antitussives and expectorants

1. SCREENING OF ANTITUSSIVES
AND EXPECTORANTS

2. CONTENTS
• Cough
• Causes
• Cough reflex
• Cough receptors
• Mechanical events in cough
• Drugs used in cough
• Screening of Antitussive activity
• Screening of Expectorants.

3. COUGH
• An essential, protective function for human airways & lungs.
• Cough reflex involves a highly orchestrated series of involuntary
muscular actions with potential for input from cortical pathways as
well.
• Ineffective cough reflex??
• Excessive cough??

6. COUGH REFLEX
• Larynx, Trachea, Carina, Bronchi, Terminal bronchioles, Alveoli
sensitive to foreign matter; corrosive chemical stimuli.
• Afferent nerve impulse respiratory passage vagus nerves
medulla of brain.
• Afferent pathway; Central pathway; Efferent pathway.

7. COUGH REFLEX
• AFFERENT PATHWAY: Sensory nerve fibres located in ciliated
epithelium of upper airways, cardiac and esophageal branches of
diaphragm.
• CENTRAL PATHWAY: Upper brainstem; Pons.

8. COUGH REFLEX
• EFFERENT PATHWAY:
• Impulse from cough centre vagus, phrenic, spinal motor nerve to diaphragm,
abdominal wall & muscles.
• Nucleus retroambigualis phrenic, spinal motor nerve inspiratory &
expiratory muscles
• Nucleus ambiguous laryngeal branches of vagus larynx.

9. COUGH REFLEX
• Termination of vagal afferents abundant in airway mucosa from
upper airways to terminal bronchioles and lung parenchyma.
• 3 broad classes of afferent nerves:
• Rapidly Adapting Receptors (RAR)
• Slowly Adapting Stretch Receptors (SARs)
• C- fibres.

10. RAPIDLY ADAPTING RECEPTORS
• Dynamic receptors respond to changes in mechanical properties.
• Sporadically active throughout respiratory cycle
• Activation
• Stimuli evoking bronchospasm/obstruction resulting from mucus
secretion/edema.
• Histamine, Capsaicin, Substance P, Bradykinin
• Stimuli evoking cough

11. SLOWLY ADAPTING STRETCH RECEPTORS
(SARs)
• Highly sensitive to mechanical forces put on lung during breathing.
• Activity increases during inspiration, peaks just prior to initiation of
expiration.
• Activation
• Central inhibition of cholinergic drive to airways.
• Decreases phrenic nerve activity
• Decreases airway smooth muscle tone.

12. C- FIBRES
• Majority of afferent nerves innervating airways and lungs are unmyelinated
C-fibres.
• PG-E2, Adrenaline, Adenosine, Capsaicin, Bradykinin sensitise C-fibres.
• Activation
• Increased airway parasympathetic nerve activity
• Chemoreflex characterized by apnea, bradycardia and hypotension.

13. MECHANICAL EVENTS OF COUGH
• INSPIRATORY PHASE: Inhalation; Generates the volume necessary
for effective cough.
• COMPRESSION PHASE: Closure of larynx combined with
contraction of muscles of chest wall, diaphragm and abdominal wall
increase in intrathoracic pressure

14. MECHANICAL EVENTS OF COUGH
• EXPIRATORY PHASE:
• Glottis opens high expiratory airflow; Coughing
• Large airway compression
• High flows- dislodge mucus from airways- allows removal from
tracheobronchial tree.

16. DRUGS FOR COUGH
1. PHARYNGEAL DEMULCENTS Lozenges, Cough drops,
Linctuses containing syrup, Glycerine.
2. EXPECTORANTS
a. BRONCHIAL SECRETION ENHANCERS Sodium/Potassium
citrate, Potassium iodide, Guaiphenesin, Ammonium chloride.
b. MUCOLYTICS Bromhexine, Ambroxol, Acetylcysteine

17. DRUGS FOR COUGH
3. ANTITUSSIVES (COUGH CENTRE SUPPRESSANTS)
a. OPIOIDS Codeine, Pholcodeine, Ethylmorphine
b. NON OPIOIDS Noscapine, Dextromethorphan
c. ANTIHISTAMINES Cholrpheniramine maleate, Diphenhydramine,
Promethazine

