Involvement of CYP2D1 in the metabolism of carteolol by male rat liver microsomes

Article abstract of DOI:10.1080/004982597239886

1. The metabolism of carteolol, a β-adrenoceptor blocking drug, was investigated in male Sprague-Dawley rat liver microsomes. 2. The formation of 8-hydroxycarteolol was the principal metabolic pathway of carteolol in vitro and followed Michaelis-Menten kinetics with a K(m) = 11·0 ± 5·4 μM and a V(max) = 1·58 ± 0·64 nmol/min/nmol P450 respectively (mean ± SD, n = 5). Eadie-Hofstee plot analysis of carteolol 8-hydroxylase activity confirmed single-enzyme Michaelis-Menten kinetics. 3. The cytochrome P450 isoforms involved in 8-hydroxylation of carteolol were investigated using selective chemical inhibitors and polyclonal anti-P450 antibodies. Quinine (K(i) = 0·06 μM)) and quinidine (K(i) = 2·0 μM)), selective inhibitors of CYP2D1, competitively inhibited 8-hydroxycarteolol formation. Furthermore, only anti-human CYP2D6 antibody inhibited this reaction. 4. These results suggest that carteolol is metabolized to 8-hydroxycarteolol by CYP2D1. The K(m) of carteolol for CYP2D1 in male rat liver microsomes was much greater than these of propranolol or bunitrolol, indicating that carteolol has a lower affinity for CYP2D1 compared with these other β-adrenoceptor blocking drugs.

Full text of DOI:10.1080/004982597239886

xenobiotica , 1997, vol. 27, no. 11, 1121±1129
Involvem ent of CYP2D1 in the m etabolism of
carteolol by m ale rat liver m icrosom es
K. UMEHARA*,S. KUDO and M . ODOMI
Tokushima Research Institute, Otsuka PharmaceuticalCo., Ltd, 463±10 Kagasuno,
Kawauchi-cho, Tokushim a 771-01, Japan
Received 5 May 1997
1
. The metabolism of carteolol, a b-adrenoceptor blocking drug, was investigated in
male Sprague-Dawley rat liver microsomes.
. The formation of 8-hydroxycarteolol was the principal metabolic pathway of
2
carteolol in vitro and followed Michaelis±Menten kinetics with a Km ¯ 11±0³5±4 lm and a
Vmax ¯ 1±58³0±64 nmol}min}nmol P450 respectively (mean³SD, n ¯ 5). Eadie±Hofstee
plot analysis of carteolol 8-hydroxylase activity con®rmed single-enzyme Michaelis±
Menten kinetics.
3. The cytochrome P450 isoforms involved in 8-hydroxylation of carteolol were
investigated using selective chemical inhibitors and polyclonal anti-P450 antibodies.
Quinine (K ¯ 0±06 lm) and quinidine (K ¯ 2±0 lm), selective inhibitors of CYP2D1,
competitively inhibited 8-hydroxycarteolol formation. Furtherm ore, only anti-hum an
CYP2D6 antibody inhibited this reaction.
i
i
4
. These results suggest that carteolol is metabolized to 8-hydroxycarteolol by
CYP2D1.The Km of carteolol for CYP2D1in male rat liver microsomeswas much greater
than those of propranolol or bunitrolol, indicating that carteolol has a lower aænity for
CYP2D1 compared with these other b-adrenoceptor blocking drugs.
Introduction
Carteolol hydrochloride is a b-adrenoceptor blocking drug and used for the
treatment of essential hypertension,angina pectoris and pulsus irregularisclinically
(
Nakano and Sawada 1976, Kaneko 1977, Toi and Yabuki 1977). Carteolol is
eliminated principally by metabolism in rat and ®ve metabolites have been identi-
ed (®gure 1 ; Mori et al. 1977). 8-Hydroxycarteolol and its glucuronide are
®
major metabolites and after oral administration of carteolol to rat these major
metabolitesare excreted in urine and faeces via the bile. Dihydroxycarteolol and its
glucuronide are highly excreted in faeces. Small amounts of 5,8-dihydroxy-3,4-
dihydrocarbostyril have also been detected in the urine and faeces.
Smith (1991) reported that many b-adrenoceptor blocking drugs are substrates
of the CYP2D subfamily. Propranolol 4-, 5- and 7-hydroxylation are catalysed by
the CYP2D subfamilyin rat (Masubuchiet al. 1993) and cytochromeP450 isoforms
in the CYP2D subfamily are known to be genetically controlled in rat and man.
Defects in expression of human CYP2D6 are found in C 7 % of the Caucasian
population (Alvan et al. 1990) and similar defects in the expression of CYP2D1 are
found in the Dark Agouti rat strain (Matsunaga et al. 1989). This genetic
polymorphism has been clinically observed in propranolol 4-hydroxylation, the
major metabolic pathway of propranolol in man (Lennard et al. 1984, Raghuram et
al. 1984, Ward et al. 1989). The CYP2D subfamily is therefore a group of key-
enzymesthat in¯uencesdrug dispositionbecausethey metabolizea numberof drugs
*
Author for correspondence.
049±8254}97 $12±00 ’ 1997 Taylor & Francis Ltd
0

