Drug Dynamics Research Section, Drug Safety and Disposition
Research Laboratories, Eisai Co., Ltd., Ibaraki, Japan
Donepezil hydrochloride (Aricept) is a drug for the treatment of
Alzheimer's disease. The absorption, distribution, metabolism, and
excretion of donepezil were investigated in male Sprague-Dawley rats
after a single oral administration. Orally administered
14C-labeled donepezil was absorbed rapidly. The plasma
level of unchanged donepezil declined more rapidly than that of
radioactivity, and the brain level of radioactivity declined almost in
parallel with the plasma level of unchanged donepezil. The ratio of
donepezil to total radioactivity in brain was 86.9 to 93.0%,
indicating low permeability of the metabolites through the blood-brain
barrier. No heterogeneous localization of radioactivity was recognized in the brain and the concentration in each part of the brain was 1.74 to 2.24 times the plasma concentration. Cumulative biliary, urinary,
and fecal excretion of radioactivity in bile duct-cannulated rats was
72.9, 24.4, and 8.84%, respectively, of the administered radioactivity
at 48 h after administration. These results indicate that the
absorption of donepezil is almost complete, and that its metabolites
are mainly excreted into feces through the bile and some of them are
subject to enterohepatic circulation. The metabolism of donepezil was
extensive in rats and involved O-demethylation, aromatic
hydroxylation, N-dealkylation,
N-oxidation, and glucuronide conjugation of
O-demethylate. The structures of the metabolites were
determined by mass spectrometry and 1H-NMR analysis.
In plasma, urine, and bile, O-glucuronides accounted for
the majority of the radioactivity, and in brain, unchanged donepezil
was mostly detected. No metabolites were found in brain. There was no
notable accumulation of radioactivity in whole blood and tissues.
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Introduction |
Donepezil hydrochloride, (±)
- 2 -[(1 - benzylpiperidin - 4 - yl) methyl] - 5, 6 - dimethoxyindan - 1 - one monohydrochloride, is a reversible
cholinesterase inhibitor that exhibits high specificity for centrally
active cholinesterase (Yamanishi, 1990
; Rho and Lipson, 1997; Rogers et
al., 1998). Cholinergic deficit is one of the major pathological
features of Alzheimer's disease. This deficit has been associated with
the loss of cognition and memory, the primary symptoms of this disorder
(Bartus et al., 1982). Donepezil HCl (also known as E2020 or Aricept)
is the first of the "new generation" acetylcholinesterase
inhibitors, and is currently used for the symptomatic treatment
of Alzheimer's disease in many countries.
After i.v. administration to rats the plasma levels of unchanged
donepezil declined biphasically with an apparent
T1/2 for the terminal phase of 3 h,
and CLtotal and
Vd(ss) were 78.6 ml/min/kg and 11.7 l/kg,
respectively. These relatively large volumes of CLtotal and Vd(ss)
indicate extensive metabolism and high tissue distribution of
donepezil. Orally administrated donepezil was absorbed rapidly. The
mean plasma levels of donepezil reached a peak at 30 min after
administration, and then declined biphasically. In the dosage groups of
1.0, 3.0, and 10 mg/kg, systemic bioavailability values were
56.1, 41.4, and 86.8%, respectively. The brain levels of donepezil
were 3.16- to 10.7-fold higher than plasma levels, but declined in
parallel, and the area under the curve
(AUC)1(0-24
h) in brain was 7.23- to 8.24-fold higher than that in
plasma (K. Matsui and T. Yoshimura, in preparation).
In this paper, the absorption, disposition, and excretion of
14C-labeled donepezil after a single oral
administration to male rats were investigated to clarify the
pharmacokinetic profiles of donepezil and its metabolites in detail.
Therefore, the primary objective of this study was to examine the
metabolism of donepezil, permeability of metabolites through the
blood-brain barrier, and accumulation of radioactivity in whole blood
and tissues.
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Materials and Methods |
Chemicals.
14C-labeled donepezil
([14C]donepezil) was synthesized at
Amersham Co., Ltd. (Amersham, UK). Its specific activity was 4.941 MBq (133.54 µCi)/mg donepezil. Its chemical structure and site of labeling are shown in Fig. 1a.
Radiochemical purity estimated by HPLC was 100%.
Unlabeled donepezil, synthesized at Eisai Co., Ltd., was used as a
standard for quantitative determination by HPLC.
