Animal Models for anti-hypertensive Drugs

Toxicological Studies on Anti-hypertensive drugs. Various Animal Models.

1. Animal Models for Antihypertensive Agents Presented by: Megh Pravin Vithalkar Roll no.07 M.Pharm Semester-1 (2020) Department of Pharmacology. Goa college of Pharmacy, Panaji-Goa

2. INTRODUCTION Hypertension, commonly referred to as “high blood pressure” , is a medical condition in which the blood pressure is chronically elevated. Hypertension and related cardiovascular diseases are the leading causes of death in many countries. The aetiology of human essential hypertension is largely unknown. It is highly likely that hypertension is a complex and multifactorial disease resulting from the interaction of multiple genetic and environmental factors. Persistent hypertension is one of the risk factors for strokes, heart attacks, heart failure and arterial aneurysm, and is a leading cause of chronic renal failure . 2

3. Hypertension can be classified as either essential or secondary . Essential hypertension indicates that no specific medical cause can be found to explain a patient’s condition. Secondary hypertension indicates that the high blood pressure is a result of (i.e. secondary to) another condition, such as kidney disease or certain tumours (especially of the adrenal gland ). T he JNC 7 (the Seventh Report of the Joint National Committee on Prevention, Detection, Evaluation, and Treatment of High Blood Pressure) has defined blood pressure 120/80 mmHg to 139/89 mmHg as “ prehypertension”. Prehypertension is not a disease category; rather, it is a designation chosen to identify individuals at high risk of developing hypertension. 3

4. Hence, New animal models of hypertension are being developed as new insights in to the pathogenesis of hypertension are revealed. 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 such as excessive salt intake , hyperactivity of rennin angiotensin-aldosterone system (RAAS) and genetic factors . A number of animal models of hypertension have been developed each having unique advantages as well as disadvantages. 4

5. IN-VIVO RAT MODELS 5

6. Rat Models of Hypertension Two-Kidney One Clip (Gold blatt hypertension, 2K1C) Ischemia of the kidneys causes elevation of blood pressure by activation of renin-angiotensin system. In rats clamping the renal artery for 4 h can activate peripheral RAAS and sympathetic nervous system and induce renal hypertension . After re-opening of the vessel, accumulated renin is released into circulation leading to acute hypertension. Renin is secreted by kidneys when sympathetic activity is increased. Renin converts angiotensinogen to angiotensin-l. Angiotensin-I is converted to angiotensin-II by angiotensin converting enzyme (ACE). Angiotensin-II is a potent vasoconstrictor and increases BP. Angiotensin-II also causes release of aldosterone leading to salt and water retention resulting in increased blood volume and hypertension. 6

7. PROCEDURE: Sprague Dawley rats (300 g) are anesthetized with hexobarbital sodium (100 mg/kg, intraperitoneally). In the left lumbar area a flank incision is made parallel to the long axis of the rat. The kidneys are identified. A PVC coated clip is placed into the left hilum of the kidney and fixed to the back muscles. The renal artery is occluded for 3.5-4 h . Ganglionic blockade is performed with pentolinium and after obtaining stable reduced blood pressure values, the ‘renal arterial clip’ is removed. Subsequently the animals are anaesthetized with pentobarbitone sodium (30-40 mg/kg, intraperitoneally). The trachea is cannulated to facilitate spontaneous respiration. 7

8. Through a pressure transducer connected to carotid artery, blood pressure is measured. Jugular vein is cannulated for administration of test compound . As a consequence of elevated plasma renin level, blood pressure rises. Test compound is administered by intravenous route. Blood pressure is monitored continuously. Increase in blood pressure after re-opening of renal artery and reduction of blood pressure after administration of test compound is determined. Percent reduction of blood pressure values under drug treatment is calculated as compared to pre-treatment values . The renal artery is constricted on only one side with the other artery (or kidney) left untouched. This results in increased plasma rennin activity. However , there is no salt and water retention because of the other normal kidney being intact. Thus, the resultant hypertension at this stage is renin-angiotensin dependent . 8

