WO2003006009A1

WO2003006009A1 - Compositions for reducing or preventing cellulite in mammalian skin

Info

Publication number: WO2003006009A1
Application number: PCT/US2002/011079
Authority: WO
Inventors: Anindita Sen; Peter Pieraccini; Yolanda Renee Lea-Currie; Dawn Marie Franklin
Original assignee: Zen-Bio, Inc.
Priority date: 2001-07-11
Filing date: 2002-04-08
Publication date: 2003-01-23

Abstract

Compositions and methods for preventing and/or treating cellulite in mammalian skin characterized by a subcutaneous accumulation of adipose tissue are provided. The skin care composition comprises conjugate linoleic acid (CLA), an AZOLE antifungal agent and a pharmaceutically acceptable carrier. In a particular embodiment, the composition comprises effective amounts of 10-trans, 12-cis conjugated linoleic acid, ketoconazole and a dermatologically acceptable carrier. Prevention and/or treatment is accomplished by topically applying a composition comprising safe and effective amounts of conjugated linoleic acid in combination with an imidazole antifungal agent. The compositions of the invention improve dermal appearance by promoting lipolysis and/or inhibiting de novo lipogenesis by adipocytes localized to targeted regions of mammalian skin.

Description

COMPOSITIONS FOR REDUCING OR PREVENTING CELLULITE IN MAMMALIAN SKTN

FIELD OF THE INVENTION The present invention relates to compositions, comprising conjugated linoleic acid and an imidazole antifungal agent, and methods that are useful for the treatment and prevention of cellulite in mammalian skin and for promoting body slimming by reducing localized fatty excesses. More specifically, the invention provides skin care compositions that can be used to inhibit lipogenesis and/or stimulate lipolysis in human adipocytes.

BACKGROUND OF THE INVENTION Cellulite is a term applied to a skin condition associated with the lumps, bumps and dimples that appear on the thighs of many women. Cellulite primarily afflicts the thighs and buttocks but may also be present on the stomach and upper arms. This condition is frequently described as "orange peel skin", "mattress phenomena" or the "cottage cheese effect." Cellulite afflictions are a stubborn problem causing emotional and psychological distress to many women. Although the etiology of cellulite is poorly understood, the main etio logical factor appears to be local accumulation of fat in a regional compartment.

Adipocytes are the principle cells implicated in fat storage by adipose tissue. It has been proposed that the anatomical structure of subcutaneous adipose tissue is the major cause of cellulite. The histological studies of subcutaneous tissues from men and women suggest that the fat lobules are larger and more vertical in women than men. As a result, these larger, less restricted lobules can express outward against the dermis causing the bumps and dimples characteristic of cellulite. The femoral subcutaneous fat deposits in women also tend to be more lipogenic and less lipolytic than abdominal subcutaneous or visceral fat due to the difference in the distribution of α and β adrenergic receptors on adipocytes in these different regions. Increased lipolysis or fat reduction of these selected subcutaneous adipose sites may lead to a reduction or the prevention of cellulite.

Among the methods for stimulating lipolysis, the most commonly known and used is that which consists in inhibiting the phosphodiesterase in order to prevent or at least limit the rate of degradation of cyclic AMP. In effect, the phosphodiesterase destroys cyclic AMP by transforming it into 5' AMP so that it cannot function as a lipolysis activator. Topical application for the treatment of cellulite of agents capable of distributing or reducing local fat accumulation by lipolytic action thereby improving the aesthetic appearance of the skin has been used. Among the common agents for treatment of cellulite as slimming agents are xanthine analogs such as caffeine or theophyUine. These agents block the antilipolytic action of adenosine, a potent endogenous inhibitor of lipolysis.

Other known methods in lipolysis stimulation are achieved by inhibiting phosphodiesterase in order to prevent or at least limit the degradation of cAMP. Xanthine based adenosine antagonists such as caffeine or theophyUine are also known to be effective phosphodiesterase inhibitors.

Other existing methods for the treatment of cellulite have been the stimulation of adenylate cyclase to increase cAMP levels (β adreiiergic agonists) or to block the antilipolytic inactivation of adenylate cyclase (α-2-adrenergic antagonists). Greenway et al. (U.S. Pat. No. 4,588,724) disclose that isoproterenol, a known β agonist (β adrenergic stimulator), is effective for the treatment of cellulite by stimulating lipolysis. Greenway et al. (U.S. Pat. Nos. 4,588,724 and 4,525,359) disclose that creams based on yohimbine, a known α-2-blocker applied to women's skin showed a decrease in thigh circumference. Soudant et al. (U.S. Pat. No. 5,194,259) disclose a Ginkgo biloba, a known α-2-blocker, as a lipolytic agent in combination with at least one other α-2-blocker in a slimming cosmetic composition.

Moreover, it has also been known to use certain oleo soluble vegetable extracts which, according to a different mechanism, can also act as a slimming agent. For instance, in U.S. Pat. No. 4,795,638 there is disclosed a thermo slimming cosmetic composition containing an oil-soluble plant extract having slimming action. Representative of these oil-soluble plant extracts are vegetable extracts including, principally, those of climbing ivy (Hedera helix), arnica (Arnica montana), rosemary (Rosmarinus officinalis N), marigold (Calendula officinalis), sage (Salvia officinalis N), ginseng (Panax ginseng), St. Johns-wart (Hypericum perforatum), ruscus (Ruscus aculeatus), meadowsweet (Filipendula ulmaria L) and orthosiphon (Ortosifon stamincus Benth), as well as mixtures of these vegetable extracts.

Conjugated linoleic acid (CLA) is a collective acronym connoting a class of positional and geometric conjugated dienoic isomers of the long chain (Cl 8) fatty acid octadecadienoic acid. As used herein, the term CLA refers to the cis and trans forms of 9,11- 10,12- and 11,13 -octadecadienoic acid. It is to be understood that as used herein the term CLA includes CLA the free fatty acid form as well as derivatives of CLA. Derivatives include those derived from substitution of the carboxyl group of the acid, such as esters (e.g., triglyceride esters, monoglyceride esters, diglyceride esters, phosphoesters), amides, salts (e.g., alkali metal, alkali earth metal, and ammonium salts), as well as forms derived from substitution of the C18 carbon chain, such as hydroxy and/or β hydroxy derivatives.

CLA is known to be anticarcinogenic and antitumorogenic. It was proposed that the anticarcinogenic effect of CLA may be attributed to its antioxidative properties Chin et al, (1993) J. Agric. Food Chem. 528: 262-271 and the antitumorogenic effects may be mediated by the inhibition of prostaglandin E2 synthesis Liu and Belury, Cancer Lett. 127: 15-22 (1998). Dietary CLA supplementation has been reported to regulate body fat and accumulation and retention in mice rats and chicks Belury et al., (\ 996) Nutr. Cancer 26: 149- 157;

Satory and Smith, (1999) J. Nutr. 129: 92-97; Park, Y., et al, (1999) Lipids 34: 235- 241.

It is an object of the present invention to provide methods for reducing or preventing cellulite in mammalian skin, particularly in human skin. It is also an object of the present invention to provide topically applied compositions comprising safe and effective amounts of conjugated linoleic acid and an imidazole antifungal agent for use a skin care composition for reducing or preventing cellulite.

These and other objects will become readily apparent from the detailed description, which follows.

SUMMARY OF THE INVENTION Compositions and methods for treating and/or preventing cellulite, and for promoting body slimming by administering a safe and effective amount of a skin care composition are provided. The disclosed composition comprises conjugated linoleic acid (CLA), and an imidazole or a triazole antifungal agent in combination with a pharmaceutically acceptable carrier. More particularly, the composition comprises effective amounts of either a mixture of conjugated linoleic acid isomers, or a single isomer, such as, but not limited to, a 9, 11 isomer, a 10, 12 isomer or an 11, 13 isomer of CLA. For example, a 10-trans, 12-cis (lOt, or a 12c) 9-cis, W-trans (9c, l it) isomer of CLA can be used in combination with an antifungal imidazole or triazole agent, such as, but not limited to, ketoconazole (imidazole) or terconazole (triazole) and an acceptable carrier. The topical administration of the disclosed skin care compositions is predicted to promote the reduction of adipose tissue accumulation and to prevent de novo lipogenesis. Thus, administration of the compositions is intended to improve dermal appearance by preventing the accumulation of subcutaneous adipose tissues, reducing adipose tissue mass, and decreasing the occurrence and severity of cellulite in mammalian skin.

