| In this paper, CYP2C9 (*1,*3,*13,*16), CYP2C8 (*1,*2,*3,*4), CYP2C19 (*1,*2) and CYP2D6*1 recombinant enzymes have been expressed through bac-to-bac expression system using recombinant plasmid of our lab; a chiral LC-MS/MS method has been developed to determine the fluoxetine (Flx) and norfluoxetine (Nflx) enantiomers based on stable-isotope labled technique, and the stereoselective metabolism of Flx catalyzed by the 11 types of enzymes expressed has been investigated in vitro.1 The expression of recombinant human CYP enzymes in Sf9 cells and activity identificationObjectives To express CYP2C9 (*1,*3,*13,*16), CYP2C8 (*1,*2,*3,*4), CYP2C19 (*1,*2) and CYP2D6*1 recombinant enzymes in Sf9 cells, identify the expression at protein level, and check the activity of the enzymes, respectively.Methods Spodoptera frugiperda (Sf9) insect cells were infected with Bacmid-CYP2C9*1,*3,*13,*16, Bacmid-CYP2D6*1, Bacmid-CYPOR, Bacmid-CYPB5 (saved by our lab) to generate recombinant baculoviruses carrying human CYP2C9, CYP2D6, CYPOR or CYPB5 gene seperately. Higher-titer baculovirus were generated after amplification, and then the titer was detected using RT-PCR and were used to express the target CYP proteins. The recombinant CYP enzymes were detected via Western blot. Co-incubating with respective classical substrate is use to identify the activity of the enzymes. Results Western blot showed that there is clear band around 55 kD, which is in coincidence with the expected size. It showed that the target enzymes have been expressed successfully. Besides, the incubation with classical substrate results showed the activity of recombinant is well.Conclusion All the recombinant enzymes have got successfully expressed in Sf9 cells, the activity is well, and can be used for in vitro experiment.2 Establishment of stereoselective analytical method for Fix and Nflx enantiomersObjectives To establish a sensitivite and high throughput analytical method to quantitively determine of Flx and Nflx in biological samples.Methods Considering the high sensitivity and selectivity of mass spectrometry, and the different molecular mass of isotope element quality, we have developed an LC-MS/MS method based on stable-isotope technique which can simultaneously determine Flx and Nflx enantiomers at trace level.Results The results indicated the LC-MS/MS method had a lineary range between 1-1000 ng·mL-1 for R-Flx and S-Flx-d5,0.1-100 ng·mL-1 for R-Nflx and S-Nflx-d5 (with R^2>0.999). The LOQ is 1 ng·mL-1 (S/N>10, RSD=5.5%) for R-Flx,1 ng·mL-(S/N>10, RSD=6.1%) for S-Flx-d5,0.1 ng·mL-1 (S/N>10, RSD=6.5%) for R-Nflx, and 0.1 ng·mL-1 (S/N>10, RSD=7.6%) for S-Nflx-d5. The mean recovery was between 96.9%-109.2%, and the intra-day and inter-day presicions were not over 10%.Conclusion The LC-MS/MS method was high sensitivity, accuracy and specifity, can be used in the quantitative determinations of Flx and Nflx in biological samples.3 Metabolism study of Fix with recombinant enzymes in vitroObjectives To determine the enzyme parameters of each recombinant enzyme catalyzing fluoxetine (Including R-Flx, S-Flx and±Flx), and investigate the interaction between the two enantiomers.Methods At optimized conditions (i.e. optimized time and enzyme concentration) incubate enzymes with Flx at different concentrations, and calculate the enzyme kinetic parameters by Graphpad Prism, using formation velocity (Y-axis) of product (Nflx) vensus concentrations (X-axis) of substrate (Flx). Fix the concentration of R-Flx or S-Flx, and then set a series of its isomer concentrations, co-incubation with CYP2C9 enzymes, determine