Hepatic metabolism of diclofenac: Role of human CYP in the minor oxidative pathways

Article abstract of DOI:10.1016/S0006-2952(99)00167-7

The aim of this study was to re-examine the human hepatic metabolism of diclofenac, with special focus on the generation of minor hydroxylated metabolites implicated in the idiosyncratic hepatotoxicity of the drug. Different experimental approaches were u

Full text of DOI:10.1016/S0006-2952(99)00167-7

Biochemical Pharmacology, Vol. 58, pp. 787–796, 1999.
© 1999 Elsevier Science Inc. All rights reserved.
ISSN 0006-2952/99/$–see front matter
PII S0006-2952(99)00167-7
Hepatic Metabolism of Diclofenac: Role of Human
CYP in the Minor Oxidative Pathways
Roque Bort,*† Katherine Mace´,‡ Alan Boobis,§ Mar´ıa-Jose´ Go´mez-Lecho´n,*
࿣
Andrea Pfeifer‡ and Jose´ Castell*†
´
*UNIDAD DE HEPATOLOG´ıA EXPERIMENTAL, CENTRO DE INVESTIGACION, HOSPITAL UNIVERSITARIO LA FE,
AVDA DE CAMPANAR 21, E-46009 VALENCIA, SPAIN; †DEPARTAMENTO DE BIOQU´ıMICA Y BIOLOG´ıA MOLECULAR,
´ ˜
FACULTAD DE MEDICINA, UNIVERSIDAD DE VALENCIA, AVDA, BLASCO IBANEZ, 20, E-46010 VALENCIA, SPAIN;
´
‡NESTLE RESEARCH CENTER, VERS CHEZ LE BLANC,
CH-1000 LAUSANNE 26, SWITZERLAND; AND §SECTION ON CLINICAL PHARMACOLOGY, IMPERIAL COLLEGE
SCHOOL OF MEDICINE, LONDON, U.K.
ABSTRACT. The aim of this study was to re-examine the human hepatic metabolism of diclofenac, with
special focus on the generation of minor hydroxylated metabolites implicated in the idiosyncratic hepatotoxicity
of the drug. Different experimental approaches were used: human hepatocytes, human microsomes, and
engineered cells expressing single human CYP (cytochromes P450). Human hepatocytes formed 3Ј-hydroxy-,
4Ј-hydroxy-, 5-hydroxy- 4Ј,5-dihydroxy-, and N,5-dihydroxydiclofenac, as well as several lactams. Formation of
4Ј- and 5-hydroxydiclofenac by human liver microsomes followed a Michaelis–Menten kinetics (Km 9 Ϯ 1 ␮M;
Vmax 432 Ϯ 15 pmol/min/mg and Km 43 Ϯ 5 ␮M; and Vmax 15.4 Ϯ 0.6 pmol/min/mg, respectively). Secondary
metabolites were detected after incubation of 5-hydroxydiclofenac with human liver microsomes, yielding
4Ј,5-dihydroxydiclofenac (Km 15 Ϯ 1 ␮M; Vmax 96 Ϯ 3 pmo1/min/mg) and small amounts of N,5-
dihydroxydiclofenac (non-Michaelis–Menten kinetics). Based on microsome studies and the incubations with
human hepatocytes and engineered cells, we estimated that in vivo CYP2C9 would be exclusively responsible for
the 4Ј hydroxylation of diclofenac (Ͼ99.5%) as well as 5-hydroxydiclofenac (Ͼ97%). CYP2C9 was exclusively
responsible for the formation of 3Ј-hydroxydiclofenac. Multiple regression analysis evidenced that the rate of
production of 5-hydroxydiclofenac in human microsomes followed the algorithm: 0.040 ϫ S-mephenytoin
4Ј-hydroxylation ϩ 0.083 ϫ tolbutamide methylhydroxylation, (multiple correlation coefficient ϭ 0.969).
However, the incubation of diclofenac with cell lines expressing different human CYP suggested that 7 isoforms
could be involved. Comparison of data obtained with CYP-expressing cells and human hepatocytes suggests that
CYP2C8 Ͼ CYP2C19 Х CYP2C18 ϾϾ CYP2B6 are the isoforms implicated in the 5-hydroxylation of diclofenac
in vivo. BIOCHEM PHARMACOL 58;5:787–796, 1999. © 1999 Elsevier Science Inc.
KEY WORDS. cytochrome P450; diclofenac; in vitro metabolism; CYP2C
Diclofenac, an arylacetic non-steroidal anti-inflammatory
drug, was developed in the late 1970s and approved for clinical
use in treating several rheumatic diseases (rheumatoid arthri-
tis, osteoarthritis, and ankylosing spondylitis) and as an anal-
gesic [1]. Early studies in rat, baboon, dog, monkey, and man
[2, 3] showed that diclofenac undergoes an extensive hepatic
metabolism involving aromatic hydroxylations and conjuga-
tions (Fig. 1). Most diclofenac metabolites are found in
animals and man, but their relative abundance differs. In man,
4Ј-OHDic,¶ 5-OHDic, 4Ј, 5-diOHDic, 3Ј-OHDic, and 3Ј-
OH-4Ј-OCH3-Dic have been identified in urine, plasma,
and/or bile [2, 4].
