RELATED APPLICATIONS

[001] This application claims the benefit of and priority to United States Provisional Application Serial Number 62/660,134 filed April 19, 2018, the disclosure of which is incorporated herein by reference in its entirety.

SEQUENCE LISTING

[002] The ASCII text file submitted herewith via EFS-Web, entitled “110697- 0l370lPCT_ST25.txt” created on April 18, 2019, having a size of 1 ,998 bytes, is hereby incorporated by reference in its entirety.

STATEMENT OF GOVERNMENT SUPPORT

[003] This invention was made with Government Support under Grant Number NSF DGE 1144804 awarded by the National Science Foundation. The Government has certain rights in the invention.

FIELD

[004] The disclosure relates generally to topical skin treatment compositions, and in particular to topical skin treatment compositing include antimicrobial peptides.

BACKGROUND

[005] The prevalence of acne vulgaris is 681.2 million (2016), a 10% increase from 2006, and acne vulgaris ranks 8 ^th on the list of most prevalent diseases in the world, affecting 8 in 10 people. Inflammatory acne dominates the acne vulgaris market. In the United States acne treatment and loss of productivity costs over $3 billion per year. Accutane, the top selling product in the prescription market was $1.2 billion (2016). One of the fastest growing markets in this space is in India and Japan.

[006] The market for dermatology therapeutics is expected to grow from $19.8 (2016) to $41.2 billion in 2026, CAGR 8%. Globally, this product market was $114 billion (2016).

[007] The topical acne market, unfortunately, is plagued by a lack of innovative treatments, particularly those that treat the inflammation that comes with acne infection. Many current products such as Proactiv ^® include the same few key ingredients, benzoyl peroxide or salicylic acid, which kill bacteria but do not prevent future outbreaks and considerably compromise skin hydration. Some natural extracts have been added to these formulations to try and alleviate skin dryness and inflammation, but these are incremental innovations to the underlying problem, the chemicals used to kill bacteria itself.

[008] Accordingly, there is a need for a safe and effective treatment of acne vulgaris, as well as other similar skin conditions.

SUMMARY

[009] Disclosed herein is a synergistic combination of natural extracts with an antimicrobial peptide. The peptide has broad-spectrum activity against many different types of bacteria, including resistant strains, has a low likelihood of inducing resistance in bacteria, helps to heal wounds, and demonstrates anti-inflammatory effects. Further, the synergy of the essential oil and the peptide requires a much lower active concentration of peptide to be delivered, leading to much lower cytotoxic effects than the current chemical-based formulations.

[0010] Aspects of the disclosure relate to a topical composition comprising (1) at least one fusion peptide comprising at least one antimicrobial peptide and at least one peptide capable of improving the stability, the activity or the stability and the activity of the antimicrobial peptide in a hydrophobic environment, (2) at least one essential oil, wherein the composition comprises the at least one fusion peptide and the at least one essential oil at concentrations capable of providing synergistic anti-inflammatory activity and/or antimicrobial activity. In some embodiments, the composition comprises the at least one fusion peptide and the at least one essential oil at concentrations that are not cytotoxic.

[0011] In some embodiments, the at least one essential oil is one of Tea Tree Oil, Manuka Oil, Orange Oil, Lemongrass Oil, Sunflower Oil, Eucalyptus Oil. Orange and Sunflower oil, or combination thereof.

[0012] In some embodiments, the composition comprises from 0.03 to 4 % (v/v) Tea Tree Oil, Manuka Oil, Orange Oil, Lemongrass Oil, Sunflower Oil, Eucalyptus Oil, or combinations thereof.

[0013] In some embodiments, the composition comprises from 0.1% to 0.5% (v/v) Manuka Oil, from 0.02% to 0.05% (v/v) Eucalyptus Oil or combinations thereof.

[0014] In some embodiments, the composition comprises from 0.1 to 15 mM the at least one fusion peptide. In some embodiments, the antimicrobial peptide comprises a synthetic LL37 peptide or variant thereof. In some embodiments, the antimicrobial peptide has an amino acid sequence as set forth in SEQ ID NO: 1. In some embodiments, the antimicrobial peptide has an amino acid sequence having at least 95% identity with SEQ ID NO: 1. In some embodiments, the fusion peptide has an amino acid sequence as set forth in SEQ ID NO: 2. In some embodiments, the fusion peptide has an amino acid sequence as set forth in SEQ ID NO: 3. In some embodiments, the at least one antimicrobial peptide is at the N-terminus. In some embodiments, the at least one fusion peptide comprises a linker at the C-terminus of the antimicrobial peptide.

[0015] In some embodiments, the at least one peptide capable of improving the stability, the activity or the stability and the activity of the antimicrobial peptide in a hydrophobic environment is a collagen binding domain. In some embodiments, the collagen binding domain is derived from human collagenase or human fibronectin.

[0016] Other aspects of the disclosure relate to a method of treating acne vulgaris, the method comprising administering to skin affected by acne vulgaris a topical composition comprising (1) at least one fusion peptide comprising at least one antimicrobial peptide and at least one peptide capable of improving the stability, the activity or the stability and the activity of the antimicrobial peptide in a hydrophobic environment, and (2) at least one essential oil, wherein the composition comprises the at least one fusion peptide and the at least one essential oil at concentrations capable of providing synergistic anti-inflammatory activity and/or antimicrobial activity. . In some embodiments, the composition comprises the at least one fusion peptide and the at least one essential oil at concentrations that are not cytotoxic.

[0017] In some embodiments, the at least one essential oil is one of Tea Tree Oil, Manuka Oil, Orange Oil, Lemongrass Oil, Sunflower Oil, Eucalyptus Oil. Orange and Sunflower oil, or combination thereof.

[0018] In some embodiments, the composition comprises from 0.03 to 4 % (v/v) Tea Tree Oil, Manuka Oil, Orange Oil, Lemongrass Oil, Sunflower Oil, Eucalyptus Oil, or combinations thereof.

[0019] In some embodiments, the composition comprises from 0.1% to 0.5% (v/v) Manuka Oil, from 0.02% to 0.05% (v/v) Eucalyptus Oil or combinations thereof.

