Assessment of Drug Drug Interaction For Silymarin

Assessment of drugdrug interaction for silymarin
Johannes Doehmer
a,
*
, Bernhard Tewes
a
, Kai-Uwe Klein
a
, Kristin Gritzko
a
,
Holger Muschick
a
, Ulrich Mengs
b
a
GenPharmTox Biotech AG, Fraunhofer Str. 9, D-82152 Planegg/Martinsried, Germany
b
Madaus GmbH, Colonia-Allee 15, D-51067 Cologne, Germany
Received 29 May 2007; accepted 30 November 2007
Available online 8 December 2007
Abstract
Silymarin was assessed for drugdrug interaction by permeability studies with Caco-2 cells, for cytochrome P450 induction with
human primary hepatocytes and for cytochrome P450 inhibition with human liver microsomes. Studies with Caco-2 cells revealed no
interference of silymarin with the permeability of nifedipine. Silymarin did not induce cytochromes P450 2C9 and 3A4 at concentrations
of 0.1; 1; and 100 lM, measured as silibinin. The inhibitory eect was tested on the nine major cytochromes P450 1A2, 2A6, 2B6, 2C8,
2C9, 2C19, 2D6, 2E1, and 3A4 at concentrations of 1 and 100 lM silymarin. At 1 lM concentration no or negligible inhibition of cyto-
chromes P450 1A2, 2A6, 2B6, 2C8, 2C9, and 2E1, minor inhibition of 3A4 (<20%), and moderate inhibition of 2C19 and 2D6 (<40%)
were observed. Inhibition constant K
i
of silymarin was determined for cytochromes P450 3A4 with 12 lM, 2C19 with 2 lM, and 2D6
with 12 lM. Only at the high concentration of 100 lM silymarin, inhibition at >50% of the cytochromes P450 2B6, 2C8, 2C9, 2C19, 2D6,
and 3A4 was observed, and no or moderate inhibition was for the cytochromes P450 1A2, 2A6, and 2E1. However, in view of the clin-
ically relevant plasma concentration of approx. 0.2 lM measured as silibinin, it is evident that there is no drugdrug interaction problem
with silymarin.
2007 Published by Elsevier Ltd.
Keywords: Silymarin; Silibinin; Cytochrome P450; Induction; Inhibition; Caco-2; Drugdrug interaction
1. Introduction
Silymarin, a avonoid complex, is the main constituent
with 7080% of the extract from seeds of the milk thistle
(Silybum marianum) and has been used for decades as a
herbal remedy and as a hepatoprotectant in therapy of
acute and chronic liver diseases (Flora et al., 1998; Gazak
et al., 2007). Silymarin consists predominantly of up to
60% of silibinin (Fig. 1), but also contains silycristin, silydi-
anin, isosilibinin and other closely related avonolignans
(Weyhenmeyer et al., 1992).
Recently, inhibition studies with silymarin or silibinin
on cytochromes P450 gave hints for potential drugdrug
interaction particularly for the inhibition of oxidation of
nifedipine (Beckmann-Knopp et al., 2000; Zuber et al.,
2002; Sridar et al., 2004).
To assess the potential of silymarin for drugdrug inter-
action several in vitro studies were performed, i.e. interfer-
ence of silymarin with nifedipine permeability in Caco-2
cells, induction of cytochromes P450 2C9 and 3A4 by
silymarin in human primary hepatocytes, and for cyto-
chrome P450 inhibition in human liver microsomes. Guide-
lines were observed in the design and performance of these
studies (Committee for Proprietary Medicinal Products,
1997; Guidance for Industry, 2006; BfArM, 2004).
2. Materials and methods
2.1. Chemicals
Silymarin (batch # 91258) was provided by Madaus
GmbH, Cologne, Germany. All silymarin concentrations
0887-2333/$ - see front matter 2007 Published by Elsevier Ltd.
doi:10.1016/j.tiv.2007.11.020
*
Corresponding author. Tel.: +49 89 895559 0; fax: +49 89 895559 18.
E-mail address: johannes.doehmer@genpharmtox.de (J. Doehmer).
www.elsevier.com/locate/toxinvit
Available online at www.sciencedirect.com
Toxicology in Vitro 22 (2008) 610617
tested correspond to the main constituent silibinin. Nifedi-
pine was purchased from Sigma Biochemikalien und
Reagenzien, Deisenhofen, Germany; proponalol from
Aldrich, ranitidine from ICN. Phenobarbital and diclofe-