The use of diclofenac has been associated with occa-
sional hepatic toxicity. Borderline elevation of enzyme
markers is found in a significant number of patients taking
the drug (15%), and a mild to severe hepatotoxicity has
been reported in a small but significant number of patients
[5–8]. Several hypotheses have been put forth to explain
this idiosyncratic adverse reaction, among them the forma-
tion of toxic metabolites and/or reactive intermediates able
to bind hepatocyte proteins [5, 6, 9–11] triggering, presum-
ably, an allergic response. Interest in diclofenac metabolism
has been increased by indirect experimental evidence
suggesting that a minor CYP-generated oxidation product
of diclofenac could be involved in the formation of drug–
protein adducts in human microsomes [12].
࿣
Corresponding author: Jose´ Castell M.D., Ph.D., Departamento de
Hepatolog´ıa Experimental, Centro de Investigacio´n, Hospital Universita-
rio La Fe, Avda de Campanar E-46009 Valencia, Spain. Tel. (96)
386-8748; FAX (96) 386-8718; E-mail: Jose.Castell@uv.es.
¶ Abbreviations: 4Ј-OHDic, 4Ј-hydroxydiclofenac [2-(2Ј,6Ј-dichloro-4Ј-
hydroxy-phenylamine)-phenyl]-acetic acid; 5-OHDic, 5-hydroxydiclo-
fenac [2-(2Ј,6Ј-dichlorophenylamine)-5-hydroxyphenyl]-acetic acid;
3Ј-OHDic, 3Ј-hydroxydiclofenac [2-(2Ј,6Ј-dichloro-3Ј-hydroxyphenyl-
amine)- phenyl]-acetic acid; 4Ј,5-diOHDic, 4Ј,5-dihydroxydiclofenac
[2-(2Ј,6Ј-dichloro-4Ј-hydroxy-phenylamine)-5-hydroxyphenyl]-acetic acid;
N,5-diOHDic, N,5-dihydroxydiclofenac [2-(2Ј,6Ј-dichlorophenylhydroxyl-
amine)-5-hydroxyphenyl]-acetic acid; 3Ј-OH-4Ј-OCH3-Dic, 3Ј-hydroxy-4Ј-
methoxydiclofenac [2-(2Ј,6Ј-dichloro-3Ј-hydroxy-4Ј-methoxyphenylamine)-
phenyl]-acetic acid; CYP, cytochrome P450; R, regression coefficient; and RT,
retention time.
4Ј-hydroxylation of diclofenac was first attributed to
CYP2C enzymes [13] and, more precisely, to CYP2C9 [14].
Received 23 October 1998; accepted 5 March 1999.

788
R. Bort et al.
FIG. 1. Structure of diclofenac and its
metabolites.
However, no detailed study has yet been performed to
identify the precise human isoform(s) involved in the
formation of the other oxidised metabolites. Findings from
our laboratory suggest that at least one minor hydroxylation
metabolic pathway could be at the root of the human
hepatic toxicity of diclofenac [15]. This led us to examine
in detail the metabolism of diclofenac and to precisely
identify the enzymes involved. By using cultured human
hepatocytes, human liver microsomes, and genetically en-
gineered cell lines expressing human CYP, it was possible
to identify the minor pathways of diclofenac biotransfor-
mation and to estimate the contribution of the different
CYP isoforms to its metabolism in vivo.
nyl]-2H-indol-2-one. The phenolic hydroxyl group of this
compound was protected by reaction with benzyl chloride to
give 1,3-dihydro-N-[(2,6-dichloro-3-benzyloxy)-phenyl]-2H-
indol-2-one. Hydrolysis of this compound with NaOH to
open the ring, and hydrogenolysis to remove the benzyl
group yielded 3Ј-OHDic. The most relevant spectral prop-
1
erties were: H-NMR (d6-acetone), d 3.9 (s, 2H, Ar-CH2-
COO-), 6.9 (s, 1H, -NH-), 7.4-6.8 (m, 6H, aromatic); (KBr
tablet), 3400 (NH stretching), 1720 (COOH carbonyl
stretching), 1585 (NH deformation), 1580 (CAC stretch-
ing); MS m/z (%), 311 (13), 293 (24), 258 (46), 230 (100).
