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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

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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.
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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 ·kg1) with or without
curcumin (0.5, 2.5 and 10 mg ·kg1) and intravenously (2 mg ·kg1) with or without curcumin (2.5 and
10 mg ·kg1) 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 2.7M.
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 ·kg1;P<0.01 for 10mg ·kg1) 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 ·kg1) was 27.2–33.5%, which was significantly enhanced (P<0.05
for 2.5 mg ·kg1;P<0.01 for 10 mg ·kg1) 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 ·kg1) 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 ·kg1) to rats
in the presence or absence of curcumin (0.5, 2.5 and 10 mg ·
kg1) 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 ·kg1) to rats
in the presence or absence of curcumin
Parameter Control Tamoxifen+ Curcumin
0.5 mg ·kg12.5 mg ·kg110 mg ·kg1
AUC0–(ng·h·ml1) 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 ·kg1;P<0.01 for
10 mg ·kg1) 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 ·kg1) was 27.2–33.5%, which was
significantly enhanced (P<0.05 for 2.5 mg ·kg1;P<0.01 for
10 mg ·kg1) 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 ·kg1) to rats in the presence or absence of curcumin
(0.5, 2.5 and 10 mg ·kg1) 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 ·kg1of 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 20M 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 ·kg1) in the absence and presence of curcumin (2.5 and
10 mg ·kg1) 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 ·
kg1) administration of tamoxifen to rats in the presence or absence of
curcumin (0.5, 2.5 and 10 mg ·kg1)(n= 6, each). Bars represent the
standard deviation. () Oral administration of tamoxifen (9 mg ·kg1); ()
the presence of 0.5 mg ·kg1of curcumin; () the presence of 2.5mg ·kg1
of curcumin; () the presence of 10 mg ·kg1of curcumin
126 Pharmazie 67 (2012)
ORIGINAL ARTICLES
Table 2: Mean (±S.D.) pharmacokinetic parameters of 4-hydroxytamoxifen after the oral administration of tamoxifen (9mg ·
kg1) to rats in the presence or absence of curcumin
Parameter Control Tamoxifen+ Curcumin
0.5 mg ·kg12.5 mg ·kg110 mg ·kg1
AUC0–(ng ·h·ml1) 284 ±62 301 ±66 315 ±70 334 ±74
Cmax (ng ·ml1) 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 ·kg1)
to rats in the presence or absence of curcumin
Parameters Control Tamoxifen+ Curcumin
2.5 mg ·kg110 mg ·kg1
AUC0−∞ (ng ·h·ml1) 1792 ±326 1898 ±371 1998 ±406
CLt(mL ·min1·kg1) 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(h1) 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 ·kg1) administration of tamoxifen to rats in the presence or absence
of curcumin (0.5, 2.5 and 10 mg ·kg1)(n= 6, each). Bars represent the
standard deviation. () Oral administration of 4-hydroxytamoxifen (9 mg ·
kg1); () the presence of 0.5 mg ·kg1of curcumin; () the presence of
2.5 mg ·kg1of curcumin; () the presence of 10 mg ·kg1of 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 ·kg1) administration of tamoxifen to rats in the presence or absence
of curcumin (2.5 and 10 mg ·kg1)(n= 6, each). Bars represent the standard
deviation. () Intravenous administration of tamoxifen (2mg ·kg1); () the
presence of 2.5 mg ·kg1of curcumin; () the presence of 10mg ·kg1of
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 ·kg1) 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 ·kg1of 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 ±2C 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 ·kg1of tamoxifen, dissolved in distilled water, 3.0ml
·kg1) without or with 0.5, 2.5 or 10 mg ·kg1of curcumin (mixed in
distilled water, 3.0ml ·kg1), and an i.v. group (2mg ·kg1of tamoxifen,
dissolved in 0.9% NaCl solution, 1.5 ml ·kg1) without or with 2.5 or 10 mg
·kg1of curcumin (mixed in distilled water, 3.0ml ·kg1). 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 8g/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·min1. 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·ml1and 0.5 ng ·ml1, 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·ml1glucose 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.).
