WO2020052994A1

WO2020052994A1 - Method of evaluating efficacy of cosmetic compositions

Info

Publication number: WO2020052994A1
Application number: PCT/EP2019/073193
Authority: WO
Inventors: Yiying Dong; Dandan HUANG; Nan Huang; Tingyan MI
Original assignee: Unilever N.V.; Unilever Plc; Conopco, Inc., D/B/A Unilever
Priority date: 2018-09-12
Filing date: 2019-08-30
Publication date: 2020-03-19

Abstract

Disclosed is an in vitro method to determine efficacy of a cosmetic ingredient against stressors, said method comprising the steps of: (i) contacting a 3D living skin-equivalent (LSE) comprising primary human keratinocytes with an oxidative environmental stressor for a pre-determined period; (ii) measuring, by any known means, the extent to which barrier function of said LSE is affected; (iii) determining the number of viable keratinocytes; (iv) contacting said LSE with said cosmetic ingredient; (v) repeating said steps (ii) and (iii); and, (vi) inferring that said ingredient is efficacious if there is improvement in cell viability and barrier function, where said step (iv) is alternatively carried out along with or prior to said step (i), wherein said living LSE is contacted with a non-environmental stressor for a pre-determined period, before step (i) or between said steps (i) and (ii) and further wherein the extent to which said barrier function is affected is measured by permeability assay using a dye.

Description

METHOD OF EVALUATING EFFICACY OF COSMETIC COMPOSITIONS

Field of the invention

The present invention relates to a method of evaluating efficacy of cosmetic compositions. More particularly, the invention relates to a method of evaluating the efficacy of such compositions to neutralize harmful effects of atmospheric pollutants on our skin and restore its natural defence mechanism.

Background of the invention

The World Health Organization (WHO) reports that outdoor air pollution originates from natural and anthropogenic sources. While natural sources contribute substantially to local air pollution in arid regions more prone to forest fires and dust storms, the contribution from human activities far exceeds natural sources.

Such human activities include fuel combustion, heat and power generation and industrial facilities (e.g. manufacturing factories, mines, and oil refineries). WHO classifies pollutants into particulate matter, black carbon, ground-level ozone and oxides of carbon, nitrogen and sulphur.

Particulate matter (PM) are inhalable particles composed of sulphate, nitrates, ammonia, sodium chloride, black carbon, mineral dust and water. Particles with a diameter of less than 10 microns (PM10), including fine particles less than 2.5 microns (PM2.5) pose the greatest risks to health, as they can enter the lungs and the bloodstream. Carbon black (soot) and dust (mineral oxides, such as iron oxides and the like) comprise much of the particulate matter in these size ranges.

WHO defines air pollution as contamination of indoor or outdoor environments by any chemical, physical, or biological agent that modifies the natural characteristics of the atmosphere. The U.S. Environmental Protection Agency (EPA) and the WHO have summarized the global extent of common atmospheric pollutants. In addition, there are innumerable reports and scientific publications pertaining to adverse effects of pollution on human skin. These adverse effects include premature ageing, development of fine lines and wrinkles, pigmented spots, hyperpigmentation, rash and inflammation.

A variety of test methods are found in books, journals, periodicals and patents. The purpose of such methods is to ascertain the efficacy of cosmetic composition. At times the purpose also is to compare the efficacy of one or more compositions or active ingredients, e.g. antioxidants. Some of these tests are conducted on human volunteers. Some others have been conducted on suitable skin-equivalents such as plastic membranes, living-skin equivalents, Vitro-Skin®, in vitro skin model and ex vivo skin. While skin-equivalents is one component of such test methods, selection of an appropriate pollutant is equally important. However, it is not always possible to perform tests with real pollutants therefore model pollutants are often used.

Exposure of our skin to some pollutants may activate cutaneous stress as the pollutants can react with skin and penetrate through the skin barrier and cause oxidative stress and inflammation by reacting with proteins, lipids and DNA molecules. Such exposure could manifest itself in the form of disorders and cosmetic conditions such as xerotic skin, sensitive skin and signs of premature or accelerated aging, such as wrinkle formation, abnormal pigmentation and dry skin. It is believed that certain pollutants may also cause acne, eczema and rashes. Therefore, some form of protection against atmospheric pollutants is necessary.

Prolonged and repetitive exposure to high levels of pollutants, such as stressors, may impair the natural defense mechanisms of our skin. Moreover, some pollutants (e.g., ozone) can induce damage via signal transduction mechanism even when there is no percutaneous penetration into deeper layers of the skin. Therefore, it is not advisable to rely solely on the natural defence and protective mechanism of our skin. Therefore, use of skincare cosmetic compositions, formulated with ingredients that are known to improve the barrier function of our skin is highly recommended. Some active cosmetic ingredients scavenge the oxidative pollutants.

On the other hand, our skin often repeatedly contacts with non-environmetal stressors, such as surfactants. Most of the skin cleansing compositions comprise at least one surfactant; usually a combination of surfactants. The combined effect of non-environmetal stressors, such as surfactants and oxidative environmental stressors such as ozone, could be detrimental to the normal functioning of our skin. Their ill effects could be neutralized, at least to some extent, using skin care ingredients, for example retinol and retinoic acid. There is need for a reliable and repeatable method of analysis to demonstrate the harmful effects mentioned hereinabove and the corresponding restorative or ameliorative effects also mentioned hereinabove.

US2011262570 A1 (P&G) discloses a biomarker panel for assessing benefits of active ingredients against damage caused by oxidative stress. The panel contains the usual antioxidant related genes. A screening method for identifying an agent effective in preventing or reversing age-related oxidative damage to skin, comprising the steps of providing a reference transcriptional profile for young skin, skin cells or a skin equivalent, providing a reference transcriptional profile for aged skin, skin cells or a skin equivalent, contacting the aged skin, skin cells or skin equivalent with a proposed agent and generating a test transcriptional profile, followed by comparing the test transcriptional profile to the reference profiles and identifying the proposed agent as effective in preventing or reversing age-related oxidative damage to skin if the test transcriptional profile is directionally shifted toward the young reference profile and/or away from the aged reference profile. The transcriptional profiles are generated with the microarray comprising immobilized oligonucleotides which hybridize specifically to nucleic acids corresponding to the genes constituting the genes which are regulated in human skin as a function of age-related oxidative stress. The panel does not have a functional or phenotypical assay to demonstrate relevance of these biomarkers to the real-life problems faced by the skin.

