EVALUATION OF ANTI-ARTHRITIC POTENTIAL OF TEST SAMPLE USING COMPLETE FREUND’S ADJUVANT INDUCED ARTHRITIS IN EXPERIMENTAL RAT

STUDY PROTOCOL

1.0 INTRODUCTION:

The disease is characterized by articular inflammation and by the formation of an inflammatory and invasive tissue, rheumatoid pannus that eventually leads to the destruction of joints. A number of strategies have been used to treat RA that target different pathological stages of the disease. The primary symptom of RA is inflammation of the synovial lining of the articular cartilage. Therefore, the majority of therapeutic strategies for RA inhibit inflammatory cytokines, such as tumor necrosis factor (TNF). However, none of the current treatments completely cure RA, therefore further studies are required to determine its exact pathology and enable the development of curative treatments

2.0 TEST SYSTEM DETAILS:

Species : Rattus norvegicus (Rat)

Strain : Wistar/Sprague dawley

Age : 8-10 Weeks

Body Wight : 180-200 g

Sex : Male or Female

No. of animals : 8 /Group

3.0 ALLOCATION OF GROUPS:

Groups	Treatment	Dose; ROA	No. of Animals
G1	Disease Control	Normal saline or 0.25% Na-CMC	8
G2	Reference Drug- Dexamethasone	0.5 mpk; p.o.* daily	8
G3	Plant Extract-1	X mpk; p.o.	8
G4	Plant Extract-2	XX mpk; p.o.	8
G5	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.

· Animals will be randomized based on body weight into five groups consisting of eight animals in each.

· On day 0 all animals (except normal control) will be injected 0.1ml of heat-killed Mycobacterium tuberculosis (Complete Freund’s adjuvant) into sub plantar of the left hind paw under mild anesthesia with Isoflurane.

· Animals of G1 served as normal control and treated (no CFA injection) with normal saline/0.25% sodium CMC

· Animals of G2 served as disease control and treated (only vehicle) with normal saline/0.25% sodium CMC

· Animals of G3 served as reference standard and treated with Dexamethasone 0.5mg/kg p.o.

· Similarly, animals of G4 and G5 treated with test sample at different dose level.

· Drug treatment will be initiated from day 0 (prophylactic) and continued for 28 days.

· The clinical disease activity (CDA) will be scored on a 4-point scale per paw: 0= normal, 1= erythema and mild swelling confined to the tarsals or ankle joint, 2= erythema and mild swelling extending from the ankle to the tarsals, 3= erythema and moderate swelling extending from the ankle to metatarsal joints, 4= erythema and severe swelling encompass the ankle, foot and digits, or ankylosis of the limb. Thus the maximum score of an animal will be 12 (only three paws are scored).

· The paw thickness and CDA will be assessed on 0, 7, 14, 21, and 28 day post CFA injection.

· Clinical observation, body weight, will be assessed on daily basis (day-0 to day 28).

· At the end of the experiment, blood will be removed via retro orbital plexus from all the animals and cytokine estimation will be performed.

· Animal will be humanely sacrificed; the fore limbs and hind limbs (paw, ankle and knee) will be removed surgically.

· Right hind will be stored in 10% buffered formalin for the histopathology whereas left hind limb and forelimbs will be processed for cytokine estimation.

5.0 END POINT PARAMETER(S):

· Incidence of arthritis

· Clinical Observation

· Body weight

· Clinical disease activity (CDA)

· Paw thickness

· Serum/paw tissue cytokines estimation- IL-6, IL-1β, TNF-α

· Histopathology of knee joint

6.0 REFERENCE(S):

6.1 Ekambaram, S., Perumal, S.S. & Subramanian, V. Evaluation of antiarthritic activity of Strychnos potatorum Linn seeds in Freund's adjuvant induced arthritic rat model. BMC Complement Altern Med 10, 56 (2010). https://doi.org/10.1186/1472-6882-10-56

6.2 Yan-li LIU , Hai-ming LIN , Rong ZOU , Jun-chao WU , Rong HAN , Laurence N RAYMOND , Paul F REID , Zheng-hong QIN. Suppression of complete Freund’s adjuvant-induced adjuvant arthritis by cobratoxin. Acta Pharmacol Sin 2009 Feb; 30 (2): 219–227

6.3 Guifang Chen, Yuxiu Song, Fang Ma , and Yuxia Ma. Anti-arthritic activity of D-carvone against complete Freund’s adjuvant-induced arthritis in rats through modulation of inflammatory cytokines. Korean J Physiol Pharmacol 2020;24(6):453-462 https://doi.org/10.4196/kjpp.2020.24.6.453

6.4 Wangin Kim, Sangbin Park, Chanhun Choi, Youg Ran Kim, Inkyu Park, Changseob Seo, Daehwan Youn, Wook Shin, Yumi Lee, Donghee Choi, Mirae Kim, Hyunju Lee, Seonjong Kim, and Changsu Na. Evaluation of Anti-Inflammatory Potential of the New Ganghwaljetongyeum on Adjuvant-Induced Inflammatory Arthritis in Rats. Evidence-Based Complementary and Alternative Medicine. Volume 2016, Article ID 1230294, 10 pages http://dx.doi.org/10.1155/2016/1230294

6.5 https://www.chondrex.com/documents/Rat%20AIA.pdf

6.6 Mbiantcha, M., Almas, J., Shabana, S.U. et al. Anti-arthritic property of crude extracts of Piptadeniastrum africanum (Mimosaceae) in complete Freund’s adjuvant-induced arthritis in rats. BMC Complement Altern Med 17, 111 (2017). https://doi.org/10.1186/s12906-017-1623-5

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