PRECLINICAL SCREENING OF ASTHMA AND CHRONIC OBSTRUCTIVE PULMONARY DISORDER

PRE CLINICAL SCREENING METHOD FOR ANTI ASTHMATICS AND COPD

1. PRECLINICAL SCREENING MODELS FOR DRUGS USED IN ASTHMA and COPD PRESENTED BY LEKHASHREE M G 1 ST SEM M PHARM DEPT OF PHARMACOLOGY PRESENTED TO DR BHARATHI K N PROFESSOR DEPT OF PHARMACOLOGY 2/21/2024 1 preclinical screening methods of asthma & COPD

2. CONTENTS Introduction – Asthma and COPD Pathogenesis Drugs used to treat asthma Preclinical models Reference 2/21/2024 2 preclinical screening methods of asthma & COPD

3. INTRODUCTION –ASTHMA Bronchial asthma is a long term inflammatory disease of the airways of the lungs ,it is characterized by variable and recurring symptom's , reversible airflow obstruction and easily triggered bronchospasm Asthma is an episodic disease manifested clinically by Paroxysms of dyspnoea Cough Wheezing During an asthma attack , the lining of the Bronchial tubes swells causing the airways to Narrow and reducing the flow of the air 2/21/2024 3 preclinical screening methods of asthma & COPD

4. PATHOGENISIS During first contact of the host with antigen sensitization takes place Antigen is captured by the antigen presenting dendritic cell It presented to the T cell which then differentiates into TH2cell The TH2 cell release mediators like IL-3,IL-4,IL-5 IL4 causes activation of b cells, it differentiate to form IgE secreting plasma cells IgE antibodies formed and binds on the mast cells 2/21/2024 4 preclinical screening methods of asthma & COPD

5. During the second contact with same antigen IgE antibodies on the surface of the mast cells – basophils are activated Cause degranulation of mast cells –basophils The released granules contain important chemical and enzymes with pro inflammatory properties , the mediators such as Histamine Serotonin Vasoactive intestinal peptide Leukotrienes B4 and D4 Prostaglandin Bronchoconstriction Increased secretion from glands 2/21/2024 5 preclinical screening methods of asthma & COPD

6. COPD (chronic obstructive pulmonary disorder) COPD is a chronic irreversible airflow obstruction , lung damage and inflammation of the air sacs (alveoli) COPD is characterized by chronic bronchitis and emphysema (destruction of walls alveoli) CAUSES- exposure to tobacco i.e. smoking, ambient air pollution sudden airway Constriction due to inhaled irritant’s bronchial asthma genetic alpha 1 antitrypsin Deficiency 2/21/2024 6 preclinical screening methods of asthma & COPD

7. 2/21/2024 7 preclinical screening methods of asthma & COPD

8. Humanized monoclonal antibody against IgE Inhibits the degranulation of mast cells Corticosteroids inhibit the phospholipase Zileution inhibit LOX enzyme Mechanism of action 2/21/2024 8 preclinical screening methods of asthma & COPD

9. SYNTHESIS OF LEUKOTRIENES 2/21/2024 9 preclinical screening methods of asthma & COPD

10. BRONCHODILATORS Beta agonists stimulates adenylate cyclase 2/21/2024 10 preclinical screening methods of asthma & COPD

11. PRECLINICAL SCREENING MODELS 2/21/2024 11 preclinical screening methods of asthma & COPD

12. SCREENING METHODS IN VIVO METHODS Broncho spasmolytic activity in anaesthetized guinea pigs Arachidonic acid/PAF induced respiratory vascular dysfunction Bronchial hyperactivity in guinea pigs Body plethysmography and respiratory parameters after histamine induced bronchoconstriction in anesthetized Guinea pigs Airways microvascular leakage Anaphylactic micro shock in Guinea pig 2/21/2024 12 preclinical screening methods of asthma & COPD

13. SCREENING METHODS IN VITRO METHODS Binding assay Histamine receptor assay Cell culture method Caltex technique WST assay Test in isolated organs Spasmolytic activity in trachea Spasmolytic activity in Guinea pig lungs Bronchia l perfusion of isolated lungs Vascular & airway responses to isolated lung Reactivity of isolated perfused guinea pig trachea 2/21/2024 13 preclinical screening methods of asthma & COPD

