Inhibition of human drug metabolizing cytochrome P450 by buprenorphine

Article abstract of DOI:10.1248/bpb.25.682

The effects of buprenorphine, a powerful mixed agonist/antagonist analgesic, on several cytochrome P450 (CYP) isoform specific reactions in human liver microsomes were investigated to predict drug interaction of buprenorphine in vivo from in vitro data. The following eight CYP-catalytic reactions were used in this study: CYP1A1/2-mediated 7-ethoxyresorufin O-deethylation, CYP2A6-mediated coumarin 7-hydroxylation, CYP2B6-mediated 7-benzyloxyresorufin O-debenzylation, CYP2C8/9-mediated tolbutamide methylhydroxylation, CYP2C19-mediated S-mephenytoin 4-hydroxylation, CYP2D6-mediated bufuralol 1′-hydroxylation, CYP2E1-mediated chlorzoxazone 6-hydroxylation, and CYP3A4-mediated testosterone 6β-hydroxylation. Buprenorphine strongly inhibited the CYP3A4- and CYP2D6-catalyzed reactions with Ki values of 14.7 μM and 21.4 μM, respectively. The analgesic also weakly inhibited specific reactions catalyzed by CYP1A1/2 (Ki=132 μM), CYP2B6 (Ki=133 μM), CYP2C19 (Ki=146 μM), CYP2C8/9 (IC₅₀>300 μM), and CYP2E1 (IC ₅₀>300 μM), but not CYP2A6 mediated pathway. In consideration of the Ki values obtained in this study and the therapeutic concentration of buprenorphine in human plasma, buprenorphine would not be predicted to cause clinically significant interactions with other CYP-metabolized drugs.

