An antimicrobial composition that selectively inhibits the growth of Propionibacterium acnes bacteria

Abstract

The present invention relates to a method and composition for preventing or treating acne by selectively inhibiting Propionibacterium acnes bacteria. The method comprises treating the skin with a combination of Propionibacterium acnes phage-derived endolysin and nucleic acid molecules encoding the same and selective essential oils.

Description

Technical Field

[0001] The present invention relates to a method and composition for preventing or treating acne by selectively inhibiting Propionibacterium acnes (P. acnes) bacteria. The method comprises treating the skin with an endolysin in combination with an essential oil compound. Background Art

[0002] Acne, also known as Acne vulgaris, is a common skin condition that affects almost all adolescents and adults at some stage in their lives. Its etiology is complex, involving abnormal keratinization, excessive sebum production, androgen function, bacterial growth and immune hypersensitivity. Although one or more of the above processes are associated with acne, a triggering factor and the exact sequence of events that lead to the formation of acne lesions are not yet fully understood. Other factors associated with acne are the presence of free radicals and the oxidative stress that subsequently causes cell damage. It has been observed that acne usually occurs in areas rich in sebaceous glands, such as the face, neck and back.

[0003] The bacterium Propionibacterium acnes is also associated with the occurrence of acne. It is one of the important and dominant bacteria that reside on the surface of human facial skin. Propionibacterium acnes is a facultative anaerobic, slow-growing, rod-shaped, Gram-positive bacterium. It is present in the sebaceous glands, and it constitutes an important part of the commensal microbiome of the skin. Propionibacterium acnes utilizes sebum and byproducts from the surrounding skin tissue as a source of energy and nutrition. This results in the release of some fatty acids, which can irritate the follicle wall and cause inflammation, leading to acne or acne vulgaris. Acne vulgaris is a chronic inflammatory disorder of the sebaceous glands of the hair follicles. Almost all adolescents are affected at some stage in their lives, with 15-20% suffering from moderate to severe forms of acne.

[0004] Acne has been treated in a variety of ways. Most treatments take weeks to months before noticeable changes are seen. Benzoyl peroxide, which has antibacterial properties, has been used in mild cases of acne. In very severe cases of acne, antibiotics such as tetracycline, erythromycin, and clindamycin have been used. Antibiotics are thought to work by several mechanisms, the most important being a reduction in the number of bacteria in and around the hair follicles. They are also thought to reduce irritating chemicals produced by white blood cells in the sebum, thereby reducing the inflammatory response. However, a disadvantage of antibiotics and other types of conventional antimicrobial treatments is that they are broad-spectrum and help kill or inhibit most bacteria on the skin.

[0005] As mentioned above, most treatments involve the use of synthetic chemicals. Many people do not like the use of synthetic chemicals because they believe that they are harmful to the human body. Some people believe that synthetic chemicals can cause side effects. Therefore, more and more people prefer to use "natural" materials, such as active substances based on herbal sources. In addition, recently, active substances based on microbial or biotechnological sources are also considered to be more "natural" than the purely synthetic methods currently used.

[0006] The skin is the largest organ of the human body and is colonized by a diverse array of microorganisms. Most microorganisms are considered to be either beneficial or opportunistic potential pathogens. Recent advances in DNA sequencing and computational biology are helping us to identify and understand the beneficial effects of these microorganisms on the skin at a higher resolution.

[0007] The interactions between skin microorganisms and human hosts can be divided into three categories: 1. commensality (one party benefits and the other party is not harmed), 2. mutualism (both parties benefit), and 3. parasitism (one organism benefits and the other party is harmed). Microorganisms that are usually associated with diseases are mainly called commensals. There is increasing evidence that commensals on the skin can help the immune system fight pathogens and maintain the homeostasis of the microbiome. An example is Staphylococcus epidermidis (a Gram-positive coagulase-negative staphylococcus), which plays an important role in acne suppression. Staphylococcus epidermidis mediates the fermentation of glycerol, which inhibits the overgrowth of Propionibacterium acnes, resulting in acne suppression.

[0008] Therefore, approaches that use broad-spectrum antibiotics that kill or inhibit the function of all microorganisms on the skin are finding less and less acceptance among healthcare practitioners. Approaches that selectively kill or inhibit microorganisms on the skin are increasingly favored by healthcare practitioners. Furthermore, the use of antibiotics is ineffective in treating antibiotic-resistant strains of P. acnes.

