EVALUATION OF ANTI-ARTHRITIC POTENTIAL OF TEST SAMPLE USING COLLAGEN ANTIBODY INDUCED ARTHRITIS IN EXPERIMENTAL MICE
Collagen antibody-induced arthritis (CAIA) is a simple mouse model of rheumatoid arthritis that can be used to address questions of pathogenic mechanisms and to screen candidate therapeutic agents. Arthritis is stimulated by the administration of a cocktail of monoclonal antibodies that are directed to conserved auto-antigenic epitopes in collagen type II, followed by endotoxin. The antibody-induced arthritis model offers several key advantages over the classic collagen-induced arthritis (CIA) model. These include rapid disease onset, high uptake rate, synchronicity, and the capacity to use genetically modified mice, such as transgenics and knockouts.
Arthrogen-CIA (Chondrex- 4 clone cocktail) is an arthritis-inducing mixture of four mAb to anti-CII resuspended in sterile Dulbecco’s PBS. All four mAb in this mixture recognize the conserved epitopes (CB11) shared by various species of CII and cross-react with homologous and heterologous CII. Three of these four mAb (IgG2a: clones F10-21, A2-10, D8-6)) recognize antigenic epitopes clustered within an 84 aa residue fragment, LyC2, of CB11, and the fourth mAb (IgG2b: clone D1-2G) reacts with LyC1.
2.0 TEST SYSTEM DETAILS:
Species : Mus musculus (Mouse)
Strain : C57BL/6; BALB/c; DBA/1
Age : 8-10 Weeks
Body Wight : 25-30 g
Sex : Male or Female
No. of animals : 8 /Group
3.0 ALLOCATION OF GROUPS:
*The dose and ROA (Routes of administration) will be decided based on the type of reference drug
Etanercept can be used as standard drug at the dose of 3mpk; SC daily
Note- Newly 5-clone cocktail can be used for the arthritis development. The arthritogenicity of the new 5-clone cocktail is two times greater than our 4-clone cocktail.The dose of the 5-clone cocktail is 1.5mg/mouse followed by 50µg LPS or 10 mg 5-clone cocktail alone
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.
· On day-0, mice will be received a single-dose intraperitoneal (i.p.) injection of a cocktail of 4 monoclonal antibodies to type II collagen (6-8 mg/mouse; Chondrex, Redmond, WA, USA) followed by i.p. injection of 50 μg of lipopolysaccharide (LPS from Escherichia coli strain 011B4) on day 3.
· Drug treatment will be initiated from day 0 (prophylactic) or day- 4 (after the arthritis development).
· On day 4, clinical disease activity will be assessed and based on the score, all the animals will be randomized into different groups.
· 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 16.
· Group G1 animal will be treated as disease control and treated with normal saline or Na- CMC.
· Animals of group G2 will be treated orally with dexamethasone at the dose of 0.5mpk.
· Group G3, G4 and G5 animals will be treated with plant extract(s) at different dose levels.
· Clinical observation, body weight, CDA and Paws thickness will be assessed daily (day-0 to day 10).
· 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 Khachigian LM1. Collagen antibody-induced arthritis. Nat Protoc. 2006;1(5):2512-6.
6.2 Zhanli Xie, Jihong Dai, Aizhen Yang and Yi Wu. A role for bradykinin in the development of anti-collagen antibody-induced arthritis. Rheumatology 2014;53:13011306
6.3 Nirmal K. Banda, Kazue Takahashi, Allyson K. Wood, V. Michael Holers and William P. Arend. Pathogenic complement activation in collagen antibody induced arthritis in mice requires amplification by the alternative pathway.
6.4 Adrian Richard Moore, Sarah Allden, Tim Bourne, Maria C Denis, Ksanthi Kranidioti, Remi Okoye, Yannis Sotsios,Zofia Stencel, Alexander Vugler, Gillian Watt, and Stevan Shaw. Collagen II antibody-induced arthritis in Tg1278TNFko mice: optimization of a novel model to assess treatments targeting human TNFα in rheumatoid arthritis. J Transl Med. 2014; 12: 285.
