Metabolism of 2,5-bis(trifluoromethyl)-7-benzyloxy-4-trifluoromethylcoumarin by human hepatic CYP isoforms: Evidence for selectivity towards CYP3A4

Article abstract of DOI:10.1080/00498250110043526

1. The metabolism of 2,5-bis(trifluoromethyl)-7-benzyloxy-4-trifluoromethylcoumarin (BFBFC) to 7-hydroxy-4-trifluoromethylcoumarin (HFC) was studied in human liver microsomes and in cDNA-expressed human liver CYP isoforms. For purposes of comparison, some limited studies were also performed with 7-benzyloxyquinoline (7BQ). 2. Initial interactive docking studies with a homology model of human CYP3A4 indicated that BFBFC was likely to be a selective substrate for CYP3A4 with a relatively high binding affinity, due to the presence of several key hydrogen bonds with active site amino acid residues. 3. Kinetic analysis of NADPH-dependent BFBFC metabolism to HFC in three preparations of pooled human liver microsomes revealed mean (±SEM) K_m and V_max = 4.6 ± 0.3 μM and 20.0 ± 3.8 pmol/min/mg protein, respectively. 4. The metabolism of BFBFC to HFC was determined in a characterized bank of 24 individual human liver microsomal preparations employing a BFBFC substrate concentration of 10 μM (i.e. around twice K_m). Good correlations (r² = 0.736-0.904) were observed between BFBFC metabolism and markers of CYP3A isoforms. 5. While 10 μM BFBFC was metabolized to HFC by cDNA-expressed CYP3A4, little or no metabolism was observed with cDNA-expressed CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP2E1. 6. The metabolism of 10 μM BFBFC in human liver microsomes was markedly inhibited by 5-50 μM troleandomycin and 0.2-5 μM ketoconazole, but stimulated by 0.2-10 μM α-naphthoflavone. The metabolism of 10 μM BFBFC in human liver microsomes was also markedly inhibited by an antibody to CYP3A4. 7. Kinetic analysis of NADPH-dependent 7BQ metabolism to 7-hydroxyquinoline (7HQ) in human liver microsomes revealed K_m and V_max = 70 μM and 3.39 nmol/min/mg protein, respectively. 8. While 80 μM 7BQ was metabolized to 7HQ by cDNA-expressed CYP3A4, only low rates of metabolism were observed with cDNA-expressed CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP2E1. 9. In summary, by correlation analysis, the use of cDNA-expressed CYP isoforms, chemical inhibition and inhibitory antibodies, BFBFC metabolism in human liver microsomes appears to be primarily catalysed by CYP3A4. BFBFC may be a useful fluorescent probe substrate for human hepatic CYP3A4, but compared with 7BQ has only a low rate of metabolism in human liver microsomes.

Full text of DOI:10.1080/00498250110043526

xenobiotica,
vol.
no.
31, 4, 187- 204
2001,
Metabolism of 2,5-bis(tri uoromethyl)-7-
benzyloxy-4-tri uoromethylcoumarin by human
hepatic CYP isoforms: evidence for selectivity
towards CYP3A4
A. B. RENWICK{, D. F. V. LEWIS{, S. FULFORD},
D. SURRY}, B. WILLIAMS}, P. D. WORBOYS},
X. CAI}, R. W. WANG}, R. J. PRICE{,
B. G. LAKE{* and D. C. EVANS}
{ TNO BIBRA International Ltd, Woodmansterne Road, Carshalton, Surrey
SM5 4DS, UK
{ School of Biological Sciences, University of Surrey, Guildford, Surrey GU2 5XH, UK
} Merck Sharp and Dohme Ltd, Neuroscience Research Centre, Terlings Park, Eastwick
Road, Harlow, Essex CM20 2QR, UK
} Drug Metabolism, Merck & Co., 126 East Lincoln Avenue, Rahway, NJ 07065, USA
Received 7 November 2000
1. The metabolism of 2,5-bis(tri uoromethyl)-7-benzyloxy-4-tri uoromethylcou-
marin (BFBFC) to 7-hydroxy-4-tri uoromethylcoumarin (HFC) was studied in human
liver microsomes and in cDNA-expressed human liver CYP isoforms. For purposes of
comparison, some limited studies were also performed with 7-benzyloxyquinoline (7BQ).
2. Initial interactive docking studies with a homology model of human CYP3A4
indicated that BFBFC was likely to be a selective substrate for CYP3A4 with a relatively
high binding anity, due to the presence of several key hydrogen bonds with active site
amino acid residues.
3. Kinetic analysis of NADPH-dependent BFBFC metabolism to HFC in three
preparations of pooled human liver microsomes revealed mean (§SEM) Km and
Vmax ˆ 4:6 § 0:3 mm and 20:0 § 3:8 pmol/min/mg protein, respectively.
4. The metabolism of BFBFC to HFC was determined in a characterized bank of 24
individual human liver microsomal preparations employing
a BFBFC substrate
concentration of 10 mm (i.e. around twice Km). Good correlations (r2 ˆ 0:736-0:904)
were observed between BFBFC metabolism and markers of CYP3A isoforms.
5. While 10 mm BFBFC was metabolized to HFC by cDNA-expressed CYP3A4, little
or no metabolism was observed with cDNA-expressed CYP1A2, CYP2A6, CYP2B6,
CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP2E1.
6. The metabolism of 10mm BFBFC in human liver microsomes was markedly
inhibited by 5-50 mm troleandomycin and 0.2-5 mm ketoconazole, but stimulated by 0.2-
10 mm ¬-naphtho avone. The metabolism of 10mm BFBFC in human liver microsomes
was also markedly inhibited by an antibody to CYP3A4.
7. Kinetic analysis of NADPH-dependent 7BQ metabolism to 7-hydroxyquinoline
(7HQ) in human liver microsomes revealed Km and Vmax ˆ 70 mm and 3.39 nmol/min/mg
protein, respectively.
8. While 80 mm 7BQ was metabolized to 7HQ by cDNA-expressed CYP3A4, only low
rates of metabolism were observed with cDNA-expressed CYP1A2, CYP2A6, CYP2B6,
CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP2E1.
9. In summary, by correlation analysis, the use of cDNA-expressed CYP isoforms,
chemical inhibition and inhibitory antibodies, BFBFC metabolism in human liver
* Author for correspondence; e-mail: blake@tnobibra.co.uk
ISSN 0049-825 4 print/ISSN 1366-5928 online # 2001 Taylor & Francis Ltd
http://www.tandf.co.uk/journals
Xenobiotica
DOI: 10.1080/0049825011004352 6

