2.1 In vitro studies
Methods for the in vitro studies are summarised briefly here; full details can be found in the Supplementary Information. All analyses were performed using ultra-high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS).
The contribution of MAO vs other Phase I and II drug metabolising enzymes on SPL026 metabolism was investigated in human whole cell hepatocytes. In vitro metabolic stability of SPL026 was determined in the presence and absence of an irreversible and combined MAO-A/MAO-B inhibitor (100 nM clorgyline and 100 nM deprenyl/selegiline). Mitochondrial preparations contain a higher proportion of MAO enzymes relative to other Phase I and II enzymes and were therefore pre-incubated with and without separate MAO-A and MAO-B inhibitor solutions (clorgyline and deprenyl, respectively) to further assess the contribution of MAO8 and its specific isoform with respect to SPL026 stability in vitro. In vitro CYP phenotyping was performed to characterise the metabolism of SPL026 by 8 human CYP isozymes: CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2D6, CYP3A4 and CYP3A5 (Table S1).
The blood:plasma ratio of SPL026 was determined using fresh human blood and plasma samples from a donor panel. Plasma protein binding was assayed using pooled donor plasma and a Rapid Equilibrium Dialysis (RED) plate (Thermo Fisher Scientific, UK).
The distribution coefficient logD7.4 measures the partition of substances in octanol and aqueous solutions in a solution at pH 7.4, i.e., approximating that of blood. SPL026 was assayed with octanol and buffer prior to extraction and analysis.