the product generating velocity. Define the product generated without adding its isomer to be 100 percent, take the percentage of product generated with adding its isomer to the former as Y-axis, and the concentration logarithm of isomer added as X-axis, investigate the relationship between them (calculate the IC50).Results The enzyme kinetic parameters of the enzymes (CYP2C 19*1, CYP2D6* 1, CYP2C9*1, CYP2C8*1) catalyzing Flx (±Flx, R-Flx and S-Flx) are as follows (for more detail, see data in section 4.3):For CYP2C19*1, Km for±Flx, R-Flx and S-Flx were 7.3±1.1μM,3.8±0.4μM,13.5±1.3μM; For CYP2D6*1, Km for±Flx, R-Flx and S-Flx were 8.1±1.0μM,4.2±0.7μM,7.5±1.2μM; For CYP2C9*1,43.4±6.2μM, 92.2±29.3μM,1660±359.4μM; For CYP2C8*1,394.8±84.0μM,153.8±32.6μM, 195±37.9μM. For CYP2C19*1, CLim for±Flx, R-Flx and S-Flx were 16.9,29.3,7.1 mL·min·1mg-1protein; For CYP2D6*1, CLint for±Flx, R-Flx and S-Flx were 2.1,4.3, 2.2 mL-min-1·mg-1protein; For CYP2C9*1,2.86* 10-2,1.89* 10-2,7.4* 10-4 mL·min-1·mg-1protein; For CYP2C8*1,3.52*10-2,3.95*10-2,3.44*10-2 mL·min-1·mg-1protein. There existed substrate inhibition in the metabolism catalyzed by CYP2C9 (*1,*2,*3,*13,*16) and CYP2C19*1, the Ksi were 451.3±84.5μM, 154.2±34.5μM,173.6±44.4μM,664.1±74.8μM,690.8±222.4μM, seperately. For CYP2C9*1,*2,*3,*13 and*16, the clearance ratio of R/S were 25.5,13.4,15.4,6.6, 15.6; For CYP2C19*1 and*2, the clearance ratio of R/S were 4.1 and 3.1; For CYP2D6,2.0; For CYP2C8*1,*2,*3 and*4, were 1.15,1.03,1.43 and 1.18. The clearance percentage of variant type to wild type were as follows:For CYP2C9*3,*13 and*16 were 33%,19%,57%(for±Flx),46%,25%,60%(for R-Flx),76%,96%, 98%(for S-Flx); For CYP2C19*2, were 27%,25%,34%for±Flx, R-Flx and S-Flx; For CYP2C8*1,*2,*3 and*4, were 95%,212%,36%(for±Flx),88%,338%,53% (for R-Flx),98%,272%,51%(for S-Flx). The results of interaction between enantiomers of Fix were as follows:The IC50 of R-Flx to S-Flx were 21.2μM,6.3μM,>57.8μM,>57.8μM and 21.2μM; while the IC50 of S-Flx to R-Flx were all over 57.0μM.Conclution CYP2C9, CYP2C19, CYP2C8, CYP2D6 can all mediate the demethylation metabolism of Flx, CYP2C19 and CYP2D6 show stronger affinity to Flx than the other two enzymes, and CYP2D6 has stongest affinity to S-Flx, maybe one reason for its main role in metabolizing S-Flx. CYP2C19 and CYP2D6 have CLint much more than CYP2C9 and CYP2C8, considering the low content of CYP2D6, we can conclude that CYP2C19 is one of the most important enzyme in metablizing Flx. There exsists substrate inhibition in metabolizing Flx, especially to CYP2C19*1 and CYP2C9 (all type), which indicates the importance of other enzyme (e.g. CYP2C8) uninhibited may increase in long-term administration. The metabolism of Flx mediated by CYP2C9, CYP2C19 and CYP2D6 showed enantioselectivity. R-Flx is more favorable, and CYP2C9 is the most significant, which suggests CYP2C9 is the one responsible for the stereoselective metabolism. Both CYP2C19 and CYP2C9 mutant have reduced the enzyme activity, which may lead to higher concentration of Flx in vivo. While, different CYP2C8s mutants have varies effect to the enzyme activity, with*2 stayed,*3 increased,*4 decreased, indicates that the polymorphism of CYP2C8 may enhance individual difference. R-Flx and S-Flx can inhibit each other's metabolism catalyzed by CYP2C9, the IC50 of R-Flx to S-Flx are smaller, we can presume the affinity between R-Flx and CYP2C9 enzyme is much stronger than its isomer, hence it can competitively occupied the activity caves easier, which lead to stronger inhibition. |
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