Supplementary Appendix

Transcription

1 Supplementary Appendix This appendix has been provided by the authors to give readers additional information about their work. Supplement to: Accurso FJ, Rowe SM, Clancy JP, et al. Effect of VX-77 in persons with cystic fibrosis and the G551D-CF TR mutation. N Engl J Med 21;363:

2 R4 Accurso et al. SUPPLEMENTARY APPENDIX Effect of VX-77 in Persons with Cystic Fibrosis Caused by the G551D CFTR Mutation Accurso FJ, Rowe SM, Clancy JP et al. CONTENTS Detailed Methodology 2 Study Design 2 Subjects 3 Treatment Adherence 4 Endpoints 4 Safety and Tolerability 4 Biomarkers of CFTR Ion Channel Function 5 Nasal Potential Difference 5 Sweat Chloride Concentration Testing 7 Pulmonary Function 8 Health-related Quality of Life 8 Responder Analyses 9 Statistical Analyses 9 Additional Results 11 Thoroughness of Outcome Data 11 Sodium Transport as Measured by Nasal Potential Difference 12 Stringent Responder Analyses 12 Additional Spirometry Assessments 13 Additional Acknowledgements 15 Supplemental References 17 Supplemental Tables and Figures 18 Supplemental Table 1 18 Supplemental Table 2 21 Supplemental Table 3 22 Supplemental Table 4 24 Supplemental Figure 1 26 Supplemental Figure 2 27 Supplemental Figure 3 29 Supplemental Figure 4 3 Supplemental Figure 5 31 Supplemental Figure 6 32 Supplemental Figure 7 33 Supplemental Figure 8 34 Version date: July 2, 21 Page 1 of 33

3 R4 Accurso et al. DETAILED METHODOLOGY Study Design Part 1 of the study consisted of two distinct groups with 1 subjects each who were randomized in a 2:2:1 ratio to receive VX-77 every 12 hours (q12h) at doses of 25 (n = 4) or 75 mg (n = 4), or placebo (n = 2) or VX-77 q12h at doses of 75 (n = 4) or 15 mg (n = 4) or placebo (n = 2). Study drug was administered in two 14-day treatment periods separated by a 7- to 28-day washout. Between periods, subjects randomized to VX-77 crossed over to the alternate dose of VX-77 whereas placebo subjects continued to receive placebo. VX-77 dose levels used in Part 2 were determined based on pharmacokinetic modeling of data from Part 1. Part 2 consisted of 19 subjects not enrolled in Part 1 who were randomized in a 2:2:1 ratio to receive VX-77 q12h at doses of 15 (n = 8) or 25 mg (n = 7) or placebo (n = 4) for 28 consecutive days. Subjects remained on their routine medications. Subjects were assigned to a treatment arm using an Interactive Voice Response system according to a concealed randomization list generated by a statistician not otherwise associated with the study. Individual doses were prepared by a non-blinded pharmacist at each study site who was otherwise not involved with the study. All subjects received the same number of tablets to maintain subject and study personnel blinding. The study sponsor and academic principal investigators were jointly responsible for study design and protocol development (Drs. Accurso, Rowe, Clancy, Mayer-Hamblett, Rose, Ordoñez, Olson, Ashlock, Campbell, and Ramsey). Site investigators and research coordinators were responsible for the collection of data, which was analyzed by the sponsor. Dr. Accurso, the Version date: July 2, 21 Page 2 of 33

4 R4 Accurso et al. principal academic author, composed the first draft of the manuscript. The sponsor and all authors had access to the data, assume responsibility for the accuracy and completeness of the data reported, and contributed to the writing of the manuscript. The principal academic author (Dr. Accurso) decided to pursue publication of the manuscript in consultation with all authors and the sponsor. Subjects Eligible subjects had a confirmed diagnosis of cystic fibrosis, 1 accompanied by either chronic sinopulmonary disease or gastrointestinal/nutritional abnormalities and a sweat chloride value at least 6 mmol/l by quantitative pilocarpine iontophoresis or a sweat sodium value at least 6 mmol/l on at least one occasion. Subjects had to have a documented G551D mutation on at least one CFTR allele. In Part 1, any known or unknown mutation except for the R117H or G A allele was permissible on the second allele. In Part 2, no limitation was placed on the second allele. At screening, subjects had to be 18 years of age; weigh 4 kg; demonstrate forced expiratory volume in one second (FEV 1 ) 4% of predicted normal for age, gender, and height before bronchodilator administration; 2 and demonstrate 92% oxygen saturation in room air. Subjects were excluded if they had a potentially confounding history of illness; ongoing acute illness, including acute respiratory infections; a pulmonary exacerbation 3 or changes in therapy for pulmonary disease within 14 days before the first dose of study drug; liver function tests greater than three times the upper limit of normal; or a history of abnormal renal function in the year prior to enrollment. Subjects were also excluded if they had a change in treatment with intranasal Version date: July 2, 21 Page 3 of 33

5 R4 Accurso et al. medication or systemic antibiotics in the 14 days prior to study drug dosing; required use of continuous (24 hours per day) supplemental oxygen; or utilized any inhibitors or inducers of cytochrome P45 3A4 (CYP3A4). The presence of the G551D-CFTR mutation on at least one allele was confirmed in each subject using CFplus cystic fibrosis expanded mutation analysis (Genzyme Corporation, Cambridge, MA). Treatment Adherence All study drug dosing that occurred at the investigational site was monitored by clinical staff. At each study visit, returned dosage units were counted and dosing diary cards reviewed. Endpoints The primary study objective was to evaluate the safety and tolerability of VX-77 in subjects with CF and at least one G551D-CFTR mutation. Secondary endpoints included assessment of biomarkers of CFTR ion channel function, pulmonary function, and health-related quality of life (HRQoL). Safety and Tolerability Safety and tolerability were evaluated by assessment of adverse events, clinical laboratory tests, vital signs, oxygen saturation by pulse oximetry, physical examination findings, and 12-lead electrocardiogram. Laboratory samples were collected for evaluation at a central laboratory and included standard hematology, urinalysis, coagulation studies, and serum chemistry. Version date: July 2, 21 Page 4 of 33

