The document provides information on respiratory and reproductive pharmacology. For respiratory pharmacology, it discusses animal models used in in-vitro and in-vivo tests, including histamine receptor binding and the effects of arachidonic acid. For reproductive pharmacology, it discusses animal models used to study mineralocorticoid activity through electrolyte excretion tests and progestational activity through progesterone receptor binding assays. The document provides details on the procedures, evaluations, and modifications of these pharmacology tests in respiratory and reproductive systems.
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
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
Preclinical Screening of Antiasthmatic DrugsShubham Kolge
Bronchial asthma is characterized by both bronchoconstriction and airway inflammation which leads to bronchial hyperresponsiveness to various stimuli. Different mediators are implicated in asthma. As the precise etiology is not known and multiple biochemical processes are triggered by different causative factors, it is difficult to have a single drug which can effectively and simultaneously act upon different mediators. This led to an intense search for potent and safe antiasthmatic drugs. This presentation intends to compile different screening methods for the evaluation of new candidate drugs with potential for the treatment of asthma. These include in vitro, in vivo, receptor binding and enzymatic methods.
In-Vitro and In-Vivo Assessment of Anti-Asthmatic Activity of Polyherbal Ayur...IOSR Journals
About 80% of asthmatic turn to alternative or complementary therapies typically in conjunction with their regular allopathic medication. The role of complementary and alternative medicine in adult asthma treatment is limited because these approaches have been insufficiently researched and their effectiveness is largely unproven. In the present study in –vivo and in-vitro effectiveness of a polyherbal Ayurvedic drug is evaluated for its anti-asthmatic activity. For in –vitro assessment of anti-asthmatic property of drug antiinflammatory, analgesic, immunomodulator effect, and antihistaminic, anti-cholinergic, mast cell stabilizing activity, anti-anaphylactic activity and bronchodilator effect were screen on animal models. Evaluation of Effect of Drug Distribution on Lung Mechanics is also evaluated using MATLAB. In a randomised,open, placebo controlled trial the effects of drug was compared with placebo medication (normal saline) in 60 adults with mild to moderate asthma as an adjunct to conventional treatment. Animal studies showed that drug possess significant mast cell stabilizing activity, immunomodulator activity, bronchodilator activity and anti-anaphylactic activity. Insignificant anti-cholinergic activity was found in the drug. There was significant improvement found in pulmonary function test (including FEV1, FVC and PEFR)in the group treated with polyherbal drug .Improvement remain constant in consecutive follow-ups signifies that there is no reverse bronchoconstriction after discontinuation of drug. This study signifies that polyherbal drug (Shirishadi ) may prove beneficial future alternative remedy for asthma and its effect is similar to that of modern contemporary drug when given through nasal route.
This presentation is on the bioassay of heparin which helps to know the potency of new heparin drug or heparin conc in individual suffering from heparin resistance diseases.
This was made by my friend Naailah and me. Hope it helps.
Ameliorating Effect of Frankincense on Red Blood Cells of Alloxan Induced-Dia...inventionjournals
International Journal of Pharmaceutical Science Invention (IJPSI) is an international journal intended for professionals and researchers in all fields of Pahrmaceutical Science. IJPSI publishes research articles and reviews within the whole field Pharmacy and Pharmaceutical Science, new teaching methods, assessment, validation and the impact of new technologies and it will continue to provide information on the latest trends and developments in this ever-expanding subject. The publications of papers are selected through double peer reviewed to ensure originality, relevance, and readability. The articles published in our journal can be accessed online.
Preclinical Screening of Antiasthmatic DrugsShubham Kolge
Bronchial asthma is characterized by both bronchoconstriction and airway inflammation which leads to bronchial hyperresponsiveness to various stimuli. Different mediators are implicated in asthma. As the precise etiology is not known and multiple biochemical processes are triggered by different causative factors, it is difficult to have a single drug which can effectively and simultaneously act upon different mediators. This led to an intense search for potent and safe antiasthmatic drugs. This presentation intends to compile different screening methods for the evaluation of new candidate drugs with potential for the treatment of asthma. These include in vitro, in vivo, receptor binding and enzymatic methods.