A. B. Renwick
188
et al.
microsomes appears to be primarily catalysed by CYP3A4. BFBFC may be a useful
 uorescent probe substrate for human hepatic CYP3A4, but compared with 7BQ has only
a low rate of metabolism in human liver microsomes.
Introduction
Isoforms of the cytochrome P450 (CYP) superfamily are known to play a major
role in the oxidative metabolism of both xenobiotics and certain endogenous
compounds (Parkinson 1996). Many studies have demonstrated that members of
the human hepatic CYP1A, CYP2A, CYP2B, CYP2C, CYP2D, CYP2E and
CYP3A subfamilies can metabolize a wide variety of therapeutic agents (Wrighton
et al
. 1993, Nelson
et al
. 1996, Parkinson 1996, Pelkonen
et al
. 1998). In recent
in vitro
years extensive use has been made of
techniques employing human liver
microsomes and cDNA-expressed human liver CYP isoforms in order to identify
which CYP isoforms will be involved in the metabolism of a new chemical entity
in vivo
et
and to gain information on the potential for
drug interactions (Wrighton
al
et al
et al
. 1993, Crespi
1999).
One requirement for conducting
. 1997, 1998, Clarke 1998, Pelkonen . 1998, Rodrigues
in vitro
metabolism studies is the availability
of suitable probe substrates for the CYP isoforms of interest. In this respect
substrates that are metabolized to highly  uorescent products are more amenable
for use in rapid screens than substrates which require chromatographic separation
of the metabolites prior to quantitation by spectroscopic, radiometric or mass
spectrometry techniques.
Various  uorescent substrates are available as metabolic probes for human
hepatic CYP isoforms. For example, 7-ethoxyresoruŽ n is often employed as a
et al
probe substrate for CYP1A2 in liver microsomes (Clarke 1998, Pelkonen
.
1998). Other studies have demonstrated that derivatives of 7-hydroxy-4-tri uor-
omethylcoumarin (HFC) may also be used as substrates for human hepatic CYP
isoforms. For example, 7-ethoxy-4-tri uoromethylcoumarin has been reported to
be metabolized by a number of CYP isoforms including CYP1A2, CYP2B6,
et al
. 1993, Code
et al
. 1997), while 7-methoxy-
CYP2C9 and CYP2E1 (Buters
4-tri uoromethylcoumarin and 7-benzyloxy-4-tri uoromethylcoumarin (BFC;
Ž gure 1) have been reported to be good substrates, respectively, for cDNA-
et al
et al
. 1999, Stresser . 2000). With
expressed CYP2C9 and CYP3A4 (Crespi
respect to CYP3A isoforms, 3-(3,4-di uorobenzyloxy)-5,5-dimethyl-4-(4-methyl-
H
sulphonylphenyl)-5 -furan-2-one (DFB) has been reported to be a speciŽ c
et al
 uorescent probe for CYP3A4/5 in human liver microsomes (Chauret
.
1999), while BFC, 7-benzyloxyresoruŽ n (7BR) and 7-benzyloxyquinoline (7BQ)
have all been used as  uorescent substrates for cDNA-expressed CYP3A4 (Crespi
et al
. 1997, 1998, 1999, Stresser
et al
. 2000). However, recent studies on the
metabolism of BFC by human hepatic CYP isoforms demonstrated that BFC was
not a speciŽ c probe for CYP3A4, but rather was metabolized by CYP1A2,
et al
. 2000).
CYP3A4 and to a lesser extent by other CYP isoforms (Renwick
While a correlation was reported between 7BR metabolism and CYP3A4 apopro-
et al
tein levels in human liver microsomes (Forrester
. 1992), studies with
inhibitory antibodies suggested that 7BR was not a speciŽ c substrate for CYP3A
et al
isoforms (Burke
. 1994). Additional molecular modelling studies and inves-
tigations with cDNA-expressed CYP isoforms have demonstrated that 7BR is

BFBFC, a speciŽ c  uorescent substrate of CYP3A4
189
Figure 1. Metabolism of BFC and BFBFC to HFC.
metabolized by a number of CYP isoforms including CYP2B6 and CYP3A4
et al et al et al
(Waxman
. 1991, Ono . 1996, Lewis . 1999a).
The aim of the present study was to develop a more speciŽ c HFC derivative as
a  uorescent probe substrate for CYP3A isoforms in human liver microsomes.
in vitro
Initial
metabolism and molecular modelling studies with a number of BFC
analogues (Lake, Lewis, Williams and Evans, unpublished observations) suggested
that 2,5-bis(tri uroromethyl)-7-benzyloxy-4-tri uoromethylcoumarin (BFBFC;
Ž gure 1) may be metabolized by human hepatic CYP3A4. The human hepatic
CYP isoform speciŽ city of BFBFC was investigated by ‘reaction phenotyping’
studies, employing correlation analysis, the use of cDNA-expressed human CYP
isoforms, chemical inhibition and inhibitory antibodies. In addition, interactive
docking studies were performed with a homology model of human CYP3A4.
Finally, some limited studies were also conducted to examine the human CYP
isoform speciŽ city of 7BQ.
Materials and methods
Materials
NADPH, NADP‡, glucose 6-phosphate, glucose 6-phosphate dehydrogenase, Tris, ¬-naphtho-
 avone, diethyldithiocarbamate, quinidine, sulphaphenazole and troleandomycin were from Sigma-
Aldrich Co. (Poole, UK), BFC, HFC, ketoconazole and S(‡)-mephenytoin were from Salford UltraŽ ne
Chemicals and Research Ltd (Manchester, UK) and 7BQ and 7HQ were from GENTEST Corporation
(Woburn, MA, USA). Samples of BFBFC (L-871,408; purity > 98%) and its 3,5-isomer, namely 3,5-
bis(tri uroromethyl)-7-benzyloxy-4-tri uoromethylcoumarin (BTBFC; L-871,420; purity > 98%), to-
gether with inhibitory antibodies against CYP2C8/9, CYP2D6 and CYP3A4, were provided by MSD
Research Laboratories (Harlow, UK). The chemicals and the inhibitory antibodies were stored at 20
and 808C, respectively. The speciŽ city and use of the CYP3A4 antibody has been described by Mei et
al. (1999). Microsomes from human B-lymphoblastoid cells containing cDNA-expressed human CYP
isoforms (GENTEST) were from Cambridge Bioscience (UK) and stored at 808C. 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. Microsomes from baculovirus-infected insect cells containing cDNA-expressed

