Influence of HIV antiretrovirals on methadone N-demethylation and transport

Biochemical Pharmacology 95 (2015) 115–125

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Influence of HIV antiretrovirals on methadone N-demethylation and transport Scott D. Campbell a, Sarah Gadel a, Christina Friedel a, Amanda Crafford a, Karen J. Regina a, Evan D. Kharasch a,b,* a b

Department of Anesthesiology, Division of Clinical and Translational Research,, Washington University in St. Louis, St. Louis, MO, USA Department of Biochemistry and Molecular Biophysics, Washington University in St. Louis, St. Louis, MO, USA

A R T I C L E I N F O

A B S T R A C T

Article history: Received 9 January 2015 Accepted 12 March 2015 Available online 20 March 2015

Drug interactions involving methadone and/or HIV antiretrovirals can be problematic. Mechanisms whereby antiretrovirals induce clinical methadone clearance are poorly understood. Methadone is Ndemethylated to 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) by CYP2B6 and CYP3A4 in vitro, but by CYP2B6 in vivo. This investigation evaluated human hepatocytes as a model for methadone induction, and tested the hypothesis that methadone and EDDP are substrates for human drug transporters. Human hepatocyte induction by several antiretrovirals of methadone N-demethylation, and CYP2B6 and CYP3A4 transcription, protein expression and catalytic activity, and pregnane X receptor (PXR) activation were evaluated. Methadone and EDDP uptake and efflux by overexpressed transporters were also determined. Methadone N-demethylation was generally not significantly increased by the antiretrovirals. CYP2B6 mRNA and activity (bupropion N-demethylation) were induced by several antiretrovirals, as were CYP3A4 mRNA and protein expression, but only indinavir increased CYP3A activity (alfentanil dealkylation). CYP upregulation appeared related to PXR activation. Methadone was not a substrate for uptake (OCT1, OCT2, OCT3, OATP1A2, OATP1B1, OATP1B3, OATP2B1) or efflux (P-gp, BCRP) transporters. EDDP was a good substrate for P-gp, BCRP, OCT1, OCT3, OATP1A2, and OATP1B1. OATP1A2- and OCT3-mediated EDDP uptake, and BCRP-mediated EDDP efflux transport, was inhibited by several antiretrovirals. Results show that hepatocyte methadone Ndemethylation resembles expressed and liver microsomal metabolism more than clinical metabolism. Compared with clinical studies, hepatocytes underreport induction of methadone metabolism by HIV drugs. Hepatocytes are not a good predictive model for clinical antiretroviral induction of methadone metabolism and not a substitute for clinical studies. EDDP is a transporter substrate, and is susceptible to transporter-mediated interactions. ß 2015 Published by Elsevier Inc.

Keywords: Methadone Hepatocytes Protease inhibitors Cytochrome P450 3A CYP3A Cytochrome P450 2B6 CYP2B6

1. Introduction Methadone is a long-duration, cost-effective opioid used for treating acute, chronic, perioperative, neuropathic, and cancer pain, in adults and children, in first- or second-line therapy, and for treating opioid addiction. Clinical advantages of methadone include effectiveness in opioid-tolerance and severe pain, rapid

* Corresponding author at: Department of Anesthesiology, Washington University in St. Louis, 660 S Euclid Ave, Campus Box 8054, St. Louis, MO 63110-1093, USA. Tel.: +1 314 362 8796; fax: +1 314 362 8571. E-mail addresses: [email protected] (S.D. Campbell), [email protected] (S. Gadel), [email protected] (C. Friedel), [email protected] (A. Crafford), [email protected] (K.J. Regina), [email protected] (E.D. Kharasch). http://dx.doi.org/10.1016/j.bcp.2015.03.007 0006-2952/ß 2015 Published by Elsevier Inc.

onset of effect, administration by multiple routes (oral, intravenous, nasal, rectal, subcutaneous, sublingual), high oral bioavailability, and lack of active metabolites. Nevertheless, increased use of methadone for pain treatment over the past decade has been accompanied by a tragic increase in adverse events. Between 1999 and 2009, the rate of fatal methadone-related overdose increased more than 5-fold, and methadone was involved in approximately one-third of opioid-related overdose deaths [1]. There is considerable variability in methadone pharmacokinetics, including metabolism and clearance, and methadone disposition is susceptible to drug interactions, which can cause inadequate analgesia, withdrawal, or toxicity. One particularly relevant class of methadone drug interactions, owing to the substantial number of HIV-infected individuals receiving methadone for opioid addiction or pain, are interactions with antiretrovirals (protease inhibitors and

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non-nucleoside reverse transcriptase inhibitors). HIV antiretrovirals cause well-known drug interactions, both untoward and therapeutically useful [2]. Methadone–antiretroviral drug interactions are well-described, albeit mechanistically unsolved [3–6]. Methadone–antiretroviral drug interactions present a mechanistic conundrum, which remains inadequately understood. Methadone in humans is cleared primarily by hepatic cytochrome P450 (CYP)-catalyzed metabolism to the pharmacologically inactive quaternary metabolite 2-ethyl-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP), with some urinary excretion of unchanged drug. Methadone N-demethylation in vitro is catalyzed most efficiently by CYP2B6 and CYP3A4 [7–11]. CYP3A4 was suggested in numerous publications and clinical guidelines to be the major determinant of methadone disposition in vivo, and also susceptible to CYP3A4-mediated clinical drug interactions [3,12–16]. HIV protease inhibitors are potent and efficacious CYP3A inhibitors [17,18], and markedly inhibited methadone N-demethylation by human liver microsomes in vitro [19]. Nevertheless, many protease inhibitors had no effect on clinical methadone disposition, and some (ritonavir, ritonavir/lopinavir, ritonavir/tipranavir, nelfinavir) even increased methadone metabolism and clearance [3,20,21]. One likely explanation is that CYP2B6 is more important for clinical methadone disposition than CYP3A4 [22,23]. Another is that antiretroviral drug effects on cDNA-expressed and human liver microsomal CYP in vitro differ from those in vivo, due to CYP induction as well as inhibition [24,25], and that expressed and microsomal CYPs in vitro can only be inhibited but not induced. A third potential consideration is that EDDP may be susceptible to drug interactions and this may affect the interpretation of clinical methadone metabolism. Hepatocytes are an intermediate model between expressed or microsomal CYPs and clinical studies, and have become an essential instrument for evaluating drug interactions [26]. Hepatocytes, importantly, can be used to examine all three mechanisms of CYP-mediated drug interactions—that is, induction as well as inhibition and inactivation. In addition, hepatocytes possess a fuller complement of biotransformation proteins, including cytosolic enzymes and drug uptake and efflux transporters, in addition to CYPs and other microsomal enzymes. Human hepatocytes were used previously to evaluate mechanisms of autoinduction and heteroinduction of methadone metabolism [5,27]. The purpose of this investigation was to determine the influence of HIV antiretrovirals on methadone metabolism in human hepatocytes, and on the expression of the major enzymes catalyzing methadone metabolism in vitro, CYP2B6 and CYP3A4. The hypothesis tested was that human hepatocytes would resemble clinical more than expressed enzyme or microsomal enzymatic drug metabolism, and can serve as a model for clinical methadone induction. The second purpose was to evaluate the uptake and efflux of methadone and EDDP by various transporters potentially involved in hepatocyte or other organ or systemic disposition. 2. Materials and methods 2.1. Chemicals and reagents Alfentanil, methadone, and 2-ethylidene-1,5-dimethyl-3,3diphenylpyrrolidene (EDDP) were obtained from the National Institute on Drug Abuse Research Resources Program, through Research Triangle Institute (Research Triangle, NC). The following were obtained through the NIH AIDS Research and Reference Reagent Program: amprenavir, ritonavir, atazanavir, nelvirapine, indinavir, lopinavir, saquinavir, and nelfinavir. Williams E media was from Lonza Walkersville (Walkersville, MD). Hepatocyte

