Temporal and electroencephalography dynamics of surreal marketing.

Event-related spectral perturbation analysis was employed in this study to explore whether surreal image designs containing metaphors could influence product marketing effects, including consumers' product curiosity, product comprehension, product preference, and purchase intention. A total of 30 healthy participants aged 21-30 years were recruited. Neurophysiological findings revealed that lower gamma, beta, and theta spectral powers were evoked in the right insula (Brodmann Area 13) by surreal marketing images. This was associated, behaviorally, with the manifestation of higher product curiosity and purchase intention. Based on previous research, the brain functions of this area include novelty, puzzle-solving, and cravings for reward caused by cognitive overload.

Sex differences in high-level appreciation of automobile design-evoked gamma broadband synchronization.

The present study was conducted to provide neuroimaging correlates for neurodesign of automobile for marketing aesthetics, using event-related spectral perturbations (ERSPs) and participant reports. Thirty men and women aged 22-27 years were presented with various 3-dimensional automobile modelling shapes (rectangular, streamlined, and round), which were cross-matched with various interior colour tones (pure hue/vivid, light, and dark tones) in the experimental conditions, i.e., rectangular exterior with a vivid tone interior. The stimuli pairs were to be rated by participants to facilitate our understanding of the emotional dimensions of automotive design qualities. Significant differences were observed in the high gamma band of 80-100 Hz in the left temporal area between the two sexes. Men elicited a stronger high gamma band signals for dark colour tone interiors and rectangular or round automobile modelling designs because of the meaningful and comprehensible signals associated with the mechanisms of working memory. In contrast, women had fewer reactions than men, and elicited higher beta-band dynamics in the anterior cingulate cortex for rectangular automobile modelling design, and higher gamma-band dynamics for light colour tone interiors, which might relate to their higher self-awareness of positive emotional reward.

Posterior cingulate cortex can be a regulatory modulator of the default mode network in task-negative state.

In recent years, the regulation of brain networks and interactions between different brain regions have become important issues in neuroscience. Effective connectivity can be employed to understand the modulatory mechanisms of brain networks. Previous studies have used the task-positive mode to examine effective connectivity between brain regions and very few studies have considered the task-negative mode to explore effective connectivity using electroencephalography (EEG). In the present study, high-density EEG experiments were conducted in 85 participants to measure EEG effective connectivity in relevant default mode network (DMN) brain regions (i.e., the medial prefrontal cortex [mPFC], posterior cingulate cortex [PCC], precuneus, and right frontal and left occipital regions) to observe the effects of different task-negative modes (eyes-open/eyes-closed state) and personality traits (introversion/extroversion). The results showed that in the eyes-closed state, the PCC had significantly increased effective connectivity and played a prominent role as a regulatory modulator of outflow to other regions mediated by alpha rhythms. The mPFC was a regulatory modulator of outflow in the eyes-open state mediated by delta rhythms. The introvert group showed stronger co-modulations in the relevant DMN regions than the extrovert group.

Temporal and spectral EEG dynamics can be indicators of stealth placement.

Stealth placement marketing, where consumers are unaware that they are being marketed to, attempts to reduce the audiences' resistance to traditional persuasive advertising. It is a form of advertising that involves targeted exposure of brands or products incorporated in other works, usually with or without explicit reference to the brands or products. Brand placement can be presented in different visual and auditory forms in video programs. The present study proposed that different 'representations' (i.e., representable or non-representable) and 'sounds' (i.e., speech or musical sound) of brand placement can affect the viewers' perception of the brand. Event-related potential results indicated significant differences in P1, N1, P2, N270, and P3. Further, event-related spectral perturbation results indicated significant differences in theta, alpha, beta, and gamma (30-100 Hz), in the right parietal, right occipital area, and limbic lobe. 'Non-representable' or 'speech sound' brand placement induced significant temporal and spectral EEG dynamics in viewers.

Right Fronto-Temporal EEG can Differentiate the Affective Responses to Award-Winning Advertisements.

