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`0090-9556/06/3403-440–448$20.00
`DRUG METABOLISM AND DISPOSITION
`Copyright © 2006 by The American Society for Pharmacology and Experimental Therapeutics
`DMD 34:440–448, 2006
`
`Vol. 34, No. 3
`6148/3091873
`Printed in U.S.A.
`
`NOVEL METABOLITES OF BUPRENORPHINE DETECTED IN HUMAN LIVER
`MICROSOMES AND HUMAN URINE
`
`Yan Chang, David E. Moody, and Elinore F. McCance-Katz
`
`Center for Human Toxicology, Department of Pharmacology and Toxicology, University of Utah, Salt Lake City, Utah
`(Y.C., D.E.M.); and Division of Addiction Psychiatry, Virginia Commonwealth University, Richmond, Virginia (E.F.M.-K.)
`
`Received June 17, 2005; accepted December 19, 2005
`
`ABSTRACT:
`
`The in vitro metabolism of buprenorphine was investigated to
`explore new metabolic pathways and identify the cytochromes
`P450 (P450s) responsible for the formation of these metabolites.
`The resulting metabolites were identified by liquid chromatogra-
`phy-electrospray ionization-tandem mass spectrometry. In addi-
`tion to norbuprenorphine, two hydroxylated buprenorphine (M1
`and M2) and three hydroxylated norbuprenorphine (M3, M4, and
`M5) metabolites were produced by human liver microsomes
`(HLMs), with hydroxylation occurring at the tert-butyl group (M1
`and M3) and at unspecified site(s) on the ring moieties (M2, M4, and
`M5). Time course and other data suggest that buprenorphine is
`N-dealkylated to form norbuprenorphine, followed by hydroxyla-
`tion to form M3; buprenorphine is hydroxylated to form M1 and M2,
`
`followed by N-dealkylation to form M3 and M4 or M5. The involve-
`ment of selected P450s was investigated using cDNA-expressed
`P450s coupled with scaling models, chemical inhibition, monoclo-
`nal antibody (MAb) analysis, and correlation studies. The major
`enzymes involved in buprenorphine elimination and norbuprenor-
`phine and M1 formation were P450s 3A4, 3A5, 3A7, and 2C8,
`whereas 3A4, 3A5, and 3A7 produced M3 and M5. Based on MAb
`analysis and chemical
`inhibition, the contribution of 2C8 was
`higher in HLMs with higher 2C8 activity, whereas 3A4/5 played a
`more important role in HLMs with higher 3A4/5 activity. Examina-
`tion of human urine from subjects taking buprenorphine showed
`the presence of M1 and M3; most of M1 was conjugated, whereas
`60 to 70% of M3 was unconjugated.
`
`Buprenorphine, a semisynthetic derivative of the alkaloid thebaine
`(Lewis, 1973), is a partial ␮-opioid agonist and ␬-opioid antagonist
`(Cowan et al., 1977). It was first developed as an analgesic for
`moderate to severe pain in the early 1970s, but is currently more
`widely used as a replacement therapy for opiate dependence. Bu-
`prenorphine has comparable effects to methadone in regard to treat-
`ment of opiate-dependent patients (Strain et al., 1996; Johnson et al.,
`2000), but has reduced risk because of the “ceiling effect” associated
`with its partial ␮-opioid agonist properties (Walsh et al., 1994, 1995).
`Absorption, distribution, metabolism, and excretion studies of bu-
`prenorphine have been carried out in humans using gas chromatog-
`raphy-mass spectrometry (Cone et al., 1984), and in animals using
`thin-layer chromatography of tritiated buprenorphine (Brewster et al.,
`1981; Pontani et al., 1985). These studies suggested that buprenor-
`phine was mainly metabolized by N-dealkylation and glucuronidation
`of both buprenorphine and norbuprenorphine. A tentative 6-O-dem-
`ethyl norbuprenorphine in free and conjugated form was observed in
`rat urine (Pontani et al., 1985), and some unknown polar metabolites
`
`This study was supported by National Institute on Drug Abuse Grants R01
`DA10100 (D.E.M.), RO1 DA 13004 (E.M.K.), and KO2 DA00478 (E.M.K.), and by
`the General Clinical Research Center at Virginia Commonwealth University
`(M01RR00065, National Center for Research Resources/National Institutes of
`Health).
`Article, publication date, and citation information can be found at
`http://dmd.aspetjournals.org.
`doi:10.1124/dmd.105.006148.
`
`were found in rat bile samples (Brewster et al., 1981). No evidence
`was given for additional metabolites in humans (Cone et al., 1984).
