GB2602359A - Topical wash composition - Google Patents

Abstract

Method for providing germicidal benefit to skin while promoting growth of commensal bacteria, comprising contacting topical surface where undesired pathogens are present with a composition. The composition comprises: 0.1-10 % betaine surfactant (e.g. cocamidopropyl betaine); 01-5 % lactic acid; 0.1-20 % polyhydric C2-C6 alcohol (e.g. glycerol); 0-10 % polyethoxy carboxylate (preferably R is C12 alkyl, K is 11, M is Na); 0.1-19 % alkyl polyethoxy amide (preferably a primary or secondary polyethoxy amide e.g, PEG 4 rapeseed amide); >8 % alkyl ether sulphate (e.g. sodium laureth sulphate); 0-20 % surfactant; 0-10 % metal salt; and has a pH 3.8-4.5. The pathogens may be bacterial species e.g. S. epidermis. Also disclosed is a method for providing microbiome therapy using the composition, and the use of the composition: as a topical prebiotic; for increasing antimicrobial peptide production/activity; for increasing adherence of at least one bacteria selected from S. epidermis, C. xerosis, S. aureus, P acnes; and for treatment of dysbiosis.

Description

TOPICAL WASH COMPOSITION

FIELD OF THE DISCLOSURE

The present disclosure relates to the use of topical wash compositions which are particularly useful in personal care applications, e.g. topical skin care, cleansing, which compositions exhibit an appreciable antimicrobial benefit whilst promoting the growth of commensal bacteria within the skin microbiome.

BACKGROUND OF THE DISCLOSURE

Washing is one of life's fundamental requirements for cleansing in terms of dirt removal and hygiene maintenance.

The skin microbiome or microflora are microorganisms that colonise the skin. Bacterial species are by far the most numerous, although fungi, viruses and mites are also found on the skin of normal healthy humans.

The microbiome includes beneficial bacteria, referred to as commensal bacteria, such as the gram-positive bacterium Staphylococcus epidermidis (S epidermidis) and l'otynehrzeterium xerosis (C.xerosis) that provide benefits to the hosts. Examples of beneficial functions include the capacity to regulate inflammation after injury, enhance expression of host innate antimicrobial peptides, and mature T cell responses.

Specific strains of S. epidermidis have been shown to produce antimicrobials such as lantibiotics and phenol soluble modulins that act directing on pathogens. Lipopeptides produced by S. epidertnidis can activate epithelial keratinocytes to increase production of antimicrobial peptides such as B-defensins. S. epidennidis can be detected by dermal dendritic cells to enhance IL-I production and consequently expansion of T-cells producing IL-17. I0 -1 -

Dysbiosis or dysbacteriosis is defined as a negative disruption of the body's microbiota on or inside the body (e.g., skin flora, gut flora or vaginal flora) that is associated with disease. This imbalance can be due to the gain or loss of community members, or changes in the relative abundance of microbes. Such imbalance can be an opportunity for infection to set in, often resulting in a prolonged reliance on chemical intervention.

Traditional daily wash formulations often do not respect the commensal bacteria and results in dysbiosis.

Eubiosis refers to the optimal balance of microflora on or inside the body.

Pre-and probiotics have the capacity to optimise, maintain and restore the microbiome to the eubiotic state in a variety of ways. Topical application of probiotic bacteria has a direct effect on the site of application by enhancing the skin natural defence barriers. Prebiotics are used to nourish the commensal bacterial in the skin microbiome.

W02020/052916 discusses the use of a topical composition comprising saccharide isomerate for microbiome balancing. This emollient was shown to function as a prebiotic, preferentially supporting the growth of S. epidermic/is over S. aureaus. This composition is reliant on the good bacteria producing metabolites, such as lactic acid, in sufficient quantities to kill the bad bacteria. It is therefore reliant on the natural defence system, which is some individuals may be compromised.

Moisture in skin plays an important role in maintaining healthy skin. The skin's outer layer is covered with a fine film of water and lipids. The skin can only perform its function as a protective barrier if it has sufficient moisture. For example, skin lipids prevent the skin from drying out, loosing elasticity, and tearing, which could allow microorganisms, including harmful bacteria, to penetrate the epidermal barrier and colonise.

Traditional daily wash formulations contain active antimicrobial agents known to cause diving of the skin and hair. This limits the frequency of use of the wash composition, and often requires the use of conditioners or moisturizers.

There exists the need in the market for a topical wash composition which at optimal skin pH delivers a mild antibacterial action whilst at the same time promoting the growth of commensal bacteria. There also exists a need for a topical wash composition that provides skin moisturisation, particularly if the topical wash composition is for use in a feminine (intimate) area.

More particularly, there is a need for a topical wash composition, in particular a rinse-off wash composition, which respects the skin surface pH of between 5.0 and 6.0 whilst delivering a mild antibacterial action and at the same time promotes the growth of commensal bacteria to maintain eubioisis.

