Publications by authors named "Steven Stout"

36 Publications

DGAT2 Inhibition Alters Aspects of Triglyceride Metabolism in Rodents but Not in Non-human Primates.

Cell Metab 2018 Jun 26;27(6):1236-1248.e6. Epub 2018 Apr 26.

Division of Cardio Metabolic Disease, Merck & Co., Inc., Kenilworth, NJ 07033, USA. Electronic address:

Diacylglycerol acyltransferase 2 (DGAT2) catalyzes the final step in triglyceride (TG) synthesis and has been shown to play a role in regulating hepatic very-low-density lipoprotein (VLDL) production in rodents. To explore the potential of DGAT2 as a therapeutic target for the treatment of dyslipidemia, we tested the effects of small-molecule inhibitors and gene silencing both in vitro and in vivo. Consistent with prior reports, chronic inhibition of DGAT2 in a murine model of obesity led to correction of multiple lipid parameters. In contrast, experiments in primary human, rhesus, and cynomolgus hepatocytes demonstrated that selective inhibition of DGAT2 has only a modest effect. Acute and chronic inhibition of DGAT2 in rhesus primates recapitulated the in vitro data yielding no significant effects on production of plasma TG or VLDL apolipoprotein B. These results call into question whether selective inhibition of DGAT2 is sufficient for remediation of dyslipidemia.
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http://dx.doi.org/10.1016/j.cmet.2018.04.004DOI Listing
June 2018

Glucagon like receptor 1/ glucagon dual agonist acutely enhanced hepatic lipid clearance and suppressed de novo lipogenesis in mice.

PLoS One 2017 24;12(10):e0186586. Epub 2017 Oct 24.

Cardiometabolic Diseases Biology- Discovery, Merck & Co., Inc., Kenilworth, NJ, United States of America.

Lipid lowering properties of glucagon have been reported. Blocking glucagon signaling leads to rise in plasma LDL levels. Here, we demonstrate the lipid lowering effects of acute dosing with Glp1r/Gcgr dual agonist (DualAG). All the experiments were performed in 25 week-old male diet-induced (60% kCal fat) obese mice. After 2 hrs of fasting, mice were injected subcutaneously with vehicle, liraglutide (25nmol/kg) and DualAG (25nmol/kg). De novo cholesterol and palmitate synthesis was measured by deuterium incorporation method using D2O. 13C18-oleate infusion was used for measuring fatty acid esterification. Simultaneous activation of Glp1r and Gcgr resulted in decrease in plasma triglyceride and cholesterol levels. DualAG enhanced hepatic LDLr protein levels, along with causing decrease in content of plasma ApoB48 and ApoB100. VLDL secretion, de novo palmitate synthesis and fatty acid esterification decreased with acute DualAG treatment. On the other hand, ketone levels were elevated with DualAG treatment, indicating increased fatty acid oxidation. Lipid relevant changes were absent in liraglutide treated group. In an acute treatment, DualAG demonstrated significant impact on lipid homeostasis, specifically on hepatic uptake, VLDL secretion and de novo synthesis. These effects collectively reveal that lipid lowering abilities of DualAG are primarily through glucagon signaling and are liver centric.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0186586PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5655430PMC
November 2017

Label-Free, LC-MS-Based Assays to Quantitate Small-Molecule Antagonist Binding to the Mammalian BLT1 Receptor.

SLAS Discov 2017 Oct 1;22(9):1131-1141. Epub 2017 Aug 1.

Discovery, Preclinical and Early Development, Merck & Co., Kenilworth, NJ, USA.

We have developed and validated label-free, liquid chromatography-mass spectrometry (LC-MS)-based equilibrium direct and competition binding assays to quantitate small-molecule antagonist binding to recombinant human and mouse BLT1 receptors expressed in HEK 293 cell membranes. Procedurally, these binding assays involve (1) equilibration of the BLT1 receptor and probe ligand, with or without a competitor; (2) vacuum filtration through cationic glass fiber filters to separate receptor-bound from free probe ligand; and (3) LC-MS analysis in selected reaction monitoring mode for bound probe ligand quantitation. Two novel, optimized probe ligands, compounds and , were identified by screening 20 unlabeled BLT1 antagonists for direct binding. Saturation direct binding studies confirmed the high affinity, and dissociation studies established the rapid binding kinetics of probe ligands and . Competition binding assays were established using both probe ligands, and the affinities of structurally diverse BLT1 antagonists were measured. Both binding assay formats can be executed with high specificity and sensitivity and moderate throughput (96-well plate format) using these approaches. This highly versatile, label-free method for studying ligand binding to membrane-associated receptors should find broad application as an alternative to traditional methods using labeled ligands.
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http://dx.doi.org/10.1177/2472555217719748DOI Listing
October 2017

Dose-dependent effects of siRNA-mediated inhibition of SCAP on PCSK9, LDLR, and plasma lipids in mouse and rhesus monkey.

J Lipid Res 2016 12 5;57(12):2150-2162. Epub 2016 Oct 5.

Cardiometabolic Disease Merck & Co. Inc., Kenilworth, NJ.

