Comparative metabolism of cinobufagin in liver microsomes from mouse, rat, dog, minipig, monkey, and human

Article abstract of DOI:10.1124/dmd.110.036830

Cinobufagin (CB), a major bioactive component of the traditional Chinese medicine Chansu, has been reported to have potent antitumor activity. In this study, in vitro metabolism of CB among species was compared with respect to metabolic profiles, enzymes involved, and catalytic efficiency by using liver microsomes from human (HLM), mouse (MLM), rat (RLM), dog (DLM), minipig (PLM), and monkey (CyLM). Significant species differences in CB metabolism were revealed. In particular, species-specific deacetylation and epimerization combined with hydroxylation existed in RLM, whereas hydroxylation was a major pathway in HLM, MLM, DLM, PLM, and CyLM. Two monohydroxylated metabolites of CB in human and animal species were identified as 1α-hydroxylcinobufagin and 5β-hydroxylcinobufagin by using liquid chromatography-mass spectrometry and two-dimensional NMR techniques. CYP3A4 was identified as the main isoform involved in CB hydroxylation in HLM on the basis of the chemical inhibition studies and screen assays with recombinant human cytochrome P450s. Furthermore, ketoconazole, a specific inhibitor of CYP3A, strongly inhibited CB hydroxylation in MLM, DLM, PLM, and CyLM, indicating that CYP3A was responsible for CB hydroxylation in these animal species. The apparent substrate affinity and catalytic efficiency for 1α- and 5β-hydroxylation of CB in liver microsomes from various species were also determined. PLM appears to have K _m and total intrinsic clearance value (V_max/K_m) similar to those for HLM, and the total microsomal intrinsic clearance values for CB obeyed the following order: mouse > dog > monkey > human > minipig. These findings provide vital information to better understand the metabolic behaviors of CB among various species. Copyright

Full text of DOI:10.1124/dmd.110.036830

0090-9556/11/3904-675–682$20.00
DRUG METABOLISM AND DISPOSITION
Copyright © 2011 by The American Society for Pharmacology and Experimental Therapeutics
DMD 39:675–682, 2011
Vol. 39, No. 4
36830/3674076
Printed in U.S.A.
Comparative Metabolism of Cinobufagin in Liver Microsomes from
□
S
Mouse, Rat, Dog, Minipig, Monkey, and Human
Xiao-Chi Ma, Jing Ning, Guang-Bo Ge, Si-Cheng Liang, Xiu-Li Wang, Bao-Jing Zhang,
Shan-Shan Huang, Jing-Kui Li, and Ling Yang
Laboratory of Pharmaceutical Resource Discovery, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian,
China (X.-C.M., J.N., G.-B.G., S.-C.L., X.-L.W., L.Y.); and School of Pharmaceutical Sciences, Dalian Medical University, Dalian,
China (X.-C.M., J.N., B.-J.Z., S.-S.H., J.-K.L.)
Received October 19, 2010; accepted January 4, 2011
ABSTRACT:
Cinobufagin (CB), a major bioactive component of the traditional CYP3A4 was identified as the main isoform involved in CB hydroxy-
Chinese medicine Chansu, has been reported to have potent anti- lation in HLM on the basis of the chemical inhibition studies and
tumor activity. In this study, in vitro metabolism of CB among screen assays with recombinant human cytochrome P450s. Fur-
species was compared with respect to metabolic profiles, en- thermore, ketoconazole, a specific inhibitor of CYP3A, strongly
zymes involved, and catalytic efficiency by using liver microsomes inhibited CB hydroxylation in MLM, DLM, PLM, and CyLM, indicat-
from human (HLM), mouse (MLM), rat (RLM), dog (DLM), minipig
ing that CYP3A was responsible for CB hydroxylation in these
(PLM), and monkey (CyLM). Significant species differences in CB animal species. The apparent substrate affinity and catalytic effi-
metabolism were revealed. In particular, species-specific deacety- ciency for 1␣- and 5␤-hydroxylation of CB in liver microsomes from
lation and epimerization combined with hydroxylation existed in various species were also determined. PLM appears to have Km
RLM, whereas hydroxylation was a major pathway in HLM, MLM, and total intrinsic clearance value (Vmax/Km) similar to those for
DLM, PLM, and CyLM. Two monohydroxylated metabolites of CB in HLM, and the total microsomal intrinsic clearance values for CB
human and animal species were identified as 1␣-hydroxylcinob- obeyed the following order: mouse > dog > monkey > human >
ufagin and 5␤-hydroxylcinobufagin by using liquid chromatogra- minipig. These findings provide vital information to better under-
phy-mass spectrometry and two-dimensional NMR techniques. stand the metabolic behaviors of CB among various species.
Introduction
one of the most intensively investigated bufadienolides because of its
natural abundance (approximately 4–6% dry weight) and potent
biological activities, such as its cardiotonic, blood pressure-stimulat-
ing, local anesthetic, antimicrobial, and anticancer activities (Hong et
al., 1992; Kamano et al., 1998; Ma et al., 2009). In recent years, more
attention has been paid to the antitumor activity of CB. It has been
well documented that CB could not only induce apoptosis against
prostate cancer cell lines (Yeh et al., 2003) but also inhibit the
migration and proliferation of human hepatic cells in vitro (Kamano et
al., 2002; Su et al., 2003).
Chansu, also called toad venom or toad poison, is prepared from the
dried white secretion of the auricular glands and the skin glands of
Chinese toads (Bufo melanostictus Schneider and Bufo bufo gargar-
zinas Gantor). Chansu has long been recognized in China and other
Asian countries as an important constituent of some famous tradi-
tional Chinese medicine (TCM) formulas, such as Liu Shen Wan
(Hong et al., 1992), Shexiang Baoxin Wan (Song et al., 2000), and Niu
Huang Xiao Yan Wan (Zhou et al., 1992). Cinobufagin (CB) (Fig. 1), the
principal constituent of Chansu (Ye et al., 2006; Zhao et al., 2006), is
During the past two decades, most studies relevant to CB were
focused on its biological activity and toxicity, but little attention was
This work was supported in part by the National Basic Research Program of
China [Grant 2009CB522808]; National Key Technology R&D Program in the 11th paid to its metabolism, which may influence its efficacy and toxicity
Five Year Plan of China [Grant 2008ZX10002-019]; National Natural Science
in vivo. CB and its analogs in Chansu have several documented
toxicities: these digoxin-like components are extremely cardiotoxic
(Gowda et al., 2003) and act rapidly to alter intracellular calcium
Foundation of China [Grants 30701088, 30630075, 30973590, 81001473]; and the
Foundation of Education Department of Liaoning Province [Grant LS2010059].