14. IN VIVO MODELS 2/21/2024 14 preclinical screening methods of asthma & COPD

15. BRONCHO SPASMOLYTIC ACTIVITY IN ANESTHETIZED GUINEA PIG (KONZETT- ROSSLER METHOD) PURPOSE AND RATIONALE 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 broncho spasmolytic effect by measuring the volume of air, which is not taken up by the lungs after bronchospasm. Animal – guniea pig ( 250-500 g) Anesthethic – urethane (1.25/kg) IP 2/21/2024 15 preclinical screening methods of asthma & COPD

16. BRONCHO SPASMOLYTIC ACTIVITY IN ANESTHETIZED GUINEA PIG PROCEDURE- Guinea pig anaesthetized with IP urethane, After this trachea is cannulated by two-way cannula. One arm of cannula is connected to respiratory pump via which guinea pig is artificially respired and another arm is connected to Statham P23 Db transducer. Artificial ventilation at frequency 60 strokes /min is maintained Excess air not taken up by lungs is measured and recorded CONT…… 2/21/2024 16 preclinical screening methods of asthma & COPD

17. Spasmogens and test substance  is administered via cannula in interna jugular vein.  The carotid artery is cannulated for measuring blood pressure . Testing Guinea-pigs receive the following spasmogen by i.v. administration: 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 2/21/2024 17 preclinical screening methods of asthma & COPD

18. After obtaining two bronchospasms of equal intensity, test compounds are administered I.V, S.C. The spasmogen is given again at the following time intervals: • 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. standard compounds are used tropine 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 2/21/2024 18 preclinical screening methods of asthma & COPD

19. BRONCHO SPASMOLYTIC ACTIVITY IN ANESTHETIZED GUINEA PIG EVALUATION- Results are expressed as percent inhibition of induced bronchospasm over the control agonistic responses . The ED50 value is calculated 2/21/2024 19 preclinical screening methods of asthma & COPD

20. BRONCHIAL HYPERACTIVITY IN GUINEA PIGS PURPOSE AND RATIONALE Symptoms like asphyctic convulsions resembling bronchial asthma in patients can be induced by inhalation of histamine or other bronchospasm inducing agents in guinea pigs. Agents are applied as aerosols produced by an ultra-sound nebulizer. The first symptoms are increased breathing frequency, forced inspiration, and finally asphyctic convulsions. The occurrence of these symptoms can be delayed by antagonistic drugs. Pre-convulsion time can be measured 2/21/2024 20 preclinical screening methods of asthma & COPD

21. The inhalation cages consist of 3 boxes each ventilated with an air flow of 1.5 l/min The animal is placed into box A to which the test drug or the standard is applied using an ultra-sound nebulizer LKBNB108 which provides an aerosol of 0.2 ml solution injection by infusion pump in 1 min Alternatively, the animal is treated orally or subcutaneously with the test drug or the standard. Box B serves as a sluice through which the animal is passed into box C. here it is exposed to an aerosol of a 0.1% solution of histamine hydrochloride provided by an ultra-sound nebulizer 2/21/2024 21 preclinical screening methods of asthma & COPD

22. Time until appearance of asphyctic convulsions is measured. Then, the animal is immediately withdrawn from the inhalation box. The aerosols are removed by applying low 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. 2/21/2024 22 preclinical screening methods of asthma & COPD

23. PURPOSE AND RATIONALE Arachidonic acid-metabolized into TXA2 and PGI2 TXA2-lungs-bronchoconstriction , which is independent from circulating platelets and leukotrienes TXA2 produced intracellularly in platelets induces a reversible thrombocytopenia PGI2- vessel wall - reduction of systolic & diastolic pressure. PAF-bronchoconstriction, thrombocytopenia, leukocytopenia, reduction of BP and increase of haematocrit. Test allows to evaluate the sites of action of drugs which interfere with the mechanisms of bronchoconstriction and thrombocytopenia. ARACHIDONIC ACID/PAF INDUCED RESPIRATORY VASCULAR DYSFUNCTION 2/21/2024 23 preclinical screening methods of asthma & COPD

24. PROCEDURE- Male guinea pigs (Pirbright White) weighing 300–600 g are anesthetized with 60 mg/kg pentobarbital sodium ( i.p. ). One of the jugular veins is cannulated for the administration of spasmogen and test compound Both external carotid arteries are cannulated; one is connected to a pressure transducer to register blood pressure, the other is used for blood withdrawal The trachea is connected to a Starling pump with an inspiratory pressure Spontaneous respiration is inhibited by intravenous injection of pancuronium (4 mg/kg) ARACHIDONIC ACID/PAF INDUCED RESPIRATORY VASCULAR DYSFUNCTION 2/21/2024 24 preclinical screening methods of asthma & COPD