Full text of DOI:10.1248/bpb.25.682

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Notes
Biol. Pharm. Bull. 25(5) 682—685 (2002)
Vol. 25, No. 5
Inhibition of Human Drug Metabolizing Cytochrome P450 by
Buprenorphine
Ken UMEHARA,* Yoshihiko SHIMOKAWA, and Gohachiro MIYAMOTO
Department of Drug Metabolism, Drug Safety Research Center, Tokushima Research Institute, Otsuka Pharmaceutical Co.,
Ltd.; 463–10 Kagasuno, Kawauchi-cho, Tokushima 771–0192, Japan.
Received October 23, 2001; accepted January 17, 2002
The effects of buprenorphine, a powerful mixed agonist/antagonist analgesic, on several cytochrome P450
CYP) isoform specific reactions in human liver microsomes were investigated to predict drug interaction of
(
buprenorphine in vivo from in vitro data. The following eight CYP-catalytic reactions were used in this study:
CYP1A1/2-mediated 7-ethoxyresorufin O-deethylation, CYP2A6-mediated coumarin 7-hydroxylation, CYP2B6-
mediated 7-benzyloxyresorufin O-debenzylation, CYP2C8/9-mediated tolbutamide methylhydroxylation, CYP2C19-
mediated S-mephenytoin 4-hydroxylation, CYP2D6-mediated bufuralol 1؅-hydroxylation, CYP2E1-mediated
chlorzoxazone 6-hydroxylation, and CYP3A4-mediated testosterone 6b-hydroxylation. Buprenorphine strongly
inhibited the CYP3A4- and CYP2D6-catalyzed reactions with Ki values of 14.7 mM and 21.4 mM, respectively. The
analgesic also weakly inhibited specific reactions catalyzed by CYP1A1/2 (Ki؍132 mM), CYP2B6 (Ki؍133 mM),
CYP2C19 (Ki؍146 mM), CYP2C8/9 (IC Ͼ300 mM), and CYP2E1 (IC Ͼ300 mM), but not CYP2A6 mediated
5
0
50
pathway. In consideration of the Ki values obtained in this study and the therapeutic concentration of buprenor-
phine in human plasma, buprenorphine would not be predicted to cause clinically significant interactions with
other CYP-metabolized drugs.
Key words buprenorphine; cytochrome P450 inhibition; in vitro drug–drug interaction; human liver microsome
Buprenorphine is a synthetic derivative of morphine alka- MATERIALS AND METHODS
loid thebaine with mixed agonist/antagonist analgesic prop-
1
)
erties. The analgesic effects are 30 times more potent than
Chemicals Buprenorphine hydrochloride was supplied
those of morphine, and it produces little physical depen- by Otsuka Pharmaceutical Co., Ltd. (Tokyo, Japan).
1
,2)
dence. Buprenorphine is used for the treatment of chronic Coumarin, tolbutamide, chlorzoxazone, glucose-6-phosphate
3
)
post-operative pain and terminal cancer patients, and re- disodium salt:hydrate, and b-NADP sodium salt were pur-
cently it has been proposed for the management of opioid ad- chased from Sigma Chemical Co. (St. Louis, MO, U.S.A.).
4
)
dicts.
Other chemicals were obtained from the following sources:
Buprenorphine is extensively metabolized in humans by a 7-ethoxyresorufin, 4-hydroxytolbutamide, S-(ϩ)-mepheny-
combination of phase I and phase II reactions to form nor- toin, (Ϯ)-4Ј-hydroxymephenytoin, 1Ј-hydoxybufuralol maleate
buprenorphine and conjugates of buprenorphine and nor- salt, (Ϯ)-bufuralol hydrochloride salt, 6-hydroxychlorzoxa-
5
)
buprenorphine (Fig. 1). Following subcutaneous, sublin- zone, and 6b-hydroxytestosterone from Salford Ultrafine
gual, and oral buprenorphine administration to human sub- Chemical & Research, Ltd. (Guildhall Close, Manchester,
5
)
jects, no free parent drug has been detected in urine. Nor- England); 7-benzyloxyresorufin from Molecular Probes (Eu-
buprenorphine is produced by N-dealkylation of the N-cyclo- gene, Oregon, U.S.A.); 7-hydroxycoumarin from Tokyo
propylmethyl group, which is a principal metabolic pathway Chemical Industry Co., Ltd. (Tokyo, Japan); resorufin,
for buprenorphine.
testosterone, and magnesium chloride hexahydrate from
Oxidative metabolism of drugs is mostly catalyzed by cy- Wako Pure Chemical Industries, Ltd. (Osaka, Japan); glu-
tochrome P450 (CYP) enzymes which comprise a large fam- cose-6-phosphate dehydrogenase (approx. 350 U/mg, 1 mg/
6
,7)
ily of hemoproteins. More than 15 isozymes have been ml) from Boehringer Mannheim GmbH (Mannheim, Ger-
identified in human liver, and several forms play important many). All other reagents and solvents were of high analyti-
8
)
roles in the metabolism of drugs. It is of great importance cal grade.
that the CYP isoforms responsible for the metabolism of
Liver Microsomes Preparation Pooled human liver mi-
drugs be identified and further investigated as to whether a crosomes from 10 donors were prepared at the Biomedical
drug has inhibitory effect on the catalytic activity of CYP
isoforms in order to consider possible drug interactions. Re-
9
)
10)
cently, Iribarne et al. and Kobayashi et al. reported that
buprenorphine N-dealkylation, which is a principal metabolic
pathway for buprenorphine, in human liver microsomes is
mainly catalyzed by CYP3A4. However, it is unclear whether
buprenorphine has an inhibitory effect on the catalytic activ-
ity of CYP isoforms. This study examines the characteristic
properties of buprenorphine on the catalytic activities of the
CYP isoforms, CYP1A1/2, CYP2A6, CYP2B6, CYP2C8/9,
CYP2C19, CYP2D6, CYP2E1, and CYP3A4 using specific
reaction probes for the enzymes.
Fig. 1. Chemical Structures of Buprenorphine and Norbuprenorphine
To whom correspondence should be addressed. e-mail: k_umehara@research.otsuka.co.jp © 2002 Pharmaceutical Society of Japan
∗