Supplement was from Life Technologies (Durham, NC). Dulbecco’s Modified Eagle Medium, Hank’s Balanced Salt Solution, and geneticin (G418) were from MediaTech (Corning, NY). Hoechst 33342 was from Invitrogen (Carlsbad, CA). Dimethyl sulfoxide (DMSO), rifampin, phenobarbital and bupropion were from Sigma (St. Louis, MO). Formic acid (Fisher Scientific, Pittsburg, PA) and acetonitrile (Sigma), were HPLC grade. Noralfentanil, d3-2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidene (d3-EDDP), d5-norfentanyl, and hydroxybupropion were from Cerilliant (Round Rock, TX). Bis-tris acrylamide gels (4–12%) and nitrocellulose membranes were from Invitrogen (Carlsbad, CA). Mouse antihuman monoclonal CYP3A4, rabbit antihuman polyclonal CYP2B6 and mouse antihuman monoclonal b-actin antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Goat anti-rabbit and goat antimouse secondary antibodies as well as SDS-cell lysis buffer and blocking buffer were from Li-COR Biosciences (Lincoln, NE). 2.2. Transfected cell lines Cells expressing human OATP1A2 were the generous gift of Dr. Markus Keiser, Department of Clinical Pharmacology, University Medicine Greifswald, Germany. A BCRP (ABCG2)-expressing cell line was constructed in Madin–Darby Canine Kidney (MDCK) cells. The full-length cDNA clone of the human ABCG2 wild-type gene was acquired from Origene (Rockville, MD) and used without further modification. MDCK cells (ATCC, Manassas, VA) were transfected with ABCG2 using Lipofectamine (Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol, and 72 h later cells were selected with 1 mg/mL geneticin in complete Dulbecco’s Modified Eagle Medium (commercial medium supplemented with 10% fetal bovine serum). Functional selection of ABCG2 clones was performed by selecting those cells exhibiting the highest level of ABCG2 expression based on exclusion of the fluorescent dye Hoechst 33342 (Sigma), as determined by fluorescence-activated cell sorting analysis. The expanded cells were incubated with 5 mg/ ml Hoeschst 33342 for 45 min, washed, and efflux allowed to continue for an additional 30 min at 37 8C. Cells were trypsinized in 0.25% trypsin-EDTA, pelleted by centrifugation, and resuspended in ice-cold Hank’s Balanced Salt Solution for flow cell analysis. The dimmest 0.5% of the Hoeschst 33342 labeled population were sorted into individual wells of a 96-well flatbottom plate and cultured to confluency for 1–3 weeks in supplemented Dulbecco’s Modified Eagle Medium containing 1 mg/mL geneticin. Expression of ABCG2 protein was then verified by Western Blot analysis. Twelve clones were selected from the 96-well plate and further expanded in a 6-well plate (TPP, Trasadingen, Switzerland). Cells were lysed using sample lysis buffer (Li-COR, Lincoln, NE) and homogenized using a QIAShredder (QIAGEN, Valencia, CA). The samples were loaded at 20 mg/well to a 4–12% Bis-Tris SDS PAGE gel (NUPAGE, Invitrogen, Carlsbad, CA) and transferred to a nitrocellulose membrane (Invitrogen, Carlsbad, CA). The membrane was blocked with Li-COR blocking buffer (LI-COR, Lincoln, NE) for 1 h at room temperature. The membrane was then incubated with blocking buffer containing a rabbit monoclonal antibody recognizing ABCG2 (Abcam, Cambridge, MA) at a 1:1000 dilution for 1 h at room temperature. The blot was washed for 30 min and further incubated with IRDye labeled anti-rabbit secondary antibody (Li-COR, Lincoln, NE). Fluorescence was detected using a Li-COR-Odyessey, and clones selected visually based on density of the band at 72 kDa. The colony with the highest expression was selected and expanded for use in the transport assay. For expression of the transporters OCT1, OCT2, OCT3, OATP1B1, OATP1B3, and OATP2B1 in human embryonic kidney (HEK) cells, sequence-verified cDNA was obtained from Open Biosystems (GE

