Winnonlin v 7

Patients were admitted to the hospital on days 1 and 15 of the study for pharmacokinetic blood sampling. A total of nine blood samples for the determination of sorafenib, sorafenib N-oxide, and sorafenib glucuronide were obtained at predefined time points (T = pre, T = 0.5 h, T = 1 h, T = 2 h, T = 4 h, T = 6 h, T = 8 h, T = 10 h, and T = 12 h). Blood samples were processed into plasma within 30 min, by vortex mixing and centrifugation for 10 min at 2500× g at 4 °C. Plasma concentrations were determined using a validated liquid chromatography tandem mass spectrometry (LC-MS/MS) method [18 (link)], at both the laboratory of Translational Pharmacology in the Erasmus MC, Rotterdam, and the laboratory of Pharmaceutics and Pharmaceutical Chemistry, Ohio State University, OH. Predefined pharmacokinetic endpoints were the dose-corrected area under the curve from preadministration time point until 12 h after sorafenib intake (AUC_{0–12 h}), maximum concentration (C_max), time until maximum concentration (T_max), and lowest plasma concentration (C_trough) and were determined using WinNonlin v. 7.0 (Phoenix, Certara, 5349 AB, Oss, The Netherlands) for sorafenib, sorafenib-N-oxide, and sorafenib glucuronide.

Inhibitory activity of the investigated and reference compounds against various subtypes of PDEs was analyzed using the PDE-Glo Phosphodiesterase Assay following the manufacturer’s instruction (Promega Corp., USA). A brief description of this procedure was presented in the supplementary materials. The values of IC₅₀, I_max, and γ parameters of the investigated and reference compounds were estimated using non-linear regression in Phoenix WinNonlin v. 7.0. Each sample was performed in quadruplicate and the relative activity of each sample was calculated as $E=\frac{\mathit{\text{sample activity}}}{\mathit{\text{control activity}}}·100\%$ . Subsequently, the following equation was fitted to the effect-concentration data: $$E=E_0-I_{\mathit{max}}·\left(\frac{C^{\gamma }}{C^{\gamma }+{{\mathit{IC}}_{50}}^{\gamma }}\right)$$ where E₀ is a baseline relative activity of each enzyme in the absence of a studied compound (fixed to 100%). I_max is the maximal inhibitory activity of a compound, C is the concentration of a compound, IC₅₀ is the concentration of a compound that produces 50% of I_max, and γ is the Hill coefficient.