Affective engineering aims to improve service/product design by translating the customer's psychological feelings. Award-winning advertisements (AAs) were selected on the basis of the professional standards that consider creativity as a prerequisite. However, it is unknown if AA is related to satisfactory advertising performance among customers or only to the experts' viewpoints towards the advertisements. This issue in the field of affective engineering and design merits in-depth evaluation. We recruited 30 subjects and performed an electroencephalography (EEG) experiment while watching AAs and non-AAs (NAAs). The event-related potential (ERP) data showed that AAs evoked larger positive potentials 250-1400 [Formula: see text]ms after stimulus onset, particularly in the right fronto-temporal regions. The behavioral results were consistent with the professional recognition given to AAs by experts. The perceived levels of creativity and "product-like" quality were higher for the AAs than for the NAAs. Event-related spectral perturbation (ERSP) analysis further revealed statistically significant differences in the theta, alpha, beta, and gamma band activity in the right fronto-temporal regions between the AAs and NAAs. Our results confirm that EEG features from the time/frequency domains can differentiate affective responses to AAs at a neural circuit level, and provide scientific evidence to support the identification of AAs.

Humor drawings evoked temporal and spectral EEG processes.

The study aimed to explore the humor processing elicited through the manipulation of artistic drawings. Using the Comprehension-Elaboration Theory of humor as the main research background, the experiment manipulated the head portraits of celebrities based on the independent variables of facial deformation (large/small) and addition of affective features (positive/negative). A 64-channel electroencephalography was recorded in 30 participants while viewing the incongruous drawings of celebrities. The electroencephalography temporal and spectral responses were measured during the three stages of humor which included incongruity detection, incongruity comprehension and elaboration of humor. Analysis of event-related potentials indicated that for humorous vs non-humorous drawings, facial deformation and the addition of affective features significantly affected the degree of humor elicited, specifically: large > small deformation; negative > positive affective features. The N170, N270, N400, N600-800 and N900-1200 components showed significant differences, particularly in the right prefrontal and frontal regions. Analysis of event-related spectral perturbation showed significant differences in the theta band evoked in the anterior cingulate cortex, parietal region and posterior cingulate cortex; and in the alpha and beta bands in the motor areas. These regions are involved in emotional processing, memory retrieval, and laughter and feelings of amusement induced by elaboration of the situation.

EEG Spectral Dynamics of Video Commercials: Impact of the Narrative on the Branding Product Preference.

Neuromarketing has become popular and received a lot of attention. The quality of video commercials and the product information they convey to consumers is a hotly debated topic among advertising agencies and product advertisers. This study explored the impact of advertising narrative and the frequency of branding product exposures on the preference for the commercial and the branding product. We performed electroencephalography (EEG) experiments on 30 subjects while they watched video commercials. The behavioral data indicated that commercials with a structured narrative and containing multiple exposures of the branding products had a positive impact on the preference for the commercial and the branding product. The EEG spectral dynamics showed that the narratives of video commercials resulted in higher theta power of the left frontal, bilateral occipital region, and higher gamma power of the limbic system. The narratives also induced significant cognitive integration-related beta and gamma power of the bilateral temporal regions and the parietal region. It is worth noting that the video commercials with a single exposure of the branding products would be indicators of attention. These new findings suggest that the presence of a narrative structure in video commercials has a critical impact on the preference for branding products.

The impact of parameters of store illumination on food shopper response.

