Pipeline: Airway Surface Liquid Modulation

1. Pipeline: Airway Surface Liquid Modulation Eric J. Sorscher, M.D.University of Alabama at Birmingham Baltimore, MD October 21, 2010

2. We Will Address: • Formation of airway surface liquid (ASL) and the mechanisms underlying mucus transport in human airways. • Key molecular defects in cystic fibrosis, including abnormalities of ASL and mucus clearance. • Emerging cystic fibrosis therapies designed to overcome defects in CFTR and enhance pathways that regulate ASL.

4. Histopathology of Human CF Airways Airway lumen White blood cell Mucus layer(gel) Airway surface (sol) liquid layer Submucosal Glandular duct Goblet cell Courtesy of Steven Rowe

5. Airway Mucus in a 2-month-old CFTR-/- Pig Courtesy of David Stoltz & Mike Welsh

6. Lung Disease in 8-month-old CF Ferret Tenacious Mucus WT CF Courtesy of John Engelhardt

7. Courtesy of Jeff Wine & Mauri Krouse

9. Cystic Fibrosis ASL Cilia Cells Filter WT (Control) Cilia ASL Cells Filter WT (VX-770) Cilia ASL Cells Filter Courtesy of Steven Rowe and Gary Tearney

10. X X

11. Courtesy of S. Aller

12. Courtesy of S. Aller

13. CaCC ENaC CFTR

15. HTS Robotics

16. Opening a CFTR Portal That is “Locked Shut”(e.g., G551D)

17. Phase 2a VX-770 Study Design for G551D Mutation Part 1 Mixed cross-over and parallel design Statistically modeled analyses based on LS mean Part 2 Parallel design with low N Statistical analyses based on median VX-770 25 mg q12h VX-770 25 mg q12h VX-770 150 mg q12h (n = 8) Group A (n = 10) Randomization (2:2:1) VX-770 75 mg q12h VX-770 75 mg q12h Randomization (n = 19) (2:2:1) VX-770 250 mg q12h (n = 7) Placebo Placebo Placebo (n = 4) VX-770 75 mg q12h VX-770 75 mg q12h Treatment (28 days) Group B (n = 10) Randomization (2:2:1) VX-770 150 mg q12h VX-770 150 mg q12h Placebo Placebo Accurso FJ, Rowe SM, Clancy JP et al. Effect of VX-770 in persons with cystic fibrosis & the G551D-CFTR mutation. N Engl J Med 2010;363(21):1991-2003. Treatment (14 days) Washout (7-28 days) Treatment (14 days)

18. Sweat Chloride Change from Baseline Individual subject response with population mean/median Part 1 Day 14 means (modeled) Part 2 Day 28 medians * P < 0.001 within-group and vs. placebo † P < 0.05 within-group and vs. placebo Accurso FJ, Rowe SM, Clancy JP et al. Effect of VX-770 in persons with cystic fibrosis & the G551D-CFTR mutation. N Engl J Med 2010;363(21):1991-2003.

19. Relative Change in FEV1 % pred (%) Individual subject response with population mean/median Part 1 Day 14 means (modeled) Part 2 Day 14 and Day 28 medians *P < 0.01 within-subject †P < 0.05 within-subject Accurso FJ, Rowe SM, Clancy JP et al. Effect of VX-770 in persons with cystic fibrosis & the G551D-CFTR mutation. N Engl J Med 2010;363(21):1991-2003.

20. NPD Change from Baseline Change in zero chloride plus isoproterenol response Individual subject response with population mean/median Part 1 Day 14 means (modeled) Part 2 Day 28 medians * P < 0.01 within-subject † P < 0.05 within-subject Accurso FJ, Rowe SM, Clancy JP et al. Effect of VX-770 in persons with cystic fibrosis & the G551D-CFTR mutation. N Engl J Med 2010;363(21):1991-2003.

