Abels, Christoph

Bertolini, Marta

Bíró, Tamás

Chéret, Jérémy

Gherardini, Jennifer

Kloepper, Jennifer E.

Oláh, Attila

Paus, Ralf

Ponce, Leslie

Soeberdt, Michael

Journal of Investigative Dermatology

University of Debrecen Electronic Archive

Accepted Manuscript
The Thyroid Hormone Analogue KB2115 (Eprotirome) Prolongs Human Hair Growth
(Anagen) EX VIVO
Attila Oláh, Jennifer Gherardini, Marta Bertolini, Jérémy Chéret, Leslie Ponce,
Jennifer Kloepper, Tamás Bíró, Michael Soeberdt, Christoph Abels, Ralf Paus
PII: S0022-202X(16)31025-9
DOI: 10.1016/j.jid.2016.03.033
Reference: JID 284
To appear in: The Journal of Investigative Dermatology
Received Date: 20 October 2015
Revised Date: 16 March 2016
Accepted Date: 28 March 2016
Please cite this article as: Oláh A, Gherardini J, Bertolini M, Chéret J, Ponce L, Kloepper J, Bíró T,
Soeberdt M, Abels C, Paus R, The Thyroid Hormone Analogue KB2115 (Eprotirome) Prolongs Human
Hair Growth (Anagen) EX VIVO, The Journal of Investigative Dermatology (2016), doi: 10.1016/
j.jid.2016.03.033.
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THE THYROID HORMONE ANALOGUE KB2115 (EPROTIROME) PROLONGS 
HUMAN HAIR GROWTH (ANAGEN) EX VIVO 
Attila Oláh1,2,3, Jennifer Gherardini1#, Marta Bertolini1#, Jérémy Chéret1, Leslie 
Ponce1, Jennifer Kloepper4, Tamás Bíró3,5, Michael Soeberdt2, Christoph Abels2, 
Ralf Paus1,6 
 
1Department of Dermatology, University of Münster, Münster, Germany; 2Dr. August 
Wolff GmbH & Co. KG Arzneimittel, Bielefeld, Germany; 3Department of Physiology, 
Faculty of Medicine, University of Debrecen, Debrecen, Hungary; 4Department of 
Dermatology, University of Lübeck, Lübeck, Germany; 5Department of Immunology, 
Faculty of Medicine, University of Debrecen, Debrecen, Hungary; 6Dermatology 
Research Center, Institute of Inflammation and Repair, University of Manchester, 
Manchester, UK. 
# These authors contributed equally. 
Running title: Eprotirome prolongs anagen 
 
Key words: hair follicle, hair cycle, anagen phase, thyroid hormone analogue 
 
Correspondence: Ralf Paus, MD; The Centre for Dermatology Research, Institute of 
Inflammation and Repair, University of Manchester, Stopford Building, Oxford Road, 
Manchester M13 9PT, UK. Tel: +1 016127551665; e-mail: ralf.paus@manchester.ac.uk 
 
Abbreviations: HF: hair follicle; TH: thyroid hormone; TR: thyroid hormone receptor; T4: 
thyroxine 
 
  
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TO THE EDITOR 
 
Premature termination of the active growth phase of the human hair follicle (HF) cycle 
(anagen) lies at the base of most clinically important hair loss disorders. Therefore, the 
development of effective, topically applied and safe anagen-prolonging agents 
constitutes one of the principle challenges in translational human hair research (Paus, 
2006; s1-s2). 
 
Previously, we had shown that thyroid hormones (THs) significantly prolong anagen 
phase, and retard the onset of apoptosis-driven HF involution (catagen) (van Beek et al., 
2008; Gáspár et al., 2010), strongly supporting the concept that TH receptors (TRs) are 
promising targets for treating HF cycle-based hair loss disorders (Billoni et al., 2000; s3). 
However, due to their potential systemic adverse effects, such as cardiovascular ones 
(Jameson, 2013; s4) and overstimulation of mitochondrial activity (Cioffi et al., 2013), 
administration of “classical” TR agonists, especially those that fully target the 
myocardially expressed TRα (Mullur et al., 2014), are difficult to justify for non-life-
threatening hair loss disorders. 
 
Therefore, in the current study we asked whether a synthetic TR-modulator, KB2115 
(eprotirome), which shows higher affinity for TRβ than for TRα (Shiohara et al., 2012) 
and thus is less likely to exert cardiovascular adverse effects than thyroxine (T4) 
(Ladenson et al., 2010), may also prolong anagen. This was tested in microdissected, 
organ-cultured human scalp HFs in serum-free medium and without addition of natural 
THs (Kloepper et al., 2010; Langan et al., 2015). The study was approved by the Ethics 
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Committee of the University of Lübeck and the University of Münster (reference No. 06-
109, 2014-041-b-N, 2015-602-f-S) and written informed consent was obtained from the 
patients (for details, see Supplementary Materials and methods section and 
Supplementary Figure S1a-f). 
 