SUMMARY OF THE DISCLOSURE

According to a first aspect of the present disclosure there is provided a method for providing a germicidal benefit to a topical surface whilst promoting the growth of commensal bacteria; the method comprising the step of contacting a topical surface upon which the presence of one or more undesired pathogens are known or suspected, with a composition comprising: a) from 0.1 wt% to 10 wt% betaine surfactant, b) from 0.1 wt% to 5.0 wt% lactic acid, c) from 0.1 wt% to 20 wt% polyhydric C2-C6 alcohol, d) from 0 wt% to 10 wt% alkyl polyethoxy carboxylate, e) from 0 wt% to 10 wt% alkyl polyethoxy amide; 0 >8 wt% alkyl ether sulphate, water, h) a pH of 3.8-4.5, i) from I 0 to 20 wt% surfactant, and from 0 wt% to 10 wt% metal salt According to a second aspect of the present disclosure there is provided a method for providing microbiome therapy to a skin or mucosal surface, the method comprising: contacting a topical surface with a composition comprising: a) from 0.1 wt% to 10 wt% betaine surfactant, b) from 0.1 wt% to 5.0 wt% lactic acid, c) from 0.1 wt% to 20wt% polyhydric C2-C6 alcohol, d) from 0 wt% to 10 wt% alkyl polyethoxy carboxylate, e) from 0 wt% to 10 wt% alkyl polyethoxy amide; 0 >8wt% alkyl ether sulphate, water, h) a pH of 3.8-4.5, i) from 10 wt% to 20 wt% surfactant, and from 0 wt% to lOwt% metal salt.

According to a third aspect of the present disclosure there is provided a topical composition for use in providing an effective antimicrobial benefit against gram positive and gram negative bacteria whilst promoting the growth of commensal bacteria when the composition is applied on a surface of a human or animal body, which composition comprises: a) from 0.1 wt% to 10 wt% betaine surfactant, b) from 0.1 wt% to 5.0 wt% lactic acid, c) from 0.1 wt% to 20 wt% polyhydric C2-C6 alcohol, d) from 0 wt% to 10 wt% alkyl polyethoxy carboxylate, e) from 0 wt% to 10 wt% alkyl polyethoxy amide; >8 wt% alkyl ether sulphate, water, h) a pH of 3.8-4.5, i) from 0 wt% to 20 wt% surfactant, and from 0 wt% to 10 wt% metal salt. 30 -4 -According to a fourth aspect of the present disclosure there is provided the use of a topical composition comprising: a) from 0.1 wt% to 10 wt% betaine surfactant, b) from 0.1 wt% to 5.0 wt% lactic acid, c) from 0.1 wt% to 20 wit% polyhydric C2-C6 alcohol, d) from 0 wt% to I 0 wt% alkyl polyethoxy carboxylate, e) from 0 wt% to 10 wt% alkyl polyethoxy amide; 0 >8 wt% alkyl ether sulphate, g) water, h) a pH of 3.8-4.5, i) from 0 wt% to 20 wt% surfactant, and from 0 wt% to lOwt% metal salt, as a prebiotic when applied on a surface of the human body.

According to a fifth aspect of the present disclosure there is provided the use of a topical composition comprising: a) from 0.1 wt% to 10 wit% betaine surfactant, b) from 0.1 wt% to 5.0 wt% lactic acid, c) from 0.1 wt% to 20 wt% polyhydric C2-C6 alcohol, d) from 0 wt% to I 0 wt% alkyl polyethoxy carboxylate, e) from 0 wt% to 10 wt% alkyl polyethoxy amide, 0 >8wt% alkyl ether sulphate, water, h) a pH of 3.8-4.5, i) from 0 wt% to 20 wt% surfactant, and from 0.1 wt% to 10 wt% metal salt, for increasing antimicrobial peptide production and / or activity when applied on a surface of a human or animal body.

According to a sixth aspect of the present disclosure there is provided the use of a topical composition comprising: a) from 0.1 wt% to 10 wt% betaine surfactant, b) from 0.1 wt% to 5.0 wt% lactic acid, c) from 0.1 wt% to 20 wt% polyhydric C2-C6 alcohol, d) from 0 wt% to 10 wt% alkyl polyethoxy carboxylate, e) from 0 wt% to 10 wt% alkyl polyethoxy amide, 0 >8wt% alkyl ether sulphate, water, h) a pH of 3.8-4.5, i) from I 0 wt% to 20 wt% surfactant, and from 0 wt% to 10 wt% metal salt, for increasing adherence of at least one of the bacteria selected from the group consisting of S. epidermis, C. xerosis, aureus and P. acnes when applied on a surface of a human or animal body.

According to a seventh aspect of the present disclosure there is provided a topical composition comprising: a) from 0.1% to 10wt% betaine surfactant, b) from 0.1% to 5.0wt% lactic acid, c) from 0.1% to 20wt% polyhydric C2-C6 alcohol, d) from 0% to I Owt% alkyl polyethoxy carboxylate, e) from 0. I % to I Owt% alkyl polyethoxy amide, 0 >8wt% alkyl ether sulphate, water, h) a pH of 3.8-4.5, i) from 0 wt% to 20 wt% surfactant, and from 0 wt% to 10wt% metal salt for use in the treatment of dysbiosis.

The uses or methods as described herein may be therapeutic or non-therapeutic. The non-therapeutic use or method may be a cosmetic cleaning or cleansing operation. The composition may be widely used within personal care or personal cleansing. -6 -

The topical wash compositions as herein described provide a combination of properties such as antiseptic, i.e., antimicrobial activity, prebiotic, cleansing, conditioning, and moisturizing, together with safety and efficacy, ease of storage and use, and aesthetic properties.

It is to be further expressly understood that the application of the topical composition may be to the skin on any part of the body, including the skin on the face, neck, chest, back, arms, axilla, hands, legs, scalp and female and male intimate areas.

The composition may be a wash-off/rinse off formulation. Such a formulation may be referred to in the art as a topical wash composition. A wash-off formulation is defined as a formulation which is intended/required to be removed from the body by washing with a solvent, such as water.

The topical composition may be a liquid hand wash or a foaming hand wash.

The composition may be formulated as a body wash. A non-limiting example of a body wash is a shower gel, foam or cream.

In some embodiments, the composition provides excellent creamy foaminess with surprisingly trouble-free rinse ability (removal with water).

