screening of anti hypertensives2003
TRANSCRIPT
EVALUATION SEMINAR ON
SCREENING OF ANTIHYPERTENSIVE AGENTS
ByMallappa. Shalavadi,Lecturer,Department of Pharmacology,HSK College of Pharmacy,Bagalkot.
CONTENTS• Definition• Types• Pathophysiology• Screening models In vitro modelsIn vivo models
WHAT IS HYPERTENSION ?• Hypertension is the most common cardiovascular
disease.• Hypertension is defined conventionally as a
sustained increase in blood pressure ≥ 140/90 mm Hg.
2- TYPESa) Primary or essential hypertension: Cause for rise in the blood pressure is unknown, several
factors implicated in its genesis:• High salt intake• Cigarette smoking• Hypersensitivity of sympathetic system
b) Secondary hypertension: Common disorders causing hypertension are:-• Cushing syndrome• Acute or chronic renal disease• Renal artery stenosis• Drugs like oral contraceptives, estrogen, steroids.
PATHOPHYSIOLOGY
SCREENING MODELS OF ANTIHYPERTENSION AGENTSIN VITRO MODELS1. α2-adrenoreceptor binding2. Electrically stimulated release of [3H]norepinephrine from
brain slices3. Inhibition of angiotensin converting enzyme in vitro4. Quantitative autoradiographic localization of angiotensin
converting enzyme 5. Angiotensin II receptor binding6. Angiotensin II induced contraction in isolated rabbit aorta7. Renin-inhibitory activity using human kidney renin and a
synthetic substrate .
8. Inhibition o endothelin converting enzyme
IN VIVO MODELS1. Acute renal hypertension2. Chronic renal hypertension in rats 3. Chronic renal hypertension in dogs4. Neurogenic hypertension in dogs 5. DOCA-salt induced hypertension in rats6. Fructose induced hypertension in rats7. Genetic hypertension in rats8. Pulmonary hypertension induced by monocrotaline9. Blood pressure in conscious rats (tail cuff method)
IN VITRO MODELSα2- ADRENORECEPTOR BINDINGPURPOSE AND RATIONALE• α2-adrenoceptors are widely distributed and are activated by
norepinephrine released from sympathetic nerve terminals• Prejunctionally mediated inhibition of the release of
neurotransmitters from many peripheral and central neurons.• α2-adrenoceptors are also present at postjunctional sites,
where they mediate actions such as smooth muscle contraction, platelet aggregation and inhibition of insulin secretion.
• Clonidine is a centrally-acting antihypertensive agent, which lowers blood pressure mostly through reducing sympathetic tone by acting at the nucleus tractus solitarius in the brain stem
• Alpha-adrenergic agonists most potently displace 3H clonidine.
• The purpose of this assay is to assess the interaction of hypotensive agents with central α2-receptors and determine possible clonidine-like mechanisms of action.
PROCEDURE• Reagents1. Tris buffer pH 7.72. [4-3H]-Clonidine hydrochloride3. Clonidine-HCl4. Test compounds: 1 mM stock solution is made up in a suitable solvent and
serially diluted, so that the final concentrations in the assay range from 10–5 to 10–8 M.
Tissue preparation ASSAY
EVALUATION• IC50 calculations are performed using log-probit
analysis.• The percent inhibition at each drug concentration is
the mean of triplicate determinations.MODIFICATIONS OF THE METHOD• Perry and U’Prichard (1981) described
[3H]rauwolscine (α-yohimbine) as a specific radioligand for brain α2-adrenergic receptors.
• Goldberg and Robertson (1983) reviewed yohimbine as a pharmacological probe for the study of the α2-adrenoreceptor.
Inhibition of angiotensin converting enzyme in vitroPURPOSE AND RATIONALE• An in vitro system can be used to screen potential
angiotensin-converting-enzyme inhibitors. • Fluorescence generated by an artificial substrate in
presence or absence of the inhibitor is measured to detect inhibitory activity.
PROCEDURE• Reagents1. 50 mM Tris-HCl buffer, pH 8.0 + 100 mM NaCl2. 10 mM potassium phosphate buffer, pH 8.3
3. Substrate: O-aminobenzoylglycyl-p-nitro-L-phenylalanyl-L-proline
4. Test compounds• Compounds are made up to a concentration of 1
mM in 50 mM Tris-HCl buffer, pH 8.0 + 100 mM NaCl or 10% methanol in Tris/NaCl if insoluble in aqueous buffer alone. This will give a final concentration in the assay of 0.1 Mm.
Enzyme preparation
Enzyme inhibition studies1. Enzyme activity is measured with a Perkin Elmer LS-5
Fluorescence Spectrophotometer or equivalent at an excitation wavelength of 357 nm and an emission wavelength of 424 nm.
2. Enzyme assay• 50 μl vehicle or inhibitor solution and 40 μl enzyme are
preincubated for 5 min, then 410 μl substrate working solution is added.
