WO2024237777A1

WO2024237777A1 - Compositions and methods for preventing and/or treating skin pigmentation

Info

Publication number: WO2024237777A1
Application number: PCT/MY2023/000005
Authority: WO
Inventors: Ursula Rho Wan CHONG; Angela Ching Ling TEOH; Nur Rizi ZAMBERI; Elaine Shu Ching LEE; Yi Peng NG; Wendy Yen Sun HEE; Juin Onn WOO; Ivy Wan Yee HOW; Ka Heng LEE; Mong Sah TOH
Original assignee: Winnox Cosmeceutics Sdn Bhd
Current assignee: Winnox Cosmeceutics Sdn Bhd
Priority date: 2023-05-18
Filing date: 2023-05-18
Publication date: 2024-11-21
Anticipated expiration: 2025-11-18
Also published as: CN121057574A; TW202446359A

Abstract

Provided herein are compositions for skin lightening and for preventing and/or treating a skin pigmentation disorder. Also provided herein are methods and uses for preventing and/or treating skin pigmentation disorders, and for inhibiting melanin production in epidermal melanocytes and/or promoting melanin degradation in dermal fibroblasts, and non-therapeutic methods of lightening skin colour.

Description

Compositions and methods for preventing and/or treating skin pigmentation

Technical field

The present disclosure relates to compositions for preventing and/or treating a skin pigmentation disorder and for non-therapeutic skin lightening. The present disclosure also relates to methods and uses for treating skin pigmentation disorders, and for inhibiting melanin production in epidermal melanocytes and/or promoting melanin degradation in dermal fibroblasts, and to non-therapeutic methods for lightening skin.

Background

Melanogenesis is a process that occurs in melanosomes by melanocytes involving a series of enzymatic and chemical reactions to produce the pigment melanin, which predominantly occurs in the epidermal layer of the skin. Skin hyperpigmentation is a common condition in humans that is primarily caused by excessive melanin synthesis and storage through uncontrolled melanogenesis. Increased melanin production can be triggered by numerous factors including excessive sun exposure, hormonal changes, skin inflammation and age, as well as many other biological processes (Nieuweboer-Krobotova, 2013).

Oxidative stress caused by the sun’s UVA and UVB rays, together with high-energy visible light, is major cause of sun-induced skin hyperpigmentation which results in small, darkened patches of skin, known as sun spots. Larger patches of hyperpigmentation, mainly on the face, called melasma, which are particularly common among women, are thought to occur when sex hormones such as estrogen and progesterone stimulate the overproduction of melanin, which can also be exacerbated through sun exposure. Post-inflammatory hyperpigmentation refers to the darkening of skin that occurs due to uncontrolled melanogenic processes resulting from an inflammatory eruption or cutaneous injury. These and other hyperpigmentary skin disorders can significantly impact an individual's appearance and may cause emotional and psychological distress and reduced quality of life.

Skin comprises multiple layers, including the epidermal (outer), dermal and hypodermal layers. The structure of these layers relies on the formation and maintenance of complex biological networks that consist of various cell types and macromolecules. Separating the epidermal and dermal skin layers is a basement membrane to which epidermal basal cells are atached. The main protein families that make up the macromolecules found in the extracellular matrices include: collagens, elastins, adhesive glycoproteins and proteoglycans (Latouf et al., 2014), which play a role in the elasticity, firmness and structure of the skin.

Because of the important issue of hyperpigmentation, there is an ever-growing demand for melanogenesis inhibitors. Many existing topical hyperpigmentation treatments are based on accelerated epidermal turnover which rely on the removal of melanin from the outer layers of the skin. Such treatments include exfoliating agents (e.g. AHA or BHA), antihyperpigmentation agents including kojic acid and hydroquinone, that act to inhibit melanin synthesis, melanosome blockers (e.g. niacinamide) which act to retard melanosome transfer from melanocytes to keratinocytes, and anti-inflammatory agents (e.g. stearyl glycyrrhetinate) which act to retard skin inflammation, as well as combinations of any of above.

Although the above-mentioned topical treatments are effective in lightening skin tone, they are often less effective in treating hyperpigmentation disorders. This is due to their mode of action primarily acting upon epidermal melanosis. In addition, many products currently available on the market for treating hyperpigmentation may contain ingredients that have been associated with possible adverse effects, including hydroquinone and kojic acid. As an alternative to the above topical treatments, facial laser treatments including Q-switched with nano-second pulse-width technology are considered effective methods for lightening or eliminating dermal melanosis, but these treatments are costly and invasive (Watanabe, 2014).

There remains a need for highly safe and effective anti-hyperpigmentation compositions which can address one or more limitations and drawbacks of the prior art.

Summary

It has now been discovered that strengthening of dermal-epidermal junctions (DEJs) contributes to reducing the movement of dermal factors associated with hyperpigmentation of melanocytes and reducing the leakage of melano somes into dermis results in a lighter skin tone. It has also been identified that the levels of collagen IV and collagen VII correlate positively with improved DEJ structural integrity. It has further been found that the active agents: N-acetyl-L-glutaminyl-L-alpha-aspartyl-L-valyl-L-lfistidine (acetyl tetrapeptide-9) and N-acetyl-L-prolyl-L-prolyl-L-tyrosyl-L-leucine (acetyl tetrapeptide- 11) in combination, or N-acetyl-L-alanyl-L-leucyl-L-histidyl-L-lysyl-L-valyl-L-lysinamide (acetyl sh- hexapeptide-5 amide acetate), enhance the production of collagen IV and collagen VII proteins in DEJs. It has also been established that a synergistic effect is achieved when administering ascorbic acid derivatives with those peptides when improving DEJ structural integrity, providing superior results in reducing skin pigmentation.

Accordingly, in a first aspect there is provided a composition for preventing and/or treating a skin hyperpigmentation disorder and/or cosmetic skin whitening, the composition including: a) N-acetyl-L-glutaminyl-L-alpha-aspartyl-L-valyl-L-histidine (acetyl tetrapeptide- 9) or salt thereof and N-acetyl-L-prolyl-L-prolyl-L-tyrosyl-L-leucine (acetyl tetrapeptide- 11) or salt thereof; and b) an ascorbic acid derivative, or salt thereof; wherein the weight ratio of N-acetyl-L-glutaminyl-L-alpha-aspartyl-L-valyl-L-histidine or salt thereof to N-acetyl-L-prolyl-L-prolyl-L-tyrosyl-L-leucine or salt thereof is in the range of from 10:1 to 1 :10.

In some embodiments, the active agents present in component a) are present in a weight ratio of N-acetyl-L-glutaminyl-L-alpha-aspartyl-L-valyl-L-histidine to N-acetyl-L-prolyl-L- prolyl-L-tyrosyl-L -leucine in the range of from 5:1 to 1:5, or optionally in a weight ratio of N-acetyl-L-glutaminyl-L-alpha-aspartyl-L-valyl-L-histidine to N-acetyl-L-prolyl-L-prolyl- L-tyrosyl-L-leucine in the range of from 2:1 to 1 :2.

In some embodiments, component b) is selected from the group consisting of 3-O-ethyl ascorbic acid, 2-O-ethyl ascorbic acid, ascorbyl glucoside, ascorbyl tetraisopalmitate, magnesium ascorbyl phosphate, sodium ascorbic phosphate, caprylyl 2-glyceryl ascorbate, 3-glyceryl ascorbate, bis-glyceryl ascorbate, hexyl 3-glyceryl ascorbate, myristyl 3-glyceryl ascorbate, and lauryl glyceryl ascorbate. In some embodiments, component b) is 3-O-ethyl ascorbic acid. In other embodiments, component b) is ascorbyl glucoside.

In some embodiments the amount of the active agents N-acetyl-L-glutaminyl-L-alpha- aspartyl-L-valyl-L-histidine and N-acetyl-L-prolyl-L-prolyl-L-tyrosyl-L-leucine that are present in component a) are each independently present in the combination in the range of from 0.0001% to 1% by weight of the composition, optionally from 0.0005% to 0.005% by weight of the composition, optionally from 0.001% to 0.002% by weight of the composition.

In some embodiments, the amount of component b) is present in the range of from 0.1% to 10% by weight of the composition, optionally from 0.2% to 8% by weight of the composition, optionally from 1% to 2% by weight of the composition.

In some embodiments, the composition does not contain any anti-hyperpigmentation agent other than components a) and b).

In some embodiments, the composition is for topical application.

In some embodiments, the composition comprises one or more pharmaceutically or cosmetically suitable excipients.

In some embodiments, the composition comprises one or more of the following: an antiinflammatory agent, a sunscreen agent, an antioxidant, a desquamation agent, and an exfoliating agent.

In some embodiments, the composition is in the form of a cream, lotion, serum, paste, wax, liquid, semisolid composition or a sprayable composition.

In a second aspect, there is provided a method of preventing, treating, and/or reducing a symptom associated with, a skin pigmentation disorder in a subject, or a method of inhibiting melanin production in epidermal melanocytes and/or promoting melanin degradation in the dermal fibroblasts of a subject, or a non-therapeutic method of skin whitening and/or lightening of a subject, comprising administering an effective amount of a composition as defined in the first aspect, to the subject.

In a third aspect, there is provided use of a composition of the first aspect as defined herein for the manufacture of a medicament for the prevention, treatment, and/or reduction of a symptom associated with, a skin pigmentation disorder, or for inhibiting melanin production in epidermal melanocytes and/or promoting melanin degradation in dermal fibroblasts.

In a fourth aspect, there is provided a composition of the first aspect as defined herein for use in prevention, treatment, and/or reduction of a symptom associated with, a skin pigmentation disorder, or for inhibiting melanin production in epidermal melanocytes and/or promoting melanin degradation in dermal fibroblasts.

In a fifth aspect, there is provided a composition for preventing and/or treating a skin hyperpigmentation disorder and/or cosmetic skin whitening, the composition including: a) N-acetyl-L-alanyl-L-leucyl- L-histidyl-L-lysyl-L-valyl-L-lysinamide

(acetyl sh-hexapeptide-5 amide acetate) or salt thereof; and b) ascorbic acid or a derivative and/or salt thereof.

In some embodiments, the component b) is selected from the group consisting of 3-O-ethyl ascorbic acid, 2-O-ethyl ascorbic acid, L-ascorbic acid, ascorbyl glucoside, ascorbyl tetraisopalmitate, magnesium ascorbyl phosphate, sodium ascorbic phosphate, caprylyl 2- glyceryl ascorbate, 3 -glyceryl ascorbate, bis-glyceryl ascorbate, hexyl 3 -glyceryl ascorbate, myristyl 3 -glyceryl ascorbate, and lauryl glyceryl ascorbate. In some embodiments, component b) is ascorbyl glucoside. In some embodiments, component b) is 3-O-ethyl ascorbic acid.

In some embodiments the amount of component a) is present in the range of from 0.0001% to 1% by weight of the composition, optionally from 0.0005% to 0.005% by weight of the composition, optionally from 0.001% to 0.002% by weight of the composition.

In some embodiments the amount of component b) is present in the range of from 0.1% to 10% by weight of the composition, optionally from 0.5% to 8% by weight of the composition, optionally from 1% to 2% by weight of the composition.

In some embodiments, the composition of the fifth aspect is for topical application.

In some embodiments, the composition of the fifth aspect comprises one or more pharmaceutically or cosmetically suitable excipients.

In some embodiments, the composition of the fifth aspect comprises one or more of the following: an anti-inflammatory agent, a sunscreen agent, an antioxidant, a desquamation agent, and an exfoliating agent.

In some embodiments, the composition of the fifth aspect is in the form of a cream, lotion, serum, paste, wax, liquid, semisolid composition or a sprayable composition.

In a sixth aspect, there is provided a method of preventing, treating, and/or reducing a symptom associated with, a skin pigmentation disorder in a subject, or a method of inhibiting melanin production in epidermal melanocytes and/or promoting melanin degradation in the dermal fibroblasts of a subject, or a non-therapeutic method of skin whitening and/or lightening of a subject, comprising administering an effective amount of N-acetyl-L-alanyl- L-leucyl-L-histidyl-L-lysyl-L-valyl-L-lysinamide (acetyl sh-hexapeptide-5 amide acetate) or salt thereof, to the subject.

In a seventh aspect, there is provided use of N-acetyl-L-alanyl-L-leucyl-L-histidyl-L-lysyl- L-valyl-L-lysinamide (acetyl sh-hexapeptide-5 amide acetate) or salt thereof, for the manufacture of a medicament for the prevention, treatment, and/or reduction of a symptom associated with, a skin pigmentation disorder, or for inhibiting melanin production in epidermal melanocytes and/or promoting melanin degradation in dermal fibroblasts.

