Acetyl Hexapeptide-8 Research Articles

Strength and targeted-sustained release properties of sodium alginate-polyacrylamide hydrogel for anti-wrinkle eye-patch

The creation of a reliable eye wrinkle solution is essential to meet consumer needs and enhance their quality of life. Researchers devised hydrogels using sodium alginate (SA) and polyacrylamide (PAM), known for their high stretchability and compressive abilities. Acetyl hexapeptide-8 (AH8) is a safe anti-wrinkle ingredient, was incorporated via β-cyclodextrin microencapsulation to ensure minimal side effects. The study conducted transdermal absorption as well with FT-IR experiments and revealed that covalent bonds conferred toughness and ductility, while hydrogen bonds facilitated slow and sustained release. The porous structure of the hybrid gel observed by SEM (average pore size of 430.7 ± 52.2 μm), porosity(60.33 ± 19.95) and swelling rate(525.82 ± 134.29 %) all indicated excellent water retention and drug delivery capabilities. Remarkably, acetyl hexapeptide-8 was first encapsulated in β-cyclodextrin microcapsules as the active anti-wrinkle substance in the study, exhibited pH sensitivity. This research aims to develop a novel anti-wrinkle eye patch using SA-PAM double-network hydrogel and expect to present promising avenues for enhancing mechanical properties and developing medical beauty products suitable for consumers with sensitive skin.

Advancing Transdermal Delivery by Zn/Ag-Electrode-Printed Iontophoretic Patch with Self-Generating Microcurrents

This study aimed to evaluate Zn/Ag-electrode-printed patches for the transdermal delivery of small molecules through iontophoresis. The Zn/Ag-electrode-printed patches interact with biological liquid electrolytes and generate suitable microcurrents for the iontophoretic delivery of small molecules across the skin. In fluorescein permeation studies, Zn/Ag-electrode-printed patches increased the transdermal depth of fluorescein into the dermis, while the permeation of fluorescein was limited when Zn/C-electrode-printed patches were tested. Further permeation experiments were conducted with 3D skin models, which showed a similar trend to the above, indicating that Zn/Ag-electrode-printed patches had a higher penetration rate compared to the blank. Studies using acetyl hexapeptide-8 as a peptide drug model and sodium ascorbyl phosphate (SAP) as a hydrophilic derivative of ascorbic acid showed that the iontophoretic patch with Zn/Ag electrodes promoted more penetration of drugs than unprinted patches. The permeation of SAP exhibited a two-phase profile with a relatively rapid permeation followed by a sustained, slower permeation. The permeation of acetyl hexapeptide-8 was slower due to its higher molecular weight, but the iontophoretic patch increased the permeation up to 1.5 times more than the unprinted patch. The microcurrent generated by the patch drives the transport of small molecule components through the skin, for the controlled and efficient delivery of therapeutic agents. The flexible design, efficient microcurrent generation, and stable electrodes make the Zn/Ag-electrode-printed patch a promising tool for transdermal drug delivery.

Developing Plant Exosomes as an Advanced Delivery System for Cosmetic Peptide.

Peptides are a promising skincare ingredient, but due to their inherent instability and the barrier function of the skin's surface, they often have limited skin absorption and penetration, which can significantly hinder their skincare benefits. To address this, a novel technique called NanoGlow has been introduced for encapsulating peptide-based cosmetic raw materials into engineered nanosized plant-derived exosomes (pExo) to achieve the goal of a healthier and more radiant skin state. In this approach, pExo served as carriers for cosmetic peptides across the intact skin barrier, enhancing their biological effectiveness in skin beauty. The NanoGlow strategy combines chemical activation and physical proencapsulation, boasting a high success rate and straightforward and stable operation, making it suitable for large-scale production. Comprehensive analysis using in vitro cellular absorption and skin penetration models has demonstrated that the nanosized pExo carriers significantly improve peptide penetration into the skin compared to free peptides. Furthermore, in vivo tissue slice studies have shown that pExo carriers efficiently deliver acetyl hexapeptide-8 to the skin's dermis, surpassing the performance of free peptides. Cosmetic skincare effect analysis has also indicated that pExo-loaded cosmetic peptides deliver superior results. Therefore, the NanoGlow technique harnesses the natural size and properties of pExo to maximize the bioavailability of cosmetic peptides, holding great promise for developing advanced peptide delivery systems in both the cosmetic and medical drug industries.

