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WO2019204729A1 - Compositions et méthodes de traitements de la peau - Google Patents

Compositions et méthodes de traitements de la peau Download PDF

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Publication number
WO2019204729A1
WO2019204729A1 PCT/US2019/028324 US2019028324W WO2019204729A1 WO 2019204729 A1 WO2019204729 A1 WO 2019204729A1 US 2019028324 W US2019028324 W US 2019028324W WO 2019204729 A1 WO2019204729 A1 WO 2019204729A1
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WO
WIPO (PCT)
Prior art keywords
peptide
oil
activity
antimicrobial
fusion
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US2019/028324
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English (en)
Inventor
Lindsay D. Lozeau
Abigail HANSON
Joshua B. MACE
Frederick T. METTERS
Nicole SHERLOCK
Terri A. Camesano
Marsha W. ROLLE
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Worcester Polytechnic Institute
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Worcester Polytechnic Institute
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Publication date
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Publication of WO2019204729A1 publication Critical patent/WO2019204729A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4723—Cationic antimicrobial peptides, e.g. defensins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/92—Oils, fats or waxes; Derivatives thereof, e.g. hydrogenation products thereof
    • A61K8/922—Oils, fats or waxes; Derivatives thereof, e.g. hydrogenation products thereof of vegetable origin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q17/00—Barrier preparations; Preparations brought into direct contact with the skin for affording protection against external influences, e.g. sunlight, X-rays or other harmful rays, corrosive materials, bacteria or insect stings
    • A61Q17/005—Antimicrobial preparations
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/40—Chemical, physico-chemical or functional or structural properties of particular ingredients
    • A61K2800/52—Stabilizers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/40—Chemical, physico-chemical or functional or structural properties of particular ingredients
    • A61K2800/59—Mixtures
    • A61K2800/592—Mixtures of compounds complementing their respective functions
    • A61K2800/5922—At least two compounds being classified in the same subclass of A61K8/18
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0014—Skin, i.e. galenical aspects of topical compositions
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/31—Fusion polypeptide fusions, other than Fc, for prolonged plasma life, e.g. albumin