A. B. Renwick
190
et al.
human CYP isoforms (all + OR) were provided by Merck Research Laboratories (West Point, PA,
USA) and were used for the studies with 7BQ.
Molecular modelling
The three-dimensional structure of CYP3A4 was derived by homology modelling using crystal-
lographic coordinates of the substrate-bound CYP102 haemoprotein domain (Li and Poulos 1997) via
methods described by Lewis et al. (1996, 1999a, b). The putative substrate, BFBFC, was constructed
from a molecular model of BFC, a known non-speciŽ c substrate of CYP3A4, which had previously been
built from pre-minimized substructural fragments using the Sybyl Fragment Library (Tripos Associ-
ates, St Louis, MO, USA). Interactive docking of these substrate molecules was undertaken via the
routines (e.g. DOCK) implemented in the Sybyl molecular modelling package (Tripos Associates) prior
to energy minimization of the enzyme-substrate complex. All molecular modelling procedures were
carried out on a Silicon Graphics Indigo2 IMPACT 10000 graphics workstation operating under
UNIX.
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 808C. Washed microsomal fractions were prepared
in 0.154m KCl containing 50 mm Tris-HCl, pH 7.4, as described (Lake 1987). Liver microsomal
fractions were assayed for total CYP content (Lake 1987) and for protein (Lowry et al. 1951) employing
bovine serum albumin as standard. The washed microsomal fractions were diluted to 10mg
1
proteinml and aliquots stored at 808C. Three separate batches, designated pools A, B and C, of
pooled human liver microsomes were prepared. Each batch was prepared by pooling liver samples from
Ž ve subjects. Pool A comprised liver samples from males aged 15, 17 and 29 years and females aged 31
and 42 years; pool B comprised liver samples from males aged 2.5 and 58 years and females aged 14, 59
and 74 years and pool C comprised liver samples from male subjects aged 44 and 50 years and female
subjects aged 11, 31 and 55 years. Studies were also conducted with a bank of 24 individual human liver
microsomal preparations (Renwick et al. 1998), which had been previously characterized for total CYP
content and CYP isoform enzyme activities. For the studies with 7BQ only, a pool of human liver
microsomes (pool D), comprising liver samples from males aged 48, 66 and 68 years and females aged 42
and 60 years, was provided by Merck Research Laboratories (Rahway, NJ, USA).
Metabolism of BFBFC by human liver microsomes
Incubations were performed in Nunc MicroWelltm (Life Technologies Ltd, Paisley, UK) poly-
styrene 96-well plates with conical wells. The NADPH-dependent metabolism of BFBFC was studied
in incubation mixtures containing 2-30 mm BFBFC, 2-50 mg, microsomal protein and 70mm phosphate
bu€er, pH 7.4, in a Ž nal volume of 100ml. Stock solutions of BFBFC were prepared in acetonitrile and
diluted 1:300 with 100 mm phosphate bu€er, pH 7.4, so that the Ž nal acetonitrile concentration in the
incubations was 0.2% (v/v). Acetonitrile was selected as the solvent for BFBFC and methanol for the
inhibitors studied (see below) in order to minimize solvent e€ects on CYP isoform activities in human
liver microsomes and cDNA-expressed human CYP isoform preparations (Chauret et al. 1998, Busby et
al. 1999). For each BFBFC substrate concentration, microsomal protein concentration and incubation
time, six wells were incubated with NADPH (tests) and two wells without NADPH (blanks). After
mixing, the plates were pre-incubated for 10min in a 378C incubator and the reaction then initiated by
the addition of 1 mm (Ž nal concentration) NADPH. Incubations were performed for 5-90 min at 378C
and were terminated by the addition of 100 ml Tris base in 60% (v/v) acetonitrile. The plates were
centrifuged at 260g for 10 min at 48C and a 150 ml aliquot removed from each well into a white Porvair
96-well plate (Perkin Elmer Ltd, BeaconsŽ eld, 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 wave-
lengths of 410 and 510 nm, respectively, employing 3 nm slits. Standard curves were constructed with
0-50 pmol HFC/well. The above incubation system was also employed to study the metabolism of
10 mm BTBFC and 20 mm BFC and the e€ect of BFBFC and BTBFC on the metabolism of 50mm BFC.
BFBFC metabolism correlation analysis
Incubations were conducted as described above with 10 mm BFBFC (added in 0.2% (v/v) acetonitrile)
and 15 mg, microsomal protein for 25 min. For each of the 24 preparations of human liver microsomes
six wells were incubated with NADPH and two wells (blanks) without NADPH.
BFBFC metabolism by cDNA-expressed CYP isoforms
For each of the B-lymphoblastoid cell cDNA-expressed CYP isoform preparations and each
incubation time, four wells were incubated with NADPH and one well (blank) without microsomal
protein. Incubation mixtures contained 10 mm BFBFC (added in 0.2% (v/v) acetonitrile), 1 mm NADPH