US2017172909 A1 (ELC Management LLC), discloses a method in which an internal stress (cortisol) is used. It is chronic and mainly impacts the dermis. The publication mainly focuses on dermal aging and senescence. The publication discloses a model which mimics stress-induced fatigue of the skin, using a stress-induced premature senescence phenotype skin model to identify and evaluate novel cosmetic materials for their efficacy in preventing, minimizing or reversing development of the stress-induced premature senescence phenotype. The concerned steps are as follows: providing a dermal equivalent skin model; incubating the dermal equivalent skin model with a stress-inducing ingredient in an amount and for a time sufficient to induce a premature senescence phenotype in the dermal equivalent skin model; incubating the dermal equivalent skin model with a test material; and ascertaining whether the test material has an efficacy for reversing the premature senescence phenotype in the skin model.

US2015276714 A1 (P&G) discloses a 2D cell model to assess the metabolic activity of cells. As cells are cultured in a medium, there are limitations in the assessment of some real-life stress-causing factors like oxidized squalene which can be applied topically only on an air-lift surface. The metabolic endpoints like oxygen consumption by the cells and acidification of the cell medium do not directly reflect the function of the skin. The method comprises the steps of providing a plurality of keratinocytes;

exposing the keratinocytes to a stressor; non-lethally detecting a metabolic indicator associated with each of glycolysis and oxidative phosphorylation to provide a response of each to the stressor; exposing the plurality of keratinocytes to a test agent; non- lethally detecting the metabolic indicators associated with each of glycolysis and oxidative phosphorylation to provide a response of each to the test agent; and identifying the test agent as a skin-care active when at least one of the responses indicates an improvement in keratinocyte metabolism relative to the corresponding response.

US20150362412 A1 (Shiseido) discloses a method to evaluate the condition of stratum corneum by tape-stripping the skin after cosmetic treatment of the skin. The sheet is contacted with a dye selected from the group consisting of fluorescein, rose bengal and their salts. The dye is then rinsed following which the staining intensity of the dye in the stratum corneum sheet is measured. Damaged stratum corneum is not as transparent and its barrier function is also adversely affected. The application also discloses a method of evaluating candidate cosmetics or cosmetic treatments and their ameliorating effect on the stratum corneum.

In the publication PLOSONE DOI: 10.1371/journal. pone.0131097 August 13, 2015, the authors Valacchi et.al., have disclosed the protective effect of defined“antioxidant” mixtures against O3 induced oxidative stress damage in human keratinocytes and understand their underlying mechanism of action. This study shows the ability of antioxidant mixtures containing pure antioxidant compounds to abolish the noxious effects of O3 in cultured human keratinocytes. Their model uses mixed pollutants/ozone as stressors.

The present invention addresses the needs by overcoming at least one drawback, disadvantage or limitation of the state of the art.

Summary of the invention

Disclosed in accordance with a first aspect of the invention is an in vitro method to determine efficacy of a cosmetic ingredient against stressors, said method comprising the steps of:

(i) contacting a 3D living skin-equivalent (LSE) comprising primary human

keratinocytes with an oxidative environmental stressor for a pre-determined period;

(ii) measuring, by any known means, the extent to which barrier function of said LSE is affected;

(iii) determining the number of viable keratinocytes;

(iv) contacting said LSE with said cosmetic ingredient;

(v) repeating said steps (ii) and (iii); and,

(vi) inferring that said ingredient is efficacious if there is improvement in cell viability and barrier function,

where said step (iv) is alternatively carried out along with or prior to said step (i), wherein said LSE is contacted with a non-environmental stressor for a pre-determined period, before step (i) or between said steps (i) and (ii) and further wherein the extent to which said barrier function is affected is measured by permeability assay using a dye.

Disclosed in accordance with a second aspect of the invention is an in vitro method to demonstrate efficacy of a cosmetic ingredient against stressors, said method comprising the steps of:

(i) contacting a LSE comprising primary human keratinocytes with an oxidative environmental stressor for a pre-determined period; (ii) measuring, by any known means, the extent to which barrier function of said LSE is affected;

(iii) determining the number of viable keratin ocytes;

(iv) contacting said LSE with said cosmetic ingredient;

(v) repeating said steps (ii) and (iii); and,

The invention will now be described in detail.

Detailed description of the invention

The skin, which consists of the epidermis and the dermis underneath, is an important organ which protects the body from dehydration and various environmental and non- environmental stress-causing factors. Being the main type of cell in the epidermis;

keratinocytes undergo proliferation and terminal differentiation to produce the cornified layer, the outermost skin barrier which is called stratum corneum. The layer is composed of dead keratinocytes and the intercellular spaces are filled with lipids which contribute to the impermeability of the barrier. When the barrier function of skin is adversely affected it leads to trans-epidermal water loss, alteration in pH of the skin and dehydration.

Surfactants are widely used as cleansing agents due to their ability to detach and remove microbes, sebum, dust and a variety of other foreign objects from the surface of our skin. At least some of the cleansing surfactants are known irritants which are associated with tightness and dryness of the skin.