Definitions

  • the disclosure relates generally to topical skin treatment compositions, and in particular to topical skin treatment compositing include antimicrobial peptides.
  • 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.
  • compositions 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.
  • the composition comprises the at least one fusion peptide and the at least one essential oil at concentrations that are not cytotoxic.
  • 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.
  • 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.
  • 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.
  • the composition comprises from 0.1 to 15 mM the at least one fusion peptide.
  • the antimicrobial peptide comprises a synthetic LL37 peptide or variant thereof.
  • the antimicrobial peptide has an amino acid sequence as set forth in SEQ ID NO: 1.
  • the antimicrobial peptide has an amino acid sequence having at least 95% identity with SEQ ID NO: 1.
  • the fusion peptide has an amino acid sequence as set forth in SEQ ID NO: 2.
  • the fusion peptide has an amino acid sequence as set forth in SEQ ID NO: 3.
  • the at least one antimicrobial peptide is at the N-terminus.
  • the at least one fusion peptide comprises a linker at the C-terminus of the antimicrobial peptide.
  • 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.
  • the collagen binding domain is derived from human collagenase or human fibronectin.
  • compositions 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.
  • the composition comprises the at least one fusion peptide and the at least one essential oil at concentrations that are not cytotoxic.
  • 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.
  • 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.
  • 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.
  • the composition comprises from 0.1 to 15 mM the at least one fusion peptide.
  • the antimicrobial peptide comprises a synthetic LL37 peptide or variant thereof.
  • the antimicrobial peptide has an amino acid sequence as set forth in SEQ ID NO: 1.
  • the antimicrobial peptide has an amino acid sequence having at least 95% identity with SEQ ID NO: 1.
  • the fusion peptide has an amino acid sequence as set forth in SEQ ID NO: 2.
  • the fusion peptide has an amino acid sequence as set forth in SEQ ID NO: 3.
  • the at least one antimicrobial peptide is at the N-terminus.
  • the at least one fusion peptide comprises a linker at the C-terminus of the antimicrobial peptide.
  • 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.
  • the collagen binding domain is derived from human collagenase or human fibronectin.
  • 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.
  • FIG. 2 is a graph depicting six essential oils’ isolated antimicrobial activity against P. acnes.
  • 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.
  • 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.
  • 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.
  • FIG. 6 depicts representative samples taken for IHC pixel intensity analysis.
  • 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.
  • 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.
  • formulation and“composition” are used herein interchangeably.
  • 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.
  • transdermal is used herein to generally include a process that occurs through the skin.
  • transdermal and percutaneous are used interchangeably throughout this specification.
  • 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.
  • collagen binding domain refers to a peptide that binds collagen.
  • “chimeric peptide”,“fusion peptide” and“fusion protein” mean a peptide comprising two or more peptides, and are used interchangeably.
  • 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.
  • 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.
  • 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.
  • 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.
  • the essential oils may also provide other anti-inflammatory and anti-oxidant purposes for the skin treatment.
  • the fusion peptide in combination with the essential oil has a synergistic effect on the anti-inflammatory and/or antimicrobial activities.
  • the essential oils may also have synergistic effects on these activities.
  • 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.
  • the antimicrobial peptide can have a broad antimicrobial spectrum.
  • the peptide is a fusion antimicrobial peptide with collagen-binding domain.
  • 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.
  • the peptide is a human-derived 37-amino acid AMP belonging to the cathelicidin family.
  • the peptide comprises an amino acid sequence as set forth in SEQ ID NO: 1, or variant thereof.
  • the peptide can have at least 99%, 98%, 97%, 96%, 95% identity with SEQ ID NO: 1.
  • 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.
  • 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.
  • the fusion peptide is 60 or less amino acid long.
  • the fusion peptide can be from 40 to 60, 45 to 60, 50 to 60, 55 to 60 amino acids long.
  • 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.
  • 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.
  • the fragment can be a 5, 6, 7, 8, 9, 10 or longer amino acid peptide.
  • the peptide and the collagen binding domain are linked via a flexible linker.
  • the linker can be from 3 to 20 amino acid long.
  • the linker can be DYKDDDDK (SEQ ID NO: 4).
  • an antimicrobial peptide is synthetic human LL37 (LLGDFFRKSKEKIGKEFKRIV QRIKDFLRNLVPRTES) (SEQ ID NO: 1).
  • the fusion peptide may comprise LL37 peptides modified with collagen binding domains.
  • the fusion peptide is cCBD-LL37
  • the fusion peptide is fCBD-LL37
  • the fusion peptides incorporate a FLAGTM 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the fusion peptide can be present in the composition at a concentration of from 0.05 micromolar to 25 micromolar.
  • 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.
  • the concentration of cCBD-LL37 can be from 0.05 micromolar to 10 micromolar.
  • 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.
  • the concentration of fCBD-LL37 can be from 0.05 micromolar to greater than 20 micromolar.
  • 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.
  • the concentration of the fusion peptide is 13mM or higher.
  • 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.
  • 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.
  • 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.
  • 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.
  • the composition comprises a fusion peptide and one or more essential oil as active agent or additive.
  • 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.
  • the composition can comprise from 0.1 to 15 mM fusion peptide.
  • 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.
  • the minimum inhibitory concentration (MIC) of the fusion peptides is reduced in the presence of low concentrations of essential oils.
  • the greater than 99% reduction in amount of the fusion peptide e.g. fCBD-LL37
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the composition comprises one or more additional active agent.
  • the additional active agent can be an anti-pain or anti-itch agent.
  • the anti-itch agent can be an antihistamine, a corticosteroid or the like.
  • the anti-pain agent can be an anaesthetic or analgesic such as benzocaine or lidocaine.
  • the additional active agent can be a plant extract having medicinal anti-itch or analgesic properties.
  • the composition is a topical formulation.
  • the formulation is liquid-based topicals including creams, ointments, gels and serums.
  • 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.
  • the composition can include a penetration enhancer.
  • the penetration enhancer can be dimethyl sulfoxide (“DMSO”) or derivatives thereof.
  • 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.
  • the composition can comprise an alkanol, such as methanol, ethanol, propanol, butanol or mixtures thereof.
  • 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.
  • 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.
  • 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.
  • the composition can comprise glycerol (also referred to herein as glycerine) at a concentration of 0-20% w/w.
  • glycerol is used at 0-4% w/w, such as 0, 1, 2, 3, or 4% w/w, and all fractions in between.
  • no glycerol is used in the composition.
  • 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.
  • the at least one thickening agent is used such that the formulation has the desired viscosity, for example from 10 and 50000 centipoise (cps).
  • the composition can comprise at least one antioxidant or chelating agents.
  • the composition can comprise a pH adjusting agent.
  • 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.
  • the composition can comprise at least one detergent, surfactant or emulsifying agents, such as sodium lauryl sulfate, polysorbates, etc.
  • the composition can comprise vitamins and minerals, including but not limited to linoleic acid, retinoic acid, etc.
  • composition can further include additional pharmaceutically acceptable excipients typically used in formulations and known to those skilled in the art.
  • excipients include, for example, humectants, emollients, and preservatives.
  • the combination of fusion peptides and essential oil is formulated as a topical cream.
  • the topical cream comprises an emulsion semisolid comprised of more than 20% water and less than 50% hydrocarbons, oils, or waxes.
  • the combination of fusion peptides and essential oil is formulated as a topical ointment.
  • the topical ointment comprises an emulsion semisolid comprised of less than 20% water and more than 50% hydrocarbons, oils or waxes.
  • the combination of fusion peptides and essential oil formulated is as a topical gel.
  • the combination of fusion peptides and essential oil formulated is as a topical serum.
  • the fusion peptides can be incorporated into nanoparticles.
  • the nanoparticles can be polymeric nanoparticles.
  • the nanoparticles can be configured for controlled release.
  • 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.
  • the fusion peptides can be delivered through microinjection.
  • the microneedle array can be used to deliver the fusion peptides.
  • 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).
  • 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).
  • the microneedle array can be surface coated with a solution comprising the fusion peptide.
  • the microneedle drug delivery device can be a patch.
  • 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.
  • 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.
  • 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.
  • MIC assays were tested in triplicate, and each step was performed at least twice to confirm reproducible results.
  • 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 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.
  • 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.
  • P. acnes were grown in 15 g/L tryptic soy broth.
  • 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.
  • 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.
  • 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.
  • the diluted liquid culture was spread on agar plates to confirm that the initial standardization was accurate.
  • 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.
  • each oil 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the glass microscope slides were loaded into a slide holder for the colorimetric staining process.
  • 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.
  • the fluorescent intensity of the IHC samples 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • CBD-LL37 Displays Antimicrobial Activity Against P. acnes
  • 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.
  • 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).

