WO2008070368A2

WO2008070368A2 - Methods and compositions for skin care

Info

Publication number: WO2008070368A2
Application number: PCT/US2007/083352
Authority: WO
Inventors: Betty Yu; Rox Anderson; Robert S. Langer; Yushan Kim; Daniel G. Anderson
Original assignee: Living Proof, Inc.
Priority date: 2006-11-01
Filing date: 2007-11-01
Publication date: 2008-06-12
Also published as: WO2008070368A3

Abstract

The present invention provides compositions and methods for diminishing signs of photodamage and/or aging. Certain of the methods comprise contacting skin with an effective amount of one or more elastase enzymes. Certain of the compositions reduce the evidence of elastotic material when contacted with skin. The compositions and methods provide for topical application as well as administration by injection Certain of the compositions comprise an elastase, e.g., an at least partially purified bacterial elastase. Certain of the methods comprise removing at least a portion of the epidermis or stratum corneum prior to contacting the skin with the composition comprising an elastase.

Description

METHODS AND COMPOSITIONS FOR SKIN CARE

Related Applications

[0001] The present application claims priority under 35 U. S. C. § 119(e) to U.S. provisional patent applications, USSN 60/855,949, filed November 1, 2006; the entire contents of which is incorporated herein by reference.

Background of the Invention

[0002] 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. In addition, the skin provides thermoregulation, serves as a permeability barrier, and functions as a sensory organ for touch, pressure, pain, itch, and temperature.

[0003] 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 μm 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.

[0004] 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.

[0005] 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 ε-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 β (TGF-β) 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 ^ α/., 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 μm in diameter.

[0007] Aging and exposure to environmental insults affect the appearance and structure of the skin. Sun-protected, naturally aged skin exhibits epidermal and dermal thinning, fragility, and fine, shallow wrinkles. There is a loss of elastic fibers in the papillary dermis, and collagen fibers become increasingly dense and more randomly oriented. Exposure to ultraviolet radiation from the sun accelerates and alters the degenerative processes associated with aging, resulting in a constellation of changes collectively known as photodamage. As much as 80% of facial aging may well be attributable to sun exposure. Photodamaged skin is characterized by loss of elasticity, deep wrinkles, increased roughness and dryness, and altered pigmentation (age spots). The skin may assume a leathery, thickened appearance characterized by deep furrows.

[0008] Many individuals desire to preserve a youthful appearance. At the same time, tanned skin is considered attractive and outdoor recreational activities are popular, resulting in significant sun exposure and consequent photodamage to skin. Various approaches have been developed or are under investigation to reduce the visible signs of photodamage (Stern, R., N. Engl. J. Med, 350:1526-1534, 2004). Use of sunscreens can help to prevent further damage. Topical retinoids (vitamin A derivatives) can reduce the severity of photoaging (U.S. Pat. No. 4,877,805). It has been proposed to treat the skin with compositions containing inhibitors of collagenase or elastase (U.S. Pat. Nos. 5,614,489; 6,884,425). Chemical peels, which involve application of a variety of different chemical compounds to skin, result in temporary improvement in the appearance of photodamaged skin in some individuals. Microdermabrasion and laser resurfacing are other alternatives. Injection of botulinum toxin (e.g., Botox®) has gained popularity as a means of reducing furrows caused by muscle hypertonicity. Skin fillers such as bovine collagen and hyaluronic acid are used for soft tissue augmentation to treat deep wrinkles. However, none of these approaches has provided a satisfactory solution to the problem of damage to skin caused by exposure to the sun. There is a need in the art for improved compositions and methods for caring for photodamaged skin. Summary of the Invention

[0009] The present invention provides compositions and methods of caring for skin, in particular photodamaged, prematurely aged, and/or aged skin. In accordance with the present invention, 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. In certain embodiments, 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. In certain embodiments, the compositions are injected into the skin. [0010] In one aspect, 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. In certain embodiments of the invention 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. In certain embodiments, 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). In certain embodiments, desmosine release may be used as a measure of elastin digestion. See Viglio et al, J. Sep. ScL, 30:202-13, 2007; incorporated herein by reference. In certain embodiments, 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. In certain embodiments 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.

[0011] In certain embodiments a composition comprising one or more elastases is topically applied. In certain embodiments the composition is topically applied for between 15 minutes and 24 hours, e.g., between 30 and 240 minutes. In certain embodiments 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. In certain embodiments the composition is topically applied to an area of skin from which at least part of the papillary dermis has been removed. In certain embodiments, two or more compositions, each comprising an elastase enzyme, are topically applied. In certain embodiments, the skin care composition further comprises one or more compounds that have skin restoring properties. In other embodiments, following treatment with the one or more elastases, the skin is contacted with a skin care composition having skin restoring properties or a physical treatment having skin restoring properties is performed.

[0012] In certain embodiments of the invention 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.

[0013] In certain embodiments 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.

[0014] In another aspect, 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.

[0015] In another aspect, 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.

[0016] In another aspect, 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.

[0017] In another aspect, 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. In certain embodiments of the method the skin is photodamaged. In certain embodiments, the skin is aged. In certain embodiments, the skin is prematurely aged. [0018] In certain embodiments of any of the inventive methods, 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. In certain embodiments of any of the inventive methods, 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.

[0019] In another aspect, 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. In certain embodiments of the invention, at least one of the elastase enzyme(s) displays selective activity towards elastotic material. In certain embodiments, 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.

[0020] In another aspect, 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. In certain embodiments, the elastase is a bacterial elastase. In one embodiment, the bacterial elastase is PA elastase. In certain embodiments, the elastase is a mammalian elatase. In certain embodiments, the elastase is a human elastase. In certain embodiments, 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. [0021] In certain embodiments, 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. In certain embodiments, the elastase is a bacterial elastase. In one embodiment, the bacterial elastase is PA elastase. In certain embodiments, the elastase is a mammalian elatase. In certain embodiments, the elastase is a human elastase. In certain embodiments, the elastase is human neutrophil elastase. In certain embodiments, 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. In certain embodiments, the pharmaceutical composition may be suitable for subcutaneous delivery. In certain embodiments, the pharmaceutical composition may be suitable for transdermal delivery. In certain embodiments, the pharmaceutical composition is an injectable composition. [0022] In another aspect, 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. In certain embodiments, 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.

[0023] In yet another aspect, 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. In certain embodiments, the enzyme may be chemically modified. For example, the enzyme may be PEGylated or conjugated to another polymer. In certain embodiments, the enzyme may be genetically engineered.

[0024] Unless otherwise stated or otherwise evident from the context, 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. Y., edition as of July 2002; Sambrook, Russell, and Sambrook, Molecular Cloning: A Laboratory Manual, 3^rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 2001; Viglio et ah, "Progress in the methodological strategies for the detection in real samples of desmosine and isodesmosine, two biological markers of elastin degradation" J. Sep. Sci. 30:202-213, 2007; and other standard references mentioned elsewhere herein. The contents of all scientific articles, books, patent applications, patents, and other publications mentioned herein are incorporated herein by reference. In the event of a conflict or inconsistency between any of the incorporated references and the instant specification, the specification shall control.

[0025] Where ranges are given, endpoints are included within the range. Furthermore, it is to be understood that unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as a range can assume any specific value or subrange within the stated range in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise. Where a percentage is recited in reference to a value that intrinsically has units that are whole numbers, any resulting fraction may be rounded to the nearest whole number.

[0026] The word "including", should be interpreted herein as "including, but not limited to", unless otherwise stated or evident from the context. The words "diminish" and "reduce" are used interchangeably herein.

Brief Description of the Drawings

[0027] 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 ⁰C 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 μg/ml Pseudomonas aeruginosa elastase (PAE) in 200 mM buffer (pH 8.8) for 24 hrs at 37 ⁰C 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) and IF (consisting of upper and lower panels on right) show additional micrographs of control and treated sections, respectively, at 4x magnification. [0028] 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 ⁰C) that were used to demonstrate the ability of elastase to degrade elastotic material in photodamaged skin sections (see Figures 1 A-IF).

[0029] Figure 3 A shows a punch of photodamaged human cheek skin that was exposed to 20 μL of 30 mM Tris buffer (pH 7.2) containing 50 μg/ml Pseudomonas aeruginosa elastase for 6 hours at 37 ⁰C 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 ⁰C 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.

[0030] Figure 4 A shows a punch of photodamaged human skin from the chin that was treated with a 20 μl intradermal injection of 30 mM Tris buffer (pH 7.2) with no elastase, exposed to buffer with no elastase for 6 hours at 37 ⁰C, 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 μl intradermal injection of 30 mM Tris buffer (pH 7.2) buffer with 50 μg/ml Pseudomonas aeruginosa elastase, exposed to buffer with no elastase for 6 hours at 37 ⁰C, and then stained for elastin using VVG stain (4x magnification). The red scale bar indicates 100 microns.

[0031] 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 ⁰C) 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).

[0032] Figure 6 shows the activity of porcine pancreatic elastase over time in the presence of common cosmetic ingredients.

[0033] Figure 7 shows the activity of porcine pancreatic elastase (PPE) over 4 hours at 37 ⁰C in Carbopol, sorbitol, glycerin, and control formulations. [0034] Figure 8 shows the activity of porcine pancreatic elastase (PPE) over 19 hours at

37 ⁰C in Carbopol, sorbitol, glycerin, and control formulations.

[0035] 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)

Placebo formulation, 4Ox.

[0036] 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)

Placebo formulation, 10x.

[0037] 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)

Placebo formulation, 4Ox.

[0038] Figure 12 shows the activity of Ps eudomonas aeruginosa elastase (PAE) over time at varying concentrations at pH 8.8.

[0039] Figure 13 shows the activity of Ps eudomonas aeruginosa elastase (PAE) over time at varying concentrations at pH 7.2.

[0040] Figure 14 shows the activity of Ps eudomonas aeruginosa elastase (PAE) over time at varying concentrations at pH 5.0.

[0041] Figure 15 shows the reaction velocity of PAE at different pH's at two different concentrations.

[0042] Figure 16 shows the activity of porcine pancreatic elastase (PPE) over time at varying concentrations at pH 8.8.

[0043] Figure 17 shows the activity of porcine pancreatic elastase (PPE) over time at varying concentrations at pH 7.2.

[0044] Figure 18 shows the reaction velocity of human neutrophil elastase (HNE) at different pH's at three different concentrations.

[0045] 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.

[0046] Figure 2OA shows human skin injected with varying concentrations of PPE and treated over varying exposure durations. The sections were stained with Verhoeff-Van

Gieson stain. Figure 2OB shows human skin with the stratum corneum barrier removed and then treated with varying concentrations of PPE over varying exposure duractions. The sections were stained with Verhoeff-Van Gieson stain. [0047] 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.

Detailed Description of Certain Embodiments of the Invention

I. Definitions

[0048] The following definitions are provided to assist in understanding the invention. [0049] "Aged" as describing skin 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.

[0050] 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. [0051] 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). [0052] 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).

[0053] The term "cosmetic" refers to a composition or substance 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.

[0054] "Cosmetically acceptable", as used herein, 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.

[0055] The term "effective amount" as used herein 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. Preferably 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.

[0056] The term "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. The term "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. In certain embodiments, the term "elastase" does not include trypsin. In certain embodiments, the term "elastase" does not include chymotrypsin. In certain embodiments, the term "elastase" does not include papain. In certain embodiments, the term "elastase" does not include bromelain. In certain embodiments, the term "elastase" does not include subtilisin. [0057] 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.

[0058] "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).

[0059] "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.

[0060] "Purified", as used herein, 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. In certain embodiments of the invention 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. [0061] The term "skin care composition" as used herein 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.

[0062] To "solubilize" means to allow or cause a substance to dissolve, i.e., to allow or cause it to go into solution.

[0063] The terms "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.

[0064] The term "topical application", as used herein, 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.

[0065] Terms such as "treat", "treating", or "treatment" as used herein refer to any type of management of the skin so as to provide a cosmetic or therapeutic benefit to the skin. [0066] "Visible signs of photodamage" 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 exposure of skin to the sun. 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.

[0067] "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

[0068] Chronic exposure to the sun results in a number of visible signs of photodamage, as described above. 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. Int., 49: 391-402, 1999; Ohnishi, Y., et al, Arch. Dermatol. Res., 292:27-31, 2000; Jenkins, G., Mechanisms of Aging and Development, 123: 801-810, 2000). Thus while the principle components of normal elastic fibers are present in photodamaged skin, the structural organization and functionality of these fibers are severely perturbed. The presence of amorphous, elastotic material, not seen in sun-protected skin, is a characteristic feature of photodamaged skin. The elastotic material accumulates primarily in the papillary dermis and may replace collagen fibers, contributing to the reduction in collagen content that is characteristic of photodamaged skin. The accumulation of elastotic material as a consequence of sun exposure is also referred to as "solar elastosis". [0069] 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. Exposure to UV light may stimulate such production (Cavarra, E., et al, Biol. Chem., 383: 199-206, 2002). There is evidence for increased synthesis of elastin mRNA in sun- exposed skin, which may contribute to accumulation of aberrant elastin. The precise structure of elastotic material remains unknown, but it differs significantly from normal elastic fibers in appearance and functional properties. Accumulation of elastotic material is believed to play a major role in the undesired alterations in the appearance and texture of photodamaged skin.

[0070] 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. While it is anticipated that a major use of the 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). The use of the 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. In most instances, 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. Without wishing to be bound by any theory, the enzyme(s) may degrade elastotic material to smaller fragments. These fragments may be further degraded, their components absorbed, or may be rinsed away.

[0071] In one embodiment, 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. In another embodiment, 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. In another embodiment, 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. In another embodiment, 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. In another embodiment, 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. In another embodiment, 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. In one embodiment 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. In the afore -mentioned embodiments, the one or more enzymes may be present in a single composition or may be present in two or more compositions.

[0072] In another embodiment 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. In another embodiment 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. In another embodiment 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. In certain embodiments the skin care composition preserves or improves the condition of the dermis. In certain embodiments the composition inhibits accumulation of elastotic material in the dermis. In certain embodiments 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. [0073] In another embodiment, 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. In another embodiment, 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. In another embodiment, 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. In certain embodiments, the skin care composition preserves or improves the condition of the dermis. In certain embodiments, the composition inhibits accumulation of elastotic material in the dermis. In certain embodiments, 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. [0074] The following sections further describe these and other aspects of the invention. III. Enzymes for Reducing Evidence of Elastotic Material

[0075] 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. In general, 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. (eds.), Handbook of Proteolytic Enzymes, Academic Press, 1998, incorporated herein by reference. See Davidson, J.M., and Giro, M. G., "Control of Elastin Synthesis: Molecular and Cellular Synthesis", in Mecham, R. (ed.), Regulation of Extracellular Matrix Accumulation, Academic Press, Inc., 1986, for discussion of elastin and methods for its preparation and analysis and other relevant information. See Beith, J.G., "Elastase: Catalytic and Biological Properties" in the same volume for discussion of elastases and methods for their preparation and analysis (including assays for elastase activity and discussion of artificial elastase substrates) as well as other relevant information. See also Methods in Enzymology, Academic Press, Volumes 5, 19, 80, and 144 for protocols describing methods for preparing elastase, methods for assessing elastase activity, etc.

[0076] 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. For purposes of description, 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. In other words, adjectives referring to an organism, cell, or tissue that naturally synthesizes a protein are used descriptively herein to refer to the origin of the amino acid sequence information rather than to denote the source for the protein used in a composition. For example, "bovine elastase" refers to a protein having the sequence of an elastase that is found naturally in bovines, and 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. Similarly, "pancreatic elastase" refers to a protein having the sequence of an elastase that is naturally found in pancreatic tissue, and 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. Unless otherwise indicated, enzymes whose name includes a number may be referred to by Roman numeral or Arabic numeral interchangeably herein. For example, 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.

A. Elastolytic enzymes

[0077] Any of a variety of different elastolytic enzymes may be used in the compositions and methods of the present invention. 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. In certain embodiments of the invention 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. [0078] 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. coagulans, B. cereus, B. pumilis, B. mycoides, B. thermoproteolyticus), 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) . See Bieth, supra and references therein; Janda, Curr Microbiol., 39(2):73-8, 1999; Jeong, K., et al, Infection and Immunity, 68(9), 5096-5106, 2000. 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. In one embodiment, elastase is obtained from staphylococci of the type sometimes found in the oral cavity (Murphy, R. A., J Dent Res 53(4): 832-4). In one embodiment, elastase is obtained from Pseudomonas aeruginosa.

[0079] 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. Unless otherwise specified, "PA elastase" as used herein 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. Thus the invention provides a skin care composition comprising PA LasB and LasA.

[0080] Genes encoding a number of other bacterial elastases have been cloned, and elastase activity of their encoded proteins has been confirmed. See, e.g., Cascon, A., et al, Infect. Immun. 68(6):3233-41, 2000, describing the cloning and recombinant expression of a gene encoding Aeromonas hydrophila elastase. Also of interest herein are elastases produced by Bacillus species, e.g., Bacillus sp. EL31410 (Xu, Y, et al., J Zhejiang Univ Sci B ^'., 6(11): 1087-94, 2005). Also of interest is 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 Gόtz, 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. Bacteriol., 172(5):2608-13, 1990). Also of interest are alkaline 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). In another embodiment, the elastase is from Clostridium histiolyticum (U.S. Pat. No. 6,146,626).

[0081] 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. One of skill in the art will be able to obtain similar information for other enzymes mentioned herein where available. 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. It will be appreciated that enzymatically active allelic or strain- specific variants of some of these enzymes may exist and are of use in the present invention. As discussed further below and well known in the art, many elastases are secreted proteins whose primary, unprocessed sequence includes a signal peptide and, in some cases, a propeptide. Unless otherwise indicated, 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.

Table 1

Gene Protein Species Gene ID Accession numbers lasA LasA P. aeruginosa 878260 NC 002516 (cDNA);

NP 250562 (protein) lasB LasB P. aeruginosa 880368 NC 002516(cDNA);

NP 252413 (protein) lasB LasB L. pneumophila 3077652 NC 002942 (cDNA);

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)

[0082] 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. In certain embodiments of the invention, 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.

[0083] In certain embodiments of the 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. (Tokyo), 99(6): 1707-12, 1986; Kawashima et al, DNA (NY), 6:161-170, 1987; Gestin et al, Comp Biochem Physiol B Biochem MoI Biol., 118(1): 181-7, 1997). In most mammals 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).

[0085] Elastolytic enzymes produced by polymorphonuclear leukocytes (neutrophils), e.g., leukocyte elastase, cathepsin G, and proteinase 3 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). However, in other embodiments the enzyme is not a leukocyte elastase. Other 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). In one embodiment 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. However, in certain embodiments of the invention the enzyme is not a cathepsin.

[0086] Also of use in certain embodiments of the invention are matrix metalloproteinases (MMPs) with elastolytic activity. 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). However, in certain embodiments of the invention, 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). [0087] 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.

Table 2

Gene Symbol/ Gene Protein name/ Species Gene Accession Number(s)

Name Alternate ID name(s)

ELAl/elastase 1, elastase 1, Homo 1990 NM_001971; NP_001962 pancreatic pancreatic sapiens

ELA2/ neutrophil Homo 1991 NM_001972; NP_001963 elastase 2 elastase; sapiens leukocyte elastase

ELA2A/ pancreatic Homo 63036 NM_033440; NP_254275 elastase 2A elastase 2 sapiens

ELA3A/ elastase 3A, Homo 10136 NM_005747; NP_005738 elastase 3A, pancreatic sapiens pancreatic

ELA3B, elastase 3B, elastase 3B, Homo 23436 NM_007352; NP_031378 pancreatic pancreatic sapiens

MMP- 12, matrix matrix Homo 4321 NM_002426; NP_002417 metallopeptidase 12 metallopeptidase sapiens

12; macrophage elastase

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,

Wegener granulomatosis autoantigen

ELAl, elastase 1, elastase 1, Bos 281139 NM_174048; NP_776473 pancreatic pancreatic taurus

Ela2, elastase 2 elastase 2, Bos 282687 NM_174714; NP_777139 pancreatic taurus

ELA3B, elastase 3B, elastase 3B, Bos 281752 AY057840; AAL23697 pancreatic pancreatic taurus

ELS 1 , pancreatic pancreatic Sus 396766 NM_213823; NP_998988 elastase I elastase I scrofa

ELA2, pancreatic pancreatic Sus 397197 NM_214109; NP_999274 elastase II elastase II scrofa

EIa 1 elastase 1, Mus 109901 NM_033612; NP_291090 pancreatic musculus Ela2a, elastase 2A elastase 2A; Mus 13706 NM_007919; NP_031945 neutrophilic musculus elastase

Ela2 elastase 2, Mus 50701 NM_015779; NP_056594 neutrophil musculus

Ela3 elastase 3, Mus 67868 NM_026419; NP_080695 pancreatic musculus

Mmp-12 matrix Mus 17381 NM_008605; NP_032631 metallopeptidase musculus

12; macrophage elastase

[0088] 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). In certain embodiments of the invention the enzyme is not found in fish or crustaceans, e.g., krill. In certain embodiments of the invention the enzyme is not found in plants. In certain embodiments the enzyme is not papain or bromelain.