nac were purchased from Sigma, rifampicin from Fluka,
testosterone from Applichem.
2.2. Hepatocytes and microsomes
Cryopreserved human heptatocytes were received from
InVitroTechnologies, Inc., article # M00995-P, lot #
BDF. Human liver microsomes containing 20 mg/ml
microsomal protein were purchased from XenoTech,
LLC. Art # H0610, lot # 0310286. Liver microsomes were
a pool from 50 donors, mixed gender. Total cytochrome
P450 content was 0.373 nmol/mg protein. The activities
as measured by marker reactions were 1700 pmol 4
0
-
hydroxylated diclofenac/min/mg protein for cytochrome
P450 2C9, and 3000 pmol 6b-hydroxylated testosterone/
min/mg protein for cytochrome P450 3A4.
2.3. Caco-2 permeability studies
Preplated Caco-2 cells were received from AdvanCell,
Barcelona, Spain, with a transepithelial electrical resistance
of 3098 and 3663.88 X cm
2
, respectively. Incubations in
DMEM/10% fetal calf serum with the test item and the ref-
erence permeability controls propanolol and ranitidine
were performed in a 24 well plate at 37 C, 5% CO
2
and
approx. 95% humidity on an orbital shaker (approx.
50 rpm). After 60 min of incubation, the samples (incuba-
tion medium) from the apical and basolateral side were
removed and analyzed via LCMS for silibinin and the per-
meability controls.
2.4. Cytochrome P450 induction studies
As much as 2.55 10
5
hepatocytes per well of a 24-well
plate were seeded in 500 ll of HIM complete medium and
incubated for 48 h at 37 C, approx. 5% CO
2
and approx.
95% humidity. After 24 h, the media was renewed. The
hepatocytes were incubated for further 48 h with 500 ll
of the respective dilutions of test items, positive and nega-
tive controls in HIM complete medium. After 24 h the
media containing the test items, positive and negative con-
trols, were renewed. Hepatocyte incubations containing the
respective concentration (0.5% (v/v)) of the solvent DMSO
but no test item served as negative controls (NC). Hepato-
cyte incubations containing no test item but the respective
reference inducers and identical concentration of the sol-
vent served as positive controls (PC). A stock solution of
silymarin at 20 mM was freshly prepared in 100% DMSO
on the day of the start of incubations. The stock solution
of test item was diluted with HIM culture medium to the
nal concentrations of 0.1; 1; and 100 lM. The nal refer-
ence inducer concentrations were 1 mM for phenobarbital,
and 10 lM for rifampicin. The nal concentrations of the
marker substrates were 50 lM for diclofenac, and
200 lM for testosterone. Three parallel incubations per
concentration of test item as well as per negative or per
positive control were tested for each of the marker reac-
tions. After the induction phase, the media were removed
and replaced by 500 ll/well S-HIMDEX,PS containing
3 mM salicylamide. After 10 min incubation at 37 C, the
media were replaced by 350 ll/well culture medium con-
taining the respective concentration of the specic sub-
strate for the enzyme marker reactions. After 3 h
incubation with the respective marker substrates at 37 C
the supernatants of all samples were collected in 2 ml tubes
which contained 350 ll of ice-cold acetonitrile. Precipitated
protein were pelleted by centrifugation for 10 min at 4 C
and ca. 14,000 rpm and 200 ll of the supernatants were
transferred to a 96-well microtiter plate for analysis by
LC/MS for the detection of the marker metabolites, 4-
OH-diclofenac and 6b-OH-testosterone, respectively.
2.5. Cytochrome P450 inhibition studies
CYP isoenzyme specic marker reactions were con-
ducted in the presence of 1 or 100 lM silymarin at
37 1 C in the presence of 2 mM NADPH, 3.3 mM
MgCl
2
, and 0.5 mg microsomal protein/ml in buer in a
96 well plate in a total volume of 200 ll. The marker sub-
strate concentrations in the incubation mixtures were cho-
