Invivo and invitro evaluation of anti-asthmatics agents.

1. Dr Manjeeta Gupta
Evaluation of anti-asthmatic drugs
7/8/2016Department of Pharmacology,MIMER Medical College Talegaon Dabhade 1

2. Asthma
 Global health problem resulting from complex
interaction between genetic & environmental
factors
 Nearly 7–10% of world population suffers from
bronchial asthma
 Among several respiratory diseases, bronchial
asthma is most common disabling syndrome
7/8/2016Department of Pharmacology,MIMER Medical College Talegaon Dabhade 2

3. 7/8/2016Department of Pharmacology,MIMER Medical College Talegaon Dabhade 3
Chronic, heterogeous,
inflammatory disorder of
airways
characterized by:
1. Airway obstruction
2. Airway inflammation
3. Bronchial hyper-
responsiveness
 In this screening method, we measure potency of anti-asthmatic agents,
by inducing bronchial hyperactivity in experimental animals (rats, mice, guinea pigs, monkeys)

4. Acute toxicity test
 Before starting any in vivo assays, it is important to study oral acute
toxicity, for selection of test dose
 Initial dose finding procedure  Albino mice (either sex, 20-25g)
Group 1 – 3 mice given 10mg/kg test drug i.p.
Group 2 – 3 mice given 100mg/kg test drug i.p.
Group 3 – 3 mice given 1000mg/kg test drug i.p.
(Monitor for 24 hrs)
 From above results we take 4 doses & administer i.p.
4 groups  1 mouse/group
 LD50  Mean of lowest dose showing death & highest dose not showing
death
7/8/2016Department of Pharmacology,MIMER Medical College Talegaon Dabhade 4

5. Ideal animal models for asthma
 Similar genetic basis to human diseases
 Similar anatomy & physiology
 Similar pathological response
 Respond to drugs with known clinical efficacy
 Predict clinical efficacy
7/8/2016Department of Pharmacology,MIMER Medical College Talegaon Dabhade 5

6. In vitro methods
1. Binding Assay
a. Histamine receptor assay
2. Cell culture methods
a. CULTEX technique
b. WST assay
3. Tests in isolated organs
a. Spasmolytic activity in guinea pig lungs
b. Vascular & airway responses to isolated lung
c. Reactivity of isolated perfused guinea pig trachea
7/8/2016Department of Pharmacology,MIMER Medical College Talegaon Dabhade 6

7. In vivo methods
1. Bronchospasmolytic activity in anaesthetized guinea pigs
2. Arachidonic acid/PAF induced respiratory vascular dysfunction
3. Anaphylactic microshock
4. Serotonin aerosol induced asphyxia
5. Histamine induced bronchoconstriction
6. Pneumatochography in guinea pigs
7. Bronchial hyperactivity in guinea pigs
8. Mast cell stabilising activity on rat mesentery
7/8/2016Department of Pharmacology,MIMER Medical College Talegaon Dabhade 7

8. 7/8/2016Department of Pharmacology,MIMER Medical College Talegaon Dabhade 8
In vitro methods

9. Histamine receptor assay
7/8/2016Department of Pharmacology,MIMER Medical College Talegaon Dabhade 9
Animal used  Guinea pig brain plasma (Male, 300-600g)
Aim  To measure inhibitory activity of test compound on
binding of 3H pyrilamine (H1 antagonist)
1. Total binding
2. Non specific binding
3. Specific binding (Total binding – Non specific binding)
4. % inhibition of 3H pyrilamine binding (100-specific
binding)

10. 7/8/2016Department of Pharmacology,MIMER Medical College Talegaon Dabhade 10
Procedure

11. CULTEX technique
 New experimental method for cultivation & exposure of cells of
respiratory tract to air borne pollutants at air/liquid interface
 Enhanced efficiency of in-vitro studies
Principle  Direct exposure of bronchial epithelial cells to
complex mixtures
Aim  To study factors influencing susceptibility of human bronchial
epithelial cells
7/8/2016Department of Pharmacology,MIMER Medical College Talegaon Dabhade 11

12. 7/8/2016Department of Pharmacology,MIMER Medical College Talegaon Dabhade 12
Bronchial epithelial cells
washed with PBS
Incubate with test drug for
24hrs
Cells exposed to clean
air/different concentrations
of smoke for 1 hr (Cell
exposure unit)
Procedure

13. WST assay
Transfer cells from cell
exposure vessel to
plates containing 2ml
fresh RPMI medium
Add 500μl RPMI &
WST-1 dye & incubate
for 1hr
Aliquots transferred
into 96 well microplate
(for absorbance at 450-
630 nm)
Measurement of
absorbance by
microplate reader
Cells are trypsinized
by adding 500μl
trypsin/ETDA soluton
Incubate at 37˚C for 4
mins
Add 25μl trypsin
inhibitor
Gently suspend cells &
dilute 100μl
suspension in 9.9ml
CASYton solution
Analyse aliquots with
Electronic cell counter
7/8/2016Department of Pharmacology,MIMER Medical College Talegaon Dabhade 13

