CYP2J2 and CYP2C19 are the major enzymes responsible for metabolism of albendazole and fenbendazole in human liver microsomes and recombinant P450 assay systems

Article abstract of DOI:10.1128/AAC.00843-13

Albendazole and fenbendazole are broad-spectrum anthelmintics that undergo extensive metabolism to form hydroxyl and sulfoxide metabolites. Although CYP3A and flavin-containing monooxygenase have been implicated in sulfoxide metabolite formation, the enzy

Full text of DOI:10.1128/AAC.00843-13

CYP2J2 and CYP2C19 Are the Major Enzymes Responsible for
Metabolism of Albendazole and Fenbendazole in Human Liver
Microsomes and Recombinant P450 Assay Systems
Zhexue Wu,a Doohyun Lee,a Jeongmin Joo,a Jung-Hoon Shin,a Wonku Kang,b Sangtaek Oh,c Do Yup Lee,c Su-Jun Lee,d Sung Su Yea,d
Hye Suk Lee,e Taeho Lee,a Kwang-Hyeon Liua
College of Pharmacy and Research Institute of Pharmaceutical Sciences, Kyungpook National University, Daegu, South Koreaa; College of Pharmacy, Yeungnam
University, Kyungpook, South Koreab; Department of Advanced Fermentation Fusion Science & Technology, Kookmin University, Seoul, South Koreac; College of
Medicine, Inje University, Busan, South Koread; College of Pharmacy and Integrated Research Institute of Pharmaceutical Sciences, The Catholic University of Korea,
Bucheon, South Koreae
Albendazole and fenbendazole are broad-spectrum anthelmintics that undergo extensive metabolism to form hydroxyl and sul-
foxide metabolites. Although CYP3A and flavin-containing monooxygenase have been implicated in sulfoxide metabolite forma-
tion, the enzymes responsible for hydroxyl metabolite formation have not been identified. In this study, we used human liver
microsomes and recombinant cytochrome P450s (P450s) to characterize the enzymes involved in the formation of hydroxyal-
bendazole and hydroxyfenbendazole from albendazole and fenbendazole, respectively. Of the 10 recombinant P450s, CYP2J2
and/or CYP2C19 was the predominant enzyme catalyzing the hydroxylation of albendazole and fenbendazole. Albendazole hy-
droxylation to hydroxyalbendazole is primarily mediated by CYP2J2 (0.34 ␮l/min/pmol P450, which is a rate 3.9- and 8.1-fold
higher than the rates for CYP2C19 and CYP2E1, respectively), whereas CYP2C19 and CYP2J2 contributed to the formation of
hydroxyfenbendazole from fenbendazole (2.68 and 1.94 ␮l/min/pmol P450 for CYP2C19 and CYP2J2, respectively, which are
rates 11.7- and 8.4-fold higher than the rate for CYP2D6). Correlation analysis between the known P450 enzyme activities and
the rate of hydroxyalbendazole and hydroxyfenbendazole formation in samples from 14 human liver microsomes showed that
albendazole hydroxylation correlates with CYP2J2 activity and fenbendazole hydroxylation correlates with CYP2C19 and
CYP2J2 activities. These findings were supported by a P450 isoform-selective inhibition study in human liver microsomes. In
conclusion, our data for the first time suggest that albendazole hydroxylation is primarily catalyzed by CYP2J2, whereas fen-
bendazole hydroxylation is preferentially catalyzed by CYP2C19 and CYP2J2. The present data will be useful in understanding
the pharmacokinetics and drug interactions of albendazole and fenbendazole in vivo.
lbendazole and fenbendazole are benzimidazole compounds the dose was excreted as hydroxyfenbendazole in the feces (12). In
A
used as broad-spectrum anthelmintics against gastrointesti-
addition, Lee et al. (11) also reported that hydroxyalbendazole was
formed in the incubation mixtures of albendazole and the
rCYP2J2 isoform. However, the involvement of the cytochrome
P450 (P450) isoform in the metabolism of albendazole and fen-
bendazole is poorly understood.
The increased use of benzimidazole drugs against systemic in-
fections, often for long periods and in combination with other
agents, suggests that identifying the specific metabolites formed by
individual P450s is essential for prediction of drug interactions
and adverse events associated with therapy (9). For example, al-
bendazole has been used for the treatment of the gastrointestinal
helminthiases and also used to cure neurocysticercosis, which re-
quires long medication periods of about 7 to 30 days (13). Al-
though it exerts broad-spectrum coverage with high cure rates
nal nematodes and the larval stages of cestodes (1). Benzimidazole
anthelmintics are extensively metabolized in domestic animals
and humans. Animal and microsomal incubation studies have
demonstrated rapid conversion of albendazole and fenbendazole
to a sulfoxide metabolite and subsequently a sulfone metabolite
(2–6). Sulfoxide metabolites are responsible for the systemic bio-
logical activity of benzimidazole drugs (7). Their metabolic pat-
terns and the resultant pharmacokinetic behaviors are relevant in
the attainment of high and sustained concentrations of pharma-
cologically active drugs/metabolites at the target parasite (8). Ev-
idence from microsomal investigations in a number of species
suggests that CYP3A4 and flavin-containing monooxygenase
(FMO) are major enzymes responsible for the formation of sul-
foxide metabolites from albendazole (4, 9, 10) and fenbendazole
(4, 5) (Fig. 1). Recently, Lee et al. (11) reported that the rates of
albendazole sulfoxide formation from albendazole by the recom-
binant CYP2J2 (rCYP2J2) isoform were significantly higher than
those by the recombinant CYP3A4 (rCYP3A4) isoform, indicat-
ing the importance of the CYP2J2 isoform in albendazole sulfox-
ide formation (Fig. 1).