sen to be close to their apparent K
m
. The incubations were
started by the addition of marker substrate after preincuba-
tion with silymarin or reference inhibitor for 510 min. The
incubation was stopped after 30 1 min by the addition of
150 ll of 43% (v/v) acetonitrile, centrifuged for 10 min at
4 C and ca. 3000 g, and the supernatant was transferred
to a new96-well microtiter plate for marker reaction specic
analysis. Marker reactions containing no test item but iden-
tical concentration of the respective solvent type of test item
incubations and FDA approved reference inhibitor control
(PC) served as respective negative control (NC). Marker
reactions containing no silymarin but identical concentra-
tions of the respective solvent type as well as a marker reac-
tion specic reference inhibitor served as positive controls
(PC). The reference inhibitor concentrations in the incuba-
tion mixtures were chosen to be close to their apparent
IC50 for the respective marker reaction. All incubations
were tested in triplicate. The nal concentrations of the mar-
ker substrate in the individual enzyme assays were as fol-
O
O
O
O
OH
OH
OCH
3
OH
CH OH
2
HO
Fig. 1. Chemical structure of silibinin.
J. Doehmer et al. / Toxicology in Vitro 22 (2008) 610617 611
lows: 5 lM 7-ethoxyresorn for CYP1A2; 5 lM coumarin
for CYP2A6; 200 lM S-mephenytoin for CYP2B6; 10 lM
paclitaxel for CYP2C8; 10 lM diclofenac for CYP2C9;
50 lM S-mephenytoin for CYP2C19; 20 lM bufuralol for
CYP2D6; 50 lM chlorzoxazone for CYP2E1; and 100 lM
testosterone for CYP3A4. The nal concentrations of the
reference inhibitors were as follows: 25 lM furafylline for
CYP1A2; 0.75 lM 8-methoxypsoralen for CYP2A6;
75 lM triethylenethiophosparamide for CYP2B6; 25 lM
ketoconazole for CYP2C8; 2 lM sulfaphenazole for
CYP2C9; 7.5 lM omeprazole for CYP2C19; 0.3 lM quini-
dine for CYP2D6; 30 lM diethyldithiocarbamate for
CYP2E1; and 0.15 lM ketoconazole for CYP3A4.
2.6. K
i
determination for cytochromes P450 2C19, 2D6, and
3A4
For further characterization of the inhibition of the S-
mephenytoin 4
0
-hydroxylation (marker reaction for
CYP2C19), bufuralol-hydroxylation (marker reaction for
CYP2D6), and testosterone 6b-hydroxylation (marker
reaction for CYP3A4) by four dierent concentrations of
silymarin: 0.1; 1; 10; and 100 mM (corresponding to the
main constituent silibinin) and six dierent concentrations
of the respective marker substrates: 25; 50; 125; 250; 500;
and 1000 lM S-mephenytoin; 10; 20; 50; 100; 200; and
400 lM bufuralol; 50; 100; 250; 500; 1000; and 2000 lM
testosterone were chosen for the determination of the K
i
values. Incubation conditions were the same as described
for the inhibition studies.
2.7. Analytics
Formation of resorun was measured photometrically at
590 nm with Fluoroskan Ascent (Labsystems). Formation
of all other marker substrate metabolites was measured
by LC/MS (AgilentSeries 1100 (Agilent or Waters Alliance
HT2790)/ Quattro Micro (Waters)). The marker metabolite
7-hydroxycoumarin for cytochrome P450 2A6 was chro-
matographed on Luna 3u C18 (Phenomenex) at 30 C with
100% water (mobile Phase A), 100% acetonitrile (mobile
Phase B), 2% formic acid in water (mobile Phase C) at a
ow rate of 0.4 ml/min, and detected in ESI+ mode with
transition (m/z) of 163.0 > 107.0. The marker metabolites
nirvanol for cytochrome P450 2B6 and 4
0
-hydroxymephe-
nytoin for cytochrome P450 2C19, were chromatographed
on Luna 3u C18 (Phenomenex) at 30 C with 100% water
(mobile Phase A), 100% acetonitrile (mobile Phase B), 2%
formic acid in water (mobile Phase C) at a ow rate of
0.4 ml/min, and detected in ESI+ mode with transition
(m/z) of 205.0 > 134.0 for nirvanol and with transition
(m/z) of 235.0 > 150.2. The marker metabolite 6a-hydroxy-
paclitaxel for cytochrome P450 2C8 was chromatographed
