Development and validation of probe drug cocktails for the characterization of CYP450-mediated metabolism by human heart microsomes.

1. The objective of our study was to develop and validate a cocktail approach to allow the simultaneous characterization of various CYP450-mediated oxidations by human heart microsomes for nine probe drug substrates, namely, 7-ethoxyresorufin, bupropion, repaglinide, tolbutamide, bufuralol, chlorzoxazone, ebastine, midazolam and dodecanoic acid. 2. The first validation step was conducted using recombinant human CYP450 isoenzymes by comparing activity measured for each probe drug as a function of (1) buffer used, (2) selectivity towards specific isoenzymes and (3) drug interactions between probes. Activity was all measured by validated LC-MSMS methods. 3. Two cocktails were then constituted with seven of the nine drugs and subjected to kinetic validation. Finally, all probe drugs were incubated with human heart microsomes prepared from ventricular tissues obtained from 12 patients undergoing cardiac transplantation. 4. Validated cocktail #1 including bupropion, chlorzoxazone, ebastine and midazolam was used to characterize CYP2B6-, 2E1-, 2J2- and 3A5-mediated metabolism in human hearts. 5. Cocktail #2 which includes bufuralol, 7-ethoxyresorufin and repaglinide failed the validation step. Substrates in cocktail #2 as well as tolbutamide and dodecanoic acid had to be incubated separately because of their physico-chemical characteristics (solubility and ionization) or drug interactions. 6. Activity in HHM was the highest towards ebastine, chlorzoxazone and tolbutamide.

Role of Arginine 117 in Substrate Recognition by Human Cytochrome P450 2J2.

The influence of Arginine 117 of human cytochrome P450 2J2 in the recognition of ebastine and a series of terfenadone derivatives was studied by site-directed mutagenesis. R117K, R117E, and R117L mutants were produced, and the behavior of these mutants in the hydroxylation of ebastine and terfenadone derivatives was compared to that of wild-type CYP2J2. The data clearly showed the importance of the formation of a hydrogen bond between R117 and the keto group of these substrates. The data were interpreted on the basis of 3D homology models of the mutants and of dynamic docking of the substrates in their active site. These modeling studies also suggested the existence of a R117-E222 salt bridge between helices B' and F that would be important for maintaining the overall folding of CYP2J2.

The inhibitory potential of Broussochalcone A for the human cytochrome P450 2J2 isoform and its anti-cancer effects via FOXO3 activation.

BACKGROUND: Broussonetia papyrifera (L.) Ventenat, a traditional medicinal herb, has been applied as a folk medicine to treat various diseases. Broussochalcone A (BCA), a chalcone compound isolated from the cortex of Broussonetia papyrifera (L.) Ventenat, exhibits several biological activities including potent anti-oxidant, antiplatelet, and cytotoxic effects. PURPOSE: The purpose of this study is to elucidate the inhibitory effect of BCA against CYP2J2 enzyme which is predominantly expressed in human tumor tissues and carcinoma cell lines. STUDY DESIGN: The inhibitory effect of BCA on the activities of CYP2J2-mediated metabolism were investigated using human liver microsomes (HLMs), and its anti-cancer effect against human hepatoma HepG2 cells was also evaluated. METHODS: Two representative CYP2J2-specific probe substrates, astemizole and ebastine, were incubated in HLMs with BCA. After incubation, the samples were analyzed using liquid chromatography-tandem mass spectrometry. To investigate the binding model between BCA and CYP2J2, we carried out structure-based docking simulations by using software and scripts written in-house. RESULTS: BCA inhibited CYP2J2-mediated astemizole O-demethylation and ebastine hydroxylase activities in a concentration dependent manner with Ki values of 2.3 and 3.7 µM, respectively. It also showed cytotoxic effects against human hepatoma HepG2 cells in a dose-dependent manner with activation of apoptosis related proteins. CONCLUSION: Overall, this was the first report of the inhibitory effects of BCA on CYP2J2 in HLMs. The present data suggest that BCA is a potential candidate for further evaluation for its CYP2J2 targeting anti-cancer activities.

New psychoactive substances: Studies on the metabolism of XLR-11, AB-PINACA, FUB-PB-22, 4-methoxy-α-PVP, 25-I-NBOMe, and meclonazepam using human liver preparations in comparison to primary human hepatocytes, and human urine.

