Multiple P450 Isoforms Research Articles

Caffeine 7-N-demethylation and C-8-oxidation mediated by liver microsomal cytochrome P450 enzymes in common marmosets

1. 3-N-Demethylation of caffeine (1,3,7-trimethylxanthine) is mediated by human cytochrome P450 1A2, whereas 7-N-demethylation and C-8-hydroxylation are reportedly catalyzed by monkey P450 2C9 and rat P450 1A2, respectively.2. Roles of marmoset P450 enzymes in caffeine oxidation were investigated using nine marmoset liver microsomes and 14 recombinantly expressed marmoset P450 enzymes.3. Predominant caffeine 7-N-demethylation and C-8-hydroxylation activities in marmoset liver microsomes were moderately (r = 0.78, p < 0.05) and highly (r = 0.82, p < 0.01) correlated with midazolam 1′-hydroxylation activities, respectively, while the former was not strongly affected by ketoconazole or α-naphthoflavone.4. Caffeine C-8-hydroxylation in liver microsomes was inhibited by ketoconazole and activated by α-naphthoflavone, suggesting main involvements of P450 3As.5. Recombinant marmoset P450 3As had high Vmax/Km values for C-8-hydroxylation, comparable to Km values for marmoset liver microsomes. Marmoset P450 1As efficiently mediated caffeine 3-N-demethylation and C-8-hydroxylation with apparently lower Km values than those of liver microsomes.6. These results collectively suggest highly active marmoset P450 3A enzymes toward caffeine 8-hydorxylaiton and involvement of multiple P450 isoforms including P450 1A in caffeine 7-N- and 3-N-demethylations in marmoset livers. Marmoset P450s have slightly different properties to human or monkey P450s regarding caffeine metabolic pathways.

Parallel assay of inkjet-printed cytochrome P450

Assaying the activities of cytochrome P450 (P450) is important for evaluating the toxicity of chemicals in drugs, food, and the environment. We developed a methodology to immobilize multiple P450 isoforms with a heightened density and assay their activities in parallel. Inkjet printing technology was applied for the deposition of P450 onto a plastic substrate (cycloolefin polymer: COP) having arrayed microwells (diameter: 50 μm, depth: 60 μm). P450 was printed into each microwell and immobilized by gelation of co-printed agarose. Activities of printed P450 isoforms were measured by introducing a substrate solution into the microwells and sealing it with an elastomer sheet, isolating individual wells. In order to avoid uncontrolled initiation of the reaction, we photo-regulated the P450 catalysis by using caged glucose-6-phosphate. The combination of inkjet printing, immobilization in microwells, and photo-regulated initiation of the enzymatic reaction enabled us to assay multiple P450 isoforms on a chip with an unprecedented density. We demonstrate the parallel assay of single nucleotide polymorphs of P450, which play important roles in different drug efficacies in individuals.

Oxidation of Endobiotics Mediated by Xenobiotic-Metabolizing Forms of Human Cytochrome P450

Cytochrome P450s (P450 or CYPs) comprise a superfamily of enzymes that catalyze the oxidation of a wide variety of xenobiotic chemicals including drugs and environmental carcinogens. Recent studies have demonstrated that endogenous chemicals are also oxidized by human P450s which mainly metabolize xenobiotics. In this review, we summarize the expected physiological significance of the biotransfornation as well as Michaelis-Menten constants (Km), maximal velocities (Vmax), Vmax/Km (intrinsic clearance) values, and/or metabolic activities for 33 endogenous substrates, including (1) arachidonic acid and fatty acids, (2) steroid hormones, such as testosterone, progesterone, and allopregnanolone, (3) amines, such as tyramine, and (4) lipid-soluble vitamins, such as retinol and vitamin D3 analogues, mediated human P450 isoforms consisting of so-called drug-metabolizing enzymes for the purpose of predicting the key enzyme(s) in vivo. Arachidonic acid is metabolized via the epoxidation and omega-hydroxylation to many biologically active eicosanoids such as epoxyeicosatrienoic acids and hydroxyeicosatetraenoic acids by multiple P450 isoforms including CYP2C, CYP2E1 and CYP4A11. CYP2D in the brain may be involved in the metabolism of neuronal amines and steroids and in the regulation of the central nervous system. CYP1A2 and CYP3A4 appear to be the major P450 enzymes catalyzing the oxidation of all-trans-retinol to all-trans-retinoic acid in human liver, and CYP3A4 is one of the vitamin D3 25-hydroxylases. Although the significance of the contribution is still unknown in detail, the collective findings provide fundamental and useful information for the biological contribution of the metabolism of endogenous substances by drug-metabolizing enzymes, P450s. In addition, genetic polymorphism of these drug-metabolizing P450s may affect the metabolism of the endobiotics. Forthermore, these findings imply that xenobiotic oxidations by P450 enzymes are affected by endobiotic molecules and that the endobiotic-xenobiotic interactions as well as drug-drug interactions or drug-food/beverage interactions may be of great importance when understanding the basis for pharmacological and toxicological actions of a number of xenobiotic chemicals.

