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Home , Acrocylindropepsin, Actinidain, Ananain, Asclepain, Aspergillopepsin II, Brachyurin

US 2003OO26794A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2003/0026794 A1 Fein (43) Pub. Date: Feb. 6, 2003

(54) SELECTIVE TREATMENT OF (52) U.S. Cl...... 424/94.2; 424/946; 424/94.63 CONDITIONS (76) Inventor: Howard Fein, Cincinnati, OH (US) (57) ABSTRACT Correspondence Address: A method of treating skin conditions by providing compo Beverly A. Lyman Sitions containing to Selectively remove Specific Wood, Herron & Evans, L.L.P. layers of skin. The depth of skin removed (that is, vertical 2700 Carew Tower Surface treated) is regulated by the type and concentration of 441 Wine Street enzyme or enzymes in the composition. The Surface area of Cincinnati, OH 45202-2917 (US) skin removed (that is, radial Surface treated) is regulated by the area of topical application. Conditions treatable by the (21) Appl. No.: 09/919,102 method include, but are not limited to, age-related condi (22) Filed: Jul. 31, 2001 tions Such as lines and wrinkles, , pigmentary disorders, follicular disorderS Such as acne, and hyperkera Publication Classification totic disorders such as warts. The inventive method and composition thus achieves the Specificity and efficacy of (51) Int. Cl." ...... A61K 38/54; A61K 38/46; more invasive methods Such as Surgery, while providing a A61K 38/48 composition that may be topically applied and is easy to use. Patent Application Publication Feb. 6, 2003. Sheet 1 of 3 US 2003/0026794 A1

FIG

FIG. 2 Patent Application Publication Feb. 6, 2003 Sheet 2 of 3 US 2003/0026794 A1

FIG. 4

FIG. 5 Patent Application Publication Feb. 6, 2003 Sheet 3 of 3 US 2003/0026794 A1