14. Spasmolytic activity in lungs
 Animal used  Albino guinea pig (either sex, 300-450g)
 Preparation  Animal sacrificed with overdose of ether
 Aim  To evaluate capability of inhibiting bronchospasm induced by histamine
& Calcium ionophores
7/8/2016Department of Pharmacology,MIMER Medical College Talegaon Dabhade 14

15. 7/8/2016Department of Pharmacology,MIMER Medical College Talegaon Dabhade 15
Procedure

16. Vascular & airway responses to isolated lung
Animal used  Sprague dawley rats (300-350g)
Anaesthetic used  Pentobarbitone sodium (50mg/kg) i.p
Aim  To measure & compare..
1. Pulmonary arterial pressure
2. Airway pressure
3. Reservoir blood level
7/8/2016Department of Pharmacology,MIMER Medical College Talegaon Dabhade 16

17. Trachea cannulated &
animal on artificial
respiration
Rat heparinized with
1000 IU & blood
withdrawn from carotid
artery
Lungs removed (median
sternotomy) &
suspended water
jacketed chamber
Pulmonary artery
catheterised
Lungs perfused with
Krebs-Henseleit
solution
Changes in parameters
recorded after addition
of test drug &
compared with baseline
7/8/2016Department of Pharmacology,MIMER Medical College Talegaon Dabhade 17
Procedure

18. Reactivity of isolated perfused trachea
 Animal used  Albino guinea pig (either sex, 300-550g)
 Preparation  Sacrificed by CO2 narcosis
 Aims 
1. To study mechanism by which epithelium affects
reactivity of tracheal musculature
2. To study effects of histamine, Calcium ionophores,
bradykinin, leukotriene, potassium channel openers on
tracheal musculature
7/8/2016Department of Pharmacology,MIMER Medical College Talegaon Dabhade 18

19. Trachea dissected & cut into individual rings
12-15 rings tied together & mounted in organ bath
(Krebs-Henseleit buffer solution)
Tissue maintained at 37˚C & bubbled with carbogen
Catheters (inlet-outlet) connected to positive & negative sides of polygraph
Response of tracheal musculature by changing inlet-outlet pressure is recorded
After 45 mins spasmogens added
When maximum contraction is reached standard drug is added (isoprenaline/aminophylline)
7/8/2016Department of Pharmacology,MIMER Medical College Talegaon Dabhade 19
Procedure

20. Readings recorded
Tissue washed
Controlled contractions induced again by addition of spasmogens
Record contractions & add test drug
Record readings
(Change in pressure in cm of water is taken as response)
7/8/2016Department of Pharmacology,MIMER Medical College Talegaon Dabhade 20

21. In vivo methods
7/8/2016Department of Pharmacology,MIMER Medical College Talegaon Dabhade 21

22. Bronchospasmolytic activity (Konzett-Rosseler method)
Animal used  Guinea pig (250-500g)
Anaesthetic used  1.25g/kg urethane i.p.
Principle  Bronchospasm causes ↓ volume of inspired air & ↑
volume of excess air
Aim  To measure volume of air not taken up by lungs after
bronchospasm
7/8/2016Department of Pharmacology,MIMER Medical College Talegaon Dabhade 22

23. Trachea is cannulated
ARM 1  connected to respiratory pump
ARM 2  connected to statham pressure tranducer
Artificial ventilation at frequency 60strokes/min is maintained
Excess air not taken up by lungs is measured
Test drug administered (through jugular vein)
7/8/2016Department of Pharmacology,MIMER Medical College Talegaon Dabhade 23
Procedure

24. BP recorded (from carotid artery)
Each animal placed in plastic containers (Histamine chamber) of 15L volume
0.25% histamine solution aerosol sprayed at 180mmHg
5 mins exposure time (Test drug given orally 1 hr before exposure)
Spasmogen challenge is repeated
Unprotected animals fall on their sides
7/8/2016Department of Pharmacology,MIMER Medical College Talegaon Dabhade 24

25. Arachidonic acid/PAF induced respiratory & vascular
dysfunction
Animal used  Guinea pigs (Male, 300-600g)
Anaesthetic used  Pentobarbitone sodium 60mg/kg i.p.
Principle  Thromboxane causes bronchoconstriction & thrombocytopenia
Prostacyclin cause ↓ SBP & ↓ DBP
Aim  To study & compare…
1. % inhibition or increase of bronchospasm
2. BP reduction (measure magnitude & duration)
3. Thrombocytopenia & haematocrit
7/8/2016Department of Pharmacology,MIMER Medical College Talegaon Dabhade 25

26. Procedure
Trachea  artificial respiration (70-75 strokes/min)
Jugular vein  test drug
Carotid artery  blood withdrawal & transducer attached for BP
measurement
7/8/2016Department of Pharmacology,MIMER Medical College Talegaon Dabhade 26
Record BP & changes in airflow
Animal given multiple intravenous injections of arachidonic
acid till 2 bronchospasms of equal intensity are obtained
Test drug administered intravenously
Repeat spasmogen injections

