This document summarizes animal models used in respiratory and reproductive pharmacology. For respiratory pharmacology, it describes in vitro tests including histamine receptor binding and effects on isolated organs. It also describes various in vivo tests in anesthetized guinea pigs to assess bronchospasmolytic activity. For reproduction pharmacology, it discusses models to evaluate inhibition of fertility and gonadotropin secretion, including suppressing organ weights in rats treated with steroids.

1. RESPIRATORY AND REPRODUCTION
PHARMACOLOGY
Baddi university of emerging science an
technology
Presented by: Manju Jakhar
M.Pharm(1ST Sem)
Pharmacology

2. CONTENT
 Respiratory pharmacology
1. Animal models for respiratory pharmacology
1.) In-vitro test:-
• Histamine(H1) receptor
• Effects on air ways:- Test in isolated organs
a) Spasmolytic activity in isolated guinea pig lung strips.
b) Spasmolytic activity in isolated trachea.
c) Bronchial perfusion of isolated lung.
d) Vascular and airway responses in the isolated lung.
e) Reactivity of the isolated perfused trachea.
2.) In-vivo test:-
• Bronchospas molytic activity in anesthetized guinea pigs. (Konzette - Rossler method)
• Effect of arachidonic acid or PAF on respiratory function in vivo.
• Bronchial hyper reactivity.
• Body plethysmography and respiratory parameters after histamine – induced
bronchoconstriction in anesthetized guinea pigs.
• Pneumotachography in anesthetized guinea pigs.
• Airway microvasular leakage.
• Isolated larynx in situ.
 Reproduction pharmacology:- Gonadotropin inhibition.
1.) Inhibition of fertility
2.) Inhibition of gonadotropin secretionin intact animals.

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 models:-
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 –
1.) 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).
2.) The homogenate is centrifuged at 4 °C for 10 min at 50 000 g. The supernatant is discarded.
3.) The pellet resuspended in buffer, centrifuged as before, and the final pellets resuspended in
Tris buffer (1 g fresh weight/5 ml).
4.) 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.

5. 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).
6.) The reaction is stopped by rapid vacuum filtration through glass fibre filters.
7.) 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
 In-vivo test models :-
1.) Bronchospasmolytic activity in anesthetized guinea pigs. (Konzette - Rossler method)
2.)Effect of arachidonic acid or PAF on respiratory function in vivo.
3.)Bronchial hyper reactivity.
4.)Body plethysmography and respiratory parameters after histamine – induced
bronchoconstriction in anesthetized guinea pigs.
5.)Pneumotachography in anesthetized guinea pigs.
6.)Airway microvasular leakage.
7.)Isolated larynx in situ.

6. 1. Bronchospasmolytic activity in anesthetized guinea pigs (Konzett-Rossler
method):-
PURPOSE –
 The method is based on registration of air volume changes of a living animal in a
closed system consisting of the respiration pump, of the trachea and the bronchi as
well as of a reservoir permitting measurement of volume or pressure of excess air.
Bronchospasm decreases the volume of inspired air and increases the volume of
excess air. Thus, the degree of bronchospasm can be quantified by recording the
volume of excess air. Administration of spasmogens like acetylcholine, histamine,
bradykinin, serotonin, ovalbumin, PAF, substance P, methacholine or leukotrienes,
results in contraction of bronchial smooth muscle.
 The method permits the evaluation of a drug’s bronchospasmolytic effect by
measuring the volume of air, which is not taken up by the lungs after bronchospasm.
PROCEDURE-
1.) Guinea-pigs of either sex weighing 250–500 g are anaesthetized with urethane.
2.) Anesthesia has to be deep enough in order to prevent influence of spontaneous respiration.
3.)The animal is artificially respired using a Starling pump with an inspiratory pressure of water.
4.) Excess air, not taken up by the lungs, is measured and recorded on a polygraph.
5.)The internal jugular vein is administration with spasmogens and test compounds.
6.)Spasmogens given by i.v administration are acetylcholine hydrochloride,methacholine, histamine
dihydrochloride , bradykinin triacetate , ovalbumin , PAF, leukotrienes , substance P

7. 7.) After obtaining two bronchospasms of equal intensity, test compounds are administered i.v., p.o.,
s.c. or intraduodenally.
8.) The spasmogen is given again at the following time intervals:
• 5, 15 and 30 min after i.v. administration.
• 15, 30 and 60 min after intraduodenal administration.
• 30 and 60 (sometimes also 120) min after p.o. administration.
9.) The standard compounds are used: -atropine sulfate , aminophylline, tolpropamine-
HCl,imipramine-HCl to inhibit spasmogens induced spasms.
EVALUATION:-
Results are expressed as percent inhibition of induced bronchospasm over the control agonistic
responses.The ED50 value is calculated.

8. 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

9. Animal models in reproductive pharmacology
1.) Inhibition of fertility :-
PURPOSE:-
 Fertility in rats can be inhibited by treatment of female with steroids inhibiting pituitary
gonadotropin secretion or competing at the progesterone receptor. The test is used for
contraceptive steroids like ethinylestradial (levonorgestral), Mestronal.
PROCEDURE :-
1.) A colony of about 100 adult female sprague dawley rats weighing between 200 and 250 is
used.
2.) Daily vaginal smeores are taken at noon for 5 days , fifteen regularly cycling females in
proesterus are selected and caged separately.
3.) The first drug dose is administered at 3:00pm (day1). At 5:00pm two vigorous males are
placed with each female.
4.) On day 2, vaginal smears are takes for sperm count at 8:00AM.
5.) The second drug dose is administered at 4:30PM.
6.) On day 3, vaginal smear are taken for sperm count at 8:00AM again.
7.) The third drug dose is administered and sperm counts are taken at 4:30PM and the males are
then removed.
8.) From the 4th to the 7th day the test drugs are administered in the morning. Controls receive
the vehicle only.

10. EVALUATION:-
Compounds which prevent conception observed by the absence of implantation sites at autopsy are
given further consideration. Minimal effective doses preventing ovulation in all animal of a test
group are determined.
2.) Inhibition of gonadotropin secretion in intact animals :-
PURPOSE:-
The young rats 80 -100g body weight are treated with gonadal steroids or their
derivatives.Suppression of gonadal weight increases indicates gonadotropin inhibition, whereas
weight increases of the sexual hormone dependent organs in castrate control groups (female or
male )indicates the estrogenic or andogenic property of the test compound.
PROCEDURE:-
1.) Immature male and female sprague- dawley rats weighing 80 – 100g are used.
2.) Groups of 10 animals per sex and dosage group are treated daily over a period of 21 days with
doses of test compound by oral or S.C. route.
3.) Controls receive the vehicle only. Testosterone may be used as standard in males and estradial in
females.
4.) After 24 hours, the animals are sacrificed and body weight are recorded.
5.) In males, testes, seminal vesicles, prostate and in females, ovaries and uterus are dissected out
and weighed.

11. EVALUATION:-
 Average organ weights are calculated for each treatment group and compared with
controls.The reference steroids testosterone and estradia, suppress gonadal weight by
pituitary gonadatropin inhibition and increase the weight of sex hormone dependent organs
by direct action on the tissues.
 Dose- response comes either pituitary inhibition or steroids action can be used for the test
compounds and the standard .The relative potency of gonadotropin inhibition can be
calculated.