Quaternary ammonium-linked glucuronidation of tamoxifen by human liver microsomes and UDP-glucuronosyltransferase 1A4

Article abstract of DOI:10.1016/j.bcp.2004.02.014

Tamoxifen (TAM), a nonsteroidal antiestrogen, is the most widely used drug for chemotherapy of hormone-dependent breast cancer in women. In the present study, we found a new potential metabolic pathway of TAM via N-linked glucuronic acid conjugation for e

Full text of DOI:10.1016/j.bcp.2004.02.014

Biochemical Pharmacology 67 (2004) 2093–2102
Quaternary ammonium-linked glucuronidation of tamoxifen by human
liver microsomes and UDP-glucuronosyltransferase 1A4
a
Teppei Kaku , Kenichiro Ogura , Takahito Nishiyama ,
a
a
a
Tomokazu Ohnuma , Kei Muro , Akira Hiratsuka
b
a,*
a
Department of Drug Metabolism and Molecular Toxicology, School of Pharmacy, Tokyo University of
Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji-shi, Tokyo 192-0392, Japan
b
Division of GI Oncology, National Cancer Center Hospital, 5-1-1 Tsukiji, Tokyo 104-0045, Japan
Received 7 January 2004; accepted 16 February 2004
Abstract
Tamoxifen (TAM), a nonsteroidal antiestrogen, is the most widely used drug for chemotherapy of hormone-dependent breast cancer in
women. In the present study, we found a new potential metabolic pathway of TAM via N-linked glucuronic acid conjugation for excretion
þ
in humans. TAM N -glucuronide was isolated from a reaction mixture consisting of TAM and human liver microsomes fortified with
UDP-glucuronic acid (UDPGA) and identified with a synthetic specimen by high-performance liquid chromatography-electrospray
ionization-mass spectrometry. However, no TAM-glucuronidating activity was detected in microsomes from rat, mouse, monkey, dog, and
2
guinea pig livers. A strong correlation (r ¼ 0:92) was observed between N-glucuronidating activities toward TAM and trifluoperazine, a
probe substrate for human UDP-glucuronosyltransferase (UGT) 1A4, in human liver microsomes from eight donors (five females, three
2
males). However, no correlation (r ¼ 0:02) was observed in the activities between 7-hydroxy-4-(trifluoromethyl)coumarin and TAM.
Only UGT1A4 catalyzed the N-linked glucuronidation of TAM among recombinant UGTs (UGT1A1, UGT1A3, UGT1A4, UGT1A6,
UGT1A9, UGT2B4, UGT2B7, UGT2B15, and UGT2B17) expressed in insect cells. Apparent K values for TAM N-glucuronidation by
m
human liver microsomes and recombinant UGT1A4 were 35.8 and 32.4 mM, respectively. These results strongly suggested that UGT1A4
could play a role in metabolism and excretion of TAM without Phase I metabolism in human liver. TAM N -glucuronide still had binding
þ
þ
affinity similar to TAM itself for human estrogen receptors, ERa and ERb, suggesting that TAM N -glucuronide might contribute to the
biological activity of TAM in vivo.
#
2004 Elsevier Inc. All rights reserved.
Keywords: Tamoxifen; UDP-glucuronosyltransferase; N-Glucuronidation; Human liver microsomes; Estrogen receptor; Antiestrogen
1
. Introduction
[1]. The antiestrogenic activity of TAM is based on com-
peting activity with b-estradiol for estrogen receptors
(ERs). However, TAM is not a pure antiestrogen and
has agonistic properties in some estrogen target tissues
[2]. The paradoxical activity of TAM remains unclear.
Also, TAM was shown to have higher binding affinity to
one isoform of human ER, ERa, than to another isoform,
ERb [3].
Tamoxifen (TAM, 1-[4-(2-dimethylaminoethoxy)phe-
nyl]-1,2-diphenylbut-1(Z)-ene) is a nonsteroidal tripheny-
lethylene antiestrogen that has been widely used for
chemotherapy and chemoprevention of breast cancer,
one of the major causes of cancer-related death in women
Abbreviations: BSA, bovine serum albumin; ESI-TOF-MS, electro-
spray ionization-time of flight-mass spectrometry; ER, estrogen receptor;
GA, glucuronic acid; HRP, horseradish peroxidase; 4-HO-TAM, 4-
hydroxytamoxifen; HPLC, high-performance liquid chromatography;
metabolite Y, 1-[4-(2-hydroxyethoxy)phenyl]-1,2-diphenylbut-1(Z)-ene;
TAM, tamoxifen; HFC, 7-hydroxy-4-(trifluoromethyl)coumarin; TLC,
thin-layer chromatography; UDPGA, UDP-glucuronic acid; UGT, UDP-
glucuronosyltransferase
The metabolism and pharmacokinetics of TAM have
been extensively studied in female patients and animals. In
the human, orally administered TAM is converted to
several metabolites, such as N-desmethyl-TAM, 4-hydro-
xytamoxifen (4-HO-TAM), N-desdimethyl-TAM, 4-
hydroxy-N-desmethyl-TAM, TAM N-oxide and the pri-
mary alcohol named metabolite Y [4–7]. Among these
Phase I metabolites, 4-HO-TAM has been considered to be
*
Corresponding author. Tel.: þ81-426-76-4516; fax: þ81-426-76-4517.
