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X.-x. Hu et al. / Drug Metabolism and Pharmacokinetics xxx (2016) 1e6
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been discovered, and 12 of them were named as *87e*93, *94A,
*94B and *95e*98.
2.3. Statistical analysis
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In the present study, we assessed the catalytic activities of 24
CYP2D6 alleles (*2, *10, *87e*98, R25Q, F164L, E215K, F219S,
V327M, D336N, V342M, R344Q, R440C, R497C) expressed in the
insect cell toward citalopram, thus proving valuable information
for further studies about CYP2D6 alleles for citalopram
metabolism.
MichaeliseMenten analysis was performed by non-linear
regression curve fitting using the computer program Prism v 5.0
(GraphPad Software Inc., San Diego, CA). Kinetic data for each
variant are presented as the mean ± S.D. of three microsomal
preparations. The one-way ANOVA was used for inter group com-
parison. Dunnett's test was used to analyze differences in catalytic
activity between CYP2D6*1 and other mutants. Statistical analyses
were performed with the SPSS package (version 19.0; SPSS Inc.,
Chicago, IL), with p < 0.05 considered to be statistically significant.
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. Materials and methods
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.1. Chemicals and materials
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. Results
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Citalopram and venlafaxine were obtained from Tokyo
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In our study, the catalytic activities of the wild-type CYP2D6*1
Chemical Industry Co., Ltd (Tokyo, Japan). Demethylcitalopram
and citalopram N-oxide were purchased from Toronto Research
Chemicals Inc. (TRC, Canada). The reduced nicotinamide adenine
dinucleotide phosphate (NADPH) were obtained from Sigma (St.
Louis, MO), P450 Cytochrome b5 microsomes and recombinant
human P450s CYP2D6 expressed in the microsomes from Spo-
doptera frugiperda (Sf) 21 insect cells were kind gifts from Beijing
Hospital. A RRHD Eclipse Plus C18 column used for high-
performance liquid chromatography (HPLC) was obtained from
the Agilent Technologies. Other reagents and organic solvents
were obtained from Chemical Industries (Beijing, China).
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and 24 allelic variants were assessed using citalopram as substrate.
MichaeliseMenten plots for each of the CYP2D6 variants are shown
in Figs. 1 and 2, and the corresponding kinetic parameters are
summarized in Tables 1 and 2. As shown in the figures and tables,
almost all of the variants exhibited changed K
compared to that of the wild-type protein. Therefore, the intrinsic
clearance (Vmax/K ) values for citalopram demethylation and
oxynitride were altered in all of the tested allelic variants, while the
clearance value of identical allelic variant for the two metabolites is
mainly consistent.
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or Vmax values as
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3.1. Demethylcitalopram
2.2. Conditions for enzymatic activity analysis
As shown in Fig. 1 and Table 1, two variants (CYP2D6*92 and
The incubation mixture consisted of recombinant microsomes
containing 5 pmol CYP2D6*1 or 5 pmol other CYP2D6 mutants,
5 pmol purified cytochrome b5 and 4.05
100 mmol/LPBS buffer (pH 7.4). Citalopram was initially prepared in
methanol solution, and the total concentration in the incubation
mixture was adjusted from 10 to 1000
of methanol was less than 0.5%. The reaction was allowed to pre-
incubate for 5 min in a Fisher shaking water bath. Then an NADPH
regenerating system was added to start the reaction at 37 C in a
final volume of 200 mL, and the mixture was incubated at the same
temperature for 30 min. Incubations were terminated by cooled
CYP2D6*96) were too week to produce demethylcitalopram,
resulting no detectable enzymatic activity. Except for CYP2D6*92
and CYP2D6*96, the remaining 22 defective alleles displayed much
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m
L citalopram in
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difference in K , Vmax or intrinsic clearance values. According to the
intrinsic clearance value compared with wild type, we classified the
remaining 22 defective alleles into three categories in regard to
demethylcitalopram. One variant (V342M) exhibited a decreased
m
M. The total concentration
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Vmaxvalue and a much lower K value with wild-type, resulting in
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higher intrinsic clearance (p < 0.01); four variants (CYP2D6*94,
E215K, F219S and V327M) showed no significant difference (1-fold)
in enzyme activity; the remaining 17 variants displayed signifi-
cantly reduced intrinsic clearance values (38e82% relative clear-
ance). As a result, eighteen of the 22 variants (CYP2D6*2, *10, *87,
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to ꢁ80 C immediately, and then add 50
mL 0.1 M HCl to the tubes
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when taking out of ꢁ80 C after 15 min approximately. Venlafaxine
(20 mL of 10 mg/mL in methanol solution) as an internal standard
*
88, *89, *90, *91, *93, *95, *97, *98, R25Q, F164L, D336N, V342M,
R344Q, R497C and R440C) had significant difference in intrinsic
clearance (Vmax/K ) values of citalopram demethylation compared
was added to the mixture followed by the addition of 0.8 mL acetic
ether. After vortexing for 2 min, the incubation mixture was
centrifuged at 12 000 rpm at 4 C for 10 min, the organic phase was
transferred into a clean tube, and it was dried under a nitrogen
stream. The resulting residue was dissolved in 100 mL mobile phase
and used for the following measurement of citalopram and its
metabolites. Incubations were performed in individual tubes for
each time and triplicate, and the data are presented by the mean
standard deviation (SD).
High-performance liquid chromatography (HPLC) was carried
out on an Agilent RRHD Eclipse Plus C18 column (3.0*100 mm,
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with the wild type (*p < 0.05, **p < 0.01).
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3.2. Citalopram N-oxide
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The MichaeliseMenten curves and the corresponding kinetic
parameter of CYP2D6 for citalopram N-oxide are shown in Fig. 2
and Table 2, respectively. Similar to the result for demethylcitalo-
pram, the kinetic parameter of citalopram N-oxide by the variants
CYP2D6*92 and CYP2D6*96 could not be determined, because of
their inactivity at all substrate concentration. Regarding to the
oxynitride of citalopram (citalopram N-oxide), the intrinsic clear-
ance of CYP2D6*R440 was increased to 138% compared with wild
type; two variants (CYP2D6*94, E215K) showed no significant dif-
ference (1-fold) in enzyme activity compared with the wild type;
The remaining 19 variants displayed significantly reduced intrinsic
clearance values compared with the wild type (13e66% relative
clearance). As a result, twenty of the 22 variants (CYP2D6*2, *10,
*87, *88, *89, *90, *91, *93, *95, *97, *98, *R25Q, F164L, F219S,
V327M, D336N, V342M, R344Q, R497C and R440C) have significant
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1.8 mm, Agilent Technologies) at 30 C. The mobile phase consisted
of acetonitrile (solvent B) and 0.05% trifluoroacetic acid in water
(solvent A) at an isocratic flow rate of 0.3 mL/min with 72% solvent
A þ 28% solvent B for 14 min. The column eluent was monitored
with a Fluorescence detector at excitation and emission wave-
lengths of 245 and 306 nm, respectively. Under these conditions,
the retention times of venlafaxine, demethylcitalopram, citalopram
and citalopram N-oxide were 4.510, 9.169,10.113 and 13.601 min,
respectively. The standard curves for citalopram and demethylci-
talopram or citalopram N-oxide were prepared using spiked incu-
bation samples with seven points.
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Please cite this article in press as: Hu X-x, et al., Effect of CYP2D6 genetic polymorphism on the metabolism of citalopram in vitro, Drug