Deoxyschizandrin, a naturally occurring lignan, is a specific probe substrate of human cytochrome P450 3A

Article abstract of DOI:10.1124/dmd.113.053884

To accurately predict the modifications done during metabolic processes by cytochrome P450 (P450) 3A enzyme, selecting substrates that best represent a broad range of substrate substitutions and that follow the Michaelis-Menten kinetic properties is highly necessary. In the present study, the oxidative pathways of deoxyschizandrin (DS), the most abundant lignan in Fructus Schisandrae fruit extract, were characterized with liver microsomes from human (HLM) and rat (RLM). Only one monohydroxylated metabolite 7(S)-hydroxylated metabolite (isoschizandrin, ISZ), was identified using liquid chromatography-mass spectrometry and nuclear magnetic resonance techniques. CYP3A4 and CYP3A5 were found to be the major isoforms involved in the monohydroxylation of DS. Also, the kinetic studies showed that DS hydroxylation obeyed Michaelis-Menten kinetics both in HLM and in RLM. However, the subsequent metabolism of ISZ was nearly nonexistent when DS was present. More importantly, the interactions between DS and three well characterized CYP3A probe substrates, testosterone (TST), midazolam (MDZ), and nifedipine (NIF), were studied. TST and MDZ were shown to compete with DS for the mutual binding site, causing K_m to be increased. The presence of DS also lowered the binding affinities for MDZ and TST. However, DS showed only slight inhibitory effects on nifedipine (NIF) oxidation even though NIF was able to inhibit DS hydroxylation in a noncompetitive fashion. These results show that DS is a good representative substrate of MDZ and TST primarily due to their shared, large binding regions on CYP3A. Therefore, DS is an attractive candidate as a novel CYP3A probe substrate for predicting the metabolic modifications in CYP3A activity. Copyright

Full text of DOI:10.1124/dmd.113.053884

Supplemental Material can be found at:
http://dmd.aspetjournals.org/content/suppl/2013/10/16/dmd.113.053884.DC1.html
1521-009X/42/1/94–104$25.00
http://dx.doi.org/10.1124/dmd.113.053884
DRUG METABOLISM AND DISPOSITION
Drug Metab Dispos 42:94–104, January 2014
Copyright ª 2013 by The American Society for Pharmacology and Experimental Therapeutics
Deoxyschizandrin, a Naturally Occurring Lignan, Is a Specific Probe
Substrate of Human Cytochrome P450 3As
Jingjing Wu, Yunfeng Cao, Yanyan Zhang, Yong Liu, James Y. Hong, Liangliang Zhu,
Guangbo Ge, and Ling Yang
Laboratory of Pharmaceutical Resource Discovery, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian,
China (J.W., Y.C., Y.Z., Y.L., L.Z., G.G., L.Y.); Department of Biopharmaceutical Sciences, University of Illinois, Chicago, Illinois
(J.Y.H.); Graduate University of Chinese Academy of Sciences, Beijing, China (J.W., L.Z.); Shanghai Institute of Planned Parenthood
Research, Shanghai, China (Y.C.)
Received July 29, 2013; accepted October 16, 2013
ABSTRACT
To accurately predict the modifications done during metabolic metabolism of ISZ was nearly nonexistent when DS was present.
processes by cytochrome P450 (P450) 3A enzyme, selecting substrates
that best represent a broad range of substrate substitutions and that
follow the Michaelis-Menten kinetic properties is highly necessary.
More importantly, the interactions between DS and three well
characterized CYP3A probe substrates, testosterone (TST), mid-
azolam (MDZ), and nifedipine (NIF), were studied. TST and MDZ
In the present study, the oxidative pathways of deoxyschizandrin were shown to compete with DS for the mutual binding site,
(DS), the most abundant lignan in Fructus Schisandrae fruit extract, causing Km to be increased. The presence of DS also lowered the
were characterized with liver microsomes from human (HLM) and
binding affinities for MDZ and TST. However, DS showed only slight
rat (RLM). Only one monohydroxylated metabolite 7(S)-hydroxylated inhibitory effects on nifedipine (NIF) oxidation even though NIF was
metabolite (isoschizandrin, ISZ), was identified using liquid able to inhibit DS hydroxylation in a noncompetitive fashion. These
chromatography–mass spectrometry and nuclear magnetic reso- results show that DS is a good representative substrate of MDZ and
nance techniques. CYP3A4 and CYP3A5 were found to be the ma-
jor isoforms involved in the monohydroxylation of DS. Also, the
kinetic studies showed that DS hydroxylation obeyed Michaelis-
Menten kinetics both in HLM and in RLM. However, the subsequent
TST primarily due to their shared, large binding regions on CYP3A.
Therefore, DS is an attractive candidate as a novel CYP3A probe
substrate for predicting the metabolic modifications in CYP3A
activity.
Introduction
To date, many in vitro probe substrates for P450 have been
established to evaluate the potential for a new drug to modify P450
activities based on the in vitro probe reactions (Kenworthy et al.,
1999; Yuan et al., 2002). However, the available information indicates
that a substrate-differential response is often observed for CYP3A4.
For example, haloperidol activates dextromethorphan N-demethyla-
tion but inhibits nifedipine oxidation (Yuan et al., 2002); erlotinib
stimulates midazolam 19-hydroxylation but inhibits formation of
6b-hydroxytestosterone and oxidized nifedipine (Dong et al., 2011).
Stresser et al. (2000) showed that the extent of substrate dependence
on the quantitative inhibition parameters (IC50) is as large as 195-fold
among the tested CYP3A4 reactions.
Several reasons could partly explain the phenomena mentioned
above. First, the presence of several distinct binding domains within
the CYP3A active site results in various substrate subgroups (Galetin
et al., 2005). Expectedly, false negative prediction of DDI will
probably occur between two different subgroups of substrates due to
the lack of mutual binding site. Second, in most cases the P450-
catalyzed reactions can be described using the Michaelis-Menten
equation, based on the premise that substrate-enzyme interactions
occur at only one site per enzyme. Then estimates can be deduced
Cytochrome P450 (P450) 3A, the most abundant human P450
enzyme, is involved in the metabolism of many structurally diverse
therapeutic agents (Wienkers and Heath, 2005). As a consequence, the
individual variability or metabolic modification in CYP3A activity has
a potential to greatly increase the risk of drug toxicity when a drug is
administrated alone or as a part of combination therapy, especially for
those with narrow therapeutic windows (Frye, 2004; Kalgutkar et al.,
2007). Thus, a “probe” compound that can be metabolized exclusively
by an individual P450 enzyme is greatly needed to evaluate metabolic
clearance rate, in order to assess alterations in P450 activities and to
adjust dosages in clinical settings with a high risk of drug-drug
interaction (DDI) (Zhou et al., 2007; Wille et al., 2008).
This work was supported by the National Key Technology Major Project of
China [Grants 2012ZX09506001-006 and 2012ZX10002011-008], and the National
Natural Science Foundation of China [Grants 81202587, 81102507, and 81072698].
dx.doi.org/10.1124/dmd.113.053884.
s This article has supplemental material available at dmd.aspetjournals.org.
