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.     

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.     

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.     

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.     

Identification of the human liver cytochrome P450 isoenzymes responsible for the 5-methylhydroxylation of the novel anti-fibrotic drug AKF-PD

Identification of cytochrome P450 isoforms (CYPs) involved in flourofenidone (5-methyl-1-(3-fluorophenyl)-2-[1H]-pyridone, AKF-PD) 5-methylhydroxylation was carried out using human liver microsomes and cDNA-expressed human CYPs (supersomes). The experiments were performed in the following in vitro models: (A) a study of AKF-PD metabolism in liver microsomes: (a) correlations study between the rate of AKF-PD 5-methylhydroxylation and activity of CYPs; (b) the effect of specific CYPs inhibitors on the rate of AKF-PD 5-methylhydroxylation; (B) AKF-PD biotransformation by cDNA-expressed human CYPs (1A2, 2D6, 2C9, 2C19, 2E1, 3A4). In human liver microsomes, the formation of AKF-PD 5-methylhydroxylation metabolite significantly correlated with the caffeine N3-demethylase (CYP1A2), chlorzoxazone 6-hydroxylase (CYP2E1), midazolam 1'- hydroxylase (CYP3A4), tolbutamide 4-hydroxylase (CYP2C9), and debrisoquin 4-hydroxylase (CYP2D6) activities. The production of AKF-PD 5-methylhydroxylation metabolite was completely inhibited by a-naphthoflavone (a CYP1A2 inhibitor) with the IC50 value of 0.12 μM in human liver microsomes. The cDNA-expressed human CYPs generated different amounts of AKF-PD 5-methylhydroxylation metabolites, but the preference of CYP isoforms to catalyze AKF-PD metabolism was as follows: 2D6?>?2C19?>?1A2?>?2E1?>?2C9?>?3A4. The results demonstrated that CYP1A2 is the main isoform catalyzing AKF-PD 5-methylhydroxylation while CYP3A4, CYP2C9, CYP2E1, CYP2C19, and CYP2D6 are engaged to a lesser degree. Potential drug-drug interactions involving CYP1A2 may be noticed when AKF-PD is used combined with CYP1A2 inducers or inhibitors.     

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.     

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.     

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.     

Identification of the human liver cytochrome P450 isoenzymes responsible for the 5-methylhydroxylation of the novel anti-fibrotic drug AKF-PD

1. Identification of cytochrome P450 isoforms (CYPs) involved in flourofenidone (5-methyl-1-(3-fluorophenyl)-2-[1H]-pyridone, AKF-PD) 5-methylhydroxylation was carried out using human liver microsomes and cDNA-expressed human CYPs (supersomes). The experiments were performed in the following in vitro models: (A) a study of AKF-PD metabolism in liver microsomes: (a) correlations study between the rate of AKF-PD 5-methylhydroxylation and activity of CYPs; (b) the effect of specific CYPs inhibitors on the rate of AKF-PD 5-methylhydroxylation; (B) AKF-PD biotransformation by cDNA-expressed human CYPs (1A2, 2D6, 2C9, 2C19, 2E1, 3A4).

2. In human liver microsomes, the formation of AKF-PD 5-methylhydroxylation metabolite significantly correlated with the caffeine N3-demethylase (CYP1A2), chlorzoxazone 6-hydroxylase (CYP2E1), midazolam 1’- hydroxylase (CYP3A4), tolbutamide 4-hydroxylase (CYP2C9), and debrisoquin 4-hydroxylase (CYP2D6) activities. The production of AKF-PD 5-methylhydroxylation metabolite was completely inhibited by a-naphthoflavone (a CYP1A2 inhibitor) with the IC50 value of 0.12 μM in human liver microsomes. The cDNA-expressed human CYPs generated different amounts of AKF-PD 5-methylhydroxylation metabolites, but the preference of CYP isoforms to catalyze AKF-PD metabolism was as follows: 2D6?>?2C19?>?1A2?>?2E1?>?2C9?>?3A4.

3. The results demonstrated that CYP1A2 is the main isoform catalyzing AKF-PD 5-methylhydroxylation while CYP3A4, CYP2C9, CYP2E1, CYP2C19, and CYP2D6 are engaged to a lesser degree. Potential drug–drug interactions involving CYP1A2 may be noticed when AKF-PD is used combined with CYP1A2 inducers or inhibitors.

     

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.     