21. VX-770 Registration Program in CF • Orphan Drug and Fast-Track Designation Three separate registration studies: • Primary trial to enroll ages 12+ with G551D mutation • Enrollment complete • Patients aged 6 to 11 with G551D mutation • Enrollment complete • Phase 2 study of CF patients homozygous for the F508del mutation • Enrollment complete Vextex press release Oct. 25, 2010 http://investors.vrtx.com/releasedetail.cfm?ReleaseID=522615

22. Correcting the F508del CFTR Processing Defect

23. VX-809 Study Design Placebo qd (n = 17) VX-809 25 mg qd (n = 18) Enrolled and randomized (n = 89) Follow-up VX-809 50 mg qd (n = 18) VX-809 100 mg qd (n = 17) VX-809 200 mg qd (n = 19) 7 28 +7 14 21 Day 0 • Subjects recruited in 2 Groups: • Group A (placebo, VX-809 25 mg, VX-809 50 mg) • Group B (placebo, VX-809 100 mg, VX-809 200 mg) • Safety evaluation was done after enrollment of Group A Clancy JP, Spencer-Green G. J Cyst Fibros 2010;9(Suppl 1):S20 http://www.clinicaltrials.gov(NCT00865904)

24. CFTR-Mediated Cl– Transport: Sweat Cl– Biomarker Treatment effect at Day 28 Direction of increased CFTR activity Sweat Cl– responder analysis at Day 28 *Combined groups A & B; ‡P=0.02 vs placebo Clancy JP, Spencer-Green G. J Cyst Fibros 2010;9(Suppl 1):S20 Vertex press release February 3, 2010 (http://investors.vrtx.com/releasedetail.cfm?ReleaseID=442429

25. Phase 2 • Randomized, double-blind, placebo-controlled study • VX-770 150 mg q12h for 16-week treatment duration • Patient population: • Homozygous for the F508del mutation • Aged ≥ 12 years • Primary endpoints • Safety • Absolute change from baseline in % predicted FEV1 through Week 16 • Estimated enrollment: 140 • Study sites in USA http://www.clinicaltrials.gov (NCT00953706)

26. 100 vehicle control m 10 M Corrector A m 10 M Corrector B 80 m m 10 M Corr A/10 M Corr B ] 60 2 A/cm m 40 [ sc I D 20 0 Fsk IBMX Genistein CFTRinh-172 -20 m m m m 10 M 100 M 20 M 20 M 0 500 1000 1500 2000 2500 time[s] Synergy of F508del CFTR Correction 1 + 1 Is Much Greater Than 2 80 ] 60 2 A/cm m [ 40 sc I D 20 0 Corr A Corr B Corr A + Corr B Bar graph illustrates synergy of end currents Current traces from FRT cell Ussing chamber recordings Courtesy of Martin Mense

27. CF Consortia Sharing of ideas and interdisciplinary collaboration to accelerate development of F508del correctors • How and why does F508del CFTR misfold? • How can CFTR misfolding be overcome? • Which interventions restore surface localization of F508del CFTR? • How can the mutant protein be activated to replete the airway surface liquid?

28. Overcoming Premature Truncation Mutations(e.g., G542X, W1282X)

29. Phase 2 Studies Have Demonstrated Ataluren Activity in Nonsense-Mutation-Mediated CF (nmCF) • Increased CFTR at the epithelial cell surface (as measured by immunofluorescence) in nmCF patients receiving ataluren for 2 weeks1 • Increased CFTR chloride channel activity (as measured by nasal transepithelial potential difference) in nmCF patients receiving ataluren for 2 weeks1 • Reduced cough (as measured by the VivoMetrics LifeShirt) in nmCFpatients receiving ataluren for 12 weeks2 • Trends in improvement in pulmonary function (as measured by spirometry) in nmCF patients receiving ataluren for 12 weeks2 1Sermet et al, AJRCCM 2010 2Wilschanski et al, ERJ [in press]

30. An International Registration-Directed Phase 3 Study in Patients with nmCF is Ongoing Open-labelExtension Study Double-blind Placebo-controlledStudy • Key Eligibility Criteria: • Nonsense mutation CF • Age  6 years • FEV1 40% and  90% predicted Ataluren10, 10, 20 mg/kg Ataluren 10, 10, 20 mg/kg N~104 R/S • Outcome Measures: • FEV1 • Exacerbation rate • HRQOL • Cough rate • NPD & Sweat Chloride • Lung CT Placebo N~104 48 Weeks • Enrollment (target = 208 subjects) is on track • Data available first half 2012 www.clinicaltrials.gov Illustration courtesy of PTC Therapeutics