Synthesis of 4؅,5-diOHDic
2,6-dichloro-4-methoxy aniline with its amino group pro-
tected by reaction with Ac2O was allowed to react with
p-bromoanisole to give N-acetyl-N-(4-methoxy)phenyl-
2,6-dichloro-4-methoxyaniline. This compound was trans-
formed to N-1, 3-dihydro-N-[(2,6-dichloro-4-methoxy)-
phenyl]-5-methoxy-2H-indol-2,3-dione first by deprotection
of the amine group with KOH, followed by reaction with
oxalyl chloride. Reduction with P/HI gave the 4Ј,5-dihy-
droxydiclofenac lactam. Hydrolysis of this compound with
NaOH to open the ring yielded 4Ј,5-diOHDic. The most
relevant spectral features of this compound were: 1H-NMR
(d6-acetone), d 3.78 (s, 2H, Ar-CH2-COO-), 7.2-6.4 (m,
5H, aromatic); i.r. (KBr tablet), 3410 (NH stretching),
3200 (OH stretching), 1695 (COOH carbonyl stretching),
1585 (NH deformation), 1580 (CAC stretching); MS: m/z
(%): 327 (46), 309 (14), 274 (22), 246 (100), 211 (24).
MATERIALS AND METHODS
Chemicals
Diclofenac, taxol, chlorzoxazone, coumarin, quinidine, trole-
andomycin, and NADPH were from Sigma. Furafylline and
sulfaphenazole were a generous gift from Dr. Alan Boobis.
6␣-Hydroxytaxol was obtained from Gentest Corp. S(ϩ)-
Mephenytoin and (Ϯ)-4Ј-hydroxymephenytoin was from Ul-
trafine Chemicals. The enzyme ␤-glucuronidase/arylsul-
phatase was from Boehringer Mannheim. Culture media
(Ham’s F-12, Lebovitz L-15) were from Life Technologies.
All other chemicals were of analytical grade. Synthesis of
4Ј-OHDic, 5-OHDic as well as their lactams and N,5-
diOHDic has been described in detail elsewhere [15, 16].
Synthesis of 3؅-OHDic
2,6-Dichloro-3-nitroaniline was treated with NaNO2 followed
by KI to give 2,6-dichloro-3-nitro-iodobenzene. This com-
pound was allowed to react with aniline under xylene reflux to
give N-phenyl-2,6-dichloro-3-nitroaniline, which was trans-
formed to 1,3-dihydro-N-[(2,6-dichloro-3-nitro)-phenyl]-2H-
indol-2,3-dione by reaction with oxalyl chloride. Reduction by
P/HI gave 1,3-dihydro-N-[(2,6-dichloro-3-amino)-phenyl]-2H
-indol-2-one, which by reaction with NaNO2 and Cu(NO3)2/
Cu2O, gave 1,3-dihydro-N-[(2,6-dichloro-3-hydroxy)-phe-
Isolation and Culture of Human Hepatocytes
Human hepatocytes were obtained from surgical liver
biopsies (2–4 g) of patients undergoing cholecystectomy,
after informed consent. Cells were isolated by microperfu-
sion of the tissue sample with collagenase, as described in
detail elsewhere [17]. Cellular viability was assessed by the
trypan blue dye exclusion test and was usually 85–90%.

Diclofenac Metabolism by Human Cytochrome P450
789
Hepatocytes were seeded on fibronectin-coated plastic dishes
(3.5 ␮g/cm2) at a density of 8 ϫ 104 viable cells/cm2 and
cultured in Ham’s F-12/Lebovitz L-15 (1:1) medium supple-
mented with 2% new-born bovine serum, 50 U/mL penicillin,
50 ␮g/mL streptomycin, 0.2% BSA, and 10Ϫ8 M insulin. One
hour later the medium was changed, and after 24 hr the cells
were shifted to serum-free hormone-supplemented medium
(10Ϫ8 M dexamethasone and insulin). The incubation with
the drug was routinely done between 24 and 48 hr of culture.
analysed by HPLC as described below. Inhibition studies
were performed using a pool of human liver microsomes and
several well-characterised specific inhibitors and substrates
of cytochrome P450, namely furafylline [22], coumarin [23],
sulfaphenazole [24], quinidine [25], chlorzoxazone [26], and
troleandomycin [27]. Inhibitors were prepared as stock
solutions in methanol and diluted 1:100 in incubation
mixtures. In the case of furafylline and troleandomycin,
microsomes were preincubated for 15 min with NADPH
and the reaction was started by the addition of diclofenac.
CYP-Engineered Cell Lines
HPLC Analysis and Identification of Diclofenac
Metabolites in Incubation Samples
Immortalised human liver epithelial cells (THLE) were
genetically manipulated to express specific CYP genes. Cell
lines expressing CYP1A2 (T5-1A2), CYP2A6 (T5-2A6),
CYP2B6 (T5-2B6), CYP2D6 (T5-2D6), CYP2E1 (T5-
2E1), and CYP3A4 (T5-3A4) were cultured on fibronec-
tin/collagen-coated flasks in serum-free medium, as previ-
ously described [18, 19]. In addition, CYP2C-expressing
cells were generated by inserting the following cDNAs by
blunt-ended cloning (kindly provided by J. A. Goldstein):
1.9 kb CYP2C8 (clone 7b), 1.85 kb CYP2C9 (clone 65;
Ile359Gly475), 2.0 kb CYP2C18 (clone 29c-1a;Asp2Thr385),
1.75 kb CYP2C19 (clone 11a) into the BamHI site of the
pCMVneo vector (kindly provided by B. Vogelstein), as
described elsewhere [20].