18. SCREENING OF ANTITUSSIVE ACTIVITY
• Antitussive activity after irritant inhalation in guinea pigs
• Cough induced by mechanical stimulation
• Cough induced by stimulation of the nervus laryngicus superior
• Cough induced by ammonia in cats
• Cough induced by sulfuric acid in dogs

19. ANTITUSSIVE ACTIVITY AFTER IRRITANT
INHALATION IN GUINEA PIGS
• PURPOSE AND RATIONALE
• Chemical activation of cough centres in brain
• Several animal species and several irritants have been used.
• Most frequent - citric acid induced cough in guinea pigs

20. ANTITUSSIVE ACTIVITY AFTER IRRITANT
INHALATION IN GUINEA PIGS
• PROCEDURE
• Guinea pigs – either gender – 300-400g
• Placed in cylindrical glass vessel with 2 tubes at either ends for influx
and efflux of aerosol.
• Latter tube side arm connecting to a tambour changes in pressure
registered
• Pinch clamp with variable screw permits regulation of sensitivity of
system

21. ANTITUSSIVE ACTIVITY AFTER IRRITANT
INHALATION IN GUINEA PIGS
• Animal exposed to aerosol of 7.5% citric acid in water for 10 min
• Number of tussive responses registered
• One hour later test substance orally/ SC
• 30 min later aerosol exposure
• Number of coughs during 10 min recorded.

22. ANTITUSSIVE ACTIVITY AFTER IRRITANT
INHALATION IN GUINEA PIGS
• EVALUATION
• Number of coughs after treatment is expressed as percentage of control
period. Using various doses, ED50 values can be calculated.
• CRITICAL ASSESSMENT
• Proven to be an effective method

23. MODIFICATIONS
• Citric cough model codeine, morphine, selective neurokinin 3
receptor antagonist, sigma-1 receptor agonists.
• Capsaicin inhalation guinea pigs GABA B agonists, NK1, NK2,
tachykinin receptor antagonist.
• Other irritants Ammonia, Nebulised sulphuric acid, Sulphur dioxide.

24. COUGH INDUCED BY MECHANICAL
STIMULATION
• RATIONALE-
• Cough can be induced by mechanical stimulation of trachea in
anaesthetized guinea pigs.
• INCLUSION CRITERIA-
• Male guinea pigs- 350-400g- maintained at temperature 21 ± 2 ͦ C; relative
humidity 55 ± 10 %; 12:12 light dark cycle; food and water ad libitum 1
week before use.

25. COUGH INDUCED BY MECHANICAL
STIMULATION- PROCEDURE
• Overnight fasting anesthesia with 25% urethane (4ml/kg IP)
induces surgical levels of analgesia without depressant effects on
respiratory function.
• Monitoring analgesia disappearance of head shaking in response to
ear pinch.
• Animals maintained at constant body temperature 37 ͦ C by means of
heated plate.

26. COUGH INDUCED BY MECHANICAL
STIMULATION- PROCEDURE
• Thin steel wire inserted into trachea through small incision near cricoid
cartilage
• Steel wire is pushed to reach bifurcation of trachea 5 min before and 30, 60
and 120 minutes after oral drug administration.
• One violent cough occurs upon each stimulation.
• Animals which respond to stimulus before dosing are selected
• Randomly assigned to receive the test drug at various doses or the standard
drug.

27. COUGH INDUCED BY MECHANICAL
STIMULATION- EVALUATION
• Evaluation of the statistical significance of the results is performed
with Student’s t-test for paired data.
• ED50 values are determined by logit transformation.

28. OTHER METHODS
• Nylon bristled stimulator thrust into the trachea in dogs
• Silver thread in decerebrated guinea pigs
• Vibration of an iron slung in trachea of a dog induced by an
electromagnet
• Electrical stimulation of trachea via bronchoscope
• Through implanted copper electrodes.

