Reductive metabolism of nabumetone by human liver microsomal and cytosolic fractions: Exploratory prediction using inhibitors and substrates as marker probes

Article abstract of DOI:10.1007/s13318-014-0190-0

The metabolic reduction of nabumetone was examined by inhibition and correlation studies using human liver microsomes and cytosol. This reduction was observed in both fractions, with the V_max values for reduction activity being approximately fourfold higher, and the V_max/K_m values approximately three-fold higher, in the microsomes than in the cytosol. The reduction of nabumetone was inhibited by 18β-glycyrrhetinic acid, an 11β-hydroxysteroid dehydrogenase (11β-HSD) inhibitor, in the microsomal fraction. The reduction activity was also inhibited by quercetin and menadione [carbonyl reductase (CBR) inhibitors], and by phenolphthalein and medroxyprogesterone acetate [potent inhibitors of aldo-keto reductase (AKR) 1C1, 1C2 and 1C4] in the cytosol. A good correlation (r² = 0.93) was observed between the reduction of nabumetone and of cortisone, as a marker of 11β-HSD activity, in the microsomal fractions. There was also an excellent relationship between reduction of nabumetone and of the AKR1C substrates, acetohexamide, and ethacrynic acid (r² = 0.92 and 0.93, respectively), in the cytosol fractions. However, a poor correlation was observed between the formation of 4-(6-methoxy-2-naphthyl)-butan-2-ol (MNBO) from nabumetone and CBR activity (with 4-benzoyl pyridine reduction as a CBR substrate) in the cytosol fractions (r² = 0.24). These findings indicate that nabumetone may be metabolized by 11β-HSD in human liver microsomes, and primarily by AKR1C4 in human liver cytosol, although multiple enzymes in the AKR1C subfamily may be involved. It cannot be completely denied that CBR is involved to some extent in the formation of MNBO from nabumetone in the cytosol fraction.

Full text of DOI:10.1007/s13318-014-0190-0

Eur J Drug Metab Pharmacokinet
DOI 10.1007/s13318-014-0190-0
ORIGINAL PAPER
Reductive metabolism of nabumetone by human liver microsomal
and cytosolic fractions: exploratory prediction using inhibitors
and substrates as marker probes
•
Kaori Matsumoto Tetsuya Hasegawa
•
•
Tomoyo Kamei Junichi Koyanagi Tamiko Takahashi
•
•
•
Masayuki Akimoto Kenji Sugibayashi
Received: 13 October 2013 / Accepted: 8 March 2014
Ó Springer International Publishing Switzerland 2014
Abstract The metabolic reduction of nabumetone was
examined by inhibition and correlation studies using
human liver microsomes and cytosol. This reduction was
observed in both fractions, with the Vmax values for
reduction activity being approximately fourfold higher, and
the Vmax/Km values approximately three-fold higher, in the
microsomes than in the cytosol. The reduction of nabum-
etone was inhibited by 18b-glycyrrhetinic acid, an 11b-
hydroxysteroid dehydrogenase (11b-HSD) inhibitor, in the
microsomal fraction. The reduction activity was also
inhibited by quercetin and menadione [carbonyl reductase
(CBR) inhibitors], and by phenolphthalein and medroxy-
progesterone acetate [potent inhibitors of aldo–keto
reductase (AKR) 1C1, 1C2 and 1C4] in the cytosol. A good
correlation (r2 = 0.93) was observed between the reduc-
tion of nabumetone and of cortisone, as a marker of 11b-
HSD activity, in the microsomal fractions. There was also
an excellent relationship between reduction of nabumetone
and of the AKR1C substrates, acetohexamide, and ethac-
rynic acid (r2 = 0.92 and 0.93, respectively), in the cytosol
fractions. However, a poor correlation was observed
between the formation of 4-(6-methoxy-2-naphthyl)-butan-
2-ol (MNBO) from nabumetone and CBR activity (with
4-benzoyl pyridine reduction as a CBR substrate) in the
cytosol fractions (r2 = 0.24). These findings indicate that
nabumetone may be metabolized by 11b-HSD in human
liver microsomes, and primarily by AKR1C4 in human
liver cytosol, although multiple enzymes in the AKR1C
subfamily may be involved. It cannot be completely denied
that CBR is involved to some extent in the formation of
MNBO from nabumetone in the cytosol fraction.
