Role of CYP3A in bromperidol metabolism in rat in vitro and in vivo

Article abstract of DOI:10.1080/004982599238281

1. The aim was to identify whether CYP3A metabolizes bromperidol (BP), an antipsychotic drug, to form 4-fluorobenzoyl-propionic acid (FBPA) in hepatic microsomes from 8-week-old male Sprague-Dawley rats and to investigate whether an inhibitor or an inducer of CYP3A affects BP pharmacokinetics in rat. 2. In an in vitro study, only troleandomycin showed marked inhibition of FBPA formation among several specific CYP isozyme inhibitors studied including troleandomycin, diethyldithiocarbamate, furafylline and quinine. Anti-rat CYP3A2 serum inhibited FBPA formation by 80%, whereas other anti-rat CYP sera (1A1, 1A2, 2B1, 2C11, 2E1) only slightly inhibited it. 3. In a pharmacokinetic study, BP half-life was prolonged to 137% of the average control value by 7-day treatment with erythromycin, a CYP3A inhibitor, and shortened to 58% of the control by 2-day treatment with dexamethasone, a CYP3A inducer. BP clearance was reduced to 68% of the control by erythromycin and was increased to 145% of control by dexamethasone. 4. These results suggested that BP biotransformation is catalysed mainly by CYP3A to form FBPA in rat and that the modification of this enzyme activity would affect the pharmacokinetics of BP.

Full text of DOI:10.1080/004982599238281

xenobiotica , 1999, vol. 29, no. 8, 839±846
Role of CYP3A in bromperidol metabolism in rat in
vitro and in vivo
M. WATANABE*, T. TATEISHI, M. TANAKA, T. KUMAI and
S. KOBAYASHI
Department of Pharmacology, St Marianna University School of Medicine, 2-16-1
Sugao, Miyamae-Ku, Kawasaki, Kanagawa 216-8511, Japan
Received 16 February 1999
1. The aim was to identify whether CYP3A metabolizes bromperidol (BP), an anti-
psychotic drug, to form 4-¯uorobenzoyl-propionic acid (FBPA) in hepatic microsomes
from 8-week-old male Sprague±Dawley rats and to investigate whether an inhibitor or an
inducer of CYP3A aåects BP pharmacokinetics in rat.
2. In an in vitro study, only troleandomycin showed marked inhibition of FBPA
formation among several speci®c CYP isozyme inhibitors studied including troleando-
mycin, diethyldithiocarbamate, furafylline and quinine. Anti-rat CYP3A2 serum inhibited
FBPA formation by 80%, whereas other anti-rat CYP sera (1A1, 1A2, 2B1, 2C11, 2E1)
only slightly inhibited it.
3. In a pharmacokinetic study, BP half-life was prolonged to 137% of the average
control value by 7-day treatment with erythromycin, a CYP3A inhibitor, and shortened to
58% of the control by 2-day treatment with dexamethasone, a CYP3A inducer. BP
clearance was reduced to 68% of the control by erythromycin and was increased to 145%
of control by dexamethasone.
4. These results suggested that BP biotransformation is catalysed mainly by CYP3A to
form FBPA in rat and that the modi®cation of this enzyme activity would aåect the
pharmacokinetics of BP.
Introduction
Bromperidol(BP), a butyrophenone-typeneuroleptic, is used in the treatment of
patients with psychiatric disease, similarly to haloperidol (HP). Although BP is a
close structural analogue of HP, with the only diåerence being bromine in BP in
place of chlorine in HP, some diåerences have been found in their treatment eåects
on speci®c symptoms in studies of patients with chronic schizophrenia. Malfroid et
al. (1978) reported that BP has a more pronounced anxiolytic eåect than HP and
Itoh (1985)foundthatBP was superior to HP in improvingschizophrenicsymptoms
in a double-blindcontrolled study. In addition, BP is reported to have a faster onset
of action than HP (Itoh 1985, Ben®eld et al. 1988).The incidence of extrapyramidal
side eåects was similar after both treatments (Malfroid et al. 1978, Denjis 1980,
Brannen et al. 1981). Therefore, BP is considered as useful as HP in the treatment
of patients with psychiatric disease.
Previous studies suggest that BP undergoes three metabolic pathways: O-
glucuronide conjugation, carbonyl reduction and oxidative N-dealkylation
(Chasseaud 1978, Heykants et al. 1978, Wong et al. 1983, Someya et al. 1991).
Although HP is reported to be N-dealkylated by CYP3A (Fang et al. 1997,
Pan et al. 1997), the isoenzymes catalysing the metabolism of BP have not been
clari®ed. Carbamazepine, an inducer of CYP3A, signi®cantly decreased plasma
* Author for correspondence; e-mail: m4wata!marianna-u.ac.jp
}
#
http: www.tandf.co.uk JNLS xen.htm http: www.taylorandfrancis.com JNLS xen.htm
Xenobiotica ISSN 0049±8254 print ISSN 1366±5928 online
}} }}
1999 Taylor & Francis Ltd
}
}
}
}

