The metabolic fate of adrenocorticotropic hormone (ACTH) fragment 4–10 (4–10) was evaluated following incorporation of a nonradioactive 127I-tag and with selective detection of I+ at m/z 127 by inductively coupled plasma mass spectrometry (ICP-MS). 127I has all the advantages of radioactive 125I as a metabolite tracer and, together with its detection in the femtogram range, has led to a successful metabolite profiling of 127I-ACTH (4–10) in vitro. The observed metabolic stability of this peptide in tissue preparations from human was plasma > kidney S9 > liver microsomes > liver cytosol, liver S9. Metabolic turnover of 127I-ACTH (4–10) was not NADPH-dependent and, together with inhibition by protease inhibitor cocktail and EDTA, is consistent with metabolism exclusively by proteases. Our preliminary studies using chemical inhibitors suggested the involvement of metalloprotease, serine peptidase, and aminopeptidase in 127I-ACTH (4–10) metabolism. The liver is the primary site of metabolic clearance of 127I-ACTH (4–10), with kidney S9 taking four times longer to produce a metabolite profile comparable to that produced by liver S9. A total of six metabolites retaining the 127I-tag was detected by ICP-MS, and their structures were elucidated using a LTQ/Orbitrap. 127I-ACTH (4–10) underwent both N- and C-terminal proteolysis to produce 127I-Phe as the major metabolite. The 127I-tag had minimal effect on the metabolic turnover and site of proteolysis of ACTH (4–10), which, together with ICP-MS providing essentially equimolar responses, suggests that the use of a 127I-tag may have general utility as an alternative to radioiodination to investigate the metabolism of peptide therapeutics

