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Introduction
Metoprolol, 1-isopropylamino-3-(4-[2-methoxyethyl]-phenoxy)-2-propanol, is a
β1 selective aryloxypropanolamine used in treatment of cardiovascular disorders such as
hyper-tension, arrhythmia, and heart failure. The drug is a lipophilic adrenoceptor
antagonist (β-blocker) with a short half life (3_4
h)[1]. It is therapeutically used as a racemic mixture.
Metoprolol is mainly eliminated by the hepatic oxidative metabolism and it undergoes extensive first pass metabolism
with about 95% of the dose being metabolized in humans. Metoprolol is metabolized to a large degree by cytochrome P-450
2D6 which is polymorphic in the human population. The appearance of major metabolites
O-desmethyl-meto-prolol, metoprolol acid, and hydroxymetoprolol varies
depending on the oxidation
phenotype[2_4].
The identification of metabolites from mammalian sources may be hindered by insufficient quantities of material. The
concept of using microorganisms, particularly the filamentous belonging to the genus
Cunninghamella, as models of mammalian metabolism has been well
documented[5_7]. The advantages of a microbial system as an
in vitro model for drug metabolism include its low cost, ease of handling,
scale-up capacity, and potential to reduce the use of mammals. A
microbial system also provides enough putative metabolites
under milder conditions than those required by chemical
systems. Thus, fermentation is used to scale up the
synthesis of metabolites for structural confirmation by nuclear
magnetic resonance (NMR)[8].
Although the metabolism of metoprolol in mammals has
previously been studied, to our knowledge, no report has been
published on the potential of filamentous fungi to metabolize
metoprolol. Cultures of 5 strains of
Cunninghamella were chosen for this investigation, because such fungi have the
ability to metabolize drugs, including
verapamil[9],
panto-prazole[10], and
indomethacin[11], in a similar manner to mammals.
The main focus of the current study was to investigate the
metabolic fate of metoprolol in cultures of the 5 strains of
Cunninghamella and demonstrate parallels of the
metabolism of metoprolol in mammalian and in microbial systems.
Materials and methods
Materials Metoprolol (purity 99.0%) was provided by
Xinhua Pharmaceutical Co (Changzhou, China);
the purity was checked by HPLC analysis. Methanol of HPLC grade
was purchased from Concord Technology (Tianjin, China).
Peptone and the yeast extract were biochemical reagents.
All other chemicals were of analytical grade.
Microorganisms C elegans AS 3.156,
C elegans AS
3.2028, C echinulata AS 3.2004, and C blakesleeana
AS 3.153 and AS 3.910, were provided by the Institute of
Microbiology, Chinese Academy of Sciences (Beijing,
China). Stock cultures were maintained on potato dextrose
agar (Aoboxing, Beijing, China) slants at 4 °C and
transferred every 6 months to maintain viability. The first-stage
microbial biotransformation was carried out in a medium
consisting of 20 g dextrose, 5 g peptone, 5 g yeast extract,
5 g NaCl, 5 g K2HPO4, and 1000 mL distilled water. The
second-stage biotransformation was carried out in a
wheat-bran medium containing 1% wheat-bran in broth. The pH
of the medium was adjusted to 6.5 with 6.0 mol/L HCl, and
then the medium was sterilized in Erlenmeyer flasks
(Pierce, Rockford, IL, USA) at 115 °C and 18 psi for 30
min and cooled before incubation.
Biotransformation procedures The microbial
metabolism was facilitated by incubating the cultures with shaking
on a rotary shaker at 28 °C. For each of the 5 strains of
Cunninghamella, the first-stage preculturing was initiated
by inoculating a 250-mL Erlenmeyer flask containing 50 mL
broth with a loop of spores obtained from a freshly growing
agar slant. After incubation for 24 h, a 1.0 mL portion from
the first-stage culture was used to inoculate a second-stage
100-mL flask containing 20 mL of broth. The second-stage
culture was incubated for 24 h before metoprolol was added
to a final concentration of 1.0 g/L. After 120 h of additional
incubation, the culture was centrifuged at
1500×g for 20 min, and the supernatant was decanted and kept at -20 °C until
analysis.
The preparative-scale biotransformation of metoprolol by
C blakesleeana AS 3.153 followed the same procedure as
the screening experiments, except for the increase of the broth
volume. Two first-stage flasks were prepared as described
earlier, and 4 second-stage flasks each containing 100 mL of
broth were incubated with 2.5 mL of the first-stage culture.
