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Efficacy and safety of the CFTR potentiator icenticaftor (QBW251) in COPD: Results from a phase 2 randomized trialSteven M. Rowe1, Ieuan Jones2, Mark T. Dransfield1, Nazmul Haque2, Stephen Gleason2, Katy A. Hayes2, Kenneth Kulmatycki2, Denise P. Yates2, Henry Danahay3, Martin Gosling3,4 David J. Rowlands2, Sarah S. Grant2 1University of Alabama at Birmingham, Department of Medicine, Birmingham, AL, USA2Novartis Institutes for BioMedical Research, Cambridge, MA, USA3Enterprise Therapeutics, Brighton, United Kingdom4Sussex Drug Discovery Centre, University of Sussex, Brighton, United Kingdom.Supplementary materialList of participating study centres and principal investigatorsPrincipal investigatorStudy centrePiotr KunaSPZOZ Uniwersytecki SzpitalKliniczny nr 1 im. Norberta BarlickiegoLodz 90-153PolandDr. Mark DransfieldUAB Lung Health CenterBirmingham AL 25394USADr. Leonard DunnClinical Research of West Florida, Inc.Clearwater FL 33765USADr. Charles FogartySpartanburg Medical ReserachSpartanburg SC 29303USADr. Stephen WatsonCarolina Research Center, Inc.Shelby NC 28150USADr. Edward KerwinClinical Research Institute of SouthernOregon, PCMedford OR 97504USADr. James PearleCalifornia Research Medical GroupFullerton CA 92835USADr. Krishna PudiUpstate Pharmaceutical ResearchGreenville SC 29615USADr. Dareen SiriSneeze, Wheeze, & Itch Associates,LLCNormal IL 61761USADr. Alexander WhiteProgressive Medical ResearchPort Orange FL 32127USADr. Joseph Boscia IIICU Pharmaceutical ResearchUnion SC 29379USADr. Asha SternResearch Carolina of HuntersvilleHuntersville NC 28078USAMethodsStudy design and participantsDuring the entire study duration, patients were maintained on their baseline COPD therapy (short-acting beta-2 agonist [SABA], long-acting beta-2 agonist [LABA], long-acting muscarinic antagonist [LAMA], and/or inhaled corticosteroids [ICS]). Prohibited medications included theophylline, chronic daily steroids or PDE4 inhibitors within four weeks of screening. In addition, as icenticaftor is a potential CYP1A2 inhibitor and CYP3A4 inducer, drugs with potential increased exposure due to CYP1A2 inhibition or decreased exposure with CYP3A4 induction were prohibited, including ciprofloxacin and verapamil (CYP1A2), and cyclosporine, sirolimus, tacrolimus, and fentanyl (CYP3A4). Medications to be withheld between 24 and 6 hours before study visits included SABAs, SAMAs, LABAs and LAMAs. Subjects with a COPD exacerbation during the treatment period or placebo follow-up period, defined as worsening of pulmonary symptoms requiring hospitalization, systemic steroids, systemic antibiotics or more than six uses of rescue inhalers or nebulizer treatments within a 24-hour period on two consecutive days, were required to discontinue. Subjects who discontinued the study for any reason completed a final clinic visit where final safety assessments were collected.Key inclusion and exclusion criteria are listed below (Table E1).Table E1. Patient key inclusion and exclusion criteriaInclusion criteriaExclusion criteriaAge: ≥35 and ≤75 yearsCOPD exacerbation or respiratory tract infection in the 6 weeks before screening, during the screening period, or during the run-in periodDiagnosis of COPD according to GOLD criteria: post-bronchodilator FEV1/FVC ratio ≤0.7 and FEV1 between 30–79% predicted ADDIN EN.CITE <EndNote><Cite><Year>2013</Year><RecNum>25</RecNum><DisplayText>(1)</DisplayText><record><rec-number>25</rec-number><foreign-keys><key app="EN" db-id="ez02vepxnt0vffe22tkxz2s2vpp2dvave2fx" timestamp="1527483770">25</key></foreign-keys><ref-type name="Web Page">12</ref-type><contributors></contributors><titles><title>Global Initiative for Chronic Obstructive Lung Disease. Global Strategy for the diagnosis, management, and prevention of chronic obstructive pulmonary disease (2013 report). </title></titles><dates><year>2013</year></dates><urls></urls></record></Cite></EndNote>(1)Baseline