Commensal bacteriotherapy of acne

Abstract

The disclosure relates to composition and methods that selectively kill or inhibit the growth of C. acnes on human skin, and uses thereof, including as a topical acne treatment.

Description

Attorney Docket No.00015-440WO1 COMMENSAL BACTERIOTHERAPY OF ACNE CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority under 35 U.S.C. §119 from Provisional Application Serial No. 63 / 680,914, filed August 8, 2024, the disclosures of which are incorporated herein by reference. TECHNICAL FIELD

[0002] The disclosure relates to compositions and methods that selectively kill or inhibit the growth of C. acnes on human skin, and uses thereof, including as a topical acne treatment. BACKGROUND

[0003] The human skin harbors diverse microbial communities which constitute an important element of innate immune barrier to defend the host from pathogens. SUMMARY

[0004] The disclosure relates to compositions and methods that selectively kill or inhibit the growth of C. acnes on human skin, and uses thereof, including as a topical acne treatment. It was observed that some bacteria on normal skin could kill C. acnes. By doing so, these bacteria or extracts from these bacteria could be used to treat acne, thus sparing the use of antibiotics. Complete analysis of strains of Cutibacterium acnes 43, as well as some other species of the genus Cutibacterium, found that these bacteria could kill a range of other C. acnes strains commonly found on human facial skin. It was shown herein that supernatant and purified proteins from strain C. acnes H1.43 was able to prevent the growth of other strains of C. acnes but did not significantly affect the growth of other commensal skin bacteria.

[0005] In a particular embodiment, the disclosure provides pharmaceutical composition comprising, consisting essential of or consisting of: Cutibacterium acnes 43 (C. acnes H1.43), a supernatant from C. acnes H1.43; and / or an antimicrobial protein purified from C. acnes H1.43; and a pharmaceutically acceptable carrier for topical administration. In a further embodiment, the supernatant from C. acnes H1.43 is a sterile supernatant. In another embodiment, the antimicrobial protein is purified from anAttorney Docket No.00015-440WO1 ammonium sulfate precipitate of C. acnes H1.43. In yet a further embodiment, the antimicrobial protein is thermosensitive over 60 °C and has a molecular weight of over 30 kDa. In another embodiment, the pharmaceutical composition is formulated as an ointment, cream, spray or gel. In yet another embodiment, the pharmaceutical composition further comprises an acne treatment selected from tretinoin, adapalene, tazarotene, benzoyl peroxide, azelaic acid, salicylic acid, or dapsone.

[0006] In a certain embodiment, the disclosure also provides a method of treating a subject having acne vulgaris, comprising: administering a therapeutically effective amount of the pharmaceutical composition of any one of the proceeding claims to subject having acne vulgaris in need of treatment thereof. In a further embodiment, the subject is treated daily with the pharmaceutical composition until the subject' acne vulgaris is resolved. In yet a further embodiment, the subject is treated with the pharmaceutical composition and one or more other treatments for acne vulgaris. In another embodiment, the one or more other treatments for acne vulgaris are selected from adapalene, tazarotene, benzoyl peroxide, azelaic acid, salicylic acid, dapsone, spironolactone, and / or oral antibiotics. In yet another embodiment, the oral antibiotics is selected from doxycycline, minocycline, tetracycline, erythromycin, azithromycin trimethoprim / sulfamethoxazole and trimethoprim.

[0007] In a particular embodiment, the disclosure further provides a method to purify an antimicrobial protein from C. acnes H1.43, comprising: isolating a supernatant from a culture of C. acnes H1.43; adding ammonium sulfate to the supernatant to achieve at least 40% saturation; isolating proteins from the ammonium sulfate treated supernatant; filtering the isolated proteins using a 30 kDa molecular weight size exclusion filter; and collecting the retained extract that did not pass through the 30 kDa molecular weight size exclusion filter, wherein the retained extract comprises the antimicrobial protein. In a further embodiment, the supernatant is isolated from the culture of C. acnes H1.43 by centrifugation. In another embodiment, the supernatant is filtered using a 0.22 µm filter prior to adding ammonium sulfate. In yetAttorney Docket No.00015-440WO1 another embodiment, the ammonium sulfate is added to the supernatant to achieve 45% saturation.

[0008] The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims. DESCRIPTION OF DRAWINGS

[0009] Figure 1 shows that the ammonium precipitate fraction from C. acnes H1.43 has antimicrobial activity against C. acnes 6919.

[0010] Figure 2 demonstrates that C. acnes H1.43 produces an antimicrobial molecule that is active against different phylotypes of C. acnes.

[0011] Figure 3presents a screening assay of the supernatant from C. acnes H1.43 in liquid culture comprising various strains of Staphylococcus. It was found that the supernatant C. acnes H1.43 does not have antimicrobial activity against S. hominis, S. epidermidis, S. warneri and S. aureus.

[0012] Figure 4 presents a screening assay of ammonium sulfate precipitated extract of C. acnes H1.43 in liquid culture comprising various strains of Staphylococcus. It was found that C. acnes H1.43 does not have antimicrobial activity against S. hominis, S. epidermidis, S. warneri and S. aureus.

[0013] Figure 5 demonstrates that C. acnes H1.43 has lipase activity. Acne-associated phylotypes include A and C (both phylotype (IA1) and F (IA2): *C type*A1. Healthy skin is colonized with more diverse populations with a higher prevalence of strains belonging to classes H (IB) and K (III): *K type *H type.

[0014] Figure 6 provides genomic analyses of C. acnes biosynthetic gene clusters (BCG). DETAILED DESCRIPTION

[0015] As used herein and in the appended claims, the singular forms "a,” "an,” and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "an agent" includes a plurality of such agents and reference to "the microorganism" includes reference to one or moreAttorney Docket No.00015-440WO1 microorganisms and equivalents thereof known to those skilled in the art, and so forth.

