US20230000750A1 - Stem cell stimulating compositions and methods - Google Patents

Stem cell stimulating compositions and methods Download PDF

Info

Publication number
US20230000750A1
US20230000750A1 US17/930,842 US202217930842A US2023000750A1 US 20230000750 A1 US20230000750 A1 US 20230000750A1 US 202217930842 A US202217930842 A US 202217930842A US 2023000750 A1 US2023000750 A1 US 2023000750A1
Authority
US
United States
Prior art keywords
defensin
skin
defensins
beta
topical formulation
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.)
Pending
Application number
US17/930,842
Inventor
Nikolay Turovets
William B. Adams
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Medicell Technologies LLC
Original Assignee
Medicell Technologies LLC
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Medicell Technologies LLC filed Critical Medicell Technologies LLC
Priority to US17/930,842 priority Critical patent/US20230000750A1/en
Assigned to MEDICELL TECHNOLOGIES, LLC reassignment MEDICELL TECHNOLOGIES, LLC CONFIRMATORY ASSIGNMENT Assignors: ADAMS, William B., TUROVETS, NIKOLAY
Publication of US20230000750A1 publication Critical patent/US20230000750A1/en
Pending legal-status Critical Current

Links

Images

Classifications

    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00Cosmetics or similar toiletry preparations
    • A61K8/18Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/64Proteins; Peptides; Derivatives or degradation products thereof
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61QSPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00Preparations for care of the skin
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61QSPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00Preparations for care of the skin
    • A61Q19/007Preparations for dry skin
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61QSPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00Preparations for care of the skin
    • A61Q19/08Anti-ageing preparations
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61QSPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00Preparations for care of the skin
    • A61Q19/10Washing or bathing preparations
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61QSPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q5/00Preparations for care of the hair
    • A61Q5/02Preparations for cleaning the hair
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2800/00Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/74Biological properties of particular ingredients
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00Cosmetics or similar toiletry preparations
    • A61K8/02Cosmetics or similar toiletry preparations characterised by special physical form
    • A61K8/14Liposomes; Vesicles
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00Cosmetics or similar toiletry preparations
    • A61K8/18Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/55Phosphorus compounds
    • A61K8/553Phospholipids, e.g. lecithin

