Identification of Uridine 5′-Diphosphate-Glucuronosyltransferases Responsible for the Glucuronidation of Mirabegron, a Potent and Selective β3-Adrenoceptor Agonist, in Human Liver Microsomes

Article abstract of DOI:10.1007/s13318-017-0450-x

Background and Objectives: Mirabegron is cleared by multiple mechanisms, including drug-metabolizing enzymes. One of the most important clearance pathways is direct glucuronidation. In humans, M11 (O-glucuronide), M13 (carbamoyl-glucuronide), and M14 (N-glucuronide) have been identified, of which M11 is one of the major metabolites in human plasma. The objective of this study was to identify the uridine 5′-diphosphate (UDP)-glucuronosyltransferase (UGT) isoform responsible for the direct glucuronidation of mirabegron using human liver microsomes (HLMs) and recombinant human UGTs (rhUGTs). Methods: Reaction mixtures contained 1–1000?μM mirabegron, 8?mM MgCl₂, alamethicin (25?μg/mL), 50?mM Tris–HCl buffer (pH 7.5), human liver microsome?(HLM) or rhUGT (1.0?mg protein/mL), and 2?mM UDP-glucuronic acid in a total volume of 200?μL for 120?min at 37?°C. HLMs from 16 individuals were used for the correlation study, and mefenamic acid and propofol were used for the inhibition study. Results: Regarding M11 formation, rhUGT2B7 showed high activity among the rhUGTs tested (11.3?pmol/min/mg protein). This result was supported by the correlation between M11 formation activity and UGT2B7 marker enzyme activity (3-glucuronidation of morphine, r²?=?0.330, p?=?0.020) in individual HLMs; inhibition by mefenamic acid in pooled HLMs (IC₅₀?=?22.8?μM); and relatively similar K_m values between pooled HLMs and rhUGT2B7 (1260 vs. 486?μM). Regarding M13 and M14 formation, rhUGT1A3 and rhUGT1A8 showed high activity among the rhUGTs tested, respectively. Conclusions: UGT2B7 is the main catalyst of M11 formation in HLMs. Regarding M13 and M14 formation, UGT1A3 and UGT1A8 are strong candidates for glucuronidation, respectively.

Full text of DOI:10.1007/s13318-017-0450-x

Eur J Drug Metab Pharmacokinet
https://doi.org/10.1007/s13318-017-0450-x
ORIGINAL RESEARCH ARTICLE
Identification of Uridine 50-Diphosphate-Glucuronosyltransferases
Responsible for the Glucuronidation of Mirabegron, a Potent
and Selective b3-Adrenoceptor Agonist, in Human Liver
Microsomes
Kentaro Konishi1
Daisuke Tenmizu1 Shin Takusagawa2
•
•
Ó Springer International Publishing AG, part of Springer Nature 2017
Abstract
mefenamic acid in pooled HLMs (IC50 = 22.8 lM); and
relatively similar Km values between pooled HLMs and
rhUGT2B7 (1260 vs. 486 lM). Regarding M13 and M14
formation, rhUGT1A3 and rhUGT1A8 showed high
activity among the rhUGTs tested, respectively.
Background and Objectives Mirabegron is cleared by
multiple mechanisms, including drug-metabolizing
enzymes. One of the most important clearance pathways is
direct glucuronidation. In humans, M11 (O-glucuronide),
M13 (carbamoyl-glucuronide), and M14 (N-glucuronide)
have been identified, of which M11 is one of the major
metabolites in human plasma. The objective of this study
was to identify the uridine 50-diphosphate (UDP)-glu-
curonosyltransferase (UGT) isoform responsible for the
direct glucuronidation of mirabegron using human liver
microsomes (HLMs) and recombinant human UGTs
(rhUGTs).
Conclusions UGT2B7 is the main catalyst of M11 for-
mation in HLMs. Regarding M13 and M14 formation,
UGT1A3 and UGT1A8 are strong candidates for glu-
curonidation, respectively.
