Metabolism of 7-benzyloxy-4-trifluoromethylcoumarin by human hepatic cytochrome P450 isoforms

Article abstract of DOI:10.1080/00498250050200113

1. The metabolism of 7-benzyloxy-4-trifluoromethylcoumarin (BFC) to 7-hydroxy-4-trifluoromethylcoumarin (HFC) was studied in human liver microsomal preparations and in cDNA-expressed human cytochrome P450 (CYP) isoforms. 2. Kinetic analysis of the NADPH-dependent metabolism of BFC to HFC in four preparations of pooled human liver microsomes revealed mean (± SEM) K(m) and V(max) of 8.3 ± 1.3 μM and 454 ± 98 pmol/min/mg protein respectively. 3. The metabolism of BFC to HFC was determined in a characterized bank of 24 individual human liver microsomal preparations employing BFC substrate concentrations of 20 and 50 μM (i.e. about two and six times K(m) respectively). With 20 μM BFC the highest correlations were observed between BFC metabolism and markers of CYP1A2 (r² = 0.784-0.797) and then with CYP3A (r² = 0.434-0.547) isoforms, whereas with 50 μM BFC the highest correlations were observed between BFC metabolism and markers of CYP3A (r²= 0.679-0.837) and then with CYP1A2 (r² = 0.421-0.427) isoforms. At both BFC substrate concentrations, lower correlations were observed between BFC metabolism and enzymatic markers for CYP2A6, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP4A9/11. 4. Using human β-lymphoblastoid cell microsomes containing cDNA-expressed CYP isoforms, 20 μM BFC was metabolized by CYP1A2 and CYP3A4, with lower rates of metabolism being observed with CYP2C9 and CYP2C19. Kinetic studies with the CYP1A2 and CYP3A4 preparations demonstrated a lower K(m) with the CYP1A2 preparation, but a higher V(max) with the CYP3A4 preparation. 5. The metabolism of 20 μM BFC in human liver microsomes was inhibited to 37-48 % of control by 5-100 μM of the mechanism-based CYP1A2 inhibitor furafylline and to 64-69 % of control by 5-100 μM of the mechanism-based CYP3A4 inhibitor troleandomycin. While some inhibition of BFC metabolism was observed in the presence of 100 and 200 μM diethyldithiocarbamate, the addition of 2-50 μM sulphaphenazole, 50-500 μM S-mephenytoin and 2-50 μM quinidine had little effect. 6. The metabolism of 20 μM BFC to HFC in human liver microsomes was also inhibited by an antibody to CYP3A4, whereas antibodies to CYP2C8/9 and CYP2D6 had no effect. 7. In summary, by correlation analysis, use of cDNA-expressed CYP isoforms, chemical inhibition and inhibitory antibodies, BFC appears metabolized by a number of CYP isoforms in human liver. BFC metabolism appears to be primarily catalysed by CYP1A2 and CYP3A4, with possibly some contribution by CYP2C9, CYP2C19 and perhaps other CYP isoforms. 8. The results also demonstrate the importance of the selection of an appropriate substrate concentration when conducting reaction phenotyping studies with human hepatic CYP isoforms.

Full text of DOI:10.1080/00498250050200113

xenobiotica, 2000, vol. 30, no. 10, 955±969
Metabolism of 7-benzyloxy-4-tri¯uoromethyl-
coumarin by human hepatic cytochrome P450
isoforms
Œ
A. B. RENWICK , D. SURRY , R. J. PRICE ,
‹
‹

‹
B. G. LAKE * and D. C. EVANS
‹
Œ
TNO BIBRA International Ltd, Carshalton SM5 4DS, UK
Merck Sharp and Dohme Ltd, Neuroscience Research Centre, Terlings Park,
Harlow CM20 2QR, UK