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... Several in vitro and in vivo studies on the bioavailability and metabolism of TAM and its metabolite 4-hydroxy-tamoxifen found that morin , silybin (Kim et al., 2010), Epigallocatechin gallate (EGCG) (Shin and hoi, 2009), myricetin (Li et al., 2011b), baicalein (Li et al., 2011a), curcumin (Cho et al., 2012), kaempferol , and quercetin (Shin et al., 2006) significantly changed the pharmacokinetics of oral TAM, resulting in reduced systemic clearance (CL/F); increased the area under the plasma concentration-time curve (AUC 0-∞) , in the peak plasma concentration (Cmax); and increased absolute and relative bioavailability, which may be the result of reducing first-pass metabolism in the intestine and liver. ...
... Among these, morin and kaempferol had no significant effect on the formation of 4-hydroxy-tamoxifen Shin et al., 2008). However, silybin, EGCG, myricetin, baicalein, curcumin, and quercetin significantly changed AUC 0-∞ and the metabolite-maternal ratio (MR) of 4-hydroxy-tamoxifen, indicating that it can significantly increase the bioavailability of TAM and affect the formation of 4-hydroxy-tamoxifen (Shin et al., 2006;Shin and Choi, 2009;Kim et al., 2010;Li et al., 2011a;Li et al., 2011b;Cho et al., 2012). These findings highlight that natural or plant products can interfere with the pharmacokinetics of TAM, as seen in Table 1. ...
... Cho et al., 2012) (Jiang et al., 2013) (Nagaraju et al., 2012) Curcumin (1E,6E)-1,7-bis(4-Hydroxy-3methoxyphenyl)hepta-1,6-diene-3,5dione (Continued on following page) Frontiers in Pharmacology | www.frontiersin.org June 2022 | Volume 13 | Article 847113 ...
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Thesis
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Curcumin, the bioactive component of turmeric (Curcuma longa L.), has been used for thousands of years in traditional medicine for the prevention or treatment of several diseases and symptoms. Nowadays, curcumin is investigated worldwide as a nutritional supplement. To overcome the central limitation of its naturally low oral bioavailability, several formulation strategies have been developed, such as its co-administration with turmeric oils or piperine to inhibit its metabolism and efflux or its incorporation into micelles, cyclodextrin complexes or phospholipid bilayers to improve its stability and solubility. So far, the different formulations have not been compared directly, in one cohort of participants and at equal doses. The present doctoral thesis aimed, for the first time, at a direct comparison of the bioavailability of curcumin in form of a native curcuma extract or seven formulations, namely polysorbate 80 micelles, g-cyclodextrin complexes, liposomes, phytosomes, submicron-particle curcumin or curcumin administered with turmeric oils or piperine, in healthy adults. The project further aimed to investigate several critical factors for curcumin bioavailability in vitro and to explain thereby the observations made in vivo. In a randomized, double-blind crossover trial with 12 healthy participants (6 females, 6 males), curcumin pharmacokinetics, namely AUC (area under the plasma concentration-time curve), Cmax (maximum plasma concentration) and tmax (time to reach Cmax) were compared after administration of a single oral dose of 207 mg curcumin in form of a native curcuma extract or one of the seven formulations. Curcumin incorporated into polysorbate 80 micelles or g-cyclodextrin complexes showed 57-fold and 30-fold improved bioavailability compared to the native extract, whereas all other formulations showed no or minor effects. tmax of the better bioavailable formulations was smaller (1 to 2 hours) compared to all others (up to 7 hours). To compare the formulations regarding their digestion characteristics and transepithelial transport, in vitro digestion experiments followed by Caco-2 cell transport assays were conducted with the formulations normalized to their curcumin content. In parallel to the effects in vivo, curcumin showed higher stability, solubility and micellization efficiency when it was incorporated into polysorbate 80 micelles (100%, 80%, 55%) or g-cyclodextrin complexes (73%, 33%, 23%), whereas curcumin permeability through Caco-2 cell monolayers was not affected by its formulation. In the next study, curcumin efflux, partially mediated by P-glycoprotein (P-gp), was investigated, because the inhibition of curcumin efflux from the intestinal cells back to the intestinal lumen is targeted by the co-administration of curcumin with turmeric oils or piperine. In LS180 (colon adenocarcinoma) cells, native curcuma extract and the seven formulations were studied regarding cellular curcumin uptake within 1 hour and efflux within further 8 hours, as well as their effects on P-gp activity. Independently from its formulation, curcumin inhibited the activity of P-gp. Cellular curcumin uptake and efflux showed significant variability between formulations but no consistent effects. Cellular uptake and efflux may thus not be important for curcumin bioavailability in vivo. Another potential factor influencing bioavailability, that was investigated for native and micellar curcumin, was the time-dependent intracellular distribution in intestinal cells. Uptake and intracellular distribution in Caco-2 cells mainly did not differ between native and micellar curcumin. After 30 minutes, both were localized in lysosomes and mitochondria, after 180 minutes in peroxisomes and native curcumin also in mitochondria. The temporary localization in lysosomes is in line with the involvement of endocytosis in cellular uptake of curcumin. Nevertheless, the intracellular localization of curcumin was not affected by its incorporation into polysorbate 80 micelles. The data generated in this doctoral project thus demonstrate that the incorporation of curcumin into polysorbate 80 micelles or g-cyclodextrin complexes successfully improve its bioavailability. The improved bioavailability of both formulations can be explained by enhanced digestive stability, solubility and micellization efficiency and appears to be independent from post-digestive processes, such as intestinal permeability, cellular uptake, cellular efflux or intracellular distribution. Consequently, the present doctoral thesis delivers relevant information for the therapeutical application of curcumin, for the development of highly bioavailable formulations, as well as the basis for further clinical research on the health beneficial effects of curcumin.
