The application of a multi‐component reaction peptide as a model regenerative active to enhance skin wound‐healing postlaser procedure in a double‐blinded placebo‐controlled clinical trial

Esthetic procedures are currently among the most effective options for consumers seeking to correct aging signs such as fine lines, wrinkles, and skin tone unevenness. Currently, there is a scientific need for an adjunct active to be paired with esthetic procedures to encourage wound recovery and address postprocedure pigmentation concerns.


| INTRODUC TI ON
Minimally invasive procedures have become very popular options for consumers seeking immediate and perceivable effects to correct signs of aging such as wrinkles, rough texture, and pigmentary concerns.2][3][4] The rise in minimally invasive procedures is fueled by the rapid introduction of new technologies that treat the skin without inducing significant visible damage.This results in reduced medical and social downtime, and can performed quickly with only local anesthetics while still providing the desired natural-looking results. 5The advance in technology creates a paradigm shift in the best practices for esthetic procedures toward a more holistic treatment routine consisting of pairing procedures with pre/postprocedure care options.
In principle, esthetic procedures serve as a "trigger" for stimulating the body's own physiological response to wound healing. 6e wound-healing event induces skin regeneration by initiating the different stages of the wound-healing cascade: inflammation, proliferation, and remodeling. 7The epidermal and dermal regeneration results in the renewal of epidermis and production of A new extracellular matrix.Since esthetic procedures initiate well-controlled skin wound healing, pre-and postprocedure care for the treated area is critical to ensure optimal results.A well-conducted survey consisting of 2000 dermatologists and 388 plastic surgeons conducted by Gold et al. provided great insights into the general pre/ postprocedure routine accompanying minimally invasive nonenergy-based procedures (defined as procedures including microdermabrasion, microneedling, threads, and chemical peels). 8The general guidelines are to avoid prolonged sun exposure and the use of stimulating products such as chemical peels, retinol, or formulations containing high alcohol content. 8Tretinoin, hydroquinone, and isopropyl alcohol are some of the commonly suggested pre-procedure products, with Aquaphor being the most frequently used postprocedure product. 8One of the key findings in that study is the consensus that it is important to apply topical treatment after minimal invasive nonenergy procedures to control the level of inflammation and prevent scarring. 8 illustrated by Gold et al., the proper scientific development of an active as part of a postprocedure product is highly important to ensure optimal clinical success. 8The objective of this study is to introduce a peptide synthesized from multicomponent reaction (referred to as MCP) as a proof of concept active for postesthetic procedure application.Originally designed to counter the effects of elastase (which induces the degradation of elastin in the skin), MCP is demonstrated to benefit the needs of skin in postprocedure settings through various in vitro evaluation methods (pigmentation, barrier recovery, stimulation of fibroblasts, and inhibition of elastase activity) and in a forearm minizone study utilizing ablative laser.

| Synthesis of MCP
The multiple-component peptide (MCP) is synthesized through a multi-component reaction by mixing isobutyraldehyde, trifluoromethylamine, and isocyanoacetate at 20° for 48 h according to the method detailed by Dalko et al. to create the final peptide of acetyltrifluoromethylphenyl valylglycine. 9Although MCP is not designed to be a commercialized active, the use of MCP is valuable to shed light on the functionalities of an active with a regenerative signature in postprocedure applications.The learnings from this study can be used to identify future actives that can enhance the wound healing and anti-aging outcome of esthetic procedures.

| In tubo enzyme inhibition experiments
To characterize the potency of the MCP molecule, the first area of focus was to study its ability to inhibit the activities of three key enzymes: collagenase, elastase, and tyrosinase.For the tyrosinase inhibition assay, a 200 U/mL solution of tyrosinase from mushroom (T3824-50KU, Sigma-Aldrich) in PBS and a 2.5 mM solution of L-tyrosine (T3754-100G, Sigma-Aldrich) in DI water was prepared.
The tyrosinase and test compound were added to a 96-well plate and incubated at 37°C for 15 min.A 1 mM and 0.1 mM solution of Kojic Acid (K3125-10G, Sigma) served as the positive control.
The L-tyrosine was then added and the absorbance at 490 nm was Conclusion: This study demonstrates the use of MCP as a proof of concept regenerative active that when incorporated into an optimized postprocedure skincare formula can improve skin healing and enhance the appearance of skin after injury with relevance to ablative aesthetic procedures.