[0089] Many enzymes of use in the present invention, including 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. In addition, 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. For example, 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 As used herein, 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. In certain embodiments of the present invention the processed form of an enzyme is used. In other embodiments the full length (unprocessed) form, optionally without the signal peptide, is used. In some embodiments of the invention a complex containing the propeptide noncovalently associated with the portion of the protein that contains the active site (e.g. , following processing) is used. In certain embodiments of the invention, sufficient propeptide dissociates from the complex or is degraded following contact with the skin to permit effective levels of activity. In certain embodiments of the invention 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.

[0090] Certain of the compositions contain a bacterial elastase and a mammalian elastase, e.g., a pancreatic elastase. In an exemplary embodiment the composition contains PA elastase and a pancreatic elastase.

B. Partial elastases

[0091] In certain embodiments of the invention 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. In certain embodiments of the invention, two or more such enzymes are used in combination to reduce evidence of elastotic material or diminish accumulation thereof. In some embodiments of the invention, 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.

C. Enzyme Variants

[0092] 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. In one embodiment, 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.

[0093] The use of recombinant DNA technology to generate nucleic acid sequences encoding variants that differ from their naturally occurring counterpart at selected or random positions is well known in the art and need not be described here (see, e.g., Sambrook, supra and Ausubel, supra). For example, PCR can be used to introduce one or more changes into a nucleotide sequence. The invention contemplates enzyme variants that differ at one or more positions that lie within a target sequence for non-enzymatic hydrolysis or for cleavage by the enzyme itself (autoproteolysis) or by enzymes that may be present in the dermis. For example, one or both of the amino acids joined by a peptide bond susceptible to cleavage, or any amino acid that lies within up to 3-4 amino acids away the bond, may be deleted or replaced by a different amino acid. Alternately, 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.

[0094] Many substitutions, deletions, and additions result in proteins that are functionally equivalent to the original protein or at least retain sufficient activity to be useful in the present invention. For example, 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. Such substitutions are referred to as "conservative substitutions". 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.

[0095] 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.

[0096] The structures of several of the elastases described herein are known in the art, in some cases complexed with substrates. Furthermore, variants of several of these enzymes have been identified or generated and their activity assessed. The availability of this information will allow one of skill in the art to predict where substitutions, deletions, or insertions are unlikely to significantly affect protein function. Nevertheless, when it is difficult to predict the effect of the substitution, deletion, or insertion in advance of creating the protein, one skilled in the art will appreciate that the effect will be evaluated using routine assays as described herein or known in the art. Thus elastases and partial elastases as described herein encompass those active variants that can be routinely made and tested and that perform adequately in the methods of the invention.

[0097] In certain embodiments of the invention 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. In certain embodiments of the invention 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. In certain embodiments of the invention at least 50%, at least 75%, at least 90%, at least 95%, or 100% of the substitutions are conservative substitutions. In certain embodiments of the invention 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.

D. Testing enzymatic activity

[0098] The elasto lytic activity of an enzyme or combination of enzymes can be tested by a variety of different methods known in the art. 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. For example, it is insoluble in 8 M urea, 6M guanidinium chloride plus dithiothreitol, hot alkali, and weak organic acids but can be partially hydrolyzed by hot oxalic acid to produce 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. (Starcher, B.C. and Galione, M. J., Anal. Biochem., 74: 441, 1976) can be used. Various methods are compared in Daamen, W.F., et al., Biomaterials, 22:1997-2005, 2001).

[0099] In certain embodiments, elastolytic activity is determined using insoluble elastin labeled with any of a variety of different fluorescent or colored labels as a substrate. For example elastin-Congo red (Sigma) may be used as the substrate. Reaction suspensions (1.1 ml in 50 mM Tris-HCl, 0.5 mM CaCl₂, 0.2 mg/ml bovine serum albumin, pH 7.5) containing 10 mg of elastin-Congo red and 0.5 μg of one or more proteins to be tested for elastase activity are incubated at 37 ⁰C 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. Alternately, one unit of elastase activity may be defined as the amount of enzyme required to solubilize 1 μg insoluble elastin-Congo Red per hour at 37°C under the test conditions when in the linear part of the activity range. A variety of other labeled elastins such as elastin-fluorescein, elastin-rhodamine, elastin-orcein, may be used (e.g. product codes ES80 (elastin-fluorescein), E164, elastin-Congo Red, The Elastin Products Company, Owensville, MO). In certain embodiments of the invention 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. In another embodiment 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). In another embodiment 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.

E. Sources

[00100] Enzymes for use in the present invention can be purified from natural sources using methods well known in the art. For example, in one embodiment 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). In another embodiment, 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" as used herein refers to organisms that naturally produce the enzyme. For example, 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.

[00101] 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.

[00102] 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. Typically 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). In some cases, 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 are also well known in the art and 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.

[00103] 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. If desired, 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. colϊ) 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. It will be appreciated that for purposes of recombinant expression a coding sequence lacking the signal sequence and/or propeptide may be used. However the propeptide may be included, particularly if it facilitates or is important for correct folding of the protein. If secretion of the recombinantly expressed enzyme is desired, a signal peptide coding sequence may be included. However, 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.

[00104] Certain of the 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.

[00105] 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. For example, conjugation with various polymers such as polyethylene glycol (PEG) is a well known method of increasing protein stability in vivo. 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. General methods for conjugation and cross-linking are described in "Cross-Linking", Pierce Chemical Technical Library, available at the Web site having URL www.piercenet.com and originally published in the 1994-95 Pierce Catalog and references cited therein; in Wong SS, Chemistry of Protein Conjugation and Crosslinking, CRC Press Publishers, Boca Raton, 1991; and G. T. Hermanson, Bioconjugate Techniques, Academic Press, Inc., 1996.

[00106] As discussed further below, 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. In general, 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. If obtained by chemical synthesis, 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. F. Selecting an Enzyme or Enzyme Combination

[00107] 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. In certain embodiments of the invention the enzyme solubilizes elastotic material while leaving normal elastic fibers substantially intact. In certain embodiments of the invention 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.

[00108] 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. In certain embodiments of the invention 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.

[00109] 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. One of skill in the art, e.g., a pathologist having knowledge of dermatopathology, will be able to distinguish normal appearing elastin fibers from elastotic material.

[00110] 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 (Berterechte, 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.

[00111] In certain embodiments, 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. [00112] In certain embodiments of the invention, 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. For example, when assessed under identical conditions of substrate amount, enzyme concentration, pH, temperature, and solvent 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. In certain embodiments of the invention 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. Exemplary conditions for PA elastase are provided in the Examples. In certain embodiments of the invention 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. Amino acids released are expressed as micromoles leucine per milligram collagenase. [00113] In certain embodiments of the invention 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 lμmole of p-toluene-sulfonyl-L-arginine methyl ester (TAME) per minute at 25 ⁰C, pH 8.2, in the presence of 1OmM calcium. In certain embodiments of the invention 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 ⁰C, pH 7.8 in the presence of calcium.

[00114] 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. In some embodiments of the invention 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. For example, in one embodiment 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.

[00115] Specificity of enzymes towards various portions of cross-linked elastin or other substrates can be evaluated using model substrates. In one embodiment the 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. M., et al., Biochim Biophys Acta 995(3): 285-90, 1989). Mecham, R. P., T. J. Broekelmann, et al (1997). Additional methods for analyzing cleavage site specificity are described in Mecham, RP, et ah, J Biol Chem 272(29): 18071-6, 1997.

[00116] Certain of the methods are performed in vivo. In one embodiment 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. In another embodiment of the method different 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.

[00117] 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.

[00118] 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. For example, 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. Skin Care Compositions Containing Elastase or Partial Elastase Enzymes [00119] 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%. In certain embodiments of the invention, 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% 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. The compositions are typically formulated by combining one or more enzymes with one or more other components that serve any of a variety of functions. It will be appreciated that many cosmetic ingredients have multiple functions in formulations and therefore could properly be included in several functional categories. Thus, it should be understood that although the components mentioned herein may be grouped based on the benefϊt(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, emulsifϊers, opacifying agents, penetrants, solvents, salts, pH adjusting agents, buffers, neutralizing agents, particulate matter, etc.

A. Carrier [00121] The enzyme(s) will typically be combined with 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. For example, the amount of water may be between 30% and 90%, between 40% and 80%, between 50% and 80%, etc. Optionally 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.

[00122] 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, inositol, palatinit (isomalt), butane- 1,4-diol, butoxytriol, and mixtures thereof. The alkoxylated derivatives of any of the above polyhydric alcohols are also suitable for use herein. In certain embodiments the polyhydric alcohol is selected from glycerine, polyethylene glycol, hexane triol, butane- 1,4- diol, butoxytriol, erythritol, xylitol, and mixtures thereof. Other 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.

[00123] In certain embodiments of the invention the composition comprises one or more elastase or partial elastase enzymes and a polyglycerol containing at least 15 carbon atoms. Examples of polyglycerols containing at least 15 carbon atoms are pentaglycerol, hexaglycerol, heptaglycerol, octaglycerol, nonaglycerol and decaglycerol. In certain embodiments the polyglycerol contains at least 21 carbon atoms, at least 27 carbon atoms, or at least 30 carbon atoms. In one embodiment, the polyglycerol is decaglycerol. In certain embodiments of the invention 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. In certain embodiments of the invention 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. Examples of polyols containing at most 6 carbon atoms are glycerol, ethylene glycol, propylene glycol, butylene glycol and sorbitol. Examples of polymeric glycols are polyethylene glycol 200 and 400. In certain embodiments 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.

B. Enzyme Stabilizers and Activity Enhancers

[00124] 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. In certain embodiments of the invention 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. In certain embodiments of the invention, the composition comprises an osmoprotectant. Suitable osmo-protectants are known in the art. See, e.g., U.S. Pub. No. 20030175232. 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. Other 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. Other 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)), may include oxidized monosaccharides, e.g. 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.

[00125] In certain embodiments of the invention, 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. In certain embodiments of the invention the inhibitor has a K₁ (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. In certain embodiments of the invention 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 K₁ 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.

[00126] 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. For example, the inhibitor could have a K₁ 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 K₁ with respect one or more other elastases (e.g., pancreatic elastase, neutrophil elastase, etc.).

[00127] 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 α-1 proteinase inhibitor, α2-macroglobulin, and the pancreatic Kunitz inhibitor, also known as aprotinin, bovine basic pancreatic trypsin inhibitor (BPTI), and trypsin-kallikrein inhibitor. Examples of 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. Tsuji et al, Biochem. Biophys. Res. Comm., 122(2): 571-576, 1984; U.S. Pat. Nos. 4,643,991; 5,008,245; 5,162,307; 5,539,123), trifluoroacetylated peptide anilides. Specific examples include Ac-Pro-Ala-Pro-Alaninal, MeOSuc-(Ala)₂-Pro-NH- C(CH(CHs)₂H-B(OH)₂, CF₃CO-LyS-AIa-NHCH₂C₆H₅, etc. See Bieth, supra, and references therein; Digenis, et al., J Med Chem., 29(8): 1468-76, 1986. Many of these compounds have peptide-like structures that mimic the enzyme substrate, e.g., they mimic a subsequence of elastin or another substrate within which cleavage occurs and will typically include at least 1 or 2 amino acids. Other compounds that structurally resemble such cleavage sites can also be used. One of skill in the art will be able to select suitable peptide or peptide-like inhibitors based on the known substrate specificities of a particular elastase or partial elastase. In certain embodiments of the invention the inhibitor is a propeptide of the enzyme. In addition to the foregoing, a number of other inhibitors in different compound classes are known. Heterocyclic inhibitors are described in Bieth, supra, and references therein. Other useful inhibitors include Nl/C4-substituted beta-lactams (Gerard, S., et al., Bioorg Med Chem., 12(l):129-38, 2004). [00128] The 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.

[00129] The 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.

[00130] The compositions may include an agent that enhances the activity of the enzyme. In certain embodiments of the invention 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).

[00131] Metalloproteinases bind a metal ion such as Zn²⁺ or Ca²⁺ in their active site. In certain embodiments of the invention the composition comprises a divalent metal ion such as Zn²⁺ or Ca²⁺ in an amount sufficient to promote activity of a metalloproteinase in the composition. In certain embodiments of the invention, the composition comprises a chelating agent such as EDTA. The chelating agent binds some or essentially all of the divalent cation {e.g., Zn²⁺ or Ca²⁺), 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. In other embodiments of the invention the composition does not contain a chelating agent.

C. pH Regulating Agents

[00132] 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. Typically 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. It will be appreciated that 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. [00133] 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-hydroxyethyl)amino]-2-hydroxypropane sulphonic acid (DIPSO), salts of phosphate, pyrophosphate, citrate, acetate, and mellitate, and mixtures thereof. Other exemplary 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, Phosphonobutanetricarboxylic Acid, Potassium Acetate, Potassium Bicarbonate, Potassium Biphthalate, Potassium Citrate, Potassium Lactate, Sodium Acetate, Sodium Aluminate, Sodium Aluminum Lactate, Sodium Bicarbonate, Sodium Citrate, Sodium Fumarate, Sodium Lactate, Sodium Phosphate, Sodium Silicate, Sodium Succinate, Sodium Trimetaphosphate, Tetrapotassium Pyrophosphate, Tetrasodium Pyrophosphate, Urea, and Zinc Glycinate. 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, Dipropylenetriamine, Disodium Fumarate, Disodium Phosphate, Disodium Pyrophosphate, Disodium Tartrate, Ethanolamine, Ethanolamine HCI, Formic Acid, Fumaric Acid, Galacturonic Acid, Glucoheptonic Acid, Glucosamine HCI, Glucuronic Acid, Glutaric Acid, Glycolic Acid, Glyoxylic Acid, Guanidine Carbonate, Hydrobromic Acid, Hydrochloric Acid, Imidazole, Isopropanolamine, Isopropylamine, Ketoglutaric Acid, Lactic Acid, Lactobionic Acid, Lithium Hydroxide, Magnesium Carbonate, Magnesium Carbonate Hydroxide, Magnesium Hydroxide, Magnesium Oxide, Maleic Acid, Malic Acid, Malonic Acid, Metaphosphoric Acid, Methylethanolamine, Methylglucamine, Mixed Isopropanolamines, Monosodium Citrate, Morpholine, Oxalic Acid, PEG-114 Methylether Polyepsilon Capralactone, Pentapotassium Triphosphate, Pentasodium Triphosphate, Phosphoric Acid, Potassium Bicarbonate, Potassium Biphthalate, Potassium Borate, Potassium Carbonate, Potassium Citrate, Potassium Hydroxide, Potassium Magnesium Aspartate, Potassium Phosphate, Potassium Tartrate, Propionic Acid, Quinic Acid, Ribonic Acid, Sebacic Acid, Sodium Aluminate, Sodium Bicarbonate, Sodium Bisulfate, Sodium Borate, Sodium Carbonate, Sodium Citrate, Sodium Fumarate, Sodium Hydroxide, Sodium Oxide, Sodium Sesquicarbonate, Sodium Silicate, Sodium Succinate, Sodium Trimetaphosphate, Strontium Hydroxide, Succinic Acid, Sulfuric Acid, Tartaric Acid, TEA- Diricinoleate/IPDI Copolymer, Tetrapotassium Pyrophosphate, Tetrasodium Pyrophosphate, Triethanolamine, Triisopropanolamine, Trisodium Phosphate, Tromethamine, Vinegar, Zinc Carbonate Hydroxide, Zinc Glycinate, Zinc Magnesium Aspartate, and mixtures thereof.

D. Additional Optional Agents

[00134] The 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. 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. (eds.), Handbook of Cosmetic Science & Technology (CRC, 2001); Milady's Skin Care and Cosmetics Ingredients Dictionary, 2^nd ed. (Milady, 2000); CTFA International Cosmetic Ingredient Dictionary and Handbook, 8^th ed., Vol. 2, edited by Wenninger and McEwen (The Cosmetic, Toiletry, and Fragrance Association, Inc., Washington, D. C, 1999); Joubert JP, Marty JP, and Chevasson, F., Handbook of Cosmetic Ingredients (2005), distributed by SoGeCos, France. [00135] Compositions of the invention may contain additional active agents. In certain embodiments 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.

[00136] In certain embodiments, the composition includes one or more vitamins to nourish the skin. Exemplary vitamins include vitamin A, vitamin Bi (thiamine), vitamin B₂ (riboflavin), vitamin B₃ (niacin), vitamin B compounds {e.g., vitamin B3 compounds such as those described in U.S. Pub. No. 20030175232 and references therein), retinoids (see below), panthenol, niacinamide, retinol, retinyl propionate, retinyl palmitate, retinoic acid, flavonoids (chalcones, chromones, flavones, isoflavones), vitamin B₄ (adenine), vitamin B5 (pantothenic acid), vitamin B₆ (pyridoxine), vitamin B₇ (biotin), vitamin B9 (folic acid), vitamin B12 (cyanocobalamin), vitamin C (ascorbic acid), vitamin D (ergocalciferol), vitamin E (tocopherol), tocopherol acetate, vitamin K, and combinations thereof. In certain embodiments, the composition includes vitamin E. In certain embodiments, 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. In certain embodiments, the composition includes approximately 0.001% to approximately 5% by weight of the vitamin. In certain embodiments, the composition includes approximately 0.0005% to approximately 3% by weight of the vitamin. In certain embodiments, the composition includes approximately 0.001% to approximately 2% by weight of the vitamin. [00137] 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. As would be appreciated by one of ordinary skill in the art, derivatives, mutants, fusion proteins, fragments, or combinations of any of these proteins may also be included in the inventive cosmetic composition. In certain embodiments, any of the twenty natural amino acids may be included in the inventive composition. In certain embodiments, 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%. In certain embodiments, 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. Plant extract 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. In certain embodiments, 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. John's bread, henna, hop, lime, orange, lemon, grapefruit, aloe, thyme, calendula, horsetail, mountain gentian, nettle, chestnut, avocado, seaward, milfoil, coltsfoot, marigold, peach, rose, senna, mint, white lilly, lavender, grape seed, bayberry, saw palmetto, tea tree, soy bean, almond, peanut, sea buckthorn seed, seaweed, tea tree, hibiscus, lemongrass, horsetail, rasberry, rose hips, and olive. Certain of these plants have been used in the past to prepare plant extract for hair care compositions. In certain embodiments, the plant extracts useful in the inventive hair care composition may be obtained commercially. 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.

[00139] In certain embodiments, the composition may include a sunscreen agent to protect the skin from the damaging ultraviolet rays of the sun. In certain embodiments, the sunscreen agent protects the skin from damaging UV-A and/or UV-B rays. In certain embodiments, 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. Exemplary 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, titanium dioxide, trolamine salicylate, zinc oxide, ecamsule, terephthalylidene dicamphor sulfonic acid, 4-methylbenzylidene camphor, bisoctrizole, methylene bis-benzotriazolyl tetramethylbutylphenol, bemotrizinol, bis-ethylhexyloxyphenol methoxyphenyl triazine, drometrizole trisiloxane, bisdisulizole disodium, disodium phenyl dibenzimidazole tetrasulfonate, diethylamino hydroxybenzoyl hexyl benzoate, octyl triazone, ethylhexyl triazone, iscotrizinol, diethylhexyl butamido triazone, polysilicone-15, hydroxyphenylbenzotriazole, isoamyl p-methoxycinnamate, benzophenone-3 (oxybenzone), benzophenone-4 (sulisobenzone), benzophenone-8 (dioxybenzone), butyl methoxydibenzoylmethane (avobenzone), cinoxate, DEA- methoxycinnamate, digalloyl trioleate, dimethicone/PEG-15 crosspolymer, l-(3,4- dimethoxyphenyl)-4,4-dimethyl- 1 ,3-pentanediene, ethylhexyl methoxycinnamate (octinoxate), ethylhexyl salicylate (octisalate), 4-(2-beta-glucopyranosiloxy) propoxy-2- hydroxybenzophenone, Helianthus annuus (sunflower) seed extract, homosalate, hydrolyzed linseed extract, lawsone, menthyl anthranilate (meradimate), octocrylene, phenylbenzimidazole sulfonic acid (ensulizole), Pinus pinaster bark extract, red petrolatum, Spirulina platensis powder, TEA-salicylate (trolamine salicylate), titanium dioxide, Vitis vinifera (grape) seed extract,and combinations thereof. The inventive may include 0.0001% to approximately 5% by weight of the sunscreen agent. In certain embodiments, the composition includes approximately 0.001% to approximately 5% by weight of the sunscreen agent. In certain embodiments, the composition includes approximately 0.0005% to approximately 3% by weight of the sunscreen agent. In certain embodiments, the composition includes approximately 0.001% to approximately 2% by weight of the sunscreen agent.