Chapter
The novel coronavirus outbreak caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was recognized in late 2019 in Wuhan, China. Subsequently, the World Health Organization declared coronavirus disease 2019 (COVID-19) as a pandemic on 11 March 2020. The proportion of potentially fatal coronavirus infections may vary by location, age, and underlying risk factors. However, acute respiratory distress syndrome (ARDS) is the most frequent complication and leading cause of death in critically ill patients. Immunomodulatory and anti-inflammatory agents have received great attention as therapeutic strategies against COVID-19. Here, we review potential mechanisms and special clinical considerations of supplementation with curcumin as an anti-inflammatory and antioxidant compound in the setting of COVID-19 clinical research.
Article
Curcumin, a yellow pigment in Asian spice, is a natural polyphenol component of Curcuma longa rhizome. Curcuminoid components include curcumin, demethoxycurcumin (DMC), and bisdemethoxycurcumin (BDMC). Previous studies established curcumin as a safe agent based on preclinical and clinical evaluations and curcuminoids have been approved by the US Food and Drug Administration (FDA) as “Generally Recognized as Safe” (GRAS). The present review collects and summarizes clinical and preclinical studies of curcumin interactions, with an emphasis on the effect of curcumin and curcumin analogs on the mRNA and protein levels of microsomal CYP450 enzymes (phase I metabolism) and their interactions with toxicants, drugs and drug probes. The literature search was conducted using keywords in various scientific databases, including Web of Science, Scopus, PubMed, and Google Scholar. Studies concerning the impact of curcumin and curcumin analogs on microsomal enzyme activity are reviewed and include oral, topical, and systemic treatment in humans and experimental animals, as well as studies from in vitro research. When taken together the data identified some inconsistent results between various studies. The findings showed significant inhibition of CYP450 enzymes by curcumin and its analogs. However such effects often differed when curcumin and curcumin analogs were coadministered with toxicant and other drugs and drug probes. We conclude from this review that herb-drug interactions should be considered when curcumin and curcumin analogs are consumed.
Article
Aims: To evaluate a possible positive association between tamoxifen treatment and the risk of developing idiopathic venous thromboembolism (VTE) in women with breast cancer in the absence of clinical risk factors for venous thromboembolism other than breast cancer itself. Methods: Using information from the large UK-based General Practice Research Database, we identified, within a cohort of more than 10000 women with breast cancer, all women who developed a first-time diagnosis of deep vein thrombosis or pulmonary embolism of uncertain cause between January 1, 1991 and December 31, 1996. In a case-control analysis, we compared their tamoxifen exposure experience prior to the thromboembolic event with that of a randomly selected group of control women with breast cancer who were matched to cases on age, year of the breast cancer diagnosis and calendar time. Results: We identified 25 cases of idiopathic VTE and 172 controls, all of whom had breast cancer, but were otherwise free from other risk factors for VTE. Past tamoxifen exposure was not materially associated with an elevated risk of developing VTE, and we therefore combined never and past users as reference group. The relative risk estimate of VTE for current tamoxifen exposure, as compared with never and past use combined, was 7.1 (95% CI 1.5-33), adjusted for body mass index, smoking status and hysterectomy status. High body mass index was an independent predictor of VTE itself. Conclusions: Our study provides evidence that current use of tamoxifen increases the risk of idiopathic venous thromboembolism.
