Preparation of human drug metabolites using fungal peroxygenases

Article abstract of DOI:10.1016/j.bcp.2011.06.020

The synthesis of hydroxylated and O- or N-dealkylated human drug metabolites (HDMs) via selective monooxygenation remains a challenging task for synthetic organic chemists. Here we report that aromatic peroxygenases (APOs; EC 1.11.2.1) secreted by the agaric fungi Agrocybe aegerita and Coprinellus radians catalyzed the H₂O₂-dependent selective monooxygenation of diverse drugs, including acetanilide, dextrorphan, ibuprofen, naproxen, phenacetin, sildenafil and tolbutamide. Reactions included the hydroxylation of aromatic rings and aliphatic side chains, as well as O- and N-dealkylations and exhibited different regioselectivities depending on the particular APO used. At best, desired HDMs were obtained in yields greater than 80% and with isomeric purities up to 99%. Oxidations of tolbutamide, acetanilide and carbamazepine in the presence of H₂¹⁸O₂ resulted in almost complete incorporation of ¹⁸O into the corresponding products, thus establishing that these reactions are peroxygenations. The deethylation of phenacetin-d₁ showed an observed intramolecular deuterium isotope effect [(k_H/k_D) _obs] of 3.1 ± 0.2, which is consistent with the existence of a cytochrome P450-like intermediate in the reaction cycle of APOs. Our results indicate that fungal peroxygenases may be useful biocatalytic tools to prepare pharmacologically relevant drug metabolites.

Full text of DOI:10.1016/j.bcp.2011.06.020

Biochemical Pharmacology 82 (2011) 789–796
Contents lists available at ScienceDirect
Biochemical Pharmacology
journal homepage: www.elsevier.com/locate/biochempharm
Preparation of human drug metabolites using fungal peroxygenases
a
b
Marzena Poraj-Kobielska a, Matthias Kinne a, , Rene Ullrich , Katrin Scheibner ,
*
´
Gernot Kayser a, Kenneth E. Hammel c, Martin Hofrichter a
a International Graduate School of Zittau (IHI Zittau), Department of Bio- and Environmental Sciences, Markt 23, 02763 Zittau, Germany
b Section of Biotechnology, Chemistry and Process Engineering, Lausitz University of Applied Sciences, Großenhainer Straße 57, 01968 Senftenberg, Germany
c USDA Forest Products Laboratory, Madison, WI 53726, USA, and Department of Bacteriology, University of Wisconsin, Madison, WI 53706, USA
A R T I C L E I N F O
A B S T R A C T
Article history:
The synthesis of hydroxylated and O- or N-dealkylated human drug metabolites (HDMs) via selective
monooxygenation remains a challenging task for synthetic organic chemists. Here we report that
aromatic peroxygenases (APOs; EC 1.11.2.1) secreted by the agaric fungi Agrocybe aegerita and
Coprinellus radians catalyzed the H2O2-dependent selective monooxygenation of diverse drugs, including
acetanilide, dextrorphan, ibuprofen, naproxen, phenacetin, sildenafil and tolbutamide. Reactions
included the hydroxylation of aromatic rings and aliphatic side chains, as well as O- and N-dealkylations
and exhibited different regioselectivities depending on the particular APO used. At best, desired HDMs
were obtained in yields greater than 80% and with isomeric purities up to 99%. Oxidations of
tolbutamide, acetanilide and carbamazepine in the presence of H218O2 resulted in almost complete
incorporation of 18O into the corresponding products, thus establishing that these reactions are
peroxygenations. The deethylation of phenacetin-d1 showed an observed intramolecular deuterium
isotope effect [(kH/kD)obs] of 3.1 ꢀ 0.2, which is consistent with the existence of a cytochrome P450-like
intermediate in the reaction cycle of APOs. Our results indicate that fungal peroxygenases may be useful
biocatalytic tools to prepare pharmacologically relevant drug metabolites.
Received 28 March 2011
Accepted 14 June 2011
Available online 23 June 2011
Keywords:
Peroxidase
Peroxygenation
Hydroxylation
O-Dealkylation
N-Dealkylation
Cytochrome P450
ß 2011 Elsevier Inc. All rights reserved.
1. Introduction
reaction rates [7,8]. More promising results have been reported
for laboratory-evolved bacterial P450s, which are capable of
Human drug metabolites (HDMs) are valuable chemicals
needed for the development of safe and effective pharmaceuticals.
They are frequently used as substrates and authentic standards in
studies of drug bioavailability, pharmacodynamics and pharma-
cokinetics [1–3]. In vivo, the C–H bonds of pharmaceuticals are
predominantly oxygenated by liver cytochrome P450-monoox-
ygenases (P450s) to yield more polar HDMs that are excreted
directly or as conjugates [4]. This directed incorporation of an
oxygen atom into a complex organic structure is one of the most
challenging reactions in synthetic organic chemistry [5] and thus
low yields and the need for laborious removal of byproducts have
prevented the cost-effective preparation of HDMs by purely
chemical methods [6].
catalyzing the H2O2-dependent hydroxylation of pharmaceuti-
cals via the ‘‘peroxide shunt’’ pathway [9]. Recent studies,
however, have demonstrated that this approach needs further
optimization [10]. Alternatively, modified hemoproteins such
as microperoxidases might be used to catalyze hydroxylations
by a P450-like oxygen transfer mechanism, but so far these
catalysts do not exhibit the necessary performance and
selectivity [11–16].
Here we have adopted a recently developed approach, using
aromatic peroxygenases (APOs)1 from the agaric basidiomycetes
Agrocybe aegerita (AaeAPO) and Coprinellus radians (CraAPO) to
produce diverse HDMs. These stable, secreted enzymes oxidize a
wide range of substrates and are promising oxidoreductases for
biotechnological applications [6,17–23].
Another approach is the in vitro preparation of HDMs with
enzymes. The obvious route is to use isolated human P450s, but
these complex multiprotein systems are membrane-bound,
poorly stable, cofactor-dependent, and generally exhibit low
1
In previous publications, the enzymes were also referred to as haloperoxidase-
peroxygenases, mushroom/fungal peroxygenases, AaP (Agrocybe aegerita peroxi-
dase/peroxygenase) or CrP (Coprinellus radians peroxygenase) [5,18,20,22]. In
February 2011, they were classified in the Enzyme Nomenclature under EC 1.11.2.1
(unspecific peroxygenase).
* Corresponding author. Tel.: +49 3583612723; fax: +49 3583612734.
E-mail address: kinne@ihi-zittau.de (M. Kinne).