Key Points
Methods Reaction mixtures contained 1–1000 lM mir-
abegron, 8 mM MgCl2, alamethicin (25 lg/mL), 50 mM
Tris–HCl buffer (pH 7.5), human liver microsome (HLM)
or rhUGT (1.0 mg protein/mL), and 2 mM UDP-glu-
curonic acid in a total volume of 200 lL for 120 min at
37 °C. HLMs from 16 individuals were used for the cor-
relation study, and mefenamic acid and propofol were used
for the inhibition study.
Mirabegron O-glucuronide, which is the main
glucuronide among direct glucuronides, was formed
mainly by UGT2B7.
UGT1A3 and UGT1A8 were the candidate isoforms
which catalyzed the formation of mirabegron
carbamoyl-glucuronide and N-glucuronide,
respectively.
Results Regarding M11 formation, rhUGT2B7 showed
high activity among the rhUGTs tested (11.3 pmol/min/mg
protein). This result was supported by the correlation
between M11 formation activity and UGT2B7 marker
enzyme activity (3-glucuronidation of morphine,
r2 = 0.330, p = 0.020) in individual HLMs; inhibition by
1 Introduction
Mirabegron [YM178, 2-(2-amino-1,3-thiazol-4-yl)-N-[4-
(2-{[(2R)-2-hydroxy-2-phenylethyl]amino}ethyl)phenyl]
acetamide] (Fig. 1) is a potent and selective agonist for the
human b3-adrenoceptor (AR) [1], and the first compound of
a new pharmacological class for the treatment of overactive
bladder (OAB) developed by Astellas Pharma Inc. (Tokyo,
Japan). Mirabegron activates b3-ARs on the detrusor
muscle of the bladder to facilitate filling of the bladder and
storage of urine without inhibiting bladder voiding
& Kentaro Konishi
kentaro.konishi@astellas.com
1
Analysis and Pharmacokinetics Research Laboratories, Drug
Discovery Research, Astellas Pharma Inc., 21, Miyukigaoka,
Tsukuba-shi, Ibaraki 305-8585, Japan
2
Clinical Pharmacology, Development, Astellas Pharma Inc.,
2-5-1, Nihonbashi-Honcho, Chuo-ku, Tokyo 103-8411, Japan

K. Konishi et al.
Fig. 1 Chemical structures of
mirabegron, M11, M13, M14,
and internal standard
Mirabegron
Internal standard
M11
M13
M14
contractions [1, 2]. Pivotal phase 3 clinical trials confirmed
that single daily oral administration of mirabegron at 25
and 50 mg is an effective treatment for OAB symptoms,
with a low occurrence of side effects [3–6]. As of March
2017, mirabegron has been launched in about 50 countries,
including Japan, the USA, Europe, and the Asia-Oceania
region.
In humans, three direct glucuronides, M11 (O-glu-
curonide of mirabegron), M13 (carbamoyl-glucuronide of
mirabegron), and M14 (N-glucuronide of mirabegron) have
been identified [7]. Of these, M11 was the most abundant
metabolite in plasma and urine, and was identified as one
of the major metabolites according to the International
Conference on Harmonization of Technical Requirements
for Registration of Pharmaceuticals for Human Use M3
(R2) guidance, since it represented 17% of total drug-re-
lated exposure in plasma in multiple-dose studies at oral
doses of 25–100 mg daily. M13 and M14 were considered
to be minor metabolites in humans, accounting for 1 and
6% of total drug-related exposure in plasma, respectively
[9]. M11 and M13 were formed following both intravenous
and oral administration, while M14 was observed only after
oral administration [10], suggesting that M14 is mainly
formed in the intestine.
