In vivo induction of human cytochrome P450 enzymes expressed in chimeric mice with humanized liver.

The induction and inhibition of human cytochrome P450 (P450) enzymes are clinically responsible for drug interactions. Although the induction of P450s is investigated using human hepatocytes in the drug development process, there are some disadvantages, such as the decline of the enzyme activity during culture. In the present study, we examined the in vivo induction potency in chimeric mice with humanized liver, which was recently established in Japan to clarify whether this chimeric mouse model would be more suitable for human induction studies. Rifampicin and 3-methylcholanthrene (3-MC) were used in vivo as typical P450 inducers in the chimeric mice. The expression levels of human CYP3A4 mRNA and CYP3A4 protein and dexamethasone 6-hydroxylase activity, specific for human CYP3A4, were increased 8- to 22-, 3- to 10-, and 5- to 12-fold, respectively, by treatment with rifampicin. In addition, the expression levels of human CYP1A2 mRNA and CYP1A2 protein were also increased 2- to 9- and 5-fold, respectively, by treatment with 3-MC. Although other human P450s are expressed in the chimeric mice, there were few effects by the treatment of rifampicin and 3-MC on the mRNA, protein, and enzyme activity of those P450s. It was demonstrated that human P450s expressed in the chimeric mice with humanized liver were induced by rifampicin and 3-MC. This chimeric mouse model may be a useful animal model to estimate and predict the in vivo induction of P450s in humans.     

Non-invasive method to detect induction of CYP3A4 in chimeric mice with a humanized liver

Chimeric mice with a humanized liver have been previously established by the transplantation of human hepatocytes to urokinase-type plasminogen activator/severe combined immunodeficiency mice. A non-invasive method to detect the induction of cytochrome P450 (CYP) 3A4 was evaluated in chimeric mice with a humanized liver. Dexamethasone (DEX) was used as a probe drug to detect induction; and rifampicin was used as a model drug to induce CYP3A4. Before and after rifampicin treatment (50 mg kg(-1), intraperitoneal injection once a day for 4 days) in the chimeric mice, DEX was subcutaneously injected and the urinary excretion of 6beta-hydroxydexamethason (6betaOHD) and DEX was determined. The metabolic ratio (6betaOHD/DEX) significantly increased after rifampicin treatment. Livers from the control and rifampicin-treated chimeric mice were stained immunohistolochemically with antibodies against CYP3A4 and CYP3A5. CYP3A4 and CYP3A5 were detected in the area of humanized liver, but staining was intense for CYP3A4 and very weak for CYP3A5. Only the staining of CYP3A4 was increased after rifampicin treatment. Formation of 6betaOHD by human liver microsomes was higher than that formed by mouse liver microsomes. Metabolite formation was catalysed by both CYP3A4 and CYP3A5 and the intrinsic clearance (V(max)/K(m)) by CYP3A4 was found to be 50-fold higher than that of CYP3A5. The results of the present study indicate that estimation of the changes of the urinary metabolic ratio (6betaOHD/DEX) in the chimeric mice with a humanized liver is a very useful tool for detecting the induction of CYP3A4 by a non-invasive method.     

Expression of human cytochromes P450 in chimeric mice with humanized liver.

Recently, a chimeric mouse line in which the liver could be replaced by more than 80% with human hepatocytes was established in Japan. Because the chimeric mouse produces human albumin (hAlb), replacement by human hepatocytes could be estimated by the hAlb concentration in the blood of chimeric mice. In this study, we investigated human major cytochrome P450 (P450) in the livers of chimeric mice by mRNA, protein, and enzyme activity using real-time polymerase chain reaction, Western blot analysis, and high-performance liquid chromatography, respectively. Chimeric mice with humanized liver generated using hepatocytes from a Japanese and white donor were used. Human P450 mRNAs were expressed in the liver of chimeric mice, and major human P450 proteins such as CYP1A2, CYP2C9, and CYP3A4 were detected. The expression of P450 mRNA and protein was correlated with the hAlb concentration in the blood. The enzyme activities such as diclofenac 4'-hydroxylase activity, dexamethasone 6-hydroxylase activity, and coumarin 7-hydroxylase activity, activities that are specific to human P450 but not to murine P450, were increased in a hAlb concentration-dependent manner. The chimeric mice with nearly 90% replacement by human hepatocytes demonstrated almost the same protein contents of human P450s and drug-metabolizing enzyme activity as those of the donor. It was confirmed that genomic DNA from the livers of the chimeric mice and that from the liver of the donor exhibited the same genotype. In conclusion, the chimeric mice exhibited a similarly efficient capacity of drug metabolism as humans, suggesting that they could be a useful animal model for drug development.     

