Materials and Methods
This prospective observational post-approval multicenter study (ClinicalTrials.gov Identifier: NCT02807415) was conducted at three CF centers of the German Center for Lung Research (DZL) in Phe508del homozygous CF patients aged 12 years and older. Prior to and 8 – 16 weeks after the initiation of lumacaftor-ivacaftor therapy anthropometry, lung function, sweat chloride concentrations, NPD and ICM were measured in all participants [9, 11]. Collection of blood and rectal biopsies for ICM, sweat test and NPD measurements were performed within 2 to 3 hours after the administration of the morning dose of lumacaftor/ivacaftor. Serum samples were stored at -30°C until further analysis.
Serum levels of lumacaftor and ivacaftor were analyzed by a validated liquid chromatography method with UV and mass detection. Briefly, 200 µl serum was deproteinised by the addition of 600 µl methanol and subsequent centrifugation at 13,000 rpm for 15 min. The supernatant was separated by an UltiMate 3000 UHPLC system (Thermo, Waltham, Ma) on a Reprosil Pur Basic C18 (3μm, 100x2mm) column with 0.1% formic acid (v/v) / methanol as mobile phase. Lumacaftor was quantified by UV-spectrophotometry at 254 nm. Ivacaftor was quantified by quadrupole time-of-flight mass spectrometry (Bruker Daltonik, Bremen, Germany) using D18 Ivacaftor as an internal standard (ivacaftor m/z= 391.2, D18-Ivacaftor m/z= 409.3). The injected volume was 4 µl. The detection limit was 2 µg/ml for lumacaftor and 0.2 µg/ml for ivacaftor.
The study protocol was approved by the ethics committees of all participating centers. Written informed consent was obtained from all patients included in the study, their parents or legal guardians.