In humans receiving a single oral dose of [14-
C]mirabegron (160 mg) as a solution, unchanged mirabe-
gron was the most abundant component of radioactivity,
accounting for about 22% of circulating radioactivity in
plasma [7]. Of the administered radioactivity, 55% was
excreted in urine as the unchanged form (approximately
25%) or metabolites (approximately 30%), and 34% was
recovered in feces almost entirely as the unchanged form.
More than half of the excreted radioactivity in urine was in
metabolites, suggesting that metabolism plays a substantial
role in mirabegron elimination. On the basis of the
metabolites found in urine, major primary metabolic
reactions of mirabegron in humans were estimated to be
amide hydrolysis, followed by glucuronidation and N-
dealkylation or oxidation of the secondary amine [7]. To
date, butyrylcholinesterase (BChE), cytochrome P450
(CYP) 3A4, and CYP2D6 have been identified as respon-
sible for the metabolism of mirabegron [8]. However, the
uridine 50-diphosphate (UDP)-glucuronosyltransferase
(UGT) isoenzymes involved in the glucuronidation of
mirabegron have not been determined.
In the present study, our primary objective was to
identify the UGT isoform responsible for M11 formation in
human liver using recombinant human UGTs (rhUGTs)
and human liver microsomes (HLMs). Our secondary
objective was to identify the isoforms responsible for M13
and M14 formation. Since direct glucuronidation is an
important clearance pathway of mirabegron in humans,
identification of the UGT isoform(s) for M11, M13, and
M14 formation is indispensable for a comprehensive
understanding of the overall metabolism of mirabegron.

Identification of UGTs Responsible for the Glucuronidation of Mirabegron
(mainly tert-butylmethylether) was removed and an aliquot
(100 lL) of the lower layer was added to 40 lL of an
internal standard solution (1 lg/mL). After filtration using
a membrane filter, 5 lL of the filtrate was injected into the
liquid chromatography–tandem mass spectrometry (LC–
MS/MS) system.
2 Materials and Methods
2.1 Chemicals and Reagents
Mirabegron (YM178; Fig. 1), the O-glucuronide of mir-
abegron (M11, YM-382984; Fig. 1), the carbamoyl-glu-
curonide of mirabegron (M13, YM-538859; Fig. 1), the N-
glucuronide of mirabegron (M14, YM-554028; Fig. 1) and
internal standard for the determination of M11, M13, and
M14 concentrations (YM-9674146; Fig. 1) were synthe-
sized at Astellas Pharma Inc (Ibaraki, Japan). Pooled HLM
(mixed gender, pool of n = 50) and a reaction phenotyping
kit of HLMs from 16 individuals were purchased from
XenoTech, LLC (Lenexa, KS, USA). rhUGTs (1A1, 1A3,
1A4, 1A6, 1A7, 1A8, 1A9, 1A10, 2B4, 2B7, 2B10, 2B15,
and 2B17) expressed by baculovirus-infected insect cells
were purchased from BD Biosciences (Woburn, MA, USA).
UGT reaction mix solutions A and B were purchased from
BD Biosciences. Mefenamic acid was purchased from
Sigma-Aldrich (St. Louis, MO, USA). 2,6-Diisopropylphe-
nol (propofol) was purchased from Kanto Chemical (Tokyo,
Japan). Acetonitrile of high-performance liquid chro-
matography (HPLC) grade was purchased from Wako Pure
Chemical Industries (Osaka, Japan). Methanol of HPLC
grade was purchased from Nacalai Tesque (Kyoto, Japan).
Purified water was prepared using a Milli-Q water purifica-
tion system from Merck Millipore (Billerica, MA, USA). All
other chemicals, including dimethyl sulfoxide (DMSO),
were of the appropriate purity commercially available.
