1. 29/12/2010 Drug Discovery and Development Process of Anti-diabetic Plants Dr. Basavaraj K. Nanjwade M. Pharm., Ph. D Professor of Pharmaceutics KLE University College of Pharmacy BELGAUM- 590010, Karnatka, India. Cell No: 0091 974243100 E-mail: bknanjwade@yahoo.co.in 28/12/20101ANRAP & BIRDEM, Dhaka, Bangladesh

2. 29/12/2010 Sources of drugs Animal Insulin (Pig, cow) Growth hormone (Man) (Creutzfeldt-Jakob) Plant Metformin ( Gallega officinalis ) Morphine (Papaver somniferum) Inorganic Arsenic, Mercury, Lithium Synthetic Chemical (Propranolol) Biological (Penicillin) Biotechnology (Human insulin) 28/12/20102ANRAP & BIRDEM, Dhaka, Bangladesh

3. 29/12/2010 Drug Discovery 28/12/20103ANRAP & BIRDEM, Dhaka, Bangladesh

4. 29/12/2010 Drug Discovery 28/12/20104ANRAP & BIRDEM, Dhaka, Bangladesh

5. 29/12/2010 The Regulatory process Differs from country to country Demands safety and quality of product Encourages efficacy and need for product Grants clinical trials certificate if volunteer and animal data OK Approves protocols and examines data 50-400 volumes (30,000-150,000 pages) Original data available Two way process; authority and company trying to produce a safe effective product Release for a specific purpose and use 28/12/20105ANRAP & BIRDEM, Dhaka, Bangladesh

6. 29/12/2010 Marketing Getting the product right (packaging; formulation) Right therapeutic slot Information on new drug Information for honest comparison Reporting problems Reporting new indications Therapeutic trends 28/12/20106ANRAP & BIRDEM, Dhaka, Bangladesh

7. 29/12/2010 Schema diagram representing anti diabetic plants data 28/12/2010ANRAP & BIRDEM, Dhaka, Bangladesh7

8. 29/12/2010 A screen shot of the database “Database on anti- diabetic plants” home page with links and dropdown search window. 28/12/2010ANRAP & BIRDEM, Dhaka, Bangladesh8

9. 29/12/2010 Annona squamosa (Sugar–Apple) 28/12/20109ANRAP & BIRDEM, Dhaka, Bangladesh

10. 29/12/2010 Nigella sativa 28/12/201010ANRAP & BIRDEM, Dhaka, Bangladesh

11. 29/12/2010 Polyherbal formulation of Annona squamosa and Nigella sativa This study was undertaken to investigate the effect of Polyherbal formulation of Annona squamosa and Nigella sativa on blood glucose, plasma insulin, tissue lipid profile, and lipidperoxidation in streptozotocin induced diabetic rats. Aqueous extract of Polyherbal formulation of Annona squamosa and Nigella sativa was administered orally (200 mg/kg body weight) for 30 days. The different doses of Polyherbal formulation on blood glucose and plasma insulin in diabetic rats were studied and the levels of lipid peroxides and tissue lipids were also estimated in streptozotocin induced diabetic rats. The effects were compared with tolbutamide. Treatment with Polyherbal formulation and tolbutamide resulted in a significant reduction of blood glucose and increase in plasma insulin. Polyherbal formulation also resulted in a significant decrease in tissue lipids and lipid peroxide formation. The decreased lipid peroxides and tissue lipids clearly showed the antihyperlipidemic and antiperoxidative effect of Polyherbal formulation apart from its antidiabetic effect. 28/12/201011ANRAP & BIRDEM, Dhaka, Bangladesh