25. 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 admi IV and spasmogen given again at intervals of 2, 10, 20 and 30 mins   Before and 30-45 sec after each dose of arachidonic acid applications, approx. 50 µl 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 if necessary ,60 mins after iv drug admins. ARACHIDONIC ACID/PAF INDUCED RESPIRATORY VASCULAR DYSFUNCTION 2/21/2024 25 preclinical screening methods of asthma & COPD

26. Blood samples are collected 30 s before and 15 s after each of the PAF applications. The number of leukocytes and hematocrit values are determined automatically Standard compounds: Dazoxiben HCl (inhibitor of thromboxane synthetase, TSI) acetylsalicylic acid- (inhibitor of cyclo-oxygenase, COI) ARACHIDONIC ACID/PAF INDUCED RESPIRATORY VASCULAR DYSFUNCTION 2/21/2024 26 preclinical screening methods of asthma & COPD

27. 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 pattern profile of the influence on bronchoconstriction, thrombocytopenia and blood pressure reduction, the mechanism of action of a test drug is conclude inhibitor of thromboxane synthetase inhibitor of cyclo-oxygenase ARACHIDONIC ACID/PAF INDUCED RESPIRATORY VASCULAR DYSFUNCTION 2/21/2024 27 preclinical screening methods of asthma & COPD

28. BODY PLETHYSMOGRAPHY AND RESPIRATORY PARAMETERS AFTER HISTAMINE INDUCED BRONCHOCONSTRICTION IN ANESTHETIZED GUINEA PIGS PURPOSE AND RATIONALE respiratory frequency and amplitude are recorded. The decrease of respiratory amplitude(diminished respiratory volume due to bronchoconstriction) and the reflectory increase of respiratory frequency after histamine inhalation are attenuated by bronchodilator drugs. This method can be used for various purposes: to evaluate the antagonism against bradykinin induced bronchoconstriction, the bronchodilator effects of K+ channel openers, or measure the effect of morphine on respiration in rats.  2/21/2024 28 preclinical screening methods of asthma & COPD

29. PROCEDURE- Guinea pigs of either sex weighing 400–600 g are anesthetized with 70 mg/kg pentobarbital i.p The trachea, pleural cavity, jugular vein, and carotid artery are prepared and cannulated The animals are mechanically ventilated with a 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. BODY PLETHYSMOGRAPHY AND RESPIRATORY PARAMETERS AFTER HISTAMINE INDUCED BRONCHOCONSTRICTION IN ANESTHETIZED GUINEA PIGS 2/21/2024 29 preclinical screening methods of asthma & COPD

30. The tracheal port is then connected with the respiration pump BODY PLETHYSMOGRAPHY AND RESPIRATORY PARAMETERS AFTER HISTAMINE INDUCED BRONCHOCONSTRICTION IN ANESTHETIZED GUINEA PIGS 2/21/2024 30 preclinical screening methods of asthma & COPD

31. Airflow rate into and out of the plethysmograph are measured as pressure difference with a No. 000 Flesch tube and a differential pressure transducer Airflow is calibrated by passing compressed air through a rotameter. The tidal volume (VT) is calculated from the flow signal. Transpulmonary pressure (P TP ) 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. P TP is a calibrated with a water monometer Signals from airflow tidal volume and transpulmonary pressure are fed into an on line computer system for calculation of pulmonary resistance R L & dynamic lung compliance C DYN BODY PLETHYSMOGRAPHY AND RESPIRATORY PARAMETERS AFTER HISTAMINE INDUCED BRONCHOCONSTRICTION IN ANESTHETIZED GUINEA PIGS 2/21/2024 31 preclinical screening methods of asthma & COPD

32. Three doses of test compound or standard are injected intravenously. Saline injections serve as controls Intravenous injections of histamine lead to a short decrease in C DYN and to a short increase in R L by approximately 200% compared with baseline. Challenges are repeated at 5-min intervals, yielding the same increase in R L during the whole 1-h experimental period. After 3 reproducible responses the test agent is administered intravenously 1 min before the histamine injection. BODY PLETHYSMOGRAPHY AND RESPIRATORY PARAMETERS AFTER HISTAMINE INDUCED BRONCHOCONSTRICTION IN ANESTHETIZED GUINEA PIGS 2/21/2024 32 preclinical screening methods of asthma & COPD

