Human Liver Microsomal Protein Research Articles

Structural Identification of Zotarolimus (ABT-578) Metabolites Generated by Human Liver Microsomes Using Ion-Trap and High-Resolution Time-of-Flight Mass Spectrometry in Combination with the Analysis of Fragmentation Patterns.

Zotarolimus (ABT-578) is a sirolimus derivative that, like sirolimus and everolimus, is an inhibitor of cell growth via inhibition of the mechanistic target of rapamycin (mTOR). Zotarolimus was developed for coating coronary stents to prevent smooth muscle cell proliferation and restenosis. Albeit zotarolimus-eluting cardiovascular devices have been on the market for years, details of zotarolimus drug metabolism in humans are still unknown. Hence, it was the goal of the present study to identify zotarolimus metabolites generated by incubation with human liver microsomes. Metabolite structures were identified using high-resolution mass spectrometry, MS/ion-trap (MSn), and comparison of fragmentation patterns of the metabolites with those of zotarolimus and other known sirolimus derivatives. Kinetic parameters such as incubation time, human liver microsomal protein concentrations, and drug concentrations were optimized before scaling up the metabolism experiments. Human liver microsomes mainly hydroxylated and/or demethylated zotarolimus. The structures of the following metabolites were identified: O-demethylated metabolites: 39-O-desmethyl, 16-O-desmethyl, and 27-O-desmethyl zotarolimus; hydroxylated metabolites: hydroxy piperidine zotarolimus, 11-hydroxy, 12-hydroxy, 14-hydroxy, 23-hydroxy, 24-hydroxy, 25-hydroxy, 45/46-hydroxy, and 49-hydroxy zotarolimus; demethylated-hydroxylated metabolites: 16-O-desmethyl, 23/24-hydroxy; 39-O-desmethyl, 23/24-hydroxy; 39-O-desmethyl, 25-hydroxy zotarolimus; 39-O-desmethyl, 11-hydroxy zotarolimus; 39-O-desmethyl, hydroxy-piperidine zotarolimus; 27-O-desmethyl, 45/46-hydroxy zotarolimus; didemethylated metabolites: 16,39-O-didesmethyl zotarolimus; 16,27-O-didesmethyl zotarolimus; 27,39-O-didesmethyl zotarolimus; and dihydroxylated metabolites: 11,24-dihydroxy zotarolimus, 12,24-dihydroxy zotarolimus, and 11,47/48-dihydroxy zotarolimus. It is concluded that zotarolimus is extensively metabolized by human liver microsomes. Twenty-four of these metabolites could be structurally identified using a combination of ion-trap MSn and high-resolution mass spectrometry.

Antimalarial Dibenzannulated Medium-Ring Keto Lactams.

We discovered dibenzannulated medium-ring keto lactams (11,12-dihydro-5H-dibenzo[b,g]azonine-6,13-diones) as a new antimalarial chemotype. Most of these had chromatographic LogD7.4 values ranging from <0 to 3 and good kinetic solubilities (12.5 to >100 μg/mL at pH 6.5). The more polar compounds in the series (LogD7.4 values of <2) had the best metabolic stability (CLint values of <50 μL/min/mg protein in human liver microsomes). Most of the compounds had relatively low cytotoxicity, with IC50 values >30 μM, and there was no correlation between antiplasmodial activity and cytotoxicity. The four most potent compounds had Plasmodium falciparum IC50 values of 4.2 to 9.4 nM and in vitro selectivity indices of 670 to >12,000. They were more than 4 orders-of-magnitude less potent against three other protozoal pathogens (Trypanosoma brucei rhodesiense, Trypanosoma cruzi, and Leishmania donovani) but did have relatively high potency against Toxoplasma gondii, with IC50 values ranging from 80 to 200 nM. These keto lactams are converted into their poorly soluble 4(1H)-quinolone transannular condensation products in vitro in culture medium and in vivo in mouse blood. The similar antiplasmodial potencies of three keto lactam-quinolone pairs suggest that the quinolones likely contribute to the antimalarial activity of the lactams.

Discovery of Potent and Fast-Acting Antimalarial Bis-1,2,4-triazines.