K E Y W O R D S
ablative laser, esthetic procedure, skin barrier, skin physiology/structure, wound healing taken every minute for 30 min.The slope of the plot (time vs. absorbance) was taken.Relative inhibition was calculated as follows: For the elastase (neutrophil elastase inhibition assay, MAK213, Sigma-Aldrich, MO) and the collagenase assays (Collagenase activity colorimetric assay kit, Cat # MAK293-1KT, Sigma-Aldrich, MO), the experiments were performed according to manufacturer's instructions.In short, neutrophil elastase hydrolyzes a specific fluorescent substrate to release the fluorescent group, which can be detected at Ex/Em = 400/505 nm.This assay evaluates the ability of MCP to inhibit the hydrolysis induced by neutrophil elastase.

| Melanocytes monoculture, melanin content quantification and gene expression
Normal human epidermal melanocytes (NHEM, C2025C, Lot# 1981687, Thermo-Fisher) were seeded in six-well plates and grown in culture medium (M254500, Thermo-Fisher) supplemented with Gibco™ Human Melanocyte Growth Supplement-2 (S0165, Thermo-Fisher) at the density of 10 000 cells/cm 2 for 3-4 days.The medium was then replaced by a culture medium with different treatments for 10 days.In this experiment, the cells were stimulated with 30 nM of PGE2 (P0409, Sigma-Aldrich), with or without 0.005% or 0.05% MCP to evaluate its inhibitory melanogenesis properties.The vehicle control (listed in the figure as control) selected for this experiment contain is PBS with 1% ethanol, as ethanol is used as the solvent for MCP.
After 10 days, RNA is extracted from the cells according to manufacturer's instructions (RNeasy Mini Column Kit, Cat# 74108, Qiagen).After RNA isolation was completed, 1 μg of RNA was used in the cDNA reactions for each sample and 10 ng of cDNA was used per reaction for qRT-PCR.To understand the changes to the key inflammatory genes related to inflammation-driven pigmentation, the gene expression of COX-2 and p38 were normalized against to TATA-binding protein (TBP, housekeeping gene).ΔΔCt values were calculated by normalization to the appropriate untreated control ΔCt value.The selection of COX-2 and p38 as the primary genes of interest is based on a previous publication demonstrating the connection of PGE2 with COX-2 and p38 MAPK in human melanocytes and suggested that this particular pathway is a promising target for treating postinflammatory hyperpigmentation. 10 the end of the experiment, NHEMs were trypsinized, collected, and centrifuged in a 1.5 mL Eppendorf tube at 180 x g for 7 min.The supernatant was discarded, and the pellet was then dissolved in a cold lysis buffer containing 1% of Triton X-100 (AC327371000, Fisher Scientific, NH) in PBS pH 7.4 for 30 min in a rotator.The cells were centrifuged at maximum speed (13 000 x g) for 30 min at 4°C and the supernatant was saved for protein BCA assay.The pellet was then suspended with 550 μL of 1 N NaOH at 80°C for 30 min and 100 μL of supernatant was placed into a 96well plate to measure the optical density (OD) of the supernatant was measured at 405 nm/475 nm (Thermo-Fisher).Total protein was measured in the soluble fraction using a BCA Assay (Thermo-Fisher).Average cellular melanin values were quantified as the ratio between total melanin and total protein from the same treatment.Statistical analyses were performed using R and GraphPad Prism 9.0.1.
For the staining of melanocytes with Fontana Masson, the procedure was conducted according to the manufacturer's instruction (ab150669, Abcam).Post-staining, the plate was imaged with a bright field image (40x) with a fluorescent microscope (Leica DM500, Wetzlar).

| MCP alters the phosphorylation of S6
Normal fibroblasts from 33-to 40-year-old female donors were treated with either DMSO control (0.1%), positive control mTOR inhibitor rapamycin (10 μM), or the indicated concentrations of MCP.
After 48 h, the cells were collected and prepared for western blot as per standard protocol and treated with primary antibodies directed against the S6 subunit and phosphorylated S6 (pS6), with GAPDH as a loading control.The lower ratio of P6S to S6 is indicative of mTOR inhibition. 11Western blot lanes containing blots for compounds unrelated to the study were removed as indicated.

