Human Hepatic Cytochrome P450s Research Articles

Inhibitory Effects of Calf Thymus DNA on Metabolism Activity of CYP450 Enzyme in Human Liver Microsomes

The present study investigated the effect of calf thymus DNA (ctDNA) on human hepatic cytochrome P450s (CYP450s) in vitro. Specific substrate probes for each isoform, CYP1A2, 2C9, 2C19, 2D6 and 3A4, were incubated using pooled human liver microsomes with or without ctDNA, and liquid chromatography coupled tandem mass spectrometry (LC-MS/MS) method was developed for the analysis of probe metabolites. Enzyme kinetics parameters Ki and IC50 values were estimated to determine the types and strength of inhibition. ctDNA could specifically inhibit the metabolism of CYP2C9 probe substrates, with the IC50 = 0.9955 µg/ml, while it was not able to inhibit CYP1A2, CYP2C19, CYP2D6 or CYP3A4 (IC50 > 100 µg/ml). The results showed that ctDNA was a potent inhibitor of CYP2C9 enzyme, and has the metabolic interaction potential with the model drugs which are metabolism substrates of CYP2C9. The inhibition mechanism study suggested ctDNA may inhibit CYP2C9 by decreasing the activity of CYP450 reductase. These findings indicated that when the medical agents catalyzed mainly by CYP2C9 were co-administered in vivo with adsorptive material in vitro, the potential inhibitory effect of ctDNA on enzyme activity and the following metabolism character changes of the former should be highly focused on.

Inhibitory effects of wogonin on catalytic activity of cytochrome P450 enzyme in human liver microsomes

Wogonin, derived from the root of Scutellaria baicalensis, is a popular herb for its anticancer, anti-inflammatory, neuroprotective and anti-convulsant effects. The purpose of this study was to investigate the effect of wogonin on human hepatic cytochrome P450s (CYP450s) in vitro. Isoform-specific substrate probes of CYP1A2, 2C9, 2C19, 2D6, 2E1 and 3A4 were incubated in human liver microsomes with or without wogonin. IC(50) and K (i) values were estimated and the types of inhibition were determined. Wogonin was a potent, competitive inhibitor of CYP1A2 (K (i) = 0.24 μM), and a weak inhibitor of CYP2C19 (IC(50) = 101.10 μM), but was not able to inhibit CYP2C9, CYP2D6, CYP2E1 and CYP3A4 (IC(50) > 200 μM). Wogonin could inhibit the activity of CYP1A2 and CYP2C19 with varying potency, while it is a strong inhibitor of CYP1A2. These findings suggested that it was necessary to study the potential pharmacokinetic drug interaction in vivo.

Metabolic bone disease in HIV infection

HIV mainly replicates in CD4þ T lymphocytes andmonocyte/macrophages causing severe immunologicalimpairment. In addition to the immune system, HIVinfection affects tissues and organs such as kidney, liver,the central nervous system, heart and bone showing acomplex pathogenesis [1].The advent and widespread use of highly activeantiretroviral therapy (HAART) in the last two decadeshasled toa markedimprovementinthe treatmentofHIVdisease even though viral infection cannot be eradicatedbecause HAART does not completely eliminate the viralreservoirs [2]. HAART has dramatically changed thecourseofHIVinfection froma fatalinfectiontoachronicand relatively manageable disease. The increased lifeexpectancyofHIVpatientsandtheeffectsofHAARThavechanged the management of HIV infection. Nowadaysmedical treatment is no longer focused solely on HIVinfection, opportunistic diseases and monitoring immunederangement, but also includes the control of metabolic,cardiovascular, liver, bone and kidney complications. Inparticular, bone alterations have been observed in thecourse of HIV disease representing a pivotal clinicalprobleminthemanagementofHIVpatientsespeciallyforapossible development of bone fractures [3]. The majorbonelesionsdetectableinHIVpatientsarerelatedtobonedemineralization (osteopenia/osteoporosis and osteoma-lacia) and osteonecrosis ([4] for a review).This report will discuss the pathogenesis, diagnosis andtreatment of major bone complications represented bybone demineralization diseases during HIV infection andHAART treatment.

