Journal of Dermatology 2020; : 1–2

doi: 10.1111/1346-8138.15512

RESEARCH NOTE

Febuxostat inhibited axillary osmidrosis risk factor ATPbinding cassette transporter C11 in vitro
Yu TOYODA,

Tappei TAKADA, Hiroshi SUZUKI

Department of Pharmacy, The University of Tokyo Hospital, Faculty of Medicine, The University of Tokyo, Tokyo, Japan

Dear Editor,
Human ATP-binding cassette transporter C11 (ABCC11) is arisk factor of axillary osmidrosis (AO) that is characterized byunpleasant body odor from the armpits. The inhibition of
ABCC11 is expected to be useful to overcome AO; however,no ABCC11 inhibitor is currently used clinically.
Humans produce body odor like other animals. Nonetheless,in human culture, strong or specific body odors are sometimes
perceived as undesirable. Among them, AO is a chronic skincondition characterized by unpleasant body odors and excessive sweating from the armpits.1 As is often the case in Asian
countries such as Japan and China, in which people withstrong body odor comprise a minor section of the population,
AO tends to be more strongly disliked and is recognized as adisease.2 However, except for surgical treatments, there has
been no causal therapy for AO. Thus, there is an unmet medical need for the development of a therapy for this unpleasantcondition.
Axillary osmidrosis risk is determined by a non-synonymous
single nucleotide polymorphism, c.538G>A (p.Gly180Arg), in the
ABCC11 gene.1,3,4 Biochemical analyses have revealed that thisamino acid substitution (variant type, Arg180) enhances proteasomal degradation of de novo synthesized ABCC11 protein,which results in the loss of the cellular function of ABCC11.3 As
genetically ABCC11-deficient subjects have little risk of AO,2and functional ABCC11 (wild-type, Gly180) is present in the axillary apocrine glands that secrete a variety of odor precursors,5 it
seems that the inhibition of ABCC11 function may be able tocontribute to the prevention and treatment of AO. However, thisnotion has not yet been demonstrated because clinical investigations have been difficult due to the absence of ABCC11 inhibitors that have been approved for clinical use. Additionally,
contrary to humans, rodents such as mice and rats have no Abcc11 gene;2 thus, it is almost impractical to examine the physiological impact of ABCC11 inhibition through in vivo experiments.
Therefore, the investigation of ABCC11 inhibitors with biologicalsafety is an important issue.
In this study, we conducted an in vitro transport assay using
ABCC11-expressing plasma membrane vesicles to explore
ABCC11 inhibitors. We identified febuxostat, a non-purine
selective inhibitor of xanthine oxidase developed for hyperuricemia management, as a novel ABCC11 inhibitor (Fig. 1).

Detailed information on our methods, including critical materials and resources we used (Table S1), is available inAppendix S1. Briefly, plasma membrane vesicles were prepared from adenovirus-mediated transiently ABCC11-expressing 293A cells. Immunoblotting and an in vitro transport assay
for [1,2,6,7-3H(N)]-dehydroepiandrosterone sulfate (DHEA-S) an
ABCC11 substrate,6 were conducted as described previously.5
To confirm the experimental system, we first examined
ABCC11 expression on the plasma membrane vesicles(Fig. 1a), ATP-dependent DHEA-S transport into the ABCC11expressing plasma membrane vesicles (Fig. 1b) and time profile for ABCC11-mediated DHEA-S transport (Fig. 1c). As
febuxostat inhibited ABCC11 in our preliminary screening, we
further examined the concentration-dependent inhibitory
effects of febuxostat on ABCC11 (Fig. 1d). The half maximal
inhibitory concentration value of febuxostat against the
ABCC11-mediated DHEA-S transport activity was 3.26 lmol/L.
Additionally, at this concentration (3.26 lmol/L), febuxostat had
little effect on the transport activity of ABCC2 (Fig. S1), which
belongs to the same protein family as ABCC11.
We herein demonstrated that febuxostat, a globally useddrug for hyperuricemia treatment, is a novel ABCC11 inhibitor(Fig. 1). To the best of our knowledge, this is the first report of
ABCC11 inhibition by a drug approved for clinical use on themarket. As human axillary apocrine glands open into the hairfollicles, the dermal administration of febuxostat onto the armpits, such as through a medical cream, may inhibit ABCC11 inhumans. For this to be effective, such treatment must ensure
appropriate levels of febuxostat in the ABCC11-expressing
apocrine glands. On the other hand, a previous study has
shown that the maximum plasma unbound concentration offebuxostat is 0.126 lmol/L when administrated to healthy subjects at a dose of 40 mg once daily for up to 12 consecutive
days,7 given that the unbound fraction of febuxostat is 0.022.
This suggests that p.o. administrated febuxostat has little inhibitory effect on ABCC11 in current clinical situations under theassumption of a uniform of febuxostat level throughout the
body. Based on this, our data should be interpreted carefully.
However, despite the limitation on its practical use, our discovery will pave the way for the first proof of concept study inhumans to investigate the effect of ABCC11 inhibition on the
AO phenotype. Such study will be worth consideration in the

