Method of improving the appearance of the skin

Abstract

The present disclosure concerns a cosmetic method of improving the appearance of the skin of a subject in need thereof. In particular, the present disclosure concerns, but is not limited to, a method of improving the appearance of the skin of a subject in need thereof comprising topically applying to the skin a composition comprising L-4-thiazolylalanine in an amount of from 0.08 wt% to 0.50 wt% of the total weight of the composition. The present disclosure also provides compositions for improving the appearance of the skin.

Description

[0001] Customer No.60723 Method of Improving the Appearance of the Skin Cross-Reference to Related Applications

[0001] This application claims priority benefit to U.S. Provisional Patent Application Serial No.63 / 482,920, filed on February 2, 2023, the entirety of which is herein incorporated by reference for all purposes. Field of the Invention

[0002] The present invention concerns a cosmetic method of improving the appearance of the skin of a subject in need thereof. More particularly, but not exclusively, this invention concerns topically applying to the skin a composition comprising L-4-thiazolylalanine in an amount of from 0.08 wt% to 0.50 wt% of the total weight of the composition. The present invention also concerns a composition for topical application comprising L-4-thiazolylalanine. Background of the Invention

[0003] The skin undergoes major structural and functional changes as a result of ageing and exposure to environmental factors such as sun and pollution. Those changes may appear as fine lines and wrinkles, skin sagging, age spots, or loss of elasticity and firmness. As the ageing process develops, dermal quality gradually deteriorates. Typically, dermal quality deterioration is marked by a decrease in skin thickness and on alterations of the extracellular matrix composition, such as a decrease of hyaluronic acid and collagen fibril density. The dermis decreases its capacity to hold water molecules, leading to a decrease of bound water, and increase of free water levels.

[0004] The first intervention for these visible signs of ageing include the use of topical anti-ageing products. The active ingredients found in many topical products such as retinol or alpha hydroxy acids (AHA) are desired by consumers for their efficacy in reducing these visible ageing signs. Retinol is known to boost collagen in the skin and is consider the gold-standard for improving skin appearance. However, frequent application of retinol to the skin, particularly of compositions comprising high concentrations of retinol (for example, in excess of 0.1 wt% retinol) for multiple consecutive days, especially for several weeks, can lead to irritation at the treatment Customer No.60723 site which may appear as redness and peeling of the skin. There is therefore a need to investigate alternative efficacious ingredients that do not irritate the skin.

[0005] Amino acids have become increasingly important constituents of cosmetics. For example, various natural amino acids have been incorporated into cosmetics as building blocks for collagen and elastin synthesis, for moisture retention, enhancing the skin barrier, reduction in sebaceous gland activity, and other functional properties. However, the use of non-natural, non-proteogenic amino acids has received little attention in the cosmetic industry. Non-proteogenic aminoacids, by definition, do not become incorporated into proteins during new protein synthesis, and therefore, it would not be predicted that topical application of non-proteogenic aminoacids would lead to new protein synthesis in the skin and to skin rejuvenation benefits. The intrinsic functionality of non-natural, non-proteogenic amino acids has been largely ignored.

[0006] L-4-thiazolylalanine is a non-natural, non-proteogenic amino acid. WO 2009 / 079126 A1 discloses a method of treating skin conditions associated with loss of elastin fibre by topical application of compositions comprising L-4-thiazolylalanine. The lysyl oxidase homolog 1 enzyme (LOXL-1) is essential for elastic fibre formation and maintenance of elastic fibre homeostasis. It is thought that L-4-thiazolylalanine promotes LOXL-1 activity which in turn promotes cross-linking of collagen and elastin through oxidative deamination of lysine or hydroxylysine side chains. Known compositions for enhancing LOXL-1 production typically comprise less than 0.01 wt% L-4-thiazolylalanine and are typically recommended for use over a relatively short period of time (typically less than 21 consecutive days of topical application) to achieve elastin fibre promotion.

[0007] Although L-4-thiazolylalanine compositions have been shown to target signs of ageing associated with elastin loss, new treatment regimes are needed to target other visible signs of ageing and other changes to the structure and functionality of the skin associated with ageing. The present invention seeks to provide an alternative or improved method of improving the cosmetic appearance of the skin. Customer No.60723 Summary of the Invention

[0008] According to a first aspect, the present invention provides a cosmetic method of improving the appearance of the skin of a subject in need thereof comprising the steps of: (i) topically applying to the skin a composition comprising Compound I of the formula: or a zwitterionic thereof, wherein the composition comprises from 0.08 wt% to 0.50 wt% of the total weight of the composition of Compound I excluding the mass of any salt; and, (ii) repeating step (i) for a continuous period of at least 24 days and until a cosmetically beneficial improvement to the skin of the subject has occurred.

[0009] It will be understood that Compound I has the chemical name L-4- thiazolyalanine and those terms may be used interchangeably herein. Optionally, L-4- thiazolyalanine may be present in zwitterionic form. Optionally, L-4-thiazolyalanine may be present in the form of a cosmetically or pharmaceutically acceptable salt of the amino acid. Thus, references to “Compound I” and “L-4-thiazolyalanine” herein are to be understood to be referring to the amino acid of the formula above, as well as zwitterionic forms and cosmetically or pharmaceutically acceptable salts thereof unless the context dictates otherwise. References to the mass or weight percent of Compound 1 or L-4-thiazolyalanine refer to the mass of the amino acid alone excluding the mass of any counter ions present in salt forms, solvate molecules or the like.

[0010] Step (ii) of the method according to the first aspect of the invention comprises repeating step (i) for a continuous period of at least 24 days, such as at least 25 days, and until a cosmetically beneficial improvement to the skin of the subject has occurred. Advantageously, step (ii) comprises repeating step (i) for a continuous Customer No.60723 period of at least 28 days, such as at least 30 days. Optionally, step (ii) comprises topically applying the composition to the skin on each consecutive day of the continuous period such that the method comprises repeating step (i) at least once per day. Alternatively, step (ii) comprises topically applying the composition intermittently throughout the continuous period. For example, the composition may be applied every other day of the continuous period. Optionally, if the composition is applied intermittently throughout the continuous period, the composition may be applied to the skin on at least 50% of the days of the continuous period, preferably at least 70% of the days of the continuous period, more preferably at least 90% of the days of the continuous period. Step (ii) of the present method may therefore optionally comprise repeating step (i) at least every other day. Alternatively, step (ii) may comprise repeating step (i) at least 5 days out of every 7 consecutive days.

[0011] The composition comprises from 0.08 wt% to 0.50 wt% of the total weight of the composition of Compound I or the zwitterionic form, excluding the mass of any salt. Preferably, the composition comprises from 0.09 wt% to 0.40 wt%, preferably from 0.10 wt% to 0.35 wt% of the total weight of the composition of Compound I or the zwitterionic form, excluding the mass of any salt. For example, the composition may comprise from 0.20 wt% to 0.32 wt% of the total weight of the composition of Compound I or the zwitterionic form, excluding the mass of any salt.

[0012] It has surprisingly been found that prolonged use of compositions comprising L-4-thiazolylalanine for a continuous period of 24 days or more and wherein the composition comprises at least 0.08 wt% L-4-thiazolylalanine does not cause, or causes negligible, skin redness or irritation. This is in contrast to existing anti-ageing treatments, in particular treatments comprising retinol compositions, which are found to cause skin irritation after prolonged use at high concentration (typically in excess of 0.1 wt% based on the total weight of the composition). Therefore, the present invention provides an effective method of improving the appearance of the skin that does not cause, or causes minimal, skin redness or irritation. Additionally, prolonged topical use of compositions comprising L-4-thiazolylalanine in an amount of from 0.08 wt% to 0.50 wt% of the total weight of the composition for a continuous period of 24 days or more results in a number of cosmetically beneficial improvements as is described herein. It has been found that many cosmetic benefits do not occur until topical application has been carried out for at least 24 days, such as about 28 days. Customer No.60723 Without wishing to be bound by theory, it is thought that a treatment with a high concentration of L-4-thiazolylalanine for prolonged periods of time causes changes in structure and function of the skin which reverses visible signs of ageing. It has been found that use of a composition comprising L-4-thiazolylalanine in an amount of from 0.08 wt% to 0.50 wt% for a continuous period of at least 24 days may provide an optimum balance between efficacy and cost of ingredients. The present inventors have found that at concentrations in excess of 0.50 wt%, there may not be a further increase in cosmetic benefit.

[0013] Optionally, the cosmetically beneficial improvement to the skin of the subject comprises at least one of: a. reduced wrinkle depth, b. increased hyaluronic acid secretion, c. increased skin hydration, d. increased epidermal thickness, e. increased skin firmness, f. increased skin smoothness, g. improvement skin barrier integrity, h. reduced redness, i. increased dermal collagen III and / or collagen I production.

[0001] Many of the improvements may result in a softer, smoother feel and a smoother appearance of the skin.

[0002] Advantageously, the improvement in the appearance of the skin may comprise reversing, ameliorating, preventing and / or treating the signs of skin ageing.

[0003] Optionally, the cosmetically beneficial improvement to the skin may not be observable until after the composition has been topically applied for a continuous period of at least 15 days, optionally a continuous period of at least 24 days.

