COMPOSITIONS AND METHODS FOR MODIFICATION OF SKIN LIPID CONTENT

Abstract: The topical use of ursolic acid compounds to alter the lipid content of mammalian skin is disclosed. The compounds can be encapsulated in liposomes and administered in this form to the skin in, for example, a lotion or a gel. The compounds are effective in, among other things, reducing the effects of aging, photoaging, and skin atrophy, including skin atrophy resulting from the topical use of retinoids and/or steroids. Compositions comprising a ursolic acid compound in combination with another therapeutically active topical compounds, such as, a retinoid or a steroid, are also disclosed.

Description: COMPOSITIONS AND METHODS FOR MODIFICATION OF SKIN LIPID CONTENT BACKGROUND OF THE INVENTION 1. Field of the Invention As known in the art, the epidermis and dermis of mammalian skin contain different cell types, perform different functions, and have different chemical compositions. A particularly important difference between these layers is their lipid concentrations. The dermis contains fibroblasts which produce collagen and other proteins, but very little lipid. The epidermis, on the other hand, contains keratinocytes which, among other things, produce lipids, but essentially no collagen. The collagen produced by the fibroblasts provides tensile strength to the skin. The lipids produced by the kerotinocytes provide a barrier between the living tissue and the outside world. The present invention relates to modifying the lipid content of skin for purposes of altering appearance, improving function, improving vitality, reversing the effects of aging, reversing the effects of photodamage, or treating disease by topical administration of ursolic acid, ursolic acid analogs, derivatives of ursolic acid, derivatives of ursolic acid analogs, or combinations thereof. Because the skin lipids are located in the epidermis, this modification of the lipid content of the skin takes place in that layer. For ease of reference, ursolic acid, ursolic acid analogs, derivatives of ursolic acid, derivatives of ursolic acid analogs, or combinations thereof will be referred to herein as simply a "ursolic acid compound". The ursolic acid compound can be encapsulated in liposomes or administered in other formulations suitable for topical administration. 2. Description of Related Art A. Patents U.S. Patent 4,857,554, Methods for Treatment of Psoriasis, is directed to treating psoriasis by applying an ointment containing ursolic acid and oleanolic acid dispersed in a petroleum jelly /lanolin carrier. U.S. Patent 4,530,934, Pharmaceutically Active Ursolic Acid Derivatives, is directed to using active derivatives of ursolic acid to treat ulcers. U.S. Patent 3,903,089, Ursolic Acid Derivatives, is directed to the synthesis of ursolic acid derivatives and analogs. U.S. Patent 5,624,909, Derivatives of Triterpenoid Acids as Inhibitors of Cell-adhesion Molecules ELAM-1 (e-selectin) and LECAM-1 (I- selectin), is directed to alleviating inflammation by administration of triterpenoid acid derivatives. U.S. Patent 5,314,877, Water-soluble Pentacyclic Triterpene Composition and Method for Producing the Same, is directed to making ursolic acid, oleanolic acid, and related triterpenoids soluble in water by formulation in cyclodextrins. U.S. Reissue Patent RE036068, Methods for Treatment of Sundamaged Human Skin with Retinoids, is directed to reversing the effects of photodamage by topical application of retinoids. U.S. Patent 5,051,449, Treatment of Cellulite with Retinoids, is directed towards retarding or reversing cellulite accumulation in skin by topical application of retinoids. U.S. Patent 5,556,844, Pharmaceutical or Cosmetic Composition Containing a Combination of a Retinoid and a Sterol, is directed towards treatment of disorders of epidermial keratinization, epithelial proliferation, or disorders of sebaceous function by topical application of retinoids. U.S. Patent 5,075,340, Retinoic Acid Glucuronide Preparations for Application to the Skin, is directed towards treatment of acne or wrinkled skin and prevention of retinoid dermatitis by topical application of retinoic acid glucoronides U.S. Patent 5,837,224, Method of Inhibiting Photoaging of Skin, is directed to reversing the effects of photodamage by topical application of agents that inhibit UVB-inducible matrix metalloproteinase. B. Publications Tokuda, H., H. Ohigashi, K. Koshimizu, and Y. Ito. 1986. Inhibitory effects of ursolic and oleanolic acid on skin tumor promotion by 12-0- tetradecanoyhlphorbol-13-acetate. Cancer Lett. 33:279-285. Ponec, M., and A. Weerheim. 1990. Retinoids and lipid changes in keratinocytes. Meth. Enzymol. 190:30-41. Griffiths, C. E. M., A. N. Russman, G. Maj udar, R. S. Singer, T. A. Hamilton, and J. J. Voorhees. 1993. Restoration of collagen formation in photodamaged human skin by tretinoin (retinoic acid). New Engl. J. Med. 329:530-5. Kligman, A. M., and J. J. Leyden. 1993. Treatment of photoaged skin with topical tretinoin. Skin Pharmacol. 6 (Suppl.l): 78-82). Huang, M.-T., C.-T. Ho, Z. Y. Wang, T. Ferraro, Y.-R. Lou, K. Stauber, W. Ma, C. Georgiadis, J. D. Laskin, and A. H. Conney. 1994. Inhibition of skin tumorigenesis by rosemary and its constituents carnosol and ursolic acid. Cancer Res. 54:701-708. Liu, J. 1995. Pharmacology of oleanolic acid and ursolic acid. J. Ethanopharmacol. 49:57-68. Manez, S., C, C. Recio, R. M. Giner, and J.-L. Rios. 1997. Effect of selected triterpenoids on chronic dermal inflammation. Eur. J. Pharmacol. 334:103-105. Ponec, M., A. Weerheim, J. Kempenaar, A. Mulder, G. S. Gooris, J. Bouwstra, and A. M. Mommaas. 1997. The formation of competent barrier lipids in reconstructed human epidermis requires the presence of Vitamin C. J. Invest. Dermatol. 