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Introduction
As an important active constituent of Ginkgo biloba
extract, ginkgolide B is a highly selective and competitive
platelet-activating factor (PAF) receptor
antagonist[1]. In recent clinical and experimental experiments, ginkgolide B has
been reported to be effective against ischemic brain
injury[2,3], platelet aggregation and
thrombosis[4], and
inflammatory[5] and central nervous system diseases, such as postischemic
neuronal damage, dementia, and Alzheimer's
disease[6,7].
Now, ginkgolide B is being developed for treating
cerebrovascular accidents. The preclinical pharmacokinetics of
ginkgolide B have been reported in our laboratory; the data
showed that 45% and 2.6% of ginkgolide B administered to
rats were excreted into urine and feces in 48
h[8], respectively, suggesting that nearly 50% of ginkgolide B may undergo
metabolism in vivo. Up to now, the detailed metabolic
pathway of ginkgolide B in rat has not been reported.
The purpose of this experiment was to identify the
metabolites of ginkgolide B in rat urine, the predominant
metabolism of ginkgolide B in rat liver microsomes
in vitro, and to identify the major cytochrome (CYP) P450 enzymes
responsible for its metabolism.
Materials and methods
Chemicals Ginkgolide B and limonin (as the internal
standard [IS]) were obtained from the National Institute for
the Control of Pharmaceutical and Biological Products
(Beijing, China). Glucose-6-phosphate dehydrogenase
(G-6-PDH; type V), α-nicotinamide adenine dinucleotide
phosphate (NADP), glucose-6-phosphate (G-6-P),
α-naphthoflavone (α-Naph), sulfaphenazole (Sul), quinidine (Qui), and
diethyldithiocarbamate (DDC) were purchased from Sigma
(St Louis, MO, USA). Ketoconazole (Ket) was kindly
provided by Nanjing Second Pharmaceutical Factory (Nanjing,
China). Ginkgolide B emulsion was presented by Beijing 303
Hospital (Beijing, China). HPLC-grade methanol was
purchased from Fisher Scientific (Fair Lawn, NJ, USA). All other
supplies were available from standard commercial sources.
Animal Ten Sprague-Dawley rats (5 male and 5 female,
200±20 g, 7 weeks of age) were obtained from the
Experimental Animal Center of China Pharmaceutical University
(Nanjing, China), and the animal experiments were approved
by the Animal Ethics Committee of China Pharmaceutical
University.
Rat urine specimen collection and sample preparation
Six Sprague-Dawley rats (3 male and 3 female) were fasted
for 10 h and then a single dose of ginkgolide B emulsion (4
mg/kg) was injected via the caudal vein. The rats were placed
in the metabolism cages and the urine was collected at 0_8 h
and immediately frozen and stored at -20 °C until analysis.
In total, 1 mL methanol was added to 20 mL rat urine in a
2 μL centrifuged tube. The tubes were mixed for 5 min and
centrifuged for 5 min at 10 000×g; 2 μL supernatant was
injected into the liquid chromatography (LC) mass
spectrometer (MS) system.
Microsomal incubations and sample preparation
The rat liver microsomes were prepared by differential
centrifugation[9], and the microsomal protein concentration was
determined by the method of Lowry et al
[10].
Substrate concentrations of 1_100 μmol/L, microsomal
protein of 0.5_2 g/L, and incubation times of 5_90 min were
used to optimize assay conditions. A typical incubation
mixture consisted of 100 mmol/L potassium phosphate buffer
(pH 7.4), an NADPH-generating system (5 mmol/L
MgCl2, 10 mmol/L G-6-P, 1 mmol/L NADP, 1 kU/L G-6-PDH), and 1 g/L
microsomal protein in a final volume of 1
mL[9]. Before the addition of the substrate, the incubation mixtures were
prewarmed for 5 min at 37 °C. Reactions was initiated by the
addition of the substrate, and the concentration of the
substrate for all incubations was 20 μmol/L. The incubations
were conducted at 37 °C for 0.5 h, and the reaction was
terminated by adding 10 μL of 10% HClO4. The samples were
centrifuged (20 000×g) at 4 °C for 10 min, and the
supernatant was separated from the precipitated protein and
concentrated by evaporation under a gentle stream of nitrogen.
The residues were then reconstituted in 200 μL methanol
followed by centrifugation at 20 000×g for 10 min; an aliquot
of 10 μL was injected into the LC-MS system. Incubations
without NADPH served as negative controls.