Metoprolol was added to yield a final concentration of 1.0
g/L. The culture was then incubated for an additional 120 h.
Two kinds of controls were conducted simultaneously
with the biotransformation procedure. Culture controls
consisting of a fermentation blank, in which the microorganisms
were grown under the same conditions without metoprolol,
were operated to eliminate the interference possibly brought
by the microorganism itself or residues of the fermentation
cultures. The substrate controls were prepared by adding
metoprolol to sterile medium and incubated without the
microorganism to determine whether metoprolol could
chemically decompose or spontaneously transform under
fermentation conditions.
Extraction of metabolites and liquid
chromatography-tandem mass spectrometry
(LC/MSn) assay A 0.5 mL aliquot of each sample was filtered through a membrane (0.45
µm pore size). The filtrate was applied to a preconditioned
Orgchem C18 cartridge (Orgchem, Troy, NY, USA). The
cartridge was washed with 1.5 mL water, and the metabolites
were eluted with 2.0 mL methanol. A 20 mL aliquot of the
eluate was directly injected into the
LC/MSn system for analysis.
The LC/MSn analysis was performed on a Thermo
Finnigan LCQ ion trap mass spectrometer (San Jose, CA,
USA) equipped with an atmospheric-pressure ionization
interface. The instrument was operated in positive
electrospray ionization (ESI) mode. The spray voltage was
set at 4.5 kV. The capillary temperature was maintained at
200 °C and the voltage was fixed at 13 V. The HPLC fluid was
nebulized by using N2 as both the sheath gas at a flow rate of
0.75 L/min and the auxiliary gas at a flow rate of 0.15 L/min. A
full-scan mass spectrum was operated to obtain the
protonated molecules (M+H)+ of each possible metabolite.
Multi-stage mass spectra (MS/MS or MS3) were produced by
collision-induced dissociation of the selected precursor ions
with helium present in the ion trap, and the relative collision
energy was set at 30%_35%. Data were collected and
analyzed with Xcalibur software (version 1.2, Thermo Finnigan,
NJ, USA). Liquid chromatography was carried out with a
Shimadzu LC-10AD solvent delivery system (Kyoto, Japan).
The samples were separated on a Diamonsil
C18 column (200 mm×4.6 mm inner diameter, 5
μm, Dikma, Beijing, China) preceded by a SecurityGuard
C18 guard column (4 mm×3.0 mm inner diameter, 5
μm, Phenomenex, Torrance, CA, USA). The mobile phase consisted of
methanol-water-formic acid (35:65:0.2,
v:v:v) at a flow rate of 0.5 mL/min.
Isolation and identification of major metabolites
To isolate the major metabolites of metoprolol in sufficient
quantities for structure elucidation, the biotransformation
was carried out on a semipreparative scale. After
biotrans-formation, the fluid was centrifuged at
1500×g for 20 min. The supernatant was condensed using a Rikakikai Eyela
Fdu1100 freeze dryer (Tokyo, Japan). The residue was
reconstituted by 10 mL methanol-water (1:1,
v:v) and directly deposited onto a
5 cm diameter column packed with Sephadex G10 gel (Zhonghuida Scientific Instrument Co, Dalian,
Liaoning, China). The sample was washed exhaustively with
methanol and the eluent was collected every 50 mL. The
solvent of each fraction was analyzed directly by
LC-MSn. Fractions containing possible metabolites were evaporated
to dryness under vacuum at 40 °C using a RE-52A rotary
evaporator (Yarong Biology Instrument, Shanghai, China).
In order to obtain sufficient quantities of metabolites for
structural identification, the sample was dissolved in a small
amount of water and then injected repeatedly onto a
semipreparative HPLC system (Shimadzu, Kyoto, Japan)
consisting of 2 LC-6AD solvent delivery units (Shimadzu,
Kyoto, Japan), a DGU-14A degasser unit (Shimadzu, Kyoto,
Japan), a SCL-10A VP system controller (Shimadzu, Kyoto,
Japan), a SPD-10A VP UV-Vis detector (Shimadzu, Kyoto,
Japan), a FRC-10A (Shimadzu, Kyoto, Japan) fraction
collector, and a CLASS-VP LC workstation (Shimadzu, Kyoto,
Japan). Separation was accomplished using a mobile phase
consisting of methanol-water (30:70,
v:v) at 8.0 mL/min on a Shim-Pack PRC-ODS column (250 mm×20 mm inner diameter,
Shimadzu, Kyoto, Japan) preceded by a GPRC-ODS pre-column (8 mm×1.5 mm inner diameter, Shimadzu, Kyoto,
Japan). The UV detector was set at 223 nm. The major peaks
with similar retention time were pooled, evaporated to
dryness, and stored at 4 °C before the structural analysis.
