Effects of curcumin on the pharmacokinetics of tamoxifen and its active metabolite, 4-hydroxytamoxifen, in rats: Possible role of CYP3A4 and P-glycoprotein inhibition by curcumin

Abstract

The effects of curcumin, a natural anti-cancer compound, on the bioavailability and pharmacokinetics of tamoxifen and its metabolite, 4-hydroxytamoxifen, were investigated in rats. Tamoxifen and curcumin interact with cytochrom P450 (CYP) enzymes and P-glycoprotein, and the increase in the use of health supplements may result in curcumin being taken concomitantly with tamoxifen as a combination therapy to treat or prevent cancer. A single dose of tamoxifen was administered orally (9 mg x kg(-1)) with or without curcumin (0.5, 2.5 and 10 mg x kg(-1)) and intravenously (2mg x kg(-1)) with or without curcumin (2.5 and 10 mg x kg(-1)) to rats. The effects of curcumin on P-glycoprotein (P-gp) and CYP3A4 activity were also evaluated. Curcumin inhibited CYP3A4 activity with 50% inhibition concentration (IC50) values of 2.7 microM. In addition, curcumin significantly (P < 0.01 at 10 microM) enhanced the cellular accumulation of rhodamine-123 in MCF-7/ADR cells overexpressing P-gp in a concentration-dependent manner. This result suggested that curcumin significantly inhibited P-gp activity. Compared to the oral control group (given tamoxifen alone), the area under the plasma concentration-time curve (AUC(0-infinity)) and the peak plasma concentration (C(max)) of tamoxifen were significantly (P < 0.05 for 2.5 mg x kg(-1); P < 0.01 for 10 mg x kg(-1)) increased by 33.1-64.0% and 38.9-70.6%, respectively, by curcumin. Consequently, the absolute bioavailability of tamoxifen in the presence of curcumin (2.5 and 10 mg x kg(-1)) was 27.2-33.5%, which was significantly enhanced (P < 0.05 for 2.5 mg x kg(-1); P < 0.01 for 10 mg x kg(-1)) compared to that in the oral control group (20.4%). Moreover, the relative bioavailability of tamoxifen was 1.12- to 1.64-fold greater than that in the control group. Furthermore, concurrent use of curcumin significantly decreased (P < 0.05 for 10 mg x kg(-1)) the metabolite-parent AUC ratio (MR), implying that curcumin may inhibit the CYP-mediated metabolism of tamoxifen to its active metabolite, 4-hydroxytamoxifen. The enhanced bioavailability of tamoxifen by curcumin may be mainly due to inhibition of the CYP3A4-mediated metabolism of tamoxifen in the small intestine and/or in the liver and to inhibition of the P-gp efflux transporter in the small intestine rather than to reduction of renal elimination of tamoxifen, suggesting that curcumin may reduce the first-pass metabolism of tamoxifen in the small intestine and/or in the liver by inhibition of P-gp or CYP3A4 subfamily.

Full text

ORIGINAL ARTICLES
School of Medicine1, Research Institute of Life Science, Gyeongsang National University, Jinju; College of Pharmacy2,
Chosun University, Gwangju, Republic of Korea
Effects of curcumin on the pharmacokinetics of tamoxifen and its active
metabolite, 4-hydroxytamoxifen, in rats: possible role of CYP3A4 and
P-glycoprotein inhibition by curcumin
Y. A. Cho1,W.Lee
2, J. S. Choi2
Received July 8, 2011, accepted August 9, 2011
Prof. Jun-Shik Choi, College of Pharmacy, Chosun University, 375 Susuk-dong, Dong-Gu, Gwangju 501-759, Republic
of Korea
jsachoi@chosun.ac.kr
Pharmazie 67: 124–130 (2012) doi: 10.1691/ph.2012.1099
The effects of curcumin, a natural anti-cancer compound, on the bioavailability and pharmacokinetics
of tamoxifen and its metabolite, 4-hydroxytamoxifen, were investigated in rats. Tamoxifen and curcumin
interact with cytochrom P450 (CYP) enzymes and P-glycoprotein, and the increase in the use of health
supplements may result in curcumin being taken concomitantly with tamoxifen as a combination therapy
to treat or prevent cancer. A single dose of tamoxifen was administered orally (9mg ·kg−1) with or without
curcumin (0.5, 2.5 and 10 mg ·kg−1) and intravenously (2 mg ·kg−1) with or without curcumin (2.5 and
10 mg ·kg−1) to rats. The effects of curcumin on P-glycoprotein (P-gp) and CYP3A4 activity were also
evaluated. Curcumin inhibited CYP3A4 activity with 50% inhibition concentration (IC50) values of 2.7␮M.
In addition, curcumin significantly (P<0.01 at 10 ␮M) enhanced the cellular accumulation of rhodamine-123
in MCF-7/ADR cells overexpressing P-gp in a concentration-dependent manner. This result suggested that
curcumin significantly inhibited P-gp activity. Compared to the oral control group (given tamoxifen alone),
the area under the plasma concentration-time curve (AUC0–∞) and the peak plasma concentration (Cmax)of
tamoxifen were significantly (P<0.05 for 2.5 mg ·kg−1;P<0.01 for 10mg ·kg−1) increased by 33.1–64.0%
and 38.9–70.6%, respectively, by curcumin. Consequently, the absolute bioavailability of tamoxifen in the
presence of curcumin (2.5 and 10 mg ·kg−1) was 27.2–33.5%, which was significantly enhanced (P<0.05
for 2.5 mg ·kg−1;P<0.01 for 10 mg ·kg−1) compared to that in the oral control group (20.4%). Moreover, the
relative bioavailability of tamoxifen was 1.12- to 1.64-fold greater than that in the control group. Furthermore,
concurrent use of curcumin significantly decreased (P<0.05 for 10mg ·kg−1) the metabolite-parent AUC
ratio (MR), implying that curcumin may inhibit the CYP-mediated metabolism of tamoxifen to its active
metabolite, 4-hydroxytamoxifen. The enhanced bioavailability of tamoxifen by curcumin may be mainly due
to inhibition of the CYP3A4-mediated metabolism of tamoxifen in the small intestine and/or in the liver and
to inhibition of the P-gp efflux transporter in the small intestine rather than to reduction of renal elimination
of tamoxifen, suggesting that curcumin may reduce the first-pass metabolism of tamoxifen in the small
intestine and/or in the liver by inhibition of P-gp or CYP3A4 subfamily.