0006-2952/$ – see front matter ß 2011 Elsevier Inc. All rights reserved.
doi:10.1016/j.bcp.2011.06.020

790
M. Poraj-Kobielska et al. / Biochemical Pharmacology 82 (2011) 789–796
2. Materials and methods
ammonium formate (pH 3.5)/acetonitrile, 95:5 for 5 min, followed
by 25-min linear gradient to 100% acetonitrile. For the
experiments with acetaminophen, acetanilide and diclofenac, a
a
2.1. Reactants
Synergi 4
length, 4
m
Fusion RP-80A column (4.6 mm diameter by 150 mm
Metoprolol, oseltamivir phosphate, 4-hydroxytolbutamide, 40-
hydroxydiclofenac, 5-hydroxydiclofenac, 3-hydroxyacetamino-
phen, omeprazole and sildenafil were obtained from Chemos GmbH
(Regenstauf, Germany). 3-Hydroxycarbamazepine, 1-hydroxyibu-
profen, 2-hydroxyibuprofen, 1-oxoibuprofen and N-desmethylsil-
denafil were purchased from Toronto Research Chemicals, Inc.
mm particle size, Phenomenex) was used. The column
was eluted at 40 8C and a flow rate of 1 ml minꢁ1 with a mixture of
aqueous phosphoric acid solution (15 mM, pH 3) and acetonitrile,
95:5, for 5 min, followed by a 20-min linear gradient to 100%
acetonitrile. For metoprolol, a Gemini-Nx 3
phase column (2 mm diameter by 150 mm length, 3
m
110A C18 reverse
m particle
m
(Toronto, Canada),
monoethylglycinexylidide from TLC PharmaChem., Inc. (Vaughan,
Canada). 17 -Ethinylestradiol, (R)-naproxen, 4-hydroxyproprano-
a
-hydroxymetoprolol, glycinexylidide and
size, Phenomenex) was used. The column was eluted at 45 8C and a
flow rate of 0.3 ml minꢁ1 with a mixture of aqueous 0.2% vol/vol
ammonium (pH 10.5) and acetonitrile, 95:5, for 1 min, followed by
a 20-min linear gradient to 80% acetonitrile, followed by 21-min
linear gradient to 100% acetonitrile. Chiral separation was done
using the HPLC apparatus above, but using a Whelk-O 5/100
Kromasil column (4.6 mm diameter by 250 mm length, Regis
Technologies (Morton Grove, USA). The isocratic mobile phase
consisted of 80% vol/vol methanol and 20% of 15 mM phosphate
buffer. The columns were operated at 40 8C and 1 ml minꢁ1 for
30 min. Retention times for products are given in Supplemental
Table 1.
a
lol, 5-hydroxypropranolol, (S)-N-desisopropylpropranolol and O-
desmethylmetoprolol were obtained from Fluka (St. Gallen,
Switzerland), Shanghai FWD Chemicals, Ltd. (Shanghai, China),
BiomolGmbH(Hamburg,Germany), SPIbio, BertinGroup(Montigny
Le Bretonneux, France), ABX Advanced Biochemical Compounds
(Radeberg, Germany) and Sandoo Pharmaceuticals & Chemicals Co.,
Ltd. (Zhejiang, China), respectively. H218O2 (90 atom%, 2% wt/vol)
was a product of Icon Isotopes (New York, USA). All other chemicals
were purchased from Sigma-Aldrich (Schnelldorf, Germany).
Phenacetin-d1 (N-(4-[1-2H]ethoxyphenyl)acetamide) was pre-
pared from acetaminophen (N-(4-hydroxyphenyl)acetamide) and
ethyl iodide-d1 as described previously for phenacetin-d3 [24]. The
reaction product of the synthesis (phenacetin-d1) was identified by
comparison of retention time, UV absorption spectra, and mass
spectra relative to authentic phenacetin [25]. Mass spectrum (m/z,
%): 180 (100, ꢁH), 138 (62, ꢁC2H2O), 110 (12, ꢁC4DH6O), 109 (96,
ꢁC3DH2O2), 108 (100, ꢁC3DH6ON), 80 (16) and 43 (18).
Aliphatic aldehydes were analyzed as their 2,4-dinitrophenyl-
hydrazones after addition of 0.2 volume of 0.1% 2,4-dinitrophe-
nylhydrazine solution in 0.6 N HCl to each reaction mixture. The
derivatized products were analyzed using the same HPLC
apparatus as above, but the Luna C18 column was eluted with
aqueous 0.1% vol/vol ammonium formate (pH 3.5)/acetonitrile,
70:30 for 5 min, followed by a 20-min linear gradient to 100%
acetonitrile. Stoichiometrical experiments on sildenafil N-demeth-
ylation were conducted in stirred reactions (0.20 ml) that
The extracellular peroxygenase of A. aegerita (AaeAPO; isoform
II, 45 kDa) was produced and purified as described previously [26].
The enzyme preparation was homogeneous by sodium dodecyl
polyacrylamide gel electrophoresis (SDS-PAGE) and exhibited an
A418/A280 ratio of 1.75. The specific activity of the AaeAPO
preparation was 117 U mgꢁ1, where 1 U represents the oxidation
contained 2 U mlꢁ1 (0.144
mM) peroxygenase, potassium phos-
phate buffer (50 mM, pH 7.0) and the substrate (0.25 mM). The
reactions were initiated with 0.0023–0.037 mM H2O2.Mass
spectroscopic determinations were made in positive or negative
ESI mode (electrospray ionization) in a mass range from 70 to 500,
step size 0.1, drying gas temperature 350 8C, capillary voltage
4000 V for positive mode and 5500 V for negative mode. The
reaction products were identified relative to authentic standards,
based on their retention times, UV absorption spectra, and mass
spectral [M+H]+ or [MꢁH]ꢁ ions.Quantifications of reactants and
products were obtained by HPLC as described above, using a linear
external standard curve (R > 0.98) of the respective compound.
The parameters were calculated as follows: total conversion
[(Sc ꢁ Ss)/Sc) ꢂ 100%; yield: (P/Sc) ꢂ 100%; regioselectivity: (P/
(Sc ꢁ Ss)] ꢂ 100% (Sc is the substrate concentration of the control,
Ss is the substrate concentration after reaction with the enzyme
and P is the product concentration).
of 1
mmol of 3,4-dimethoxybenzyl alcohol (veratryl alcohol) to 3,4-
dimethoxybenzaldehyde (veratraldehyde) in 1 min at 23 8C [26].
The Coprinellus radians peroxygenase (CraAPO) was produced and
purified as described previously [27]. It was homogeneous by SDS-
PAGE, exhibited an A419/A280 ratio of 1.04 and had a specific activity
of 25.8 U mgꢁ1 in the above assay.
2.2. Product identification
Typical reaction mixtures (0.2–1.0 ml) contained purified
peroxygenase (1.0–2.0 U mlꢁ1 = 0.4–0.8
mM), substrate to be
oxidized (0.5–2.0 mM), potassium phosphate buffer (50 mM, pH
7.0) and ascorbic acid (4.0–6.0 mM, to inhibit further oxidation of
any phenolic products that were released [6,17]). The reactions
were started by the addition of limiting H2O2 (2.0–4.0 mM) and
stirred at room temperature. They were stopped by addition of
sodium azide (1 mM) or trichloroacetic acid (TCA) (5%) after 3 min.