Investigation of drug-drug interactions caused by human pregnane X receptor-mediated induction of CYP3A4 and CYP2C subfamilies in chimeric mice with a humanized liver

The induction of cytochrome P450 (P450) enzymes is one of the risk factors for drug-drug interactions (DDIs). To date, the human pregnane X receptor (PXR)-mediated CYP3A4 induction has been well studied. In addition to CYP3A4, the expression of CYP2C subfamily is also regulated by PXR, and the DDIs caused by the induction of CYP2C enzymes have been reported to have a major clinical impact. The purpose of the present study was to investigate whether chimeric mice with a humanized liver (PXB mice) can be a suitable animal model for investigating the PXR-mediated induction of CYP2C subfamily, together with CYP3A4. We evaluated the inductive effect of rifampicin (RIF), a typical human PXR ligand, on the plasma exposure to the four P450 substrate drugs (triazolam/CYP3A4, pioglitazone/CYP2C8, (S)-warfarin/CYP2C9, and (S)-(-)-mephenytoin/CYP2C19) by cassette dosing in PXB mice. The induction of several drug-metabolizing enzymes and transporters in the liver was also examined by measuring the enzyme activity and mRNA expression levels. Significant reductions in the exposure to triazolam, pioglitazone, and (S)-(-)-mephenytoin, but not to (S)-warfarin, were observed. In contrast to the in vivo results, all the four P450 isoforms, including CYP2C9, were elevated by RIF treatment. The discrepancy in the (S)-warfarin results between in vivo and in vitro studies may be attributed to the relatively small contribution of CYP2C9 to (S)-warfarin elimination in the PXB mice used in this study. In summary, PXB mice are a useful animal model to examine DDIs caused by PXR-mediated induction of CYP2C and CYP3A4.     

Application of chimeric mice with humanized liver for predictive ADME

Much effort to extrapolate the in vivo pharmacokinetics of drugs in human from experimental animals or in vitro studies has been made by many researchers. A urokinase-type plasminogen activator+/+/severe combined immunodeficient transgenic mouse line, in which the liver could be replaced by more than 80% with human hepatocytes, was established recently in Japan. This chimeric mouse line is remarkable because the replacement is higher than any other chimeric mouse reported previously. Since the liver is the critical organ involved in the pharmacokinetics of drugs, human liver is essential for the development of new drugs. To predict the human drug metabolism and pharmacokinetics, human hepatocytes and liver microsomes are recognized as better tools and are frequently used. Thus, chimeric mice with humanized liver would have great advantages in studies on drug metabolism and pharmacokinetics. We have evaluated chimeric mice for studies on absorption, distribution, metabolism, and excretion (ADME). In the liver of the chimeric mice, human phase I and phase II enzymes were clarified to be expressed and to have a similar drug metabolizing capacity as the donor. Human specific metabolites could be detected in the serum, suggesting that the chimeric mice might be used as a human ADME model for both in vitro and in vivo studies. For predicting human drug interactions, enzyme induction and inhibition are serious problems. By the treatment with typical inducers, human CYP1A2 and CYP3A4 expressed in the liver of the chimeric mice had induction potencies. After the treatment with quinidine, a specific inhibitor of human CYP2D6, the area under the curve (AUC) of a CYP2D6 metabolite, 4'-hydroxydebrisoquin, was significantly decreased in the chimeric mice but not in the control mice. Therefore, it was indicated that the chimeric mice could be used for assessing the drug interactions via enzyme induction and inhibition. As well as drug metabolism, the drug excretion was demonstrated to be humanized because cefmetazole was mainly excreted in urine both in the chimeric mice and human but in feces in control uPA-/-/SCID mice. In this review, basic researches on ADME in the chimeric mice with humanized liver are summarized and the application of the chimeric mice for predictive ADME is proposed.     