SREBP cleavage-activating protein (SCAP) is a key protein in the regulation of lipid metabolism and a potential target for treatment of dyslipidemia. SCAP is required for activation of the transcription factors SREBP-1 and -2. SREBPs regulate the expression of genes involved in fatty acid and cholesterol biosynthesis, and LDL-C clearance through the regulation of LDL receptor (LDLR) and PCSK9 expression. To further test the potential of SCAP as a novel target for treatment of dyslipidemia, we used siRNAs to inhibit hepatic SCAP expression and assess the effect on PCSK9, LDLR, and lipids in mice and rhesus monkeys. In mice, robust liver Scap mRNA knockdown (KD) was achieved, accompanied by dose-dependent reduction in SREBP-regulated gene expression, de novo lipogenesis, and plasma PCSK9 and lipids. In rhesus monkeys, over 90% SCAP mRNA KD was achieved resulting in approximately 75, 50, and 50% reduction of plasma PCSK9, TG, and LDL-C, respectively. Inhibition of SCAP function was demonstrated by reduced expression of SREBP-regulated genes and de novo lipogenesis. In conclusion, siRNA-mediated inhibition of SCAP resulted in a significant reduction in circulating PCSK9 and LDL-C in rodent and primate models supporting SCAP as a novel target for the treatment of dyslipidemia.
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http://dx.doi.org/10.1194/jlr.M071498DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5321219PMC
December 2016

Potential mechanism of enhanced postprandial glucagon-like peptide-1 release following treatment with a diacylglycerol acyltransferase 1 inhibitor.

Pharmacol Res Perspect 2015 Dec 30;3(6):e00193. Epub 2015 Nov 30.

Merck Research Laboratories 2000 Galloping Hill Road Kenilworth New Jersey 07033.

Studies have demonstrated that blockade of diacylglycerol acyltransferase 1 (DGAT1) leads to prolonged release of glucagon-like peptide 1 (GLP-1) after meal challenge. The current study was undertaken to investigate the mechanism of action underlying the elevated levels of GLP-1 release following pharmacological inhibition of DGAT1. We utilized a potent, specific DGAT1 inhibitor, compound , to investigate the changes in intestinal lipid profile in a mouse model after oral administration of the compound and challenge with tracer containing fatty meal. [C]-oleic acid and LC-MS were employed to trace the fate of dietary fatty acids provided as part of a meal challenge in lean mice. Lipid profiles in plasma, proximal to distal segments of intestine, and feces were evaluated at various times following the meal challenge to study the kinetics of fatty acid absorption, synthesis into complex lipids, and excretion. Pharmacological inhibition of DGAT1 led to reduction of postprandial total and newly synthesized triglyceride (TG) excursion and significant increases in TG and FFA levels in the distal portion of intestine enriched with enteroendocrine L cells. Enhanced levels of FFA and cholesteryl ester were observed via fecal fat profiling. DGAT1 inhibition leads to enhancement of carbon flow to the synthesis of phosphatidylcholine within the intestine. DGAT1 inhibition markedly increases levels of TG and FFA in the distal intestine, which could be the predominant contributor to the prolonged and enhanced postprandial GLP-1 release. Inactivation of DGAT1 could provide potential benefit in the treatment of dysmetabolic diseases.
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http://dx.doi.org/10.1002/prp2.193DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4777249PMC
December 2015

Evaluation of CETP activity in vivo under non-steady-state conditions: influence of anacetrapib on HDL-TG flux.

J Lipid Res 2016 Mar 9;57(3):398-409. Epub 2015 Dec 9.

Merck Research Laboratories, Merck & Co. Inc., Kenilworth, NJ 07033.

Studies in lipoprotein kinetics almost exclusively rely on steady-state approaches to modeling. Herein, we have used a non-steady-state experimental design to examine the role of cholesteryl ester transfer protein (CETP) in mediating HDL-TG flux in vivo in rhesus macaques, and therefore, we developed an alternative strategy to model the data. Two isotopomers ([(2)H11] and [(13)C18]) of oleic acid were administered (orally and intravenously, respectively) to serve as precursors for labeling TGs in apoB-containing lipoproteins. The flux of a specific TG (52:2) from these donor lipoproteins to HDL was used as the measure of CETP activity; calculations are also presented to estimate total HDL-TG flux. Based on our data, we estimate that the peak total postprandial TG flux to HDL via CETP is ∼ 13 mg · h(-1) · kg(-1) and show that this transfer was inhibited by 97% following anacetrapib treatment. Collectively, these data demonstrate that HDL TG flux can be used as a measure of CETP activity in vivo. The fact that the donor lipoproteins can be labeled in situ using well-established stable isotope tracer techniques suggests ways to measure this activity for native lipoproteins in free-living subjects under any physiological conditions.
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http://dx.doi.org/10.1194/jlr.M063842DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4766989PMC
March 2016

Discovery and Pharmacology of a Novel Class of Diacylglycerol Acyltransferase 2 Inhibitors.

J Med Chem 2015 Dec 20;58(23):9345-53. Epub 2015 Nov 20.