on Luna 3u C18 (Phenomenex) at 30 C with 100% water
(m/z) of 870.2 > 524.9. The marker metabolite 4
0
-hydrox-
ydiclofenac for cytochrome P450 2C6 was chromato-
graphed on Luna 3u C18 (Phenomenex) at 30 C with
100% water (mobile Phase A), 100% acetonitrile (mobile
Phase B), 2% formic acid in water (mobile Phase C) at a
ow rate of 0.4 ml/min, and detected in ESI mode with
SIR (m/z) of 265.9. The marker metabolite hydroxybufura-
lol for cytochrome P450 2D6 was chromatographed on
Luna 3u C18 (Phenomenex) at 30 C with 100% water
(m/z) of 278.2 > 186.1. The marker metabolite 6-hydroxy-
chlorzoxazone for cytochrome P450 2E1 was chromato-
graphed on Luna 3 u C8 (Phenomenex) at 30 C with
water: acetonitrile (75:25), 0.375% H
3
PO
4
(mobile Phase),
at a ow rate of 0.5 ml/min, and detected by absorption
at 298 nm. The marker metabolite 6b-hydroxytestosterone
for cytochrome P450 3A4 was chromatographed on Luna
C8 (Phenomenex) at 30 C with 100% water (mobile Phase
A), 100% acetonitrile (mobile Phase B), 2% formic acid in
water (mobile Phase C) at a ow rate of 0.4 ml/min, and
detected in ESI+ mode with transition (m/z) of
305.0 > 287.0.
2.8. Data analysis
Data analysis of the CYP inhibition experiment was per-
formed using standard software: MS-EXCEL by Micro-
soft, Inc., SigmaPlot 8.02 for Windows, Enzyme
Kinetics 1.1 Module for Sigma Plot 2001 by SPSS, Inc.,
MassLynxTMVers. 3.5 and QuanLynxTM by Waters,
Ltd. The calibration curves were determined by calculation
of the regression line (y = ax + y0, least squares, weighted
1/y), and these calculations were performed using standard
software: Sigma Plot 8.02 for Windows by SPSS, Inc. The
correlation coecient, y-intercept, slope, and the data plot
are reported. From the measured signals (e.g. peak areas)
the reaction velocities for each concentration of marker sub-
strate and test item were determined (see Formula (1)) using
the reference metabolite calibration curves. As the samples
were stopped by the addition of 150 ll acetonitrile to
200 ll incubation mix, the dilution factor of 1.75 was used
to adjust the concentrations of the test item samples to the
calibration samples.
reaction velocity
dilution factor concentration of metabolite lM
incubation time min protein concentrationmg=ml 1000
1
612 J. Doehmer et al. / Toxicology in Vitro 22 (2008) 610617
The resulting data were tted using the Enzyme Kinetics
1.1 Module for Sigma Plot 2001 (SPSS, Inc.) to competi-
tive, non-competitive, uncompetitive, and mixed as well as
full and partial inhibition models. The ts were ranked
according to R
2
. Additionally, the AICc, Sy.x, and conver-
gence for each tting model are listed in Tables 35.
The Michaelis-Menten-, Lineweaver-Burk-, Eadie-Hof-
stee-, and Dixon-plots for the model with the highest R
2
that pass the Convergence criterion were generated. These
parameters, the plots as well as the resulting apparent K
i
are reported (Figs. 46).
3. Results
The Caco-2 cell permeability study revealed that silyma-
rin, measured for its major constituent silibinin, may be clas-
sied as to be of moderate permeability with an apparent
permeability coecient (P
app
) of 5.52 10
6
cm/s (Table
1). To assess for permeability interference, the permeability
of nifedipine in Caco-2 cells was determined in the absence
and presence of 10 and 50 lM silymarin (Fig. 2). Nifedipine
in the absence of silymarin had an apparent permeability
coecient (P
app
) of about 60 10
6
cm/s, and thus
appeared to be highly permeable. Permeability of nifedipine
Table 1
Caco-2 permeability of silymarin and control compounds
Sample Mean Papp SD (10
6
cm/s) Permeability
Silibinin 5.52 1.07 Medium
Propranolol 38.98 0.71 High
Ranitidine 0.05
a
Low
a
Single value.
Permeability of nifedipine in presence of silymarin
0.00
10.00
20.00
30.00
40.00
50.00
60.00
70.00
80.00
Sil 0 M Sil 10 M Sil 50 M
P
a
p
p

