Synthesis of novel vitamin K2 analogues with modification at the Ω-terminal position and their biological evaluation as potent steroid and xenobiotic receptor (SXR) agonists

Article abstract of DOI:10.1021/jm200025f

Vitamin K₂ is a ligand for a nuclear receptor, steroid and xenobiotic receptor (SXR), that induces the gene expressions of CYP3A4. We synthesized vitamin K₂ analogues with hydroxyl or phenyl groups at the Ω-terminal of the side chain. The up-regulation of SXR-mediated transcription of the target gene by the analogues was dependent on the length of the side chain and the hydrophobicity of the Ω-terminal residues. Phenyl analogue menaquinone-3 was as active as the known SXR ligand rifampicin.

Full text of DOI:10.1021/jm200025f

BRIEF ARTICLE
pubs.acs.org/jmc
Synthesis of Novel Vitamin K2 Analogues with Modification at the
ω-Terminal Position and Their Biological Evaluation as Potent Steroid
and Xenobiotic Receptor (SXR) Agonists
Yoshitomo Suhara,† Masato Watanabe,§ Kimie Nakagawa,§ Akimori Wada,|| Yoichi Ito,† Kazuyoshi Takeda,‡
Kazuhiko Takahashi,† and Toshio Okano*,§
†Laboratory of Environmental Sciences, and ‡Department of Medicinal Chemistry, Yokohama College of Pharmacy, 601, Matano-cho,
Totsuka-ku, Yokohama 245-0066, Japan
§Department of Hygienic Sciences, and Department of Organic Chemistry for Life Sciences, Kobe Pharmaceutical University,
4-19-1, Motoyamakita-machi, Higashinada-ku, Kobe 658-8558, Japan
S Supporting Information
b
ABSTRACT: Vitamin K2 is a ligand for a nuclear receptor, steroid and xenobiotic receptor (SXR), that induces the gene expressions
of CYP3A4. We synthesized vitamin K2 analogues with hydroxyl or phenyl groups at the ω-terminal of the side chain. The up-
regulation of SXR-mediated transcription of the target gene by the analogues was dependent on the length of the side chain and the
hydrophobicity of the ω-terminal residues. Phenyl analogue menaquinone-3 was as active as the known SXR ligand rifampicin.
’ INTRODUCTION
homologues are confined to only the isoprene units.14 To evaluate
the role of the side chain of vitamin K, we first investigated SXR-
mediated transcriptional activity of new vitamin K2 analogues in
comparison with vitamin K2 homologues. We anticipated that
the introduction of a hydrophilic or hydrophobic group at the ω-
terminal position of vitamin K homologues (Figure 2) would
result in significant changes in the activity of the analogues. Some
of the new analogues might have more potent activity compared
with the natural homologues. At the same time, we hoped to gain
insights into the mechanism that converts vitamin K analogues
to MK-4 if the new compounds were useful as substrates. In this
study, we report the synthesis of new vitamin K analogues and
we present the results of their effects on SXR-mediated gene
transcription.
Vitamin K is a well-known cofactor for the γ-carboxylation
reaction that converts specific glutamic acid residues to γ-carbox-
yglutamic acid residues in proteins related to blood coagulation
and bone formation.1 Vitamin K has two major homologues: the
plant-derived vitamin K1 (1) (phylloquinone, PK) and the bacter-
ium-derived vitamin K2 (2) (menaquinone-n, MK-n) (Figure 1).2
Recently, additional biological actions of menaquinone-4 (MK-4)
that are independent of the vitamin K-dependent γ-carboxyla-
tion reaction have been reported.3ꢀ5 One of important actions
is the activation of the steroid and xenobiotic receptor (SXR)
(also known as PXR,6 PAR,7 NR1I2) to regulate transcription of
extracellular matrix-related genes that may contribute to collagen
assembly.8 MK-4 and known SXR ligands rifampicin (RIF) and
hyperforin up-regulated the target gene, CYP3A4, and other bone
markers such as alkaline phosphatase and MGP.9 Those findings
indicated that MK-4 affected bone homeostasis by acting on
γ-carboxylation and by modulating MK-4-dependent transcrip-
tional regulation. Furthermore, there is consistent evidence that
other dietary vitamin K homologues can be converted into MK-
4.10 We recently confirmed that MK-4 was converted from
dietary PK and then was accumulated in various tissues at high
concentrations.11 In certain tissues containing high concentra-
tions of lipids, such as brain tissue, MK-4 would be a preferred
form of vitamin K.