Department of Discovery Chemistry, ‡Chemistry Modeling, and Informatics, §In Vitro Pharmacology, ∥In Vivo Pharmacology, ⊥Pharmacokinetics, Pharmacodynamics, and Drug Metabolism, #Basic Pharmaceutical Sciences, and ○Atherosclerosis, Merck & Co., Inc. , 2000 Galloping Hill Road, Kenilworth, New Jersey 07033, United States.

DGAT2 plays a critical role in hepatic triglyceride production, and data suggests that inhibition of DGAT2 could prove to be beneficial in treating a number of disease states. This article documents the discovery and optimization of a selective small molecule inhibitor of DGAT2 as well as pharmacological proof of biology in a mouse model of triglyceride production.
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http://dx.doi.org/10.1021/acs.jmedchem.5b01345DOI Listing
December 2015

Identification of DGAT2 Inhibitors Using Mass Spectrometry.

J Biomol Screen 2016 Feb 24;21(2):117-26. Epub 2015 Sep 24.

Department of Pharmacology, Merck Research Laboratories, Boston, MA, USA

Mass spectrometry offers significant advantages over other detection technologies in the areas of hit finding, hit validation, and medicinal chemistry compound optimization. The foremost obvious advantage is the ability to directly measure enzymatic product formation. In addition, the inherent sensitivity of the liquid chromatography/mass spectrometry (LC/MS) approach allows the execution of enzymatic assays at substrate concentrations typically at or below substrate Km. Another advantage of the LC/MS approach is the ability to assay impure enzyme systems that would otherwise be difficult to prosecute with traditional labeled methods. This approach was used to identify inhibitors of diacylglycerol O-acyltransferase-2 (DGAT2), a transmembrane enzyme involved in the triglyceride (TG) production pathway. The LC/MS approach was employed because of its increased assay window (compared with control membranes) of more than sevenfold compared with less than twofold with a conventional fluorogenic assay. The ability to generate thousands of dose-dependent IC50 data was made possible by the use of a staggered parallel LC/MS system with fast elution gradients. From the hit-deconvolution efforts, several structural scaffold series were identified that inhibit DGAT2 activity. Additional profiling of one chemotype in particular identified two promising reversible and selective compounds (compound 15 and compound 16) that effectively inhibit TG production in mouse primary hepatocytes.
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http://dx.doi.org/10.1177/1087057115607463DOI Listing
February 2016

Discovery of Novel Allosteric Non-Bisphosphonate Inhibitors of Farnesyl Pyrophosphate Synthase by Integrated Lead Finding.

ChemMedChem 2015 Nov 18;10(11):1884-91. Epub 2015 Sep 18.

Novartis Institutes for BioMedical Research, Basel, 4002, Switzerland.

Farnesyl pyrophosphate synthase (FPPS) is an established target for the treatment of bone diseases, but also shows promise as an anticancer and anti-infective drug target. Currently available anti-FPPS drugs are active-site-directed bisphosphonate inhibitors, the peculiar pharmacological profile of which is inadequate for therapeutic indications beyond bone diseases. The recent discovery of an allosteric binding site has paved the way toward the development of novel non-bisphosphonate FPPS inhibitors with broader therapeutic potential, notably as immunomodulators in oncology. Herein we report the discovery, by an integrated lead finding approach, of two new chemical classes of allosteric FPPS inhibitors that belong to the salicylic acid and quinoline chemotypes. We present their synthesis, biochemical and cellular activities, structure-activity relationships, and provide X-ray structures of several representative FPPS complexes. These novel allosteric FPPS inhibitors are devoid of any affinity for bone mineral and could serve as leads to evaluate their potential in none-bone diseases.
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http://dx.doi.org/10.1002/cmdc.201500338DOI Listing
November 2015

Effect of Error Propagation in Stable Isotope Tracer Studies: An Approach for Estimating Impact on Apparent Biochemical Flux.

Methods Enzymol 2015 19;561:331-58. Epub 2015 Aug 19.

Merck Research Laboratories, Kenilworth, New Jersey, USA.

Stable isotope tracers are widely used to quantify metabolic rates, and yet a limited number of studies have considered the impact of analytical error on estimates of flux. For example, when estimating the contribution of de novo lipogenesis, one typically measures a minimum of four isotope ratios, i.e., the precursor and product labeling pre- and posttracer administration. This seemingly simple problem has 1 correct solution and 80 erroneous outcomes. In this report, we outline a methodology for evaluating the effect of error propagation on apparent physiological endpoints. We demonstrate examples of how to evaluate the influence of analytical error in case studies concerning lipid and protein synthesis; we have focused on (2)H2O as a tracer and contrast different mass spectrometry platforms including GC-quadrupole-MS, GC-pyrolysis-IRMS, LC-quadrupole-MS, and high-resolution FT-ICR-MS. The method outlined herein can be used to determine how to minimize variations in the apparent biology by altering the dose and/or the type of tracer. Likewise, one can facilitate biological studies by estimating the reduction in the noise of an outcome that is expected for a given increase in the number of replicate injections.
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http://dx.doi.org/10.1016/bs.mie.2015.06.021DOI Listing
June 2016

Predictors of substance abuse treatment outcome in hospitalized veterans.