[
1
0
^
-
6

c
m
/
s
]

Fig. 2. Permeability of nifedipine in the presence and absence of
silymarin.
Table 2
Summary of cytochrome P450 induction
Sample Concentration (lM) Fold induction of cytochrome P450
2C9 3A4
PC
a
a
1.8 6.2
NC 1.0 1.0
Silymarin 0.1
b
0.8 1.0
Silymarin 1
b
0.7 0.9
Silymarin 100
b
c
0.3
a
PC: CYP2C9: phenobarbital 1 mM CYP3A4: rifampicin 10 lM.
b
Silymarin concentrations belong to the main constituent silbinin.
c
No marker metabolite could be detected and therefore no enzyme
activity could be determined.
Inhibitory Effect of Silymarin
-20
0
20
40
60
80
100
1A2 2A6 2B6 2C8 2C9 2C19 2D6 2E1 3A4
Cytochrome P450 Isoenzyme
I
n
h
i
b
i
t
i
o
n

[
%
]
1 M 100 M
Fig. 3. Inhibitory eects of silymarin on the major drug related
cytochromes P450.
Michaelis-Menten
[Substrate] ( M)
0 200 400 600 800 1000 1200
0
20
40
60
80
100
120
I =0
I = 0.1
I = 1
I = 10
I = 100
Eadie-Hofstee
Rate (pmol/mg/min)/[Substrate] ( M)
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8
0
20
40
60
80
100
120
I = 0
I = 0.1
I = 1
I = 10
I = 100
Fig. 4. Inhibition of cytochrome P450 2C19 by silymarin with S-

mephenytoin as substrate: (A) Michaelis-Menten plot; (B) Eadie-Hofstee
plot.
did not change in the presence of 10 or 50 lM silymarin
(Fig. 2).
The induction study on human primary hepatocytes
with 1 and 100 lM silymarin did not indicate any induction
of CYP2C9 or CYP3A4 (Table 2).
The inhibition study on human liver microsomes did not
indicate interference of 1 lM silymarin with the marker
reactions for the cytochromes P450 1A2, 2A6, 2B6, 2C8,
2C9, 2E1, and only minor or moderate inhibition with
cytochromes P450 2C19, 2D6, and 3A4 (Fig. 3).
To substantiate the nding on minor or moderate inhi-
bition of the cytochromes P450 2C19, 2D6, and 3A4, a
detailed enzyme kinetic study was performed (Figs. 46)
yielding inhibition constants K
i
for cytochromes P450
2C19 with 2 lM, 2D6 with 12 lM, and 3A4 with 12 lM
(Tables 35).
Inhibition ranging only between 50% and 100% was
observed in the presence of 100 lM silymarin for all cyto-
chromes P450 tested, except for cytochromes P450 2A6 and
2E1 (Fig. 3).
4. Discussion
Flavonoids are widely distributed in plants and are reg-
ularly consumed as vegetables, fruits, and beverages such
as tea and red wine. It is well known that avonoids may
interfere with the metabolism of drugs by induction or inhi-
bition of cytochromes P450 (Ubeda et al., 1995; Dai et al.,
1997; Fuhr, 1998). Thus, silymarin as a complex mixture of
avonoids may potentially cause drugdrug interaction.
Although the long term administration of silymarin is
considered safe with no or minimal adverse eects (Sridar
et al., 2004), inhibitory eects of silymarin on cytochromes
P450 indicated a potential for drug interaction (Beckmann-
Knopp et al., 2000; Sridar et al., 2004). To substantiate
these ndings, the potential of silymarin to interfere with
drugs was investigated in vitro on uptake, and induction
and inhibition of cytochromes P450.
The interference in uptake of silymarin with nifedipine
from the gut was considered in a clinical trial (Fuhr et al.,
Michaelis-Menten
[Substrate] ( M)
0 100 200 300 400 500
0
20
40
60
80
100
120
140
160
180
200
Eadie-Hofstee
0 2 4 6 8
0
20
40
60
80
100
120
140
160
180
200
I = 0
I = 0.1
I = 1
I = 10
I = 100
I = 0
I = 0.1
I = 1
I = 10
I = 100
Fig. 5. Inhibition of cytochrome P450 2D6 by silymarin with bufuralol as