ABBREVIATIONS: 19-OH-MDZ, 19-Hydroxymidazolam; ABT, 1-aminobenzotriazole; CLint, intrinsic clearance; DS, deoxyschizandrin; ESI,
electrospray ionization; HLM, human liver microsomes; HPLC, high-performance liquid chromatography; ISZ, isoschizandrin; Km, apparent
affinity; KHB, Krebs-Henseleit buffer; MDZ, midazolam; MS, mass spectrometry; NIF, nifedipine; NMR, nuclear magnetic resonance; P450,
cytochrome P450; RLM, Wistar rat liver microsomes; TST, testosterone; UFLC, ultra-fast liquid chromatography; Vmax, apparent maximum reaction
velocity.
94

Deoxyschizandrin Hydroxylation by CYP3A
95
diet (from the Experimental Animal Center of Dalian Medical University) at
a temperature of 20–25°C with a 12-hour light-dark cycle and relative humidity
of 50 6 10%. All procedures involving animals complied with the Laboratory
Animal Management Principles of China.
using maximum velocity (Vmax) and apparent affinity (Km) parameters
to predict both clearance and DDI in vivo (Lin and Lu, 1997; Houston
and Kenworthy, 2000). Yet, due to the relatively large substrate-
binding cavity of CYP3A, two or more molecules of smaller substrates
are postulated to simultaneously occupy the active site cavity (Yano
et al., 2004). In response, more complex and rational mechanistic
analyses are required (Galetin et al., 2003); otherwise, false positive or
negative prediction of DDI will result from classic Michaelis-Menten
analysis. Finally, the uniqueness of catalytic site within the substrate-
binding cavity of CYP3A that help yield a single metabolite is
indispensable to accurately quantify the total intrinsic metabolic
clearance.
Unfortunately, until now, most of the commonly used in vitro probes
are reported to demonstrate non-Michaelis-Menten kinetic properties,
such as autoactivation for testosterone, a probe used in 50% of reported
studies, and autoinhibition for midazolam (Houston and Kenworthy,
2000). Therefore, a criteria-based screening for better probe substrates
that demonstrate Michaelis-Menten kinetics with a single metabolite and
excellent substrate substitution properties are highly sought after, since
they can serve as the most representative in vitro probes for CYP3A
metabolic activity.
Enzyme Source
Pooled human liver microsomes were obtained from BioreclamationIVT
(Baltimore, MD). cDNA-expressed recombinant human CYP3A4 and CYP3A5
were obtained from Cypex Ltd. (Dundee, UK). cDNA-expressed CYP1A2,
CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2D6, CYP2E1, CYP3A1, and
CYP3A2 derived from baculovirus-infected insect cells coexpressing NADPH-
P450 reductase were obtained from BD Gentest Corp. (Woburn, MA). cDNA-
expressed CYP2C19 in Escherichia coli coexpressing NADPH-P450 reductase
was purchased from New England Biolabs Ltd. (Beijing, China). Pooled Wistar
male rat liver microsomes (RLM; n = 10) were prepared from liver tissue by
differential ultracentrifugation as described previously (Liu et al., 2009), and
the Lowry method was adopted to determine the concentration of microsomal
protein by using bovine serum albumin as a standard (Lowry et al., 1951).
Pooled mouse microsomes (MLM), pig microsomes (PLM), and male New
Zealand rabbit microsomes (RaLM) were purchased from Research Institute for
Liver Diseases (Shanghai, China). All microsomal samples and recombinant
human P450 isoforms were stored at –80°C until use.
Previously, our laboratory has screened the CYP3A substrates from
a large number of Traditional Chinese Medicines that are classified as
superior drugs (Wu et al., 2012). Fructus Schisandrae has been used
for thousands of years, owing to its diverse pharmacological effects
(Wei et al., 2010). Many structurally similar dibenzocyclooctadiene
lignans from Fructus Schisandrae were found to be metabolized by
CYP3A4. For example, schizandrin has been proven to undergo C-8
hydroxylation and subsequent demethylation by CYP3A4 in vitro
(Cao et al., 2010). Also, our preliminary experiments indicated that
deoxyschizandrin (DS), another most abundant lignan from Fructus
Schisandrae, was metabolized to a single metabolite via CYP3A4.
Besides, it is worthy to note the similarity of chemical structures
between DS and schizandrin, which differs by a single hydroxyl group
present on the cyclooctene ring. Therefore, we hypothesized that DS
might be a novel CYP3A substrate that meets the desired criteria for
an in vitro probe.
Incubation Conditions
The optimal conditions for microsomal incubation were determined in the
linear range for the formation of metabolite from DS or isoschizandrin (ISZ).
The incubation mixture, with a total volume of 200 ml, consisted of 100 mM
potassium phosphate buffer (pH 7.4), NADPH-generating system (1 mM
NADP+, 10 mM glucose-6-phosphate, 1 IU/ml of glucose-6-phosphate
dehydrogenase, and 4 mM MgCl2), and liver microsomes. In all experiments,
DS or ISZ (20 mM previously dissolved in methanol) was serially diluted to the
required concentrations, where the final methanol concentration did not exceed
0.5% (v/v) in the mixture. After 3 minutes of incubation at 37°C, the reaction
was initiated by adding NADPH-generating system, and further incubation
followed for 10 minutes at 37°C in a shaking water-bath. The reaction was
terminated by the addition of methanol (200 ml). The mixture was kept on ice
until it was centrifuged at 20,000g for 10 minutes at 4°C. Aliquots of
supernatants were stored at –30°C until analysis. Control incubations without
NADPH or without substrate or without microsomes were carried out to ensure
that the formation of metabolite was microsome- and NADPH-dependent. All
incubations throughout the study were carried out in three independent
experiments performed in duplicate with standard deviation (S.D.) values
generally below 10%, and results were expressed as mean 6 S.D.
The aim of the present study was therefore to find further support
that DS may serve as a specific probe substrate for CYP3A in vitro,
through identifying its metabolite, metabolic pathway, and specificity
to CYP3A. Then, we further assessed DS’s metabolic kinetic prop-
erties and its substitution as a substrate for three commonly used sub-
group probes, testosterone, midazolam, and nifedipine.
Liver Perfusion Studies
Male Wistar rats were anesthetized with intraperitoneal administration of
sodium pentobarbital (50 mg/kg). The surgical procedure was based on
previously described methods with minor modification (Liu et al., 2000);
erythrocyte-free Krebs-Henseleit buffer (KHB) was oxygenated with 95% O2
and 5% CO2. After anesthesia, the abdomen was opened with a U-section. The
hepatic artery and infrahepatic vena cava were ligated, and the portal vein was
cannulated by a 14-gauge needle double catheter for infusion. The venous
perfusate outflow was allowed to drain back into the reservoir. KHB without
DS perfusate passed the liver at a flow rate of 15 ml/min at 37°C for 20 minutes
for equilibration. Then, KHB perfusate, containing DS (50 mM) from a second
reservoir with 200 ml, was used for recirculation for 30 minutes. The reservoir
perfusate was sampled at the end of the experiment and stored at –20°C before
Materials and Methods
Chemicals
Deoxyschizandrin (purity . 98.0%) was purchased from China Chengdu
Scholar Bio-Tech. Co., Ltd. 1-Aminobenzotriazole (ABT), sulfaphenazole,
quinidine, clotrimazole, clomethiazole, furafylline, 8-methoxypsoralen, ome-
prazole, testosterone (TST), 6b-hydroxytestosterone, nifedipine (NIF), oxidized
nifedipine, midazolam (MDZ), 19-hydroxymidazolam (19-OH-MDZ), glucose-
6-phosphate dehydrogenase, NADP+, and D-glucose-6-phosphate were pur-
chased from Sigma-Aldrich (St. Louis, MO). Ketoconazole was obtained from
ICN Biomedicals Inc. (Aurora, OH). Montelukast was from Beijing Aleznova assay.