2.3 Correlation Analysis for Individual HLMs
M11 formation activity was measured in HLMs prepared
from 16 individual human livers. The activity of each UGT
isoform in each HLM determined by isoform-specific
reaction markers (UGT1A1, 3-glucuronidation of 17b-
estradiol; UGT1A4, glucuronidation of trifluoperazine;
UGT1A9, glucuronidation of propofol; UGT2B7, 3-glu-
curonidation of morphine) was provided by XenoTech,
LLC. Although morphine is known not to be a selective
substrate of UGT2B7, we considered it reasonable to
support the contribution of UGT2B7 because morphine
3-glucuronide is known to be mainly formed by UGT2B7
[12, 13]. The assays were performed as described in the
Sect. 2.2 with 100 lM mirabegron. Correlation between
M11 formation activity and each isoform-specific reaction
marker activity was determined by correlation analysis
using GraphPad Prism version 5 (GraphPad Software Inc.,
San Diego, CA, USA). Correlation coefficient (r) was
calculated by Pearson test and a p value of less than 0.05
was considered to be statistically significant.
2.4 Effects of Typical UGT Inhibitors
on Mirabegron Glucuronidation in Pooled HLM
2.2 In Vitro Assay of Mirabegron Glucuronidation
Incubations for mirabegron glucuronidation by HLMs and
rhUGTs were conducted as described below. Protein con-
centration and incubation time were 1.0 mg protein/mL
and 120 min, respectively. Typical incubation mixtures
contained 1–1000 lM mirabegron for kinetic studies,
8 mM MgCl2, alamethicin (25 lg/mL), 50 mM Tris–HCl
buffer (pH 7.5), enzyme preparation (HLM or rhUGT), and
2 mM UDP-glucuronic acid (UDPGA) in a total volume of
200 lL. It is known that HLM and rhUGTs are fully
activated by the addition of alamethicin [11]. Mirabegron
For UGT inhibitors, mefenamic acid and propofol were
selected. Although these are not selective inhibitors of
UGT2B7 and UGT1A9, respectively [14–20], they were
used to obtain supportive data. The assay procedure is
described in the Sect. 2.2 with 100 lM mirabegron and
mefenamic acid (0–100 lM) or propofol (0–100 lM). The
concentration range of inhibitors was set as sufficient to
inhibit at least UGT2B7 or UGT1A9, respectively, from
the previous literature described above. The percentages of
glucuronidation rate relative to the vehicle control and IC50
values were calculated.
was dissolved in DMSO to prepare a 200 mM mirabegron
solution and the solution was diluted with DMSO to pre-
pare the various concentrations. An aliquot of 1 lL mir-
abegron solution was added to the incubation mixture.
After the incubation mixture without UDPGA was allowed
to stand on ice, the mixture was preincubated for 10 min at
37 °C, and then the reaction was started by adding
UDPGA. After incubation for 120 min, the reaction was
terminated by precipitating proteins via the addition of
2 mL of tert-butylmethylether. The samples were cen-
trifuged for 15 min at 5 °C (18709g). The upper layer
2.5 LC–MS/MS Analysis for Assessment of M11,
M13, and M14 Formation
For the calibration curve, M11, M13, and M14 were dis-
solved in 50% methanol, DMSO, and methanol, respec-
tively, to prepare 1 mg/mL solutions and the solutions were
diluted with 50% methanol to prepare the various con-
centrations. The concentrations of M11, M13, and M14
were determined according to a previous report with minor

K. Konishi et al.
The residual activity (% of control) of M11 formation
was calculated using Microsoft Excel 2007 for Windows
(Microsoft Corporation). The equation is described below:
modifications [21]. Briefly, an LC–MS/MS system com-
posed of an LC-20AD series HPLC (Shimadzu, Kyoto,
Japan) coupled with a triple quadrupole mass spectrometer
using multiple reaction monitoring in positive electrospray
ionization mode (4000QTRAP; AB Sciex, Foster City, CA,
USA) was used. A Synergi Fusion-RP (2.0 9 150 mm
column with 4 lm particle size; Phenomenex, Inc., Tor-
rance, CA, USA) connected to a SecurityGuard Standard,
Fusion-RP (4 9 2.0 mm guard column; Phenomenex, Inc.)
was used. Mobile phase A consisted of a 75 mM ammo-
nium acetate solution, 0.15% formic acid, and purified
water (1:1:8, v/v/v) and mobile phase B was acetonitrile.