Customer behavior in sales areas is strongly influenced by the perception of surroundings and feelings of well-being. By using dynamic retail solutions of basic, accent and dramatic lighting it is possible to attract attention, create a unique in-store environment and give customers a reason to stay and return to the store. The simplest and also the most successful method to reach customer attention in food selection (buying) process is through eye-catchingly illuminated visuals of products. Visual senses has evolved to top ranks in the sensory hierarchy, therefore visual stimuli have a tendency to overcome all other senses. The paper deals with a comprehensive interdisciplinary research of the influence of light and color on the emotional state of consumers (valence) on the food market. It integrates the measurement of light intensity, color temperature or emitted color spectrum in grocery stores, recognition of emotional response and the time of its occurrence among respondents due to different lighting types and color in simulated laboratory conditions. The research is focused on accent lighting in the segment of fresh unpackaged food. Using a mobile 16-channel electroencephalograph (EEG equipment) from EPOC company and a mini camera we observed the response time and the emotional status (valence), in order to reveal true consumer preferences in different lighting conditions (color temperature and color rendering index) and their non-traditional color (yellow, purple, red, blue and green) for the selected food type. The paper suggests possibilities for rational combination of the effective, efficient and energy-saving accent lighting, by which the retailer can achieve not only an eye-catching and attractive presentation of merchandised products, but also significant savings within operating their stores.

The conduct of in vitro studies to address time-dependent inhibition of drug-metabolizing enzymes: a perspective of the pharmaceutical research and manufacturers of America.

Time-dependent inhibition (TDI) of cytochrome P450 (P450) enzymes caused by new molecular entities (NMEs) is of concern because such compounds can be responsible for clinically relevant drug-drug interactions (DDI). Although the biochemistry underlying mechanism-based inactivation (MBI) of P450 enzymes has been generally understood for several years, significant advances have been made only in the past few years regarding how in vitro time-dependent inhibition data can be used to understand and predict clinical DDI. In this article, a team of scientists from 16 pharmaceutical research organizations that are member companies of the Pharmaceutical Research and Manufacturers of America offer a discussion of the phenomenon of TDI with emphasis on the laboratory methods used in its measurement. Results of an anonymous survey regarding pharmaceutical industry practices and strategies around TDI are reported. Specific topics that still possess a high degree of uncertainty are raised, such as parameter estimates needed to make predictions of DDI magnitude from in vitro inactivation parameters. A description of follow-up mechanistic experiments that can be done to characterize TDI are described. A consensus recommendation regarding common practices to address TDI is included, the salient points of which include the use of a tiered approach wherein abbreviated assays are first used to determine whether NMEs demonstrate TDI or not, followed by more thorough inactivation studies for those that do to define the parameters needed for prediction of DDI.

Assessment of the CYP3A-mediated drug interaction potential of anacetrapib, a potent cholesteryl ester transfer protein (CETP) inhibitor, in healthy volunteers.

In this study, midazolam was used as a probe-sensitive CYP3A substrate to investigate the effect of anacetrapib on CYP3A activity, and ketoconazole was used as a probe-inhibitor to investigate the effect of potent CYP3A inhibition on the pharmacokinetics of anacetrapib, a novel cholesteryl ester transfer protein inhibitor in development for the treatment of dyslipidemia. Two partially blinded, randomized, 2-period, fixed-sequence studies were performed. Safety, tolerability, and midazolam and anacetrapib plasma concentrations were assessed. All treatments were generally well tolerated. The geometric mean ratios (90% confidence interval) of midazolam with anacetrapib/midazolam alone for AUC0-infinity and Cmax were 1.04 (0.94, 1.14) and 1.15 (0.97, 1.37), respectively. Exposure to anacetrapib was increased by ketoconazole--specifically, the geometric mean ratios (90% confidence interval) of anacetrapib with ketoconazole/anacetrapib alone for AUC0-infinity and Cmax were 4.58 (3.68, 5.71) and 2.37 (2.02, 2.78), respectively. The study showed that anacetrapib does not inhibit or induce CYP3A activity. Furthermore, anacetrapib appears to be a moderately sensitive substrate of CYP3A.

Characterization of mice lacking the multidrug resistance protein MRP2 (ABCC2).