9. Chronic Renal Hypertension in Rats ( 1-kidney-1-clip method) As discussed earlier, ischemia of the kidneys induces hypertension. Various modifications of the technique have been described for several animal species. The 1-kidney-1-clip method is one of the most effective modifications in rats in which one kidney is removed . Constriction of renal artery is done on one side and the contralateral kidney is removed. There is an increase in BP within a few hours. Since there is no other kidney, there is no pressure diuresis and natriuresis , so there is rapid salt and water retention . Plasma rennin activity is usually normal. Hypertension soon becomes volume dependent . 9

10. Sprague Dawley rats (200 to 250 g) are anesthetized with pentobarbitone sodium (50 mg/ kg, intraperitoneally). In the left lumbar area a flank incision is made parallel to the long axis of the rat, exposing the kidneys. The kidney is retracted and renal artery identified. The renal artery is dissected, cleaned and a U shaped silver clip is slipped around it near the aorta. The size of the clip is adjusted so that the internal gap ranges from 0.25 to 0.38 nm . The right kidney is removed after tying off the renal pedicel. Four to five weeks after clipping, blood pressure is measured and rats are divided into different groups receiving different doses of the test drugs. Test compounds are administered for 3 days . Pre-drug and 2 h post drug blood pressure readings are taken . Antihypertensive activity of test drug is determined by comparing treatment blood pressure value with day 1, pre-drug B.P. 10

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12. Blood Pressure in Pithed Rats The pithed rat model is devoid of neurogenic reflex control that may modulate the primary drug effect. It is frequently used to evaluate drug action on the cardiovascular system . Male Wistar rats (250 to 350 g) are anesthetized with halothane. The carotid artery is cannulated for monitoring blood pressure and blood sampling. The trachea is cannulated and the animal is maintained on artificial respiration using a ventilation pump (60 cycles/ min). The jugular vein is also cannulated for the administration of test drug. Pithing is done by inserting a steel rod, 2.2 mm in diameter and 11 cm in length, through the orbit and foramen magnum down the whole length of the spinal canal. 12

13. Inspired air is oxygen-enriched by providing a flow of oxygen across a T-piece attached to the air inlet of the ventilation pump. Thirty minutes after pithing, a 0.3 ml blood sample is withdrawn from the carotid cannula and analysed for pO2, pCO 2 , pH and bicarbonate concentration using blood gas analyser. Through the carotid artery blood pressure and cardiac frequency is recorded. To measure a1 and a2 antagonism , first dose response curves are registered with phenylephrine, a selective a1-agonist (0.1-30 mcg/kg , intravenously) and BHT 920, a selective a2-agonist (11000 mcg/kg , intravenously). The test drug is administered intravenously and the agonist dose response curves are repeated 15 min later. The curve of blood pressure response to agonist is obtained. Dose response curves are plotted on a logarithmic probit scale . 13

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15. Tail Cuff Method in Rats The indirect tail cuff method allows the measurement of blood pressure without any surgical procedure . The principle used in this method is that the pulse is obliterated when the cuff is inflated well above suspected systolic blood pressure. The pulse reappears as the pressure in the cuff is slowly released and it falls below the systolic blood pressure. The method is analogous to sphygmomanometer in humans. The indirect tail cuff method is widely used to evaluate the influence of antihypertensive drugs in spontaneously and experimentally induced hypertensive rats . 15

16. Male Spontaneously hypertensive rats (300 to 350 g) are anesthetized with 0.8 ml of 4% chloralhydrate solution. Both kidneys are exposed. A silver clip (0.2 mm diameter) is placed onto both renal arteries, kidneys are reposed and wound is closed by suture. After 5 to 6 weeks, operated animals attain renal hypertension with systolic blood pressure of 170 to 200 mm Hg . To measure blood pressure, a tubular inflatable cuff is placed around the base of the tail and a piezoelectric pulse detector is positioned distal to the cuff. The cuff is inflated well above suspected systolic blood pressure until the pulse is obliterated. 16

17. Thereafter, pressure in the cuff is slowly released and as the pressure reaches the systolic blood pressure, the pulse reappears which is detected and subsequently recorded on a polygraph. The test substance is administered intraperitoneally once a day over a period of 5 days . Blood pressure and heart rate is measured ( pre dose and 2 h post drug) at days 1, 3 and 5 . Percent decrease in systolic blood pressure after administration of the test drug and the duration of effect is determined. Scores for percentage decrease in systolic blood pressure and for the duration of the effect are allotted. 17

