WO2008070368A2 - Procédés et compositions pour le soin de la peau - Google Patents

Procédés et compositions pour le soin de la peau Download PDF

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Publication number
WO2008070368A2
WO2008070368A2 PCT/US2007/083352 US2007083352W WO2008070368A2 WO 2008070368 A2 WO2008070368 A2 WO 2008070368A2 US 2007083352 W US2007083352 W US 2007083352W WO 2008070368 A2 WO2008070368 A2 WO 2008070368A2
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WIPO (PCT)
Prior art keywords
skin
elastase
composition
oil
enzyme
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PCT/US2007/083352
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English (en)
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WO2008070368A3 (fr
Inventor
Betty Yu
Rox Anderson
Robert S. Langer
Yushan Kim
Daniel G. Anderson
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Living Proof, Inc.
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Publication of WO2008070368A2 publication Critical patent/WO2008070368A2/fr
Publication of WO2008070368A3 publication Critical patent/WO2008070368A3/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
    • A61K38/482—Serine endopeptidases (3.4.21)
    • A61K38/486—Elastase (3.4.21.36 or 3.4.21.37)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
    • A61K8/66—Enzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A61Q19/08—Anti-ageing preparations

Definitions

  • the skin is the primary interface between the body and the environment and plays an essential protective role, shielding the body from injury, pathogenic agents, the sun, extremes of temperature, and other environmental factors.
  • the skin provides thermoregulation, serves as a permeability barrier, and functions as a sensory organ for touch, pressure, pain, itch, and temperature.
  • Skin is composed of two major layers: the epidermis and the underlying dermis, which are distinct in terms of their architecture, physiology, and function.
  • the epidermis is a stratified epithelium composed of four layers: the stratum basale, stratum spinosum, stratum granulosum, and the outermost stratum corneum.
  • the stratum basale contains a single layer of cuboidal keratinocytes attached to a basement membrane. Above this layer is the spinous layer, characterized by presence of numerous desmosomes.
  • the stratum granulosum overlies the stratum spinosum and consists of keratinocytes that contain basophilic granules of keratohyalin as well as lamellar granules in the intercellular compartment.
  • the stratum corneum is the most superficial layer and is composed of anucleated, flattened, fully keratinized cells (corneocytes) fused together to form a plate-like structure.
  • the intercellular space is occupied by ordered lipid lamellae that contain specialized proteins and lipids, such as ceramides, fatty acids, and cholesterols, which are secreted from lamellar bodies in the stratum granulosum.
  • the resulting "bricks and mortar” structure provides the stratum corneum with the ability to perform its protective and moisture retaining functions.
  • the thickness of the epidermis ranges from about 75 to 150 ⁇ m except on the soles and palms, where it is about 0.4 to 0.6 mm.
  • the dermoepidermal junction (DEJ) is an undulating basement membrane composed primarily of collagen that separates the epidermis from the dermis.
  • the dermis is a dense, fibroelastic connective tissue that lies beneath the epidermis and provides a strong and flexible supporting layer. It is composed of cells ⁇ e.g., fibroblasts), ground substance, and a fibrous network containing collagenous and elastic fibers and also contains blood vessels, nerves, hair follicles, smooth muscle, glands and lymphatic tissue. Collagen, primarily types I, III, V, and VI, forms the majority of the fibrous component, making up about 75% of the dry weight of the dermis and imparting firmness and tensile strength.
  • Elastin accounts for about 2-4% of dermal proteins and, together with a variety of elastin-associated proteins, forms an interconnecting network of insoluble elastic fibers that are interwoven among the collagen bundles and provide normal skin with its elasticity and resilience.
  • the major component of the mature fiber is a covalently cross-linked polymer of the protein elastin. Elastin is secreted from the cell as a soluble monomer called tropoelastin. In the extracellular space, lysine residues within tropoelastin are specifically modified to form covalent cross-links between tropoelastin chains.
  • This cross-linked polymer has a high degree of reversible distensibility, including the ability to deform to large extensions with small forces.
  • Tropoelastin is composed primarily of the amino acids alanine, valine, glycine, pro line, and lysine.
  • the gene encoding tropoelastin has been cloned from a variety of different organisms including human, bovine, and chick (reviewed in Indik et ah, Am J Med Genet., 34(l):81-90, 1989).
  • the tropoelastin molecule is characterized by a series of tandem repeats, each of which includes a lysine-containing cross-linking region followed by a hydrophobic motif (Indik, supra).
  • Cross-linking is initiated by the extracellular enzyme(s) lysyl oxidase, which catalyzes the oxidative deamination of lysyl ⁇ -amino groups. Subsequent reactions lead to formation of the tetrafunctional cross-links desmosine and isodesmosine. It has long been believed that during development, fibrillin rich microfibrils provide a scaffold or template for elastin assembly by binding and aligning tropoelastin monomers so that lysine-containing regions are in register for cross-linking (Kozel et al., J. Biol. Chem., 278 (20) : 18491-18498 , 2003).
  • the resulting fibers have an outer microfibrillar mantle and an inner core of amorphous, cross-linked elastin.
  • the microfibrillar component contains a variety of proteins such as fibrillin- 1, -2, and -3, micro fibrillar-associated proteins -1, -3, and -4, and latent transforming growth factor ⁇ (TGF- ⁇ ) binding proteins. Further information about elastin and its structure is found in Debelle L. and Tamburro A.M., Int. J. Biochem. Cell Biol. 31 : 261-272, 1999 and in Keilty ⁇ ⁇ /., J. Cell. ScL, 115: 2817-2828, 2001. [0006] The dermis can be divided into two regions.
  • the papillary dermis conforms to the shape of the overlying epidermis.
  • the reticular dermis lies below the papillary dermis and forms the majority of the dermal layer, giving it most of its elasticity and strength.
  • Elastic fibers of the papillary dermis are oriented parallel (elaunin fibers) or perpendicular (oxytalan fibers) to the DEJ and are thinner than the elastic fibers of the reticular dermis.
  • Oxytalan fibers lack the elastin core while elaunin fibers contain a small amount of elastin.
  • Mature elastin fibers are found primarily arranged in bundles in the reticular dermis and measure about 1-3 ⁇ m in diameter.
  • the present invention provides compositions and methods of caring for skin, in particular photodamaged, prematurely aged, and/or aged skin.
  • skin is contacted with a composition comprising one or more enzymes that reduce evidence of elastotic material, accumulation of which is typically associated with sun exposure.
  • the dermis is contacted with the composition. Signs and/or the appearance of photodamage or premature aging are thereby diminished.
  • the compositions may be topically applied to improve the condition or appearance of human skin.
  • the compositions are injected into the skin.
  • the invention provides a method for diminishing signs of photodamage or aging comprising the step of: contacting skin with an effective amount of one or more elastases that individually or in combination reduce evidence of elastotic material, whereby signs or appearance of photodamage or aging are diminished.
  • the effective amount is sufficient to reduce histopathologic evidence of elastotic material in a sample of heavily photodamaged skin by at least 0.5% (e.g., between 0.5% and 1%), by at least 1% (e.g., between 1% and 5%), by at least 5% (e.g., between 5% and 10%), by at least 10% (e.g., between 10% and 25%), by at least 25% (e.g., between 25% and 50%), at least 50% (e.g., between 50% and 77%), at least 75% (e.g., between 75% and 90%), or more (e.g., between 90% and 100%) within a time period of 4 hours or less.
  • an effective amount is sufficient to reduced elastotic material in skin by at least 1% (e.g., between 1% and 5%), by at least 5% (e.g., between 5% and 10%), by at least 10% (e.g., between 10% and 25%), by at least 25% (e.g., between 25% and 50%), at least 50% (e.g., between 50% and 77%), at least 75% (e.g., between 75% and 90%), or more (e.g., between 90% and 100%) over an extended time period of 1 month, 2 months, or 6 months with repeated treatements (e.g., daily, weekly, every other week).
  • desmosine release may be used as a measure of elastin digestion.
  • the composition is applied to the surface of the skin. In certain embodiments, the composition is injected into the skin. For example, daily use of the treatment with elastase may reduce the elastotic material by at least 0.0025% within a 24 hour period.
  • the elastase is a mammalian elastase (e.g., human elastase, porcine elastase). In certain embodiments the elastase is a bacterial elastase, e.g., Pseudomonas elastase.
  • a composition comprising one or more elastases is topically applied.
  • the composition is topically applied for between 15 minutes and 24 hours, e.g., between 30 and 240 minutes.
  • the composition is topically applied to an area of skin from which at least a portion of the epidermis or stratum corneum has been removed, e.g., wherein 1, 2, 3, or all four layers of the epidermis have been removed.
  • the composition is topically applied to an area of skin from which at least part of the papillary dermis has been removed.
  • two or more compositions, each comprising an elastase enzyme are topically applied.
  • the skin care composition further comprises one or more compounds that have skin restoring properties.
  • the skin is contacted with a skin care composition having skin restoring properties or a physical treatment having skin restoring properties is performed.
  • the method comprises the step of removing at least a portion of the epidermis of an area of skin prior to contacting the dermis with the composition. Removal may be accomplished using, e.g. , dermabrasion, microdermabrasion, laser treatment, tape stripping, or by applying a chemical peeling agent to the surface of the skin. In certain embodiments the step of removing comprises removing the stratum corneum to a depth of at least 50% of its thickness, or at least 90% of its thickness. In certain embodiments the step of removing comprises removing at least the upper three layers of the epidermis. In certain embodiments the step of removing comprises removing at least a portion of the papillary dermis.
  • any of the methods further comprises contacting the skin with an inhibitor of at least one of the enzymes after the skin has been contacted with said enzyme for a suitable period of time. In certain embodiments any of the methods further comprises contacting the skin with an inhibitor of one or more endogenous enzymes. In certain embodiments of the invention the inhibitor inhibits an endogenous metalloproteinase, elastase, or collagenase.
  • the invention provides a method for improving the appearance of photodamaged or aged skin comprising the step of: contacting skin with a composition comprising an effective amount of one or more enzymes that individually or in combination diminishes evidence of elastotic material, whereby the appearance of the photodamaged or aged skin is improved.
  • the invention provides a method of caring for photodamaged or aged skin comprising the step of: contacting the dermis of skin with a composition comprising an effective amount of one or more enzymes that individually or in combination diminish evidence of elastotic material.
  • the invention provides a method of caring for skin comprising the step of: contacting the skin with a composition comprising an effective amount of one or more elastases that individually or in combination inhibits accumulation of elastotic material, whereby development of signs of photodamage or aging is inhibited.
  • the invention provides a method of improving the appearance of a subject's skin comprising steps of: optionally, removing at least a portion of the epidermis; and contacting the skin with a composition comprising an effective amount of one or more enzymes that individually or in combination have sufficient activity to reduce evidence of elastotic material.
  • the skin is photodamaged.
  • the skin is aged.
  • the skin is prematurely aged.
  • the subject is between 20 and 90 years of age. In certain embodiments of any of the inventive methods, the subject is between 20 and 30 years of age. In certain embodiments of any of the inventive methods, the subject is between 30 and 40 years of age.
  • the subject is between 40 and 50 years of age. In certain embodiments of any of the inventive methods, the subject is between 50 and 60 years of age. In certain embodiments of any of the inventive methods, the subject is between 60 and 70 years of age. In certain embodiments of any of the inventive methods, the subject is between 70 and 80 years of age. In certain embodiments of any of the inventive methods, the subject is between 80 and 90 years of age.
  • the invention provides a cosmetic skin care composition in the form of a cosmetically acceptable gel, lotion, ointment, paste, or cream, comprising: an effective amount of one or more elastase enzymes that diminish evidence of elastotic material following contact with skin; and a dermatologically acceptable carrier.
  • a cosmetic skin care composition in the form of a cosmetically acceptable gel, lotion, ointment, paste, or cream, comprising: an effective amount of one or more elastase enzymes that diminish evidence of elastotic material following contact with skin; and a dermatologically acceptable carrier.
  • at least one of the elastase enzyme(s) displays selective activity towards elastotic material.
  • the cosmetic skin care composition further comprises at least one, two, or three ingredients selected from the group consisting of: polyhydric alcohols, osmoprotectants, elastase inhibitors, pH regulating agents, oils, emollients, surfactants, vitamins, colorants, polymers, antioxidants, preservatives, chelating agents, proteins, amino acids, plant extracts, sunscreen agents, humectants, thickening agents, emulsifiers, and fragrances.
  • polyhydric alcohols osmoprotectants, elastase inhibitors, pH regulating agents, oils, emollients, surfactants, vitamins, colorants, polymers, antioxidants, preservatives, chelating agents, proteins, amino acids, plant extracts, sunscreen agents, humectants, thickening agents, emulsifiers, and fragrances.
  • the invention provides a cosmetic skin care composition in the form of a cosmetically acceptable gel, cream, ointment, lotion, or paste, the composition comprising: an at least partially purified bacterial elastase in an amount effective to diminish evidence of elastotic material when contacted with photodamaged skin; and a cosmetically acceptable carrier.
  • the elastase is a bacterial elastase.
  • the bacterial elastase is PA elastase.
  • the elastase is a mammalian elatase.
  • the elastase is a human elastase.
  • the elastase is human neutrophil elastase. In certain embodiments, the elastase is a porcine elastase.
  • the composition may further comprise at least one, two, or three ingredients selected from the group consisting of: polyhydric alcohols, osmoprotectants, elastase inhibitors, pH regulating agents, oils, emollients, surfactants, vitamins, colorants, polymers, antioxidants, preservatives, chelating agents, proteins, amino acids, plant extracts, sunscreen agents, humectants, thickening agents, emulsifiers, and fragrances.
  • the invention provides a pharmaceutical composition, the composition comprising: an at least partially purified elastase in an amount effective to diminish evidence of elastotic material when injected into skin; and a pharmaceutically acceptable carrier.
  • the elastase is a bacterial elastase.
  • the bacterial elastase is PA elastase.
  • the elastase is a mammalian elatase.
  • the elastase is a human elastase.
  • the elastase is human neutrophil elastase.
  • the elastase is a porcine elastase.
  • the pharmaceutical composition may further comprise at least one, two, or three ingredients selected from the group consisting of: polyhydric alcohols, osmoprotectants, elastase inhibitors, pH regulating agents, oils, emollients, surfactants, vitamins, colorants, polymers, antioxidants, preservatives, chelating agents, proteins, amino acids, plant extracts, sunscreen agents, humectants, thickening agents, emulsifiers, and fragrances.
  • the pharmaceutical composition may be suitable for subcutaneous delivery.
  • the pharmaceutical composition may be suitable for transdermal delivery.
  • the pharmaceutical composition is an injectable composition.
  • the invention provides a method of selecting an enzyme with desirable properties for a skin care composition comprising steps of: (a) contacting each of a plurality of enzymes with elastotic material and with elastin not derived from skin that has been subjected to photodamage; and (b) assessing the degradative activity of each enzyme towards elastotic material relative to its degradative activity towards elastin not derived from skin subjected to photodamage; and (c) selecting an enzyme that displays a greater selectivity for elastotic material versus elastin not derived from skin that has been subjected to photodamage than at least some of the other enzymes as a preferred enzyme for a skin care composition.
  • the invention provides a method of selecting an enzyme with desirable properties for a skin care composition comprising steps of: (a) contacting each of a plurality of enzymes with elastotic material and with elastin not derived from aged skin; and (b) assessing the degradative activity of each enzyme towards elastotic material relative to its degradative activity towards elastin not derived from aged skin; and (c) selecting an enzyme that displays a greater selectivity for elastotic material versus elastin not derived from aged skin than at least some of the other enzymes as a preferred enzyme for a skin care composition.
  • the invention provides a method of engineering an enzyme with desirable properties for a skin care composition comprising steps of: (a) making one or more amino acid changes to the sequence of a naturally occurring elastolytic enzyme, thereby producing a modified elastolytic enzyme; (b) comparing the degradative activity of the modified elastolytic enzyme towards elastotic material with its degradatives activity towards elastin that has not been subjected to photodamage or aging; and (c) selecting the enzyme as a preferred enzyme for a skin care composition if it displays enhanced selectivity towards elastotic material relative to that of the naturally occurring elastolytic enzyme.
  • the enzyme may be chemically modified.
  • the enzyme may be PEGylated or conjugated to another polymer.
  • the enzyme may be genetically engineered.
  • the invention makes use of standard methods of molecular biology, chemistry, immunohistochemistry cosmetic formulation techniques, and dermatologic evaluation and uses art-accepted meanings of terms. Techniques and materials useful in certain aspects of the invention are found in Ausubel, F., (ed.), Current Protocols in Molecular Biology, Current Protocols in Immunology, Current Protocols in Protein Science, and Current Protocols in Cell Biology, all John Wiley & Sons, N.
  • Figure IA shows a section of photodamaged human cheek skin that was exposed to 200 mM Tris buffer (pH 8.8) with no elastase for 24 hrs at 37 0 C and then stained for elastin using Verhoeff van Gieson (VVG) stain (4x magnification).
  • Figure IB shows a section of photodamaged human cheek skin that was exposed to 250 ⁇ g/ml Pseudomonas aeruginosa elastase (PAE) in 200 mM buffer (pH 8.8) for 24 hrs at 37 0 C and then stained for elastin using VVG stain (4x magnification).
  • Figure 1C shows a portion of the section of Figure IA at 10x magnification.
  • Figure ID shows a portion of the section of Figure IB at 10x magnification.
  • Figures IE consisting of upper and lower panels on left
  • IF consisting of upper and lower panels on right
  • Figure 2 is a graph that confirms the elastase activity of Ps eudomonas aeruginosa elastase preparations under the same conditions of pH and temperature (pH 8.8, 37 0 C) that were used to demonstrate the ability of elastase to degrade elastotic material in photodamaged skin sections (see Figures 1 A-IF).
  • Figure 3 A shows a punch of photodamaged human cheek skin that was exposed to 20 ⁇ L of 30 mM Tris buffer (pH 7.2) containing 50 ⁇ g/ml Pseudomonas aeruginosa elastase for 6 hours at 37 0 C after it was heat-stripped and then stained for elastin using VVG stain (4X magnification).
  • Figures 3B, 3C, and 3D show portions of the section of Figure 3 A at 10x, 4Ox, and 4Ox magnification, respectively.
  • Figure 3E shows a section of heat-stripped photodamaged human skin that was exposed to buffer solution for 6 hrs at 37 0 C and then stained for elastin using VVG stain (4X magnification).
  • Figures 3F and 3G show portions of the section of Figure 3 E at 1OX and 4OX magnification respectively.
  • the red scale bar indicates 100 microns. Note the dense staining for elastin in the samples exposed to buffer without elastase.
  • Figure 4 A shows a punch of photodamaged human skin from the chin that was treated with a 20 ⁇ l intradermal injection of 30 mM Tris buffer (pH 7.2) with no elastase, exposed to buffer with no elastase for 6 hours at 37 0 C, and then stained for elastin using VVG stain (4x magnification).
  • Figure 4B shows a portion of the same section as Figure 4 A at 4Ox magnification.
  • Figure 4C shows a punch of photodamaged human skin from the chin that was treated with a 20 ⁇ l intradermal injection of 30 mM Tris buffer (pH 7.2) buffer with 50 ⁇ g/ml Pseudomonas aeruginosa elastase, exposed to buffer with no elastase for 6 hours at 37 0 C, and then stained for elastin using VVG stain (4x magnification).
  • the red scale bar indicates 100 microns.
  • Figure 5 is a graph that confirms the elastase activity of Pseudomonas aeruginosa elastase preparations under the same conditions of pH and temperature (pH 7.2, 37 0 C) that were used to demonstrate the ability of elastase to degrade elastotic material when topically applied to photodamaged skin from which the epidermis was removed (see Figures 3A-3G and Figures 4A-4C).
  • Figure 6 shows the activity of porcine pancreatic elastase over time in the presence of common cosmetic ingredients.
  • Figure 7 shows the activity of porcine pancreatic elastase (PPE) over 4 hours at 37 0 C in Carbopol, sorbitol, glycerin, and control formulations.
  • Figure 8 shows the activity of porcine pancreatic elastase (PPE) over 19 hours at
  • Figure 9 shows the in situ activity of the Carbopol formulation, (a) PPE formulation, 4x objective lens, (b) PPE formulation, 4Ox. (c) Placebo formulation, 4x. (d)
  • Figure 10 shows the in situ activity of the sorbitol formulation, (a) PPE formulation, 4x objective lens, (b) PPE formulation, 10x. (c) Placebo formulation, 4x. (d)
  • Figure 11 shows the in situ activity of the glycerin formulation, (a) PPE formulation, 4x objective lens, (b) PPE formulation, 4Ox. (c) Placebo formulation, 4x. (d)
  • Figure 12 shows the activity of Ps eudomonas aeruginosa elastase (PAE) over time at varying concentrations at pH 8.8.
  • PAE Ps eudomonas aeruginosa elastase
  • Figure 13 shows the activity of Ps eudomonas aeruginosa elastase (PAE) over time at varying concentrations at pH 7.2.
  • Figure 14 shows the activity of Ps eudomonas aeruginosa elastase (PAE) over time at varying concentrations at pH 5.0.
  • PES Ps eudomonas aeruginosa elastase
  • Figure 15 shows the reaction velocity of PAE at different pH's at two different concentrations.
  • Figure 16 shows the activity of porcine pancreatic elastase (PPE) over time at varying concentrations at pH 8.8.
  • PPE porcine pancreatic elastase
  • Figure 17 shows the activity of porcine pancreatic elastase (PPE) over time at varying concentrations at pH 7.2.
  • PPE porcine pancreatic elastase
  • Figure 18 shows the reaction velocity of human neutrophil elastase (HNE) at different pH's at three different concentrations.
  • Figure 19 shows human skin sections treated with varying concentrations of porcine pancreatic elastase (PPE) over varying durations and stained with Verhoeff-Van
  • Gieson stain a stain for elastic fiber.