Squalene monohydroperoxide (SQOOH) is a primary oxidized sebum product formed from squalene (SQ) by solar UV radiation. Squalene is the single most abundant unsaturated constituent of skin lipids. Cytotoxicity of SQOOH is reported in skin keratinocytes as well as model 3D living skin equivalent (LSE). SQOOH is also regarded as a reliable biomarker of environmental pollution studies. Squalene (SQ), the most abundant unsaturated skin lipid, is oxidized upon exposure to solar UV. The primary oxidized lipid from SQ is squalene monohydroperoxide (SQOOH), which has been shown to have detrimental effects on keratinocytes by way of studies conducted on 3D skin models. Often, our skin comes in contact with non-environmental stressors like surfactants and abrasive agents which are found in cosmetics. On the other hand, there are oxidative environmental stressor like peroxides which contact our skin. It is believed that their combined effect is more detrimental. Therefore, it is useful to study and understand such detrimental effect on the barrier properties of skin and the viability of cells, such as keratinocytes. While individually such stressors are known to affect the barrier, the potential synergistic effect is of practical relevance to formulation scientists and cosmetologists as their combination could result in higher incidences of sensitive or sensitised skin, as reported in epidemiological studies. It is useful to determine how and to what extent certain cosmetic ingredients could be used to reverse, prevent, nullify, neutralise or at least delay the unwanted effects.

As used herein the term“comprising” encompasses the terms“consisting essentially of” and“consisting of. Where the term“comprising” is used, the listed steps or options need not be exhaustive. Unless otherwise specified, numerical ranges expressed in the format "from x to y" are understood to include x and y. In specifying any range of values or amounts, any particular upper value or amount can be associated with any particular lower value or amount. Except in the examples and comparative

experiments, or where otherwise explicitly indicated, all numbers are to be understood as modified by the word“about”. All percentages and ratios contained herein are calculated by weight unless otherwise indicated. As used herein, the indefinite article “a” or“an” and its corresponding definite article“the” means at least one, or one or more, unless specified otherwise. The various features of the present invention referred to in individual sections above apply, as appropriate, to other sections mutatis mutandis. Consequently, features specified in one section may be combined with features specified in other sections as appropriate. Any section headings are added for convenience only, and are not intended to limit the disclosure in any way. The examples are intended to illustrate the invention and are not intended to limit the invention to those examples per se.

The term cosmetic composition means a composition suitable for topical application on mammalian skin. Preferably the cosmetic composition is a skin-care composition, a skin cleansing composition, a hair care composition, a shampoo, an antiperspirant or a deodorant. Non-limiting examples of specific cosmetic compositions include lotions (e.g. hand lotion and body lotion), skin-care products (e.g., face and neck lotions, serums, sprays), sunless tanners, cosmetics (e.g., foundation, concealer, blush, lipstick, lip gloss), depilatories, shampoos, conditioning shampoos, hair conditioners, hair dyes, body washes, moisturizing body washes, shower gels, skin cleansers, cleansing milks, in-shower body moisturizers, shaving preparations, after-shaves, razor moisturizing/lubricating strips, razor shave-gel bars, bar soaps and baby-care products.

The term in vitro means that the method in accordance with the invention is not carried out on human volunteers, for example, on the forearms of human volunteers.

The term cosmetic ingredient means any ingredient for any beneficial effect against pollutants, preferably against oxidative environmental stressors. Non-limiting examples thereof include polymers and extracts of natural products such as extract of roots or leaves of any particular plant.

Living skin equivalent means the skin equivalent comprises viable keratinocytes and a barrier layer differentiated into stratum corneum.

Human skin acts like a natural shield which protects our body from external influences. However, at times, and under certain conditions, the skin may no longer perform this function fully and efficiently. There is plethora of evidence indicating that atmospheric pollutants affect the normal functioning of human skin. Particulate pollutants tend to top the list. Formulation scientists have explored and continue to explore newer and more effective cosmetic compositions to protect the skin from particulate pollutants, including the compositions which can neutralize the detrimental effects of such pollutants. However, as discussed at length under the section of background and prior art, there is need for a more robust and reliable method for demonstrating the efficacy of such compositions. The present invention addresses such a need, at least in part.

The method according to the invention Step (i) of the method in accordance with the invention is contacting a 3D living skin- equivalent comprising primary human keratinocytes with an oxidative environmental stressor for a pre-determined period.

The method in accordance with the invention is carried out on a 3D living skin-equivalent (LSE) which is also known as human skin-equivalent (HSE), i.e. a material that resembles the skin of human beings. The living skin-equivalent comprises viable keratinocytes. It also comprises a barrier layer differentiated into stratum corneum. Currently, several LSE are commercially available for such applications. Suitable examples include EpiKutis^®, MelaKutis^®, EpiSkin ®, SkinEthic RHE®, SkinEthic RHPE®, EpiDerm®, T-skin®, MelanoDerm®, EpiDermFT®. These HSEs could be divided into two major categories: epidermal and full-thickness models.

It is preferred that oxidative environmental stressor generates Reactive Oxygen Species (ROS) in the skin-equivalent upon contact therewith. Preferably the oxidative

environmental stressor is ozone, an oxide of nitrogen, a peroxide, ultraviolet radiation or PM2.5 or PM10.

"Oxidative Stressor" means an environmental that causes the formation of undesirable reactive oxygen species in a cell. Some other non-limiting examples of oxidative stressors include cigarette smoke, engine exhaust, diesel exhaust, smog and radiation from monitor or television. The pre-determined period could be variable, and it would depend on the features of the living skin-equivalent and the nature of the non-environmental stressor. However, the pre- determined period is sufficiently long to induce the intended changes in the living skin- equivalent but is not long enough to irreversibly affect the barrier function and cell viability which would prevent any further analysis using the same LSE. For example, in some cases, this pre-determined period is preferably from 1 to 60 minutes, more preferably 5 to 40 minutes and most preferably from 10 to 30 minutes. However, for the oxidative environmental stressor the amount, rather than time, is more important. The amount of the oxidative environmental stressor which contacts the 3D living skin- equivalent model comprising primary human keratinocytes is also variable, and it would also depend on the features of the living skin-equivalent and the nature of the oxidative environmental stressor. However, the amount is sufficient to induce the intended changes in the living skin-equivalent but is not enough to irreversibly affect the barrier function and cell viability which would prevent any further analysis using the same living skin- equivalent

The next step (ii) involves measuring, by any known means, the extent to which barrier function of the skin-equivalent is affected. Preferably this is measured by permeability assay using a dye on the LSE. Preferably this assay is Lucifer Yellow Assay and the dye is Lucifer Yellow. In this case, contact of the 3D living skin-equivalent model with said non-environmental stressor and the oxidative environmental stressor increases intensity of fluorescence in the basal medium due to increased penetration of Lucifer yellow, while contact with the cosmetic ingredient decreases the intensity, where the intensity is directly proportional to the barrier function.