[00140] 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, imidazolidinylurea, sulfur dioxide, and combinations thereof. Exemplary 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-demethoxycurcumin, Bis-Hydroxyethyl Tocopherylsuccinoylamido, Hydroxypropane, Butylated Xylenol, 4-Butylresorcinol, Caffeic Acid, Calcium Ascorbate, Calophyllum Inophyllum Seed Oil, Camellia Sinensis Catechins, Camellia Sinensis Leaf Extract, Camellia Sinensis Leaf Oil, Carnosic Acid, Carotenoids, Chitosan Ascorbate, Chitosan Glycolate, Chitosan Salicylate, Chlorogenic Acids, Cimicifuga Dahurica Root Extract, Citrus Medica Vulgaris Fruit Extract, Coptis Chinensis Root Extract, Crotonaldehyde, Curcumin, Cyamopsis Tetragonoloba (Guar) Symbiosome Extract, Cysteine, Cysteine HCI, Decyl Mercaptomethylimidazole, Demethoxycurcumin, Diamylhydroquinone, Di-t-Butylhydroquinone, Dicetyl Thiodipropionate, Dicyclopentadiene/t-Butylcresol Copolymer, Digalloyl Trioleate, Dilauryl Thiodipropionate, Dimethoxy Di-p-Cresol, Dimethylmethoxy Chromanol, Dimyristyl Thiodipropionate, Dioleyl Tocopheryl Methylsilanol, Diosmine, Disodium Ascorbyl Sulfate, Disodium Rutinyl Disulfate, Distearyl Thiodipropionate, Ditridecyl Thiodipropionate, Dodecyl Gallate, Dunaliella Bardawil Powder, Ellagic Acid, Epigallocatechin Gallate, Epimedium Sagittatum Extract, Ergothioneine, Eriobotrya Japonica Leaf Protoplasts, Erythorbic Acid, Ethylbisiminomethylguaiacol Manganese Chloride, Ethyl Ferulate, Ethylhexyl Ferulate, Ethylhexyl Gallate, Ferulic Acid, Foeniculum Vulgare (Fennel) Seed Extract, Forsythia Suspensa Fruit Extract, Fragilaria Pinnata Extract, Furfuryl Palmitate, Glucosylrutin, Glycine Max (Soybean) Symbiosome Extract, Glycine Soja (Soybean) Oil, Glycyrrhiza Glabra (Licorice) Root Extract, Grifola Frondosa Fruit Body Extract, Haematococcus Pluvialis Extract, Haematococcus Pluvialis Oil, Haematococcus Pluvialis Powder, Hesperetin Laurate, Hesperidin Methyl Chalcone, Hydrolyzed Gardenia Florida Extract, Hydroquinone, p- Hydroxyanisole, Hydroxydecyl Ubiquinone, Hydroxylamine HCI, Hydroxylamine Sulfate, Isooctyl Thioglycolate, Isoquercitrin, Kojic Acid, Kojyl Glucoside, Kojyl Methylenedioxycinnamate, Kou-Cha Ekisu, Lactobacillus/Rice Bran/Saccharomyces/Camellia Sinensis Leaf Extract Ferment, Lactobacillus/Wasabia Japonica Root Ferment Extract, Lens Culinaris (Lentil) Symbiosome Extract, Ligusticum Striatum Root Extract, Lupinus Subcarnosus Symbiosome Extract, Lycium Chinense Fruit Extract, Lycopene, Madecassicoside, Magnesium Ascorbate, Magnesium Ascorbate/PCA, Magnesium Ascorbyl Phosphate, Malachite Extract, Malpighia Punicifolia (Acerola) Fruit Extract, Manganese Dioxide, Medicago Sativa (Alfalfa) Symbiosome Extract, Melaleuca Alternifolia (Tea Tree) Leaf Oil, Melatonin, Melia Azadirachta Conditioned Media/Culture, Methoxy PEG-7 Ascorbic Acid, Methoxy-PEG-7 Rutinyl Succinate, Methoxytrimethylphenyl Dihydroxyphenyl, Propanol, Methylene Di-t-Butylcresol, Methylsilanol Ascorbate, Monascus/Rice Ferment, Murraya Exotica Leaf Extract, Nordihydroguaiaretic Acid, Octadecyl Di-t-butyl-4-hydroxyhydrocinnamate, Octanicotinoyl Epigallocatechin Gallate, Olea Europaea (Olive) Bud Extract, Olea Europaea (Olive) Fruit Unsaponifϊables, Oxycoccus Palustris Seed Oil, Palmitoyl Camellia Sinensis Extract, Palmitoyl Grape Seed Extract, PEG/PPG-2/5 Tocopheryl Ether, PEG/PPG-5/10 Tocopheryl Ether, PEG/PPG-5/20 Tocopheryl Ether, PEG/PPG-5/30 Tocopheryl Ether, PEG/PPG-30/10 Tocopheryl Ether, PEG/PPG-50/20 Tocopheryl Ether, PEG/PPG-70/30 Tocopheryl Ether, PEG/PPG- 100/70 Tocopheryl Ether, Pentaerythrityl Tetra-di-t-butyl Hydroxyhydrocinnamate, Perilla Ocymoides Seed Extract, Phenylthioglycolic Acid, Phloroglucinol, Pikea Robusta Extract, Pinus Pinaster Bark Extract, Pinus Radiata Bark Extract, Piper Nigrum (Black Pepper) Seed, Pisum Sativum Symbiosome Extract, Platycodon Grandiflorum Root Extract, Polygonum Cuspidatum Root Extract, Potassium Ascorbyl Tocopheryl Phosphate, Potassium Sulfite, PPG-2 Tocophereth-5, PPG-5 Tocophereth-2, PPG-IO Tocophereth-30, PPG-20 Tocophereth-50, PPG-30 Tocophereth-70, PPG-70 Tocophereth-100, PPG-5 Tocopheryl Ether, Propyl Gallate, Prunus Cerasus (Bitter Cherry) Extract, Pyridoxine Serinate, Quercetin, Quercetin Caprylate, Resveratrol, Rhodochrosite Extract, Rosmarinic Acid, Rosmarinus Officinalis (Rosemary) Flower Extract, Rosmarinus Officinalis (Rosemary) Leaf Extract, Rutin, Ryoku-Cha Ekisu, Salnacedin, Sargassum Pallidum Extract, Sarothamnus Scoparius Extract, Sedum Rosea Root Extract, Smithsonite Extract, Sodium Ascorbate, Sodium Ascorbyl/Cholesteryl Phosphate, Sodium Ascorbyl Phosphate, Sodium Bisulfite, Sodium Erythorbate, Sodium Metabisulfite, Sodium Phosphono-Pyridoxylidenerhodanine, Sodium Sulfite, Sodium Thioglycolate, Sodium Tocopheryl Phosphate, Sorbityl Furfural, TBHQ, Tetrabutyl Ethylidinebisphenol, Tetradibutyl Pentaerithrityl Hydroxyhydrocinnamate, Tetrahexyldecyl Ascorbate, Tetrahydrobisdemethoxydiferuloylmethane, Tetrahydrodemethoxydiferuloylmethane, Tetrahydrodiferuloylmethane, Tetramethylchromanol Glucosides, Thioctic Acid, Thiodiglycol, Thiodiglycolamide, Thiodiglycolic Acid, Thioglycolic Acid, Thiolactic Acid, Thiosalicylic Acid, Thiotaurine, Tococysteamide, Tocophereth-5, Tocophereth-10, Tocophereth-12, Tocophereth-18, Tocophereth-50, Tocopherol, Tocophersolan, Tocopheryl Acetate, Tocopheryt Linoleate, Tocopheryl Linoleate/Oleate, Tocopheryl Nicotinate, Tocopheryl Succinate, Tocoquinone, Toluene, o-Tolyl Biguanide, Totarol, Tremella Fuciformis Extract, Trifolium Pratense (Clover) Symbiosome Extract, Trigonella Foenum Symbiosome Extract, Tripropylene Glycol, Tris(Nonylphenyl) Phosph ite, Trisodium Fructose Diphosphate, Ubiquinone, Uuron-Cha Ekisu, Vaccinium Myrtillus Bud Extract, Vaccinium Myrtillus Stem Extract, Vaccinium Vitis-Idaea Leaf Protoplasts, Vaccinium Vitis- Idaea Seed Oil, Vicia Sativa Symbiosome Extract, Vitis Vinifera (Grape) Seed Extract, Xylyl Dibutylbenzofuranone, and Zinc Dibutyldithiocarbamate. 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 Cyclodextrin, Methyl Dihydroxybenzoate, Oxyquinoline, Oxyquinoline Sulfate, Pentapotassium Triphosphate, Pentasodium Aminotrimethylene Phosphonate, Pentasodium Ethylenediamine Tetramethylene Phosphonate, Pentasodium Pentetate, Pentasodium Triphosphate, Pentetic Acid, Phosphonobutanetricarboxylic Acid, Phytic Acid, Potassium Citrate, Potassium EDTMP, Potassium Gluconate, Potassium Polyphosphate, Potassium Trisphosphonomethylamine Oxide, Ribonic Acid, Sodium Chitosan Methylene Phosphonate, Sodium Citrate, Sodium Diethylenetriamine Pentamethylene Phosphonate, Sodium Dihydroxyethylglycinate, Sodium EDTMP, Sodium Gluceptate, Sodium Gluconate, Sodium Glycereth-1 Polyphosphate, Sodium Hexametaphosphate, Sodium Lauroyl Ethylenediamine Triacetate, Sodium Metaphosphate, Sodium Metasilicate, Sodium Phytate, Sodium

Polydimethylglycinophenolsulfonate, Sodium Polyphosphate, Sodium Trimetaphosphate, TEA-Cocamide Diacetate, TEA-EDTA, TEA-Polyphosphate, Tetrahydroxyethyl Ethylenediamine, Tetrahydroxypropyl Ethylenediamine, Tetrahydroxypropyl Ethylenediamine Dioleate, Tetrapotassium Etidronate, Tetrapotassium Pyrophosphate, Tetrasodium Dicarboxymethyl Aspartate, Tetrasodium EDTA, Tetrasodium Etidronate, Tetrasodium Glutamate Diacetate, Tetrasodium Iminodisuccinate, Tetrasodium Pyrophosphate, Tripotassium EDTA, Trisodium Dicarboxymethyl Alaninate, Trisodium EDTA, Trisodium Ethylenediamine Disuccinate, Trisodium Fructose Diphosphate, Trisodium HEDTA, Trisodium NTA, and Trisodium Phosphate. The inventive may include 0% to approximately 5% by weight of the preservative, antioxidant, or chelating agent. In certain embodiments, the composition includes approximately 0.0001% to approximately 5% by weight of the preservative, antioxidant, or chelating agent. In certain embodiments, the composition includes approximately 0.0005% to approximately 3% by weight of the preservative, antioxidant, or chelating agent. In certain embodiments, the composition includes approximately 0.001% to approximately 2% by weight of the preservative, antioxidant, or chelating agent.

[00141] Compositions may comprise an emollient (substance that softens and/or soothes the skin). In certain embodiments 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. Branched chain hydrocarbon emollients for use herein include isododecane, isohexadecane, isoeicosane, isooctahexacontane, isohexapentacontahectane, isopentacontaoctactane, and mixtures thereof. Other suitable 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. Other 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 (C₆-C ₁₃) fatty acids with linear (C₆-C_2O) fatty alcohols; esters of branched (C6-C13) carboxylic acids with linear (C₆-C₂O) fatty alcohols, esters of linear (C₆- C is) fatty acids with branched alcohols, especially 2-ethylhexanol; esters of linear and/or branched fatty acids with polyhydric alcohols (such as dimerdiol or trimerdiol, for example) and/or guerbet alcohols; triglycerides based on (C₆-Ci₀) fatty acids; esters such as dioctyl adipate, diisopropyl dimer dilinoleate; propylene glycols/dicaprylate or waxes such as beeswax, paraffin wax or microwaxes, alone or in combination with hydrophilic waxes, such as cetylstearyl alcohol, for example; fluorinated and perfluorinated oils; fluorinated silicone oils; mixtures of the aforementioned compounds. 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 oil. 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. In certain embodiments, 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. In certain embodiments, the oil is a fluorinated oil. Examples of 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. In certain embodiments, 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.

