Antioxidants with proven efficacy and elastin-conserving vitamin C-A new approach to free radical defense.

BACKGROUND: This paper describes the background research and validation related to the formulation of a novel antioxidant product. Two defined outcomes were sought. Firstly, a combined efficacy of antioxidant ingredients in quenching free oxygen radicals. Secondly, the investigation into whether a vitamin C derivative sodium salt was elastin conserving in contrast to current vitamin C/l-ascorbic acid variations that have been reported to negatively affect elastin constitution and regeneration. MATERIALS AND METHODS: A leading l-ascorbic acid antioxidant available on the market was compared with the experimental new product in two studies. In the first experiment, the products were compared to assess their antioxidant properties. The evaluated products TOPICAL ANTIOXIDANT 1 and TOPICAL ANTIOXIDANT 2 were applied to human skin cultures (25-30 mg/cm2 ) for a total of 72 h of treatment and exposed to oxidative stress. The generation of free radicals was semi-quantitatively assessed by measuring the fluorescence intensity of the deacetylation and oxidation of the probe dichlorofluorescein diacetate (DCFH-DA). In the second experiment, an ex vivo skin model (derived from patients undergoing facelift procedures) was used to assess elastin preservation. Three skin explants were topically subjected to the two formulations daily for 7 days. The skin was then prepared and fixed for immunofluorescent assessment after staining with CD44 and tropoelastin antibodies. Images were then analyzed using ImageJ. RESULTS: A full description of the different components selected for the new formulation is presented. In the first study, the experimental formulation performed with absolute equivalence to the comparator in its radical quenching capacity; both showed extremely effective antioxidant function. In the second study, the comparator negatively affected the existing elastin with areas of breakdown and diminished staining. In contrast, the new formulation showed good conservation of healthy elastin in all sections demonstrating elastin preservation. CONCLUSION: A new antioxidant formulation was carefully designed with multiple actives that show an equivalent antioxidant capacity to a leading product on the market. More importantly, the vitamin C component shows direct elastin conservation and improvement as opposed to the comparator, which had negative effects on elastin preservation. This is in keeping with little-known literature reports on vitamin C and its negative effects on elastin and validates the use of a sodium salt derivative, which appears to have protective effects on elastin. These findings support the overall regenerative extracellular matrix changes seen with TriHex® technology in other products.

Advances in Pigmentation Management: A Multipronged Approach.

BACKGROUND: Key cellular players regulating human skin pigmentation include melanocytes in the epidermis that synthesize melanin, neighboring keratinocytes that receive and distribute melanin in the upper layers, and fibroblasts in the dermis that affect overlying melanocytes and keratinocytes. In addition, endocrine factors from the blood supply (endothelial cells) and inflammation-related factors play a role. Thus, new strategies for affecting pigmentation need to consider these multiple cell lines to adequately cover various causes and disease processes associated with hyperpigmentation. METHODS: Pathophysiologic mechanisms and cellular pathways involved in melanogenesis were thoroughly reviewed with particular emphasis on the cellular interplay involved in the process. A complex system of interlinking and independent pathways was defined and described demonstrating differing pathways for altered pigmentary disorders - melasma associated with endothelial cell interactions; post inflammatory hyperpigmentation associated with keratinocyte inflammatory mediators (PGE2 in particular); and photodamage involving all 4 cell types. In vitro validation studies were then undertaken to define differing cell group gene expression profiles with selected peptides and other active agents. Melanocytic production of pigment was then tested with these agents to identify key potential players capable of limiting pigmentation. RESULTS: Hexapeptide-12 and lactoferrin (melanocytes), Hexapeptide-11 (in keratinocytes), and phosphatidylserine (endothelial cells) were identified as major inhibitors of melanogenesis based on their gene expression profiles. This was confirmed by secondary melanin production tests performed on melanocytic lines. Additional active agents were also identified as inhibitors of melanocytic production of melanin, and together, these constituents formed the basis for a novel formulation for use in pigmentary disorders. CONCLUSION: A comprehensive scientific narrative of the various facets relating to pigmentation has been presented including differing pathways affecting varied cell lines that effect pigment production. Based on this concept, actives were tested using gene expression studies as well as in vitro melanogenic model testing in different cell lines. Using this novel multi-faceted approach, we have selected and validated a series of active agents to be used in a formulation targeting the complex problem of hyperpigmentation. J Drugs Dermatol. 2022;21(11):1206-1220. doi:10.36849/JDD.7013.

