2.3. Single‐dose pharmacokinetics in rat and dog

Two days prior to drug dosing, rats were anesthetized with a mixture of ketamin (90 mg/kg) and intraperitoneal xylazine 2% (10 mg/kg), and a catheter was implanted under aseptic conditions into the jugular vein to allow for serial blood sampling. After recovery from general anesthesia, animals were housed individually with free access to water and food during the recovery period and the entire duration of the experiment. Pharmacokinetic experiments were started after 2 days of recovery. For intravenous dosing, macitentan was formulated as 55:45 (v/v) mixture of 50 mmol/L TRIS‐HCl buffer (pH 8.3) and PEG400. For oral experiments, macitentan was given as an aqueous suspension in 7.5% modified gelatin. Serial blood samples of 0.25 mL each were taken over a period of 32 hour and collected in vials fortified with EDTA. Plasma was generated by centrifugation and stored at −20°C pending analysis.

Experiments in the Beagle dog were performed using three male animals in a cross‐over design with a wash‐out period of 7 days between dosing events. For intravenous administration, macitentan was formulated as solution in 1:1:2 (v/v/v) mixture of PEG400, N‐methylpyrrolidone and 50 mmol/L TRIS‐HCl buffer (pH 8.3). For oral purposes, macitentan was given as an aqueous suspension in 7.5% succinylated gelatin (B. Braun, Melsungen, Germany). Serial blood samples of 2 mL each were taken over a period of 32 hour and transferred into vials fortified with EDTA as anticoagulant. Plasma was generated by centrifugation and stored at −20°C pending analysis.