Selection of CRO for preclinical toxicity and formulation development studies

FORMULATION STUDY PACKAGE –I (PRODUCT-I*)

1. Development of prototypes formulation of Product-I 2. Compatibility studies with Excipients 3. Finalization of composition 4. Characterization and evaluation of capsule 5. Optimization of the formulation (Description of composition of product) 6. Description of Manufacturing process and flow chart (Manufacturing formula) 7. In process quality control checks and reports 8. Process validation of Pilot scale batches 9. Specifications for finished product (Release) test and procedures 10. Justification for specifications 11. Certificate of analysis (CoA) of respective batches 12. Stability specification 13. Analytical Method validation protocol & reports. The methods which are to be intended use 14. Packing material identification pack presentation & product package interaction studies and stability studies (with HDPE bottle will contain 60 capsules) and specifications of packing material 15. Testing and evaluation of packing materials 16. Batch packing record 17. Labeling information 18. Stability data: Accelerated and Long term (24 months) 19. Compilation of the data in TTD. 20. Stability studies of APPI (accletated and long term) Note: Phytopharmaceuticals are defined under definition of new drugs as per ND& CT Rules, 2019. Firm has to submit application in Form CT10/CT/12/CT-13 for permission for manufacturing of the test batches for conduct of the clinical trial before obtaining license in Form 29 from the respective state licensing authority. * Note: Product I is controlled substances under NDPS act, the firm shall intimate and take necessary approvals from the concern state/Central authorities regarding transportation, storage and testing.

FORMULATION STUDY PACKAGE–II (PRODUCT-II)

1. Development of prototypes formulation of Product-II 2. Compatibility studies with Excipients 3. Finalization of composition 4. Characterization and evaluation of capsule 5. Optimization of the formulation (Description of composition of product) 6. Description of Manufacturing process and flow chart (Manufacturing formula) 7. In process quality control checks and reports 8. Process validation of Pilot scale batches 9. Specifications for finished product (Release) test and procedures 10. Justification for specifications 11. Certificate of analysis (CoA) of respective batches 12. Stability specification 13. Analytical Method validation protocol & reports. The method which are to be intended use 14. Packing material identification pack presentation & product package interaction studies and stability studies (with HDPE bottle will contain 60 capsules) and specifications of packing material 15. Testing and evaluation of packing materials 16. Batch packing record 17. Labeling information 18. Stability data: Accelerated and Long term (24 months) 19. Compilation of the data in TTD. 20. Stability studies of APPI (accletated and long term) Note: Phytopharmaceuticals are defined under definition of new drugs as per ND & CT Rules, 2019. Firm has to submit application in Form CT10/CT/12/CT-13 for permission for manufacturing of the test batches for conduct of the clinical trial before obtaining license in Form 29 from the respective state licensing authority.

PRECLINICALSTUDY PACKAGE-I

Product- 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 Note: *#Test and IIIM reference controls are controlled substances (under NDPS Act), the firm shall intimate and take necessary approvals to the concern state/Central authorities regarding storage, transportation and testing.

PRECLINICALSTUDY PACKAGE-II

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 1. Normal Control 2. Control 3. Reference Control 4. IIIM Reference Control-1* 5. IIIM Reference Control-2* 6. Test Dose-1 7. Test Dose-2 8. Test Dose-3 *IIIM Reference Control 1 and 2 will be provided by CSIR-IIIM along with test compound

PRECLINICALSTUDY PACKAGE-III

Product- II- Efficacy of Test compounds in Monoiodoacetate Model of Osteoarthritis Pain in Rat Animal Welfare: Studies to be done as per IAEC (CPCSEA) approved Protocols (1)- Test System Species: Rat Strain: Wistar Sex, Age: Male/Female, 6-8 weeks Procedure: Animals is to be anaesthetized and given single intra-articular injection of 1 mg of monosodium iodoacetate (MIA) through the infrapatellar ligament of the right knee. Sterile saline: used for injections in normal control animals Treatment start: 5/7 days post MIA injection. Treatment end: Animals to be terminated 28/56 day post MIA injection. Efficacy Readout: • Body weights • Mechanical hyperalgesia • Histopathology • 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 Test Dose -1 10 5 Test Dose-2 10 6 Test Dose -3 10

PRECLINICALSTUDY PACKAGE-IV

Regulatory safety studies of Phytopharmaceutical leads (Product- I*) in Mice and Rat The study to be done in following three phases: Report of each study should include all results, raw data, interpretations, observations, conclusions. (a) 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). (b) 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. (c) 28-days repeated dose oral toxicity study of Phytopharmaceutical lead (Product- I*) in rat as per Appendix III of Schedule Y (GLP study) (d) 28-days repeated dose oral toxicity study of Phytopharmaceutical lead (Product- I*) in mice as per Appendix III of Schedule Y (GLP study) *Test lead is controlled substances under NDPS act, the firm shall intimate and take necessary approvals to the concern state/Central authorities regarding storage, transportation and testing.

PRECLINICALSTUDY PACKAGE-V

Regulatory safety studies of Phytopharmaceutical lead (Product- II) in Mice and Rat The study to be done in following three phases: Report of each study should include all results, raw data, interpretations, observations, conclusions. (a) Acute single dose oral toxicity of Phytopharmaceutical lead(Product- II) in mice and rat by oral route as per Appendix III of Schedule Y (GLP study). (b) 10-days dose-range finding (DRF) study (non-GLP study) of Phytopharmaceutical lead (Product- II) in rat to identify a dose-range for 28-days toxicity study. (c) 90-days repeated doseoral toxicity study of Phytopharmaceutical lead (Product- II) in rat as per Appendix III of Schedule Y (GLP study) (d) 90-days repeated dose oral toxicity study of Phytopharmaceutical lead (Product- II) in mice as per Appendix III of Schedule Y (GLP study)

Aurex Laboratories

Dabur Research Foundation