[0020] In some embodiments, the composition comprises from 0.1 to 15 mM the at least one fusion peptide. In some embodiments, the antimicrobial peptide comprises a synthetic LL37 peptide or variant thereof. In some embodiments, the antimicrobial peptide has an amino acid sequence as set forth in SEQ ID NO: 1. In some embodiments, the antimicrobial peptide has an amino acid sequence having at least 95% identity with SEQ ID NO: 1. In some embodiments, the fusion peptide has an amino acid sequence as set forth in SEQ ID NO: 2. In some embodiments, the fusion peptide has an amino acid sequence as set forth in SEQ ID NO: 3. In some embodiments, the at least one antimicrobial peptide is at the N-terminus. In some embodiments, the at least one fusion peptide comprises a linker at the C-terminus of the antimicrobial peptide.

[0021] In some embodiments, the at least one peptide capable of improving the stability, the activity or the stability and the activity of the antimicrobial peptide in a hydrophobic environment is a collagen binding domain. In some embodiments, the collagen binding domain is derived from human collagenase or human fibronectin.

BRIEF DESCRIPTION OF THE DRAWINGS

[0022] These and other characteristics of the present disclosure will be more fully understood by reference to the following detailed description in conjunction with the attached drawings by way of non-limiting examples of exemplary embodiments, in winch:

[0023] FIG. 1 is a graph showing soluble activity of LL37 and and /CBD-LL37 against P. acnes, starting at a concentration of 15 mM. MICs were determined to be 1.88mM and 7.5mM for LL37 and fCBD-LL37, respectively.

[0024] FIG. 2 is a graph depicting six essential oils’ isolated antimicrobial activity against P. acnes.

[0025] FIG. 3A and FIG. 3B depict enhancement of /CBD-LL37 antimicrobial activity by inclusion of sub-inhibitory concentrations of manuka oil and eucalyptus oil, respectively.

[0026] FIGs. 4A-4C depict negative control sections stained with Eosin, Hoechst, and anti-LL37 IHC, respectively, where no peptide was included in the assessed formulations applied to porcine skin samples ex vivo.

[0027] FIGs. 5A-5C show experimental sections stained with Eosin, Hoechst and anti-LL37 IHC, respectively, where peptide /CBD-LL37 solubilized in water versus in the delivery vehicle, were applied to porcine skin samples ex vivo.

[0028] FIG. 6 depicts representative samples taken for IHC pixel intensity analysis.

[0029] FIG. 7 shows Pixel Intensity Analysis of the IHC images for the Tween® 80-only negative control, the /CBD-LL37 peptide control (solubilized in water), and the experimental /CBD-LL37 solubilized in the eucalyptus oil (0.05%) delivery vehicle.

[0030] While the above-identified drawings set forth presently disclosed embodiments, other embodiments are also contemplated, as noted in the discussion. This disclosure presents illustrative embodiments by way of representation and not limitation. Numerous other modifications and embodiments can be devised by those skilled in the art which fall within the scope and spirit of the principles of the presently disclosed embodiments.

DETAILED DESCRIPTION

[0031] The term“topical formulation” is used herein to generally include a formulation that can be applied to skin or a mucosa. Topical formulations may, for example, be used to confer therapeutic benefits or cosmetic benefits to a patient. Topical formulations can be used for both topical and transdermal administration of substances. The term“formulation” and“composition” are used herein interchangeably.

[0032] The term“topical administration” is used herein to generally include the delivery of a substance, such as a therapeutically active agent, to the skin or a localized region of the body.

[0033] The term“transdermal” is used herein to generally include a process that occurs through the skin. The terms“transdermal” and“percutaneous” are used interchangeably throughout this specification.

[0034] The term“transdermal administration” is used herein to generally include administration through the skin. Transdermal administration is often applied where systemic delivery of an active agent is desired, although it may also be useful for delivering an active agent to tissues underlying the skin with minimal systemic absorption.

[0035] The term“collagen binding domain” as used herein refers to a peptide that binds collagen.

[0036] As used herein“chimeric peptide”,“fusion peptide” and“fusion protein” mean a peptide comprising two or more peptides, and are used interchangeably.

[0037] The term“delivery vehicle” refers to compositions or devices for delivery of peptides to the treatment area. It should be noted that as used herein, the term“delivery vehicle” includes essential oils by themselves in the essential oil/peptide compositions, as well as other delivery means for delivering the essential oil/peptide compositions to the treatment area.

[0038] Aspects of the disclosure relates to chimeric peptides, compositions and methods of treating mild to moderate acne vulgaris, severe acne vulgaris, psoriasis, rosacea, dermatitis, mild to moderate skin abrasions; potentially any skin disease that involves bacterial infection or inflammation leading to itching.

[0039] Aspects of present disclosure provide an antimicrobial peptide being co-delivered with a natural agent, an essential oil such as Manuka, Eucalyptus or Tea Tree oil, which serves a beneficial purpose in reducing the required concentration of peptide required to kill bacteria, including P. acnes, by acting synergistically. In some aspects, the antimicrobial peptide can be co-administered topically with a natural agent to treat mild to moderate acne vulgaris, severe acne vulgaris, psoriasis, rosacea, dermatitis, mild to moderate skin abrasions; potentially any skin disease that involves bacterial infection or inflammation leading to itching. In some embodiments, the essential oils may also provide other anti-inflammatory and anti-oxidant purposes for the skin treatment.

[0040] In some embodiments, the fusion peptide in combination with the essential oil has a synergistic effect on the anti-inflammatory and/or antimicrobial activities. For example, if in combination with human LL37, which is known to have inherent anti-inflammatory activity, or fusion peptides that include LL37 peptide sequence (e.g., fCBD-LL37), the essential oils may also have synergistic effects on these activities.

[0041] Aspects of the present disclosure relate to compositions comprising a chimeric antimicrobial peptide (also referred herein as fusion peptide or chimeric peptide) with at least one collagen-binding domain (CBD) and an essential oil and methods for using the same. In some embodiments, the antimicrobial peptide can have a broad antimicrobial spectrum.

[0042] In some embodiments, the peptide is a fusion antimicrobial peptide with collagen-binding domain. In some embodiments, the antimicrobial peptide is a cationic peptide with less than 100, less than 90, less than 80, less than 70, less than 60, less than 50 amino acids. In some embodiments, the peptide is a human-derived 37-amino acid AMP belonging to the cathelicidin family. In some embodiments, the peptide comprises an amino acid sequence as set forth in SEQ ID NO: 1, or variant thereof. In some embodiments, the peptide can have at least 99%, 98%, 97%, 96%, 95% identity with SEQ ID NO: 1.