  • Figure 2OA shows human skin injected with varying concentrations of PPE and treated over varying exposure durations. The sections were stained with Verhoeff-Van
  • Figure 21 shows photomicrographs from tape stripping treatment followed by topical application of elastase on human facial skin.
  • Figure 21 A shows normal SC and epidermis with H&E staining (10Ox magnification).
  • Figure 2 IB shows normal SC and epidermis with VVG staining of elastin fibers (10Ox magnification).
  • Figure 21C shows SC and epidermis treated with elastase with H&E staining (10Ox magnification).
  • Figure 2 ID shows SC and epidermis treated with elatase with VVG staining (10Ox magnification).
  • Figure 2 ID shows a minor reduction of aberrant elastin fibers in the papillary dermis. Fibers were unchanged in the reticular dermis.
  • Aged refers to a variety of alterations in the appearance and structure of the skin that occur as a consequence of aging which may be more severe the older skin is. These alterations include wrinkles ⁇ e.g., folds, furrows, or creases in the skin), alterations and irregularities in pigmentation ⁇ e.g., age spots, sallowness), pebbly texture and appearance, increased roughness and dryness, reduced skin tone and elasticity, accumulation of elastotic material, loss of collagen, etc. It will be appreciated that not all of these changes need be present in order for an individual's skin to be considered aged. In certain embodiments, aged may refer to prematurely aged skin, that is, skin that looks and or feels older than what would be typical for skin of that chronological age.
  • the term "in combination” or “in combination with” when referring to the use of two or more agents, e.g., two or more enzymes encompasses situations in which the agents are present in the same composition, situations in which the agents are present in different compositions that are contacted with the skin either during all or part of the same time period, and situations in which the agents are present in different compositions that are contacted with the skin sequentially but within 24 hours of each other optionally with an intervening step of removing or neutralizing one or more of the compositions or agents.
  • the terms “approximately” or “about” in reference to a number generally include numbers that fall within a range of 5% in either direction (greater than or less than) of the number unless otherwise stated or otherwise evident from the context ⁇ e.g., where such number would impermissibly exceed 100% of a possible value).
  • the term "contacting” is intended to encompass any manner of locally providing, delivering, or making available to the skin, e.g., to the dermis, and excludes such systemic routes of administration as intravenous, pulmonary, and oral. Methods of contacting include, without limitation, injection into the skin (e.g., intradermal injection), and topical application to a skin surface (optionally following partial or complete removal of superficially located skin layer(s) as described herein).
  • compositions or substances that improves, preserves, restores, or enhances the appearance of the body, especially that of the skin, e.g., that beautifies or promotes attractiveness or youthful appearance.
  • a composition or substance is administered for "cosmetic purposes” if it is administered in order to improve, preserve, restore, promote, or enhance the appearance of the body, e.g., to beautify or promote attractiveness or youthful appearance.
  • Cosmetically acceptable means that the component or composition to which the term applies is suitable for contact with the skin of humans and, preferably, that of other mammals without undue toxicity or damage.
  • “Pharmaceutically acceptable”, as used herein, means that the component or composition to which the term applies is suitable for use in humans and, preferably, other mammals, without undue toxicity or damage.
  • an effective amount means an amount of a compound or composition sufficient to result in a benefit, such as an improved skin appearance and/or reduced amount of photodamage, including independently or in any combination the benefits described herein.
  • an effective amount is also "safe", i.e., low enough to avoid serious side effects, i.e., to provide a reasonable benefit to risk ratio, within the scope of sound judgment of the skilled artisan such as a cosmetologist, aesthetician, dermatologist or other clinician, etc.
  • elastase refers to an enzyme that possesses the ability to solubilize mature, cross-linked elastin under suitable conditions of pH, temperature, solvent, etc., such conditions being ones compatible with stability of mature, cross-linked elastin, e.g., physiological conditions. Suitable solvents include aqueous solutions such as water or physiological saline. Elastases include, but are not limited to, any enzyme referred to as an elastase herein and other enzymes known in the art to be capable of solubilizing mature, cross-linked elastase. The term further encompasses any enzyme that possesses equivalent ability to solubilize mature, cross-linked elastin as does a known elastase.
  • elastolytic enzyme and "elastase” are used interchangeably herein. At times the term “elastolytic enzyme” is used to refer to an enzyme that has the ability to solubilize mature, cross-linked elastin but that is not typically referred to in the art as an "elastase", e.g., it has acquired a name in the art indicative of ability to hydrolyze a different substrate.
  • a "partial elastase” is an enzyme that possesses the ability to cleave one or more cross-links or peptide bonds in mature, cross-linked elastin but does not possess the ability to solubilize mature, cross-linked elastin under physiological conditions.
  • Partial elastases include, but are not limited to, any enzyme referred to as a partial elastase herein, and other enzymes that possess equivalent ability to cleave one or more cross-links or peptide bonds in mature, cross-linked elastin. It will be appreciated that an elastase or a partial elastase need not be specific for elastin but may, and often does, possess the ability to hydrolyze other proteins.
  • the term “elastase” does not include trypsin.
  • the term “elastase” does not include chymotrypsin.
  • the term “elastase” does not include papain.
  • the term “elastase” does not include bromelain. In certain embodiments, the term “elastase” does not include subtilisin.
  • the term “elastotic material” is used herein consistently with its meaning in the art, to refer to material that stains positively for elastin (e.g., using antibodies to elastin or a variety of art-recognized stains for elastin such as the Verhoeff van Gieson stain). In certain embodiments, elastotic materials accumulates in the dermis of skin that has been chronically exposed to the sun.
  • “Mature, cross-linked elastin” refers to insoluble elastin composed of molecules of tropoelastin cross-linked via isodesmosine and desmosine cross-links to form a material that is insoluble in most typical solvents, e.g., aqueous solutions such as water or physiological saline, or organic solvents, across a broad pH range. Structural and physical properties of mature, cross-linked elastin have been extensively characterized (Debelle, L., et al, Eur J Biochem., 258(2):533-9, 1998; Debelle, L., Alix, A.J., J. Biochimie, 81(10):981-94, 1999, and references therein).
  • Photodamage refers to a variety of alterations in the appearance and structure of the skin that occur as a consequence of exposure to the sun, and which may be particularly severe if the skin has been experienced significant and extended exposure. These alterations include wrinkles (e.g., folds, furrows, or creases in the skin), alterations and irregularities in pigmentation (e.g., age spots, sallowness), pebbly texture and appearance, increased roughness and dryness, reduced skin tone and elasticity, accumulation of elastotic material, loss of collagen, etc. It will be appreciated that not all of these changes need be present in order for an individual's skin to be considered photodamaged. The severity of photodamage will vary depending on a variety of factors such as amount of sun exposure, age, genetic makeup, health, and habits of the individual, exposure to other environmental conditions such as dry air, pollution, cigarette smoke, etc.
  • wrinkles e.g., folds, furrows, or creases in the skin
  • alterations and irregularities in pigmentation e.g
  • Purified means that a substance is separated from one or more other entities or substances with which it was previously found before being purified.
  • a substance may be partially purified, substantially purified, or pure.
  • a substance such as a nucleic acid or polypeptide is considered pure when it is removed from substantially all other substances other than a solvent and any ions contained in the solvent, i.e., it constitutes at least about 90%, more preferably at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater than 99% of the dry weight of the composition.
  • a partially or substantially purified substance may be removed from at least 50%, at least 60%, at least 70%, or at least 80% by dry weight of the material with which it is found before being purified, other than a solvent and any ions contained in the solvent.
  • a purified protein constitutes at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or even more, by dry weight, of the total protein in a composition.
  • Methods for assessing purity are known in the art and include chromatographic methods, immunological methods, electrophoretic methods, etc. Any of the proteins described herein may be purified.
  • skin care composition refers to any product that is contacted with the skin, optionally after partial or complete removal of one or more layers of the epidermis and, where appropriate, partial or complete removal of the upper layer of the dermis.
  • To "solubilize” means to allow or cause a substance to dissolve, i.e., to allow or cause it to go into solution.
  • subject and “individual” are used interchangeably herein to refer to an individual whose skin is contacted with a composition in accordance with the present invention.
  • Preferred subjects are mammals such as humans or other mammals with skin that is susceptible to photodamage.
  • Topical application means to apply or transfer a composition onto the skin, optionally after partial or complete removal of one or more layers of the epidermis and, where appropriate, partial or complete removal of the upper layer of the dermis.
  • Topical application may be accomplished using the hands, gauze, cotton swabs, foam applicators, spatulas, brushes, or other spreading implements, or any other convenient material or device. Any conventionally used method of topical application may be employed.
  • a composition may, for example, be spread, dabbed, smoothed, wiped, or sprayed onto the skin.
  • These signs include, but are not limited to, the development of textural discontinuities such as wrinkles (e.g., fine wrinkles and/or coarse deep wrinkles), furrows, creases, skin lines, crevices, bumps, large pores (e.g., associated with adnexal structures such as sweat gland ducts, sebaceous glands, or hair follicles), unevenness or roughness, loss of skin elasticity, sagging, loss of skin firmness, loss of skin tightness, loss of skin recoil from deformation, discoloration, blotching, sallowness, hyperpigmented skin regions such as age spots and freckles, keratoses, telangiectasia, spider vessels, etc.
  • wrinkles e.g., fine wrinkles and/or coarse deep wrinkles
  • furrows creases
  • skin lines crevices, bumps
  • large pores e.g., associated with adnexal structures such as sweat gland ducts, sebaceous glands
  • Visible signs of aging include, but are not limited to, all outward visible and/or tactilely perceptible manifestations, as well as any other macroscopic or microscopic changes attributable at least in part to aging of skin. These signs include, but are not limited to, the development of textural discontinuities such as wrinkles (e.g., fine wrinkles and/or coarse deep wrinkles), furrows, creases, skin lines, crevices, bumps, large pores (e.g., associated with adnexal structures such as sweat gland ducts, sebaceous glands, or hair follicles), unevenness or roughness, loss of skin elasticity, sagging, loss of skin firmness, loss of skin tightness, loss of skin recoil from deformation, discoloration, blotching, sallowness, hyperpigmented skin regions such as age spots and freckles, keratoses, telangiectasia, spider vessels, etc. II. Overview
  • Photodamage may also be associated with a variety of functional abnormalities such as reduced resistance of the skin to trauma, decrease in immune competence, sensory decrease, easy distensibility and delayed return to normal contour, etc.
  • the most prominent histopathologic sign of photodamage is the accumulation of so-called elastotic material in the dermis.
  • the fibrillary elastic fibers found in sun-protected skin are replaced by large quantities of abnormal, thickened, disorganized fibers and amorphous masses that stain positively for elastin and various elastin-associated proteins (Suwabe, H., et al, Path.
  • elastotic material as a consequence of sun exposure is also referred to as "solar elastosis”.
  • the process by which elastotic material is formed remains unclear but may involve degradation of existing elastic fibers and possibly dysregulation of elastin and fibrillin synthesis and/or modification (Sellheyer, K., J. Cutaneous Path., 30:123-127, 2003). Histochemically, depletion of intact microfibrils and elastic fibers has been observed in photodamaged skin. Degradation of elastic fibers may be promoted by elastin degrading enzymes produced by dermal fibroblasts and/or by neutrophils that infiltrate sun-exposed skin.
  • the present invention is based in part on the inventors' discovery that elastotic material found in photodamaged skin is susceptible to removal by enzymes that degrade normal, i.e., non-photodamaged, elastin. As described in more detail in the Examples below, injection or topical administration of a composition containing elastase resulted in dramatic reduction in the amount of elastotic material evident in photodamaged skin. This discovery opens the way for a new generation of skin care compositions effective to reduce signs of photodamage and/or to inhibit development or progression of signs of photodamage.
  • compositions and methods of the present invention will be to reduce the visible signs of photodamage and/or to inhibit development or progression of signs of photodamage, they may also be used to ameliorate elastosis in a variety of different conditions not necessarily associated with exposure to the sun (e.g., aging).
  • Other conditions associated with increased accumulation and/or elastotic degradation of dermal elastic fibers include elastoderma, linear focal elastosis, late-onset focal dermal elastosis, acquired pseudoxanthoma elasticum, and Favre-Racouchot syndrome (Lewis, K.G, et al., J. Am. Acad. Dermatol., 51 :1-21, 2004).
  • compositions and methods may also reduce and/or inhibit the signs of aging in skin.
  • the compositions may thus be used for cosmetic or therapeutic purposes.
  • the compositions are contacted with human skin, particularly the skin of the face (e.g., forehead, cheeks, eye area, nose, mouth area, chin), ears, neck, shoulders, arms, back of the hands, or other areas that are frequently exposed to the sun.
  • the enzyme(s) may degrade elastotic material to smaller fragments. These fragments may be further degraded, their components absorbed, or may be rinsed away.
  • the invention provides a method for diminishing signs of photodamage or aging comprising the step of: contacting the skin with an effective amount of one or more enzymes that individually or in combination reduce evidence of elastotic material, whereby signs of photodamage or aging are diminished.
  • the invention provides a method for diminishing signs of photodamage or aging comprising the step of: contacting the skin with a composition comprising an effective amount of two or more enzymes that in combination are capable of solubilizing mature, cross-linked elastin, wherein neither enzyme is an elastase, whereby signs of photodamage or aging are diminished.
  • the invention provides a method comprising the step of contacting skin with an effective amount of one or more enzymes that individually or in combination reduce evidence of elastotic material, whereby the appearance of the skin is improved.
  • the invention provides a method for diminishing signs of photodamage, aging, or other skin damage comprising the step of: contacting skin with an effective amount of two or more enzymes that in combination are capable of solubilizing mature, cross-linked elastin, wherein neither enzyme is an elastase, whereby the appearance of the skin is improved.
  • the invention provides a method for inhibiting development or progression of photodamage or aging comprising the step of contacting the skin with an effective amount of one or more enzymes that individually or in combination inhibit accumulation of elastotic material, whereby the appearance of the skin is preserved.
  • the invention provides a method for inhibiting development or progression of signs of photodamage or aging comprising the step of: contacting skin with an effective amount of two or more enzymes that in combination are capable of solubilizing mature, cross-linked elastin, wherein neither enzyme is an elastase, whereby the appearance of the skin is preserved.
  • the invention provides a method for treating skin comprising contacting photodamaged or aged skin with an effective amount of one or more enzymes that degrades elastotic material.
  • the one or more enzymes may be present in a single composition or may be present in two or more compositions.
  • the invention provides a cosmetic skin care composition comprising an effective amount of one or more enzymes that individually or in combination reduce evidence of elastotic material.
  • the invention provides a skin care composition comprising an effective amount of one or more enzymes that individually or in combination inhibit development or progression of signs of photodamage.
  • the invention provides a skin care composition comprising an effective amount of one or more enzymes that individually or in combination inhibit development or progression of signs of aging.
  • the skin care composition preserves or improves the condition of the dermis.
  • the composition inhibits accumulation of elastotic material in the dermis.
  • the composition reduces the rate of accumulation of elastotic material in the dermis.
  • the composition reduces evidence of elastotic material in the dermis.
  • the invention provides a pharmaceutical composition comprising an effective amount of one or more enzymes that individually or in combination reduce evidence of elastotic material in the skin.
  • the invention provides a composition comprising an effective amount of one or more enzymes that individually or in combination inhibit development or progression of signs of photodamage.
  • the invention provides a composition comprising an effective amount of one or more enzymes that individually or in combination inhibit development or progression of signs of aging.
  • the skin care composition preserves or improves the condition of the dermis.
  • the composition inhibits accumulation of elastotic material in the dermis.
  • the composition reduces the rate of accumulation of elastotic material in the dermis. In certain embodiments, the composition reduces evidence of elastotic material in the dermis.
  • the methods and compositions of the invention employ enzymes that either individually or in combination are capable of reducing evidence of elastotic material in skin.
  • the enzymes are proteases (also referred to as proteinases or proteolytic enzymes) that cleave, e.g., hydrolyze, peptide bonds.
  • Suitable enzymes include elastolytic enzymes, partial elastases, and enzymes that are capable of cleaving soluble elastin substrates but may not necessarily be capable of cleaving mature, cross-linked elastin.
  • Many of the enzymes of use in the present invention are described in Barrett, A., Rawlings, N., and Woessner, J.
  • Enzymes of use in various embodiments of this invention may be purified from natural sources, produced in vitro or in vivo in suitable expression systems using recombinant nucleic acid technology ⁇ e.g., by recombinant host cells or in transgenic animals or plants), or synthesized through chemical means.
  • a protein having the sequence of a naturally occurring protein may be referred to herein according to the name of the organism and/or the cell or tissue type in which the protein naturally occurs, regardless of whether any particular preparation of the protein used in a composition or method of the invention is obtained from the natural source or is produced using recombinant techniques or chemical synthesis.
  • bovine elastase refers to a protein having the sequence of an elastase that is found naturally in bovines
  • a composition comprising bovine elastase is one that comprises such a protein, regardless of whether the protein material present in the composition was prepared from bovine tissue.
  • pancreatic elastase refers to a protein having the sequence of an elastase that is naturally found in pancreatic tissue
  • a composition comprising pancreatic elastase is one that comprises such a protein, regardless of whether the protein was prepared from pancreatic tissue.
  • the invention includes embodiments of each composition and method described herein in which one, more than one, some, most, or all of the enzymes present in or used in the composition or method is obtained from natural sources, recombinantly produced, or chemically synthesized. In the case of compositions or methods that use more than one enzyme, all combinations in which different sources and/or methods are used to obtain the enzymes are encompassed in the invention.
  • enzymes whose name includes a number may be referred to by Roman numeral or Arabic numeral interchangeably herein.
  • elastase I and elastase 1 refer to the same enzyme.
  • the terms "protein sequence”, "polypeptide sequence” or "amino acid sequence” as used herein can refer to the polypeptide material itself and are not restricted to the sequence information (i.e., the succession of letters or three letter codes chosen among the letters and codes used as abbreviations for amino acid names) that describes a polypeptide.
  • a polypeptide sequence presented herein is presented in an N- terminal to C-terminal direction unless otherwise indicated.
  • Elastolytic enzymes fall into several classes based on their mechanism of action and include a variety of serine proteases, cysteine proteases, and metalloproteases. Elastases are classified under Enzyme Classification Number EC. 3.4 in accordance with the Recommendations of the International Union of Biochemistry and Molecular Biology (IUBMB).
  • Examples include mammalian elastases such as pancreatic elastase I (EC 3.4.21.36), pancreatic elastase II (EC 3.4.21.71), leukocyte (neutrophil) elastase (EC 3.4.21.37), macrophage elastase (EC 3.4.21.65), and bacterial elastases such as LasB, produced by Pseudomonas aeruginosa and Legionella pneumophila, among others (EC 3.4.24.26). These enzymes and other are discussed further below.
  • mammalian elastases such as pancreatic elastase I (EC 3.4.21.36), pancreatic elastase II (EC 3.4.21.71), leukocyte (neutrophil) elastase (EC 3.4.21.37), macrophage elastase (EC 3.4.21.65), and bacterial elastases such as LasB, produced by Pseudomonas aeruginos
  • the elastase is capable of cleaving mature, cross-linked elastin into fragments of between 1 kilodalton (kD) and 10 kD in size when incubated with the elastin for 24 hours or less under suitable conditions for enzyme activity and in which enzyme concentration is not limiting. In certain embodiments of the invention the elastase is capable of cleaving at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, or at least 10% of the peptide bonds in mature, cross-linked elastin when incubated with the elastin for 24 hours or less under conditions suitable for enzyme activity and in which enzyme concentration is not limiting.
  • Elastases produced by bacteria belonging to a variety of different genera are of use in the invention. Most bacterial elastases are serine proteaseas or zinc metalloproteases. Bacteria that produce elastolytic enzymes include Aeromonas (e.g., Aeromonas hydrophila), Pseudomonas (e.g., P. aeruginosa, P. pseudomallei, P. chrysogenuni), Actinomyces, Mycobacterium (e.g., M. tuberculosis), Clostridium (e.g., C. histolyticum), Bacillus (e.g., B. anthracis, B. licheniformis, B.
  • Aeromonas hydrophila Aeromonas hydrophila
  • Pseudomonas e.g., P. aeruginosa, P. pseudomallei, P. chrysogenuni
  • Actinomyces e.g., M
  • Flavobacterium e.g., F. elastolyticum, F. immotum
  • Streptomyces e.g., S.fradiae, S. griseus
  • Sporangium Staphylococcus (e.g., S. epidermidis)
  • Vibrio e.g., Vibrio cholerae, V. vulnificans
  • Elastase enzymes can be obtained from cultures of any of these microorganisms or, if the coding sequences are known, by recombinant expression or chemical synthesis.
  • elastase is obtained from staphylococci of the type sometimes found in the oral cavity (Murphy, R. A., J Dent Res 53(4): 832-4).
  • elastase is obtained from Pseudomonas aeruginosa.
  • Pseudomonas aeruginosa (PA) elastase is of interest herein.
  • This bacterium has two genes, las A and lasB, that encode the elastolytic enzymes Las A and LasB. Both of these proteins have been well characterized.
  • LasB also referred to as elastase or pseudolysin, is the major PA elastase.
  • PA elastase refers to PA LasB.
  • LasA has relatively low elastolytic activity but is important as an enzyme that enhances the elastolytic activity of LasB (Kessler, E., et al., JBiol Chem., 272, 9884-9889, 1997).
  • the present invention contemplates use of LasB, use of LasA, or use of LasA in combination with another elastase (e.g., another bacterial elastase such as LasB) to diminish evidence of photodamage.
  • another elastase e.g., another bacterial elastase such as LasB
  • the invention provides a skin care composition comprising PA LasB and LasA.
  • alkaline elastase YaB an extracellular serine protease of the alkalophilic Bacillus strain YaB (Kaneko, R., J Bacteriol. 1989 September; 171(9): 5232-5236).