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19. Salt-sensitive Dahl Rats The kidneys have the ability to excrete easily the daily salt load without allowing a marked Rise in extracellular volume. Chronic ingestion of excess salt produces hypertension in rats, which mimics human hypertension morphologically. The salt sensitive Dahl rats develop severe and fatal hypertension when fed high salt diets, whereas salt resistance Dahl rats do not develop such severe hypertension upon salt loading. Also when fed normal salt diets, the salt sensitive rats become hypertensive, demonstrating that this is a model of genetic hypertension , with the extra feature of salt sensitivity . 19

20. Sprague Dawley rats (250 to 300 g) are used for this study. The drinking water is replaced with 8% NaCl saline solution. High Dahl salt diet is prepared in the laboratory by mixing salt with the regular diet. The animals are fed the prepared diet and 8% NaCl solution ad libitum. Blood pressure changes are recorded in the different experimental groups at present time points. Blood pressure starts to rise after 1 week and reaches systolic values between 160-180 mm Hg after 4 weeks. The animals are divided into two groups: sham control (untreated) and treated (administered the drug orally for 1 month) groups. After the completion of the experimental duration, animals of both groups (treated and sham control) are sacrificed. Their hearts are removed and total cardiac mass, weight of left and right ventricle is Measured and compared, Upon salt feeding (8% NaCl ), blood pressure rises steeply, to levels slightly higher than found in spontaneously hypertensive rats (up to 32 %). The ability of the test drug to reverse these changes is studied. 20

21. Fructose-induced Hypertension In Rats Feeding a high-fructose diet induces hypertension and insulin-resistance in Sprague-Dawley rats. Fructose feeding also causes insulin resistance, hyperinsulinemia and hypertriglyceridemia in normal rats. Fructose feeding induces hypertension in normal or high-salt fed animals and it is associated with an increased expression of the angiotensin-II Type-1 (AT-1) receptor in adipose tissue. AT-1 receptors play a role in the pathophysiology of metabolic and hemodynamic abnormalities induced by fructose feeding. 21

22. Wistar rats (200 to 250 g) are housed per cage on a 12 h light and dark cycle and fed water and diet ad libitum. Drinking water consists of 10% fructose solution . Fluid intake, food intake and body weight of each rat are measured every week during the course of drug treatment. Systolic blood pressure and pulse rate is measured using the tail-cuff method. Blood samples are collected before and every second week during treatment and plasma glucose, insulin, triglycerides is measured . The animals are divided into two groups: control (untreated) and treated (administered the drug orally for 1 month) groups. The above mentioned parameters are compared between the two groups. One way or two way analysis of variance followed by Newman- Keuls test is used for the statistical analysis. 22

23. DOCA-Salt Rats Mineralocorticoid induces hypertension by causing increase in plasma and extracellular volume. The administration of deoxy corticosterone acetate (DOCA), a mineralocorticoid in combination with a high salt diet and unilateral nephrectomy induces hypertension. 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. In addition, altered activity of RAAS leads to increased sympathetic activity. In this model rennin levels are low as opposed to the other models, where renin levels are high . 23

24. Male Sprague Dawley rats (250 to 300 g) are anesthetized with ether. The left kidney is removed through a flank incision. Deoxy corticosterone acetate (20 mg/kg) is dissolved in olive oil and injected subcutaneously to rats, twice weekly for four weeks. Drinking water is replaced with 1% NaCl solution. Blood pressure starts to rise after 1 week and systolic value reaches around 160 to 180 mm H g after 4 weeks. DOCA pellets or implants in silastic devices can also be used instead of repeated injections. Animals are randomly allocated to control (untreated) or treated (Test drug is administered orally for one month) groups. Blood pressure is measured in the different experimental Groups at present time points throughout the experimental duration . After the completion of the experimental duration, the animals are sacrificed and their hearts weighed, Renal (proteinuria and glomerulosclerosis) and blood pressure changes are studied and compared between the two groups to evaluate the antihypertensive effect of the test drug. 24

25. Spontaneously Hypertensive Rats (SHR) By breeding a strain of spontaneously hypertensive Wistar rats with a female having slightly raised blood pressure, Okamoto and Aoki obtained a strain of rats with spontaneous hypertension, the SHR . SHR was developed by meticulous genetic inbreeding that uniformly resulted in 100% of the progeny having naturally occurring hypertension. Since then, several expert panels have reported that SHR is an excellent model of experimental hypertension that could serve as a counterpart for clinical essential hypertension as well as model for complications of hypertension. In these rats, blood pressure rises around 5 to 6 weeks of age and steadily increases to reach systolic blood pressure of 180 to 200 mm Hg . 25