109:348-355. Griffiths, C. E. M. 1999. Drug treatment of photoaged skin. Drugs & Aging 14:289-301. Japanese Patent Publication No. 11-5727, published January 12, 1999, describes the use of ursolic acid in combination with retinols in a final cosmetic product to increase dermal collagen. As discussed above, collagen is located and produced in the dermis by fibroblasts. The present invention, on the other hand, is concerned with modifying the content of lipids located and produced in the epidermis by kerotinocytes 3. Epidermal Lipid Composition and Alterations During Differentiation The epidermis of skin contains a number of lipids that are altered during differentiation as follows (see Downing et al., 1993, p210-221, In: Dermatology in General Medicine): (i) Phospholipids: Most common are phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, and sphingomyelin. Phospholipids are the most abundant lipids in the basal layers of the epidermis, but decrease towards the surface of the skin so much so that they are one of the least abundant lipids in the cornified layer. Thus, the phospholipid content of keratinocytes decreases as they differentiate. Conversely, high phospholipid levels are associated with keratinocyte proliferation, (ii) Free fatty acids: These are primarily saturated and range from 14 to 28 carbons (myristic = 14, palmitic = 16, stearic = 18, arachidic = 20, behenic = 22, lignoceric = 24, cerotic=26). The most common fatty acids in skin are the 22-carbon (15 wt.%) and 24 carbon (27 wt.%) species, (iii) Triglycerides: These are minor lipid components that serve as intermediates in the transfer of fatty acids from phospholipids to glucosylceramides. (iv) Glucosylceramides A,B,C-l,C-3,D-l,D-2, C-2: Glucosylceramide A also known as acylglucosylceramide is the major form, comprising 56 wt.% of this group of lipids. The acyl group in glucosylceramide A is often linoleic acid which is bound to the hydroxyl group of the ω-hydroxyacid (Abraham, Wertz and Downing, 1985, J. Lipid Res. 26:761-766). (v) Ceramides 1-7: Ceramides are the major lipids in the stratum corneum. They result from deglycosylation of glucosylceramides at the end of the epidermal differentiation process. Ceramide 1 is derived from Glucosylceramide A, Ceramide 2 is derived from Glucosylceramide B, Ceramide 3 is derived from Glucosylceramide C-l, Ceramide 4 is derived from Glucosylceramide C-3, Ceramide 5 is derived from Glucosylceramide D-l, Ceramide 6-A is derived from Glucosylceramide D-2, and Ceramide 6-B is derived from Glucosylceramide C-2. Although Glucosylceramide A comprises 56 wt.% of the glucosyleramides, Ceramide 1 comprises only 8 wt.% of the extractable ceramides because most of it is converted to the ω-hydroxyceramide which permits covalent binding to glutamates of the cornified cell envelope protein, thus, forming a protective barrier around each corneocyte. Ceramide 2 comprises 42 wt.% of this group. Ceramide 6 (phytosphingosine) and ceramide 7 (6-hydroxy-4- sphingenine) together comprise 20 wt.%. (vi) Cholesterol: This Hpid increases as keratinocytes differentiate so that it comprises 30 mol% of stratum corneum lipids (Schaefer and Redelmeier, 1996, Skin Barrier). (vii) Cholesterol sulfate: This Hpid increases ceU cohesiveness by forming interceUular cholesterol sulfate calcium bridges, (viii) Cholesterol esters: During the latter stages of epidermal differentiation, phospholipids are degraded liberating fatty acids which are utilized to produce cholesterol esters. Examination of Hpids in serial sections through pig skin showed the foUowing (Cox and Squier, 1986, J. Invest. Dermatol. 87:741-744): (i) increases of both glucosylceramides and ceramides towards the surface layers, but decreases of glucosylceramides concomitant with increases of ceramides at the outermost layers, (n) decreases of phosphoHpids towards the surface layers of the skin (iii) progressive increases of triglycerides, cholesterol and cholesterol esters towards the surface layers of the skin, (iv) progressive increase of cholesterol sulfate and then a sudden decrease at the outermost layer (related to desquamation by a sulfatase). Ponec and Weerheim (1990, Meth. Enzymol. 190:30-41) reviewed the Hterature and state that normal epidermal terminal differentiation is marked by depletion of phospholipids, with increase of sterols and certain classes of sphingolipids, with the final stratum corneum lipid products of differentiation consisting mainly of ceramides and nonpolar Hpids. OveraU, the flow of fatty acids during differentiation appears to be from phosphoHpids, to triglycerides, to ceramides, and finally hydroxyceramides (Swartzendruber et al., 1987, J. Invest. Dermatol. 88:709-713; Ponec et al., 1997, J. Invest. Dermatol. 109:348-355). Thus the metaboHc Hpid flow during differentiation appears to be towards formation of hydroxyceramides in the stratum corneum. Hydroxyceramides are linked to involucrin via its numerous glutamate residues (20%) during cornification, resulting in the highly effective barrier function of the skin (Swartzendruber et al., 1987, J. Invest. Dermatol. 88:709-713). 4. Agents Shown to Alter Epidermal Lipids Retinoic acid is able to reverse the alterations of Hpid synthesis that occur during differentiation, resulting in a 3-4-fold increase in phospholipids, a 3-fold decrease in sphingolipids (most notably, ceramides), a 9-fold decrease of acy leer amides, a near 2-fold decrease of cholesterol and cholesterol sulfate, a 6-fold decrease of lanosterol, and a 3-fold decrease of FFA in living skin equivalents (Ponec and Weerheim, 1990, Meth. Enzymol. 190:30-41). Thus, it would appear that there are marked differences between the terminal differentiation that occurs naturaUy in skin, and the ceUular reprogramming that occurs as a result of treatment with retinoic acid. Vitamin C (50 ug/ml) has been found to result in increases of glucosylceramides and ceramides, most notably ceramides 6 and 7 in Hving skin equivalents (Ponec et al., 1997, J. Invest. Dermatol. 109:348-355). These increases were accompanied by increased barrier function. Since Vitamin E had no effect on Hpid composition even though it is hydrophobic, it was concluded that the main role of Vitamin C is as a donor of hydroxyl groups to sphingoid bases and fatty acids for the formation of protein-bound hydroxyceramides (Ponec et al., 1997, J. Invest. Dermatol. 109:348-355). 5. Effect of Aging on Epidermal Lipids AU major species of epidermal Hpids are decreased during the aging process. Particular attention has been paid to the reductions of the ceramide fraction since this results in a notable loss of barrier function with age (reviewed in Rogers et al., 1996, Arch. Dermatol. Res. 288:765-770). However, the percentage ratio of each of the major classes of Hpids is unchanged during aging, even though total epidermal Hpids are decreased by 30% in the aged (Rogers et al., 1996, Arch. Dermatol. Res. 288:765-770). The most important change of epidermal lipids that occurs with age is related to altered ratios of free fatty acids that result in reductions in ceramide 1 Hneolate (Rogers et al., 1996, Arch. Dermatol. Res. 288:765- 770). Reductions of ceramide 1 Hneolate have been Hnked to dry skin, atopic dermatitis, and acne (reviewed in Rogers et al., 1996, Arch. Dermatol. Res. 288:765-770). 