840
M. Watanabe et al.
concentrations of BP in a previous clinical study (Otani et al. 1997), which suggests
that BP is N-dealkylated by CYP3A and that the modi®cation of this enzyme activity
by an inhibitor or an inducer may aåect BP pharmacokinetics. The present authors,
therefore, studied whether CYP3A catalysed BP N-dealkylation and whether the
pharmacokinetics of BP was actually aåected when the CYP3A activity in liver
microsomes was altered by an inhibitor or an inducer of CYP3A in rat.
Materials and methods
In vitro experiments
Preparation of liver microsomes. Male Sprague±Dawley rats (n 10, body weight of 272±305 g) were
5
purchased from Nippon Clea (Tokyo, Japan). Animals were killed by decapitation, and the livers were
immediately removed and homogenized with 3 vols 1.15% KCl. Hepatic microsomes were prepared by
sequential centrifugation of the homogenate at 9000g for 20 min and of the resultant supernatant at
105000g for 60 min, both at 4 C. The precipitates were mixed and diluted with distilled water (about
Ê
}
10 mg ml). The microsome samples were separated into approximately 200 tubes, immediately frozen in
liquid nitrogen, and stored at 80 C until use. Protein concentrations were determined by the method
2
Ê
of Lowry et al. (1951).
Chemicals. The chemicals used were: NADP+ , glucose 6-phosphate and glucose 6-phosphate de-
hydrogenase(OrientalYeast, Tokyo, Japan); HP, BP and reduced BP (RBP) (Yoshitomi Pharmaceutical
Co., Ltd, Osaka,Japan); 4-¯uorobenzoyl-propionicacid (FBPA) and SKF-525A (ResearchBiochemicals
International, Natick, MA, USA); troleandomycin and quinine (Sigma Chemical Co., St Louis, MO,
USA); diethyldithiocarbamate (DDC) (Wako Pure Chemical Industries, Osaka, Japan). All other
chemicals were purchased from Wako Pure Chemical Industries and were of analytical grade.
Incubationconditions. The incubation mixture used to assay the formation of FBPA from BP consisted of
100 mm Na-K phosphate buåer (pH 7.4), 0.05 mm EDTA, 0.8 mg microsomal protein, and a substrate
(100 lm BP). The reaction was started by the addition of an NADPH-generating system (5 mm
magnesium chloride, 0.5 mm NADP+ , 5 mm glucose 6-phosphate and 1 unit of glucose 6-phosphate
dehydrogenase) in a ®nal volume of 1 ml. After incubation for 30 min at 37 C, the reaction was
Ê
terminated by the addition of 1 ml acetonitrile. The incubation (30 min) and amount of microsomal
protein (0.8 mg) were determined by results showing that FBPA formation rate increased linearly in a
range of 0.4±2.0 mg microsomal protein and 15±60 min of incubation.
FBPA measurement. A solution of 20 nmol HP (internal standard) in 100 ll methanol was added to each
mixture. The denatured proteins were removed by centrifugation, and 50 ll 2 N HCl was added to
1.5 ml supernatant. The mixture was extracted with 5 ml dichloromethane for 30 s on a vortex mixer.
After centrifugation, the organic layer was separatedand evaporated. The residue was reconstitutedwith
300 ll of the HPLC mobile phase, and 100 ll was injected into the chromatographic system. BP, RBP,
FBPA and HP were measured in the reaction medium by high-performance liquid chromatography
(HPLC) using a slightly modi®ed method of Fang and Gorrod (1993). Brie¯y, the chromatographic
system comprised of a CCPM-II solvent pump (TOSOH, Tokyo, Japan) and a MCPD-3600 spectro-
multi-channel photo detector (OTUKA ELECTRONICS, Osaka, Japan) set to measure at 220 and
245 nm wavelengths. The HPLC system used a 5 lm TSK CN-80Ts column (silica gel with bonded
cyanopropyl groups, 250 4.6 mm i.d.) (TOSOH, Tokyo, Japan). Mobile phase A(acetonitrile) and
3
mobile phase B (30 mm ammonium acetate buåer adjusted to pH 5.0 with acetic acid) were used in a
}
gradient program at 1.2 ml min: A was 20% at 0±1 min, 20±30% linearly from 1 to 15 min, and 20%
at 15±25 min.
Inhibition experiments. The eåect of various speci®c and non-speci®c inhibitors of CYP on the rate of
FBPA production from BP were examined to identify the CYP isozyme(s) that catalysed BP N-
de-alkylation. Troleandomycin (a CYP3A inhibitor), diethyldithiocarbamate (a CYP2E1 inhibitor),
furafylline (a CYP1A2 inhibitor), quinine (a CYP2D inhibitor) or SKF-525A were added to the
incubation mixture as a speci®c or non-speci®c inhibitor of a CYP isozyme at 20, 40 and 100 lm (2, 4 and
10 lm for quinine). Troleandomycin, diethyldithiocarbamate, furafylline, quinine and SKF-525A were
pre-incubated with an NADPH-generating system for 10, 3, 10, 2 and 5 min respectively before the
reaction was started (Schenkman et al. 1972, Kobayashi et al. 1989, Kunze and Trager 1993, Bourrie et
al. 1996, Nunoya et al. 1996). Also examined was the inhibitory eåect of quinine on dextromethorphan
O-demethylation activity (Kerry et al. 1993) and it was compared with the eåect on BP N-dealkylation.

