Screening methods of anti hypertensive agents

screening methods of antihypertensive drugs

1. 1 SCREENING METHODS OF ANTI HYPERTENSIVE AGENTS Dr.Swaroopa, 1 st year pg , Department of pharmacology

2. 2 CONTENTS INTRODUCTION REGULATION OF HYPERTENSION SCREENING MODELS CONCLUSION REFERENCES

3. 3 INTRODUCTION Hypertension is a complex multifactorial disease and one of the leading causes of mortality and morbidity ( 92 million disability-adjusted life years worldwide were attributable to high blood pressure) Hypertension doubles the risk of cardiovascular diseases, including coronary heart disease (CHD), congestive heart failure (CHF), ischemic and haemorrhagic stroke, renal failure, and peripheral arterial disease. The prevalence of hypertension increases with age, among individuals age ≥60, it is 65.4%. Harrison’s principles of Internal medicine, 20 th Edition, Hypertensive Vascular Disease, SK Gupta, Drug screening methods, Antihypertensive agents.

4. 4 Contd …… As the etiology of essential hypertension is not specific and is multifactorial, the use of experimental animal models may provide valuable information regarding many aspects of the disease, Which include etiology , pathophysiology, complications and treatment.

5. HYPERTENSION Hypertension is defined as a sustained increase in blood pressure of 140/90 mmHg or higher, a criterion that characterizes a group of patients whose risk of hypertension-related cardiovascular disease is high enough to merit medical attention. Isolated systolic hypertension : defined as systolic blood pressure greater than 140mmHg with diastolic blood pressure less than 90 mmHg, is largely confined to people older than 60 years.

6. 6 Classification Blood Systolic BP pressure( mmHg) Diastolic BP Normal <120 <80 Prehypertension 120-139 80-89 Hypertension, stage1 140-159 90-99 Hypertension, stage 2 >160 >100 Hypertensive crisis >180 >110 Goodman and Gilman, 13 th edition, The Pharmacological Basis of Therapeutics, Section III, Treatment of Hypertension, pg 507 – 526 AMERICAN HEART ASSOCIATION CRITERIA FOR HYPERTENSION IN ADULTS

7. 7 Bertram G Katzung , Antihypertensive agents. Basics & Clinical Pharmacology, McGraw Hill, New Delhi, 14 th edition;2018, pg-173 – 193

8. 8 Screening methods of Antihypertensive agents: 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 like excessive salt intake, Hyperactivity of Renin-Angiotensin-Aldosterone-System(RAAS) and genetic factors.

9. 9 ANIMALS USED Earlier, most studies on experimental hypertension were carried out on Dogs. Currently, Rat is the preferred species. Spontaneous Hypertensive Rat(SHR), the genetic strain of hypertensive rat, is the animal of choice.

10. 10 SCREENING MODELS IN VIVO MODELS:- Acute renal hypertension in Rats Chronic Renal Hypertension in Rats Chronic Renal Hypertension in Dogs Neurogenic Hypertension in Dogs DOCA-salt Induced Hypertension in Rats Fructose Induced Hypertension in Rats Genetic Hypertension in Rats Hypertension Induced by Chronic NO-synthase Inhibition Portal Hypertension in Rats Vogel & Vogel, drug discovery and evaluation ,chapter A2, methods to induce experimental Hypertension, pg. no -239 -250

11. 11 Pulmonary Hypertension Induced by Monocrotaline Endothelin Receptor Antagonism in Porcine Isolated Hearts IN VITRO MODELS:

12. 12 Methods of inducing renovascular hypertension Goldblatt Method :- Goldblatt et.al (1934) Three variants of hypertension are produced by this method. Two kidney one clip method(2K1C) One kidney one clip method(1K1C) Two kidney two clip method(2K2C)

13. 13 Acute Renal Hypertension in Rats/2K1C Principle used : Ischemia of the kidneys causes elevation of blood pressure by activation of renin-angiotensin system. Clamping of renal artery for 4h Accumulated renin released into circulation after releasing the vessel Protease renin catalyses the first & rate limiting step in the formation of angiotensin2 Acute hypertension Vogel & Vogel, drug discovery and evaluation ,chapter A2, methods to induce experimental Hypertension , pg no -239 .