[0009] Therefore, there is a need to find new methods to treat problems such as acne in a selective manner. Therefore, the inventors began to work on providing a "natural" solution to the acne problem. They adopted a method to test new active substances that kill or selectively inhibit the growth of Propionibacterium acnes but not other organisms such as Staphylococcus epidermidis, Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus), and conducted experiments on them. They finally found a combination of a Propionibacterium acnes phage-derived endolysin and an essential oil compound, which was found to interact synergistically to selectively inhibit the growth of Propionibacterium acnes.

[0010] US2016346294 (Vyome Biosciences), entitled "Treatments for resistant acne", relates generally to new molecules, compositions and formulations for treating bacterial infections in general and more specifically bacterial infections with antibiotic-resistant pathogens. Among them, Staphylococcus capitis (S. capitis) (no specific mention of subspecies) is exemplified, which is considered a pathogen rather than an antimicrobial agent.

[0011] EP23488316B1 (Unilever, 2013) discloses a method for disinfecting a surface comprising a combination of thymol and terpineol and a wash-off composition comprising the same.

[0012] The above two publications do not teach that the combination of P. acnes phage-derived endolysin and essential oil compounds can be used to selectively inhibit the growth of P. acnes.

[0013] One object of the present invention is to provide selective killing or inhibition of Propionibacterium acnes for the treatment of acne.

[0014] Another object of the present invention is to provide a solution for treating acne by means of more natural or nature-based active substances. Summary of the invention

[0015] According to a first aspect of the present invention, there is provided an antimicrobial composition for selectively inhibiting the growth of Propionibacterium acnes bacteria, comprising

[0016] (i) an endolysin derived from a Propionibacterium acnes phage or a nucleic acid molecule encoding the same; and

[0017] (ii) an essential oil compound of the general formula 1 having the following structure

[0018]

[0019] wherein R1 is H, OH or OR, wherein R is an alkyl chain having 1 to 5 carbon atoms;

[0020] R2 is a C1 to C6 straight chain alkyl group; or a C3 to C6 branched chain alkyl group; or a C5 to C6 cycloalkyl group or heterocycloalkyl group; or a C6 aryl group.

[0021] According to another aspect of the present invention, there is provided a method for controlling or eradicating Propionibacterium acnes from the skin, comprising the step of applying the composition of the present invention to a desired skin surface. DETAILED DESCRIPTION

[0022] By reading the following detailed description and the appended claims, these and other aspects, features and advantages will become apparent to those of ordinary skill in the art. For the avoidance of doubt, any feature of one aspect of the present invention may be used in any other aspect of the present invention. The word "comprising" is intended to mean "including" but not necessarily "consisting of" or "composed of". In other words, the steps or options listed do not have to be exhaustive. It should be noted that the embodiments given in the following description are intended to illustrate the present invention, but are not intended to limit the present invention to these embodiments themselves. Similarly, unless otherwise indicated, all percentages are weight / weight percentages.

[0023] Except in the operating and comparative examples, or where otherwise explicitly indicated, all numbers in this specification indicating the amount of material or reaction conditions, the physical properties of the material and / or the use thereof should be understood to be modified by the word "about". Unless otherwise indicated, numerical ranges expressed in the format "x to y" should be understood to include x and y. When multiple preferred ranges are described in the format "x to y" for a particular feature, it should be understood that all ranges combining different endpoints are also intended.

[0024] Compositions according to the present invention can be in the form of a leave-on format or wash-off format for cleaning or disinfecting surface areas of mammals (particularly humans) such as skin and / or hair. Such compositions include any product applied to the human body that is also used to improve appearance, cleanliness, odor control or overall aesthetics. Compositions of the present invention can be in the form of a liquid, lotion, cream, foam or gel or toner, or applied with appliances or via a facial mask, pad or patch. "Skin" as used herein refers to the skin including face and body (e.g., neck, chest, back, arms, underarms, hands, legs, buttocks and scalp). It is particularly useful for treating acne in any other part of the face or body that forms acne.

[0025] The composition according to the present invention comprises an endolysin derived from a bacteriophage of Propionibacterium acnes and a nucleic acid molecule encoding the same. Particularly preferred compositions comprise an endolysin derived from a bacteriophage of Propionibacterium acnes.

[0026] Propionibacterium acnes (P. acnes) refers to Propionibacterium acnes, also known as Cutibacterium acnes.

[0027] The present inventors have investigated various methods for inhibiting P. acnes and have realized the use of bacterial phages and enzymes derived from bacterial phages for this purpose. One group of phage-derived enzymes are peptidoglycan (PG) hydrolases called endolysins. Endolysins are novel muralytic hydrolases encoded by double-stranded DNA phages that degrade the PG layer of the bacterial cell wall, allowing progeny phages to escape later in the infection cycle. Purified endolysins can cause "lysis from the outside" when exposed to PG externally. Endolysins from phages that infect Gram-positive pathogens were the inventors' motivation and hypothesis as an element of the composition of the present invention based on their antimicrobial properties (with particular substrate specificity and high activity when added externally).

