Novel Protease Inhibitors for Hepatitis C

1. Novel Protease Inhibitors for Hepatitis C Brittany Holt Kelly Kerr 4/30/09

2. Project Goals • Determine manufacturing and commercialization efforts, including the FDA approval process for a new orally available hepatitis C NS3/NS4A protease inhibitor (SCH 503034, Boceprevir) • Determine a novel molecule that can prevent the production and release of infectious HCV particles. • Predict the effectiveness of the new molecule. Compare this to the effectiveness of drugs that are currently being developed. • Modify molecules based on binding affinity tests

3. HCV Background

4. Hepatitis C: Background Information • 4.1 million Americans are affected • 150-200 million people worldwide • Genotype 1 is the most common genotype in America, Japan, and Europe • 10,000-12,000 Americans die annually • Current medications lead to a sustained virological response of only 40-50% among patients with Genotype 1 HCV • Current medications have many severe side effects

5. HCV Life Cycle

6. Viral Diversity Demands Drug Diversity • Potent drugs already on market • Dozens currently being evaluated So why another drug? • Multiple HCV strains • HCV inhibitors select for mutants

7. Modeling versus Laboratory Research Study of actual virions is very difficult. • Lack of suitable cell cultures • Extremely small size • Trouble isolating virions from serum HCV proteins in databases!

8. Target Selection Criteria Tool Available: Modeling Software Therefore we must select… • a viral protein • essential for virion production • that has a known active site for its function.

9. Target: Adsorption • E1 and E2 • glycoproteins • associate with • cell surface • molecules • high variability

10. Target: Endocytosis/Fusion/Uncoating Clathrin-coated pit: Entry Acidic endosomes: Fusion Uncoating: HCV RNA release

11. Target: Translation RNA travels to the rough ER 5’NTR binds to ribosomal subunits & initiator tRNA creating the Translational active complex (HCV polyprotein synthesis)

12. Target: Posttranslational Processing

13. Target: Replication • NS4B • attaches to ER and • forms membranous • web • NS3 helicase • unwinds RNA • NS5B • replicates RNA • NS5A • ?

14. Target: Assembly and Release • Viral components • Cellular factors • Lipid droplets • Precise mechanisms are not well understood

15. NS3 Protease • NS3 protease is a viral protein • NS3P cleaves several essential proteins • NS4A, NS4B, NS5A, and NS5B • Active site: Catalytic triad • NS3 needs NS4A (another viral protein)

16. Catalytic Triad Mechanism • Acylation • peptide bond is cleaved • ester linkage is formed between polyprotein carbonyl C and NS3 protease • Deacylation • ester linkage is hydrolized • enzyme is regenerated

17. Acylation

18. Deacylation

19. Hydrogen bonding of Boceprevir to NS3/NS4A protease

20. Manufacturing and Commercialization Solution Procedure

21. FDA Clinical Trials • Investigational New Drug Application (IND) • Sponsors must show the FDA results of preclinical (animal) testing • Submit proposal for clinical trials • FDA decides on safety of proposed trials • Phase 1 • 20-80 volunteers • Goals: • Determine side effects • Determine how the drug is metabolized and excreted • Phase 2 • Evidence of safety must be shown • 20-300 patients with HCV • Goals: • Determine if the drug works on HCV • Compare the drug with a placebo or another medication • Evaluate safety • Study side effects • Phase 3 • Evidence of effectiveness must be shown • 200-3,000 patients with HCV • Goals: • Effectiveness in different populations • Dosage amounts • Effectiveness with other drugs

22. Gantt Chart – Implementation of Boceprevir

23. Gantt Chart Analysis • There is a competitive edge to producing the first drug that makes it through clinical trials • Gain reputation for safety and effectiveness • Retain a significant market share • Increased sales

24. Synthesis of Boceprevir

25. Alternate Pathways for Synthesis of Boceprevir

26. Choosing Schemes to Model Schemes 1 and 4 were modeled in Super Pro Comparisons between 1, 2, & 3 were not made • Detailed instructions were given for the Scheme 1 • Can include every operation for Scheme 1 • Estimated batch times and costs for Schemes 2 and 3 would be unfairly low • Reagents used in Scheme 1 are more readily obtainable • Cost should be more accurate • Cost should be lower

27. Production Goals • Tablets with 250 mg of active ingredient (SCH503034) • Roughly 7.5 million pills per year • Enough for 1% of HCV infected Americans to take once a day for 24 weeks • Minimal batch time • Reduce time equipment spends idle • Reduce losses that would occur if serious error

28. Superpro Designer Modeling

29. Super Pro - Flow Chart

30. Super Pro - Flow Chart

31. Gantt Chart – Processing Schedule (Synthesis of Boceprevir)

32. Gantt Chart – Processing Schedule • One batch requires 149 hrs and over 200 operations • Effective batch time = 57.8 hrs • Use AOT schedule • Bottleneck is in Reactor 1 • Options for optimization • Make process continuous • Add a second R1

33. Cost Estimates

34. Histogram –Preclinical to Manufacturing Implementation and FCI

35. Should development continue? • Money spent up to this point • $509,642,508 • Research and Development • Preclinical (Animal) Trials • Clinical Trials • Phase 1 • Phase 2 • Part of Phase 3

36. Comparison with current medication costs

37. Results • Development and Manufacturing will take approximately 14 years • The most likely total cost from research and development through plant construction will cost $528 million • This is less than the average cost for development of a drug, $802 million, as reported in the Journal of Health Economics • This cost is expensive, but relative to other drugs the cost of research and commercialization for boceprevir is reasonable • 1 month of current treatments: $4,225 • 1 month of boceprevir: $2553 with 1 year payback

38. Predict new molecule that will work better than Boceprevir

39. Selecting Molecules • Selection Criteria • Compare to Boceprevir • Ki • Inhibition constant: [L] at which ½ of the sites are occupied • Selectivity against human neutrophilelastase (HNE) • Ki (HNE)/ Ki (HCV) • Pharmacokinetics • Bioavailability: Fraction that reaches systemic circulation (4-11%) • AUC: Area under the concentration time curve (0.12 uMh )

40. Solution Procedure

41. Proposed Molecules

42. NS3/NS4A Complex

43. Binding of Drug to Protease

44. Docking Server Ligands

45. Docking Server Results

46. Results • 16 potential drugs have been developed • All bind to catalytic triad • Potential drug #9 • Lower Ki • Higher HNE/HCV • We recommend that potential drug #9 be further evaluated using different docking software, and then be synthesized and undergo animal trials • We also recommend that further research continue on potential drugs 5, 7, 8, 10 and 15 • Increase selectivity • Keep high binding affinity

47. Questions?

48. Docking Server • molecular modeling internet service • developed by Virtua Drug Ltd • capable of calculating the site, geometry, and energy of molecules interacting with proteins

49. Genetic algorithms • Genotype - translation, orientation, and conformation • Phenotype - atomic coordinates of the ligand • Fitness - total interaction energy of the ligand with the protein

50. Genetic algorithms • Individuals that have low fitness die • New individuals are created by inheriting genes from either of two parents • Mutations also occur randomly • DockingServer can predict where and how a ligand will bind to be most energetically favorable