12. 29/12/2010 INTRODUCTION Diabetes mellitus is syndrome, initially characterized by a loss of glucose homeostasis resulting from defects in insulin secretion, insulin action both resulting impaired metabolism of glucose and other energy yields fuels such as lipids and protein. Experimental diabetes in animals has provided considerable insight into the physiologic and biochemical derangement of the diabetic state. Many of the derangement have been characterized in hyperglycemic animals. Significant changes in lipid metabolism and structure also occur in diabetes. In these cases the structural changes are clearly oxidative in nature and are associated with development of vascular disease in diabetes. In diabetic rats, increased lipidperoxidation was also associated with hyperlipidemia. Liver, an insulin dependent tissue that plays a pivotal role in glucose and lipid homeostasis and it is severely affected during diabetes. 28/12/201012ANRAP & BIRDEM, Dhaka, Bangladesh

13. 29/12/2010 Animals Male Wistar albino rats (weighing 160–200 g) were procured from Venkateshwara Enterprise, Bangalore and they kept in under standard environmental conditions (12 h light/dark cycles at 25–28 0 C, 60– 80% relative humidity) in clean and dry cages and maintained in well-ventilated animal house. Animals were divided into 8 groups of five each and were fed with standard diet and water ad libitum. The study was approved by the Institutional Animal Ethics Committee. 28/12/201013ANRAP & BIRDEM, Dhaka, Bangladesh

14. 29/12/2010 Preparation of drug The seeds of Nigella sativa obtained from Prgati Ayurvedic Drug store Belgaum and matured fruit of Annona squamosa from local market of Belgaum and they were authenticated from Botanical Survey of India, Pune (Maharastra). The extracts of the both antidiabetic plants were mixed and polyherbal formuation was prepared (Table 1). Five hundred grams of each plant (chopped into small pieces) was extracted individually and were soaked overnight in 1:l of water. This suspension was filtered and the filtrates were pooled and the solvents were evaporated in a rotavapor at 40–50 0 C under reduced pressure and lyophilized. 28/12/201014ANRAP & BIRDEM, Dhaka, Bangladesh

15. 29/12/2010 Chemicals Streptozotocin and other biochemicals used in this experiment were purchased from Sigma Chemical Company Inc., St. Louis, Mo, and USA. Enzyme linked immunosorbant assay (ELISA) kit for insulin assay was purchased from Boehringer Mannheim, Germany. Tolbutamide was a generous gift sample from Sun Pharmaceuticals Limited, Baroda, India. All other chemicals used were of analytical grade. 28/12/201015ANRAP & BIRDEM, Dhaka, Bangladesh

16. 29/12/2010 Drug administration Polyherbal formulation of Annona squamosa and Nigella sativa was suspended in distilled water and administered orally through intragastric tube at the following doses of 50, 100 and 200 mg/kg body weight. 28/12/201016ANRAP & BIRDEM, Dhaka, Bangladesh

17. 29/12/2010 Streptozotocin-induced diabetes Rats were made diabetic by single administration of streptozotocin (60 mg/kg/i.p) dissolved in 0.1 M- citrate buffer, pH 4.5. Forty-eight hours later, blood samples were collected and glucose levels were determined to confirm the development of diabetes. Only those animals which showed hyperglycemia (blood glucose levels > 250 mg/dl) were used in the experiment. 28/12/201017ANRAP & BIRDEM, Dhaka, Bangladesh

18. 29/12/2010 Experimental design In the experiment, a total of 42 rats (30 diabetic surviving rats, 12 normal rats) were used. The rats were divided into seven groups of six rats each after the induction of streptozotocin diabetes. 28/12/201018ANRAP & BIRDEM, Dhaka, Bangladesh

19. 29/12/2010 Experimental design 1.Group1: Normal treated rats. 2. Group2: Normal rats given aqueous solution of Polyherbal formulation (200 mg/kg body weight) daily using an intragastric tube for 30 days. 3. Group 3: Diabetic control rats. 4. Group 4: Diabetic rats given aqueous solution of Polyherbal formulation (50 mg/kg body weight) daily using an intragastric tube for 30 days. 28/12/201019ANRAP & BIRDEM, Dhaka, Bangladesh