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Healthcare, UK) or GeneCopoeia (Rockville, MD). The following clones were used for transfection; SLC22A1 (OCT1: 8992080), SLC22A2 (OCT2: 5186229), SLC22A3 (OCT3: 9052800), SLCO1B1 (OATP1B1: EX-I0042-M02), SLCO1B3 (OATP1B3: EX-U0996-M02), and SLCO2B1 (OATP2B1: EX-U0477-MO2). HEK-293 cells (ATCC, Manassas, VA) were plated at a density of 2.0  105 cells/well in 500 mL of Dulbecco’s Modified Eagle’s Medium (DMEM) + 10% fetal bovine serum (FBS, Invitrogen, Carlsbad, CA) without antibiotics, in a 24-well plate to achieve a confluency of 90–95% on the day of transfection. For transfection, 2.0 mL Lipofectamine 2000 (Invitrogen) was added to 50 mL of Opti-MEM (Invitrogen) in a sterile polystyrene tube (BD Falcon, Franklin Lakes, NJ). The diluted reagent was incubated for 5 min at room temperature and then added to second polystyrene tube that contained 0.8 mg of the appropriate plasmid that had been diluted in 50 mL of Opti-MEM. The DNA and lipid mixture was incubated for 20 min at room temperature to allow DNA–lipid complexes to form. The cells were then overlaid with the diluted DNA–Lipofectamine 2000 complexes and incubated overnight at 37 8C, 95% humidity and 5% CO2. After 24 hr, the cells were trypsinized and diluted 1:10 with DMEM that contained 10% FBS and geneticin (400 mg/mL, OCT1, OATP1B1, OATP1B3, OATP2B1) or blasticidin (5 mg/mL, OCT2, OCT3) and seeded onto a fresh 24-well plate and incubated overnight as above. The media was then replaced with DMEM + 10% FBS and geneticin (400 mg/mL, OCT1, OATP1B1, OATP1B3, OATP2B1) or blasticidin (5 mg/mL, OCT2, OCT3). Cell lines exhibiting uptake of a transporterspecific substrate at least 2-fold greater than untransfected HEK-293 cells were used for uptake assays. OATP1A2-expressing HEK cells have been described previously [28]. MDCK cells (untransfected and transfected with human P-gp) were kindly provided by Professor Piet Borst (Netherlands Cancer Institute, Amsterdam, The Netherlands) and were maintained according to the instructions provided. Both the transfected and untransfected cell lines were cultured in supplemented DMEM without antibiotics, and were maintained in culture no more than 2 months. Expression of ABCB1 protein was verified by Western Blot analysis. Cells were lysed using sample lysis buffer (Li-COR, Lincoln, NE) and homogenized using a QIAShredder (QIAGEN, Valencia, CA). The samples were loaded at 20 mg/well to a 3–8% Tris-Acetate SDS PAGE gel (NUPAGE, Invitrogen, Carlsbad, CA) and transferred to a nitrocellulose membrane (Invitrogen, Carlsbad, CA). The membrane was blocked with Li-COR blocking buffer (LICOR, Lincoln, NE) for 1 h at room temperature. The membrane was then incubated with blocking buffer containing a mouse monoclonal antibody recognizing ABCB1 (Santa Cruz Biotechnology Inc., Santa Cruz, CA) at a 1:200 dilution for 1 h at room temperature. The blot was washed for 30 min and further incubated with IRDye labeled anti-mouse secondary antibody (Li-COR, Lincoln, NE). Fluorescence was detected using a Li-COR-Odyessey, and clones selected visually based on density of the band at 170 kDa. 2.3. Hepatocyte induction, methadone metabolism and CYP activity Freshly plated human hepatocytes were generously provided by Life Technologies (Durham, NC). Hepatocytes were isolated from 3 (typical for such induction studies, and based on FDA Guidance1) donor livers, plated, and overlaid with Matrigel prior to shipping (see Table 1 for donor descriptions). Shipping media was removed and changed to supplemented William’s E Media upon arrival and the cells allowed to equilibrate for 24 h at 37 8C, 5% CO2, 95% humidity. Experiments were conducted essentially as described previously [5,27]. For induction, cells were incubated

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in media containing drug (or vehicle control, 0.1% DMSO) for 72 h, with media/drug changed each day. Each incubation was performed in triplicate wells from the same liver. Methadone N-demethylation was determined by EDDP formation, CYP2B6 activity as bupropion metabolism to hydroxybupropion, and CYP3A4/5 activity as alfentanil N-dealkylation to noralfentanil, at saturating substrate concentrations. After the 72 h induction period, hepatocytes were incubated with drug-free media for 1 h in a shaker incubator (60 rpm, 37 8C), then successively with media containing 500 mM racemic bupropion (40 min, and then media was sampled for analysis), drug-free media (1 h), 500 mM racemic methadone (40 min, and then media was sampled for analysis), and then 200 mM alfentanil (40 min, and then media was sampled for analysis). Metabolite analysis was performed on an API 3200 triple-quadrupole mass spectrometer (EDDP and noralfentanil) and API 4000 QTRAP mass spectrometer (hydroxybupropion), using d3-EDDP, d5-noralfentanil; and d6-8hydroxybupropion as internal standards, as described previously [5,27]. Fold induction was calculated for each substrate by comparing the metabolic activity following drug treatment to that following the treatment with 0.1% DMSO. After each experiment, hepatocytes were treated with RNAlater (QIAGEN, Valencia, CA) and frozen for later quantification of mRNA and CYP protein. After the metabolic activity assays, hepatocyte mRNA was isolated and measured by quantitative PCR as described [5,27]. cDNA copy number was calculated and fold induction then determined by comparing drug treated sample to vehicle control. Hepatocyte CYP3A4 protein content was determined by Western blot analysis. One of the triplicate wells for each drug concentration from the hepatocyte test plates were lysed using 1 SDS protein loading buffer (Li-COR Biosciences, Lincoln, NE) and homogenized using a QIAShredder (Qiagen, Valencia, CA). Total protein concentration for the standards and hepatocyte samples were determined by the Bradford method (BIORAD, Hercules, CA), using bovine serum albumin as a reference. Equal amounts of cell lysate (15 mg total protein) were loaded into each lane of a 4–12% gradient NuPAGE gel (Invitrogen) for analysis and transferred onto a nitrocellulose membrane. CYP3A4 supersomes (BD Gentest, San Jose, CA) were used as the reference standard. Blots were incubated overnight at 4 8C with blocking buffer (Li-COR Biosciences). Primary and secondary antibodies were diluted according to the manufacturer’s specifications in blocking buffer containing 0.1% Tween 20 (Sigma, St. Louis, MO). Nitrocellulose sheets were then incubated with primary antibodies to CYP3A4 and b-Actin (Santa Cruz Biotechnologies, Santa Cruz, CA) for 1 h, and washed three times for 10 min in 1 PBS + 0.1% Tween 20 at room temperature before the application of secondary antibodies (Li-COR Biosciences) for 30 min at room temperature. The membranes were washed again five times for 10 min in 1 PBS + 0.1% Tween 20. The intensities of both the CYP3A4 and b-actin bands for each sample were measured by densitometry (Odyssey Imaging Software, LiCOR Biosciences) and ratio of CYP3A4 to reference protein (bactin) was calculated. Fold induction was calculated by comparing the CYP3A4/b-Actin band density ratio for each drug treatment to the CYP3A4/b-actin band density ratio for the vehicle control. 2.4. Pregnane X receptor (PXR) reporter gene assay The PXR reporter gene assay was kindly performed by Puracyp, Inc (Carlsbad, CA) using their proprietary method, as described [27]. 2.5. Transport assays

1

Guidance for industry: Drug interaction studies - Study design, data analysis, implications for dosing, and labeling recommendations, Food and Drug Administration Center for Drug Evaluation and Research (CDER), February 2012.