The purified metabolites were dissolved in
D2O for the NMR analysis. The NMR measurements were carried out
at 600 MHz on a Bruker ARX 600 NMR spectrometer (Bruker,
Faellanden, Switzerland). Chemical shifts were reported as
parts per million relative to tetramethylsilane as the internal
standard.
Results
Screening of cultures Five strains of
Cunninghamella were screened and all could transform metoprolol to some
extent. The percentages of transformation were 10.1%
(C elegans AS 3.156), 12.6% (C
elegans AS 3.2028), 82.7%
(C blakesleeana AS 3.153), 15.3% (C blakesleeana
AS
3.910), and 3.69% (C echinulata AS 3.2004). Since
C blakes-leeana AS 3.153 transformed the highest proportion of
metoprolol, it was selected for further investigation. The
type of medium, pH, and concentration of the substrate were
also investigated using an L9
(34) orthogonal table (Table 1). According to Table 1, the first choice for biotransformation
should be at pH 4.5 in a wheat-bran medium with a substrate
concentration of 1.0 g/L
(A2B2C1). However, the factor of pH
was found to have no influence on the final yield. Finally
biotransformation was carried out at pH 6.5 in a wheat-bran
medium with a final substrate concentration of 1.0 g/L.
Identification of metoprolol metabolites
The LC/MSn chromatograms of the culture control showed no
spontaneous formation of possible metabolites of metoprolol under
the same conditions. The substrate control contained only
metoprolol. As shown in Figure 1, with the exception of
metoprolol, 7 possible metabolites were detected in the
C blakesleeana AS 3.153 cultures, compared with the control
cultures.
Following the transformation of metoprolol by
C blakesleeana AS 3.153, 2 major metabolites (M1 and M2)
were isolated by semipreparative HPLC, and their structures
were identified by a combination analysis of
LC/MSn and NMR spectra. The other metabolites were tentatively
identified based on their retention time and
MSn information (Table 2).
Parent drug The identification of metoprolol (M0) was
confirmed by comparison of the retention time and mass
spectra (Figure 2) with the authentic reference. The
compound eluting at 10.5 min was identified as metoprolol. In
order to elucidate the structures of metabolites through the
mass spectra, the cleavage pathways of metoprolol were
studied first. The MS/MS product ion spectra corresponding to
the precursor ion of M0 at m/z 268 and its possible cleavage
pathways are shown in Figure 2. The fragment ion at
m/z 250 was 18 Da lower than that at m/z 268, which
was due to the loss of H2O, and the fragment ion at
m/z 218 was formed by further neutral loss of
CH3OH, while the fragment ions at
m/z 226 and m/z 176 were due to the loss of
CH3CH=CH2 according to each precursor ions. The fragment at
m/z 191 was due to neutral loss of
NH3 and H2O based on the
fragment ion at m/z 226. The fragment at
m/z 116 was attributed to the elimination of the 4-(2-methoxyethyl)-phenoxy
substituent from the structure, whereas the fragment ion at
m/z 98 was associated with further loss of
H2O.
Metabolite M1 The retention time of M1
([M+H]+ at m/z 254) obtained by the
LC/MSn analysis was 7.57 min. The protonated molecule of M1 was 14 Da lower than that of M0,
and its fragment ions at m/z 236, 212, and 177 were also 14 Da
lower than that at m/z 250, 226, and 191 of M0, respectively,
while the ions at m/z 116 and 98 were the same as that of M0.