Novel 3,3'-disubstituted-5,5'-bi(1,2,4-triazine) compounds with potent in vitro activity against Plasmodium falciparum parasites were recently discovered. To improve the pharmacokinetic properties of the triazine derivatives, a new structure-activity relationship (SAR) investigation was initiated with a focus on enhancing the metabolic stability of lead compounds. These efforts led to the identification of second-generation highly potent antimalarial bis-triazines, exemplified by triazine 23, which exhibited significantly improved in vitro metabolic stability (8 and 42 μL/min/mg protein in human and mouse liver microsomes). The disubstituted triazine dimer 23 was also observed to suppress parasitemia in the Peters 4-day test with a mean ED50 value of 1.85 mg/kg/day and exhibited a fast-killing profile, revealing a new class of orally available antimalarial compounds of considerable interest.

Pharmacokinetic, metabolic stability, plasma protein binding and CYP450s inhibition/induction assessment studies of N-(2-pyridylmethyl)-2-hydroxiymethyl-1-pyrrolidinyl-4-(3-chloro-4-methoxy-benzylamino)-5-pyrimidine-carboxamide as potential type 5 phosphodiesterase inhibitors

ABSTRACTN-(2-pyridylmethyl)-2-hydroxiymethyl-1-pyrrolidinyl-4-(3-chloro-4-methoxy-benzylamino)-5-pyrimidine-carboxamide (NHPPC) is a new potential of type 5 phosphodiesterase (PDE5) inhibitors, synthesized from the avanafil analogue for the treatment of erectile dysfunction. The targets of this article were to assess plasma protein binding, liver microsomal metabolic stability, inhibition and induction on cytochrome P450 isozymes and the pharmacokinetics of NHPPC. Equilibrium dialysis technique was applied to determine Plasma protein binding (PPB) and NHPPC was evaluated in male Sprague–Dawley rats and Beagle dogs in vivo pharmacokinetic. The NHPPC was highly bound to plasma proteins in rats, dogs and human tested and the mean values for PPB rate were 96.2%, 99.6% and 99.4%, respectively. After in vitro liver microsomes incubated for 60 min, the percent remaining of NHPPC was 42.8%, 0.8% and 42.0% in rats, dogs and human, respectively. In vitro intrinsic clearance was found to be 0.0233, 0.1204 and 0.0214 mL/min/mg protein in rat, dog and human liver microsomes of NHPPC, respectively. NHPPC showed no significant inhibitory effects on major CYP450 enzymes, and had no significant induction potential on CYP1A2 and CYP3A4. Following oral administration in rats and dogs, tmax was 6 and 0.5 h, respectively. The clearance for NHPPC was 1.19 and 1.46 L/h/kg in rats and dogs, respectively. And absolute bioavailability in rat and dog were approximately 34.5% and 53.1%, respectively. These results showed that NHPPC has a good development prospect.

Global Proteomic Analysis of Human Liver Microsomes: Rapid Characterization and Quantification of Hepatic Drug-Metabolizing Enzymes.

Many genetic and environmental factors lead to interindividual variations in the metabolism and transport of drugs, profoundly affecting efficacy and toxicity. Precision dosing, that is, targeting drug dose to a well characterized subpopulation, is dependent on quantitative models of the profiles of drug-metabolizing enzymes (DMEs) and transporters within that subpopulation, informed by quantitative proteomics. We report the first use of ion mobility-mass spectrometry for this purpose, allowing rapid, robust, label-free quantification of human liver microsomal (HLM) proteins from distinct individuals. Approximately 1000 proteins were identified and quantified in four samples, including an average of 70 DMEs. Technical and biological variabilities were distinguishable, with technical variability accounting for about 10% of total variability. The biological variation between patients was clearly identified, with samples showing a range of expression profiles for cytochrome P450 and uridine 5'-diphosphoglucuronosyltransferase enzymes. Our results showed excellent agreement with previous data from targeted methods. The label-free method, however, allowed a fuller characterization of the in vitro system, showing, for the first time, that HLMs are significantly heterogeneous. Further, the traditional units of measurement of DMEs (pmol mg-1 HLM protein) are shown to introduce error arising from variability in unrelated, highly abundant proteins. Simulations of this variability suggest that up to 1.7-fold variation in apparent CYP3A4 abundance is artifactual, as are background positive correlations of up to 0.2 (Spearman correlation coefficient) between the abundances of DMEs. We suggest that protein concentrations used in pharmacokinetic predictions and scaling to in vivo clinical situations (physiologically based pharmacokinetics and in vitro-in vivo extrapolation) should be referenced instead to tissue mass.