1
122
K. Umehara et al.
Figure 1. Proposed metabolic pathway of carteolol in rat.
other than b-adrenoceptorblockingdrugs and genetic polymorphismis found in the
expression of these proteins.
Although carteolol is extensively metabolized in rat, the enzymatic system
involved in carteolol metabolism has not yet been studied. In the current study, the
biotransformation of carteolol to 8-hydroxycarteolol, the major metabolic pathway
in vivo, has been characterized in vitro using rat liver microsomes.This reaction was
inhibited by quinine and an anti-human CYP2D6 antibody, indicating that a rat
CYP2D isoform (CYP2D1)is involvedin carteolol 8-hydroxylation.In addition,we
have examined the aænity of carteolol for CYP2D1 in comparison with other b-
adrenoceptor blocking drugs.
M aterials and m ethods
Chemicals and reagents
Carteolol hydrochlorideand 8-hydroxycarteolol hydrochloridewere obtained from Otsuka Pharm a-
ceutical Co (Tokyo, Japan). p-Aminobenzoic acid, quinidine sulphate dihydrate and sodium N,N-
diethyldithiocarbamate trihydrate were purchased from Wako Pure ChemicalIndustries(Osaka, Japan).
b-NADPH,b-NADH,troleandomycinand sulphaphenazolewere purchased from Sigma ChemicalCo.
(
St Louis, MO, USA). Furafylline was purchased from Ultra®ne Chemical Co. (Manchester, UK).
Cimetidinewas purchasedfrom Research BiochemicalsInc. (Natick,MA, USA).Quininewas purchased
from Extrasynthese (Genay, France). Anti-rat CYP1A2, 2C11, 2E1 and 3A2 and anti-human CYP2D6
antibodieswere obtained from DaiichiPure Chemicals(Tokyo, Japan). Polyclonalantibodiesagainst the
puri®ed rat CYP1A2, 2C11, 2E1 or 3A2 were raised in goat and rabbit as reported previously (Imaoka
et al. 1990, Isogai et al. 1993, Neville et al. 1993). Human CYP2D6 was puri®ed from microsomes
derived from human AHH-1 TK­}® cells expressing human CYP2D6 (Gonzalez et al. 1988) and
polyclonal antibody against this protein was raised in rabbit. This antibody cross-reacted with rat
CYP2D isoforms (personal communication with M. Isogai of Daiichi Pure Chemicals). All other
reagents and solvents were of analytical or hplc grade and were used without further puri®cation.
Animals
Male Sprague-Dawleyrats (140±220 g) obtained from Charles River, Inc. (Japan), were housed in an
air-conditioned room at constant temperature (21±25 °C) and humidity (50±70%) under a 12-h
light}dark cycle (light: 07 :00±19:00 hours). The animals were fed a standard laboratory diet (MF,