Evaluative amplification refers to the tendency for people to rate positive minorities more positively than non-minorities, but at the same time rate negative minorities more negatively than non-minorities. Past studies of evaluative amplification in the impressions of AfricanAmericans and women have focused on ratings of overall favorability. The present study examined differential amplification across personality domains by incorporating race and gender labels into the classic impression-formation paradigm of Solomon Asch (1946). The results showed that Conscientiousness was amplified in ratings of African-American targets and Extraversion was amplified in ratings of female targets. These findings suggest that evaluative amplification may be restricted to certain personality domains that depend on the demographic group being evaluated. As such, race and gender appear to operate as peripheral, rather than central traits. Implications of the results for the major theories of evaluative amplification are discussed. [96] --------------[97 ] INTRODUCTION Ample research indicates that perceptions of African-Americans and women have become increasingly favorable since the Civil Rights movement (Dovidio & Gaertner, 1986; Schuman, Steeh, & Bobo, 1985). Indeed, many studies today find that Whites often evaluate Black targets who show socially desirable or highly competent behaviors more favorably than they evaluate similar Whites (e.g., Dienstbier, 1970; Hass, Katz, Rizzo, Bailey, & Eisenstadt, 1991). Similarly, men are often found to rate competent women more favorably than competent men (Deaux & Farris, 1975). However, these same studies report that when Black or female targets show socially undesirable or incompetent behaviors, they are criticized more sharply than nonminorities. Thus, people are said to amplify both positive and negative evaluations of members of stigmatized groups. Such evaluative amplification has long been a focus of stereotyping research, although its scope and etiology are not yet fully understood (see Jackson, Sullivan & Hodge, 1993; Jussim, Coleman, & Lerch, 1987; Katz, 1981; Linville & Jones, 1980). From the earliest amplification studies (Gergen & Jones, 1963) to the more recent (e.g., Hart & Morry, 1997), interracial evaluations have been measured using trait-ratings of the targets' personalities. Raters in amplification studies are asked to judge the extent to which the targets appear "warmhearted," "easily-angered" (Gergen & Jones, 1963), "coarse," "persistent," "foresighted," "striving" (Feldman, 1972), "careful," "serious" (Weitz, 1972), "motivated," "unfriendly" (Linville, 1980), "hardworking," "considerate," and "dependable" (Bailey, 1991). Despite collecting detailed impressions of the targets' personalities in this way, researchers typically aggregate all of the trait-ratings into a single favorability index for analysis. This practice reflects a valence-oriented approach to understanding amplification, which focuses on perceptions of the "goodness" and "badness" of the targets. A number of important issues arise when we take a more content-oriented approach (see Peabody, 1990) to evaluative amplification. By disaggregating the overall favorability scores into separate personality domains for analysis, we may investigate whether certain personality domains are amplified, whereas others are not (see Hart & Morry, 1997). Such knowledge would help to delineate the scope of evaluative amplification, and it may also shed light on why it occurs. If amplification occurs across all personality domains, this would suggest that it arises from prejudices, defined as one's overall positive or negative attitudes toward a group (Katz, 1981). As some authors suggest, raters may amplify negative traits due to residual, old-fashioned prejudices, and at the same time, amplify positive traits in an effort to prove that they themselves are egalitarian (Dovidio & Gaertner, 1986; Katz & Hass, 1988). These motivations would lead people to exaggerate their ratings of minorities regardless of which personality domain they were evaluating. If, however, amplification occurs on some personality domains and not others, this would suggest that it arises from stereotypic beliefs that African-Americans and women possess certain personality traits to a greater or lesser degree than European-Americans and/or men. The Five Factor Model of personality (Costa & McCrae, 1992; Goldberg, 1990) may help guide a more content-oriented look at evaluative amplification. Using this model, we may explore differential amplification across the domains of Neuroticism (or Emotional Stability), Extraversion, Openness, Agreeableness, and Conscientiousness. These five factors have also been found in trait-ratings of acquaintances and strangers (Funder & Colvin, 1988; Watson, 1989), thus making them relevant to the study of interracial impressions. However, any attempt to analyze amplification across personality domains requires that we control what personality information is given about the targets more carefully than has been done in past research. For example, Dienstbier (1970) described the positive target as "a likable and socially successful high school junior who was college bound upon his graduation, and whose ideas were 'a lot like those of most people his age'" and the negative target as a "high school dropout who was a somewhat greedy, shy, and rebellious social misfit whose ideas were generally "too far out for most of the other people his age." (p. 201). These descriptions likely gave information on different personality domains for the positive and negative targets. To examine differential amplification across personality domains, the personality information about the targets would ideally be more symmetrical. The classic impression-formation paradigm of Solomon Asch (1946; Study 6) offers a way to achieve this symmetry. Recall that Asch found that impressions of others are strongly influenced by a primacy effect. Specifically, when the first few traits appearing in a list describing a target (what Asch called "S-traits" or "stimulus" traits) were positive, then the target was perceived more positively than when the first few traits were negative even if the two lists contained the identical traits and was merely reversed in its order. [97] --------------[98 ] By capitalizing on the primacy effect, we described positive and negative targets in the present study using the identical traits. To portray a target positively, the positive traits were listed first. To portray a target negatively, the negative traits were listed first. However, both targets were described with the same list of traits. To further manipulate the targets' group membership, race or gender labels were simply listed among the S-traits. This created a conceptual replication of the traditional 2 (group membership) by 2 (positive, negative portrayal) amplification paradigm. The dependent measures in the Asch paradigm are already the same as in amplification studies: participants evaluate the targets on a series of additional personality traits (which Asch referred to as "R-traits" or "rating" traits). From this we may operationally define evaluative amplification in the present study as an interaction of group membership and order of presentation showing the exaggeration of the primacy effect in the impressions of minority targets. Additionally, this paradigm allows us to separate the R-traits into their respective personality domains and investigate whether some of them show greater or lesser amplification than others. Of course, incorporating race and gender into the Asch (1946) paradigm begs the question whether group membership is a central or peripheral trait. Asch originally defined a central trait as one that "is more important, contributes more substantively to, or is more highly correlated with, the final impression than a peripheral trait" (p. 268). When Asch inserted the central traits "warm" or "cold" into the list of S-traits in his studies, impressions of the targets were very positive and very negative, respectively. Subsequent theory and research has defined central traits more narrowly. Peabody (1990) suggested that central traits affect impressions across all personality domains, whereas peripheral traits affect impressions only on the domain to which they belong. In support of this position, Peabody (1990) showed that the "central" traits "warm" and "cold," which load on the Agreeableness domain (Goldberg, 1990), affected only ratings on other Agreeableness traits in Asch's studies and were thus "peripheral" in nature. Peabody's reasoning leads to the conclusion that perhaps no personality traits, short of "good" or "bad," are truly central. However, race and gender may indeed have a central impact on impressions. If race and gender activate global prejudicial biases then group membership may well affect impressions across all personality domains in the manner of a central trait. If however, race and gender communicate circumscribed personality information in the form of stereotypes, then their effect on impressions may indeed be peripheral. Based on the above, a number of outcomes are possible when race and gender are inserted into the Asch paradigm. First, race and/or gender may have a unidirectional effect on impressions in the way Asch believed the central traits "warm" and "cold" to have. This outcome would be expected to occur if the majority of our sample shows either old-fashioned prejudice (McConohay, 1986), or consistent reverse-discrimination (Dutton, 1976). Second, and perhaps more likely, race and/or gender may have a bi-directional, or amplifying effect on impressions, as would be expected by amplification theory. In either case, if these effects occur across all personality domains, we would conclude that race and gender are central traits. If, however, these effects are restricted to certain personality domains, we wo