In Vitro Inhibition of Multiple Cytochrome P450 Isoforms by Xanthone Derivatives from Mangosteen Extract

Mangosteen is a xanthone-containing fruit found in Southeast Asia for which health claims include maintaining healthy immune and gastrointestinal systems to slowing the progression of tumor growth and neurodegenerative diseases. Previous studies have identified multiple xanthones in the pericarp of the mangosteen fruit. The aim of the current study was to assess the drug inhibition potential of mangosteen in vitro as well as the cytochrome P450 (P450) enzymes responsible for the metabolism of its individual components. The various xanthone derivatives were found to be both substrates and inhibitors for multiple P450 isoforms. Aqueous extracts of the mangosteen pericarp were analyzed for xanthone content as well as inhibition potency. Finally, in vivo plasma concentrations of alpha-mangostin, the most abundant xanthone derivative found in mangosteen, were predicted using Simcyp and found to be well above their respective in vitro K(i) values for CYP2C8 and CYP2C9.

Antibodies as a Probe in Cytochrome P450 Research

Cytochrome P450 (P450) is the superfamily of enzymes responsible for biotransformation of endobiotics and xenobiotics. However, their large isoform multiplicity, inducibility, diverse structure, widespread distribution, polymorphic expression, and broad overlapping substrate specificity make it difficult to measure the precise role of each individual P450 to the metabolism of drugs (or carcinogens) and hamper the understanding of the relationship between the genetic/environmental factors that regulate P450 phenotype and the responses of the individual P450s to drugs. The antibodies against P450s have been useful tools for the quantitative determination of expression level and contribution of the epitope-specific P450 to the metabolism of a drug or carcinogen substrate in tissues containing multiple P450 isoforms and for implications in pharmacogenetics and human risk assessment. In particular, the inhibitory antibodies are uniquely suited for reaction phenotyping that helps to predict human pharmacokinetics for clinical drug-drug interaction potential in drug discovery and development.

Identification of cytochrome P450 isoforms involved in the metabolism of loperamide in human liver microsomes.

The purpose of the present study was to elucidate the cytochrome P450 (P450) isoform(s) involved in the metabolism of loperamide (LOP) to N-demethylated LOP (DLOP) in human liver microsomes. Three established approaches were used to identify the P450 isoforms responsible for LOP N-demethylation using human liver microsomes and cDNA-expressed P450 isoforms: (1) correlation of LOP N-demethylation activity with marker P450 activities in a panel of human liver microsomes, (2) inhibition of enzyme activity by P450-selective inhibitors, and (3) measurement of DLOP formation by cDNA-expressed P450 isoforms. The relative contribution of P450 isoforms involved in LOP N-demethylation in human liver microsomes were estimated by applying relative activity factor (RAF) values. The formation rate of DLOP showed biphasic kinetics, suggesting the involvement of multiple P450 isoforms. Apparent Km and Vmax values were 21.1 microM and 122.3 pmol/min per milligram of protein for the high-affinity component and 83.9 microM and 412.0 pmol/min per milligram of protein for the low-affinity component, respectively. Of the cDNA-expressed P450 s tested, CYP2B6, CYP2C8, CYP2D6, and CYP3A4 catalyzed LOP N-demethylation. LOP N-demethylation was significantly inhibited when coincubated with quercetin (a CYP2C8 inhibitor) and ketoconazole (a CYP3A4 inhibitor) by 40 and 90%, respectively, but other chemical inhibitors tested showed weak or no significant inhibition. DLOP formation was highly correlated with CYP3A4-catalyzed midazolam 1-hydroxylation (rs=0.829; P<0.01), CYP2B6-catalzyed 7-ethoxy-4-trifluoromethylcoumarin O-deethylation (rs=0.691; P<0.05), and CYP2C8-catalyzed paclitaxel 6alpha-hydroxylation (rs=0.797; P<0.05). CYP2B6, CYP2C8, CYP2D6, and CYP3A4 catalyze LOP N-demethylation in human liver microsomes, and among them, CYP2C8 and CYP3A4 may play a crucial role in LOP metabolism at the therapeutic concentrations of LOP. Coadministration of these P450 inhibitors may cause drug interactions with LOP. However, the clinical significance of potential interaction of LOP metabolism by CYP2C8 and CYP3A4 inhibitors should be studied further.

The involvement of CYP3A4 and CYP2C9 in the metabolism of 17 alpha-ethinylestradiol.