Contribution of Human Hepatic Cytochrome P450 Isoforms to the Metabolism of Psychotropic Drugs

The metabolic activities of six psychotropic drugs, diazepam, clotiazepam, tofisopam, etizolam, tandospirone, and imipramine, were determined for 14 isoforms of recombinant human hepatic cytochrome P450s (CYPs) and human liver microsomes by measuring the disappearance rate of parent compounds. In vitro kinetic studies revealed that Vmax/Km values in human liver microsomes were the highest for tofisopam, followed by tandospirone>clotiazepam>imipramine, diazepam, and etizolam. Among the recombinant CYPs, CYP3A4 exhibited the highest metabolic activities of all compounds except for clotiazepam and imipramine. The metabolism of clotiazepam was catalyzed by CYP2B6, CYP3A4, CYP2C18, and CYP2C19, and imipramine was metabolized by CYP2D6 most efficiently. In addition, the metabolic activities of diazepam, clotiazepam, and etizolam in human liver microsomes were inhibited by 2.5 microM ketoconazole, a CYP3A4 inhibitor, by 97.5%, 65.1%, and 83.5%, respectively, and the imipramine metabolism was not detected after the addition of 1 or 10 microM quinidine, a CYP2D6 inhibitor. These results suggest that the psychotropic drugs investigated are metabolized predominantly by CYP3A4, except that CYP2D6 catalyzes the metabolism of imipramine. In addition, this approach based on the disappearance rate appears to be useful for the identification of the responsible CYP isoform(s) of older drugs, for which metabolic profiles have not been reported.

Organophosphorothionate pesticides inhibit the bioactivation of imipramine by human hepatic cytochrome P450s

The drug–toxicant interaction between the antidepressant imipramine (IMI) and three organophosphorothionate pesticides (OPTs), to which humans may be chronically and simultaneously exposed, has been investigated in vitro. Concentrations of IMI (2–400 μM) and OPTs (≤10 μM) representative of actual human exposure have been tested with recombinant human CYPs and human liver microsomes (HLM). The different CYPs involved in IMI demethylation to the pharmacologically active metabolite desipramine (DES) were CYP2C19 > CYP1A2 > CYP3A4. The OPTs significantly inhibited (up to >80%) IMI bioactivation catalyzed by the recombinant CYPs tested, except CYP2D6, and by HLM; the inhibition was dose-dependent and started at low pesticide concentrations (0.25–2.5 μM). The OPTs, having lower K m values, efficiently competed with IMI for the enzyme active site, as in the case of CYP2C19. However, with CYP1A2 and CYP3A4, a time- and NADPH-dependent mechanism-based inactivation also occurred, consistently with irreversible inhibition due to the release of the sulfur atom, binding to the active CYP during OPT desulfuration. At low IMI and OPT concentrations, lower IC50 values have been obtained with recombinant CYP1A2 (0.7–1.1 μM) or with HLM rich in 1A2-related activity (2–10.8 μM). The K i values (2–14 μM), independent on substrate concentrations, were quite low and similar for the three pesticides. Exposure to OPTs during IMI therapeutic treatments may lead to decreased DES formation, resulting in high plasma levels of the parent drug, eventual impairment of its pharmacological action and possible onset of adverse drug reactions (ADRs).

Inhibition of human hepatic cytochrome P450s and steroidogenic CYP17 by nonylphenol.

Effect of nonylphenol on aminopyrine N-demethylase activity, a typical drug-metabolizing enzyme activity, by ten kinds of human hepatic cytochrome P450s (CYP) and on progesterone 17alpha-hydroxylase activity by steroidogenic CYP17 was investigated. When determined at 2 mM substrate concentration, nonylphenol (1 mM) most efficiently inhibited aminopyrine N-demethylation by CYP2C9 and CYP2C19, by 61% and 59%, respectively, followed by CYP2D6, CYP1A2, CYP2C18 and CYP2C8 (46-51%), whereas inhibition of the activities by other CYPs was less than 27%. Additionally, nonylphenol competitively inhibited diclofenac 4'-hydroxylation by CYP2C9 and S-mephenytoin 4'-hydroxylation by CYP2C19 with Ki values of 5.3 and 37 microM, respectively. Furthermore, nonylphenol exhibited a competitive inhibition of progesterone 17alpha-hydroxylase activity by CYP17 with Ki value of 62 microM. These results suggest that nonylphenol inhibits human hepatic CYPs, especially CYP2C9 and CYP2C19, and steroidogenic CYP17 activities.

