Involvement of microsomal NADPH-cytochrome P450 reductase in metabolic reduction of drug ketones

Article abstract of DOI:10.1002/bdd.1946

Recently, it was found that the carbonyl group of 1-[3-(4-phenoxyphenoxy)-2-oxopropyl]indole-5-carboxylic acid (5), an inhibitor of the pro-inflammatory enzyme cytosolic phospholipase A₂α, is easily reduced by rat liver S9 fractions in vitro. Determination of the inhibitory potency of certain putative inhibitors of carbonyl reducing enzymes on the transformation of the ketone derivative 5 to its alcohol 6 by recombinant microsomal NADPH-cytochrome P450 reductase and by recombinant cytosolic carbonyl reductase-1 now reveals that these compounds show a lack of specificity for these two enzymes in part. Thus, an assignment of the roles of different carbonyl reductases in metabolic keto reduction by the use of inhibitors is problematic. In addition, the ability of NADPH-cytochrome P450 reductase and carbonyl reductase-1 to reduce the ketone groups of the drugs haloperidol and daunorubicin was examined. Under the conditions applied, a pronounced reductive metabolism was only observed for daunorubicin in the presence of microsomal NADPH-cytochrome P450 reductase. Similarly, in rat liver S9 fractions a marked reduction of daunorubicin was seen, while haloperidol was only slightly metabolized to its alcohol. After separation of the S9 homogenate into a microsomal and a cytosolic fraction, it became evident that the ketone groups of daunorubicin, haloperidol and compound 5 were mainly reduced by cytosolic enzymes. However, since microsomes also catalysed these carbonyl reductions to some extent, it can be concluded that microsomal NADPH-cytochrome P450 reductase can contribute to metabolic keto reductions in xenobiotics.

Full text of DOI:10.1002/bdd.1946

BIOPHARMACEUTICS & DRUG DISPOSITION
Biopharm. Drug Dispos. 36: 398–404 (2015)
Published online 14 April 2015 in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/bdd.1946
SHORT COMMUNICATION
Involvement of microsomal NADPH-cytochrome P450
reductase in metabolic reduction of drug ketones
*
Matthias Lehr , Jörg Fabian, and Walburga Hanekamp
Institute of Pharmaceutical and Medicinal Chemistry, University of Münster, Germany
ABSTRACT: Recently, it was found that the carbonyl group of 1-[3-(4-phenoxyphenoxy)-
2-oxopropyl]indole-5-carboxylic acid (5), an inhibitor of the pro-inflammatory enzyme cytosolic
phospholipase A2α, is easily reduced by rat liver S9 fractions in vitro. Determination of the inhibitory
potency of certain putative inhibitors of carbonyl reducing enzymes on the transformation of the ke-
tone derivative 5 to its alcohol 6 by recombinant microsomal NADPH-cytochrome P450 reductase
and by recombinant cytosolic carbonyl reductase-1 now reveals that these compounds show a lack
of specificity for these two enzymes in part. Thus, an assignment of the roles of different carbonyl re-
ductases in metabolic keto reduction by the use of inhibitors is problematic. In addition, the ability of
NADPH-cytochrome P450 reductase and carbonyl reductase-1 to reduce the ketone groups of the
drugs haloperidol and daunorubicin was examined. Under the conditions applied, a pronounced re-
ductive metabolism was only observed for daunorubicin in the presence of microsomal NADPH-
cytochrome P450 reductase. Similarly, in rat liver S9 fractions a marked reduction of daunorubicin
was seen, while haloperidol was only slightly metabolized to its alcohol. After separation of the S9
homogenate into a microsomal and a cytosolic fraction, it became evident that the ketone groups
of daunorubicin, haloperidol and compound 5 were mainly reduced by cytosolic enzymes. However,
since microsomes also catalysed these carbonyl reductions to some extent, it can be concluded that
microsomal NADPH-cytochrome P450 reductase can contribute to metabolic keto reductions in xe-
nobiotics. Copyright © 2015 John Wiley & Sons, Ltd.