[00142] 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. Examples of humectants useful in the inventive compositions include acetyl glucosamine, ascorbic acid, diglycerin, methylglucamine, methylpropanediol, phytantriol, raffϊnose, 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, Agave Atrovirens extract, alanyl glutamine, Alcaligenes Polysaccharides, Algae extract, Aloe Barbadensis Leaf Extract, Aloe Barbadensis Leaf Polysaccharides, amidinoproline, Bacillus/Rice Bran Extract/Soybean Extract Ferment Filtrate, Betaine, Bittern, Black Strap Powder, 2,3-Butanediol, Caprylyl Glycol/Glycerin/Polyacrylic Acid Copolymer, Carnitine HCI, Chitosan Lauroyl Glycinate, Cholesterol/HDI/Pullulan Copolymer, Coix Lacryma-Jobi (Job's Tears) Shell Extract, Coleus Forskohlii Root Extract, Cryptomeria Japonica Leaf Extract, Cucumis MeIo (Melon) Fruit Water, Diglycereth-7 Malate, Diglycerin, Diglycol Guanidine Succinate, Dunaliella Bardawil Extract, Earthworm Conditioned Soil, Earthworm Conditioned Soil Extract, Erythritol, Ethylhexyl Hydroxystearoyl Hydroxystearate, Fructose, Glucose, Glucosyl Hesperidin, Glucuronolactone, Glycereth-7 Glycolate, Glycerin, glyceryl citrate crosspolymer, Glyceryl Dimaltodextrin, Glycol, Glycosyl Trehalose, Hesperetin Laurate, 1,2,6-Hexanetriol, Honey, Hydrogenated Honey, Hydrogenated Isocetyl Olivate, Hydrogenated Starch Hydrolysate, Hydrolyzed Wheat Protein/PEG-20 Acetate Copolymer, Hydroxypropyltrimonium Hyaluronate, Impatiens Balsamina Extract, Inositol, Isostearyl Acetyl Glutaminate, Jojoba Amino Acids, Lactic Acid, Lactitol, Lactobaccillus/Phoenix Dactylifera (Date) Fruit Ferment Extract, lactose, Lauramidobutyl Guanidine Acetate, Lauramidobutyl Guanidine HCI, Laur/MyrisUPalmitamidobutyl Guanidine Acetate, Lauryl Malamide, Lonicera Caerulea Fruit Juice, Lonicera Caerulea Fruit Water, Lysophosphatidylethanolamine, Malpighia Emarginata (Acerola) Seed Extract, Maltitol, Maltose, Mannitol, Mannose, Methoxy PEG-7, Methoxy PEG-IO, Methoxy PEG- 16, Methoxy PEG-25, Methoxy PEG-40, Methoxy PEG- 100, Momordica Charantia Fruit Extract, Myrciaria Dubia Seed Extract, Myristamidobutyl Guanidine Acetate, Myrist/Palmitamidobutyl Guanidine Acetate, Palmitamidobutyl Guanidine Acetate, PEG-4, PEG-6, PEG-7, PEG-8, PEG-9, PEG-IO, PEG-12, PEG-14, PEG- 16, PEG-18, PEG-20, PEG-32, PEG-33, PEG-40, PEG-45, PEG-55, PEG-60, PEG-75, PEG- 80, PEG-90, PEG-100, PEG-135, PEG-150, PEG-180, PEG-200, PEG-220, PEG-240, PEG- 800, PEG- 15 Butanediol, PEG-3 Methyl Ether, PEG-4 Methyl Ether, PEG-8 Methyl Ether Dimethicone, PEG-5 Pentaerythrityl Ether, PEG/PPG-28/21 Acetate Dimethicone, PEG/PPG-23/23 Butyl Ether Dimethicone, PEG/PPG-24/18 Butyl Ether Dimethicone, Persea Thunbergii Extract, Polydimethylsiloxy PPG- 13 Butyl Ether Silsesquioxane, Polyglyceryl Sorbitol, Polyquaternium-64, Polyquaternium-65, Potassium Dextrin Octenylsuccinate, Potassium PCA, PPG-12 Butyl Ether Dimethicone, PPG-6-Sorbeth-245, PPG-6-Sorbeth-500, PPG-2 Tocophereth-5, Propylene Glycol, Prunus Mume Fruit Extract, Pseudoalteromonas Ferment Extract, Rosa Canina Seed Extract, Saccharomyces/Prunus Extract Ferment Filtrate, Schizophyllan, Sea Water, Sodium Acetylated Hyaluronate, Sodium Dextrin Octenylsuccinate, Sodium Glucuronate, Sodium PCA, Sorbeth-6, Sorbeth-20, Sorbeth-30, Sorbeth-40, Sorbitol, Sorbityl Silanediol, Stearyl Acetyl Glutaminate, Stearyl Palmitate, Sucrose, TEA-Dextrin Octenylsuccinate, Trehalose, Triglycereth-7 Citrate, Trimethylamine Oxide, Trioxaundecanedioic Acid, Tripropylene Glycol, Urea, urea-d-glucuronic acid, xylitol, xyloglucan, and xylose. 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. [00143] 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. In certain embodiments, 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. In certain embodiments, the thickening agent is a natural polymeric thickener. In certain embodiments, the thickening agent is a cellulosic thickener. Examples of 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. Other specific exemplary 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, Isoprene/Pentadiene Copolymer, Lauryl Dimethicone PEG- 15 Crosspolymer, Polianthes Tuberosa Extract, Polysilicone-16, Pyrus Malus (Apple) Fiber, Rosa Multiflora Flower Wax, Sodium Polyacrylate, Sodium Trimethylpentene/MA Copolymer, Alcohol, Alcohol Denat., Benzyl Alcohol, Butoxydiglycol, Butoxyethanol, Butylene Glycol, CD Alcohol 19, Ceteareth-22, C7-8 Isoparaffϊn, C8-9 Isoparaffm, C9-11 Isoparaffm, C9-13 Isoparaffm, C9-14 Isoparaffϊn, ClO- 11 Isoparaffin, ClO-12 Isoparaffin, Cl 1-14 Isoparaffin, Decane, Decene, Deodorized Kerosene, Diethylene Glycol, Dimethyl Ether, Dimethyl Isosorbide, Dimethyl Sulfone, Dipropylene Glycol, Dodecene, Ethoxydiglycol, Ethoxyethanol, Ethyl Perfluorobutyl Ether, Glycereth-7, Glycereth-1 2, Glycereth-20, Glycereth-26, Glycereth-31 , Glycerin, Glycofurol, Glycol, Heptane, Hexadecene, Hexane, 1,2,6-Hexanetriol, Hexyl Alcohol, Hexylene Glycol, Isobutoxypropanol, Isopentane, Isopropyl Alcohol, Methoxydiglycol, Methoxyethanol, Methoxyethanol Acetate, Methoxyisopropanol, Methyl Hexyl Ether, Methyl Perfluorobutyl Ether, Octadecene, Octene, Pentane, Polyglyceryl Sorbitol, Propanediol, Propyl Alcohol, Propylene Carbonate, Propylene Glycol, SD Alcohol 1, SD Alcohol 3-A, SD Alcohol 3-B, SD Alcohol 3-C, SD Alcohol 23-A, SD Alcohol 23-F, SD Alcohol 23-H, SD Alcohol 27-B, SD Alcohol 30, SD Alcohol 31-A, SD Alcohol 36, SD Alcohol 37, SD Alcohol 38-B, SD Alcohol 38-C, SD Alcohol 38-D, SD Alcohol 38-F, SD Alcohol 39, SD Alcohol 39-A, SD Alcohol 39-B, SD Alcohol 39-C, SD Alcohol 39-D, SD Alcohol 40, SD Alcohol 40-A, SD Alcohol 40-B, SD Alcohol 40-C, SD Alcohol 46, Sorbeth-6, Sorbeth-20, Sorbeth-30, Sorbeth-40, Tetradecene, Triethylene Glycol, Turpentine, Acetamide MEA, Acrylamides Copolymer, Acrylamide/Sodium Acrylate Copolymer, Acrylamide/Sodium Acryloyldimethyltaurate Copolymer, Acrylates/ Acetoacetoxyethyl Methacrylate Copolymer, Acrylates/Beheneth-25 Methacrylate Copolymer, Acrylates/C 10-30 Alkyl Acrylate Crosspolymer, Acrylates/Ceteth-20 Itaconate Copolymer, Acrylates/Ceteth-20 Methacrylate Copolymer, Acrylates/Laureth-25 Methacrylate Copolymer, Acrylates/Palmeth-25 Acrylate Copolymer, Acrylates/Palmeth-25 Itaconate Copolymer, Acrylates/Steareth-50 Acrylate Copolymer, Acrylates/Steareth-20 Itaconate Copolymer, Acrylates/Steareth-20 Methacrylate Copolymer, Acrylates/Stearyl Methacrylate Copolymer, Acrylates/Vinyl Isodecanoate Crosspolymer, Acrylic Acid/Acrylonitrogens Copolymer, Adipic Acid/Methyl DEA Crosspolymer, Agar, Agarose, Alcaligenes Polysaccharides, Algin, Alginic Acid, Almondamide DEA, Almondamidopropyl Betaine, Aluminum/Magnesium Hydroxide Stearate, Ammonium Acrylates/Acrylonitrogens Copolymer, Ammonium Acrylates Copolymer, Ammonium Acryloyldimethyltaurate/Vinyl Formamide Copolymer, Ammonium Acryloyldimethyltaurate/VP Copolymer, Ammonium Alginate, Ammonium Chloride, Ammonium Polyacryloyldimethyl Taurate, Ammonium Sulfate, Amylopectin, Apricotamide DEA, Apricotamidopropyl Betaine, Arachidyl Alcohol, Arachidyl Glycol, Arachis Hypogaea (Peanut) Flour, Ascorbyl Methylsilanol Pectinate, Astragalus Gummifer Gum, Attapulgite, Avena Sativa (Oat) Kernel Flour, Avocadamide DEA, Avocadamidopropyl Betaine, Azelamide MEA, Babassuamide DEA, Babassuamide MEA, Babassuamidopropyl Betaine, Behenamide DEA, Behenamide MEA, Behenamidopropyl Betaine, Behenyl Betaine, Bentonite, Butoxy Chitosan, Caesalpinia Spinosa Gum, Calcium Alginate, Calcium Carboxymethyl Cellulose, Calcium Carrageenan, Calcium Chloride, Calcium Potassium Carbomer, Calcium Starch Octenylsuccinate, C20-40 Alkyl Stearate, Canolamidopropyl Betaine, Capramide DEA, Capryl/Capramidopropyl Betaine, Carbomer, Carboxybutyl Chitosan, Carboxymethyl Cellulose Acetate Butyrate, Carboxymethyl Chitin, Carboxymethyl Chitosan, Carboxymethyl Dextran, Carboxymethyl Hydroxyethylcellulose, Carboxymethyl Hydroxypropyl Guar, Carnitine, Cellulose Acetate Propionate Carboxylate, Cellulose Gum, Ceratonia Siliqua Gum, Cetearyl Alcohol, Cetyl Alcohol, Cetyl Babassuate, Cetyl Betaine, Cetyl Glycol, Cetyl Hydroxyethylcellulose, Chimyl Alcohol, Cholesterol/HDI/Pullulan Copolymer, Cholesteryl Hexyl Dicarbamate Pullulan, Citrus Aurantium Dulcis (Orange) Peel Extract, Cocamide DEA, Cocamide MEA, Cocamide MIPA, Cocamidoethyl Betaine, Cocamidopropyl Betaine, Cocamidopropyl Hydroxysultaine, Coco-Betaine, Coco- Hydroxysultaine, Coconut Alcohol, Coco/Oleamidopropyl Betaine, Coco-Sultaine, Cocoyl Sarcosinamide DEA, Cornamide/Cocamide DEA, Cornamide DEA, Croscarmellose, Cyamopsis Tetragonoloba (Guar) Gum, Decyl Alcohol, Decyl Betaine, Dehydroxanthan Gum, Dextrin, Dibenzylidene Sorbitol, Diethanolaminooleamide DEA, DiglycoVCHDM/Isophthalates/SIP Copolymer, Dihydroabietyl Behenate, Dihydrogenated Tallow Benzylmonium Hectorite, Dihydroxyaluminum Aminoacetate, Dimethicone/PEG-15 Crosspolymer, Dimethicone Propyl PG-Betaine, DMAPA Acrylates/ Acrylic Acid/Acrylonitrogens Copolymer, Erucamidopropyl Hydroxysultaine, Ethylene/Sodium Acrylate Copolymer, Gelatin, Gellan Gum, Glyceryl Alginate, Glycine Soja (Soybean) Flour, Guar Hydroxypropyltrimonium Chloride, Hectorite, Hyaluronic Acid, Hydrated Silica, Hydrogenated Potato Starch, Hydrogenated Tallow, Hydrogenated Tallowamide DEA, Hydrogenated Tallow Betaine, Hydroxybutyl Methylcellulose, Hydroxyethyl Acrylate/Sodium Acryloyldimethyl Taurate Copolymer, Hydroxyethylcellulose, Hydroxyethyl Chitosan, Hydroxyethyl Ethylcellulose, Hydroxyethyl Stearamide-MIPA, Hydroxylauryl/Hydroxymyristyl Betaine, Hydroxypropylcellulose, Hydroxypropyl Chitosan, Hydroxypropyl Ethylenediamine Carbomer, Hydroxypropyl Guar, Hydroxypropyl Methylcellulose, Hydroxypropyl Methylcellulose Stearoxy Ether, Hydroxypropyl Starch, Hydroxypropyl Starch Phosphate, Hydroxypropyl Xanthan Gum, Hydroxystearamide MEA, Isobutylene/Sodium Maleate Copolymer, Isostearamide DEA, Isostearamide MEA, Isostearamide MIPA, Isostearamidopropyl Betaine, Lactamide MEA, Lanolinamide DEA, Lauramide DEA, Lauramide MEA, Lauramide MIPA, Lauramide/Myristamide DEA, Lauramidopropyl Betaine, Lauramidopropyl Hydroxysultaine, Laurimino Bispropanediol, Lauryl Alcohol, Lauryl Betaine, Lauryl Hydroxysultaine, Lauryl Sultaine, Lecithinamide DEA, Linoleamide DEA, Linoleamide MEA, Linoleamide MIPA, Lithium Magnesium Silicate, Lithium Magnesium Sodium Silicate, Macrocystis Pyrifera (Kelp), Magnesium Alginate, Magnesium/ Aluminum/Hydroxide/Carbonate, Magnesium Aluminum Silicate, Magnesium Silicate, Magnesium Trisilicate, Methoxy PEG-22/Dodecyl Glycol Copolymer, Methylcellulose, Methyl Ethylcellulose, Methyl Hydroxyethylcellulose, Microcrystalline Cellulose, Milkamidopropyl Betaine, Minkamide DEA, Minkamidopropyl Betaine, MIPA- Myristate, Montmorillonite, Moroccan Lava Clay, Myristamide DEA, Myristamide MEA, Myristamide MIPA, Myristamidopropyl Betaine, Myristamidopropyl Hydroxysultaine, Myristyl Alcohol, Myristyl Betaine, Natto Gum, Nonoxynyl Hydroxyethylcellulose, Oatamide MEA, Oatamidopropyl Betaine, Octacosanyl Glycol Isostearate, Octadecene/MA Copolymer, Oleamide DEA, Oleamide MEA, Oleamide MIPA, Oleamidopropyl Betaine, Oleamidopropyl Hydroxysultaine, Oleyl Betaine, Olivamide DEA, Olivamidopropyl Betaine, Oliveamide MEA, Palmamide DEA, Palmamide MEA, Palmamide MIPA, Palmamidopropyl Betaine, Palmitamide DEA, Palmitamide MEA, Palmitamidopropyl Betaine, Palm Kernel Alcohol, Palm Kernelamide DEA, Palm Kernelamide MEA, Palm Kernelamide MIPA, Palm Kernelamidopropyl Betaine, Peanutamide MEA, Peanutamide MIPA, Pectin, PEG-800, PEG-Crosspolymer, PEG-150/Decyl Alcohol/SMDI Copolymer, PEG- 175 Diisostearate, PEG- 190 Distearate, PEG- 15 Glyceryl Tristearate, PEG- 140 Glyceryl Tristearate, PEG- 240/HDI Copolymer Bis-Decyltetradeceth-20 Ether, PEG-100/IPDI Copolymer, PEG- 180/Laureth-50/TMMG Copolymer, PEG-10/Lauryl Dimethicone Crosspolymer, PEG- 15/Lauryl Dimethicone Crosspolymer, PEG-2M, PEG-5M, PEG-7M, PEG-9M, PEG-14M, PEG-20M, PEG-23M, PEG-25M, PEG-45M, PEG-65M, PEG-90M, PEG-115M, PEG- 160M, PEG- 120 Methyl Glucose Trioleate, PEG-180/Octoxynol-40/TMMG Copolymer, PEG- 150 Pentaerythrityl Tetrastearate, PEG-4 Rapeseedamide, PEG-150/Stearyl Alcohol/SMDI Copolymer, Phaseolus Angularis Seed Powder, Polianthes Tuberosa Extract, Polyacrylate-3, Polyacrylic Acid, Polycyclopentadiene, Polyether-1, Polyethylene/Isopropyl Maleate/MA Copolyol, Polyglyceryl-3 Disiloxane Dimethicone, Polyglyceryl-3 Polydimethylsiloxyethyl Dimethicone, Polymethacrylic Acid, Polyquaternium-52, Polyvinyl Alcohol, Potassium Alginate, Potassium Aluminum Polyacrylate, Potassium Carbomer, Potassium Carrageenan, Potassium Chloride, Potassium Palmate, Potassium Polyacrylate, Potassium Sulfate, Potato Starch Modified, PPG-2 Cocamide, PPG-I Hydroxy ethyl Caprylamide, PPG-2 Hydroxyethyl Cocamide, PPG-2 Hydroxyethyl Coco/Isostearamide, PPG-3 Hydroxyethyl Soyamide, PPG- 14 Laureth-60 Hexyl Dicarbamate, PPG- 14 Laureth-60 Isophoryl Dicarbamate, PPG- 14 Palmeth-60 Hexyl Dicarbamate, Propylene Glycol Alginate, PVP/Decene Copolymer, PVP Montmorillonite, Pyrus Cydonia Seed, Pyrus Malus (Apple) Fiber, Rhizobian Gum, Ricebranamide DEA, Ricinoleamide DEA, Ricinoleamide MEA, Ricinoleamide MIPA, Ricinoleamidopropyl Betaine, Ricinoleic Acid/Adipic Acid/ AEEA Copolymer, Rosa Multiflora Flower Wax, Sclerotium Gum, Sesamide DEA, Sesamidopropyl Betaine, Sodium Acrylate/Acryloyldimethyl Taurate Copolymer, Sodium Acrylates/Acrolein Copolymer, Sodium Acrylates/Acrylonitrogens Copolymer, Sodium Acrylates Copolymer, Sodium Acrylates Crosspolymer, Sodium Acrylates/Vinyl Isodecanoate Crosspolymer, Sodium Acrylate/Vinyl Alcohol Copolymer, Sodium Carbomer, Sodium Carboxymethyl Chitin, Sodium Carboxymethyl Dextran, Sodium Carboxymethyl Beta-Glucan, Sodium Carboxymethyl Starch, Sodium Carrageenan, Sodium Cellulose Sulfate, Sodium Chloride, Sodium Cyclodextrin Sulfate, Sodium Hydroxypropyl Starch Phosphate, Sodium Isooctylene/MA Copolymer, Sodium Magnesium Fluorosilicate, Sodium Oleate, Sodium Palmitate, Sodium Palm Kernelate, Sodium Polyacrylate, Sodium Polyacrylate Starch, Sodium Polyacryloyldimethyl Taurate, Sodium Polygamma-Glutamate, Sodium Polymethacrylate, Sodium Polystyrene Sulfonate, Sodium Silicoaluminate, Sodium Starch Octenylsuccinate, Sodium Stearate, Sodium Stearoxy PG-Hydroxyethylcellulose Sulfonate, Sodium Styrene/Acrylates Copolymer, Sodium Sulfate, Sodium Tallowate, Sodium Tauride Acrylates/ Acrylic Acid/Acrylonitrogens Copolymer, Sodium Tocopheryl Phosphate, Solanum Tuberosum (Potato) Starch, Soyamide DEA, Soyamidopropyl Betaine, Starch/ Acrylates/ Acrylamide Copolymer, Starch Hydroxypropyltrimonium Chloride, Stearamide AMP, Stearamide DEA, Stearamide DEA-Distearate, Stearamide DIBA-Stearate, Stearamide MEA, Stearamide MEA-Stearate, Stearamide MIPA, Stearamidopropyl Betaine, Steareth-60 Cetyl Ether, Steareth-100/PEG-136/HDI Copolymer, Stearyl Alcohol, Stearyl Betaine, Sterculia Urens Gum, Synthetic Fluorphlogopite, Tallamide DEA, Tallow Alcohol, Tallowamide DEA, Tallowamide MEA, Tallowamidopropyl Betaine, Tallowamidopropyl Hydroxysultaine, Tallowamine Oxide, Tallow Betaine, Tallow Dihydroxyethyl Betaine, Tamarindus Indica Seed Gum, Tapioca Starch, TEA-Alginate, TEA-Carbomer, TEA- Hydrochloride, Trideceth-2 Carboxamide MEA, Tridecyl Alcohol, Triethylene Glycol Dibenzoate, Trimethyl Pentanol Hydroxyethyl Ether, Triticum Vulgare (Wheat) Germ Powder, Triticum Vulgare (Wheat) Kernel Flour, Triticum Vulgare (Wheat) Starch, Tromethamine Acrylates/ Acrylonitrogens Copolymer, Tromethamine Magnesium Aluminum Silicate, Undecyl Alcohol, Undecylenamide DEA, Undecylenamide MEA, Undecylenamidopropyl Betaine, Welan Gum, Wheat Germamide DEA, Wheat Germamidopropyl Betaine, Xanthan Gum, Yeast Beta-Glucan, Yeast Polysaccharides, Zea Mays (Corn) Starch, Abietyl Alcohol, Acrylates/C 10-30 Alkyl Acrylate Crosspolymer, Adipic Acid/PPG- 10 Copolymer, Ally! Methacrylates Crosspolymer, Alumina Magnesium Metasilicate, Aluminum Behenate, Aluminum Benzoate, Aluminum Caprylate, Aluminum Dilinoleate, Aluminum Dimyristate, Aluminum Distearate, Aluminum Isostearate, Aluminum Isostearates/Laurates/Palmitates, Aluminum Isostearates/Laurates/Stearates, Aluminum Isostearates/Myristates, Aluminum Isostearates/Palmitates, Aluminum Isostearates/Stearates, Aluminum Lanolate, Aluminum/Magnesium Hydroxide Stearate, Aluminum Myristate, Aluminum Myristates/Palmitates, Aluminum Starch Octenylsuccinate, Aluminum Stearate, Aluminum Stearates, Aluminum Tristearate, Arachidyl Alcohol, Arachidyl Behenate, Arachidyl Glycol, Beeswax, Behenamide, Behenamidopropyl Dimethylamine Behenate, Behenyl Alcohol, Behenyl Methacrylate/Perfluorooctylethyl, Methacrytate Copolymer, Bispolyethylene Dimethicone, Butadiene/ Acrylonitrile Copolymer, Butylene/Ethylene Copolymer, Butylene/Ethylene/Styrene Copolymer, Butylene Glycol Cocoate, Butylene Glycol Montanate, Butyrospermum Parkii (Shea Butter), C29-70 Acid, C23-43 Acid Pentaerythritol Tetraester, C8-12 Acid Triglyceride, C 12- 18 Acid Triglyceride, Calcium Behenate, Calcium Laurate, Calcium Montanate, Calcium Myristate, Calcium Stearate, Calcium Undecylenate, C9-11 Alcohols, C12-13 Alcohols, C12-15 Alcohols, C12-16 Alcohols, C14-15 Alcohols, C20-22 Alcohols, C30-50 Alcohols, C40-60 Alcohols, C18-38 Alkyl C24-54 Acid Ester, C20-24 Alkyl Dimethicone, C24-28 Alkyl Dimethicone, C8-10 Alkyl Ethyl Phosphate, C 1-5 Alkyl Galactomannan, C 18-38 Alkyl Hydroxystearoyl Stearate, C20-24 Alkyl Methicone, C24-28 Alkyl Methicone, C30-45 Alkyl Methicone, Candelilla Wax Hydrocarbons, Camauba Acid Wax, C 10-30 Cholesterol/Lanosterol Esters, Cellobiose Octanonanoate, Ceresin, Cerotic Acid, Cetearyl Alcohol, Cetearyl Dimethicone/Vinyl Dimethicone Crosspolymer, Cetyl Alcohol, Cetyl Glycol, Chimyl Alcohol, Chlorinated Paraffin, Cholesterol, Cholesteryl Acetate, Cholesteryl Hydroxystearate, Cholesteryl Isostearate, Cholesteryl Macadamiate, Cholesteryl Stearate, C 10-40 Hydroxyalkyl Acid Cholesterol Esters, C 10-40 Isoalkyl Acid Cholesterol Esters, C 10-40 Isoalkyl Acid Octyldodecanol Esters, C 10-40 Isoalkyl Acid Phytosterol Esters, C 10-40 Isoalkyl Acid Triglyceride, Coconut Alcohol, C30-38 Olefϊn/Isopropyl Maleate/MA Copolymer, Copal, Corn Starch Modified, C6-14 Perfluoroalkylethyl Acrylate/HEMA Copolymer, C6-14 Polyolefϊn, Cyclocarboxypropyloleic Acid, Decene/Butene Copolymer, Decyl Alcohol, 7- Dehydrocholesterol, Dibehenyl Fumarate, Dibenzylidene Sorbitol, Dihydrogenated Tallow Benzylmonium Hectorite, Dilinoleic Acid/Ethylenediamine Copolymer, Dilinoleic Acid/Sebacic Acid/Piperazine/Ethylenediamine Copolymer, Dimer Dilinoleyl Diisostearate, Dimer Dilinoleyl Dimer Dilinoleate, Dimer Dilinoleyl Hydrogenated Rosinate, Dimethicone Crosspolymer, Dimethicone/Phenyl Vinyl Dimethicone Crosspolymer, Dimethicone/Polyglycerin-3 Crosspolymer, Dimethicone/Vinyl Dimethicone Crosspolymer, Dimethicone/Vinyltrimethylsiloxysilicate Crosspolymer, Dimethyldibenzylidene Sorbitol, Dipentaerythrityl Hexacaprylate/Hexacaprate, Dipentaerythrityl Hexaheptanoate/Hexacaprylate/Hexacaprate, Dipentaerythrityl Hexahydroxystearate, Dipentaerythrityl Hexahydroxystearate/Hexastearate/Hexarosinate, Dipentaerythrityl Hexaoctanoate/Hexabehenate, Dipentaerythrityl Pentahydroxystearate/Pentaisostearate, Dipentaerythrityl Tetrahydroxystearate/Tetraisostearate, Diphenyl Dimethicone/Vinyl Diphenyl Dimethicone/Silsesquioxane Crosspolymer, Divinyldimethicone/Dimethicone Crosspolymer, Dodecanedioic Acid/Cetearyl Alcohol/Glycol Copolymer, Ericerus PeIa Wax, Erucamide, Ethylcellulose, Ethylene/ Acrylic Acid Copolymer, Ethylene/ Acrylic AcidNA Copolymer, Ethylenediamine/Dimer Tallate Copolymer Bis-Hydrogenated Tallow Amide, Ethylenediamine/Stearyl Dimer Dilinoleate Copolymer, Ethylenediamine/Stearyl Dimer Tallate Copolymer, Ethylene Dihydrogenated Tallowamide, Ethylene Dioleamide, Ethylene Distearamide, Ethylene/Octene Copolymer, Ethylene/Propylene Copolymer, Ethylene/Propylene/Styrene Copolymer, Ethylhexyl C 10-40 Isoalkyl Acidate, Euphorbia Cerifera (Candelilla) Wax, Glyceryl Abietate, Glyceryl Arachidate, Glyceryl Diisostearate/Hydrogenated Rosinate, Glyceryl Stearate Diacetate, Glycol/Butylene Glycol Montanate, Glycol Dibehenate, Glycol Diethylhexanoate, Glycol Dilaurate, Glycol Dioleate, Glycol Distearate, Hexacosyl Glycol, Hexadecyleicosanoic Acid, Hexanediol Distearate, Hexyldecyloctadecanol, Hydroabietyl Alcohol, Hydrogenated Apricot Kernel Oil, Hydrogenated Butylene/Ethylene/Styrene Copolymer, Hydrogenated Canola Oil, Hydrogenated Castor Oil, Hydrogenated Castor Oil Hydroxystearate, Hydrogenated Castor Oil Isostearate, Hydrogenated Castor Oil Laurate, Hydrogenated Castor Oil Stearate, Hydrogenated Castor Oil Triisostearin Esters, Hydrogenated C6-14 Olefin Polymers, Hydrogenated Cottonseed Oil, Hydrogenated C 12- 18 Triglycerides, Hydrogenated Ethylene/Propylene/Styrene Copolymer, Hydrogenated Fish Oil, Hydrogenated Grapeseed Oil, Hydrogenated Japan Wax, Hydrogenated Lard, Hydrogenated Lard Glyceride, Hydrogenated Lard Glycerides, Hydrogenated Menhaden Oil, Hydrogenated Methyl Abietate, Hydrogenated Microcrystalline Wax, Hydrogenated Olive Oil, Hydrogenated Palm Glycerides, Hydrogenated Palm Kernel Glycerides, Hydrogenated Palm Kernel Oil, Hydrogenated Palm Oil, Hydrogenated Peanut Oil, Hydrogenated Pistachio Seed Oil, Hydrogenated Polyisobutene, Hydrogenated Rapeseed Oil, Hydrogenated Rice Bran Wax, Hydrogenated Rosin, Hydrogenated Safflower Seed Oil, Hydrogenated Shea Butter, Hydrogenated Soybean Oil, Hydrogenated Soy Glycerides, Hydrogenated Styrene/Butadiene Copolymer, Hydrogenated Styrene/Methyl Styrene/Indene Copolymer, Hydrogenated Sunflower Seed Oil, Hydrogenated Sweet Almond Oil, Hydrogenated Tallow Alcohol, Hydrogenated Tallow Amide, Hydrogenated Tallow Glycerides, Hydrogenated Vegetable Glycerides, Hydrogenated Vegetable Oil, Hydrogenated Wheat Germ Oil, Hydroxyoctacosanyl Hydroxystearate, Hydroxypropylcellulose, Isobutylene/Isoprene Copolymer, Isocetyl Alcohol, Isocetyl Stearoyl Stearate, Isostearyl Alcohol, Isostearyl Stearoyl Stearate, Jojoba Alcohol, Lanolin Alcohol, Lanolin Wax, Lard Glyceride, Lard Glycerides, Lauryl Alcohol, Lauryl Dimethicone/Polyglycerin-3 Crosspolymer, Linoleamide, Lithium Oxidized Polyethylene, Lithium Stearate, Magnesium Cocoate, Magnesium Lanolate, Magnesium Myristate, Magnesium Palmitate, Magnesium Stearate, Magnesium Tallowate, Mellisic Acid, Methoxy PEG-17/Dodecyl Glycol Copolymer, Methoxy PEG- 22/Dodecyl Glycol Copolymer, Methyl Dehydroabietate, Methyl Dihydroabietate, Methyl Hydrogenated Rosinate, Methyl Methacrylate Crosspolymer, Methyl Rosinate, Methylstyrene/Vinyltoluene Copolymer, Microcrystalline Wax, Montan Acid Wax, Montan Wax, Myrica Cerifera (Bayberry) Fruit Wax, Myricyl Alcohol, Myristyl Alcohol, Neopentyl Glycol Dicaprate, Neopentyl Glycol Dicaprylate/Dicaprate, Neopentyl Glycol Dicaprylate/Dipelargonate/Dicaprate, Neopentyl Glycol Diethylhexanoate, Neopentyl Glycol Diheptanoate, Neopentyl Glycol Diisostearate, Neopentyl Glycol Dilaurate, Nylon- 611/Dimethicone Copolymer, Octacosanyl Glycol, Octadecene/MA Copolymer, Octyldodecyl Stearoyl Stearate, Oleic/Linoleic/Linolenic Polyglycerides, Oleostearine, Oleyl Alcohol, Oleyl Palmitamide, Ouricury Wax, Oxidized Beeswax, Oxidized Microcrystalline Wax, Oxidized Polyethylene, Oxidized Polypropylene, Ozokerite, Palm Alcohol, Palm Kernel Alcohol, Paraffin, PEG- 18 Castor Oil Dioleate, PEG-IO Dimethicone Crosspolymer, PEG- 12 Dimethicone Crosspolymer, PEG-2 Dirosinate, PEG-3 Dirosinate, PEG-5 Hydrogenated Castor Oil Isostearate, PEG-IO Hydrogenated Castor Oil Isostearate, PEG-20 Hydrogenated Castor Oil Isostearate, PEG-30 Hydrogenated Castor Oil Isostearate, PEG-40 Hydrogenated Castor Oil Isostearate, PEG-50 Hydrogenated Castor Oil Isostearate, PEG-58 Hydrogenated Castor Oil Isostearate, PEG-50 Hydrogenated Castor Oil Succinate, PEG-5 Hydrogenated Castor Oil Triisostearate, PEG-IO Hydrogenated Castor Oil Triisostearate, PEG- 15 Hydrogenated Castor Oil Triisostearate, PEG-20 Hydrogenated Castor Oil Triisostearate, PEG-30 Hydrogenated Castor Oil Triisostearate, PEG-40 Hydrogenated Castor Oil Triisostearate, PEG-60 Hydrogenated Castor Oil Triisostearate, PEG-5 Lanolinamide, PEG-5 Oleamide Dioleate, Pentaerythrityl