6. Effect of Photodamage on Epidermal Lipids Long-term (3 week) daily treatment with either UVA (50 J/cm 2 ) or UVB (124 mJ/cm 2 ) has been shown to result in an approximate 2-fold increase of total epidermal Hpids in human skin, with increases in the triglyceride, free fatty acid, alkane, squalene, and ceramide fractions (Wefers et al., 1991, J. Invest. Dermatol. 96:959-962). No changes were found in the sterol, cholesterol, cholesterol ester or cholesterol sulfate fractions (Wefers et al., 1991, J. Invest. Dermatol. 96:959-962). PhosphoHpids were not examined in this study. In contrast to these results, shortly after exposure (24-48 hr), UVA (50 J/cm 2 ) resulted in a decrease of the ceramide fraction of Hving skin and an increase in the relative proportion of phosphoHpids (Robert et al., 1999, Int. J. Radiat. Biol. 75:317- 26). Similarly, shortly after exposure (24-48 hrs), UV-B (0.15 J/cm 2 ) resulted in a marked depletion of ceramides (HoUeran et al., 1997, Photoderm. Photoimmunol. Photomed. 13:117-128). However, unlike UV-A, short-term exposure to UV-B also resulted in a marked (>2-fold) depletion of phosphoHpids (HoUeran et al, 1997, above). 7. Methods to Treat Aged and Photodamaged Skin Retinoic acid is weU known as an agent for treatment of photoaged skin. Topical retinoic acid has been shown to restore coUagen I levels that are reduced in photodamaged skin (Griffiths et al., 1993, New Engl. J. Med. 329:530-5). Restoration of coUagen I levels correlate with a reduction of fine wrinkles in skin (Griffiths et al., 1993, New Engl. J. Med. 329:530-5). Although retinoids have been shown to alter Hpids in cultured skin equivalents (Ponec and Weerheim, 1990, Meth. Enzymol. 190:30-41), there are no reports indicating that retinoids reverse aging or photodamage by altering Hpid levels. In part, this may be because retinoids reduce ceramide levels in skin equivalents (Ponec and Weerheim, 1990, Meth. Enzymol. 190:30-41), and reduce the thickness of the stratum corneum when applied topicaUy to human skin (KHgman and Leyden, 1993, Skin Pharmacol. 6, Suppl.1:78-82), which could exacerbate the depletion of ceramides and barrier function that occurs in the aged. 8. Pharmacological Uses of Ursolic Acid UrsoHc acid is pentacyclic triterpene compound known to have a number of pharmacological effects (reviewed in Liu, 1995, J. Ethanopharmacol. 49:57-68). Ursolic acid is closely related to steroids since both are derived from the cycHzation of squalene (Suh et al., 1998, Cancer Res. 58:717-723). It is found in the waxy coating of fruit and in the leaves of many plants, such as heather and rosemary. It is insoluble in most common solvents and as a result it is not widely used. In fact, commercial extraction processes for plant leaves fail to recover measurable levels of ursoHc acid. UrsoHc acid has been characterized as an inhibitor of Hpoxygenase and cyclooxygenase in inflammatory ceUs (Najid et al., 1992, FEBS 299:213-217; Suh et al., 1998, Cancer Res. 58:717-723). As such, ursoHc acid is expected to have usefulness as an anti-inflammatory agent. UrsoHc acid has been shown to inhibit chronic dermal inflammation induced by phorbal esters in an animal model (Manez et al., 1997, Eur. J. Pharmacol. 334:103- 105). UrsoHc acid has also been shown to inhibit induction of inducible nitric oxide synthase in macrophages (Suh et al., 1998, Cancer Res. 58:717- 723), which may contribute to its anti-inflammatory activity. UrsoHc acid has also been shown to induce differentiation and growth arrest of several types of ceUs, suggesting that it may be useful as a chemotherapeutic differentiation agent (Es-Saady et al., 1996, Cancer Lett. 106:193-197; Hsu et al., 1997, Cancer Lett. 111:7-13; Es-Saady et al., 1996, Anticancer Res. 16:481-486; Paik et al., 1998, Arch. Pharm. Res. 21:398- 405). UrsoHc acid has also been shown to induce apoptosis in tumor ceUs (Baek et al., 1997, Int. J. Cancer 73:725-728). Both ursoHc acid and oleanoHc acid, a closely related structural analog of ursolic acid, have been shown to inhibit tumor promotion induced in mouse skin by phorbal esters (Tokuda et al., 1986, Cancer Lett. 33:279-285; Huang et al., 1994, Cancer Res. 54:701-708). Both compounds have also been shown to prevent Hpid peroxidation, which may inhibit free radical damage during cancer initiation and promotion (Balanehru and Nagarajan, 1991, Biochem. Int. 24:981-990). Ursolic acid also downregulates matrix metalloproteinases (Cha et al., 1998, Oncogene 16:771-778) and elastase (Ying et al., 1991, Biochem. J. 277:521-526) which may provide a mechanism for preventing tumor invasion (Cha et al., 1996, Cancer Res. 56:2281-84), and, inflammation related damage in skin (Ying et al., 1991, Biochem. J. 277:521-526). UrsoHc acid and a number of triterpenoid derivatives have been shown to have hypolipidemic and anti-atherosclerotic properties (reviewed in Liu, 1995, J. Ethanopharmacol. 49:57-68). UrsoHc acid and oleanoHc acid lowered blood cholesterol and β-lipoprotein levels 40-50% in animal models of atherosclerosis (reviewed in Liu, 1995, J. Ethanopharmacol. 49:57-68). Consistent with this prior art understanding, topical ursolic acid has been proposed for use in the treatment of psoriasis, a condition characterized by hyperproliferation and inflammation of the epidermis (US patent 4,857,554). In fact, these prior results that ursoHc acid and its analogs decrease Hpid production and may be used in treatment of the hyperproliferation of psoriasis teach away from the present invention, and make the discovery of the opposite effects unexpected and novel. Contrary to the findings in the literature and the understanding of the prior art, we have discovered that ursolic acid increases the production of Hpids, especiaUy ceramides and phosphoHpids, by keratinocytes of the skin. SUMMARY OF THE INVENTION The present invention provides a method for altering the Hpid content of mammalian skin by administering an effective amount of a ursolic acid compound to the skin of a mammal (e.g., a human) in need of such a treatment, e.g., to skin which is aged, photoaged, atrophied, etc. As discussed above, because the Hpids of the skin are located in the epidermal