Tokushima Research Institute, Otsuka Pharmaceutical Co., Ltd,
Tokushima, Japan (S.K., H.O., G.M.); and
Department of Pharmacology and
Therapeutics, Graduate School of Clinical Pharmacy, Kumamoto
University, Kumamoto, Japan (T.I.)
Cytochrome P-450 (CYP) isoforms responsible for the cleavage of
Hantzsch pyridine ester at the 3-position of pranidipine were studied
in vitro using cDNA-expressed human CYP enzymes. CYP1A1, 1A2, 2D6, and
3A4 cleaved the ester with a catalytic activity of 5.5, 0.93, 13.1, and
22.4 nmol/30 min/nmol P-450, respectively. CYP2A6, 2B6, 2C8, 2C9, 2C19,
and 2E1 were not involved in the de-esterification. The
Km and Vmax
values for the de-esterification were 11.8 µM and 0.47 nmol/min/nmol
P-450 in the CYP2D6-catalyzed reaction and 8.7 µM and 0.84 nmol/min/nmol P-450 in the CYP3A4-catalyzed reaction. The intrinsic
clearance
(Vmax/Km) of the
de-esterification by CYP3A4 was 2-fold greater than that by CYP2D6.
Quinidine almost completely inhibited the CYP2D6-mediated
de-esterification at the concentration of 1 × 10
6
M. Ketoconazole and troleandomycin inhibited the CYP3A4-mediated reaction in a dose-related manner. The results indicate that although the multiple CYP isoforms can catalyze the de-esterification, CYP3A4
and 2D6 are the major isoforms.
 |
Introduction |
Pranidipine,
(±)-methyl 3-phenyl-2(E)-propenyl
1,4-dihydro-2,6-dimethyl-4-(3-nitrophenyl)-3,5-pyridinedicarboxylate
(Fig. 1), is a potent and long-acting 1,4-dihydropyridine calcium
antagonist (Nakayama et al., 1990
, 1991a; Mori et al., 1993), which is
being developed as a therapeutic agent for the treatment of essential hypertension and angina pectoris (Broadhurst et al., 1991; Nakayama et
al., 1991b).
Several metabolic pathways of pranidipine have been identified in
animals and humans. These include the dehydrogenation of the
1,4-dihydropyridine to the pyridine ring, hydrolysis of the carboxyl
acid ester, hydroxylation of the methyl group accompanied by lactone
formation, and glucuronide conjugation of the phase I reactants (Sasabe
et al., 1993; Fujita et al., 1994).
In humans, methyl
2,6-dimethyl-4-(3-nitrophenyl)-3-carboxy-5-pyridinecarboxylate
(OPC-13463)1 is one of the main metabolites of pranidipine
(Fig. 1), and the plasma level of the metabolite is approximately five
times higher than that of unchanged pranidipine (Sasabe et al., 1993;
Fujita et al., 1994). Two possible metabolic pathways exist for the
production of OPC-13463 from pranidipine: one is mediated via the
formation of (±)-methyl
1,4-dihydro-2,6-dimethyl-4-(3-nitrophenyl)-3-carboxy-5-pyridine carboxylate (MOP-13031), and the other is via that of dehydrogenated pranidipine (Fig. 1). Our recent findings have revealed that cytochrome P-450 (CYP) 3A4 plays an important role in the biotransformation of pranidipine to dehydrogenated pranidipine and of MOP-13031 to
OPC-13463 (Kudo et al., 1998). The intrinsic clearance of
MOP-13031 to OPC-13463 by the enzyme is much lower than that of
pranidipine to dehydrogenated pranidipine, suggesting that the primary
route for OPC-13463 formation is via hydrolysis of dehydrogenated
pranidipine (Fig. 1). However, no information is available regarding
the enzyme(s) involved in the metabolic conversion of dehydrogenated
pranidipine to OPC-13463.
The aims of this study were to determine whether the biotransformation
of dehydrogenated pranidipine to OPC-13463 was mediated by CYP
isoform(s) with 10 human cDNA-expressed CYPs, and if so, to establish
which CYP isoform(s) catalyze the biotransformation reaction.
| |
Materials and Methods |
Chemicals and Reagents.
Dehydrogenated pranidipine, OPC-13463, and
3,4-dihydro-5-methoxy-2(1H)-quinolinone were provided by Otsuka
Pharmaceutical Co., Ltd. (Tokyo, Japan). Quinidine, 
-NADPH
tetrasodium salt, and -NADH disodium salt were purchased from Sigma
Chemical Co. (St. Louis, MO). Ketoconazole and troleandomycin were
obtained from Biomol Research Laboratories Inc. (Plymouth Meeting, PA). All other reagents and solvents were of an analytical grade.
