CA2552442A1 - Compositions and delivery methods for the treatment of wrinkles, fine lines and hyperhidrosis - Google Patents

Abstract

The present invention describes compositions and methods for treating, preventing and improving the appearance of skin, particularly, treating, preventing, ameliorating, reducing and/or eliminating fine lines and/or wrinkles of skin, wherein the compositions include limonoid constituents which inhibit acetylcholine release at neuromuscular junctions of skeletal muscle so as to relax the muscles involved with wrinkling, folding and creasing of skin, e.g., facial movement and expression. The limonoids preferably include the plant alkaloids toosendanin and azadirachtin. The compositions, which also are used to treat hyperhidrosis, are preferably applied to the skin, or are delivered by directed means to a site in need thereof.

Description

-1a Compos'rtior~s and Delivery Methods for the Treatment of Wrlnttles, Eine Lines and Hyperhidrosis FIELD C.~IL THE INVENTION
[0001 j The present invention generally relates to compositions and methods for shin and personal care involving components as active agents that exert their effects at the neuromuscular junction of muscle and nerve cells and tissue. Such compositions and methods are particularly suitable .or the treatment and prevention of wrinkles, tine lines end hyperhidrosis of skin. Mare particularly, the present invention relates to compositions including giant or fruit constituents which can work at the level of the muscle and affect muscle relaxation as a consequence of the use, preferably topical use, of these compositions. Such constituents include, far example, limonaids, e.g., plant alkaloids; from the Maliaecae family, suds as toosendanin from Melia Toosendan Sieb. Et. Zucc. (the Chinaberry treej and azadirachtin from Melia Azadirachta (the Neem tree). The invention further relates to methods of delivery for such campos'rtlons so as to allow the active components to more readily reach the muscle layer and preferably function at the level of the neuromuscular junction of faaal muscles, more preferably facial expression muscles, to treat, including preventing, reducing, ameliorating, andlor eliminating, signs of dermatolagical aging, for example, wrinkles, fine lines, folds and furrows in the skin; to treat, prevent, or reduce hyperhidrnsis; andlor to improve the aesthetic appearance of the skin.

[0002] A number of treatments and compositions for topical appiica~on are promoted in the art for mitigating dermatological cor!ditions of the skin which frequently are associated with natural or snvironrnental causes, such as the process of aging (i.e., intrinsic agingj; exposure to the sun, or the resulting ultraviolet radiation therefrom (i.e., extrinsic aging), and the like. Other factors, such as improper care and/or diet, stress, nutritional deficiencies, repeated facial movement and' genetic predisposition also contribute to the development of adverse skin conditions, such as fine fines, frown fines, folds, furrows and wrinkles.

[0003] Popular culture and trends, the fashion industry, and many arms of the current media promote and encourage a youthful appearance that reflects skin, particularly facial skin, which is free of wrinkles, fine fines and the tike. In today's world, both women and men desire to maintain a youthful, wrinkle-free appearance for as Long as possible and consequently seek to prevent, reduce, improve, or eliminate the signs of aging (and/or sun exposure) which reveal themselves in the forms of fine lines, furrows, and wrinkles, for example. The products commonly available to date for this purpose typically contain agents that act on the skirt by merely moisturizing;
promoting cell growth, swelling the extraceltutar matrix (by, for example, increasing or promoting the synthesis of collagen, elastin or glycosaminoglycans) which comprises skin. Calcium channel inhibitory agents that act on calcium channels of subcutaneous muscle and/or nerve tissue have also been described as components of compositions to relax and/or decontract cutaneous andlor subcutaneous tissue whose contraction is associated with wrinkle formation (U.S. Patent No. 6,344,461 B1 to L..
Breton et at.).