Keywords Nabumetone Á Carbonyl reductase Á Aldo–
keto reductase Á 11b-Hydroxysteroid dehydrogenase Á
In vitro metabolism
1 Introduction
Nabumetone is a non-steroidal anti-inflammatory drug
which has proven to be effective in the treatment of
rheumatoid arthritis and osteoarthritis (Friedel et al. 1993;
Davies 1997; Friedel and Todd 1988). Nabumetone is a
pro-drug that undergoes extensive first-pass metabolism to
6-methoxy-2-naphthylacetic acid (6-MNA), the major cir-
culating metabolite, which is a potent inhibitor of prosta-
glandin synthesis (Mangan et al. 1987). Two main
metabolic pathways of nabumetone appear to be operating:
reduction of the ketone to an alcohol and oxidative
cleavage of the side chain to yield acetic acid derivatives.
All animals studied, including humans, use both pathways
(Haddock et al. 1984). Tsuchiya et al. (1988) investigated
the route of conversion and confirmed the existence of two
metabolic pathways from nabumetone to 6-MNA; one is
K. Matsumoto Á T. Hasegawa Á T. Kamei Á J. Koyanagi Á
T. Takahashi Á M. Akimoto (&)
Faculty of Pharmaceutical Sciences, Josai International
University, 1 Gumyo, Togane, Chiba 283-8555, Japan
e-mail: makimoto@jiu.ac.jp
K. Sugibayashi
Faculty of Pharmaceutical Sciences, Josai University,
1-1 Keyakidai, Sakado, Saitama 350-0295, Japan
via 4-(6-methoxy-2-naphthyl)-butan-2-ol (MNBO),
a
reductive metabolite of nabumetone, to 6-MNA (60 %) and

Eur J Drug Metab Pharmacokinet
the other results in 6-MNA (40 %) in rats. In our previous
study, cytochrome P450 (CYP) 2C9 was identified as the
major isoform involved in the oxidation of 6-MNA, which
is a pharmacologically active metabolite of nabumetone,
by human liver microsomes (Matsumoto et al. 2011a).
Recent studies have suggested that metabolism of nabum-
etone to 6-MNA is primarily mediated by CYP1A2 (Tur-
peinen et al. 2009). Carbonyl reduction is also the most
common phase I metabolic reaction in the majority of
xenobiotic aldehydes and ketones. Carbonyl reduction of
aldehyde and ketone moieties may be catalyzed by alcohol
dehydrogenases, aldo–keto reductases (AKRs), short-chain
dehydrogenases/reductases (SDRs) including carbonyl
reductase (CBR) and 11b-hydroxysteroid dehydrogenase
(11b-HSD), and quinone reductases (Matsunaga et al.
2006). In contrast to CYPs, however, the absence of
commercially available recombinant systems and the lack
of specific inhibitors mean that further discrimination of
individual isoforms has not yet been achieved. Therefore,
individual isoforms involved in the metabolic reductions of
most carbonyl compounds in human liver have not yet been
well characterized.
2 Materials and methods
2.1 Chemicals
Nabumetone (4-(6-methoxy-2-naphthyl)-2-butanone) and
rac-MNBO (4-(6-methoxy-2-naphthyl)-butan-2-ol) were
supplied by Sanwa Kagaku Kenkyusho (Mie, Japan). (?)-
and (-)-MNBO were obtained by chromatographic sepa-
ration (CHIRALPAK AS-H column, 10 9 250 mm, 5 lm
particle size, Daicel, Tokyo, Japan) of rac-MNBO. (?)-
MNBO was converted to the diastereomeric MTPA esters
1
with (R)- and (S)-MTPACl. H NMR spectra of the dia-
stereomers were measured and each signals was assigned
by COSY. The absolute configuration of (?)-MNBO was
determined to be S according to the modified Mosher’s
method (Ohtani et al. 1991). Naproxen was purchased from
Tokyo Kasei Kogyo (Tokyo, Japan). NADPH (b-nicotin-
amide adenine dinucleotide phosphate, reduced form) and
NADP (b-nicotinamide adenine dinucleotide phosphate)
were purchased from Alexis (San Diego, CA). Glucose
6-phosphate was purchased from Oriental Yeast (Tokyo,
Japan). Glucose 6-phosphate dehydrogenase was purchased
from MP Biomedicals (Aurora, OH). Hydroxyhexamide
was synthesized from acetohexamide using sodium boro-
hydride. All other chemicals were of the highest purity
available.
