BJD

M E D I C A L D E R MA T O L O GY British Journal of Dermatology

Nonclinical and human pharmacology of the potent and

selective topical retinoic acid receptor-c agonist trifarotene*
J. Aubert,1 D. Piwnica,1 B. Bertino,1 S. Blanchet-Re
thor
e,1 I. Carlavan,1 S. De

ret,1 B. Dreno iD ,2,3 B. Gamboa,1
A. Jomard, A.P. Luzy, P. Mauvais, C. Mounier, J. Pascau, I. Pelisson, T. Portal,1 M. Rivier,1 P. Rossio,1
1 1 4 1 1 1

E. Thoreau,1 E. Vial1 and J.J. Voegel iD 1

1
Research Department, Galderma R&D, Les Templiers, 2400 Route des Colles, 06410 Biot, France
2
Department of Dermatology, Nantes University Hospital, Nantes, France
3
CIC, Inserm U892-CNRS 6299, Nantes, France
4
Pharma & Life Sciences Xpert, Antibes, France

Linked Editorial: Balak. Br J Dermatol 2018; 179:231–232.

Summary

Correspondence Background First- and third-generation retinoids are the main treatment for acne.
Johannes J. Voegel. Even though efficacious, they lack full selectivity for retinoic acid receptor (RAR)
E-mail: johannes.voegel@galderma.com c, expressed in the epidermis and infundibulum.
Objectives To characterize the in vitro metabolism and the pharmacology of the
Accepted for publication
7 February 2018
novel retinoid trifarotene.
Materials and methods In vitro assays determined efficacy, potency and selectivity on
Funding sources RARs, as well as the activity on the expression of retinoid target genes in human
The studies have been financed by Galderma R&D, keratinocytes and ex vivo cultured skin. In vivo studies investigated topical comedo-
Sophia Antipolis, France. lytic, anti-inflammatory and depigmenting properties. The trifarotene-induced
gene expression profile was investigated in nonlesional skin of patients with acne
Conflicts of interest
With the exception of B.D. and P.M. the authors
and compared with ex vivo and in vivo models. Finally, the metabolic stability in
are employees of Galderma R&D. human keratinocytes and hepatic microsomes was established.
Results Trifarotene is a selective RARc agonist with > 20-fold selectivity over RARa
J.A. and D.P. contributed equally to this study. and RARb. Trifarotene is active and stable in keratinocytes but rapidly metabolized
by human hepatic microsomes, predicting improved safety. In vivo, trifarotene
*Plain language summary available online
0
01% applied topically is highly comedolytic and has anti-inflammatory and
DOI 10.1111/bjd.16719 antipigmenting properties. Gene expression studies indicated potent activation of
known retinoid-modulated processes (epidermal differentiation, proliferation,
stress response, retinoic acid metabolism) and novel pathways (proteolysis, trans-
port/skin hydration, cell adhesion) in ex vivo and in vivo models, as well as in human
skin after 4 weeks of topical application of trifarotene 0
005% cream.
Conclusions Based on its RARc selectivity, rapid degradation in human hepatic
microsomes and pharmacological properties including potent modulation of epi-
dermal processes, topical treatment with trifarotene could result in good efficacy
and may present a favourable safety profile in acne and ichthyotic disorders.

What’s already known about this topic?

• All-trans and 13-cis retinoic acid, which binds all three retinoic acid receptors
(RARs), and adapalene and tazarotene, which interact preferentially with RARb
and RARc, are first-line treatments for acne and are also used in other inflam-
matory skin diseases.
• Topical formulations of these retinoids avoid significant systemic retinoid-related
side-effects.

442 British Journal of Dermatology (2018) 179, pp442–456 © 2018 British Association of Dermatologists
 Pharmacology of trifarotene, Aubert et al. 443

What does this study add?

• Trifarotene is the first fourth-generation retinoid with potent and selective RARc
agonist activity, potentially associated with an improved efficacy/safety ratio com-
pared with less selective RAR agonists.
• The pharmacological potency of trifarotene translates from in vitro models to topi-
cally treated rodent and human skin in vivo. The modulated pathways collectively
are expected to translate into strong clinical efficacy in acne.
• Based on its rapid degradation in human hepatic microsomes, trifarotene is
expected to be rapidly eliminated in the blood stream, thereby potentially provid-
ing good safety, which should be particularly useful for the treatment of patients
with ichthyosis with application on large surface areas.
• Based on the favourable metabolic and pharmacological characteristics of tri-
farotene, it is worth investigating the clinical efficacy of this fourth-generation reti-
noid in acne and lamellar ichthyosis.