Article, publication date, and citation information can be found at
stores from cardiomyocytes even in small doses (Bick et al., 2002). In
http://dmd.aspetjournals.org.
addition, as potent Naϩ-Kϩ-ATPase inhibitors, CB and its analogs
doi:10.1124/dmd.110.036830.
have been shown to be associated with a high mortality rate (Ko et al.,
1996; Brubacher et al., 1999), and some of them are even more potent
□S The online version of this article (available at http://dmd.aspetjournals.org)
contains supplemental material.
ABBREVIATIONS: TCM, traditional chinese medicine; CB, cinobufagin; HLM, human liver microsomes; MS, mass spectrometry; ABT, 1-amino-
benzotriazole; DAD, diode array detector; LC, liquid chromatography; P450, cytochrome P450; UFLC, ultrafast liquid chromatography; ESI,
electrospray ionization; MLM, Swiss-Hauschka mouse liver microsome(s); DLM, beagle dog liver microsome(s); PLM, minipig liver microsome(s);
CyLM, cynomolgus monkey liver microsome(s); DCB, 16-deacetylcinobufagin; RLM, Sprague-Dawley rat liver microsomes; 1-HCB, 1␣-hydroxy-
lcinobufagin; 5-HCB, 5␤-hydroxylcinobufagin.
675

676
MA ET AL.
old) were obtained from the Department of Animal Science, Third Military
Medical University (Chongqing, China). These animals were euthanized by
intravenous injection of pentobarbital sodium (150 mg/kg b.wt.); tissue sam-
ples were harvested within 5 min after death. All liver specimens were stored
in liquid nitrogen until microsome preparation. All procedures involving
animals complied with the Laboratory Animal Management Principles of
China. Microsomes were prepared from liver tissue by differential ultracen-
trifugation as described previously (Liu et al., 2009), and the method of Lowry
et al. (1951) was adopted to determine the concentration of microsomal protein
by using bovine serum albumin as a standard.
Pooled human liver microsomes (prepared from five male and five female
human liver microsomal samples) were obtained from BD Gentest (Woburn,
MA). cDNA-expressed recombinant human CYP1A2, CYP2A6, CYP2B6,
CYP2C8, CYP2C9, CYP2D6, CYP2E1, and CYP3A4 derived from baculo-
virus-infected insect cells coexpressing NADPH-P450 reductase were also
obtained from BD Gentest. cDNA-expressed CYP2C19 in Escherichia coli
coexpressing NADPH-P450 reductase was purchased from New England
Biolabs (Beijing) Ltd. (Beijing, China). All microsomal samples and recom-
binant human P450 isoforms were stored at Ϫ80°C until use.
Incubation Conditions. The incubation mixture, with a total volume of 200
␮l, consisted of 100 mM potassium phosphate buffer (pH 7.4), an NADPH-
generating system (1 mM NADPϩ, 10 mM glucose 6-phosphate, 1 unit/ml
glucose-6-phosphate dehydrogenase, and 4 mM MgCl2) and liver microsomes.
In all experiments, CB (25 mM dissolved in acetonitrile previously) was
serially diluted to the required concentrations, and the final concentration of
acetonitrile did not exceed 1% (v/v) in the mixture. After preincubation at 37°C
for 3 min, the reaction was initiated by addition of the NADPH-generating
system and further incubated at 37°C in a shaking water bath. The reaction was
terminated by the addition of ice-cold acetonitrile (100 ␮l). The mixture was
kept on ice until it was centrifuged at 20,000g for 10 min at 4°C. Aliquots of
supernatants were stored at Ϫ20°C until analysis. Control incubations without
the NADPH-generating system or without substrate or without microsomes
were performed to ensure that metabolite formation was microsome- and
NADPH-dependent. All incubations throughout the study were performed in
three experiments performed in duplicate with S.D. values generally less
than 10%.
UFLC-DAD and UFLC-ESI-MS Analysis. The UFLC system was
equipped with a CBM-20A communications bus module, an SIL-20ACHT
autosampler, two LC-20AD pumps, a DGU-20A3 vacuum degasser, a CTO-
20AC column oven, and an SPD-M 20A diode array detector. A Shim-pack
XR-ODS (750 ϫ 2.0 mm, 2.2 ␮m; Shimadzu, Kyoto, Japan) analytical column
with an ODS guard column (5 mm ϫ 2.0 mm, 2.2 ␮m; Shimadzu) was used
to separate CB and its metabolites. The mobile phase consisted of CH3CN (A)
and water containing 0.5% (v/v) formic acid (B), with the following gradient
profile: 0 to 2 min, 96 to 76% B; 2 to 5 min, 76 to 64% B; 5 to 10 min, 64 to
49% B; 10 to 13 min, 49 to 5% B; and 13 to 16 min, balanced to 96% B. The
flow rate was 0.4 ml/min, and the column temperature was kept at 40°C. CB
and its metabolites were detected at 294 nm and quantified according to the
calibration curves of authentic standards.
A Shimadzu LC-MS-2010EV instrument with an ESI interface was used for
identification of CB and its metabolites. Mass detection was performed in both
positive ion mode (ESIϩ) and negative ion mode (ESIϪ) from m/z 100 to 800.
