Steady-state plasma concentration of donepezil enantiomers and its stereoselective metabolism and transport in vitro

Article abstract of DOI:10.1002/chir.22153

The aim of the present study was to elucidate the differences in the plasma concentration of two enantiomers of donepezil in Chinese patients with Alzheimer's disease (AD) and investigate in vitro stereoselective metabolism and transport. Donepezil enantiomers were separated and determined by LC-MS/MS using D5-donepezil as an internal standard on a Sepax Chiralomix SB-5 column. In vitro stereoselective metabolism and transport of donepezil were investigated in human liver microsomes and MDCKII-MDR1 cell monolayer. Pre-dose (Css-min) plasma concentrations were determined in 52 patients. The mean plasma level of (R)-donepezil was 14.94 ng/ml and that of (S)-donepezil was 23.37 ng/ml. One patient's plasma concentration of (R)-donepezil was higher than (S)-donepezil and the ratio is 1.51. The mean plasma levels of (S)-donepezil were found to be higher than those of (R)-donepezil in 51 patients and the ratio of plasma (R)- to (S)-donepezil varies from 0.34 to 0.85. In the in vitro microsomal system, (R)-donepezil degraded faster than (S)-donepezil. V_max of (R)-donepezil was significantly higher than (S)-donepezil. The P-gp inhibition experiment shown that the P_app of the two enantiomers was higher than 200 and the efflux ratios were 1.11 and 0.99. The results of the P-gp inhibition identification experiment showed IC50 values of 35.5 and 20.4 μM, respectively, for the two enantiomers. The results indicate that donepezil exhibits stereoselective hepatic metabolism that may explain the differences in the steady-state plasma concentrations observed. Neither (R)- nor (S)-donepezil was a P-gp substance and the two enantiomers are highly permeable through the blood-brain barrier. 2013 Wiley Periodicals, Inc.

Full text of DOI:10.1002/chir.22153

CHIRALITY 25:498–505 (2013)
Steady-State Plasma Concentration of Donepezil Enantiomers and Its
Stereoselective Metabolism and Transport In Vitro
WAN LILI, GUO CHENG, * ZHOU ZHIYONG, YU QI, LI YAN, LI DAN, ZHENG XUELI, AND ZHONG YUAN2
1
1
1
1
1
1
2
1
2
Department of Pharmacy, Shanghai Sixth People’s Hospital, Shanghai Jiao Tong University, Shanghai 200233, P. R. China
Department of Geriatrics, Shanghai Sixth People’s Hospital, Shanghai Jiao Tong University, Shanghai 200233, P. R. China
ABSTRACT
The aim of the present study was to elucidate the differences in the plasma con-
centration of two enantiomers of donepezil in Chinese patients with Alzheimer’s disease (AD)
and investigate in vitro stereoselective metabolism and transport. Donepezil enantiomers were
separated and determined by LC-MS/MS using D5-donepezil as an internal standard on a Sepax
Chiralomix SB-5 column. In vitro stereoselective metabolism and transport of donepezil were in-
vestigated in human liver microsomes and MDCKII-MDR1 cell monolayer. Pre-dose (Css-min)
plasma concentrations were determined in 52 patients. The mean plasma level of (R)-donepezil
was 14.94 ng/ml and that of (S)-donepezil was 23.37 ng/ml. One patient’s plasma concentration
of (R)-donepezil was higher than (S)-donepezil and the ratio is 1.51. The mean plasma levels of
(
S)-donepezil were found to be higher than those of (R)-donepezil in 51 patients and the ratio
of plasma (R)- to (S)-donepezil varies from 0.34 to 0.85. In the in vitro microsomal system, (R)-
donepezil degraded faster than (S)-donepezil. Vmax of (R)-donepezil was significantly higher than
(
S)-donepezil. The P-gp inhibition experiment shown that the Papp of the two enantiomers was
higher than 200 and the efflux ratios were 1.11 and 0.99. The results of the P-gp inhibition iden-
tification experiment showed IC50 values of 35.5 and 20.4 mM, respectively, for the two enantio-
mers. The results indicate that donepezil exhibits stereoselective hepatic metabolism that may
explain the differences in the steady-state plasma concentrations observed. Neither (R)- nor
(
S)-donepezil was a P-gp substance and the two enantiomers are highly permeable through
the blood–brain barrier. Chirality 25:498–505, 2013. © 2013 Wiley Periodicals, Inc.
KEY WORDS: donepezil; enantiomers; plasma concentration; stereoselective; metabolism;
transport
INTRODUCTION
as two enantiomers about a chiral center adjacent to the car-
bonyl group (Fig. 1), and the relative kinetic and clinical proper-
ties of the (R)- and (S)-enantiomers of the clinically used are
results from racemic mixture. Matsui et al. found evidence of
Donepezil (DNP) is a centrally acting reversible acetyl cho-
linesterase inhibitor. Its main therapeutic use is in the treat-
ment of Alzheimer’s disease (AD) where it is employed to
increase cortical acetylcholine. There is a large variability in
the clinical response to DNP, which may be mainly due to sin-
gle nucleotide polymorphisms in genes, encoding drug-
metabolizing enzymes, transporters, and targets. The varied
therapeutic responses to donepezil in the treatment of AD pa-
tients provide a strong rationale for the development of corre-
lation measures that can predict the therapeutic response to
donepezil in AD patients. Apolipoprotein E (ApoE) gene sta-
7
enantiomeric selective kinetics in healthy volunteers. These
authors found that the mean plasma (S)-donepezil peak concen-
trations were 2.6-fold higher than for (R)-donepezil. Hence
there is scope to consider further the kinetics of the enantio-
mers in AD patients. (2) CYP450’s presence in brain tissue pro-
8,9
vides an opportunity for local drug metabolism which may not
be reflected in plasma drug concentrations. (3) In addition to
passive diffusion, two major families of transporters affect the in-
flux and efflux of molecules across the blood–brain barrier
1
2–4
tus, cytochrome P450(CYP) 2D6 polymorphism, and the
(
BBB): the adenosine-triphosphate binding cassette (ABC)
gene-encoding P-glycoprotein (P-gp), and ABCB1 polymor5-
10–12
and the solute carrier (SLC) transporters.
