Human P450 Isozymes Research Articles

Biotransformation of Bisphenol by Human Cytochrome P450 2C9 Enzymes: A Density Functional Theory Study.

Bisphenol A (BPA, 2,2-bis-(4-hydroxyphenyl)propane) is used as a precursor in the synthesis of polycarbonate and epoxy plastics; however, its availability in the environment is causing toxicity as an endocrine-disrupting chemical. Metabolism of BPA and their analogues (substitutes) is generally performed by liver cytochrome P450 enzymes and often leads to a mixture of products, and some of those are toxic. To understand the product distributions of P450 activation of BPA, we have performed a computational study into the mechanisms and reactivities using large model structures of a human P450 isozyme (P450 2C9) with BPA bound. Density functional theory (DFT) calculations on mechanisms of BPA activation by a P450 compound I model were investigated, leading to a number of possible products. The substrate-binding pocket is tight, and as a consequence, aliphatic hydroxylation is not feasible as the methyl substituents of BPA cannot reach compound I well due to constraints of the substrate-binding pocket. Instead, we find low-energy pathways that are initiated with phenol hydrogen atom abstraction followed by OH rebound to the phenolic ortho- or para-position. The barriers of para-rebound are well lower in energy than those for ortho-rebound, and consequently, our P450 2C9 model predicts dominant hydroxycumyl alcohol products. The reactions proceed through two-state reactivity on competing doublet and quartet spin state surfaces. The calculations show fast and efficient substrate activation on a doublet spin state surface with a rate-determining electrophilic addition step, while the quartet spin state surface has multiple high-energy barriers that can also lead to various side products including C4-aromatic hydroxylation. This work shows that product formation is more feasible on the low spin state, while the physicochemical properties of the substrate govern barrier heights of the rate-determining step of the reaction. Finally, the importance of the second-coordination sphere is highlighted that determines the product distributions and guides the bifurcation pathways.

Mechanistic aspects of reactive metabolite formation in clomethiazole catalyzed biotransformation by cytochrome P450 enzymes.

Clomethiazole (CLM), a sedative and anticonvulsant drug, is commonly employed for the treatment of alcohol withdrawal syndromebecause it suppresses cytochrome P450 (P450) activity associated with the generation of free radicals and liver damage. The catalyzed biotransformation of thiazole-containing drugs by P450 is known to afford reactive metabolites. These metabolites can alter the biological functions of macromolecules and result in toxicity and adverse drug interactions. Multitargeted molecular modeling and quantum chemical DFT calculations were performed to explore the binding modes and molecular mechanisms underlying the mechanism-based inactivation (MBI) of P450 by CLM. The mechanistic details associated with reactive metabolite formation from further metabolic processes were extensively assessed. Seven possible routes were proposed for CLM-P450 biotransformation including CLM hydroxylation, sulfoxidation, N-oxidation, CN epoxidation (oxaziridine formation), and CC epoxidation. The results revealed a degree of preference for the C-N epoxidation pathway because of the low energy requirements of its rate-determining step (8.74 and 10.07 kcal mol-1 for LS and HS states, respectively). A kinetic competition for the CLM-methyl hydroxylation pathway was detected because the H-abstraction energy barrier was relatively comparable to the thermodynamically prevailing oxaziridine formation rate-determining step (12.58 and 14.52 kcal mol-1 for quartet and doublet states, respectively). Our studies assessed the mechanisms of covalent nucleophilic epoxide adduct formation through nucleophilic addition, hydrolysis of epoxidation products, and nonenzymatic degradation. CLM was shown to display P450-inhibitory activity by forming covalent adducts rather than further metabolization to reactive metabolites. The outcomes of molecular docking allowed assessing the binding profile of CLM with three human P450 isozymes, namely, CYP2E1, CYP3A4, and CYP2D6.

Mechanism of Melatonin Metabolism by CYP1A1: What Determines the Bifurcation Pathways of Hydroxylation versus Deformylation?

