Screening Models For Drugs Used in Asthma

1. SCREENING MODELS FOR
ANTI-ASTHMATICS AGENTS
Guided by:-
Dr. Saumya Das
Professor (H.O.D, Pharmacology)
NIET (Pharmacy Institute)
Presented by:-
Sanidhya Jain
M. Pharma (Pharmacology)
SEM- 1
NOIDA INSTITUTE OF ENGINEERING AND TECHNOLOGY
(PHARMACY INSTITUTE)

2. INTRODUCTION
“Hyper responsiveness of smooth muscles in respiratory tract is
termed as asthma.
It is the most common disabling syndrome.
Narrowing of air tube, increase in secretion, mucosal oedema and
mucus plugging are common symptoms.
Inflammation starts with “Mast cells” present in lungs which follows
up by the release of intracellular granules like “Histamine” and by the
release of phospholipids like “PGs”, “LTs”, “Interleukins” and “TNF-
alpha”.
Approaches for treatment:-
LTs receptor antagonist like Montelukast and Zafirlukast.
By using Mast cell stabilizer like Nedocromil, Ketotifen.
By using Anti-IgE antibody like Omalizumab.
By using “Bronchodilators” like Ephidrine, Theophylline, Tiotropium
etc.

3. SCREENING MODELS
In vitro tests-:
Histamine receptor binding assay.
Effects on air ways-:
Spasmolytic activity in isolated guinea pig lung strips.
Spasmolytic activity in isolated trachea.
In vivo tests-:
Bronchospasmolytic activity in anesthetized guinea pigs
(Konzett-Rössler method).
Effect of arachidonic acid or PAF on respiratory function.

4. IN VITRO MODELS:-
HISTAMINE RECEPTOR BINDING ASSAY
PURPOSE AND RATIONALE
Histamine is a key player in asthmatic attacks. In this assay,
we measure the inhibitory activities of test compounds on the
binding of 3H-pyrilamine to the plasma membrane
preparation from guinea pig brain to determine their affinity to
the histamine H1 receptor.
PROCEDURE
 Guinea pig brains are homogenized in ice-cold Tris buffer
(pH 7.5).
 At 4°C, the homogenate is centrifuged.
 The supernatant is discarded, and the pellet is re-
suspended in buffer before being centrifuged as before.
 The final pellets are re-suspended in tris(hydroxymethyl)-
aminomethane (Tris buffer). 1 g fresh weight/5 ml.

5.  Aliquots of 1 ml are frozen at -70 °C {to divide (as a
solution or suspension) into equal parts}.
 Now, for 30 minutes, 50 µl 3H-pyrilamine, 50 µl test
compound, and 100 µl membrane suspension from guinea
pig whole brain are incubated in a shaking bath at 25 °C.
 Saturation experiments are carried out, and total binding in
the presence of incubation buffer is determined, as is non-
specific binding in the presence of mepyramine or doxepin.
EVALUATION OF RESULTS
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. EFFECTS ON AIR WAYS:-
SPASMOLYTIC ACTIVITY IN ISOLATED GUINEA PIG LUNG STRIPS
PURPOSE AND RATIONALE
Drugs are tested for their ability to inhibit bronchospasm caused by
histamine or calcium ionophores using this method.
Calcium ionophores stimulate leukotriene release via the 5-
lipoxygenase pathway. Leukotrienes are potent bronchoconstrictor
that appear to act on smooth muscles.
This method is used to detect test compound H1 and leukotriene-
receptor blocking properties.
PROCEDURE
 Euthanasia is performed on albino guinea pigs of either sexes
weighing 300-450 g.
 The lungs are removed after the chest cavity is opened.
 Lungs are cut into 5 cm strips and immersed in physiological
saline solution.