29. COUGH INDUCED BY STIMULATION OF
NERVUS LARYNGICUS SUPERIOR
• PURPOSE
• Stimulation of nervus laryngicus superior induces coughing
• Antitussive agents with predominant central action suppress coughing reflex.
• INCLUSION CRITERIA
• Cats either gender 2-3 kg

30. COUGH INDUCED BY STIMULATION OF NERVUS
LARYNGICUS SUPERIOR- PROCEDURE
• Anaesthetised 40 mg/kg IP; Pentobarbital
• Placed on a heated operating table; Extremities secure.
• Fur is shaved ventrally at neck
• Small incisions made at both sides of larynx
• Superior laryngeal nerves prepared carefully.

31. COUGH INDUCED BY STIMULATION OF NERVUS
LARYNGICUS SUPERIOR- PROCEDURE
• Trachea is exposed and cannulated after a median incision
• Connected to Fleisch tube (Size 00)
• Femoral artery cannulated via Statham pressure transducer for
registration of BP.
• Femoral vein cannulated for intravenous application of test
substances.

32. COUGH INDUCED BY STIMULATION OF NERVUS
LARYNGICUS SUPERIOR- PROCEDURE
• Small hook electrodes- attached to each laryngeal nerve
• Wave impulse Frequency 50 Hz; Impulse width 0.5 ms; Amplitude
0.2 to 1 Volt; Duration 1-10s every 5 min
• The intensity of the forced expiration is measured by the Fleisch
pneumotachograph and recorded simultaneously with blood pressure
on a polygraph.

33. COUGH INDUCED BY STIMULATION OF NERVUS
LARYNGICUS SUPERIOR- PROCEDURE
• Control response to 3 stimuli recorded before IV application of test
compound.
• Stimuli repeated every 5 min after test compound/ standard is injected
• Suppression of forced expiration is recorded over 1 hr
• Then, next dose/ standard drug is given.

34. COUGH INDUCED BY STIMULATION OF NERVUS
LARYNGICUS SUPERIOR- EVALUATION
• Total or partial suppression of the forced expiration are recorded over
time and expressed as percentage of control.
• Intensity and duration of the effect are compared with the standard.

35. OTHER METHODS
• Electrical stimulation of the dorsolateral region of the medulla with
bipolar needle electrodes oriented by means of a stereotactic
instrument.
• Cough in conscious dogs by stimulation of the vagus nerve in a
surgically prepared skin loop.

36. SCREENING OF EXPECTORANTS

37. SCREENING OF EXPECTORANTS
• In vitro studies of mucus secretion
• Acute studies of mucus secretion
• Studies of mucus secretion with chronic cannulation
• Broncheoalveolar lavage
• Ciliary Activity
• Studies of mucociliary transport
• Culture of tracheal epithelial cells
• Alveolar macrophages.

38. IN VITRO STUDIES OF MUCUS SECRETION
• PURPOSE
• Mucus secretion has been studied in isolated tracheas from ferrets and
dogs.
• PROCEDURE
• Ferrets- either gender- 0.6-1.5kg
• Anaesthetized with sodium barbital IP.

39. IN VITRO STUDIES OF MUCUS SECRETION-
PROCEDURE
• Trachea exposed, cannulated with special Perspex cannula, 5 mm
below larynx.
• Animal sacrificed chest opened along midline.
• Trachea exposed to carina; Adjacent tissues removed.

40. IN VITRO STUDIES OF MUCUS SECRETION-
PROCEDURE
• Trachea- laryngeal end down- mounted in water jacketed organ bath.
• Bathed on its submucosal site with Krebs-Henseleit solution+ 0.1%
glucose at 37 ͦ C; Bubbled with 95% O2 and 5% CO2
• Lumen of trachea- air-filled.
• Plastic catheter inserted into lower cannula forms airtight seal
secretions withdrawn and collected periodically.

41. IN VITRO STUDIES OF MUCUS SECRETION-
PROCEDURE
• Volume of secretions weight difference of catheter lengths with/
without secretions.
• Portions of Ferret trachea cut longitudinally along posterior wall;
flattened; pinned to Perspex chamber simultaneous measurement of
mucus secretion and changes in tissue volume.
• Promotion of mucus secretion electrical field stimulation at 50-
100V 20 Hz, 1-2ms through pins holding tissue.