Carteolol hydroxylation by CYP 2D1
1123
Oriental Yeast Co., Tokyo, Japan) and water ad libitum. Five individual rats were used for all
experiments, aged 7 weeks.
Microsome preparation
The rats were killed by decapitation.The livers were immediatelyremoved,washed in cold saline and
1
±15 % potassium chloride, weighed, minced and homogenized in 3 vol cold 1±15% potassium chloride
using a Potter±Elvehjemglass±Te¯ontissue homogenizer.The homogenatewas centrifugedat 9000 g for
2
6
0 min at 4 °C. To prepare livermicrosomes,the 9000 g supernatantwas then centrifugedat 100000 g for
0 min at 4 °C. The microsomal pellet was resuspended in 7±5 ml 0±05 m potassium phosphate buåer
(
pH 7±4) and the microsomes stored at ®80 °C. Cytochrome P450 content was measured spectro-
photometorical ly according to Omura and Sato (1964) and the protein concentration was determined
using Bio-Rad DC Protein Assay Kits (CA, USA).
Assay of carteolol 8-hydroxylase activity
The reaction mixtures contained0±1 m potassium phosphatebuåer (pH 7±4), 5 mm b-NADPH,5 mm
b-NADHand carteololhydrochloridein a ®nal incubationvolumeof 0±5 ml. Carteololhydrochloridewas
dissolved in water. The tubes were preincubatedfor 2 min at 37 °C ; all reactions started by the addition
of microsomal protein (0±11±0±29 mg) and were carried out in air at 37 °C in a shaking water bath for
1
5
0±20 min.For all experimentsthe reaction was stopped by adding0±5 ml 20 % (w}v) Na CO containing
# $
00 lg}ml p-aminobenzoic acid as an internal standard and 20 mg}ml sodium bisulphite. Under the
conditions used, the formation of 8-hydroxycarteolol increased linearly with time (5±30 min) and with
protein concentration (0±03±0±55 mg). In experiments to determine the kinetic parameters for 8-
hydroxycarteolol formation, carteolol concentrations of 2±5±100 lm were studied.
To examine the eåects of cytochrome P450 inhibitors on the formation of 8-hydroxycarteolol,
substrate concentration of 10 lm similar to the respective Km, was selected. Compounds screened for
possible inhibitory eåects are listed in table 2 and four inhibitor concentrations, generally 0±1, 1, 10 and
1
00 lm, were used. The percent of activity remainingwas calculated relative to controls run on the same
day without inhibitors (100%). Inhibitors in methanol were added to incubations in a volume of 10 ll.
Respective controls were incubated with the same volume of methanol. The reaction mixtures with or
without inhibitors were preincubated at 37 °C for 2 min and the reaction started by the addition of
microsomalprotein. The inhibitionconstant (K ) for quinine and quinidineand the nature of inhibition
i
were characterized.Three substrate concentrations (Km, 2¬Km and 4¬Km) were used to determ ineKi.
The immunoinhibiti on of the 8-hydroxylationof carteolol was examinedby preincubationof rat liver
microsomes (0±14 mg) with 50 ll of the antiserum of various titers diluted with normal goat or rabbit
serum and 0±1 m phosphate buåer (pH 7±4) for 30 min at room temperature. To this mixture, 10 lm
carteolol hydrochloride,5 mm b-NADPH, and 5 mm b-NADH were added in a ®nal volume of 0±5 ml.
The reaction was conducted for 20 min at 37 °C.
The reaction mixtures were extracted with 5 ml ethyl acetate, the upper organic layer transferred to
a clean glass tube and dried under a stream of nitrogen.The residue was dissolved in 100 ll mobile phase
and analysed by a Waters hplc system. An aliquot (20±30 ll) was injected on a TSK-gel ODS-80Ts
column (4±6¬250 mm, 5 lm, Tosoh Co., Tokyo, Japan). The mobile phase consisted of aceto-
nitrile: water:acetic acid (10±5: 89±5 :1 mainly used) and was pumped at 0±8 ml}min using a 510 Solvent
Delivery Module (Waters Associates, Milford, USA). The UV absorbance was monitored at 254 nm
with a Model 484 or 486 detector (Waters). The retention times for 8-hydroxycarteolol, p-aminobenzoic
acid and carteolol were 10±4, 11±7, and 17±7 min respectively.
Data analysis
Estimatesof apparentKm and Vmax were calculatedby plotting the reciprocalvelocityof the formation
of metabolite against the reciprocal concentration of substrate (Lineweaver±Bu rk plots). The results
obtained were expressed as mean³SD. Monophasicity was con®rmed with Eadie±Hofstee plots. The
method of Dixon (1953) was used to calculate inhibition constants (Ki).
Results
Carteolol metabolism by rat liver microsomes
When carteolol was incubated with male rat liver microsomesin the presence of
the NADPH,8-hydroxycarteolol was formed mainly (®gure 2). The formation of 8-
hydroxycarteolol was consistent with Michaelis±Menten kinetics and the Eadie±
Hofstee plot of carteolol 8-hydroxylase activity demonstrated monophasic