`However, a recent study by Picard et al. (2005) using liquid chroma-
`tography-tandem mass spectrometry identified the presence of two
`hydroxylated metabolites, one of buprenorphine and one of norbu-
`prenorphine, in human liver microsomes (HLMs) and urine samples
`from patients treated with buprenorphine. Buprenorphine N-dealkyla-
`tion is mainly catalyzed by cytochrome P450 (P450) 3A4 (Iribarne et
`al., 1997; Kobayashi et al., 1998), with involvement of P450 3A5 and
`2C8 (Moody et al., 2002; Picard et al., 2005). The involvement of
`specific P450s in production of the hydroxylated metabolites was
`limited to a finding that trace amounts of hydroxy-buprenorphine
`were produced by P450 3A4-, 3A5-, and 3A7-transfected cell lines
`(Picard et al., 2005).
`In our previous study, we observed a higher rate of buprenorphine
`elimination than of norbuprenorphine formation in HLMs, suggesting
`that there might be some other routes for metabolism of buprenor-
`phine or its metabolites (Chang and Moody, 2005). In this paper, we
`report a study of the metabolism of buprenorphine in HLMs and
`analysis of human urine from subjects treated with buprenorphine.
`The identification of new metabolites was achieved by liquid chro-
`matography-electrospray ionization-tandem mass spectrometry (LC-
`ESI-MS/MS), and the involvement of P450s in the formation of new
`metabolites was clarified using cDNA-expressed human P450s and
`correlations with a panel of HLMs. The contribution of each enzyme
`was estimated by inhibitory analysis using monoclonal antibodies
`
`ABBREVIATIONS: HLM, human liver microsome; P450, cytochrome P450; MAb, monoclonal antibody; NADPH GS, NADPH generating system;
`LC-ESI-MS/MS, liquid chromatography-electrospray ionization-tandem mass spectrometry; MS, mass spectrometer; SRM, selected reaction
`monitoring; SIM, selected ion monitoring; CID, collision-induced dissociation; RAF, relative activity factor.
`
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`NOVEL METABOLITES OF BUPRENORPHINE
`
`441
`
`(MAbs) and chemical inhibitors in phenotyped HLMs, and also was
`predicted by relative activity factor (RAF) and immunoquantification
`scaling approaches. Based on our results, an extended biotransforma-
`tion profile is proposed for buprenorphine.
`
`Materials and Methods
`
`Materials. Buprenorphine (for incubation), D-glucose 6-phosphate monoso-
`dium salt, glucose-6-phosphate dehydrogenase, ␤-NADP sodium salt, EDTA
`disodium salt, MgCl2, ␤-glucuronidase (from Helix pomatia, which also has
`sulfatase activity),
`trimethoprim, 8-methoxypsoralen, sulfaphenazole, and
`quinidine were obtained from Sigma-Aldrich (St. Louis, MO). Furafylline was
`obtained from Synergy House (Manchester, UK). Buprenorphine (for analy-
`sis), d4-buprenorphine, norbuprenorphine, and d9-norbuprenorphine were pur-
`chased from Cerilliant (Round Rock, TX). Ketoconazole was obtained from
`ICN Biomedicals Inc. (Aurora, OH). 10-Hydroxybuprenorphine, buprenor-
`phine N-oxide, and 10-oxobuprenorphine were provided by Reckitt Benckiser
`Healthcare Limited (Hull, UK). The liver samples were obtained from Tissue
`Transformation Technologies (Edison, NJ). Insect cell cDNA-expressed hu-
`man P450s (Supersomes) and 15 phenotyped HLMs were purchased from BD
`Gentest (Woburn, MA). Inhibitory MAbs to human P450 3A4/5 and 2C8 were
`provided by the National Cancer Institute of the National Institutes of Health
`(Bethesda, MA). All aqueous reagents were prepared in purified water (spe-
`cific resistance ⬎18.2 m⍀/cm) obtained by a Milli-Q Plus water purification
`system (Millipore, Billerica, MA).
`In Vitro Incubations of Buprenorphine with HLMs. Microsomes were
`prepared from human liver by differential centrifugation as described by
`Nelson et al. (2001). The first centrifugation was at 9000g; the homogenization
`buffer contained 0.25 M sucrose, and 10 strokes of homogenization were used.
`HLMs prepared in our laboratory are not thoroughly phenotyped; to enhance
`the probability of having a representative amount of different P450 enzymes,
`pooled HLMs (n ⫽ 5) were used for initial metabolite identification studies.