This suggested that the
1-phenoxy-3-([1-methylethyl]amino)-2-propanol moiety was unchanged. Therefore, M1 was
supposed as O-desmethylmetoprolol. To further verify its
struc-ture, NMR (1H and 13C) analysis was carried out and the
results were as follows: 1H NMR
(D2O): δ 0.96 (6H, t, J1=5.28
Hz, J2=4.99 Hz, H-3''), 2.62 (1H, m, H-2''), 2.69 (2H, t, J=6.68 Hz,
H-6'), 2.75 (1H, m, H-3), 2.77(1H, m, H-3), 3.67(2H, t, J=6.66 Hz,
H-7'), 3.87 (1H, m, H-2), 3.96 (2H, dd, J=5.28
Hz, 18.5 Hz, H-1), 6.85 (2H, d, J=8.30 Hz, H-3'), 7.12(2H, d, J=8.30 Hz, H-4');
13C NMR (D2O): δ 23.6
(C3''), 23.6 (C3''), 39.6
(C6'), 50.9 (C3),
51.0(C2''), 65.4 (C7'), 71.1
(C1), 73.2(C2), 117.6
(C3'), 117.6 (C3'),
132.9 (C4'), 132.9 (C4'), 134.6
(C5'), and 159.4 (C2'). Compared
with the data of metoprolol in previous
studies[12], M1 was finally identified as
O-desmethyl-metoprolol.
Metabolite M2 The retention time of M2
([M+H]+ at m/z 268), obtained by the
LC/MSn analysis, was 5.87 min. The protonated molecule of M2 was the same as that of M0. The
MS/MS spectrum of M2 showed product ions similar to those
in M0, except for the absence of m/z 218 and 176, which
indicated that changes took place in the methoxyethyl group.
To further verify its structure, NMR (1H and
13C) analysis was carried out and the results were as follows:
1H NMR(D2O): δ 1.25 (6H, t,
J1=4.99 Hz, J2=5.58 Hz, H-3''), 3.12 (1H, m,
H-3), 3.21 (1H, m, H-3), 3.38 (3H, m, H-2'', 6'), 3.98 (1H, dd, J=
4.82 Hz, 9.91 Hz, H-1), 4.03 (1H, dd, J=3.73 Hz, 10.1 Hz, H-1),
4.18 (1H, d, J=4.45 Hz, H-2), 6.86 (2H, d, J=8.16 Hz, H-3'),
7.13(2H, d, J=8.14 Hz, H-4'); 13C NMR
(D2O): δ 20.6 (C3''), 21.0
(C3''), 46.2(C7'),
49.4(C3), 53.8(C2''),
68.3(C1), 72.2(C2), 117.5
(C3'), 117.5 (C3'), 133.1
(C4'), 133.1 (C4'), 133.2
(C5'), 159.2 (C2'), and 184.0
(C7'). The downfield shift of C7'
from 65.4 to 184.0 ppm suggested there was a carboxy group in the structure of
M2. Compared with the data of metoprolol in previous
studi-
es[12], M2 was finally identified as metoprolol acid.
Metabolites M3_M7 The precursor ion of M3 at
m/z 284 was 16 Da higher than that of M0, and its fragment at
m/z 207 was also 16 Da higher than that at
m/z 191 of M0, while the same fragment ion at
m/z 116 was in both the MS/MS spectra of M0 and M3, indicating that the
3-([1-methylethyl]amino)-2-propanol moiety was intact, hence, M3 was deduced as
a-hydroxymetoprolol. The protonated molecule at
m/z 226 (M4) and its fragment ion at
m/z 74 were 42 Da lower than that of M0 and its fragment ion at
m/z 116, respectively, while there was the same fragment ion at
m/z 191, indicating that the metabolism took place at the
3-([1-methylethyl]amino)-2-propanol moiety. Furthermore, the protonated molecule at m/z
226 was identical to the fragment ion at
m/z 226 of M0, and MS3 product scan was performed on the fragment ion
at m/z 226 of M0. The MS3 spectrum was the same as that of
MS2 spectrum of M4, which gave further proof of the structure of
M4. Many compounds were found to be N-dealkylated by
CYP2D6[13], including other beta-1 selective
aryloxy-propano-lamine, such as
propranolol[14] and
atenolol[15]. N-dealkylation has become an established metabolic pathway, which could
also occur on metoprolol. Hence, M4 was tentatively
identified as N-desalkylmetoprolol. However, further
investigation is needed.
The protonated molecule of M5 was at m/z
241; according to its fragmentation, M5 was supposed to be deaminated
metoprolol. To obtain further information, the analysis was
operated in negative ESI mode. A peak was detected with
the deprotonated molecule at m/z 239. Following these
results, M5 was finally identified as deaminated metoprolol.