6 R4 Accurso et al. Biomarkers of Ion Channel Function Nasal potential difference Nasal potential difference (NPD) was used to directly measure ion movement across the nasal epithelium. 4 NPD was performed at each site using standardized procedures from the Cystic Fibrosis Foundation Therapeutics, Inc. Therapeutic Development Network (CFFT-TDN) and results were interpreted by a single central reader who was blinded to treatment assignments. The pre-specified NPD endpoint was the change in the transepithelial electrical potential after perfusion with chloride-free and isoproterenol solutions (zero chloride plus isoproterenol response), which assesses CFTR-specific chloride transport. 4 For NPD measurement, electrodes were placed under the inferior turbinate of the nasal mucosa and in the subcutaneous space of the forearm while the nasal mucosa was bathed in a series of solutions: Ringer s followed by amiloride to estimate sodium ion transport, zero chloride followed by activation of CFTR with isoproterenol to gauge CFTR-dependent chloride ion transport, and adenosine triphosphate (ATP) to stimulate CFTR-independent chloride ion transport. Measurements of interest from the procedure were: the average potential difference (PD) measurements at the start of the procedure at.5, 1., 1.5, 2., and 3. cm with the inferior meatus past the anterior tip of the inferior turbinate (average basal PD); the absolute most polarizing basal PD measurements (maximum basal PD); baseline PD at the start of amiloride perfusion (end Ringer s PD); the change in PD from the end of Ringer s solution to the end of amiloride perfusion (amiloride response); the relative change in PD from the end of Ringer s solution to the end of amiloride perfusion (percentage change in amiloride response); the change Version date: July 2, 21 Page 5 of 33

7 R4 Accurso et al. in PD from the end of amiloride perfusion to the end of zero chloride perfusion (zero chloride response); the change in PD from the end of zero chloride perfusion to the end of isoproterenol perfusion (isoproterenol response); the net change in PD from the end of amiloride perfusion to the end of isoproterenol perfusion (zero chloride plus isoproterenol response); and the change in PD from the end of isoproterenol perfusion to the end of ATP perfusion (ATP response). The total change in potential difference (end of Ringer s perfusion to the end of isoproterenol perfusion) was also calculated. All perfusion solutions were distributed from a central site. The zero chloride plus isoproterenol response was the measurement most indicative of CFTRmediated chloride diffusion potential in the nasal epithelium and was the primary pharmacodynamic outcome assessment used. NPD was performed at each site using standardized procedures from the Cystic Fibrosis Foundation Therapeutics, Inc. Therapeutics Development Network (CFFT-TDN) and all NPD operators were trained and pre-qualified for the performance of the NPD procedure. NPD measurements in each nostril were collected at each time point and read by a single central reader blinded to treatment arm. Analyses were performed using the average of these two measurements. To assess the change in each NPD parameter between study time points, the calculated change between the time points were summarized as an average across nostrils. To compute the average across nostrils for the change from baseline, the within nostril changes were computed first. Interpretability of NPD tracings was assessed for the zero chloride plus isoproterenol response, and included stability in the NPD tracing at the chloride-free and the ATP solution switches (3 sec with ± 1 mv) and no large shifts or breaks in the tracing that did not return to pre-shift Version date: July 2, 21 Page 6 of 33

8 R4 Accurso et al. baselines during the zero chloride plus isoproterenol period (up to a 5 mv shift was allowed as long as the shift returned to baseline). Sweat Chloride Concentration Testing The sweat test, the major diagnostic tool of cystic fibrosis, measures sweat electrolyte levels including chloride using quantitative pilocarpine iontophoresis. The sweat chloride concentration of a patient with cystic fibrosis is typically greater than 6 mmol/l, while a concentration between 4 and 6 mmol/l is considered diagnostically indeterminate, and a concentration less than 4 mmol/l is considered normal. 4 Sweat chloride concentrations were used to measure CFTR function in the sweat gland and were determined according to the standardized protocol of the CFFT-TDN, using the Macroduct 37 collection system (Wescor, Inc. Logan, Utah). Sweat samples were analyzed and interpreted at a central laboratory in a blinded fashion. To ensure consistency and reproducibility in this multi-centered study, sweat secretions were collected at each site according to the standardized procedure of the CFFT-TDN using the Macroduct Model 37 Collection System (Wescor, Inc. Logan, Utah), with standardized solutions provided to each site. Two sweat collections, one from each arm were obtained from each subject at each time point. Sweat samples were immediately frozen and sent to a central laboratory in a blinded fashion. After thawing, sweat chloride concentration was determined by chloridometer Labconco Digital Chloridometer (Labconco Corporation, Kansas City, Missouri). Analyses were performed using the chloride value obtained from the collection with the greater amount of sweat at each time point. The minimum collection volume for a sweat chloride value to be included was 15 μl. Version date: July 2, 21 Page 7 of 33

9 R4 Accurso et al. Pulmonary Function Spirometry was used to assess pulmonary function, with studies scheduled at baseline and posttreatment prior to bronchodilator use (at least 4 hours since last short-acting β-agonist or anticholinergic and 12 hours since last long-acting treatment) and according to American Thoracic Society guidelines. 5 The principal measure of clinical efficacy was change from baseline in absolute FEV 1 (measured in L) and percent predicted (%predicted) FEV 1. For change from baseline analyses, time points where spirometry was performed asynchronously with bronchodilator use (e.g., baseline measurements performed pre-bronchodilator and time point performed post-bronchodilator) were excluded. Forced expiratory volume in one second (FEV 1 ), forced vital capacity (FVC), and forced expiratory flow over the middle half of the FVC (FEF ) were recorded as a volume (L) and/or a rate (L/sec) and as percent predicted for age, gender, and height. 2 The ratio of FEV 1 to FVC was also calculated. Health-related Quality of Life Subjects in Part 2 completed the Cystic Fibrosis Questionnaire Revised (CFQ-R), a validated disease-specific instrument that measures health-related quality of life (QoL) for persons with CF. 6 The CFQ-R consists of 5 items across 9 QoL dimensions (physical functioning, role, vitality, emotional functioning, social, body image, eating disturbances, treatment burden, and health perceptions) and 3 symptom scales (weight, respiratory symptoms, and digestive symptoms). Responses are provided on a 4-point Likert scale and rescaled within each domain to a score range from zero to 1 points, with higher scores representing better health. The CFQ-R was administered prior to the morning dose of study drug. Version date: July 2, 21 Page 8 of 33