In an eighth aspect, there is provided N-acetyl-L-alanyl-L-leucyl-L-histidyl-L-lysyl-L-valyl- L-lysinamide (acetyl sh-hexapeptide-5 amide acetate) or salt thereof for use in prevention, treatment, and/or reduction of a symptom associated with, a skin pigmentation disorder, or for inhibiting melanin production in epidermal melanocytes and/or promoting melanin degradation in dermal fibroblasts.

In a ninth aspect, there is provided a method of preventing, treating, and/or reducing a symptom associated with, a skin pigmentation disorder in a subject, or a method of inhibiting melanin production and/or promoting melanin degradation in the dermal fibroblasts of a subject, or a non-therapeutic method of skin whitening and/or lightening of a subject, comprising administering an effective amount of a composition of the fifth aspect as defined herein, to the subject.

In a tenth aspect, there is provided use of a composition of the fifth aspect as defined herein, for the manufacture of a medicament for the prevention, treatment, and/or reduction of a symptom associated with, a skin pigmentation disorder, or for inhibiting melanin production in epidermal melanocytes and/or promoting melanin degradation in dermal fibroblasts.

In an eleventh aspect, there is provided a composition of the fifth aspect as defined herein for use in prevention, treatment, and/or reduction of a symptom associated with, a skin pigmentation disorder, or for inhibiting melanin production in epidermal melanocytes and/or promoting melanin degradation in dermal fibroblasts.

In some embodiments the method, use or composition for use as defined herein when used to prevent and/or treat a skin hyperpigmentation disorder, the disorder is selected from the group consisting of skin hyperpigmentation, melasma, solar lentigo, nevus of Ota, nevus of Ito and congenital dermal melanocytosis.

Detailed Description

Definitions

Unless specifically defined otherwise, all technical and scientific terms used herein shall be taken to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., chemistry, biology, and the like).

The present disclosure refers to the entire contents of certain documents being incorporated herein by reference. In the event of any inconsistent teaching between the teaching of the present disclosure and the contents of those documents, the teaching of the present disclosure takes precedence.

It is to be understood that if any prior art publication is referred to herein, such reference does not constitute an admission that the publication forms a part of the common general knowledge in the art.

As used herein, the term “and/or”, e.g., “X and/or Y” shall be understood to mean either "X and Y" or "X or Y" and shall be taken to provide explicit support for both meanings or for either meaning.

As used herein, the term about, unless stated to the contrary, refers to +/- 10%, of the designated value.

Throughout this specification, unless specifically stated otherwise or the context requires otherwise, reference to a single step, composition of matter, group of steps or group of compositions of matter shall be taken to encompass one and a plurality (i.e. one or more) of those steps, compositions of matter, groups of steps or groups of compositions of matter. Thus, as used herein, the singular forms "a", "an" and "the" include plural aspects unless the context clearly dictates otherwise. For example, reference to "a" includes a single as well as two or more; reference to "an" includes a single as well as two or more; reference to "the" includes a single as well as two or more and so forth.

Unless otherwise indicated, terms such as "first," "second," etc. are used herein merely as labels, and are not intended to impose ordinal, positional, or hierarchical requirements on the items to which these terms refer. Moreover, reference to a “second” item does not require or preclude the existence of lower-numbered item (e.g., a “first” item) and/or a higher- numbered item (e.g., a “third” item).

As used herein, the phrase “at least one of’, when used with a list of items, means different combinations of one or more of the listed items may be used and only one of the items in the list may be needed. The item may be a particular object, thing, or category. In other words, “at least one of’ means any combination of items or number of items may be used from the list, but not all of the items in the list may be required. For example, “at least one of item A, item B, and item C” may mean item A; item A and item B; item B; item A, item B, and item C; or item B and item C. In some cases, “at least one of item A, item B, and item C” may mean, for example and without limitation, two of item A, one of item B, and ten of item C; four of item B and seven of item C; or some other suitable combination.

As used herein, the word “comprise” and other forms of the word, such as “comprising” and “comprises,” means including but not limited to, and is not intended to exclude, for example, other additives, components, integers, or steps.

As used herein, the term “subject” refers to any organism that is susceptible to a disease or condition. For example, the subject can be an animal, a mammal, a primate, a livestock animal (e.g., sheep, cow, horse, pig), a companion animal (e.g., dog, cat), or a laboratory animal (e.g., mouse, rabbit, rat, guinea pig, hamster). In one embodiment, the subject is a mammal. In one embodiment, the subject is human. In one embodiment, the subject is a non-human animal.

As used herein, the term “treating” includes alleviation of symptoms associated with a specific disorder or condition. As used herein, the term “prevention” includes prophylaxis of the specific disorder or condition.

As used herein, the term “anti-hyperpigmentation agent”, refers to an agent that possesses melanogenic inhibitory activity. Without wishing to be bound by theory, the inhibitory activity may, for example, act through inhibition of tyrosinase activity or its protein expression. Tyrosinase is a copper-containing enzyme present in plant and animal tissues that catalyses the production of melanin and other pigments from tyrosine by oxidation.

The disclosure also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations or any two or more of said steps or features.

Each embodiment of the present disclosure described herein is to be applied mutatis mutandis to each and every other embodiment unless specifically stated otherwise, or required otherwise by context.

Compositions

As discussed above, the present disclosure relates to compositions useful for preventing and/or treating a skin pigmentation disorder, and/or which are useful for non-therapeutic (e.g. cosmetic skin whitening and/or lightening), for example by inhibiting melanin production in epidermal melanocytes and/or promoting melanin degradation in the dermal fibroblasts. The compositions contain a specific peptide or combination of peptides which, when used in combination with ascorbic acid or a derivative and/or salt thereof, provides for unpexcetedly effective reduction of epidermal and dermal melanin.

The applicants have surprisingly found that anti-aging agents associated with enhancing skin smoothness and firmness, can be effective for reducing skin pigmentation and are particularly effective when used in combination with ascorbic acid derivatives. A correlation between the increased expression of collagens IV and VII, strengthening of dermal- epidermal junctions and reduced skin pigmentation has been identified. Taken together, it has been found that anti-melanogenic agents as disclosed herein promote the fibroblasts to produce collagens IV and VII to strengthen dermal-epidermal junctions, thereby inhibiting dermal factor-inducing melanogenesis in the melanocytes and leakage of melanosome from epidermis to dermis.

Advantageously, the present inventors have shown that N-acetyl-L-glutaminyl-L-alpha- aspartyl-L-valyl-L-histidine and N-acetyl-L-prolyl-L-prolyl-L-tyrosyl-L-leucine and 3-O- ethyl ascorbic acid work synergistically to inhibit melanogenesis activity in the epidermis to diminish melanin content in melanosome internalised fibroblasts and to increase collagen IV and collage VII expression, which strengthens dermal-epidermal junctions, in a skin model of dermal melanosis.

The composition of the first aspect contains the peptides N-acetyl-L-glutaminyl-L-alpha- aspartyl-L-valyl-L-histidine (acetyl tetrapeptide-9) and N-acetyl-L-prolyl-L-prolyl-L- tyrosyl-L-leucine (acetyl tetrapeptide- 11). These peptides can be prepared by any suitable route, and are known from, for example, (US2019/0336429).

This peptide can be prepared by any suitable means, of obtained from commercial sources. Commercial sources for obtaining this peptide includes, but not limited to BASF (Trade name: Replexium®).

The chemical structure of N-acetyl-L-glutaminyl-L-alpha-aspartyl-L-valyl-L-histidine is depicted below:

The chemical structure of N-acetyl-L-prolyl-L-prolyl-L-tyrosyl-L-leucine is depicted below:

In some embodiments, acetyl tetrapeptide-9 is used. In some embodiments, a salt form of acetyl tetrapeptide-9 is used.

In some embodiments, acetyl tetrapeptide- 11 is used. In some embodiments, a salt form of acetyl tetrapeptide- 11 is used. Suitable salts include those formed with organic or inorganic acids or bases. Exemplary acid addition salts include, but are not limited to, sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucuronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, and pamoate (i.e., l,l'-methylene-bis-(2 -hydroxy-3 -naphthoate)) salts. Exemplary base addition salts include, but are not limited to, ammonium salts, alkali metal salts, for example those of potassium and sodium, alkaline earth metal salts, for example those of calcium and magnesium, and salts with organic bases, for example dicyclohexylamine, N-methyl-D- glucomine, morpholine, thiomorpholine, piperidine, pyrrolidine, a mono-, di- or tri-lower alkylamine, for example ethyl-, tert-butyl-, diethyl-, diisopropyl-, triethyl-, tributyl- or dimethyl -propylamine, or a mono-, di- or trihydroxy lower alkylamine, for example mono- , di- or triethanolamine.

The composition of the first aspect includes an ascorbic acid derivative and/or salt thereof. In some embodiments an ascorbic acid derivative is used. In some embodiments an ascorbic acid salt is used. In some embodiments, a salt of an ascorbic acid derivative is used.

Examples of ascorbic acid derivatives and/or salts thereof include 3-O-ethyl ascorbic acid (3-EAA), 2-O-ethyl ascorbic acid, ascorbyl glucoside, ascorbyl tetraisopalmitate, magnesium ascorbyl phosphate, sodium ascorbic phosphate, caprylyl-2-glyceryl ascorbate, 3-glyceryl ascorbate, bis-glyceryl ascorbate, hexyl 3-glyceryl ascorbate, myristyl 3-glyceryl ascorbate and lauryl glyceryl ascorbate.

In some embodiments of the first aspect, the ascorbic acid derivative and/or salt thereof is 3- O-ethyl ascorbic acid (3-EAA). The chemical structure of 3-O-ethyl ascorbic acid is depicted below:

3-O-Ethyl ascorbic acid can be prepared by any suitable means, of obtained from commercial sources. Commercial sources for obtaining 3-O-ethyl ascorbic acid include, but are not limited to, Nippon Fine Chemical Co. Ltd. (Trade name: Ethyl ascorbic acid), Corum Inc. (Trade name: Et-VCTM), Kimika, LLC (Trade name: Neosome OL EAA), Sigma- Aldrich and Naturalis SRL (Trade name: Nio-VCS).

In some embodiments of the first aspect, the ascorbic acid derivative and/or salt thereof is ascorbyl glucoside. The chemical structure of ascorbyl glucoside is provided below:

Ascorbyl glucoside can be prepared by any suitable means, of obtained from commercial sources. Commercial sources for obtaining ascorbyl glucoside include, but are not limited to Sigma-Aldrich, DKSH (Trade name: AA2G), SpecChem (Trade name: SpecWhite 02), Macrocare (Trade name: V2G), Kimika, LLC (Trade name: iVit Glucoside C), etc.

The weight range of N-acetyl-L-glutaminyl-L-alpha-aspartyl-L-valyl-L-histidine (acetyl tetrapeptide-9) or salt thereof to N-acetyl-L-prolyl-L-prolyl-L-tyrosyl-L-leucine (acetyl tetrapeptide- 11) or salt thereof is in the range of from 10:1 to 1:10.

In some embodiments, the weight range of acetyl tetrapeptide-9 to acetyl tetrapeptide- 11 is in the range of from 10:1 to 1:10, or from 9:1 to 1:9, or from 8:1 to 1:8, or from 7:1 to 1 :7, or from 6:1 to 1:6, or from 5:1 to 1:5, or from 4:1 to 1 :4, or from 3:1 to 1:3, or from 2:1 to 1 :2, or about 1:1. In some embodiments, the weight ratio of acetyl tetrapeptide-9 to acetyl tetrapeptide- 11 is in the range of from 1 :1 to 1 :1.5, or from 1:1.1 to 1:1.3, or about 1 :1.2.

In some embodiments, acetyl tetrapeptide-9 is present in the composition in an amount in the range of from 0.00001% to 1% by weight of the composition, or from 0.00001% to 0.01% by weight of the composition, or from 0.00035% to 0.0013% by weight of the composition, or from 0.0009% to 0.0011% by weight of the composition, or about 0,001% by weight of the composition.

In some embodiments, acetyl tetrapeptide- 11 is present in the composition in an amount in the range of from 0.00001% to 1% by weight of the composition, or from 0.00001% to 0.01% by weight of the composition, or from 0.000525% to 0.00165% by weight of the composition, or from 0.0009% to 0.0014% by weight of the composition, or about 0,0012% by weight of the composition.

In some embodiments, acetyl tetrapeptide-9 is present in the composition in an amount in the range of from 0.00001% to 1% by weight of the composition, and acetyl tetrapeptide- 11 is present in the composition in an amount in the range of from 0.00001% to 1% by weight of the composition. In some embodiments, acetyl tetrapeptide-9 is present in the composition in an amount in the range of from 0.00001% to 0.01% by weight of the composition, and acetyl tetrapeptide- 11 is present in the composition in an amount in the range of from 0.00001% to 0.01% by weight of the composition. In some embodiments, acetyl tetrapeptide-9 is present in the composition in an amount in the range of from 0.00035% to 0.0013% by weight of the composition, and acetyl tetrapeptide- 11 is present in the composition in an amount in the range of from 0.000525% to 0.00165% by weight of the composition. In some embodiments, acetyl tetrapeptide-9 is present in the composition in an amount in the range of from 0.0009% to 0.0011% by weight of the composition, and acetyl tetrapeptide- 11 is present in the composition in an amount in the range of from 0.0009% to 0.0014% by weight of the composition. In some embodiments, acetyl tetrapeptide-9 is present in the composition in an amount of about 0.001% by weight of the composition, and acetyl tetrapeptide- 11 is present in the composition in an amount of about 0.0012% by weight of the composition.