Two HPLC methods were developed to analyse diclofenac
metabolites. In method A, samples from microsome incu-
bations were precipitated with acetonitrile and centrifuged
for 10 min at 8,000 g. After dilution with 20 mM phosphate
buffer pH 7.4 to reach 25% (v/v) acetonitrile, samples were
HPLC analysed using a C-18 reverse-phase Kromasil 5 ␮m
20 ϫ 0.46-cm column. The mobile phase (75% triethanol-
amine 0.02% in 20 mM phosphate buffer pH 7.4, 25%
acetonitrile) was delivered at 1 mL/min, and the effluents
were monitored at 282 nm. Carprofen (8 ␮M) was used as
an internal standard during the analysis. Metabolites were
identified on the basis of their retention times and UV
spectra, and compared with chemically synthesised metab-
olites. A second method was developed to fit the criteria for
MS analysis. Aliquots of the culture media were enzymat-
ically deconjugated with 50 mU/mL ␤-glucuronidase and
30 mU/mL arylsulphatase in 0.1 M acetate buffer pH 4.5 for
4 hr at 37° and extracted several times with ethyl acetate.
The organic phase was dried under nitrogen atmosphere,
dissolved in methanol and injected into a 150 ϫ 2.1 mm
C-18 reverse-phase column (Symmetry). The mobile phase
(20 mM ammonia acetate buffer pH 6.5: methanol; 63:37,
v/v) was delivered at 0.3 mL/min. The column effluent was
monitored at 282 nm using a photodiode detector (M996,
Waters) and a mass spectrometer coupled on line to the
HPLC (Therma-Beam௡ detector, Waters). The UV and
MS spectra of the major metabolites were compared with
those of chemically synthesised samples.
Metabolism by Cultured Cells
Cells were incubated with diclofenac (Na salt) at a con-
centration showing no cytotoxic effect on cells. Samples of
the culture media were withdrawn and immediately acidi-
fied with acetate buffer 0.1 M, pH 4.5. The metabolites
present in the culture medium were enzymatically decon-
jugated and subjected to HPLC analysis as described below.
Microsome Incubations and Inhibition Studies
Samples of human liver were from the human liver bank
maintained by the Section on Clinical Pharmacology,
Imperial College School of Medicine, London. Local Eth-
ical Committee permission and coroner’s approval were
obtained to use the samples in the studies described here.
The hepatic microsomal fraction was isolated following
homogenisation of the samples in 0.25 M potassium phos-
phate buffer, pH 7.25 containing 1 mM EDTA and 0.15 M
KCl, by differential ultracentrifugation [21]. The microso-
mal pellet was resuspended and stored at Ϫ80° in 0.25 M
potassium phosphate buffer, pH 7.25 containing 30% v/v
glycerol. Assays were performed in a buffer containing 75
mM Tris–hydrochloride buffer pH 7.4, 3 mM magnesium
chloride, and 500 ␮g of microsomal protein and the drug.
Samples were preincubated for 5 min at 37° and the
reaction started by the addition of NADPH (final concen-
tration 1.2 mM). The reaction was stopped by the addition
of 1 mL cold acetonitrile. Controls were incubated either
without NADPH or without microsomes. Samples were
Data Analysis
Experiments were done at least in triplicate, and the statistical
significance of the data was analysed. Enzyme kinetic param-
eters were calculated by Lineweaver–Burk transformation.
Correlation matrix and stepwise regression analysis were per-
formed using a commercial statistical program (Statgraphics).
RESULTS
Metabolism of Diclofenac by Human Hepatocytes
Human hepatocytes were incubated with a non-cytotoxic
concentration of diclofenac (100 ␮M), and aliquots of

790
R. Bort et al.
FIG. 2. HPLC chromatogram of di-
clofenac and metabolites formed by
human hepatocytes. Diclofenac (100
␮M) was incubated with cultured
hepatocytes for 24 hr and the super-
natant analysed by HPLC/UV/MS
(method B; Materials and Methods).
The column effluent was monitored at
282 nm with a photodiode detector
and the identification achieved by UV
and mass spectra. In addition, a chro-
matogram at t ؍ 0 is shown in the
inset.
culture medium were withdrawn at regular time intervals
and analysed by HPLC/UV/MS. The chromatogram shown
in Fig. 2 is an analysis of a representative sample showing
the presence of several diclofenac-related metabolites in
culture medium. The peaks were identified on the basis of
their retention times, UV and MS, by comparison with
chemically synthesised probes. The major metabolite found
in the human hepatocyte culture was 4Ј-OHDic (RT: 11.0
min). Two minor monohydroxylated metabolites 5-OHDic
(RT: 14.6 min) and 3Ј-OHDic (RT: 12.9 min) were also
found. The peak eluting at 3.6 min showed the UV spectra
characteristics and the RT of chemically synthesised 4Ј,5-
diOHDic. Peaks at 25.5 and 34.0 min were identified as the
lactam of 4Ј-OHDic and 5-OHDic. A minor metabolite
found at RT 17.6 min showed a mass spectrum compatible
with a monohydroxylated derivative (m/z: 311 [mass peak],
293, 258, 230, and 194). The UV spectra and retention
time did not fit any of the synthesised monohydroxylated
derivatives of diclofenac. The peak at RT 10.4 min showed
a displacement of the UV absorption maximum to lower
values (268 nm) and spectra comparison with chemically
synthesised N,5-diOHDic confirmed the identity of the
peak. A peak with RT 24.9 min was detected in samples of
culture supernatant showing the following m/z features: 309
(mass peak), 265, 229, 201, 195, and 166; this fragmenta-
tion pattern and the UV spectra are in agreement with the
diclofenac iminoquinone described by Miyamoto et al. [11].