The composition may be formulated as a leave-on composition. A leave-on formulation is defined as a formulation which is not required to be removed from the human body after application. The formulation may be in the form of a cream or lotion. Non-limiting examples include a hand sanitizer and a serum.

The composition may be formulated as a hair care product, such as shampoo, conditioner, rinse or other hair or scalp treatment. The formulation may be a medicated shampoo.

In an embodiment, the one or more undesired pathogens is/are bacterial species.

As used herein, the term "animal" includes, but is not limited to mammals which includes but is not limited to rodents, aquatic mammals, domestic animals such as dogs and cats, farm animals such as sheep, pigs, cows and horses, and humans. Wherein the terms animal or mammal or their plurals are used, it is contemplated that it also applies to any animals that are capable of the effect exhibited or intended to be exhibited by the context of the passage.

A healthy vagina is dominated by Lactobacillus, which is a non-spornu -positive bacilli that produces lactic acid, resulting in an acidic environment (pH 3-4). Vaginal pH correlates with total lactate concentration as the vaginal mucosa is also a rich source of lactic acid, a by-product of estrogen-regulated anaerobic glucose metabolism. The normal vaginal flora, acidic vaginal pH, and vaginal discharge are all components of the innate defence mechanisms that protect against vulvov aginal infections. The importance of vaginal lactic acid needs to be emphasized, as it correlates with vaginal health, inhibits the growth of bacteria associated with bacterial vaginosis, and possibly plays a role in the local immune defence.

Protection against dysbiosis is particularly important for pregnant women as it may lead to urinary track infections, upper genital tract infections, postpartum endometritis, pneumonia and puerperal sepsis.

Due to the risks associated with internal washing/douching, external feminine washes are considered more appropriate for intimate health, particularly those containing lactic acid, with an acidic pH that augments skin homeostasis and may serve as a helpful adjunct therapy in women with vaginal infections or taking antibiotics.

Accordingly, the disclosure may be specially formulated as a feminine (intimate) wash formulation for the external skin and mucous membranes of, e.g., the vulva or intimate area. Such a formulation may also be referred to in the art as a personal wash -g -composition. Advantageously, the formulation provides, at an appropriate pH for this sensitive area of the body, a combination of gentle cleansing, moisturising, freshness, and antibacterial protection whilst promoting the growth of the skin's microbiome, particularly the growth of S. epidermic/is.

When used as a feminine wash formulation it has been found that the composition provides / maximum./ optimal antibacterial benefits and furthermore is also gentle and clinically tested to be mild. The rinse-off formulation is also clinically proven to be moisturizing and helps protect from dryness and does not harm, indeed promotes the intimate natural microflora. The fommlation utilizes a natural, bio-similar active, lactic acid and provides mild antibacterial action at an appropriate pH for this area of the body. The formulation further provides balance to the intimate area by sustaining an optimal skin pH, moisturized skin and balanced vaginal flora.

The composition of the present disclosure provides as a primary technical benefit the reduction of undesired microorganisms, particularly in the reduction of both gram positive and gram negative microorganisms, while at the same time providing secondary benefits including promoting the growth of commensal bacteria on the skin, as well as skin conditioning and/or skin cleansing. Further ancillary benefits may be provided by the presence of one or more for the optional constituents which may be included in formulations or compositions according to the present disclosure.

In some embodiments the betaine surfactant is present in an amount of from 1 to 5 wt%, for example about 4.45wt%. In foam compositions for intimate personal use, the betaine surfactant may be present in an amount of from 0.1 to 12wt%, and for example about 0.6wt%.

Alkyl betaines are known surfactants which are mainly produced by carboxyalkylation, for example carboxymethylation of aminic compounds. Typical examples are the carboxymethylation products of hexyl methyl amine, hexyl dimethyl amine, octyl dimethyl amine, decyl dimethyl amine, dodecyl methyl amine, dodecyl dimethyl amine, dodecyl ethyl methyl amine, Cp/m cocoalkyl dimethyl amine, myristyl dimethyl amine, cetyl dimethyl amine, stearyl dimethyl amine, stearyl ethyl methyl amine, oleyl dimethyl amine, C16/18 tallow alkyl dimethyl amine and technical mixtures thereof. Alkyl amidobetaines which represent carboxyalkylation products of amidoamines are also suitable. Typical examples are reaction products of fatty acids containing 6 to 22 carbon atoms, namely caproic acid, caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, palmitoleic acid, stearic acid, isostearic acid, oleic acid, elaidic acid, petroselic acid, linoleic acid, linolenic acid, elaeostearic acid, arachic acid, gadoleic acid, behenic acid and erucic acid and technical mixtures thereof, with N,N-dimethylaminoethyl amine, N,N-dimethylaminoproply amine, N,N-diethylaminoethyl amine and N,Ndiethylaminoproply amine which are condensed with sodium chloroacetate.

In some embodiments the betaine is cocoamidopropyl betaine.

The lactic acid may be present in an amount of from 0.2 wt% to 8 wt%, for example between 0.3 wt% to 7 wt%, for example between 0.4 wt% to 5 wt%, for example between 2 wt% to 5 wt%, e.g. about 2.5 wt%. In foam compositions for intimate personal use, the lactic acid may be present in an amount of between 0.2 wt% to 5wt%, for example between 0.3 wt% to 51,-,4%, for example between 0.4 wt% to 5wt%, for example between 2 wt% to 5w1%, for example about 2.7.5wt?/.

In some embodiments the polyhydric C2-C6 alcohol is present in an amount of between 2 wt% to 18wt.%, for example between 6 wt% to 17wt%, for example between 8 wt% to 16w-t%, for example between 10 wt% to 15 wt%.

The polyhydric C2-Co alcohol may comprise glycerol.

