EVALUATION OF PROTECTIVE EFFECT OF TEST SAMPLE AGAINEST 4-ETHOXYMETHYLENE-2-PHENYL-2-OXAZOLIN-5-ONE INDUCED ATOPIC DERMATITIS IN MICE

                                                                       STUDY PROTOCOL

EVALUATION OF PROTECTIVE EFFECT OF TEST SAMPLE AGAINEST 4-ETHOXYMETHYLENE-2-PHENYL-2-OXAZOLIN-5-ONE INDUCED ATOPIC DERMATITIS IN MICE

1.0 INTRODUCTION:

Atopic dermatitis (AD) is a chronic inflammatory skin disease characterized by lesions from severe pruritus, epidermal hyperplasia, edema, erythema and erythematous plaque. AD can persist for an entire lifetime. It strongly impairs the quality of life of affected patients. Atopic skin inflammation is regulated by diverse inflammatory cytokines like Interleukin 4 (IL-4), IL-5 and IL-13 are hyperproduced in AD patients compared with healthy patients, and IL-4 and Interferon-γ are clearly increased in acute AD.

2.0  TEST SYSTEM DETAILS:

Species             : Mus musculus (Mouse)

Strain                : Swiss albino or C57bl/6 or Balb/c

Age                   : 6-8 weeks

Body Wight        : 20-25 g

Sex                   : Male or Female

No. of animals    : 8 /Group

Total animals     : 56 + 8 extra = 64

3.0  ALLOCATION OF GROUPS:

Groups	Treatment	Dose; ROA	No. of Animals
G1	Normal Control	Normal saline or 0.25% Na-CMC	8
G2	Disease Control	Normal saline or 0.25% Na-CMC	8
G3	Reference Drug- Dexamethasone	1 mpk; p.o.	8
G4	Plant Extract-1	X mpk; p.o.	8
G5	Plant Extract-2	XX mpk; p.o.	8
G6	Plant Extract-3	XXX mpk; p.o.	8

*The dose and ROA (Routes of administration) will be decided based on the type of reference drug

 

4.0 METHODOLOGY:

· The study protocol (Form B) shall be approved from the IAEC before commencing the experiment.

· Animals shall be procured from the CPCSEA authorized vendor.

· Animals shall be quarantined for 1 week as per the in house SOP.

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

· The mice will be sensitized with 1.5% oxazolone by application to the clipped abdomen (100µl) 7 days before challenge with 0.1, 0.5 or 1.5% oxazolone at the ear (20µl/ear).

· Before challenge (0 hour), the ear thickness will be measured using a digital vernier caliper and post challenge at 6, 24, 48 and 72 hr.

· The increase in ear thickness will be determined by subtracting the ear thickness of day 0 (before challenge or acetone application) from the respective time point thickness.

· Group G1 & G2 animal will be treated as normal control and disease control and treated with normal saline or Na- CMC.

· Animals of group G3 will be treated orally with dexamethasone at the dose of 1mpk.

· Group G4, G5 and G6 animals will be treated with test compound at different dose levels.

· Animals will be treated daily orally with vehicle or test sample at different dose level throughout the experiment.

5.0 END POINT PARAMETER(S):

·       Ear Thickness

·       Ear Biopsy weight

·       MPO activity in ear tissue

·       Histopathology of skin tissue (extent of the lesion, severity of hyperkeratosis, number and size of Pustules, height of epidermal hyperplasia, severity of inflammation in the dermis and soft tissue).

 

6.0  REFERENCE(S):

6.1 Luting Zeng, Yingqin Liu, Congcong Xing, Yijie Huang, Xin Sun, and Guangchen Sun. Saponin from Periploca forrestii Schltr Mitigates OxazoloneInduced Atopic Dermatitis via Modulating Macrophage Activation. Mediators of Inflammation Volume 2020, Article ID 4346367, 13 pages https://doi.org/10.1155/2020/4346367.

6.2 Young Bok Lee, Su Jin Kim, Sae Mi Park, Kyung Ho Lee, Hyung Jin Han, Dong Soo Yu, So Youn Woo , Seong Taek Yun , Se-Yeong Hamm , Hong Jig Kim , Jin-Wou Kim. Immunomodulatory Effects of Deokgu Thermomineral Water Balneotherapy on Oxazolone-Induced Atopic Dermatitis Murine Model. Ann Dermatol Vol. 28, No. 2, 2016.

6.3 Sullim Lee, Hyun Jegal, Sim-Kyu Bong, Kyeong-No Yoon, No-June Park, Myoung-Sook Shin Min Hye Yang, Yong Kee Kim, and Su-Nam Kim. Anti-Atopic Effect of Acorn Shell Extract on Atopic Dermatitis-Like Lesions in Mice and Its Active phytochemicals. Biomolecules 2020, 10 (1), 57 https://doi.org/10.3390/biom10010057

6.4 Shishu Goindi, Gautam Kumar, and Amanpreet Kaur. Novel flexible vesicles based topical formulation of levocetirizine: in vivo evaluation using oxazolone-induced atopic dermatitis in murine model. J Liposome Res, 2014; 24(3): 249–257.

                                 END OF DOCUMENT

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1. List of All SOPs and Documents for In-vivo Laboratory

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3. List of Pharmacological Study Models with details about disease Inducing agent, Standard or Reference Drug and related parameters.

4.List of OECD Guidelines or Toxicological Studies

5.List of Pharmacopeial Test or Quality Control Test or Batch Testing Parameters (In-vivo)

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