[0043] In some embodiments, the fusion peptide comprises (1) at least one antimicrobial peptide and (2) at least one peptide capable of improving the stability, the activity or the stability and the activity of the antimicrobial peptide in a hydrophobic environment. In some embodiments, the fusion peptide consists of at least one antimicrobial peptide and at least one peptide capable of improving the stability, the activity or the stability and the activity of the antimicrobial peptide in a hydrophobic environment. In some embodiments, the fusion peptide is 60 or less amino acid long. For example, the fusion peptide can be from 40 to 60, 45 to 60, 50 to 60, 55 to 60 amino acids long. In some embodiments, the peptide is a peptide derived from extracellular matrix binding domain, such as collagen binding domain. In some embodiments the peptide is about 5, 6, 7, 8, 9, 10 or more amino acid long. In some embodiments the peptide is at the C-terminus of the antimicrobial peptide. In some embodiments, the fusion peptide further comprised a peptide linker between the antimicrobial peptide and the peptide. In some embodiments, the fusion peptide consists of at least one antimicrobial peptide, a linker and at least one peptide capable of improving the stability, the activity or the stability and the activity of the antimicrobial peptide in a hydrophobic environment.

[0044] In some embodiments, the fusion peptide comprises one or more antimicrobial peptides and one or more extracellular matrix binding domain. In some embodiments, the fusion peptide comprises one or more antimicrobial peptides and one or more collagen binding domain. In some embodiments, the fusion peptide is a synthetic LL37 peptide fused with at least one collagen binding domain. In some embodiments, the collagen binding domain can be collagen binding domains derived from collagenase (cCBD) or fibronectin (fCBD). In some embodiments, the collagen binding domain can be collagen binding domains derived from human collagenase or human fibronectin. In some embodiments, the collagen binding domain can be a fragment of collagenase or of fibronectin. In some embodiments, the fragment can be a 5, 6, 7, 8, 9, 10 or longer amino acid peptide. In some embodiments, the peptide and the collagen binding domain are linked via a flexible linker. In some embodiments, the linker can be from 3 to 20 amino acid long. For example, the linker can be DYKDDDDK (SEQ ID NO: 4).

[0045] In some embodiments, an antimicrobial peptide is synthetic human LL37 (LLGDFFRKSKEKIGKEFKRIV QRIKDFLRNLVPRTES) (SEQ ID NO: 1). In some embodiments, the fusion peptide may comprise LL37 peptides modified with collagen binding domains. In some embodiments, the fusion peptide is cCBD-LL37

(LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTESDYKDDDDKTKKTLRT (SEQ ID NO: 2), where the collagenase-derived cCBD has an amino acid sequence of TKKTLRT. In some embodiments, the fusion peptide is fCBD-LL37

(LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTESDYKDDDDKCQDSERTFY (SEQ ID NO: 3), where the fibronectin-derived fCBD has an amino acid sequence of CQDSERTFY. In some embodiments, the fusion peptides incorporate a FLAG™ domain (DYKDDDDK) (SEQ ID NO: 4) between the LL37 sequence and CBD, which can act as an epitope tag and as a flexible linker sequence.

[0046] In some embodiments, the collagen binding domain can increase the stability and retention of AMPs on collagen by limiting the peptide exposure to proteases, providing high affinity, specific binding onto collagen and combinations thereof.

[0047] In some embodiments, the composition of the fusion peptide allows the fusion peptide to remain on the skin for a desired period of time, i.e. allows the peptide to remain on the skin for a long enough period so that the peptide can be effective. For example, the period of time can be between 1 to 14 days, 17 to 21 days, 1 to 30 days, 7 to 14 days, 7 to 30 or more. For example, the period of time can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more.

[0048] In some embodiments, the fusion peptide can be cCBD-LL37 (SEQ ID NO: 2) or fCBD- LL37 (SEQ ID NO: 3). In some embodiments, the fusion peptide can have at least 99%, 98%, 97%, 96%, 95% identity with SEQ ID NO: 2 or SEQ ID NO: 3. In some embodiments, the fusion peptides have an antimicrobial activity against target bacteria, such as P. acnes, are effective in treating target bacteria, such as P. acnes, at non-cytotoxic concentrations and are of a molecular size that allows skin penetration. In some embodiments, the fusion peptide has anti-inflammatory activity or antibacterial and anti-inflammatory activities.

[0049] In some embodiments, the fusion peptide can be present in the composition at a concentration of from 0.05 micromolar to 25 micromolar. For example, the composition can comprise 0.05 to 1, 0.05 to 5, 0.05 to 10, 0.05 to 15, 0.05 to 20, 0.05 to 25, 1 to 5, 1 to 10, 1 to 15, 1 to 20, 1 to 25, 5 to 10, 5 to 15, 5 to 20, 5 to 25, 10 to 15, 10 to 20, 10 to 25, 15 to 20, 15 to 25, 20 to 25 micromolar, and any ranges in between, of a fusion peptide. In some embodiments, the concentration of cCBD-LL37 can be from 0.05 micromolar to 10 micromolar. For example, the composition can comprise 0.05 to 1, 0.05 to 2, 0.05 to 3, 0.05 to 4, 0.05 to 5, 0.05 to 10, 1 to 5, 1 to 10, 5 to 10, 5 to 15, 5 to 20, 10 to 15, 10 to 20, 15 to 20 micromolar, and any ranges in between, of fusion peptide cCBD-LL37. In some embodiments, the concentration of fCBD-LL37 can be from 0.05 micromolar to greater than 20 micromolar. For example, the composition can comprise 0.05 to 1, 0.05 to 5, 0.05 to 10, 0.05 to 15, 0.05 to 20, 0.05 to 25, 1 to 5, 1 to 10, 1 to 15, 1 to 20, 1 to 25, 5 to 10, 5 to 15, 5 to 20, 5 to 25, 10 to 15, 10 to 20, 10 to 25, 15 to 20, 15 to 25, 20 to 25 micromolar, and any ranges in between, of fusion peptide fCBD-LL37. In some embodiments, the concentration of the fusion peptide is 13mM or higher.

[0050] In some embodiments, the fusion peptide has a minimum inhibitory concentration (MIC) equal to or lower than 10 m M. In some embodiments, the fusion peptide has a minimum inhibitory concentration between 0.05 - 10 micromolar.

[0051] In some embodiments, the fusion peptide is able to enter hair follicle pores and into the sebaceous gland. In some embodiments, the has a size smaller than 7000 Daltons and is able to enter hair follicle pores and into the sebaceous gland.

[0052] Experiments performed demonstrate:

[0053] Essential oils demonstrate antimicrobial activity on their own against both P. acnes and Methicillin-resistant S. aureus strains. Oils tested included Tea Tree Oil, Manuka Oil, Orange Oil, Lemongrass Oil, Sunflower Oil, Eucalyptus Oil. Orange and Sunflower oil did not exhibit antimicrobial activity against P. acnes at any concentration tested, all others did. According to aspects of the disclosure the essential oil is used as a delivery vehicle.