`The incubation mixture (final volume 500 ␮l) contained incubation buffer (0.1
`M phosphate buffer, pH 7.4 with 1.0 mM EDTA and 5.0 mM MgCl2), a
`NADPH-generating system (NADPH GS) composed of 10 mM glucose
`6-phosphate, 1.2 mM NADP, and 1.2 units of glucose-6-phosphate dehydro-
`genase, 0.5 mg/ml microsomal protein, and 10 ␮M buprenorphine or norbu-
`prenorphine. The reaction was initiated by adding the NADPH GS and incu-
`bated at 37°C in a shaking water bath for the specified times. For qualitative
`studies, after a 30-min incubation, the mixture was adjusted to pH ⬎10 with 50
`␮l of 1 N NaOH, followed by extraction with a mixture of n-butyl chloride and
`acetonitrile (4:1, v/v). For quantitative studies, the reaction was terminated by
`the addition of 200 ␮l of ice-cold methanol, and the samples were stored at
`⫺75°C until analysis.
`In Vitro Incubations of Buprenorphine with Recombinant Human
`P450s. The metabolism of buprenorphine and norbuprenorphine was evaluated
`in microsomes prepared from insect cells transfected with cDNAs encoding for
`human P450s 1A2, 2A6, 2B6, 2C8, 2C9*1, 2C18, 2C19, 2D6*1, 2E1, 3A4,
`3A5, and 3A7. Supersomes that coexpressed cytochrome b5 were used where
`available; this was not the case for 1A2, 2C18, and 3A5. Buprenorphine or
`norbuprenorphine (10 ␮M) was incubated at 37°C for 20 min with 25 pmol of
`P450 in the incubation buffer described above. Control insect cell microsomes
`were used at the mean protein concentration averaged over all of the Super-
`somes. All reactions were initiated by addition of the NADPH GS and stopped
`by the addition of 200 ␮l of ice-cold methanol, after which the samples were
`stored at ⫺75°C until analysis.
`Inhibition of Buprenorphine Metabolism Using MAbs. The role of P450
`3A4/5 and 2C8 was measured by the addition of the P450 target-specific MAb,
`either alone or in combination, to the reaction mixture, using the procedure
`proposed by Yang et al. (1999). The recommended volumes (10 ␮l) of MAbs
`specific for P450 3A4/5 or 2C8 were mixed with phenotyped HLMs in 0.5 ml
`of incubation buffer and preincubated for 5 min at 37°C. Tubes were then
`placed on ice, buprenorphine was added (final concentration 10 ␮M), and the
`reaction was initiated by addition of the NADPH GS. The reaction continued
`for specified times at 37°C and was terminated with 200 ␮l of ice-cold
`methanol. Ten microliters of egg lysozyme was used as a control.
`Chemical Inhibition Studies. The effect of the selective P450 inhibitors on
`buprenorphine metabolism was first studied in pooled HLMs. Subsequently,
`
`more extensive studies were performed in phenotyped HLMs using the selec-
`tive P450 3A4/5 inhibitor ketoconazole (2 ␮M) (Newton et al., 1995; Sai et al.,
`2000) and the selective P450 2C8 inhibitor trimethoprim (100 ␮M) (Wen et al.,
`2002). The inhibitor and buprenorphine (final concentration 10 ␮M) were
`added to the reaction mixture, and the reaction was initiated by the addition of
`the NADPH GS in a 37°C shaking water bath. The reaction continued for
`specified times and was terminated by the addition of 200 ␮l of methanol. The
`incubation sample with no inhibitor served as control.
`Correlation Studies. HLMs from 15 individual donors, along with data for
`P450-specific enzyme activities, provided by BD Gentest, were used to study
`the relationship between the metabolism of buprenorphine and the metabolism
`of selective P450 substrates. The ability of HLMs from each donor to metab-
`olize buprenorphine was correlated with the P450-specific enzyme activities
`for each sample. The assay was performed with 10 ␮M buprenorphine and
`incubated for the specified times.
`In Vivo Metabolism of Buprenorphine. Twenty-four-hour postdose urine
`samples were collected from seven subjects who had been maintained on a
`daily sublingual dose of 16 mg of buprenorphine for at least 21 days. A 1-ml
`aliquot of each urine sample was adjusted to pH 5 with sodium acetate buffer
`(0.1 M) and treated with 5000 units of ␤-glucuronidase (containing sulfatase).
`The mixture was incubated at 50°C for 16 h. Another aliquot of the urine
`samples was analyzed without hydrolysis. Blank urine samples also underwent
`hydrolysis to control for interference arising from endogenous materials.