Received 22 April 2013 Returned for modification 9 June 2013
Accepted 13 August 2013
Published ahead of print 19 August 2013
Address correspondence to Kwang-Hyeon Liu, dstlkh@knu.ac.kr, or Taeho Lee,
tlee@knu.ac.kr.
Z.W. and D.L. contributed equally to this article.
Copyright © 2013, American Society for Microbiology. All Rights Reserved.
doi:10.1128/AAC.00843-13
Hydroxy metabolite formation is one of the most common
biotransformation pathways in drug metabolism. Fenbendazole is
metabolized to hydroxyfenbendazole in rat liver microsomes (5).
In cattle following intravenous administration, more than 50% of
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Albendazole and Fenbendazole Metabolism by CYP2J2
FIG 1 Proposed metabolic pathways of albendazole (A) and fenbendazole (B) in human liver microsomes.
against helminthic infections, multiple studies have indicated a CA). Human P450s 2A6, 2B6, 2C8, 2C9, 2C19, 2E1, 2J2, and 3A4 are
coexpressed with human P450 reductase and cytochrome b5; however,
P450s 1A2 and 2D6 are coexpressed only with human P450 reductase.
Information regarding protein content, P450 content, and enzymatic ac-
tivity is supplied by the manufacturer (BD Biosciences).
lack of understanding of the causes of the large variations in the
concentrations of the albendazole metabolite in human blood (14,
15) and possible adverse effects, such as infantile eczema, associ-
ated with albendazole treatment during pregnancy (16, 17). Inves-
tigation of the detailed metabolism of anthelminthic agents would
be helpful for the assessment of the risks and benefits of routine
treatment with these drugs. Therefore, the objective of this study
was to identify and kinetically characterize the P450 isoforms re-
sponsible for the formation of the corresponding hydroxyl metab-
olites from albendazole and fenbendazole. The information from
these studies will allow a better understanding of the factors af-
fecting benzimidazole pharmacokinetics and drug interactions.
Preparation of hydroxyalbendazole and hydroxyfenbendazole
standards. Hydroxyalbendazole and hydroxyfenbendazole were synthe-
sized by three-step reactions from 5-chloro-2-nitroaniline using 3-mer-
captopropan-1-ol and 4-mercaptophenol, respectively (Fig. 2) (18–21).
(i)ProcedureforsynthesisofcompoundsA1andB1. Toasolutionof
5-chloro-2-nitroaniline (523 mg, 3.00 mmol) in acetonitrile (15 ml) was
added thiol [3-mercaptopropan-1-ol for 3-(3-amino-4-nitrophenylthio)