Am J Addict 2013 Jul-Aug;22(4):358-65

Department of Psychiatry, School of Medicine, Emory University, Atlanta, GA, USA.

Background And Objectives: Historically patients consulted for the substance abuse treatment from the medical surgical floors have a very low show rate for the substance abuse treatment. The authors performed retrospective chart review to find predictors of substance abuse treatment outcome in hospitalized veterans at Atlanta VA Medical Center.

Methods: The medical records from all the patients who were admitted to the medical/surgical floor with substance abuse consults from January-December 2009 were reviewed. A total of 235 consults were received. Those records were examined to find the predictors for substance abuse treatment.

Results: Multiple variables were tested for significance - patient demographics, housing status, employment, reason for hospitalization, toxicology screens, co-morbid psychiatric and medical conditions, physician visits, and patients on waiting list. All variables were given cut-off point for the p-value of .10. These variables were then included in the logistic regression model. It was found that homelessness (χ2 = 16.14 and p < .0001) was the only individual variable that showed a statistically significant correlation with starting the program. It was found that homelessness (χ2 = 19.21 and p < .0001) was the only individual variable that showed statistically significant correlation with completing the program.

Conclusions And Scientific Significance: Our study supports that for veterans with substance abuse, housing was the only consistent predictor to enter intensive outpatient program (IOP), complete IOP, and start aftercare. Our study demonstrates the need for and potential benefit of providing stable housing for the homeless veterans.
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http://dx.doi.org/10.1111/j.1521-0391.2013.12050.xDOI Listing
May 2014

New methodologies for studying lipid synthesis and turnover: looking backwards to enable moving forwards.

Biochim Biophys Acta 2014 Mar 22;1842(3):402-13. Epub 2013 May 22.

Molecular Biomarkers, Merck, 2015 Galloping Hill Road, Kenilworth, NJ 07033, USA.

Our ability to understand the pathogenesis of problems surrounding lipid accretion requires attention towards quantifying lipid kinetics. In addition, studies of metabolic flux should also help unravel mechanisms that lead to imbalances in inter-organ lipid trafficking which contribute to dyslipidemia and/or peripheral lipid accumulation (e.g. hepatic fat deposits). This review aims to outline the development and use of novel methods for studying lipid kinetics in vivo. Although our focus is directed towards some of the approaches that are currently reported in the literature, we include a discussion of the older literature in order to put "new" methods in better perspective and inform readers of valuable historical research. Presumably, future advances in understanding lipid dynamics will benefit from a careful consideration of the past efforts, where possible we have tried to identify seminal papers or those that provide clear data to emphasize essential points. This article is part of a Special Issue entitled: Modulation of Adipose Tissue in Health and Disease.
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http://dx.doi.org/10.1016/j.bbadis.2013.05.019DOI Listing
March 2014

Use of [13C18] oleic acid and mass isotopomer distribution analysis to study synthesis of plasma triglycerides in vivo: analytical and experimental considerations.

Anal Chem 2013 Jul 10;85(13):6287-94. Epub 2013 Jun 10.

Merck Research Laboratories, Merck & Co., Inc., Kenilworth, New Jersey 07033, USA.

We have previously reported on a liquid chromatography-mass spectrometry method to determine the disposition of [(13)C18]-oleic acid following intravenous and oral administration in vivo. This approach has enabled us to study a variety of aspects of lipid metabolism including a quantitative assessment of triglyceride synthesis. Here we present a more rigorous evaluation of the constraints imposed upon the analytical method in order to generate accurate data using this stable-isotope tracer approach along with more detail on relevant analytical figures of merit including limits of quantitation, precision, and accuracy. The use of mass isotopomer distribution analysis (MIDA) to quantify plasma triglyceride synthesis is specifically highlighted, and a re-evaluation of the underlying mathematics has enabled us to present a simplified series of equations. The derivation of this MIDA model and the significance of all underlying assumptions are explored in detail, and examples are given of how it can successfully be applied to detect differences in plasma triglyceride synthesis in lean and high-fat diet fed mouse models. More work is necessary to evaluate the applicability of this approach to triglyceride stores with slower rates of turnover such as in adipose or muscle tissue; however, the present report provides investigators with the tools necessary to conduct such studies.
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http://dx.doi.org/10.1021/ac400363kDOI Listing
July 2013

Tracking fatty acid kinetics in distinct lipoprotein fractions in vivo: a novel high-throughput approach for studying dyslipidemia in rodent models.

J Lipid Res 2013 Jan 5;54(1):276-81. Epub 2012 Oct 5.

Merck Research Laboratories, Rahway, NJ 07065, USA.