`LC-ESI-MS/MS Analysis. The quantification of buprenorphine and nor-
`buprenorphine (or semiquantification of hydroxylated metabolites) in incuba-
`tion samples and urine samples was performed using a modification of our
`previously described LC-ESI-MS/MS method (Moody et al., 2002). The in-
`cubation samples were made basic (pH ⬎10) by the addition of 50 ␮l of 1 N
`NaOH and extracted with a 4-ml mixture of n-butyl chloride and acetonitrile
`(4:1, v/v); the organic layer was dried under N2. The final residue was
`reconstituted to a volume of 75 ␮l using the initial mobile phase, and 20 ␮l was
`injected into the liquid chromatograph.
`Mass spectrometric analysis was performed on a TSQ 7000 or TSQ Quan-
`tum spectrometer (Thermo Electron, San Jose, CA) equipped with a triple-
`quadrupole MS and an ESI source operated at 4.5 kV. The MS was set to scan
`for positive ions. Quantification was performed by selected reaction monitor-
`ing (SRM) transitions m/z 468 to m/z 396 (buprenorphine), m/z 414 to m/z 101
`(for TSQ 7000) (norbuprenorphine), m/z 472 to m/z 400 (d4-buprenorphine),
`and m/z 423 to m/z 110 (d9-norbuprenorphine). The semiquantification of
`hydroxylated metabolites by SRM is described in detail under Results. MS/MS
`conditions used were 3.0 mTorr argon collision gas and 45 eV collision
`potential. When the Quantum was used, we found that norbuprenorphine had
`better sensitivity when the survivor molecular ion was monitored (i.e., 22 eV
`collision potential with m/z 414 to m/z 414) (Huang et al., 2006). The liquid
`chromatograph was a Hewlett-Packard Series 1100 HPLC (Agilent Technol-
`ogies, Palo Alto, CA). The chromatographic separations were conducted on a
`3 ␮M YMC ODS-AQ column (2.0 ⫻ 50 mm cartridge) (Waters, Milford,
`MA). The mobile phase was Milli-Q H2O (A) and CH3CN (B), both containing
`0.1% formic acid. The gradient elution went from 97% A at 1 min to 80% A
`at 3 min, holding for 5 min, then decreased to 20% A at 10 min, holding for
`2 min.
`Qualitative studies were performed on an Inertsil C18 column (250 ⫻ 2.1
`mm i.d.), packed with 3-␮m particles (Metachem Technologies, Inc., Torrance,
`CA). Isocratic elution was performed at 81% A with a flow rate of 0.25
`ml/min. The screening of metabolites by mass spectrometry was based on
`full-scan, selected ion monitoring (SIM), constant neutral loss scan, precursor
`ion scan, and product ion scan. The constant neutral loss scan of 54 u was used
`to detect the metabolites that undergo a loss of the cyclopropylmethyl group.
`The precursor ion scans of m/z 396 and m/z 101 were used to detect the
`metabolites that can produce typical fragment ions at m/z 396 and m/z 101
`under the collision-induced dissociation (CID) conditions. The product ion
`scan was used to identify the metabolites.
`
`Results
`In our previous studies on in vitro metabolism of buprenorphine, we
`focused on use of substrate concentrations that approached therapeutic
`plasma concentrations (Moody et al., 2002; Chang and Moody, 2005).
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`FIG. 1. The SIM chromatograms of the hydroxylated buprenorphine metabolites M1 and M2 (A) and the hydroxylated norbuprenorphine metabolites M3, M4, and M5 (B)
`after incubation of 10 ␮M buprenorphine for 30 min with HLM at 1.0 mg protein/ml. The dashed line is the control sample incubated with heat-inactivated HLMs.
`
`Since the purpose of this study was to identify new metabolites, we
`have used a higher concentration, 10 ␮M, for in vitro experiments to
`enhance our ability to detect what might be minor metabolites. This
`concentration, which is less than the reported Km for buprenorphine
`metabolism (Kobayashi et al., 1998), still meets the criterion sug-
`gested by Bjornsson et al. (2003) for P450 phenotyping studies. The
`in vivo relevance will be shown from studies in human urine.
`Buprenorphine Elimination and Norbuprenorphine Formation
`in HLMs. When buprenorphine (10 ␮M) was incubated with pooled
`HLMs (n ⫽ 5), norbuprenorphine formation only accounted for 46%
`and 37% of buprenorphine elimination at 20 min and 60 min incuba-
`tion time, respectively (data not shown). Higher buprenorphine elim-
`ination compared with norbuprenorphine formation suggested that
`other biotransformation pathways for buprenorphine or its metabolites
`exist in HLMs.