Predicting drug pharmacokinetics in humans from in vitro metabolism studies

The pharmaceutical industry is committed to market safer drugs with fewer side effects, predictable pharmacokinetic properties and quantifiable drug-drug interactions. There is an increasing need to develop robust, enhanced-throughput in vitro assays, which accurately extrapolate to humans. The major drug metabolizing human hepatic cytochrome P450s (CYPs; CYP1A2, 2C9, 2C19, 2D6 and 3A4) have been co-expressed functionally in Escherichia coli with human NADPH-cytochrome P450 reductase and validated as surrogates to their counterparts in human liver microsomes (HLM) with respect to their kinetic and inhibition properties. Using these recombinant enzymes, fully automated in vitro assays to assess CYP inhibition and determine the enzymology of drug oxidation have been developed and validated. IC50 values determined for a series of test compounds in HLM and recombinant CYPs were similar (r2 = 0.9, P < 0.001). There was a good correlation between the sum of individual CYP intrinsic clearance (Clint) and HLM CIint (r2 = 0.8, P< 0.001) for ten prototypic substrates for which clearance was CYP-dependent. Several in vitro incubation milieu (e.g. CYPs, HLM, human hepatocytes) are routinely used and the level of non-specific binding was investigated with respect to effects on Km and Ki determinations. There were clear correlations between binding and lipophilicity (logD7.4) for a selection of bases (r2 = 0.98, P < 0.001) and acids (r2 = 0.79, P < 0.001) that may allow prediction of this property. Our laboratory has shown that recombinant enzymes are suitable for ‘frontline’ predictive human metabolism studies in early drug discovery.

Predicting drug pharmacokinetics in humans from in vitro metabolism studies.

The pharmaceutical industry is committed to market safer drugs with fewer side effects, predictable pharmacokinetic properties and quantifiable drug-drug interactions. There is an increasing need to develop robust, enhanced-throughput in vitro assays, which accurately extrapolate to humans. The major drug metabolizing human hepatic cytochrome P450s (CYPs; CYP1A2, 2C9, 2C19, 2D6 and 3A4) have been co-expressed functionally in Escherichia coli with human NADPH-cytochrome P450 reductase and validated as surrogates to their counterparts in human liver microsomes (HLM) with respect to their kinetic and inhibition properties. Using these recombinant enzymes, fully automated in vitro assays to assess CYP inhibition and determine the enzymology of drug oxidation have been developed and validated. IC(50) values determined for a series of test compounds in HLM and recombinant CYPs were similar (r(2)=0.9, P<0.001). There was a good correlation between the sum of individual CYP intrinsic clearance (Cl(int)) and HLM Cl(int) (r(2)=0.8, P<0.001) for ten prototypic substrates for which clearance was CYP-dependent. Several in vitro incubation milieu (e.g. CYPs, HLM, human hepatocytes) are routinely used and the level of non-specific binding was investigated with respect to effects on K(m) and K(i) determinations. There were clear correlations between binding and lipophilicity (logD(7.4)) for a selection of bases (r(2)=0.98, P<0.001) and acids (r(2)=0.79, P<0.001) that may allow prediction of this property. Our laboratory has shown that recombinant enzymes are suitable for "frontline" predictive human metabolism studies in early drug discovery.

Coexpression of Genetically Engineered Fused Enzyme between Yeast NADPH–P450 Reductase and Human Cytochrome P450 3A4 and Human Cytochrome b5 in Yeast

Human hepatic cytochrome P450 3A4 (CYP3A4) was expressed in yeast Saccharomyces cerevisiae. While the expression level was high as compared with other human hepatic cytochrome P450s, CYP3A4 showed almost no catalytic activity toward testosterone. Coexpression of CYP3A4 with yeast NADPH–P450 reductase did not give a full activity. Low monooxygenase activity of CYP3A4 was attributed to the insufficient reduction of heme iron of CYP3A4 by NADPH–P450 reductase. To enhance the efficiency of electron transfer from NADPH–P450 reductase to CYP3A4, a fused enzyme was constructed between CYP3A4 and yeast NADPH–P450 reductase. The rapid reduction of the heme iron of the fused enzyme by NADPH was observed. The fused enzyme showed a high testosterone 6β-hydroxylation activity with a sigmoidal velocity saturation curve. However, the coupling efficiency between NADPH utilization and testosterone 6β-hydroxylation was only 10%. Finally, coexpression of the fused enzyme and human cytochrome b5 was examined. A significant decrease in the Km value and a remarkable increase in the coupling efficiency were observed. Substrate-induced spectra revealed that the dissociation constant of the fused enzyme for testosterone significantly decreased with coexpression of human cytochrome b5. These results strongly suggest that human cytochrome b5 directly interacts with the CYP3A4 domain of the fused enzyme and modifies the tertiary structure of substrate binding pocket, resulting in tight binding of the substrate and high coupling efficiency.