In some cases, H2O2 was added continuously with a syringe pump
and the reactions were stopped after 4–6 h, when chromatograph-
ic analyses showed that product formation was complete.
Reaction products of interest were obtained, generally with
baseline resolution, by high performance liquid chromatography
(HPLC) using an Agilent Series 1200 instrument equipped with a
diode array detector (DAD) and an electrospray ionization mass
spectrometer (MS) (Agilent Technologies Deutschland GmbH,
Bo¨blingen, Germany). Unless otherwise stated, reverse phase
(RP) chromatography of reaction mixtures was performed on a
2.3. Enzyme kinetics
The kinetics of propranolol hydroxylation as well as metoprolol
and phenacetin O-dealkylation were analyzed in stirred microscale
reactions (0.10 ml, 23 8C) that contained 0.40
mM peroxygenase
(AaeAPO), potassium phosphate buffer (50 mM, pH 7.0), ascorbic
acid (4 mM) and 0.010–5.000 mM substrate. The reactions were
initiated with 2.0 mM H2O2. Reactions with propranolol or
phenacetin were stopped with 0.010 ml of 50% TCA solution after
5 s, and reactions with metoprolol were stopped with 0.2 volume
of 0.1% 2,4-dinitrophenylhydrazine solution in 0.6 N HCl after 10 s.
The resulting products were quantified by HPLC as described above
using external standard curves prepared with authentic standards.
The kinetics of acetanilide hydroxylation were analyzed in
Luna C18 column (2 mm diameter by 150 mm length, 5
m
m
stirred reactions (0.5 ml, 23 8C) that contained 0.08 mM AaeAPO,
particle size, Phenomenex (Aschaffenburg, Germany), which was
eluted at 0.35 ml minꢁ1 and 40 8C with aqueous 0.01% vol/vol
potassium phosphate buffer (50 mM, pH 7.0), ascorbic acid (4 mM)
and 0.010–5.000 mM substrate. The reactions were initiated with

M. Poraj-Kobielska et al. / Biochemical Pharmacology 82 (2011) 789–796
791
2.0 mM H2O2 and stopped with 0.050 ml of sodium azide solution
(1 mM) after 5 s. The initial velocity of acetaminophen formation
was then determined from the increase in absorbance at 290 nm
Using AaeAPO and two of the above substrates, we investigated
the source of the oxygen introduced during hydroxylation. When
the oxidation of acetanilide (II) was conducted with H218O2 in
place of H2O2, mass spectral analysis of the resulting acetamino-
phen showed that the principal [MꢁH]ꢁ ion had shifted from its
natural abundance m/z of 150 to m/z 152. Likewise, the
hydroxylation of 3-hydroxycarbamazepine under these conditions
resulted in a shift of the [M+H]+ ion from its natural abundance m/z
of 253 to m/z 255. Accordingly, H2O2 was the source of the
introduced oxygen in both reactions.
(e
= 1100 Mꢁ1 cmꢁ1) using a Cary 50 UV/visible spectrophotome-
ter. The kinetic parameters were determined by nonlinear
regression using the Michaelis–Menten model in the ANEMONA
program [28].
2.4. 18O-labeling experiments
The reaction mixtures (0.20 ml, stirred at room temperature)
Also using AaeAPO, we determined apparent kinetic constants
for two of these aromatic hydroxylations. For propranolol, the Km
contained 2 U mlꢁ1 (0.08
mM) of AaeAPO, potassium phosphate
buffer (50 mM, pH 7.0) and 0.5 mM substrate (tolbutamide,
acetanilide, or carbamazepine). Reactions with tolbutamide or
acetanilide were initiated with a single addition of 2.0 mM (final
concentration) H218O2. For reactions with carbamazepine, the
same quantity of H218O2 was added continuously with a syringe
pump over 6 h. A portion of each completed reaction was then
analyzed by HPLC/MS as described in Section 2.2. For each m/z
value, the average total ion count within the metabolite (4-
hydroxytolbutamide, acetaminophen and 3-hydroxycarbamaze-
pine) peak was used after background correction to generate the
ion count used for mass abundance calculations [18].
was 442
were 1,310
m
M and the kcat was 59 sꢁ1; for acetanilide, the constants
m
M and 1,925 sꢁ1
.
3.2. Aliphatic hydroxylation
Both peroxygenases catalyzed the hydroxylation of aliphatic
side chains. Thus, ibuprofen (IV) was predominantly oxidized to 2-
hydroxyibuprofen, with the CraAPO-catalyzed reaction exhibiting
a higher yield and regioselectivity. Similarly, tolbutamide (VII) was
oxidized to 4-hydroxytolbutamide (Table 1). A mass spectral
experiment with tolbutamide and H218O2 as the substrates showed
that the resulting 4-hydroxytolbutamide exhibited a principal
[MꢁH]ꢁ ion with an m/z of 289 rather than 287, again showing that
H2O2 was the source of the introduced oxygen (Fig. 3).
2.5. Determination of intramolecular isotope effect
The reaction mixture (0.2 ml) contained purified AaeAPO
(2 U mlꢁ1, 0.08
m
M), potassium phosphate buffer (50 mM, pH
3.3. O-Dealkylation
7.0), phenacetin-d1 and ascorbic acid (4.0 mM). The reaction was
started by the addition of H2O2 (2.0 mM) and stirred at room
temperature. The reaction was stopped by the addition of 10%
sodium azide (10 mM) after 10 s. The reaction products were
analyzed as described above. For each m/z value, the average total
ion count within the acetaminophen peak was used after
background correction to generate the ion count used for mass
abundance calculations.
Both APOs cleaved alkyl aryl ether linkages in various drugs to
yield phenols [18]. Thus, metoprolol (VIII) was selectively
demethylated to O-desmethylmetoprolol, naproxen (IX) to O-
desmethylnaproxen and dextromethorphan (X) to dextrorphan.
Yields and regioselectivities were generally greater with AaeAPO.
The above phenolic reaction products all tended to undergo further
oxidation to quinones and/or coupling products because APOs
exhibit general peroxidase activity that generates phenoxyl
radicals from phenols [5,6,17,18], but this undesired reaction
was partially inhibitable via addition of the radical scavenger
ascorbate to the reactions. DNPH derivatization of the reaction
mixtures showed that the methyl group was released as
formaldehyde in each case. A kinetics analysis done with AaeAPO
and one of the substrates, metoprolol, showed that the reaction
3. Results
The peroxygenases from Agrocybe aegerita (AaeAPO) and
Coprinellus radians (CraAPO) oxidized diverse pharmaceuticals
(I–XX, Fig. 1), showing different selectivity in some cases. The
products, most of which we obtained with baseline resolution by
HPLC, were monooxygenated or dealkylated compounds that
correspond in most cases to previously identified HDMs (Table 1).