CT scan demonstrated significant radiographic emphysema (>25% whole lung) or with a diagnosis of severe bronchiectasis or another concomitant pulmonary diseaseSmoking history (current or former): ≥10 pack-years Unchanged underlying COPD treatment regimen for ≥4 weeks prior to screening and during the placebo run-in period Symptoms of chronic bronchitis (cough and sputum production on most days for a minimum of 3 months during the last year)Evidence of global air trapping (≥15%) on a baseline end-expiratory computed tomography (CT) scan Lung clearance index 2.5 (LCI) ≥8 at screening, adapted from trials in CF patients (LCI >8 indicates small airway dysfunction)FEV1, forced expiratory volume in 1 second; FVC, forced vital capacityRandomization and maskingOn Day 1, eligible patients were randomized using an interactive response technology system to treatment with either 300 mg b.i.d. icenticaftor or placebo (2:1) orally. Two patients received 450 mg b.i.d. icenticaftor before protocol amendment reducing the dose to 300 mg b.i.d. icenticaftor. Subjects were stratified according to smoking status (smoker or ex-smoker). The placebo run-in and placebo follow-up periods were single-blinded, with only the patients being blinded, while the treatment period with icenticaftor or placebo was double-blinded, with patients, investigator staff, persons performing the assessments, and data analysts blinded to the identity of study treatments. Novartis was unblinded to completed subjects data at the time of the interim analysis and at the end of study (EOS).Procedures and outcomesSpirometry, in accordance with American Thoracic Society/European Respiratory Society guidelines, was performed with the patient in a sitting position. National Health and Nutrition Examination Survey III reference ranges (NHANES III) were used. Spirometry and all pulmonary function assessments performed at all sites were reviewed centrally. Spirometry was performed in the following order: pre-bronchodilator spirometry, post-bronchodilator spirometry (assessed 15 minutes after 200 ug of salbutamol/albuterol), diffusing capacity of the lungs for carbon monoxide (DLCO), slow vital capacity, multiple breath nitrogen washout (MBNW). Spontaneous sputum was used to quantify colony forming units (CFUs) as a potential efficacy marker. The data on following key respiratory pathogens was collected: Haemophilus parainfluenza, H. influenza, Pseudomonas aeruginosa, Moraxella catarrhalis, and Streptococcus pneumonia. An expiratory HRCT was performed at Screening and on Day 29 to assess the change from baseline in quantitative air trapping. Centralized review of the HRCT image data was performed by MedQIA, Los Angeles, California. Historical HRCT scans were also used at the Screening visit for assessing (i) extent of emphysema if acquired within 6 months prior to Screening visit (ii) air trapping if acquired within 3 months of the Screening visit (only applicable to patient who were re-screened). HRCT scans, regardless of origin (at Screening or historical) must have exhibited <25% total lung emphysema extent and <15% total lung air trapping. The total radiation exposure for a single patient completing the study did not exceed 10 millisievert (mSv) which was less than the annual limits allowed in the EU and US, 20 and 50 mSv, respectively.Proposed revision for HRCT section:A central imaging CRO, MedQIA, Los Angeles, CA provided HRCT accreditation, site-specific imaging protocols and centralized imaging review for the inclusion criteria and exploratory efficacy analysis of quantitative air trapping. Briefly, all clinical centers performed as part of the quality assessment COPD Gene phantom imaging at all time points (screening and Day 29). At the screening visit, thin section volumetric chest CT was performed at Total Lung Capacity (TLC) and Residual Volume (RV) was used for evaluation of the inclusion criteria and at Day 29, the