Public Interest in Acetyl Hexapeptide-8: Longitudinal Analysis.

Acetyl hexapeptide-8, also known as Argireline, is a topical, short-acting, synthetic peptide that has recently gained popularity for its antiwrinkle effects. This agent has emerged as a more accessible alternative to botulinum neurotoxin. This study evaluates the public interest in acetyl hexapeptide-8 in the United States from 2013 to 2023, as described by search volume on Google, the most-used search engine. We analyzed the longitudinal relative monthly search volume from January 1, 2013, to January 1, 2023, for acetyl hexapeptide-related terms. We compared the internet search trends for "Botox" during this period to "Argireline." The terms "Argireline" and "Botox in a Bottle" both had substantial increases in search volume in 2022. Although its search volume is drastically increasing, "Argireline" was less searched than "Botox," which had a stable, up-trending search volume over the past decade. The increasing interest in acetyl hexapeptide-8 may be due to its cost-effectiveness and use as a botulinum neurotoxin alternative. Affordability, over-the-counter availability, and ease of self-application of the agent suggest its potential to enhance accessibility to cosmetic dermatologic care.

Enhanced Skin Permeation of Anti‐Aging Peptides Using Bioactive Ionic Liquids at Low Concentrations

AbstractDelaying aging is an eternal goal for humanity. Acetyl hexapeptide‐8 (AHP‐8) is an effective skin anti‐aging drug that locally interrupts the signal transmission of muscle contractions and promotes collagen production, thus smoothening wrinkles. However, its high molecular weight and strong hydrophilicity limit its skin permeation capacity. To solve this problem, an ionic liquid with excellent bioactivity, MAC, is synthesized from l‐malic acid and l‐carnitine and used as a permeation enhancer to improve the anti‐aging effects of AHP‐8. The ratio of MAC's components is optimized based on density functional theory calculations and cytotoxicity experiments. Low concentrations of MAC significantly increase AHP‐8 transdermal delivery, reaching a maximum at 5‐10 wt% MAC. The pro‐permeation mechanism of MAC is investigated using molecular dynamics simulations, and the penetration enhancement and anti‐aging performance of AHP‐8 are demonstrated by conducting cellular, animal, and clinical experiments. The results show that MAC/AHP‐8 outperforms AHP‐8 in terms of collagen and hyaluronic acid synthesis, anti‐inflammation, anti‐oxidation, and inhibition of muscle contraction, thus significantly reducing the number and area of wrinkles and increasing skin moisture and elasticity. This study presents MAC as a simple and efficient transdermal vehicle for the clinical application of AHP‐8.

Polydioxanone Bioactive Sutures-Acetyl Hexapeptide-8 (Argireline): An Intelligent System for Controlled Release in Facial Harmonization.

We propose a new facial lifting protocol using polydioxanone (PDO) threads embedded in acetyl hexapeptide-8 (Argireline [Arg]). We assume that Arg reinforces the effects of PDO threads, as it is a mimetic of botulinum toxin. Because the PDO suture is hydrolyzable, this assumption is analyzed by instrumental analysis. To demonstrate the capacity of the PDO suture as a system for the controlled release of acetyl hexapeptide-8 to apply in deep wrinkles of the upper third. Three segments of 1-cm long 21G PDO threads immersed in 1 mL of Arg. PDO threads were observed under an optical, electron microscope at 24, 48, and 72 h later. They were also weighed before and after being soaked in Arg, and employing ultraviolet (UV)-visible spectroscopy, the release rate of Arg from the PDO suture was measured. Finally, was insert the thread PDO-Arg following a protocol designed especially for deep static wrinkles in the upper third. The electronic weighing revealed that the PDO thread enjoys capillarity by the peptide, doubling its weight every 24 h. UV spectra revealed that PDO thread is a well-controlled release system for Arg, allowing its sustained release for 1 h. Optical and electronic photomicrographs confirm the swelling of the PDO thread by absorbing Arg by its capillarity, but this hydrophilicity does not lead to its premature physical degradation. The PDO thread system with Arg is an intelligent bioactive system useful in facial harmonization. It recommend conduct clinical trial to verify his superior lifting effect.