The temperature of the column was kept at 40 °C and the
flow rate was 0.5 mL/min. The gradient program was set as
0.00 min (B: 20%) to 4.00 min (B: 40%) in linear mode,
and 4.01 min to 5.00 min was set as isocratic (B: 20%).
The monitoring ions selected were m/z 573.1–146.1 for
M11, m/z 617.1–113.0 for M13, m/z 573.1–146.1 for M14,
and m/z 559.1–246.0 for IS. Retention time of M11, M13,
M14, and internal standard was about 1.8, 2.7, 1.3, and
2.5 min, respectively. Quantitation of the analytes was
performed using Analyst version 1.5 (AB Sciex).
Residual activity of M11 formation ð%Þ
¼ M11 formation activity at each inhibitor concentration=
M11 formation activity in control samples  100:
The values of inhibitor concentration showing the half
formation activity (IC50) were determined by fitting of the
relationship between the residual activity and the inhibitor
concentration using WinNonlin version 6.1 (Pharsight
Corp.) using Eq. (3). When the IC50 value was not
calculated because of high residual activity at all
concentrations of inhibitor or above the highest
concentration tested, the result was expressed as
‘‘IC50[the maximum concentration of the inhibitor’’:
n
E ¼ Emin þ ðEmax À EminÞ=f1 þ ð½IŠ=IC50Þ g;
ð3Þ
where E is the residual activity, Emax is maximum of
residual activity, Emin is minimum of residual activity, I is
inhibitor concentration, and n is the Hill coefficient.
2.6 Kinetic Analyses for M11, M13, and M14
Formation
3 Results
3.1 M11, M13, and M14 Formation in rhUGTs
Microsoft Excel 2007 for Windows (Microsoft Corporation,
Redmond, WA, USA) was used to calculate M11, M13, or
M14 formation activity. The equation is described as below:
UGT1A9 and UGT2B7 showed the highest activity for
M11 formation (13.4 and 11.3 pmol/min/mg protein,
respectively), followed by UGT1A4, 1A8, 1A1, 2B4,
2B17, 1A3, 1A7, and 1A6 (Fig. 2a). No other UGT isoform
(UGT1A10, 2B10, or 2B15) showed M11 formation
activity. Regarding M13 formation, UGT1A3 and
UGT1A9 showed the highest activity (4.03 and
0.220 pmol/min/mg protein, respectively), followed by
UGT1A1, 1A7, 2B15, 1A8, and 2B7 (Fig. 2b). No other
UGT isoform (UGT1A4, 1A6, 1A10, 2B4, 2B10, and
2B17) showed M13 formation activity. M14 formation was
catalyzed mainly by UGT1A8, 1A10, and 1A9 (0.576,
0.184, and 0.177 pmol/min/mg protein, respectively) fol-
lowed by UGT1A1, 1A3, 1A4, 1A7, and 2B7 (Fig. 2c). No
other UGT isoform (UGT1A6, 2B4, 2B10, 2B15, and
2B17) showed M14 formation activity.
[MxŠ
Mx formation activity ¼
 1000
MW Â RT Â [P]
where Mx is M11, M13 or M14 formation activity in
pmol/min/mg protein, [Mx] is M11, M13 or M14
concentration in ng/mL, MW is the molecular weight
of free form of M11, M13 or M14, RT is the reaction time
in min, [P] is the final protein concentration in reaction
mixtures in mg protein/mL.
The enzyme kinetic parameters (Michaelis–Menten
constant Km and maximum velocity Vmax) were determined
by fitting the data to the Michaelis–Menten equation
(Eq. 1) according to a nonlinear least-squares regression
analysis using WinNonlin version 6.1 (Pharsight Corp.;
Mountain View, CA, USA) [22]:
V ¼ Vmax  ½SŠ=ðKm þ ½SŠÞ;
where V is formation activity and [S] is substrate
concentration.