31. CaCC ENaC CFTR

32. Denufosol is a Novel Ion Channel Regulator Under Development for the Treatment of CF Lung Disease Denufosol’s Mechanism of Action • Increase of chloride secretion via calcium-activated chloride channels (CaCCs) • Inhibition of sodium absorption via epithelial Na+ channels (ENaCs) • Stimulation of ciliary beat frequency Yerxa et al., JPET 302:871-880, 2002 Courtesy of Jack Stutts, PhD, UNC at Chapel Hill, manuscript in preparation

33. Mean Change From Baseline FEV1 140 120 100 80 60 40 20 0 -20 -40 0 4 12 24 28 36 48 Mean (SE) Change From Baseline FEV1 (mL)* Denufosol Placebo Denufosol only, open-label phase Placebo switched to denufosol Mean (SE) Change From Baseline FEV1 (ml)* Treatment Week ITT population *Values displayed are mean observed values at each time point without adjustment for discontinuations or covariates. RB Moss, RD Anbar, RW Wilmott, M Barnes, AE Schaberg, TA Durham, FJ Accurso. Phase 3 study of denufosol tetrasodium for the treatment of cystic fibrosis. Am J Respir Crit Care Med 2009; 179: A1189.

34. TIGER-2 Phase 3 Clinical Trial (08-110) Planned Trial Size: 450 RANDOMIZATION Primary Endpoint Key Exclusion Criteria • Change in FEV1 in liters at 48-week endpoint • FEV1≥75% and ≤110% predicted normal for age, gender and height • Age 5 or older Denufosol 60 mg TID N = 225 Secondary Endpoints • Other pulmonary function tests, pulmonary exacerbation, antibiotic use, incidence of hospitalization/ER visits, health resource utilization, quality of life Placebo TID N = 225 Key Exclusion Criteria • Participated in TIGER-1 • Standard CF meds allowed, except hypertonic saline • Based on TIGER-1, made three modifications to enhance TIGER-2 – extended trial length; added upper limit on lung function entry criteria; and increased number of patients enrolled • Patients who complete TIGER-2 are eligible for three-year open-label, denufosol trial 4-week Follow-up Period 48-week Treatment Period www.clinicaltrials.gov (NCT00625612) Illustration courtesy of Inspire Pharmaceuticals

36. Newborn Screening

37. Continuing Clinical Studies of Hypertonic Saline Aerosolization ISIS Trial: Some Firsts • First clinical trial of chronic maintenance therapy in infants • Opportunity to intervene early in CF especially with NBS • First multi-center trial using pulmonary exacerbation and infant PFTs as endpoints in this age group • Model for subsequent trials assessing other early intervention strategies Courtesy of Stephanie Davis & Margaret Rosenfeld

38. ISIS 001: Pilot Data2-week Safety Trial 20 infants enrolled 5 with pre-post HS PFTs 1 withdrew after <1 min 1 of 19 (5%) intolerant of test dose (95% CI 0, 26%) 1 not evaluable due to acute respiratory infection None of 17 intolerant of repeated doses (95% CI 0, 20%) Courtesy of Stephanie Davis & Margaret Rosenfeld

39. ISIS 001: Current Recruitment Status Courtesy of Stephanie Davis & Margaret Rosenfeld

40. Summary • Powerful new capabilities have positioned the field to address questions about clinical CF in ways that were not possible even 12 months ago. • Novel CFTR modulators, animal models, and other advances in basic CF science will allow us to discern mechanistic features of airway surface liquid generation, pulmonary ion transport, infection, and inflammation. • New human clinical trials will address the magnitude of CFTR correction necessary to improve the surface liquid and overcome pulmonary injury; including the potential for ameliorating structural lung damage.

42. Conclusion • The CF field is benefiting from a tremendous wealth of new capabilities. These will provide an even better understanding of airway surface liquid and pathobiology in the disease. • Numerous interventions to restore airway surface liquid and mucus clearance in cystic fibrosis are rapidly advancing through the clinical testing phase, and the pipeline for the future is robust. • What has made all of this possible is a spirit of collaboration, sharing of ideas, working together, and sincere concern about a cause so important to all of us.

43. International Collaboration CF Colleagues Worldwide CFF Therapeutics Development Network Patients and their Families

44. Rosey, age 19 months