2-[(1-benzylpiperidin-4-yl) ethyl]-5, 6-dimethoxyindan-1-one
monohydrochloride, which was used as an internal standard (IS), was
synthesized at Eisai Co., Ltd. The chemical structure of IS compound is
shown in Fig. 2b.
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Fig. 2.
Blood levels of radioactivity after a
single oral administration of
[14C]donepezil (1 mg/kg) to intact
rats.
Each point represents the mean ± S.E. of four animals.
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Synthetic reference compounds (M1, M2, M3, M4, M5, and M6), were
synthesized at Eisai Co., Ltd., and used as thin-layer chromatography (TLC), mass spectrometry (MS), and NMR reference standards. Other reagents and solvents used in this study were of analytical or HPLC
grade and were obtained commercially (Wako Pure Chemicals, Osaka, Japan).
Animals and Administration.
Male Sprague-Dawley rats, obtained from Charles River Co., Ltd.
(Yokohama, Japan), aged 9 to 10 weeks, were used in this study. The
animals were fasted overnight before dosing, with access to water ad
libitum. Feeding was resumed 12 h after administration of
donepezil. [14C]donepezil was dissolved in
distilled water and administered orally by gavage. The dose was set at
1.0 mg/kg, based on toxicity.
Determination of Radioactivity.
Radioactivity was measured by an LSC-3500 liquid scintillation counter
(Aloka Co., Ltd., Tokyo, Japan). The counting efficiency was corrected
according to the channel ratio method by an external standard curve.
The detection limit of radioactivity was set at 40 dpm/sample after
subtraction of the background level (20 dpm/sample). Radioactivity was
expressed as nanograms × equivalents per gram or milliliter.
Quantitative Determination of Donepezil in Plasma and Brain.
Plasma and brain samples were obtained at 30 min and 4, 8, 12, 24, and
48 h after administration, using four animals at each time point.
The levels of unchanged donepezil in plasma and brain were determined
by HPLC with UV detection. A 5-µm Nucleosil 5C18 column of 4.6 mm
i.d. × 250 mm length (Chemo Scientific Co., Ltd., Tokyo, Japan)
analytical column was eluted with acetonitrile/5 m M SDS (pH 2.5)
(52:48, v/v) at a flow rate of 1.0 ml/min at 40°C. Donepezil was
detected using a UV spectrophotometer at 315 nm. In this study, the
assay methods were validated within the range 1.0 to 200 ng/ml in
plasma, and 2.0 to 500 ng/ml of homogenate in brain.
Measurement of Radioactivity in Blood.
Blood samples were collected from a tail vein immediately before
administration, and at 15 min and 0.5, 1, 1.5, 2, 3, 4, 6, 8, 10, 12, 14, 24, 36, 48, and 72 h after oral administration of
[14C]donepezil to four rats, and the
radioactivity in each sample was determined.
Measurement of Urinary and Fecal Excretion of Radioactivity.
Four rats were individually housed and fed in metabolic cages. Urine
and feces samples were collected each day for the first 7 days. Urinary
and fecal excretion were determined by measuring the radioactivity in
each sample. For urine samples, the metabolic cages were washed with
purified water at the time of collection and washing water was added to
the urine samples.
Measurement of Biliary Excretion of Radioactivity.
On the day before administration, under ether anesthesia, the rats
underwent cannulation of the bile duct with a PE-50 polyethylene tube.
Four bile duct-cannulated rats were given
[14C]donepezil orally and were individually
fixed in a Bollman cage (Toriumi et al., 1994). Bile, urine, feces, and
blood samples were collected after dosing. Biliary, urinary, and fecal
excretions were determined by measuring the radioactivity in each
sample. Bile samples were collected for 48 h using a fraction
collector. Urine and feces samples were collected to a dish placed
under the Bollman cage and the wire net placed on the dish,
respectively, each day for the first 2 days. Blood samples (50 µl)
were collected from a tail vein with a heparinized micropipette
immediately before administration, and at 15 min and 0.5, 1, 1.5, 2, 3, 4, 6, 8, 10, 12, 24, and 72 h, and the radioactivity in blood
samples was determined.
Tissue Distribution of Radioactivity.
Rats were anesthetized with ether and exsanguinated from the descending
aorta using heparinized disposable plastic syringes at 30 min and 4, 8, 24, 48, and 168 h after administration, using four animals at each
time point. The rats examined at 168 h were those used to study
urine plus fecal excretion.