layer, the alteration of the lipid content of the skin takes place in that layer. In another aspect, the present invention provides a method for reversing certain aspects of the photoaging or aging process in mammalian skin and, in yet another aspect, the present invention provides a method for improving function, increasing barrier function, improving vitaHty, or treating lipid deficient diseases of mammalian skin, which comprises topical appHcation of: (a) an effective amount of a ursoHc acid compound in a suitable medium for topical administration, e.g., a lotion, gel, or the like; (b) an effective amount of a ursolic acid compound encapsulated in Hposomes; and/or (c) an effective amount of a ursoHc acid compound encapsulated in Hposomes and incorporated into a suitable medium for topical administration, e.g., a lotion, gel, or the like. As discussed below, because ursolic acid compounds are highly insoluble in many solvents, including water, administration of such compounds in Hposomes is a particularly preferred embodiment of the invention. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows micrographs of cultured normal human epidermal keratinocytes (NHEK) that were: (a) Untreated, (b) treated with 1% (volume/volume) Empty Liposomes, (c & d) treated with 1% 4 mM UrsoHc Acid (URA) Liposomes, which are identical to Empty Liposomes except for incorporation of ursoHc acid at a concentration of 4 mM - the final concentration of ursoHc acid in media is 40 uM. Details are described in Example 1. Figure 2 shows the amount of phosphatidylchoHne per cell relative to Untreated cultured normal human epidermal keratinocytes (NHEK), cultured NHEK treated with 1% (volume/volume) Empty Liposomes, or cultured NHEK treated with 1% 4 mM Ursolic Acid Liposomes. Details are described in Example 1. Figure 3 shows the amount of phosphatidylethanolamine per cell relative to Untreated cultured normal human epidermal keratinocytes (NHEK), cultured NHEK treated with 1% (volume/volume) Empty Liposomes, or cultured NHEK treated with 1% 4 mM Ursolic Acid Liposomes. Details are described in Example 1. Figure 4 shows the amount of free fatty acid per ceU relative to Untreated cultured normal human epidermal keratinocytes (NHEK), cultured NHEK treated with 1% (volume/volume) Empty Liposomes, or cultured NHEK treated with 1% 4 M UrsoHc Acid Liposomes. DetaUs are described in Example 1. Figure 5 shows the amount of total ceramides per ceU relative to Untreated cultured normal human epidermal keratinocytes (NHEK), cultured NHEK treated with 1% (volume/volume) Empty Liposomes, or cultured NHEK treated with 1% 4 mM UrsoHc Acid Liposomes. Details are described in Example 1 Figure 6 shows the amount of total glycosylceramides per ceU relative to Untreated cultured normal human epidermal keratinocytes (NHEK), cultured NHEK treated with 1% (volume/volume) Empty Liposomes, or cultured NHEK treated with 1% 4 mM UrsoHc Acid Liposomes. Details are described in Example 1. Figure 7 shows the amount of cholesterol per ceU relative to Untreated cultured normal human epidermal keratinocytes (NHEK), cultured NHEK treated with 1% (volume/volume) Empty Liposomes, or cultured NHEK treated with 1% 4 mM UrsoHc Acid Liposomes. Details are described in Example 1. Figure 8 shows the amount of extractable ceramides in human skin foUowing treatment with 0.3% or 1% UrsoHc Acid Liposomes in a hydrogel Lotion for 3 and 11 days, relative to adjacent areas treated with Empty Liposome Lotion. These concentrations of UrsoHc Acid Liposomes resulted in a final concentration of ursoHc acid in the hydrogel Lotion of lOμM and 30μM, respectively. DETAILED DESCRIPTION OF THE INVENTION The compounds and compositions of the present invention effectively and efficiently increase the phospholipid content of normal human epidermal keratinocytes. Furthermore, the compounds and compositions of the present invention increase the free fatty acid content and the ceramide and glycosylceramide content of normal human epidermal keratinocytes. Increases of phosphoHpid, free fatty acid, ceramide and glycosylceramide content are due to the effects of ursoHc acid and are over and above any effects of the lipid carrier. Increasing the lipid content of epidermal keratinocytes has several consequences. First, increasing the phospholipid content prevents ceU senescence and stimulates proliferation, which is manifested as increased ceUular viabiHty and increased epidermal thickening. Second, increasing the free fatty acid content relates to increases of several Hpid fractions including phospholipids, triglycerides, glucoceramides, ceramides and acylceramides. Increasing the Hpid content of epidermal keratinocytes reverses the Hpid reductions associated with aging. Third, increasing the glucoceramide, ceramide and acylceramide content of keratinocytes results in an improved barrier function. Improved barrier function, in turn, reduces atopic dermatitis and protects the skin and body from the effects of many agents including ultraviolet irradiation, toxic chemicals, toxins, and irritants. Fourth, preventing ceU senescence and increasing barrier function reverses the effects of aging. Fifth, increases of phospholipids directly reverse the depleting effect of UV-B on phosphoHpids in skin, resulting in increased viabiHty as manifested by cellular proliferation, and reducing the effects of photoaging. The users of this invention who benefit from its discovery include, among others, those suffering from ichthyosis and ichthyosiform dermatoses, those with acute dry skin, those with skin atrophy and retinoid dermatoses, those with acne and those with aging and photoaging skin. These conditions all have in common a reduction of skin Hpids, particularly those produced by keratinocytes and especially ceramides, a reduction in the thickness and uniformity of intact stratum corneum and a loss of skin barrier function. As a consequence, the users of this invention share the pathological conditions of increased transepidermal water loss and a sensation of dry skin. The active compound according to the present invention is a ursoHc acid compound. Examples of such compounds are set forth in Table 1, it being understood that the invention is not Hmited to the examples of this table but includes aU ursolic acid compounds which achieve the beneficial, lipid altering effects of the invention when appHed to mammalian skin. The table shows the chemical