In some embodiments, the composition comprises an ascorbic acid derivative and/or a salt thereof in a range of from 0.01 to 20% by weight of the composition, or from 0.1% to 10% by weight of the composition, or from 0.1% to 9%, or from 0.1% to 8%, or from 0.1% to 7%, or from 0.1% to 6%, or from 0.1% to 5%, or from 0.2% to 10%, or from 0.2% to 9%, or from 0.2% to 8%, or from 0.5% to 8%, or from 0.5% to 5%, or from 0.5% to 3%, or from 0.5% to 2.5%, or from 0.5% to 2%, or from 1% to 2%, or about 0.5%, or about 1%.

In some embodiments, the composition of the present disclosure comprises 3-O-ethyl ascorbic acid in an amount within the range of from 0.1% to 10% by weight of the composition, or from 0.1% to 9%, or from 0.1% to 8%, or from 0.1% to 7%, or from 0.1% to 6%, or from 0.1% to 5%, or from 0.2% to 10%, or from 0.2% to 9%, or from 0.2% to 8%, or from 0.5% to 8%, or from 0.5% to 5%, or from 0.5% to 3%, or from 0.5% to 2.5%, or from 0.5% to 2%, or from 1% to 2%. In some embodiments, the composition of the present disclosure comprises an amount of ascorbyl glucoside in the range of from 0.1 % to 10% by weight of the composition, or from 0.1% to 9%, or from 0.1% to 8%, or from 0.1% to 7%, or from 0.1% to 6%, or from 0.1% to 5%, or from 0.2% to 10%, or from 0.2% to 9%, or from 0.2% to 8%, or from 0.5% to 8%, or from 0.5% to 5%, or from 0.5% to 3%, or from 0.5% to 2.5%, or from 0.5% to 2%, or from 1% to 2%.

In some embodiments, acetyl tetrapeptide-9 is present in the composition in an amount in the range of from 0.0009% to 0.0011% by weight of the composition, and acetyl tetrapeptide- 11 is present in the composition in an amount in the range of from 0.0009% to 0.0014% by weight of the composition, and 3-O-ethyl ascorbic acid is present in an amount in the range of from 0.5% to 2% by weight.

In some embodiments, acetyl tetrapeptide-9 is present in the composition in an amount of about 0.001% by weight of the composition, acetyl tetrapeptide- 11 is present in the composition in an amount of about 0.0012% by weight of the composition, and 3-O-ethyl ascorbic acid is present in an amount of about 0.5% by weight, or about 1% by weight.In some embodiments, acetyl tetrapeptide-9 is present in the composition in an amount in the range of from 0.0009% to 0.0011% by weight of the composition, and acetyl tetrapeptide- 11 is present in the composition in an amount in the range of from 0.0009% to 0.0014% by weight of the composition, and ascorbyl glucoside is present in an amount in the range of from 0.5% to 2% by weight.

In some embodiments, acetyl tetrapeptide-9 is present in the composition in an amount of about 0.001% by weight of the composition, acetyl tetrapeptide- 11 is present in the composition in an amount of about 0.0012% by weight of the composition, and ascorbyl glucoside is present in an amount of about 0.5% by weight, or about 1% by weight.

In some embodiments, the composition comprises independently from 0.0005 % to 0.005% by weight of each of the active agents N-acetyl-L-glutaminyl-L-alpha-aspartyl-L-valyl-L- histidine (acetyl tetrapeptide-9) and N-acetyl-L-prolyl-L-prolyl-L-tyrosyl-L-leucine (acetyl tetrapeptide-11) as component a) and comprises from 0.2% to 8% of 3-O-ethyl ascorbic acid as component b) and optionally wherein the weight ratio of component a) to component b) is in the range of from 1 : 16,000 to 1 :40, or from 1 : 1 ,600 to 1 :400, or about 1 : 1 ,000. In another aspect, there is provided a composition for preventing and/or treating a skin hyperpigmentation disorder and/or cosmetic skin whitening, the composition including: a) N -acetyl-L-alany 1-L-leucy 1- L-histidy 1-L-ly sy 1-L-valy 1-L-ly sinamide

(acetyl sh-hexapeptide-5 amide acetate); and b) ascorbic acid or a derivative and/or salt thereof.

Advantageously, the present inventors have shown that N-acetyl-L-alanyl-L-leucyl- L- histidyl-L-lysyl-L-valyl-L-lysinamide (acetyl sh-hexapeptide-5 amide acetate) and ascorbyl glucoside, or salt thereof, work synergistically to inhibit melanogenesis activity in the epidermis, concomitantly to diminish melanin content in melanosome internalised fibroblasts in dermis, and increase collagen IV and collagen VII expression, which strengthens dermal-epidermal junctions, in a skin model of dermal melanosis.

In some embodiments, acetyl sh-hexapeptide-5 amide acetate is used. In some embodiments, a salt form of acetyl sh-hexapeptide-5 amide acetate is used.

The chemical structure of N-acetyl-L-alanyl-L-leucyl- L-histidyl-L-lysyl-L-valyl-L- lysinamide (acetyl sh-hexapeptide-5 amide acetate) is depicted below:

This peptide can be prepared by any suitable route, and is known from, for example, (US2022/0062145A1).

This peptide can be prepared by any suitable means, of obtained from commercial sources. Commercial sources for obtaining this peptide includes, but not limited to Lipotrue (Trade name: Versillin™). In some embodiments, acetyl sh-hexapeptide-5 amide acetate is present in the composition in an amount in the range of from 0.00001% to 1% by weight of the composition, or from 0.00001% to 0.01% by weight of the composition, or from 0.0004% to 0.0015% by weight of the composition, or from 0.00035% to 0.0013% by weight of the composition, or from 0.0009% to 0.0011% by weight of the composition, or about 0,001% by weight of the composition.

The composition includes an ascorbic acid derivative and/or salt thereof. In some embodiments an ascorbic acid derivative is used. In some embodiments an ascorbic acid salt is used. In some embodiments, a salt of an ascorbic acid derivative is used.

Examples of ascorbic acid derivatives and/or salts thereof include 3-O-ethyl ascorbic acid (3-EAA), 2-O-ethyl ascorbic acid, ascorbyl glucoside, ascorbyl tetraisopalmitate, magnesium ascorbyl phosphate, sodium ascorbic phosphate, caprylyl-2-glyceiyl ascorbate, 3-glyceryl ascorbate, bis-glyceryl ascorbate, hexyl 3-glyceryl ascorbate, myristyl 3-glyceryl ascorbate and lauryl glyceryl ascorbate.

In some embodiments, the ascorbic acid derivative and/or salt thereof is 3-O-ethyl ascorbic acid (3-EAA).

In some embodiments, the ascorbic acid derivative and/or salt thereof is ascorbyl glucoside.

In some embodiments, the composition of the present disclosure comprises 3-O-ethyl ascorbic acid in an amount within the range of from 0.1% to 10% by weight of the composition, or from 0.1% to 9%, or from 0.1% to 8%, or from 0.1% to 7%, or from 0.1% to 6%, or from 0.1% to 5%, or from 0.2% to 10%, or from 0.2% to 9%, or from 0.2% to 8%, or from 0.5% to 8%, or from 0.5% to 5%, or from 0.5% to 3%, or from 0.5% to 2.5%, or from 0.5% to 2%, or from 1% to 2%. In some embodiments, the composition of the present disclosure comprises an amount of ascorbyl glucoside in the range of from 0.1% to 10% by weight of the composition, or from 0.1% to 9%, or from 0.1% to 8%, or from 0.1% to 7%, or from 0.1% to 6%, or from 0.1% to 5%, or from 0.2% to 10%, or from 0.2% to 9%, or from 0.2% to 8%, or from 0.5% to 8%, or from 0.5% to 5%, or from 0.5% to 3%, or from 0.5% to 2.5%, or from 0.5% to 2%, or from 1% to 2%.

In some embodiments, acetyl sh-hexapeptide-5 amide acetate is present in the composition in an amount in the range of from 0.0009% to 0.0011% by weight of the composition, and 3-O-ethyl ascorbic acid is present in an amount in the range of from 0.5% to 2% by weight.

In some embodiments, acetyl sh-hexapeptide-5 amide acetate is present in the composition in an amount of about 0.001% by weight of the composition, and 3-O-ethyl ascorbic acid is present in an amount of about 0.5% by weight, or about 1% by weight.

In some embodiments, acetyl sh-hexapeptide-5 amide acetate is present in the composition in an amount in the range of from 0.0009% to 0.0011% by weight of the composition, and ascorbyl glucoside is present in an amount in the range of from 0.5% to 2% by weight.

In some embodiments, acetyl sh-hexapeptide-5 amide acetate is present in the composition in an amount of about 0.001% by weight of the composition, and ascorbyl glucoside is present in an amount of about 0.5% by weight, or about 1% by weight.

In some embodiments, the composition comprises independently from 0.0005 % to 0.005% by weight of each of the active agents N-acetyl-L-alanyl-L-leucyl- L-histidyl-L-lysyl-L- valyl-L-lysinamide (acetyl sh-hexapeptide-5 amide acetate) as component a) and comprises from 0.2% to 8% of ascorbyl glucoside as component b) and optionally wherein the weight ratio of component a) to component b) is in the range of from 1 :16,000 to 1 :40, or from 1 : 1 ,600 to 1 :400, or about 1 : 1 ,000.

In some embodiments of the compositions, the compositions may include other components that strengthen integrity of the dermal-epidermal junction. Examples include palmitoyl tripeptide-38, palmitoyl tripeptide-5, camosie, palmitoyl tripeptide-1, palmitoyl tetrapeptide-7, cyclotetrapeptide-24 aminocyclohexane carboxylate, tripeptide- 10 citrulline and palmitoyl pentapeptide-4. In some embodiments, the composition may include a retinoid and/or derivative thereof (e.g. triretinoin, retinol, retinaldehydes, retinyl esters, hydroxypinacolone retinoate).

In some embodiments, the composition may include niacinamide and/or a derivative thereof.

In some embodiments, the composition may include adenosine and/or a derivative thereof.

In some embodiments, the composition may include a Centella asiatica extract.

Whilst in some embodiments, other anti-hyperpigmentation agents and/or other skin lightening agents may be used with components a) and b), it has also been found that a comparatively simple formulation containing a) and b) as the only anti-hyperpigmentation agents provides unexpectedly effective results. Thus, in some other embodiments, the composition does not contain any anti-hyperpigmentation agent other than a) and b). In some embodiments, the composition does not contain any skin lightening agent other than a) and b). In some embodiments, the composition consists of or consists essentially of components a) and b), and one or more carriers and/or excipients.

The compositions of the present disclosure may be formulated as appropriate for any desired method of administration. For example, the composition of the present disclosure may be formulated as a topical composition, suitable to be applied topically to the skin.

The compositions of the present disclosure additionally may also contain other additives. For example, the compositions of the present disclosure may include known additional cosmetic or pharmaceutical agents. In some embodiments, the composition consists of or consists essentially of components a) and b), one or more carriers and/or excipients, and optionally one or more additional cosmetic or pharmaceutical agents which are not antihyperpigmentation agents or skin lightening agents.

The CTFA Cosmetic Ingredient Handbook, Second Edition, 1992 describes a wide variety of non-limiting cosmetic and pharmaceutical ingredients commonly used in the skin care industry, which may be suitable for use in the compositions of the present disclosure. Examples of suitable formulation ingredients include: antioxidants, binders, biological additives, buffering agents, colorants, thickeners, pearlescent agents, polymers, astringents, silicones, odor absorbers, gelling agents (e.g. hydrophilic or lipophilic gelling agents), fragrance, humectants, fillers, solvents, opacifying agents, skin-lightening agents, skin tanning agents, perfumes, conditioners, exfoliating agents, solubilisers (e.g. methylpropanediol), sunscreens, vitamins, colouring agents, pH adjusters (e.g. sodium hydroxide), preservatives (e.g. ethyhexylglycerin, phenoxyethanol, or hydroxyacetophenone), rheological modifiers, foaming agents, surfactants, emollients, natural extracts, essential oils, skin sensates, scalp soothing agents, and scalp healing agents.