Retinoids are an important class of nuclear receptor agonists
used in dermatology. They are classified into three genera-
tions. Retinol, tretinoin (all-trans retinoic acid, ATRA) and iso-
tretinoin (13-cis retinoic acid) belong to the first generation;
etretinate and its metabolite acitretin to the second generation;
and tazarotene, through its metabolite tazarotenic acid, and
adapalene, to the third generation.1,2 First- and third-genera-
tion retinoids are mainstay topical treatments for acne, and
target comedones and microcomedones.3,4 In addition, third-
generation retinoids exert anti-inflammatory activity on the
innate immune system.5–7
Adapalene is a selective agonist of nuclear receptors retinoic
acid receptor (RAR) b and RARc, with lower affinity on
RARa. Its activity on proliferation and differentiation can be
blocked by a RAR b-c antagonist.8 Like adapalene, tazarotenic
acid is a selective agonist of RARb and RARc.9 Both adapalene
and tazarotenic acid modulate abnormal keratinocyte differen-
tiation, hyperproliferation, and the expression of inflammatory
markers, and both result in low systemic exposure after topical
administration.8–12 Even though RARc activity has been shown
to be associated with skin irritation in rodent models,13 we
and others14 have hypothesized that a fully RARc-selective
compound should retain strong efficacy for the modulation of
epidermal biology while potentially minimizing side-effects.
Our hypothesis is based on the observation that (i) RARb is
strongly and rapidly induced in human dermal fibroblasts trea-
ted with retinoic acid,15–17 (ii) retinoic acid-induced glandular
metaplasia in mouse skin is linked to the dermal expression of
RARb,18 and (iii) RARb is induced in vivo in retinoid-treated
rat skin, likely due to dermal induction (our unpublished
observations). We therefore hypothesized that dermal RARb,
induced and activated by topical application of nonselective
retinoids, might significantly participate in retinoid-induced
skin irritation. A selective RARc agonist would avoid these
RARb-mediated effects. Moreover, a molecule with further
increased hepatic instability, compared with first- and third-
generation retinoids, is expected to demonstrate a favourable
systemic safety profile, allowing in addition to acne the topical
treatment of patients with diseases like lamellar ichthyosis
where application on large surface areas is required.19–23
The present report describes for the first time the in vitro
metabolism and the nonclinical and clinical pharmacology of
the fourth generation, i.e. the fully RARc-selective, retinoid
trifarotene [see Figure S1 (Supporting Information) for a
depiction of the chemical structure of trifarotene].24
Materials and methods
Retinoic acid receptor profiling
RAR activity and selectivity of trifarotene and reference reti-
noids were evaluated using transactivation assays with RAR
and retinoid X receptor (RXR) reporter gene technology
(details of all materials and methods are given in File S1; see
Supporting Information).25
Stability testing in keratinocytes and hepatic microsomes
Metabolic stability of retinoids in human keratinocyte mono-
cultures and hepatic microsomes was evaluated by measuring
the loss of the parent product by HPLC/MS/MS (high-perfor-
mance liquid chromatography tandem mass spectrometry) as a
function of time (for details see File S1).
Gene expression in keratinocytes, reconstructed
epidermis and ex vivo cultured skin
The activity of retinoids on the expression of target genes in
DK-7 human immortalized keratinocytes was evaluated using
Multiplexed Molecular Profiling (High Throughput Genomics,
Tucson, AZ, U.S.A.) assays according to the manufacturer’s
instructions. The expression of target genes in reconstructed
human epidermis (RHE) and ex vivo cultured human skin was