Hence there is
phism have been used to predict the therapeutic outcomes,
scope for interaction between these systems that may determine
the net concentration of the enantiomers at the site of action in
the brain and the clinical response.
Few studies are available on the stereoselective concentra-
tion of DNP and its clinical outcomes, and no human studies
are available on enantiomeric plasma concentration in AD
patients. If the metabolism of the different stereoisomers is
although these results were heterogeneous.
The plasma concentration of a particular drug is deter-
mined by the absorption, distribution, metabolism, and excre-
tion of a drug. Therefore, the concentration of DNP may be
influenced by the patient’s genetic characteristics, such as
CYP2D6 and ABCB1, on the metabolism and transport of
DNP. Correlations may exist between plasma concentration
and pharmacological response, and monitoring the plasma
concentration of the drug will be beneficial for patient man-
agement. But the plasma concentration of DNP was not
*
Correspondence to: Guo Cheng, Shanghai Sixth People’s Hospital, Shanghai
Jiao Tong University, 600 Yishan Rd., Shanghai 200233, China; E-mail:
guoc66@gmail.com
6
related to its therapeutic responses.
Received for publication 15 August 2012; Accepted 10 December 2012
DOI: 10.1002/chir.22153
Published online 25 June 2013 in Wiley Online Library
(wileyonlinelibrary.com).
Given the complexity of the relationship between plasma
DNP concentration and its polymorphic effect, the authors
aimed to consider three issues: (1) (R) - and (S) - DNP existed
©
2013 Wiley Periodicals, Inc.

STEREOSELECTIVE METABOLISM AND TRANSPORT OF DONEPEZIL
499
Fig. 1. Representative MRM chromatograms.
handled by different enzymes, which are either polymorphic
or can be induced or inhibited, and if their pharmacodynamic
effects have differences either in strength or in quality,
and 99.3%, respectively). D5-donepezil was obtained from TLC
PharmaChem (Vaughan, Ontario, Canada). 6-O-desmethyl donepezil
was purchased from International Laboratory USA (San Francisco, CA,
USA). HPLC-grade methanol, acetonitrile, ammonium formate, and
formic acid were purchased from Tedia Inc. (CA, USA). Water was puri-
fied by a Milli-Q system. b-Nicotinamide adenine dinucleotide phosphate
13
enantiospecific analysis is urgently needed —and enantiospecific
analysis may be more clinically relevant than racemic concentra-
tions. However, it is unknown whether enantioselective pharmaco-
kinetics results from significantly different liver metabolism.
Whether donepezil enantiomers are substrates and/or
inhibitors of P-gp transport processes, or any other transporter
system, has not been evaluated. Based on the above informa-
tion, the authors aimed to evaluate the stereoselective plasma
concentration of DNP in Chinese AD patients and to elucidate
the reason for the differences in plasma concentration of the
two enantiomers on the basis of stereoselective metabolism
and transport.
(NADPH), fluorescein, digoxin, and cyclosporine A were purchased from
Sigma-Aldrich (St. Louis, MO, USA). All other chemicals and solvents
were analytical grade and purchased from commercial sources. Human
liver microsomes were obtained from the Research Institute for Liver Dis-
eases (Shanghai) Co., Ltd (Beijing, China). A Madin-Darby canine kidney
MDCKII-MDR1 cell line was purchased from the Netherlands Cancer In-
stitute (Amsterdam, Netherlands). A 24-well Millicell culture plate was
purchased from Millipore (Bedford, MA, USA). Cell culture medium
(
(
Dulbecco’s Modified Eagle Medium [DMEM], with GlutaMAX, 10%
v/v) Fetal Bovine Serum, 0.5% (v/v) penicillin/streptomycin 10,000
units/ml and transport medium DMEM supplemented with L-glutamine,
5 mM HEPES, Pyridoxine HCl but without sodium pyruvate and phenol
2
red, pH 7.4) were purchased directly from Gibco.
MATERIALS AND METHODS
Materials
Relationship between steady-state plasma concentration of
donepezil enantiomers and therapeutic response to patient. The
study protocol was approved by the Committee on Human Research,
Shanghai Sixth People’s Hospital and written informed consent was
obtained from all participants or their legal representative. Fifty-two
elderly subjects who had been treated with 5, 7.5, or 10 mg of DNP for
six months participated in this study. Demographic and clinical character-
istics of patients at baseline are listed in Table 3. At the 6-month follow-up,
Racemic donepezil was obtained from the National Institute for the
Control of Pharmaceutical and Biological Products (Beijing, China).
The two enantiomers of DNP were separated and prepared using a pro-
ductive high-performance liquid chromatography (HPLC) method on a
Chiralpak OD column (0.46 cm i.d. ꢀ 25 cm l). The mobile phase
consisted of n-hexane, isopropanol, triethylamine (87:12.9:0.1), and their
stereochemical purities were ascertained by stereospecific HPLC resolu-
tion (stereochemical purities of (S)-donepezil and (R)-donepezil were 98.6
TABLE 1. Accuracy (%) and precision (RSD, %) of the chiral LC-MS/MS method for measurement of donepezil enantiomers (n = 5)
(
ꢁ)-(R)-Donepezil (R)-Donepezil
(S)-Donepezil
Conc.
ng/ml)
(
Conc. found
Accuracy (%)
RSD (%)
Conc. found
Accuracy (%)
RSD (%)
Intra-day
0
1
1
.5
0
00
0.45 ꢂ 0.03
9.29 ꢂ 0.86
90.90
92.87
103.75
6.02
9.30
11.52
0.46 ꢂ 0.03
10.73 ꢂ 1.26
97.13 ꢂ 11.39
92.26
107.30
97.13
6.39
11.78
11.72
103.73 ꢂ 1 1.94
Inter-day
0
1
1
.5
0
00
0.46 ꢂ 0.03
9.65 ꢂ 0.85
91.92
96.46
105.89
7.47
8.84
9.42
0.48 ꢂ 0.04
10.85 ꢂ 1.02
95.88 ꢂ 10.45
95.47
108.56
95.88
9.10
9.41
10.90
105.89 ꢂ 9.98
Chirality DOI 10.1002/chir

5
00
LILI ET AL.
