Identification of Metabolites and Study on Enzyme Phenotype of Compound P109, Analogues of Valbenazine in Human Liver Microsomes

Valbenazine (VBZ) is a selective synaptic vesicle monoamine transporter 2 (VMAT2) inhibitor that counteracts the increased activity of the dopamine system by inhibiting VMAT2 activity, reducing the uptake and storage of monoamines from the cytoplasm into presynaptic vesicles, and decreasing the concentration of monoamines released into the synapse. VBZ is the first drug approved by the US FDA for using in adult patients with tardive dyskinesia. Compound P109 is a structural modification product of VBZ, which is substituted by cyclopropane methyl at position 9. It has similar pharmacological activity and pharmacological mechanism as VBZ. In order to study its metabolites in the human liver microsome incubation system, a Waters Eclipse Plus-C18 column (150 mm×2.1 mm×3.5 μm) was used for separation, and 0.1% formic acid water (A)-0.1% formic acid acetonitrile (B) as the mobile phase for gradient elution. The high performance liquid chromatography coupled with four stage electrostatic field Orbitrap mass spectrometry (UHPLC-Q ExactiveTM Orbitrap MS) was used to analyze samples in positive ion mode. Nine compounds (including P109 and its 8 metabolites) were screened, and retention time, accurate mass measurement results and fragmentation patterns were all characterized. By using the Xcaliber4.2 workstation, the accurate molecular mass was used to infer the possible element composition, and then combined with the mass spectrometry fragment information for data processing. The molecular formula prediction module was used to predict the molecular formula of the parent ion and the fragment ion, and to identify the structure of in vitro metabolites of P109. The results showed that P109 undergoes three main metabolic pathways via human liver microsomes: 1) Oxidation reaction occurs to produce compounds 2, 4 and 9; 2) Hydrolysis reaction occurs to produce metabolites 1, 3, 7 and 8; 3) Decyclopropanemethyl reaction occurs to produce metabolite 5. The reaction phenotypes of seven cytochrome P450 isoenzymes (CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP3A4) were studied using specific chemical inhibitor inhibition method. The content of P109 metabolites was determined by HPLC-MS/MS, and the data was analyzed by Excel software. The results showed that oxidative metabolites and dehydrogenation metabolites were mainly metabolized by CYP3A4, and the secondary metabolic subtype was CYP2C8. The decyclopropane-methyl metabolites were mainly metabolized by CYP2C8, and the secondary metabolites were CYP3A4 and CYP2D6. Hydrolysis of metabolites involved multiple subtypes of enzymes. This study can help to clarify the metabolic pathways of drugs in vivo, which is of great significance to the research of pharmacokinetics, pharmacodynamics, and drug safety evaluation. At the same time, it provides theoretical support for extrapolating the results of in vitro experiments to in vivo experiments.