Isotopic tracers have been used to examine lipid trafficking for many years, and data from those studies have typically yielded novel insight regarding the pathophysiology of dyslipidemia. Previous experimental designs were suitable for studies in humans because relatively large volumes of plasma could be regularly sampled. We have expanded on the earlier logic by applying high-throughput analytical methods that require reduced sample volumes. Specifically, we have examined the possibility of coupling gel-based separations of lipoproteins (e.g., lipoprint) with LC-MS/MS analyses of complex lipid mixtures as a way to routinely measure the labeling profiles of distinct lipids in discrete lipoprotein subfractions. We demonstrate the ability to measure the incorporation of [U-(13)C]oleate into triglycerides (TG), PLs (PL), and cholesterol esters (CE) in VLDL, LDL, and HDL particles in mice. Although rodent models of dyslipidemia are inherently different from humans because of alterations in enzyme activities and underlying metabolism, rodent models can be used to screen novel compounds for efficacy in altering a given biochemical pathway and therein enable studies of target engagement in vivo. We expect that it is possible to translate our approach for application in other systems, including studies in humans.
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http://dx.doi.org/10.1194/jlr.D030791DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3520534PMC
January 2013

AAV8-mediated long-term expression of human LCAT significantly improves lipid profiles in hCETP;Ldlr(+/-) mice.

J Cardiovasc Transl Res 2011 Dec 6;4(6):801-10. Epub 2011 Aug 6.

Atherosclerosis, Cardiovascular Diseases, Merck Research Laboratories, RY80T-A100, 126 E. Lincoln Ave, Rahway, NJ 07065, USA.

Lecithin:cholesterol acyltransferase (LCAT) is the key circulating enzyme responsible for high-density lipoprotein (HDL) cholesterol esterification, HDL maturation, and potentially reverse cholesterol transport. To further explore LCAT's mechanism of action on lipoprotein metabolism, we employed adeno-associated viral vector (AAV) serotype 8 to achieve long-term (32-week) high level expression of human LCAT in hCETP;Ldlr(+/-) mice, and characterized the lipid profiles in detail. The mice had a marked increase in HDL cholesterol, HDL particle size, and significant reduction in low-density lipoprotein (LDL) cholesterol, plasma triglycerides, and plasma apoB. Plasma LCAT activity significantly increased with humanized substrate specificity. HDL cholesteryl esters increased in a fashion that fits human LCAT specificity. HDL phosphatidylcholines trended toward decrease, with no change observed for HDL lysophosphatidylcholines. Triglycerides reduction appeared to reside in all lipoprotein particles (very low-density lipoprotein (VLDL), LDL, and HDL), with HDL triglycerides composition highly reflective of VLDL, suggesting that changes in HDL triglycerides were primarily driven by the altered triglycerides metabolism in VLDL. In summary, in this human-like model for lipoprotein metabolism, AAV8-mediated overexpression of human LCAT resulted in profound changes in plasma lipid profiles. Detailed lipid analyses in the lipoprotein particles suggest that LCAT's beneficial effect on lipid metabolism includes not only enhanced HDL cholesterol esterification but also improved metabolism of apoB-containing particles and triglycerides. Our findings thus shed new light on LCAT's mechanism of action and lend support to its therapeutic potential in treating dyslipidemia.
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http://dx.doi.org/10.1007/s12265-011-9309-8DOI Listing
December 2011

Effect of methadone maintenance treatment on heroin craving, a literature review.

J Addict Dis 2011 Jan;30(1):27-38

Atlanta, VA Medical Center, Emory University, School of Medicine, Decatur, GA 30033, USA.

Despite agreement that methadone maintenance treatment (MMT) is an effective and safe option for treatment of heroin dependence, there have been controversies about its effect on heroin craving. A systematic literature review of the PubMed database was used to find studies eligible for inclusion in the study. The authors present the results of 16 articles that met all inclusion criteria. Overall, 7 studies reported that methadone could reduce heroin craving, 4 studies reported that patients in MMT are still at risk of having heroin craving, 1 study reported that methadone could increase heroin craving, and 4 studies reported that methadone has a neutral effect on heroin craving. One may speculate from these data that methadone may help with heroin craving, but patients in MMT may still be at risk of cue-induced heroin cravings. Methadone provides a helpful tool for reducing some components of craving and risk of relapse for patient receiving MMT.
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http://dx.doi.org/10.1080/10550887.2010.531672DOI Listing
January 2011

Allosteric non-bisphosphonate FPPS inhibitors identified by fragment-based discovery.

Nat Chem Biol 2010 Sep 15;6(9):660-6. Epub 2010 Aug 15.

Center for Proteomic Chemistry and Global Discovery Chemistry, Novartis Institutes for Biomedical Research, Basel, Switzerland.

Bisphosphonates are potent inhibitors of farnesyl pyrophosphate synthase (FPPS) and are highly efficacious in the treatment of bone diseases such as osteoporosis, Paget's disease and tumor-induced osteolysis. In addition, the potential for direct antitumor effects has been postulated on the basis of in vitro and in vivo studies and has recently been demonstrated clinically in early breast cancer patients treated with the potent bisphosphonate zoledronic acid. However, the high affinity of bisphosphonates for bone mineral seems suboptimal for the direct treatment of soft-tissue tumors. Here we report the discovery of the first potent non-bisphosphonate FPPS inhibitors. These new inhibitors bind to a previously unknown allosteric site on FPPS, which was identified by fragment-based approaches using NMR and X-ray crystallography. This allosteric and druggable pocket allows the development of a new generation of FPPS inhibitors that are optimized for direct antitumor effects in soft tissue.
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http://dx.doi.org/10.1038/nchembio.421DOI Listing
September 2010

Assay development and screening of human DGAT1 inhibitors with an LC/MS-based assay: application of mass spectrometry for large-scale primary screening.