The present invention further relates to a skin care composition comprising from about 0.01% to about 40%, by weight, conjugated linoleic acid and about 0.01% to about 40% by weight ketoconazole in a package for said skin care composition. In particular embodiments, the skin composition of the invention compare from about .025% - 10% conjugated linoleic acid and imidazole antifungal agent. Unless otherwise indicated, all percentages and ratios used herein are by weight of the total composition. All weight percentages, unless otherwise indicated, are on an actives weight basis. All measurements made are at approximately 25° C, unless otherwise designated. In an alternative embodiment, the invention provides compositions suitable for use in the cellulite treatment/preventive use disclosed herein. Suitable skin care compositions for use in the methods described herein include compositions generally comprising at least one isomer of conjugated linoleic acid (CLA) in combination with an imidazole or triazole antifungal agent, (referred to herein as an AZOLE agent) and a pharmaceutically acceptable carrier. Generally speaking, suitable compositions will comprise CLA and AZOLE at concentrations ranging from about 0.01% to about 40% by weight. In addition, the compositions contemplated by the invention may also include an additional agent selected from the group consisting of phosphodiestrase inhibitors, oleosoluble vegetable extracts, herbal extracts, botanical extracts, hydroxy acids, a desquamatory agent, a sunscreen agent, an anti-oxidant, a retinoid or a mixture of any of the above-listed agents.

In a second alternative embodiment, the invention also disclose the use of a composition comprising at least one conjugated linoleic acid isomer, and an AZOLE (e.g., an imidazole or triazole) antifungal agent and a pharmaceutically acceptable carrier to stimulate lipolysis in adipoctes localized in a subcutaneous region of mammalian skin that is characterized by adipose tissue accumulation. This embodiment of the invention can be practiced for the purpose of treating or preventing the occurrence of cellulite in the skin region selected for treatment. For example, a composition of the invention formulated for topical application to the skin could be applied to the hip and/or thigh region of a female human patient's leg. In a third alternative embodiment, the invention discloses the use of the compositions described herein to inhibit de novo lipogenesis in adipocytes localized , in a subcutaneous region of mammalian skin that is characterized by adipose tissue accumulation, and which is therefore, likely to become a site where cellulite is likely to appear. This embodiment of the invention can also be utilized to promote body slimming by reducing localized fatty excesses.

The invention disclosed herein also discloses the use of conjugated linoleic acid and an antifungal imidazole agent to manufacture topical skin care compositions suitable for use in anyone of the methods disclosed herein.

In particular embodiments, the skin care compositions of the invention comprise from- about 0.010% to about 40% of the active agents (i.e., conjugated linoleic acid and antifungal imidazole). For example, a skin care composition of the invention may comprise about 0.01%, 0.015%, 0.02%, 0.025%, 0.030%, 0.050%,

0.075%, 0.10%, 0.15%, 0.20%, 0.25%, 0.30%, 0.50%, 0.75%, 0.80%, 1%, 2.5%, 5%, 7.5%, 8.5%, 9.5%, 10%, 12%, 15%, 17.5%, 20%, 25%, 30%, 35%, 38% or 40% of each of the active ingredients (e.g. CLA and AZOLE) disclosed herein. It is to be understood that the amount of conjugated linoleic acid and imidazole antifungal agent can be present in the combination at the same relative percentage by weight or at different relative percentages by weight. The compositions of the invention may be provided with information about and/or instructions on the use of the combination to prevent and/or treat cellulite. As used herein the term "safe and effective amount" means an amount of a compound or composition sufficient to significantly induce a positive benefit, preferably a positive skin appearance or feel benefit, including independently the benefits disclosed herein, but low enough to avoid serious side effects, i.e., to provide a reasonable benefit to risk ratio, within the scope of sound judgment of the skilled artisan.

BRIEF DESCRIPTION OF THE DRAWINGS Figures 1A and IB provide graphic representations of the effects of conjugated linoleic acid (CLA) and ketoconazole (KETO) either alone or in various combinations on lipolysis of human adipocytes isolated from two different human patients. The bar graphs provided in Figures 1A and IB indicate the amount of glycerol released from adipocytes in response to stimulation with: CLA alone, lμM CLA (column 1), 10 μM CLA (column 2), 100 μM CLA (column 3); KETO alone 1 μM KETO (column 4), 10 μM KETO (column 5), 100 μM KETO (column 6); or combinations comprising CLA and KETO at various concentrations 10 μM CLA and 10 μM KETO (column 7), 100 μM CLA and 10 μM KETO (column 8), 10 μM CLA and 100 μM KETO (column 9), 100 μM CLA and 100 μM KETO (column 10), 100 μM CLA and 0.1 μM KETO

(column 11), 0.1 μM CLA and 100 μM KETO (column 12), 100 μM CLA and 1 μM KETO (column 13), 1 μM CLA and 100 KETO (column 14), and 0.1 μM CLA and 0.1 μM KETO (column 15).

Figure 2 is a graphic representation of the effects of conjugated linoleic acid (i.e., 10- trans, \2-cis conjugated linoleic acid (CLA)) the AZOLE ketoconazole (KETO)), and combinations comprising CLA and KETO on lipolysis of human adipocytes isolated from two alternative sources of adipose tissue (dark bars indicate adipocytes obtained from adipose tissue obtained from a thigh/hip region; light bars indicate adipocytes obtained from abdominal tissue). The concentration of each agent is indicated in the figure legend. All concentrations are in μM. L011901H adipocytes were obtained from subcutaneous adipose tissue obtained from a patient undergoing thigh/hip liposuction. L011701 adipocytes were obtained from subcutaneous adipose tissue obtained from a patient undergoing abdominal liposuction. The data is displayed as fold increase in glycerol release relative to the concentration of glycerol released by adipocytes cultured in the presence of vehicle only.

Figure 3 is a graphic representation of the effects of linoleic acid, a 9-cis, 1 l-trans CLA isomer, a IQ-trans, 12-cis CLA isomer on de no o lipogenesis in cultured human adipocytes. The data is displayed as percentage of ¹⁴C-glucose incorporation into total cellular lipids based on vehicle only controls.

Figure 4 provides a graphic representation of the effects of a combination of CLA and KETO on lipolysis in human adipocytes obtained from subcutaneous human adipose tissue (dark bars indicate adipocytes obtained from adipose tissue obtained from a thigh/hip region; light bars indicate adipocytes obtained from abdominal tissue). The data is displayed as fold increase in glycerol release relative to the amount of glycerol that is released in response to stimulation with a negative control sample. CLA designates conjugated linoleic acid, KETO designates ketoconazole, D3MX designates Isobutylmethylxanthine. All of the values defining concentrations are in μM.

DETAILED DESCRIPTION OF THE INVENTION The present invention now will be described more fully hereinafter. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art. Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.

Compositions and methods for controlling or reducing localized fatty excesses or cellulite are provided. In one embodiment, the compositions comprise conjugate linoleic acid (CLA), and an antifungal AZOLE in combination with a pharmaceutically acceptable carrier. Conjugate linoleic acid or CLA is a mixture of isomers that can be formed from 9 cis, 12 czs-octadecadienoic acid (linoleic acid) which can, theoretically, be autoxidized or alkali-isomerized into different conjugated geometric isomers including, but not limited to, 9 cis, 11 cis; 9 cis, 11 trans; 9 trans, 11 cis; 9 trans, 11 trans; 10 cis, 12 cis; 10 cis, 12 trans; 10 trans, 12 cis and 10 trans,

12 trαrø; 11 cw, 13 cis; 11 cis, 13 trans; 11 tr rø, 13 ciy; or 11 trans, 13 trαrø.

Dietary CLA, particularly the 10 cis, 12 trans isomer, has direct effects on the proliferation and differentiation of cultured murine adipocytes Satroy, D. L. and Smith, S. B., J Nutr. (1999) 129:92-97; Park, Y., et al, (1999) Lipids 34:235-241. Animals fed standard preparations of CLA consistently gain less weight than non- CLA fed controls. This can be a commercial disadvantage, in that it is often desirable to increase weight gain and rate of gain in animals raised to be food sources. This effect of CLA can be seen in numerous papers including, for example, Wong, M. W., et al, Anticancer Research 17:987-994 (1997); Hayek, M. G., et al, J. Nutr. 129:32- 38 (1999); West, D. B., et al, (1998) Am. J. Physiol 275 (Regulatory hitegrative Comp. Physiol. 44): R667-R672; Cesano, A., etal, (1998) Anticancer Research 18:1429-1434.