| Ex vivo tape-stripping model
Fresh ex vivo skin (n = 2 lots, one Hispanic and one Caucasian female donors, ages 37 and 40 years) was acquired 1-day postabdominoplasty (BioIVT Inc.).Tissue was defatted and cleaned of blood residue.The tissue was then subjected to 30 tape strips using fresh pieces of duct tape.Following tape stripping, 1.2 cm skin biopsy punches of both untreated and tape-stripped regions were created.
The tape-stripped punches were then either left untreated or topically treated with 0.45 mL/cm 2 of 0.5% MCP or aquaphor.Samples were cultured at air-liquid interface in Dulbecco's modified Eagle's medium (DMEM) at 37°C and 5% CO 2 (10% fetal bovine serum and 1% penicillin-streptomycin in DMEM).Aquaphor and 0.5% MCP were applied to the designated explants daily.Following the 7-day culture period, all biopsies were processed for histological and immunohistochemical analyses.Skin explants were processed for hematoxylin and eosin staining and immunohistochemical staining was performed using a Leica Bond automated immunostainer (Reveal Biosciences).Tissue samples were embedded as formalin-fixed paraffin-embedded (FFPE) blocks.Heat-induced antigen retrieval was performed using Leica Bond Epitope Retrieval Buffer and endogenous peroxidase was blocked for 20 min.Nonspecific antibody binding was blocked for 30 min using Novolink Protein Block and sections were incubated with the primary antibodies Mouse Monoclonal to Anti-Filaggrin (Thermo-Fisher) and Rabbit Polyclonal to Anti-Transglutaminase 3 (Thermo-Fisher).Anti-Filaggrin and Anti-Transglutaminase 3 were detected using Novocastra Bond Refine Polymer Detection and visualized with 3′3 diaminobenzidine (DAB).
All sections were then counterstained with a hematoxylin nuclear stain and imaged with a fluorescent microscope (Leica DM500, Wetzlar).Staining intensity was reported by calculating the optical density using MATLAB R2020a.

| Laser-wounded reconstructed skin model
To evaluate the effect of MCP in a reconstructed skin model, this experiment is similarly to a previously published model by He et al. 12 The bio-fabricated skin used in this study was reconstructed as previously described using normal human epidermal keratinocytes and dermal fibroblasts. 12,13A mixture of collagen and fibroblasts were used to construct the dermal compartment.Human normal keratinocytes were seeded on top of this layer on Day 4. The tissue was cultured in a submerged condition with minimal essential medium containing 10% fetal calf serum and transferred to the culture at air-liquid interface for another 7 days before harvest.Laser resurfacing was conducted using the CO2RE® system, Syneron (Candela).The energy level was set to Deep mode (fractional coverage 5%, core energy 30 mJ, core energy fluence 170 J/cm 2 ).The tissue was treated with the laser for 0. underwent standard hematoxylin and eosin staining.Cryosection tissue slices were processed followed by the method used in the previous study. 14The tissue was immunolabeled using the following primary antibodies: anti-filaggrin (MA5-13440, Thermo-Fisher) and anti-Ki67 (MA7240, Dako).Photomicrographs were captured using a Nikon eclipse Ti fluorescence microscope.Statistical analyses were performed using GraphPad Prism.

| Laser-induced mini-zone wound healing model clinical study
A randomized double-blinded placebo-controlled clinical study was performed to investigate the wound-healing efficacy of MCP using an ablative laser-induced wound clinical model on the forearm.The selection of the volar forearm as the chosen test site for inducing superficial wounds through ablative laser is supported by specific advantages.First, the volar forearm presents a consistently even surface, which facilitates meticulous control over both the layout and depth of the wounds.This property is essential for maintaining accuracy during the wound creation process.Second, the volar forearm exhibits a notable uniformity in both color and texture, thereby aiding in the standardization of observations.Third, but not least, the volar forearm's accessibility ensures ease of postprocedure care 11, 14, and 18.The evaluations included the investigator's assessment of wound-healing efficacy parameters: erythema, edema, epithelial confluence, crusting/scabbing, smoothness, and general wound appearance. 15Details of the grading criteria for each of the parameters can be found in Table S1.Digital photographs were taken using a Nikon D7000 digital SLR camera for documentation purposes.Subjective tolerance to products and adverse events were monitored throughout the study for safety assessment.The grader, study principal investigator, and the data scientists directly involved in this study were blinded to the differently treated sites.
Statistical analysis was performed on the intent-to-treat (ITT) population that included all subjects who were randomized and participated in at least one postbaseline evaluation.The statistical analysis baseline was the Day 1 time point for investigator's assessments of efficacy parameters.Mean of the change from the analysis baseline (defined as postbaseline value minus baseline value) was estimated at all applicable postbaseline time points for each applicable parameter.The null hypothesis, that the mean change from baseline is zero, was tested using a Wilcoxon signed-rank test and applied at all applicable postbaseline time points.All statistical analysis was performed using SAS software version 9.3.