Pharmaceutical (Beijing, China). Triethylenethiophosphoramide was purchased
from Acros Organics (Geeleu, Belgium). All other reagents were of high-
performance liquid chromatography (HPLC) grade or of the highest grade
commercially available.
Ultra-Fast Liquid Chromatography–Diode Array Detector and Electrospray
Ionization–Mass Spectroscopy Analysis
The ultra-fast liquid chromatography (UFLC) system was employed. A
Hypersil ODS (C18) analytical column (100 mm ꢀ 2.1 mm, 3 mm; Thermo
Scientific, Pittsburgh, PA) with a Hypersil ODS (C18) guard column (150 nm
Animals
Wistar rats (n = 10, male, 180–220 g) were purchased from Dalian Medical ꢀ 2.1 mm, 3 mm; Thermo Scientific) was used to separate DS, MDZ, TST,
University (Dalian, China). The animals had free access to tap water and pellet NIF, ISZ, and their respective metabolites. As for DS, MDZ, TST, and NIF, the

96
Wu et al.
mobile phase consisted of methanol (A pump) and water (B pump) with the
following gradient profile: 0–9 minutes, 50–5% B; 9–12 minutes, 5% B; 12–
16.5 minutes, balanced to 50% B; as for ISZ, the mobile phase consisted of
CH3OH (A) and water with the following gradient profile: 0–12 minutes, 55–
45% B; 12–14 minutes, 45–5% B; 14–18.5 minutes, balanced to 55% B. The
flow rate was 0.25 ml/min and the column temperature was kept at 40°C. The
respective metabolites of DS, MDZ, TST, and NIF were detected at the detector
wavelength of 250 nm, 254 nm, 245 nm, and 237 nm, respectively. Shimadzu
LC-MS-2010EV (Kyoto, Japan) instrument with an electrospray ionization
(ESI) interface was used for identification of DS and its metabolite. Mass
detection was performed in both positive-ion mode (ESI+) and negative ion
mode (ESI–) from m/z 100 to 800. The detector voltage was set at +1.75 kV
and –1.55 kV for positive and negative ion detections, respectively. The curved
desolvation line temperature (CDL) and the block heater temperature were both
set at 250°C. Other mass spectrometry (MS) detection conditions were as
follows: interface voltage, 4 kV; CDL voltage, 40 V; nebulizing gas (N2) flow
was 1.5 l/min and the drying gas (N2) pressure was set at 0.06 MPa. Data
processing was performed using the LC-MS Solution software, version 3.41.
DS, MDZ, TST, NIF, and their respective metabolites were quantified by the
standard curve of authentic standards, which was linear from 0.1 to 30 mM,
with correlation coefficient of .0.999. The quantitative method displayed good
sensitivity. The limit of detection for DS hydroxylated metabolite is 0.5 ng. The
method also displayed good reproducibility, with the intraday and interday
variances both less than 3%.
Therefore, inhibitory effects of clotrimazole (0.001, 0.01, 0.1 mM),
ketoconazole (1 mM), and furafylline (50 mM) toward DS metabolism in
RLMs were examined.
Correlation Study
The formation rate of the metabolite described for DS (2 mM, near Km value)
was determined in a panel of HLM prepared from 12 different human organ
donors. These values were compared with the catalytic activities of CYP1A2,
2A6, 2C8, 2C9, 2C19, 2D6, 2E1, and 3A4. Isoform-specific reaction markers
and the methods involved were as follows: phenacetin O-deethylation
(CYP1A2), coumarin 7-hydroxylation (CYP2A6), paclitaxel 6a-hydroxylation
(CYP2C8), diclofenac 49-hydroxylation (CYP2C9), S-mephenytoin 49-hydrox-
ylation (CYP2C19), dextromethorphan O-demethylation (CYP2D6), chlorzox-
azone 6-hydroxylation (CYP2E1), and testosterone 6b-hydroxylation (CYP3A4).
The correlation parameter was expressed by the linear regression coefficient
(r2). A P value less than 0.05 was considered statistically significant.
Assay with Recombinant P450s
Ten cDNA-expressed human P450 isoforms coexpressing NADPH-P450
reductase and cytochrome b5 (CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9,
CYP2C19, CYP2D6, CYP2E1, CYP3A4, and CYP3A5) were used. The
incubations were carried out as described for the human liver microsomal
study. To investigate the contribution of each P450 isoform, DS (50 mM) was
incubated with each of the recombinant P450s (40–80 nM) at 37°C for
20 minutes. An ultra-fast liquid chromatography–diode array detector was
employed to monitor possible metabolites.
Metabolite Purification
The metabolite (ISZ) was biosynthesized using mixed liver microsomes
from rat and human (90% RLM and 10% HLM) and purified for structure
elucidation and quantitative analysis. In brief, the incubation system was scaled
up to 50 ml. DS (20 mM) was incubated with mixed liver microsomes (10 mg
protein/ml) and NADPH-generating system (1 mM NADP+, 10 mM glucose-6-
phosphate, 1 IU/ml of glucose-6-phosphate dehydrogenase, and 4 mM MgCl2)
for 90 minutes at 37°C. Under these conditions, about 54% of DS was
converted to the metabolite. Methanol (25 ml) was added to the reaction
mixture to precipitate the protein. After centrifugation at 9000g for 10 minutes,
the supernatant was separated and extracted with chloroform (50 ml ꢀ3). The
organic layer was combined and dried in vacuum. Then the residue was
dissolved in methanol (1 ml) and the metabolite was isolated and purified by
semipreparative HPLC with a YMC-Pack ODS-A column (10ꢀ250 mm, 5mm;
YMC Europe GmbH, Dinslaken, Germany) and eluted with MeOH-H2O (55:
45, v/v) to give the compound M (8 mg). The purity of the metabolite was
about 98% (HPLC).
Kinetic Study
To estimate kinetic parameters, DS (0.2–50 mM) was incubated with the
pooled HLM (0.05 mg protein/ml), pooled RLM (0.2 mg protein/ml),
recombinant CYP3A4 or CYP3A5 (10 nM), CYP3A1 (0.1 mg CYP/ml), or
CYP3A2 (0.075 mg P450/ml) for 10 min, respectively. To compare DS kinetic
parameters with those of the well known CYP3A4 probes TST, NIF, and MDZ
were simultaneously incubated with the pooled HLM, RLM, or recombinant
CYP3A4 or CYP3A5 for 10 minutes. On the basis of a previous report, reaction
mixtures with human microsomes were incubated at 0.25, 0.25, 0.25 mg/ml of
microsomal protein for TST, NIF and MDZ, respectively (Patki et al., 2003).
Likewise, the kinetic study of ISZ metabolism was also conducted in HLM and
RLM. ISZ (5–300 mM) was incubated with HLM (0.4 mg/ml) or with RLM
(0.4 mg/ml) at 37°C for 30 minutes with NADPH-generating system.
Formation of metabolites with liver microsomes was linear with respect to
incubation time and microsomal protein concentration over ranges relevant to
this study. All incubations were carried out in duplicate. The apparent Km and
Vmax values were calculated from nonlinear regression analysis of experimental
data according to the Michaelis-Menten equation with Origin software, version
7.5. Kinetic constants were reported as the value 6 S.D. of the parameter
estimates.