26. The SHR develops many features of hypertensive end organ damage including cardiac hypertrophy, cardiac failure and renal dysfunction. However , they do not exhibit gross vascular problems. Apart from depressed endothelium dependent relaxations, they have no tendency to develop strokes, and do not develop macroscopic atherosclerosis or vascular thrombosis . The SHR stroke prone (SHRSP) is a further developed sub-strain, with even higher levels of blood pressure, and a strong tendency to die from stroke . The SHR have been widely used to evaluate genetic factors in hypertension, yielding a wide variety of genes that seem to co-segregate in various crosses which is not always confirmed.   Other models of genetically determined spontaneous hypertension include the New Zealand strain of genetic hypertension (GH-Smirk), the stroke prone and stroke resistant sub strains of SHR (SHRSP and SHRSR ), arterio-lipoidosis prone SHR (SHRLP), the obese SHR stain, the Milan hypertensive (MFIS), Dahl’s salt susceptible (S) and salt resistant (R) strains, and Smirk’s genetic hypotensive strain of rats. Most abundant experience has been gained with the Wistar Kyoto strain . 26

27. DOG MODEL OF HYPERTENSION 27

28. Chronic Renal Hypertension Partial constriction of renal arteries in dogs produced hypertension. This method has been modified and is now known as the ‘wrapping’ technique . A sheet of cellophane is placed around the kidney and held in place by silk sutures tied loosely around the renal hilus . Both kidneys are wrapped or one kidney is wrapped and other is removed. A fibro collagenous shell is formed around the kidney in 3-5 days because of reaction of the tissue to the foreign material. This shell compresses renal parenchyma leading to decreased renal vascular pressure. This expands the extracellular volume leading to increased peripheral resistance and hence increased BP.   28

29. Dogs (8 to 12 kg) are anesthetized intravenously with 15 mg/kg thiopental. Midline abdominal incision is made and one kidney is exposed. It is wrapped in cellophane and then replaced. The contra lateral kidney is exposed and artery, vein and ureter are ligated and this kidney is removed. The abdomen is then closed and sutured back. The animals are treated with antibiotics post surgery and allowed to recover. After six weeks of surgery, blood pressure is measured. Blood pressure is recorded either by indirect tail cuff method or by direct measurement through the carotid artery. Test drugs are administered for 5 days. On day 1 readings are taken every 2 h, just before, and 2 & 4 h after oral treatment. On day 3 and 5 blood pressure is recorded before, 2 & 4 h after drug treatment. The starting value is the average of the two readings before application of the drug. Subsequent readings are subtracted from this value and recorded as fall of blood pressure at the various recording times. 29

30. MONKEY MODEL OF HYPERTENSION 30

31. Renin Inhibition in Monkeys Blood pressure is mainly regulated by the renin angiotensin system and can be influenced by inhibition of renin. Renin is an aspartyl protease that hydrolyses angiotensinogen to release angiotensin-I. Angiotensin-I is subsequently converted to angiotensin-II by angiotensin converting enzyme. Renin inhibitors developed so far have a high specificity for primate renin and cause only weak inhibition of renin in sub primate species. This suggests that most common laboratory animals such as rats and dogs are not suitable for in vivo evaluation of renin inhibitors. 31

32. Marmosets (Callithrix jacchus ) of 300 to 400 g are fed pellet diet supplemented with fruits. The animals are anesthetized two days prior to the experiment and catheters are implanted in the femoral artery for measurement of blood pressure . Lateral tail vein is cannulated for the administration of test compounds. Furosemide (5 mg/kg) is injected intravenously 30 min before the experiment in order to stimulate renin release. During the experiment, the marmosets are sedated with diazepam (0.3 mg/kg, intraperitoneally) and kept in restraining boxes. Mean blood pressure is recorded continuously, and heart rate is measured at fixed intervals. The test compound and standard drug are injected by intravenous infusion or administered orally. Blood pressure is recorded after 30 min of intravenous infusion and 30 min after stopping the infusion. Changes from pre-treatment values after various doses of drug are compared. Dose-response curves can be established. 32