Correspondence: Yu Toyoda, Ph.D., Department of Pharmacy, The University of Tokyo Hospital, Faculty of Medicine, The University of Tokyo,
7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan. Email: ytoyoda-tky@umin.ac.jp
Received 6 April 2020; accepted 18 June 2020.

© 2020 Japanese Dermatological Association1

Y. Toyoda et al.

ACKNOWLEDGMENTS: This study was supported by JSPS
KAKENHI (grant no. 19K16441) to Y. T. The authors thank Mr Hiroki
Saito for his technical assistance. Author contributions are as follows:
Y. T. was responsible for conceptualization, methodology, resources,
validation, formal analysis, investigation, original draft preparation,
review and editing, funding acquisition, and project administration; T. T.
was also responsible for review and editing; H. S. supervised the study.

CONFLICT OF INTEREST: The authors have a patentpending related to the work reported in this article.

REFERENCES

Figure 1. Febuxostat inhibited ATP-binding cassette transporter C11 (ABCC11) in vitro. (a) Expression of ABCC11 on
plasma membrane vesicles. Plasma membrane vesicles were
subjected to immunoblot analysis using an ABCC11 antibody
or anti-Na+/K+-ATPase antibody. Na+/K+-ATPase, loading control. (b) [1,2,6,7-3H(N)]-dehydroepiandrosterone sulfate (DHEAS) transport activities. Plasma membrane vesicles were incubated with or without ATP for 5 min. (c) Time-dependent
increase in DHEA-S transport by ABCC11. DHEA-S transport
into membrane vesicles was measured at the indicated periods
with or without ATP; the ATP-dependent DHEA-S transport
was calculated by subtracting the DHEA-S transport activity inthe absence of ATP from that in the presence of ATP. (d) Concentration-dependent inhibition of ABCC11-mediated DHEA-S
transport by febuxostat. The DHEA-S transport activities were
measured in the presence of febuxostat at the indicated concentrations for 5 min. Data are expressed as the mean  standard deviation. (b,c) n = 3 and (d) 4. Statistical analyses for
significant differences were performed using Bartlett’s test, followed by a parametric Tukey–Kramer multiple comparison test(**P < 0.01 vs the other groups; NS, not significantly different
among groups in [b]) or Dunnett’s test (**P < 0.01 vs 0 lmol/Lcontrol in [d]); statistical analyses for significant differences
between groups in each time point were performed using a
two-sided t-test (††P < 0.01 in [c]). M.W., molecular weight.
near future. We believe that our results shed light on a new
possibility of overcoming AO by drug repurposing.

2

1 Inoue Y, Mori T, Toyoda Y et al. Correlation of axillary osmidrosis toa SNP in the ABCC11 gene determined by the Smart Amplification
Process (SmartAmp) method. J Plast Reconstr Aesthet Surg 2010;
63: 1369–1374.
2 Ishikawa T, Toyoda Y, Yoshiura K, Niikawa N. Pharmacogenetics ofhuman ABC transporter ABCC11: new insights into apocrine gland
growth and metabolite secretion. Front Genet 2013; 3: 306.
3 Toyoda Y, Sakurai A, Mitani Y et al. Earwax, osmidrosis, and breast
cancer: why does one SNP (538G>A) in the human ABC transporter
ABCC11 gene determine earwax type? FASEB J 2009; 23:
2001–2013.
4 Martin A, Saathoff M, Kuhn F, Max H, Terstegen L, Natsch A.
A functional ABCC11 allele is essential in the biochemical formation of human axillary odor. J Invest Dermatol 2010; 130:
529–540.
5 Toyoda Y, Takada T, Gomi T, Nakagawa H, Ishikawa T, Suzuki H.
Clinical and molecular evidence of ABCC11 protein expression inaxillary apocrine glands of patients with axillary osmidrosis. Int J Mol
Sci 2017; 18: 417.
6 Chen ZS, Guo Y, Belinsky MG, Kotova E, Kruh GD. Transport of bile
acids, sulfated steroids, estradiol 17-beta-D-glucuronide, and leukotriene C4 by human multidrug resistance protein 8 (ABCC11). Mol
Pharmacol 2005; 67: 545–557.
7 Khosravan R, Grabowski BA, Wu JT, Joseph-Ridge N, Vernillet L.
Pharmacokinetics, pharmacodynamics and safety of febuxostat, a
non-purine selective inhibitor of xanthine oxidase, in a dose escalation study in healthy subjects. Clin Pharmacokinet 2006; 45:
821–841.