The multidrug resistance protein Mrp2 is an ATP-binding cassette (ABC) transporter mainly expressed in liver, kidney, and intestine. One of the physiological roles of Mrp2 is to transport bilirubin glucuronides from the liver into the bile. Current in vivo models to study Mrp2 are the transporter-deficient and Eisai hyperbilirubinemic rat strains. Previous reports showed hyperbilirubinemia and induction of Mrp3 in the hepatocyte sinusoidal membrane in the mutant rats. In addition, differences in liver cytochrome P450 and UGT1a levels between wild-type and mutant rats were detected. To study whether these compensatory mechanisms were specific to rats, we characterized Mrp2(-/-) mice. Functional absence of Mrp2 in the knockout mice was demonstrated by showing increased levels of bilirubin and bilirubin glucuronides in serum and urine, a reduction in biliary excretion of bilirubin glucuronides and total glutathione, and a reduction in the biliary excretion of the Mrp2 substrate dibromosulfophthalein. To identify possible compensatory mechanisms in Mrp2(-/-) mice, the expression levels of 98 phase I, phase II, and transporter genes were compared in liver, kidney, and intestine of male and female Mrp2(-/-) and control mice. Unlike in Mrp2 mutant rats, no induction of Mrp3 in Mrp2(-/-) mice was detected. However, Mrp4 mRNA and protein in liver and kidney were increased approximately 6- and 2-fold, respectively. Phenotypic analysis of major cytochrome P450-mediated activities in liver microsomes did not show differences between wild-type and Mrp2(-/-) mice. In conclusion, Mrp2(-/-) mice are a new valuable tool to study the role of Mrp2 in drug disposition.

Evidence for the bioactivation of zomepirac and tolmetin by an oxidative pathway: identification of glutathione adducts in vitro in human liver microsomes and in vivo in rats.

Although zomepirac (ZP) and tolmetin (TM) induce anaphylactic reactions and form reactive acyl glucuronides, a direct link between the two events remains obscure. We report herein that, in addition to acyl glucuronidation, both drugs are subject to oxidative bioactivation. Following incubations of ZP with human liver microsomes fortified with NADPH and glutathione (GSH), a metabolite with an MH+ ion at m/z 597 was detected by LC/MS/MS. On the basis of collision-induced dissociation and NMR evidence, the structure of this metabolite was determined to be 5-[4'-chlorobenzoyl]-1,4-dimethyl-3-glutathionylpyrrole-2-acetic acid (ZP-SG), suggesting that the pyrrole moiety of ZP had undergone oxidation to an epoxide intermediate, followed by addition of GSH and loss of the elements of H2O to yield the observed conjugate. The oxidative bioactivation of ZP most likely is catalyzed by cytochrome P450 (P450) 3A4, since the formation of ZP-SG was reduced to approximately 10% of control values following pretreatment of human liver microsomes with ketoconazole or with an inhibitory anti-P450 3A4 IgG. A similar GSH adduct, namely 5-[4'-methylbenzoyl]-1-methyl-3-glutathionylpyrrole-2-acetic acid (TM-SG), was identified when TM was incubated with human liver microsomal preparations. The relevance of these in vitro findings to the in vivo situation was established through the detection of the same thiol adducts in rats treated with ZP and TM, respectively. Taken together, these data suggest that, in addition to the formation of acyl glucuronides, oxidative metabolism of ZP and TM affords reactive species that may haptenize proteins and thereby contribute to the drug-mediated anaphylactic reactions.

Utility of recombinant cytochrome p450 enzymes: a drug metabolism perspective.

An important role of human cytochrome P450s (P450s) has been well recognized in the area of drug metabolism and pharmacokinetics. It has become possible in recent years to express catalytically active forms of these enzymes in various host systems. The resulting recombinant human P450s are either purified for studies of protein structure and the mechanism of catalysis or isolated in microsomal forms to serve the purposes of P450 phenotyping, metabolic stability screening and inhibitory potential evaluation. Intact mammalian cells expressing human enzymes may also be used to test the mutagenic and toxicity potential of drug candidates. The issue remains, however, that the data derived from recombinant P450s are not always consistent with those generated from human tissue preparations. The aim of this communication is to discuss applications of recombinant P450s in the drug discovery and development setting, with an emphasis on comparison of recombinant and human liver microsomal systems.