Application of Chimeric Mice with Humanized Liver for Predictive ADME

Much effort to extrapolate the in vivo pharmacokinetics of drugs in human from experimental animals or in vitro studies has been made by many researchers. A urokinase-type plasminogen activator^+/+/severe combined immunodeficient transgenic mouse line, in which the liver could be replaced by more than 80% with human hepatocytes, was established recently in Japan. This chimeric mouse line is remarkable because the replacement is higher than any other chimeric mouse reported previously. Since the liver is the critical organ involved in the pharmacokinetics of drugs, human liver is essential for the development of new drugs. To predict the human drug metabolism and pharmacokinetics, human hepatocytes and liver microsomes are recognized as better tools and are frequently used. Thus, chimeric mice with humanized liver would have great advantages in studies on drug metabolism and pharmacokinetics. We have evaluated chimeric mice for studies on absorption, distribution, metabolism, and excretion (ADME). In the liver of the chimeric mice, human phase I and phase II enzymes were clarified to be expressed and to have a similar drug metabolizing capacity as the donor. Human specific metabolites could be detected in the serum, suggesting that the chimeric mice might be used as a human ADME model for both in vitro and in vivo studies. For predicting human drug interactions, enzyme induction and inhibition are serious problems. By the treatment with typical inducers, human CYP1A2 and CYP3A4 expressed in the liver of the chimeric mice had induction potencies. After the treatment with quinidine, a specific inhibitor of human CYP2D6, the area under the curve (AUC) of a CYP2D6 metabolite, 4′-hydroxydebrisoquin, was significantly decreased in the chimeric mice but not in the control mice. Therefore, it was indicated that the chimeric mice could be used for assessing the drug interactions via enzyme induction and inhibition. As well as drug metabolism, the drug excretion was demonstrated to be humanized because cefmetazole was mainly excreted in urine both in the chimeric mice and human but in feces in control uPA^?/?/SCID mice. In this review, basic researches on ADME in the chimeric mice with humanized liver are summarized and the application of the chimeric mice for predictive ADME is proposed.     

Evaluation of mRNA expression of human drug-metabolizing enzymes and transporters in chimeric mouse with humanized liver

The hepatic mRNA expression of human drug-metabolizing enzymes and transporters in chimeric mise with almost-completely humanized liver (replacement index: 71-89%) was investigated. The mRNAs of 58 human phase I enzymes, 26 human phase II enzymes, 23 human transporters, and five mouse Cyps were measured in the chimeric mice with humanized liver generated using hepatocytes from a Japanese donor. The mRNA expression of 52 human phase I enzymes, which includes 20 human CYPs, 26 human phase II enzymes and 21 human transporters was ascertained in the chimeric mouse liver. Among them, the expression of the target mRNAs vital for liver function such as the metabolism and secretion of endogenous compounds appeared to be maintained. The central value for the expression ratio in all target genes in chimeric mouse liver to the donor liver was 0.46, which was lower than the substitution rate of chimeric mouse liver by donor liver. The ratio of mouse Cyp mRNA expression of chimeric mouse liver to that of control mouse liver was 0.19 or less, except for that of Cyp2b10. There were good correlations between the mRNA expression levels of human hepatic albumin gene, the values of the rate of replacement of mouse liver by human liver, and the human blood albumin concentration in the chimeric mice. The chimeric mice with humanized liver may be a useful tool for the evaluation of drug-drug interactions such as the inhibition and induction of drug-metabolizing enzymes and transporters.     

