SCREENING OF DIURETICS_075702.pptx presentation
- 1. SCREENING OF DIURETICS PRESENTER : DR C R SUHAAS MODERATOR : DR EESHA B RAO 1
- 2. CONTENT 1. Introduction 2. Types of Diuretics 3. Models used to evaluate Diuretic agents 4. IN-VITRO models 1. Carbonic anhydrase inhibition 2. Patch clamp technique 3. Isolated Tubule Preparation 5. IN-VIVO models 1. Lipschitz test 2. Saluretic activity in rats 3. Stop flow technique 4. Micropuncture Technique 2
- 3. INTRODUCTION Diuretics are pharmacological agents that cause loss and Na and water Saluretics are agents that facilitate the removal of salt Natriuretics are agents that facilitate the removal of mainly Na+ 3
- 4. TYPES OF DIURETICS CLASS SITE OF ACTION EXAMPLES Osmotic diuretics Lumen of the Nephron Mannitol Carbonic anhydrase inhibitors Proximal tubule Acetazolamide, dorzolamide Loop diuretics Thick ascending limb of loop of henle Furosemide, Torsemide Thiazide diuretics Early distal tubule Hydrochlorthiazide, chlorthalidone Potassium sparing diuretics Collecting duct Amiloride, Spironolactone 4
- 5. MODELS USED TO EVALUATE DIURETIC AGENTS IN VITRO 1. Carbonic anhydrase inhibition 2. Isolated tubule preparation 3. Patch clamp technique IN VIVO 1. Lipschitz test 2. Saluretic activity in rats 3. Stop flow technique 4. Clearance method 5. Micro-puncture technique 5
- 6. Carbonic Anhydrase Inhibition PRINCIPLE: Carbonic anhydrase is a Zn containing enzyme By inhibiting the action of carbonic anhydrase, these inhibitors prevent the reabsorption of salt, bicarbonate and water PROCEDURE: Analytic method based on the catalysis of the conversion of CO2 to H2CO3 by the enzyme with resulting decrease in pH being monitored calorimetrically. 6
- 7. Carbonic Anhydrase Inhibition (contd…) MATERIALS: 1. Phenol red indicator 2. 1 M Sodium bicarbonate buffer (pH 9.8) 3. Enzyme carbonic anhydrase from dog blood 4. Reaction vessel ASSAY: CO2 is allowed to flow at a rate of 30-45mL/min Phenol red indicator, enzyme and buffer are added to the reaction vessel 7
- 8. Carbonic Anhydrase Inhibition (contd…) Tu (uncatalysed time) time for colour change to occur in absence of the enzyme Te (catalysed time) time for colour change to occur in presence of the enzyme Enzyme rate = Tu – Te Ti Enzyme rate in the presence of various concentrations of inhibitors Evaluation: %𝑖𝑛ℎ𝑖𝑏𝑖𝑡𝑖𝑜𝑛 = 1 − 𝑇𝑢−𝑇𝑒 − 𝑇𝑖 −𝑇𝑐 𝑇𝑢−𝑇𝑐 𝑥 100 8
- 9. Patch Clamp technique PRINCIPLE: The technique allows the study of ion channels It involves the use of patch of electrodes with reasonably big tip (>1mm) and a smooth surface Various technique models for a patch clamp as cell-attached mode, cell- excised mode and whole-cell mode PROCEDURE: Patch clamp electrode is pressed against a cell membrane and suction is applied to pull the cell membrane inside the electrode tip Suction causes the cell to form a tight, high resistance seal with the rim of the electrode (gigaseal) 9
- 10. Patch Clamp technique (contd…) Cell-attached mode Patch electrode remains sealed to the cell membrane, permitting the recording of currents through single ion channels from the patch of membrane surrounded by the tip of electrode Whole-cell mode To the cell-attached configuration, additional suction is applied to rupture the cell membrane, thus providing access to the intracellular space of the cell Soluble contents of the cell are replaced by those of the electrode It records current through all channels from the entire cell membrane at once 10
- 11. Patch Clamp technique (contd…) EVALUATION: Concentration response curve of the drugs which inhibits ion channels can be obtained Single ion channels can be studied by cell-attached and cell-excised techniques Co-transport system can only be studied by whole-cell patch clamp technique 11