[0016] Also, the use of “or” means “and / or” unless stated otherwise. Similarly, “comprise,” “comprises,” “comprising” “include,” “includes,” and “including” are interchangeable and not intended to be limiting.

[0017] It is to be further understood that where descriptions of various embodiments use the term “comprising,” those skilled in the art would understand that in some specific instances, an embodiment can be alternatively described using language “consisting essentially of” or “consisting of.”

[0018] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. Any methods and reagents similar or equivalent to those described herein can be used in the practice of the disclosed methods and compositions.

[0019] As used herein, an “active drug substance” refers to any substance, compound, or composition that is capable of or intended to have an effect in the treatment, amelioration, or cure of dysbiosis in a subject; or of any disease, disorder, or condition related to dysbiosis in a subject, or that is otherwise intended to affect the health, comfort, or appearance of a subject. In some embodiments, an active drug substance as disclosed herein may comprise one or more of a bacterium, a killed bacterium, a bacterial culture, a bacterial extract, a growth medium, a lyophilized bacterium, a lyophilized killed bacterium, a lyophilized bacterial culture, a lyophilized bacterial extract, a lyophilized growth medium, or any equivalent or derivative thereof, or any combination of the foregoing. In some embodiments, an active drug substance may be provided for use in treating, ameliorating, or curing dysbiosis in a subject; or any disease, disorder, or condition related to dysbiosis in a subject, or otherwise in affecting the health, comfort, or appearance of a subject. In some embodiments, an active drug substance may be provided for the manufacture of a medicament for use in treating, ameliorating, or curing dysbiosis in a subject; or any disease, disorder, orAttorney Docket No.00015-440WO1 condition related to dysbiosis in a subject, or otherwise in affecting the health, comfort, or appearance of a subject. Where a substance, compound, or composition is provided primarily for the purpose of altering or improving the appearance of a subject, without regard to any effects said substance, compound, or composition may have on the treatment, amelioration, or cure of dysbiosis in said subject, the term “active cosmetic substance” may optionally be used. The disclosure contemplates the use of active drug substances for cosmetic purposes and vice versa.

[0020] As used herein, the term "biocompatible" refers to a composition that does not have clinically significant toxic or injurious effects, locally or systemically. The term "biocompatible" does not exclude the possibility that a composition may have one or more effects on the health, appearance, or comfort of a subject which do not rise to the level of clinical significance or are acceptable in comparison to the severity of other symptoms or conditions.

[0021] The term "contacting" refers to exposing a tissue to a composition. In one embodiment, the contacting can be contact with the skin (e.g., topical treatment). Such contacting can include contacting with a composition comprising a topical prebiotic, probiotic and / or postbiotic composition such that the composition can kill or inhibit C. acnes on the skin.

[0022] Cutibacterium acnes (C. acnes; formerly Propionibacterium acnes; C. acnes and P. acnes are used interchangeably herein) is a slow-growing, aerotolerant anaerobic, Gram-positive bacterium linked to skin acne. These bacteria can also cause chronic blepharitis and endophthalmitis, the latter particularly following intraocular surgery. Although C. acnes is a member of the normal skin commensal bacterial flora, it plays a critical role in the development of inflammatory acne when the microorganism overgrows within the pilosebaceous unit. Inflammatory acne is the most common disease of human skin afflicting up to 80% of individuals through their lives.

[0023] The term "cosmetically acceptable carrier,” "cosmetically acceptable excipient" or "cosmetically acceptable ingredient" includes any and all solvents, diluents, emulsifiers, binders,Attorney Docket No.00015-440WO1 buffers, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like, or any other such compound as is known by those of skill in the art to be useful in preparing cosmetic compositions or formulations. It is understood in the art that excipients may serve multiple purposes within a formulation. It is further additionally understood that the disclosure of any excipient for any particular purpose (such as, as an emulsifier, emollient, preservative, etc.) does not restrict the use of the excipient to that particular purpose. The use of such media and agents for cosmetic compositions or formulations is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in pharmaceutical, cosmetic, or personal care compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions. In addition, various adjuvants such as are commonly used in the art can be included. In some embodiments, the excipients can include a topical pharmaceutically- and cosmetically-acceptable emollient. As used herein, "emollients" refer to materials used for the prevention or relief of dryness, as well as for the protection of the skin. Sagarin, Cosmetics, Science and Technology, 2nd Edition, Vol. 1, pp. 32-43 (1972), which is incorporated herein by reference in its entirety, contains numerous examples of suitable materials for use as emollients.

[0024] As used herein, the term "dysbiosis" means an imbalance or maladaptation of the flora or microbiota within one or more tissues, compartments, sub-compartments, or locations of the body, and particularly within the various domains and subdomains of the skin. Such dysbiosis is characterized by a change in the composition of the local microbiome, in terms of the species / strains which are present and / or the relative abundance or proportion of the species / strains which are present, in which the change has a definable effect on the host organism. The effect on the host organism can result from microbiome-mediated changes in electrolyte balance, biofilm formation, epithelial, mesothelial, or endothelial barrier integrity, or the release from the microbiome of metabolites which directly (e.g., as toxins or effectors) orAttorney Docket No.00015-440WO1 indirectly (e.g., as pre-cursors to toxins or effectors) affect the health, appearance, or comfort of the host.