In some embodiments the alkyl polyethoxy carboxylate is present in an amount of between 2 wt% to 7 wt%, for example about 4.9wt% -10-In some embodiments the alkyl polyethoxy amide is present in an amount of up to 8 wt%, such as up to 5 wt%, such as up to 4 wt%, such as up to 3wt%, In some embodiments the alkyl polyethoxy amide is present in an amount of greater than 0.1 wt%, such as greater than 0.2 wt%, such as greater than 0.5 wt%, such as greater than 1.0 wt%, such as greater than 1.5 wt%, The alkyl polyethoxy amide may be a primary alkyl polyethoxy amide, a secondary alkyl polyethoxy amide, or a combination thereof.

In some embodiments, the primary alkyl polyethoxy amide has the formula RO(CFLCH20)(CH2CONH2 wherein R is a C-C" alkyl, k is an integer from 0 to 20.

In some embodiments the alkyl polyethoxy carboxylate the R comprises Cu alkyl; K is 11 and M is Na.

In some embodiments, the secondary alkyl polyethoxy amide comprises or consists of PEG 4 rapeseed amide.

In some embodiments the alkyl ether sulphate is present in an amount of >12 wt%.

In some embodiments the metal salt is present in an amount of between 0 wt% to 5wt°,"0" for example between 0.2 wt% to 3 wt%, for example about 2w1.%.

The metal salt may comprise an alkali and / or alkaline metal halide. Suitable examples of alkali and / or alkaline metal halides include sodium and.1 or calcium chloride.

In some embodiments the alkyl ether sulphate comprises sodium laureth sulphate (SLS).

Notably sulphur containing surfactants, such as SLS, are inexpensive and are very effective foaming agents but are often associated with skin irritation. Surprisingly it has been found that the formulations disclosed herein when used for intimate personal use provide satisfactory cleaning and germ kill whilst avoiding issues caused by irritancy from these sulphur containing compounds In more detail it has been observed that the composition may be characterized in exhibiting at least a Ilogio reduction of various microorganisms / bacteria, such as E. Coll when tested according to the standardized test protocols of ASTM E2315-053 "Standard Guide for Assessment of Antimicrobial Activity Using a Time-Kill Procedure." pH It is contemplated that certain embodiments of inventive formulations may also provide a disinfecting or sanitizing benefit which is aided due to the low pH, particularly wherein the compositions are at a pH of 3.8-4.5.

Viscosity The compositions of the disclosure may be viscous and exhibit a viscosity of at least 500cps at room temperature as measured using a Brookfield viscometer, Type 3 spindle.

The compositions may exhibit viscosities in the range of at least about 500 cps as measured under these conditions. In some embodiments the topical compositions of the disclosure exhibit a viscosity in the range of about 1000 to about 10,000 cps, such as from about 1500 to 7000 cps, and such as from about 1500 to about 4500 cps.

Optional constituents A sequestrant may optionally be present. The overall level of sequestsa.nt present. (when present) may be within the range of 0.1 wt% to 10 wt%.

Particularly preferred sequestrants tor use with the invention are irninodisuccinc acid or its salts and/or DTPA (diethylene triamine pentaacetic acid) such as Dequest 2066 (Trade -12 -Mark; product available from Solutia Inc., St Louis 6366-6760" USA), EDT, and Dissolvine (GILDA / glutamate diacetate) EDE' (Trade Mark; product available from Akzo Nobel, Ciillingham, UK), The inventive compositions may optionally include further constituents useful in improving one or more aesthetic characteristics of the compositions or in improving one or more technical characteristics of the compositions.

Exemplary further optional constituents include colouring agents, fragrances and fragrance solubilizers, viscosity modifying agents includim?, one or more thickeners, pH adjusting agents and buffers including organic and inorganic salts, optical brighteners, °pacifying agents, hydrotropes, and preservatives, as well as other optional constituents providing improved technical or aesthetic characteristics known to the relevant art. When present, the total amount of such one or more optional constituents present in the inventive compositions do not exceed about 10 %wt._ more specifically do not exceed 5 %wt, and more specifically do not exceed 2.5 %wt.

These aspects and advantages of the disclosure are discussed in more detail particularly in reference to one or more of the examples set forth below.

BRIEF DESCRIPTION OF TUT FIGURES

FIG. I: illustrates the effect of Test Formulation El compared to Comparative Examples on commensal bacterial enumeration; FIG.2: illustrates the effect of Test Formulation El compared to Comparative Examples on bacterial adherence on reconstructed human epidermis; F1G.3illustrates the skin pH altering effect of Test Formulation E2; FIG.4: illustrates the effect of Test Formulation E2 on the gene expression of antimicrobial genes in Staphylococcus species within a subject's skin microbiome.

FIGS: illustrates the effect of Test Formulation E2 on the antimicrobial function of coagulase negative (CoNS) Streptococci species of bacteria within subject's skin 30 microbiome.

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DETAILED DESCRIPTION

TABLE 1: Exemplary Formulations Raw Material El E2 % w/w concentration % w/w concentration Deionised water 49.86 51.84 Tetrasodi um Glutamate Diacetate 0.40 0.40 Sodium Lauryl Ether Sulfate (70% active) 12.16 12.16 Sodium Laureth-11 carboxylate 4.85 4.85 PEG 4 rapeseedamide 1.70 1.70 Cocoamidopropyl Betaine (29% active) 4.45 4.45 Glycerin 10.00 10.00 Benzoic acid, sorbic acid, benzyl - 0.60 alcohol L-Lactic Acid (50% solution) 4.50 2.50 Sodium Hydroxide (30% solution) 2.08 1.25 Sodium Chloride (20% solution) 10.00 10.00 Fragrance/Perfume 0.25 100.00 100,00 -14-IN VITRO EVALUATION OF TEST FORMULATION El EXAMPLE 1: A comparison of the effect of the Test Formulation El and Comparative Examples on bacterial enumeration The effect of a 10% solution of Test Formulation El on bacterial growth of S. epidermis, C. xerosis,S mums and P. cranes was compared at 6, 24 and 48 hours against three Comparative Examples.