Immediately after blood collection, tissues and/or organs were removed.
The brain, liver, and kidneys were removed, and their weights were
measured. The radioactivity of weighed samples of tissues and/or
organs were determined.
Isolation of Metabolites in Urine and Feces.
Metabolites of donepezil were isolated from rat urine and feces
collected for 24 h after oral administration. The urine or fecal
homogenate with water was applied to a column (40 mm i.d. × 600 mm) of
Amberlite XAD-2 (Yoshimura et al., 1985; Teegarden et al., 1991; Street
et al., 1996). The column was washed with distilled water and
metabolites were eluted with methanol. The fractions were evaporated at
40°C under reduced pressure and the residues were dissolved in
methanol. An aliquot was subjected to TLC on Kieselgel
60F254 plates (E. Merck, Darmstadt,
Germany) and developed in chloroform/methanol/25%
ammonium-water (100:20:1, v/v/v, TLC system A). The position of the
radioactive spots was confirmed using X-ray film, and the silica gel
corresponding to the radioactive spots was scraped off and extracted
with methanol. The metabolite fractions were evaporated at 40°C under
reduced pressure.
The most polar metabolites (which corresponded to the radioactive spot
on the origin in the TLC system A) were subjected to TLC again and
developed in n-butanol/acetic acid/water (4:1:1, v/v/v, TLC
system B). The silica gel corresponding to the radioactive spots was
again scraped off, extracted with methanol, and evaporated at 40°C
under reduced pressure. The metabolite fractions were then purified by HPLC.
Identification of Metabolites by MS and NMR Analysis.
The structures of purified metabolites were determined by using MS and
NMR analysis. Fast atom bombardment mass spectra (Naitoh et al., 1997;
Dieden et al., 1997; Nickmilder et al., 1997) were recorded using a
JEOL model JMS-HX100 mass spectrometer (Japan Electron Optics Lab,
Tokyo, Japan). Unless specified otherwise, m-nitrobenzyl
alcohol was used as the matrix. Electron impact mass spectra (Chung et
al., 1994) were determined using a JMS-DX300. 1H-NMR spectrometry was conducted using a JEOL
model JNM-GX400. The spectra were recorded in
CD3OD, with the CHD2OD peak
(
3.35) as reference.
Levels of Metabolites in Tissues and Excreta.
The amounts of metabolites were quantitated in the following biological
samples: plasma, brain, liver, and kidneys at 0.5 h after
administration; urine, feces, and bile for 24 h after administration.
After the addition of distilled water, the brain, liver, kidneys, and
feces (respectively about 2-10 g) were weighed and then homogenized
using a mechanical homogenizer with a stainless steel blade. After
extraction 4 times with 15 ml of methanol, the pooled methanol phases
were evaporated under reduced pressure at about 35°C. To the
semisolid residue 1 ml of methanol was added, the sample was vortexed
and centrifuged, and the resulting supernatant was analyzed. About 2 ml
of plasma, urine, and bile were each extracted with methanol in the
same manner and evaporated under reduced pressure to obtain the samples
for analysis. An aliquot was subjected to TLC (Kieselgel
60F254; E. Merck) and developed on TLC system A. The radioactive spots observed were confirmed by radioluminography
(BAS-2000; Fuji Film Co., Ltd., Fuji, Japan)(Okuyama et al., 1994). The
silica gel corresponding to the radioactive spots was scraped off, and
2 ml of methanol and scintillator (ACS II; Amersham) were added. The
radioactivity was measured to calculate the ratio of metabolites in
each sample. For polar metabolites, a second aliquot was subjected to
TLC and developed on TLC system A, then the silica gel corresponding to
the radioactive spots on the origin was scraped off and extracted with
10 ml of methanol, evaporated under reduced pressure, and subjected to
TLC system B. The relative amounts of metabolites in tissues and
excreta were corrected by the extraction ratio for each sample.
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Results |
Blood Levels of Radioactivity in Intact and Bile Duct-Cannulated
Rats.
Mean blood levels of radioactivity after a single oral administration
of [14C]donepezil (1 mg/kg) are shown in Fig.