Incubation of nabumetone with human liver subcellular
fractions led to the observation of both the preferential
contribution of microsomal and cytosolic enzymes, and of
the pronounced inhibition of ketone reduction by potent
inhibitors including quercetin, menadione, ethacrynic acid,
and 18b-glycyrrhetinic acid (Matsumoto et al. 2011b).
Comprehensive screening based on inhibition and corre-
lation studies for evaluating involvement of individual
2.2 Enzyme preparations
Individual human liver microsomes (HG3, HG74, HG95,
HH13, HH47, HK37 and HG103) and individual human
liver cytosolic fractions (HH18, HG42, HG43, HK34,
HG64, HH31, HH47 and HH35) were purchased from BD
Gentest Co. (Woburn, MA). Pooled human liver micro-
somes and cytosol were also purchased from BD Gentest
Co. The pooled human liver microsomes and cytosol were
obtained from 20 to 10 donors, respectively.
isoforms offers
a quick and easy-to-use approach.
Recently, Skarydova et al. (2013) investigated the meta-
bolic reduction of nabumetone, mediated mainly by AKRs
and CBR, through incubation with eight recombinant car-
bonyl reducing enzymes which they had prepared. How-
ever, a new chemical entity usually has little information
on the predominant metabolic process during the drug
discovery stage. A combination of inhibitory activities, and
the correlation with commercially available substrates
based on the formation rates of metabolites, appears to be a
useful approach for estimating the contribution of indi-
vidual enzymes. In addition, this approach might eliminate
more time consuming, cumbersome and complicated
approaches. Therefore, we attempted to compare the pre-
dictive performance of our approach with the results of
Skarydova et al. (2013), because their method using
recombinant systems is likely to be accurate.
2.3 Metabolism of nabumetone in human liver
microsomes and cytosol
Metabolism of nabumetone in human liver microsomes
and cytosol were evaluated in the presence of NADPH.
A typical incubation mixture (0.5 ml of total volume)
contained 50 mM phosphate buffer (pH 7.4), an
NADPH-generating system (0.5 mM NADP?, 5 mM
glucose 6-phosphate, 5 mM MgCl2, 1 U/ml glucose
The primary objective of the present study was to esti-
mate enzymes possibly involved in the metabolic reduction
of nabumetone (Fig. 1) through inhibition and correlation
studies using human liver microsomes and cytosol, and to
compare these results with the recombinant system repor-
ted by Skarydova et al. (2013).
6-phosphate
dehydrogenase)
containing
0.5 mM
NADPH, nabumetone, and human liver microsomes or
cytosol (0.1 mg/ml). The reaction was initiated by
adding 250 ll of the NADPH-generating system after a
3 min pre-incubation of the microsomes at 37 °C. The
reaction mixtures were incubated for 20 min, and

Eur J Drug Metab Pharmacokinet
O
OH
Fig. 1 Metabolic reduction of
nabumetone
reduction
H3 CO
H3CO
Nabumetone
MNBO
reactions were terminated by the addition of 100 ll of
10 % trichloroacetic acid and 500 ll of acetonitrile
containing 10 lM of naproxen as an internal standard.
