Human Source Cyp450s High Human Cyp3a4 And Mdr1 Expression System And The Nuclear Receptor-mediated Induction Of The Role Of The Research System And Application

With the explosive growth in the field of genomics and combinatorial chemistry, synthesizing candidate compounds have been accelerated greatly. It is an important part of new drug screening to evaluate pharmacodynamics, pharmacokinetics and toxicity of compound candidates. Drug metabolism is an indispensable part in the process of new drug research and development. Every year a large number of drug candidates are eliminated due to poor pharmacokinetic parameters and metabolic characteristics. Therefore, early evaluation can contribute to get safe and effective drugs, and to reduce the elimination rate during drug design and development.Evaluation of drug metabolism has become mature increasingly. Nowadays, it is widely used in the research of drug metabolism, including induction and inhibition of drug metabolizing enzyme, enzyme types involved in drug metabolism, active metabolites formation and so on.These studies will also provide important evidences to predict drug interactions during coadministration.Evaluation of drug metabolism include in vivo and in vitro. As simple and feasible methods, in vitro is widespread attention, compared to in vivo. Evaluation of metabolism in vitro includes recombinant enzymes, liver slices, liver cells.Recombinant enzyme is a method that can express and putify human drug metabolizing enzymes through genetic and cellular engineering to obtain pure, single drug metabolizing enzymes. They can be used to study the profile of drugs and xenobiotics metabolism, and analyze metabolites from drugs and xenobiotics catalyzed by single enzyme. The probe substrate and the selective inhibitor of the single metabolizing enzyme can be characterized with the recombinant enzymes, which provide information for the dtug interaction. Since drug metabolizing enzymes have species differences, application of animal or tissueit to predict characteristics of drug pharmacokineticsis is limit, and even lead to error messages. Recombinant enzymes can make up for shortcomings from animal or isolated tissue to some extent, avoid interference to results from other enzymes in the course of study, and increase relevance and credibility of research. Recombinant drug metaboliszing enzymes provide an important and desirable model in vitro and effective analytical tools for the evaluation of drug metabolism and the clinical rational application of drugsCytochrome P450 (CYP450), the most important I phase drug metabolizing enzymes, mainly catalyze the redox reactions. CYP450 include three gene families (CYP1, CYP2, CYP3), among which CYP3A4, CYP2D6, CYP2C9, CYP2C19, CYP2E1 and CYP1A2 are mainly involved in drug metabolism. CYP3A4 is the most important metabolizing enzyme related to the biotransformation of endogenous and exogenous compounds including drugs, and metabolizesmore than 50% of the clinical drugs. P-glycoprotein (P-gp), one of ATP-dependent membrane transporter, is highly expressed in intestine, liver and kidney and can affect the absorption, distribution and excretion.Pregnane X receptor (PXR) and constitutive androstane receptor (CAR), the key nulear receptors, can interact with the retinoid X receptor a in response to structurally diverse compounds, and regulate the transcriptional activity of their target genes, such as CYP3A4 and multi-drug resistance 1 (MDR1). Construction of research system of PXR/CAR-mediated induction of CYP3A4 and MDR1 may provide effective evaluated system for the study of drug metabolism in vitro and mechanism.Baicalin, baicalein, chlorogenic acid, and ginsenoside Rf derived from herbal products are widely present in vegetables, fruit and herbal medicines with multiple beneficial effects, including anti-tumor, anti-inflammatory, anti-hypertensive and anti-fatigue. It is reported that the mentioned above compounds had induction or inhibition on some CYP450s isozymes, but the detailed molecular mechanisms underlying their action are not well explored.In the present study, molecular and cell biology methods were used to establish