structure common to this famfly of compounds. The reference compound, ursoHc acid, contains the substituents at the indicated sites as shown in the second Hne of the table. Each of the analogs/derivatives Hsted below ursoHc acid differs from the ursolic acid structure only where indicated. Blank cells indicate that the substituents at those sites are identical to ursoHc acid. As can be appreciated from the diagram, these compounds share great similarity in the A, B, and C rings of the pentacycHc structure. An addition at sites on any of these rings, such as the hemisuccinate at the R 2 position of ring A in carbenoxolone, reduces the potency of the compound despite the increase in solubility that may be achieved by the addition. On the other hand, many modifications of the E ring, including larger alkyl groups at the Re position and substitution of a pentyl for a hexyl ring, can be tolerated. Oxo additions at the R 4 position increase the mineralocorticoid activity of the compound and are to be preferred only when anti-diuresis and water retention is desirable or not harmful. As discussed above, the methods and compositions of the present invention employ a ursoHc acid compound (which, as defined above, can be a combination (mixture) of compounds) as an active ingredient for various uses. In a preferred embodiment, the active ingredient is given topically in an acceptable formulation. A particularly preferred formulation is the incorporation of the ursoHc acid compound into liposomes. A variety of different types of lipids at various concentrations can be used to form the liposomes, examples of which can be found in Liposome Technology, ed. Gregory Gregoriadis, CRC Press Inc., Boca Raton, Florida 1984. The present invention also relates to the incorporation of the ursoHc acid compound, either alone or incorporated in Hposomes, into lotions, gels, creams or other acceptable formulations conducive to uptake of active ingredients into the epidermis. Liposomal formulations are preferred because ursoHc acid is highly insoluble in many solvents, particularly water, and common emulsifiers such as LECINOL S-10 have Httle effect. In accordance with the invention, this insolubility problem is addressed by taking advantage of the flat, planar structure of ursoHc acid to stack it between the lipid tails in the phosphoHpid bflayer membranes of liposomes. Due to the charged head- group of the phospholipids, Hposomes containing ursolic acid are readily soluble in water. However, only a Hmited number of sites within the tails of the lipid bflayer of the Hposome membrane are available for stacking ursolic acid. The preferred ratio of ursolic acid to lipid components must be determined by experimentation. For example, the preferred ratio of ursoHc acid to phosphatidylcholine and cholesterol is approximately 1.5 (range 1.0 to 3.0):10:1.9 (w/v). At lower concentrations of ursoHc acid, the effects on lipid production by the epidermal layer of mammalian skin are not easfly evoked. At the higher ranges of ursolic acid concentration, other compounds that partition into the liposome membrane cannot be included in the preparation, because they displace the ursoHc acid and lead to its precipitation. For example, inclusion of the HpophiHc preservative phenoxyethanol at the recommended concentration of 1% leads to a ursoHc acid precipitate forming in the preparation. Therefore it is difficult to achieve an effective concentration of ursoHc acid in Hposomes and an effective concentration of a HpophiHc preservative. The preferred preservatives that are compatible with ursoHc acid Hposomes are water-soluble preservatives that do not partition into the Hposome membrane. One such water-soluble preservative that can be used in the practice of the invention is potassium sorbate. Other water soluble preservatives can be found in: Cosmetic and Drug Preservation. Principles and Practices. Ed. J. J. Kabara. Marcel Dekker, Inc. New York, 1984. Similarly, other additives to a liposomal ursolic acid composition should not displace the ursoHc acid from the liposome, and the preferred form of such additives should be water soluble or otherwise sequestered from the Hposomes. The dose regimen wiU depend on a number of factors which may be readfly determined, such as severity and responsiveness of the condition to be treated, but will normaUy be one or more treatments per day, with treatment lasting from several days to several months, or untfl the desired response is obtained, or a cure is effected, or a remediation of a condition or a diminution of a disease state is achieved. One of ordinary skill may readfly determine optimum dosages, dosing methodologies and repetition rates. In general, the unit dosage for compositions according to the present invention will contain from 0.1 mM to 10 mM of the ursoHc acid compound in Hposomes or an alternative carrier, with said Hposomes or carrier comprising from 1 wt.% to 90 wt.% of a lotion or alternative topical formulation. In some instances, it may be preferable to apply said Hposomes or alternative formulations full strength to skin. If desired, the ursoHc acid compound can be combined with other active ingredients in the formulation. For example, it is known that retinoids and topical steroids have the undesirable side effect of skin atrophy. This side effect can be ameHorated by co-administration of a ursoHc acid compound with these agents. The co-administration can be performed using a single vehicle, e.g., a lotion, containing both active ingredients or by means of separate vehicles, e.g., separate lotions, which can be administered either simultaneously or sequentiaUy, with either agent being administered first. As another example, a ursolic acid compound can be combined with a sunblock to form a sunscreen product. The uses of and useful and novel features of the present methods and compositions wiU be further understood in view of the following non- Hmiting examples. Example 1 Preparation of Liposomes Ursolic acid-containing Hposomes and empty liposomes containing no ursolic acid were prepared as foUows: 0.393 g phosphatidylchoHne (DOOSAN) and 0.077 g cholesterol (AVANTI) were dissolved in 20 ml ethanol, and split into two 10 ml aHquots. Sixty mg ursolic acid (SIGMA- ALDRICH) was then dissolved in one aHquot which was then used to make UrsoHc Acid Liposomes. No additions were made to the aliquot designated as Empty Liposomes. Seven ml of