Methadone Induces the Expression of Hepatic Drug-Metabolizing Enzymes through the Activation of Pregnane X Receptor and Constitutive Androstane Receptor

Tolson, Antonia H.; Li, Haishan; Eddington, Natalie D.; Wang, Hongbing

doi:10.1124/dmd.109.027854

Cited by 27 publications

(26 citation statements)

References 30 publications

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“…The criteria adopted from previous publications (Sinz et al, 2006;Faucette et al, 2007;Chu et al, 2009) classify compounds that achieve more than 40% of RIF-normalized hPXR activation in reporter assays as strong activators of hPXR, whereas drugs exhibiting between 15 and 40% of RIF response are moderate activators; those exhibiting less than 15% of RIF response are recognized as nonactivators. Consistent with our previous report, MD was classified as a moderate-to-strong activator (Tolson et al, 2009). It is worth noting that BUP and DIP, with high structural similarity (see Fig.…”

Section: Effects Of Bup On the Activation Of Hpxr In Hepg2supporting

confidence: 73%

Differential Activation of Pregnane X Receptor and Constitutive Androstane Receptor by Buprenorphine in Primary Human Hepatocytes and HepG2 Cells

Li¹,

Hassan²,

Tolson³

et al. 2010

J Pharmacol Exp Ther

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Buprenorphine is a partial -opioid receptor agonist used for the treatment of opioid dependence that has several advantages over methadone. The principal route of buprenorphine disposition has been well established; however, little is known regarding the potential for buprenorphine to influence the metabolism and clearance of other drugs by affecting the expression of drug-metabolizing enzymes (DMEs). Here, we investigate the effects of buprenorphine on the activation of pregnane X receptor (PXR) and constitutive androstane receptor (CAR), as well as the induction of DMEs, in both HepG2 cells and human primary hepatocytes (HPHs). In HepG2 cells, buprenorphine significantly increased human PXR-mediated CYP2B6 and CYP3A4 reporter activities. CYP2B6 reporter activity was also enhanced by buprenorphine in HepG2 cells cotransfected with a chemical-responsive human CAR variant. Real-time reverse transcription-polymerase chain reaction analysis revealed that buprenorphine strongly induced CYP3A4 expression in both PXR-and CAR-transfected HepG2 cells. However, treatment with the same concentrations of buprenorphine in HPHs resulted in literally no induction of CYP3A4 or CYP2B6 expression. Further studies indicated that buprenorphine could neither translocate human CAR to the nucleus nor activate CYP2B6/CYP3A4 reporter activities in transfected HPHs. Subsequent experiments to determine whether the differential response was due to buprenorphine's metabolic stability revealed a dramatically differential rate of elimination for buprenorphine between HPHs and HepG2 cells. Taken together, these studies indicate that metabolic stability of buprenorphine defines the differential induction of DMEs observed in HepG2 and HPHs, and the results obtained from PXR and CAR reporter assays in immortalized cell line require cautious interpretation.

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Section: Effects Of Bup On the Activation Of Hpxr In Hepg2supporting

confidence: 73%

Differential Activation of Pregnane X Receptor and Constitutive Androstane Receptor by Buprenorphine in Primary Human Hepatocytes and HepG2 Cells

Li¹,

Hassan²,

Tolson³

et al. 2010

J Pharmacol Exp Ther

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“…To investigate whether activation of hCAR1ϩA reflects the chemical selectivities of the reference hCAR1 activation, a series of 22 compounds has been tested in HepG2 cells cotransfected with hCAR1ϩA and CYP2B6 reporter construct. These compounds include known hCAR activators (CITCO, PB, ART, PHN, EFV, NVP, CMZ, BHA, DAP, MCB, and MD) (Maglich et al, 2003;Wang et al, 2004;Burk et al, 2005;Faucette et al, 2007;Li et al, 2009;Tolson et al, 2009), hCAR deactivators (PK11195, OA and CLZ) Stanley et al, 2006;Li et al, 2008), selective rodent CAR activator and/or CYP2B inducers (TCPOBOP, MLZ, and FLU) (Tzameli et al, 2000;Huang et al, 2004), as well as typical activators of other nuclear receptors including: RIF for PXR, CDCA for farnesoid X receptor, HOC for liver X receptor, 3MC for aryl hydrocarbon receptor, and WY-14643 for peroxisome proliferator-activated receptor ␣. Figure 3A demonstrates that hCAR1ϩA was significantly activated by 11 of the 22 tested compounds at least 2-fold over the control in cell-based reporter assay.…”

Section: Resultsmentioning

confidence: 99%

A Single Amino Acid Controls the Functional Switch of Human Constitutive Androstane Receptor (CAR) 1 to the Xenobiotic-Sensitive Splicing Variant CAR3