Role of CYP3A in bromperidol metabolism
841
Immuno-inhibition experiments. Anti-rat CYP1A1, 1A2, 2B1, 2C11, 2E1 and 3A2 antibodies were
purchased from Daiichi Pure Chemicals Co., Ltd (Tokyo, Japan). The immuno-inhibition of the FBPA
formation was examined by pre-incubation of rats liver microsomes (0.8 mg) with various anti-rat CYP
sera (0.2 mg IgG) at room temperaturefor 30 min before the reaction was startedwith 100 lm BP and an
NADPH-generating system. Individual pre-immune serawere pre-incubated under the same conditions
as a control for the various anti-rat CYP sera.
In vivo experiments
Animals and procedures. The treatment regiment consisted of either erythromycin (100 mg kg per day)
}
or dexamethasone (80 mg kg per day) suspended in corn oil and administered to male Sprague±Dawley
}
rats (n 6 in each treatment group, with body weight of 273±308 g) by intraperitoneal injection (i.p.) for
5
7 or 2 days respectively during the eighth week of age. The respective treatment regimens were chosen
}
because the 7-day treatment with 100 mg kg erythromycin has previously been shown signi®cantly to
increase the serum levels of cyclosporin, a substrate for CYP3A, in rat (Murray et al. 1992) and because
}
the 2-day treatment with 80 mg kg dexamethasone has been shown signi®cantly to induce CYP3A in
hepatic microsomes (Kolars et al. 1992). Corn oil vehicle was administered at the same time to another
}
group (n 6) serving as control. Rats received a single intravenous injection of 0.5 mg kg BP (Serenace
5
Injection, Dainippon Pharmaceutical Co., Ltd, Osaka, Japan) in the tail vein 1 h after the last
administration of erythromycin or corn oil and 24 h after the last administration of dexamethasone.
Determinationof BP and pharmacokineticparameters. At 0.25, 0.5, 1, 2, 4, 6 and 8 h after injection of BP,
blood was collected from the tail vein on the reverseside of the one used for injection. The concentration
of BP in plasma was measured by enzyme immunoassay (MARKIT-M Bromperidol, Dainippon).
Pharmacokinetic parameters for BP were obtained from plasma concentration±time pro®les. Area under
the curve (AUC) was determined by the trapezoidal rule. The values were extrapolated to in®nity by
dividing the last measured serum concentration by b, the slope of the terminal elimination phase (BP
concentration at 2, 4, 6 and 8 h after the administration) obtained by least-squares linear regression. t" #
/
}
In 2 b. The volume of distribution (V ) and clearance (Cl) were determined by
d
was calculated as t
" # 5
/
}
}
the following respective ratios: dose (AUC b) and dose AUC.
3
Statistical analysis
Results of the in vitro studies are expressed as means from duplicate experiments,
and results of the in vivo studies are expressed as mean SEM. Means of the latter
6
!
were compared with one-way analysis of variance followed by Scheåe test. p 0.05
was considered as statistically signi®cant.
Results
In vitro study
Figure 1 shows a chromatogram of 1.25 nmol of the standard compounds
including FBPA, HP, BP and RBP. FBPA formation followed Michaelis±Menten
} }
were 18.9 lm and 333.0 pmol min mg of protein
kinetics and the Km and V
respectively.
max
The reaction resulting in FBPA formation from BP (BP N-dealkylation activity)
was inhibited by 100 lm SKF-525A to 6.9% of the control and was minimal in the
absence of an NADPH-generating system (data not shown). Figure 2 shows the
eåect of four chemical inhibitors on BP N-dealkylation activity. Troleandomycin
(20 lm) inhibited BP N-dealkylation to 38% of the control, whereas 100 lm DDC
or furafylline only marginally inhibited the N-dealkylation. Of quinine, which
reduced dextromethorphan O-demethylation to 44% of the control, 2 lm slightly
inhibited BP N-dealkylation to 82% of the control.
Figure 3 shows the eåect of six anti-rat CYP sera on BP N-dealkylation activity.
Anti-rat CYP3A2 serum showed an 80% decrease in BP N-dealkylation activity,
whereas the other anti-rat CYP sera could not inhibit activity.