  • Another elastase of interest is produced by Staphylococcus epidermidis and encoded by the sepA gene (Teufel, P. and G ⁇ tz, F., J Bacteriol., 175(13): 4218-4224, 1993).
  • Another elastase of interest is Legionella pneumophila zinc metalloprotease, also known as LasB as it is closely related to PA LasB (Black, W.J., et al., J.
  • elastases purified from Micrococcus luteus (Clark, DJ, Protein Expr Purif., 18(l):46-55, 2000) and Myxococcus xanthus (Dumont, L, 1 : Eur. J. Biochem., 223(3):775-82, 1994).
  • the elastase is from Clostridium histiolyticum (U.S. Pat. No. 6,146,626).
  • Table 1 provides Gene IDs and cDNA and/or protein accession numbers for certain of these enzymes, which can be used to readily obtain the corresponding sequences from public databases such as Genbank, SwissProt, PDB, etc. available through Entrez at the National Center for Biotechnology Information (NCBI) at www.ncbi.nlm.nih.gov/entrez.
  • NCBI National Center for Biotechnology Information
  • Gene IDs, gene symbols and names, and protein names as used in the tables herein are according to the nomenclature used by the NCBI as of July 29, 2006. Alternate names are sometimes used in the literature.
  • nucleic acid sequences listed under the accession numbers provided herein typically include coding sequences for the signal peptide and, where relevant, propeptide, and protein sequences listed under the accession numbers provided herein typically include the signal peptide and, where relevant, the propeptide. It will be appreciated that these portions of the sequence may be removed in the active form.
  • YP 095682 protein
  • ahpB elastase A. hydrophila N/A AF 193422 cDNA
  • AAF07184 protein
  • sepA extracellular S. epidermidis 2,2A26 ⁇ A NC_002976 cDNA
  • elastase AAW53096 protein
  • the enzymes mentioned above represent only some of the bacterial elastases of use in various embodiments of the present invention. Many predicted bacterial proteins having significant sequence similarity over part or all of their sequence to known bacterial protease sequences, e.g., bacterial elastase sequences, have been identified through cDNA and genome sequencing efforts, but in some cases their elastolytic activity remains to be experimentally confirmed. Sequence comparison algorithms known in the art can or have been used to identify such putative proteolytic enzymes.
  • an enzyme of use in the present invention is one whose sequence is a "match" with an expectation value (E value) of 10 ⁇ 10 or less, 10 ⁇ 20 or less, 10 ⁇ 50 or less, or 10 ⁇ 100 or less, when the sequence of a protein selected from the group consisting of PA LasA, PA LasB, P. pneumophila LasB, Aeromonas hydrophila elastase, and S.
  • epidermidis elastase is used as a query to search the "GenBank CDS translations+PDB+SwissProt+PIR+PRF excluding environmental samples" database of nonredundant protein sequences available from the NCBI using the BLAST, Gapped BLAST, or PSI-BLAST algorithm (Altschul, S.F., et ah, Nucleic Acids Res. 25:3389-3402, 1997; Schaffer et al, Nucleic Acids Res. 29:2994-3005, 2001) with the BLOSUM62 substitution matrix with word size 3, gap penalties as follows: existence: 11, extension: 1, and default values for any other parameters.
  • Identified coding sequences can be used to produce the protein, e.g., using recombinant expression methods.
  • the ability of the protein to solubilize or cleave mature, cross-linked elastin or a soluble elastase substrate in vitro or to remove elastotic material in skin ⁇ e.g., photodamaged skin, aged skin) may be assessed as described elsewhere herein in order to identify enzymes useful in the present invention.
  • the elastolytic enzyme is a mammalian elastase.
  • the elastase may be from any mammalian organism ⁇ e.g., porcine, bovine, rat, mouse, canine, non-human primate, human).
  • Elastolytic enzymes are found in a wide variety of mammalian cells and tissues including pancreas, polymorphonuclear leukocytes (neutrophils), macrophages, monocytes, and various other normal or tumor cells in culture. Elastolytic enzymes are also found in many non-mammalian species including avians and fish (Bieth, supra).
  • Humans have six elastase genes which encode the structurally similar proteins elastase I, II, HA, HB, IIIA, and IIIB. In certain embodiments of the invention, an elastase encoded by a human elastase gene is used. [0084] Several elastase enzymes are commonly expressed to high levels selectively in the mammalian exocrine pancreas.
  • Elastases I and II were the first pancreateic elastases to be identified, and cDNAs encoding them have been cloned from a variety of organisms including human, bovine, and rat (see, e.g., MacDonald et al, Biochemistry, 21(6):1453-63, 1982; Swift, G.H., et al J Biol Chem., 259(22): 14271-8, 1984; Shirasu et al, J. Biochem.
  • elastase I is the major pancreatic protease with elastin-degrading activity.
  • Elastase I is a single polypeptide chain of about 240 amino acids cross-linked by 4 disulfide bridges. It has a hydrophobic core and exhibits extensive sequence homology with other serine proteinases, such as trypsin and chymotrypsin.
  • Elastase III enzymes have also been identified, and cDNAs encoding them have been cloned (Shen et al, Biochemistry, 26: 3447-3452, 1987; Tani et al, J Biol Chem., 263(3):1231-9, 1988).
  • Elastolytic enzymes produced by polymorphonuclear leukocytes are of use in certain embodiments of the invention (Barrett, A.J. Meth. Enzymol. 80, 581, 1981; Boudier, C, et al, Am J Respir Cell MoI Biol, 4(6):497-503, 1991; Ying, Q-LO, Simon SR, Am J Respir Cell MoI Biol, 26:356-351 2002).
  • the enzyme is not a leukocyte elastase.
  • elastolytic enzymes of use in various embodiments of the invention include cathepsins K, S, L and V, which are produced by macrophages (Yasuda, Y., et al, JBiol Chem., 279(35):36761-70, 2004).
  • the elastase is metalloproteinase produced by human alveolar macrophages (Shapiro, S. D., et al, J Biol Chem 268(32): 23824-9, 1993).
  • Leukocyte elastase is a serine protease while cathepsins are cysteine proteases.
  • the enzyme is not a cathepsin.
  • MMPs matrix metalloproteinases
  • Mammalian elastolytic metalloproteinases include MMP-2, MMP-7 (matrilysin), MMP-9, and MMP- 12, which are produced by cell types such as macrophages and/or fibroblasts (Mecham, R.P., et al, J. Biol. Chem., 272(29): 18071-18076, 1997; Wallace, S.Y., et al, Arterioscler. Thromb. Vase. Biol, 25:372-378, 2005).
  • the elastase is not a mammalian metalloproteinase.
  • Platelet elastase, or any of various elastases produced by certain tumor cell lines are also of use in certain embodiments of the invention (Kao, R.T., Stern, R., Cancer Res., 46(3): 1355-8, 1986).
  • Table 2 provides a summary of some mammalian elasto lytic enzymes of use in the present invention together with relevant Gene ID and/or accession numbers for the mRNA or cDNA and the protein.
  • ELA3A/ elastase 3A Homo 10136 NM_005747; NP_005738 elastase 3A, pancreatic sapiens pancreatic
  • CTSG cathepsin G cathepsin G Homo 1511 NM_001911; NP_001902 sapiens
  • CTSK cathepsin K cathepsin K Homo 1513 NM_000396; NP_000387 sapiens
  • CTSS cathepsin S cathepsin S Homo 1520 NM_004079; NP_004070 sapiens
  • CTSL2 cathepsin L2 cathepsin L2, Homo 1515 NM_001333; NP_001324 cathepsin V sapiens
  • CTSL cathepsin L cathepsin L Homo 1514 NM_001912; NP_001903 sapiens
  • PRTN3 proteinase 3 proteinase 3, Homo 5657 NM_002777; NP_002768 erine proteinase, sapiens neutrophil,
  • the present invention also contemplates use of elastolytic enzymes found in fish; fungi; parasites such as nematodes, trematodes, and helminths; and snake venoms. See Bieth, supra, and references therein. See also Okumura et al, J Med Microbiol, 53(Pt 5):351-4, 2004, describing elastases found in Aspergillus species; Aztori et al, Parasite. 6(1):9-16, 1999, and Salter, J.P, et al, J Biol Chem., 277(27):24618-24, 2002, describing elastase genes of the trematode genus Schistosoma).
  • the enzyme is not found in fish or crustaceans, e.g., krill.
  • the enzyme is not found in plants.
  • the enzyme is not papain or bromelain.
  • pancreatic elastase and many bacterial elastases are secreted proteins and, as such, are typically synthesized in nature as precursors containing a signal sequence at their amino terminus that is cleaved off as part of the process of forming the mature protein.
  • many of these proteases are synthesized as inactive proenzymes or zymogens that typically include a pro-domain (also referred to as propeptide), that is covalently attached to the amino and/or carboxy terminus of the mature enzyme sequence and controls the activity of the enzyme, maintaining it in an inactive state until removed.
  • the propeptide can be required for the proper folding of the protease into an active enzyme but is typically removed following secretion.
  • pancreatic elastase is secreted as an inactive precursor and activated by cleavage in the duodenum.
  • PA elastase is synthesized with an ⁇ 18 kDA propeptide which is cleaved autocatalytically in the periplasm and forms a complex with the
  • the term "processed form” refers to a polypeptide that has been cleaved such that its signal peptide and, where applicable, its propeptide, are no longer covalently attached to the portion of the protein that contains the active site residues.
  • the processed form of an enzyme is used.
  • the full length (unprocessed) form optionally without the signal peptide, is used.
  • a complex containing the propeptide noncovalently associated with the portion of the protein that contains the active site is used.
  • sufficient propeptide dissociates from the complex or is degraded following contact with the skin to permit effective levels of activity.
  • an activating agent e.g., a chemical compound or protease that cleaves the zymogen to activate it or that cleaves the noncovalently associated propeptide is admixed with a composition containing the zymogen before contacting the skin with the composition, or is delivered to the skin shortly thereafter.
  • compositions contain a bacterial elastase and a mammalian elastase, e.g., a pancreatic elastase.
  • a mammalian elastase e.g., a pancreatic elastase.
  • the composition contains PA elastase and a pancreatic elastase.
  • At least one of the enzymes used to diminish evidence of photodamage or aging is not itself able to solubilize mature, cross- linked elastin but is able to cleave at least one peptide bond in mature, cross-linked elastin.
  • two or more such enzymes are used in combination to reduce evidence of elastotic material or diminish accumulation thereof.
  • a partial elastase and an elastase are used in combination. Partial elastases may be found among the bacterial metalloproteinases, eukaryotic matrix metalloproteinases, serine proteases, cysteine proteases, and aspartyl proteases.
  • Suitable enzymes for use in the present invention include naturally occurring variants (e.g., strain-specific or allelic variants), variants identified after mutagenizing cells that produce an elastase, and engineered variants of naturally occurring enzymes.
  • the sequence of a variant has one or more amino acid additions, substitutions, or deletions relative to that of a naturally occurring enzyme.
  • bacteria are mutagenized and screened to identify those that produce an elastase having desired properties.
  • the sequence of the gene encoding the elastase will typically vary from that of the unmutagenized cell.
  • amino acids joined by a peptide bond susceptible to cleavage may be deleted or replaced by a different amino acid.
  • an amino acid insertion may be made. Such alteration(s) may prevent recognition of the site by a protease that would otherwise cleave it and result in a more stable enzyme with a longer duration of action and/or a product with a longer shelf life or a greater range of storage conditions.
  • the elastase activity of a useful variant of a naturally occurring elastase may range from between 50 and 100%, between 75% and 100%, or between 90% and 100% of that of the naturally occurring elastase. Variants with higher activity may also be obtained and would be of use. Lower activity could be compensated for by increasing the amount of enzyme used.
  • Amino acids have been divided into various groups based properties such as hydrophilic/hydrophobic character, side chain size, etc. Substitutes for an amino acid within the sequence may be selected from other members of the class to which the amino acid belongs.
  • the non-polar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, glycine, proline, phenylalanine, tryptophan and methionine.
  • the polar (hydrophilic), neutral amino acids include serine, threonine, cysteine, tyrosine, asparagine, and glutamine.
  • the positively charged (basic) amino acids include arginine, lysine and histidine.
  • the negatively charged (acidic) amino acids include aspartic acid and glutamic acid.
  • Substitutions may be selected with respect to: (i) maintaining the structure of the peptide backbone in the area of the substitution, for example, as a sheet or helical conformation; (ii) maintaining the charge or hydrophobicity of the molecule; or (iii) maintaining the bulk of the side chain.
  • the substitutions that in general may be expected to induce greater changes are those in which: (a) glycine and/or proline is substituted by another amino acid or is deleted or inserted; (b) a hydrophilic residue, e.g.
  • serine or threonine is substituted for (or by) a hydrophobic residue, e.g., leucine, isoleucine, alanine, or phenylalanine,; (c) a cysteine residue is substituted for (or by) any other residue; (d) a residue having an electropositive side chain, e.g., lysine, arginine, or histidine, is substituted for (or by) a residue having an electronegative charge, e.g., glutamate or aspartate, or (e) a residue having a bulky side chain, e.g., phenylalanine, is substituted for one (or by) one not having such a side chain, e.g., glycine.
  • Most deletions and insertions are not expected to produce dramatic changes in the characteristics of the protein.
  • the sequence of a variant can be obtained by making no more than a total of 5, 10, 15, or 20 amino acid additions, deletions, or substitutions to the sequence of a naturally occurring enzyme.
  • the sequence of a variant is at least 80%, at least 85%, at least 90%, or at least 95% identical to the sequence of a naturally occurring enzyme over a window of comparison at least 80%, at least 85%, at least 90%, or at least 95%, of the length of the naturally occurring enzyme (optionally excluding any signal peptide or propeptide), when the two sequences are aligned to achieve maximal percent identity allowing the introduction of gaps.
  • Percent identity may be calculated by aligning the sequences, counting the number of identical residues in the two sequences within a window of comparison, dividing the result by the length of the window of comparison, and multiplying by 100.
  • the window of comparison may begin at any position within either protein.
  • the length of the window of comparison is calculated without counting any gaps that may have been introduced to maximize percent identity.
  • a variety of algorithms are available that can calculate percent identity, e.g., those implemented in the BLASTP program(Altschul, supra), the Smith- Waterman algorithm (Smith, T. F. & Waterman, M. S., J. MoI. Biol. 147, 195-197, 1981), etc.
  • At least 50%, at least 75%, at least 90%, at least 95%, or 100% of the substitutions are conservative substitutions.
  • no more than 10% of the amino acids are deleted or substituted and the number of additions is less than 10% of the number of amino acids in the original sequence.
  • Elastin is widely distributed in various connective tissues. It is found abundantly in the ligamentum nuchae of grazing ungulates and the thoracic aorta of warm-blooded vertebrates. It is prominent in the walls of the major arteries and arterioles and is an important constituent of lung tissue and the deeper layers of the skin. Mature, cross-linked elastin can be prepared from any of these natural sources, or others, by a variety of different methods. The material remains insoluble under a variety of harsh extraction conditions that facilitate its purification.
  • soluble peptides (Bieth, supra).
  • Exemplary methods for preparing mature, cross-linked elastin are well known in the art. For example, the neutral extraction method of Partidge (Partridge, et al. , Biochem, J, 61 : 11 , 1955), the alkaline extraction method of Lowry et al. (Lowry, E.A., et al. J. Biol. Chem., 139: 795, 1941), or the method of Starcher et al.
  • elastolytic activity is determined using insoluble elastin labeled with any of a variety of different fluorescent or colored labels as a substrate.
  • elastin-Congo red (Sigma) may be used as the substrate.
  • Reaction suspensions 1.1 ml in 50 mM Tris-HCl, 0.5 mM CaCl 2 , 0.2 mg/ml bovine serum albumin, pH 7.5
  • 10 mg of elastin-Congo red and 0.5 ⁇ g of one or more proteins to be tested for elastase activity are incubated at 37 0 C for 2 h. Reactions are stopped by adding 0.1 ml of 120 mM EDTA followed by immediate cooling and centrifugation.
  • the degree of elastin solubilization is determined by measuring the absorbance at 495 nm of the clear supernatant (Kessler, E., et al., JBiol Chem., 272(15):9884-9, 1997).
  • One unit of elastase activity may be defined as the amount of enzyme required to solubilize 20 mg of insoluble elastin-Congo Red under the test conditions within a period of 48 hours.
  • one unit of elastase activity may be defined as the amount of enzyme required to solubilize 1 ⁇ g insoluble elastin-Congo Red per hour at 37°C under the test conditions when in the linear part of the activity range.
  • elastin-fluorescein elastin-fluorescein
  • elastin-rhodamine elastin-orcein
  • product codes ES80 elastin-fluorescein
  • E164 elastin-Congo Red
  • the Elastin Products Company Owensville, MO
  • the elastase has an activity of between 50 and 300 U/mg, e.g., about 260 U/mg using either or both definitions of the unit of activity.
  • plates e.g., Petri dishes containing appropriately buffered agar impregnated with elastin (optionally labeled) are used (Rust, supra; Yagci, A., et al., New Microbiol. 25(2):223-9, 2002).
  • the gels contain a number of wells into which the material to be tested for elastase activity is loaded. Elastolytic enzymes diffuse into the agar and hydro lyze the elastin. The size of the zone of clearing around any particular well is indicative of the amount of elastase activity of its contents.
  • Suitable plates are commercially available ⁇ e.g., the Alphasin® plate from the Elastin Products Company).
  • elastin-coated plates are prepared by drying radiolabeled elastin on the entire surface of each plates and adding a solution containing an enzyme to be tested under appropriate conditions for elasto lysis to occur. After a period of incubation, supernatants are collected, and solubilized elastin is quantified by scintillation counting.
  • Enzymes for use in the present invention can be purified from natural sources using methods well known in the art.
  • PA elastase is prepared using the method of Morihara (Morihara, Journal Biochemistry, Vol. 240: 3295, 1965) or either of the methods of Rust, L., et al. (Methods Enzymol, 235:554-62, 1994).
  • porcine elastase is prepared by crystallization from the euglobin fraction of porcine pancreas by the method of Lewis et al. (Lewis, U.J., et al., J. Biol. Chem., 22, 705, 1956).
  • Natural sources refers to organisms that naturally produce the enzyme.
  • enzymes can be purified from pancreatic tissue or from neutrophils or fibroblasts harvested from an organism.
  • Bacteria that naturally produce elastase may be cultured and the elastase harvested from the bacterial cells or culture medium.
  • Enzymes may also be produced using standard recombinant expression methods. Chemical synthesis, including conventional solid phase peptide synthesis and/or methods involving chemical ligation of synthesized peptides may also be used (see, e.g., Kent, S., J Pept ScL, 9(9):574-93, 2003 and U.S. Pub. No. 20040115774, incorporated herein by reference). It will be appreciated that a protein obtained by certain of these methods may contain amino acids that do not naturally occur in proteins and/or may contain amino acid analogs known in the art. Recombinant expression systems and chemical synthesis are appropriate methods for producing enzyme variants with sequences different from those occurring in nature.
  • Recombinant expression methods encompass any method of protein preparation that involves introducing exogenous nucleic acid into, or modifying endogenous nucleic acid of, a host cell or a precursor of the host cell.
  • the host cell is one that does not naturally produce the protein or produces it at a different, e.g., lower, level.
  • Such methods are well known in the art and are described, for example, in Sambrook, et ah, supra, and Ausubel, supra.
  • Typical methods involve expression of a particular gene in a host cell by introduction of an exogenous nucleic acid sequence that encodes the protein into the cell or activation of the endogenous gene (U.S. Pat. No. 5,641,670, incorporated herein by reference).
  • the host cell may be genetically modified, treated, or selected to improve its properties as a host for expression of the protein of interest. Suitable modifications or treatments include, e.g. , causing the cell to express one or more proteins naturally involved in processing the protein of interest or a precursor thereof, reducing or eliminating expression of proteolytic enzymes ⁇ e.g., using insertional mutagenesis, RNA interference, chemical inhibitors), etc.
  • Recombinant expression methods may utilize cultured cells that have been transiently or stably transfected or infected with an expression vector, transgenic animals, transgenic plants, plants transiently transfected or infected with a plant viral vector, etc.
  • Recombinant expression methods also include techniques in which in vitro transcription and translation are used to prepare a protein. In other embodiments synthesis of a protein of interest is induced by exposure to various chemical agents or environmental conditions.
  • An advantage of using a recombinant expression system is that the protein can be produced in host cells that are free of potential human pathogens. Nucleic acid sequences, e.g., cDNAs, encoding many enzymes of use in the present invention are known in the art.
  • Methods for identifying coding sequences for additional enzymes include (i) screening cDNA libraries at low stringency using known cDNA or oligonucleotide probes that encode at least a portion of a known enzyme, (ii) sequencing an enzyme of interest purified from natural sources and using the genetic code to deduce a coding sequence therefor; (iii) PCR-based screening in which PCR primers designed to hybridize to mRNA of an enzyme of a first strain or species are used to amplify mRNA from another strain or species, or (iv) screening expression libraries using antibodies to known enzymes.
  • the coding sequence for an enzyme of interest, and optionally 5' and/or 3' untranslated region(s) may be inserted into an expression vector and proteins expressed and purified using standard methods.
  • the recombinant proteins thus produced can be characterized by, for example, polyacrylamide gel electrophoresis (PAGE) analysis, N- terminal sequencing, and/or assays for enzymatic activity, e.g. , ability to cleave a known elastase substrate.
  • Well known expression systems utilize prokaryotic (e.g., E. col ⁇ ) and/or eukaryotic (e.g., yeast, insect, mouse, or human) cells.
  • Suitable yeast cells suitable for expression of the proteins of the invention include Saccharomyces cerevisiae and Pichia pastoris, insect cells (e.g., Sf9), plant cells, and animal cells, e.g., mammalian cells such as CHO, Rl.1, B-W, L-M, African Green Monkey Kidney cells (e.g. COS-I, COS-7, BSC-I, BSC-40 and BMT-IO), BHK cells, and cultured human cells.