Effects of a Topical Anti-aging Formulation on Skin Aging Biomarkers.

Objective: Following previous clinical trials, an antiaging product (Restorative Skin complex [RSC]; Alastin Skin Care Carlsbad, a Galderma company), was investigated for its effects on Klotho gene regulation, telomere length, and histological biopsy changes to provide a comprehensive picture of the mechanism and efficacy of its anti-aging effect. Methods: Neonatal human fibroblasts were used for telomere length studies to examine the effect of the full RSC formulation and the amino acid components Tripeptide-1 and Hexapeptide-12 (TriHex™) on these cellular aging mechanisms. In addition, RNA sequencing was conducted using human keratinocytes specifically investigating Klotho and related genes. This was supplemented by a clinical study using biopsy samples. Results: TriHex™ significantly upregulated the Klotho gene and related FGF23, FGFR1 and FOXO3B anti-aging genes. Significant telomere shortening reduction over control was demonstrated with the RSC formulation at four weeks and with TriHex™ at six weeks for all percentiles tested. Previous clinical studies demonstrated that the use of the antiaging regimen for 12 weeks produced a statistically significant improvement in scores for all evaluated parameters. Restaining of previous biopsy blocks from the clinical trial revealed positive ECM changes, stimulation of collagen, fibrillin, CD44 and elastin. Limitations: The study was limited by a relatively small numbers of patients in the clinical trial and the non-competitive nature of the trial. Conclusion: RSC anti-aging formulation and its TriHex™ components demonstrated significant reduction in telomere shortening, upregulation of Klotho and FOXO3 genes and biopsy validation of anti-aging efficacy. This new science supplements previous trials that demonstrated clinical efficacy of the formulation.

Designing topical hyaluronic acid technology-Size does matter .

INTRODUCTION: Hyaluronic acid (HA) plays an important role in cellular and extracellular matrix (ECM) homeostasis. Recent studies demonstrate that low molecular weight (MW) HA has pro-inflammatory characteristics while high MW HA is considered anti-inflammatory and regenerative. In formulating a topical HA product, the possibility of creating a focused high MW HA technology was posed, combining external surface high MW HA constituents with active agents promoting fibroblast production of high MW in the depths of the dermis. METHODS: Human dermal fibroblasts and keratinocytes were treated with various agents, and RNA sequencing (RNA-seq) was conducted to identify genes involved in HA synthesis. HA production by fibroblasts was assessed by collecting the culture supernatant, concentrating the protein, and conducting polyacrylamide gel electrophoresis (PAGE). The gel was stained with Stains-All to identify bands relative to known HA products of different MWs. Subsequently, the supernatants were treated with hyaluronidase to confirm the bands corresponded to HA. RESULTS: The RNA-seq results revealed a variety of agents upregulated HA-related genes. However, a potent upregulation of HA synthesis gene was observed by hexapeptide-11 in the keratinocytes and a newly identified proprietary octapeptide in the fibroblasts. PAGE demonstrated not only robust production of HA by octapeptide, but significantly, the HA produced was ~2 Mega Daltons in size. Octapeptide was the most potent stimulator among the tested agents. CONCLUSION: Comprehensive in vitro testing identified a group of active agents that stimulated high MW HA production. This novel approach to HA topical application with exclusively high MW HA production should maximize hydration capacity while encouraging regenerative activity within the ECM. Multi-center trials are underway.

Developing a Topical Adjunct to Injectable Procedures.