[0014] Optionally, the method comprises altering the dermal matrix assemble. For example, the dermal matrix assemble may be altered via the production of hyaluronic acid and pro-collagen I and upregulation of matrix genes such as VCAN, HAS1, and collagen type VI (COL6A6). Optionally, the method comprises decreasing inflammation. It may be that a decrease in inflammation is observed as a decrease in inflammatory proteins, such as enzymes, produced by the skin and / or a down- regulation of inflammatory genes. Customer No.60723

[0015] Wrinkles and fine lines are a feature commonly associated with aged skin. The improvement in the appearance of the skin may result in a reduction in the appearance of such wrinkles and fine lines, such as a reduction in the depth, length or density per unit area of skin of wrinkles and fine lines. Advantageously, the cosmetically beneficial improvement to the skin of the subject comprises reducing the wrinkle depth of the skin. It has been found that the method according to the first aspect of the invention advantageously reduces the depth of deep wrinkles. A deep wrinkle is defined as having a wrinkle depth of greater than 110 µm. It has surprisingly been found that an extended treatment period in combination with a composition comprising L-4-thiazolylalanine in an amount of at least 0.08 wt% of the total weight of the composition is required to achieve a reduction in the depth of deep wrinkles. While treatments with compositions comprising L-4-thiazolylalanine for shorter durations, e.g. for 14 days, and / or in lower amounts, e.g. of 0.05 wt% or less, have not been found to have a significant effect on the decrease in deep wrinkles, it has surprisingly been found that on performing the method of the invention a reduction in the depth of deep wrinkles can be achieved. Without wishing to be bound by any theory, it is postulated that application of significant amounts of L-4-thiazolylalanine, i.e.0.08 wt% or more, over a prolonged period has a cumulative effect that only results in a substantive benefit in reducing the depth of deep wrinkles after continued treatment for more than three weeks, i.e.24 days or more. Accordingly, the method of the present invention may comprise reducing wrinkle depth, wherein the wrinkle depth is greater than 110 µm prior to treatment (i.e. prior to performing step (i) of the method). It may be that no significant difference in deep wrinkle depth is observable until a composition according to the present invention is applied to the skin for a continuous period of more than 14 days. The present method may be particularly effective in targeting deep wrinkles which are crow’s feet wrinkles. It will be understood that the term ‘crow’s foot’ wrinkle is commonly used in the art and refers to a branching wrinkle at the outer corner of the eye. Advantageously, the average depth of deep wrinkles (having a depth greater than 110 µm prior to treatment) may be decreased by at least 3%, such as at least 5% or preferably at least 7% after treatment according to a method of the present invention (i.e. after performing step (ii) of the method according to the first aspect of the invention). The depth of a winkle may be determined by obtaining a polymer silicone skin print (Silflo, Monaderm, Customer No.60723 Monaco) of the subject’s crow’s feet region and examined using SIA (Skin Image Analyzer, Courage+Khazaka electronic GmbH, Germany) as described herein. Additionally or alternatively, the average relief amplitude (Rz) of deep wrinkles is advantageously reduced by at least 5%, such as at least 10%, by the method of the invention, e.g. after repeating step (i) for at least 24 days, especially at least 28 days. Advantageously, the depth of deep wrinkles are reduced by at least 5 µm, such as at least 10 µm, by the method of the invention, e.g. after repeating step (i) for at least 24 days, especially at least 28 days.

[0016] Advantageously, the method comprises increasing the hyaluronic acid secretion of the skin cells. It has been found that compositions comprising L-4- thiazolylalanine, particularly compositions comprising L-4-thiazolylalanine in an amount of from 0.08 wt% to 0.50 wt% of the total weight of the composition, increase the amount of hyaluronic acid secreted by skin cells. It may be that a particularly pronounced increase in hyaluronic acid secretion is observed after the composition has been applied to the skin for a continuous period of at least 24 days, such as at least 28 days. Hyaluronic acid has a number of beneficial effects on the skin, such as improving skin hydration. Hyaluronic acid plays an important role in maintaining hydration levels due to its high potential to attract and hold water molecules at the dermis and at the epidermis by establishing hydrogen bonds with water molecules, forming bound water. Optionally, an increase in hyaluronic acid secretion is accompanied by an increase in skin hydration, such as an increase in total water content of the epidermis and / or the stratum corneum. Advantageously, wherein the cosmetically beneficial improvement to the skin comprises increased hyaluronic acid secretion, the amount of hyaluronic acid secreted after step (ii) exceeds the amount of hyaluronic acid secreted prior to step (i) by at least 10%, such as at least 15% or at least 20%. The secretion of hyaluronic acid by skin cells may be measured via homogeneous time resolved fluorescence assay using labelled-hyaluronic acid binding protein (HABP) where the intensity of the labelled-HABP is proportional to the concentration of hyaluronic acid in tested samples (Cisbio), as described herein.

[0017] Advantageously, the method comprises increasing skin hydration. It will be understood that skin hydration refers to the water content contained within the skin. Skin with poor hydration may feel dry or rough. At the stratum corneum, water content depends upon three major factors: the water quantity diffusing from the Customer No.60723 underlying layers up to the stratum corneum, the stratum corneum’s ability to retain water and the water loss amount due to evaporation (transepidermal water loss). Advantageously, the increased skin hydration comprises an increase in the total water content of the epidermis and / or the stratum corneum. The total water content may be measured by Confocal Raman spectroscopy (CRS) as described herein. Thus, when the cosmetically beneficial improvement to the skin comprises increased skin hydration, the increased skin hydration may comprise an increase in total water content of the epidermis and / or stratum corneum such that the total water content of the epidermis and / or stratum corneum after step (ii) exceeds the total water content of the epidermis and / or stratum corneum prior to step (i). The total water content of the epidermis and / or stratum corneum after step (ii) may exceed the total water content of the epidermis and / or stratum corneum prior to step (i) by at least 10%, at least 20% or at least 50%. For example, the total water content of the epidermis and / or stratum corneum after step (ii) may exceed the total water content of the epidermis and / or stratum corneum prior to step (i) by at least 100%.

[0018] Advantageously, the method comprises increasing the epidermal thickness. Advantageously, the treatment may result in the epidermal thickness increasing by at least 2 µm, such as at least 4 µm compared to the thickness of the epidermis prior to treatment. The increase in epidermal thickness may be expressed as a percentage increase of the thickness of the epidermis before and after treatment. For example, the epidermal thickness may increase by at least 5%, such as at least 10%, after treatment for at least 24 days compared to baseline thickness. Thus, the method of the present invention may advantageously comprise increasing the epidermal thickness, wherein the epidermal thickness after step (ii) of the method is at least 5% greater than the epidermal thickness prior to step (i) of the method. The thickness of the epidermis may be evaluated using a calibrated ocular micrometer (CaseViewer2.2) at a magnification of 40x as described herein.

[0019] Advantageously, the method comprises improving skin firmness and / or smoothness. It has been found that treating the skin for at least 24 days, optionally 28 days, with a composition comprising L-4-thiazolylalanine results in firmer and smoother skin.

[0020] Skin firmness may be evaluated via the deformation of the skin caused by a puff of air fired at and perpendicular to the skin surface (referred to in the art as an Customer No.60723 ‘air puff’ method). Changes in firmness of the skin surface change the volume, surface area, and maximum depth of deformation created by the air puff. Firming effects may be measured using a DynaSKIN ((EOTECH SA, Marcousis, France) instrument in conjunction with the fringe projection system, DermaTOP (EOTECH SA, Marcousis, France) as described herein and also described in RORH M, SHRADER A – FOITS, Fast optical in vivo topometry of human skin: a classical method in modern efficacy testing, A history of Fringe Projection in Cosmetics, SOFW-Journal, 2009, 135, 8. Non-contact mechanical pressure in combination with fringe projection may be used to quantify and visualise the skin surface response in 3- dimensions (Kearney et al. - Evaluation of skin firmness by the DynaSKIN, a novel non-contact compression device, and its use in revealing the efficacy of a skincare regimen featuring a novel anti-ageing ingredient, acetyl aspartic acid, Skin Research and Technology 2017; 23: 155–168). It has been found that after 14 days of treatment, no change in volume, surface area or maximum depth is observed when a force is applied to the skin surface compared to the volume, surface area or maximum depth of deformation to the skin when a force is applied to the skin surface prior to treatment, suggesting no significant change in skin firmness after 14 days of treatment. However, it has surprisingly been found that after at least 24 days of treatment with a composition comprising L-4-thiazolylalanine in an amount of from 0.08 wt% to 0.50 wt% of the total weight of the composition a significant decrease in volume (-15%, p<0.05), surface area (-5%, p<0.05), and maximum depth (-11%, p<0.05) over baseline has been observed which is indicative of increased skin firmness. Without wishing to be bound by theory, it is suggested that the method according to the present invention, specifically a method comprising topical application of a composition comprising L-4-thiazolylalanine in an amount of from 0.08 wt% to 0.50 wt% of the total weight of the composition and for a period of time of at least 24 days, may have a cumulative effect on skin firmness. Advantageously, the present method comprises increasing skin firmness wherein at least one of a volume, surface area or maximum depth of deformation to the skin when a force is applied to the skin surface after step (ii) of the present method , is less than the volume, surface area or maximum depth of deformation to the skin when an equal force is applied to the skin surface prior to step (i) of the present method. Customer No.60723

[0021] Smoothing effects may be determined directly in vivo, using DermaTOP (EOTECH SA, Marcousis, France) to acquire a 2D and 3D image of wrinkles, as disclosed herein. This system calculates cutaneous relief parameters by averaging 50 vertical profiles along the zone of interest. It will be understood that skin smoothness may conversely be described as skin roughness. An increase in skin smoothness will correspond to a decrease in skin roughness. The average roughness (Ra) of the skin, which is the mean average absolute values of peak and valley heights of the skin surface to determine the smoothing effect (FIG.6A). Rz is the average of five single roughness depths (difference from highest peak to lowest valley) measured within the sample section (FIG.6B). Rt is the height difference between the highest peak and the lowest valley over the entire measurement section (FIG.6B). Optionally, at least one of average roughness, Ra, average relief amplitude, Rz, and maximum relief amplitude, Rt, of the skin after step (ii) is less than the average roughness, Ra, average relief amplitude, Rz, or maximum relief amplitude, Rt, of the skin prior to step (i). Advantageously, the present method has been shown to decrease Ra by at least 5%, preferably at least 8%. Advantageously, the present method has been shown to decrease Rz by at least 5%, preferably at least 8%. Advantageously, the present method has been shown to decrease Rt by at least 3%, preferably at least 6%.

[0022] Advantageously, the method comprises improving the skin barrier integrity. Advantageously, the skin barrier integrity comprises a reduction in transepidermal water loss (TEWL). It will be understood that TEWL results in a loss of moisture from the skin. Thus, a reduction in transepidermal water loss may result in smoother feeling skin. Alternatively, the method may comprise maintaining the skin barrier integrity. It has surprisingly been found that the present method does not result in loss of TEWL in contrast to other topical compositions. Thus, the present invention may allow cosmetic signs of ageing to be improved whilst maintaining or improving the skin barrier integrity. Thus, when the cosmetically beneficial improvement to the skin comprises an improvement in skin barrier integrity, the improvement in skin barrier integrity may comprise maintenance of, or reduction in, transepidermal water loss. The transepidermal water loss after step (ii) may be less than or substantially equal to transepidermal water loss prior to step (i). Substantially equal will be understood as being a transepidermal water loss after step (ii) that is no more than 5% greater or no more than 5% less than the transepidermal water loss prior to step (i). Customer No.60723

[0023] Advantageously, the method comprises reducing skin redness. The redness of the skin after to step (ii) of the present method, may be less than the redness of the skin prior to step (i) of the present method. It will be understood that skin redness may be as a result of dilation of the capillaries in the upper layers of the skin. Optionally, the decreased redness comprises constriction of the capillaries of the upper layers of the skin.