33. EVALUATION- Inhibition of histamine induced bronchoconstriction by various doses of test compound and standard is recorded ED50 values for inhibition in R L are calculated. the time course of histamine antagonism can be evaluated. BODY PLETHYSMOGRAPHY AND RESPIRATORY PARAMETERS AFTER HISTAMINE INDUCED BRONCHOCONSTRICTION IN ANESTHETIZED GUINEA PIGS 2/21/2024 33 preclinical screening methods of asthma & COPD

34. AIRWAYS MICROVASCULAR LEAKAGE PURPOSE AND RATIONALE Plasma exudation in guinea-pig airways in vivo can be determined by Evans Blue dye and is fairly correlated with radiolabeled 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 response 2/21/2024 34 preclinical screening methods of asthma & COPD

35. PROCEDURE- Female Dunkin-Hartley guinea pigs weighing 380–600 g are anesthetized with an initial dose of 1.5 g/kg urethane injected i.p. A tracheal cannula is inserted into the lumen of the cervical trachea, a polyethylene catheter into the left carotid artery to monitor blood pressure and heart rate and another polyethylene catheter into the external jugular vein for administration of 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. AIRWAYS MICROVASCULAR LEAKAGE 2/21/2024 35 preclinical screening methods of asthma & COPD

36. 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 test compound (bradykinin receptor antagonist)is given intravenously. Ten min later, Evans Blue dye(20 mg/ml) is injected i.v. for 1 min. After 1 min, bronchoconstriction and microvascular leakage is induced by injection of bradykinin AIRWAYS MICROVASCULAR LEAKAGE 2/21/2024 36 preclinical screening methods of asthma & COPD

37. Six min after induction of leakage, the thoracic cavity is opened, and a cannula is inserted into the aorta through a ventriculostomy. 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-: lower part of the trachea, main bronchi, the proximal 5 mm portion, and the distal intrapulmonary airways ,tissues are blotted dry and weighed. Evans blue dye is extracted in 2 ml formamide at 40 o C for 24hrs, and measured in spectrophotometer at 620 nm AIRWAYS MICROVASCULAR LEAKAGE 2/21/2024 37 preclinical screening methods of asthma & COPD

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 group AIRWAYS MICROVASCULAR LEAKAGE 2/21/2024 38 preclinical screening methods of asthma & COPD

39. Histamine is released from various sites in anaphylactic response Introduction of foreign particles in the body can cause microshock A microshock is apparently one that is interrupted before death and is repeatable PROCEDURE- Guinea pigs (200-300g) are sensitized with subcutaneous injection of egg albumin After 3 weeks the animals are exposed to an aerosol of 5% albumin They are removed from the chamber as soon as they become dyspneic 2/21/2024 preclinical screening methods of asthma & COPD 39 ANAPHYLATIC MICROSHOCK IN GUINEA PIG

40. The time from the start of exposure to severe dyspnea is referred as preconvulsion time . If no shock is seen for more than 6min, the animal is considered as protected and preconvulsion time is taken as infinite. After this control exposure the Guinea pig is treated with test drug and pre convulsion time is measured The degree of protection (p) is calculated from the formula 2/21/2024 preclinical screening methods of asthma & COPD 40 ANAPHYLATIC MICROSHOCK IN GUINEA PIG

41. IN VITRO MODELS 2/21/2024 41 preclinical screening methods of asthma & COPD

42. HISTAMINE H 1 RECEPTOR BINDING PURPOSE AND RATIONALE This method evaluates affinity of test compare to histamine - H1 receptor to measure their inhibitory activity on binding of pyrilamine (H1 antagonist) to guinea pig brain plasma membrane preparation. PROCEDURE Guinea pig (wt. 300 - 600 gm) is sacrificed by CO2 necrosis. 2/21/2024 42 preclinical screening methods of asthma & COPD

43. PROCEDURE Brains from guinea-pigs are homogenized in ice-cold Tris buffer (pH 7.5) Homogenate is centrifuged for 10 minute at 4 o C at 50,000 rpm. Supernatant is discarded and pellet is resuspended in buffer centrifuge again Aliquots of 1ml are frozen at –70 °C 50 ml 3H pyrilamine, 50 ml test Compound, 100 ml membrane suspension from guinea pig whole Brain per sample are incubated in a shaking bath at 25 C for 30 min incubation buffer used is Tris HCl buffer (50mM, pH 7.5) with 11 concentrations of 3H pyrilamine (0.1-50 × 10 -9 m) Saturation experiments are performed . 2/21/2024 43 preclinical screening methods of asthma & COPD