Raloxifene was metabolized predominantly by CYP3A4 in human liver microsomes to a pair of carbon-carbon (RD1–2) and ether (RD3–4) linked homodimers in an nicotinamide adenine dinucleotide phosphate-dependent manner. The major homodimer formed by human liver microsomes (RD3) was different from the major homodimer formed by peroxidases (RD1). RD1, 3 and 4 were identified by both mass spectrometry (MS) and nuclear magnetic resonance (NMR) as symmetrical carbon-carbon (both carbon 7 from benzo[b]thiopen-6-ol) linked homodimer, asymmetrical ether (oxygen from 4-hydroxyphenyl and carbon 7 from benzo[b]thiopen-6-ol) linked homodimer and asymmetrical ether (oxygen and carbon 7 from benzo[b]thiopen-6-ol) linked homodimer, respectively. The structures of the homodimers RD1, 3 and 4 provided evidence for free radical metabolism of raloxifene by predominantly CYP3A4 in human liver microsomes to oxygen-centered phenoxy radicals from 4-hydroxyphenyl and benzo[b]thiopen-6-ol moieties. Further delocalization to ortho carbon-centered radical was only observed for benzo[b]thiopen-6-ol derived phenoxy radical.

OBJECTIVE:To determine whether out-of-hospital administration of hypertonic fluids would improve survival after severe injury with hemorrhagic shock. BACKGROUND:Hypertonic fluids have potential benefit in the resuscitation of severely injured patients because of rapid restoration of tissue perfusion, with a smaller volume, and modulation of the inflammatory response, to reduce subsequent organ injury. METHODS:Multicenter, randomized, blinded clinical trial, May 2006 to August 2008, 114 emergency medical services agencies in North America within the Resuscitation Outcomes Consortium. INCLUSION CRITERIA:injured patients, age ≥ 15 years with hypovolemic shock (systolic blood pressure ≤ 70 mm Hg or systolic blood pressure 71-90 mm Hg with heart rate ≥ 108 beats per minute). Initial resuscitation fluid, 250 mL of either 7.5% saline per 6% dextran 70 (hypertonic saline/dextran, HSD), 7.5% saline (hypertonic saline, HS), or 0.9% saline (normal saline, NS) administered by out-of-hospital providers. Primary outcome was 28-day survival. On the recommendation of the data and safety monitoring board, the study was stopped early (23% of proposed sample size) for futility and potential safety concern. RESULTS:: A total of 853 treated patients were enrolled, among whom 62% were with blunt trauma, 38% with penetrating. There was no difference in 28-day survival-HSD: 74.5% (0.1; 95% confidence interval [CI], -7.5 to 7.8); HS: 73.0% (-1.4; 95% CI, -8.7-6.0); and NS: 74.4%, P = 0.91. There was a higher mortality for the postrandomization subgroup of patients who did not receive blood transfusions in the first 24 hours, who received hypertonic fluids compared to NS [28-day mortality-HSD: 10% (5.2; 95% CI, 0.4-10.1); HS: 12.2% (7.4; 95% CI, 2.5-12.2); and NS: 4.8%, P < 0.01]. CONCLUSION:Among injured patients with hypovolemic shock, initial resuscitation fluid treatment with either HS or HSD compared with NS, did not result in superior 28-day survival. However, interpretation of these findings is limited by the early stopping of the trial. CLINICAL TRIAL REGISTRATION:Clinical Trials.gov, NCT00316017.