Melatonin, a widely applied cosmetic active ingredient, has a variety of uses as a skin protector through antioxidant and anti-inflammatory functions as well as giving the body UV-induced defenses and immune system support. In the body, melatonin is synthesized from a tryptophan amino acid in a cascade of reactions, but as melatonin is toxic at high concentrations, it is metabolized in the human skin by the cytochrome P450 enzymes. The P450s are diverse heme-based mono-oxygenases that catalyze oxygen atom-transfer processes that trigger metabolism and detoxification reactions in the body. In the catalytic cycle of the P450s, a short-lived high-valent iron(IV)-oxo heme cation radical is formed that has been proposed to be the active oxidant. How and why it activates melatonin in the human body and what the origin of the product distributions is, are unknown. This encouraged us to do a detailed computational study on a typical human P450 isozyme, namely CYP1A1. We initially did a series of molecular dynamics simulations with substrate docked into several orientations. These simulations reveal a number of stable substrate-bound positions in the active site, which may lead to differences in substrate activation channels. Using tunneling analysis on the full protein structures, we show that two of the four binding conformations lead to open substrate-binding pockets. As a result, in these open pockets, the substrate is not tightly bound and can escape back into the solution. In the closed conformations, in contrast, the substrate is mainly oriented with the methoxy group pointing toward the heme, although under a different angle. We then created large quantum cluster models of the enzyme and focused on the chemical reaction mechanisms for melatonin activation, leading to competitive O-demethylation and C6-aromatic hydroxylation pathways. The calculations show that active site positioning determines the product distributions, but the bond that is activated is not necessarily closest to the heme in the enzyme-substrate complex. As such, the docking and molecular dynamics positioning of the substrate versus oxidant can give misleading predictions on product distributions. In particular, in quantum mechanics cluster model I, we observe that through a tight hydrogen bonding network, a preferential 6-hydroxylation of melatonin is obtained. However, O-demethylation becomes possible in alternative substrate-binding orientations that have the C6-aromatic ring position shielded. Finally, we investigated enzymatic and non-enzymatic O-demethylation processes and show that the hydrogen bonding network in the substrate-binding pocket can assist and perform this step prior to product release from the enzyme.

Synthesis, CYP 450 evaluation, and docking simulation of novel 4-aminopyridine and coumarin derivatives.

Four series of novel compounds based on 4-aminopyridine, glatiramer acetate, pyrone, and coumarin backbones were sufficiently synthesized and identified by spectroscopic methods. CYP enzyme inhibition assays of five predominate human P450 isozymes indicate that all compounds, except for 4-hydrazide pyridine 1c, seem to be less toxic than 4-aminopyridine. Further investigation of the compounds using molecular docking experiments revealed different, the same, or stronger binding modes for most of the synthesized compounds, with both polar and hydrophobic interactions with the 1WDA and 1J95 receptors compared to benzoyl l-arginine amide and 4-aminopyridine, respectively. These results introduce the synthesized compounds as K+ channel blockers that could be considered for in vivo CNS disease studies.

An Automated High-Throughput Metabolic Stability Assay Using an Integrated High-Resolution Accurate Mass Method and Automated Data Analysis Software.

Advancement of in silico tools would be enabled by the availability of data for metabolic reaction rates and intrinsic clearance (CLint) of a diverse compound structure data set by specific metabolic enzymes. Our goal is to measure CLint for a large set of compounds with each major human cytochrome P450 (P450) isozyme. To achieve our goal, it is of utmost importance to develop an automated, robust, sensitive, high-throughput metabolic stability assay that can efficiently handle a large volume of compound sets. The substrate depletion method [in vitro half-life (t1/2) method] was chosen to determine CLint The assay (384-well format) consisted of three parts: 1) a robotic system for incubation and sample cleanup; 2) two different integrated, ultraperformance liquid chromatography/mass spectrometry (UPLC/MS) platforms to determine the percent remaining of parent compound, and 3) an automated data analysis system. The CYP3A4 assay was evaluated using two long t1/2 compounds, carbamazepine and antipyrine (t1/2 > 30 minutes); one moderate t1/2 compound, ketoconazole (10 < t1/2 < 30 minutes); and two short t1/2 compounds, loperamide and buspirone (t½ < 10 minutes). Interday and intraday precision and accuracy of the assay were within acceptable range (∼12%) for the linear range observed. Using this assay, CYP3A4 CLint and t1/2 values for more than 3000 compounds were measured. This high-throughput, automated, and robust assay allows for rapid metabolic stability screening of large compound sets and enables advanced computational modeling for individual human P450 isozymes.