Determination of phase I metabolic enzyme activities in liver microsomes of Mrp2 deficient TR- and EHBR rats.

The canalicular multispecific organic anion transporter/multidrug resistance protein 2 (cMOAT/Mrp2) plays a major role in the transport of anionic xenobiotics across the bile canalicular membrane. Transport deficient rats (TR-) and Eisai-hyperbilirubinemic rats (EHBR), defective in Mrp2, are mutants of Wistar and Sprague Dawley (SD) rats, respectively. In this study, Phase I metabolic enzyme activities in liver microsomes prepared from these mutant male and female rats were compared to their corresponding non-mutant rats. The total cytochrome P450 contents and NADPH-cytochrome P450 reductase activity in male and female TR- rats were significantly higher than in Wistar rats. In male TR- rats, ethoxyresorufin O-deethylation (EROD), pentoxyresorufin O-deethylation (PROD), testosterone 2alpha, 7alpha and 16 alpha-hydroxylase activities were higher, but testosterone 6beta-hydroxylase activity and the rate of androstenedione formation were lower than in Wistar rats. Female TR- rats had higher 7alpha-hydroxylase activity, but EROD activity was lower in female Wistar rats. Similar studies conducted in EHBR versus SD rats demonstrated increased total cytochrome P450 content in male and female EHBR rats; NADPH-cytochrome P450 reductase activity was not significantly affected. Decreased PROD activity and the rate of androstenedione formation were observed in male and female EHBR rats. Furthermore, testosterone 6beta-hydroxylase activity was lower in male EHBR rats than in male SD rats while testosterone 7alpha-hydroxylase activity was significantly higher in male and female EHBR rats. Thus, in addition to Mrp2 deficiency, differential expression of CYP isoforms and their potential impact on the metabolism and pharmacokinetics of compounds should be considered when interpreting data from these rat strains.

Coimmunoprecipitation of UDP-glucuronosyltransferase isoforms and cytochrome P450 3A4.

Coimmunoprecipitation was used to investigate protein-protein interactions between several UDP-glucuronosyltransferase (UGT) isoforms and cytochrome P450 3A4. Solubilized human liver microsomes were incubated with specific antibodies to UGT2B7, UGT1A6, UGT1A1, and CYP3A4, and the immunoprecipitates were run on SDS-polyacrylamide gel electrophoresis. Western blots showed that UGT2B7, UGT1A6, UGT1A1, and CYP3A4 were successfully immunoprecipitated with the specific antibodies for each enzyme. Upon immunoprecipitating UGT2B7, the corresponding immunoblot showed that UGT1A6, UGT1A1, and CYP3A4 were immunoprecipitated. Similar studies found that different UGT isoforms or CYP3A4 immunoprecipitated along with the original immunoprecipitating enzyme. These data suggest that UGT isoforms may form complexes (dimers, tetramers, etc.) with each other in the endoplasmic reticulum and nuclear envelope. In addition, the UGT isoforms tested here may have interacted with CYP3A4 in the endoplasmic reticulum, suggesting that these enzymes may cooperate in the excretion of compounds in a multistep metabolic process.

Cytochrome P450 3A4 is the major enzyme involved in the metabolism of the substance P receptor antagonist aprepitant.