20. 29/12/2010 Experimental design 5. Group 5: Diabetic rats given aqueous solution of Polyherbal formulation (100 mg/kg body weight) daily using an intragastric tube for 30 days. 6. Group 6: Diabetic rats given aqueous solution of Polyherbal formulation (200 mg /kg body weight) daily using an intragastric tube for 30 days. 7. Group 7: Diabetic rats given aqueous solution of Tolbutamide (250 μg/kg bodyweight) daily use an intragastric tube for 30 days. 28/12/201020ANRAP & BIRDEM, Dhaka, Bangladesh

21. 29/12/2010 Experimental design At the end of 30 days, all the rats were killed by decapitation under pentobarbitone sodium (60 mg/kg) anesthesia. Blood was collected in tubes containing potassium oxalate and sodium fluoride solution for the estimation of blood glucose and plasma was separated for the assay of insulin. 28/12/201021ANRAP & BIRDEM, Dhaka, Bangladesh

22. 29/12/2010 Table 1: Polyherbal Formulation of Annona Squamosa and Nigella Sativa (Composition and Concentration). Sl. No.Botanical Name Common Name FamilyPart usedConc. (%) 1Annona squamosa SharifaAnnonnaceaeMatured fruits 50 2Nigella sativa KalonjiRanunculaceaeSeeds 50 28/12/201022ANRAP & BIRDEM, Dhaka, Bangladesh

23. 29/12/2010 Table 2: Changes in blood glucose and plasma insulin levels of control and experimental animals GroupFasting blood glucose (mg/dl) Plasma insulin (IU/ml) Normal81.04 ± 2.2911.26 ± 0.96 Diabetic control262.24 ± 22.233.48 ± 0.69 Diabetic + Polyherbal formulation (50 mg/kg) 209.58 ± 12.465.59 ± 0.34 Diabetic + Polyherbal formulation (100 mg/kg) 155.58 ± 11.696.03 ± 0.45 Diabetic + Polyherbal formulation (200 mg/kg) 104.16 ± 6.567.15 ± 0.45 Diabetic + Tolbutamide (250 mg/kg) 110.65 ± 9.356.32 ± 0.48 28/12/201023ANRAP & BIRDEM, Dhaka, Bangladesh

24. 29/12/2010 Pharmaceutical Product Quality Cannot Be Tested in - It Is Built in Pharmaceutical product quality is assured by – comprehensive development program – extensive manufacturing and environmental controls – rigorous validation procedures and requirements The high quality thus built into the final product is ensured through in-process controls and verified in a series of confirmatory tests before each manufactured batch is released to the market 28/12/201024ANRAP & BIRDEM, Dhaka, Bangladesh

25. 29/12/2010 Building-in of Quality Starts Early. Development Builds-in Quality The chemistry, manufacturing and controls (CMC) aspect of drug development is focused on producing medicines suitable for human use with specified quality, safety and efficacy characteristics The drug development program is geared towards – thorough understanding of the drug product’s performance – identification of drug product’s critical characteristics (which would be monitored on a batch-by-batch basis) – demonstration of drug’s safety and efficacy – ultimately leads to the review and approval of the drug 28/12/201025ANRAP & BIRDEM, Dhaka, Bangladesh

26. 29/12/2010 Relationship between Safety, Efficacy and Quality Every drug product (with its specifications) has been thoroughly tested in clinical trials for safety and efficacy – Specifications for release and stability testing may be equal to or tighter than the specifications for clinical trial batches Therapeutic indication and QC are considerations in establishing specifications 28/12/201026ANRAP & BIRDEM, Dhaka, Bangladesh

27. 29/12/2010 Drug Development Process 28/12/201027ANRAP & BIRDEM, Dhaka, Bangladesh

28. 29/12/2010 Drug Development Process 28/12/201028ANRAP & BIRDEM, Dhaka, Bangladesh

29. 29/12/2010 Quality is Always Part of the Picture - Built-In and Built-Up Quality Control and Quality Assurance Pre-INDPhase IPhase IIPhase III Commercial Manufacturing Specification/Manufacturing Development for the Product Less establishedFully established 28/12/201029ANRAP & BIRDEM, Dhaka, Bangladesh