Definitions

  • the field of the invention is topical cosmetic formulations using defensins to reduce the appearance of wrinkles, pores, irregularity in texture of a skin surface, oiliness, brown spots, and red spots in non-injured skin.
  • Defensins are small cystein-rich proteins, usually only 14-85 amino acids long. Defensins can be found in invertebrates, vertebrates, and plants, and have been shown to be active against many bacteria, fungi, and viruses. In fact, much of the body of research has focused on the antimicrobial properties of defensins. However, in recent years, some research has explored other roles defensins may play in human skin, such as in wound healing or hair growth.
  • PCT Patent Application WO 2014/004339 A2 by Applicant ELC Management LLC teaches the use of a resveratrol-containing cosmetic composition to stimulate endogenous production of cellular beta defensins in skin cells.
  • the inventors tested the composition on Normal Human Epidermal Keratinocytes (“NHEK”) in vitro and measured the presence of beta defensin in NHEK. The resulting stimulated quantities of defensin were very small (approximately 0.001 ng/ml).
  • LGR5+ and LGR6+ were activated by pro-inflammatory conditions and other factors already present in the wound or due to the topical application of defensins.
  • the wound healing formulation used by Lough et al. contained concentrations of defensins at antimicrobially active concentrations (e.g. about 10 5 ng/ml).
  • concentrations of defensins at antimicrobially active concentrations e.g. about 10 5 ng/ml.
  • this high concentration of defensin may make any resulting cosmetic composition more allergenic and more costly.
  • defensin containing and defensin stimulating compositions are known, there is a need for a cosmetic topical formulation for use on healthy skin that makes use of defensins for reducing the visual appearance of age on the skin.
  • the inventive subject matter is directed towards various topical formulations, methods of manufacture of the topical formulation in which the topical formulation includes sub-antimicrobially effective concentrations of at least one defensin, and methods of applying the topical formulation to the healthy skin of users to reduce the apparent age of the users' skin.
  • a topical cosmetic formulation includes a defensin in a cosmetically acceptable carrier.
  • Preferred topical cosmetic formulations are ready-to-use and contain the defensin at a sub-antimicrobially effective concentration, wherein the concentration is ineffective to inhibit growth of a microbial pathogen in a therapeutically effective manner.
  • Still further preferred topical formulations may further comprise a blend of two or more different defensins, wherein the combined concentration of defensins in the formulation is a sub-antimicrobially effective concentration.
  • methods of recruiting LGR6+ stem cells may include a step of providing a topical formulation containing a sub-antimicrobial concentration of at least one defensin and a further step of applying the formulation to non-injured skin 1) to reduce at least one of wrinkle depth, wrinkle length, wrinkle width, pore size, irregularity in texture of a skin surface, oiliness, brown spots, and red spots 2) to improve skin brightness, 3) to lighten skin, and/or 4) to correct aging skin markers such as skin firmness, sagging contours, lines, wrinkles, enlarged pores, impurities, brightness or uneven skin tone, surface imperfections, evenness, overall clarity, age spots, hydration, dryness, smooth texture, radiance and redness in non-injured skin.
  • inventive compositions, methods, and uses employ at least one of alpha-defensin 1, alpha-defensin 5, alpha-defensin 6, neutrophil defensin 1, neutrophil defensin 2, neutrophil defensin 3, neutrophil defensin 4, theta-defensin, beta-defensin 1, beta-defensin 2, beta-defensin 3, and beta-defensin 4.
  • alpha-defensin 5 and beta-defensin 3 are employed.
  • the defensin may comprise a synthetic defensin, a human defensin, recombinant defensin, a monkey defensin, a mouse defensin, a rat defensin, a bovine defensin, a sheep defensin, a horse defensin, a rabbit defensin, a swine defensin, a dog defensin, and/or a cat defensin.
  • a synthetic defensin a synthetic defensin, a human defensin, recombinant defensin, a monkey defensin, a mouse defensin, a rat defensin, a bovine defensin, a sheep defensin, a horse defensin, a rabbit defensin, a swine defensin, a dog
  • contemplated concentrations may be between 0.01 and 100 ng/ml, or between 1 and 30 ng/ml, including the end points of each range. Additionally, especially preferred embodiments of the inventive subject matter employ defensin concentrations of about 22 ng/ml and about 4.4 ng/ml in ready-to-use formulations.
  • defensins may be effective 1) to substantially reduce wrinkle depth, wrinkle length, wrinkle width, pore size, irregularity in texture of a skin surface, oiliness, brown spots, and red spots 2) to improve skin brightness, 3) to lighten skin, and/or 4) to correct aging skin markers such as skin firmness, sagging contours, lines, wrinkles, enlarged pores, impurities, brightness or uneven skin tone, surface imperfections, evenness, overall clarity, age spots, hydration, dryness, smooth texture, radiance and redness in non-injured skin.
  • defensins used in preferred embodiments of the inventive subject matter have a purity greater than 95% as shown by HPLC, and the sequence and proper disulfide bond formation of the defensins can be confirmed by tandem MS/MS.
  • defensins may be encapsulated in liposomes or other nanoparticles.
  • defensins may also be associated with a carrier, in particular a protein carrier such as albumin (e.g., human serum albumin, bovine serum albumin, egg albumin, and recombinant albumin produced by rice, other plants, bacteria or yeast), also encapsulated in liposomes where desirable.
  • albumin e.g., human serum albumin, bovine serum albumin, egg albumin, and recombinant albumin produced by rice, other plants, bacteria or yeast
  • the topical cosmetic formulations may also include supplements to provide nutrition and support for LGR6+ stem cells.
  • Typical supplements include human serum albumin, bovine serum albumin, egg albumin, recombinant albumin produced by rice, other plants, bacteria or yeast, plant hydrolysate, beta-cyclodextrin, glutamine, phospholipids, fibronectin, hyaluronate, hyaluronic acid, plant hydrolysate, L-alanyl-L-glutamine, gelatin, recombinant gelatin, Epidermal Growth Factor (EGF), vitamin E, Tocopheryl Nicotinate, and ubiquinone, coenzyme Q10, antioxidants.
  • EGF Epidermal Growth Factor
  • kits with exfoliating masks include a mask, a cream treatment formulation, and a serum treatment formulation.
  • inventive subject matter provide methods of treating scars, sunburn, bruises, and other skin disorders in which the epidermal layers of the skin are largely intact.
  • Exemplary methods include the steps of providing a topical formulation having at least one defensin at a sub-antimicrobially effective concentration and applying the formulation to non-injured skin under a protocol effective to substantially reduce the appearance of scars, sunburn, bruises, etc.
  • FIG. 1 shows side-by-side photographs that show reduction in wrinkles.
  • FIG. 2 shows side-by-side photographs that show a reduction in the appearance of brown spots.
  • topical formulations provided 1) significant reduction in wrinkles, pore size, irregularity in skin texture, oiliness, brown spots, and red spots, 2) improvement in skin brightness, 3) skin lightening, and/or 4) correction of aging skin markers such as skin firmness, sagging contours, lines, wrinkles, enlarged pores, impurities, brightness or uneven skin tone, surface imperfections, evenness, overall clarity, age spots, hydration, dryness, smooth texture, radiance and redness.
  • a ready-to-use topical cosmetic formulation comprises at least one defensin present in a topical cosmetic formulation at a sub-antimicrobially effective concentration.
  • topical formulations may contain one defensin, a combination of two defensins, or a combination of three or more defensins.
  • the defensins used may be of the same or different types and subtypes.
  • suitable defensins may include one or more of alpha-defensin 1, alpha-defensin 5, alpha-defensin 6, neutrophil defensin 1, neutrophil defensin 2, neutrophil defensin 3, neutrophil defensin 4, theta-defensin, beta-defensin 1, beta-defensin 2, beta-defensin 3, and beta-defensin 4.
  • Especially preferred topical formulations contain alpha-defensin 5 and beta-defensin 3.
  • each defensin may be present in equal quantities by mass or at mass ratios specified to achieve a desired result, such as 1:1.5, 1:2, 1:4, 1:5, etc.
  • a desired result such as 1:1.5, 1:2, 1:4, 1:5, etc.
  • ready-to-use indicates that the defensin-containing topical formulation is in a form that is presented for sale and application. It is contemplated that ready-to-use formulations can comprise a fully combined solution, cream, gel, serum, lotion, etc. Alternatively, the defensin can be packaged in a separate container (e.g., in a vial that pumps a defensin solution with a cream that the user blends before applying to unbroken skin) and combined with another topical formulation at the time of use/application.
  • the phrase “sub-antimicrobially effective concentration” means concentration(s) of defensins which are characterized by an inability to inhibit the proliferation of microbes in an established infection.
  • ready-to-use topical formulations do not include concentrations greater than 1 ⁇ g/ml.
  • the concentration of defensins lies between 0.01 and 100 ng/ml, and even more typically between 1 and 30 ng/ml, wherein contemplated concentrations include the end points of each range.
  • the topical cosmetic formulations have defensin concentrations of about 22 ng/ml and 4.4 ng/ml.
  • non-injured skin refers to skin in which dermis and hypodermis are substantially intact. Therefore, viewed from a different perspective, non-injured skin will appear intact to the unaided eye, with no breach sufficiently large or deep to result in bleeding.
  • non-injured (or “healthy”) skin includes aged skin and skin with first degree sunburn, environmental exposure, bruising, or partially ablated stratum corneum.
  • Non-injured (or healthy) skin also excludes skin displaying persistent infection with pathogens that result in visible symptoms and signs of infection.
  • defensins from both natural and synthetic sources may be suitable for incorporation into topical formulations.
  • defensins may be obtained from plants (e.g., Arabidopsis, pea, tobacco, and spruce), mammals, or other animals.
  • Exemplary defensins derived from natural sources may include human defensins, monkey defensins, mouse defensins, rat defensins, bovine defensins, sheep defensins, horse defensins, rabbit defensins, swine defensins, dog defensins, and/or cat defensins.
  • the defensins are synthetic defensins.
  • Synthetic defensins include defensins produced by chemical synthesis (e.g., solid phase synthesis) or by recombinant technologies (e.g., produced by recombinant bacteria, yeast, tissue cultures, plants or animals).
  • defensin analogues such as hapivirins and diprovirins may be used in some embodiments of the inventive subject matter.
  • the defensins can also be modified to increase their activity and specificity for cosmetic improvements to the appearance of skin.
  • defensins may be unfolded and refolded under controlled conditions to ascertain proper disulfide bond formation (which can be monitored by MS analysis and/or CD spectroscopy).
  • chemical modifications e.g., using non-natural amino acids to increase half-life time, or derivatized proteinogenic amino acids to increase lipophilicity are contemplated to tailor the defensins to a particular need.
  • defensins Regardless of the source of the defensins, it should be appreciated that specific activity of defensins is dependent on various factors, including isomeric form and tertiary structure of the final protein.
  • orthogonal protecting groups can be used to protect selected cysteine residues, which can then be individually deprotected and bonded with the matching target cysteine residue, leading to coordinated non-random disulfide bond formation.
  • Use of such protecting groups in the synthetic strategy can give rise to defensins with a specific activity that is comparable to the specific activity of the native defensin. Any suitable characterization and quality control measures may be employed.
  • the specific activity of defensins incorporated into the inventive topical formulations is measured by purity as determined by HPLC.
  • the defensin is between 80% and 100% pure, more typically the defensin is at least 90% pure, or at least 95% pure, or at least 99% pure, or at least 99.9% pure. Additionally, proper amino acid sequence and disulfide bond formation can be confirmed by tandem MS/MS, for example.
  • concentrations are cosmetically effective (i.e., improves in the visual appearance of healthy skin, and especially I) reduces wrinkles, pore size, irregularity in skin texture, oiliness, brown spots, and/or red spots. 2) improves skin brightness, 3) lightens skin, and/or 4) corrects aging skin markers such as skin firmness, sagging contours, lines, wrinkles, enlarged pores, impurities, brightness or uneven skin tone, surface imperfections, evenness, overall clarity, age spots, hydration, dryness, smooth texture, radiance and redness).
  • the cosmetic formulation is intended to be used for improvement in visual appearance of healthy aged skin.
  • the concentrations employed in the inventive subject matter are not intended to be therapies for the treatment of cuts, third degree burns, or other injuries where there is a loss of integrity in epidermis and dermal layers (typically associated with bleeding and/or scab formation).
  • the total concentration of defensins (single type or combination of distinct defensins) in the final cosmetic formulation as applied to the skin will be between 0.01 ng/ml and 100 ng/ml, or between 0.1 ng/ml and 100 ng/ml, or between 1 ng/ml and 100 ng/ml, or between 2 ng/ml and 80 ng/ml, or between 4 ng/ml and 60 ng/ml, or between 1 ng/ml and 30 ng/ml.