E-mail address: hiratuka@ps.toyaku.ac.jp (A. Hiratsuka).
0006-2952/$ – see front matter # 2004 Elsevier Inc. All rights reserved.
doi:10.1016/j.bcp.2004.02.014

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T. Kaku et al. / Biochemical Pharmacology 67 (2004) 2093–2102
an active metabolite of TAM because of its higher affinity
toward ERs than the parent drug and other side-chain
metabolites [8]. Interestingly, 4-HO-TAM was partially
converted in vivo to its geometrical isomer, cis-4-HO-
TAM, a much less potent antiestrogen that may have weak
estrogenic properties [9].
capable of catalyzing N-glucuronidation of TAM to reveal
a new potential excretion pathway of the anti-cancer drug.
Binding affinity of TAM N -glucuronide to human estro-
gen receptors, ERa and ERb, was also investigated.
þ
OurpreviousstudyonPhaseIImetabolismof4-HO-TAM
showed that the geometrical isomers of 4-HO-TAM were
selectively glucuronidated in the manner of cis @ trans by
human liver microsomes and sulfated in the manner of
trans @ cis by human liver cytosol [10]. We also demon-
strated through the use of recombinant enzymes that such
sulfation and glucuronidation were catalyzed by human
sulfotransferase isoform SULT1A1 and UDP-glucuronosyl-
transferase (UGT) isoform UGT2B15, respectively [10].
A pharmacokinetic study in humans showed that the
major route of TAM excretion was via the feces [11]. In
females, more than 60% of the administered radiolabeled
TAM was excreted as unchanged drug, with most of the
remaining fecal radioactivity due to Phase II conjugated
metabolites. Only 9–14% of radiolabeled TAM was elimi-
nated into urine. Lien et al. [12] reported in an extensive
study on the distribution of TAM and its metabolites in
human biological fluids that bile and urine were rich in
hydroxylated, conjugated metabolites (4-HO-TAM, 4-HO-
N-desmethyl-TAM, and metabolite Y), whereas unconju-
gated 4-HO-TAM and unmetabolized TAM were the pre-
dominant species in feces. However, treatment of the fecal
extract from one patient with b-glucuronidase increased
the concentration of TAM and TAM metabolites, indicat-
ing the existence of glucuronic acid conjugates [12]. They
also noted that significant amounts of ‘‘conjugated TAM,’’
which released TAM by treatment of b-glucuronidase,
could be recovered from bile, although they provided no
further information on conjugated TAM.
2. Materials and methods
2.1. Chemicals
Bovine serum albumin (BSA), b-estradiol, eugenol,
b-glucuronidase (Type VII-A from Escherichia coli,
100 U/ml), D-saccharic acid 1,4-lactone, TAM and UDP-
glucuronic acid (UDPGA) were purchased from Sigma
Chemicals Co. 7-Hydroxy-4-(trifluoromethyl)coumarin
(HFC) was purchased from Kanto Chemicals Co. Ala-
methicin, trifluoperazine and [ C]UDPGA (300 mCi/
mmol) were purchased from ICN Pharmaceuticals, Inc.
Microsomes prepared from insect cells expressing recom-
binant human UGT1A1, UGT1A3, UGT1A6 and UGT2B7
in Sf9 cells were purchased from PanVera Co., and recom-
binant human UGT1A4, UGT1A9, UGT2B4, UGT2B15,
and UGT2B17 expressed in BTI-TN-5B1-4 cells were
purchased from Gentest Co. All other reagents were of
the highest grade commercially available.
14
2.2. High performance liquid chromatography-
electrospray ionization-time of flight-mass spectrometry
(HPLC-ESI-TOF-MS) conditions
HPLC was performed with an HPLC system (Waters
2695, Waters) using a reverse phase column (Capcell Pak
C18 AQ, 2:0 mm  150 mm, 5 mm particles) (Shiseido).
The mobile phase was composed of methanol and 100 mM
ammonium acetate buffer, pH 5.0. Separation was carried
out by a linear gradient from 75 to 100% methanol in
15 min at a flow rate of 0.2 ml/min. The mass spectrometer
used was a Micromass model LCT. For a positive ESI-
Entero-hepatic circulation of unmetabolized TAM as
well as hydroxylated TAM metabolites in patients who
were administered TAM was observed [11]. Therefore, we
hypothesized a possible excretion pathway of TAM
þ
TOF-MS spectrum of synthetic TAM N -glucuronide, the
þ
whereby TAM could be excreted into bile via TAM N -
sample was dissolved in methanol and infused via a syringe
pump at a flow rate of 1 ml/min into the ion source. The
positive ion electrospray needle voltage was 2500 V.
glucuronide. N-Glucuronidation has been known to pro-
duce N-linked glucuronide as urinary and biliary metabo-
lites for more than 30 nitrogen-containing drugs and
chemicals [13]. In particular, N-glucuronidation has been
2.3. NMR spectroscopy
extensively studied in the metabolism of H antihistamine
1
1
and antidepressant drugs with an aliphatic tertiary amine
group, such as chlorpheniramine, diphenhydramine, keto-
tifen, amitriptyline, and imipramine [14–17]. Luo et al.