44. EVALUATION The following parameters are calculated: total binding of 3 H-pyrilamine non-specific binding: binding of 3 H-pyrilamine in the presence of mepyramine or doxepin specific binding = total binding – non-specific binding % inhibition of 3H-pyrilamine binding: 100 – specific binding as percentage of control value 2/21/2024 44 preclinical screening methods of asthma & COPD

45. SPASMOLYTIC ACTIVITY IN GUINEA PIG LUNGS PURPOSE AND RATIONALE- to check the spasmolytic activity induced by histamine and calcium ionophores Animal used – albino Guinea pig (300-450g) PROCEDURE Animal sacrificed with an overdose of ether , the chest cavity is opened and the lung are removed, cut into strips of 5cm in PSS Mounted in organ bath ,bubbled with carbogen(temp at 37 c)tissue left to equilibrate for 30-60 min Prior to testing , carbachol is added to check their ability to contract 2/21/2024 45 preclinical screening methods of asthma & COPD

46. SPASMOLYTIC ACTIVITY IN GUINEA PIG LUNGS After 20 min , two prevalues are obtained by adding the spasmogen histamine dihydrochloride 10 –6 g/ml for 5 min, or Ca – ionophore 5 × 10 –6 g/ml for 5 min, or Record contractile force at its maximum level 20 min eq. period, the spasmogen is administered again, five minutes later test compound is added in cumulative doses from 10 –8 to 10 –4 g/ml at 5 -10 min intervals Contractile response is determined 2/21/2024 46 preclinical screening methods of asthma & COPD

47. SPASMOLYTIC ACTIVITY IN GUINEA PIG LUNGS EVALUATION % inhibition of spasmogen induced contraction is calculated 2/21/2024 47 preclinical screening methods of asthma & COPD

48. SPASMOLYTIC ACTIVITY IN TRACHEA PURPOSE AND RATIONALE- The isolated tracheal chain of guinea pigs can be used to test for β-blocking activity this model can be used to test compounds which inhibit bronchospasms. It is used to detect β-sympathomimetic, H1-receptor blocking and leuko-triene receptor blocking properties of test drugs. 2/21/2024 48 preclinical screening methods of asthma & COPD

49. PROCEDURE Albino guinea pigs of either sex weighing 300–550 g are sacrificed by CO 2 narcosis -trachea dissected into individual rings (2-3 cartilaginous rings wide) 12- 15 rings tied together with silk threads-mounted in Organ bath-containing Krebs- Henseleit sol.-maintained at 37 C-0.5g tension-gassed with carbogen. Isometric contractions recorded via strain-gauge transduced on a polygraph-45mins equilibration-before administering spasmogen The following spasmogen are used: • carbachol (2 × 10 –7 g/ml) • histamine (10 –7 g/ml) • leukotriene LTD4 (10 –9 –10 –8 g/ml) SPASMOLYTIC ACTIVITY IN TRACHEA 2/21/2024 49 preclinical screening methods of asthma & COPD

50. When maximum contraction, after 10-12 min. ,standard drug (isoprenaline/aminophylline) added. The b ronchial responses allowed to plateau and are recorded. The tissue is rinsed thoroughly and control contractions are induced again by adding spasmogen . After initial spasm-test drug –contraction recorded at max. SPASMOLYTIC ACTIVITY IN TRACHEA 2/21/2024 50 preclinical screening methods of asthma & COPD

51. EVALUATION The percent inhibition of spasmogen induced contractions is calculated. From dose-response curves ED50 values can be calculated. SPASMOLYTIC ACTIVITY IN TRACHEA 2/21/2024 51 preclinical screening methods of asthma & COPD

52. BRONCHIA L PERFUSION OF ISOLATED LUNGS PURPOSE AND RATIONALE- 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 2/21/2024 52 preclinical screening methods of asthma & COPD

53. PROCEDURE Guinea pigs weighing about 200 g are sacrificed by a head blow. The chest is opened, the trachea cut at the upper end and removed with the lung The trachea is attached to the cannula of an perfusion apparatus. -one lung perfused-other tied off-lower part of lower lobe cut off-rest lung surface-scratched deeply The perfusion fluid has the following composition in percentage of anhydrous salts: NaCl 0.659, NaHCO 3 0.252, KCl 0.046, CaCl 2 0.005, MgCl 2 0.0135, NaH 2 PO 4 0.01, Na 2 HPO 4 0.008, glucose 5%, pH 8.0 BRONCHIA L PERFUSION OF ISOLATED LUNGS 2/21/2024 53 preclinical screening methods of asthma & COPD