1. Introduction
Tamoxifen belongs to a class of compounds known as selective
estrogen receptor modulators which act as estrogen receptor
agonists in some tissues and as antagonists in other tissues
(Furr and Jordan 1984; Park and Jordan 2002). Tamoxifen is
an estrogen receptor agonist in bone, the cardiovascular sys-
tem, and the endometrium, but acts as an antagonist in breast
tissue (Buchanan et al. 2007). Tamoxifen is clinically used for
treating and preventing breast cancer (Jaiyesimi et al. 1995).
Tamoxifen has a relatively low toxicity and is less harm-
ful than most chemotherapeutics. The main adverse effects
of tamoxifen in humans are increased risks of endometrial
cancer and thromboembolic diseases (Fornander et al. 1993;
Meier and Jick 1998). Orally administered tamoxifen undergoes
extensive hepatic metabolism with subsequent biliary excre-
tion (Buckley and Goa 1989). In humans, the main pathway
in tamoxifen biotransformation proceeds via N-demethylation
catalyzed mostly by cytochrome P450 (CYP) 3A4 enzymes
(Jacolot et al. 1991; Mani et al. 1993). Another important
drug metabolite, 4-hydroxytamoxifen, is produced in humans
by CYP2C9 and CYP3A4 (Crewe et al. 1997; Mani et al.
1993). 4-Hydroxytamoxifen has shown 30- to 100-fold greater
potency than tamoxifen in suppressing estrogen-dependent cell
proliferation (Borgna and Rochefort 1981; Coezy et al. 1982).
A secondary metabolite of tamoxifen, endoxifen, exhibits a
potency similar to 4-hydroxytamoxifen (Johnson et al. 2004;
Stearns et al. 2003). Thus, tamoxifen is referred to as a prodrug
that requires activation to exert its effects.
Tamoxifen also acts as a substrate for P-glycoprotein (P-gp)
(Gant et al. 1995; Rao et al. 1994). P-gp co-localized with
CYP3A in the polarized epithelial cells of excretory organs
such as the liver, kidney and intestine (Sutherland et al. 1993;
124 Pharmazie 67 (2012)

ORIGINAL ARTICLES
Log concentration of ketoconazole (µM)
0.0010.010.1110
% inhibition of CYP3A4
0
20
40
60
80
100
(A) (B)
Log concentration of curcumin (µM)
0.11101001000
% inhibition of CYP3A4
0
20
40
60
80
100
Fig. 1: Inhibitory effects of ketoconazole (A) and curcumin (B) on CYP3A4 activity All experiments were done in duplicate, and results are expressed as the percent of inhibition
Turgeon et al. 2001) to eliminate foreign compounds out of
the body. A substantial overlap in substrate specificity exists
between CYP3A4 and P-gp (Wacher et al. 1995). P-gp and
CYP3A modulators might be able to affect the oral bioavail-
ability of tamoxifen. The low bioavailability of oral tamoxifen
is mainly due to first-pass metabolism in the intestine or in the
liver, and P-gp mediated efflux in the intestine.
Curcumin is the major yellow pigment in turmeric, curry, and
mustard and has been widely used medicinally (Govindarajan
1980). Studies on the chemopreventive efficacy of curcumin
have shown that it possesses both antiinitiating and antipro-
moting activities in several experimental systems (Deshpande
et al. 1998; Huang et al. 1994). Curcumin inhibited carcino-
genesis in various tissues, including skin (Huang et al. 1997),
colorectal (Rao et al. 1995), oral (Tanaka et al. 1994), forestom-
ach (Singh et al. 1998) and mammary (Singletary et al. 1998)
cancers. In vitro and animal studies have suggested that cur-
cumin may have antitumor (Aggarwal and Shishodia 2006; Choi
et al. 2006), antioxidant, anti-ischemic (Shukla et al. 2008) and
anti-inflammatory properties (Srivastava et al. 1995).
Appiah-Opong et al. (2008) reported that curcumin inhibits
human CYP3A4 and 2C9, while Thapliyal et al. (2001) found
that curcumin inhibits human CYP1A1 and 1A2. Thus, the
inhibitory effects of curcumin against human CYP enzymes
remain somewhat controversial. Curcumin is an inhibitor of P-gp
in the KB/MDR cell line (Efferth et al. 2002), but the inhibitory
effect of curcumin against P-gp is ambiguous elsewhere. There-
fore, we re-evaluated the inhibition of CYP enzyme activity
and P-gp activity by curcumin using CYP inhibition assays
and rhodamine-123 retention assays in P-gp-overexpressing
MCF-7/ADR cells. Tamoxifen and curcumin interact with CYP
enzymes and P-gp, and the increase in the use of health supple-
ments may result in curcumin being taken concomitantly with
tamoxifen to treat or prevent cancer. It is important to assess the
potential pharmacokinetic interactions after the concurrent use
of tamoxifen and curcumin in order to ensure the effectiveness
and safety of the drug therapy. However, there a few studies
investigated the effect of some flavonoids on the bioavailability
of tamoxifen in rats (Kim et al. 2010; Shin et al. 2006; Shin and
Choi 2009). Consequently, it could be expected that curcumin
would change the pharmacokinetics of drugs, substrates of P-gp
and/or CYP3A4, if they are concomitantly used. Curcumin and
tamoxifen could be prescribed for the treatment or prevention of
cancer as a combination therapy. However, the possible effects
of curcumin on the bioavailability of tamoxifen have not been
studied in vivo.
Therefore, the aim of this study was to investigate effect of
curcumin, herbal anti-cancer compound, on inhibitory effect of
P-gp, CYP3A4 activity, bioavailability, and pharmacokinetics of
tamoxifen and its active metabolite, 4-hydroxytamoxifen, after
oral and intravenous administration of tamoxifen in rats.