FIG. 6

FIG. 7 US 2003/0026794 A1 Feb. 6, 2003

SELECTIVE ENZYME TREATMENT OF SKIN treating various skin disorders, especially those involving CONDITIONS Some type of keratinization disorder. While most of these products are available over-the-counter, Some topical skin FIELD OF THE INVENTION care formulations that contain enzymes are available by 0001. The invention relates to the use of skin treatment prescription only. These include Accuzyme(E) () and compositions containing enzymes that Selectively target one GranuleXOR (), whose application is limited for debri or more layers of skin. dement of wounds. 0006 There are, however, other skin conditions whose BACKGROUND OF THE INVENTION treatment involves more than just removal of the outer layers of the . Such conditions may affect, and hence 0002 The skin, the largest organ of the human body, is of require removal of, either deeper epidermal layers, full interest from biological, medical and cosmetic points of thickness epidermis, or even dermal and/or Subcutaneous View. Many products exist which purport to target one or layers. Current treatment methods for these conditions usu more of these aspects of skin care. Confounding Such ally consist of methods that destroy the skin at the treatment treatment is the fact that conditions affecting the skin may be Site. These methods include cryotherapy (freezing with Specific to the Skin, Such as psoriasis and atopic dermatitis, liquid nitrogen), Surgical removal, electroSurgery (tissue or may be manifestations of a general disease, Such as destruction with electricity), laser Surgery (tissue destruction general allergic reactions. with light), burning, and chemical destruction with caustic 0003. There are a variety of over-the-counter and pre agents Such as trichloroacetic acid and phenol. Most of these Scription products that contain enzymes which are used for methods are non-Selective in terms of tissue destruction, and the purpose of skin care. Reported applications for these damage or destroy not only the affected Site, but other products include Softening skin, treating skin conditions healthy layers and Surfaces of Skin as well. These methods Such as dryneSS, wrinkles, and acne, and/or removing devi also incur pain, Scarring, pigmentary alterations, delayed talized or necrotic skin. In general, enzyme-containing prod wound healing, and lesion recurrence. ucts have been directed to conditions in which it is desirable 0007. Therefore, it would be desirable to combine the to remove the upper layers of the skin. In this process, beneficial features of the above known skin treatment com termed exfoliation, the skin that has been removed is even positions and methods, Such as the convenience and ease of tually replaced with newly generated Skin from Surrounding use of a topically applied Skin care product, with the and StructureS. applicability of a more invasive method, to achieve Selective 0004 One class of enzymes used in skin-related products destruction of affected layers of skin. Such a combination is proteolytic enzymes, also called or peptidases, has not, until now, been disclosed. which function to hydrolyze, or break down, . Pro SUMMARY OF THE INVENTION teins known as adhesion molecules are present in the extra cellular matrix of skin where they act to bind and anchor 0008. The invention is directed to a method for treating a keratinocytes (cells that are present in the Outermost layer of patient having a condition involving the epidermal, and/or skin). When proteolytic enzymes are applied to the Surface dermal, and/or Subcutaneous layer of skin using a compo of skin, they hydrolyze adhesion molecules, resulting desir Sition containing at least one enzyme that affects one or more ably in exfoliation of the upper skin layers. Generally, most particular layers of skin. In one embodiment, a physiologi of the topically applied enzyme-containing products contain cally acceptable formulation containing an effective amount proteolytic enzymes, and would thus be directed to appli of an enzyme that Selectively affects one or more Skin layers is administered topically to treat the condition. In another cations that involve treating the epidermis (the Outermost embodiment, the enzyme-containing formulation may be layer of skin). administered directly to an underlying skin layer. The 0005 There are several types of proteolytic enzymes. enzyme, naturally occurring or Synthetic, may belong to the Papain, a proteolytic enzyme that is isolated from unripe class of , , , , fruit, is effective in hydrolyzing esters and hydro , and/or . The enzymes may be produced phobic proteins, and has been used in various types of using recombinant techniques and may contain one or more topical formulations. Papain-containing formulations may modified amino acids while retaining a desired level of also contain other proteolytic enzymes Such as , activity. Also included in the invention are enzymes that which is isolated from . The formulations may have the desired therapeutic activity but that are not cur additionally contain denaturing chemicals. Such as rently classified in one of the above classifications. urea, and/or other chemicals. Such as alpha hydroxyacid and salicylic acid, both of which lower the pH of the skin and 0009. The method may be used to treat a wide variety of cause exfoliation. AS another example, proteolytic enzymes conditions which include, but are not limited to, neoplasms, obtained from the bacterium MicrococcuS Sedentarius have pigmentary disorders, infectious disorders, follicular disor been shown to degrade calluses in vitro. AS Still another ders, hyperkeratotic disorders, inflammatory disorders, Vas example, U.S. Pat. No. 5,981,256 discloses the use of a cular disorders, aging disorders including photo-aging dis recombinant Stratum corneum chymotryptic enzyme orders, deposition disorders, connective tissue disorders, (SCCE) for pharmaceutical and cosmetic skin care. SCCE, cutaneous cystic disorders, dandruff, Seborrheic dermatitis, a proteolytic enzyme, is responsible for the degrada dry skin, corns, calluses, warts, freckles, acne, wrinkles, tion of desmosomal proteins, which play a role in the cysts, eczema, insect bites, lupus, Varicose veins, tattoos, intercellular cohesion in the epidermis. SCCE activity and/or Scars. results in cell shedding from the surface of the cornified 0010. The inventive composition contains at least one surface layer. Hence, recombinant SCCE could be used for enzyme in a pharmaceutically acceptable formulation and an US 2003/0026794 A1 Feb. 6, 2003 amount effective to remove the affected Skin layer or layers. 0018. In another embodiment, a method for treating a The amount of enzyme can range from about 1x10% to condition affecting skin is disclosed by applying a compo about 80%” of the formulation, more particularly from Sition to the affected skin, where the composition contains at about 1x10%YY to about 10% of the formulation. least one enzyme at a concentration Selective for regulating 0.011 The invention is additionally directed to a compo the depth of skin treatment, and is applied to an area of skin Sition in a pharmaceutically acceptable formulation for Selective for regulating a radial Surface of Skin treatment. Selectively treating skin, and that contains one or more 0019. These and other embodiments will be further hydrolases in an amount ranging from about 1x10% by appreciated from the following detailed description and weight to about 80% by weight of the formulation, more examples. particularly from about 1x10% to about 10% of the formulation. The may be one or more of an BRIEF DESCRIPTION OF THE DRAWINGS esterase, a glycosidase, a peptidase Such as , trypsin, papain, bromelain, , etc., a phosphatase, a 0020 FIG. 1 is a photograph of skin showing a sebor thiolase, a phospholipase, an amidase, a deaminase, and/or rheic keratosis lesion. a ribonuclease. 0021 FIG. 2 is a photograph of the skin lesion of FIG. 0012. Thus, in one embodiment, a method for treating a 1 immediately after treatment with one embodiment of the patient having a condition affecting at least one layer of skin inventive composition. is disclosed. A physiologically acceptable formulation con 0022 FIG. 3 is a photograph of the skin lesion of FIG. taining at least one enzyme Selective for a layer of skin 1 three weeks post treatment. affected by the condition is applied in Situ in an amount and for a duration effective to remove the skin layer or layers and 0023 FIG. 4 is a photograph of the skin lesion of FIG. treat the condition. 1 Six months post treatment. 0013 In another embodiment, a composition contains at 0024 FIG. 5 is a photograph of a histologic section of least one , Such as trypsin, , papain, skin with a Seborrheic keratosis lesion. bromelain, dispase, , and/or V8 protease, at a 0025 FIG. 6 is a photograph of the histologic section of concentration in the range of about 1x10%YY to about skin shown in FIG. 5 two hours after treatment with one 10% in a pharmaceutically acceptable formulation to embodiment of the inventive composition. Selectively effect an epidermal layer of skin. The composi tion may also contain other components, such as exfoliants, 0026 FIG. 7 is a photograph of the histologic section of drugs, and/or cytotoxic agents. skin shown in FIG. 5 twenty four hours after treatment. 