Nuclear Magnetic Resonance Spectrometry
1H and 13C nuclear magnetic resonance (NMR) spectra were obtained at
600 MHz on a Bruker AV-600 spectrometer (Bruker, Newark, Germany).
Compound M was dissolved in CDCl3 and experiments were conducted at
21°C. Chemical shifts are reported in parts per million (ppm) with reference to
tetramethylsilane.
Interaction Studies
Incubation Conditions. Recombinant human P450s at final concentration
of 20 nM were incubated with various concentrations of a pair of CYP3A
substrates in 100 mM potassium phosphate buffer (pH 7.4) with 1 mM EDTA,
6 mM MgCl2, and an NADPH-generating system consisting of 1 mM NADP+,
10 mM glucose-6-phosphate, 1 IU/ml of glucose-6-phosphate dehydrogenase,
and 4 mM MgCl2 in a total volume of 0.1 ml. Incubations were carried out in
a 37°C shaking water bath for 10 minutes. The substrates (concentration ranged
at least from 1/5 Km to 5 Km) were added to each incubation in either methanol
or phosphate buffer depending on the solubility. The final concentration of the
organic solvent (methanol) in incubation media was #0.5% (v/v). The range of
the inhibitor concentrations applied was from 0.5 to100 mM in most studies.
The reaction was terminated by 100 ml of ice-cold methanol. Samples were
then centrifuged at 20,000g for 15 minutes and further analyzed by UFLC.
Inhibition Kinetics Analysis. Inhibition constant (Ki) values were de-
termined by using various concentrations of substrates in the presence or
absence of inhibitors with Origin software, version 7.5. When DS was used as
Chemical Inhibition Study
Chemical inhibition studies were performed by adding different human P450
inhibitors to the incubation mixture of DS (2 mM) before the addition of
NADPH-generating system. The selection of a 2 mM concentration was based
on the Km value. The selective inhibitors and their concentrations were as
follows (Bjornsson et al., 2003): montelukast (5 mM) for CYP2C8 (Walsky
et al., 2005), sulfaphenazole (10 mM) for CYP2C9, omeprazole (20 mM) for
CYP2C19, quinidine (10 mM) for CYP2D6, clomethiazole (50 mM) for
CYP2E1, and ketoconazole (1 mM) for CYP3A4. Inhibition by furafylline
(10 mM) for CYP1A2, 8-methoxypsoralen (2.5 mM) for CYP2A6, triethylenethio-
phosphoramide (50 mM) for CYP2B6 (Rae et al., 2002) and ABT (500 mM) for
broad P450s (Emoto et al., 2003) were examined by adding DS after preincubation
with NADPH-generating system at 37°C for 10 minutes.
Clotrimazole (Turan et al., 2001) was found to be a selective inhibitor of rat
CYP3A subfamily. Ketoconazole is a broad inhibitor for rat P450s. Furafylline
(Eagling et al., 1998) was able to inhibit both rat CYP1A and rat CYP2C. the substrate, a single-site kinetic model was used to calculate Ki values by

Deoxyschizandrin Hydroxylation by CYP3A
97
nonlinear regression using the equations for competitive inhibition (eq. 1),
noncompetitive inhibition (eq. 2), or mixed inhibition (eq. 3) (Copeland, 2000;
Liu et al., 2010).
analyses (Table 1). And the most distinctive spectra changes were
involved in the C-7 region. The doublet of C-7-methyl (C-17) proton
signal at 1.00 ppm (3H, d) in DS was replaced by the singlet and
shifted downfield to 1.1 ppm (3H, d) in the metabolite. Moreover, the
13C-NMR spectrum of the metabolite showed C-7 downfield shift of
18.9 ppm, compared with DS. The above observations clearly
indicated hydroxylation at C-7. 1H- and 13C-NMR spectra of the
metabolite coincided with those of isoschizandrin standard (Ikeya
et al., 1991). Additionally, no conjugates were observed in the UDP-
glucuronic acid (UDPGA)-, 39-phosphoadenosine-59-phosphosulfate
(PAPS)-, and S-adenosyl-L-methionine (SAM)-generating systems,
when DS was incubated with either HLM or S9 fractions (data not
Vmax
S
v ¼
v ¼
ð1Þ
ð2Þ
ð3Þ
Kmð1 þ I=KiÞ þ S
Vmax
S
ðKm þ SÞð1 þ I=KiÞ
Vmax
S
v ¼
Kmð1 þ I=KiÞ þ Sð1 þ I=aKiÞ
Here v is the velocity of the reaction; S and I are the substrate and inhibitor
concentrations, respectively; Ki is the inhibition constant describing the affinity shown).
of the inhibitor for the enzyme; and Km is the substrate concentration at half of
the Vmax of the reaction. The a value determines the degree to which the
binding of inhibitor changes the affinity of the enzyme for substrate. When a is
very large (a . 1), binding of inhibitor prevents binding of the substrate and
the mixed-model becomes identical to competitive inhibition. The type of
inhibition was determined from the fitting of data to the enzyme inhibition
models. Goodness of fit to kinetic and inhibition models was assessed from the
r2 values and parameter S.D. estimates. Kinetic constants are reported as the
mean 6 S.D. of the parameter estimate.
Biotransformation of Isoschizandrin by Liver Microsomes. To
elucidate the subsequent biotransformation of ISZ (7S, 8R), the natural
enantiomer of schizandrin (7R, 8R) ISZ was biosynthesized by the
incubation of DS and NADPH-generating system in mixed liver
microsomes. Four product peaks were observed when ISZ (100 mM)
was incubated with the hepatic microsomes from human or rat (0.4 mg
protein/ml) along with the NADPH-generating system for more than
30 minutes (Fig. 1). Each metabolite of ISZ was below the detection
limit when the incubation time is less than 25 minutes. These four
Two-site kinetic models were used to calculate Ki values by using the
equations for a substrate with substrate-inhibition kinetics (eq. 4), and a substrate metabolites (M-1, M-2, M-3, and M-4) were identified as demethy-
with sigmoidal kinetics (eq. 5) (Galetin et al., 2003).
lated ISZ by UFLC-ESI-MS (Supplemental Table 1), and the
formation of these metabolites was time-, NADPH-, and microsome-
dependent (data not shown). Demethylation was identified as the
major metabolic pathway of ISZ, while hydroxylation was the key
metabolic pathway of schizandrin (Cao et al., 2010). Furthermore, no
conjugates were observed in the UDP-glucuronic acid (UDPGA)-, 39-
phosphoadenosine-59-phosphosulfate (PAPS)-, and S-Adenosyl-L-
methionine (SAM)-generating systems, when ISZ was incubated with
either HLM or S9 fractions (data not shown).
Chemical Inhibition Studies. The effect of various chemical
inhibitors on the metabolism of DS was investigated in pooled HLM
and RLM (Fig. 2). ABT, a broad P450 inactivator, inhibited DS
monohydroxylation completely, suggesting that P450s were the enzymes
responsible for DS hydroxylation. Among the ten selective inhibitors
tested, ketoconazole, the selective inhibitor of CYP3A, inhibited ISZ
formation from DS by 100% (P , 0.05). In our experiments, the other
putative inhibitors were not active in reducing DS metabolism in our
in vitro test systems. These data together demonstrate that DS metabolism
is catalyzed by CYP3A.
