animal models for respiratory and reproduction pharmacology.

1. RESPIRATORY AND REPRODUCTION
PHARMACOLOGY
BADDI UNIVERSITY OF EMERGING SCIENCE AND TECHNOLOGY
PRAJJWAL
M.PHARMA(1ST SEM)
PHARMACOLOGY

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).

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