54. The trachea is attached to the cannula of a perfusion apparatus which pumps the solution at a constant rate into a manometric tube connected with the perfused organ. Resistance to the flow (bronchoconstriction) results in an increase in the height of the column of fluid in the manometer After the lung is attached to a T-shaped cannula, the pump is set in motion and the fluid, after filling the lung, flows out of the system through the third opening of the cannula. By gentle pressure air bubbles are forced out of the lung into the overflow. BRONCHIA L PERFUSION OF ISOLATED LUNGS 2/21/2024 54 preclinical screening methods of asthma & COPD

55. Histamine HCl is added 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 Broncho dilating agents versus the standard is calculated with a 3 + 3 point assay including confidence limits. BRONCHIA L PERFUSION OF ISOLATED LUNGS 2/21/2024 55 preclinical screening methods of asthma & COPD

56. VASCULAR & AIRWAY RESPONSES TO ISOLATED LUNG PURPOSE AND RATIONALE- The isolated perfused rat lung allows the simultaneous registration of pulmonary vascular and airway responses to various drug Animal - Male Sprague-Dawley rats PROCEDURE rats weighing 300–350 g are intraperitoneally anesthetized with pentobarbital sodium (50 mg/kg). The trachea is cannulated with a short section of polyethylene tubing, connected to a rodent ventilator, and ventilated with room air enriched with 95% O 2 /5% CO 2 , with a tidal volume of 4–5 ml/kg and 2 cm H 2 O positive end-respiratory pressure . 2/21/2024 56 preclinical screening methods of asthma & COPD

57. The rats are heparinized with 1 000 units of intravenous heparin and are rapidly exsanguinated by withdrawing blood from the carotid artery The lung is exposed by median sternotomy, and a ligature is placed around the aorta to prevent systemic loss of blood The main pulmonary artery is catheterized, and the lung is removed and suspended in a warmed (39 °C), humidified (100%) water-jacketed chamber The perfusate solution (15 ml of heparinized blood and 5 ml modified Krebs- Henseleit solution) is placed in a reservoir The lungs are perfused with a peristaltic roller pump at a flow rate of 8–14 ml/min to maintain a physiological baseline pulmonary arterial perfusion pressure of 15 ±0.5 mm Hg. VASCULAR & AIRWAY RESPONSES TO ISOLATED LUNG 2/21/2024 57 preclinical screening methods of asthma & COPD

58. Pulmonary arterial perfusion pressure, airway pressure, and reservoir blood level are continuously monitored, electronically averaged and recorded with a polygraphy EVALUATION Changes (increase or decrease) in pulmonary arterial pressure and in airway pressure after injection of test compounds are measured in mm Hg and compared with baseline values. VASCULAR & AIRWAY RESPONSES TO ISOLATED LUNG 2/21/2024 58 preclinical screening methods of asthma & COPD

59. CULTEX technique is new experimental system for cultivation and exposure of cells intermediately at the air/ liquid interface It allows direct exposure of bronchial epithelial cells to gases, ultrafine particles or mixture of both 2/21/2024 preclinical screening methods of asthma & COPD 59 CELL CULTURE METHODS – CULTEX TECHNIQUE

60. 2/21/2024 preclinical screening methods of asthma & COPD 60 CELL CULTURE METHODS – CULTEX TECHNIQUE Cultured bronchial epithelial cells in plates washed with PBS Test group bronchial epithelial cells are incubated with test drugs for 24 hours Cells exposed to the clean air or different concentration of side steam smoke for 1hr

61. Parameters that can be estimated are number of cells , metabolic activity and glutathione concentrations Cell viability measurements are carried out using WST assay an electronic cell counting 2/21/2024 preclinical screening methods of asthma & COPD 61 CELL CULTURE METHODS – CULTEX TECHNIQUE

62. 2/21/2024 preclinical screening methods of asthma & COPD 62 CELL CULTURE METHODS – WST ASSAY

63. REFERNCE Drug Discovery and Evaluation: pharmacological assays by H Gerhard Vogel Gupta SK. (2016). Drug Screening Methods. 3rd ed. Jaypee Brothers Medical Publisher (P) Ltd, pp.683-696 2/21/2024 63 preclinical screening methods of asthma & COPD