First, we probed whether KB2115 stimulated mitochondrial function in the HF epithelium 
by assessing how low concentrations (1 and 100 pM) of KB2115 influence the 
expression of mitochondrially encoded cytochrome C oxidase I (MTCO1; a key enzyme 
of respiratory complex IV). We had previously documented that the immunoreactivity 
intensity of MTCO1 correlates well with respiratory complex IV activity and even 
mitochondrial biogenesis (Knuever et al., 2012; Poeggeler et al., 2010; Vidali et al., 
2014); moreover classical THs up-regulate complex IV activity as well as MTCO1 
immunoreactivity in human HF epithelium (van Beek et al., 2008). This showed that 
KB2115 did not significantly alter MTCO1 immunoreactivity (Supplementary Figure 
S2a-b) suggesting that the tested picomolar concentrations may not exert major effects 
on energy metabolism. 
 
Preliminary results from microarray analysis (data not shown), followed-up by qRT-PCR 
for four selected TH-responsive, mitochondrial biology-related genes (Peroxisome 
Proliferator-Activated Receptor Gamma Coactivator 1 [PGC1]-α and -β as well as 
sarco/endoplasmic reticulum Ca2+-ATPase 2 [SERCA2]) (s5-s7) and mitochondrial 
creatine kinase 1A (CMKT1A) showed that KB2115 induced significantly smaller (if any) 
up-regulation of the above genes in human HFs, compared to T4 (Supplementary 
Figure S3). This supports the concept that KB2115 is less likely to exert unwanted 
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systemic, mitochondrial activity-related adverse effects than T4 (see Supplementary 
Discussion 1 for details). 
 
Therefore, we studied the effects of these (presumably “mitochondrially safe”) 
concentrations of KB2115 on the spontaneous anagen-catagen transition of human 
scalp HFs ex vivo. Quantitative hair cycle histomorphometry (Kloepper et al., 2010) 
showed that KB2115 treatment significantly increased the percentage of anagen HFs 
compared to vehicle controls (Figure 1a). Given that KB2115 thus effectively countered 
the tendency of organ-cultured HFs to spontaneously enter into catagen (Kloepper et al., 
2010; Langan et al., 2015), our findings support the concept that KB2115 may prevent 
or reduce premature catagen entry leading to telogen effluvium in vivo. 
 
The anagen-prolonging effect of KB2115 was supported by the finding that the 
percentage of Ki67+ (i.e. proliferating) cells in the hair matrix was also significantly 
increased; whereas, the percentage of apoptotic (TUNEL+) hair matrix cells remained 
unchanged (Figure 1b-c). 
 
KB2115 also slightly, but non-significantly increased hair bulb melanin content compared 
to vehicle control HFs, as assessed by quantitative Masson-Fontana histochemistry 
(Figure 2a-b). Moreover, KB2115 significantly increased the intrafollicular activity of 
tyrosinase in situ (s8; Figure 2c-d), the rate-limiting enzyme of melanogenesis (s9). 
Since none of the tested KB2115 concentrations significantly altered the histochemically 
detected hair bulb melanin content or tyrosinase activity in situ to controls, when 
exclusively anagen VI HFs were compared with each other (data not shown), this 
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pigmentary effect of KB2115 most likely reflects prolonged anagen-associated 
intrafollicular melanogenesis (Kloepper et al., 2010; s8). However, it deserves further 
scrutiny whether long-term administration of KB2115 may also stimulate HF 
pigmentation in a hair cycle-independent manner and thus may also exert “anti-graying” 
actions. 
 
Finally, we asked, whether and how KB2115 impacts on the two best-studied human 
hair cycle-regulatory growth factors, i.e. transforming growth factor-β2 (TGF-β2), which 
promotes catagen, and insulin-like growth factor-1 (IGF-1), which maintains anagen 
(Fischer et al., 2014; Gáspár et al., 2010; s10-s11). This showed that KB2115 
significantly decreased TGF-β2 protein expression in the outer root sheath (Fig. 2e-f). 
Surprisingly, intrafollicular IGF-1 protein expression was slightly decreased 
(Supplementary Fig. 4a-b), suggesting that KB2115 may prolong anagen in an IGF-1-
independent manner. 
 
That the synthetic TR-modulator, KB2115, which is expected to show reduced adverse 
effects compared to T4, promotes human HF growth ex vivo corroborates the 
recognized anagen-prolonging effect of endogenous THs (van Beek et al., 2008; Billoni 
et al., 2000). The apparently much lower impact of KB2115 than of T4 on mitochondrial 
activity parameters in human HF keratinocytes in situ suggests a favorable profile of 
systemic adverse effects, which may be further reduced by employing a HF-targeting 
topical application vehicle (s12-s13). Also unlike T4 (van Beek et al., 2008), KB2115 did 
not increase HF pigmentation under the current assay conditions. 
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Taken together, our ex vivo pilot data suggest that KB2115 is a promising candidate for 
the future treatment of hair loss disorders characterized by premature catagen entry, 
such as various forms of telogen effluvium and androgenetic alopecia (Paus, 2006; s1) 
ideally after topical application, and that KB2115 may serve as a lead compound for the 
development of other hair growth-promoting agents with reduced toxicity compared to 
T4. However, given that systemic KB2115 treatment may lead to cartilage damage in 
dogs (s14), further rigorous toxicological and pharmacokinetic screening is required 
before KB2115 is applied topically to human skin (see Supplementary Discussion 
section 2 for details).  
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CONFLICT OF INTEREST 
This study was supported by an industry research grant (see Acknowledgement), and 
two of the authors (MS and CA) are employees of the sponsor. AO was employed by the 
sponsor between 02/01/2014 and 01/31/2015, and RP has previously served in a 
consultancy function for the sponsor. MS and CA are named as inventors on a patent 
claiming the use of eprotirome in the prevention and/or treatment of hair disorders. 
 