We did not observe the presence of 3Ј-OH-4Ј-OCH3-Dic
in the culture medium of human hepatocytes after 24-hr
incubation with diclofenac.
Diclofenac Metabolism by Human Liver Microsomes
Incubation of diclofenac with human microsomes yielded
only 4Ј-OHDic and 5-OHDic. The formation of both
metabolites followed a Michaelis–Menten kinetics (Fig. 3).
The following kinetic parameters were obtained: Km 9 ␮M
and Vmax 432 pmol/min/mg for 4Ј-hydroxylation; Km 43
␮M and Vmax 15.4 pmol/min/mg for 5-hydroxylation.
Incubation of 5-OHDic with human liver microsomes
showed that the formation of 4Ј,5-diOHDic observed in
hepatocyte cultures was subsequent to CYP oxidation of
5-OHDic (Km 15 ␮M and Vmax 96 pmol/min/mg). This
dihydroxylated metabolite was not formed when micro-
somes were incubated with 4Ј-OHDic. In microsome incu-
bations with 5-OHDic, it was also possible to detect the
formation of N,5-diOHDic. The rate of formation of the
hydroxylamine did not follow typical Michaelis–Menten
kinetics as it did not reach a plateau upon an increased
concentration of substrate, suggesting that N,5-diOHDic
could be partially formed non-enzymatically.
Identification of CYP Involved in the Oxidative
Metabolism of Diclofenac
In order to identify the CYP involved in the formation of
the oxidised metabolites, incubations were carried out with
a pool of microsomes from a human liver bank in the
presence of specific CYP inhibitors (Fig. 4). Incubation
with furafylline, a potent inhibitor of CYP1A2, [22] did not
show any effect on the reactions studied, suggesting that

Diclofenac Metabolism by Human Cytochrome P450
791
FIG. 3. Kinetic data of hydroxylation by human microsomes. 5-OHDic (1–100 ␮M) and diclofenac (5–200 ␮M) were incubated with
a pool of human liver microsomes for 15 and 60 min, respectively. The reaction was stopped by addition of 1 mL acetonitrile and
analysed by HPLC (method A; Materials and Methods).
CYP1A2 is not responsible for either 4Ј- or 5-hydroxyla-
tions. Similarly, an excess of coumarin, a well-known
specific substrate of CYP2A6 [23], failed to inhibit diclofe-
nac hydroxylation. The CYP2C9 specific inhibitor sulfa-
phenazole [24] clearly inhibited 4Јhydroxylation of diclofe-
nac and 5-OHDic, while 5-hydroxylation of diclofenac was
not affected even at high concentrations of this inhibitor
(no effect at 100 ␮M; data not shown). Interestingly,
quinidine, a substrate for CYP3A4 but also a potent
inhibitor of CYP2D6 [24], clearly failed to inhibit any
hydroxylation, which indicates that CYP2D6 is not likely
to be involved in the metabolism of diclofenac. Surpris-
ingly, an increased formation of 5-OHDic was observed.
Chlorzoxazone, a specific substrate for CYP2E1 [26], caused
no inhibition of diclofenac hydroxylation. Finally, the
CYP3A4/5 inhibitor troleandomycin [27] slightly inhibited
5-hydroxylation.
Incubation experiments were also conducted with micro-
FIG. 4. Effects of various substrates and inhibitors of cyto-
somes from a well-characterised liver bank. The rates of
metabolite formation were determined in 16 liver micro-
some preparations and correlated to specific P450 activities,
namely CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP2E1,
and CYP3A4. As shown in Table 1 and Fig. 5, there was a
good correlation between tolbutamide methylhydroxylase
(CYP2C9) activity and the formation of 4Ј-OH and 4Ј,5-
chrome P450 on diclofenac and 5-OHDic hydroxylations. Di-
clofenac (200 ␮M) and 5-OHDic (50 ␮M) were incubated with
a pool of five different human liver microsomes in the presence or
absence of two concentrations of various chemicals. Each bar
represents the mean of duplicate incubations with less than 10%
deviation. Forming rates of uninhibited samples for 4؅-OHDic
(black bar), 5-OHDic (white bar) and 4,5-diOHDic (dashed bar)
were 372, 12.2, and 101.4 pmol/min/mg protein, respectively.