Merck and Co., Inc., 126 East Lincoln Avenue, Rahway, NJ 08535, USA
Received 14 April 2000
1. The metabolism of 7-benzyloxy-4-tri¯uoromethylcoumarin (BFC) to 7-hydroxy-4-
tri¯uoromethylcoumarin (HFC) was studied in human liver microsomal preparations and
in cDNA-expressed human cytochrome P450 (CYP) isoforms.
2. Kinetic analysis of the NADPH-dependent metabolism of BFC to HFC in four
of
³
preparations of pooled human liver microsomes revealed mean ( SEM) K and V
m
max
³
8.3 1.3 lm and 454 98 pmol min mg protein respectively.
³
}
}
3. The metabolism of BFC to HFC was determined in a characterized bank of 24
individual human liver microsomal preparations employing BFC substrate concentrations
of 20 and 50 lm (i.e. about two and six times K respectively). With 20 lm BFC the highest
m
#
correlations were observed between BFC metabolism and markers of CYP1A2 (r
#
¯
0.434±0.547) isoforms, whereas with 50 lm BFC
0.784±0.797) and then with CYP3A (r
the highest correlations were observed between BFC metabolism and markers of CYP3A
¯
#
#
(r
0.679±0.837) and then with CYP1A2 (r
0.421±0.427) isoforms. At both BFC
¯
¯
substrate concentrations, lower correlations were observed between BFC metabolism and
enzymatic markers for CYP2A6, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and
}
CYP4A9 11.
4. Using human b-lymphoblastoid cell microsomes containing cDNA-expressed CYP
isoforms, 20 lm BFC was metabolized by CYP1A2 and CYP3A4, with lower rates of
metabolism being observed with CYP2C9 and CYP2C19. Kinetic studies with the
CYP1A2 and CYP3A4 preparations demonstrated a lower K with the CYP1A2
m
preparation, but a higher V
with the CYP3A4 preparation.
max
5. The metabolism of 20 lm BFC in human liver microsomes was inhibited to 37±48%
of control by 5±100 lm of the mechanism-based CYP1A2 inhibitor furafylline and to
64±69% of control by 5±100 lm of the mechanism-based CYP3A4 inhibitor troleando-
mycin. While some inhibition of BFC metabolism was observed in the presence of 100 and
200 lm diethyldithiocarbamate, the addition of 2±50 lm sulphaphenazole, 50±500 lm S-
mephenytoin and 2±50 lm quinidine had little eåect.
6. The metabolism of 20 lm BFC to HFC in human liver microsomes was also
}
inhibited by an antibody to CYP3A4, whereas antibodies to CYP2C8 9 and CYP2D6 had
no eåect.
7. In summary, by correlation analysis, use of cDNA-expressed CYP isoforms,
chemical inhibition and inhibitory antibodies, BFC appears metabolized by a number of
CYP isoforms in human liver. BFC metabolism appears to be primarily catalysed by
CYP1A2 and CYP3A4, with possibly some contribution by CYP2C9, CYP2C19 and
perhaps other CYP isoforms.
8. The results also demonstrate the importance of the selection of an appropriate
substrate concentration when conducting reaction phenotyping studies with human hepatic
CYP isoforms.
!
*Author for correspondence; e-mail: blake bibra.co.uk
}
}}
’
http: www.tandf.co.uk journals
Xenobiotica ISSN 0049-8254 print ISSN 1366-5928 online
}
2000 Taylor & Francis Ltd

956
A. B. Renwick et al.
Introduction
The cytochrome P450 (CYP) superfamily of isoforms is known to have a major
role in the oxidative metabolism of xenobiotics and certain endogenous compounds
including fatty acids, steroids and vitamins (Parkinson 1996a, b). In human liver
microsomes members of the CYP1A, CYP2A, CYP2B, CYP2C, CYP2D, CYP2E
and CYP3A subfamilies have been shown to metabolize a wide variety of drugs
(Wrighton et al. 1993a, 1995, Nelson et al. 1996, Parkinson 1996a, b, Pelkonen et al.
1998). Knowledge of the CYP isoforms responsible for the metabolism of a new
therapeutic agent is required for a number of reasons. For example, such information
can help predict likely drug interactions and identify whether some individuals will
be poor metabolizers of the drug as a consequence of known genetic polymorphisms
(Parkinson 1996a, b, Pelkonen et al. 1998).
In recent years extensive use has been made of in vitro techniques employing
human liver microsomes and cDNA-expressed human liver CYP isoforms
(Wrighton et al. 1993a, 1995, Crespi et al. 1997, 1998, Clarke 1998, Pelkonen et al.
1998, Rodrigues 1999). Using these systems, it is possible to identify which CYP
isoforms will be involved in the metabolism of a new chemical entity and from CYP
isoform inhibition studies to obtain information on the potential for in vivo drug
interactions. One requirement for conducting such studies is the availability of
suitable probe substrates for the CYP isoforms of interest. For example, rapid
screening for CYP isoform inhibition may be performed in a 96-well plate format
using either liver microsomes or cDNA-expressed CYP isoforms and substrates that
are metabolized to highly ¯uorescent products (Crespi et al. 1997, 1998, 1999).
Recently, the present authors have been developing some more rapid procedures
for the determination of CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and
CYP3A4 isoform enzyme activities in human liver microsomes. As part of this work,
the metabolism of 7-benzyloxy-4-tri¯uoromethylcoumarin (BFC) has been
examined using a 96-well plate format coupled to ¯uorimetric quantitation of the 7-
hydroxy-4-tri¯uoromethylcoumarin (HFC) product. Using a bank of characterized
#
human liver microsomes, good correlations (r
0.77±0.84) were observed between
¯
BFC metabolism and the metabolism of the CYP3A isoform substrates testosterone
and midazolam (Surry and Evans, unpublished data). Others have reported that
BFC is a selective substrate for cDNA-expressed CYP3A4 (Crespi et al. 1999). The
availability of selective ¯uorescent CYP3A4 substrates would be very useful for
rapid CYP isoform metabolism and inhibition screening studies. Currently, many
substrates used as CYP3A4 probes are not readily amenable for use in rapid screens
as the assays often involve chromatographic separation of the metabolites before
quantitation by spectroscopic, radiometric or mass spectrometry techniques. One
exception appears to be 3-(3,4-di¯uorobenzyloxy)-5,5-dimethyl-4-(4-methyl-
sulphonylphenyl)-5H-furan-2-one (DFB), which has been reported to be a speci®c
}
¯uorescent probe for CYP3A4 5 in human liver microsomes (Chauret et al. 1999).
The objective of this study was to identify which CYP isoforms are responsible
for BFC metabolism in human liver in order to ascertain if BFC is a speci®c
substrate for CYP3A isoforms. CYP isoform `reaction phenotyping’ (Parkinson
1996a, b, Rodrigues 1999) was performed by correlation analysis, the use of cDNA-
expressed human CYP isoforms, chemical inhibition and inhibitory antibodies.