842
M. Watanabe et al.
Figure 1. Chromatogram of a mixture of standard compounds (1.25 nmol each). HP, haloperidol; BP,
bromperidol; RBP, reduced bromperidol; FBPA; 4-¯uorobenzoylpropionic acid. The left panel
was monitored at 245 nm; the right at 220 nm.
Figure 2. Eåects of various speci®c cytochrome P450 inhibitors on 4-¯uorobenzoylpropionic acid
(FBPA) formation in the hepatic microsomes of rat. Each point represents the mean of duplicate
determinations.
Figure 3. Eåects of various anti-CYP serum on 4-¯uorobenzoylpropionicacid (FBPA) formationin the
hepatic microsomes of rat. The immuno-inhibition of the FBPA formation was examined by pre-
incubation of rat liver microsomes (0.8 mg) with various anti-rat CYP sera (+ ) at room
temperature for 30 min before the reaction was started. Individual pre-immune serum (* ) was
pre-incubated under the same conditions as a controlfor the various anti-rat CYP sera. Each value
represents the mean of duplicate determinations.

Role of CYP3A in bromperidol metabolism
843
Figure 4. Eåect of erythromycin and dexamethasone on plasma bromperidol concentration. Six male
}
Sprague±Dawley rats were pretreated intraperitoneally with either erythromycin (100 mg kg per
}
day for 7 days, + ) or dexamethasone (80 mg kg per day for 2 days, E ) as a suspension in corn oil.
The corn oil vehicle alone (D ) was administered at the same condition as to the control group
}
(n 6). Rats received a single intravenous injection of 0.5 mg kg bromperidol in the tail vein.
5
Table 1. Eåect of erythromycin and dexamethasone on the pharmacokinetic parameters of
bromperidol.
t
AUC
(ng h ml)
Cl
(ml h kg)
V
d
/
("h#)
(ml kg)
}
} }
}
[
Control (n 6)
5
Erythromycin
(n 6)
5
Dexamethasone
2.43 0.12
176.7 11.5
2.89 0.20
10.3 1.0
6
6
6
6
3.34 0.20*
274.4 38.1* 1.95 0.19
9.3 1.0
6
6
6
6
1.42 0.08uu 127.2 13.8u 4.20 0.51u
8.6 1.1
6
6
6
6
(n 6)
5
Pharmacokinetic parameters for bromperidol were obtained from plasma
concentration±time pro®les (®gure 4).
t" #, elimination half-life; AUC, area under the plasma bromperidol
/
concentration; Cl, total body clearance; V , volume of distribution.
d
!
!
p
*p 0.05, ** p 0.01, signi®cantly diåerent from control.
u
uu
!
!
p
0.01,
0.001, signi®cantly diåerent from erythromycin.
Pharmacokinetic study
Plasma BP concentration±time pro®les are shown in ®gure 4; pharmacokinetic
parameters obtained from the pro®les are summarized in table 1. The t and AUC
/
" #
for BP were increased by erythromycin pretreatment to 137 and 155% of the
control, respectively and were reduced by dexamethasone pretreatment to 58 and
72% of the control respectively. Clearance of BP was reduced by erythromycin
pretreatment to 68% of the control and increased by dexamethasone pretreatment
to 145% of the control. These parameters were signi®cantly diåerent between
erythromycin and dexamethasone pretreatment groups.
Discussion
In the present study, CYP3A catalysed BP to FBPA in hepatic microsomes
of rat, and an inducer and an inhibitor of CYP3A altered BP pharmacokinetics
in rat. FBPA production was inhibited by SKF-525A (a non-speci®c inhibitor of