’ CHEMISTRY
Synthesis of Vitamin K Analogues. Menaquinones 3ꢀ6
were generous gifts of Eisai Co., Ltd., and the ω-hydroxylated
vitamin K analogues 7ꢀ9 were synthesized according to our
previously reported method.15,16 The requisite analogues 10 and
11, with phenyl groups introduced at the ω-terminal position,
were obtained by the synthetic method as shown in Scheme 1.
Geranyl acetate (12a) and farnesyl acetate (12b) served as
starting materials for 10 and 11 and were modified in a two-step
procedure to introduce the tetrahydropyranyl (THP) ether, pro-
viding 13a and 13b.15,16 The acetate was hydrolyzed with 1 N
NaOH (aq) to give side chain structures 14a and 14b in
quantative yield. Conversion of the terminal THP group into
This background information prompted us to explore the
biological activities of new vitamin K analogues. To develop new
analogues, we focused on modification of the side chain moiety.
No studies have referred to the biological role of the side chain
part of vitamin K even though many reports have described the
biological action of PK or MK-4.3ꢀ5,12,13 We predicted that the
side chain should play an important role for the various biological
activities, since the structural differences among vitamin K
Received: January 11, 2011
Published: May 11, 2011
r
2011 American Chemical Society
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|

Journal of Medicinal Chemistry
BRIEF ARTICLE
Scheme 1. Synthesis of 10 and 11a
Figure 1. Structures of vitamin K homologues: phylloquinone (1) and
menaquinones (2).
a Reagents and conditions: (a) two steps according to refs 15 and 16; (b)
1 N NaOH (aq), quant; (c) PhMgBr, CuI, 75ꢀ85%; (d) 2-methyl-1,
4-naphthoquinone, Na2S2O4, Et2O, quant; (e) 15a or 15b, BF3 Et2O,
3
55ꢀ65%.
Figure 2. Menaquinones and their analogues.
the phenyl group by Grignard reaction by a reported procedure17
gave 15a and 15b in good yields.
We first tried to couple 15a or 15b with 1-acetoxy-2-methyl-
4-naphthalenol in the presence of BF3 Et2O according to a
3
reported procedure,16 but the resultant derivatives were quite
unstable and easily decomposed. Therefore, we adopted another
coupling method.14 The 1,4-naphthoquinone (16) was vigor-
ously stirred with a 10% sodium hydrosulfite (aq) solution and
Et2O to form the hydroquinone derivative, to which an amount
of 2 equiv of isoprene unit 15a or 15b was successively coupled in
Figure 3. Transcriptional activity with a one-hybrid luciferase assay
with SXR-GAL4. HepG2 cells were treated with new vitamin K2
analogues and natural homologues at 5.0 ꢁ 10ꢀ6 M. The histogram
data are expressed as the mean obtained from three independent
experiments. The error bars indicate the SD. Significant difference is
as follows: /, p < 0.05; //, p < 0.01; ##, p < 0.01 (by Student’s t test).
the presence of a catalytic amount of BF3 Et2O. Finally, the
3
resulting hydroquinone derivatives were immediately oxidized to
quinone under atmospheric conditions. Thus, the desired vita-
min K analogues 10 and 11 were obtained in 55ꢀ65% yield from
the oxidation of the quinone, respectively. When an isoprene unit
in the side chain part was shorter than geraniol, the chemical yield
was not satisfactory. Hence, in this study we were unable to
obtain the phenyl analogue menaquinone-1, which had a phenyl
group at the ω-terminal position of 3.
SXR, while the CYP3A4 promoter assay evaluated SXR-
mediated transcription.
To evaluate the effects of ligand binding, we tested all the
analogues in the SXR-GAL4 assay at 5.0 ꢁ 10ꢀ6 M (Figure 3).
For purposes of comparison, samples treated with DMSO or
EtOH served as baseline, untreated controls, while the sample
treated with RIF, a well-known ligand for SXR, served as a positive
control.19 Among the natural vitamin K homologues, the tran-
scriptional activity of 4, 5, and 6 exceeded that of 3 in a manner
that appeared related to the length of the side chain. For the
analogues that contained a hydroxyl group in the side chain, 7, 8,
and 9, almost no enhancement in ligand binding activity occurred.