SUPPORTING INFORMATION
Additional Supporting Information may be found in the online
version of this article:
Appendix S1. Supplementary Methods.
Figure S1. Febuxostat hardly affected ATP-binding cassettetransporter C2 (ABCC2) function.
Table S1. Key resources.

© 2020 Japanese Dermatological Association

Supporting Information

Febuxostat inhibited axillary osmidrosis risk factor ATP-binding cassettetransporter C11 in vitro
Yu Toyoda1*, Tappei Takada1, Hiroshi Suzuki1
1

Department of Pharmacy, The University of Tokyo Hospital, Faculty of Medicine, The Universityof Tokyo, Tokyo, Japan* Correspondence: ytoyoda-tky@umin.ac.jp (YT)

Appendix S1: Supplementary Methods
Supplementary Table S1: Key resources
Supplementary Figure S1: Febuxostat hardly affected ABCC2 function
Supplementary References

Appendix S1: Supplementary Methods
Materials
Critical materials and resources used in this study are summarized in Supplementary Table S1. Stocksolution of febuxostat (Tokyo Chemical Industry, Tokyo, Japan) was prepared with dimethyl sulfoxide(DMSO). Recombinant adenoviruses for the expression of human ABCC11 wild-type (NCBI
accession; NM_033151) or EGFP as a control were from our previous study.1 After purification by a
CsCl gradient ultracentrifugation method, the adenovirus titer was determined using an Adeno-X™
Rapid Titer Kit (Clontech Laboratories, Palo Alto, CA, USA) according to the manufacturer’s
instructions. The adenoviruses were stored at −80°C until use. All other chemicals used werecommercially available and of analytical grade.
Cell culture
Human embryonic kidney 293 (HEK293)-derived 293A cells were maintained in Dulbecco’s Modified
Eagle’s Medium (Nacalai Tesque, Kyoto, Japan) supplemented with 10% fetal bovine serum (Biowest,
Nuaillé, France), 1% penicillin-streptomycin (Nacalai Tesque), 2 mM L-Glutamine (Nacalai Tesque),and 1 × Non-Essential Amino Acid (Life Technologies, Tokyo, Japan) at 37°C in a humidified
atmosphere of 5% (v/v) CO2 in air. Adenovirus infection was performed as described previously.1
Preparation of ABCC11-expressing plasma membrane vesicles
Plasma membrane vesicles were prepared from 293A cells infected with the ABCC11-expressing or
EGFP-expressing (control) adenovirus as described previously.1 Obtained plasma membrane vesicles
were rapidly frozen in liquid N2 and stored at −80°C until use. Protein concentration of plasma
membrane vesicles was quantified using a BCA Protein Assay Kit (Pierce, Rockford, IL, USA) withbovine serum albumin as a standard according to the manufacturer’s protocol.
Immunoblotting
Expression of ABCC11 protein in plasma membrane vesicles was examined by immunoblotting as
described previously,1, 2 with minor modifications. Briefly, the prepared samples were
electrophoretically separated on poly-acrylamide gels and transferred to a Hybond® ECLTM
nitrocellulose membrane (GE Healthcare, Buckinghamshire, UK) by electroblotting at 15 V for 70 min.
For blocking, the membrane was incubated in Tris-buffered saline containing 0.05% Tween 20 and 5%
skim milk (TBST-skim milk), at 4°C overnight. Blots were probed with a rat monoclonal anti-ABCC11
antibody (M8I-74; Abcam, Cambridge, MA, USA; diluted 200 fold) and a rabbit polyclonal antiNa+/K+-ATPase α antibody (sc-28800; Santa Cruz Biotechnology, Santa Cruz, CA, USA; diluted 1,000
fold) followed by incubation with a goat anti-rat immunoglobulin G (IgG)-horseradish peroxidase(HRP)-conjugated antibody (NA935V; GE Healthcare; diluted 2,000 fold) and a donkey anti-rabbit
IgG-HRP-conjugated antibody (NA934V; GE Healthcare; diluted 3,000 fold), respectively. All
antibodies were used in TBST-skim milk. HRP-dependent luminescence was developed using the
ECLTM Prime Western Blotting Detection Reagent (GE Healthcare) and detected using a multi-imaging
Analyzer Fusion Solo 4TM system (Vilber Lourmat, Eberhardzell, Germany).
Vesicle transport assay
Experiments to study the in vitro transport of [1,2,6,7-3H(N)]-dehydroepiandrosterone sulfate (DHEAS) (PerkinElmer, Waltham, MA, USA), an ABCC11 substrate,3 into ABCC11-expressing and control
