Introduction What’s already known?

1. Non-clinical and human pharmacology of the potent and selective topical RARγ agonist trifarotene J. Aubert1, D. Piwnica1, B. Bertino1, S. Blanchet-Réthoré1, I. Carlavan1, S. Déret1, B. Dreno2,3, B. Gamboa1, A. Jomard1, A.P. Luzy1, P. Mauvais4, C. Mounier1, J. Pascau1, I. Pelisson1, T. Portal1, M. Rivier1, P. Rossio1, E. Thoreau1, E. Vial1, J.J. Voegel1 1 Research Department, Galderma R&D, 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 British Journal of Dermatology. DOI: 10.111/bjd.16719

2. Introduction What’s already known? • All-trans and 13-cis retinoic acid (RA), binding all three retinoic acid receptors (RARs), and adapalene and tazarotene interacting preferentially with RARb and RARg are first line treatments of acne and are also used in other inflammatory skin diseases. • Topical formulations of these retinoids avoid significant systemic retinoid-related side effects, but they lack full selectivity for RARγ expressed in the epidermis and infundibulum.

3. Objective • To characterize the non-clinical and human pharmacology, as well as the in vitro metabolism, of the potent and selective topical RARγ agonist trifarotene.

4. Methods • In vitro transactivation assays to determine efficacy, potency and selectivity on RARs and RXR. • Analysis of the expression of retinoid target genes in human keratinocytes and ex vivo cultured skin. • Investigation of topical comedolytic, anti-inflammatory and depigmenting properties in in vivo pharmacology studies.

5. Methods • Comparison of large scale gene expression profiles ex vivo, in vivo and in non-lesional skin of acne patients, to describe trifarotene-mediated modulation of diverse biological processes. • Analysis of the metabolic stability of trifarotene in human keratinocytes and hepatic microsomes.

6. Results – Retinoic acid receptor profiling • Trifarotene is the first RARg selective retinoid advanced to human clinical trials EC50 - The concentration that produces 50% of the maximal response Efficacy and potency on RAR are similar for adapalene, tazarotene and trifarotene.

7. Results – Retinoic acid receptor profiling • Trifarotene is the first retinoid ligand rapidly degraded in hepatic microsomes but stable in keratinocytes • Rapid degradation in human hepatic microsomes is expected to result in rapid elimination of trifarotene from the blood stream. • This would be particularly important when treating large body surfaces topically, in diseases such as lamellar ichthyosis, Half-life (t ½ ) of retinoids in keratinocytes and hepatic microsomes

8. Results – Comedolytic activity in the rhino mouse model • Trifarotene shows full comedolytic activity in the rhino mouse at concentrations ten-fold lower than comparator molecules trifarotene 0.01% placebo tazarotene 0.1% tretinoin 0.05% Following 11 once daily topical applications, the number of comedones was counted in paraffin-embedded histology specimens Results are expressed as mean number of comedones per mm of epidermis ± SEM (3 sections/animal, N = 6) Percentage reductions compared with the non-treated condition are indicated. * = P-value < 0.05

9. Results – Translation of observations from in vivo and ex vivo models to topically-treated skin of acne patients • Large scale gene expression profiles ex vivo, in vivo and in non-lesional skin of acne patients were analysed • Of the overlapping forty-seven genes, 41 were modulated in the same direction across all three studies and analysed further

10. Results –Topically applied trifarotene efficiently modulates known- and novel retinoid pathways in skin of acne patients Trifarotene modulated genes expression in diverse biological processes including epidermal differentiation, keratinisation, desquamation, cornification and inflammation. Novel pathways identified involved proteolysis, cell adhesion and transport/skin hydration. mRNA Protein Trifarotene Vehicle humanstudy KLK6 KLK6 humanstudy KLK8 KLK8 humanstudy humanstudy KLK10 KLK10 humanstudy humanstudy

11. Discussion • In line with the described effects of retinoids, trifarotene 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. • Within biological factors influencing cellular proliferation, FGFR2 was down-regulated by trifarotene. • This observation was not previously described for retinoids. • A potential pathogenic role of FGFR2 in acne has been suggested in the literature.

12. Discussion • Inflammatory acne is often accompanied by post-inflammatory hyperpigmentation (PIHP), especially in Black and Asian patients. The rapid in vivo anti-pigmenting activity of trifarotene is therefore another useful characteristic of this molecule. • Of note, topical all-trans retinoic acid is already recommended to reduce PIHP secondary to acne.

13. Discussion • Three novel trifarotene-modulated pathways were identified in our comparative large scale gene expression analysis: cell adhesion, transport/skin hydration and proteolysis. • With respect to proteolysis, trifarotene induced the expression of several serine proteases, including KLK6, KLK8, KLK10 and PRSS27. • These proteases are involved in the desquamation process and are up-regulated in psoriasis. • Upregulation of these proteases may provide a partial molecular explanation for the effect of trifarotene on the renewal of terminally differentiated corneocytes in human skin.

14. Conclusions – What does this study add? • Trifarotene is the first 4th generation retinoid with potent and selective RARγ agonist activity, potentially associated with an improved efficacy/safety ratio compared to less selective RAR agonists. • The pharmacological potency of trifarotene translates from in vitro models to topically 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 from the blood stream, potentially providing a good safety profile. This should be particularly useful for the treatment of patients with ichthyosisrequiring applications on large body surface areas.

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