The 500 MHz H NMR spectra were recorded on a
Bruker model DRX500 at 300 K. The synthetic TAM
þ
N -glucuronide was dissolved in dimethyl sulfoxide-d6
to a concentration of 1 mg/0.5 ml.
þ
[14] reported fecal excretion of cyclizine N -glucuronide
and suggested possible entero-hepatic circulation of the
þ
þ
drug via N -glucuronide metabolites. However, although
2.4. Chemical synthesis of TAM N -glucuronide
TAM has an N,N-dimethylaminoalkyl side chain in its
structure, there is no information on N-glucuronidation
of TAM and TAM metabolites.
In the present study, we investigated whether human
liver microsomes and recombinant UGT isoforms were
Methyl(2,3,4-tri-O-acetyl-a-D-glucopyranosyl bromi-
de)uronate (160 mg, 0.4 mmol) and TAM (100 mg,
0.27 mmol) were dissolved in dichloromethane (0.2 ml)
and stirred for 24 h at room temperature. The organic

T. Kaku et al. / Biochemical Pharmacology 67 (2004) 2093–2102
2095
solvent was removed by evaporation, and the residue was
dissolved in 1 ml methanol. The methanolic solution was
alkalinized with 0.3 ml of 0.5 M aqueous sodium bicarbo-
nate and stirred for 12 h at room temperature to hydrolyze
protecting acetyl groups and carboxyl methyl ester. After
the addition of water (25 ml), the mixture was extracted
three times with the same volume of ether to remove
unreacted TAM. The aqueous layer was adjusted with
supernatant was collected. The supernatant was centri-
fuged at 105,000 Â g for 60 min at 4 8C, and the resultant
pellet was resuspended in 1 ml of the same buffer and used
as the microsomal fraction. Protein concentrations of
microsomal fractions were measured by the method of
Bradford [19] using BSA as a standard.
2.7. Enzyme assay
0
.1N HCl to pH 5.0 and loaded onto a preparative C18
column (1 cm  5 cm, 55–105 mm, Millipore), which was
Determinations of glucuronidating activity of human
liver microsomes and insect cell microsomes expressing
UGT isoforms toward TAM, trifluoperazine, eugenol, and
HFC were carried out as described previously [20] with the
following modifications. Human liver microsomes were
activated with alamethicin (50 mg/mg protein) for 15 min
in an ice bath as described by Fisher et al. [21]. This step
was omitted for the insect cell microsomes, because ala-
methicin did not affect glucuronidating activity of insect
microsomes. The reaction was performed for 1 h at 37 8C
in a mixture consisting of 50 mM Tris–HCl buffer, pH 7.4,
pre-equilibrated with water. Unbound inorganic salts were
washed out with water, and the bound TAM N -glucur-
onide was eluted with methanol. Evaporation of the metha-
þ
1
nolic eluate yielded 11.3 mg of white powder (6.7%). H
NMR (500 MHz, dimethyl sulfoxide-d ): d 0.85 (t, 3H, J
6
7
3
.4 Hz, CH CH ), 2.37 (q, 2H, J 7.4 Hz, CH CH ), 3.12 (s,
2 3 2 3
0
H, N–CH ), 3.15 (m, 1H, H-4 ), 3.18 (s, 3H, N–CH ), 3.27
3
3
0
0
(m, 1H, H-3 ), 3.40 (d, 1H, J 10.1 Hz, H-5 ), 3.54 (m, 1H,
0
H-2 ), 3.77–3.87 (m, 2H, N–CH CH –O), 4.35 (br.t, 2H, J
5
2
2
0
.0 Hz, N–CH CH –O), 4.64 (d, 1H, J 8.8 Hz, H-1 ), 5.45
2 2
0
0
(bs, 1H, OH-3 ), 5.97 (bs, 1H, OH-2 ), 6.68 (d, 2H, J
containing 10 mM MgCl , microsomal protein (50 mg) and
2
14
8
ArH, meta to NCH CH O–), 7.08 (bs, 1H, OH-4 ), 7.11–
.7 Hz, ArH, ortho to NCH CH O–), 6.77 (d, 2H, J 8.7 Hz,
0
2 mM [ C]UDPGA (0.25 mCi) in a final volume of 50 ml.
The reaction was started by the addition of the substrate
(dissolved in 2.5 ml dimethyl sulfoxide) and terminated by
the addition of 50 ml of methanol. After centrifugation at
12,000 Â g for 5 min, 30 ml of the supernatant was applied
2
2
2
2
7
.40 (m, 10H, ArH). 13C NMR (125.7 MHz, dimethyl
sulfoxide-d ): d 13.27, 28.56, 47.99, 49.13, 61.25,
6
6
1
1
1.95, 70.03, 71.37, 76.05, 77.27, 94.61, 113.70, 126.25,
26.68, 127.97, 128.28, 128.92, 129.33, 131.31, 135.66,
37.77, 140.93, 141.71, 143.18, 155.45, 169.97. ESI-TOF-
onto a silica gel 60 F2 thin layer plate (Merck Ltd.)