  • Numerous vectors are available for expressing heterologous proteins in such cells. These vectors usually supply a promoter and other regulatory elements such as enhancers, splice acceptor and/or donor sequences, and polyadenylation signals.
  • Some vectors also include an in-frame sequence encoding a short peptide "tag" such as a 6X His, FLAG, HA, Myc, or other tag useful for affinity purifying a protein that includes the tag.
  • Vectors include DNA or RNA plasmids or viruses.
  • Recombinant molecules can be introduced into host cells, for example, via transformation, transient or stable transfection, infection, electroporation, lipofection, etc. Once the recombinant proteins are expressed, they may be isolated and purified by standard methods including chromatography (e.g., ion exchange, affinity, and sizing column chromatography), centrifugation, precipitation, differential solubility, by any other standard technique for the purification of proteins or, as appropriate, the specific methods mentioned above.
  • chromatography e.g., ion exchange, affinity, and sizing column chromatography
  • centrifugation precipitation
  • differential solubility by any other standard technique for the purification of proteins or, as appropriate, the specific methods mentioned
  • a coding sequence lacking the signal sequence and/or propeptide may be used.
  • the propeptide may be included, particularly if it facilitates or is important for correct folding of the protein.
  • a signal peptide coding sequence may be included.
  • the signal peptide need not be the one that is found in the naturally occurring precursor but may be any signal peptide capable of directing secretion in the host cell being employed.
  • enzymes of use in the present invention are commercially available from companies such as The Elastin Products Company, Innovative Research, Worthington, Sigma- Aldrich, United States Biological, Biodesign International, and a range of other suppliers. Enzymes obtained from these suppliers may be formulated together with other suitable components to prepare a composition useful for cosmetic or therapeutic purposes, optionally after additional purification steps.
  • the invention contemplates a variety of different modifications that may contribute to the activity or stability of the enzyme or confer any of a variety of desirable properties.
  • conjugation with various polymers such as polyethylene glycol (PEG) is a well known method of increasing protein stability in vivo.
  • PEG polyethylene glycol
  • the elastase could be attached to any of a wide variety of other polymers known in the art. Methods for modifying proteins are well known in the art.
  • compositions of the present invention may be formulated by combining one or more enzymes with one or more other components that serve any of a variety of functions.
  • an enzyme to be used in the composition will have been at least partially purified prior to being combined with at least some of the other components. If obtained from tissue or cultured cells, an enzyme will typically have been removed from most of the cellular material present in the tissue or cultured cells (as well as most of the noncellular material present in the tissue) prior to being formulated in a composition of the present invention. If obtained from cell culture medium, the enzyme will typically have been removed from most of the cells, cell debris, nutrients, metabolites, etc., present in the culture medium.
  • the enzyme may have been removed from synthesis reagents and/or from free amino acids.
  • the enzyme may, for example, be purified such that it constitutes at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or more, by dry weight of a preparation to be combined with other components of the composition.
  • the enzyme may, for example, be purified such that it constitutes at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or more, by dry weight of the protein present in a preparation to be combined with other components of the composition.
  • the present invention contemplates in some embodiments the use of enzymes that display selectivity towards elastotic material, e.g., they preferentially solubilize elastotic material while having lesser activity towards normal elastic fibers under the same conditions.
  • the enzyme solubilizes elastotic material while leaving normal elastic fibers substantially intact.
  • the enzyme exhibits specificity for elastotic material versus normal elastic fibers, e.g., the enzyme has greater ability to degrade elastotic material than to degrade normal, mature elastin fibers.
  • the invention also contemplates use of enzymes that display selectivity towards elastotic material or elastin relative to collagen fibers, e.g., the enzyme has greater ability to degrade elastotic material or elastin than to degrade collagen fibers.
  • a composition contacted with photodamaged skin in accordance with the invention does not significantly affect the dermal collagen fiber network, i.e., the collagen network remains largely intact.
  • Evidence for the ability of a composition to remove elastotic material and/or to exhibit specificity may be obtained in a number of different ways. Visual assessment may be used. For example visual observation of the presence of normal appearing elastic and/or collagen fibers in the dermis of photodamaged skin following contact with a composition comprising an enzyme or combination of enzymes in an amount effective to significantly or substantially diminish evidence of elastotic material provides evidence that the composition is selective for elastotic material.
  • a pathologist having knowledge of dermatopathology, will be able to distinguish normal appearing elastin fibers from elastotic material.
  • Automated image analysis systems can be used to quantify the amount of elastotic material relative to that of normal elastic fibers or collagen fibers in samples of skin that have been contacted with an enzyme-containing composition in accordance with the present invention.
  • Automated image analysis systems useful for quantifying elastin, collagen, elastase activity, etc. are known in the art (Bertermü, B., et al, Br J Dermatol., 133(6):836- 41, 1995; Boudier, supra, Har-Shai, Y., et al, Plast Reconstr Surg., 102(7):2466-70, 1998; Ghomrasseni, S., et al., J Eur Acad Dermatol Venereol., 15(4):305-l 1, 2001).
  • the automated image analysis methods may be employed to analyze cryostat sections, sections taken from skin punches, etc.
  • the samples will typically be analyzed using light microscopy, fluorescence microscopy, electron microscopy, etc., after appropriate processing to allow detection of the proteins of interest.
  • Sections may be stained with chemical agents or antibodies specific for different proteins such as elastin and collagen. Suitable methods may include computerized threshold selection and elimination of non-elastic dark elements, calculation of fiber diameters, calculation of average area and/or volume occupied by fibers of various types, calculation of average number of fibers of different types, calculation of amount of different proteins based on staining intensity, etc.
  • desmosine release may be used as a measure of digestion of elastotic material.
  • Exemplary methods of detecting the degradation of elastotic material are described in Viglio et ah, J. Sep. Sci. 30:202-213, 2007. Any methods described by Viglio et al. may be used to assess the degradation of elastotic material in skin.
  • the enzyme has greater activity towards elastin than towards collagen.
  • the enzyme may have at least 5 -fold, at least 10-fold, or at least 25-fold greater ability to solubilize mature, cross-linked elastin than to solubilize triple helical, cross-linked collagen under selected conditions, e.g., physiological conditions.
  • the amount (by weight) of mature, cross-linked elastin solubilized by the enzyme may be at least 5, at least 10, or at least 25-fold as great as the amount of mature, triple helical collagen solubilized by the enzyme.
  • the collagen may be any collagen that assembles into a triple helical structure, e.g., collagen I.
  • the enzyme does not substantially affect the collagen fibers present in the dermis of photodamaged skin under conditions in which evidence of elastotic material is substantially diminished.
  • the selected conditions may be those conditions under which the enzyme is known in the art to be active towards elastin. Such conditions will vary depending on the particular enzyme being tested.
  • the enzyme has less than 10%, less than 5%, or less than 1%, of the collagenase activity of an average crude or chromatographically purified collagenase from Clostridium histiolyticum (Worthington Biochemical, Product Code: CLSPA).
  • Collagenase activity can be assayed using any method known in the art, e.g., using a modification of Mandl wherein collagenase is incubated for five hours with native collagen and the extent of collagen breakdown is determined using the Moore and Stein, J. Biol. Chem., 176, 367, (1948) colorimetric ninhydrin method.
  • the trypsin activity of the enzyme is less than 15 U/mg, less than 7.5 U/mg, or less than 1.5 U/mg, where one unit hydro lyzes l ⁇ mole of p-toluene-sulfonyl-L-arginine methyl ester (TAME) per minute at 25 0 C, pH 8.2, in the presence of 1OmM calcium.
  • TAME p-toluene-sulfonyl-L-arginine methyl ester
  • the chymotrypsin activity of the enzyme is less than 3.5 U/mg, less than 1.75 U/mg, or less than .35 U/mg, where one unit hydro lyzes one micromole of benzoyl-L-tyrosine ethyl ester per minute at 25 0 C, pH 7.8 in the presence of calcium.
  • One aspect of the present invention is the identification of preferred enzymes for removal of elastotic material.
  • the invention provides a variety of methods for identifying a preferred enzyme or enzyme combination. Some of the methods are performed in vitro and involve incubating an enzyme or combination of enzymes with elastotic material and also, in a separate vessel or container, incubating the same enzyme or combination of enzymes with a relevant elastin-containing substrate. The ability of an enzyme or combination of enzymes to solubilize or partially digest elastotic material is compared with the ability of that enzyme or combination of enzymes to solubilize or partially digest non-photodamaged elastin, e.g. , insoluble elastin prepared as described above. Materials for performing the methods may be obtained in any suitable manner.
  • elastotic material is prepared from photodamaged skin. Procedures described above for preparing mature, cross- linked elastin, or modifications thereof, are used to prepare elastotic material in certain embodiments of the invention, but other procedures may be used instead. Any preparation of insoluble elastin may be used for purposes of making the comparison.
  • the elastin may, but need not be, prepared from skin.
  • Other elastin-containing substrates may also be used.
  • elastic fibers that contain a significant amount of elastin (e.g., at least 50% elastin by dry weight) are used.
  • the elastic fibers may also contain elastin- associated proteins such as fibrillins, etc.
  • elastase has higher activity towards cross-linked elastin, which can be demonstrated as previously described (Yasutake, A. and J. C. Powers, Biochemistry 20(13): 3675-9, 1981). Specificity of elastase I and II towards different regions of elastin has been compared as described (Vered, M., et al, Int J Pept Protein Res 25(1): 76-84, 1985). Cleavage specificity of PA elastase and pancreatic elastase has been analyzed (Saulnier, J.
  • an enzyme or combination of enzymes is contacted with skin, e.g., by injection or topical application. Following exposure to the enzyme or combination of enzymes, the skin is stained for elastin. The relative ability of the enzyme composition to remove elastotic material and normal elastic fibers is assessed visually or using an automated method. An enzyme or enzyme combination that selectively removes elastotic material while leaving normal elastic fibers relatively intact is selected as a preferred enzyme for use in the present invention. As described in Example 2, following removal of elastotic material present in photodamaged using PA elastase, fine, normal appearing elastic fibers are observed. This observation suggests that PA elastase may selectively degrade elastotic material.
  • enzymes or combinations of enzymes are tested to determine their effect on elastic fibers in non-photodamaged and in photodamaged skin.
  • Enzymes or enzyme combinations that display less degradative effect on non-photodamaged skin relative to that displayed by other enzymes or enzyme combinations while displaying an equivalent ability to degrade elastotic material from photodamaged skin are selected as preferred for use in the compositions and methods of the present invention.
  • the invention contemplates comparing a variety of different elastases, combinations of elastases, combinations of an elastase and a partial elastase, and combinations of partial elastases in order to identify enzymes and/or enzyme combinations that are capable of effectively removing elastotic material while preserving normal elastic fibers. Any elastase or partial elastase known in the art or discovered in the future may be tested.
  • the invention also includes a method of identifying preferred components and characteristics of a composition comprising an elastase or partial elastase such that normal elastic fibers and other components of the skin ⁇ e.g., collagenous fibers, glycosaminoglycans) are preserved while elastotic material is degraded.
  • any of the above methods may involve exposing the elastotic material, elastin containing substrate, or skin, to an enzyme or combination of enzymes for varying periods of time, at varying concentrations and temperatures, and in solvents having a range of different pH values, salt concentrations, and other components, in order to identify, preferred conditions. IV.
  • the invention provides skin care compositions that contain one or more elastases or partial elastases.
  • Such compositions may be cosmetic compositions or pharmaceutical compositions.
  • the one or more elastases and/or partial elastases can be present in total in an amount of about 0.000001% to about 75.0% by weight of the skin care composition, e.g., about 0.00001% to about 50.0%, or about 0.0001% to about 25.0%.
  • the composition contains between 0.000001% and 0.00001%, between 0.00001% and 0.001%, between 0.001% and 0.01%, between 0.01% and 0.1%, or between 0.1% and 1% elastases and/or partial elastases by weight. In certain embodiments of the invention the composition contains between about 0.01% to about 50%, e.g., about 0.1% to about 25.0%, e.g., about 0.5% to about 5% 5% elastases and/or partial elastases by weight. [00120] Skin care compositions of the present invention may contain other components in addition to one or more enzymes that either individually or in combination diminish evidence of elastotic material.
  • compositions are typically formulated by combining one or more enzymes with one or more other components that serve any of a variety of functions.
  • many cosmetic ingredients have multiple functions in formulations and therefore could properly be included in several functional categories.
  • the components mentioned herein may be grouped based on the benef ⁇ t(s) they provide or according to their proposed mode of action or purpose for including them in the composition, some of these ingredients can in some instances provide more than one cosmetic and/or therapeutic benefit or may have multiple modes of action or effects. Therefore, agents are grouped together for the sake of convenience and the classifications are not intended to limit the agent to any particular function, mode of action, etc.
  • Non-limiting examples of these categories include carriers, stabilizers, solubilizers, thickening agents, surfactants, anti-oxidants, anti-microbials, anti-fungals, preservatives, antioxidants, chelating agents, sunscreens, vitamins, colorants (pigments or dyes), proteins, peptides, amino acids, plant extracts, humectants, fragrances, perfumes, oils, emollients, lubricants, butters, thickeners, viscosity modifiers, polymers, surfactants, detergents, emulsif ⁇ ers, opacifying agents, penetrants, solvents, salts, pH adjusting agents, buffers, neutralizing agents, particulate matter, etc.
  • the enzyme(s) will typically be combined with a cosmetically acceptable carrier, e.g., a liquid solvent or diluent such as water.
  • a cosmetically acceptable carrier e.g., a liquid solvent or diluent such as water.
  • the amount of water may range between 20% and about 99% by weight.
  • the amount of water may be between 30% and 90%, between 40% and 80%, between 50% and 80%, etc.
  • the carrier can comprise a suitable amount of an alcohol, e.g., ethanol, isopropanol, benzyl alcohol.
  • the composition may comprise a polyhydric alcohol in an amount that may range from 0.1% to 50%, from 0.5% to 40%, from 1% to 30%, or from 1% to 15%.
  • the ratio (weight/weight %) of polyhydric alcohol to water usually ranges from 1 :2 to 1 : 100, e.g., from 1 :3 to 1 :50, from 1 :5 to 1 :25, or from 1 :6 to 1 :10.
  • Polyhydric alcohol or "polyols” as used herein means an organic compound comprising two or more alcohol functions or alkoxylated derivatives thereof.
  • the polyhydric alcohol may have a number average molecular weight of less than 50,000, e.g., less than 35,000, e.g., less than 2,000.
  • Exemplary polyhydric alcohols include polyvinylalcohol, polyalkylene glycols, such as alkylene polyols and their derivatives, including propylene glycol, dipropylene glycol, polypropylene glycol, polyethylene glycol and derivatives thereof, sorbitol, hydroxypropyl sorbitol, erythritol, threitol, pentaerythritol, xylitol, glucitol, mannitol, hexylene glycol, butylene glycol (e.g., 1,2-butylene glycol and 1,3-butylene glycol), hexane triol (e.g., 1,2,6-hexanetriol), 2,4,4-trimethyl-pentanediol, neopentyl glycol, glycerine, ethoxylated glycerine, propane- 1,3 diol, propoxylated glycerine, in
  • the alkoxylated derivatives of any of the above polyhydric alcohols are also suitable for use herein.
  • the polyhydric alcohol is selected from glycerine, polyethylene glycol, hexane triol, butane- 1,4- diol, butoxytriol, erythritol, xylitol, and mixtures thereof.
  • Examples include (a) monosaccharides, e.g., ribose, ribulose, arabinose, xylose, xylulose, lyxose, allose, altrose, glucose, mannose, gulose, idose, galactose, talose, psicose, fructose, sorbose, tagatose, and mixtures thereof, (b) disaccharides, e.g., lactose, maltose, isomaltose, cellose, trehalose, sucrose, and mixtures thereof, (c) oligosaccharides, e.g., malto-oligosaccharide, cello- oligosaccharide, and mixtures thereof; and (d) mixtures thereof.
  • monosaccharides e.g., ribose, ribulose, arabinose, xylose, xylulose, lyxose
  • the composition comprises one or more elastase or partial elastase enzymes and a polyglycerol containing at least 15 carbon atoms.
  • polyglycerols containing at least 15 carbon atoms are pentaglycerol, hexaglycerol, heptaglycerol, octaglycerol, nonaglycerol and decaglycerol.
  • the polyglycerol contains at least 21 carbon atoms, at least 27 carbon atoms, or at least 30 carbon atoms.
  • the polyglycerol is decaglycerol.
  • the polyglycerol contains a maximum of 20, 30, 50, 75, or 100 carbon atoms.
  • Polyglycerols can be linear, branched or cyclic compounds in various embodiments of the invention. Polyglycerols can be used either individually or the composition can comprise multiple polyglycerols differing in moleulcar weight and/or structure, etc.
  • the composition further comprises a polyol.
  • the polyol can be a monomeric polyol containing, e.g., at most 6 carbon atoms or a polymeric glycol.
  • polyols containing at most 6 carbon atoms are glycerol, ethylene glycol, propylene glycol, butylene glycol and sorbitol.
  • polymeric glycols are polyethylene glycol 200 and 400.
  • the composition is an oil-in-water emulsion. In other embodiments the composition is a water-in-oil emulsion.
  • Suitable polyglycerols, polyols, and other components are described in U.S. S.N. 10/922,790, which is incorporated herein by reference in its entirety.
  • the present invention contemplates compositions prepared as described in U.S. S.N. 10/922,790, but wherein the composition comprises one or more elastase or partial elastase enzymes.
  • compositions of the present invention comprising an enzyme may include an agent that serves as an enzyme stabilizer in an amount effective to reduce the rate at which the enzyme permanently loses activity (e.g. , becomes denatured or degraded) prior to use of the composition.
  • an agent that serves as an enzyme stabilizer in an amount effective to reduce the rate at which the enzyme permanently loses activity (e.g. , becomes denatured or degraded) prior to use of the composition.
  • presence of the stabilizer increases the time in which the enzyme loses 50% of its initial elasto lytic or elastin cleaving activity by at least 25%, 50%, 75%, or 100% relative to the time in which the enzyme would lose 50% of its initial activity in the absence of the stabilizer.
  • a polyhydric alcohol may serve as a stabilizer.
  • the composition comprises an osmoprotectant. Suitable osmo-protectants are known in the art.
  • Examples include alanine, glycine, serine, proline, carnitine, taurine, trimethylamineoxide, tricine, dimethyl proline, gamma-butyro betaine, beta-alanine betaine, valine betaine, lysine betaine, ornithine betaine, alanine betaine, glutamic acid betaine, and phenyalanine betaine.
  • stabilizers include formate, acetate, propionate, glycolate, glycerate, malonate, succinate, adipate, malate, tartarate, sulfo-succinate, sulfate, phosphate, citrate, isethionate, glycerol phosphate, benzoate, glutarate, pimelate, suberate, phytate, and mixtures thereof, preferably formate, glycolate, adipate, malonate, sulfate, phosphate, succinate, and mixtures thereof.
  • suitable ingredients which are typically incorporated in their salt form, wherein the cation is any monovalent ion (e.g.
  • an alkaline metal such as sodium, potassium, lithium, ammonium, tris[hydroxymethyl]aminomethane (TRIS), acetamide monoethanolamine (AMEA), and triethanolamine (TEA)
  • TIS tris[hydroxymethyl]aminomethane
  • AMEA acetamide monoethanolamine
  • TAA triethanolamine
  • ribonic acid ribulonic acid, arabinonic acid, xylonic acid, xylulonic acid, lyxonic acid, allonic acid, altronic acid, gluconic acid, mannonic acid, gulonic acid, idonic acid, galactonic acid, talonic acid, glucoheptonic acid, psiconic acid, fructonic acid, sorbonic acid, tagatonic acid, glucuronic acid, and mixtures thereof; oxidized disaccharides, e.g. lactobionic acid, maltobionic acid, isomaltobionic acid, cellobionic acid, and mixtures thereof; oxidized oligosaccharides, e.g.
  • oxidized malto-oligosaccharide oxidized cello- oligosaccharide, and mixtures thereof
  • oxidized polysaccharides e.g. oxidized cellulose; chitin; gum arabic; gum karaya; gum xanthan; oxidized gum guar; oxidized locust bean gum; oxidized agars; oxidized algins; oxidized gellan gum; and mixtures thereof, and mixtures of any of the foregoing.
  • the stabilizing agent is a protease inhibitor that inhibits the activity of an elastase or partial elastase present in the composition.
  • the enzyme thus remains in an inactive, but activatable state. Removal of the inhibitor or reversal of the inhibition restores activity.
  • the amount of the inhibitor may fall within any of the ranges recited above for the enzyme. In certain embodiments, the amount ranges from 0.00005% to 5%, e.g., from 0.005% to 2%, such as from 0.025% to 1% by weight of the composition.
  • the inhibitor has a K 1 (i.e., inhibitor concentration at which 50% inhibition is observed, which is given by [E][I]/[EI] where [E] is enzyme concentration, [I] is inhibitor concentration, and [EI] is the concentration of the enzyme-inhibitor complex) between 10 "8 M and 10 "5 M, e.g., between 5x10 7 M and 5x10 6 M, or between 10 "7 M and 10 "6 M.
  • the ratio of inhibitor to enzyme in the composition on a molar basis is between .5 and 50, between 1 and 20, or between 1 and 10.
  • the K 1 of the inhibitor may be selected so as to achieve effective inhibition while the composition is being stored prior to use, while permitting sufficient dissociation of the inhibitor from the enzyme to allow effective activity following contact of the composition with the skin. It will be appreciated that the inhibitor will generally be a reversible inhibitor.
  • the inhibitor may be selective or may be capable of inhibiting a broad range of enzymes. Agents that act as general or selective inhibitors of serine, cysteine, or metalloproteases are known in the art.
  • the inhibitor may have specificity for a group of elastases or for a single elastase or partial elastase.