ð1Þ
3.2 Correlation Analyses for M11 Formation Using
Individual HLMs
A significant correlation was observed between M11 for-
mation activity and the marker enzyme activities for
UGT2B7 (r2 = 0.330, p = 0.020; Fig. 3a) and UGT1A4
(r2 = 0.649, p = 0.0002; Fig. 3b), although there was no
correlation between M11 formation activity and that for
The value of intrinsic clearance (CLint) was calculated
using Microsoft Excel 2007 for Windows (Microsoft Cor-
poration) using the following equation:
CLint¼Vmax=Km:
ð2Þ

Identification of UGTs Responsible for the Glucuronidation of Mirabegron
Fig. 2 M11, M13, and M14
formation activities in rhUGTs.
a–c M11, M13, and M14
formation activities in each
rhUGT, respectively. Each
rhUGT (1 mg protein/mL) was
incubated with 100 lM
mirabegron and 2 mM UDPGA
at 37 °C for 120 min. Mean and
SD of triplicate samples are
represented. UGT uridine 50-
diphosphate-
glucuronosyltransferase
Fig. 3 Correlation between
M11 formation activity and
respective UGT marker enzyme
activity in HLMs from 16
individual human livers. a–
d The correlation between M11
formation activity and
UGT2B7, UGT1A4, UGT1A1,
and UGT1A9 marker enzyme
activity, respectively. Individual
HLMs (1 mg protein/mL) were
incubated with 100 lM
mirabegron and 2 mM UDPGA
at 37 °C for 120 min. UGT
marker enzyme activities were
provided by the supplier. Data
represent the mean of triplicate
samples and lines are from
linear regression analysis
UGT1A1 (r2 = 0.099, p = 0.235; Fig. 3c) and UGT1A9
(r2 = 0.031, p = 0.511; Fig. 3d).
not clearly inhibit M11 formation even at the maximum
concentration of 100 lM and the IC50 value was estimated
to be[100 lM (Fig. 4b).
3.3 Effects of Typical UGT Inhibitors on M11
Formation in Pooled HLM
3.4 Kinetics of M11 Formation in Pooled HLM
and rhUGT2B7
M11 formation in pooled HLM was inhibited by mefe-
namic acid and the IC50 value was estimated to be
22.8 6.0 lM (Fig. 4a). On the other hand, propofol did
M11 formation activities in pooled HLM were fitted to the
Michaelis–Menten equation shown in Fig. 5a. Km values

K. Konishi et al.
A
B
120
100
80
60
40
20
0
120
100
80
60
40
20
0
0.1
1
10
100
1000
0.1
1
10
100
1000
Mefenamic acid (μM)
Propofol (μM)
Fig. 4 Effect of UGT inhibitors on M11 formations in pooled HLM.
Pooled HLM (1 mg protein/mL) was incubated with 100 lM
mirabegron, UGT inhibitor (a mefenamic acid, b propofol), and
2 mM UDPGA at 37 °C for 120 min. Formation activity of each (%
of control) represents mean and SD of triplicate samples
for M11 formation were 214 22 pmol/min/mg protein
and 0.169 lL/min/mg protein, respectively. On the other
hand, M11 formation activities in rhUGT2B7 were also
fitted to Michaelis–Menten equation shown in Fig. 5b. Km
values for M11 formation were estimated as 486 40 lM.
The calculated Vmax and CLint values for M11 formation
were 34.0 1.3 pmol/min/mg protein and 0.070 lL/min/
mg protein, respectively.