Adipate/Caprate/Caprylate/Heptanoate, Pentaerythrityl Dioleate, Pentaerythrityl Distearate, Pentaerythrityl Hydrogenated Rosinate, Pentaerythrityl

Isostearate/Caprate/Caprylate/Adipate, Pentaerythrityl Rosinate, Pentaerythrityl Stearate/Caprate/Caprylate/Adipate, Pentaerythrityl

Stearate/Isostearate/Adipate/Hydroxystearate, Pentaerythrityl Tetraabietate, Pentaerythrityl Tetrabehenate, Pentaerythrityl Tetrabenzoate, Pentaerythrityl Tetracaprylate/Tetracaprate, Pentaerythrityl Tetracocoate, Pentaerythrityl Tetraethylhexanoate, Pentaerythrityl Tetraisononanoate, Pentaerythrityl Tetraisostearate, Pentaerythrityl Tetralaurate, Pentaerythrityl Tetramyristate, Pentaerythrityl Tetraoleate, Pentaerythrityl Tetrapelargonate, Pentaerythrityl Tetrastearate, Pentaerythrityl Trioleate, Phthalic Anhydride/Butyl Benzoic Acid/Propylene Glycol Copolymer, Phthalic Anhydride/Glycerin/Glycidyl Decanoate Copolymer, Phthalic Anhydride/Trimellitic Anhydride/Glycols Copolymer, Phytosteryl/Isostearyl/Cetyl/Stearyl/BehenylDimer Dilinoleate, Phytosteryl Isostearyl Dimer Dilinoleate, Piperylene/Butene/Pentene Copolymer, Polybutene, Polybutylene Terephthalate, Poly C 10-30 Alkyl Acrylate, Polycyclopentadiene, Polydipentene, Polyethylene, Polyethylene Terephthalate, Polyglyceryl-3 Polyricinoleate, Polyglyceryl-4 Polyricinoleate, Polyglyceryl-5 Polyricinoleate, Polyglyceryl-10 Polyricinoleate, Polyisobutene, Polyisoprene, Polypentene, Polyperfluoroethoxymethoxy Difluoromethyl Distearamide, Polypropylene, Polysilicone-4, Polysilicone-5, Polysilicone- 1 7, Polystyrene, Polyvinyl Butyral, Polyvinyl Laurate, Potassium Linoleate, Potassium Oxidized Microcrystalline Wax, Potassium Palm Kernelate, Potassium PEG-50 Hydrogenated Castor Oil Succinate, Potassium Rapeseedate, Potassium Soyate, Propylene Glycol Dicaprate, Propylene Glycol Dicaproate, Propylene Glycol Dicaprylate, Propylene Glycol Dicocoate, Propylene Glycol Diisononanoate, Propylene Glycol Diisostearate, Propylene Glycol Dilaurate, Propylene Glycol Dioleate, Propylene Glycol Dipelargonate, Propylene Glycol Distearate, Propylene Glycol Diundecanoate, Prunus Amygdalus Dulcis (Sweet Almond) Oil Unsaponifϊables, PVM/MA Decadiene Crosspolymer, PVP/Decene Copolymer, Rhus Succedanea Fruit Wax, Rosa Multiflora Flower Wax, Rosin, Silica Dimethicone Silylate, Silica Dimethyl Silylate, Simmondsia Chinensis (Jojoba) Seed Wax, Sodium Acrylate/Sodium Acryloyldimethyl Taurate Copolymer, Sodium Linoleate, Sodium Olivate, Sodium Palmate, Sodium Peanutate, Sodium PVM/MA/Decadiene Crosspolymer, Sodium Rapeseedate, Sodium Rosinate, Sodium Soyate, Spent Grain Wax, Stearamide, Stearamide DEA-Distearate, Stearamide DIBA- Stearate, Stearamide MEA-Stearate, Steareth-10 Allyl Ether/ Acrylates Copolymer, Steareth- 60 Cetyl Ether, Stearone, Stearoxymethicone/Dimethicone Copolymer, Stearyl Alcohol, Stearyl Erucamide, Stearyl Erucate, Stearyl Glycol, Stearyl Glycol Isostearate, Stearyl Linoleate, Stearyl Methacrylate/Perfluorooctylethyl Methacrylate Copolymer, Stearyl Stearate, Stearyl Stearoyl Stearate, Styrene/Methacrylamide/ Acrylates Copolymer, Synthetic Beeswax, Synthetic Candelilla Wax, Synthetic Carnauba, Synthetic Japan Wax, Synthetic Wax, Tallow Alcohol, Tallow Amide, TDI Oxidized Microcrystalline Wax, TEA-Rosinate, Tetradecyleicosanoic Acid, Tetradecyleicosanol, Tetradecyloctadecanoic Acid, Tetradecyloctadecanol, Tourmaline, Triarachidin, Tricontanyl PVP, Tridecyl Alcohol, Trierucin, Triethylene Glycol Hydrogenated Rosinate, Trifluoropropyl Dimethicone/PEG-10 Crosspolymer, Trifluoropropyl Dimethicone,Trifluoropropyl Divinyldimethicone Crosspolymer, Trifluoropropyl Dimethicone/Vinyl Trifluoropropyl Dimethicone/Silsesquioxane Crosspolymer, Triheptanoin, Triheptylundecanoin, Trihydroxystearin, Triisononanoin, Triisopalmitin, Triisostearin, Triisostearyl Trilinoleate, Trilaurin, Trilinoleic Acid, Trilinolein, Trilinolenin, Trimethylpentanediol/Isophthalic Acid/Trimellitic Anhydride Copolymer, Trimethylsiloxysilicate/Dimethiconol Crosspolymer, Trimyristin, Triolein, Tripalmitin, Tripalmitolein, Triricinolein, Trisebacin, Tristearin, Undecyl Alcohol, Vinyl Dimethicone/Lauryl Dimethicone Crosspolymer, Vinyl Dimethicone/Methicone Silsesquioxane Crosspolymer, VP/Eicosene Copolymer, VP/Hexadecene Copolymer, Zinc Laurate, Zinc Myristate, Zinc Neodecanoate, Zinc Palmitate, Zinc Rosinate, and Zinc Stearate. As would be appreciated by one of skill in the art, certain ingredients found in other categoreis of agents described herein may be used as thickening agents in the inventive compositions. Also suitable for use herein are gums, e.g., xanthan, karaya, gellan, wellan, arabic, carrageanan, biosaccharide gum-1, and locust bean. Also suitable are alginate and agarose.

[00144] Surfactants, emulsifϊers, and the like 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. In certain embodiments, the surfactant is an anionic surfactant. In other embodiments, the surfactant is a cationic surfactant. In yet other embodiments, the surfactant is a nonionic surfactant. In certain embodiments, the surfactant is a zwitterionic surfactant. In certain embodiments, the surfact is an amphoteric surfactant. [00145] The surfactant used in the inventive composition may be chosen based on its HLB (Hydrophile-Lipophile Balance) value. In certain embodiments, 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. Methods of determining HLB are well known in the art and any of such methods may be used for HLB determination. A description of the HLB System and methods for HLB determination are described in "The HLB System: a time saving guide to emulsifier selection," ICI Americas Inc., Wilmington, Delaware, 1976.

[00146] 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 sulfosuccinic acid, dioctyl esters of sodium sulfosuccinic acid), olefin sulfonates, hydroxy-alkanesulfonates, beta-alkyloxy alkane sulfonates (e.g., potassium-β-methoxydecanesulfonate, sodium 2-methoxytridecanesulfonate, potassium 2-ethoxytetradecylsulfonate, sodium 2-isopropoxyhexadecylsulfonate, lithium 2-t- butoxytetradecylsulfonate, sodium β-methoxyoctadecysulfonate, ammonium β-n-propoxy- dodecylsulfonate), dioctyl esters of sodium sulfosuccinic acid, alkyl ethoxylated sulfates, alkyl sulfates, aliphatic secondary and tertiary amines (e.g., sodium 3- dodecylaminopropionate, N-alkyltaurines, stearamido propyl dimethyl amine, diethyl amino ethyl stearamide, dimethyl stearamine, dimethyl soyamine, soyamine, myristyl amine, tridecyl amine, ethyl stearylamine, N-tallowpropane diamine, ethoxylated (5 moles E. O) stearylamine, dihydroxy ethyl stearylamine, and arachidylbehenylamine), 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 ammonium chloride, dieicosyl dimethyl ammonium chloride, didocosyl dimethyl ammonium chloride, di(hydrogenated tallow) dimethyl ammonium acetate, dihexadecyl dimethyl ammonium chloride, dihexadecyl dimethyl ammonium acetate, ditallow dipropyl ammonium phosphate, ditallow dimethyl ammonium nitrate, and di(coconutalkyl benzyl ammonium chloride); aliphatic phosphonium compounds, aliphatic sulfonium compounds, alkyl amino sulfonates, alkyl betaines (e.g. , 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- octadecylammonio]-butane- 1 -carboxylate; 5-[S-3-hydroxypropyl-S-hexadecylsulfonio]-3- hydroxy-pentanel-sulfate; 3-[P,P-diethyl-P-3,6,9-trioxatetradexoxylphosphonio]-2-hydroxy- propane- 1 -p hosphate; 3-[N,N-dipropyl-N-3-dodecoxy-2-hydroxypropylammonio]-propane- 1 -phosphate; 3-(N,N-dimethyl-N~hexadecylammonio)propane- 1 -sulfonate; 3-(N ,N- dimethyl-N-hexadecylammonio)-2-hydroxy-propane- 1 -sulfonate; 4-[N,N-di-(2-hydroxy- ethyl)-N-(2-hydroxydodecyl)ammonio]-butane- 1 -carboxyl ate; 3-[S-ethyl-S-(3-dodecoxy-2- hydroxypropyl)sulfonio]-propane- 1 -phosphate; 3- [P,P-dimethyl-P-dodecylphosphonio]- propane- 1 -phosphonate; and 5-[N,N-di(3-hydroxypropyl)-N-hexadecylammonio]-2- hydroxypentane-1 -sulfate, sodium 3-dodecylaminopropane sulfonate; alkyl amphosulfonates; alkyl amphosulfosuccinates; oleoamphopropylsulfonate, and cocoamphopropylsulfonate; polyethylene oxide condensates; long chain tertiary phosphine oxides; long chain dialkyl sulfoxides; silicone copolyols (e.g., dimethicone copolyols), stearamide diethanolamide (DEA), cocamide monoethanolamide (MEA), glyceryl monoleate, sucrose stearate, Cetheth- 2, Poloxamer 181, hydrogenated tallow amide DEA, polyoxyethylene 4 sorbitol beeswax derivative (ATLAS 6-1702), polyoxyethylene 2 cetyl ether (BRIJ 52), polyoxyethylene 2 stearyl ether (BRIJ 72), polyoxyethylene 2 oleyl ether (BRIJ 92), polyoxyethylene 2 oleyl ether (BRIJ 93), sorbitan monopalmitate (SPAN 40), sorbitan monostearate (SPAN 60), sorbitan tristearate (SPAN 65), sorbitan monoleate, NF (SPAN 80) sorbitan trioleate (SPAN 85), fluorinated alkyl quaternary ammonium iodide; mixed mono- and bis-perfluoroalkyl phosphates, ammonium salts; mixed mono- and bis-fluoroalkyl phosphate, ammonium salts, complexed with aliphatic quaternary methosulfates; perfluoroalkyl sulfonic acid, ammonium salts; mixed telomer phosphate diethanolamine salts; amine perfluoroalkyl sulfonates; ammonium perfluoroalkyl sulfonates; potassium perfluoroalkyl sulfonates; potassium fluorinated alkyl carboxylates; ammonium perfluoroalkyl sulfonates; and ammonium perfluoroalkyl carboxylates; sodium dioctyl sulfosuccinate; magnesium dioctyl sulfosuccinate; ammonium dioctyl sulfosuccinate; sodium dodecyl sulfate; magnesium dodecyl sulfate; ammonium dodecyl sulfate; cocoamidopropyl betaine sodium laureth sulfate; sodium dinonyl sulfo succinate; sodium decyl sulfate; sodium alpha olefin sulfonate; sodium laureth sulfate; magnesium laureth sulfate; ammonium laureth sulfate; cocoamidopropyl betaine; polyethoxylated glycol ether of glyceryl isostewarate; polyethoxylated glycol ether of glyceryl monooleate; PEG-30 glyceryl isostearate; polyoxyethylene glycerol monoleate; and PEG 75 lanolin.