each mixture were injected through a 3O 2G needle into 10 ml cold 1XPBS. The resulting mixture was dialyzed for 2 hr in 2 liters 1XPBS, and then overnight in a fresh batch of 1XPBS. Liposomes were coUected and ursoHc acid content was measured by high performance thin layer chromatography (HPTLC) using RP-18 F254S HPTLC plates (MERCK). UrsoHc acid was dissolved in methanol to make a series of standards. Liposomes were appHed directly to HPTLC plates. The mobile phase was 100% methanol. Chromatograms were visualized by spraying with antimony trichloride foUowed by heating at 100°C for 5 min. Stained chromatograms were photographed and spots were analyzed by QUANTISCAN image analysis software. UrsoHc Acid Liposomes contained 4 mM ursoHc acid. Cell Culture and Treatments Normal human epidermal keratinocytes (NHEK) were obtained from CLONETICS BIOWHITTAKER and cultured in KGM-2 media (CLONETICS-BIOWHITTAKER) according to the manufacturer's instructions. Four days before beginning treatments, 10 5 NHEK were plated in each of six 10 cm 2 CORNING cell culture dishes in 10 ml KGM-2 media. Two days later and on the first day of treatments, media was replaced with 10 ml fresh media. On the first day of treatments, 100 ul Empty Liposomes were added to each of two dishes, and 100 ul 4 mM Ursolic Acid Liposomes were added to each of two dishes. Thus, the Empty Liposome and 4 M Ursolic Acid Liposome treatments each received 1% Hposomes, and the final concentration of ursoHc acid in the UrsoHc Acid Liposome treatment was 40 uM. All treatments received fresh media and Hposomes on the fourth day foUowing the initiation of treatments. CeUs were harvested 8 days foUowing the initiation of treatments. Media was removed and ceUs were washed once with 10 ml CLONETICS- BIOWHITTAKER Hank's Buffered SaHne Solution (HBSS), then 5 ml HBSS was added, and ceUs were photographed using a NIKON microscope equipped with camera Hnked to a NORTHERN EXPOSURE computer imaging system. FoUowing photograph, HBSS was removed, 6 ml CLONETICS Trypsin/EDTA was added, and ceUs were incubated at 37°C for 6 min untfl they detached from dishes. Then, 6 ml CLONETICS Trypsin NeutraHzation Solution was added, and the ceUs were mixed thoroughly and transferred to a 15 ml conical tube. One-half ml of suspended ceUs were added to 19.5 ml Isoton II and counted on a model ZBI COULTER counter. The remainder of cells were then pelleted by centrifugation at 178 X g for 5 min. Supernatant was removed and ceUs were then resuspended in 5 ml 1XPBS, transferred to a PYREX test tube with teflon-lined Hd, and pelleted for lipid extraction. Lipid Extraction Method Lipids were extracted using procedures developed by Ponec and Weerheim (1990, Meth. Enzymol. 190:30-41), that were a modification of procedures developed by BHgh and Dyer (1959, Canad. J. Biochem. Physiol. 37:911-917). Pelleted ceUs were extracted by mixing with 2 ml chlorform: methanol (2:1) for 60 min at room temperature (RT) on a LABQUAKE rotary mixer. Cellular debris was pelleted, the supernatant collected, and the peUet re-extracted with 2 ml chloroform:methanol:deionized water (1:2:0.5) for 60 min at 37°C, followed by 2 ml chloroform:methanol (1:2) for 30 min at RT, 2 ml chloroform: methanol (2:1) for 30 min at RT, and 2 ml chloroform at for 15 min at RT. The combined extracts were mixed with 200 ul 2.5% KC1 by vortexing, and then mixed with 2 ml deionized water for 10 min at RT. Both the upper aqueous layer and the bottom extract layer were transferred to clean tubes, and the aqueous layer was re-extracted with 4 ml chloroform by mixing for 10 min at RT. This chloroform extract was combined with the previous extracts. The combined extracts were placed in a 50°C water bath and dried under a stream of nitrogen. The peUet was dissolved in 500 ul chloroform(2):methanol(l) and stored in a teflon lined vial under nitrogen at -20°C. For analysis of Hpids in human skin, Hpids were extracted from subjects using a modification of the protocol described by Bonte, F., A. Saunois, P. Pinguet, and A. Meybeck, in Arch. Dermatol. Res. 289:78-82, 1997. FoUowing treatments, the area on the forearm to be extracted was first rinsed with tap water, dried thoroughly and then tape-stripped once with SCOTCH™ 810 MAGIC™ tape. The excised top 1-inch of 50 ml polypropylene conical tubes were used as reservoirs for the solvents during extraction of subjects. The reservoirs were placed and held firmly on the arm and 1 ml of cyclohexane:ethanol (4:1) was added and stirred gently for one minute. Solvent was then removed and placed into a pyrex tube with a teflon-Hned Hd. One ml of cyclohexane:ethanol (1:1) was then added to the reservoir, stirred for one minute, and then removed and added to the tube containing the first extract. The tubes were then dried at 50°C under nitrogen gas as described above. The dried extracts were dissolved in 200 μl of chloroform:methanol (2:1). The Hpid solution was then placed into a smaU storage tube with a teflon-Hned lid, purged with nitrogen gas and stored at -20°C. Thin Layer Chromatography Methods Standard solutions were prepared that contained 5 ug/ul each of phosphatidylcholine (PC), phosphatidylethanolamine (PE), cholesterol (CH), total ceramides (α-hydroxy ceramides + non-hydroxy ceramides)(Cer), total glycosylceramides (GlyCer), and oleic acid, a commonly used reference material for free fatty acids (FFA). These were then serially diluted to 2.5 ug/ul, 1.25 ug/ul, 0.625 ug/ul, 0.313 ug/ul, 156 ng/ul, and 78 ng/ul, and 5 ul of each were run on 250 um thick Silica TLC plates with polyester support (SIGMA-ALDRICH) for PC and PE, or, on 150 um thick Sflica HPTLC plates with glass support (SIGMA-ALDRICH) for FFA, CH, Cer, and GlyCer. TLC plates were developed by running chloroform:methanol:deionized water (65:30:5) 60 mm as the mobile phase. HPTLC plates were developed using the foUowing sequence of mobile phases for FFA and CH: (i) chloroform run 15 mm, (ii) chloroform-acetone- methanol (76:8:16) run 10 mm, (iii) chloroform -hexyl acetate-acetone- methanol (86:1:10:4) run 70 mm, (iv) chloroform-acetone-methanol (76:4:20) run 20 mm, (v) chloroform-diethyl ether-hexyl acetate-ethyl acetate- acetone -methanol (72:4:1:4:16:4) run 75 mm, and, (vi) hexane-diethyl ether- ethyl acetate (80:16:4) run 90 mm (Ponec and Weerheim, 1990, Meth. Enzymol. 