10 R4 Accurso et al. Responder Analyses Pre-defined significant response criteria were established for the NPD zero chloride plus isoproterenol response (at least a 5 mv change relative to perfusion with amiloride) and for sweat chloride (decrease from baseline by at least 2 mmol/l). After completion and analysis of data from Part 1 of the study, additional more stringent significant response levels were established for analysis after completion of Part 2 and applied retrospectively to Part 1 data. These criteria were: for NPD, an increase of at least 1 mv in amiloride response plus zero chloride plus isoproterenol response relative to Ringer s solution alone (starting basal PD); for sweat chloride, a post-treatment sweat chloride concentration that has decreased from baseline by at least 3 mmol/l. Similarly, two significant response levels were defined for FEV 1 (greater than 5% increase in FEV 1 from baseline or a greater than 1% increase from baseline in percent predicted FEV 1 ). For the CFQ-R, a minimal clinically important difference (MCID) of at least 4 points has been established for the respiratory domain. 7 All responder analyses were performed on data at the Day 14 time point using the Part 1 and Part 2 combined dataset. Statistical Analyses Calculation of 9% confidence interval (CI) estimates for the true adverse event rate under different scenarios based on a sample size of eight subjects per dosing group suggested that a sample size of 2 subjects was adequate to examine the safety of VX-77 in Part 1 and was deemed feasible given the frequency of the G551D mutation in the CF population. 8 Version date: July 2, 21 Page 9 of 33

11 R4 Accurso et al. Analyses were performed on the population of all randomized subjects who received at least one dose of study drug. Continuous data were summarized using summary statistics. In Part 1, due to the crossover plus parallel study design (placebo-treated subjects remained on placebo for the second treatment period), a mixed model was applied including baseline, dose, and period as fixed effects and subject as a random effect and with change from baseline as the dependent variable. Statistical comparisons for Part 1 were performed using model-based least square means. subjects were counted twice, once for each treatment period. In Part 2, due to the parallel design and small sample size, statistical comparisons were performed using nonparametric tests. For Part 1 and Part 2 pooled data analyses, a mixed effect model was utilized and the least square mean change from baseline was obtained from the model. In Part 2, the sign-rank test was used to assess change from baseline within each dose group and the Wilcoxon test was used to assess change from baseline between dose groups. Since P values from these tests were only used for exploratory purposes, multiplicities were not adjusted. Missing data was not imputed. Statistical analyses were performed using SAS Version 9.1 (SAS Institute Inc., Cary, North Carolina). Responder analyses were performed on secondary measures. For NPD, a significant response post-treatment was pre-defined as a zero chloride plus isoproterenol change of at least 5 mv from baseline. 4 For sweat chloride, a significant response post-treatment was pre-defined as a decrease from baseline in sweat chloride concentration of at least 2 mmol/l. 4 Additional response criteria were defined post-hoc as described in this supplementary appendix (see Responder Analyses section above). Adherence was recorded as the percentage of scheduled tablet administrations fully completed by the subject. Version date: July 2, 21 Page 1 of 33

12 R4 Accurso et al. ADDITIONAL RESULTS Thoroughness of Outcome Data For NPD, in Part 1 the procedure was performed at baseline and Day 14 during each treatment period. Interpretable readings were obtained from 156 of 16 (96.3%) tracings from each nostril for each perfusion (Ringer s, amiloride, zero chloride, isoproterenol, and ATP). Two baseline tracings were not performed in one subject and two tracings were partially unreadable. In Part 2, NPD was performed at baseline, Day 14, and Day 28. Interpretable readings were obtained from 112 of 114 (98.2%) tracings from each nostril for each perfusion; only single nostril tracings were interpreted in two NPD measurements. Sweat chloride was measured in Part 1 of the study on both arms at baseline, Day 7, and Day 14 during each treatment period. Acceptable sweat chloride volumes for analysis were obtained for 213 of 24 (88.8%) collections performed, with 27 tests in six subjects either not performed (n = 14) or having an insufficient quantity for analysis (n = 13). In eight cases, data from both arm measurements were missing for that time point. In Part 2, sweat chloride was measured on both arms at baseline, Day 3, Day 14, Day 21, and Day 28. Viable readings were obtained for 18 of 19 (94.7%) collections performed, with 1 tests in four subjects having a volume insufficient for analysis. In one case, data was missing for both arm measurements for that time point. Lung function was assessed at each study visit (Part 1: baseline and days 7 and 14 in each treatment period; Part 2: baseline and days 3, 14, 21, and 28). In Part 1, all 12 scheduled spirometry tests were performed, with 64 of 8 (8.%) paired values valid for change from Version date: July 2, 21 Page 11 of 33

13 R4 Accurso et al. baseline analyses. Data excluded from analysis were assessments performed asynchronously with bronchodilator use. In Part 2, all 95 scheduled spirometry tests were completed, with all data valid for analysis. Sodium Transport Measured by Nasal Potential Difference Sodium channel function is hyperabsorptive in CF and is reflected in a high basal PD and a large response to amiloride. Changes from baseline to end of treatment in NPD sodium channel parameters are shown in the Supplementary Table 3. Results for the VX and 15-mg groups in Part 1 were significantly different versus placebo for all basal values and amiloride responses. Trend analysis was significant for all endpoints except the percent change in amiloride response. There were no statistically significant findings for sodium transport measurements in Part 2. Stringent Responder Analyses Results for the predefined responder criteria for NPD and sweat chloride in subjects with available data using the combined Part 1 and Part 2 dataset at Day 14 are given in the main text. For NPD, using the response level of at least a -1 mv change from baseline in the amiloride response plus the zero chloride plus isoproterenol response, 2/7 (28.6%), 4/15 (26.7%), 7/16 (43.8%), and 4/7 (57.1%) subjects in the VX-77 25, 75, 15, and 25 mg groups, respectively, were responders and 1/12 (8.3%) subject in the placebo group was a responder. For sweat chloride after 14 days of treatment, using the response threshold of a decrease from baseline of at least 3 mmol/l, 2/8 (25.%), 9/13 (69.2%), 12/14 (85.7%), and 3/7 (42.9%) subjects were Version date: July 2, 21 Page 12 of 33