propan-1-ol (compound A1) or 4-mercaptophenol for 4-(3-amino-4-ni-
trophenylthio)phenol (compound B1), 3.30 mmol] and K2CO3 (498 mg,
3.6 mmol) at room temperature. The reaction mixture was refluxed for 5
h. After cooling to room temperature, the K2CO3 was filtered off. The
filtrate was concentrated in vacuo. The subsequent residue was purified by
column chromatography to give compound A1 (96%) or B1 (97%). Com-
pound A1: orange solid; 1H nuclear magnetic resonance (NMR) (400
MHz, dimethyl sulfoxide [DMSO]-d6) ␦ 7.86 (d, J ϭ 9.1 Hz, 1H), 7.44 (s,
2H), 6.84 (d, J ϭ 2.0 Hz, 1H), 6.49 (dd, J ϭ 9.1, 2.1 Hz, 1H), 4.62 (t, J ϭ 5.2
Hz, 1H), 3.54 to 3.49 (m, 2-H), 3.02 (t, J ϭ 7.3 Hz, 2H), 1.85 to 1.73 (m,
2H). Compound B1: orange solid; 1H NMR (400 MHz, DMSO-d6) ␦
10.05 (s, 1H), 7.85 (d, J ϭ 9.1 Hz, 1H), 7.43 (s, 2H), 7.38 (d, J ϭ 8.3 Hz,
2H), 6.90 (d, J ϭ 8.2 Hz, 2H), 6.48 (d, J ϭ 1.8 Hz, 1H), 6.26 (dd, J ϭ 9.1,
2.0 Hz, 1H).
MATERIALS AND METHODS
Chemicals and reagents. Hydroxyebastine was obtained from Toronto
Research Chemicals (North York, ON, Canada). Albendazole, astemizole,
danazol, diethyldithiocarbamate, ebastine, fenbendazole, furafylline,
ketoconazole, mebendazole, quercetin, quinidine, sulfaphenazole, tran-
ylcypromine, ticlopidine, triethylenethiophophoramide (thio-TEPA),
␤-NADP (␤-NADPϩ), MgCl2, potassium phosphate (KH2PO4), D-glu-
cose-6-phosphate (G6P), and glucose-6-phosphate dehydrogenase
(G6PDH) were purchased from Sigma-Aldrich (St. Louis, MO). Solvents
were high-pressure liquid chromatography (HPLC) grade (Fisher Scien-
tific Co., Pittsburgh, PA), and the other chemicals were of the highest
quality available. Pooled human liver microsomes (H161), human liver
microsomes (HLMs) from 14 different single donors, and 10 human re-
(ii) Procedure for synthesis of compounds A2 and B2. To a solution
of a nitro compound [compound A1 for 3-(3,4-diaminophenylthio)pro-
combinant P450 isoforms (1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, 2J2, pan-1-ol (compound A2) or compound B1 for 4-(3,4-diaminophenylthio)
and 3A4; Supersomes) were purchased from BD Biosciences (San Jose, phenol (compound B2), 2.00 mmol] in ethanol (20 ml) was added tin(II)
FIG 2 Synthesis of hydroxyalbendazole and hydroxyfenbendazole. MeCN, acetonitrile; EtOH, ethanol; AcOH, acetic acid.
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Wu et al.
chloride dehydrate (2.48 g, 11.0 mmol). The reaction mixture was re- carbamate (5 and 25 ␮M) for CYP2E1, danazol (1 and 5 ␮M) for CYP2J2,
fluxed for 12 h. After cooling to room temperature, the reaction mixture and ketoconazole (0.2 and 1 ␮M) for CYP3A. In all of the experiments the
was basified to pH 9 using a 3 N sodium hydroxide solution. Ethyl acetate inhibitors were dissolved and serially diluted to the required concentra-
(50 ml) was added and then filtered through Celite, followed by washing tions with methanol, and the final concentration of methanol did not
with water and ethyl acetate. The aqueous layer was separated and then exceed 1%. Except for the addition of P450 isoform-selective inhibitors,
extracted twice with ethyl acetate. The combined organic layers were all other incubation conditions were similar to those described above. For
washed with brine, dried over MgSO4, filtered, and concentrated in vacuo the assays with mechanism-based inhibitors (furafylline and ticlopidine),
to give compound A2 (88%) or B2 (92%). Compound A2: yellow oil; 1H the reaction mixture was preincubated for 15 min at 37°C in the presence
NMR (400 MHz, DMSO-d6) ␦ 6.59 (t, J ϭ 1.1 Hz, 1H), 6.44 (d, J ϭ 1.1 Hz, of inhibitor and an NADPH-generating system before the addition of
2H), 4.53 (br s, 4H), 4.44 (t, J ϭ 5.2 Hz, 1H), 3.46 to 3.41 (m, 2H), 2.70 (t, substrate (albendazole or fenbendazole). After adding the internal stan-
J ϭ 7.3 Hz, 2H), 1.64 to 1.56 (m, 2H). Compound B2: brown solid; 1H dard and centrifugation as described above, aliquots of the supernatant
NMR (400 MHz, DMSO-d6) ␦ 9.43 (s, 1H), 7.02 (d, J ϭ 8.7 Hz, 2H), 6.68 were analyzed on an LC-MS/MS system.
(d, J ϭ 8.7 Hz, 2H), 6.56 (dd, J ϭ 1.6, 0.6 Hz, 1H), 6.49 to 6.43 (m, 2H),
4.64 (br s, 2H), 4.56 (br s, 2H).
Correlation experiments. Albendazole (5 ␮M) or fenbendazole (5
␮M) was incubated with HLMs from 14 different livers to examine the
(iii) Procedure for synthesis of hydroxyalbendazole and hydroxy- formation of hydroxyalbendazole and hydroxyfenbendazole from al-
fenbendazole. To a solution of diamino compound (compound A2 for bendazole and fenbendazole, respectively, relative to specific P450 activ-
hydroxyalbendazole or compound B2 for hydroxyfenbendazole, 1.00 ity. The activity of each P450 isoform was determined using LC-MS/MS as
mmol) in acetic acid-ethanol (8:1, 45 ml) was added 1,3-bis(methoxycar- previously described (22). Isoform-specific reaction markers were used to
bonyl)-2-methyl-2-thiopseudourea (234 mg, 1.10 mmol) at room tem- determine the activity of each P450: phenacetin O-deethylation with 50