- 12. Isolated tubule Reabsorption PRINCIPLE: Measurement of change in concentration of solutes in perfusion fluid It is the preferred strategy if the target location and mechanism of the diuretic are to be determined PROCEDURE: Technique can be used in the kidney segments of Wrister Albino Rat, mouse, hamster, rabbit, etc A thin (1mm) kidney tubule fragment is extracted and then put through a development assembly A micropipette hole is drilled into a suitable tubule end to allow for perfusion 12
- 13. Isolated tubule Reabsoprption (contd…) The other end of the tubule is sucked into the collecting pipette The oil inside the collecting pipette prevents evaporation The accumulated fluid is collected at periodic intervals by inserting a narrow calibrated pipette in the collecting pipette To approximate the in-vivo situation, an isotonic rabbit serum is perfused while the tubule is immersed in a bath of rabbit serum EVALUATION: The absolute volume of reabsorption is determined from the change in the concentration of an impermeable marker like (3H)Inulin, (125I) isothalamate in the collecting fluid Leak around the perfusion pipette is detected from the appearance of the marker in external bath 13
- 14. IN-VIVO METHODS 1. Diuretic activity in Rats (LIPSCHITZ TEST) 2. Sal-uretic activity test in rats 3. Stop flow technique 4. Micro-puncture technique 14
- 15. Diuretic activity in rats (LIPSCHITZ TEST) PRINCIPLE: Based on water and Na+ excretion in test animal and compared to rats treated with standard drug (usually high dose of urea) PROCEDURE: Male Wister rats weighing 100-200g are selected They are divided into 3 groups Test (given test drug) Control (Given normal saline) Standard (given standard drug) 15
- 16. Diuretic activity in rats (LIPSCHITZ TEST) (contd…) The test group may be further divided into 3 groups with the dose given in accordance with acute toxicity studies (mild, moderate, severe) The Rats are placed in a metabolic cage Wired mesh at the bottom Funnel to collect urine Stainless steel sieve to retain faeces and allow only the urine to pass through Rats are fed a standard diet (Altromin pellets) and water 15 hours before the experiment food and water are withdrawn Urine excretion is recorded at 5 hrs and 24 hrs Na+ is estimated by flame photometry and urine volume is calculated for each group 16
- 17. Diuretic activity in rats (LIPSCHITZ TEST) (contd…) EVALUATION: Results are expressed in LIPSCHITZ values for both urine excretion and for electrolytes 𝐿𝐼𝑃𝑆𝐶𝐻𝐼𝑇𝑍 𝑉𝐴𝐿𝑈𝐸 = 𝑈𝑟𝑖𝑛𝑒 𝑜𝑢𝑡𝑝𝑢𝑡 𝑖𝑛 𝑡𝑒𝑠𝑡 𝑎𝑛𝑖𝑚𝑎𝑙 𝑈𝑟𝑖𝑛𝑒 𝑜𝑢𝑡𝑝𝑢𝑡 𝑖𝑛 𝑠𝑡𝑑 𝑑𝑟𝑢𝑔 𝑡𝑟𝑒𝑎𝑡𝑒𝑑 𝑎𝑛𝑖𝑚𝑎𝑙 LV >= 1 Positive effect LV >= 2 Potent diuretic activity For studying prolonged effect, 24hr urine sample is collected and analysed 17
- 18. Saluretic activity in Rats PRINCIPLE: Excretion of electrolytes is important for the treatment of peripheral edema, Congestive heart failure, Hypertension, etc,. So it’s important to develop diuretics with saluretic and K+ sparing effects Test in rats is designed to determine the Na+, K+, Cl-, water content and osmolarity of urine 18
- 19. Saluretic activity in Rats (contd…) PROCEDURE: Male Wister rats weighing 100-200g are fed with standard diet and water 15hrs prior to experiment food is withdrawn, but not water Animals are placed in a metabolic cage Urine excretion is measured ever hour upto 5 hours and the collected urine is analysed for Na+, K+ and Cl- Two groups of rats are used for test and standard drug Furosemide, hydrochlorthiazide, triamterene and amiloride are used as standards 19
- 20. Saluretic activity in Rats (contd…) EVALUATION: For Saluretic activity: Na+ + Cl- excreted is calculated For natriuretic activity: 𝑁𝑎 + 𝐾 + is calculated Natriuretic effect > 2 Potassium sparing effect > 10 For estimating Carbonic anhydrase inhibition: 𝐶𝑙 − 𝑁𝑎 + +𝐾 + is calculated CA Inhibition can be exclude at ratio between 1 to 0.8. With decreasing ratio slight to strong Carbonic anhydrase inhibitor can be assumed 20