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Abstract

Des aspects de l'invention concernent une composition topique comprenant (1) au moins un peptide de fusion comprenant au moins un peptide antimicrobien et au moins un peptide capable d'améliorer la stabilité, l'activité ou la stabilité et l'activité du peptide antimicrobien dans un environnement hydrophobe, (2) au moins une huile essentielle, la composition comprenant l'au moins un peptide de fusion et l'huile essentielle ou les huiles essentielles à des concentrations capables de fournir une activité anti-inflammatoire et/ou une activité antimicrobienne synergique.
PCT/US2019/028324 2018-04-19 2019-04-19 Compositions et méthodes de traitements de la peau Ceased WO2019204729A1 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
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WO2021202476A1 (fr) * 2020-03-31 2021-10-07 Innate Immunity LLC Peptide de recombinaison pour le traitement du feu bactérien
US20230090691A1 (en) * 2021-09-01 2023-03-23 Spinart, LLC Compositions and methods for promoting and/or maintaining vaginovulval and perianal tissue vitality and tissue health
CN115836996A (zh) * 2022-09-08 2023-03-24 科丝美诗(中国)化妆品有限公司 一种含麦卢卡树提取物的祛痘组合物及应用

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US20130237613A1 (en) * 2007-08-29 2013-09-12 Agency For Science, Technology And Research Sugar-based surfactant microemulsions containing essential oils for cosmetic and pharmaceutical use
US20140276493A1 (en) * 2013-03-15 2014-09-18 Braden King-Fung Leung Wound healing compositions
US20150005266A1 (en) * 2006-12-04 2015-01-01 Molecular Design International, Inc. Topical compositions for treatment of skin conditions
US20150064118A1 (en) * 2004-06-04 2015-03-05 Camurus Ab Lipid depot formulations
US20150202350A1 (en) * 2012-06-26 2015-07-23 Marsha Rolle Matrix scaffold with antimicrobial activity
US9629856B2 (en) * 2010-03-03 2017-04-25 Anteis Sa Compositions and methods for the treatment of skin diseases and disorders using antimicrobial peptide sequestering compounds

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US20150064118A1 (en) * 2004-06-04 2015-03-05 Camurus Ab Lipid depot formulations
US20150005266A1 (en) * 2006-12-04 2015-01-01 Molecular Design International, Inc. Topical compositions for treatment of skin conditions
US20130237613A1 (en) * 2007-08-29 2013-09-12 Agency For Science, Technology And Research Sugar-based surfactant microemulsions containing essential oils for cosmetic and pharmaceutical use
US9629856B2 (en) * 2010-03-03 2017-04-25 Anteis Sa Compositions and methods for the treatment of skin diseases and disorders using antimicrobial peptide sequestering compounds
US20150202350A1 (en) * 2012-06-26 2015-07-23 Marsha Rolle Matrix scaffold with antimicrobial activity
US20140276493A1 (en) * 2013-03-15 2014-09-18 Braden King-Fung Leung Wound healing compositions

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021202476A1 (fr) * 2020-03-31 2021-10-07 Innate Immunity LLC Peptide de recombinaison pour le traitement du feu bactérien
US20230090691A1 (en) * 2021-09-01 2023-03-23 Spinart, LLC Compositions and methods for promoting and/or maintaining vaginovulval and perianal tissue vitality and tissue health
CN115836996A (zh) * 2022-09-08 2023-03-24 科丝美诗(中国)化妆品有限公司 一种含麦卢卡树提取物的祛痘组合物及应用

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