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Raucy J.L.,Puracyp Inc. | Lasker J.M.,Puracyp Inc.
Drug Metabolism Reviews | Year: 2013

The evolution of scientific information relating to the regulation of xenobiotic disposition has extended to the discovery of an intricate group of receptor systems now recognized as master regulators. These ligand-activated transcription factors are commonly designated as "nuclear receptors", and include CAR (NR1I3), PXR (NR1I2), PPAR (NR1C1, NR1C2, and NR1C3) and AhR (HLHE76). As regulators of gene expression, activation of these receptors can elicit a plethora of drug-drug interactions. The aforementioned nuclear receptors bind a wide range of structurally-unrelated ligands, such as steroid hormones, bile acids, and small drug-type molecules. A pivotal nuclear receptor with regards to regulation of drug-drug interactions is the pregnane X receptor (PXR). Gene expression profiling has demonstrated that PXR regulates over 60 human genes that are involved not only in physiological functions but also in the metabolism of xenobiotics. Moreover, chemical library screening suggests that about 10% of the compounds comprising the U. S. Food and Drug Administration 1 and 2, Sigma-Aldrich LOPAC collection, Biomol, and Tocris/TimTec bioactive collection libraries exhibit some form of PXR binding. For these reasons, efficient, rapid and economical systems have been developed to identify nuclear receptor ligands. Cell-based assays encompassing transiently and stably-transfected cells and mammalian two-hybrid systems are currently being employed by the pharmaceutical industry to screen compounds for binding to and/or activation of nuclear receptors. Overall, these systems have the ability to predict in vivo responses to receptor activation that culminate in drug-drug interactions and adverse drug effects. © 2013 Informa Healthcare USA, Inc.


Raucy J.L.,Puracyp Inc. | Lasker J.M.,Puracyp Inc.
Current Drug Metabolism | Year: 2010

The screening of new drug candidates for nuclear receptor activation can identify agents with the potential to produce drug-drug interactions or elicit adverse drug effects. The nuclear receptors of interest are those that control the expression of drug metabolizing enzymes and drug transporters, and include the constitutive androstane receptor (CAR, NR1I3), the pregnane X receptor (PXR, NR1I2) and the aryl hydrocarbon receptor (AhR). This review will focus on the methods currently used to assess activation of these receptors. Assessment of nuclear receptor activation can be accomplished using direct or indirect approaches. Indirect methods quantify specific gene products that result from nuclear receptor activation while direct approaches measure either the binding of ligands to the receptors or the transcriptional events produced by ligand binding. Assays that directly quantify nuclear receptor activation are growing in popularity and, importantly, are amenable to high throughput screening (HTS). Several ligand binding assays are currently being utilized, including radioligand competition binding, where compounds compete with radiolabelled ligand for binding to PXR or CAR, such as the scintillation proximity binding assay that measures the reaction of ligands with receptor-coated beads. A fluorescence resonance energy transfer assay has also been developed, where the fluorescent signal is generated via the ligand-dependent interaction between the fluores-cently-labeled ligand binding domain of a nuclear receptor and co-activator proteins. Other in vitro activation assays include transient-and stably-transfected cell lines incorporating an expression vector for PXR, CAR or AhR plus a reporter gene vector containing response elements. The methods focused on in this review will be limited to the more direct in vitro approaches that are amenable to high throughput screening. © 2010 Bentham Science Publishers Ltd.


Pinne M.,Puracyp Inc. | Ponce E.,Puracyp Inc. | Raucy J.L.,Puracyp Inc.
PLoS ONE | Year: 2016

The pregnane X receptor (PXR/SXR, NR1I2) and constitutive androstane receptor (CAR, NR1I3) are nuclear receptors (NRs) involved in the regulation of many genes including cytochrome P450 enzymes (CYPs) and transporters important in metabolism and uptake of both endogenous substrates and xenobiotics. Activation of these receptors can lead to adverse drug effects as well as drug-drug interactions. Depending on which nuclear receptor is activated will determine which adverse effect could occur, making identification important. Screening for NR activation by New Molecular Entities (NMEs) using cell-based transactivation assays is the singular high throughput method currently available for identifying the activation of a particular NR. Moreover, screening for species-specific NR activation can minimize the use of animals in drug development and toxicology studies. With this in mind, we have developed in vitro transactivation assays to identify compounds that activate canine and rat PXR and CAR3. We found differences in specificity for canine and rat PXR, with the best activator for canine PXR being 10 μM SR12813 (60.1 ± 3.1-fold) and for rat PXR, 10 μM dexamethasone (60.9 ± 8.4 fold). Of the 19 test agents examined, 10 and 9 significantly activated rat and canine PXR at varying degrees, respectively. In contrast, 5 compounds exhibited statistically significant activation of rat CAR3 and 4 activated the canine receptor. For canine CAR3, 50 μM artemisinin proved to be the best activator (7.3 ± 1.8 and 10.5 ± 2.2 fold) while clotrimazole (10 μM) was the primary activator of the rat variant (13.7 ± 0.8 and 26.9 ± 1.3 fold). Results from these studies demonstrated that cellbased transactivation assays can detect species-specific activators and revealed that PXR was activated by at least twice as many compounds as was CAR3, suggesting that there are many more agonists for PXR than CAR. © 2016 Pinne et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.