7.  The lung strips are then placed in an organ bath containing
a nutritive solution.
 The bath is kept at 37 °C and bubbled with carbogen. The
tissue is left to equilibrate for 30-60 minutes with a pre-load
of 0.5 g-3 g.
 Before testing, carbachol is added to the bath to assess
the ability of the lung strips to contract.
 Two pre-values are obtained 20 minutes later by adding
the spasmogen (either histamine or calcium ions) to the
bath and recording the contractile force at its maximum
level.
 The spasmogen is administered again after 20 minutes of
equilibration.
 The test compound is then added in cumulative doses at 5
or 10 minute intervals for the next 5 minutes. The
contractile response is calculated.

8. EVALUATION
The percent inhibition of spasmogen induced
contraction is calculated.
Modification:
 Inhibition of prostaglandin synthesis:-
The procedure is identical to the one described above,
with the exception that prostaglandin synthesis is
inhibited by the addition of indomethacin at a
concentration of 10-6 g/ml prior to the administration of
spasmogen.
Kleinstiver and Eyre measured bronchoactivity using
lung parenchyma strips from various species (1979).

9. SPASMOLYTIC ACTIVITY IN ISOLATED TRACHEA
PURPOSE AND RATIONALE
Guinea pig isolated tracheal chain can be used to test
for β-blocking activity. This model can also be used to
test compounds that inhibit bronchospasms.
A β-receptor blocking agent must be added to assess a
compound's ability to inhibit carbachol-induced
bronchospasm via β-receptor activation.
If β-receptor activation causes bronchial smooth muscle
relaxation, the spasmolytic effect will be reduced after -
receptor blocker administration.

10. PROCEDURE
 Albino guinea pigs weighing 300-550 g of either sex are
euthanized.
 The trachea is dissected and divided into individual rings
(each part is 2–3 cartilaginous rings wide).
 12-15 rings are tied together with silk threads and mounted
in an organ bath containing Krebs-Henseleit solution at 37
°C and 0.5 g tension.
 Before adding the spasmogen, 40-45 minutes are allowed
for equilibration (either histamine or carbachol or calcium
ions are used).
 After 10-12 minutes, when the contraction has reached its
maximum (initial spasm), the standard drug, such as
isoprenaline (1 mg/ml) or aminophylline (10 mg/ml), is
administered.

11.  Bronchial responses are allowed to plateau before being
recorded.
 The tissue is thoroughly rinsed, and control contractions
are induced once more by adding spasmogen.
 The test drug is then added, and the contractile force is
measured at its maximum.
Determination of mechanism of action
Testing for β-sympathomimetic effect:- After obtaining the
initial carbachol-induced spasm, propranolol is given 5
minutes before the test drug is added.
EVALUATION
 The percent inhibition of carbachol or other spasmogen
induced contractions is calculated.
 From dose-response curves ED50 values can be
calculated.

12. IN VIVO TESTS:-
BRONCHO-SPASMOLYTIC ACTIVITY IN
ANESTHETIZED GUINEA PIGS (KONZETT-RÖSSLER
METHOD)
PURPOSE AND RATIONALE
The method is based on changes in the air volume of a living
animal in a closed system comprised of the respiration pump,
the trachea, and the bronchi, as well as a reservoir that
allows for the measurement of excess air volume or
pressure.
Bronchospasm reduces the amount of inspired air while
increasing the amount of expired air. Thus, the volume of
excess air can be used to quantify the degree of
bronchospasm.

13. 1.25 g/kg i.p. urethane is used to anesthetize
250-500 g guinea pigs of either sex.
Bronchospasm is best treated with
pentobarbital and alcuronium chloride.
After anesthesia, it is essential to avoid any
influence on spontaneous respiration.
A two-way cannula is used to cannulate the
trachea, with one arm connected to the
respiratory pump and the other to a Statham
P23 Db transducer.
A Starling pump with an inspiratory pressure
of 90-120 mm and a frequency of 60 strokes
per minute is used to artificially respire the
animal.
Excess air that is not absorbed by the lungs
is measured and recorded.
For the administration of spasmogens and
test compounds, the internal jugular vein is
cannulated.
PROCEDURE