ꢀ
ꢁ
S
bS2
2
gSI
dKSKi
2I
Vmax
S
þ
þ
KS KS
S2
v ¼
ð4Þ
2SI
I2
1 þ
þ
2 þ
þ
þ
KS KS
dKSKi Ki Ki2
In eq. 4, b represents change in product formation from substrate-enzyme-
substrate (SES) complex due to the second substrate binding; g represents
change in product formation from inhibitor-enzyme-substrate (IES) complex
due to inhibitor binding; and d represents change in affinity of substrate-
enzyme or enzyme-substrate (SE/ES) complex for the inhibitor molecular.
ꢀ
ꢁ
S
S2
Vmax
S2
þ
2
KS «KS
v ¼
ð5Þ
2S
I
2SI
S2I
1 þ
þ
2 þ
þ þ
Ki dKiKS dKiKS
2
KS «KS
In eq. 5, d represents change in affinity of SE/ES and SES complexes for the
inhibitor molecule; « represents change in affinity of SE/ES complex for the
second substrate molecular.
To explore whether the metabolic enzymes responsible for DS
hydroxylation in rat were also CYP3A isoenzymes, inhibition studies
in RLM were conducted with clotrimazole as a selective inhibitor for
Results
Identification of DS Hydroxylation. A single new peak was eluted CYP3A, furafylline as the inhibitor for CYP1A and CYP2C, and
at 6.8 minutes by UFLC when DS (50 mM) was incubated with HLM ketoconazole as a broad inhibitor for rat P450s. As illustrated in Fig. 2,
and RLM (0.2 mg/ml) in the presence of NADPH for 20 minutes (Fig. furafylline (50 mM) slightly inhibited the formation of ISZ in rat,
1). Similar profiles were also observed in MLM, PLM, and RaLM whereas ketoconazole (1 mM) was able to inhibit the formation of
(Supplemental Fig. 1). The peak was not detected in control samples ISZ nearly completely, and clotrimazole (0.1 mM) also inhibited the
without microsomes, NADPH, or DS (data not shown). To identify the hydroxylation of DS completely. This result implies that CYP3A also
in vivo metabolic profile of DS, the rat liver perfusion was conducted plays an important role in the hydroxylation of DS in rat.
to imitate the in vivo situation. As expected, one predominant metabolite
Assay with Recombinant Human P450 Isoforms. To elucidate
was detected in the perfusate after 30 minutes (Fig. 1). Mass spectra P450 isoforms involved in the metabolism of DS in human, the
with positive ion ESI was dominated by [M + K]+. The metabolite (M) activity of DS hydroxylation was determined in ten cDNA-expressed
was identified by UFLC–diode array detector–ESI-MS to show an m/z P450 isoforms (Fig. 3). After incubation at 37°C for 20 minutes,
value of 471, a value that corresponds well to DS (416 plus 39) hydroxylated metabolite ISZ was observed exclusively in CYP3A4
incorporated with the m/z 16 of the oxygen substitution (Supplemental and CYP3A5. No metabolites were observed in the incubation with
Table 1). The monohydroxylated metabolite of DS was biosynthesized CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6,
and purified for structure elucidation, as well as for use as a standard or CYP2E1 (less than 0.01 pmol/min per pmol P450). The formation
in different analyses. The metabolite structure determination rates of ISZ in CYP3A4 and CYP3A5 were 34.9 6 1.4 and 15.1 6 1.2
was carried out by NMR, using both 1H-NMR and 13C-NMR pmol/min per pmol P450, respectively. These results indicated that

98
Wu et al.
Fig. 1. Representative UFLC profiles of DS, ISZ, and their metabolites in human liver microsomes, rat liver microsomes, and rat liver perfusate. DS (50 mM) was incubated
with liver microsomes (0.3 mg/ml) at 37°C for 20 minutes (A) from different species with NADPH-generating system; the recirculating perfusate was obtained after a bolus
introduction of DS (50 mM) to the perfusate over 30 minutes. Meanwhile, ISZ (100 mM) was incubated with liver microsomes (0.4 mg/ml) at 37°C for 40 minutes from
different species and schizandrin (100 mM) was incubated with human liver microsomes (0.3 mg/ml) at 37°C for 20 minutes with NADPH-generating system (B).
CYP3A4 and CYP3A5 both contribute to DS in HLMs, with CYP3A5 DS (Fig. 3). Therefore, CYP3A was the major P450 isoform involved
being metabolically less active than CYP3A4.
in the formation of ISZ in HLM.
Recombinant human P450 isoforms were also employed to identify
Kinetic Study of DS. The kinetic parameters of ISZ formation in
P450 isoforms involved in the subsequent metabolism of ISZ in HLM, RLM, and recombinant P450s were determined. The formation
human. After incubation at 37°C for 40 minutes, four demethylated rate of ISZ was linear up to 0.05 mg/ml and 0.2 mg/ml microsomal
metabolites (M-1, M-2, M-3, and M-4) of ISZ were found in CYP3A4 protein and in HLM and RLM for 10-minutes’ incubation, re-
and CYP3A5 (Supplemental Fig. 2), while no metabolites were spectively. DS (0.2–50 mM) was incubated with pooled HLM, RLM,
observed in the incubations with CYP1A2, CYP2A6, CYP2B6, recombinant CYP3A4, or recombinant CYP3A5. In parallel, TST
CYP2C8, CYP2C9, CYP2C19, CYP2D6, or CYP2E1 (less than (5–300 mM), NIF (2–200 mM), and MDZ (0.5–500 mM) were also
0.01 pmol/min per pmol P450).
treated in the same incubation system. Over the whole concentration
Correlation Study. For DS hydroxylation to ISZ, a strong range tested, sigmoidal kinetics and substrate inhibition kinetics were
correlation was observed with a CYP3A marker, testosterone observed for TST 6b-hydroxylation and MDZ 19- hydroxylation,
6b-hydroxylation (r2 = 0.85, P , 0.001). DS hydroxylation also cor- respectively, in accordance with the previous reports (Houston and
related with other CYP3A markers, such as phenacetin O-deethylation Kenworthy, 2000). NIF oxidation was observed to obey the Michaelis-
(CYP1A2, r2 = 0.31), coumarin 7-hydroxylation (CYP2A6, r2 = 0.52), Menten kinetics even though both sigmoidal and convex curves were
paclitaxel 6a-hydroxylation (CYP2C8, r2 = 0.65), diclofenac 49- reported for this substrate (Houston and Kenworthy, 2000). Impor-
hydroxylation (CYP2C9, r2 = 0.05), S-mephenytoin 49-hydroxylation tantly, DS C7-hydroxylation followed Michaelis-Menten kinetics, as
(CYP2C19, r2 = 0.11), dextromethorphan O-demethylation (CYP2D6, depicted by linear Eadie-Hofstee plots in HLM, RLM, and recombinant
r2 = 0.31), and chlorzoxazone 6-hydroxylation (CYP2E1, r2 = 0.29). P450s (Fig. 4). By comparing the kinetic parameters (Table 2), DS
The recombinant CYP1A2, CYP2A6, and CYP2C8 failed to metabolize showed higher affinity to CYP3A and metabolic capacity than that of

Deoxyschizandrin Hydroxylation by CYP3A
99
TABLE 1
Proton and carbon NMR chemical shift assignments for deoxyschizandrin and its metabolite (M)
All spectra were recorded on a Bruker ARX-600 spectrometer, in CDCl3.