In-Vitro and In-Vivo Assessment of Anti-Asthmatic Activity of Polyherbal Ayur...IOSR Journals
About 80% of asthmatic turn to alternative or complementary therapies typically in conjunction with their regular allopathic medication. The role of complementary and alternative medicine in adult asthma treatment is limited because these approaches have been insufficiently researched and their effectiveness is largely unproven. In the present study in –vivo and in-vitro effectiveness of a polyherbal Ayurvedic drug is evaluated for its anti-asthmatic activity. For in –vitro assessment of anti-asthmatic property of drug antiinflammatory, analgesic, immunomodulator effect, and antihistaminic, anti-cholinergic, mast cell stabilizing activity, anti-anaphylactic activity and bronchodilator effect were screen on animal models. Evaluation of Effect of Drug Distribution on Lung Mechanics is also evaluated using MATLAB. In a randomised,open, placebo controlled trial the effects of drug was compared with placebo medication (normal saline) in 60 adults with mild to moderate asthma as an adjunct to conventional treatment. Animal studies showed that drug possess significant mast cell stabilizing activity, immunomodulator activity, bronchodilator activity and anti-anaphylactic activity. Insignificant anti-cholinergic activity was found in the drug. There was significant improvement found in pulmonary function test (including FEV1, FVC and PEFR)in the group treated with polyherbal drug .Improvement remain constant in consecutive follow-ups signifies that there is no reverse bronchoconstriction after discontinuation of drug. This study signifies that polyherbal drug (Shirishadi ) may prove beneficial future alternative remedy for asthma and its effect is similar to that of modern contemporary drug when given through nasal route.
This presentation is on the bioassay of heparin which helps to know the potency of new heparin drug or heparin conc in individual suffering from heparin resistance diseases.
This was made by my friend Naailah and me. Hope it helps.
Ameliorating Effect of Frankincense on Red Blood Cells of Alloxan Induced-Dia...inventionjournals
International Journal of Pharmaceutical Science Invention (IJPSI) is an international journal intended for professionals and researchers in all fields of Pahrmaceutical Science. IJPSI publishes research articles and reviews within the whole field Pharmacy and Pharmaceutical Science, new teaching methods, assessment, validation and the impact of new technologies and it will continue to provide information on the latest trends and developments in this ever-expanding subject. The publications of papers are selected through double peer reviewed to ensure originality, relevance, and readability. The articles published in our journal can be accessed online.
The kidneys play a vital role in the excretion of waste products and toxins such as urea, creatinine and uric acid, regulation of extracellular fluid volume, serum osmolality and electrolyte concentrations, as well as the production of hormones like erythropoietin and 1,25 dihydroxy vitamin D and renin.
Specimen collection requirements are dependent on the procedure or test requested. Generally, for serum creatinine and blood urea nitrogen (BUN) levels, no additional patient preparation is required, and a random blood sample suffices. However, the effect of recent high protein ingestion may increase serum creatinine and urea levels to a significant extent. Also, hydration status can have a considerable impact on BUN measurement.
For timed urine collections such as the 24-hour urine creatinine clearance, it is essential that urine be collected accurately over the required period as under or over collection will affect final results. Hence, a 5 to 8-hour timed collection is preferable to a 24-hour collection.
There are several clinical laboratory tests that are useful in investigating and evaluating kidney function. Clinically, the most practical tests to assess renal function is to get an estimate of the glomerular filtration rate (GFR) and to check for proteinuria (albuminuria).
Tests of renal function can be used to assess overall renal function by direct measurement or estimation of the glomerular filtration rate. Estimation of the GFR is utilized to determine the presence of renal impairment.
formulation and evaluation of insulin loaded nanoparticles.pptxTenzinPema20
Formulation of insulin nanoparticles for treatment of diabetes.
The action was sustained for a long period of time around 5 hrs as compared to 2 hrs in case of parenteral insulin administration.
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
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.
Ureases (EC 3.5.1.5), functionally, belong to the superfamily of amidohydrolases and phosphodiesterase.
Nickel containing metalloenzyme.
Ureases are found in numerous bacteria, fungi, algae, plants, and some invertebrates, as well as in soils, as a soil enzyme.
Not synthesized by animals.
James B. Sumner in 1926, Noble Prize in Chemistry in 1946.
Urease catalyzes the hydrolysis of urea to from ammonia and
Carbon dioxide
Similar to respiratory and reproduction pharmacology (20)
The Roman Empire A Historical Colossus.pdfkaushalkr1407
The Roman Empire, a vast and enduring power, stands as one of history's most remarkable civilizations, leaving an indelible imprint on the world. It emerged from the Roman Republic, transitioning into an imperial powerhouse under the leadership of Augustus Caesar in 27 BCE. This transformation marked the beginning of an era defined by unprecedented territorial expansion, architectural marvels, and profound cultural influence.