Key words: NADPH-cytochrome P450 reductase; carbonyl reductase-1; haloperidol; daunorubicin;
keto-reduction
Introduction
(3 and 4, respectively) (Figure 1) [1,2]. The
reduction of aldehyde and ketone functions of xe-
nobiotic compounds is thought to be catalysed
mainly by two classes of enzymes: the short chain
dehydrogenase/reductase (SDR) and the aldo-
keto reductase (AKR) [3–5]. In addition, NADPH-
cytochrome P-450 reductase was reported to be
capable of reducing ketones to alcohols [6].
In past years, 1-heteroarylpropan-2-one deriva-
tives were developed as inhibitors of cytosolic
phospholipase A2α and fatty acid amide hydro-
lase, two enzymes involved in inflammatory pro-
cesses [7,8]. Because the carbonyl group of such
The most important metabolic pathway of xenobi-
otics is the oxidative biotransformation by
cytochrome P450 (CYP) enzymes. However, re-
duction processes also play a significant role in
phase I metabolism. For example, a variety of drugs
bearing a carbonyl group such as haloperidol (1)
and daunorubicin (2) are reduced to alcohols
*Correspondence to: Institute of Pharmaceutical and Medicinal
Chemistry, University of Münster, Germany.
E-mail: lehrm@uni-muenster.de
Received 13 October 2014
Revised 17 February 2015
Accepted 2 March 2015
Copyright © 2015 John Wiley & Sons, Ltd.

METABOLIC KETO-REDUCTION
399
Figure 1. Metabolism of haloperidol, daunorubicin and 1-[3-(4-phenoxyphenoxy)-2- oxopropyl]indole-5-carboxylic acid (5) by car-
bonyl reducing enzymes
compounds is readily reduced to an alcohol in
rat liver homogenate in vitro as well as in mice
in vivo resulting in a loss of their inhibitory
potency [7], our interest was focused on this met-
abolic reaction. Recently, it was found that in a
mixture of human recombinant CYP enzymes
(CYP1A2, 2C8, 2C9, 2C19, 2D6, 3A4) and
NADPH-cytochrome P450 reductase, the latter-
enzyme was responsible for the observed pro-
nounced reduction of 1-[3-(4-phenoxyphenoxy)-
2-oxopropyl]indole-5-carboxylic acid (5) to
1-[2-hydroxy-3-(4-phenoxyphenoxy)propyl]indole-
5-carboxylic acid (6) (Figure 1) [9]. In rat liver S9
fractions, compound 5 was even completely me-
tabolized to the alcohol 6. Here, cytosolic SDRs
(cSDRs) are held mainly to be responsible for this
reaction, because the keto-reduction could be
inhibited by certain compounds, which were re-
ported in the literature to be inhibitors of cSDR en-
zymes [10,11].
In the present study, first the specificity of these
inhibitors was re-evaluated by measuring their
effect on the transformation of compound 5 to
compound 6 by NADPH-cytochrome P450 reduc-
tase and by one of the most important cSDRs, the
carbonyl reductase-1. Furthermore, the study
explored whether these two enzymes are able to
reduce the carbonyl-bearing drugs haloperidol
and daunorubicin to the corresponding alcohols.
Materials and Methods
Materials
Inhibitors and other reagents and solvents used
were purchased from standard suppliers; micro-
somal NADPH-cytochrome P450 reductase was
obtained from Corning Life Sciences (Amster-
dam, The Netherlands; product no. 456244) and
consisted of a microsomal preparation of insect
cell-expressed human P450 reductase and hu-
man cytochrome b5 in 0.5 ml 100 mM potassium
phosphate buffer, pH 7.4; the protein content
was 2.5 mg/0.5 ml, NADPH-cytochrome P450
reductase activity present in the preparation
was 400 nmol/min/mg protein and cytochrome
b5 content was 750 pmol/mg protein. The
preparation was used as delivered. Recombinant
human carbonyl reductase-1 produced in E. coli
(20 μg/20 μl) by ProSpec was purchased from
Hoelzel Diagnostika (Cologne, Germany; prod-
uct no. enz-415); the preparation (20 μl) was
diluted with 480 μl of a 9:1 mixture of Tris-HCl
buffer (20 mM, pH 8.5 at 20 °C) and glycerol prior
to usage. The amount of NADPH-cytochrome
P450 reductase and carbonyl reductase-1, respec-
tively, used in the incubation procedures led to
about a 60–70% conversion of compound 5 to its
alcohol metabolite 6.