Social-Networking Websites (SNWs) are rapidly becoming a central media for social exchange. A basic question is how well are people able to get to know each other through these websites? In this study, we explore characteristics of the profile photographs and their association with impression agreement. Using a specially designed social networking website, we examined 1,316 first-impressions of profile owners who had posted photographs as part of a complete profile. The results suggest that photographs in which the profile owners were smiling, outdoors, and shown with others were associated with higher impression agreement. Several gender interactions suggested that other aspects of the photographs, including head covering and apparent weight, also affected impression agreement depending on the gender of the profile owner and visitors. These results were interpreted in light of the literature on interpersonal perception.

With the recent publication of the FDA guidance on metabolites in safety testing (MIST), a reflection is provided that describes the impact of this guidance on the processes of drug metabolite identification and quantification at various stages of drug development. First, a retrospective analysis is described that was conducted on 12 human absorption, metabolism, and excretion (AME) trials with the application of these MIST criteria. This analysis showed that the number of metabolites requiring identification, (semi)-quantification, and coverage in the toxicology species would substantially increase. However, a significant proportion of these metabolites were direct or indirect conjugates, a class of metabolites that was specifically addressed in the guidance as being largely innocuous. The nonconjugated metabolites were all covered in at least one toxicology animal species, with no need for additional safety evaluation. Second, analytical considerations pertaining to the efficient identification of metabolites are discussed. Topics include software-assisted detection and structural identification of metabolites, the emerging hyphenation of ultraperformance liquid chromatography (UPLC) with radioactivity detection, and the various ways to estimate metabolite abundance in the absence of an authentic standard. Technical aspects around the analysis of metabolite profiles are also presented, focusing on precautions to be taken in order not to introduce artifacts. Finally, a tiered approach for metabolite quantification is proposed, starting with quantification of metabolites prior to the multiple ascending dose study (MAD) in humans in only specific cases (Tier A). The following step is the identification and quantification of metabolites expected to be of pharmacological or toxicological relevance (based on MIST and other complementary criteria) in selected samples from the MAD study and preclinical studies in order to assess metabolite exposure coverage (Tier B). Finally, a metabolite quantification strategy for the studies after the MAD phase (Tier C) is proposed.

A need still exists for a liquid chromatography/tandem mass spectrometry (LC/MS/MS) method that can detect broad classes of glutathione (GSH) conjugates and provide characterization of their structures. We now describe the development of a method that multiplexes high-resolution accurate mass analysis with isotope pattern triggered data-dependent product ion scans, for simultaneous detection and structural elucidation of GSH conjugates within a single analysis using a LTQ/Orbitrap. This method was initially developed to detect GSH conjugates generated from incubating 10 microM test compound with pooled human liver microsomes fortified with NADPH-regenerating system and a 2:1 ratio of 5 mM glutathione and [(13)C(2) (15)N-Gly]glutathione. The GSH conjugates were detected by isotope search of mass defect filtered and control subtracted full scan accurate MS data using MetWorks software. This was followed by elucidation of reactive intermediate structures using chemical formulae for both protonated molecules and their product ions from accurate masses in a single analysis. The mass accuracies measured for the precursor and product ions by the Orbitrap were <2 ppm in external mass calibration mode. Successful detection and characterization of GSH conjugates of acetaminophen, tienilic acid, clozapine, ticlopidine and mifepristone validated this method. In each case, the detected GSH conjugates were within the top five hits by isotope search. This method also has a broader detection capability since it is independent of the collision-induced dissociation behavior of the GSH conjugates. Furthermore, this method is amenable to a broad class of reactive intermediate trapping agents as exemplified by the simultaneous detection and structural elucidation of the cyano-N-methylene iminium ion conjugates of verapamil and its O-desmethyl metabolites, which we report for the first time. In addition to the chemically tagged reactive intermediates, this method also provides information on stable metabolites from the full scan accurate MS data.