| MCP functions as enzyme inhibitors for elastase and tyrosinase
To better characterize the efficacy of the MCP molecule, the first area of focus is to study its ability to act as an enzyme inhibitor.To

| MCP counteracts PGE2-induced melanogenesis via mapk pathway
Following the interesting tyrosinase inhibition behavior seen with MCP, the next area of focus is to study its behavior in a melanocyte model in which the cells were challenged with PGE2 to induce melanogenesis. 10In this model, human melanocytes (NHEM) were treated with PGE2 (30 nM) to result in an increased melanin content in NHEM compared with the vehicle control group (Figure 2).

| MCP dose-dependently inhibits mTOR
In order to explore the potential mechanism of action for MCP on the cellular level, we examined the ability of MCP to inhibit the mTOR pathway through quantification of the pS6/S6 ratio.
Inhibition of the mTOR pathway by topical rapamycin attenuates histological signs of skin aging and significantly reduces senescent cell numbers with a concomitant increased presence of Collagen VII, suggesting mTOR to be a promising pathway for anti-aging intervention. 16We therefore examined the ability if MCP to reduce the S6/phosphorylated S6 ratio within primary human fibroblasts, an indicator of mTOR inhibition.As anticipated, 48 h of fibroblast treatment with prototypical mTOR inhibitor rapamycin resulted in a near complete reduction of pS6 expression, as determined by western blot (Figure 3A).MCP exhibited a dose-dependent increase in mTOR inhibition within this system, with the highest effect occurring at 0.1% (Figure 3A,B).The MCP-treated fibroblasts did not result in significant increase in fibroblast proliferation or motility (data not shown).

| MCP enhances the kinetics of skin barrier repair post tape stripping
The effects of MCP on the regeneration of stratum corneum following physical damage (as simulated by tape stripping) were evaluated using hematoxylin and eosin (H&E) staining and immunohistochemical staining.Figure 4 demonstrates the recovery of stratum corneum throughout the culture period for all treatment groups.Compared to the untreated control, histology confirms complete removal of the stratum corneum immediately following the tape strip treatment on Day 1 (Figure S1).After 3 days in culture, there is an observed regrowth of stratum corneum in all untreated and treated tape strip groups (Figure 4A).By Day 7, the treatment groups had similar levels of regenerated stratum corneum thickness (Figure 4A).It was observed that tape-strip control samples displayed paraketosis as demonstrated by the retention of nuclei in the stratum corneum, which was not observed in tape-stripped explants treated with 0.5% MCP.
To assess the maturation of the regenerated stratum corneum, immunohistochemical staining was conducted to evaluate the effect of MCP on biomarkers related to the reformed skin barrier.Staining against filaggrin demonstrates that tape-stripped skin followed by the treatment of 0.5% MCP shows a similar level of expression as untreated control tissue while tape strip alone demonstrates reduced expression of Filaggrin (Figure 4B).The reduction in filaggrin expression indicates disturbed epidermal maturation and keratinocyte differentiation, altered skin lipid composition and organization, and reduced natural moisturizing factor. 17

| MCP enhancing skin repair in a laser-wounded reconstructed skin model
To evaluate the benefits of MCP to accelerate skin repair in a condition more similar to aesthetic procedure treatment, we have created | 905 LIAO et al.

