Screening Methods of Diuretic Activity On Animal Model

They are the agents which increase the rate of urine flow and sodium excretion and are used to adjust the volume of body fluids. It is very useful for critical condition like- hypertension, heart failure, renal failure etc.

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BIOLOGICAL EVALUATION OF DIURETIC AGENTS

In vitro Methods

1. Carbonic Anhydrase Inhibition In Vitro

In vivo Methods

1. Diuretic activity in rats (LIPSCHITZ test).
2. Saluretic activity in rats
3. Diuretic and saluretic activity in dogs

In vitro Methods: Carbonic Anhydrase Inhibition In Vitro

PURPOSE AND RATIONALE

Acetazolamide first non mercurial diuretics.

• MOA- inhibition of carbonic anhydrase .
• It is zinc containing enzyme that catalyzes reversible hydration of CO2 to H2CO3 (dissociates into HCO3– and H+).
• Its primary work to enhance H+ secretion into urine.
• At least three isoenzymes, designated as I, II and II or A, B and C, are known to exist.
• Enzyme source are red cells, a rich source of the same isoenzymes found in the eye.

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PROCEDURE:

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.

Equipment-

          – Reaction vessel

          – Monostat bench mounted flowmeter

          – 30% CO2

Assay:

• CO2( flow rate 30 (45)ml/min).
• Solutions 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

Evalution:

Percent inhibitor of carbonic anhydrase is calculated according to the following formula---

In vitro Methods: Carbonic Anhydrase Inhibition In Vitro

Tu = (uncatalyzed time) =time for the color change in the absence of enzyme.

Te = (catalyzed time) =     time for the color change in the presence of the enzyme.

Tu – Te =                          enzyme rate

Ti =                                   enzyme rate in the presence of various concentrations of inhibitor.

In VIVO Methods

1.) Diuretic activity in rats (LIPSCHITZ Test)

PURPOSE AND RATIONALE:

Lipschitz (1943) describe this method. 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.

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PROCEDURE:

• Animal - Wistar rats (100–200 g)
• In metabolic cages 3 animals per group 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. 
• 15 hours prior to the experiment food and water are withdrawn.
• 2 groups of three animals are used for one dose of the test compound orally (dose 50 mg/kg in 5.0 ml water/kg body weight).
• Two groups of 3 animals orally 1 g/kg urea.
• Additionally, 5 ml of 0.9% NaCl solution per 100 g body weight are given.
• Urine excretion is recorded after 5 and after 24 h.
• The sodium content of the urine is determined by flame photometry. Active compounds are tested again with lower doses.

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EVALUATION:

1- For each group Urine volume excreted per 100 g body weight is calculated.

2- Results are expressed as the “Lipschitz-value”, 

i.e., the ratio T/U

Where 

T = response of the test compound,

U =  urea treatment.

3-  Positive effect if value is 1.0 and more.

4-  Potent diuretics, values is 2.0 and more can be found.

5- Calculating this index for the 24 h excretion period as well as for 5 h indicates the duration of the diuretic effect.

Saluretic drugs, (hydrochlorothiazide)= around 1.8,

Where as loop diuretics (or high ceiling diuretics) like furosemide, bumetanide = 4.0 and more.

2.) Saluretic Activity in Rats

PURPOSE AND RATIONALE:

Excretion of electrolytes is as important as the excretion of water for treatment of peripheral edema and in congestive heart failure as well as for treatment of hypertension. Potassium loss has to be avoided.

As a consequence, saluretic drugs and potassium-sparing diuretics were developed.

The diuresis test in rats was modified in such a way that potassium and chloride are determined in addition to water and sodium. Ratios between electrolytes can be calculated indicating carbonic anhydrase inhibition or a potassium sparing effect.

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Diuretic activity in rats (LIPSCHITZ Test)

PROCEDURE:

• Animal - Male Wistar rats (100–200 g).
• Fifteen hours prior to the test, food but not water is withdrawn.
• Test compounds (dose 50 mg/kg orally in 0.5 ml/100 g) body weight starch suspension.
• Three animals are placed in one metabolic cage 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.
• 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.
• Furosemide (25 mg/kg ), hydrochlorothiazide (25 mg/kg), triamterene (50 mg/kg), or amiloride (50 mg/kg) as standards.

Evaluation:

• The sum of Na+ and Cl- excretion is calculated as parameter for saluretic activity.
• The ratio Na+/K+ is calculated for natriuretic activity. value greater than 2.0 indicate a favorable natriuretic effect. ratios greater than 10.0 indicated a potassium- sparing effect (ion quotient) is calculated to estimate carbonic anhydrase inhibition.
• Carbonic anhydrase inhibition can be excluded at ratios between 1.0 and 0.8 with decreasing ratios slight to strong carbonic anhydrase inhibition can be assumed.

3.)  Diuretic and Saluretic Activity in Dogs

PURPOSE AND RATIONALE

• Dogs used to study renal physiology and the action of diuretics.
• Renal physiology of the dog is claimed to be closer to man than that of rats.
• Oral absorbability of diuretic substances can appropriately be studied in dogs.
• Using catheters, interval collections of urine can be made with more reliability than in rats.
• Simultaneously, blood samples can be withdrawn to study pharmacokinetics.

PROCEDURE:

• Beagle dogs have to undergo intensive training to be accustomed to accept and hourly catheterization without any resistance. 
• The dogs are placed in metabolic cages.
• At least 4 dogs as controls receiving water only, as standard controls (1 g/kg urea or 5 mg/kg furosemide) or the test drug group.
• 24 hours prior to the experiment food but not water is withheld.
• On the morning of the experiment, the urine bladder is emptied with a plastic catheter.
• The dogs receive 20 ml/kg body weight water, followed by hourly doses of 4 ml/kg body weight drinking water.
• The bladder is catheterized twice in an 1 h and the urine collected for analysis of initial values. Then, the test compound or the standard is applied either orally or i.v.
• Hourly catheterization is repeated over the next 6 h. Without further water dosage the animals are placed in metabolic cages overnight.
• 24 hours after dosage of the test compound, the dogs are catheterized once more and this urine together with the urine collected over night in the metabolic cage registered.
• All urine samples are analyzed by flame photometry for sodium and potassium and by argentometry (Chloride Titrator Aminco) for chloride content.

EVALUATION:

• Urine volume, electrolyte concentrations and osmolality are averaged for each group.
• The values are plotted against time to allow comparison with pretreatment values as well as with water controls and standards.
• The non-parametric U-test is used for statistical analysis.