The detector voltage was set at ϩ1.75 and Ϫ1.55 kV for positive and negative
ion detection, respectively. The curved desolvation line temperature and the
block heater temperature were both set at 250°C. Other MS detection condi-
tions were as follows: interface voltage, 4 kV; curved desolvation line voltage,
40 V; nebulizing gas (N2) flow, 1.5 l/min; and drying gas (N2) pressure, 0.06
MPa. Data processing was performed using LC-MS Solution software (version
3.41).
FIG. 1. Structure of cinobufagin.
than the well know poisonous cardiac glycoside ouabain (Bick et al.,
2002). Therefore, a study on the metabolic/clearance pathway(s) of
CB in humans is necessary for clinical risk assessment of this toxic
compound and CB-containing TCMs.
To date, the metabolism of CB has been studied mostly in rats.
Previous studies revealed that most of the in vivo metabolites isolated
from rat bile are mono- or dihydroxylated derivatives of 3-epi-deace-
tylcinobufagin (Ma et al., 2007; Ning et al., 2010), suggesting that
deacetylation and epimerization are major metabolic pathways in rats
(Toma et al., 1987; Zhang et al., 1992). It should be noted that the
biotransformation of CB, including epimerization of 3␤-hydroxyl,
deacetylation of the C-16 ester, and introduction of hydroxyl, could
reduce its bioactivity significantly. On the other hand, significant
differences in metabolic pathways among different species may exist,
and the various metabolites and their concentrations can potentially
affect in vivo bioactivities and toxicities. All these reasons encouraged
us to investigate the metabolic behaviors of CB among different
species.
The specific aims of the current study were to reveal the metabolic
pathway(s) of CB in humans and different experimental animals
including mouse, rat, dog, minipig, and monkey and to compare the
similarities and differences in the enzymes involved and their bio-
transformation activities on CB in liver microsomes from various
species. Moreover, the anticancer activities of two hydroxylated me-
tabolites of CB in human liver microsomes (HLM) were measured
after structure elucidation by two-dimensional NMR.
Materials and Methods
Chemicals. CB was isolated from Chansu by one of the authors (J.N.) and
unambiguously identified by NMR and MS techniques. The purity was greater
than 98% as determined by high-performance liquid chromatography-DAD.
1-Aminobenzotriazole (ABT), sulfaphenazole, quinidine, clomethiazole, fura-
fylline, 8-methoxypsoralen, omeprazole, glucose-6-phosphate dehydrogenase,
NADPϩ, and D-glucose 6-phosphate were purchased from Sigma-Aldrich (St.
Louis, MO). Ketoconazole was obtained from MP Biomedicals (Salon, OH).
Montelukast was from Beijing Aleznova Pharmaceutical (Beijing, China).
Triethylenethiophosphoramide was purchased from Acros Organics (Geel,
Belgium). All other reagents were either of LC grade or of the highest grade
commercially available.
Preparation and Purification of Hydroxylated Metabolites by Biotrans-
formation. Mucor polymorphosporus AS 3.3443 was purchased from the
Enzyme Sources. Swiss-Hauschka (ICR) mice (n ϭ 20, male, 18–20 g) and
Sprague-Dawley rats (n ϭ 10, male, 180–220 g) were purchased from Dalian
Medical University (Dalian, China). The animals had free access to tap water China General Microbiological Culture Collection Center (Beijing, China). All
and a pellet diet. All of the mice and rats were euthanized by decapitation, and
livers were rapidly excised and pooled for microsome preparation. Cynomol-
gus monkeys (n ϭ 3, male, 2.7–3.0 kg, 4 years old) were provided by the
Animal Center of the Chinese Academy of Military Medical Sciences (Beijing,
China). Colony-bred Chinese Bama minipigs (n ϭ 3, male, 10–12 kg, 6
months old) and beagle dogs (n ϭ 3, male, approximately 10 kg, 12 months
culture and biotransformation experiments using filamentous fungi were per-
formed in potato medium, and each liter of potato medium contained 200 g of
potato and 20 g of glucose. Preparative scale biotransformation of CB by M.
polymorphosporus AS 3.3443 was carried out in a 1000-ml Erlenmeyer flask
containing 400 ml of the medium. The flasks were placed on the rotary
shakers, operating at 180 rpm at 28 to 30°C. The substrate (10 mg) was added

COMPARATIVE METABOLISM OF CINOBUFAGIN IN VITRO
677
to 350 ml of precultured medium for 36 h. In total, 300 mg of substrate were
used. The incubation was continued for 4 additional days under the fermen-
CYP3A4, the incubation conditions were optimized to ensure that formation
rates of 1-HCB and 5-HCB were in the linear range in relation to incubation
tation conditions. Culture controls consisted of fermentation blanks in which time and protein concentration. CB (5, 10, 25, 50, 75, 100, 150, 200, and 250
microorganisms grew without substrate but with the same amount of acetone.
Substrate controls contained the sterile medium with the same amount of
substrate and were incubated under the above conditions.
␮M) was incubated with the pooled HLM (0.125 mg protein/ml), MLM (0.1
mg protein/ml), DLM (0.12 mg protein/ml), PLM (0.125 mg protein/ml), and
CyLM (0.1 mg protein/ml) for 10 min or incubated with recombinant CYP3A4
(4 nM) for 20 min. All incubations were performed in three independent
experiments in duplicate. The apparent Km and Vmax values were calculated
from nonlinear regression analysis of experimental data according to the
Michaelis-Menten equation, and the results were graphically represented by
Eadie-Hofstee plots. Kinetic constants were reported as the value Ϯ S.E. of the
parameter estimate.
Cytotoxicity Study of Cinobufagin Metabolites. Human hepatoma cell
line Bel-7402 was maintained in GIBCO RPMI 1640 medium (Invitrogen,
Carlsbad, CA) supplemented with 10% (v/v) fetal bovine serum (Invitrogen)
and 1% penicillin/streptomycin. For cytotoxicity detection, the cells were
harvested by 0.25% trypsin and culture-seeded in 96-well cell culture plates.
Appropriate dilutions of the compounds tested were added into the cultures.