| MCP promotes wound healing in laser wound-healing model
Using an established laser wound-healing model in humans, the efficacy of MCP in promoting the wound-healing process was tested directly in an IRB-approved, placebo-controlled, double-blinded, and randomized clinical study.Uniform wound test sites were created on the volar forearm and randomized to receive either 10% MCPcontaining formulation, placebo treatment, or left untreated.During the course of 18-day period, the wound healing process was monitored and clinically evaluated through the investigator's assessment.
As shown in Figures 7 and 8, the wound sites treated with 10% MCP showed statistically better wound-healing progress than those treated with placebo for crusting/scabbing on Day 7 and epithelial confluence at Days 11 and 14.No product-related adverse event was reported, and there were no statistically significant changes in subjective tolerance parameters (burning, stinging, itching, tightness, tingling, and pain).Under the current protocol of pairing of the 10% MCP in the ablative laser procedure, no adverse events and local intolerance were reported throughout the study.Thus, we can conclude the use of MCP is safe to accompany ablative laser treatment and does not risk inducing allergic reactions.

| DISCUSS ION
9][20] In general, esthetic procedures are highly efficacious treatments providing appreciable results in a short timeframe with relatively few cases of adverse events. 8,18,21,22However, understanding the root causes of these adverse events enables the development of an optimized skincare routine to accompany esthetic procedures.Gowd et al. and Li et al. conducted excellent literature reviews on the potential complications that may arise in microneedling and laser procedures, respectively. 22,23For radiofrequency and micro-roller devices, erythema, edema, postinflammatory hyperpigmentation, and tram tracking are some of the more commonly observed advent events. 22The importance of a welldesigned postprocedure product is further illustrated by a case report study published by Soltani-Arabshahi et al. 24 In this study, the subjects experienced delayed type 2 hypersensitivity to the vitamin C serum formula applied postmicroneedling and led to the significant development of facial granulomas. 24Although the root cause investigation was inconclusive, the author hypothesized that incompatible fragrances, preservative systems, and the potential of introducing immunogenic particles are some of the key reasons for the development of allergic granulomatous reactions. 24As for ablative and nonablative procedures, hyper/hypo-pigmentation, scarring, acne, erythema, contact dermatitis, and line of demarcation are some of the more commonly seen adverse events. 23e potential for these adverse events highlights the opportunity for postprocedure products to enhance the treatment outcome.
6][27][28][29] Although it is difficult to establish a consensus on the optimal postcare routine considering the diversity in procedure practices, many of these post care products have brought about the enhancement in the level of postprocedure edema and erythema as well as boosting the level of anti-aging/scar appearance. 25,28,29 this study, the key objective is to evaluate the benefits of MCP as a proof of concept active in some of the key criteria that can positively influence the outcome of aesthetic procedure.
Given that many types of procedures involve the compromisation of the skin barrier to induce rejuvenation, one of the key functions evaluated for MCP is the ability to induce barrier repair.To assess this property, this study conducted in vitro experiments in a tape-stripping ex vivo skin model and a laser wounded reconstructed skin model, followed by a forearm laser minizone study.
To assess the kinetics of barrier repair, this study focuses on the expression of filaggrin and transglutaminase postbarrier damage.Transglutaminase is a key regulatory enzyme that functions to crosslink cornified envelope proteins such as involucrin and loricrin during late-stage barrier differentiation. 30Similarly, the crosslinking of filaggrin is an essential element of a mechanically robust cornified envelope. 17Following tape stripping on ex vivo skin, the application of 0.5% MCP enhanced the expression of transglutaminase and filaggrin (Figure 4).This suggests the functional implication of MCP being beneficial to barrier restoration.