14 R4 Accurso et al. considered responders in the VX-77 25, 75, 15, and 25 mg dose groups, respectively; no placebo subject (n = 11) achieved responder status. For respiratory function, the number of subjects with available data considered to be treatment responders at Day 14 according to the criterion of greater than 5% increase relative to baseline in FEV 1 was 3/12 (25.%), 3/8 (37.5%), 9/16 (56.3%), 1/16 (62.5%), and 6/7 (85.7%) in the placebo, VX-77 25, 75, 15, and 25 mg groups, respectively. The number of subjects who achieved responder status according to the second response level of greater than 1% relative increase from baseline FEV 1 was 2/12 (16.7%), 2/8 (25.%), 7/16 (43.8%), 7/16 (43.8%), and 4/7 (57.1%) in the placebo, VX-77 25, 75, 15, and 25 mg groups, respectively. There was no pre-specified response criteria set for CFQ-R. However, 2/4 (5.%), 5/8 (62.5%), and 6/7 (85.7%) subjects in the placebo, VX mg, and VX mg groups, respectively, achieved the MCID of an improvement of at least 4 points for the Respiratory Symptoms domain 7 at Day 14. After 28 days, 2/4 (5.%), 5/8 (62.5%), and 5/7 (71.4%) subjects in the placebo, VX mg, and VX mg groups, respectively, achieved the MCID. Additional Spirometry Assessments Results for other lung function parameters (forced vital capacity [FVC], forced expiratory flow in the middle half of the FVC [FEF ], and the ratio of FEV 1 to FVC) are presented in Supplementary Figures 3, 4, and 5. Version date: July 2, 21 Page 13 of 33

15 R4 Accurso et al. For FVC, statistically significant within-subject changes from baseline to Day 14 were observed in the VX and 15 mg groups in Part 1 of the study (mean +.18 L [95% CI: +.2 to +.34 L] and +.22 L [+.1 to +.42 L] for VX and 15 mg, respectively; P =.3 and P =.4, respectively). Neither the VX mg group (+.15 L [-.7 to.37 L]) nor the placebo group (-.3 L [-.31 to +.25 L]) was significantly changed after 14 days compared to baseline (P =.15 and P =.81, respectively). In Part 2, median changes from baseline to Day 28 in FVC were not statistically significant (VX mg: +.18 L [range: -.29 to +.48 L], P =.8; VX mg: +.9 L [-.9 to +.43 L], P =.16; placebo: +.1 L [+.7 to +.3], P =.13) However, median change from baseline to Day 21 was significant for the VX mg group (+.22 L [range: -.11 to +.54 L]; P =.2) and at all time points other than Day 28 in the VX mg group (P =.3). For FEF 25-75, mean changes from baseline to Day 14 in Part 1 were statistically significant in the VX and 15 mg groups (VX mg: +.23 L/s [95% CI: +.1 to +.37 L/s], P =.3; VX mg: +.22 L/s [+.4 to +.4 L/s], P =.2). The VX mg group did not significantly improve (+.7 L/s [-.12 to +.27 L/s], P =.43). The improvements in the VX and 15 mg groups were also significant compared to placebo (P =.3 and P =.5, respectively); mean change in the placebo group was -.7 L/s (95% CI: -.29 to +.14 L/s, P =.47). Similar to FVC, median within-subject changes from baseline to Day 28 in FEF during Part 2 of the study were not statistically significant (VX mg: +.5 L/s [range: to L/s], P =.46; VX mg: +.17 L/s [-.5 to +1.9 L/s], P =.6; placebo: +.27 L/s [+.8 to +.41 L/s], P =.13), although changes from baseline to Days 3 (P =.3), 14 (P =.2), and 21 (P =.2) were significant in the VX mg group. At Day 14, the Version date: July 2, 21 Page 14 of 33

16 R4 Accurso et al. VX mg group had a +.44 L/s (range: +.1 to L/s) change, which was also significant versus placebo (P =.5). At the same time point, the improvement in the VX mg group was +.6 L/s (-.6 to L/s, P =.38 within-subject, P =.56 vs placebo) and in the placebo group was +.9 L/s (+.7 to +.11 L/s, P =.13 within-subject). There were no statistically significant changes from baseline for the mean FEV 1 to FVC ratio (FEV 1 /FVC) at Day 14 for any study group in Part 1. Similarly, there were no statistically significant differences in the median change from baseline at Day 28 for any study group in Part 2 of the study. However, at Day 14 and Day 21, median (range) changes in FEV1/FVC ratio from baseline were significant in the VX mg group (P =.3). At Day 14, the FEV 1 /FVC ratio in the VX mg group increased by.4 ( to.7) and at Day 21 the ratio had improved by.5 (-.1 to.6) from baseline. ADDITIONAL ACKNOWLEDGEMENTS The authors would like to thank the following sub-investigators for their contributions: Monica Federico, M.D., Theresa Laguna, M.D., and Ruth DeVoogd, P.N.P. (University of Colorado Denver); Pamela Zeitlin, M.D., Ph.D. (Johns Hopkins); Felix Ratjen, M.D., Ph.D. and Tanja Gonska, M.D. (Hospital for Sick Children; Toronto); Elizabeth Tullis, M.D. (Michael's Hospital, Toronto); Joanne Billings, M.D. (University of Minnesota); Robert C. Stern, M.D. and Steven D. Strausbaugh, M.D. (Case Western Reserve University); George Retsch-Bogart, M.D. (University of North Carolina, Chapel Hill); Lindo T. Spencer, M.D. (Children s Hospital Boston); James Kreindler, M.D., Suzanne Beck, M.D., and Samuel Goldfarb, M.D. (Children s Hospital of Philadelphia); Christopher H. Goss M.D., M.Sc. (University of Washington, Seattle). Version date: July 2, 21 Page 15 of 33