BFC metabolism by human CYP isoforms
Materials and methods
Materials
957
NADPH, Tris, diethyldithiocarbamate, quinidine, sulphaphenazole and troleandomycin were
obtained from Sigma-Aldrich Co. Ltd (Poole, Dorset, UK) and BFC, HFC, furafylline and S(-)-
mephenytoin from Salford Ultra®ne Chemicals and Research Ltd. (Manchester, UK). Microsomes from
human b-lymphoblastoid cells containing cDNA-expressed human CYP isoforms (Gentest Corp.,
Woburn, MA, USA) were obtained from Cambridge Bioscience (Cambridge, UK) and stored at ±80 °C.
The samples of human b-lymphoblastoid cell microsomes comprised control cell microsomes (i.e. no
transfected human CYP isoform cDNA but contains native CYP1A1 activity) and cell microsomes
containing CYP1A2, CYP2A6-OR (i.e. CYP2A6 cDNA plus human NADPH-cytochrome P450
reductase (OR) cDNA), CYP2B6, CYP2C8-OR, CYP2C9-OR, CYP2C19, CYP2D6-OR,
}
CYP2E1-OR and CYP3A4-OR. Inhibitory antibodies against CYP2C8 9, CYP2D6 and CYP3A4
were provided by MSD Research Laboratories (Harlow, Essex, UK) and stored at
speci®city and use of the CYP3A4 antibody has been recently described (Mei et al. 1999).
80 °C. The
®
Human liver microsomes
Samples of human liver (surplus to transplant requirements) were transported on ice to TNO BIBRA
from other institutions and were stored at ±80 °C. Washed microsomal fractions were prepared in 0.154 m
KCl containing 50 mm Tris-HCl pH 7.4 as described previously (Lake, 1987). Liver microsomal
fractions were assayed for protein (Lowry et al. 1951) employing bovine serum albumin as standard. The
}
washed microsomal fractions were diluted to 10 mg protein ml and aliquots stored at ±80 °C. Four
separate batches, designated pools A, B, C and D, of pooled human liver microsomes were prepared.
Each batch was prepared by pooling liver samples from ®ve subjects. Pool A comprised liver samples
from males aged 15, 29 and 44 years and females aged 12 and 42 years; pool B comprised liver samples
from males aged 26, 29 and 41 years and females aged 14 and 42 years; pool C comprised liver samples
from male subjects aged 15, 17 and 29 years and female subjects aged 31 and 42 years and pool D
comprised liver samples from male subjects aged 2.5 and 58 years and female subjects aged 14, 59 and 74
years. Studies were also conducted with a bank of 24 individual human liver microsomal preparations that
had been previously characterized for total CYP content and CYP isoform enzyme activities (Renwick et
al. 1998).
Metabolism of BFC by human liver microsomes
Incubations were performed in Nunc MicroWell (Life Technologies Ltd., Paisley, Scotland, UK)
TM
polystyrene 96-well plates with conical wells. The NADPH-dependent metabolism of BFC was studied
in incubation mixtures containing 2±100 lm BFC, 2±50 lg of microsomal protein and 70 mm phosphate
buåer pH 7.4 in a ®nal volume of 100 ll. Stock solutions of BFC were prepared in methanol and diluted
1 in 300 with 100 mm phosphate buåer pH 7.4 so that the ®nal methanol concentration in the incubations
}
was 0.2% (v v). For each BFC substrateconcentration, microsomal protein concentration and incubation
time, 6 wells were incubated with NADPH (tests) and 2 wells without NADPH (blanks). After mixing,
the plates were pre-incubated for 10 min in a 37 °C incubator and the reaction then initiated by the
addition of 1 mm (®nal concentration) NADPH. Incubations were performed for 5±30 min at 37 °C and
}
were terminated by the addition of 100 ll of Tris base in 60% (v v) acetonitrile. The plates were
centrifuged at 260 g for 10 min at 4 °C and a 120 ll aliquot removed from each well into a white Porvair
96-well plate (Perkin Elmer Ltd., Beacons®eld, Bucks, UK). All ¯uorescence measurements were
performed with a Perkin Elmer model LS-50B luminescence spectrophotometer equipped with a Perkin-
Elmer model LS50 plate reader. Fluorescence measurements were made at excitation and emission
wavelengths of 410 and 510 nm respectively, employing 3 nm slit widths. Standard curves were
}
constructed with 0±50 pmol HFC well.
BFC metabolism correlation analysis
Incubations were conducted as described above with either 20 or 50 lm BFC and 10 lg microsomal
protein for 15 min. For each of the 24 preparations of human liver microsomes 6 wells were incubated
with NADPH and 2 wells (blanks) without NADPH.
BFC metabolism by cDNA-expressed CYP isoforms
For each of the b-lymphoblastoid cell cDNA-expressed CYP isoform preparations and each
incubation time, 4 wells were incubated with NADPH and 1 well (blank) without NADPH. Incubation
}
mixtures contained 20 lm BFC (added in 0.2% (v v) methanol), 1 mm NADPH and 100 mm phosphate
buåer pH 7.4 in a ®nal volume of 100 ll. After a 10 min pre-incubation at 37 °C, the reaction was initiated
with 100 lg of b-lymphoblastoid cell microsomal protein (equivalent to 2.3±25.5 pmol of each CYP
isoform) and incubations terminated after 10 and 40 min. A direct ¯uorescence monitoring procedure
was used for the kinetic studies with the cDNA-expressed CYP1A2 and CYP3A4 preparations. The