As used herein the term "AZOLE antifungal agent" refers to agents having five-membered ring structures that contain two nitrogen atoms. More specifically the term encompasses imidazole and triazole agents such as: bifonazole, butoconazole, croconazole, clotrimazole, miconazole, fluconazole, econazole, fenticonazole, flutriazole, itraconazole, ketoconazole, omoconazole, oxiconazole, sertaconazole, sulconazole, terconazole, and tioconazole. Generally speaking the efficacy of all antifungal imidazole (AZOLE) agents is principally attributed to inhibition of several cytochrome P450 enzyme systems such as 14α-demethylase (P45014DM), 11 β-hydroxylase, C-17, 20-lyase, cholesterol side- chain cleavage enzyme). The 14-α-demethylase enzyme is involved in the sterol biosynthesis pathway and the synthesis of ergosterol from lanosterol. Ergosterol is a necessary component of the fungal cytoplasmic membrane. Presumably the antifungal activity (e.g., fungistatic or fungicidal) of this class of agents is therefore attributed to alterations of fungal cell membranes, resulting in increased membrane permeability, secondary metabolic effects, and growth inhibition. In addition some imidazole agents, such as miconazole, seems to be attributed to direct physiochemical effects on the fungal cell membrane.

Ketoconazole (KETO) which is the representative imidazole used in the examples presented herein, is structurally similar to miconazole and clotrimazole (Sonino N, (1987) New Eng. J. ofMed. 317: 812-817; Sohn C. A., (1982) Clin. Pharm., 1:211 -22A. KETO is an approved and marketed antifungal drug. The process of making ketoconazole is well known and described. This compound has good oral bioavailability. Ketoconazole is a white odorless, tasteless powder. It is stable at room temperature and requires acidity for dissolution and absorption. Ketoconazole is commercially available from Sigma Chemical Company (St Louis, MO).

Ketoconazole has been used as an oral and topical antimycotic agent (US Patent No. 6,080,744) with broad spectrum activity (against fungi, yeast and dermatophytes) and low toxicity. Chemically, ketoconazole is a cw-l-acetyl-4-[4-{[2- (2,4-dichlorophenyl)-2-(lH-imidazol-l-ylmethyl)-l,3-dioxolan-4-yl] methoxy}phenyl] piperazine. The mechanism of action of ketoconazole is by inhibiting the synthesis of ergosterol in fungi and cholesterol in mammalian cells. As mentioned above, ergosterol is the main sterol in fungal cell membranes. It is to be understood that although the examples provided herein utilize KETO, the invention contemplates the use of any of the above-listed imidazole antifungal agents in combination with CLA, and the use of KETO for the experiments disclosed herein is not intended to limit the scope of the invention.

Ketoconazole is known to be a PPARγ activator which lowers blood glucose in diabetic animals and man. PPARγ activators have shown clinical utility as anti- diabetic drugs. However, these drugs have a side effect of causing significant weight gain. Differentiation of preadipocytes to adipocytes by ketoconazole has not been shown to be associated with the lipid accumulation normally seen with other PPARγ activators. Long term treatment with ketoconazole does not cause significant weight gain unlike other PPARγ activators. Glucocorticoids like cortisol and cortisone are essential for abundant lipid accumulation during the differentiation of preadipocytes to adipocytes. A cortisol anti-activator effect of any molecule would have significant effect on lipid accumulation in the adipocyte. Ketoconazole may have properties similar to a cortisol anti-activator (Marin, US Patent # No. 5,849,740, 1998).

More particularly, the compositions of the invention comprise an effective amount of conjugated linoleic acid in combination with an imidazole antifungal agent. In particular embodiments, the skin care compositions of the invention comprise from about 0.010% to about 40% of the active agents (i.e., conjugated linoleic acid and antifungal imidazole). For example, a skin care composition of the invention may comprise about 0.01%, 0.015%, 0.02%, 0.025%, 0.030%, 0.050%, 0.075%, 0.10%, 0.15%, 0.20%, 0.25%, 0.30%, 0.50%, 0.75%, 0.80%, 1%, 2.5%, 5%, 7.5%, 8.5%, 9.5%, 10%, 12%, 15%, 17.5%, 20%, 25%, 30%, 35%, 38% or 40% of each of the active ingredients (e.g. CLA and AZOLE) disclosed herein. In a particular embodiment the invention provides a composition comprising from 0.025% - 10% conjugated linoleic acid (e.g. trans-10, cis-12 CLA) and 0.025% - 10% ketoconzaole. The composition may comprise a single CLA isomer, such as a lOt, 12c-CLA or a 9, 11 isomer or mixture. Alternatively, the composition can comprise a mixture of two or more CLA isomers. For example, a composition according to the invention could comprise a lOt, 12c-CLA isomer in combination with an 11, 13 isomer such as 11 -cis, 13-trans (1 lc, 13t). For topical formulation both the CLA and AZOLE are each provided at a concentration of at least about 0.01%. For oral administration, ketoconazole should be given at 100 mg - 1000 mg and the lOt, 12c-CLA isomer at 1000 mg- 5000 mg. By "effective amount" is an amount sufficient to provide cellulite reduction or prevention. It is accordingly an object of this invention to provide a composition which can reduce or eliminate cellulite or fat build-ups. Cellulite, as noted above, results from an accumulation of fatty materials and water imprisoned in a matrix made up of more or less water tight compartments. This matrix is comprised of elements of fundamental matter and more particularly of proteoglycons which are polymeric.

As discussed in more detail below, the compositions of the invention can be formulated for oral or topical administration. For oral administration, the composition is administered in a safe and effective dosage for cellulite prevention or reduction and for the treatment of obesity. Oral administration of the composition results in decreased weight gain. Generally, for topical use, the composition is presented in the form of a cream or oil for topical administration, usually in the form of a cream. Thus, the methods of the invention encompass application of the composition used for local slimming and for fighting cellulite and for an entire body slimming effect if used orally.

The composition according to the invention was conceived for fighting conditions of external appearance and figure, such as cellulite, general or local obesity, relaxing or ptosis of the skin and excessive secretion of fat (seborrhoea), which reveal profound bodily dysfunctions. Thus, the compositions of the invention demonstrate slimming and "rejuvenating" effects on appearance. By using the cream of the invention, positive results maybe obtained in terms of slimming and of reducing cellulite that has accumulated in a localized area of the body. That is, the composition is useful for preventing local fat and cellulite accumulation. The skin becomes toned and fortified and the user feels no need, from an aesthetic point of view, to use another cream as a supplementing thereof.

The compositions used in the present invention can comprise, consist of, or consist essentially of the essential elements and limitations of the invention described herein, as well any of the additional or optional ingredients, components, or limitations described herein.

References herein to a "patient" are intended to refer to human subjects with a desire to treat or prevent cellulite. References herein to "animals" can be, but are not limited to, a rodent, a mammal (such as a bovine, an ovine, a caprine, a primate and a human), and an avian animal (such as a chicken, a duck, a turkey, and a quail).

The improved efficacy resulting from the use of a composition comprising CLA and an AZOLE is demonstrated by the synergistic effect that a combination comprising a lOt, 12c-CLA isomer and ketoconazole has on the stimulation of lipolysis (breakdown of triglycerides) by cultured human adipocytes. As disclosed herein, a composition comprising a combination of CLA and Keto enhances the in vitro lipolysis relative to the amount of activity observed when either agent is used alone. This new understanding permits one skilled in the art to produce compositions that comprise specific CLA isomers combined with an AZOLE that promote a desirable effect when administered while reducing or eliminating one or more undesirable effects. The compositions of the invention may be administered orally or applied topically.

The compositions of the present invention are exemplified herein by combinations comprising an indicated CLA isomer and ketoconazole, but it is understood that suitable compositions may also contain other CLA isomers, as well as other fatty acids, or alternative AZOLE agents. The isomers can be extracted from natural sources or prepared using enzymatic or biological methods known to those skilled in the art. When making preparations of the invention, the source of the isomers is not critical, one should merely determine that the lOt, 12c isomers is provided in the composition at a percentage of at least about 0.01% to about 40% and that ketoconazole is present at a percentage of at least about 0.01% to about 40%.

The commercial CLA can be made from oils having at least 50% linoleic acid and which can contain 95%. linoleic acid or more. The cost of CLA isomers increases with increasing purity. Bulk conjugated linoleic acid isomers in a significantly purified form (98%+pure) are commercially available from Matreya, Inc. (Pleasant Gap, Pa.). However, since the source of the isomer is not critical, it is economically advantageous to use the least expensive source of CLA to make preparations according to the invention. The compositions can comprise a (lOt, 12c) isomer or a (9c, 1 It) isomer of

CLA isomer mixed with other CLA isomers. Although, preferably the composition comprises only a single CLA isomer. The isomers are heat stable and can be used as is, or dried and powdered. Purified isomers of CLA are also commercially available from Matreya. In the method of the present invention for reducing cellulite, an effective amount of a CLA/AZOLE composition prepared as a topical skin care composition is administered to the patient. Since CLA is a natural food ingredient and antifungal imidazole agents are commonly used ingredients in anti-fungal topical applications both of the active agents of the disclosed compositions are relatively non-toxic and the amount of CLA/AZOLE that can be administered is not critical as long as it is enough to be effective to achieve the desired outcome noted herein.