high expression system of human CYP450s isozymes and research system of nuclear receptors-mediated induction of CYP3A4 and MDR1. Then the inhibition of CYP2E1 recombinant enzyme by baicalein and baicalin was studied with the high expression system of human CYP450s isozymes. Moreover, the research system of nuclear receptors-mediated induction of CYP3A4 and MDR1 was also used to study the induction and molecular mechanism of CYP3A4 and MDR1 by baicalin, baicalein, chlorogenic acid and ginsenoside Rf. The present study will provide a good experimental model in vitro for research of drug metachnism in vitro, and also provide valuable experimental evidences for the clinical application of the above compounds.1. Construction and application of high expression system of human CYP450s isoenzymes1.1 As the primary electron donor, cytochrome P450 oxidoreductase (CYPOR) play an important role on the activity of CYP450 isoenzymes. The 2106bp in length cDNA fragment of CYPOR was amplified by RT-PCR, and inserted into prokaryotic expression vector pET-22b(+). Thus the recombinant expressive vector pET-22b(+)-CYPOR was constructed. After transformation into E.coli BL21with pET-22b(+)-CYPOR and induction with IPTG, recombinant target protein with Mr 75kD or so was expressed. Compared with negative control, CYPOR protein had higher enzymatic activity with 1309.64 nmol/mg-minute, and could be used for the following study.1.2 Baculovirus-insect cell (Bac-to-Bac) system is an ideal eukaryotic expression system due to good security, high expression and procession post-translation. The high expression system of CYP450s was established using Bac-to-Bac, through which some CYP450 isoenzymes such as CYP2E1, CYP2D6 and CYP3A4 were expressed. The results of enzyme activity determination demonstrated that CYP2E1 had higher enzyme activity. The expression and activity of CYP2E1 recombinant enzyme closely related to many factors, thus optimization was an important aspect to obtain the highest expression and activity of recombinant enzymes. Specifically, when the MOI value was 0.1, the activity of CYP2E1 recombinant enzyme was highest, and then gradually decreased with the MOI value increasing. In comparision with 48h,96h and 120h, CYP2E1 enzyme activity was highest at 72h when Sf9 cells were infected with recombinant virus. Since the concentrationof CYPOR and CYb5 had a great effect on the CYP2E1 enzyme activity, the appropriate concentration should be selected to increase enzyme catalytic activity. Some conditions such as MOI value, infection time, and the concentration of CYPOR and CYb5 have been optimized to improve the activity of CYP2D6 and CYP3A4 recombinant enzymes, but more suitable factors need to be further studied.1.3 The inhibition of activity of CYP2E1 by baicalin and baicalein was studied using CYP2E1 recombinant enzyme with high activity. The results showed that baicalin had the most potent inhibition with an IC50 value of 5.702μM, while the IC50 value of baicalein was over 50μM. The results indicated that baicalin had a stronger inhibition on CYP2E1, while the inhibition by baicalein was weak.2. Construction and application of research system of nuclear receptors-mediated induction of human CYP3A4 and MDR12.1 The DNA fragment of CYP3A4 promoter/enhancer and retinoid X receptor (RXRa) gene were amplified by RT-PCR, and inserted into expression vectors pGL4.10 and pCDNA3.1(-), respectively. Thus pGL4.10-CYP3A4 promoter reporter gene expression vector and pCDNA3.1(-)-RXRa eukaryotic expression vector were constructed successfully.2.2 The effects of baicalin, baicalein, chlorogenic acid, and ginsenoside Rf on the mRNA expression of CYP3A4 and MDR1 were investigated using real time PCR because transcriptional regulation is the key mechanism for gene expression and modulation. As a result, it was different that baicalin, baicalein, chlorogenic acid and ginsenoside Rf showed induction on CYP3A4 and MDR1 at the level of transcription. Baicalein was found to be the strongest inducer of CYP3A4 and MDR1 among the four bioactive ingredients, while chlorogenic acid and ginsenoside Rf only showed induction of CYP3A4. However, baicalin had no effect