RV scan to assess the change from baseline in quantitative air trapping. The total radiation exposure for a single patient completing the study did not exceed 10 millisievert (mSv) which was less than the annual limits allowed in the EU and US, 20 and 50 mSv, respectively. The independent (blinded and de-identified) imaging analysis included semi-automated evaluation of lung and lobar volumes which were then subject to a CAD-based quantitative assessment to determine percent total lung and lobar regions with pixel intensity thresholds of less than ?950 HU (TLC) and ?856 HU (RV) for emphysema and air trapping extent, respectively. To satisfy HRCT inclusion, TLC must have exhibited <25% total lung emphysema extent and RV > 15% total lung air trapping. Change from baseline statistical analysis was performed on the RV derived whole lung and lobar percentages of air trapping that were evaluated with the same CAD based quantitative analysis. Exploratory 3D renderings were constructed to further visualize regional patterns of response. Pharmacokinetic assessmentsPlasma samples were obtained from all patients and pharmacokinetics were evaluated in all patients who received icenticaftor. Icenticaftor was analyzed by a validated liquid chromatography tandem mass spectrometry (LC-MS/MS) method with a lower limit of quantification (LLOQ) of 1 ng/mL. Concentrations below the LLOQ were reported as “0” (zero) and missing data was labeled as such in the Bioanalytical Data Report. The following PK parameters were calculated: Cmax, Clast, Tmax, Tlast, and AUClast on Day 1 and on Day 28. Patient-reported outcomes (PRO)The PRO measurements included St. George’s Respiratory Questionnaire (SGRQ) and Baseline/Transition Dyspnea Index (BDI/TDI) in the same order. The SGRQ is a disease-specific instrument designed to measure the impact on overall health, daily life, and perceived well-being in patients with obstructive airways disease. It contains 51 items divided into 3 components: “Symptoms” concerned with respiratory symptoms, their frequency and severity; “Activity” concerned with activities that cause or are limited by breathlessness; and “Impacts” which covered a range of aspects concerned with social functioning and psychological disturbances resulting from airway disease.The TDI is an 8-point scale PRO designed to measure functional impairment, magnitude of task and magnitude of effort. It was administered to the patient by investigator staff during interview. Patients were interviewed by an assessor experienced in the use of such questionnaires. Every attempt was made to ensure that it was the same assessor that completed all the TDI assessments for an individual patient. BDI/TDI was conducted after the SGRQ assessment.Statistical analysisSample size and powerA sample size of approximately 90 patients was required for the study (icenticaftor, n=60; placebo, n=30) in order to appropriately power for both LCI and FEV1. For LCI, assuming the true effect was 2 over placebo (SD: 3.8), an analysis had 80% power for declaring statistically important effect at Day 29. For pre-bronchodilator FEV1, assuming the true effect at Week 4 was 0.1 L over placebo (SD: 0.2 L), a Bayesian analysis utilizing a weakly informative prior had 90% power for declaring a statistically important effect. The expected placebo response was evaluated by summarizing 1200 subjects from similar historical Novartis COPD trials. A meta-analytic predictive prior was used to quantify the placebo in the new trial. ADDIN EN.CITE <EndNote><Cite><Author>Neuenschwander</Author><Year>2010</Year><RecNum>49</RecNum><DisplayText>(2)</DisplayText><record><rec-number>49</rec-number><foreign-keys><key app="EN" db-id="ez02vepxnt0vffe22tkxz2s2vpp2dvave2fx" timestamp="1534484599">49</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Neuenschwander, B.</author><author>Capkun-Niggli, G.</author><author>Branson, M.</author><author>Spiegelhalter, D. J.