[0024] Advantageously, the method comprises increased dermal collagen III and / or collagen I production. Advantageously, the amount of collagen I secreted after step (ii) exceeds the amount of collagen I secreted prior to step (i) by at least 15%, such as at least 20%. Additionally or alternatively, the amount of collagen III secreted after step (ii) exceeds the amount of collagen III secreted prior to step (i) by at least 15%, such as at least 20%. Collagen I and / or collagen III production may be assessed by HTRF assay using labelled pro-collagen I and / or collagen III antibodies (Cisbio) as described herein. Advantageously, the amount of collagen III secreted after step (ii) exceeds the amount of collagen III secreted prior to step (i) by at least 15%, such as at least 20%. Advantageously, the method comprises increasing the ratio of collagen III / I after treatment (i.e. after step (ii)) compared to before treatment (i.e. prior to step (i)) by at least 40%, at least 60% or at least 75%.

[0025] Optionally, the method further comprises: (iii) further repeating step (i) in order to maintain the cosmetically beneficial improvement in the appearance of the skin of the subject. Step (i) may be repeated for as long as necessary for there to be observed an improvement in the appearance of the skin of a subject. Step (i) may also be repeated as long as necessary to maintain the improvement in the skin. Furthermore, step (i) may be repeated after a pause in administration of the composition, for example because of non-compliance such as if the subject forgets to apply the composition for several days, or if a significant improvement in the appearance of the skin is maintained for several weeks or months. Step (i) may be repeated if after pausing the administration of the composition, the subject experiences a decline in the appearance of the skin. In such a scenario, repeating step (i) may lead to a return of the skin to the improved condition or appearance.

[0026] The method according to the first aspect of the invention may be used to prevent degradation of the skin before the ageing process has become visible, or to Customer No.60723 slow the onset or progression of imperfections associated with ageing. Thus, according to the present invention there is provided a cosmetic method of preventing the degradation of the skin before the ageing process has begun or is obvious visually, or at least slow the onset or progression of skin imperfections associated with ageing skin. For example, the method may prevent, or decrease the rate of onset or progression of at least one of the following imperfections associated with ageing skin: increased wrinkle depth, reduced hyaluronic acid secretion, decreased skin hydration, decreased epidermal thickness, decreased skin firmness, decreased skin smoothness, decreased skin barrier integrity, increased redness, decreased dermal collagen III and / or collagen I production. It will be understood that such a method may include any step described in relation to the first aspect of the invention, except that those method steps are performed before signs of ageing of the skin are visible, or at least when the signs of ageing of the skin are minimal. For example, the method may include the steps of: (i) topically applying to the skin of a subject who’s skin has no or minimal visual signs of ageing the composition according to any aspect of the invention, especially in reference to the first aspect of the invention, and, (ii) repeating step (i) to maintain the appearance of the skin of the subject.

[0027] Optionally, the skin to be treated is the skin of at least one of the face, scalp, neck or décolletage. Advantageously, the skin is the skin of the face and / or neck. Optionally, the area of the skin of the face to be treated is at least one of the under eye area, the crow’s feet area, the forehead area or the upper-lip area.

[0028] Optionally, the subject is female. Optionally, the subject is male.

[0029] Optionally, the subject is of from 21 to 65 years of age, for example in the range of from 45 to 65 years of age. Optionally, the subject is in the range of from 50 to 60 years of age. Optionally, the subject is female and is from 21 to 65 years of age.

[0030] According to a second aspect of the invention, there is provided a composition for topical application comprising Compound I of the formula:

[0002] Customer No.60723 or a zwitterionic form or cosmetically acceptable salt thereof, wherein the composition comprises from 0.08 wt% to 0.50 wt% of the total weight of the composition of Compound I or the zwitterionic form excluding the mass of any salt. It will be understood that the composition according to the second aspect of the invention may have any feature of the composition described in relation to the method of the first aspect of the invention.

[0031] Optionally, the composition comprises from 0.09 wt% to 0.40 wt%, preferably from 0.10 wt% to 0.35 wt% of the total weight of the composition of Compound I or the zwitterionic form excluding the mass of any salt. For example, the composition may comprise from 0.20 wt% to 0.32 wt% of the total weight of the composition of Compound I or the zwitterionic excluding the mass of any salt.

[0032] According to a third aspect of the invention, the composition according to the second aspect of the invention is advantageously for use in a therapeutic treatment of a skin condition, wherein the skin condition is associated with at least one of: a. increased wrinkle depth, b. reduced hyaluronic acid secretion, c. decreased skin hydration, d. decreased epidermal thickness, e. decreased skin firmness, f. decreased skin smoothness, g. decreased skin barrier integrity, h. increased redness, i. decreased dermal collagen III and / or collagen I production.

[0033] Advantageously, the composition may be for use in treating erythema.

[0034] Optionally, the composition of the second aspect of the invention may be for use in a treatment of a skin condition, wherein the treatment comprises the steps of: (i) topically applying to the skin the composition; and, (ii) repeating step (i) for a continuous period of at least 24 days and until an improvement in the skin condition of the subject has occurred.

[0035] Optionally, the treatment comprises any of the steps disclosed in relation to the method of the first aspect of the invention. For example, step (ii) may comprise repeating step (i) for a continuous period of at least 28 days. Customer No.60723

[0036] According to a fourth aspect of the invention, is use of the composition according to the second aspect of the invention in a method according to the first aspect of the invention.

[0037] It will of course be appreciated that features described in relation to one aspect of the present invention may be incorporated into other aspects of the present invention. For example, the method of the invention may incorporate any of the features described with reference to the composition of the invention and vice versa. Description of the Drawings

[0038] Embodiments of the present invention will now be described by way of example only with reference to the accompanying schematic drawings of which: FIG.1A is a graph showing the percentage change in hyaluronic acid secretion of 3D tissue models for tissues treated with an L4 formula comprising 0.3 wt% L-4-thiazolylalanine or a vehicle control after 3 days of treatment. Tissues treated with the L4 formula secreted significantly more hyaluronic acid than the vehicle control (19.7% + 8.8% increase, p<0.05). Data represents the average percent change (+SD) versus the vehicle over three experiments; FIG.1B is a graph showing the percentage change in pro-collagen I secretion of 3D tissue models for tissues treated with an L4 formula comprising 0.3 wt% L-4-thiazolylalanine or a vehicle control after 3 days of treatment. Tissues treated with the L4 formula secreted significantly more pro-collagen I than the vehicle control over three experiments (25.6% + 12.0% increase, p<0.05). Data represents the average percent change (+SD) versus the vehicle over three experiments; FIG.2 is a graph showing the percentage change in F295 / F340 excitation-emission observed after treatment of the skin for 28 days with one of formula base (labelled ‘placebo’), L4 formula or retinol formula. F295 / F340 excitation was significantly stimulated in skin treated with the retinol formula or the L4 formula, indicating epidermal turnover stimulation; Customer No.60723 FIG.3 is a graph showing change in hyaluronic acid secretion vs. placebo (formula base) (as indicated by HABP staining of histological samples) after treatment for 28 days in in vivo study. A significant increase of HABP staining was observed in skin treated with L4 formula compared to the formula base (42.69±20.7% increase vs base alone, p<0.05); FIG.4A is a graph showing percentage change in collagen III (as indicated by Herovici staining of histological samples which stains young collagen (collagen III) in blue and mature collagen (collagen I) in red) in a treatment group treated with the retinol formula and a treatment group treated with L4 formula compared to a treatment group treated with formula base, after 28 days of treatment. Treatment with L4 formula resulted in significant increase in collagen III compared to formula base (24.79±8.2% vs. base, p<0.05); FIG.4B is a graph showing percentage change in collagen III / I (as indicated by Herovici staining of histological samples which stains young collagen (collagen III) in blue and mature collagen (collagen I) in red) in a treatment group treated with the retinol formula and a treatment group treated with L4 formula compared to a treatment group treated with formula base, after 28 days of treatment; FIG.5A is a graph showing changes in volume of skin deformation created by air puff in a firming evaluation of skin treated with L4 formula or formula base (‘vehicle’) for 28 days over baseline. At day 28, the skin treated with the L4 formula had a significant decrease in volume vs. baseline (-15%, p<0.05); FIG.5B is a graph showing changes in average surface area of skin deformation created by air puff in a firming evaluation of skin treated with L4 formula or formula base (‘vehicle’) for 28 days over baseline. At day 28, the skin treated with the L4 formula had a significant decrease in surface area vs baseline (-5%, p<0.05); FIG.5C is a graph showing maximum depth of skin deformation created by air puff in ae firming evaluation of skin treated with L4 formula or Customer No.60723 formula base (‘vehicle’) for 28 days over baseline. At day 28, the skin treated with the L4 formula had a significant decrease in maximum depth vs baseline (-11%, p<0.05); FIG.6A is a graph showing how average roughness (Ra) is calculated. Ra is defined as the mean absolute values of peak and valley heights of the skin surface; FIG.6B is a graph showing how maximum relief amplitude (Rt) and the average relief (Rz) is calculated. Rt is the height difference between the highest peak and the lowest valley over the entire measurement section. Rz is the average of five single roughness depths (difference from highest peak to lowest valley) measured within the sample section; FIG.6C is a graph showing the average roughness (Ra) percentage change vs baseline for skin treated with L4 formula or formula base (‘vehicle’) at 14 days and 28 days of treatment. L4 formula use demonstrated a significant decrease (-8%, p<0.05 vs. baseline) in the average roughness (Ra) of the skin after 14 days compared to baseline. After 28 days, L4 formula use exhibited a directional decrease (-8%, p=0.052) in Ra vs. baseline; FIG.6D is a graph showing the average relief (Rz) percentage change vs baseline for skin treated with L4 formula or formula base (‘vehicle’) at 14 days and 28 days of treatment. L4 formula use exhibited a significant decrease (p<0.05) in average relief (Rz) over baseline after 14 and 28 days (-8% and -9%, respectively); FIG.6E is a graph showing the maximum relief amplitude (Rt) percentage change vs baseline for skin treated with L4 formula or formula base (‘vehicle’) at 14 days and 28 days of treatment. L4 formula resulted in a directional decrease (p=0.0783) in maximum relief (Rt) over baseline at day 14 (-6%, Figure 6E); FIG.7A and FIG.7B are skin prints of the crow’s foot deep wrinkles of subjects treated with L4 formula (FIG.7A) or formula base (FIG.7B). The skin prints were analysed with a Skin Image Analyser at day zero (D0) of treatment and day 28 (D28) of treatment. The darker the Customer No.60723 region on the image, the deeper the wrinkle. The average depth of deep wrinkles was significantly less after 28 days of treatment for those subjects treated with L4 formula compared to those subjects treated with formula base; FIG.8A is a graph showing the percentage change in ptosis volume vs baseline after 28 days of treatment with L4 formula or formula base (‘vehicle’); FIG.8B is a graph showing the percentage change in volume of skin deformation vs baseline after 28 days of treatment with L4 formula or formula base (‘vehicle’); FIG.9A is a graph showing water content increase, in arbitrary units (a.u.), at the stratum corneum for placebo formula, L4 formula (‘Protinol’) and PCA formula treatment, with statistically significant increase in relation to placebo marked with “*”. Water content was measured at day 15 and day 30; FIG.9B is a graph showing water content increase, in arbitrary units (a.u.), at the epidermis for placebo formula, L4 formula (‘Protinol’) and PCA formula treatment, with statistically significant increase in relation to placebo marked with “*”. Water content was measured at day 15 and day 30; FIG.10 is a graph showing trans-epidermal water loss (TEWL) measured overtime for 4 weeks of the forearms of subjects treated with 0.3 wt% L4 formula. Data represent Means±SE, n=11. *: p<0.05 vs vehicle; FIG.11A is a graph showing erythema evaluated using a visual 0 – 4 scale with increasing severity during a scalp study comprising daily application of 0.3 wt% L4 for 28 days. Data represent Means±SE; n=11 subjects *: p<0.05 vs untreated or vehicle; and, FIG.11B is a graph showing desquamation evaluated using a visual 0 – 4 scale with increasing severity during a scalp study comprising daily application of 0.3 wt% L4 for 28 days. Data represent Means±SE; n=11 subjects *: p<0.05 vs untreated or vehicle. Detailed Description Customer No.60723 Compositions