On the other hand, the phenyl analogues 10 and 11 exhibited
extremely strong activity in comparison with 4 and 5; 11 had
activity comparable to that for RIF. In separate studies, we
confirmed that the transcriptional activity as determined by
SXR-GAL4 assay showed dose dependency (data not shown).
Similar tendencies were exhibited in the transcriptional activ-
ity of the CYP3A4 promoter as shown in Figure 4. Apparent
activity of 3ꢀ6 gradually increased with respect to the length of
’ RESULTS
Evaluation of Transcriptional Activities. All the synthetic
ligands and the vitamin K homologues were tested in assays that
measured the SXR-mediated transcriptional activity. Luciferase-
based assays of SXR-GAL4 and CYP3A4 promoter were carried
out with HepG2 human hepatocellular carcinoma cells. SXR is
well-known to control the gene expression of CYP3A4. The cells
were transfected with pM-SXR, pGVP2-GAL2 luciferase reporter
vector, and pRL-CMV using Lipofectamine18 in the SXR-GAL4
assay. On the other hand, the cells were treated with pcDNA3.1-
FLAG-SXR, pGL4.10-CYP3A4pro luciferase reporter vector,
and pRL-CMV using Lipofectamine in the CYP3A4 promoter
assay. The SXR-GAL4 assay measured the binding affinity to
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Journal of Medicinal Chemistry
BRIEF ARTICLE
Figure 4. Transcriptional activity with luciferase assay including SXRE
with CYP3A4 promoter. HepG2 cells were treated with new vitamin K2
analogues and natural homologues at 1.0 ꢁ 10ꢀ6 M. The histogram data
indicate the mean obtained from three independent experiments. The
error bars indicate the SD. Significant difference is as follows: ///,
p < 0.001; ###, p < 0.001 (by Student’s t test).
Figure 5. Docked model of 11 bound to SXR (PXR) model (PDB code
1M13, displayed as purple ribbon). 11 is displayed as sticks colored by
atom type, while Leu209 is depicted as atom type colored CPK models.
the side chain, and 7, 8, and 9 showed higher induction activity
than vehicle controls but slightly lower than 6. Again, 10 and 11
had the highest activity among the analogues, with 11 showing
comparable activity with the known substrate RIF.
RXR and binding to SXREs. With respect to ligand binding
(Figure 3), the activity of natural vitamin K2 homologues 3ꢀ6
increased in a manner dependent on the length of side chain moiety,
being greatest with 6. Hydroxylated 7, 8, and 9 exhibited only weak
activity. Among all the phenyl analogues, 10 and 11 showed the
most potent activity, with 11 exhibiting strong activity compar-
able to that of the established SXR ligand refampicin.
Similar tendencies were noted in the assay that evaluated
heterodimerization and binding to SXREs (Figure 4). Compounds
10 and 11 effectivelyincreasedtheexpressionof a well-characterized
SXR target gene, CYP3A4, in a human hepatocyte cell line,
compared with other analogues.
Since the SXR-GAL4 assay measured SXR ligand-binding
domain (LBD) driven transcription, we then conducted molec-
ular modeling of 11, exhibiting potent transcriptional activities,
with LBD of SXR. Docking study indicated an additional hydro-
phobic interaction between the ω-terminal phenyl group of 11
and a part of constituent amino acid (Leu209) of SXR compared
with other analogues.
Docking Studies. To better understand the binding modes of
10 and 11 to SXR at the atomic level, we performed molecular
docking studies on 11 with SXR using the ASEDock docking
program, part of the MOE suite (see Computational Details). The
docking results positioned 11 within the SXR binding pocket,
and the compound with the lowest binding energy displayed an
affinity of ꢀ29.1 kcal/mol. In this model, the side chain part and
the naphthalene moiety of 11 showed hydrophobic interactions
with Leu209, Leu240, Met243, Phe281, Phe288, Trp299, Met323,
Leu411, and Met246. Especially, a strong interaction between
Leu209 andthe phenyl group of the side chain moiety was observed
(Figure 5). Moreover, the carbonyl groups exerted hydrophilic
interaction with Ser247, Gln285, and His407, although no hydro-
gen bonding was exhibited, providing the complex with an
increased predicted stability fully compatible with the experi-
mental biological assays. The binding mode of 10 was similar to
that of 11.