792
R. Bort et al.
TABLE 1. Regression analysis between specific CYP activities and diclofenac metabolism
Simple regression analysis of metabolite formation
4؅-hydroxylation
of diclofenac
5-hydroxylation
of diclofenac
4؅-hydroxylation
of 5-OHDic
Specific reaction
CYP
Ethoxyresorufin-O-deethylation
Tolbutamide methylhydroxylation
S-Mephenytoin 4Ј-hydroxylation
Debrisoquine-4-hydroxylation
Chlorzoxazone-6-hydroxylation
Midazolam-1-hydroxylation
1A2
2C9
2C19
2D6
2E1
0.079
0.960*
0.327
Ϫ0.025
0.189
0.378
0.507
0.146
0.932*
0.288
0.040
0.185
0.384
0.673†
0.749*
0.193
0.127
0.624†
3A
Stepwise regression analysis of 5-OHDic formation
Std.
Error
Specific reaction
Coefficient
t‡
P
F‡
VIF‡
S-Mephenytoin 4Ј-hydroxylation
Tolbutamide methylhydroxylation
0.083
0.040
0.009
0.017
4.590
4.990
0.0004
0.0002
9.37
7.29
1.187
1.259
*P Ͻ 0.001.
†P Ͻ 0.01.
‡t, Student’s t-test; F, F-statistic; VIF, variance inflation factor.
diOHDic (R ϭ 0.960 and 0.932, respectively). A very poor
correlation was obtained in sample-to-sample regression in
the case of debrisoquine-4-hydroxylase (CYP2D6, R ϭ
Ϫ0.025 and 0.040). In accordance with the above-men-
tioned results with CYP inhibitors, there was a poor
correlation (R ϭ 0.378 and 0.384) with midazolam-1-
hydroxylation (CYP3A4/5) and CYP1A2 catalysed
ethoxyresorufin-O-dethylation (R ϭ 0.079 and 0.146). In
the case of 5-OHDic, there was not a clear outcome when
simple correlation analysis was performed with each CYP
activity assayed. The data from simple regression analysis
(Table 1) indicated, in order of decreasing regression
coefficient, that CYP2C19 (S-mephenytoin 4Ј-hydroxyla-
tion; R ϭ 0.749), CYP2C9 (tolbutamide methylhydroxyla-
tion; R ϭ 0.673), CYP3A4 (midazolam-1-hydroxylation;
R ϭ 0.624), and CYP1A2 (ethoxyresorufin-O-deethyla-
tion; R ϭ 0.507) could be partially involved in 5-OHDic
formation. Data was further analysed by stepwise multiple
regression analysis to better accommodate the experimental
results obtained for 5-OHDic formation. Previously, the
Kolmogorov–Smirnov test was performed to ensure the
normality of all the data sets; also, a variance inflation
factor below 1.5 guaranteed the absence of multicollinear-
ity. The stepwise regression analysis (Table 1) selected only
two activities corresponding to CYP2C19 and CYP2C9,
while the other two activities were rejected even under less
stringent conditions (F-to-enter Ͻ 4.0). The results of this
mathematical statistical analysis were highly significant
(P Ͻ 0.0001), with a multiple correlation coefficient
(R ϭ 0.969). The resulting algorithm: “diclofenac-5-hy-
droxylation ϭ 0.040 ϫ S-mephenytoin 4Ј-hydroxylation ϩ
0.083 ϫ tolbutamide methylhydroxylation”, accounted for
the activity observed in the individual microsome prepara-
tions with an accuracy of 93.8%.
Metabolism of Diclofenac by CYP-Expressing Cells
Diclofenac and 5-OHDic were incubated with genetically
engineered cells expressing different human cytochromes
(CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9,
CYP2C18, CYP2C19, CYP2D6, CYP2E1, and CYP3A4),
and the formation of diclofenac metabolites was measured
by HPLC. The results clearly confirmed that CYP2C9 was
responsible for the 4Ј-hydroxylation of diclofenac and
FIG. 5. Simple regression analysis of tolbutamide methylhy-
droxylation and the rates of formation of 4؅-OHDic and 4؅,5-
diOHDic. Diclofenac (200 ␮M) and 5-OHDic (50 ␮M) were
incubated with different human liver microsome samples. Form-
ing rates of 4؅-OHDic (black circles) and 4,5-diOHDic (black
squares) were determined by HPLC (method A; Materials and
Methods). Data are expressed as the means of duplicate incuba-
tions with less than 10% deviation.

Diclofenac Metabolism by Human Cytochrome P450
793
FIG. 6. Rate of diclofenac metabolism by CYP-engineered cell lines. Diclofenac or 5-hidroxydiclofenac were incubated at 300, 100, 30,
and 3 ␮M with CYP-engineered cells. After 24-hr incubation, cell supernatant was withdrawn and the content of 4؅-OHDic (A),
4؅,5-diOHDic (B), 5-OHDic (C), and 3؅-OHDic (D) analysed by HPLC (method A; Materials and Methods).