4 Discussion and Conclusion
We conducted several in vitro studies to identify the UGT
isoforms responsible for the metabolism of mirabegron in
human liver. Several studies were required because it is
generally difficult to draw conclusions from a single study,
due to the limited information about selective UGT sub-
strates/inhibitors and the expression level differences of
each isoform in human liver. Among in vitro studies, the
use of rhUGTs is considered most reliable for the identi-
fication of UGT isoforms, as it is described in the draft
guidance for industry published from the US Food and
Drug Administration (FDA) (CDER, 2012). In the present
study, therefore, we placed the greatest emphasis on the
results of studies using rhUGTs.
The results of study using rhUGTs suggested that various
UGT isoforms were involved in mirabegron glucuronida-
tion (Fig. 2). Regarding M11 formation, UGT1A9 and
UGT2B7 showed the highest activity among the rhUGTs
tested, whereas M11 formation activities via UGT1A1 and
UGT1A4 were negligible. A correlation study using indi-
vidual HLMs indicated the involvement of UGT2B7,
whereas the involvement of UGT1A1 and UGT1A9 was
non-significant (Fig. 3). Mefenamic acid has inhibitory
effects on UGT2B7, UGT1A1, and/or UGT1A9, and the
results of the inhibition study using mefenamic acid sug-
gested the involvement of these UGT isoforms (Fig. 4a).
The involvement of UGT1A1 and UGT1A9 was, however,
Fig. 5 M11 formation by pooled HLM and rhUGT2B7. Michaelis–
Menten plots (a for pooled HLM and b for rhUGT2B7) and Eadie–
Hofstee plots (inset figures) are shown. Pooled HLM or rhUGT2B7
(1 mg protein/mL) was incubated with 1–1000 lM mirabegron and
2 mM UDPGA at 37 °C for 120 min. Mean and SD of triplicate
samples are represented in Michaelis–Menten plots and mean of
triplicate samples are represented in Eadie–Hofstee plots. UGT
uridine 50-diphosphate-glucuronosyltransferase, v formation activity,
s substrate concentration
for M11 formation were estimated as[1000
(1260 205) lM (values in parentheses are extrapolated
values). The extrapolated Vmax and calculated CLint values

Identification of UGTs Responsible for the Glucuronidation of Mirabegron
did not inhibit the M11 formation supported the idea that
M11 formation in HLMs was mainly via UGT2B7, and not
UGT1A9. Furthermore, Km values for M11 formation in
pooled HLM and rhUGT2B7 were relatively similar (1260
[extrapolation value] vs. 486 lM, respectively) (Fig. 5).
Taken together, these results indicate that the main con-
tributor to M11 formation in humans is UGT2B7, whereas
the contribution of UGT1A1 and UGT1A9 seems to be low
(Table 1).
Table 1 Summary of the study results for M11 formation
M11 formation
UGT1A1
UGT1A4
UGT1A9
UGT2B7
rhUGTs study
Correlation study
Inhibition study
Kinetics study
Negative
Negative
Positive
ND
Negative
Positive
ND
Positive
Negative
Negative
ND
Positive
Positive
Positive
Positive
ND
Positive: possibility of contribution to M11 formation, negative: less
possibility of contribution to M11 formation, ND no data
In the correlation studies, a significant correlation was
observed between M11 formation activity and the marker
activity of UGT1A4 in individual HLMs (Fig. 3b). How-
ever, rhUGT1A4 did not show a significant activity for
M11 formation (Fig. 2a). Although no clear reason has
been identified, one possibility for this discrepancy may
reflect inter-correlation between UGT1A4 and 2B7 activi-
ties in individual HLMs, which were used for the correla-
tion experiment (r = 0.683; data provided by XenoTech).