The contribution of human cytochrome P450 (P450) isoforms to the metabolism of aprepitant in humans was investigated using recombinant P450s and inhibition studies. In addition, aprepitant was evaluated as an inhibitor of human P450s. Metabolism of aprepitant by microsomes prepared from baculovirus-expressed human P450s was observed only when CYP1A2, CYP2C19, or CYP3A4 was present in the expression system. Incubation with CYP1A2 and CYP2C19 yielded only products of O-dealkylation, whereas CYP3A4 catalyzed both N- and O-dealkylation reactions. The metabolism of aprepitant by human liver microsomes was inhibited completely by ketoconazole or troleandomycin. No inhibition was observed with other P450 isoform-selective inhibitors. Aprepitant was evaluated also as a P450 inhibitor in human liver microsomes. No significant inhibition of CYP1A2, CYP2B6, CYP2C8, CYP2D6, and CYP2E1 was observed in experiments with isoform-specific substrates (IC50 > 70 microM). Aprepitant was a moderate inhibitor of CYP3A4, with Ki values of approximately 10 microM for the 1'- and 4-hydroxylation of midazolam, and the N-demethylation of diltiazem, respectively. Aprepitant was a very weak inhibitor of CYP2C9 and CYP2C19, with Ki values of 108 and 66 microM for the 7-hydroxylation of warfarin and the 4'-hydroxylation of S-mephenytoin, respectively. Collectively, these results indicated that aprepitant is both a substrate and a moderate inhibitor of CYP3A4.

Validation of (-)-N-3-benzyl-phenobarbital as a selective inhibitor of CYP2C19 in human liver microsomes.

(-)-N-3-Benzyl-phenobarbital (NBPB) was reported to be a potent and selective inhibitor of CYP2C19. To validate the selectivity of NBPB toward CYP2C19 in human liver microsomes, the inhibitory effects on major cytochrome P450 isoform-specific reactions were evaluated in the present study. In human liver microsomes, NBPB showed potent competitive inhibition on CYP2C19-mediated S-mephenytoin 4'-hydroxylation with an IC(50) value of 0.25 microM and K(i) value of 0.12 microM, whereas weak inhibition was observed for CYP1A2-, CYP2A6-, CYP2B6-, CYP2C8-, CYP2C9-, CYP2D6-, and CYP3A4-mediated reactions with IC(50) values >100, >100, 62, 34, 19, >100, and 89 microM, respectively. Importantly, its selectivity toward CYP2C19 among the CYP2C subfamily was demonstrated. Therefore, NBPB can be used as a potent and selective inhibitor to establish the relative contribution of CYP2C19 for in vitro reaction phenotyping studies. This compound can also serve as a positive control inhibitor of CYP2C19 for routine screening of P450 reversible inhibition when human liver microsomes are used as the enzyme source.

Cytochrome P450 in vitro reaction phenotyping: a re-evaluation of approaches used for P450 isoform identification.

Marker substrates, chemical inhibitors, and inhibitory antibodies are important tools for the identification of cytochrome P450 (P450) isoform responsible for the metabolism of therapeutic agents in vitro. In view of the versatile and nonspecific nature of P450 enzymes, many of the marker substrates and chemical inhibitors used for P450 in vitro reaction phenotyping are isoform selective but not specific. Recently, the use of marker substrate and chemical inhibitors in CYP2D6 in vitro reaction phenotyping was questioned by Granvil et al. (2002). In comparison of a panel of 15 recombinant P450 enzymes, they found that in addition to CYP2D6, CYP1A1 is also capable of catalyzing the formation of 4-hydroxylated metabolite of debrisoquine and that the intrinsic clearance of debrisoquine by CYP2D6-mediated 4-hydroxylation is only about twice that by CYP1A1. In their study, they have also demonstrated that quinidine inhibits both CYP2D6- and CYP1A1-mediated debrisoquine 4-hydroxylation. In view of these important findings, we have reevaluated various approaches used to identify P450 isoform(s) responsible for the metabolism of therapeutic agents. While acknowledging the value of inhibitory antibodies in P450-phenotyping studies, it is our opinion that in well conducted in vitro experiments, isoform-selective chemical inhibitors can also provide valuable and reliable information. Hopefully, future efforts may produce even better P450 isoform-selective marker substrates and inhibitors.

Pharmacokinetics and interactions of a novel antagonist of chemokine receptor 5 (CCR5) with ritonavir in rats and monkeys: role of CYP3A and P-glycoprotein.