We considered that the ability of the ligands to be absorbed
into cells also affects transcriptional activities. Since uptake of the
ligands into cells is generally proportional to lipophilicity, intro-
duction of a phenyl group to the molecule would enhance the
absorption of 10 and 11. In contrast, introduction of a hydrophilic
group to the molecule, such as for 7ꢀ9, would reduce the
absorption to cells. Thesefindings suggest that vitamin K analogues
with modification at the ω-terminal group can leadto stronger SXR
agonists that are more potent than known SXR-ligand such as
Taxol, RIF, hyperforin, phenobarbital, etc.
Currently, clinical applications of vitamin K include use of MK-
4 as an effective treatment for osteoporosis and preventing
fractures, and it is frequently prescribed for osteoporosis in
Japan.22 PK has been used as a therapeutic agent for vitamin K
deficiency syndromes such as hypoprothrombinemia in newborn
babies and in antibiotic-treated patients.23,24 The daily dosage of
MK-4 for osteoporotic patients is 45 mg, and the body concen-
tration corresponds to 10ꢀ6 M. Moreover, the tissue levels are
much higher (>10ꢀ5 M).25,26 These concentrations are well
correlated with those used in our in vitro transcriptional assay.
Recent studies have demonstrated that SXR also up-regulated
the expression of osteoblastic markers and the factors related
’ DISCUSSION
In the present report, we describe the synthesis and the pharma-
cological profile related to SXR-mediated transcriptional activities of
new vitamin K analogues modified at the ω-terminal group.
SXR is a master gene orchestrating the expression of a large
family of genes involved in uptake, metabolism, and disposal of a
number of endo- and xenobiotics, including drugs, bile acids,
steroid hormones, and metabolic intermediates in mammalian
cells.20 Following ligand binding, SXR forms a heterodimer
with the retinoid X receptor (RXR) that binds to SXR response
elements (SXREs), located in the 50-flanking regions of SXR
target genes, resulting in their transcriptional activation.21
To determine if vitamin K analogues have a role in regulating
SXR, weevaluatedthe transcriptionalactivityofthe new analogues
modified at the ω-terminal group by two luciferase-based assay
methods. The one-hybrid luciferase assay with SXR-GAL4 per-
mitted us to examine only the effects of ligand-binding affinity to
SXR in the transcription and excluded any influence of hetero-
dimerization with RXR and binding to SXREs. The second
luciferase-based assay including SXRE with CYP3A4 promoter
allowed us to examine the influences of heterodimerization with
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Journal of Medicinal Chemistry
BRIEF ARTICLE
to bone formation such as a small leucine-rich proteoglycan
named tsukushi, an extracellular matrix protein matrilin-2, CD14
antigen,27 and Msx2 gene.28 Therefore, strong SXR agonists
based on vitamin K analogues may be a source for entirely new
therapeutic drugs for osteoporosis in aspects of transcriptional
activity and coenzyme activity of γ-carboxylative action.
Cloning and Construction of cDNAs. Human SXR expression
vector (pcDNA3.1-FLAG-SXR) was generated by PCR using human
genomic DNA as templates, respectively, and then inserted in-frame to
pcDNA3.1(þ) vector (Invitrogen) atEcoRI and XhoI sites. The CYP3A4
luciferase reporter plasmid pGL3-CYP3A4pro was constructed in our
laboratory. The CYP3A4 promoter, from base pairs ꢀ362 to þ53, and
a distal enhancer module of CYP3A4 promoter, from base pairs ꢀ7876
to ꢀ7208, were generated by polymerase chain reaction (PCR) using
the DNA template isolated from the human genome DNA. These
products were then subcloned into the pGL4.10 vector, a promoterless
luciferase reporter vector, at the NheI and BamHI sites. Finally, pGL4.10-
CYP3A4pro, a CYP3A4 luciferase reporter plasmid containing ꢀ7876
to ꢀ7208 and ꢀ362 to þ53 bp of the CYP3A4 proximal promoter, was
prepared.18
Luciferase Assay. Human hepatoma cell line HepG2 cells were
maintained in Eagle’s modified medium (Nakalai Tesque) supplemen-
ted with 1% penicillin, 1% streptomycin, and 10% fetal calf serum (FCS)
(Gibco BRL). Luciferase-based assay of SXR-GAL4 was performed
using HepG2 cells (2 ꢁ 105 cells/well on six-well plates) transfected
with 0.1 μg of pM-SXR (constructed in our laboratory), 1.0 μg of
pGVP2-GAL2 (extended from Prof. Keiichi Ozono, Osaka University),
and 0.05 μg of pRL-CMV (Promega) using Lipofectamine (Invitrogen).