Identification of metabolites using LC-MS and LC
ion-trap-time-of-flight MS in rat urine and microsomal
incubations Urine and incubations samples were analyzed by LC
quadrupole MS (LC/ESI-MS; Quadrupole MS, Shimadzu,
Kyoto, Japan) and LC ion-trap-time-of-flight MS
(LC-IT-TOFMS; Shimadzu, Japan). LC/ESI-MS was performed for the
profile of metabolites of ginkgolide B, and mass
spectrometric conditions were optimized to obtain maximum sensitivity;
the curved desolvation line voltage was fixed as that in
Tuning, the probe high voltage was set at 4.5 kV, Q-array
voltage of DC _35 V, and RF 150 V. Mass spectra were
obtained at a dwell time of 1 s in scan mode. The accuracy
masses, formulas, and the possible structure of the
metabolite were obtained by using an LC-IT-TOF-MS equipped with
an electrospray ionization source (negative-ionization mode).
The probe voltage was 4.5 kV, and both the curved
desolvation line temperature and the block heater temperature were 200
°C. The ion accumulation time and isolation time were set at 30
and 20 ms, respectively. Argon was used as collision gas
and its inflow rate to the ion trap was set 50% during
collision induced dissociation (CID). The CID energy and times
were 50% and 30 ms, respectively.
Full-scan quadrupole MS spectra were first acquired for
molecular ions of parent compounds and relevant metabolites.
Subsequently, the ion trap mass analyzer could be used for
the MSn analysis and could give the maximum amount of
structural information by CID of appropriate molecular ions
that were selected; the accuracy masses, formulas of the
molecular ions, and their fragments were obtained by using
the TOF analyzer.
The separation of metabolites was achieved on ODS and
HPLC columns (Shim-pack, 5 μm, 2.0 mm ID×150 mm;
Shimadzu, Japan) using a LC2010 series chromatography
system (Shimadzu, Japan) consisting of 2 pumps, an
autosampler, and a column oven. A gradient elution mode
was adopted using 2 mobile phases: 0.04% of triethylamine
water (v/v) and methanol. The flow rate was 0.2 mL/min. The
column was equilibrated with 25% ginkgolide B at 0 min.
After an injection of the sample, the methanol content was
linearly increased to 85% at 8 min. Then the percentage of
ginkgolide B was linearly decreased within 2 min to 25% of
the initial composition, and held to equilibrate the column
for 2 min before the application of the next samples. Data
acquisition and processing were accomplished using
Shimadzu LCMS solution version 3.40 for the
LC-IT-TOF-MS system.
Characterization of P450 isoforms involved in the
formation of ginkgolide B metabolites in rat liver microsomes
The inhibition of the ginkgolide B metabolism was evaluated
using various selective chemical inhibitors that contained
α-Naph for CYP1A2, Qui for CYP2D6, DDC for CYP2E, Sul
for CYP2C9, and Ket for CYP3A. The final concentration of
ginkgolide B was 20 μmol/L, and the concentration range of
inhibitors was 2.5_20 μmol/L for Qui, 12.5_100 μmol/L for
α-Naph and Sul, 6.25_50 μmol/L for DDC, and 0.5_5
μmol/L for Ket in microsomal
incubations[11]. Rat liver microsomes (1
g/L) were pre-incubated with various concentrations of
inhibitors for 5 min at 37 °C, followed by the addition of buffer.
The incubation mixtures were prewarmed for 5 min in the
NADPH-regenerating system before the reactions were
initiated by the addition of the substrate; then the incubations
were conducted for 0.5 h and terminated as described earlier.
As the inhibitors were dissolved in methanol, pure methanol
was included in the control incubations (without inhibitors)
to rule out any effects of the solvent on the microsomal
activity. The final concentration of the organic solvent in
the incubation system was 1%.
After identifying the major metabolic enzyme involved
in the metabolism of ginkgolide B in vitro, the microsomes
were incubated with various concentrations of ginkgolide
B (0.5-20 μmol/L) in the absence and presence of the various
concentrations of the inhibitors; all of the incubations were
carried out as described before. Then the Lineweaver-Burk
plot of ginkgolide B oxide activity in the absence or
presence of the inhibitors was compared with the enzyme
kinetics characteristics of the inhibition types
(Vmax and Km). The
Lineweaver-Burk plot and linear regressions were applied
for the inhibition study and for the calculation of
Ki values.