© 2018 British Association of Dermatologists British Journal of Dermatology (2018) 179, pp442–456
444 Pharmacology of trifarotene, Aubert et al.
evaluated using reverse transcriptase-polymerase chain reac-
tion. Detailed methods are provided in File S1.
In vivo topical comedolytic activity
The comedolytic properties of trifarotene were assessed in the
rhino mouse model26,27 after 11 daily topical applications. The
dose–response of cream formulations of trifarotene was com-
pared with ATRA 0
05% (Retacnylâ, Galderma International,
France) and tazarotene 0
1% (Zoracâ, Pierre Fabre Dermatology,
Boulogne, France). Detailed methods are provided in File S1.
In vivo topical anti-inflammatory activity
The anti-inflammatory activity of trifarotene 0
1%, tazarotene
0
1% and tazarotenic acid 0
1% was assessed after a single
topical administration in the TPA (12-O-tetradecanoylphorbol-
13-acetate)-induced ear oedema mouse model.28,29 Detailed
methods are provided in File S1.
In vivo topical depigmenting and antipigmenting activity
The activity of trifarotene was investigated in studies lasting
6 weeks in the SKH2 mouse. The depigmentation activity of
trifarotene 0
01% was compared with that of adapalene 0
1%
and ATRA 0
01% on the mouse tail. The antipigmenting activ-
ity on ultraviolet radiation (UVR)-induced pigmentation of
the tail was determined according to the model described by
Warren.30 Detailed methods are provided in File S1.
Sample collection for large-scale gene expression
profiling
Clinical investigations were conducted in accordance with the
Declaration of Helsinki principles, and the ICH Guideline for
Good Clinical Practice. The clinical study received approval from
the ethics committee of Brest, France (reference CPP Ouest 6 -
755). Subjects provided written informed consent prior to biop-
sies. Subjects with moderate inflammatory acne on the back at
the screening visit, defined by scores of between 2 and 4 for the
whole back, with at least one area scored at 2 and with a maxi-
mum of three nodules using the ECLA (Acne Lesion Score Scale)
scale,31 were included in the study. Nineteen applications of a
dose of 2 mg cm 2 of trifarotene 0
005% (50 lg g 1) and
vehicle cream on the back for 4 weeks (every day except
Table 1 Retinoic acid receptor profiling
RARa RARb
1 1
EC50 (nmol L ) Efficacy (%) EC50 (nmol L )
ATRA 0
9 105 0
9
Adapalene67 22 100 2 100
Tazarotenic acid 11 104 2
5
Trifarotene 498 63 125
ATRA, all-trans retinoic acid.
British Journal of Dermatology (2018) 179, pp442–456
weekends) were performed. Biopsies of nonlesional areas of tri-
farotene- and vehicle-treated skin were made under local anaes-
thesia at day 27. Biopsies were stored in RNAlater TissueProtect
Tubes (Qiagen, Les Ulis, France).
Skin biopsies were also collected from ex vivo organ-cultured
human skin and fuzzy rats topically treated with trifarotene.
Detailed methods are provided in File S1.
Large-scale gene expression profiling
Samples from all three studies were homogenized with a
Potter-Elvehjem tissue grinder in lysis buffer for RNA extrac-
tion (Qiagen). Total RNA was extracted using miRNeasy
extraction kits (Qiagen) according to the manufacturer’s pro-
tocol. RNA quantity was measured using a Thermo ScientificTM
Nano Drop 8000 spectrophotometer (Thermo Fisher, Illkirch,
France). RNA quality was monitored using a 2100 Bioanalyzer
(Agilent Technologies, Waldbronn, Germany). Probes were
synthesized and then hybridized on Affymetrix U133 Plus 2.0
chips (GeneChipTM, Santa Clara, CA, U.S.A.). All chips were
normalized using the robust multi-array average (RMA)
method.32 Only Affymetrix identifiers with expression ≥ 26 in
at least one condition were selected. Data analysis was per-
formed on Array Studio software (Omicsoft Corporation, Cary,
NC, U.S.A.). Mean expression levels were obtained by calculat-
ing the geometric means of the RMA-normalized data for
involved and noninvolved sample group. A two-sided paired
t-test was performed using Array Studio (Omicsoft Corpora-
tion), to determine which genes were significantly differen-
tially expressed between trifarotene- and vehicle-treated skin.
The Benjamini–Hochberg false discovery rate (FDR) multiple
testing correction was applied.33 The raw data are available at
NCBI GEO, accession number GSE 107232.
Ontology analysis
Gene Ontology (GO) category enrichment analysis was per-
formed using DAVID 6
7 (http://david.abcc.ncifcrf.gov/).
Immunohistochemistry
Five-lm sections were prepared from paraffin-embedded
biopsies, then deparaffinized and rehydrated. Antigen retrieval
was performed using heat at pH = 8. The list of antibodies
RARc
Efficacy (%) EC50 (nmol L 1) Efficacy (%)
82 0
7 89
9 100
74 11 86
58 7
7 85
© 2018 British Association of Dermatologists
 Pharmacology of trifarotene, Aubert et al. 445