Deoxyschizandrin
M (Isoschizandrin)
HMBC (H→C)
No.
d1H
d
13C
d1H
d
13C
NOESY
1
151.5
140.1
152.8
107.1
139.1
35.6
151.3
132.8
151.9
110.4
140.5
48.2
2
3
4
5
6
6.54 (s)
a: 2.55 (dd,
J = 13.8, 7.8 Hz)
b: 2.50 (dd,
6.64 (s)
C-1, C-2, C-3, C-5, C-6, C-16
H-6, H-17
a: 2.68 (d, J = 13.2 Hz)
b: 2.62 (d, J = 13.2 Hz)
C-4, C-5, C-7, C-8, C-16, C-17
C-7, C-10, C-17, C-18
H-4, H-8, H-17
J = 13.8, 1.8 Hz)
1.81 (m)
1.91 (m)
7
8
9
40.7
33.7
39.1
1.83 (m)
1.73 (m)
a: 2.22 (m)
47.9
73.8
37.1
H-6, H-9, H-11, H-18
H-8, H-11, H-18
a: 2.28 (dd,
J = 13.2, 9.6 Hz)
b: 2.06 (br d,
J = 13.2 Hz)
b: 2.18 (m)
C-7, C-8, C-10, C-11, C-15, C-18
10
11
12
13
14
15
16
17
18
133.9
110.4
151.6
139.7
151.4
123.3
122.3
12.6
140.1
107.1
153.1
138.1
151.4
121.7
123.3
18.9
6.54 (s)
6.56 (s)
C-9, C-10, C-12, C-13, C-14, C-15 H-8, H-9
1.00 (d, J = 7.2 Hz)
0.74 (d, J = 6.6 Hz)
1.1 (d, J = 7.2 Hz)
1.07 (s)
C-6,C-7,C-8
C-7,C-8
H-4, H-6
H-8, H-9
21.8
20.7
OMe 3.59 ꢀ2, 3.87, 3.88,
60.9 ꢀ2, 60.5 ꢀ2, 3.60 ꢀ2, 3.87, 3.89,
61.0, 60.9,
3.90 ꢀ2 (each 3H, s) 60.6 ꢀ2, 55.9 ꢀ2
3.89, 3.90 (each 3H, s)
55.9 ꢀ2
Abbreviations: br, broad; d, doublet; m, multiplet; s, singlet; HMBC, heteronuclear multiple-bond correlation spectroscopy; J, coupling constant; NOESY, nuclear Overhauser effect spectroscopy.
TST, NIF, and MDZ in HLM and RLM. Vmax of DS 7-hydroxylation TST and 3.8 for MDZ. Also, inhibitions of DS by TST and MDZ in
by CYP3A4 was nearly 2.5-fold higher than that by CYP3A5, while CYP3A5 were both best fitted to competitive inhibition model with Ki
DS had similar binding affinity to both enzymes (Km values of 3.2 and values of 40.2 6 8.9 mM and 14.5 6 3.3 mM, respectively. The
4.1 mM, respectively), and yielded 3-fold clearance difference. Similar inhibition of TST by DS was fitted to a two-site kinetic model for
trends were also observed for TST and NIF, but the 19-OH-MDZ sigmoidal kinetics, yielding a larger Ki value of 21.1 6 3.3 mM in
formation rate in CYP3A4 was almost 6-fold lower than that in CYP3A4 than the Ki value of 16.3 6 1.3 mM in CYP3A5. Here, affinity
CYP3A5. Moreover, the intrinsic clearance of DS by CYP3A2 was interaction factor d yielded low values altogether, with 0.73 in CYP3A4
nearly 2.5-fold higher than that by CYP3A1 (Supplemental Table 2 and 0.58 in CYP3A5. The inhibition of MDZ by DS was fitted to the
and Supplemental Fig. 3).
two-site kinetic model for substrate inhibition kinetics, yielding Ki
Kinetic Study of ISZ. In the same fashion as the DS kinetic study, values of 17.3 6 2.2 mM in CYP3A4 and 10.4 6 1.8 mM in CYP3A5,
the kinetic parameters of demethylated ISZ production in pooled HLM and yielding low affinity interaction factor d values of 0.26 in CYP3A4
and RLM were determined. The formation rates of demethylated and 0.41 in CYP3A5.
metabolites were linear up to 0.4 mg/ml microsomal protein after
In contrast, Dixon and Lineweaver-Burk plots show that inhibition
30 minutes incubation (ISZ 5–300 mM). In the range of concentrations of DS by NIF was best fitted to a noncompetitive inhibition model
tested, ISZ demethylation in liver microsomes complied with (Supplemental Fig. 4), in which the inhibition kinetic parameter (Ki)
Michaelis-Menten kinetics, as evidenced by linear Eadie-Hofstee plot was calculated to be 24.8 6 2.1 mM and 27.8 6 4.2 mM for CYP3A4
(Fig. 5). It is noteworthy that DS exhibited 20–50 times higher affinity and CYP3A5, respectively. Also, DS showed negligible inhibitory
to CYP3A in HLM than did ISZ (Table 3 and Supplemental Table 3). effects on NIF oxidation in both CYP3A4 and CYP3A5 (Supplemen-
Also, due to the low affinity of ISZ to CYP3A, the total intrinsic tal Fig. 5).
clearance (CLint) of ISZ via demethylation only accounts for less than
5% of DS hydroxylation clearance in both HLM and RLM individually.
Based on the above results, the proposed primary metabolic pathways
Discussion
of DS in HLM and RLM are shown in Fig. 6.
In the present study, we identify that C-7- hydroxylation of DS is
Interaction Studies. In Table 3, the interaction studies of DS in the highly selectively catalyzed by CYP3A in liver microsomes from
presence of the three CYP3A substrates (MDZ, TST, and NIF) are human and rat. DS hydroxylation was proven to obey Michaelis-
demonstrated. The best model was selected mainly based on the Menten kinetics both in HLM, RLM, and recombinant enzymes. For
criteria described as r2 values and parameter S.D. estimates (Supple- rat, the most commonly used animal species for pharmacokinetic
mental Table 4). Dixon and Lineweaver-Burk plots here show that studies, CYP3A1 and CYP3A2 predominantly catalyzed DS hydrox-
inhibition of DS by both TST and MDZ were best fitted to the mixed ylation (Supplemental Fig. 3). The similarities observed in metabolite
inhibition–type models, in which the inhibition kinetic parameter (Ki) in profiles and kinetic properties between rat and human suggest that rat
the inhibiting CYP3A4 activity was calculated to be 74.1 6 10.1 mM is a suitable surrogate animal model for further preclinical pharma-
for TST and 6.6 6 1.3 mM for MDZ. The a values yielded were 5.8 for cokinetic studies for DS. Primary results indicated mouse, pig, and

100
Wu et al.
Fig. 3. The formation of metabolite M (ISZ) catalyzed by various recombinant
human P450s. DS (50 mM) was incubated with various recombinant human P450s
(40–80 nM) at 37°C for 20 minutes. Data represent the mean of three experiments
carried out in duplicate.
example, TST was used in nearly 50% of reported screening studies
for DDI potentials (Yuan et al., 2002), but due to its atypical
kinetic, its result is difficult to extrapolate to an in vivo setting
(Foti et al., 2010). Thus, it is vital to find a substrate with classic
Michaelis-Menten kinetic that can serve as an ideal probe for
CYP3A activity.