After removal of the protein by centrifugation at
10,0009g for 5 min, a 500 ll portion of the supernatant
was applied to cartridges. Solid phase extraction was
performed using Bond Elut Certify II cartridges
(200 mg, Varian, Harbor City, CA). Each cartridge was
first conditioned with 6 ml of 5 % methanol, at a flow
rate of 2 ml/min. After application of the samples at
2 ml/min, the cartridges were washed with 4 ml of
water. The cartridges were eluted with 6 ml of a freshly
prepared mixture of solvents (hexane and ethyl acetate,
1:1 v/v). The purified extract eluted from the solid-
phase extraction was transferred to a test tube, and the
solvents from the eluate were evaporated to dryness on
a heating block at 40 °C. The residue was dissolved in
5 ml of mobile phase, and 20 ll was injected into the
high-performance liquid chromatograph (HPLC) col-
umn. The HPLC system was a Shimadzu LC-10AVP
were used: n-benzylimidazole (1 mM) for CYP, methim-
azole (1 mM) for flavin-containing monooxygenase (FMO)
(Grothusen et al. 1996), TlCl3 (64 lM) for NADPH-cyto-
chrome P450 reductase, dicumarol (0.1 mM) for
NAD(P)H:quinone oxidoreductase, barbituric acid
(0.1 mM) for aldehyde dehydrogenase, pyrazol (0.1 mM)
for alcohol dehydrogenase (Tani et al. 2005), quercetin
(0.1 mM) for CBR, menadione (0.5 mM) for CBR and
11b-HSD, 18b-glycyrrhetinic acid (200 lM) for 11b-HSD
(Maser et al. 2000; Atalla and Maser 2001; Lee et al. 2008;
Diederich et al. 2000; Maser et al. 2003), ethacrynic acid
(1 mM) for CBR and AKRs (Maser et al. 2000; Barski
et al. 2008), phenolphthalein (20 lM) and flufenamic acid
(20 lM) for AKR1C1, 1C2, 1C3 and 1C4, medroxypro-
gesterone acetate (20 lM) for AKR1C1, 1C2 and 1C4 and
5b-cholanic acid-3a,7a-diol (50 lM) for AKR1C2 (Hara
et al. 1990; Deyashiki et al. 1992; Atalla et al. 2000; Higaki
et al. 2003; Steckelbroeck et al. 2006; Blech et al. 2010;
Tong et al. 2010). Nabumetone (50 lM) was incubated
with NADPH or NADH for 20 min at 37 °C.
equipped with
a RF-10AXL fluorescence detector
(Kyoto, Japan). The samples were injected into the
HPLC system and separated on a YMC-Pack ODS-A
column (4.6 9 150 mm, 5 lm particle size, YMC,
Kyoto, Japan). The temperature of the column oven was
set at 30 °C. The mobile phase consisted of acetonitrile:
20 mM K2HPO4 (pH 3.0) (50:50, v/v), and the flow rate
was 1.0 ml/min. The peaks were monitored at an exci-
tation wavelength of 280 nm and an emission wave-
length of 350 nm (Mikami et al. 2000; Kobylinska et al.
2003).
2.5 Correlation between 11b-HSD activity and MNBO
formation from nabumetone in microsomes
MNBO formation rates were correlated with 11b-HSD
activities in seven human liver microsome fractions.
Nabumetone (50 lM) was incubated for 20 min at
37 °C. The activity of 11b-HSD was determined with
the substrate cortisone. A typical incubation mixture
(200 ll total volume) contained 50 mM phosphate buf-
fer (pH 7.4), NADPH (4 mM), cortisone (100 lM), and
human liver microsomes (0.4 mg/ml). The reaction was
initiated by adding NADPH solution after a 3 min pre-
incubation of human liver microsomes at 37 °C. The
reaction mixtures were incubated for 60 min, and reac-
tions were terminated by the addition of 300 ll of ice-
cold acetonitrile and 100 ll of 50 lM 6a-methylpred-
nisolone as an internal standard. The reduction product
(cortisol) was determined by HPLC according to the
method of Piwowarska et al. (2009) with slight modi-
fication. The samples were injected into the HPLC
system and separated on an YMC-Pack ODS-A column
(4.6 9 150 mm, 5 lm particle size, YMC, Kyoto,
Japan). The temperature of the column oven was set at
Separation of enantiomers was performed using a
CHIRALPAK AS-RH column (4.6 9 150 mm, 5 lm par-
ticle size, Daicel, Tokyo, Japan). The temperature of the
column oven was set at 40 °C. The mobile phase consisted
of water:acetonitrile (55:45, v/v), and the flow rate was
1.0 ml/min. The UV detection was set at 320 nm.