</author></authors></contributors><auth-address>Novartis Pharma, Basel, Switzerland. beat.neuenschwander@</auth-address><titles><title>Summarizing historical information on controls in clinical trials</title><secondary-title>Clin Trials</secondary-title></titles><periodical><full-title>Clin Trials</full-title></periodical><pages>5-18</pages><volume>7</volume><number>1</number><keywords><keyword>Clinical Trials, Phase IV as Topic/*methods</keyword><keyword>*Control Groups</keyword><keyword>*Databases, Factual</keyword><keyword>Humans</keyword><keyword>Meta-Analysis as Topic</keyword><keyword>Models, Statistical</keyword><keyword>Research Design</keyword><keyword>Sample Size</keyword></keywords><dates><year>2010</year><pub-dates><date>Feb</date></pub-dates></dates><isbn>1740-7753 (Electronic)&#xD;1740-7745 (Linking)</isbn><accession-num>20156954</accession-num><urls><related-urls><url>;(2) It was believed to be normally distributed with a mean change of 0.02 L worse (SD of 0.05 L). This approach allows for uncertainty in the belief about placebo due to trial-to-trial variability by discounting the prior and allowed Novartis to reduce the sample size by 14 placebo subjects. The Bayesian sample size calculations were performed using the freely available software ADDIN EN.CITE <EndNote><Cite><Author>Fisch</Author><Year>2015</Year><RecNum>41</RecNum><DisplayText>(3)</DisplayText><record><rec-number>41</rec-number><foreign-keys><key app="EN" db-id="ez02vepxnt0vffe22tkxz2s2vpp2dvave2fx" timestamp="1527660003">41</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Fisch, Roland.</author><author>Jones, Ieuan.</author><author>Jones, Julie.</author><author>Kerman, Jouni.</author><author>Rosenkranz, Gerd Karl.</author><author>Schmidli, Heinz.</author></authors></contributors><titles><title>Bayesian Design of Proof-of-Concept Trials</title><secondary-title>Therapeutic Innovation &amp; Regulatory Science</secondary-title></titles><periodical><full-title>Therapeutic Innovation &amp; Regulatory Science</full-title></periodical><pages>155-162</pages><volume>49</volume><number>1</number><dates><year>2015</year></dates><urls></urls></record></Cite></EndNote>(3).The study also assessed additional spirometric parameters including forced expiratory flow (FEF25–75), forced expiratory volume in 6 seconds (FEV6), inspiratory capacity (IC) and residual volume (RV); however, the study was not appropriately powered for these assessments. Interim analysisA total of 3 interim analyses (IAs) were performed during the study. The IA, unblinded to the study statistician, included data from 28 patients with FEV1, and was used to assess LCI and FEV1 variability to confirm sample size assumptions. The second unblinded IA included data from 42 patients, and was used to assess the probability of futility for LCI and FEV1. Futility criteria (≥60% posterior probability that the treatment effect was worse than placebo) were not met and the study continued as planned. The third IA included data from 63 patients, and was used to support internal decision-making evaluation for further development. Population setsEfficacy data were analyzed in patients who received any treatment and had available pharmacodynamic data with no major relevant protocol deviations. Patients were excluded from the PD analysis if they did not complete the 28 day treatment period (withdrawal of consent, AEs, PDs related to inclusion/exclusion), if >2 days elapsed between the last dose of icenticaftor and the Day 29 assessments, or if a COPD exacerbation occurred in the 2-weeks before or after the Day 29 visit. CFU analysis population also required ≥1 pathogen isolated at baseline and ≥1 post-randomization assessment available. The percentage of eradication at EOS was compared between treatments using a Bayesian logistic model with non-informative priors. For safety analysis, all AE data were displayed by treatment, dose and patient. Absolute values and change from