2. In intact rats, the mean blood level of radioactivity reached a peak
(61.1 ± 6.26 ng eq/ml) at 0.5 h after administration, and
then declined with two small peaks at 6 and 14 h. The blood level
of radioactivity decreased to 8.1% of the maximum 72 h after
administration. AUC(0-72 h) was 1346 ± 66.8 ng eq · h/ml. In bile duct-cannulated rats, the mean
blood level of radioactivity reached a peak (107 ± 29.9 ng eq/ml)
at 1.0 h after administration, and then declined biphasically. AUC(0-72 h) was 657 ± 38.0 ng
eq · h/ml, which was 48.8% of that in intact rats.
Plasma and Brain Levels of Unchanged Donepezil and Radioactivity.
Plasma and brain levels of unchanged donepezil and radioactivity after
a single oral administration of [14C]donepezil
(1 mg/kg) are shown in Fig. 3.
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Fig. 3.
Plasma and brain levels of donepezil and
radioactivity after a single oral administration of
[14C]donepezil (1 mg/kg) to rats.
Each points represents the mean ± S.E. of four animals.
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The plasma and brain levels of unchanged donepezil were determined by
the HPLC-UV method, and were found to be reach a maximum at 0.5 h
after dosing (47.5 ± 12.3 ng/ml and 234 ± 61.8 ng/g, respectively). In plasma, the concentration of unchanged drug declined
more rapidly than that of radioactivity. Consequently, the ratios of
unchanged donepezil to total level of radioactivity in plasma at 0.5, 4, and 8 h after administrations were 32.7, 9.42, and 4.31%,
respectively. Radioactive analysis of 0.5-h plasma samples showed that
in plasma most of the radioactivity was associated with
O-glucuronides, and unchanged donepezil accounted for 31.0% of radioactivity (Table 6).
In contrast, the brain level of radioactivity declined almost in
parallel with that of unchanged donepezil, and the ratios of unchanged
donepezil to total radioactivity in the brain at 0.5, 4, and 8 h
after administration were 93.0, 87.9, and 86.9%, respectively. This
result was further confirmed by the radioactive analysis of 0.5-h brain
samples (Table 6). Brain level of unchanged donepezil changed almost in
parallel with that in plasma.
Tissue Distribution of Radioactivity.
Tissue distribution of radioactivity at various times after a single
oral administration of [14C]donepezil is
summarized in Table 1.
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TABLE 1
Tissue distribution of radioactivity after a single oral administration
of [14C]donepezil (1 mg/kg) to rats
Mean and S.E. are calculated for four rats at each time point.
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At 30 min after administration, which is
tmax of plasma radioactivity
(Cmax; 136 ng ± 26.7 ng · eq/ml),
excluding the gastrointestinal tissues at the administration
site, the highest concentrations of radioactivity were detected in the
liver, pancreas, hypophysis, adrenals, kidneys, and bone marrow. These
were 11.4 to 31.9 times the plasma concentration. Relatively high
levels were also found in the spleen, lung, submaxillary gland,
harderian gland, urinary bladder, and prostate gland, which were 5.51 to 9.96 times the plasma concentration. Almost all other tissues had
higher levels than that in plasma. In brain as the target organ, the
cerebrum, hypothalamus, hippocampus, striatum, cerebellum, and
hypophysis radioactivities were measured separately. Except for
hypophysis, no heterogeneous localization of radioactivity was
recognized, and the concentration in each part of the brain was 1.74 to
2.24 times the plasma level. At this time point, brain, liver, and kidneys contained 0.19 ± 0.05, 14.0 ± 2.62, and 1.48 ± 0.34% of the dose, respectively.
At 4 h after administration, the mean plasma level of
radioactivity decreased to 91.6 ± 20.0 ng · eq/ml, but the
levels in some tissues (e.g., hypophysis, harderian gland, submaxillary gland, pancreas, and testis) reached a maximum at this time. High concentrations of radioactivity, which were 15.0 to 56.8 times the
plasma concentration, were detected in the pancreas, hypophysis, harderian gland, adrenals, submaxillary gland, and liver. Relatively high levels, which were 4.10 to 8.86 times the plasma concentration, were also found in the kidneys, testis, prostate gland, spleen, bone
marrow, and lung. At this time point, brain, liver, and kidneys contained 0.06 ± 0.01, 4.20 ± 0.36, and 0.71 ± 0.12%
of the dose, respectively.