844
M. Watanabe et al.
CYP), and FBPA was hardly detected in the absence of an NADPH-generating
system, both of which suggest that one or more members of the CYP subfamilies
N-dealkylate(s) BP to FBPA. The eåects of various chemical inhibitors and anti-
bodies on N-dealkylation activity of BP were examined to investigate whether
CYP3A members are responsible for this reaction. Among the CYP inhibitors used
in this study, troleandomycin was the most eåective inhibitor of BP dealkylation
activity. Diethyldithiocarbamate (a CYP2E1 inhibitor), furafylline (a CYP1A in-
hibitor) and quinine (a CYP2D inhibitor) had little eåect on BP N-dealkylation.
Similar results were obtainedin the immuno-inhibitionstudy.Only anti-rat CYP3A
serum inhibited BP N-dealkylation (80%), whereas other anti-rat CYP sera
were essentially without eåects. These results suggest that CYP3A members play
a primary role in the N-dealkylation from BP to FBPA in rat.
In rat liver microsomes, quinine is approximately 50 times more potent than
quinidine as a CYP2D inhibitor (Kobayashi et al. 1989). Quinine (2 lm) slightly
inhibited BP N-dealkylation in this study, whereas the same concentration of
quinine inhibited dextromethorphan O-demethylation activity, a probe of CYP 2D
activity (Kupfer et al. 1984, Kerry et al. 1993), to 44% of the control. Suzuki et al.
(1997) reported that thioridazine, which increased the debrisoquine metabolic ratio
(Spina et al. 1991), co-administration with BP did not aåect the plasma con-
centration of BP. This previous study and the ®ndings of our present study suggest
that CYP2D may play a minor role in BP N-dealkylation. Some researchers
considered the CYP2D6 isoenzyme to be involved in the metabolism of HP in
human (Tyndale et al. 1991, Llerena et al. 1992). Recent studies, however, have
reported that both the N-dealkylation of HP and the oxidation of RHP back to HP
are mainly catalysed by CYP3A4 and not CYP2D6 in human (Fang et al. 1997, Pan
et al., Kudo and Odomi 1998).
The present authors also investigated whether an inhibitor or an inducer of
CYP3A alters BP pharmacokinetics in rat. In previous studies, treatment with
}
100 mg kg erythromycin for 7 days or with 80 mg kg dexamethasone for 2 days
aåected CYP3A activity (Kolars et al. 1992, Murray et al. 1992). In our present
}
study, the t of BP was signi®cantly prolonged by treatment of erythromycin, a
/
" #
CYP3A inhibitorand was signi®cantly shortened by treatment of dexamethasone, a
CYP3A inducer. Since it was not evaluated which CYP isoform was involved in the
N-dealkylation of BP at the low concentration observed in the pharmacokinetic
studies, it is diæcult to know whether CYP3A plays a major role in the intact rat.
However, BP metabolism was aåected by an inducer and an inhibitor of CYP3A. If
other isoforms rather than CYP3A play a main role in BP metabolism, BP
pharmacokinetics should be unaåected by an inducer or an inhibitor of CYP3A. BP
clearance was reduced by erythromycin and increased by dexamethasone. These
results indicate that in vivo alteration of CYP3A activity aåects BP metabolism and
pharmacokinetics, and suggest that the transformation from BP to FBPA, for which
CYP3A is responsible, is one of the major steps in the metabolism of BP in rat. In
previous clinical studies, carbamazepine, an inducer of CYP3A, signi®cantly
decreased plasma concentrations of BP (Otani et al. 1997). These previous studies
and the ®ndings of the present study suggest that CYP3A contributes more than
CYP2D to the metabolism and pharmacokinetics of BP.
In conclusion, it has been shown that CYP3A N-dealkylated BP to form FBPA
in rat and that alteration of this enzyme activity aåected BP pharmacokinetics in rat.
Although a simplistic extrapolation of these results to humans is inappropriate, the

Role of CYP3A in bromperidol metabolism
845
results imply that the modi®cation of in vivo CYP3A activity aåects BP pharmaco-
kinetics. Since a number of drugs and xenobiotics are reported to aåect in vivo
CYP3A activity (Ameer et al. 1997, Thummelet al. 1998) and since therapeutic and
toxic eåects of BP are dependenton its plasma concentration, precaution is required
in co-administering such a drug or xenobiotic with BP.
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846
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