  • compositions include defensins at a concentration of at least 0.01 ng/ml, at least 0.1 ng/ml, at least 1 ng/ml, or at least 4 ng/ml, but no more than 200 ng/ml, no more than 100 ng/ml, no more than 75 ng/ml, or no more than 50 ng/ml.
  • the defensin is associated with a cosmetically acceptable protein to increase stability and/or delivery characteristics.
  • the association is preferably non-covalent (e.g., electrostatic, ionic, hydrophobic, etc.), however, covalent attachment to a side group of the protein is not excluded.
  • Exemplary proteins include lactoferrin, transferrin, and albumin (e.g., human serum albumin, bovine serum albumin, and egg albumin, recombinant albumin).
  • the defensins and protein carriers may be in various ratios, including equimolar, sub-, and supramolar ratios. Additionally, combinations of two or more protein carriers may be used.
  • one defensin may be associated with one carrier, and the other defensin may be associated with a different carrier. Therefore, any combination of defensins and carriers are contemplated.
  • the defensins can be encapsulated in cosmetically acceptable formulations, and especially formulations using a lipid membrane.
  • cosmetically acceptable formulations and especially formulations using a lipid membrane.
  • liposomes, microcapsules, nanocapsules, microparticles, nanoparticles, microparticle delivery systems are especially contemplated.
  • a description of some cosmetically acceptable cosmetic delivery systems can be found in Maherani et al, “ Liposomes: A Review of Manufacturing Techniques and Targeting Strategy ,” Current Nanoscience; 7:436-452 (2011).
  • a preferred method of liposome manufacturing is shear method.
  • Preferred cosmetic delivery systems resemble naturally occurring membranes, are flexible, and can penetrate interstitial spaces between cells.
  • cosmetic delivery systems may have monolayer, bilayer (e.g. unilammellar vesicle or ULV), or multi layer structures (e.g. multilammerlar vesicle or MLV). Additionally, multilayer liposomes, microcapsules, microsomes, and nanaocapsules may have nested structures (e.g. multivesicular vesicle or MVV).
  • Cosmetic delivery systems used in the topical formulations can range in size from 500 nm to 10 microns. In the preparation of cosmetic delivery systems, all cosmetically acceptable lipid compositions are contemplated, especially pharmaceutically acceptable lipids.
  • preferred cosmetic delivery systems comprise amphipathic or amphiphilic molecules such as phospholipids or combinations of phospholipids (e.g., phosphatidylcholine, phosphatidylethanolamine, phosphatidic acid, phosphatidylserine, and phosphoinositides). Additionally, in some instances contemplated cosmetic delivery systems can contain additive(s) such as sterols, polyethylene glycol, cholesterol, dicethylphosphate, stearyl amine, etc. With respect to the amount of delivery systems incorporated in each ready-to-use formulation, the cosmetic delivery system content will typically be adjusted to achieve a sub-antimicrobial concentration of defensins within a preferred range.
  • Unilamellar vesicles/liposomes can be produced using high shear techniques. These vesicles have a greater Zeta Potential than the typical liposome, which allows for smaller, more uniform particle size with increased stability. Zeta Potential is an indicator of the electronic charge on the surface of any macroscopic material that is in contact with a liquid. This can be used to predict and control the stability of suspensions; the higher the Zeta Potential, the greater the stability of the molecule because the charged particles are able to repel and overcome their innate affinity to assemble.
  • defensins protein carriers, liposomes, or other membranaceous structures have a molecular weight that exceeds transmembrane delivery, and even delivery across the stratum corneum. Nevertheless, as is discussed in more detail, the defensins have a profound effect on stem cell activity in dermal and hypodermal layers. While not wishing to be bound by any theory or hypothesis, the inventors contemplate that the liposomal formulations have the ability to transport the defensins via an interstitial route and/or to invade the hair follicle to a depth and concentration sufficient to activate LGR6+ cells.
  • the use of cosmetic delivery systems is thought to aid in the delivery of defensins as one would not expect defensins per se to penetrate the stratum corneum of the skin (acting as a barrier to molecules with molecular weights greater than 500 Da).
  • defensins when associated with albumin (65-70 kDa) and/or liposomes, it becomes at least conceptually even more difficult for the defensin composition to penetrate unbroken skin.
  • albumin 65-70 kDa
  • liposomes it becomes at least conceptually even more difficult for the defensin composition to penetrate unbroken skin.
  • the inventors hypothesize that the mechanism for delivering defensins is different in unbroken skin when compared with broken or injured skin.
  • the inventors advantageously provide additional ingredients that nourish and support the recruited stem cells in healthy skin.
  • albumin e.g., human serum albumin, bovine serum albumin), egg albumin (albumen), recombinant albumin, plant hydrolysate, and ⁇ -cyclodextrin, glutamine, phospholipids (liposomes), fibronectin, hyaluronate, plant hydrolysate, L-alanyl-L-glutamine, gelatin, Vitamin E (tocopheryl nicotinate), ubiquinone (coenzyme Q 10 ), gelatin, recombinant gelatin, hyaluronic acid, Epidermal Growth Factor can provide nutrition and support to the stem cells.
  • albumin e.g., human serum albumin, bovine serum albumin
  • egg albumin albumin
  • recombinant albumin e.g., plant hydrolysate, and ⁇ -cyclodextrin
  • glutamine phospholipids (liposomes),
  • cosmetic treatment of healthy skin may further be assisted by supplemental procedures, and especially contemplated procedures include chemical and/or mechanical exfoliation.
  • chemical exfoliation may be performed using one or more proteases (for example papain, Lactobacillus/Pumpkin Ferment Extract, Lactobacillus/Punica Granatum Fruit Ferment Extract), alpha-hydroxy acids, etc.
  • mechanical exfoliation may be performed using sugar crystals, cellulosic plant matter, tape stripping, frozen CO 2 , polymeric beads, and silica granules.
  • inventive subject matter is thus also directed to methods and use of sub-antimicrobially active concentrations of defensins in topical cosmetic formulations to recruit LGR6+ stem cells to the interfollicular space in non-injured skin.
  • a user will be instructed to apply the topical formulation to non-injured skin under a protocol effective 1) to reduce at least one of wrinkle depth, wrinkle length, wrinkle width, pore size, irregularity in texture of a skin surface, oiliness, brown spots, and red spots in non-injured skin, 2) to improve skin brightness, 3) to lighten skin, and/or 4) to correct aging skin markers such as skin firmness, sagging contours, lines, wrinkles, enlarged pores, impurities, brightness or uneven skin tone, surface imperfections, evenness, overall clarity, age spots, hydration, dryness, smooth texture, radiance and redness.
  • defensins will preferably be applied at least once daily (or twice daily) for a period of at least one week, or two weeks, three weeks, six weeks, or even longer.
  • the total quantity of applied formulations is such that the formulation is absorbed into the skin.
  • topical formulations are typically applied at 0.1-500 mg per cm 2 , 0.1-500 mg per cm 2 , 0.5-300 mg per cm 2 , 5-500 mg per cm 2 , or 100-500 mg per cm 2 .
  • the inventors discovered that the cosmetic formulations that included the defensins 1) reduces at least one of wrinkle depth, wrinkle length, wrinkle width, pore size, irregularity in texture of a skin surface, oiliness, brown spots, and red spots in non-injured skin, and/or 2) improves skin brightness, and/or 3) lightens skin, and/or 4) corrects at least one aging skin markers such as skin firmness, sagging contours, lines, wrinkles, enlarged pores, impurities, brightness or uneven skin tone, surface imperfections, evenness, overall clarity, age spots, hydration, dryness, smooth texture, radiance and redness.
  • cosmetic formulations may be prepared using any number of ingredients and formulations known in the art, preferred topical formulations include those that are ready-to-use and can be applied by a user. Therefore, with respect to cosmetically acceptable carriers, all cosmetically acceptable carriers are contemplated and include creams, oil-in-water emulsions, water-in-oil emulsions, foams, mousses, ointments, lotions, suspensions, serum, gels, etc.
  • Table 1 below shows an exemplary cosmetic delivery liposome formulation including two types of defensins. These liposomes are typically incorporated into a cosmetic formulation at a fraction of about 1.0 wt % to 10.0 wt % for most skin care applications.
  • topical cosmetic formulations can be offered together as a kit.
  • kits include a mask formulation and a defensin-containing cream or serum treatment formulation. Even more preferred kits include a mask formulation and both the defensin-containing cream and serum treatment formulations.
  • concentrated defensin preparations can be added to cosmetic base formulations such that the concentration of the defensin in the ready-to-use product is at a targeted sub-antimicrobially effective concentration.
  • defensins can be incorporated into concentrated preparations as solutions, associated with carrier proteins, and more typically as liposomal formulations.
  • concentrated defensin preparations can be added to the cosmetic base formulations in proportions as given below:
  • Mixture a) is melted at approximately 70° C. and mixture b) is heated to approximately 70° C. and added to mixture a) while stirring. Stirring is continued until the lotion has cooled down to approximately 30° C . Then c) and d) are added while stirring, and the lotion is homogenized.
  • Mixture a) is dissolved at approximately 50° C .
  • Mixture b) is dispersed at room temperature and added to a) while stirring. Then, composition c) is added while stirring.
  • cetearyl alcohol (and) ceteareth-20 8.00% cocoglycerides 2.00% cetearyl alcohol 2.00% dicaprylyl ether 8.00% oleyl erucate 7.00% phenoxyethanol, methylparaben, 0.30% ethylparaben, butylparaben, propylparaben, isobutylparaben b) water, distilled 62.40% phenoxyethanol, methylparaben, 0.30% ethylparaben, butylparaben, propylparaben, isobutylparaben glycerin 5.00% c) Concentrated defensin preparation 5.00%
  • Mixture a) is melted at approximately 70° C. and mixture b) is heated to approximately 70° C. and added to mixture a) while stirring. Stirring is continued until the cream has cooled down to approximately 30° C . Then, composition c) is added while stirring and the cream is homogenized.
  • diisostearoyl polyglyceryl-3 dimer dilinoleate 3.00% beeswax 0.60% castor oil, hydrated 0.40% paraffinum subliquidum 5.00% isohexadecane 10.00% PPG-15 stearyl ether 2.00% dimethicone 0.50% phenoxyethanol, methylparaben, 0.30% ethylparaben, butylparaben, propylparaben, isobutyparaben b) water, distilled 68.40% phenoxyethanol, methylparaben, 0.30% ethylparaben, butylparaben, propylparaben, isobutylparaben glycerin 3.00% MgSO 4 *7H 2 O 1.00% c) Concentrated defensin preparation 5.00% d) silica dimethyl silylate 0.50%
  • Mixture a) is heated to approximately 80° C.
  • mixture b) is brought to 80° C. and added to a) while stirring. Stirring is continued until the cream has cooled down to approximately 30° C.
  • c) and d) are added, and the cream is homogenized.
  • Clinical Study 1 Clinical Study 2
  • Clinical Study 2 double-blinded study
  • Both studies were performed under the supervision of Dr. Gregory Keller, M.D, F.A.C.S at the Plastic Surgery Clinic in Santa Barbara, California.
  • the design for both studies was substantially similar.
  • Both clinical studies examined 10 subjects over a duration of 6 weeks. Each subject was given a cream, a serum, and a masque. Half of the subjects (Study 2) were given formulations of the cream, serum, and masques containing defensins. The other half of subjects were given placebo formulations of the cream, serum, and masque that were identical in composition to the test group, except the formulations did not contain defensins.
  • the inventors measured individual skin health scores for each participant before and after treatment using the QuantifiCareTM 3D LifeVizTM Imaging Clinical System and protocol developed by QuantifiCare Inc. (www.quant Stamm.com).
  • the faces of each participants were scanned using the 3D LifeVizTM system and given a value for each the following categories: wrinkle depth, length, and width; pore depth; skin evenness; skin oiliness; skin brown spots; and skin red spots.
  • the resulting values for each participant were then compared against a population with the same gender, age, and skin type, using QuantifiCare's Reference Population Database.
  • the resulting skin heath score for each participant was a percentile ranking of skin health when compared with a population of people with the same age, gender, and skin type.
  • Table 2 shows the average age of each study group when compared to a population with corresponding age, gender, and skin type. Age was calculated using the wrinkle parameter, which is a combination of depth, length, and width of wrinkles in the forehead and cheeks.
  • FIG. 1 shows the before (A) and after (B) images acquired by a 3D LifeVizTM system for participant 3-PC (63 year old female) of Clinical Study 1. Participant 3-PC was given the test formulation. Measurements by the 3D LifeVizTM system showed that participant 3-PC saw a reducing in visible skin age estimation (based on the skin evenness value calculated by the 3D LifeVizTM system) from 64 years to 37 years. Photos have not been retouched.
  • FIG. 2 shows before (A) and after (B) images acquired by a 3D LifeVizTM system with the brown-spot filter for participant 8-IK (female) of Clinical Study 1. Participant 3-PC was given the test formulation. Images show a reduction in brown spots on the face as a result of treatment. Photos have not been retouched.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Dermatology (AREA)
  • Birds (AREA)
  • Epidemiology (AREA)
  • Gerontology & Geriatric Medicine (AREA)
  • Cosmetics (AREA)
  • Medicinal Preparation (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Biophysics (AREA)
  • Molecular Biology (AREA)

