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N-of-1 studies may be useful in studying rare CFTR mutations in patients with CF.
The safety profile of ivacaftor was consistent with that of prior clinical trials.
Ivacaftor is effective in patients with residual-function CFTR mutations.
Ivacaftor shows benefit in patients with cystic fibrosis (CF) and CFTR mutations associated with residual CF transmembrane conductance regulator (CFTR) function. Here we further assess the effect of ivacaftor in such patients using an N-of-1 study design.
Patients aged ≥12 years with CF with clinical or molecular evidence of residual CFTR function were randomized to 1 of 4 treatment sequences for two 4-week, double-blind crossover cycles (each divided into 2 weeks of ivacaftor treatment and placebo) followed by 8 weeks of open-label ivacaftor treatment. The primary endpoint was absolute change from cycle baseline of percent predicted forced expiratory volume in 1 s (ppFEV1) after 2 weeks of treatment with ivacaftor relative to placebo.
Absolute change (SD) from study baseline in ppFEV1 favored ivacaftor by 2.3 (1.0) percentage points (95% credible interval, 0.4–4.1) after 2 weeks of treatment. Absolute mean change (SD) from open-label baseline (defined as day 1 of the open-label ivacaftor treatment period) in ppFEV1 after 8 weeks of treatment was 4.7 (4.2) percentage points (P<.0001). Safety of ivacaftor was consistent with that observed in prior studies.
Ivacaftor improved lung function during the double-blind and open-label treatment periods in patients with CF and CFTR mutations associated with residual CFTR function (ClinicalTrials.gov, NCT01685801).
Ivacaftor (Kalydeco®) is a cystic fibrosis transmembrane conductance regulator (CFTR) potentiator that enhances chloride transport in multiple mutant CFTR forms in vitro, including the G551D-CFTR mutation, other severe gating mutations, and certain mutations associated with residual CFTR function [
]. Clinical studies first established the efficacy and safety of ivacaftor in patients with CF and a G551D mutation or other severe gating mutations (ie, G1244E, G1349D, G178R, G551S, S1251N, S1255P, S549N, or S549R) [
Residual CFTR function can result from a variety of molecular defects diverse in type and severity, which may include reduced and/or variable synthesis of CFTR channels, impaired channel gating, altered channel conductance, and/or moderate defects in processing and trafficking [
]. Patients with CF who have a residual function mutation have variable disease expression and age at onset of clinical manifestations. Patients are typically diagnosed after early infancy, have sweat chloride concentrations <90 mmol/L, and show delayed presentation of pulmonary disease and a greater likelihood of pancreatic sufficiency than patients with other CFTR mutations [
]. Ivacaftor improved lung function and patient-reported outcomes in a large-scale Phase 3 trial (EXPAND; NCT02392234) in patients heterozygous for F508del and a second allele associated with residual CFTR function [
]. Ivacaftor also improved lung function, sweat chloride concentration, and patient-reported outcomes in a Phase 3 trial in adult patients with ≥1 copy of the R117H residual-function mutation (KONDUCT; NCT01614457) [
]. Consistent with these studies, in a small real-world observational study, patients with residual function mutations who received ivacaftor had improved lung function compared with patients who did not receive ivacaftor [
The aim of this study was to assess whether patients with clinical or molecular evidence of residual CFTR function responded to ivacaftor therapy using an N-of-1 study design. We explored whether this design might be useful when evaluating investigational agents with in vitro evidence for activity in patients with extremely rare mutations.
2.1 Study design and patients
This was a Phase 2, randomized, double-blind, placebo-controlled, multiple, within-patient, crossover study (ClinicalTrials.gov identifier, NCT01685801). Patients were randomized 1:1:1:1 to receive ivacaftor at the recommended dose (150 mg every 12 h) or matched placebo in 1 of 4 treatment sequences (Fig. 1).