190:30-41), or, the foUowing sequence of mobile phases for Cer and GlyCer: (i) chloroform-methanol-water (40:10:1) run 30 mm, (n) chloroform-methanol-glacial acetic acid (190:9:1) run 75 mm, (iii) hexane- diethyl ether-glacial acetic acid (80:20:10) run 75 mm, and (iv) petroleum ether run 85 mm (Kennedy et al., 1996, Pharmaceut. Res. 13:1162-1167). TLC plates were dried, stained with iodine, photographed, and analyzed using a QUANTASCAN computer imaging system. HPTLC plates were dried, sprayed with phosphomolybdic acid, baked at 120°C for 2 min for FFA and CH, or, sprayed with 10% copper sulfate in 8% phosphoric acid for Cer and GlyCer, and analyzed similar to TLC plates. Intensity of standard staining was found to be linear up to at least 0.625 ug/ul, and this was therefore used as a standard on chromatographic runs with samples. Samples were run using either a standard amount of extract (5 ul), foUowed by normaHzation of results to ceU number, or by loading an amount of extract equivalent to a standard number of cells (2.5 X 10 4 ceUs). Both techniques produced similar results. Results obtained using extracts from a standard number of cells are shown in this example. TLC's and HPTLC's were run, stained and quantified in a fashion identical to that described above for standards. Statistical analysis was done by Student- Newman-Keuls ANOVA using the INSTAT software. Results and Pharmacological Applications Visual observation and photographic record (Figure 1) showed NHEK ceUs treated with 1% 4 mM UrsoHc Acid Liposomes were highly vacuolated relative to Untreated cells or ceUs treated with Empty Liposomes. Vacuolation can result from several conditions including accumulation of Hpid (Robert et al., 1999, Int. J. Radiat. Biol. 75:317-326). In Hght of the Hpid analysis below, it is beHeved that the vacuoles observed here resulted from Hpid accumulation. It should be noted that there was Httle or no increase in vacuolation of NHEK treated with Empty Liposomes, despite the fact that Hposomes contain phosphatidlychoHne and cholesterol. Thus, the vacuolation observed here is specific for UrsoHc Acid Liposome treated ceUs, indicating that ursoHc acid is responsible for induction of vacuolation. Although the photographs in Figure 1 were taken after 8 days of treatment, visual observation indicated that significant vacuolation of Ursolic Acid Liposome treated ceUs was apparent as soon as 2 days after the initiation of treatments. Analysis of Hpid content by TLC and HPTLC indicated that UrsoHc Acid Liposomes resulted in a statisticaUy significant 2-fold increase of phosphatidylchoHne (PC) in NHEK relative to Untreated NHEK (Figure 2). There was no increase of PC in NHEK treated with Empty Liposomes despite the fact that these Hposomes contain PC. There was a statisticaUy significant 1.3-fold increase of phosphatidylethanolamine (PE) in NHEK treated with UrsoHc Acid Liposomes (Figure 3). Treatment of NHEK with Empty Liposomes resulted in a 13% decrease of PE, although this was not statistically significant. Empty Hposomes resulted in a 1.15-fold increase of free fatty acids (FFA) while UrsoHc Acid Liposomes resulted in 1.3-fold increases of FFA; only the increase of FFA induced by UrsoHc Acid Liposomes was statisticaUy significant (Figure 4). Ursolic acid Hposomes dramaticaUy increased total ceramides and glycosylceramides compared to untreated ceUs (Figures 5 and 6). Neither Empty Liposomes nor UrsoHc Acid Liposomes increased cholesterol levels in ceUs (Figure 7). These findings in keratinocytes are unexpected in view of the reports in the prior art that ursoHc acid reduces cholesterol levels and is hypolipidemic. Treatment of human subjects with lotion containing 10 μM or 30μM Hposomal ursoHc acid resulted in induction of ceramides, with increases of hydroxy-ceramides generaUy greater than non-hydroxy-ceramides (Figure 8). In fact, foUowing 3 days of treatment, non-hydroxy ceramides were decreased approximately 12% by 10 μM or 30μM Hposomal ursoHc acid. In contrast, hydroxy ceramides were increased by approximately 18% foUowing 3 days of treatment with either 10 μM or 30μM Hposomal ursolic acid. FoUowing 11 days of treatment, both non-hydroxy and hydroxy ceramides were increased approximately 30% by lOμM Hposomal ursolic acid. However, at this same time period, non-hydroxy and hydroxy ceramides were increased only 7% and 18% respectively, by 30 μM ursoHc acid. Thus, whereas 30μM Hposomal ursolic acid was a more optimal concentration for induction of ceramides in cultured NHEK, lOμM Hposomal ursolic acid was a more optimal concentration for induction of ceramides in human skin. However, it should be noted that whereas cell culture treatments were done only once every third day, treatment of human skin was done twice daily. Thus, the ability of Hposomal ursolic acid to induce ceramides in human skin may have been saturated by this treatment regimen. Previous studies have shown that alterations of specific lipids can be associated with functional and aesthetic changes in skin. For example, treatment of skin with retinoids results in increases of phosphoHpids, a reduction of ceramides, and reduced senescence of epidermal ceUs. Thus, the viabiHty and vitality of skin is increased while the barrier function is reduced. Furthermore, it is known that Hpid accumulation in epidermal ceUs is associated with a restoration of a youthful appearance. Similar to retinoids, the compounds of the present invention result in increased levels of phosphoHpids in ceUs. However, incorporation of the compounds of the present invention into liposomes results in accumulation of additional Hpids, including total ceramides and glycosylceramide s which are extremely important for barrier function. Thus, unlike results with retinoids wherein ceramide levels are decreased (Ponec and Weerheim, 1990, Meth. Enzymol. 190:31-40), the barrier function of skin wiU not be compromised by the compositions of the present invention. In addition, the compositions will stimulate phospholipid synthesis and thereby increase ceU viability. Thus, the topical administration of a ursoHc acid compound in accordance with the invention will serve to counteract the effects of aging and photoaging and to treat diseases of dry skin related to impaired barrier function and dysfunctional stratum corneum. TABLE 1 Representative Ursolic Acid Compounds TABLE 1 (continued) Representative Ursolic Acid Compounds Ursolic acid is the reference compound. Blank cells indicate that the site is identical to ursolic acid.