Content and activity of human liver microsomal protein and prediction of individual hepatic clearance in vivo.

The lack of information concerning individual variation in content and activity of human liver microsomal protein is one of the most important obstacles for designing personalized medicines. We demonstrated that the mean value of microsomal protein per gram of liver (MPPGL) was 39.46 mg/g in 128 human livers and up to 19-fold individual variations existed. Meanwhile, the metabolic activities of 10 cytochrome P450 (CYPs) were detected in microsomes and liver tissues, respectively, which showed huge individual variations (200-fold). Compared with microsomes, the activities of liver tissues were much suitable to express the individual variations of CYP activities. Furthermore, individual variations in the in vivo clearance of tolbutamide were successfully predicted with the individual parameter values. In conclusion, we offer the values for MPPGL contents in normal liver tissues and build a new method to assess the in vitro CYP activities. In addition, large individual variations exist in predicted hepatic clearance of tolbutamide. These findings provide important physiological parameters for physiologically-based pharmacokinetics models and thus, establish a solid foundation for future development of personalized medicines.

Significantly Different Covalent Binding of Oxidative Metabolites, Acyl Glucuronides, and S-Acyl CoA Conjugates Formed from Xenobiotic Carboxylic Acids in Human Liver Microsomes.

Xenobiotic carboxylic acids may be metabolized to oxidative metabolites, acyl glucuronides, and/or S-acyl-CoA thioesters (CoA conjugates) in vitro, e.g., in hepatocytes, and in vivo. These metabolites can potentially be reactive species and bind covalently to tissue proteins and are generally considered to mediate adverse drug reactions in humans. Acyl glucuronide metabolites have been the focus of reactive metabolite research for decades, whereas drug-CoA conjugates, which have been shown to be up to 40-70 times more reactive, have been given much less attention. In an attempt to dissect the contribution of different pathways to covalent binding, we utilized human liver microsomes supplemented with NADPH, uridine 5'-diphosphoglucuronic acid (UDPGA), or CoA to evaluate the reactivity of each metabolite separately. Seven carboxylic acid drugs were included in this study. While ibuprofen and tolmetin are still on the market, ibufenac, fenclozic acid, tienilic acid, suprofen, and zomepirac were stopped before their launch or withdrawn. The reactivities of the CoA conjugates of ibuprofen, ibufenac, fenclozic acid, and tolmetin were higher compared to those of their corresponding oxidative metabolites and acyl glucuronides, as measured by the level of covalent binding to human liver microsomal proteins. The highest covalent binding was observed for ibuprofenyl-CoA and ibufenacyl-CoA, to levels of 1000 and 8600 pmol drug eq/mg protein, respectively. In contrast and in agreement with the proposed P450-mediated toxicity for these drug molecules, the reactivities of oxidative metabolites of suprofen and tienilic acid were higher compared to the reactivities of their conjugated metabolites, with NADPH-dependent covalent binding of 250 pmol drug eq/mg protein for both drugs. The seven drugs all formed UDPGA-dependent acyl glucuronides, but none of these resulted in covalent binding. This study shows that, unlike studies with hepatocytes or in vivo, human liver microsomes provide an opportunity to investigate the reactivity of individual metabolites.

Identification of human liver microsomal proteins adducted by a reactive metabolite using shotgun proteomics.