At 8 h after administration, the highest concentration of
radioactivity, which was 47.7 times the plasma concentration (62.7 ± 6.40 ng · eq/ml), was observed in the pancreas. The
concentration of radioactivity in almost all tissues was lower than
that at 4 h after administration.
At 24 h after administration, the highest concentration of
radioactivity, which was 57.3 times the plasma concentration (8.85 ± 0.63 ng · eq/ml), was observed in the pancreas. As this ratio was similar to that at 8 h, the pancreas levels of radioactivity were tending to decrease at a similar rate to the plasma levels. Relatively high levels, which were 8.07 to 20.7 times the plasma concentration, were also found in the adrenals, liver, and harderian gland.
At 48 h after administration, the mean plasma level of
radioactivity was below the quantification limit. In contrast, high concentrations of radioactivity were still observed in the pancreas, liver, adrenals, testis, and harderian gland, but all of these concentrations were less than 2.31% of the maximum values obtained.
At 168 h after administration, no radioactivity was detected in
any tissues except for testis and liver, the concentrations of which
had declined to 0.93 and 0.06%, respectively, of the maximum.
Urinary and Fecal Excretion of Radioactivity.
Cumulative urinary and fecal excretions of radioactivity after a single
oral administration of [14C]donepezil are shown
in Fig. 4. Twenty-four hours after
administration, 91.2 ± 0.72% of the administered dose was
recovered in the excreta, of which 36.9 ± 0.81% was in urine and
54.3 ± 0.32% in feces. A total of 98.9 ± 0.77% of the
administered dose was recovered in the excreta, of which 39.2 ± 0.65% was in urine and 59.7 ± 0.64% in feces, by the end of the
study period.
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Fig. 4.
Cumulative urinary and fecal excretion of
radioactivity after a single oral administration of
[14C]donepezil (1 mg/kg) to rats.
Each point represents the mean ± S.E. of four animals.
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Biliary, Urinary, and Fecal Excretion of Radioactivity in Bile
Duct-Cannulated Rats.
Cumulative biliary, urinary, and fecal excretion of radioactivity after
a single oral administration of [14C]donepezil
to bile duct-cannulated rats are shown in Fig.
5. One rat was excluded from the mean as
it had an extremely low excretion rate in bile and higher blood
radioactivity levels compared with the other animals.
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Fig. 5.
Cumulative binary, urinary, and fecal
excretion of radioactivity after a single oral administration of
[14C]donepezil (1 mg/kg) to bile
duct-cannulated rats.
Each point represents the mean ± S.E. of three animals.
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In the bile, 70.1, 72.2, and 72.9% of administered radioactivity were
excreted 12, 24, and 48 h after administration, respectively. In
the urine and feces concurrently collected, 24.4 and 8.84%, respectively, of the administered radioactivity was excreted 48 h
after administration. At this time, 97.3% of the administered radioactivity was recovered from the urine and bile. These results indicate that over 90% of donepezil was absorbed.
Structures of Isolated Metabolites.
The assignment of peaks in the 1H-NMR spectrum
for metabolites is shown in Tables 2,
3, and 4,
and the assignment of major fragment ions in the electron impact mass
spectrum is shown in Table 5.
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TABLE 2
1H-NMR assignment of metabolite (1)
Reference: CHD2OD/3.35. s, singlet; dd, double
doublet; ddd, double double doublet; J, coupling constant; m,
multiplet; br, broad.
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TABLE 3
1H-NMR assignment of metabolite (2)
Reference: CHD2OD/3.35. s, singlet; dd, double doublet; ddd,
double double doublet; J, coupling constant; m, multiplet; br, broad.
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TABLE 4
1H-NMR assignment of metabolite (3)
Reference: CHD2OD/3.35. s, singlet; dd, double doublet; ddd,
double double doublet; J, coupling constant; m, multiplet; br, broad.
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Metabolites M1 and M2 were identified as the O-demethylate
derivatives based on 1H-NMR and MS data, and M3
and M4 were identified as the aromatic hydroxylate and
N-dealkylate, respectively.
Metabolites M5 and M6 were identified as axial and equatorial forms of
N-oxide, respectively, based on 1H-NMR
and MS data. The axial configuration of 1-CH2 was
determined by Nuclear Overhauser Effect (NOE) correlation spectroscopy
(mixing time, 100 ms) in CDCl3. NOE cross-peaks
were observed between 1-CH2 (4.97) and 3a and
5a (1.74). As for the equatorial configuration of
1-CH2, NOE cross-peaks were observed between
1-CH2 (4.75) and 2a (6a) and 2e (6e) (3.6
2) (data not shown).