Chen¹,

Tompkins²,

Li³

et al. 2009

J Pharmacol Exp Ther

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The constitutive androstane receptor (CAR) is constitutively activated in immortalized cell lines independent of xenobiotic stimuli. This feature of CAR has limited its use as a sensor for xenobiotic-induced expression of drug-metabolizing enzymes. Recent reports, however, reveal that a splicing variant of human CAR (hCAR3), which contains an insertion of five amino acids (APYLT), exhibits low basal but xenobiotic-inducible activities in cell-based reporter assays. Nonetheless, the underlying mechanisms of this functional shift are not well understood. We have now generated chimeric constructs containing various residues of the five amino acids of hCAR3 and examined their response to typical hCAR activators. Our results showed that the retention of alanine (hCAR1ϩA) alone is sufficient to confer the constitutively activated hCAR1 to the xenobiotic-sensitive hCAR3. It is noteworthy that hCAR1ϩA was significantly activated by a series of known hCAR activators, and displayed activation superior to that of hCAR3. Moreover, intracellular localization assays revealed that hCAR1ϩA exhibits nuclear accumulation upon 6-(4-chlorophenyl) imidazo[2,1-b][1,3]thiazole-5-carbaldehyde-O-(3,4-dichlorobenzyl) oxime (CITCO) treatment in COS1 cells, which differs from the spontaneous nuclear distribution of hCAR1 and the nontranslocatable hCAR3. Mammalian two-hybrid and glutathione S-transferase pulldown assays further demonstrated that hCAR1ϩA interacts with the coactivator SRC-1 and GRIP-1 at low level before activation, while at significantly enhanced level in the presence of CITCO. Thus, the alanine residue in the insertion of hCAR3 seems in charge of the xenobiotic response of hCAR3 through direct and indirect mechanisms. Activation of hCAR1ϩA may represent a sensitive avenue for the identification of hCAR activators.

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“…hPXR governs the transactivation of multiple drug-metabolizing genes such as CYP3A4, CYP2B6, and UDP-glucuronosyltransferase 1A1, as well as drug transporters such as multidrug resistance 1. The CYP2B6 reporter construct containing both the proximal phenobarbital-responsive enhancer module and the distal xenobiotic-responsive enhancer module is highly responsive to chemical-mediated hPXR activations (Tolson et al, 2009). Here, we investigated the ability of 17 drugs to activate hPXR-mediated CYP2B6 reporter expression in HepG2 cells.…”

Section: Resultsmentioning

confidence: 99%

“…The CYP2B6 reporter construct, containing both the phenobarbital-responsive enhancer module and the distal xenobiotic-responsive enhancer module [CYP2B6-2.2 kilobases (kb)], were generated as described previously (Wang et al, 2003;Tolson et al, 2009). The pSG5-hPXR expression plasmid was acquired from Dr. Steve Kliewer (University of Texas, Southwestern Medical Center, Dallas, TX).…”

Section: Principal Component Analysis Of Scut Database Molecules Andmentioning

confidence: 99%

Identification and Validation of Novel Human Pregnane X Receptor Activators among Prescribed Drugs via Ligand-Based Virtual Screening

Pan

Li²,

Gregory³

et al. 2010

Drug Metab Dispos

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ABSTRACT:Human pregnane X receptor (hPXR) plays a key role in regulating metabolism and clearance of endogenous and exogenous substances. Identification of novel hPXR activators among commercial drugs may aid in avoiding drug-drug interactions during coadministration. We applied ligand-based computational approaches for virtual screening of a commonly prescribed drug database (SCUT). Bayesian classification models were generated with a training set comprising 177 compounds using Fingerprints and 117 structural descriptors. A cell-based luciferase reporter assay was used for evaluation of chemical-mediated hPXR activation in HepG2 cells. All compounds were tested at 10 M concentration with rifampicin and dimethyl sulfoxide as positive and negative controls, respectively. The Bayesian models showed specificity and overall prediction accuracy up to 0.92 and 0.69 for test set compounds. Screening the SCUT database with this model retrieved 105 hits and 17 compounds from the top 25 hits were chosen for in vitro testing. The reporter assay confirmed that nine drugs, i.e., fluticasone, nimodipine, nisoldipine, beclomethasone, finasteride, flunisolide, megestrol, secobarbital, and aminoglutethimide, were previously unidentified hPXR activators. Thus, the present study demonstrates that novel hPXR activators can be efficiently identified among U.S. Food and Drug Administrationapproved and commonly prescribed drugs, which should lead to detection and prevention of potential drug-drug interactions.

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Methadone Induces the Expression of Hepatic Drug-Metabolizing Enzymes through the Activation of Pregnane X Receptor and Constitutive Androstane Receptor

Cited by 27 publications

References 30 publications

Differential Activation of Pregnane X Receptor and Constitutive Androstane Receptor by Buprenorphine in Primary Human Hepatocytes and HepG2 Cells

Differential Activation of Pregnane X Receptor and Constitutive Androstane Receptor by Buprenorphine in Primary Human Hepatocytes and HepG2 Cells

A Single Amino Acid Controls the Functional Switch of Human Constitutive Androstane Receptor (CAR) 1 to the Xenobiotic-Sensitive Splicing Variant CAR3

Identification and Validation of Novel Human Pregnane X Receptor Activators among Prescribed Drugs via Ligand-Based Virtual Screening

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