Covalent modification of cellular proteins by chemically reactive compounds/metabolites has the potential to disrupt biological function and elicit serious adverse drug reactions. Information on the nature and binding patterns of protein targets are critical toward understanding the mechanism of drug induced toxicity. Protein covalent binding studies established in liver microsomes can quantitively estimate the extent of protein modification, but they provide little information on the nature of the modified proteins. In this article, we describe a label-free shotgun proteomic workflow for the identification of target proteins modified in situ by reactive metabolites in human liver microsome incubations. First, we developed a shotgun proteomic workflow for the characterization of the human liver microsomal subproteome, which consists of predominately membrane-bound proteins. Human liver microsomes were solubilized with a combination of MS-compatible organic solvents followed by protein reduction, alkylation, and tryptic digestion. The unmodified samples were analyzed by UHPLC-MS/MS, and the proteins were identified by database searching. This workflow led to the successful identification of 329 human liver microsomal subproteome proteins with 1% FDR (false discovery rate). The same method was then applied to identify the modifications of human liver microsomal proteins by a known reactive metabolite 2-(methylsulfonyl)benzo[d]thiazole (2), either after incubation directly with 2 or with its parent compound 2-(methylthio)benzo[d]thiazole (1). A total of 19 modified constituent peptides which could be mapped to 18 proteins were identified in human liver microsomes incubated directly with 2. Among these, 5 modified constituent peptides which could be mapped to 4 proteins were identified in incubation with 1, which is known to generate 2 in human liver microsomal incubations. This label-free workflow is generally applicable to the identification and characterization of proteins adducted with reactive metabolites in complex matrices and may serve as a valuable tool to understand the link between protein targets and clinically relevant toxicities.

Structural identification of imatinib cyanide adducts by mass spectrometry and elucidation of bioactivation pathway

Recent publications have reported that imatinib forms cyanide and methoxylamine adducts in vitro but without detail structural identification. The current work reports the identification of seven cyanide adducts that elucidate the bioactivation pathways and may provide hints for observed clinical adverse effects of the drug. Imatinib was incubated with human liver microsomal proteins in the presence of a NADPH-regeneration system and the trapping agents reduced GSH, potassium cyanide and methoxylamine. Samples were analyzed by high-performance liquid chromatography (HPLC) coupled with a LTQ-Orbitrap data collection system. Chemical structures were determined and/or postulated based on data-dependent high-resolution tandem mass spectrometric (MS(n)) exact mass measurements in both positive and negative scan modes, as well as in combination with hydrogen-deuterium exchange (HDX). GSH and methoxylamine conjugates were either not detected or were in insufficient quantities for characterization. However, seven cyanide conjugates were identified, indicating that the piperazine and p-toluidine partial structures in imatinib can become bioactivated and subsequently trapped by the nucleophile cyanide ion. The reactive intermediates were postulated as imine and imine-carbonyl conjugate (α,β-unsaturated) structures on the piperazine ring, and imine-methide on the p-toluidine partial structure. Chemical structures of seven cyanide adducts of imatinib have been identified or proposed based on high-resolution MS/MS data. Mechanisms for the formation of the conjugates were also proposed. The findings may help to understand the mechanism of hepatotoxicity of imatinib in humans.

A Calibrated Human PBPK Model for Benzene Inhalation with Urinary Bladder and Bone Marrow Compartments

A physiologically-based pharmacokinetic (PBPK) model of benzene inhalation based on a recent mouse model was adapted to include bone marrow (target organ) and urinary bladder compartments. Empirical data on human liver microsomal protein levels and linked CYP2E1 activities were incorporated into the model, and metabolite-specific conversion rate parameters were estimated by fitting to human biomonitoring data and adjusting for background levels of urinary metabolites. Human studies of benzene levels in blood and breath, and phenol levels in urine were used to validate the rate of human conversion of benzene to benzene oxide, and urinary benzene metabolites from Chinese benzene worker populations provided model validation for rates of human conversion of benzene to muconic acid (MA) and phenylmercapturic acid (PMA), phenol (PH), catechol (CA), hydroquinone (HQ), and benzenetriol (BT). The calibrated human model reveals that while liver microsomal protein and CYP2E1 activities are lower on average in humans compared to mice, the mouse also shows far lower rates of benzene conversion to MA and PMA, and far higher conversion of benzene to BO/PH, and of BO/PH to CA, HQ, and BT. The model also differed substantially from existing human PBPK models with respect to several metabolic rate parameters of importance to interpreting benzene metabolism and health risks in human populations associated with bone marrow doses. The model provides a new methodological paradigm focused on integrating linked human liver metabolism data and calibration using biomonitoring data, thus allowing for model uncertainty analysis and more rigorous validation.