The composition of the present disclosure may also comprise other skin conditioning agents, including, but not limited to, vitamins or vitamin derivatives and macro or micro capsules containing vitamins or their derivatives, cosmetic peptides, plant extracts, polyphenol compounds, sunscreen additives, sunscreen agents, sensorial additives or texturisers, natural extracts and essential oils.

In some embodiments, the composition of the present disclosure comprises one or more of the following: an anti-inflammatory agent, a sunscreen agent, an antioxidant, a desquamation agent, and an exfoliating agent. The compositions may be formulated with one or more other components that are advantageous for treatment of skin conditions, for example, hyaluronic acid and/or aloe vera gel.

In some embodiments, the composition comprises a pharmaceutically or cosmetically acceptable carrier or vehicle, and/or comprises one or more excipients.

The pharmaceutically or cosmetically acceptable vehicle may be any pharmaceutically or cosmetically acceptable vehicle, including pharmaceutically and/or dermatologically acceptable carriers, including surfactants. Pharmaceutically and/or dermatologically acceptable carriers are preferably compatible with skin, nails, mucous membranes, tissues and/or hair, and may include any conventionally used pharmaceutical or dermatological carriers meeting these requirements. The carriers and/or vehicles may aid in formulating the ascorbic acid or a derivative and/or a salt thereof, or anti-hyperpigmentation agent, into suitable pharmaceutical or cosmetic formulations. Such carriers may be readily selected by one of ordinary skill in the art. Carriers include, but are not limited to, emollients, emulsifiers, and rheological modifiers.

Emollients suitable for use in the vehicle or carrier for the compositions of the disclosure include, for example, Cl 2- 15 alkyl benzoates, PPG-3 myristyl ether, paraffinum liquidum and dimethicone. Emulsifiers suitable for use in the vehicle or carrier for the compositions of the disclosure include, for example, hydroxyethyl acrylate/sodium acryloyldimethyl taurate copolymer, Ceteareth-20 (cetostearyl alcohol, which is polyethylene glycol ether of cetearyl alcohol). Rheological modifiers suitable for use in the vehicle or carrier for the compositions of the disclosure include, for example, carbomer, glyceryl polyacrylate, and acrylates/C 10-30 alkyl acrylate crosspolymer. Other suitable carriers include, but are not limited to, mineral oil, liquid petroleum, white petroleum, propylene glycol, polyoxyethylene and/or polyoxypropylene compounds, emulsifying wax, sorbitan monostearate, polysorbate 60 (polyoxyethylene(20) sorbitan monostearate), cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol, sodium hyaluronate, hyaluronic acid, and water.

For topical administration, the compositions of the present disclosure may be provided in any suitable form, for example the compositions may be formulated as ointments, liquids, lotions, pastes, foams, sprays, serums, masks, gels, hydrogels or transdermal patches, leave- on and rinse-off lotions or toners, skin ampoules, skin cleansers, facial and eye make-up removers, leave on hair conditioners or hair vitamins, hair styling aids, shower gels/creams, toilet bars, antiperspirants, deodorants, depilatories, all colour cosmetics including, but not limited to, lipsticks and foundations, sunless tanners or sunscreen lotions, shampoos, conditioners, hair tonics, mousses, gels, liposomes, or other topically suitable forms.

Ointments and creams may, for example, be formulated with an aqueous or oily base with the addition of suitable thickening, emulsifying and/or gelling agents (e.g. emulsion of oil in water, emulsion of water in oil or emulsion of water in silicone). Lotions may be formulated with an aqueous or oily base and generally also contain one or more emulsifying agents, stabilising agents, dispersing agents, suspending agents, thickening agents, colouring agents or preservatives (e.g., methyl paraben, propyl paraben, phenoxyethanol). Ointments, creams, lotions and the like may also be formulated to comprise Cl-3-alkoxylated oils and waxes (e.g., ethoxylated vegetable oils, ethoxylated jojoba oil/wax, ethoxylated lanolin oil, ethoxylated coconut oil, ethoxylated cocoa butter).

Any cosmetic product that contains water (i.e., creams, gels, lotions, etc) has the potential for bacteria, yeast, and mould to grow, and may in some embodiments require a preservative. Exemplary preservatives for the compositions of the present disclosure include ethylhexylglycerine, phenoxyethanol, chlorphensin and sodium metabisulfite. Exemplary emollients include Cl 2- 15 alkyl benzoate, PPG-3 myristyl ether, isopropyl lauroyl sarcosinate, glycerin and bis-PEG-18 methyl ether dimethyl silane. An exemplary humectant is 1,2-hexane diol or betaine. An exemplary rheological modifier includes hydroxyethyl acrylate, sodium acryloyldimethyl taurate copolymer, or mixtures thereof.

In some embodiments, the compositions of the present disclosure comprise one or more of the following: water, an alkyl benzoate, PPG-3 myristyl ether, hydroxy aery late/sodium acryloyldimethyl taurate copolymer, ethylhexylglycerin, phenoxythanol, methylpropanediol, betaine, sclerotium gum, sodium polyacryloyldimethyl taurate, disodium EDTA, sodium hyaluronate, bis-PEG-18 methyl ether dimethyl silane, PEG- 12 dimethicone, glycerin, chlorphenesin, sodium metabisulfite, lactic acid, dicaprylyl carbonate, and polyacrylate crosspolymer-6.

The compositions of the present disclosure may be compositions suitable for topical use. In some embodiments, the composition is for topical application.

The topical use could for example be by way of incorporating components a) and b) in a leave-on or in a rinse off product that is applied to the human body primarily for improving skin appearance and general aesthetic benefit. “Skin" as used herein, is meant to include the external surface of mammals, especially humans and includes skin, scalp and hair. The use of the compositions of the present disclosure may for example be by way of incorporation in a leave-on composition.

The compositions can be in the form of a liquid, lotion, cream, foam, scrub, gel, soap bar, toner, or applied with an implement or via a face mask, pad or patch in any form of materials. Non-limiting examples of such compositions include leave-on skin lotions and creams in the forms of oil/silicone in water emulsion, water in oil/silicone emulsion and multiple emulsions, serums, shampoos, conditioners, shower gels, toilet bars, antiperspirants, deodorants, depilatories, lipsticks, foundations, mascara, sunscreen lotions, ointments, sprays, pastes, mousses, foams, gels, liposomes, or other topically suitable forms.

In some embodiments, the compositions of the present disclosure are in the form of a cream, lotion, paste, wax, liquid, semisolid composition or a sprayable composition

In the case where the compositions are in the form of a spray, the carri^r^isvstfitably water, and the compositions may further comprise preservative such as benzyl alcohol (for example, in an amount of from 0.1 - 5 wt%), co-solvents such as propylene glycol (for example, in an amount of from 1 - 20 wt%), and surfactants such as PEG-40 hydrogenated castor oil (for example, in an amount of from 1 - 20 wt%).

In the case where the compositions are in the form of a lotion, they may for example further comprise water in the presence of a suitable emulsifying agent. Examples of suitable emulsifying agents are ceteareth-20, PEG- 100 stearate, glyceryl stearate, sodium cetearyl sulfate. The compositions may further comprise emollients in the form of esters and/or hydrocarbons and/or silicones and/or plant oils and fragrances, preservatives (such as benzyl alcohol at levels of, for instance, 0.1 10%) and humectant and/or excipients (such as glycerol, for instance at levels of 0.5 - 10% and cyclomethicone at levels of between 0.1 - 20%).

When the compositions are in the form of an ointment, they may for example further comprise a conventional ointment base to which the active ingredient is added. The ointment base may be a paraffin, such as soft paraffin, or a combination of soft and liquid paraffin. Other ointment bases may also be used such as polyalkylene glycol (such as polyethylene or polypropylene glycol) base. Other possible components of an ointment composition include emulsifying wax (for example, in an amount of between 1 - 40%, preferably 5 - 40%), and optionally one or more preservatives.

When the compositions are in the form of a liquid soap, any known liquid soap may be used. A preferred liquid soap with key composition saponified fatty acids derived from palm kernel oil or coconut oil. The pH of the liquid soap base may be adjusted to that which is suitable for topical use.

When the compositions are an anhydrous cream, any suitable cream base may be used. A preferred cream base is petrolatum cream and/or natural plant oils and waxes with introduction of consistency factors.

When the compositions are a shampoo or conditioner, they may for example further comprise one or more of a surfactant, a thickening agent, a pH adjuster/buffer, an aesthetic additive, water, a conditioner, a preservative and moisturisers/vitamins in addition to the active ingredients. Shampoos may also include other active components known to those skilled in the art as having some advantage when included in a shampoo formula or in a medicinal combination for treating a hyperpigmentation disorder. A shampoo may comprise a primary surfactant to provide flash foam for cleaning the hair by removing dirt and other impurities. A secondary surfactant may be included to provide stable foam and to reduce the harshness of the primary surfactant. A surfactant may be used that includes a charged, hydrophilic head group and a long, hydrophobic alkyl chain tail. Surfactants are configured to reduce surface tension of an interphase between dirt and hair and allowing the dirt to be transported into an aqueous medium to be rinsed free from the hair and scalp. Examples of surfactants that may be contained in a shampoo in accordance with certain embodiments include sodium laureth sulphate, ammonium laureth sulfate, and sodium cocoyl isethionate. Examples of co-surfactants include cocamide MEA and cocoamidopropyl betaine. Suitable thickening or suspending agents include carbomer and PEG 150 distearate. The thickening agent may be included to stabilise the shampoo during storage and/or to prevent the settling or dumping of pigments and silicone. Suitable pH adjusters or buffers include citric acid, tartaric and sodium hydroxide. The pH adjuster or buffer is configured to cause the shampoo to be gentle to the skin. A lower pH may cause hair to be compact and to shine and to protect the surfactant from hydrolysis, and as such, the pH

Advantageously, components a) and b) can be prepared in stabilised topical composition and applied on the skin.

Accordingly, components a) and/or b) may for example be provided in an encapsulated format within the compositions. Advantageously, the encapsulation technology may be used to protect and enhance transdermal delivery. Suitable encapsulation technologies may be selected from the group consisting of liposomes, niosomes, nano-emulsions, and cyclodextrin inclusion technologies. Suitable encapsulation technologies may be produced by any means known in the art and are not limited to the present disclosure.

An encapsulation material may for example comprise an emulsifier and a co-emulsifier. The emulsifier may be selected from glyceryl esters of different fatty chains, including, but not limited to, glyceryl citrate, glyceryl lactate, glyceryl myristate, glyceryl laurate, glyceryl linoleate, glyceryl oleate or any combination thereof or any mixed glyceryl esters of fatty acids including citrate, lactate, myristate, laurate, linoleate and/or oleate. In one embodiment, the emulsifier is glyceryl citrate/lactate/linoleate/oleate (a mixed ester of citric acid, lactic acid, linoleic acid and oleic acid with glycerol). In some embodiments, the co-emulsifier is a di or polyglyceryl ester selected from, but not limited to, diglyceryl monooleate, polyglyceryl monooleate, polyglyceryl monolinoleate, or any combination thereof. In particular embodiments, the co-emulsifier is diglyceryl monooleate.

Where used, the stabiliser can for example be selected from organic substances of any kind that may solubilise the emulsifiers. Suitable examples include alcohols or glycols, including, but not limited to, ethanol, isopropanol, propylene glycol, pentylene glycol, caprylyl glycol, especially pentylene glycol.

Therapeutic and Non-Therapeutic Methods and Uses

The present disclosure further relates to methods and uses for treating skin pigmentation disorders, and for reducing melanin in dermal fibroblasts, using the compositions and agents described herein. The compositions may be used to treat, control, prevent and ameliorate skin hyperpigmentation in a safe and non-invasive way. As discussed above, it has now been discovered that fibroblast cells in the dermal layer of skin are involved in uptake of melanosomes, and that melanin can be retained in fibroblast cells and may be degraded at a slower rate, thus contributing to a persistently darker skin tone. Advantageously, the novel findings by the present inventors have emerged as a strategy for treating hyperpigmentation by inhibiting dermal factor inducing melanogenesis in the melanocytes through strengthening dermal-epidermal junctions. Accordingly, it has been identified that ascorbic acid derivatives such as 3-O-ethyl ascorbic acid and ascorbyl glucoside, in combination with certain peptides, provides superior anti-hyperpigmentation properties, providing superior results in diminishing melanin content in fibroblast cells. For example, 3-O-ethyl ascorbic acid and N-acetyl-L-glutaminyl-L-alpha-aspartyl-L-valyl-L-histidine and N-acetyl-L- prolyl-L-prolyl-L-tyrosyl-L-leucine have been shown to synergistically inhibit melanogenesis activity in the epidermis and reduced melanin content in melanosome internalised fibroblasts in a human skin model of dermal melanosis.