54
having a preabsorbent sample-spotting area, and developed
with n-butanol/acetone/glacial acetic acid/30% (w/v)
ammonia/water (70:50:9:8:60, v/v/v/v). The radioactivity
of the glucuronide was measured by radioluminography
with a BAS 2000 bioimaging analyzer (Fuji Photo Films
Co., Ltd.). The addition of the b-glucuronidase inhibitor, D-
saccharic acid 1,4-lactone (1 mM), had no effect on UGT
activity. The concentration (5%, v/v) of dimethyl sulfoxide
did not affect the UGT activities of human and insect
microsomes toward all substrates used in the present study.
Determinations of TAM N-glucuronidating activity of liver
microsomes from rats, mice, monkeys, dogs, and guinea
pigs were performed under the same reaction conditions as
described above. Data were expressed as the arithmetic
mean values ꢁ S:D: obtained from at least three replicated
incubations. For determination of apparent kinetic con-
stants for glucuronidation of TAM, substrate concentra-
tions ranging from 10 to 400 mM were used. The kinetic
constants were determined by extrapolation from Michae-
lis–Menten plots.
þ
MS m/z (relative intensity): 548.2629 [M] (100%),
3
C H NO [M] .
72.2281 (33%). Calculated mass is 548.2648 for
þ
3
2
38
7
2
.5. Human liver samples
Liver samples from eight cancer patients (five females,
three males) were obtained from the National Cancer
Center Hospital, Tokyo, Japan. Informed consent was
obtained from each patient prior to study entry. The present
study was approved by an ethics committee of the National
Cancer Center Hospital. All patients had undergone partial
hepatectomy to remove liver metastases of colon cancer.
Pathologically and histologically normal liver samples
used in the study were obtained from normal portions of
removed tissue. All of the fresh samples were rapidly
frozen in liquid nitrogen and stored at ꢀ80 8C before use.
2
.6. Preparation of liver microsomes
þ
Preparation of liver microsomes from human and male
2.8. Identification of TAM N -glucuronide formed by
human liver microsomes in the presence of UDPGA
and female Sprague–Dawley rats (n ¼ 3 each), male ddy
mice (n ¼ 3), male cynomolgus monkeys (n ¼ 2), male
beagle dogs (n ¼ 2), and male Hartley guinea pigs (n ¼ 3)
was performed as follows: approximately 1 g of liver was
homogenized in 4 ml of 50 mM Tris–HCl buffer, pH 7.4,
containing 10 mM MgCl2 [18]. The homogenate was
centrifuged at 10,000 Â g for 30 min at 4 8C, and the
þ
For identification of TAM N -glucuronides formed by
human liver microsomes, the reaction was performed on a
large scale for 2 h. The final volume of the reaction mixture
was increased up to 2 ml, and the TAM concentration was
0.4 mM. The reaction was started by the addition of the

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T. Kaku et al. / Biochemical Pharmacology 67 (2004) 2093–2102
substrate (dissolved in 100 ml of dimethyl sulfoxide) and
terminated by the addition of 2 ml of methanol. After the
termination, the reaction mixture was centrifuged at
reader Model SAFIRE (Tecan Japan Co., Ltd.) at 450 nm
according to the manufacturer’s instructions. The relative
binding affinity of each competitor is taken at the ratio of
IC50 values (concentration of ligand required to reduce the
specific b-estradiol binding by 50%) to that of DES. Data
were expressed as the arithmetic mean values ꢁ S:D:
obtained from at least three replicated assays.
2
000 Â g for 10 min. The supernatant was lyophilized,
and the residue was dissolved with 200 ml methanol for
the analysis by HPLC-ESI-TOF-MS as indicated above.
For treatment with b-glucuronidase, chromatographic frac-
þ
tions containing N -glucuronide were pooled, and the
solvent was evaporated to dryness in vacuo at 40 8C.
The residue obtained was dissolved in 100 ml of 4 mM
sodium phosphate buffer (pH 6.8) containing 10 units of b-
glucuronidase and incubated for 1 h at 37 8C. The incuba-
tion mixture was filtered through a disc filter (Kanto) and
subjected to HPLC performed under the same conditions as
described above.
3. Results
þ
3.1. Chemical synthesis of TAM N -glucuronide
A simple reaction of TAM with methyl(2,3,4-tri-O-
acetyl-a-D-glucopyranosyl bromide)uronate in dichloro-
methane gave the N-linked glucuronide. NMR spectrum
of the glucuronide showed the signal for the anomeric
proton on the sugar ring at d 4.64 ppm as a doublet with a
coupling constant of 8.8 Hz. The chemical shift and cou-
2
.9. Estrogen receptor binding
þ
The competitive binding affinities of TAM, TAM N -
þ
glucuronide, and diethylstilbestrol (DES) to b-estradiol for
human ERa and ERb were assayed using a Ligand Screen-
ing System (TOYOBO Co., Ltd.). Briefly, purified recom-
binant human ERa or ERb was incubated in a microplate
with various concentrations of these ligands in the presence
of b-estradiol (12.5 nM) at 4 8C for 1 h. After incubation,
unbound b-estradiol was allowed to compete with anti-b-
estradiol antibody and horseradish peroxidase (HRP)-
labeled b-estradiol at 4 8C for 1 h. After washing the plate,
the remaining peroxidase-labeled b-estradiol bound on the
well was measured by densitometry using a microplate
pling constant were characteristic of N -linked b-glucur-
onides [13,14,17]. Separation of N,N-dimethyl proton
signals as singlets at d 3.12 and 3.18 ppm indicated that
these methyl groups were unequivalent due to the forma-
tion of the fourth N–C bond.