Evaluation of mRNA expression of human drug-metabolizing enzymes and transporters in chimeric mouse with humanized liver

The hepatic mRNA expression of human drug-metabolizing enzymes and transporters in chimeric mise with almost-completely humanized liver (replacement index: 71–89%) was investigated. The mRNAs of 58 human phase I enzymes, 26 human phase II enzymes, 23 human transporters, and five mouse Cyps were measured in the chimeric mice with humanized liver generated using hepatocytes from a Japanese donor. The mRNA expression of 52 human phase I enzymes, which includes 20 human CYPs, 26 human phase II enzymes and 21 human transporters was ascertained in the chimeric mouse liver. Among them, the expression of the target mRNAs vital for liver function such as the metabolism and secretion of endogenous compounds appeared to be maintained. The central value for the expression ratio in all target genes in chimeric mouse liver to the donor liver was 0.46, which was lower than the substitution rate of chimeric mouse liver by donor liver. The ratio of mouse Cyp mRNA expression of chimeric mouse liver to that of control mouse liver was 0.19 or less, except for that of Cyp2b10. There were good correlations between the mRNA expression levels of human hepatic albumin gene, the values of the rate of replacement of mouse liver by human liver, and the human blood albumin concentration in the chimeric mice. The chimeric mice with humanized liver may be a useful tool for the evaluation of drug–drug interactions such as the inhibition and induction of drug-metabolizing enzymes and transporters.     

Human arylacetamide deacetylase is responsible for deacetylation of rifamycins: rifampicin, rifabutin, and rifapentine

Rifamycins such as rifampicin, rifabutin, and rifapentine are used for the treatment of tuberculosis and induce various drug-metabolizing enzymes. Rifamycins have been reported to be mainly deacetylated by esterase(s) expressed in human liver microsomes (HLM) to 25-deacetylrifamycins, but the responsible enzyme remained to be determined. In this study, we found that recombinant human arylacetamide deacetylase (AADAC) could efficiently deacetylate rifamycins, whereas human carboxylesterases, which are enzymes responsible for the hydrolysis of many prodrugs, showed no activity. The involvement of AADAC in the deacetylation of rifamycins in HLM was verified by the similarities of the K_m and K_i values and the inhibitory characteristics between recombinant AADAC and HLM. Rifamycins exhibited potent cytotoxicity to HepG2 cells, but their 25-deacetylated metabolites did not. Luciferase assay using a reporter plasmid containing CYP3A4 direct repeat 3 and everted repeat 6 motifs revealed that 25-deacetylrifamycins have lesser potency to transactivate CYP3A4 compared with the parent drugs. Supporting these results, HepG2 cells infected with a recombinant adenovirus expressing human AADAC showed low cytotoxicity and induction potency of CYP3A4 by rifamycins. In addition, CYP3A4 induction in human hepatocytes by rifamycins was increased by transfecting siRNA for human AADAC. Thus, we found that human AADAC was the enzyme responsible for the deacetylation of rifamycins and would affect the induction rate of drug-metabolizing enzymes by rifamycins and their induced hepatotoxicity.     

Nortriptyline E-10-hydroxylation in vitro is mediated by human CYP2D6 (high affinity) and CYP3A4 (low affinity): implications for interactions with enzyme-inducing drugs.

