ul><ul><li>Contraction and relaxation of agonist responses of artery are assessed. </li></ul><ul><li>Cumulative Conc. responce of KCl, Angiotension II, and NOR-Epinephrine are plotted. </li></ul><ul><li>Both the responses are plotted as a function of negative log of agonist Conc. </li></ul><ul><li>To compare the differences in mean responses, t-test is applied. </li></ul>
  20. 21. ACE INHIBITION IN ISOLATED GUINEA PIG ILEUM ANGIOTENSIN CONVERTING ENZYME 1. Inactive Angiotensin I Active Angiotensin II 2. Active Peptide Bradykinin Inactive products A ctivity of Angiotensin II ACE Inhibition Activity of Bradykinin The Guinea Pig Ileum responds powerfully for both Angiotensin II, and Bradykinin.
  21. 22. <ul><li>PROCEDURE </li></ul><ul><li>Guinea Pig of either sex (300 – 500gm) is selected. </li></ul><ul><li>Animals are sacrificed by stunning, and abdomen is opened. </li></ul><ul><li>A chord is tied around the starting of intestine. </li></ul><ul><li>The intestine is gradually removed from the bottom and the mesentry is cut away. </li></ul><ul><li>When reached the colon, the intestine is cut halfway by passing Tyrode solution to clean the surface. </li></ul><ul><li>The distal pieces (more sensitive) are fixed in the tissue clamp and brought into organ bath of 37 O C (oxygenated) </li></ul><ul><li>Angiotensin I is added (10ng/ml) after 30mts of equilibrium in bath solution and contraction is recorded. </li></ul><ul><li>5min after the addition of ACE Inhibitor (test drug), the diminished contraction is recorded. </li></ul><ul><li>The opposite response can be observed while using Bradykinin. </li></ul>
  22. 23. β 1 SYMPATHOLYTIC ACTIVITY IN GUENIA PIG ATRIA <ul><li>β agonist (Isoprenaline) the frequency of the force of contraction of right atria </li></ul><ul><li>. (+ve ionotropic) </li></ul><ul><li>potentiates the contraction of electrically stimulated left atria. </li></ul><ul><li>β1 sympatholytic Drugs isoprenaline induced ionotropic action. </li></ul><ul><li>β1 receptor blocking activity can be evaluated by this technique. </li></ul>
  23. 24. <ul><li>Procedure: </li></ul><ul><li>Pirbright- Guineapig of either sex (250-300gm) is used. </li></ul><ul><li>The animal is sacrificed by stunning and exsanguination. </li></ul><ul><li>Heart is removed and right/ left atrium is mounted in 50ml organ bath containing Krebs – Henseleit buffer with aeration (95%O 2 , 5% CO 2 ) at 37 o C. </li></ul><ul><li>Contractions are recorded using lever transducer. </li></ul><ul><li>Right atrium </li></ul><ul><ul><li>Isoprenaline is administered in the organ bath after 30mins to induce ionotropy. </li></ul></ul><ul><ul><li>Cumulative dose is maintained starting from 0.5µgm/ml and consecutive doses at 3 min intervals. </li></ul></ul><ul><ul><li>The bath is flushed for 3-5 times after the stable max plateau is achieved. </li></ul></ul><ul><ul><li>The test drug is added into the organ bath. </li></ul></ul><ul><ul><li>5min later, again isoprenaline is added in above Concentrations & observe the response. </li></ul></ul>
  24. 25. OBSERVATION 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
  25. 26. Left atrium (Electricaly stimulated) <ul><li>LA is stimulated by square wave stimulator(2 impulses at 15V, dur- 1min) </li></ul><ul><li>After equilibrium, repeat the same as above using Isoprenaline at concentrations of 0.05-0.1 5µgm/ml. </li></ul><ul><li>The bath is flushed for 3-5 times after the stable max. plateau is achieved. </li></ul><ul><li>The test drug is added into the organ bath, 3min later again isoprenaline is added in above same doses. </li></ul><ul><li>If it has β1 receptor blocking activity, the ISP induced activity is inhibited. </li></ul><ul><li>IC50 values are determined from the individual dose response. </li></ul>

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