Below, we have classified the major reactions according to the
chemical moiety that was oxidized on the targeted drug. In many
cases, a single oxidation predominated but, as noted in Table 1,
more than one of these reactions sometimes occurred on a single
substrate. A complete overview of all reactants investigated,
confirmed products and hypothetical products for which we lacked
authentic standards (drawn in brackets), is presented in Supple-
mentary Table 2.
exhibited an apparent Km for it of 2,330
96 sꢁ1
mM and an apparent kcat of
.
Analogously to the demethylations discussed above, phenace-
tin (XI) was de-ethylated to give acetaminophen and acetaldehyde
(Table 1). Since phenacetin has a symmetrical site at its
a
-carbon, it
is suitable substrate to determine whether
a
a catalyzed
etherolytic reaction exhibits an intramolecular deuterium isotope
effect. LC/MS analysis of DNPH-derivatized reactions showed that
the AaeAPO-catalyzed cleavage of N-(4-[1-2H]ethoxyphenyl)ace-
tamide (phenacetin-d1) resulted in a preponderance of [2H]acetal-
dehyde 2,4-dinitrophenylhydrazone (m/z 224, [MꢁH]ꢁ) over
natural abundance acetaldehyde 2,4-dinitrophenylhydrazone
(m/z 223, [MꢁH]ꢁ) (Fig. 4). The observed mean intramolecular
deuterium isotope effect [(kH/kD)obs] from three experiments was
3.1 ꢀ 0.2. The apparent Km of AaeAPO for phenacetin was 998 mM and
3.1. Aromatic hydroxylation
Both APOs catalyzed the hydroxylation of aromatic rings, in
some cases with high regioselectivity. For example, AaeAPO
preferentially oxidized propranolol (I) to 5-OH-propranolol [17],
acetanilide (VI) to acetaminophen (paracetamol; Fig. 2), carba-
mazepine (III) to 3-OH-carbamazepine and diclofenac (IV) to 40-
OH-diclofenac [17]. CraAPO catalyzed the same reactions but with
lower regioselectivity. Both enzymes also oxidized tamoxifen (V)
to produce low yields of 4-OH-tamoxifen and other products,
which we were unable to resolve sufficiently by HPLC to permit
quantifications.
the apparent kcat was 33 sꢁ1
.
3.4. N-Dealkylation
The APOs catalyzed the oxidative N-dealkylation of several
drugs that contain secondary or tertiary amine groups. For
example, AaeAPO regioselectively N-dealkylated sildenafil (XII)
at the tertiary N in its N-methyl piperazine ring to give N-

792
M. Poraj-Kobielska et al. / Biochemical Pharmacology 82 (2011) 789–796
Propranolol
(ß-blocker)
IV Diclofenac
I
II
Carbamazepine
(anticonvulsant)
Tamoxifen
(anti-cancer)
Acetanilide
(anti-inflammatory)
III
V
(anti-inflammatory)
N
O
H2N
Cl
O
O
N
H
O
NH
OH
N
NH
Cl
OH
O
VIIbuprofen
VIITolbutamide
(Na+-channel blocker)
(anti-inflammatory)
Naproxen
IX
Metoprolol
(ß-blocker)
VIII
O
O
O
OH
(anti-inflammatory)
S
NH
O
NH
O
OH
O
O
O
NH
XDextromethorphan
(antitussive)
OH
Phenacetin
XI
(anti-inflammatory)
NH
N
H
Sildenafil
XII
Lidocaine
XIII
(neuraminidase inhibitor)
(anesthetic)
O
O
O
N
N
N
N
O
O
N
N
O
NH
S
N
H
O
XIV 4-Dimethylaminoantipyrine
O
(antipyrretic)
XV
Oseltamivir
(antiviral)
Sulfadiazine
(antibacterial)
XVII
N
Ketoprofen
(anti-inflammatory)
O
XVI
O
N
NH
S
O
N
O
N
O
O
N
O
O
O
H2N
NH
OH
H2N
Felbamate
(anticonvulsant)
XXI
Chlorpromazine
XX
XVIII Gemfibrozil
(antipsychotic)
Cl
(lipid-lowering agent)
Omeprazole
XIX
O
(proton pump inhibitor)
NH2
O
O
OH
N
O
O
O
S
O
O
S
N
N
H
NH2
O
17α-Ethinylestradiol
XXII
N
N
(contraceptive)
Reichstein substance S
(prohormone)
XXIII
OH
O
HO
H
OH
H
H
HO
O
Fig. 1. Chemical structures of pharmaceuticals tested as peroxygenase substrates.
desmethylsildenafil and formaldehyde in high yield. By contrast,
CraAPO was ineffective in this oxidation (Table 1). A quantitative
analysis of AaeAPO-catalyzed sildenafil oxidation in the presence
of the limiting H2O2 showed that one equivalent of N-desmethyl-
sildenafil was formed per equivalent of oxidant supplied (Table 2).
Among the other N-dealkylations listed in Table 1 are the
conversion of lidocaine (XIII) to monoethylglycinexylidide and
glycinexylidide and of 4-dimethylaminoantipyrine (XIV) to 4-
aminoantipyrine.
3.5. Ester cleavage
Ester cleavage by a peroxygenase can be regarded as a special
case of O-dealkylation. Thus, CraAPO selectively cleaved oselta-

M. Poraj-Kobielska et al. / Biochemical Pharmacology 82 (2011) 789–796
793
Table 1
Products identified by mass spectroscopy after oxidation of pharmaceuticals by AaeAPO and CraAPO in the presence of H2O2. The m/z value for the major observed diagnostic
ion, consumed substrate (SC), the yield (Y) and the regioselectivity (RS) of the product is shown in each case.
Substrate
Reaction product
Discussed as HDM
in Ref no.
m/z
AaeAPO (%)
CraAPO (%)
SC
23
Y
RS
91
SC
11
Y
RS
I
5-OH-Propranolol
[9,29]
[M+H]+ 276
[M+H]+ 276
[M+H]+ 218
21
6
2
3
53
13
22
4-OH-Propranolol
[9,29,46]
[9,29]
–
N-Desisopropyl-propranolol
Traces
II
89
20
Acetaminophen
[47–49]
[47]
[MꢁH]ꢁ 150
[MꢁH]ꢁ 166
80
5
90
5
13
1
65
2
3-OH-Acetaminophen
IIIb
IV
V
22
78
25
15
15
3-OH-Carbamazepine
40-OH-Diclofenac
[50]
[M+H]+ 253
[M+H]+ 312
13
68
61
87
6
5
40
30
[51,52]
4-OH-Tamoxifen
N-Desmethyltamoxifen
Endoxifen
[53–55]
[53,54]
[53,54]
[M+H]+ 388
[M+H]+ 358
[M+H]+ 374
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
VI
87
98
2-OH-Ibuprofen
1-OH-Ibuprofen
1-Oxo-Ibuprofen
[56,57]
[57]
[M+H]+ 223
[M+H]+ 223
[M+H]+ 221
21
7
24
8
74
13
75
62
–
–
n.a.