In addition to, or instead of, the permeability assay, the extent to which said barrier function of said skin-equivalent is affected is preferably measured by biomarker analysis. In this case the preferred biomarker is filaggrin. Further, in such a case, contact of the 3D living skin-equivalent model with said non- environmental stressor and the oxidative environmental stressor downregulates the biomarker while contact with the cosmetic ingredient upregulates it, where the extent of the upregulation is directly proportional to the barrier function. All other alternative means of measuring the barrier function of the skin are within the knowledge of persons skilled in the art.

The next step (iii) involves determining the number of keratinocytes remaining viable after the contact. Such viability analysis could be performed by any method known in the art. Preferably the cell viability is measured by MTT assay. The MTT assay is a

colorimetric assay for assessing cell metabolic activity. NAD(P)H-dependent cellular oxidoreductase enzymes may, under defined conditions, reflect the number of viable cells present. Contact of the LSE with the non-environmental stressor and the oxidative environmental stressor reduces the number of viable cells, whereas contact with the cosmetic ingredient increases the number.

Keratinocytes are generally recognized as the predominant cell type in the epidermis, typically constituting about 95% of the cells. Keratinocytes are formed by differentiation from epidermal stem cells residing in the lower part of the stratum basale layer of the epidermis. Keratinocytes divide and differentiate as they move upward through the layers of the epidermis (e.g., stratum spinosum and stratum granulosum) to eventually become corneocytes in the stratum corneum. During the differentiation process, keratinocytes produce more and more keratin ("cornification") and eventually permanently withdraw from the cell cycle to form the corneocytes that make up the hard, outer layer of the stratum corneum. Corneocytes are eventually shed off through desquamation as new cells come in. When oxidative stress reduces the metabolism of keratinocytes, the rate at which the keratinocytes divide and differentiate may be reduced or even stop. This, in turn, may reduce the rate at which lost corneocytes are replaced in the stratum corneum and ultimately lead to an undesirable decrease in the barrier properties of the skin. Oxidative stress is caused by excessive production of reactive oxygen species (ROS) in comparison to the normal ability of the cellular mechanisms to respond to such changes and detoxify or repair as necessary. ROS are generally linked with toxic effects as they generate free radicals such as peroxides and superoxides. Free radicals are highly reactive. They can cause significant damage to the cellular structural membranes, lipids, proteins and nucleic acids. Free radicals are also linked to inflammatory conditions. Then in the following step (iv), the model is contacted with the cosmetic ingredient. The cosmetic ingredient is contacted with the 3D living skin-equivalent model in an amount and for a time effective to induce the desired changes in the skin model. For example, the cosmetic ingredient may be used topically or systemically in the range of from about 0.000001 % to about 5%, including all amounts therebetween, such as about 0.1 %, by total weight of the composition applied. Alternatively, the step (iv) is carried out along with or prior to the step (i).

In step (v), the steps (ii) and (iii) are repeated. In other words, the extent to which the barrier function of the skin-equivalent is affected, and the number of viable keratinocytes is measured again.

Finally, in step (vi), it is inferred that the ingredient is efficacious if there is improvement in cell viability and barrier function. What amounts to an improvement in this context is well within the knowledge of the skilled person.

Contact of the living skin-equivalent model with a non-environmental stressor for a pre- determined period is important. This contact is before step (i) or (alternatively) between the steps (i) and (ii). Before step (i), means prior to, and between said steps (i) and (ii), means after contact with the oxidative environmental stressor. It is preferred that the living skin-equivalent model is contacted with the non-environmental stressor for a pre- determined period before contact with the oxidative environmental stressor.

This pre-determined period could also be variable, and it would depend on the features of the living skin-equivalent and the nature of the non-environmental stressor. However, the pre-determined period is sufficiently long to induce the intended changes in the living skin-equivalent but is not long enough to irreversibly affect the barrier function and cell viability which would prevent any further analysis using the same living skin-equivalent. For example, in some cases, this pre-determined period is preferably from 1 to 60 minutes, more preferably 5 to 40 minutes and most preferably from 10 to 30 minutes. In this case the time, rather than the amount is important. Similarly, the amount of the non-environmental stressor which contacts the 3D living skin-equivalent model comprising primary human keratinocytes is also variable, and it would also depend on the features of the living skin-equivalent and the nature of the oxidative environmental stressor. However, the amount is sufficient to induce the intended changes in the living skin-equivalent but is not enough to irreversibly affect the barrier function and cell viability which would prevent any further analysis using the same living skin-equivalent.

It is preferred that the non-environmental stressor is a surfactant, an abrasive agent, a desquamation agent, an organic sunscreen or an antiperspirant. However, the non- environmental stressor is the one that induces stress upon contact with skin. More preferably the non-environmental stressor is a surfactant. The preferred surfactant is a non-ionic or anionic surfactant. An example of anionic surfactant is sodium dodecyl sulphate.

Preferably the cosmetic ingredient is an antioxidant. It is preferred that the antioxidant is one or more of vitamin C or its derivatives, e.g., including ascorbyl glucoside, phenols, polyphenols such as tannins, ellagic acid and tannic acid, tea extracts such as green tea extracts; anthocyanins; rosemary extracts; phenol acids, stilbenes in particular resveratrol, derivatives of sulphur amino acids such as S-carboxymethyl-cysteine; ergothioneine; N- acetylcysteine, carotenoids, retinoic acid, retinol, flavonoids, vitamin E, sulfated polysaccharides or lignans.

Upon such contact the step (ii) and (iii) of the method of the invention as disclosed hereinbefore are repeated, in that order, to determine the effect of the ingredient on cell viability and barrier function. Based on the observations, certain inferences are drawn. It is inferred that the ingredient is efficacious if there is improvement in cell viability (of the keratinocytes) and the barrier function.