Conference Paper
Curcumin (diferuloylmethane), the naturally occurring yellow pigment in turmeric and curry, is isolated from the rhizomes of the plant Curcuma longa Linn. Curcumin inhibits tumorigenesis during both initiation and promotion (post-initiation) periods in several experimental animal models. Topical application of curcumin inhibits benzo[a]pyrene (B[a]P)-mediated formation of DNA-B[a]P adducts in the epidermis. It also reduces 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced increases in skin inflammation, epidermal DNA synthesis, ornithine decarboxylase (ODC) mRNA level, ODC activity, hyperplasia, formation of c-Fos, and c-Jun proteins, hydrogen peroxide, and the oxidized DNA base 5-hydroxymethyl-2'-deoxyuridine (HmdU). Topical application of curcumin inhibits TPA-induced increases in the percent of epidermal cells in synthetic (S) phase of the cell cycle. Curcumin is a strong inhibitor of arachidonic acid-induced edema of mouse ears in vivo and epidermal cyclooxygenase and lipoxygenase activities in vitro. Commercial curcumin isolated from the rhizome of the plant Curcuma longa Linn contains 3 major curcuminoids (approximately 77% curcumin, 17% demethoxycurcumin, and 3% bisdemethoxycurcumin). Commercial curcumin, pure curcumin, and demethoxycurcumin are about equipotent as inhibitors of TPA-induced tumor promotion in mouse skin, whereas bisdemethoxycurcumin is somewhat less active. Topical application of curcumin inhibits tumor initiation by B[a]P and tumor promotion by TPA in mouse skin. Dietary curcumin (commercial grade) inhibits B[a]P-induced forestomach carcinogenesis, N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG)-induced duodenal carcinogenesis, and azoxymethane (AOM)-induced colon carcinogenesis. Dietary curcumin had little or no effect on 4-(methylnitosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced lung carcinogenesis and 7,12-dimethylbenz[a]anthracene (DMBA)-induced breast carcinogenesis in mice. Poor circulating bioavailability of curcumin may account for the lack of lung and breast carcinogenesis inhibition. (C) 1998 Wiiey-Liss, Inc.
Article
4-Hydroxytamoxifen is 100 times more potent as an oestrogen receptor antagonist than the parent drug. The aim of this study was to identify the cytochrome P450 enzymes involved in the 4-hydroxylation of tamoxifen by human liver microsomes. Microsomes from each of 10 human livers tested catalysed the reaction [ranges 0.6 to 2.9 pmol/mg protein/min (1 μM) and 6 to 25 pmol/mg protein/min (18 μM)]. Three of the livers with the lowest tamoxifen 4-hydroxylase activity were from poor CYP2D6 metabolisers. Inhibition of activity by quinidine (1 μM), sulphaphenazole (20 μM) and ketoconazole (2 μM) ranged from 0-80%, 0-80% and 12-57%, respectively. The proportion of activity inhibited by quinidine correlated positively with total microsomal tamoxifen 4-hydroxylase activity (rs = 0.89, p < 0.01). Recombinant human CYPs 2D6, 2C9 and 3A4 but not CYPs 1A1, 1A2, 2C19 and 2E1 displayed significant 4-hydroxylase activity. These findings indicate that the 4-hydroxylation of tamoxifen is catalysed almost exclusively by CYPs 2D6, 2C9 and 3A4 in human liver microsomes. However, there was marked between-subject variation in the contribution of these isoforms.