ACKNOWLEDGEMENT 
This work was supported by Dr. August Wolff GmbH & Co. KG Arzneimittel (Bielefeld, 
Germany). 
  
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REFERENCES 
 
van Beek N, Bodó E, Kromminga A, Gáspár E, Meyer K, Zmijewski MA, et al. Thyroid 
hormones directly alter human hair follicle functions: anagen prolongation and 
stimulation of both hair matrix keratinocyte proliferation and hair pigmentation. J 
Clin Endocrinol Metab. 2008;93(11):4381–8. 
Billoni N, Buan B, Gautier B, Gaillard O, Mahé YF, Bernard BA. Thyroid hormone 
receptor beta1 is expressed in the human hair follicle. Br J Dermatol. 
2000;142(4):645–52. 
Cioffi F, Senese R, Lanni A, Goglia F. Thyroid hormones and mitochondria: with a brief 
look at derivatives and analogues. Mol Cell Endocrinol. 2013;379(1-2):51–61. 
Fischer TW, Herczeg-Lisztes E, Funk W, Zillikens D, Bíró T, Paus R. Differential effects 
of caffeine on hair shaft elongation, matrix and outer root sheath keratinocyte 
proliferation, and transforming growth factor-β2/insulin-like growth factor-1-mediated 
regulation of the hair cycle in male and female human hair follicles in vitro. Br J 
Dermatol. 2014;171(5):1031–43. 
Gáspár E, Hardenbicker C, Bodó E, Wenzel B, Ramot Y, Funk W, et al. Thyrotropin 
releasing hormone (TRH): a new player in human hair-growth control. FASEB J. 
2010;24(2):393–403. 
Jameson JL, editor. Harrison’s Endocrinology. 3rd ed., McGrawHill: New York; 2013. 
Kloepper JE, Sugawara K, Al-Nuaimi Y, Gáspár E, van Beek N, Paus R. Methods in hair 
research: how to objectively distinguish between anagen and catagen in human hair 
follicle organ culture. Exp Dermatol. 2010;19(3):305–12. 
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Knuever J, Poeggeler B, Gáspár E, Klinger M, Hellwig-Burgel T, Hardenbicker C, et al. 
Thyrotropin-releasing hormone controls mitochondrial biology in human epidermis. 
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human mitochondria. FASEB J. 2010;24(5):1525–31. 
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FIGURE LEGENDS 
 
Figure 1 – KB2115 promotes the anagen phase, and increases proliferation of matrix 
keratinocytes in human HFs 
(a) Results of microscopic hair cycle staging of three independent donors. Ratio of 
anagen, early, mid and late catagen HFs was calculated in each case. Data are shown 
as mean±SEM of the three donors. (b) Percentages of proliferating (Ki67 positive) and 
apoptotic (TUNEL positive) matrix keratinocytes were determined. Data are expressed 
as mean±SEM of N=36-37 HFs from three donors. *p<0.05 and ***p<0.001 vs. control 
group, respectively. (c) Representative pictures of Ki67 (red) and TUNEL (green) double 
staining following the indicated treatments. Scale bar: 50 µm. Nuclei were 
counterstained by 4’-6-diamidino-2-phenylindole (DAPI; blue). 
 
Figure 2 – KB2115 tends to increase melanin synthesis, significantly increases 
tyrosinase activity, and decreases TGF-β2 immunoreactivity 
(a, c, e) Signal intensities (determined as arbitrary units) of Masson-Fontana (MF) 
histochemistry (a), tyrosinase activity assay (c) and TGF-β2 immunofluorescence (e) 
were normalized to the control. Data are expressed as mean±SEM of N=37-42 (a), 
N=28-39 (c) or N=29-36 (e) HFs of three donors. **p<0.01, ***p<0.001 vs. control group. 
(b, d, f) Representative images of Masson-Fontana (black) histochemistry (b), 
tyrosinase activity (red) assay (d) and TGF-β2 (green) immunofluorescence (f). Nuclei 
were counterstained by hematoxylin (blue) (b) or by DAPI (blue) (d, f). Scale bar: 50 µm. 
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The Thyroid Hormone Analogue KB2115 (Eprotirome) Prolongs Human Hair Growth (Anagen) Ex Vivo

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The Thyroid Hormone Analogue KB2115 (Eprotirome) Prolongs Human Hair Growth (Anagen) Ex Vivo

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