[0028] Therefore, it is useful that the endolysin contained is a recombinant form of the endolysin of the Propionibacterium acnes phage. The endolysin is preferably cloned from the endolysin gene sequence (gene ID: NC_018851; 855 nucleotide base pairs long, 284 amino acids, protein ID: 97935.1) of the Propionibacterium phage 29399B_C (GenBank: JX262225.1), which is codon-optimized for expression in E. coli and cloned into the commercially available pET303 / CT-His expression vector.

[0029] The nucleotide sequence of a particularly useful endolysin is shown in the sequence ID SEQ ID1 listed below:

[0030] ATGCGTTATATTCCGGCAGCACATCATTCAGCAGGTAGCAATCATCCGGTTAATCGTGTTGTTATTCATGCAACCTGTCCGGATGTTGGTTTTCCGAGCGCAAGCCGTAAAGGTCGTGCAGTTAGCACCGCAAACTATTTTGCAAGCCCGAGCAGCGGTGGTAGCGCACATTATGTTTGTGATATTGGTGAAACCGTTCAGTGTCTGAGCGAAGGCACCATTGGTTGGCATGCACCGCCTAATCCGCATAGCCTGGGTATTGAAATTTGTGCAGATGGTGGTAGCCATGCAAGCTTTCGTGTTCCGGGTCATGCATATACCCGTGAACAGTGGCTGGATCCGCGTGTTTGGCCTGCAGTTGAAAAAGCAGCAATTCTGTGTCGTCGTCTGTGCGATAAATACAATGTTCCGAAACGTAAACTGAGCGCAGCAGATCTGAAAGCAGGTCGTCGTGGTGTTTGTGGTCATGTTGATGTTACCGATGCATGGCATCAGAGCGATCATGATGATCCGGGTCCGTGGTTTCCGTGGGATCGTTTTATGGCAGTTGTTAATGGTCATAACGAAAGCGGTGAACTGACCGTTGCAGATGTTAAAGCACTGCATGATCAGATTAAACAGCTGAGCGCACAGCTGGCAGGTAGCGTTAATAAACTGCATCATGATGTTGGTGTTGTTCAGGTTCAGAATGGTGATCTGGGTAAACGTGTTGATGCACTGAGCTGGGTTAAAAATCCGGTTACCGGTAAACTGTGGCGTACCAAAGATGCACTGTGGTCAGTTTGGTATTATGTTCTGGAATGTCGTAGCCGTATTGATCGTCTGGAAAGCGCAGTTAATGGTCTGAAAAAACTCGAGCACCACCACCACCACCACTGAGATCCGGCTGCTAA。

[0031] A particularly preferred aspect relates to an endolysin in which the stop codon has been removed from the 3' end of the gene to accommodate a 6X histidine tag.

[0032] Peptide sequence of endolysin with C-terminal histidine tag (x6)

[0033] MRYIPAAHHSAGSNHPVNRVVIHATCPDVGFPSASRKGRAVSTANYFASPSSGGSAHYVCDIGETVQCLSEGTIGWHAPPNPHSLGIEICADGGSHASFRVPGHAYTREQWLDPRVWPAVEKAAILCRRLCDKYNVPKRKLSAADL KAGRRGVCGHVDVTDAWHQSDHDDPGPWFPWDRFMAVVNGHNESGELTVADVKALHDQIKQLSAQLAGSVNKLHHDVGVVQVQNGDLGKRVDALSWVKNPVTGKLWRTKDALWSVWYYVLECRSRIDRLESAVNGLKKLEHHHHHH.

[0034] Nucleic acid molecules for endolysins optionally included in the compositions of the present invention may also comprise fragments, variants and fusions of the endolysins, which are capable of specifically binding to and / or lysing cells of P. acnes.

[0035] The composition of the present invention comprises an essential oil compound having the general structure

[0036]

[0037] wherein R1 is H, OH or OR, wherein R is an alkyl chain having 1 to 5 carbon atoms;

[0038] R2 is a C1 to C6 straight chain alkyl group; or a C3 to C6 branched chain alkyl group; or a C5 to C6 cycloalkyl group or heterocycloalkyl group; or a C6 aryl group.

[0039] The most preferred essential oil compounds according to the compound of formula 2 for use in the composition of the present invention are selected from: thymol, carvacrol, (E)-2(prop-1-enyl)phenol, 2-propylphenol, 4-pentylphenol, 4-sec-butylphenol, 2-benzylphenol or eugenol or a combination thereof.

