This document discusses various in vitro and in vivo models for screening diuretic agents. It describes the Lipschitz test for evaluating diuretic activity in rats by measuring urine excretion and sodium content compared to a urea control. It also discusses methods to evaluate saluretic activity in rats by analyzing urine electrolytes and ratios. Clearance methods to assess renal function and site of action are described using conscious dogs under water diuresis and hydropenia conditions. The stop flow technique is explained to study transport along the nephron by clamping the ureter and collecting sequential urine samples.
In this slide contains diabetics, classification, symptoms, complication, invivo and invitro screening models of anti diabetics.
Presented by: GEETHANJALI ADAPALA (Department of pharmacology).
RIPER, anantapur
PRECLINICAL SCREENING MODELS
In vitro methods
• Patch clamp technique in kidney cells
• Perfusion of isolated kidney tubules
• Isolated perfused kidney
In vivo methods
• Diuretic activity in rats (LIPSCHITZ test)
• Saluretic activity in rats
• Diuretic and saluretic activity in dogs
• Clearance methods
• Micro puncture techniques in the rat
• Stop-flow technique
In this slide contains diabetics, classification, symptoms, complication, invivo and invitro screening models of anti diabetics.
Presented by: GEETHANJALI ADAPALA (Department of pharmacology).
RIPER, anantapur
PRECLINICAL SCREENING MODELS
In vitro methods
• Patch clamp technique in kidney cells
• Perfusion of isolated kidney tubules
• Isolated perfused kidney
In vivo methods
• Diuretic activity in rats (LIPSCHITZ test)
• Saluretic activity in rats
• Diuretic and saluretic activity in dogs
• Clearance methods
• Micro puncture techniques in the rat
• Stop-flow technique
In-vitro and in-vivo methods of diuretics & antihypertensive final.pptxAishwaryaPatil697206
This ppt contains in-vitro and in-vivo preclinical methods of diuretics and antihypertensive drugs. It contains classification as well as mechanism of action.
DIURETICS
Diuertics are the drugs used to increase the urine output by excretion of Na+ and water from the kidney.
Primary effect: Reduce absorption of sodium and chlorine ions from the filtrate.
Secondary effect: Increased water loss along with the excretion of sodium and chlorine.
CLASSIFICATION
Based on mechanism of action and site of action
Acting on PCT
a. Carbonic anhydrase inhibitor
Acetazolamide
Dorazolamide
Metazolamide
b. Xanthine derivative
Aminophylline
Theophylline
2. Act on loop of Henlee
a. Osmotic Diuretic
Mannitol
Glycerin
Urea
b. Loop diuretic/ High ceiling
Furosemide
Torsemide
Ethacrynic acid
3. Drug acting on DCT
a. Thaizide diuretic
Chlorthiazide
Hydrochlorthiazide
Hydroflumethazide
Bendroflumethazide
Benzthiazide
Cyclopenthiazide
b. Thiazide like diuretic
Chlorthalidone
Indapamide
Metolazine
4. Drugs acting on collecting duct
a. Aldosterone antagonist
Spironolactone
b. Directly acting
Amiloride
Triamterine
Major application of diuretics ;
Used in congestive heart failure
Essential hypertension
Acute and chronic heart failure
Currently used screening methods are based on effect of drug on water and electrolyte metabolism in rats.
SCREENING METHODS
IN VIVO METHODS :
Diuretic activity in rats [LIPSCHITZ TEST]
Saluretic and diuretic activity in dogs
Saluretic activity in rats
Clearance methods
Stop flow technique
Micro puncture technique in rat.
IN VITRO METHODS :
Carbonic anhydrase inhibition in vitro
Patch clamp technique in kidney cells
Isolated perfused kidney
Perfusion of isolated kidney tubules
1. CARBONIC ANHYDRASE INHIBITION IN-VITRO
PURPOSE AND RATIONALE
Carbonic anhydrase is a Zn containing enzyme.
H2CO3 H20+CO2
Inhibition of CARBONIC anhydrase in PCT causes
● Decreased H+ ion formation
● Decreased Na+/H+ antiport
●Increased Na+and HCO3- in lumen
●increased excretion of Na+HCO3-
●Increased production of alkaline urine
PROCEDURE
The analytical method is based on the catalysis of the conversion of CO2 to H2CO3 by the enzyme , with resulting decrease in pH being monitored colorimetrically.