The 0.2 mL incubation mixtures were added to 10
μL of 1 mg/L IS (limonin) solution, extracted with 1.2 mL
acetoacetate and centrifuged at 3000×g
for 10 min. Then the organic layer was removed and evaporated under a stream of
nitrogen at 45 °C. The residue was re-dissolved in 200
μL of mobile phase, and an aliquot (5 μL) was injected into the
LCMS system. The metabolites in the incubation mixture
were determined using a LC2010 series chromatography
system coupled to a quadrupole mass spectrometer (Shimadzu,
Kyoto, Japan). MS spectrometer was operated in negative
ion electrospray mode and analyte detection was performed
in the selected ion monitoring mode. The LC separation
condition was identical with the described above. Data
acquisition and processing were accomplished using Shimadzu
LCMS solution version 2.02 worked on Windows 2000. As
M1 standards were not available, the relative quantification
of the metabolite concentrations in the incubation were
compared using metabolite peak area ratios to the IS obtained for
the respective samples.
Results
In vivo identification of metabolites of ginkgolide B in
rat urine The full-scan mass spectrum was obtained from
urine sample after ginkgolide B was intravenously
administered to the rats and compared with the blank urine samples
(Figure 1) to discover the main possible metabolites in rat
urine. The [M-H]- ions of the possible metabolites were at
m/z 439 (M1), m/z 441
(M2), and m/z 457 (M3), respectively. Thus,
the molecular weights of M1,
M2, and M3 were 440, 442, and
458, respectively. No significant differences were detected
in the formation of the metabolites between the male and
female rats. The accuracy mass data, formulas, and the
possible structure of the metabolites were obtained by using a
LC-IT-TOF-MS (Table 1). The [M-H]- ions of
M1, M2, and M3 were increased by 16, 18, and 34 Da, respectively, compared
to that of ginkgolide B. The measured accuracy masses of
the m/z 439 metabolite of ginkgolide B indicated the charge
of +O moiety from the parent compound, which is consistent
with its being the corresponding hydroxyl metabolite. The
measured accuracy masses of the m/z 441 metabolite of
ginkgolide B indicated the charge of
+H2O moiety from the parent compound. Relative to ginkgolide B,
biotransformation to m/z 457 resulted in a charge of
+H2O2. As ginkgolide B readily dissolved in the basic solution, recovered
quantitatively on subsequent
acidification[12], and was present in 2
forms in vivo by using 3H-ginkgolide B: the original
ginkgolide B with its ring closed and the second form with one of the
rings open[13], we can tentatively presume that
M2 was a hydrolysis metabolite of ginkgolide B, and
M3 was a hydrolysis product of
M1.
The most abundant product ion of m/z 367.1422 was formed
by the loss of 2CO from the molecular ion at
m/z 423.1334. The main fragment ions of
m/z 439.1253 and 457.1385 in the
MS2 spectra were m/z 383.13 by the loss of 2CO (Figure 2). This
suggests that ginkgolide B and M1,
M2, and M3 have a similar mass schizolysis rule and structure core. According to
the above analysis, we can tentatively presume the proposed
metabolic pathway of ginkgolide B in rats (Figure 3).
In vitro identification metabolism of ginkgolide B in rat
microsomes incubations Following the incubation of
ginkgolide B with rat liver microsomes, ginkgolide B was
rapidly metabolized, and only 1 metabolite
(M1) was isolated in the incubation (Figure 4). Their MS spectra were obtained
using an electrospray ionization interface under
negative-ion mode (Figure 5). Their accuracy mass data and formulas
were obtained by using a LC-IT-TOF-MS (Table 2). The
accuracy masses suggested elemental composition of the
main fragment MS2 ions of ginkgolide B, and its metabolite
were compared in rat liver microsomes obtained by the CID
of the corresponding [M-H]- ions (Table 3).
Compared to ginkgolide B
(C20H24O10,
m/z 423.1326), biotransformation to
M1
(C20H24O11,
m/z 439.1258) resulted in charge of +O moiety. Furthermore, the main fragment
MS2 ions of ginkgolide B (m/z 367.1423) and
M1 (m/z 383.1369), respectively, by using and ion trap mass analyzer, indicated
the loss of the 2CO group of ginkgolide B and its metabolites,
which demonstrated ginkgolide B and M1 have a similar mass
schizolysis rule and structure core. According to above
analysis, we could tentatively presume that
M1 was a hydroxyl metabolite of ginkgolide B (Figure 6).