2. Investigations and results
2.1. Inhibitory effect of CYP3A4 activity
The inhibitory effect of curcumin on CYP3A4 activity is shown
in Fig. 1. Curcumin inhibited CYP3A4 activity and the 50%
inhibition concentration (IC50) value of curcumin on CYP3A4
activity is 2.7 ␮M.
2.2. Rhodamine-123 retention assay
As shown in Fig. 2, accumulation of rhodamine-123, a P-gp
substrate, was raised in MCF-7/ADR cells over-expressing P-gp
compared with that in MCF-7 cells lacking P-gp. The concurrent
use of curcumin enhanced the cellular uptake of rhodamine-123
in a concentration-dependent manner and showed a statistically
significant (P<0.01) increase over the concentration range of
10 ␮M. This result suggested that curcumin significantly inhib-
ited P-gp activity.
2.3. Effect of curcumin on the pharmacokinetics of oral
tamoxifen
Mean arterial plasma concentration-time profiles of tamoxifen
following oral administration of tamoxifen (9 mg ·kg−1) to rats
in the presence or absence of curcumin (0.5, 2.5 and 10 mg ·
kg−1) are shown in Fig. 3, the corresponding pharmacokinetic
parameters are shown in Table 1. The presence of curcumin sig-
nificantly altered the pharmacokinetic parameters of tamoxifen.
Pharmazie 67 (2012) 125

ORIGINAL ARTICLES
Table 1: Mean (±S.D.) pharmacokinetic parameters of tamoxifen after the oral administration of tamoxifen (9 mg ·kg−1) to rats
in the presence or absence of curcumin
Parameter Control Tamoxifen+ Curcumin
0.5 mg ·kg−12.5 mg ·kg−110 mg ·kg−1
AUC0–∞(ng·h·ml−1) 1832 ±359 2051 ±418 2439 ±494*3004 ±558**
Cmax (ng·ml-1) 126 ±22 146 ±29 175 ±33*215 ±41**
Tmax (h) 1.17 ±0.41 1.33 ±0.52 1.33 ±0.52 1.33 ±0.52
t1/2 (h) 11.3 ±2.76 11.7 ±2.86 11.9 ±2.84 12.4 ±3.12
A.B. (%) 20.4 ±4.52 22.9 ±4.77 27.2 ±5.02*33.5 ±5.80**
R.B. (%) 100 112 133 164
AUC0–∞: area under the plasma concentration-time curve from 0 h to infinity; Cmax: peak plasma concentration; Tmax: time to reach; t1/2 : terminal half-life; A.B.: absolute bioavailability; R.B.: relative
bioavailability
*P<0.05, **P<0.01, significant difference compared to the control
Compared to the control group (given oral tamoxifen alone),
curcumin significantly (P<0.05 for 2.5 mg ·kg−1;P<0.01 for
10 mg ·kg−1) increased the AUC0–∞and the Cmax of tamoxifen
by 33.1–64.0% and 38.9–70.6%, respectively. Consequently, the
absolute bioavailability (A.B.) of tamoxifen in the presence of
curcumin (2.5 and 10 mg ·kg−1) was 27.2–33.5%, which was
significantly enhanced (P<0.05 for 2.5 mg ·kg−1;P<0.01 for
10 mg ·kg−1) compared to that in the oral control group (20.4%).
The relative bioavailability (R.B.) of tamoxifen was 1.12- to
1.64-fold greater than that in the control group. However, there
were no significant differences in Tmax and the t1/2 of tamoxifen
in the presence of curcumin.
2.4. Effect of curcumin on the pharmacokinetics of
4-hydroxytamoxifen
Mean plasma concentration-time profiles of 4-
hydroxytamoxifen after oral administration of tamoxifen
(9 mg ·kg−1) to rats in the presence or absence of curcumin
(0.5, 2.5 and 10 mg ·kg−1) are shown in Fig. 4, while the
correlated pharacokinetic parameters are shown in Table 2. The
metabolite-parent AUC ratio (MR) was significantly (P<0.05
for 10 mg ·kg−1of curcumin) decreased in the presence of
MCF- 7 0 1 3 10 100
Relative cellular uptake (Arbitrary unit)
0
2000
4000
6000
8000
10000
12000
(µM)
MCF-7/ADR
**
**
Curcumin
Verapamil
Fig. 2: Rhodamine-123 retention. MCF-7/ADR cells were preincubated with
curcumin for 30 min and incubation of MCF-7/ADR cells with 20␮M R-123
for 90 min. Verapamil (100 ␮M) was used as a positive control. The values
were divided by total protein contents of each sample. Data represents
mean ±SD of 6 separate samples (significantly different from control
MCF-7, ** P<0.01)
curcumin compared with that in the control group, indicating
that curcumin may effectively inhibit the CYP3A4-mediated
metabolism of tamoxifen in the small intestine and/or in
the liver. These results suggest that the production of 4-
hydroxytamoxifen was considerably inhibited by curcumin.
The AUC, Cmax,t
1/2 and Tmax of 4-hydroxytamoxifen were not
significantly altered by the presence of curcumin.
2.5. Effects of curcumin on the pharmacokinetics of
intravenous tamoxifen
The mean arterial plasma concentration–time profiles of tamox-
ifen following the intravenous administration of tamoxifen
(2 mg ·kg−1) in the absence and presence of curcumin (2.5 and
10 mg ·kg−1) are shown in Fig. 5. The relevant pharmacokinetic
parameters are listed in Table 3. The plasma concentrations of
tamoxifen declined in a poly-exponential fashion in all rats stud-
ied. The pharmacokinetic parameters of intravenous tamoxifen
listed in Table 3 were comparable among three groups of rats,
suggesting that the effects of oral curcumin on the pharmacoki-
netics of intravenous tamoxifen were almost negligible.