Method to demonstrate efficacy of a cosmetic ingredient

In another aspect is disclosed an in vitro method to demonstrate efficacy of a cosmetic ingredient against stressors, said method comprising the steps of: (i) contacting a LSE comprising primary human keratinocytes with an oxidative environmental stressor for a pre-determined period;

(iii) determining the number of viable keratinocytes;

(iv) contacting said LSE with said cosmetic ingredient;

(v) repeating said steps (ii) and (iii); and,

where said step (iv) is alternatively carried out along with or prior to said step (i), wherein said LSE is contacted with a non-environmental stressor for a pre-determined period, before step (i) or between said steps (i) and (ii) and further wherein the extent to which said barrier function is affected is measured by permeability assay using a dye. The method of the invention is useful to determine either the efficacy of a cosmetic composition as a whole, or of an active ingredient in the composition. Preferably the ingredient is an antioxidant such as, for example, retinoic acid.

The method in accordance with this invention can be used to distinguish between efficacy of a first composition from a second composition or alternatively could be useful to distinguish between efficacy of two or more ingredients, so as to choose one over the other. In this case, the steps are first carried out on a first composition or a first ingredient, as the case may be, and then repeated with a second different composition or the second ingredient, to thereby compare the efficacy.

The demonstration could be useful for any consumer promotion event, or a consumer demonstration such as in a mall or supermarket or at a consumer fair. The

demonstration may also be useful for claim support and advertising. The Cosmetic Composition

The cosmetic compositions comprise a cosmetically acceptable carrier and other ingredients usually present in cosmetic compositions. Their selection depends on the type and the nature of the cosmetic composition.

Cosmetically acceptable carriers suitable for use in this invention may include mineral oils, di and triglyceride oils, silicone oils, synthetic or natural esters, and alcohols. Total amounts of these materials may range from about 0.1 to about 50%, and preferably, from about 0.1 to about 30%, and most preferably, from about 1 to about 20% by weight of the cosmetic composition.

Silicone oils are divided into the volatile and non-volatile variety. The term "volatile" as used herein refers to those materials which have a measurable vapor pressure at ambient temperature (e.g. 25°C). Volatile silicone oils are preferably chosen from cyclic or linear polydimethylsiloxanes containing from about 3 to about 9, and preferably, from about 4 to about 5 silicon atoms.

Linear volatile silicone materials generally have viscosities of less than about 5 centistokes at 25°C. while cyclic materials typically have viscosities of less than about 10 centistokes.

Nonvolatile silicone oils useful as a carrier material in the cosmetic composition include polyalkyl siloxanes, polyalkylaryl siloxanes and polyether siloxane copolymers. The essentially non-volatile polyalkyl siloxanes useful herein include, for example, polydimethylsiloxanes (like dimethicone) with viscosities of from about 5 to about 100,000 centistokes at 25°C.

Silicone oils (especially, Dimethicone 35 to 75 centistokes suitable for use are often made commercially available from Dow Corning and are preferred. Among suitable esters are: alkenyl or alkyl esters of fatty acids having 10 to 20 carbon atoms like isopropyl palmitate, isopropyl isostearate, isononyl isonanonoate, oleyl myristate, isopropyl myristate, oleyl stearate, and oleyl oleate;

ether-esters such as fatty acid esters of ethoxylated fatty alcohols;

Polyhydric alcohol esters such as ethylene glycol mono- and di-fatty acid esters, diethylene glycol mono- and di-fatty acid esters, polyethylene glycol (200-6000) mono- and di-fatty acid esters, propylene glycol mono- and di-fatty acid esters, polypropylene glycol 2000 monooleate, polypropylene glycol 2000 monostearate, ethoxylated propylene glycol monostearate, glyceryl mono- and di-fatty acid esters, polyglycerol poly-fatty esters, ethoxylated glyceryl monostearate, 1 ,3-butylene glycol monostearate, 1 ,3-butylene glycol distearate, polyoxyethylene polyol fatty acid ester, sorbitan fatty acid esters, and polyoxy- ethylene sorbitan fatty acid esters;

wax esters such as beeswax, spermaceti, myristyl myristate, stearyl stearate; and

sterol esters, of which soya sterol and cholesterol fatty acid esters are examples thereof.

Emulsifiers are preferably present in the cosmetic composition of the present invention. Total concentration of the emulsifiers may range from about 0.1 to about 40%, and preferably, from about 1 to about 20%, and most preferably, from about 1 to about 7% by weight of the cosmetic composition. The emulsifier may be selected from the group consisting of anionic, nonionic, cationic and amphoteric actives. Particularly preferred nonionic actives are those with a C10 to C20 fatty alcohol or acid hydrophobe condensed with from about 2 to about 100 moles of ethylene oxide or propylene oxide per mole of hydrophobe; C2 to C10 alkyl phenols condensed with from 2 to 20 moles of alkylene oxide; mono- and di- fatty acid esters of ethylene glycol; fatty acid monoglyceride; sorbitan, mono- and d-Cs to C20 fatty acids; and polyoxyethylene sorbitan as well as combinations thereof. Alkyl polyglycosides and saccharide fatty amides (e.g. methyl gluconamides) are also suitable nonionic emulsifiers. Preferred anionic emulsifiers include alkyl ether sulfate and sulfonates, alkyl sulfates and sulfonates, alkylbenzene sulfonates, alkyl and dialkyl sulfosuccinates, Cs to C20 acyl isethionates, Cs to C20 alkyl ether phosphates, alkylethercarboxylates and combinations thereof.

Cationic emulsifiers that may be used include, for example,

palmitamidopropyltrimonium chloride, distearyldimonium chloride and mixtures thereof. Useful amphoteric emulsifiers include cocoamidopropyl betaine, C12 to C20 trialkyl betaines, sodium lauroamphoacetate, and sodium laurodiamphoacetate or a mixture thereof.

Other generally preferred emulsifiers include glyceryl stearate, glycol stearate, stearamide AMP, PEG-100 stearate, cetyl alcohol as well as emulsifying/thickening additives like hydroxyethylacrylate/sodium acryloyldimethyl taurates

copolymer/squalane and mixtures thereof.