The Effect of Elasticity Improvement from Acetyl Hexapeptide-8 Ampoule on UVB-Induced Mouse Skin Damage

Acetyl hexapeptide-8 (AH-8) is a well-known component of anti-aging products. AH-8 appears in a variety of cosmetic products, its skin penetration is sparsely studied and controversially discussed. This study investigated ampoule containing AH-8 as an active ingredient. The elasticity improving effect of 3% AH-8 ampoule was tested <i>in vivo</i>. To test the stability of the formulated ampoules, we performed the centrifugation, cycling and measured viscosity, and pH changes. 6 weeks-old female C3H mice were purchased from Orient Bio, Korea Inc. (Seongnam, Korea). After one week of acclimatization, the mice were randomly assigned to experimental groups (n=4 for each group); Control, UVB irradiation, Vehicle ampoule, and 3% AH-8 ampoule group. Irradiated with UVB to mouse skin, then 3% AH-8 ampoule were applied into irradiated sites for 6 weeks. Changes in the amount of TGF-β1 and collagen were measured using western blot. As a result, it was confirmed that the 3% AH-8 ampoule increased the expression of TGF-β1 and collagen. In summary, we showed that AH-8 ampoule had extremely high capability of improving elasticity when compared to vehicle ampoule containing ginkgo leaf and green tea extracts. This study revealed the efficacy of AH-8 for increasing skin elasticity and anti-aging in terms of a cosmetic active ingredient.

Efficacy of Acetyl Hexapeptide-8 Ampoule on Improvement of Elasticity in C3H Mice Skin

Purpose: Acetyl hexapeptide-8 (AH-8) had been reportedly effective in improving keratinocytes. Ampoules containing AH-8 as an active ingredient were prepared, and their efficacy for improving skin elasticity was evaluated.Methods: Ampoules containing 3% and 5% AH-8 were prepared, and their efficacy was evaluated. C3H mice were pretreated with different AH-8 ampoule concentrations and then irradiated with UVB for 6 weeks. Changes in the number of collagen and elastic fibers from paraffin-embedded skin tissues were quantitatively measured using an image analysis program.Results: Our data showed that the skin of C3H mice with UVB-irradiated markedly decreased the collagen levels compared to the control. Moreover, pretreatment of 3% and 5% AH-8 ampoules on UVB irradiation increased collagen production. Furthermore, the elastin production was decreased in the UVB-irradiated group compared to the control and the elastin level in the skin of C3H mice was similar in both pretreated with 5% AH-8 on the UVB irradiation and control groups.Conclusions: This study has proven that HA-8 ampoules had a high capability of improving elasticity when compared to vehicle ampoule-containing ginkgo leaf and green tea extracts. Moreover, this study revealed the efficacy of AH-8 for increasing skin elasticity in terms of a cosmetic active ingredient.