After incubation at 37°C for 72 h, the survival rates of the cancer cells were
evaluated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium method
(Mizutani et al., 1995). The activity is shown as the IC50 value, a parameter that
stands for the concentration (micromolar) of the compound tested to give 50%
inhibition of cell growth. The data are expressed as the mean Ϯ S.D (n ϭ 3).
The culture was filtered, and then the filtration was extracted five times with
the same volume of ethyl acetate. The organic phase was collected and
concentrated to dryness in vacuo. The residues were applied to a silica gel
column (45 g, 200–300 mesh, Ø 2.5 ϫ 35 cm) and eluted with petroleum ether
(60–90°C)-ethyl acetate (in a gradient manner from 100:1 to 1:1, at a flow rate
of 1.5 ml/min) to afford fractions I to VII). Fraction V was separated by
preparative high-performance liquid chromatography with an YMC ODS-A
column (10 ϫ 250 mm, 5 ␮m) and eluted with methanol-H2O (55:45, v/v) to
give compounds M-1 (6 mg) and M-2 (5 mg).
NMR Spectroscopy. All NMR experiments were performed on a Varian
INOVA-500 NMR spectrometer. 1H and 13C NMR spectra (at 500 and 125
MHz, respectively) were measured at room temperature (22°C). Chemical
shifts are given on the ␦ scale and were referenced to tetramethylsilane at ␦ ϭ
0 ppm for 1H and 13C.
Chemical Inhibition Study. The hydroxylation of CB in HLM in the
absence or presences of selective inhibitors for different P450 isoforms were
measured to explore the enzyme(s) involved in this biotransformation. In brief,
CB (20 ␮M, relevant to the Km values) was incubated in HLM (0.125 mg of
protein/ml) with an NADPH-generating system in the absence (control) or
presence of known P450 isoform-specific inhibitors/substrates. The selective
inhibitors and their concentrations were as follows (Bjornsson et al., 2003):
montelukast (2 ␮M) for CYP2C8 (Walsky et al., 2005), sulfaphenazole (10
␮M) for CYP2C9, omeprazole (20 ␮M) for CYP2C19, quinidine (10 ␮M) for
CYP2D6, clomethiazole (50 ␮M) for CYP2E1, and ketoconazole (1 ␮M) for
CYP3A4. Inhibition by furafylline (10 ␮M) for CYP1A2, 8-methoxypsoralen
(2.5 ␮M) for CYP2A6, triethylenethiophosphoramide (50 ␮M) for CYP2B6
(Rae et al., 2002), and ABT (500 ␮M) for broad P450s (Emoto et al., 2003)
was examined by adding CB after preincubation with an NADPH-generating
system at 37°C for 20 min. In addition, furafylline (10 ␮M), sulfaphenazole
(10 ␮M), and ketoconazole (1 ␮M) were used to explore the inhibitory effect
toward CB (20 ␮M) hydroxylation in MLM, DLM, PLM, and CyLM.
Assay with Recombinant P450s. Nine cDNA-expressed human P450
isoforms coexpressing NADPH-P450 reductase (CYP1A2, CYP2A6,
CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4)
were used to screen the involved isoform(s) for hydroxylation of CB in HLM.
The incubations were performed under the above conditions with HLM. To
generate adequate metabolites for detection, a relatively high substrate con-
centration (50 ␮M) was selected and incubated with each of the recombinant
P450s (40–80 nM) at 37°C for 30 min. UFLC with a DAD was used to
quantify the metabolites of CB.
Results
Biotransformation of Cinobufagin by Liver Microsomes from
Different Species. Two product peaks were observed when CB (100
␮M) was incubated with the hepatic microsomes from mouse, dog,
minipig, monkey, or human (0.3 mg protein/ml) along with the
NADPH-generating system for 30 min (Fig. 2). These two peaks (M-1
and M-2) were identified as the monohydroxylated metabolites of CB
by UFLC-ESI-MS, and the formation of monohydroxylcinobufagin
was time-, NADPH-, and microsome-dependent (data not shown). An
extra dihydroxylcinobufagin (M-3) can be detected in MLM under the
same incubation conditions, and this minor metabolite can also be
found in other species by increasing the microsome concentration or
extending the incubation time. In contrast, 16-deacetylcinobufagin
(DCB) was the major metabolite of CB in RLM without an NADPH-
generating system (Fig. 3A), whereas at least eight metabolites could
be detected in RLM with an NADPH-generating system (Fig. 3B),
indicating that complex metabolic pathways are involved in CB
metabolism in rat.
The metabolites of CB in RLM with an NADPH-generating system
were characterized by UFLC-DAD-ESI-MS. With positive ion ESI,
Kinetic Study. To estimate kinetic parameters of hydroxylation of CB in
liver microsomes from human and other species, as well as recombinant CB and its metabolites can produce intact molecular ions [M ϩ H]ϩ
FIG. 2. Representative UFLC profiles of CB and
its metabolites in liver microsomes from human,
minipig, dog, monkey, and mouse. CB (100 ␮M)
was incubated with liver microsomes (0.3 mg/ml)
from different species and an NADPH-generating
system at 37°C for 30 min. mAU, milli-arbitrary
units.

678
MA ET AL.
FIG. 3. Representative UFLC profiles of CB and
its metabolites (peaks 1–8) in rat. CB (100 ␮M)
was incubated with RLM (0.3 mg/ml) at 37°C for
30 min without an NADPH-generating system
(A) and with an NADPH-generating system (B).
mAU, milli-arbitrary units.