Time (h)
0 4 8 12 16 20 24 28 32 36
Plasma concentration of tamoxifen (ng/mL)
1
10
100
1000
Fig. 3: Mean plasma concentration-time profiles of tamoxifen after oral (9 mg ·
kg−1) administration of tamoxifen to rats in the presence or absence of
curcumin (0.5, 2.5 and 10 mg ·kg−1)(n= 6, each). Bars represent the
standard deviation. (䊉) Oral administration of tamoxifen (9 mg ·kg−1); (◦)
the presence of 0.5 mg ·kg−1of curcumin; () the presence of 2.5mg ·kg−1
of curcumin; () the presence of 10 mg ·kg−1of curcumin
126 Pharmazie 67 (2012)

ORIGINAL ARTICLES
Table 2: Mean (±S.D.) pharmacokinetic parameters of 4-hydroxytamoxifen after the oral administration of tamoxifen (9mg ·
kg−1) to rats in the presence or absence of curcumin
Parameter Control Tamoxifen+ Curcumin
0.5 mg ·kg−12.5 mg ·kg−110 mg ·kg−1
AUC0–∞(ng ·h·ml−1) 284 ±62 301 ±66 315 ±70 334 ±74
Cmax (ng ·ml−1) 13.3 ±2.74 13.5 ±3.49 13.7 ±3.51 13.9 ±3.52
Tmax (h) 2.17 ±0.42 2.33 ±0.52 2.33 ±0.52 2.33 ±0.52
t1/2 (h) 15.3 ±3.66 15.9 ±4.18 16.4 ±4.21 16.8 ±4.23
MR (%) 15.5 ±3.16 14.7 ±3.05 12.9 ±2.41 11.1 ±2.14*
AUC0–∞: area under the plasma concentration-time curve from 0 h to infinity; Cmax: peak plasma concentration; Tmax : time to reach; t1/2: terminal half-life; MR: metabolite-parent ratio
*P<0.05, significant difference compared to the control
Table 3: Mean (±S.D.) pharmacokinetic parameters of tamoxifen after an intravenous administration of tamoxifen (2 mg ·kg−1)
to rats in the presence or absence of curcumin
Parameters Control Tamoxifen+ Curcumin
2.5 mg ·kg−110 mg ·kg−1
AUC0−∞ (ng ·h·ml−1) 1792 ±326 1898 ±371 1998 ±406
CLt(mL ·min−1·kg−1) 17.9 ±3.32 16.9 ±3.19 16.2 ±3.10
t1/2 (h) 9.0 ±1.56 9.1 ±1.63 9.2 ±1.68
Kel(h−1) 0.077 ±0.015 0.076 ±0.013 0.075 ±0.013
R.B. (%) 100 106 111
AUC0−∞ : area under the plasma concentration-time curve from 0 h to infinity; CLt: total body clearance; t1/2: terminal half-life; Kel: elimination rat constant; R.B.: relative bioavailability
3. Discussion
Based on the broad overlap in substrate specificities as well
as co-localization in the small intestine, the primary site of
absorption for orally administered drugs, CYP3A4 and P-gp, are
recognized as a concerted barrier to drug absorption (Cummins
et al. 2002; Wolozin et al. 2000). CYP enzymes contribute signif-
icantly to first-pass metabolism and oral bioavailability of many
drugs. The first-pass metabolism of compounds in the intestine
limits the absorption of toxic xenobiotics and may ameliorate
Time (h)
0 4 8 12162024283236
Plasma concentration of 4-hydroxytamoxifen
(ng/mL)
1
10
100
Fig. 4: Mean plasma concentration-time profiles of 4-hydroxytamoxifen after an oral
(9 mg ·kg−1) administration of tamoxifen to rats in the presence or absence
of curcumin (0.5, 2.5 and 10 mg ·kg−1)(n= 6, each). Bars represent the
standard deviation. (䊉) Oral administration of 4-hydroxytamoxifen (9 mg ·
kg−1); (◦) the presence of 0.5 mg ·kg−1of curcumin; () the presence of
2.5 mg ·kg−1of curcumin; () the presence of 10 mg ·kg−1of curcumin
side effects. Moreover, induction or inhibition of intestinal CYPs
may be responsible for significant herbal products-drug interac-
tions when one agent decreases or increases the bioavailability
and absorption rate constant of a concurrently administered drug
(Kaminsky and Fasco 1991; Kim et al. 2010; Shin et al. 2006;
Shin and Choi 2009).
Tamoxifen and its primary metabolites undergo extensive oxi-
dation, principally by CYP3A4 and CYP2C9 (Crewe et al.
1997; Mani et al. 1993). Tamoxifen and its metabolites, N-
desmethyltamoxifen and 4-hydroxytamoxifen, are substrates for
the efflux of P-gp as well (Gant et al. 1995; Rao et al. 1994).
Time (h)
0 4 8 12162024283236
Plasma concentration of tamoxifen (ng/mL)
1
10
100
1000
10000
Fig. 5: Mean plasma concentration-time profiles of tamoxifen after an intravenous
(2 mg ·kg−1) administration of tamoxifen to rats in the presence or absence
of curcumin (2.5 and 10 mg ·kg−1)(n= 6, each). Bars represent the standard
deviation. (䊉) Intravenous administration of tamoxifen (2mg ·kg−1); (◦) the
presence of 2.5 mg ·kg−1of curcumin; () the presence of 10mg ·kg−1of
curcumin
Pharmazie 67 (2012) 127

ORIGINAL ARTICLES
CYP3A and P-gp inhibitors might interact with tamoxifen and
its metabolites and thus contribute to substantial alteration of
their pharmacokinetics. Curcumin is a popular herbal product
marketed to treat or prevent cancer in various tissues (Singh
et al. 1998; Singletary et al. 1998; Rao et al. 1995; Tanaka et al.
1994). Despite its popularity, limited information is available on
the safety, interactions with other drugs, or the mechanisms of
interactions of curcumin. As shown in Fig. 1, curcumin inhibited
CYP3A4 activity with an IC50 value of 2.7 ␮M. The cell-based
P-gp activity test using rhodamine-123 also showed that cur-
cumin (10 ␮M, P<0.01) significantly inhibited P-gp activity
(Fig. 2). The phase I and phase II metabolizing enzymes are
expressed with P-gp in the liver, kidney and intestine (Sutherland
et al. 1993; Turgeon et al. 2001), regulating the bioavailability
of many orally ingested compounds. Therefore, the inhibitors
against both metabolizing enzyme CYP3A4 and P-gp may have
a large impact on the pharmacokinetics of those compounds.