The metabolite ISZ is a naturally occurring (+)-enantiomer of
schizandrin. The asymmetric carbon at position 7 yields Z(cis)-
(+)-schizandrin (7R, 8R) and E(trans)-(+)-isoschizandrin (7S, 8R).
These cis- and trans-lignans possess similar biologic properties, and
the pharmacological activity resides mainly at the C7-hydroxyl (Ikeya
et al., 1988; Warshawsky and Meyers, 1990). Then the further
metabolism of ISZ in microsomes and recombinant P450s was also
investigated. Demethylation was identified as the major metabolic
pathway of ISZ, in which CYP3A4 and CYP3A5 were involved in the
transformation indicated by the recombinant P450s assays. It was
almost unrealistic to biosynthesize and purify these demethylated
metabolites for structure elucidation, because the metabolite formation
Fig. 2. Effects of selective P450 inhibitors on the formation of metabolite ISZ (M)
in human liver microsomes (A) and rat liver microsomes (B). Results are the mean
6 S.D. from three experiments carried out in duplicate.
rabbit also showed similar metabolic profiles in vitro, which implied rate was close to the limits of quantification. In comparison to the
these species could be involved in further in vivo studies. Michaelis-Menten kinetic profiles in HLM, Vmax and Vmax/Km values
To date, it is known that most P450-catalyzed reactions can be for schizandrin hydroxylation were 5- to 9-fold larger and at least 40%
described using the Michaelis-Menten equation, and the existing larger, respectively, than those for ISZ demethylation (Cao et al.,
mathematical models used for the prediction of DDI or clearance are 2010). This hints that the metabolic stability improves when Z (cis)-
all based on Michaelis-Menten kinetic (Ito et al., 1998). Therefore, (+)-schizandrin is transformed into E (trans)-(+)-ISZ. Analysis of the
a probe substrate with Michaelis-Menten kinetic was considered to be structure-metabolism relationship for the three lignans isolated from
a “pragmatic” substrate. In the case of substrates with non-Michaelis- Fructus Schisandrae suggests that the dibenzocyclooctadiene lignans
Menten kinetic, two or more molecules of substrates can simulta- with six methoxy groups on two benzene rings is the most potent
neously occupy the active site cavity and the kinetic parameters CYP3A substrate. This is observed through its single-site kinetic
change with substrate occupancy. As a result, more complex mechanistic property, by which hydroxylation or demethylation occur either at C7
analyses are demanded; otherwise, false positive or negative pre- or C8, such as with DS, ISZ, and schizandrin (Cao et al., 2010).
diction of DDI will result from classic Michaelis-Menten analysis. However, the lignans are prone to be CYP3A inhibitors when the
However, the most commonly used substrates of CYP3A recommended methoxyl groups on phenyl are replaced with methylenedioxy ring
by FDA (http://www.fda.gov/Drugs/DevelopmentApprovalProcess/ (Iwata et al., 2004).
DevelopmentResources/DrugInteractionsLabeling/ucm093664.htm) do
Notably, the subsequent ISZ demethylations were not observed
not obey the classic Michaelis-Menten kinetics, e.g., TST (Kenworthy when DS was incubated with HLM even at a very high substrate
et al., 2001), MDZ, NIF (Houston and Kenworthy, 2000), triazolam concentration (DS, 100 mM) within the linear reaction time range
(Hallifax et al., 2005), and dextromethorphan (Witherow and Houston, (data not shown). These data implied that the subsequent metabolism
1999). Regardless of the complex mechanism of non-Michaelis- of ISZ might be abolished when DS was incubated with HLMs and
Menten kinetics, ignoring it and truncating the data can lead to RLM. This can be explained by the fact that DS exhibited 20- to 50-
erroneous estimates of kinetic parameters (Lin et al., 2001). For fold higher affinity to CYP3A in HLMs than did ISZ. Another

Deoxyschizandrin Hydroxylation by CYP3A
101
Fig. 4. Kinetic plots of DS metabolism after incubation with pooled human liver microsomes (A), pooled rat liver microsomes (B), and recombinant CYP3A4 (C) and
CYP3A5 (D). Eadie-Hofstee plots are shown for DS hydroxylation. Lines represent linear regressions of Eadie-Hofstee transformed data best-fits to a Michaelis-Menten
model (A–D).
explanation is the extremely low total CLint of ISZ, which accounts for Observations here together suggest that DS was metabolized to
even less than 5% of DS hydroxylation clearance in HLM (Fig. 6). A a single metabolite, ISZ, via CYP3A with high selectivity. The
third is the fact that the logD values for DS and ISZ were calculated to existence of single catalytic/metabolic site for the probe could
4.7 and 3.39, respectively (ChemAxon), which suggested that ISZ is potentially decrease the chance of erroneous judgment on prediction
a poor substrate compared with DS, owing to a change in lipophilicity of DDI potentials, because the P450 modifiers might play seesaw
driven by the introduction of a hydroxyl group. And the predominant effects on each metabolic site. Quinidine, a CYP3A4 substrate, can be
metabolite was identified in the rat liver perfusate that mimics the in metabolized to two or more metabolites, 3-hydroxyquinidine and
vivo situation. Furthermore, when either DS or ISZ was incubated quinidine N-oxide, despite exhibiting Michaelis-Menten kinetics
with HLM or S9 fraction, no subsequent conjugates of ISZ or DS were (Nielsen et al., 1999). MDZ can also be metabolized to at least two
observed in the UDPGA-, PAPS- or SAM-generating systems. metabolites, 19-hydroxymidazolam and 4-hydroxymidazolam, and
TABLE 2
Kinetic parameters of DS, NIF, TST metabolism in HLM, RLM, and recombinant human CYP3A4 and CYP3A5
Km values were in mM; Vmax values were in pmol/min per milligram for liver microsomes, or in pmol/min per pmol P450 for CYP3A4; CLint (Vmax/Km) values were in ml/mg per minute for liver
microsomes, or in ml/pmol per minute for CYP3A4 and CYP3A5. Each value was the mean 6 S.D. of three determinations performed in duplicate.
DS
NIF
TST
MDZ
Enzyme
Source
M (Isoschizandrin)
OX-NIF
6b-HTS
19-OH-MDZ
a
Vmax
Km
CLint
Vmax
Km
CLint
Vmax
S50
n
CLmax
Vmax
Ks
Ksi
CLint
HLM
RLM
623 6 9.9 1.6 6 0.1 389 4461 6 59 15.3 6 1.0 297 5484 6 90 53.1 6 5.1 1.4 6 0.1 56.8
168 6 2.4 1.4 6 0.3 120 1360 6 16 20.3 6 0.8 67.0 2563 6 78 61.2 6 4.0 1.2 6 0.1 26.7 96.9 6 3.8 6.1 6 1.7 206 6 41 16.1
35.7 6 0.7 10.2 6 0.7 3.50 101 6 1.1 35.1 6 3.2 1.2 6 0.1 1.80 5.9 6 0.2 1.3 6 0.2 114 6 17 4.50
119 6 4.6 3.5 6 0.4 228 6 43 33.7
CYP3A4 36.7 6 0.3 3.2 6 0.1 11.5
CYP3A5 16.1 6 2.4 4.1 6 0.1 3.90 18.4 6 0.3 15.8 6 0.8 1.20 44.7 6 1.3 89.5 6 5.2 1.3 6 0.1 0.29 34.2 6 2.5 4.1 6 0.8 173 6 52 8.30
6b-HTS, 6b-hydroxytestosterone; OX-NIF, oxidized nifedipine
a
TST clearance was obtained by Vmax/Km corrected with Hill coefficient (Houston and Kenworthy, 2000).