Social-Networking Websites (SNWs) like Facebook and MySpace are playing an increasingly prevalent role in everyday social interactions. But very little is known about the effectiveness of the various profile elements in conveying information about the personality of the profile owner. Here we examine 5,303 impressions made on the basis of a specially designed social networking website (http://www.YouJustGetMe.com) and on the basis of Facebook profiles. Our findings suggest that profile owners are generally seen by others as they see themselves; that when raters are judging unknown targets, rater-target agreement is stronger for female (vs. male) targets and for female (vs. male) raters; and several specific elements of profiles are associated with increased or diminished levels of rater-target impression agreement. The findings are important because they are the first to show how impression agreement may be affected by specific elements in SNW profiles.

Martel, Guillaume MD; Al-Sabti, Hilal MD; Mulder, David S. MD; Sirois, Christian MD; Evans, David C. MD Author Information

The formation of drug-protein adducts carries a risk of clinical toxicities that may not be predicted by preclinical safety studies. The process of minimizing the potential for metabolic activation at the lead optimization stage could therefore be viewed as a step for building quality into future generations of drug products.

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

1. The in vitro cooperativity exhibited by cytochrome P450 (CYP) 3A4 is influenced by the nature of the recombinant system in which the phenomenon is studied. Diclofenac, piroxicam and R-warfarin were used as model substrates, and quinidine was the effector. 2. The 5-, 5'- and 10-hydroxylation of diclofenac, piroxicam and R-warfarin, respectively, were enhanced five- to sevenfold by quinidine in human liver microsomal incubations. Whereas these cooperative drug interactions were apparent in incubations with CYP3A4 expressed in human lymphoblast cells, similar phenomena were not observed with the enzyme expressed in insect cells. 3. Insect cell microsomes were treated with a detergent and CYP3A4 was solubilized into a buffer medium. In incubations with CYP3A4 'freed' from its host membrane, the 5-hydroxylation of diclofenac increased with increasing quinidine concentrations, reaching a maximal eightfold elevation relative to controls. The metabolism of piroxicam and warfarin was similarly enhanced by quinidine. 4. Kinetically, enhancement by quinidine of the 5-hydroxylation of diclofenac in incubations with solubilized CYP3A4 was characterized by increases in the rate of metabolism with little change in the substrate-binding affinity. Conversely, the 3-hydroxylation of quinidine was not affected by diclofenac. 5. The data suggest that certain properties of CYP3A4 are masked by expression of the protein in insect cells and reinforce the concept that the enzyme possesses multiple binding domains. The absence of cooperative drug interactions with quinidine when CYP3A4 was expressed in insect cells might be due to an absence of enzyme conformation changes on quinidine binding, or the inability of quinidine to gain access to a putative effector-binding domain. 6. Caution should be exercised when comparing models for CYP3A4 cooperativity derived from different recombinant preparations of the enzyme.