0.014. In another embodiment, a composition for treating a patient having a skin condition affecting at least one DETAILED DESCRIPTION OF THE epidermal, and/or dermal layer, and/or Subcutaneous layer INVENTION contains a hydrolase and is topically applied. The hydrolase 0027. The invention is directed to compositions and is present in an amount in the range of about 1x10% by methods to Selectively treat, alleviate, or prevent conditions weight to about 10% by weight. in mammals that affect one or more layers of skin. For 0.015. In other embodiments, methods are disclosed to example, the compositions are chosen and formulated to treat Skin by topically applying to an outermost layer of skin Selectively remove part or all of an epidermal layer of skin, a composition containing a protease in a biologically accept and/or part or all of a dermal layer of skin. The compositions able formulation in an amount and formulation to Selectively contain one or more enzymes from the , remove at least one epidermal layer containing a skin , , , , and hydrolase classes condition; by providing a composition containing a protease of enzymes to remove affected Skin layers. In one embodi in a biologically acceptable formulation in an amount and ment, the composition and formulation Selectively targets formulation to Selectively remove at least one dermal layer one or more epidermal layers. Alternatively, other areas of containing a skin condition; and by providing a composition the Skin, Such as the deeper dermal and/or Subcutaneous containing a lipid hydrolyzing enzyme in a biologically layers, may also be treated by increasing the concentration of an enzyme in the formulation, or by adding other enzymes acceptable formulation in an amount and formulation to or agents that will promote and/or demonstrate efficacy in Selectively remove at least one Subcutaneous layer contain deeper layers of the skin. For treatment of at least an ing a skin condition. epidermal layer, the composition may be applied topically 0016. In another embodiment, a method to target skin and/or may be injected. For Selective treatment of a dermal treatment of an affected area is disclosed. A composition and/or Subcutaneous layer, the composition may be admin containing at least one enzyme in an amount and formula istered directly to the desired layer, for example, by injec tion effective to Selectively target Skin removal is provided tion. In any route of administration, the composition and to the affected area. method avoids conventional destructive techniqueS Such as 0.017. In another embodiment, a method of treating signs cryotherapy or Surgery, while providing comparable efficacy of aging in Skin, Such as Xerosis, rhytids, loSS of Skin tone, in treating numerous types of skin conditions. acitinic damage, fine lines, and dySpigmentation, is dis 0028. In one embodiment, the enzyme is a protease or a closed. A protease-containing biologically acceptable com mixture of proteases in a Suitable formulation that is admin position is provided to an outermost layer of affected skin in istered to the affected skin. In this embodiment, the inventive an amount and formulation to Selectively target the affected method involves one or more administrations of protease skin layers. containing formulations for alleviation, treatment, and/or US 2003/0026794 A1 Feb. 6, 2003 prevention of skin conditions. Depending upon the layer or ichthyosis Vulgaris, lamellar ichthyosis, lichen Simplex layers of skin to be treated, different proteases are used in the chronicus, palmoplantar keratoderma, Xerosis (dry skin), composition. For example, if only the epidermis is to be X-linked ichthyosis and papillomatosis. This group also treated (that is, one or more epidermal layers), the compo includes corns and calluses, both of which show thickening Sition may contain trypsin and/or papain. If only the dermis of the Skin as a result of friction or pressure. is to be treated, the composition may contain collagenase and/or elastase and may be administered directly into the 0035 A sixth group is inflammatory disorders. These dermis. If epidermal and dermal layers are to be treated, the include, but are not limited to, atopic dermatitis, dermatitis, composition may be applied topically and may contain eczema, insect bites, lichen planus, lupus erythematosus, trypsin and collagenase; trypsin and elastase; papain and lymphomatoid papulosis, pityriasis lichenoides, psoriasis, collagenase, papain and elastase, trypsin, papain, and col Sarcoid, Scleroderma, Seborrheic dermatitis, acne Vulgaris lagenase, trypsin, papain, and elastase, or trypsin, papain, and dandruff. collagenase, and elastase. 0036) A seventh group is vascular disorders. These 0029) Skin conditions to be treated can be categorized include, but are not limited to, cherry angioma, heman into Several groups, as the nature of each condition can be gioma, pigmented purpuric dermatoses, Stasis dermatitis, quite variable. One group of conditions is cosmetically telangectasia, Varicose veins, vascular malformations and undesirable, but is non-pathological, Such as natural or vascular neoplasms. artificial altered skin pigmentation. Another group of con 0037. An eighth group is photo-aging disorders. These ditions is pathological but is readily treated or controlled, include, but are not limited to, actinic damage, photo-aging, Such as Skin infections. Still another group of conditions is photo-damage, poikiloderma, rhytid (wrinkles) and Solar pathological and more invasive, Such as neoplasms affecting elastosis. the skin. Within this last group are neoplasms having various degrees of aggressiveness, ranging from the relatively non 0038 A ninth group is deposition disorders. These aggressive basal cell carcinoma, to the aggressive malignant include, but are not limited to, amyloidosis, cutaneous melanoma. The types and examples of each group are as mucinosis, , myxedema, tattoos and Xanthomas. follows. 0039. A tenth group is connective tissue disorders. These 0.030. One group is neoplastic disorders. These include, include, but are not limited to, connective tissue nevus, but are not limited to, actinic keratosis, apocrine gland dermal atrophy, fibrous papule, hypertrophic Scars, keloids, neoplasm, Bowenoid papulosis, Bowen's disease, basal cell lichen Sclerosis, morphea, rhinophyma, cicatrix (Scars) and carcinoma, dermatofibroma, dermatosis papulosa nigrans, Striae. eccrine gland neoplasm, fibroepithelial polyp (skin tag), 0040. An eleventh group is cutaneous cystic disorders. keratoacanthoma, Kaposi's Sarcoma, lentigo maligna, len These include, but are not limited to, epidermal inclusion tigo maligna melanoma, melanoma, melanocytic nevus, cyst and milium. neurofibroma, Sebaceous gland neoplasm, Sebaceous gland hyperplasia, Seborrheic keratosis, Squamous cell carcinoma, 0041 Advantageously, the inventive method can also Squamous cell carcinoma in situ, Stucco keratoses, Syrin encompass use of the enzyme composition as an exfoliant, goma, and tricholemmoma. that is, for use as a cosmetic peel. Such a cosmetic peel would be used to reduce the appearance of wrinkles, 0.031) A second group is pigmentary disorders. These improve skin tone and texture, and/or reduce signs of skin include, but are not limited to, café au lait macule, chloasma, aging. The method also encompasses use of the enzyme ephilide (freckle), lentigo (age spot), melasma, Mongolian composition as a depilatory agent to remove exceSS hair or Spot, nevus of Ito, nevus of Ota, post-inflammatory hyper unwanted hair. pigmentation, and post-inflammatory hypopigmentation. 0042 Enzymes are grouped into various classes, depend 0032. A third group is infectious disorders. Infectious ing on the types of reactions catalyzed and their mechanism disorders encompass those caused by any infectious agent of action. Oxidoreductases catalyze oxidation/reduction and include bacterial, parasitic, fungal, Mycobacteria, and reactions. Examples of oxidoreductases include dehydroge Viral pathogens. Infectious disorders include, but are not nases, oxidases, reductases, peroxidases, catalases, oxyge limited to, condyloma accuminatum (genital wart), dermato nases and hydroxylases. Transferases transfer functional phytosis, Verruca plana (flat wart), Verruca Vulgaris (com groups between donors and acceptors. Examples of trans mon wart), molluscum contagiosum, onychomycosis, ferases include transaldolase and transketolase; acyl, pediculosis capitis, pediculosis pubis, tinea, Scabies, and methyl, glucosyl, and phosphoryltransferases, kinases, and tinea Versicolor. phosphomutases. Hydrolases are a special class of trans 0033. A fourth group is follicular disorders. These ferases in which the donor group is transferred to water. include, but are not limited to, acne Vulgaris, acne keloidalis Examples of hydrolases include esterases, glycosidases, nuchae, comedone, folliculitis, hair follicle neoplasms, kera proteases, phosphatases, thiolases, lipases, phospholipases, tosis pilaris, pseudo-folliculitis barbae, and rosacea. amidases, deaminases, ceramidases, and nucleases. Lyases add or remove water, ammonia, and/or carbon dioxide. 0034. A fifth group is hyperkeratotic disorders. Hyperk Examples of lyases include decarboxylases, aldolases, eratosis is a pathologic disease State characterized by reten hydratases, dehydratases, Synthases, and lyases. Isomerases tion of keratinocytes, normal descquamation does not occur. catalyze isomerization reactions. Examples of isomerases These include, but are not limited to, acquired ichthyosis, include racemases, epimerases, isomerases, and Some acanthosis nigricans, epidermal nevus, hyperkeratotic der mutases. Ligases are involved in Synthetic reactions. matitis of palms and Soles, ichthyosis, prurigo nodularis, Examples of ligases include Synthetases and carboxylases. US 2003/0026794 A1 Feb. 6, 2003