plasma membrane vesicles were performed by using a rapid filtration technique,1, 4 with a minor
modification. Briefly, the plasma membrane vesicles were incubated with 100 nM of [1,2,6,7-3H(N)]DHEA-S in the reaction mixture (10 mM Tris/HCl, 250 mM sucrose, 10 mM MgCl2, 10 mM creatine
phosphate, 1 mg/mL creatine phosphokinase, and 50 mM ATP or AMP as a substitute of ATP at pH27.4) for the indicated periods at 37°C with or without febuxostat at the indicated concentrations. Then,the radioactivity derived from the incorporated DHEA-S was measured. To reduce background signals
derived from nonspecifically adsorbed radiolabeled DHEA-S on the filter membrane (MF-Millipore
Membrane, HAWP02500; 0.45 µm pore size and 25 mm diameter; Millipore, Tokyo, Japan) for thetrapping of plasma membrane vesicles, the filter membranes were incubated with 2 μM of cholesterol(Wako Pure Chemical Industries, Tokyo, Japan) in ice-cold Stop buffer (250 mM Sucrose, 0.1 M NaCl,
2 mM EDTA, and 10 mM Tris-HCl, pH 7.4) that contained 0.2% (v/v) DMSO before use. In thistransport experiment, the transport activity in each group was calculated as incorporated clearance[mL/mg protein/min = incorporated level of DHEA-S (disintegrations per minute (DPM)/mg
protein/min) / DHEA-S level in the incubation mixture (DPM/mL)]. ATP-dependent DHEA-S
transport was calculated by subtracting the DHEA-S transport activity in the absence of ATP from thatin the presence of ATP; ABCC11-mediated DHEA-S transport activity was calculated by subtracting
the ATP-dependent DHEA-S transport activity for control plasma membrane vesicles from that for
ABCC11-expressing plasma membrane vesicles.
To examine the effect of febuxostat on the function of ABCC2 (also known as MRP2), a similar
vesicle transport assay was conducted. For this purpose, we used ABC Transporter Vesicles for human
ABCC2 and control (GenoMembrane, Yokohama, Japan) of which function was confirmed in ourprevious study,5 and estradiol 17β-D-glucuronide, [estradiol-6,7-3H(N)] (PerkinElmer) (100 nM in thereaction mixture that was incubated for 5 min at 37°C) was employed as an ABCC2 substrate.
Calculation of the half-maximal inhibitory concentration (IC50) values
To calculate the IC50 values of febuxostat against DHEA-S transport by ABCC11, the DHEA-S
transport activities were measured in the presence of febuxostat at several concentrations. Then, theABCC11-mediated transport activities were expressed as a percentage of the control (100%). Basedon the calculated values, fitting curves were obtained according to the following formula using theleast-squares methods with Excel 2019 (Microsoft, Redmond, WA, USA) as described previously:6
× Cn
EPredicted value [%] = 100 − � max
�ECn + Cn �
50where, Emax is the maximum effect, EC50 is the half maximal effective concentration, C is theconcentration of test compound, and n is the sigmoid-fit factor. Finally, based on the results, IC50 was
calculated.
Statistical analyses
All statistical analyses were performed using Excel 2019 with Statcel4 add-in software (OMS
publishing Inc., Saitama, Japan). Different statistical tests were used for different experiments as
described in the figure legends. Briefly, when analyzing multiple groups, the similarity of variance
between groups was compared using Bartlett’s test. When passing the test for homogeneity of variance,a parametric Tukey–Kramer multiple-comparison test for all pairwise comparisons or a Dunnett’s test
for comparisons with a control group was used. In the case of a single pair of quantitative data, after
comparing the variances of a set of data using an F-test, an unpaired Student’s t-test was performed.
Statistical significance was defined in terms of P values less than 0.05 or 0.01.
Data Availability Statement
Data supporting the results of this study are included in this published article and its Supplementary
Information or are available from the corresponding author on reasonable request.