Since curcumin may competitively inhibit P-gp and CYP3A4
this study examined the effect of curcumin on the bioavailability
and pharmacokinetics of tamoxifen.
As CYP3A9 in rats is corresponds to the ortholog of CYP3A4
in humans (Kelly et al. 1999), rat CYP3A2 is similar to human
CYP3A2 (Bogaards et al. 2000; Guengerich et al. 1986). Human
CYP2C9 and 3A4 and rat CYP2C11 and 3A1 have 77 and 73%
protein homology, respectively (Lewis 1996). Rats were selected
as an animal model in this study to evaluate the potential phar-
macokinetic interactions mediated by CYP3A4, although there
should be some difference in enzyme activity between rat and
human (Cao et al. 2006). The presence of curcumin (2.5 and
10 mg ·kg−1) significantly increased the AUC0–∞and Cmax of
tamoxifen (Table 1). Since orally administered tamoxifen is a
substrate for CYP3A4-mediated metabolism and P-gp-mediated
efflux in the intestine and/or in the liver, curcumin might be
effective to obstruct this metabolic pathway. These results are
consistent with a report by Shin et al. (2006) where the oral
coadministration of quercetin increased the Cmax and the AUC
of tamoxifen in rats and with a report by Kim et al. (2010) where
the presence of silybinin significantly increased the AUC0–∞and
Cmax of tamoxifen, a P-gp and CYP3A substrate, in rats. This is
also supported by the finding that the presence of epigallocat-
echin gallate significantly enhanced the oral bioavailability of
tamoxifen in rats (Shin and Choi 2009).
Curcumin did not increase the AUC0–∞of 4-hydroxytamoxifen
compared to the control group. However, these results are not
consistent with reports by Kim et al. (2010) and Shin et al.
(2009) showing that silybinin significantly increased AUC of
4-hydroxytamoxifen in rats. The metabolite-parent AUC ratio
(MR) was significantly reduced in the presence of curcumin
(Table 2), this result suggested that curcumin was capable of
altering the production of 4-hydroxytamoxifen, which is mainly
formed by CYP3A4 (Crewe HK et al. 1997; Mani et al. 1993).
These results are consistent with Shin et al. and Kim et al. who
reported that quercetin, silybinin and epigallocatechin gallate
significantly decreased MR of tamoxifen, a P-gp and CYP3A
substrate, in rats. (Kim et al. 2010; Shin et al. 2006; Shin and
Choi 2009)
We selected two concentrations (2.5 and 10 mg ·kg−1of cur-
cumin) because those significantly increased the AUC of oral
tamoxifen. In Table 3, the pharmacokinetic parameters of intra-
venous tamoxifen are compared among three groups of rats data,
suggesting that the effects of oral curcumin on the pharmacoki-
netics of intravenous tamoxifen were almost negligible.
Those studies in conjunction with our present findings sug-
gest that the combination of tamoxifen and CYP3A4 and P-gp
inhibitors may result in a significant pharmacokinetic drug
interaction. Therefore, the enhanced bioavailability of tamox-
ifen may be mainly due to inhibition of the CYP3A4-mediated
metabolism in the liver and/or in the intestine and to inhibition
of the P-gp efflux transporter in the small intestine by curcumin.
Although being potentially an adverse effect, this interaction
may provide a therapeutic benefit whereby it enhances bioavail-
ability and lowers the dose administered. The present study
raises awareness about potential drug interactions with concomi-
tant use of curcumin and tamoxifen, but further evaluation in
clinical studies is necessary.
In conclusion, the presence of curcumin enhanced the oral
bioavailability of tamoxifen in rats. The enhanced bioavalability
of tamoxifen may be mainly due to inhibition of the CYP3A4-
mediated metabolism of tamoxifen in the intestinal and/or in
the liver and to inhibition of the P-gp efflux transporter in the
small intestine rather than to reduction of renal elimination of
tamoxifen by curcumin. If the results obtained from the rats’
model is confirmed in clinical trials, the tamoxifen dose should
be adjusted for potential drug interactions when tamoxifen is
used with curcumin.
4. Experimental
4.1. Chemicals and apparatus
Tamoxifen, 4-hydroxytamoxifen, curcumin and butylparaben (p-
hydroxybenzoic acid n-butyl ester) were purchased from Sigma-Aldrich
Co. (St. Louis, MO, USA). HPLC-grade methanol and acetonitrile were
acquired from Merck Co. (Darmstadt, Germany). All other chemicals in
this study were of reagent grade and were used without further purification.
Rhodamine was from Calbiochem (USA), the CYP inhibition assay kit was
from GENTEST (Woburn, MA, US).
Apparatus used in this study included an HPLC equipped with a Waters
1515 isocratic HPLC Pump, a Waters 717 plus autosampler and a WatersTM
474 scanning fluorescence detector (Waters Co., Milford, MA, USA), an
HPLC column temperature controller (Phenomenex Inc., CA, USA), a
Bransonic®Ultrasonic Cleaner (Branson Ultrasonic Co., Danbury, CT,
USA), a vortex-mixer (Scientific Industries Co., NY, USA), and a high-speed
micro centrifuge (Hitachi Co., Tokyo, Japan).
4.2. Animal experiments
Male Sprague-Dawley rats (weighing 270–300g) were purchased from the
Dae Han Laboratory Animal Research Co. (Choongbuk, Korea), and were
given access to a commercial rat chow diet (No. 322-7-1; Superfeed Co.,
Gangwon, Korea) and tap water. The animals were housed, two per cage,
and maintained at 22 ±2◦C and 50–60% relative humidity under a 12:12 h
light-dark cycle. The experiments were initiated after acclimation under
these conditions for at least 1 week. The Animal Care Committee of Chosun
University (Gwangju, Korea) approved the design and the conduct of this
study. The rats were fasted for at least 24h prior to the experiments and
each animal was anesthetized lightly with ether. The left femoral artery and
vein were cannulated using polyethylene tubing (SP45, i.d. 0.58 mm, o.d.
0.96 mm; Natsume Seisakusho Co. LTD., Tokyo, Japan) for blood sampling
and i.v. injection, respectively.