Metabolites M7, M8, M9, and M10 were identified as the
O-demethylate of M3, the hydroxylate of M4, and the
di-O-demethylate and di-O-demethylate of M7,
respectively, based on 1H-NMR and MS data.
Metabolites M11 and M12 were identified as the glucuronide conjugate of
M1 and M2, respectively, and M13 and M14 were identified as the
glucuronide conjugates of M9 based on 1H-NMR and
MS data.
The structure of metabolites M1, M2, M3, M4, M5, and M6 were confirmed
by comparison with the respective reference standard compounds.
Levels of Metabolites in Tissues and Excreta.
The amount of metabolites in tissues and excreta after a single oral
administration of [14C]donepezil are shown in
Tables 6,
7, and 8.
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TABLE 6
Levels of metabolites in plasma and tissues after a single oral
administration of [14C]donepezil (1 mg/kg) to rats
Mean and S.E. are calculated for four rats at each time point. BQL,
<40 dpm/sample; 0.135 ng eq/sample.
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TABLE 7
Levels of metabolites in excreta after a single oral administration of
[14C]donepezil (1 mg/kg) to rats
Mean and S.E. are calculated for three rats at each time point. BQL,
<40 dpm/sample; 0.135 ng eq/sample.
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TABLE 8
Levels of metabolites in bile after a single oral administration of
[14C]donepezil (1 mg/kg) to bile duct-cannulated rats
Mean and S.E. are calculated for three rats at each time point. BQL,
<40 dpm/sample; 0.135 ng eq/sample.
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The amount of metabolites was determined by TLC. As the glucuronide
conjugates of M1, M2, and M9 (M11, M12, and M13 and M14, respectively)
could not be separated by TLC, they were handled as a composite of the
O-glucuronide fraction. Unknown metabolites, UM1, UM2, and
UM3, were also separated and their relative amounts were determined.
At 0.5 h after dosing, the majority of radioactivity was found as
unchanged donepezil in the brain, liver, and kidneys, whereas in
plasma, O-glucuronides were associated to 55.1% of
radioactivity (Table 6).
In urine, O-glucuronides accounted for 23.0% of the dose,
followed by M4, M1, donepezil, M7-sulfate, and M2; 1.52% of the dose
was found as unchanged donepezil (Table 7).
In feces, M9 was the main metabolite followed by
O-glucuronides, donepezil, M7-sulfate, UM1, M4, and M1;
4.21% of the dose was found as unchanged donepezil (Table 7). In
contrast, in bile the majority of the radioactivity was found as
O-glucuronides, and unchanged donepezil accounted for 0.16%
of the dose (Table 8). These results indicate that biliary excretion of
unchanged donepezil is negligible and O-glucuronides
excreted via bile are mostly hydrolyzed to liberate their aglycons in
the gastrointestinal tract.
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Discussion |
Absorption, distribution, metabolism, and excretion of
[14C]donepezil were studied after a single oral
administration (1 mg/kg) to rats.
The difference between the blood levels of radioactivity in intact and
bile duct-cannulated rats versus time indicate that drug-related
materials undergo enterohepatic circulation as is known to occur with
many drugs (Dickinson et al., 1979; Rubio et al., 1980; Watari et al.,
1984; Greenbelt and Engelking, 1988; Komura et al., 1992; Hasselstrom
and Sawe, 1993; Kawakami et al., 1993). In bile, the majority of the
radioactivity was found as O-glucuronides, and unchanged
donepezil accounted for 0.16% of the dose. As direct conjugation of
donepezil was not found, it is believed that its metabolite(s) appear
to be mainly involved in enterohepatic circulation. Based on the
results of fecal and biliary radioactivity measurements,
O-glucuronides excreted via bile are mostly hydrolyzed in
the gastrointestinal tract to liberate their aglycons (Simons et al.,
1992; Ouellet, 1995; Sandouk et al., 1998). In feces, the main
metabolite was M9, which might result from enterohepatic circulation
and/or metabolism by enterobacterial flora of M13 and M14.
