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Dr Manjeeta Gupta
Evaluation of anti-asthmatic drugs
7/8/2016Department of Pharmacology,MIMER Medical College Talegaon Dab...
Asthma
 Global health problem resulting from complex
interaction between genetic & environmental
factors
 Nearly 7–10% o...
7/8/2016Department of Pharmacology,MIMER Medical College Talegaon Dabhade 3
Chronic, heterogeous,
inflammatory disorder of...
Acute toxicity test
 Before starting any in vivo assays, it is important to study oral acute
toxicity, for selection of t...
Ideal animal models for asthma
 Similar genetic basis to human diseases
 Similar anatomy & physiology
 Similar patholog...
In vitro methods
1. Binding Assay
a. Histamine receptor assay
2. Cell culture methods
a. CULTEX technique
b. WST assay
3. ...
In vivo methods
1. Bronchospasmolytic activity in anaesthetized guinea pigs
2. Arachidonic acid/PAF induced respiratory va...
7/8/2016Department of Pharmacology,MIMER Medical College Talegaon Dabhade 8
In vitro methods
Histamine receptor assay
7/8/2016Department of Pharmacology,MIMER Medical College Talegaon Dabhade 9
Animal used  Guinea ...
7/8/2016Department of Pharmacology,MIMER Medical College Talegaon Dabhade 10
Procedure
CULTEX technique
 New experimental method for cultivation & exposure of cells of
respiratory tract to air borne pollutant...
7/8/2016Department of Pharmacology,MIMER Medical College Talegaon Dabhade 12
Bronchial epithelial cells
washed with PBS
In...
WST assay
Transfer cells from cell
exposure vessel to
plates containing 2ml
fresh RPMI medium
Add 500μl RPMI &
WST-1 dye &...
Spasmolytic activity in lungs
 Animal used  Albino guinea pig (either sex, 300-450g)
 Preparation  Animal sacrificed w...
7/8/2016Department of Pharmacology,MIMER Medical College Talegaon Dabhade 15
Procedure
Vascular & airway responses to isolated lung
Animal used  Sprague dawley rats (300-350g)
Anaesthetic used  Pentobarbiton...
Trachea cannulated &
animal on artificial
respiration
Rat heparinized with
1000 IU & blood
withdrawn from carotid
artery
L...
Reactivity of isolated perfused trachea
 Animal used  Albino guinea pig (either sex, 300-550g)
 Preparation  Sacrifice...
Trachea dissected & cut into individual rings
12-15 rings tied together & mounted in organ bath
(Krebs-Henseleit buffer so...
Readings recorded
Tissue washed
Controlled contractions induced again by addition of spasmogens
Record contractions & add ...
In vivo methods
7/8/2016Department of Pharmacology,MIMER Medical College Talegaon Dabhade 21
Bronchospasmolytic activity (Konzett-Rosseler method)
Animal used  Guinea pig (250-500g)
Anaesthetic used  1.25g/kg uret...
Trachea is cannulated
ARM 1  connected to respiratory pump
ARM 2  connected to statham pressure tranducer
Artificial ven...
BP recorded (from carotid artery)
Each animal placed in plastic containers (Histamine chamber) of 15L volume
0.25% histami...
Arachidonic acid/PAF induced respiratory & vascular
dysfunction
Animal used  Guinea pigs (Male, 300-600g)
Anaesthetic use...
Procedure
Trachea  artificial respiration (70-75 strokes/min)
Jugular vein  test drug
Carotid artery  blood withdrawal ...
Anaphylactic Microshock
Animals used  Guinea pig (200-300g) sensitized with s.c injection of
egg albumin
Rabbit (2000-300...
7/8/2016Department of Pharmacology,MIMER Medical College Talegaon Dabhade 28
Procedure
Serotonin aerosol induced asphyxia
Animal used  Guinea pig (200-300g)
Principle  Serotonin causes bronchoconstriction  ...
7/8/2016Department of Pharmacology,MIMER Medical College Talegaon Dabhade 30
Procedure
Histamine induced bronchocostriction
Animal used  Guinea pig (400-600g)
Anaesthetic used  Pentobarbitone 70mg/kg i.p.
Pr...
Procedure
Trachea  artificial respiration (60 strokes/min)
Jugular vein  test drug
Carotid artery  blood withdrawal & t...
7/8/2016Department of Pharmacology,MIMER Medical College Talegaon Dabhade 33
Histamine intravenous
injection (↓LC & ↑PR by...
Pneumatography in guinea pigs
7/8/2016Department of Pharmacology,MIMER Medical College Talegaon Dabhade 34
Animal used  G...
Mast cell stabilising activity on rat mesentery
(Kaley & Weiner)
Animal used  Male albino rats
Aim  To study % of degran...
Non human primate models
Animals used  Rhesus monkeys
Cynomologus monkey
Spasmogens  House dust mite, ascaris, pollen an...
Alternatives to animal models
 Cell culture
 Patient sputum culture
 BAL fluid sampling
 Direct reprogramming of patie...
References
 Drug screening methods by S.K. Gupta
 Animal models of asthma: reprise or reboot? Biochemical pharmacology
...
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Evaluation methods of anti-asthmatics

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Invivo and invitro evaluation of anti-asthmatics agents.

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Evaluation methods of anti-asthmatics

  1. 1. Dr Manjeeta Gupta Evaluation of anti-asthmatic drugs 7/8/2016Department of Pharmacology,MIMER Medical College Talegaon Dabhade 1
  2. 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. 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. 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. 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. 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. 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. 8. 7/8/2016Department of Pharmacology,MIMER Medical College Talegaon Dabhade 8 In vitro methods
  9. 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. 10. 7/8/2016Department of Pharmacology,MIMER Medical College Talegaon Dabhade 10 Procedure
  11. 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. 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. 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. 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. 15. 7/8/2016Department of Pharmacology,MIMER Medical College Talegaon Dabhade 15 Procedure
  16. 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. 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. 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. 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. 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. 21. In vivo methods 7/8/2016Department of Pharmacology,MIMER Medical College Talegaon Dabhade 21
  22. 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. 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. 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. 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. 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. 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. 28. 7/8/2016Department of Pharmacology,MIMER Medical College Talegaon Dabhade 28 Procedure
  29. 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. 30. 7/8/2016Department of Pharmacology,MIMER Medical College Talegaon Dabhade 30 Procedure
  31. 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. 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. 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. 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. 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. 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 7/8/2016Department of Pharmacology,MIMER Medical College Talegaon Dabhade 36
  37. 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. 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

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