Screening model for asthma copd,

1. Preclinical
screening models
for drugs used in
Asthma and COPD
Presented By:- Ms. Hiran Suthar
M.Pharm(Department of Pharmacology)
SEM 1
L.M. College of
Pharmacy
Date: 07/10/17
1

2. Asthma & COPD:- key points
 Asthma: chronic airway inflammation, progressive
airway remodelling, Airway hyper-responsiveness
(AHR) & various degrees of reversible airflow
obstruction.
 Mail reason is allergic: mediated via IgE antibody
reaction.
 COPD: chronic obstructive pulmonary disease,
accelerated rate of age-related lung function decline (
structural changes in lung).
 Mucous gland enlargement, epithelial thickening,
mucous hyper-secretion and occlusion by inflammatory
mucous exudates.
2

3. Treatment options
 Long acting Bronchodilators: Beta-
agonists(salmeterol),
anticholinergics(ipratropium),
corticosteroids(prednisone)
 So for successful preclinical screening of drugs
having these kinds of activity and to check if
they’re effective first we have to induce the
opposite conditions in animals i.e
bronchoconstriction to check for effectiveness of
bronchodilators.
3

4. Types of models
 in-vitro : histamine receptor binding
 Effects on airways:-
a) tests on isolated organs
b) in-vivo
 Anti-tussive activity
 Effects on tracheal cells, bronchial mucous
secretion and transport
4

5. Effect on airways: tests in
isolated organs
1. Spasmolytic activity in isolated guinea pig
lung strips
2. Spasmolytic activity in trachea
3. Reactivity of isolated perfused trachea
4. Bronchial perfusion of isolated lung
5. Vascular and airway responses in the
isolated lung
5

6. In-vivo tests
1. Bronchospasmolytic activity in anesthetized guinea
pig (Konzett-Rossler method)
2. Effect of arachidonic acid or PAF on respiratory
function
3. Bronchial hyperreactivity
4. Body plethysmography and respiratory parameters
after histamine-induced bronchoconstriction in
anesthetized guinea pigs
5. Pneumotachography in anesthetized guinea pigs
6. Airway microvascular leakage
7. Isolated larynx in situ
6

7. 1)Spasmolytic activity in isolated guinea
pig lung strips
 Purpose and rationale:- to check the spasmolytic activity induced by histamine and
calcium ionophores.
 Procedure:
 Animal: albino guinea pigs
 Sex: m/f
 Weight: 300-450g
 Method: sacrificed by ether overdoses. Lungs isolated. Cut into strips of 5cm in PSS.
Mounted in organ bath. Bubbled with carbogen, temp maintained at 37o. Tissue left to
equilibrate for 30-60 mins.
 Prior to testing carbachol added to check their ability to contract.
 20 mins later, two prevalues are obtained by adding the spasmogen & record
contractile force at maximum level.
 20 mins eq. period- spasmogen again given. 5 mins later- test compound added in
cumulative doses at 5-10 mins interval. Contractile response determined.
 histamine dihydrochloride 10–6 g/ml for 5 min, or
 • Ca – ionophore 5 × 10–6 g/ml for 5 min, or
 • Leukotriene LTC4 10–9–10–8 g/ml for 10 min, or
 • Leukotriene LTD4 10–9–10–8 g/ml for 10 min
 Evaluation: %inhibition of spasmogen induced contraction is calculated.
7

8. 2)Spasmolytic activity in isolated trachea
 P&R:- for beta blocking activity. Test compounds which inhibit bronchospasms. Beta sympathomimetic,
H1 blocking and leuko-triene blocking
 Procedure:
 Animal: albino GP
 Sex: m/f
 Weight :300-550g
 Method: sacrificed-co2 narcosis-trachea dissected into individual rings (2-3 cartilaginous rings wide) 12-
15 rings tied together with silk threads-mounted in O.B-containing Krebs-Henseleit sol.-maintained at
37o-0.5g tension-gassed with carbogen.
 Isometric contractions recorded via strain-gauge transduced on a polygraph-45mins quilibration-before
administering spasmogen
 The following spasmogens are used:
 • carbachol (2 × 10–7 g/ml)
 • histamine (10–7 g/ml)
 • Ca – ionophore for release of leukotrienes
 • leukotriene LTC4 (10–9–10–8 g/ml)
 • leukotriene LTD4 (10–9–10–8 g/ml)
 When maxcontraction-after 10-12 min-standard drug (isoprenalin/aminophylline) added-bronchil
responses allowed to plateau-rinsed-controolled contractions induced agin.
 After initial spasm-test drug –contraction recorded at max.
 Evaluation: %inhibition of spasmogen induced contractions. From dose response curved ED50 can be
calctd.
8