1
124
K. Umehara et al.
Figure 2. Reversed-phase hplc-U V pro®le (254 nm) of an ethyl acetate extract from an incubation of
carteolol and male rat liver microsomes for 10 min.
Figure 3. Typical Eadie±Hofsteeplot for the 8-hydroxylationof carteolol by male rat liver microsomes.
Each value of duplicate determinationsusing microsomes from a single rat liver is shown.
Michaelis±Menten kinetics (®gure 3). Table 1 presents the cytochrome P450
content of microsomes and kinetic constants for carteolol 8-hydroxylase activity.
Apparent Km and Vmax were 11±0³5±4 lm and 1±58³0±64 nmol}min}nmol P450
respectively (mean³SD, n ¯ 5).
Eåect of cytochrome P450 inhibitors and antibodies
Selective cytochrome P450 inhibitors were used in this study to determine the
potential roles of cytochromeP450 isoforms on the formation of 8-hydroxycarteolol
(
table 2). Both quinine and quinidine,known to be selective inhibitors of CYP2D1

Carteolol hydroxylation by CYP 2D1
1125
Table 1. Michaelis±Menten parametersfor 8-hydroxycarteolol formationby malerat livermicrosomes.
Vmax
Animal P450 content
no.
K
(lm)
(nmol}min}
m
(nmol}mg)
nmol P450)
1
1±05
1±03
0±94
1±04
1±15
1±04
0±07
6±2
17±9
10±7
14±6
5±3
1±01
2±46
1±44
1±99
0±99
1±58
0±64
2
3
4
5
Mean
SD
11±0
5±4
Table 2. Eåect of various compoundson carteolol 8-hydroxylase activity in male rat liver microsomes.
Control activity
%
Compound
0±1 lm
1 lm
113
10 lm
100 lm
Furafylline
Sulphaphenazole
Cimetidine
120
90
98
90
99
96
115
102
54
108
98
Diethyldithiocarbamate
Quinidine
Quinine
102
98
108
63
101
10
93
Ð ‹
6
78
89
26
108
12
111Œ
Troleandomycin
106
The control carteolol 8-hydroxylase activities were 0±449, 0±449, 0±298, 0±498, 0±288, 0±240 and
±498 nmol}min}nmol P450 for furafylline, sulphaphenazole, cimetidine, diethyldithiocarbamate, qui-
0
nidine, quinine and troleandomycin inhibitory assay respectively. Values are the average of duplicate
determinationsusing microsomes from a single rat liver (control ¯ 100%).
‹
Not determined.
Œ n ¯ 1.
Figure 4. Dixon plots for the inhibitionof carteolol 8-hydroxylase activity by quinine and quinidinein
male rat liver microsomes. Each experiment was performed in duplicate using microsomes from
a single liver and each value is shown.(A) Quinine,(B) quinidine.Concentrationsof carteolol were
1
0 (E ), 20 (_ ) and 40 lm (+ ).