TABLE 2. Stability of donepezil enantiomers in human plasma (n = 3)
(R)-Donepezil
(S)-Donepezil
Stability
Observed concentration (ng/ml)
Bias (%)
Observed concentration (ng/ml)
Bias (%)
Stability in plasma for 4 h at room temperature
0
1
1
.5 ng/ml
0 ng/ml
00 ng/mlL
0.47 ꢂ 0.05
10.02 ꢂ 0.74
101.94 ꢂ 4.48
ꢁ5.33
0.22
1.94
0.45 ꢂ 0.02
10.91 ꢂ 1.49
101.29 ꢂ 13.24
ꢁ10.44
9.06
1.29
Autosampler rack for 24 h at room temperature
0
1
1
.5 ng/mL
0 ng/mL
00 ng/mL
0.46 ꢂ 0.03
10.46 ꢂ 0.65
108.60 ꢂ 13.02
ꢁ7.94
4.46
8.6
0.44 ꢂ 0.02
10.91 ꢂ 1.49
101.29 ꢂ 13.24
ꢁ11.24
9.06
1.29
ꢃ
Freezing for 30 days at ꢁ80 C
0.5 ng/ml
10 ng/ml
100 ng/ml
0.45 ꢂ 0.02
9.96 ꢂ 0.68
ꢁ9.65
ꢁ0.44
5.04
0.47 ꢂ 0.03
10.95 ꢂ 0.40
93.53 ꢂ 10.84
ꢁ5.07
9.5
ꢁ6.47
105.04 ꢂ 12.52
ꢃ
Freezing for 90 days at ꢁ80 C
0.5 ng/ml
10 ng/ml
100 ng/ml
0.44 ꢂ 0.03
9.29 ꢂ 0.56
98.37 ꢂ 7.23
ꢁ11.71
ꢁ7.11
ꢁ1.63
0.45 ꢂ 0.03
11.32 ꢂ 0.98
102.20 ꢂ 3.97
ꢁ9.77
13.16
2.2
Two freeze/thaw cycles
0.5 ng/ml
10 ng/ml
100 ng/ml
0.45 ꢂ 0.03
10.29 ꢂ 0.81
100.91 ꢂ 4.69
ꢁ10.7
2.87
0.47 ꢂ 0.04
10.16 ꢂ 0.84
100.20 ꢂ 6.73
ꢁ6.05
1.59
0.2
0.91
clinical assessments—including evaluation of cognitive and functional
status, compliance, and drug-related adverse events—were recorded.
For each recruited AD patient, the clinical evaluations were performed for
the degree of functional and cognitive impairment with the Mini-Mental
State Examination (MMSE) screening test at baseline before DNP
treatment. All patients started DNP therapy at 5 mg/day. After 3 months of
treatment, the treating physicians decided whether to maintain the same
dose (38 patients) or to increase it to 7.5 (2 patients) or 10 mg/day (12
patients), based on clinical evaluations. After treatment with a stable dose
of DNP for at least 6 months, a second assessment (MMSE) tof he patients
was made and blood samples (minimum plasma steady-state concentration)
were collected before the next dose was administered.
acetonitrile containing D5-donepezil (internal standard) 15 ng/ml, then
vortexed again for 10 sec. After centrifugation at 15,000 rpm (11,500 ꢀ g)
for 6 min, 100 ml of supernatant was transferred to a sample vial, and 5 ml
was injected into the LC-MS/MS for quantitative analysis.
Determination of the Elution Order. The elution order of the individ-
ual DNP enantiomers was established by analyzing the pure enantiomers
14
of DNP using the conditions described by Radwan et al. The pure enan-
tiomers were separated and prepared using a productive HPLC method
on a Chiralpak OD column and their stereochemical purities were
ascertained by stereospecific HPLC resolution (stereochemical purities
of (S)-donepezil and (R)-donepezil were 98.6 and 99.3%, respectively).
Method Validation. A calibration curve was generated to confirm the
linear relationship between the peak areas ratios (analyte/internal stan-
dard) plotted against the respective concentrations (0.5, 1, 2, 5, 10, 20,
50, 100 ng/ml) of each DNP enantiomer. To determine the within-day
precision of the method, three samples of plasma with concentrations of
0.5, 10, and 100 ng/ml were analyzed five times on the same day. To de-
termine the between-day precision and accuracy, samples of the three
concentrations of plasma were run on five different days. For the evalua-
tion of recovery, five replicates of blank plasma samples spiked with stan-
dard solutions of DNP (0.5, 10, and 100 ng/ml of each enantiomer in
plasma) were submitted to the extraction procedure described above
and the concentrations of these samples were compared with the
Separation and Quantitation of (R)-Donepezil and (S)-Donepezil.
DNP enantiomers were separated and determined by LC-MS/MS using
D5-donepezil as an internal standard. The LC–MS–MS system was an
Agilent 1200 liquid chromatography and a 6410B triple quadrupole mass
spectrometer with an electrospray ionization source. Data were analyzed
by MassHunter software (Agilent Corporation, MA, USA). Chromatogra-
phy was performed on a Sepax Chiralomix SB-5 column (2.1 mm ꢀ 150
ꢃ
mm i.d., 3.5 mm; Sepax Corporation, USA) maintained at 35 C using gra-
dient elution with 5-mM ammonium formate and 0.1% formic acid in puri-
fied water (pH 4.0) as solvent A and acetonitrile as solvent B. The
gradient program was then as follows: 0–2 min, 90% A; 2–10 min, 90%!
5
4% A; and 10.1–15 min, 10% A. The flow rate was set at 0.4 ml/min.