J Biomol Screen 2010 Jul 18;15(6):695-702. Epub 2010 May 18.

Center for Proteomic Chemistry, Novartis Institutes for Biomedical Research, Cambridge, MA 02139, USA.

Many attractive targets for therapeutic intervention are enzymes that catalyze biological reactions involving small molecules such as lipids, fatty acids, amino acid derivatives, nucleic acid derivatives, and cofactors. Some of the reactions are difficult to detect by methods commonly used in high-throughput screening (HTS) without specific radioactive or fluorescent labeling of substrates. In addition, there are instances when labeling has a detrimental effect on the biological response. Generally, applicable assay methodologies for detection of such reactions are thus required. Mass spectrometry (MS), being a label-free detection tool, has been actively pursued for assay detection in HTS in the past several years. The authors have explored the use of multiparallel liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS) for high-throughput detection of biochemical reactions. In this report, we describe in detail the assay development and screening with a LC/MS-based system for inhibitors of human diacylglycerol acyltransferase (DGAT1) with a chemical library of approximately 800,000 compounds. Several strategies and process improvements have been investigated to overcome technical challenges such as data variation and throughput. Results indicated that, through these innovative approaches, the LC/MS-based screening method is both feasible and suitable for high-throughput primary screening.
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http://dx.doi.org/10.1177/1087057110370210DOI Listing
July 2010

A comparison of four models of delay discounting in humans.

Behav Processes 2009 Jun 27;81(2):256-9. Epub 2008 Dec 27.

Department of Psychology, Jacksonville State University, Jacksonville, AL 36265, USA.

The present study compared four prominent models of delay discounting: a one-parameter exponential decay, a one-parameter hyperbola [Mazur, J.E., 1987. An adjusting procedure for studying delayed reinforcement. In: Commons, M.L., Mazur, J.E., Nevin, J.A., Rachlin, H. (Eds.), Quantitative Analyses of Behavior: The Effect of Delay and of Intervening Events on Reinforcement Value, vol. 5. Erlbaum, Hillsdale, NJ, pp. 55-73], a two-parameter hyperboloid in which the denominator is raised to a power [Green, L., Myerson, J., 2004. A discounting framework for choice with delayed and probabilistic rewards. Psychol. Bull. 130, 769-792], and a two-parameter hyperbola in which delay is raised to a power [Rachlin, H., 2006. Notes on discounting. J. Exp. Anal. Behav. 85, 425-435]. Sixty-four college undergraduates made choices between hypothetical monetary rewards, one immediate and one delayed, and the fit of the four models to their data was assessed. All four equations accounted for a large proportion of the variance at both the group and the individual levels, but the exponents of both two-parameter models were significantly less than 1.0 at the group level, and frequently so at the individual level. Taken together, these results strongly suggest that more than one parameter is needed to accurately describe delay discounting by humans. Notably, both the Rachlin and the Green and Myerson models accounted for more than 99% of the variance at the group level and for 96% of the variance in the median individual. Because both models provide such good descriptions of the data, model selection will need to be based on other grounds.
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http://dx.doi.org/10.1016/j.beproc.2008.12.017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2674118PMC
June 2009

Overshadowing as a function of trial number: dynamics of first- and second-order comparator effects.

Learn Behav 2003 Feb;31(1):85-97

State University of New York, Binghamton, New York 13902-6000, USA.

In two conditioned lick suppression experiments with rats, we examined the permanence of the overshadowing effect as a function of the number of compound reinforced training trials. In Experiment 1, robust overshadowing was observed following 4 compound-US pairings but dissipated with 36 pairings. Overshadowing decreased because responding to the overshadowed stimulus increased, not because responding by the control group decreased. This dissipation was stimulus specific and not attributable to a response ceiling. Experiment 2 extended the generality of the effect to a sensory preconditioning design and further demonstrated that overshadowing lost through many compound-US pairings was restored by posttraining extinction of the training context. The results are explicable in terms of the extended comparator hypothesis (Denniston, Savastano, & Miller, 2001) under the assumption that the impacts of first- and second-order comparator processes grow differentially as a function of number of trials.
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http://dx.doi.org/10.3758/bf03195972DOI Listing
February 2003

Mass spectrometric techniques for label-free high-throughput screening in drug discovery.

Anal Chem 2007 Nov 29;79(21):8207-13. Epub 2007 Sep 29.

Discovery Technologies, Novartis Institute for Biomedical Research, 250 Massachusetts Avenue, Cambridge, Massachusetts 02139, USA.