14.  Spasmogens (such as acetylcholine hydrochloride,
methacholine, histamine dihydrochloride, bradykinin
triacetate, and others) are administered intravenously to
guinea pigs.
 After obtaining two bronchospasms of equal intensity, test
compounds are administered intravenously, orally,
subcutaneously, or intraduodenally.
 The spasmogen is administered again at the following
intervals:
I. 5, 15, and 30 minutes after intravenous administration of
the drug;
II. 15, 30, and 60 minutes after intraduodenal administration
of the drug and
III. 30, 60, and 120 minutes after p.o. administration of the
drug.
EVALUATION
Results are expressed as percent inhibition of induced
bronchospasm over the control agonistic responses. The
ED50 value is calculated.

15. • Forced insufflation was proposed as a simple
but accurate inhalation procedure for studying
the activity of anti-asthmatic drugs in guinea
pigs.
• Changes in the cross-sectional area of
conducting airways in anaesthetized dogs after
cumulative doses of ipratropium with and
without gallamine, a selective M2 muscarinic
receptor blocker, and after metaproterenol were
measured using high-resolution computed
tomography.
MODIFICATIONS

16. EFFECT OF ARACHIDONIC ACID OR PAF ON
RESPIRATORY FUNCTION
PURPOSE AND RATIONALE
 TXA2 and prostaglandlin are formed from the
breakdown of arachidonic acid (PGI2).
 Bronchoconstriction is caused by TXA2 produced in
the lung.
 In contrast to arachidonic acid, PAF as an inducer
causes platelet-dependent bronchoconstriction.
The test allows for the evaluation of drug sites of action
that interfere with the mechanisms of
bronchoconstriction and thrombocytopenia challenged
by spasmogens, arachidonic acid, and PAF.

17. PROCEDURE
 Anesthetized male guinea pigs (Pirbright White) weighing
300-600 g with 60 mg/kg pentobarbital sodium (i.p.).
 One of the jugular veins is cannulated to administer
spasmogen and the test compound.
 One external carotid artery is cannulated for a pressure
transducer and the other for blood withdrawal.
 A Starling pump is connected to the trachea (pressure
pump).
 Intravenous administration of pancuronium or gallamine
inhibits spontaneous respiration.
 Excess air is routed to a transducer with a bronchotimer,
which converts changes in air flow to an electrical signal
and records the changes.
 The animal is now given multiple intravenous injections of
the same dose of arachidonic acid until two equal-intensity
bronchospasms are obtained.

18.  The test compound is given intravenously, and the
spasmogen is given again at 2, 10, and 20 minutes after
the drug is given.
 After each bronchoconstriction, the lung must be passively
dilated.
 A platelet analyzer is used to count the number of
thrombocytes immediately before and 30-45 seconds after
each arachidonic acid application.
 If PAF is used as an inducer, it is injected intravenously 60
minutes before and 5 minutes after the drug is
administered.
 Blood samples are taken 30 seconds before and 15
seconds after each PAF application, and the number of
leukocytes and hematocrit values are calculated.

19. EVALUATION
 Percent inhibition or increase of bronchospasm, reduction
of blood pressure, thrombocytopenia, leukocytopenia and
hematocrit following test drug administration are
calculated.
 The mechanism of action of a test drug is concluded:
I. inhibitor of thromboxane synthetase.
II. inhibitor of cyclo-oxygenase.
MODIFICATIONS
 Kagoshima et al. tested the suppressive effects of a PAF-
antagonist on asthmatic responses in guinea pigs actively
sensitised with ovalbumin using a modification.
 The oscillation method was used to assess both the
immediate and late asthmatic response.

20. REFERENCES
 H. Gerhard Vogel (Ed.) (2002) “Drug Discovery and Evaluation –
Pharmacological Assays”. 2nd edition. Springer-Verlag Berlin
Heidelberg Publishers, New York, pp 351-383
 Gupta SK. (2016). Drug Screening Method. 3rd edition. Jaypee
Brothers Medical Publishers (P) Ltd, pp 683-696