Expanded P-gp-transfected, ABCG2-transfected, or untransfected cells were seeded at a density of 2.5  105 cells/ml on

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118 Table 1 Liver donor demographics. Donor ID

Sex

Race

Age

Smoker

Alcohol use

Concomitant medications

Hu 1284 Hu 1290 Hu 1331

Male Female Male

Caucasian Caucasian Caucasian

51 52 75

No No Yes

No Yes, Rarely Yes

alprazolam escitalopram Not reported duloxetine cyclobenzaprine

96 well membrane inserts (Transwell, Corning, Corning, NY) in supplemented DMEM media, which was changed on day 3 to supplemented media containing 2 mM sodium butyrate (Sigma, St. Louis, MO) to enhance expression of the ABCG2 protein [29]. Sodium butyrate was not used to enhance the expression in either the MDCK-P-gp or associated untransfected MDCK. Transport studies were performed on day 5. On the day of the assay cell monolayers were washed twice by the addition and aspiration of HBSS (MediaTech, Corning, NY) containing 1 mM CaCl2/0.1 mM MgSO4/0.1 mM MgCl2 (HBSSCM). Wells receiving drug plus inhibitor cells were pre-incubated with 0.2 mM K0143 (Tocris, Ellisville, MO) in HBSSCM for 30 min prior to addition of drug (or drug plus inhibitor), whereas cells that were incubated with drug (without inhibitor) were equilibrated with HBSSCM for 30 min prior to the start of the assay. Cells were then incubated with 2 mM drug (or 2 mM drug plus inhibitor) for 2 h at 37 8C. At the conclusion of the incubation both the donor and receiver chambers for each well were sampled and analyzed by LC/MS/MS as described below. Following the assay, monolayer integrity was checked using lucifer yellow. Briefly, 100 ml of a 100 mg/ml lucifer yellow solution in HBSSCM was added to the apical compartment of each well and 200 ml HBSSCM (without lucifer yellow) was added to the basolateral chamber. After incubating the cells for 1 h at 37 8C/95% humidity/5%CO2 the receiver (basolateral) chamber was sampled and the lucifer yellow-related fluorescence (excitation 485 nm, emission 535 nm) was determined using a BioTEK SynergyMX spectrophotometer (Winooski, VT). Wells were rejected if the permeability of the lucifer yellow was above 2%. For uptake assays (OCT1, OCT2, OCT3, OATP1A2, OATP1B1, OATP1B3, OATP2B1), transfected and wild-type HEK cells were plated on day 1 at 225,000 cells/ml, 1 ml per well of 24-well poly-Dlysine coated plates (BD Biosciences, Bedford, MA). To maximize transporter expression, on day 2 transfected and untransfected cells were treated with 10 mM sodium butyrate (Sigma) for 18–24 h and uptake assays were performed on day 3. Cells were washed 3 with 1 ml room temperature HBSSCM to remove any media. The final wash was left on the cells to allow equilibration in HBSSCM for 5– 40 min at 37 8C. For samples containing inhibitors, a 5 min preincubation was performed with the inhibitor in HBSSCM at 37 8C. After equilibration, the wash solution was aspirated and 250 ml of HBSSCM containing the compound of interest (or compound and inhibitor) was added and uptake proceeded for 5 min at 37 8C. The assay was stopped by washing 3 with ice cold HBSSCM, aspirating the final wash and any remaining buffer. Cells were lysed by adding 1 ml of methanol containing internal standard (norfentanyl-d5, 0.1 mg/ml final concentration) to each well and scraping to remove the cells. The entire volume of methanol was transferred to a deep well plate and cell debris was pelleted 5 min at 1000 g. Five hundred microliters of methanol was transferred to a new deep well plated and evaporated to dryness under nitrogen. Samples were resuspended in 200 ml of water and subjected to LC/MS/MS. Preliminary experiments evaluated time-dependency of uptake, and all experiments were conducted in the linear portion of the time course. Methadone and EDDP achiral analysis was performed on an API 4000 QTRAP triple-quadrupole mass spectrometer (Applied Biosystems/MDS Sciex, Foster City, CA), with Shimadzu XR HPLC system (Shimadzu, Columbia, MD) and C18 Sunfire column (2.1  50 mm, 3.5 mm, Waters Corp, Milford, MA). Injection

volume was 10 ml and the oven temperature was 40 8C. The HPLC mobile phase (0.3 ml/min) was (A) 4.5 mM ammonium acetate pH 4 and (B) acetonitrile. The gradient program for methadone was 15% B for 0.5 min, linear to 95% B over 0.25 min, held at 95% B for 1.75 min, returned linearly to 5% B over 6 s, then re-equilibrated for 1.5 min. Retention times were 2.2 and 2.4 min, respectively, for methadone and the internal standard, norfentanyl-d5. The gradient program for EDDP analysis was 5% B for 0.5 min, linear to 95% B over 0.5 min, held at 95% B for 1.5 min, returned linearly to 5% B over 6 s, then re-equilibrated for 1.5 min. Retention times were 2.4 and 2.5 min respectively, for EDDP and the internal standard, norfentanyl-d5. The mass spectrometer was operated in positive ion mode, ion spray voltage was optimized to 5500, curtain gas 20, ion source gas 1 at 30, ion source gas 2 at 40, collision gas medium, the source temperature was set at 450 8C and unit mass resolution was utilized. Multiple reaction monitoring transitions for the analytes and standards were m/z 310.2!265.1, 278.2!234.2 and 238.2!84.1 for methadone, EDDP and norfentanyl-d5 internal respectively. Samples were quantified using area ratios and standard curves prepared using individual methadone or EDDP calibration standards in blank media, as appropriate for the analyte quantified, to which was added the internal standard, and then analyzed as above. Interday coefficients of variation were less than 15%. Apparent permeability (Papp) for both the wild type and transfected MDCK cells was calculated using the following equation, where (Vr) is the volume of buffer in the receiver, (A) is the surface area of the PVDF membrane (0.14 cm2), (t) is the incubation time in seconds and (d) and (r) are the drug concentrations of the donor and receiver wells respectively at time t.

P app ¼

Vr ½Drugr  ð A  t Þ ½Drugd

The directional ratio (R) for each cell line was calculated by dividing the apparent permeability (Papp) for the basolateral-toapical direction by the Papp for the apical-to-basolateral direction.