4.3. Drug administration
The rats were divided into seven groups (n=6 each group): an oral con-
trol group (9 mg ·kg−1of tamoxifen, dissolved in distilled water, 3.0ml
·kg−1) without or with 0.5, 2.5 or 10 mg ·kg−1of curcumin (mixed in
distilled water, 3.0ml ·kg−1), and an i.v. group (2mg ·kg−1of tamoxifen,
dissolved in 0.9% NaCl solution, 1.5 ml ·kg−1) without or with 2.5 or 10 mg
·kg−1of curcumin (mixed in distilled water, 3.0ml ·kg−1). Oral tamoxifen
was administered intragastrically using a feeding tube, and curcumin was
administered in the same manner 30 min prior to the oral administration
of tamoxifen. Tamoxifen for i.v. administration was injected through the
femoral vein within 1 min and curcumin was administered in the same man-
ner 30 min prior to the i.v. administration of tamoxifen. A 0.4 ml-aliquot of
blood sample was collected into heparinized tubes from the femoral artery at
0 (to serve as control), 0.017 (only for the i.v. group), 0.25, 0.5, 1, 2, 3, 4, 6,
8, 12, 24 and 36 h after tamoxifen administration. The blood samples were
centrifuged at 13,000 rpm for 5min, and the plasma samples were stored at
–40 ◦C until analysed by HPLC.
4.4. HPLC Analysis
The plasma concentrations of tamoxifen and 4-hydroxytamoxifen were
determined by HPLC using a method reported by Fried et al. (1994) after
128 Pharmazie 67 (2012)

ORIGINAL ARTICLES
a slight modification. Briefly, a 50-␮l aliquot of 8␮g/ml butylparaben, as
an internal standard, and a 0.2-ml aliquot of acetonitrile were mixed with
a 0.2-ml aliquot of the plasma sample. The resulting mixture was then
vortex-mixed vigorously for 2 min and centrifuged at 13,000 rpm for 10 min.
A 50-␮l aliquot of the supernatant was injected into the HPLC system.
Chromatographic separations were achieved using a Symmetry®C18 col-
umn (4.6 ×150 mm, 5 ␮m, Waters Co.), and a ␮BondapakTM C18 HPLC
Precolumn (10 ␮m, Waters Co.). The mobile phase consisted of 20 mM
dipotassium hydrogen phosphate (pH 3.0, adjusted with phosphoric acid)-
acetonitrile (60: 40, v/v). The flow-rate of the mobile phase was maintained
at 1.0 ml·min−1. Chromatography was performed at a temperature of 30 ◦C
regulated by an HPLC column temperature controller. The fluorescence
detector was operated at excitation wavelength of 254nm with an emission
wavelength of 360nm. A homemade post-column photochemical reactor
was supplied with a bactericidal ultraviolet lamp (Sankyo Denki Co, Japan),
and Teflon®tubing (i.d. 0.01¨
, o.d. 1/16”, 2 m long) was crocheted and fixed
horizontally with a stainless steel frame under the lamp at a 10 cm dis-
tance. Tamoxifen, 4-hydroxytamoxifen and butylparaben were eluted with
retention times of 26.1, 7.3 and 14.5 min, respectively. The lower limit of
quantification for tamoxifen and 4-hydroxytamoxifen in the rat plasma was
5ng·ml−1and 0.5 ng ·ml−1, with coefficients of variation below 4.5 and
1.5%, respectively.
4.5. CYP Inhibition assay
The assays of inhibition on human CYP3A4 enzyme activities were per-
formed in multiwell plates using CYP inhibition assay kit (GENTEST,
Woburn, MA) as described previously (Crespi et al. 1997). Briefly, human
CYP enzymes were obtained from baculovirus-infected insect cells. CYP
substrates (7-BFC for CYP3A4) were incubated with or without curcumin
in enzyme/substrate buffer consisting of 1 pmol of P450 enzyme and
NADPH generating system (1.3 mM NADP, 3.54mM glucose 6-phosphate,
0.4 U·ml−1glucose 6-phosphate dehydrogenase and 3.3 mM MgCl2)in
potassium phosphate buffer (pH 7.4). Reactions were terminated by adding
a stop solution after the 45-min incubation. Metabolite concentrations were
measured by a spectrofluorometer (Molecular Device, Sunnyvale,CA, USA)
at an excitation wavelength of 409nm and an emission wavelength of
530 nm. Positive control (1 ␮M ketoconazole for CYP3A4) was run on the
same plate and produced 99% inhibition. All experiments were carrid out
in duplicate, and the results are expressed as the percentage of inhibition.
4.6. Rhodamine-123 retention assay
MCF-7/ADR cells were seeded in 24-well plates. At 80% confluence, the
cells were incubated in FBS-free DMEM for 18 h. The culture medium was
changed to Hanks’ balanced salt solution and the cells were incubated at
37 ◦C for 30 min. After the cells were incubated with 20 ␮M in the presence
of curcumin (0, 1, 3 and 10 ␮M) for 90 min, the medium was completely
removed. The cells were then washed three times with ice-cold phosphate
buffer (pH 7.0) and lysed in lysis buffer. The rhodamine-123 fluorescence in
the cell lysates was measured at excitation and emission wavelengths of 480
and 540 nm, respectively. Fluorescence values were normalized to the total
protein content of each sample and are presented as the ratio to controls.
4.7. Pharmacokinetic analysis
The plasma concentration data were analyzed by non-compartmental
method using WinNonlin software version 4.1 (Pharsight Co., Mountain
View, CA, USA). The elimination rate constant (Kel) was calculated by
log-linear regression of tamoxifen and 4-hydroxytamoxifen concentration
data during the elimination phase. The terminal half-life (t1/2) was calcu-
lated by 0.693/Kel. The peak plasma concentration (Cmax ) and time to reach
peak plasma concentration (Tmax) of tamoxifen and 4-hydroxytamoxifen
in plasma was obtained by visual inspection of the data from the
concentration–time curve. The area under the plasma concentration-time
curve (AUC0–t) from time zero to the time of last measured concentration
(Clast) was calculated by the linear trapezoidal rule. The AUC zero to infin-
ity (AUC0–∞) was obtained by the addition of AUC0–t and the extrapolated
area determined by Clast/Kel . The absolute bioavailability (A.B.) was calcu-
lated by AUCoral/AUCi.v. ×Dosei.v./Doseoral , and the relative bioavailability
(R.B.) was calculated by AUCcontrol/AUCwith curcumin. The metabolite-parent
ratio (MR) was estimated by (AUC4-hydroxytamoxifen/AUCtamoxifen) ×100.