  • the inhibitor could have a K 1 with respect to an elastase or partial elastase present in a composition of the invention that is at least 10-fold, at least 50-fold, at least 100-fold, at least 1, 000-fold, or at least 10,000-fold greater than its K 1 with respect one or more other elastases (e.g., pancreatic elastase, neutrophil elastase, etc.).
  • elastases e.g., pancreatic elastase, neutrophil elastase, etc.
  • the inhibitor may be a protein, peptide, small molecule, etc. It may be naturally occurring or artificial (i.e., having a structure invented by man). A variety of naturally occurring proteins and peptides inhibit elastases of various types. A naturally occurring inhibitor may come from the same species as the elastase that it inhibits or from a different species. Examples of naturally occurring inhibitors from mammals include ⁇ -1 proteinase inhibitor, ⁇ 2-macroglobulin, and the pancreatic Kunitz inhibitor, also known as aprotinin, bovine basic pancreatic trypsin inhibitor (BPTI), and trypsin-kallikrein inhibitor.
  • BPTI bovine basic pancreatic trypsin inhibitor
  • Suitable inhibitors from microorganisms include elastatinal, elasnin, chymostatin, leupeptin, phosphoramidon, etc. (Bieth, supra). Streptomyces serine protease inhibitor (Adekoya, OA, et al., J Struct Biol. 2006 Feb;153(2):129-44) and BTL.009 (U.S. Pat. No. 6,294,648) are also of interest.
  • Synthetic reversible inhibitors include a variety of peptide aldehydes, peptide boronic acid derivatives, acyl carbazates, peptidyl carbamates or thiocarbamates (see, e.g., K.
  • elastase inhibitor could be attached to any of a wide variety of polymers known in the art, optionally via a spacer or linker. In certain embodiments a homobifunctional or heterobifunctional linker is used to attach an inhibitor to a polymer. In certain embodiments of the invention the inhibitor is a polymer bound elastase inhibitor such as those described in U.S. Pat. Nos. 5,162,307.
  • compositions may include a suitable amount of an alkali earth metal salt effective to enhance the stability of the enzyme.
  • the suitable amount may be less than 5%, e.g., less than 1%, less than 0.1%, less than 0.05%, less than 0.02%, less than 0.015%, or less than 0.01% by weight of the composition.
  • Alkali earth metals include magnesium, calcium, and strontium.
  • compositions may include an agent that enhances the activity of the enzyme.
  • the agent increases catalytic activity of the enzyme even under optimal reaction conditions.
  • examples include tripeptide compounds, GIu- Arg- Pro-amide (ERPm), D-Pro-Thr-Trp-amide (dPTWm) and thioproline-Thr-Trp (tPTW) (Hiwasa, T, et al, FEBS Lett. 386(l):47-50, 1996).
  • Metalloproteinases bind a metal ion such as Zn 2+ or Ca 2+ in their active site.
  • the composition comprises a divalent metal ion such as Zn 2+ or Ca 2+ in an amount sufficient to promote activity of a metalloproteinase in the composition.
  • the composition comprises a chelating agent such as EDTA.
  • the chelating agent binds some or essentially all of the divalent cation ⁇ e.g., Zn 2+ or Ca 2+ ), thereby inhibiting activity of the enzyme.
  • a metal ion may be added to the composition prior to contacting with the skin, or a sufficient amount of metal ion may be naturally present in the skin to promote activity.
  • the composition does not contain a chelating agent.
  • compositions of the invention may further comprise a pH regulating agent in an amount that is sufficiently effective to adjust the pH of the composition to fall within a range suitable for activity of the enzyme and/or to adjust the pH of the skin environment to one that is suitable for activity of the enzyme.
  • a pH regulating agent in an amount that is sufficiently effective to adjust the pH of the composition to fall within a range suitable for activity of the enzyme and/or to adjust the pH of the skin environment to one that is suitable for activity of the enzyme.
  • the desired pH will range from 5.5 to 9, e.g., from 6 to 8.5, e.g., from 6.5 to 8, e.g., from 7 to 8.
  • the amount of the pH regulator to be added will at least in part depend on the quantity and type of other ingredients selected to make the compositions as well as on the pH at which the enzyme(s) in the composition display suitable levels of activity.
  • the concentration of the pH regulating agent in the final composition may range from about 0.01% to about 10% by weight. In certain embodiments, the concentration of the agent is about 0.1% to about 10% by weight. In certain embodiments, the concentration of the agent is about 1% to about 10% by weight. In certain embodiments, the concentration of the agent is about 0.1% to about 5% by weight. In certain embodiments, the concentration of the agent is about 0.1% to about 3% by weight. In certain embodiments, the concentration of the salt is about 1% to about 5% by weight.
  • Exemplary pH regulating agents include a variety of acceptable acids and bases (e.g., hydrochloric acid, phosphoric acid, amines, hydroxides (e.g., sodium salts of hydroxides), etc.)
  • the inventive compositions may comprise a buffering agent in an amount that is sufficient to maintain a desired pH.
  • Buffering agents that are suitable for use herein include histidine, 1 ,4-piperizinediethanesulfonic acid (PIPES), tris[hydroxymethyl]aminomethane (TRIS), [bis-(2-hydroxyethyl)imino]-tris- [(hydroxymethyl)methane] (BIS-TRIS), l-[4-(2-hydroxyethyl)-l-piperazinyl]e- thane-2- sulfonic acid (HEPES), N-(2-acetamido)-2-iminodiacetic acid (ADA), 2-[(2-amino-2- oxoethyl)amino]ethanesulfonic acid (ACES), N,N-bis(2-hydroxethyl)-taurine (BES), morpholinopropanesulfonic acid (MOPS), N- [2-hydroxy-l,l-bis(hydromethyl)ethyl] -taurine (TES), 3-[bis(2-
  • pH regulating agents useful in the present invention include aluminum glycinate, aluminum lactate, ammonium acetate, ammonium carbonate, ammonium hexafluorophosphate, Ammonium Lactate, Ammonium Phosphate, Boric Acid, Calcium Carbonate, Calcium Phosphate, Cyclohexylamine, Diammonium Citrate, Diammonium Phosphate, Diethanolamine Bisulfate, Diethylamine, Diethyl Ethanolamine, Disodium Fumarate, Disodium Phosphate, Disodium Pyrophosphate, Ectoin, Ethanolamine HCI, Glycine, Hydroxyethylpiperazine Ethane Sulfonic Acid, Lauryl p-Cresol Ketoxime, Lithium Fluoride, Magnesium Acetate, M EA-Borate, MIPA-Borate, PEG-114 Methylether Polyepsilon Capralactone, Phosphonobutane
  • Exemplary pH adjusting agents useful in accordance with the present invention include Acetic Acid, Acetyl Mandelic Acid, Adipic Acid, Aluminum Triformate, 2-Aminobutanol, Aminoethyl Propanediol, Aminomethyl Propanediol, Aminomethyl Propanol, Ammonia, Ammonium Bicarbonate, Ammonium Carbamate, Ammonium Carbonate, Ammonium Glycolate, Ammonium Hydroxide, Ammonium Phosphate, Ascorbic Acid, Azelaic Acid, Benzoic Acid, Bis-Hydroxyethyl Tromethamine, Calcium Citrate, Calcium Dihydrogen Phosphate, Calcium Hydroxide, Calcium Oxide, Citric Acid, Diethanolamine, Diethanolamine Bisulfate, Diisopropanolamine, Diisopropylamine, Dimethyl MEA, Dioleoyl Edetolmonium Methosulfate, Dipotassium Phosphate, Diprop
  • compositions of the present invention may, in some embodiments, further contain additional components known or believed to provide a benefit when present in skin care products, e.g., topically applied skin care products.
  • additional components e.g., topically applied skin care products.
  • Such components are referred to as being "active”.
  • Other components may be inactive but may play a role in providing bulk, desirable texture, spreadability, etc.
  • Any optional component(s) should be physically and chemically compatible with the enzyme(s) of the composition (i.e., should not significantly impair stability), and should not otherwise unduly impair product stability, aesthetics or performance. Suitable components are known in the art and are only briefly described here. See, e.g., Barel, A., Payne, M., Maibach, H.
  • compositions of the invention may contain additional active agents.
  • the composition contains at least one additional active agent in an amount ranging from 0.00001% to 0.01%, 0.01% to .1%, .1% to 1%, or 1% to 10%.
  • Additional active agents may include vitamins, proteins, amino acids, small molecules, drugs, plant extracts, sunscreen agents, etc.
  • the composition includes one or more vitamins to nourish the skin.
  • vitamins include vitamin A, vitamin Bi (thiamine), vitamin B 2 (riboflavin), vitamin B 3 (niacin), vitamin B compounds ⁇ e.g., vitamin B3 compounds such as those described in U.S. Pub. No.
  • retinoids see below
  • panthenol niacinamide
  • retinol retinyl propionate
  • retinyl palmitate retinoic acid
  • flavonoids chalcones, chromones, flavones, isoflavones
  • vitamin B 4 adenine
  • vitamin B5 pantothenic acid
  • vitamin B 6 pyridoxine
  • vitamin B 7 biotin
  • vitamin B9 folic acid
  • vitamin B12 cyanocobalamin
  • vitamin C ascorbic acid
  • vitamin D ergocalciferol
  • vitamin E tocopherol acetate
  • vitamin K and combinations thereof.
  • the composition includes vitamin E.
  • the composition include a one of the B vitamins ⁇ e.g., vitamin B5).
  • the inventive composition may include 0.0001% to approximately 5% by weight of the vitamin.
  • the composition includes approximately 0.001% to approximately 5% by weight of the vitamin.
  • the composition includes approximately 0.0005% to approximately 3% by weight of the vitamin.
  • the composition includes approximately 0.001% to approximately 2% by weight of the vitamin.
  • the inventive compositions may include a protein, peptide, or amino acid. Such components may be added to the inventive composition to nourish the skin, impart a desired characteristic to skin, or impart a desired characteristic to the composition (e.g. , thickening the composition).
  • Exemplary proteins that may be added to the composition include elastin, fibrillin, fibronectin, laminin, keratin, proteoglycans, wheat protein, gelatin, collagen, silk, soy protein, wheat protein, rice protein, corn protein, jojoba protein, milk protein, whey protein, casein, albumin, egg protein, and fragments thereof.
  • derivatives, mutants, fusion proteins, fragments, or combinations of any of these proteins may also be included in the inventive cosmetic composition.
  • any of the twenty natural amino acids may be included in the inventive composition.
  • the amino acid used in the inventive compostion is selected from the group consisting of phenylalanine, valine, tryptophan, threonine, isoleucine, methionine, cysteine, homocysteine, histidine, arginine, lysine, leucine, proline, serine, aspartate, glutamate, glutamine, and combinations thereof.
  • Proteins, peptides, or amino acids may be added to the inventive composition up to 10% by weight of the composition. In certain embodiments, the proteins, peptides, or amino acids are included in the composition in a range from about 0.0001% to about 10%.
  • the proteins, peptides, or amino acids are included in the composition in a range from about 0.01% to about 5%. In certain embodiments, the proteins, peptides, or amino acids are included in the composition in a range from about 0.1% to about 3%. In certain embodiments, the proteins, peptides, or amino acids are included in the composition in a range from about 0.1% to about 2%. In certain embodiments, the proteins, peptides, or amino acids are included in the composition in a range from about 5% to about 10%. In certain embodiments, the proteins, peptides, or amino acids are included in the composition in a range from about 1% to about 5%. [00138] In certain embodiments, the compositions may include an extract from a plant.
  • Extracts may be added to the inventive composition to nourish the skin, provide a fragrance or color to the composition, impart a desired characteristic to the treated skin, or impart a desired characteristic to the composition.
  • Extracts may be prepared from any part of a plant, including leaves, fruit, flower, grass, vegetable, nut, root, stem, bark, and the like.
  • An extract may be prepared by using any solvent and optionally heat.
  • the extraction solvents are typically polar organic solvents including, for example, lower alcohols such as methanol, ethanol, and the like, propylene glycol, 1,3-butylene glycol and glycerine, and water. These solvents may be used singly or in combination. Any extraction technique known in the art may be used to prepare the plant extract.
  • the plant extract is simply ground plant material.
  • the extent of grinding of the plant material may be determined by the formulator. Any plant may be used to prepare a plant extract. Exemplary plants that can be used to prepare plant extracts include, but are not limited to, birch, rosemary, arnica, hamamelis, camomile, sage, St.
  • the plant extract When the plant extract is in a liquid form, the plant extract is typically used in an amount ranging from 0.001 to 10.0% by weight of the total composition, calculated as a residue obtained after distillation of extraction solvent therefrom. In certain embodiments, the plant extract is used in an amount ranging from 0.01 to 1.0% by weight of the total compositon, calculated as a residue obtained after distillation of extraction solvent therefrom.
  • the composition may include a sunscreen agent to protect the skin from the damaging ultraviolet rays of the sun.
  • the sunscreen agent protects the skin from damaging UV-A and/or UV-B rays.
  • the sunscreen agent absorbs light in the wavelength range from approximately 150 nm to approximately 400 nm. In certain embodiments, the sunscreen agent absorbs light in the wavelength range from approximately 250 nm to approximately 390 nm.
  • sunscreen agents useful in the inventive hair care compositions include /?-aminobenzoic acid (PABA), PABA-derivatives (e.g., allantoin PABA, butyl PABA, dimethyl PABA ethyl cetearyldimonium tosylate, ethyl dihydroxypropyl PABA, ethylhexyl dimethyl PABA, N- ethyl-3-nitro PABA, ethyl PABA, glyceryl PABA, PEG-25 PABA, pentyl dimethyl PABA, and triP ABA panthenol), avobenzone, cinoxate, dioxybenzone, homosalate, menthyl anthranilate, octocrylene, octyl methoxycinnamate, octyl salicylate, oxybenzone, padimate O, phenylbenzimidazole sulfonic acid, sulisobenzone,
  • the inventive may include 0.0001% to approximately 5% by weight of the sunscreen agent.
  • the composition includes approximately 0.001% to approximately 5% by weight of the sunscreen agent.
  • the composition includes approximately 0.0005% to approximately 3% by weight of the sunscreen agent.
  • the composition includes approximately 0.001% to approximately 2% by weight of the sunscreen agent.
  • the inventive compositions may include preservatives, antioxidants, and/or chelating agents to extend the shelf- life and/or prevent the degradation of the components of the composition.
  • Antioxidants used in the compositions include reducing agents and/or free radical scavengers.
  • Exemplary preservative, antioxidants, and chelating agents useful in the inventive hair care compositions include vitamin C (ascorbic acid), glutathione, lipoic acid, uric acid, carotenes ⁇ e.g., beta-carotene), alpha-tocopherol (vitamin E), ubiquinol (coenzyme Q), melatonin, ethylenediamine tetraacetic acid (EDTA) and salts thereof, citric acid and salts thereof, EGTA, aminotrimethylene phosphonic acid, resveratrol, flavonoids, lycophene, propyl gallate, tertiary butylhydroquinone, butylated hydroxyanisole, butylated hydroxytoluene, benzoates, nitrites, nitrates, sulfites, calcium propionate, sodium bisulfite, potassium hydrogen sulfite, benzyl alcohol, methyl paraben, propyl paraben, imi
  • antioxidants include Acer Palmatum leaf extract, acetamidocaproic acid, acetyl benzoyloxy prasterone, Acetyl Cysteine, Agrimonia Eupatoria Root Extract, Aminoethanesulfmic Acid, Aminopropyl Ascorbyl Phosphate, Aminopropyl tocopheryl phosphate, anserine, Arbutin, Alpha- Arbutin, argon, ascorbic acid, ascorbic acid aolypeptide, Ascorbyl Dipalmitate, Ascorbyl Glucoside, Ascorbyl Methylsilanol Pectinate, Ascorbyl Palmitate, Ascorbyl Stearate, Ascorbyl Tetraisopalmitate, Ascorbyl Tocopheryl Maleate, Asiaticoside, Avena Sativa (Oat) Kernel Extract, Bacillus/Rice Bran Extract/Soybean Extract Ferment Filtrate, BHA, BHT, Bis
  • Exemplary chelating agents useful in accordance with the present invention include Acrylic Acid/Acrylamidomethyl Propane Sulfonic Acid Copolymer, Beta- Alanine Diacetic Acid, Aminotrimethylene Phosphonic Acid, Calcium Disodium EDTA, Citric Acid, Citrus Medica Vulgaris Fruit Extract, Cyclodextrin, Cyclohexanediamine Tetraacetic Acid, Diammonium Citrate, Diammonium EDTA, Diethylenetriamine Pentamethylene Phosphonic Acid, Dipotassium EDTA, Disodium Azacycloheptane Diphosphonate, Disodium EDTA, Disodium Pyrophosphate, EDTA, Etidronic Acid, Galactaric Acid, Galacturonic Acid, Alpha-Glucan, Gluconic Acid, Glucuronic Acid, HEDTA, Humic Acids, Hydroxypropyl Cyclodextrin, Lauroyl Ethylenediamine Triacetic Acid, Methyl Cyclodextr
  • the inventive may include 0% to approximately 5% by weight of the preservative, antioxidant, or chelating agent.
  • the composition includes approximately 0.0001% to approximately 5% by weight of the preservative, antioxidant, or chelating agent.
  • the composition includes approximately 0.0005% to approximately 3% by weight of the preservative, antioxidant, or chelating agent.
  • the composition includes approximately 0.001% to approximately 2% by weight of the preservative, antioxidant, or chelating agent.
  • compositions may comprise an emollient (substance that softens and/or soothes the skin).
  • the composition contains an emollient in an amount ranging from 0.01% to 10%, e.g., from 0.1% to 8%, e.g., from 0.5% to 5%.
  • Suitable emollient materials include branched chain hydrocarbons having an average molecular weight of from about 100 to about 15,000, e.g., from about 100 to 1000. Other suitable compounds are described in U.S. Pub. No. 20030175232.
  • Examples include methyl isostearate, isopropyl isostearate, isostearyl neopentanoate, isononyl isononanoate, isodecyl octanoate, isodecyl isononanoate, tridecyl isononanoate, myristyl octanoate, octyl pelargonate, octyl isononanoate, myristyl myristate, myristyl neopentanoate, myristyl octanoate, myristyl propionate, isopropyl myristate and mixtures thereof.
  • Others include vegetable oils and hydrogenated vegetable oils, e.g. safflower oil, coconut oil, cottonseed oil, menhaden oil, palm kernel oil, palm oil, peanut oil, soybean oil, rapeseed oil, linseed oil, rice bran oil, olive oil, pine oil, sesame oil, sunflower seed oil, meadowfoam seed oil, shea butter, partially and fully hydrogenated oils from the foregoing sources, and mixtures thereof.
  • vegetable oils and hydrogenated vegetable oils e.g. safflower oil, coconut oil, cottonseed oil, menhaden oil, palm kernel oil, palm oil, peanut oil, soybean oil, rapeseed oil, linseed oil, rice bran oil, olive oil, pine oil, sesame oil, sunflower seed oil, meadowfoam seed oil, shea butter, partially and fully hydrogenated oils from the foregoing sources, and mixtures thereof.
  • Branched chain hydrocarbon emollients for use herein include isododecane, isohexadecane, isoeicosane, isooctahexacontane, isohexapentacontahectane, isopentacontaoctactane, and mixtures thereof.
  • emollients for use herein are perhydroxysqualene, perflouropolyethers, methylglucosesesquistearate, tributylcitrate, glycerol stearylcitrate, butylene glycol dicaprylatedicaprate, capric caprylic triglyceride, lanolin, allantoin, mineral oil, stearyl alcohol, Slippery Elm, Chickweed, Flax Seed, Marshmallow root, Comfrey, and mixtures thereof.
  • exemplary emollients include include silicone oils; phenylsilicones; silicone resins; silicone gums; mineral oils such as paraffin oil or vaseline oil; oils of animal origin such as perhydrosqualene, lanolin; oils of plant origin such as liquid triglycerides, e.g., sunflower oil, corn oil, soybean oil, rice oil, jojoba oil, babusscu oil, pumpkin oil, grapeseed oil, sesame oil, walnut oil, apricot oil, macadamia oil, avocado oil, sweet almond oil, lady's- smock oil, castor oil, triglycerides of caprylic/capric acids, olive oil, peanut oil, rapeseed oil, and coconut oil; synthetic oils such as purcellin oil, isoparaffins, linear and/or branched fatty alcohols and fatty acid esters, preferably guerbet alcohols having 6 to 18, preferably 8 to 10, carbon atoms; esters of linear (C 6 -C 13 )
  • Exemplary plant derived oils useful as emollients include almond oil, apricot kernel oil, arnica oil, avocado oil, babusscu oil, black cumin seed oil, borage seed oil, castor oil, coconut oil, colza oil, corn oil, cotton seed oil, cowslip oil, evening primrose seed oil, grapeseed oil, hazelnut oil, hemp seed oil, jojoba oil, kukui nut oil, lady's-smock oil, linseed oil, macadamia nut oil, menhaden oil, neem seed oil, olive oil, palm oil, palm seed oil, peanut oil, pine oil, pomegranate seed oil, pumpkin seed oil, rape oil, rapeseed oil, rice oil, rice palm oil, rosehip seed oil, safflower oil, seabuckthorn oil, sesame oil, sesame seed oil, shea nut oil, soya oil, soybean oil, sunflower oil, tamanu oil, vitamin E oil, wheat germ oil, and walnut
  • Exemplary animal-derived oils include squalene, perhydrosqualene, and lanolin.
  • Exemplary synthetic oils include purcellin oil, isoparafiins, linear or branched hydrocarbons, linear or branched fatty alcohols, linear or branched fatty acids, and linear or branched fatty acid esters.
  • Examplary mineral oils include paraffin oil and vaseline oil.