`Mass Spectrometric Analysis of Buprenorphine. Under the CID-
`MS/MS conditions, the characterized product ions generated from
`protonated molecular ions of buprenorphine (m/z 468) were at m/z
`414, m/z 396, and m/z 101 (Moody et al., 2002). A [M ⫺ 54]⫹ peak
`at m/z 414 (referred to as the a-moiety) showed the removal of a
`cyclopropylmethyl group. The peak at m/z 396 (referred to as the
`b-moiety) was formed by combination of the loss of a methyl group
`and cleavage of a tert-butyl group instead of loss of the cyclopropyl-
`methyl group and a water molecule, which was confirmed by the
`presence of a high, abundant product ion at m/z 400, produced from
`d4-buprenorphine (m/z 472) (data not shown). This assignment was
`consistent with previous work reported by Polettini and Huestis
`(2001). At the low mass range, a fragment ion at m/z 101 (referred
`to as
`the c-moiety) was assigned to the alkyl
`side chain
`⫹ at C-7, and it can lose a water molecule to form
`HOC(CH3)C(CH3)3
`the fragment ion at m/z 83. Another fragment ion at m/z 55 corre-
`sponds to the cyclopropylmethyl group.
`Identification of in Vitro Phase I Metabolites of Buprenorphine.
`In HLMs, the major metabolite, norbuprenorphine, formed by N-
`dealkylation of buprenorphine, has been studied in great detail. In the
`current study, different scan modes of the triple quadrupole MS were
`used to screen for unknown metabolites. A constant neutral loss scan
`
`of 54 u and a precursor ion scan of m/z 396 and m/z 101 showed the
`presence of hydroxylated buprenorphine and norbuprenorphine. In
`initial experiments, norbuprenorphine was found to readily form an
`adduct ion with acetonitrile (plus 41 u) which shows better response
`on the mass spectrometer used than the protonated molecular ion. As
`such, the acetonitrile adduct ion was used to determine structurally
`related metabolites of norbuprenorphine. The m/z 484 and m/z 471
`ions correspond to the hydroxylated buprenorphine protonated mo-
`lecular ion and hydroxylated norbuprenorphine adduct ion with ace-
`tonitrile. There are four peaks in the SIM chromatogram at m/z 484
`and three peaks at m/z 471 (Fig. 1). At retention times 9.83 min (M1)
`and 12.13 min (M2) (Fig. 1A), and retention times 6.34 min (M3),
`7.87 min (M4), and 9.96 min (M5) (Fig. 1B), the peaks are absent in
`the chromatograms of the corresponding blank control samples. Peaks
`at retention times 14.90 min (I1) and 16.62 min (I2) in the SIM
`chromatogram of m/z 484 were also present in the control samples
`incubated with heat-inactivated microsomes, and their amounts did
`not change with changes in incubation time, suggesting that these two
`peaks are probably inert impurities.
`When HLMs were incubated with buprenorphine, the microsomal
`protein precipitated with methanol, and the supernatant was directly
`injected into the LC-MS/MS, the same, and no additional, metabolites
`were observed. Selected ion monitoring of other possible metabolites,
`such as O-demethyl, N-oxide, and di-hydroxyl metabolites, showed
`negative results. The oxidative degradation compounds of buprenor-
`phine found in sublingual tablets, i.e., 10-hydroxybuprenorphine, bu-
`prenorphine N-oxide, and 10-oxobuprenorphine, were not detected in
`microsomal samples using comparisons with the reference com-
`pounds. 6-O-Demethyl norbuprenorphine, which was tentatively iden-
`tified in rat bile (Pontani et al., 1985), was not identified in HLMs.
`The structure of the metabolites has been proposed by interpreting
`their product ion mass spectra and comparison with that of parent
`drug. The CID product ion mass spectrum of M1 (Fig. 2A) presented
`the strongest peak at m/z 396, suggesting that the b-moiety is intact.
`The m/z 414 ion in the CID product ion scan of buprenorphine shifted
`by 16 for M1 and, meanwhile, the m/z 55 ion was present, indicating
`that the cyclopropylmethyl group was intact and hydroxylation had
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`FIG. 2. CID product ion mass spectra of M1 (A) and M2 (B), and deduced structures.
`
`occurred at the a-moiety. The absence of m/z 101 ion confirmed that
`the addition of a hydroxyl group was on the c-moiety. In consideration
`of the spatial hindrance and molecular stability, the hydroxylation
`occurred at the tert-butyl group.