Eligible patients were aged ≥12 years with a confirmed diagnosis of CF, defined as (1) a sweat chloride concentration ≥60 mmol/L or 2 CF-causing mutations, and (2) chronic sinopulmonary disease or gastrointestinal and/or nutritional abnormalities. Patients also had a percent predicted forced expiratory volume in 1 s (ppFEV1) of ≥40% at screening and clinical evidence of residual CFTR function, defined as any 1 of the following: residual exocrine pancreatic function (eg, fecal elastase-1 >200 μg/g), screening sweat chloride concentration ≤80 mmol/L, or aged ≥12 years at diagnosis with ≥1 copy of a CFTR missense or splicing mutation associated with residual CFTR function (see Supplementary Methods).
Patients with ≥1 copy of a CFTR mutation encoding prespecified gating mutations were not eligible. Complete exclusion criteria are available in Supplementary Methods.
This study was conducted in accordance with the Declaration of Helsinki. The study protocol was approved by the National Jewish Health Institutional Review Board, and all patients provided written informed consent and assent before participation.
The study comprised a 2-week screening period, a segmented crossover period, an 8-week open-label period, and a follow-up visit. The crossover period included two 4-week cycles during which patients received study drug, each followed by a 4- to 8-week washout period. During each 4-week crossover cycle, patients received 2 weeks each of active treatment and placebo in 1 of 4 treatment sequences that were randomly assigned. In the 8-week open-label period that followed the second crossover washout period, all patients received ivacaftor. Patients who completed the study, including assessments through the follow-up visit, and met a responder criterion were offered enrollment in a Phase 3, open-label extension trial of ivacaftor (KONTINUE; NCT01707290).
Study visits occurred on days 1, 15, and 29 of each crossover cycle and days 1, 15, 29, and 57 of the open-label period; a follow-up visit occurred 2 weeks after patients received the last dose of study drug, or, if discontinuation occurred during a washout period, within 2 weeks of the decision to discontinue.
The primary efficacy endpoint was absolute change from cycle baseline in ppFEV1 after 2 weeks of treatment with ivacaftor relative to placebo. Efficacy endpoints used cycle baseline to calculate change from baseline and to assess repeated “on-off” effects of study drug. Secondary efficacy endpoints included absolute change from cycle baseline in lung clearance index2.5 (LCI2.5; ie, turnovers required to reduce the concentration of nitrogen to 2.5% of its starting concentration) after 2 weeks of treatment with ivacaftor relative to placebo; absolute change from baseline after 8 weeks of open-label treatment in ppFEV1, LCI2.5, sweat chloride, and body weight; and correlations between absolute change from baseline in ppFEV1 and LCI after 2 and 8 weeks of treatment. Safety evaluations included incidence of treatment-emergent adverse events (AEs), clinical laboratory parameters, and vital signs. A treatment-emergent AE was defined as any AE that developed or worsened at or after the first dose of study medication.
2.4 Statistical analysis
Enrollment was planned for a maximum of 40 patients based on operational considerations. Primary and secondary efficacy analyses were conducted in the intention-to-treat population, which included all randomized patients who received ≥1 dose of study drug.
For the primary efficacy endpoint of absolute change from cycle baseline in ppFEV1 after 2 weeks of treatment with ivacaftor relative to placebo, a Bayesian hierarchical model with noninformative priors was the primary analysis, based on pooled data from the 2 crossover cycles. The absolute change from baseline was defined as the difference of the post-baseline value minus the baseline value. Posterior mean and SD were estimated for each patient and for the overall population; posterior 95% credible interval was reported.
As a supportive analysis to the primary efficacy endpoint, a mixed-effects model was conducted, with the absolute change from cycle baseline in ppFEV1 as the dependent variable; sequence, treatment, and visit as fixed effects; and patient nested within sequence as the random effect. Relative change from baseline was analyzed using the same model but with relative change from cycle baseline in ppFEV1 as the dependent variable. Estimated mean treatment effect with 95% confidence intervals (CIs) and 2-sided P values was provided.
Bayesian hierarchical model and mixed-effects model analyses were performed for the secondary efficacy measure of change from baseline in LCI2.5 after 2 weeks of treatment. Pearson correlation coefficient was used to assess the correlation between absolute change from baseline in ppFEV1 at weeks 2 and 8 during the open-label period. P values <.05 were considered statistically significant.