Dimethocaine, a synthetic cocaine derivative: Studies on its in vitro metabolism catalyzed by P450s and NAT2

Dimethocaine (DMC), a synthetic derivative of cocaine, is distributed and consumed as “new psychoactive substance” (NPS) without any safety testing at the forefront. It is mainly metabolized by N-acetylation, N-deethylation or hydroxylation. Therefore, the aim of the presented study was to determine the human NAT and P450 isozymes involved in this major metabolic steps, to measure the kinetics of the reactions, and to estimate the contribution on in vivo hepatic clearance. For these studies, cDNA-expressed NATs and P450s were used and formation of metabolites after incubation was measured using LC–MS or LC–MSn. For N-acetylation, NAT2 could be shown to be the only isoform catalyzing the reaction in vitro hence assuming to be the only relevant enzyme for in vivo acetylation. Kinetic profiles of all P450 catalyzed metabolite formations followed classic Michaelis–Menten behavior with enzyme affinities (Km values) between 3.6 and 220μM. Using the relative activity factor approach, the net clearances for deethylation of DMC were calculated to be 3% for P450 1A2, 1% for 2C19, <1% for 2D6, and 96% for 3A4. The net clearances for hydroxylation of DMC were calculated to be 32% for P450 1A2, 5% for 2C19, 51% for 2D6, and 12% for 3A4. Furthermore, these data were confirmed by chemical inhibition tests in human liver microsomes. As DMC is metabolized via two main steps and different P450 isoforms were involved in the hepatic clearance of DMC, a clinically relevant interaction with single P450 inhibitors should not be expected. However, a slow acetylation phenotype or inhibition of NAT2 could lead to decreased N-acetylation and hence leading to an increased risk of side effects caused by this arylamine.

Identification of Key Licorice Constituents Which Interact with Cytochrome P450: Evaluation by LC/MS/MS Cocktail Assay and Metabolic Profiling

Licorice has been shown to affect the activities of several cytochrome P450 enzymes. This study aims to identify the key constituents in licorice which may affect these activities. Bioactivity assay was combined with metabolic profiling to identify these compounds in several complex licorice extracts. Firstly, the inhibition potencies of 40 pure licorice compounds were tested using an liquid chromatography/tandem mass spectrometry cocktail method. Significant inhibitors of human P450 isozymes 1A2, 2C9, 2C19, 2D6, and 3A4 were then selected for examination of their structural features by molecular docking to determine their molecular interaction with several P450 isozymes. Based on the present in vitro inhibition findings, along with our previous in vivo metabolic studies and the prevalence of individual compounds in licorice extract, we identified several licorice constituents, viz., liquiritigenin, isoliquiritigenin, together with seven isoprenylated flavonoids and arylcoumarins, which could be key components responsible for the herb-drug interaction between cytochrome P450 and licorice. In addition, hydrophilic flavonoid glycosides and saponins may be converted into these P450 inhibitors in vivo. These studies represent a comprehensive examination of the potential effects of licorice components on the metabolic activities of P450 enzymes.

The Role of Human Hepatic Cytochrome P450 Isozymes in the Metabolism of Racemic 3,4-Methylenedioxyethylamphetamine and Its Single Enantiomers

The 3,4-methylenedioxy-methamphetamine (MDMA)-related designer drug 3,4-methylenedioxyethylamphetamine (MDEA, Eve) is a chiral compound that is mainly metabolized by N-deethylation and demethylenation during phase I metabolism. The involvement of several cytochrome P450 (P450) isozymes in these metabolic steps has been demonstrated by inhibition assays using human liver microsomes. However, a comprehensive study on the involvement of all relevant human P450s has not been published yet. In addition, the chirality of this drug was not considered in these in vitro studies. The aim of the present work was first to elucidate the contribution of the relevant human P450 isozymes in the demethylenation as well as in the N-dealkylation of racemic MDEA and its single enantiomers and secondly to compare these findings with recently published data concerning the enantioselective metabolism of MDMA. Racemic MDEA and its single enantiomers were incubated using heterologously expressed human P450s, and the corresponding metabolites dihydroxyethylamphetamine and methylenedioxyamphetamine were determined by gas chromatography-mass spectrometry after chiral derivatization with S-heptafluorobutyrylprolyl chloride. The highest contributions to both metabolic steps as calculated from the enzyme kinetic data were obtained for CYP3A4 and CYP2D6 at substrate concentrations corresponding to plasma concentrations of recreational users after intake of racemic MDEA. Both metabolic reactions were found to be enantioselective with a general preference for the S-enantiomers, which was particularly pronounced in the case of CYP2C19. In conclusion, different pharmacokinetic properties of MDEA enantiomers observed in vivo are therefore partially caused by P450-dependent enantioselective metabolism.