ASSAY
■ CO2 flow rate is adjusted to 30 to 45 ml/min
■ 400 µl phenol red indicator solution
■ 100µ l enzyme.
■ 200 µl H2O or appropriate drug concentration after 3min of equilibriation.
■ 100 µl carbonate/bicarbonate buffer is added.
■ The following parameters are determined in duplicate samples :
Tu = [uncatalysed time]=time for the colour change to occur in the absence of enzyme.
Te =[Catalysed time]=time for the colour change to occur in presence of enzyme.
Tu – Te = enzyme rate
Ti = enzyme rate in the presence of various concentrations of inhibitor.
EVALUATION
Percentage inhibition of carbonic anhydrase is evaluated
% evaluation =1
Each kidney contains over 1 million tiny structures called nephrons. Each nephron has a glomerulus, the site of blood filtration. The glomerulus is a network of capillaries surrounded by a cuplike structure, the glomerular capsule (or Bowman’s capsule). As blood flows through the glomerulus, blood pressure pushes water and solutes from the capillaries into the capsule through a filtration membrane. This glomerular filtration begins the urine formation process.Inside the glomerulus, blood pressure pushes fluid from capillaries into the glomerular capsule through a specialized layer of cells. This layer, the filtration membrane, allows water and small solutes to pass but blocks blood cells and large proteins. Those components remain in the bloodstream. The filtrate (the fluid that has passed through the membrane) flows from the glomerular capsule further into the nephron.The glomerulus filters water and small solutes out of the bloodstream. The resulting filtrate contains waste, but also other substances the body needs: essential ions, glucose, amino acids, and smaller proteins. When the filtrate exits the glomerulus, it flows into a duct in the nephron called the renal tubule. As it moves, the needed substances and some water are reabsorbed through the tube wall into adjacent capillaries. This reabsorption of vital nutrients from the filtrate is the second step in urine creation.The filtrate absorbed in the glomerulus flows through the renal tubule, where nutrients and water are reabsorbed into capillaries. At the same time, waste ions and hydrogen ions pass from the capillaries into the renal tubule. This process is called secretion. The secreted ions combine with the remaining filtrate and become urine. The urine flows out of the nephron tubule into a collecting duct. It passes out of the kidney through the renal pelvis, into the ureter, and down to the bladder.The nephrons of the kidneys process blood and create urine through a process of filtration, reabsorption, and secretion. Urine is about 95% water and 5% waste products. Nitrogenous wastes excreted in urine include urea, creatinine, ammonia, and uric acid. Ions such as sodium, potassium, hydrogen, and calcium are also excreted
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New Directions in Targeted Therapeutic Approaches for Older Adults With Mantl...i3 Health
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Mantle cell lymphoma (MCL) is a rare, aggressive B-cell non-Hodgkin lymphoma (NHL) accounting for 5% to 7% of all lymphomas. Its prognosis ranges from indolent disease that does not require treatment for years to very aggressive disease, which is associated with poor survival (Silkenstedt et al, 2021). Typically, MCL is diagnosed at advanced stage and in older patients who cannot tolerate intensive therapy (NCCN, 2022). Although recent advances have slightly increased remission rates, recurrence and relapse remain very common, leading to a median overall survival between 3 and 6 years (LLS, 2021). Though there are several effective options, progress is still needed towards establishing an accepted frontline approach for MCL (Castellino et al, 2022). Treatment selection and management of MCL are complicated by the heterogeneity of prognosis, advanced age and comorbidities of patients, and lack of an established standard approach for treatment, making it vital that clinicians be familiar with the latest research and advances in this area. In this activity chaired by Michael Wang, MD, Professor in the Department of Lymphoma & Myeloma at MD Anderson Cancer Center, expert faculty will discuss prognostic factors informing treatment, the promising results of recent trials in new therapeutic approaches, and the implications of treatment resistance in therapeutic selection for MCL.
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Knee anatomy and clinical tests 2024.pdfvimalpl1234
This includes all relevant anatomy and clinical tests compiled from standard textbooks, Campbell,netter etc..It is comprehensive and best suited for orthopaedicians and orthopaedic residents.
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
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.
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
8.
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.