perature. The reaction mixture was refluxed for 3 h. After cooling to room ␮M phenacetin (CYP1A2), coumarin 7-hydroxylation with 5 ␮M cou-
temperature, the reaction mixture was concentrated in vacuo. The subse- marin (CYP2A6), bupropion hydroxylation with 50 ␮M bupropion
quent residue was diluted with dichloromethane and washed with water. (CYP2B6), paclitaxel 6=-hydroxylation with 10 ␮M paclitaxel (CYP2C8),
The organic layer was dried over MgSO4 and evaporated under vacuum. tolbutamide 4-methylhydroxylation with 100 ␮M tolbutamide
The residue was purified by column chromatography to give the desired (CYP2C9), S-mephenytoin 4-hydroxylation with 100 ␮M S-mepheny-
hydroxyalbendazole or hydroxyfenbendazole. Methyl-{5-[(3-hydroxy- toin (CYP2C19), dextromethorphan O-demethylation with 5 ␮M dextro-
propyl)thio]-1H-benzo[d]imidazol-2-yl}carbamate (hydroxyalbenda- methorphan (CYP2D6), chlorzoxazone 6-hydroxylation with 50 ␮M
zole): white solid; 1H NMR (400 MHz, DMSO-d6) ␦ 11.60 (br s, 2H), 7.42 chlorzoxazone (CYP2E1), ebastine hydroxylation with 1 ␮M ebastine
(d, J ϭ 1.4 Hz, 1H), 7.34 (d, J ϭ 8.2 Hz, 1H), 7.10 (dd, J ϭ 8.2, 1.8 Hz, 1H), (CYP2J2), and midazolam 1=-hydroxylation with 5 ␮M midazolam
4.49 (t, J ϭ 5.2 Hz, 1H), 3.75 (s, 2H), 3.50 to 3.44 (m, 2H), 2.91 (t, J ϭ 7.3 (CYP3A). The correlation coefficients between the rates of hydroxyal-
Hz, 2H), 1.73 to 1.60 (m, 1H); liquid chromatography (LC)-mass spec- bendazole or hydroxyfenbendazole formation from albendazole or fen-
trometry (MS) (electrospray ionization [ESI]) m/z 282 ([M ϩ 1]ϩ). bendazole, respectively, and the activity of each P450 isoform were calcu-
Methyl-{5-[(4-hydroxyphenyl)thio]-1H-benzo[d]imidazol-2-yl} carba- lated by parametric regression analysis (SAS version 8.01; SAS Institute,
mate (hydroxyfenbendazole): white solid; 1H NMR (400 MHz, DMSO- Cary, NC). A P value of Յ0.05 was considered to indicate statistical sig-
d6) ␦ 11.61 (br s, 2H), 9.67 (s, 1H), 7.33 (d, J ϭ 8.3 Hz, 1H), 7.28 (d, J ϭ 1.5 nificance.
Hz, 1H), 7.20 (d, J ϭ 8.7 Hz, 2H), 7.01 (dd, J ϭ 8.3, 1.8 Hz, 1H), 6.76 (d,
LC-MS/MS analysis of hydroxyalbendazole and hydroxyfenbenda-
zole. The concentrations of hydroxyalbendazole and hydroxyfen-
J ϭ 8.7 Hz, 2H), 3.74 (s, 3H); LC-MS (ESI) m/z 316 ([M ϩ H]ϩ).
Metabolism of albendazole and fenbendazole in HLMs and with re- bendazole in HLM cultures were quantified using LC-MS/MS as de-
combinant P450s. The optimal conditions for microsomal incubation for scribed elsewhere, with some modification (2, 11). The system
the formation of metabolites of albendazole and fenbendazole were de- consisted of a Thermo Vantage triple-quadrupole mass spectrometer
termined in the linear range. In all experiments, albendazole and fen- (Thermo Fisher Scientific, San Jose, CA), coupled with a Thermo Ac-
bendazole were dissolved and serially diluted to the required concentra- cela HPLC system (Thermo Fisher Scientific). The compounds were
tions with methanol, and the final concentration of organic solvent did separated on a reversed-phase column (Cosmosil C18; 2.0 mm [inner
not exceed 1%.
diameter] by 75 mm; particle size, 2.5 ␮m; Nacalai Tesque, Inc., Kyoto,
The incubation mixture, containing either 5 ␮l of microsomes (5 mg Japan) with an isocratic mobile phase consisting of acetonitrile and
of protein/ml of stock) or 5 ␮l of recombinant P450 (diluted to 50 water (30/70, vol/vol) containing 0.1% formic acid. The mobile phase
pmol/ml with phosphate buffer, pH 7.4) and various concentrations of was eluted at a flow rate of 0.2 ml/min. For identification of hydroxy-
albendazole and fenbendazole (0 to 100 ␮M), was reconstituted in 100 albendazole and hydroxyfenbendazole, mass spectra were recorded by
mM phosphate buffer (pH 7.4) and preincubated for 5 min at 37°C. To electrospray ionization in positive mode. The operating conditions
initiate the reaction, an NADPH-generating system (including 3.3 mM were optimized by flow injection of an analyte and were as follows:
G6P, 1.3 mM ␤-NADPϩ, 3.3 mM MgCl2, and 500 units/ml G6PDH) and capillary temperature, 350°C; vaporizer temperature, 300°C; sheath
the reaction mixtures (final volume, 100 ␮l) were incubated for 20 min at gas pressure, 35 arbitrary units; auxiliary gas, 10 arbitrary units; nitro-
37°C in a thermo-shaker (AllSheng, Hangzhou, China). The reaction was gen gas flow rate, 8 liters/min; spray voltage, 4,000 V; collision energy,
terminated by placing the incubation tubes on ice and by immediately 20 eV. Quadrupoles Q1 and Q3 were set on unit resolution. Quantita-
adding 50 ␮l of acetonitrile. After adding the internal standard (IS; tion was performed by selective reaction monitoring (SRM) of the
mebendazole, 1 ␮M), the incubation mixtures were centrifuged at 10,000 ϫ protonated precursor ion [M ϩ H]ϩ and the related product ion for
g for 5 min at 4°C, and aliquots of the supernatant were injected into an hydroxyalbendazole and hydroxyfenbendazole using the internal stan-
LC-tandem MS (LC-MS/MS) system.