4.8. Statistical analysis
Statistical analysis was conducted using one-way ANOVA followed by a
posteriori testing with the use of the Dunnett correction. Differences were
considered to be significant at a level of P<0.05. All mean values are
presented with their standard deviation (mean ±S.D.).
References
Aggarwal BB, Shishodia S (2006) Molecular targets of dietary agents for
prevention and therapy of cancer. Biochem Pharmacol 71: 1397–1421.
Appiah-Opong R, de Esch I, Commandeur JN, Andarini M, Vermeulen NP
(2008) Structure-activity relationships for the inhibition of recombinant
human cytochromes P450 by curcumin analogues. Eur J Med Chem 43:
1621–1631.
Bogaards JJ, Bertrand M, Jackson P, Oudshoorn MJ, Weaver RJ, van
Bladeren PJ, Walther B (2000) Determining the best animal model for
human cytochrome P450 activities: a comparison of mouse, rat, rabbit,
dog, micropig, monkey and man. Xenobiotica 30: 1131–1152.
Borgna JL, Rochefort H (1981) Hydroxylated metabolites of tamoxifen are
formed in vivo and bound to estrogen receptor in target tissues. J Biol
Chem 256: 859–868.
Buchanan CM, Buchanan NL, Edgar KJ, Little JL, Malcolm MO, Ruble KM,
WacherVJ, Wempe MF (2007) Pharmacokinetics of tamoxifen after intra-
venous and oral dosing of tamoxifen-hydroxybutenyl-beta-cyclodextrin
formulations. J Pharm Sci 96: 644–660.
Buckley MT, Goa KL (1989) Tamoxifen: a reappraisal of its pharma-
codynamic and pharmacokinetic properties, and therapeutic use. Clin
Pharmacokin 37: 451–490.
Cao X, Gibbs ST, Fang L, Miller HA, Landowski CP, Shin HC, Lennernas
H, Zhong Y, Amidon GL, Yu LX, Sun D (2006) Why is it challenging to
predict intestinal drug absorption and oral bioavailability in human using
rat model. Pharm Res 23: 1675–1686.
Choi H, Chun YS, Kim SW, Kim MS, Park JW (2006) Curcumin
inhibits hypoxia-inducible factor-1 by degrading aryl hydrocarbon recep-
tor nuclear translocator: a mechanism of tumor growth inhibition. Mol
Pharmacol 70: 1664–1671.
Coezy E, Borgna JL, Rochefort H (1982) Tamoxifen and metabolites in
MCF7 cells: correlation between binding to estrogen receptor and inhi-
bition of cell growth. Cancer Res 42: 317–323.
Crespi CL, Miller VP, Penman BW (1997) Microtiter plate assays for inhi-
bition of human, drug-metabolizing cytochromes P450. Anal Biochem
248: 188–190.
Crewe HK, Ellis SW, Lennard MS, Tucker GT (1997) Variable contribu-
tion of cytochromes P450 2D6, 2C9 and 3A4 to the 4-hydroxylation of
tamoxifen by human liver microsomes. Biochem Pharmacol 53: 171–178.
Cummins CL, Jacobsen W, Benet LZ (2002) Unmasking the dynamic inter-
play between intestinal P-glycoprotein and CYP3A4. J Pharmacol Exp
Ther 300: 1036–1045.
Deshpande SS, Ingle AD, Maru GB (1998) Chemopreventive
efficacy of curcumin-free aqueous turmeric extract in 7,12-
dimethylbenz[a]anthracene-induced rat mammary tumorigenesis.
Cancer Lett 123: 35–40.
Efferth T, Davey M, Olbrich A, Rücker G, Gebhart E, Davey R (2002)
Activity of drugs from traditional Chinese medicine toward sensitive
and MDR1- or MRP1-overexpressing multidrug-resistant human CCRF-
CEM leukemia cells. Blood Cells Mol Dis 28: 160–168.
Fornander T, Hellstrom AC, Moberger B (1993) Descriptive clinicopatho-
logic study of 17 patients with endometrial cancer during or after adjuvant
tamoxifen in early breast cancer. J Nat Cancer Inst 85: 1850–1855.
Freid KM, Wainer IW (1994) Direct determination of tamoxifen and its four
major metabolites in plasma using coupled column high-performance
liquid chromatography. J Chromatogr B Biomed Appl 655: 261–268.
Furr BJ, Jordan VC (1984) The pharmacology and clinical uses of tamoxifen.
Pharmacol Ther 25: 127–205.
Gant TW,O’Connor CK, Corbitt R, Thorgeirsson U, Thorgeirsson SS (1995)
In vivo induction of liver P-glycoprotein expression by xenobiotics in
monkeys. Toxicol Appl Pharmacol 133: 269–276.
Govindarajan VS (1980) Turmeric-chemistry, technology, and quality. Crit
Rev Food Sci Nutr 12: 199–301.
Guengerich FP, Martin MV, Beaune PH, Kremers P, Wolff T, Waxman DJ
(1986) Characterization of rat and human liver microsomal cytochrome
P-450 forms involvedin nifedipine oxidation, a prototype for genetic poly-
morphism in oxidative drug metabolism. J Biol Chem 261: 5051–5060.
Huang MT, Lou YR, Ma W, Newmark HL, Reuhl KR, Conney AH (1994)
Inhibitory effects of dietary curcumin on forestomach, duodenal, and
colon carcinogenesis in mice. Cancer Res 54: 5841–5847.
Huang MT, Newmark HL, Frenkel K (1997) Inhibitory effects of curcumin
on tumorigenesis in mice. J Cell Biochem 27: 26–34.