[0004] Compositions containing active agents and ingredients for use in personal care products to affect, prevent, and/or reduce hyperhidrosis, such as antiperspirants and deodorants, are also widely employed in the general population. Such compositions are provided to reduce and prevent perspiration and body malodor associated with human perspiration and sweating, particularly underarm malodor, and ideally should contain constituents that are effective in the treatment of sweating following topical application to the skin. While antiperspirants combat auxiliary body malodors by inhibiting perspiration through, for example, the action of astringent salts such as aluminum and zirconium salts, deodorants neutralize objectionable odors resulting from the degradation of the components of sweat which are attacked by chemicals and microbes, thereby producing foul-smelling fatty acids.
[0003 By the regular use of antiperspirants or deodorants, a considerable number of users have experienced pain and/or irritation in the sensitive skin or body surface to which the antiperspirant or deodorant is applied, due to the active salts in these compositions, as well as to skin sensitivity. As is appreaated by the practitioner, deodorants are generally less irritating to the skin than antiperspirants because deodorants do not inhibit sweat, but rather neutralize the degradation products of sweat Despite this, however, deodorants can also be irritating to sensitive skin, particularly for those whose skin is compromised in certain areas (e.g., the underarm area) as a result of shaving. Also, deodorants are difficult to use in other sweat prone areas (e.g., feet, palms, groin). In view of the widespread use of such personal care products, effective antiperspirant and deodorant compositions are desired to inhibit, prevent, or reduce perspiration and body matodor with less irritafion to the body and stein of the user.
[0006 Many of the compositions described for various topical treatments of the skin include components, such as alpha-hydroxyacids ("AHAs"), that may provide only supe~ciat effectiveness, or that may cause adverse side effects to the user's skin, for example, irritation. Indeed, some commonly used compositions and methods for reduang fine tines and/or wrinkles contain AHAs, such as gtycolic acid, lactic acid, tartaric acid and citric acid, and salts thereof, or they contain tretinoin, also known as alt-trans retinoic acid or retinot (Vitamin A), or ascorbic acid (Vitamin C), all of which, without further mitigation, can induce the above-mentioned disadvantageous effects, often due to a lowering of the pH of the skin, among other factors.
To provide maximum effectiveness and to avoid or prevent the foregoing types of adverse reactions, new and alternative components of compositions and formulations for the treatment and care of the skin are desired, [0007) Human skin comprises two major compartments: a superfiicial outer compartment; the epidermis, and a deeper compartment, the dermis.
The outermost epidermal skin layers typically provide a certain degree of protection to the body, although fine lines and wrinkles can be readily visualized in this portion of the skin. The epidermis and dermis bear the brunt of harmful effects of photodamage. The natural human epidermis functions to provide the essential protectant role of the skin in the human body. The dermis, which provides a solid and nutritional support to the epidermis, comprises mainly fibrobtasts and an extracellular matrix composed primarily of a substance that includes ootlagen, elastin and glycosaminoglycans ("GAGs"). In addition, the dermis contains leukocytes, mast cells, tissue macrophages, blood vessels, muscle dells and nerve ftbers.
[0008] The dermal muscles of the face are controlled by motor nerve afferences of the facial nerve. The interlobular septa of the hypoderm contain within them fibers that institute a striated muscle tissue, i:e., the panniculus camosus. It is known that a subpoputation of dermal fcbrobtasfs, called myofibrobtasts, has contractile characteristics similar to those of muscle cells and tissue.
[0009] Botutinurn toxin, (also known by the trade name, Botox'~, Allergen, Irvine, Catif.), is currently in vogue for treating wrinkles and trne lines, but was initially used to treat spasms, This toxin acts on states of muscular spasticity by specifically inhibiting neurotransmission in nerve cells, thereby causing contracted muscles to relax (e.g., A. Blitzer et al., 1993, Arch. Otolaryngol. Head Neck Surg., 119:11&122; U.S. Patent No.