Raucy J.L.,Puracyp Inc.
Methods in Pharmacology and Toxicology | Year: 2014

Analysis of pregnane X receptor (PXR, NR1I2) activation to determine induction of drug metabolizing enzymes and transporters and predict drug-drug interactions (DDIs) is a wildly used technique among in vitro assays. Direct assessment of PXR activation is a cell-based assay that requires two major components, the PXR and a reporter gene linked to the promoter and enhancer regions of the CYP3A gene. Because of species differences in the ligand binding region of PXR, the receptor from the species of interest should be used when assessing activation. At present, PXR activation determined in stable cell lines can be assessed in medium (96-well) to high throughput (384 to 1,536-well) systems. Assays involving stable cell lines allow for simultaneous detection of PXR activation, CYP3A metabolism and cytotoxicity in a single well of a multi-well plate. In this manner, compounds that are toxic and are both inducers and inhibitors of CYP3A are readily identified. Here, we provide comprehensive step-by-step instructions for the application of screening for human PXR activation using commercially available stable cell lines harboring the PXR and a luciferase reporter gene linked to the promoters of the human CYP3A gene. These instructions provide detailed information on how to thaw, culture, passage and seed the cells in 96 well plates to use for screening of new drug entities to determine their ability to activate PXR. Instructions will also be provided for assessing not only nuclear receptor activation but also cytotoxicity and CYP3A4 metabolism simultaneously in the stable transformants. Finally, methods are provided for interpreting the results generated in the cell lines and a mechanistic model described for predicting clinical drug-drug interactions. The basic protocol provided here for identifying new drugs with the ability to activate human PXR and subsequently cause P450 enzyme induction can be miniaturized for higher throughput and extended to PXR from other species and additional nuclear receptors. © 2014 Springer Science+Business Media New York.


Pinne M.,Puracyp Inc | Raucy J.L.,Puracyp Inc
Expert Opinion on Drug Discovery | Year: 2014

Introduction: Adverse drug effects and drug-drug interactions (DDIs) can be elicited by the activation of several nuclear receptors (NRs). Of the NRs that regulate expression of drug metabolizing enzymes and transporters and alter cellular processes, the most important are pregnane X receptor, constitutive androstane receptor and aryl hydrocarbon receptor. Screening for the activation of these receptors can be achieved during drug discovery by using various high-throughput analyses including ligand binding and transactivation assays. Areas covered: This review focuses on the importance of screening for NR activation during drug discovery and includes a discussion of the various assays to evaluate activation of NRs by xenobiotics. It also describes screening for species-specific NR activation to attenuate the use of animals in toxicology studies and to identify complications associated with drug metabolism and clearance that may occur during pharmacokinetic analyses. Expert opinion: Given the potential for adverse drug effects and DDIs during all phases of drug elimination, NR screening should occur early in drug discovery. Such screening could be used in structure-activity relationship studies to guide chemists in altering compound structures to eliminate the NR-binding and activation properties on priority compounds. Early screening can also reduce the risk of adverse drug effects, identify novel therapeutic agents and decrease the number of animals used in drug development. Overall, performing these types of assays described here could decrease drug development costs, alleviate the liability associated with drugs that activate NR and prevent unsafe drugs from entering the marketplace. © 2014 Informa UK, Ltd.


Hsu M.-H.,Scripps Research Institute | Savas U.,Scripps Research Institute | Lasker J.M.,Scripps Research Institute | Lasker J.M.,Hackensack University Medical Center | And 2 more authors.
Journal of Pharmacology and Experimental Therapeutics | Year: 2011