102
Wu et al.
Fig. 5. Kinetic plots of ISZ metabolism after incubation with pooled human liver microsomes (A) and pooled rat liver microsomes (B). Eadie-Hofstee plots are shown for
both ISZ demethylation. Lines represent linear regressions of Eadie-Hofstee transformed data best-fits to a Michaelis-Menten model (A and B).
modulators could exert different effects on each metabolite formation potencies of DS in CYP3A4 and CYP3A5 were evident in binding
(Xia et al., 2009; Dong et al., 2011). As reported, erlotinib and affinity for MDZ, where low d values of 0.26 and 0.41 were observed
panaxytriol, an active component in Shenmai injection (Zeng et al., for CYP3A4 and CYP3A5, respectively.
2013), both showed significant activation on MDZ 19-hydroxylation,
However, inhibition studies using NIF showed different inhibition
whereas the activation but not inhibition made it difficult for the in results. NIF noncompetitively inhibited DS hydroxylation in both
vivo extrapolation. Moreover, either glucuronidation of hydroxylmi- CYP3A4 and CYP3A5. Moreover, DS inhibition of NIF oxidation
dazolam or direct glucuronidation of MDZ occurs in vitro and in vivo was negligible in both CYP3A4 and CYP3A5, even at high DS
(Klieber et al., 2008; Hyland et al., 2009; Seo et al., 2010). The concentrations up to 100 mM. Similarly to the previous findings
multiple metabolic pathways may partly compensate for the decrease reported by Wang et al. (2000) on the TST-NIF interaction, the
in MDZ metabolic clearance caused by the addition of the inhibitor unusual DS-NIF interaction seen in this study could also be explained
and would also make it difficult for the in vivo quantification. Thus, by assuming that NIF has more freedom of movement, and that it can
the in vivo DDI prediction from the in vitro data for MDZ is lack of bind to multiple metabolic sites, including the active site of CYP3A4/5
rationality at least in theory, despite the reported wealth of in vitro to which DS binds and is fixed. Consequently, NIF can inhibit the
CYP3A inhibition data using MDZ and in vivo DDI data for MDZ.
hydroxylation of DS, but the inhibition of NIF oxidation by DS cannot
On the other hand, using in vitro DDI studies as screening tools to be demonstrated kinetically. Regardless of the possible mechanisms,
evaluate the potential DDI in vivo is based on the assumption that the the results in these inhibition studies reveal a complex interaction
drugs being analyzed are competing for the same enzyme catalytic between NIF and CYP3A even though Michaelis-Menten kinetics was
site. (Wang et al., 2000). Thus, substrate interaction studies were observed.
conducted in which DS substrate substitution was analyzed in the
Overall, an assumption that the CYP3A preferential binding domain
presence of the three substrates of CYP3A. The kinetics of DS in to which DS binds is large can be inferred from its high affinity to the
CYP3A4 were best fitted to mix models of inhibition resulting from CYP3A active sites and the fact that it competes strongly with both
competition yielding large a values of 5.8 and 3.8 in the presence of MDZ and TST for a mutual binding site an assumption that the
TST and MDZ, respectively (Copeland, 2000); DS also exhibited preferential binding domain of CYP3A is large, to which DS binds,
competitive inhibition type in CYP3A5. DS markedly decreased the can be inferred by its high affinity to the CYP3A active sites and the
affinity for TST by CYP3A and TST-CYP3A complex, where low d fact that DS competes strongly with both MDZ and TST for the mutual
values of 0.73 and 0.58 were observed for CYP3A4 and CYP3A5, binding site. Furthermore, during the screening and development
respectively, and a low d value suggested that the affinity of inhibitor process, a drug-candidate can be preclassified, with in vitro methods
to the enzyme (or enzyme-substrate complex) increased but the as recommended here, by evaluating different types of kinetic profiles
affinity of substrate to the enzyme decreased. Also, similar inhibitory and by studying the similarity between binding regions of the
TABLE 3
Kinetic parameters for the in vitro inhibitory effects of pairs of CYP3A substrates in recombinant human CYP3A4 and CYP3A5
Each value was the mean 6 S.D. of three determinations performed in duplicate.
CYP3A4
CYP3A5
Substrates
DS
Inhibitors
Model*
Ki (mM)
a
d
Goodness of fit (R2)
Model*
Ki (mM)
d
Goodness of fit (R2)
NIF
MDZ
TST
DS
Eq. 2
Eq. 3
Eq. 3
Eqs. 1–5
Eq. 4
24.8 6 2.1
6.6 6 1.3
74.1 6 10.1
–
–
–
–
0.990
0.994
0.988
Eq. 2
Eq. 1
Eq. 1
Eqs. 1–5
Eq. 4
27.8 6 4.2
14.5 6 3.3
40.2 6 8.9
–
–
–
0.968
0.972
0.977
3.8 6 0.8
5.8 6 1.2
NIF
MDZ
TST
N/A
N/A
17.3 6 2.2
21.1 6 3.3
–
–
0.26 6 0.1
0.73 6 0.2
0.960
0.989
10.4 6 1.8
6.8 6 1.3
0.41 6 0.2
0.89 6 7.8
0.988
0.979
Eq. 5
Eq. 5
N/A, Not available; *, inhibitory kinetic models as described in Materials and Methods.

Deoxyschizandrin Hydroxylation by CYP3A
103
Fig. 6. The proposed primary metabolic pathways of DS in
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investigational drug and of the probe substrate. This can be then
followed by predicting the possible doses in order to modify P450
activities for in vivo DDI risk assessment.
In this study, hydroxylation of DS at the C-7 site to produce a single
metabolite, ISZ, via CYP3A metabolism is shown as the exclusive
metabolic pathway in human liver microsomes. Additionally, the
kinetic profile obeyed Michaelis-Menten kinetics. More importantly,
DS is shown to compete with both TST and MDZ for a mutual binding
site. Thus, DS is considered a good representative of MDZ and TST
substrate subgroups and possesses the three criteria for a pragmatic in
vitro probe: high-selectivity for CYP3A with a single metabolite,
Michaelis-Menten kinetic properties, and a broad range of substrate
substitutions. Conclusively, DS serves as an ideal candidate for
a novel and specific CYP3A probe substrate that can predict the
modifications in CYP3A-derived metabolism.
Acknowledgments
The authors thank Dr. Di Hu for analysis of NMR data.
Authorship Contributions
Participated in research design: Wu, Cao, Zhang, Liu, Hong, Zhu, Ge,
Yang.
Conducted experiments: Wu, Cao, Zhang.
Performed data analysis: Wu, Cao, Zhang, Liu, Hong, Zhu, Ge, Yang.
Wrote or contributed to the writing of the manuscript: Wu, Cao, Zhang, Liu,
Hong.
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Address correspondence to: Dr. Ling Yang, Laboratory of Pharmaceutical
Resource Discovery, Dalian Institute of Chemical Physics, Chinese Academy of
Sciences, 457 Zhongshan Road, Dalian, China. E-mail: yling@dicp.ac.cn