The Role of Human Hepatic Cytochrome P450 Isozymes in the Metabolism of Racemic 3,4-Methylenedioxy-Methamphetamine and Its Enantiomers

The entactogen, 3,4-methylenedioxy-methamphetamine (MDMA), is a chiral drug that is mainly metabolized by N-demethylation and demethylenation. The involvement of cytochrome P450 (P450) isozymes in these metabolic steps has been studied by inhibition assays with human liver microsomes and, in part, with heterologously expressed human P450 isozymes. However, a comprehensive study on the involvement of all relevant human P450s has not been published yet. In addition, the chirality of this drug was not considered in these in vitro studies. The aim of the present work was to study the contribution of human P450 isozymes in the N-demethylation and demethylenation of racemic MDMA and its single enantiomers. MDMA and its enantiomers were incubated using heterologously expressed human P450s, and the metabolites were quantified by gas chromatography-mass spectrometry after derivatization with S-heptafluorobutyrylprolyl chloride. The highest contribution for the N-demethylation as calculated from the enzyme kinetic data, were obtained for CYP2B6 (R,S-MDMA), CYP1A2 (R-MDMA), and CYP2B6 (S-MDMA). In the case of the demethylenation, the isozyme with the highest contribution to net clearance for R,S-MDMA, R-MDMA, and S-MDMA was CYP2D6. For the first time, marked enantioselectivity was observed for N-demethylation and demethylenation by CYP2C19 with a preference for the S-enantiomers. In addition, CYP2D6 showed preference for S-MDMA in the case of demethylenation. None of the other isozymes showed major preferences for certain enantiomers. In conclusion, therefore, the different pharmacokinetic properties of the MDMA enantiomers may be caused by enantioselective metabolism by CYP2C19 and CYP2D6.

Novel Metabolic Pathways of p-n-Nonylphenol Catalyzed by Cytochrome P450 and Estrogen Receptor Binding Activity of New Metabolites

Nonylphenol, which is used industrially as a surfactant, is an endocrine-disrupting chemical (EDC) which has estrogenic activity. The novel biotransformation of nonylphenol was investigated, based on our previously reported ipso-metabolism of para-substituted phenols by cytochrome P450 (P450). Three novel metabolites of nonylphenol, i.e., nonylquinol, 4'-hydroxynonanophenone (CO-NP) as benzyl-oxidized nonylphenol, and hydroquinone, were detected in a rat liver microsome reaction mixture. On the other hand, production of 1-(4'-hydroxyphenyl)nonan-1-ol (OH-NP), namely benzyl-hydroxylated nonylphenol, was detected in a human liver microsome reaction mixture. The formation of all these metabolites was suppressed by the addition of P450 inhibitor. This showed that all nonylphenol metabolism was catalyzed by P450. To identify which P450 isoenzyme is involved in each reaction, fourteen human P450 (CYP) isozymes, CYP1A1, 1A2, 2A6, 2B6, 2C8, 2C9, 2C18, 2C19, 2D6, 2E1, 3A4, 3A5, 3A7, and CYP4A11, were examined. CYP1A1, 1A2, and CYP2B6 effectively catalyzed the production of nonylquinol. CYP2B6 also catalyzed the benzyl-hydroxylation to give OH-NP. Hydroquinone was formed mainly from OH-NP, not via CO-NP. We examined the estrogenic activity of these new metabolites by estrogen receptor (ER)-binding reporter gene assay. Nonylquinol, OH-NP and hydroquinone have no ER-binding activity. However, CO-NP showed the same level of estrogen receptor binding activity as nonylphenol. Moreover, the amount of CO-NP formed was small. Therefore, the novel metabolic pathways led overall to metabolic inactivation, as concerns the estrogenic activity of nonylphenol through the ER.