The methods of the present invention may take several embodiments. In the preferred embodiment, the CLA/AZOLE mixture is administered in a pharmaceutical or cosmetic composition containing a safe and effective dose of the CLA and AZOLE. A pharmaceutically or cosmetically acceptable carrier may additionally be provided.

In some embodiments, the formulations of the invention comprise a pharmaceutically acceptable carrier. By "pharmaceutically acceptable carrier" is intended a carrier that is conventionally used in the art to facilitate the storage, administration, and/or the healing effect of the therapeutic ingredients. A carrier may also reduce any undesirable side effects of the CLA and/or the AZOLE agent present in the composition. A suitable carrier should be stable, i.e., incapable of reacting with other ingredients in the formulation. It should not produce significant local or systemic adverse effects in recipients at the dosages and concentrations employed for treatment. Such carriers are generally known in the art. Suitable carriers for this invention are those conventionally used large stable macromolecules such as albumin, for example, human serum albumin, gelatin, collagen, polysacchari.de, monosaccharides, polyvinyl-pyrrolidone, polylactic acid, polyglycolic acid, polymeric amino acids, fixed oils, ethyl oleate, liposomes, glucose, sucrose, lactose, mannose, dextrose, dextran, cellulose, sorbitol, polyethylene glycol (PEG), and the like. Slow- release carriers, such as hyaluronic acid, may also be suitable. See particularly Prisell et al (1992) Int. J. Pharmaceu. 85:51-56, and U.S. Patent No. 5,166,331. Other acceptable components in the composition include, but are not limited to, pharmaceutically acceptable agents that modify isotonicity including water, salts, sugars, polyols, amino acids, and buffers. Examples of suitable buffers include phosphate, citrate, succinate, acetate, and other organic acids or their salts and salts that modify the tonicity such as sodium chloride, sodium phosphate, sodium sulfate, potassium chloride, and can also include the buffers listed above.

The method for formulating a pharmaceutical composition is generally known in the art. A thorough discussion of formulation and selection of pharmaceutically acceptable carriers, stabilizers, and isomolytes can be found in Remington's Pharmaceutical Sciences (18 ed.; Mack Pub. Co.: Eaton, Pennsylvania, 1990), herein incorporated by reference.

In the preferred embodiment of the invention, a cosmetically acceptable vehicle is comprised either of water or of a water/solvent blend. The solvent is optimally chosen from propylene glycol, ethanol, butylene glycol, and polyethylene glycols of various molecular weights. Vehicles other than water can include liquid or solid emollients, solvents, humectants, thickeners and powders. An especially preferred nonaqueous carrier is a polydimethyl siloxane and/or a polydimethyl phenyl siloxane. Silicones of this invention may be those with viscosities ranging anywhere from about 10 to 10,000,000 centistokes at 25°C. Especially desirable are mixtures of low and high viscosity silicones. These silicones are available from the General Electric Company under trademarks Vicasil, SE and SF and from the Dow Corning Company under the 200 and 550 Series. Amounts of silicone which can be utilized in the compositions of this invention range anywhere from 5% to 95%, preferably from 25% to 90% by weight of the composition. The cosmetically acceptable vehicle will usually form from 5% to 99.9%, preferably from 25% to 80% by weight of the emulsion, and can, in the absence of other cosmetic adjuncts, form the balance of the composition. The compositions used in the present invention also contain a dermatologically acceptable carrier. The phrase "dermatologically-acceptable carrier", as used herein, means that the carrier is suitable for topical application to the skin, has good aesthetic properties, is compatible with the active ingredients of the present invention and any other components, and will not cause any untoward safety or toxicity concerns. A safe and effective amount of carrier is from about 50% to about 99.99%, preferably from about 99.9% to about 80%, more preferably from about 98% to about 90%, most preferably from about 95% to 90% of the composition.

The carrier can be in a wide variety of forms. For example, emulsion carriers, including, but not limited to, oil-in-water, water-in-oil, water-in-oil-in-water, and oil- in-water-in-silicone emulsions, are useful herein. These emulsions can cover a broad range of viscosities, e.g., from about 100 cps to about 200,000 cps. These emulsions can also be delivered in the form of sprays using either mechanical pump containers or pressurized aerosol containers using conventional propellants. These carriers can also be delivered in the form of a mousse. Other suitable topical carriers include anhydrous liquid solvents such as oils, alcohols, and silicones (e.g., mineral oil, ethanol, isopropanol, dimethicone, cyclomethicone, and the like); aqueous-based single phase liquid solvents (e.g., hydro-alcoholic solvent systems); and thickened versions of these anhydrous and aqueous-based single phase solvents (e.g., where the viscosity of the solvent has been increased to form a solid or semi-solid by the addition of appropriate gums, resins, waxes, polymers, salts, and the like). Examples of topical carrier systems useful in the present invention are described in the following four references all of which are incorporated herein by reference in their entirety: "Sun Products Formulary" Cosmetics & Toiletries, Vol. 105, pp. 122-139 (December 1990); "Sun Products Formulary", Cosmetics & Toiletries, Vol. 102, pp. 117-136 (March 1987); U.S. Pat. No. 4,960,764 to Figueroa et al, issued Oct. 2, 1990; and U.S. Pat. No. 4,254,105 to Fukuda et al, issued Mar. 3, 1981.

The carriers of the skin care compositions can comprise from about 50% to about 99% by weight of the compositions used in the present invention, preferably from about 75% to about 99%, and most preferably from about 85% to about 95%. Preferred cosmetically and/or pharmaceutically acceptable topical carriers include hydroalcoholic systems and oil-in-water emulsions. When the carrier is a hydro- alcoholic system, the carrier can comprise from about 0% to about 99% of ethanol, isopropanol, or mixtures thereof, and from about 1% to about 99% of water. More preferred is a carrier comprising from about 5% to about 60% of ethanol, isopropanol, or mixtures thereof, and from about 40% to about 95% of water. Especially preferred is a carrier comprising from about 20% to about 50% of ethanol, isopropanol, or mixtures thereof, and from about 50% to about 80% of water. When the carrier is an oil-in-water emulsion, the carrier can include any of the common excipient ingredients for preparing these emulsions. A more detailed discussion of suitable carriers is found in U.S. Pat. No. 5,605,894 to Blank et al, and in PCT application WO 97/39733, published Oct. 30, 1997, to Oblong et al, both herein incorporated by reference in their entirety.

The compositions used in the present invention may optionally comprise additional materials including slimming agents as well as additional actives useful in providing cellulite control. For example, a second anti-cellulite agent can be included in the composition. Among these agents are phosphodiesterase inhibitors (e.g., xanthine derivatives such as theophyUine, caffeine, theobromine or salts thereof such as aminophylline) and certain oleosoluble vegetable extracts, including, principally, those of climbing ivy (Hedera helix), arnica (Arnica montana), rosemary (Rosmarinus officinalis N), marigold (Calendula officinalis), sage (Salvia officinalis N), ginseng (Panax ginseng), St. Johns-wart (Hypericum perforatum), ruscus (Ruscus aculeatus), meadowsweet (Filipendula ulmaria L) and orthosiphon (Ortosifon stamincus Benth), as well as mixtures of these vegetable extracts, all of which are disclosed in U.S. Pat. No. 4,795,638, herein incorporated by reference. A Xanthines can be employed at a concentration such that it is present in a proportion from about 0.05% to about 20%> by weight of the composition.