-5-

6,344,461 81 to L. Breton et al.). Botutinum toxin also can act on wrinkles of the glabella (wrinkles between the eyebrows) when injected subcutaneously.
(See, J.D. Carruthers, 1992, J. Dermatol. Surg. Oncol., 18:17-21; U.S.
Patent No. 6,344,461 B1 to L. Breton et al.). However, the full extent of adverse effects related to long-term use of botuiinum toxin and products or treatments containing this material are still not well established. Botulinum toxin treatment has been associated with a number of side effects including, transient fatigue, dysphagia, neck weakness, hoarseness, and localized pain. 1n addition, many patients who preliminarily respond to botulinum toxin subsequently become non-responsive to treatment. (See, e.g., published U.S. Patent Application No. US2002100812914 to Hawrot).
[0010 New ingredients for use as effective, active agents in compositions and formulations for application, preferably topical application, to the skin are advantageous to the industry and the consumer for a variety of uses in the areas of skin care treatment and therapy, and personat use products.
[0011 J Plant extracts and substances serving a variety of diverse purposes include limonoid constituents of the Maliaecae and Rutaceae families. Limonoids are a group of chemically related triterpene derivatives which are among the bitter principles found in citrus fruits such as lemons, lime, orange and grapefruit. They are also present as glucose derivatives in mature fruit tissues and seeds, and are one of the major secondary metabolites present in citrus. More specific examples of such plant materials include the plant alkaloids toosendan (from Melia Toosendan Sieb.
Et. Zucc), (i.e., Chinaberry) and azadirachtin (from Melia Azadirachta (i.e., Neem). Limonoids have been known to have insecticidal properties (e.g., U.S. Patent No. 6,372,239 to Wu et al.; U.S. Patent No. 5,885,600 to M.
Blum et al.), as weft as anti-neoplastic and anti-carcinogenic effects in laboratory animals (e.g., U.S. Patent No. 5,370,873 to t.J. Udeinya; U.S.

Patent Nos. 6,239,114 and 6,251,400 to N. Guthrie et a!.). The furan moiety attached to the D-ring is specifically responsible for detoxifying the chemical carcinogen glutathione-S-transferase enzyme system (Lam et al., 1994, Food Technology, 48:104-108).
[0012] Citrus fruit tissues~and the byproducts of juice processing, such as peels and molasses ace sources of limonoid gfucosides and citrus seeds contain high concentrations of both limonoid agtycones and glucosides. Limonoid aglycones in the fruit tissues gradually disappear during the (ate stages of fruit growth and maturation. Thirty eight limonoid aglycones have been isolated from citrus. The limonoids are present in three different forms, namely, the dilactone is present as the open D-ring form (monolactone), the iimonoate A-ring lact~e and the glucoside form (See, e.g., U.S. Patent No. 6,239;114 to N. Guthrie et at.). Only the monolactones and glucasides are present in fru~ tissues. (Hasegawa S. et al., 1994, In: Food Phytochemicats for Cancer Prevention t; Eds M-T.
Huang et aI, American Chemical Society, 198-207).
[0013] Procedures for the extraction and isolation of both agiycones and glucosides have been established to obtain concentrated sources of various limonoids (Lam, L,K.T. et al., 1994, !n: Food Phytochemicals for Cancer Prevention, Eds. M. Huang, T. Osawa, C. Ho and R. T. Rosen, ACS
Symposium Series 546, p 209).
[0014] Safe, effective and new components of compositions to treat, prevent, reduce, inhibit, and/or improve the dermatological signs of aging, as well as hyperhidrosis, would be advantageous for the formulation of treatments and products for the skin. As described herein, novel 'and beneficial methods and compositions, as well as their mode of action, for the treatment of wrinktes and the like, as well as for personal care products for the skin, are provided by the present invention.