1
126
K. Umehara et al.
Figure 5. Eåect of antisera on carteolol 8-hydroxylase activity in male rat liver microsomes.Results are
presented as the remainingactivity relative to control samplestreated with non-im munizedserum
and are the average of duplicate determinationsusing microsomesfrom a single liver. The control
carteolol 8-hydroxylase activities were 0±737, 0±604, 0±673, 0±717 and 0±819 nmol}min}nmol P450
for anti-rat CYP1A2,2C11, 2E1, 3A2, and anti-humanCYP2D6inhibitoryassay respectively.D ,
Anti-rat CYP1A2 (0±26±3 mg protein}nmol P450); + , anti-rat CYP2C11 (0±35±5 mg prot-
ein}nmol P450); E , anti-hum an CYP2D6 (0±5±5 mg protein}nmol P450); * , anti-rat CYP2E1
(
0±44±1 mg protein}nmol P450); and _ , anti-rat CYP3A2 (0±26±7 mg protein}nmol P450).
(
Kobayashiet al. 1989), inhibitedthe 8-hydroxylationof carteolol by approximately
0 % at 10 lm . Apparent K for quinine and quinidine,calculatedfrom Dixon plots,
9
i
were 0±06 and 2±0 lm respectively (®gure 4). The type of inhibition as judged from
Dixon plots was competitive. Cimetidine inhibited the carteolol 8-hydroxylase
activity by 46 % at 100 lm. Furafyllin (an inhibitor of CYP1A2; Sesardic et al.
1
990), sulphaphenazole (an inhibitor of CYP2C6; Correia 1995), diethyldithio-
carbamate (an inhibitorof CYP2E1; Brady et al. 1991, Guengerichet al. 1991), and
troleandomycin (an inhibitor of CYP3A2; Wrighton et al. 1985, Sonderfan et al.
1
987) had no eåect on carteolol 8-hydroxylase activity in male rat liver microsomes.
Male rat liver microsomes were incubated with polyclonal antibodies against
various cytochrome P450 isoforms. As shown in ®gure 5, preincubationof male rat
liver microsomeswith anti-human CYP2D6antibody result in signi®cantinhibition
of the 8-hydroxylase activity. Anti-rat CYP1A2,CYP2C11,CYP2E1 and CYP3A2
antibodies did not inhibit the reaction.
Discussion
Despite the fact that carteolol is eliminatedprincipallyby metabolism in rat and
the formation of 8-hydroxycarteolol is the initial step (Mori et al. 1977), there is no
informationidentifyingthe enzymesinvolvedin carteolol metabolismor the kinetics
of metabolism.As the expression and activity of cytochromeP450 isoformsin¯uence
the drug disposition, identifying the enzymes catalysing carteolol metabolism will
contribute to our understanding of factors in¯uencing carteolol disposition. In the
current study, we have shown that the biotransformation of carteolol is catalysed by
male rat liver microsomesand identi®edcytochromeP450 isoform(s)responsiblefor
its metabolism.

