The document discusses in vivo evaluation techniques for diuretics, detailing various screening methods and classifications of diuretics such as thiazide, loop, and potassium-sparing diuretics. It describes the Lipschitz test for measuring diuretic activity in rats, including methodology and evaluation criteria, as well as saluretic activity testing in both rats and dogs/cats. References from established pharmacology literature are also provided to support the content.

1. PRESENTED BY
Swapnil S. Tirmanwar
Pharmacognosy
(M. Pharm 1st year)
PRIYADARSHINI J. L. COLLEGE OF
PHARMACY,
Electronic zone, MIDC, Hingna road
NAGPUR-440016
2022-2023
In vivo evaluation techniques, for Diuretics
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2. CONTENTS
• INTRODUCTION
• CLASSIFICATION
• SCREENING METHODS FOR DIURETICS:
1. IN-VITRO METHOD
2. IN-VIVO METHOD
• REFERENCES
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3. CLASSIFICATION
THIAZIDE DIURETICS
• CHLOROTHIAZIDE
• HYDROCHLOROTHIAZIDE
LOOP DIURETICS
• FUROSEMIDE
• ETHACRYNIC ACID
POTASSIUM SPARING DIURETICS
• SPIRONOLACTONE
• AMILORIDE
• TRIAMTERENE
CARBONIC ANHYDRASE INHIBITORS
• ACETAZOLAMIDE
• DORZOLAMIDE
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4. • DIURETIC PROMOTES THE REMOVAL OF EXCESS WATER, SALTS,
POISON & ACCUMULATED METABOLIC PRODUCTS SUCH AS UREA
FROM THE BODY.
• INCREASES RATE OF URINE FLOW AND SODIUM EXCRETION ARE
USED TO ADJUST THE VOLUME AND/OR COMPOSITION OF BODY
FLUIDS IN A VARIETY OF CLINICAL SITUATIONS.
• ELIMINATION OF EXCESS URINE (MORE THAN NORMAL LEVELS) IS
TERMED AS DIURESIS.
• SALURETICS ARE AGENTS THAT FACILITATE THE REMOVAL INCREASE
THE RATE & FLOW OF URINE OF SALT OR ESPECIALLY SODIUM ION.
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Introduction

5. EVALUATION MODELS
(SCREENING METHODS FOR
DIURETICS)
• IN-VITRO MODELS
ISOLATED TUBULE PREPARATION
CARBONIC ANHYDROUS PREPARATION
PATCH CLAMP TECHNIQUE
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6. • IN-VIVO MODELS
DIURETIC ACTIVITY IN RAT (LIPSCHITZ TEST).
 SAL URETIC ACTIVITY IN RATS.
 DIURETIC & SAL URETIC ACTIVITY IN DOG CATS.
STOP FLOW TECHNIQUE.
CLEARANCE METHOD.
MICRO PUNCTURE TECHNIQUE.
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7. DIURETIC ACTIVITY IN RATS
(LIPSCHITZ TEST )
PURPOSE AND RATIONALE:
• LIPSCHITZ TEST IS FOR TESTING OF DIURETIC ACTIVITY IN RATS & HAS
BEEN DESCRIBED BY LIPSCHITZ ET AL IN 1943.
• THE TEST IS BASED ON WATER AND SODIUM EXCRETION IN TEST
ANIMALS AS COMPARED TO THE RATS TREATED WITH HIGH DOSE OF
UREA (CONTROL).
• THE "LIPSCHITZ VALUE" =
EXCRETION BY TEST ANIMALS/EXCRETION BY UREA CONTROL
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9. METHODOLOGY:
• SPECIES - MALE WISTAR RATS (100-200 GM).
• SEX - MALE RATS.
• THREE ANIMALS IN EACH GROUP BOTH IN TEST AND CONTROL (4 GROUPS).
• INDUCING AGENT :UREA(1 GM/KG)
• STD. DRUG :FUROSEMIDE
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10. PROCEDURE:
• 3 ANIMALS PER GROUP (6) ARE PLACED IN METABOLIC CAGES PROVIDED WITH A
WIRE MESH BOTTOM AND FUNNEL TO COLLECT THE URINE.
• SS SIEVES ARE PLACED IN THE FUNNEL TO RETAIN FECES BUT ALLOW URINE TO
PASS.
• RATS ARE FED WITH STD DIET AND WATER 17-24 HRS.
• PRIOR TO THE EXPERIMENT FOOD AND WATER IS WITHDRAWN.
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11. PROCEDURE:
• TEST COMPOUND IS APPLIED ORALLY AT A DOSE OF 50MG/KG IN 5MLWATER/KG
BODY WEIGHT.
• 2 GROUPS OF 3 ANIMALS ACT AS CONTROL AND ARE TREATED WITH UREA.
• ADDITIONALLY,5ML OF 0.9% NACL SOLUTION/100G BODY WEIGHT ARE GIVEN BY
GAVAGE.
• URINE EXCRETION IS RECORDED AFTER 5 AND 24 HRS.
• THE NA CONTENT OF URINE IS DETERMINED BY FLAME PHOTOMETRY.
