EVALUATION OF THE EFFICACY OF TEST FORMULATIONS IN MOUSE MODEL OF MONOBENZONE-INDUCED VITILIGO

 EVALUATION OF THE EFFICACY OF TEST FORMULATIONS IN MOUSE MODEL OF MONOBENZONE-INDUCED VITILIGO

 

1.0  TEST SYSTEM DETAILS:

Species                   : Mus musculus (Mice)

Age                        : 4-6 weeks

Sex                        : Male/Female

No. of animals        : 10 /Group

Total animals         : 60

 

2.0   ALLOCATION OF GROUPS:

Group No.	Group Description	Disease Induction agent administered	Treatment administered	Dose Volume and Route
G1	Normal Control	50 mg of Vaseline on the dorsal back skin (2 × 2 cm area),  once a day for 50-consecutive days	0.5% MC, q.d.	10 ml/kg, p.o.
G2	Disease Control	50 mg of 40% Monobenzone cream on the dorsal back skin (2 × 2 cm area) , once a day for 50-consecutive days	0.5% MC, q.d.
G3	Reference Control		Tacrolimus-0.1% ointment applied topically, b.i.d. + 0.5% methylcellulose administered orally b.i.d.
G4	Treated orally with low dose of TF		TF -X1 mg/kg, in 0.5% MC., q.d.
G5	Treated orally with intermediate dose of TF		TF -X2 mg/kg, in 0.5% MC., q.d.
G6	Treated orally with high dose of TF		TF -X3 mg/kg, in 0.5% MC., q.d..

 

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

 

3.0  METHOD:

·         After completion of quarantine, healthy animals will be selected for the study. Subsequently, they will be randomized based on body weight and allocated into 6, different groups consisting of 10 animals each.

·         Post-randomization, animals will be acclimatized for 5 days in an experimental room earmarked for the experiment.

·         For the induction of vitiligo, animals allocated to groups G2-G6 will be applied 50 mg of 40% Monobenzone cream on the depilated dorsal back skin in an area of 2 × 2 cm, once a day for 50-consecutive days. The animals assigned to the Normal-control group (G1) will be applied 50 mg of Vaseline on the dorsal back skin in an area of 2 × 2 cm, once a day for 50-consecutive days.

·         Treatments to be administered:

Ø Animals of the Group G1 and G2, designated as normal -control and disease-control respectively, will be administered 0.5% MC, p.o., q.d.

Ø Animals of group G3 will be treated with reference drug, Tacrolimus (Ointment of strength 0.1%), topically. The animals will additionally receive 0.5% MC, q.d. by the oral route.

Ø Animals of group G4-G6 will be treated with TF, orally at different dose levels ranging from 10-1000 mg/kg, q.d.  

Ø Monobenzone administration will be stopped at Day 50 and the animals will continue to receive the treatments for 15 additional days.

·         The extent of depigmentation will be evaluated in animals once a week. The body of the animals will be divided into 4 anatomic regions including head/neck, trunk, limbs and tail. The depigmentation score will be awarded as the percentage of the anatomic site: 0%, 0; >0-10%, 1 point; >10-25%, 2 points; >25-50%, 3 points; >50-75%, 4 points; and >75-100%, 5 points.

·         On day 66, the animals will be sacrificed under overdose of thiopentone anaesthesia. After suitable anaesthesia but before the animal dies, blood will be collected and serum will be separated for estimation of biochemical parameters.

·         After completion of the procedure, the skin of the animals will be excised. One portion of the skin will be fixed in 10% neutral buffered formalin for the histopathological analysis, whereas the other portion will be snap frozen in liquid nitrogen and stored at -80°C for biochemical and gene expression analysis.

 

4.0  PARAMETERS TO BE EVALUATED:

·         Depigmentation scoring

·         Serum levels of TYR, MIF, MAO, TNF-α, IL-6, IL-13, IFN-γ, MDA, MPO and SOD

·         Gene expression analysis of RAB27A, CXCL5, CXCL10, CXCR4 and TGF-β in skin tissues

·         Histological analysis of skin tissues (H&E and Fontana-Masson staining)

·         Immunofluorescence analysis of skin tissues for CD8+ T cells

 

5.0  REFERENCES:

1.        Zhu, Y., Wang, S., Lin, F., Li, Q. & Xu, A. The therapeutic effects of EGCG on vitiligo. Fitoterapia 99, 243–251 (2014).

2.        Moreira, C. G. et al. Pre-clinical evidences of Pyrostegia venusta in the treatment of vitiligo. J. Ethnopharmacol. 168, 315–325 (2015).

3.        Bian, Y., Yu, H., Jin, M. & Gao, X. Repigmentation by combined narrow-band ultraviolet B/adipose-derived stem cell transplantation in the mouse model: Role of Nrf2/HO-1-mediated Ca2+ homeostasis. Mol. Med. Rep. 25, 1–9 (2022).

                                                            END OF THE DOCUMENT

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