and significant acetonitrile adducts [M ϩ CH3CN ϩ H]ϩ that can be NMR spectrum of M-1 displayed a carbon signal at ␦ 29.3 (C-1)
used for molecular mass determination (Table 1). Most metabolites shifted downfield to ␦ 71.8 (CH). The other carbon signals of M-1
can be assigned by comparison of retention times, UV spectra, and were similar to that of cinobufagin. In the heteronuclear multiple-bond
mass spectra with authentic standards (Ye et al., 2006; Ma et al., correlation spectroscopy spectrum, the carbon signal at ␦ 71.8 corre-
2007), whereas peaks 1 (5.28 min) and 3 (5.61 min) were tentatively lated with the proton signals of ␦ 0.99 (H-19) and ␦ 1.93 (H-2). In the
assigned as monohydroxylated deacetylcinobufagin and peak 2 (5.40 1H-1H correlation spectroscopy spectrum, the proton signal at ␦1.93
min) was tentatively assigned as 3-ketohydroxylated deacetylcinob- (H-2) correlated with the proton signals at ␦ 3.62 (H-1) and ␦ 4.00
ufagin by UFLC-ESI-MS. It is evident from Fig. 3 and Table 1 that (H-3); these data suggested that the hydroxyl group should be at the
DCB was the most abundant metabolite of CB in RLM, whereas C-1 site. In the nuclear Overhauser effect spectroscopy spectrum, the
hydroxylated and epimerized derivatives of cinobufagin and DCB nuclear Overhauser enhancements between the proton signal of ␦ 3.62
were also detected. Of note, two monohydroxylated derivatives (peaks (H-1) and the signals at ␦ 0.99 (19-Me) and ␦ 1.61 (5-H) suggested the
5 and 6) in RLM corresponded well to the monohydroxylated metab- ␣-configuration of 1-OH. Thus, M-1 can be assigned as 1␣-
olites of CB in HLM (M-1 and M-2, respectively).
Preparation and Identification of Monohydroxylated Metabo-
hydroxylcinobufagin (1-HCB), which is a novel compound.
The H NMR spectrum of M-2 revealed that the proton signal of
1
lites. To elucidate their absolute structures and the metabolic labile H-5 at ␦ 1.69 disappeared by comparison with NMR data for cinob-
sites of CB in HLM, two monohydroxylated metabolites of CB were ufagin. The 13C NMR spectrum of M-2 showed that the carbon signal
biosynthesized by microorganisms and characterized by 2D NMR. of C-5 shifted downfield to ␦ 73.5. Furthermore, the NMR data of M-2
Eighteen strains of filamentous fungi (from seven genera) were ini- agreed well with the spectral data of cinobufotalin reported in previ-
tially screened by LC-MS for their biotransformation capability (from ous studies (Ye et al., 2006; Ma et al., 2008). Taken together, these
CB to monohydroxycinobufagin). Among the cultures screened, M. data indicated that M-2 was 5␤-hydroxylcinobufagin (5-HCB).
polymorphosporus AS 3.3443 was found to be able to convert cinob-
Chemical Inhibition Studies. Selective inhibitors of nine major
ufagin into several monohydroxylated metabolites. Of more impor- P450 isoforms were used to screen the P450 isoform(s) responsible for
tance, two major biotransformed products by this strain were charac- the formation of two monohydroxylated metabolites in HLM (Fig. 4).
terized by UFLC-DAD-ESI-MS and they were found to have ABT, a broad-specificity P450 inactivator, inhibited the formation of
retention times, UV spectra, and mass spectra identical to those of 1-HCB and 5-HCB completely, suggesting that P450s were respon-
M-1 and M-2 of CB in HLM (Supplemental Data).
sible for the CB hydroxylation in HLM. Among the nine P450 isoform
These two products biotransformed by microorganism were also selective inhibitors tested, ketoconazole inhibited the formation of
1
1
isolated and characterized by H NMR and 13C-NMR. The H NMR 1-HCB and 5-HCB completely, whereas inhibitors of other P450
and 13C NMR spectral data of two monohydroxylated metabolites are isoforms did not exhibit significant inhibition (less than 30% inhibi-
listed in Table 2. Compared with NMR data of cinobufagin, the 13C tion, p Ͼ 0.05) toward the formation of these two metabolites. These

COMPARATIVE METABOLISM OF CINOBUFAGIN IN VITRO
679
TABLE 1
Retention times, molecular weights, and MS data for metabolites of CB in RLM
a
Peak No.
tR
Mol. Wt.
Identification
Molecular Ions (ESIϩ, m/z)
min
ϩ
1
2
3
4
5
6
7
8
5.28
416
414
416
400
458
458
400
442
Monohydroxyl-deacetylcinobufagin
3-Ketohydroxyl-deacetylcinobufagin
Monohydroxyl-deacetylcinobufagin
16-Deacetylcinobufagin
417 ͓M ϩ H͔
ϩ
458 ͓M ϩ CH3CN ϩ H͔
ϩ
5.40
5.61
6.95
7.35
7.60
8.32
10.59
415 ͓M ϩ H͔
ϩ
ϩ
ϩ
ϩ
ϩ
ϩ
ϩ
456 ͓M ϩ CH3CN ϩ H͔
ϩ
417 ͓M ϩ H͔
458 ͓M ϩ CH3CN ϩ H͔
ϩ
401 ͓M ϩ H͔
442 ͓M ϩ CH3CN ϩ H͔
ϩ
1␣-Hydroxylcinobufagin
459 ͓M ϩ H͔
500 ͓M ϩ CH3CN ϩ H͔
ϩ
5␤-Hydroxylcinobufagin
459 ͓M ϩ H͔
500 ͓M ϩ CH3CN ϩ H͔
ϩ
3-epi-Deacetylcinobufagin
3-epi-Cinobufagin
401 ͓M ϩ H͔
442 ͓M ϩ CH3CN ϩ H͔
ϩ
443 ͓M ϩ H͔
484 ͓M ϩ CH3CN ϩ H͔
tR, retention time.
findings suggested that the formation of 1-HCB and 5-HCB was species mentioned above, whereas ketoconazole (1 ␮M) could inhibit
mainly catalyzed by CYP3A. the formation of 1-HCB and 5-HCB nearly completely, implying that
To explore whether the metabolic enzymes responsible for CB CYP3A played an important role in the hydroxylation of CB among
hydroxylation in mouse, dog, minipig, and monkey were also CYP3A these species.
isoenzymes, furafylline and sulfaphenazole, the selective inhibitors of
Assays with Recombinant Human P450 Isoforms. To further
CYP1A and CYP2C, respectively, as well as ketoconazole, a potent verify the P450 isoform(s) involved in the metabolism of CB in
selective inhibitor for CYP3A, were used. The results are illustrated in humans, the oxidation activity of CB was determined using nine
Fig. 5. It was evident that furafylline (10 ␮M) and sulfaphenazole (10 cDNA-expressed P450 isoforms. After incubation at 37°C for 30 min,
␮M) slightly inhibited the formation of 1-HCB and 5-HCB in all two hydroxylated metabolites were formed exclusively by CYP3A4;
TABLE 2
1H NMR (500 MHz, DMSO) and 13C NMR (125 MHz, DMSO) spectral data for M-1 and M-2
1␣-Hydroxylcinobufagin (M-1)
5␤-Hydroxylcinobufagin (M-2)
No.