[0025] The terms "inhibiting" or "inhibiting effective amount" refers to the amount of a pharmaceutical composition comprising, consisting essentially of or consisting of Cutibacterium acnes 43 (C. acnes H1.43), a supernatant from C. acnes H1.43, and / or an antimicrobial protein purified from C. acnes H1.43 that is sufficient to cause, for example, inhibition of C. acnes growth, proliferation or presence on the skin. The term "inhibiting" also includes preventing or ameliorating a sign or symptoms of a disorder (e.g., acne, sore, and the like).

[0026] The term "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" includes any and all solvents, cosolvents, diluents, emulsifiers, binders, adsorbents, permeation enhancers, surfactants, stabilizers, preservatives, cheating agents, thickeners, smoothing agents, abrasives, polymers, humectants, emollients, moisturizers, buffers, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like, or any other such compound as is known by those of skill in the art to be useful in preparing pharmaceutical formulations. As used herein, an excipient or pharmaceutically acceptable excipient, or a cosmetically acceptable excipient, may comprise any component of a formulation that is not itself, or is not intended to be, an active drug product or primary cosmetic product. It is understood in the art that excipients may serve multiple purposes within a formulation. It is further additionally understood that the disclosure of any excipient for any particular purpose (such as, as an emulsifier, emollient, preservative, etc.) does not restrict the use of the excipient to that particular purpose. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions. In addition, various adjuvants such as are commonly used in the art can be included. These and other such compounds are described in the literature, e.g., in theAttorney Docket No.00015-440WO1 Merck Index, Merck & Company, Rahway, NJ. Considerations for the inclusion of various components in pharmaceutical compositions are described, e.g., in Gilman et al. (Eds.) (1990); Goodman and Gilman's: The Pharmacological Basis of Therapeutics, 8th Ed., Pergamon Press.

[0027] The term "purified" as used herein refers to a protein, polypeptide, peptide or compound that is substantially free of other proteins, lipids, and polynucleotides (e.g., cellular components with which an in vivo produced peptide would naturally be associated). Typically, the protein, polypeptide, peptide or other compound is at least 70%, 80%, or most commonly 90% or more pure by weight. For example, in one embodiment, an antimicrobial protein purified from C. acnes H1.43 is substantially free of other cells or components. The level of purification can vary from about 50-100%.

[0028] The term "subject" as used herein, refers to a human, other mammal, or a non-human animal including, but not limited to, a dog, cat, horse, donkey, chicken, duck, goose, turkey, guinea fowl, emu, ostrich, parrot, canary, mynah bird, pheasant, quail, partridge, peafowl, mule, cow, domestic buffalo, camel, llama, alpaca, bison, yak, goat, sheep, pig, elk, deer, domestic antelope, or a non-human primate selected or identified for treatment, inhibition, or amelioration of a disease, disorder, or condition, or any symptom thereof, including cosmetic conditions, associated with an alteration in the microbiome of the subject, especially with respect to the microbiome of the skin, intestine, or mucosa. Particular diseases or disorders may include without limitation any diseases or disorders disclosed herein, such as atopic dermatitis, eczema, pyotraumatic dermatitis, pyoderma, superficial pyoderma, folliculitis, rosacea, Netherton syndrome, acne, wounds (including abrasions, radiation damage, and burns), psoriasis, mastitis, icthyosis, lichen formation, and sebhorreic dermatitis, or any combination thereof.

[0029] The term "therapeutically effective amount" as used herein for treatment of a subject afflicted, for example, with acne vulgaris means an amount of a pharmaceutical composition disclosed herein that is sufficient to ameliorate a sign or symptom of theAttorney Docket No.00015-440WO1 disease or disorder. For example, a therapeutically effective amount can be measured as the amount sufficient to decrease a subject's symptoms of acne.

[0030] As used herein, the term “topical” can include administration to the skin externally, as well as shallow injection (e.g., intradermally and intralesionally) such that a pharmaceutical composition described herein comes in direct contact with skin and dermal layer.

[0031] As mentioned, C. acnes plays a role in the development of inflammatory acne when the microorganism overgrows within the pilosebaceous unit. Acne has many different symptoms including comedones, papules, pustules, nodules, cysts and pilosebaceous inflammation. Among these, inflammatory lesions of acne are of serious concern to patients because they may lead to acne scarring, thereby inducing adverse psychological effects. The genome of the bacterium has been sequenced and a study has shown several genes can generate enzymes for degrading skin and proteins that may be immunogenic (e.g., activating the immune system). This bacterium is largely commensal and part of the skin flora present on healthy adult human skin. Typically, the organism is just barely detectable on the skin of healthy preadolescents. It lives primarily on, among other things, fatty acids in sebum secreted by sebaceous glands in the follicles. It may also be found throughout the gastrointestinal tract.

[0032] Reduction in C. acnes survival correlates with clinical improvement of acne in patients. Systemic antibiotics have been used to treat acne for several decades and are still widely prescribed for acne patients. Topical antibiotics are also helpful, and the oxidizing agent benzoyl peroxide (BPO) has been one of the most frequently used topical medications for acne treatment. Topical drug therapies are often used as the first line treatment for patients suffering from mild to moderate acne. However, current antibiotic treatments have major drawbacks. Systemic antibiotics non-specifically disrupts microbial ecosystem including normal biotic environments of the body, and promote antibiotic resistance. Topical antibiotics are very poor at killing C. acnes on the skin surface.Attorney Docket No.00015-440WO1

[0033] In some embodiments, a pharmaceutical composition described herein comprise, consists essential of or consists of Cutibacterium acnes H143 (referred to as C. acnes H1.43, C. acnes H1.43.H1 or C. acnes H1.43). In another embodiment,a pharmaceutical composition described herein comprise, consists essential of, or consists of a supernatant from C. acnes H1.43. In yet another embodiment, a pharmaceutical composition described herein comprise, consists essential of, or consists of an antimicrobial protein purified from C. acnes H1.43.