In addition to diminishing melanin content in fibroblast cells, the compositions comprising ascorbic acid derivatives and/or salts thereof and peptides, may also act to promote depigmentation via alternative mechanisms, including, but not limited to, autophagic activity, which accelerates melanin clearance at both epidermal and dermal skin layers, and phagocytic activity by melanophages, which aid in melanosome clearance at both epidermal and dermal skin layers.

Melanosomes were shown to be internalised by dermal fibroblasts via an actin-dependent endocytosis process. In normal skin physiology, internalised melanosomes in keratinocytes can be differentiated and shed or removed via exfoliating agents like glycolic acid, salicylic acid etc. However, unlike keratinocytes in the epidermis, internalised melanosomes in fibroblasts are retained within the dermis and degrade at a slower rate which may contribute to persistent darker skin spots. The present inventors have thus determined that promoting the clearance of melanin in fibroblasts and strengthening the dermal-epidermal junctions through increased collagen IV and collagen VII expression has emerged as a new strategy for treating hyperpigmentation.

Advantageously, application of the composition of the present disclosure in a method as defined herein demonstrates minimal negative effects on tissue viability of reconstituted skin and cell viability of the fibroblasts, which indicates that the inhibitory activity to achieve melanin reduction mediated by the compositions of the present disclosure are not due to cytotoxic effects.

Accordingly, in one aspect, there is provided a method of preventing, treating, and/or reducing a symptom associated with, a skin pigmentation disorder in a subject, comprising administering an effective amount of a composition as defined herein to the subject.

There is also provided a method of reducing melanin in dermal fibroblasts of a subject, comprising administering an effective amount of a composition as defined herein to the subject.

In another aspect, there is provided a use of a composition as defined herein for the manufacture of a medicament for the prevention, treatment, and/or reduction of a symptom associated with, a skin pigmentation disorder in a subject.

There is also provided a use of a composition as defined herein for the manufacture of a medicament for reducing melanin in dermal fibroblasts of a subject.

In another aspect, there is provided a composition as defined herein for use in the prevention, treatment, and/or reduction of a symptom associated with, a skin pigmentation disorder. 1

In another aspect, there is provided a composition as defined herein for use in reducing melanin in dermal fibroblasts.

In some embodiments, the composition is for promoting skin lightening. In some embodiments, the composition is for treating skin hyperpigmentation. In some embodiments, the composition enhances the dermal-epidermal junction through promoting protein expression of collagen IV and/or collagen VII.

The composition of the present disclosure can be used to treat skin pigmentation disorders including, but not limited to, skin hyperpigmentation, melasma, solar lentigo, nevus of Ota, nevus of Ito and congenital dermal melanocytosis, post-inflammatory hyper- or hyperpigmentation, progressive pigmentary purpura, a canthosis nigricans, and incontinentia pigmenti.

In some embodiments, the skin pigmentation disorder is selected from the group consisting of skin hyperpigmentation, melasma, solar lentigo, nevus of Ota, nevus of Ito, and congenital dermal melanocytosis.

In some embodiments, the composition is applied topically, to the skin of a subject.

In some embodiments, the subject is a human subject. In some embodiments the subject is an adult subject. In some embodiments the subject is a human subject of at least 30 years age, at least 35 years age, at least 40 years age, at least 45 years age, at least 50 years age, at least 55 years age, or at least 60 years age. In some embodiments, the subject is male. In some embodiments, the subject is female.

The present disclosure also relates to non-therapeutic methods for lightening skin, for example, the composition of the present disclosure may be applied in cosmetic uses to lighten skin colour.

Accordingly, in one aspect, there is provided a non-therapeutic method of skin lightening of a subject, comprising administering an effective amount of a composition as defined herein to the subject.

In some embodiments, the composition is applied topically to the skin of the subject.

The compositions of the present disclosure may for example be a topical composition that is to be applied on an external surface of a subject. In particular embodiments, the external surface may be one or more of the scalp, face, arms, legs or torso of a subject. Advantageously, the compositions of the present disclosure demonstrate effective skin bioavailability which allows skin penetration to lighten skin tone, which has been demonstrated in human skin penetration and efficacy studies and the biomimetic skin model of dermal melanosis.

In some embodiments, the composition of the present disclosure is administered once daily, or twice daily, or three times daily, or four times daily, for example for a period of time sufficient to bring about a desired level of improvement in hyperpigmentation. Alternatively, the compositions may be used less frequently if that will provide the required effects. For example, it may be applied once every other day or once, twice, three or four times a week, or on an ad hoc or as needed basis.

For example, a user may topically administer a composition of the present disclosure directly to the skin where decreased pigmentation is desired by gently massaging the composition of the present disclosure into the desired area. In some embodiments, the composition of the present disclosure is left on the skin or other area where decreased pigmentation is desired between applications.

Topical application of the compositions of the present disclosure may continue for any suitable period of time. For example, within a few days to a few weeks, a user may notice a reduction in pigmentation. Advantageously, topical treatment with the composition of the present disclosure may demonstrate anti-hyperpigmentation effects by significantly reducing the skin tone of identified hyperpigmentation in, for example, as short as 4- weeks of product application.

It should be appreciated that the frequency with which the compositions of the present diclosure should be applied will vary depending on the desired effect. In some embodiments, the degree of therapeutic and/or cosmetic enhancement will vary and may be directly proportional with the total amount of compositions used. Topical application of the compositions may be administered in any amount and at any frequency sufficient to bring about the desired level of improvement in the hyperpigmentation.

For example, the compositions may be applied in amounts ranging from about 1 ppm to about 10,000 ppm of peptides, or from about 2 ppm to about 2,000 ppm, or from about 3 ppm to about 1,000 ppm, or from about 4 ppm to about 500 ppm, or from about 5 ppm to about 100 ppm, or from about 5 ppm to about 50 ppm, or from about 10 ppm to about 25 ppm, or from about 10 ppm to about 20 ppm.

When topically applied, the compositions of the present disclosure may be applied to the area to be treated, for example, the face, by spraying, dabbing, swabbing, rubbing or massaging. For example, in one embodiment, a composition of the present disclosure may be provided in a liquid, lotion, cream, foam, serum, ointment, foundation, scrub, gel, soap bar, toner, or applied with an implement or via a face mask, pad or patch in any form of materials or other leave-on product formulation, which preferably may be applied on at least a daily basis.

Preparation of Compositions

The compositions of the present disclosure may be prepared by any suitable method known to a person skilled in the art.

In some embodiments, the compositions are prepared as compositions suitable for topical use.

In some embodiments, the compositions are prepared as emulsions. For example, the compositions of the present disclosure may be prepared by combining an aqueous phase and a lipid phase, followed by homogenisation.

The compositions may comprise suitable solvents or agents which act to enhance the absorption of other ingredients into the skin, humectants and anti-irritants, gums for producing a gel base that may have skin smoothing and soothing properties, emulsion stabilising and viscosity controlling agents, agents which act to prevent deterioration of the formulation, moisture attracting agents, emollients, hair/skin-conditioning agents, agents which help improve the overall texture and finish of skin care formulae, gelling agents, preservatives, agents which reduce the appearance of large pores, age spots and hyperpigmentation, including lactic acids, and subjecting this mixture to homogenisation.

In some embodiments, the compositions are prepared as compositions suitable for topical use, by combining suitable solvents which may enhance the absorption of other ingredients into skin, such as methylpropanediol, humectants and anti-irritants, such as betaine, natural gums such as sclerotium gum, emulsion stabilizing and viscosity agents such as sodium polyacryloyldimethyl taurate, anti-deterioration, agents such as disodium EDTA, moisture attractants, such as sodium hyaluronate, water-dispersible silicones, such as bis-PEG-18 methyl ether dimethyl silane, which may act as emollients and with added foam-boosting properties, hair/skin conditioning agents, such as PEG- 12 dimethicone, other suitable emollients selected from Cl 2- 15 alkyl benzoate, PPG-3 myristyl ether and glycerin, gelling agents selected from hydroxy ethyl acrylate/sodium acryloyldimethyl taurate copolymer, agents which reduce the appearance of large pores, age spots and hyperpigmentation, including lactic acids, preservatives selected from ethylhexylglycerine and phenoxyethanol, antioxidants including sodium metabisulfite, biocides including chlorphensin with deionised water, and subjecting this mixture to homogenisation.

Those skilled in the art will appreciate that the disclosure herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the disclosure includes all such variations and modifications.

Examples

The present disclosure is further illustrated by the following non-limiting examples.

Example 1: Investigation of dermal-epidermal junction integrity, its role in skin hyperpigmentation and identification of relevant biomarkers.

1.1 Objective

To understand the role of dermal-epidermal junction (DEJ) integrity in skin hyperpigmentation, and to identify biomarkers that may be useful indicators of neuregulin- induced skin hyperpigmentation.

1.2 Methodology

1.2.1 Investigation of the role of DEJ integrity in skin hyperpigmentation by comparing old and young skin

Four ex vivo skin explants from two young and two old donors were obtained from Genoskin® for this study. The skins were equilibrated in the provided tissue culture media at 37°C under 5% CO2 for one hour prior to commencing the experiment. After equilibration, the culture medium was replaced with fresh culture medium. Neuregulin (R&D Systems, Abingdon, UK), a dermal melanogenic factor, was then added to the explant cultures at final concentration of 500 ng/ml and incubated overnight. The culture media was then replaced with fresh maintenance medium and neuregulin was again added to the culture medium. The culturing process was repeated three times before the skin explants were harvested for melanin quantification using a Fontana-Masson staining kit (Abeam, Cambridge, UK).

1.2.2 Identification of DEJ integrity biomarkers that correlate with neuregulin-induced hyperpigmentation in ex vivo young skin treated with metalloproteinase-3 (MMP-3)

Ex vivo skin explants from two young donors were obtained from Genoskin® for this study. The explants were equilibrated in the provided proprietary tissue culture medium for one hour prior to commencing the experiment. After equilibration, culture medium was replaced with fresh culture medium. A concentration of 500 ng/mL of neuregulin and 500 ng/ml of matrix metalloproteinase-3 (MMP-3) (R&D Systems, Abingdon, UK) were then added to the explant cultures and incubated overnight. The process was repeated three times before the skin explants were harvested for melanin quantification using Fontana-Masson histological staining. Collagen IV and collagen VII proteins were also stained using an immunofluorescent staining method and target protein expression levels were quantified using NIS-Elements imaging software (Nikon, Japan).

1.3 Results and Discussion

It was hypothesised that older skin has a higher prevalence of dermal melanogenic factor- induced skin hyperpigmentation because of impairment of DEJs that act to facilitate the crossing of neuregulin to exert its melanogenesis-promoting effect in the melanocytes in the epidermis. It was reported that the DEJ proteins are significantly reduced in older skin (Langton et al., 2016). In order to investigate if DEJ impairment is playing a role in promoting dermal melanogenic factor-induced skin hyperpigmentation, both old and young skin were treated with neuregulin, which is one of the dermal melanogenic factors, in the culture media to induce skin hyperpigmentation. Table 1 shows that old skins treated with neuregulin have a significantly higher melanin intensity than their young skin counterpart. This indicates that the integrity of DEJ does play a role in regulating the movement of dermal factors into the epidermis. Biomarkers involved in DEJ impairment that correlate with neuregulin-induced skin hyperpigmentation were determined. MMP-3 (stromelysin- 1), can degrade a broad range of substrates including extracellular matrix components, collagen IV and collagen VII in the DEJ. MMP-3 was used in this study to induce DEJ impairment in young skin to facilitate an investigation of potential biomarkers. The evidence provided in Table 1.2 indicates that the test skin sample treated with MMP-3 and induced by neuregulin show an increase in melanin levels. It was also discovered that collagen IV and VII expression was significantly reduced by MMP-3. This observation validated that these two proteins can be used as biomarkers to assess DEJ integrity.

Table 1.1. Effect of neuregulin on melanin intensity of old and young human skin explants.

Melanin level (% of control) Old versus Young

Treatment Old skin Young skin (P value)

(50-70 years old) (25-35 years old)

Untreated Control 100 100

<0.05*

Neuregulin-treated 134.21 ± 7.91^### 103.85 ± 1.32

Data are expressed in mean ± S.E.M of two independent experiments (n=2). ###p<0.001 when compared to untreated control by students t-test. Statistical significance was indicated by a P value of *p<0.05 when comparing between old and young skin by students t-test.

Table 1.2. Effect of MMP-3 on neuregulin-induced melanin levels and expression of collagen IV and collagen VII in young skin explants.

Expression level (% of untreated control)

Treatment Neuregulin-induced

Collagen IV Collagen VII

Melanin

Untreated Control 100 100 100

MMP3 -treated 140.61 ± 11.60* 57.32 ± 7.32**** 59.68 ± 4.01****

Data are expressed in mean ± S.E.M of two independent experiments (n=2). Statistical significance was indicated by a P value of *p<0.05, **p<0.01, ****p<0.0001 when compared to untreated control using One-Way ANOVA. 1.4 Conclusion

DEJ integrity plays a significant role in neuregulin-induced skin hyperpigmentation and collagen IV and collagen VII are useful biomarkers to assess DEJ integrity.