Human cDNA-Expressed CYP.
Microsomes derived from human AHH-1 TK ± cells expressing human
CYP were purchased from Gentest Corp. (Woburn, MA). The following microsomes were used in this study: control microsomes containing a
vector; CYP1A1 and P-450 reductase; CYP1A2; CYP2A6 and P-450 reductase;
CYP2B6; CYP2C8 and P-450 reductase; CYP2C9-cys and P-450 reductase;
CYP2C19; CYP2D6-val and P-450 reductase; CYP2E1 and P-450 reductase;
and CYP3A4 and P-450 reductase. The functional viability of each CYP
isoform was previously assessed (Kudo et al., 1997; Kudo and Odomi,
1998).
Enzyme Assays.
In the experiments to identify CYP isoform(s) involved in the cleavage
of the ester linkage of dehydrogenated pranidipine at the 3-position, a
0.5-ml reaction mixture containing 1 mg/ml microsomal protein
expressing 1 of 10 different forms of CYPs, 5 mM -NADPH, 2.5 mM
-NADH, and three different concentrations of dehydrogenated
pranidipine at 1, 10, or 100 µM in 100 mM phosphate buffer (pH 7.4)
was incubated for 30 min at 37°C.
The formation rate of OPC-13463 was determined over the dehydrogenated
pranidipine concentration range of 1 to 100 µM in a reaction mixture
containing 1 mg/ml microsomal protein expressing CYP2D6 or 3A4.
Dehydrogenated pranidipine was dissolved in N', N'-dimethylformamide (DMF), and the amount of DMF added to
the incubation mixture was 1% in all incubations. The incubation
mixture (0.45 ml), including substrate and cofactors, was preincubated, and then the reaction was initiated by the addition of a 50-µl microsomal suspension. Incubation was performed in an atmosphere of air
for 30 min. Under these conditions, the reaction rate was linear for at
least 30 min at an enzyme concentration of up to 1 mg of microsomal
protein/ml of incubate. The apparent
Km and Vmax values were calculated from a
nonlinear regression analysis with a computer program, WinNonlin
Standard (Version 1.5, Scientific Consulting, Inc., Apex, NC).
The effects of the following selective CYP inhibitors on cleavage of
the ester group in the 3-position of dehydrogenated pranidipine were
determined: quinidine (CYP2D6) (Inaba et al., 1985; Guengerich et al.,
1986b; Otton et al., 1988), troleandomycin (CYP3A4) (Newton et al.,
1995; Kudo et al., 1997), and ketoconazole (CYP3A4) (Baldwin et al.,
1995). The incubation volume was 0.5 ml and the concentration of
dehydrogenated pranidipine was 10 µM. The inhibitors were
preincubated with CYP2D6- or CYP3A4-expressed microsomes and cofactors
for 10 min, and the reaction was initiated by the addition of the substrate. The concentration ranges of the inhibitors were set up as
follows: 1 × 104~1 × 108M for
quinidine; 1 × 104~1 × 107 M
for troleandomycin; and 1 × 105~1 × 108 M for ketoconazole. To determine whether the
de-esterification of dehydrogenated pranidipine required P-450
activation, microsomal incubations were conducted with both NADPH and
NADH present, or neither present, in the reaction mixture.
Reactions were quenched by adding 0.5 ml of methanol that contained 0.5 µg/ml of 3,4-dihydro-5-methoxy-2(1H)-quinolinone, which
was used as an internal standard. Reaction-terminated samples were
centrifuged at 9500g for 10 min at 4°C, and the
supernatant was evaporated to dryness at 65°C for 2 h with a
model AES201 Automatic Environmental Speedvac (Savant Instruments Inc.,
Holbrook, NY). Extracted samples were dissolved in 100 µl of methanol
and then centrifuged at 140g for 10 min. Thirty microliters
of the resultant supernatant was analyzed by a high-performance liquid chromatography (HPLC) assay as described below.
HPLC Analysis.