Article
Synopsis Tamoxifen, a non-steroidal antioestrogen, represents a significant advance in treatment of female breast cancer. In trials of tamoxifen as postsurgical adjuvant treatment of early breast cancer, disease-free survival is consistently prolonged, representing an enhanced quality of life in association with tamoxifen’s favourable adverse effect profile. Moreover, overview analysis indicates a survival benefit of approximately 20% at 5 years for all women, most clearly evident in women over 50 years, while a survival benefit independent of menopausal, nodal or oestrogen receptor status has been demonstrated in some individual trials. Thus, for postmenopausal women, tamoxifen is clearly optimal adjuvant treatment, although the relative benefit of adjuvant chemotherapy in node-negative patients requires clarification. A survival benefit for women under 50 has not been clearly demonstrated in overview analysis, but is not precluded by these rather limited data, and adjuvant treatment of premenopausal women with tamoxifen may also warrant serious consideration. Response rates to tamoxifen in advanced breast cancer are around 30 to 35%, increasing with patient selection for oestrogen receptor positivity. Tamoxifen must be regarded as first-line endocrine treatment in postmenopausal women, and may represent an alternative to first-line ovarian ablation in premenopausal women. An emergent role in primary therapy of elderly and frail patients with operable disease is apparent. Tamoxifen is also of benefit following surgery in male breast cancer, and may have a role as first-line endocrine treatment. Tamoxifen also has a potential role in other hormone-sensitive malignancies such as pancreatic carcinoma, and in treatment of benign breast disease. Finally, tamoxifen has a place in treatment of male and female infertility. Tamoxifen is very well tolerated, and discontinuation of therapy because of adverse effects is rarely necessary. The most frequent adverse effects are related to the drug’s anti-oestrogenic activity, and include hot flushes, nausea and/or vomiting, vaginal bleeding or discharge, and menstrual disturbances in premenopausal patients. Thus, tamoxifen continues to play a major role in management of female breast cancer in both early and advanced stages of disease, with a place also in treatment of male breast cancer and of infertility. Pharmacodynamic Studies Tamoxifen is the trans isomer of a triphenylethylene derivative, and is administered orally as the citrate salt. Tamoxifen exhibits complex pharmacological properties, acting as an oestrogen antagonist, or partial or full agonist, depending on the target tissue and the species studied. Predominantly antioestrogenic and partial oestrogenic effects are apparent in humans and in rats, antioestrogenic effects in the chick, and fully oestrogenic effects in the mouse and in the dog. A number of metabolites are active; the N-demethyl metabolite may contribute to the activity of the parent compound in vivo. The tumour growth inhibitory actions of tamoxifen have been investigated in vitro principally using the oestrogen-responsive MCF-7 human breast cancer cell line; in addition there has been extensive research using the DMBA-induced rat mammary carcinoma model. The precise mechanism of tamoxifen’s antitumour activity remains elusive. The classical account involves competitive blockade by tamoxifen of sites on the oestrogen receptor; however, recently the emphasis of research has shifted to address effects of tamoxifen on tumour growth of which postulated mediators include polypeptide growth factors, a specific anti-oestrogen binding site, protein kinase C, and calmodulin. Tamoxifen has complex effects at the hypothalamic-pituitary level, the clinical significance of which is unclear. An elevation in plasma oestrogen levels has been observed in premenopausal patients. Tamoxifen generally exerts weak oestrogen-like effects in postmenopausal patients, with reduction in circulating gonadotrophin and prolactin levels and increases in serum pregnancy zone protein and sex hormone-binding globulin. Other effects reported, of uncertain clinical significance, include reduction in functional antithrombin III activity and increases in serum high density lipoprotein cholesterol; tamoxifen appears not to exert adverse effects on bone mineral content. Pharmacokinetic Studies Data regarding the pharmacokinetics of tamoxifen in humans are incomplete. Following administration of a single oral dose, maximum plasma concentrations of the parent drug and the demethyl metabolite are achieved within several hours. However, with chronic administration, steady-state concentrations are not attained for 3 to 4 weeks. Tamoxifen is highly plasma protein bound at therapeutic concentrations. It undergoes extensive hepatic metabolism; N-demethylation is the principal metabolic pathway, with subsequent sidechain deamination to the primary alcohol. The fraction of the dose excreted as unchanged drug in urine is negligible. Biliary excretion is the main route of elimination; elimination appears to be biphasic, with an initial phase of 7 to 14 hours, and a terminal phase of around 7 days. Therapeutic Trials During the past decade, tamoxifen has been the subject of extensive clinical research in the treatment of female breast cancer. Over 40 trials of tamoxifen as postsurgical adjuvant therapy of early breast cancer are currently in progress. Results published so far consistently indicate prolonged disease-free survival with tamoxifen monotherapy vs no adjuvant treatment or tamoxifen as treatment of first relapse. Furthermore, significant benefit in overall survival is now apparent. An overview analysis of 28 adjuvant trials in a total of 16,513 women reported a reduction in 5-year mortality of 16% among women of all ages. The effect was most apparent in women over 50 years, in whom a highly significant 5.7% absolute difference from controls in 5-year survival was observed. Although a clear benefit in survival was not demonstrated for women under 50, it was not precluded by the limited available data, which involved only 1062 younger women receiving tamoxifen alone without chemotherapy. More prolonged adjuvant therapy