Also useful are herbal and/or botanical extracts such as those disclosed in U.S. Pat. Nos. 5,705,170 and 5,667,793, both of which are herein incorporated by reference. Mixtures of any of above additional materials may also be used. The compositions used in the present invention may optionally comprise additional skin actives. Non-limiting examples of such skin actives include hydroxy acids such as salicylic acid; desquamatory agents such as zwitterionic surfactants; sunscreens such as 2-ethylhexyl-p-methoxycinnamate, 4,4'-t-butyl methoxydibenzoyl-methane, octocrylene, phenyl benzimidazole sulfonic acid; sun-blocks such as zinc oxide and titanium dioxide; anti-inflammatory agents; corticosteroids such as hydrocortisone, methylprednisolone, dexamethasone, triamcinolone acetconide, and desoxametasone; anesthetics such as benzocaine, dyclonine, lidocaine and tetracaine; antipruitics such as camphor, menthol, oatmeal (colloidal), pramoxine, benzyl alcohol, phenol and resorcinol; anti-oxidants/radical scavengers such as tocopherol and esters thereof; chelators; retinoids such as retinol, retinyl palmitate, retinyl acetate, retinyl propionate, and retinal; hydroxy acids such as glycolic acid; keto acids such as pyruvic acid; N-acetyl-L-cysteine and derivatives thereof; benzofuran derivatives; and skin protectants. Mixtures of any of the above mentioned skin actives may also be used. A more detailed description of these actives is found in U.S. Pat. No. 5,605,894 to Blank et al. (previously incorporated by reference). Preferred skin actives include hydroxy acids such as salicylic acid, sunscreen, antioxidants and mixtures thereof. Other conventional skin care product additives may also be included in the compositions used in the present invention. For example, urea, guanidine, glycerol, petrolatum, mineral oil, sugar esters and polyesters, polyolefins, methyl isostearate, ethyl isostearate, cetyl ricinoleate, isononyl isononanoate, isohexadecane, lanolin, lanolin esters, cholesterol, pyrrolidone carboxylic acid/salt (PCA), trimethyl glycine (betaine), tranexamic acid, amino acids (e.g., serine, alanine), panthenol and its derivatives, collagen, hyaluronic acid, elastin, hydrolysates, primrose oil, jojoba oil, epidermal growth factor, soybean saponins, mucopolysaccharides, and mixtures thereof may be used. Other suitable additives or skin actives are discussed in further detail in PCT application WO 97/39733, published Oct. 30, 1997, to Oblong et al, herein incorporated by reference in its entirety.

The compositions used in the present invention are generally prepared by conventional methods such as are known in the art of making topical compositions. Such methods typically involve mixing of the ingredients in one or more steps to a relatively uniform state, with or without heating, cooling, application of vacuum, and the like. Non-limiting examples of the product form can be a gel, emulsion, lotion, cream, ointment, solution, liquid, etc.

The methods of the present invention are useful for preventing cellulite, especially in the subcutaneous, dermis and epidermis tissues of mammalian skin. The methods of the present invention involve topically applying to the skin an effective amount of the skin care composition of the present invention. The amount of the composition which is applied, the frequency of application and the period of use will vary widely depending upon the concentration of CLA (e.g. 10-trans, \2-cis conjugated linoleic acid), AZOLE (e.g., ketoconazole) and/or other component of a given composition and the degree of cellulite fading desired.

The skin care compositions used in the present invention can be administered by chronic topical application. By "chronic topical application" is meant continued topical application of the composition over an extended period during the subject's lifetime, preferably for a period of at least about one week, more preferably for a period of at least about two weeks, even more preferably for a period of at least one month, even more preferably for at least about three months, even more preferably for at least about six months, and more preferably still for at least about one year. While benefits are obtainable after various maximum periods of use (e.g., five, ten or twenty years), it is preferred that chronic application continue throughout the subject's lifetime to maintain and/or increase the benefits achieved. Typically applications would be on the order of one to four times per day over such extended periods, however application rates can be more than four times per day, especially on areas particularly prone to agglomerations of fat and water such as the thighs and buttocks. A wide range of quantities of the compositions used in the present invention can be employed to provide a skin appearance and/or feel benefit. Quantities of the present compositions which are typically applied per application are, in mg composition/cm.sup.2 skin, from about 0.1 mg/cm.sup.2 to about 10 mg/cm.sup.2. The method of treating cellulite is preferably practiced by applying a composition in the form of a skin lotion, cream, gel, cosmetic, or the like which is intended to be left on the skin for some aesthetic, prophylactic, therapeutic or other benefit (i.e., a "leave-on" composition). After applying the composition to the skin, it is preferably left on the skin for a period of at least about 15 minutes, more preferably at least about 30 minutes, even more preferably at least about 1 hour, most preferably for at least several hours, e.g., up to about 12 hours.

Another approach to ensure a continuous exposure of the skin to at least a minimum level of conjugated linoleic acid and antifungal imidazole is to apply the compound by use of a patch. Such an approach is particularly useful for problem skin areas needing more intensive treatment. The patch can be occlusive, semi-occlusive or non-occlusive. For example, a composition comprising lb-trans, 12-cis conjugated linoleic acid and ketoconazole could be contained within a patch that is contacted with the skin; or alternatively, a composition comprising CLA and AZOLE could be applied to the skin prior to the application of a patch. The patch can also include additional actives such as chemical initiators for exothermic reactions such as those described in PCT application WO 9701313 to Burkett et al. Preferably the patch is applied at night as a form of night therapy.

The preferred xanthine employed in the invention is caffeine and/or theophyUine due to their availability and optimum efficacy. Caffeine and theophyUine can be, and preferably are naturally-derived, in order to keep with a "natural" character of the inventive compositions.

The xanthine is employed in the inventive method preferably in an amount of at least 0.05%, generally in the amount of from 0.05% to 20%, preferably in the amount of from 0.10% to 10%, optimally in the amount of from 0.5% to 3.0% by weight of the composition in order to maximize efficacy at optimum cost.

Another preferred ingredient employed in the inventive method is an hydroxy acid. The presence of the α hydroxy acid facilitates the increase in the strength and firmness of dental and epidermal layers of the skin. Even more preferably, the hydroxy acid is chosen from lactic acid, glycolic acid, mandelic acid, and mixtures thereof to optimize the efficacy of compositions by increasing percutaneous absorption. In the most preferred embodiment of the invention, in order to maximize the perfonnance of hydroxy acid, inventive compositions contain the L- form of an hydroxy acid. Preferably the amount of the hydroxy acid component present in the composition according to the invention is from 1.5% to 20%, more preferably from 1.5% to 15%, and most preferably from 3.0% to 12.0% by weight of the composition.

An oil or oily material may be present, together with an emulsifier to provide either a water-in-oil emulsion or an oil-in-water emulsion, depending largely on the average hydrophilic-lipophilic balance (HLB) of the emulsifier employed.

Various types of active ingredients may be employed in the method of the present invention. Actives are defined as skin benefit agents other than emollients and other than ingredients that merely improve the physical characteristics of the composition. Although not limited to this category, general examples include sunscreens, tanning agents, skin anti-wrinkling agents, anti-inflammatory agents, skin lighteners and moisturizers.

Sunscreens include those materials commonly employed to block ultraviolet light. Illustrative compounds are the derivatives of PABA, and cinnamate. For example, octyl methoxycinnamate and 2-hydroxy-4-methoxybenzophenone (also known as oxybenzone) can be used. Octyl methoxy-cinnamate and 2-hydroxy-4- methoxy benzophenone are commercially available under the trademarks, Parsol MCX and Benzophenone-3, respectively. The exact amount of sunscreen employed in the emulsions can vary depending upon the degree of protection desired from the sun's UV radiation.

Suitable anti-inflammatory compounds include but are not limited to rosmarinic acid, glycyrrizinate derivatives, αbisabolol, azulene and derivatives thereof, asiaticoside, sericoside, ruscogenin, escin, esculin, quercetin, rutin, betulinic acid and derivatives thereof, catechin and derivatives thereof.

Suitable vasoactive compounds include but are not limited to papaverine, yohimbine, visnadin, khellin, bebellin, nicotinate derivatives.

Surfactants, which are also sometimes designated as emulsifiers, may be incorporated into the cosmetic compositions of the present invention. Surfactants can comprise anywhere from about 0.5% to about 30%, preferably from about 1% to about 15%) by weight of the total composition. Surfactants may be cationic, nonionic, anionic, or amphoteric in nature and combinations thereof may be employed.

Illustrative of the nonionic surfactants are alkoxylated compounds based upon fatty alcohols, taffy acids and sorbitan. These materials are available, for instance, from the Shell Chemical Company under the "Neodol" designation. Copolymers of polyoxypropylene-polyoxyethylene, available under the Pluronic trademark sold by the BASF Corporation, are sometimes also useful. Alkyl polyglycosides available from the Henkel Corporation similarly can be utilized for the purposes of this invention. Anionic-type surfactants may include fatty acid soaps, sodium lauryl sulphate, sodium lauryl ether sulphate, alkyl benzene sulphonate, mono and/or dialkyl phosphates and sodium fatty acyl isethionate. Amphoteric surfactants include such materials as dialkylamine oxide and various types of betaines (such as cocoamido propyl betaine).

Emollients are often incorporated into cosmetic compositions of the present invention. Levels of such emollients may range from about 0.5% to about 50%, preferably between about 5%_> and 30% by weight of the total composition. Emollients may be classified under such general chemical categories as esters, fatty acids and alcohols; polyols and hydrocarbons.

Esters may be mono- or di-esters. Acceptable examples of fatty di-esters include dibutyl adipate, diethyl sebacate, diisopropyl dimerate, and dioctyl succinate. Acceptable branched chain fatty esters include 2-ethyl-hexyl myristate, isopropyl stearate and isostearyl palmitate. Acceptable tribasic acid esters include triisopropyl trilinoleate and trilauryl citrate. Acceptable straight chain fatty esters include lauryl palmitate, myristyl lactate, oleyl eurcate and stearyl oleate. Preferred esters include coco-caprylate/caprate(a blend of coco-caprylate and coco-caprate), propylene glycol myristyl ether acetate, diisopropyl adipate and cetyl octanoate.

