Topical application of RTA 408 lotion activates Nrf2 in human skin and is well-tolerated by healthy human volunteers

Abstract

Background: Topical application of the synthetic triterpenoid RTA 408 to rodents elicits a potent dermal cytoprotective phenotype through activation of the transcription factor Nrf2. Therefore, studies were conducted to investigate if such cytoprotective properties translate to human dermal cells, and a topical lotion formulation was developed and evaluated clinically.

Methods: In vitro, RTA 408 (3-1000 nM) was incubated with primary human keratinocytes for 16 h. Ex vivo, RTA 408 (0.03, 0.3, or 3 %) was applied to healthy human skin explants twice daily for 3 days. A Phase 1 healthy volunteer clinical study with RTA 408 Lotion (NCT02029716) consisted of 3 sequential parts. In Part A, RTA 408 Lotion (0.5 %, 1 %, and 3 %) and lotion vehicle were applied to individual 4-cm(2) sites twice daily for 14 days. In Parts B and C, separate groups of subjects had 3 % RTA 408 Lotion applied twice daily to a 100-cm(2) site for 14 days or a 500-cm(2) site for 28 days.

Results: RTA 408 was well-tolerated in both in vitro and ex vivo settings up to the highest concentrations tested. Further, RTA 408 significantly and dose-dependently induced a variety of Nrf2 target genes. Clinically, RTA 408 Lotion was also well-tolerated up to the highest concentration, largest surface area, and longest duration tested. Moreover, significant increases in expression of the prototypical Nrf2 target gene NQO1 were observed in skin biopsies, suggesting robust activation of the pharmacological target.

Conclusions: Overall, these data suggest RTA 408 Lotion is well-tolerated, activates Nrf2 in human skin, and appears suitable for continued clinical development.

Figures

Fig. 1

Effect of RTA 408 on mRNA Expression of Nrf2 Target Genes in Primary Human Keratinocytes. Freshly isolated primary human keratinocytes were incubated with RTA 408 (3–1000 nM) or vehicle (DMSO, 0.1 % v/v) for 16 h and analyzed for mRNA expression of Nrf2 target genes. Data were normalized to the housekeeping gene PPIB and are presented as mean fold vehicle control ± standard error of the mean (S.E.M.). *p < 0.05 vs. vehicle control

Fig. 2

Effect of RTA 408 on the mRNA Expression of Nrf2 Target Genes in Human Skin Explants. Human skin explants from a healthy donor were cultured. RTA 408 (0.03, 0.3, or 3 %) or vehicle (sesame oil) was applied topically up to twice daily for 3 days and skin was then processed and analyzed for mRNA expression of Nrf2 target genes. Data were normalized to the average of housekeeping genes RPL13A and PPIB and are presented as mean fold vehicle control ± S.E.M. *p < 0.05 vs. vehicle control

Fig. 3

Effect of RTA 408 on the Protein Expression of NQO1 in Human Skin Explants. Human skin explants from a healthy donor were cultured. RTA 408 (0.03, 0.3, or 3 %) or vehicle (sesame oil) was applied topically up to twice daily for 3 days and the skin was fixed in formalin. NQO1 protein was evaluated using standard immunohistochemical techniques. a. Representative photomicrographs (20X) are presented for each treatment group. b. Staining intensity was determined and presented as mean fold vehicle control ± S.E.M. *p < 0.05 vs. vehicle control

Fig. 4

Effect of RTA 408 Lotion on the Protein Expression of NQO1 in Human Skin Biopsies from Phase 1 Clinical Trial. Skin biopsies were collected from healthy volunteers one day after the final dose in Parts a, b, and c of the Phase 1 clinical trial. Part a evaluated 3 concentrations of RTA 408 Lotion (0.5, 1, and 3 %) compared to lotion vehicle applied topically to 12 healthy subjects BID for 14 days to a small skin surface area on the lower back (4 cm2 for each concentration). Part b was open-label and assessed the RTA 408 Lotion (3 %) applied topically to 10 healthy subjects BID for 14 days to skin on the lower back (100 cm2). Part c was open-label and assessed the RTA 408 Lotion (3 %) applied topically to 10 healthy subjects BID for 28 days to skin on the lower back (500 cm2). Representative photomicrographs (20X) from each part are presented on the left with corresponding quantified staining intensities presented on the right. Dots represent individual data points for each subject. Bars present data as mean fold vehicle or untreated control ± S.E.M. *p < 0.05 vs. vehicle or untreated control