Preparation and Evaluation of the Effect of Acetyl Hexapeptide-8 Ampoule for Scalp Treatment

Purpose: The topical application of scalp-care cosmetics, infused with functional ingredients, can offer an optimal cosmetic approach to prevent problems associated with aging. In terms of the development of anti-aging cosmetics, we studied the use of acetyl hexapeptide-8 (AH-8) for improving elasticity of the scalp.Methods: Ampoules containing 3% and 5% concentrations of AH-8 as an active ingredient were prepared and their safety and efficacy were evaluated. HaCaT cells were used to evaluate the safety of AH-8 ampoules by measurement of in vitro cytotoxicity. In <i>in vivo</i> experiments, we tested the AH-8 ampoules by cumulative irritation test (CIT) on healthy adults, 20–50 years of age. Relative expressions of superoxide dismutase 2 (<i>SOD2</i>), forkhead box O1 (<i>FOXO1</i>), actinin alpha 1 (<i>ACTN1</i>), collagen type XVII alpha 1 chain (<i>COL17A1</i>), and integrin subunit beta 4 (<i>ITGB4</i>) were assessed using quantitative real-time PCR (Q-PCR).Results: AH-8 ampoules showed significant cytotoxicity to HaCaT cells in a dose-dependent manner. CIT assessment in 30 adults revealed no skin reactions to both 3% and 5% AH-8 ampoules. After treatment of HaCaT cells with the AH-8 ampoules, mRNA levels of <i>SOD2</i> and <i>FOXO1</i>, which are directly implicated as antioxidant factors, were increased. Of the factors that improve elasticity, <i>ACTN1</i> mRNA levels had the greatest increase when treated with the 5% AH-8 ampoules (1.75 µg/mL). Also, the AH-8 ampoules increased mRNA levels of <i>COL17A1</i>, and <i>ITGB4</i> in HaCaT cells.Conclusions: In this study, we proved that 3% and 5% AH-8 ampoules are safe for use as a scalp-care cosmetic. AH-8 ampoule had efficacy in anti-aging and improving elasticity of the scalp. This comprehensive test showed that AH-8 can be developed as a cosmetic for anti-aging and improvement of scalp elasticity.

Quantitation of Acetyl Hexapeptide-8 in Cosmetics by Hydrophilic Interaction Liquid Chromatography Coupled to Photo Diode Array Detection

Bioactive peptides are gaining more and more popularity in the research and development of cosmetic products with anti-aging effect. Acetyl hexapeptide-8 is a hydrophilic peptide incorporated in cosmetics to reduce the under-eye wrinkles and the forehead furrows. Hydrophilic interaction liquid chromatography (HILIC) is the separation technique of choice for analyzing peptides. In this work, a rapid HILIC method coupled to photodiode array detection operated at 214 nm was developed, validated and used to determine acetyl-hexapeptide-8 in cosmetics. Chromatography was performed on a Xbridge® HILIC BEH analytical column using as mobile phase a 40 mM ammonium formate water solution (pH 6.5)-acetonitrile mixture 30:70, v/v at flow rate 0.25 mL min−1. The assay was linear over the concentration range 20 to 30 μg mL−1 for the cosmetic formulations and 0.004 to 0.007% (w/w) for the cosmetic cream. The limits of quantitation for acetyl hexapeptide-8 were 1.5 μg mL−1 and 0.002% (w/w) for the assay of cosmetic formulations and cosmetic creams, respectively. The method was applied to the analysis of cosmetic formulations and anti-wrinkle cosmetic creams.

Chemical and biological properties of anti-wrinkle peptide Argireline

Argireline, a peptide with the sequence: Ac-Glu-Glu-Met-Gln-Arg-Arg-NH2, also known as Acetyl Hexapeptide-8, reduces facial lines and wrinkles by destabilization of the formation of the SNARE complex (SNAP Receptor, soluble N-ethylmaleimide sensitive factor (NSF) attachment protein receptor), thus preventing muscle contraction. It is a biosafe cosmetic alternative to the botulinum toxin. The method of choice in bioactive peptide analysis is reversed-phase high performance liquid chromatography coupled with mass spectrometry (LC-MS). The aim of this work as to present the properties of Argireline and the analysis of cosmetic products containing this peptide. Previous reports on possible Argireline transformations in cosmetic formulations have not confirmed deacetylation, whereas the oxidation of methionine residue was detected by our team. As the biological activity of the oxidized Argireline is not known, further biological studies, as well as efficient analytical procedures for transformation monitoring and quality control in cosmetic products, are necessary.