on either CYP3A4 or MDR1 gene expression. Baicalein, chlorogenic acid, and ginsenoside Rf were observed to up-regulate the mRNA level of CYP3A4 in a dose-dependent manner, and the maximal induction was achieved at the concentration of 20,0.1 and 10μM, respectively. Furthermore, CYP3A4 activity and protein levels were also increased by baicalein-/chlorogenic acid-/ginsenoside Rf.2.3 PXR and CAR are considered to be the key transcriptional regulators of CYP3A4 and MDR1. The research system of nuclear receptors-mediated induction of CYP3A4 and MDR1 was established by reportor gene assay with cotransfection, and was confirmed with rifampin and CITCO which were the typical ligands of pregnane X receptor (PXR) and constitutive Androstane receptor (CAR), respectively. As a result, rifampin significantly enhanced PXR-mediated CYP3A4 and MDR1 gene transcription, while CITCO also upregulated the transcription level of CYP3A4 and MDR1 genes through the activation of CAR receptor. The results suggested that the the research system of nuclear receptors-mediated induction of CYP3A4 and MDR1 was constructed successfully, and can be used for drugs induction of CYP3A4 and MDR1 in vitro. The molecular mechanisms of induction of CYP3A4 and MDR1 by baicalein, chlorogenic acid and ginsenoside Rf were investigated through the construced research system. The results showed that baicalein significantly induced the expression of CYP3A4 and MDR1 mRNA by activating PXR and CAR. Chlorogenic acid and ginsenoside Rf induced CAR but not PXR-mediated CYP3A4 expression, and demonstrated effects on MDR1 via neither the CAR nor PXR pathways.2.5 The response elements of the CYP3A4 promoter consist of two copies of the DR3 and ER6, and DR4 is an important response element in the MDR1 promoter. All of these elements could be recognized by CAR and PXR after heterodimerization with RXR. Baicalein significantly increased the binding of CAR/RXRa heterodimers to the DR3 and ER6 response elements of CYP3A4, and to the DR4(I) response element of MDR1. However, no effect on the binding of PXR/RXRa heterodimers to the DR4(I) and ER6 elements of MDR1 and CYP3A4 except DR3 sequences was observed.2.6 It is reported that most common used oral drugs were metabolized by CYP3A in human and most of them including tacrolimus, a new type of immunosuppressive drug, were also cosubstrates of both CYP3A and P-gp. Since baicalein had induction on CYP3A and P-gp, coadministration interaction should be concerned between those drugs and pharmaceutics from baicalein. Our results showed that the blood concentration of tacrolimus was decreased (Cmax reduced by 57%, AUC reduced by 55%) by multiple dosing of baicalein. As a result, the dosage of tacrolimus should be increased to maintain effective plasma concentration and avoid the occurring of organ rejection when this drug was used in combination with baicalein.In conclusion, the high expression system of CYP450s isoenzymes and research system of nuclear receptors-mediated induction of CYP3A4 and MDR1 were established in the present study, which provide a good experimental model in vitro for research of the induction/inhibition and related mechanisms of drug metabolizing enzymes and transporter proteins, and drug interactions. The inhibition of activity of CYP2E1 by baicalin and baicalein was studied using CYP2E1 recombinant enzyme with high activity. The results indicated that baicalin had a stronger inhibition on CYP2E1, while the inhibition by baicalein was weak. Moreover, the research system of nuclear receptors-mediated induction of CYP3A4 and MDR1 was also used to study the induction and molecular mechanism of CYP3A4 and MDR1 by baicalin, baicalein, chlorogenic acid and ginsenoside Rf. The results showed that baicalein significantly induced the expression of CYP3A4 and MDR1 mRNA by activating PXR and CAR. Chlorogenic acid and ginsenoside Rf induced CAR but not PXR-mediated CYP3A4 expression, and demonstrated effects on MDR1 via neither the CAR nor PXR pathways.The results of baicalin, baicalein, chlorogenic acid and ginsenoside Rf also provide a reference for prevention of drug interactions and improving drug efficacy and safety.