0.043 AS used herein, the term enzyme encompasses both and maintenance of the Structural integrity of the epidermis. naturally occurring and Synthetic enzymes. For example, an Congenital absence of cellular adhesion proteins, Such as in enzyme may be Synthesized using recombinant techniques, the disease epidermolysis bullosa, results in extreme epider as known to one skilled in the art. The term enzyme also mal fragility, which manifests clinically as blisters and encompasses enzymes that contain one or more modified erosions. Similarly, autoantibodies directed against these amino acids, while Still retaining desirable activity. Enzymes cell Surface adhesion proteins, Such as in bullous pemphig may also be modified to alter other properties Such as, but oid and pemphigus Vulgaris, will also result in epidermal not limited to, Stability, target Site affinity, Substrate Speci blisters and erosions. Proteases that hydrolyze cellular adhe ficity, allosteric control, and required co-factors. In various Sion proteins have the potential to degrade both cell-Surface embodiments, the enzyme may be isolated or may be added proteins and those in the extracellular matrix. Without these in a less than pure form, as long as its desired property is inter- and extracellular attachments, the epidermis disinte retained. grates in a process known as acantholysis. Thus, treating 0044 AS previously described, hydrolases are used in skin with proteaseS results in removal of one or more layers one embodiment of the invention. Hydrolases are designated of skin. Moreover, cell adhesion proteins are located in the as Class 3 hydrolases according to the guidelines of the epidermis, which advantageously makes them readily acces Nomenclature Committee of the International Union of Sible to topical enzymatic therapy. Biochemistry and , and can be subdivided 0046. In another embodiment of the invention, the com into four groups depending upon the type of bond which is position contains one or more esterases, glycosidases, and/or hydrolyzed by the enzymes. These groups are (1) Class 3.1, nucleases. Esterases hydrolyze the various lipids that form which act on ester bonds (esterases), examples of which cellular membranes of skin cells and the various lipids that include the lipid hydrolyzing enzymes Such as lipases, form the epidermal intercellular lamellar lipid structures. phospholipases (phospholipase A, phospholipase A, phos Glycosidases hydrolyze the glycosidic bonds of skin pro pholipase C, phospholipase D), Sphingomylenases, choles teins and lipids that have been naturally modified by gly terol ester hydrolases, and ceramidases; (2) Class 3.2, which cosylation. Nucleases hydrolyze functional and non-func act on glycosidic bonds (glycosidases); (3) Class 3.3, which tional nuclear material of viable and non-viable skin cells. act on ether bonds, and (4) Class 3.4, which act on These hydrolases and nucleosidases are used alone or in bonds (proteases, also called peptidases), examples of which combinations as needed to Selectively treat affected Skin include trypsin, chymotrypsin, papain, collagenase, elastase, layers. exopeptidases (carboxypeptidase A, carboxypeptidase B), aminopeptidases, , bromelain, chymotrypsin 0047 The concentration of the enzyme, or mixture of C, , trypsin, , , , enzymes, in the composition is in the range from about alpha-lytic , prolyl oligopeptidase, 1x10%YY to about 80%Y, more specifically from about , plasma , tissue kallikrein, pancreatic 1x10%YY to about 9%YY. In one embodiment, the con elastase, leukocyte elastase, , cerevisin, hypoder centration of the enzyme, or mixture of enzymes, is in the min C, endopeptidase La, alpha-, leucyl endopeptidase, range from about 1x10%YY to about 10%Y, more spe , , , , endopeptidase K, thermo cifically from about 0.1%YY to about 3.0%YY. If the enzyme mycolin, , endopeptidase So, tissue plasminogen or enzymes is intended for topical administration to treat a activator, pancreatic endopeptidase E, II, condition with a single application, a concentration in the urine , B, , chymopa higher range would be useful. Such a formulation may pain, , , , , cathep contain an enzyme, or a mixture of enzymes, in the range Sin L, , , cathepsin T, glycyl endopepti from about 1%YY to about 5%YY. Alternatively, if the dase, , , , , fruit enzyme, or mixture of enzymes, is intended to achieve bromelain, legumain, histoly Sain, A, , gas gradual tissue destruction, a concentration in the lower range tricsin, , , retropepsin, aspergillopepsin would be useful, such as from about 1x10°%” to about I, aspergillopepsin II, , , 1.0%YY. Arbitrary concentration classifications for the , , , Saccharopep enzyme, or mixture of enzymes, in the composition are from Sin, rhodotorulapepsin, phySaropepsin, , about 1x10% to about 1x10%YY and classified as a , pycnoporopepsin, Scytalidopepsin A, low strength composition, from about 1x10°%” to about Scytalidopepsin B, Xanthomonoapepsin, pseudomonapepsin, 1%” and classified as an intermediate strength composi , atrolysin, microbial collagenase, leucolysin, tion, and from about 1%YY to about 10%Y, classified as a interstitial collagenase, , matrix metalloprotein high Strength composition. ases, dispase, thermolysin, and V8 protease. Although not included in the above classification, nucleases, Such as 0048. A composition containing a lower concentration of deoxyribonuclease and ribonuclease, nucleosidases, and the enzyme, or mixture of enzymes, would likely be avail nucleoside phosphorylases are also encompassed by the able over the counter for self-administration by the patient. invention. A composition containing a higher concentration of the enzyme, or mixture of enzymes, would likely be available 0.045. In one embodiment of the invention, a composition only by prescription, or would be applied to the patient by contains one or more proteases. One function of proteases is a physician or other health care professional, rather than to hydrolyze cellular adhesion proteins, which are found on either the Surface of keratinocytes, the predominant cell type being Self-administered. in the epidermis, or within the Surrounding extracellular 0049. The formulation may be administered at various matrix. Examples of cellular adhesion proteins include des intervals, depending upon factorS Such as the type of con mogleins, lamins, integrins, bullous pemphigoid antigen 1 dition, the Severity of the condition, the outcome desired, the and 2, and others. They are responsible for adhesion of cells concentration of the enzyme or mixture of enzymes, etc. The US 2003/0026794 A1 Feb. 6, 2003