The majority of radioactivity in 30-min plasma was
O-glucuronides, whereas in the liver, kidneys, and brain,
over 70% of radioactivity was attributable to unchanged donepezil. In
brain, approximately 90% of radioactivity in brain was found as
unchanged donepezil, and O-glucuronides were BQL,
suggesting low permeability of O-glucuronides through the
blood-brain barrier.
Tissue distribution studies showed that the concentration of
radioactivity in most organs and/or tissues was higher than that in
plasma, and at 168 h, the concentration of radioactivity in all
organs and/or tissues examined was BQL except for testis and liver
(0.93 and 0.06% of the maximum, respectively), suggesting that
drug-related materials do not accumulate markedly.
The proposed metabolic pathways of donepezil in rats are shown in Fig.
6. Based on the metabolite profile in
excreta, the metabolism involves mainly three types of reactions: 1)
O-demethylation at each methoxy group of the dimethoxyindan
moiety, followed by O-glucuronide conjugation, 2)
N-dealkylation at the piperidine ring, 3)
N-oxidation at the piperidine ring, and to a less extent 4)
aromatic hydroxylation followed by sulfate conjugation.
A similar O-demethylation occurred in the biotransformation
of KB-2796, which has a 2,3,4-trimethoxybenzyl moiety (Kawashima and
Satomi, 1991); 4-desmethyl KB-2796 was mostly present in urine, followed by 3-desmethyl KB-2796. Also in the metabolic studies of
trimetazidine (Jackson et al., 1996), 3 and 4-desmethyl trimetazidine was more present in urine 2-desmethyl trimetazidine. In the case of
laudanosine (in dog, rabbit, and humans), has dimethoxybenzyl moiety
(Canfell and Castagnoli, 1986), it was proposed that
O-demethylation occurred at two methoxy groups equally. In
donepezil, because the demethylate at 6"-position of the dimethoxyindan
(M1) was present in larger amounts than that at 5"-position of the
dimethoxyindan (M2) in urine and feces, donepezil might be demethylated
position selectively. The potency of M1 (and M3) in
acetylcholinesterase inhibition activity is comparable with donepezil
and about 140-times higher than that of M2 (data not shown). From these
results, the 6"-position methoxy of the dimethoxyindan moiety is
supposed to play an important role for biological activity. However,
the transfer of M1 and M3 into the brain as the target organ appeared
to be low; it was supposed that the contribution of M1 and M3 to the pharmacological activity of donepezil is negligible.
As a result of the metabolism by oxidative N-dealkylation of
the piperidine ring in donepezil, M4 and M8 were found. A similar N-dealkylation occurred in risperidone, and the major
metabolic pathway was the oxidative dealkylation at the piperidine
nitrogen in rats and dogs (Meuldermans and Hendrickx, 1994). In the
metabolic studies of alfentanil, the piperidine nitrogen dealkylation
to noralfentanil was the major metabolic pathway and this metabolite formation was catalyzed by CYP3A4 in human liver microsomes (Labroo and
Paine, 1997). Moreover, fentanyl undergoes extensive metabolism to
norfentanyl by piperidine N-dealkylation and norfentanyl
formation were significantly correlated with CYP3A4 activity,
suggesting a prominent role for CYP3A4 in human liver microsomes
(Labroo and Thummel, 1995). Also, metabolite M4 of donepezil formed
mainly by CYP3A4 and to a lesser extent by CYP2C9 (K. Matsui, S. Taniguchi, and T. Yoshimura, in preparation).
Phase II metabolism occurred after phase I metabolism of donepezil.
Glucuronide conjugates of M1, M2, and M9 (O-demethylate) were detected, but not of M7 (aromatic hydroxylate), whereas the sulfate conjugation occurred in only M7.
In summary, results of the present study indicate that after oral
administration of [14C]donepezil, over 90% of
radioactivity was absorbed rapidly and extensively metabolized. Taking
into account levels of metabolites in excreta, the major metabolic
pathways for donepezil involve O-demethylation followed by
glucuronide conjugation and N-dealkylation. Although the
tissue distribution of [14C]donepezil was
relatively high, in brain most of radioactivity was unchanged donepezil
and no metabolites were found in brain. There was no notable
accumulation of radioactivity in whole blood and tissues.
Abbreviations used are:
AUC, area under the
curve;
TLC, thin-layer chromatography;
MS, mass spectrometry;
NOE, Nuclear Overhauser Effect;
BQL, below quantification limit;
IS, internal standard;
EI, electron impact.
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Abstract
Introduction