958
A. B. Renwick et al.
Figure 1. Michaelis±Menten plots of BFC metabolism to HFC in four pooled human liver microsomal
preparations. Incubations (n 6 wells for each BFC substrate concentration) were performed
¯
}
with 10 lg protein well of the pool A (D), B (E), C (*) and D (+) microsomal preparations with
2±100 lm BFC for 15 min.
incubations were performed in a Molecular-Devices (Wokingham, UK) Gemini Fluorescence Plate
Reader with readings being made every 45 seconds at excitation and emission wavelengths of 430 and
538 nm respectively. Following each ¯uorescence reading the plates were agitated for 3 seconds.
Incubation mixtures contained 0.24±250 lm BFC (added in 5 ll of acetonitrile), 8.9±9.9 pmol CYP
isoform in Dulbecco’s phosphate buåered saline pH 7.4 in a ®nal volume of 250 ll. For each of the CYP
isoform preparations and BFC substrate concentration 2 wells were incubated with NADPH and 2 wells
(blanks) without NADPH. After a 10 min pre-incubation at 37 °C the reaction was initiated by the
addition of 2 mm (®nal concentration) NADPH. Incubations were performed at 37 °C for 15 min and the
rate determined over the linear portion of the reaction curve. Standard curves were constructed with
}
0±100 pmol HFC well.
BFC metabolism inhibition studies
Incubations were conducted as described above with 20 lm BFC, 10 lg human liver microsomal
protein and 70 mm phosphate buåer pH 7.4 for 15 min. For each inhibitor concentration 6 wells were
incubated with NADPH and 2 wells (blanks) without NADPH. To study the eåect of the mechanism-
based inhibitors furafylline (CYP1A2), diethyldithiocarbamate (CYP2E1) and troleandomycin
(CYP3A4), the compounds were pre-incubated for 30 min at 37 °C with 1 mm NADPH and liver
microsomes before the addition of BFC and a further aliquot of 1 mm NADPH. In the studies with the
inhibitors sulphaphenazole (CYP2C9) and quinidine (CYP2D6) and the substrate S-mephenytoin
(CYP2C19), the compounds were pre-incubated with liver microsomes for 10 min at 37 °C before the
joint addition of BFC and 1 mm NADPH. Stock solutions of the inhibitors and S-mephenytoin were
prepared in dimethyl sulphoxide and added to give a ®nal solvent concentration in the incubation
}
}
mixtures of 0.25% (v v). The eåects of antibodies to CYP2C8 9, CYP2D6 and CYP3A4 were studied
in incubation mixtures containing 20 lm BFC, 1 mm NADPH, 10 lg human liver microsomal protein,
0.005, 0.05 or 0.5 ll of each antibody and 70 mm phosphate buåer pH 7.4 for 15 min. For each
concentration of each inhibitor 6 wells were incubated with NADPH and 2 wells (blanks) without
NADPH. The antibodies were pre-incubated with the liver microsomes and buåer for 10 min at 37 °C
before the joint addition of BFC and NADPH.
Results
Kinetics of BFC metabolism
Initial BFC metabolism studies were conducted with a substrate concentration
of 20 lm. The pool D human liver microsomal preparation was used as this