New psychoactive substances (NPS) are an increasing problem in clinical and forensic toxicology. The knowledge of their metabolism is important for toxicological risk assessment and for developing toxicological urine screenings. Considering the huge numbers of NPS annually appearing on the market, metabolism studies should be realized in a fast, simple, cost efficient, and reliable way. Primary human hepatocytes (PHH) were recommended to be the gold standard for in vitro metabolism studies as they are expected to contain natural enzyme clusters, co-substrates, and drug transporters. In addition, they were already successfully used for metabolism studies of NPS. However, they also have disadvantages such as high costs and limited applicability without special equipment. The aims of the present study were therefore first to investigate exemplarily the phase I and phase II metabolism of six NPS (XLR-11, AB-PINACA, FUB-PB-22, 4-methoxy-α-PVP, 25-I-NBOMe, and meclonazepam) from different drug classes using pooled human S9 fraction (pS9) or pooled human liver microsomes combined with cytosol (pHLM/pHLC) after addition of the co-substrates for the main metabolic phase I and II reactions. Second to compare results to published data generated using primary human hepatocytes and human urine samples. Results of the incubations with pS9 or pHLM/pHLC were comparable in number and abundance of metabolites. Formation of metabolites, particularly after multi-step reactions needed a longer incubation time. However, incubations using human liver preparations resulted in a lower number of total detected metabolites compared to PHH, but they were still able to allow the identification of the main human urinary excretion products. Human liver preparations and particularly the pooled S9 fraction could be shown to be a sufficient and more cost-efficient alternative in context of metabolism studies also for developing toxicological urine screenings. It might be recommended to use the slightly cheaper pS9 fraction instead of a pHLM/pHLC combination. As formation of some metabolites needed a long incubation time, two sampling points at 60 and 360min should be recommended.

Heme Modification Contributes to the Mechanism-Based Inactivation of Human Cytochrome P450 2J2 by Two Terminal Acetylenic Compounds.

The mechanism-based inactivation of human CYP2J2 by three terminal acetylenic compounds: N-(methylsulfonyl)-6-(2-propargyloxyphenyl)hexanamide (MS), 17-octadecynoic acid (OD), and danazol (DZ) was investigated. The loss of hydroxyebastine (OHEB) carboxylation activity in a reconstituted system was time- and concentration-dependent and required NADPH for MS and OD, but not DZ. The kinetic constants for the mechanism-based inactivation of OHEB carboxylation activity were: KI of 6.1 µM and kinact of 0.22 min-1 for MS and KI of 2.5 µM and kinact of 0.05 min-1 for OD. The partition ratios for MS and OD were ∼10 and ∼20, respectively. Inactivation of CYP2J2 by MS or OD resulted in a loss of the native heme spectrum and a similar decrease in the reduced CO difference spectrum. A heme adduct was observed in the MS-inactivated CYP2J2. The possible reactive metabolite which covalently modified the prosthetic heme was characterized by analysis of the glutathione conjugates formed by MS or OD following oxygenation of the ethynyl moiety. Liquid chromatography-mass spectrometry showed that inactivation by MS or OD did not lead to modification of apoprotein. Interaction of CYP2J2 with DZ produced a type II binding spectrum with a Ks of 2.8 µM and the IC50 for loss of OHEB carboxylation activity was 0.18 µM. In conclusion, heme modification by MS and OD was responsible for the mechanism-based inactivation of CYP2J2. The results suggest that the ethynyl moiety of MS and OD faces the heme iron, whereas the isoxazole ring of DZ is preferentially oriented toward the heme iron of CYP2J2.

Dried Blood Spots Combined With Ultra-High-Performance Liquid Chromatography-Mass Spectrometry for the Quantification of the Antipsychotics Risperidone, Aripiprazole, Pipamperone, and Their Major Metabolites.