[00147] Many surfactants useful in the inventive compositionss are described in McCutcheon's Detergents and Emulsifϊers, 1984 Annual, published by Allured Publishing Corporation, which is incorporated herein by reference. Other useful surfactant in the inventive compositions include those described in U.S. Patent 2,396,278; 2,438,091; 2,486,921; 2,486,922; 2,528,378; 2,658,072; 3,155,591; 3,332,880; 3,929,678; 3,959,461; 3,993,744; 3,993,745; 4,122,029; 4,176,176; 4,265,878; 4,275,055; 4,387,090; 4,421,769; each of which is incorporated herein by reference. The 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. In certain embodiments, the composition contains surfactant in the range from about 0.5% to about 15% by weight. In certain embodiments, the composition contains surfactant in the range from about 2% to about 10% by weight. [00148] Certain 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. Without wishing to be bound by any theory, once evidence of the elastotic material is reduced by a composition of the present invention comprising an elastase or partial elastase, 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. Contacting the skin with an inhibitor of an endogenous, skin-associated elastase may facilitate this process by reducing degradation of the newly synthesized elastin by endogenous elastase(s). [00149] In certain embodiments of the invention the inhibitor of an endogenous, skin- associated elastase has a K₁ 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 K₁ 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. U.S. Pat. Nos. 5,618,792; 5,807,829; 5,861,380; 5,869,455; 5,874,585; and 5,891,852, each of which is incorporated herein by reference, describe small molecule inhibitors and their method of synthesis that are selective for human neutrophil elastase (NE). In certain embodiments of the invention the elastase inhibitor inhibits both neutrophil elastase and PR-3 (see, e.g., U.S. Pat. No. 6,777,403, incorporated herein by reference). Many examples of irreversible inhibitors are known in the art. 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. 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. In certain embodiments of the invention the composition comprises a compound or combination of compounds that inhibits synthesis of an endogenous, skin-associated elastase. For example, 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. Thus 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. In certain embodiments of the invention, 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. In certain embodiments, the MMP inhibitor is a small molecule. [00150] The inventive composition may comprise a penetration enhancer. As used herein, 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. A variety of 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. Examples of 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. Certain penetration enhancers described in U.S. Pat. No. 6,946,144 (incorporated herein by reference) and references therein are of use. In certain embodiments of the invention the penetration enhancer enhances penetration of molecules having a molecular weight of between 20 kD and 100 kD, e.g., between 20 kD and 50 kD. [00151] The inventive compositions may optionally include a fragrance or perfume. Fragrance ingredient, as defϊend 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. The function comprises aroma chemicals, essential oils, natural extracts, distillates and isolates, oleoresins, etc." In certain embodiments, 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. Exemplary 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, Allyl Caproate, Aloe Barbadensis Leaf, Aloe Barbadensis Leaf Water, Amidinoproline, Aminomethyl Propanediol, Ammonia, Ammonium Chloride, Ammonium Glycyrrhizate, Amyl Acetate, Amyl Benzoate, Amyl Cinnamal, Amylcinnamyl Alcohol, Amyl Salicylate, Amyris Balsamifera Bark Oil, Anethole, Angelica Archangelica Root Extract, Angelica Archangelica Root Oil, Aniba Rosaeodora (Rosewood) Wood Extract, Aniba Rosaeodora (Rosewood) Wood Oil, Anisaldehyde, Anise Alcohol, p-Anisic Acid, Anthemis Nobilis Flower Extract, Anthemis Nobilis Flower Oil, Apium Graveolens (Celery) Extract, Arginine, Arnica Montana Flower Extract, Artemisia Annua Extract, Artemisia Princeps Leaf Water, Artemisia Tridentata Oil, Ascorbic Acid, Ascorbyl Palmitate, Asparagine, Aspartic Acid, Astragalus Gummifer Gum, Azelaic Acid, Backhousia Citriodora Oil, Beeswax, Benzaldehyde, Benzoic Acid, Benzophenone, Benzophenone-2, Benzophenone-6, Benzyl Acetate, Benzyl Alcohol, Benzyl Benzoate, Benzyl Benzoyloxybenzoate, Benzyl Cinnamate, 3-Benzylidene Camphor, Benzyl Laurate, Benzyl Salicylate, Betula Alba Bark Extract, Betula Alba Extract, Betula Alba Leaf Extract, Betula Alba Oil, BHA, BHT, Bisabolol, Bixa Orellana Extract, Bixa Orellana Seed Extract, Boesenbergia Pandurata Root Oil, Borneo), Boswellia Carterii Extract, Boswellia Carterii Oil, Boswellia Serrata Gum, Boswellia Serrata Oil, 2,3-Butanediol, Butoxydiglycol, Butoxyethanol, Butoxyethyl Acetate, Butyl Acetate, n- Butyl Alcohol, t-Butyl Alcohol, 4-t-Butyl Benzaldehyde, Butyl Benzoate, 4-t-Butylbenzoic Acid, 2-t-Butylcyclohexyl Acetate, 2-t-Butylcyclohexyloxybutanol, Butylene Glycol, Butyl Lactate, Butyl Methacrylate, Butyl Oleate, Butylparaben, Butylphenyl Methylpropional, Butyl Stearate, Butyric Acid, Butyrolactone Caffeic Acid, Caffeine, Calcium Acetate, Calcium Alginate, Calendula Officinalis Flower Extract, Calendula Officinalis Flower Oil, Callitris Introtropica Wood Oil, Callitris Quadrivalvis Gum, Camellia Oleifera Leaf, Camellia Sinensis Leaf Extract, Camellia Sinensis Leaf Water, Camphene, Camphor, Camphylcyclohexanol, Cananga Odorata Flower Oil, Cananga Odorata Flower Wax, Canarium Commune Gum Oil, Canarium Luzonicum Gum Nonvolatiles, Capric Acid, Caproic Acid, Gamma-Caprolactone, Caprylic Acid, Caprylic Alcohol, Caprylic/Capric Triglyceride, Caprylyl Butyrate, Capsaicin, Capsicum Annuum Extract, Capsicum Frutescens Resin, Caramel, Carmine, Carthamus Tinctorius (Safflower) Seed Oil, Carum Carvi (Caraway) Fruit Oil, Carum Carvi (Caraway) Seed Extract, Carum Carvi (Caraway) Seed Oil, Carom Petroselinum (Parsley) Seed Oil, Carvone, Beta-Caryophyllene, Cedrol, Cedrus Atlantica (Cedarwood) Bark Oil, Cedrus Deodara Wood Oil, Cellulose Gum, Ceratonia Siliqua (Carob) Fruit Extract, Ceratonia Siliqua Gum, Cetyl Acetate, Cetyl Alcohol, Cetyl Palmitate, Chamaecyparis Obtusa Oil, Chamaecyparis Obtusa Water, Chamomilla Recutita (Matricaria) Flower Extract, Chamomilla Recutita (Matricaria) Flower Oil, Chamomilla Recutita (Matricaria) Flower Water, Chamomilla Recutita (Matricaria) Leaf Extract, Chondrus Crispus (Carrageenan), Chouji Yu, CI 75470, Cichorium Intybus (Chicory) Leaf Extract, Cichorium Intybus (Chicory) Root Extract, Cinnamal, Cinnamomum Camphora (Camphor) Bark Oil, Cinnamomum Camphora (Camphor) Leaf Extract, Cinnamomum Cassia Leaf Oil, Cinnamomum Zeylanicum Bark Oil, Cinnamyl Acetate, Cinnamyl Alcohol, Cistus Ladaniferus Extract, Cistus Ladaniferus Oil, Cistus Ladaniferus Resin, Cistus Monspeliensis Extract, Citral, Citric Acid, Citronella), Citronellol, Citronellyl Acetate, Citronellyl Methylcrotonate, Citrus Aurantifolia (Lime) Oil, Citrus Aurantium Amara (Bitter Orange) Flower Water, Citrus Aurantium Amara (Bitter Orange) Oil, Citrus Aurantium Amara (Bitter Orange) Peel Extract, Citrus Aurantium Bergamia (Bergamot) Fruit Oil, Citrus Aurantium Dulcis (Orange) Flower Oil, Citrus Aurantium Dulcis (Orange) Flower Water, Citrus Aurantium Dulcis (Orange) Fruit Extract, Citrus Aurantium Dulcis (Orange) Fruit Water, Citrus Aurantium Dulcis (Orange) Oil, Citrus Grandis (Grapefruit) Fruit Water, Citrus Grandis (Grapefruit) Peel Oil, Citrus Grandis/Paradisi Fruit Water, Citrus Limon Leaf Oil, Citrus Medica Limonum (Lemon) Fruit Extract, Citrus Medica Limonum (Lemon) Fruit Water, Citrus Medica Limonum (Lemon) Peel Oil, Citrus Medica Vulgaris Peel Oil, Citrus Nobilis (Mandarin Orange) Fruit Extract, Citrus Nobilis (Mandarin Orange) Peel Extract, Citrus Nobilis (Mandarin Orange) Peel Oil, Citrus Reticulata Leaf Oil, Citrus Tangerina (Tangerine) Peel Oil, Citrus Unshiu Peel Powder, Cnidium Officinale Root Water, Cochlearia Armoracia (Horseradish) Root Extract, Cocos Nucifera (Coconut) Oil, Cocos Nucifera (Coconut) Water, Cod Liver Oil, Coffea Arabica (Coffee) Extract, Coffea Arabica (Coffee) Seed Extract, Coffea Arabica (Coffee) Seed Oil, Coleus Forskohlii Root Oil, Commiphora Myrrha Oil, Commiphora Myrrha Resin, Copaifera Officinalis (Balsam Copaiba) Resin, Coriandrum Sativum (Coriander) Extract, Coriandrum Sativum (Coriander) Fruit Oil, Corylus Avellana (Hazel) Leaf Water, Corylus Avellana (Hazel) Seed Oil, Coumarin, Crataegus Oxyacantha Flower Water, m-Cresol, o-Cresol, p-Cresol, Crocus Sativus Flower Extract, Crotonaldehyde, Crotonic Acid, Cryptocarya Crassinervia Bark Oil, Cryptocarya Massoy Bark Extract, Cuminum Cyminum (Cumin) Seed Extract, Cuminum Cyminum (Cumin) Seed Oil, Cupressus Sempervirens Oil, Curcuma Longa (Turmeric) Root Extract, Cyamopsis Tetragonoloba (Guar) Gum, Cyclamen Aldehyde, Cyclohexylethyl Acetate, Cyclohexylethyl Butyrate, Cyclopentadecanone, Cymbopogon Flexuosus Oil, Cymbopogon Martini Oil, Cymbopogon Nardus (Citronella) Oil, Cymbopogon Schoenanthus Oil, p- Cymene, Cyperus Esculentus Root Oil, Cysteine, Cystine, Cytisus Scoparius Flower Extract, Dalea Spinosa Seed Oil, Alpha-Damascone, Daucus Carota Sativa (Carrot) Root Water, Daucus Carota Sativa (Carrot) Seed Oil, Delta-Decalactone, Decanal, Decane, Decenal, Decenol, Decyl Alcohol, Denatonium Benzoate, Diacetone Alcohol, Dianthus Caryophyllus Flower Oil, Dibutyl Phthalate, Dibutyl Sebacate, Diethoxynonadiene, Diethyl Adipate, Diethylamine, Diethyl Caprylamide, Diethylene Glycol, Diethylhexyl Phthalate, Diethylhexyl Sebacate, Diethyl Oxalate, Diethyl Phthalate, Diethyl Sebacate, Diethyl Succinate, Dihydrocitronellol, Dihydrocoumarin, Dihydrojasmonate, Dihydro Pentamethylindanone, Diisobutyl Adipate, Diisopropyl Adipate, Dimethyl Brassylate, Dimethyl Carbonate, 2,4-Dimethyl-3-Cyclohexene Carboxaldehyde, Dimethyl Decadienal, Dimethyl Hexahydronaphthyl Dihydroxymethyl Acetal, Dimethylhydroxy Furanone, D imethyloctahyd ro-2-Naphthaldehyde, 2, 6-D iethyl-7-Octen-2-ol, Dimethyl Phenylpropanol, Dimethyl Phthalate, Dimethyl Succinate, Diphenyl Ether, Diphenyl Methane, Dipropylene Glycol, Dipteryx Odorata Seed Extract, Disodium Phosphate, Disodium Succinate, Dodecene, Eicosane, Elettaria Cardamomum Seed Extract, Elettaria Cardamomum Seed Oil, Erythorbic Acid, Ethoxydiglycol, Ethoxyethanol, Ethoxyethanol Acetate, Ethyl Acetate, Ethyl Benzoate, Ethyl Butyl Valerolactone, Ethylcellulose, Ethyl Cinnamate, Ethyl Cyclohexyl Propionate, Ethyl 2, 2-Dimethylhyd rocin namal, Ethylene Brassylate, Ethylene Dodecanedioate, Ethyl Ether, Ethyl Hexanediol, Ethylhexyl Ethylhexanoate, Ethylhexyl Palmitate, Ethylhexyl Salicylate, Ethyl Lactate, Ethyl Laurate, Ethyl Linoleate, Ethyl Linolenate, Ethyl Menthane Carboxamide, Ethyl Methacrylate, Ethyl Methylphenylglycidate, Ethyl Myristate, Ethyl Nicotinate, Ethyl Oleate, Ethyl Palmitate, Ethylparaben, Ethyl Pelargonate, Ethyl Phenethyl Acetal, Ethyl Phenylacetate, Ethyl Pyruvate, Ethyl Ricinoleate, Ethyl Stearate, Ethyl Thioglycolate, Ethyl Trimethylcyclopentene Butenol, Ethyl Vanillin, Eucalyptol, Eucalyptus Citriodora Oil, Eucalyptus Globulus Leaf Oil, Eucalyptus Globulus Leaf Water, Eugenia Caryophyllus Bud Oil, Eugenia Caryophyllus (Clove) Flower Extract, Eugenia Caryophyllus (Clove) Flower Oil, Eugenia Caryophyllus (Clove) Leaf Oil, Eugenia Operculata Leaf Powder, Eugenol, Eugenyl Acetate, Euphorbia Cerifera (Candelilla) Wax, Evernia Furfuracea (Treemoss) Extract, Evernia Prunastri (Oakmoss) Extract, Farnesene, Farnesol, Farnesyl Acetate, Ferula Galbaniflua (Galbanum) Resin Oil, Ficus Carica (Fig) Fruit Water, Foeniculum Vulgare (Fennel) Fruit Extract, Foeniculum Vulgare (Fennel) Fruit Powder, Foeniculum Vulgare (Fennel) Oil, Foeniculum Vulgare (Fennel) Water, Formic Acid, Fucus Vesiculosus Extract, Fumaric Acid, Furfural, Fusanus Spicatus Wood Oil, Galactoarabinan, Gardenia Florida Oil, Gaultheria Procumbens (Wintergreen) Leaf Extract, Gaultheria Procumbens (Wintergreen) Leaf Oil, Gentiana Lutea Root Extract, Geraniol, Geranium Maculatum Oil, Geranyl Acetate, Gluconic Acid, Gluconolactone, Glucose Pentaacetate, Glutamic Acid, Glutamine, Glutaral, Glutaric Acid, Glutathione, Glycerin, Glyceryl Oleate, Glyceryl Rosinate, Glyceryl Stearate, Glycine, Glycine Soja (Soybean) Oil, Glycol, Glycyrrhiza Glabra (Licorice), Glycyrrhizic Acid, Glyoxal, Gnaphalium Leontopodium Water, Gossypium Herbaceum (Cotton) Fruit Water, Guaiazulene, Hakka Yu, Hay Water, Helichrysum Stoechas Extract, Heliotropine, 2-Heptylcyclopentanone, Heterotheca Inuloides Flower Extract, Hexadecanolactone, H examethyl indanopyran, Hexanal, 3-Hexenol, Hexyl Alcohol, Hexyl Cinnamal, Hexylene Glycol, Hexyl Salicylate, Hibiscus Abelmoschuus Extract, Hinokitiol, Hippuric Acid, Histidine, Homosalate, Humulus Lupulus (Hops) Cone Oil, Humulus Lupulus (Hops) Extract, Hydroabietyl Alcohol, Hydrogenated Ethylbicycloheptane Guaiacol, Hydrogenated Lanolin, Hydrogenated Polydecene, Hydrogenated Rosin, Hydroquinone, p-Hydroxyanisole, 4-Hydroxybenzoic Acid, Hydroxycitronellal, Hydroxyisohexyl 3-Cyclohexene Carboxaldehyde, Hydroxymethoxybenzyl Pelargonamide, Hyptis Suaveolens Seed Oil, Hyssopus Officinalis Extract, Hyssopus Officinalis Leaf Oil, Illicium Verum (Anise) Oil, Iris Florentina Root Extract, Isoamyl Acetate, Isoamyl Alcohol, Isoamyl Allylglycolate, Isoamyl Cinnamate, Isoamyl Laurate, Isobornyl Acetate, Isobutenyl Methyltetrahydropyran, Isobutyl Acetate, Isobutyl Benzoate, Isobutyl Methyl Tetrahydropyranol, Isobutyl Palmitate, Isobutyl Pelargonate, Isobutyric Acid, Isododecane, Isoeugenol, Isoleucine, Isolongifolene Epoxide, Isolongifolene Ketone-Exo, Alpha-Isomethyl lonone, Isopentanal, Isopentylcyclohexanone, Isopropyl Acetate, Isopropyl Alcohol, Isopropyl Benzoate, Isopropyl Cyclohexylpropionate, Isopropyl Laurate, Isopropyl Myristate, Isopropyl Palmitate, Isopropylphenylbutanal, Isopulegol, Jasminum Officinale (Jasmine) Extract, Jasminum Officinale (Jasmine) Flower Extract, Jasminum Officinale (Jasmine) Flower Water, Jasminum Officinale (Jasmine) Oil, Juglans Regia (Walnut) Leaf Extract, Juglans Regia (Walnut) Shell Extract, Juniperus Communis Fruit Extract, Juniperus Communis Fruit Oil, Juniperus Mexicana Oil, Juniperus Oxycedrus Wood Oil, Juniperus Oxycedrus Wood Tar, Juniperus Scopulorum Oil, Juniperus Virginiana Oil, Keihi Ekisu, Keihi Yu, Ketoglutaric Acid, Kinginka Ekisu, Krameria Triandra Root Extract, Kunzea Ericoides Leaf Oil, Lactic Acid, Laminaria Cloustoni Extract, Laminaria Digitata Extract, Laminaria Hyperborea Extract, Laminaria Japonica Extract, Laminaria Ochroleuca Extract, Laminaria Saccharina Extract, Lantana Camara Leaf Water, Lauraldehyde, Lauramine Oxide, Laurie Acid, Laurus Nobilis Leaf, Laurus Nobilis Leaf Extract, Laurus Nobilis Oil, Lauryl Alcohol, Lauryl Lactate, Lavandula Angustifolia (Lavender) Extract, Lavandula Angustifolia (Lavender) Flower Extract, Lavandula Angustifolia (Lavender) Flower Powder, Lavandula Angustifolia (Lavender) Flower Water, Lavandula Angustifolia (Lavender) Oil, Lavandula Angustifolia (Lavender) Water, Lavandula Hybrida Extract, Lavandula Hybrida Oil, Lavandula Spica (Lavender) Extract, Lavandula Stoechas Extract, Lemon Ekisu, Leptospermum Petersonii Oil, Leptospermum Scoparium Oil, Leucine, Levisticum Officinale Oil, Levulinic Acid, Limonene, Linalool, Linalyl Acetate, Linoleic Acid, Linolenic Acid, Linum Usitatissimum (Linseed) Seed Oil, Lippia Citriodora Flower Water, Lippia Citriodora Water, Liquidambar Styraciflua Oil, Litsea Cubeba Fruit Oil, Longifolene, Lysine, Maleic Acid, Malic Acid, Malonic Acid, Marrubium Vulgare Extract, Massoy Bark Oil, Medicago Sativa (Alfalfa) Extract, MEK, Melaleuca Alternifolia (Tea Tree) Leaf Oil, Melaleuca Ericifolia Leaf Oil, Melaleuca Leucadendron Cajaput Oil, Melilotus Officinalis Extract, Melissa Officinalis Leaf Extract, Melissa Officinalis Leaf Oil, Mentha Aquatica Water, Mentha Arvensis Extract, Mentha Arvensis Leaf Oil, Mentha Arvensis Powder, p-Menthan-7-ol, Mentha Piperita (Peppermint) Leaf, Mentha Piperita (Peppermint) Leaf Extract, Mentha Piperita (Peppermint) Leaf Water, Mentha Piperita (Peppermint) Oil, Mentha Pulegium Oil, Mentha Viridis (Spearmint) Extract, Mentha Viridis (Spearmint) Leaf Oil, Menthol, Menthone Glycerin Acetal, Menthoxypropanediol, Menthyl Acetate, Menthyl Lactate, Menthyl Salicylate, Methionine, Methoxydiglycol, Methoxyethanol, Methoxyethanol Acetate, Methoxyindane, Methoxyisopropanol, Methoxytrimethylheptanol, Methyl Acetate, p-Methyl Acetophenone, Methylal, Methyl Alcohol, Methyl Anthranilate, 4-Methylbenzaldehyde, Methyl Benzoate, Methyl Benzodioxepinone, Methylbenzyl Acetate, Methyl 4-t-B utyl benzoate, Methyl Caproate, Methyl Caprylate, Methylcellulose, 6-Methyl Coumarin, Methylcyclohexenyl Butanol, Methylcyclopentadecenone, Methyldihydrojasmonate, Methyl Diisopropyl Propionamide, Methyl Eugenol, Methyl Hexyl Ether, Methyl Hydrogenated Rosinate, Methyl Lactate, Methyl Lactic Acid, Methyl Laurate, Methyl Linoleate, Methyl 3-Methyl resorcylate, Methyl Myristate, Methyl Nicotinate, Methyl 2-Octynoate, Methyl Oleate, Methyl Palmitate, Methylparaben, Methyl Pelargonate, Methyl Phenylbutanol, Methyl Rosinate, Methyl Salicylate, Methyl Stearate, MIBK, Michelia Alba Flower Oil, Michelia Alba Leaf Oil, Michelia Champaca Oil, Mimosa Tenuiflora Bark Extract, Mimosa Tenuiflora Leaf Extract, Mineral Oil, Mixed Cresols, Mixed lonones, Monarda Didyma Oil, Musa Sapientum (Banana) Flower Water, Musk Ketone, Myrcenol, Myrica Gale Extract, Myristic Acid, Myristica Fragrans (Nutmeg) Extract, Myristica Fragrans (Nutmeg) Kernel Oil, Myristyl Alcohol, Myrocarpus Fastigiatus Oil, Myroxylon Balsamum (Balsam ToIu) Resin, Myroxylon Pereirae (Balsam Peru) Oil, Myroxylon Pereirae (Balsam Peru) Resin, Myrrhis Odorata Extract, Myrtus Communis Leaf Water, Myrtus Communis Oil, 2-Naphthol, Narcissus Poeticus Extract, Narcissus Poeticus Flower Wax, Nasturtium Officinale Extract, Nelumbium Speciosum Flower Water, Nelumbo Nucifera Flower Water, Neohesperidin Dihydrochalcone, Nepeta Cataria Extract, Nicotiana Tabacum (Tobacco) Leaf Extract, Nindou Ekisu, Nitrous Oxide, Gamma-Nonalactone, Nonyl Acetate, Nopyl Acetate, Ocimum Basilicum (Basil) Extract, Ocimum Basilicum (Basil) Oil, Octadecane, Octyldodecanol, Olax Dissitifiora Root Oil, Olea Europaea (Olive) Fruit Oil, Oleic Acid, Oleth-2, Oleth-3, Oleth-4, Oleth-5, Oleth-6, Oleth-7, Oleth-8, Oleth-9, Oleth-10, Oleth-11, Oleth-12, Oleth-15, Oleth- 16, Oleth-20, Oleth-23, Oleth-24, Oleth-25, Oleth-30, Oleth-35, Oleth-40, Oleth-44, Oleth- 50, Oleth-82, Oleth-106, Oleyl Alcohol, Olibanum, Opoponax Oil, Orange Yu, Origanum Heracleoticum Flower Oil, Origanum Majorana Flower Oil, Origanum Majorana Leaf Extract, Origanum Majorana Leaf Oil, Origanum Vulgare Flower Extract, Ormenis Multicaulis Extract, Ormenis Multicaulis Oil, Oryza Sativa (Rice) Bran Water, Osmanthus Fragrans Flower Extract, Palmitic Acid, Panax Ginseng Root Water, Pandanus Amaryllifolius Leaf Extract, Paraffin, PEG-2 Hydrogenated Castor Oil, PEG-5 Hydrogenated Castor Oil, PEG-6 Hydrogenated Castor Oil, PEG-7 Hydrogenated Castor Oil, PEG-IO Hydrogenated Castor Oil, PEG- 16 Hydrogenated Castor Oil, PEG-20 Hydrogenated Castor Oil, PEG-25 Hydrogenated Castor Oil, PEG-30 Hydrogenated Castor Oil, PEG-35 Hydrogenated Castor Oil, PEG-40 Hydrogenated Castor Oil, PEG-45 Hydrogenated Castor Oil, PEG-50 Hydrogenated Castor Oil, PEG-54 Hydrogenated Castor Oil, PEG-55 Hydrogenated Castor Oil, PEG-60 Hydrogenated Castor Oil, PEG-80 Hydrogenated Castor Oil, PEG-100 Hydrogenated Castor Oil, PEG-200 Hydrogenated Castor Oil, PEG-IO Sorbitan Laurate, PEG-40 Sorbitan Laurate, PEG-44 Sorbitan Laurate, PEG-75 Sorbitan Laurate, PEG-80 Sorbitan Laurate, PEG-3 Sorbitan Oleate, PEG-6 Sorbitan Oleate, PEG-3 Sorbitan Stearate, PEG-4 Sorbitan Stearate, PEG-6 Sorbitan Stearate, PEG-40 Sorbitan Stearate, PEG-60 Sorbitan Stearate, Pelargonic Acid, Pelargonium Graveolens Extract, Pelargonium Graveolens Flower Oil, Pelargonium Graveolens Water, Pelargonium Graveolens Wax, Pentadecalactone, Pentadecyl Alcohol, Pentamethylheptenone, Perillaldehyde, Persea Gratissima (Avocado) Fruit Water, Phenethyl Acetate, Phenethyl Alcohol, Phenol, Phenoxyethanol, Phenylalanine, Phenyl Benzoate, Phenylisohexanol, Phenylmethylpentanal, o-Phenylphenol, Phenylpropanol, Phenyl Salicylate, Phosphoric Acid, Phytol, Picea Excelsa Leaf Oil, Pimenta Acris (Bay) Leaf Oil, Pimpinella Anisum (Anise) Fruit Extract, Pinus Palustris Oil, Pinus Palustris Tar Oil, Pinus Pumilio Oil, Pinus Strobus Cone Extract, Pinus Sylvestris Cone Extract, Pinus Sylvestris Cone Oil, Pinus Sylvestris Leaf Oil, Piper Betle Leaf Oil, Piper Nigrum (Black Pepper) Fruit Oil, Pistacia Lentiscus (Mastic) Gum, Pogostemon Cablin Oil, Polianthes Tuberosa Extract, Polysorbate 20, Polysorbate 21, Polysorbate 60, Polysorbate 61, Polysorbate 80, Polysorbate 81, Pongamol, Potassium Acetate, Potassium Sorbate, PPG-2-Buteth-2, PPG-2-Buteth-3, PPG-3-Buteth-5, PPG-4- Buteth-4, PPG-5-Buteth-5, PPG-5-Buteth-7, PPG-7-Buteth-4, PPG-7-Buteth-10, PPG-9- Buteth-12, PPG-10-Buteth-9, PPG-12-Buteth-12, PPG-12-Buteth-16, PPG-15-Buteth-20, PPG-17-Buteth-17, PPG-19-Buteth-19, PPG-20-Buteth-30, PPG-24-Buteth-27, PPG-26- Buteth-26, PPG-28-Buteth-35, PPG-30-Buteth-30, PPG-33-Buteth-45, PPG-36-Buteth-36, PPG-38-Buteth-37, PPG-2 Methyl Ether, Proline, Propionic Acid, Propyl Acetate, Propyl Alcohol, Propyl Benzoate, Propylene Glycol, Propylene Glycol Alginate, Propylene Glycol Butyl Ether, Propylene Glycol Stearate, Propyl Gallate, Propylparaben, Prunus Amygdalus Amara (Bitter Almond) Kernel Oil, Prunus Amygdalus Dulcis (Sweet Almond) Oil, Prunus Armeniaca (Apricot) Fruit Water, Prunus Armeniaca (Apricot) Kernel Oil, Prunus Cerasus (Bitter Cherry) Seed Oil, Prunus Serotina (Wild Cherry) Bark Extract, Prunus Serotina (Wild Cherry) Fruit Extract, Punica Granatum Bark Extract, Punica Granatum Extract, Pyrocatechol, Pyrogallol, Pyrus Cydonia Seed Extract, Pyrus Malus (Apple) Fruit Water, Pyruvic Acid, Quillaja Saponaria Bark Extract, Quillaja Saponaria Root Extract, Quinine, Raspberry Ketone, Raspberryketone Glucoside, Resorcinol, Rhamnose, Rhododendron Chrysanthum Leaf Extract, Rhododendron Ferrugineum Extract, Ricinus Communis (Castor) Seed Oil, Rosa Canina Flower, Rosa Canina Flower Oil, Rosa Centifolia Flower Extract, Rosa Centifolia Flower Oil, Rosa Damascena Extract, Rosa Damascena Flower Oil, Rosa Damascena Flower Water, Rosa Damascena Flower Wax, Rosa Eglentaria Extract, Rosa Gallica Flower Oil, Rosa Moschata Oil, Rosa Multiflora Fruit Extract, Rose Flower Oil, Rosmarinus Officinalis (Rosemary) Flower Wax, Rosmarinus Officinalis (Rosemary) Leaf Extract, Rosmarinus Officinalis (Rosemary) Leaf Oil, Rosmarinus Officinalis (Rosemary) Leaf Water, Rosmarinus Officinalis (Rosemary) Water, Rubus Fruticosus (Blackberry) Fruit Extract, Rubus Fruticosus (Blackberry) Leaf Extract, Rubus Idaeus (Raspberry) Fruit Water, Rubus Idaeus (Raspberry) Leaf Wax, Ruta Graveolens (Rue) Oil, Saccharin, Salicylic Acid, Salvia Lavandulaefolia Oil, Salvia Officinalis (Sage) Leaf Water, Salvia Officinalis (Sage) Oil, Salvia Sclarea (Clary) Oil, Sambucus Nigra Oil, Sambucus Nigra Wax, Santalum Album (Sandalwood) Oil, Sassafras Officinale Root Oil, Satureia Hortensis Extract, Schinus Molle Oil, Sciareolide, Serine, Sesamum Indicum (Sesame) Seed Oil, Sisymbrium Trio Seed Oil, Beta-Sitosterol, Sodium Acetate, Sodium Benzoate, Sodium Citrate, Sodium Glutamate, Sodium Hexametaphosphate, Sodium Saccharin, Solanum Lycopersicum (Tomato) Fruit Water, Sorbic Acid, Sorbitan Oleate, Sorbitan Stearate, Sorbitol, Spartium Junceum Flower Extract, Stearic Acid, Stearyl Alcohol, Sterculia Urens Gum, Styrax Benzoin Gum, Styrax Benzoin Resin Extract, Succinic Acid, Sucrose Octaacetate, Synthetic Wax, Tagetes Minuta Flower Oil, Taimu Yu, Tanacetum Cinerariifolium (Pyrethrum) Flower Extract, Tannic Acid, Tarchonanthus Camphoratus Oil, Tar Oil, Tartaric Acid, Taurine, TBHQ, Terpineol, 4- Terpineol, Terpineol Acetate, Tetrahydrofurfuryl Acetate, Tetrahydrofurfuryl Alcohol, Tetramethyl Cyclopentene Butenol, Theobroma Cacao (Cocoa) Seed Butter, Theobromine, Thiamine HCI, Thio lactic Acid, Threonine, Thuja Occidentalis Leaf Oil, Thymol, Thymus Mastichina Oil, Thymus Vulgaris (Thyme) Extract, Thymus Vulgaris (Thyme) Leaf, Thymus Vulgaris (Thyme) Oil, Thymus Zygis Oil, Tilia Americana Flower Extract, Tilia Cordata Flower Water, Tilia Cordata Oil, Tocopherol, Torreya Californica (California Nutmeg) Oil, Triacetin, Tribenzoin, Tricalcium Phosphate, Tricaprin, Tricaprylin, Tricyclodecenyl Propionate, Tridecyl Alcohol, Triethanolamine, Triethyl Citrate, Triethylene Glycol, Triethylhexanoin, Trifolium Pratense (Clover) Flower Extract, Trigonella Foenum-Graecum Seed Extract, Trimethylamine Oxide, Trimethylhexanol, Tromethamine, Tryptophan, Turpentine, Tyrosine, Uikyo Yu, Gamma-Undecalactone, Undecanoic Acid, Undecyl Alcohol, Undecylenal, Undecylenic Acid, Undecylenyl Alcohol, Ursolic Acid, Valeriana Officinalis Root, Valine, Vanilla Planifolia Fruit, Vanilla Tahitensis Fruit, Vanillin, Vanillyl Butyl Ether, Verbena Officinalis Extract, Vetiveria Zizanoides Root Oil, Viburnum Prunifolium Extract, Viola Odorata Extract, Viola Odorata Leaf Extract, Viola Odorata Leaf Wax, Viola Odorata Oil, Viola Tricolor Water, Vitis Vinifera (Grape) Leaf Oil, Ximenia Americana Seed Oil, Xylene, Xylose, Yucca Aloifolia Extract, Yucca Filamentosa Extract, Yucca Schidigera Extract, Yucca Vera Extract, Yukari Yu, Zanthoxylum Acanthopodium Fruit Oil, Zanthoxylum Americanum Bark Extract, Zanthoxylum Piperitum Oil, Zea Mays (Corn) Oil, Zingiber Officinale (Ginger) Root Extract, Zingiber Officinale (Ginger) Root Oil, and Zingiber Officinale (Ginger) Water. 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.