1H NMR
13C NMR
71.8 d
HMBC (H 3 C)
NOESY
1H NMR
13C NMR
1
3.62 brs
C-2, C-10
H-5, Me-19
1.25
1.70
1.48
24.8 t
27.2 t
2Ha Hb
1.93
1.68
4.00 (brs)
1.78
32.0 t
C-1, C-3, C-4
H-3
H-1
Ha-2
H-5
1.52
3
66.7 d
32.8 t
C-2, C-4, C-5
C-2, C-3, C-5, C-10
3.02 (brs)
2.10 brd
66.5 d
36.5 t
4Ha Hb
1.82
5
1.63
1.32
1.73
1.20
1.33
1.87
1.73
30.1 d
25.0 t
C-4, C-6, C-10
C-5, C-8, C-10
Me-19
H-5
—
73.5 s
33.8 t
6Ha Hb
1.15
1.52
0.92
1.39
1.98
1.80
7Ha Hb
19.8 t
C-8, C-9
H-8
22.2 t
8
9
10
32.8 d
39.9 d
40.5 s
20.6 t
C-9,C-11, C-14
C-10, C-11, C-12, C-19
Me-19
Ha-11
31.5 d
41.1 d
40.4 s
21.0 t
11Ha Hb
1.17
1.40
1.48
1.65
C-8, C-9, C-12, C-13
C-9, C-11, C-13, C-18
H-9
H-19
H-18
1.24
1.50
1.41
1.65
12Ha Hb
38.7 t
38.8t
13
14
15
16
17
18
19
20
21
22
23
24
25
26
44.6 s
71.9 s
44.3 s
71.9 s
3.75 (s)
59.3 d
74.6 d
48.9 d
16.9 q
18.7 q
116.1 s
152.2 d
148.6 d
112.9 d
160.9 s
169.4 s
20.2 q
C-14, C-16, C-17
C-13, C15, C-25
C-13, C-14, C-16, C-20
C-12,C-13,C-14
C-1,C-5,C-10
H-16
H-15, H-17
H-16
H-8, H-21
H-1, H-5, H-8
3.75 (s)
59.3 d
74.5 d
48.8 d
16.5 q
16.8 q
116.1 s
152.2 d
148.4 d
112.9 d
160.7 s
169.3 s
20.2 q
5.45 (d, 9.5)
2.85 (9.5)
0.71 (s)
5.42 (d, 9.5)
3.35 (d, 9.5)
0.62 (s)
0.99 (s)
0.91 (s)
7.48 (s)
7.81 (brs)
6.24 (d, 9.5)
C-20
C-20, C-23
C-22, C-24
Me-18
H-23, H-26
H-22
7.35 (s)
7.80 (brs)
6.23 (d, 9.5)
1.82 (s)
C-25
Me-18
1.81 (s)
DMSO, dimethyl sulfoxide; HMBC, heteronuclear multiple-bond correlation spectroscopy; NOESY, nuclear Overhauser effect spectroscopy.

680
MA ET AL.
FIG. 6. Michaelis-Menten plots of CB metabolism in human liver microsomes. CB
(2.5–250 ␮M) was incubated with HLM (0.125 mg/ml) at 37°C for 10 min with an
NADPH-generating system. An Eadie-Hofstee plot is shown as an inset to illustrate
monophase kinetics. Data points represent the mean of triplicate determinations.
FIG. 4. Effects of selective P450 inhibitors on the formation of two monohydroxy-
lated metabolites (M-1 and M-2) in HLM. Results are the mean Ϯ S.D. from three
experiments performed in duplicate. TEPA, triethylenethiophosphoramide; N.D.,
not detected.
Cytotoxicity Studies of Cinobufagin Metabolites. Taking into
account the fact that 1␣- and 5␤-hydroxylation of CB by CYP3A is a
major metabolic pathways in human liver, we evaluated the bioactiv-
ities of these two monohydroxylated metabolites (1-HCB and 5-HCB)
compared with those of CB (Fig. 7). In vitro cytotoxicity of 1-HCB,
5-HCB, and CB was evaluated by using the Bel-7402 cell line. The
IC50 values were 0.37 Ϯ 0.05 (M-1), 8.62 Ϯ 0.42 (M-2), and 4.9 ϫ
10Ϫ2 Ϯ 0.16 ϫ 10Ϫ2 ␮M (CB), respectively. These results showed
that 1␣- or 5␤-hydroxylation of CB was capable of reducing the
cytotoxic activities (increasing the IC50 values approximately 7.5- and
175-fold for 1-HCB and 5-HCB, respectively) against Bel-7402 cells,
suggesting that these hydroxylation pathways might serve as the
deactivation routs.
no metabolites were observed in the incubation with CYP1A2,
CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and
CYP2E1 (less than 0.01 pmol per min/pmol P450). The formation
rates of CYP3A4 for 1-HCB and 5-HCB were 14.01 Ϯ 0.90 and
26.02 Ϯ 1.70 pmol per min/pmol P450, respectively.
