In Vitro Metabolic And Metabolomics Study Of TAK788 Based On UPLC Q-TOF Technology

Lung cancer is the leading cause of cancer in morbidity and mortality,with non-small cell lung cancer（NSCLC）accounts for approximately 85%.Molecular targeted therapy is the best treatment for advanced NSCLC patients with positive driver genes,and the epidermal growth factor receptor（EGFR）gene is the most common and thoroughly studied molecular target for lung cancer.Mobocertinib（TAK788）is a new generation of irreversible,mutation-selective,small molecule EGFR inhibitor developed by Takeda Corporation of Japan,which is used to treat NSCLC with exon 20 insertion mutation of EGFR.At present,the related reports of TAK788 are mostly related to its clinical anti-tumor activity.In this study,we conducted in vitro metabolism and metabolomics research on TAK788,which will provide a useful reference for the follow-up pharmacological experiments and drug interaction.At first,the structure of TAK788 synthesized in the laboratory was confirmed by UV,IR,NMR and high-resolution MS,and its chemical structure was verified.The stability under the conditions of oxidation,strong acid and alkali as well as high temperature were investigated.It was found that the compound had the characteristics of acid resistance,alkali resistance and high temperature resistance,but it was easy to oxidize,which should be paid attention to in the process of drug storage and preparation development.Then,a rapid trace quantitative analysis method of TAK788 based on UPLC Q-TOF technology was established,which was capable of detecting the content of TAK788 within 10min.A Waters BEH C₁₈（2.1 mm×50 mm,1.7μm）column was used.The mobile phase A was acetonitrile and the mobile phase B was 0.1%formic acid water,and gradient elution method was as follows:time（min）:A/B（v/v）,T₀:2/98,T_0.1:2/98,T₇:100/0,T₈:100/0,T₉:2/98,T₁₀:2/98.The flow rate was 0.2 m L?min^-1,the column temperature was 40℃,and the detection wavelength was in the range of 200～600 nm.The sample chamber temperature was 6℃and the injection volume was 2μL.The optimal mass-to-charge ratio of monitored ions was 293.7m/z,the optimal cone voltage was 25 V,and the optimal scanning time was 1 s.The results of instrument suitability show that the method had good resolution and linear relationship within the initial concentration range of 1.5～60μM.The RSD value of precision was 0.40%,the RSD value of recovery was 1.32%～3.24%,which met the requirements for quantitative analysis.The in vitro metabolism of TAK788 was studied by the trace quantitative analysis method established by LC-MS.The in vitro experiments showed that the method was specific and the linear relationship of TAK788 concentration in liver microsomes was good after internal standard correction,and the correlation coefficients of the weighted regression equations were all greater than 0.99.The RSD values of intra-day precision and inter-day precision were less than 15%.The matrix effect was within±10%,the extraction recovery rate was more than 85%and the repeatability was good.The RSD values of the stability of the sample placed at room temperature for 8 h,autosampler at 6℃for 72 h and three times of freeze-thaw at-20℃were all within 10%.The results above demonstrated that the developed UPLC Q-TOF assay met the requirements for quantitative analysis of biological samples.Through UPLC Q-TOF detection,the half-life of TAK788 in SD rat liver microsomes was19.10±1.05 h,and the intrinsic clearance rate in liver microsomes was 0.0385±0.0022m L·h^-1·mg protein^-1,and the intrinsic clearance rate in vivo was 0.0343±0.0019 m L·h^-1·kg^-1.The half-life of TAK788 in human liver microsomes was 22.05±0.98 h,the intrinsic clearance rate in liver microsomes was 0.0334±0.0015 m L·h^-1·mg protein^-1,and the intrinsic clearance rate in vivo was 0.0163±0.0007 m L·h^-1·kg^-1.The results showed that TAK788 was metabolized for a long time and with low drug metabolism in SD rats and human liver microsomes.At last,based on the above data,a non-targeted metabolomics study of TAK788 in SD rat liver microsomes was conducted and 10 potential biomarkers such asα-D-glucose,L-valine and hypoxanthine were identified,involving seven metabolic pathways such as arachidonic acid metabolism and glycolysis/gluconeogenesis,which provided references for drug interaction research and individualized drug treatment in subsequent clinical trials of TAK788.