Patent Application
20220288038
Humans produce body odor like other animals; nonetheless, in our daily life, strong or specific body odors are sometimes perceived as an undesired constitution. Axillary osmidrosis (AO) is a condition characterized by unpleasant body odors and excessing sweating from the armpits (Wu et al., 1994). As is often the case with Asian countries where persons with strong body odor comprise a minor population, AO tends to be more strongly disliked and recognized as a disease (Toyoda et al., 2016). However, excepting surgical treatments, there was no causal therapy for AO.
Accumulating evidence suggests that ATP-binding cassette transporter C11 (ABCC11) is an AO risk factor (Nakano et al., 2009; Toyoda et al., 2009; Inoue et al., 2010; Martin et al., 2010). Although physiological substrates of ABCC11 remain unclarified, given that ABCC11-deficient subjects have little risk of AO (Ishikawa et al., 2013), together with the presence of functional ABCC11 in the axillary apocrine glands that secrete a variety of odor precursors (Toyoda et al., 2017), inhibition of ABCC11 function can contribute to the prevention and treatment of AO. However, little information has been available for ABCC11 inhibitors; no agent is approved yet for this unpleasant constitution. Here, we serendipitously identified febuxostat a well-used agent for hyperuricemia—as an ABCC11 inhibitor (
Using an in vitro transport system with ABCC11-expresssing plasma. membrane vesicles (
We herein demonstrated that febuxostat—an approved drug on the market—is a novel ABCC11 inhibitor. Given that axillary apocrine glands open into the hair follicles, dermal administration of febuxostat via the armpits like a medical cream inhibits ABCC11 in humans, if such treatment could ensure appropriate levels of febuxostat in the ABCC11-expressing apocrine glands. Since febuxostat is a relatively safe drug, this notion will be worth considering as a future issue. Besides, investigation of ABCC11 inhibitory activities of febuxostat analogues would be encouraged. We believe that our results may shed light on a possibility of overcoming of AO in terms of drug repurposing.
Critical materials and resources used in this study were summarized in Supplementary Table S1. Plasma membrane vesicle preparation from adenovirus mediated-transiently ABCC11-expressing 293A cells, immunoblotting, and in vitro [1,2,6,7-3H(N)]-DHEAS transport assay with ABCC11-expressing plasma membrane vesicles were conducted as described previously (Toyoda et al., 2017). Detailed description of Materials and Methods is provided in Supplementary Information.
The authors declare that they have no conflict of interest.
Conceptualization, YT; methodology, YT; resources, YT; validation, YT; formal. analysis, YT; investigation, YT; writing—original draft preparation, YT; writing—review and editing, YY, TT, and ITS; supervision, I-IS; project administration, YT; funding acquisition, YT.
The study performed by the authors was supported by JSPS KAKENHI Grant Numbers 19K16441 (to YT).
The Supplementary Material for this article can be found online.
Data supporting the findings of this study are included in this published article and its Supplementary Information or are available from the corresponding author on reasonable request.
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Inoue, Y., Mori, T., Toyoda, Y., Sakurai, A., Ishikawa, Mitani, Y., et al. (2010). Correlation of axillary osmidrosis to a SNP in the ABCC11 gene determined by the Smart Amplification Process (SmartAmp) method. J Plast Reconstr Aesthet Sorg 63(8), 1369-1374. doi: 10.1016/j.bjps.2009.06.029.
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Martin, A., Saathoff, M., Kuhn, F., Max, H., Terstegen, L., and Natsch, A. (2010). A functional ABCC11 allele is essential in the biochemical formation of human axillary odor. J Invest Dermatol 130(2), 529-540. doi: 10.1038/jid.2009.254.
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Toyoda, Y., Takada, T., Gomi, T., Nakagawa, H., Ishikawa, T., and Suzuki, H. (2017). Clinical and Molecular Evidence of ABCC11 Protein Expression in Axillary Apocrine Glands of Patients with Axillary Osmidrosis. Int J Mol Sci 18(2), 417. doi: 10.3390/ijms18020417.
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Critical materials and resources used in this study were summarized in Supplementary Table S1. Stock solution of febuxostat (Tokyo Chemical Industry, Tokyo, japan) was prepared with diethyl sulfoxide. Recombinant adenoviruses for the expression of the human ABCC11 WT (NCBI accession; NM_033151) or EGFP as a control were from our previous study (Toyoda et al., 2017). After the purification by a CsCl gradient ultracentrifugation method, the adenovirus titer was determined by using an. Adeno-X™ Rapid Titer Kit (Clontech Laboratories, Palo Alto, Calif., USA) according to manufacturer's instruction; the adenoviruses were stored at −80° C. until use. All the other chemicals used were commercially available and of analytical grade.
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), and 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 (Toyoda et al., 2017).
Membrane vesicles were prepared from 293A cells infected with the ABCC11-expressing or EGFP-expressing (control) adenovirus as described previously (Toyoda et al., 2017). Obtained plasma membrane vesicles were rapidly frozen in liquid N2 and kept at −80° C. until used. Protein concentration of the plasma membrane vesicles was quantified using a BCA Protein Assay Kit (Pierce, Rockford, Ill., USA) with bovine serum albumin (BSA) as a standard according to the manufacturer's protocol.