MCP's induction of filaggrin expression is further confirmed in the by accelerating the skin cornification process.These in vitro findings were confirmed by the laser minizone study, where the course of 18 days MCP was able to reduce the level of wound scabbing and enhance the level of epithelial confluency over the untreated and the placebo formula (Figures 7 and 8).
This study also investigated the potential benefits of MCP in an experimental condition that utilized PGE2 to trigger inflammation and melanin production.We demonstrated that PGE2 induced upregulated of key inflammatory genes such as COX-2 and p38 MAPK in NHEM and MCP showed anti-inflammatory effects by counteracting these genes in melanocytes.We also observed that PGE2 had a significant effect on stimulating melanin production in NHEM, where 0.05% MCP significantly counteracted the melanin content in PGE2-stimulated melanocytes.These findings, coupled with the efficacy of MCP working as a tyrosinase enzyme inhibitor, suggest the potential for skin tone improvement benefits when paired with esthetic procedures.
The physiological consequences of mTOR pharmacological inhibition within the skin are multifaceted.Animal studies and in vitro evaluation have suggested that rapamycin administration and mTOR inhibition improve clinical wound healing, 31 however, the biological activity of nonclassical mTOR inhibitors may not necessarily mirror those of rapamycin, as seen from the ability of mTOR inhibitor INK128 to promote wound healing via inflammatory pathways. 32Rapamycin also reduces epigenetic aging within keratinocytes 33 and is a potent inhibitor of cellular senescence development. 34 5 s and left with a pattern size of 9 mm.After laser treatment (Day 0), skin models were transferred to six-well plates.The medium was changed on Day 2 and Day 4, and samples were harvested on Day 0, Day 2, Day 4, and Day 7 for H&E and immunostaining.The tissue was treated with or without 10 μL of 0.5% MCP solution topically from Day 0 to Day 2, whereas the laser alone group received 10 μL of 2% ethanol solution (vehicle for MCP solution).The reconstructed skin tissues were fixed in neutral formalin solution.The tissue was embedded, paraffin sectioned, and and continuous observation, contributing to its appropriateness as a preferred test site.Precautions were diligently implemented to mitigate the potential for scarring or any other adverse outcomes.Notably, a key criterion for participant exclusion encompassed individuals displaying compromised wound-healing tendencies, including a susceptibility to keloid formation.Prior to the onset of the clinical study, the study protocol was approved by the Institutional Review Board (protocol number C16-C174, IntegReview IRB), and all volunteers provided informed consent.Healthy men and women volunteers 25-50 years of age with Fitzpatrick skin type I to III and uniform skin color on the volar forearm were recruited to participate in the clinical study at SGS Clinical Studies (Dallas) and 20 subjects completed the study.Laser wounds were created on the volar forearm by a board-certified dermatologist using an erbium/CO 2 laser.The schematic of randomization was according to the predetermined protocol by SGS Clinical Studies to balance out any potential location bias.The laser was set for 2 passes using 5 W of CO 2 followed by 1 pass of erbium at 1.7 J/cm 2 which made the wound deep to the dermal level.Wound sites were randomized into three groups: treatment with 10% MCP, placebo treatment, or no treatment.The selection of the 10% MCP concentration stemmed from a well-tolerated outcome in a prior human repeat insult patch test, devoid of local intolerance or adverse events.To conduct the clinical study, 10% MCP was incorporated in an inverse emulsion formula based on dimethicone and water.Subjects used provided cleanser to clean the wounds once daily and applied the products to designed wound sites at approximately 2 mg/cm 2 three times a day.All wounds were covered with nonadhesive wound covering pad, gauze, and compression wraps for the first 7 days.Clinical evaluations were performed at baseline prelaser, immediately postlaser, Days 1, 4, 7, do so, we investigated three key enzymes: collagenase, elastase, and tyrosinase.These enzymes are key regulatory enzymes on matrix turnover (collagenase and elastase) and skin pigmentation (tyrosinase).Of the different enzymes tested, MCP showed concentration-dependent inhibition effects of elastase and tyrosinase (Figure1A,B) but no significant effect on collagenase (data not shown).The inhibition properties on elastase and tyrosinase have potential implications for the protective effects for consumers presented with loss of skin elasticity and pigmentation challenges.