All patients in the safety population (i.e., patients who received ≥1 dose of study drug) were included in the intent-to-treat population. All AEs were coded by system organ class and preferred terminology using the Medical Dictionary for Regulatory Activities version 15.0 and summarized descriptively.
From September 2012 to April 2014, 24 patients from multiple centers were enrolled at a single center in the United States (Fig. 1) and randomized to the study; all patients received ≥1 dose of study drug. Overall, 21 patients (87.5%) completed the study through the follow-up visit, 1 patient (4.2%) discontinued at the end of cycle 1 during the washout period (due to an AE of infective pulmonary exacerbation [PEx] of CF), and 2 patients (8.3%) discontinued after cycle 2 because of nonadherence to the study drug regimen. Baseline demographics and clinical characteristics are summarized in Supplementary Table 1. Mean (SD) age was 37.3 (13.9) years, ppFEV1 was 67.8 (22.6) percentage points, BMI was 24.2 (4.8) kg/m2, and sweat chloride concentration was 64.7 (25.7) mmol/L.
The primary endpoint was met and showed a positive treatment difference in absolute change from study cycle baseline in ppFEV1 after 2 weeks of treatment, derived from posterior means (SD) of ivacaftor over placebo of 2.3 (1.0) percentage points, with a 95% credible interval of 0.4 to 4.1 (Table 1).
Table 1Lung Function Effects After 2 Weeks of Treatment in Crossover Cycles.
Placebo (n = 24)
Ivacaftor (n = 24)
Bayesian hierarchical model
Treatment difference (ivacaftor vs placebo): posterior mean (SD) of absolute change in ppFEV1 from baseline, percentage points
Cycle baseline was defined as the most recent nonmissing measurement collected prior to initial administration of study drug in each treatment cycle, with the additional requirement that the cycle 2 baseline needed to be from an assessment after 14 days of washout. If cycle 2 baseline was missing, cycle 1 baseline was used. CI, confidence interval; ppFEV1, percent predicted forced expiratory volume in 1 s.
Mean absolute change in ppFEV1 from baseline, percentage points
Treatment difference: ivacaftor vs placebo, percentage points (95% CI)
2.1 (0.7, 3.6)
Mean relative change in ppFEV1 from baseline, percentage points
Treatment difference: ivacaftor vs placebo, percentage points (95% CI)
4.0 (1.6, 6.4)
a Cycle baseline was defined as the most recent nonmissing measurement collected prior to initial administration of study drug in each treatment cycle, with the additional requirement that the cycle 2 baseline needed to be from an assessment after 14 days of washout. If cycle 2 baseline was missing, cycle 1 baseline was used.CI, confidence interval; ppFEV1, percent predicted forced expiratory volume in 1 s.
In addition, analysis from a supportive mixed-effects model demonstrated a treatment difference (95% CI) of 2.1 (0.7, 3.6) percentage points with ivacaftor vs placebo (P=.004; Table 1), corroborating the results from the primary analysis using a Bayesian hierarchical model. A treatment difference (95% CI) with ivacaftor vs placebo was also observed for the mean relative change from study cycle baseline in ppFEV1 (4.0 [1.6, 6.4] percentage points; P=.002; Table 1).
The within-group mean (SD) absolute change from open-label period baseline in ppFEV1 after the 8-week open-label treatment period was 4.7 (4.2) percentage points (Supplementary Table 2), and the mean (SD) relative change from baseline was 7.8 (7.5) percentage points (P=.0001). The change in lung function observed after 2 weeks of treatment in the open-label period (mean [SD] absolute change from open-label period baseline in ppFEV1 after 2 weeks of treatment in the open-label period, 3.7 [3.7] percentage points) was generally similar to the change in ppFEV1 observed with ivacaftor over placebo after 2 weeks of treatment during the crossover periods (Fig. 2).
Absolute changes from open-label period baseline in ppFEV1 at weeks 2 and 8 of open-label treatment were highly correlated (Pearson correlation coefficient = 0.7; P=.0004; Supplementary Figure 1), indicating that lung function responses observed after 2 weeks were related to responses after 8 weeks of ivacaftor treatment. Absolute changes in ppFEV1 during the 2-week crossover periods and the 8-week open-label period by treatment sequence to which patients were randomized in the crossover period are shown in Fig. 3, with the absolute mean change overlaid onto individual patient changes (for the 21 patients who completed the study).