used for detection is detailed in File S1. Labelling was revealed Table 2 Half-life of retinoids in human keratinocytes and hepatic
using a detection kit (alkaline phosphatase/fast red substrate) microsomes
and nuclei were counterstained with haematoxylin. Slides
were observed and scanned using a NanoZoomer (Hamamatsu T1/2: human T1/2: human
Retinoid keratinocytes hepatic microsomes
Photonics, Hamamatsu City, Japan).
Adapalene > 24 h > 60 min
Tazarotene NA (not stable in 7 min
Results
medium)
Tazarotenic acid > 24 h 57 min
Trifarotene is a pure and potent retinoic acid receptor-c Trifarotene > 24 h 5 min
agonist
*
NA, not applicable.
RAR profiling assays showed that trifarotene is RARc-selective
with an efficacy of 81% and a half maximal effective concentration
(EC50) of 7
7 nmol L 1, which is 16-fold and 65-fold lower than tazarotene was metabolized in keratinocytes and more rapidly
the EC50 on RARb and RARa, respectively (Table 1). The mea- in hepatocytes. The active compound, tazarotenic acid, was
sured EC50 of ATRA on RARc was 0
7 nmol L 1 (89% efficacy), 10-fold more stable than trifarotene in human hepatic micro-
that of adapalene was 9 nmol L 1 (100% efficacy) and that of somes, with a t1/2 of 57 min. Adapalene had a t1/2 in hepatic
tazarotenic acid 11 nmol L 1 (86% efficacy). Efficacy and microsomes of > 60 min.
potency on RARc are therefore similar for adapalene, tazarotene
and trifarotene. Moreover, trifarotene was inactive on RXR (the Trifarotene is a potent modulator of target gene
chemical structure of trifarotene and details of pharmacological expression in keratinocytes, reconstructed epidermis and
profiling are given in Figures S1–S3; see Supporting Information). ex vivo cultured human skin
Gene expression of a set of target genes was assessed in DK-7
Trifarotene is stable in keratinocytes and unstable in
immortalized keratinocytes. The mean EC50 on the combined
hepatic microsomes
target genes was determined at 0
09 nmol L 1 for trifarotene,
Trifarotene was stable in human keratinocytes for more than 1
2 nmol L 1 for tazarotenic acid and 12 nmol L 1 for ATRA
24 h and very rapidly metabolized in human liver micro- (Fig. 1a). Table S1 (see Supporting Information) provides
somes, with a half-life (t1/2) of 5 min (Table 2). The prodrug individual EC50 values for each retinoid response gene.

(a)
Trifarotene Tazaarotenic acid ATTRA
Mean EC50 = 0.09 nmol L–1
Mean EC50 = 1·2 nmol L–1 Mean EC50 = 12 nmol L–1

(b) mol L–1

EC50 = 3 µm EC50 = 1.9 µmol L–1 EC50 = 30 µmol L–1

Fig 1. In vitro pharmacology of trifarotene. (a) Dose–response curves of trifarotene, tazarotenic acid and all-trans retinoic acid (ATRA) in DK-7
human immortalized keratinocytes. Individual EC50 values for each induced retinoid response gene are reported in Table S1 (see Supporting
Information). (b) Topical activity of trifarotene on gene markers in human reconstructed epidermis. Genes for which the expression was
significantly modulated by trifarotene: KRT2, KRT4, KRT10, KRT19, TGM3 and DSC1 (differentiation process); IL1B, LCN2 and PTGES (inflammation
process). The geometric mean EC50 of fold modulations (FM) is reported. Mean EC50 values include induced and repressed genes. For repressed
genes (FM < 1), the transformed 1/FM was used.

© 2018 British Association of Dermatologists British Journal of Dermatology (2018) 179, pp442–456
446 Pharmacology of trifarotene, Aubert et al.
In reconstructed human epidermis (RHE), expression of
KRT2, KRT4, KRT10, KRT19, TGM3 and DSC1, involved in epider-
mal differentiation, and of IL1B, LCN2 and PTGES, involved in
inflammation, was modulated by the topical application of
retinoids. The mean EC50 on the combined target genes of tri-
farotene and tazarotenic acid was 3 lmol L 1 and
1
9 lmol L 1, respectively, and that of ATRA was
30 lmol L 1 (Fig. 1b).
Finally, in an ex vivo human skin explant model with a skin
barrier similar to human skin, trifarotene modulated genes
involved in keratinization, desquamation, cornification and cell
adhesion (Table 3). The mean EC50 on the combined target
genes was 0
0048% for trifarotene and 0
013% for tazarotenic
acid (Fig. S4; see Supporting Information).
Trifarotene is an efficient comedolytic agent in the rhino
mouse
The comedolytic activity of retinoids was evaluated in the
well-established rhino mouse model (Fig. 2a, b)8,34 (for
Table 3 Topical activity on gene markers in human ex vivo cultured skin from three donors. Mean fold modulations, normalized on the nontreated
condition, are indicated for each dose applied, of either trifarotene or tazarotenic acid
British Journal of Dermatology (2018) 179, pp442–456
details see File S1). As expected, following 11 daily applica-
tions, the trifarotene placebo cream had no comedolytic activ-
ity (6
1 comedones mm 1) and was well tolerated. ATRA
0
05% cream had a marked comedolytic activity, with a 61%
reduction of comedones (2
4 comedones mm 1). At day 12 it
also produced an increase of 240% in epidermal thickness
(60 lm) and increased transepidermal water loss (TEWL) by
285% (26 g h 1 m 2) (Fig. 2c, d). Both parameters are typi-
cally associated with retinoid activity in vivo.
The dose of 0
1% of tazarotene, required to reach full effi-
cacy (Fig. S5; see Supporting Information), resulted in a 99%
reduction of comedones (0
07 comedones mm 1); it
increased epidermal thickness and TEWL by 254% (62 lm)
and 451% (37 g h 1 m 2), respectively.
Trifarotene showed dose-dependent comedolytic activity,
being fully efficacious at 0
01% (98% reduction, 0
15 come-
dones mm 1), a concentration 10-fold lower than that
required for tazarotene to achieve similar results. At the same
dose trifarotene increased the epidermis thickness by 275%
(66 lm) and the TEWL by 285% (26 g h 1 m 2).
© 2018 British Association of Dermatologists
 Pharmacology of trifarotene, Aubert et al. 447