Traces
VIIa
25
82
20
73
4-OH-Tolbutamide
[58]
[MꢁH]ꢁ 287
15
60
VIIIa
O-Desmethylmetoprolol
[59]
[59]
[M+H]+ 254
[M+H]+ 284
17
2
20
2
4
1
5
1
a-OH-Metoprolol
IXb
X
60
17
34
10
15
16
O-Desmethylnaproxen
[60,61]
[62,63]
[MꢁH]ꢁ 215
57
16
95
95
9
85
53
80
Dextrorphan
[M+H]+ 258
8
XI
Acetaminophen
[64,65]
[64]
[M+H]+ 152
[M+H]+ 168
23
2
66
5
13
3-OH-Acetaminophen
–
XII
82
60
80
45
N-Desmethylsildenafil
[66]
[M+H]+ 461
82
99
4
5
XIII
Monoethylglycinexylidide
Glycinexylidide
[67]
[67]
[M+H]+ 207
[M+H]+ 179
25
18
41
30
32
5
70
11
XIV
68
–
38
80
4-Aminoantipyrine
[68]
[69]
[M+H]+ 204
16
23
19
71
48
88
XVb
Oseltamivir carboxylate
[M+H]+ 285
–
a
Mass spectral data indicate the formation of corresponding carbonyls as described previously [19] (see supplementary Material).
b
A syringe pump was used for hydrogen peroxide supply. (n.d.) = not determined due to poor resolution; (ꢁ) = not detected; (n.a.) = not applicable.
mivir (XV) to oseltamivir carboxylate and acetaldehyde in high
yield. Interestingly, oseltamivir was not converted by AaeAPO.
neither one predominated in the end-product mixture (data not
shown).
3.6. Additional observations
4. Discussion
For some products of APO-catalyzed oxidations no authentic
standards were available. However, the mass shifts we observed
after these reactions (see supplementary material) indicate the
regioselective oxidation of ketoprofen (XVI), sulfadiazine (XVII),
gemfibrozil (XVIII), omeprazole (XIX) and chlorpromazine (XX).
We also noted some limitations on substrates for the APOs. No
Our results show that two fungal peroxygenases (APOs)
catalyzed the hydroxylation or dealkylation of diverse pharma-
ceuticals, in some cases with high regioselectivity. Furthermore,
most of the oxidations matched those catalyzed by human liver
P450s, e.g. propranolol to 5-hydroxypropranolol (CYP2D6) [29],
tolbutamide to 4-hydroxytolbutamide (CYP2C9) [30], naproxen
to O-desmethylnaproxen (CYP1A2) [31], acetanilide to acet-
aminophen (CYP1A2) [31,32] and sildenafil to N-desmethylsil-
denafil (CYP3A4) [33]. The catalyzed reactions: aromatic
hydroxylation, aliphatic hydroxylation, O-dealkylation of ethers
and esters and N-dealkylation of amines, are all typical of P450s
[34,35]. Our data agree with previous work that suggests the
product formation was observed from felbamate (XXI), 17a-
ethinylestradiol (XXII), or Reichstein substance S (XXIII). More-
over, we found that the APOs generally failed to discriminate
between chiral centers in the pharmaceutical substrates. For
example, chiral HPLC separation of the two O-desmethylnaproxen
entantiomers that resulted from naproxen oxidation showed that

794
M. Poraj-Kobielska et al. / Biochemical Pharmacology 82 (2011) 789–796
Table 2
500
400
300
200
100
0
Stoichiometry of sildenafil (XII) oxidation by AaeAPO.
H2O2 added
M)
N-Desmethylsildenafil
Ratio
(m
produced (
mM)
N-desmethylsildenafil/H2O2
2.3
2.12
4.27
0.92
0.93
0.96
1.12
0.88
O
O
4.6
9.2
8.75
NH
II
NH
OH
18.3
36.6
20.49
32.37
APO
H2O2
The initial sildenafil concentration was 250 mM.
H2O
extracellular APOs share characteristics with intracellular P450s,
which also catalyze H2O2-dependent oxidations by a pathway
termed the peroxide shunt [36–38]. These peroxygenations are
thought to be initiated when the enzyme heme is oxidized by
H2O2 to give an iron species (oxo-ferryl iron, compound I) that
carries one of the peroxide oxygens, which subsequently
oxidizes a C–H bond in the substrate [39].
0
10 20 30 40
200 400 600
Reaction time (s)
According to the above model, oxygen incorporation from H2O2
should be quantitative when the substrate is oxidized. Our data on
APO-catalyzed pharmaceutical oxidations agree with this picture
since 100% of the oxygen present in newly generated phenolic or
Fig. 2. Time course of AaeAPO-catalyzed para-hydroxylation of acetanilide (*, II) to
acetaminophen (&).
benzylic reaction products
– e.g. in the phenol group of
acetaminophen or in the alcohol moiety of 4-hydroxytolbutamide
– was 18O-labeled when the experiment was conducted with
H218O2. Moreover, the stoichiometrical result we obtained using
sildenafil as the substrate – one equivalent of N-desmethylsilde-
nafil formed per equivalent of H2O2 supplied – agrees with the
two-electron oxidation expected from a peroxygenative mecha-
nism (Table 2).
Also consistent with a P450-like mechanism is the intramolec-
ular deuterium isotope effect we observed for phenacetin-d1
oxidation by AaeAPO, in that our value of (kH/kD)obs around 3 is
close to the values of (kH/kD)obs near 2 that have been observed for
the P450-catalyzed O-dealkylation of this substrate. A value of 3 is
considerably smaller than the intrinsic isotope effect near 10
expected for a hydrogen abstraction mechanism, and may indicate
that phenacetin oxidation by APOs proceeds instead via electron
transfer, as proposed earlier for the P450-catalyzed reaction [40].
By contrast, the O-dealkylation of 1,4-dimethoxybenzene-d3 by
AaeAPO probably does proceed via hydrogen abstraction, because
it exhibits a much higher (kH/kD)obs near 12 [18,40].
O
O
NH
NH
HN
S
HN
S
O
O
O
O
APO
H2O2
VII
VIIa
VII
H2O
OH
VIIb
VIIa
m/z
Fig. 3. HPLC elution profiles after conversion of tolbutamide (VII) in the presence of
ascorbic acid:(front profile) controlwithoutenzyme;(backgroundprofile)completed
reaction with AaeAPO. Insets of mass spectra showing the incorporation of 18O from
H218O2 into the alcohol group of 4-hydroxytolbutamide (VIIa) after hydroxylation of
tolbutamide byAaeAPO are asfollows. Upper: MS oftheproductobtainedwithnatural
abundance H2O2. Lower: MS of the product obtained with 90 atom% H218O2. The peak
VIIb showed a mass as expected for 4-oxo-tolbutamide, a potential oxidation product
of 4-hydroxytolbutamide (see supplemental Table 2) [19].