baseline values for HRCT were listed by treatment, time point and patient.ResultsTable E2. Change from baseline for spirometry and lung volume measurements on Day 29 (Bayesian analysis)RawPosterior distributionPosterior distribution of treatment difference versus Placebo (Icenticaftor–placebo)ParameterTreatmentNMean ± SDMean ± SDMean ± SD90% CrIProbability (icenticaftor better than placeboFVC pre (L)Placebo23 0.01 ± 0.36 0.01 ± 0.08 0.06 ± 0.09?0.09, 0.210.73Icenticaftor 51 0.07 ± 0.35 0.07 ± 0.05FVC post (L)Placebo24 0.00 ± 0.22 0.01 ± 0.05 0.02 ± 0.07?0.09, 0.12 0.62Icenticaftor51 0.03 ± 0.28 0.03 ± 0.04 FEV6?pre?(L)Placebo23?0.00 ± 0.31 0.01 ± 0.06 0.06 ± 0.08 ?0.08, 0.180.78Icenticaftor51 0.06 ± 0.30 0.07 ± 0.04 FEV6?post?(L)Placebo24?0.03 ± 0.19 ?0.02 ± 0.05 0.05 ± 0.06 ?0.05, 0.150.82Icenticaftor51 0.04 ± 0.26 0.04 ± 0.04 FEF25–75%?pre?(L/s)Placebo23?0.02 ± 0.15 ?0.02 ± 0.03 0.05 ± 0.04 ?0.02, 0.110.88Icenticaftor51 0.03 ± 0.16 0.03 ± 0.02 FEF25–75%?post?(L/s)Placebo24?0.02 ± 0.16 ?0.02 ± 0.04 0.06 ± 0.05 ?0.02, 0.14 0.91Icenticaftor51 0.05 ± 0.21 0.04 ± 0.03 RV (L)Placebo24?0.03 ± 0.44 ?0.02 ± 0.10 0.05 ± 0.12 ?0.15, 0.25 0.34Icenticaftor48 0.01 ± 0.51 0.03 ± 0.07 IC (L)Placebo24?0.06 ± 0.16 ?0.05 ± 0.06 0.05 ± 0.07 ?0.06, 0.16 0.78Icenticaftor50 0.00 ± 0.31 0.00 ± 0.04 b.i.d, twice daily; CrI, credible interval; FEF, forced expiratory flow; FEV, forced expiratory volume; FVC, forced vital capacity; IC, inspiratory capacity; LS, least squares; RV, residual volumeTable E3. Summary of sputum microbiology (pharmacodynamic analysis set)Type of organismIsolate VisitPlaceboIcenticaftorHaemophilus influenzae1Baseline1419285.7 (1725636.85)419652.2 (461411.15)Day 29432550.0 (496992.31)767260.0 (1290194.99)EOS771692.3 (1592153.85)1450024.3 (1777665.15)Pseudomonas aeruginosaBiotype 2 – dry1Baseline-12000.0 (11313.71)Day 291400.0 (-)17000.0 (18384.78)EOS-40000.0 (-)Pseudomonas aeruginosaSmall colony variant1Baseline-2400.0 (-)Table E4. Incidence of adverse events (AEs) by preferred term, arranged by frequency in the total column (safety set)ParameterPlaceboN=28, n (%)IcenticaftorN=64, n (%)TotalN=92, n (%)Patients with AEs11 (39.3)26 (40.6)37 (40.2)Chronic obstructive pulmonary disease2 (7.1)4 (6.3)6 (6.5) Diarrhea2 (7.1)2 (3.1)4 (4.3) Dyspnea1 (3.6)2 (3.1)3 (3.3) Nasopharyngitis1 (3.6)2 (3.1)3 (3.3) Nausea2 (7.1)1 (1.6)3 (3.3) Constipation1 (3.6)1 (1.6)2 (2.2) Cough02 (3.1)2 (2.2) Non-cardiac chest pain02 (3.1)2 (2.2) Productive cough02 (3.1)2 (2.2) Vomiting02 (3.1)2 (2.2)AEs that occurred in >2% of study population are presented AE, adverse events; b.i.d., twice dailyFigure E1. Radar plot demonstrating probability of a positive effect with icenticaftor for 6 different endpoints at Day 29*Data presented as mean (90% credible interval). For CFU, data presented as odds of response (90% credible interval)*For CFU, data presented were at EOS BD, bronchodilator; CFU, colony forming units; EOS, end of study; FEV1, forced expiratory volume in 1 second; LCI, lung clearance indexPharmacokineticsAfter single and multiple oral doses of icenticaftor 300 mg b.i.d. to COPD patients, absorption of icenticaftor was rapid with median Tmax at 1.27 hr and 1.98 hr, respectively, as shown in Table E5. Although AUClast was only calculated to 8 hours, icenticaftor exposure (Cmax and AUC) increased after multiple-dose administration to less than 2-fold on Day 28. The mean plasma concentration-time profiles of icenticaftor following single and multiple doses is shown in Figure E2.Table E5. Pharmacokinetic parameters following single and multiple oral doses of icenticaftor 300 mg twice dailyDayStatisticAUClast(hr*ng/mL)Clast(ng/mL)Cmax(ng/mL)Tlast(hr)Tmax(hr)Day 1 N5757575757Mean (SD)3830 (3280)241 (445)1250 (840)CV% mean85.618567.2Geo-mean3040142969CV% geo-mean80.097.396.7Median307012410808.001.27min-max312-2400037.2-257052.9-46607.77-8.080.983-8.03Day 28 N5353535353Mean (SD)6840 (4490)552 (569)1640 (916)CV% mean65.7103.056.0Geo-mean57103941380CV% geo-mean65.492.767.1Median535039014808.001.98min-max1820-2240088.3-3380372-36407.73-8.080.933-7.97AUClast, area under the plasma concentration-time curve calculated to the last quantifiable concentration; Clast, last observed quantifiable concentration; Cmax, observed maximum concentration; Tlast, time point corresponding to the last quantifiable concentration; Tmax, time to reach maximum concentrationFigure E2. Arithmetic mean (SD) concentration-time profiles of icenticaftor after administration of single and multiple oral doses of 300 mg twice dailyAfter single and multiple oral doses of icenticaftor 450 mg b.i.d. to COPD patients, absorption was rapid with median Tmax ranging from 2 to 4 hours. The AUClast, Cmax and Clast was calculated from two patients on Day 1 and for one patient on Day 28 is presented in Table E6. The plasma concentration-time profiles of icenticaftor 450 mg following single and multiple oral doses is shown in Figure E3.Table E6. Pharmacokinetic parameters for two subjects after a single and one subject after multiple oral doses of icenticaftor 450 mg twice dailyDayAUClast(hr*ng/mL)Clast(ng/mL)Cmax(ng/mL)Tlast(hr)Tmax(hr)116800185029008.052.03117600210030408.024.002844700564064607.954.03AUClast, area under the plasma concentration-time curve calculated to the last quantifiable concentration; Clast=last observed quantifiable concentration; Cmax, observed maximum concentration; Tlast, time point corresponding to the last quantifiable concentration; Tmax, time to reach maximum concentrationFigure E3. Concentration-time profiles of icenticaftor after administration of single and multiple oral doses of 450 mg twice dailyPatient-reported outcomesBaseline Dyspnea Index and Transition Dyspnea IndexAn increase in mean change from baseline TDI scores is considered improvement. On Day 29, the mean TDI scores of functional impairment, magnitude of task, magnitude of effort and adjusted total score for icenticaftor (placebo) were 0.10 (0.42), 0.18 (0.58), 0.06 (0.33) and 0.32 (1.33), respectively. The mean scores increased in both placebo and icenticaftor groups as compared with baseline, but relative to placebo, there was no improvement noted with icenticaftor. On Day 29, the estimated mean difference (icenticaftor to placebo) in change from baseline TDI was ? 1.00 (90% CI: ? 1.81, ? 0.19), suggesting no improvement with icenticaftor as compared with placebo.St. George’s Respiratory QuestionnaireA decrease in scores from baseline is considered improvement, with a 4-point change representing a clinically meaningful improvement. The estimated mean difference (icenticaftor to placebo) in change from baseline for the SGRQ total score did not suggest improvement with icenticaftor as compared with placebo. For the SGRQ-Symptoms and SGRQ-Impacts domains, the estimated mean difference (icenticaftor to placebo) in change from baseline suggests a numerical trend for improvement with icenticaftor as compared with placebo. Activity of icenticaftor on WT CFTRAuthors: Giles Pergl-Wilson, Henry Danahay and Martin GoslingMethods:Cell culture:Human bronchial epithelial cells from wt or homozygous F508del CFTR donors were cultured using a modification of the method described by Gray and colleagues ADDIN EN.CITE <EndNote><Cite><Author>Gray</Author><Year>1996</Year><RecNum>50</RecNum><DisplayText>(4)</DisplayText><record><rec-number>50</rec-number><foreign-keys><key app="EN" db-id="ez02vepxnt0vffe22tkxz2s2vpp2dvave2fx" timestamp="1534755880">50</key></foreign-keys><ref-type name="Journal Article">17</ref-type><contributors><authors><author>Gray, T. E.</author><author>Guzman, K.</author><author>Davis, C. W.</author><author>Abdullah, L. H.</author><author>Nettesheim, P.</author></authors></contributors><auth-address>Laboratory of Pulmonary Pathobiology, National Institutes of Environmental Health Sciences, Research Triangle Park, NC 27709, USA.