17 R4 Accurso et al. SUPPLEMENTAL REFERENCES 1. Farrell PM, Rosenstein BJ, White TB, et al. Guidelines for diagnosis of cystic fibrosis in newborns through older adults: Cystic Fibrosis Foundation consensus report. J Pediatr 28;153:S4-S Hankinson JL, Odencrantz JR, Fedan KB. Spirometric reference values from a sample of the general U.S. population. Am J Respir Crit Care Med 1999;159: Rosenfeld M, Emerson J, Williams-Warren J, et al. Defining a pulmonary exacerbation in cystic fibrosis. J Pediatr 21;139: Rowe SM, Accurso F, Clancy JP. Detection of cystic fibrosis transmembrane conductance regulator activity in early-phase clinical trials. Proc Am Thorac Soc 27;4: Standardization of Spirometry, 1994 Update. American Thoracic Society. Am J Respir Crit Care Med 1995;152: Quittner AL, Buu A, Messer MA, Modi AC, Watrous M. Development and validation of The Cystic Fibrosis Questionnaire in the United States: a health-related quality-of-life measure for cystic fibrosis. Chest 25;128: Quittner AL, Modi AC, Wainwright C, Otto K, Kirihara J, Montgomery AB. Determination of the minimal clinically important difference scores for the Cystic Fibrosis Questionnaire-Revised respiratory symptom scale in two populations of patients with cystic fibrosis and chronic Pseudomonas aeruginosa airway infection. Chest 29;135: Cystic Fibrosis Foundation Patient Registry: 27 Annual Data Report to the Center Directors. Bethesda, Maryland; 28. Version date: July 2, 21 Page 16 of 33

18 R4 Accurso et al. Supplemental Table 1. Frequency of occurrence for all adverse events reported during the study (listed alphabetically by MedRA term). (n = 8) VX mg (n = 8) Part 1 Part 2 VX mg (n = 16) VX mg (n = 8) (n = 4) VX mg (n = 8) VX mg (n = 7) Adverse event, n (%) Abdominal distension 1 (25) Abdominal pain 1 (13) Abdominal pain, upper 1 (13) 1 (13) 1 (6) 2 (25) Abnormal dreams 1 (13) 1 (6) 1 (13) Activated partial thromboplastin 1 (13) time prolonged Allergy to arthropod bite 1 (14) Arthralgia 1 (6) 1 (13) Blood creatinine increased 1 (14) Blood glucose increased 2 (13) 1 (13) Blood pressure increased 1 (13) Bowel sounds abnormal 1 (13) Breath sounds abnormal 1 (13) 1 (6) Cardiac murmur 1 (14) Catheter related complication 1 (13) Chest discomfort 1 (25) Chills 1 (13) Chronic sinusitis 1 (25) Constipation 1 (13) Contusion 1 (13) Cough 3 (38) 2 (13) 1 (13) 1 (25) 1 (13) 3 (43) Cough decreased 1 (6) 1 (13) Cystic fibrosis lung* 2 (25) 1 (6) 1 (13) 1 (13) 1 (14) Decreased appetite 1 (25) Dermatitis, contact 1 (25) Diarrhea 1 (13) 1 (25) 2 (25) Dysgeusia 1 (13) Dysphonia 1 (6) Dyspnea 2 (25) Ecchymosis 1 (13) Epigastric discomfort 1 (13) Epistaxis 1 (14) Erythema 2 (25) 1 (14) Excoriation 1 (14) Fatigue 1 (14) Flatulence 1 (25) Flushing 1 (13) Genital pruritus, female 1 (13) Glucose present in urine 1 (6) 1 (13) Glycosuria 1 (13) Version date: July 2, 21 Page 17 of 33

19 R4 Accurso et al. Hematoma 1 (13) Headache 1 (13) 2 (13) 1 (14) Increased bronchial secretion 1 (13) 1 (14) Increased upper airway secretion 1 (13) 1 (14) Increased viscosity of bronchial secretion 1 (13) Infusion site hemorrhage 1 (13) Infusion site reaction 1 (13) Injection site edema 1 (13) Joint crepitation 1 (13) Lymphadenopathy 1 (14) Medical device complication 1 (6) Mucosal erosion 1 (13) Nasal congestion 1 (13) 1 (14) Nasal discomfort 1 (13) Nasal dryness 1 (6) 1 (13) Nasal edema 1 (6) 2 (25) 1 (13) Nasal septum disorder 1 (13) Nasal turbinate abnormality 1 (6) 1 (13) Nausea 1 (13) 3 (19) 1 (13) 1 (13) Nephrolithiasis 1 (6) Oral candidiasis 1 (13) Oral pain 1 (6) Pain 3 (19) 1 (13) Painful defecation 1 (13) Painful respiration 1 (6) Pharyngeal edema 1 (13) Pharyngeal erythema 2 (29) Pharyngitis 1 (6) 1 (13) Pharyngolaryngeal pain 1 (13) 1 (6) 2 (29) Photosensitivity reaction 1 (14) Postnasal drip 1 (13) Productive cough 2 (25) 2 (13) 1 (13) 1 (25) 1 (13) Proteinuria 1 (13) Pruritus 1 (13) Pyrexia 4 (25) 2 (25) Pyuria 1 (13) Rales 1 (6) 1 (13) Rash 1 (6) 1 (13) 1 (13) 1 (14) Rash, macular 1 (13) Rash, maculo-papular 3 (19) 1 (13) Rash, papular 1 (13) 1 (6) Respiratory tract congestion 1 (13) Rhinorrhea 3 (19) 1 (13) Rhonchi 1 (13) Sinus congestion 2 (13) 2 (25) 1 (13) 1 (14) Sinus headache 2 (13) 1 (13) 1 (14) Sinusitis 1 (14) Skin laceration 1 (14) Somnolence 1 (6) 1 (14) Sputum abnormal 1 (6) 1 (13) 1 (14) Version date: July 2, 21 Page 18 of 33