14. 14 Procedure: Male Sprague-Dawley rats weighing 300 g are anesthetized by intraperitoneal injection of 100 mg/kg hexobarbital sodium. A PVC-coated clip is placed onto the left hilum of the kidney and fixed to the back muscles. The renal artery is occluded for 3.5–4 h. The animals are anesthetized by intraperitoneal injection of 30–40 mg/kg pentobarbital sodium. The trachea is cannulated to facilitate spontaneous respiration . Voge l& vogel , drug discovery and evaluation ,chapter A2, methods to induce experimental Hypertension, pg no -239

15. 15 The cannula in the carotid artery is connected to a pressure transducer to measure systolic and diastolic blood pressures. For administration of the test compound, a jugular vein is cannulated . After obtaining stable reduced blood pressure values , ganglionic blockade is performed with pentolinium (10mg/kg i.v. ) and the renal arterial clip is removed. This leads to a rise in blood pressure as a consequence of elevated plasma renin level. Within 15 min a stable hypertension is achieved (control = 100%). Voge l& vogel , drug discovery and evaluation ,chapter A2, methods to induce experimental Hypertension, pg no -239

16. 16 The test substance is then administered by intravenous injection at doses o f 10 and 100 µg/kg. Blood pressure is monitored continuously until an increased level to the starting level is obtained. Ten–twelve animals are used per compound . Vogel & vogel , drug discovery and evaluation ,chapter A2, methods to induce experimental Hypertension, pg no -239

17. 17 Increase in blood pressure is determined after reopening of the renal artery. R eduction in blood pressure is determined after administration of the test drug. Percent inhibition of hypertensive blood pressure values under drug treatment are calculated as compared to pretreatment hypertension values. Duration of the effect is determined [min ]. Statistical significance is assessed by the paired t-test. EVALUATION : Vogel & Vogel, drug discovery and evaluation ,chapter A2, methods to induce experimental Hypertension, pg. no -239 -250

18. 18 MODIFICATIONS OF THE METHOD: cardiogenic hypertensive chemo reflex Sharp and transient increase in systemic arterial blood pressure associated with reflex bradycardia can be elicited by injection of 5-hydroxytryptamine, cyanide, nicotine or lobeline into the coronary artery blood stream of dogs (Berthold et al. 1989) 5-HT proved to be the most powerful agent for its initiation Vogel & Vogel, drug discovery and evaluation ,chapter A2, methods to induce experimental Hypertension, pg. no -239 -250

19. 19 Chronic Renal Hypertension in Rats/1K1C method: This is One of the most effective modifications in rats. Procedure: Male Sprague-Dawley rats weighing 200–250 g are anaesthetized with 50 mg/kg i.p . pentobarbital. The fur on the back is shaved and the skin disinfected. In the left lumbar area a flank incision is made parallel to the long axis of the rat . The renal pedicel is exposed with the kidney retracted to the abdomen. The renal artery is dissected clean and a U-shaped silver clip is slipped around it near the aorta. Vogel & Vogel, drug discovery and evaluation ,chapter A2, methods to induce experimental Hypertension, pg. no -239 -250

20. 20 The right kidney is removed through a flank incision after tying off the renal pedicle. The skin incisions are closed by wound clips . 4–5 weeks after clipping blood pressure is measured and rats with values higher than 150 mm Hg are selected for the experiments. Blood pressure readings are taken on each of 3 days prior to drug treatment . Drugs are administered orally in volumes of 10 ml/kg. Compounds are administered for 3 days and predrug and postdrug (2h) blood pressure readings are taken. The rats are divided into 4 animals per dose and each animal is used as his own control Vogel & Vogel, drug discovery and evaluation ,chapter A2, methods to induce experimental Hypertension, pg. no -239 -250

21. 21 Activity is determined by comparing treatment blood pressure values with the control blood pressure value (Day 1, predrug blood pressure ). Comparisons are made using the paired t-test for evaluation of statistical significance. EVALUATION Vogel & Vogel, drug discovery and evaluation ,chapter A2, methods to induce experimental Hypertension, pg. no -239 -250

22. MODIFICATIONS OF THE METHOD Duan et al. (1996) induced renal hypertension in male Hartley guinea pigs by a two-step procedure consisting of ligation of the left caudal renal artery and right nephrectomy. Arterial blood pressure and heart rate were monitored in conscious animals. ACE-inhibitors reduced blood pressure in sham-operated and in renal hypertensive guinea pigs , whereas renin inhibitors were effective only in renal hypertensive animals Vogel & Vogel, drug discovery and evaluation ,chapter A2, methods to induce experimental Hypertension, pg. no -239 -250

