Screening of antitussives and expectorants

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??

4. 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 .

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

6. 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.

7. 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 .

8. 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

9. 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.

10. 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.

11. 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

12. 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.

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

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

15. 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

16. 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

17. 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

18. 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.

19. 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

20. 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.

21. 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.

22. 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.

23. 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.

24. COUGH INDUCED BY MECHANICAL STIMULATION- EVALUATION Evaluation of the statistical significance of the results is performed with Student’s t -test for paired data . ED 50 values are determined by logit transformation.

25. 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.

26. 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

27. 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.

28. 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.

29. 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.

30. 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.

31. 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 .

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

33. SCREENING OF EXPECTORANTS

34. 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.

35. 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 .

36. 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.

37. 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% O 2 and 5% CO 2 Lumen of trachea- air-filled. Plastic catheter  inserted into lower cannula forms airtight seal secretions withdrawn and collected periodically.

38. 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.

39. 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;

40. 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.

41. 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% )

42. 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.

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

44. 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.

45. 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.

46. 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.

47. 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

48. 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

49. 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.

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

51. THANK YOU