Copyright © 2015 John Wiley & Sons, Ltd.
Biopharm. Drug Dispos. 36: 398–404 (2015)
DOI: 10.1002/bdd

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M. LEHR ET AL.
Inhibition of NADPH-cytochrome P450
reductase- and carbonyl reductase-1-catalysed
reduction of compound 5 by carbonyl reductase
inhibitors, and metabolism of haloperidol and
daunorubicin by these enzymes
Inhibition of carbonyl reductase-1-catalysed for-
mation of compound 6 from compound 5 and me-
tabolism of haloperidol (1) and daunorubicin (2)
by NADPH-cytochrome P450 reductase and car-
bonyl reductase-1 was measured similarly (for de-
tails see Supplementary Material).
Inhibition of NADPH-cytochrome P450 reductase-
catalysed formation of the alcohol derivative 6 from the
ketone derivative 5. To a 1.57 mM DMSO solution of
compound 5 (2 μl) was added DMSO (0.5 μl) in the
case of the controls or a DMSO solution of the appro-
priate inhibitor (see Table 1) (0.5 μl). After treatment
with a 19.5:0.5 (v/v) mixture of phosphate buffered
saline and 0.1 M aqueous MgCl2 solution (92.5 μl),
the NADPH-cytochrome P450 reductase prepara-
tion (5 μl) was added. Then the enzyme reactions
were started by the addition of a 15 mM solution of
NADPH in a 19.5:0.5 (v/v) mixture of phosphate
buffered saline and 0.1 M aqueous MgCl2 solution
(25 μl). The final volume was 125 μl, the final concen-
tration of compound 5 was 25 μM, and the final con-
centration of NADPH was 3 mM. Incubations were
carried out at 37 °C for 1 h. Then the reactions were
stopped by the addition of acetonitrile (250 μl). The
samples were allowed to stand at room temperature
for 10 min and then centrifuged at 2000 × g and room
temperature for 5 min. For calculation of the relative
amount of compound 5 metabolized, a mixture of a
1.57 mM DMSO solution of compound 5 (2 μl),
DMSO (0.5 μl), phosphate buffered saline (117.5 μl)
and the NADPH-cytochrome P450 reductase prepa-
ration (5 μl) was incubated in the same manner. The
supernatants were analysed directly for metabolite
formation by HPLC/UV-MS (for details see Supple-
mentary Material).
Inhibition of the reduction of compound 5 in rat
liver S9 fractions by carbonyl reductase inhibi-
tors, and metabolism of haloperidol and daunoru-
bicin by rat liver S9 fractions
Inhibition of the reduction of compound 5 to com-
pound 6 in rat liver S9 fractions by ethacrynic acid
and diphenyleneiodonium chloride and metabolism
of haloperidol and daunorubicin by rat liver S9 frac-
tions was measured according to a published proce-
dure [9] (for details see Supplementary Material).
Metabolism of compound 5, haloperidol and daunoru-
bicin by rat liver microsomes and rat liver cytosol.
Rat liver S9 fractions were separated into micro-
somes and cytosol by centrifugation at 100 000 × g
for 60 min. Metabolism of compound 5, haloperidol
and daunorubicin by microsomes and by cytosol
was measured in a similar way as for the experi-
ments with S9 fractions [9] (for details see Supple-
mentary Material).