Example 2: Investigation of the combination of acetyl tetrapeptide-11 and acetyl tetrapeptide-9, or acetyl SH-hexapeptide-5 amide acetate in reducing melanin levels in skin with impaired DEJ

2.1 Objective

To investigate the effects of acetyl tetrapeptide-11, acetyl tetrapeptide-9 and acetyl sh- hexapeptide-5 amide acetate in reducing melanin in skin explants with impaired DEJ. 2.2 Methodology

2.2.1 Formulation preparation

The test and control formulations were prepared by premixing the ingredients of each Phases A and B (Table 2.1), then combining the Phase A and Phase B solutions and homogenising at 2500 rpm for 5 minutes. Then, the premixed ingredients of Phase C was added into the solution containing both Phases A and B and stirred at 100-200 rpm for 30 minutes.

Table 2.1. Formulation ingredients listed by phase

Phase Ingredient Formula 1 (%) Formula 2 (%) Formula 3 (%)

A Deionised water qs 100 qs 100 qs 100

Acetyl tetrapeptide- 11 - 0.0012

Acetyl tetrapeptide-9 - 0.001

Acetyl SH-Hexapeptide-5 - - 0.0005

Amide Acetate

B Cl 2- 15 Alkyl Benzoate 2 2 2

PPG-3 Myristyl Ether 3 3 3

Hydroxyethyl Acrylate/Sodium 2 2 2

Acryloyldimethyl Taurate

Copolymer (and) Squalane

(and) Polysorbate 60

C Ethylhexylglycerin 0.3 0.3 0.3

Phenoxyethanol 0.4 0.4 0.4

2.2.2 Topical treatment of formulation on the neuregulin-induced hyperpigmentation in ex vivo skin with impaired DEJ

Ex vivo skin explants from two young donors were obtained from Genoskin® for this study. The skins were equilibrated in the provided tissue culture medium for one hour prior to commencing the experiment. After equilibration, the culture medium was replaced with fresh culture medium. Then, 500 ng/ml of neuregulin (dermal melanogenic factor) and 150 ng/ml of metalloproteinase-3 (MMP-3, to degrade DEJ) were added to the culture medium and the explants incubated overnight. The culture medium was replaced with fresh culture medium supplemented with 500 ng/ml of neuregulin and 500 ng/ml of MMP-3. Topical treatments listed in Table 2.1 were applied to the skin and left for four hours at 37°C under 5% CO2. After four hours, the treatment product was washed off with phosphate buffered saline (PBS) and the skin explants were incubated overnight at 37°C under 5% CO2. The product treatment and wash step were repeated without MMP-3 in the next day before the skin explants were harvested for melanin quantification using Fontana-Masson staining.

Collagen IV and Collagen VII were also stained using an immunofluorescent staining method and protein expression was quantified using NIS-Elements imaging software (Nikon, Japan).

2.3 Results and Discussion As shown in Table 2.2, Formula 2 (containing active agents acetyl tetrapeptides 9 and 11) and Formula 3 (containing active agent acetyl SH-hexapeptide 5 amide acetate) significantly reduced the neuregulin-induced melanin levels in skin explants with impaired DEJ when compared with its negative control formulation base (Formula 1). Concomitantly, significant elevation of collagen VII expression was observed in the Formula 2 and Formula 3 -treated skin samples. When compared to the control sample (Formula 1), a correlation between the melanin reduction effect observed with each Formula 2 and Formula 3, and collagen VII expression was established. Some elevation of collagen IV expression was also observed in the Formula 2 and Formula 3 -treated skin samples when compared to the control sample (Formula 1).

Table 2.2. Comparison of active agents, Formula 2 and Formula 3 (AA2G and ASH5AA) efficacy for melanin reduction in skin with neuregulin-induced skin hyperpigmentation and the effect on collagen IV and VII expression in a skin explant with DEJ impairment.

Expression level (% of Formula 1) Treatment

Neuregulin-induced Melanin Collagen IV Collagen VII

Formula 1 100 100 100

Formula 69.90 ± 7.67* 125.49 ± 44.10 149.64 ± 8.64*

Formula ? 68.26 ± 2.51* 131.19 ± 33.85 167.53 ± 20.90*

Data are expressed in mean ± S.E.M of two independent experiments (n=2). Statistical significance was indicated by a P value of *p<0.05, **p<0.01, ****p<0.0001 when compared to Formula 1 using One-Way ANOVA.

2.4 Conclusion

The combination of active agents acetyl tetrapeptide- 11 and acetyl tetrapeptide-9, or acetyl sh-hexapeptide-5 amide acetate are useful active ingredients in reducing skin melanin levels and to promote significant collagen VII expression in human skin explants obtained from young donors with MMP-3 induced DEJ impairment.

Example 3: Evaluation of the efficacy a topically administered aqueous composition comprising of active agents 3-EAA, acetyl tetrapeptide-11 and acetyl tetrapeptide-9 for the treatment of hyperpigmentation in clinical and ex vivo samples 3.1 Objective

To investigate the efficacy of a topical formulation comprising of active agents 3-O-ethyl ascorbic acid (3-EAA), acetyl tetrapeptide- 11 and acetyl tetrapeptide-9 for the purpose of reducing hyperpigmentation as determined by measuring the melanin levels in neuregulin- induced skin hyperpigmentation of ex vivo human skin explants with MMP-3-induced DEJ impairment and in treating the skin of clinical trial participants.

3.2 Methodology

3.2.1 Formulation preparation

The test and control formulations were prepared by premixing the Phase A ingredients (Table 3.1) by adding each ingredient sequentially into the aqueous solution and stirring at 100-200 rpm for 15 minutes until fully dissolved. All ingredients in Phase B were mixed until fully dissolved, then added into the Phase A solution and stirred at 100-200 rpm for 5 minutes. Lastly, the preservatives (aminomethyl propanol and chlorphenesin, glycerin, phenoxyethanol) were added to the Phase A/B solution and stirred at 100-200 rpm for 5 minutes.

Table 3.1. Formulation ingredients listed by phase

3.2.2 Topical treatment of formulation on the neuregulin-induced hyperpigmentation in ex vivo skin with impaired DEJ

This experiment was performed as described in method 2.2.2 in the present specification using the formulations listed in Table 3.1.

3.2.3 Clinical trial

The efficacy of product formulation 4 and 5 for the reduction of hyperpigmentation was assessed in a clinical trial with 12 trial participants. The participants were women with an age range of 41 to 54 years old and having obvious skin pigmentation. In this study, recruited participants were instructed to apply formula 5 topically twice daily for one month. The skin tone of the defined skin area of treatment was recorded photographically using a VISIA Complexion Analysis system (Canfield, USA) at baseline (To), and after 4 weeks from the first day of product application (T4_W). Additionally, the skin tone of the pigmented treatment zone was marked and further analysed by determining individual topology angle (ITA°) differences between a representative clear skin region and compared to the pigmented skin treatment zone of the same subjects. A lower A ITA° indicated lightening of the treatment zone with improved skin homogeneity.

3.3 Results and Discussion

As shown in Table 3.2, topical treatment with Formula 5 significantly reduced the melanin levels of neuregulin-induced skin hyperpigmentation in skin explants with MMP-3 -induced DEJ impairment when compared to an untreated control. Concomitantly, a significant increase of collagen IV and VII expression was observed in skin explants treated with Formula 5 as compared to untreated skin explants. An inverse correlation of melanin levels compared to collagen IV and VII levels after treatment was observed.

Table 3.2. Evaluation of Formula 5 (3-EAA, AT-11 and AT-9) efficacy for melanin reduction in skin with neuregulin-induced skin hyperpigmentation and the effect on collagen IV and VII expression in a skin explant with DEJ impairment. Expression level (% of untreated control)

Treatment Neuregulin-induced „ „ _r, , . ...

, . Collagen IV Collagen VII

Melanin

Untreated control 100 100 100

Formula 4 91.56 ± 19.86 112.23 ± 3.18 126.41 ± 17.81*

Formula s 64.96 ± 10.02** 156.83 ± 29.07**** 182.52 ± 51.79****

Data are expressed in mean ± S.E.M of two independent experiments (n=2). Statistical significance was indicated by a P value of *p<0.05, **p<0.01, ****p<0.0001 when compared to untreated control using One-Way ANOVA.

To evaluate the anti-hyperpigmentation efficacy in a clinical setting, results illustrated in Table 3.2 showed that topical treatment of Formula 5 reduced the skin tone difference between the established pigmented skin test zone as compared to a clear control skin region. Skin tone was measured immediately prior to topical administration and 4 weeks post administration of Formula 5.

Table 3.3 Effects of formula 5 on lightening of pigmented spot which determined by difference of Individual Topology Angle (A ITA°) between clear skin region and pigmented skin region of same subject.

Formula 5 Duration

Baseline (To) 4-weeks (Tiw)

A ITA⁰ 2.14 ± 0.43 1.57 ± 0.38

Statistical analysis (vs To) - P < 0.05*

% Improvement - 26.6

All values are expressed as mean ± SEM of group of test trial participant (n=12). Statistical significance was indicated by a P value of *p<0.05, when compared to baseline (To) by applying the Wilcoxon Matched Pairs Signed Rank Test.

3.4 Conclusion

Topical administration of Formula 5, comprising of 3-EAA, AT-11 and AT-9 was efficacious in reducing melanin levels in human skin explants with neuregulin-induced skin hyperpigmentation in DEJ impairment as compared to an untreated control. This combination of active agents is a useful treatment for skin hyperpigmentation and has been shown to lighten skin tone in human clinical trial participants after 4 weeks.

Example 4: Evaluation of the efficacy and reproducibility of a topically administered aqueous composition comprising of active agents 3-EAA, acetyl tetrapeptide- 11 and acetyl tetrapeptide-9 for the treatment of hyperpigmentation in skin explants

4.1. Objective

To evaluate the reproducibility of and validate the anti-hyperpigmentation activity of a topically administered formulation comprising of active agents: 3-EAA, AT-11 and AT-9, in ex vivo human skin explant samples with MMP-3 -induced DEJ impairment. 4.2. Methodology

4.2.1 Formulation preparation

The test and control formulations were prepared by premixing the Phase A ingredients (Table 4.1) by adding each ingredient sequentially into the aqueous solution and stirring at 100-200 rpm for 15 minutes until fully dissolved. All ingredients in Phase B were mixed until fully dissolved, then added into the Phase A solution and stirred at 100-200 rpm for 5 minutes. Lastly, the preservatives (chlorphenesin, and phenoxyethanol) were added to the Phase A/B solution and stirred at 100-200 rpm for a further 5 minutes.

Table 4.1. Formulation ingredients listed by phase

4.2.2 Topical treatment of formulation on the neuregulin-induced hyperpigmentation in ex vivo skin with impaired DEJ

This experiment was performed as described in method 2.2.2 in the present specification using the formulations listed in Table 4.1.

4.3. Results and Discussion

As shown in Table 4.2, topical treatment of Formula 7 significantly reduced the melanin levels in skin explants with neuregulin-induced DEJ impairment when compared to control Formula 6 (without active agents). Concomitantly, significant elevation of Collagen IV and VII expression was observed in skin explants treated with Formula 7 as compared to skin explants treated with Formula 6. Formula 7, comprising the combination of active agents 3- EAA, AT-11 and AT-9, demonstrated significant efficacy in melanin reduction in skin with DEJ impairment and enhanced expression of proteins crucial for function and stability of the extracellular matrix, namely, collagen IV and collagen VII.

Table 4.2. Evaluation of Formula 5 (3-EAA, AT-11 and AT-9) efficacy and reproducibility for melanin reduction in skin with neuregulin-induced skin hyperpigmentation and the effect on collagen IV and VII expression in a skin explant with DEJ impairment.

Expression level (% of untreated control)

Treatment Neuregulin-induced

Collagen IV Collagen VII

Melanin

Untreated control 100 100 100

Formula 6 86.36 ± 26.69 98.05 ± 0.81 102.15 ± 24.22

Formula 7 31.69 ± 12.52*** 130.93 ± 15.89**** 186.72 ± 4.49****

Data are expressed in mean ± S.E.M of two independent experiments (n=2). Statistical significance was indicated by a P value of *p<0.05, **p<0.01, ****p<0.0001 when compared to the untreated control using One-Way ANOVA.

4.4 Conclusion

Topical administration of Formula 7, comprising of 3-EAA, AT-11 and AT-9 was reproducibly efficacious in reducing melanin levels in human skin explants with neuregulin- induced skin hyperpigmentation and DE J impairment as compared to an untreated control. The efficacy of Formula 7 for lightening skin was confirmed in a repeat experiment. This composition of active agents when administered topically in the concentration tested is a useful treatment for skin hyperpigmentation.