BACKGROUND: Risperidone, aripiprazole, and pipamperone are antipsychotic drugs frequently prescribed for the treatment of comorbid behavioral problems in children with autism spectrum disorders. Therapeutic drug monitoring (TDM) could be useful to decrease side effects and to improve patient outcome. Dried blood spot (DBS) sample collection seems to be an attractive technique to develop TDM of these drugs in a pediatric population. The aim of this work was to develop and validate a DBS assay suitable for TDM and home sampling. METHODS: Risperidone, 9-OH risperidone, aripiprazole, dehydroaripiprazole, and pipamperone were extracted from DBS and analyzed by ultra-high-performance liquid chromatography-tandem mass spectrometry using a C18 reversed-phase column with a mobile phase consisting of ammonium acetate/formic acid in water or methanol. The suitability of DBS for TDM was assessed by studying the influence of specific parameters: extraction solution, EDTA carryover, hematocrit, punching location, spot volume, and hemolysis. The assay was validated with respect to conventional guidelines for bioanalytical methods. RESULTS: The method was linear, specific without any critical matrix effect, and with a mean recovery around 90%. Accuracy and imprecision were within the acceptance criteria in samples with hematocrit values from 30% to 45%. EDTA or hemolysis did not skew the results, and no punching carryover was observed. No significant influence of the spot volume or the punch location was observed. The antipsychotics were all stable in DBS stored 10 days at room temperature and 1 month at 4 or -80°C. The method was successfully applied to quantify the 3 antipsychotics and their metabolites in patient samples. CONCLUSIONS: A UHPLC-MS/MS method has been successfully validated for the simultaneous quantification of risperidone, 9-OH risperidone, aripiprazole, dehydroaripiprazole, and pipamperone in DBS. The assay provided good analytical performances for TDM and clinical research applications.

A New Marmoset P450 4F12 Enzyme Expressed in Small Intestines and Livers Efficiently Metabolizes Antihistaminic Drug Ebastine.

Common marmosets (Callithrix jacchus) are attracting attention as animal models in preclinical studies for drug development. However, cytochrome P450s (P450s), major drug-metabolizing enzymes, have not been fully identified and characterized in marmosets. In this study, based on the four novel P450 4F genes found on the marmoset genome, we successfully isolated P450 4F2, 4F3B, 4F11, and 4F12 cDNAs in marmoset livers. Deduced amino acid sequences of the four marmoset P450 4F forms exhibited high sequence identities (87%-93%) to the human and cynomolgus monkey P450 4F homologs. Marmoset P450 4F3B and 4F11 mRNAs were predominantly expressed in livers, whereas marmoset P450 4F2 and 4F12 mRNAs were highly expressed in small intestines and livers. Four marmoset P450 4F proteins heterologously expressed in Escherichia coli catalyzed the ω-hydroxylation of leukotriene B4 In addition, marmoset P450 4F12 effectively catalyzed the hydroxylation of antiallergy drug ebastine, a human P450 2J/4F probe substrate. Ebastine hydroxylation activities by small intestine and liver microsomes from marmosets and cynomolgus monkeys showed greatly higher values than those of humans. Ebastine hydroxylation activities by marmoset and cynomolgus monkey small intestine microsomes were inhibited (approximately 60%) by anti-P450 4F antibodies, unlike human small intestine microsomes, suggesting that contribution of P450 4F enzymes for ebastine hydroxylation in the small intestine might be different between marmosets/cynomolgus monkeys and humans. These results indicated that marmoset P450 4F2, 4F3B, 4F11, and 4F12 were expressed in livers and/or small intestines and were functional in the metabolism of endogenous and exogenous compounds, similar to those of cynomolgus monkeys and humans.

The modulation of carbonyl reductase 1 by polyphenols.

Carbonyl reductase 1 (CBR1), an enzyme belonging to the short-chain dehydrogenases/reductases family, has been detected in all human tissues. CBR1 catalyzes the reduction of many xenobiotics, including important drugs (e.g. anthracyclines, nabumetone, bupropion, dolasetron) and harmful carbonyls and quinones. Moreover, it participates in the metabolism of a number of endogenous compounds and it may play a role in certain pathologies. Plant polyphenols are not only present in many human food sources, but are also a component of many popular dietary supplements and herbal medicines. Many studies reviewed herein have demonstrated the potency of certain flavonoids, stilbenes and curcuminoids in the inhibition of the activity of CBR1. Interactions of these polyphenols with transcriptional factors, which regulate CBR1 expression, have also been reported in several studies. As CBR1 plays an important role in drug metabolism as well as in the protection of the organism against potentially harmful carbonyls, the modulation of its expression/activity may have significant pharmacological and/or toxicological consequences. Some polyphenols (e.g. luteolin, apigenin and curcumin) have been shown to be very potent CBR1 inhibitors. The inhibition of CBR1 seems useful regarding the increased efficacy of anthracycline therapy, but it may cause the worse detoxification of reactive carbonyls. Nevertheless, all known information about the interactions of polyphenols with CBR1 have only been based on the results of in vitro studies. With respect to the high importance of CBR1 and the frequent consumption of polyphenols, in vivo studies would be very helpful for the evaluation of risks/benefits of polyphenol interactions with CBR1.