The human cytochrome P450 (CYP) isoforms mediating nortriptyline 10-hydroxylation have been identified using kinetic studies on heterologously expressed human CYPs and chemical inhibition studies on human liver microsomes. Nortriptyline was metabolized to E-10-hydroxynortriptyline by human lymphoblast-expressed CYPs 2D6 (Km 2.1 microM) and 3A4 (Km 37.4 microM) with high and low affinity, respectively, whereas CYPs 1A2, 2A6, 2B6, 2C9, 2C19, and 2E1 had no detectable activity. Human liver microsomal nortriptyline E-10-hydroxylation displayed biphasic kinetics. The high-affinity component (Km 1.3 +/- 0.4 microM, n = 11 livers) was selectively inhibited by the CYP 2D6 inhibitor quinidine, whereas the CYP3A4 inhibitor ketoconazole selectively inhibited the low-affinity component (K(m) 24.4 +/- 7 microM, n = 11 livers). Inhibition by ketoconazole increased with increasing substrate concentration, whereas the reverse was true for quinidine. The Vmax of the low-affinity component in human liver microsomes was significantly correlated (r2 = 0.84) with the relative activity factor for CYP3A4, a measure of the amount of catalytically active enzyme. A simulation of the relative contribution of CYPs 2D6 and 3A4 to net nortriptyline hydroxylation rate suggested that the relative contribution of CYP3A4 is only 20% even at the higher end of the therapeutic range. Induction of CYP3A4 will increase its importance and increase the net metabolic rate, whereas inhibition of CYP3A4 will be of little importance due to its minimal relative contribution under uninduced conditions. The identification of CYP3A4 as a low-affinity nortriptyline E-10-hydroxylase explains the ability of poor metabolizers of debrisoquin to hydroxylate nortriptyline, as well as the increased in vivo clearance via this pathway caused by CYP3A4-inducing drugs such as pentobarbital, carbamazepine, and rifampin.     

Metabolite-intermediate complexation and inhibition of microsomal CYP3A in rat liver by diltiazem

1. The formation of metabolite intermediate (MI)-complexes between cytochrome P450 (CYP) and the alkylamine-substituted drugs diltiazem (DTZ) and desipramine (DES) and their effect on CYP activities was investigated in rat liver. 2. Dexamethasone and phenobarbitone pretreatment enhanced MI-complexation by DTZ (36% and 11% of total CYP complexed, respectively), whereas beta-naphthoflavone induction was without effect. All three treatments decreased MI-complexation produced by DES. 3. After a preincubation step in NADPH-supplemented microsomes DTZ and DES were effective inhibitors of the activities of CYPs 3A and 2C11 (testosterone 6beta- and 16alpha-hydroxylations, respectively). 4. Although MI-complexation by DTZ was more extensive in microsomes from dexamethasone-induced rats, the apparent inhibition potency of the drug toward CYP activity was unchanged. By comparison, inhibition of CYP activity by DES was less pronounced than in control liver. 5. These findings indicate that drug-mediated MI-complexation of CYPs does not necessarily potentiate the inhibitory effect on monooxygenase activity, although the duration of inhibition is longer. The extent of inhibition produced by stable drug metabolites may be similar to that from MI-complexation. but their duration of action is limited by diffusion from the active site of the enzyme.     

CYP3A inductive potential of the rifamycins, rifabutin and rifampin, in the rabbit.

Rifabutin is effective in the treatment and prevention of Mycobacterium avium infection in people with HIV infection. Rifabutin is structurally related to another rifamycin, rifampin, a well-known inducer of the human P-450 isoform 3A. The rabbit isoform CYP3A6 and the human isoform CYP3A4 have similar P-450 predominance and substrate specificity and are both induced by rifampin. Our goal was to predict the CYP3A induction capacity of rifabutin and to determine if ex vivo CYP3A induction potential of rifamycins is predictive of that obtained in vivo. We determined the in vivo and ex vivo CYP3A6 induction by 4 days of treatment with rifabutin (100 mg/kg), rifampin (100 mg/kg), or vehicle (DMSO) in the rabbit. The ex vivo measures were CYP3A6 activity (N-demethylation of erythromycin and hydroxylation of triazolam) and CYP3A content in rabbit hepatic microsomes preparations. The in vivo measures were oral clearance of triazolam and its formation clearance to its hydroxylated metabolites, alpha-hydroxytriazolam and 4-hydroxytriazolam. Rifampin increased CYP3A6 activity by 2- to 3-fold in hepatic microsomes compared to vehicle. Rifabutin increased CYP3A content 1.7-fold, but did not significantly increase microsomal CYP3A6 activity. Oral triazolam clearance and formation clearances to the two hydroxylated metabolites were 2- to 3-fold greater in rabbits treated with rifampin. These clearances were unaffected by rifabutin administration. Ex vivo enzyme activities correlated with in vivo changes in clearance of triazolam and the formation clearance to its hydroxylated metabolites. Rifabutin is a weaker inducer of CYP3A6 than rifampin. These data suggest that ex vivo enzyme activity is a viable approach to predict in vivo inductive potential of CYP3A inducers.     