5-OHDic (Fig. 6, A and B, respectively). Preliminary
experiments on the 5-hydroxylation of diclofenac revealed
that as many as seven cytochromes were potentially in-
volved (Fig. 6C). To get a better estimation of the rele-
vance of each CYP in these hydroxylation pathways,
diclofenac was incubated at various concentrations (100,
30, and 3 ␮M). Analysis of the 5-hydroxylation rate
indicated that, despite the fact that CYP1A2, CYP2C9,
and CYP3A4 can 5-hydroxylate diclofenac at 300 ␮M,
CYP2B6, CYP2C8, CYP2C18, and CYP2C19 were the
isoenzymes able to do it more efficiently at a pharmacoki-
netically relevant concentration (3 ␮M) [28]. In the case of
3Ј-hydroxylation, only CYP2C9 was able to catalyse its
formation at any concentration of diclofenac (Fig. 6D).
To estimate the participation of the various CYP in the
5-hydroxylation in vivo, we compared the rates of formation
of the different diclofenac metabolites in CYP-engineered
cells and in 24-hrs cultured human hepatocytes. In parallel,
individual CYP activities were measured with specific
substrates in both cell systems, and the ratio activity in cell
line/activity in hepatocyte was determined (Table 2). Based
on this factor, we estimated the participation of each of the
individual CYP in the metabolism of diclofenac in the
human hepatocyte (Fig. 7). The results strongly suggest that
TABLE 2. Cytochrome P450 activities in 24-hr cultured human hepatocytes versus CYP-expressing THLE cells
CYP-expressing
Hepatocyte/CYP
CYP
Hepatocytes
THLE cells
cell ratio
Methoxyresorufin-O-deethylase*
Benzoxyresorufin-O-deethylase*
Taxol-6-hydroxylase†
Diclofenac-4Ј-hydroxylase†
S-Mephenytoin-4Ј-hydroxylase†
Testosterone-6␤-hydroxylase*
1A2
2B6
2C8
2C9
2C19
3A4
5.5 Ϯ 3.1
2.4 Ϯ 1.8
3.4 Ϯ 1.2
149 Ϯ 35
11.9 Ϯ 3.8
177 Ϯ 98
28.5 Ϯ 5.5
58.9 Ϯ 3.5
24 Ϯ 1
669 Ϯ 55
78 Ϯ 12
0.134
0.041
0.142
0.223
0.153
0.680
260.4 Ϯ 90.6
Activities are expressed in pmol/min/mg cellular protein.
*See Ref. 17.
†See Ref. 20.

794
R. Bort et al.
pathways of diclofenac were identified by the combined use
of human liver microsomes and CYP-engineered human-
derived cells. The inhibition and regression experiments
(Figs. 4 and 5 and Table 1) using human liver microsomes
suggested a predominant role of CYP2C9 in the hydroxy-
lation of diclofenac at the 4Ј position, clearly sustained by
the observations made with the CYP-expressing cell lines.
In the case of 4Ј-OHDic formation, the small contribution
of other members of the CYP2C subfamily (i.e. CYP2C8
and CYP2C19) as well as other CYP (CYP1A2 and
CYP2A6) was negligible compared to that of CYP2C9,
suggesting this isozyme plays the major role in vivo. The
experiments concerning the formation of 4Ј,5-diOHDic
clearly demonstrated that this metabolite was formed by the
CYP2C9-catalysed 4Ј-hydroxylation of 5-OHDic, and not
by 5-hydroxylation of 4Ј-OHDic (Fig. 8). The kinetics of
the 4Ј-hydroxylation of diclofenac, 5-OHDic, and diclofe-
nac analogues [16] are very similar, thus supporting a key
role of CYP2C9 in this reaction. The 3Ј-hydroxylation
pathway could not be properly investigated in microsomes
because of the negligible formation of this metabolite.
However, the studies with CYP-expressing cell lines
brought to light the involvement of CYP2C9 in its forma-
tion. Studies on the 5-hydroxylation of diclofenac by
microsomes were less convincing. Microsome studies using
chemical inhibitors were not clear; however, simple corre-
lation analysis with specific CYP activities identified
CYP2C9, CYP2C19, CYP3A4, and CYP1A2 as potentially
involved in 5-OHDic formation. The stepwise regression
analysis partially confirmed this hypothesis: it revealed that
CYP2C9 and CYP2C19 were involved, while the role of
CYP3A4 and CYP1A2 was not significant. Indeed, the
correlation coefficient value did not significantly improve
when these two activities were forced to enter the mathe-
matical model. The formation of 5-OHDic could be accu-
rately predicted by a linear combination of the two CYP
activities, S-mephenytoin 4Ј-hydroxylase and tolbutamide
methylhydroxylase.