Suitable fatty alcohols and acids include those compounds having from 10 to 20 carbon atoms. Especially preferred are such compounds such as cetyl, myristyl, palmitic and stearyl alcohols and acids.

Among the polyols which may serve as emollients are linear and branched chain alkyl polyhydroxyl compounds. For example, propylene glycol, sorbitol and glycerin are preferred. Also useful may be polymeric polyols such as polypropylene glycol and polyethylene glycol. Butylene and propylene glycol are also especially preferred as penetration enhancers.

Exemplary hydrocarbons which may serve as emollients are those having hydrocarbon chains anywhere from 12 to 30 carbon atoms. Specific examples include mineral oil, petroleum jelly, squalene and isoparaffins.

Another category of functional ingredients within the cosmetic compositions of the present invention are thickeners. A thickener will usually be present in amounts anywhere from 0.1% to 20% by weight, preferably from about 0.5% to 10% by weight of the composition. Exemplary thickeners are cross-linked polyacrylate materials available under the trademark Carbopol from the B. F. Goodrich Company. Gums maybe employed such as xanthan, carrageenan, gelatin, karaya, pectin and locust bean gum. Under certain circumstances the thickening function may be accomplished by a material also serving as a silicone or emollient. For instance, silicone gums in excess of 10 centistokes and esters such as glycerol stearate have dual functionality. Cellulosic derivatives may also be employed, e.g., hydroxypropyl cellulose (Klucel HI.RTM.). Many cosmetic compositions, especially those containing water, must be protected against the growth of potentially harmful microorganisms. Preservatives are, therefore, necessary. Suitable preservatives include alkyl esters of p- hydroxybenzoic acid, hydantoin derivatives, propionate salts, and a variety of quaternary ammonium compounds. Particularly preferred preservatives of this invention are methyl paraben, propyl paraben, imidazolidinyl urea, sodium dehydroxyacetate and benzyl alcohol. Preservatives will usually be employed in amounts ranging from about 0.5%> to 2%> by weight of the composition.

Powders may be incorporated into the cosmetic composition employed in the invention. These powders include chalk, talc, Fullers earth, kaolin, starch, smectite clays, chemically modified magnesium aluminum silicate, organically modified montmorillonite clay, hydrated aluminum silicate, fumed silica, aluminum starch octenyl succinate and mixtures thereof.

Other adjunct minor components may also be incorporated into the cosmetic compositions. These ingredients may include coloring agents, opacifiers and perfumes. Amounts of these materials may range anywhere from 0.001% up to 20% by weight of the composition.

The method of the present invention is useful for reducing or preventing the appearance of cellulite, for improving the firmness and elasticity of skin and generally to enhance the quality and flexibility of skin.

The following examples will more fully illustrate the embodiments of this invention, but the invention is not limited thereto. AU parts, percentages and proportions referred to herein and in the appended claims are by weight unless otherwise indicated. The following examples further describe and demonstrate embodiments within the scope of the present invention. The examples are given solely for the purpose of illustration and are not to be construed as limitations of the present invention, as many variations thereof are possible without departing from the spirit and scope of the invention.

MATERIALS AND METHODS Materials Type I collagenase was obtained from Worthington Biochemical (Lakewood, NJ). Bovine serum albumin (BSA), dexamethasone, biotin, pantothenate, Krebs-Ringer buffer, isoproterenol, and isobutyl methylxanthine were purchased from Sigma (St. Louis, MO). Human recombinant insulin was obtained from Boehringer Mannhaim (Indianapolis, IN). The fetal bovine serum was from HyClone (Logan, Utah). The Dublecco Modified Eagles Medium (DMEM) Ham's F-10 Nutrient Broth (1:1 Mixture) and phosphate buffered saline (PBS) was obtained from Biologos (Naperville, IL). All tissue culture flasks and plates were obtained from Corning (Corning, NY).

Methods

Cell Culture: Preparation and maintenance of preadipocytes. The adipocyte precursor cells (preadipocytes) are isolated from subcutaneous adipose tissue as described. The plates are kept at 37°C with 5% CO₂ until ready for use. Differentiation into adipocytes should be initiated immediately. If cells are to be maintained as preadipocytes, they should be fed with preadipocyte medium every other day. Preadipocytes are flat, phase-dark spindle-shaped cells. The cells have a similar appearance in culture to fibroblasts or smooth muscle cells. Greater than 80% of the preadipocytes will differentiate to adipocytes using differentiation medium (DM-2/10). The differentiation efficiency varies depending on the donor. Tissue preparation: The subcutaneous adipose was acquired from elective surgeries (liposuction) with the patient's consent as approved by the hospital histitution Review Boards. The tissues used were from female patients. Other than obesity in some instances, the patients were in good health; no diabetes or other complications were reported. Tissues isolated from either the abdomen or the thigh/hip regions were transported to the laboratory in saline solution and in separate containers within two hours post-surgery. The tissue was washed several times with 2 volumes of Krebs-

Ringer-Bicarbonate (KRB; Sigma Chemical Co., St. Louis, MO) to remove blood.

The tissue was then digested with one volume of collagenase type I (1 g/liter of KRB with 1% BSA) for 60 minutes at 37° C with intermittent shaking. The floating adipocytes were separated from the stromal- vascular fraction by centrifugal force (300 g) for 5 minutes.

Preparation and maintenance of adipocytes: Preadipocytes are differentiated into adipocytes. The plates are kept at 37°C with 5% CO₂ until ready for use. The adipocytes should be fed with adipocyte medium (AM-1) every 3 days. The adipocytes should remain healthy and responsive for at least three weeks. Adipocytes are rounded, lipid-filled cells. Cultured adipocytes contain multiple vesicles termed "locules". These locules are the site of lipid storage and can be visualized by counterstaining with Oil red O.

Differentiation of adipocytes: The stromal cells were trypsinized and plated in multiple- well plates at 40,000 cells/cm for 16 hours to allow attachment. The medium was then changed to Dulbecco's modified Eagle's -Ham's F-10 medium (vol/vol, 1:1) supplemented with 3% fetal bovine serum, 15mM HEPES (pH 7.4), biotin (33 μM), pantothenate (17 μM, Sigma, MO), human recombinant insulin (100 nM, Boerhinger Mannheim, IN), dexamethasone (1 μM), l-methyl-3-isobutylxanthine (0.2 mM), and a PPARγ agonist (1 μM) for 3 days. During the remaining 9-10 day adipocyte differentiation period, the cells were fed every 3 days with the same medium without l-methyl-3-isobutylxanthine and PPARγ agonist supplementation.

Lipolysis Assay: Day 21 differentiated human adipocytes plated in 96 well plates (Falcon, etc.) generated as described (Halvorsen et al, Metabolism. 50(4):407-13, 2001), were used. The medium was removed completely and 150 μl of the test compound resuspended in KRB added to each well. The plates were incubated at

37°C for 5 hrs. 100 μl of KRB from each well was transferred into the corresponding well in another 96-well plate. Distilled water (40 ml) was added to the glycerol assay reagent (GPO-Trinder Reagent A, Sigma, St. Louis MO) and mixed gently by inversion several times. 100 μl of this reagent was added to each well of this new plate. The solutions were mixed well either by pipetting up and down several times or by using the mix function on the plate reader. Incubated at room temperature for 15 minutes. The optical density of each well was measured at 540 nm and converted to glycerol concentration by use of the glycerol standard. The increase in absorbance at 540 nm is directly proportional to glycerol concentration of the sample.

Lipogenesis assay: Stromal Vascular (SV) cells were seeded at a density of 3 x 10⁴/cm² and continuously treated with increasing concentrations (3, 10, or 30 uM) of either linoleic acid, cis-9, tran-11 CLA, or trans-10, cis-12 CLA. A set of control cultures received vehicle (BSA) plus TZD. AU cultures received differentiation media (days 1-3), adipocyte media (days 4-9), and low-glucose (~ 5mM) adipocyte media (days 10-12) prior to measuring de novo lipogenesis. On day 12, C-labeled glucose incorporation into the lipid fraction of the cultures was measured for 2 h and, following lipid extraction, the radioactivity in the lipid fraction was determined by scintillation counting.

Example 1 Lipolytic activities of Active Compounds

The lipolytic activities of 10-trans, 12-cis conjugated linoleic acid, ketoconazole, Isoproterenol and Isobutylmethylxanthine were evaluated in the above-described assay. The results are summarized in Table 1. The data summarize the activity of the various compounds at different concentrations relative to the level of lipolysis observed in the control sample. The data indicate that CLA stimulates lipolysis (e.g. the breakdown of stored triglycerides into fatty acids and glycerol) in human adipocytes.