Injectable procedures have come to play an enormous part in everyday aesthetic medical practice. Whether its use is directed at volumizing with fillers, decreasing volume using enzymes, skin-tightening using multi-needle approaches, or neuromuscular blockade, the injectable route is the means of delivery in all these cases, making injectable procedures the most common aesthetic procedure performed. As with all procedures, expected and unexpected consequences may follow including bruising, swelling, discomfort, and the possibility of infection. This paper outlines the scientific process and validation of a product designed as an adjunct to injection therapy and the scientific deep dive needed to encompass both symptomatic and adjunctive purposes. On the symptomatic side, bruising, swelling, and pain were considered, while volumetric enhancement, regeneration, and anti-microbial/biofilm effects were desired outcomes from the adjunctive perspective. Utilizing peptides and active agents aimed at reducing excess residual iron and stimulating macrophage absorption of red blood cells, we were able to achieve efficient resolution of bruising. In addition, peptides were included to stimulate collagen, elastin, and hyaluronic acid in synergy with the injectable. Anti-inflammatory, antimicrobial, and antibiofilm agents were added to aid in the safety profile of the injectable. In vivo testing of bruising resolution demonstrated that at day 2/3, participants using the study product (INhance Post-Injection Serum with TriHex Technology®, Alastin Skincare, Inc. Carlsbad, CA) had 73% less bruise color intensity and statistically significant improvement over the bland moisturizer. Overall, 81% of subjects applying the study topical product had less bruising at day 2/3 compared to the bland moisturizer. J Drugs Dermatol. 2020;19(4):398-404. doi:10.36849/JDD.2020.5016.

Gene Expression Studies Pertaining to Extracellular Matrix Integrity and Remodeling: Nuances and Pitfalls of In Vitro Investigations

Anti-aging strategies using topicals with active agents demand validation and proof of efficacy. One investigation in this realm involves gene expression testing. This study undertakes gene expression analysis of Alastin Skincare Regenerating Skin Nectar (RSN) using an in vitro human skin model. The current study is similar to other published human skin model studies, but with additional time periods beyond 24-hours (which are more appropriate for testing peptides) and a suitable control for the Alastin non-aqueous product. Results show the Alastin product upregulates a large array of genes within areas of skin renewal, extracellular matrix remodeling, barrier function, and inflammation after 72 hours. The study provides gene expression data that support the clinical success of the product. It also demonstrates the difficulty and vulnerabilities in assessing efficacies of products with certain in vitro investigations when the nuances of that product are not considered. J Drugs Dermatol. 2019;18(12):1255-1259.

Non-Surgical Fat Reduction and Topical Modulation of Adipose Tissue Physiology

Non-surgical fat reduction procedures have gained in popularity over the past few years and remain in great demand. The process results in accumulation of breakdown products, lipid droplets, that are slowly absorbed over a period of months. This paper outlines the physiological process whereby lipid droplets are absorbed through a process of autophagy (lipophagy) involving a repackaging of these droplets to smaller sizes so that macrophages can then cope with digestion of these very large particles. Furthermore, a fat compartment is described within the dermis surrounding the tail of the hair follicle, which is attracting much attention due to its unique phenotype, function, and connection to the deeper subcutaneous fat compartment. This provides an entry route for direct signaling to the subcutaneous fat. Related to these novel concepts, peptides can be designed in liposomal delivery systems to target lipid droplet breakdown via the hair follicle entry route. This concept is elucidated in this paper. J Drugs Dermatol. 2019;18(4):375-380.

Clinical efficacy and safety of a multimodality skin brightener composition compared with 4% hydroquinone.

There are numerous common skin disorders involving hyperpigmentation, including solar lentigines, postinflammatory hyperpigmentation, melasma, freckles, and dyschromia from photoaging. While these conditions are of an aesthetic nature, there is great interest in newer, safer, and more effective treatment modalities. Topical hydroquinone (HQ) has been the gold standard of skin lighteners for many years. However, regulatory authorities around the world are now questioning its safety. A randomized, double-blind, half-face study was conducted in females with moderate to severe facial hyperpigmentation to assess the efficacy and tolerability of 3 new skin brightener formulations containing SMA-432, a prostaglandin E2 inhibitor, compared with 4% HQ. Each subject was assigned 2 of the 4 test materials and was instructed to apply the product on the assigned side of the face twice daily for 12 weeks. Evaluation visits were conducted at baseline and at 4, 8, and 12 weeks. At each visit, subjects were evaluated by a blinded investigator for clinical efficacy and tolerability using grading scales. Standardized digital photography and Chroma Meter assessments were also taken. Self-assessment questionnaires were completed at weeks 4, 8, and 12. Sixty-eight Caucasian subjects (136 half faces) completed the study. All test materials significantly reduced Overall Hyperpigmentation and improved the Investigator's Global Hyperpigmentation Improvement rating at weeks 4, 8, and 12 compared with baseline. SMA-432 exhibited a dose-dependent improvement in hyperpigmentation. There were no major tolerability issues with any of the test materials. Self-assessments were generally favorable for all test materials. At the completion of the trial, subjects rated one of the tested multimodality brightener compositions as the most favorable product and 4% HQ as the least favorable. This study demonstrated that the new non-HQ-containing skin brightener formulations were as effective and equally well tolerated as the gold standard, 4% HQ, in females with facial hyperpigmentation.