2.4 Inhibition of MNBO formation from nabumetone
by chemical inhibitors
Inhibition studies were performed by incubating pooled
human liver microsomes and cytosol with chemical
inhibitors. The following chemical inhibitors and substrates

Eur J Drug Metab Pharmacokinet
40 °C. The mobile phase consisted of water/acetonitrile
(70:30, v/v), and the flow rate was 1.0 ml/min. The UV
detection was set at 252 nm.
microsomes
7000
6000
5000
4000
3000
2000
1000
0
2.6 Correlation between CBR and/or AKR1C activities
and MNBO formation from nabumetone in cytosol
cytosol
MNBO formation rates were correlated with CBR and/or
AKR1C activities (Matsunaga et al. 2006; Nakayama et al.
1985) in liver cytosol derived from 6–8 human. Nabume-
tone (50 lM) was incubated for 20 min at 37 °C. The
activities of ethacrynic acid (AKR1C1 and 1C4), metyra-
pone (CBR1 and AKR1C4), loxoprofen (CBR1, AKR1C1,
1C2 and 1C4) and 4-benzoylpyridine (CBR) reductase
were determined according to the method of Ohara et al.
(1995) and acetohexamide (AKR1C1, 1C2 and 1C4)
reductase were determined according to the method of
Imamura and Shimada (2004).
0
50
100
150
200
250
300
Concentration of nabumetone (µM)
Fig. 2 Michaelis–Menten plots for MNBO formation from nabum-
etone in human microsomes and cytosol. Each point represents the
mean SD of three measurements
NADPH or NADH as the cofactor in the cytosol fraction
(data not shown).
3.2 Inhibition of MNBO formation from nabumetone
by chemical inhibitors
2.7 Data analysis
The results are presented as the mean of estimates obtained
in triplicate experiments. The apparent Michaelis–Menten
parameters for the formation of MNBO from nabumetone
were estimated for Eadie–Hofstee plots.
MNBO formation from nabumetone in human liver
microsomes and cytosol is shown in Figs. 3 and 4,
respectively. The reductive formation to MNBO from
nabumetone in the microsomes was not inhibited by n-
benzylimidazole (P450), methimazole (FMO), TlCl3
(NADPH-cytochrome P450 reductase) or dicumarol
(NAD(P)H:quinone oxidoreductase), whereas it was
inhibited by 18b-glycyrrhetinic acid and menadione (11b-
HSD) (Fig. 3). The mean levels of inhibition elicited by
18b-glycyrrhetinic acid and menadione were 78.5 and
52.3 %, respectively, compared with the control value.
MNBO formation from nabumetone in the human liver
cytosol was not inhibited by barbituric acid (aldehyde
dehydrogenase), pyrazol (alcohol dehydrogenase) or dicu-
marol, whereas it was inhibited by quercetin and menadi-
one (CBR), ethacrynic acid (CBR and AKRs) and
phenolphthalein, flufenamic acid and medroxyprogesterone
acetate (AKR1C) (Fig. 4). The mean values of inhibition
elicited by the various inhibitors (compared with the con-
trol value) were: quercetin 64.0 %, menadione 77.4 %,
ethacrynic acid 78.5 %, phenolphthalein 47.1 %, flufen-
amic acid 43.8 % and medroxyprogesterone acetate
56.2 %.
Analyses for correlations and statistical significances
were carried out using EXCEL statistics software (Esumi
Co. Ltd., Tokyo, Japan).