Optimized Assays for Human UDP-Glucuronosyltransferase (UGT) Activities: Altered Alamethicin Concentration and Utility to Screen for UGT Inhibitors

The measurement of the effect of new chemical entities on human UDP-glucuronosyltransferase (UGT) marker activities using in vitro experimentation represents an important experimental approach in drug development to guide clinical drug-interaction study designs or support claims that no in vivo interaction will occur. Selective high-performance liquid chromatography-tandem mass spectrometry functional assays of authentic glucuronides for five major hepatic UGT probe substrates were developed: β-estradiol-3-glucuronide (UGT1A1), trifluoperazine-N-glucuronide (UGT1A4), 5-hydroxytryptophol-O-glucuronide (UGT1A6), propofol-O-glucuronide (UGT1A9), and zidovudine-5'-glucuronide (UGT2B7). High analytical sensitivity permitted characterization of enzyme kinetic parameters at low human liver microsomal and recombinant UGT protein concentration (0.025 mg/ml), which led to a new recommended optimal universal alamethicin activation concentration of 10 μg/ml for microsomes. Alamethicin was not required for recombinant UGT incubations. Apparent enzyme kinetic parameters, particularly for UGT1A1 and UGT1A4, were affected by nonspecific binding. Unbound intrinsic clearance for UGT1A9 and UGT2B7 increased significantly after addition of 2% bovine serum albumin, with minimal changes for UGT1A1, UGT1A4, and UGT1A6. Eleven potential UGT and cytochrome P450 inhibitors were evaluated as UGT inhibitors, resulting in observation of nonselective UGT inhibition by chrysin, mefenamic acid, silibinin, tangeretin, ketoconazole, itraconazole, ritonavir, and verapamil. The pan-cytochrome P450 inhibitor, 1-aminobenzotriazole, minimally inhibited UGT activities and may be useful in reaction phenotyping of mixed UGT and cytochrome P450 substrates. These methods should prove useful in the routine assessments of the potential for new drug candidates to elicit pharmacokinetic drug interactions via inhibition of human UGT activities and the identification of UGT enzyme-selective chemical inhibitors.

Optimization of the SDS-PAGE gel slicing approach for identification of human liver microsomal proteins via MALDI-TOF mass spectrometry

Proteomics often exploits sequential cutting of the SDS-PAGE gel lane into slices with subsequent identification of proteins by peptide mass fingerprinting (PMF). In this paper, the influence of slice thickness on protein identification was investigated. Following the separation of the human liver microsomal fraction, the 37 to 75 kDa range of the gel lane was cut into 20 or 40 slices of 0.5 mm and 0.25 mm thickness, respectively. Identification of proteins after trypsinolysis was performed by MALDI-TOF mass spectrometry. A twofold reduction of slice thickness did not influence the number of peaks in PMF-spectra. It was established that at a tolerance of 0.15 Da the number of identified proteins in the 40 slices series exceeded by more than twofold the number of identifications in 20 slices series. It was shown that the decrease of slice thickness leads to a considerable enhancement of peptide peak’s intensity and, also, to the changes of PMF-spectra which resulted gaining additional peptides for enrichment of sequence coverage.

Quantification of Human Uridine-Diphosphate Glucuronosyl Transferase 1A Isoforms in Liver, Intestine, and Kidney Using Nanobore Liquid Chromatography–Tandem Mass Spectrometry