Electrospray ionization (ESI) was performed in the positive ion mode
and quantified by the selected reaction monitoring transitions as proton-
ated precursor/product ion transition of m/z 380/91 for (R)- and (S)-
donepezil and 385.2/96.1 for IS (D5-DNP); the nebulizer and drying gas
were high-purity nitrogen and drying gas; gas temperature and gas flow
were set at 350 C and 8.0 l/min. Other ESI source conditions were: neb-
ulizer pressure, 40 psi; capillary voltage, 4000 V; dwell time, 200 ms; and
EMV, 200.
TABLE 3. Demographic and clinical characteristics of the AD
patients
AD patients (n = 52)
ꢃ
Age (years)
Male/female
80.34 ꢂ 4.65( 69–90)
38/14
MMSE at the baseline
MMSE at the steady-state
ΔMMSE
22.46 ꢂ 2.36 (15–26)
23.12 ꢂ 2.92 (16–28)
0.65 ꢂ 2.44 (ꢁ6–5)
Plasma Extraction of (R)- and (S)-Donepezil in Plasma. Blood was
ꢃ
centrifuged and the plasma stored at ꢁ80 C until analysis. Plasma sam-
ples (30 ml) were mixed with 5 mM of ammonium formate and 0.1% formic
acid buffer solution, vortexed for 10 sec, and then combined with 120 ml of
MMSE: Mini-Mental State Examination.
ΔMMSE: steady-state MMSE – baseline MMSE.
Chirality DOI 10.1002/chir

STEREOSELECTIVE METABOLISM AND TRANSPORT OF DONEPEZIL
501
Metabolism of DNP enantiomers in human liver microsomes. The
test for the metabolism of (S)-donepezil and (R)-donepezil (final concen-
tration 0.5 mM) was conducted using 1 mg/ml of microsomal protein in
ꢃ
0
.1 M potassium phosphate buffer with 5.0 mM MgCl
2
(pH 7.4) at 37 C.
The reactions were initiated by adding the NADPH regenerating system.
In a final incubation volume of 150 ml. The reactions were stopped by
adding 450 ml of ice-cold acetonitrile at 0, 10, 20, 30, 60, 90 and 120 min
after incubation respectively. The stability of the substrates was tested
by incubating without the NADPH-regenerating system. System kinetic
studies were performed with eight substrate concentrations, ranging
from 0.5 to 50 mM, of (R)-donepezil and (S)-donepezil; the reaction time
was 90 min. Linearity of the metabolites, 6-OD-DNP, formation rate was
established with respect to microsomal protein and incubation time. Reac-
tions were performed in duplicate. The samples were aliquoted and
ꢃ
stored at ꢁ80 C until analyzed.
Fig. 2. Mean plasma donepezil (R)/(S) concentration ratio for 52 patients.
Statistical Analysis
Nonlinear regression of substrate concentration versus reaction veloc-
ity curves wasre analyzed using GraphPad Prism. A P-value less than 0.05
was considered significant following correction for multiple testing within
the linear regression with a Holm test. Intrinsic metabolic clearance
equivalent amount concentrations of standard solutions. The quantifica-
tion limit was defined as the lowest concentration of each DNP enantio-
mer analyzed with precision and accuracy (error of 20% or lower).
Absolute recovery was determined by comparing the peak areas of the
two enantiomers in plasma samples at three QC concentration levels with
the peak areas of solutions of the two enantiomers.
The matrix effect was calculated by comparing the peak areas of each
DNP enantiomer or IS in the post-extraction spiked blank plasma with
those of the corresponding DNP enantiomer or IS solutions. The differ-
ence between peak areas was an indication of the presence or absence
of a matrix effect.
m
(Clint) was calculated by the following equation: Clint = Vmax/K .
Stereoselective transport study of DNP
The MDCKII-MDR1 cells were cultured to confluency, trypsined, and
seeded onto 24-multiwell Insert Systems with PET (polyethylene tere-
2
phthalate) membranes (1-mm pore size and 0.3 cm surface area) at an op-
timized density of 250,000 cells/ml in cell culture medium DMEM, with
GlutaMAX, 10% (v/v) Fetal Bovine Serum. Following an overnight attach-
ment period (24 h after seeding), the cell media was replaced with fresh
media in both apical and basolateral sides. The cell monolayers were
In short-term stability tests, samples were analyzed after 4 h at room
temperature. The freeze-thaw stability was evaluated by analyzing sam-
ples after undergoing two freeze-thaw cycles. Long-term stability was
15–17
used for transport studies 3 days after seeding.
P-gp Substrate and Inhibitor Identification Studies
Before the transport study, all apical and basolateral sides were washed
ꢃ
studied by assaying samples after storage for 60 and 90 days at ꢁ80 C.
Postprocessing stability was evaluated after the prepared samples had
ꢃ
with 0.7 ml of donor working solution PBS buffer (pH 7.4) for 30 min at
been kept in the auto-injector for 24 h at 25 C. The stability of two enan-
ꢃ
ꢃ
37 C, in 5% CO
2
incubator.
tiomers in stock solutions for one month at 4 C and at room temperature
for 6 h was also investigated.
P-gp substrate identification study. MDCKII-MDR1 cell monolayers
ꢃ
were preincubated (at 37 C, in 5% CO
2
incubator) in transport media,
with or without specific P-gp inhibitor cyclosporine A (10 mM), for
Stereoselective metabolism study of DNP
4
0
0 min prior to addition of test article. For A!B directional transport,
Assays for the Oxidative Metabolism of DNP. Concentrations of the
metabolite 6-O-demethylated donepezil (6-OD-DNP) were determined by
LC-MS/MS using D5-donepezil as an internal standard on a Zorbax XDB-
C18 column (2.1 mm ꢀ 150 mm i.d., 3.5 mm; Agilent Corporation, MA,
USA). The gradient program was then as follows: 0–1 min, 85%!85% A;
.2 ml of donor working solution with test article or positive control was
added to the A compartment and then 0.7 ml of the transport media as
the receiver working solution to the
B
compartment. For B!A
directional transport, 0.7 ml of donor working solution with test article
or positive control was added to the B compartment and 0.2 ml of trans-
1
–3 min, 85%!30% A; 3–5 min, 30–85% A, 5–10 min, 85% A. The flow rate
port media as receiver working solution to the A compartment. The cells
was set at 0.3 ml/min. ESI was performed in the positive ion mode and
quantified by the selected reaction monitoring transitions. As protonated
precursor/product ion transition of 6-OD-DNP is 366.2/91 and the transi-
tion of IS is 385.2/96.1 (D5-donepezil). Other conditions were same as for
the determination of DNP enantiomers mentioned above.