In addition, antimicrobial compounds could be included in the cosmetic compositions of this invention to protect against the growth of potentially harmful microorganisms. However, preservatives are regulated ingredients with upper limitations defined by regulatory agencies. In addition, many preservatives are skin sensitizers and it is preferable to use the lowest concentration of preservative and protect against potentially harmful microorganisms which can spoil the product and pose a consumer safety risk. Suitable traditional antimicrobial compounds for cosmetic compositions of this invention are alkyl esters of para-hydroxybenzoic acid. Other antimicrobial compounds which have more recently come into use include isothiazolinones, DMDM hydantoin derivatives, propionate salts, and a variety of quaternary ammonium compounds, e.g., iodopropynyl butyl carbamate, phenoxyethanol, methyl paraben, propyl paraben, imidazolidinyl urea, sodium dehydroacetate and benzyl alcohol. It i9s preferred that compositions of the invention comprise 0.02%, 0.06% or 0.15% preservative.

Thickening agents may optionally be included in cosmetic compositions of the present invention. Particularly useful are the polysaccharides. Examples include starches, natural/synthetic gums and cellulosics. Representative of the starches are chemically modified starches such as sodium hydroxypropyl starch phosphate and aluminum starch octenylsuccinate. Tapioca starch is often preferred. Suitable gums include xanthan, sclerotium, pectin, karaya, arabic, agar, guar, carrageenan, alginate and combinations thereof. Suitable cellulosics include hydroxypropyl cellulose,

hydroxypropyl methylcellulose, ethylcellulose and sodium carboxy methylcellulose. Synthetic polymers are yet another class of effective thickening agent. This category includes crosslinked polyacrylates such as the Carbomers, polyacrylamides such as Sepigel® 305 and taurate copolymers such as Simulgel EG® and Aristoflex®AVC, the copolymers being identified by respective I NCI nomenclature as Sodium

Acrylate/Sodium Acryloyldimethyl Taurate and Acryloyl Dimethyltaurate/Vinyl

Pyrrolidone Copolymer. Another preferred synthetic polymer suitable for thickening is an acrylate-based polymer made commercially available by Seppic and sold under the name Simulgel® INS100.

Amounts of the thickener, when used, may range from about 0.001 to about 5%, and preferably, from about 0.1 to about 3%, and most preferably, from about 0.1 to about 1.5% by weight of the cosmetic composition.

Conventional humectants may be employed in the compositions of the present invention. These are generally polyhydric alcohol-type materials. Typical polyhydric alcohols include glycerol (i.e., glycerine or glycerin), propylene glycol, dipropylene glycol, polypropylene glycol, polyethylene glycol, sorbitol, hydroxypropyl sorbitol, hexylene glycol, 1 ,3-butylene glycol, isoprene glycol, 1 ,2,6-hexanetriol, ethoxylated glycerol, propoxylated glycerol and mixtures thereof. Most preferred is glycerin, propylene glycol or a mixture thereof. The amount of humectant may range anywhere from 0.5 to 25%, preferably between 1 and 20% by weight of the cosmetic

composition.

Fragrances, colorants, fixatives and abrasives may optionally be included in cosmetic composition of the present invention. Each of these substances may range from about 0.05 to about 5%, preferably between 0.1 and 3% by weight. The cosmetic compositions may include opacifiers like T1O2 and ZnO and colorants like iron oxide red, yellow and black. Such opacifiers and colorants typically have a particle size from 50 to 1200 nm, and preferably, from 50 to 350 nm. Cosmetic compositions of the present invention may include vitamins. Illustrative vitamins are Vitamin A (retinol) as well as retinol esters like retinol palmitate and retinol propionate, Vitamin B₂, Vitamin B3 (niacinamide), Vitamin B_Q, Vitamin C, Vitamin E,

Folic Acid and Biotin. Derivatives of the vitamins may also be employed. For instance, Vitamin C derivatives include ascorbyl tetraisopalmitate, magnesium ascorbyl phosphate and ascorbyl glycoside. Derivatives of Vitamin E include tocopheryl acetate, tocopheryl palmitate and tocopheryl linoleate. DL-panthenol and derivatives may also be employed. Total amount of vitamins when present in cosmetic compositions according to the present invention may range from 0.001 to 10%, preferably from 0.01% to 1%, optimally from 0.05 to 0.5% by weight of the cosmetic composition.

Azelaic acid, ubiquinone, dihydroxyacetone (DHA) and mixtures thereof may also be used as actives in the cosmetic composition of this invention. Such compounds, when used, typically make up from about 0.2 to 10%, and preferably, from about 0.5 to 5% by weight of the cosmetic composition.

Other optional actives suitable for use in this invention include resveratrol, resorcinols like 4-ethyl resorcinol, 4-hexyl resorcinol, 4-phenylethyl resorcinol, dimethoxytoluyl propyl resorcinol, 4-cyclopentyl resorcinol, 4-cyclohexylresorcinol, alpha-an/or beta- hydroxyacids, phenylethyl resorcinol (Symwhite® 377 from Symrise), undecylenol phenylalanine (Seppi White® from Seppic) mixtures thereof or the like. Such actives, when used, collectively make up from about 0.001 to about 12% by weight of the cosmetic composition.

A variety of other herbal extracts may optionally be included as actives in cosmetic compositions of this invention. The extracts may either be water soluble or water- insoluble carried in a solvent which respectively is hydrophilic or hydrophobic. Water and ethanol are the preferred extract solvents. Illustrative extracts include those from green tea, yarrow, chamomile, licorice, aloe vera, grape seed, citrus unshui, willow bark, sage, thyme and rosemary. Soy extracts may be used and especially when it is desirable to include retinol.

Also, optionally suitable for use include materials like chelators (e.g., EDTA), Cs to C22 fatty acid substituted saccharides, lipoic acid, retinoxytrimethylsilane (available from Clariant Corp. under the Silcare® 1 M-75), dehydroepiandrosterone (DHEA) and combinations thereof. Ceramides (including Ceramide 1 , Ceramide 3, Ceramide 3B and Ceramide 6) as well as pseudoceramides may also be useful. Occlusives like Oilwax® LC are often desired. Amounts of these materials may range from about 0.000001 to about 10%, preferably from about 0.0001 to about 3% by weight of the cosmetic composition.