23. 23 Chronic Renal Hypertension in Dogs first described by Goldblatt et al. (1934) in dogs. The method has been modified as the “wrapping” technique (Abram and Sobin 1947). PROCEDURE Dogs weighing 8–12 kg are anesthetized with i.v. injection of 15 mg/kg thiopental . Anesthesia is maintained with a halothane-oxygen mixture. Under aseptic conditions, a midline abdominal incision is made One kidney is exposed and wrapped in cellophane and then replaced . The contralateral kidney is exposed. Vogel & Vogel, drug discovery and evaluation ,chapter A2, methods to induce experimental Hypertension, pg. no -239 -250

24. 24 The artery, vein and ureter are ligated and the kidney is removed. The abdomen is closed by sutures and clips On the day of surgery and for 3 days following, the dogs are given antibiotics. Body temperature is measured twice daily for 4 days following surgery. Six weeks following surgery, blood pressure is measured using a tail-cuff method. For recording, the tail cuff is attached to a polygraph. Only animals with a systolic blood pressure higher than 150 mm Hg are considered to be hypertensive and can participate in studies evaluating potential antihypertensive compounds. Vogel & Vogel, drug discovery and evaluation ,chapter A2, methods to induce experimental Hypertension, pg. no -239 -250

25. 25 Blood pressure is recorded either by the indirect tail-cuff method or by direct measurement via an implanted arterial cannula. On day 1 readings are made every 2 h, just before, and 2 and 4 h after oral treatment with the potential antihypertensive compound. Drug administration is repeated for 5 days. On days 3 and 5 blood pressure readings are taken before and 2 and 4 h after treatment. At least 3 dogs are used per dose and compound. Vogel & Vogel, drug discovery and evaluation ,chapter A2, methods to induce experimental Hypertension, pg. no -239 -250

26. 26 EVALUATION The starting value is the average of the 2 readings before application of the drug . Each of the following readings is subtracted from this value and recorded as fall of blood pressure at the various recording times. Vogel & Vogel, drug discovery and evaluation ,chapter A2, methods to induce experimental Hypertension, pg. no -239 -250

27. 27 MODIFICATIONS OF THE METHOD Technique according to Grollman (1944). The kidney is exposed through a lumbar incision , The renal capsule is removed by gentle traction, A figure-8ligature is applied being tight enough to deform the kidney but not tight enough to cut the tissue. 2. Technique according to Abrams and Sobin 1947 Renal hypertension may be induced in the rat by encapsulating both kidneys with latex rubber capsules Vogel & Vogel, drug discovery and evaluation ,chapter A2, methods to induce experimental Hypertension, pg. no -239 -250

28. 28 Moulds are formed from plastic using a rat kidney as a model . The capsules are prepared by dipping the moulds in liquid latex allowing them to dry in the air. Three applications of latex are applied before the capsules are toughened by placing them under warm running tap water. The kidney is exposed by lumbar incision, the renal capsule gently removed and the capsule applied. Vogel & Vogel, drug discovery and evaluation ,chapter A2, methods to induce experimental Hypertension, pg. no -239 -250

29. 29 Vasodilator and depressor reflexes, originating in the baroreceptor areas of the carotid sinus and aortic arch, play an important part in the regulation of blood pressure. Neurogenic Hypertension in Dogs Stimulation of the afferent buffer fibres exerts an inhibitory influence on the vasomotor center, and their sectioning leads to a persistent rise in blood pressure. Acute neurogenic hypertension can be induced in dogs Principle : Vogel & Vogel, drug discovery and evaluation ,chapter A2, methods to induce experimental Hypertension, pg. no -239 -250

30. 30 PROCEDURE Adult dogs of either sex weighing 10–15 kg are anesthetized using 15 mg/kg sodium thiopental, 200 mg/kg sodium barbital and 60 mg/kg sodium pentobarbital i.v. A femoral vein and artery are cannulated using polyethylene tubing to administer compounds i.v and record arterial pressure and heart rate, respectively Left ventricular pressure is recorded via the left common carotid artery (post- deafferentiation ) using a Millar micro tip pressure transducer. Vogel & Vogel, drug discovery and evaluation ,chapter A2, methods to induce experimental Hypertension, pg. no -239 -250

