Expression Of Human UGT1A4, UGT2B7, CYP2D6～(?)1 And CYP2D6～(?)10 By Bac-to-Bac System And Their Applications To Study On The Related Drugs Metabolism

The recombinant human metabolizing enzyme is useful for the drugs and xenobiotics metabolism in vitro. They can be used to screen the profile of drugs and xenobiotics metabolism and avoid the substrate overlapping by using liver microsome. The probe substrate and the selective inhibitor of the single metabolizing enzyme can be characterized with the recombinant enzymes and this can provide information for the drug interaction or predict the potential drug candidate. The recombinant enzymes have been expressed in several systems including E.coli, yeast, mammalian cells, and baculovirus infected insect cells.UDP-Glucuronosyltransferases (UGTs) are glycoproteins localized in the endoplasmic reticulum that catalyze the conjugation of a broad variety of endogenous and exogenous lipophilic aglycon substrates (such as bilirubn, steroid hormone, drugs, insecticide, etc.) with glucuronic acid. UGTs are gene superfamily of phase II drug-metabolizing enzymes, they are responsible for the glucuronidation of a significant number of different function group (e.g. -OH, -COOH, -NH2, -SH). Glucuronidation is a major reaction in the elimination of lipophilic compounds from the body and the most pathway in phase II metabolism. UGT1 A4 and UGT2B7 were expressed by baculovirus infected Sf9 cells in this experiment and the activities were characterized by probe substrates. Then the enantioselectivity of propranolol glucuronidation was studied by the recombinant UGT1A3, UGT1A4, UGT1A6, UGT1A9 and UGT2B7.Multiple forms of cytochrome P450 enzymes play important roles in the oxidation of structurally diverse xenobiotics and endobiotics. The resulting recombinant human P450s are either purified for studying protein structure and the mechanism of catalysis or P450 phenotyping, metabolic stability screening and inhibitory potential evaluation. We focus on the co-expression of the recombinant CYPOR and CYP2D6*1 or CYP2D6*10. The recombinant subenzymes were used to study the metabolism of imipramine and propranolol.E.coli, yeast, mammalian cell and baculovirus infected insect cells were the main expression system used for enzyme. Baculovirus infected insect cells were effective eukaryotic expression system for human drug metabolizing enzymes. UGT and CYP450 were successfully expressed by this system in our laboratory.I. Expression of human UGT1A4 with baculoirus infected insect Sf9 cells and the imipramine metabolism via the recombinant UGTl A4UGTs catalyze the transfer of glucuronic acid from uridine 5'diphosphate glucuronic acid(UDPGA) to compounds with amine, hydroxyl, and carboxylic acid moieties. N-glucuronidation is an important pathway for elimination of many tertiary amine therapeutic agents used in humans. UGT1A4 has been reported to be specific for glucuronidating primary, secondary, and tertiary amines, forming N-glucuronides. To further investigate the drugs metabolized by UGTl A4, the Bac-to-Bac expression system was used to express the recombinant UGTl A4 with His-tag on the C-terminal. The His-tagged recombinant UGTl A4 expressed in Spodopteraβ-ugiperda (Sf9) cells were detected using anti-His antibody and the molecular weight of the recombinant protein was approximately 55 kDa. The enzyme activity towards imipramine in cell homogenate protein was found to be 83.14±15pmol/min/mg protein (n=3) with0.5mmol/L imipramine by HPLC, but was not detectable in blank Sf9 cells.To study the profile of imipramine N-glucuronidation using homogenates of recombinant UGT1A4 from baculovirus-infected Sf9 cells, imipramine N-glucuronide was biosynthesized by incubating imipramine with recombinant UGT1A4 and then purified with solid-phase cartridges. A reversed phase-high performance liquidchromatography (RP-HPLC) assay