May 2002
683
Research Institute, Human and Animal Bridge Discussion metabolism (data not shown).
Group (HAB, Chiba, Japan). Human liver samples were
For the determination of the Ki values, the ranges of
legally procured from National Disease Research Inter- 7-ethoxyresorufin and buprenorphine concentrations were
change (NDRI, Pennsylvania, U.S.A.) through the interna- 0.25—1 mM and 50—150 mM, respectively; those of 7-benzyl-
tional partnership between NDRI and HAB. The study was oxyresorufin and buprenorphine concentrations were 0.75—
conducted in accordance with the Declaration of Helsinki. 3 mM and 30—300 mM; those of S-mephenytoin and buprenor-
Human livers were homogenized with 0.25 M sucrose con- phine concentrations were 100—400 mM and 30—300 mM;
taining 3 mM Tris and 0.1 mM EDTA (pH 7.4), and micro- those of bufuralol and buprenorphine concentrations were
somes were isolated by differential centrifugation using an 20—80 mM and 10—100 mM; and those of testosterone and
ordinary method. Washed microsomes were resuspended in buprenorphine concentrations were 50—200 mM and 10—
1
00 mM Tris–HCl buffer (pH 7.4) containing 1 mM EDTA 100 mM, respectively.
and 20% glycerol at protein concentrations of 20 mg/ml in
the livers.
Data Analysis Apparent K and V values for the for-
m max
mation of metabolites were calculated by a nonlinear regres-
Determination of Human CYP Activities 7-Ethoxyre- sion analysis using a computer program, WinNonlin Stan-
sorufin O-deethylase activity by CYP1A1/2, coumarin 7-hy- dard (Version 2.1, Scientific Consulting, Inc., Apex, NC,
2
1)
droxylase activity by CYP2A6, 7-benzyloxyresorufin O- U.S.A.). The method of Dixon was used to calculate inhi-
debenzylase activity by CYP2B6, tolbutamide methylhydrox- bition constants (Ki).
ylase activity by CYP2C8/9, S-mephenytoin 4-hydroxylase
activity by CYP2C19, bufuralol 1Ј-hydroxylase activity by RESULTS AND DISCUSSION
CYP2D6, chlorzoxazone 6-hydroxylase activity by CYP2E1,
and testosterone 6b-hydroxylase activity by CYP3A4 were
Multiple drug therapy is a common therapeutic practice,
particularly for patients with various diseases. Whenever 2 or
1
1)
determined as described.
Standard incubation mixtures of 0.5 ml contained microso- more drugs are administered concurrently, there is the possi-
mal protein (0.1—0.5 mg), 0.1 M potassium phosphate buffer bility of drug interaction. Many drug interactions are clini-
(
pH 7.4), 0.1 mM EDTA, NADPH generating system (2.5 mM cally caused by inhibition of drug metabolizing enzymes, cy-
b-NADP, 25 mM glucose-6-phosphate, 2 units of glucose-6- tochrome P450s (CYPs), leading to decreased metabolic
phosphate dehydrogenase, and 10 mM magnesium chloride), clearance and increased exposure to the inhibited drug. The
and substrates with or without buprenorphine. Buprenor- inhibition of CYP enzymes should thus be examined to as-
phine was dissolved in dimethyl sulfoxide and organic sol- sess the potential for drug interactions.
vent concentration was 1 (v/v) % in the reaction system.
The current study examined the in vitro ability of
Product formation was determined using HPLC and a fluo- buprenorphine to inhibit the metabolism of substrates for
rescence spectrophotometer. Assay methods were validated CYP1A1/2, CYP2A6, CYP2B6, CYP2C8/9, CYP2C19,
in this study. Calibration curves for resorufin, 7-hydroxy- CYP2D6, CYP2E1, and CYP3A4 (Table 1 and Fig. 2).
coumarine, 4-hydroxytolbutamide, 4-hydroxymephenytoin, Buprenorphine had strong inhibitory effects on CYP3A4-
1
Ј-hydroxybufuralol, 6-hydrochlorzoxazone, and 6b-hydroxy- mediated testosterone 6b-hydroxylation and CYP2D6-medi-
testosterone were established with respective calibration ated bufuralol 1Ј-hydroxylation with Ki values of 14.7 mM
ranges of 0.2—200 nM (gϭ0.9996), 0.025—5 mM (gϭ1.0000), and 21.4 mM, respectively. The type of inhibition was consid-
0
5
3
.05—10 mM (gϭ0.9998), 0.025—5 mM (gϭ0.9996), 0.025— ered to be competitive from Dixon plots. In addition,
mM (gϭ0.9995), 0.25—100 mM (gϭ0.9994), and 0.03— buprenorphine had weak inhibitory effects on CYP1A1/
0 mM (gϭ0.9999).
2-mediated 7-ethoxyresorufin O-deethylation (Kiϭ132 mM),
The substrate concentrations used to estimate the kinetic CYP2B6-mediated 7-benzyloxyresorufin O-debenzylation
parameters for each assay were 0.01—10 mM 7-ethoxyre- (Kiϭ133 mM), CYP2C19-mediated S-mephenytoin 4-hydrox-
sorufin, 0.1—100 mM coumarin, 0.03—10 mM 7-benzyloxyre- ylation (Kiϭ146 mM), CYP2C8/9-mediated tolbutamide
sorufin, 25—1000 mM tolbutamide, 5—500 mM S-mepheny- methylhydroxylation (IC Ͼ300 mM), and CYP2E1-mediated
5
0
toin, 5—200 mM bufuralol, 10—400 mM chlorzoxazone, and chlorzoxazone 6-hydroxylation (IC Ͼ300 mM), while it did
5
0
1
0—250 mM testosterone. For the determination of the resid- not inhibit CYP2A6-mediated coumarin 7-hydroxylation at
ual activity in the presence of buprenorphine (30—300 mM), concentrations up to 300 mM. Buprenorphine is a substrate of
9
,10)
the concentrations of substrates were 0.5 mM 7-ethoxyre- CYP3A4,
and thus may have the strongest inhibitory ef-
sorufin, 2 mM coumarin, 1 mM 7-benzyloxyresorufin, 100 mM fect on CYP3A4 catalyzed reactions.
tolbutamide, 100 mM S-mephenytoin, 20 mM bufuralol, 100
The lowest Ki value (CYP3A4; Kiϭ14.7 mM) was used to
mM chlorzoxazone, and 100 mM testosterone. Selective CYP estimate conservatively the potential for inhibitory drug in-
inhibitors were used in this study to validate that the assays teractions with buprenorphine during clinical use of the drug.
1
2)
13)
were working properly. 7,8-Benzoflavone, furafylline,
orphenadrine,
promine, quinidine, diethyldithiocarbamate, and keto- dose of 4 mg in healthy volunteers was 24.40 to 55.83 ng/ml
conazole, which are inhibitors of CYP1A1, 1A2, 2B6, (0.0522 to 0.119 mM), 1.93 to 7.20 ng/ml (0.00413 to 0.0154
Peak plasma concentration of buprenorphine after an intra-
1
4)
15)
16)
quercetin,
sulfaphenazole,
tranylcy- venous dose of 1.2 mg, a sublingual dose of 4 mg, or a buccal
1
7)
18)
19)
2
0)
2
C8, 2C9, 2C19, 2D6, 2E1, and 3A4 activities, inhibited the mM), and 0.25 to 3.90 ng/ml (0.000535 to 0.00834 mM), re-
22)
respective enzyme activities (data not shown). Diethyldithio- spectively. Oral dose of 40 mg or subcutaneous dose of
carbamate is also known to be a specific inhibitor of 2 mg to a patient produced peak levels of 0.57 to 4.69 ng/ml
1
2)
CYP2A6 and the present study confirmed the potent in- (0.00122 to 0.0100 mM) and 0.2 to 8.74 ng/ml (0.000428 to
hibitory capability of this compound on CYP2A6 mediated 0.0187 mM), respectively. Plasma concentration of buprenor-
23)