Topical application of Zanthoxylum piperitum extract improves lateral canthal rhytides by inhibiting muscle contractions

Facial wrinkles are the predominant phenotypes of skin aging. To date, one of the most effective ways to improve wrinkles is botulinum toxin type A (BoNT/A) injection, which inhibits muscle contractions by reducing acetylcholine release from neurons. However, since BoNT/A is a hazardous neurotoxin, the injection can only be performed by medical doctors and the procedure is only possible through invasive injection, causing inconveniences such as pain. To overcome these inconveniences, we tried to find a way to reduce wrinkles non-invasively via mechanisms similar to BoNT/A. We first designed in vitro assays to test BoNT/A-like muscle contraction inhibition in two different model systems. By using the assays, we identified Zanthoxylum piperitum (Z. piperitum) fruit extract as a BoNT-like reagent (27.7% decrease of muscle contraction rates by 1000 ppm of Z. piperitum extract treatment). Next, we determined mechanisms of how Z. piperitum extract decreases muscle contraction rates and found that the extract treatment inhibits electrical signal transduction in neurons. We also showed that among known components of Z. piperitum extract, quercitrin is responsible for muscle contraction inhibition. We further identified that Z. piperitum extract has synergistic effects with acetyl hexapeptide-8 and BoNT/A light chain, which are well-known BoNT-like peptides. Finally, we showed that topical treatment of the Z. piperitum extract indeed decreases facial wrinkles and treatment of Z. piperitum extract with acetyl hexapeptide-8 has a tendency to improve wrinkles synergistically (14.5% improvement on average). The synergistic effect of the combination is expected to improve wrinkles effectively by implementing the BoNT/A mechanisms in a non-invasive way.

Trending Anti-Aging Peptides

The development of synthetic peptides for skin care dates to the 1980s. The cosmetic industry periodically launches new peptides, as they are promising and appealing active ingredients in the growing and innovative cosmetics market. In this study, trends in the use of peptides in anti-aging products were analyzed by comparing the composition of the products marketed in 2011 with products launched or reformulated in 2018. The scientific and marketing evidence for their application as active ingredients in anti-aging cosmetics was also compiled from products’ labels, suppliers’ technical data forms and online scientific databases. The use of peptides in anti-aging cosmetics increased by 7.2%, while the variety and the number of peptide combinations in products have increased by 88.5%. The most used peptides in antiaging cosmetic formulations are, in descending order, Palmitoyl Tetrapeptide-7, Palmitoyl Oligopeptide and Acetyl Hexapeptide-8. In 2011, the majority of peptides were obtained from synthesis, while in 2018, biotechnology processing was the dominant source. This study provides an overview of the market trends regarding the use of peptides in anti-aging products, providing meaningful data for scientists involved in the development of new peptides to identify opportunities for innovation in this area.

Skin scars and wrinkles temporary camouflage in dermatology and oncoesthetics: focus on acetyl hexapeptide-8

The aim of study is to evaluate the aesthetic outcome of specific formulated cosmeceutical product to mask and reduce the appearance surgical scars or unappealing skin tags in chronic diseases, such as cancer In a spontaneous, anecdotal, retrospective study, 26 patients with skin disorders appealed to Second Opinion Medical Network (Modena, Italy), required masking and improving the skin appearance. To evaluate the aesthetic improvement of skin imperfections, a gelcream containing 10% of acetyl hexapeptide-8 (registered trademark Argireline®) was selected, that can be applied directly upon the lesion, followed by a light massage in the treated area for a few minutes The skin quality parameters (hydration, elasticity, sebum), photographs and investigators clinical assessment have been performed before and after the treatment and demonstrated that this cream significantly improved the skin values and the self-image expectation of each patient. No allergic reactions were documented during the period treatment The topical administration of this cosmeceutical cream is a safe and effective alternative to the invasive procedures, to improve the quality of life in patients with some skin disorders such as cancer, surgical scars, hidradenitis, aging wrinkles

Anti-Wrinkle Efficacy of Cross-Linked Hyaluronic Acid-Based Microneedle Patch with Acetyl Hexapeptide-8 and Epidermal Growth Factor on Korean Skin.

