| Human cytochrome P450(CYP) is a superfamily of monooxygenases with a heme molecule. CYP plays an important role in the metabolism of various endogenous and exogenous compounds and the activation of environmental carcinogens.There are three families of CYP enzymes that are mainly responsible for the metabolism of drugs—CYP1, CYP2and CYP3, which include CYP1A2, CYP2A6, CYP2A13, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4and so on. Among the various CYPs, CYP3A4is the most abundant enzyme in human liver, and also the most related with drug metabolism.CYP3A4metabolizes about50%clinically used drugs. CYP3A4has a broad spectrum of substrates, and the structure and affinity of substrates are various. Testosterone and midazolam are two classical substrates that are chosen for CYP3A4activity determination. There are some typical metabolizing pathways with CYP3A4, for example, nicotine C-oxydation, voriconazole N-oxydation, fentanyl N-dealkylation, fluoxetine O-dealkylation, bromperidol and haloperidol dehydrogenation, clonazepam nitroreduction and terfenadine C-hydroxylation (Guergerich FP et al.,1999).Human cytochrome P450oxidoreductase (POR) is an important flavoprotein anchoring on endoplasmic reticulum (ER)(Williams CH et al.,1962; Phillips AH et al.,1962; Pandey AV et al.,2010; Miller WL et al.,2011). POR accepts the electrons from NADPH and electrons are then transferred to FAD and FMN sequentially. Reduced FMN finally transfers the electrons to CYP to help it catalyze the endogenous or exogenous compounds (Porter TD et al.,1991; Pandey AV et al.,2010; Porter TD et al.,2011).Human cytochrome b5(cyt b5) is a cytochrome b family protein anchoring on endoplasmic reticulum. Cyt b5contains two heme groups, and transfers electrons for multiple oxygenases anchoring on ER, such as CYP450.With the development of molecular biological techniques, heterologous expression of recombinant human enzymes is more and more broadly applied in the scientific research. Heterologous expression system can effectively and abundantly express recombinant human drug-metabolizing enzymes (DMEs) with high catalytic activity, so that we can investigate one single DME in vitro. During the process of study, various expression systems are compared. Among them, Baculovirus Expression System not only posses the merit of high expression level, but also has the correct glycosylation, phosphorylation and tissue localization, etc. Moreover, the biological character of the expressed protein is similar with that of the native protein. Therefore, we can use the Baculovirus Expression System to establish a high predictive ability metabolic model, which has a great significance on clinical medicine.In this study, we used Bac-to-Bac(?) Baculovirus Expression System to tri-express CYP3A4, POR and cyt b5. With the optimization of conditions for the tri-expression, we obtained the microsomes with high metabolic activity. On the basis of this, we use the microsomes to investigate the pharmacokinetics in metabolizing several substrates of CYP3A4and furthermore, to study the drug-drug interactions of these substrates.1. Construction and tri-expression of human CYP3A4, POR and cyt b5Currently, there are several expression systems used for drug-metabolizing enzymes expression, such as E.coli expression system, yeast expression system, mammalian cells expression system and Bac-to-Bac(?) Baculovirus Expression System. In our study, we used the Bac-to-Bac(?) Baculovirus Expression System to tri-express CYP3A4, POR and cyt b5.In the experiment, we designed the primers, and obtained the genes of CYP3A4, POR and cyt b5with polymerase chain reaction (PCR). After double digestion of the PCR products, we ligated them with the vector pFastBacTM1, respectively. Products of liagation are transformed into competent DH55α E.coli cells, and monoclonal colonies were picked to determine whether they are correctly constructed with PCR, double ligation and sequencing. Correctly constructed pFastBacTM1-CYP3A44, pFastBacTM1-POR, and pFastBacTM1-cyt b5were then transformed into competent DH10BacTM Escherichia coli cells. After transposition in DH10BacTM E.coli, we obtained recombinant Bacmid-CYR3A4, Bacmid-POR and Bacmid-cyt bs, respectively. Then, those bacmids were used to transfect Spodoptera frugiperda9(Sf9) cells respectively, to obtain the first passage of baculovirus (PI), which was amplified to P2, then P3. Generally, P3was suitable for protein expression.The obtained baculoviruses, V-CYP3A4, v-POR and v-cyt b5were added to one flask of Sf9cells with a certain proportion (such as V-CYP3A4:v-POR:v-cyt b5=1:1:1) to transfect cells and tri-express proteins. At the same time, to obtain the protein with higher metabolic activity, we used a new vector pFastBacTMual to construct pFastBacTMual-POR-CyP3A4. With the same method above, we obtained the P3of V-POR-CYP3A4, and then added it