R¼

P appB ! A P appA ! B

The relative efflux ratio (RER) was calculated by the dividing the directional ratio value for the transfected cell line by the directional ratio of the untransfected MDCK cells.

RER ¼

Rtransfected Runtransfected wild-type

2.6. Statistical analysis Hepatocyte incubations were generally performed in triplicate, and results presented as the mean  SD for each of the three livers. A 2-fold increase in enzyme activity, mRNA expression, or protein expression was considered positive induction [30]. Transport experiments were generally performed a minimum of three times. Transport rate vs substrate concentration data were

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analyzed by non-weighted non-linear regression analysis (SigmaPlot 12.5, Systat Software, San Jose, CA) using a simple Michaelis– Menten model or a Michaelis–Menten model with product inhibition (as appropriate). Results are the parameter estimate  standard error of the estimate. Effects of HIV antiretrovirals on transport were analyzed by analysis of variance (after log transformation when appropriate) with post hoc Dunnet’s testing (significance assigned at p < 0.05). 3. Results 3.1. Effects on hepatocyte CYP activity and methadone metabolism HIV antiretroviral effects on human hepatocyte CYP2B6 and CYP3A4/5 activities, and on methadone N-demethylation, were determined after 72 h, in cells from three humans (Fig. 1). CYP2B6 was induced more than 2-fold by amprenavir, lopinavir, nelfinavir and ritonavir, at both 1 and 10 mM, and by 10 mM nevirapine, with variability between livers. CYP3A4/5 activity was induced more than 2-fold only by indinavir, in one liver. Across all three livers, the positive controls for CYP2B6, and CYP3A4, 1 mM phenobarbital

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and 10 mM rifampin, respectively [30], induced CYP2B6 an average of 10- and 4-fold, and both induced CYP3A activity an average of 12-fold. Assuming that hepatocytes were fully induced by phenobarbital [30], CYP2B6 induction by nelfinavir and ritonavir was approximately 50% and 80% of maximum, respectively. Methadone N-demethylation was not increased consistently in all livers at a 2-fold level of induction by any of the HIV drugs tested, at either concentration, however small increases were observed for maraviroc, nelfinavir and nevirapine in some livers. After the three substrate incubations, mRNA transcripts for CYPs 2B6, 3A4 and 3A5 were quantified (Fig. 2). CYP2B6 mRNA expression was increased more than 2-fold by amprenavir, nevirapine and ritonavir. CYP3A4 mRNA was increased 2-fold or more by amprenavir, atazanavir, lopinavir, nelfinavir, and ritonavir, with substantial variability between livers. None of the compounds increased CYP3A5 mRNA more than 2-fold. Phenobarbital and rifampin (positive controls) increased CYP2B6 and CYP3A4 mRNA 5- to 17-fold. Phenobarbital increased CYP3A5 mRNA 3-fold in one liver. To determine if HIV drugs increased protein synthesis, CYP3A4 was evaluated by Western blot (Fig. 3). CYP3A4 protein was

Fig. 1. HIV antiretroviral drug effects on CYP2B6 and CYP3A4/5 activities and methadone metabolism in human hepatocytes. Media containing the indicated concentration of drug or DMSO (control) was added to fresh plated human hepatocytes each day for 3 d. Activity of CYP2B6 (bupropion hydroxylation) and CYP3A4/5 (alfentanil metabolism to noralfentanil) and methadone N-demethylation (EDDP formation) was then determined. Fold induction results represent normalization to the 0.1% DMSO control. Antiretroviral drugs were evaluated at 1 mM (white bars) and 10 mM (grey bars), respectively. Phenobarbital (1 mM) and rifampin (10 mM) were evaluated as positive induction controls (black bars). Each data point is the mean  SD of triplicate determinations. Results are shown for each of the three livers evaluated (Hu 1284, 1290, 1331, left to right, for each inducer tested, as open bars, upward hatched bars, and downward hatched bars, respectively). The dotted line shows 2-fold induction.

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Fig. 2. Effects of HIV antiretroviral drugs on CYP mRNA expression in human hepatocytes. mRNA content was determined by quantitative PCR. Quantitative PCR for GAPDH was run in parallel as an internal control and the calculated fold induction corrected for differences in GAPDH number. Antiretroviral drugs were evaluated at 1 mM (white bars) and 10 mM (grey bars), respectively. Fold induction results represent normalization to the 0.1% DMSO vehicle control. Phenobarbital (1 mM) and rifampin (10 mM) were evaluated as positive induction controls. CYP2B6 mRNA, CYP3A4 mRNA, and CYP3A5 mRNA induction averaged (SD) 8  2-fold, 17  10-fold, and 2  1 fold, respectively, by phenobarbital, and 5  3-fold, 15  9-fold, and 1  1-fold, respectively, by rifampin. Results are shown for each of the three livers evaluated (Hu 1284, 1290, 1331, left to right, for each inducer tested, as open bars, upward hatched bars, and downward hatched bars, respectively). The dotted line shows 2-fold induction. Each data point is a single determination.

increased more than 2-fold by amprenavir, atazanavir, indinavir, nelvinavir, nevirapine, and ritonavir, with much greater induction by amprenavir, atazanavir and ritonavir. Both positive controls induced CYP3A4 protein more than 60-fold. CYP2B6 protein expression was similarly evaluated, using multiple CYP2B6 antibodies from various sources. Nevertheless, an increase in CYP2B6 protein expression could not be detected, even for phenobarbital and rifampin (data not shown). Western blot analysis of hepatocyte CYP2B6 protein expression was previously shown to be not as sensitive or quantitative, and to underestimate induction, compared with mRNA and enzyme activity [31]. 3.2. Effects on CYP regulation A reporter gene assay assessed the ability of the HIV drugs to function as a PXR agonist (Fig. 4). Compounds were incubated with HepG2 cells transfected with both a PXR response element and the luciferase gene, and luciferase activity was used to measure transcription of genes under control of the PXR response element.

With the exception of lopinavir and nelfinavir, all compounds increased PXR-related luminescence when incubated at 10 mM, and amprenavir, maraviroc, nevirapine and ritonavir were sufficiently potent to induce a 2-fold response at 1 mM. The positive controls (rifampin, mifepristone, androstanol) all increased PXR-dependent response. 3.3. Methadone and EDDP transport To assess the potential for transport to influence methadone and EDDP concentrations in media, MDCK cells transfected with the major hepatocyte efflux proteins P-gp and BCRP, and HEK cells transfected with the uptake proteins OCT1, OCT2, OCT3, OATP1A2, OATP1B1, OATP1B3, and OATP2B1 were used to determine the transport of methadone and EDDP (Table 2). Methadone was not a substrate for any of the uptake or efflux transporters (relative transport ratios all less than 2). The positive controls had uptake and efflux ratios consistent with those reported previously [32–34].