Induction of human cytochrome P450 3A4 by the irreversible myeloperoxidase inactivator PF-06282999 is mediated by the pregnane X receptor

1. 2-(6-(5-Chloro-2-methoxyphenyl)-4-oxo-2-thioxo-3,4-dihydropyrimidin-1(2H)-yl) acetamide (PF-06282999) is a member of the thiouracil class of irreversible inactivators of human myeloperoxidase enzyme and a candidate for the treatment of cardiovascular disease. PF-06282999 is an inducer of CYP3A4 mRNA and midazolam-1′-hydroxylase activity in human hepatocytes, which is consistent with PF-06282999-dose dependent decreases in mean maximal plasma concentrations (C_max) and area under the plasma concentration time curve (AUC) of midazolam in humans following 14-day treatment with PF-06282999.

2. In the present study, the biochemical mechanism(s) of CYP3A4 induction by PF-06282999 was studied. Incubations in reporter cells indicated that PF-06282999 selectively activated human pregnane X receptor (PXR). Treatment of human HepaRG cells with PF-06282999 led to ～14-fold induction in CYP3A4 mRNA and 5-fold increase in midazolam-1′-hydroxylase activity, which was nullified in PXR-knock out HepaRG cells. TaqMan® gene expression analysis of human hepatocytes treated with PF-06282999 and the prototypical PXR agonist rifampin demonstrated increases in mRNA for CYP3A4 and related CYPs that are regulated by PXR.

3. Docking studies using a published human PXR crystal structure provided insights into the molecular basis for PXR activation by PF-06282999. Implementation of PXR transactivation assays in a follow-on discovery campaign should aid in the identification of back-up compounds devoid of PXR activation and CYP3A4 induction liability.     

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.     

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.     

Predictive value of comparative molecular field analysis modelling of naphthalene inhibition of human CYP2A6 and mouse CYP2A5 enzymes.

The objects of this study were first to compare how well the recently constructed structure-inhibition activity relationship models of mouse CYP2A5 and human CYP2A6 predict the interaction of naphthalene in liver microsomes and secondly to study if these CYP enzymes actually oxidize naphthalene. The CoMFA model of CYP2A5 predicted the IC(50) value of naphthalene to be 42 microM (18-115 microM 95% CL) whereas in the in vitro experiment the result was 74 microM (65-83 microM) with the corresponding values for CYP2A6 being 41 microM (18-112 microM) and 25 microM (21-30 microM), respectively. Naphthalene appeared to be a competitive inhibitor both for mouse and human liver microsomal coumarin 7-hydroxylase, which is the specific probe activity for CYP2A5 and CYP2A6. The K(i)-value for the mouse enzyme was between 12-26 microM and for the human enzyme 1.2-5.6 microM. A 1-h in vitro incubation of naphthalene with human and pyrazole treated mouse liver microsomes produced more 1-naphthol than 2-naphthol. Antibody against the purified CYP2A5 inhibited 50-60% of the formation of 1-naphthol and 30-40% of the formation of 2-naphthol. These results indicate that in silico CoMFA models predict relatively well the interaction of naphthalene with CYP2A5 and CYP2A6 and that these CYPs actually oxidize naphthalene in vitro. CoMFA CYP2A5 and CYP2A6 models are thus useful as a technique for elucidating the interaction and potency of untested chemicals with these CYPs.     

Metabolism of Zaleplon by human hepatic microsomal cytochrome P450 isoforms

1. The metabolism of Zaleplon (CL-284,846; ZAL) has been studied in human liver microsomal preparations and in cDNA-expressed human cytochrome P450 (CYP) isoforms. 2. Human liver microsomes catalysed the NADPH-dependent N -deethylation of ZAL to DZAL (CL-284,859), but not to two other known in vivo metabolites, namely M1 (CL345,644) and M2 (CL-345,905). Sigmoidal kinetic plots were observed for ZAL deethylation indicating positive cooperativity. 3. The metabolism of ZAL to DZAL was determined in a characterized bank of 24 human liver microsomalpreparations.Good correlations (r² = 0.734-0.937) were observed with caffeine 8-hydroxylase, diazepam 3-hydroxylase, dextromethorphan N-demethylase and testosterone 2β-, 6β- and 15β-hydroxylase activities, which are allcatalysed by CYP3A isoforms. In contrast, poor correlations (r² 0.152-0.428) were observed for enzymatic markers for CYP1A2, CYP2A6, CYP2C9 10, CYP2D6, CYP2E1 and CYP4A9 11. 4. The metabolism of ZAL to DZAL in human liver microsomes was inhibited to 6-15% of control by 5-50 μM of the mechanism-based CYP3A inhibitor troleandomycin. Whereas some inhibition of DZAL formation was observed in the presence of 200 μM diethyldithiocarbamate, 5-50 μM furafylline, 2-20 μM sulphaphenazole, 50-500 μM S-mephenytoin and 1-10 μM quinidine had little effect. 5. Using human B-lymphoblastoid cell microsomes containing cDNA-expressed CYP isoforms, ZAL was metabolized to DZAL by CYP3A4, but not to any great extent by CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP2E1. 6. In contrast with ZAL, the NADPH-dependent N-deethylation of M2 to M1 proceeded at only a very low rate with both human liver microsomes and cDNA-expressed CYP3A4. 7. In summary, by correlation analysis, chemical inhibition studies and the use of cDNA-expressed CYPs, ZAL N -deethylation to DZAL in human liver appears to be catalysed by CYP3A isoforms.     