QUANTITATIVE ANALYSIS OF CYTOCHROME P450 ISOZYMES BY MEANS OF UNIQUE ISOZYME-SPECIFIC TRYPTIC PEPTIDES: A PROTEOMIC APPROACH

A novel matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry method has been developed to quantitate cytochrome P450 (P450) isozymes based on their unique isozyme-specific tryptic peptides. It was shown that the molar ratio of P450 isozyme-specific peptides is linearly proportional to the mass peak area ratio of corresponding peptides not only in simple two-peptide mixtures, but also in complex digest mixtures. This approach is applicable both to in-gel (as shown for CYP2B1 and CYP2B2) and in-solution digests (as shown for CYP1A2, CYP2E1, and CYP2C19) and does not require introduction of stable isotopes or labeling with isotope-coded affinity tagging. The relative and absolute quantitation can be performed after developing corresponding calibration curves with synthesized P450 isozyme-specific peptide standards. The absolute quantitation of human P450 isozymes was performed by using CYP2B2 isozyme-specific peptide (1306.7 Da) as the universal internal standard. The utility of this approach was demonstrated for two highly homologous (>97%) rat liver CYP2B1 and CYP2B2 and three human P450 isozymes belonging to two different families and three different subfamilies: CYP1A2, CYP2E1, and CYP2C19. In summary, we have demonstrated that MALDI TOF-based peptide mass fingerprinting of different cytochrome P450 isozymes can provide not only qualitative but quantitative data, too.

METABOLISM OF MELATONIN BY HUMAN CYTOCHROMES P450

In humans, the pineal hormone melatonin (MEL) is principally metabolized to 6-hydroxymelatonin (6-HMEL), which is further conjugated with sulfate and excreted in urine. MEL O-demethylation represents a minor reaction. The exact role of individual human cytochromes P450 (P450s) in these pathways has not been established. We used a panel of 11 recombinant human P450 isozymes to investigate for the first time the 6-hydroxylation and O-demethylation of MEL. CYP1A1, CYP1A2, and CYP1B1 all 6-hydroxylated MEL, with CYP2C19 playing a minor role. These reactions were NADPH-dependent. CYP2C19 and, to some extent CYP1A2, O-demethylated MEL. The K(m) (microM) and V(max) (k(cat), pmol min(-1) pmol(-1) P450) for 6-hydroxylation were estimated as 19.2 +/- 2.01 and 6.46 +/- 0.22 (CYP1A1), 25.9 +/- 2.47 and 10.6 +/- 0.32 (CYP1A2), and 30.9 +/- 3.76 and 5.31 +/- 0.21 (CYP1B1). These findings confirm the suggestion of others that CYP1A2 is probably the foremost hepatic P450 in the 6-hydroxylation of MEL and a single report that CYP1A1 is also able to mediate this reaction. However, this is the first time that CYP1B1 has been shown to 6-hydroxylate MEL. The IC50 for the CYP1B1-selective inhibitor (E)-2,4,3',5'-tetramethoxystilbene was estimated to be 30 nM for MEL 6-hydroxylation by recombinant human CYP1B1. Comparison of brain homogenates from wild-type and cyp1b1-null mice revealed that MEL 6-hydroxylation was clearly mediated to a significant degree by CYP1B1. CYP1B1 is not expressed in the liver but has a ubiquitous extrahepatic distribution, and is found at high levels in tissues that also accumulate either MEL or 6-HMEL, such as intestine and cerebral cortex, where it may assist in regulating levels of MEL and 6-HMEL.

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.