[0054] According to aspect of the disclosure, the composition can comprise less than 50%, less than 40%, less than 30%, less than 20%, less than 20%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1% of essential oils. In some embodiments, the composition comprises a fusion peptide and one or more essential oil as active agent or additive. For example, the composition can comprise from 0.03 to 4 % (v/v) Tea Tree Oil, Manuka Oil, Orange Oil, Lemongrass Oil, Sunflower Oil, Eucalyptus Oil, or combinations thereof. In some embodiments, the composition can comprise from 0.1 to 15 mM fusion peptide.

[0055] In some embodiments, the combination of essential oils comprises from 0.1% to 0.5% (v/v) Manuka Oil, from 0.02% to 0.05% (v/v) Eucalyptus Oil or combinations thereof.

[0056] In some embodiments, the minimum inhibitory concentration (MIC) of the fusion peptides is reduced in the presence of low concentrations of essential oils. In some embodiments, the greater than 99% reduction in amount of the fusion peptide (e.g. fCBD-LL37) can be required to kill bacteria when the fusion peptide is combined with <less than 1% essential oils. For example, the minimum inhibitory concentration of the fusion peptide may be reduced from 7.5 mM to 0.23 mM when in combination with an essential oil. In some embodiments, the minimum inhibitory concentration of the fusion peptide is reduced to from 7.5mM to 0.032mM in presence of from 0.1% to 0.5% (v/v) Manuka Oil, from 0.02% to 0.05% (v/v) Eucalyptus Oil or combinations thereof.

[0057] According to aspects of the disclosure, the minimum inhibitory concentration of )CBD- LL37 and cCBD-LL37 is reduced in the presence of low concentrations of essential oils, Manuka and Eukalyptus oil against P. acnes, which demonstrates the synergistic effect of the combination of peptides with these oils.

[0058] In some embodiments, the combination of the fusion peptide and at least one essential oil described herein provide a synergistic bacteriostatic/bactericidal and/or anti-inflammatory activity for topical application, i.e. the dosages of the fusion peptide and the essential oil(s) are lower than what would be expected to be clinically efficacious than when administered individually.

[0059] It has been surprisingly discovered that the formulation comprising less than 0.5 micromolar fusion peptide and as low as 0.1% essential oil has efficacy in treating acne (Figure 3) that is comparable to that obtained with formulations containing 7.5 micromolar (Figure 3) or 10 micromolar fusion peptide (Figure 1) or with formulations containing 1% essential oil (Figure 2).

[0060] Histology and immunohistochemistry and quantitative pixel analyses demonstrate that the oils and peptides are able to diffuse on skin: /CBD-LL37 peptide mixed with 0.05% (v/v) eucalyptus oil diffused into the hair follicles better than fCBD-LL37 dissolved in water. In addition, the /CBD-LL37 peptide alone and formulated with oils can be delivered to the hair follicles.

[0061] The combinations of peptide and essential oils can be used in both the topical treatment and prevention of acne as well as other dermatological diseases (such as rosacea, dermatitis, psoriasis, etc.). In some embodiments, the composition comprises (1) at least one essential oil, and (2) at least one fusion peptide comprising at least one antimicrobial peptide, optionally a linker, and at least one peptide capable of improving the stability and/or the activity of the antimicrobial peptide in a hydrophobic environment. This includes formulations that are either oil- or water-based, with a low concentration of antimicrobial peptide combined with a generally regarded as safe essential oil. This treatment would kill a variety of bacteria as well as reduce inflammation in a non-toxic way.

[0062] In some embodiments, the composition comprises one or more additional active agent. For example, the additional active agent can be an anti-pain or anti-itch agent. In some embodiments, the anti-itch agent can be an antihistamine, a corticosteroid or the like. In some embodiments, the anti-pain agent can be an anaesthetic or analgesic such as benzocaine or lidocaine. In some embodiments, the additional active agent can be a plant extract having medicinal anti-itch or analgesic properties.

[0063] In some embodiments, the composition is a topical formulation. In some embodiments, the formulation is liquid-based topicals including creams, ointments, gels and serums. In some embodiments, the topical composition comprises excipients that allow for improved properties of drying time, increased transdermal flux and greater pharmacokinetic absorption in vivo, desired viscosity, good adherence to the skin, and ready spreadability, while maintaining stability over time.

[0064] In some embodiments, the composition can include a penetration enhancer. The penetration enhancer can be dimethyl sulfoxide (“DMSO”) or derivatives thereof. The DMSO may be present in an amount by weight of 1% to 70%, between 25% and 60%, such as 25, 30, 40, 45, 50, 55, or 60% w/w.

[0065] In some embodiments, the composition can comprise an alkanol, such as methanol, ethanol, propanol, butanol or mixtures thereof. In some embodiments, the alkanol is present at about 1 to about 50% w/w, for example 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50% w/w, and all fractions in between. In some embodiments, no alkanol is used in the composition. [0066] In some embodiments, the composition can comprise a polyhydric alcohol, such as a glycol. Suitable glycols include ethylene glycol, propylene glycol, butylene glycol, dipropylene glycol, hexanetriol and a combination thereof. In some embodiments, the propylene glycol is used at about at 1-15% w/w, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15% w/w, and all fractions in between. In some embodiments, no polyhydric alcohol is used in the formulation.

[0067] In some embodiments, the composition can comprise glycerol (also referred to herein as glycerine) at a concentration of 0-20% w/w. In some embodiments, glycerol is used at 0-4% w/w, such as 0, 1, 2, 3, or 4% w/w, and all fractions in between. In some embodiments, no glycerol is used in the composition.

[0068] In some embodiments, the composition can comprise at least one thickening agent. The at least one thickening agent can be an acrylic polymer, an acrylic polymer derivative, a cellulose polymer, a cellulose polymer derivative such as hydroxypropylcellulose (HPC), polyvinyl alcohol, poloxamers, polysaccharides or mixtures thereof. In some embodiments, the at least one thickening agent is used such that the formulation has the desired viscosity, for example from 10 and 50000 centipoise (cps).

[0069] In some embodiments, the composition can comprise at least one antioxidant or chelating agents.

[0070] In some embodiments, the composition can comprise a pH adjusting agent. For example, the pH adjusting agent can be a base or an acid, an acid salt, or mixtures thereof. The pH adjusting agent can be present in an amount sufficient to adjust the pH of the composition to between about pH 4.0 to about 10.0, for example about pH 7.0 to about 9.5.