[0039] Compound I, L-4-thiazolylalanine, also referred to as protinol, is a non- natural, non-proteinogenic amino acid. L-4-thiazolylalanine is a modified histidine with a sulfur substituting the nitrogen on its ring structure and has the chemical structure:

[0040] Non-natural, non- are not coded in the human genome or incorporated to any side chains during post-translational modifications. In nature, NPAAs serve as essential building blocks of polypeptide chains or metabolites in bacteria, fungi, plants, and marine organisms with many NPAA as analogs of natural amino acids, which do incorporate into protein. Although these NPAA do not incorporate into proteins, their structural similarity to proteinogenic amino acids create an opportunity to investigate synthetic NPAA that are non-toxic or non-irritating salts of the natural amino acids. It will be understood that the term “amino acid” refers generally to an organic molecule which contains an amino group and a carboxyl group in the same molecule, typically located within no more than 5 bonds apart. The term “α-amino acid” refers to an amino acid in which the amino group and carboxyl group are attached to the same carbon atom. The term “β-amino acid” refers to an amino acid in which the amino group and carboxyl group are attached to adjacent carbon atoms. It will be understood that “natural amino acid” refers to any amino acid synthesized by an organism in nature, including without limitation, the standard amino acids L-alanine, L-valine, L-leucine, L-isoleucine, L- proline, L-tryplophan, L-phenylalanine, L-methionine, glycine, L-serine, L-tyrosine, L-threonine, L-cysteine, L-cystine, L-asparagine, L-glutamine, L-aspartic acid, L- glutamic acid, L-lysine, L-arginine, L-histidine, and other non-standard amino acids, such as L-ornithine, and derivatives, such as L-4-hydroxyproline. “Non-natural amino acid” refers broadly to any amino acid other than a “natural amino acid,” and includes, without limitation, α-amino acids having side chains not found in natural Customer No.60723 amino acids, as well as β-amino acids. “Non-natural amino acids” also refers to amino acids having the same structure as natural amino acids, albeit in a different stereochemical configuration. “Non-proteogenic” amino acid broadly refers to any amino acid which is not capable of being incorporated into peptides or proteins by a living organism and includes non-natural amino acids.

[0041] Optionally, compositions according to the present invention comprise L-4- thiazolylalanine in an amount of from 0.08 wt% to 0.50 wt% of the total weight of the composition, an amount of from 0.09 wt% to 0.40 wt% of the total weight of the composition, optionally in an amount of from 0.10 wt% to 0.35 wt% of the total weight of the composition. For example, compositions according to the present invention may comprise 0.3 wt% L-4-thiazolylalanine based on the total weight of the composition.

[0042] As will be evident to one skilled in the art that L-4-thiazolylalanine may be present in zwitterionic form. Further, the invention embraces the use of cosmetically or pharmaceutically acceptable (e.g., non-toxic and / or non-irritating) salts of the amino acids. Zwitterions, salts and prodrugs may be collectively referred to herein as “derivatives” of the free amino acid. The salts may be either inorganic or organic acid or base addition salts. Suitable acid salts include but are not limited to acetate, adipate, alginate, citrate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, camphorate, camphorsulfonate, digluconate, cyclopentanepropionate, dodecylsulfate, ethanesulfate, glucoheptonate, glycerophosphate, hemisulfate, heptonate, hexanoate, fumarate, hydrochloride, hydrobromide, hydroiodide, 2- hydroxy-ethanesulfate, lactate, maleate, methanesulfonale, nicotinate, 2- naphthalenesulfonate, oxalate, pamoate, pectinate, persulfate, 3-phenylpropionate, picrate, pivalale, propionate, succinate, tartrate, thiocyanate, tosylate, and undecanoate. Special mention may be made of hydrochloride salts. Base addition salts include those formed with metal cations such as zinc, calcium, bismuth, barium, magnesium, aluminum, copper, cobalt, nickel, cadmium, sodium, potassium, and the like, or with a cation formed from ammonia, N,N-dibenz.ylethylenediamine, D-glucosamine, tetraethylammonium, or ethylenediamine, etc. The nitrogen-containing groups, including the amino functional group and nitrogen- containing side chains (e.g., helerocycles), can be quarternized with loweralkyl halides, such as methyl, ethyl, propyl, and butyl chloride, bromides, and iodides; dialkyl sulfates like dimethyl, diethyl, dibutyl, and Customer No.60723 diamyl sulfates, long chain halides such as decyl, lauryl, myristyl and stearyl chlorides, bromides and iodides, aralkyl halides such as benzyl and phenethyl bromides, to name a few.

[0043] The invention also embraces the use of prodrug forms of the amino acids to improve dermal penetration. As used herein, prodrug refers to a compound which is converted in vivo to the amino acid of the invention. The prodrug may be derived from the amino group, the carboxyl group, or both. Suitable prodrug derivatives for modifying these functional groups are well known in the art and described, for example, in Textbook of Drug Design and Discovery, Third Edition (2002), Chapter 14, pp.411- 458, which is hereby incorporated by reference herein.

[0044] L-4-thiazolylalanine or a derivative thereof may be associated with other molecules in the formulations of the invention and / or non-chemically bound to other molecules, e.g. by electrostatic or van der Waals forces. For example L-4- thiazolylalanine or a derivative thereof may be present in a solvated form wherein L- 4-thiazolylalanine or a derivative are non-chemically bound to with solvent molecules. Other ingredients

[0045] Compositions according to the present invention may contain one or more further cosmetic actives and / or excipients. Such actives and excipients include but are not limited to, fillers, emulsifying agents, antioxidants, surfactants, film formers, chelating agents, gelling agents, thickeners, emollients, humectants, moisturizers, vitamins, minerals, viscosity and / or rheology modifiers, sunscreens, keratolytics, depigmenting agents, retinoids, hormonal compounds, alpha-hydroxy acids, alpha- keto acids, anti-mycobacterial agents, antifungal agents, antimicrobials, antivirals, analgesics, lipidic compounds, anti-allergenic agents, H1 or H2 antihistamines, anti- inflammatory agents, anti-irritants, antineoplastics, immune system boosting agents, immune system suppressing agents, anti-acne agents, anesthetics, antiseptics, insect repellents, skin cooling compounds, skin protectants, skin penetration enhancers, exfollients, lubricants, fragrances, colorants, depigmenting agents, hypopigmenting agents, preservatives, stabilizers, pharmaceutical agents, photostabilizing agents, sunscreens, and mixtures thereof. Optionally, the composition according to the present invention comprises glycerin. Optionally, glycerin is present in an amount of from 1 wt% to 12 wt%, preferably in an amount of from 3 wt% to 10 wt%. Customer No.60723

[0046] The composition can be formulated as a water-in-oil or an oil-in-water emulsion, lotion, cream, serum, spray, stick or other forms suitable for topical application. Cosmetically beneficial improvement

[0047] The present invention advantageously provides a cosmetic benefit to the user, as described herein. For example, the present invention may improve the appearance of skin.

[0048] Advantageously, the present invention provides a cosmetic benefit to the skin including at least one of: a. reduced wrinkle depth, b. increased hyaluronic acid secretion, c. increased skin hydration, d. increased epidermal thickness, e. increased skin firmness, f. increased skin smoothness, g. improvement skin barrier integrity, h. reduced redness, i. ,increased dermal collagen III and / or collagen I production.

[0049] Compositions according to the present invention may improve epidermal and dermal parameters that change during ageing. Compositions according to the present invention may improve epidermal and dermal parameters caused by photodamage to the skin. Advantageously, compositions according to the present invention do not cause redness or irritation to the skin.

[0050] Cosmetic methods and uses envisaged by the present invention include methods of improving the appearance of the skin, especially of facial skin, for example by reducing wrinkle depth and / or improving the texture and tone of the skin. The cosmetic methods and uses envisaged by the present invention may additionally or alternatively include methods of improving the appearance of the skin by having anti-wrinkle effects. The composition may increase hyaluronic acid production in the skin, which is an extracellular matrix associated with the retention of skin moisture. As a consequence, the compositions of the present invention may maintain or increase skin hydration. Customer No.60723

[0051] A further improvement in the appearance of the skin may include a decrease in transepidermal water loss (TEWL). Alternatively, the TEWL may be maintained at an amount equal to or substantially equal to the TEWL prior to treatment. An increase in TEWL impairs the functions of the enzymes within the skin and this results in visibly dry and aged skin. High or increased TEWL values are indicative of disturbances in the skin’s barrier function and are frequently correlated with low levels of hydration of the stratum corneum.

[0052] Other improvements to the appearance of the skin may include a reduction in skin roughness or an increase in skin smoothness, a decrease in wrinkle depth, length or density, an improvement in wrinkle smoothness and / or an improvement in skin elasticity. Wrinkle formation is associated with the regulation of collagen synthesis and degradation.

[0053] Photoaging is characterized by the degradation of collagen and the accumulation of abnormal elastin in the superficial dermis. The present invention may further provide an improvement in skin imperfections or disorders associated with UV damage to the skin, or ageing affects associated with UV exposure to the skin. The present invention may provide an anti-oxidant effect which limits, reverses or prevents damage to the skin caused by UV irradiation.

[0054] The present invention may further provide an improvement in skin irritation or redness. The present invention may provide an improvement in erythema. The present invention may itself not cause, or may cause negligible, skin irritation or redness.