BFC metabolism by human CYP isoforms
959
Figure 2. Variability in the metabolism of 20 (A) and 50 (B) lm BFC to HFC in a panel of 24 individual
human liver microsomal preparations. Incubations (n 6 wells for each microsomal preparation)
¯
}
were performed with 10 lg protein well for 15 min.
preparation was found to have the highest rate of BFC metabolism. BFC was
metabolized by human liver microsomes in the presence of NADPH to HFC. The
formation of HFC was linear with respect to incubation time for at least 15 min and
}
microsomal protein concentration up to at least 15 lg well (data not shown).
Using all four pooled human liver microsomal preparations, the metabolism of
BFC was investigated over a substrate concentration range of 2±100 lm (®gure 1).
Kinetic analysis of Michaelis±Menten plots revealed K of 6.7, 6.0, 8.7 and 11.8 lm
m
for the pool A, B, C and D human liver microsomal preparations respectively (®gure
} }
were 253, 498, 359 and 705 pmol min mg protein
respectively. For the four pooled human liver microsomal preparations mean
1). Corresponding V
max
³
respectively.
³ ³ } }
were 8.3 1.3 lm and 454 98 pmol min mg protein
max
(
SEM) K and V
m

960
A. B. Renwick et al.
Table 1. Correlation analysis for BFC metabolism to HFC with various CYP isoform-dependent
enzyme activities in human liver microsomes.
#
r
a
CYP isoform
Enzyme activity
20 lm BFC 50 lm BFC
±
1A2
Total CYP
0.460
0.797
0.784
0.012
0.085
0.336
0.330
0.078
0.036
0.510
0.497
0.484
0.547
0.434
0.272
0.632
0.421
0.427
0.033
0.207
0.401
0.267
0.092
0.114
0.814
0.786
0.760
0.837
0.679
0.208
7-ethoxyresoru®n O-deethylase
caåeine N3-demethylase
coumarin 7-hydroxylase
2A6
2B6
2C9
2C19
2D6
2E1
S-mephenytoin N-demethylase
tolbutamide methylhydroxylase
S-mephenytoin 4«-hydroxylase
dextromethorphan O-demethylase
chlorzoxazone 6-hydroxylase
testosterone 6b-hydroxylase
testosterone 2b-hydroxylase
testosterone 15b-hydroxylase
diazepam 3-hydroxylase
}
3A4 5
caåeine 8-hydroxylase
lauric acid 12-hydroxylase
}
4A9 11
a
Total CYP content and the CYP isoform enzyme activities shown were
correlated with data obtained from the metabolism of either 20 or 50 lm BFC
(®gure 2) in a panel of 24 individual human liver microsomal preparations.
BFC metabolism correlation analysis
The metabolism of BFC was examined with a characterized bank of 24 human
liver microsomal preparations employing substrate concentrations of 20 and 50 lm
(i.e. approximately two and six times K respectively). BFC was metabolized to
m
HFC by all 24 liver microsomal preparations examined (®gure 2). Microsomal BFC
}
}
metabolism varied from 9 to 702 and from 35 to 1924 pmol min mg protein at
substrate concentrations of 20 and 50 lM respectively.
The rates of BFC metabolism were correlated with obtained for total CYP
content and a range of CYP isoform enzyme activities (table 1). With 20 lm
#
substrate the highest correlations (r
0.797 and 0.784) were observed between
¯
BFC metabolism and 7-ethoxyresoru®n O-deethylase (®gure 3A) and caåeine N3-
demethylase activities. Lower correlations were observed between BFC metabolism
and testosterone 6b-hydroxylase (®gure 3B) and other CYP3A isoform enzyme
#
activities (r
0.434±0.547). In contrast, with 50 lm BFC substrate the highest
¯
correlations were with testosterone 6b-hydroxylase (®gure 3D) and other CYP3A
#
isoform enzyme activities (r
between BFC metabolism and 7-ethoxyresoru®n O-deethylase (®gure 3C; r
0.679±0.837). Lower correlations were observed
#
¯
¯
#
0.421) and caåeine N3-demethylase (r
0.427) activities.
¯
Metabolism of BFC by cDNA-expressed CYP isoforms
In initial studies the metabolism of 20 lm BFC was examined in Gentest b-
lymphoblastoid cell microsomes containing human cDNA-expressed CYP1A2,
CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and
CYP3A4, together with microsomes from control (wild type) cells. High rates of
BFC metabolism to HFC were observed with the cDNA-expressed CYP1A2 and
CYP3A4 preparations (®gure 4). BFC was also metabolized to HFC by the cDNA-
expressed CYP2C9 and CYP2C19 preparations, whereas a low rate of BFC

BFC metabolism by human CYP isoforms
961
Figure 3. Correlation between BFC metabolism to HFC and 7-ethoxyresoru®n O-deethylase (A, C)
and testosterone 6b-hydroxylase (B, D) enzyme activities in a characterized panel of 24 human
liver microsomal preparations. Incubations (n 6 wells for each microsomal preparation) were
¯
performed with both 20 (A, B) and 50 (C, D) lm BFC substrate concentrations.
metabolism was observed with the CYP2B6 preparation. Only trace amounts of
BFC metabolism was observed with the CYP2A6, CYP2C8, CYP2D6 and CYP2E1
preparations (®gure 4) and with the control b-lymphoblastoid cell preparation (data
not shown).
The kinetics of BFC metabolism were investigated over a substrate concentration
range of 0.24±250 lm using b-lymphoblastoid cell microsomes containing cDNA-
expressed CYP1A2 and CYP3A4 (®gure 5). For the cDNA-expressed CYP1A2 and
CYP3A4 preparations, K were 4.1 and 26.0 lM respectively, with corresponding
V
m
} }
of 11.1 and 61.2 pmol min pmol CYP isoform respectively.
max