[0071] In some embodiments, the composition can comprise at least one detergent, surfactant or emulsifying agents, such as sodium lauryl sulfate, polysorbates, etc.

[0072] In some embodiments, the composition can comprise vitamins and minerals, including but not limited to linoleic acid, retinoic acid, etc.

[0073] If desired, the composition can further include additional pharmaceutically acceptable excipients typically used in formulations and known to those skilled in the art. Such excipients include, for example, humectants, emollients, and preservatives.

[0074] In some embodiments, the combination of fusion peptides and essential oil is formulated as a topical cream. In some embodiments, the topical cream comprises an emulsion semisolid comprised of more than 20% water and less than 50% hydrocarbons, oils, or waxes.

[0075] In some embodiments, the combination of fusion peptides and essential oil is formulated as a topical ointment. In some embodiments the topical ointment comprises an emulsion semisolid comprised of less than 20% water and more than 50% hydrocarbons, oils or waxes.

[0076] In some embodiments, the combination of fusion peptides and essential oil formulated is as a topical gel.

[0077] In some embodiments, the combination of fusion peptides and essential oil formulated is as a topical serum.

Delivery systems

[0078] Nanoparticles

[0079] In some embodiments, the fusion peptides can be incorporated into nanoparticles. In some embodiments, the nanoparticles can be polymeric nanoparticles. In some embodiments, the nanoparticles can be configured for controlled release. In some embodiments, the nanoparticles comprise one or more polymers that are biodegradable and non-toxic. Examples of biodegradable polymers include, but are not limited to, natural polymers such as gelatin, sodium alginate, agarose, casein, zein, chitosan, glycol chitosan, N, N trimethyl chitosan, starch, cellulose and wheat gluten, hyaluronic acid, or combinations thereof.

[0080] Microneedle Arrays

[0081] In some embodiments, the fusion peptides can be delivered through microinjection. In some embodiments, the microneedle array can be used to deliver the fusion peptides.

[0082] In some embodiments, the delivery system comprises a microneedle arrays to deliver a formulation through a biological barrier, such as the stratum comeum of human skin and into the patient's lower skin tissues (e.g., epidermis, dermis, or subcutaneous skin layers). In some embodiments, the microneedle array can be surface coated with a solution comprising the fusion peptide. In some embodiments, the microneedle drug delivery device can be a patch.

Methods of treatment

[0083] The compounds described herein can be used to treat acne by applying the composition to affected areas of the skin, such as on the face, neck, back, and chest. The composition can be applied one or more times daily, for example, one to three times daily, for a time sufficient to ameliorate the signs of acne.

EXAMPLES

[0084] The systems, compositions and methods of the present disclosure are described in the following Examples, which are set forth to aid in the understanding of the disclosure, and should not be construed to limit in any way the scope of the disclosure as defined in the claims which follow thereafter. The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the embodiments of the present disclosure, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for.

Design Verification Methods

[0085] A three-step process of experiments was identified that allowed for verification of the conceptual delivery vehicle design. This process included a series of three minimum inhibitory concentration (MIC) assays, that informed subsequent MIC assays; the results of which allowed selection of preferred components. The first step was to characterize the soluble, antimicrobial activity of /CBD-LL37 against P. acnes. The second step was to investigate the antimicrobial activity of each of a total of six essential oils against P. acnes. Finally, the third step was to test the combined /CBD-LL37 and oil activity, informed by the second MIC assay, to ensure that within the delivery vehicle (essential oil). /CBD-LL37 would be active within a safe therapeutic dose. In order to verify and optimize the aspects of the delivery vehicle design, MIC assays were tested in triplicate, and each step was performed at least twice to confirm reproducible results.

MIC Assay Universal Experiment Conditions

[0086] Each MIC assay in the three step design verification process required the use of P. acnes liquid bacterial culture. Additionally, steps two and three involve essential oils and bacterial media, which required emulsification using polysorbate 80, also known as Tween® 80. These components are universal across each assay, and are described individually below.

P. acnes Bacterial Culture

[0087] As P. acnes (ATCC® 6919, VPI0389 (strain), 70005466 (lot #)) is an aerotolerant anaerobe, it requires minimal exposure to oxygen. Therefore, bacterial cultures were stored in an anaerobic chamber, a BD GasPak EZ chamber (Becton Dickinson, Franklin Lakes, NJ), along with anaerobic gas sachets that remove oxygen from the environment within the chamber. The bacteria were cultured using 15 g/L tryptic soy broth and plated on tryptic soy agar plates. P. acnes is slow growing, therefore four liquid cultures of ~5 mL each were inoculated one week prior to use in an experiment. Adequate bacterial growth was verified by assessing liquid culture turbidity.

[0088] To prepare the lxlO ⁶ CFU/mL concentration of bacteria needed for each MIC assay, a 0.5 McFarland standard correction was conducted. The optical density of the 0.5 McFarland standard tube represents a bacteria concentration of lxl 0 ⁸ CFU/mL. In order to adjust the P. acnes liquid culture to the desired concentration, a cycle of pipetting bacteria out of the culture tube and pipetting media in was repeated until visual comparison with a Wickerham card proved that the optical density was close to the liquid in the 0.5 McFarland standard tube. Then, to lower the standardized P. acnes culture to lxl 0 ⁶ CFU/mL, it was diluted 1 : 100 in broth in a petri dish, which was used for the MIC assays. To prove that the McFarland standardization procedure was done accurately, the lxl 0 ⁶ CFU/mL culture was further diluted to 10 ⁴ and 10 ³ , respectively, and spread onto n=2 tryptic soy agar plates for colony counts.

Media Preparation

[0089] P. acnes were grown in 15 g/L tryptic soy broth. In order to ensure a uniform solution of bacteria with each of the essential oils, 0.5% Tween® 80 (Sigma- Aldrich) was used as an emulsifier and surfactant. The 0.5% Tween® 80 solution was prepared by diluting the polysorbate 80 in tryptic soy broth and vortexing to mix the solution. Due to its viscosity, the Tween® 80 was heated in a 37 °C water bath prior to use to ensure more efficient pipetting.

Peptide Preparation

[0090] Stock solutions of the modified /CBD-LL37 peptide as well as unmodified LL37 were diluted with Dulbecco’s Phosphate-Buffered Saline (DPBS) to reach the desired concentration for the relevant assay being performed. Each peptide stock was stored in water, pH 3.5 supplemented with 5 mM ethylenediaminetetraacetic acid at -20°C at a concentration of 670 mM.