Kinetic Study. Over the whole concentration range tested, hy-
droxylation of CB in liver microsomes from humans and four com-
mon animal species, as well as in recombinant CYP3A4, obeyed
Michaelis-Menten kinetics, as evidenced by the Eadie-Hofstee plot
(Fig. 6). The kinetic parameters including Km, Vmax, and the intrinsic
clearance (Vmax/Km) for CB hydroxylation were determined and are
listed in the Table 3. In human liver microsomes, the Km values for the
formation of 1-HCB and 5-HCB were 12.5 and 20.4 ␮M, respectively,
whereas the Vmax values for the formation of 1-HCB and 5-HCB were
0.59 and 1.17 nmol per min/mg, respectively. The Km values for the
formation of 1-HCB and 5-HCB in recombinant CYP3A4 were 16.0
and 32.6 ␮M, respectively, and Vmax values for the formation of
1-HCB and 5-HCB were 19.22 and 45.81 pmol per min/pmol P450,
respectively. In liver microsomes from four experimental animals, the
Km values for 1-HCB formation ranged from 3.3 to 34.2 ␮M, whereas
the Km values for 5-HCB formation ranged from 3.4 to 20.4 ␮M. The
intrinsic clearance (CLint) for the formation of 1-HCB and 5-HCB
differed greatly among different species, with a range of 14.5 to 595.0
and 54.6 to 644.6 ␮l per min/mg protein, respectively.
Discussion
In the past 30 years, the rat has been widely used in pharmacoki-
netic and toxicological studies of Chansu and cinobufagin (Toma et
al., 1987; Zhang et al., 1992; Gowda et al., 2003; Xu et al., 2007;
Liang et al., 2008; Jiang et al., 2009). However, until now, the
differences in the metabolic pathway and metabolic behavior of CB
between human and common experimental animals have not been
revealed. In this study, a comparison of metabolic profiles, enzymes
involved, and catalytic efficiency for CB metabolism in liver micro-
somes from different species were performed. Our results revealed
that complex pathways including deacetylation, epimerization, and
hydroxylation were involved in metabolism of CB in RLM, whereas
species-specific deacetylation of CB was an important metabolic
pathway that was well characterized previously (Zhang et al., 1992).
In sharp contrast, P450-mediated hydroxylation was thought to be the
major pathway responsible for the elimination of CB in human liver,
because none of the hydrolyzed, epimerized, and glucuronidated
metabolites were formed, and the CB concentration was not decreased
in the HLM incubation (data not shown). Except for rat, comparable
metabolic profiles were observed when CB was incubated with liver
microsomes from human, monkey, minipig, dog, and mouse along
with an NADPH-generating system. These results revealed the large
difference in metabolic profiles between rat and other species and
could explain why the major metabolites of CB in rat, namely deace-
tylcinobufagin and 3-epi-deacetylcinobufagin, cannot be observed in
the serum of the dog with oral or intravenous administration of CB
(Toma et al., 1987).
FIG. 5. Effects of furafylline, sulfaphenazole, and ketoconazole on the formation of
two monohydroxylated metabolites (M-1 and M-2) in CyLM, PLM, DLM, and
MLM. Results are the mean Ϯ S.D. from three experiments performed in duplicate.
N.D., not detected.
For further exploration of the similarity of the enzymes responsible
for hydroxylation of CB in liver microsome from various species,

COMPARATIVE METABOLISM OF CINOBUFAGIN IN VITRO
681
TABLE 3
Kinetic parameters of CB metabolism in HLM, CyLM, DLM, PLM, MLM, and recombinant CYP3A4
Km values are micromolar concentrations, Vmax values are nanomoles per minute per milligram for liver microsomes or picomoles per minute per picomole of P450 for CYP3A4, and
max/Km values are microliters per milligram per minute for liver microsomes or microliters per picomoles per minute for CYP3A4. The range of substrate concentrations was 5 to 250 ␮M.
V
Each value is the mean Ϯ S.D. of three determinations performed in duplicate.
M-1 (1-HCB)
M-2 (5-HCB)
Species
Vmax
Km
V
max/Km
Vmax
Km
V
max/Km
Human
Monkey
Dog
Minipig
Mouse
CYP3A4
0.59 Ϯ 0.01
2.12 Ϯ 0.04
1.28 Ϯ 0.03
0.25 Ϯ 0.02
8.39 Ϯ 0.15
19.22 Ϯ 0.68
12.5 Ϯ 0.8
34.2 Ϯ 2.4
3.3 Ϯ 0.5
17.2 Ϯ 3.8
14.1 Ϯ 1.3
16.0 Ϯ 2.6
47.2
62.0
387.9
14.5
595.0
1.2
1.17 Ϯ 0.02
1.84 Ϯ 0.03
0.44 Ϯ 0.01
0.65 Ϯ 0.05
6.51 Ϯ 0.09
45.81 Ϯ 1.97
20.4 Ϯ 1.3
8.5 Ϯ 0.9
3.4 Ϯ 0.5
11.9 Ϯ 2.5
10.1 Ϯ 0.8
32.6 Ϯ 5.1
57.4
216.5
129.4
54.6
644.6
1.4
chemical inhibition studies with use of selective inhibitors were used. metabolites. Both CyLM and MLM displayed high catalytic activities
It is evident from chemical inhibition studies and assays with recom- for the formation of 1-HCB and 5-HCB, which could be attributed to
binant P450s that both 1␣- and 5␤-hydroxylation of CB in HLM were the higher total P450 levels per milligram of liver microsomal protein
catalyzed predominantly by CYP3A4, whereas 1␣- and 5␤-hydroxy- in monkeys and mice compared with that in humans (Martignoni et
lation of CB in liver microsomes from monkey, minipig, dog, and al., 2006). In comparison, PLM exhibited Km and total intrinsic
mouse were also CYP3A-mediated. These findings suggested the clearance values similar to those of HLM. These results indicated that
potential similarity in the metabolic enzymes for CB hydroxylation in minipig might be a good choice to serve as a surrogate model for drug
liver microsomes from species mentioned above. In contrast, a spe- metabolism and pharmacokinetics studies of CB.