The kinetics data we report here for AaeAPO action on a
variety of pharmaceuticals suggest that APOs may be useful
alternatives to P450s for the regioselective preparation of HDMs.
Although some P450s are known to bind pharmaceutical
substrates more strongly than APOs, exhibiting Km values between
1 and 70
mM, they generally exhibit relatively low kcat values in the
vicinity of 0.2 sꢁ1 or less [40,41]. As a result, the catalytic
efficiencies [kcat/Km] of AaeAPO for pharmaceutical oxidations lie
in the same range as those of the P450s, as exemplified by our
values for propranolol hydroxylation (1.32 ꢂ 105 Mꢁ1 sꢁ1), meto-
prolol O-dealkylation (4.1 ꢂ 104 Mꢁ1 sꢁ1) and phenacetin O-deal-
kylation (3.3 ꢂ 104 Mꢁ1 sꢁ1). The kcat/Km value we obtained for
acetanilide (1.5 ꢂ 106 Mꢁ1 sꢁ1) is much higher than the corre-
sponding value found for the P450-catalyzed reaction [32].
APOs have some advantages over P450s, including currently
available laboratory-evolved P450s, where reaction yields are
concerned [17]. For example, the transformation of diclofenac to 4-
hydroxydiclofenac by mutants of P450cam (CYP101A1) resulted in
total conversions between 15 and 44% and yields around 10% [42].
By contrast, wild-type AaeAPO gave a total conversion of 78% and a
yield of 68% for this reaction. In addition, fungal APOs may be
better than P450s in practical applications for several other
reasons: (i) they utilize an inexpensive co-substrate, H2O2; (ii) they
do not require costly co-reactants such as pyridine nucleotides,
kD
AaeAPO
kH
*
m/z
Fig. 4. Cleavage of N-(4-[1-2H]ethoxyphenyl)acetamide (phenacetin-d1) by AaeAPO.
The mass spectrum of the reaction mixture containing the products acetaldehyde
2,4-dinitrophenylhydrazone ([MꢁH]ꢁ, 123) and [2H] acetaldehyde 2,4-
dinitrophenylhydrazone ([MꢁH]ꢁ, 126) obtained from the oxidation of
phenacetin-d1 is shown. The mass spectrum is one of three used to calculate the
observed mean intramolecular isotope effect.

M. Poraj-Kobielska et al. / Biochemical Pharmacology 82 (2011) 789–796
795
[12] Veeger C. Does P450-type catalysis proceed through a peroxo-iron intermedi-
ate?. A review ofstudies withmicroperoxidase. J Inorg Biochem2002;91:35–45.
[13] Caputi L, Di Tullio A, Di Leandro L, De Angelis F, Malatesta F. A new micro-
peroxidase from Marinobacter hydrocarbonoclasticus. Biochim Biophys Acta
2005;1725:71–80.
[14] Prieto T, Marcon RO, Prado FM, Caires AC, Di Mascio P, Brochsztain S, et al.
Reaction route control by microperoxidase-9/CTAB micelle ratios. Phys Chem
Chem Phys 2006;8:1963–73.
[15] Dorovska-Taran V, Posthumus MA, Boeren S, Boersma MG, Teunis CJ, Rietjens
IM, et al. Oxygen exchange with water in heme-oxo intermediates during
H2O2-driven oxygen incorporation in aromatic hydrocarbons catalyzed by
microperoxidase-8. Eur J Biochem 1998;253:568–659.
flavin reductases, or ferredoxins; (iii) they are secreted enzymes
and thus can be cost-effectively produced; (iv) they are stable and
water-soluble due to their high degree of glycosylation [43,44].
There are also disadvantages associated with APO-catalyzed
oxidations. One is that the enzymes have relatively narrow
catalytic clefts (albeit considerably wider than those of other
heme peroxidases), that prevent access of markedly bulky
substrates to the active site [45]. The negative results we obtained
with felbamate, 17a-ethinylestradiol, and Reichstein substance S
[16] Dallacosta C, Monzani E, Casella L. Reactivity study on microperoxidase-8. J
Biol Inorg Chem 2003;8:770–6.
may reflect this limitation. Another problem is the high general
peroxidase activity of APOs, which necessitates the inclusion of a
radical scavenger such as ascorbate in reactions when the desired
products are phenols. Ultimately, protein engineering of APOs may
address these limitations and the recent crystallization of an APO
provides the first information needed to begin this work [43,45]. In
the meantime, the most fruitful approach is likely to involve
empirical comparisons of APOs to determine their individual
substrate specificities. Recent phylogenic investigations have
shown that APOs are widespread in the fungal kingdom [44]
and some new representatives have already been isolated from
Coprinopsis verticillata, Marasmius rotula and other fungi [27]. The
above developments open the possibility that APOs may serve as a
‘‘monooxygenation toolbox’’ for the selective production of HDMs
and other fine chemicals.
[17] Kinne M, Poraj-Kobielska M, Aranda E, Ullrich R, Hammel KE, Scheibner K, et al.
Regioselective preparation of 5-hydroxypropranolol and 40-hydroxydiclofe-
nac with a fungal peroxygenase. Bioorg Med Chem Lett 2009;19:3085–7.
[18] Kinne M, Poraj-Kobielska M, Ralph SA, Ullrich R, Hofrichter M, Hammel KE.
Oxidative cleavage of diverse ethers by an extracellular fungal peroxygenase. J
Biol Chem 2009;284:29343–9.
[19] Kinne M, Zeisig C, Ullrich R, Kayser G, Hammel KE, Hofrichter M. Stepwise
oxygenations of toluene and 4-nitrotoluene by a fungal peroxygenase. Bio-
chem Biophys Res Commun 2010;397:18–21.
[20] Hofrichter M, Ullrich R, Pecyna M, Liers C, Lundell T. New and classic families
of secreted fungal heme peroxidases. Appl Microbiol Biotechnol 2010;87:
871–97.
[21] Kluge M, Ullrich R, Dolge C, Scheibner K, Hofrichter M. Hydroxylation of
naphthalene by aromatic peroxygenase from Agrocybe aegerita proceeds via
oxygen transfer from H2O2 and intermediary epoxidation. Appl Microbiol
Biotechnol 2009;81:1071–6.
[22] Aranda E, Kinne M, Kluge M, Ullrich R, Hofrichter M. Conversion of dibenzothio-
phene by the mushrooms Agrocybe aegerita and Coprinellus radians and their
extracellular peroxygenases. Appl Microbiol Biotechnol 2008;82:1057–66.
[23] Aranda E, Ullrich R, Hofrichter M. Conversion of polycyclic aromatic hydro-
carbons, methyl naphthalenes and dibenzofuran by two fungal peroxy-
genases. Biodegradation 2009;21:267–81.