ꢃ
incubated (37 C, in 5% CO
ꢁ
2
incubator) for 90 min. Samples were stored at
ꢃ
20 C until analyzed by LC-MS/MS.
P-gp inhibitor identification study. MDCKII-MDR1 cell monolayers
ꢃ
were preincubated (at 37 C, in 5% CO
2
incubator) in transport media with
Fig. 3. Concentration-time curves of two donepezil enantiomers at 500 nM after 120-min incubation in human liver microsomes.
Chirality DOI 10.1002/chir

5
02
LILI ET AL.
TABLE 4. (R)/(S) enantiomeric ratio at steady-state concentrations following repeated dosing of donepezil in 52 patients enrolled in
maintenance program
5
mg (38)
7.5 mg (2)
10 mg (12)
Parameter
Mean
%CV
Mean
%CV
Mean
%CV
Css-min, R
Css-min, S
Ration
11.74 ꢂ 6.47
18.41 ꢂ 10.45
0.64 ꢂ 0.18
55.16
54.58
28.76
3.06 ꢂ 0.18
6.65 ꢂ 0.89
0.46 ꢂ 0.02
5.76
1.34
4.42
21.79 ꢂ 7.99
33.98 ꢂ 12.48
0.64 ꢂ 0.09
36.66
36.74
13.72
Css-min: minimum plasma concentration pre-dose (ng/ml).
a series of positive control P-gp inhibitor cyclosporine A or test compound
concentrations for 40 min prior to addition of the substrate. For B!A di-
rectional transport, 0.7 ml of donor working solution with a series of test
articles or positive control cyclosporine A concentrations and 1 mM of sub-
strate digoxin (containing 100-mM fluorescein as well) was added to the B
200 nm/s), or high (>200 nm/s). Fluorescein, a fluorescent marker for the
paracellular pathway, was used as an internal control in every monolayer to
verify tight junction integrity during the assay. Cell monolayers with fluores-
cein Papp > 50 nm/sec were discarded.
compartment and then 0.2 ml of the transport media as receiver working
Mass balance. Mass balance was calculated from: (M
where M = mass of drug in donor compartment at time = t; M
of drug in receiver compartment at time = t; M = mass of drug in donor
D
+ M
R
)/ M
O
ꢃ
solution to the A compartment. The cells incubated (37 C, in 5% CO
2
in-
D
R
= mass
ꢃ
cubator) for 90 min. Samples were stored at ꢁ20 C until analyzed by LC-
O
MS/MS.
compartment at time zero. For non-radio-labeled compounds, studies
with mass balance of <50% or >130% are repeated.
Sample Analysis
Efflux ratio. Monolayer flux ratios were derived using the following
equation: flux ratio = Papp [B!A] (nm/sec)/Papp [A!B] (nm/sec). The
flux ratios generated in the presence and absence of a P-gp inhibitor were
compared to identify whether the test article was a P-gp substrate. A com-
pound was considered to be a P-gp substrate when the flux ratio in the ab-
sence of the inhibitor was 3.0 and was significantly reduced (1) in the
presence of the inhibitor.
For the substrate study, a 50-ml aliquot sample of (S)-donepezil, (R)-
donepezil, and amprenavir was taken from both donor and receiver com-
partments separately and put into 96-well assay plates, which were pre-
added with 150-ml internal standard solution (containing 25 ng/ml D5).
To compartment A samples were added 50-ml 1% HSA of DMEM, and to
B compartment were added 50-ml FaSSsif, followed by adding 300 ml of in-
ternal standard solution (200 ng/ml tolbutamide in acetonitrile) to precip-
itate protein, then centrifuged 10 min at 13,000 rpm, diluted with water
before analysis. The concentrations of the test article or substrate probe
amprenavir (m/z 506.4–418.0) were determined by LC-MS/MS. These ra-
tios of analyte peak area to internal standard area were used to calculate
the passive permeability and efflux ratio of the test compound.
IC50 determination by fitting inhibitor concentration vs. %(Papp).
The IC50 was determined by fitting inhibitor concentration vs. %(Papp
[
[
B!A]i/Papp [B!A]) plot using Graphpad Prism (version 5) where (Papp
B!A]i/Papp [B!A]i) represents the Papp of the probe without an
inhibitor.
For the inhibition study, a 50-ml sample aliquot was taken from both do-
nor and receiver compartments and placed into 96-well assay plates,
which were pre-added with 150 ml of internal standard solution
RESULTS
(
containing 200 ng/ml of tolbutamide [m/z 271.1–155.0]). The concentra-
Plasma Concentration of Donepezil Enantiomers in AD
Patients
tions of the substrate probe digoxin were determined by LC-MS/MS.
These ratios of digoxin (m/z 781.1–651.1) peak area to internal standard
area were used to calculate the passive permeability of digoxin from the B
compartment to the A compartment.
Enantioseparation and quantification of (R)- and (S)-donepezil.
R-donepezil and (S)-donepezil were baseline separated and
no endogenous interference peaks eluted at retention times
(
7.97 min for (R)-donepezil and 9.09 min for (S)-donepezil)
LC-MS/MS Analysis Method
in all samples (Fig. 1). The validation of the method showed
that the calibration curves were linear, ranging from 0.5 to
1
The LC–MS–MS system used was an Agilent 1200 liquid chromatogra-
phy and AB Sciex API4000 triple quadrupole mass spectrometer with an
electrospray ionization source. Chromatography was performed on a
Thermo Betasil C18 (2.1 mmꢀ 50mm i.d., 5 mm; Thermo Fisher Scientific,
00 ng/ml in plasma for each enantiomer with correlation
ꢃ
USA) maintained at 35 C, using gradient elution with 0.1% formic acid in pu-
rified water as solvent A and 0.1% formic acid acetonitrile as solvent B. The
gradient program was then as follows: 0–0.3 min, 90% A; 0.3–3.4 min, 90%!