`The CID-MS/MS spectrum of M2 presents characteristic product
`ions at m/z 430 and m/z 412, and a strong fragment ion at m/z 101 (Fig.
`2B). The presence of m/z 430 and m/z 101 ions suggests that the
`cyclopropylmethyl group and the alkyl side chain at C-7 position are
`intact; the addition of a hydroxyl group might occur at one of the ring
`moieties.
`Three peaks were observed in the SIM chromatogram of m/z 471
`(Fig. 1B). The characteristic fragment ion m/z 101 corresponding to
`the alkyl side chain at C-7 was absent in the product ion scan of M3
`(Fig. 3A), whereas it was present in the product ion scan of M4 (Fig.
`3B) and M5 (Fig. 3C). This finding suggests that the hydroxylation of
`M3 is similar to that of M1, and the addition of oxygen is on the
`tert-butyl group. The hydroxylation of M4 and M5 is similar to that of
`M2, and the hydroxyl group is on one of the ring moieties, but the
`exact hydroxyl position could not be determined.
`The Time Course of Hydroxylated Metabolite Formation in
`HLMs. After incubation of 10 ␮M buprenorphine with HLMs, the
`amount of M1, M2, M3, M4, and M5 was determined by SRM of m/z
`484 to 396 (M1), m/z 484 to m/z 101 (M2), m/z 471 to 202 (M3), and
`m/z 471 to 101 (M4 and M5) transitions, respectively. The amount
`was expressed as peak area ratio in comparison with internal standard
`d4-buprenorphine because no standard compound was available. The
`rate of formation of M1 was greater than that of M3 and M5, as
`indicated by the slope of the curves at earlier incubation times. The
`amount of M1 decreased after 10 min, suggesting that it might
`undergo further metabolism (Fig. 4A). Only M3 was detected in
`HLMs incubated with 10 ␮M norbuprenorphine, and it increased
`linearly up to 60 min (Fig. 4B). The metabolites M2 and M4 were not
`detected by SRM.
`Screening of 12 cDNA-Expressed Human P450s in the Metab-
`olism of Buprenorphine. Consistent with our previous study using 21
`
`nM buprenorphine (Moody et al., 2002), incubation of 10 ␮M bu-
`prenorphine with 12 human baculovirus insect cell-expressed P450
`isoenzymes (25 pmol) showed that the 3A family and 2C8 were the
`major enzymes involved in buprenorphine elimination and norbu-
`prenorphine formation (data not shown). The most efficient enzyme
`for M1 formation was P450 3A5, followed by 2C8, 3A4, and 3A7.
`The formation of M3 and M5 was mediated by P450 3A4, with a
`smaller contribution of 3A7 and 3A5. No metabolism was observed
`with other P450s and control insect microsomes (Fig. 5A). Incubation
`of 10 ␮M norbuprenorphine with P450s only produced M3, which
`was mainly mediated by 3A4 and, to a much lesser extent, by 3A5
`(Fig. 5B).
`The Contribution of Individual P450s to Buprenorphine Me-
`tabolism in HLMs. MAb Analysis and Chemical Inhibition. Based on
`our P450 screening data, together with previously reported results
`(Moody et al., 2002; Picard et al., 2005), P450 3A4, 3A5, 3A7, and
`2C8 are the major enzymes involved in the elimination of buprenor-
`phine. In addition, a preliminary experiment in pooled HLMs using
`other selective P450 inhibitors, 5 ␮M furafylline (1A2), 5 ␮M 8-me-
`thoxypsoralen (2A6), 20 ␮M sulfaphenazole (2C9), and 10 ␮M quin-
`idine (2D6), did not show any significant inhibition on buprenorphine
`metabolism. Therefore, the study on the contribution of individual
`P450s focused on 3A4/5 and 2C8. The individual contribution of
`3A4/5 and 2C8 was determined by measuring metabolite(s) formation
`and buprenorphine elimination in phenotyped HLMs after the addition
`of MAbs (see Yang et al., 1999; Krausz et al., 2001 for specificity of
`MAbs) or chemical inhibitors. Based on time course results, norbu-
`prenorphine and M1 formation were evaluated at 10 min and all others
`at 30 min. The percentage of inhibition observed with the addition of
`a MAb or chemical inhibitor determined its contribution to the total
`metabolism (Table 1). In the current study, two phenotyped HLMs
`with different relative activities of 3A4/5 and 2C8 were used. HLM
`452013 had higher 2C8 and lower 3A4/5 activity, whereas HLM
`452164 had higher 3A4/5 and lower 2C8 activity. In HLM 452013,
`the contributions of 2C8 to the elimination of buprenorphine, and the
`
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`FIG. 3. CID product ion mass spectra of M3 (A), M4 (B), and M5 (C), and deduced structures.