20 R4 Accurso et al. Sputum decreased 1 (6) 1 (13) Sputum discolored 1 (13) Tension headache 1 (14) Thermal burn 1 (13) Tonsillar hypertrophy 1 (25) Tympanic membrane hyperemia 1 (6) Upper respiratory tract congestion 1 (13) Upper respiratory tract infection 1 (6) 2 (29) Vomiting 1 (6) 1 (13) 1 (14) Vulvovaginal mycotic infection 1 (13) 1 (14) Wheezing 1 (13) White blood cells in urine 1 (6) 1 (14) TOTAL 5 (63) 4 (5) 13 (81) 7 (88) 4 (1) 6 (75) 6 (86) Severity Mild 4 (5) 3 (38) 8(5) 5 (63) 3 (75) 5 (63) 5 (71) Moderate 1 (13) 1 (13) 4 (25) 1 (25) 1 (13) 1 (14) Severe 1 (6) 2 (25) * Medical Dictionary for Regulatory Activities (MedDRA) term; reported by investigator as pulmonary exacerbation MedDRA term; reported by investigator as clicking in knee Version date: July 2, 21 Page 19 of 33

21 R4 Accurso et al. Supplemental Table 2. Sodium ion transport parameters of nasal potential difference measurement. Change from baseline in basal potential difference and amiloride response (mv unless otherwise indicated). Parameter Starting basal PD (Ringer s solution) Average basal PD Maximum basal PD Amiloride response Amiloride response (percent) n = a (-17.3, -1.) n = (-12.4, +1.) n = (-16.1, -.5) n = (-1., +1.1) n = (-14.9, +15.) Part 1 (Baseline to Day 14) n, Mean (95% CI) VX mg n = (-7.5, +9.3) n = 7-1. (-8., +6.) n = 7-1. (-9., +7.) n = (-5.6, +5.2) n = (-17.7, +14.8) VX mg n = bc (+2.8, +14.2) n = fg (+.3, +9.9) n = hi (+.1, +11.) n = l (-7.2, +.2) n = o (+2.8, +24.6) VX mg n = de (+7.5, +23.6) n = ag (+1.1, +15.7) n = jk (+2.7, +19.2) n = mn (-13.7, -3.5) n = (-16.5, +13.4) Part 2 (Baseline to Day 28) n, Median (range) VX mg VX mg n = 4 n = 8 n = ( ) ( ) ( ) n = 4 n = 8 n = ( +11.8) ( ) ( ) n = 4 n = 8 n = ( ) ( ) ( ) n = 4 n = 8 n = ( ) ( ) ( ) n = 4 n = 8 n = ( ) ( ) ( ) PD, potential difference a P =.3 within-subject; b P =.6 within-subject; c P =.2 versus placebo; d P =.1 within-subject; e P <.1 versus placebo; f P =.4 withinsubject; g P =.1 versus placebo; h P =.5 within-subject; i P =.8 versus placebo; j P =.1 within-subject; k P =.4 versus placebo; l P =.2 versus placebo; m P =.3 within-subject; n P =.3 versus placebo; o P =.2 within-subject Version date: July 2, 21 Page 2 of 33

22 R4 Accurso et al. Supplemental Table 3. CFQ-R. Change from baseline. Values given in median (range). Each domain is scored on a 1-point scale. A minimal clinically important difference (MCID) of 4 points has been determined for the Respiratory Symptoms domain. 7 DOMAIN (n = 4) Body Image (-11.1, 22.2) Digestive Symptoms -5.6 (-22.2, ) Eating Disturbances (-11.1, 11.1) Emotional Functioning 13.3 (-6.7, 2.) Health Perceptions 5.6 (-11.1, 11.1) Physical Functioning (, 4.2) Respiratory Symptoms 2.8 (-5.6, 11.1) Role (, 8.3) Social 2.8 (-5.6, 5.6) Day 14 Day 28 VX mg (n = 8) (-22.2, ) (, 22.2) (, 11.1) (-26.7, ) (-22.2, 22.2) 2.1 (-4.2, 16.7) 5.6 b (, 16.7) (-8.3, 8.3) (-44.4, 11.1) VX mg (n = 7) (-11.1, 22.2) (-11.1, 22.2) (-11.1, ) (-2., 26.7) (-33.3, 11.1) 4.2 (, 8.3) 5.6 c (-11.1, 11.1) (, 8.3) -5.6 (-16.7, 5.6) (n = 4) VX mg (n = 8) -5.6 (-11.1, 22.2) (-22.2, 11.1) 5.6 a (-22., ) (, 22.2) -5.6 (-11.1, ) (, ) 3.3 (, 2.) (-6.7, 6.7) (-11.1, 11.1) (-22.2, 22.2) (, 4.2) (-8.3, 25.) b (-5.6, 11.1) (, 16.7) (, ) (-8.3, 8.3) 2.8 (-11.1, 5.6) (-11.1, 5.6) VX mg (n = 7) (-11.1, 44.4) (-11.1, 33.3) (-11.1, ) 6.7 (-6.7, 2.) (-11.1, 11.1) (, 12.5) 11.1 d (-5.6, 33.3) (-8.3, 8.3) (-16.7, 11.1) Version date: July 2, 21 Page 21 of 33

23 R4 Accurso et al. Treatment Burden (-11.1, 11.1) -5.6 (-22.2, 11.1) (-22.2, 22.2) -5.6 (, ) (-22.2, 11.1) (-22.2, 11.1) Vitality 4.2 (-16.7, 8.3) 4.2 (-8.3, 16.7) (-8.3, 16.7) -8.3 (-16.7, ) (-16.7, 25.) (, 16.7) Weight (, 33.3) (, 33.3) (-33.3, 33.3) (, 33.3) (, 33.3) (-33.3, 33.3) a P =.13 within-subject; P =.1 vs. placebo b P =.6 within-subject c P =.16 within-subject d P =.8 within-subject Version date: July 2, 21 Page 22 of 33