15% A; and 3.5–3.8 min, 90% A. The flow rate was set at 0.4 ml/min.
Electrospray ionization (ESI) was performed in the positive ion mode and
quantified by the selected reaction monitoring transitions as protonated pre-
cursor/product ion transition of m/z 380/91 for (R)- and (S)-donepezil,
3
85.2/96.1 for IS (D5-DNP), 506.4/418.0 for amprenavir, 271.1/155.0 for
tolbutamide (IS), and 781.1/651.1 for digoxin. The DP and CE for (R)- and
S)-donepezil, D5-DNP were 95 and 52; for amprenavir, 94 and 18; for
(
tolbutaniride, 42 and 26; and for digoxin, 100 and 12.
Data Calculation
Apparent permeability. The apparent permeability (Papp) values were cal-
culated using the following equation: Papp =(dQ/dt)ꢀ (1/C
0
)ꢀ (1/A), where
dQ/dt is the permeability rate, C is the initial concentration in the donor com-
0
partment, and A is the surface area of the filter, which corresponds to the sur-
face area of the cell monolayer. The Papp value had the dimension of a rate
Fig. 4. Concentration-time curves of metabolism (6-O-desmethyl
donepezil) of two donepezil enantiomers at 500 nM after 120-min incubation
in human liver microsomes.
(nm/sec). Calculated Papp was ranked as low (<100 nm/s), moderate (100–
Chirality DOI 10.1002/chir

STEREOSELECTIVE METABOLISM AND TRANSPORT OF DONEPEZIL
503
and the correlation coefficients (r) were 0.9997. Validation of
the method showed that the method is selective and reliable.
Results of incubation with a single enantiomer of DNP at
0
.5 mM in liver microsomes indicated that the reversion of
the (S)- to the (R)- or (R)- to the (S)-enantiomer does not
seem to occur.
In the absence of the NADPH-regenerating system, (R)-
and (S)-donepezil kept intact (less than 93.82% depletion)
within 2-h incubations with liver microsomes (Fig. 3). As
shown in Figure 3, at a concentration of 0.5 mM, 2-h incuba-
tion of either single enantiomer without NADPH regenera-
tion resulted in minimal loss of substrate (R)- donepezil or
(
S)-donepezil (final concentration >93%). In the presence of
the NADPH-regenerating system, both (R)-donepezil and
S)-donepezil enantiomers showed significant metabolism to
(
approximately 60% of the initial 0.5 mM concentration, respec-
tively. Figure 4 also suggests that the (R)-donepezil degraded
more rapidly than the (S)-donepezil.
Fig. 5. 6-O-Desmethyl donepezil formation curves made with eight differ-
ent concentrations of two donepezil enantiomers incubated for 90 min each
with human liver microsomes.
For enzyme kinetic analysis, Figure 5 shows 6-OD-DNP
formation curves generated with eight different concentrations
of (R)- or (S)-donepezil at an incubation time of 90min. Meta-
bolic rate constants were determined and the Michaelis–
Menten plots are shown in Figure 6. Vmax of (R)-donepezil is
significantly higher than (S)-donepezil. Data are given in
Table 5.
coefficients from 0.9983 to 0.9991. The accuracies and preci-
sions were between 6.02% and 11.78% (Table 1). The LOQ
was found to be 0.5 ng/ml for both DNP enantiomers and was
determined with high precision and accuracy, not exceeding
2
0% and 80–120%, respectively. The average absolute recovery
for (R)-donepezil and (S)-donepezil were 80.08% and 73.02% in
the concentrations of plasma QC samples respectively. The
matrix effect was evaluated by preparing spiked samples of
Transport of (R)- and (S)-enantiomers of DNP in MDCKII-
MDR1 cell monolayer
0
.1, 2, and 20 ng/ml (R)-donepezil and (S)-donepezil and inter-
In this study we tested the Papp value, efflux ratio, and in-
hibition identification of (R)- and (S)-donepezil across the
MDCKII-MDR1 cell monolayer. For all cell monolayers,
the fluorescein Papp was <50 nm/sec. The Papp value, efflux
ratio, and recovery of (R)- and (S)-donepezil and reference
compound amprenavir across the MDCKII-MDR1 cell
monolayer with or without P-gp inhibitor are listed in
Table 6. Inhibitor identification of (R)- and (S)- donepezil
and CsA for P-gp are shown in Figure 6. The data of the
permeability experiment show that (R)- and (S)-donepezil
are both high-permeability compounds (Papp > 200 nm/
sec). The efflux ratios for (R)- and (S)-donepezil were 1.11
and 0.99, respectively, less than 3.0. With the addition of cy-
closporine A, a potent P-gp inhibitor, the efflux ratios were
nal standard in six different plasma post-extraction matrix
samples. The mean value of matrix effect in human plasma of
(
8
R)-donepezil and (S)-donepezil and IS were 86.65, 83.99, and
7.71, respectively.
Stability test results of two enantiomers in plasma were
analyzed basing of three replicates of QC samples under the
conditions of short-term temperature stability, long-term
stability, post-preparation stability, and freeze–thaw stability.
These results (Table 2) demonstrated that the test com-
pounds were stable in human plasma. Results for stock and
working solution stability were stable during 1-month storage
ꢃ
at 4 C and 6 h at room temperature. Validation of the method
shows that the method is selective and reliable.