The results were further expanded by the CYP-express-
ing cell line experiments. Although some showed a certain
capacity to metabolise diclofenac at 300 ␮M, only
CYP2C8, CYP2C18, CYP2C19, and CYP2B6 were able to
5-hydroxylate at pharmacokinetically relevant concentra-
tions of the drug (3 ␮M). The involvement of the tolbut-
amide methylhydroxylase activity predicted by the stepwise
regression analysis was further differentiated from the re-
sults obtained with the CYP-engineered cell lines. In
agreement with inhibition studies using human liver mi-
crosomes in which sulfaphenazole (specific inhibitor of
CYP2C9) did not cause any significant change in the
production of 5-OHDic, CYP-expressing cell lines con-
firmed the significant contribution of CYP2C8 at high or
low concentration of the drug, while CYP2C9 only partic-
ipated at concentrations between 100–300 ␮M. The ability
of CYP2C8, in addition to CYP2C9, to methylhydroxylate
tolbutamide [29] may explain this fact.
FIG. 7. Estimation of the contribution of the different CYP to
the metabolism of diclofenac in human hepatocytes. The per-
centage of contribution of each CYP to the in vivo formation of
5-OHDic was estimated by applying a factor that relates the
levels of individual CYP in the cell lines and hepatocytes to the
data shown in Fig. 6.
in the human liver CYP2C9 would be exclusively respon-
sible for the 4Ј-hydroxylation of diclofenac (participation
greater than 99.5%) as well as 5-OHDic (Ͼ97%), while the
isoforms CYP2C8, CYP2C19, CYP2C18, and CYP2B6 are
those expected to catalyse the formation of 5-OHDic.
CYP2C9 is the only isoform involved in the 3Ј-hydroxyla-
tion of diclofenac.
DISCUSSION
Diclofenac is metabolised in man by CYP-mediated oxida-
tion as well as conjugation with glucuronic acid. The major
metabolite found in plasma and urine of human volunteers
is 4Ј-OHDic [3]. In addition, several minor metabolites are
also present, whose origin could also be hepatic. Analysis by
HPLC/UV/MS of culture media of human hepatocytes
incubated with diclofenac revealed the existence of several
monohydroxylated metabolites, 4Ј-OHDic, 3Ј-OHDic and
5-OHDic, as well as the dihydroxylated derivatives 4Ј,5-
diOHDic and N,5-diOHDic (Fig. 1). Based on the exper-
iments with human liver microsomes (Fig. 3), it became
evident that the reduced formation of 5-OHDic by human
hepatocytes (and hence its reduced presence in human
urine) has a kinetic basis: the formation of 5-OHDic by
human microsomes has much less favourable kinetic pa-
rameters than that of 4Ј-OHDic (Vmax/Km relative ratio 48
vs 0.38). The formation of 3Ј-OHDic could not be detected
in microsome experiments but was detected in cultured
hepatocytes in relative abundance, similar to what is
observed in vivo. On the contrary, we could not detect the
formation of 3Ј-OH-4Ј-OCH3-Dic described by Faigle et al.
[4] in culture medium of human hepatocytes, even using an
MS-selected ion-monitoring mode. This would suggest a
non-hepatocyte formation of the metabolite, for instance
by the ubiquitous cathecol-O-methyltransferase. In culture
medium of hepatocytes, we could detect the presence of
N,5-diOHDic. This metabolite seems to be involved in the
toxicity exerted by diclofenac to hepatocytes [15].
The cytochromes involved in the different oxidative
By comparing the data on metabolic rates obtained with

Diclofenac Metabolism by Human Cytochrome P450
795
FIG. 8. Metabolic pathways of
diclofenac in human hepato-
cytes.
CYP-expressing cells and human hepatocytes, an estima-
tion of the degree of participation of each CYP in the
metabolism of diclofenac in the human liver can be made.
As suggested by Remmel et al. [30], one possible approach is
to determine the activity of each CYP using a specific
substrate in the cell line and hepatocytes (Table 2).
Knowing the rate of formation of a given metabolite by cell
line and the specific CYP activity, the rate of production by
the CYP present in the human hepatocyte can be inferred.
This parallelism was carried out for the various CYP
implicated in the formation of the different diclofenac
metabolites. Based on these estimations, CYP2C8 Ͼ
CYP2C19 Х CYP2C18 were identified as the enzymes that
would contribute most actively to the in vivo 5-hydroxyla-
tion of diclofenac (Figs. 7 and 8). Apart from the predom-
inant role of CYP2C9 in the metabolisation of diclofenac
to 4Ј-OHDic and 3Ј-OHDic, and indirectly to 4Ј,5-diOHDic,
the participation of the polymorphic CYP2C19 in the forma-
tion of 5-OHDic and, indirectly, N,5-diOHDic confers
CYP2C19 relevance in terms of explaining the variability of
the hepatotoxicity exerted by diclofenac in man.
The authors wish to thank J. Hidalgo for his help in the statistical
analysis and to Miss E. Belencho´n for her technical help in cell cultures.
This research was supported by the European Union (BMH4-CT96-
0254) and the Fondo de Investigaciones Sanitarias (97/1961). R. B.
was holder of a research fellowship from the Conseller´ıa de Cultura de
la Generalitat Valenciana.

796
R. Bort et al.
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Biochemical characterization and comparison with in vivo. In
Vitro Cell Dev Biol 26: 67–74, 1990.
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