- 21. Stop flow technique PRINCIPLE: Useful in localization of transport process along the length of the nephron Ureter is clamped, during which the GFR is grossly reduced Contact time for tubular fluid in respective nephron segment increases and concentration of the constituents of the tubular fluid approximate the static head situation After releasing clamp, rapid passage of the tubular fluid modify composition of tubular fluid only slightly Urine is sampled sequentially The initial samples correspond to the distal nephron segments, while the latest to the glomerular fluid 21
- 22. Stop flow technique (contd…) PROCEDURE: Performed in different animals during anaesthesia Ureter is clamped allowing static column of the urine to remain in contact with the tubular segments for longer than usual time period Clamp is released and urine is sampled sequentially Substances examined are administered along with inulin before the application of ureteral occlusion 22
- 23. Stop flow technique (contd…) EVALUATION: Concentration of inulin and the substance under study is measured in each sample Fractional excretion of substance and inulin are plotted against cumulative urine volume 23
- 24. Micropuncture Technique PRINCIPLE: Micropunture technique allows localization of tubular site of action Measure the change in tubular fluid reabsorption rates and electrolyte concentration and thereby infer mechanism of action PROCEDURE: Animals are selected according to the site where micropuncture is going to be performed 24 Bowmen’s capsule and Loop of Henle Rat PCT and DCT Dog and Rat Collecting Duct Rat and Hamster
- 25. Micropuncture Technique (Contd…) Rats weighing 250g are used for this procedure They are anaesthetized with intra-peritoneal injection of Thiopentone 15 hrs before the experiment food is stopped, but water is continued After anaesthetizing the rats, they are tracheotomized and then placed on a table with a thermostat Carotid artery and the jugular veins are cannulated to allow for BP measurement and blood sample collection The retroperitoneum can be accessed by a flank incision The area is then encapsulated in a tiny plastic item with cotton and then bathed in liquid paraffin oil at room temperature 25
- 26. Micropuncture Technique (Contd…) Body temperature is continuously monitored when cannula is put in the ureter After 3 hrs a single large dose of inulin is given as a bolus The 0.85% of NaCl solution is given at an flow rate of 2.5 mL/min and the dose is appropriately calculated as per 100g of body weight After 45mins of iv administration, a thin elongated channel begins to gradually leak Renal cortex and medulla intracellular liquid samples are directly collected using glass capillaries (8-10mm external diameter) A micromanipulator and microscopic observation is employed Lissamine green is administered intravenously to detect the distal tubules 26
- 27. Micropuncture Technique (Contd…) EVALUATION: The following parameters are evaluated Inulin Clearance (GFR) Single nephron GFR Fractional delivery of water, sodium and potassium concentration in proximal and distal renal tubules as well as in urine Fluid reabsorption is assessed by comparing the insulin concentration in the perfusate to that in the collected samples 27
- 28. Conclusion IN-VITRO METHODS: 1. Carbonic anhydrase inhibition: Inhibition of CA enzyme 2. Isolated Tubule Preparation: Measurement of change in concentration of solutes in the perfusion fluid 3. Patch clamp technique: Measurement of current across the ion channels IN-VIVO METHODS: 1. Lipschitz test: Measurement of urine volume and sodium excretion 2. Saluretic activity in rats: Measurement of electrolyte excretion 3. Stop flow technique: Allow urine to remain in the nephron for a long period of time and then collection of urine 4. Micropuncture technique: Measures the changes in tubular reabsorptive rates and electrolytes concentration 28
- 29. References 1. Goodman and Gillman, 2. Drug discovery and evaluation_Pharmacological Assay, 4th edition 3. Hirani R, Raval KY. A comparative review on In-vivo and In-vitro screening models for diuretic agents. IP Int J Comprehensive Adv Pharmacol 2023;8(2):86-90. 4. Drug screening methods, 3rd edition 29