The empire's roots lie in the city of Rome, founded, according to legend, by Romulus in 753 BCE. Over centuries, Rome evolved from a small settlement to a formidable republic, characterized by a complex political system with elected officials and checks on power. However, internal strife, class conflicts, and military ambitions paved the way for the end of the Republic. Julius Caesar’s dictatorship and subsequent assassination in 44 BCE created a power vacuum, leading to a civil war. Octavian, later Augustus, emerged victorious, heralding the Roman Empire’s birth.
Under Augustus, the empire experienced the Pax Romana, a 200-year period of relative peace and stability. Augustus reformed the military, established efficient administrative systems, and initiated grand construction projects. The empire's borders expanded, encompassing territories from Britain to Egypt and from Spain to the Euphrates. Roman legions, renowned for their discipline and engineering prowess, secured and maintained these vast territories, building roads, fortifications, and cities that facilitated control and integration.
The Roman Empire’s society was hierarchical, with a rigid class system. At the top were the patricians, wealthy elites who held significant political power. Below them were the plebeians, free citizens with limited political influence, and the vast numbers of slaves who formed the backbone of the economy. The family unit was central, governed by the paterfamilias, the male head who held absolute authority.
Culturally, the Romans were eclectic, absorbing and adapting elements from the civilizations they encountered, particularly the Greeks. Roman art, literature, and philosophy reflected this synthesis, creating a rich cultural tapestry. Latin, the Roman language, became the lingua franca of the Western world, influencing numerous modern languages.
Roman architecture and engineering achievements were monumental. They perfected the arch, vault, and dome, constructing enduring structures like the Colosseum, Pantheon, and aqueducts. These engineering marvels not only showcased Roman ingenuity but also served practical purposes, from public entertainment to water supply.
Ethnobotany and Ethnopharmacology:
Ethnobotany in herbal drug evaluation,
Impact of Ethnobotany in traditional medicine,
New development in herbals,
Bio-prospecting tools for drug discovery,
Role of Ethnopharmacology in drug evaluation,
Reverse Pharmacology.
Palestine last event orientationfvgnh .pptxRaedMohamed3
An EFL lesson about the current events in Palestine. It is intended to be for intermediate students who wish to increase their listening skills through a short lesson in power point.
Welcome to TechSoup New Member Orientation and Q&A (May 2024).pdfTechSoup
In this webinar you will learn how your organization can access TechSoup's wide variety of product discount and donation programs. From hardware to software, we'll give you a tour of the tools available to help your nonprofit with productivity, collaboration, financial management, donor tracking, security, and more.
Unit 8 - Information and Communication Technology (Paper I).pdfThiyagu K
This slides describes the basic concepts of ICT, basics of Email, Emerging Technology and Digital Initiatives in Education. This presentations aligns with the UGC Paper I syllabus.
Operation “Blue Star” is the only event in the history of Independent India where the state went into war with its own people. Even after about 40 years it is not clear if it was culmination of states anger over people of the region, a political game of power or start of dictatorial chapter in the democratic setup.
The people of Punjab felt alienated from main stream due to denial of their just demands during a long democratic struggle since independence. As it happen all over the word, it led to militant struggle with great loss of lives of military, police and civilian personnel. Killing of Indira Gandhi and massacre of innocent Sikhs in Delhi and other India cities was also associated with this movement.
2024.06.01 Introducing a competency framework for languag learning materials ...Sandy Millin
http://sandymillin.wordpress.com/iateflwebinar2024
Published classroom materials form the basis of syllabuses, drive teacher professional development, and have a potentially huge influence on learners, teachers and education systems. All teachers also create their own materials, whether a few sentences on a blackboard, a highly-structured fully-realised online course, or anything in between. Despite this, the knowledge and skills needed to create effective language learning materials are rarely part of teacher training, and are mostly learnt by trial and error.
Knowledge and skills frameworks, generally called competency frameworks, for ELT teachers, trainers and managers have existed for a few years now. However, until I created one for my MA dissertation, there wasn’t one drawing together what we need to know and do to be able to effectively produce language learning materials.
This webinar will introduce you to my framework, highlighting the key competencies I identified from my research. It will also show how anybody involved in language teaching (any language, not just English!), teacher training, managing schools or developing language learning materials can benefit from using the framework.
Instructions for Submissions thorugh G- Classroom.pptxJheel Barad
This presentation provides a briefing on how to upload submissions and documents in Google Classroom. It was prepared as part of an orientation for new Sainik School in-service teacher trainees. As a training officer, my goal is to ensure that you are comfortable and proficient with this essential tool for managing assignments and fostering student engagement.