High-throughput screening (HTS) is an important tool for finding active compounds to initiate medicinal chemistry programs in pharmaceutical discovery research. Traditional HTS methods rely on fluorescent or radiolabeled reagents and/or coupling assays to permit quantitation of enzymatic target inhibition or activation. Mass spectrometry-based high-throughput screening (MS-HTS) is an alternative that is not susceptible to the limitations imposed by labeling and coupling enzymes. MS-HTS offers a selective and sensitive analytical method for unlabeled substrates and products. Furthermore, method development times are reduced without the need to incorporate labels or coupling assays. MS-HTS also permits screening of targets that are difficult or impossible to screen by other techniques. For example, enzymes that are challenging to purify can lead to the nonspecific detection of structurally similar components of the impure enzyme or matrix of membraneous enzymes. The high selectivity of tandem mass spectrometry (MS/MS) enables these screens to proceed with low levels of background noise to sensitively discover interesting hits even with relatively weak activity. In this article, we describe three techniques that we have adapted for large-scale (approximately 175,000 sample) compound library screening, including four-way parallel multiplexed electrospray liquid chromatography tandem mass spectrometry (MUX-LC/MS/MS), four-way parallel staggered gradient liquid chromatography tandem mass spectrometry (LC/MS/MS), and eight-way staggered flow injection MS/MS following 384-well plate solid-phase extraction (SPE). These methods are capable of analyzing a 384-well plate in 37 min, with typical analysis times of less than 2 h. The quality of the MS-HTS approach is demonstrated herein with screening data from two large-scale screens.
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http://dx.doi.org/10.1021/ac062421qDOI Listing
November 2007

Sometimes-competing retrieval (SOCR): a formalization of the comparator hypothesis.

Psychol Rev 2007 Jul;114(3):759-83

Department of Psychology, Jacksonville State University, Jacksonville, AL, USA.

Cue competition is one of the most studied phenomena in associative learning. However, a theoretical disagreement has long stood over whether it reflects a learning or performance deficit. The comparator hypothesis, a model of expression of Pavlovian associations, posits that learning is not subject to competition but that performance reflects a complex interaction of encoded associative strengths. That is, subjects respond to a cue to the degree that it signals a change in the likelihood or magnitude of reinforcement relative to that in the cue's absence. Initially, this performance-focused view was supported by studies showing that posttraining revaluation of a competing cue often influences responding to the target cue. However, recently developed learning-focused accounts of retrospective revaluation have revitalized the debate concerning cue competition. Further complicating the picture are phenomena of cue facilitation, which have been addressed less frequently than cue competition by formal models of conditioning of either class. The authors present a formalization and extension of the comparator hypothesis, which results in sharpened differentiation between it and the new learning-focused models.
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http://dx.doi.org/10.1037/0033-295X.114.3.759DOI Listing
July 2007

Retraction.

Q J Exp Psychol (Hove) 2006 Dec;59(12):2207

Binghamton, NY, USA.

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http://dx.doi.org/10.1080/17470210601095086DOI Listing
December 2006

When more is less: extending training of the blocking association following compound training attenuates the blocking effect.

Learn Behav 2006 Feb;34(1):21-36

University of Sevile, Seville, Spain.

Three conditioned lick suppression experiments with rats were performed to assess the influence, following compound training of two stimuli (A and X) with the same outcome (AX-O trials), of extending training of the blocking association (i.e., A-O) on responding to the target stimulus (X) at test. In Experiment 1, backward blocking was attenuated when the blocking association was extensively trained. Experiment 2 showed that forward blocking was also attenuated by extensive further training of the blocking association following the AX-O trials. Experiment 3 contrasted candidate explanations of the results of Experiments 1 and 2 and demonstrated that these results are consistent with the framework of the extended comparator hypothesis (Denniston, Savastano, & Miller, 2001).
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1809472PMC
http://dx.doi.org/10.3758/bf03192868DOI Listing
February 2006

Trial order and retention interval in human predictive judgment.

Mem Cognit 2005 Dec;33(8):1368-76

Valdosta State University, Vakldosta, Georgia, USA.

The influences of order of trial type and retention interval on human predictive judgments were assessed for a cue that was reinforced on half of its training presentations. Subjects observed 10 cue-outcome presentations (i.e., reinforced trials) and 10 cue-alone presentations (i.e., nonreinforced trials) in one of three different orders: all nonreinforced trials followed by all reinforced trials(latent inhibition), reinforced and nonreinforced trials interspersed (partial reinforcement), or al lreinforced trials followed by all nonreinforced trials (extinction). Ratings were based mainly on the most recent event type (i.e., a recency effect) when the test occurred immediately after training but were based mainly on initial event types (i.e., a primacy effect) when the test occurred after a 48-h delay. The subjects tested both immediately and with a long retention interval did not exhibit this shift to primacy (i.e., the recency effect persisted). These results demonstrate noncatastrophic forgetting and the flexible use of trial order information in predictive judgments.
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http://dx.doi.org/10.3758/bf03193369DOI Listing
December 2005

Interaction of retention interval with CS-preexposure and extinction treatments: symmetry with respect to primacy.