Jacolot F, Simon I, Dreano Y, Beaune P, Riche C, Berthou F (1991) Identi-
fication of the cytochrome P450 IIIA family as the enzymes involved in
the N-demethylation of tamoxifen in human liver microsomes. Biochem
Pharmacol 41: 1911–1919.
Pharmazie 67 (2012) 129

ORIGINAL ARTICLES
Jaiyesimi IA, Buzdar AU, Decker DA, Hortobagyi GN (1995) Use of
tamoxifen for breast cancer: twenty-eight years later. J Clin Oncol 13:
513–529.
Johnson MD, Zuo H, Lee KH, Trebley JP, Rae JM, Weatherman RV, Desta
Z, Flockhart DA, Skaar TC (2004) Pharmacological characterization of 4-
hydroxy-N-desmethyl tamoxifen, a novel active metabolite of tamoxifen.
Breast Cancer Res Treat 85: 151–9.
Kaminsky LS, Fasco MJ (1991) Small intestinal cytochromes P450. Crit
Rev Toxocol 21: 407–422.
Kelly PA, Wang H, Napoli KL, Kahan BD, Strobel HW (1999) Metabolism
of cyclosporine by cytochromes P450 3A9 and 3A4. Eur J Drug Metab
Pharmacokinet 24: 321–328.
Kim CS, Choi SJ, Park CY, Li C, Choi JS (2010) Effects of silybinin
on the pharmacokinetics of tamoxifen and its active metabolite, 4-
hydroxytamoxifen in rats. Anticancer Res 30: 79–85.
Lewis DFV (1996) Cytochrome P450. Substrate specificity and metabolism.
In: Cytochromes P450. Structure, Function, and Mechanism. Taylor &
Francis: Bristol., pp. 122–123.
Mani C, Gelboin HV, Park SS, Pearce R, Parkinson A, Kupfer D
(1993) Metabolism of the antimammary cancer antiestrogenic agent
tamoxifentamoxifen. I. Cytochrome P450-catalysed N-demethylation
and 4-hydroxylation. Drug Metab Dispos 21: 645–656.
Meier CR, Jick H (1998) Tamoxifen and risk of idiopathic venous throm-
boembolism. Br J Clin Pharmacol 45: 608–612.
Park WC, Jordan VC (2002) Selective estrogen receptor modulators
(SERMS) and their roles in breast cancer prevention. Trends Mol Med 8:
82–88.
Rao CV, Rivenson A, Simi B, Reddy BS (1995) Chemoprevention of colon
carcinogenesis by dietary curcumin, a naturally occurring plant phenolic
compound. Cancer Res 55: 259–266.
Rao US, Fine RL, Scarborough GA (1994) Antiestrogens and steroid hor-
mones: substrates of the human P-glycoprotein. Biochem Pharmacol 48:
287–292.
Shin SC, Choi JS (2009) Effects of epigallocatechin gallate on the oral
bioavailability and pharmacokinetics of tamoxifen and its main metabo-
lite, 4-hydroxytamoxifen, in rats. Anti-Cancer Drugs 20: 584–588.
Shin SC, Choi JS, Li X (2006) Enhanced bioavailability of tamoxifen after
oral administration of tamoxifen with quercetin in rats. Int J Pharm 313:
144–149.
Shukla PK, Khanna VK, Ali MM, Khan MY, Srimal RC (2008) Anti-
ischemic effect of curcumin in rat brain. Neurochem Res 33: 1036–1043.
Singh SV, Hu X, Srivastava SK, Singh M, Xia H, Orchard JL, Zaren HA
(1998) Mechanism of inhibition of benzo[a]pyrene-induced forestomach
cancer in mice by dietary curcumin. Carcinogenesis 19: 1357–1360.
Singletary K, MacDonald C, Iovinelli M, Fisher C, Wallig M (1998)
Effect of the beta-diketones diferuloylmethane (curcumin) and diben-
zoylmethane on rat mammary DNA adducts and tumors induced by
7,12-dimethylbenz[a]anthracene. Carcinogenesis 19: 1039–1043.
Srivastava KC, Bordia A, Verma SK (1995) Curcumin, a major component
of food spice turmeric (Curcuma longa) inhibits aggregation and alters
eicosanoid metabolism in human blood platelets. Prostaglandins Leukot
Essent Fatty Acids 52: 223–227.
Stearns V, Johnson MD, Rae JM, Morocho A, Novielli A, Bhargava P,
Hayes DF, Desta Z, Flockhart DA (2003) Active tamoxifen metabo-
lite plasma concentrations after coadministration of tamoxifen and the
selective serotonin reuptake inhibitor paroxetine. J Natl Cancer Inst 95:
1758–1764.
Sutherland L, Ebner T, Burchell B (1993) The expression of UDP-
glucuronosyltransferases of the UGT1 family in human liver and kidney
and in response to drugs. Biochem Pharmacol 45: 295–301.
TanakaT, Makita H, Ohnishi M, Hirose Y, WangA, Mori H, Satoh K, Hara A,
Ogawa H (1994) Chemoprevention of 4-nitroquinoline 1-oxide-induced
oral carcinogenesis by dietary curcumin and hesperidin: comparison with
the protective effect of beta-carotene. Cancer Res 54: 4653–4659.
Thapliyal R, Maru GB (2001) Inhibition of cytochrome P450 isozymes by
curcumins in vitro and in vivo. Food Chem Toxicol 39: 541–547.
Turgeon D, Carrier JS, Lévesque E, Hum DW, Bélanger A (2001) Rela-
tive enzymatic activity, protein stability, and tissue distribution of human
steroid-metabolizing UGT2B subfamily members. Endocrinology 142:
778–787.
WacherVJ, Wu CY,Benet LZ (1995) Overlapping substrate specificities and
tissue distribution of cytochrome P450 3A and P-glycoprotein: implica-
tions for drug delivery and activity in cancer chemotherapy. Mol Carcinog
13: 129–134.
Wolozin B, Kellman W, Ruosseau P, Celesia GG, Siegel G (2000)
Decreased prevalence of Alzheimer disease associated with 3-hydroxy-
3-methyglutaryl coenzyme A reductase inhibitors. Arch Neurol 57:
1439–1443.
130 Pharmazie 67 (2012)