[0055] The present invention may further provide a skin healing effect.

[0056] The present invention may further provide an improvement in skin sagging or reduction in ptosis volume. Subjects

[0057] The subjects are human and may be male or female. Preferably, the subject is female.

[0058] Optionally, the subject is in the range of from 21 to 65 years of age, for example in the range of from 45 to 65 years of age. Optionally, the subject is in the range of from 50 to 60 years of age. Customer No.60723

[0059] Preferably, the subject prior to treatment has skin showing visible signs of ageing. For example, mild to moderate wrinkles and fine lines of crow’s feet and / or subjects with loose skin on the face.

[0060] Optionally, the skin of the subject to be treated is the skin of at least one of the face, scalp, neck and décolletage. Most preferably, the skin of the subject is the skin of the face and neck. Optionally, the area of the skin of the face to be treated is at least one of the under eye area, the crow’s feet area, the forehead area or the upper-lip area. Treatment regime

[0061] The method of the present invention is performed for a continuous period of at least 24 days, for example at least 28 days. Optionally, the method comprises topically applying the composition to the skin at least once per day. Optionally, the composition is applied to the skin twice per day. For example, the composition according to the present invention may be applied in the morning and in the evening. Optionally, the method comprises topically applying the composition to the skin at least one every other day. Optionally, the method comprises topically applying the composition at least five days out of every seven consecutive days. Optionally, the composition may be topically applied at the same time each day. Optionally, the composition may be applied to the skin so that the area of skin to be treated is provided with at least 0.5 µg of composition per cm2of skin, optionally at least 1 µg of composition per cm2of skin, optionally at least 2 µg of composition per cm2of skin.

[0062] The method may further comprise topical application for a period of time after the cosmetic benefit has been achieved. Once the appearance of the skin has been improved, the composition may continue to be topically applied to maintain the improved appearance of the skin, or to improve the appearance of the skin still further. It may be that the composition is topically applied indefinitely. It may be that the frequency at which the composition is topically applied decreases once an improvement is observed in the appearance of the skin.

[0063] In some embodiments, the composition of the invention is topically applied to a subject in combination with a further treatment. The further treatment may be the use of one or more of a cleanser, exfoliator or moisturiser. Customer No.60723 Examples

[0064] Both in vitro and in vivo studies were conducted to determine the efficacy of compositions comprising L-4-thiazolylalanine for improving the appearance of the skin, and in particular to improve the visible signs of ageing on the skin. There was no observation of skin redness or irritation following use of a formula comprising L-4- thiazolylalanine. Example 1 - In vitro analysis using 3D human skin equivalents Materials and Methods

[0065] EFT400 (MatTek, Ashland, MA) 3D tissues were cultured in media supplied by manufacturer. The model contained single donor cells forming an epidermal layer with stratified normal human epidermal keratinocytes (NHEK), a dermal layer with normal human dermal fibroblasts (NHDF), and an observable basal membrane under histological assessment. Tissues (n=3) were treated topically with 30 µL in 3 replicates of a composition (herein referred to as ‘L4 formula’) comprising 0.3 wt% L-4-thiazolylalanine in a vehicle containing 60 wt% propylene glycol, 30 wt% ethanol and 10 wt% water, or a vehicle control composition (herein referred to as ‘vehicle control’) comprising 60 wt% propylene glycol, 30 wt% ethanol, and 10 wt% water. Topical application of L4 formula or vehicle control to the tissue was repeated every 24 hours for 3 days. The fresh treatment was applied after rinsing tissues with phosphate-buffered saline (PBS). After treatment for three days, tissue viability was assessed by MTT assay. Conditioned media from all time points were analyzed for pro-collagen I and hyaluronic acid production via homogeneous time resolved fluorescence (HTRF) technology (Cisbio, Bedford, MA).

[0066] Gene Expression from EFT4003D tissues was also assessed. EFT4003D tissues (n=3) were cultured in media supplied by manufacturer and treated with the same treatment as the previously described 3D tissue study (topical application of L4 formula comprising 0.3 wt% L-4-thiazolylalanine (L4) in a vehicle containing 60% propylene glycol, 30% ethanol, and 10% water, or topical application with vehicle control only). After 24 hours of treatment, tissues were rinsed with PBS and flash frozen with liquid nitrogen. Tissues were homogenized with a benchtop, bead homogenizer (FastPrep 24, MP Biomedicals, Irvine, USA) in RLT buffer (a lysis buffer containing 30-50% guanidinium thiocyanate and obtained from Qjagen) with Customer No.60723 B-mercaptoethanol and RNA extracted from the lysate using manufacturer protocols (RNeasy Mini Kit, Qiagen; Valencia, USA). cDNA synthesis was completed using 0.3 µg of extracted RNA, pooled and perform in replicates in a RT2First Strand Kit (Qiagen; Valencia, USA). A custom-designed RT2Profiler PCR Array (Qiagen, UK) was used to evaluate the expression of genes relevant to skin biology.

[0067] All statistical tests were two-sided at significance level α=0.05. Grade and image data for treated sides of the face, adjusted for the untreated side, were analyzed by ANOVA (treated – untreated) or ANCOVA (untreated as covariate). An ANOVA mixed model for repeated measurements was fitted to raw clinical data obtained from instrumentation, included the factors: product as fixed, time as fixed, and product x time interaction. Results of in vitro evaluation

[0068] Viability testing via MTT assay indicated no significant loss of tissue viability in any of the treated groups. In vitro studies on 3D tissue support changes in dermal matrix via hyaluronic acid and pro-collagen I production and decrease in inflammatory genes. Conditioned media from the lower chamber were pooled for measurement of pro-collagen I and hyaluronic acid secreted by the 3D tissues after three days of treatment. The presence of hyaluronic acid (‘HA’) was measured via Homogeneous Time Resolved Fluorescence (HTRF) assay using labelled-hyaluronic acid binding protein (HABP) where the intensity of the labelled-HABP is proportional to the concentration of hyaluronic acid in tested samples (Cisbio). As shown in FIG. 1A, tissues treated with the L4 formula comprising 0.3 wt% L-4-thiazolylalanine secreted significantly more hyaluronic acid than the vehicle control (19.7% + 8.8% increase, p<0.05, Figure 1A).

[0069] Detection of pro-collagen I was performed by HTRF assay using labelled pro- collagen 1 antibodies (Cisbio). As shown in FIG.1B, tissue treated with L4 formula comprising 0.3 wt% L-4-thiazolylalanine secreted significantly more pro-collagen I than the vehicle control over three experiments (25.6% + 12.0% increase, p<0.05, Figure 1B). Increased secretion of hyaluronic acid and pro-collagen I in the 3D tissue models supports the ability of L-4-thiazolylalanine to effect changes in the skin matrix. Customer No.60723

[0070] Gene expression was measured from 3D tissue samples after 24 hours of exposure to treatment. Samples were evaluated for the expression of genes relevant to skin biology. Protein and gene expression data suggest that L-4-thiazolylalanine contributes to the dermal matrix assembly via the production of HA and pro-collagen I and upregulation of matrix genes such as VCAN, HAS1, and collagen type VI (COL6A6). TIMP metallopeptidase inhibitor 2 (TIMP2) was also upregulated by L-4- thiazolylalanine. L4 downregulates MMP3, in addition to TNF, both of which have been linked to collagen degradation in skin, further demonstrating the ability of L-4- thiazolylalanine to support the dermal matrix in skin. Matrix metallopeptidase 9 (MMP9), a metalloproteinase that is upregulated during wound healing, is also upregulated with L-4-thiazolylalanine exposure suggesting L-4-thiazolylalanine contributes to matrix remodelling. L-4-thiazolylalanine appears to significantly downregulate gene associated with inflammation such as TNF, interleukin 8 (CXCL8 or IL-8), IL-6, interleukin 1, beta (IL-1B), and prostaglandin-endoperoxide synthase 2 (PTGS2) suggesting that the efficacy of dermal changes exhibited by L-4- thiazolylalanine are not initiated by an inflammatory response.

[0071] Table 1 lists the gene expression of tissue samples treated with L4 formula and having a fold regulation greater than |1.50| over tissue treated with control formula (vehicle). Compared to the control formula, L4 formula treated samples exhibited a significant fold increase (p<0.05) in versican (VCAN), hyaluronan synthase 1 (HAS1), and matrix metallopeptidase 9 (MMP9), genes contributing to matrix structure and remodelling. This supports the protein expression levels of collagen and hyaluronic acid seen in 3D tissue. The array also revealed a significant downregulation (p<0.05) of genes associated with inflammation: interleukin 8 (CXCL8), interleukin 1 beta (IL1B), interleukin 6 (IL6), prostaglandin-endoperoxide synthase 2 (PTGS2), and tumor necrosis factor (TNF). This suggest that L-4- thiazolylalanine upregulates genes associated with dermal changes and downregulates genes associated with inflammation. Evaluation of L4 formula in the in vitro 3D tissue model demonstrates that L-4-thiazolylalanine has a notable effect on the gene expression and protein production of key dermal matrix components. Table 1: Gene expression of tissues treated with L4 formula with a fold regulation greater than |1.50| over tissue treated with control formula. Customer No.60723 Gene Fold regulation Protein VCAN 2.25 Veriscan Example 2 - In vivo treatments and biopsy Materials and Methods

[0072] An in vivo study was conducted at Product Investigations, Inc (Conshohocken, PA), with the study protocol approval by the Greater Delaware Valley Investigational Review Board. After informed consent approval, Caucasian female (n=30) subjects with Fitzpatrick skin type I-III, between 21 and 65 years of age participated in the study. The ventral aspects of each subjects forearm were treated topically with test formulations (2 ug / cm2) for 4 weeks and placed under a semi-occlusive patch which was taped to the skin. Three treatments were applied in randomized fashion: base formula (not containing L-4-thiazolylalanine)(herein referred to as ‘formula base’), base formula further containing 0.1 wt% retinol (herein referred to as ‘retinol formula’), and base formula further containing 0.3 wt% L-4-thiazolylalanine (herein referred to as ‘L4 formula’). The base formula also contained 10 wt% glycerin. Formulation containing 0.1 wt % retinol (retinol formula) was used as a benchmark control, as this concentration has been tested to be efficacious while not irritating to participants over long-term studies (DiNatale L, Idkowiak-Baldys J, Zhuang Y, Customer No.60723 Gonzalez A, Stephens TJ, Jiang LI, et al. Novel Rotational Combination Regimen of Skin Topicals Improves Facial Photoaging: Efficacy Demonstrated in Double-Blinded Clinical Trials and Laboratory Validation. Front Med (Lausanne).2021;8:724344). Applications were repeated daily (Monday-Friday) and remained patched throughout the weekend. At the end of treatments a 2 mm skin punch biopsy was taken from each treatment site.