Acknowledgments
We thank M. Kluge, C. Dolge, A. Karutz, N. Lemanska, M. Brandt,
U. Schneider and A. Elsner for useful discussions and technical
assistance. Financial support of the European Social Fund (project
080935557), the German Environmental Foundation (DBU, project
13225-32) and the German Ministry for Education and Research
(BMBF, Cluster Integrierte Bioindustrie Frankfurt) is gratefully
acknowledged.
[24] Garland WA, Hsiao KC, Pantuck EJ, Conney AH. Quantitative determination of
phenacetin and its metabolite acetaminophen by GLC-chemical ionization
mass spectrometry. J Pharm Sci 1977;66:340–4.
[25] Nottebaum LJ, McClure TD. Methods, systems, and computer program pro-
ducts for producing theoretical mass spectral fragmentation patterns of
chemical structures. USA: Syngenta Participations AG/ipcom; 2010.
[26] Ullrich R, Nu¨ske J, Scheibner K, Spantzel J, Hofrichter M. Novel haloperoxidase
from the agaric basidiomycete Agrocybe aegerita oxidizes aryl alcohols and
aldehydes. Appl Environ Microbiol 2004;70:4575–81.
[27] Anh DH, Ullrich R, Benndorf D, Svatos A, Muck A, Hofrichter M. The coprophi-
lous mushroom Coprinus radians secretes a haloperoxidase that catalyzes
aromatic peroxygenation. Appl Environ Microbiol 2007;73:5477–85.
[28] Hernandez A, Ruiz MT. An EXCEL template for calculation of enzyme kinetic
parameters by non-linear regression. Bioinformatics 1998;14:227–8.
[29] Masubuchi Y, Hosokawa S, Horie T, Suzuki T, Ohmori S, Kitada M, et al.
Cytochrome P450 isozymes involved in propranolol metabolism in human
liver microsomes. The role of CYP2D6 as ring-hydroxylase and CYP1A2 as N-
desisopropylase. Drug Metab Dispos 1994;22:909–15.
[30] Miners JO, Birkett DJ. Use of tolbutamide as a substrate probe for human
hepatic cytochrome P450 2C9. Methods Enzymol 1996;272:139–45.
[31] Miners JO, Coulter S, Tukey RH, Veronese ME, Birkett DJ. Cytochromes P450,
1A2, and 2C9 are responsible for the human hepatic O-demethylation of R- and
S-naproxen. Biochem Pharmacol 1996;51:1003–8.
[32] Liu G, Gelboin HV, Myers MJ. Role of cytochrome P450 IA2 in acetanilide 4-
hydroxylation as determined with cDNA expression and monoclonal anti-
bodies. Arch Biochem Biophys 1991;284:400–6.
[33] Ku HY, Ahn HJ, Seo KA, Kim H, Oh M, Bae SK, et al. The contributions of
cytochromes P450 3A4 and 3A5 to the metabolism of the phosphodiesterase
type 5 inhibitors sildenafil, udenafil, and vardenafil. Drug Metab Dispos
2008;36:986–90.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bcp.2011.06.020.
References
[1] Nelson SD. Metabolic activation and drug toxicity. J Med Chem 1982;25:
753–65.
[2] Baillie TA, Cayen MN, Fouda H, Gerson RJ, Green JD, Grossman SJ, et al. Drug
metabolites in safety testing. Toxicol Appl Pharmacol 2002;182:188–96.
[3] Peters FT, Dragan CA, Wilde DR, Meyer MR, Zapp J, Bureik M, et al. Biotechno-
logical synthesis of drug metabolites using human cytochrome P450 2D6
heterologously expressed in fission yeast exemplified for the designer drug
metabolite 40-hydroxymethyl-alpha-pyrrolidinobutyrophenone. Biochem
Pharmacol 2007;74:511–20.
[4] Gibson GG, Skett P. Introduction to drug metabolism. Cheltenham, UK: Nelson
Thornes Publishers; 2001.
[5] Ullrich R, Hofrichter M. Enzymatic hydroxylation of aromatic compounds. Cell
Mol Life Sci 2007;64:271–93.
[6] Kinne M, Ullrich R, Hammel KE, Scheibner K, Hofrichter M. Regioselective
preparation of (R)-2-(4-hydroxyphenoxy)propionic acid with a fungal perox-
ygenase. Tetrahedron Lett 2008;49:5950–3.
[7] Urlacher VB, Lutz-Wahl S, Schmid RD. Microbial P450 enzymes in biotechnol-
ogy. Appl Microbiol Biotechnol 2004;64:317–25.
[8] Eiben S, Kaysser L, Maurer S, Kuhnel K, Urlacher VB, Schmid RD. Preparative
use of isolated CYP102 monooxygenases—a critical appraisal. J Biotechnol
2006;124:662–9.
[9] Otey CR, Bandara G, Lalonde J, Takahashi K, Arnold FH. Preparation of human
metabolites of propranolol using laboratory-evolved bacterial cytochromes
P450. Biotechnol Bioeng 2006;93:494–9.
[34] Ortiz de Montellano P. Cytochrome P450—structure, mechanism and bio-
chemistry. New York: Kluwer Academic/Plenum Publishers; 2005.
[35] Guengerich FP. Oxidative cleavage of carboxylic esters by cytochrome P-450. J
Biol Chem 1987;262:8459–62.
[36] Shoji O, Fujishiro T, Nakajima H, Kim M, Nagano S, Shiro Y, et al. Hydrogen
peroxide dependent monooxygenations by tricking the substrate recognition
of cytochrome P450BSb. Angew Chem Int Ed 2007;46:3656–9.
[37] Guengerich FP. Common and uncommon cytochrome P450 reactions related
to metabolism and chemical toxicity. Chem Res Toxicol 2001;14:611–50.
[38] Ortiz de Montellano PR, de Voss JJ. Substrate oxidation by cytochrome P450
enzymes. New York: Kluwer Academic/Plenum Publishers; 2005.
[39] Rittle J, Green MT. Cytochrome P450 compound I: capture, characterization,
and C–H bond activation kinetics. Science 2010;330:933–7.
[40] Yun CH, Miller GP, Guengerich FP. Rate-determining steps in phenacetin
oxidations by human cytochrome P450 1A2 and selected mutants. Biochem-
istry 2000;39:11319–2.
[41] Upthagrove AL, Nelson WL. Importance of amine pKa and distribution coeffi-
cient in the metabolism of fluorinated propranolol derivatives. Preparation,
identification of metabolite regioisomers, and metabolism by CYP2D6. Drug
Metab Dispos 2001;29:1377–88.
[10] Sawayama AM, Chen MM, Kulanthaivel P, Kuo MS, Hemmerle H, Arnold FH. A
panel of cytochrome P450 BM3 variants to produce drug metabolites and
diversify lead compounds. Chemistry 2009;15:11723–9.