Inhibition of drug-metabolizing enzyme activity in human hepatic cytochrome P450s by bisphenol A.

Effect of bisphenol A on drug-metabolizing enzyme activities by human hepatic cytochrome P450s (CYP) was investigated. We measured aminopyrine N-demethylation by eleven kinds of cDNA-expressed CYPs. CYP2C19 and CYP2B6 catalyzed most efficiently the aminopyrine N-demethylation, followed by CYP2C8 and CYP2D6. Bisphenol A (1 mM) most efficiently inhibited aminopyrine N-demethylation by CYP2C8 and CYP2C19 by 82% and 85%, respectively, whereas inhibition of the activities by CYP 2B6 and 2D6 was less than 40%. Bisphenol A exhibited a noncompetitive-type inhibition of aminopyrine N-demethylase activity by CYP2C8 with Ki value of 97 microM. Additionally, we investigated the inhibitory effect of bisphenol A on CYP2C19-mediated S-mephenytoin 4-hydroxylation. Bisphenol A exhibited a mixed-type inhibition with Ki value of 113 microM. These results suggest that bisphenol A inhibits human hepatic CYP activities, especially CYP2C8 and CYP2C19.

Roles of human hepatic cytochrome P450s 2C9 and 3A4 in the metabolic activation of diclofenac.

Recently, it was shown that diclofenac was metabolized in rats to reactive benzoquinone imines via cytochrome P450-catalyzed oxidation. These metabolites also were detected in human hepatocyte cultures in the form of glutathione (GSH) adducts. This report describes the results of further studies aimed at characterizing the human hepatic P450-mediated bioactivation of diclofenac. The reactive metabolites formed in vitro were trapped by GSH and analyzed by LC/MS/MS. Thus, three GSH adducts, namely, 5-hydroxy-4-(glutathion-S-yl)diclofenac (M1), 4'-hydroxy-3'-(glutathion-S-yl)diclofenac (M2), and 5-hydroxy-6-(glutathion-S-yl)diclofenac (M3), were identified in incubations of diclofenac with human liver microsomes in the presence of NADPH and GSH. The formation of the adducts was taken to reflect the intermediacy of the corresponding putative benzoquinone imines. While M2 was the dominant metabolite over a substrate concentration range of 10-50 microM, M1 and M3 became equally important products at >/=100 microM diclofenac. The formation of M2 was inhibited by sulfaphenazole or an anti-P450 2C9 antibody (5-10% of control values). The formation of M1 and M3 was inhibited by troleandomycin, ketoconazole, or an anti-P450 3A4 antibody (30-50% of control values). In studies in which recombinant P450 isoforms were used, M2 was generated only by P450 2C9-catalyzed reaction, while M1 and M3 were produced by P450 3A4-catalyzed reaction. Good correlations were established between the extent of formation of M2 and P450 2C9 activities (r = 0.93, n = 10) and between the extent of formation of M1 and M3 and P450 3A4 activities (r = 0.98, n = 10) in human liver microsomal incubations. Taken together, the data suggest that the biotransformation of diclofenac to M2 is P450 2C9-dependent, whereas metabolism of the drug to M1 and M3 involves mainly P450 3A4. Although P450s 2C9 and 3A4 both catalyze the bioactivation of diclofenac, P450 2C9 is capable of producing the benzoquinone imine intermediate at lower drug concentrations which may be more clinically relevant.