Dose-Response Relationships of Tissue Distribution and Induction of Cyp1A1 and Cyp1A2 Enzymatic Activities Following Acute Exposure to 2,3,7,8-Tetrachlorodibenzo-p-Dioxin (TCDD) in Mice

Tissue disposition of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) has been shown to be dose-dependent in rats. However, no reported studies in mice have demonstrated dose- and time-dependent distribution of TCDD and the potential sensitivities of target tissues to enzyme induction. The objectives of this study were to determine in mice the effects of dose (0, 0.1, 1, or 10 μg [³H]TCDD/kg) and time (7, 14, 21, and 35 days post-treatment) on tissue distribution (18 tissues) and enzyme induction (CYP1A1 in liver, skin, and lung and CYP1A2 in liver). Distribution of TCDD-derived radioactivity in all tissues was dose- and time-dependent with nonlinear distribution. Liver-to-adipose tissue concentration ratios range from 0.6 to 3.1 (low to high dose at Day 7) demonstrating a dose-dependent shift in the disposition of TCDD. In contrast to liver, relative concentrations of percentage dose/g and percentage dose/total tissue decreased with increasing doses in all other tissues. At Day 7 and lowest dose, all tissues contained <3% dose/g except for thyroid, adrenals, skin, liver, and adipose tissue which had 3, 6, 6, 15, and 24% dose/g, respectively. Induction of EROD activity, a marker for CYP1A1, was dose-dependent in liver, lung, and skin but did not parallel tissue concentrations of TCDD. At the highest dose, fold induction of EROD activity was two times greater in lung than liver, while the concentration in liver was 100 times greater than that in lung. Fold inductions of EROD activity in liver and skin were similar but the concentration was 20 times greater in liver than that in skin. Induction of hepatic acetanilide-4-hydroxylase (ACOH) activity, a CYP1A2 marker, was dose-dependent. Results of the present study demonstrated dose and time dependency in tissue distribution and induction of CYP1A1 and CYP1A2 as well as tissue sensitivities for enzyme induction in the female B6C3F1 mouse. These results provide important considerations for high- to low-dose extrapolations in risk assessments and use of sensitive markers of enzyme induction as surrogates for estimating exposure and in predicting risk.     

In vitro identification of metabolic pathways and cytochrome P450 enzymes involved in the metabolism of etoperidone

1. In vitro studies have been carried out to investigate the metabolic pathways and identify the hepatic cytochrome P450 (CYP) enzymes involved in etoperidone (Et) metabolism. 2. Ten in vitro metabolites were profiled, quantified and tentatively identified after incubation with human hepatic S9 fractions. Et was metabolized via three metabolic pathways: (A) alkyl hydroxylation to form OH-ethyl-Et (M1); (B) phenyl hydroxylation to form OH-phenyl-Et (M2); and (C) N-dealkylation to form 1-m-chlorophenylpiperazine (mCPP, M8) and triazole propyl aldehyde (M6). Six additional metabolites were formed by further metabolism of M1, M2, M6 and M8. 3. Kinetic studies revealed that all metabolic pathways were monophasic, and the pathway leading to the formation of OH-ethyl-Et was the most efficient at eliminating the drug. On incubation with microsomes expressing individual recombinant CYPs, formation rates of M1-3 and M8 were 10-100-fold greater for CYP3A4 than that for other CYP forms. The formation of these metabolites was markedly inhibited by the CYP3A4-specific inhibitor ketoconazole, whereas other CYP-specific inhibitors did not show significant effects. In addition, the production of M1-3 and M8 was strongly correlated with CYP3A4-mediated testosterone 6 β -hydroxylase activities in 13 different human liver microsome samples. 4. Dealkylation of the major metabolite M1 to form mCPP (M8) was also investigated using microsomes containing recombinant CYP enzymes. The rate of conversion of M1 to mCPP by CYP3A4 was 503.0 ± 3.1 pmole nmole^?1 min^?1. Metabolism of M1 to M8 by other CYP enzymes was insignificant. In addition, this metabolism in human liver microsomes was extensively inhibited by the CYP3A4 inhibitor ketoconazole, but not by other CYP-specific inhibitors. In addition, conversion of M1 to M8 was highly correlated with CYP3A4-mediated testosterone 6 β -hydroxylase activity. 5. The results strongly suggest that CYP3A4 is the predominant enzyme-metabolizing Et in humans.