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Fig. 3. HIV antiretroviral drug effects on CYP protein expression in human hepatocytes. Subsequent to the determination of catalytic activity, cell then were lysed. CYP3A4 protein content was determined by Western blot analysis, loading 15 mg of total protein into each well of a 4–12% Bis-Tris gel. The intensities of both the CYP3A4 and b-actin bands for each sample were measured by densitometry using Odyssey Imaging System and Image Studio Software 2.0 (Li-COR Biosciences) and ratio of 3A4 to reference protein (b-Actin) calculated. Fold induction was calculated by comparing the CYP3A4/b-actin band density ratio for each drug treatment to the CYP3A4/b-actin band density ratio for the vehicle control. Phenobarbital (1 mM) and rifampin (10 mM) were evaluated as positive controls, which increased CYP3A4 expression an average of 66  42-fold and 79  50-fold, respectively. Results are shown for each of the three livers evaluated (Hu 1284, 1290, 1331, left to right, for each inducer tested, as open bars, upward hatched bars, and downward hatched bars, respectively). The dotted line shows 2-fold induction. Each data point is a single determination.

In contrast to methadone, EDDP was a substrate for efflux by both P-gp and BCRP, with relative efflux ratios of 2.2 and 4.8, respectively. In addition, EDDP was a substrate for uptake by OCT1, OCT3, OATP1A2, and OATP1B1, but not OCT2, OATP1B3 or OATP2B1. EDDP uptake by OCT1, OCT3, and OATP1A2, the most active EDDP transporters, was evaluated further. Uptake was concentrationdependent and saturable (Fig. 5). Apparent Km, Vmax and CLint values were 4.5  3.4 mM, 11.1  3.9 pmol/min/mg protein, and 2.5  2.1 ml/ min/ng for OCT1; 10.5  8.0, 18.2  9.7, and 1.7  1.6 for OCT3; and 4.0  0.7, 28.0  1.5, and 7.0  1.3 for OATP1A2. The influence of HIV antiretrovirals, and the non-selective inhibitor cyclosporin, on EDDP uptake by OCT1, OCT3, and OATP1A2 was next evaluated (Fig. 6). Of the three uptake transporters, OATP1A2 was most affected, with EDDP uptake inhibited by the non-selective inhibitor cyclosporin, and by

Fig. 4. HIV antiretroviral drug effects on PXR activation. PXR activation was assessed using a reporter gene assay, using a proprietary method and cell line (Puracyp, Inc). HepG2 cells were stably transfected with the PXR response element and a luciferase gene. Cells were plated and incubated in the presence of the indicated concentration of drug, or 0.1% DMSO for 48 h, and luciferase activity and cell viability then determined. Results (mean  SD, n = 3) are expressed as fold increase in luminescence for each incubation condition compared to that for the 0.1% DMSO control and are corrected for cell viability. Positive controls rifampin, mifepristone, and androstanol (all 10 mM) increased PXR activation 17  1-fold, 12  1-fold, and 3  1fold, respectively.

amprenavir, atazanavir, efavirenz, lopinavir, nelfinavir, ritonavir, and saquinavir. OCT3-mediated EDDP uptake was inhibited only by amprenavir, atazanavir, efavirenz, and nelfinavir, and only at 10 mM. EDDP uptake by OCT1 was not affected by any of the drugs tested. The influence of HIV antiretrovirals and cyclosporin on BCRP-mediated efflux of EDDP was also evaluated (Fig. 7). Apparent inhibition was observed with low (1 mM) concentrations of cyclosporin, amprenavir, atazanavir, efavirenz, lopinavir, and maraviroc, with apparent stimulation by some compounds at higher concentrations. 4. Discussion 4.1. Methadone metabolism This investigation evaluated effects of various HIV antiretrovirals on methadone N-demethylation using plated human hepatocytes. We previously showed that the non-nucleoside reverse transcriptase inhibitor efavirenz induced CYP2B6 and CYP3A4 mRNA expression and catalytic activities, and methadone Ndemethylation, in primary human hepatocytes [5]. In contrast, none of the HIV drugs tested, after incubation with hepatocytes for 3 d, upregulated methadone metabolism, based on quantification of EDDP in the culture medium. The positive controls phenobarbital and rifampin, evaluated to verify that a particular donor’s hepatocytes had induction potential, did substantially induce methadone N-demethylation 4- to 5-fold, as previously observed [5]. Another study also found no significant effect of nelfinavir (4d) on EDDP formation by hepatocytes, while rifampin increased EDDP formation approximately 10-fold, although CYP expression and activity were not evaluated [35]. Thus, the present and prior findings demonstrate that of the HIV antiretrovirals and other compounds tested, only efavirenz, phenobarbital, and rifampin induced methadone N-demethylation in human hepatocytes, based on EDDP measurement in the culture medium. In contrast, antiretroviral effects on hepatocyte expression and activity of CYP2B6 and CYP3A were greater than effects on methadone metabolism, and in general agreement with previous observations, with some important differences [2,36–38]. Of the antiretrovirals tested, ritonavir was the most effective inducer of CYP2B6 mRNA and catalytic activity, demonstrating upregulation of CYP2B6 expression without apparent enzyme inhibition, thus

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Table 2 Methadone and EDDP transport by uptake and efflux proteins. Substratea

Methadone EDDP

Net uptake ratio (transfected/wild type)c

Net efflux ratio (transfected/ wild type)b BCRP

P-glycoprotein

OCT1

OCT2

OCT3

OATP1A2

OATP1B1

OATP1B3

OATP2B1

0.9  0.2 (6) 5.1  0.6 (6)

1.5  0.6 (12) 2.2  0.1 (3)

1.8  0.9 (3)d 7.5  0.4 (3)

1.4  0.6 (3) NDe (3)

1.2  0.1 (3) 5.4  0.2 (3)

1.4  0.3 (6) 17.2  0.6 (3)

1.7  0.1 (3) 2.4  0.6 (3)

1.3  0.1 (3) 1.7  0.4 (3)

1.0  0.1 (3) 1.3  0.1 (3)

