ABSTRACT
Atopic dermatitis is a chronic inflammatory skin condition that is characterized by skin inflammation, itching, and redness. Although various treatments can alleviate symptoms, they often come with side effects, highlighting the need for new treatments. Here, we discovered a new peptide-based therapy using the intra-dermal delivery technology (IDDT) platform developed by Remedi Co., Ltd (REMEDI). The platform screens and identifies peptides derived from proteins in the human body that possess cell-penetrating peptide (CPP) properties. We screened over 1000-peptides and identified several derived from the Speckled protein (SP) family that have excellent CPP properties and have anti-inflammatory effects. We assessed these peptides for their potential as a treatment for atopic dermatitis. Among them, the RMSP1 peptide showed the most potent anti-inflammatory effects by inhibiting the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and signal transducer and activator of transcription 3 (STAT3) signaling pathways while possessing CPP properties. To further improve efficacy and stability, we developed a palmitoylated version called Pal-RMSP1. Formulation studies using liposomes (Pal-RMSP1 LP) and micelles (Pal-RMSP1 DP) demonstrated improved anti-inflammatory effects in vitro and enhanced therapeutic effects in vivo. Our study indicates that nano-formulated Pal-RMSP1 could have the potential to become a new treatment option for atopic dermatitis.
Subject(s)
Dermatitis, Atopic , Nanoparticles , Humans , Dermatitis, Atopic/drug therapy , NF-kappa B/metabolism , Peptides/pharmacology , Peptides/therapeutic use , Anti-Inflammatory Agents/pharmacologyABSTRACT
Anti-pigmentation peptides have been developed as alternative skin-lightening agents to replace conventional chemicals that have adverse effects on the skin. However, the maximum size of these peptides is often limited by their low skin and cell penetration. To address this issue, we used our intra-dermal delivery technology (IDDT) platform to identify peptides with hypo-pigmenting and high cell-penetrating activity. Using our cell-penetrating peptides (CPPs) from the IDDT platform, we identified RMNE1 and its derivative RMNE3, "DualPep-Shine", which showed levels of α-Melanocyte stimulating hormone (α-MSH)-induced melanin inhibition comparable to the conventional tyrosinase inhibitor, Kojic acid. In addition, DualPep-Shine was delivered into the nucleus and regulated the gene expression levels of melanogenic enzymes by inhibiting the promoter activity of microphthalmia-associated transcription factor-M (MITF-M). Using a 3D human skin model, we found that DualPep-Shine penetrated the lower region of the epidermis and reduced the melanin content in a dose-dependent manner. Furthermore, DualPep-Shine showed high safety with little immunogenicity, indicating its potential as a novel cosmeceutical ingredient and anti-pigmentation therapeutic agent.
Subject(s)
Basic Helix-Loop-Helix Transcription Factors , Cell-Penetrating Peptides , Melanins , Melanocytes , Microphthalmia-Associated Transcription Factor , Nerve Tissue Proteins , Skin Lightening Preparations , Skin Pigmentation , Transcription, Genetic , Melanins/antagonists & inhibitors , Skin Pigmentation/drug effects , Microphthalmia-Associated Transcription Factor/genetics , Transcription, Genetic/drug effects , alpha-MSH/antagonists & inhibitors , alpha-MSH/metabolism , Humans , Cell-Penetrating Peptides/chemistry , Cell-Penetrating Peptides/pharmacology , Skin Lightening Preparations/chemistry , Skin Lightening Preparations/pharmacology , Melanoma, Experimental , Nerve Tissue Proteins/chemistry , Nerve Tissue Proteins/pharmacology , Basic Helix-Loop-Helix Transcription Factors/chemistry , Basic Helix-Loop-Helix Transcription Factors/pharmacology , Keratinocytes/drug effects , Keratinocytes/metabolism , Melanocytes/drug effects , Melanocytes/metabolism , Epidermis/drug effects , Epidermis/metabolismABSTRACT
The ocular surface is continuously exposed to various environmental factors, and innate and adaptive immunity play crucial roles in ocular surface diseases (OSDs). Previously, we have reported that the topical application of RCI001 affords excellent anti-inflammatory and antioxidant effects in dry eye disease and ocular chemical burn models. In this study, we examined the inhibitory effects of RCI001 on the Rac1 and NLRP3 inflammasomes in vitro and in vivo. Following RCI001 application to RAW264.7 and Swiss 3T3 cells, we measured Rac1 activity using a glutathione-S-transferase (GST) pull-down assay and G-protein activation assay kit. In addition, we quantified the expression of inflammatory cytokines (interleukin [IL]-1ß, IL-6, and tumor necrosis factor [TNF]-α) in lipopolysaccharide (LPS)-stimulated RAW264.7 cells using ELISA and real-time PCR. In the mouse ocular alkali burn model, RCI001 was administered via eye drops (10 mg/mL, twice daily) for 5 days, and 1% prednisolone acetate (PDE) ophthalmic suspension was used as a positive control. Corneal epithelial integrity (on days 0-5) and histological examinations were performed, and transcript and protein levels of Rac1, NLRP3, caspase-1, and IL-1ß were quantified using real-time PCR and western blotting in corneal tissues collected on days 3 and 5. We observed that RCI001 dose-dependently inhibited Rac1 activity and various inflammatory cytokines in LPS-stimulated murine macrophages. Furthermore, RCI001 restored corneal epithelial integrity more rapidly than corticosteroid treatment in chemically injured corneas. Compared to the saline group, activation of Rac1 and the NLRP3 inflammasome/IL-1ß axis was suppressed in the RCI001 group, especially during the early phase of the ocular alkali burn model. Topical RCI001 suppressed the expression of activated Rac1 and inflammatory cytokines in vitro and rapidly restored the injured cornea by inhibiting activation of Rac1 and the NLRP inflammasome/IL-1ß axis in vivo. Accordingly, RCI001 could be a promising therapeutic agent for treating OSDs.
Subject(s)
Anti-Inflammatory Agents , Burns, Chemical , Inflammasomes , 3T3 Cells , Animals , Anti-Inflammatory Agents/pharmacology , Anti-Inflammatory Agents/therapeutic use , Burns, Chemical/drug therapy , Cytokines/metabolism , Eye Burns/drug therapy , Inflammasomes/metabolism , Lipopolysaccharides/pharmacology , Mice , NLR Family, Pyrin Domain-Containing 3 Protein/metabolism , RAW 264.7 CellsABSTRACT
The coumarins decursin and decursinol angelate, which are found in Angelica gigas Nakai, have a variety of biological functions. Here, we show that treatment with these compounds improves wound healing by HaCaT human keratinocytes. Wound healing was increased by treatment with up to a threshold concentration of decursin, decursinol angelate, a mixture of both, and a nano-emulsion of these compounds, but inhibited by treatment with higher concentrations. Immunoblotting and fluorescence imaging of cells expressing an epidermal growth factor receptor (EGFR) biosensor demonstrated that these compounds did not stimulate wound healing by inducing EGFR phosphorylation. Rather, transcriptional analysis revealed that decursin and decursinol angelate improved wound healing by upregulating the expression of genes encoding extracellular matrix remodeling proteins, inflammatory cytokines, and growth factors.