substrate: (A) Michaelis-Menten plot; (B) Eadie-Hofstee plot.
Michaelis-Menten
[Substrate] ( M)
0 500 1000 1500 2000 2500
0
1000
2000
3000
4000
5000
6000
7000
Eadie-Hofstee
0 20 40 60 80 100 120
0
1000
2000
3000
4000
5000
6000
7000
I = 0
I = 0.1
I = 1
I = 10
I = 100

I = 0
I = 0.1
I = 1
I = 10
I = 100
Fig. 6. Inhibition of cytochrome P450 3A4 by silymarin with testosterone
as substrate: (A) Michaelis-Menten plot; (B) Eadie-Hofstee plot.
2007). Therefore, the Caco-2 assay was applied to check for
interference of silymarin with nifedipine. The results of the
Caco-2 assay clearly indicated no interference of silymarin
with nifedipine permeability (Fig. 2). Nifedipine by itself
is highly permeable with an apparent permeability factor
(P
app
) of about 60 10
6
cm/s, whereas silymarin had a
moderate P
app
of about 5 10
6
cm/s. (Table 1). The per-
meability of nifedipine was not changed in the presence of
10 and 50 lM silymarin (Fig. 2). This supports the results
fromthe above mentioned clinical trial that uptake of nifed-
ipine is not inuenced by silymarin at plasma concentra-
tions distinctly less than 1 lM, measured as silibinin
(Fuhr et al., 2007).
Induction of cytochrome P450 activity may cause drug
interaction. The drugs of concern to interfere with silymarin,
e.g. nifedipine, are substrates for cytochromes P450 2C9 and
Table 3
Determination of the K
i
of silymarin for CYP2C19 (S-mephenytoin 4
0
-hydroxylation)
Rank by R
2
Equation R
2
AICc Sy.x Con. K
i
(lM) SE
1 Mixed (partial) 0.980 269.3 4.74 Yes 2.19 0.33
2 Mixed (full) 0.980 267.0 4.71 Yes 2.19 0.32
3 Competitive (full) 0.978 273.4 4.93 Yes 1.61 0.14
4 Competitive (partial) 0.978 275.7 4.96 Yes 1.61 0.14
5 Noncompetitive (partial) 0.963 319.6 6.44 Yes 10.00 1.29
6 Noncompetitive (full) 0.963 318.0 6.43 Yes 10.74 0.96
7 Uncompetitive (full) 0.942 354.9 8.01 Yes 8.23 0.94
8 Uncompetitive (partial) 0.942 357.2 8.06 Yes 8.24 1.33
The best t results were obtained using a mixed (partial) or mixed (full) inhibition model. The resulting K
i
were 2.19 0.33 lM and 2.19 0.32 lM,
respectively. The parameters for these two inhibition models were calculated to be
Mixed (partial): Mixed (full):
V
max
: 104.2 V
max
: 104.2
K
m
: 67.2 K
m
: 67.2
K
i
: 2.2 K
i
: 2.2
a: 20.6 a: 20.6
b: 1.52E 10
m
V max
1
bI
aK
i

1
I
aK

1
Km
S
1
I
K
i

1
I
aK
i
0
@
1
A
m
V max
Km
S
1
I
K
i

1
I
aK

Table 4
i
of silymarin for CYP2D6 (bufuralol hydroxylation)
Rank by R
2
Equation R
2
AICc Sy.x Con. K
i
(lM) SE
3 Mixed (full) 0.942 439.7 11.40 Yes 18.09 5.92
The best t results were obtained using a mixed (partial) or noncompetitive (partial) inhibition model. The resulting K
i
were 11.65 3.22 lM and
17.87 2.95lM, respectively. The parameters for these two inhibition models were calculated to be
Mixed (partial): Noncompetitive (partial):
V
max
: 171.0 V
max
: 173.0
K
m
: 21.4 K
m
: 22.7
K
i
: 11.6 K
i
: 17.9
a: 1.76 b: 0.204
b: 0.23
m
V max
1
bI
aK
i