Delivery Vehicle Activity Assay

[0091] In order to verify the choice to use /CBD-LL37 as a potential acne treatment within the delivery vehicle, it was important first to verify its ability to kill P. acnes. This assay utilized unmodified LL37 as a control due to its published activity against P. acnes. The MIC assay, performed in a 96-well plate, followed a protocol described by Lozeau et al. Appropriate positive and negative controls were included in each assay, including a growth control containing only P. acnes, and a sterility control containing growth medium only. Each peptide was tested in triplicate and serially diluted at a 1 :2 ratio with 15 g/L tryptic soy broth. After completing the serial dilutions, liquid P. acnes culture was added to each well at a concentration of lxlO ⁶ CFU/mL, as determined by the 0.5 McFarland standard protocol described above. In the end, each well contained 100 pl total volume, 50 pL of peptide dilution plus 50 pL of liquid bacterial culture. Inoculated plates were incubated for 72 hours before being read for absorbance at 590 nm. Seventy -two hours was chosen as an appropriate incubation time period due to the slow doubling time of P. acnes. Additionally, in accordance to the McFarland standard protocol, the diluted liquid culture was spread on agar plates to confirm that the initial standardization was accurate. Essential Oil Antimicrobial Activity Assay

[0092] Each of the six chosen essential oils was primarily chosen due to their previously reported antimicrobial properties. Identifying the MIC of each oil against P. acnes indicated whether or not the oil would be likely to inhibit /CBD-LL37 activity within the delivery vehicle.

[0093] Prior to being pipetted into the 96-well plate, each oil was first diluted in tryptic soy broth supplemented with 0.5% Tween® 80 to a concentration of 8% v/v. The final volume of each well was 100 pL, containing 50 pL oil and 50 pL liquid bacterial culture. This resulted in a starting oil concentration of 4% v/v, which was chosen because it is higher than each of the published oil MICs against P. acnes. After being pipetted into the wells, the oils were serially diluted 1 :2 with tryptic soy broth supplemented with Tween® 80.

Oil-Peptide Combination

[0094] The primary goal of oil-peptide combination experiments was to evaluate the activity of the peptide when combined with the oil component in the delivery vehicle against P. acnes. This could be reflected by a lower MIC value of the peptide-oil combination compared with that of peptide alone; using a lower concentration of the peptide would improve the safety profile of the delivery vehicle.

[0095] The starting peptide concentration for /CBD-LL37 was determined based on the delivery vehicle activity assay MIC result. Despite testing MICs of 6 different oils, the two with the lowest MIC results from the oil MIC assay were chosen for combination testing with the peptide, which were eucalyptus and manuka oil. Two sub-inhibitory concentrations for both eucalyptus and manuka oil were evaluated in order to ensure that the oils could not create false positive results by killing the bacteria on their own. Each experimental well was set up as follows: 50 pL bacteria, 25 pL diluted essential oil, 25 pL peptide. Since the manuka MIC against P. acnes was 1% v/v, the concentrations chosen to test in combination were 0.5% v/v and 0.1% v/v; similarly, since eucalyptus had an MIC of 0.0625% v/v, it was tested at 0.05% v/v and 0.02% v/v. Since the oils made up one quarter of the well volume, oil aliquots were first made at four times the desired final concentration. The peptides were added to the 96-well plate and serially diluted 1 :2. Following the peptide additions, the oils were added and the bacteria procedure was corrected to lxlO ⁶ CFU/mL. In addition to the growth and sterility controls, a peptide-only control was included to corroborate the results found from the first MIC assay with the same P. acnes cultures used to test the combination effects. The results of this assay determined which delivery vehicle formulation to move forward with for the skin penetration studies.

Skin Diffusion Assay [0096] Once a delivery vehicle was determined for /CBD-LL37. the significant challenge of topically delivering a large protein molecule through the skin barrier needed to be addressed. In order to assess peptide diffusion, an accurate yet cost-effective ex vivo skin model was needed. Porcine ear skin was identified as most comparable to human forehead skin in both hair follicle density and stratum comeum thickness.

[0097] A set of pig ears was obtained from Adams Farm in Athol, MA to complete skin penetration studies. This allowed for a visualization of the peptide location after application to confirm whther or not the delivery vehicle facilitates penetration of the peptide into the sebaceous glands of skin, in accordance with the third objective. It should be ensured that the peptide could be effectively delivered to the pilosebaceous unit so that it could be active against the P. acnes bacteria in the sebaceous gland. The three-step process that guided diffusion assessment included skin retrieval through dissection, application of test formulations and control solutions to the skin, and histological evaluation of skin samples, including Eosin staining, Hoechst staining and immunohistochemistry to detect LL37 peptide in the skin sample.

Skin Model Dissection

[0098] Skin from two porcine ears was separated from the cartilage using surgical tools into roughly 2 cm x 0.5 cm sections. To isolate the skin, the perimeter of the ear was removed with surgical scissors to expose the layers of skin and cartilage in the ear. Then, the skin was pulled away from the cartilage with forceps while the connective tissue between the skin and cartilage was severed using a scalpel. This process was carried out until most of the skin-cartilage barrier was severed, at which point the skin flap was removed completely from the ear.

[0099] The large skin specimen was cut into approximately 2 cm by 0.5 cm samples. Formulations, mixed with a black tissue marking dye (Ted Pella Inc.) that persists through tissue processing, were applied to these sections for five and thirty minutes, with sets at both room temperature and 37 °C, and subsequently used for histology experiments. Once formulation incubation time on the skin samples was complete, the samples were placed in histology cassettes in 10% neutral buffered formalin overnight to fix the samples.

Histology

[00100] After the skin samples had been fixed in formalin, the cassettes were processed using Sakura’s Tissue-Tek VIP 6 AI Vacuum Infiltration Processor. After processing, the tissue samples were embedded in paraffin wax, sectioned into 5pm thick slices, and mounted on glass slides in preparation for staining. Skin samples were stained with Eosin, a colorimetric staining method, to reveal the detailed structure of the tissue. Sister skin sections were used for Hoechst staining and immunohistochemistry.

