Screening of Diuretics In Vivo- In Vitro Methods

1. PRESENTED BY
GANGURDE S.K.
Roll No-06
( M.Pharm-Sem I)
(Pharmacology)
UNDER GUIDANCE
Prof. PAGAR H.J.
M.Pharm.
Pharmacology
DR.VITHALRAO VIKHE PATIL FOUNDATION’S
COLLEGE OF PHARMACY, AHMEDNAGAR.2018-19
1

2. CONTENTS
 Introduction.
 Classification.
 Screening Methods for Diuretics: -
1. In-Vitro Models.
2. In-Vivo Models.
 References.

3. INTRODUCTION
 Increase the rate & flow of urine.
 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 of
salt or especially sodium ion.

5. Evaluation models
In-Vitro Models
 Isolated tubule preparation.
 Carbonic anhydrase inhibition.
 Patch clamp technique.

6.  Diuretic Activity in Rat (Lipschitz test).
 Saluretic activity in rats.
 Diuretic & Saluretic Activity in Dog.
 Stop flow technique.
 Clearance method.
 Micropuncture technique.

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

9. Methodology: -
 Species - Male Wistar rats (100–200 gm).
 Sex - Male rats.
 Three animals in each group both in test and control (4 Groups).
Procedure: -
 3 animals per group 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 17-24 hrs prior to the experiment food and water is withdrawn.

10. 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.

11. 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
& analyzed

12. SALURETIC ACTIVITY IN RATS
PURPOSE AND RATIONALE :
➢ Diuresis 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

13. METHODOLOGY: -
 Species-Wistar rats (100-200g).
 Sex – Male.
 Three animals in each group one group acts as a test & another one as a control.
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 upto 5 hrs.

14. Continue…
 The 5 hr urine is analyzed by flame photometry for Na & K+ & by
Argentometry for chloride.
 To evaluate compounds with prolonged effect 24 hr urine is analyzed.
 Furosemide(25mg/kg) or Hydrochlorthiazide(25mg/kg) is used as
standard.
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.

15. CLEARANCE METHOD
PRINCIPLE :
➢ Method for evaluation of renal function & provide information about site of
action of diuretics.
➢ A drug that acts solely in the proximal convoluted tubule, by causing the
delivery of the increased amounts of filtrate to the loop of Henle and the distal
convolution, would augment the clearance of solute free water (CH2O) during
water diuresis and the reabsorption of solute free water (TCH2O) during water
restriction.
➢ The drugs that inhibits sodium reabsorption in Henle`s loop would impair
both CH2O and TCH2O
➢ Drugs that act only in distal tubule would reduce CH2O but not TCH2O

16. PROCEDURE
 Clearance experiments are performed either in conscious or anaesthetized beagle
dogs under conditions of water diuresis and hydropenia.
 Water diuresis is induced by oral administration of 50 ml of water per kg body
weight and maintained by continuous infusion into jugular vein of 2.5% glucose
solution and 0.58% NaCl solution at 0.5 ml/min per kg body weight.
When water diuresis is well established, the glucose infusion is discontinued and
control urine samples are collected by urethral catheter.
 Blood samples are obtained in the middle of each clearance period. After the
control period, compounds to be tested are administered and further clearance tests
are performed.
 Hydropenia is induced by withdrawing the drinking water 48 h before
experiment.
 On the day before the experiment 0.5 U/kg body weight of vasopressin in oil is
injected intramuscularly.
 On the day of the experiment 20 mU/kg vasopressin is injected i.v., followed by
infusion of 50 mU/kg per hour vasopressin.

17.  To accomplish constant urine flow 5% NaCl solution is infused at 1 ml/min
per kg body weight up to i.v. administration of a compound to be tested,
followed by i.v. infusion of 0.9% NaCl solution at a rate equal to the urine
flow.
 Glomerular filtration rate (GFR) and renal plasma flow (RPF) are measured
by the clearance of inulin and para-aminohippurate, respectively.
 Therefore, appropriate infusion of inulin and para-aminohippurate are
initiated.
 Inulin and para-aminohippurate are measured.

18. EVALUATION
 The following parameters may be determined: water and
electrolyte excretion, GFR, RPF, CH2O, TCH2O and plasma
rennin activity.
 Results of test compound are compared statistically with
control and standard drug treated animals.

19. STOP FLOW TECHNIQUE
Purpose and Rationale
 This procedure is of considerable value in the localization of transport processes
along the length of the nephron.
 During clamping of the ureter, glomerular filtration is grossly reduced.
 The contact time for the tubular fluid in the respective nephron segments increases,
and the concentration of the constituents of tubular fluid should approximate the
static-head situation.
 After release of the clamp, the rapid passage of the tubular fluid should modify the
composition of the fluid only slightly.
 The first samples should correspond to the distal nephron segment, the latest to
glomerular fluid.
 However, with introduction of the micropuncture technique, the stop-flow method
appears less attractive.

20. PROCEDURE
 This test can be performed on different animals.
 The ureter of an animal undergoing intense osmotic diuresis is clamped for several
minutes allowing a relatively static column of urine to remain in contact with the
various tubular segments for longer than the usual periods of time.
 Thus, the operation of each segment on the tubular fluid is exaggerated.
 Then the clamp is released, and the urine is sampled sequentially.
 Small serial samples are collected rapidly, the earliest sample representing fluid
which had been in contact with the most distal nephron segment.
 Substances examined are administered along with inulin before the application of
urethral occlusion.
 However, tubular segments downstream from the proximal segments may modify
the tubular fluid during its egress.

21. EVALUATION
 Each sample the concentration of a glomerular marker, such as
inulin, and the concentration of the under study are measured.
 Fractional excretion of the substance and the glomerular marker
are plotted versus the cumulative urinary volume.

22. REFERENCES
 H. Gerhard Vogel “Drug Discovery and Evaluation”
,Pharmacological Assays springer publication London , 2nd
edition ,2002, pg (316-331).
 http://www.pharmatutor.org/articles/screening-diuretic-agents-
overview
Gerard A. Mckay, John L. Reid, Mathew R. Walters, “Clinical
Pharmacology & therapeutics”, 8th Edition,42pg.
K.D.Tripathi, “Essential of Medical Pharmacology”,6th edition,
pg580.