Uridine-disphosphate glucuronosyl transferase (UGT) enzymes catalyze the formation of glucuronide conjugates of phase II metabolism. Methods for absolute quantification of UGT1A1 and UGT1A6 were previously established utilizing stable isotope peptide internal standards with liquid chromatography-tandem mass spectrometry (LC-MS/MS). The current method expands upon this by quantifying eight UGT1A isoforms by nanobore high-performance liquid chromatography (HPLC) coupled with a linear ion trap time-of-flight mass spectrometer platform. Recombinant enzyme digests of each of the isoforms were used to determine assay linearity and detection limits. Enzyme expression level in human liver, kidney, and intestinal microsomal protein was determined by extrapolation from spiked stable isotope standards. Intraday and interday variability was <25% for each of the enzyme isoforms. Enzyme expression varied from 3 to 96 pmol/mg protein in liver and intestinal microsomal protein digests. Expression levels of UGT1A7, 1A8, and 1A10 were below detection limits (<1 pmol/mg protein) in human liver microsome (HLMs). In kidney microsomes the expression of UGT1A3 was below detection limits, but levels of UGT1A4, 1A7, 1A9, and 1A10 protein were higher relative to that of liver, suggesting that renal glucuronidation could be a significant factor in renal elimination of glucuronide conjugates. This novel method allows quantification of all nine UGT1A isoforms, many previously not amenable to measurement with traditional methods such as immunologically based assays. Quantitative measurement of proteins involved in drug disposition, such as the UGTs, significantly improves the ability to evaluate and interpret in vitro and in vivo studies in drug development.

Simultaneous Absolute Protein Quantification of Transporters, Cytochromes P450, and UDP-Glucuronosyltransferases as a Novel Approach for the Characterization of Individual Human Liver: Comparison with mRNA Levels and Activities

The purpose of the present study was to determine the absolute protein expression levels of multiple drug-metabolizing enzymes and transporters in 17 human liver biopsies, and to compare them with the mRNA expression levels and functional activities to evaluate the suitability of the three measures as parameters of hepatic metabolism. Absolute protein expression levels of 13 cytochrome P450 (P450) enzymes, NADPH-P450 reductase (P450R) and 6 UDP-glucuronosyltransferase (UGT) enzymes in microsomal fraction, and 22 transporters in plasma membrane fraction were determined using liquid chromatography/tandem mass spectrometry. CYP2C9, CYP2E1, CYP3A4, CYP2A6, UGT1A6, UGT2B7, UGT2B15, and P450R were abundantly expressed (more than 50 pmol/mg protein) in human liver microsomes. The protein expression levels of CYP3A4, CYP2B6, and CYP2C8 were each highly correlated with the corresponding enzyme activity and mRNA expression levels, whereas for other P450s, the protein expression levels were better correlated with the enzyme activities than the mRNA expression levels were. Among transporters, the protein expression level of organic anion-transporting polypeptide 1B1 was relatively highly correlated with the mRNA expression level. However, other transporters showed almost no correlation. These findings indicate that protein expression levels determined by the present simultaneous quantification method are a useful parameter to assess differences of hepatic function between individuals.

Metabolism of thalidomide by human liver microsome cytochrome CYP2C19 is required for its antimyeloma and antiangiogenic activities in vitro

In this study, we used a system of human liver microsomes to investigate the antimyeloma and antiangiogenic activities of thalidomide. Myeloma cells and human umbilical vein endothelial cells (HUVECs) were treated with thalidomide alone or thalidomide incubated with human liver microsomal protein. We found that thalidomide alone had no direct effect on several multiple myeloma cell lines (U266, NCI-H929, RPMI 8226, LP-1, CZ-1) or on HUVECs in vitro. However, when incubated with human liver microsomal protein, thalidomide (100 µg/ml) caused a decrease of 34.9-46.7% in cell viability in myeloma cells and 12% in HUVECs. Cell cycle analysis and apoptosis detection indicated that the decreases in cell viability were correlated with the induction of apoptosis. Thalidomide incubated with microsomal protein also influenced HUVEC migration and tube formation. These effects were partially reversed by omeprazole (10 µmol/l), a potent inhibitor of CYP2C19, suggesting that CYP2C19 is required for thalidomide to exhibit its antimyeloma and antiangiogenic activities.