[0040] The structures of these compounds are shown below:

[0041] The structure of thymol is shown below:

[0042] Thymol

[0043]

[0044] Carvacrol

[0045]

[0046] (E)-2(Propan-1-enyl)phenol

[0047]

[0048] 2-Propylphenol

[0049] 4-Amylphenol

[0050]

[0051] 4-Sec-Butylphenol

[0052] 2-Benzylphenol

[0053]

[0054] Eugenol

[0055]

[0056] Among the above essential oil compounds, thymol, carvacrol, 4-pentylphenol or 4-sec-butylphenol is more preferably used in the composition of the present invention. The most preferred essential oil compound is thymol.

[0057] The essential oil compound is preferably included in the composition at 0.001% to 1%, preferably 0.005% to 1%, more preferably 0.005% to 0.5% by weight of the composition.

[0058] Further more preferably, other essential oil compounds of terpene base are included in the composition of the present invention. The most preferred terpene base compound is terpineol. Terpineol is preferably selected from: α-terpineol, β-terpineol, γ-terpineol or a mixture thereof. It is particularly preferred that the terpineol is α-terpineol. Terpineol can be added to the antimicrobial composition in purified form.

[0059] The structure of the terpineol compound is as follows:

[0060]

[0061] The antimicrobial composition preferably comprises from 0.01% to 1% terpineol, more preferably from 0.05% to 0.5% terpineol by weight of the composition.

[0062] According to a particularly preferred aspect of the present invention, the composition comprises a mixture of thymol and terpineol.

[0063] Without wishing to be bound by theory, the inventors believe that the combination of essential oils and endolysins synergistically targets two different substrates that are critical to bacterial defense. They believe that for Gram-positive bacteria such as P. acnes, endolysins interfere with the outermost coating of the bacteria (i.e., the cell wall), thereby providing a rapid pathway for thymol to target lipid moieties in the bacterial inner cell membrane. Together, this explains the rapid and specific lysis of P. acnes.

[0064] The composition of the present invention preferably includes a topical acceptable carrier. Preferred topical acceptable carriers may comprise an anhydrous base, gel, lotion, cream or emulsion.

[0065] The composition of the present invention is preferably a rinse-off composition, and this is achieved by including from 1% to 80% by weight of a surfactant. Typically, the surfactant can be selected from well-known textbooks such as "Surface Active Agents" Vol. 1, Schwartz & Perry, Interscience 1949, Vol. 2, Schwartz, Perry & Berch, Interscience 1958 and / or "McCutcheon's Emulsifiers and Detergents" current edition, published by Manufacturing Confectioners Company or "enside Taschenbuch", H. Stache, 2nd edition, Carl Hauser Verlag, 1981) described in the surfactant. Any type of surfactant can be used, i.e. anionic, cationic, nonionic, zwitterionic or amphoteric surfactants.

[0066] A particularly preferred surfactant is soap. Soap is a suitable surfactant for personal washing applications of the compositions of the present invention. The soap is preferably a C8-C24 soap, more preferably a C10-C20 soap, and most preferably a C12-C16 soap. The soap may or may not have one or more carbon-carbon double or triple bonds. The cation of the soap may be an alkali metal, an alkaline earth metal, or ammonium. Preferably, the cation of the soap is selected from sodium, potassium, or ammonium. More preferably, the cation of the soap is sodium or potassium.

[0067] Soap can be obtained by saponification of fats and / or fatty acids. The fats or oils commonly used for soap making can be such as tallow, tallow stearines, palm oil, palm stearin, soybean oil, fish oil, castor oil, rice bran oil, sunflower oil, coconut oil, babassu oil, palm kernel oil, etc. In the above method, the fatty acid is derived from an oil / fats selected from coconut, rice bran, groundnut, tallow, palm, palm kernel, cottonseed, soybean, castor, etc.

[0068] Typical fatty acid mixtures are composed of 5%-30% coconut fatty acids and 70%-95% fatty acids from hardened rice bran oil. Fatty acids derived from other suitable oils / fats such as peanut, soybean, tallow, palm, palm kernel, etc. can also be used in other desired proportions. The most preferred soap is laurate soap. When present in the solid form of the present invention, the soap is present in an amount of 30% to 90% by weight of the composition, preferably 50% to 85%, more preferably 55% to 75%. When present in the liquid form of the composition, the soap is present in an amount of 0.5% to 20% by weight of the composition, preferably 1% to 10%.