In a similar way, luciferase assay of CYP3A4 promoter was conducted
using HepG2 cells transfected with 0.25 μg of pcDNA3.1-FLAG-SXR,
0.25 μg of pGL4.10-CYP3A4pro luciferase reporter vector (Toyo Ink
Co., Ltd.), and 0.1 μg of pRL-CMV using Lipofectamine. Twenty-four
hours after transfection, cells were treated with RIF (Nakalai Tesque,
Kyoto, Japan), vitamin K compounds, or vehicles (EtOH, DMSO) for
48 h in fresh media. The amount of each sample was 2 μL against 2 mL
of culture medium. Luciferase activities were determined by a Lumat
LB9507 luminometer (Berthold Technologies) using the dual-luciferase
assay system (Toyo Ink). Firefly luciferase activity was normalized to
that of Renilla luciferase, which was used as a transfection control. The
experiments were repeated three times with similar results.
’ CONCLUSION
We report for the first time the synthesis of new vitamin K2
analogues with ω-terminalmodifications, and we demonstrate their
effects on the induction of transcription. The phenyl derivative 11
exhibited the most potent activity in separate assays that eval-
uated ligand binding and the role of heterodimerization in the
induction of transcription, and this compound was as effective as
the known substrate RIF. The compounds would be useful not
only in the development of new biologically active agents but also
in the evaluation of still unknown biological roles of vitamin K.
’ EXPERIMENTAL SECTION
High-resolution ESI-MS (ESI-HRMS) was performed with a Micro-
mass Q-TOF mass spectrometer. 1H NMR spectra were recorded at 500
MHz and 13C NMR spectra were recorded at 125 MHz using CDCl3 as a
solvent unless otherwise specified. Chemical shifts are given in parts per
million (δ) using tetramethylsilane (TMS) as the internal standard.
Column chromatography was carried out on silica gel 60 (70ꢀ230
mesh), and preparative thin layer chromatography (TLC) was run on
silica gel 60F254. Unless otherwise noted, all reagents were purchased
from commercial suppliers.
Synthesis of 10 and 11. Toa solution ofmenadion (16) (K3) (150
mg, 871 μmol) in ether (20 mL) was added a 10% Na2S2O4 aqueous
solution (20 mL), and the mixture was stirred vigorously at 30 °C for 1 h
under argon. After the yellow ether layer turned colorless, the mixture
was extracted with AcOEt (50 mL ꢁ 3). The combined organic layer was
washed with brine (50 mL ꢁ 3), dried over MgSO4, and concentrated to
afford crude hydroquinone. The residue was immediately dissolved in
AcOEt (1 mL) and dioxane (1 mL). Then 15a (396 mg, 1.72 mmol) or
15b (519 mg, 1.72 mmol) and boron trifluoride ether complex (50 μL)
were added. The mixture was stirred at 70 °C for 3 h under argon and
cooled to room temperature. The mixture was poured into iceꢀwater and
extracted with AcOEt (50 mL ꢁ 3). The combined organic layer was
washed with water (100 mL) and brine (100 mL), dried over MgSO4,
and concentrated. The residue was purified by preparative TLC on silica
gel (n-hexane/AcOEt = 20:1) to afford 10 or 11 as a yellow oil (10, 218
mg, 65%; 11, 217 mg, 55%). Data for 10: 1H NMR (500 MHz, CDCl3)
δ 8.10ꢀ8.07 (2H, m), 7.70ꢀ7.68 (2H, m), 7.28ꢀ7.11 (5H, m), 5.17
(1H, t, J = 7.0 Hz), 5.03 (1H, t, J = 7.0 Hz), 3.38 (2H, d, J = 7.0 Hz), 3.21
(2H, s), 2.19 (3H, s), 2.14ꢀ2.10 (2H, m), 2.05ꢀ2.02 (2H, m), 1.80
(3H, s), 1.50 (3H, s); 13C NMR (125 MHz, CDCl3) δ 185.5, 184.5,
146.1, 143.4, 140.4, 137.4, 134.6, 133.4, 133.3, 132.19, 132.15, 128.8,
128.1, 126.3, 126.2, 126.0, 125.9, 119.3, 46.2, 39.6, 26.5, 26.0, 16.4, 15.8,
12.7; ESI-HRMS (M þ Hþ) m/z calcd for C27H28O2 385.2167. Found
Computational Details. Calculations were performed on a Dell
Precision T3500 workstation. Conformational analyses o ligands, mena-
quinones, and their analogues were conducted using MOE29 and
MMFF94x molecular mechanics force field. Docking of the minimized
energy structure of 11 into the crystal structure of the human pregnane X
receptor in complex with hyperforin (PDB code 1M13)30 was carried out
with the ASEDock docking program, part of the MOE suite.