Colorimetric Staining

[00101] Once mounted, the glass microscope slides were loaded into a slide holder for the colorimetric staining process. First, slides were soaked in three rounds of xylene solution for three minutes each to deparaffmize the sections. Slides were then rehydrated via two rounds of soaking in 100% ethanol solution for three minutes each, followed by soakings in 95% ethanol and 70% ethanol, each for three minutes. Slides were then inserted into the Eosin counterstain for 30 seconds, which colors eosinophilic structures (such as cell cytoplasms and extracellular proteins such as collagen) in shades of red and pink. Two rinses (3 minutes each) were then completed in 95% ethanol, followed by another three rinses (3 minutes each) in 100% absolute ethanol. Three additional rinses in xylene (2 minutes each) were completed before the slides were coverslipped using xylene-based Permount mounting medium and left to dry before imaging.

Hoechst and Anti-LL37 Immunohistochemistry (IHC)

[00102] Rabbit anti-LL37 polyclonal primary antibodies and goat anti-rabbit Alexa Fluor 488 secondary antibodies were used to locate the peptides within the skin samples. A Hoechst nuclear counterstain was applied in conjunction with the fluorescent antibodies as a counterstain to label cell nuclei and visualize tissue structures. Briefly, samples were deparaffmized in xylene and ethanol rinses. Samples were then boiled under pressure with an antigen retrieval solution (SigmaAldrich) for five minutes. Three PBS rinses (5 minutes each) were completed, followed by blocking with 5% v/v normal goat serum in PBS for 30 minutes at room temperature. After blocking, l0pg/mL primary rabbit polyclonal anti(human)-LL37 (Anaspec) in 3% v/v goat serum was added and samples were incubated overnight at 7 °C. Three PBS rinses were again completed. Alexa Fluor 488 goat-anti-rabbit antibody (LifeTechnologies), diluted 1 :50 in 3% v/v goat serum was then added for 1 hour at room temperature. Samples were kept in the dark from this point forward. A Hoecsht nuclear counterstain was then applied, and an addition three PBS rinses were conducted prior to adding coverslips using Prolong Gold mounting medium. All slides were imaged at the same time within three days of staining and stored at 4°C.

IHC Pixel Intensity Analysis

[00103] In order to quantitatively assess the fluorescent intensity of the IHC samples, the fluorescent intensity of three different regions within each tissue specimen was determined. These regions were the dermal tissue, the epidermis, and the hair follicles (Fig. 6). Pixel intensity is defined as the sum of the brightness of each pixel within a given region, divided by the total number of pixels within the region. This analysis allowed quantitative comparison of the fluorescence within the tissue and follicles between different skin samples and formulations.

[00104] Three samples per region per slide were analyzed. Using the average pixel intensity and the variance associated with each group, a one-way ANOVA was performed in order to determine if there was a statistically significant difference between the mean pixel intensity for each region (as shown in Fig. 7).

Delivery Vehicle Activity

[00105] The MICs for soluble LL37 and CBD-LL37 against P. acnes were found to be approximately 1.88 mM and 7.5 pM, respectively, demonstrated by the concentration at which the OD (590) for each peptide crosses the sterility control line, representing 100% killing. This result verified that the modified /CBD-LL37 peptide was active against P. acnes bacteria, and indicated the highest concentration of the peptide necessary in the formulation to kill P. acnes (Fig. 1). Therefore, this concentration of the modified /CBD-LL37 peptide was used for the oil-peptide combination assays.

Essential Oil Antimicrobial Activity

[00106] The MICs for each of the six essential oils against P. acnes were determined (n=2) (Fig. 2, Table 1). Based on their effectiveness, manuka (MIC of 1%) and eucalyptus (MIC of 0.063%) oils were chosen to investigate in the combined activity assay.

[00107] Table 1. Results of Essential Oil Antimicrobial Activity Assays

[00108] Eucalyptus oil was chosen because it performed the best out of all six oils in requiring the lowest concentration to be effective against P. acnes. Manuka oil was chosen because, although lemongrass oil performed slightly better, it has additional benefits for skin health, a vital component for a clinical skincare application.

Oil-Peptide Combination Activity

[00109] To determine whether or not the peptides retained their antimicrobial activity in combination with the essential oils in the delivery vehicle within a safe therapeutic window, a checkerboard dilution MIC assay was performed. Based on the MIC for /CBD-LL37 of 7.5 mM, the initial starting peptide concentration for both modified peptides in the serial dilutions was 7.5 mM. However, there was 100% visible growth reduction at all subsequent peptide concentrations. Due to this surprising and extremely significant result, the next assay instead began with 1 pM peptide to effectively determine the new MICs.

[00110] The results indicate that the combined activity of the peptide and oil significantly lowers the required concentration of peptide and oil to be active against P. acnes. For both sub-inhibitory concentrations of each oil, 100% killing was observed with all concentrations of the modified /CBD-LL37 peptide as low as 0.032 pM (Fig. 3A and Fig. 3B). This means that, when paired with the essential oils, CBD-LL37 experienced a near 250-fold decrease in its MIC.

[00111] A peptide-only control was used in conjunction with the growth and sterility controls to directly compare the antimicrobial activity of the modified peptide with and without the oil component. The peptide-only control resulted in MIC values that were consistent with the values found during the initial peptide only MIC assays.

Skin Diffusion

[00112] Results of the combined essential oil and /CBD-LL37 peptide demonstrated that eucalyptus 0.05% v/v performed the best out of the four formulations evaluated; therefore, this delivery vehicle was selected for use in the skin penetration studies. Figs. 4A-4C depictthe Eosin-stained, Hoechst-stained, and IHC-stained negative control slides, where no peptide was included in the assessed formulations. These Eosin-stained and Hoechst-stained negative controls allowed visualization of the hair follicle morphology, and the IHC slides provided a baseline fluorescence to compare the results of the experimental formulations.

[00113] Eosin stain (Fig. 4A), Hoechst stain (Fig. 4B ), and IHC (Fig. 4C) of both negative controls used - 0.05% Eucalyptus oil only (Top) and water + Tween® 80 only (Bottom). IHC results display baseline tissue autofluorescence for comparison to IHC with fCBD-LL37, and Eosin and Hoechst results display tissue morphology, including hair follicles, which are labeled.

[00114] Experimental diffusion assay results comparing CBD-LL37 solubilized in water versus in the delivery vehicle are shown in Figs. 5A-5C. Increased fluorescence can be observed in the soluble /CBD-LL37 images compared to the negative controls portrayed above; the fluorescence on the epidermal layer and inside the hair follicle indicates that the peptide was able to diffuse into the hair follicle, following the path of least resistance. The additional increase in fluorescence observed in the delivery vehicle formulation compared to the water soluble /CBD-LL37 demonstrates that qualitatively, the delivery vehicle/essential oil was able to enhance penetration and absorption of the peptide into the hair follicle.