Results and Discussion
Recently, it was shown that compound 5 can be re-
duced to compound 6 by a mixture of human
Table 1. Effect of different carbonyl reductase inhibitors on reduction of compound 5 to compound 6 by rat liver S9 fractions,
human recombinant NADPH-cytochrome P450 reductase and human recombinant carbonyl reductase-1
Inhibition of the formation of 6 from 5 (%)a
Inhibitor
Conc. (μM)
Rat liver S9 fractions
NADPH-CYP450 reductase
Carbonyl reductase-1
Quercetin
200
200
1000
5000
20
39 10
41 2.6
28 7.5
48 3.2
85 1.2
64 3.1
17 8.2
n.s.b
87 1.5
n.s.b
Menadione
Ethacrynic acid
n.s.b
Alpha-lipoic acid
Diphenyleneiodonium chloride
Phenolphthaleine
Flufenamic acid
7
1.2
75 6.4
n.s.b
n.s.b
20
n.s.b
n.s.b
n.s.b
13 7.0
n.s.b
20
2
n.s.b
n.s.b
18β-Glycyrrhetinic acid
n.s.b
n.s.b
aValues are means standard deviations of independent determinations (n = 3).
bn.s.: not significant.
Copyright © 2015 John Wiley & Sons, Ltd.
Biopharm. Drug Dispos. 36: 398–404 (2015)
DOI: 10.1002/bdd

METABOLIC KETO-REDUCTION
401
recombinant CYP enzymes as well as by rat liver
S9 fractions [9]. In the microsomal CYP prepara-
tion the accessory protein NADPH-cytochrome
P450 reductase, which catalyses electron transfer
from the co-factor NADPH to the CPYs, was re-
sponsible for the carbonyl reduction. This was
shown, among other things, by the observation
that an excessive conversion of ketone 5 to the al-
cohol 6 took place in a preparation solely contain-
ing NADPH-cytochrom P450 reductase.
effectively (Table 1). Thus, these results confirmed
our previous findings.
Nevertheless, we wanted to repeat the inhibi-
tion experiments with pure carbonyl reductase-1,
which is one of the main cSDRs in the organism
[18] and NADPH-cytochrome P450 reductase.
From both enzymes human recombinant forms
are commercially available. First, it was verified
that carbonyl reductase-1 is also able to reduce
compound 5 to compound 6. In the following in-
hibition experiments, the activities of the two en-
zymes were chosen in a way that about 60–70%
of substrate 5 was converted to product 6. The
concentrations of the inhibitors were selected on
the basis of inhibition data published in the litera-
ture [10,11]. The results obtained (Table 1) were
very surprising. Quercetin, menadione and
ethacrynic acid, which have been reported to be
inhibitors of cSDRs, showed a pronounced inhibi-
tion of NADPH-cytochrome P450 reductase,
while the cSDR carbonyl reductase-1 only was
inhibited by quercetin and not by menadione
and ethacrynic acid. Vice versa, the NADPH-
cytochrome P450 reductase inhibitor alpha-lipoic
acid inhibited carbonyl reductase-1 with a much
higher potency than NADPH-cytochrome P450
reductase itself. For diphenyleneiodonium chlo-
ride, published to be an inhibitor of NADPH-
cytochrome P450 reductase, neither an effect on
this enzyme nor on carbonyl reductase-1 could be
measured. The AKR inhibitor phenolphthaleine
slightly affected NADPH-cytochrome P450 reduc-
tase, while the AKR inhibitor flufenamic acid
showed no inhibitory effect on the formation
of compound 6. The mSDR inhibitor 18β-
glycyrrhetinic acid also was ineffective. Taken to-
gether, these results indicate that it is difficult to as-
sign the roles of carbonyl reducing enzymes with
the aid of the published inhibitors, because these
inhibitors lack specificity in part or do not have
an effect on these enzymes at all.
The enzymes contributing to the reductive
transformation of compound 5 in rat liver S9 frac-
tions were evaluated with the aid of enzyme
inhibitors [9]. For the reduction of ketone functions
of xenobiotics in the organism, mainly two classes
of enzymes are thought to be responsible, namely
the AKRs and the SDRs [3–5]. Therefore, the effect
of published inhibitors of these enzymes [10,11] on
the reduction of compound 5 was determined in
this former study [9]. Since the AKR inhibitors
flufenamic acid and phenolphthalein did not affect
the formation of the alcohol 6 from ketone 5
(Table 1), a contribution of AKRs in this metabolic
reaction was ruled out. Also, 18β-glycyrrhetinic
acid, an inhibitor of microsomal SDRs (mSDRs)
showed no inhibition at a concentration reported
in the literature to be effective. In contrast, mena-
dione and quercetin, which have been assigned
as inhibitors of cytosolic forms of SDR (cSDR), sig-
nificantly inhibited the reduction of the ketone
group of compound 5. Therefore, it was assumed
that cSDRs play an important role in the reductive
metabolism of compound 5 in rat liver S9 frac-
tions. Although quercetin was reported to be an
inhibitor of NADPH-cytochrome P450 reductase
too [12], the primary role of this enzyme in the
metabolism of compound 5 was excluded, be-
cause the NADPH-cytochrome P450 reductase in-
hibitor alpha-lipoic acid [13,14] only slightly
inhibited the formation of the alcohol 6 in S9
fractions.