[0069] Alternatively, the surfactant is a nonionic surfactant, such as C8-C22, preferably C8-C16 fatty alcohol ethoxylate, containing 1-8 ethylene oxides, preferably selected from: primary alkyl sulfates, secondary alkyl sulfonates, alkylbenzene sulfonates or ethoxylated alkyl sulfates. The composition may further comprise anionic surfactants, such as alkyl ether sulfates, preferably those with 1 to 3 ethylene oxide groups, from natural or synthetic sources, and / or sulfonic acids. Particularly preferred is sodium lauryl ether sulfate. Alkyl polyglucosides may also be present in the composition, preferably those with a carbon chain length of C6-C16. Suitable surfactant concentrations in liquid forms for cleaning applications are generally greater than 0.5% but less than 10% by weight of the composition, preferably 1% to 5%. In solid compositions, the surfactant is present in an amount of 5% to 40% by weight of the composition, preferably 10% to 30%.

[0070] Depending on the form of the composition, water may preferably be present in an amount of 10% to 90% by weight of the composition. In solid compositions, water may be present in an amount of 10%-30%, while in liquid or semi-solid compositions, water may be present in an amount of 40%-90%.

[0071] The compositions of the present invention are particularly suitable for washoff methods where the contact time of the antimicrobial active with the surface is short, ie less than 5 minutes, preferably less than 2 minutes, even more preferably less than 1 minute and in many cases less than 15 seconds.

[0072] When the composition according to the present invention is a leave-on composition, it preferably comprises one or more of fragrance, surfactant, organic sunscreen, inorganic sunscreen, emollient, humectant, extender pigment and preservative.

[0073] Sunscreens include those that block harmful ultraviolet light. Preferred sunscreens are derivatives of para-aminobenzoic acid (PABA), cinnamate, and salicylate. For example, avobenzophenone ( 1789), octyl methoxycinnamate and 2-hydroxy-4-methoxybenzophenone (also known as oxybenzone). Octyl methoxycinnamate and 2-hydroxy-4-methoxybenzophenone are respectively known as MCX and benzophenone-3 are commercially available. (a benzylidene camphor derivative) and drometrizole trisiloxane (a benzotriazole). Further examples include octocrylene, phenylbenzimidazole sulfonic acid (also known as ), ethylhexyl salicylate, diethylhexyl naphthoate, bimotrizinole (trademark S) and bisoctrizole ( M). Inorganic sunscreens include oxides that reflect or scatter the sun's rays such as titanium dioxide and zinc oxide. The amount of sunscreen present in the composition may vary depending on the degree of protection from UV radiation desired. Preferably, the composition comprises 0.01% to 15% by weight, more preferably 0.1% to 10% by weight of sunscreen, and most preferably 0.5-%7.5%.

[0074] Illustrative examples of the types of fragrances that can be used include those containing terpenes and terpene derivatives, such as those described in Bauer, K., et al., Common Fragrance and Flavor Materials, VCH Publishers (1990). Other examples include myrcene, dihydromyrcenol, citral, tagetone, cis-geranyl geraniol, citronellal, and mixtures thereof.

[0075] The carrier acts as a diluent or dispersant for the ingredients of the composition. The carrier may be aqueous based, anhydrous or an emulsion, with water-in-oil or oil-in-water emulsions generally being preferred. If water is used, it generally constitutes the balance of the composition, most preferably 40% to 80% by weight of the composition.

[0076] In addition to water, organic solvents may optionally be included as carriers to aid any other carriers in the compositions of the present invention. Examples include alkanols such as ethanol and isopropanol.

[0077] Other suitable organic solvents include ester oils such as isopropyl myristate, cetyl myristate, 2-octyldodecyl myristate, avocado oil, almond oil, olive oil, and neopentyl glycol dicaprate. Typically, such ester oils help emulsify the composition, and are generally used in an effective amount to produce a stable, and most preferably, water-in-oil emulsion.

[0078] If necessary, emollients can also be used as carriers. Preferred alcohols are 1-hexadecanol (i.e., cetyl alcohol). Other emollients include silicone oils and synthetic esters. Suitable silicone oils include cyclic or linear polydimethylsiloxanes containing 3-9, preferably 4-5 silicon atoms. Non-volatile silicone oils that can be used as emollients include polyalkylsiloxanes, polyalkylarylsiloxanes, and polyethersiloxane copolymers. Non-volatile polyalkylsiloxanes can be used polydimethylsiloxanes. Silicone elastomers can also be used. The ester emollients that can be used optionally are:

[0079] (i) Alkenyl or alkyl esters of fatty acids having 10 to 20 carbon atoms. Examples thereof include isoarachidyl neopentanoate, isononyl isononanoate, oleyl myristate, oleyl stearate and oleyl oleate;