[0034] The disclosure provides whole cell preparations comprising a substantially homogeneous or substantially pure preparation of Cutibacterium acnes H1.43. The disclosure also provides fractions derived from such whole cells comprising agents that reduce C. acnes growth or viability in the skin.

[0035] In some embodiments, the compositions described herein comprise a probiotic composition comprising C. acnes H1.43. In other embodiments, the probiotic organism comprises a mixture of strains including C. acnes H1.43. Such formulations typically comprise sufficient quantities of C. acnes H1.43 as to provide a final density of 10 10 CFU / cm when applied to the skin of a subject. Such formulations may comprise concentrations of from about 10 to about 10 CFU / g, or alternatively, from 10 to about 10 CFU / g, or alternatively, from about 10 to about 10 CFU / g. Such formulations may comprise multiple strains of C. acnes H1 and may further comprise Lactococcus lactis, Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus acidophilus, and / or other such species or strains as are known in the art to form a part of the normal healthy cutaneous or mucosal flora. In some embodiments, C. acnes H1.43 comprise 100% of the bacterial cells in a formulation. In some further embodiments, C. acnes H1.43 comprises 90-100%, 85-95%, 70-80%, 75-85%, 60-70%, 65-75%, 50-60%, 55-65%, 40-50%, 45-55%, 30-40%, 35-45%, 20-30%, 25-35%, 10-20%, 15- 20%, 1-10%, 5-15%, or less than 1% of the bacterial cells in a given formulation, wherein in some embodiments the remainder of the colony forming units are provided by S. epidermidis, Lactococcus lactis, Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus acidophilus, and / or other such strains as are knownAttorney Docket No.00015-440WO1 in the art to form a part of the normal healthy cutaneous or mucosal flora.

[0036] In some embodiments, the composition is obtained through preparation of an autologous sample. In this embodiment, a skin sample is obtained from a subject to be treated and C. acnes H1.43 alone or in combination with other probiotic strains are isolated and cultured to increase their population(s). The cultures are then prepared to be administered to the subject as a pharmaceutical or probiotic composition. As used herein, an autologous transplant refers to the transplantation of bacterial strains from one site to another on the same subject or to the same site, regardless of whether the strains are cultured prior to administration or not. In some embodiments, the bacterial strains obtained from the subject are expanded in culture and then transplanted back to the subject.

[0037] As used herein, an allogeneic transplant refers to the transplantation of bacterial strains from one subject to another subject, or to the administration to a subject of a composition comprising bacterial strains that were not collected from upon or within their own body.

[0038] Such collection can be carried out by swabbing, scraping, wiping, or cutting and removing tissue on which resides one of the bacterial strains as described herein; optionally growing and isolating single colonies from agar plates or otherwise using methods known in the art; optionally growing expanded cultures of the isolated bacteria or crude swabs, wipes, scrapes, tissue, or other isolate in liquid or solid culture according to methods known in the art; optionally harvesting bacteria from said expanded culture by centrifugation, filtration, gravity settling, scraping, or by other means known in the art; formulating the bacteria or the crude isolate with a thickener, carrier, or excipient; and contacting the subject in an area determined to be in need of the transplant, with said formulation.

[0039] The probiotic compositions or pharmaceutical compositions of the disclosure can include prebiotic compounds and agents. As used herein, a prebiotic compound comprises a polysaccharide, hydrolysate, salt, herbal extract, or any other compound sufficient to foster the growth of an associated probiotic strain when used inAttorney Docket No.00015-440WO1 combination with that strain, such as yeast hydrolysate in concentrations of less than about 40% (w / w), microcrystalline cellulose in concentrations of less than about 10% (w / w), and / or sucrose in concentrations of less than about 10% (w / w). Other examples of prebiotics that may be adapted for use with cutaneous bacteria include inulin, glucooligosaccharides, isomaltooligosaccharides, lactosucrose, polydextrose, soybean oligosaccharides, and xylooligosaccharides, and those disclosed in Gibson, G.R. and Roberfroid, M,(Eds.) Handbook of Prebiotics, CRC press (2008); Roberfroid, M., J. Nutr. 137(3):830S-837 (2007) and Slavin, J. Nutrients 5(4):1417-1435 (2013), each of which is incorporated herein by reference in its entirety.

[0040] In some embodiments a method of the disclosure comprises contacting a subject with a probiotic and / or prebiotic composition as described herein. In some embodiments, such contacting comprises an autologous transplant. In some embodiments such contact comprises an allogeneic transplant, wherein elements of the cutaneous or mucosal flora are transplanted to a first subject in need thereof from a second subject (the donor). For example, in some embodiments, bacterial strains as disclosed above are identified and isolated from a second subject, amplified in an appropriate culture medium under such conditions as are known in the art to be conducive to bacterial growth, followed by harvest of the bacterial cells, mixing of the harvested cells at a predetermined concentration according to the disclosure with a predetermined formulation, and application of the mixture to the affected area of the first subject. In some embodiments such composition comprises a standardized formulation, such as a formulation in which the concentrations of ingredients are fixed and are not varied from subject to subject. In some embodiments, the formulation is developed individually for each subject, based on criteria including but not limited to: the composition of the subject’s own cutaneous or mucosal flora; the subject’s disease state and treatment history; the nature and severity of the subject’s condition; the nature and severity of concurrent cutaneous or mucosal infections; the presence of other antimicrobial compounds including systemic antibiotics within theAttorney Docket No.00015-440WO1 subject’s body; and other criteria such as are known to or would readily be apparent to those of skill in the art.