Example 5: Evaluation of the efficacy of a topically administered emulsion composition comprising of active agents ascorbyl glucoside (AA2G) and acetyl sh- hexapeptide-5 amide acetate (ASH5AA) for the treatment of skin hyperpigmentation

5.1. Objective

To investigate the efficacy of a topical formulation comprising of AA2G and ASH5AA for the purpose of reducing hyperpigmentation as determined by measuring the melanin levels in neuregulin-induced skin hyperpigmentation of ex vivo human skin explants with MMP- 3 -induced DE J impairment.

5.2. Methodology

5.2.1 Formulation preparation

The test and control formulations were prepared by premixing Phase A and Phase B ingredients (Table 5.1) independently in separate containers until dispersed homogeneously at 70°C to 75°C. Then, Phase B was added into Phase A and stirred at 3500 rpm for 15 minutes. Phase C was mixed until fully dissolved and added into the Phase A/B solution at 60°C and stirred at 3500 rpm for 5 minutes. Then, Phase D was mixed until fully dissolved and add into the Phase A/B/C solution at 45°C and stirred at 100-200 rpm for 5 minutes. Lastly, ingredients in Phase E were added sequentially into Phase A/B/C/D solution and stirred at 100-200 rpm for 30 minutes.

Table 5.1. Ingredients of formulation listed by phase

5.2.2 Topical treatment of formulation on the neuregulin-induced hyperpigmentation in ex vivo skin -with impaired DEJ This experiment was performed as described in method 2.2.2 in the present specification using the formulations listed in Table 5.1.

5.3. Results and Discussion

As shown in Table 5.2, topical administration of Formula 8 or Formula 9 significantly reduced the melanin levels in neuregulin-induced skin hyperpigmentation in skin explants with MMP-3-induced DEJ impairment when compared to an untreated control. However, Formula 9 (comprising active agents AA2G and ASH5AA) showed a greater reduction of melanin than observed with Formula 8 (formula without active agents, that is ‘base formula’) treated samples. In addition, Formula 9 significantly increased expression of the Collagen IV and VII when compared the base formula (Formula 8). Taken together, the inverse correlation of melanin levels to collagen IV and VII levels was strongest in samples treated with Formula 9.

Table 5.2. Evaluation of Formula 9 (AA2G and ASH5AA) efficacy for melanin reduction in skin with neuregulin-induced skin hyperpigmentation and the effect on collagen IV and VII expression in a skin explant with DEJ impairment.

Expression level (% of untreated control)

Treatment Neuregulin-induced

Collagen IV Collagen VII

Melanin

Untreated control 100 100 100

Formula s 67.30 ± 5.70**** 123.70 ± 22.79** 122.62 ± 21.83**

Formula 9 55.33 ± 24.10**** 159.14 ± 3.83**** 183.94 ± 35.92****

5.4 Conclusion

A topically administered formulation comprising of ascorbyl glucoside and acetyl sh- hexapeptide-5 amide acetate was useful in reducing melanin levels in human skin explants with neuregulin-induced skin hyperpigmentation and DEJ impairment as compared to an untreated control.

Example 6: Synergistic anti-hyperpigmentation effect of 3-EAA with Acetyl tetrapeptide-11 and Acetyl tetrapeptide-9 in skin explant ex-vivo

6.1. Objective

To investigate the possible synergistic effect of 3-EAA when administered topically in combination with Acetyl tetrapeptide-11 and Acetyl tetrapeptide-9 for reducing hyperpigmentation in ex vivo human skin explants with neuregulin-induced skin hyperpigmentation and DEJ impairment.

6.2. Methodology

6.2.1 Formulation preparation The test and control formulations were prepared by premixing the Phase A ingredients (Table 6.1) by adding each ingredient sequentially into the aqueous solution and stirring at 100-200 rpm for 15 minutes until folly dissolved. All ingredients in Phase B were mixed until folly dissolved, then added into the Phase A solution and stirred at 100-200 rpm for 5 minutes. Lastly, the preservatives (Aminomethyl Propanol and Chlorphenesin, Glycerin, Phenoxyethanol) were added to the Phase A/B solution and stirred at 100-200 rpm for a further 5 minutes.

Table 6.1. Formulation ingredients listed by phase

6.2.2 Topical treatment of formulation on the neuregulin-induced hyperpigmentation in ex vivo skin with impaired DEJ

This experiment was performed as described in method 2.2.2 in the present specification using the formulations listed in Table 6.1. 63. Results and Discussion

As demonstrated in Table 6.2, all formulations tested significantly reduced melanin and significantly enhanced collagen IV and collagen VII expression in human skin explants with impaired DE J as compared to the untreated control. As expected, Formula 12 comprised of acetyl tetrapeptide- 11 and acetyl tetrapeptide-9 or Formula 13 comprised of 3-EAA showed significant melanin reduction in DEJ-impaired skin. Interestingly, Formula 11 which comprised of half of the dose concentration of the active agent, either 3-EAA or acetyl tetrapeptide- 11 and acetyl tetrapeptide-9, showed higher melanin reduction than Formula 13 or Formula 12, respectively. Taken together, this indicated that the combination of 3-EAA, acetyl tetrapeptide- 11 and acetyl tetrapeptide-9 work synergistically in reducing melanin in human skin explant with impaired skin DEJ.

Table 6.2. Topical efficacy of formula on reduction of neuregulin-induced skin melanin and collagen IV and VII expression in skin explant.

Neuregulin-induced expression level

Active Level (%)

(% of untreated control)

Treatment Acetyl Acetyl

3-EAA tetrapepti tetrape Total Melanin Collagen IV Collagen VII de- 11 ptide-9

Untreated

100 100 100 control

Formula 93.07 ± 161.05 ± 167.41 ±

10 48.93 30.57**** 44.29****

Formula 36.93 ± 234.46 ± 241.96 ±

0.5000 0.0006 0.0005 0.5011

11 3 27**** 11 29**** 48 02****

Formula 56.96 ± 210.92 ± 246.64 ±

0.0012 0.001 0.0022

12 19 35**** 24 59**** 10 go****

Formula 62.81 ± 190.32 ± 215.95 ±

1.0000 - - 1

13 19.92*** 40.22*** 30.51*** Data are expressed in mean ± S.E.M of two independent experiments (n=2). Statistical significance was indicated by a P value of *p<0.05, **p<0.01, ****p<0.0001 when compared to untreated control using One-Way ANOVA. 6.4 Conclusion

The active agent 3-o-ethyl ascorbic acid (3-EAA) works synergistically when combined with Acetyl tetrapeptide- 11 and Acetyl tetrapeptide-9 when reducing melanin levels in human skin explants with neuregulin-induced skin hyperpigmentation and DEJ impairment as compared to an untreated control.

Example 7: Comparison of aqueous and emulsion formulations with ascorbic acid derivatives for reducing dermal melanin in vitro as a holistic anti-hyperpigmentation approach

7.1. Objective

To investigate and compare the efficacy of selected active agents combined with ascorbic acid derivatives for the purpose of reducing dermal melanin in an in vitro skin dermal melanosis model.

7.2. Methodology

7.2.1 Preparation of aqueous formulation

The test and control formulations were prepared by premixing the Phase A ingredients (Table 7.1) by adding each ingredient sequentially into the aqueous solution and stirring at 100-200 rpm for 15 minutes until fully dissolved. All ingredients in Phase B were mixed until fully dissolved, then added into the Phase A solution and stirred at 100-200 rpm for 5 minutes. Lastly, the preservatives (Aminomethyl Propanol and Chlorphenesin, Glycerin, Phenoxyethanol) were added to the Phase A/B solution and stirred at 100-200 rpm for a further 5 minutes.

Table 7.1. Formulation ingredients listed by phase - aqueous formula

7.2.2 Preparation of emulsion formulation

The test and control formulations were prepared by premixing Phase A and Phase B ingredients (Table 7.2) independently in separate containers until dispersed homogeneously at 70°C to 75°C. Then, Phase B was added into Phase A and stirred at 3500 rpm for 15 minutes. Phase C was mixed until fully dissolved and added into the Phase A/B solution at 60°C and stirred at 3500 rpm for 5 minutes. Then, Phase D was mixed until fully dissolved and add into the Phase A/B/C solution at 45°C and stirred at 100-200 rpm for 5 minutes. Lastly, ingredients in Phase E were added sequentially into Phase A/B/C/D solution and stirred at 100-200 rpm for 30 minutes.

Table 7.2. Formulation ingredients listed by phase - emulsion formula

7.2.3 Preparation of fibroblast cells -with internalised melanosome

The mouse melanoma cells B16-F10 (ATCC, USA) were seeded at a concentration of 5 x 10⁵ cells in a T75 tissue culture flask and maintained in complete Dulbecco’s Modified Eagle’s Media (DMEM; Nacalai, Japan) supplemented with 10% foetal bovine serum (FBS), sodium pyruvate (1 mM), L-glutamine (2mM), streptomycin (50 pg/ml) and penicillin (50 U/ml) at 37°C in an atmosphere of 5% CO2 for 5 days. Then, the culture media were centrifuged at 300 xg to sediment the cells debris and the melanosomes where harvested in the supernatant, and referred to as: melanosome conditioned media.

In a separate flask, human skin fibroblast HS-68 cells (ATCC, USA) were seeded 7 x 10⁵ in a T25 tissue culture flask and maintained in DMEM media supplemented with 10% FBS, sodium pyruvate (1 mM), L-glutamine (2mM), streptomycin (50 pg/ml) and penicillin (50 U/ml) at 37°C in an atmosphere of 5% CO2 for 24 hours. Then, the culture media were replaced with melanosome conditioned media and cultured for a further 24 hours. Lastly, the cell viability of treated fibroblast cells was determined by using a MTT assay (Nacalai Tesque, Japan). Besides, the fibroblast cells treated with melanosome conditioned media were treated with trypsin and harvested by centrifugation at 10,000 xg for lOmin. The supernatant was removed, and the cell pellets collected. The harvested cells were lysed by re-suspending with IM NaOH (Merck, Germany) and incubated at 80°C for 1 hour. Lastly, the lysed samples were centrifuged at 3000 xg for 5 min, the supernatant were collected for melanin and protein quantification.

7.2.4 Topical treatment of reconstituted skin and fibroblast cells with internalised melanosome in a co-culture model

Human skin fibroblast HS-68 cells were seeded in a 12- well hanging top plate at cell concentration of lx 10⁵ cells/well and cultured for 24h under 5% CO2 and 37 °C. Then, the culture media were replaced with melanosome conditioned media and cultured for another 24h. On the same day, reconstituted skin with melanocytes (Melanoderm; Mattek, USA) were maintained with long life maintenance media (LLMM from Mattek, USA) and cultured at 37°C and 5% CO2 overnight. On the next day, the melanosome conditioned media were discarded and rinsed with PBS. The Melanoderm reconstituted skin was then added to the 12-well hanging top plate containing melanosome conditioned media treated fibroblast cells for co-culturing. The co-culture system was maintained in LLMM media and topical application of each test and control formulation were administered to the skin and incubated for two hours. The formulation was then washed off with PBS. The media was refreshed, and the topical treatments applied every 2 days for total duration of 7 days. Lastly, fibroblast cells were treated with trypsin and harvested for melanin quantification. Cell viability of treated skin and fibroblast cells were determined by using MTT assay.

7.2.5 Melanin and total protein quantification

Briefly, 150 pL of collected supernatant containing melanin were treated with 45 pL of hydrogen peroxide (Merck, Germany) and incubated at room temperature under dark conditions. Then, the samples were centrifuged at 3000 xg for 5 min, supernatant harvested and the fluorescence intensity of the collected supernatant were measured at excitation wavelength of 470 nm and emission wavelength of 550 nm by using Cytation 5 multi-mode reader (Biotek Instrument, USA). The amount of melanin in the samples was calculated from a standard curve (0-100 pg/ml) of freshly prepared melanin in a serial dilution (Sigma, USA). In addition, the total protein was quantified by Bradford assay and CBB solution (Nacalai Tesque, Japan) according to the manufacturer’s instructions. Lastly, the melanin content was normalised against the total protein content and expressed as total melanin (pg) in one mg of protein.

7.3. Results and Discussion

As shown in Table 7.3, the aqueous formulation, Formula 15 comprising of acetyl tetrapeptide- 11 and acetyl tetrapeptide-9 only, showed insignificant reduction of melanin content in the fibroblast cells. In contrast, Formula 14, which comprised both the ascorbic acid derivative (3-EAA) and the active agents acetyl tetrapeptide- 11 and acetyl tetrapeptide- 9, demonstrated significant reduction of melanin content in fibroblast cells.