9. 3)Reactivity of isolated perfused
trachea
 P&R:- how epithelium affects the reactivity of tracheal musculture? Contractile
agoninsts added to serosal(extraluminal)or mucosal surface(intraluminal)
 Procedure
 Animal: Guinea pig
 Sex: male
 Method:-4cm trachea removed after sacrificed-cleaning in modified krebs henseleit sol
at 37o containting(millimolar) NaCl 113.0, KCl 4.8, CaCl2 2.5,
 KH2PO4 1.2, MgSO4 1.2, NaHCO3 25.0, and glucose 5.7
 (pH 7.4) being gassed with 95% O2/5% CO2-trachea attached to stainless steel
perfusion holder- extended to its insitu length-placed in organ chamber-370-25ml KHS
 This soln-pumped at constant rate 30ml/min through lumen-responses of tracheal
musculature obt by measuring changes in inlet-outlet ΔP between the side holes of
indwelling catheters, while the trachea as perfused at a constant rate of modified
Krebs-Henseleit solution. The inlet and outlet catheters are connected to the positive
and negative sides, respectively, of a differential pressure transducer
 Agonists are added in step-wise increasing, cumulative concentrations.
 Two consecutive dose-response curves are obtained after the addition either to the
serosal or mucosal compartment.
 The second dose-response curve is obtained 1.5 h after the end of the first, the
preparation being washed every 15 min during the intervening period.
 Evaluation:Responses are quantified as ΔP in centimeters of H2O
9

10. 4)Bronchial perfusion of isolated lung
 P&R:- The method consists in perfusing fluid down the trachea through the bronchi, and
allowing it to escape from the alveoli through scratches on the surface of the lungs.
 Bronchoconstriction results in a reduced rate of flow, bronchodilatation is indicated by an
increased flow.
 Procedure
 Animal: guinea pigs
 Weight : 200g
 Method:- sacrificed by head blow-trachea cut at upper end & removed with lung-trachea
attached to cannula of perfusion apparatus-one lung perfused-other tied off-lower part of
lower lobe cut off-rest lung surface-scratched deeply
 Perfusion fluid in percentage of anhydrous salts: NaCl 0.659, NaHCO3 0.252, KCl
0.046, CaCl2 0.005, MgCl2 0.0135, NaH2PO4 0.01, Na2HPO4 0.008, glucose 5%, pH
8.0
 Temp 37o-lung enclosed in glass cylinder-
 Trachea attached to cannula of perfusion apparatus-pumps sols at constant rate into
manometric tube connctd with perfused organ-bronchoconstriction( resistence to flow)
result in increase in ht of coumn of fluid in manomenter.
 Histamine HCl is added in a concentration of 1 : 2 500 000 as soon as the perfusion
starts and the flow is adjusted to obtain a constant progressive increase in pressure.
 The drugs are injected near the cannula when the perfusion pressure reaches a level of
500–650 ml of water. The volume injected is always 0.1 ml.
 Evaluation: activity ratios of bronchodilating ratio vs standard calc.
10

11. 5)Vascular and airway responses in
isolated lung
 P&R:- isolated perfused rat lung allows simultaneous registrtn of pulmonary
vascular and airway responses to various drugs
 Procedure:
 Animal: sprague-dawley rats
 Sex: m
 Weight: 300-350g
 Method: rats anesthetized via i.p. by pentobarbital sodium-trachea cannulated
with short section of polyethylene tubing-connected to rodent ventilator-
ventilated with room air enriched 95% O2/5% CO2, with a tidal volume of 4–5
ml/kg and 2 cm H2O positive end-respiratory pressure.
 Rats-heparinized with 1000 units of iv heparin-rapidly exsanguinated by
withdrawing blood from carotid artery-lung exposed-main pulmonary artery
catheterized-lung removed-suspended in warmed 39o humidified water
jacketed chamber-perfusate sol placed in reservoir-mixed constantly-lungs
perfused with peristaltic roller pump at a flow rate of 8-14 ml/min to maintain
physiological baseline pulmonary arterial pressure of 15 ±0.5 mm Hg.
Pulmonary arterial perfusion pressure, airway pressure, and reservoir blood
level are continuously monitored, electronically averaged and recorded with a
polygraph.
 Evaluation:- changes in pulmonary arterial pressure after injection of test drug
are measured in mm hg and compared with baseline values.
11

12. 6)Invivo tests
Bronchospasmolytic activity in anesthetized
guinea pig (Konzett-Rossler method)
 P&R:- 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.
 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.
12