[0041] The ability of a pharmaceutical composition of the disclosure to inhibit the activity of a second bacterial composition (e.g., C. acnes) can be determined by contacting, e.g., the pharmaceutical composition with, for example, a C. acnes or other pathogenic bacteria and measuring the growth or viability of the C. acnes or bacteria before and after contacting with the pharmaceutical composition.

[0042] Any of a variety of methods known in the art can be used to administer a pharmaceutical composition to a subject. For example, a pharmaceutical composition disclosed herein is formulated for topical administration (e.g., as a lotion, cream, spray, gel, or ointment). Such topical formulations are useful in treating acne vulgaris. Examples of formulations include topical lotions, creams, soaps, wipes, and the like.

[0043] In another embodiment, a bandage or dressing is provided comprising a pharmaceutical composition described herein. In various embodiments, a bandage or dressing is provided, the major constituents of which includes a matrix and a pharmaceutical composition disclosed herein that inhibits the growth of C. acnes and / or other pathogenic bacteria on the skin.

[0044] A pharmaceutical composition disclose herein may be formulated in any dosage form that is suitable for topical administration for local or systemic effect, including emulsions, solutions, suspensions, creams, gels, hydrogels, ointments, dusting powders, dressings, elixirs, lotions, tinctures, pastes, foams, films, aerosols, irrigations, sprays, suppositories, bandages, and dermal patches. The topical formulation comprising a probiotic disclosed herein may also comprise liposomes, micelles, microspheres, nanosystems, and mixtures thereof.

[0045] A “pharmaceutically acceptable carrier” is intended to include solvents, dispersion media, coatings, antibacterial and antifungal agents (as needed so long as they are not detrimental to Cutibacterium acnes 43), isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art.Attorney Docket No.00015-440WO1 Except insofar as any conventional media or agent is incompatible with the pharmaceutical composition, use thereof in the therapeutic compositions and methods of treatment is contemplated. Supplementary active compounds can also be incorporated into the compositions.

[0046] Pharmaceutically acceptable carriers and excipients suitable for use in the topical formulations disclosed herein include, but are not limited to, aqueous vehicles, water-miscible vehicles, non-aqueous vehicles, stabilizers, solubility enhancers, isotonic agents, buffering agents, antioxidants, local anesthetics, suspending and dispersing agents, wetting or emulsifying agents, complexing agents, sequestering or chelating agents, penetration enhancers, cryoprotectants, lyoprotectants, thickening agents, and inert gases.

[0047] A pharmaceutical composition disclosed herein may be formulated in the forms of ointments, creams, sprays and gels. Suitable ointment vehicles include oleaginous or hydrocarbon vehicles, including such as lard, benzoinated lard, olive oil, cottonseed oil, and other oils, white petrolatum; emulsifiable or absorption vehicles, such as hydrophilic petrolatum, hydroxystearin sulfate, glycerol and anhydrous lanolin; water-removable vehicles, such as hydrophilic ointment; water-soluble ointment vehicles, including polyethylene glycols of varying molecular weight; emulsion vehicles, either water-in-oil (W / O) emulsions or oil-in- water (O / W) emulsions, including cetyl alcohol, glyceryl monostearate, lanolin, and stearic acid (see, Remington: The Science and Practice of Pharmacy). These vehicles are emollient but generally require addition of antioxidants and preservatives.

[0048] Suitable cream base can be oil-in-water or water-in-oil. Cream vehicles may be water-washable, and contain an oil phase, an emulsifier, and an aqueous phase. The oil phase is also called the "internal" phase, which is generally comprised of petrolatum and a fatty alcohol such as cetyl or stearyl alcohol. The aqueous phase usually, although not necessarily, exceeds the oil phase in volume, and generally contains a humectant. The emulsifier in a cream formulation may be a nonionic, anionic, cationic, or amphoteric surfactant.Attorney Docket No.00015-440WO1

[0049] Gels are semisolid, suspension-type systems. Single- phase gels contain material substantially uniformly throughout the liquid carrier. Suitable gelling agents include crosslinked acrylic acid polymers, such as carbomers, carboxypolyalkylenes, Carbopol ; hydrophilic polymers, such as polyethylene oxides, polyoxyethylene- polyoxypropylene copolymers, and polyvinylalcohol; cellulosic polymers, such as hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose phthalate, and methylcellulose; gums, such as tragacanth and xanthan gum; sodium alginate; and gelatin. In order to prepare a uniform gel, dispersing agents such as alcohol or glycerin can be added, or the gelling agent can be dispersed by trituration, mechanical mixing, and / or stirring.

[0050] In another embodiment, a pharmaceutical composition disclosed herein can be formulated either alone or in combination with one or more additional therapeutic agents, including, but not limited to, tretinoin, adapalene, tazarotene, benzoyl peroxide, azelaic acid, salicylic acid, or dapsone.

[0051] If desired, a suitable therapy regime can combine administration of a pharmaceutical composition of the disclosure with one or more additional treatments for acne vulgaris. The treatments for acne vulgaris can be administered, simultaneously, but may also be administered sequentially with a pharmaceutical composition disclosed herein. Examples of treatments for acne vulgaris include, but are not limited to, adapalene, tazarotene, benzoyl peroxide, azelaic acid, salicylic acid, dapsone, spironolactone, and / or oral antibiotics. Examples of oral antibiotics include, but are not limited to, doxycycline, minocycline, tetracycline, erythromycin, azithromycin trimethoprim / sulfamethoxazole and trimethoprim. Generally, the oral antibiotic is administered in a bactericidal amount. A "bactericidal amount" is an amount sufficient to achieve a bacteria-killing concentration in the subject receiving the treatment. In accordance with its conventional definition, an "antibiotic," as used herein, is a chemical substance that, in dilute solutions, inhibits the growth of, or kills microorganisms.Attorney Docket No.00015-440WO1 Also encompassed by this term are synthetic antibiotics (e.g., analogs) known in the art.