In the present study two more carbonyl reduc-
tase inhibitors were first tested for their effect on
the metabolism of compound 5 in S9 fractions.
The cSDR inhibitor ethacrynic acid [15] signifi-
cantly blocked the reductive reaction, while the
NADPH-cytochrome P450 reductase inhibitor
diphenyleneiodonium chloride [16,17] did not
show any inhibitory potency at a concentration re-
ported to be sufficient to inhibit this enzyme
Like compound 5, haloperidol and daunorubi-
cin are drugs with metabolically sensitive carbonyl
groups. Enzymes that have actually been de-
scribed to reduce daunorubicin at the C13 position
to yield the hydroxy metabolite daunorubicinol (4)
(Figure 1) are carbonyl reductase-1 [19–23],
carbonyl reductase-3 [23] and certain AKRs
[19,22,24–27]. Haloperidol has been reported to
be reduced by AKRs with low affinity and low
Copyright © 2015 John Wiley & Sons, Ltd.
Biopharm. Drug Dispos. 36: 398–404 (2015)
DOI: 10.1002/bdd

402
M. LEHR ET AL.
efficiency and by an unspecified carbonyl reduc-
tase with low affinity but high efficiency [19,28].
With purified NADPH-cytochrome P450 reduc-
tase and carbonyl reductase-1 to hand, we were
also interested to what extent daunorubicin and
haloperidol are reduced by these two enzymes.
The results are shown in Table 2. Under the
conditions applied, about 60–70% of the propan-
2-one 5 was converted to the alcohol 6 by both en-
zymes. NADPH-cytochrome P450 reductase only
reduced a small amount of haloperidol (1) to its al-
cohol 3 as determined by HPLC/UV-MS studies,
while it extensively metabolized daunorubicin
(2) to the alcohol metabolite daunorubicinol (4)
(for HPLC chromatograms see Supplementary
Material). To our knowledge, a reduction of the keto
group of daunorubicin by NADPH-cytochrome
P450 reductase has not been reported before. After
incubation of haloperidol with carbonyl reductase-
1 under these conditions no carbonyl reduction
was seen, and only a slight transformation of dau-
norubicin to its alcohol form occurred in the pres-
ence of this enzyme.
Finally, the rate of metabolism of haloperidol
and daunorubicin in rat liver S9 fractions was
studied. Only little metabolic transformation of
haloperidol appeared in this environment. One
minor metabolite found could be identified as
the alcohol derivative 3. In contrast, compound
5 and daunorubicin were extensively metabo-
lized giving rise to one main metabolite in each
case, which were identified by LC/MS as
daunorubicinol (4) and the alcohol 6, respectively
(for HPLC-chromatograms see Supplementary
Material). Similar results have been obtained in
the experiments with recombinant microsomal
NADPH-cytochrome P450 reductase described
above. To clarify, whether the latter enzyme is
the key player in the reduction of daunorubicin,
haloperidol and compound 5 in rat liver homog-
enate, S9 fraction was separated into microsomes
and cytosol by centrifugation at 100 000 × g and
the substances were incubated with these prepa-
rations. While the protein content in the experi-
ments with S9 fractions had been 2.0 mg/ml
measured with the Bradford method, in the incu-
bation solutions of the isolated microsomes and
the cytosol it was 0.16 mg/ml and 1.7 mg/ml,
respectively.