(a) Number of comedones

8

Number of comedones mm–1

–1% Nontreated
ns
–14% Placebo
6 *
Trifarotene 0·001%
–45%
Trifarotene 0·0025%
*
4 Trifarotene 0·005%
–61%
*
–77% Trifarotene 0·01%
2 *
Tretinoin 0·05%
–98% –99%
*
Tazarotene 0·1%
*
0
(b)

Fig 2. Efficacy of trifarotene in the rhino

mouse model. (a) Following 11 daily topical
applications with placebo, trifarotene 0
001%,
0
0025%, 0
005% and 0
01%, Retacnyl cream
(tretinoin) 0
05% or Zorac cream 0
1%
(tazarotene), the number of comedones was
counted on skin histology sections stained
with haematoxylin–phloxin–saffron. Results
are expressed as mean number of comedones
mm 1 of epidermis  SEM (3 sections per/
animal, n = 6). Percentage reductions
compared with the nontreated condition are
indicated. (b) Images of representative skin
histology sections: placebo (upper left panel),
trifarotene 0
01% (upper right panel), (c) Epidermal thickness
80 275%
Retacnyl cream 0
05% (lower left panel) and 254%
Nontreated
Epidermal thickness (µm)

*
223% 240% *
Zorac cream 0
1% (lower right panel). 215%
* * * Placebo
Inverted triangles, epidermis; arrows, 60 159%
Trifarotene 0·001%
*
sebaceous glands; triangles, utriculi. (c) On Trifarotene 0·0025%
the same sections, epidermal thickness was 40
36% Trifarotene 0·005%
also evaluated and expressed in mean ns
Trifarotene 0·01%
lm  SEM. Percentage increases compared 20 Tretinoin 0·05%
with the nontreated condition are indicated. Tazarotene 0·1%
(d) Transepidermal water loss (TEWL) was 0
monitored on restrained animals at the end of
the treatment using a Tewameter device and (d) 50 TEWL
451% Nontreated
expressed in mean g h 1 m 2  SEM *
40 Placebo
(n = 6). Percentage increases compared with
TEWL (g h–1 m–2)

271%
285% 285% Trifarotene 0·001%
the nontreated condition are indicated. All 30 238% 230%
* *
*
*
Trifarotene 0·0025%
statistical analyses were performed using *
Trifarotene 0·005%
multiple comparisons by Wilcoxon test and 20
22% Trifarotene 0·01%
Bonferroni P-value correction. As Retacnyl ns
Tretinoin 0·05%
10
0
05% and Zorac 0
1% placebos were not Tazarotene 0·1%
available for testing, all results are presented 0
vs. the nontreated group. *P-value < 0
05.

Trifarotene demonstrates anti-inflammatory properties Trifarotene shows depigmenting and antipigmenting

in vivo
The anti-inflammatory effects of retinoids, known to be
mediated in part by anti-AP-1 activity,35 were evaluated in
the TPA-induced ear oedema mouse model.8,36 All tested
compounds, including the RARc-selective agonist trifarotene,
showed potent anti-inflammatory characteristics in vivo, result-
ing in ear oedema reduction of 73%, 99% and 95% for tri-
farotene, tazarotene and tazarotenic acid, respectively
(Fig. 3a).
activity in vivo
Depigmenting activity of retinoids has been observed in SKH2
mice.37 Unlike other retinoids, and potentially due to a
weaker penetration in hyperkeratotic tail skin, adapalene 0
1%
showed no significant depigmenting activity after 6 weeks of
topical application on the SKH2 mouse tail (Fig. 3b). In con-
trast, trifarotene and ATRA showed significant depigmenting
activity at 0
01% ( 1
1 and 1, respectively, on the pigmen-
tation score at day 43). After UVR induction, in the same

© 2018 British Association of Dermatologists British Journal of Dermatology (2018) 179, pp442–456
448 Pharmacology of trifarotene, Aubert et al.