[0052] Pharmaceutical compositions disclosed herein can be used concurrently with other antibacterial agents including sulfa drugs such as sulfamethizole, sulfisoxazole, sulfamonomethoxine, sulfamethizole, salazosulfapyridine, silver sulfadiazine and the like; quinoline antibacterial agents such as nalidixic acid, pipemidic acid trihydrate, enoxacin, norfloxacin, ofloxacin, tosufloxacin tosilate, ciprofloxacin hydrochloride, lomefloxacin hydrochloride, sparfloxacin, fleroxacin and the like; antiphthisics such as isoniazid, ethambutol (ethambutol hydrochloride), p- aminosalicylic acid (calcium p-aminosalicylate), pyrazinamide, ethionamide, protionamide, rifampicin, streptomycin sulfate, kanamycin sulfate, cycloserine and the like; antiacidfast bacterium drugs such as diaphenylsulfone, rifampicin and the like; antiviral drugs such as idoxuridine, acyclovir, vidarabine, ganciclovir and the like; anti-HIV agents such as zidovudine, didanosine, zalcitabine, indinavir sulfate ethanolate, ritonavir and the like; antispirocheteles; antibiotics such as tetracycline hydrochloride, ampicillin, piperacillin, gentamicin, dibekacin, kanendomycin, lividomycin, tobramycin, amikacin, fradiomycin, sisomycin, tetracycline, oxytetracycline, rolitetracycline, doxycycline, ampicillin, piperacillin, ticarcillin, cephalothin, cephapirin, cephaloridine, cefaclor, cephalexin, cefroxadine, cefadroxil, cefamandole, cefotoam, cefuroxime, cefotiam, cefotiam hexetil, cefuroxime axetil, cefdinir, cefditoren pivoxil, ceftazidime, cefpiramide, cefsulodin, cefinenoxime, cefpodoxime proxetil, cefpirome, cefozopran, cefepime, cefsulodin, cefinenoxime, cefinetazole, cefminox, cefoxitin, cefbuperazone, latamoxef, flomoxef, cefazolin, cefotaxime, cefoperazone, ceftizoxime, moxalactam, thienamycin, sulfazecin, aztreonam or a salt thereof, griseofulvin, lankacidin-group and the like.

[0053] In yet a further embodiment, a pharmaceutical composition disclosed herein can be combined with one or more steroidal drugs known in the art, including, but not limited to, spironolactone, aldosterone, beclometasone, betamethasone, deoxycorticosterone acetate, fludrocortisone acetate,Attorney Docket No.00015-440WO1 hydrocortisone (cortisol), prednisolone, prednisone, methylprenisolone, dexamethasone, and triamcinolone.

[0054] In yet a further embodiment, a pharmaceutical composition disclosed herein can be combined with one or more anti- fungal agents, including, but not limited to, amorolfine, amphotericin B, anidulafungin, bifonazole, butenafine, butoconazole, caspofungin, ciclopirox, clotrimazole, econazole, fenticonazole, filipin, fluconazole, isoconazole, itraconazole, ketoconazole, micafungin, miconazole, naftifine, natamycin, nystatin, oxyconazole, ravuconazole, posaconazole, rimocidin, sertaconazole, sulconazole, terbinafine, terconazole, tioconazole, and voriconazole.

[0055] For use in the therapeutic applications described herein, kits and articles of manufacture are also described herein. Such kits can comprise a carrier, package, or container that is compartmentalized to receive one or more containers such as vials, tubes, and the like, each of the container(s) comprising one of the separate elements to be used in a method described herein. Suitable containers include, for example, bottles, vials, syringes, and test tubes. The containers can be formed from a variety of materials such as glass or plastic.

[0056] For example, the container(s) can comprise a pharmaceutical composition disclosed herein, optionally in combination with another treatment for acne vulgaris as disclosed herein. Such kits optionally comprise a composition disclosed herein with an identifying description or label or instructions relating to its use in the methods described herein.

[0057] The disclosure provides a more selective and potent method for killing and / or inhibiting the growth of C. acnes. The disclosure provides strains of skin commensal bacteria that produce selective antimicrobial activity against pathogenic bacterial strains, but not against other members of skin microflora. Therefore, antimicrobial therapy using commensal bacterial strains would selectively kill target strains of microorganisms. The capacity for selective killing of pathogenic bacteria over the normal microflora is highly desirable because it may help to maintain homeostasis and shape the normal bacterial community. InAttorney Docket No.00015-440WO1 this case, antimicrobial therapy using commensal strains of bacteria would have a low risk of generating a resistant mutant against antibiotics. This disclosure may be used as a live bacterial application of C. acnes H1.43 or as a sterile extract of the C. acnes H1.43 bacteria (e.g., supernatant) or as purified active antimicrobial protein.

[0058] In some embodiments, the composition comprises a cream, ointment, oil suspension or unguent wherein Cutibacterium acnes H1.43 (C. acnes H1.43), a supernatant from C. acnes H1.43, and / or an antimicrobial protein purified from C. acnes H1.43, as described above are incorporated within a moisturizer or emulsion such as those described below and in Nakatsuji, T. et al. (2016), Nature Medicine. In some embodiments, the composition comprises a patch or poultice wherein the bacteria are combined with a suitable excipient and are incorporated within a fabric, gel matrix, or polymer sheet. Suitable excipients and carriers for topical administration are known in the art and include thickeners, emulsifiers, fatty acids, polysaccharides, polyols, and polymers and copolymers, including, without limitation, alginate, microcrystalline cellulose, polylactic acid, polylactic-co-glycolic acid, petrolatum, and numerous others known in the art.