2 F
To eliminate the melanin content caused by increased NHEM cell number, the final melanin level was calculated by dividing the protein content in each treatment group.PGE2 + 0.05% MCP also showed significant reduction of melanin content compared with the PGE-treated group (Figure2A-E).The increase in melanin content in PGE2 cells was also visualized via Fontana-Mason staining.As shown in Figure2, cells treated with PGE2 have an increased melanin level than control cells (Figure2A,B).Treatment with PGE2+ 0.05% MCP effectively inhibited the upregulation of melanin synthesis caused by PGE2 and kept it at a similar level as controls (Figure2D,E).Previous publications showed that PGE2 upregulated the expression of COX-2 and p38 MAPK in human melanocytes and suggested that this particular pathway is a promising target for the pharmacologic or genetic approaches to treat postinflammatory hyperpigmentation (PIH).10PIH is a relatively common complication in energy-based esthetic procedures, particularly, in skin of color subjects.Therefore, having an active with functionality to manage pigmentation is highly important to improve the skin tone evenness.In Figure2F,G, the stimulated effects of PGE2 on the expression of COX-2 and p38 were significantly increased compared with the untreated control group.The elevated expression of both COX-I G U R E 1 (A) In tubo evaluation of MCP for the inhibition of elastase enzyme.*p < 0.05, **p < 0.01 compared with untreated control and DMSO vehicle.(B) In tubo evaluation of MCP for the inhibition of tyrosinase enzyme.*p < 0.05, **p < 0.01 compared with a negative control.and p38 was significantly reduced by adding 0.05% MCP to PGE2, but not in PGE2 + 0.005% MCP-treated groups.Using NHEM, we demonstrated that PGE2 induced the expression of COX-2 and p38 while 0.05% MCP significantly suppressed PGE2-induced COX-2 and p38 expression.
Staining againstTransglutaminase 3 demonstrates that tape-stripped skin treated with 0.5% MCP leads to a slight trending increase in expression compared with tape-stripped alone (Figure4B).Based on the histological result, it is hypothesized that MCP acts to enhance the keratinocyte differentiation and encourage the cornification process, while is not functioning by encouraging epithelial proliferation.The benefits of MCP in encouraging keratinization and upper terminal differentiation help to reform a mature barrier in skin subjected to barrier disruption.
a wound-healing model by exposing the reconstructed skin to an ablative laser treatment and compared normal wound healing from in tissue with laser resurfacing.For each group, we investigated skin barrier function protein (Filaggrin) and tissue proliferation marker (Ki67).The laser created 56 small wounds per tissue and the wound morphology was tracked on Day 0, Day 4, and Day 7. Figure 5B presents a macroscopic view of the wound during the recovery period using color 3D rendering of the wound volume.One of the key outputs of this assay is to study the extent of cell migration into the dermis layer through hematoxylin and eosin (H&E) staining.Lasertreated tissue demonstrated to have delayed healing in the dermis region over the course of 4 days postinjury, therefore, allowing the keratinocytes to migrate deep into the dermis region (Figure 5F,I).In comparison, laser-treated tissue with the application of 0.5% MCP post damage showed a significantly reduced level of keratinocyte infiltration into the dermis (Figure 5G,J), suggesting that MCP was able to enhance the recovery of the kinetics of skin repair post damage.In the normal control group, expression of the proliferation marker (Ki67) was observed in very few nuclei along the basal cell layer (Figure6A).There was increased expression of Ki67 within basal cells during epidermal wound healing (Figure6B,C,G).By Day 2 post laser procedure, there was a general increase in Ki67 expression for both laser control and laser with MCP treatment compared with the control group.There was no significant difference between the laser alone group and laser with MCP treatment.MCP treatment induced significantly higher filaggrin expression compared with both control and laser control groups(Figure6D,F).Relevant protein expression was quantified.In Figure6H, a clear trend toward increased expression (p < 0.001, compared with the laser control) was detected in MCP-treated tissue.F I G U R E 3 MCP modulates the mTOR pathway by altering the phosphorylation of S6. (A) Representative western blot images (lanes for compounds unrelated to the study removed, indicated by double black lines) and (B) quantification of S6 and phosphorylated S6 (P-S6) ribosomal protein normalized to GADPH of normal human fibroblasts following raw material treatment.*p < 0.01 compared with DMSO control (one-way ANOVA).