2. I N D E X
Respiratory pharmacology
1. Animal models for respiratory pharmacology
1. In-vitro test
Histamine(H1) receptor
1) In-vivo test
Effect of arachidonic acid or PAF
Reproduction pharmacology
1. Animal model for reproduction pharmacology
1) In-vivo test
Electrolyte excretion
2) In-vitro test
Progestational activity
3. RESPIRATORY PHARMACOLOGY
• It is a application of pharmacology to treatment of cardiopulmonary disease & critical area.
• It is used in treatment of respiratory disorders.
• There are several types of respiratory disease.
1. Asthma
2. Chronic bronchitis
3. Chronic obstructive pulmonary disease(COPD)
• Types of respiratory agent
1. Anti-asthmatic combination
2. Anti-histamines
3. Anti-tussive
4. Bronchodilators
5. Expectorants
6. Miscellaneous respiratory agents
7. Selective phosphodilesterase-4 inhibitors
4. ANIMAL MODELS IN RESPIRATORY PHARMACOLOGY
In-vitro test
1. Histamine (H1) receptor binding
PURPOSE & RATIONAL -
• Histamine consider to play a major role in asthmatic attacks.
• Used to determine the affinity of test compounds to the histamine H1 receptor by measuring
their inhibitory activities on the binding of the H1 antagonist 3H-pyrilamine to a plasma
membrane preparation from guinea pig brain.
PROCEDURE –
• Brain of guinea-pigs are homogenized in ice-cold tris buffer (pH 7.5) in a Potter homogenizer (1
g brain in 30 ml buffer).
• The homogenate is centrifuged at 4 °C for 10 min at 50 000 g.
• The supernatant is discarded, the pellet resuspended in buffer, centrifuged as before, and the
final pellets resuspended in Tris buffer (1 g fresh weight/5 ml).
5. • In the competition experiment, 50 μl 3H-pyrilamine (one constant concentration of 2×10–9 M), 50 μl
test compound (>10 concentrations, 10–5–10–10 M) and 100 μl membrane suspension from guinea pig
whole brain (approx. 10 mg wet weight/ml) per sample are incubated in a shaking bath at 25 °C for 30
min. Incubation buffer: 50 mM Tris-HCl buffer, pH 7.5.
• Total binding is determined in the presence of incubation buffer, non-specific binding is determined in
the presence of mepyramine or doxepin (10–5 M).
• The reaction is stopped by rapid vacuum filtration through glass fibre filters.
• Membrane-bound is separated from the free radioactivity and retained membrane-bound radioactivity
on the filter is measured after addition of 3 ml liquid scintillation cocktail per sample in a liquid
scintillation counter.
EVALUATION OF RESULT –
• The following parameters are calculated:
• total binding of 3H-pyrilamine
• non-specific binding: binding of 3H-pyrilamine in
• the presence of mepyramine or doxepin
• specific binding = total binding – non-specific binding
• % inhibition of 3H-pyrilamine binding:100 – specific binding as percentage of control value
6. • The dissociation constant (Ki) and the IC50 value of the test drug are determined from the
competition experiment of 3H-pyrilamine
In-vivo test
1. Effect of arachidonic acid or PAF
PURPOSE & RATIONAL –
• Arachidonic acid is metabolized into thromboxane (TXA2) and prostacyclin (PGI2).
• TXA2 produced in the lung (intracellularly in platelets induces a reversible thrombocytopenia)
leads to bronchoconstriction, which is independent from circulating platelets and leukotrienes.
• PGI2 produced in the vessel wall leads to the reduction of systolic and diastolic blood pressure.
• All three effects are inhibited by drugs which block cyclo-oxygenase.
• PAF as inducer leads to bronchoconstriction, which is platelet-dependent.
• In addition, PAF induces thrombocytopenia, leukocytopenia, reduction of blood pressure and
increase of hematocrit.
• These effects are reversible, but more persistent than arachidonic acid, and quickly result in
tachyphylaxis.
7. • The test allows to evaluate the sites of action of drugs, which interfere with the mechanisms of
broncho-constriction and thrombocytopenia in an in vivo-model.
PROCEDURE –
• Male guinea pigs (Pirbright White) weighing 300–600 g are anesthetized with 60 mg/kg
pentobarbital sodium (i.p.).
• One of the jugular veins is cannulated for the administration of spasmogenic and test compound.
• Both external carotid arteries are cannulated; one is connected to a pressure transducer to
register blood pressure, the other is used for blood withdrawal.