Ethinylestradiol (EE) is one of the key constituents of oral contraceptives. Major metabolites of EE in humans are the glucuronide and sulfate conjugates, EE-3-O-glucuronide (EE-G) and EE-3-O-sulfate (EE-S). In the present study, transport of EE-G and EE-S by the human multidrug resistance proteins MRP1, MRP2, and MRP3 was investigated using inside-out membrane vesicles, isolated from Sf9 cells expressing human MRP1, MRP2, or MRP3. Vesicular uptake studies showed that EE-G was not a substrate for MRP1, whereas an ATP-dependent and saturable transport of [(3)H]EE-G was observed in MRP2 (K(m) of 35.1 +/- 3.5 microM) and MRP3 (K(m) of 9.2 +/- 2.3 microM) containing vesicles. EE-S was not transported by either MRP1, MRP2, or MRP3. However, low concentrations of EE-S stimulated MRP2-mediated uptake of ethacrynic acid glutathione. EE-S also stimulated MRP2 and MRP3-mediated uptake of 17beta-estradiol-17beta-D-glucuronide. Interestingly, EE-S stimulated strongly MRP2- and MRP3-mediated uptake of EE-G by increasing its apparent transport affinity, whereas no reciprocal stimulation of EE-S uptake by EE-G was observed. These data indicate that EE-S allosterically stimulates MRP2- and MRP3-mediated transport of EE-G and is not cotransported with EE-G. Our studies demonstrate specific active transport of a pharmacologically relevant drug conjugate by human MRP2 and MRP3, involving complex interactions with other organic anions. We also suggest that caution needs to be taken when using only competition studies as screening tools to identify substrates or inhibitors of MRP-mediated transport.

Ligand-mediated activation of the pregnane X receptor (PXR, NR1I2) is postulated to affect both hepatic and intestinal gene expression, because of the presence of this nuclear receptor in these important drug metabolizing organs; as such, activation of this receptor may elicit the coordinated regulation of PXR target genes in both tissues. Induction of hepatic and intestinal drug metabolism can contribute to the increased metabolism of drugs, and can result in adverse or undesirable drug-drug interactions. 2(S)-((3,5-bis(Trifluoromethyl)benzyl)-oxy)-3(S)phenyl-4-((3-oxo-1,2,4-triazol-5-yl)methyl)morpholine (L-742694) is a potent activator of the rat PXR and was characterized for its effects on hepatic and intestinal gene expression in female Sprague-Dawley rats by DNA microarray analysis. Transcriptional profiling in liver and small intestine revealed that L-742694 and dexamethasone (DEX) induced the prototypical battery of PXR target genes in liver, including CYP3A, Oatp2, and UGT1A1. In addition, both DEX and L-742694 induced common gene expression profiles that were specific to liver or small intestine, but there was a distinct lack of coordinated gene expression of genes common to both tissues. This pattern of gene regulation occurred in liver and small intestine independent of PXR, constitutive androstane receptor, or hepatic nuclear factor-4alpha expression, suggesting that other factors are involved in controlling the extent of coordinated gene expression in response to a PXR agonist. Overall, these results suggest that ligand-mediated activation of PXR and induction of hepatic, rather than small intestinal, drug metabolism genes would contribute to the increased metabolism of orally administered pharmaceuticals.

Stimulation by quinidine of warfarin metabolism in vitro was first demonstrated with liver microsomal preparations. We report herein that this drug interaction is reproducible in an animal model but that it exhibits profound species differences. Thus, using rabbit liver microsomes and a kinetic model incorporating two binding sites, the hepatic intrinsic clearance of R-warfarin via the 10-hydroxylation pathway (CL(int)(W)) was projected to be 6 +/- 1 and 128 +/- 51 microl/min/g liver, respectively, in the absence and presence of 21 microM unbound quinidine. These estimates were consistent with the results from studies in which rabbit livers (n = 5) were perfused in situ with R-warfarin or R-warfarin plus quinidine. The CL(int)(W) increased from 7 +/- 3 to 156 +/- 106 microl/min/g liver after increasing the hepatic exposure of unbound quinidine from 0 to 21 microM. In contrast, when liver microsomes or intact livers from rats were examined, R-warfarin metabolism was inhibited by quinidine, the CL(int)(W) decreasing to 26% of the control value after exposure of perfused rat livers (n = 5) to 22 microM unbound quinidine. The third example involved monkey liver microsomes, in which the rate of 10-hydroxylation of R-warfarin was little affected in the presence of quinidine (<2-fold increase). In all three species, the 10-hydroxylation of R-warfarin was catalyzed primarily by members of CYP3A, based on immuno- and chemical inhibition analyses. These findings not only highlight the variability of drug interactions among different species but also suggest that changes in hepatic clearance resulting from stimulation of cytochrome P450 activity may be projected based on estimates generated from corresponding liver microsomal preparations.