Interestingly, in the experiments with the micro-
somes
a reduction of daunorubicin (2) to
daunorubicinol (4) was not detected. Thus, it
could be concluded that the activity of micro-
somal NADPH-cytochrome P450 reductase in this
preparation is obviously much lower than that ap-
plied in the incubation solutions with the recombi-
nant enzyme, and that the impact of this enzyme
on metabolic reduction of daunorubicin could be
overestimated when performing reduction studies
solely with pure enzymes. In contrast, a high re-
duction rate of daunorubicin was seen in the cyto-
solic preparation. As for the S9 fractions, about
70–80% of this compound was transformed to
daunorubicinol. Compound 5 was quantitatively
metabolized in the cytosol resulting in high con-
centrations of alcohol 6. In the microsomal prepa-
ration compound 5 was reduced too, but only by
10%. In this context it has to be mentioned that
in earlier experiments it was found that this com-
pound was nearly quantitatively transformed to
Table 2. Metabolism of 1-heteroarylpropan-2-one 5, haloperidol and daunorubicin by rat liver S9 fractions, rat liver microsomes,
rat liver cytosol, human recombinant NADPH-cytochrome P450 reductase, and human recombinant carbonyl reductase-1
Compound
Metabolic stability (%)a
Rat liver
NADPH-CYP450 reductase
Carbonyl reductase-1
(cytosolic)
(microsomal)
S9 fractions
Microsomes
Cytosol
Compound 5
Haloperidol (1)
Daunorubicin (2)
0
89 2.6
90 2.6
96 4.2
0
36 2.9
90 8.0
23 4.2
30 4.9
100
95 3.2
84 6.7
28 4.0
91 6.0
21 1.2
aPercentage of parent compound remaining after incubation in the presence of the co-factor NADPH; means standard deviations, n = 3, in case of
the experiments with rat liver S9 fractions n = 6.
Main metabolites determined by HPLC/UV-MS: alcohol derivative 6 in case of compound 5, alcohol derivative 3 in case of haloperidol,
daunorubicinol (4) in case of daunorubicin.
Copyright © 2015 John Wiley & Sons, Ltd.
Biopharm. Drug Dispos. 36: 398–404 (2015)
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METABOLIC KETO-REDUCTION
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its alcohol 6 by rat liver microsomes. This can be
explained by the fact that in these experiments
[7] the concentration of the microsomes was about
14-fold higher than in the present case. Under the
conditions applied, haloperidol was metabolized
by S9 fractions, isolated microsomes as well as cy-
tosol only to a minor extent. However, in all prep-
arations small amounts of the reduced form of
haloperidol (3) could be detected by LC/MS. Sim-
ilar to the results obtained with compound 5, the
amount of the alcohol 3 was about 10-fold higher
in the incubations performed with S9 fractions
and cytosol, respectively, than in that with the
microsomes.
Taken together it was shown that the metabolic
reduction of the ketone group of haloperidol, dau-
norubicin and compound 5 in liver homogenate is
predominantly catalysed by cytosolic enzymes.
Nevertheless, since the microsomes also catalyse
this reaction in the case of haloperidol and com-
pound 5 to some extent, it can be concluded that
microsomal NADPH-cytochrome P450 reductase
can also contribute to metabolic keto reduction
of xenobiotics in principal.
Conflict of Interest
The authors have declared that there is no conflict
of interest.
References
1. Kudo S, Ishizaki T. Pharmacokinetics of haloperi-
dol: an update. Clin Pharmacokinet 1999; 37: 435–456.
2. Pirolli D, Giardina B, Mordente A, Ficarra S, De Rosa
MC. Understanding the binding of daunorubicin
and doxorubicin to NADPH-dependent cytosolic re-
ductases by computational methods. Eur J Med Chem
2012; 56: 145–154. doi:10.1016/j.ejmech.2012.08.023.
3. Oppermann UC, Maser E. Molecular and struc-
tural aspects of xenobiotic carbonyl metabolizing
enzymes. Role of reductases and dehydrogenases
in xenobiotic phase I reactions. Toxicology 2000;
144: 71–81.
4. Malátková P, Maser E, Wsól V. Human carbonyl
reductases. Curr Drug Metab 2010; 11: 639–658.