[0080] (ii) ether esters, such as fatty acid esters of ethoxylated fatty alcohols;

[0081] (iii) Polyol esters. 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, glycerol mono- and di-fatty acid esters, polyglycerol poly-fatty acid esters, ethoxylated glycerol monostearate, 1,3-butylene glycol monostearate, 1,3-butylene glycol distearate, polyoxyethylene polyol fatty acid esters, sorbitan fatty acid esters, and polyoxyethylene sorbitan fatty acid esters are satisfactory polyol esters;

[0082] (iv) wax esters such as beeswax, spermaceti, stearyl stearate and arachidyl behenate; and,

[0083] (v) Sterol esters, of which cholesterol fatty acid esters are examples.

[0084] Emollients, when present, generally comprise from 0.1% to 50% by weight of the composition including all ranges subsumed therein.

[0085] A fatty acid having 10 to 30 carbon atoms may also be included as a carrier. Examples of such fatty acids include pelargonic acid, lauric acid, myristic acid, palmitic acid, stearic acid, isostearic acid, oleic acid, linoleic acid, arachidic acid, behenic acid or erucic acid and mixtures thereof.

[0086] Polyol-type wetting agents can also be used in the composition. Wetting agents generally help to improve the efficacy of emollients, reduce scales on the skin surface, promote the removal of accumulated scales and improve skin feel. Typical polyols include glycerol, polyalkylene glycols, more preferably alkylene polyols and derivatives thereof, including propylene glycol, dipropylene glycol, polypropylene glycol, polyethylene glycol and derivatives thereof, sorbitol, hydroxypropyl sorbitol, hexylene glycol, 1,3-butylene glycol, 1,2,6-hexanetriol, ethoxylated glycerol, propoxylated glycerol and mixtures thereof. For best results, the wetting agent is preferably propylene glycol or sodium hyaluronate. Other wetting agents that can be used include hydroxyethyl urea. The amount of the wetting agent can be 0.2%-25% by weight, and preferably 0.5-15% by weight of the composition.

[0087] Moisture retention can be improved by using petrolatum or paraffin. Thickeners can also be used as part of the carrier in the composition. Typical thickeners include cross-linked acrylates (e.g. 982), hydrophobically modified acrylates (e.g. 1382), cellulose derivatives and natural gums. Among the useful cellulose derivatives are sodium carboxymethylcellulose, hydroxypropyl methylcellulose, hydroxypropyl cellulose, hydroxyethyl cellulose, ethyl cellulose and hydroxymethyl cellulose. Natural gums suitable for use in the present invention include guar gum, xanthan gum, sclerotium, carrageenan, pectin and combinations of these gums. The amount of thickener may be in the range of 0.001% to 5%, preferably 0.01% to 0.5% by weight of the composition.

[0088] Surfactants may also be present. When present, the total amount of surfactant is 2% to 40%, preferably 4% to 20%, and optimally 5% to 12% by weight of the composition. The surfactant is selected from anionic, nonionic, cationic and amphoteric surfactants. Particularly preferred nonionic surfactants are C 60 having a condensation ratio of 2 to 100 moles of ethylene oxide or propylene oxide per mole of hydrophobe. 10-20 Those of fatty alcohols or acid hydrophobes; mono- and di-fatty acid esters of ethylene glycol; fatty acid monoglycerides; sorbitan, mono- and di-C8-C20 fatty acids; block copolymers (ethylene oxide / propylene oxide); and polyoxyethylene sorbitan and combinations thereof. Alkyl polyglycosides and carbohydrate fatty amides (e.g., methyl glucamide) are also suitable nonionic surfactants.

[0089] Preferred anionic surfactants include soaps, alkyl ether sulfates and sulfonates, alkyl sulfates and sulfonates, alkyl benzene sulfonates, alkyl and dialkyl sulfosuccinates, C 8-20 Acyl isethionate, acyl glutamate, C 8-20 Alkyl ether phosphates and combinations thereof.

[0090] Various other ingredients may also be used in the composition. Actives are defined as skin benefit agents other than emollients and ingredients that merely improve the physical properties of the composition. Although not limited to this class, general examples include body pigments such as talc and silica, as well as alpha-hydroxy acids, beta-hydroxy acids, and zinc salts.

[0091] Beta-hydroxy acids include salicylic acid. Zinc oxide and zinc pyrithione are examples of useful zinc salts.