Contribution of human hepatic cytochrome P450s and steroidogenic CYP17 to the N-demethylation of aminopyrine

1. Aminopyrine N -demethylase activity was determined for 11 forms of human hepatic cytochrome P450s (P450s) expressed in yeast Saccharomyces cerevisiae and for human steroidogenic CYP17 expressed in Escherichia coli. 2. Among the hepatic P450s, the N-demethylation of aminopyrine was catalysed most efficiently by CYP2C19, followed by CYP2C8, 2D6, 2C18 and 1A2, whereas the activity with CYP2E1 was negligible. The kinetics of the N-demethylation process by CYP1A2, 2C8, 2C19 and 2D6 were studied by fitting to Michaelis-Menten kinetics by LineweaverBurkplots. CYP2C19 exhibited the highest affinityanda high capacity forthe aminopyrine N-demethylation. CYP2C8 showed the highest Vmax, followed by CYP2C19, 2D6 and 1A2, whereas the K m for CYP2C8, 2D6 and 1A2 were 10-17 times higher than that for CYP2C19. Accordingly, the Vmax/Km for CYP2C19 was more than nine times higher than that of other P450s. 3. Human steroidogenic CYP17 also catalysed aminopyrine N-demethylation and the activity was comparable with that for CYP3A4 which is a dominant P450 in human liver. The activity was increased 1.5-fold by the addition of cytochrome b, whereas the activity was not affected by the addition of Mg. 4. These results suggest that several human hepatic P450s, especially CYP2C19, and steroidogenic CYP17 exhibit aminopyrine N-demethylase activity.

Fully automated analysis of activities catalysed by the major human liver cytochrome P450 (CYP) enzymes: assessment of human CYP inhibition potential

1. Fully automated inhibition screens for the major human hepatic cytochrome P450s have been developed and validated. Probe assays were the fluorometric-based ethoxyresorufin O -deethylation for CYP1A2 and radiometric analysis of erythromycin N demethylation for CYP3A4, dextromethorphan O -demethylation for CYP2D6, naproxen O -demethylation for CYP2C9 and diazepam N -demethylation for CYP2C19. For the radiometric assays 99.7% of C-labelled substrate was routinely extracted from incubations by solid-phase extraction. 2. Furafylline, sulphaphenazole, omeprazole, quinidine and ketoconazole were identified as specific markers for the respective CYP1A2 (IC=6muM), CYP2C9 (0.7muM), CYP2C19 (6muM), CYP2D6 (0.02muM) and CYP3A4 (0.2muM) inhibition screens. 3. For the radiometric methods, a two-point IC estimate was validated by correlating the IC obtained with a full (seven-point) assay (r=0.98, p 0.001). The two-point IC estimate is useful for initial screening, while the full IC method provides more definitive quantitation, where required. 4. IC determined for a series of test compounds in human liver microsomes and cytochrome P450 cDNA-expressed enzymes were similar (r=0.89, p 0.001). In particular, the CYP1A2, CYP2D6 and CYP3A4 screens demonstrated the flexibility to accept either enzyme source. As a result of incomplete substrate selectivity, expressed enzymes were utilized for analysis of CYP2C9 and CYP2C19 inhibition. Good agreement was demonstrated between IC determined in these assays to IC published by other laboratories using a wide range of analytical techniques, which provided confidence in the universality of these inhibition screens. 5. These automated screens for initial assessment of P450 inhibition potential allow rapid determination of IC. The radiometric assays are flexible, sensitive, robust and free from analytical interference, and they should permit the identification and eradication of inhibitory structural motifs within a series of potential drug candidates.

Contribution of human hepatic cytochrome P450 isoforms to regioselective hydroxylation of steroid hormones

1. Hydroxylation activities toward steroid hormones were determined for eleven forms of human hepatic cytochrome P450s expressed in yeast Saccharomyces cerevisiae cells. Microsomes were prepared from the yeast cells and assayed for their regioselectivity of hydroxylation toward progesterone, pregnenolone, dehydroepiandrosterone (DHEA) and oestrone. 2. 6β-Hydroxylation of progesterone was catalysed most efficiently by CYP3A4, followed by CYP2D6. CYP3A4 showed the highest progesterone 16α-hydroxylation activity, followed by CYP1A1 and CYP2D6. 16 α-Hydroxylation of pregnenolone was catalysed efficiently by CYP1A1 and CYP3A4. Only CYP3A4 exhibited 16α-hydroxylase activities toward DHEA and oestrone. 3. Addition of nifedipine, a typical substrate of CYP3A4, inhibited the 6β- and 16α-hydroxylation of progesterone by CYP3A4. 4. These results suggest that CYP3A4 and CYP1A1 are responsible for the hydroxylation of these endogenous steroids, as well as xenobiotics, in human liver.

Microsomal ethanol oxidizing system activity by human hepatic cytochrome P450S

Microsomal ethanol oxidizing system activity by human hepatic cytochrome P450S