Alteration in the Expression of Cytochrome P450s (CYP1A1, CYP2E1, and CYP3A11) in the Liver of Mouse Induced by Microcystin-LR

Microcystins (MCs) are cyclic heptapeptide toxins and can accumulate in the liver. Cytochrome P450s (CYPs) play an important role in the biotransformation of endogenous substances and xenobiotics in animals. It is unclear if the CYPs are affected by MCs exposure. The objective of this study was to evaluate the effects of microcystin-LR (MCLR) on cytochrome P450 isozymes (CYP1A1, CYP2E1, and CYP3A11) at mRNA level, protein content, and enzyme activity in the liver of mice the received daily, intraperitoneally, 2, 4, and 8 µg/kg body weight of MCLR for seven days. The result showed that MCLR significantly decreased ethoxyresorufin-O-deethylase (EROD) (CYP1A1) and erythromycin N-demthylase (ERND) (CYP3A11) activities and increased aniline hydroxylase (ANH) activity (CYP2E1) in the liver of mice during the period of exposure. Our findings suggest that MCLR exposure may disrupt the function of CYPs in liver, which may be partly attributed to the toxicity of MCLR in mice.     

In vivo drug metabolism model for human cytochrome P450 enzyme using chimeric mice with humanized liver

We previously clarified that major human drug metabolizing enzymes were expressed in a chimeric urokinase-type plasminogen activator (uPA)+/+/severe combined immunodeficient (SCID) mouse line established recently, in which the liver could be replaced by more than 80% with human hepatocytes. In the present study, we investigated the in vivo drug metabolism of a CYP2D6 substrate, debrisoquin (DB), in chimeric mice with high (High) or low (Low) human albumin (hAlb) concentrations and in control uPA-/-/SCID mice. The hAlb in the mouse blood is one of the indices of humanized liver because the chimeric mice produce hAlb. After oral administration of DB at 2.0 mg/kg, the AUC0-8 value of a major CYP2D6 metabolite of DB, 4'-hydroxydebrisoquin (4-OH DB), in High was 3.6-fold higher than those of Low and uPA-/-/SCID mice. By pre-treatment with a typical CYP2D6 inhibitor, quinidine, the AUC0-8 value of 4-OH DB in High was decreased although such values in Low and uPA-/-/SCID mice did not change. The in vitro kinetic analyses and the Ki values of quinidine on the DB 4'-hydroxylase activity in liver microsomes also supported the humanization of the chimeric mice. In conclusion, the chimeric mice exhibited a humanized profile of drug metabolism and the inhibition of P450.     

The unique regulation of brain cytochrome P450 2 (CYP2) family enzymes by drugs and genetics

Cytochrome P450 (CYP) enzymes in the brain may have a role in the activation or inactivation of centrally acting drugs, in the metabolism of endogenous compounds, and in the generation of damaging toxic metabolites and/or oxygen stress. CYPs are distributed unevenly among brain regions, and are found in neurons, glial cells and at the blood-brain interface. They have been observed in mitochondrial membranes, in neuronal processes and in the plasma membrane, as well as in endoplastic reticulum. Brain CYPs are inducible by many common hepatic inducers, however many compounds affect liver and brain CYP expression differently, and some CYPs which are constitutively expressed in liver are inducible in brain. CYP induction is isozyme-, brain region-, cell type- and inducer-specific. While it is unlikely that brain CYPs contribute to overall clearance of xenobiotics, their punctate, region- and cell-specific expression suggests that CNS CYPs may create micro-environments in the brain with differing drug and metabolite levels (not detected or predicted by plasma drug monitoring). Coupled with the sensitivity of CNS CYPs to induction, this may in part account for inter-individual variation in response to centrally acting drugs and neurotoxins, and may have implications for individual variation in receptor adaptation and cross-tolerance to different drugs. In addition, genetic variation in brain CYPs, depending on the type of polymorphism (structural versus regulatory), will alter enzyme activity. These aspects of brain CYP expression regulation and genetic influences are illustrated in this review using mRNA, protein, and enzyme activity data for CYP2D1/6, CYP2E1 and CYP2B1/6 in rat and human brain. The role of CYP-mediated metabolism in the brain, a highly heterogeneous and complex organ, is a new and relatively unexplored field of scientific enquiry. It holds promise for furthering our undestanding of inter-individual variability in response to centrally acting drugs as well as risk for neurological diseases and pathogies.     