0
.67 and 0.73. The resulta suggested that (R)- and (S)-
donepezil were not a P-gp substrate. (A compound is con-
sidered to be a P-gp substrate when the flux ratio in the ab-
sence of an inhibitor is 3.0 and is significantly reduced [1]
in the presence of an inhibitor.) The results of the P-gp
Quantification of (R)-donepezil and (S)-donepezil in plasma. Pre-
dose (Css-min) plasma (R)-donepezil concentrations of 52
Chinese AD patients (Table 3) dosing of 5, 7.5, and 10 mg
was 11.74, 3.06, and 21.79 ng/ml, while that of (S)-donepezil
was 18.41, 6.65, and 33.98 ng/ml. In contrast to the other 51
patients, one patient’s plasma concentration of (R)- donepezil
was higher than (S)-donepezil and the ratio is 1.51. The mean
plasma levels of (S)-donepezil were found to be higher than
those of (R)-donepezil in 51 patients with a mean ration of
(
R)- to (S)-donepezil of 0.62 (range 0.28–0.87; Fig. 2). The
data for the (R)/(S) enantiomeric ratio at minimum steady-
state plasma concentrations following difference chronic
dosing donepezil in 52 patients enrolled in a maintenance
program are shown in Table 4.
Metabolism of (R)- and (S)-enantiomers of DNP in human
liver microsomes
Assays for the Oxidative Metabolism of DNP. Calibration curves
showed good linearity in the range of 1–500 nM for 6-OD-DNP
Fig. 6. Inhibition identification of cyclosporine A and two donepezil
enantiomers.
Chirality DOI 10.1002/chir

5
04
LILI ET AL.
TABLE 5. Apparent kinetic constants of donepezil enantiomers degradation in human liver microsomes
–1
–1
–1
–1
Sample
V
max (nmol min mg protein)
K
m
(nM)
C
lint (ml min mg protein)
(
(
R)-Donepezil
S)-Donepezil
345.35 ꢂ 42.50
207.40 ꢂ 25.89
28.75 ꢂ 5.48
46.28 ꢂ 7.94
12.38 ꢂ 3.84
4.60 ꢂ 1.35
inhibitionidentification experiment suggest that (R)- and
S)-donepezil are weak inhibitors of P-gp; the IC50 was
5.5 and 20.4 mM, individually. The mass balance for all
was acceptable (72.7–107.3%), suggesting that there was
minimal loss of substrate to plastic surfaces and cells under
the experimental conditions.
donepezil transformations may be the main reasons for
the difference in the plasma levels. The difference may be
further influenced by other factors, such as genetic poly-
morphism in CYP450 isoenzymes involved in drug metabo-
lism, so further study should be carried out to confirm the
primary roles of the CYPs 3A4 and 2D6 in DNP
stereoselective metabolism. Stereoselective metabolism
and stereoselective inhibition might play a role in varied
plasma concentrations of the two enantiomers and if differ-
ences in the CYP2D6 or CYP3A4 genotype account for the
population variability in biotransformation of DNP, genetic
effects of the enzymes in the metabolism of racemic DNP
can be studied further to compare the degree of
stereoselectivity of different CYP2D6 or CYP3A4 genotypes.
The enantiomers’ concentration ratio reversion may be caused
by the different metabolism of the enzyme genotypes.
(
3
DISCUSSION
In this study, a chiral LC-MS/MS method has been built,
validated, and applied to investigate the plasma steady-state
concentration in patients with respect to stereoselectivity.
The result was consistent with previously reported data,
indicating that healthy adults given racemic DNP are ex-
posed to much higher concentrations of (S)-donepezil than
of (R)-donepezil. The CV of DNP enantiomers in plasma
ranged from 50.83% to 51.14% and the R/S ratio was 24.21 in pa-
tient subjects. A large inter-individual variability in the plasma
concentration of DNP among Chinese AD patients was ob-
Some chiral drugs are stereoselectively distributed to the
brain.11 This may be due to stereoselective plasma protein
binding or stereoselective transport across the BBB. In this
study, we investigated the transport of two enantiomers in
MDCK-MDR1 cells. (R)- and (S)- donepezil are both high-
permeability compounds but not P-gp substrate and weak in-
hibitors. Heterogeneous results have reported that ABCB1
polymorphisms may play a role in DNP disposition and clini-
cal outcome, and the single nucleotide polymorphisms in the
ABCB1 gene, associated with decreased P-gp activity, may
lead to increased DNP CNS levels due to a reduced efflux
5
served, the same as was reported in Italian AD patients. The
inter-individual differences in plasma concentration were related
to the clinical response to DNP and were mainly caused by ge-
6–8
netic polymorphisms affecting drug disposition. The fact that
the R/S ratio of inter-individual variability was not significantly
different suggests that the CYP450 enzyme polymorphisms
were likely to influence DNP enantiomers metabolism in the
same way. However, for one of the 52 patients whose concentra-
tion of (R)-donepezil was higher than for (S)-donepezil (R/S
ratio 1.51), further studies will be needed to investigate whether
a significant association was accrued between enantiomers
concentration ratio reversion in plasma and clinical response.
In order to explore the possible intrinsic factors that con-
tribute to the difference in plasma concentrations between
the two enantiomers, we performed an in vitro study on
the metabolism of DNP in human liver microsomes. The
most unusual metabolic pathway, and one that is unique
to chiral drugs, is the phenomenon of enantiomer inver-
5
of the drug from the CNS to the blood compartment. The
transport of DNP across the BBB is mediated by organic cat-
19
ion transporters such as choline transport systems, and
OCT1B1, OCT1B2, and OCT1B3 have been considered as
critical molecular determinants of the pharmacokinetics of
DNP, and association of genetic polymorphisms in with
alterations in the pharmacokinetics properties. Therefore,
further study is necessary to seek stereoselective and
consistent associations of in vitro transport activity with
in vivo alteration of the pharmacokinetics of DNP. Eventually,
pharmacogenomic information should prove useful for more
effective pharmacotherapy.
1
8
sion ; the conversion was negligible for DNP in human
liver microsomes in our study. The intrinsic clearance of
the (R)-donepezil was higher than the (S)-donepezil and
significant differences were found for the stereoselective
degradation of DNP. The difference between (S)- and (R)-
In this study, we applied chiral separation technique in
therapeutic drug monitoring. The results indicate that DNP
had a stereoselective difference in steady-state plasma
TABLE 6. The Papp values of cyclosporine A applied in combination with two donepezil enantiomers across
MDCKII-MDR1 monolayers
–6
P
app (ꢀ 10 cm/sec)
Efflux
ratio
Permeability
classification
P-gp
substrate
Group
A!B
B!A
Amprenavir
Without inhibitor
4.41
26.00
20.26
29.64
21.07
27.30
60.91
20.39
22.41
19.93
20.81
19.83
13.80
0.78
1.11
0.67
0.99
0.73
high
high
high
+
–
–
+
cyclosporine A
Without inhibitor
cyclosporine A
Without inhibitor
Cyclosporine A
(
(
R)-Donepezil
S)-Donepezil
+
+
+
P-gp substrate, – not P-gp substrate.