1
I
aK

1
Km
S
1
I
K
i

1
I
aK
i
0
@
1
A
m
V max
1
Km
S

1
I
K
i

1
Ib
K
i

0
@
1
A
3A4. This was tested for silymarinat concentrations of 0.1; 1;
and 100 lM for cytochromes P450 2C9 and 3A4. No induc-
tion was detected for these cytochromes P450 (Table 2).
Thus, cytochrome P450 induction at least for these cyto-
chromes P450 as a cause for drug interaction can be ruled
out for the concentrations tested. In addition, the results of
the induction experiments suggest that the higher concentra-
tions of silymarin actually decrease cytochrome P450 2C9
and 3A4 activities. Thus, even higher concentrations than
100 lM of silymarin which may be discussed for hepatic or
biliary concentrations of up to 200 lM (Beckmann-Knopp
et al., 2000) are unlikely to cause induction.
Inhibition studies for silymarin were carried out for the
most relevant drug related cytochromes P450, i.e. cyto-
chromes P450 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6,
2E1, and 3A4. At a concentration of 1 lM silymarin no
or negligible inhibition was observed, except for the cyto-
chromes P450 2C19, 2D6, and 3A4 (Fig. 3), which were
submitted to a more detailed kinetic studies for the deter-
mination of their K
i
values to study the moderate inhibition
of these cytochromes P450 in more detail (Figs. 46; Tables
46). All these results might be of clinical relevance as the
applied concentration of 1 lM is close to the maximum
plasma concentrations of silibinin recently measured in a
clinical trial and in human pharmacokinetic studies (Wey-
henmeyer et al., 1992; Fuhr et al., 2007).
The K
i
values determined for the cytochromes P450
2C19, 2D6, and 3A4 compare favorably with those previ-
ously reported (Table 6). The inhibition kinetics shown as
Eadie-Hofstee diagrams suggest a concentration dependent
mixed-partial type of inhibition (Figs. 46). All K
i
values
are well above clinically relevant plasma concentrations
of silbinin, and therefore, concerns because of mecha-
nism-based inhibition for cytochrome P450 2C9 and 3A4
as raised by Sridar et al., 2004 may be considered to be
of no relevance.
Table 5
i
of silymarin for CYP3A4 (testosterone 6b-hydroxylation)
Rank by R
2
Equation R
2
AICc Sy.x Con. K
i
(lM) SE
2 Mixed (full) 0.952 966.6 403.26 Yes 12.01 4.53
The best t results were obtained using a mixed (partial) or mixed (full) inhibition model. The resulting K
i
were 12.01 4.61 lM and 12.01 4.53 lM,
respectively. The parameters for these two inhibition models were calculated to be
Mixed (partial): Mixed (full):
V
max
: 5781.4 V
max
: 5781.5
K
m
: 55.3 K
m
: 55.3
K
i
: 12.0 K
i
: 12.0
a: 2.4 a: 2.4
b: 9.33E 11
m
V max
1
bI
aK
i