to Sf9cells with v-cyt b5(with the ratio of1:1) to tri-express CYP3A4, POR and cyt b5.To improve the expression level of protein of interest, we probed and optimized the culturing conditions of Sf9cells in shaker flasks. After a lot of practice, we found that when the temperature and rotation speed of the shaker were27℃and90r/min, respectively, the cell density and culture volume are5×105cells/ml and80-120ml per 250ml shaker flasks, respectively, the amount of Pluronic(?) F-68is0.1%, and the culture time is72h, the condition is the most suitable for Sf9culture and also for expression of our proteins of interest.2. Study of pharmacokinetics of different substrates metabolized by CYP3A4CYP3A4is among the most significant human drug-metabolizing enzymes, which mainly exist in liver and small intestine. CYP3A4accounts for about30-40%of total CYPs in human liver (Paine MJ et al.,2000), metabolizing about38kinds of more than150species of clinical medicine (Miners JO et al.,1996), including antibiotics, antifungal agents, antidepressants, antihistamines, calcium antagonists, sedative-hypnotics, and hormones, etc (Crespi CL et al.,1997). Therefore, the study of metabolism on single enzyme of CYP3A4played an important role on drug metabolism and clinical pharmacology.In this experiment, we use Bac-to-Bac Baculovirus Expression System to tri-express CYP3A4, POR and cyt b5. After preparation of microsomes, we used the expressed products to metabolize testosterone and midazolam. With the kinetic curve fit, we obtained two metabolic kinetic curves as well as the metabolic kinetic parameters, respectively. In addition, we also investigated the methodology of the substrates and their products, incubation conditions and High Performance Liquid Chromatography (HPLC) conditions. By the analysis of each metabolic kinetic, we finally obtained the metabolic kinetic parameters, for testosterone, the Km, Vmax and CLint values are119.6±13.79μmol/L,0.52±0.026μmol/min/g protein and4.34mL/min/g protein, respectively; for midazolam, the Km, Vmax and CLint values are6.79±0.56μmol/L,0.11±0.0029μmol/min/g protein and15.56mL/min/g protein, respectively.In the sduty, we prepared microsomes from tri-expressed CYP3A4, POR and cyt b5in Sf9cells, and used these microsomes to metabolize testosterone and midazolam. After investigation of the methodology of the substrates and their products, incubation conditions and HPLC conditions, we studied the metabolic kinetics of these two substrates.3. Application of CYP3A4in drug-drug interaction studyBecause of the broad spectrum of substrates of CYP3A4, many adverse drug reactions (ADR) are caused by drug-drug interactions of substrates metabolized by CYP3A4(Guengerich FP et al.,1997). Therefore, heterologous expression of CYP3A4to study the drug-drug interactions between its substrates can provide reliable prediction for clinical drug-drug interactions in vivo (Masimirembwa CM et al.,1999).In our experiment, we investigated the inhibition of testosterone metabolism by ketoconazole and the interaction of testosterone and midazolam with the prepared microsomes. Finally, we found that, with the increase of concentration, ketaconazole had a gradually increased effect of inhibition on the formation of6β-hydroxy testosterone by CYP3A4. With Dixon graphical method, we obtained that the inhibition constant Ki was0.0594±0.00586μM. In the study of the interaction between testosterone and midazolam, we found that when the concentration of testosterone was lower than Km value, midazolam had obvious inhibition on testosterone6β-hydroxylation, and this inhibition was enhanced with the decrease of the substrate concentration. However, when the concentration of testosterone reached200μM, only high concentration of midazolam (over40μM) inhibited testosterone metabolism, lower concentration of midazolam induced testosterone metabolism, instead. In the other experiment, we found that testosterone had obvious inhibition on different concentrations of midazolam, and this inhibition was enhanced with the decrease of the substrate concentration.The results of our study suggested that the tri-expressed CYP3A4, POR and cyt b5microsomes using Bac-to-Bac(?) Baculovirus Expression System had a high metabolic activity and that the optimized conditions were suitable for the expression of CYP3A4and could be further extended to express other drug-metabolizing enzymes, like other CYPs or UGTs. After further optimization, this kind of expression method could also be applied to large-scale expression of drug-metabolizing enzymes and to industrial production. CYP3A4has a broad spectrum of substrates, by its in vitro drug-drug interaction study of the substrates or inhibitors, we can predict the potential drug-drug interactions in vivo, so that some clinical adverse drug reactions can be avoided. |
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