13. Procedure
 Animal:- guinea pig
 Sex: m/f
 Weight;- 250-500g
 Method: animal anesthetized with i.p.urethane.
 Anesthesia should be deep enough to prevent influence of
spontaneous respiration.
 Trachea cannulated by two way cannula: one arm connected to
respiratory pump –other to a Statham P23 transducer.
 Animal artificially respired using starling pump with inspiratory
pressure set at 90-120mm of water, tidal volume: 3ml/100 g body
weight, frequency: 60 strokes/min.
 Excess air , not taken up by lungs measured and recorded on
polygraph
 Internal jugular vein cannulated-admins of spasmogens and test
cmpd
 Carotid artery cannulated to measure BP
13

14. Testing
 Spasmogens(i.v)
 acetylcholine hydrochloride (20–40 μg/kg), or
 methacholine (20–40 μg/kg), or
 histamine dihydrochloride (5–20 μg/kg), or
 bradykinin triacetate (10–20 μg/kg), or
 ovalbumin (1 mg/kg), or
 PAF (25–50 ng/ml), or
 leukotrienes LTC4, LTD4 (about 1 μg/kg), or
 substance P (0.5 μg/kg).
 After 2 bronchospasms of equal intensity- test drug given
i.v./p.o./s.c. or intraduodenally.
 Spasmogen given again 5, 15 and 30 min after i.v. administration
of the drug
 15, 30 and 60 min after intraduodenal administration
 of the drug
 30 and 60 (sometimes also 120) min after p.o. administration
 of the drug.
14

15. Standard compounds used
 atropine sulfate (0.01 mg/kg, i.v.) to inhibit
acetylcholine or methacholine induced spasms
 aminophylline (6 mg/kg, i.v.) to inhibit bradykinin
induced spasms
 tolpropamine-HCl (0.2 mg/kg) to inhibit histamine
induced spasms
 imipramine-HCl (3–5 mg/kg) to inhibit
serotoninkreatinin- sulphate induced spasms.
15

16. Evaluation
 Results are expressed as percent inhibition of
induced bronchospasm over the control
agonistic responses.
 The ED50 value is calculated.
16

17. 7)Effect of Arachidonic acid or PAF on
respiratory function in vivo
 P&R:- Arachidonic acid-metabolized into TXA2 and
PGI2.
 TXA2-lungs-bronchoconstriction(w or w/o
thrombocytopenia)
 PGI2-reduction of systolic & diastolic pressure.
 PAF-bronchoconstriction, thrombocytopenia,
leukocytopenia, reduction of BP and increase of
hematocrit.
 Test allows to evaluate the sites of action of drugs
which interfere with the mechanisms of
bronchoconstriction and thrombocytopenia.
17

18. Procedure
 Animal:- guinea pigs(pirbright White)
 Sex: m
 Weight: 300-600g
 Method: anesthetized with pentobarbital sodium (i.p.)-one jugular
vein-cannulated for admin of spasmogen and test drug-both
external carotid arteries-cannulated: one connected to a pressure
transducer to register BP-other used for blood withdrawal
 Trachea-connected to Starling pump with inspiratory pressure 80
mm hg of water, tidal vol 10ml/kg body weight, frequency 70-75
strokes/min
 Spontaneous respiration-inhibited by i.v. Of pancuronium or
gallamine
 Excess air not taken up by lungs is conducted to a transducer with
bronchotimer which translates changes in air flow to an electrical
signal.
 Animal gets multiple iv injection of same dose of Arachidonic acid
till 2 bronchospasms of equal intensity are obtained. Test is adm.
Iv and spasmogen given again at intervals of 2, 10, 20 and 30
mins after.
18

19. Procedure
 Before and 30-45 sec after each dose of AA 50
microlitre blood is collected in Na-EDTA coated tubes.
No. of thrombocytes are determined with platelet
analyzer .
 PAF as inducer- i.v-60 mins before, 5 mins and 60
mins after iv drug admins.
 Blood samples to be collected 30 s before and 15 s
after each PAF application
 No. of leukocytes and hematocrit are determined
automatically.
19

20. Continued…
 STANDARD COMPOUNDS
 dazoxiben HCl (inhibitor of thromboxane synthetase,TSI)
 acetylsalicylic acid- (inhibitor of cyclo-oxygenase, COI)
 Evaluation:-% inhibition or increase of bronchospasm,
reduction in BP, thrombocytopenia, leukocytopenia and
hematocrit after test drug admin calculated in comparison to
control values before drug treatment.
 From the patern of profile of influence on bronhoconstriction,
TCP and BP reduction, the mechanism of test drug is
concluded.
 inhibitor of thromboxane synthetase
 inhibitor of cyclo-oxygenase
 other effect = no profile
20

