In Vivo Pharmacokinetics

Single-dose PK studies in mouse, rat, dog, and monkey were performed at Nuvisan GmbH (Grafing, Germany) according to established practices and operating procedures.

These animal experiments were approved by the District Government of Upper Bavaria and conducted in compliance with German and European Animal Welfare Laws and Regulations in an Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC)-accredited facility.

Single-dose intravenous (i.v., 0.2 mg/kg) and oral (p.o., 0.5 mg/kg) PK studies with M8891 were performed in NMRI mice (n = 3 per dose group), Beagle dogs (n = 2 per dose group), and cynomolgus monkeys (n = 3). Wistar rats (n = 3 per dose group) were dosed with M8891 at 2 mg/kg i.v. and 5 mg/kg p.o., respectively. A solution of PEG200 40% in water was used for both i.v. and the p.o. routes. For these standard PK assessments, the size of each dose group was selected to allow variability of exposure to be captured while minimizing animal use.

Pre-dosing blood samples (dog and monkey) as well as multiple consecutive samples up to 24 h post-dose (all species) were taken and used for plasma isolation.

Details of the bioanalytic methods used can be found in the Supplementary Materials.

Maximum plasma concentration (C_max) and time to reach maximum plasma concentration (t_max) were taken from the observed data. Area under the curve (AUC), clearance (CL), volume of distribution at steady-state (V_ss), and elimination half-life (t_1/2) were calculated by non-compartmental analysis (NCA) using the custom-made software “DDS-Tox” developed by Nuvisan GmbH, which provides results comparable to Phoenix WinNonlin (Certara, L.P., Princeton, New Jersey, USA). AUC values were calculated by NCA using the linear up/log down trapezoidal method. More details on calculation methods can be found in the Supplementary Materials.