We evaluated the metabolic profiles of several CYP3A4 substrates which were metabolized by the humanized CYP3A transchromosomic mouse (CYP3A-HAC/Cyp3a KO mouse) liver microsome. It is confirmed that this mouse has humanCYP3A gene cluster (CYP3A4, CYP3A5, CYP3A7, CYP3A43) (Kazuki, Y. et al., a poster presentation at the 16th North American ISSX Meeting, 2009). As we reported, those were observed that the metabolic functions of human CYP3A4/CYP3A5 introduced were kept in CYP3A-HAC/Cyp3a KO mice (Tsukazaki et al, a poster presentation at the 9th ISSX International Meeting, 2010).
In the present study, we estimated a simultaneous quantification method for human CYPs3A4, 3A7, 2B6, 2C9 and 2C19 in adult and fetal CYP3A-HAC/Cyp3a KO mouse liver microsomes. Each CYP specific digested fragment was assigned by a high resolution LC-MS/MS system (AB SCIEX Triple TOF 5600 equipped with ekisigent nano flow LC-system), and specific digested fragments for each human CYPs were determined by Peak View™. Human CYPs in mouse liver microsome were quantified by MRM method using a high sensitive LC-MS/MS system (AB SCIEX QTRAP® 5500 equipped with Shimadzu LC-20A system). The specificity and calibration curves were estimated by wild type mouse liver microsome samples which were spiked each human CYP independently. Microsome was digested by trypsin, and after digested samples were extracted using the OASIS HLB µElution plate.
Human CYP3A4 was found of 8.47 ± 0.706 pmol/mg protein from adult CYP3A-HAC/Cyp3a KO mouse liver microsome, but CYPs3A7, 2B6, 2C9 and 2C19 were not detected by the AB SCIEX Triple TOF 5600. The other hand, both of CYPs3A7 and 3A4 were detected from fetal CYP3A-HAC/Cyp3a KO mouse liver microsome (5.37 ± 0.660, 0.153 ± 0.0338 pmol/mg protein, respectably). All isoformes of human CYPs were not detected from wild type mouse liver microsomes.
Furthermore, we measured DHEA hydroxylation activity using Triple TOF 5600 equipped with conventional HPLC system. Fetal CYP3A-HAC/cyp3a KO mouse liver microsome was catalyzed 16-alpha hydoroxylation of DHEA, mainly caused by CYP3A7. The other hand, only 7-beta hydroxy DHEA, mainly caused by CYP3A4 was detected from liver microsome of adult CYP3A-HAC/cyp3a KO mouse.
In conclusion, we established the quatification method on human CYPs 3A4, 3A7, 2B6, 2C9 and 2C19 spiked on mouse liver microsome Furthermore, we could detected not only CYPs 3A4 and 3A7 in CYP3A-HAC/Cyp3a KO mice but also some metabolites formed by those enzymes. It shows that human CYP3As introduced in CYP3A-HAC/Cyp3a KO mouse is functional..