`
`same for MAb analysis and chemical inhibition, and the contribution
`of 3A4/5 was higher than that of 2C8. In both HLMs, the contribution
`of 3A4/5 was higher than that of 2C8 for the formation of M3 and M5.
`No significant difference was observed by increasing the amount of
`MAbs from 10 ␮l to 20 ␮l.
`Scaling of cDNA-Expressed P450 Activities. RAFs were deter-
`mined using the average of the enzyme activities for the 15 pheno-
`typed HLMs used in this study divided by the enzyme activities
`provided by BD Gentest for the cDNA-expressed P450s (Crespi,
`1995; Venkatakrishnan et al., 2000). The immunoquantification abun-
`dances were from another previously described (Neff and Moody,
`2001) BD Gentest data bank of seven HLMs; the abundance of 2C8,
`which was not provided, was estimated from 2C9 abundance and the
`finding of Lapple et al. (2003) that the average content of 2C8 is
`64.2% of 2C9. The predicted contributions of individual P450s are
`shown in Table 2. Using RAFs, P450 3A contributed the most to
`buprenorphine elimination (78.1%) and norbuprenorphine formation
`(48.4%), followed by 2C8, with a contribution of 14.5% and 36.4%,
`respectively. For the formation of M1, 2C8 was predicted to contrib-
`ute most (70.2%), followed by 3A (29.2%). The estimated contribu-
`tion of 3A increased and 2C8 decreased when the immunoquantitative
`data were used (Table 2).
`Correlation Study. The rates of formation of metabolites and
`buprenorphine elimination were determined in 15 individual HLMs,
`and the data were correlated with the P450 phenotyped activities
`provided by the vendor. The results for correlations with 3A and 2C8
`
`FIG. 4. The time course of M1 (⽧), M3 (f), and M5 (Œ) formation in HLMs
`incubated with 10 ␮M buprenorphine (A), and the time course of M3 formation in
`HLMs incubated with 10 ␮M norbuprenorphine (B). The microsomal protein
`content is 0.5 mg/ml. The amount of metabolites was expressed as peak area ratio
`of metabolites to internal standard d4-buprenorphine. Each point is the mean of
`duplicate experiments.
`
`formation of norbuprenorphine and M1 were 4.8 to 11.9 times higher
`than that of 3A4/5 according to MAb analysis, and 1.6 to 3.5 times
`higher based on chemical inhibition. In HLM 452164, the data are the
`
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`

`

`NOVEL METABOLITES OF BUPRENORPHINE
`
`445
`
`TABLE 2
`Relative activity factor (RAF) versus immunoquantitation scaling of cDNA-
`expressed P450 activity for buprenorphine (Bup) utilization and formation of
`norbuprenorphine (Nor) and M1
`
`P450
`
`1A2
`2A6
`2B6
`2C8
`2C9
`2C19
`2D6
`2E1
`3A
`
`Bup
`
`2.1
`4.8
`0.0
`14.5
`0.0
`0.1
`0.4
`0.0
`78.1
`
`RAF
`
`Nor
`
`0.3
`0.0
`0.0
`36.4
`5.1
`0.1
`0.0
`9.7
`48.4
`
`Immunoquantitation
`
`M1
`
`Bup
`
`Nor
`
`M1
`
`% contribution
`0.4
`1.1
`0.0
`1.3
`0.1
`0.0
`70.2
`6.4
`0.0
`0.0
`0.1
`0.3
`0.0
`0.3
`0.0
`0.0
`29.2
`90.5
`
`0.2
`0.0
`0.0
`21.9
`1.0
`0.4
`0.0
`0.8
`75.6
`
`0.3
`0.0
`0.3
`47.5
`0.0
`0.5
`0.0
`0.0
`51.4
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`investigated (data not shown). The only other significant correlations
`observed were M1 formation with 2E1 activity, chlorzoxazone 6-hy-
`droxylation (r ⫽ 0.550).