Inhibition study with chemical inhibitors
When the rat liver microsomes were incubated using various
concentrations of the microsomal protein for various time periods, 1
g/L microsomal protein and the 30 min time frame were found to
be optimal incubation conditions. The effect of various
substrate concentrations of 1-100 μmol/L on the rate of the
metabolite formation demonstrated that the
Km for M1 was approximately 4
mmol/L. A substrate concentration of 20 μmol/L was chosen for further experiments, considering the
concentration levels in liver tissues (approximately 20
μmol/g liver tissue) in vivo[8], and the linearity and the sensitivity of
the detection of M1 and the
Km value of the M1 metabolite.
The effects of the inhibitors on the formation of
M1 are presented in Figure 7. Qui, the specific inhibitor of CYP2D6,
could inhibit the formation of M1, while other inhibitors had
no significant inhibitory effects on the
M1 formation. After identifying CYP2D6 involved in the metabolism of ginkgolide
B in vitro, the microsomes were incubated with various
concentrations of ginkgolide B (0.5-20 μmol/L) in the absence
and presence of Qui (0-20 μmol/L). The Lineweaver-Burk
plot of ginkgolide B oxide activity in the rat liver microsomes
in the absence or presence of Qui is presented in Figure 8.
The Lineweaver-Burk plot of ginkgolide B oxide activity in
the rat liver microsomes in the absence or presence of Qui
was parallel, which consistent with the enzyme kinetics
characteristics of the uncompetitive inhibition types (decreased
in Vmax and Km). The value of
Ki was estimated to be 8 μmol/L using the Lineweaver-Burk plot and linear regressions.
Discussion
For the first time, LC-IT-TOF-MS with electrospray
ionization in negative-ion mode was used for the analysis of
ginkgolide B and its metabolites in urine and microsomal
incubation. The measurement of the accuracy masses of
ions of the parent compounds and metabolites were useful
at unambiguously establishing that M1
(m/z 439.124;
C20H24O11),
M2 (m/z 441.1434;
C20H26O11), and
M3 (m/z 457.1385;
C20H26O12) had attached a mass equivalent to a O,
H2O, and H2O2 moiety relative to the corresponding ginkgolide B
(m/z 423.1334;
C20H24O10), respectively. Furthermore, abundant
main fragment MS2 ions of ginkgolide B and
M1 in the urine and the microsome incubation sample, respectively, by
using ion trap mass analyzer, further indicated the loss of 1 and
2 carbon oxide groups of ginkgolide B and its metabolites,
which further demonstrated they had a similar mass
schizolysis rule and structure core. It identified that
M1 is precisely a hydroxyl metabolite of ginkgolide B. Comparing
M1 in the urine and M1 in the microsome incubation, we
found that they had identical retention times and accuracy
masses of [M-H]- ions, formulas, and
MS2 fragments; thus, we confirmed that they were same compound. Therefore,
the proposed metabolic pathway of ginkgolide B is
metabolized to its hydroxyl metabolite (Figures 4 and 6). Furthermore,
the hydroxyl metabolite was not ginkgolide C, based on its
retention time being different to that of
M1 under the same chromatographic conditions.
In microsomal incubations, various concentrations of
ginkgolide B on the rate of the metabolite formation
demonstrated that the Km for
M1 was estimated to be approximately 4
μmol/L. In chemical inhibition studies, Qui, a CYP2D6
inhibitor, could uncompetitively inhibit the formation of
M1, and its Ki value was estimated to be 8
μmol/L, while other inhibitors for CYP3A (Ket), CYP2C9 (Sul), CYP1A2
(α-Naph), and CYP2E1 (DDC) had no effect on the formation of
M1. The results suggest that CYP2D6 is the major CYP450 isozyme
involved in the conversion of ginkgolide B to hydroxyl
metabolite. Pharmacokinetic drug interactions caused by
metabolic processes are regarded as one of the most
important factors affecting the concentration of drugs. Therefore,
the inhibition of the above metabolic pathway can lower the
formation rate of the hydroxyl metabolite and increase the
concentration of ginkgolide B, suggesting the possibilities
of metabolic interactions of ginkgolide B with other
therapeutic agents, such as inhibitors and substrates of CYP2D6.
In conclusion, the metabolic pathway ginkgolide B was
identified in rats using a high-resolution hybrid
LC-IT-TOFMS, which was further confirmed by rat liver microsome
incubation in vitro, and the CYP2D6 isozyme was identified as
the major CYP450 enzyme responsible for ginkgolide B
metabolism in the rat liver microsomes. These results are
important for further understanding the disposition of
ginkgolide B in rats.
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