31. 31 Pmax is recorded by speeding up the chart paper. Cardiac output is determined by introducing a Swan-Ganz catheter into the right heart and pulmonary artery via a jugular vein. 5 ml of cold 5% dextrose is injected into the right atrium and an Edwards Cardiac output computer is used to calculate the cardiac output from the temperature change in the pulmonary artery . All recordings are made with a polygraph. Vogel & Vogel, drug discovery and evaluation ,chapter A2, methods to induce experimental Hypertension, pg. no -239 -250

32. 32 Both of the carotid arteries are cleared up to the bifurcation of the internal and external carotid arteries. The carotid sinus nerves are isolated, ligated and sectioned and a bilateral vagotomy is performed to produce neurogenic hypertension (mean arterial pressure more than 150 mm Hg). The dog is allowed to equilibrate for approximately 30 min and a bolus of the test compound is administered by intravenous injection. Heart rate, arterial pressure, left ventricular pressure, Pmax and are monitored for 90 min. minimum of 3 dogs are used for each compound. Vogel & Vogel, drug discovery and evaluation ,chapter A2, methods to induce experimental Hypertension, pg. no -239 -250

33. 33 EVALUATION Changes of the cardiovascular parameters are expressed as percentage of the values before administration of the drug. MODIFICATIONS OF THE METHOD Neurogenic hypertension through baroreceptor denervation has also been described in rabbits (Angell James 1984) and in rats (Krieger 1984). Vogel & Vogel, drug discovery and evaluation ,chapter A2, methods to induce experimental Hypertension, pg. no -239 -250

34. 34 CRITICAL ASSESSMENT OF THE METHOD The neurogenic hypertension is useful for acute experiments. I t is less useful for chronic experiments since the elevated blood pressure caused by buffer nerve section is more labile than that caused by renal ischemia. Vogel & Vogel, drug discovery and evaluation ,chapter A2, methods to induce experimental Hypertension, pg. no -239 -250

35. 35 DOCA-salt Induced Hypertension in Rats ( desoxycorticosteroneacetate ) Mineralocorticoid-induced hypertension sodium retaining properties of the steroid increases in plasma and extracellular volume The hypertensive effect is increased by salt loading and unilateral nephrectomy in rats. Vogel & Vogel, drug discovery and evaluation ,chapter A2, methods to induce experimental Hypertension, pg. no -239 -250

36. 36 Male Sprague Dawley rats weighing 250–300 g are anesthetized with ether. PROCEDURE Through a flank incision the left kidney is removed. The rats are injected twice weekly with 20 mg/kg s.c. desoxycorticosteroneacetate in olive oil for 4 weeks. Drinking water is replaced with a 1% NaCl solution. B Blood pressure starts to rise after one week and reaches systolic values between 160 and 180 mm Hg after 4 weeks Vogel & Vogel, drug discovery and evaluation ,chapter A2, methods to induce experimental Hypertension, pg. no -239 -250

37. 37 MODIFICATIONS OF THE METHOD The regimen to induce DOCA-salt hypertension modified by many authors (Stanton 1971). DOCA-salt hypertension can also be achieved without nephrectomy ( Bockman et al. 1992 ). Using kininogen-deficient Brown Norway Katholiek (BN- Ka ) rats, Majima et al. (1991, 1993) showed suppression of rat desoxycorticosterone -salt hypertension by the kallikrein-kinin system. Li et al. (1996) examined small-artery structure on a wire myograph and quantified endothelin-1 messenger RNA by Northern blot analysis in DOCA-salt hypertensive rats after administration of an ACE-inhibitor , a calcium channel antagonist and a nitric oxide synthase inhibitor. Vogel & Vogel, drug discovery and evaluation ,chapter A2, methods to induce experimental Hypertension, pg. no -239 -250

38. 38 Fructose Induced Hypertension in Rats The increased intake of either sucrose or glucose was shown to enhance the development of either spontaneous hypertension or salt hypertension in rats (Hall and Hall 1966). Hwang et al. (1987) first reported that hypertension could be induced in normal rats by feeding a high-fructose diet. F Fructose feeding was also found to cause insulin resistance, hyperinsulinemia, and hypertriglyceridemia in normal rats ( Zavaroni et al. 1980; Tobey et al. 1982). Tobey et al. 1982). Dai and McNeill (1995) studied the concentration- and duration-dependence of fructose induced hypertension in rats. Vogel & Vogel, drug discovery and evaluation ,chapter A2, methods to induce experimental Hypertension, pg. no -239 -250