dard (1 ␮M mebendazole) to establish peak area ratios. Ions was de-
Chemical inhibition studies with HLMs. Pooled HLMs (H161) and a tected by monitoring the transitions of m/z 282 ¡ 250 for hydroxyal-
P450-selective inhibitor were added to an incubation mixture similar to bendazole, m/z 316 ¡ 284 for hydroxyfenbendazole, and m/z 296 ¡
that described above. The concentration of albendazole or fenbendazole 264 for mebendazole (IS). The analytical data were processed by Xcali-
was 5 ␮M. The P450 isoform-selective inhibitors used were furafylline (1 bur (version 2.1) software. The lower limits of quantification for the
and 5 ␮M) for CYP1A2, tranylcypromine (1 and 5 ␮M) for CYP2A6, two analytes were 1.0 nM. The interassay precision for the analyte was
thio-TEPA (1 and 5 ␮M) for CYP2B6, quercetin (2 and 10 ␮M) for less than 15%.
CYP2C8, sulfaphenazole (1 and 5 ␮M) for CYP2C9, ticlopidine (0.2 and 1
Data analysis. Results are presented as the means Ϯ standard devia-
␮M) for CYP2C19, quinidine (0.2 and 1 ␮M) for CYP2D6, diethyldithio- tions (SDs) of estimates obtained from three different liver microsome
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Albendazole and Fenbendazole Metabolism by CYP2J2
FIG 3 Effects of P450 isoform-specific inhibitors on the metabolism of albendazole (A) and fenbendazole (B) by human liver microsomes. Albendazole
or fenbendazole (5 ␮M) was incubated with pooled human liver microsomes in the presence of various inhibitors. Error bars represent standard
deviations (n ϭ 3). The P450 isoform-selective inhibitors used were as follows: furafylline (1 and 5 ␮M) for CYP1A2, tranylcypromine (1 and 5 ␮M) for
CYP2A6, thio-TEPA (1 and 5 ␮M) for CYP2B6, quercetin (2 and 10 ␮M) for CYP2C8, sulfaphenazole (1 and 5 ␮M) for CYP2C9, ticlopidine (0.2 and 1
␮M) for CYP2C19, quinidine (0.2 and 1 ␮M) for CYP2D6, diethyldithiocarbamate (5 and 25 ␮M) for CYP2E1, danazol (1 and 5 ␮M) for CYP2J2, and
ketoconazole (0.2 and 1 ␮M) for CYP3A.
preparations in triplicate experiments. The apparent kinetic parameters strong CYP2J2 (24) inhibitor, respectively (Fig. 3A). The selective
of hydroxyalbendazole and hydroxyfenbendazole metabolism were deter-
mined by fitting the unweighted kinetic data from HLMs and recombi-
nant P450s to a one-enzyme Michaelis-Menten equation, a sigmoidal
(Hill) equation model {V ϭ Vmax ϫ [S]n/(Knm ϩ [S]n)}, and a substrate
inhibition model {V ϭ Vmax/(1 ϩ Km/[S] ϩ [S]/Ksi)}, where the calcu-
lated parameters were the maximum rate of metabolite formation (Vmax),
the Michaelis constant (Km), intrinsic clearance (CLint; which is equal to
CYP3A inhibitor (22) ketoconazole (1 ␮M) also inhibited
(ϳ45%) albendazole hydroxylation. This inhibition was due to
nonselective inhibition of CYP2J2 activity by ketoconazole. To
confirm these data, we evaluated inhibition of CYP2J2 activity by
ketoconazole. Ketoconazole showed moderate inhibition of
CYP2J2-mediated ebastine hydroxylation, with a 50% inhibitory
concentration (IC50) of 2.1 ␮M in HLMs (our unpublished data).
Similar inhibition of CYP2J2 activity by ketoconazole was
observed in the CYP2J2-mediated formation of metabolites of
terfenadine and astemizole in HLMs (24). Second, recombinant
CYP2J2 metabolized albendazole more efficiently than did other
P450s, with few contributions from CYP2C19 and CYP2E1 (Fig.