The in vivo and in vitro metabolism and the detectability in urine of 3',4'-methylenedioxy-alpha-pyrrolidinobutyrophenone (MDPBP), a new pyrrolidinophenone-type designer drug, studied by GC-MS and LC-MS(n.).

3',4'-Methylenedioxy-alpha-pyrrolidinobutyrophenone (MDPBP), a designer drug of the pyrrolidinophenone-type, was first seized in Germany in 2009. It was also identified in 'legal high' samples investigated in the UK. Therefore, the aim of the presented work was to identify its in vivo and in vitro phase I and II metabolites using gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-ion trap mass spectrometry (LC-MS(n) ). Furthermore, detectability of MDPBP in rat and human urine using standard urine screening approaches (SUSA) by GC-MS and LC-MS(n) was studied. The metabolites were isolated either directly or after enzymatic cleavage of conjugates by solid-phase extraction (C18, HCX). The metabolites were then analyzed and structures proposed after GC-MS (phase I) and LC-MS(n) (phase II). Based on these identified metabolites, the following main metabolic steps could be proposed: demethylenation followed by methylation of one hydroxy group, aromatic and side chain hydroxylation, oxidation of the pyrrolidine ring to the corresponding lactam as well as ring opening to the corresponding carboxylic acid. Furthermore, in rat urine after a typical user's dose as well as in human urine, mainly the metabolites could be detected using the authors' SUSA by GC-MS and LC-MS(n) . Thus, it should be possible to monitor an application of MDPBP assuming similar toxicokinetics in humans. Finally, CYP2C19 and CYP2D6 could be identified as the isoenzymes mainly responsible for demethylenation.

Functional studies of N-terminally modified CYP2J2 epoxygenase in model lipid bilayers.

CYP2J2 epoxygenase is a membrane bound cytochrome P450 that converts omega-3 and omega-6 fatty acids into physiologically active epoxides. In this work, we present a comprehensive comparison of the effects of N-terminal modifications on the properties of CYP2J2 with respect to the activity of the protein in model lipid bilayers using Nanodiscs. We demonstrate that the complete truncation of the N-terminus changes the association of this protein with the E.coli membrane but does not disrupt incorporation in the lipid bilayers of Nanodiscs. Notably, the introduction of silent mutations at the N-terminus was used to express full length CYP2J2 in E. coli while maintaining wild-type functionality. We further show that lipid bilayers are essential for the productive use of NADPH for ebastine hydroxylation by CYP2J2. Taken together, it was determined that the presence of the N-terminus is not as critical as the presence of a membrane environment for efficient electron transfer from cytochrome P450 reductase to CYP2J2 for ebastine hydroxylation in Nanodiscs. This suggests that adopting the native-like conformation of CYP2J2 and cytochrome P450 reductase in lipid bilayers is essential for effective use of reducing equivalents from NADPH for ebastine hydroxylation.

Myocardial pharmacokinetics of ebastine, a substrate for cytochrome P450 2J, in rat isolated heart.

BACKGROUND AND PURPOSE: It is well established that cytochrome P450 2J (CYP2J) enzymes are expressed preferentially in the heart, and that ebastine is a substrate for CYP2J, but it is not known whether ebastine is metabolized in myocardium. Therefore, we investigated its pharmacokinetics in the rat isolated perfused heart. EXPERIMENTAL APPROACH: Rat isolated hearts were perfused in the recirculating mode with ebastine for 130 min. The concentrations of ebastine and its metabolites, hydroxyebastine and carebastine, were measured using liquid chromatography with a tandem mass spectrometry. The data were analysed by a compartmental model. The time course of negative inotropic response was linked to ebastine concentration to determine the concentration-effect relationship. KEY RESULTS: Ebastine was metabolized to an intermediate compound, hydroxyebastine, which was subsequently further metabolized to carebastine. No desalkylebastine was found. The kinetics of the sequential metabolism of ebastine was well described by the compartmental model. The EC(50) of the negative inotropic effect of ebastine in rat isolated heart was much higher than free plasma concentrations in humans after clinical doses. CONCLUSIONS AND IMPLICATIONS: The kinetics of ebastine conversion to carebastine via hydroxyebastine resembled that observed in human liver microsomes. The results may be of interest for functional characterization of CYP2J activity in rat heart.