In vitro metabolism of nobiletin, a polymethoxy-flavonoid, by human liver microsomes and cytochrome P450

Cytochrome P450 enzymes (CYPs) in the liver metabolize drugs prior to excretion, with different enzymes acting at different molecular motifs. At present, the human CYPs responsible for the metabolism of the flavonoid, nobiletin (NBL), are unidentified. We investigated which enzymes were involved using human liver microsomes and 12 cDNA-expressed human CYPs. Human liver microsomes metabolized NBL to three mono-demethylated metabolites (4'-OH-, 7-OH- and 6-OH-NBL) with a relative ratio of 1:4.1:0.5, respectively, by aerobic incubation with nicotinamide adenine dinucleotide phosphate (NADPH). Of 12 human CYPs, CYP1A1, CYP1A2 and CYP1B1 showed high activity for the formation of 4'-OH-NBL. CYP3A4 catalyzed the formation of 7-OH-NBL with the highest activity and of 6-OH-NBL with lower activity. CYP3A5 also catalyzed the formation of both metabolites but considerably more slowly than CYP3A4. In contrast, seven CYPs (CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP2E1) were inactive for NBL. Both ketoconazole and troleandomycin (CYP3A inhibitors) almost completely inhibited the formation of 7-OH- and 6-OH-NBL. Similarly, α-naphthoflavone (CYP1A1 inhibitor) and furafylline (CYP1A2 inhibitor) significantly decreased the formation of 4'-OH-NBL. These results suggest that CYP1A2 and CYP3A4 are the key enzymes in human liver mediating the oxidative demethylation of NBL in the B-ring and A-ring, respectively.     

Use of mRNA expression to detect the induction of drug metabolising enzymes in rat and human hepatocytes

It is important to investigate the induction of cytochrome P450 (CYP) enzymes by drugs. The most relevant end point is enzyme activity; however, this requires many cells and is low throughput. We have compared the CYP1A, CYP2B and CYP3A induction response to eight inducers in rat and human hepatocytes using enzyme activities (CYP1A2 (ethoxyresorufin), 2B (benzoxyresorufin for rat and bupropion for human) and CYP3A (testosterone)) and Taqman™ Low Density Array (TLDA) analysis. There was a good correlation between the induction of CYP1A2, CYP2B6 and CYP3A4 enzyme activities and mRNA expression in human hepatocytes. In contrast, BROD activities and mRNA expression in rat hepatocytes correlated poorly. However, bupropion hydroxylation correlated well with Cyp2b1 expression in rat hepatocytes. TLDA analysis of a panel of mRNAs encoding for CYPs, phase 2 enzymes, nuclear receptors and transporters revealed that the main genes induced by the 8 compounds tested were the CYPs. AhR ligands also induced UDP-glucuronosyltransferases and glutathione S-transferases in rat and human hepatocytes. The transporters, MDR1, MDR3 and OATPA were the only transporter genes significantly up-regulated in human hepatocytes. In rat hepatocytes Bsep, Mdr2, Mrp2, Mrp3 and Oatp2 were up-regulated. We could then show a good in vivo:in vitro correlation in the induction response of isolated rat hepatocytes and ex-vivo hepatic microsomes for the drug development candidate, EMD392949. In conclusion, application of TLDA methodology to investigate the potential of compounds to induce enzymes in rat and human hepatocytes increases the throughput and information gained from one assay, without reducing the predictive capacity.