Topical Probiotic Formulation Promotes Rapid Healing in Dog Keratinocyte Cells: A Promising Approach for Wound Management.

The use of probiotics has gained increasing attention as a strategy for wound healing to decrease microbial resistance to disinfectants and antibiotics. This study aimed to investigate the potential of a non-medicinal topical cocktail of probiotic bacteria (CPB) in promoting wound healing in dogs using in vitro scratch assay. Canine Progenitors Epidermal Keratinocytes (CPEK) were exposed to a prototype product formulated with CPB (PPP), non-formulated CPB, and the vehicle. The viability of CPB and CPEK cells was first evaluated in the co-culture model. Then, wound closure was analyzed over time. The CPB required a minimum concentration of 75 CFU/mL for better viability with CPEK. While the CPEK preserved 100% of their viability when PPP was diluted to up to 75,000 CFU/mL. At higher concentrations, the viability of CPEK was reduced by the concomitant effect of the non-formulated CPB and the vehicle. The formulated and non-formulated CPB and the vehicle seem to lead to a dose-dependent increase in cell migration compared to the control. Importantly, at the concentration of 750,000 CFU/mL, the PPP showed a 20% increase in wound closure. Taken together, our findings suggest the potential beneficial effects of the probiotic-based topical cocktail (PPP) on wound healing. However, to confirm and validate these effects, further experiments are necessary to provide more robust evidence and allow us to confidently establish the potential beneficial effects of the probiotic bacteria (CPB) in promoting wound healing.

Receptors and Cofactors That Contribute to SARS-CoV-2 Entry: Can Skin Be an Alternative Route of Entry?

To prevent the spread of SARS-CoV-2, all routes of entry of the virus into the host must be mapped. The skin is in contact with the external environment and thus may be an alternative route of entry to transmission via the upper respiratory tract. SARS-CoV-2 cell entry is primarily dependent on ACE2 and the proteases TMPRSS2 or cathepsin L but other cofactors and attachment receptors have been identified that may play a more important role in specific tissues such as the skin. The continued emergence of new variants may also alter the tropism of the virus. In this review, we summarize current knowledge on these receptors and cofactors, their expression profile, factors modulating their expression and their role in facilitating SARS-CoV-2 infection. We discuss their expression in the skin and their possible involvement in percutaneous infection since the presence of the virus has been detected in the skin.

Visualisation of penetration of topical antifungal drug substances through mycosis-infected nails by matrix-assisted laser desorption ionisation mass spectrometry imaging.

BACKGROUND: Matrix-assisted laser desorption ionisation mass spectrometry imaging (MALDI-MSI) is a mass spectrometry-based technique, which can be applied for compound-specific imaging of pharmaceuticals in tissues samples. MALDI-MSI technology is widely used to visualise penetration and distribution profile through different tissues but has never been used with nail tissue. OBJECTIVES: This study used MALDI-MSI technology to visualise distribution profile and penetration into ex vivo human mycosis-infected toenails of three antifungal active ingredients amorolfine, ciclopirox and naftifine contained in topical onychomycosis nail treatment preparations, marketed as Loceryl® , Ciclopoli® and Exoderil® . METHODS: Three mycosis-infected toenails were used for each treatment condition. Six and twenty-four hours after one single topical application of antifungal drugs, excess of formulation was removed, nails were cryo-sectioned at a thickness of 20 µm, and MALDI matrix was deposited on each nail slice. Penetration and distribution profile of amorolfine, ciclopirox and naftifine in the nails were analysed by MALDI-MSI. RESULTS: All antifungal actives have been visualised in the nail by MALDI-MSI. Ciclopirox and naftifine molecules showed a highly localised distribution in the uppermost layer of the nail plate. In comparison, amorolfine diffuses through the nail plate to the deep layers already 6 hours after application and keeps diffusing towards the lowest nail layers within 24 hours. CONCLUSIONS: This study shows for the first-time distribution and penetration of certain antifungal actives into human nails using MALDI-MSI analysis. The results showed a more homogeneous distribution of amorolfine to nail and a better penetration through the infected nails than ciclopirox and naftifine.

Membrane transporter data to support kinetically-informed chemical risk assessment using non-animal methods: Scientific and regulatory perspectives.