The Metabolism of Clopidogrel Is Catalyzed by Human Cytochrome P450 3A and Is Inhibited by Atorvastatin

The prodrug clopidogrel (Plavix) is activated by cytochrome p450 (p450) to a metabolite that inhibits ADP-induced platelet aggregation. Clopidogrel is frequently administered to patients in conjunction with the CYP3A4 substrate atorvastatin (Lipitor). Since clinical studies indicate that atorvastatin inhibits the antiplatelet activity of clopidogrel, we investigated whether CYP3A4 metabolized clopidogrel in vitro. Microsomes prepared from dexamethasone-pretreated rats metabolized clopidogrel at a rate of 3.8 nmol min(-1) nmol of p450(-1), which is 65 and 1270% faster than the rate of metabolism by microsomes from control and beta-napthoflavone-treated rats, respectively. To identify the human p450s responsible for clopidogrel oxidation, genetically engineered microsomes containing a single human p450 isozyme were tested for their ability to oxidize clopidogrel. CYP3A4 and 3A5 metabolized clopidogrel at a significantly higher rate than eight other p450 isozymes, suggesting that CYP3A4 and 3A5 are primarily responsible for in vivo clopidogrel metabolism. Clopidogrel interacts with human CYP3A4 with a spectral dissociation constant (K(s)), K(m), and V(max) of 12 microM, 14 +/- 1 microM and 6.7 +/- 1 nmol min(-1) nmol p450(-1), respectively. Atorvastatin lactone, the physiologically relevant substrate, inhibits clopidogrel with a K(i) of 6 microM. When clopidogrel and atorvastatin are present at equimolar concentrations, clopidogrel metabolism is inhibited by greater than 90%. Since CYP3A4 and 3A5 metabolize clopidogrel faster than other human p450 isozymes and are the most abundant p450s in human liver, they are predicted to be predominantly responsible for the activation of clopidogrel in vivo.

Effects of olopatadine, a new antiallergic agent, on human liver microsomal cytochrome P450 activities.

Olopatadine, a new histamine H(1) receptor-selective antagonist, is a tricyclic drug containing an alkylamino moiety. Some compounds containing a similar alkylamino group form a cytochrome p450 (p450) -iron (II)-nitrosoalkane metabolite complex [metabolic intermediate complex (MIC)], thereby causing quasi-irreversible inhibition of the p450. There was concern that olopatadine might also form MICs, therefore, the present investigation was undertaken to explore this possibility. We identified the enzymes catalyzing olopatadine metabolism and investigated the effect of olopatadine on human p450 activities. During incubation with human liver microsomes in the presence of a NADPH-generating system, olopatadine was metabolized to two metabolites, M1 (N-monodemethylolopatadine) and M3 (olopatadine N-oxide) at rates of 0.330 and 2.50 pmol/min/mg protein, respectively. Troleandomycin and ketoconazole, which are both selective inhibitors of CYP3A, significantly reduced M1 formation but specific inhibitors of other p450 isozymes did not decrease M1 formation. Incubation of olopatadine with cDNA-expressed human p450 isozymes confirmed that M1 formation was almost exclusively catalyzed by CYP3A4. The formation of M3 was enhanced by N-octylamine and was inhibited by thiourea. High specific activity of M3 formation was exhibited by cDNA-expressed flavin-containing monooxygenase (FMO)1 and FMO3. Olopatadine did not inhibit p450 activities when it was simultaneously incubated with substrates for different p450 isozymes. Also, p450 activities in human liver microsomes were unaffected by pretreatment with olopatadine or M1. Furthermore, spectral analysis revealed that neither olopatadine nor M1 formed an MIC. Therefore, it is unlikely that olopatadine will cause drug-drug interactions involving p450 isozymes.

Displacement of histamine from liver cells and cell components by ligands for cytochromes P450.

Intracellular histamine (HA) and cytochrome P450 monooxygenases (P450) each have been proposed as mediators of cell function, growth, and proliferation. The P450 family of heme enzymes is found in virtually all cells and generates, transforms, or inactivates steroids and other lipids that participate in cell regulation. We previously demonstrated a second messenger role for HA in blood platelets and the formation of a HA-P450 heme complex when exogenous HA was added to microsomes isolated from rat liver cells or to purified human P450 isozymes. Employing a radioimmunoassay, we now demonstrate that rat liver slices, microsomes derived from the livers of adult male rats and mast cell-deficient mice, and hepatoma cells, all contain endogenous HA. HA release from microsomes into the incubation medium, as determined by radioimmunoassay, is enhanced in the presence of carbon monoxide, steroids, and certain drugs, all agents that unite either directly with the iron atom or bind elsewhere within the heme cavity. Rat liver slices preincubated with (3)H-HA release labeled amine into the medium in the presence of those same ligands. These findings provide evidence of an in situ HA-P450 complex and offer further support that the imidazole, HA, is a physiological, intracellular modulator of cytochromes P450 in liver cells, and perhaps of these and other heme proteins in tissues in general.