39. 39 PROCEDURE Groups of 8 male Wistar rats weighing 210–250 g are used. The are housed two per cage on a 12-h light 12- h dark cycle and are allowed free access to standard laboratory diet (Purina rat chow) and drinking fluid Drinking fluid consists either of tap water or 10%- fructose solution Vogel & Vogel, drug discovery and evaluation ,chapter A2, methods to induce experimental Hypertension, pg. no -239 -250

40. 40 Blood samples are collected before and every second week during treatment for determination of plasma glucose, insulin, and triglycerides. Body weight, food intake and fluid intake of each rat are measured every week during treatment. Using the tail-cuff method, systolic blood pressure and pulse rate is measured before and every week during treatment. Vogel & Vogel, drug discovery and evaluation ,chapter A2, methods to induce experimental Hypertension, pg. no -239 -250

41. 41 EVALUATION The duration of treatment 6 weeks. Statistical analysis is performed using a one-way or two-way analysis of variance, followed by the Newman- Keuls test. Vogel & Vogel, drug discovery and evaluation ,chapter A2, methods to induce experimental Hypertension, pg. no -239 -250

42. 42 MODIFICATIONS OF THE METHOD Reaven et al. (1988, 1989) found an attenuation of fructose-induced hypertension by exercise training and an inhibition by somatostatin treatment. Brands et al. (1991, 1992) found an increase of arterial pressure during chronic hyperinsulinemia in conscious rats . Hall et al. (1995) reported the effects of 6 weeks of a high-fat diet on cardiovascular, renal, and endocrine functions in chronically instrumented conscious dogs. Body weight increased by approximately 16.9 kg, whereas MAP, cardiac output, and heart rate increased by 28%, 77%, and 68%, respectively Vogel & Vogel, drug discovery and evaluation ,chapter A2, methods to induce experimental Hypertension, pg. no -239 -250

43. 43 Genetic Hypertension in Rats Inherited hypertension in rats has been described by Smik and Hall 1958; Phelan 1968 as genetically hypertensive (GH) rats (Simpson and Phelan 1984). Okamoto et al. (1963, 1966) reported the development of a strain of spontaneously hypertensive rats from mating one Wistar male rat with spontaneously occurring high blood pressure with a female with slightly elevated blood pressure. Vogel & Vogel, drug discovery and evaluation ,chapter A2, methods to induce experimental Hypertension, pg. no -239 -250

44. 44 Hypertension in these rats is clearly hereditary and genetically determined, thus comparable to primary hypertension in humans . Cardiac hypertrophy (Sen et al. 1974) and cellular ionic transport abnormalities have been observed ( Yamori 1984). By inbreeding over several generations a high incidence of hypertension with blood pressure values of 200 mm Hg or more was achieved. These strains were called “Spontaneously hypertensive rats ( Akamoto -Aoki)” = SHR or “Wistar–Kyoto rats” =WKY Vogel & Vogel, drug discovery and evaluation ,chapter A2, methods to induce experimental Hypertension, pg. no -239 -250

45. 45 Inbred strains being salt-hypertension-sensitive and salt-hypertension-resistant (RD) have been developed by Dahl et al. Another hypertensive strain derived from Wistar rats was produced by brother-sister mating in the group of Bianchi et al. (1974, 1986) at the University of Milan called “Milan hypertensive strain” = MHS. These rats show a cell membrane defect resulting in abnormal kidney function. Salvati et al. (1990) studied the diuretic effect of bumetanide in isolated perfused kidneys of Milan hypertensive rats Vogel & Vogel, drug discovery and evaluation ,chapter A2, methods to induce experimental Hypertension, pg. no -239 -250

46. 46 Several substrains of spontaneous hypertensive rats were separated by the group of Okamoto et al. (1974) including the stroke-prone strain SHR = SHRSP. These rats have an increased sympathetic tone and show a high incidence of hemorrhagic lesions of the brain with motor disturbances followed by death Vogel & Vogel, drug discovery and evaluation ,chapter A2, methods to induce experimental Hypertension, pg. no -239 -250

47. 47 Bohlender et al. (1996) reconstructed the human renin-angiotensin system in transgenic rats overexpressing the human angiotensin gene TGR( hOGEN ) 1623 by chronically injecting human recombinant renin intravenously using Alzet pumps. Zolk et al. (1998) described the effects of quinapril, losartan and hydralazine on cardiac hypertrophy and β- adrenergic neuroeffector mechanisms in transgenic TGR(mREN2)27 rats. Vogel & Vogel, drug discovery and evaluation ,chapter A2, methods to induce experimental Hypertension, pg. no -239 -250