42. IN VITRO STUDIES OF MUCUS SECRETION-
PROCEDURE
• Epithelium coated with powdered tantalum dust mucus secretion
from submucosal glands through gland ducts tantalum traps mucus.
• Hemispherical hillocks formed.
• Surface photographed through dissecting microscope; hillock
diameters measured
• Secretion volume per unit area calculated;

43. • Drugs can be added to submucosal bath.
• EVALUATION
• Secretory response after electrical stimulation in the presence/ absence
of drugs is recorded after 45, 90 & 135 minutes.

44. ACUTE STUDIES OF MUCUS SECRETION
• PURPOSE
• To study the influence of drugs, methods of collecting bronchial mucus.
• PROCEDURE
• Rabbits 2.5- 3.5kg anaesthetized IP 1.1 to 1.4 g/kg urethane.
• Trachea exposed; Half opened 2 cm below cricoid cartilage
• T-cannula one arm- inserted into trachea.
• Perpendicular arm connected to air outlet of humidifier (temp 35-
38 ͦ C; humidity 80% )

45. ACUTE STUDIES OF MUCUS SECRETION-
PROCEDURE
• Other arm collection tube
• Rabbit restrained in supine position 60 ͦ inclined board with head
downwards.
• Mucus secretion vagal stimulation/ ammonium chloride given by
stomach tube/ pilocarpine IP.

46. EVALUATION
• Time response curves after stimulants of mucus secretion are
compared with data from untreated animals.

47. STUDIES OF MUCUS SECRETION WITH
CHRONIC CANNULATION
• PURPOSE
• Several techniques have been developed for chronic collection of mucus
(Wardell et al 1970; Yankell et al 1970; Scuri et al 1980)
• PROCEDURE
• Beagle dogs 9-11kg
• Anaesthetised IV injection 35-40 mg/kg pentobarbital sodium.

48. STUDIES OF MUCUS SECRETION WITH
CHRONIC CANNULATION- PROCEDURE
• Cervical trachea exposed midline skin incision; blunt dissection of
muscles.
• Transection of segment of 10 rings length with intact blood and nerve
supply.
• Cephalic & caudal parts of trachea- anastomosed with interrupted gut
sutures to re-establish patent airway.

49. STUDIES OF MUCUS SECRETION WITH
CHRONIC CANNULATION- PROCEDURE
• Isolated segment- loosened slightly- turned 180 ͦ to reverse cilia
movement
• Funnel shaped silicone canula attached to outer surface of proximal
end of tracheal segment with surgical mesh and sutured in place
• After cannulation segment placed in pocket below sternohyoid
muscle; cannula brought to surface and exteriorized.

50. STUDIES OF MUCUS SECRETION WITH
CHRONIC CANNULATION- PROCEDURE
• Isolated segment closed at caudal end with interrupted gut sutures.
• Mucosal surface of cervical end is sutured; Interrupted skin sutures.
• Muscles, Skin normally sutured.
• In 2-3 weeks, skin heals over small stoma subcutaneous pouch of
functioning tracheal tissue

51. STUDIES OF MUCUS SECRETION WITH
CHRONIC CANNULATION- PROCEDURE
• A balloon may be placed into the pouch
• Pressure changes in balloon contraction of smooth tracheal
muscles physostigmine injection/ vagal stimulation/ relaxation after
atropine injection recorded demonstrating parasympathetic
innervation

52. STUDIES OF MUCUS SECRETION WITH
CHRONIC CANNULATION- EVALUATION
• Parasympathomimetic stimulation (0.5mg/kg pilocarpine SC)
increases flow rate of tracheal fluids.
• Pressure changes in balloon after injection of parasympathomimetic/
sympathomimetic drugs are compared with baseline values.

53. REFERENCES
• Textbook of Medical Physiology, Guyton, 11th edition
• Davidson’s Principles and Practice of Medicine, 21st edition.
• Ganong’s review of medical physiology, 23rd edition.
• Essentials of Medical Pharmacology, K. D. Tripathi, 6th edition
• Drug Discovery and Evaluation of Pharmacological Assays- H.
Gerhard. Vogel- 3rd edition
• Polverino et al.: Anatomy and neuro-pathophysiology of the cough
reflex arc. Multidisciplinary Respiratory Medicine 2012 7:5.

54. THANK YOU