Activators of AMP-activated protein kinase (AMPK) increase. CYP4F2. A 24-h treatment of either primary human hepatocytes or the human hepatoma cell line HepG2 with 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR), which is converted to 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranosyl 5′-monophosphate, an activator of AMPK, caused an average 2.5- or 7-fold increase, respectively, of CYP4F2 mRNA expression but not of CYP4A11 or CYP4F3, CYP4F11, and CYP4F12 mRNA. Activation of CYP4F2 expression by AICAR was significantly reduced in HepG2 cells by an AMPK inhibitor, 6-[4-(2-piperidin-1- yl-ethoxy)-phenyl)]-3-pyridin-4-yl-pyrrazolo[ 1,5-a]-pyrimidine (compound C) or by transfection with small interfering RNAs for AMPKα isoforms α1 and α2. A 2.5-fold increase in CYP4F2 mRNA expression was observed upon treatment of HepG2 cells with 6,7-dihydro-4-hydroxy-3-(2′-hydroxy[1, 1′-biphenyl]-4-yl)-6-oxo-thieno[2,3-b]pyridine-5-carbonitrile (A-769662), a direct activator for AMPK. In addition, the indirect activators of AMPK, genistein and resveratrol increased CYP4F2 mRNA expression in HepG2 cells. Pretreatment with compound C or 1,2-dihydro-3H-naphtho[2,1-b]pyran-3-one (splitomicin), an inhibitor of the NAD + activated deacetylase SIRT1, only partially blocked activation of CYP4F2 expression by resveratrol, suggesting that a SIRT1/AMPK-independent pathway also contributes to increased CYP4F2 expression. Compound C greatly diminished genistein activation of CYP4F2 expression. 7H-benz[de]benzimidazo[2,1-a]isoquinoline-7-one-3-carboxylic acid acetate (STO-609), a calmodulin kinase kinase (CaMKK) inhibitor, reduced the level of expression of CYP4F2 elicited by genistein, suggesting that CaMKK activation contributed to AMPK activation by genistein. Transient transfection studies in HepG2 cells with reporter constructs containing the CYP4F2 proximal promoter demonstrated that AICAR, genistein, and resveratrol stimulated transcription of the reporter gene. These results suggest that activation of AMPK by cellular stress and endocrine or pharmacologic stimulation is likely to activate CYP4F2 gene expression. Copyright © 2011 by The American Society for Pharmacology and Experimental Therapeutics.


PubMed | Puracyp Inc.
Type: Journal Article | Journal: Current drug metabolism | Year: 2011

The screening of new drug candidates for nuclear receptor activation can identify agents with the potential to produce drug-drug interactions or elicit adverse drug effects. The nuclear receptors of interest are those that control the expression of drug metabolizing enzymes and drug transporters, and include the constitutive androstane receptor (CAR, NR1I3), the pregnane X receptor (PXR, NR1I2) and the aryl hydrocarbon receptor (AhR). This review will focus on the methods currently used to assess activation of these receptors. Assessment of nuclear receptor activation can be accomplished using direct or indirect approaches. Indirect methods quantify specific gene products that result from nuclear receptor activation while direct approaches measure either the binding of ligands to the receptors or the transcriptional events produced by ligand binding. Assays that directly quantify nuclear receptor activation are growing in popularity and, importantly, are amenable to high throughput screening (HTS). Several ligand binding assays are currently being utilized, including radioligand competition binding, where compounds compete with radiolabelled ligand for binding to PXR or CAR, such as the scintillation proximity binding assay that measures the reaction of ligands with receptor-coated beads. A fluorescence resonance energy transfer assay has also been developed, where the fluorescent signal is generated via the ligand-dependent interaction between the fluorescently-labeled ligand binding domain of a nuclear receptor and co-activator proteins. Other in vitro activation assays include transient- and stably-transfected cell lines incorporating an expression vector for PXR, CAR or AhR plus a reporter gene vector containing response elements. The methods focused on in this review will be limited to the more direct in vitro approaches that are amenable to high throughput screening.


The pregnane X receptor (PXR/SXR, NR1I2) and constitutive androstane receptor (CAR, NR1I3) are nuclear receptors (NRs) involved in the regulation of many genes including cytochrome P450 enzymes (CYPs) and transporters important in metabolism and uptake of both endogenous substrates and xenobiotics. Activation of these receptors can lead to adverse drug effects as well as drug-drug interactions. Depending on which nuclear receptor is activated will determine which adverse effect could occur, making identification important. Screening for NR activation by New Molecular Entities (NMEs) using cell-based transactivation assays is the singular high throughput method currently available for identifying the activation of a particular NR. Moreover, screening for species-specific NR activation can minimize the use of animals in drug development and toxicology studies. With this in mind, we have developed in vitro transactivation assays to identify compounds that activate canine and rat PXR and CAR3. We found differences in specificity for canine and rat PXR, with the best activator for canine PXR being 10 M SR12813 (60.1 3.1-fold) and for rat PXR, 10 M dexamethasone (60.9 8.4 fold). Of the 19 test agents examined, 10 and 9 significantly activated rat and canine PXR at varying degrees, respectively. In contrast, 5 compounds exhibited statistically significant activation of rat CAR3 and 4 activated the canine receptor. For canine CAR3, 50 M artemisinin proved to be the best activator (7.3 1.8 and 10.5 2.2 fold) while clotrimazole (10 M) was the primary activator of the rat variant (13.7 0.8 and 26.9 1.3 fold). Results from these studies demonstrated that cell-based transactivation assays can detect species-specific activators and revealed that PXR was activated by at least twice as many compounds as was CAR3, suggesting that there are many more agonists for PXR than CAR.

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