1 1 Method 1.1.1. Bacterial suspension Before each analysis, an amplification of each bacterial strain was performed using liquid and/or solid medium. After amplification, for each bacterial strain, a bacterial suspension was prepared using the assay medium and adjusted to an optical density at 600nm (0D6o01m) of 1. A mix of each bacterial strain was then prepared at I.67x107CFU./m1 for each strain.

1.1.2. Culture and treatment The mix of bacteria was transferred into 24-well plates containing (or not) the test compounds at a concentration of 10%. The bacteria were then incubated for 48 hours. AU experimental conditions were performed in n=3.

1.1.3 Bacterial enumeration Bacterial enumeration was performed using qPCR-Taqman.

1. 2. Results The results are shown in FIG. 1 (a)-(e).

A reduction or an increase in 2 log of bacterial growth was considered to be significant.

Surprisingly, and in contrast to the Comparative formulations, when the bacterial mix was exposed to Test Formulation El there was an immediate increase in the proliferation -15 -of S. epidermic/is. This result suggests that this formulation can selectively inhibit the growth of the bad bacteria (AS: auretts and P. acnes) whilst promoting the growth of the good bacteria (S. epidermidis) over at least a 48 hour time period.

It is speculated that the lactic acid in this formulation is acting as an anti-microbial against S. atireus and P. acnes, and a prebiotic for S. epidermic/is.

EXAMPLE 2: A comparison of the effect of the Formulation El and Comparative Examples on bacterial adherence on reconstructed human epidermis (RHE) The effect of Test Formulation El on bacterial adhesion of S. epidermis, C. xerosis, S. 10 aureus and P. aenes on RHE was compared against 3 Comparative Examples.

2.1 Method 2 I. I Culture and treatment A 0.5 cm2 section of RI-IF was treated (topical application 50 FIUME) with the test compounds or ultrapure water (control or non-infected condition) and pre-incubated for 24 hours. The topical treatments were renewed, and the bacterial mix was then added (infected condition) or not (non-infected condition) on the RUE and incubated for 4 hours. Bacteria mix removed in order to eliminate non-attached bacteria.

2.1.2. Bacterial adhesion Remaining (adherent) bacteria was quantified by gene analysis using qPCR-Taqman. 20 2.2 Results In the positive control condition, bacteria were present on the RHE regardless of the tested bacterial strain within the bacterial mix. Nevertheless, the bacterial proportion differed considerably within the bacterial mix. Bacterial adhesion capacity could be classified as follows (from the highest to the lowest): C. xero.sis>S. epidermis> S. (wrens P. acnes.

The results are shown in FIG. 2 (a)-(d). -16-

In response to the topical application of Test Formulation El (tested at 0.001% and 0.003%), a concentration-dependent increase of bacterial adhesion was observed for S auretts (148% and 202% of the infected control), S. epidermis (142% and 161% of the infected control) and P. acnes (279% and 369% of the infected control). At 0.003% an increase of 147% of the infected control was observed for the adhesion of C. rerosis.

In conclusion, the result suggest that Test Formulation El markedly promotes adhesion of all four tested bacterial species on the RI-1E. It is hypotheses that the adherence of all four bacterial species is advantageous because it provides a two-prong antimicrobial action against S. aurezts and P. acnes. The first prong is achieved via the antimicrobial effect of the topical wash composition on the adhered S. aurezts and P. acnes. The second prong is via the antimicrobial effect of the adhered commensal bacteria; S. epidermis and xerosts against the adhered S. aureus and P. acnes.

EXAMPLE 3: A comparison of the effect of the Inventive Formulation El and Comparative Examples on a gene expression profile in reconstructed human 15 epidermis (RHE) The effect of Test Formulation El on a gene expression profile in reconstructed human epidermis was assessed compared to a Comparative Example.

Extracted mRNA was analysed using a PCR array against 16 target genes, and one housekeeping gene (GADPH) selected for their importance in antibacterial activity and 20 inflammation.

Genes coding for antimicrobial peptides and markers involved in innate immunity tested were: CAMP, DEFB103A, DEFB4A, P13, RNASE7, SI00A7, TER2. Genes coding for cytokines/chemokines were: CCL5, CXCL1, CXCL6, IL8, IL23A, and also a marker of the oxidative stress response: HMOX I. -17-3.1 Method 3.1.1 Culture and treatment 0.5 cm2 sections of tenday old RHE was topically treated or not (control) with the test compounds or PBS solution (control PBS) (25 L WHE) The RUE were then incubated for 48 hours. All experimental conditions were performed in n=3, except for control conditions n=2.

The effects on gene expression were evaluated using reverse transcriptase quantitative (RT-q) PCR technology 3.1.2 Differential expression analysis TABLE 2: Classification Protocol 3.2 Results Neutrophils are known to play an essential role in the eliminating S. aureus from the host, and specifically from the skin. This has been shown partly to be as a result of Saureus stimulating production of neutrophil chemoattractants such as CXCL I, and IL-8 (CXCL8) by human keratinocytes. As shown in Table 4, Test Formulation El was >300% Strong stimulation Relative expression of control (% of control) Classification of effects Ind:Maffl. . . . . Shaken:" . . . . Slight simulation >150% and <200% >50% and <65% Moderate inhibition >30% and <50% Inhibition Strong iriftibition -18 -found to upregulate, by over 200%, the expression of both CXCL1 and IL8 at concentrations of 0.001% and 0.003%. It is hypothesised that the Test Formulation El enhances the keratinocyte's natural defence system, by recruiting neutrophils to the site of infection.