4A). We noted that the contributions of the CYP2C19 and
CYP2E1 isoforms to albendazole hydroxylation appeared to be
minor, as the CLints for hydroxyalbendazole formation by
CYP2C19 and CYP2E1 were 3.9- and 8.1-fold lower, respectively,
than the CLint for CYP2J2 (Table 1). Moreover, S-mephenytoin
4-hydroxylation (a marker of CYP2C19 activity) and chlorzoxa-
zone 6-hydroxylation activity (a marker of CYP2E1 activity) in 14
individual HLMs exhibited no correlation with the rate of forma-
tion of hydroxyalbendazole from albendazole (P Ͼ 0.05) (Table
2). Third, the rates of hydroxyalbendazole formation from al-
bendazole were significantly correlated (P Ͻ 0.001) with CYP2J2-
mediated ebastine hydroxylation activity (a marker of CYP2J2 ac-
tivity (25–27). The significant correlation between CYP3A activity
and albendazole hydroxylation may not be associated with the
actual involvement of CYP3A in albendazole hydroxylation. In-
Vmax/Km), the Hill coefficient (n), and the substrate inhibition constant
(Ksi) and where [S] is the substrate concentration. Percent inhibition was
calculated by the ratio of metabolite formation with and without the spe-
cific inhibitor. Calculations were performed using WinNonlin software
(Pharsight, Mountain View, CA).
RESULTS AND DISCUSSION
In this study, we present a detailed characterization of the P450
isoforms responsible for the formation of hydroxyalbendazole
and hydroxyfenbendazole from albendazole and fenbendazole,
respectively, using human liver P450 enzymes (Fig. 1). These re-
sults demonstrate that albendazole is metabolized to hydroxyal-
bendazole mainly by CYP2J2 and fenbendazole is metabolized to
hydroxyfenbendazole by CYP2C19 and CYP2J2. These data pro-
vide an important resource for accumulation of focused clinical
information that will facilitate understanding of the pharmacoki-
netics, therapeutic efficacies, drug interactions, and safety profile
of albendazole and fenbendazole.
We provide evidence that albendazole hydroxylation is cata-
lyzed predominantly by CYP2J2. First, the rates of hydroxyal-
bendazole formation were reduced by 1 ␮M ticlopidine (ϳ40%),
5 ␮M danazol (ϳ60%), and 25 ␮M diethyldithiocarbamate
(ϳ65%), a selective CYP2C19 (23), a selective CYP2E1 (22), and a stead, the correlation is likely derived from the significant corre-
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Wu et al.
FIG 4 Representative plots of the formation of hydroxyl metabolites from albendazole (A) and fenbendazole (B) by cDNA-expressed human P450 isoforms.
Human cDNA-expressed P450s 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, 2J2, and 3A4 were incubated with albendazole or fenbendazole (0.2 and 2 ␮M). Error
bars represent standard deviations (n ϭ 3).
lation between the activity of CYP3A and CYP2J2 (Pearson r ϭ (33), CYP2J2-mediated hydroxyebastine carboxylation (25), and
0.60; P ϭ 0.023) in the bank of HLMs tested. Several studies have CYP2E1-mediated p-nitrophenol oxidation (34), and such pro-
also reported cross correlations among P450 isoform activities files are suggestive of multiple substrate-binding sites (or multiple
(28–32).
regions within a single active site). Although this observation may
The kinetics of formation of hydroxyalbendazole from al- have no clinical relevance because the expected concentrations of
bendazole by recombinant CYP2C19 (rCYP2C19) and rCYP2J2 albendazole in human plasma after a person takes the usual dosage
were characterized by the Hill equation (Fig. 5A and C). The (400 mg, twice daily) (35) are much lower than the substrate in-
Eadie-Hofstee plots of hydroxyalbendazole formation revealed hibition constants (Ksi ϭ 3.17 ␮M) that we obtained here, it does
concave relationships (data not shown), indicating negative (n ϭ offer insight into the characteristics of the enzyme. The kinetic
0.56 and 0.82 for CYP2C19 and CYP2J2, respectively) cooperativ- parameters estimated from the recombinant P450 isoforms are
ity (33). The rate of hydroxyalbendazole formation by recombi- shown in Table 1. The intrinsic clearance value (0.34 ␮l/min/pmol
nant CYP2E1 versus albendazole concentrations was character- P450) of hydroxyalbendazole formation by CYP2J2 was markedly
ized by an initial rapid increase at lower concentrations, followed higher than the values for CYP2C19 (0.042 ␮l/min/pmol P450)
by a progressive decline at higher substrate concentrations (Fig. and CYP2E1 (0.088 ␮l/min/pmol P450) (Table 1), indicating that
5B). Comparison of the goodness-of-fit values generated from CYP2J2 is the major metabolizing enzyme responsible for hy-
these data indicates that a substrate inhibition equation enzyme droxyalbendazole formation.
model provided a better fit than did other models. The corre-
The specific hepatic P450 enzymes involved in the formation of
sponding Eadie-Hofstee plot indicated a hook in the upper region hydroxyfenbendazole from fenbendazole have not been identified
(data are not shown), which is characteristic of substrate inhibi- so far. Both ticlopidine and danazol markedly inhibited (Ͼ60%)
tion. Similar substrate inhibition profiles have previously been hydroxyfenbendazole formation (Fig. 3B). The inhibition by ke-
observed for CYP1A2-mediated KR-32570 O-demethylation toconazole, a CYP3A inhibitor, might be derived from its inhibi-
TABLE 1 Kinetics of hydroxyalbendazole and hydroxyfenbendazole formation from albendazole and fenbendazole, respectively, by cDNA-
expressed P450 isoformsb
Vmax
Vmax/Km
(␮l/min/pmol P450)
Substrate and P450
(pmol/min/pmol P450)
Km (␮M)
na
Ki (␮M)
Albendazole
CYP2C19
CYP2E1
0.28
0.25
0.65
6.69
2.83
1.91
0.56
0.82
0.042
0.088
0.34
3.17
CYP2J2
Fenbendazole
CYP2C19
CYP2D6
0.91
0.13
0.62
0.34
0.57
0.32
60.7
98.6
2.68
0.23
1.94
0.78
CYP2J2
a n represents the Hill coefficient.
b The values are estimated from nonlinear least regression analysis using WinNonlin software (n ϭ 3 experiments).