30. 29/12/2010 Drug Development Cycle 28/12/201030ANRAP & BIRDEM, Dhaka, Bangladesh

31. 29/12/201031 Examples of QA & QC Considerations During Drug Development Evolution of documentation systems – SOP – change control – trend analysis Evolution of QA and QC systems – internal audits – supplier audits – document review (e.g., SOP, batch records, specifications, data) 28/12/2010ANRAP & BIRDEM, Dhaka, Bangladesh

32. 29/12/2010 Chemistry Manufacturing Controls Evolve During Drug Development The goal is to have process and product performance determined by the time of validation, although some level of validation occurs along the continuum and eventually leads to the full-scale validation. 28/12/201032ANRAP & BIRDEM, Dhaka, Bangladesh

33. 29/12/201033 Examples of CMC Considerations During Drug Development Selection of appropriate technology and raw materials Optimization – of formulation and device – of manufacturing process – of specifications and analytical methods Careful selection and control of container closure systems Identification and control of critical manufacturing process parameters Process capability established Technical transfer to larger scale, i.e., scale-up Process validation 28/12/2010ANRAP & BIRDEM, Dhaka, Bangladesh

34. 29/12/2010 Process Registration Requirements Sponsor is required to describe how the product was developed Companies need to optimize, justify and register the entire “recipe” – ranges temperatures mixing times hold times etc. – quantities active ingredient excipients – raw material specifications – in-process limits – in-process methods – product specifications – etc. 28/12/201034ANRAP & BIRDEM, Dhaka, Bangladesh

35. 29/12/201035 Validation 28/12/2010ANRAP & BIRDEM, Dhaka, Bangladesh

36. 29/12/2010 What is Validation? Documented evidence that the manufacturing process consistently produces product that meets predetermined specifications – Defines product quality – Developed and validated based on a thorough understanding of the critical process parameters – Parameters are carefully controlled within the validated ranges to ensure a consistent manufacturing process. Manufacturing process validation consists of successfully manufacturing at least three full-scale batches in succession, which pass all in-process and product quality attributes 28/12/201036ANRAP & BIRDEM, Dhaka, Bangladesh

37. 29/12/2010 Validation is Always Part of the Picture Ongoing Validation (DOE, IQ, OQ, PQ, PV)* Pre-INDPhase IPhase IIPhase III Commercial Manufacturing * DOE = Design of Experiment IQ = Installation Qualification OQ = Operational Qualification PQ = Performance Qualification PV = Process Validation Specification Development Final process validation Re-validation The extent of IQ, OQ, PQ, validation, etc. depends on complexity of product 28/12/201037ANRAP & BIRDEM, Dhaka, Bangladesh

38. 29/12/2010 Role of QC Tests Each batch of orally inhaled and nasal drug products (OINDP), manufactured by the validated process, is tested to the critical QC attributes as defined during development – Confirms consistent performance The Delivered Dose Uniformity test for OINDP is one of several confirmatory QC tests of the finished product – a result of a long and careful development and characterization process – QC tests confirm the quality built-in through a well-understood and well-controlled manufacturing process 28/12/201038ANRAP & BIRDEM, Dhaka, Bangladesh

39. 29/12/2010 Pre-Approval Inspection Confirms Facility is Ready – Sponsor can do what they submit in the NDA – Process is validated or validation protocols are in place Validation required prior to launch – Thorough documentation review – Quality systems are established and capable – Confirms specifications are met Compliance versus Review Division – Specifications may change based on NDA review 28/12/201039ANRAP & BIRDEM, Dhaka, Bangladesh