• The trachea is connected to a Starling pump with an inspiratory pressure set of 80 mm H2O.
• Spontaneous respiration is inhibited by intravenous injection of pancuronium (4 mg/kg).
• Excess air, not taken up by the lungs, is conducted to a transducer with broncho timer which
translates changes in air flow to an electrical signal.
• Animals receive multiple I.V. injections of the same dose of arachidonic acid (Sigma, 250–600
μg/kg prepared from a stock solution 10 mg/ml ethanol, 1 : 20 dilution with Na2CO3) until two
bronchospasms of equal intensity are obtained.
8. • The test compound is administered intravenously and the spasmogen is given again at the
following intervals: 2, 10, 20 and, if necessary, 30 min after administration of the drug.
• Before and 30–45 s after each of the arachidonic acid applications, approx. 50 μl blood are
collected into Na-EDTA-coated tubes.
• The number of thrombocytes is determined with a platelet analyzer in 10 μl samples of whole
blood.
• PAF (0.03–0.04 μg/kg in 0.9% saline + 0.1% human serum albumin) as inducer is injected
intravenously 60 min before and if necessary, 60 min after i.v. drug administration.
• Blood samples are collected 30 s before and 15 s after each of the PAF applications. The number
of leukocytes and hematocrit values are determined automatically.
• Standard compounds:
• dazoxiben HCl (inhibitor of thromboxane synthetase , TSI)
• acetylsalicylic acid- (inhibitor of cyclo-oxygenase , COI0)
EVALUATION –
• Percent inhibition or increase of bronchospasm, reduction of blood pressure, thrombocytopenia,
9. leukocytopenia and hematocrit following test drug administration are calculated in comparison to
control values before drug treatment.
• For the reduction of blood pressure, both the magnitude [mm Hg, systolic and diastolic] and the
duration [min] are determined.
• Even a sole increase in duration of blood pressure reduction is considered as an increase of the
effect.
• From the pattern profile of the influence on bronchoconstriction, thrombocytopeniamand blood
pressure reduction, the mechanism of action of a test drug is concluded:
• inhibitor of thromboxane synthetase
• inhibitor of cyclo-oxygenase
• other effect = no profile
• The inherent action of the test substance on blood pressure is determined before arachidonic acid-
or PAF-administration.
MODIFICATIONS OF THE METHODS –
• Kagoshima et al. (1997) used a modification of the Konzett-Rossler method to test the suppressive
effects of a PAF-antagonist on asthmatic responses in guinea pigs actively sensitized with
ovalbumin.
10. REPRODUCTIVE PHARMACOLOGY
• Reproductive pharmacology is an area which has flourished since the middle of the twentieth
century.
• Increasing specificity of treatment for the various disorders and discomfort of reproductive
system function has increased the quality of life the of many women and reduced health and
economic costs.
• Drugs used in treatment of reproductive disorders-
• Follicle stimulating hormone
• Human cholinergic gonadotropin
• Equine chronic gonadotropin
• Estradiol compound
• Progesterone and synthetic progesterone
• Testosterone
• Prostaglandins
11. ANIMAL MODELS IN REPRODUCTIVE PHARMACOLOGY
In-vivo method (Mineralocorticoids activity)
1. Electrolyte excretion
PURPOSE & RATIONAL –
• Mineralocorticosteroids enhance sodium retention and potassium excretion.
• The sodium excretion in adrenalectomized rats is dose-dependently decreased. This parameter
can be used for mineralocorticoid activity of test compounds (Kagawa et al. 1952).
PROCEDURE –
• Male Sprague-Dawley rats weighing 140–160 g are adrenalectomized. They are maintained on
1% NaCl solution as drinking fluid.
• Fourth day, food and drinking fluid are withdrawn. On the following day, each rat is given 5 ml
water by stomach tube; one hour later 5 ml 0.9% NaCl orally.
• Test compounds may be injected S.C. in 0.2 ml of vehicle suspension. The rats are lightly
anesthetized with ether to induce emptying of the bladder and placed in metabolic cages, 2 rats
per cage, for 4 h, again anesthetized with ether and removed from the cages.
12. • Urine volume is recorded and cages rinsed over the collection cylinders with a distilled water
spray. Collections are diluted to 100 ml and appropriate dilutions analyzed for sodium with a
flame photometer.
• Sodium is expressed as percent of excretion of control animals.
• Standard compound Desoxy-corticosterone acetate(1 and 40 μg per rat) is used.