E. Formulations and Methods of Manufacture Thereof

[00152] The 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. In certain embodiments, the compositions are cosmetic compositions. In certain embodiments, 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. 8, Noyes Publications; 2nd ed., 2000. For example, 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. Generally, the aqueous phase, and when present, the oil phase would be individually prepared, with materials having similar phase partitioning being added in any order. If the final product is an emulsion, the two phases will then be combined with vigorous stirring. Any ingredients in the formulation with high volatility, or which are susceptible to hydrolysis at high temperatures, can be added with gentle stirring towards the end of the process, post emulsification if applicable. 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%. When present, 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.

[00153] In certain embodiments of the invention one or more of the components of the composition, e.g., one or more active agents such as an elastase, is encapsulated in liposomes, microcapsules, nanocapsules, vesicles, or other similar structures, or microparticles or nanoparticles are impregnated with the agent and release it by diffusion or as they degrade. 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. Various 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.) may also be used. See, e.g., Rosen, M. R., ed., Delivery System Handbook for Personal Care and Cosmetic Products : Technology, Applications and Formulations (William Andrew Publishing, 2005). 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. For injection a suitable article would be a needle optionally with a syringe. The 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. For example, 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. In certain embodiments of the invention 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. In certain embodiments of the invention 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. [00154] 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. Examples of 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, propionate, salicylate, succinate, sulfate, tartrate, thiocyanate, tosylate and undecanoate. 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. This invention also envisions the quaternization of any basic nitrogen-containing groups of the compounds disclosed herein. Water or oil-soluble or dispersible products may be obtained by such quaternization. [00155] In one aspect, the composition is an injectable form of a pharmaceutical composition. An injectable composition of the present invention may be in the form of a solution or suspension. 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.

[00156] Sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, 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 .

[00157] 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. In certain embodiments, 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. In the case of sterile powders for the preparation of sterile injectable solutions, 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.

V. Methods of Use A. Primary Treatment

[00158] In certain embodiments, 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). In certain embodiments, the skin may be chronologically aged skin. In certain embodiments, the skin is prematurely aged skin. The invention contemplates contacting skin of any of these types with a composition of the invention. In some embodiments, the skin is heavily photodamaged. For purposes of this invention 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. Optionally the degree of photodamage or aging is classified according to a classification scheme. [00159] As noted above, visible signs of photodamage typically begin to be detectable in young adult life, e.g., between about 20-30 years of age, becoming more and more evident thereafter, especially in individuals who experience significant amounts of sun exposure. In one embodiment, 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. [00160] Various methods of administration are envisioned. In certain embodiments of the invention the compositions are topically applied. In accordance with the present invention once a composition is topically applied to the skin surface, components of the composition may penetrate to deeper layers of the skin. For example, in certain embodiments of the invention the composition is contacted with the epidermis and the enzyme diffuses or is transported into the dermis.

[00161] In other embodiments of the invention the compositions are injected intradermally, e.g., using standard techniques for intradermal injection. If desired, 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.

[00162] In other embodiments of the invention the compositions are injected subcutaneous Iy, e.g., using standard techniques for subcutaneous injection. If desired, 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. [00163] 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. Also envisioned are "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.

[00164] In certain embodiments of the invention the enzymes are provided tethered to a water insoluble substrate, e.g., a textile. See, e.g., U.S. Pat. No. 6,410,017, incorporated herein by reference. 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 μg/cm² to about 1000 μg/cm². 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. [00165] In certain embodiments of the invention 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". Without wishing to be bound by any theory, 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". Other surface removal methods include tape stripping (e.g., using an adhesive tape) or occlusion under a moisture- impermeant barrier. It will be appreciated that many of these methods are often employed in their own right to improve the appearance and/or structure of the skin. In certain embodiments of the invention, applying a composition of the present invention following removal of one or more layers of the skin using any suitable method provides an additional benefit beyond that obtained simply by using the surface removal method. [00166] Surface removal methods and suitable compositions and instrumentation for performing the methods are well known in the art and need not be described in detail here. Chemical peeling agents include various α- and β-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. Surg., 107: 22, 2001) Fulton, J.E., and Porumb, S., Am. J. Clin. Dermatol, 5(3): 179-187 (2004); Rubin, M., Procedures in Cosmetic Dermatology Series: Chemical Peels (Saunders, 2005).

[00167] 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).

[00168] In certain embodiments of the invention 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.

[00169] In certain embodiments of the invention the SC is substantially removed, and a composition of the invention is topically applied to the surface of the skin following such removal. In certain embodiments of the invention 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. In other embodiments of the invention 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. For example, the 3 upper layers of the epidermis may be substantially removed while leaving the stratum basale substantially intact. In other embodiments of the invention 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. In certain embodiments of the invention, 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.

[00170] One of skill in the art will be able to select appropriate compositions and methods to remove the desired depth or amount of epidermis, DEJ, or papillary dermis. For example, one of skill in the art would know that glycolic acid peels are milder than phenol peels, and that the relative proportions of different components can be varied to achieve a desired extent of removal. Furthermore, one of skill in the art would be able to apply the surface removal compositions and methods for varying periods of time to remove the desired amount of skin. 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.

[00171] 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. [00172] In certain embodiments of the invention rather than applying an inventive composition comprising an elastase or partial elastase after a chemical peeling procedure, 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. It will be appreciated that 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.

[00173] It will be appreciated that certain surface removal methods are often employed in their own right to improve the appearance and/or structure of the skin. In certain embodiments of the invention, applying a 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.

[00174] The 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. The skilled artisan will appreciate that certain factors can influence the amount and timing required to provide a visible improvement in the skin, including but not limited to the severity of the elastosis, previous and concurrent treatments, age of the subject, etc. Generally, treatment of a subject with an inventive composition can include a single treatment or, in many cases, can include a series of treatments. It is understood that 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.

[00175] As discussed above, a number of products are known to exfoliate the skin. In certain embodiments of the invention 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. As used herein, 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. Alternately or additionally, 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.

[00176] In certain embodiments of the invention 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. In certain embodiments of the invention 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.

B. Supplementary Treatments

[00177] In certain embodiments of the invention, following contacting the skin with a composition comprising one or more elastases or partial elastases for a period of time to diminish evidence of elastotic material, 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". For purposes of convenience, 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.

[00178] In certain embodiments of the invention the supplementary treatment comprises physically removing the enzyme, e.g., by rinsing with an appropriate solution. In certain embodiments of the invention 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. 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. For example, it may be desirable to provide a gradual diminishment in the evidence of elastotic material over the course of several treatments rather than a rapid diminishment using fewer treatments. By spreading the treatments over time, a gradual replacement of the elastotic material by normal elastic and collagenous fibers may occur. Furthermore, application of 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.

[00179] In certain embodiments of the invention, 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 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.

[00180] In certain embodiments of the invention, 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. As used herein, the term "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. In certain embodiments of the invention, 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. In fact, 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. However, 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. Typically 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. [00181] 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. 6,974,799, incorporated herein by reference), one or more peptides 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). It will be appreciated that any of the peptides may be modified, e.g., by addition an acetyl or amide group, fatty acid chain, etc. 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. 20040162232, incorporated herein by reference, describing elastin digest compositions. The composition 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", as used herein, 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. 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. Such compounds are known and are commercially available from, e.g., Sigma Chemical Company (St. Louis, Mo.), and Boerhinger Mannheim (Indianapolis, Ind.). Other retinoids which are useful herein are described in U.S. Pat. Nos. 4,677,120; 4,885,311; 5,049,584; 5,124,356; each of which is incorporated herein by reference. Others are tocopheryl-retinoate [tocopherol ester of retinoic acid (trans- or cis-), adapalene {6-[3-(l-adamantyl)-4-methox- yphenyl]-2-naphthoic acid}, and tazarotene (ethyl 6-[2-(4,4-dimethylthioch- roman-6-yl)-ethynyl]nicotinate). Exemplary amounts of retinoids range from 0.005% to 2%, e.g., 0.01% to 2%. 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%.

[00182] In certain embodiments of the invention, following 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 soothing composition. As used herein, 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 are known in the art and 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.

VI. Assessing Effectiveness [00183] 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 profϊlometry, 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. In one embodiment, at least one of the aforementioned parameters is improved, e.g., elasticity or fϊrmess 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.

[00184] The effectiveness of the skin care compositions and methods of the present invention can also be assessed in 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. In certain embodiments of the invention, 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. Alternately or additionally, in certain embodiments of the invention "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. In certain embodiments of the invention, 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. For example, portions of skin located within up to 2 cm of each other could be used as control and test samples. In one embodiment, punches or sections taken from adjacent areas of skin are used as test and control samples. [00185] In certain embodiments, the effectiveness of the compositions and methods of the present invention can also be assessed in tissue samples or living tissue using biochemical testing. For example, the degradation of elastin may be monitored directly. In other embodiments, a surrogate of elastin degradation may be monitored to assess the inventive method or composition. In certain embodiments, 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. 30:202-13, 2007. These methods of 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.

Equivalents and Scope

[00186] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. The scope of the present invention is not intended to be limited to the above Description, but rather is as set forth in the appended claims. The 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. As used 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 term "comprising" should be interpreted to mean that the composition or method is not limited to the recited ingredients or steps. In the claims articles such as "a,", "an" and "the" may mean one or more than one unless indicated to the contrary or otherwise evident from the context. "Signs of photodamage" refers to any one or more signs of photodamage, e.g., at least one sign of photodamage. Claims or descriptions that include "and/or" or "or" between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. 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. Furthermore, it is to be understood that 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. Where the claims recite a composition, it is to be understood that methods of administering the composition according to any of the methods disclosed herein, and methods of using the composition for any of the purposes disclosed herein, and methods of making the composition according to any of the methods of making disclosed herein are included in the invention, unless otherwise indicated or unless it would be evident to one of ordinary skill in the art that a contradiction or inconsistency would arise.