-7-SUMMARY OF THE INVENTION
[0015] The present invention provides compositions and methods comprising plant-derived constituents newly found to be effective to treat, including prevent, reduce, ameliorate, andlor eliminate, signs and results of dermatologicat aging of skin, espeaally wrinkles and fine lines, and/or to improve the aesthetic appearance of skin. Further these constituents are provided for use in compositions and methc?ds to treat, including prevent, ameliorate, and/or reduce, hyperhidrosis (or sweating or perspiration) and its associated effects, such as malodor. More specifically, the active constituents (also refen~ed to as active agents, components, ingredients, reagents, or compounds herein)~are Iimonoids, obtained from plants and the fike, of the Mafiaecae family. Nonlimiting examples of timonoids for use~in accordance with this invention include the plant alkaloids toosendanin from Metta Toosendan Sieb. Et Zucc (more commonly known as the Chinaberry tree, Ku Lian Pi extract, or Melia azedarach); and azadirachtin from Melia Azadirachta (more commonly known as the Neem tree or Azacdiracta indica). These plant materials have been newly found to provide treatment for wrinkles, fine lines and. other signs of dermatologicat aging (i.e., intrinsic aging) or sunlight exposure of the slan (i.e., extrinsic aging), as well for use in the treatment of hypefiidrosis, by virtue of their effect at the neuromuscular junction (NMJ) of muscle and nerve cells and tissue; arid at the ftbers of the sweat glands to inhibit acetylcholine release, e.g., at the NMJ, thereby relaxing or decontracting muscles, e.g., facial expression muscles.
[0016] It is to be understood that, as used herein, the terms treating and treatment include and encompass preventing, reduang, ameliorating, improving, alleviating, andlor eliminating the dermatological effects of aging and sun exposure, with particular regard to wrinkles, fine lines, folds, furrows, creases of the skin, and the tike. The present invention further _$_ encompasses the treatment, as defined above, of umarionette" tines that run on either side of the mouth, as well as lines on the forehead, and the perpendicular fines between the brows. The present compositions and methods are also suitable for use in treating, as defined above, dermatological conditions of the skin in numerous areas of the body, including, without limitation, the face, forehead, neck, arms, hands, legs, knees, feet, chest, back, groin, buttocks, and the Like.
[00171 It is another aspect of the present invention to provide compositions, formulations and methods containing materials newly determined to be useful in the treatment of wrinkles, fine tines, folds, furrows and other signs of aging skin, in addition to treating, preventing or reducing hyperhidrosis. These materials exert their effectiveness according to this invention by preferably working at the neuromuscular junctions (NMJ), such as in the muscle and nerve cell or tissue layer of a site of application, e.g., skin of the face, neck, arms, feet, or hands, or in the demlal layer of the skin where sweat glands are located. More specfically, the limonoid constituents (i.e., chemical actives, such as toosendanin or azadirachtin, or a combination thereof) block or inhibit the release of acetylcholine at the NMJ
of skeletal muscle, which, in tum, results in the relaxation or decontractson of contracted muscles. Contracted muscles are associated with the fom~a#ion of fne fines, wrinkling and the like.
j0018~ According to the present invention the materials, in general, comprise plant alkaloids, or limonoids of the Matiaecae family. More specifically, the Itmonoids comprise toosendanin and azadirachtin, which were not previously known to inhibit acetytcholine release in NMJ of skeletal muscles, thereby relaxing the facial expression muscles, for example, to treat, prevent, reduce, ameliorate, and/or eliminate aesthetically displeasing wrinkles, frown lines, fine Lines, folds, furrows, or neck bands, etc. that can arise from aging, prolonged sun exposure of the skin, ultraviolet radiation _g_ associated with sun or photoexposure, or in particular, hyperactive facial expression muscles.
[0019] In addition, because it is understood that the contraction or hypercontraction of certain muscles, particularly facial muscles, is related to the appearance of wrinkles, fine lines, etc., the relaxation of such muscles, and/or the control or modulation of the contraction of such muscles, by the newly-determined action of the limonoids as disclosed herein can serve a pivotal function in the treatment, prevention, reduction, amelioration, or elimination of wrinkles, fine lines and the like.
[0020] In accordance with this invention the limonoid materials comprise compositions which include, without limitation, topicahy applied sunscreens, anti-oxidants, anti-inflammatories, cosmetics, inducting makeups, anti-aging formulations, e.g, cxeams for fine lines andlor wrinkles, topicals, skin permeants antiperspirants, deodorants and the like. Also in accordance with this invention, ingredients, components, or compounds that are formulated in such compositions in a variety of product forms, e.g., transdermals, such as patches, and the like, are encompassed, particularly for topical administration.
j0021 ] Another aspect of the present invention. provides the compositions comprising the limonoid materials preferably for topical administration without inducing significant irr<ttation. Further, such compositions are preferably delivered by, but not limited to, the use of targeted delivery systems, for example, liposomes, micraspheres, transdermal patches, and the tike, so that the actives can more readily reach and affect the muscle layer of the area of application, e.g., face or neck, or the dennai layer of the skin, where sweat glands are located, e.g., underarm, foot, hand, etc. Compositions comprising iirnonoid constituents, including liposome formulations, can be administered by direct injec~on subcutaneously, intradermally, or through iontophoresis, to deposit the active agents at the sites requiring muscle relaxation or decontraction.
[0022] In another of its aspects, the present invention provides limonoid-containing compositions and methods thereof which can improve the aesthetic appearance of the skin by treating, including preventing, ameliorating and/or reducing at least one of the following: dermatologicaf aging, especially chronological, actinic or hormonal aging. The improvement preferably results following topical application of a product or formulation containing one or more of the limonoid constituents as described herein.
[0023] Another aspect of this invention provides a method of reducing, preventing, treating, or ameliorating wrinkles, fine lines, creases, frown lines, or other signs of dermatological aging, or photoexposure of skin, comprising: applying a composition comprising an acetytcfwline antagonist at a site comprising a neuromuscular junction (NMJ} in an amount effective to reduce or block acetylcholine release at the neuromuscular junction (NMJ), wherein the reduction or block of acetylcholine release at the neuromuscular junction (NMJ) concomitantly reduces or blocks muscle cell or tissue contraction (or contractility}, thereby treating, preventing, reducing, ameliorating, or eliminating the formation of wrinkles, fine lines, creases;