BFBFC, a speciŽ c  uorescent substrate of CYP3A4
191
and 100mm phosphate bu€er, pH 7.4, in a Ž nal volume of 100 ml. After a 10-min pre-incubation at
378C, the reaction was initiated with 100 mg, B-lymphoblastoid cell microsomal protein and incubations
terminated after 40 min.
BFBFC metabolism inhibition studies
Incubations were conducted as described above with 10mm BFBFC, 15 mg, human liver microsomal
protein and 70mm phosphate bu€er, pH 7.4, for 25 min. For each inhibitor concentration six wells were
incubated with BFBFC and two wells (blanks) without substrate. To study the e€ect of the mechanism-
based inhibitors, diethyldithiocarbamate (CYP2E1) and troleandomycin (CYP3A4), the compounds
were pre-incubated for 30 min at 378C with 1 mm NADPH and liver microsomes prior to the addition of
BFBFC and a further aliquot of 1mm NADPH. In the studies with the inhibitors ¬-naphtho avone
(CYP1A2), sulphaphenazole (CYP2C9), quinidine (CYP2D6) and ketoconazole (CYP3A4) and the
substrate S-mephenytoin (CYP2C19), the compounds were pre-incubated for 10 min at 378C with 1mm
NADPH and liver microsomes prior to the addition of BFBFC. Stock solutions of the inhibitors and S-
mephenytoin were prepared in methanol and added to give a Ž nal solvent concentration (i.e. acetonitrile
and methanol) in the incubation mixtures of either 0.4 or 0.45% (v/v). The e€ects of antibodies to
CYP2C8/9, CYP2D6 and CYP3A4 were studied in incubation mixtures containing 10 mm BFBFC,
1 mm NADPH, 15 mg, human liver microsomal protein, 70 mm phosphate bu€er, pH 7.4, and dilutions
of each antibody (0.001, 0.01, 0.05 or 0.5 ml/well) for 25min. For each concentration of each antibody
six wells were incubated with BFBFC and two wells (blanks) without substrate. The antibodies
were pre-incubated with the liver microsomes and bu€er for 10min at 378C prior to the addition of
BFBFC.
Metabolism of 7BQ by human liver microsomes and cDNA-expressed CYP isoforms
Incubations were conducted with either 50 mg human liver microsomal protein or 10 pmol cDNA-
expressed human CYP isoforms with 5-800 mm 7BQ, 1.3 mm NADP‡, 3.5 mm glucose 6-phosphate, 0.28
units glucose 6-phosphate dehydrogenase, 3.3 mm MgCl2 and 200 mm phosphate bu€er, pH 7.4, in a
Ž nal volume of 200ml. The incubations were performed for either 10 min (cDNA-expressed CYP
isoforms) or 20 min (human liver microsomes) in a shaking incubator at 378C. The formation of 7HQ
was monitored at excitation and emission wavelengths of 420 and 530 nm, respectively.
Results
Molecular modelling of BFBFC with CYP3A4
Molecular modelling studies with BFBFC were performed with a model of
et al
. 1996, 1999b). Figure 2
human hepatic CYP3A4 described previously (Lewis
shows the preferred orientation of BFBFC within the putative active site of
CYP3A4, where a combination of º-stacking and hydrogen-bonded interactions
with complementary amino acid residues position the molecule for oxidation at the
experimentally observed site, namely, at the benzylic position. In particular, a
tyrosine residue (TYR 266) in the putative I helix distal to the haem moiety forms
a key hydrogen bond with one of the tri uoromethyl groups. In addition, there is
hydrogen bonding between an asparagine sidechain (ASN 74), located within
substrate recognition site 1 (Gotoh 1992) and lying within the haem environment,
and both the second tri uoromethyl group on the substrate and the oxygen of the
coumarin nucleus. Adjacent to this asparagine, a phenylalanine sidechain (PHE
-
72) forms a º º interaction with the coumarin nucleus, and a further hydrogen
bond between a tyrosine residue (TYR 69) with the tri uoromethyl moiety of the
coumarin nucleus serves to ‘anchor’ this substructure within the CYP3A4 reactive
centre, as indicated in Ž gure 2. In contrast, molecular modelling studies with the
3,5-isomer of BFBFC, namely BTBFC, revealed a greatly diminished ability to
form hydrogen bonds with the tri uoromethyl groups in the putative active site of
CYP3A4 (data not shown).

A. B. Renwick
192
et al.
Figure 2. Interaction of BFBFC with the putative active site of human CYP3A4. The putative active
site of CYP3A4 is shown with BFBFC docked in the preferred orientation for oxidation at the
benzylic position. Hydrogen bonds between BFBFC and key CYP3A4 active site amino acid
residues are shown by the dotted lines. The central iron atom of the haem moiety of CYP3A4 is
also indicated. For further details see the Results.
Kinetics of BFBFC metabolism in human liver microsomes
Initial BFBFC metabolism studies were conducted with a substrate concentra-
m
tion of 25 M. BFBFC was metabolized by human liver microsomes in the
presence of NADPH to HFC (Ž gure 1). The formation of HFC was linear with
respect to incubation time for at least 25 min and microsomal protein concentra-
m
tion up to at least 20 g, protein/well (data not shown).
Using three pooled human liver microsomal preparations, the metabolism of
mm
BFBFC was investigated over a substrate concentration range of 2-30
(Ž gure
K
ˆ
mm
3). Kinetic analysis of Eadie-Hofstee plots revealed
4:1, 4.5 and 5.1
for
m
the pool A, B and C human liver microsomal preparations, respectively. Corre-
Vmax
Vmax
ˆ
sponding
observed
13:3, 26.4 and 20.2 pmol/min/mg protein, respectively. The
appeared to correlate with the total CYP content of the pooled
liver microsomal preparations, which were 0.39, 0.72 and 0.51 nmol/mg protein for
the A, B and C pools, respectively. For the three pooled human liver microsomal
§
Km
Vmax
ˆ
§
mm
§
preparations mean ( SEM)
and
4:6 0:3
and 20:0 3:8 pmol/
mm
min/mg protein, respectively. A BFBFC substrate concentration of 10
K
(i.e. approximately twice m) was selected for the subsequent studies described
below.
Comparison of BFBFC metabolism with BFC and BTBFC
The rate of metabolism of BFBFC in human liver microsomes was compared
with both BFC and the 3,5-isomer of BFBFC, namely BTBFC. The studies were
mm
performed with substrate concentrations of 10
for BFBFC (i.e. around twice
K
mm
K
for BFC (i.e. around twice the for BFC in human
m
m) and BTBFC and 20
et al
. 2000). Both BFC (Ž gure 4A) and BFBFC (Ž gure
liver microsomes; Renwick
4B) were metabolized to HFC in a time-dependent manner, whereas only trace
amounts of BTBFC metabolism was observed (Ž gure 4B). Under the incubation
¹
conditions employed, the rate of BFBFC metabolism was only 6% of that of BFC