The mechanisms of pharmacokinetic interactions of a novel anti-human immunodeficiency virus (anti-HIV-1) antagonist of chemokine receptor 5 (CCR5) [2-(R)-[N-methyl-N-(1-(R)-3-(S)-((4-(3-benzyl-1-ethyl-(1H)-pyrazol-5-yl)piperidin-1-yl)methyl)-4-(S)-(3-fluorophenyl)cyclopent-1-yl)amino]-3-methylbutanoic acid (MRK-1)] with ritonavir were evaluated in rats and monkeys. MRK-1 was a good substrate for the human (MDR1) and mouse (Mdr1a) multidrug resistance protein transporters and was metabolized by CYP3A isozymes in rat, monkey, and human liver microsomes. Both the in vitro MDR1-mediated transport and oxidative metabolism of MRK-1 were inhibited by ritonavir. Although the systemic pharmacokinetics of MRK-1 in rats and monkeys were linear, the oral bioavailability increased with an increase in dose from 2 to 10 mg/kg. The area under the plasma concentration-time curve (AUC) of MRK-1 was increased 4- to 6-fold when a 2 or 10 mg/kg dose was orally coadministered with 10 mg/kg ritonavir. Further pharmacokinetic studies in rats indicated that P-glycoprotein (P-gp) inhibition by ritonavir increased the intestinal absorption of 2 mg/kg MRK-1 maximally by approximately 30 to 40%, and a major component of the interaction likely resulted from its reduced systemic clearance via the inhibition of CYP3A isozymes. Oral coadministration of quinidine (10 and 30 mg/kg) increased both the extent and the first-order rate of absorption of MRK-1 (2 mg/kg) by approximately 40 to 50% and approximately 100 to 300%, respectively, in rats, thus further substantiating the role of P-gp in modulating the intestinal absorption of MRK-1 in this species. At the 10 mg/kg MRK-1 dose, however, the entire increase in its AUC upon coadministration with ritonavir or quinidine could be attributed to a reduced systemic clearance, and no effects on intestinal absorption were apparent. In contrast to rats, the effects of P-gp in determining the intestinal absorption of MRK-1 appeared less significant in rhesus monkeys at either dose.

Cytochrome P450 3A4-mediated bioactivation of raloxifene: irreversible enzyme inhibition and thiol adduct formation.

Raloxifene is a selective estrogen receptor modulator which is effective in the treatment of osteoporosis in postmenopausal women. We report herein that cytochrome P450 (P450)3A4 is inhibited by raloxifene in human liver microsomal incubations. The nature of the inhibition was irreversible and was NADPH- and preincubation time-dependent, with K(I) and k(inact) values estimated at 9.9 microM and 0.16 min(-1), respectively. The observed loss of P450 3A4 activity was attenuated partially by glutathione (GSH), implying the involvement of a reactive metabolite(s) in the inactivation process. Subsequently, GSH adducts of raloxifene were identified in incubations with human liver microsomes; substitution with GSH occurred at the 5- or 7-position of the benzothiophene moiety or at the 3'-position of the phenol ring, with the 7-glutathionyl derivative being most abundant based on LC/MS and NMR analyses. These adducts are postulated to derive from addition of GSH to raloxifene arene oxides followed by dehydration and aromatization. Alternatively, raloxifene may be oxidized to an extended quinone intermediate, which then is trapped by GSH conjugation. The bioactivation of raloxifene most likely is catalyzed by P450 3A4, since the formation of GSH adducts was almost abolished when liver microsomes were pretreated with ketoconazole or with an inhibitory anti-P450 3A4 IgG. The GSH adducts also were detected in incubations of raloxifene with rat or human hepatocytes, while the corresponding N-acetylcysteine adducts were identified in the bile and urine from rats treated orally with the drug at 5 mg/kg. Taken together, these data indicate that P450 3A4-mediated bioactivation of raloxifene in vitro is accompanied by loss of enzyme activity. The significance of these findings with respect to the clinical use of raloxifene remains to be determined.