The study followed the principles of the Declaration of Helsinki and Good Clinical Practice and the protocol was approved by an independent Ethics Committee (Ethikkommission der Ärztekammer Schleswig-Holstein, Bad Segeberg, Germany). Written informed consent was obtained from all subjects prior to study start.
Study design and subjects
This was a double-blind, randomised, placebo-controlled, single-ascending-dose study in which each dose level was investigated in a new group of eight healthy male subjects (6 on active drug, 2 on placebo). Doses of 0.2, 1, 5, 25, 100, 300 and 600 mg of macitentan were compared with placebo and the next higher dose was only administered after review of the safety and tolerability findings of subjects receiving the preceding dose. Subjects could only receive one treatment and were not allowed to re-enter another dose group. Subjects were healthy as assessed by medical history, physical examination, ECG, vital signs and clinical laboratory tests. They could not participate if they smoked, had a prior history of drug or alcohol abuse, were allergic to any drugs, were using any medication or had participated in another clinical trial during the 3-month period preceding the screening examination.
A screening examination was performed within 2 weeks of study drug administration. Subjects were admitted to the research unit approximately 12 h before study drug administration, which was performed under fasted conditions. The subjects remained in the research unit until 48 h after dosing. Later study assessments were done on an ambulatory basis. During the study days, frequent recording of vital signs and 12-lead ECG, evaluation of adverse events, blood and urine sampling for clinical laboratory testing and physical examination took place. The end-of-study examination took place after the 48-h (dose groups 0.2 to 100 mg) or 144-h (dose groups 300 and 600 mg) blood sample for pharmacokinetic and pharmacodynamic purposes had been taken. An interim pharmacokinetic analysis after the 25-mg dose group had been completed revealed an unexpected long t1/2 of macitentan. In order to fully characterise the elimination phase, the protocol was amended to increase the time period of follow-up after dosing from 48 h to 144 h. This change in protocol was effective for the two higher dose groups only.
Sampling and bioanalytics
Plasma macitentan, its main metabolite, ACT-132577, and endothelin-1 concentrations were determined in 4-ml venous blood samples taken predose and at 0.33, 0.67, 1, 1.5, 2, 3, 4, 5, 6, 8, 10, 12, 16, 24, 30, 36, 48, 72, 96, 120 and 144 h after dosing. The 72- to 144-h blood samples were only taken from subjects having received the 300 and 600 mg doses. After collection, the tubes containing EDTA as anticoagulant were centrifuged for 10 min at 1,500 g and 4°C. The plasma was separated and stored at −20°C pending analysis.
Serum total bile salts were measured in venous blood samples taken predose and 1, 2, 3 and 4 h after dosing. After collection, the tubes containing no anticoagulant were left at room temperature for 30 min and subsequently centrifuged for 10 min at 1,500 g and 4°C. The separated serum was stored at −20°C pending analysis. No blood samples for total bile salt measurements were taken at later time points because subjects consumed a meal after the 4-h blood sample, which was expected to affect these measurements.
Prior to the start of urine collection and at the end of each collection interval, each subject was requested to completely empty his bladder. Starting at dosing, total urine over four consecutive 12-h intervals was collected in polyethylene bottles, which were kept in the refrigerator. An aliquot of 10 ml was taken and stored at −20°C until analysis.
Plasma concentrations of macitentan and its active metabolite ACT-132577 were determined using a validated liquid chromatography coupled to tandem mass spectrometry method (LC-MS/MS). The assay was linear in the concentration range 1–2,000 ng/ml and the limit of quantification was 1.0 ng/ml for both analytes. The performance of the method was monitored using quality control samples at concentrations of 3, 100 and 1,500 ng/ml. At these concentrations, precision (%CV) was ≤7.1% for macitentan and ≤6.7% for ACT-132577, whereas bias was ≤1.3% and ≤5.3% for macitentan and ACT-132577 respectively.
Plasma concentrations of endothelin-1 were determined using a commercially available radioimmunoassay (R & D Systems Europe Ltd, Abingdon, UK; detection limit 0.25 pg/ml).
Total serum bile salts were measured using a commercially available enzymatic assay (Sigma Bile Acids Method No#450, Trinity Biotech, Darmstadt, Germany; detection limit 1 μmol/l).
The presence of bromine atoms in the chemical structure of macitentan (Fig. 1) facilitated the detection of both parent and possible metabolites in plasma and urine. For this, 50 μl of plasma from the 2-, 6- and 48-h blood samples from a subject in the 600-mg dose group were pooled and proteins precipitated with an acetonitrile/ethanol mixture (50/50, v/v). Similarly, 50 μl of urine from each collection interval from two subjects in the 600-mg dose group were pooled. Following centrifugation of both plasma and urine at 3,360 g for 10 min at 8°C, 20 μl of the extracted sample were injected onto the HPLC column (Atlantis dC18; Waters, Milford, MA, USA). Detection was performed with a LTQ Quantum mass spectrometer (ThermoFinnigan, San Jose, CA, USA) and metabolites identified with the help of the Metabolite ID software (ThermoFinnigan, San Jose, CA, USA).
Safety and tolerability variables were analysed descriptively. For this, subjects treated with placebo were pooled.
The pharmacokinetic variables Cmax, tmax, AUC and t1/2 of macitentan and its metabolite as well as the AUC of endothelin-1 (AUC0–48h) and total bile salts (AUC0–4h) were determined by non-compartmental methods, as previously described . Dose proportionality of macitentan pharmacokinetics was only explored by comparing log-transformed Cmax values using a power model described by Gough et al. , as for most dose groups no reliable estimate of AUC0−∞ could be determined because of the unexpected long t1/2 of macitentan. The variables AUC0–48h and AUC0–4h for endothelin-1 and total bile salts respectively were compared between the different macitentan doses and placebo using analysis of variance (ANOVA, factor treatment). A statistically significant difference was accepted at P < 0.05.