[0059] In some embodiments, the method as described herein comprises supporting the health of the skin of a subject. In further embodiments, the method comprises providing a treatment for acne vulgaris. In some embodiments, the treatment comprises the steps of administering to acne a pharmaceutical composition disclosed herein. Determination of the appropriate mode of administration of a given formulation (ointment, gel, patch, etc.) can be done by one of ordinary skill in the art of treating skin infections. In some further embodiments, the pharmaceutical composition of the disclosure is re-applied at regularly timed intervals. In some embodiments, the pharmaceutical composition is reapplied every three days. In some embodiments, the pharmaceutical composition is reapplied every two days. In some embodiments, the pharmaceutical composition is reapplied every two days. In some embodiments, the pharmaceutical composition is reapplied daily. In some embodiments, the pharmaceuticalAttorney Docket No.00015-440WO1 composition is reapplied more than once per day. In some embodiments, the pharmaceutical composition is reapplied weekly. In some embodiments, the pharmaceutical composition is only applied a single time.

[0060] The following examples are exemplary and are not intended to limit the claimed invention, but rather illustrate methods of compositions of the disclosure. EXAMPLES

[0061] C. acnes H1.43 growth conditions. C. acnes H1.43 are grown in reinforced clostridial media (RCM), vortexed and plated onto brucella blood agar plates supplemented with Vitamin K, hemin and 5% sheep's blood and incubated for 5 days at 37°C.

[0100] Ammonium sulfate precipitation of C. acnes H1.43. 40 mL of the supernatant from a 200 mL C. acnes culture wase transferred to 5 separate 50 mL tubes. The tubes were centrifuged at 4,000 x g for 20 minutes at room temperature to pellet any remaining bacteria. After centrifugation, the supernatant was carefully collected from each of the 5 tubes and passed through a 0.22 µm filter. Ammonium sulfate was then added to the filtered supernatant to achieve 45% saturation (277 g / L of bacterial supernatant). The resulting mixture was rotated at ambient temperature for 1-2 hours to precipitate the proteins. The tubes were then centrifuged at 4,500 x g for 30 minutes at ambient temperature to pellet the protein precipitate. The supernatant was discarded to ensure minimal salt residue by inverting the tube. The protein pellet was resuspended in 30 mL of either distilled water, PBS, culture media, or phosphate buffer. The resuspended solution was filtered using a 0.22 µm filter and then either stored at -20 °C, or further extracted using the following extraction steps. 15 mL of the filtered solution was placed into two falcon tubes and then filtered using a 30 kDa molecular weight filter and centrifuged at 4,500 x g at ambient temperature for 10 minutes. The flow-through was discarded and the extract, on the top of the filter, was retained. Finally, 5 mL of distilled water, PBS, culture media, or phosphate buffer were added to the retained extract in each tube, resulting in a total volume of 10 mL and achieving a 20X concentration of the extract.Attorney Docket No.00015-440WO1

[0062] In vitro antimicrobial assays. For the radial diffusion agar assays, melted TSB or RCM agar (12 mL) is mixed with C. acnes (1 x 10 CFU) and poured into a 10 cm square petri square. When the agar is solidified, a 10 µl aliquot of bacteria is inoculated onto a single grid. The plates are incubated at 37 °C for 3 days in a 2.5L AnaeroPack (Thermo Fisher Scientific) to allow visible growth of bacteria. Antibacterial activity was determined by a clear zone of inhibition within the agar that surrounds the colony. The size of the inhibitory zone is recorded as a measure of antimicrobial activity (+ slight, ++ moderate, or +++ potent) (e.g., see FIG. 2).

[0063] For the liquid culture assay, conditioned supernatant from overnight cultures of C. acnes is harvested and sterile filtered (0.22 µm). 50% of conditioned supernatant is mixed with 50% fresh TSB or RCM containing 1 x 10 CFU / mL of C. acnes, in a 96 well round bottom plate. C. acnes plates are incubated standing at 37 °C in an anaerobic chamber. Bacterial growth is measured by OD and positive antimicrobial strains are identified as those that suppressed bacterial growth to less than 50% (I ) of growth measured for negative control strains. Bacterial survival is measured by counting the number of CFU on Brucella blood agar plates. At selected times post-treatment with bacterial supernatant or extracts, the number of CFU is determined by serial dilution in phosphate-buffered saline (PBS) and plating onto appropriate agar media. Bacterial survival is measured as the total number of CFU per milliliter.

[0064] Purification of antimicrobials produced by C. acnes. the use of an ion exchange column is a common technique in protein purification and separation. The method relies on the principle of reversible binding of charged molecules (such as proteins) to a solid-phase matrix containing charged functional groups. Proteins with opposite charges to those on the matrix will bind to the column, while uncharged or similarly charged molecules will flow through or be easily washed away. The bound proteins can then be eluted by altering the conditions, such as changing the pH or ionic strength of the buffer, causing the proteins to dissociate from the column. Different buffers with different pH were tested in the studies presented herein. It was found that the best condition toAttorney Docket No.00015-440WO1 purify the antimicrobial protein from C. acnes H1.43 was to use the cation exchanger column HiTrap SP XL in association with a step elution of 100 mM NaCl in succinate buffer at pH 5.

[0101] Protein purification from C. acnes H1.43 ammonium sulfate precipitate: Column Preparation: the ion exchange column is equilibrated with an appropriate buffer to ensure the column is ready for use. 1 to 2 mL of the protein purification from C. acnes H1.43 ammonium sulfate precipitate was loaded into the column. The column was washed with 9 mL of the same buffer used during equilibration. This step removed unbound impurities and ensured a clean baseline before elution. A gradient of NaCl was used under the selected buffer conditions to elute the selectively bound proteins (3 to 9 mL). By increasing the ionic strength, the proteins were released from the column. The eluted fractions were collected containing the antimicrobial protein of interest. It was found that the antimicrobial protein from C. acnes H1.43 ammonium sulfate precipitate was thermosensitive over 60 °C, with a molecular weight of over 30 kDa.