cific B-type esterase(s) localized in the microsomal fraction of rat
The structure-activity relationship studies of bufadienolides re-
liver was responsible for the hydrolysis of CB (Zhang et al., 1992). vealed that biotransformation of CB including epimerization, deacety-
The large difference in metabolic profiles and metabolic enzymes lation, and hydroxylation could reduce its bioactivities, including
involved between rat and human suggested that rat was not a suitable anticancer activity and cardiotonic activity (Kamano, 1970; Kamano
surrogate animal model for pharmacokinetic, toxicological, and phar- et al., 1998). This study also demonstrated that 1␣- and 5␤-hydroxy-
macological studies of CB, and the in vivo data obtained from rat lation of CB by CYP3A would reduce its cytotoxicity activity. In this
should be used cautiously for interspecies extrapolation.
case, the effects on CYP3A may lead to large variations in exposure
Although animal models have been commonly used in preclinical of CB, which can influence the in vivo potency or cause severe
studies to evaluate pharmacokinetics and toxicity in humans, it should adverse reactions. Because CB is a substrate of CYP3A, more adverse
be pointed out that a suitable animal models for in vivo drug metab- drug-drug reactions or undesirable effects may occur when CB is
olism and pharmacokinetics studies should have the relevant metab- coadministrated with drugs that are substrates, inducers, or inhibitors
olism behaviors compared with those of humans, including identical of CYP3A. It has been reported that many compounds including
metabolic profiles, equivalent or similar metabolic enzymes, and xenobiotics and endogenous steroids could cause marked modifica-
closed catalytic efficiency (Martignoni et al., 2006). CYP3A is the tions in CYP3A-mediated metabolism (Nakamura et al., 2002; Us-
most important isoform involved in the metabolism of CB in humans mani et al., 2003; Zhou et al., 2007; Zhou, 2008). Their effects on
and the above-mentioned animal species, but various CYP3A iso- CYP3A, including altering kinetic behaviors or activating or inhibit-
forms are expressed in various species with different substrate spec- ing the biotransformation of other substrates, would make pharmaco-
ificities and catalytic efficiency. In this study, a comparative study of kinetic behaviors more complicated. It also should be noted that the
CB metabolism in humans and four experimental animals with similar metabolism of CB could be affected by other bufadienolides coexist-
profiles was performed to determine the catalytic efficiency of CB ing in Chansu, because of their similar structures and their potential
hydroxylation by liver microsomes from various species. None of the inhibitory effect on CYP3A4 (Shimada et al., 2006; Li et al., 2009).
animal species is completely similar to humans with respect to en- The inhibition of CYP3A by other bufadienolides in Chansu can lead
zyme activities, and catalytic efficiencies for the formation of 1-HCB to accumulation of CB at a relatively high level in vivo, which may
and 5-HCB are diversified. HLM, PLM, and MLM have similar Km cause some serious adverse reactions. Therefore, more attention
values for the formation of 1-HCB (M-1), but none of the experimen- should be paid to the interactions of CB with its coadminstrated drugs
tal animal species have closed Km values for the formation of 5-HCB and other bufadienolides in the clinic.
(M-2). Intrinsic clearance values (Vmax/Km) for 1-HCB and 5-HCB in
In summary, the metabolic pathways of CB in humans and five
dogs were much higher than those in humans, because of the high experimental animals were elucidated in the present study. The spe-
affinity of DLM (low Km values) for the formation of these two cies-specific deacetylation and epimerization together with hydroxy-
FIG. 7. The metabolic pathways of CB in liver
microsomes from human, monkey, dog, mini-
pig, and mouse.

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lation are biotransformation pathways in RLM, whereas hydroxyla-
tion of CB at C-1 and C-5 sites by CYP3A is a major metabolic
pathway in liver microsomes from human, monkey, dog, minipig, and
mouse. The metabolic behaviors of CB among different species have
been elucidated with respect to the similarities in metabolic profiles,
enzymes involved, and catalytic efficacy, which would contribute to
animal selection in toxicological and pharmacokinetic studies of CB
or other TCMs containing CB.
Acknowledgments
We thank Professor Hong Wei (Third Military Medical University) for
providing the livers of monkey, minipig, and dog.
Authorship Contributions
Nakamura H, Nakasa H, Ishii I, Ariyoshi N, Igarashi T, Ohmori S, and Kitada M (2002) Effects
of endogenous steroids on CYP3A4-mediated drug metabolism by human liver microsomes.
Drug Metab Dispos 30:534–540.
Participated in research design: Ma, Ge, Liang, and Yang.
Conducted experiments: Ning and Ge.
Ning J, Wu TH, Tian Y, Wang CY, Tian G, Zhang BJ, Liu KX, and Ma XC (2010) Identification
of cinobufagin metabolites in the bile of rats. Xenobiotica 40:48–54.
Rae JM, Soukhova NV, Flockhart DA, and Desta Z (2002) Triethylenethiophosphoramide is a
specific inhibitor of cytochrome P450 2B6: implications for cyclophosphamide metabolism.
Drug Metab Dispos 30:525–530.
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venom using high-performance liquid chromatography and liquid chromatography-mass spec-
trometry. Biomed Chromatogr 20:1321–1327.
Song H, Guo T, Bi K, Wang H, and Zhang R (2000) Determination of resibufogenin and
cinobufagin in heart-protecting musk pill by HPLC. Biomed Chromatogr 14:130–132.
Su YH, Yin XC, Xie JM, Gao B, and Ling CQ (2003) Inhibition effects of three kinds of
bufotoxins on human SMMC-7721 and BEL-7402 hepatoma cells lines. Dier Junyi Daxue
Xuebao 24:393–395.
Performed data analysis: Ma, Ning, and Ge.
Wrote or contributed to the writing of the manuscript: Ning, Ma, Ge, Liang,
Wang, and Yang.
Other: Zhang, Huang, and Li contributed to the preparation of authentic
standards.
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Address correspondence to: Dr. Ling Yang, Laboratory of Pharmaceutical
Resource Discovery, Dalian Institute of Chemical Physics, Chinese Academy of
Sciences, Dalian, 116023 China. E-mail: yling@dicp.ac.cn