[0092] It is necessary to protect the compositions, especially those containing water, from harmful microorganisms. Antimicrobial compounds (such as triclosan) and preservatives may be required. Suitable preservatives include alkyl esters of parahydroxybenzoic acid, hydantoin derivatives, propionates and various quaternary ammonium compounds. Particularly preferred preservatives are methylparaben, propylparaben, phenoxyethanol and benzyl alcohol. The preservative is 0.1%-2% by weight of the composition.

[0093] The packaging may be a patch, bottle, tube, roller-ball applicator, propellant-driven aerosol device, squeeze container, or jar with a lid.

[0094] According to another aspect of the present invention, a method for controlling or eradicating Propionibacterium acnes from the skin is provided, comprising the step of applying the composition of the present invention to the desired skin surface. The method is preferably non-therapeutic. The method is particularly useful for reducing or eliminating acne on the skin.

[0095] The invention will now be illustrated with the aid of the following non-limiting examples.

[0096] Example

[0097] Recombinant endolysin and thymol derived from Propionibacterium acnes phage; and thymol + terpineol combination The synergistic effect of combination.

[0098] Propionibacterium acnes strain 6919 (ATCC, USA) was grown on RCM agar under anaerobic conditions from a glycerol stock. A loop of culture from the glycerol stock was streaked on a freshly prepared RCM agar plate and incubated anaerobically at 37°C for 4-5 days. After incubation, the culture was removed from the plate and resuspended in saline and then centrifuged at 8000 rpm for 6 minutes at 4°C. The obtained precipitate was resuspended in 10 mM HEPES buffer (pH 7.0). An OD of 0.5 was prepared in HEPES buffer.600 A 1:10 dilution of the 0.5 OD adjusted culture was used for the contact kill assay, which is equivalent to 10 6 cells / ml.

[0099] The assay was performed in a total volume of 200 μl. 14 μl of purified endolysin (final concentration 100 μg / ml) was used for the assay. 10% w / v of the essential oil was freshly prepared in absolute ethanol. The contact assay was performed in 10 mM HEPES buffer (pH 7.0). As a control, 181 μl of a 1:10 dilution of 0.6 OD 600 The conditioned culture was mixed with 19 μl HEPES buffer to a volume of 200 μl. Similarly, for endolysins and essential oils, the required amount of protein was mixed with Propionibacterium acnes culture for individual and combined treatments. The mixture was mixed and incubated at 37°C in a shaking incubator for 2 hours. After incubation, the samples were neutralized by serial 1:10 dilutions in D / E broth and then diluted with saline. 100 μl of each dilution was spread on a fresh RCM agar plate and allowed to dry. The plates were sealed with parafilm and incubated at 37°C in an anaerobic chamber for 4-5 days. After incubation, colonies were counted and titers were estimated in log CFU / ml. The relative log reduction for individual and combined treatments was calculated relative to the control, and the data are listed in the following table:

[0100]