(a) TPA inflammation

Fig 3. Anti-inflammatory and antipigmenting
20
TPA + vehicle activities of trifarotene in vivo. The anti-
Ear oedema (1/100 mm)

TPA + trifarotene 0·1% inflammatory activity of vehicle, trifarotene

15 0
1%, tazarotene 0
1% and tazarotenic acid
TPA + tazarotene 0·1%
0
1% was evaluated after a single topical co-
TPA + tazarotenic acid 0·1%
application with TPA 0
01% on Balb/c mouse
10
ears. The ear oedema (increase in ear
–75%
***
thickness) was measured using a micrometer
5 –95% (Oditest) 6 h after application and expressed
–99%
***
*** in mean 1/100 mm increase  SEM (n = 5).
Percentage decreases compared with the
0
vehicle-treated condition are indicated.
(b) Pigmentation Statistical analyses were performed using
Student’s t-test: *0
01 < P-value < 0
05;
0·0
Trifarotene 0·01% **0
005 < P-value < 0
01; ***P-value
ns
Tretinoin 0·01% < 0
005. (b) The depigmenting activity of
Pigmentation score

–0·5 vehicle, trifarotene 0
01%, tretinoin 0
01%

Adapalene 0·1%
and adapalene 0
1% was evaluated after 6-
* Vehicle
week daily topical applications on the tail of
–1·0
SKH2 mice. Results are expressed as mean
***
pigmentation score [from 0 (naturally
–1·5 pigmented) to 4 (totally depigmented)] 
SEM (n = 5). Statistical analyses at day 43
were performed using Student’s t-test:
–2·0
D1 D8 D15 D22 D29 D36 D43
*0
01 < P-value < 0
05; **0
005 < P-value
< 0
01; ***P-value < 0
005. (c) The
(c) Pigmentation + UV antipigmenting activity of trifarotene 0
003%
6
UV + trifarotene 0·003% was evaluated after 6-week daily topical
UV + vehicle applications on the tail of SKH2 mice in
4
Pigmentation score

***

Trifarotene 0·003%
conjunction with ultraviolet (UV) B
irradiations (90 mJ cm 2 three times a week
2 Vehicle
for 6 weeks). Results are expressed as mean
pigmentation score [from 4 (totally
0
pigmented) to 4 (totally depigmented)] 
SEM (n = 5). Statistical analyses at day 42
–2
*** were performed using Student’s t-test:
*** *0
01 < P-value < 0
05; **0
005 < P-value
–4
D1 D8 D15 D22 D29 D36 D42
< 0
01; ***P-value < 0
005. TPA, 12-O-
tetradecanoylphorbol-13-acetate.

mouse model, the antipigmenting activity of trifarotene was
again highly significant (Fig. 3c).
Translational transcriptomics with trifarotene, including
human skin in vivo, demonstrates potent activation of
known and novel retinoid-modulated pathways
The pharmacodynamic effects of trifarotene were investigated in
noninflammatory skin of patients with acne after 4 weeks of
topical treatment with trifarotene 0
005% cream. Retinoid-
modulated genes observed in the human study were compared
with transcriptomics data obtained after topical treatment in
human ex vivo skin culture and in the fuzzy rat in vivo. Forty-one
genes were modulated in the same direction in all three models,
and formed the basis for biological interpretation (Fig. 4a and
Table 4). Trifarotene regulated the expression of genes involved
in known retinoid-induced pathways, including retinoid
metabolism (CYP26A1, STRA6, DHRS9, CRABP2), epidermal differ-
entiation (KRT4, ELF3, PPARD, ID1), proliferation (FOSL1, P2RY2,
RIT1, CCNG2, ZBTB20, FGFR2, BTC) and response to stress
(S100A9, GPX2, IL1RN, CXCR2, TYMS, F3). Interestingly, three reti-
noid-modulated pathways were identified that have not yet been
described: proteolysis (PRSS27, KLK6, KLK8, KLK10, CTSD, SER-
PINA12, MME) (Fig. 4b), transport/skin hydration (PADI1, AQP3,
RHCG, ATP11B) (Fig. 4c) and cell adhesion (PRRG4, EFHD2,
CHL1, SPON1, DST) (Fig. 4d). For the proteolysis pathway, the
induction of KLK6, KLK8 and KLK10 expression was confirmed by
immunohistochemistry (Fig. 4e).
Discussion
We described the pharmacology of trifarotene, the first potent
RARc-selective agonist entering clinical trials. The molecule
was designed to present good metabolic stability in cultured

British Journal of Dermatology (2018) 179, pp442–456 © 2018 British Association of Dermatologists
 Pharmacology of trifarotene, Aubert et al. 449

(a) Noninflammatory Fuzzy rat

skin of in vivo
acne patients 0 72 (312 genes)
(214 genes)
1

47
95 192
Ex vivo cultured
human skin 0
(334 genes)

(b)

