Selection of CRO for Preclinical Toxicity and Formulation Development studies

STUDY-1

Study- I- Efficacy of Test compounds in rat model of bone cancer pain produced by breast cancer cells Animal Welfare: Studies to be done as per IAEC (CPCSEA) approved Protocols (1). Test System Species: Rat Strain: Wistar Sex, Age: Female, 6-8 weeks Procedure In anesthetized animals 1-cm-long skin incision to be made over the third trochanter. The rectus femoris muscle will be gently incised to expose the shaft of femur. A 23-gauge needle will be drilled vertically in the shaft of femur at the third trochanter level, allowing it to reach the intramedullary canal of the femur. 10 μl volume of Walker 256 cells (105 cells) or Hank's solution (sham group) will be injected. After slow injectiondrill hole will be sealed using bone wax. The muscle and skin will be stitched and disinfected. Treatment start: 3 days post surgery. Treatment end: 21 days. Efficacy Readout: • Body weights • mechanical allodynia • Behavioral tests • Observations: General clinical signs Analysis: One way ANOVA (95 % confidence level) Group Treatment No. of animals / group 1 Normal control 10 2 Disease Control 10 3 Reference Control 10 4 IIIM Reference Control-1*# 10 5 IIIM Reference Control-2*# 10 6 Test Dose -1# 10 7 Test Dose-2# 10 8 Test Dose -3# 10 *IIIM Reference Control 1 and 2 will be provided by CSIR-IIIM along with test compound

STUDY-2

Product- I- Efficacy of Test compounds in Cisplatin-induced Pain in Rat Animal Welfare: Studies to be done as per IAEC (CPCSEA) approved Protocols (1). Test System Species: Rat Strain: Sprague-Dawley Sex, Age: Male/Female, 5-7 weeks Procedure Animals will be dosed with ten intraperitoneal injections of cisplatin every three days. The dose of cisplatin will be 2 mg/kg alternately with 1 mg/kg resulting in a cumulative dose of 15 mg/kg. Before each injection, 2 ml of saline will be injected subcutaneously to avoid cisplatin nephrotoxicity. The Normal control group will be injected saline. Treatment start: from 1st day or 12th day onwards cisplatin injection. Treatment end: 21 days. Efficacy Readout: • Body weights • mechanical allodynia • Thermal allodynia • Clinical Observations: General clinical signs Analysis: One way ANOVA (95 % confidence level) Group Treatment No. of animals / group 1 Normal control 10 2 Control 10 3 Reference Control 10 4 IIIM Reference Control-1* 10 5 IIIM Reference Control-2* 10 6 Test Dose -1 10 7 Test Dose-2 10 8 Test Dose -3 10 *IIIM Reference Control 1 and 2 will be provided by CSIR-IIIM along with test compound.

STUDY-3

Product- I*, Acute Toxicology Studies (Two Species: Mice and Rat) Acute single dose oral toxicity of Phytopharmaceutical lead (Product- I*) in mice and rat by oral route as per Appendix III of Schedule Y (GLP study). Report of study should include all results, raw data, interpretations, observations, conclusions. *Test and IIIM reference controls are controlled substances (under NDPS), the firm shall intimate and take necessary approvals to the concern state/Central authorities regarding storage, transportation and testing.

STUDY-4

Product- I*, 10 days Dose ranging finding (DRF) studies 10-days dose-range finding (DRF) study (non-GLP study) of Phytopharmaceutical lead (Product- I* ) in rat to identify a dose-range for 28-days repeated dose toxicity study. *Test and IIIM reference controls are controlled substances (under NDPS), the firm shall intimate and take necessary approvals to the concern state/Central authorities regarding storage, transportation and testing.

STUDY-5

Product- I*, Sub Acute Toxicology Studies (28 days) in rat 28-days repeated dose oral toxicity study of Phytopharmaceutical lead (Product- I*) in rat as per Appendix III of Schedule Y (GLP study) Report of study should include all results, raw data, interpretations, observations, conclusions. *Test and IIIM reference controls are controlled substances (under NDPS), the firm shall intimate and take necessary approvals to the concern state/Central authorities regarding storage, transportation and testing.

Dabur Research Foundation