Abstract LB-105: Single nucleotide polymorphisms (SNPs) in the CYP19A1 gene are strongly associated with CYP1A2 expression: First evidence for epistasis in the CYP superfamily

Abstract CYP1A2 is a member of the cytochromes P450 enzymes responsible for the oxidative metabolism of a wide array of endo- and xenobiotics, and has been demonstrated to be the major isoform responsible for oxidation of estrone and estradiol. To date, SNPs identified in this gene do not adequately explain the 60-80-fold variation in activity observed in Caucasians. We hypothesized that CYP1A2 could be a target of epistatic regulation (i.e., gene-gene interactions where SNPs in one gene influence the expression of another). CYP1A2 and CYP19A1 both lie on chromosome 15, and since CYP19A1 (aromatase) is responsible for the production of estrogens, and CYP1A2 is the primary catabolic P450, we hypothesized that these genes could be coordinately regulated, and SNPs influencing levels of CYP19A1 could also be related to expression levels of CYP1A2. Two TagSNPs (rs749292 A&amp;lt;G and rs727479 A&amp;lt;C) in CYP19A1 were genotyped using Beckman Single Base Extension Kit and TaqMan MGB probe, respectively. Levels of CYP1A2 protein in human liver microsomes from 100 donors were assessed by Western blotting and correlations between CYP19A1 SNPs and CYP1A2 protein amounts were determined. CYP1A2 expression was significantly higher in specimens with CYP19A1 (rs727479) AA homozygous compared with CC genotype (P&amp;lt;0.001), a SNP associated with increased CYP19A1 activity. CYP1A2 expression also is related CYP19A1 (rs749292) polymorphism and AA genotype carriers have higher CYP1A2 expression compared with GG genotype carriers. Moreover, in silico analyses shows the presence of over 300 putative estrogen-responsive elements upstream of the CYP1A2 transcription start sit. SNPs in CYP19A1 are significantly associated with CYP1A2 protein expression. This is the first evidence of epistasis in the P450 enzymes and suggests a role of estrogens in the regulation of CYP1A2. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-105.

Metabolic activation of indole-containing prostaglandin D2 receptor 1 antagonists: Impacts of glutathione trapping and glucuronide conjugation on covalent binding

Some DP1 receptor antagonists from an indole-containing series were shown to cause in vitro covalent binding to protein in rat and human liver microsomes. Glutathione trapping experiments along with in vitro labeling assays confirmed that the presence of a strong electron withdrawing group was necessary to abrogate in vitro covalent binding, leading to the discovery of MK-0524. Hepatocyte incubations and in vivo studies showed that acyl-glucuronide formation did not translate into covalent binding.

Glucuronidation of Antiallergic Drug, Tranilast: Identification of Human UDP-Glucuronosyltransferase Isoforms and Effect of Its Phase I Metabolite

Tranilast is an oral antiallergic agent widely used in Japan. Recently, in Western populations, hyperbilirubinemia induced by tranilast was suspected during clinical trials. Tranilast has been reported to be mainly metabolized to a glucuronide and a phase I metabolite, 4-demethyltranilast (N-3). In the present study, we investigated the in vitro metabolism of tranilast in human liver and jejunum microsomes and recombinant UDP-glucuronosyltransferases (UGTs). The glucuronidation of tranilast was clarified to be mainly catalyzed by UGT1A1 in human liver and intestine. The K(m) values of tranilast glucuronosyltransferase activity were 51.5, 50.6, and 38.0 microM in human liver microsomes, human jejunum microsomes, and recombinant UGT1A1, respectively. The V(max) values were 10.4, 42.9, and 19.7 pmol/min/mg protein in human liver microsomes, human jejunum microsomes, and recombinant UGT1A1, respectively. When the intrinsic clearance was calculated using the in vitro kinetic parameters, microsomal protein content, and weight of tissues, tranilast glucuronosyltransferase activity was 2.5-fold higher in liver than in intestine. Tranilast glucuronosyltransferase activity was strongly inhibited by bilirubin, a typical UGT1A1 substrate, and N-3, indicating that the phase I metabolite could affect the tranilast glucuronosyltransferase activity. In the case of N-3 formation, the K(m) and V(max) values were 37.1 microM and 27.6 pmol/min/mg protein in human liver microsomes. The bilirubin glucuronosyltransferase activity was strongly inhibited by both tranilast and N-3, suggesting that tranilast-induced hyperbilirubinemia would be responsible for the inhibition by tranilast and N-3 of the bilirubin glucuronosyltransferase activity, as would the UGT1A1 genotype.