All results are the mean  SD (n). a Methadone and EDDP concentrations were 2 mM in efflux experiments and 1 mM in uptake experiments. b Net efflux ratio for the P-gp positive control 2 mM loperamide was 12.4  6.6 (12); net efflux ratios for the BCRP positive controls 2 mM prazosin and imatinib were 5.5  1.8 (12) and 4.2  1.6 (18). c Net uptake ratios for the positive control 10 mM estrone-3-sulfate were 17.5  5.2 (11), 2.8  0.1 (3), 3.2  1.0 (3), 10.3  1.1 (3) and 2.0  0.8 (3) for OATP1A2, OATB1B1, OATP1B3, OATB2B1, and OCT2. Net uptake ratios for the positive control 10 mM tetraethylammonium (TEA) were 26  18 (3) and 29  2 (3) for OCT1 and OCT3. d Uptake ratio was 1.7  0.5 (3) at 10 mM methadone. e Not detectable at 1 mM. Uptake ratio was 0.4  0.1 (3) at 10 mM EDDP.

causing net induction. In contrast, ritonavir reportedly inhibited CYP2B6 activity in human liver microsomes [39]. Lopinavir, nelfinavir, and nevirapine also induced CYP2B6 activity. Mild lopinavir induction of CYP2B6 occurred without commensurate increase in mRNA, as observed previously [37]. Conversely, several antiretrovirals upregulated CYP3A transcripts and/or protein expression, without increased catalytic activity, reflecting welldescribed mechanism-based inhibition of newly synthesized CYP3A protein [17,18]. Unlike Liu et al. [38], who reported robust induction of both CYP3A4 mRNA and catalytic activity by amprenavir, despite being an effective CYP3A inhibitor [17,18,40], we found induction of CYP3A4 mRNA and protein but not CYP3A activity. Similarly, atazanavir induced CYP3A4 transcripts and protein expression but not catalytic activity, while Liu et al. described increased activity [38]. In general, CYP3A5 mRNA induction was less than that of CYP3A4, and that observed previously [38]. Upregulation of CYP transcription by antiretrovirals appears generally related to PXR activation, although lopinavir does cause some constitutive androstane receptor activation [37]. Although an effective PXR activator, maraviroc did not increase CYP3A or CYP2B6 transcripts, protein expression or catalytic activity, consistent with lack of effects on clinical CYP3A activity or drug interactions [41]. The positive controls rifampin and phenobarbital did show that CYP3A and CYP2B6 were inducible in all three livers. Although there are some differences between this and prior reports, possibly related to donor and experimental differences, the overall general conclusion is that in human hepatocytes, several HIV drugs (protease inhibitors

Fig. 5. EDDP uptake by OCT1, OCT3, and OATP1A2. Uptake was assessed using HEK cells stably transfected with OCT1, OCT3, or OATP1A2. EDDP concentration was 1– 40 mM. EDDP uptake is the observed rate in transfected HEK cells, from which was subtracted the rate in untransfected cells. The observed rate with untransfected HEK cells is also shown. Each data point is the mean  SD (n = 6–9). Lines represent rates predicted using Michaelis–Menten kinetic parameters derived from non-linear regression analysis of the data.

and nevirapine) variably activated PXR, induced CYP2B6 transcription and catalytic activity, and upregulated CYP3A transcription and protein expression without net induction of catalytic activity. These observations on CYP regulation in hepatocytes are consistent with clinical studies [24,25,42,43]. 4.2. Methadone and EDDP transport There is considerable interest in the potential role of active transport in methadone disposition, particularly hepatic and renal elimination, and in brain access. The efflux ratio for methadone observed in human P-gp overexpressing cells (1.6, which is below the cut-off of 2 considered for substrate activity) resembled that described by others [44,45]. Together this shows that methadone is a weak substrate for human P-gp, if at all, and certainly compared with loperamide, and methadone transport by human P-gp is far less than by rodent P-gp [46]. Weak human P-gp methadone transport is consistent with results of clinical drug interaction studies [47,48], but inconsistent with reports of P-gp genetic influences on methadone dose requirements in patients [49,50]. Similarly, methadone was not a substrate for BCRP efflux transport, or the uptake transporters OCT1-3, and OATPs 1A2, 1B1, 1B3, and 2B1. Identification of the protein(s) responsible for methadone transport, and mechanism(s) by which ritonavir and nelfinavir [20,51], but not efavirenz or lopinavir [5,21], increase methadone renal clearance remains unattained. Similarly, transporters responsible for methadone pharmacodynamic drug interactions at the blood brain barrier [5,8,20], also remain unidentified. Identification of EDDP as a substrate for uptake by OCT1, OCT3, and particularly OATP1A2, and efflux by P-gp and BCRP, is novel. Uptake by OATP1A2 but not OATP1B1 or 1B3 is consistent with the preference of OATP1A2 and OATP1B1/3 for basic and acidic compounds, respectively [52]. EDDP is pharmacologically inactive, but is the primary methadone metabolite and used as an index of N-demethylation; thus susceptibility to potential transporterbased interactions may further add to the complexity of methadone disposition. For example, EDDP was quantified in the hepatocyte media, and transport might have influenced its concentration. Several antiretrovirals (e.g. ritonavir, lopinavir, nelfinavir, saquinavir, amprenavir, atazanavir) are effective BCRP inhibitors [2,53], and amprenavir, atazanavir, and lopinavir did inhibit BCRP-mediated EDDP efflux. Similarly, several antiretrovirals inhibited OCT3- and particularly OATP1A2-mediated EDDP uptake. Antiretroviral or other drug interactions may influence EDDP concentrations in hepatocyte media in vitro, and EDDP transport by hepatocytes and renal tubular cells and disposition in plasma and urine in vivo. Substantially greater transport of EDDP than methadone by OCT1, OCT3, OATP1A2, P-gp, and BCRP is notable. EDDP is considered an unusual metabolite, with physicochemical properties considerably

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Fig. 7. Effects of HIV antiretroviral drugs on EDDP efflux by BCRP. Efflux of EDDP (2 mM) was determined in the absence or presence of 1–10 mM antiretroviral drug, using ABCG2-transfected BCRP-expressing cells on 96 well Transwell inserts, as described in Section 2. Results (mean  SD, n = 2–3) are expressed relative to the transfected/wild type transport ratio with EDDP alone. *Significantly different vs EDDP alone (p < 0.05) **Significantly different vs EDDP alone (p < 0.001).