þ
3.2. Identification of TAM N -glucuronide formed by
human liver microsomes
þ
Enzymatic formation of TAM N -glucuronide by human
liver microsomes was first characterized by HPLC (Fig. 1)
(A)
TAM
(B)
(C)
TAM
0
5
10
15
20 0
5
10
15
20 0
5
10
15
20
Retention time (min)
Retention time (min)
Retention time (min)
þ
Fig. 1. HPLC separation of TAM and TAM N -glucuronide formed by human liver microsomes and recombinant UGT1A4. Human liver microsomes (from
donor F4 in Table 1) (A) or recombinant UGT1A4 (B) was incubated at 37 8C for 2 h with 0.4 mM TAM in the presence of 2 mM UDPGA. Details are
described in the text. Chromatogram C represents the elution profile of synthetic TAM N -glucuronide.
þ

T. Kaku et al. / Biochemical Pharmacology 67 (2004) 2093–2102
2097
100
371.2249 + H
548.2459
+
HOOC
HO
O
N
O
HO
OH
50
Calculated Mr Mass: 548.2648
372.2039
309.6028
394.2062
0
100
200
300
400
500
600
700
800
m/z
þ
Fig. 2. Representative mass spectrum of TAM N -glucuronides formed from TAM by human liver microsomes. The spectrum was taken at the retention time
of 9.4 min in Fig. 1A.
2
and HPLC-ESI-TOF-MS (Fig. 2). Fig. 1 shows represen-
tative HPLC chromatograms of enzymatically formed
correlation (r ¼ 0:92) between TAM N-glucuronidating
activity and trifluoperazine N-glucuronidating activity
2
þ
TAM N -glucuronide by human microsomes and recom-
binant human UGT1A4 in the presence of UDPGA. The
(Fig. 3A). In contrast, no correlation (r ¼ 0:02) was
observed between HFC O-glucuronidation and TAM
N-glucuronidation (Fig. 3B). In addition, there was no
þ
TAM N -glucuronide formed in the reaction mixture had
a retention time of 9.4 min (chromatograms A and B),
which was identical to that of the synthetic specimen
2
correlation (r ¼ 0:01) between trifluoperazine and HFC
conjugating activities.
(
chromatogram C). Treatment with b-glucuronidase of
þ
the N -glucuronide eluted from the HPLC column
afforded TAM, which was identified by HPLC-MS (data
not shown). MS analysis of the eluted TAM N -glucur-
onide showed a mass spectrum (Fig. 2) very similar to that
of the synthetic specimen. The mass spectrum showed
3.4. TAM N-glucuronidation by human UGT isoforms
expressed in insect cells
þ
Recombinant human UGT isoforms (UGT1A1,
UGT1A3, UGT1A4, UGT1A6, UGT1A9, UGT2B4,
þ
an [M ] ion at m/z 548.2459, corresponding to TAM
þ
N -glucuronide and a fragment ion at m/z 372.2039,
corresponding to the parent drug TAM þ H with loss of
Table 1
N-Glucuronidation of TAM by human liver microsomes
the glucuronic acid moiety (176 amu).
Donor
Sex
Age
Specific activities (pmol/min/mg protein)
TAM Trifluoperazine HFC
3.3. TAM N-glucuronidation by human liver
microsomes
F1
F
71
46
29
50
52
54
56
67
27.0 ꢁ 4.5 59.9 ꢁ 5.8
39.1 ꢁ 3.1 71.3 ꢁ 6.4
32.0 ꢁ 2.7 66.6 ꢁ 7.1
36.0 ꢁ 7.2 68.0 ꢁ 2.1
40.4 ꢁ 1.6 72.2 ꢁ 6.4
37.3 ꢁ 5.1 67.1 ꢁ 7.0
36.1 ꢁ 2.1 69.5 ꢁ 4.1
23.2 ꢁ 1.8 50.8 ꢁ 2.5
2757 ꢁ 163
3917 ꢁ 189
4440 ꢁ 248
4074 ꢁ 192
2940 ꢁ 123
5475 ꢁ 251
4942 ꢁ 161
4104 ꢁ 105
F2
F3
F
The rates of N-linked glucuronide formation of TAM
were determined using liver microsomes from eight donors
F
F4
F
F5
F
(five females, F1–F5; three males, M1–M3). Pretreatment
M1
M2
M3
M
M
M
of human liver microsomes with alamethicin increased
activity approximately 36%, while the addition of Triton
X-100 (400 mg/mg protein) into the reaction mixture rather
inhibited activity. As shown in Table 1, TAM N-glucur-
onidating activity varied, ranging from 23.2 to 40.4 pmol/
min/mg protein. The activities toward standard substrates,
trifluoperazine and HFC, also varied among the liver
microsomes from the eight donors, ranging from 50.8 to
TAM (100 mM) was incubated for 1 h with human liver microsomes from
five females (F1–F5) and three males (M1–M3) in the presence of 2 mM
[14C]UDPGA in a final volume of 50 ml of 50 mM Tris–HCl buffer (pH
7.4) containing 10 mM MgCl
2
. Trifluoperazine (200 mM) and HFC
(
50 mM) were used as standard substrates for determining microsomal
activity under the same conditions as stated above except for incubation
time (30 min) for HFC. Radioactive glucuronides formed were determined
by TLC-radioluminography as described in the text. Data are expressed as
the arithmetic mean values ꢁ S:D: of at least three experiments.