Humans are continuously exposed to low levels of thousands of industrial chemicals, most of which are poorly characterised in terms of their potential toxicity. The new paradigm in chemical risk assessment (CRA) aims to rely on animal-free testing, with kinetics being a key determinant of toxicity when moving from traditional animal studies to integrated in vitro-in silico approaches. In a kinetically informed CRA, membrane transporters, which have been intensively studied during drug development, are an essential piece of information. However, how existing knowledge on transporters gained in the drug field can be applied to CRA is not yet fully understood. This review outlines the opportunities, challenges and existing tools for investigating chemical-transporter interactions in kinetically informed CRA without animal studies. Various environmental chemicals acting as substrates, inhibitors or modulators of transporter activity or expression have been shown to impact TK, just as drugs do. However, because pollutant concentrations are often lower in humans than drugs and because exposure levels and internal chemical doses are not usually known in contrast to drugs, new approaches are required to translate transporter data and reasoning from the drug sector to CRA. Here, the generation of in vitro chemical-transporter interaction data and the development of transporter databases and classification systems trained on chemical datasets (and not only drugs) are proposed. Furtheremore, improving the use of human biomonitoring data to evaluate the in vitro-in silico transporter-related predicted values and developing means to assess uncertainties could also lead to increase confidence of scientists and regulators in animal-free CRA. Finally, a systematic characterisation of the transportome (quantitative monitoring of transporter abundance, activity and maintenance over time) would reinforce confidence in the use of experimental transporter/barrier systems as well as in established cell-based toxicological assays currently used for CRA.

A pilot, layerwise, ex vivo evaluation of the antifungal efficacy of amorolfine 5% nail lacquer vs other topical antifungal nail formulations in healthy toenails.

BACKGROUND: Studies investigating the penetration of amorolfine through the nail have shown the highest concentration in the uppermost layer and measurable antifungal activity even in the lower layers of the nail. OBJECTIVES: This pilot, ex vivo study compared the penetration of antifungal concentrations of amorolfine 5% nail lacquer in different layers of healthy, human cadaver toenails with that of terbinafine 10% nail solution, ciclopirox 8% nail lacquer and naftifine 1% nail solution. Moreover, the effect of nail filing prior to application on the penetration of amorolfine 5% was assessed. METHODS: Unfiled (n = 3) and filed (n = 3) nails were used for each antimycotic agent and amorolfine 5% nail lacquer, respectively. Twenty-four hours after topical application, the nails were sliced (10 µm), solubilised and added to agar plates seeded with Trichophyton rubrum. Zones of growth inhibition were measured. RESULTS: Only amorolfine penetrated the nails at sufficient concentrations to inhibit growth of T rubrum at different nail depths. In contrast, the comparators did not show antifungal efficacy. Nail filing resulted in larger zones of inhibition for amorolfine compared with those of intact nails. CONCLUSIONS: Unlike its comparators, a single application of amorolfine 5% nail lacquer resulted in antifungal efficacy within the nail plate. Nail filing increased the antifungal efficacy of amorolfine 5% nail lacquer.

Pretreatment of skin using an abrasive skin preparation pad, a microneedling device or iontophoresis improves absorption of methyl aminolevulinate in ex vivo human skin.

BACKGROUND: Pretreatment of skin to remove scales/crusts and roughen the surface is essential to enhance the penetration of topically applied methyl aminolevulinate (MAL) prior to photodynamic therapy and to permit daylight to access all parts of the skin lesions. Numerous procedures of skin preparation are currently available. This study compared the in vitro penetration of MAL into ex vivo human skin pretreated with skin preparation pad abrasion or a microneedling device, and evaluated the effectiveness of an iontophoretic device in delivering MAL into ex vivo human skin. METHODS: Human skin samples, obtained from aesthetic surgeries, were used in this study. The thickness of the skin samples ranged between 1.44-2.87mm. Pretreatment of samples was performed with 10 passages of the Ambu® Unilect™ 2121M (Ambu A/S, Denmark) skin preparation pad, 8 rolling repetitions using the microneedling device Dermaroller® HC 902 (Dermaroller GmbH, Germany), or by an iontophoresis device (Feeligreen SA, France) for 1.5h. The effect of these pretreatment procedures on the penetration of MAL into the skin was assessed. RESULTS: Penetration in the total skin, liquid receptor and total penetration was most increased by skin preparation pad treatment, followed by microneedling and iontophoresis. Overall, MAL total penetration was increased up to 103-fold by skin preparation pad treatment, 4-fold by microneedling and 1.8-fold by iontophoresis. CONCLUSIONS: Abrasion with skin preparation pad was shown to be superior to microneedling and iontophoresis for increasing MAL penetration in ex vivo human skin.