Abstract

The inventive subject is directed towards ready-to-use topical cosmetic formulations that include at least one defensin present in sub-antimicrobially effective concentrations. Surprisingly, even at such low concentrations, defensins recruit LGR6+ stem cells from hair follicles to the interfollicular space. Including defensins in the inventive topical cosmetic formulations may reduce one or more of wrinkle depth, wrinkle length, wrinkle width, pore size, irregularity in texture of a skin surface, oiliness, brown spots, and red spots in non-injured skin, thus reducing apparent age.

Description

    INCORPORATION BY REFERENCE
  • This application is a continuation of U.S. application Ser. No. 17/815,972, filed Jul. 29, 2022 which is a continuation of U.S. application Ser. No. 15/383,423, filed Dec. 19, 2016 which is a continuation of International Application No. PCT/US2015/036049, filed Jun. 16, 2015 which claims priority to U.S. Provisional Application No. 62/014,044, which was filed Jun. 18, 2014, and which is incorporated by reference herein.
  • FIELD OF THE INVENTION
  • The field of the invention is topical cosmetic formulations using defensins to reduce the appearance of wrinkles, pores, irregularity in texture of a skin surface, oiliness, brown spots, and red spots in non-injured skin.
  • BACKGROUND
  • The following background discussion includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.
  • Since their initial discovery in the 1960's, there has been much research surrounding the role of defensins. Defensins are small cystein-rich proteins, usually only 14-85 amino acids long. Defensins can be found in invertebrates, vertebrates, and plants, and have been shown to be active against many bacteria, fungi, and viruses. In fact, much of the body of research has focused on the antimicrobial properties of defensins. However, in recent years, some research has explored other roles defensins may play in human skin, such as in wound healing or hair growth.
  • PCT Patent Application WO 2014/004339 A2 by Applicant ELC Management LLC teaches the use of a resveratrol-containing cosmetic composition to stimulate endogenous production of cellular beta defensins in skin cells. In order to illustrate this effect, the inventors tested the composition on Normal Human Epidermal Keratinocytes (“NHEK”) in vitro and measured the presence of beta defensin in NHEK. The resulting stimulated quantities of defensin were very small (approximately 0.001 ng/ml). The inventors theorized that the stimulation of beta defensins in keratinocytes by resveratrol-containing compositions would be effective for treating acne, inhibiting microbial growth on the skin, and improving skin barrier repair. Because of the low stimulated quantities of beta defensin, and because the compositions do not actually contain defensins, it is questionable that these compositions would produce cosmetically meaningful results. These and all other extrinsic materials discussed herein are incorporated by reference in their entirety. Where a definition or use of a term in an incorporated reference is inconsistent or contrary to the definition of that term provided herein, the definition of that term provided herein applies and the definition of that term in the reference does not apply.
  • On the other hand, defensins have been shown to play a significant role in wound healing. In the journal article titled “Stimulation of the follicular bulge LGR5+ and LGR6+ stem cells with the gut-derived human alpha defensin 5 results in decreased bacterial presence, enhanced wound healing, and hair growth from tissues devoid of adnexal structures” (Plast. Reconstr. Surg. 2013;132(5):1159-71), Lough et al. report a human alpha defensin 5-containing formulation that was shown under experimental conditions to recruit LGR5+ and LGR6+ stem cells to third-degree burns in mice, which accelerated healing of the wound. However, these findings leave many questions. For example, in the experimental conditions described by Lough et al., it is unclear if LGR5+ and LGR6+ were activated by pro-inflammatory conditions and other factors already present in the wound or due to the topical application of defensins. Furthermore, the wound healing formulation used by Lough et al. contained concentrations of defensins at antimicrobially active concentrations (e.g. about 105 ng/ml). However, this high concentration of defensin may make any resulting cosmetic composition more allergenic and more costly.
  • Therefore, even though defensin containing and defensin stimulating compositions are known, there is a need for a cosmetic topical formulation for use on healthy skin that makes use of defensins for reducing the visual appearance of age on the skin.
  • SUMMARY OF THE INVENTION
  • The inventive subject matter is directed towards various topical formulations, methods of manufacture of the topical formulation in which the topical formulation includes sub-antimicrobially effective concentrations of at least one defensin, and methods of applying the topical formulation to the healthy skin of users to reduce the apparent age of the users' skin.
  • In one aspect of the invention, a topical cosmetic formulation includes a defensin in a cosmetically acceptable carrier. Preferred topical cosmetic formulations are ready-to-use and contain the defensin at a sub-antimicrobially effective concentration, wherein the concentration is ineffective to inhibit growth of a microbial pathogen in a therapeutically effective manner. Still further preferred topical formulations may further comprise a blend of two or more different defensins, wherein the combined concentration of defensins in the formulation is a sub-antimicrobially effective concentration.
  • The inventors further contemplate methods of using defensins at sub-antimicrobially effective concentrations in topical formulations to recruit LGR6+ stem cells to an interfollicular space in non-injured skin. It should be appreciated that methods of recruiting LGR6+ stem cells may include a step of providing a topical formulation containing a sub-antimicrobial concentration of at least one defensin and a further step of applying the formulation to non-injured skin 1) to reduce at least one of wrinkle depth, wrinkle length, wrinkle width, pore size, irregularity in texture of a skin surface, oiliness, brown spots, and red spots 2) to improve skin brightness, 3) to lighten skin, and/or 4) to correct aging skin markers such as skin firmness, sagging contours, lines, wrinkles, enlarged pores, impurities, brightness or uneven skin tone, surface imperfections, evenness, overall clarity, age spots, hydration, dryness, smooth texture, radiance and redness in non-injured skin.
  • It is preferred that the inventive compositions, methods, and uses employ at least one of alpha-defensin 1, alpha-defensin 5, alpha-defensin 6, neutrophil defensin 1, neutrophil defensin 2, neutrophil defensin 3, neutrophil defensin 4, theta-defensin, beta-defensin 1, beta-defensin 2, beta-defensin 3, and beta-defensin 4. In especially preferred topical compositions and methods, alpha-defensin 5 and beta-defensin 3 are employed. It should be appreciated that the defensin may comprise a synthetic defensin, a human defensin, recombinant defensin, a monkey defensin, a mouse defensin, a rat defensin, a bovine defensin, a sheep defensin, a horse defensin, a rabbit defensin, a swine defensin, a dog defensin, and/or a cat defensin.
  • With respect to the sub-antimicrobially effective concentration of the first defensin in the ready-to-use topical cosmetic formulation, contemplated concentrations may be between 0.01 and 100 ng/ml, or between 1 and 30 ng/ml, including the end points of each range. Additionally, especially preferred embodiments of the inventive subject matter employ defensin concentrations of about 22 ng/ml and about 4.4 ng/ml in ready-to-use formulations.
  • The inventors unexpectedly found that, even at these low concentrations, defensins may be effective 1) to substantially reduce wrinkle depth, wrinkle length, wrinkle width, pore size, irregularity in texture of a skin surface, oiliness, brown spots, and red spots 2) to improve skin brightness, 3) to lighten skin, and/or 4) to correct aging skin markers such as skin firmness, sagging contours, lines, wrinkles, enlarged pores, impurities, brightness or uneven skin tone, surface imperfections, evenness, overall clarity, age spots, hydration, dryness, smooth texture, radiance and redness in non-injured skin. Without wishing to be bound by any particular theory, the effectiveness of defensins at these sub-antimicrobially effective concentrations may be due to the activation and/or recruitment of LGR6+ stem cells. Typically, defensins used in preferred embodiments of the inventive subject matter have a purity greater than 95% as shown by HPLC, and the sequence and proper disulfide bond formation of the defensins can be confirmed by tandem MS/MS.
  • Depending on the nature of the topical formulation, it should be recognized that defensins may be encapsulated in liposomes or other nanoparticles. In preferred formulations, defensins may also be associated with a carrier, in particular a protein carrier such as albumin (e.g., human serum albumin, bovine serum albumin, egg albumin, and recombinant albumin produced by rice, other plants, bacteria or yeast), also encapsulated in liposomes where desirable.
  • The inventors further contemplate that the topical cosmetic formulations may also include supplements to provide nutrition and support for LGR6+ stem cells. Typical supplements include human serum albumin, bovine serum albumin, egg albumin, recombinant albumin produced by rice, other plants, bacteria or yeast, plant hydrolysate, beta-cyclodextrin, glutamine, phospholipids, fibronectin, hyaluronate, hyaluronic acid, plant hydrolysate, L-alanyl-L-glutamine, gelatin, recombinant gelatin, Epidermal Growth Factor (EGF), vitamin E, Tocopheryl Nicotinate, and ubiquinone, coenzyme Q10, antioxidants.
  • The inventors have appreciated that the topical cosmetic formulations of the inventive subject matter can be included in kits with exfoliating masks. Especially preferred kits include a mask, a cream treatment formulation, and a serum treatment formulation.
  • Further aspects of the inventive subject matter provide methods of treating scars, sunburn, bruises, and other skin disorders in which the epidermal layers of the skin are largely intact. Exemplary methods include the steps of providing a topical formulation having at least one defensin at a sub-antimicrobially effective concentration and applying the formulation to non-injured skin under a protocol effective to substantially reduce the appearance of scars, sunburn, bruises, etc.
  • Various objects, features, aspects and advantages of the inventive subject matter will become more apparent from the following detailed description of preferred embodiments, along with the accompanying drawing figures in which like numerals represent like components.
  • BRIEF DESCRIPTION OF THE DRAWING
  • FIG. 1 shows side-by-side photographs that show reduction in wrinkles.
  • FIG. 2 shows side-by-side photographs that show a reduction in the appearance of brown spots.
  • DETAILED DESCRIPTION
  • The inventors unexpectedly discovered that sub-antimicrobial concentrations of defensins can achieve numerous desirable effects on non-injured skin when applied in ready-to-use topical formulations. Among other things, such topical formulations provided 1) significant reduction in wrinkles, pore size, irregularity in skin texture, oiliness, brown spots, and red spots, 2) improvement in skin brightness, 3) skin lightening, and/or 4) correction of aging skin markers such as skin firmness, sagging contours, lines, wrinkles, enlarged pores, impurities, brightness or uneven skin tone, surface imperfections, evenness, overall clarity, age spots, hydration, dryness, smooth texture, radiance and redness.
  • In one preferred embodiment, a ready-to-use topical cosmetic formulation comprises at least one defensin present in a topical cosmetic formulation at a sub-antimicrobially effective concentration. Of course it should be appreciated that topical formulations may contain one defensin, a combination of two defensins, or a combination of three or more defensins. The defensins used may be of the same or different types and subtypes. For example suitable defensins may include one or more of alpha-defensin 1, alpha-defensin 5, alpha-defensin 6, neutrophil defensin 1, neutrophil defensin 2, neutrophil defensin 3, neutrophil defensin 4, theta-defensin, beta-defensin 1, beta-defensin 2, beta-defensin 3, and beta-defensin 4. Especially preferred topical formulations contain alpha-defensin 5 and beta-defensin 3. When two more defensins are used in combination, each defensin may be present in equal quantities by mass or at mass ratios specified to achieve a desired result, such as 1:1.5, 1:2, 1:4, 1:5, etc. Notably, it should be appreciated that the total concentrations of defensins used in contemplated ready-to-use formulations are ineffective at inhibiting substantial proliferation of microbes in established skin infections in a therapeutically effective manner.
  • As used herein, the term “ready-to-use” indicates that the defensin-containing topical formulation is in a form that is presented for sale and application. It is contemplated that ready-to-use formulations can comprise a fully combined solution, cream, gel, serum, lotion, etc. Alternatively, the defensin can be packaged in a separate container (e.g., in a vial that pumps a defensin solution with a cream that the user blends before applying to unbroken skin) and combined with another topical formulation at the time of use/application.