folds, frown lines, or other signs of demzatotogical aging, hyperactive facial expression muscles, or photoexposure. Preferably, the acefylcholine antagonist is a limonoid constituent, more preferably a limonoid constituent of the Maliaecae family, and most preferably, toosendanin or azadirachtin.
[.Q024] A further aspect of this invention provides a method of reducing, preventing, treating, or ameliorating hypefiidrosis or perspiration, particularly, excessive perspiration, comprising: applying'a composition comprising an acetylcholine antagonist at a site comprising a neuromuscular junction (NMJ), preferably in the fibers of a sweat gland, in an amount effective to reduce or block acetylcholine release at the neuromuscular junction (NMJ), wherein the reduction or block of acetylcholine release at the neuromuscular junction (NMJ) concomitantly reduces or blocks contraction (or contractility) of cells in the dermis associated with the sweat gland, thereby reducing, preventing, treating, or ameliorating hypefiidrosis or perspiration. Preferably, the acetytcholine antagonist is a limonoid constituent, more preferably a limonoid constituent of the Maliaecae family, and most preferably, the Iimonoids toosendanin or azadirachtin.
[0025] Further aspects, features and advantages of the present invention will be better appreciated upon a reading of the detailed description of the invention.
DESCRIPTION OF THE INVENTION
[0026) The present invention provides novel compositions and methods comprising components newly found to be effective to treat, including prevent, reduce, ameliorate, inhibit, alleviate, and/or eliminate signs and results relating to the dermatological aging of skin due to chronological and/or hormonal aging, or due or to photoexposure (e.g., to the sun, or ultraviolet radiation) of skin, espeaally wrinkles, creases, furrows, folds and fine lines; and/or to improve the aesthetic appearance of skin. These components are further provided for use in compositions and methods to treat, including prevent, ameliorate, inhibit and/or reduce, hyperhidrosis (sweating or perspiration) and its associated effects, such as malodor and unaesthetic appearance. .
[00271 More specifically, the components (also referred to as active agents, constituents, ingredients, reagents, substances, or compounds herein} of this invention are limonoids, obtained from plants and the tike, preferably of the Maliaecae family. Noniimiting examples of timonoids for use in accordance with this invention include the plant alkaloids toosendanin from Metia Toosendan Sieb. Et. Zucc (more commonly known as the Chinaberry tree or Melia azedarach); and azadirachtin from Melia Azadirachta (more commonly known as the Neem tree or Azacdiracta indica). Azadirachtin is a limonoid of the tetranortriterpenoid type. These plant materials have been newly determined to be effective constituents in compositions and methods for treating, preventing, reducing; inhibiting, improving and/or eliminating wrinkles, fine lines, creases, folds, fun-ows, and other signs of dermatologicat aging due to chronological andlor hormonal aging, or due to sunlight exposure of the skin, as welt for use in the treatment of hyperhidrosis.
[0028] According to the present invention, yet without wishing to be bound by theory, the limonoid constituents exert their effects by their ability to inhibit, block, reduce, or prevent the release of acetylcholine at the neuromuscular junction (NMJ) of skeletal muscle cells and tissue, thereby relaxing muscles, for example, muscles assodated with fadal movement or expressions, or the fibers of the sweat glands. Both nerve and muscle cells (skeletal muscle cells) are electrically excitable and the junctions between these types of cells are known as NMJ. It has been known for many years that the stimulation of a motor nerve innervating a skeletal muscle also causes the release of acetylcholine and that acetylcholine; in tum, stimulates the skeletal muscle to contract. Thus, acetylcholine has been identified as the neurotransmitter at the neuromuscular junction. (see, e.g., B. Alberts et al., 1989, Molecular Biology of the Cell, 2"d Ed., Chapter 19;
The Nervous System, Garland Publishing, Inc., NY., pp. 1075 et seq.).
Provided by the present invention are Iimonoid constituents which are newly described herein to inhibit, reduce, block, or prevent the release of acetyicholine at the NMJ of the skeletal muscle and thus relax the muscles, preferably those associated with facial expression or movement, particularly when employed in the compositions and methods of the invention. By also being able to inhibit aceiyichotine release in the fbers of the sweat glands, compositions and methods comprising the limonoid constituents as described herein can reduce, treat, and/or prevent excessive sweating or perspiration.
[0029] The ability of the limonoid constituents to inhibit acetylcholine release by muscle cells results in a modulation of motor contraction so as to relax the muscle fibers in cutaneous or subcutaneous muscle and/or nerve tissue, thereby attenuating wrinkles, as well as fine lines, folds, furrows, and the tike. By relaxing or preventing the contraction or hypercontraction of the cutaneous or subcutaneous muscle cells of areas such as the face, or hands, feet, etc., the Iimonoids and compositions containing limonoids can effectively smooth out the Landscape of the skin in those areas where muscle contraction is involved in the formation of wrinkles and the like.
Thus, if muscle cell contractility is associated with the formation of wrinkles, fine lines, etc., the relaxation or decontraction of the contractility of cutaneous or subcutaneous musde tissue by the timonoid actives in the compositions of this invention can serve to Loosen or slacken the contracting muscle tissue and alleviate, reduce, ameliorate, inhibit, or eradicate the wrinkles, fine lines, etc. Contraction of cutaneous or subcutaneous muscle cells or tissue of skin can elicit wrinkling, fine lines, and the like, which constitute at feast some of the visible dermatologicai effects of aging due to chronological and/or hormonal aging, and/or due to photoexposure, as described herein.
[0030] Thus, in one of its embodiments, the present invention encompasses compositions, formulations and methods containing components, preferably, limonoid constituents, newly determined to be useful in the treatment of wrinkles, fine fines, folds, furrows and other signs of aging and/or photoexposure of skin, in addition to treating, preventing or reducing hypefiidrosis. Skin in a variety of areas of the body is amenable for treatment andlor receipt of the compositions of the present invention, including the face, forehead, neck, amps, legs, hands, feet, torso (chest), back, and the tike.

