SCREENING OF DIURETIC AGENTS-AN OVERVIEW
*Nilesh Sovasia, Prof.Sanjeev Thacker, Arshad Hala
Seth G.L.Bihani S.D.College Of Technical Education,
Institute Of Pharmaceutical Science & Drug Research,
Diuretic agents are very useful for several critical conditions like hypertension, heart failure, renal failure, nephrotic syndrome, and cirrhosis.The various methods for screening of Diuretic agents provides useful tool to evaluate the safety and effectiveness of the drugs.It is also useful for determining the dose lavel of particular class of diuretic agents.
REFERENCE ID: PHARMATUTOR-ART-1472
Diuretics increase the rate of urine flow and sodium excretion and are used to adjust the volume and/or composition of body fluids in a variety of clinical situations, including hypertension, heart failure, renal failure, nephrotic syndrome, and cirrhosis.
By definition, diuretics are drugs that increase the rate of urine flow; however, clinically useful diuretics also increase the rate of excretion of Na+ (natriuresis) and of an accompanying anion, usually Cl-. NaCl in the body is the major determinant of extracellular fluid volume, and most clinical applications of diuretics are directed toward reducing extracellular fluid volume by decreasing total-body NaCl content. A sustained imbalance between dietary Na+ intake and Na+ loss is incompatible with life. A sustained positive Na+ balance would result in volume overload with pulmonary edema, and a sustained negative Na+ balance would result in volume depletion and cardiovascular collapse. Although continued administration of a diuretic causes a sustained net deficit in total-body Na+, the time course of natriuresis is finite because renal compensatory mechanisms bring Na+ excretion in line with Na+ intake, a phenomenon known as diuretic braking. These compensatory, or braking, mechanisms include activation of the sympathetic nervous system, activation of the renin-angiotensin-aldosterone axis, decreased arterial blood pressure (which reduces pressure natriuresis), hypertrophy of renal epithelial cells, increased expression of renal epithelial transporters, and perhaps alterations in natriuretic hormones such as atrial natriuretic peptide Historically, the classification of diuretics was based on a mosaic of ideas such as site of action (loop diuretics), efficacy (high-ceiling diuretics), chemical structure (thiazide diuretics), similarity of action with other diuretics (thiazidelike diuretics), effects on potassium excretion (potassium-sparing diuretics), etc. However, since the mechanism of action of each of the major classes of diuretics is now well understood.
Diuretics not only alter the excretion of Na+ but also may modify renal handling of other cations (e.g., K+, H+, Ca2+, and Mg2+), anions (e.g., Cl-, HCO3-, and H2PO4-), and uric acid. In addition, diuretics may alter renal hemodynamics indirectly.1
BIOLOGICAL EVALUATION OF DIURETIC AGENTS:
1.Diuretic and Saluretic Activity
1.1 In vitro Methods
1.1.1 Carbonic Anhydrase Inhibition in vitro:
PURPOSE AND RATIONALE
Acetazolamide (Diamox®) 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 CO2 to form H2CO3 which dissociates non-enzymatically into HCO3- and H+.Although many methods to measure carbonic anhydrase activity have been developed, the micro method described by Maren (1960) is relatively simple, sensitive and reliable. The enzyme source are red cells, a rich source of the same isoenzymes found in the eye.1,4
Materials and solutions:
· Phenol red indicator solution
12.5 mg phenol red/liter 2.6 mM NaHCO3, pH 8.3 + 218 mM Na2CO3
· 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.
– Reaction vessel
– Monostat bench mounted flowmeter
– 30% CO2 – M&G Gases, Branchburg, NJ, USA
CO2 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 μl enzyme
200 μl H2O or appropriate drug concentration after
3 min for equilibration:
100 μl carbonate/bicarbonate buffer is added.
The following parameters are determined in duplicate samples:
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
Percent inhibition of carbonic anhydrase is calculated according to the following formula:
% Inhibition = 1- (Tu - Te) - (Ti - Te)
Tu - Te
• Compound IC50 [M]
• Acetazolamide 9.0 × 10–9
• Chlorothiazide 9.0 × 10–7
CRITICAL ASSESSMENT OF THE METHOD
Determination of carbonic anhydrase inhibition is of value to characterize the activity spectrum of sulfonamide diuretics.
MODIFICATIONS OF THE METHOD
Landolfi et al. (1997) reported a modified procedure for the measurement of carbonic anhydrase activity. The measure of carbonic anhydrase activity is based on the rate of CO2 hydration by the enzyme. Such transformation was monitored by a procedure which consists in the measure of time necessary for the pH of an appropriate buffer to decrease from 8 to 7.5 in the presence of a constant CO2 flow: such time period is dosedependently reduced by the addition of the enzyme and further modified in the presence of carbonic anhydrase inhibitory compounds.1,4
1.1.2 Patch Clamp Technique In Kidney Cells
PURPOSE AND RATIONALE
In the different parts of the kidney (proximal tubules, distal tubules, collecting ducts) fluid is reabsorbed and substances may be transported either from the tubule lumen to the blood side (reabsorption) or vice versa (secretion). Besides active transport and coupled transport systems, ion channels play an important role in the function of kidney cells. The various modes of the patch clamp technique (cell-attached, cell-excised, whole-cell mode)allow the investigation of ion channels.
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