  • As also used herein, the phrase “sub-antimicrobially effective concentration” means concentration(s) of defensins which are characterized by an inability to inhibit the proliferation of microbes in an established infection. Typically, ready-to-use topical formulations do not include concentrations greater than 1 μg/ml. In preferred embodiments, the concentration of defensins lies between 0.01 and 100 ng/ml, and even more typically between 1 and 30 ng/ml, wherein contemplated concentrations include the end points of each range. In even more preferred embodiments, the topical cosmetic formulations have defensin concentrations of about 22 ng/ml and 4.4 ng/ml. As used herein, when the term “about” is used in conjunction with a numeral, “about” means a range of plus or minus ten percent of the numerical value given, including end points. The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein.
  • General topical formulations may include any and all formulations suitable for cosmetic topical use, especially on non-injured skin. As used herein, the term “non-injured” skin refers to skin in which dermis and hypodermis are substantially intact. Therefore, viewed from a different perspective, non-injured skin will appear intact to the unaided eye, with no breach sufficiently large or deep to result in bleeding. Thus, non-injured (or “healthy”) skin includes aged skin and skin with first degree sunburn, environmental exposure, bruising, or partially ablated stratum corneum. Non-injured (or healthy) skin also excludes skin displaying persistent infection with pathogens that result in visible symptoms and signs of infection.
  • With respect to the source of defensins, the inventors contemplate that defensins from both natural and synthetic sources may be suitable for incorporation into topical formulations. For example, defensins may be obtained from plants (e.g., Arabidopsis, pea, tobacco, and spruce), mammals, or other animals. Exemplary defensins derived from natural sources may include human defensins, monkey defensins, mouse defensins, rat defensins, bovine defensins, sheep defensins, horse defensins, rabbit defensins, swine defensins, dog defensins, and/or cat defensins.
  • Due to their relatively low quantities in a living organism and small molecular weight, it is generally preferred, however, that the defensins are synthetic defensins. Synthetic defensins include defensins produced by chemical synthesis (e.g., solid phase synthesis) or by recombinant technologies (e.g., produced by recombinant bacteria, yeast, tissue cultures, plants or animals). The inventors further contemplate that defensin analogues such as hapivirins and diprovirins may be used in some embodiments of the inventive subject matter. Still further, the inventors further contemplate that the defensins can also be modified to increase their activity and specificity for cosmetic improvements to the appearance of skin. For example, defensins may be unfolded and refolded under controlled conditions to ascertain proper disulfide bond formation (which can be monitored by MS analysis and/or CD spectroscopy). Alternatively, chemical modifications (e.g., using non-natural amino acids to increase half-life time, or derivatized proteinogenic amino acids to increase lipophilicity) are contemplated to tailor the defensins to a particular need.
  • Regardless of the source of the defensins, it should be appreciated that specific activity of defensins is dependent on various factors, including isomeric form and tertiary structure of the final protein. Thus, and especially where the defensin is synthetic, orthogonal protecting groups can be used to protect selected cysteine residues, which can then be individually deprotected and bonded with the matching target cysteine residue, leading to coordinated non-random disulfide bond formation. Use of such protecting groups in the synthetic strategy can give rise to defensins with a specific activity that is comparable to the specific activity of the native defensin. Any suitable characterization and quality control measures may be employed. Typically, the specific activity of defensins incorporated into the inventive topical formulations is measured by purity as determined by HPLC. In exemplary embodiments, the defensin is between 80% and 100% pure, more typically the defensin is at least 90% pure, or at least 95% pure, or at least 99% pure, or at least 99.9% pure. Additionally, proper amino acid sequence and disulfide bond formation can be confirmed by tandem MS/MS, for example.
  • With respect to suitable concentration of defensins in cosmetic formulations presented herein, it is contemplated that all concentrations are deemed appropriate so long as such concentrations are cosmetically effective (i.e., improves in the visual appearance of healthy skin, and especially I) reduces wrinkles, pore size, irregularity in skin texture, oiliness, brown spots, and/or red spots. 2) improves skin brightness, 3) lightens skin, and/or 4) corrects aging skin markers such as skin firmness, sagging contours, lines, wrinkles, enlarged pores, impurities, brightness or uneven skin tone, surface imperfections, evenness, overall clarity, age spots, hydration, dryness, smooth texture, radiance and redness). Thus, it should be noted that the cosmetic formulation is intended to be used for improvement in visual appearance of healthy aged skin. In other words, the concentrations employed in the inventive subject matter are not intended to be therapies for the treatment of cuts, third degree burns, or other injuries where there is a loss of integrity in epidermis and dermal layers (typically associated with bleeding and/or scab formation). Consequently, the total concentration of defensins (single type or combination of distinct defensins) in the final cosmetic formulation as applied to the skin will be between 0.01 ng/ml and 100 ng/ml, or between 0.1 ng/ml and 100 ng/ml, or between 1 ng/ml and 100 ng/ml, or between 2 ng/ml and 80 ng/ml, or between 4 ng/ml and 60 ng/ml, or between 1 ng/ml and 30 ng/ml. Thus, preferred compositions include defensins at a concentration of at least 0.01 ng/ml, at least 0.1 ng/ml, at least 1 ng/ml, or at least 4 ng/ml, but no more than 200 ng/ml, no more than 100 ng/ml, no more than 75 ng/ml, or no more than 50 ng/ml.
  • In most instances the defensin is associated with a cosmetically acceptable protein to increase stability and/or delivery characteristics. In this context, it should be noted that the association is preferably non-covalent (e.g., electrostatic, ionic, hydrophobic, etc.), however, covalent attachment to a side group of the protein is not excluded. Exemplary proteins include lactoferrin, transferrin, and albumin (e.g., human serum albumin, bovine serum albumin, and egg albumin, recombinant albumin). The defensins and protein carriers may be in various ratios, including equimolar, sub-, and supramolar ratios. Additionally, combinations of two or more protein carriers may be used. For example, in a formulation in which two defensins are used, one defensin may be associated with one carrier, and the other defensin may be associated with a different carrier. Therefore, any combination of defensins and carriers are contemplated.
  • In still further contemplated aspects, the defensins (and carrier proteins) can be encapsulated in cosmetically acceptable formulations, and especially formulations using a lipid membrane. For example, liposomes, microcapsules, nanocapsules, microparticles, nanoparticles, microparticle delivery systems, are especially contemplated. A description of some cosmetically acceptable cosmetic delivery systems can be found in Maherani et al, “Liposomes: A Review of Manufacturing Techniques and Targeting Strategy,” Current Nanoscience; 7:436-452 (2011). A preferred method of liposome manufacturing is shear method. Preferred cosmetic delivery systems resemble naturally occurring membranes, are flexible, and can penetrate interstitial spaces between cells. It is further contemplated that cosmetic delivery systems may have monolayer, bilayer (e.g. unilammellar vesicle or ULV), or multi layer structures (e.g. multilammerlar vesicle or MLV). Additionally, multilayer liposomes, microcapsules, microsomes, and nanaocapsules may have nested structures (e.g. multivesicular vesicle or MVV). Cosmetic delivery systems used in the topical formulations can range in size from 500 nm to 10 microns. In the preparation of cosmetic delivery systems, all cosmetically acceptable lipid compositions are contemplated, especially pharmaceutically acceptable lipids. In most instances preferred cosmetic delivery systems comprise amphipathic or amphiphilic molecules such as phospholipids or combinations of phospholipids (e.g., phosphatidylcholine, phosphatidylethanolamine, phosphatidic acid, phosphatidylserine, and phosphoinositides). Additionally, in some instances contemplated cosmetic delivery systems can contain additive(s) such as sterols, polyethylene glycol, cholesterol, dicethylphosphate, stearyl amine, etc. With respect to the amount of delivery systems incorporated in each ready-to-use formulation, the cosmetic delivery system content will typically be adjusted to achieve a sub-antimicrobial concentration of defensins within a preferred range. Unilamellar vesicles/liposomes can be produced using high shear techniques. These vesicles have a greater Zeta Potential than the typical liposome, which allows for smaller, more uniform particle size with increased stability. Zeta Potential is an indicator of the electronic charge on the surface of any macroscopic material that is in contact with a liquid. This can be used to predict and control the stability of suspensions; the higher the Zeta Potential, the greater the stability of the molecule because the charged particles are able to repel and overcome their innate affinity to assemble.
  • It should be noted that defensins, protein carriers, liposomes, or other membranaceous structures have a molecular weight that exceeds transmembrane delivery, and even delivery across the stratum corneum. Nevertheless, as is discussed in more detail, the defensins have a profound effect on stem cell activity in dermal and hypodermal layers. While not wishing to be bound by any theory or hypothesis, the inventors contemplate that the liposomal formulations have the ability to transport the defensins via an interstitial route and/or to invade the hair follicle to a depth and concentration sufficient to activate LGR6+ cells. Viewed from another perspective, the use of cosmetic delivery systems is thought to aid in the delivery of defensins as one would not expect defensins per se to penetrate the stratum corneum of the skin (acting as a barrier to molecules with molecular weights greater than 500 Da). Moreover, when associated with albumin (65-70 kDa) and/or liposomes, it becomes at least conceptually even more difficult for the defensin composition to penetrate unbroken skin. Thus, the inventors hypothesize that the mechanism for delivering defensins is different in unbroken skin when compared with broken or injured skin.
  • In another aspect, the inventors advantageously provide additional ingredients that nourish and support the recruited stem cells in healthy skin. For example, albumin (e.g., human serum albumin, bovine serum albumin), egg albumin (albumen), recombinant albumin, plant hydrolysate, and β-cyclodextrin, glutamine, phospholipids (liposomes), fibronectin, hyaluronate, plant hydrolysate, L-alanyl-L-glutamine, gelatin, Vitamin E (tocopheryl nicotinate), ubiquinone (coenzyme Q10), gelatin, recombinant gelatin, hyaluronic acid, Epidermal Growth Factor can provide nutrition and support to the stem cells.
  • In yet further contemplated aspects, cosmetic treatment of healthy skin may further be assisted by supplemental procedures, and especially contemplated procedures include chemical and/or mechanical exfoliation. For example, chemical exfoliation may be performed using one or more proteases (for example papain, Lactobacillus/Pumpkin Ferment Extract, Lactobacillus/Punica Granatum Fruit Ferment Extract), alpha-hydroxy acids, etc. while mechanical exfoliation may be performed using sugar crystals, cellulosic plant matter, tape stripping, frozen CO2, polymeric beads, and silica granules.
  • Based on the above and further data (not shown), the inventive subject matter is thus also directed to methods and use of sub-antimicrobially active concentrations of defensins in topical cosmetic formulations to recruit LGR6+ stem cells to the interfollicular space in non-injured skin. Most typically, a user will be instructed to apply the topical formulation to non-injured skin under a protocol effective 1) to reduce at least one of wrinkle depth, wrinkle length, wrinkle width, pore size, irregularity in texture of a skin surface, oiliness, brown spots, and red spots in non-injured skin, 2) to improve skin brightness, 3) to lighten skin, and/or 4) to correct aging skin markers such as skin firmness, sagging contours, lines, wrinkles, enlarged pores, impurities, brightness or uneven skin tone, surface imperfections, evenness, overall clarity, age spots, hydration, dryness, smooth texture, radiance and redness. For example, defensins will preferably be applied at least once daily (or twice daily) for a period of at least one week, or two weeks, three weeks, six weeks, or even longer. Beneficially, the total quantity of applied formulations is such that the formulation is absorbed into the skin. For example, topical formulations are typically applied at 0.1-500 mg per cm2, 0.1-500 mg per cm2, 0.5-300 mg per cm2, 5-500 mg per cm2, or 100-500 mg per cm2.