EVALUATION –
• Percent reduction of sodium excretion compared with controls is calculated for each dosage group.
• Dose response curves are compared with the dose-response curve of deoxycorticosterone acetate to
calculate potency ratios.
MODIFICATIONS OF THE METHOD –
• Simpson and Tait (1952) measured both urinary sodium and potassium and used the sodium-to-
potassium ratio as an index of electrolyte activity of corticoids.
• Nikisch et al. (1991) infused glucocorticoid-substituted adrenalectomized rats with saline-glucose
solution containing aldosterone and measured sodium and potassium concentrations in 1 h
fractions of urine.
13. In-vitro method(Progestational activity)
1. Gestagen activity binding
PURPOSE & RATIONAL –
• Progesterone receptor may be obtained from uterine tissue or cultured cells.
• Ligands used
1) Uteri from estrogen primed rabbits (Ojasoo and Raynaud 1978; Boonkasemsanti et al. 1989; Phillips et
al. 1990; Cook et al. 1992),
2) castrated and estrogen treated mice or rats (Philibert and Raynaud 1977; Li et al. 1997),
3) MCF-7 cells derived from human breast tumour (Bergink et al. 1983; Kloosterboer et al. 1988a,b;
Kloosterboer et al. 1994),
4) breast cancer T47D cells (Meyer et al. 1990),
5) the quail fibroblast cell line QT6 (Schowalter et al. 1991) or human uteri obtained after hysterectomy
(Jänne et al. 1976; Pollow et al. 1989a,b, 1992),
6) Tritium labelled progesterone or R 5 020 ([6,7-3H]17,21-dimethyl-19- nor-pregna-4,9-diene-3,20-dione).
14. PROCEDURE –
• Relative binding affinities, Human uteri obtained after hysterectomy are snap frozen in liquid nitrogen
and stored at –80 °C until use.
• For cytosol preparations, uterine tissues are minced and homogenized with an Ultra-Turrax at 0–4 °C
in ice-cold buffer composed of 10 mM KH2PO4, 10 mM K2HPO4, 1.5 mM EDTA, 3 mM NaN3, 10%
glycerol, pH 7.5 (PENG buffer).
• Homogenates are then centrifuged at 105 000 g at 4 °C for 30 min.
• The supernatant is taken as cytosol. The cytosol preparations are incubated with 3H-R 5 020 as
radioligand at a concentration of 8 nmol/l and increasing concentrations (1 × 10–10 to 1 × 10–5 mol/l) of
the competitor steroids overnight at 4 °C.
• Then, unbound steroids are adsorbed by incubating with 0.5 ml of DCC (0.5% Norit A, 0.05% dextran
T400 in PENG buffer) for 10 min at 4 °C. After centrifugation (10 min at 1500 g at 4 °C) 0.5 ml of the
supernatant is withdrawn and counted for radioactivity.
EVALUATION –
• For calculation of relative binding affinity, the percentage of radioligand bound in the presence of
competitor compared to that bound in its absence is plotted against the concentration of unlabeled
steroid.
15. • A standard curve for the unlabeled radioligand (progesterone) is constructed with of 9–10
concentrations; 5 or 6 concentrations of the competitor are tested.
• The ratio of unlabeled radioligand and competitor for 50% competition multiplied by 100 is
calculated for relative binding affinity.
• Association rate (k+1) is calculated by the slope of the line
k+1 t = (2.3 / E0 – R0) log (E R0 / R E0)
where, E0 and E represent free steroid,
R0 and R free receptor at time t = 0 and time t, respectively.
• Dissociation rate (k–1) is calculated from the slope of the line
k–1= –2.3 log B / B0
where, B0 and B represent bound steroid at time t = 0 and time t, respectively.
MODIFICATIONS OF THE METHOD –
• The binding of the progesterone agonist R 5 020 and of the progesterone antagonist RU486 to the
progesterone receptor from calf uterus was characterized by Hurd and Moudgil (1988).
16. • A high affinity ligand and novel photoaffinity labeling reagent for the progesterone receptor
([3H]DU41 165) was described by Pinney et al. (1990).
• Different DNA-binding properties of the calf uterine estrogen and progesterone receptors were
explained by different dimerization constants (Skafar 1991).
• The complete amino acid sequence of the human progesterone receptor has been deduced from
cloned cDNA by Misrani et al. (1987).
• Allan et al. (1992) studied conformational changes in the ligand binding domain induced by
various progestins and antiprogestins.
• Comparative pharmacology of newer progestogens has been reviewed by Kuhl (1996).