with tamoxifen appeared to be more effective, but not significantly so. In several recently published trials an overall survival benefit was observed, independently of menopausal and oestrogen-receptor status, although other trials have reported a highly significant correlation of treatment effect with oestrogen receptor status. In a comparison with adjuvant irradiation menopause in premenopausal patients, a trend towards increased overall survival with tamoxifen was apparent. Adjuvant trials comparing cytotoxic chemotherapy with chemotherapy plus tamoxifen have provided evidence of significant benefit in overall survival with the combination regimen only in women 50 years of age or older, with some indication of an adverse effect of the regimen including tamoxifen on survival of women younger than 50 whose primary tumours were oestrogen or progesterone receptor-negative. The inclusion of tamoxifen has significantly prolonged disease-free survival in most trials, particularly in patients who were postmenopausal, those oestrogen receptor-positive, and those with a greater degree of nodal involvement. Further enhancement of disease-free survival was apparent in a trial which extended the duration of adjuvant treatment to a third year, with the benefit accruing principally to patients 50 years and older, irrespective of the degree of nodal involvement. Trials of chemoendocrine adjuvant therapy with or without concomitant tamoxifen have not so far conclusively indicated benefit with the addition of tamoxifen, but further follow-up is required. The addition of tamoxifen to chemotherapy may be disadvantageous in younger receptor-negative patients. A trial comparing tamoxifen plus radiotherapy with radiotherapy alone in 961 postmenopausal patients demonstrated significantly greater relapse-free survival at 6 years with tamoxifen, especially in oestrogen receptor-positive patients, those with well-differentiated primary tumours, with 4 or more nodes involved, and patients aged 50 to 59 years. There was, however, no significant difference in overall survival according to treatment regimen. In treatment of advanced breast cancer, response rates to tamoxifen and ovarian ablation in premenopausal patients are similar, at 20 to 40%, with a similar duration of response. In postmenopausal patients, the response rate, which increases with patient selection for oestrogen receptor positivity, is similar to that observed with oestrogens, progestins and androgens, and with the aromatase inhibitor aminoglutethimide, but the incidence of adverse effects reported with other additive endocrine therapies is significantly greater than that observed with tamoxifen. Use of combination therapy with fluoxymesterone has provided no advantage over tamoxifen alone in 1 study while a greater number of adverse effects of combination treatment were reported. Trials comparing tamoxifen plus cytotoxic chemotherapy with tamoxifen or chemotherapy alone in postmenopausal patients have not demonstrated any advantage of concurrent treatment, over sequential tamoxifen and chemotherapeutic regimens. Uncontrolled trials have indicated tamoxifen to be of benefit in primary treatment of elderly and frail patients. One recent report of a randomised comparison with surgery described similar rates of local relapse or progression with either treatment, but a second randomised study reported a higher rate of local progression with tamoxifen and a requirement for subsequent surgery in 40% of patients receiving tamoxifen. A trial comparing tamoxifen with radiotherapy in such patients reported a similar incidence of disease progression at 6 months with either therapy, with no difference in time to development of distant metastases or in survival at 30 months’ follow-up. Response rates to tamoxifen in a small number of male patients with breast cancer were around 40%, with a benefit in survival apparent in a trial of tamoxifen following surgery. Survival benefit was also demonstrated in 2 small trials of tamoxifen in pancreatic carcinoma, while responses in small trials in prostatic, ovarian, renal and colorectal carcinomas have not been impressive, and preliminary data regarding the use of tamoxifen in malignant melanoma are inconclusive. Ovulation is achieved in around 70% of patients with anovulatory infertility following tamoxifen treatment, with rates of subsequent pregnancy, where assessed, ranging from 15 to 60%. Administration of tamoxifen to men with idiopathic oligospermia has been associated with subsequent pregnancy rates in partners of 20 to 34%. There is at present considerable interest in the potential of tamoxifen in treatment of benign breast disease. Resolution of symptoms was apparent in 70 to 90% of patients with mastalgia receiving tamoxifen, in 2 preliminary controlled trials, and further investigations are in progress. Adverse Effects Tamoxifen is in general very well tolerated, and discontinuation of therapy because of adverse effects is rarely necessary. The most frequent adverse effects include hot flushes, nausea and/or vomiting, and vaginal bleeding or discharge, while menstrual disturbances occur in a significant proportion of premenopausal patients. Haematological changes such as thrombocytopenia and leucopenia are reported infrequently, but the relationship to treatment is uncertain. CNS disturbances including dizziness, lethargy and depression have also been described, although infrequently. Disease ‘flare’ is apparent in a number of patients early in therapy, but is not necessarily an indication for treatment withdrawal. Thromboembolic events have been reported very rarely, and in contrast, potentiation of the anticoagulant effect of warfarin; ocular disturbances, generally in association with very high dosages, have also been described in rare instances. Dosage and Administration The recommended dosage of tamoxifen, in advanced breast cancer and as adjuvant therapy, is 10mg twice daily, administered orally, increasing to 20mg twice daily if no response. The optimal duration of adjuvant therapy remains to be established, but at present it appears that longer, or indeed indefinite, treatment may be desirable. Tamoxifen should not be administered during pregnancy.