1
I
aK

1
Km
S
1
I
K
i

1
I
aK
i
0
@
1
A
m
V max
Km
S
1
I
K
i

1
I
aK

Table 6
Inhibition by silymarin or silibinin of cytochrome P450 isoforms (CYP) in comparison with published data
CYP Own data Beckmann-Knopp et al. Zuber et al. Sridar et al.
1A2 No inhib.
1
No inhib. n.d. n.d.
2A6 No inhib.
1
IC
50
: 541688 lM n.d. n.d.
2B6 No inhib.
1
n.d. n.d. No inhib.
2
2C8 No inhib.
1
n.d. n.d. n.d.
2C9 No inhib.
1
K
i
: 1819 lM n.d. K
i
: 5 lM
2C19 K
i
: 2.2 lM IC50: 309424 lM n.d. n.d.
2D6 K
i
: 11.6 lM K
i
: 112135 lM K
i
: 8.2 lM No inhib.
2
2E1 No inhib.
1
IC50: 460799 lM K
i
: 28.7 lM No inhib.
2
3A4 K
i
: 12.0 lM K
i
: 912 lM K
i
: 4.910 lM K
i
: 32166 lM
No inhib.
1
: at 1 lM silymarin; no inhib.
2
: up to 100 lM silibinin; n.d.: not done.
Inhibition studies with 100 lM silymarin revealed negli-
ble inhibition of the cytochromes P450 2A6, and 2E1, a
moderate inhibition of 50% of cytochrome P450 1A2,
and extensive inhibition of more than 80% for cytochromes
P450 2B6, 2C8, 2C9, 2C19, 2D6, and 3A4. However, the
concentration of silymarin applied was more than 100-fold
over the maximum plasma concentration under therapeutic
condition, and therefore the observed inhibitions are of no
clinical relevance.
When comparing the inhibition results reported here,
with those reported previously (Beckmann-Knopp et al.,
2000; Sridar et al., 2004) several discrepancies were obvi-
ous. Most of all, none of these reports covered a concentra-
tion of silymarin or silibinin as low as 1 lM. The inhibition
studies presented by Beckmann-Knopp et al., 2000, cov-
ered a concentration range of 3.7300 lM. According to
the studies presented by Sridar et al., 2004, the concentra-
tion range tested was 550 lM silibinin for cytochrome
P450 2C9 and 25250 lM for cytochrome P450 3A4, which
is in any case far above the observed maximum plasma
concentration in patients, which is less than 1 lM, and
are therefore of no clinical relevance. Furthermore, Sridar
et al., 2004 reported no inhibition of cytochromes P450 2B6
and 2D6 by silibinin at concentrations of up to 100 lM.
This discrepancy might be explained by the applied test sys-
tems. Sridar et al., 2004 made use of puried and reconsti-
tuted cytochromes P450, whereas a certied and highly
controlled preparation of human liver microsomes was
used in our studies. Particularly for cytochromes P450
2D6, the validity of our results is supported by our detailed
kinetic studies (Figs. 46). The lack of an inhibitory eect
on cytochrome P450 2E1 conrms results of previous stud-
ies (Miguez et al., 1994; Beckmann-Knopp et al., 2000; Sri-
dar et al., 2004).
Inhibition of CYP3A4 by silymarin was recently studied
in a clinical trial conducted at the University of Cologne
(Fuhr et al., 2007). In a controlled, open cross-over trial
with 16 male volunteers, the CYP3A4 substrate nifedipine
(Adalat
) was given orally with or without pre-treatment

by administration of silymarin (Legalon
). The relative
bioavailability for the AUC of nifedipine in the presence
of silymarin compared with nifedipine alone was 1.13
(90% condence interval: 0.971.32). This result supports
our general assumption that inhibition studies with 1 lM
silymarin are of clinical relevance, in this case particularly
for cytochrome P450 3A4, where only a minor inhibition
was observed for 1 lM silymarin (Fig. 3).
Intentionally, all studies were performed with a standard-
ized silymarin extract to be as close as possible to the clini-
cally applied form of silymarin. The same accounts for the
chosen concentrations. In conclusion, results on cyto-
chrome P450 inhibition studies with silymarin may indicate
drug interaction, but clinically relevant concentrations
should be considered for assessment of drug interaction.
Conict of interest statement
All studies were funded by Madaus GmbH, Cologne,
Germany. However, the experiments were designed and
carried out independently by GenPharmTox Biotech AG,
without the inuence of the sponsor, and observing GLP
regulations.
Acknowledgements
The technical assistance by Birgit Ammermann and Na-
dine Herrmann is appreciated. These studies were spon-
sored by Madaus GmbH, Cologne, Germany.
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Assessment of Drug Drug Interaction For Silymarin

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Assessment of drugdrug interaction for silymarin

Fig. 4. Inhibition of cytochrome P450 2C19 by silymarin with S-

Fig. 5. Inhibition of cytochrome P450 2D6 by silymarin with bufuralol as

) was given orally with or without pre-treatment

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