5. Skarydová L, Wsól V. Human microsomal car-
bonyl reducing enzymes in the metabolism of xe-
nobiotics: well-known and promising members of
the SDR superfamily. Drug Metab Rev 2012; 44:
173–191. doi:10.3109/03602532.2011.638304.
6. Chen C, Lefers RC, Brough EL, Gurka DP. Metabo-
lism of alcohol and ketone by cytochrome P-450 ox-
ygenase: fluoren-9-ol in equilibrium with fluoren-
9-one. Drug Metab Dispos 1984; 12: 421–426.
7. Drews A, Bovens S, Roebrock K, et al. 1-
(5-Carboxyindol-1-yl)propan-2-one inhibitors of
human cytosolic phospholipase A2α with reduced
lipophilicity: synthesis, biological activity, meta-
bolic stability, solubility, bioavailability, and topical
in vivo activity. J Med Chem 2010; 53: 5165–5178.
doi:10.1021/jm1001088.
8. Forster L, Ludwig J, Kaptur M, et al. 1-Indol-1-
yl-propan-2-ones and related heterocyclic com-
pounds as dual inhibitors of cytosolic phospholi-
pase A2α and fatty acid amide hydrolase. Bioorg
Med Chem 2010; 18: 945–952. doi:10.1016/j.
bmc.2009.11.028.
9. Fabian J, Hanekamp W, Thomas MH, Olivier JL,
Lehr M. Investigations on the metabolic stability
of cytosolic phospholipase A2α inhibitors with 1-
indolylpropan-2-one structure. Chem Biol Interact
2013; 206: 356–363. doi:10.1016/j.cbi.2013.10.005.
10. Rosemond MJ. Measurement of xenobiotic carbonyl
reduction in human liver fractions. Curr Protoc
Toxicol 2005; 4.17.1–4.17.7. doi:10.1002/0471140856.
tx0417s25
Conclusions
The results obtained in the inhibition experiments
indicate that many of the chemical inhibitors used
to ascertain the roles of different carbonyl reduc-
tases in metabolic processes lack specificity for
these enzymes. Furthermore, the investigations
on the metabolic reduction of the ketone groups
of haloperidol (1), daunorubicin (3) and the
propan-2-one derivative 5 by recombinant micro-
somal NADPH-cytochrome P450 reductase, re-
combinant cytosolic carbonyl reductase-1, rat
liver S9 fractions as well as its microsomal and cy-
tosolic fractions indicate that the ketone groups of
daunorubicin, haloperidol and compound 5 are
mainly reduced by cytosolic enzymes. However,
since microsomes also catalysed these carbonyl re-
ductions at least to some extent, it can be con-
cluded that microsomal NADPH-cytochrome
P450 reductase can contribute to metabolic keto
reductions in xenobiotics. Further studies should
be performed to clarify the roles of the different
carbonyl reducing enzymes for the reductive me-
tabolism of ketone compounds.
11. Tong Z, Chandrasekaran A, Li H, et al. In vitro me-
tabolism and identification of human enzymes in-
volved in the metabolism of methylnaltrexone.
Drug Metab Dispos 2010; 38: 801–807. doi:10.1124/
dmd.110.032169.
Copyright © 2015 John Wiley & Sons, Ltd.
Biopharm. Drug Dispos. 36: 398–404 (2015)
DOI: 10.1002/bdd

404
M. LEHR ET AL.
12. Dudka J, Jodynis-Liebert J, Korobowicz E, et al. Ac-
tivity of NADPH-cytochrome P-450 reductase of
the human heart, liver and lungs in the presence
of (-)-epigallocatechin gallate, quercetin and res-
veratrol: an in vitro study. Basic Clin Pharmacol
Toxicol 2005; 97: 74–79.
13. Dudka J. Decrease in NADPH-cytochrome P450
reductase activity of the human heart, liver and
lungs in the presence of alpha-lipoic acid. Ann
Nutr Metab 2006; 50: 121–125.
14. Slepneva IA, Sergeeva SV, Khramtsov VV. Revers-
ible inhibition of NADPH-cytochrome P450 reduc-
tase by alpha-lipoic acid. Biochem Biophys Res
Commun 1995; 214: 1246–1253.