The HPLC apparatus included two model 510 HPLC pumps (Waters, Milford,
MA), a model 712 auto sample processor (Waters), a model 680 solvent
programmer (Waters), a model 486 tunable absorbance detector (Waters),
and a Chromatopac C-R7A (Shimadzu, Kyoto, Japan). A TSK-gel
ODS-80TS column (4.6 mm i.d. x 150 mm, Tosoh,
Tokyo, Japan) equipped with a TSK-gel guardgel
ODS-80TS guard column (3.2 mm i.d. x 15 mm;
Tosoh) was used for the analysis. The mobile phase used for OPC-13463
was a solution of 10 mM phosphate buffer (pH 7.4) containing 10%
acetonitrile as solution A and the buffer containing 70% acetonitrile
as solution B. A linear gradient from 0 to 100% solution B over 45 min
was used for the determination of the compound. The flow rate was 1.0 ml/min, and UV detection was performed at 254 nm. The retention times
for the metabolite and the internal standard were 12.4 and 19.1 min, respectively.
The calibration curve for OPC-13463 was established by an internal
standard method, based on the peak area ratio between the metabolite
and internal standard, with a calibration range of 0.02 to 2 µg/ml in
an incubation medium containing cofactors and control microsomes at 1 mg/ml. The correlation coefficient of the curve was 0.9999, and the
quantitation limit was set at 0.02 µg/ml. Precision ranged from 0.5 to 4.5% relative S.D. and mean accuracy ranged from 1.2 to 8.0%
relative error of the mean. The recovery rates of OPC-13463 and the
internal standard were 103.1 to 110.0% and 83.5 to 93.0%,
respectively. No incubation mixture-derived constituents were found to
interfere with the determination of OPC-13463 in six independent
sources of the blank sample, and nonenzymatic hydrolysis of the
carboxylic acid ester of dehydrogenated pranidipine did not occur under
the analytical conditions described above.
| |
Results |
Enzyme Kinetics.
The formation rates of OPC-13463 by CYP1A1, 1A2, 2A6, 2B6, 2C8, 2C9,
2C19, 2D6, 2E1, and 3A4 at substrate concentrations of 1, 10, and 100 µM are shown in Fig. 2. CYP1A1, 1A2,
2D6 and 3A4 were all active in the de-esterification of dehydrogenated
pranidipine, with CYP3A4 showing the highest rates, followed by 2D6,
1A1, and 1A2. CYP2D6 and 3A4 were catalytically active at 1 µM
substrate, whereas CYP1A1 only showed appreciable activity at
concentration levels of 10 µM or higher, and CYP1A2 was only
effective at a substrate concentration of 100 µM (Fig. 2). CYP2A6,
2B6, 2C8, 2C9, 2C19, and 2E1 showed no detectable de-esterification
activity at any substrate concentration.
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Fig. 2.
Catalytic properties of human
cDNA-expressed CYPs on the cleavage of dehydrogenated pranidipine
ester.
|
|
Based on these data, we conducted in vitro experiments to assess the
kinetic parameters for the de-esterification of dehydrogenated pranidipine to OPC-13463 with substrate concentrations ranging from 1 to 100 µM in CYP2D6- or 3A4-expressed microsomes. Eadie-Hofstee plots
for OPC-13463 formation by either enzyme were monophasic. The apparent
Km and
Vmax values estimated with a nonlinear
regression analysis for the data on substrate concentration versus
initial velocity were 11.8 µM and 0.47 nmol/min/nmol P-450 for
CYP2D6, and 8.7 µM and 0.84 nmol/min/nmol P-450 for CYP3A4,
respectively. The intrinsic clearance, calculated as
Vmax/Km,
was 39.8 µl/min/nmol P-450 for CYP2D6 and 96.6 µl/min/nmol P-450
for CYP3A4, indicating that the value for CYP3A4 was about 2-fold
greater than that for CYP2D6.
Inhibition Studies.
The effects of CYP2D6- or 3A4-specific inhibitors on the
respective CYP-mediated de-esterifications of dehydrogenated
pranidipine to OPC-13463 were examined. The mean percentage of
inhibition data derived from the duplicate experiments are listed in
Table 1. Quinidine at a concentration of
1 × 106 M inhibited more than 92% of the
CYP2D6-mediated catalytic activity. Troleandomycin and ketoconazole
inhibited the formation of OPC-13463 catalyzed by CYP3A4 in a dose- or
concentration-dependent manner.
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TABLE 1
Effect of selective inhibitors on the de-esterification of
dehydrogenated pranidipine catalyzed by CYP2D6 or 3A4
|
|
Effects of Cofactors on De-Esterification Activity.