`In Vivo Studies. Human urine samples with or without hydrolysis
`by ␤-glucuronidase (containing sulfatase) were analyzed by LC-ESI-
`MS/MS. The product ion mass spectrum and the retention time on the
`LC when compared with those in HLM incubations demonstrated that
`buprenorphine can be metabolized to form M1 and M3 in vivo (data
`not shown). Semiquantitation of M1 and M3 was determined from
`peak area ratios of metabolite to d4-buprenorphine, and those deter-
`mined in hydrolyzed urine compared with those determined in non-
`hydrolyzed urine (Table 4). The negligible amount of M1 in the
`nonhydrolyzed urine sample in comparison to hydrolyzed samples
`suggests that M1 is significantly conjugated with glucuronide in vivo.
`The smaller difference between hydrolyzed M3 and nonhydrolyzed
`M3 (mean ⫽ 68.5%, range 53–100% of unconjugated) suggests that
`it is excreted, for the most part, as the unconjugated form (Table 4).
`Nonhydrolyzed urine was also extracted by solid-phase extraction
`(Huang et al., 2006) to directly examine the conjugated buprenorphine
`and metabolites. Neutral loss scans of 176 (glucuronide conjugates)
`and 80 (sulfonate conjugates), and SRM (transition of molecular ion
`of interest to ⫺176 and ⫺80) were performed. Glucuronide conju-
`gates were identified for buprenorphine, norbuprenorphine, M1, and
`M3. Only norbuprenorphine showed evidence for a sulfonate conju-
`gate at approximately 1% of its glucuronide conjugate (data not
`shown).
`
`FIG. 5. Metabolism in cDNA-expressed human P450s. A, the formation of M1,
`M3, and M5 incubated with 10 ␮M buprenorphine; B, the formation of M3
`incubated with 10 ␮M norbuprenorphine. The incubation time was 20 min and
`cDNA-expressed P450 content was 25 pmol. Data represent the mean of duplicate
`incubations. Because the nanomoles of P450 added and incubation times were
`constant, results for new metabolite formation are just given as peak area ratios.
`Control insect cell microsomes were used at the mean protein concentration aver-
`aged over all of the Supersomes.
`
`activities are shown in Table 3. Significant correlations ( p ⬍ 0.05)
`between testosterone 6␤-hydroxylation catalyzed by P450 3A were
`observed with buprenorphine elimination and the formation of each
`metabolite. For the formation of M1 and M5, the significant correla-
`tions were only observed by excluding three HLMs with the highest
`3A activities. The only significant correlation with paclitaxel 6␣-
`hydroxylation, catalyzed by 2C8, was with norbuprenorphine forma-
`tion. The power of these correlation experiments depends, in part, on
`the extent of the inter-HLM variation in activity; the greater the range
`within a liver bank, the more power it has to establish a significant
`correlation. It should be noted that the range of 3A activity (highest
`activity HLM/lowest activity HLM) in the 15 HLMs was 15.8; that of
`2C8 activity was only 5.4. Correlations with other P450 activities
`(activity range in parentheses), 1A2 (15.3), 2A6 (14.3), 2B6 (20), 2C9
`(4.9), 2C19 (171), 2D6 (7.9), 2E1 (3.2), and 4A11(37.7), were also
`
`TABLE 1
`The effect of immuno- and chemical inhibition on the metabolism of buprenorphine
`The testosterone 6␤ -hydroxylase activities (3A4/5) of HLMs 452013 and 452164 are 890 and 4100, respectively; the paclitaxel 6␣ -hydroxylase activities (2C8) are 380 and 78,
`respectively. The activities are expressed as pmol product per (mg protein ⫻ minute). The recommended volume (10 ␮l) of MAbs specific for P450 3A4/5 or 2C8 was used. The
`concentrations of ketoconazole and trimethoprim were 2 ␮M and 100 ␮M, respectively. Results are the mean of duplicate incubations. Formation of norbuprenorphine and M1 were determined
`after 10-min incubations; all others were determined after 30-min incubations.
`
`Inhibitor
`
`Elimination of
`Buprenorphine
`
`A. HLM 452013: higher 2C8, lower 3A4/5
`Anti-3A4/5
`Anti-2C8
`Ketoconazole
`Trimethoprim
`B. HLM 452164: lower 2C8, higher 3A4/5
`Anti-3A4/5
`Anti-2C8
`Ketoconazole
`Trimethoprim
`
`12
`77
`18
`28
`
`76
`3
`77
`7
`
`Formation of
`
`Norbuprenorphine
`
`M1
`
`M3
`
`% inhibition
`
`7
`83
`14
`32
`
`60
`18
`59
`13
`
`14
`67
`4
`14
`
`48
`18
`48
`16
`
`74
`14
`89
`38
`
`95
`0
`95
`38
`
`M5
`
`87
`12
`100
`0
`
`94
`0
`90
`7
`
`P