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Vol. 25, No. 5
Table 1. Effect of Buprenorphine on the Cytochrome P450 Catalyzed Oxidase Activities in Human Liver Microsomes
%
Control activity
Km
Vmax
CYP
Reaction
(mM)
(pmol/min/mg)
3
0 mM
100 mM
300 mM
CYP1A1/2
CYP2A6
CYP2B6
CYP2C8/9
CYP2C19
CYP2D6
CYP2E1
CYP3A4
7-Ethoxyresorufin O-deethylation
Coumarin 7-hydroxylation
7-Benzyloxyresorufin O-debenzylation
Tolbutamide methylhydroxylation
S-Mephenytoin 4-hydroxylation
Bufuralol 1Ј-hydroxylation
0.519
1.73
1.59
442
86.0
21.7
115
136
15.0
163
95.3
82.6
89.4
87.7
86.4
42.1
83.4
51.1
74.2
69.1
57.3
62.4
69.7
22.3
74.7
21.1
12.3
86.1
32.0
54.8
39.0
0
4.83
84.9
8.54
61.0
2600
5540
Chlorzoxazone 6-hydroxylation
Testosterone 6b-hydroxylation
58.4
9.4
The control activities were 7.25, 74.8, 1.44, 8.52, 2.39, 10.9, 109, and 1490 pmol/min/mg for CYP1A1/2, CYP2A6, CYP2B6, CYP2C8/9, CYP2C19, CYP2D6, CYP2E1, and
CYP3A4 catalyzed reactions, respectively. Enzyme incubations and metabolite analysis were carried out in duplicate.
Fig. 2. Dixon Plots for the Inhibition of CYP1A1/2-Mediated 7-Ethoxyresorufin O-Deethylase (A), CYP2B6-Mediated 7-Benzyloxyresorufin O-Debenzy-
lase (B), CYP2C19-Mediated S-Mephenytoin 4-Hydroxylase (C), CYP2D6-Mediated Bufuralol 1Ј-Hydroxylase (D), and CYP3A4-Mediated Testosterone
6b-Hydroxylase (E) Activites by Buprenorphine in Human Liver Microsomes
Each experiment was performed in duplicate using pooled microsomes and each value is shown.

May 2002
685
phine is 120 times less than the apparent Ki value determined
in the in vitro studies, suggesting buprenorphine to possibly
have little or no inhibitory effect on the metabolism of drugs
oxidized by CYP1A1/2, CYP2B6, CYP2C8/9, CYP2C19,
CYP2D6, CYP2E1, and CYP3A4 in vivo. Actually, there are
no clinical reports that buprenorphine causes drug–drug in-
teractions with other CYP-metabolized drugs.
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2
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