Effects of NADPH and NADH on the de-esterification activity of
microsomes expressing CYP2D6 or 3A4 are given in Table
2. In the CYP2D6-catalyzed reaction,
OPC-13463 was only formed in reactions that contained NADPH and NADH.
In the CYP3A4-catalyzed reaction, OPC-13463 was formed at the rate of
0.70 nmol/min/nmol P-450 when both NADPH and NADH were in the reaction
mixture and at a rate of 0.05 nmol/min/nmol P-450 when both cofactors
were absent, indicating that the cofactors, particularly NADPH, are essential for the catalytic activity.
| |
Discussion |
OPC-13463, a major metabolite of pranidipine (Fig. 1), is produced
via two distinguishable metabolic processes, the dehydrogenation of the
Hantzsch dihydropyridine ring and the hydrolysis of the carboxylic acid
ester at the 3-position of the ring. These reactions (i.e., ring
oxidation and ester cleavage) appear to be generally common and
important metabolic pathways in the biotransformation of
1,4-dihydropyridine calcium antagonists, e.g., nifedipine (Kleinbloesem et al., 1984), nicardipine (Rush et al., 1986), felodipine (Hoffmann and Andersson, 1987), nitrendipine (Krol et al., 1987), nisoldipine (Scherling et al., 1988), and amlodipine (Beresford et al., 1988).
CYP has been shown to catalyze both the dehydrogenation of the
dihydropyridine ring, and the oxidative cleavage of the carboxylic acid
esters of the Hantzsch pyridine esters to the corresponding carboxylic
acids in vitro (Guengerich, 1987; Guengerich et al., 1988) and in vivo
(Funaki et al., 1989). However, any CYP isoform(s) involved in
the cleavage of the ester linkage has, to our knowledge, not been
characterized, particularly in humans.
To identify any CYP isoform(s) involved in the ester cleavage reaction
of pranidipine, 10 isoforms of cDNA-expressed human CYP were examined
in this study. The results indicated that the cleavage of the ester
linkage was catalyzed by multiple CYP isoforms that included CYP1A1,
1A2, 2D6, and 3A4, but not CYP2A6, 2B6, 2C8, 2C9, 2C19, or 2E1. Isoform
specificity for the de-esterification of
2,6-dimethyl-4-phenyl-3,5-pyridinedicarboxylic acid diethyl ester from
rat liver P-450 has also been determined (Guengerich, 1987; Guengerich
et al., 1988).
OPC-13463 formation activity was greatest with CYP3A4, followed by 2D6,
1A1, and 1A2, in that order (Fig. 2). Kinetic parameters were only
estimated for the CYP2D6- and 3A4-catalyzed reactions because only
these two isoforms had appreciable catalytic activity at low substrate
concentrations (Fig. 2). The Eadie-Hofstee plots for OPC-13463
formation from either of the isoforms was monophasic, indicating a
single enzyme catalytic reaction. Substrate inhibition, such as
that observed in the CYP3A4-catalyzed oxidation of nifedipine (Guengerich et al., 1986a) did not occur in these experiments up to a
substrate concentration of 100 µM, nor did side reactions. In
fact, methylhydroxylated OPC-13463 (methyl
2-hydroxy-4-(3-nitrophenyl)-6-methyl-3-carboxy-5-pyridinecarboxylate), 2-hydroxymethyl dehydrogenated pranidipine and demethylated
dehydrogenated pranidipine were not detected at all in any
chromatographic observations.
The intrinsic clearance
(Vmax/Km)
of the de-esterification of dehydrogenated pranidipine by CYP3A4 has an
approximate value close to that for the dehydrogenation of pranidipine
by the same enzyme (Table 3) and was 35 times greater than that for the CYP3A4-catalyzed conversion of
MOP-13031 to OPC-13463 (Fig. 1). In addition, Kudo et al. (1998) have
observed that pranidipine is not converted to MOP-13031 by P-450. Thus,
it was thought that the major metabolic pathway for OPC-13463 formation
should follow the sequence pranidipine to dehydrogenated pranidipine to
OPC-13463, and that the conversion of pranidipine to MOP-13031 mediated
possibly by esterase would be of a minor concern, if any, in the
formation of OPC-13463.
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TABLE 3
Comparison of kinetic parameter data on pranidipine dehydrogenation
and de-esterification of dehydrogenated pranidipine
|
|
Microsomes derived from human AHH-1 TK ± cells expressing human
CYPs were used in this study. It was unclear whether any esterase activity was present in this subcellular fraction. Thus, it was essential to confirm that CYP(s) was(were) involved in catalyzing the
de-esterification of dehydrogenated pranidipine. Therefore, chemical
inhibition experiments were conducted with specific CYP inhibitors. The
results confirmed that CYPs were involved in the catalytic reaction.