N,N-diethyl-2-[4-(phenylmethyl)phenoxy] ethanamine (DPPE) a chemopotentiating and cytoprotective agent in clinical trials: interaction with histamine at cytochrome P450 3A4 and other isozymes that metabolize antineoplastic drugs.

N,N-diethyl-2-[4-(phenylmethyl)phenoxy]ethanamine HCl (DPPE), an intracellular histamine (HA) antagonist with chemopotentiating and cytoprotective properties, is currently in phase 2 and 3 clinical trials in breast and prostate cancer. DPPE modulates growth at in vitro concentrations that antagonize HA binding to cytochromes P450 in rat liver microsomes. HA inhibits P450 metabolism of some drugs. Recent in vitro studies in human colon cancer cells have linked DPPE enhancement of paclitaxel, doxorubicin and vinblastine cytotoxicity to inhibition of the P-glycoprotein (P-gp) pump. Many substrates of P-gp are also substrates of CYP3A4, a P450 isozyme that metabolizes a variety of antineoplastic agents and is highly expressed in some malignant tissues. Therefore, we assessed whether (a) DPPE and HA interact at CYP3A4 and other P450 human isozymes, and (b) DPPE inhibits the catalytic activity of CYP3A4. Using spectral analysis, we measured DPPE and HA binding to insect microsomes that express human P450 isozymes 1A1, 2B6, 2D6 or 3A4. Employing thin-layer chromatography, we assessed the metabolism of DPPE by each isozyme and DPPE inhibition of testosterone metabolism by CYP3A4 and by rat liver microsomes. (1) DPPE evoked "type I" (substrate site binding) absorbance-difference spectra with CYP2D6 (K(S) = 4.1 +/- 0.4 microM), CYP3A4 (K(S) = 31 +/- 15 microM) and CYP1A1 (K(S) = 40 +/- 9 microM), but not with CYP2B6. (2) In correspondence with the binding studies, DPPE was metabolized by CYP2D6, CYP3A4 and CYP1A1; no metabolism occurred with CYP2B6. (3) HA evoked "type II" (heme iron binding) absorbance-difference spectra with all four isozymes, with K(S) values in the range 80-600 microM. DPPE inhibited HA (600 microM) binding to CYP2D6 (IC50 = 4 microM, 95% CI= 1.8-8.9 microM) and CYP1A1 (IC50 = 135 microM: 95% CI = 100-177 microM), but stimulated HA (500 and 1000 microM) binding to CYP3A4 (EC50 = 155 microM, 95% CI = 104-231 microM). DPPE did not affect HA binding to CYP2B6. (4) DPPE inhibited the metabolism of testosterone by CYP3A4. The concentration/effect curve was biphasic: DPPE inhibited metabolism by 30% at the first site (IC50 = 3 microM, 95% CI = 0.5-25.5 microM), and an additional 70% inhibition occurred at the second site (IC50 = 350 microM, 95% CI = 215-570 microM). A similar result was observed with rat liver microsomes. DPPE is a substrate for CYP3A4, CYP2D6 and CYP1A1, but not CYP2B6. DPPE inhibits testosterone metabolism by interacting at two sites on CYP3A4, the first correlating with its K(S) value to bind the substrate site and the second, with its EC50 value to enhance HA binding to the heme iron. We postulate that (1) the inhibitory effect of DPPE on CYP3A4 activity is mediated directly at the substrate site and indirectly by its enhancement of the binding of HA to the heme moiety; (2) in tumor cells that express high constitutive levels of CYP3A4, potentiation of chemotherapy cytotoxicity by DPPE results, in part, from inhibition of CYP3A4-mediated metabolism and P-gp-mediated efflux of antineoplastic drugs; (3) in normal cells that express low constitutive levels of the isozyme, cytoprotection by DPPE results, in part, from induction of CYP3A4 and P-gp, resulting in an increase both in metabolism and efflux of antineoplastic drugs.