  • Using the compositions presented herein, the inventors discovered that the cosmetic formulations that included the defensins 1) reduces at least one of wrinkle depth, wrinkle length, wrinkle width, pore size, irregularity in texture of a skin surface, oiliness, brown spots, and red spots in non-injured skin, and/or 2) improves skin brightness, and/or 3) lightens skin, and/or 4) corrects at least one aging skin markers such as skin firmness, sagging contours, lines, wrinkles, enlarged pores, impurities, brightness or uneven skin tone, surface imperfections, evenness, overall clarity, age spots, hydration, dryness, smooth texture, radiance and redness. Furthermore, observed effects (data not shown) included treatment of a scar, such that the appearance of the scar is reduced after application of the topical formulation. The inventors contemplate that other non-wound conditions such as sunburn and discoloration may be treated according to the inventive uses and methods.
  • EXAMPLES
  • While the cosmetic formulations may be prepared using any number of ingredients and formulations known in the art, preferred topical formulations include those that are ready-to-use and can be applied by a user. Therefore, with respect to cosmetically acceptable carriers, all cosmetically acceptable carriers are contemplated and include creams, oil-in-water emulsions, water-in-oil emulsions, foams, mousses, ointments, lotions, suspensions, serum, gels, etc.
  • Example Topical Cosmetic Cream Formulation Ingredient List
  • Water (Aqua), Carthamus Tinctorius (Safflower) Oleosomes, Butyrospermum Parkii (Shea) Butter, Macadamia Integrifolia Seed Oil, Niacinamide, Yeast Extract, Ammonium Acryloyldimethyltaurate/VP Copolymer, Helianthus Annuus (Sunflower) Seed Oil, Phospholipids, Alpha-Defensin 5, Beta-Defensin 3, Hyaluronic Acid, Ophiopogon Japonicus Root Extract, Hydrolyzed Candida Saitoana Extract, Sea Whip Extract,Lycium Chinense Fruit Extract, Vaccinium Angustifolium Fruit Extract, Vaccinium Marcrocarpon Fruit (Cranberry) Fruit Extract, Rosmarinus Officinalis (Rosemary) Leaf Extract, Panthenol, Albumin, Tocopheryl Acetate, Ubiquinone, L-Alanyl-L-Glutamine, Leuconostoc/Radish Root Ferment Filtrate, Gelatin, SH Oligopeptide-1, Xanthan Gum, Phytic Acid, Polysorbate 20, Caprylic/Capric Triglyceride, Phenoxyethanol, Caprylyl Glycol, Ethylhexylglycerin, Hexylene Glycol, Potassium Sorbate, Sodium Chloride, Fragrance.
  • Example Topical Cosmetic Serum Formulation Ingredient List
  • Water (Aqua), Cyclopentasiloxane, Glycerin, Niacinamide, Sinorhizobium Meliloti Ferment Filtrate, Dimethicone, Polysorbate 20, Dimethicone/Vinyl Dimethicone Crosspolymer, Lauryl PEG-9 Polydimethylsiloxyethyl Dimethicone, Ammonium Acryloyldimethyltaurate/VP Copolymer, Phospholipids, Alpha-Defensin 5, Beta-Defensin 3, Palmitoyl Tripeptide-38, Sodium Hyaluronate, Arabidopsis Thaliana Extract, Sea Whip Extract, Ergothioneine, Helianthus Annuus (Sunflower) Seed Oil, Rosmarinus Offinalis (Rosemary) Leaf Extract, SH Oligopeptide-1, Tocopheryl Acetate, Ubiquinone, Leuconostoc/Radish Root Ferment Filtrate, Albumin, Gelatin, L-Alanyl-L-Glutamine, Caprylic/Capric Triglyceride, Cetyl Hydroxyethylcellulose, Lecithin, Hydroxypropyl Cyclodextrin, Phytic Acid, Phenoxyethanol, Caprylyl Glycol, Ethylhexylglycerin, Hexylene Glycol, Sodium Chloride.
  • Example Mask
  • Butylene Glycol, PEG-8, Tapioca Starch, Sucrose, Titanium Dioxide, Hydroxyethyl Acrylate/Sodium Acryloyldimethyl Taurate Copolymer, Squalane, Polysorbate 60, Carica Papaya (Papaya) Fruit, Papain, Aloe Barbadensis Leaf Juice, Lactobacillus/Pumpkin Ferment Extract, Lactobacillus/Punica Granatum Fruit Ferment Extract, Sea Whip Extract, Cananga Odorata Flower Oil, Citrus Aurantium Dulcis (Orange) Peel Oil, Caprylic/Capric Triglyceride, Lactic Acid, Phenoxyethanol, Caprylyl Glycol, Ethylhexylglycerin, Hexylene Glycol.
  • Example Liposome Formulation
  • Table 1 below shows an exemplary cosmetic delivery liposome formulation including two types of defensins. These liposomes are typically incorporated into a cosmetic formulation at a fraction of about 1.0 wt % to 10.0 wt % for most skin care applications.
  • TABLE 1
    Exemplary Liposome Formulation
    Component Concentration
    Water (protease-free) to 100%
    Albumin 0.1-1.0 mg/ml
    L-alanyl-L-glutamine 0.1-1.0 mg/ml
    Gelatin 2-200 μg/ml
    Matrix proteins 1-100 ng/ml
    Human alpha-defensin 5 1-200 ng/ml
    Human beta-defensin 3 1-200 ng/ml
    Growth factors (e.g., EGF, FGF-2) 0.1-100 ng/ml
    Phospholipids 2-20 wt %
    Antioxidants 0.3-3 wt %
  • In yet a further aspect of the inventive subject matter, topical cosmetic formulations can be offered together as a kit. Preferred kits include a mask formulation and a defensin-containing cream or serum treatment formulation. Even more preferred kits include a mask formulation and both the defensin-containing cream and serum treatment formulations.
  • In the manufacture of cosmetic defensin formulations, it is contemplated that concentrated defensin preparations can be added to cosmetic base formulations such that the concentration of the defensin in the ready-to-use product is at a targeted sub-antimicrobially effective concentration. Depending on the desired formulation, defensins can be incorporated into concentrated preparations as solutions, associated with carrier proteins, and more typically as liposomal formulations. Such concentrated defensin preparations can be added to the cosmetic base formulations in proportions as given below:
  • Body Lotion (Oil-in-water)
  • a) PEG-7 hydrogenated castor oil 2.00%
    PEG-20 glyceryl laurate 1.00%
    cocoglycerides 3.00%
    cetearyl alcohol 1.00%
    cetearyl isononanoate 4.00%
    octyl stearate 4.00%
    phenoxyethanol, methylparaben, 0.30%
    ethylparaben, butylparaben,
    propylparaben, isobutylparaben
    b) water, distilled 73.40%
    phenoxyethanol, methylparaben, 0.30%
    ethylparaben, butylparaben,
    propylparaben, isobutylparaben,
    glycerin 3.00%
    c) Concentrated defensin preparation 5.00%
    d) acrylamides copolymer, mineral oil 3.00%
    C13-C14 isoparaffin, polysorbate 85
  • Mixture a) is melted at approximately 70° C. and mixture b) is heated to approximately 70° C. and added to mixture a) while stirring. Stirring is continued until the lotion has cooled down to approximately 30° C . Then c) and d) are added while stirring, and the lotion is homogenized.
  • Gel-Lotion
  • a) acrylamides copolymer, mineral oil, 5.00%
    C13-14 isoparaffin, polysorbate 85
    myreth-3 myristate 4.00%
    b) water, distilled 85.00%
    phenoxyethanol (and) methylparaben (and) 0.50%
    ethylparaben (and) butylparaben (and)
    propylparaben (and) isobutylparaben
    xanthan gum 0.50%
    c) Concentrated defensin preparation 5.00%
  • Mixture a) is dissolved at approximately 50° C . Mixture b) is dispersed at room temperature and added to a) while stirring. Then, composition c) is added while stirring.
  • Oil-in-water Cream
  • a) cetearyl alcohol (and) ceteareth-20 8.00%
    cocoglycerides 2.00%
    cetearyl alcohol 2.00%
    dicaprylyl ether 8.00%
    oleyl erucate 7.00%
    phenoxyethanol, methylparaben, 0.30%
    ethylparaben, butylparaben,
    propylparaben, isobutylparaben
    b) water, distilled 62.40%
    phenoxyethanol, methylparaben, 0.30%
    ethylparaben, butylparaben,
    propylparaben, isobutylparaben
    glycerin 5.00%
    c) Concentrated defensin preparation 5.00%
  • Mixture a) is melted at approximately 70° C. and mixture b) is heated to approximately 70° C. and added to mixture a) while stirring. Stirring is continued until the cream has cooled down to approximately 30° C . Then, composition c) is added while stirring and the cream is homogenized.
  • Water-in-oil Cream
  • a) diisostearoyl polyglyceryl-3 dimer dilinoleate 3.00%
    beeswax 0.60%
    castor oil, hydrated 0.40%
    paraffinum subliquidum 5.00%
    isohexadecane 10.00%
    PPG-15 stearyl ether 2.00%
    dimethicone 0.50%
    phenoxyethanol, methylparaben, 0.30%
    ethylparaben, butylparaben,
    propylparaben, isobutyparaben
    b) water, distilled 68.40%
    phenoxyethanol, methylparaben, 0.30%
    ethylparaben, butylparaben,
    propylparaben, isobutylparaben
    glycerin 3.00%
    MgSO4 *7H2O 1.00%
    c) Concentrated defensin preparation 5.00%
    d) silica dimethyl silylate 0.50%
  • Mixture a) is heated to approximately 80° C. , mixture b) is brought to 80° C. and added to a) while stirring. Stirring is continued until the cream has cooled down to approximately 30° C. , then c) and d) are added, and the cream is homogenized.
  • Shampoo
  • Sodium polyoxyethylene lauryl ether sulfate 15.0%
    Alkyl polyglucoside 4.0%
    N-ethanol-N-methyl dodecanoic acid amide 3.0%
    EDTA-Na2 0.3%
    Malic acid to adjust pH to 6.0 q.s.
    Preservative 0.5%
    Concentrated defensin preparation 10.0%
    Purified water balance
    Total 100.0%
  • All ingredients are mixed together and the volume is brought to about 90 ml. The pH is then adjusted and the volume is finally adjusted to 100 ml (all percentages are weight %).
  • Body Wash
  • Sodium polyoxyethylene lauryl ether sulfate 16.0%
    Sodium polyoxyethylene 5.0%
    N-ethanol-N-methyl palm kernel oil fatty acid
    amide 2.5%
    Glycerin 3.0%
    Cationized cellulose 0.1%
    Ethylene glycol distearate 3.0%
    EDTA-Na2 0.3%
    Citric acid to adjust pH to 5.7 q.s.
    Preservative 0.5%
    Concentrated defensin preparation 7.5%
    Purified water balance
    Total 100.0
  • All ingredients are mixed together and the volume is brought to about 90 ml. The pH is then adjusted and the volume is finally adjusted to 100 ml (all percentages are weight %).
  • Face Wash
  • Sodium polyoxyethylene lauryl ether sulfate 20.0%
    N-ethanol-N-methyl dodecanoic acid amide 4.8%
    Glycerin 3.0%
    Hydroxyethyl cellulose 0.3%
    Ethylene glycol distearate 1.5%
    EDTA-Na2 0.3%
    Citric acid to adjust pH to 6.0 q.s.
    Preservative 0.5%
    Concentrated defensin preparation 10.0%
    Purified water balance
    Total 100.0
  • All ingredients are mixed together and the volume is brought to about 90 ml. The pH is then adjusted and the volume is finally adjusted to 100 ml (all percentages are weight %).
  • The inventors tested a preferred formulation of the inventive subject matter in two clinical studies, Clinical Study 1 and Clinical Study 2 (double-blinded study). Both studies were performed under the supervision of Dr. Gregory Keller, M.D, F.A.C.S at the Plastic Surgery Clinic in Santa Barbara, California. The design for both studies was substantially similar. Both clinical studies examined 10 subjects over a duration of 6 weeks. Each subject was given a cream, a serum, and a masque. Half of the subjects (Study 2) were given formulations of the cream, serum, and masques containing defensins. The other half of subjects were given placebo formulations of the cream, serum, and masque that were identical in composition to the test group, except the formulations did not contain defensins.
  • The inventors measured individual skin health scores for each participant before and after treatment using the QuantifiCare™ 3D LifeViz™ Imaging Clinical System and protocol developed by QuantifiCare Inc. (www.quantificare.com). The faces of each participants were scanned using the 3D LifeViz™ system and given a value for each the following categories: wrinkle depth, length, and width; pore depth; skin evenness; skin oiliness; skin brown spots; and skin red spots. The resulting values for each participant were then compared against a population with the same gender, age, and skin type, using QuantifiCare's Reference Population Database. The resulting skin heath score for each participant was a percentile ranking of skin health when compared with a population of people with the same age, gender, and skin type.
  • For example, Table 2 (below) shows the average age of each study group when compared to a population with corresponding age, gender, and skin type. Age was calculated using the wrinkle parameter, which is a combination of depth, length, and width of wrinkles in the forehead and cheeks.
  • TABLE 2
    Average Age Ranking For Clinical Study
    2 Before and After Treatment
    Average Age Ranking
    Group Before Treatment After 6 Weeks
    Placebo 77% 74%
    Test Group 61% 81%
  • FIG. 1 shows the before (A) and after (B) images acquired by a 3D LifeViz™ system for participant 3-PC (63 year old female) of Clinical Study 1. Participant 3-PC was given the test formulation. Measurements by the 3D LifeViz™ system showed that participant 3-PC saw a reducing in visible skin age estimation (based on the skin evenness value calculated by the 3D LifeViz™ system) from 64 years to 37 years. Photos have not been retouched.
  • FIG. 2 shows before (A) and after (B) images acquired by a 3D LifeViz™ system with the brown-spot filter for participant 8-IK (female) of Clinical Study 1. Participant 3-PC was given the test formulation. Images show a reduction in brown spots on the face as a result of treatment. Photos have not been retouched.
  • It should be apparent to those skilled in the art that many more modifications besides those already described are possible without departing from the inventive concepts herein. The inventive subject matter, therefore, is not to be restricted except in the scope of the appended claims. Moreover, in interpreting both the specification and the claims, all terms should be interpreted in the broadest possible manner consistent with the context. In particular, the terms “comprises” and “comprising” should be interpreted as referring to elements, components, or steps in a non-exclusive manner, indicating that the referenced elements, components, or steps may be present, or utilized, or combined with other elements, components, or steps that are not expressly referenced. Where the specification claims refers to at least one of something selected from the group consisting of A, B, C . . . and N, the text should be interpreted as requiring only one element from the group, not A plus N, or B plus N, etc. Moreover, as used in the description herein and throughout the claims that follow, the meaning of “a,” “an,” and “the” includes plural reference unless the context clearly dictates otherwise. Also, as used in the description herein, the meaning of “in” includes “in” and “on” unless the context clearly dictates otherwise.

