EVALUATION OF THE ANTI-INFLAMMATORY ACTIVITY OF TEST FORMULATIONS IN MOUSE MODEL OF CIGARETTE SMOKE EXTRACT-INDUCED PULMONARY INFLAMMATION

 EVALUATION OF THE ANTI-INFLAMMATORY ACTIVITY OF TEST FORMULATIONS IN MOUSE MODEL OF CIGARETTE SMOKE EXTRACT-INDUCED PULMONARY INFLAMMATION

1.0  TEST SYSTEM DETAILS:

Species                   : Mus musculus (Mice)

Strain                     : C57BL/6

Age                        : 6-8 weeks

Body Wight            : 20- 25g

Sex                         : Male/Female

No. of animals        : 8 /Group

Total animals          : 56

Study duration        : 60 days

 

2.0   ALLOCATION OF GROUPS:

Group No.	Group Description	Disease Induction agent administered	Treatment administered	Dose Volume and Route
G1	Normal Control	Normal saline (intranasal) × 28 days	0.5% MC, p.o.	10 ml/kg, p.o.
G2	Disease Control	50 μL of cigarette smoke extract (CSE, intranasal) × 28 days	0.5% MC, p.o.
G3	Reference Control		Roflumilast-10 mpk, q.d. in 0.5% MC
G4	Treated with low dose of Test-X1		Test-X1 mg/kg, b.i.d.  in 0.5% MC
G5	Treated with intermediate dose 1 of Test-X2		Test-X2 mg/kg,    b.i.d. in 0.5% MC
G6	Treated with intermediate dose 2 of Test-X3		Test-X3 mg/kg,   b.i.d. in 0.5% MC
G7	Treated with high dose of Test-X4		Test-X4 mg/kg,  b.i.d. in 0.5% MC

X1, X2, X3, and X4 are defined as the incremental doses of the test formulations. MC-Methyl Cellulose, p.o.-per os. The dose range will be from 10 mg/kg to 1000 mg/kg, q.d.: quaque die; bid: bis in die

3.0  METHOD:

Healthy animals will be selected, randomized based on body weight and allocated into 7, different groups consisting of 8 animals each.

Animals will be anaesthetized by ketamine & xylazine. Subsequently, they will be held in a vertical position and 50 μL of cigarette smoke extract (CSE) will be placed on the edge of the nostril and inhaled by the mouse. The extract will be instilled for 28 days.

Animals of the Group G1 will serve as normal control and administered 0.5% MC, p.o.

Disease control (G2) animals will receive 0.5% MC, p.o.

Animals of group G3 will be treated with reference drug Roflumilast 10 mg/kg, p.o.

Animals of group G4-G7 will be treated with Test Formulations (X1, X2, X3 and X4) at different dose levels ranging from 10-1000 mg/kg, b.i.d.

On last day, the animals will be euthanized by overdose of thiopentone. After suitable anaesthesia but before the animal dies, blood will be collected from the retro-orbital plexus. Subsequently, tracheostomy will be performed and the trachea will be cannulated with a polyethylene catheter. Bronchoalveolar lavage fluid will be collected by flushing the airways with 4 x 0.5 ml HBSS containing 10mM EDTA. Lungs will be subsequently harvested. The left lung will be fixed in 10% neutral buffered formalin for histopathological and the right lung will be stored at -80°C for biochemical and molecular evaluations.

 

4.0  END POINT PARAMETER(S):

·       Total and differential leukocyte counts in BALF

·       TNF-α and Muc5AC

·       Serum Cotinine Levels

·       Protein kinase A (PKA) & Protein kinase C (PKC) activity

·       Histone deacetylase (HDAC) activity

·       SOD and IL-6 in serum

·       Protein expression

·       Histological Analysis of lung tissue.

5.0  REFERENCE(S):

  I.          Margaret K. Elliott, Joseph H. Sisson, William W. West, Todd A. Wyatt Differential in vivo effects of whole cigarette smoke exposure versus cigarette smoke extract on mouse ciliated tracheal epithelium. Exp Lung Res. 2006, 32 (3-4), 99–118.

II.          David J. Lamb, Nicole Parker, Kristina Ulrich, Roddy Walsh, Mike Yeadon and Steven M. Evans, Characterization of a Mouse Model of Cigarette Smoke Extract-Induced Lung Inflammation. Journal of Pulmonary & Respiratory Medicine, 2012, 2,3.

III.    Kyoung-Hee Lee, Jinwoo Lee, Jiyeong Jeong, Jisu Woo, Chang-Hoon Lee and Chul-Gyu Yoo, Cigarette smoke extract enhances neutrophil elastase-induced IL-8 production via proteinase-activated receptor-2 upregulation in human bronchial epithelial cells. Experimental & Molecular Medicine, 2018, 50,79.

IV.    Zhi-Hui He, Ping Chen, Yan Chen, Sheng-Dong He, Ji-Ru Ye, Hong-Liang Zhang, Jun Cao, Comparison between cigarette smoke-induced emphysema and cigarette smoke extract-induced emphysema. Tobacco Induced Diseases, 2015, 13,6.

                                                                  END THE DOCUMENT

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