Comparison of the Effect of Skin Preparation Pads on Transepidermal Water Loss in Ex Vivo Human Skin.

INTRODUCTION: Pre-treatment of the skin to remove scales and crusts prior to photodynamic therapy (PDT) is essential to enhance the uptake of topically applied methyl aminolevulinate (MAL) and to improve treatment efficacy. This study compared the effect of two different skin preparation pads on skin integrity in ex vivo human skin. METHODS: Ex vivo human skin samples from three donors were pre-treated in triplicates with PREPSTER™ (PR) skin preparation pad (6, 8, and 10 passages) or Ambu Unilect™ (A-UN) skin preparation pad (6, 8, and 10 passages). In addition, skin samples were pre-treated with tape strippings (10 adhesive tape strips) as a reference method for comparison. Transepidermal water loss (TEWL) was measured on intact skin and following skin barrier impairment using skin preparation pads and tape stripping. Histological analysis was performed to verify the impairment of the stratum corneum (SC) barrier function in samples from intact skin (control), 10 tape strippings (reference method), 10 passages of PR, and 10 passages of A-UN. RESULTS: TEWL increased with the increasing number of passages of skin preparation pads, with 2.4- and 3.3-fold increases following 10 passages of A-UN and PR, respectively, versus a 2.2-fold increase with 10 tape strippings (reference). Histological analysis showed only partial removal of the SC, with no damage observed on the epidermis, regardless of the procedure used. CONCLUSION: Pre-treatment of skin using PR and A-UN skin preparation pads markedly increases TEWL, indicating slight impairment of the SC barrier function. Comparison of both skin preparation pads showed that PR pad consistently induced significantly higher TEWL than A-UN pad (p < 0.05), regardless of the number of passages. Both skin preparation pads are thought to increase the uptake of MAL and can therefore be used for the preparation of skin prior to PDT. FUNDING: Nestlé Skin Health - Galderma R&D.

Fixed-Combination Gels of Adapalene and Benzoyl Peroxide Provide Optimal Percutaneous Absorption Compared to Monad Formulations of These Compounds: Results from Two In Vitro Studies.

INTRODUCTION: Adapalene 0.1%/benzoyl peroxide 2.5% (0.1% A/BPO) and adapalene 0.3%/BPO 2.5% (0.3% A/BPO) gels are fixed-combination options for the topical treatment of acne. However, the active compounds of these combinations are also available as monads, to be used in association or as monotherapy. These two in vitro studies determined the effect of different treatment regimens on the percutaneous absorption of adapalene (0.1% and 0.3%) gels and BPO 2.5% gel in ex vivo human skin. METHODS: In vitro percutaneous absorption studies were conducted using full-thickness human skin from six donors. Treatment regimens included the application of 0.1% A/BPO, 0.3% A/BPO, or four free-combination regimens of the monads. Skin samples were incubated for 24 h. Concentrations of adapalene and BPO equivalent (BPO-eq) (i.e. benzoic acid after chemical transformation of BPO) were measured using high-performance liquid chromatography. Comparison of regimens was performed using a bioequivalence criterion (estimated ratio bewteen 0.8 and 1.25). RESULTS: The fixed combination 0.3% A/BPO regimen demonstrated more than three times higher absorption of adapalene versus the fixed-combination 0.1% A/BPO. Based on the bioequivalence acceptance criterion, all four free-combination regimens were different from 0.1% A/BPO and 0.3% A/BPO, with higher adapalene release delivered by the fixed combinations versus the free combinations. For BPO-eq, the results showed that the free-combination regimens where adapalene 0.1% was applied first were different from 0.1% A/BPO, with lower BPO-eq release delivered by these regimens compared to the fixed combination. The regimen adapalene 0.3% for 10 h followed by BPO 2.5% delivered lower BPO-eq release compared to the fixed combination. CONCLUSION: The fixed-combination A/BPO gels provide optimal percutaneous absorption of the active compounds compared to free combinations of adapalene 0.1%, adapalene 0.3%, and BPO 2.5%. The higher concentration of adapalene in the 0.3% A/BPO gel and the resulting higher absorption may explain higher clinical efficacy.