Claims (20)

What is claimed is:
1. A method of improving skin texture in a subject in need thereof, the method comprising:
applying a topical formulation comprising a first defensin to a non-injured skin of the subject,
recruiting LGR6+ stems cells to an interfollicular space in the non-injured skin of the subject,
wherein the topical formulation reduces wrinkle depth, wrinkle length, wrinkle width, pore size, irregularity in texture of a skin surface, oiliness, brown spots, or red spots in the non-injured skin of the subject.
2. The method of claim 1, wherein the first defensin is present in the topical formulation at a sub-antimicrobially effective concentration.
3. The method of claim 1, wherein the first defensin comprises alpha-defensin 1, alpha-defensin 5, alpha-defensin 6, neutrophil defensin 1, neutrophil defensin 2, neutrophil defensin 3, neutrophil defensin 4, theta-defensin, beta-defensin 1, beta-defensin 2, beta-defensin 3, or beta-defensin 4.
4. The method of claim 1, wherein the first defensin comprises a synthetic defensin, a human defensin, recombinant defensin, a monkey defensin, a mouse defensin, a rat defensin, a bovine defensin, a sheep defensin, a horse defensin, a rabbit defensin, a swine defensin, a dog defensin, or a cat defensin.
5. The method of claim 1, wherein the first defensin is present in the topical formulation at a concentration between 1 picogram per milliliter and 100 milligram per milliliter.
6. The method of claim 1, wherein the first defensin is present in the topical formulation at a concentration between 1 and 30 ng/ml.
7. The method of claim 1, wherein the first defensin is encapsulated in a liposome or nanoparticle.
8. The method of claim 8, wherein the first defensin is associated with a protein carrier.
9. The method of claim 10, wherein the protein carrier comprises human serum albumin, recombinant albumin, bovine serum albumin, or egg albumin.
10. The method of claim 1, wherein the topical formulation further comprises a supplement for LGR6+ stem cells, wherein the supplement comprises human serum albumin, bovine serum albumin, egg albumin, plant hydrolysate, beta-cyclodextrin, glutamine, phospholipids, fibronectin, hyaluronate, plant hydrolysate, L-alanyl-Lglutamine, gelatin, vitamin E, recombinant albumin, hyaluronic acid, epidermal growth factor, fibroblast growth factor, recombinant gelatin, Tocopheryl Nicotinate, Coenzyme Q10, ubiquinone, tocopheryl acetate, leuconostoc/radish root ferment filtrate, fibronectin, L-alanyl-L-glutamine, vitamin B complex, carthamus tinctorius (sunflower) oleosomes, C12-15 alkyl benzoate, sodium acyloyldimethyltaurate/VP crosspolymer, resveratrol, retinyl palmitate, ascorbyl palmitate, phenoxyethanol, caprylyl glycol, ethylhexylglycerin, hexylene glycol, panthenol, folic acid, green tea extract, chamomile extract, saccharide isomerate, borago offincinalis seed oil, tocopheryl, saccharomyces lysate extract, carrageenan, or phenoxyethanol.
11. The method of claim 1, wherein the first defensin comprises alpha-defensin 5 or beta-defensin 3.
12. The method of claim 1, wherein the topical formulation further comprises a second defensin.
13. The method of claim 12, wherein the second defensin is different from the first defensin, and wherein the second defensin is present in the topical formulation at a sub-antimicrobially effective concentration.
14. The method of claim 12, wherein the second defensin comprises a synthetic defensin, a human defensin, recombinant defensin, a monkey defensin, a mouse defensin, a rat defensin, a bovine defensin, a sheep defensin, a horse defensin, a rabbit defensin, a swine defensin, a dog defensin, or a cat defensin.
15. The method of claim 12, wherein the second defensin comprises alpha-defensin 1, alpha-defensin 5, alpha-defensin 6, neutrophil defensin 1, neutrophil defensin 2, neutrophil defensin 3, neutrophil defensin 4, theta-defensin, beta-defensin 1, beta-defensin 2, beta-defensin 3, or beta-defensin 4.
16. The method of claim 12, wherein the second defensin is present in the topical formulation at a concentration between 1 picogram per milliliter and 100 milligram per milliliter.
17. The method of claim 12, wherein the second defensin is present in the topical formulation at a concentration between 1 and 30 ng/ml.
18. The method of claim 1, wherein the subject has a skin condition, and wherein the skin condition comprises a decrease in skin hydration, a decrease in skin elasticity, a decrease in skin extensibility, a decrease in skin firmness, skin laxity, skin aging, skin thinning, or a decrease in a barrier property of skin.
19. The method of claim 1, wherein the applying the topical formulation is effective to activate an inflammatory pathway, activate a wound healing pathway, stimulate LGR6+ cell activation, recruit stem cells to replace depleted interfollicular epidermis stem cells, increase the number of interfollicular epidermal stem cells, increase the number of epidermal cells, prevent water loss in skin, improve tight junctions of epithelial cells, stimulate cell migration, stimulate skin collagen structure, reconstruct skin collagen structure, stimulate cell redistribution in an affected area and surrounded tissues, inhibit apoptosis, stimulate angiogenesis, stimulate chemotaxis, stimulate an immune response, or stimulate repair of a cell membrane.
20. The method of claim 1, wherein the subject has psoriasis, eczema, acne, EGRF inhibitor related cutaneous skin toxicities, or sun burned skin.
US17/930,842 2014-06-18 2022-09-09 Stem cell stimulating compositions and methods Pending US20230000750A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US17/930,842 US20230000750A1 (en) 2014-06-18 2022-09-09 Stem cell stimulating compositions and methods

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
US201462014044P 2014-06-18 2014-06-18
PCT/US2015/036049 WO2015195677A1 (en) 2014-06-18 2015-06-16 Stem cell stimulating compositions and methods
US15/383,423 US11491096B2 (en) 2014-06-18 2016-12-19 Stem cell stimulating compositions and methods
US17/815,972 US20220401337A1 (en) 2014-06-18 2022-07-29 Stem cell stimulating compositions and methods
US17/930,842 US20230000750A1 (en) 2014-06-18 2022-09-09 Stem cell stimulating compositions and methods

Related Parent Applications (1)

Application Number Title Priority Date Filing Date
US17/815,972 Continuation US20220401337A1 (en) 2014-06-18 2022-07-29 Stem cell stimulating compositions and methods

Publications (1)

Publication Number Publication Date
US20230000750A1 true US20230000750A1 (en) 2023-01-05

Family

ID=54936047

Family Applications (5)

Application Number Title Priority Date Filing Date
US15/383,423 Active US11491096B2 (en) 2014-06-18 2016-12-19 Stem cell stimulating compositions and methods
US17/815,972 Pending US20220401337A1 (en) 2014-06-18 2022-07-29 Stem cell stimulating compositions and methods
US17/930,842 Pending US20230000750A1 (en) 2014-06-18 2022-09-09 Stem cell stimulating compositions and methods
US17/930,840 Pending US20230000749A1 (en) 2014-06-18 2022-09-09 Stem cell stimulating compositions and methods
US17/930,845 Abandoned US20230000751A1 (en) 2014-06-18 2022-09-09 Stem cell stimulating compositions and methods

Family Applications Before (2)

Application Number Title Priority Date Filing Date
US15/383,423 Active US11491096B2 (en) 2014-06-18 2016-12-19 Stem cell stimulating compositions and methods
US17/815,972 Pending US20220401337A1 (en) 2014-06-18 2022-07-29 Stem cell stimulating compositions and methods

Family Applications After (2)

Application Number Title Priority Date Filing Date
US17/930,840 Pending US20230000749A1 (en) 2014-06-18 2022-09-09 Stem cell stimulating compositions and methods
US17/930,845 Abandoned US20230000751A1 (en) 2014-06-18 2022-09-09 Stem cell stimulating compositions and methods

Country Status (13)