27. Anaphylactic Microshock
Animals used  Guinea pig (200-300g) sensitized with s.c injection of
egg albumin
Rabbit (2000-3000g) sensitized with 2% histamine
aerosol
Ova transgenic mouse model (mouse sensitized with
ova i.p  Gold standard model
Aim  Measure degree of protection (p) = [1- (C/T)] * 100
Where, C = control animals
T = treated animals
(C ≤ 40 secs ≥ 165 secs are excluded)
7/8/2016Department of Pharmacology,MIMER Medical College Talegaon Dabhade 27

28. 7/8/2016Department of Pharmacology,MIMER Medical College Talegaon Dabhade 28
Procedure

29. Serotonin aerosol induced asphyxia
Animal used  Guinea pig (200-300g)
Principle  Serotonin causes bronchoconstriction  asphyxia & death
Aim  To evaluate % protection = (1- T1/T2)* 100
where, T1 = mean of control pre-convulsion time 2 days
before & 2 days after administration of drug
T2 = Pre-convulsion time with administration of drug
7/8/2016Department of Pharmacology,MIMER Medical College Talegaon Dabhade 29

30. 7/8/2016Department of Pharmacology,MIMER Medical College Talegaon Dabhade 30
Procedure

31. Histamine induced bronchocostriction
Animal used  Guinea pig (400-600g)
Anaesthetic used  Pentobarbitone 70mg/kg i.p.
Prniciple  Bronchodilators attenuate ↓ respiratory amplitude
& ↑ respiratory frequency after histamine inhalation
Aims  To study & calculate…
1. Respiratory frequency
2. Respiratory amplitude
3. Time required for antagonism against bradykinin induced
bronchoconstriction
4. Bronchodilator effects of Potassium channel openers
7/8/2016Department of Pharmacology,MIMER Medical College Talegaon Dabhade 31

32. Procedure
Trachea  artificial respiration (60 strokes/min)
Jugular vein  test drug
Carotid artery  blood withdrawal & transducer attached for BP
measurement
Other parameters:
Airflow rate  Differential pressure transducer
Tidal volume & trans pulmonary pressure
Pulmonary resistance (PR) & dynamic lung compliance (LC)
Systemic BP  Statham pressure transducer
7/8/2016Department of Pharmacology,MIMER Medical College Talegaon Dabhade 32

33. 7/8/2016Department of Pharmacology,MIMER Medical College Talegaon Dabhade 33
Histamine intravenous
injection (↓LC & ↑PR by
200%)
Repeat after 5mins
After 3 reproducible
responses
Test drug given
intravenously 1min before
histamine injection
Inhibition of histamine
induced bronchoconstriction
recorded

34. Pneumatography in guinea pigs
7/8/2016Department of Pharmacology,MIMER Medical College Talegaon Dabhade 34
Animal used  Guinea pig (300-400g)
Anaesthetic used  Urethane (1.5g/kg, i.p.)
Aim  To measure respiratory & circulatory parameters
Procedure Trachea is cannulated (connected to pnematograph)
Catheter placed in oesophagus (Oesophageal transducer)
with tip inside thorax (registers intrathoracic pressure)
Cephalic vein & carotid artery cannulated (Gould
pressure transducer)
Test & control readings recorded

35. Mast cell stabilising activity on rat mesentery
(Kaley & Weiner)
Animal used  Male albino rats
Aim  To study % of degranulated mast cells
Procedure
7/8/2016Department of Pharmacology,MIMER Medical College Talegaon Dabhade 35
Small intestine with mesentery (Petri dish with PBS)
Mesentery incubated with different concentrations of disodium
cromoglycate
Challenged with 1μg/ml 48/80 (standard granulator) for 10mins
Mesentery stained with 0.1% toluidine blue for 20-30mins
Mount pieces on slide

36. Non human primate models
Animals used  Rhesus monkeys
Cynomologus monkey
Spasmogens  House dust mite, ascaris, pollen antigens
Aims  To study…
1. Early & late phase bronchoconstriction response
2. Airway eosinophilia
3. Acute hyperactivity response
4. Human proteins & monoclonal antibody therapeutics
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37. Alternatives to animal models
 Cell culture
 Patient sputum culture
 BAL fluid sampling
 Direct reprogramming of patient fibroblasts (patient derived
induced pluripotent stem cells)
 Newer in vitro techiques – Human tissue explants
Precision cut lung slices
7/8/2016Department of Pharmacology,MIMER Medical College Talegaon Dabhade 37

38. References
 Drug screening methods by S.K. Gupta
 Animal models of asthma: reprise or reboot? Biochemical pharmacology
 Evaluation of antiasthmatic activity of a polyherbal formulation containing four
plant extracts. Journal of current pharmaceutical research
 Measuring the lung function in the mouse: the challenge of size. Respiratory
research
 Various screening methods of anti-allergic activity. International journal of
pharmaceutical sciences and nanotechnology
 Vogel
7/8/2016Department of Pharmacology,MIMER Medical College Talegaon Dabhade 38