72.2 pmol/min/mg and from 2757 to 5475 pmol/min/mg,
respectively. Correlation analysis showed a very strong

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T. Kaku et al. / Biochemical Pharmacology 67 (2004) 2093–2102
80
70
60
50
40
6000
5000
4000
3000
2000
20
30
40
50
20
30
40
50
TAM N-glucuronidation
TAM N-glucuronidation
(A)
(pmol/min/mg protein)
(B)
(pmol/min/mg protein)
Fig. 3. Correlation between glucuronidation of TAM and trifluoperazine or HFC by human liver microsomes from eight donors. Glucuronidating activities
from eight donors (F1–F5 and M1–M3) toward TAM, trifluoperazine and HFC listed in Table 1 were used. Correlation efficiencies were as follows: Panel A,
2
2
TAM- and trifluoperazine-glucuronidating activities (r ¼ 0:92) and Panel B, TAM- and HFC-glucuronidating activities (r ¼ 0:02).
UGT2B7, UGT2B15, and UGT2B17) independently
expressed in insect cells were examined for their ability
to catalyze glucuronidation of TAM in comparison with
activity toward the reference substrates, trifluoperazine and
HFC. Of these recombinant human enzymes, only
UGT1A4 showed TAM and trifluoperazine N-glucuroni-
dating activity (Table 2). In contrast, all UGT isoforms
except for UGT1A4 exhibited glucuronidating activity
toward HFC, which was measured according to the man-
ufacturer’s instructions using the indicated substrates.
(nmol/min/mg protein) of 2:6 ꢁ 0:2 (male rat), 2:5 ꢁ 0:3
(female rat), 3:4 ꢁ 0:7 (male mice), 4:0 ꢁ 0:6 (male mon-
keys), 9:9 ꢁ 0:8 (male dogs), and 8:9 ꢁ 0:6 (male guinea
pigs) at the substrate concentration of 50 mM.
3.6. Kinetic parameters for TAM N-glucuronidation by
human liver microsomes and recombinant UGT1A4
Kinetic analysis of TAM N-glucuronidation was per-
formed using human liver microsomes (from donor F4) and
recombinant UGT1A4. Similar apparent K values were
m
3.5. Species difference in liver microsomal
TAM N-glucuronidation
observed for TAM N-glucuronidation by human micro-
somes (35:8 ꢁ 6:0 mM) and recombinant UGT1A4
(
32:4 ꢁ 4:5 mM). The insect cell microsomes containing
None of the liver microsomes from the experimental
animals showed detectable TAM N-glucuronidating activ-
ity (less than 5 pmol/min/mg protein), whereas these
microsomes showed to have HFC-glucuronidating activity
recombinant UGT1A4 catalyzed TAM N-glucuronidation
with a Vmax value of 401:5 ꢁ 20:8 pmol/min/mg protein
which was approximately 4.5-fold higher than that of
human microsomes (89:3 ꢁ 12:5 pmol/min/mg protein).
Table 2
N-Glucuronidation of TAM by recombinant human UGTs expressed in insect cells
Human UGT isoform
Specific activities (pmol/min/mg protein)
TAM
Trifluoperazine
HFC
a
UGT1A1
UGT1A3
UGT1A4
UGT1A6
UGT1A9
UGT2B4
UGT2B7
UGT2B15
UGT2B17
N.D.
N.D.
N.D.
N.D.
242.3 ꢁ 15.3
56.5 ꢁ 1.5
N.D.
218.2 ꢁ 5.8
N.D.
N.D.
504.3 ꢁ 10.6
N.D.
N.D.
886.0 ꢁ 76.5
1972.1 ꢁ 127
N.D.
N.D.
379.4 ꢁ 10.2
N.D.
N.D.
361.0 ꢁ 18.5
N.D.
N.D.
N.D.
N.D.
287.6 ꢁ 11.5
b
31.9 ꢁ 9.2 (409.0 ꢁ 10.1)
1
4
TAM (100 mM) was incubated for 1 h with insect microsomes expressing each UGT isoform in the presence of 2 mM [ C]UDPGA in a final volume of 50 ml
of 50 mM Tris–HCl buffer (pH 7.4) containing 10 mM MgCl . Trifluoperazine (200 mM) and HFC (50 mM) were used as standard substrates for determining
2
microsomal activity under the same conditions as stated above except for incubation time (30 min) for HFC. Activity toward eugenol (200 mM) was also
determined as the indicated substrate for UGT2B17 with the incubation time of 30 min. Radioactive glucuronides formed were determined by TLC-
radioluminography as described in the text. Data are expressed as the arithmetic mean values ꢁ S:D: of at least three experiments.
a
Not detectable (less than 5 pmol/min/mg protein).
b
Activity toward eugenol.