[0101] The data in the above table show that endolysin and thymol, as well as its combination with thymol and terpineol, have synergistic antimicrobial activity against P. acnes at a combined exposure of 2 hours over a wide range of concentrations. Sequence Listing <110> Unilever Intellectual Property Holdings Ltd. <120> An antimicrobial composition for selectively inhibiting the growth of Propionibacterium acnes bacteria <130> J2323 <160> 2 <170> PatentIn version 3.5 <210> 1 <211> 893 <212> DNA <213> Artificial Sequence <220> <223> Nucleotide sequence of endolysin <400> 1 atgcgttata ttccggcagc acatcattca gcaggtagca atcatccggt taatcgtgtt 60 gttattcatg caacctgtcc ggatgttggt tttccgagcg caagccgtaa aggtcgtgca 120 gttagcaccg caaactattt tgcaagcccg agcagcggtg gtagcgcaca ttatgtttgt 180 gatattggtg aaaccgttca gtgtctgagc gaaggcacca ttggttggca tgcaccgcct 240 aatccgcata gcctgggtat tgaaatttgt gcagatggtg gtagccatgc aagctttcgt 300 gttccgggtc atgcatatac ccgtgaacag tggctggatc cgcgtgtttg gcctgcagtt 360 gaaaaagcag caattctgtg tcgtcgtctg tgcgataaat acaatgttcc gaaacgtaaa 420 ctgagcgcag cagatctgaa agcaggtcgt cgtggtgttt gtggtcatgt tgatgttacc 480 gatgcatggc atcagagcga tcatgatgat ccgggtccgt ggtttccgtg ggatcgtttt 540 atggcagttg ttaatggtca taacgaaagc ggtgaactga ccgttgcaga tgttaaagca 600 ctgcatgatc agattaaaca gctgagcgca cagctggcag gtagcgttaa taaactgcat 660 catgatgttg gtgttgttca ggttcagaat ggtgatctgg gtaaacgtgt tgatgcactg 720 agctgggtta aaaatccggt taccggtaaa ctgtggcgta ccaaagatgc actgtggtca 780 gtttggtatt atgttctgga atgtcgtagc cgtattgatc gtctggaaag cgcagttaat 840 ggtctgaaaa aactcgagca ccaccaccac caccactgag atccggctgc taa 893 <210> 2 <211> 292 <212> PRT <213> Artificial Sequence <220> <223> Polypeptide sequence of Endolysin with C-terminal Histidine tag (x6) <400> 2 Met Arg Tyr Ile Pro Ala Ala His His Ser Ala Gly Ser Asn His Pro 1 5 10 15 Val Asn Arg Val Val Ile His Ala Thr Cys Pro Asp Val Gly Phe Pro 20 25 30 Ser Ala Ser Arg Lys Gly Arg Ala Val Ser Thr Ala Asn Tyr Phe Ala 35 40 45 Ser Pro Ser Ser Gly Gly Ser Ala His Tyr Val Cys Asp Ile Gly Glu 50 55 60 Thr Val Gln Cys Leu Ser Glu Gly Thr Ile Gly Trp His Ala Pro Pro 65 70 75 80 Asn Pro His Ser Leu Gly Ile Glu Ile Cys Ala Asp Gly Gly Ser His 85 90 95 Ala Ser Phe Arg Val Pro Gly His Ala Tyr Thr Arg Glu Gln Trp Leu 100 105 110 Asp Pro Arg Val Trp Pro Ala Val Glu Lys Ala Ala Ile Leu Cys Arg 115 120 125 Arg Leu Cys Asp Lys Tyr Asn Val Pro Lys Arg Lys Leu Ser Ala Ala 130 135 140 Asp Leu Lys Ala Gly Arg Arg Gly Val Cys Gly His Val Asp Val Thr 145 150 155 160 Asp Ala Trp His Gln Ser Asp His Asp Asp Pro Gly Pro Trp Phe Pro 165 170 175 Trp Asp Arg Phe Met Ala Val Val Asn Gly His Asn Glu Ser Gly Glu 180 185 190 Leu Thr Val Ala Asp Val Lys Ala Leu His Asp Gln Ile Lys Gln Leu 195 200 205 Ser Ala Gln Leu Ala Gly Ser Val Asn Lys Leu His His Asp Val Gly 210 215 220 Val Val Gln Val Gln Asn Gly Asp Leu Gly Lys Arg Val Asp Ala Leu 225 230 235 240 Ser Trp Val Lys Asn Pro Val Thr Gly Lys Leu Trp Arg Thr Lys Asp 245 250 255 Ala Leu Trp Ser Val Trp Tyr Tyr Val Leu Glu Cys Arg Ser Arg Ile 260 265 270 Asp Arg Leu Glu Ser Ala Val Asn Gly Leu Lys Lys Leu Glu His His 275 280 285 His His His His 290

Claims

1. An antimicrobial composition for selectively inhibiting the growth of Propionibacterium acnes bacteria, comprising (i) an endolysin derived from a Propionibacterium acnes phage, wherein the amino acid sequence of the endolysin is encoded by the nucleotide sequence of SEQ ID NO: 1; and (ii) Thymol.

2. A composition as claimed in claim 1, wherein the endolysin is a recombinant form of a Propionibacterium acnes bacteriophage endolysin.

3. The composition as claimed in claim 1, wherein the endolysin is cloned from the endolysin gene sequence of Propionibacterium phage 29399B_C, which is codon-optimized for expression in Escherichia coli (E. coli) and cloned into the commercially available pET303 / CT-His expression vector.

4. A composition as claimed in claim 3, wherein the stop codon is removed from the 3' end of the gene to accommodate a 6X histidine tag.

5. The composition as claimed in claim 1, wherein the composition further comprises terpineol.

6. A composition as claimed in claim 5 comprising a mixture of terpineol and the essential oil compound thymol.

7. The composition as claimed in claim 1, further comprising a carrier acceptable for topical administration.

8. A composition as claimed in claim 7, wherein the topically acceptable carrier comprises a non-aqueous base, gel, cream or emulsion.

9. A composition as claimed in claim 7 wherein the topically acceptable carrier comprises an emulsion.

10. Use of the composition of any one of claims 1 to 9 in the preparation of a medicament for controlling or eradicating Propionibacterium acnes from the skin.

11. Use of the composition of any one of claims 1 to 9 in the preparation of a medicament for reducing or eliminating acne on the skin.

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