Use of recombinant human ferrochelatase as a sensitive bioassay for N-alkylprotoporphyrin IX formed after interaction of porphyrinogenic xenobiotics with rat liver microsomes

Several porphyrinogenic xenobiotics elicit mechanism-based inactivation of cytochrome P450 (CYP) isozymes, leading to the formation of N-alkylprotoporphyrin IX (N-alkylPP), a potent inhibitor of ferrochelatase, the terminal enzyme in heme biosynthesis. Recognizing their role in experimental porphyria, our long term objective is the establishment of an appropriate in vitro system for the detection and quantification of N-alkylPPs, formed in human liver after the administration of potential porphyrinogenic compounds. In a previous study, we used a combination of thin-layer chromatography and UV-visible spectrophotometry to isolate and identify N-alkylPPs after incubating porphyrinogenic compounds with rat liver microsomes. However, the overall yield of N-alkylPPs was low, and it was concluded that in vitro systems, such as human lymphoblastoid microsomal preparations containing single cDNA-expressed human cytochrome P450 (CYP) isozymes, do not contain sufficient CYP for in vitro studies designed to isolate N-alkylPP. In the present study we demonstrate that purified recombinant human ferrochelatase (FC) provides an extremely sensitive bioassay system for N-alkylPPs and is capable of detecting N-alkylPP in the 10(-6) nmol range. Therefore, we propose that this bioassay system might allow the use of human lymphoblastoid microsomal preparations containing single cDNA-expressed human CYP isozymes to detect N-alkylPP produced after mechanism-based (catalysis-based) CYP inactivation. If this is found to be correct it will facilitate identification of potentially porphyrinogenic drugs prior to administration to humans.

Homology modeling and substrate binding study of human CYP2C9 enzyme.

The CYP2C subfamily of human liver P450 isozymes is of major importance in drug metabolism. The most abundant 2C isozyme, CYP2C9, regioselectively hydroxylates a wide variety of substrates. A major obstacle to understanding this specificity in human CYP2C9 is the absence of a 3D structure. A 3D model of CYP2C9 was built, assessed, and used to characterize explicit enzyme-substrate complexes using methods previously developed in our laboratory. The 3D model was assessed by determining its stability to unconstrained molecular dynamics and by comparison of specific properties with those of known protein structures. The CYP2C9 model was then used to characterize explicit enzyme complexes with three structurally and chemically diverse substrates: (S)-naproxen, phenytoin, and progesterone. Each substrate was found to bind to the enzyme with a favorable interaction energy and to remain in the binding site during unconstrained molecular dynamics. Moreover, the mode of binding of each substrate led to calculated preferred hydroxylation sites consistent with experiment. Binding-site residues identified for the models included Arg 105 and Arg97 as key cationic residues, as well as Asn 202, Asp 293, Pro 101, Leu 102, Gly 296, and Phe 476. Site-specific mutations are proposed for further integrated computational and experimental study.

薬物動態研究・トランスポーター研究とドラッグデザイン

Recently, attempts have been made to synthesize rapidly very large numbers of compounds and identify promising drug candidates in terms of both their pharmacological activity and pharmacokinetic features during the early stages of screening (high throughput screening). In drug development, therefore, the importance of the extrapolation and prediction of pharmacokinetics in humans has increased and physiologically-based pharmacokinetics has played an important role in this.It has become possible to predict in vivo pharmacokinetics in humans if accurate data are available on in vitro drug metabolism, plasma membrane transport, and protein binding and if these data can be combined with physiological parameters such as hepatic blood flow etc. Recent findings are summarized for the quantitative prediction of the hepatic metabolic clearance from in vitro studies using human liver microsomes or P450 isozyme recombinant systems. Furthermore, a method is proposed to predict pharmacokinetic alterations caused by drug-drug interactions based on in vitro metabolic inhibition studies using human liver microsomes or human enzyme expression systems.Biliary or urinary excretion of drugs has been shown to be mediated by various transporters. Some transporters have been shown to be involved also in the efflux of various compounds through the blood-brain barrier (BBB). Examples of the recent studies showing the relevance of transporters in pharmacokinetics are given and the prediction of BBB permeability is also discussed. In future, these transport characteristics of the drug and the possibility of drug-drug interactions involving transport will also need to be taken into consideration for more strategic and efficient drug development.