BackgroundHyaluronic acid (HA)-based microneedle patch has recently been studied for wrinkle improvement. Cross-linked HA (CLHA) is widely used in dermal fillers. Acetyl hexapeptide-8 (AHP-8) and epidermal growth factor (EGF) are used for cosmetic ingredients.ObjectiveThis study aimed to verify the efficacy of the CLHA/HA-based patch with microstructure (microneedle patch) containing AHP-8 or EGF.MethodsA total of 52 Korean females were enrolled in a double-blind, randomized, controlled, split-face trial. The subjects were divided into 3 groups: (1) microneedle patch alone, (2) microneedle patch/AHP-8, and (3) microneedle patch/EGF. The treatment was applied on the periorbital and nasolabial fold area for 4 hours to completely dissolve the microstructures once per week for 29 days. Evaluations, including photodamage scoring, image analysis with Antera 3D® (Miravex, Ireland), skin hydration measurement, and adverse effect assessments, were performed at each visit.ResultsFifty subjects (96.2%) completed this clinical study. On day 29 after application, statistically significant improvements in wrinkle and skin hydration were observed in all groups (p<0.01). Treatment with microneedle patch/AHP-8 and microneedle patch/EGF showed statistically significant improvements in wrinkle compared with microneedle patch alone (p<0.05). No serious adverse effects were noted.ConclusionCombination of CLHA-based microneedle patch and functional cosmetic ingredients can improve wrinkle with minimal discomfort.

히알루론산의 각질 유동성 향상을 통한 주름 개선 펩타이드 피부 흡수 촉진

Acetyl hexapeptide 8 (AH8) is a synthetic peptide for anti-wrinkle cosmetics ingredient. It was developed as a mimetic of botox, patternd after N –terminal end of the protein synatosomal-associated protein 25 (SNAP25), a substrate of botulinum toxin. While AH8 has good efficacy and safety profiles, the permeation through the skin is poor. Therefore, we tried to enhance the transdermal delivery of AH8 by using of hyaluonic acid (HA), a linear polysaccharide of N-acetyl glucosamine and glucuronic acid. To investigate the effect of HA on AH8 penetration, we analyzed paraffin sections of MicropigⓇ skin. Fluorescence labeled AH8 was applied to micropig skin with or without HA. The absorption of AH8 was limited to the stratum corneum (SC) without HA. On the other hand, AH8 penetrated to the dermis with HA. Especially, low molecular weight HA (5 kDa) was most efficient compared to 500 kDa HA and 2000 kDa HA. Experiments using fourier-transform infrared (FTIR) spectroscopy revealed that lower molecular weight HA had a tendency to increase the fluidity of the SC lipids more, which means enhancing the skin penetration. Therefore, HA could be expected to enhance the anti-wrinkle effect of AH8.

Observational studies with confirmation of safety, tolerance and efficacy of a facial sterile solution for the skin rejuvenation containing hyaluronic acid, acetyl hexapeptide-8, carnitine and pyruvic acid in association to laser procedure

Observational studies with confirmation of safety, tolerance and efficacy of a facial sterile solution for the skin rejuvenation containing hyaluronic acid, acetyl hexapeptide-8, carnitine and pyruvic acid in association to laser procedure

Multiple W/O/W emulsions as dermal peptide delivery systems

Dermal delivery of peptides is a difficult task that needs to be solved by formulators in the pharmaceutical as well as in the cosmetic industry. Since multiple W/O/W emulsions are interesting vehicles for delivery of peptides, the influences of different plant-derived oils, namely avocado, wheatgerm and olive oil, on the formation and properties of multiple emulsions were investigated. The multiple W/O/W emulsions were characterized in terms of long-term stability, rheological properties, droplet size and internal structure. Moreover, the in vitro skin permeation of acetyl hexapeptide-8 (AH-8) from the multiple emulsions was investigated by Franz-type diffusion cells with porcine skin. The use of olive oil led to the most stable multiple emulsion with enhanced skin permeation of AH-8 compared to the other multiple emulsions. Moreover, a significantly increased skin permeation of AH-8 at acidic compared to physiological conditions revealed a strong dependency of AH-8 permeation on the chosen pH value.

