STUDY PROTOCOL
Species : Mus musculus (Mouse)
Strain : Swiss albino or C57bl/6 or Balb/c
Age : 6-10 weeks
Body Wight : 20-25 g
Sex : Male or Female
No. of animals : 8 /Group
Total animals : 56 + 6 extra = 62
3.0 ALLOCATION OF GROUPS:
|
Group No. |
Group Description |
Disease Induction agent administered |
Treatment administered |
Dose Volume and Route |
|
G1 |
Normal Control |
None |
0.5% MC, p.o., q.d |
10 mL/kg, p.o. |
|
G1 |
Disease
Control
|
20 μL of TPA solution (2.5 μg/ear of TPA dissolved in acetone)
on the right pinna and 20 µL of acetone on the left pinna (This will serve as the
vehicle control)
|
0.5% MC, p.o., q.d.+ Acetone on right pinna |
Oral: 10 mL/kg Topical application (acetone) : 20 µL |
|
G3 |
Reference Control |
0.5% MC, p.o. bid + Dexamethasone - 0.2 mg (dissolved in acetone) on right
ear, q.d. |
Oral: 5 mL/kg Topical application: 20 µL |
|
|
G4 |
Treated orally with low dose of Test Formulation-1 + Topically with Oil formulation of Test Formulation-1x |
Test Formulation-1-X1 mg/kg, q.d. in 0.5% MC
+ 20 µL of Test Formulation-1x oil on
right ear, q.d. |
Oral: 10 mL/kg Topical application (Oil) : 20 µL |
|
|
G5 |
Treated orally with intermediate dose 1 of Test Formulation-1 + Topically with Oil formulation of |
Test Formulation-1 -X2
mg/kg, q.d. in 0.5% MC + 20 µL of Test Formulation-1x oil
on right ear, q.d. |
||
|
G6 |
Treated orally with of Test Formulation-2 + Topically with Oil formulation
of |
Test Formulation-2 -Y1 mg/kg, q.d. in 0.5% MC + 20 µL of Test Formulation-2x oil
on right ear, q.d. |
||
|
G7 |
Treated orally with high dose of Test Formulation-2 + Topically with Oil formulation of |
Test Formulation-2 -Y2 mg/kg, q.d. in 0.5% MC + 20 µL of Test Formulation-2x oil
on right ear, q.d. |
Abbreviations: MC-Methyl Cellulose, p.o.-per os. q.d..: quaque die. X1, X2 and Y1, Y2 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.
4.0 METHOD:
· Healthy animals will be selected, randomized based on body weight and divided into 7 different groups consisting of 8 animals each.
· Treatment will be given as follows:
· Group G1 will serve as normal control and treated with 0.5% MC, p.o., q.d.
· Group G2 will serve as disease control and treated with 0.5% MC, p.o., q.d., and acetone (20 µL) on the right ear.
· Animals of group G3 will be treated with dexamethasone, which will be solubilized in acetone and will be topically applied at the dose of 0.2 mg in a total volume of 20 µL on the right pinna. They will additionally receive 0.5 % MC, p.o., q.d.
· Mice of Group G4 to G5 will be treated with TF-1 at dose levels ranging from 10 to 1000 mg/kg, p.o. q.d. They will be additionally administered the oil formulation of TF-1x , topically in a total volume of 20 µL.
· Animals allocated to groups G6 to G7 will be treated with TF-2 at dose levels ranging from 10 to 1000 mg/kg, p.o. q.d. They will be additionally administered the oil formulation of TF-2x, topically in a total volume of 20 µL.
· For the induction of disease 20 µL of TPA solution prepared in acetone will be applied topically on the right ear of the animal at the concentration of 2.5 mg/ear every alternate day till the 10th day by using a micropipette. Acetone will be applied topically on the left ear of the animal, which will serve as the vehicle control.
· Ear thickness will be measured every day using a digital Vernier caliper
· Animals will receive the treatments for ten consecutive days.
· At the end of the experiment (i.e. on day 10), mice will be killed by an overdose of thiopentone, 6 hours after the last drug treatment, and punch biopsies will be taken from the pinnae and weighed in order to measure edema.
· Punch biopsies will be then fixed in formalin for histopathological analysis.
· Alternatively, biopsies will be frozen in liquid N2 and homogenized. Cytokines in skin homogenates were measured by ELISA according to the manufacturer’s specifications, and eicosanoids and MPO.
5.0 ENDPOINT PARAMETER(S):
· Ear Thickness
· Ear Biopsy weight
· MPO activity in ear tissue
· Cytokines in the ear homogenates: IFN-γ, IL-6, IL-1β, IL-17A, IL-17C, IL-17 E, IL-22, TNF-α.
· Gene Expression in ear homogenates: Expression of IFN-γ, IL-1, IL-4, IL-6, IL-22, IL-23, MCP-1, GM-CSF, KRT-17.
· Histopathology of skin tissue
6.0 REFERENCE(S):
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