Itraconazole and rifampin alter significantly the disposition and antihistamine effect of ebastine and its metabolites in healthy participants.

The present study was performed to elucidate the effects of itraconazole and rifampin on the pharmacokinetics and pharmacodynamics of ebastine, a nonsedative H1 receptor antagonist. In a 3-way crossover sequential design with 2-week washouts, 10 healthy participants were pretreated with itraconazole for 6 days, rifampin for 10 days, or neither. After oral administration of 20 mg ebastine, blood and urine samples were collected for 72 and 24 hours, respectively, and histamine-induced wheal and flare reactions were measured to assess the antihistamine response for 12 hours. Itraconazole pretreatment decreased the oral clearance of ebastine to 10% (P < .001) and increased the AUC(infinity) of the active metabolite, carebastine, by 3-fold (P < .001). On the other hand, rifampin pretreatment decreased the AUC(infinity) of carebastine to 15% (P < .001), with an enormous reduction in the oral bioavailability of ebastine and significantly reduced histamine-induced skin reactions. From these results, the disposition of ebastine and carebastine seems to be significantly altered by coadministration of itraconazole or rifampin. The antihistamine response after ebastine dosing would be decreased following rifampin pretreatments.

Biotechnological synthesis of the designer drug metabolite 4'-hydroxymethyl-alpha-pyrrolidinohexanophenone in fission yeast heterologously expressing human cytochrome P450 2D6--a versatile alternative to multistep chemical synthesis.

1-(4-Methylphenyl)-2-pyrrolidin-1-ylhexan-1-one (4'-methyl-alpha-pyrrolidinohexanophenone, MPHP) is a new designer drug that appeared on the illicit drug market. It is mainly metabolized to 4'-hydroxymethyl-alpha-pyrrolidinohexanophenone (HO-MPHP) followed by oxidation to the respective carboxylic acid. For studies on the quantitative involvement of human cytochrome P450 (CYP) isoenzymes in the initial hydroxylation, a reference standard of HO-MPHP was needed. Therefore, the aim of this study was to synthesize this metabolite using a biotechnological approach. MPHP.HNO(3) (250 micromol) was incubated with 1 L culture of the fission yeast (Schizosaccharomyces pombe) strain CAD64 heterologously co-expressing human CYP reductase and CYP2D6. After centrifugation, the product was isolated from the incubation supernatants by solid-phase extraction. Further product cleanup was achieved by semi-preparative high-performance liquid chromatography (HPLC). After extraction of HO-MPHP from the respective eluent fractions, it was precipitated as its hydrochloric salt. The final product HO-MPHP.HCl was obtained in a yield of 138 micromol (43 mg, 55%). Its identity was confirmed by full scan gas chromatography-mass spectrometry (after trimethylsilylation), (1)H-NMR, and (13)C-NMR. The product purity as estimated from HPLC-ultraviolet analysis was greater than 99%. The described biotechnological approach proved to be a versatile alternative to the chemical synthesis of HO-MPHP.

An unambiguous assay for the cloned human sigma1 receptor reveals high affinity interactions with dopamine D4 receptor selective compounds and a distinct structure-affinity relationship for butyrophenones.

The ability of the sigma(1) receptor to interact with a huge range of drug structural classes coupled with its wide distribution in the body has contributed to it being implicated as a possible therapeutic target for a broad array of disorders ranging from substance abuse to depression to Alzheimer's disease. Surprisingly, the reported affinity values for some sigma(1) receptor ligands vary more than 50-fold. The potential of the sigma(1) receptor as a pharmacotherapeutic target prompted us to develop an unambiguous assay system for measuring the affinity of ligands to the cloned human sigma(1) receptor. In the course of characterizing this system and determining the true affinity values for almost three dozen compounds, it was discovered that some dopamine D(4) receptor selective compounds bind sigma(1) receptors with high affinity. A systematic analysis of haloperidol-like compounds revealed a clear structure-affinity relationship amongst clinically relevant butyrophenones. The antidepressant fluvoxamine, the drug of abuse methamphetamine, and the neurosteroid progesterone were amongst the many ligands whose interactions with the sigma(1) receptor were confirmed with our screening assay.

Identification of cytochrome P450 enzymes involved in the metabolism of the new designer drug 4'-methyl-alpha-pyrrolidinobutyrophenone.