BFBFC, a speciŽ c  uorescent substrate of CYP3A4
193
Figure 3. Kinetics of BFBFC metabolism to HFC in three pooled human liver microsomal
preparations. Incubations (n ˆ 6 wells for each BFBFC substrate concentration) were performed
with 15mg, protein/well of pools A (*), B (*) and C (&) microsomal preparations with 2-30 mm
BFBFC for 25 min.
Figure 4. Comparison of the metabolism of BFC, BFBFC and BTBFC to HFC in human liver
microsomes. Incubations (n ˆ 6 wells for each substrate and time point) were performed with
20 mm BFC (A, ~), 10mm BFBFC (B, *) and 10mm BTBFC (B, &) with 15 mg/well microsomal
protein for 5-90min. Results are mean of data obtained with the pool B and C human liver
microsomal preparations.
€
(Ž gure 4A and B). In some additional studies the e ect of BFBFC and BTBFC on
mm
the metabolism of 50
BFC (i.e. a substrate concentration where metabolism is
et al
predominantly catalysed by CYP3A4; Renwick
. 2000) in human liver
¹
microsomes was studied. BFC metabolism was only inhibited to 64% of control
mm
(mean of studies with pool B and C liver microsome preparations) by either 100
mm
BFBFC or 100
BTBFC (data not shown).

A. B. Renwick
194
et al.
Figure 5. Variability of BFBFC metabolism to HFC in a panel of 24 characterized human liver
microsomal preparations. Incubations (n ˆ 6 wells for each microsomal preparation) were
performed with 10 mm BFBFC and 15 mg/well microsomal protein for 25min.
BFBFC metabolism correlation analysis
mm
The metabolism of 10
BFBFC was examined with a characterized bank of
et al
24 human liver microsomal preparations (Renwick
. 1998). BFBFC was
metabolized to HFC by 22 of the 24 liver microsomal preparations examined
(Ž gure 5). Microsomal BFBFC metabolism varied from 1 to 101 pmol/min/mg
protein.
The rates of BFBFC metabolism were correlated with values for total CYP
content and a range of CYP isoform enzyme activities (table 1). Good correlations
r2 ˆ
-
(
0:736 0:904) were observed between BFBFC metabolism to HFC and a
number of enzyme activities catalysed by CYP3A isoforms including testosterone
r2 ˆ
r2 ˆ
6- -hydroxylase (
0:904; Ž gure 6H). A good correlation (
0:834) was also
observed between BFBFC metabolism and total CYP content (table 1) whereas a
lower correlation (
CYP2B6 catalysed -mephenytoin N-demethylase activity (Ž gure 6C). Only low
correlations (
markers of enzyme activities catalysed by CYP1A2, CYP2A6, CYP2C9,
CYP2C19, CYP2D6, CYP2E1 and CYP4A9/11 isoforms (Ž gure 6 and table 1).
r2 ˆ
0:532) was observed between BFBFC metabolism and
S
r2 ˆ
-
0:076 0:328) were observed between BFBFC metabolism and
Metabolism of BFBFC by cDNA-expressed CYP isoforms
mm
The metabolism of 10
BFBFC was examined in GENTEST B-lympho-
blastoid cell microsomes containing cDNA-expressed CYP1A2, CYP2A6,
CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A4,
together with microsomes from control (wild-type) cells. BFBFC was metabolized
to HFC by the cDNA-expressed CYP3A4 preparation (Ž gure 7), whereas only
very low rates of BFBFC metabolism were observed with the cDNA-expressed
CYP2B6 preparation and with the control cell microsomes (which contain native
CYP1A1). In contrast, BFBFC was not metabolized to HFC by the cDNA-
expressed CYP1A2, CYP2A6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and
CYP2E1 preparations (Ž gure 7).

BFBFC, a speciŽ c  uorescent substrate of CYP3A4
195
Figure 6. Correlation between BFBFC metabolism to HFC and some CYP isoform enzyme activities
in a panel of 24 characterized human liver microsomal preparations. Incubations (n ˆ 6 wells for
each microsomal preparation) were performed with 10mm BFBFC and 15 mg/well microsomal
protein for 25 min. The metabolism of BFBFC was compared with enzymatic markers of
CYP1A2 (A), CYP2A6 (B), CYP2B6 (C), CYP2C9 (D), CYP2C19 (E), CYP2D6 (F), CYP2E1
(G) and CYP3A4 (H).