Article
Background: Tamoxifen, a selective estrogen receptor modulator (SERM), is converted to 4-hydroxy-tamoxifen and other active metabolites by cytochrome P450 (CYP) enzymes. Selective serotonin reuptake inhibitors (SSRIs), which are often prescribed to alleviate tamoxifen-associated hot flashes, can inhibit CYPs. In a prospective clinical trial, we tested the effects of coadministration of tamoxifen and the SSRI paroxetine, an inhibitor of CYP2D6, on tamoxifen metabolism. Methods: Tamoxifen and its metabolites were measured in the plasma of 12 women of known CYP2D6 genotype with breast cancer who were taking adjuvant tamoxifen before and after 4 weeks of coadministered paroxetine. We assessed the inhibitory activity of pure tamoxifen metabolites in an estradiol-stimulated MCF7 cell proliferation assay. To determine which CYP isoforms were involved in the metabolism of tamoxifen to specific metabolites, we used CYP isoform-specific inhibitors. All statistical tests were two-sided. Results: We separated, purified, and identified the metabolite 4-hydroxy-N-desmethyl-tamoxifen, which we named endoxifen. Plasma concentrations of endoxifen statistically significantly decreased from a mean of 12.4 ng/mL before paroxetine coadministration to 5.5 ng/mL afterward (difference = 6.9 ng/mL, 95% confidence interval [CI] = 2.7 to 11.2 ng/mL) (P = .004). Endoxifen concentrations decreased by 64% (95% CI = 39% to 89%) in women with a wild-type CYP2D6 genotype but by only 24% (95% CI = 23% to 71%) in women with a variant CYP2D6 genotype (P = .03). Endoxifen and 4-hydroxy-tamoxifen inhibited estradiol-stimulated MCF7 cell proliferation with equal potency. In vitro, troleandomycin, an inhibitor of CYP3A4, inhibited the demethylation of tamoxifen to N-desmethyl-tamoxifen by 78% (95% CI = 65% to 91%), and quinidine, an inhibitor of CYP2D6, reduced the subsequent hydroxylation of N-desmethyl-tamoxifen to endoxifen by 79% (95% CI = 50% to 108%). Conclusions: Endoxifen is an active tamoxifen metabolite that is generated via CYP3A4-mediated N-demethylation and CYP2D6-mediated hydroxylation. Coadministration of paroxetine decreased the plasma concentration of endoxifen. Our data suggest that CYP2D6 genotype and drug interactions should be considered in women treated with tamoxifen.
Article
Context Increasing evidence suggests that cholesterol plays a role in the pathophysiology of Alzheimer disease (AD). For instance, an elevated serum cholesterol level has been shown to be a risk factor for AD.Objective To determine whether patients taking 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins), which are a group of medicines that inhibit the synthesis of cholesterol, have a lower prevalence of probable AD.Design The experiment uses a cross-sectional analysis comparing the prevalence of probable AD in 3 groups of patients from hospital records: the entire population, patients receiving 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (hereafter referred to as the statins), and patients receiving medications used to treat hypertension or cardiovascular disease.Patients The subjects studied were those included in the computer databases of 3 different hospitals for the years October 1, 1996, through August 31, 1998.Main Outcome Measures Diagnosis of probable AD.Results We find that the prevalence of probable AD in the cohort taking statins during the study interval is 60% to 73% (P<.001) lower than the total patient population or compared with patients taking other medications typically used in the treatment of hypertension or cardiovascular disease.Conclusions There is a lower prevalence of diagnosed probable AD in patients taking 2 different 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors—lovastatin and pravastatin. While one cannot infer causative mechanisms based on these data, this study reveals an interesting association in the data, which warrants further study.