method may include application once a day, twice a day, any of the formulations that contain the enzyme. For once every other day, etc., as described in U.S. Pat. No. example, gels or liquids may be useful in Some instances in 5,981,256 which is expressly incorporated by reference which rapid penetration is desired, Such as when treatment herein in its entirety. occurs at certain intervals or in treatment of pediatric popu 0050. In one embodiment, the inventive method relates to lations. A moisturizing cream base may be useful in other use of an enzyme-containing composition Suitable for topi applications, Such as in the treatment of Xerosis (dry skin) cal application for the treatment of skin disorders or condi and/or in the treatment of geriatric populations. One or more tions affecting an epidermal layer of skin. This composition exfoliants, Such as common chemical exfoliants including may include additional enzymes. A composition containing Salicylic acid, lactic acid, alpha-hydroxy acids, beta-hydroxy the protease papain could be present in a relatively low acids, urea, and/or proteases, may also be added to the concentration and/or be formulated to affect only epidermal inventive composition. Other co-additives include divalent layers of skin, or could be present in a relatively higher cation chelatorS Such as ethylenediaminetetraacetic acid concentration and/or formulated to affect Some or all epi (EDTA) and ethylene glycol-bis (B-aminoethyl ether)-N,N, dermal layers, or even dermal layers, of skin. N',N'-tetraacetic acid (EGTA), both of which sequester 0051. In alternative embodiments, the composition con extracellular calcium, a necessary for the function tains an enzyme Such as collagenase that affects a dermal of cellular adhesion molecules and hence which may be layer of skin, or an enzyme Such as lipase that affects a considered as exfoliants, Since removal of calcium facilitates Subcutaneous layer of skin. These compositions may also the activity of a protease in hydrolyzing the epidermal contain additional enzymes, Such as . proteins. Other coadditives include exfoliants Such as Sali cylic acid, lactic acid, alpha-hydroxy acids, beta-hydroxy 0.052 In another embodiment, the inventive method relates to the use of a composition Suitable for topical acids, and/or urea, immunomodulating drugs. Such as glu application as a cosmetic facial peel and that contains an cocorticoids, tacrolimus, cycloSporin, interferon, ascomycin enzyme or a mixture of enzymes for the treatment of skin (SDZ ASM 981), imiquimod, or any of their respective conditions associated with aging, including photoaging. derivatives and combinations thereof, cytotoxic agents Such Such conditions include rhytides, loSS of Skin tone, fine as podophyllin, podophylox, and/or cantharadin, and caustic lines, and dySpigmentation. Skin aging disorders may also agents. be accelerated or complicated by photoaging disorders 0055 Conventional skin destructive methods, such as related to actinic damage, photo-damage, Solar elastosis and topical application of liquid nitrogen and trichloroacetic poikiloderma. These conditions may involve layers of the acid, destroy tissue indiscriminately. The depth of tissue skin ranging from only the few outer layers of the epidermis, damage caused by these agents is a function of exposure to all epidermal layers, to epidermal and dermal layers. time and/or concentration of the active agent. Conceivably, Hence, a variety of enzymes may be incorporated in the these conventional agents, if used in a Sufficient concentra formulation to treat these conditions. The enzymes may tion and for a Sufficient exposure duration, would destroy not include proteases, Such as trypsin and papain, and cerami only full-thickness Skin, but could produce damage extend dases, either alone or in combination, for treatment of ing into the deeper Soft tissue and even bone. In contrast to mainly the Outer epidermal layers of the skin. These conventional destructive agents, the inventive enzyme-con enzymes may also include phospholipases, lipases and col taining compositions have the ability to produce Selective lagenases to include treatment of the deeper epidermal tissue destruction limited to one or more layers of the skin, layerS and the dermis, depending upon the Severity of the Such as epidermis. For example, when the composition condition. contains a protease and is topically applied, the enzyme may 0.053 Any type of suitable, physiologically acceptable Spread along the skin Surface where it can interact with and enzyme formulation may be used, as is known to one of Skill destroy the epidermis only, or the epidermis and a portion of in the art. Examples of Such formulations include, but are not the dermis, or full-thickness skin. The skin layers affected limited to, creams, ointments, lotions, emulsions, foams, can be changed by altering the particular enzyme(s) used. aerosols, liniments, gels, Solutions, Suspensions, pastes, The depth of Skin affected can be changed by altering the Sticks, sprays, or Soaps. Additionally, the inventive compo enzyme formulation, enzyme concentration, and/or expo Sition may be formulated So that it is encapsulated within a Sure duration. bead, Sphere, capsule, microbead, microSphere, microcap Sule, liposome, etc., as is known to one skilled in the art. 0056. In the method, the composition may be applied at Such formulations may advantageously release the compo or adjacent to the affected Site or Sites. To limit the exposure to affected Skin and to protect unaffected Skin, or skin in Sition over a period of time (time release formulations). The which treatment is not desired, the composition may be encapulated formulation may also be prepared as a concen formulated in a Viscous material to form an ointment or other trate or in a dry State or in a powder-like consistency to formulation in which inadvertent spread is prevented. Skin increase the shelf life of the enzyme preparation. Such may also be protected from the composition through the use formulations are diluted or reconstituted prior to adminis of physical barrierS Such as plastic wrap, petrolatum, petro tration and can be prepared using methods known to one leum jelly, etc. The composition may be formulated in a skilled in the art. foam or gel, or within a device which could be cut precisely 0.054 The composition containing an enzyme or mixture to the shape of the lesion. Alternatively, the composition of enzymes may also contain other compounds that have may be applied at or adjacent to Sites not yet affected, but desirable therapeutic, cosmetic, and/or aesthetic properties, Sought to be treated for preventative or other reasons. The that either do not affect or only minimally affect the activity application may be performed in any manner that is Suitable of the enzyme. These So-called co-additives may be used in to the individual and/or the type of composition, and may US 2003/0026794 A1 Feb. 6, 2003