15. Atalla A, Maser E. Characterization of enzymes
participating in carbonyl reduction of 4-
methylnitrosamino-1-(3-pyridyl)-1-butanone
(NNK) in human placenta. Chem Biol Interact 2001;
130–132: 737–748.
16. Tew DG. Inhibition of cytochrome P450 reductase
by the diphenyliodonium cation. Kinetic analysis
and covalent modifications. Biochemistry 1993; 32:
10209–10215.
17. McGuire JJ, Anderson DJ, McDonald BJ,
Narayanasami R, Bennett BM. Inhibition of
NADPH-cytochrome P450 reductase and glyceryl
trinitrate biotransformation by diphenyleneiodonium
sulfate. Biochem Pharmacol 1998; 56: 881–893.
18. Persson B, Kallberg Y. Classification and nomen-
clature of the superfamily of short-chain
dehydrogenases/reductases (SDRs). Chem Biol In-
teract 2013; 202: 111–115.
19. Ohara H, Miyabe Y, Deyashiki Y, Matsuura K,
Hara A. Reduction of drug ketones by dihydrodiol
dehydrogenases, carbonyl reductase and aldehyde
reductase of human liver. Biochem Pharmacol 1995;
50: 221–227.
20. Slupe A, Williams B, Larson C, et al. Reduction of
13-deoxydoxorubicin and daunorubicinol anthra-
quinones by human carbonyl reductase. Cardiovasc
Toxicol 2005; 5: 365–376.
22. Kassner N, Huse K, Martin HJ, et al. Carbonyl re-
ductase 1 is a predominant doxorubicin reductase
in the human liver. Drug Metab Dispos 2008; 36:
2113–2120. doi:10.1124/dmd.108.022251.
23. Bains OS, Karkling MJ, Grigliatti TA, Reid RE,
Riggs KW. Two nonsynonymous single nucleotide
polymorphisms of human carbonyl reductase 1
demonstrate reduced in vitro metabolism of dau-
norubicin and doxorubicin. Drug Metab Dispos
2009; 37: 1107–1114. doi:10.1124/dmd.108.024711.
24. Blanco JG, Leisenring WM, Gonzalez-Covarrubias
VM, et al. Genetic polymorphisms in the carbonyl
reductase 3 gene CBR3 and the NAD(P)H:quinone
oxidoreductase 1 gene NQO1 in patients who de-
veloped anthracycline-related congestive heart
failure after childhood cancer. Cancer 2008; 112:
2789–2795. doi:10.1002/cncr.23534.
25. Takahashi RH, Bains OS, Pfeifer TA, Grigliatti TA,
Reid RE, Riggs KW. Aldo-keto reductase 1C2 fails
to metabolize doxorubicin and daunorubicin
in vitro. Drug Metab Dispos 2008; 36: 991–994.
doi:10.1124/dmd.108.020388.
26. O’Connor T, Ireland LS, Harrison DJ, Hayes JD.
Major differences exist in the function and tissue-
specific expression of human aflatoxin B1 aldehyde
reductase and the principal human aldo-keto
reductase AKR1 family members. Biochem J 1999;
343: 487–504.
27. Martin HJ, Breyer-Pfaff U, Wsol V, Venz S, Block S,
Maser E. Purification and characterization of
akr1b10 from human liver: role in carbonyl reduc-
tion of xenobiotics. Drug Metab Dispos 2006; 34:
464–470.
28. Breyer-Pfaff U, Nill K. High-affinity stereoselective
reduction of the enantiomers of ketotifen and of
ketonic nortriptyline metabolites by aldo-keto re-
ductases from human liver. Biochem Pharmacol
2000; 59: 249–260.
Supporting information
21. Matsunaga T, Shintani S, Hara A. Multiplicity of
mammalian reductases for xenobiotic carbonyl
compounds. Drug Metab Pharmacokinet 2006; 21:
1–18.
Additional supporting information may be found
in the online version of this article at the publisher’s
web-site.
Copyright © 2015 John Wiley & Sons, Ltd.
Biopharm. Drug Dispos. 36: 398–404 (2015)
DOI: 10.1002/bdd