TEST FORNIULATION El Genes 0.001% % 0.003% % cycles control cycles control

GADPH GADPH

Housekeeping GAPDH 18.35 100 18.21 100 18.35 18.25 Keratinocyte FLG 18.46 126 18.45 109 differentiation 18.27 18.44 KRT10 15.93 95 15.93 81 15.92 16.13 CAMP 33.73 110 33.13 133 33.66 33.49 DEFB103A 22.64 106 22.02 145 22.54 22.02 DEFB4A 27.65 126 27.29 139 27.52 27.36 Antimicrobial peptide, innate immunity P13 17.61 153 17.08 200 17.59 17.11 RNASE7 21.98 98 21.57 120 21.98 21.58 SIO0A7 24.34 113 23.96 143 -19-24 46 23.91 TLR2 28.37 28.29 28.52 28.37 CCL5 CXCL1 30.28 30.33 28.64 28.94 * '..*& ** * * 29.63 29.90 28.84 28.85 Cytokines/Chemokines CXCL6 34.57 34.84 34.88 35.00 1L8 28.62 28.31 29 15 29.19 I 40 1L32A 32.14 31.60 30.85 31.02 Oxidative and cellular stress response IIMOX1 22.25 22.23 22.27 22.47 COMPARATIVE FORMULA 1 Genes 0.0011Y0 ?A 0.003% % cycles control cycles control -20 -

GAD PH GAD PH

Housekeeping

GAPDH 18.79

18.88 18.72 18.91 FLG 22.77 22.86 KRT10 20.62 20.54 CAMP 32.72 32.82 DEFB103A 21.88 21.91 DEFB4A 29.71 29.89 P13 21.15 20.60 37 20.44 19.28 12 19.24 305 34.78 63 35.03 23.74 65 23.66 31.72 12 31.16 21.62 12 21.64 Keratinocyte differentiation Antimicrobial peptide, innate immunity RNASE7 21.91 154 23.09 21.85 23.15 S100A7 27.14 2 28.84 27.49 28.87 TLR2 CCL5 29.68 71 31.11 29.58 30.85 29.86 176 31.09 29.85 31.74 CXCL1 29.87 195 34.69 29.56 33.60 Cytokines/Chemokines CXCL6 32.82 213 34.20 33.19 34.62 IL8 25.16 t.et,k\c, 29.48 -21 -20 29.22 IL32A 30.84 418 33.50 64 31.31 33.76 Oxidative and cellular IIMOX I 22.82 128 23.51 74 stress response 22.88 23.50

CLINICAL EVALUATION

A four (4) week clinical evaluation of the lactic acid containing Inventive Formulation E2 was performed to assess the effect on the skin defences.

EXAMPLE 4: Maintenance of the acid skin mantle The importance of skin pH to skin defence against pathogens has been well studied, particularly through maintaining an acid mantle on the skin. Acidification of the skin barrier with leave-on products has been demonstrated to have a number of benefits for skin. These include helping strengthen skin through improved barrier strength, stratum corneum integrity and improved lipid processing. Skin pH is also known to help commensal bacteria to thrive and remain attached and as a result crowd out any pathogenic bacteria.

The pH of the skin was measured at baseline (before washing), at T=0 immediately after washing and at T=1 hour to T=6 hours after washing. As shown in FIG. 3, Test Formulation E2 maintained the pH within the normal skin pH range whereas the Comparative Example resulted in an instant alkalisation of the skin.

As shown in Table 5 over a 2 week period of' use of Test Formulation E2 the hand pH dropped to 5.12 (p<0.001) and after 4 weeks dropped further to 4.93 (p<0.001). This clinical study demonstrates that the lactic-acid containing Test Formulation E2 preserved and improved the acid mantle of the hands.

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Table 5: Skin pH

EXAMPLE 5: Deposition of lactic acid residues on the skin The effect of the daily use over a four-week period of the Inventive Formulation E2 on the deposition of lactic acid residues on the skin was assessed.

5.1 Method Twenty (20) human subjects were provided with the Inventive Formulation E2 and the ASTM El 874-14 "Standard Test Method for recovery of Microorganisms from Skin Using the Scup Scrub Technique" was modified to collect samples of skin. Cup scrub samples were taken from the left volar arm of each subject. The samples were analysed by ultra-performance chromatography (UPLC) to measure the presence of lactic acid residues.

Washing protocol: Both hands were washed up to the elbows.

7 days prior to first collection, the subjects washed with a control wash for a minimum of 20 seconds each day.

At the baseline clinic assessment, the subjects washed with the Test Formulation E2 and a cup scrub sample was taken after 1 hour and 24 hours. The subjects continued to wash 20 daily with Test Formulation E2 and further cup scrub samples were taken at Weeks 2 and 4.

Mean A 4.93 5.12 5.33 5.48 Baseline A 5.48 5.48 5.48 % change -1 0% -3% <0.001 <0.001 0.064 A v. Baseline p= -7% ittest fled paired -23 - 5.2 Results The amount of lactic acid recovered from the surface of the left volar forearm of each subject was calculated as micrograms of lactic acid per cm2. For each subject, the within change from baseline to each timepoint (1 hour, 24 hours, 2 weeks and 4 weeks) was calculated, and is presented in Table 6.