additionally involve an application device. The composition and 2.5% bromelain is formulated as a Solution in phosphate may be applied directly or indirectly, Such as by a dressing, buffered saline, pH 7.4 using methods known to one skilled bandage, covering, etc. in the art. The Solution is applied topically to the Site of the 0057 The duration and timing of treatment intervals and lesion, using a cotton-tipped applicator. The composition is enzyme concentration in the composition can vary. Variables reapplied, at intervals of three minutes, five additional times. include the extent and type of lesion, the physical properties of the lesion, how long it takes for the lesion to be no longer EXAMPLE 3 Visible, physician and patient preference, patient compli 0064. The patient is diagnosed as having a lentigo, a ance, etc. AS one example, a thick Scaly lesion Such as a pigmentary disorder, on the right cheek. An enzyme com verruca (wart) may require prolonged or repeated treatment position is formulated and applied to the lesion as described relative to a flat non-Scaly lesion. AS another example, a in Example 1. physician administering the inventive composition may pre fer a single treatment of a more concentrated dose than EXAMPLE 4 multiple treatments of less concentrated doses. 0065. The patient is diagnosed as having verruca plana, 0.058. The treatment regimen may also vary, and the an infectious disorder, on the Sole of the left foot. An enzyme treatment time may be extended or shortened by varying the composition is formulated and applied to the lesion as enzyme concentration and the formulation of the composi tion (e.g., a single application in contact with the lesion for described in Example 2. fifteen minutes, or three applications in contact with the lesion for five minutes each). The treatment method may EXAMPLE 5 require Short time intervals where the lesion, Such as icthyo 0066. The patient is diagnosed as having acne keloidalis sis, actinic keratosis, and the like, is located primarily in the nuchae, a follicular disorder, on the neck. A composition is epidermis, the Outermost layer of skin. Longer times, and formulated as a cream of 0.5% trypsin and 0.2% papain higher enzyme concentrations, are necessary to treat lesions using methods known to one skilled in the art. The compo Such as cysts, connective tissue disorders and the like, which Sition is applied topically to the lesion by spreading the are located in the deeper layers of the skin and thus require cream in a thin layer. The composition is applied once every the enzyme to penetrate deeper through a greater Volume of 24 hours until the condition disappears. tissue. 0059) Depending upon the above parameters, healing of EXAMPLE 6 treated Skin may take up to Several weeks. After healing, 0067. The patient is diagnosed as having atopic derma treatment efficacy may be determined by Visual inspection of titis on the left wrist and right ankle. A composition is the Site by the physician and/or patient. One criterion of prepared as described in Example 5. The composition is efficacy would be lack of visibility of the condition. For applied to the lesions by Spreading the cream in a thin layer. more intensive treatment, a biopsy or repeat biopsy made be performed, for example, to histologically verify destruction of a malignancy. EXAMPLE 7 0068 The patient is diagnosed as having pigmented 0060. The following examples are directed to different purpuric dermatosis, a vascular disorder, on the lower embodiments of the invention and are illustrative, rather extremities. A composition is formulated as an ointment of than limiting. 0.2% trypsin and 0.1% papain using methods known to one skilled in the art. The composition is applied topically to the EXAMPLE 1. lesion as a thin film. The composition is reapplied every 0061 A patient was diagnosed as having a seborrheic twelve hours. keratosis, a benign neoplasm, on the left lower extremity as shown in FIG. 1. A composition of 2.5% trypsin, was EXAMPLE 8 formulated as a solution in phosphate buffered saline, pH 7.4 using methods known to one skilled in the art. The Solution 0069. The patient is diagnosed as having actinic damage, was applied topically to the Site of the lesion, using a a photoaging disorder, to the entire facial region. A compo cotton-tipped applicator. The Solution was reapplied, at Sition is formulated and applied to the entire face as intervals of three minutes, five additional times. described in Example 5. 0.062 Immediately following the above-described treat EXAMPLE 9 ment protocol, an erosion was present at the treatment Site as shown in FIG. 2. The treated area was cleansed with soap 0070 The patient has a tattoo, a deposition within the and water and dressing was applied. At three weeks post skin, on the upper exterior portion of the right arm. A treatment, the lesion was completely eliminated without composition is formulated and applied as described in evidence of scarring or residual tissue as shown in FIG. 3. Example 1. At Six months post-treatment, there was no evidence of recurrence of the seborrheic keratosis as shown in FIG. 4. EXAMPLE 10