T. Kaku et al. / Biochemical Pharmacology 67 (2004) 2093–2102
2099
0
0
0
0
0
0
0
.08
.07
.06
.05
.04
.03
.02
0.012
0
.010
.008
0
0.006
.004
.002
0
0
0
.01
-
0.04
A)
-0.02
0
0.02
-
0.04
0.06
0.08
0.1
-0.04
(B)
-0.02
0
0.02
0.04
0.06
0.08
0.1
1
(
1/S (µM)
-1
/S (µM)
1
Fig. 4. Double reciprocal plot for N-linked glucuronidation by human liver microsomes and recombinant UGT1A4. Liver microsomes from donor F4 (Panel
A) or recombinant UGT1A4 (Panel B) were incubated with various concentrations of TAM at 37 8C for 1 h in the presence of 2 mM [14C]UDPGA. For
determination of kinetic parameters, substrate concentrations ranging 10–400 mM were used. Data are expressed as arithmetic mean values of at least three
replicated incubations.
þ
Fig. 5. Competitive binding affinities of DES, TAM, and TAM N -glucuronide for ERa and ERb. Purified recombinant human ERa (Panel A) or ERb (Panel
þ
B) was incubated with various concentrations of DES (*), TAM (&), and TAM N -glucuronide (~) in the presence of b-estradiol (12.5 nM) at 4 8C for 1 h.
The amount of unbound b-estradiol was assayed by absorptiometry based on competition with HRP-labeled b-estradiol to anti-b-estradiol antibody as
described in the text.
No substrate inhibition was observed at the substrate
concentrations used (Fig. 4).
glucuronide, taken at the ratio of IC50 values to that of
DES, were 100, 31.7, and 38.4 for ERa and 100, 35.0, and
38.9 for ERb, respectively.
þ
3.7. Binding affinity of TAM N -glucuronide for
human ERs
4. Discussion
þ
The binding affinities of TAM and TAM N -glucuronide
for human ERa and ERb were determined by competitive
binding analysis in comparison with that of DES. Based on
the concentrations at which binding between b-estradiol
and ERs is reduced to 50% of binding in the absence of a
The present study provides the first evidence of TAM N-
glucuronidation in human liver microsomes in vitro. TAM
N -glucuronide formed in the reaction mixture consisting
þ
of human liver microsomes and TAM in the presence of
UDPGA was identified with the synthetic specimen by
HPLC-ESI-TOF-MS. Substrate specificity of nine iso-
forms of human hepatic UGTs indicated that N-glucuro-
nidation in human liver microsomes was catalyzed only by
UGT1A4. Also revealed was that the activity of TAM N-
glucuronidation of human liver microsomes from eight
individuals strongly correlated with activity to trifluoper-
azine, which was known to be a probe substrate for
þ
competing ligand, TAM and TAM N -glucuronide had
similar affinities for both ERs (Fig. 5). Among the che-
micals tested, DES competed with b-estradiol at the lowest
IC50 values of 18:3 ꢁ 1:0 nM and 31:4 ꢁ 0:9 nM for
human ERa and ERb binding, respectively. TAM and
þ
TAM N -glucuronide competed at similar concentrations
of 57:8 ꢁ 1:6 nM and 47:6 ꢁ 1:8 nM for ERa and
8
The relative binding affinity of DES, TAM, and TAM N -
9:7 ꢁ 3:1 nM and 80:8 ꢁ 2:7 nM for ERb, respectively.
þ
UGT1A4 [22]. The similar K values for N-glucuronidation
m

2100
T. Kaku et al. / Biochemical Pharmacology 67 (2004) 2093–2102
by human liver microsomes (35.8 mM) and by recombinant
UGT1A4 (32.4 mM) also indicated that UGT1A4 was
responsible for human hepatic microsomal N-glucuronida-
tion of TAM. The higher Vmax value for TAM N-glucur-
onidation by recombinant UGT1A4 indicated that the
baculovirus expression system, used for the expression
of UGT1A4, could produce a larger amount of UGT1A4
protein in the insect cell microsomes than that existed in
human liver microsomes.
At least 17 UGT mRNAs are known to exist in the
human. These are divided into two families, UGT1 and
UGT2, consisting of nine and eight isoforms, respectively,
on the basis of amino acid sequence identity. Among these
isoforms, five isoforms of the UGT1A subfamily, UGT1A1
many species [42]. In the rat, the reason for lack of N-
glucuronidating activity is suggested to result from a defect
of an exon encoding the N-terminal part of the UGT
isoform corresponding to human UGT1A4. The mechan-
þ
ism for the lack of N -glucuronidation in other species
remains unclear. Therefore, especially, quaternary ammo-
nium-linked glucuronidation is highly species-dependent
and may be an important elimination pathway for many
tertiary amines in humans.