Article
Aims To evaluate a possible positive association between tamoxifen treatment and the risk of developing idiopathic venous thromboembolism (VTE) in women with breast cancer in the absence of clinical risk factors for venous thromboembolism other than breast cancer itself. Methods Using information from the large UK-based General Practice Research Database, we identified, within a cohort of more than 10 000 women with breast cancer, all women who developed a first-time diagnosis of deep vein thrombosis or pulmonary embolism of uncertain cause between January 1, 1991 and December 31, 1996. In a case-control analysis, we compared their tamoxifen exposure experience prior to the thromboembolic event with that of a randomly selected group of control women with breast cancer who were matched to cases on age, year of the breast cancer diagnosis and calendar time. Results We identified 25 cases of idiopathic VTE and 172 controls, all of whom had breast cancer, but were otherwise free from other risk factors for VTE. Past tamoxifen exposure was not materially associated with an elevated risk of developing VTE, and we therefore combined never and past users as reference group. The relative risk estimate of VTE for current tamoxifen exposure, as compared with never and past use combined, was 7.1 (95% CI 1.5–33), adjusted for body mass index, smoking status and hysterectomy status. High body mass index was an independent predictor of VTE itself. Conclusions Our study provides evidence that current use of tamoxifen increases the risk of idiopathic venous thromboembolism.
Article
The antiestrogen tamoxifen (Tam or Nolvadex, ICI)—Z-1-[4-[2-(dimethylamino) ethoxy]phenyl]-1,2-diphenyl-1-butene is widely used in treatment of hormone-dependent breast cancer. The drug is extensively metabolized by cytochrome P450 dependent hepatic mixed function oxidase in man, yielding mainly the N-desmethyl metabolite (DMT). This study has been carried out to determine the P450 enzyme involved in the N-oxidative demethylation of Tam in microsomal samples from 25 human livers (23 adults, two children). This metabolic step was inhibited by carbon monoxide up to 75%. Tam was demethylated into DMT with an apparent Km of 98 ± 10 μM; rates varied between 37 and 446 pmol/min/mg microsomal protein. These metabolic rates were strongly correlated with 6β-hydroxylation of testosterone (r = 0.83) and erythromycin N-demethylase (r = 0.75), both activities known to be associated with P450 IIIA enzyme. To further assess whether or not the Tam demethylation pathway is catalysed by the same P450, the inhibitory effect of TST on this reaction was determined. The competitive inhibition had an apparent Ki of 100 ± 10 μM. Drugs such as erythromycin, cyclosporin, nifedipine and diltiazem were shown to inhibit in vitro the metabolism of tamoxifen. Furthermore the P450 IIIA content of liver microsomal samples, measured by Western blot technique using a monoclonal P450NF (nifedipine) antibody, was strongly correlated with DMT formation (r = 0.87). Tam N-demethylase activity was inhibited by more than 65% with polyclonal anti-human anti-P450NF. All these in vitro observations establish that a P450 enzyme of the IIIA sub-family is involved in the oxidative demethylation of tamoxifen in human liver.
Article
A rapid, rugged and fully automated method has been developed for the determination of tamoxifen and its major metabolites in plasma. The system is based upon an in-line extraction process combined with column switching to a coupled analytical column. The plasma sample is deproteinated by the addition of acetonitrile before injection onto a semi-permeable surface (SPS) cyano guard column (1.0 × 0.46 cm I.D.). After washing the guard column briefly with water, the sample is eluted with a mobile phase composed of 35% acetonitrile in 20 mM potassium phosphate buffer (pH 3). The eluent is directed through a cyano analytical column (25 × 0.46 cm I.D.) and a photochemical reactor where the analytes are converted to highly fluorescent phenanthrene derivatives. Tamoxifen, 4-hydroxytamoxifen, N-desdimethyltamoxifen, N-desmethyltamoxifen and tamoxifen-ol are eluted in that order at a flow-rate of 1.0 ml/min. The method has been validated for use in a clinical study utilizing tamoxifen in the treatment of recurrent cerebral astrocytomas.