Similarly, the emulsion formula demonstrated the same trend as the aqueous formulation as shown in Table 7.4. That is, Formula 17, which comprised of acetyl sh-hexapeptide-5 amide acetate only showed insignificant reduction of melanin content in fibroblast cells. Again, in contrast, Formula 16, which comprised of the ascorbic acid derivative (ascorbyl glucoside) and acetyl sh-hexapeptide-5 amide acetate, showed a significant reduction of melanin content in fibroblast cells.

Taken together, anti-hyperpigmentation results were more efficacious when the ascorbic acid derivatives were administered together with the other active agents, namely acetyl tetrapeptide- 11 and acetyl tetrapeptide-9 or, acetyl sh-hexapeptide-5 amide acetate. Coadministration resulted in significant reduction of dermal melanin.

Table 7.3. Effects of selected aqueous formula on melanin content in melanosome internalised fibroblast cells of biomimetic skin model of dermal melanosis.

Active Level (%) Melanin Skin tissue Fibroblast cell

Acetyl Acetyl content in viability (% of viability (% of

Treatment 3- tetrapept tetrape fibroblast control) control) ? ide- 11 ptide-9 (% of control)

Untreated

100 100 100 control

Formula 14 1.0000 0.0012 0.001 70.01 ± 2.99* 89.07 ± 10.95 101.40 ± 5.82

Formula 15 - 0.0012 0.001 82.84 ± 22.49 83.89 ± 6.62 105.00 ± 7.48

All data were normalised with untreated control group and expressed in mean ± S.E.M of three independent experiments (n=3). Statistical significance was indicated by a P value of *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 when compared to the untreated control group by one-way ANOVA. Table 7.4. Effects of selected emulsion formula on melanin content in melanosome internalised fibroblast cells of biomimetic skin model of dermal melanosis.

Active Level (%) Melanin

Acetyl sh- content in Skin tissue Fibroblast cell

Treatment Ascorbyl

Hexapeptide-5 fibroblast viability (% viability (% of glucoside

Amide Acetate (% of control) of control) control)

Untreated

100 100 100 control Formula 16 2.0000 0.0012 61.74 ± 9.64* 97.49 ± 7.35 103.2 ± 4.56

Formula 17 0.0012 82.25 ± 22.12 91.58 ± 1.87 106.9 ± 5.42

All data were normalised with untreated control group and expressed in mean ± S.E.M of three independent experiments (n=3). Statistical significance was indicated by a P value of *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 when compared to the untreated control by one-way ANOVA.

7.4 Conclusion

Peptides (acetyl tetrapeptide- 11 and acetyl tetrapeptide-9 or acetyl sh-hexapeptide-5 amide acetate) show a reduction in dermal melanin, but the reduced melanin level is not significant when compared to an untreated control in a skin dermal melanosis model in vitro. However, when each of the active agents were combined with an ascorbic acid derivative (3-EAA or Ascorbyl glucoside), the reduction in melanin levels was significant in the skin dermal melanosis model in vitro. This demonstrates that these formulations a useful and are promising treatments for skin lightening or skin hyperpigmentation disorders.

References

Ando, H. et al. Dermal fibroblasts internalize phosphatidylserine-exposed secretory melanosome clusters and apoptotic melanocytes. Int. J. Mol. Sci. 21, 1-14 (2020).

Lattouf R, Younes R, Lutomski D, Naaman N, Godeau G, Senni K, Changotade S. (2014). Picrosirius red staining: a useful tool to appraise collagen networks in normal and pathological tissues. J Histochem Cytochem. Oct;62(10):751-8. doi: 10.1369/0022155414545787. Epub 2014 Jul 14. PMID: 25023614.

Langton, A. K., Halai, P., Griffiths, C. E. M., Sherratt, M. J., & Watson, R. E. B. (2016). The impact of intrinsic ageing on the protein composition of the dermal-epidermal junction.

Mechanisms of Ageing and Development, 156, 14-16. https://doi.Org/10.1016/J.MAD.2016.03.006

Nieuweboer-Krobotova L. (2013). Hyperpigmentation: types, diagnostics and targeted treatment options. J Eur Acad Dermatol VenereoL Jan;27 Suppl 1 :2-4. doi: 10.1111/jdv.12048. PMID: 23205538.

Watanabe, S. (2014) Facial Dermal Melanocytosis. Austin J Dermatolog 1(2), 1006.

Claims

1. A composition including: a) N-acetyl-L-glutaminyl-L-alpha-aspartyl-L-valyl-L-histidine (acetyl tetrapeptide- 9), or salt thereof and N-acetyl-L-prolyl-L-prolyl-L-tyrosyl-L-leucine (acetyl tetrapeptide- 11), or salt thereof; and b) an ascorbic acid derivative, or salt thereof; wherein the weight ratio of N-acetyl-L-glutaminyl-L-alpha-aspartyl-L-valyl-L-histidine or salt thereof to N-acetyl-L-prolyl-L-prolyl-L-tyrosyl-L-leucine or salt thereof is in the range of from 10:1 to 1:10.

2. A composition as claimed in claim 1, wherein the active agents comprised in component a) are present in a weight ratio of N-acetyl-L-glutaminyl-L-alpha-aspartyl-L- valyl-L-histidine to N-acetyl-L-prolyl-L-prolyl-L-tyrosyl-L-leucine in the range of from 5:1 to 1 :5, or optionally in a weight ratio of N-acetyl-L-glutaminyl-L-alpha-aspartyl-L-valyl-L- histidine to N-acetyl-L-prolyl-L-prolyl-L-tyrosyl-L-leucine in the range of from 2:1 to 1 :2.

3. A composition as claimed in claim 1 or 2, wherein component b) is selected from the group consisting of 3-O-ethyl ascorbic acid, 2-O-ethyl ascorbic acid, ascorbyl glucoside, ascorbyl tetraisopalmitate, magnesium ascorbyl phosphate, sodium ascorbic phosphate, caprylyl 2-gly ceryl ascorbate, 3 -glyceryl ascorbate, bis-gly ceryl ascorbate, hexyl 3 -glyceryl ascorbate, myristyl 3 -glyceryl ascorbate, and lauryl glyceryl ascorbate.

4. A composition as claimed in claim 3, wherein component b) is 3-O-ethyl ascorbic acid or ascorbyl glucoside.

5. A composition as claimed in any one of claims 1 to 4, wherein N-acetyl-L- glutaminyl-L-alpha-aspartyl-L-valyl-L-histidine and N-acetyl-L-prolyl-L-prolyl-L-tyrosyl- L-leucine are each independently present in the combination in an amount in the range of from 0.0001% to 1% by weight of the composition, optionally from 0.0005% to 0.005% by weight of the composition, optionally from 0.001% to 0.002% by weight of the composition.

6. A composition as claimed in any one of claims 1 to 5, wherein component b) is present in an amount in the range of from 0.1% to 10% by weight of the composition, optionally from 0.2% to 8% by weight of the composition, optionally from 1% to 2% by weight of the composition.

7. A composition as claimed in any one of claims 1 to 6, wherein the composition does not contain any anti-hyperpigmentation agent other than components a) and b).

8. A composition as claimed in any one of claims 1 to 7, wherein the composition is suitable for topical application.

9. A composition as claimed in any one of claims 1 to 8, wherein the composition comprises one or more pharmaceutically or cosmetically suitable excipients.

10. A composition as claimed in any one of claims 1 to 9, wherein the composition comprises one or more of the following: an anti-inflammatory agent, a sunscreen agent, an antioxidant, a desquamation agent, and an exfoliating agent.

11. A composition as claimed in any one of claims 1 to 10, wherein the composition is in the form of a cream, lotion, serum, paste, wax, liquid, semisolid composition or a sprayable composition.

12. A method of preventing, treating, and/or reducing a symptom associated with, a skin pigmentation disorder in a subject, or a method of inhibiting melanin production in epidermal melanocytes and/or promoting melanin degradation in the dermal fibroblasts of a subject, or a non-therapeutic method of skin whitening and/or lightening of a subject, comprising administering an effective amount of a composition as claimed in any one of claims 1 to 11, to the subject.

13. Use of a composition as claimed in any one of claims 1 to 11, for the manufacture of a medicament for the prevention, treatment, and/or reduction of a symptom associated with, a skin pigmentation disorder, or for inhibiting melanin production in epidermal melanocytes and/or promoting melanin degradation in dermal fibroblasts.

14. A composition as claimed in any one of claims 1 to 11 for use in prevention, treatment, and/or reduction of a symptom associated with, a skin pigmentation disorder, or for inhibiting melanin production in epidermal melanocytes and/or promoting melanin degradation in dermal fibroblasts.

15. A composition including : a) N-acetyl-L-alanyl-L-leucyl- L-histidyl-L-lysyl-L-valyl-L-lysinamide

16. A composition as claimed in claim 15, wherein component b) is selected from the group consisting of 3-O-ethyl ascorbic acid, 2-O-ethyl ascorbic acid, L-ascorbic acid, ascorbyl glucoside, ascorbyl tetraisopalmitate, magnesium ascorbyl phosphate, sodium ascorbic phosphate, caprylyl 2-glyceryl ascorbate, 3 -glyceryl ascorbate, bis-gly ceryl ascorbate, hexyl 3 -glyceryl ascorbate, myristyl 3 -glyceryl ascorbate, and lauryl glyceryl ascorbate.

17. A composition as claimed in claim 16, wherein component b) is ascorbyl glucoside or 3-O-ethyl ascorbic acid.

18. A composition as claimed in any one of claims 15 to 17, wherein component a) is present in an amount in the range of from 0.0001% to 1% by weight of the composition, optionally from 0.0005% to 0.005% by weight of the composition, optionally from 0.001% to 0.002% by weight of the composition.

19. A composition as claimed in any one of claims 15 to 18, wherein component b) is present in an amount in the range of from 0.1% to 10% by weight of the composition, optionally from 0.5% to 8% by weight of the composition, optionally from 1% to 2% by weight of the composition.

20. A composition as claimed in any one of claims 15 to 19, wherein the composition is suitable for topical application.

21. A composition as claimed in any one of claims 15 to 20, wherein the composition comprises one or more pharmaceutically or cosmetically suitable excipients.

22. A composition as claimed in any one of claims 15 to 21, wherein the composition comprises one or more of the following: an anti-inflammatory agent, a sunscreen agent, an antioxidant, a desquamation agent, and an exfoliating agent.

23. A composition as claimed in any one of claims 15 to 22, wherein the composition is in the form of a cream, lotion, serum, paste, wax, liquid, semisolid composition or a sprayable composition.

24. A method of preventing, treating, and/or reducing a symptom associated with, a skin pigmentation disorder in a subject, or a method of inhibiting melanin production in epidermal melanocytes and/or promoting melanin degradation in the dermal fibroblasts of a subject, or a non-therapeutic method of skin whitening and/or lightening of a subject, comprising administering an effective amount of N-acetyl-L-alanyl-L-leucyl-L-histidyl-L- lysyl-L-valyl-L-lysinamide (acetyl sh-hexapeptide-5 amide acetate) or salt thereof, to the subject.

25. Use of N-acetyl-L-alanyl-L-leucyl-L-histidyl-L-lysyl-L-valyl-L-lysinamide (acetyl sh-hexapeptide-5 amide acetate) or salt thereof, for the manufacture of a medicament for the prevention, treatment, and/or reduction of a symptom associated with, a skin pigmentation disorder, or for inhibiting melanin production and/or promoting melanin degradation in dermal fibroblasts.

26. N-acetyl-L-alanyl-L-leucyl-L-histidyl-L-lysyl-L-valyl-L-lysinamide (acetyl sh- hexapeptide-5 amide acetate) or salt thereof for use in prevention, treatment, and/or reduction of a symptom associated with, a skin pigmentation disorder, or for inhibiting melanin production and/or promoting melanin degradation in dermal fibroblasts.

27. A method of preventing, treating, and/or reducing a symptom associated with, a skin pigmentation disorder in a subject, or a method of inhibiting melanin production in epidermal melanocytes and/or promoting melanin degradation in the dermal fibroblasts of a subject, or a non-therapeutic method of skin whitening and/or lightening of a subject, comprising administering an effective amount of a composition as claimed in any one of claims 15 to 23, to the subject.

28. Use of a composition as claimed in any one of claims 15 to 23, for the manufacture of a medicament for the prevention, treatment, and/or reduction of a symptom associated with, a skin pigmentation disorder, or for inhibiting melanin production in epidermal melanocytes and/or promoting melanin degradation in dermal fibroblasts.

29. A composition as claimed in any one of claims 15 to 23 for use in prevention, treatment, and/or reduction of a symptom associated with, a skin pigmentation disorder, or for inhibiting melanin production in epidermal melanocytes and/or promoting melanin degradation in dermal fibroblasts.

30. A method, use, or composition for use, as claimed in any one of claims 12-14 and 24 to 29, wherein the skin pigmentation disorder is selected from the group consisting of skin hyperpigmentation, melasma, solar lentigo, nevus of Ota, nevus of Ito and congenital dermal melanocytosis.