48. 48 CRITICAL ASSESSMENT OF THE METHOD On the basis of available data no preference can be given to a particular strain Transgenic rats with well defined genomes are gaining more importance. Vogel & Vogel, drug discovery and evaluation ,chapter A2, methods to induce experimental Hypertension, pg. no -239 -250

49. 49 MODIFICATIONS OF THE METHOD Pijl et al. (1994) described streptozotocin -induced diabetes mellitus in spontaneously hypertensive rats as a pathophysiological model for the combined effects of hypertension and diabetes . Rosenthal et al. (1997) used rats of the CohenRosenthal diabetic hypertensive strain to examine the effects of an ACE-inhibitor, an ATII antagonist and a calcium antagonist on systolic pressure and spontaneous blood glucose levels . Holycross et al. (1997) used hypertensive SHHF/ Mcc-facp rats to study plasma renin activity during development of heart failure Vogel & Vogel, drug discovery and evaluation ,chapter A2, methods to induce experimental Hypertension, pg. no -239 -250

50. 50 Linz et al. (1997) compared the outcome of lifelong treatment with the ACE inhibitor Ramipril in young prehypertensive stroke-prone spontaneously hypertensive rats and age-matched normotensive Wistar–Kyoto rats . Lifelong ACE inhibition doubled the lifespan in hypertensive rats matching that of normotensive Ohkubo et al. (1990) generated transgenic mice with elevated blood pressure by introduction of the rat renin and angiotensinogen genes. Vogel & Vogel, drug discovery and evaluation ,chapter A2, methods to induce experimental Hypertension, pg. no -239 -250

51. 51 Hypertension Induced by Chronic NO-Synthase Inhibition Chronic blockade of NO synthesis in the rat produces systemic hypertension and glomerular damage ( Baylis et al. 1992). This was recommended by Ribeiro et al. (1992) as a model of hypertension. The detrimental sequels of chronic NO synthase inhibition in rats can be inhibited by treatment with ACE inhibitors. Vogel & Vogel, drug discovery and evaluation ,chapter A2, methods to induce experimental Hypertension, pg. no -239 -250

52. 52 PROCEDURE Male Wistar rats at an age of 7–8 weeks weighing 210± 10 g were placed at random in metabolic cages, divided in four to six groups of six to eight rats each. Group 1 (control) had free access to tap water and food. Groups 2–4 were treated with 0.02% L-NAME water solution for 6 weeks in a daily dose of 25 mg/kg. Vogel & Vogel, drug discovery and evaluation ,chapter A2, methods to induce experimental Hypertension, pg. no -239 -250

53. 53 Groups 3 and 4 received the angiotensin receptor antagonists fonsartan (10 mg/kg) or lorsatan (30 mg/kg) for 6 weeks daily per stomach tube. Groups 5 and 6 received fonsartan and lorasatan alone. At the end of the study, 24-h urine samples were collected and retrobulbar blood samples were taken in short inhalation anesthesia. Vogel & Vogel, drug discovery and evaluation ,chapter A2, methods to induce experimental Hypertension, pg. no -239 -250

54. 54 For clearance evaluation rats were anesthetized with 50 mg/kg thiopentone i.p . In order to determine glomerular filtration rate and renal plasma flow, clearances of inulin and para- aminohippurate were performed. EVALUATION Results are presented as arithmetical means ± SEM . A one-way ANOVA was calculated with SYSTAT for Windows (SYSTAT, Evanston, Ill., USA) followed by multiple pairwise comparisons according to Tukey. Vogel & Vogel, drug discovery and evaluation ,chapter A2, methods to induce experimental Hypertension, pg. no -239 -250

55. 55 MODIFICATIONS OF THE METHOD Arnal et al. (1993) measured cardiac weight of rats in hypertension induced by NO synthase blockade. Linz et al. (1999) reviewed the interactions between ACE, kinins and NO. Sampaio et al. (2002) reported that hypertension plus diabetes mimics the cardiomyopathy induced by NO inhibition in rats . Rossi et al. (2003) found that chronic inhibition of NO synthase induces hypertension and cardiomyocyte mitochondrial and myocardial remodeling in the absence of hypertrophy Vogel & Vogel, drug discovery and evaluation ,chapter A2, methods to induce experimental Hypertension, pg. no -239 -250