Fig 4. Comparative analysis of trifarotene

(Trif)-induced gene modulation in three
pharmacological studies: ex vivo cultured
human skin, fuzzy rat in vivo and
noninflammatory skin of patients with acne.
(a) Venn diagram including data of three
experimental studies, depicting genes
modulated with a fold modulation > 1
6 and
a Benjamini–Hochberg false discovery
rate < 0
05. Forty-seven genes were found in
the union of all three studies. Of these, 41
were modulated in the same direction in all
three studies, and used for further analysis.
(b) Genes involved in proteolysis and
modulated by topical trifarotene application in
all three studies. Expression levels are
provided in arbitrary units (AU). Continued
over (c) Genes involved in skin hydration and
transport, and modulated by topical
trifarotene application in all three studies.
Expression levels are provided in AU. (d)
Genes involved in cell adhesion and
modulated by topical trifarotene application in
all three studies. Expression levels are
provided in AU. (e) Immunohistochemical
staining of three kallikrein members on skin
sections obtained from vehicle- and
trifarotene-treated noninflammatory skin of
patients with acne. Ctrl, control.

keratinocytes, while being rapidly metabolized in human liver
microsomes. The in vitro metabolic profile of trifarotene is
expected to result in low systemic levels, while retaining
strong cutaneous activity. These properties are particularly
important when treating large body surface areas topically in
diseases such as lamellar ichthyosis, as potential systemic safety
issues may thus be reduced.
The potent pharmacological efficacy of trifarotene was con-
firmed in the pluristratified RHE model. Although presenting
only limited barrier properties, the RHE model is routinely
used for the topical efficacy evaluation of skin-targeted
compounds.38,39 Importantly, topically applied trifarotene was
active in ex vivo cultured human skin, with a stratum corneum
barrier close to human skin in vivo. Interestingly, in this assay
trifarotene was threefold more potent than tazarotene, which
is in agreement with the demonstrated overall higher in vitro
potency of trifarotene compared with tazarotenic acid, the
active metabolite of the prodrug tazarotene.
In vivo, trifarotene eliminated almost all comedones from the
classical retinoid-responsive rhino mouse model, with a dose
10 times lower than that required for tazarotene and ATRA to
achieve the same effects.

© 2018 British Association of Dermatologists British Journal of Dermatology (2018) 179, pp442–456
450 Pharmacology of trifarotene, Aubert et al.
(c)
(d)
In line with the described effects of retinoids,40–42 tri-
farotene demonstrated strong anti-inflammatory properties,
which in addition to the comedolytic activity is expected to
contribute to its efficacy in inflammatory skin diseases such as
acne.
Inflammatory acne is often accompanied by post-inflamma-
tory hyperpigmentation (PIHP), especially in Black and Asian
subjects. The rapid in vivo antipigmenting activity of trifarotene
is therefore another useful characteristic of this molecule. Of
British Journal of Dermatology (2018) 179, pp442–456
Fig 4. Continued
note, toATRA is already recommended to reduce PIHP sec-
ondary to acne.43,44
To evaluate if the potent in vitro and in vivo pharmacological
activities of trifarotene translate to the clinical setting, topical
trifarotene 0
005% cream was applied to noninflammatory
skin of patients with acne, followed by large-scale gene
expression profiling in skin biopsies. Transcriptomics was per-
formed in parallel in ex vivo cultured human skin, and in the
fuzzy rat in vivo model. Forty-one genes modulated in all three
© 2018 British Association of Dermatologists