[0073] To conduct histological analysis, the skin samples were fixed in 10% formalin followed by embedding in paraffin following standard protocol. After processing, skin samples were cut into 6 µm sections for immunohistochemistry analysis. Next, samples were processed for hematoxylin and eosin (H&E) following standard protocol. H&E staining of biopsy samples was performed to evaluate the effect of L- 4-thiazolylalanine on viable epidermal thickness.3D-Histech Pannoramic-250 microscope slide-scanner was used to obtain histological images. Thickness of viable epidermis was evaluated using a calibrated ocular micrometer (CaseViewer2.2) at a magnification of 40x. The measurements were performed using only the inter-rete ridge regions of the epidermis, from the basement membrane to the top of the granular layer at 3 randomly selected points across each section. For each section, the average was calculated then the average ± SD of all subjects were calculated. Herovici staining was used to evaluate dermal collagen. Briefly, the slides were incubated in Van Giesan stain solution for 10 minutes, followed by 1% acetic acid for 30 seconds, and dehydration through alcohol and xylene before mounting. For dermal hyaluronic acid assessment, sections were incubated with biotinylated HABP antibody (Sigma, St. Louis, MO). Slides were scanned and analyzed using Definiens Tissue Studio software, version 64.4.0 (Definiens, Munich, Germany) and Matlab software (MathWorks, Natick, MA), respectively.

[0074] To conduct skin fluorescence measurements, eleven healthy females between 21 and 65 years of age, with Fitzpatrick phototype I-III, participated in a daily application regimen with 2µg / cm2of each product over a 28 day period on their volar forearms. All subjects read and signed an Informed Consent document in a study that was conducted at the Avon Innovation Center in Suffern, NY. Three treatments were applied in randomized fashion: base formula with no active ingredients (not containing L-4-thiazolylalanine) (‘formula base’), base formula containing 0.1 wt% retinol (retinol formula), and base formula containing 0.3 wt% L-4-thiazolylalanine Customer No.60723 (L4 formula). The base formula also contained 10 wt% glycerin. SPEX SkinSkan spectrofluorometers (JY Horiba, Edison, NJ) with a Xenon arc lamp excitation source was used for in vivo fluorescence measurements as disclosed in Maidhof R, Liebel F, Hwang C, Ruvolo E, Lyga J. UV fluorescence excitation spectroscopy as a noninvasive predictor of epidermal proliferation and clinical performance of cosmetic formulations. Photodermatol Photoimmunol Photomed.2019;35(6):408-14. For each treated site, skin was washed and no formula was present, excitation spectrum was scanned from 250 nm to 370 nm and emission was set at 380 nm. Excitation-emission spectra at 295 nm (F295) for cell proliferation.

[0075] All statistical tests were two-sided at significance level α=0.05. Grade and image data for treated sides of the face, adjusted for the untreated side, were analyzed by ANOVA (treated – untreated) or ANCOVA (untreated as covariate). An ANOVA mixed model for repeated measurements was fitted to raw clinical data obtained from instrumentation, included the factors: product as fixed, time as fixed, and product x time interaction. Results of histological analysis

[0076] Histological analysis of biopsies obtained from treated sites was performed to assess changes in epidermal thickness and hyaluronic acid secretion as well as dermal collagen after four weeks of treatment. H&E staining of biopsy samples was performed to evaluate the effect of L-4-thiazolylalanine on viable epidermal thickness.

[0077] Both treatment with the L4 formula and the retinol formula resulted in a significant increase (p<0.001) in epidermal thickness compared to treatment with the formula base, validating epidermal renewal efficacy of the L4 formula and retinol formula. The average epidermal thickness of skin treated with the retinol formula was 57.57±16.5 μm, the average epidermal thickness of skin treated with the L4 formula was 46.01±6.1 μm, and the average epidermal thickness of skin treated with the formula base was 41.75±4.5 μm.

[0078] To determine if the changes in epidermal thickness correlated with skin fluorescence, epidermal turnover was measured by evaluating F295 / F340 excitation- emission spectra. As shown in FIG.2, F295 / F340 excitation was significantly stimulated in skin treated with the retinol formula, but also in the L4 formula treatment group (46.05±19.9% and 24.03±9.6% increases respectively, both p<0.05). Customer No.60723 No significant change in F295 / F340 excitation was observed in the formula base treatment group (-10.76±9%). Skin fluorescence measurements suggest that the increase in epidermal thickness may be attributed to skin turnover.

[0079] The effect on epidermal hyaluronic acid (HA) level was examined using HA- binding protein (HABP) staining of dermal structures (Figure 3). L4 exhibited a significant increase of HABP staining and therefore HA secretion over baseline (42.69±20.7% increase vs base alone, p<0.05). Samples from subjects treated with retinol formula showed directional (p=0.16) increase in HABP staining (29.07±20.7%) compared to base formula treatment alone, and no statistical difference compared to L4 formula treatment. Production of HA from L-4- thiazolylalanine is supported by protein and gene expression in 3D tissue.

[0080] To evaluate the effect of treatments on dermal collagen changes and quality, sections of the biopsy samples were stained with Herovici stain. As shown in FIG. 4A, treatment with the retinol formula resulted in significant increase in collagen III compared to treatment with formula base (23.37±8.3% vs. base, p<0.05). L4 formula treatment group showed similar efficacy (24.79±8.2% vs. base, p<0.05). The level of collagen III and collagen I change in the skin with age and during wound healing, where the lowest level of the ratio of collagen III / I occurs in aged skin. During normal wound healing of the skin, collagen III is deposited in the early stages later to be replace with collagen I. This suggests the importance of collagen III and the ratio of collagen III / I as essential elements of dermal matrix quality. With both the retinol formula and L4 formula treatments, increase in collagen III in papillary dermis led to concomitant improvement in the ratio of collagen III / I (FIG.4B), implicating stimulation of dermal matrix remodelling. Histological analysis of L4 formula-treated skin confirms the efficacy of L-4-thiazolylalanine on increasing epidermal and dermal matrix components compared to retinol and base formulation. The significant increase of collagen III in L4 formula treated biopsy samples indicates new tissue formation similar to wound healing, supported by the upregulation of MMP9. Example 3 - Clinical Study Materials and Methods

[0081] Clinical evaluation of L-4-thiazolylalanine was performed via split-face study (n=28) versus baseline and formula base control. The objective was to evaluate the Customer No.60723 smoothing / wrinkle, firming, and sagging effects after 14 and 28 days of twice-daily use. A clinical efficacy study was conducted for 28 days in Lyon, France that evaluated the performance of the L4 formula versus a formula base control. The formula base did not comprise L-4-thiazolylalanine. Both the L4-formula and formula base further comprised 3 wt% glycerin. The study performed was in accordance with Decree n° 2017-884 of May 9, 2017 modifying some regulatory requirements concerning researches involving human subjects. Therefore, the study does not require the Ethics Committee Approval or the Competent Authority Authorization. After free informed, written consent, healthy Caucasian women (n=28), ages 45 to 65 years old with Fitzpatrick phototype I-III, used both products (L4 formula and formula base) twice daily on the facial skin in a split-face design, following randomization. Specific inclusion criteria included subjects with mild to moderate wrinkles and fine lines on crow’s feet and subjects with loose skin on the face (lower cheek). All participants were given the same cleanser and SPF 30 cream to use a week prior and for the duration of the study in replacement of their usual cleanser and cream. During the study, the SPF is only used as needed in case of excessive UV exposure of more than 10 minutes. Performance was evaluated for firming, smoothing and wrinkles, and sagging after 14 and 28 days. Baseline readings were collected on all instruments at day 0, before product use and a week after using provided cleanser and SPF cream. Firming measurements

[0082] Firming effects were measured using a DynaSKIN (EOTECH SA, Marcousis, France) instrument in conjunction with the fringe projection system, DermaTOP (EOTECH SA, Marcousis, France). Non-contact mechanical pressure in combination with fringe projection to quantify and visualise the skin surface response in 3- dimensions (Kearney et al. - Evaluation of skin firmness by the DynaSKIN, a novel non-contact compression device, and its use in revealing the efficacy of a skincare regimen featuring a novel anti-ageing ingredient, acetyl aspartic acid, Skin Research and Technology 2017; 23: 155–168; RORH M, SHRADER A – FOITS, Fast optical in vivo topometry of human skin: a classical method in modern efficacy testing, A history of Fringe Projection in Cosmetics, SOFW-Journal, 2009, 135, 8). The system measures firmness of the skin (cheek) by blowing gentle air perpendicular to the skin Customer No.60723 surface and transform the deformation information into a 3D map of skin surface. DynaSKIN measures firmness as the correlation of the size of the skin’s maximum resistance to an applied force, which can provide a quantitative measure of skin structural integrity and a consumer-relevant measure, as it mirrors the way consumers judge firmness on their own skin, by pushing or pressing on the skin surface. This technique provides qualitative and quantitative data for real tactile perception of firmness and laxity of the skin and its sag via its measurement of depth, surface area, and volume of the skin deformation. Smoothing and wrinkle measurements

[0083] Smoothing / wrinkle effects were assessed directly in vivo, using DermaTOP (EOTECH SA, Marcousis, France) to acquire a 2D and 3D image of wrinkles on each crow’s foot. This system calculate cutaneous relief parameters by averaging 50 vertical profiles along the zone of interest. The average roughness (Ra) of the skin, which is the mean average absolute values of peak and valley heights of the skin surface to determine the smoothing effect (FIG.6A). From this image, average relief amplitude (Rz) and maximum relief amplitude (Rt) is also calculated. Rz is the average of five single roughness depths (difference from highest peak to lowest valley) measured within the sample section. Rt is the height difference between the highest peak and the lowest valley over the entire measurement section (FIG.6B). These relief parameters contribute to determine the anti-wrinkle effect of the product.

[0084] Anti-wrinkle effects are also measured on crow’s feet using a polymer silicone skin print (Silflo, Monaderm, Monaco) of the subject’s crow’s feet region and examined using SIA (Skin Image Analyzer, Courage+Khazaka electronic GmbH, Germany). Oblique lighting at 35° reveal shadows on the replica surface which is captured by a digitized picture and studied with the QuantiRides® software (Monaderm, Monaco). Deep wrinkles greater than 110 μm were analyzed where the minimal surface of a furrow to be considered is arbitrarily fixed to 0.03 mm². Sagging Measurements

[0085] Sagging effects were determined with a FaceScan instrument (GFM, Berlin, Germany), which uses interference fringe projection images of the whole face to create a 3D digital image. Variations in face ptosis volume is calculated by comparing Customer No.60723 changes the baseline 3D images with identical 3D images captured at specific timepoints. Results of clinical study

[0086] Clinical evaluation of L4 formula resulted in firmer and smoother skin from 28 days of treatment with L4 formula over the formula base. Without wishing to be bound by theory, it is suggested that L-4-thiazolylalanine promotes structural and functional changes in the skin which are observed as significantly firmer, smoother skin after 28 days of application compared to base formula.