was employed to directly measure the concentration of imipramine and its metabolite, imipramine N-glucuronide, with />-nitrophenol as the internal Standard. The validated method was applied to characterize the activity of recombinant UGT1A4 and carry out kinetic study on imipramine glucuronidation in vitro. The high concentration of imipramine inhibited glucuronidation, so the formula V^Vmax'S/iKm+S+Sf/Ki) was used to calculate the parameters by using MATLAB software. The values of apparent Km, K\, and Fmax for imipramine glucuronidation via UGT1A4 were 1.39±0.09 mmol/L, 6.24±0.45 mmol/L and 453.81±32.12 pmol/min/mg cell homogenate (h=3), respectively. As a specific substrate of UGTl A4, imipramine was used as a convenient method to characterize the activity of recombinant UGT1A4 by using HPLC. Furthermore, the profile of imipramine glucuronidation was evaluated by using recombinant UGTl A4 in vitro.II. Expression of human UGT2B7 with baculovirus infected insect Sf9 cells and the naproxen metabolism via the recombinant enzymeThe recombinant UGT2B7 was expressed with baculovirus infected insect cells and the procedure was the same as that for UGT1A4. The His-tagged UGT2B7 was characterized with anti-His antibody. The activity was detected using the naproxen as substrate. The metabolite, naproxen acyl glucuronide, was confirmed with LC-MS.Naproxen was not hydrophilic and was solublized in methanol as store solution. The incubation solution containing high concentration of methanol may inhibit the activity of enzyme. So the effect of methanol in incubation solution on the activity of UGT2B7 was studied, the concentration of naproxen being 20 μmol/L and 50 μmol/L, separately. As a result, the methanol concentration higher than 3% showed obvious inhibition to the activity of UGT2B7. For the dose-course curve, as the concentration of naproxen was higher than 100μmol/L, the glucuronide produced will decrease. The inhibition may be caused by substrate inhibition. The UGT2B7 was successfully expressed with baculovirus infected insect Sf9 cells. The recombinant enzyme can be used to screen glucuronidation for many xenobiotics and endobiotics in vitro.III. Study of the propranolol glucuronidation and its enantioselectivity by the recombinant UGT1A3, UGT1A4, UGTl A6, UGT1A9 and UGT2B7 in vitroThe glucuronidation of propranolol was studied with the recombinant human UGT1A3, UGT1A4, UGT1A6, UGT1A9 and UGT2B7, which were expressed with the recombinant baculovirus in insect Sf9 cells in our lab. The propranolol glucuronides produced in incubation mixtures were assayed by RP-HPLC with fluorescent detection and the excitation and emission wavelength were set at 310 nm and 339 nm, respectively. Of these recombinant isoenzymes, UGT1A9 and UGT2B7 show the activity to catalyze propranolol glucuronidation. UGT1A9 prefers to catalyze S-(-)-isomer than R-(+)-isomer and the clearance ratio of S-(-)-isomer to R-(+)-isomer is 3.4 in racemic incubation mixtures. Whereas UGT2B7 catalyzes S-isomer slightly less than R-isomer and the clearance ratio of S-(-)-isomer to R-(+)-isomer is 0.74 in racemic incubation mixtures. It has been reported that the ratio of Vmaii/Km for the glucuronide conjugate of S-(-)-propranolol was from 2.1- to 4.9-fold greater than for the conjugate of the R-(+)-enantiomer in human. It is suggested that human UGTl A9 is responsible for the stereoselectivity to propranolol glucuronidation in human.IV. The co-expression of CYP2D6 and CYPOR by the baculovirus infected Sf9 cells and the relative metabolism researchCYP2D6 play an important role on drugs (antidepressant, antiarrhythmia and etc.) and xenobiotics metabolism. CYP2D6*10 is an important subtype in Asian people and 51.3% Chinese were classified with this subtype. Compared with CYP2D6*10, the amino acid P at 34 site was substituted with S and the amino acid S at 486 site was substituted