The role of specific cytochrome P450 (P450) isoforms in the metabolism of ethinylestradiol (EE) was evaluated. The recombinant human P450 isozymes CYP1A1, CYP1A2, CYP2C9, CYP2C19, and CYP3A4 were found to be capable of catalyzing the metabolism of EE (1 microM). Without exception, the major metabolite was 2-hydroxy-EE. The highest catalytic efficiency (Vmax/Km) was observed with rCYP1A1, followed by rCYP3A4, rCYP2C9, and rCYP1A2. The P450 isoforms 3A4 and 2C9 were shown to play a significant role in the formation of 2-hydroxy-EE in a pool of human liver microsomes by using isoform-specific monoclonal antibodies, in which the inhibition of formation was approximately 54 and 24%, respectively. The involvement of CYP3A4 and CYP2C9 was further confirmed by using selective chemical inhibitors (i.e., ketoconazole and sulfaphenazole). The relative contribution of each P450 isoform to the 2-hydroxylation pathway was obtained from the catalytic efficiency of each isoform normalized by its relative abundance in the same pool of human liver microsomes, as determined by quantitative Western blot analysis. Collectively, these results suggested that multiple P450 isoforms were involved in the oxidative metabolism of EE in human liver microsomes, with CYP3A4 and CYP2C9 as the major contributing enzymes.

HIV-1 protease inhibitors with picomolar potency against PI-resistant HIV-1 by modification of the P1' substituent.

Transposition of the pyridyl nitrogen from the P(3) substituent to the P(1)' substituent in HIV-1 protease inhibitors (PI) affords compounds such as 3 with an improved inhibitory profile against multiple P450 isoforms. These compounds also displayed increased potency, with 3 inhibiting viral spread (CIC(95)) at <8 nM for every strain of PI-resistant HIV-1 tested. The poor to modest bioavailability of these compounds may correlate in part to their aqueous solubility.

HIV protease inhibitors with picomolar potency against PI-Resistant HIV-1 by extension of the P3 substituent.

A biaryl pyridylfuran P(3) substituent on the hydroxyethylene isostere scaffold affords HIV protease inhibitors (PI's) with picomolar (IC(50)) potency against the protease enzymes from PI-resistant HIV-1 strains. Inclusion of a gem-dimethyl substituent afforded compound 3 with 100% oral bioavailability (dogs) and more than double the t(1/2) of indinavir. Inhibition of multiple P450 isoforms is dependent on the regiochemistry of the pyridyl nitrogen in these compounds.

Detection of Cytochrome P450 Induction. Development of Non-Invasive Assay.

Cytochrome P450s (P450s) are induced by drugs, environmental chemicals, diseases, and genetic factors. Changes in the levels of P450 have a significant effect on the pharmacokinetics or pharmacodynamics of drugs and endogenous substrates. In experimental animals such as a rat, induction of P450 is detected in hepatic microsomes by drug metabolism assay and Western blotting with antibodies. Assay system of multiple P450 isoforms with Western blotting has been established but it is difficult to apply this method to human subjects because human hepatic tissue must be isolated to assay P450 induction. We tried to develop a less-invasive assay for detection of P450 induction. Detection system of P450 mRNA induction in human lymphocytes in peripheral blood was established using by a competitive PCR or a real-time PCR. Induction of CYP2E1 and CYP3A4 by diabetes and rifampicin, respectively, was detected by this method.

Expression of cytochrome P450 isoforms in rat hepatic stellate cells

The current study evaluated the expression and the inducibility of cytochrome P450 isoforms in rat hepatic stellate cells (HSCs). Immunoblotting study revealed that HSCs expressed several P450s and CYP2C11, 3A2, and 2D1 were major isoforms. The levels of CYP2B1, 2C11, 2D1, 2E1, and 3A2 in HSCs were 14 -38% of those in hepatocytes. CYP1A2 content was similar in each cell type. These P450 levels in HSCs gradually decreased during culture as seen in hepatocytes; the level of CYP3A2 rapidly, whereas that of CYP2D1 slowly decreased. Phenobarbital, a typical inducer of CYP3A2 and 2B1 increased CYP3A2 level as well, but had less potency in the induction of CYP2B1 in HSCs. These results indicate that multiple P450 isoforms were present in HSCs, but their content and inducibility were different between HSCs and hepatocytes.

Molecular recognition and electron transfer in mitochondrial steroid hydroxylase systems

Mitochondrial monooxygenase systems are involved in the biosynthesis of glucocorticoids, mineralocorticoids, bile acids, and 1,25-dihydroxyvitamin D. The reactions are catalyzed by specific P450 enzymes that receive reducing equivalents via NADPH-ferredoxin oxidoreductase (adrenodoxin reductase) and ferredoxin (adrenodoxin). Although the three-dimensional structures of the individual components have not yet been solved, methods of expressing recombinant forms of these enzymes in Escherichia coli have allowed the use of site-directed mutagenesis to investigate the roles of specific amino acids in protein binding interactions, electron transfer, and catalysis. These studies have identified key charged residues in NADPH-ferredoxin oxidoreductase, ferredoxin, and P450scc, which are involved in electrostatic interactions critical for recognition, high-affinity binding, and electron transfer. The finding that the binding sites on ferredoxin for NADPH-ferredoxin oxidoreductase and P450 show significant overlap supports the proposed function for ferredoxin as a mobile electron shuttle between the reductase and P450 enzymes and is consistent with ferredoxin's role in serving multiple P450 isoforms.