  • the oil is a glyceryl ester which is primarily a fatty acid mono-, di-, or triglyceride modified by reaction with other alcohols, for example, acetylated castor oil, glyceryl stearate, glyceryl dioleate, glyceryl distearate, glyceryl trioctanoate, glyceryl distearate, glyceryl linoleate, glyceryl myristate, glyceryl isostearate, PEG castor oils, PEG glyceryl oleates, PEG glyceryl stearates, PEG glyceryl tallowates, etc.
  • other alcohols for example, acetylated castor oil, glyceryl stearate, glyceryl dioleate, glyceryl distearate, glyceryl trioctanoate, glyceryl distearate,
  • the oil is a fluorinated oil.
  • fluorinated oils include fluoro guerbet esters or perfluropolyethers. Suitable perfluoropolyethers are disclosed in U.S. Patents 5,183,589, 4,803,067, and 5,183,588, all of which are hereby incorporated by herein reference.
  • the oil is a sorbitan derivative.
  • Exemplary sorbitan derivatives include PEG sorbitan beeswax, PEG sorbitan isostearate, PEG sorbitan lanolate, PEG sorbitan laurate, PEG sorbitan oleate, PEG sorbitan palmitate, PEG sorbitan stearate, polysorbates, sorbitan trioleates, sorbitan sesquioleates, sorbitan stearates, sorbitan tristearates, etc.
  • the emollient is used in the composition in an amount ranging from about 1% to about 50% by weight. In certain embodiments, the emollient is used in the hair care composition in an amount ranging from about 1% to about 20% by weight. In certain embodiments, the emollient is used in the hair care composition in an amount ranging from about 1% to about 10% by weight.
  • Compositions may include a humectant (substance that attracts moisture to the skin).
  • a humectant is a hydrogroscopic substance. It is typically a chemical compound containing hydrophilipic groups such as hydroxyl groups, amines, carboxylates, etc. Humectants are typically found in skin care compositions to provide a moisturizing quality to the composition. The humectants attracts and holds moisture in the skin.
  • humectants useful in the inventive compositions include acetyl glucosamine, ascorbic acid, diglycerin, methylglucamine, methylpropanediol, phytantriol, raff ⁇ nose, riboflavin, maltose, glycerin, glycerol, hyaluronic acid, atelocollagen, propylene glycol, glyceryl triacetate, polyols, sorbitol, sorbityl acetate, sorbityl furfural, sorbityl silanediol, xylose, tromethamine, zinc glucoheptonate, xylitol, maltitol, polydextrose, quillaia, lactic acid, sodium lactate, triacetin, urea, acetyl arginine, acetyl hydroxyproline, Adansonia Digitata fruit extract, Adenophora Stricta root extract, Ag
  • the humectant is used in the hair care composition in an amount ranging from about 0.01% to about 30% by weight. In certain embodiments, the humectant is used in the hair care composition in an amount ranging from about 0.1% to about 20% by weight. In certain embodiments, the humectant is used in the hair care composition in an amount ranging from about 1% to about 25% by weight. In certain embodiments, the humectant is used in the hair care composition in an amount ranging from about 1% to about 10% by weight. In certain embodiments, the humectant is used in the hair care composition in an amount ranging from about 1% to about 5% by weight.
  • Compositions may contain one or more emulsifiers, surfactants, silicone oils, or gums such as those described in U.S. Pub. No. 20030175232.
  • Compositions may contain a thickening agent, e.g., a polymeric thickener. Exemplary amounts range from 0.1% to 10%, e.g., from 0.5% to 8%, such as from 1% to 5%.
  • Exemplary polymeric thickeners suitable for use herein may have a number average molecular weight of greater than 50,000, preferably greater than 100,000. Non-ionic thickening agents and anionic thickening agents, and mixtures thereof may be used.
  • Suitable non-ionic thickening agents include polyacrylamide polymers, crosslinked poly(N-vinylpyrrolidones), polysaccharides, natural or synthetic gums, polyvinylpyrrolidone, and polyvinylalcohol.
  • Suitable anionic thickening agents include acrylic acid/ethyl acrylate copolymers, carboxyvinyl polymers and crosslinked copolymers of alkyl vinyl ethers and maleic anhydride, polyacrylamide/ AMPS polymers such as polyacrylamide and isoparaffin and laureth-7, and acrylic acid/ethyl acrylate copolymers and the carboxyvinyl polymers, including hydrophobically modified derivatives thereof.
  • the thickening agent is a polysaccharide. In certain embodiments, the thickening agent is a protein. In certain embodiments, the thickening agent is a low melting point wax. Examples of low melting point waxes include emulsifying wax, and fatty alcohols having the formula R-OH, wherein R is a straight or branched chain, unsaturated or unsaturated alkyl moiety having from about 4 to 35, more preferably about 6 to 22, carbon atoms. Examples of fatty alcohols include stearyl alcohol, cetearyl alcohol, behenyl alcohol, and the like. In certain embodiments, the thickening agent is a synthetic polymeric thickener.
  • Examples of synthetic polymeric thickeners include polymers of acrylic acid, methacrylic acid and their simple esters, which may be co-polymerized with one or more organic groups such as ethoxylated or propoxylated polymeric moieties.
  • Examples of such synthetic polymeric thickeners include acrylamides copolymer, acrylates/behenth-25 methacrylate copolymer, acrylates ClO 30 alkyl acrylate crosspolymer, acrylates ceteth-20 itaconate copolymer, acrylates/steareth-50 acrylate copolymer, acrylates/stearyl methacrylate copolymer, acrylates/vinyl isodecanoate crosspolymer, or mixtures theref.
  • the thickening agent is a natural polymeric thickener.
  • the thickening agent is a cellulosic thickener.
  • cellulosic thickeners include cellulose gum as well as alkyl and hydroxylalkyl derivatives of celluloses and methyl or ethyl cellulose, such as hydroxyethylcellulose, hydroxypropylcellulose, hydroxyethyl ethylcellulose, hydroxybutyl celluose, or mixtures thereof.
  • viscosity modifying agents include carrageenan, quince mucilage, carboxyvinyl polymer, xanthane gum, acrylates/bis-hydroxypropyl dimethicone crosspolymer, ammonium phosphatidyl rapeseedate, C40-60 acid, Citrus Aurantium Dulcis (Orange) Peel Extract, Cocamide Methyl MEA, Diallyloxyneohexyl Zirconium Tridecanoate, Dimethyldibenzylidene Sorbitol, Ethylhexyl AcrylateNP/Dimethicone Methacrylate Copolymer, Glycereth-7/IPDI Copolymer, Hydrogenated Didodecene, Hydrogenated Polydodecene, Hydrogenated Styrene/Isoprene Copolymer, Hydrogenated Tridodecene, Hydroxypropyl Dimethiconylpropyl Acrylates Copolymer
  • Adipate/Caprate/Caprylate/Heptanoate Pentaerythrityl Dioleate, Pentaerythrityl Distearate, Pentaerythrityl Hydrogenated Rosinate, Pentaerythrityl
  • ingredients found in other categoreis of agents described herein may be used as thickening agents in the inventive compositions.
  • gums e.g., xanthan, karaya, gellan, wellan, arabic, carrageanan, biosaccharide gum-1, and locust bean.
  • alginate and agarose are also suitable.
  • Surfactants may be used in the inventive compositions. Such agents may work to make the final composition homogenous or help to solubilize certain ingredients of the composition.
  • the surfactant is an anionic surfactant.
  • the surfactant is a cationic surfactant.
  • the surfactant is a nonionic surfactant.
  • the surfactant is a zwitterionic surfactant.
  • the surfact is an amphoteric surfactant.
  • the surfactant used in the inventive composition may be chosen based on its HLB (Hydrophile-Lipophile Balance) value.
  • the surfactant has an average HLB of less than or equal to about 7. In certain embodiments, the surfactant has an average HLB of less than or equal to about 10. In certain embodiments, the surfactant has an average HLB of greater than or equal to about 10. In certain embodiments, the surfactant has an average HLB of ranging from about 10 to about 15. In certain embodiments, the surfactant has an average HLB of ranging from about 10 to about 12. In certain embodiments, the surfactant has an average HLB of ranging from about 12 to about 18. In certain embodiments, the surfactant has an average HLB of ranging from about 14 to about 16. The HLB System was introduced in the late 1940's by ICI Americas Inc.
  • Examplary surfactants useful in the present invention include sodium dioctyl sulfo succinate, sodium dodecyl sulfate, cocoamidopropyl betaine, and sodium laureth sulfate, alkyl and alkyl ether sulfates (e.g., sodium coconut alkyl Methylene glycol ether sulfate; lithium tallow alkyl triethylene glycol ether sulfate; sodium tallow alkyl hexaoxyethylene sulfate), succinamates, sulfosuccinamates (e.g., disodium N-octadecyl-sulfosuccinamate, tetrasodium N-(l,2-dicarboxyethyl)-N-octadecylsulfosuccinamate, diamyl ester of sodium sulfosuccinic acid, dihexyl ester of sodium sulf
  • alkyl amphoglycinates e.g., cocoamphoglycinate, lauroamphocarboxyglycinate, , cocoamphocarboxyglycinate
  • alkyl amphopropionates e.g., isostearoamphopropionate, cocoamphocarboxypropionic acid
  • alkyl ethoxylated sulfates alkyl sulfates
  • aliphatic quaternary ammonium compounds e.g., tallow propane diammonium dichloride, dialkyldimethylammonium chlorides, ditallowdimethyl ammonium chloride, ditallowdimethyl ammonium methyl sulfate, dihexadecyl dimethyl ammonium chloride, di(hydrogenated tallow) dimethyl ammonium chloride, dioctadecyl dimethyl
  • coco dimethyl carboxymethyl betaine lauryl dimethyl carboxymethyl betaine, lauryl dimethyl alphacarboxyethyl betaine, cetyl dimethyl carboxymethyl betaine, lauryl bis-(2-hydroxyethyl) carboxy methyl betaine, stearyl bis-(2-hydroxypropyl) carboxymethyl betaine, oleyl dimethyl gamma-carboxypropyl betaine, lauryl bis-(2- hydroxypropyl) alpha-carboxyethyl betaine), sulfo betaines (e.g., coco dimethyl sulfopropyl betaine, stearyl dimethyl sulfopropyl betaine, lauryl dimethyl sulfoethyl betaine, lauryl bis(2- hydroxyethyl) sulfopropyl betaine), alkyl amido betaines, 4- [N ,N-di(2 -hydroxy ethyl)-N- o
  • inventive compositions contain a surfactant in the range of from about 0.01% to about 20% by weight. In certain embodiments, the composition contains a surfactant in the range from about 0.1% to about 10% by weight.
  • compositions of the present invention comprise an inhibitor of an endogenous, skin-associated elastase.
  • Endogenous in this context means that the elastase is naturally produced by the individual to whom a composition of the present invention is delivered.
  • Skin-associated in this context means that the elastase is produced by cells that are present either as permanent residents or transiently in the skin. Such cells include dermal fibroblasts, neutrophils, monocytes, macrophages, or other immune system cells.
  • Endogenous, skin-associated elastases include neutrophil elastase, proteinase 3, fibroblast elastase, cathepsins, matrix metalloproteinases (MMPs) with elastolytic activity such as MMP-2, MMP-7 (matrilysin), MMP-9, and MMP-12.
  • MMPs matrix metalloproteinases
  • new elastin may be synthesized in the skin and deposited in the form of normal elastic fibers. This may contribute to improving the condition of the skin, e.g., desirably increasing its elasticity.
  • the inhibitor of an endogenous, skin-associated elastase has a K 1 with respect to an elastase or partial elastase present in a composition of the invention that is at least 10-fold, at least 50-fold, at least 100-fold, at least 1, 000-fold, at least 10, 000-fold greater than its K 1 with respect an endogenous, skin- associated elastase such as neutrophil elastase.
  • the inhibitor may be a small molecule, peptide, protein, etc. It may be a naturally occurring compound or synthetic. It may be a reversible or irreversible inhibitor of an endogenous, skin-associated elastase. It may be selective for the endogenous, skin-associated elastase.
  • Exemplary inhibitors include eglin, eglin c and variants thereof (U.S. Pat. No. 7,001,884, incorporated herein by reference), Kunitz domain peptides such as DPI-14 (U.S. Pat. No. 6,989,369, incorporated herein by reference); compounds of the N-acylaminoamide family such as those described in U.S. Pat. No. 6,998,129 (incorporated herein by reference), etc.
  • the irreversible inhibitor may be a compound that forms a stable bond with a catalytically important amino acid residue of the enzyme, e.g., serine or histidine of the catalytic triad.
  • a catalytically important amino acid residue of the enzyme e.g., serine or histidine of the catalytic triad.
  • examples include diisopropylphosphorylfluoridate, alkyl isocyanates, sulfonyl fluorides, and peptide chloromethylketones. See, e.g., Bieth, supra and U.S. Pat. No. 5,614,489, incorporated herein by reference.
  • the composition comprises a compound or combination of compounds that inhibits synthesis of an endogenous, skin-associated elastase.
  • the enzyme may inhibit activity of a transcription factor such as AP-I or NF -KB.
  • Certain compositions of the present invention comprise an inhibitor of an endogenous, skin-associated collagenase. Many such inhibitors are known in the art.
  • the invention provides a skin care composition comprising a non- endogenous elastase and an inhibitor of an endogenous skin-associated elastase.
  • the invention further provides a skin care composition comprising a non-endogenous elastase and an inhibitor of a non-endogenous skin-associated collagenase.
  • the composition comprises an inhibitor of an MMP enzyme found in the skin.
  • the inhibitor may be selective for a particular class of MMPs, or it may be non-selective.
  • the MMP inhibitor is a small molecule.
  • the inventive composition may comprise a penetration enhancer.
  • a penetration enhancer is an agent or combination of agents that increases delivery of a second agent across the SC or a portion thereof, such that an increased amount of the second agent reaches the dermis relative to the amount of the second agent that would reach the dermis in the absence of the penetration enhancer under otherwise identical conditions.
  • penetration enhancers are known in the art.
  • the penetration enhancer may extract, solubilize, or degrade SC lipids.
  • Exemplary penetration enhancers include various oils, fatty acids, lipases, alcohols, surfactants, etc.
  • pentration enhancers include benzyloxy ethanol, benzyl alcohol, phenoxy ethanol, phenoxy isopropanol, methyl phenoxy ethanol, benzyl glycerol, N-benzyl formide, N-methylpyrrolidone, N-ethyl pyrrolidone, cinnamyl alcohol, phenethyl alcohol, p-methyl benzyl alcohol, butyl cellosolve, methyl carbitol, ethyl carbitol, propyl carbitol, butyl carbitol, diethyleneglycol, diethyl ether, and dipropyleneglycol diethyl ether.
  • the inventive compositions may optionally include a fragrance or perfume.
  • Fragrance ingredient as def ⁇ end by the International Fragrance Association, is "any basic substance used in the manufacture of fragrance materials for its odorous, odor-enhancing, or blending properties. Fragrance ingredients may be obtaiend by chemical synthesis from synthetic, fossil, or natural raw materials, or by physical operations from natural sources.
  • a fragrance is any natural or synthetic substance or substances used solely to impart an odor to a cosmetic product. Any perfume or fragrance used in the cosmetics industry may be used in the inventive compositions. In certain embodiments, the perfume or fragrance is commercially available. In certain embodiments, the perfume or fragrance may be blended from raw ingredients used in the cosmetics industry.
  • fragrance ingredients include Abies Alba Leaf Oil, Abies Balsamea (Balsam Canada) Resin, Abies Pectinata Oil, Abies Sibirica Oil, Acacia Catechu Gum, Acacia Senegal Gum, Acetaldehyde, Acetanilid, Acetic Acid, 1-Acetonaphthone, 2- Acetonaphthone, Acetone, Acetyl Hexamethyl Indan, Acetyl Hexamethyl Tetralin, Acetyl Tributyl Citrate, Acetyl Triethyl Citrate, Achillea Millefolium Extract, Achillea Millefolium Flower Water, Achillea Millefolium Oil, Achyrocline Satureiodes Flower Oil, Actinidia Chinensis (Kiwi) Fruit Water, Adipic Acid, Agar, Agropyron Repens Root Extract, Alanine, Albizia Julibrissin Bark Extract, Alcohol, Alcohol Denat., Algae Extract, Algin, All
  • the perfume or fragrance may be used in the composition in an amount ranging from 0.0001% to 10% by weight. In certain embodiments, the perfume or fragrance is used in the composition in an amount ranging from 0.01% to 1% by weight. In certain embodiments, the perfume or fragrance is used in the composition in an amount ranging from 0.001% to 0.01% by weight. In certain embodiments, the perfume or fragrance is used in the composition in an amount ranging from 0.001% to 0.1% by weight.
  • compositions of the invention can be provided in any form that delivers an effective amount of the enzyme(s) and, if desired, any other active agent to the skin.
  • the compositions are cosmetic compositions.
  • the compositions are pharmaceutical compositions. Suitable forms include, but are not limited to, gels, hydrogels, creams, ointments, lotions, pastes, suspensions, emulsions, sprays, patches (e.g., hydrogel patch), masks, powders, etc. Appropriate ingredients and methods of preparing such formulations are known in the art and need not be detailed here. See, e.g., Flick, E., Cosmetic and Toiletry Formulations, Vol.
  • ointments, pastes, creams, or gels may comprise animal, vegetable, or synthetic fats, waxes, paraffins, petrolatum, starch, tragacanth or other gums, cellulose or derivatives thereof, polyethylene oxides such as polyethylene glycols, silicones, bentonites, silica, talc, zinc oxide, or mixtures thereof.
  • Solutions or emulsions may comprise solvent, stabilizer, solubilizer, emulsifier, etc., such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, oils (e.g., cottonseed oil, groundnut oil, olive oil, castor oil, sesame seed oil), glycerol fatty esters, fatty acid esters of sorbitan, PEG, etc., and mixtures thereof.
  • solvent stabilizer, solubilizer, emulsifier, etc.
  • solvent such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, oils (e.g., cottonseed oil, groundnut oil, olive oil, castor oil, sesame seed oil), glycerol fatty esters, fatty
  • Emulsions will comprise one or more oil phases in one or more aqueous phases, each oil phase comprising a single oily component or a mixture of oily components in miscible or homogenous form.
  • the total level of oil phase components will typically be from 0.1% to 60%, e.g., 1% to 30%, e.g., from 1% to 10%, e.g., from 2% to 10%.
  • the oil phase(s) may comprise preferably comprise an emollient, a silicone oil, or mixtures thereof; they may also comprise additional oily components such as a natural or synthetic oil selected from mineral, vegetable, and animal oils (e.g. lanolin), fats and waxes, fatty acid esters, fatty alcohols, fatty acids and mixtures thereof. Examples include saturated and unsaturated fatty alcohols such as behenyl alcohol, cetyl alcohol and stearyl alcohol and hydrocarbons such as mineral oils or petrolatum. Further examples suitable for use herein are disclosed in WO98/22085.
  • Suspensions may comprise liquid diluents such as water, ethanol or propylene glycol, suspending agents such as ethoxylated isostearyl alcohols, polyoxyethylene sorbitol esters, microcrystalline cellulose, aluminum hydroxide or metahydroxide, bentonite, agar, gums such as tragacanth, and mixtures thereof.
  • suspending agents such as ethoxylated isostearyl alcohols, polyoxyethylene sorbitol esters, microcrystalline cellulose, aluminum hydroxide or metahydroxide, bentonite, agar, gums such as tragacanth, and mixtures thereof.
  • one or more of the components of the composition e.g., one or more active agents such as an elastase
  • one or more active agents such as an elastase
  • liposomes, polymeric microparticles, nanoparticles, microcapsules, and nanocapsules, and methods for encapsulating agents therein or forming particles impregnated with the agents are well known in the art and need not be described in detail herein.
  • polymers e.g., biocompatible polymers, which may be biodegradable, can be used.
  • the active agent may be released as the polymer degrades.
  • Useful polymers include poly(lactic-co-gly colic acid), polyanhydrides, ethylene vinyl acetate, polyglycolic acid, chitosan, polyorthoesters, polyethers, polylactic acid, and poly (beta amino esters).
  • Peptides, proteins such as collagen and albumin, and dendrimers e.g., PAMAM dendrimers, peptide dendrimers, etc.
  • PAMAM dendrimers e.g., PAMAM dendrimers, peptide dendrimers, etc.
  • the invention provides a skin care composition comprising a liposome, microparticle, nanoparticle, microcapsule, or nanocapsule comprising an elastase or partial elastase.
  • the inventive compositions may be packaged with a suitable article for contacting the composition with the skin, e.g., for topical application such an article could be an applicator brush, pad, wipe, cloth, towelette, sponge, puff, scrubber, etc. In some embodiments such an article will be impregnated with the inventive composition.
  • a suitable article would be a needle optionally with a syringe.
  • compositions described herein may be provided in a single container or may be packaged in a kit comprising multiple containers or compartments.
  • the contents of the containers or compartments may be applied separately to the skin or may be mixed shortly before application.
  • one compartment may contain the enzyme, a carrier, and one or more stabilizers.
  • a second compartment may contain a gel, lotion, or cream that by itself lacks elastolytic activity.
  • a compartment may contain an agent that activates the enzyme and/or provides an appropriate pH or other suitable condition for activity of the enzyme.
  • Any of the compartments may contain additional active agents or compositions containing them, which may be applied either prior to or following use of a composition comprising an elastase or partial elastase.
  • the kit includes a first container having therein a composition comprising an elastase or partial elastase and a second container or set of containers having chemical peeling agents therein.
  • the composition is provided as a lyophilized powder which is admixed with other ingredients shortly before topical application in the form of a gel, paste, cream, lotion, or the like.
  • the kit may contain instructions for use of the composition.
  • Any of the enzymes or other components of the compositions of the present invention may be provided as pharmaceutically or cosmetically acceptable salts. Acceptable salts include those derived from various inorganic and organic acids and bases.
