Topical SCD-153, a 4-methyl itaconate prodrug, for the treatment of alopecia areata

Abstract

Alopecia areata is a chronic hair loss disorder that involves autoimmune disruption of hair follicles by CD8⁺ T cells. Most patients present with patchy hair loss on the scalp that improves spontaneously or with topical and intralesional steroids, topical minoxidil, or topical immunotherapy. However, recurrence of hair loss is common, and patients with extensive disease may require treatment with oral corticosteroids or oral Janus kinase (JAK) inhibitors, both of which may cause systemic toxicities with long-term use. Itaconate is an endogenous molecule synthesized in macrophages that exerts anti-inflammatory effects. To investigate the use of itaconate derivatives for treating alopecia areata, we designed a prodrug of 4-methyl itaconate (4-MI), termed SCD-153, with increased lipophilicity compared to 4-MI (CLogP 1.159 vs. 0.1442) to enhance skin and cell penetration. Topical SCD-153 formed 4-MI upon penetrating the stratum corneum in C57BL/6 mice and showed low systemic absorption. When added to human epidermal keratinocytes stimulated with polyinosinic-polycytidylic acid (poly I:C) or interferon (IFN)γ, SCD-153 significantly attenuated poly I:C-induced interleukin (IL)-6, Toll-like receptor 3, IL-1β, and IFNβ expression, as well as IFNγ-induced IL-6 expression. Topical application of SCD-153 to C57BL/6 mice in the resting (telogen) phase of the hair cycle induced significant hair growth that was statistically superior to vehicle (dimethyl sulfoxide), the less cell-permeable itaconate analogues 4-MI and dimethyl itaconate, and the JAK inhibitor tofacitinib. Our results suggest that SCD-153 is a promising topical candidate for treating alopecia areata.

Keywords: alopecia areata; anagen; cytokine; cytotoxicity; double-stranded RNA; hair cycle; hair growth; immunosuppression; inflammation; interferon; interleukin; itaconate; keratinocyte; pharmacokinetics; polyinosinic–polycytidylic acid; prodrug; stability; telogen; topical.

Fig. 1.

SCD-153 exhibits solid-state stability at room temperature and forms 4-MI in skin homogenate and plasma. (a) The synthetic scheme of SCD-153, a prodrug of 4-MI with enhanced lipophilicity (CLogP 1.159 vs. 0.1442) for improved skin and cell penetration. (b) SCD-153 remained stable in the solid state within error of 100% after 84 days at room temperature (n = 2 per time point). (c) SCD-153 readily converted to 4-MI in mouse skin homogenate (n = 4 per time point), mouse plasma (n = 2 per time point), human skin homogenate (n = 4 per time point), and human plasma (n = 3 per time point). Chromatograms show the presence of significant 4-MI levels at 60 minutes after the addition of SCD-153 to skin homogenate or plasma. Data are expressed as mean ± SEM.

Fig. 2.

SCD-153 shows minimal systemic absorption and forms 4-MI in the epidermis and dermis of C57BL/6 mice. (a) Following topical application of 5% SCD-153 in DMSO to C57BL/6 mice, levels of SCD-153 and its metabolite 4-MI were significantly higher in the skin than in the plasma (n = 3 per time point). (b) Levels of SCD-153 were significantly higher in the stratum corneum than in the rest of the epidermis and dermis, while 4-MI was the predominant form of the drug present in the epidermis and dermis (n = 3 per time point). Data are expressed as mean ± SEM.

Fig. 3.

SCD-153 shows time- and dose-dependent cytotoxicity on NHEKs. Viability (%) of NHEKs incubated with SCD-153, DMI, and 4-MI at various concentrations and durations. NHEKs showed decreased viability after 8 hours of incubation with SCD-153, at drug concentrations above 100 μM (n = 2 per compound at each concentration). Separate but chemically identical samples of SCD-153 were prepared at the Institute of Organic Chemistry and Biochemistry (IOCB) and the Sun Pharma Advanced Research Company (SPARC). Data are expressed as mean ± SEM.

Fig. 4.

SCD-153 inhibits poly I:C- and IFNγ- induced expression of proinflammatory cytokines in NHEKs. (a) SCD-153 caused dose-dependent inhibition of poly I:C-induced IL-6 and TLR3 expression in NHEKs at 8 hours of incubation ( n = 2 per condition for incubation time of 8 hours, n  = 1 per condition for other incubation times). (b) SCD-153 caused dose-dependent inhibition of poly I:C-induced IL-6, IL-1β, and IFNβ expression and IFNγ-induced IL-6 expression in NHEKs ( n = 2 per condition). P values were calculated by one-way ANOVA followed by Dunnett's multiple comparisons test. * P < 0.05, ** P < 0.01, *** P  < 0.001 compared to poly I:C or IFNγ alone. Data are expressed as mean ± SEM.

Fig. 5.

Topical application of 3% SCD-153 induces early telogen to anagen transition in C57BL/6 mice. (a) Female C57BL/6 mice underwent hair clipping at 8.5 weeks after birth (during the telogen phase of the hair growth cycle), followed by four doses of topical treatment on the dorsal right side with vehicle (DMSO), 3% 4-MI, or 3% SCD-153 on days 1, 3, 5, and 7 after hair clipping. (b) Number and (c) Photographs of mice showing skin erythema and scaling on day 9, as well as hair growth on day 18. (d) Difference in mean pixel intensity between the treated and untreated sides of mice measured from photographs that were converted to 8-bit grayscale images, with negative change in mean pixel intensity suggestive of hair growth and skin darkening. SCD-153 in this experiment was prepared at SPARC. Dots on the bar graph represent measurements of individual mice (n = 6 per group). P values were calculated by one-way ANOVA followed by Tukey's multiple comparisons test. * P < 0.05. Data are expressed as mean ± SEM.

Fig. 6.

Topical application of 5% SCD-153 induces early telogen to anagen transition in C57BL/6 mice. (a) Female C57BL/6 mice underwent hair clipping at 8.5 weeks after birth (during the telogen phase of the hair growth cycle), followed by two doses of topical treatment on the dorsal right side with vehicle (DMSO), 40% dimethyl itaconate, 5% tofacitinib, or 5% SCD-153 on days 1 and 3 after hair clipping. (b) Number and (c) Photographs of mice showing skin erythema and scaling on days 5 and 9, as well as hair growth on day 15. (d) Difference in mean pixel intensity between the treated and untreated sides of mice measured from photographs that were converted to 8-bit grayscale images, with negative change in mean pixel intensity suggestive of hair growth and skin darkening. Separate but chemically identical samples of SCD-153 were prepared at the IOCB and SPARC. Dots on the bar graph represent measurements of individual mice (n = 5 for 5% tofacitinib group, n = 7 for all other groups). P values were calculated by one-way ANOVA followed by Tukey's multiple comparisons test. ^***P < 0.001. Data are expressed as mean ± SEM.