þ
TAM N -glucuronide showed affinities very similar to
TAM itself for human ERa and ERb as a competitor to
endogenous b-estradiol, suggesting that the formation of
TAM N -glucuronide may not be only for a deactivation
þ
pathway. In contrast, in the case of 4-HO-TAM, O-
glucuronidation greatly reduced the relative binding
affinity of 4-HO-TAM for MCF-7 cytosolic ERs to
1/1000, which indicated that O-glucuronidation of 4-
HO-TAM represented solely a deactivation pathway
[45]. However, no information is available on N-glucur-
onidation of 4-HO-TAM. Our preliminary results indi-
cated that 4-HO-TAM also received N-glucuronidation
by human liver microsomes. These data will be pub-
lished elsewhere.
[23,24], UGT1A3 [25], UGT1A4 [23], UGT1A6 [26],
and UGT1A9 [27], and seven isoforms of the UGT2B
subfamily, UGT2B4 [28–30], UGT2B7 [29,31–33],
UGT2B10 [30], UGT2B11 [34], UGT2B15 [35,36],
UGT2B17 [37,38], and UGT2B28 [39], are known to be
expressed in the human liver. Therefore, we used these five
isoforms of the UGT1A subfamily and four of the isoforms
of the UGT2B subfamily, which are currently commer-
cially available. It should be noted that we have no
evidence of a role in TAM N-glucuronidation by human
hepatic UGT2B10, UGT2B11, and UGT2B28, all of which
were commercially unavailable and were not tested in this
study.
N-Glucuronidation of primary, secondary, and tertiary
amines are known to be catalyzed mainly by UGT iso-
forms, UGT1A3 and/or UGT1A4. N-Glucuronidation of
antihistamic and antidepressant drugs, clozapine, chlor-
promazine, loxapine, amitriptyline, imipramine, and (R)-
and (S)-ketotifens were identified to be catalyzed by both
isoforms UGT1A3 and UGT1A4 [40–42]. Known Km
values for N-glucuronidation by UGT1A4 of these drugs
are 27, 93, 98, 100, 310, and 59, 85 mM, respectively.
However, these drugs are poor substrates for UGT1A3 (Km
values ranged from 96 to 514 mM). Only trifluoperazine
has been shown to be a probe substrate specific for
UGT1A4 [22]. In the present study, TAM was conjugated
to form a quaternary ammonium-linked glucuronide only
by UGT1A4, with a Km value of 32.4 mM. The data
presented here shows that TAM is specifically conjugated
by UGT1A4, and TAM can be a new probe substrate for
this isoform.
TAM is known as a mixed antiestrogen in which the
biocharacter (agonist versus antagonist activity) varies
among different tissues, cells, and promoters [46]. In view
of the structure–binding activity relationship, it is reported
that antiestrogens, such as 4-HO-TAM and raloxifene,
require an alkylaminoethoxy side chain to block estrogen
action [47]. However, the N,N-dimethyl-alkyl side chain of
TAM is not essential for binding to ERs because N-
demethylation or cleavage of the side chain from TAM
reduces but not abolishes ER binding affinity [6]. The
crystal structures of 4-HO-TAM:ER and raloxifene:ER
complexes demonstrated that the antiestrogenic side chain
interacts with aspartate at position 351 in the ER [48].
Mechanistic studies of antiestrogen action using ER
mutants indicated that the amino acid residue Asp351 in
the ERa was critical for interactions with the antiestro-
genic side chain of the antiestogens [48]. Therefore, it is
possible that the N-glucuronidation of TAM might mod-
ulate the antiestrogenic activity of TAM. Further study will
þ
be required to determine whether TAM N -glucuronide
acts as an antagonist or agonist for ER transcriptional
activity. Furthermore, it should be determined whether
þ
Liver microsomes from Sprague–Dawley rats, ddy mice,
cynomolgus monkeys, beagle dogs, and Hartley guinea
N -glucuronide has biological significance in TAM
therapy.
þ
þ
pigs failed to produce detectable amount of TAM N -
Detection and identification of TAM N -glucuronide
from biological fluids of patients administered TAM are
now in progress in our laboratory.
glucuronide under the same incubation conditions. This
glucuronidation unique to humans is similar to findings by
Kassahun et al. [43] on olanzapine N-glucuronidation that
was studied in CD-1 mice, Fischer 344 rats, beagle dogs,
and rhesus monkeys. Other than human UGT1A3 and
UGT1A4, only two rabbit UGT isoforms have been shown
to catalyze glucuronidation of tertiary amines [44],
whereas N-glucuronidation of primary amines occurs in
Acknowledgments
We gratefully acknowledge Dr. Yasuo Shida and Dr.
Chiseko Sakuma of the Analysis Center, Tokyo University

T. Kaku et al. / Biochemical Pharmacology 67 (2004) 2093–2102
2101
human UDP-glucuronosyltransferases and liver microsomes. Drug
Metab Dispos 2000;28:869–72.
of Pharmacy and Life Science, for expert technical assis-
tance with mass and NMR spectrometry.
[
[
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the UDP-glucuronosyltransferase 1A locus is an early event in human
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