Carteolol hydroxylation by CYP 2D1
1127
Carteolol was metabolized to 8-hydroxycarteolol in the microsomal incubates,
dependenton the presence of NADPH.Almost no other metaboliteswere observed
in any microsomalincubates, indicatingthat the formation of 8-hydroxycarteolol is
probably the initial step in the metabolism of carteolol in vitro. In all ®ve livers
studied,the formation of 8-hydroxycarteolol was consistentwith Michaelis±M enten
kinetics. Eadie±Hofstee analysis of carteolol 8-hydroxylase activity con®rmed
single-enzyme Michaelis±Menten kinetics, indicating that only one cytochrome
P450 isoform is involved in the 8-hydroxylation of carteolol.
Characterizations of the cytochrome P450 isoform involved in 8-hydroxylation
of carteolol were investigated using selective chemical inhibitors and polyclonal
anti-P450 antibodies. Quinine and quinidine competitively inhibited the carteolol
8
-hydroxylationat low concentrations.The inhibitionconstant (K ) for quinineand
i
quinidinewere 0±06 and 2±0 lm respectively.The inhibitoryeåect of quinine on this
hydroxylation was 33-fold more potent than that of quinidine.Quinine is reported
to be a more potent inhibitor of debrisoquine 4-hydroxylation, a typical CYP2D
subfamily catalysed reaction, than quinidine in male rat liver microsomes (Koba-
yashi et al. 1989). Similar results were observed in the 8-hydroxylation of carteolol.
Steroselective inhibition by quinine and quinidine has been described for com-
pounds whose metabolism is catalysed by CYP2D1 (Kobayashi et al. 1989). The
involvementof CYP2D1 in this hydroxylation reaction is further supported by the
observation that only anti-human CYP2D6 antibody inhibited this reaction.
On the other hand, as it is reported that quinidine inhibits CYP3A activity at
higher concentrations (Newton et al. 1995), there is a possibility that CYP3A2 is
involved in carteolol 8-hydroxylation. However, the inhibition of carteolol 8-
hydroxylase activity by troleandomycin or anti-rat CYP3A2 antibody were not
observed, indicating that CYP3A2 is not involved in this reaction.
Cimetidine, a speci®c inhibitor of CYP2C11, also inhibited carteolol 8-
hydroxylase activity at 100 lm. However, cimetidine is reported to be a speci®c
inhibitor of CYP2C11 only at low concentrations (% 50 lm) and at high concen-
trations is less speci®c (Chang et al. 1992). Furthermore, anti-rat CYP2C11
antibody did not inhibit this activity. These observations suggested that CYP2C11
is not involved in the carteolol 8-hydroxylase activity.
Enzymes of the CYP2D gene subfamily are known to metabolize only
compounds with one or more basic nitrogen atoms (Koymans et al. 1992).
Hydroxylation is usually directed towards the para position of their aromatic rings
or to alkyl substituents situated therein (Smith 1991). b-adrenoceptor blocking
drugs, being arylalkylamine s, such as propranolol, bunitrolol, bufurolol and
metoprolol,are typical substrates of the CYP2D subfamily (Brosen 1990, Suzuki et
al. 1992). Rat CYP2D isoforms catalyse propranolol 4-, 5- and 7-hydroxylation
(
M asubuchi et al. 1993) and bunitrolol 4-hydroxylation (Suzuki et al. 1992). In the
current study, we have demonstratedthat carteolol aromatichydroxylationat the C-
para position of the substrate, to give 8-hydroxycalteolol, is catalysed by CYP2D1
8
in male rat liver microsomes.Carteolol is also a b-adrenoceptor blocking drug, and
has two basic nitrogen atoms. Therefore, carteolol would be predicted to be a typical
substrate of the CYP2D subfamily, a prediction con®rmed by our current studies.
Masubuchi (1993) reported that the Km and Vmax for propranolol 4-hydroxy-
lation in male Wistar rat liver microsomes were 0±225 lm and 0±230 nmol}min}mg
respectively, and Suzuki (1992) reported that the Km and Vmax for bunitrolol
4
-hydroxylation in male Sprague-Dawley rat liver microsomes were 0±84 lm and

1
2
128
K. Umehara et al.
±05 nmol}min}nmol P450 respectively.In contrast, the Km for carteolol (11 lm) in
our studies was much greater than those for propranolol or bunitrolol, indicating
that carteolol has a lower aænity for the CYP2D1 compared with these other b-
adrenoceptor blocking drugs.
In summary,our results from these studiesindicatedthat carteololis metabolized
to 8-hydroxycarteolol by CYP2D1 in male Sprague-Dawley rat liver microsomes.
The aænity of carteolol for CYP2D1is lower among b-adrenoceptorblocking drugs
by comparison of their respective Km.
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