Considering the possibility and the results of study using
rUGT1A4, the correlation observed for UGT1A4 was
considered to be an artifact or coincidence, and the con-
tribution of UGT1A4 to M11 formation in humans was
judged to be low.
denied, for the following reasons: (1) the study using
rUGT1A1 suggested that the contribution of UGT1A1 was
low (Fig. 2a), and no correlation was seen between M11
formation and 17b-estradiol 3-glucuronidation activities in
individual HLMs (Fig. 3a); (2) no correlation was observed
between M11 formation and propofol glucuronidation
activities in individual HLMs (Fig. 3c), and propofol did
not inhibit M11 formation in HLMs at a concentration up to
100 lM (Fig. 4b). Propofol was reported to inhibit not only
UGT1A9 but also UGT2B7. However, the IC50 value for
UGT1A9 inhibition (approximately 50–100 lM) was lower
than that for UGT2B7 inhibition (over 400 lM) [18, 20].
The finding that propofol at a concentration up to 100 lM
M11 (YM-382984)
M14 (YM-554028)
UGT2B7
UGT1A8
M13 (YM-538859)
UGT1A3
BChE
Mirabegron
oxidation +
glucuronidation
oxidation +
glucuronidation
N-dealkylation
M16 (YM-208876)
M12 (YM-538858)
M8 (YM-538853)
M15 (YM-9636324)
Fig. 6 Overall metabolic pathways of mirabegron. UGT uridine 50-diphosphate-glucuronosyltransferase, BChE butyrylcholinesterase

K. Konishi et al.
It was reported that fatty acids were released during the
incubation and influenced an increase in the Km value
regarding UGT2B7 substrates [23]. Although bovine serum
albumin, which binds to fatty acids, was not added in the
incubation buffer in the study, rhUGT2B7 activity was
shown to be high (Fig. 2), meaning that even under the
condition that the reaction via UGT2B7 was somewhat
inhibited, the UGT2B7 contribution to M11 formation was
supported. In addition, considering that steady-state maxi-
mum mirabegron plasma concentration following multiple
50 mg doses (approximately 50 ng/mL; 0.125 lM) [9], is
far from the Km value, M11 formation activity may not
saturate in clinical settings.
the overall metabolic pathways of mirabegron (Fig. 6).
This study will aid the understanding of mirabegron dis-
position and safety in humans.
Acknowledgements The authors would like to sincerely thank Mr.
Tadashi Hashimoto, Dr. Kiyoshi Noguchi, and Dr. Takashi Usui for
their useful suggestions about the experiments and Dr. Toshifumi
Shiraga, Dr. Tsuyoshi Minematsu, Dr. Yasuhisa Nagasaka, Dr.
Takafumi Iwatsubo, Dr. Yoichi Naritomi, Mr. Aiji Miyashita, and Dr.
Kenji Tabata for their contribution to this study.
Author Contributions Participated in research design Mr. Kentaro
Konishi, Dr. Daisuke Tenmizu, and Dr. Shin Takusagawa. Conducted
experiments Mr. Kentaro Konishi. Performed data analysis Mr.
Kentaro Konishi, Dr. Daisuke Tenmizu, and Dr. Shin Takusagawa.
Wrote or contributed to the writing of the manuscript Mr. Kentaro
Konishi, Dr. Daisuke Tenmizu, and Dr. Shin Takusagawa
For M13 and M14 formation, the results of studies using
rhUGTs suggested that UGT1A3 and UGT1A8 were
responsible in humans, respectively (Fig. 2b, c). As
described in Sect. 1, M11 and M13 were formed following
both intravenous and oral administration, while M14 was
observed only after oral administration [10]. From this
observation, M14 appears to be formed mainly in the
human intestine, rather than in the human liver. Indeed, in
studies using rhUGTs, M14 formation was suggested to be
catalyzed by UGT1A8, which is known to be predomi-
nantly expressed in human intestine [24, 25].
Compliance with Ethical Standards
Conflict of interest Mr. Kentaro Konishi, Dr. Daisuke Tenmizu and
Dr. Shin Takusagawa are employees of Astellas Pharma, Japan.
Funding This study was sponsored by Astellas Pharma, Japan.
Editorial support was funded by Astellas Pharma, Japan.
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