PRATEEK DIURETICS

Views:
 
Category: Education
     
 

Presentation Description

screening procedures for diuretics and saluretic drugs

Comments

Presentation Transcript

PowerPoint Presentation:

PRESENTED BY PRATEEK DIXIT SCREENING PROCEDURE OF DIURETICS

PowerPoint Presentation:

CARBONIC ANHYDRASE INHIBITION PATCH CLAMP TECHNIQUE IN KIDNEY CELLS PERFUSION OF ISOLATED KIDNEY TUBULES IN VIVO METHODS LIPSCHITZ TEST IN VITRO METHODS

IN VITRO METHOD :

CARBONIC ANHYDRASE INHIBITION -: PRINCIPLE:- Acetazolamide was one of the first synthetic non-mercurial diuretics. The mode of action was found to be inhibition of carbonic anhydrase . Carbonic anhydrase is a zinc-containing enzyme that catalyzes the reversible hydration (or hydroxylation) of CO 2 to form H 2 CO 3 . IN VITRO METHOD

Mechanisms of Action: Carbonic anydrase inhibitors:

Mechanisms of Action: Carbonic anydrase inhibitors CAIs work on cotransport of Na + , HCO 3 - and Cl - that is coupled to H + countertransport . Acts to block carbonic anhydrase (CA), CA converts HCO 3 - + H + to H 2 O + CO 2 in tubular lumen CO 2 diffuses into cell (water follows Na + ), CA converts CO 2 + H 2 O into HCO 3 - + H + H + now available again for countertransport with Na+, etc) Na + and HCO 3 - now transported into peritubular capillary

Site of Action of CAIs:

Site of Action of CAIs

PROCEDURE:-:

MATERIAL AND SOLUTION Phenol red indicator solution: 12.5 mg phenol red/liter 2.6 mM NaHCO 3 , pH 8.3 + 218 Mm H 2 CO 3 1 M sodium carbonate/bicarbonate buffer, pH 9.8 Enzyme: Carbonic anhydrase from dog blood; Blood is collected into a heparinized tube and diluted 1:100 with deionized water. PROCEDURE:-

EQUIPMENTS:- :

Reaction vessel – custom made by Labglass Inc., Vineland, NJ, USA. Monostat bench mounted flowmeter . 30% CO2 – M&G Gases, Branchburg, NJ, USA. EQUIPMENTS:-

ASSAY :

CO 2 flow rate is adjusted to 30 (45) ml/min. The following solutions are added to the reaction vessel: 400 μl phenol red indicator solution. 100 μ l enzyme. 200 μl H 2 O or appropriate drug concentration after 3 min for equilibration. 100 μl carbonate/bicarbonate buffer is added. ASSAY

PowerPoint Presentation:

The following parameters are determined in duplicates Tu = ( uncatalyzed time ) = time for the color change to occur in the absence of enzyme. Te = (catalyzed time) = time for the color change to occur in the presence of the enzyme. Tu – Te = enzyme rate. Ti = enzyme rate in the presence of various concentrations of inhibitor.

EVALUATION:-:

Percent inhibition of carbonic anhydrase is calculated according to the following formula: EVALUATION:-

IN VIVO METHODS :-:

IN VIVO METHODS :- Diuretic activity in rats (LIPSCHITZ test) PRINCIPLE:- The test is based on water and sodium excretion in test animals and compared to rats treated with a high dose of urea. The “ Lipschitz - value” is the quotient between excretion by test animals and excretion by the urea control.

PROCEDURE:

PROCEDURE Male Wistar rats weighing 100–200 g are used. Three animals per group are placed in metabolic cages provided with a wire mesh bottom and a funnel to collect the urine. Stainless-steel sieves are placed in the funnel to retain feces and to allow the urine to pass.

PowerPoint Presentation:

The rats are fed with standard diet ( Altromin ® pellets) and water ad libitum Three animals are placed in one metabolic cage. Fifteen hours prior to the experiment food and water are withdrawn.

PowerPoint Presentation:

Urine excretion is recorded after 5 and after 24 h. Additionally, 5 ml of 0.9% NaCl solution per 100 g body weight are given by gavage . For screening procedures two groups of three animals are used for one dose of the test compound.

PowerPoint Presentation:

Two groups of 3 animals receive orally 1 g/kg urea. For screening procedures two groups of three animals are used for one dose of the test compound. The sodium content of the urine is determined by flame photometry. Active compounds are tested again with lower doses.

PowerPoint Presentation:

-: EVALUATION:- Urine volume excreted per 100 g body weight is calculated for each group. Results are expressed as the “ Lipschitz -value”, i.e., the ratio T/U, in which T is the response of the test compound, and U, that of urea treatment. Indices of 1.0 and more are regard with potent diuretics, Lipschitz values of 2.0 and more can be found as a positive effect. Calculating this index for the 24 h excretion period as well as for 5 h indicates the duration of the diuretic effect. Dose-response curves can be established using various doses.

Saluretic activity in rats:

PRINCIPLE:- Excretion of electrolytes is as important as the excretion of water for treatment of peripheral edema and ascites in congestive heart failure as well as for treatment of hypertension. Potassium loss has to be avoided. The diuresis test in rats was modified in such a way that potassium and chloride as well as osmolality are determined in addition to water and sodium. Saluretic activity in rats

PowerPoint Presentation:

Male Wistar rats weighing 100–200 g fed with standard diet ( Altromin ® pellets) and water are used. Fifteen hours prior to the test, food but not water is withdrawn. Test compounds are applied in a dose of 50 mg/kg orally in 0.5 ml/100 g body weight starch suspension. PROCEDURE

PowerPoint Presentation:

Three animals are placed in one metabolic cage provided with a wire mesh bottom and a funnel to collect the urine. Two groups of 3 animals are used for each dose of a test drug. Urine excretion is registered every hour up to 5 h.

PowerPoint Presentation:

The 5-h urine is analyzed by flame photometry for sodium and potassium and argentometrically by potentiometrical end point titration (Chloride- Titrator Aminco ) for chloride. To evaluate compounds with prolonged effects the 24 h urine is collected and analyzed.

EVALUATION:-:

EVALUATION :- The sum of Na+ and Cl – excretion is calculate the ratio Na+/K+ is calculated for natriuretic activity. Values greater than 2.0 indicate a favorable natriuretic effect. Ratios greater than 10.0 indicate a potassium-sparing effected as parameter for saluretic activity.

authorStream Live Help