’ ASSOCIATED CONTENT
Supporting Information. 1H and 13C NMR spectra of 10
S
b
and 11; HPLC data of 3ꢀ11 used in biological assays; synthesis
procedures of 15a and 15b. This material is available free of
charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
*Phone: þ81-78-441-7564. Fax: þ81-78-441-7565. E-mail:
t-okano@kobepharma-u.ac.jp.
1
385.2165. Data for 11: H NMR (500 MHz, CDCl3) δ 8.09ꢀ8.07
(2H, m), 7.69ꢀ7.67 (2H, m), 7.27ꢀ7.24 (2H, m), 7.18ꢀ7.14 (3H, m),
5.18 (1H, t, J = 7.0 Hz), 5.06 (1H, t, J = 7.0 Hz), 5.02 (1H, t, J = 7.0 Hz),
3.37 (2H, d, J = 7.0 Hz), 3.24 (2H, s), 2.19 (3H, s), 2.09ꢀ2.04 (4H, m),
2.01ꢀ1.95 (4H, m), 1.80 (3H, s), 1.56 (3H, s), 1.49 (3H, s); 13C NMR
(125 MHz, CDCl3) δ 185.5, 184.5, 146.2, 143.4, 140.5, 137.5, 135.0,
134.2, 133.4, 133.3, 132.19, 132.15, 128.8, 128.1, 126.4, 126.3, 126.2,
125.8, 124.1, 119.1, 46.2, 39.7, 39.6, 26.6, 26.5, 26.0, 16.4, 16.0, 15.7,
12.7; ESI-HRMS (M þ Hþ) m/z calcd for C32H36O2 453.2793. Found
453.2795.
’ ACKNOWLEDGMENT
We are grateful to Dr. Atsuko Takeuchi and Dr. Chisato Tode
for the spectroscopic measurements at Kobe Pharmaceutical
University, Japan. We also thank Eisai Co., Ltd. for generously
providing the intermediate of the vitamin K analogues. Y.S.
acknowledges Takeda Science Foundation. This study was
supported in part by a Grant-in-Aid for Scientific Research
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BRIEF ARTICLE
(KAKENHI) (C) (Grant No. 20590113 to Y.S.) from the Japan
Society for the Promotion of Science.
conversion of vitamin K analogues into menaquinone-4. Substrates
essential to the synthesis of menaquinone-4 in cultured human cell lines.
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’ ABBREVIATIONS USED
SXR, steroid and xenobiotic receptor; PXR, pregnane X recep-
tor; PAR, proteinase-activated receptor; CYP3A4, cytochrome
P450 3A4; MGP, matrix Gla protein; RXR, retinoid X receptor;
SXRE, SXR response element; MOE, Molecular Operating En-
vironment; ESI-HMRS, electrospray ionization high-resolution
mass spectrometry; TMS, tetramethylsilane; TLC, thin layer
chromatography; PCR, polymerase chain reaction; PDB, Pro-
tein Data Bank
(17) Mechelke, M. F.; Wiemer, D. F. Synthesis of farnesol analogues
through Cu(I)-mediated displacements of allylic THP ethers by
Grignard reagents. J. Org. Chem. 1999, 64, 4821–4829.
(18) Tirona, R. G.; Lee, W.; Leake, B. F.; Lan, L. B.; Cline, C. B.;
Lamba, V.; Parviz, F.; Duncan, S. A.; Inoue, Y.; Gonzalez, F. J.; Schuetz,
E. G.; Kim, R. B. The orphan nuclear receptor HNF4alpha determines
PXR- and CAR-mediated xenobiotic induction of CYP3A4. Nat. Med.
2003, 9, 220–224.
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