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Albendazole and Fenbendazole Metabolism by CYP2J2
TABLE 2 Correlation between rates of hydroxyalbendazole and hydroxyfenbendazole formation from albendazole and fenbendazole, respectively,
with P450 activities in 14 human liver microsome samplesa
r (P valueb)
Activity
P450 isoform
Albendazole
Fenbendazole
Phenacetin O-deethylation
Coumarin 7-hydroxylation
Bupropion hydroxylation
Paclitaxel 6␣-hydroxylation
Tolbutamide 4-methylhydroxylation
S-Mephenytoin 4-hydroxylation
Dextromethorphan O-demethylation
Chlorzoxazone 6-hydroxylation
Ebastine hydroxylation
CYP1A2
CYP2A6
CYP2B6
CYP2C8
CYP2C9
CYP2C19
CYP2D6
CYP2E1
CYP2J2
CYP3A
0.24 (0.40)
0.09 (0.75)
0.23 (0.41)
0.49 (0.07)
0.44 (0.11)
0.30 (0.29)
0.03 (0.90)
0.50 (0.07)
0.91 (Ͻ0.01)
0.60 (0.02)
0.41 (0.15)
0.09 (0.75)
0.02 (0.93)
0.50 (0.07)
0.39 (0.17)
0.78 (Ͻ0.01)
0.22 (0.45)
0.44 (0.11)
0.59 (Ͻ0.03)
0.63 (0.02)
Midazolam 1=-hydroxylation
a Albendazole and fenbendazole were used at 5 ␮M each. Data were analyzed using Pearson’s parametric correlation test. The activity of each P450 isoform was determined using
the respective specific substrate probe reaction, as described previously (22).
b A P value of Ͻ0.05 was considered to be statistically significant.
tory potential against CYP2J2 activity (IC50 ϭ 2.1 ␮M). Human formation by CYP2D6 were 11.7- and 8.4-fold lower than those
recombinant CYP2C19 and CYP2J2 formed hydroxyfenbenda- for CYP2C19 and CYP2J2, respectively (Table 1), and the rate of
zole from fenbendazole (Fig. 4B). We also noted that the contri- dextromethorphan O-demethylation (a marker of CYP2D6 activ-
bution of the CYP2D6 isoform to fenbendazole hydroxylation ity) in 14 individual HLMs exhibited no correlation with the rate
appeared to be minor, as the CLint values for hydroxyfenbendazole of hydroxyfenbendazole formation from fenbendazole (P ϭ 0.45)
FIG5 Kinetics for hydroxyalbendazole (A to C) or hydroxyfenbendazole (D to F) formation from albendazole or fenbendazole by recombinant human CYPC19,
CYP2D6, CYP2E1, and/or CYP2J2. An increasing concentration of substrate (0 to 100 ␮M) was incubated with human cDNA-expressed P450s and an
NADPH-generating system at 37°C for 20 min. The velocity (pmol/min/pmol P450) versus the albendazole or fenbendazole concentration was fit to a Hill or
substrate inhibition equation (see “Data analysis” in Materials and Methods). Each data point represents the mean of triplicate incubations.
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Wu et al.
FIG 6 Abundance-adjusted simulations of the relative contributions of CYP2J2, CYP2C19, CYP2E1, and CYP2D6 to albendazole and fenbendazole hydroxy-
lation.
(Table 2). The rates of hydroxyfenbendazole formation from fen- form liver contents reported by Shimada et al. (38), Yamazaki
bendazole were significantly correlated (P Ͻ 0.05) with the
CYP2C19-mediated S-mephenytoin hydroxylation and CYP2J2-
mediated ebastine hydroxylation activities. The significant corre-
lation (P ϭ 0.02) between fenbendazole hydroxylase activity and
CYP3A activity might also be derived from the cross correlation
between CYP2J2 and CYP3A activity (P ϭ 0.05) in the bank of
HLMs evaluated.
The kinetics of the formation of hydroxyfenbendazole from
fenbendazole by rCYP2C19 and rCYP2J2 was characterized using
the substrate inhibition equation (Fig. 5D and F). The Eadie-Hof-
stee plots of hydroxyfenbendazole formation also showed a hook
in the upper region of this plot (data not shown), which is char-
acteristic of substrate inhibition. The rate of hydroxyfenbendazole
formation from fenbendazole by recombinant CYP2D6 was char-
acterized by the Hill equation (Fig. 5E). The Eadie-Hofstee plots of
hydroxyfenbendazole formation revealed concave relationships
(data not shown), indicating negative (n ϭ 0.78) cooperativity.