3 Results
3.1 Kinetic analyses of MNBO formation
from nabumetone by human liver microsomes
and cytosol
The kinetics of the NADPH-dependent metabolism of
10–300 lM nabumetone to the metabolite MNBO was
examined using pooled human liver microsomes and
cytosol. Michaelis–Menten kinetics for MNBO activity
was observed in human liver microsomes and cytosol
(Fig. 2), and the results of the kinetic analysis are shown in
Table 1. The Vmax value for MNBO formation in the
human liver microsomes was approximately fourfold
higher than that in the human liver cytosol, and the Vmax
/
Km values in human liver microsomes were approximately
threefold higher than those in human liver cytosol. Both
subcellular fractions produce both enantiomers. The for-
mation ratios of (S)-/(R)-MNBO were 74/26 and 67/33 in
microsomes and cytosol, respectively. The reduction of
nabumetone was observed on incubating with either
3.3 Correlation between 11b-HSD activity and MNBO
formation from nabumetone
The correlation between 11b-HSD activity and MMBO
formation from nabumetone was assessed in liver

Eur J Drug Metab Pharmacokinet
Table 1 Michaelis–Menten kinetic parameters of MNBO formation from nabumetone in human liver microsomes and cytosol
Subcellular fraction
Km (lM)
Vmax (pmol/min/mg protein)
Vmax/Km (ll/min/mg protein)
(S)-/(R)-MNBO
Microsomes
Cytosol
35.0 5.7
27.1 2.0
6,136.7 306.1
1,592.8 105.3
177.5 20.6
59.1 7.4
74/26
67/32
120
100
80
activity and MNBO formation from nabumetone
(r2 = 0.93, P \ 0.0005).
3.4 Correlation between CBR and/or AKR1C activities
and MNBO formation from nabumetone
60
Correlation between CBR activity and MNBO formation
from nabumetone were compared in the liver cytosol from
6–8 human samples. Enzymatic reduction of acetohexam-
ide, ethacrynic acid, metyrapone and loxoprofen reductase
was used for assaying the CBR and/or AKR1C activities in
cytosolic fractions. Figure 6 shows a good correlation
between the reduction of acetohexamide (r2 = 0.92,
P \ 0.0005), ethacrynic acid (r2 = 0.93, P \ 0.005),
metyrapone (r2 = 0.93, P \ 0.005) and loxoprofen
(r2 = 0.86, P \ 0.01) and MNBO formation from nab-
umetone. In contrast, a poor correlation was observed
between CBR activity with 4-benzoyl pyridine reduction as
a substrate and MNBO formation from nabumetone
(r2 = 0.24).
40
20
0
NBI
MTZ
TC
DM
GA
MD
Inhibitors
Fig. 3 Effect of enzyme inhibitors on MNBO formation from
nabumetone in human microsomes. Each bar represents the
mean SD (n = 3). NBI: 1 mM n-benzylimidazole (P450), MTZ:
1 mM methimazole (FMO), TC: 64 lM TlCl3 (NADPH-cytochrome
P450 reductase), DM: 0.1 mM dicumarol (NAD(P)H:quinone oxido-
reductase), GA: 200 lM 18b-glycyrrhetinic acid (11b-HSD), MD:
0.5 mM menadione (11b-HSD)
4 Discussion
microsomes from seven human samples. A well-known
reaction, cortisol formation from cortisone, was used for
assaying the 11b-HSD activity in the microsomal fractions.