Table 1 : Effect of test compound on Lipolysis

Substance Fold increase over control

1 μM CLA 2.7 lO μM CLA 4.9

100 μM CLA 6.3

1 μM Ketoconazole 1.4

10 μM Ketoconazole 4.9

100 nM Isoproterenol 1.94

10 nM Isobutylmethylxanthine 4.7

100 nM Isobutylmethylxanthine 3.3

The data presented in Table 2 summarizes the relative activity level (e.g., stimulation of glycerol release expressed as fold increase over an appropriate control) of combinations comprising CLA and KETO compared to the activity of other know stimulators of lipolysis in cultured human adipocytes.

Table 2: Comparison of CLA Isoforms With Known Stimulators of lipolysis

Example 2 Effect of Conjugated Linoleic Acid and/or Ketoconazole on Lipolysis

Lipolysis experiments were performed on adipocytes isolated from 2 different patients (LOl 1701 and LOl 1901H). The effect of 10-trans, 12-cis conjugated linoleic acid (CLA) and ketoconazole (KETO) either alone or in the combinations defined in Table 3 were evaluated. All of the values used to define the compositions listed in table 3 refer to μM concentrations. The lipolysis assay was done according to the above-described method and the results are graphically displayed in Figures 1 A and IB.

Table 3 CLA and KETO Compositions

Agent Column Agent Column

1 CLA 1 lO CLA/lOO KETO 9

10 CLA 2 lOO CLA/lOO KETO 10

100 CLA 3 100 CLA/0.1 KETO 11

1 KETO 4 0.1 CLA/100 KETO 12

10 KETO 5 100 CLA/1 KETO 13

100 KETO 6 1 CLA/100 KETO 14

10 CLA/10 KETO 7 0.1 CLA/0.1 KETO 15

100 CLA/ 10 KETO 8

The data in Figure 1 demonstrates that particular combinations of CLA and

KETO consistently release higher levels of glycerol (e.g., lipolysis). More specifically, stimulation with combinations comprising combination #8 (100 μM CLA and 10 μM KETO), combination #9 (10 μM CLA and 100 μM KETO) and combination #13 (100 μM CLA and 1 μM KETO) resulted in the highest levels of glycerol release. Thus, the lipolytic effects of CLA demonstrated in Example 1 is augmented when CLA is used in combination with the AZOLE ketoconazole. Further, the lipolytic effect of the combination is at least as efficacious than the effect of using either agent alone. Example 3 Effect of CLA on Lipolysis in Cultured Human Adipocytes

Mature adipocytes plated in 96-well plates were washed once with Krebs- Ringer buffer supplemented with lg / liter glucose and were incubated with or without lipolytic reagent (120 μl per well) for 3 hours at 37°C. The conditioned media (100 μl/ well) were removed and the amount of released glycerol determined using the triglyceride reagent A (GPO-Trinder, Sigma, St. Louis, MO) following manufacturer's instruction.

Lipolysis was assayed by measuring the glycerol released from the mature adipocytes obtained from two different sources (e.g., LOl 1901 H and LOl 1701) in response to the control agent Isoproterenol (Iso), conjugated linoleic acid (CLA) alone, ketoconazole (KETO) alone, and compositions comprising CLA and KETO in combination. The concentrations of the active ingredients present in each sample are indicated in the figure legend as uM. The results indicate that both CLA and KETO alone stimulate the break down of stored triglycerides into fatty acids and glycerol in cultured human adipocytes. These data also established that the combination of the two agents is more efficacious than either agent alone, and that the stimulatory effects of the all of the agents tested is more effective on adipocytes isolated from subcutaneous tissue obtained from the hip/thigh region as opposed to subcutaneous adipose tissue obtained from the abdominal region.

Example 4 Effect of CLA and Ketoconazole on Lipolysis in Cultured Human Adipocytes The effect of compositions comprising CLA and ketoconazole (KETO) on lipolysis were also evaluated in the above-described assay, using human adipocytes obtained from subcutaneous adipose tissue removed during liposuction surgery of either the abdomen (light bars) or the thigh hip region (dark bars) of female patients. The results presented in figure 4 indicate that a combination of CLA and KETO is more effective in stimulating lipolysis than either compound alone. More specifically, the results indicate that combinations comprising either: 50 μM CLA and 5 μM KETO; 5 μM CLA and 50 μM KETO; 50 μM CLA and 50 μM KETO or 50 μM CLA and 0.5 μM KETO were the most efficacious. Example 5 Effects of CLA on Lipogenesis in Cultured Human Adipocytes

The effect of linoleic acid, cis-9, trans-11 CLA, and trans-10, cis-12 CLA on lipogenesis in cultured human adipocytes was evaluated using the above-described lipogenesis assay. More specifically, lipogenesis was assayed by determining the level of C-glucose incorporation into total cellular lipid [(pmol/(L 10 )] during de novo lipogenesis of cultured human adipocytes. The results are presented in Figure 3. Data are expressed as percentage of vehicle (BSA) controls. Means (+_ SEM, n+6) not sharing a letter differ, p<0.05. The data indicate that CLA blocks the uptake of fat in the form of triglycerides by cultured human adipocytes.

Example 6 Exemplary Skin Creams

The following embodiments describe two alternative examples of a skin cream incorporating the compositions of the present invention. The compositions are formed by combining and mixing the ingredients of each column using conventional technology and then applying about 0.5g to about 50 g of the mixture to the skin.

Example 1

Ingredient % Weight

Glycerine 6.9333

CLA 10,12 25.00

An Azole 10.000

Permethyl 101 A¹ 3.000

Sepigel² 2.500

Q2-1403³ 2.000

Isopropyl Isosterate 1.330

Arlatone 2121⁴ 1.000

Cetyl Alcohol CO- 1695 0.720

SEFA Cottonate⁵ 0.670

Tocopherol Acetate 0.500

Panthenol 0.500 Adol 62⁶ 0.480

Kobo Titanium Dioxide 0.400

Sodium Hydroxife 50% Aqueous 0.0125

Fiery 5⁷ 0.1150

Disodium EDTA 0.100

Glydant Plus⁸ 0.100

Myrj 59⁹ 0.100

Emersol 132¹⁰ 0.100

Color 0.00165

Purified Water q.s. to 100

Example 2

Ingredient % Weight

Glycerine 6.9333

CLA 10,12 10.000

Ketoconazole 10.000

Permefhyl 101 A¹ 4.000

Q2-1403³ 2.000

Isopropyl Isostearate 1.330

Arlatone 2121⁴ 1.000

Cetyl Alcohol CO-1695 0.720

SEFA Cottonate⁵ 0.670

Carbopol 945^π 0.500

Tocopherol Acetate 0.500

Panthenol 0.500

Adol 62⁶ 0.480

Kobo Titanium Dioxide 0.400

Sodium Hydroxide 50% Aqueous 0.250

Fiery 5⁷ 0.150

Disodium EDTA 0.100

Glydant Plus⁸ 0.100 Myrj 59^y 0.100 Emersol 132 10 0.100

Carbopol 1382 12 0.100 Color 0.00165 Purified Water q.s. to 100

^sohexadecane, Presperse Inc., South Plainfield, NJ

² Polyacrylamide(and)C1314 Isoparaffin(and)Laureth7, Seppic Corporation,

Fairfield, NJ

³dimethicone(and)dimethiconol, Dow Corning Corp., Midland, MI ⁴Sorbitan Monostearate and Sucrococoate, ICI Americas h e, ⁵Sucrose ester of fatty acid, Procter and Gamble, Cincinnati, OH ⁶Stearyl Alcohol, Procter and Gamble, Cincinnati, OH. ⁷Fiery 5 n/a, Procter and Gamble, Cincinnati, OH.

⁸DMDM Hydantoin (and) Iodopropynyl Butylcarbamate, Lonza Inc., Fairlawn, NJ ⁹PEG100 Stearate, ICI Americas Inc., Wilmington, DE. ¹⁰Stearic acid, Henkel Corp., Kankakee, IL

¹¹ Carbomer, BF Goodrich, Cleveland, OH

¹² Carbomer, BF Goodrich, Cleveland, OH

All publications and patent applications mentioned in the specification are indicative of the level of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claims.

Claims

THAT WHICH IS CLAIMED:

1. Use of a skin care composition comprising: a) at least one conjugated linoleic acid isomer; b) an AZOLE antifungal agent, and, c) a pharmaceutically acceptable carrier to prevent the occurrence of cellulite localized regions of mammalian skin characterized by fat accumulation.