In an analysis of absolute change in LCI2.5 from baseline at 2 weeks, the posterior mean (SD) difference between ivacaftor and placebo was −0.42 (0.22). The mixed-effects model showed an estimated treatment effect of −0.2 (95% CI, −1.3, 0.9; P=.686). We estimated but did not test differences from baseline over the open-label treatment period for LCI2.5, sweat chloride concentration (Supplementary Table 2), BMI, and weight. In the open-label ivacaftor treatment, the mean (SD) absolute difference from study baseline in LCI2.5 was −1.1 (2.6) at week 2 and −1.6 (2.3) at week 8; the mean (SD) absolute difference from baseline in sweat chloride concentration was −15.4 (13.0) mmol/L at week 2 and −15.7 (14.8) mmol/L at week 8. The mean (SD) absolute difference from baseline in BMI and weight were 0.5 (0.7) kg/m2 and 1.8 (1.9) kg, respectively, at 8 weeks in the open-label period.
Overall, 23 patients (95.8%) reported AEs during ivacaftor treatment, with the most common AEs being cough (33.3%), upper respiratory tract infection (33.3%), and fatigue (16.7%) (Table 2). Most AEs were mild or moderate in severity. The rates of AEs were 75.0% with ivacaftor and 70.8% with placebo during the crossover period and 95.2% with ivacaftor during the open-label period. The most common AEs by treatment group and study period are summarized in Table 2.
Table 2Adverse Events (occurring in ≥10% of patients) Overall and by Treatment Group and Study Period in the Safety Population.
AEs that started or increased in severity during the period from the first dose of study drug through completion of the follow-up visit were considered treatment emergent, except for AEs that started during the washout period and were beyond 14 days from the last dose date of the preceding cycle. A patient with multiple events within a system organ class or within a preferred term was counted only once within each system organ class or preferred term, respectively. AEs were coded from MedDRA, version 15.0. AE, adverse event; CF, cystic fibrosis; PEx, pulmonary exacerbation.
Ivacaftor Overall (N = 24)
Placebo (n = 24)
Ivacaftor (n = 24)
Ivacaftor (n = 21)
Patients with any AE
Upper respiratory tract infection
Infective PEx of CF
a AEs that started or increased in severity during the period from the first dose of study drug through completion of the follow-up visit were considered treatment emergent, except for AEs that started during the washout period and were beyond 14 days from the last dose date of the preceding cycle. A patient with multiple events within a system organ class or within a preferred term was counted only once within each system organ class or preferred term, respectively. AEs were coded from MedDRA, version 15.0.AE, adverse event; CF, cystic fibrosis; PEx, pulmonary exacerbation.
During the crossover period, 1 serious treatment-emergent AE of infective PEx of CF occurred during treatment with ivacaftor; the event was not considered related to the study drug. Treatment interruption due to AEs occurred in 1 patient after receiving placebo during the crossover period (infective PEx of CF) and in 1 patient during the open-label period (urticaria, which was considered possibly related to study drug). No patients withdrew from the study due to AEs while receiving ivacaftor therapy. No clinically important changes in laboratory values or vital signs were observed, and no deaths occurred during the study.
Ivacaftor treatment improved lung function as measured by ppFEV1 during the 2-week crossover and 8-week open-label treatment periods in patients with CF with clinical or molecular evidence of a residual CFTR function. Furthermore, short-term lung function response correlated with longer-term response observed after 8 weeks of open-label treatment. Similar trends in response were observed for LCI2.5 measures, although changes in LCI2.5 did not reach statistical significance. Changes in extrapulmonary outcomes, including CFTR activity (as measured by sweat chloride concentration) and nutritional status (as measured by weight and BMI), were also observed with ivacaftor treatment during the 8-week open-label period.