962
A. B. Renwick et al.
Figure 4. Metabolism of 20 lm BFC by b-lymphoblastoid cell microsomes containing cDNA-expressed
human CYP isoforms. Incubations (n 4 wells for each microsomal preparation) were performed
¯
with 100 lg protein well for 10 and 40 min.
}
Figure 5. Kinetics of metabolism of 0.24±250 lm BFC by b-lymphoblastoid cell microsomes containing
cDNA-expressed CYP1A2 (E) and CYP3A4 (_). Incubations (n 2 wells for each BFC
¯
substrate concentration) were performed with 9.9 and 8.9 pmol CYP well respectively for 15 min.
}
Inhibition of BFC metabolism
The eåect of some known CYP inhibitors and one substrate on the metabolism
of 20 lm BFC in human liver microsomes (pool D) was studied. For the mechanism-
based inhibitors (Newton et al. 1995) furafylline, diethyldithiocarbamate and
troleandomycin, the compounds were pre-incubated with NADPH and the liver
microsomes before addition of BFC. Treatment with 5±100 lm furafylline and
5±100 lm troleandomycin inhibited BFC metabolism to 37±48 and 64±69% of
control respectively (®gure 6A). While 10±50 lm diethyldithiocarbamate had little
eåect on BFC metabolism, the addition of 100 and 200 lm diethyldithiocarbamate

BFC metabolism by human CYP isoforms
963
Figure 6. Inhibition of 20 lm BFC metabolism to HFC in human liver microsomes. To study the
eåects of 5±100 lm furafylline, 10±200 lm diethyldithiocarbamate and 5±100 lm troleandomycin
(A), but not 2±50 lm sulphaphenazole, 50±500 lm S-mephenytoin and 2±50 lm quinidine (B), the
compounds were pre-incubated with liver microsomes and NADPH at 37 °C before addition of
BFC. Incubations (n 6 wells for each inhibitor concentration) were performed with 10 lg
¯
}
protein well of the pool D human liver microsomal preparation for 15 min and the results
expressed as percentage of control (no inhibitor added) incubations. Control rates of metabolism
}
}
of BFC to HFC were 345 and 424 pmol min mg protein for the mechanism-based inhibitors
(A) and the other compounds (B) respectively.
inhibited enzyme activity to 67 and 43% of control respectively (®gure 6A). In
contrast, treatment with 2±50 lm sulphaphenazole, 50±500 lm S-mephenytoin and
2±50 lm quinidine had little eåect on the metabolism of BFC in human liver
microsomes (®gure 6B).
Also examined were the eåects of three available inhibitory antibodies on BFC
metabolism in human liver microsomes. Using two pooled human liver microsomal
}
preparations (pools C and D) treatment with either 0.005, 0.05 or 0.5 ll well of
inhibitory antibodies to CYP2C8 9 and CYP2D6 had no eåect on BFC metabolism
}
}
(®gure 7A and B). In contrast, the addition of 0.005, 0.05 or 0.5 ll well (i.e. 0.005,

964
A. B. Renwick et al.
Figure 7. Inhibition of 20 lm BFC metabolism to HFC in human liver microsomes by antibodies to
}
}
CYP2C8 9, CYP2D6 and CYP3A4. The antibodies (0.005, 0.05, 0.5 ll well) were pre-incubated
with the pool C (A) and pool D (B) human liver microsomal preparations at 37 °C before addition
of NADPH and BFC. Incubations (n 6 wells for each inhibitory antibody concentration) were
}
¯
performed with 10 lg microsomal protein well for 15 min and the results are expressed as
percentage of control (no antibody added) incubations. Control rates of BFC metabolism to HFC
by the pool C (A) and pool D (B) human liver microsomal preparations were 341 and
}
677 pmol min mg protein respectively.
}
}
0.05 and 0.5 ll 100 ll incubation mixture) of an inhibitory antibody to CYP3A4
reduced BFC metabolism to 68, 53 and 50% of control (mean of both human liver
microsomal preparations) respectively (®gure 7A and B). Additional studies with
the pool D microsomal preparation demonstrated that tolbutamide methyl-