[00187] Where elements are presented as lists, e.g., in Markush group format, it is to be understood that each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. It should it be understood that, in general, where the invention, or aspects of the invention, is/are referred to as comprising particular elements, features, etc., certain embodiments of the invention or aspects of the invention consist, or consist essentially of, such elements, features, etc. For purposes of simplicity each of these various embodiments have not in all cases been specifically set forth in haec verba herein. [00188] 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. For example, 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. Thus the composition may be administered with the intent to reduce signs of photodamage, e.g., to reduce evidence of elastosis. In certain embodiments 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. 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.

[00189] In addition, it is to be understood that 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.

Exemplification

Example 1

Reduced Evidence of Elastotic Material in Sections of Photodamaged Human Skin Exposed to Elastase

[00190] The ability of Pseudomonas aeruginosa elastase to reduce evidence of elastotic material in photodamaged human skin was tested using excess facial skin tissue as the photodamaged skin model. Pseudonomas aeruginosa elastase (PE) was purchased from the Elastin Products Company.

[00191] Elastase activity was validated at a pH of 8.8 and 7.2 in two separate experiments, where modified versions of the method described by Shorten (Shotton, D. M., "Elastase." Methods Enzymol 19: 113-140, 1970) were used. Fluorescein labeled bovine elastin substrate was suspended in a 20OmM Tris buffer solution at the pH value corresponding to each experiment. The substrate suspensions were incubated with different concentrations of PE over a period of 24 hours. Fluorometric measurements of the supernatant materials were taken at different timepoints for the two different experiments. The resulting PE activity is shown in Fig. 2, where the mass of elastin digested was calculated based on an approximate elastin-fluorescein molecular weight of 7OkDa and the fluorescence calibration curve obtained with fluorescein alone at the same pH.

[00192] In one set of experiments, 60 micron cryotomed sections of 7mm frozen facial skin punches were thawed and exposed to the active and control solutions. The active and control sections belonging to the same group consist of neighboring sections from the skin punch. Five groups were evaluated. For each group the sections were each placed on a microscope slide, and exposed to 250 ug/ml PA elastase in the pH 8.8 Tris buffer for the active case and to the buffer only for the control case. The slides were placed in a cell culture plate with the cover and incubated overnight at 37°C. An internal control consisting of the PA elastase in substrate suspension, along with a control consisting of the substrate suspension with PA elastase buffer only were also exposed to the same conditions. [00193] Following the exposure period, the skin sections were rinsed off with the buffer and fixed in a formalin solution using standard methods. The slides were then submitted for elastic staining using the Verhoeff van Gieson (VVG) method.

[00194] 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. In contrast, sections treated with elastase (Figs. IA, 1C, and IE) show greatly reduced elastin staining, which appeared localized to lower dermis. The presence of elastin fibrils (normal elastin) is revealed in the sections exposed to the PA elastase suggesting preservation of the normal elatic fibers along with reduced evidence of elastotic material. Note in particular the presence of fine reticular black stained fibers in Fig. IE.

Example 2

Reduced Evidence of Elastotic Material in Photodamaged Human Skin By Topical Application of Elastase

[00195] Following the experiments described in Example 1 the feasibility of removing aberrant elastin in larger skin samples through topical exposure was evaluated. The buffer system used in these experiments was 200 mM Tris at pH 7.2. The PA elastase activity is shown in Fig. 5. At this pH, the activity of the PA elastase appears to be higher than that observed at pH 8.8. [00196] Eight mm diameter punches of photo-damaged facial skin were heat stripped to remove the epidermis. The heat stripping process involved exposure of the skin punch to the buffer solution for 2 minutes at 60⁰C. The epidermis was then 'peeled' off using a spatula. 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. Thus in this experiment 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.

[00197] Figs. 3 and 4 show the results of the histology, where elastin is stained black. Table 3 below 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. However, within each group, evidence of aberrant elastin degradation is evidenced by the reduced fraction of black stained fibers (compare punch 1 with punch 2, and punch 3 with punch 4) when the skin is exposed to elastase. Moreover, a zone of elastase action is visible in IH-I A, 4Ox (Fig. 3D), where the elastin staining increases with the distance from the elastase exposure surface for the heat stripped skin sample.

In another set of experiments, 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.

[00198] In summary, the results clearly demonstrate the ability of topically applied or injected elastase to reduce evidence of elastotic material in photodamaged skin. Table 3

Punch No Label Facial location Application process Solution

1 1H1-1A Cheek Topical to heat stripped skin active

2 2Hl -2P Cheek Topical to heat stripped skin control 3 3H1-CA Chin Injection active 4 4Hl -CP Chin Injection control

Example 3

Preparation of Compositions

[00199] The following nonlimiting exemplary compositions are formulated as gel/hydrogel patches and/or as face masks. Amounts are listed as weight percents.

Table 4

Composition I II III IV

1. Trimethyl glycine 10 2 — —

2. Bicine — — 8 —

3. Carnitine — — — 12

4. Sodium formate 1.0 — — —

5. Sodium succinate — — 1.0 —

6. Potassium glycolate — — — 1.5

7. Calcium chloride dihydrate 0.01 0.01 0.01 —

8. Glycerin — 5 — 12

9. Polyethylene glycol 200 — — 2 —

10 . Hexylene glycol — 5 — 2

11 . Polyvinylalcohol (31,000 mwt) 5 — 3 10

12 . Polyvinylalcohol (205,000 mwt) 5 — 8 6

13 . Agarose — 2.0 1.0 0.5

14 . Xanthan/Locust bean — 0.5 1.0 0.75

15 . Potassium Phosphate 0.1 — — —

16 . Tris — 0.1 — 0.1

17 . Elastase #1 0.1 0.1 .25 0.01

18 . Elastase #2 0.05 — — —

19 . pH 7.0 7.5 6.0 8.0

20 . Water as needed to 100

[00200] In certain embodiments Elastase #1 is PA elastase (LasB). In certain embodiments Elastase 2 is a second bacterial elastase, e.g., PA LasA. In some embodiments 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. Example 4

Evaluation of elastase activity in presence of common cosmetic ingredients [00201] 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).

[00202] The measured enzyme activity in the presence of the cosmetic ingredient is shown in Figure 6. While elastase activity is maintained in the presence of all these ingredients, the enzyme remains the most active in the presence of Tris buffer alone. [00203] In addition to evaluating elastase activity in vitro in these single component systems, simple formulations were also evaluated using both in vitro assay and in situ models, where formulation composition and exposure duration were observed to effect the extent of aberrant elastin digestion in cryotomed sections of human photodamaged skin. [00204] Selective degradation of the elastotic material located in the papillary dermis was observed using the carbopol formulation. The lag time in elastase activity observed using this formulation in the in vitro activity screen suggests that selectivity may be tuned through manipulating enzyme kinetics or the composition of the formulation.

Example 5

Evaluation of elastase activity in presence of thickening agents

[00205] To ensure that elastase retains its elastolytic activity when incorporated into comositions with thickening agents, in vitro assays as well as in situ studies were performed. Sorbitol and glycerol wer chosen as thickening agents due to their known protein-stabilizing properties and their known elastase stabilizing properties. Carbopol Ultrez 21 was chosen for its thickening properties in high-electrolyte solutions.

[00206] The following formulations were prepared and tested:

[00207] Carbopol Formulation

0.23% Carbopol Ultrez 21

5.27% 30 mM Tris-HCl

94.5% 30 mM Tris base

1 U/mL Porcine Pancreas elastase (PPE)

Final pH 7.2

Refrigerate at 4 ⁰C to remove bubbles, which may interfere with PPE activity

[00208] Sorbitol Formulation

80% 30 mM Tris (pH 7.2)

20% Sorbitol

1 U/mL Porcine Pancreas elastase (PPE)

[00209] Glycerin Formulation

80% 30 mM Tris (pH 7.2)

20% Glycerin

1 U/mL Porcine Pancreas elastase (PPE)

[00210] Control Formulation (Tris)

100% 30 mM Tris (pH 7.2)

1 U/mL Porcine Pancreas elastase (PPE)

Methods

[00211] 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 ⁰C with light shaking. [00212] A portion (20 μL) 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 ⁰C 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.

Results

[00213] Using the in vitro screen, over an initial period of 3 hours, the glycerin formulation exhibited elastolytic activity that matched the control formulation. See Figure

11. The sorbitol formulation exhibited somewhat decreased activity, while the carbopol formulation exhibited the greatest decrease in elastolytic activity.

[00214] Over 19 hours, all four formulations reached the same level of elastin cleavage. It should be noted that the Tris-only control formulation showed some fluorescence. This may be due to some hydro lytic cleavage of the fluorescein from elastin. This effect was more pronounced at elevated temperatures.

[00215] Skin section treated with the Carbopol formulation (see Figure 9) showed that the formulation clears only the aberrant elastin fibers in the papillary dermis, while the deeper normal fibers still remain. The activity of elastase seems to have been slightly lowered compared to the control formulation. Note that these skin sections were exposed to the respective formulations for four hours. This timeframe corresponds to the initial lag time in elastase activity for the Carbopol formulation. Additional studies are needed to delineate the mechanism underlying the observed "selectivity" to account for the delay in elastase activity versus a Carbopol-induced chemical selectivity for the aberrant elastin in the papillary dermis.

[00216] Some skin sections treated with the sorbitol formulation (see Figure 10) showed selectivity for digesting aberrant elastin fibers, while others appear to be more digested in the reticular dermis. The activity of elastase seems to be between the control formulation and the

Carbopol formulation. These results are consistent with the in vitro assays where elastase activity was decreased relative to the control formulation.

[00217] The glyerin formulation was found to have effects very similar to that of the control formulation. Both formulations fully cleared all elastin with no evidence of selectivity for aberrant elastin. These results are also consistent with the in vitro assay data, where the glycerin formulation exhibits similar activity as the control formulation. [00218] These studies suggest that elastin degradationis tunable, potentially through the manipulation of dosage of enzyme and formulation.

Example 6 In vitro assay screen

[00219] 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.

Example 7

In situ activity screen

[00220] Full thickness photodamaged facial skin was frozen and cryotomed for the in situ enzyme activity screen.

[00221] The cryotomed sections were transferred to individual glass microscope slides. 20 μL 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 ⁰C 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). Fifteen minutes prior to the end of the duration time, the slide was removed from the micro-humidity chamber, and the skin section was allowed to dry to the slide. The sample is then rinsed with Tris buffer to stop elastase activity, immersed in formalin solution for 8-24 hours followed by immersion in 70% ethanol. Slides was then dried and processed for histological evaluation. [00222] Histological evaluation of treated skin sections shows a decrease in the intensity of the elastin stain with an increase in enzyme concentration or exposure duration for each elastase evaluated. Trypsin and alpha-chymotrypsin did not show an effect on reducing aberrant elastin. The effect of concentration and exposure duration can be visualized in Figure 19, wherein elastin is stained black in the images. Decreased staining is observed with increased exposure duration and concentration. Example 8

Ex vivo evaluation of enzyme activity

[00223] For the ex vivo evaluation of enzyme activity, live human skin was excised and maintained in RPMI medium. Cell viability was noted in the H&E slides of the treated samples.

[00224] Full thickness skin samples were exposed to porcine pancreatic elastase (PPE) using three different delivery routes that included injection, and topical application following skin pretreatment with either dermabrasion or tape stripping. At a dosage of 100 U/mL, decreases in the aberrant elastin content of photodamaged skin were observed when PPE was either injected or applied topically following dermabrasion. Marginal differences between the placebo and the active formulation were observed following tape stripping at his concentration. These findings demonstrate the feasilibity of aberrant elastin removal in live full thickness skin when treated with PPE.

[00225] 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.

Example 9

Ex vivo evaluation of enzyme activity with tape stripping

[00226] The surface of excised human photodamaged skin was blotted dry. The adhesive side of 3M transpore tape was applied to the skin surface and then removed. This process was repeated 20 times in the tape stripping procedure. The tape stripped skin surface was then exposed to 100 U/mL for 4 hours at 37 ⁰C. Using VVG staining for elastic fibers, a minor decrease in elastotic material was observed in the histology sections.

Claims

ClaimsWhat is claimed is:

1. A method of treating skin comprising the step of contacting skin with an effective amount of one or more elastases.

2. A method of treating skin 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.

3. A method of diminshing signs of premature aged skin comprising the step of contacting the premature aged skin with an effective amount of one or more elastases that individually or in combination improve the appearance of skin.

4. The method of claim 1, 2, or 3, wherein the effective amount is sufficient to reduce histopathologic evidence of elastotic material in a sample of heavily photodamaged skin by at least 1% within a time period of 4 hours or less.

5. The method of claim 1, 2, or 3, wherein the effective amount is sufficient to reduce histopathologic evidence of elastotic material in a sample of heavily photodamaged skin by at least 5% within a time period of 4 hours or less.

6. The method of claim 1, 2, or 3, wherein the effective amount is sufficient to reduce histopathologic evidence of elastotic material in a sample of heavily photodamaged skin by at least 10% within a time period of 4 hours or less.

7. The method of claim 1, 2, or 3, wherein the effective amount is sufficient to reduce histopathologic evidence of elastotic material in a sample of heavily photodamaged skin by at least 25% within a time period of 4 hours or less.

8. The method of claim 1, 2, or 3, wherein the effective amount is sufficient to reduce histopathologic evidence of elastotic material in a sample of heavily photodamaged skin by at least 50% within a time period of 4 hours or less.

9. The method of claim 1, 2, or 3, wherein the effective amount is sufficient to reduce histopathologic evidence of elastotic material in a sample of heavily photodamaged skin by at least 75% within a time period of 4 hours or less.

10. The method of claim 1, 2, or 3, wherein the effective amount is sufficient to reduce histopathologic evidence of elastotic material in a sample of heavily photodamaged skin by at least 90% within a time period of 4 hours or less.

11. The method of claim 1 , 2, or 3, wherein the elastase is a mammalian elastase.

12. The method of claim 1, 2, or 3, wherein the elastase is a bacterial elastase.

13. The method of claim 1, 2, or 3, wherein the elastase is a serine protease.

14. The method of claim 1, 2, or 3, wherein the elastase is not trypsin, chymotrypsin, papain, bromelain, or subtilisin.

15. The method of claim 1, 2, or 3, wherein the one or more elastases are individually or in combination capable of degrading non-photodamaged insoluble elastin.

16. The method of claim 1, 2, or 3, wherein the elastase is Pseudomonas elastase.

17. The method of claim 1, 2, or 3, wherein the elastase is porcine elastase.

18. The method of claim 1, 2, or 3, wherein the elastase is human elastase.

19. The method of claim 1, 2, or 3, wherein the one or more elastases are topically applied.

20. The method of claim 1, 2, or 3, wherein the one or more elastases are topically applied for between 15 minutes and 24 hours.

21. The method of claim 1 , 2, or 3 , wherein the one or more elastases are topically applied to an area of skin from which at least 50% by volume of the stratum corneum has been removed.

22. The method of claim 1 , 2, or 3, wherein the one or more elastases are topically applied to an area of skin from which at least 50% by depth of the stratum corneum has been substantially removed.

23. The method of claim 1, 2, or 3, wherein the one or more elastases are topically applied to an area of skin from which the stratum corneum has been substantially removed.

Ill

24. The method of claim 1, 2, or 3, wherein the one or more elastases are topically applied to an area of skin from which at least three layers of the epidermis have been substantially removed.

25. The method of claim 1, 2, or 3, wherein the one or more elastases do not significantly degrade elastotic material in the dermis when applied to skin having an intact stratum corneum.

26. The method of claim 1, 2, or 3, wherein the one or more elastases do not significantly degrade elastotic material in the dermis when applied to skin from which the stratum corneum has been removed to a depth less than 50% its full thickness.

27. The method of claim 1, 2, or 3, wherein the one or more elastases do not significantly degrade elastotic material in the dermis when applied to skin from which the stratum corneum has been removed to a depth less than 90% its full thickness.

28. The method of claim 1, 2, or 3, wherein the step of contacting comprises injecting the skin with an effective amount of one or more elastases.

29. The method of claim 1, 2, or 3, further comprising administering one or more compounds that have skin restoring properties to the subject's skin.

30. The method of claim 1, 2, or 3, comprising contacting the skin with a composition comprising one or more elastase enzymes.

31. The method of claim 30, comprising contacting the skin with a composition comprising at least two elastase enzymes.

32. The method of claim 30, wherein the composition further comprises one or more components 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, emulsifϊers, and fragrances.

33. The method of claim 1, 2, or 3, further comprising the step of: removing at least a portion of the epidermis of an area of skin prior to contacting the dermis with the composition.

34. The method of claim 33, wherein the step of removing comprises performing a procedure selected from the group consisting of: dermabrasion, microdermabrasion, laser treatment, or applying a chemical peeling agent to the surface of the skin.

35. The method of claim 33, wherein the step of removing comprises removing the stratum corneum to a depth of at least 50% of its thickness.

36. The method of claim 33, wherein the step of removing comprises removing the stratum corneum to a depth of at least 90% of its thickness.

37. The method of claim 33, wherein the step of removing comprises removing at least the upper three layers of the epidermis.

38. The method of claim 1, 2, or 3, further comprising the step of: contacting the skin with a skin care composition having skin restoring properties or performing a physical treatment having skin restoring properties.

39. The method of claim 1, 2, or 3, further comprising contacting the skin with an inhibitor of at least one of the elastase enzymes after the skin has been contacted with said enzyme for a suitable period of time.

40. The method of claim 1 or 2, further comprising the step of: contacting the skin with an inhibitor of one or more endogenous enzymes present in the skin.

41. A method for improving the appearance of photodamaged skin comprising the step of: contacting the dermis of photodamaged skin with an effective amount of one or more enzymes that individually or in combination diminish evidence of elastotic material, whereby the appearance of the photodamaged skin is improved.

42. A method of caring for photodamaged skin comprising the step of: contacting the dermis of photodamaged skin with an effective amount of one or more enzymes that individually or in combination diminish evidence of elastotic material.

43. A method of caring for skin comprising the step of: contacting the skin with an effective amount of one or more elastases that individually or in combination inhibit accumulation of elastotic material, whereby development of signs of photodamage or aging is inhibited.

44. A method of improving the appearance of a subject's skin comprising steps of: removing at least a portion of the epidermis; and contacting the skin with an effective amount of one or more enzymes that individually or in combination reduce evidence of elastotic material.

45. The method of claim 44, wherein the step of removing comprises applying a chemical peeling agent or performing microdermabrasion or dermabrasion.

46. The method of claim 44, wherein the skin is photodamaged skin.

47. The method of claim 44, wherein the subject is between 20 and 90 years of age.

48. 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 photodamaged skin; and a cosmetically acceptable carrier.

49. The cosmetic skin care composition of claim 48, wherein at least one elastase enzyme displays selective activity towards elastotic material.

50. The cosmetic skin care composition of claim 48, further comprising an ingredient 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.

51. 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.

52. The composition of claim 51 , wherein the bacterial elastase is PA elastase.

53. The skin care composition of claim 51 , further comprising an ingredient 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.

54. A pharmaceutical composition comprising: an effective amount of one or more elastase enzymes that diminish evidence of elastotic material following contact with skin; and a pharmaceutically acceptable carrier.

55. The pharmaceutical composition of claim 54, wherein at least one elastase enzyme displays selective activity towards elastotic material.

56. The pharmaceutical composition of claim 54, further comprising 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.

57. The pharmaceutical composition of claim 54, wherein the composition is in injectable form.

58. The pharmaceutical composition of claim 54, wherein the composition is suitable for transdermal administration.

59. A cosmetic composition comprising an effective amount of one or more elastase enzymes; and a cosmetically acceptable carrier.

60. A pharmaceutical compostion comprising an effective amount of one or more elastase enzymes; and a pharmaceutically acceptable carrier.

61. The composition of claim 48, 51, 54, 59, or 60, wherein the elastase enzyme is not trypsin, chymotrypsin, papain, bromelain, or subtilisin.

62. 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.

63. 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 degradative activity towards normal elastin that has not been subjected to photodamage; and

(c) selecting the enzyme as a preferred enzyme for a skin care composition if it displays enhanced selectivity towards elastotic material versus normal elastin relative to that of the naturally occurring elastolytic enzyme.