EXAMPLE 2 0071. The patient has hypertrophic scars, a connective tissue disorder, on the outside portion of the left thigh. A 0.063. The patient is diagnosed as having an actinic composition is formulated and applied as described in keratosis behind the right ear. A composition of 2.5% trypsin Example 1. US 2003/0026794 A1 Feb. 6, 2003

EXAMPLE 11 2. The method of claim 1 wherein said layer of skin is Selected from the group consisting of an epidermal layer, a 0.072 The patient is diagnosed as having an epidermal dermal layer, a Subcutaneous layer, and combinations inclusion cyst, a cutaneous cystic disorder, on the left cheek. thereof. A composition is formulated and applied as described in 3. The method of claim 1 wherein said enzyme is a Example 1. hydrolase. EXAMPLE 12 4. The method of claim 3 wherein said hydrolase is Selected from the group consisting of esterases, glycosi 0073. The patient is diagnosed as having facial wrinkles dases, proteases, phosphatases, thiolases, phospholipases, and loss of skin tone as a result of aging. A composition is amidases, ceramidases, lipases, deaminases, nucleases and formulated and applied as described in Example 6. combinations thereof. 5. The method of claim 1 wherein said enzyme is selected EXAMPLE 13 from the group consisting of trypsin, chymotrypsin, papain, 0.074 The patient is diagnosed as having ichthyosis Vul bromelain, dispase, thermolysin, ceramidase, lipase, gly garis, a hyperkeratotic disorder, on the lower extremities. A cosidase, deoxyribonuclease, ribonuclease and combina composition is formulated as a cream of 0.001% trypsin tions thereof to treat an epidermal layer. using methods known to one skilled in the art. The compo 6. The method of claim 1 wherein said enzyme is selected Sition is applied topically to the lesions by spreading the from the group consisting of collagenase, elastase, and cream in a thin layer. The composition is applied twice every combinations thereof to treat a dermal layer. 24 hours until the condition resolves. 7. The method of claim 1 wherein said enzyme is a lipase to treat a Subcutaneous layer. EXAMPLE 1.4 8. The method of claim 1 where the condition is selected 0075 To demonstrate the histological result of treatment from the group consisting of neoplasms, pigmentary disor by the invention, Skin with Seborrheic keratosis treated as ders, infectious disorders, follicular disorders, hyperkera described in FIG. 1 was removed from the patient by shave totic disorders, inflammatory disorders, vascular disorders, exision. A thin Section of the skin was prepared and pro photo-aging disorders, deposition disorders, connective tis cessed using Standard methods for histologic examination. Sue disorders, and cutaneous cystic disorders. 9. The method of claim 1 wherein the enzyme is selected As shown in FIG. 5 (100x magnification) the seborrheic from the group consisting of trypsin, chymotrypsin, papain, keratosis is intact and there is no other significant . collagenase, , elastase, eXopeptidases, aminopepti 0.076 The remaining shaved skin section was placed in a dases, endopeptidases, bromelain, , metri petri dish. A saline solution containing 2.5%” was applied din, trypsin, thrombin, plasmin, enteropeptidase, alpha-lytic to the surface of the lesion and incubated at 37 C. After two endopeptidase, prolyl oligopeptidase, brachyurin, plasma hours, another Section of the skin was processed for histo kallikrein, tissue kallikrein, pancreatic elastase, leukocyte logic examination. As shown in FIG. 6 (100x magnification) elastase, chymase, cerevisin, hypodermin C, endopeptidase there is near complete Sub-epidermal detachment and La, alpha-renin, leucyl endopeptidase, tryptase, kexin, Sub marked epidermal acantholysis. tilisin, oryzin, endopeptidase K, thermomycolin, thermitase, 0077. After 24 hours, incubation at 37° C. a section of the endopeptidase So, tissue plasminogen activator, pancreatic skin was processed for histologic examination. AS shown in endopeptidase E, pancreatic elastase II, urine plasminogen FIG. 7 (100x magnification) the epidermis was completely activator, , facain, , asclepain, clos destroyed with only the Stratum corneum remaining. tripain, Streptopain, actinidain, , cathepsin H, calpain, cathepsin T, , cathepsin S, 0078. It should be understood that the embodiments of caricain, ananain, Stem bromelain, , legu the present invention shown and described in the Specifica main, histolysain, pepsin B, gastricSin, chymosin, cathepsin tion are only preferred embodiments of the inventor who is D, retropepsin, , aspergillopepsin II, peni skilled in the art and are not limiting in any way. For cillopepsin, rhizopuspepsin, endothiapepsin, mucorpepsin, example, the inventive composition and method may have candidapepsin, , rhodotorulapepsin, physa Veterinary applications for treatment of skin conditions in ropepsin, acrocylindropepsin, polyporopepsin, pycnopo non-human animals. The dose, formulations, and other vari ropepsin, Scytalidopepsin A, Scytalidopepsin B, Xanth ables would be altered depending upon the particular Species omonoapepsin, pseudomonapepsin, cathepsin E, atrolysin, to be treated, as would be known by one skilled in the art. microbial collagenase, leucolysin, interstitial collagenase, Therefore, various changes, modifications or alterations to neprilysin, matrix metalloproteinases dispase, thermolysin, these embodiments may be made or resorted to without V8 protease, ribonuclease, deoxyribonuclease. and combi departing from the Spirit of the invention and the Scope of nations thereof. the following claims. 10. The method of claim 1 wherein said administering is Selected from the group consisting of topical application, , and combinations thereof. What is claimed is: 11. A composition comprising at least one protease 1. A method for treating a patient having a condition Selected from the group consisting of trypsin, chymotrypsin, affecting at least one layer of Skin comprising administering papain, bromelain, dispase, thermolysin, V8 protease, and in Situ a physiologically acceptable formulation containing combinations thereof at a concentration in the range of about at least one enzyme Selective for a layer of Skin affected by 1x10% to about 10% in a pharmaceutically accept Said condition in an amount and for a duration effective to able formulation to selectively effect an epidermal layer of remove Said layer and treat Said condition. skin. US 2003/0026794 A1 Feb. 6, 2003