Furthermore, the CYP-related catalytic reaction only occurred when
NADPH and NADH were present in the incubation (Table 2), implying that
the de-esterification activity by the microsomes used in this study
originates from CYPs and not from esterase, thereby suggesting the
oxidative cleavage of the ester linkage rather than a simple
hydrolysis. Based upon these findings, we wish to propose the
hypothetical mechanistic scheme for oxidative ester cleavage shown in
Fig. 3. The proposed scheme involves a CYP-mediated hydroxylation of the carbon 
to the ester oxygen, as
described earlier by other workers (Guengerich, 1987; Guengerich et
al., 1988; Peng et al., 1995).
CYP3A4, which has very broad substrate specificity due to a large and
flexible binding site, can catalyze a variety of oxidative reactions
including hydroxylation, dehydrogenation, N- and
O-dealkylation, N- and S-oxidation,
hydroxyl oxidation, quinone formation, and epoxidation (Guengerich,
1995; Surapaneni et al., 1997; Kudo and Odomi, 1998). In this study, we
have observed that CYP3A4 can also catalyze the oxidative cleavage of a
carboxylic acid ester. To our knowledge, this is the first report of
the involvement of CYP3A4 in the cleavage of the ester linkage,
although the roles of CYP2E1 and 2B4 responsible for the oxidative
cleavage of a series of aliphatic esters have been noted in rabbit
liver microsomes (Peng et al., 1995).
In the CYP2D6-mediated catalytic reactions, it has been suggested that
CYP2D6 requires ion-pair formation between a substrate and the enzyme
for effective catalytic activity (Smith, 1991; Smith and Jones, 1992)
and, hypothetically, a positively charged basic nitrogen (pKa > 7.5) located 5 to 7 Å from the site of oxidation for typical CYP2D6
substrates (Strobl and Wolff, 1991; Smith and Jones, 1992). Although
the structural modeling may be considered as tentative (Islam et al.,
1991), a molecular template appears to be of value in
determining drug substrates for the enzyme. This appears to be the case
for dehydrogenated pranidipine. The weakly basic pyridine nitrogen
(Fig. 1), which would be partially protonated at physiological pH, is
estimated to be 5.84 Å from the site of oxidation by the
three-dimensional structural model. The activity of CYP2D6 toward
oxidative ester cleavage would be applicable, not only to pranidipine
but also to other 1,4-dihydropyridine calcium antagonists because all
of the compounds biotransformed to the pyridine ring by dehydrogenation
appear to meet the defined structural requirement for the specificity
of the CYP2D6-catalyzed de-esterification.
Dehydrogenated pranidipine has two ester groups, one at the 3- and the
other at 5-position of the pyridine ring (Fig. 1). The intramolecular
distances between the nitrogen atom and the -carbon atom at the
3-and 5-positions of the pyridine ring are almost the same (5.84 Å and
5.78 Å, respectively). Nevertheless, CYP2D6, as well as CYP3A4,
selectively cleaved the ester linkage at the 3-position rather than at
the 5-position. The selectivity for de-esterification at the 3-position
over the 5-position is due probably to a difference in radical
stability in differentiating substituents of the cinnamylmethyl and
methyl groups.
In conclusion, the overall results obtained from the present in vitro
study demonstrate that the metabolic cleavage of Hantzsch pyridine
ester of pranidipine is mediated via the multiple CYP isoforms,
including CYP1A1, 1A2, 2D6 and 3A4. However, of the four isoforms,
CYP3A4 is the most catalytically effective for the de-esterification reaction.
We thank Dr. Jun Matsubara and Dr. Takeshi Hasegawa for their
assistance in modeling dehydrogenated pranidipine.
Abbreviations used are:
OPC-13463, methyl
2,6-dimethyl-4-(3-nitrophenyl)-3-carboxy-5-pyridinecarboxylate;
MOP-13031, (±)-methyl
1,4-dihydro-2,6-dimethyl-4-(3-nitrophenyl)-3-carboxy-5-pyridine
carboxylate;
CYP, cytochrome P-450;
DMF, N',
N'-dimethylformamide;
HPLC, high-performance liquid
chromatography.
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Abstract
Introduction