A. B. Renwick
196
et al.
Table 1. Correlation analysis for BFBFC metabolism to HFC with various CYP isoform-dependent
enzyme activities in human liver microsomes.
CYP isoform
Enzyme activitya
r2
-
total CYP
7-ethoxyresoruŽ n O-deethylase
ca€eine N3-demethylase
0.834
0.189
0.232
0.181
0.532
0.328
0.169
0.220
0.109
0.904
0.897
0.897
0.893
0.844
0.736
0.076
1A2
2A6
2B6
2C9
2C19
2D6
2E1
coumarin 7-hydroxylase
S-methenytoin N-demethylase
tolbutamide methylhydroxylase
S-mephenytoin 40-hydroxylase
dextromethorphan O-demethylase
chlorzoxazone 6-hydroxylase
testosterone 6- -hydroxylase
testosterone 2- -hydroxylase
testosterone 15- -hydroxylase
diazepam 3-hydroxylase
3A4/5
ca€eine 8-hydroxylase
dextromethorphan N-demethylase
lauric acid 12-hydropxylase
4A9/11
a Levels of total CYP content and CYP isoform enzyme activities were
determined in a panel of 24 individual human liver microsomal preparations
(Renwick et al. 1998). These values (plotted on the x axis) were then plotted
against the data obtained for the metabolism of 10 mm BFBFC (Ž gure 5) by the
same panel of human liver microsomes (plotted on the y-axis) and the various
correlations determined.
Inhibition of BFBFC metabolism
€
S
The e ect of some known CYP inhibitors and one substrate ( -mephenytoin)
mm
on the metabolism of 10
BFBFC in human liver microsomes (pool B) was
et al
studied. For the mechanism-based inhibitors (Newton
. 1995) diethyldithio-
carbamate and troleandomycin, the compounds were pre-incubated with NADPH
and the liver microsomes for 30 min prior to the addition of BFBFC and another
mm
aliquot of NADPH. While treatment with 10-50
diethyldithiocarbamate had
€
mm
little e ect on BFBFC metabolism, the addition of 5
troleandomycin inhibited
BFBFC metabolism to < 2% of control, with a complete inhibition of enzyme
mm
activity being observed with 10-50
troleandomycin (Ž gure 8A). The addition of
mm S
mm €
quinidine had little e ect on BFBFC
either 50-500
-mephenytoin or 2-20
metabolism, whereas some inhibition of enzyme activity was observed by treat-
mm
ment with 2-20
sulphaphenazole (Ž gure 8B). In contrast, BFBFC metabolism
mm
was markedly stimulated to up to 325% of control by treatment with 0.2-10
¬-
naphtho avone (Ž gure 8B). Ketoconazole was a potent inhibitor of BFBFC
metabolism, inhibiting enzyme activity to 46% of control at a concentration of
mm
0.2 , with a complete inhibition of enzyme activity being observed by the
mm
addition of 0.5-5
ketoconazole (Ž gure 8B).
€
The e ect of three available inhibitory antibodies to CYP2C8/9, CYP2D6 and
CYP3A4 on BFBFC metabolism by human liver microsomes was also investi-
gated. Using two pooled human liver microsomal preparations (pools B and C),
m
m
m
treatment with 0.001-0.5 l/well (i.e. 0.001-0.5 l/100 l incubation mixture) of
€
either the CYP2C8/9 or the CYP2D6 antibody had little e ect on BFBFC
m
metabolism (Ž gure 9A and B). In contrast, the addition of 0.01 l/well of the
€
CYP3A4 antibody had a marked e ect on BFBFC metabolism, with HFC

BFBFC, a speciŽ c  uorescent substrate of CYP3A4
197
Figure 7. Metabolism of BFBFC by B-lymphoblastoid cell microsomes containing cDNA-expressed
human CYP isoforms. Incubations (n ˆ 4 wells for each microsomal preparation) were
performed with 10 mm BFBFC and 100 mg/well B-lymphoblastoid cell microsomal protein for
40 min.
Figure 8. Inhibition of BFBFC metabolism in human liver microsomes. To study the e€ects of 10-
50 mm diethyldithiocarbamate and 5-50 mm troleandomycin (A), but not 0.2-10mm ¬-naphtho-
 avone, 2-20 mm sulphaphenazole, 50-500 mm S-mephenytoin, 2-20 mm quinidine and 0.2-5 mm
ketoconazole (B), the compounds were pre-incubated with liver microsomes and NADPH for
30 min at 378C prior to the addition of 10 mm BFBFC. Incubations (n ˆ 6 wells for each inhibitor
concentration) were performed with 15 mg protein/well of the pool B liver microsomal
preparation for 25 min and the results are expressed as percentage of control (no inhibitor
added) incubations. Control rates of metabolism of BFBFC to HFC were 9.1 and 10.1 pmol/
min/mg protein for the mechanism-based inhibitors (A) and the other compounds (B) respect-
ively.

A. B. Renwick
198
et al.
Figure 9. E€ect of inhibitory antibodies on BFBFC metabolism in human liver microsomes.
Inhibitory antibodies (0.001, 0.01, 0.05 and 0.5 ml/well) to CYP2C8/9, CYP2D6 and CYP3A4
were pre-incubated with the pool B (A) and C (B) liver microsomal preparations for 10 min at
378C prior to addition of 10 mm BFBFC. Incubations (n ˆ 6 wells for each inhibitory antibody
concentration) were performed with 15 mg/well microsomal protein for 25 min and the results are
expressed as percentage of control (no antibody added) incubations. Control rates of BFBFC
metabolism to HFC by the pool B (A) and C (B) human liver microsomal preparations were 18.3
and 13.5 pmol min/mg protein, respectively.
¶
m
formation being inhibited by 98% by the addition of 0.05 and 0.5 l/well of the
CYP3A4 inhibitory antibody (Ž gure 9A and B). Additional studies demonstrated
that CYP2C9 catalysed tolbutamide methylhydroxylase was inhibited to 25% of
m
m
control by the addition of 0.5 l/100 l incubation mixture of the CYP2C8/9
m
m
antibody, whereas no inhibition was observed by the addition of (1.0 l/100 l
incubation mixture of the CYP3A4 antibody (data not shown).
Kinetics of 7BQ metabolism in human liver microsomes
The NADPH-dependent metabolism of 7BQ to 7HQ was studied with the
pool D liver microsomal preparation over a substrate concentration range of
mm
Km
Vmax
ˆ
mm
70 and 3.39 nmol/min/mg
5-800
(Ž gure 10). Observed
and
protein, respectively.
Metabolism of 7BQ by cDNA-expressed CYP isoforms
mm
K
7BQ (i.e. a substrate concentration similar to the
The metabolism of 80
m
for 7BQ metabolism in human liver microsomes) was studied in baculovirus-
infected insect cell microsomes containing cDNA-expressed CYP1A2, CYP2A6,
CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A4 (Ž gure