[0065] Screening the antimicrobial activity of the supernatant and purified protein from the C. acnes H1.43 ammonium sulfate precipitate v. C. acnes phylotypes and Streptococcus strains. It was found in the agar plate radial diffusion assay using the sterile supernatant from C. acnes H1.43 inhibited the growth of C. acnes 6919 (see FIG. 1). In a liquid culture of C. acnes 6919 it was found that the ammonium sulfate precipitate of C. acnes H1.43 inhibited the growth of C. acnes 6919 int a dose dependent manner (see FIG. 1). In a larger screen of C. acnes phylotypes, it was found that C. acnes H1.43 strongly inhibited the growth of seven C. acnes phylotypes (e.g., see FIG. 2).

[0066] In liquid cultures of various Streptococcus strains, it was found that the supernatant of C. acnes H1.43 did not inhibit the growth of the Streptococcus strains (e.g., see FIG. 3), which was further confirmed in agar plate radial diffusion assays. Similarly, the ammonium sulfate precipitate of C. acnes H1.43 did not inhibit the growth of liquid cultures of various Streptococcus strains (e.g., see FIG. 4). Accordingly, the antimicrobial activity of protein from C. acnes H1.43 has been shown to be highlyAttorney Docket No.00015-440WO1 specific to other phylotype strains of C. acnes, but does not have general antibiotic activity, as demonstrated by the failure to inhibit the growth of the tested Streptococcus strains.

[0067] A number of embodiments of the disclosure have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the disclosure. Accordingly, other embodiments are within the scope of the following claims.

Claims

Attorney Docket No.00015-440WO1 WHAT IS CLAIMED IS:

1. A pharmaceutical composition comprising: Cutibacterium acnes 43 (C. acnes H1.43), a supernatant from C. acnes H1.43; and / or an antimicrobial protein purified from C. acnes H1.43; and a pharmaceutically acceptable carrier for topical administration.

2. The pharmaceutical composition of claim 1, wherein the supernatant from C. acnes H1.43 is a sterile supernatant.

3. The pharmaceutical composition of claim 1 or claim 2, wherein the antimicrobial protein is purified from an ammonium sulfate precipitate of C. acnes H1.

43.

4. The pharmaceutical composition of claim 1 or 2, wherein the antimicrobial protein is thermosensitive over 60 °C and has a molecular weight of over 30 kDa.

5. The pharmaceutical composition of claim 1 or 2, wherein the pharmaceutical composition is formulated as an ointment, cream, spray or gel.

6. A composition comprising a thickened topical formulation of Cutibacterium acnes 43 (C. acnes H1.43) and optionally, a prebiotic compound, a protectant, humectant, emollient, abrasive, salt, and / or surfactant; and wherein the composition is formulated for the topical treatment of disorders of dysbiosis or acne of the skin, scalp, or mucosae.

7. The composition according to claim 6, wherein the Cutibacterium acnes 43 (C. acnes H1.43) is provided in a live form.

8. The composition according to claim 6, wherein the Cutibacterium acnes 43 (C. acnes H1.43) is provided in a lyophilized or freeze-dried or spray dried form.Attorney Docket No.00015-440WO1 9. The composition according to Claim 8, wherein the Cutibacterium acnes 43 (C. acnes H1.43) can be reconstituted into a live form.

10. The composition of claim 1 or 6, wherein the composition further comprises an acne treatment selected from tretinoin, adapalene, tazarotene, benzoyl peroxide, azelaic acid, salicylic acid, or dapsone.

11. A method of treating a subject having acne vulgaris, comprising: administering a therapeutically effective amount of the pharmaceutical composition of claim 1 or composition of claim 6 to the subject having acne vulgaris in need of treatment thereof.

13. The method according to claim 11, wherein the composition is applied topically.

14. The method according to claim 11, wherein the composition is formulated as a cream, ointment, unguent, spray, powder, oil, thickened formulation or poultice.

15. The method of claim 11, wherein the subject is treated daily with the pharmaceutical composition until the subject's acne vulgaris is resolved.

16. The method of claim 11, wherein the subject is treated with the composition and one or more other treatments for acne vulgaris.

17. The method of claim 16, wherein the one or more other treatments for acne vulgaris are selected from adapalene, tazarotene, benzoyl peroxide, azelaic acid, salicylic acid, dapsone, spironolactone, and / or oral antibiotics.

18. The method of claim 17, wherein the oral antibiotics is selected from doxycycline, minocycline, tetracycline, erythromycin, azithromycinAttorney Docket No.00015-440WO1 19. A method to purify an antimicrobial protein from C. acnes H1.43, comprising: isolating a supernatant from a culture of C. acnes H1.43; adding ammonium sulfate to the supernatant to achieve at least 40% saturation; isolating proteins from the ammonium sulfate treated supernatant; filtering the isolated proteins using a 30 kDa molecular weight size exclusion filter; and collecting the retained extract that did not pass through the 30 kDa molecular weight size exclusion filter, wherein the retained extract comprises the antimicrobial protein.

20. The method of claim 19, wherein the supernatant is isolated from the culture of C. acnes H1.43 by centrifugation.

21. The method of claim 19 or claim 20, wherein the supernatant is filtered using a 0.22 µm filter prior to adding ammonium sulfate.

22. The method of claim 19 or claim 20, wherein the ammonium sulfate is added to the supernatant to achieve 45% saturation.

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