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.