Figure 5 shows a good correlation between 11b-HSD
Nabumetone is established as a non-acidic, non-steroidal
anti-inflammatory drug. It is an example of a pro-drug,
because nabumetone itself exists only for a short time in
120
100
80
60
40
20
0
BA
PZ
DM
QC
MD
EA
PP
FA
MPA CDCA
Inhibitors
Fig. 4 Effect of enzyme inhibitors on MNBO formation from
nabumetone in human cytosol. Each bar represents the mean SD
(n = 3). BA: 0.1 mM barbituric acid (aldehyde dehydrogenase), PZ:
0.1 mM pyrazol (alcohol dehydrogenase), DM: 0.1 mM dicumarol
(NAD(P)H:quinone oxidoreductase), QC: 0.1 mM quercetin (CBR),
MD: 0.5 mM menadione (CBR), EA: 1 mM ethacrynic acid (CBR
and AKRs), PP: 20 lM phenolphthalein (AKR1C1, 1C2, 1C3 and
1C4), FA: 20 lM flufenamic acid (AKR1C1, 1C2, 1C3 and 1C4),
MPA: 20 lM medroxyprogesterone acetate (AKR1C1, 1C2 and 1C4),
CDCA: 50 lM 5b-cholanic acid-3a,7a-diol (AKR1C2)

Eur J Drug Metab Pharmacokinet
5
4
3
2
1
0
from the sequential demonstration that (1) nabumetone was
metabolized by human liver microsomes; (2) NADPH was
the favored cofactor; (3) the 11b-HSD substrate menadione
was highly efficient in inhibiting this metabolism; (4) the
11b-HSD inhibitor 18b-glycyrrhetinic acid decreased
nabumetone metabolism in a dose-dependent fashion (data
not shown); (5) good correlation was observed between
MNBO formation rate and cortisone reduction rate
(r2 = 0.93). It is clear that nabumetone is a substrate for
11b-HSD. 11b-HSD has been shown to be capable of
catalyzing the reductive metabolism of a variety of xeno-
biotic compounds (Matsunaga et al. 2006).
r2=0.93
Nabumetone reduction was assessed using selective
chemical inhibitors of AKR isoforms and CBR/SDR. The
reduction of nabumetone was most effectively inhibited
by ethacrynic acid, which inhibits CBR and AKRs. For-
mation of MNBO was also inhibited (by up to 52.9 % of
control) by phenolphthalein, which inhibits AKR1C iso-
forms with greater selectivity for AKR1C4 than for
AKR1C1, AKR1C2 or AKR1C3 (Higaki et al. 2003).
Medroxyprogesterone acetate, which inhibits AKR1C1,
AKR1C2 and AKR1C4, and AKR1C4 with high speci-
ficity (Hara et al. 1990), inhibited the formation of
MNBO by up to 43.8 % of control. Fufenamic acid, a
selective inhibitor of AKR1C1, AKR1C2 and AKR1C3
and a weak inhibitor of AKR1C4 (Deyashiki et al. 1992),
also inhibited the formation of MNBO. The AKR1C2-
selective inhibitor 5b-cholanic acid-3a,7a-diol (Deyashiki
et al. 1992) did not appear to significantly affect the
reduction of nabumetone. Significant correlations were
observed between the nabumetone reductive activity and
reduction of acetohexamide (r2 = 0.92), a marker for the
AKR1C subfamily, or ethacrynic acid (r2 = 0.93), a
marker for AKR1C1 and 1C4, in cytosol. Some residual
activity done by different enzymes not related to nab-
umetone reduction may be existed because the regression
lines did not go through the origin in Fig. 6. The lack of
an inhibitory effect of barbituric acid (an inhibitor of
aldehyde dehydrogenase), dicumarol and pyrazol (alcohol
dehydrogenase and NAD(P)H:quinone oxidoreductase) on
the reduction of nabumetone confirmed that in humans,
the AKR1C subfamily are the primary AKRs capable of
reducing xenobiotic ketones. In addition, MNBO was
detected when nabumetone was incubated with human
liver cytosol in the presence of either NADPH or NADH
as the cofactor. The rate of nabumetone reduction of the
human liver cytosol with NADH as a cofactor was nearly
1.1 times higher than with NADPH. The dehydrogenation
of (S)-1-indanol catalyzed by AKR1C4 proceeded at
similar rates when either NADPH or NADH were used as
the cofactor (Hara et al. 1990). The efficient reduction of
nabumetone with NADH as a cofactor was observed,
which might suggest involvement of AKR1C4. Together,
0
100
200
300
400
500
Cortisone reduction rate
β -HSD activity (pmol/min/mg protein)
11
Fig. 5 Correlation between 11b-HSD activity and MNBO formation
from nabumetone by liver microsomes obtained from seven human
samples
the plasma, while its active metabolite, 6-MNA (which
exerts an anti-inflammatory effect) remains in the plasma
for a long period of time (Kendall et al. 1989). The exis-
tence of metabolic pathway from an intermediate com-
pound, MNBO, to 6-MNA has been confirmed in rats
(Tsuchiya et al. 1988). MNBO has no anti-inflammatory
properties and is a weak inhibitor of cyclooxygenase
(Nobilis et al. 2003).