24 R4 Accurso et al. Supplemental Table 4. Complete table of statistical comparisons. All comparisons on change from baseline results using modelbased means (Part 1) or medians (Part 2). Parameter Part 1 Part 2 n Day 14 n Day 14 Day 28 Withinsubject Versus placebo Versus placebo Withinsubject Withinsubject Versus placebo NPD zero chloride plus isoproterenol response 8.41 n/a 4.63 n/a.88 n/a VX mg n/a n/a n/a n/a VX mg n/a n/a n/a n/a VX mg VX mg n/a n/a Sweat chloride 7.53 n/a 4.63 n/a.38 n/a VX mg 8 <.1 <.1 n/a n/a n/a n/a VX mg 13 <.1 <.1 n/a n/a n/a n/a VX mg 7 <.1 < VX mg n/a n/a Actual measured FEV 1 (in L) 6.76 n/a 4.13 n/a.38 n/a VX mg n/a n/a n/a n/a VX mg n/a n/a n/a n/a VX mg VX mg n/a n/a FEV 1 %predicted relative to baseline (%) 6.88 n/a 4.13 n/a.13 n/a VX mg n/a n/a n/a n/a VX mg n/a n/a n/a n/a Version date: July 2, 21 Page 23 of 33

25 R4 Accurso et al. VX mg VX mg n/a n/a CFQ-R Respiratory Domain (Part 2 only) 15 n/a n/a 4.75 n/a.75 n/a VX mg n/a n/a VX mg n/a n/a n/a, not applicable; NPD, nasal potential difference; FEV 1, forced expiratory volume in one second; CFQ-R, Cystic Fibrosis Questionnaire - Revised Version date: July 2, 21 Page 24 of 33

26 R4 Accurso et al. Supplemental Figure 1. Study design. Part 1 Part 2 VX mg VX mg VX mg Group A randomization VX mg VX mg Randomization VX mg VX mg VX mg Treatment (28 days) Group B randomization VX mg VX mg Treatment (14 days) Washout (7-28 days) Treatment (14 days) Version date: July 2, 21 Page 25 of 33

27 R4 Accurso et al. Supplemental Figure 2A. Subject disposition. (A) Part 1; (B) Part 2. Part 1 24 Patients were screened for eligibility 3 Were not enrolled 2 Withdrew consent 1 Failed to meet enrollment criteria (ongoing acute illness) 11 Were enrolled in Group A and randomized 1 Were enrolled in Group B and randomized 2 Were assigned to receive placebo in both treatment periods 4 Were assigned to receive VX mg in the first treatment period and VX mg in the second treatment period 5 Were assigned to receive VX mg in the first treatment period and VX mg in the second treatment period 2 Were assigned to receive placebo in both treatment periods 4 Were assigned to receive VX mg in the first treatment period and VX mg in the second treatment period 4 Were assigned to receive VX mg in the first treatment period and VX mg in the second treatment period 1 Was not treated due to failure to meet enrollment criteria (required prohibited medication) 2 Completed all study visits and were included in the safety analysis 4 Completed all study visits and were included in the safety analysis 4 Completed all study visits and were included in the safety analysis 2 Completed all study visits and were included in the safety analysis 4 Completed all study visits and were included in the safety analysis 4 Completed all study visits and were included in the safety analysis Version date: July 2, 21 Page 26 of 33

28 R4 Accurso et al. Supplemental Figure 2B. Subject disposition. (A) Part 1; (B) Part 2. Part 2 22 Patients were screened for eligibility 2 Were not enrolled 1 Withdrew consent 1 Failed to meet enrollment criteria (ongoing acute illness) 2 Underwent randomization 5 Were assigned to receive placebo 8 Were assigned to receive VX mg 7 Were assigned to receive VX mg 1 Was not treated due to withdrawal of consent prior to dosing 4 Completed all study visits and were included in the safety analysis 8 Completed all study visits and were included in the safety analysis 7 Completed all study visits and were included in the safety analysis Version date: July 2, 21 Page 27 of 33

29 R4 Accurso et al. Supplemental Figure 3. Effect of washout on biomarker response. Panel A shows mean (range) NPD zero chloride plus isoproterenol response (mv) at baseline and Day 14 of the first treatment period of Part 1; the washout time point represents baseline of treatment period 2. Panel B shows the individual subject NPD zero chloride plus isoproterenol response (mv) labeled by treatment group for baseline and Day 14 of treatment period 1 and baseline of treatment period 2 (washout). Panel C shows the mean (range) sweat chloride concentration (mmol/l) at baseline and Day 14 of the treatment period 1 of Part 1; the washout time point represents baseline of treatment period 2. Panel D shows the individual subject sweat chloride concentration (mmol/l) labeled by treatment group for baseline and Day 14 of treatment period 1 and baseline of treatment period 2 (washout). The dashed line represents the 6 mmol/l cutoff for diagnosis of CF disease. The maximum number of subjects providing data were n = 4, n = 4, n = 8, and n = 4 for the placebo, VX , 75-, and 15-mg groups, respectively. A Potential difference (mv) VX mg VX mg VX mg Baseline Day 14 Washout B Potential difference (mv) VX mg VX mg VX mg Baseline Day 14 Washout C 12 D Sweat chloride (mmol/l) VX mg VX mg VX mg Sweat chloride (mmol/l) VX mg VX mg VX mg Baseline Day 14 Washout Baseline Day 14 Washout Version date: July 2, 21 Page 28 of 33

30 R4 Accurso et al. Supplemental Figure 4. Sweat chloride concentration. Panel A shows the sweat chloride concentration at Day 14 for individual subjects (symbols) in each study group and the group mean (lines) in Part 1 of the study. Panel B shows the sweat chloride concentration at Day 14 and Day 28 for individual subjects (symbols) in each study group and the group means (lines) in Part 2 of the study. The dashed line represents the 6 mmol/l cutoff for diagnosis of CF disease. A 12 B Sweat chloride (mmol/l) Sweat chloride (mmol/l) VX mg VX mg VX mg VX mg VX mg VX mg VX mg Day 14 Day 28 Version date: July 2, 21 Page 29 of 33

