缬苯那嗪类似物P109人肝微粒体中代谢产物鉴定及酶表型研究

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

化合物P109是缬苯那嗪结构类似物,其在9位进行了环丙烷甲基取代,具有与缬苯那嗪相似的药物活性与药理作用机制。为研究P109在人肝微粒体孵育体系中的代谢产物,采用Waters Eclipse Plus-C18色谱柱(150 mm×2.1 mm×3.5μm)分离,以0.1%甲酸水(A)-0.1%甲酸乙腈(B)为流动相进行梯度洗脱,利用超高效液相色谱-高性能台式四极杆-轨道阱质谱(UHPLC-Q Exactive^(TM) Orbitrap MS)技术,在正离子模式下分析样品,筛选出9个化合物(包括P109及其8个代谢产物),并进行结构鉴定。结果表明,P109经人肝微粒体主要发生氧化、水解、脱环丙烷甲基的Ⅰ相代谢反应。代谢产物酶表型研究表明,P109的主要代谢酶亚型为CYP3A4和CYP2C8,次要代谢酶亚型为CYP2D6。本研究初步阐明了P109在人肝微粒体中的代谢规律及代谢产物酶表型,可为新药筛选、毒理学研究提供支持。

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 Exactive;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(CYP1 A2, CYP2 B6, CYP2 C8, CYP2 C9, CYP2 C19, CYP2 D6, CYP3 A4) 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 CYP3 A4, and the secondary metabolic subtype was CYP2 C8. The decyclopropane-methyl metabolites were mainly metabolized by CYP2 C8, and the secondary metabolites were CYP3 A4 and CYP2 D6. 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.