The involvement of human hepatic cytochrome P450 (P450) isoenzymes in the metabolism of the new designer drug 4'-methyl-alpha-pyrrolidinobutyrophenone (MPBP) to 4'-(hydroxymethyl)-alpha-pyrrolidinobutyrophenone (HO-MPBP) was studied using insect cell microsomes with cDNA-expressed human P450s and human liver microsomes (HLM). Incubation samples were analyzed by liquid chromatography-mass spectrometry. Only CYP2D6, CYP2C19, and CYP1A2 were capable of catalyzing MPBP 4'-hydroxylation. According to the relative activity factor approach, these enzymes accounted for 54, 30, and 16% of net clearance. At 1 microM MPBP, the chemical inhibitors quinidine (CYP2D6), fluconazole (CYP2C19), and alpha-naphthoflavone (CYP1A2) reduced metabolite formation in pooled HLM by 83, 53, and 47%, respectively, and at 50 microM MPBP by 41, 47, and 45%, respectively. In experiments with HLM from CYP2D6 and CYP2C19 poor metabolizers, HO-MPBP formation was found to be 78 and 79% lower in comparison with pooled HLM, respectively. From these data, it can be concluded that polymorphically expressed CYP2D6 is mainly responsible for MPBP hydroxylation.

Biotechnological synthesis of drug metabolites using human cytochrome P450 2D6 heterologously expressed in fission yeast exemplified for the designer drug metabolite 4'-hydroxymethyl-alpha-pyrrolidinobutyrophenone.

The aim of this study was evaluating the principle feasibility of biotechnological synthesis of drug metabolites using heterologously expressed human cytochrome P450 (CYP) enzymes. Human CYP2D6 expressed in fission yeast (Schizosaccharomyces pombe) strain CAD58 was used as model enzyme and the designer drug 4'-methyl-alpha-pyrrolidinobutyrophenone (MPBP) as model drug. For synthesis of 4'-hydroxmethyl-alpha-pyrrolidinobutyrophenone (HO-MPBP), 250 micromol of MPBP.HNO(3) were incubated with one litre of CAD58 culture (10(8)cells/mL, pH 9, 48 h, 30 degrees C). HO-MPBP was isolated by liquid-liquid extraction and precipitated as its hydrochloride salt. Identity and purity of the product were tested by HPLC with ultraviolet (UV) detection, GC-MS, and (1)H-NMR. CAD58 was further characterized regarding the influence of incubation pH (5-10), cell density (10(7)-10(8)cells/mL), and incubation time (0-120 h) on metabolite formation using the substrates dextromethorphan and MPBP. The preparative experiment yielded 40 mg (141mumol) of HO-MPBP.HCl with a purity of >98%. In the characterization experiments, the metabolite formation rate peaked at pH 8. A linear relationship was observed between cell density and metabolite formation (R(2)>0.996). The rate of metabolite formation was slower in the earlier stages of incubation but then increased. For HO-MPBP, it became constant in the time interval of 2.5-34 h (R(2)>998).

Multistructure 3D-QSAR studies on a series of conformationally constrained butyrophenones docked into a new homology model of the 5-HT2A receptor.

The present study is part of a long-term research project aiming to gain insight into the mechanism of action of atypical antipsychotics. Here we describe a 3D-QSAR study carried out on a series of butyrophenones with affinity for the serotonin-2A receptor, aligned by docking into the binding site of a receptor model. The series studied has two peculiarities: (i) all the compounds have a chiral center and can be represented by two enantiomeric structures, and (ii) many of the structures can bind the receptor in two alternative orientations, posing the problem of how to select a single representative structure for every compound. We have used an original solution consisting of the simultaneous use of multiple structures, representing different configurations, binding conformations, and positions. The final model showed good statistical quality (n = 426, r2 = 0.84, q2LOO = 0.81) and its interpretation provided useful information, not obtainable from the simple inspection of the ligand-receptor complexes.

The effect of CYP2J2, CYP3A4, CYP3A5 and the MDR1 polymorphisms and gender on the urinary excretion of the metabolites of the H-receptor antihistamine ebastine: a pilot study.