(e)
KLK6
KLK8
KLK10
Fig 4. Continued
topical models provided a robust set of trifarotene-biomarkers,
including genes involved in retinoid-modulated processes such
as retinoid metabolism,45 and epidermal differentiation and
proliferation.46,47
Within biological factors influencing cellular proliferation,
FGFR2 was downregulated by trifarotene. This observation, not
previously described for retinoids, is of particular interest, as a
potential pathogenic role of FGFR2 in acne has been sug-
gested.48,49 Naturally occurring gain-of-function germline
mutations of the FGFR2 gene have been demonstrated to cause
Apert syndrome, characterized by skin manifestations includ-
ing severe acne and oily skin.50,51 Of note, a case report with
oral isotretinoin describes the resolution of acneiform lesions
in Apert syndrome.52
Importantly, three novel pathways were identified in our
comparative large-scale gene expression analysis: (1) cell
adhesion, (2) transport/skin hydration and (3) proteolysis:
1 Cell adhesion: Genes associated with the cell adhesion
process may take part in the renewal of epidermal cells.53 As
such, the downregulation of dystonin by trifarotene may pro-
mote the weakening of hemidesmosomes, inducing the migra-
tion of keratinocytes, thereby supporting the anticomedolytic
activity of the drug.
2 Transport/skin hydration: By activating the expression of
aquaporin 3 (AQP3) and peptidyl arginine deiminase 1 (PADI1),
trifarotene may improve skin hydration and impact cutaneous
barrier function. Water channel AQP3 transports both water and
small neutral solutes, such as the humectant glycerol,54 important
for maintaining hydration of the stratum corneum. PADI1 is
© 2018 British Association of Dermatologists
Pharmacology of trifarotene, Aubert et al. 451
Vehicle Trifarotene
KLK6
KLK8
KLK10
associated with late stages of epidermal differentiation, where it
catalyses deimination of filaggrin and keratin K1.55
The upregulation of Rh family C glycoprotein (RHCG), an
ammonium transporter, suggests that trifarotene may impact
the NH4+/NH3 balance, and thereby cutaneous pH homeosta-
sis. Furthermore, a change in the ammonium ion content may
indirectly affect the natural barrier structure, for example by
regulating glutamine synthetase and the production of
glutamine.56,57
SLC2A1 or GLUT-1 in humans is the most common trans-
porter of glucose, the major energy source for cells.
3 Proteolysis: To date, the downregulation of membrane
metalloendopeptidase (MME) observed with trifarotene has
not been described for other retinoids. Of note, upregulated
expression of MME has been associated with the degradation
of elastin fibres, resulting in wrinkling or sagging of the
skin.58,59 MME downregulation may therefore constitute
another previously not yet recognized pathway involved in the
positive effects of retinoids on skin ageing.60,61
Interestingly, trifarotene induced the expression of several
serine proteases, including kallikreins (KLK6, KLK8, KLK10)
and serine protease PRSS27. The involvement of these pro-
teases in the desquamation process62,63 and their upregulated
expression in psoriasis64 has been described previously.
Whereas discordant results, i.e. induced and repressed expres-
sion, respectively, have been described for other kallikrein
members (KLK5 and KLK7) in cultured keratinocytes treated
with retinoids,65,66 we demonstrated consistent upregulation
of KLK6, KLK8 and KLK10 in three independent experimental
British Journal of Dermatology (2018) 179, pp442–456
 Table 4 Gene expression profile induced by trifarotene across three experimental models: ex vivo cultured human skin, fuzzy rat in vivo and noninflammatory skin of patients with acne. Genes commonly
modulated in all three models are grouped according to their main biological function provided by DAVID tools. References for genes known to be regulated by retinoids are indicated in brackets
452 Pharmacology of trifarotene, Aubert et al.

British Journal of Dermatology (2018) 179, pp442–456

© 2018 British Association of Dermatologists
 Pharmacology of trifarotene, Aubert et al. 453

© 2018 British Association of Dermatologists British Journal of Dermatology (2018) 179, pp442–456
454 Pharmacology of trifarotene, Aubert et al.
models on the mRNA level and in human skin in vivo on the
protein level. This observation may provide a partial molecular
explanation for the effect of trifarotene on the renewal of ter-
minally differentiated corneocytes in human skin.
This report is limited to pharmacology and does not describe
in vivo pharmacokinetics and toxicology data, or data on human
safety and cutaneous tolerance. At this point predictions of
improved safety of topically applied trifarotene are therefore
hypothetical and based only on rapid metabolism in hepatic
microsomes in vitro and strong selectivity of trifarotene for
RARc. Future investigations with a suitably formulated drug
product will have to investigate safety and tolerance as well as
clinical efficacy in dermatological diseases and in skin ageing.
Today, based on the favourable metabolic and pharmaco-
logical characteristics of trifarotene described in this report,
this fourth-generation retinoid is under investigation for clini-
cal efficacy in acne and lamellar ichthyosis.
Acknowledgments
The excellent scientific and experimental contributions of G.
Duvert, G. F
eraille, C. Feret, P. Fogel, M. Freylinger, D.
Froude, C. Menigot, T. Minier, K. Nicolas, P. Reiniche and D.
Spiesse are gratefully acknowledged. The authors acknowledge
the writing assistance of Karl Patrick G€
oritz, SMWS, Scientific
and Medical Writing Services France.
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Supporting Information
Additional Supporting Information may be found in the online
version of this article at the publisher’s website:
File S1. Materials and Methods. In vitro retinoic acid recep-
tor (RAR) and retinoid X receptor (RXR) profiling; In vitro
assessment of the activity of trifarotene on the expression of
retinoid target genes; In vivo assessment of the comedolytic,
anti-inflammatory and depigmenting activity of trifarotene;
Samples for large-scale gene expression analysis; Antibodies
and dilutions for immunohistochemistry on human skin
sections.
Table S1 Activity in DK-7 human immortalized ker-
atinocytes in culture.
Fig. S1. Chemical structures.
Fig. S2. Pharmacological activity on retinoic acid receptors
(RARa, RARb, RARc).
Fig. S3. Pharmacological activity on retinoid X receptors
(RXRa, RXRb).
Fig. S4. Topical activity on gene markers in human skin
ex vivo.
Fig. S5. Dose–response of tazarotene and tazarotenic acid in
the rhino mouse model.
Powerpoint S1 Journal Club Slide Set.
© 2018 British Association of Dermatologists