BFC metabolism by human CYP isoforms
965
hydroxylase activity was inhibited to 66 and 25% of control by the addition of 0.05
}
and 0.5 ll 100 ll incubation mixture respectively of the CYP2C8 9 antibody (data
not shown). In contrast, no inhibition of tolbutamide methylhydroxylase activity
}
}
was observed by the addition of 0.01±1.0 ll 100 ll incubation mixture of the
CYP3A4 antibody (data not shown).
Discussion
In this study the metabolism of BFC to HFC by human liver microsomal
preparations and cDNA-expressed CYP isoforms has been investigated. BFC
metabolism was observed in all 24 individual and the four pooled human liver
microsomal preparations examined. Previous studies have demonstrated that other
HFC derivatives are also substrates for human hepatic CYP isoforms. For example,
7-methoxy-4-tri¯uoromethylcoumarin has been reported as a good substrate for
cDNA-expressed CYP2C9 (Crespi et al. 1999) and 7-ethoxy-4-tri¯uoro-
methylcoumarin is metabolized by a number of CYP isoforms including CYP1A2,
CYP2B6, CYP2C9 and CYP2E1 (Buters et al. 1993, Code et al. 1997).
Investigations into the kinetics of BFC metabolism in human liver microsomes
³
revealed a mean ( SEM, n 4) K
14.2 3.4 lm (n 3) observed in the present authors’ previous investigations (Surry
³
8.3 1.3 lm. This is similar to the
¯
¯
m
³
¯
and Evans, unpublished data). Higher rates of BFC metabolism (and hence V
max
values) were observed when the 96-well plates were agitated during the incubations
(Surry and Evans, unpublished data). In the present investigations, with the
exception of the kinetic studies with cDNA-expressed CYP1A2 and CYP3A4, a
static incubation system was employed.
In this study `reaction phenotyping’ for BFC metabolism was performed by
correlation analysis, the use of cDNA-expressed CYP isoforms, chemical inhibition
and three available inhibitory antibodies. BFC metabolism correlation analysis was
conducted with substrate concentrations of 20 and 50 lm (i.e. about two and six
times K respectively). At a substrate concentration of 20 lm, high correlations
#
m
0.797 and 0.784) were observed between BFC metabolism and the activities of
(r
¯
7-ethoxyresoru®n O-deethylase and caåeine N3-demethylase (table 1). These two
enzyme activities are considered to be catalysed by CYP1A2 in human liver (Fuhr
et al. 1992, Wrighton et al. 1993a, Tassaneeyakul et al. 1994, Parkinson 1996a, b,
#
Pelkonen et al. 1998). Some correlations (r
0.434±0.547) were also observed
¯
between BFC metabolism and testosterone 2b-, 6b- and 15b-hydroxylases, diazepam
3-hydroxylase and caåeine 8-hydroxylase activities. These enzyme activities are
considered to be catalysed by CYP3A isoforms in human liver (Tassaneeyakul et al.
1994, Parkinson 1996a, b, Yang et al. 1999). Lower correlations were observed
#
between BFC metabolism and tolbutamide methylhydroxylase (r
0.336) and S-
¯
#
mephenytoin 4«-hydroxylase (r
by CYP2C9 and CYP2C19 respectively (Wrighton et al. 1993b, Parkinson 1996a, b,
0.330) activities, which are known to be catalysed
¯
#
Miners and Birkett 1998). No correlations (r
0.012±0.085) were observed
¯
between BFC metabolism and coumarin 7-hydroxylase, S-mephenytoin
N-demethylase, dextromethorphan O-demethylase and chlorzoxazone 6-hydrox-
ylase activities, which are catalysed by CYP2A6, CYP2B6, CYP2D6 and CYP2E1
respectively in the human liver (Wrighton et al. 1993a, Heyn et al. 1996, Pelkonen
et al. 1998).