Formulation optimization, skin irritation, and efficacy characterization of a novel skin-lightening agent.

BACKGROUND: Skin-lightening preparations are used by people all over the world for a diverse range of dermatologic indications. The gold standard treatment for skin lightening is with hydroquinone but has been controversial because of the presence of several side effects. Therefore, there has been a constant search for developing new treatment alternatives. Furthermore, the new amendments and bans on animal testing by ECVAM have made the three-dimensional models like EpiDerm(™) and MelanoDerm(™) increasingly popular. OBJECTIVE: This work aims at the formulation development for a new skin-lightening agent, SMA-012, followed by testing for skin irritation and efficacy. METHODS: Formulation parameters such as concentration of SMA-012, amount of ethanol, effect of permeation enhancers and pH were first optimized using Franz cell experiments. Tape stripping and underlying skin assays were performed to analyze the amounts of SMA-012 in different layers of skin. The irritation potential and efficacy of the screened formulation were evaluated using Epiderm(™) and Melanoderm(™) models. RESULTS: Skin permeation experiments suggested that concentrations of 0.1% SMA-012, 35% ethanol, and pH of 8.5 to be the best formulation characteristics. This particular formulation was found to be nonirritant for short-term exposure, when tested in Epiderm(™) model and also significantly effective in decreasing the amount of melanin in pigmented skin equivalent models. CONCLUSION: SMA-012 shows a good promise as a skin-lightening agent for cosmetic and therapeutic applications. Additionally, our study demonstrates the application of skin equivalent models as alternatives to animal testing in studying the regulation of skin pigmentation.

Clinical efficacy and safety of a multimodality skin brightener composition compared with 4% hydroquinone.

There are numerous common skin disorders involving hyperpigmentation, including solar lentigines, postinflammatory hyperpigmentation, melasma, freckles, and dyschromia from photoaging. While these conditions are of an aesthetic nature, there is great interest in newer, safer, and more effective treatment modalities. Topical hydroquinone (HQ) has been the gold standard of skin lighteners for many years. However, regulatory authorities around the world are now questioning its safety. A randomized, double-blind, half-face study was conducted in females having moderate to severe facial hyperpigmentation to assess the efficacy and tolerability of 3 new skin brightener formulations containing SMA-432, a prostaglandin E2 inhibitor, compared with 4% HQ. Each subject was assigned 2 of the 4 test materials and was instructed to apply the product on the assigned side of the face twice daily for 12 weeks. Evaluation visits were conducted at baseline and at 4, 8, and 12 weeks. At each visit, subjects were evaluated by a blinded investigator for clinical efficacy and tolerability using grading scales. Standardized digital photography and Chroma Meter assessments were also taken. Self-assessment questionnaires were completed at weeks 4, 8, and 12. Sixty-eight Caucasian subjects (136 half faces) completed the study. All test materials significantly reduced overall hyperpigmentation and improved the Investigator's Global Hyperpigmentation Improvement rating at weeks 4, 8, and 12 compared with baseline. SMA-432 exhibited a dose-dependent improvement in hyperpigmentation. There were no major tolerability issues with any of the test materials. Self-assessments were generally favorable for all test materials. At the completion of the trial, subjects rated one of the tested multimodality brightener compositions as the most favorable product and 4% HQ as the least favorable. This study demonstrated that the new non-HQ-containing skin brightener formulations were as effective and equally well tolerated as the gold standard, 4% HQ, in females with facial hyperpigmentation.