In this study, we investigated the in vitro formation of
MNBO in human liver subcellular fractions and charac-
terized the enzymes involved in the carbonyl reduction of
nabumetone. To identify the human liver enzymes involved
in the metabolic clearance of nabumetone, enzyme inhi-
bition studies were performed using an in vitro metabolism
system deemed to reflect in vivo metabolism. The effects
of enzyme inhibitors on metabolic clearance of nabume-
tone were assessed in terms of MNBO formation.
The kinetic parameters in human liver microsomes
showed that the capacity for carbonyl reduction was
approximately three times greater than that in human liver
cytosol. In vitro metabolism of nabumetone in human liver
microsomes with NADPH was inhibited by 18b-glycyrrh-
etinic acid and menadione (inhibitors of 11b-HSD), but not
by n-benzylimidazole (an inhibitor of CYP), methimazole
(an inhibitor of FMO), TlCl3 (an inhibitor of NADPH-
cytochrome P450 reductase), or dicumarol (an inhibitor of
NAD(P)H:quinine oxidoreductase). Nabumetone was
reduced to the two MNBO enantiomers by human liver
microsomes and cytosol. The use of chemical inhibitors in
inhibition studies inhibited (S)- and (R)-MNBO at
approximately the same ratio (data not shown).
The results of the in vitro metabolism studies presented
here have several implications. These could be inferred

Eur J Drug Metab Pharmacokinet
Fig. 6 Correlation between
CBR and/or AKR1C activities
and MNBO formation from
nabumetone by liver cytosol
obtained from 6–8 human
samples
2.5
2
2.5
2
1.5
1
1.5
1
r2=0.92
r2=0.93
0.5
0
0.5
0
0
0.5
1
1.5
0
0.5
1
1.5
Ethacrynuc acid reduction rate
Acetohexamide reduction rate
AKR1C activities (nmol/min/mg protein)
2.5
2.5
2
2
1.5
1.5
1
r2=0.93
r2=0.86
1
0.5
0.5
0
0
0
2
4
6
0
2
4
6
Loxoprofen reduction rate
Metyrapone reduction rate
CBR and/or AKR activities (nmol/min/mg protein)
2.5
2
r2=0.24
1.5
1
0.5
0
0
20
40
60
4-Benzoylpyridine reduction rate
CBR activity (nmol/min/mg protein)
these results indicate that multiple isoforms in the
AKR1C subfamily are responsible for nabumetone
reduction, with the predominant contribution probably
from AKR1C4, which accounts for the largest amount of
enzyme activity in human liver (Penning et al. 2000;
Steckelbroeck et al. 2006).
In correlation studies, however, poor correlation was
observed in the cytosol between the formation of MNBO
from nabumetone and CBR activity with 4-benzoyl pyri-
dine reduction as a substrate (r2 = 0.24). However, some
contribution of CBR could not be excluded. Quercetin and
menadione, inhibitors of CBR, caused a significant

Eur J Drug Metab Pharmacokinet
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expressed in human liver, and its expression is an order of
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ckelbroeck et al. 2006). These observations suggest that
CBR could be involved to some extent in the metabolism
of nabumetone to MNBO. The absence of commercially
available AKR and CBR isoforms and the lack of specific
inhibitors did not allow further discrimination of the indi-
vidual isoforms. Therefore, the ability of other enzymes to
convert nabumetone to MNBO could not be completely
excluded.
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In conclusion, the present investigation provides evidence
that nabumetone is metabolized by 11b-HSD in human
liver microsomes. AKR1C4 appears to play a major role in
the carbonyl reduction of nabumetone, although multiple
enzymes in the AKR1C subfamily may be involved in the
reduction. In addition, CBR appears to be responsible for
the formation of MNBO from nabumetone. These results
are in accordance with those of Skarydova et al. (2013).
We suggest that a combination of inhibition and correlation
studies is a useful approach for estimating the contribution
of individual enzymes.
Conflict of interest None.
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