Table 6: Lactic acid deposition (mg/cm2) N = 20 *From a Wilcoxon Signed Rank test under a null hypothesis that the change from baseline = 0 After 4 weeks, the lactic acid concentration was significantly higher than at baseline (p<0.0198). The number of subjects with higher lactic acid concentrations compared to baseline increased at each timepoint, such that at 4 weeks 75% of subjects have a higher concentration compared to their baseline value. The data demonstrates that the rinse-off Absolute Change Absolute Change Absolute Change Absolute Change Absob 5.04 2.23 -2.81 5.54 0.50 6.10 1.06 7.66 (1.749) (1.853) (6.128) (4.986) (5.173) (4.563) (7.4861 (2.708) 1.6 0.5 -7.3 1,0 -5.4 1.8 -4.4 0.00 5.24 1.79 -2.42 2.99 -0.98 4.48 0.65 5.60 10.7 6.8 -0.4 20.3 12.6 24 17.1 37.0 <0 0001 0 2815 0.4359 Mean Minimum Median p-value* (SD) Maximum -24 -Test Formulation E2 formulation can help to maintain the acid mantle of the skin via the deposition of lactic acid residues on the skin surface.

EXAMPLE 6: Analysis of antimicrobial peptide gene expression and efficacy analysis Antimicrobial peptide (AMP) sampling was taken from the right volar forearm of each subject at baseline, 15 minutes, and week 4. There were two sets of AMP sampling conducted at each time point; one for collection of bacterial DNA and a second for the collection of live bacteria Each set was conducted in duplicate, for a total of 4 swabs collected at each time point. One set of swabs was sampled from the lateral volar forearm, and the second set of swabs was sampled from the medial volar forearm.

6.1 AMP gene expression Analysis of skin swabs was performed for the expression of DNA of 5 AMP genes expressed by S. horn/ills or S. epidermidis.

6.1.1 Method PCR primers for the antimicrobial genes listed in Table 7 were designed and validated 20 against standard, qPR was performed The p-value was determined by both-end paired nonparametric t-test.

Table 7-POI Primers

S humility Lantibiotic A S hominis Lant b otic B Pep 5 S. epidermidis Esp S. epidermidis -25 -S. epidennidis Epi 6.1.2 Results the results are shown in Figure 4: (a) S. hominis -Lantibiotic A; (b) S. hominis -Lantibiotic B; (c) S. epidermic/is -pep5; (d) S. epidermic/is -esp.

An increase in the absolute abundance of DNA for S. epidennidis -esp was seen by week 4. A trend towards an increasing abundance of Lantibiotic A, Lantibiotic B, and Pep 5 was also observed. This increase reflects an increase in the commensal bacteria residing within the skin microbiome, that can protect against colonisation and/or infection by pathogenic bacteria, such as S. aureits.

6.2 AMP efficacy The species of Staphylococci that do not give a positive coagulase test are considered Coagulase negative Staphylococci (CoNS). Traditionally, only S. aureus is considered coagulase positive, whilst S. epidermic/is, and S. hominis are coagulase negative Staphylococci.

Analysis of skin swabs was performed to determine the capacity of culturable CoNS to inhibit the growth of S. aureits.

6,2.1 Method A functional bioassay was performed from individual isolates of CoNS obtained from each swab. Each isolate was grown in liquid culture overnight. This culture media was then separate from the bacteria by ultrafiltration. S. auretts was then added to the sterile filtered conditioned media from each CoNS isolate and the growth of S.attrems -26 -determined. The capacity of each CoNS to inhibit at least 50% growth of S. aureus was measured at 24 hours and compared to known positive and negative controls. If a minimum of 10 individual isolates was cultured in a swab the proportion of isolates that inhibited &mucus was recorded.

6.2.2 Results The results are shown in Figure 5. Dots shown at 100% are swabs from which all isolated CoNS clones inhibited S. aureus.

An increase in the frequency of CoNS isolates recovered on swabs with the capacity to inhibit the growth of S. aureus was seen by week 4. These data are consistent with the observation of increased antimicrobial genes seen in the VCR data from analysis of duplicate swabs.

In conclusion, subjects in the clinical evaluation showed improvement in the host defense function of their skin microbiome after 4 weeks of treatment. This is hypothesised to represent an increase in the abundance of commensal bacteria that express genes encoding AMPs, proteins or other beneficial metabolites.

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Claims (1)