56. 56 Portal Hypertension in Rats Portal hypertension is associated with hyperdynamic splanchnic circulation and reduced vascular resistance ( Vorobioff et al. 1983). Tanoue et al. (1991) developed a method for inducing portal hypertension and esophageal varices in rats – partial ligation of the portal vein after devascularization of the circumference of the left renal vein and complete ligation of the portal vein on the fifth day thereafter. Tsugawa et al. ( 2000) used this model to study the role of nitric oxide and endothelin-1 in rat portal hypertension. Vogel & Vogel, drug discovery and evaluation ,chapter A2, methods to induce experimental Hypertension, pg. no -239 -250

57. 57 Male Sprague-Dawley rats were anesthetized with 50 mg/kg Nembutal intraperitoneally . PROCEDURE The portal vein was isolated and stenosis created by a single ligature of 3–0 silk placed around the portal vein and a 20-gauge blunt-tipped needle after devascularization of the left renal vein. This devascularization is indispensable in preventing the development of excess collateral vessels, which inhibit the formation of esophageal varices and the portal hypertensive state. 3–0 silk was also placed at the area of partial ligation (loose ligation), and both ends were then drawn out through the abdominal wall. Vogel & Vogel, drug discovery and evaluation ,chapter A2, methods to induce experimental Hypertension, pg. no -239 -250

58. 58 Five days after the operation, the ends of the silk that had been placed in the flank were simultaneously pulled to induce complete portal vein ligation. Two weeks later, this portal hypertension model was completed. Portal venous pressure, blood flow volume in the intra-abdominal viscera, plasma NO and plasma endothelin-1 were measure EVALUATION Results were expressed as mean ± standard deviation. The Student’s t-test was used to determine significance between portal hypertension rats and sham-operated controls. Vogel & Vogel, drug discovery and evaluation ,chapter A2, methods to induce experimental Hypertension, pg. no -239 -250

59. 59 MODIFICATIONS OF THE METHOD Portal hypertension by portal vein ligation without devascularization of the left renal vein was used by Lee et al. (1985), Braillon et al. (1986), Oren et al. (1995 ), Fernandez et al. (1996), Moreno et al. (1996), Dieguez et al. (2002) used a surgical technique based on the development of a triple stenosing ligation to worsen the complications inherent to the prehepatic chronic portal hypertension. Jaffe et al. (1994) and Li et al. (1998) used injection of different sized microspheres into the portal vein of male Wistar rats to induce portal hypertension. Vogel & Vogel, drug discovery and evaluation ,chapter A2, methods to induce experimental Hypertension, pg. no -239 -250

60. 60 METHODS TO MEASURE BP IN RATS Direct method: It is an invasive method . Femoral/carotid cannula is inserted into an anaesthetized animal. On day of screening, cannula is connected to a pressure transducer then to the pre-amplifier. BP is recorded on the polygraph.

61. 61 Indirect /Non invasive method : Tail cuff method: It is a common and convenient method. Tail cuff is inflated and deflated Pulsations disappear when cuff is inflated. Pulsations start appearing when pressure in the cuff equals systolic pressure while deflating. The cuff is attached to a tail cuff sphygmomanometer or pressure transducer and BP is recorded on a chart.

62. 62 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 D iazoxide are effective in Renal hypertensive rats. Calcium channel blockers, ACE Inhibitors and AT-1 antagonists decrease BP in nephrectomised SHR. Diuretics, are active in mineralocorticoid or salt induced hypertension. Sympathomimetic Drugs decrease BP in both endocrine and neurogenic hypertension.

63. 63

64. 64 CONCLUSION The most important point from direct comparison of these animal models is that, despite the well-known heterogeneity of hypertension, the outcome of hypertension can be similar in some respects. Not all classes of antihypertensives are equally effective in all rat models of hypertension. These models of hypertension provide ample opportunity not only to investigate the mechanisms involved in the pathogenesis of hypertension, but also to learn about the critical balance between stress and its overcoming which eventually determines prognosis.

65. 65 REFERENCES Vogel & Vogel , 3 rd edition, Drug discovery and evaluation ,chapter A2, methods to induce experimental Hypertension , pg. no -239 - 250. SK. Gupta, Drug screening methods, chapter16, Antihypertensive agents, pg.no 266-276. www.slideshare.net

66. 66 Thank you