4.3. Methadone disposition in vitro vs in vivo

Fig. 6. Effects of HIV antiretroviral drugs on EDDP uptake by OCT1, OCT3, and OATP1A2. Uptake was assessed using HEK cells stably transfected with OCT1, OCT3, or OATP1A2. EDDP uptake (2 mM) was assessed with EDDP alone or with 1–10 mM antiretroviral drug. EDDP uptake is the observed rate in transfected HEK cells, from which was subtracted the rate in untransfected cells. Each data point is the mean  SD (n = 6). *Significantly different vs EDDP alone (p < 0.05). **Significantly different vs EDDP alone (p < 0.001).

different from those of the parent drug [54]. Conversion of methadone to EDDP reduces lipophilicity (cLogD decreases 6 log units from 2.3 to 3.5) and topological polar surface area (from 20.3 to 3.0). The high polarity of EDDP renders it unfavorable for lipophilic diffusion from hepatocytes into the circulation [54], potentially accounting for low plasma metabolite/parent (EDDP/ methadone) concentration ratios, and also increasing dependence on hepatocyte transport for transfer into the circulation. These physicochemical differences may also underlie greater transporter affinity for EDDP compared with methadone.

One major objective of this investigation was to use human hepatocytes to reconcile apparent differences between in vitro methadone metabolism and drug interactions (expressed CYPs and human liver microsomes) and clinical methadone metabolism, specifically, the discrepancy between CYP isoform(s) responsible for methadone metabolism. Methadone N-demethylation in vitro, both by human CYPs and liver microsomes, is catalyzed most efficiently by CYP2B6 and CYP3A42 [7–11]. In contrast, clinical drug interaction studies show that CYP2B6, rather than CYP3A4, is the predominant CYP responsible for methadone disposition. Specifically, neither CYP3A induction [55], nor strong CYP3A inhibition [8,20,21,51,56], altered methadone N-demethylation or clearance, while CYP2B6 induction or inhibition did modulate methadone metabolism and disposition [10,20,57]. There is similar discordance between antiretroviral effects on methadone metabolism in vitro and in vivo. The HIV protease inhibitors amprenavir, indinavir, nelfinavir, ritonavir, saquinavir, atazanavir, tipranavir, and lopinavir are strong CYP3A4 inhibitors in vitro [17,18], and ritonavir, indinavir, and saquinavir inhibited human liver microsomal methadone N-demethylation [19]. Nevertheless, atazanavir, indinavir, ritonavir/indinavir and ritonavir/saquinavir had no effect on clinical methadone clearance and plasma concentrations, and, moreover, ritonavir, ritonavir/lopinavir, ritonavir/tipranavir, and nelfinavir actually increased clearance [3,20,21,51,56]. In addition to the explanation that CYP2B6 is a more important determinant than CYP3A4 of clinical methadone metabolism and clearance, another possible factor is that antiretroviral effects on expressed and liver microsomal CYP3A in vitro may differ from those in vivo, due to CYP induction in vivo as well as inhibition [24,25]. Use of hepatocytes as an intermediate model between clinical and expressed or microsomal methadone metabolism to resolve the in vitro–in vivo discordance, was therefore attractive. Nevertheless, a major conclusion of this investigation is that hepatocytes underreported induction of methadone N-demethylation compared with clinical studies. Rifampin upregulation of

2 Preliminary experiments in human hepatocytes found that inhibition of CYP3A (by ritonavir) and CYP2B6 (by clopidogrel) diminished metabolism of both methadone enantiomers, suggesting a role for both CYP isoforms in human hepatocytes as well as in microsomes (S. Campbell and E. Kharasch, unpublished observation).

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hepatocyte CYP2B6 and CYP3A4 activities and methadone Ndemethylation, here and previously [5,35], did reflect clinical induction of methadone metabolism and clearance [8], but this was not generalizable to the several HIV drugs evaluated. A second major conclusion is that methadone N-demethylation in hepatocytes resembled metabolism by expressed and human liver microsomal CYPs more than clinical metabolism. Hepatocyte Ndemethylation was unaffected when CYP2B6 was induced and CYP3A4 activity unchanged, whereas coordinate upregulation of CYPs 2B6 and 3A4 did increase methadone metabolism. Thus, hepatocytes did not resolve the in vitro–in vivo disconnect between CYP3A4 and methadone metabolism, and do not appear to be a robust predictor of clinical methadone drug interactions. Several factors may explain hepatocytes underreporting induction of methadone N-demethylation. Hepatocytes in general appear less sensitive to CYP induction by HIV protease inhibitors compared with effects in vivo [24,25,36,38]. Additional factors [24,25], and others include: (a) different in vitro and in vivo drug exposure profiles (tonic vs phasic concentrations, intracellular concentration differences, metabolic depletion of intracellular inducer or substrate), (b) different time courses of induction, (c) different in vitro vs in vivo expression profiles of nuclear receptors mediating induction, (d) diminished intrinsic sensitivity of cultured hepatocytes, and (e) differences between plated vs sandwich-cultured hepatocytes. In addition to these CYP-related factors, other possibilities include in vitro and in vivo differences in the expression and activity of influx and efflux transporters, and their potential induction or inhibition by antiretrovirals, which might influence hepatic (or renal) methadone or EDDP transport. There are potential limitations to the experiments presented. CYP2B6 could not be reliably quantified by Western blot, as observed previously [27,31], since experiments were performed before mass spectrometric methods were available [27]. Hepatocytes were not genotyped for CYP polymorphisms. Activity of multidrug resistance transporters was not a focus of this investigation. Methadone enantiomers metabolism and transport were not evaluated because there were minimal antiretroviral effects on racemate metabolism, and further enantiomer evaluations were therefore not indicated, and, similarly, methadone racemate transport was negligible and lack of stereoselectivity in methadone transport was previously described [44]. In summary, this investigation evaluated inductive effects of several antiretrovirals on methadone N-demethylation to EDDP in human hepatocytes, along with CYP2B6 and CYP3A4 transcripts, protein expression and catalytic activity, and PXR activation. Several drugs caused net induction of CYP2B6 activity, and/or upregulation of CYP3A4 protein expression without change in CYP3A catalytic activity, without apparent induction of methadone N-demethylation. Compared with clinical studies, human hepatocytes underreport the induction of methadone metabolism by HIV drugs. Hepatocytes are not a good predictive model for clinical antiretroviral induction of methadone metabolism and not a substitute for clinical studies. Methadone was not a substrate for uptake (OCT1, OCT2, OCT3, OATP1A2, OATP1B1, OATP1B3, OATP2B1) or efflux (P-gp, BCRP) transporters, but EDDP was an excellent substrate for P-gp, BCRP, OCT1, OCT3, and OATP1A2, and EDDP transport was inhibited by several antiretrovirals. The major clinical implication of this investigation is that clinical drug interaction studies remain the best methodology for assessing methadone–antiretroviral drug interactions. Acknowledgements Supported by National Institutes of Health (Bethesda, MD) grants R01-DA14211, R01-GM63674, R01-DA25931, and K24DA00417.

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