• Samples are mixed by drawing fluid back up into the pipette and by pipetting into the cuvette.
• For the initial control run of the day, the auto zero is pushed immediately after placing the sample in the cuvette.
EVALUATION• The individual fluorescence slope is measured and %
inhibition is calculated as follows:
• Inhibitor concentrations on either side of the IC50 should be tested to generate a dose-response curve.
• The IC50 is calculated using Litchfield-Wilcoxon log probit analysis.
IN VIVO MODELS
1. Acute renal hypertension in ratsPURPOSE AND RATIONALE• Ischemia of the kidneys causes elevation of blood pressure by
activation of the renin-angiotensin system.• In rats acute renal hypertension is induced by clamping the
left renal artery for 4 h. • After reopening of the vessel, accumulated renin is released
into circulation. • The protease renin catalyzes the first and rate-limiting step in
the formation of angiotensin II leading to acute hypertension.• The test is used to evaluate antihypertensive activities of
drugs.
PROCEDURE
• EVALUATION• Increase in blood pressure after reopening of the
renal artery and reduction in blood pressure after administration of the test drug are determined [mm Hg].
• 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.
2. DOCA-salt induced hypertension in rats
PURPOSE AND RATIONALE• Mineralocorticoid-induced hypertension is thought
to be due to the sodium retaining properties of the steroid causing increases in plasma and extracellular volume.
• The hypertensive effect is increased by salt loading and unilateral nephrectomy in rats.
PROCEDURE Male Sprague Dawley rats weighing 250–300 g are
anesthetized with ether.
Through a flank incision the left kidney is removed.
The rats are injected twice weekly with 20 mg/kg s.c. desoxycorticosterone-acetate in olive oil for 4 weeks.
Drinking water is replaced with a 1% NaCl solution.
Blood pressure starts to rise after one week and reaches systolic values between 160 and 180 mm Hg after 4 weeks.
MODIFICATIONS OF THE METHOD• DOCA-salt hypertension can also be achieved
without nephrectomy (Bockman et al. 1992).• DOCA pellets or implants in silastic devices
(Ormsbee and Ryan 1973; King and Webb 1988) were used instead of repeated injections.
3. Fructose induced hypertension in ratsPURPOSE AND RATIONALE• Increases in dietary carbohydrate intake can raise blood
pressure in experimental animals. • The increased intake of either sucrose or glucose was shown
to enhance the development of either spontaneous hypertension or salt hypertension in rats
• Hypertension could be induced in normal rats by feeding a high-fructose diet.
• Fructose feeding was also found to cause insulin resistance, hyperinsulinemia, and hypertriglyceridemia in normal rats
• Dai and McNeill (1995) studied the concentration- and duration-dependence of fructose-induced hypertension in rats.
PROCEDURE
EVALUATION• Since maximum effects on the chosen parameters
are achieved after 6 weeks, the duration of treatment can be limited to this time.
• Statistical analysis is performed using a one-way or two-way analysis of variance, followed by the Newman-Keuls test.
MODIFICATIONS OF THE METHOD• 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.
4. Genetic hypertension in rats• Inherited hypertension in rats has been described
by Smik and Hall 1958; Phelan and Smirk 1960; Laverty and Smirk 1961; Phelan 1968 as genetically hypertensive (GH) rats.
• 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. 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.
• Hypertension in these rats is clearly hereditary and genetically determined, thus comparable to primary hypertension in humans.
• Cardiac hypertrophy and cellular ionic transport abnormalities have been observed
CRITICAL ASSESSMENT OF THE METHOD• The use of spontaneously hypertensive rats to
detect potential antihypertensive compounds is well established.
• On the basis of available data no preference can be given to a particular strain.
• The most abundant experience has been gained with the Wistar-Kyoto strain.
• Transgenic rats with well defined genomes are gaining more importance.
5. Pulmonary hypertension induced by monocrotaline
PURPOSE AND RATIONALE• The pyrrolizidine alkaloid monocrotaline, derived
from Crotalaria spectabilis, is hepatotoxic and pneumotoxic in the rat.
• A single injection of monocrotaline leads to progressive pulmonary hypertension resulting in right ventricular hypertrophy and cardiac failure
• Rats given monocrotaline develop severe right ventricular hypertrophy often accompanied by ascites and pleural efflusions.
• Amelioration by angiotensin-converting enzyme inhibitors and by penicillamine has been demonstrated.
REFERANCES• H. Gerhard Vogel., Wolfgang H.Vogel., Bernward A.
Schölkens., Jürgen Sandow., Günter Müller., Wolfgang F. Vogel, Drug discovery and evaluation, 2nd ed. Springer-Verlag Berlin Heidelberg, 2002;26-172.
• Harsh mohan, Text book of Pathology, 5th ed., Jaypee, 2005;708-709.
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