1. CLAIMS1. A method for providing a germicidal benefit to a skin or mucosal surface surface whilst promoting the growth of commensal bacteria; the method comprising the step of: contacting a topical surface upon which the presence of one or more undesired pathogens, are known or suspected, with a composition comprising: a) from 0.1 wt% to 1 0 wt% betaine surfactant, b) from 0.1 wt °A to 5.0 wt% lactic acid, c) from 0. I wt % to 20 wt% polyhydric C2-C6 alcohol, d) from 0 wt% to I 0 wt% alkyl polyethoxy carboxylate, e) from 0.1 wt% to 10 wt% alkyl polyethoxy amide, f) >8 wt% alkyl ether sulphate, (r) water, h) a pH of 3.8-4.5, i) from 0 wt% to 20 wt% surfactant, and.0 from 0 wt% to 10 wt% metal salt.The method according to claim 1, wherein the one or more undesired pathogens are bacterial species.The method according to claim I or 2, wherein the alkyl polyethoxy carboxylate is present in an amount of from 2 wt% to 7 wt%, The method according to any preceding claim, wherein in the alkyl polyethoxy carboxylate R comprises is a C12 alkyl; K is 11 and M is Na.5. The method according to any preceding claim, wherein the betaine surfactant is present in an amount from 3 wt% to 7wt%.The method according to any preceding claim, wherein the betaine comprises cocoamidopropyl betaine.-28 -The method according to any preceding claim, wherein the lactic acid is present in an amount of from 0.2 wt% to 8 wt%.The method according to claim 7, wherein the lactic acid is present in an amount of from 0.4 wt% to 5 wt%.The method according to claim 8, wherein the lactic acid is present in an amount of from 2 wt% to 5 wt% 10. The method according to claim 9, wherein the lactic acid is present in an amount of about 2.5 wt%.11. The method according to any preceding claim, wherein the polyhyd c C2-C6 alcohol comprises glycerol.12. The method according to any preceding claim, wherein the alkyl polyethoxy amide comprises a primary alkyl polyethoxy amide.13. The method according to claim 12, wherein the primary alkyl polyethoxy amide is of the formula RO(CH2CH20)kCH2CONH2 wherein R is a Cs-C.22 alkyl, k is an integer from 0 to 20.14. The method according to of claims 1 to 11, wherein the alkyl polyethoxy amide comprises a secondary alkyl polyethoxy amide.15. The method according to claim 14, wherein the alkyl polyethoxy amide comprises PEG 4 rapeseed amide.16. The method according to any preceding claim, wherein the alkyl ether sulphate comprises sodium laureth sulphate.-29 - 17. The method according to any preceding claim, wherein the commensal bacteria is AS: epidermis.18. The method according any preceding claim, wherein the topical surface is a dermal surface, such as an intimate dermal / external surface.19. The method according to any preceding claim, wherein the method further comprises the step of rinsing off the composition.20. A method for providing microbiome therapy to a skin or mucosal surface, the method comprising: contacting a topical surface with a composition comprising: a) from 0.1 wt% to 10 wt% betaine surfactant, b) from 0.1 wt% to 5.0 wt% lactic acid, c) from 0.1 wt% to 20 wt% polyhydric C2-C6 alcohol, d) from 0 wt% to 10 wt% alkyl polyethoxy carboxylate, e) from 0 wt% to 10 wt% alkyl polyethoxy amide; 0 >8 wt% alkyl ether sulphate, g) water, h) a pH of 3.8-4.5, i) from 0 wt% to 20 wt% surfactant, and j) from 0 wt% to I Ow-t% metal salt.21. A topical composition for use in providing an effective antimicrobial benefit against gram positive and gram negative bacteria whilst promoting the growth of commensal bacteria when applied on a surface of a human or animal body, in which the composition comprises: a) from 0. I wt% to 10 wt% betaine surfactant, b) from 0.1 w-t% to 5.0 wt% lactic acid, c) from 0. I wt% to 20 wt% polyhydric C9-C6 alcohol, d) from 0 wt% to I 0 wt% alkyl polyethoxy carboxylate, -30 -from 0.1 wt% to 10 wt% alkyl polyethoxy amide, >8 wt% alkyl ether sulphate, water, a pH of 3.8-4.5, from 0 wt% to 20 wt% surfactant, and from 0 wt% to 10 wt% metal salt.22. Use of a topical composition comprising: a) from 0.1 wt% to 10 wt% betaine surfactant, b) from 0.1 wt% to 5.0 wt% lactic acid, c) from 0.1 wt% to 20 wt% polyhydric C2-C6 alcohol, d) from 0 wt% to 10 wt% alkyl polyethoxy carboxylate, e) from 0.1 wt % to 10 wt% alkyl polyethoxy amide, 0 >8 wt% alkyl ether sulphate, g) water, h) a pH of 3.8-4.5, i) from 0 wt% to 20 wt% surfactant, and j) from 0 wt% to 10 wt% metal salt as a prebiotic when applied on a surface a human or animal body.23. Use of a topical composition comprising: a) from 0.1 wt% to 10 wt% betaine surfactant, b) from 0.1 wt % to 5.0 wt% lactic acid, c) from 0.1 wt % to 20 wt% polyhydric C2-C6 alcohol, d) from 0 wt% to lOwt% alkyl polyethoxy carboxylate, e) from 0.1 wt% to lOwt% alkyl polyethoxy amide, 0 >8 wt% alkyl ether sulphate, g) water, h) a pH of 3.8-4.5, i) from 0 wt% to 20 wt% surfactant, and -31 -j) from 0 wt% to 10 wt% metal salt, for increasing antimicrobial peptide production and / or activity when applied on a surface of a human or animal body.24. Use of a topical composition comprising: a) from 0.1 wt% to 10 wt% betaine surfactant, b) from 0.1 wt% to 5.0 wt% lactic acid, c) from 0.1 wt% to 20 wt% polyhydric C2-C6 alcohol, d) from 0 wt% to 10 wt% alkyl polyethoxy carboxylate, e) from 0.1 wt% to 10 wt% alkyl polyethoxy amide, f) >8 wt% alkyl ether sulphate, g) water, h) a pH of 3.8-4.5, i) from 0 wt% to 20 wt% surfactant, and j) from 0 wt% to 10 wt% metal salt for increasing adherence of at least one of the bacteria selected from the group consisting of S. epidermis, C. xerosis, S. aureus and P. acnes when applied on a surface a human or animal body.25. A topical composition comprising: a) from 0.1 wt% to 10 wt% betaine surfactant, b) from 0.1 wt% to 5.0 wt% lactic acid, c) from 0.1 wt% to 20 wt% polyhydric C2-(76 alcohol, d) from 0 wt% to 10 wt% alkyl polyethoxy carboxylate, e) from 0.1 wt% to 10 wt% alkyl polyethoxy amide, 0 >8wt% alkyl ether sulphate, g) water, h) a pH of 3.8-4.5, i) from 0 wt% to 20 wt% surfactant and j) from 0 wt% to 10 wt% metal salt for use in the treatment of dysbiosis.-32 -