3

Supplementary Table S1. Key resources.
REAGENT or RESOURCE
Antibodies
Rat monoclonal anti-MRP8 (ABCC11) antibody[M8I-74]
Rabbit polyclonal anti-Na+/K+-ATPase α antibody
Goat anti-rat IgG-horseradish peroxidase (HRP)conjugate
Donkey anti-rabbit IgG-horseradish peroxidase(HRP)-conjugate
Chemicals, Peptides, and Recombinant Proteins
ABC Transporter Vesicles (Human MRP2)ABC Transporter Vesicles (Control)
Cholesterol
Clear-sol II
Dehydroepiandrosterone sulfate, sodium salt,[1,2,6,7-3H(N)] (60.0 Ci/mmol)
Dimethyl Sulfoxide
Estradiol 17β-D-glucuronide,[estradiol-6,7-3H(N)] (41.8 Ci/mmol)
Febuxostat
Critical Commercial Assays
PierceTM BCA Protein Assay Reagent A
PierceTM BCA Protein Assay Reagent B
Virus strains
ABCC11-expressing adenovirus
EGFP-expressing adenovirus
Recombinant DNA
The complete human ABCC11 cDNA
Experimental Models: Cell Lines
293A
Software and Algorithms
Excel 2019
Statcel4 add-in software

SOURCEIDENTIFIER

Abcam
Santa Cruz
Biotechnology
GE Healthcare

Cat# ab91452;
RRID: AB_2049125
Cat# sc-28800;
RRID: AB_2290063
Cat# NA935V; RRID: AB_772207

GE Healthcare

Cat# NA934V; RRID: AB_772206

GenoMembrane
GenoMembrane
Wako Pure Chemical
Industries
Nacalai Tesque
PerkinElmer

Cat# GM0001
Cat# GM0003
Cat# 034-03002; CAS: 57-88-5

Nacalai Tesque
PerkinElmer

Cat# 13445-74; CAS: 67-68-5
Cat# NET1106

Tokyo Chemical
Industry

Cat# F0847; CAS: 144060-53-7

Thermo Fisher Scientific
Thermo Fisher Scientific

Cat# 23223
Cat# 23224

Toyoda et al, 2017
Toyoda et al, 2017

N/A
N/A

Toyoda et al, 2009

NCBI Reference Sequence:
NM_033151

Invitrogen

R70507Microsoft
OMS publishinghttps://www.microsoft.com/ja-jp/
http://www.oms-publ.co.jp/

Cat# 09136-83
Cat# NET860

4

Supplementary Figure S1.

E217βG transport[μL/mg protein/min]

14
12

c

10
b

8
6
4
2

0
ATP
Feb

c

a/b

a

-

+
-

Mock

-

+
-

+
+

ABCC2

Supplementary Figure S1. Febuxostat hardly affected ABCC2 function.
The estradiol 17β-D-glucuronide, [estradiol-6,7-3H(N)] (E217βG) transport activities were measured
in the presence or absence of febuxostat (Feb) at 3.26 μM for 5 min. Data are expressed as the mean ±SD. n = 3. Statistical analyses for significant differences were performed using Bartlett’s test, followed
by a parametric Tukey–Kramer multiple-comparison test. Different letters indicate significant
differences between groups (P < 0.05).

5

Supplementary References
1
2

3
4
5
6

Toyoda Y, Takada T, Gomi T, Nakagawa H, Ishikawa T, Suzuki H. Clinical and Molecular
Evidence of ABCC11 Protein Expression in Axillary Apocrine Glands of Patients with Axillary
Osmidrosis. Int J Mol Sci 2017; 18: 417.
Toyoda Y, Sakurai A, Mitani Y et al. Earwax, osmidrosis, and breast cancer: why does one
SNP (538G>A) in the human ABC transporter ABCC11 gene determine earwax type? FASEB
journal : official publication of the Federation of American Societies for Experimental Biology
2009; 23: 2001-2013.
Chen ZS, Guo Y, Belinsky MG, Kotova E, Kruh GD. Transport of bile acids, sulfated steroids,
estradiol 17-beta-D-glucuronide, and leukotriene C4 by human multidrug resistance protein 8(ABCC11). Mol Pharmacol 2005; 67: 545-557.
Toyoda Y, Takada T, Suzuki H. Inhibitors of Human ABCG2: From Technical Background to
Recent Updates With Clinical Implications. Front Pharmacol 2019; 10: 208.
Toyoda Y, Takada T, Suzuki H. Halogenated hydrocarbon solvent-related cholangiocarcinoma
risk: biliary excretion of glutathione conjugates of 1,2-dichloropropane evidenced byuntargeted metabolomics analysis. Sci Rep 2016; 6: 24586.
Miyata H, Takada T, Toyoda Y, Matsuo H, Ichida K, Suzuki H. Identification of Febuxostat asa New Strong ABCG2 Inhibitor: Potential Applications and Risks in Clinical Situations. Front
Pharmacol 2016; 7: 518.

6