966
A. B. Renwick et al.
However, with 50 lm BFC substrate a diåerent pattern of correlations was
#
apparent (table 1). The highest correlations (r 0.679±0.837) were obtained
between BFC metabolism and the CYP3A isoform markers and some correlation
¯
#
(r
0.632) was also observed with total CYP content. This latter observation may
¯
be attributable to CYP3A isoforms being relatively abundant in the human liver
(Wrighton et al. 1993, Lecoeur et al. 1994, Shimada et al. 1994, Rodrigues 1999).
The good correlations with CYP3A isoform enzyme activities are in agreement with
previous investigations (Surry and Evans, unpublished data) where a BFC substrate
concentration of 50 lm was also used. Weaker correlations were obtained with the
#
#
enzymatic markers of CYP1A2 (r
0.421±0.427) and CYP2C9 (r
0.401). The
¯
¯
higher aænity of BFC for CYP1A2 and CYP3A4 at low and high substrate
concentrations respectively is contrasted in ®gure 3 with data for 7-ethoxyresoru®n
O-deethylase and testosterone 6b-hydroxylase activities.
The eåect of chemical inhibitors and inhibitory antibodies on BFC metabolism
in human liver microsomes was also investigated. Previous work has demonstrated
that furafylline, sulphaphenazole, S-mephenytoin, quinidine, diethyldithio-
carbamate and troleandomycin may be considered as either inhibitors or substrates
of human hepatic CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A4
respectively (Wrighton et al. 1993a, Chang et al. 1994, Clarke et al. 1994, Baldwin
et al. 1995, Newton et al. 1995, Bourrie et al. 1996, Ono et al. 1996, Parkinson 1996a,
b, Rodrigues 1999). BFC metabolism (20 lm substrate) to HFC was inhibited to
37±48% of control by 5±100 lm furafylline and to 64±69% of control by 5±100 lm
troleandomycin. Other work has demonstrated that at the concentrations examined
furafylline and troleandomycin may be considered relatively speci®c mechanism-
based inhibitors of CYP1A2 and CYP3A4 respectively (Newton et al. 1995).
However, both furafylline and troleandomycin only inhibited a fraction of the total
metabolism of BFC (®gure 6A), suggesting that CYP1A2, CYP3A4 and possibly
other CYP isoforms can metabolize BFC in human liver microsomes. The inhibition
of BFC metabolism by high concentrations of diethyldithiocarbamate (®gure 6A) is
unlikely to re¯ect metabolism by CYP2E1, but rather that this compound also
inhibits other CYP isoforms (Chang et al. 1994, Ono et al. 1996). The involvement
of CYP3A4 in BFC metabolism was also demonstrated by the studies with the three
available inhibitory antibodies. At the concentrations examined the CYP3A4
antibody inhibited a fraction of total BFC metabolism in human liver microsomes,
}
whereas the CYP2C8 9 and CYP2D6 antibodies were without any eåect (®gure 5).
The metabolism of BFC by cDNA-expressed CYP isoforms was also examined.
At a substrate concentration of 20 lm, the highest rates of BFC metabolism were
observed with cDNA-expressed CYP1A2 and CYP3A4, then with CYP2C9 and
CYP2C19, with a low level of BFC metabolism being catalysed by CYP2B6. Only
trace amounts of BFC metabolism were observed with the other cDNA-expressed
CYP isoforms examined and with the control b-lymphoblastoid cell microsomes.
Kinetic studies with cDNA-expressed CYP1A2 and CYP3A4 revealed a lower K
for CYP1A2, but a higher V for CYP3A4.
m
max
Overall, the results of the reaction phenotyping studies demonstrate that BFC
can be metabolized by a number of CYP isoforms in human liver. At low substrate
concentrations BFC is a more speci®c substrate for CYP1A2 than for CYP3A4,
whereas the reverse is true at high substrate concentrations. BFC may also be a
substrate for CYP2C9 and CYP2C19, as indicated by the studies with the cDNA-
expressed CYP isoforms and to some extent by correlation analysis. However,

BFC metabolism by human CYP isoforms
967
CYP2C9 would only appear to have a minor role in the metabolism of BFC in
}
human liver microsomes, as neither sulphaphenazole nor the CYP2C8 9 inhibitory
antibody had any marked eåect on the metabolism of 20 lm BFC. The studies with
the cDNA-expressed isoforms indicated that BFC could be metabolized by
CYP2C19 and possibly also by CYP2B6. However, compared with the levels of
CYP3A4 and even CYP1A2, these two CYP isoforms are present at only low levels
in human liver microsomes (Wrighton et al. 1993, Lecoeur et al. 1994, Shimada et
al. 1994, Rodrigues 1999). Thus, overall BFC metabolism in human liver is likely to
be primarily catalysed by CYP1A2 and CYP3A4.
These studies also demonstrate the importance of the selection of appropriate
substrate concentrations for reaction phenotyping studies with human hepatic CYP
isoforms. For therapeutic agents, ideally such studies should be performed with
pharmacologically relevant substrate concentrations. For example, lansoprazole 5-
hydroxylation is predominantly catalysed by CYP2C19 in vivo, yet in studies with
human liver microsomes at high substrate concentrations lansoprazole 5-hydroxy-
}
lation is dominated by CYP3A4 5, a low-aænity high-capacity enzyme (Pearce et
al. 1996). Other studies have demonstrated that diazepam N-demethylation can be
catalysed by CYP2C19 and CYP3A isoforms, with CYP2C19 predominating at low
substrate concentrations (Yasumori et al. 1993, Andersson et al. 1994, Yang et al.
1999).
In summary, BFC appears to be a useful substrate for CYP isoform metabolism
studies employing either human liver microsomes or cDNA-expressed CYP
isoforms (Crespi et al. 1999). Like 7-ethoxyresoru®n (Kennedy and Jones 1994),
BFC is metabolized to a highly ¯uorescent product that permits the assay of enzyme
activity in a 96-well plate format using only small amounts of tissue (e.g. 10 lg
human liver microsomal protein). However, unlike 7-ethoxyresoru®n, which is
considered a relatively speci®c marker for CYP1A2 in human liver microsomes
(Wrighton et al. 1993a, Parkinson 1996a, b, Pelkonen et al. 1998), BFC appears to
be a substrate for a number of CYP isoforms. BFC metabolism in human liver
appears to be primarily catalysed by CYP1A2 and CYP3A4, with possibly some
contribution by CYP2C9, CYP2C19 and perhaps other CYP isoforms.
Acknowledgements
The authors are grateful to MSD Research Laboratories, Harlow, Essex, for the
provision of a research grant. They thank Drs T. A. Baillie and T. H. Rushmore,
Merck Research Laboratories, West Point, PA, for the generous gifts of the
}
CYP2C8 9, CYP2D6 and CYP3A4 inhibitory antibodies.
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