[0087] The greatest effect of L-4-thiazolylalanine was seen in the firming evaluation. Firming was evaluated via the deformation of the skin due to a puff of air being fired at and perpendicular to the skin surface. Changes in firmness of the skin surface change the volume, surface area, and maximum depth of deformation created by the air puff. After 14 days, there was no significant difference in the volume, surface area, or maximum depth for skin treated with the L4 formula or skin treated with the formula base over baseline. At day 28, it was observed that skin treated with the L4 formula had a significant decrease in volume (-15%, p<0.05), surface area (-5%, p<0.05), and maximum depth (-11%, p<0.05) over baseline (FIGs 5A-C). Additionally, the L4 formula supported a significant decrease in surface area of skin deformation over formula base (-10%, p<0.05, Figure 5B). This suggests that L-4- thiazolylalanine has a significant firming effect on facial skin after 28 days of use. The formula base also exhibited a significant decrease in volume (-8%, p<0.05) and depth (-11%, p<0.05) vs. baseline which may be attributed to increased hydration by the traditional moisturizer base at the skin surface. However, the formula base demonstrated an increase in the deformation of the skin surface area (+5%, p<0.05) vs. baseline which, combined with a significant decrease in depth of deformation, suggests a tightening of the skin via tensing effect of the base formula.

[0088] Smoothing and anti-wrinkle effects are clinical outcomes that target the visible signs of ageing. For smoothing and wrinkle effects, subject’s crow’s feet area were examined directly in vivo for changes to cutaneous relief parameters after formula use. As shown in FIG.6C, L4 formula use demonstrated a significant decrease (-8%, p<0.05 vs. baseline, FIG.6C) in the average roughness (Ra) of the skin after 14 days. Ra is the mean absolute values of peak and valley heights of the skin surface area (Figure 6A). After 28 days, L4 formula use exhibited a directional decrease (-8%, Customer No.60723 p=0.052) in Ra vs. baseline. The formula base control did not exhibit any significant changes to the Ra (p=0.3212). A decrease in the Ra over baseline suggests smoother skin after use. Moreover, as shown in FIG.6B, L4 formula use exhibited a significant decrease (p<0.05) in average relief (Rz, Figure 6B) over baseline after 14 and 28 days (-8% and -9%, respectively, Figure 6D). A decrease in Rz supports a decrease in wrinkled skin. Additionally, as shown in FIG.6E, L4 formula resulted in a directional decrease (p=0.0783) in maximum relief (Rt) over baseline at day 14 (-6%, Figure 6E). The formula base had no significant difference in Rz and Rt of the crow’s feet area at any time point further supporting the smoothing and anti-wrinkle effects of L-4- thiazolylalanine.

[0089] As an additional measure of anti-wrinkle effects, the crow’s feet region for each subject was analyzed via skin print and Skin Image Analyser after 14 and 28 days for deep wrinkles (depth > 110 µm). The skin print measure for wrinkles further supports the efficacy of L-4-thiazolylalanine to reduce wrinkle depth after four weeks of treatment over base formula. There was no significant difference in the depth of deep wrinkles measured for L4 formula or formula base versus baseline after 14 days. However, at day 28, the average depth of deep wrinkles measured for L4 formula was significantly less than the formula base (-9%, p<0.05, FIG.7A and FIG.7B). L4 formula also demonstrated a directional decrease in deep wrinkle depth over baseline (-7%, p=0.0709). This further supports the greater anti-wrinkle effect of L-4- thiazolylalanine over formula base after 28 days.

[0090] Improvements in sagging were determined directly in vivo on the whole face after 14 and 28 days by calculating changes in face ptosis volume. A decrease in ptosis volume indicates a remodelling effect. As shown in FIG.8A, neither L4 formula nor formula base induced any significant variation in this measurement over baseline at either time point. However, after 28 days of use, L4 formula use resulted in a significantly lower (-5%, p<0.05) ptosis volume compared to the formula base (FIG.8B), supporting the greater remodelling effect of L-4-thiazolylalanine. The clinical evaluation of L-4-thiazolylalanine on facial skin indicates an improvement in firming, smoothing, wrinkle and sagging reduction, after 28 days of use, further supporting L-4-thiazolylalanine as a viable ingredient to target visible signs of ageing without redness or irritation. Customer No.60723 Example 4 – in vivo confocal Ramen spectroscopy study of skin collagen and water content

[0091] Skin moisture, which may be regarded as skin water content, is important for the normal functioning of the skin, but also for providing a healthier and younger looking skin. Collagen content is also important for maintaining the health and appearance of the skin. The total water content of the epidermis, and of three different water types at the dermis, considering free, weakly bound and strongly bound water was determined following treatment and was measured using confocal Raman spectroscopy. Collagen content, along with collagen-water interactions, was measured at papillary dermis. Materials and Methods

[0092] Confocal Raman spectroscopy (CRS) is a non-invasive optical method based on the inelastic scattering of light that can be applied in vivo and in real time for the study of the skin’s molecular composition with high spatial resolution (Rostron, Paul & Gerber, Dina. (2016). Raman Spectroscopy, a review. International Journal of Engineering and Technical Research.6.50-64). Confocal Raman spectroscopy (CRS) was applied to determine water content, from the skin surface up to the dermis, with high spatial resolution. CRS may be used to measure the water content at the epidermis and at the dermis in vivo.

[0093] The study was performed using three different formulations provided by AVON Products, Inc. assessed in vivo using CRS. The three different formulations were a placebo (herein referred to as ‘placebo formula’) containing 7 wt% glycerin, a formula comprising 0.3 wt% L-4-thiazolylalanine and 7 wt% glycerin (herein referred to as ‘L4 formula’), and a formula comprising 2 wt% sodium pyrrolidone carboxylic acid and 7 wt% glycerin (herein referred to as ‘PCA formula’) on the human skin. A monocentric and double-blind study was performed at the laboratory of Vibrational Spectroscopy DermoPROBES – Efficacy tests on Skin, Hair, Nails and Drugs. The study was submitted and approved by the Ethics Committee under number 5.112.705. The study was conducted following the Good Clinical Practices. All participants signed the Free and Informed Consent Form.

[0094] Participants were selected from DermoProbes laboratory database, following inclusion and non-inclusion criteria: male or female, aged between 50 – 60 years old, Customer No.60723 non-pregnant, phototype II or III according to the Fitzpatrick’s classification, and with intact skin at the inner forearms.

[0095] Sixteen study subjects were selected and had three regions at their forearms, with a square area of 4 cm2, delimited. Each area received either one of the three formulations. Sites were assigned by a randomization process, as a method to avoid any possible bias.

[0096] Formulations were topically applied once a day, always in the afternoon and by a trained specialist to guarantee study quality. Treatment with the formulations took place on 30 consecutive days. Assessment of the forearm skin parameters was performed at baseline, or basal time, (T0), and after 15 (T15) and 30 (T30) days of continuous daily treatment. Each formulation was applied with a calibrated pipette to 10 µL, equivalent to 10 mg (2.5 mg / cm2).

[0097] Before collection of Raman measurements, which happened in a temperature and humidity-controlled environment, participants remained for 30 minutes in an acclimatized room, with room temperature at 23 ± 2 °C, and humidity set at 40 – 50%, to stabilize skin.

[0098] Raman spectroscopy was performed with a confocal Confocal Raman System (Confocal Raman Rivers Diagnostics® - Model 3510 Skin Composition Analyzer) coupled to a laser excitation of 671 nm was used. Laser light was centered at the skin surface with a microscope objective (40x) located under the quartz window and the Raman signal was collected with a Charge Coupled Device (CCD) camera. The laser power at the skin surface was set to approximately 20 ± 2 mW. Spectral acquisition occurred in the high frequency region between 2500 – 3800 cm-1. Skin depths analyzed were between 0 – 110 µm, with steps of 2 – 10 µm.

[0099] Epidermal total water content was determined based on the Area Under the Curve (AUC) within the spectral range between 3350 cm-1and 3550 cm-1, after normalization by the protein band at 2936 cm-1.

[0100] At the dermis, Strongly-hydrogen bound water (DDAA-OH), Weakly- hydrogen bound water (DA-OH) and unbound water (free water) were determined based on the AUC from Raman peaks 3277 cm-1, 3458 cm-1, and 3604 cm-1, respectively (Choe, C., Lademann, J., & Darvin, M. E. (2016). Depth profiles of hydrogen bound water molecule types and their relation to lipid and protein interaction in the human stratum corneum in vivo. The Analyst, 141(22), 6329–6337. Customer No.60723 doi:10.1039 / c6an01717g; Choe, C., Schleusener, J., Lademann, J., & Darvin, M. E. (2018). Human skin in vivo has a higher skin barrier function than porcine skin ex vivo-comprehensive Raman microscopic study of the stratum corneum. Journal of Biophotonics, 11(6), e201700355).

[0101] During the analysis, all data obtained at day 15 (T15) and at day 30 (T30) were subtracted from basal data (T0), thus providing water content changes, in arbitrary units (a.u.).

[0102] Collagen content changes upon topical administration of the formulations was analyzed based on 1322 cm-1Raman peak intensity. This peak corresponds to CH3CH2twisting and wagging mode of collagen, and to deforming modes of collagen and of nucleic acids (Liu, H., Liu, H., Deng, X., Chen, M., Han, X., Yan, W., & Wang, N. (2016). Raman spectroscopy combined with SHG gives a new perspective for rapid assessment of the collagen status in the healing of cutaneous wounds. Analytical Methods, 8(17), 3503–3510). Collagen-water interactions determination

[0103] Formation of hydrogen bonds between carbonyl and proline or hydroxyproline was determined by the ratio between the intensity of the Raman peaks 938 cm-1and 922 cm-1(I938 / I922) Transition from weak to strong hydrogen bonds was determined by the ratio between the intensity of the Raman peaks 1658 cm-1and 1668 cm-1(I1658 / I1668). (Nguyen, T. T., Gobinet, C., Feru, J., -Pasco, S. B., Manfait, M., & Piot, O. (2012). Characterization of Type I and IV Collagens by Raman Microspectroscopy: Identification of Spectral Markers of the Dermo-Epidermal Junction. Spectroscopy: An International Journal, 27, 421–427). Results

[0104] Total water content increased at the stratum corneum (FIG.9A) and the epidermis (FIG.9B) for skin treated with the L4 formula or the PCA formula after 15 days of treatment and the increase was maintained after 30 days of treatment. At T15, water content at the stratum corneum was of 58.25 a.u. for placebo formula, 115.48 a.u. for L4 formula, and 129.97 a.u. for PCA formula. At T30, values were of 5.42 a.u. for placebo formula, 107.14 a.u. for L4 formula, and 114.63 a.u. for PCA formula. In addition, water content decreased from T15 to T30. Highest decrease Customer No.60723 occurred for placebo formula, of 52.83 a.u., followed by PCA formula, of 15.34 a.u., and last by L4 formula, of 8.34 a.u. Statistically significant increases occurred for L4 formula and PCA formula at both times in relation to placebo formula.