21. 8)Bronchial hyperreactivity
 P&R:-symptom of asphyctic convulsions resembling
bronchial asthma can be induced by inhalation of
histamine or other agents in Guinea Pig.
 Agents applied as aerosols produced by an ultra
sound nebulizer. First symptoms are increasing
breathing frequency, forced inspiration and finally
asphyctic convulsions. Occurences of these can be
delayed by antagonistic drugs. Pre-convulsion time
can be measured.
21

22. Procedure Animal: albino guinea pigs (10)
sex: male
 Weight : 300-400g
 Method: inhalation cages: 3 boxes- each ventilated with airflow of 1.5
l/min
 Animal placed in box A-test/standard is applied using ultra sound
nebulizer LKBNB108-aerosol of 0.2ml solution injection by infusion
pump in 1 min
 Alternatively animal is treated orally or s.c. with test or standard.
 Box B acts as a sluice through which animal passes into box C
 Here it is exposed to aerosol of 0.1% sol of histamine HCl provided by
an ultra sound nebulizer.
 Time until asphyctic convulsions is measured.
 Then animal is immediately withdrawn. Aerosols are removed by
applying pressure
 Evaluation:- Percent of increase of pre-convulsion time is calculated
versus controls.
 ED50 values can be found, i.e. 50% of increase of pre-convulsion time
22

23. 9)Body plethysmography and respiratory parameters after
histamine induced bronchoconstriction in anesthetized
guinea pigs.
 P&R:- respiratory freqeuncy and amplitude are
recorded. Thedecrease of respiratory
amplitude(diminished respiratory volume due to
bronchoconstriction) and the reflectory increase of
respiratory frequency after histamine inhalation are
attenuated by bronchodilatatory drugs.
 This method can be used for various purposes: to
evaluate the antagonism against bradykinin induced
bronchoconstriction, the bronchodilating effects of K+
channel openers, or effect of morphine on respiration in
rats.
23

24. Procedure
 Animal: guinea pig
 Sex: m/f
 Weight: 400-600g
 Method: anesthetized with i.p. pentobarbital
 Trachea, pleural cavity, jugular vein and carotid artery are
prepared and cannulated.
 Animals are mechanically ventilated by Starling pump
delivering inspiration volume representing tracheal pressure
of 8cm water at rate of 60 strokes/min.
 Succinylcholine chloride 1mg/kg given i.v. to prevent
interference from spontaneous respiration.
 The guinea pigs are placed inside a whole body
plethysmograph and tracheal, pleural, venous and arterial
catheters are connected to onset ports in the wall of the
plethysmograph box.
 The tracheal port is then connected with the respiration
pump.
24

25. 25

26. 26

27. Procedure
 Airflow rate into and out of the plethysmograph are measured as
pressure difference with a No. 000 Fleisch tube and a differential
pressure transducerAirflow is calibrated by passing compressed air
through a rotameter.
 The tidal volume (VT) is calculated from the flow signal.
 Transpulmonary pressure (PTP) is measured with a differential
pressure transducer with one side attached to a catheter inserted into
the right pleural cavity and the other side connected to a side port of
the tracheal cannula.
 PTP is calibrated with a water manometer.
 Signals from airflow tidal volume and transpulmonary pressure are fed
into an on-line computer system for calculation of pulmonary
resistance (RL) and dynamic lung compliance (CDYN).
 These parameters are calculated for each breath with a sampling rate
of 100 s–1 for each circle.
 Flow and pressure signals for computation are obtained from a
PLUGYS measuring system
 Systemic arterial pressure is measured using a Statham pressure
transducer
 Heart rate is computed from pressure pulses
27

28. Procedure
 3 doses of test/standard injected i.v. saline injections serve
as control
 i.v. histamine lead to short decrease in Cdyn and short
increase in Rl by approx 200% compared with baseline
 Challenges repeated at 5 min interval yielding same
increase in Rl during 1 hr period. After 3 reproducible
responses test is given i.v 1 min before histamine injection
 Evaluation:- Inhibition of histamine induced
bronchoconstriction by various doses of test compound and
standard is recorded.
 ED50 values for inhibition in RL are calculated.
 Furthermore, the time course of histamine antagonism can
be evaluated.
28

29. 9)Pneumotachography in
anesthetized guinea pigs
 P&R:- The use of a pneumotachograph
based on the principle of the Fleisch-tube and
of additional pressure transducers allows
simultaneous measurements of several
respiratory and circulatory parameters in
anesthetized guinea pigs
29