Claims: What is claimed is: 1. A method for altering the lipid content of the skin of a mammal in need of such alteration comprising topicaUy administering a ursolic acid compound to said skin in an amount effective to alter the Hpid content of the epidermis of said skin. 2. The method of Claim 1 wherein the skin of the mammal is skin that exhibits aging as a result of a reduction in the Hpid content of its epidermal layer. 3. The method of Claim 1 wherein the skin of the mammal is skin that exhibits photoaging as a result of a reduction in the Hpid content of its epidermal layer. 4. The method of Claim 1 wherein the skin of the mammal is skin that has atrophied as a result of a reduction in the Hpid content of its epidermal layer. 5. The method of Claim 4 wherein the reduction in the Hpid content of the epidermal layer is a result of the application of at least one retinoid to the skin. 6. The method of Claim 4 wherein the reduction in the Hpid content of the epidermal layer is a result of the appHcation of at least one steroid to the skin. 7. The method of Claim 1 wherein the skin of the mammal suffers from ichthyosis or ichthyosiform dermatoses. 8. The method of Claim 1 wherein the skin of the mammal suffers from acne. 9. The method of Claim 1 wherein the mammal has the sensation of dry skin as a result of a reduction in the Hpid content of the skin's epidermal layer. 10. The method of Claim 1 wherein the ursolic acid compound is administered in a formulation and the concentration of the ursoHc acid compound in that formulation is equal to or less than 30 micromolar. 11. The method of Claim 1 wherein the ursoHc acid compound is encapsulated in Hposomes. 12. The method of Claim 11 wherein the ursoHc acid compound is administered in a preparation that includes a water soluble preservative. 13. The method of Claim 12 wherein the water soluble preservative is potassium sorbate. 14. The method of Claim 1 wherein the ursolic acid compound is ursolic acid. 15. The method of Claim 1 wherein the content in the epidermal layer of the skin of at least one member of the ceramide Hpid famfly is increased. 16. The method of Claim 1 wherein the free fatty acid content of the epidermal layer of the skin is increased. 17. The method of Claim 1 wherein the ursoHc acid compound is administered in combination with at least one other active compound which is not a ursoHc acid compound. 18. The method of Claim 17 wherein the at least one other active compound is a retinoid. 19. The method of Claim 17 wherein the at least one other active compound is a steroid. 20. The method of Claim 1 wherein the mammal is a human. 21. A method for treating skin of a mammal which exhibits aging as a result of a reduction in the Hpid content of its epidermal layer comprising topicaUy administering a ursoHc acid compound to said skin in an amount effective to reduce the effects of aging exhibited by said skin. 22. A method for treating skin of a mammal which exhibits photoaging as a result of a reduction in the Hpid content of its epidermal layer comprising topicaUy administering a ursoHc acid compound to said skin in an amount effective to reduce the effects of photoaging exhibited by said skin. 23. A method for treating skin of a mammal which is atrophied as a result of a reduction in the lipid content of its epidermal layer comprising topically administering a ursoHc acid compound to said skin in an amount effective to reduce the atrophy of said skin. 24. The method of Claim 23 wherein the atrophied mammalian skin is a result of treatment with at least one retinoid. 25. The method of Claim 23 wherein the atrophied mammalian skin is a result of treatment with at least one steroid. 26. A method for treating skin of a mammal suffering from acne comprising topicaUy administering a ursoHc acid compound to said skin in an amount effective to reduce said acne. 27. A method for treating skin of a mammal suffering from ichthyosis or ichthyosiform dermatoses comprising topicaUy administering a ursoHc acid compound to said skin in an amount effective to reduce said ichthyosis or ichthyosiform dermatoses. 28. A method of treating a mammal suffering from the sensation of dry skin as a result of a reduction in the Hpid content of its epidermal layer comprising topicaUy administering a ursolic acid compound to skin of said mammal in an amount effective to reduce said sensation of dry skin. 29. A composition for topical administration to the skin of a mammal comprising (i) a ursoHc acid compound encapsulated in Hposomes and (ii) a medium suitable only for topical administration. 30. The composition of Claim 29 further comprising a water soluble preservative. 31. The composition of Claim 30 wherein the water soluble preservative is potassium sorbate. 32. The composition of Claim 29 wherein the concentration of the ursolic acid compound in the composition is equal to or less than 30 micromolar. 33. A composition for topical administration to the skin of a mammal comprising (i) a ursolic acid compound encapsulated in liposomes, (n) at least one other therapeuticaUy active topical compound which is not a ursoHc acid compound, and (iii) a medium suitable for topical administration. 34. The composition of Claim 33 wherein the at least one other therapeuticaUy active topical compound is a retinoid. 35. The composition of Claim 33 wherein the at least one other therapeuticaUy active topical compound is a steroid. 36. The composition of Claim 33 further comprising a water soluble preservative. 37. The composition of Claim 36 wherein the water soluble preservative is potassium sorbate. 38. The composition of Claim 33 wherein the concentration of the ursolic acid compound in the composition is equal to or less than 30 micromolar.

Inventors: BROWN DAVID A, YAROSH DANIEL B, BROWN, DAVID, A., YAROSH, DANIEL, B.
Applicants: APPLIED GENETICS INC DERMATICS, BROWN DAVID A, YAROSH DANIEL B, APPLIED GENETICS INCORPORATED DERMATICS, BROWN, DAVID, A., YAROSH, DANIEL, B.
Application year: 2000
Main classification: A61K31
IPC codes: A61K8, A61K9, A61K31, A61K45, A61P17, A61Q19