2. The use according to claim 1, wherein the conjugated linoleic acid isomer is selected from the group consisting of 9 cis, 11 cis conjugated linoleic acid; 9 cis, 11 trans conjugated linoleic acid; 9 trans, 11 cis conjugated linoleic acid; 9 trans, 11 trans conjugated linoleic acid; 10 cis, 12 cis conjugated linoleic acid; 10 cis, 12 trans conjugated linoleic acid; 10 trans, 12 cis conjugated linoleic acid; 10 trans, 12 trans conjugated linoleic acid; 11 cis, 13 cis conjugated linoleic acid; 11 cis, 13 trans conjugated linoleic acid; 11 trans, 13 cis conjugated linoleic acid and 11 trans, 13 trans conjugated linoleic acid.

3. The use according to claim 2, wherein the AZOLE antifungal agent is selected from the group consisting of bifonazole, butonazole, clotrimazole, croconazole, econazole, fenticonazole, fluconazole, itraconazole, ketoconazole, miconazole, omoconazole, oxiconazole, sertaconazole, sulconazole, terconazole, and tioconazole.

4. The use according to claim 3, wherein the concentration of conjugated linoleic acid and the AZOLE antifungal agent are present in a concentration ranging from about 0.01 % to about 40% by weight.

5. The use according to claim 4, wherein the composition further comprises an additional active selected from the group consisting of phosphodiesterase inhibitors, oleosoluble vegetable extracts, herbal extracts, botanical extracts and mixtures thereof.

6. The use according to claim 4, wherem the composition further comprises an additional skin active selected from the group consisting of hydroxy acids, desquamatory agents, sunscreens, anti-oxidants, retinoids and mixtures thereof.

7. The use according to claim 6, wherein the hydroxy acid is salicylic acid; the desquamatory agent is selected from the group consisting of zwitterionic surfactants and mixtures thereof; the sun-block is selected from the group consisting of zinc oxide, titanium dioxide and mixtures thereof; the sunscreen is selected from the group consisting of 2-ethyU exyl-p-methoxycinnamate, 4,4'-t-butyl methoxydibenzoyl-methane, phenyl benzimidazole sulfonic acid, octocrylene and mixtures thereof; the anti-oxidant is selected from the group consisting of tocopherol, esters thereof and mixtures thereof; and the retinoid is selected from the group consisting of retinol, retinyl acetate, retinyl propionate, and mixtures thereof; agents, sunscreens, anti-oxidants, retinoids and mixtures thereof.

8. A composition comprising at least one isomer of conjugated linoleic acid, an AZOLE antifungal agent and a pharmaceutically acceptable carrier.

9. The composition according to claim 8, wherein the concentration of the at least one isomer of conjugated linoleic acid and the AZOLE agent are present at a concentration ranging from about 0.01% to about 40% by weight.

10. The composition according to claim 9, wherein the conjugated linoleic acid isomer is selected from the group consisting of 9 cis, 11 cis conjugated linoleic acid; 9 cis, 11 trans conjugated linoleic acid; 9 trans, 11 cis conjugated linoleic acid; 9 trans, 11 trans conjugated linoleic acid; 10 cis, 12 cis conjugated linoleic acid; 10 cis, 12 trans conjugated linoleic acid; 10 trans, 12 cis conjugated linoleic acid; 10 trans, 12 trans conjugated linoleic acid; 11 cis, 13 cis conjugated linoleic acid; 11 cis, 13 trans conjugated linoleic acid; 11 trans, 13 cis conjugated linoleic acid and 11 trans, 13 trans conjugated linoleic acid.

11. The composition according to claim 10, wherein the AZOLE antifungal agent is selected from the group consisting of bifonazole, butonazole, clotrimazole, croconazole, econazole, fenticonazole, fluconazole, itraconazole, ketoconazole, miconazole, omoconazole, oxiconazole, sertaconazole, sulconazole, terconazole and tioconazole.

12. The composition of claim 11 , wherein the at least one isomer of conjugated linoleic acid comprises 10-trans, 12-cis conjugated linoleic acid and the AZOLE antifungal agent is ketoconazole and further wherein the conjugated linoleic acid and ketoconazole are present at a concentration at least about 0.025% by weight.

13. The composition according to claim 11, wherein the composition further comprises an additional agent selected from the group consisting of phosphodiesterase inhibitors, oleosoluble vegetable extracts, herbal extracts, botanical extracts and mixtures thereof.

14. The composition according to claim 13, wherein the composition further comprises an additional agent selected from the group consisting of hydroxy acids, desquamatory agents, sunscreens, anti-oxidants, retinoids and mixtures thereof.

15. Use of a topical skin care composition comprising: a) at least one conjugated linoleic acid isomer; b) an AZOLE antifungal agent, and, c) a pharmaceutically acceptable carrier to stimulate lipolysis in adipocytes localized in a subcutaneous region of mammalian skin characterized by adipose tissue accumulation.

16. The use according to claim 15, wherein the mammal is a human and the subcutaneous region of skin characterized by adipose accumulation is a femoral fat deposit.

17. The use according to claim 15, wherein the conjugated linoleic acid isomer is selected from the group consisting of 9 cis, 11 cis conjugated linoleic acid 9 cis, 11 trans conjugated linoleic acid; 9 trans, 11 cis conjugated linoleic acid; 9 trans, 11 trans conjugated linoleic acid; 10 cis, 12 cis conjugated linoleic acid; 10 cis, 12 trans conjugated linoleic acid; 10 frans, 12 cis conjugated linoleic acid; 10 trans, 12 trans conjugated linoleic acid; 11 cis, 13 cis conjugated linoleic acid; 11 cis, 13 trans conjugated linoleic acid; 11 trans, 13 cis conjugated linoleic acid and 11 trans, 13 trans conjugated linoleic acid.

18. The use according to claim 16, wherein the AZOLE antifungal agent is selected from the group consisting of bifonazole, butonazole, clotrimazole, croconazole, econazole, fenticonazole, fluconazole, itraconazole, ketoconazole, miconazole, omoconazole, oxiconazole, sertaconazole, sulconazole, terconazole and tioconazole.

19. The use according to claim 18, wherein the at least one isomer of conjugated linoleic acid comprises 10-trans, 12-cis conjugated linoleic acid and the AZOLE antifungal agent is ketoconazole and further wherein the conjugated linoleic acid and ketoconazole are present at a concentration at least about 0.025% by weight.

20. Use of a topical skin care composition comprising: a) at least one conjugated linoleic acid isomer; b) an AZOLE antifungal agent , and, c) a pharmaceutically acceptable carrier as a slimming agent which inhibits de novo lipogenesis in adipocytes localized in a subcutaneous region of mammalian skin characterized by adipose tissue accumulation.

21. The use according to claim 20, wherein the mammal is a human and the subcutaneous region of skin characterized by adipose accumulation is a femoral fat deposit .

22. The use according to claim 20, wherein the conjugated linoleic acid isomer is selected from the group consisting of 9 cis, 11 cis conjugated linoleic acid ; 9 cis, 11 trans conjugated linoleic acid; 9 trans, 11 cis conjugated linoleic acid; 9 trans, 11 trans conjugated linoleic acid; 10 cis, 12 cis conjugated linoleic acid; 10 cis, 12 trans conjugated linoleic acid; 10 trans, 12 cis conjugated linoleic acid; 10 trans, 12 trans conjugated linoleic acid; 11 cis, 13 cis conjugated linoleic acid; 11 cis, 13 trans conjugated linoleic acid; 11 trans, 13 cis conjugated linoleic acid and 11 trans, 13 trans conjugated linoleic acid.

23. The use according to claim 22, wherein the AZOLE antifungal agent is selected from the group consisting of bifonazole, butonazole, clotrimazole, croconazole, econazole, fenticonazole, fluconazole, itraconazole, ketoconazole, miconazole, omoconazole, oxiconazole, sertaconazole, sulconazole, terconazole and tioconazole.

24. The use according to claim 18, wherein the at least one isomer of conjugated linoleic acid comprises 10-trans, 12-cis conjugated linoleic acid and the AZOLE antifungal agent is ketoconazole and further wherein the conjugated linoleic acid and ketoconazole are present at a concentration at least about 0.025%) by weight.

25. A topical skin care composition comprising an effective amounts of 10-trans, 12-cis conjugated linoleic acid and an AZOLE antifungal agent sufficient to inhibit de novo lipogenesis in adipocytes localized to a subcutaneous region of mammalian skin characterized by adipose tissue accumulation.

26. Use of conjugated linoleic acid and an imidazole antifungal to manufacture a topical slimming composition for the prevention of cellulite and reduction of fatty excesses in mammalian skin characterized by an accumulation of subcutaneous adipose tissue.