[11] Osman AM, Koerts J, Boersma MG, Boeren S, Veeger C, Rietjens IM. Micro-
peroxidase/H2O2-catalyzed aromatic hydroxylation proceeds by
chrome-P-450-type oxygen-transfer reaction mechanism. Eur
1996;240:232–8.
a
cyto-
J Biochem

796
M. Poraj-Kobielska et al. / Biochemical Pharmacology 82 (2011) 789–796
[42] Weis R, Winkler M, Schittmayer M, Kambourakis S, Vink M, Rozzell JD,
et al. A diversified library of bacterial and fungal bifunctional cytochrome
P450 enzymes for drug metabolite synthesis. Adv Synth Catal 2009;351:
2140–6.
[55] Stearns V, Johnson MD, Rae JM, Morocho A, Novielli A, Bhargava P, et al. Active
tamoxifen metabolite plasma concentrations after coadministration of tamox-
ifen and the selective serotonin reuptake inhibitor paroxetine. J Natl Cancer
Inst 2003;95:1758–64.
[43] Ullrich R, Liers C, Schimpke S, Hofrichter M. Purification of homogeneous
forms of fungal peroxygenase. Biotechnol J 2009;4:1619–26.
[44] Pecyna MJ, Ullrich R, Bittner B, Clemens A, Scheibner K, Schubert R, et al.
Molecular characterization of aromatic peroxygenase from Agrocybe aegerita.
Appl Microbiol Biotechnol 2009;84:885–97.
[45] Piontek K, Ullrich R, Liers C, Diederichs K, Plattner DA, Hofrichter M.
Crystallization of a 45 kDa peroxygenase/peroxidase from the mushroom
Agrocybe aegerita and structure determination by SAD utilizing only
[56] Hamman MA, Thompson GA, Hall SD. Regioselective and stereoselective
metabolism of ibuprofen by human cytochrome P450 2C. Biochem Pharmacol
1997;54:33–41.
[57] Holmes E, Loo RL, Cloarec O, Coen M, Tang H, Maibaum E, et al. Detection of
urinary drug metabolite (xenometabolome) signatures in molecular epidemi-
ology studies via statistical total correlation (NMR) spectroscopy. Anal Chem
2007;79:2629–40.
[58] Hansen LL, Brøsen K. Quantitative determination of tolbutamide and its
metabolites in human plasma and urine by high-performance liquid chroma-
tography and UV detection. Ther Drug Monit 1999;21:664.
the haem iron. Acta Crystallogr Sect
2010;66:693–8.
F Struct Biol Cryst Commun
[46] Fitzgerald JD, O’Donnell SR. Pharmacology of 4-hydroxypropranolol, a metab-
olite of propranolol. Br J Pharmacol 1971;43:222–35.
[59] Hoffmann K-J, Gyllenhaal O, Vessman J. Analysis of a-hydroxy metabolites of
metoprolol in human urine after phosgene/trimethylsilyl derivatization. Biol
[47] Greenberg LA, Lester D. The metabolic fate of acetanilid and other aniline
derivates. J Pharmacol Exp Ther 1946;88:87–98.
[48] Berthou F, Flinois JP, Ratanasavanh D, Beaune P, Riche C, Guillouzo A.
Evidence for the involvement of several cytochromes P-450 in the first steps
of caffeine metabolism by human liver microsomes. Drug Metab Dispos
1991;19:561–7.
[49] Yoshitomi S, Ikemoto K, Takahashi J, Miki H, Namba M, Asahi S. Establishment
of the transformants expressing human cytochrome P450 subtypes in HepG2,
and their applications on drug metabolism and toxicology. Toxicol In Vitro
2001;15:245–56.
Mass Spectrom 1987;14:543–8.
[60] Segre EJ. Naproxen metabolism in man. J Clin Pharmacol 1975;15:316–23.
[61] Bougie D, Aster R. Immune thrombocytopenia resulting from sensitivity
to metabolites of naproxen and acetaminophen. Blood 2001;97:3846–50.
[62] Jacqz-Aigrain E, Funck-Brentano C, Cresteil T. CYP2D6- and CYP3A-dependent
metabolism of dextromethorphan in humans. Pharmacogenetics 1993;3:
197–204.
[63] Broly F, Libersa C, Lhermitte M, Bechtel P, Dupuis B. Effect of quinidine on the
dextromethorphan O-demethylase activity of microsomal fractions from hu-
man liver. Br J Clin Pharmacol 1989;28:29–36.
[50] Pearce RE, Vakkalagadda GR, Leeder JS. Pathways of carbamazepine bioactiva-
tion in vitro. I. Characterization of human cytochromes P450 responsible for
the formation of 2- and 3-hydroxylated metabolites. Drug Metab Dispos
2002;30:1170–9.
[64] Brodie BB, Axelrod J. The fate of acetophenetidin (phenacetin) in man and
methods for the estimation of acetophenetidin and its metabolites in biologi-
cal material. J Pharmacol Exp Ther 1949;97:58–67.
[65] Fura A, Shu Y-Z, Zhu M, Hanson RL, Roongta V, Humphreys WG. Discovering
drugs through biological transformation: role of pharmacologically active
metabolites in drug discovery. J Med Chem 2004;47:4339–51.
[66] Hyland R, Roe EGH, Jones BC, Smith DA. Identification of the cytochrome P450
enzymes involved in the N-demethylation of sildenafil. Br J Clin Pharmacol
2001;51:239–48.
[67] Orlando R, Piccoli P, De Martin S, Padrini R, Floreani M, Palatini P. Cytochrome
P450 1A2 is a major determinant of lidocaine metabolism in vivo: effects of
liver function. Clin Pharmacol Ther 2004;75:80–8.
[68] Banerjee NC, Miller GE, Stowe CM. Excretion of aminopyrine and its meta-
bolites into cows’ milk. Toxicol Appl Pharmacol 1967;10:604–12.
[69] McClellan K, Perry CM. Oseltamivir: a review of its use in influenza. Drugs
2001;61:263–83.
´
´
´
[51] Bort R, Mace K, Boobis A, Gomez-Lechon M-J, Pfeifer A, Castell J. Hepatic
metabolism of diclofenac: role of human CYP in the minor oxidative pathways.
Biochem Pharmacol 1999;58:787–96.
[52] Bort R, Ponsoda X, Jover R, Gomez-Lechon MJ, Castell JV. Diclofenac toxicity to
hepatocytes: a role for drug metabolism in cell toxicity. J Pharmacol Exp Ther
1999;288:65–72.
[53] Jin Y, Desta Z, Stearns V, Ward B, Ho H, Lee K-H, et al. CYP2D6 Genotype,
antidepressant use, and tamoxifen metabolism during adjuvant breast cancer
treatment. J Natl Cancer Inst 2005;97:30–9.
[54] Vitseva O, Flockhart DA, Jin Y, Varghese S, Freedman JE. The effects of
tamoxifen and its metabolites on platelet function and release of reactive
oxygen intermediates. J Pharmacol Exp Ther 2005;312:1144–50.