12. The composition of claim 11 wherein Said protease is Sin II, penicillopepsin, rhizopuspepsin, endothiapepsin, Selected from the group consisting of naturally occurring mucorpepsin, candidapepsin, Saccharopepsin, rhodotorula proteases, Synthetic proteases, and combinations thereof. pepsin, physaropepsin, acrocylindropepsin, polyporopepsin, 13. The composition of claim 11 wherein the concentra pycnoporopepsin, Scytalidopepsin A, Scytalidopepsin B, tion of protease is in the range of about 1x10"%” to about Xanthomonoapepsin, pseudomonapepsin, cathepsin E, atrol 10% w/v. ySin, microbial collagenase, leucolysin, interstitial collage 14. The composition of claim 11 wherein the concentra nase, neprilysin, matrix metalloproteinases, dispase, ther tion of protease is in the range of about 0.1%” to about molysin, V8 protease and combinations thereof. 3.0% w/v. 24. A method to treat Skin comprising topically applying 15. The composition of claim 11 wherein the formulation to an outermost layer of skin a composition containing a is Selected from the group consisting of creams, ointments, protease in a biologically acceptable formulation in an lotions, liniments, gels, Solutions, Suspensions, pastes, amount and formulation to Selectively remove at least one Sticks, sprayS, beads, Spheres, microbeads, microSpheres, epidermal layer containing a skin condition to thereby treat liposomes, and combinations thereof. Said skin. 16. The composition of claim 11 further containing a 25. The method of claim 24 wherein said protease is co-additive Selected from the group consisting of exfoliants, Selected from the group consisting of trypsin, papain, bro immunomodulating drugs, cytotoxic drugs and combina melain, dispase, thermolysin, and combinations thereof. tions thereof. 26. A method to treat Skin comprising providing a com 17. The composition of claim 16 wherein said exfoliant is position containing a protease in a biologically acceptable Selected from the group consisting of ethylendiaminetetrace formulation in an amount and formulation to Selectively tic acid (EDTA), ethylene glycol-bis (B-aminoethyl ether)- remove at least one dermal layer containing a skin condition N.N,N',N'-tetra acetic acid (EGTA), salicylic acid, lactic to thereby treat Said skin. acid, alpha-hydroxy acids, beta-hydroxy acids, urea and 27. The method of claim 26 wherein said composition is combinations thereof. provided by an administration method selected from the 18. The composition of claim 16 wherein said immuno group consisting of topical, injection, and combinations modulating drug is Selected from the group consisting of thereof. glucocorticoids, tacrolimus, cycloSporin, interferon, asco 28. The method of claim 26 wherein said protease is mycin (SDZASM 981), imiquimod, or any of their respec Selected from the group consisting of , tive derivatives and their combinations thereof. elastases, and combinations thereof. 19. The composition of claim 16 wherein said cytotoxic 29. A method to treat skin comprising providing a com agent is Selected from the group consisting of podophyllin, position containing a lipid hydrolyzing enzyme in a biologi podophylox, cantharadin, and combinations thereof. cally acceptable formulation in an amount and formulation 20. A topically applied composition for treating a patient to Selectively remove at least one Subcutaneous layer con having a skin condition affecting at least one epidermal, taining a skin condition to thereby treat Said skin. and/or dermal layer, and/or Subcutaneous layer comprising a 30. A method to target skin treatment of an affected area hydrolase in a pharmaceutically acceptable formulation, Said comprising providing to Said affected area a composition enzyme in an amount in the range of about 1x10% by containing at least one enzyme in an amount and formula weight to about 10% by weight of said formulation. tion effective to Selectively target Skin removal in Said 21. The composition of claim 20 wherein said enzyme is affected area. a hydrolase Selected from the group consisting of esterases, 31. The method of claim 30 wherein said affected area is glycosidases, proteases, phosphatases, thiolases, phospholi Selected from the group consisting of epidermis, dermis, pases, amidases, ceramidases, lipases, deaminases, Subcutaneous layer, and combinations thereof. nucleases and combinations thereof. 32. A method of treating aging in at least one layer of skin 22. The composition of claim 21 wherein said hydrolase comprising providing a protease-containing biologically is Selected from the group consisting of naturally occurring acceptable composition to an outermost layer of affected hydrolases, Synthetic hydrolases, and combinations thereof. skin in an amount and formulation to Selectively target Said 23. The composition of claim 21 wherein Said protease is affected Skin layers. Selected from the group consisting of collagenases, trypsin, papain, bromelain, , chymotrypsin, chymot 33. The method of claim 32 wherein said signs are rypsin C, elastases, eXopeptidases, endopeptidases, metri Selected from the group consisting of Xerosis, rhytids, loss of din, thrombin, plasmin, enteropeptidase, alpha-lytic skin tone, acitinic damage, fine lines, dySpigmentation, and endopeptidase, prolyl oligopeptidase, brachyurin, plasma combinations thereof. kallikrein, tissue kallikrein, pancreatic elastase, leukocyte 34. A method for treating a condition affecting skin elastase, chymase, cerevisin, hypodermin C, endopeptidase comprising applying a composition to the affected skin, the La, alpha-renin, leucyl endopeptidase, tryptase, kexin, Sub composition containing at least one enzyme at a concentra tilisin, oryzin, endopeptidase K, thermomycolin, thermitase, tion Selective for regulating depth of skin treatment and endopeptidase So, tissue plasminogen activator, pancreatic applied to an area Selective for regulating a radial Surface of endopeptidase E, pancreatic elastase II, urine plasminogen skin treatment. activator, cathepsin B, papain, ficain, chymopapain, ascle 35. The method of claim 34 wherein the enzyme is a pain, cloStripain, Streptopain, actinidain, cathepsin L, cathe protease. psin H, calpain, cathepsin T, glycyl endopeptidase, cathepsin 36. The method of claim 34 wherein the composition is S, caricain, ananain, Stem bromelain, fruit bromelain, legu topically applied. main, histolysain, pepsin A, pepsin B, gastricSin, chymosin, 37. The method of claim 34 wherein the composition cathepsin D, retropepsin, aspergillopepsin I, aspergillopep comprises a protease and is topically applied. US 2003/0026794 A1 Feb. 6, 2003

38. The method of claim 34 wherein the composition is tration is used to treat a condition affecting an epidermal applied one time. layer. 39. The method of claim 34 wherein the enzyme is a 41. The method of claim 34 wherein a relatively long protease to treat an epidermal layer. duration of exposure and a relatively high enzyme concen 40. The method of claim 34 wherein a relatively short tration is used to treat a condition affecting a dermal layer. duration of exposure and a relatively low enzyme concen k k k k k