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12. EVALUATION :
• URINE VOLUME EXCRETED PER 100 G BODY WEIGHT IS CALCULATED FOR EACH
GROUP.
• RESULTS ARE EXPRESSED AS LIPSCHITZ VALUE (THE RATIO OF T/U)
• LIPSCHITZ VALUE =
T (RESPONSE OF TEST COMPOUND)/U (RESPONSE OF UREA TREATMENT)
• LIPSCHITZ VALUE≥ 1 INDICATES POSITIVE EFFECT
• LIPSCHITZ VALUE≥ 2 INDICATES POTENT DIURETIC ACTIVITY
• FOR STUDYING PROLONGED EFFECT, 24 HR URINE SAMPLE
COLLECTED& ANALYSED
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13. SALURETIC ACTIVITY IN RATS
PURPOSE AND RATIONALE:
• SALURETIC TEST IN RATS WAS DESIGNED TO DETERMINE SODIUM, POTASSIUM,
CHLORIDE AND WATER CONTENT & OSMOLARITY OF URINE
• RATIO BETWEEN ELECTROLYTES CAN BE CALCULATED, INDICATING CARBONIC
ANHYDRASE INHIBITION OR K+ SPARING EFFECT
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14. METHODOLOGY:
• SPECIES-WISTAR RATS (100-200G).
• SEX-MALE.
• THREE ANIMALS IN EACH GROUP ONE GROUP ACTS AS A TEST & ANOTHER ONE
AS A CONTROL.
• INDUCING AGENT :UREA(1 GM/KG)
• STD. DRUG : FUROSEMIDE(25MG/KG) OR OR HYDROCHLOROTHIAZIDE(25MG/KG)
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15. PROCEDURE:
• MALE WISTAR RATS FED WITH STD DIET AND WATER ARE USED.
• 15 HRS. PRIOR TO THE TEST FOOD BUT NOT WATER IS WITHDRAWN.
• TEST COMPOUNDS ARE APPLIED 50MG/KG ORALLY IN 0.5ML/100G BODY WEIGHT STARCH
SUSPENSION.
• 3 ANIMALS ARE PLACED IN 1 METABOLIC CAGE WITH A WIRE MESH BOTTOM AND A FUNNEL TO
COLLECT THE URINE.
• 2 GROUPS OF 3 ANIMALS ARE USED FOR EACH DOSE OF THE TEST COMPOUND.
• URINE EXCRETION IS REGISTERED EVERY HR. UP TO 5 HRS.
• THE 5 HR URINE IS ANALYSED BY FLAME PHOTOMETRY FOR NA & K+ & BY ARGENTOMETRY FOR
CHLORIDE.
• TO EVALUATE COMPOUNDS WITH PROLONGED EFFECT 24 HR URINE IS ANALYSED.
• FUROSEMIDE(25MG/KG) OR HYDROCHLOROTHIAZIDE(25MG/KG) IS USED AS STANDARD.
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16. EVALUATION
• THE SUM OF NA AND CHLORIDE EXCRETION IS A MEASURE OF SALURETIC ACTIVITY.
• THE RATIO OF NA/K IS CALCULATED FOR NATRIURETIC ACTIVITY.
• VALUES >2 INDICATE NATRIURETIC EFFECT. RATIOS>10 INDICATE K SPARING EFFECT.
• THE RATIO OF CHLORIDE/ NA + K IS CALCULATED FOR CARBONIC ANHYDRASE INHIBITION.
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17. SALURETIC ACTIVITY IN DOGS/CATS
PRINCIPLE :
IT IS BASED ON PRINCI8PLE THAT THE HIGH DOSE OF
UREA INDUCES DIURESIS AS WELL AS SALURETIC EFFECT
• ANIMALS : DOGS OR CATS
• INDUCING AGENT : UREA
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18. PROCEDURE:
• GIVE ANESTHESIA TO ANIMAL WITH PHENOBARBITONE(35-50 MG
/KG)
• OPEN THE LOWER ABDOMEN ANANNULA FOR DOSING THE TEST/S
DRUG AND SALINE SOLUTION.
• TREAT EACH ANIMAL OF GROUP WITH UREA.
• ADMINISTER STANDARD DRUG AND TEST/S IN DECIDED DOSES AT
PER DECIDED FREQUENCY.
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19. EVALUATION
• COLLECT AND MEASURE THE URINE VOLUME.
• MEASURE SODIUM AND POTASSIUM CONC. BY FLAME PHOTOMETRY.
• COMPARE RESULTS OF CONTROL, STANDARD DRUG AND TEST
SAMPLE/S GROUPS
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20. REFERENCES
• K.D.TRIPATHI, "ESSENTIAL OF MEDICAL PHARMACOLOGY", 6TH
EDITION,PG580.
• PHARMACOGNOSY AND PHYTOCHEMISTRY: A COMPREHENSIVE
APPROACH, EBOOK: S. L. DEORE, S. S. KHADABADI, B.A. BAVISKAR.
• GERARD A. MCKAY, JOHN L. REID, MATHEW R. WALTERS, "CLINICAL
PHARMACOLOGY & THERAPEUTICS", 8TH EDITION, 42PG.
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