6.5 https://www.chondrex.com/collagen-antibody-arthritis
6.6 Oestergaard S1, Rasmussen KE, Doyle N, Varela A, Chouinard L, Smith SY, Qvist P, Karsdal MA. Evaluation of cartilage and bone degradation in a murine collagen antibody-induced arthritis model. Scand J Immunol. 2008 Mar;67(3):304-12.
6.7 Adrian Richard Moore, Sarah Allden, Tim Bourne, Maria C Denis, Ksanthi Kranidioti, Remi Okoye, Yannis Sotsios, Zofia Stencel, Alexander Vugler, Gillian Watt and Stevan Shaw. Collagen II antibody-induced arthritis in Tg1278TNFko mice: optimization of a novel model to assess treatments targeting human TNFα in rheumatoid arthritis. Journal of Translational Medicine 2014, 12:285.
|
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
Etanercept can be used as standard drug at the dose of 3mpk; SC daily
Note- Newly 5-clone cocktail can be used for the arthritis development. The arthritogenicity of the new 5-clone cocktail is two times greater than our 4-clone cocktail.The dose of the 5-clone cocktail is 1.5mg/mouse followed by 50µg LPS or 10 mg 5-clone cocktail alone
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.
· On day-0, mice will be received a single-dose intraperitoneal (i.p.) injection of a cocktail of 4 monoclonal antibodies to type II collagen (6-8 mg/mouse; Chondrex, Redmond, WA, USA) followed by i.p. injection of 50 μg of lipopolysaccharide (LPS from Escherichia coli strain 011B4) on day 3.
· Drug treatment will be initiated from day 0 (prophylactic) or day- 4 (after the arthritis development).
· On day 4, clinical disease activity will be assessed and based on the score, all the animals will be randomized into different groups.
· 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 16.
· Group G1 animal will be treated as disease control and treated with normal saline or Na- CMC.
· Animals of group G2 will be treated orally with dexamethasone at the dose of 0.5mpk.
· Group G3, G4 and G5 animals will be treated with plant extract(s) at different dose levels.
· Clinical observation, body weight, CDA and Paws thickness will be assessed daily (day-0 to day 10).
· 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 Khachigian LM1. Collagen antibody-induced arthritis. Nat Protoc. 2006;1(5):2512-6.
6.2 Zhanli Xie, Jihong Dai, Aizhen Yang and Yi Wu. A role for bradykinin in the development of anti-collagen antibody-induced arthritis. Rheumatology 2014;53:13011306
6.3 Nirmal K. Banda, Kazue Takahashi, Allyson K. Wood, V. Michael Holers and William P. Arend. Pathogenic complement activation in collagen antibody induced arthritis in mice requires amplification by the alternative pathway.
6.4 Adrian Richard Moore, Sarah Allden, Tim Bourne, Maria C Denis, Ksanthi Kranidioti, Remi Okoye, Yannis Sotsios,Zofia Stencel, Alexander Vugler, Gillian Watt, and Stevan Shaw. Collagen II antibody-induced arthritis in Tg1278TNFko mice: optimization of a novel model to assess treatments targeting human TNFα in rheumatoid arthritis. J Transl Med. 2014; 12: 285.
6.5 https://www.chondrex.com/collagen-antibody-arthritis
6.6 Oestergaard S1, Rasmussen KE, Doyle N, Varela A, Chouinard L, Smith SY, Qvist P, Karsdal MA. Evaluation of cartilage and bone degradation in a murine collagen antibody-induced arthritis model. Scand J Immunol. 2008 Mar;67(3):304-12.
6.7 Adrian Richard Moore, Sarah Allden, Tim Bourne, Maria C Denis, Ksanthi Kranidioti, Remi Okoye, Yannis Sotsios, Zofia Stencel, Alexander Vugler, Gillian Watt and Stevan Shaw. Collagen II antibody-induced arthritis in Tg1278TNFko mice: optimization of a novel model to assess treatments targeting human TNFα in rheumatoid arthritis. Journal of Translational Medicine 2014, 12:285.
END OF DOCUMENTS
You may like to read theses links:
Hi. I’m Writer of Researchsop.com. ’ ’ Please share these SOPs to all concern pharma people for their development. I like to fullfill the need of curious people. These things inspire me to make things looks better.
0 comments:
Post a Comment