40. 29/12/2010 Foreign Marketing Data Several Ayurvedic, Siddha and Unani drugs have been marketed for centuries in India. The drugs are also dispensed to the patients by recognized and qualified physicians of the complimentary systems. Most of these drugs have been found to be safe, It may be worthwhile for the, FDA to accord adequate weightage to the data already available in these countries in determining whether a “drug has been used under particular conditions to a material extent and for a material time” to qualify for inclusion in an OTC drug monograph. 28/12/2010ANRAP & BIRDEM, Dhaka, Bangladesh40

41. 29/12/2010 Foreign Market Data Since the FDA is proposing to make it available the facility for OTC marketing agencies in the United States, who are already marketing their products, the same exemptions can be made available to overseas marketing firms who have safely-marketed their products in their respective countries. 28/12/2010ANRAP & BIRDEM, Dhaka, Bangladesh41

42. 29/12/2010 Chemistry Manufacturing Controls (CMC) information for the botanical drug products Botanical, drugs. are derived from vegetable matter and are usually complex mixtures. The chemical constituents of such a mixture are not, always defined and in most cases even the active constituent in a botanical drug is not defined nor its biological activity well characterized. 28/12/201042ANRAP & BIRDEM, Dhaka, Bangladesh

43. 29/12/2010 Chemistry Manufacturing Controls (CMC) information for the botanical drug products Therefore the CMC documentation would have to be substantially liberal in comparison to that’ of synthetic or highly purified drugs. Simple or combination tests like spectroscopic, chromatographic, fingerprints,, chemical and or biological assays can be the main reliability criteria to understand the product. While these tests may not generate the necessary specifics that are desirable, it can atleast bring about a rational approach to Quality Control. 28/12/201043ANRAP & BIRDEM, Dhaka, Bangladesh

44. 29/12/2010 Applicability of Combination Drug Regulations It would be practical not to confine the botanical drug products that are derived from a single part of a plant such as leaves, stems, roots seeds under the fixed combination drug category. The current requirement of Botanical drugs composed of multiple parts of a single plant species under the combination requirement should also be revised and exemption accorded. 28/12/201044ANRAP & BIRDEM, Dhaka, Bangladesh

45. 29/12/2010 Studies on bioavailability and drug interactions Bioavailability and Pharmacokinetic studies are cumbersome and extremely difficult to generate in complex formulations, where a number of herbs are involved. It would be desirable to have” a very practical view in this area. Well-controlled clinical trials can substitute the bioavailability and Pharmacokinetic studies. 28/12/201045ANRAP & BIRDEM, Dhaka, Bangladesh

46. 29/12/2010 Studies on bioavailability and drug interactions Ayurvedic and Siddha pharmacopoeia are full of formulations which have a. combination of several herbs and most of them can never be launched in the next decade or two if requirements are not simplified by the FDA. 28/12/201046ANRAP & BIRDEM, Dhaka, Bangladesh

47. 29/12/2010 Natural Vs Cultivable source of Plants In a Country like India where the source of raw materials for Drug preparations is predominantly from the forests (80%) a practical solution has to be found in connection with the source of the raw material for quality enforcements. When a medicinal plant is cultivated a lot of the Quality aspects are directly under the control of the grower. 28/12/201047ANRAP & BIRDEM, Dhaka, Bangladesh

48. 29/12/2010 Natural Vs Cultivable source of Plants However, when the plant material is collected from the forests, it would be reasonable to presume that the origin of the source is, from nature and variations if any would be natural. The document should highlight separate guidelines for natural and cultivable sources for manufacturers using plants from the above two sources 28/12/201048ANRAP & BIRDEM, Dhaka, Bangladesh

49. 29/12/2010 Conclusions Pharmaceutical quality is built-in through the entire drug development process – validation is key element of ensuring quality – in-process controls assure quality during manufacturing – Specifications established based on thorough understanding of process 28/12/201049BIRDEM & ANRAP, Dhaka, Bangladesh49ANRAP & BIRDEM, Dhaka, Bangladesh

50. 29/12/2010 THANK YOU Cell No: 0091 974243100 E-mail: bknanjwade@yahoo.co.in 28/12/201050ANRAP & BIRDEM, Dhaka, Bangladesh