AIMS: To determine the effect of gender and the genetic polymorphisms of CYP2J2, CYP3A4, CYP3A5 and MDR1 on the urinary excretion of the H(1) antihistamine ebastine in healthy subjects. METHODS: Eighty-nine Caucasians were studied. The presence of polymorphisms in genes known to be involved in ebastine metabolism and transport (CYP2J2*2,*3,*4,*6,*7, CYP3A4*1B, CYP3A5*3, *6 and MDR1(ABCB1)(C3435T)) was assessed by means of PCR-restriction fragment length polymorphism and sequencing methods. Genotype was correlated with the urinary excretion of the main ebastine metabolites (desalkylebastine and carebastine) under basal conditions and after administration of grapefruit juice. RESULTS: Women excreted statistically greater amounts of desalkylebastine in urine (mean +/- SD (95% confidence intervals, 95% CI), 23.0 +/- 19.5 (18.1, 27.9) micromol) than men (12.4 +/- 11.0 (7.9, 16.9)), (mean difference: 10.6 (2.4, 18.7), P < 0.005). The CYP2J2, CYP3A4 and CYP3A5 analysed polymorphisms did not greatly affect ebastine metabolite excretion. The MDR1(C3435T) polymorphism was found to affect both the urinary excretion of the active metabolite carebastine (32.3 +/- 18.3 (23.1, 41.4), 22.8 +/- 14.7 (18.6, 27.0) and 21.5 +/- 15.3 (14.7, 28.3) for CC, CT and TT carriers, respectively; P < 0.05) and the grapefruit juice-induced inhibition of its transport/formation (mean fold-decrease +/- SD (95% CI), 1.5 +/- 0.8 (1.0, 2.0), 1.1 +/- 0.9 (0.7, 1.4) and 0.9 +/- 0.4 (0.6, 1.2) for CC, CT and TT carriers, respectively; P = 0.01). CONCLUSIONS: Gender and the presence of the MDR1(C3435T) polymorphism both influence the excretion of ebastine metabolites in urine.

Studies on the metabolism and toxicological detection of the new designer drug 4'-methyl-alpha-pyrrolidinobutyrophenone (MPBP) in rat urine using gas chromatography-mass spectrometry.

The aim of the presented study was to identify the metabolites of the new designer drug 4'-methyl-alpha-pyrrolidinobutyrophenone (MPBP) in rat urine using GC-MS techniques. After enzymatic hydrolysis, extraction and various derivatizations, seven metabolites of MPBP could be identified suggesting the following metabolic steps: oxidation of the 4'-methyl group to the corresponding alcohol and further oxidation to the respective carboxy compound, hydroxylation of the pyrrolidine ring followed by dehydrogenation to the corresponding lactam or reduction of the keto group to the 1-dihydro compound. A previously published GC-MS-based screening procedure for pyrrolidinophenones involving enzymatic hydrolysis and mixed-mode solid-phase extraction of urine samples allowed detection of MPBP metabolites. Assuming similar metabolism and dosages in humans, an intake of MPBP should be detectable via its metabolites in urine.

Identification and functional characterization of novel CYP2J2 variants: G312R variant causes loss of enzyme catalytic activity.

CYP2J2 plays important roles in the metabolism of therapeutic drugs, such as astemizole and ebastine, as well as endogenous fatty acids. This study aimed to identify CYP2J2 genetic variants in Koreans and to characterize their functional consequences. From direct sequencing of the CYP2J2 gene, 12 genetic variations, including the two novel nonsynonymous mutations G312R and P351L, were identified from 93 Korean subjects. The two novel CYP2J2 variants were co-expressed with NADPH-cytochrome P450 reductase in Sf9 cells and their catalytic activities were quantified. The recombinant CYP2J2 G312R variant showed almost complete loss of enzymatic activity, as determined by CYP2J2-catalysed astemizole O-demethylation and ebastine hydroxylation. The CYP2J2 P351L variant showed enzymatic activities that were comparable with the wild-type CYP2J2. The reduced CO spectra of the recombinant CYP2J2 proteins suggested no CO binding to the heme in CYP2J2 G312R. In addition, molecular modelling of the three-dimensional structure consistently predicted that there might be spatial hindrance between heme and the bulky side chain of the R312 residue in CYP2J2 G312R variant. The CYP2J2 G312R variant was not found in 192 Chinese, 99 African-Americans, 100 Caucasians and 159 Vietnamese subjects. Two of the 192 Chinese subjects (0.52%) were heterozygous for CYP2J2 P351L. Twelve CYP2J2 variants, including two novel nonsynonymous variants, were identified in a Korean population. The G312R variant is the first nonfunctional CYP2J2 allele to be identified, and is expected to influence the disposition of its substrate therapeutics, as well as endogenous compounds.