30. Procedure
 Animal : guinea pigs (Pirbright white)
 Weight : 300-400 g
 Method:
 Shaved ventrally at neck-placed on heated operating table-fixed
at upper extremities
 Metal cannula with blunted tip-inserted into trachea-secured with
a loop.
 Thin plastic catheter-inserted into oesophagus-tip located inside
thorax to register intrathoracic pressure.
 Cephalic vein on one side and carotid artery on other side is
cannulated.
 Tracheal cannula-connected to Fleisch tube (pneumotachograph)
by pieces of tubing.
 Fleisch tube connected with a sensitive differential pressure
transducer-range of 2cm water.
30

31. Procedure
 One side of another pressure differential transducer with 20
ml water range connected to oesohageal catheter and other
side is open to room air.
 Both transducers connected to separate pre-amplifier
 For recording of the arterial blood pressure a Gould
pressure transducer is used.
 The signals for airflow and esophageal pressure are
monitored at the output of the preamplifier with a digital 2-
channel oscilloscope.
 To obtain various respiratory and circulatory parameters
from the three primary signals they are calculated by certain
formulae by an analogue computer
31

32. Parameters presented at the output of
instrument as analogue electrical
signals
 Circulation
 Systolic blood pressure, diastolic blood
pressure, mean blood pressure
 Respiration
 Tidal volume, respiratory volume per minute,
respiratory rate
 Pulmonary mechanics
 Airway resistance, dynamic compliance, end-
respiratory work
32

33. Evaluation
 For each individual experiment the data of the last
 5 min before the first substance application are
averaged and used as controls.
 The response values after substance application
are then expressed as percentages of the controls.
 In this way each animal serves as its own control.
 For an analysis of the results the response values
are averaged over certain time intervals.
33

34. 10)Airway microvascular leakage
 P&R:- Plasma exudation in guinea-pig airways in vivo
can be determined by Evans Blue dye and is fairly
correlated with radiolabelled albumin
 This method can be used to study the antagonism
against bradykinin- and platelet-activating factor-
induced airway microvascular leakage and vagal
stimulation- induced airway responses
34

35. Procedure
 Animal: Dunkin-Hartley guinea pigs
 Sex: Female
 Weight : 380-600 g
 Method:
 Anesthetized with urethane i.p.
 Tracheal cannula inserted in the lumen of cervical trachea,
a polyethylene catheter into the left carotid artery (BP &
HR) and another one into external jugular vein ( for
administering drugs)
 The animals are connected to a constant volume
mechanical ventilator and then given an injection of 1.0–1.5
mg/kg suxamethonium i.v. to prevent interference with
spontaneous respiration.
 A tidal volume of 10 ml/kg and a frequency of 60
strokes/min are used.
35

36. Procedure
 Lung resistance is measured as an index of airway
function and monitored throughout the experiment.
 Transpulmonary pressure is measured with a pressure
transducer with one side attached to a catheter
inserted into the right pleural cavity and the other side
attached to the side port of the intratracheal cannula.
 Airflow is measured by a pneumotachograph
connected to a pressure transducer.
 The signals of the transducers are used for
instantaneous calculation of lung resistance by an
appropriate computer program.
36

37. Procedure
 Test compound ( bradykinin antagonist) given via i.v.
 Ten minutes later, Evans blue dye is injected i.v for 1 min.
 After 1 min, bronchoconstriction and microvascular leakage is
induced by injection of bradykinin.
 6 mins after the leakage, thoracic cavity is opened, cannula is
inserted into the aorta.
 Perfusion is performed to remove intravascular dye from
systemic circulation
 Blood and Perfusion liquid are expelled through an incision in
right and left atrium
 Right ventricle is opened-pulmonary circulation perfused with
saline.
 Lungs are removed-airways divided into 4 components-tissues
are blotted dry and weighed.
 Evans blue dye is extracted in 2 ml formamide at 40oC for
24hrs, and measured in spectrophotometer at 620 nm
37

38. Evaluation
 Evans Blue dye concentration, expressed as
ng/mg tissue, as well as lung resistance are
compared by statistical means (unpaired
Student’s t-test or Mann- Whitney U test)
between treated groups and controls
receiving the challenge only.
38

39. References!
1) Drug Discovery
and Evaluation: pharmacological assays by H
Gerhard Vogel
2) Preclinical models of asthma and COPD
(stevenson and Belvisi)
3) Screening methods in pharmacology: by
Robert Turner.
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