Expression of ABCC11 protein in the plasma membrane vesicles was examined by immunoblotting as described previously (Toyoda et al., 2009; Toyoda et al,, 2017) with minor modifications. In brief, the prepared samples were electrophoretically separated on poly-acrylamide gels and then transferred to Hybond® ECL™ 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 the anti-ABCC11 antibody (M8I-74; Abeam, Cambridge, Mass., USA; diluted 200 fold) and a rabbit anti-Na+/K+-ATPase α antibody (sc-28800; Santa Cruz Biotechnology, Santa Cruz, Calif., 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 ECL™ Prime Western Blotting Detection Reagent (GE Healthcare) and detected using a multi-imaging Analyzer Fusion Solo 4™ system (Vilber Lourmat, Eberhardzell, Germany).
Experiments to study the in vitro transport of [1,2,6,7-3H(N)]-dehydroepiandrosterone sulfate (DHEAS) (Perkin-Elmer Japan, Tokyo, Japan), an ABCC11 substrate (Chen et al., 2005), into ABCC11-expressing and control vesicles were performed by using a rapid filtration technique (Toyoda et al., 2017; Toyoda et al., 2019) with a minor modification. In brief, the membrane vesicles were incubated with 100 nM of [1,2,6,7-3H(N)]-DHEAS in the reaction mixture (10 mM Tris/HCl, 250 mM sucrose, 10 mM MgCl2, 10 mM creatine phosphate, 1 mg/mL creatine phosphokinase, pH 7.4, and 50 mM ATP or AMP as a substitute of ATP) for indicated periods at 37° C. with or without febuxostat at indicated concentrations. Then, the radioactivity derived from the incorporated DHEAS was measured. To reduce background signals derived from nonspecifically adsorbed radiolabeled DHEAS on filter membrane (MU-Millipore Membrane, HAWP02500; 0.45 μm pore size and 25 mm diameter; Millipore, Tokyo, Japan) for the trapping 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) before use. In this transport experiment, the transport activity in each group was calculated as incorporated clearance [μL/mg protein/min=incorporated level of DHEAS (disintegrations per minute (DPM)/mg protein/min)/DHEAS level in the incubation mixture (DPM/μL)]. ATP-dependent DHEAS transport was calculated by subtracting the DHEAS transport activity in the absence of ATP from that in the presence of ATP.
To address IC50 values of febuxostat against the DHEAS transport by ABCC11, the DHEAS transport activities were measured in the presence of febuxostat at several concentrations. Then, the ABCC11-mediated transport activities were expressed as a. percentage of control (100%). Based on the calculated values, fitting curves were obtained according to the following formula using the least-squares methods with the Excel 2019 (Microsoft, Redmond, Wash., USA) as described previously (Miyata et al., 2016):
Predicted value [%]=100−(Emax×Cn/EC50n+Cn)
where, Emax is the maximum effect, EC50 is the half maximal effective concentration, C is the concentration of test compound, and n is the sigmoid-fit factor. Finally, based on the results, IC50 was calculated.
All statistical analyses were performed by using Excel 2019 (Microsoft Corp., Redmond, Wash., USA) 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 Dunnett's test was used. In the case of a single pair of quantitative data, after comparing the variances of a set of data by F-test, unpaired Student's t-test was performed. Statistical significance was defined in terms of P values less than 0.05 or 0.01.
Chen, Z. S., Guo, Y., Belinsky, M. G., Kotova, E., and Kruh, G. D. (2005). Transport of bile acids, sulfated steroids, estradiol 17-beta-D-glucuronide, and leukotriene C4 by human multidrug resistance protein 8 (ABCC11). Mol Pharmacol 67(2), 545-557. doi: 10.1124/mol104.007138.
Miyata, H., Takada, T., Toyoda, Y, Matsuo, H., Ichida, K., and Suzuki, H. (2016). Identification of Febuxostat as a New Strong ABCG2 Inhibitor: Potential Applications and Risks in Clinical Situations. Front Pharmacol 7, 518. doi: 10.3389/fphar.2016.00518.
Toyoda, Y., Sakurai, A., Mitani, Y., Nakashima, M., Yoshiura, K., Nakagawa, H., et al. (2009). Earwax, osmidrosis, and breast cancer: why does one SNP (538G>A) in the human ABC transporter ABCC11 gene determine earwax type? FASEB J 23(6), 2001-2013. doi: 10.1096/fj.09-129098.
Toyoda, Y., Takada, T., Gomi, T., Nakagawa, H., Ishikawa, T., and Suzuki, H. (2017). Clinical and Molecular Evidence of ABCC11 Protein Expression in Axillary Apocrine Glands of Patients with Axillary Osmidrosis. Int J Mol Sci 18(2), 417. doi.: 10.3390/ijms18020417.
Toyoda, Y., Takada, T., and Suzuki, H. (2019). Inhibitors of Human ABCG2: From Technical Background to Recent Updates With Clinical implications. Front Pharmacol 10, 208. doi: 10.3389/fphar.2019.00208.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/JP2020/031672 | 8/21/2020 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 62890053 | Aug 2019 | US |