  • Suitable acid salts include acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptanoate, glycerophosphate, glycolate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oxalate, palmoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, prop
  • Salts derived from appropriate bases include alkali metal (e.g., sodium and potassium), alkaline earth metal (e.g., magnesium), ammonium and N(Ci_ 4 alkyl) 4 + salts.
  • alkali metal e.g., sodium and potassium
  • alkaline earth metal e.g., magnesium
  • ammonium e.g., sodium and potassium
  • N(Ci_ 4 alkyl) 4 + salts e.g., sodium and potassium
  • alkaline earth metal e.g., magnesium
  • ammonium e.g., sodium and potassium
  • N(Ci_ 4 alkyl) 4 + salts e.g., sodium and potassium
  • ammonium e.g., sodium and potassium
  • N(Ci_ 4 alkyl) 4 + salts e.g., sodium and potassium
  • ammonium e.g., sodium and potassium
  • N(Ci_ 4 alkyl) 4 + salts e.g., sodium and potassium
  • Solutions or suspensions can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
  • the injectable preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
  • Injectable compositions may be provided in the form of sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the preparation of sterile injectable solutions or dispersions.
  • Sterile, fixed oils are conventionally employed as a solvent or suspending medium.
  • any bland fixed oil may be employed including synthetic mono- or di-glycerides.
  • Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically-acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions.
  • These oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant, such as carboxymethyl cellulose or similar dispersing agents that are commonly used in the formulation of cosmetically or pharmaceutically acceptable preparations such as emulsions and suspensions.
  • Other commonly used surfactants, such as Tweens, Spans and other emulsifying agents or bioavailability enhancers which are commonly used in the manufacture of pharmaceutically acceptable solid, liquid, or other formulations may also be used .
  • Preferred injectable formulations are sterile, if possible, and are stable under the conditions of manufacture and storage. They may be preserved against the contaminating action of microorganisms such as bacteria and fungi. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. Isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride may be included in the composition.
  • Sterile injectable solutions can be prepared by incorporating the enzyme(s) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
  • solutions for injection are free of endotoxin.
  • Dispersions may be prepared by incorporating the enzyme(s) into a sterile vehicle which contains a basic dispersion medium and the other desired ingredients from those enumerated above.
  • suitable methods of preparation are vacuum drying and freeze-drying, which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • the methods of the present invention involve contacting the skin with a composition comprising an enzyme in an amount effective to diminish evidence of elastotic material and/or to inhibit accumulation of elastotic material.
  • the skin will typically be photodamaged skin.
  • the degree of photodamage can vary.
  • Several grading schemes have been developed to quantify the degree of photodamage.
  • One widely used classification divides skin into four types, as delineated by Glogau (Sem. Cutan. Med. Surg., 15(3): 134-138, 1996). Briefly, these categories are Type I (early photoaging); Type II (early to moderate photoaging); Type III (advanced photoaging); and Type IV (severe photoaging).
  • the skin may be chronologically aged skin.
  • the skin is prematurely aged skin.
  • the invention contemplates contacting skin of any of these types with a composition of the invention.
  • the skin is heavily photodamaged.
  • Types III and IV or their equivalents under other classification schemes are considered heavily photodamaged.
  • the method of the invention may include identifying a subject having photodamaged skin and/or skin characteristics suitable for administration of an inventive composition.
  • the method may comprise examining the skin of a subject (e.g., visually, pathologically, microscopically, etc.) to determine the degree of photodamage or aging and selecting an area that exhibits signs of photodamage or aging for administration of an inventive composition.
  • the degree of photodamage or aging is classified according to a classification scheme.
  • the composition is administered to or by an individual between 20 and 30 years old. In other embodiments, the composition is administered to or by an individual between 30 and 40, between 40 and 50, between 50 and 60, between 60 and 70, between 70 and 80, between 80 and 90, or older.
  • the signs of photodamage may be in combination with signs of aging. The two may be indistinguishable.
  • Various methods of administration are envisioned. In certain embodiments of the invention the compositions are topically applied.
  • components of the composition may penetrate to deeper layers of the skin.
  • the composition is contacted with the epidermis and the enzyme diffuses or is transported into the dermis.
  • compositions are injected intradermally, e.g., using standard techniques for intradermal injection.
  • the skin may be cleaned and excess fat removed with such agents as acetone, rubbing alcohol, or Septisol, or a combination of these agents prior to topical application or injection of a composition of the invention.
  • the surface of the skin may be disinfected prior to injection using standard methods.
  • compositions are injected subcutaneous Iy, e.g., using standard techniques for subcutaneous injection.
  • the skin may be cleaned and excess fat removed with such agents as acetone, rubbing alcohol, or Septisol, or a combination of these agents prior to injection of a composition of the invention.
  • the surface of the skin may be disinfected prior to injection using standard methods.
  • the composition may be topically applied to a contiguous area of skin, e.g. , the cheeks, the area around the eyes or mouth, the entire face, the face and neck, etc.
  • spot treatments in which the composition is topically applied or injected into smaller, localized regions, such as areas of deep wrinkles, bumps, or other textural discontinuities.
  • the enzymes are provided tethered to a water insoluble substrate, e.g., a textile.
  • a water insoluble substrate e.g., a textile.
  • the substrate may be woven or nonwoven and may be fibrous or non- fibrous. It may have abrasive qualities. It may be natural or synthetic.
  • the substrate is contacted with the surface of the skin, thereby providing the enzyme to the skin.
  • the substrate may be wiped or rubbed across the skin.
  • the substrate may be in the form of a personal care wipe.
  • the amount of enzyme on the substrate may be, for example, from about 0.01 ⁇ g/cm 2 to about 1000 ⁇ g/cm 2 .
  • the invention provides a substrate having an elastase or partial elastase tethered thereto.
  • the invention further provides a substrate impregnated or coated with a composition of the present invention comprising an elastase or partial elastase.
  • one or more layers of the epidermis is removed through some or all of its thickness prior to topical application of a composition of the invention. Such removal may comprise removing outer layers or flakes composed of dead skin cells and/or other components of the epidermis. This process is referred to herein and in the art as "exfoliation".
  • removing one or more layers of the epidermis through some or all of the thickness of such layer or layers improves access of the enzyme to the dermis, where elastotic material is chiefly present in photodamaged skin.
  • a variety of methods known in the art can be used to remove one or more layers of the epidermis through part or all of the thickness of such layer(s). Certain of these methods may also be used to remove the DEJ and, optionally, part or all of the papillary dermis in certain embodiments of the invention. Suitable methods include, but are not limited to, dermabrasion, microdermabrasion, chemical peels, laser resurfacing, and the like, which will collectively be referred to as "surface removal methods".
  • Chemical peeling agents include various ⁇ - and ⁇ -hydroxy acids such as glycolic, citric, malic, lactic, salicylic, beta hydroxybutanoic, tropic, and trethocanic acids; trichloroacetic acid (TCA); phenol, resorcinol, etc. Often a combination of agents is used.
  • Examples of the chemical peel compositions include Jessner solution (salicylic acid 14%, lactic acid 14%, and resorcinol 14% in alcohol), Jessner + TCA (Monheit peel), Baker-Gordon phenol peel, etc. See, e.g., Butler, P.E.M., Plast, Reconstr.
  • Exfoliation can be achieved using mechanical methods such as gentle massage or scrubbing with a material such as loofah, small particulates such as salt, sand, or grape seed, etc.
  • Microdermabrasion is a more intense procedure in which the stratum corneum (SC) is partially or completely removed by light abrasion.
  • Different methods include mechanical abrasion using jets of zinc or aluminum oxide crystals, or a roughened surface such as a brush or wand, optionally with light suction. See, e.g., Hill, P., Milady's Aesthetician Series: Microdermabrasion (Milady, 2005).
  • the SC is partially removed, and a composition of the invention is topically applied to the resulting skin surface following such removal.
  • Partial removal of the SC can entail removal of from anywhere between 1% and 99%, e.g., between 10% and 90%, by volume, by dry weight, or by thickness over a given skin area, with all intervening ranges and individual values being among the options.
  • the SC typically includes between 10 and about 22 layers of corneocytes, e.g., 18-21 layers. In certain embodiments of the invention, on average between 10% and 90% of these cells removed, and a composition of the invention is topically applied to the surface of the skin following such removal.
  • the SC also contains intercellular lipids, primarily cholesterols (20- 30%), ceramides (40-60%), and fatty acids (20-30%). In certain embodiments of the invention between 10% and 99% by weight of one, more, or all of these lipid types are removed over a given skin area.
  • the SC is substantially removed, and a composition of the invention is topically applied to the surface of the skin following such removal.
  • at least the upper two or the upper three layers of the epidermis are substantially removed, and a composition of the invention is topically applied to the surface of the skin following such removal.
  • all four layers of the epidermis are substantially removed, and a composition of the invention is topically applied to the surface of the skin following such removal.
  • the 3 upper layers of the epidermis may be substantially removed while leaving the stratum basale substantially intact.
  • the DEJ is substantially removed.
  • “Substantially removed” when referring to removal of a layer of skin means that at least 80%, at least 85%, at least 90%, or at least 95% of the layer (by volume or dry weight in various embodiments of the invention) is removed over a given area of skin. "Substantially intact” when referring to a layer of skin means that at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% of the layer (by volume or dry weight in various embodiments of the invention) remains intact.
  • the papillary dermis is partly or substantially removed. For example, up to 25%, or up to 50% by volume or depth of the papillary dermis may be removed. In other embodiments, the papillary dermis is substantially intact. In other embodiments the papillary dermis is substantially removed.
  • Neutralization of a chemical peeling agent is an important step once the proper depth of the peel is achieved, which may be determined either by the frost (change in coloration of the skin to a whitish tint signifying keratin agglomeration) or how much time has elapsed. Neutralization can be achieved by cold water or wet, cool towels applied to the face following the frost. Other neutralizing agents that can be used include bicarbonate spray or soapless cleanser.
  • a composition of the invention may be applied immediately (i.e., within about 0.1-10 minutes) after the surface removal procedure is performed, or within up to 2, up to 4, up to 8, up to 12, up to 16, up to 20, or up to 24 hours thereafter. In other embodiments, the composition of the invention is applied up to 48 hours or up to 72 hours after the surface removal procedure.
  • the skin may be prepared with multiple surface removal treatments, e.g. , the subject may self-apply glycolic acid pads impregnated with 10%-20% glycolic acid over a period of days or weeks prior to treatment with a composition of the invention.
  • one or more elastases or partial elastases is included in the chemical peeling composition.
  • the chemical peeling agent is applied according to the standard methods.
  • the elastase or partial elastase enhances effectiveness of the chemical peel.
  • the chemical peeling agent employed in these embodiments should be one that is compatible with stability of the enzyme(s).
  • the invention therefore provides a skin care composition comprising (i) any of the afore-mentioned chemical peeling agents or others known in the art; and (ii) an elastase or partial elastase.
  • composition of the present invention in conjunction with removal of one or more layer of the epidermis using any suitable method provides an additional benefit beyond that obtained simply by using the surface removal method.
  • "In conjunction with” means that the composition of the present invention is applied within up to 72 hours following a surface removal procedure.
  • Many surface removal procedures and treatments entail multiple repetitions over a period of time. For example, a surface removal treatment or procedure may be performed on a daily, weekly, biweekly, or monthly basis, on an "as-needed" basis depending on the condition of the skin, or at random time intervals.
  • a composition of the present invention may be applied in conjunction with one, some, most (e.g., at least 50%), or substantially all (e.g., >90%) of such surface removal procedures or treatments.
  • inventive compositions and methods can be employed at various time intervals and over different periods of time as desired to enhance the appearance of the skin, e.g., multiple times per day (e.g., in the morning and evening), daily, every other day, once a week, once a month, bimonthly, etc. for between about 1 to 10 weeks or longer, e.g., indefinitely.
  • multiple times per day e.g., in the morning and evening
  • daily every other day
  • treatment of a subject with an inventive composition can include a single treatment or, in many cases, can include a series of treatments.
  • the specific dose, number of repeat treatments, and other aspects of the treatment regimen for any particular subject may depend upon a variety of factors including the activity of the specific enzyme(s) employed, the degree of photodamage, the desired amount of improvement, whether the composition is administered to inhibit progression of photodamage, etc. For example, if the composition is administered to an individual who exhibits no or minimal evidence of photodamage, e.g., primarily to inhibit photodamage, a smaller amount may be used.
  • the composition is not applied to the surface of the skin primarily for purposes of exfoliation.
  • the composition may be one that is relatively ineffective at achieving exfoliation when applied to the intact epidermis for up to, e.g., 15, 30, 45, 60, 90, or 120 minutes.
  • the composition may be relatively ineffective at achieving exfoliation when applied to the intact epidermis for up to 2, 3, 4, 8, 12, or 24 hours.
  • a composition is "relatively ineffective at achieving exfoliation” if a competent cosmetologist or dermatologist having at his or her disposal the skin care products commercially available as of the date of this application and intending primarily to exfoliate the skin of a typical subject, would be unlikely to select the composition to achieve this purpose.
  • a composition is "relatively ineffective at achieving exfoliation” if when applied to the epidermis for a period of time not exceeding 2 hours in amounts that are tolerated without undue irritation or discomfort and without mechanical abrasion, optionally repeating the treatment up to 3 times, the composition removes less than 5% of the epidermis by depth.
  • the composition is not contacted with skin for therapeutic purposes, e.g., to treat burns or wounds or to debride skin, or in order to expand healthy skin so as treat diseases or disorders occurring in areas adjacent to the healthy skin.
  • the composition is not contacted with healthy skin adjacent to (e.g., within 1 cm of) an area affected by undesired skin loss such as due to surgery, injury or the like (where deliberate removal of all or part of the epidermis as described above is not considered undesired skin loss) or with skin intended for use as a skin graft.
  • the skin is contacted with any of a variety of other compositions that may serve various purposes.
  • Such treatments will be referred to herein as “supplementary treatments", and the compositions are "supplementary compositions".
  • the composition comprising one or more enzymes in an amount effective to reduce evidence of elastotic material may be referred to as a "primary composition”.
  • the supplementary composition may be provided in any convenient formulation, e.g., rinses, gels, creams, lotions, etc., and may contain any of the additional active or inactive agents described herein.
  • the composition may be a "leave -on" composition, meaning that it is not removed following application.
  • the supplementary compositions may be prepared using conventional cosmetic preparation methods and ingredients, many of which are described above.
  • the supplementary treatment comprises physically removing the enzyme, e.g., by rinsing with an appropriate solution.
  • the supplementary treatment comprises contacting the skin with a supplementary composition comprising a neutralizing agent such as an inhibitor of one or more elastases or partial elastases that was present in the primary composition or an agent that alters the pH to a range at which the enzyme has low or no activity.
  • a neutralizing agent such as an inhibitor of one or more elastases or partial elastases that was present in the primary composition or an agent that alters the pH to a range at which the enzyme has low or no activity.
  • Any suitable method of inhibiting or neutralizing the enzyme(s) such as altering ionic strength, application of surfactants or an oil phase, etc., can be used. Doing so may reduce or essentially prevent further activity of the enzyme(s), thereby providing control over the effect of the primary composition.
  • the inhibitor allows tailoring of the treatment depending on the extent of photodamage by providing control over the time during which the elastase or partial elastase is active. Suitable inhibitors are discussed above.
  • the skin is contacted with a composition comprising an inhibitor of the activity or synthesis of an endogenous, skin-associated elastase or collagenase.
  • the amount may be effective to inhibit elastase activity in the skin associated with endogenous, skin-associated elastase or collagenase by between 20% and 100%, between 30% and 90%, between 40% and 80%, or by any intermediate range or value. Suitable inhibitors are described above. Without wishing to be bound by any theory, inhibiting synthesis or activity of one or more endogenous, skin-associated elastases or collagenases may facilitate replacement of the elastotic material with newly synthesized elastic and/or collagenous fibers.
  • the skin after contacting the skin with a composition comprising one or more elastases or partial elastases for a period of time to reduce evidence of elastotic material, the skin is contacted with a skin restoring composition.
  • skin restoring composition includes, but is not limited to, a composition that (i) stimulates synthesis of one or more components of skin, e.g., one or more proteins that make up the extracellular matrix component of skin (collagen, elastin, laminin, fibronectin, microfibrillar proteins) or glycosaminoglycans; (ii) stimulates proliferation of keratinocytes; and/or (iii) augments the skin by providing extracellular matrix components or fragments thereof (e.g. , proteins such as collagen, elastin and/or glycosaminoglycans) directly to the skin.
  • contacting the skin with a skin restoring composition following contacting the skin with a composition comprising one or more elastases or partial elastases so as to reduce evidence of elastotic material provides an additional benefit above that which might be obtained simply by contacting the skin with the skin restoring composition.
  • the skin restoring composition may be relatively ineffective when used by itself, possibly as a consequence of the presence of extensive elastotic material in the skin.
  • diminishing the elastotic material in accordance with the present invention may improve the ability of the skin restoring composition to effectively improve the appearance and/or structure of skin, e.g. , to restore a youthful or non-photodamaged appearance, to enhance natural skin reparative processes, etc.
  • the skin restoring composition is contacted with the skin between 5 minutes and 24 hours following contacting the skin with a primary composition.
  • the skin restoring composition may be applied once or more than once.
  • the invention contemplates once or twice daily, every other day, or weekly treatments with a skin restoring composition to maintain and/or enhance the benefit provided by a primary composition.
  • the skin restoring composition may contain e.g. , peptides of general sequence X- Thr-Thr-Lys-Y, wherein for example X is lysine and Y is serine (U.S. Pat. No. 6,620,419, incorporated herein by reference), one or more tri- and tetrapeptides (U.S. Pat. No.
  • polypeptides selected from the group consisting of pentapeptides, derivatives of pentapeptides, and mixtures thereof (U.S. Pat. No. 6,492,326, incorporated herein by reference), and/or at least one polypeptide having an amino acid sequence of from 3 to 12 amino acids in length or an N-acyl derivative thereof, wherein in certain embodiments the polypeptide comprises Val-Gly-Val-Ala-Pro-Gly (SEQ ID NO: 1) and optionally the composition comprises ceramide (U.S. Pub. No. 20040120918, incorporated herein by reference).
  • the skin restoring composition may comprise one or more collagens naturally found in the skin (e.g., collagen I, IV, or V), elastin, or a fragment or combination of fragments of collagen or elastin (fragments being at least 3 contiguous amino acids in length).
  • the composition may comprise tropocollagen and/or tropoelastin or fragment(s) of either. Fragments may be prepared by proteolytic or chemical cleavage of a protein or by recombinant methods. See, e.g., U.S. Pub. No.
  • compositions may comprise L-fucose and fucose-rich polysaccharides, which have been demonstrated to increase elastin biosynthesis (Robert, L., et al, Biomed Pharmacother, 58(2): 123-8, 2004).
  • Other skin restoring agents include retinoids and hydroquinones.
  • Retinoid includes all natural and/or synthetic analogs of Vitamin A or retinol-like compounds which possess the biological activity of Vitamin A in the skin as well as the geometric isomers and stereoisomers of these compounds.
  • retinol examples include retinol, retinol esters (e.g., C2-C22 alkyl esters of retinol, including retinyl palmitate, retinyl acetate, retinyl propionate), retinal, retinoic acid (including all-trans retinoic acid and/or 13-cis- retinoic acid), and combinations thereof.
  • retinol retinol esters
  • retinyl esters e.g., C2-C22 alkyl esters of retinol, including retinyl palmitate, retinyl acetate, retinyl propionate
  • retinal examples include retinoic acid (including all-trans retinoic acid and/or 13-cis- retinoic acid), and combinations thereof.
  • retinoic acid including all-trans retinoic acid and/or 13-cis- retinoic acid
  • Exemplary amounts of retinol are from 0.01% to 0.15%. Exemplary amounts of retinol esters are from 0.01% to 2%. Exemplary amounts of retinoic acids are from 0.01% to 0.25%. Exemplary amounts of tocopheryl-retinoate, adapalene, and tazarotene are from 0.01% to 2%.
  • a skin soothing composition is one that reduces irritation, redness, or inflammatory effects that may result from a treatment such as microdermabrasion, chemical peel, or treatment with a composition of the invention.
  • the skin soothing composition comprises a skin soothing agent.
  • a large number of skin soothing agents include steroidal and non-steroidal anti-inflammatory compounds, local anesthetics, chamomile, panthenol and derivatives (e.g., ethyl panthenol), aloe vera, pantothenic acid and its derivatives, allantoin, bisabolol, and dipotassium glycyrrhizinate.
  • Soothing compositions may include any of the emollients, humectants, moisturizers, etc., mentioned above.
  • any method known in the art to assess the effectiveness of the skin care compositions and methods of the present invention may be used. Typical approaches for use in living subjects include evaluations by dermatologists, aestheticians, or cosmetologists, self-assessment questionnaires, optical or mechanical prof ⁇ lometry, etc. Parameters that may be assessed include elasticity, firmness, skin smoothness or roughness, scaliness, discoloration, altered pigmentation, or blotchiness, evidence of fine lines and wrinkles.
  • At least one of the aforementioned parameters is improved, e.g., elasticity or f ⁇ rmess is increased, skin smoothness is increased, skin roughness is decreased, evidence of fine lines or wrinkles is decreased, scaliness, altered pigmentation, blotchiness, or discoloration is decreased, etc. In other embodiments, any two or more of the aforementioned parameters are improved.
  • tissue samples e.g., cadaveric skin or skin taken from a living subject.
  • the skin sample may or may not have been subjected to various processing and/or preservation steps such as cryopreservation, formaldehyde fixation, etc.
  • contacting a composition with skin in accordance with the present invention significantly reduces histopathologic evidence of photodamage, by which is meant that a person of ordinary skill in the art, e.g., a trained pathologist having knowledge of dermatopathology and applying art-recognized methods for visualizing elastin ⁇ e.g., appropriate stains and microscopic examination), would repeatably and reliably conclude that a visually detectable difference in the amount of elastotic material exists between a sample that has been contacted with a composition in accordance with the present invention and a suitable control sample.