The protonated molecule of M6 at m/z 270 was 16 Da higher
than that of M1. The fragment ions of M6 at
m/z 252, 228, and 193 were also 16 Da higher than the fragment ions of M1
at m/z 236, 212, and 177, respectively, which suggested that
M6 was a hydroxyl metabolite of M1. Similar metabolic
pathways also exist in humans, dogs, horses, and
rats[2,3].
For M7, the protonated molecule
([M+H]+) was at m/z 416, and 162 Da higher than that of M1. The MS/MS spectra
gave only 1 major fragment at m/z 254, which may be due to
the neutral loss of glucose. Therefore, the
MS3 product scan was performed on m/z
254, and 3 major fragment ions at m/z 236, 212, and 177 were observed (data not shown), which
was similar to the MS/MS spectra of M1. The retention time
of M7 (5.05 min) was ahead of other metabolites, which was
possibly due to its increasing polarity. These data
suggested that M7 was the glucoside conjugate of M1. The
eluate of M7 was collected and evaporated to dryness under
a gentle stream of nitrogen. The residue was incubated with
β-D-glucosidase (100 kU/L) at 37 °C for 24 h. The
incubation solution was analyzed by
LC/MSn. The peak of M7 disappeared; instead, a peak with the same MS spectra and
retention time as that of M1 was detected, which provided
further evidence that M7 was the metabolite conjugated with
glucoside.
Microbial transformation of metoprolol and
comparison with mammalian metabolism In the present
study, metoprolol was transformed by C
blakesleeana AS
3.153 to 7 metabolites: O-desmethylmetoprolol (M1),
meto-prolol acid (M2), a-hydroxymetoprolol (M3),
N-desalkyl-metoprolol (M4), deaminated metoprolol (M5),
hydroxyl-O-desmethylmetoprolol (M6), and glucoside conjugate of
O-desmethylmetoprolol (M7). As shown in Figure 3, the
structures of the metabolites and proposed biotransformation
pathways by C blakesleeana are compared to those that
have been identified in mammals[2,3]. After 120-h incubation
by C blakesleeana, about 80% metoprolol was metabolized
mainly to 3 metabolites, which was consistent with that in
mammals. The yields of these metabolites were M1 (21.2%),
M2 (59.4%), and M3 (4.5%), respectively.
Discussion
We reported a successful biotransformation of metoprolol
by C blakesleeana AS 3.153 in this study.
After centrifu-gation, the layer of mycelium was analyzed and trace amounts
of metoprolol and its metabolites (M1 and M2) were detected.
About 96% of the substrate and metabolites were obtained
in the supernatant after centrifugation according to the
initial amount of metoprolol. Thus, the analysis was
performed only in the supernatant. After 120-h incubation in
wheat-bran broth, about 80% of the drug was metabolized to
7 metabolites. As shown in Figure 3, 5 of the metabolites
were essentially similar to those obtained in mammalian
metabolism studies, whereas 2 novel metabolites,
N-desalkyl-metoprolol and the glucoside conjugate of
O-desmethyl-metoprolol, were identified. It has been well recognized that
conjugation is an important metabolic pathway of many
compounds both in mammals and in
microorganisms[16_18]. A glucoside conjugate was detected in the present study,
which conformed to previous studies that the glucosidation
of drugs can be formed by microbial
models[10,18,19]. In humans, metoprolol was metabolized to
O-desmethylmeto-prolol and metoprolol acid or
a-hydroxymetoprolol,
depending on the cytochrome P450 oxidation phenotype.
The 3 primary metabolites of metoprolol in rats, dogs, and
horses were O-desmethyl-metoprolol, metoprolol acid, and
a-hydroxymetoprolol[2,3]. The fungus
C blakesleeana converted metoprolol to 3 major metabolites, which showed
similarities with the metabolism of metoprolol in mammals.
In conclusion, 7 metabolites of metoprolol were formed
by C blakesleeana AS 3.153. The ability of
C blakesleeana to mimic the mammalian metabolism and to perform novel
biotransformations clearly demonstrated that microbial
systems could predict potential routes of the mammalian
biotransformation of drug candidates, and could also be used for the
understanding of chemistry and biology significance of drug
metabolism. Because of the capacity of the microbial
metabolism, substrate concentrations used are much higher
than those employed in other cell or tissue models and
consequently allow for easier detection and isolation. Thus, the
models can be scaled up easily for the preparation of
metabolites for structure confirmation by NMR and further
pharmacological and toxicological studies.
Acknowledgement
We thank Ms Yu-ya WANG for her assistance on the
interpretation of the NMR spectrum.
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