Lack of effect of selected sunscreens applied on ex vivo human skin for 5-methyl-aminolevulinic acid penetration and protoporphyrin IX photoactivation.

BACKGROUND: Photodynamic therapy (PDT) is a successful treatment for non-melanoma skin cancers. Methyl-aminolevulinate (MAL) is metabolized to protoporphyrin IX (PpIX) which accumulates in the skin lesion and which generates a painful photochemical toxic reaction upon red light exposure. PDT using daylight (DL) exposure is now used to reduce pain and subjects are advised to protect the areas with sunscreen. This work investigated the effect of sunscreen on MAL penetration and PpIX photoactivation in ex vivo human skin. METHODS: To measure skin penetration of MAL, particle-free sunscreens were applied on ex vivo human skin samples mounted on diffusion cells before application of Metvix cream containing [14C]-MAL for 2.5h. To circumvent the absence of skin penetration of PpIX, skin samples were first treated with microneedles and mounted on diffusion cells before the application of PpIX solution for 1h followed by sunscreens. Skin samples were then exposed to solar simulator for 1h. Concentrations of [14C]-MAL or PpIX were measured in both total skin and receptor liquid. RESULTS AND CONCLUSIONS: The results showed that the in vitro skin penetration of MAL and the PpIX photoactivation on ex vivo human skin samples are not modified by pretreatments of ex vivo human skin with sunscreens. This study demonstrates that neither in vitro skin penetration of MAL nor PpIX photoactivation were modified by pretreatments with Cetaphil SPF 30 Dermacontrol and Actinica® Lotion SPF 50+. This supports the efficacy and safety of MAL DL-PDT in the clinical situation.

Characterization of ABC transporters in human skin.

BACKGROUND: Most identified drug transporters belong to the ATP-binding cassette (ABC) and solute carrier (SLC) families. Recent research indicates that these transporters play an important role in the absorption, distribution and excretion of drugs, and are involved in clinically relevant drug-drug interactions for systemic drugs. However, very little is known about the role of drug transporters in human skin, especially in the disposition of topically applied drugs, and their involvement in drug-drug interactions. The aim of this work was to characterize the ABC transporters in human skin. METHODS: Expressions of ABCB1 multidrug resistance protein 1 (MDR1) also known as P-gp, ABCC1 and ABCC2 multidrug resistance-associated protein 1 and 2 (MRP1 and MRP2), and ABCG2 brest cancer resistance protein (BCRP) in human skin tissues were analyzed by quantitative real-time polymerase chain reaction (RT-PCR). The modulations of ABCB1 and ABCC1 expressions were analyzed after ex vivo treatment of human skin with rifampicin and dexamethasone. The localization of the major transporter MRP1 in human skin was analyzed by immunohistochemistry. Finally, functional analysis of MRP1 in human skin was performed using different specific substrates and inhibitors. RESULTS: The expressions of ABCB1, ABCC1, ABCC2, and ABCG2 were all detected in human skin, of which the expression of ABCC1 was considered the most important. The comparison of human skin with human hepatocytes and kidneys shows that the expression of ABCC1 increased 15-fold in skin than in hepatocytes. Immunohistochemistry revealed marked expressions of MRP1 within the hair follicle, sweat gland and muscle, as well as moderate expression in the basal epidermis. Functional analysis demonstrated that the skin absorptions of rhodamine 123, [3H]-vinblastine, and [3H]-LTC4 were markedly decreased in the presence of MRP1 inhibitors (verapamil and MK571), thus supporting the role of MRP1 in the uptake of compounds from the epidermal compartment as well as their secretion into the bloodstream and sweat ducts. CONCLUSIONS: The present findings are the first to demonstrate the involvement of MRP1 in drug uptake in human skin.