with T.The oxidoreduction catalyzed by CYP450s was connected with CYPOR, which can deliver the electron. Partly CYP450s need cytochrome b5 to catalyze the reaction. There is no hemin in insect cell, so it is necessary to add the hemin to improve the expression level of the recombinant enzymes in Sf9 cells. It is convenient toco-express NADPH-cytochrome P450 oxidoreductase (CYPOR) and cytochrome P4502D6 (CYP2D6) in Sf9 cells with recombinant viruses.The wide type gene of CYPOR was obtained by RT-PCR from the normal human liver tissue and connected with the pFastBac to construct the recombinant plasmid named as pFastBac-CYPOR. The recombinant virus for CYPOR was obtained according to the procedure for UGTl A4.The CYP2D6*10 gene was a gift from Dr Zhu Gejian. The CYP2D6*1 gene was obtained by site directed mutagenesis. And then the recombinant CYP2D6*1 and CYP2D6*10 virus were obtained according to the procedure for UGT1A4.The cells infected with the recombinant viruses were collected after certain time and homogenized by sonification. Then homogenate protein activity was determined with dextromethorphan as substrate. The multiple of infection (MOI) ratio of the recombinant CYP2D6 virus to CYPOR virus was adjusted by detecting the activity of the homogenate protein of different rarios. The appropriate time of infection was determined by measuring the activity of the homogenate protein at different infection time.The CYP2D6 content in the microsomes was determined by a reduced carbon monoxide (CO) difference spectrum. The CYP2D6*1 is 0.21nmol /mg protein and the CYP2D6*10 is 0.16nmol/mg protein. There is significant difference between the recombinant CYP2D6*1 and CYP2D6*10 as the activity to catalyze the dextromethorphan was concerned. The Km and Fmax are 26.67±2.7^mol/L (n=3) and 666.7±56.78 pmol/nmol CYP2D6/min (n=3) for CYP2D6*1 to catalyze dextromethaphan. The Km and Fmax are 111.36±10.89μmol/L (n=3) and 222.2±20.12 pmol/ nmol CYP2D6/ min (n=3) for CYP2D6*10 to catalyze dextromethorphan. There is significant difference between CYP2D6 *1 and CYP2D6*10 for Fmax and Km (P<0.01). The clearance ratio of CYP2D6*1 is 25.0 and the clearance ratio of CYP2D6*10is2.0.The expressed CYP2D6*1 and CYP2D6*10 were useful tools to screen the metabolism profile of many xenobiotics and endobiotics in vitro. CYP2D6 plays animportant role to catalyze imipramine and propranolol to produce hydroxyl and desmethyl metabolites. In order to study the profile of this reaction, the recombinant CYP2D6*1 and CYP2D6*10 were used to incubate with them and the metabolites were characterized with LC-MS.As a result, the metabolite of imipramine was hydroxyl imipramine and desipramine. There is not significant difference between CYP2D6*1 and CYP2D6*10 as the enzyme kinetics for imipramine was concerned. For CYP2D6*1> Km is 10.15±0.91 umol/L (n=3), VmaK is 0.40±0.05 nmol/nmol CYP2D6*l/min (n=3);For CYP2D6*10, Km is 9.31±0.87 μmol/L (n=3), Fmax is 0.42±0.03 nmol/nmol/min (n=3). For proprnaolol, there were two metabolites when the substrate is 0.20umol/L. The metabolites were characterized by LC-MS, which were hydroxyl and N-desisopropylation propranolol. There is not significant difference between CYP2D6*1 and CYP2D6*10 as the activity of catalyzing proprnolol at low concentration is concerned. However, there is enantioselectivity between two enantiomers. The hydroxyl propranolol produced from R-(+)~isomer was more than that from S-(-)-isomer by CYP2D6*1 and CYP2D6*10 and there is no difference for N-desisopropylation propranolol between R-(+)- and S-(-)- isomer.The recombinant CYP2D6*1 and CYP2D6*10 can be used to screen the metabolism profile for many drugs and xenobiotics. Many of the metabolites are of pharmacologic effect and the CYP2D6 polymorphism may have effect on drug activity. It is important to study this difference with the recombinant polymorphic enzymes.