31 R4 Accurso et al. Supplemental Figure 5. Forced vital capacity (FVC). Panel A shows mean (range) change from baseline to Day 14 in FVC in L during Part 1 of the study for each study group. Panel B shows median (range) FVC change from baseline during Part 2 of the study for each study group in L. Panel C shows the relative change from baseline in %predicted FVC from baseline to Day 14 for individual subjects (symbols) in each study group and the least-squares means (lines) from the mixed model in Part 1 of the study. Panel D shows the relative change in %predicted FVC from baseline to Day 14 and Day 28 for individual subjects (symbols) in each study group and the medians (lines) in Part 2 of the study. Statistically significant differences are indicated with the superscripted letters. A Change in FVC (L) VX mg VX mg VX mg +.22 a +.18 b B Change in FVC (L) b VX mg VX mg c c b C -.4 Baseline Day 14 D -.4 Baseline Day 3 Day 14 Day 21 Day 28 Relative change in %predicted FVC (%) d c +1.8 VX mg VX mg VX mg Relative change in FVC %predicted (%) c VX mg VX mg VX mg VX mg Day 14 Day 28 a P =.4 within-subject b P =.3 within-subject c P =.2 within-subject d P =.1 within-subject Version date: July 2, 21 Page 3 of 33

32 R4 Accurso et al. Supplemental Figure 6. Forced expiratory flow in the middle part of the FVC range (FEF 25-75% ). Panel A shows mean (range) change from baseline to Day 14 in FEF 25-75% in L/s during Part 1 of the study for each study group. Panel B shows median (range) FEF 25-75% change from baseline during Part 2 of the study for each study group in L/s. Panel C shows the relative change from baseline in %predicted FEF 25-75% from baseline to Day 14 for individual subjects (symbols) in each study group and the least-squares means (lines) from the mixed model in Part 1 of the study. Panel D shows the relative change in %predicted FEF 25-75% from baseline to Day 14 and Day 28 for individual subjects (symbols) in each study group and the medians (lines) in Part 2 of the study. Statistically significant differences are indicated with the superscripted letters. A Change in FEF 25-75% (L/s) VX mg VX mg VX mg +.23 ab +.22 cd B Change in FEF 25-75% (L/s) e VX mg VX mg ab a Baseline Day Baseline Day 3 Day 14 Day 21 Day 28 C D Relative change in FEF 25-75% (%) f Relative change in %predicted FEF 25-75% (%) a VX mg VX mg VX mg -6 VX mg VX mg VX mgvx mg Day 14 Day 28 a P =.2 within-subject b P =.5 versus placebo c P =.3 within-subject d P =.2 versus placebo e P =.3 within-subject f P =.1 within-subject Version date: July 2, 21 Page 31 of 33

33 R4 Accurso et al. Supplemental Figure 7. Ratio of FEV 1 /FVC. Panel A shows mean (range) change from baseline to Day 14 in FEV 1 /FVC ratio during Part 1 of the study for each study group. Panel B shows median (range) FEV /FVC ratio 1 change from baseline during Part 2 of the study for each study group. Panel C shows the relative change from baseline in %predicted FEV 1 /FVC ratio from baseline to Day 14 for individual subjects (symbols) in each study group and the least-squares means (lines) from the mixed model in Part 1 of the study. Panel D shows the relative change in %predicted FEV /FVC ratio 1 from baseline to Day 14 and Day 28 for individual subjects (symbols) in each study group and the medians (lines) in Part 2 of the study. Statistically significant differences are indicated with the superscripted letters. A.15 B.15 Change in FEV 1 /FVC ratio VX mg VX mg VX mg Change in FEV 1 /FVC ratio VX mg VX mg a a Baseline Day Baseline Day 3 Day 14 Day 21 Day 28 C D 2 2 Relative change in FEV 1 /FVC ratio (%) VX mg VX mg VX mg Relative change in FEV 1 /FVC ratio (%) a VX mg VX mg VX mg VX mg Day 14 Day 28 a P =.3 within-subject Version date: July 2, 21 Page 32 of 33

34 R4 Accurso et al. Supplemental Figure 8. Pooled data analyses. All panels show mean (95% CI) relative change from baseline to Day 14 in analysis of combined Part 1 and Part 2 data for the indicated outcome parameter. Panel A shows change from baseline in the NPD zero chloride plus isoproterenol response in mv. Panel B shows change from baseline in sweat chloride concentration in mmol/l. Panel C shows percentage change from baseline in %predicted FEV 1. Panel D shows percentage change from baseline in %predicted FEF 25-75%. Panel E shows percentage change from baseline in %predicted FVC. Panel F shows percentage change from baseline in the %predicted ratio of FEV 1 to FVC. Statistically significant differences are indicated with the superscripted letters. A C Change from baseline in zero chloride plus isoproterenol response (mv) VX mg VX mg VX mg VX mg (n = 12) (n = 7) (n = 15) (n = 16) (n = 7) a -4.6 bc -7.6 de B D Change in sweat chloride (mmol/l) VX mg VX mg VX mg VX mg (n = 12) (n = 8) (n = 14) (n = 15) (n = 7) fg -42. fg -46. fg fh Relative change in %predicted FEV 1 (%) fi 12. j VX mg VX mg VX mg VX mg (n = 1) (n = 7) (n = 14) (n = 16) (n = 7) Relative change in %predicted FEF 25-75% (%) VX mg VX mg VX mg VX mg (n = 1) (n = 7) (n = 14) (n = 16) (n = 7) E F Relative change in %predicted FVC (%) m +8.1 b +6.8 n Relative change in FEV 1/FVC %predicted ratio (%) VX mg VX mg VX mg VX mg (n = 1) (n = 7) (n = 14) (n = 16) (n = 7) -6 VX mg VX mg VX mg VX mg (n = 1) (n = 7) (n = 14) (n = 16) (n = 7) a P =.3 within-subject b P =.2 within-subject c P =.4 versus placebo d P =.1 within-subject e P =.8 versus placebo f P <.1 within-subject g P <.1 versus placebo h P =.2 versus placebo i P =.5 versus placebo j P =.8 within-subject k P =.3 within-subject l P =.4 within-subject m P =.2 within-subject n P =.5 within-subject Version date: July 2, 21 Page 33 of 33