The safety profile of ivacaftor in patients with residual CFTR function was similar to that reported in previous studies [
]. The Phase 3 EXPAND trial in patients with the F508del-CFTR mutation and a mutation associated with residual CFTR function demonstrated benefit in spirometry and patient-reported outcomes with ivacaftor treatment [
]. While our findings are consistent with those from EXPAND and KONDUCT, improvements in spirometry at 2 weeks were less robust in the present study (2.3 percentage points) compared with those observed in EXPAND and in adult patients in KONDUCT at the same time point (both ≈4 percentage points) [
]. This difference may have been related to the lack of washout period within the crossover cycles, which may have increased the potential for carryover or rebound effects, confounded baseline values, and had an impact on outcomes. However, after the 8-week open-label treatment period in this study, the improvement in ppFEV1 (4.7 percentage points; P<.0001) was consistent with spirometry results observed at the same time point in EXPAND (≈5 percentage points). Although ivacaftor improved lung function in 2 case studies of patients with R117H and P67L residual function CFTR mutations [
]. In a randomized, crossover N-of-1 study series, the effect of ivacaftor in patients with residual CFTR function was unclear and not generalizable; however, it should be noted that the patient population of this previous study differed from the population discussed here, and these differences could account for the differences in study results [
]. The present study enrolled patients with confirmed residual-function CFTR mutations that included those with R117H-CFTR mutation, had a more selective sweat chloride concentration window of eligibility (60–80 mmol/L vs 40–80 mmol/L), and had an older age of study eligibility (≥12 years of age vs ≥8 years of age) [
We designed this trial using a multiple within-patient crossover design to explore its feasibility, based on challenges associated with performing randomized studies in rare patient populations where patient access and adequate study powering can be problematic [
]. Results of this analysis were confirmed using the supportive mixed-effects model analysis. This approach, in which each patient served as his or her own control, enabled the determination of treatment response with limited patient enrollment and reduced overall variability. However, no clear association between individual genotypes and response in any of the efficacy outcome measures was observed.
Two main limitations of this trial design were noted. First, patients from 15 different CF centers across the United States were evaluated at a single center, and enrollment was lower than planned—partly attributable to the study requirements—which may limit generalizability of our findings. Second, we saw changes in pulmonary function that were greater after 8 weeks of continuous ivacaftor treatment than after 2 weeks, suggesting that maximal clinical response may not have been achieved within 2 weeks. This observation contrasted with previous reports of ivacaftor in patients with CF and a G551D or other CFTR gating mutation, as well as those with a mutation associated with residual CFTR function; in both groups, improvements in lung function were detected within 2 weeks of therapy initiation [
]. As previously noted, the difference in timing of achieving a clinical response (8 weeks in our study vs 2 weeks in previous studies) may have been due to the lack of washout within each crossover cycle, further complicating the interpretation of results. Taken together, these results suggest that the current design may have underestimated the full extent of the potential benefit of ivacaftor on lung function in this population. Despite the trial-design limitations, the overall data suggest that ivacaftor therapy improved lung function in patients with CF and CFTR mutations associated with residual CFTR function.
Recently, the FDA expanded the indication of ivacaftor to include patients with CF aged ≥6 months who have ≥1 mutation in the CFTR gene that is responsive to ivacaftor based on clinical data and/or in vitro assay [
]. In vitro responsiveness was defined as an increase of ≥10% above baseline in CFTR-mediated chloride transport in Fischer rat thyroid (FRT) cells expressing mutant CFTR. Data from the present study supported the approach recently adopted by the FDA in basing approvals on thorough in vitro and mechanistic data along with robust safety profiling. There is growing interest in ex vivo assessments, using FRT cells, human bronchial epithelial cells, and rectal organoids as additional approaches, that have the potential to inform the responsiveness of some ultra-rare mutations in CF [
]. In particular, ex vivo assays and N-of-1 studies may provide utility when assessing treatment benefits in patients carrying a rare mutation with no F508del mutation on the second allele. In very small and/or heterogeneous populations, elements of this study design may be considered, in combination with additional approaches, when assessing treatment benefit.