BFBFC, a speciŽ c  uorescent substrate of CYP3A4
199
Figure 10. Kinetics of 7BQ metabolism to 7HQ in the pool D human liver microsomal preparation.
Duplicate incubations were performed with 5-800 mm 7BQ substrate and 50mg, human liver
microsomal protein for 20 min.
Figure 11. Metabolism of 7BQ by baculovirus-infected insect cell microsomes containing cDNA-
expressed human CYP isoforms. Duplicate incubations were performed with 80mm 7BQ
substrate and 10 pmol of the cDNA-expressed CYP isoforms for 10 min.
11). 7BQ was metabolized to 7HQ by the cDNA-expressed CYP3A4 preparation,
whereas only very low rates of 7BQ metabolism were observed with the control cell
microsomes and the other cDNA-expressed CYP isoforms.
Discussion
In this study the metabolism of BFBFC to HFC (Ž gure 1) by human liver
microsomal preparations and cDNA-expressed CYP isoforms has been investi-
gated. For purposes of comparison some more limited studies were performed
with 7BQ.

A. B. Renwick
200
et al.
In a previous study conducted in our laboratory BFC was shown not to be a
et al
. 2000).
speciŽ c substrate for CYP3A4 in human liver microsomes (Renwick
However, in this investigation initial molecular modelling studies demonstrated
that BFBFC could be docked in the putative active site of our CYP3A4 model
et al
. 1996, 1999b) in an orientation for oxidation at the experimentally
(Lewis
observed site, namely at the benzylic site (Ž gure 2). In this position, the substrate
-
is held in place by a number of key hydrogen bonds, and a º º stacking interaction,
with nearby amino acid residues. No molecular modelling studies were performed
with 7BQ.
BFBFC metabolism to HFC was observed in three preparations of pooled liver
microsomes. Additional studies would be required to determine if BFBFC could
be metabolized to products other than HFC in human liver microsomes. Inves-
tigations into the kinetics of BFBFC metabolism to HFC in human liver micro-
§
n ˆ
Km
ˆ
§
mm
K
§
somes revealed a mean ( SEM,
3)
4:6 0:3 . The
for BFBFC
m
mm
metabolism is thus similar to that of BFC, where values of 8:3 1:3
(mean-
§
n ˆ
et al
§
mm
§
n ˆ
SEM,
4; Renwick
. 2000) and 14:2 3:4
(mean SEM,
3; Surry
and Evans, unpublished data) were previously observed. Despite the similarity in
K
m, the rate of BFBFC metabolism to HFC in human liver microsomes is much
lower than that of BFC (Ž gure 4). The 3,5-isomer of BFBFC, namely BTBFC,
was also synthesized. However, this compound is clearly not suitable as a CYP
isoform  uorescent substrate, as it was not metabolized to HFC in human liver
microsomes to any signiŽ cant extent (Ž gure 4). Moreover, neither BTBFC nor
BFBFC were potent inhibitors of BFC metabolism in human liver microsomes.
In this study ‘reaction phenotyping’ (Parkinson 1996, Rodrigues 1999) for
BFBFC metabolism was performed by correlation analysis, the use of cDNA-
expressed CYP isoforms, chemical inhibition and the use of inhibitory antibodies.
mm
K
The studies were conducted with 10
BFBFC (i.e. around twice m). The best
r2 ˆ
-
correlations (
0:736 0:904) with BFBFC metabolism to HFC were obtained
with testosterone 2- -, 6- - and 15- -hydroxylases, diazepam 3-hydroxylase, ca eine
€
N
8-hydroxylase and dextromethorphan -demethylase (table 1). These enzyme
activities are considered to be catalysed by CYP3A isoforms in human liver
et al
et al
et al
et al
. 1994, Parkinson
0:834) was also observed between
(Wrighton
1996, Yang
. 1993, Gorski
. 1994, Tassaneeyakul
r2 ˆ
. 1999). A good correlation (
BFBFC metabolism and total CYP content. This may be attributable to the fact
that CYP3A isoforms are relatively abundant in human liver microsomes
et al
. 1993, Shimada
contrast to the CYP3A isoform markers, poorer correlations (
were observed between BFBFC metabolism and the enzymatic markers of
CYP1A2, CYP2A6, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and
CYP4A9/11 studied (table 1).
et al
(Wrighton
. 1994, Clarke 1998, Rodrigues 1999). In
r2 ˆ
-
0:076 0:532)
mm
The metabolism of 10
BFBFC by cDNA-expressed CYP isoforms was also
examined. While BFBFC was extensively metabolized to HFC by the cDNA-
expressed CYP3A4 preparation, no detectable BFBFC metabolism was observed
with the CYP1A2, CYP2A6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and
CYP2E1 preparations (Ž gure 7). Although detectable BFBFC metabolism was
observed with the control B-lymphoblastoid cell microsomes (which contain native
CYP1A1) and cDNA-expressed CYP2B6, the rates of BFBFC metabolism were
only 0.5 and 2.0%, respectively, of that of cDNA-expressed CYP3A4 when
expressed per unit of protein. The activity of the CYP2B6 preparation was only