</auth-address><titles><title>Mucociliary differentiation of serially passaged normal human tracheobronchial epithelial cells</title><secondary-title>Am J Respir Cell Mol Biol</secondary-title></titles><periodical><full-title>Am J Respir Cell Mol Biol</full-title></periodical><pages>104-12</pages><volume>14</volume><number>1</number><keywords><keyword>Bronchi/*cytology/secretion/ultrastructure</keyword><keyword>*Cell Differentiation/drug effects</keyword><keyword>Cells, Cultured</keyword><keyword>*Cilia</keyword><keyword>Epidermal Growth Factor/pharmacology</keyword><keyword>Epithelial Cells</keyword><keyword>Epithelium/secretion/ultrastructure</keyword><keyword>Humans</keyword><keyword>Immunoblotting</keyword><keyword>Microscopy, Electron</keyword><keyword>Microscopy, Electron, Scanning</keyword><keyword>Mucous Membrane/cytology</keyword><keyword>Trachea/*cytology/secretion/ultrastructure</keyword><keyword>Tretinoin/pharmacology</keyword></keywords><dates><year>1996</year><pub-dates><date>Jan</date></pub-dates></dates><isbn>1044-1549 (Print)&#xD;1044-1549 (Linking)</isbn><accession-num>8534481</accession-num><urls><related-urls><url>;(4). Briefly, cells (1 x 106) were seeded in plastic T-162 flasks and were grown in bronchial epithelial cell growth medium (BEGM; Lonza) supplemented with bovine pituitary extract (52 μg/mL), hydrocortisone (0.5 μg/mL), human recombinant epidermal growth factor (0.5 ng/mL), epinephrine (0.5 μg/mL), transferrin (10 μg/mL), insulin (5 μg/mL), retinoic acid (0.1 μg/mL), triiodothyronine (6.5 μg/mL), gentamicin (50 μg/mL), and amphotericin B (50 ng/mL). Cells were seeded onto collagen-coated polyester inserts (Transwell and Snapwell; Costar) in differentiation media containing 50% DMEM in BEGM with the same supplements as above but without triiodothyronine and a final retinoic acid concentration of 50 nM (all-trans retinoic acid). Cells were maintained submerged for the first 7 days in culture. After 7 days submerged, the cells were exposed to air liquid interface (ALI) for the remainder of the culture period. Cells were used between days 14 and 28 after establishment of the ALI. At all stages of culture, cells were maintained at 37°C in 5% CO2 in an air incubator unless otherwise stated.Short-circuit current (ISC) measurements:Snapwell inserts were mounted in Vertical Diffusion Chambers (Harvard Apparatus), bathed with continuously gassed physiological salt solution (5% CO2 in O2; pH 7.4) maintained at 37°C. Cells were voltage clamped to 0 mV and transepithelial resistance measured. Sequentially, amiloride (10 μM) followed by FSK EC20 and then cumulative concentrations of icenticaftor (or vehicle) were added to both the apical and basolateral chambers. At the end of the experiment, CFTRinh172 (30 μM apical) was added to inhibit all CFTR mediated anion transport.Results: CFTR mutationEC50 (uM)NNone0.072 ± 0.04513F508del/F508del0.032 ± 0.015118EC50 values are expressed as mean ± standard deviation (SD). EC20 values for FSK were calculated to cause and increase in ISC equivalent to 20% of the maximum IFSK value as determined by the sigmoidal curve fit of the FSK concentration responseReferences ADDIN EN.REFLIST 1. Global Initiative for Chronic Obstructive Lung Disease. Global Strategy for the diagnosis, management, and prevention of chronic obstructive pulmonary disease (2013 report). . 2013.2. Neuenschwander B, Capkun-Niggli G, Branson M, Spiegelhalter DJ. Summarizing historical information on controls in clinical trials. Clin Trials 2010; 7: 5-18.3. Fisch R, Jones I, Jones J, Kerman J, Rosenkranz GK, Schmidli H. Bayesian Design of Proof-of-Concept Trials. Therapeutic Innovation & Regulatory Science 2015; 49: 155-162.4. Gray TE, Guzman K, Davis CW, Abdullah LH, Nettesheim P. Mucociliary differentiation of serially passaged normal human tracheobronchial epithelial cells. Am J Respir Cell Mol Biol 1996; 14: 104-112. ................
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