Glucuronidation of DRF-6574, hydroxy metabolite of DRF-4367 (a novel COX-2 inhibitor) by pooled human liver, intestinal microsomes and recombinant human UDP-glucuronosyltransferases (UGT): Role of UGT1A1,1A3 and 1A8

DRF-4367 is a novel COX-2 inhibitor, which showed good efficacy in several animal models of inflammation. In a comparative in vitro metabolism in various liver microsomes, DRF-4367 forms a hydroxy metabolite (DRF-6574) mediated by CYP2D6 and 2C19 isoenzymes. DRF-6574 readily undergoes Phase-II metabolism and forms glucuronide and sulfate conjugates both in vitro and in vivo. The objective of the present study was two folds: to study the glucuronidation of DRF-6574 in human liver and intestinal microsomes and to identify the recombinant human liver and intestinal UDP-glucuronosyltransferase (UGT) enzymes responsible for glucuronidation of DRF-6574. Of twelve recombinant UGTs tested, two hepatic UGTs viz., UGT1A1 and 1A3 and an extra hepatic UGT i.e., UGT1A8 showed the catalytic activity. The enzyme kinetics in pooled human liver, intestinal and recombinant UGT microsomes showed a typical Michaelis-Menten plot. The apparent Km and Vmax value for DRF-6574 was found to be 116 +/- 24 microM and 2.07 +/- 0.12 microg/min/mg protein and 142 +/- 17 microM and 3.83 +/- 0.15 microg/min/mg protein in pooled human liver and intestinal microsomes, respectively. The intrinsic clearance (Vmax/Km) value for DRF-6574 was estimated to be 0.043 and 0.065 ml/min/mg protein, respectively in pooled human liver and intestinal microsomes. Moreover we have determined the Km and Vmax and intrinsic clearance values for specific UGTs viz., UGT 1A1, 1A3 and 1A8. The apparent Km and Vmax values are 23 +/- 7.2 microM, 3.44 +/- 0.17 microg/min/mg protein for UGT1A1, 60 +/- 7.9 microM, 3.67 +/- 0.11 microg/min/mg protein for UGT1A3, 96 +/- 8.0 microM, 2.95 +/- 0.06 microg/min/mg protein for UGT1A8. The intrinsic clearance values (Vmax/Km) estimated were 0.367, 0.148, 0.074 ml/min/mg protein for UGT1A1, 1A3 and 1A8, respectively. The intrinsic clearance value in UGT1A8 was very close to that in human intestinal and liver microsomes. The formation of DRF-6574 glucuronide by human liver, intestinal and UGT1A1, 1A3 and 1A8 microsomes was effectively inhibited by phenylbutazone.

CYP2D6 CATALYZES 5-HYDROXYLATION OF 1-(2-PYRIMIDINYL)-PIPERAZINE, AN ACTIVE METABOLITE OF SEVERAL PSYCHOACTIVE DRUGS, IN HUMAN LIVER MICROSOMES

1-(2-Pyrimidinyl)-piperazine (1-PP) is an active metabolite of several psychoactive drugs including buspirone. 1-PP is also the major metabolite in the human circulation and in rat brains following oral administration of buspirone. This study was conducted to identify the enzyme responsible for the metabolic conversion of 1-PP to 5-hydroxy-1-(2-pyrimidinyl)-piperazine (HO-1-PP) in human liver microsomes (HLMs). The product HO-1-PP was quantified by a validated liquid chromatography-tandem mass spectrometry method. In the presence of NADPH, 1-PP (100 microM) was incubated separately with human cDNA-expressed cytochrome P450 isozymes (including CYP2D6, 3A4, 1A2, 2A6, 2C9, 2C19, 2E1, and 2B6) at 37 degrees C. CYP2D6 catalyzed the formation of HO-1-PP from 1-PP. This catalytic activity was >95% inhibited by quinidine, a CYP2D6 inhibitor. HO-1-PP formation rates correlated well with the bufuralol 1-hydroxylase (CYP2D6) activities of individual HLMs. The formation of HO-1-PP followed a Michaelis-Menten kinetics with a K(m) of 171 microM and V(max) of 313 pmol/min x mg protein in HLMs. Collectively, these results indicate that polymorphic CYP2D6 is responsible for the conversion of 1-PP to HO-1-PP.