BFBFC, a speciŽ c  uorescent substrate of CYP3A4
201
around 1.0% of that of CYP3A4 when calculated per unit of cDNA-expressed
CYP.
€
The e ect of chemical inhibitors and inhibitory antibodies on BFBFC meta-
bolism in human liver microsomes was also investigated. Previous work has
S
demonstrated ¬-naphtho avone, sulphaphenazole, -mephenytoin, quinidine,
diethyldithiocarbamate and troleandomycin may be considered either inhibitors
or substrates of human hepatic CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP2E1
et al
et al et al
. 1994, Newton .
and CYP3A4, respectively (Wrighton
. 1993, Chang
et al
. 1998, Rodrigues 1999). In
1995, Parkinson 1996, Clarke 1998, Pelkonen
addition, at low concentrations, ketoconazole is an inhibitor of human hepatic
et al
CYP3A4 (Newton
. 1995). BFBFC metabolism to HFC was inhibited by
mm
598% by 5-50
mm
of the CYP3A4 mechanism-based inhibitor troleandomycin and
mm
¬-naphtho avone
by 0.5-5
ketoconazole (Ž gure 8). In contrast, 0.2-10
stimulated BFBFC metabolism in human liver microsomes. Other studies have
demonstrated that ¬-naphtho avone and some other  avonoids can stimulate the
et al
metabolism of certain CYP3A substrates in human liver microsomes (Ueng
.
et al
€
. 1998). BFBFC metabolism was essentially una ected by 10-
1997, Pelkonen
mm
mm S
mm
50
diethyldithiocarbamate, 50-500
-mephenytoin and 5-20
quinidine,
mm
whereas some inhibition was observed with 5-20
sulphaphenazole. However,
€
the relatively small e ect of sulphaphenazole was not supported by the other
studies (i.e. correlation analysis, use of cDNA-expressed CYP isoforms and
inhibitory antibodies). The involvement of CYP3A4 in BFBFC metabolism in
human liver microsomes was also demonstrated by the studies with the inhibitory
m
antibodies. At concentrations of 0.01-0.5 l/well the CYP3A4 antibody inhibited
€
BFBFC metabolism by 64-100%, whereas little e ect was observed with the
CYP2C8/9 and CYP2D6 antibodies (Ž gure. 9).
Overall, the results of the reaction phenotyping studies demonstrate that
BFBFC is primarily metabolized by CYP3A4 in human liver microsomes. Good
correlations were obtained between BFBFC metabolism and known CYP3A4-
dependent enzyme activities. BFBFC was metabolized by cDNA-expressed
CYP3A4 and BFBFC metabolism was inhibited by ketoconazole, troleandomycin
and a CYP3A4 inhibitory antibody. These studies were performed with a BFBFC
mm
substrate concentration of 10 . Additional investigations would be required to

ascertain whether BFBFC would be a low a nity substrate for other CYP
isoforms at higher substrate concentrations. Like BFBFC, in some limited studies
with human cDNA-expressed CYP isoforms, 7BQ was found to be a relatively
speciŽ c substrate for CYP3A isoforms. Additional reaction phenotyping studies
would be required to conŽ rm the CYP isoform speciŽ city of this substrate.
The aim of the current study was to develop a  uorescent substrate with a high
speciŽ city for CYP3A4 in human liver microsomes. Although there are many
et al
. 1998, Kenworthy
. 1999), most are unsuitable for rapid screening purposes as they require
CYP3A4 probe substrates available (Clarke 1998, Pelkonen
et al
chromatographic separation of the metabolites prior to quantitation by spectro-
scopic, radiometric or mass spectrometry techniques. While cDNA-expressed
CYP3A4 can be assayed in rapid 96-well plate format inhibition screens with
et al
. 1997,
 uorescent substrates such as BFC and 7-benzyloxyresoruŽ n (Crespi
1998, 1999), these substrates lack the required speciŽ city for studies with human
et al et al et al et
liver microsomes (Waxman
al et al
. 1991, Burke
. 1994, Ono . 1996, Lewis
. 1999a, Renwick . 2000). One exception is DFB, which has been recently

A. B. Renwick
202
et al.
reported to be a speciŽ c  uorescent probe for CYP3A4 inhibition studies utilizing
et al
human liver microsomes in a 96-well plate format (Chauret
. 1999).
for BFBFC metabolism in human liver microsomes of 4.6
mm et al
Km
mm
The
is
. 1999) and
observed in the present study for 7BQ. However, the
somewhat lower than the 26
reported for DFB (Chauret
mm
lower than the 70
Vmax
et al
. 1999) and 7BQ (this study) are 1.0 and
reported
for DFB (Chauret
V
ˆ
3.39 nmol/min/mg protein, respectively, which are much greater than the
max
20:0 pmol min/mg protein observed for BFBFC. Thus although BFBFC is a
speciŽ c substrate for CYP3A4, it has only a low turnover which may limit the
usefulness of this  uorescent probe. Although the rate of BFBFC metabolism
appears low compared with DFB and 7BQ, it may be worthwhile examining
whether BFBFC is also a suitable  uorescent probe substrate for rapid CYP3A4
inhibition screening studies using human liver microsomes. One potential advan-
et al
. 1999), is that NADPH does not interfere
tage of BFBFC over DFB (Chauret
with the measurement of the HFC product. The use of BFBFC as a  uorescent
substrate for CYP isoform induction studies is currently being investigated in our
laboratories.
In summary, the present results demonstrate that BFBFC is metabolized to
HFC in human liver microsomes. From the results of reaction phenotyping studies
BFBFC appears to be a speciŽ c  uorescent probe substrate for human hepatic
CYP3A4. This study also demonstrates the usefulness of human CYP isoform
homology models in the design of new speciŽ c CYP substrates. Finally, based on
limited studies with cDNA-expressed CYP isoforms 7BQ also appears to be a
relatively speciŽ c substrate for CYP3A4.
Acknowledgements
The authors are grateful to MSD Research Laboratories, Harlow, Essex, for
the provision of a research grant. They thank Dr T. A. Baillie, Ms Q. Mei, Dr T.
H. Rushmore and Dr M. Shou, Merck Research Laboratories, West Point, PA, for
the generous gifts of the CYP2C8/9, CYP2D6 and CYP3A4 inhibitory antibodies
and the cDNA-expressed human CYP isoforms.
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