[00115] Eosin stain (Fig. 5A), Hoechst stain (Fig. 5A), and IHC stain (Fig. 5A) of fCBD-LL37 were solubilized in water (Top) and in delivery vehicle formulation (Bottom). Qualitatively, the formulation IHC shows the greatest fluorescent intensity within the hair follicles (labeled).

IHC Pixel Intensity Analysis

[00116] A quantitative analysis of pixel intensity of the IHC images was performed. The average intensity of the three regions of interest within each skin sample show an increase of /CBD-LL37 within the 0.05% eucalyptus delivery vehicle, when compared to Tween® 80-only and /CBD-LL37 only controls (Fig. 7).

[00117] The mean pixel intensities for each region and their associated variances were used to conduct a one-way ANOVA in order to determine if there was a statistically significant difference in the pixel intensity for each region. A significant difference in fluorescence would indicate a significant difference in peptide delivery accomplished between the no-oil control and essential oil delivery vehicle.

Analysis of Results

[00118] The following sections include a detailed analysis of the results, placing them in context with past published studies, as well as presenting the potential limitations of each experiment. CBD-LL37 Displays Antimicrobial Activity Against P. acnes

[00119] Establishing the antimicrobial effects of /CBD-LL37 against Gram-positive P. acnes was crucial to the success of an effective delivery vehicle design. This indicated effective performance against P. acnes, and that /CBD-LL37 was a plausible candidate for treating acne.

Using unmodified LL37 as a control in the soluble peptide antimicrobial activity procedure was essential because it ensured that the methods used to test antimicrobial activity were working as intended, since /CBD-LL37 had not been tested against P. acnes previously.

Essential Oils Kill P. acnes Independently

[00120] Identifying independent antimicrobial activity indicated that essential oils would likely not inhibit peptide efficacy within the delivery vehicle. Published studies report antimicrobial activity for tea tree oil, lemongrass oil, orange oil, sunflower oil, eucalyptus oil, manuka oil against various bacteria, including Gram-positive strains similar to P. acnes ( Table 2). The antimicrobial activity of essential oils is largely due to their hydrophobicity, which allow them to easily diffuse through the phospholipid bilayer of bacterial cell membranes and disturb cellular structures. Altered permeability of bacterial cells leads to leakage of cellular contents, leading to ultimate microbial cell death.

[00121] However, three of the six essential oils that were evaluated (manuka, orange, and sunflower) had no published data regarding their MICs against P. acnes specifically. Of the remaining three oils, only the results from lemongrass oil were close to the published value. The MIC found for tea tree oil (4% v/v) was higher than the published value (0.25 - 1% v/v), and the MIC found for eucalyptus oil was lower than the published value (4% v/v). This discrepancy could be due to a number of factors, including differing levels of quality and purity between the oils used in this project and in the literature studies. The specific strains of P. acnes used in this study and in the literature studies could also have influenced the concentrations of oils required to kill the bacteria. Despite the differences between published and measured values, this does not affect the validity of the results because each oil-only MIC assay trial yielded consistent results.

[00122] Table 2. Essential oil MIC values compared to published values

Essential Oils Enhance /CBD-LL37 Activity Against P. acnes

[00123] When tested in combination, the results were surprising. Not only did manuka and eucalyptus oils allow for peptide efficacy within a safe therapeutic window, but essential oils combined with peptide exhibited enhanced activity at concentrations up to 30-fold lower than CBD-LL37’s MIC, 7.5 mM.

[00124] Since the concentrations of the manuka and eucalyptus oils used in this assay were lower than the isolated MICs of the oils found through the essential oil activity assays, the oils alone did not kill as well as in the combination formulations ( Table 3). It has been shown that hydrophobic environment causes LL37 to gain a more helical structure, making it more active. With this in mind, the modified /CBD-LL37 peptide is expected to work similarly, thus forming the basis for the combination therapy incorporated into the delivery vehicle design.

[00125] Table 3. Comparison of Isolated MICs and Combination Therapy MICs

Essential Oils Enhance Delivery of CBD-LL37 to the Hair Follicles

[00126] Peptide diffusion studies using the ex vivo porcine ear skin model were successful, and results indicate that delivery vehicle may improve the ability of /CBD-LL37 to diffuse into the hair follicles and actively eliminate P. acnes where it grows.

[00127] The pixel intensity analysis data were analyzed for statistical significance using a one- tailed T-test and an analysis of variance (ANOVA). The one-tailed T-test was used to analyze the differences in pixel intensity, and thereby diffusion, between the /CBD-LL37 which was solubilized in DI water and the /CBD-LL37 which was in the delivery vehicle with 0.05% v/v eucalyptus oil. Within a 95% confidence interval, the one-tailed T-test revealed a statistically significant (P<0.05) difference between the pixel intensities for each of the epidermal layers, hair follicles, and dermal tissues of these two sample groups ( Table 4).

[00128] Table 4. One-tailed T-test of pixel intensity results between the /CBD-only control and the CBD in the delivery vehicle

[00129] The data quantitatively confirm that the delivery vehicle significantly improves delivery of /CBD-LL37 not only through the skin, but also into the hair follicles ( Table 4).

[00130] The ANOVA test was used to analyze the differences between the pixel intensities of each tissue region using the values for every tissue sample ( Table 5). A one-way ANOVA was used because the three groups are not related to one another, and their results do not depend on one another.

[00131] Table 5. One-way ANOVA comparing the pixel intensity data of each tissue group

[00132] These data show that /CBD-LL37 diffuses most effectively into the hair follicles and the epidermis, while not entering a significant portion of the dermal tissue. This indicates that /CBD-LL37 in general will likely be effective at specifically targeting the P. acnes bacteria in the hair follicles and sebaceous glands while avoiding any potential off-target effects. This also indicates that /CBD-LL37 cannot non-specifically diffuse past the epidermis, and thus is not diffusing through the dermal tissue. Instead, ^CBD-LL37 is following the path of least resistance to enter the hair follicles, which are open to the surface of the skin.

[00133] All patents, patent applications, and published references cited herein are hereby incorporated by reference in their entirety. It should be emphasized that the above-described embodiments of the present disclosure are merely possible examples of implementations, merely set forth for a clear understanding of the principles of the disclosure. Many variations and modifications may be made to the above-described embodiment(s) without departing substantially from the spirit and principles of the disclosure. It can be appreciated that several of the above- disclosed and other features and functions, or alternatives thereof, may be desirably combined into many other different systems or applications. All such modifications and variations are intended to be included herein within the scope of this disclosure.