  • "significantly reduce histopathologic evidence of elastotic material” means that an automated image analysis system capable of quantitating the amount of elastotic material present in a sample detects less than 90%, less than 75%, less than 50%, less than 25%, or less than 10% as much elastotic material in a sample that has been contacted with a composition in accordance with the present invention than in a suitable control sample.
  • contacting a composition with skin in accordance with the present invention reduces histopathologic evidence of photodamage sufficiently such that a person of ordinary skill in the art, e.g., a trained pathologist having knowledge of dermatopathology, would repeatably and reliably conclude that the amount of elastotic material present in a sample that has been contacted with a composition in accordance with the present invention is 90%, 75%, 50%, 25%, or 10% less than the amount of elastotic material in a suitable control sample.
  • a suitable control sample will be one having a similar amount of photodamage and typically from the same region of the body. The control and test samples may be from the same individual.
  • the effectiveness of the compositions and methods of the present invention can also be assessed in tissue samples or living tissue using biochemical testing.
  • the degradation of elastin may be monitored directly.
  • a surrogate of elastin degradation may be monitored to assess the inventive method or composition.
  • the release of desmosine and/or isodesmosine may be used to assess degradation of elastin. Methods for detecting desmosine and/or isdesmosine are described in Viglio et al., J. Sep. Sci.
  • detecting desmosine and/or isodesmosine include immunochemical methods (e.g. , ELISA, RIA), chromatographic methods (e.g., HPLC, HPLC combined with mass spectrometry), and electrophorectic methods.
  • immunochemical methods e.g. , ELISA, RIA
  • chromatographic methods e.g., HPLC, HPLC combined with mass spectrometry
  • electrophorectic methods e.g., electrophorectic methods.
  • compositions of the present invention can comprise, consist essentially of, or consist of, one or more enzymes and, in certain embodiments of the invention, any one or more other ingredients described herein.
  • Consisting essentially of means that the composition or component may include additional ingredients, but only if the additional ingredients do not materially alter the basic and novel characteristics of the claimed compositions or methods.
  • the invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process.
  • the invention also includes embodiments in which more than one, most (more than half), almost all (more than 90%), or all of the group members are present in, employed in, or otherwise relevant to a given product or process.
  • the invention encompasses all variations, combinations, and permutations in which one or more elements, clauses, descriptive terms, limitations, etc., from one or more of the dependent claims is introduced into another claim that is dependent on the same base claim. Any claim that is dependent on another claim can be modified to include one or more elements, clauses, descriptive terms, limitations, etc., found in any other claim that is dependent on the same base claim.
  • Methods of the invention may comprise providing a suitable subject and contacting the skin of the subject with a composition of the invention.
  • a “suitable subject” may be any subject whose skin may reasonably be expected to benefit from the composition.
  • a suitable subject may be one whose skin exhibits evidence of photodamage.
  • the inclusion of a step of "providing a subject whose skin exhibits evidence of photodamage" in certain methods of the invention is intended to indicate that the composition is administered to a subject whose skin exhibits evidence of photodamage, e.g., elastosis.
  • the composition may be administered with the intent to reduce signs of photodamage, e.g., to reduce evidence of elastosis.
  • the composition is administered upon the recommendation of a medical or cosmeto logical practitioner, e.g., a dermatologist or cosmetologist, who may or may not be the individual who actually administers the composition.
  • a medical or cosmeto logical practitioner e.g., a dermatologist or cosmetologist
  • Each method of the invention includes an embodiment in which a step of providing is not explicitly included and an embodiment in which a step of providing is included.
  • Each method of the invention also includes an embodiment in which a step of identifying the subject as exhibiting evidence of photodamage (e.g., elastosis) is included and an embodiment in which identifying is not included.
  • any one or more embodiments of the present invention may be explicitly excluded from any one or more of the claims for any reason or no reason.
  • Any specific enzyme or combination thereof, any class of enzymes (where class can be expressed in terms of source, activity, functional characteristics, etc.), any additional active or inactive agents in a composition, any method of contacting with the skin, any method of removing part or all of one or more layers of skin, any skin disorder or condition or characteristic(s) thereof, can be excluded from any one or more claims, for any reason or no reason.
  • Pseudomonas aeruginosa elastase was tested using excess facial skin tissue as the photodamaged skin model.
  • Pseudonomas aeruginosa elastase (PE) was purchased from the Elastin Products Company.
  • Figs. 1A-1F show the VVG staining results for the sections treated with the control and the active PA elastase solutions.
  • the internal controls verified elastase activity, where the supernatant from the active suspension yield fluorescence values that were 1.3 fold higher than the corresponding placebo (i.e., no elastase) case.
  • the abundant presence of aberrant elastin is observed in the control slides (Figs. IB, ID, and IF), where the elastin stains black.
  • sections treated with elastase Figs. IA, 1C, and IE
  • the remaining dermis was then exposed to 20ul of either the control or the active (50 ug/ml PA elastase in buffer) solutions for two hours.
  • the solution was applied to the remaining surface of the skin.
  • Internal controls consisting of the fluorescein- elastin substrate and 20 ul aliquots of the control and active solutions were also prepared and incubated at 37°C with the exposed skin samples.
  • Positive controls consisting of skin punches treated with a 20 uL intradermal injection of either the control or the active solution. These positive controls were incubated with the heat stripped skin samples. Following two hours, the samples were removed and rinsed with buffer solution prior to fixing the samples in 10% formalin. After 12 hours, the samples were transferred to a 70% ethanol solution and submitted for routine histology.
  • Figs. 3 and 4 show the results of the histology, where elastin is stained black.
  • Table 3 describes the sample and the corresponding label assignments given to the images. The magnification of the image is indicated after the label (4x, 10x or 4Ox). The red scale bar indicates 100 microns. Dense elastin staining is visible in the control samples (Figs. 3E, 3F, 3G, 4A, and 4B) whereas staining is much lighter in the sections exposed to elastase (Fig. 3A-3D, 4C). Note that skin punches from the chin (Fig. 4) are less photodamaged when compared to the cheek samples (Fig. 3) based on the elastin staining.
  • the skin punches were completely digested upon incubation with 200 uL of either the control or the active solution at the same elastase concentration of 50 ug/mL for 15 hours.
  • compositions are formulated as gel/hydrogel patches and/or as face masks. Amounts are listed as weight percents.
  • Elastase #1 is PA elastase (LasB).
  • Elastase 2 is a second bacterial elastase, e.g., PA LasA.
  • the composition includes an elastase inhibitor. It will be appreciated that many of the above components fall into one or more of the various classes described herein and could be substituted for other member(s) of those classes or omitted entirely, and that other components may be included instead or in addition.
  • Elastase activity was evaluated in the presence of common cosmetic ingredients such as PEG-6 caprylic/capric glycerides, isoceteth-20, polysorbate 80, PEG-7 glyceryl cocoate, and xanthum gum. Elastase activity was evaluated using fluorescein-labeled elastin in an in vitro model. Each cosmetic ingredient listed in the table below was introduced to Tris buffer at pH 7.2 with the indicated percentage of cosmetic ingredient and 0.5 U/mL of porcine pancreas elastase (PPE).
  • PPE porcine pancreas elastase
  • the activity of elastase was determined in each formulation using a suspension of elastin tagged with fluorescein. Water-soluble fluorescein is released when the elastin is cleaved. Fluorescence was measured by excitation at 490 nm and emission at 520 nm. Each of the above formulations was screened for elastase activity over 19 hours at 37 0 C with light shaking. [00212] A portion (20 ⁇ L) of each of the above formulations was applied to a cryotomed section of photodamaged facial skin. Adjacent skin sections received active and placebo applications. The sections were placed in micro-humidity chambers and incubated at 37 0 C for 4 hours. The sections were then rinsed in Tris buffer at pH 7.2 to remove any remaining active elastase, then fixed in formalin and stained with VVG elastin stain.
  • the sorbitol formulation exhibited somewhat decreased activity, while the carbopol formulation exhibited the greatest decrease in elastolytic activity.
  • porcine pancreatic elastase The activity of porcine pancreatic elastase was evaluated under a variety of conditions using fluoroscein-labeled elastin degradataion in vitro. The effects of pH, concentration, and exposure duration were evaluated. These data are shown in Figures 12-18.
  • the cryotomed sections were transferred to individual glass microscope slides. 20 ⁇ L of solution was added to immerse the entire cross-section in media. Sections were covered with micro-humidity chambers to prevent dehydration of sample and loss of enzyme activity. Each slide was then placed in a Petri dish with a moist tissue and covered. The slides were then incubated at 37 0 C for the indicated duration (4 hours or 16 hours) with the indicated concentration of porcine pancreatic elastase (PPE) (1 U/mL, 0.1 U/mL, 0.01 U/mL, or 0.001 U/mL) at the indicated pH (8.8, 7.2, or 5.0).
  • PPE porcine pancreatic elastase
  • Elastin stains of non- viable human skin are shown in Figures 2OA and 2OB illustrating the removal of aberrant elastin from injection and topical administration post microdermabrasion, respectively. Placebo-treated control are shown as well.

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Abstract

La présente invention propose des compositions et procédés pour diminuer les signes de photo-endommagement et/ou vieillissement. Certains des procédés comprennent la mise en contact de la peau avec une quantité efficace d'une ou plusieurs enzymes élastases. Certaines des compositions réduisent l'évidence d'une matière élastotique lors d'une mise en contact avec la peau. Les compositions et procédés proposent une application topique ainsi qu'une administration par injection. Certaines des compositions comprennent une élastase, par exemple une élastase bactérienne au moins partiellement purifiée. Certains des procédés comprennent l'enlèvement d'au moins une partie de l'épiderme ou de la couche cornée avant mise en contact de la peau avec la composition comprenant une élastase.
PCT/US2007/083352 2006-11-01 2007-11-01 Procédés et compositions pour le soin de la peau WO2008070368A2 (fr)

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Cited By (31)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010093065A1 (fr) * 2009-02-10 2010-08-19 (주)파이온텍 Composition cosmétique contenant une poudre de dermabrasion d'origine végétale et capable de libération de principe actif
US8163861B2 (en) 2009-01-15 2012-04-24 Living Proof, Inc. Beta-amino ester compounds and uses thereof
WO2012094638A1 (fr) * 2011-01-07 2012-07-12 Skinmedica, Inc. Compositions de modification de mélanine et procédés d'utilisation
WO2013009997A1 (fr) 2011-07-12 2013-01-17 Burn-Off, Llc Composition et procédé d'utilisation de la composition, efficace pour minimiser les effets nocifs associés aux individus souffrant d'une intoxication alcoolique
DE102013208492A1 (de) * 2013-05-08 2014-11-13 Beiersdorf Ag Wässrige Gelzubereitungen mit Diethylether
WO2016153797A1 (fr) * 2015-03-26 2016-09-29 Elc Management Llc Compositions pour l'augmentation de teneur en lipides de kératinocytes
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DE102017203797A1 (de) 2017-03-08 2018-09-13 Beiersdorf Ag Verwendung von Calciumsalzen in kosmetischen Zubereitungen
KR101899345B1 (ko) 2017-02-10 2018-09-17 대봉엘에스 주식회사 TES Trioleate를 포함하는 자외선에 대한 피부 보호용 조성물
WO2018172948A1 (fr) * 2017-03-21 2018-09-27 Ratti Karan Composition d'éclaircissement et de réduction de l'hyperpigmentation de la peau
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US10245220B2 (en) 2015-07-22 2019-04-02 Biosynthis Process for pongamol enrichment of karanja oil
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WO2020121017A1 (fr) * 2018-12-09 2020-06-18 Afrasiabian Hamidollah Synthèse de crème à partir de médiateurs de plaquettes avec des activateurs de pénétration à base d'herbes permettant d'augmenter la teneur en collagène de la peau
WO2020139851A1 (fr) * 2018-12-26 2020-07-02 Clear Lake Research, Llc Administration de bienfaits pour la santé par l'intermédiaire d'une formulation de constituants
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WO2020243742A3 (fr) * 2019-05-29 2021-02-25 Aqua Vitae Laboratories, Llc. Nouvelles applications et formulations de milieux de culture de fertilité embryonnaire humains modifiés, optimisés avec des biguanides et/ou des équivalents fonctionnels
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WO2022156876A1 (fr) * 2021-01-19 2022-07-28 Beiersdorf Ag Formulations de protéines
US11400032B2 (en) 2013-12-20 2022-08-02 Colgate-Palmolive Company Tooth whitening oral care product with core shell silica particles
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US11602495B2 (en) 2013-12-20 2023-03-14 Colgate-Palmolive Company Core shell silica particles and use for malodor reduction
WO2023182971A1 (fr) * 2022-03-21 2023-09-28 Tintoria Piana, US Inc. Compositions contenant une teneur élevée en proanthocyanidines et leurs procédés de préparation et d'utilisation
CN116982553A (zh) * 2023-06-29 2023-11-03 广西壮族自治区南宁良凤江国家森林公园 一种黄果厚壳桂组织培养方法
CN117357692A (zh) * 2023-12-06 2024-01-09 成都中医药大学 一种原位固化成型水凝胶及其制备方法和用途
CN117797055A (zh) * 2024-02-29 2024-04-02 江西瑞秀朗科技有限公司 一种改善皮肤老化的复方精油和含有其的护肤品组合物

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE19627232A1 (de) * 1996-07-05 1998-01-08 Basf Ag Definierte Enzymmischungen für kosmetische Zwecke
US6943024B2 (en) * 2003-09-24 2005-09-13 Dan-Ning Hu Epidermal melanocyte culture formulations

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE19627232A1 (de) * 1996-07-05 1998-01-08 Basf Ag Definierte Enzymmischungen für kosmetische Zwecke
US6943024B2 (en) * 2003-09-24 2005-09-13 Dan-Ning Hu Epidermal melanocyte culture formulations

Cited By (52)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8163861B2 (en) 2009-01-15 2012-04-24 Living Proof, Inc. Beta-amino ester compounds and uses thereof
US8362175B2 (en) 2009-01-15 2013-01-29 Living Proof, Inc. Beta-amino ester compounds and uses thereof
US8748551B2 (en) 2009-01-15 2014-06-10 Living Proof, Inc. β-amino ester compounds and uses thereof
US9175114B2 (en) 2009-01-15 2015-11-03 Living Proof, Inc. Beta-amino ester compounds and uses thereof
WO2010093065A1 (fr) * 2009-02-10 2010-08-19 (주)파이온텍 Composition cosmétique contenant une poudre de dermabrasion d'origine végétale et capable de libération de principe actif
US9044404B2 (en) 2011-01-07 2015-06-02 Allergan, Inc. Melanin modification compositions and methods of use
WO2012094638A1 (fr) * 2011-01-07 2012-07-12 Skinmedica, Inc. Compositions de modification de mélanine et procédés d'utilisation
CN103442702A (zh) * 2011-01-07 2013-12-11 阿勒根公司 黑色素改良组合物及其使用方法
US8778315B2 (en) 2011-01-07 2014-07-15 Allergan, Inc. Melanin modification compositions and methods of use
CN103442702B (zh) * 2011-01-07 2016-02-10 阿勒根公司 黑色素改良组合物及其使用方法
US9186350B2 (en) 2011-07-12 2015-11-17 Gdb Patent Holdings, Llc Composition, and method of using the composition, effective for minimizing the harmful effects associated with individuals suffering from alcohol intoxication
US10028991B2 (en) 2011-07-12 2018-07-24 Gdb Patent Holdings, Llc Composition, and method of using the composition, effective for minimizing the harmful effects associated with individuals suffering from alcohol intoxication
WO2013009997A1 (fr) 2011-07-12 2013-01-17 Burn-Off, Llc Composition et procédé d'utilisation de la composition, efficace pour minimiser les effets nocifs associés aux individus souffrant d'une intoxication alcoolique
WO2014180819A1 (fr) 2013-05-08 2014-11-13 Beiersdorf Ag Préparation de gel aqueuse contenant du diéthyléther
DE102013208492A1 (de) * 2013-05-08 2014-11-13 Beiersdorf Ag Wässrige Gelzubereitungen mit Diethylether
US11400032B2 (en) 2013-12-20 2022-08-02 Colgate-Palmolive Company Tooth whitening oral care product with core shell silica particles
US11602495B2 (en) 2013-12-20 2023-03-14 Colgate-Palmolive Company Core shell silica particles and use for malodor reduction
US11951196B2 (en) 2013-12-20 2024-04-09 Colgate-Palmolive Company Core shell silica particles and use for malodor reduction
WO2016153797A1 (fr) * 2015-03-26 2016-09-29 Elc Management Llc Compositions pour l'augmentation de teneur en lipides de kératinocytes
CN107427455A (zh) * 2015-03-26 2017-12-01 Elc 管理有限责任公司 用于增加角质形成细胞的脂质含量的组合物
US10245220B2 (en) 2015-07-22 2019-04-02 Biosynthis Process for pongamol enrichment of karanja oil
KR101765574B1 (ko) 2015-08-19 2017-08-07 부경대학교 산학협력단 양어 사료 조성물
GR20160100328A (el) * 2016-06-22 2018-03-09 Pharmacros Ad Αντιγηραντικη κρεμα προσωπου-λαιμου-σωματος
KR101899345B1 (ko) 2017-02-10 2018-09-17 대봉엘에스 주식회사 TES Trioleate를 포함하는 자외선에 대한 피부 보호용 조성물
DE102017203797A1 (de) 2017-03-08 2018-09-13 Beiersdorf Ag Verwendung von Calciumsalzen in kosmetischen Zubereitungen
WO2018172948A1 (fr) * 2017-03-21 2018-09-27 Ratti Karan Composition d'éclaircissement et de réduction de l'hyperpigmentation de la peau
WO2018236897A1 (fr) * 2017-06-20 2018-12-27 Kado Jessica Formulation topique pour la liaison à des récepteurs cannabinoïdes dermatologiques
US11779529B2 (en) 2017-12-12 2023-10-10 Colgate-Palmolive Company Personal care composition
US11298306B2 (en) 2017-12-12 2022-04-12 Colgate-Palmolive Company Personal care composition
WO2020121017A1 (fr) * 2018-12-09 2020-06-18 Afrasiabian Hamidollah Synthèse de crème à partir de médiateurs de plaquettes avec des activateurs de pénétration à base d'herbes permettant d'augmenter la teneur en collagène de la peau
WO2020139851A1 (fr) * 2018-12-26 2020-07-02 Clear Lake Research, Llc Administration de bienfaits pour la santé par l'intermédiaire d'une formulation de constituants
WO2020243742A3 (fr) * 2019-05-29 2021-02-25 Aqua Vitae Laboratories, Llc. Nouvelles applications et formulations de milieux de culture de fertilité embryonnaire humains modifiés, optimisés avec des biguanides et/ou des équivalents fonctionnels
US11065188B2 (en) 2019-05-29 2021-07-20 Av Laboratories Llc Applications and formulations of optimized, modified human embryonic fertility culture media with biguanides and/or functional equivalents
CN110548129A (zh) * 2019-09-05 2019-12-10 复旦大学附属中山医院 一种复方氨基酸双肽注射液及其制备方法和应用
CN110548129B (zh) * 2019-09-05 2023-04-07 复旦大学附属中山医院 一种复方氨基酸双肽注射液及其制备方法和应用
CN111544564A (zh) * 2020-04-29 2020-08-18 娇时日化（杭州）股份有限公司 一种婴儿尿布性皮炎药膏及其制备方法
CN111544564B (zh) * 2020-04-29 2023-07-28 娇时日化（杭州）股份有限公司 一种婴儿尿布性皮炎药膏及其制备方法
WO2022156876A1 (fr) * 2021-01-19 2022-07-28 Beiersdorf Ag Formulations de protéines
IT202100011825A1 (it) * 2021-05-07 2022-11-07 Effegilab Srl Kit per la prevenzione ed il trattamento dell’invecchiamento cutaneo e sistemico e metodo cosmetico
CN113599310B (zh) * 2021-09-18 2023-02-10 上海新高姿化妆品有限公司 一种保湿组合物及其在化妆品中的应用
CN113599310A (zh) * 2021-09-18 2021-11-05 上海新高姿化妆品有限公司 一种保湿组合物及其在化妆品中的应用
CN114432166A (zh) * 2022-02-17 2022-05-06 广州欧正化妆品技术研究院有限公司 一种硅素提物银耳多糖的保湿面膜液
CN114432166B (zh) * 2022-02-17 2023-09-01 广州欧正化妆品技术研究院有限公司 一种含有硅素提取银耳多糖的保湿面膜液
WO2023182971A1 (fr) * 2022-03-21 2023-09-28 Tintoria Piana, US Inc. Compositions contenant une teneur élevée en proanthocyanidines et leurs procédés de préparation et d'utilisation
CN114984103A (zh) * 2022-07-12 2022-09-02 赣南师范大学 一种橙皮提取物及其制备方法和应用
CN114984103B (zh) * 2022-07-12 2023-10-03 赣南师范大学 一种橙皮提取物及其制备方法和应用
CN115770318A (zh) * 2022-12-19 2023-03-10 中国人民解放军海军军医大学 一种复合多孔止血敷贴及其应用
CN116982553A (zh) * 2023-06-29 2023-11-03 广西壮族自治区南宁良凤江国家森林公园 一种黄果厚壳桂组织培养方法
CN116982553B (zh) * 2023-06-29 2024-04-19 广西壮族自治区南宁良凤江国家森林公园 一种黄果厚壳桂组织培养方法
CN117357692A (zh) * 2023-12-06 2024-01-09 成都中医药大学 一种原位固化成型水凝胶及其制备方法和用途
CN117357692B (zh) * 2023-12-06 2024-02-02 成都中医药大学 一种原位固化成型水凝胶及其制备方法和用途
CN117797055A (zh) * 2024-02-29 2024-04-02 江西瑞秀朗科技有限公司 一种改善皮肤老化的复方精油和含有其的护肤品组合物

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