Findings from this study were consistent with those from prior studies showing benefit with ivacaftor in patients with CF and a residual-function CFTR mutation. Treatment with ivacaftor improved total CFTR activity and pulmonary function in patients aged ≥12 years with CF and clinical or molecular evidence of residual CFTR function and demonstrated a safety profile consistent with that shown in previous studies. Further, this study demonstrated that the N-of-1 crossover study design may play a role in future clinical studies of rare diseases.
JAN contributed to study design; data collection, interpretation, and analysis; and manuscript conception, writing, and revision. MH contributed to data analysis and interpretation and to manuscript conception, writing, and revision. LL contributed to data analysis and interpretation and to critical revision of the manuscript. CSC and MCJ contributed to data collection and interpretation and to critical revision of the manuscript.
Role of the funding source
This study was funded by Vertex Pharmaceuticals Incorporated, which participated in the design, statistical analysis, and interpretation of the data, and provided editorial and writing assistance. All authors had full access to the study data, and the corresponding author had the final responsibility for the decision to submit for publication.
Vertex is committed to advancing medical science and improving patient health. This includes the responsible sharing of clinical trial data with qualified researchers. Proposals for the use of these data will be reviewed by a scientific board. Approvals are at the discretion of Vertex and will be dependent on the nature of the request, the merit of the research proposed, and the intended use of the data.
Declaration of Competing Interest
JAN reports grants and nonfinancial support from Vertex Pharmaceuticals Incorporated during the conduct of the study and personal fees from Vertex Pharmaceuticals Incorporated outside of the submitted work. MH is an employee of Vertex Pharmaceuticals Incorporated and may own stock or stock options in that company. LL is a former employee of Vertex Pharmaceuticals Incorporated and may own stock or stock options in that company. CSC and MCJ have nothing to disclose.
We thank the patients who participated in this study and the following individuals who referred patients for screening or assisted with the conduct of the clinical trial: Teka Siebenaler, RRT, and Jordan M. Dunitz, MD, of the University of Minnesota; Dawn J. Baker, ARNP, CCRC, and Pamela M. Schuler, MD, of the University of Florida; Scott C. Sanborn, MD, of Woodland Hills Medical Center; Mark W. Rolfe, MD, of Tampa General Hospital; Daniel T. Layish, MD, of Central Florida Pulmonary Group; Emily A. DiMango, MD, of Columbia University; Cheryl Kushner, ARNP, of Joe DiMaggio Children's Hospital; Moira L. Aitken, MD, David P. Nichols, MD, and Alan Genatossio, RN, of the University of Washington; Joseph M. Pilewski, MD, Michael M. Myerburg, MD, and Carolyn Walker, RN, BSN, of the University of Pittsburgh; Elika Rad, MS, RN, NP-C, of Stanford University; Holly Carveth, MD, of the University of Utah; Adupa Rao, MD, and Lynn K. Fukushima, RN, MSN, CCRC, of the University of Southern California; Peter J. Murphy, MD, and Janelle Sorensen, RN, of the University of Nebraska; G. Marty Solomon, MD, of the University of Alabama at Birmingham; and Milene T. Saavedra, MD, Jennifer L. Taylor-Cousar, MD, Sara J. Brayshaw, RN, Cathy S. Chacon, RN, and Churee Pardee, MSN, RN, of National Jewish Health and the University of Colorado, Denver.
Editorial coordination and support were provided by Tejendra R. Patel, PharmD. TRP is an employee of Vertex Pharmaceuticals Incorporated and may own stock or stock options in that company. Statistical support and critical review of the manuscript were provided by David Rodman, PhD. DR is a former employee of Vertex Pharmaceuticals Incorporated and may own stock or stock options in that company. Statistical review was provided by Daniel Campbell, PhD, safety review was provided by Lily Lee, PhD, and critical review of the data was provided by Kelly Naegelin. DC, LL, and KN are employees of Vertex Pharmaceuticals Incorporated and may own stock or stock options in that company. Medical writing and editorial support were provided by Michelle Yochum, PhD, and Katherine Mills-Lujan, PhD, CMPP, of ArticulateScience LLC, and were funded by Vertex Pharmaceuticals Incorporated.
Source of funding
This study was sponsored by Vertex Pharmaceuticals Incorporated.