Conventional buffers/pH modifiers may be used. These include commonly employed additives like sodium hydroxide, potassium hydroxide, hydrochloric acid, citric acid and citrate/citric acid buffers. In an especially preferred embodiment, the pH of the cosmetic composition of this invention is from about 4 to about 8, and preferably, from about

4.25 to about 7.75, and most preferably, from about 6 to about 7.5, including all ranges subsumed therein. The cosmetic composition of this invention may be a solid stick or bar. Viscosity of the cosmetic composition of this invention is, however, preferably from about 1 ,000 to about 120,000 cps, and most preferably, from about 5,000 to 80,000 cps, taken at ambient temperature NS and a shear rate of 1 s.sup.-1 with a strain controlled parallel plate rheometer made commercially available from suppliers like T.A. Instruments under the Ares name.

The cosmetically acceptable carrier may constitute from 10 to 99.9%, preferably from 50 to 99% by weight of the composition, and can, in the absence of other personal care adjuncts, form the balance of the composition.

When the composition is a deodorant or an antiperspirant, the compositions preferably comprise a conventional deodorant base as the cosmetically acceptable carrier. By a deodorant is meant a product in the stick, roll-on, or propellant medium which is used for personal deodorant benefit e.g. application in the under-arm area which may or may not contain anti-perspirant actives. Alternatively, the cosmetic composition of the invention is a rinse-off cosmetic

composition. Preferably the rinse-off composition of the invention is a face wash.

Alternatively, it is a body wash. Further alternatively it is in the form of a wipe, which comprises a suitable carrier such as cotton or sponge. Yet further alternatively it is a mist.

“Hair Care Composition" as used herein, is meant to include a composition for topical application to hair. Non-limiting examples of such compositions include leave-on hair lotions, creams, arid wash-off shampoos, conditioners, shower gels, or a toilet bar. When the composition of the invention is a hair care composition, it preferably is a wash-off composition, especially shampoo or a conditioner.

When the composition of the invention is a shampoo, it preferably comprises other ingredients which are generally included in such compositions. A shampoo preferably comprises 1 to 20 wt%, more preferably 2 to 16 wt%, furthermore preferably from 3 to 16 wt% anionic surfactants, e.g. an alkyl sulphate and/or ethoxylated alkyl sulfate surfactant. Preferred alkyl sulfates are Cs-is alkyl sulfates, more preferably C12-18 alkyl sulfates, preferably in the form of a salt with a solubilising cation such as sodium, potassium, ammonium or substituted ammonium.

The shampoo composition further preferably comprises a suspending agent. Suitable suspending agents are polyacrylic acids, cross-linked polymers of acrylic acid, copolymers of acrylic acid with a hydrophobic monomer, copolymers of carboxylic acid- containing monomers and acrylic esters, cross-linked copolymers of acrylic acid and acrylate esters, heteropolysaccharide gums and crystalline long chain acyl derivatives. The long chain acyl derivative is desirably selected from ethylene glycol stearate, alkanolamides of fatty acids having from 16 to 22 carbon atoms and mixtures thereof. Ethylene glycol distearate and polyethylene glycol distearate are preferred long chain acyl derivatives, since these impart pearlescence to the composition. Polyacrylic acid is available commercially as Carbopol® 420, Carbopol® 488 or Carbopol® 493.

Polymers of acrylic acid cross-linked with a polyfunctional agent may also be used; they are available commercially as Carbopol® 910, Carbopol® 934, Carbopol® 941 and Carbopol® 980. Suitable cross-linked polymers of acrylic acid and acrylate esters are Pemulen® TR1 and Pemulen® TR2. A suitable hetero polysaccharide gum is xanthan gum, for example that available as Kelzan. The invention will now be described in detail with the following non-limiting examples.

Examples

Example 1 : Lucifer yellow permeation assay

LSE (Epikutis®) was recovered with pre-warmed EpiRecovery® medium for around 20 hours.

Thereafter, the LSE was topically treated with 0.1% sodium dodecyl sulphate (SDS) (100 pL) for 30 minutes except for the control experiment in which it was treated with

Dulbecco’s phosphate buffered saline (DPBS) for 30 minutes. After removal of SDS, the LSE was washed with DPBS.

For the experiments involving SDS and squalene and SDS and squalene

hydroperoxide (SQOOH), LSE was then topically covered with 3 pL of SQ or SQOOH (1 % in SQ v / v), respectively, after the treatment with SDS.

For the experiments involving SDS, squalene hydroperoxide (SQOOH) and retinoic acid, 2 pM retinoic acid was added into the basal medium. Medium was then refreshed after 24 hours and all LSE samples were harvested at 48 hours after treatment with retinoic acid.

For Lucifer yellow permeation assay, the LSEs were washed with DPBS once. Then, 100 pL of lucifer yellow (1 mM in DPBS) was applied topically and incubated in a 37 °C incubator for appropriate time. LSE medium was sampled every 30 minutes for fluorescence intensity (excitation 425 nm, emission 528 nm) until adequate separation was observed among treatments. The fluorescence intensity of permeated lucifer yellow in different groups are summarized in Table 1. Table 1

Note:

• The data is presented as % of the control in mean ± SEM (n=3).

• One-way ANOVA followed by T ukey’s test was performed for statistical

analysis. Treatment without common letter annotation are significantly different. The data in Table 1 clearly demonstrates that intensity of fluorescence decreases dramatically in response to retinoic acid. This observation confirms the beneficial restorative/neutralizing effect of retinoic acid.

Example 2: Immunohistochemistry staining

Several epidermal differentiation and proliferation biomarkers are analyzed using immunohistochemistry to further investigate the functional and structural change of epidermis under stress challenge. Filaggrin (FLG) and Loricrin (LOR) are epidermal differentiation markers. They are indicators of full development of epidermal structure and barrier function. They are found in the upper epidermis (stratum corneum and stratum granulosum), and the intensity of staining, i.e., extent of staining, indicates the amount of these two proteins.