Country Link
US (5) US11491096B2 (en)
EP (2) EP3157504B1 (en)
CY (2) CY1122333T1 (en)
DK (2) DK3586821T3 (en)
ES (2) ES2903108T3 (en)
HR (2) HRP20211983T1 (en)
HU (1) HUE057737T2 (en)
LT (2) LT3586821T (en)
PL (2) PL3586821T3 (en)
PT (2) PT3157504T (en)
RS (1) RS62981B1 (en)
SI (1) SI3586821T1 (en)
WO (1) WO2015195677A1 (en)

Families Citing this family (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DK3586821T3 (en) * 2014-06-18 2022-01-17 Medicell Tech Llc STEM CELL STIMULATING COMPOSITIONS AND METHODS
DK3389620T3 (en) 2015-12-15 2022-12-19 Medicell Tech Llc STEM CELL STIMULATING COMPOSITIONS AND METHODS FOR TREATING MELASMA
US11260013B2 (en) 2016-07-15 2022-03-01 Medicell Technologies, Llc Compositions and methods for providing hair growth
CN109692129B (en) * 2019-01-30 2022-06-21 威海同丰海洋生物科技有限公司 Shaving cream containing defensins and preparation method thereof
CN109718127B (en) * 2019-01-30 2022-08-09 荣成市吉海生物科技有限公司 Nonionic bactericidal anti-inflammatory facial cleanser and preparation method thereof
CN109692130B (en) * 2019-01-30 2022-06-21 威海同丰海洋生物科技有限公司 Mask liquid containing defensins and preparation method thereof
JP6735887B2 (en) * 2019-08-22 2020-08-05 富士フイルム株式会社 Cell senescence inhibitor of mesenchymal stem cells, skin external composition for cell senescence inhibition of mesenchymal stem cells, functional food and drink for cell senescence inhibition of mesenchymal stem cells, and method for inhibiting cell senescence of mesenchymal stem cells
IT202100018542A1 (en) * 2021-07-14 2023-01-14 Clever Bioscience S R L Liposomes containing synergistic antimicrobial combinations based on selected peptides and fatty acids

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090048167A1 (en) * 2005-06-17 2009-02-19 Yitzchak Hillman Disease treatment via antimicrobial peptides or their inhibitors
US20090202458A1 (en) * 2003-12-17 2009-08-13 Avon Products, Inc. si-RNA-Mediated Gene Silencing Technology To Inhibit Tyrosinase And Reduce Pigmentation
DE102009044970A1 (en) * 2009-07-23 2011-01-27 Henkel Ag & Co. Kgaa Use of human beta-defensin to influence the natural pigmentation process
US20130017239A1 (en) * 2010-03-24 2013-01-17 Lipotec S.A. Lipid nanoparticle capsules
US20170157015A1 (en) * 2014-06-18 2017-06-08 Medicell Technologies, Llc Stem cell stimulating compositions and methods
US20210244794A1 (en) * 2015-12-15 2021-08-12 Medicell Technologies, Llc Stem Cell Stimulating Compositions for Treatment of Melasma
US11260013B2 (en) * 2016-07-15 2022-03-01 Medicell Technologies, Llc Compositions and methods for providing hair growth

Family Cites Families (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3689668A (en) * 1967-10-18 1972-09-05 Emile Leon Piette Method of alleviating wrinkles on skin
US5242902A (en) * 1989-09-06 1993-09-07 The Regents Of The University Of California Defensin peptide compositions and methods for their use
FR2767832B1 (en) * 1997-08-29 2001-08-10 Genset Sa DEFENSINE DEFENSINE POLYPEPTIDE DEF-X, GENOMIC DNA AND CDNA, COMPOSITION CONTAINING THEM AND APPLICATIONS TO DIAGNOSIS AND THERAPEUTIC TREATMENT
US6984622B2 (en) * 1998-03-25 2006-01-10 The Regents Of The University Of California Use of lipopolysaccharides to manage corneal infections and wounds
CA2480141A1 (en) * 2002-03-28 2003-10-09 Exponential Biotherapies, Inc. Oxygenating agents for enhancing host responses to microbial infections
DE102005014687A1 (en) * 2005-03-29 2006-10-12 Henkel Kgaa Composition containing β-defensin 2
US7528107B2 (en) * 2005-11-30 2009-05-05 Auburn University Methods of treating inflammation in mammalian tissues comprising administering human alpha-defensins
WO2008133928A2 (en) * 2007-04-27 2008-11-06 The Gi Company, Inc. Mucin glycoproteins and their use for treatment of epithelial lesions and mucin dependent disorders
ES2336995B1 (en) * 2008-10-13 2011-02-09 Lipotec, S.A. COSMETIC OR DERMOPHARMACEUTICAL COMPOSITION FOR SKIN CARE, HAIR LEATHER AND NAILS.
US9034302B2 (en) * 2011-04-11 2015-05-19 L'oreal Mineral sunscreen composition and process for protecting skin from photodamage and aging
WO2012155048A1 (en) * 2011-05-11 2012-11-15 Wisconsin Alumni Research Foundation Liposome-encapsulated hydrogels for use in a drug delivery system
CN103732239A (en) * 2011-06-02 2014-04-16 加利福尼亚大学董事会 Blockade of inflammatory proteases with theta-defensins
US20130310327A1 (en) * 2012-05-18 2013-11-21 Rapid Pathogen Screening, Inc. Histatin for Corneal Wound Healing and Ocular Surface Disease
WO2014004339A2 (en) 2012-06-25 2014-01-03 Elc Management Llc Method for stimulating cellular beta defensins

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090202458A1 (en) * 2003-12-17 2009-08-13 Avon Products, Inc. si-RNA-Mediated Gene Silencing Technology To Inhibit Tyrosinase And Reduce Pigmentation
US20090048167A1 (en) * 2005-06-17 2009-02-19 Yitzchak Hillman Disease treatment via antimicrobial peptides or their inhibitors
DE102009044970A1 (en) * 2009-07-23 2011-01-27 Henkel Ag & Co. Kgaa Use of human beta-defensin to influence the natural pigmentation process
US20130017239A1 (en) * 2010-03-24 2013-01-17 Lipotec S.A. Lipid nanoparticle capsules
US20170157015A1 (en) * 2014-06-18 2017-06-08 Medicell Technologies, Llc Stem cell stimulating compositions and methods
US20210244794A1 (en) * 2015-12-15 2021-08-12 Medicell Technologies, Llc Stem Cell Stimulating Compositions for Treatment of Melasma
US11260013B2 (en) * 2016-07-15 2022-03-01 Medicell Technologies, Llc Compositions and methods for providing hair growth

Also Published As

Publication number Publication date
HRP20191935T1 (en) 2020-05-15
RS62981B1 (en) 2022-03-31
PL3586821T3 (en) 2022-03-14
HUE057737T2 (en) 2022-06-28
EP3586821B1 (en) 2021-12-01
EP3586821A1 (en) 2020-01-01
WO2015195677A1 (en) 2015-12-23
EP3157504A1 (en) 2017-04-26
EP3157504B1 (en) 2019-08-07
US11491096B2 (en) 2022-11-08
PL3157504T3 (en) 2020-03-31
CY1125101T1 (en) 2023-06-09
CY1122333T1 (en) 2021-01-27
US20230000751A1 (en) 2023-01-05
PT3586821T (en) 2022-04-11
LT3586821T (en) 2022-03-25
HRP20211983T1 (en) 2022-08-19
US20170157015A1 (en) 2017-06-08
US20220401337A1 (en) 2022-12-22
LT3157504T (en) 2019-11-11
ES2754523T3 (en) 2020-04-20
PT3157504T (en) 2019-11-11
WO2015195677A4 (en) 2016-01-21
DK3586821T3 (en) 2022-01-17
SI3586821T1 (en) 2022-04-29
EP3157504A4 (en) 2018-01-03
ES2903108T3 (en) 2022-03-31
DK3157504T3 (en) 2019-11-04
US20230000749A1 (en) 2023-01-05

Similar Documents

Publication Publication Date Title
US20230000750A1 (en) Stem cell stimulating compositions and methods
US11806384B2 (en) Stem cell stimulating compositions for treatment of melasma
CN108882951B (en) Moisturizing composition and application thereof
US20190167765A1 (en) Human serum albumin-based topical ointment for treatment of acne, psoriasis, egfr-induced toxicity, premature skin aging and other skin conditions
US12115241B2 (en) Compositions and methods for modifying hair color
KR20070081192A (en) Cosmetic compostion comprising liposome incorporating oryzanol, rice bran oil and phospholipid
KR102333531B1 (en) Cosmetic composition for improving skin barrier comprising high content of beta-sitosterol
JP2023547984A (en) Tissue-derived matrix composition and method thereof
EP2961481B1 (en) Topical antimicrobial dermatological composition
KR102582633B1 (en) Cosmetic composition for skin regeneration and wrinkle improvement containing epidermal growth factor liposomes and growth factor complex
EP2306999B1 (en) Compositions for treating rosacea comprising chitosan and a dicarboxylic acid amide
CN113491643A (en) Antipruritic use of angiogenin
TW201924656A (en) Methods and compositions for modulating melanogenesis
KR20210012350A (en) Artificial layer cream composition having superior moisture retention and skin penetration effect by nanoliposome treatment method
KR20110040768A (en) Use of a human prolactin

Legal Events

Date Code Title Description
AS Assignment

Owner name: MEDICELL TECHNOLOGIES, LLC, CALIFORNIA

Free format text: CONFIRMATORY ASSIGNMENT;ASSIGNORS:TUROVETS, NIKOLAY;ADAMS, WILLIAM B.;SIGNING DATES FROM 20220629 TO 20220710;REEL/FRAME:061433/0637

STPP Information on status: patent application and granting procedure in general

Free format text: NON FINAL ACTION MAILED

STPP Information on status: patent application and granting procedure in general

Free format text: RESPONSE TO NON-FINAL OFFICE ACTION ENTERED AND FORWARDED TO EXAMINER

STPP Information on status: patent application and granting procedure in general

Free format text: FINAL REJECTION MAILED

STPP Information on status: patent application and granting procedure in general

Free format text: DOCKETED NEW CASE - READY FOR EXAMINATION

STPP Information on status: patent application and granting procedure in general

Free format text: NON FINAL ACTION MAILED

STPP Information on status: patent application and granting procedure in general

Free format text: RESPONSE TO NON-FINAL OFFICE ACTION ENTERED AND FORWARDED TO EXAMINER

STPP Information on status: patent application and granting procedure in general

Free format text: FINAL REJECTION MAILED

STPP Information on status: patent application and granting procedure in general

Free format text: DOCKETED NEW CASE - READY FOR EXAMINATION