F I G U R E 4
MCP enhances barrier repair following tape stripping in an ex vivo skin model.(A) Hematoxylin and eosin staining for undamaged control skin, tape-stripped skin, and tape-stripped skin +0.5% MCP at Day 3 and Day 7. (B) Filaggrin and TGM3 immunostaining for undamaged control skin, tape-stripped skin, and tape stripped skin +0.5% MCP at Day 7. Scale bar: 20 μm.

F I G U R E 5
Assessment of MCP in a laser reconstructed skin model.(A) Reconstructed SoftSkin model was subjected to fractional ablative laser treatment.(B) Induced laser wound presented in a circular pattern with an average diameter of 200 μm.(C, E, H) Transverse section of untreated control tissue at Day 0 (C), Day 2 (E), and Day 4 (H).(D, F, I) Transverse section of laser-treated tissue at Day 0 (D), Day 2 (F), and Day 4 (I).Transverse section of laser +0.5%MCP at Day 2 (G) and Day 4 (J).Scale bar: 100 μm.

F I G U R E 6 F I G U R E 8
Immunostaining of the laser-wounded reconstructed skin for (A-C) Ki67 and (D-F) filaggrin 4 days postlaser injury.The treatment groups are (A, D) untreated control, (B, E) laser treated, and (C, F) laser +0.5% MCP, respectively.(G, H) Quantification of (G) Ki67/ DAPI and (H) relative filaggrin expression intensity.Scale bar: 100 μm.**p < 0.01 vs. untreated control, ***p < 0.001 vs. untreated control and laser-treated tissue.laser-wounded reconstructed skin model.In this model, the kinetics of skin recovery postlaser injury is tracked through histological changes and the expression of Ki67 and filaggrin on Day 2. On the other hand, MCP does not induce a statistically significant level of cell proliferation postinjury but prevents parakeratosis and enhance the level of filaggrin expression.This suggests the mechanism of action for MCP is to enhance the kinetics of keratinocyte differentiation over proliferation and to encourage barrier repair F I G U R E 7 Wound appearance of 10% MCP, placebo-treated and-untreated wound at Day 1, Day 7, and Day 14 following laser-induced injury.Scale bar: 10 mm.Clinical score of (A) crusting/scabbing and (B) epithelial confluency over 14 days.*p < 0.05 vs. untreated group at respective timepoint.

5 |
Topical rapamycin administration is associated with an improvement in aged skin clinical signs, with daily application resulting in improved dermal collagen organization, and reduction in p16INK4A  -positive cells in the epidermis, indicative of senolytic/senomorphic activity.16These findings led us to examine if MCP possessed modulatory properties on mTOR, with our study indicating MCP to be an efficacious mTOR inhibitor.These data open up the possibility of an additional mechanism of action for MCP, which may in part explain the skin regenerative potential of this peptide and is a topic of interest for further studies concerning MCP and senescence/skin regeneration.These data may at least be in part explained by our finding that MCP acts as an mTOR inhibitor, as seen by the reduction of phosphorylated S6/ S6 ratio in NHF.As the connection between cellular senescence and skin aging is well documented, we believe the functionality of MCP to reverse the senescent cell phenotype can potentially improve the signing of aging when applied after aesthetic procedure treatments.Although this study successfully elucidated the multifaceted function of MCP, its ability to enable the anti-aging outcome of the procedure remains to be established.This is the next step for the development of MCP as a regenerative active to be paired with esthetic procedures.The design of this experiment also did not include skin of color subjects, which makes it difficult to extrapolate the efficacy of MCP for skin of color subjects during esthetic procedures.From the in vitro evaluation standpoint, although this study clearly illustrated the multiple functionalities of the active, we did not include a comprehensive gene panel to evaluate the effects of MCP on cells or reconstructed skin.CON CLUS IONS This study introduces the use of MCP as a model active that serves to accelerate the healing kinetics and augment the level of antiaging outcome of skin undergoing aesthetic procedures.Although esthetic procedures are effective in addressing a wide range of skin conditions including dyschromia, roughness, and wrinkles, all procedures inevitably induce a certain level of damage to the treated area.This creates a significant need for an active to facilitate the repair and regeneration of the treated skin area.Furthermore, the transient opening of skin barrier from the esthetic procedures enabled actives of interest to penetrate more effectively and potentially augment the level of anti-aging outcome achievable by the procedure alone.In this study, MCP is found to be an interesting active that can potentially augment the outcome of esthetic procedure by four key mechanisms: (1) induction of barrier restoration, (2) tampering the level of inflammation and the potential of postinflammatory-hyperpigmentation, (3) reduction of elastase activity, and (4) modulation of the mTOR pathway.This study affirms the positive benefits of MCP in a forearm minizone study, where a simple formula containing 10% MCP showed significant improvement in epithelial confluence and reduced the level of crusting/scabbing appearance to the wound during the healing process.This study highlights the potential of a regenerative active to positively impact skin treated with esthetic procedures as part of an optimized postprocedure care routine.protocol was reviewed and approved by the Institutional Review Board (Protocol C16-C174, IntegReview, Austin, TX) prior to study enrollment.O RCI D I-Chien Liao https://orcid.org/0009-0000-7228-0943Richard Betts https://orcid.org/0000-0002-7999-2158R E FE R E N C E S