[0105] Without wishing to be bound by theory, it is suggested that the increase in water content could be related to skin turnover. This is in contrast to the placebo formula treatment which resulted in an initial increase in total water content at 15 days of treatment, followed by a decline at 30 days of treatment. It is thought that glycerin’s humectant effect may attract water from the dermis up the skin surface resulting in an increase in water content which may explain the increased water content at day 15. However, glycerin is known to cause an increase in transepidermal water loss which is indicative of skin barrier disruption, and may account for the observed decrease in water content at the stratum cornea and epidermis after 30 days of treatment with placebo formula.

[0106] Water at the dermis can be differently bounded via hydrogen bonds according to its molecular structure. It can be tightly bounded (DAA-OH), strongly bounded (DDAA-OH), weakly bounded (DA-OH), or very weakly bounded (DDA-OH). It can also be present as the free form, not bounded to any other molecule. The L4 formula was found to promote an increase in dermal free water and dermal weakly bound water after 30 days of treatment.

[0107] An increase in dermal strongly bound water was observed after treatment with the L4 formula after 15 days and further increase was observed after 30 days of treatment. Example 5 – Skin barrier integrity Materials and Methods TEWL study

[0108] An in vivo study was performed at the Avon Innovation Center (Suffern, New York) where female subjects (N=11), ages 25-60 years old of Fitzpatrick skin type I- III were selected and informed consented. Subjects were instructed to apply a 2µg / cm2dose daily of a formulation containing 0.3 wt% L-4-thiazolylalanine to their volar forearms daily for 28 consecutive days. Trans-epidermal water loss measurements (Derma Lab, Cortex Technology, Denmark) were taken at baseline and weekly thereafter. Customer No.60723 Clinical scalp study

[0109] A study was performed at Zurko Research S.L. (Madrid, Spain) where 11 male and female subjects, ages 40-70 years old were recruited with dry irritated scalp, non- dandruff. After signing an informed consent, subjects applied a serum containing 0.3 wt% L-4-thiazolylalanine to their scalp daily after showering for 28 consecutive days. Clinical evaluation was performed for efficacy and tolerance at baseline, day 14 and 28. Erythema intensity grading scale: 0: No redness; 1: Light redness; 2: Red, but not deep red; 3: Very red; 4: Extremely red. Desquamation intensity grading scale: 0: No desquamation 1: Very slight desquamation 2: Slight desquamation 3: Moderate desquamation 4: Severe desquamation.

[0110] All the data analysis was performed using the JMP statistical software (www.jmp.com, Cary, NC, USA) using the LS Means Tukey HSD test with significance set at p < 0.05. For the clinical scalp studies, Wilcoxon Signed-Rank Test was used to evaluate the efficacy of the treatment along time. The effect of the treatment on the biometric measurements was interpreted comparing each evaluation time in relation to baseline and between references. A significance value of 0.05 was established (95% confidence interval). Results

[0111] transepidermal water loss (TEWL) was evaluated. As shown in FIG.10, an average TEWL value at baseline was 7.05 ± 1.18 and after 4 weeks the value was 6.12 ± 0.45, meaning no significant change in TEWL values was measured when the skin was treated with a formula comprising L-4-thiazolylalanine. Unlike other known anti- ageing ingredients which are known to increase TEWL because of excessive proliferation, L-4-thiazolylalanine did not appear to disrupt the skin barrier.

[0112] In a dermatologist graded study where subjects applied daily a 0.3 wt% L-4- thiazolylalanine serum to their dry, flaky, irritated scalps for 28 days, significant improvements were seen. The L-4-thiazolylalanine treatment significantly reduced erythema from baseline by 41% after 14 days and by 77% after 28 days. When the amount of desquamation was graded it had significantly reduced by 25% after 14 days and by 42% after 28 days (FIGs.11A and 11B). Customer No.60723

[0113] Where in the foregoing description, integers or elements are mentioned which have known, obvious or foreseeable equivalents, then such equivalents are herein incorporated as if individually set forth. Reference should be made to the claims for determining the true scope of the present invention, which should be construed so as to encompass any such equivalents. It will also be appreciated by the reader that integers or features of the invention that are described as preferable, advantageous, convenient or the like are optional and do not limit the scope of the independent claims. Moreover, it is to be understood that such optional integers or features, whilst of possible benefit in some embodiments of the invention, may not be desirable, and may therefore be absent, in other embodiments.

Claims

Customer No.60723 Claims 1. A cosmetic method of improving the appearance of the skin of a subject in need thereof comprising the steps of: (i) topically applying to the skin a composition comprising Compound I of the formula: or a zwitterionic salt thereof, whereinthe composition comprises from 0.08 wt% to 0.50 wt% of the total weight of the composition of Compound I or the zwitterionic form excluding the mass of any salt; and, (ii) repeating step (i) for a continuous period of at least 24 days and until a cosmetically beneficial improvement to the skin of the subject has occurred.

2. The method according to claim 1, wherein step (ii) comprises repeating step (i) at least once per day.

3. The method according to claim 1, wherein step (ii) comprises repeating step (i) at least every other day.

4. The method according to claim 1, wherein step (ii) comprises repeating step (i) at least five days out of every seven consecutive days.

5. The cosmetic method of any preceding claim, wherein the cosmetically beneficial improvement to the skin of the subject comprises at least one of: a. reduced wrinkle depth, b. increased hyaluronic acid secretion, c. increased skin hydration,Customer No.60723 d. increased epidermal thickness, e. increased skin firmness, f. increased skin smoothness, g. improvement skin barrier integrity, h. reduced redness, i. increased dermal collagen III and / or collagen I production.

6. The cosmetic method of any preceding claim, wherein the cosmetically beneficial improvement to the skin comprises a reduced wrinkle depth, wherein the wrinkle depth is greater than 110 µm prior to step (i).

7. The cosmetic method of any preceding claim, wherein the cosmetically beneficial improvement to the skin comprises increased hyaluronic acid secretion, wherein the amount of hyaluronic acid secreted after step (ii) exceeds the amount of hyaluronic acid secreted prior to step (i) by at least 10%.

8. The cosmetic method of any preceding claim, wherein the cosmetically beneficial improvement to the skin comprises increased skin hydration, wherein the total water content of the epidermis and / or stratum corneum after step (ii) exceeds the total water content of the epidermis and / or stratum corneum prior to step (i).

9. The cosmetic method of any preceding claim, wherein the cosmetically beneficial improvement to the skin comprises increased epidermal thickness, wherein the epidermal thickness after step (ii) is at least 5% greater than the epidermal thickness prior to step (i).

10. The cosmetic method of any preceding claim, wherein the cosmetically beneficial improvement to the skin comprises increased skin firmness, wherein at least one of a volume, surface area or maximum depth of deformation to the skin when a force is applied to the skin surface after step (ii), is less than theCustomer No.60723 volume, surface area or maximum depth of deformation to the skin when an equal force is applied to the skin surface prior to step (i) of the present method.

11. The cosmetic method of any preceding claim, wherein the cosmetically beneficial improvement to the skin comprises increased skin smoothness, wherein at least one of average roughness, Ra, average relief amplitude, Rz, and maximum relief amplitude, Rt, of the skin after step (ii) is less than the average roughness, Ra, average relief amplitude, Rz, or maximum relief amplitude, Rt, of the skin prior to step (i).

12. The cosmetic method of any preceding claim, wherein the cosmetically beneficial improvement to the skin comprises an improvement in skin barrier integrity, wherein the improvement in skin barrier integrity comprises maintenance of or reduction in transepidermal water loss in which transepidermal water loss after step (ii) is less than or substantially equal to transepidermal water loss prior to step (i).

13. The cosmetic method of any preceding claim, wherein the cosmetically beneficial improvement to the skin comprises reduced redness, wherein the redness of the skin after step (ii) is less than the redness of the skin prior to step (i).

14. The cosmetic method of any preceding claim, wherein the cosmetically beneficial improvement to the skin comprises increased dermal collagen I and / or collagen III production, wherein the amount of collagen I and / or collagen III secreted after step (ii) exceeds the amount of collagen I and / or collagen III secreted prior to step (i) by at least 15%.

15. The cosmetic method of any preceding claim, wherein step (ii) comprises repeating step (i) for a continuous period of at least 28 days.

16. The cosmetic method of any preceding claim, wherein the method further comprises:Customer No.60723 (iii) further repeating step (i) in order to maintain the cosmetically beneficial improvement in the appearance of the skin of the subject.

17. The cosmetic method according to any preceding claim, wherein the composition comprises from 0.09 wt% to 0.40 wt%, preferably from 0.10 wt% to 0.35 wt% of the total weight of the composition of Compound I or the zwitterionic form excluding the mass of any salt.

18. The cosmetic method according to any preceding claim, wherein the skin is the skin of at least one of the face, scalp, neck or décolletage.

19. The cosmetic method of any preceding claim, wherein the subject is female.

20. The cosmetic method of any preceding claim, wherein the subject is in the range of from 21 to 65 years of age.

21. A composition for topical application comprising Compound I of the formula: or a zwitterionic form orwherein the composition comprises from 0.08 wt% to 0.50 wt% of the total weight of the composition of Compound I or the zwitterionic form excluding the mass of any salt.

22. The composition according to claim 21, for use in a therapeutic treatment of a skin condition, wherein the skin condition is associated with at least one of: a. increased wrinkle depth, b. reduced hyaluronic acid secretion, c. decreased skin hydration, d. decreased epidermal thickness,Customer No.60723 e. decreased skin firmness, f. decreased skin smoothness, g. decreased skin barrier integrity, h. increased redness, i. decreased dermal collagen III and / or collagen I production.

23. The composition according to claim 21 or claim 22, for use in treating erythema.

24. The composition according to any one or claims 21 to 23, wherein the therapeutic treatment of a skin condition comprises the steps of: (iii) topically applying to the skin the composition; and, (iv) repeating step (i) for a continuous period of at least 24 days and until an improvement in the skin condition of the subject has occurred.

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