Ki67 is a proliferation marker. It is in the nucleus of epidermal basal cells. It is quantified by counting the positive stained cells amongst the epidermal cells.

LSE was treated with or without stress/cosmetic ingredient as mentioned in Example 1. After the treatment, LSE samples were fixed in 10% neutral buffered formalin solution, dehydrated and embedded in paraffin. Paraffin blocks were sliced into 5 pm sections. The slides then went through epitope retrieval and proper blocking steps to reduce the non-specific background noises. After several rounds of washes with PBS, primary antibody (filaggrin, loricrin or Ki67) was added onto the samples which were then incubated according to manufacturer's protocol. After several rounds of washes with PBS, the sections were incubated with horseradish peroxidase secondary antibodies conjugates for 30 minutes. Sections were then washed and completely covered by 3- amino-9-ethylcarbazole (AEC) single solution and incubated for 10 minutes and then rinsed four times in PBC. Slides were finally rinsed under tap water and then covered by cover slips using an aqueous mounting medium. Immunohistochemistry staining quantification method

Immunohistochemistry staining images were quantified using Inform 2.0 software (Perkin Elmer). Staining intensity of Filaggrin was quantified by measuring total optical density of red chromogen. The Ki67 positive cells were quantified by counting the number of cells stained with red chromogen in nuclei. The Ki67 protein (also known as MKi67) is a cellular marker for proliferation. It is strictly associated with cell

proliferation. During interphase, the Ki67 antigen can be exclusively detected within the cell nucleus, whereas in mitosis most of the protein is relocated to the surface of the chromosomes.

Results

As far as filaggrin is concerned, it was observed that the intensity of staining decreased upon contact with SDS and the addition of SQOOH further reduced the intensity. This observation indiciated the extent of damage caused to the barrier function of the living skin equivalent (LSE) when challenged with squalene monohydroperoxides (SQOOH) and sodium dodecyl sulphate (SDS) to mimic the damage caused by an oxidative environmental stressor and a non-environmental stressor. It was observed that treatment with retinoic acid increased the filaggrin level indiating again its benficial restorative effects. The observations are tabulated in Table 2. Table 2

Positive Ki67 staining cells were greatly reduced by a single stressor as well as the combination of stressors, especially under the combination of SDS and SQOOH as no Ki67 positive cell was found. On the other hand, retinoic acid mildly increased the Ki67 positive cells. The observations are tabulated in Table 3.

Table 3

Example 3: Model viability assessment (MTT assay)

LSE were treated with or without stressors as mentioned in Example 1. After the treatment, LSE inserts were placed in a 24-well plate containing 100 pL MTT solution (0.5 mg/mL) in each well and incubated in a 37°C for four hours. LSE was then cut off from inserts and incubated in 0.5 mL acidic isopropanol at 4°C for overnight. The optical density (OD) was read (100 pL/well) in a plate spectrophotometer at 570 nm

The data is summarised in Table 4.

Table 4

Note:

• The data is presented as % of the control in mean ± SEM (n=3).

• One-way ANOVA followed by T ukey’s test was performed for statistical

analysis. Treatment without common letter annotation are significantly different.

The data in table 4 indicates that SDS alone or SDS plus squalene only slightly decreased the viability but the combination of SDS and squalene monohydroperoxides (SQOOH) significantly decreased the viability. On the other hand, retinoic acid restored the viability effectively.

Claims

1 . An in vitro method to determine efficacy of a cosmetic ingredient against stressors, said method comprising the steps of:

(i) contacting a 3D living skin-equivalent (LSE) comprising primary human

(iii) determining the number of viable keratinocytes;

(iv) contacting said LSE with said cosmetic ingredient;

(v) repeating said steps (ii) and (iii); and,

2. A method as claimed in claim 1 wherein said non-environmental stressor is a surfactant, an abrasive agent, a desquamation agent, an organic sunscreen or an antiperspirant.

3. A method as claimed in claim 2 wherein said surfactant is a non-ionic or anionic

surfactant.

4. A method as claimed in any of claims 1 to 3 wherein said oxidative environmental

stressor generates Reactive Oxygen Species (ROS) in said LSE upon contact therewith.

5. A method as claimed in claim 4 wherein said oxidative environmental stressor is ozone, an oxide of nitrogen, a peroxide, ultraviolet radiation, PIV _.s or PM10.

6. A method as claimed in any of claims 1 to 5 wherein said cosmetic ingredient is an

antioxidant.

7. A method as claimed in claim 6 wherein said antioxidant is one or more of vitamin C or its derivatives, phenols, polyphenols, tea extracts; anthocyanins, rosemary extracts, phenol acids, stilbenes, derivatives of sulphur amino acids, metal complexes or esters, carotenoids, retinoic acid, retinol, flavonoids, vitamin E, sulfated polysaccharides or lignans.

8. A method as claimed in claim 1 to 7 wherein said assay is Lucifer Yellow Assay and said dye is Lucifer Yellow.

9. A method as claimed in claim 8 wherein contact of said LSE with said non-environmental stressor and said oxidative environmental stressor increases intensity of fluorescence, while contact with said cosmetic ingredient decreases the intensity, where the intensity is directly proportional to the barrier function.

10. A method as claimed in any of claims 1 to 9 wherein the extent to which said barrier

function is affected is measured by biomarker analysis in addition to or instead of said permeability assay.

1 1. A method as claimed in any of claims 1 to 10 wherein said cell viability of said

keratinocytes is measured by MTT assay.

12. A method as claimed in claim 1 1 wherein contact of said LSE with said non- environmental stressor and said oxidative environmental stressor reduces the number of viable cells, whereas contact with said cosmetic ingredient increases the number.

13. An in vitro method to demonstrate efficacy of a cosmetic ingredient against stressors, said method comprising the steps of:

(i) contacting a LSE comprising primary human keratinocytes with an oxidative environmental stressor for a pre-determined period;

(iii) determining the number of viable keratinocytes;

(iv) contacting said LSE with said cosmetic ingredient; (v) repeating said steps (ii) and (iii); and,