Learn Behav 2004 Aug;32(3):335-47

State University of New York, Binghamton, New York 13902-6000, USA.

Imposition of a retention interval between cue-outcome pairings and testing can alleviate the retardation of conditioned responding induced by pretraining exposure to the cue (i.e., the CS-preexposure effect). However, recent studies have reported an enhanced effect of CS-preexposure treatment with longer retention intervals (De la Casa & Lubow, 2000, 2002; Lubow & De la Casa, 2002). In a series of conditioned barpress suppression studies with rats, we examined the effects of imposing a retention interval just prior to testing following either CS-preexposure (cue alone before cue-outcome pairings) or extinction (cue alone after cue-outcome pairings) treatments. Experiment 1 replicated in a different preparation recent reports of CS-preexposure treatment effects increasing with longer retention intervals. Experiment 2 showed that spontaneous recovery of stimulus control of behavior after extinction can be obtained with the same parameters as those used to observe the augmented effect of CS-preexposure treatment. In Experiment 3, both the augmented effect of CS-preexposure treatment and spontaneous recovery from extinction were found when we used, in place of a retention interval, an associative priming manipulation.
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http://dx.doi.org/10.3758/bf03196032DOI Listing
August 2004

Breast cancer and depression.

Oncology (Williston Park) 2004 Jul;18(8):1021-34; discussion 1035-6, 1047-8

Emory University, School of Medicine, Atlanta, Georgia 30322, USA.

Major depression and depressive symptoms, although commonly encountered in patients with medical illnesses, are frequently underdiagnosed and undertreated in women with breast cancer. Depression and its associated symptoms diminish quality of life, adversely affect compliance with medical therapies, and reduce survival. Treatment of depression in women with breast cancer improves their dysphoria and other depressive symptoms, enhances quality of life, and may increase longevity. In this review, studies that investigate pathophysiologic alterations in patients with cancer and comorbid depression are discussed, and the few studies on treatment of depression and related symptoms in women with breast cancer are examined.
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July 2004

Trial number and compound stimuli temporal relationship as joint determinants of second-order conditioning and conditioned inhibition.

Learn Behav 2004 May;32(2):230-9

State University of New York, Binghamton, New York 13902-6000, USA.

Two conditioned lick suppression experiments with rats used feature-negative training (A-footshock trials intermixed with nonreinforced XA presentations) to analyze the role of the number of XA compound presentations and the temporal relationship of the elements within the compound (simultaneous or serial) as determinants of the resulting behavioral control. Second-order conditioning (i.e., excitatory behavioral control by X) was observed to decline as the number of XA compound trials was increased. This decline was more rapid if X and A were presented simultaneously, as opposed to serially (i.e., X before A; Experiment 1). Conditioned inhibition to X, as assessed by a summation test (Experiment 1) and a retardation test (Experiment 2), increased with the number of XA trials and did so more quickly for simultaneous than for serial pairings of X and A. The results help to clarify previously discrepant findings regarding factors that promote excitation versus inhibition with this protocol.
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http://dx.doi.org/10.3758/bf03196024DOI Listing
May 2004

Effect of amount of context extinction on revaluation of a target CS.

Behav Processes 2004 Apr;66(1):7-16

Department of Psychology, State University of New York at Binghamton, Binghamton, NY 13902-6000, USA.

In two experiments using rats in a conditioned lick suppression paradigm the effects of posttraining extinction of the conditioning context on responding to the target conditioned stimulus (CS) which had previously been paired with a footshock was examined in a neutral context. Experiment 1 replicated Marlin's [Learn. Motiv. 13 (1981) 526] observation that this treatment can reduce responding to the target CS. This finding is contrary to other reports that context extinction enhances responding to the target CS. Experiment 2 examined the amount of posttraining context extinction as a variable potentially controlling this effect. It found the mediated extinction effect following one, but not 10 h of context extinction.
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http://dx.doi.org/10.1016/j.beproc.2003.11.003DOI Listing
April 2004

The basic laws of conditioning differ for elemental cues and cues trained in compound.

Psychol Sci 2004 Apr;15(4):268-71

Department of Psychology, State University of New York at Binghamton, 13902-6000, USA.

The cue-duration effect (i.e., longer cues result in less conditioned responding than shorter cues) was examined as a function of whether cues were trained alone or in compound. Compound (AX) or elemental (X) cues of either long or short duration were paired with the unconditioned stimulus. In testing with X alone, the cue-duration effect was observed with elementally trained cues, but not with compound cues. Instead, stronger responding resulted from training with long compound cues relative to short compound cues (i.e., a reversed cue-duration effect). Moreover, an overshadowing effect (i.e., decreased responding due to compound conditioning) was observed when conditioning was conducted with a short cue, but compound conditioning resulted in enhanced responding when it was conducted with a long cue (i.e., reversed overshadowing). These findings are consistent with other recent demonstrations that some laws of learning that apply to elementally trained cues do not similarly apply to cues trained in compound.
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http://dx.doi.org/10.1111/j.0956-7976.2004.00664.xDOI Listing
April 2004