Chirality DOI 10.1002/chir

STEREOSELECTIVE METABOLISM AND TRANSPORT OF DONEPEZIL
505
7
. Matsui K, Oda Y, Nakata H, Yoshimura T. Simultaneous determination of
donepezil (Aricept) enantiomers in human plasma by liquid chromatogra-
phy–electrospray tandem mass spectrometry. J Chromatogr B Biomed Sci
Appl 1999;729:147–155.
concentration in AD patients and is related to the stereochem-
istry in the liver metabolism. Neither (R)- nor (S)-donepezil
was a P-gp substance and the two enantiomers are highly
permeable through the BBB. Therefore, further studies will
be required to investigate the impact of polymorphisms of
organic cation transporters on DNP enantiomers’ disposition
and clinical outcome.
8. Haduch A, Bromek E, Daniel WA. The effect of psychotropic drugs on
cytochrome P450 2D (CYP2D) in rat brain. Eur J Pharmacol 2011;51–58.
9. Haining RL, Nichols-Haining M. Cytochrome P450-catalyzed pathways in
human brain: metabolism meets pharmacology or old drugs with new
mechanism of action? Pharmacol Ther 2007;113:537–545.
1
0. Ronaldson PT, Lee G, Dallas S. Involvement of P-glycoprotein in the trans-
port of saquinavir and indinavir in rat brain microvessel endothelial and
microglia cell lines. Pharm Res 2004;21:811–818.
LITERATURE CITED
1
1. Choong E, Dobrinas M, Carrupe PA, Eap CB. The permeability P-
glycoprotein: a focus on enantioselectivity and brain distribution. Expert
Opin Drug Metab Toxicol 2010;6:953–965.
2. Koepsell H. Polyspecific organic cation transporters: their functions and
interactions with drugs. Trends Pharmacol Sci 2004;25:375–381.
1
2
3
. Patterson CE, Todd SA, Passmore AP. Effect of apolipoprotein E and
butyrylcholinesterase genotypes on cognitive response to cholinesterase
inhibitor treatment at different stages of Alzheimer’s disease.
Pharmacogenomics J 2011;11:444–450.
. Varsaldi F, Miglio G, Scordo MG, Dahl ML, Villa LM, Biolcati A,
Lombardi G. Impact of the CYP2D6 polymorphism on steady-state plasma
concentrations and clinical outcome of donepezil in Alzheimer’s disease
patients. Eur J Clin Pharmacol 2006;62:721–726.
. Seripa D, Bizzarro A, Pilotto A, D’onofrio G, Vecchione G, Gallo AP,
Cascavilla L, Paris F, Grandone E, Mecocci P, Santini SA, Masullo C,
Pilotto A. Role of cytochrome P4502D6 functional polymorphisms in the
efficacy of donepezil in patients with Alzheimer’s disease. Pharmacogenet
Genomics 2011;21:225–230.
. Pilotto A, Franceschi M, D’Onofrio G, Bizzarro A, Mangialasche F,
Cascavilla L, Paris F, Matera MG, Pilotto A, Daniele A, Mecocci P,
Masullo C, Dallapiccola B, Seripa D. Effect of a CYP2D6 polymorphism
on the efficacy of donepezil in patients with Alzheimer disease. Neurology
1
1
1
3. Rentsch KM. The importance of stereoselective determination of drugs in
the clinical laboratory. J Biochem Biophys Methods 2002;54:1–9.
4. Radwan MA, Abdine HH, Al-Quadeb BT, Aboul-Enein HY, Nakashima KJ.
Stereoselective HPLC assay of donepezil enantiomers with UV detection
and its application to pharmacokinetics in rats. Chromatogr B Analyt
Technol Biomed Life Sci 2006;830:114–119.
5. Wang Q, Rager JD, Weinstein K, Kardos PS, Dobson GL, Li J, Hidalgo IJ.
Evaluation of the MDR-MDCK cell line as a permeability screen for the
blood-brain barrier. Int J Pharm 2005;88:349–359.
1
4
1
6. Agarwal S, Pal D, Mitra AK. Both P-gp and MRP2 mediate transport of
lopinavir, a protease inhibitor. Int J Pharm 2007;339:139–147.
17. Carrara S, Reali V, Misiano P, Dondio G, Bigogno C. Evaluation of in vitro
brain penetration: optimized PAMPA and MDCKII-MDR1 assay compari-
son. Int J Pharm 2007;345:125–133.
18. Campo VL, Bernardes LS, Carvalho I. Stereoselectivity in drug metabo-
lism: molecular mechanisms and analytical methods. Cur Drug Metab
2009;10:188–205.
2
009;73:761–767.
5
. Magliulo L, Dahl ML, Lombardi G, Fallarini S, Villa LM, Biolcat A, Scordo
MG. Do CYP3A and ABCB1 genotypes influence the plasma concentra-
tion and clinical outcome of donepezil treatment? Eur J Clin Pharmacol
2
010;67:47–54.
6
. Yang YH, Wu SL, Chou MC, Lai SL, Chen SH, Liu CK. Plasma concentra-
tion of donepezil to the therapeutic response of Alzheimer’s disease in
Taiwanese. J Alzheimers Dis 2011;391–397.
19. Kim MH, Maeng HJ, Yu KH, Lee KR, Tsuruo T, Kim DD, Shim CK,
Chung SJ. Evidence of carrier-mediated transport in the penetration of
donepezil into the rat brain. J Pharm Sci 2010;99:1548–1566.
Chirality DOI 10.1002/chir