Topical delivery of acetyl hexapeptide-8 from different emulsions: Influence of emulsion composition and internal structure

Acetyl hexapeptide-8 (AH-8) is a well-known component of anti-aging products and was recently explored as a promising topical treatment of blepharospasm. Although AH-8 appears in a variety of cosmetic products, its skin penetration is sparsely studied and controversially discussed. Therefore, the aim of the present study was to investigate the influence of the vehicle type on the AH-8 delivery to the skin. Besides skin permeation experiments with Franz type diffusion cells, the spatial distribution of AH-8 in the stratum corneum after a real in-use application was investigated by in vitro tape stripping on porcine ear skin. By applying LC–MS/MS for quantification of AH-8, we demonstrated that a multiple water-in-oil-in-water (W/O/W) emulsion can significantly increase penetration of AH-8 into porcine skin compared to simple O/W and W/O emulsions. The internal structure of the developed multiple emulsion was confirmed by electron microscopic investigations and NMR self diffusion studies. In general, a clear superiority of water-rich W/O/W and O/W emulsions over an oil-rich W/O emulsion in terms of dermal delivery of AH-8 was found. This enhanced delivery of AH-8 could be explained by an increased absorption of the water-rich emulsions into the skin, confirmed by combined ATR-FTIR and tape stripping experiments.

In vitro skin penetration of acetyl hexapeptide-8 from a cosmetic formulation

There is a concern that peptides in cosmetic creams marketed as anti-aging/anti-wrinkle may penetrate into the deep layers of the skin and potentially stimulate biological activity. Claims for one cosmetic peptide, acetyl hexapeptide-8 (Ac-EEMQRR-amide), suggest interference with neuromuscular signaling as its anti-wrinkle mechanism of action. Therefore, the skin penetration of commercially available Ac-EEMQRR-amide from a cosmetic formulation (oil-in-water (O/W) emulsion) was determined in hairless guinea pig (HGP) and human cadaver skin assembled into in vitro diffusion cells. An O/W emulsion containing 10% Ac-EEMQRR-amide was applied to skin at a dose of 2 mg/cm2. After a 24-h exposure, the skin surface was washed to remove unabsorbed peptide. Skin disks were tape stripped to determine the amount of peptide in the stratum corneum. Removal of the stratum corneum layers was verified by confocal microscopy. The epidermis was heat separated from the dermis and each skin fraction was homogenized. Skin penetration of Ac-EEMQRR-amide was measured in skin layers by hydrophilic interaction liquid chromatography with tandem mass spectrometry using electrospray ionization (ESI) in the positive mode. Stable isotopically labeled hexapeptides were used as internal standards for the quantitation of native hexapeptides to correct for matrix effects associated with ESI. The results (percent of applied dose) showed that the majority of the Ac-EEMQRR-amide was washed from the surface of both HGP and human skin. Ac-EEMQRR-amide that penetrated skin remained mostly in the stratum corneum of HGP (0.54%) and human (0.22%) with the peptide levels decreasing as each layer was removed by tape stripping. Total Ac-EEMQRR-amide found in the epidermis of HGP and human skin was similar at 0.01%. No peptide was detected in the dermis or buffer collected underneath the skin for both human and HGP. There was no hexapeptide metabolite (H2N-EEMQRR-amide) detected in any layers of HGP skin, human skin or buffer collected underneath the skin. This skin penetration data will be useful for evaluating the safety of cosmetic products containing small peptide cosmetic ingredients.