The intrinsic clearance values of hydroxyfenbendazole formation
by CYP2C19 (2.68 ␮l/min/pmol P450) and CYP2J2 (1.94 ␮l/min/
pmol P450) were much higher than the intrinsic clearance value of
hydroxyfenbendazole formation by CYP2D6 (0.23 ␮l/min/pmol
P450) (Table 1), indicating that CYP2C19 and CYP2J2 are the
major metabolizing enzymes responsible for hydroxyfenbenda-
zole formation.
To determine the relative contributions of the three P450
isoforms to hydroxylation metabolism at different concentra-
tions in human liver microsomes, we estimated the percentage
of the net reaction rate from the abundance-adjusted simula-
tion of each P450 isoform in human liver microsomes (Fig. 6).
For this, we normalized the metabolic rates of the expressed
P450 isoforms according to the abundance of the respective
isoforms in the human liver, as described by Rodrigues (36) or
Kim et al. (37). Briefly, the reaction rates measured with indi-
vidual cDNA-expressed P450 isoforms were normalized with
respect to the nominal specific content of the corresponding
P450 in native human liver microsomes. In this study, we
et al. (39), Yamazaki et al. (40), and Lasker et al. (41); i.e.,
2.73% for CYP2C19, 1.16% for CYP2J2, 1.5% for CYP2D6, and
6.6% for CYP2E1. In turn, the normalized rates for each
cDNA-expressed P450 were summed, yielding a total normal-
ized rate (TNR; Αfi · Vi), and the normalized rate for each P450
isoform (fi · Vi) was expressed as a percentage of the net reac-
tion rate {100 ϫ [(fi · Vi)/(Αfi · Vi)]}, where fi indicates the
fraction of the content of each P450 isoform in the human liver,
and Vi is Vmax,i · [Si]/(Km,i ϩ [Si]), where Vmaxi is the Vmax for
each isoform, [Si] is the substrate concentration for each iso-
form, and Km,i is the Km for each isoform. This simulation
shows that CYP2C19 and CYP2J2 are the major P450 isoforms
responsible for hydroxy metabolite formation from albenda-
zole over the whole concentration range tested (0 to 50 ␮M).
The percentage of the net reaction by CYP2E1 (about 40%)
decreased rapidly with increasing albendazole concentration
and reached 10% at 50 ␮M. CYP2C19 and CYP2J2 are the
major P450 isoforms responsible for hydroxy metabolite for-
mation from fenbendazole.
Taken together, these results suggest that CYP2J2 is one of the
major enzymes involved in the hydroxylation of both albendazole
and fenbendazole. CYP2J2 is expressed in the human brain, heart,
kidney, liver, lung, placenta, and skeletal muscle (42) and is re-
garded as a minor hepatic drug-metabolizing enzyme. However,
recent studies have shown CYP2J2 to be involved in the metabo-
lism of endogenous arachidonic acid (43), as well as drugs, includ-
ing amiodarone (24) astemizole (44), apixaban (45), cyclosporine
(11), ebastine (25), mesoridazine (11), tamoxifen (11), and thio-
ridazine (11). Thus, CYP2J2 could be a target of the interactions of
the drugs metabolized by this enzyme. In addition, the identifica-
tion of CYP2J2 as the catalyst of hydroxylation of albendazole may
allow use of albendazole as a CYP2J2 probe substrate due to the
nonavailability of a specific probe for measuring CYP2J2 activity
in vitro and in vivo, despite identification of an increasing number
of drugs for which CYP2J2 is responsible for metabolism. Our
adapted the data for immunologically determined P450 iso- data indicate that albendazole hydroxylation is a specific in vitro
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Albendazole and Fenbendazole Metabolism by CYP2J2
synthesis in three steps and spectral determination of methyl-5-[(o-, m-,
and p-substituted-phenylthio]-2-benzimidazolecarbamates. J. Heterocy-
clic Chem. 41:273–276.
reaction marker of CYP2J2 and may have utility as a phenotyping
tool to study the role of this enzyme in drug metabolism.
19. Lee T, Lee D, Lee IY, Gong YD. 2010. Solid-phase synthesis of thia-
zolo[4,5-b]pyridine derivatives using Friedlander reaction. J. Combinato-
rial Chem. 12:95–99.
20. Lee T, Park JH, Lee DH, Gong YD. 2009. Traceless solid-phase synthesis
of 2,4,6-trisubstituted thiazolo[4,5-d]pyrimidine-5,7-dione derivatives. J.
Combinatorial Chem. 11:495–499.
ACKNOWLEDGMENT
This work was supported by a grant from the Korea Health Technology
R&D Project, Ministry of Health & Welfare, Republic of Korea
(A111345).
21. Lee T, Park JH, Jeon MK, Gong YD. 2009. Solid-phase synthesis of
1,3,6-trisubstituted-1H-thiazolo[4,5-c][1,2]thiazin-4(3H)one-2,2-
dioxide derivatives using traceless linker. J. Combinatorial Chem. 11:288–
293.
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