[0031 ] The plant-derived materials exert their effectiveness according to this invention by preferably working at the neuromuscular junctions (NMJ), such as in and around muscle and nerve cells and tissue at a site of application, e.g., the skin of face, neck, arms, feet, hands, or chest, or in the dermis layer of the skin where sweat glands are located. According to this invention, the materials, in general, comprise limonoids, which are plant alkaloids, of the Mafiaecae family. More specfically, the limonoids comprise toosendanin and azadirachtin, which are plant alkaloids. Prior to the present invention, these materials were not previously known or recognized to inhibit acetylcholine release at NMJ of skeletal muscles, thereby relaxing muscles associated with aesthetically displeasing wrinkles, frown lines, fine lines, folds, furrows, ceases, neck bands, and the tike, that can arise from aging due to chronological andlor hormonal aging, prolonged or overexposure of the skin to the sun, ultraviolet radiation assoaated with phofioexposure; or over-exercised expression muscles.
[Q032j In general, for the purposes of the present invention, a substance, such as a limonoid constituent of the described compositions, is recognized as being a muscle relaxant when it elicits a relaxation effect on contracted muscle cells or orr tissue, such as cutaneous or subcutaneous muscle tissue, and/or exhibits an inhibitory effect on acetylcholine release at the NMJ. Contracted musde ~tls or tissue is associated with formation of wrinkles, frne lines, etc. Relaxation or decontraction of contracted muscle, such as by the action of limonoids to inhibit or block acetylcholine release at the NMJ, serves to smooth out the landscape, or microrelief, of the skin, thereby effecting the prevention, amelioration, reductiort, and/or eradication of wrinkling and fine line, etc. formation caused by contracted muscle tissue in skin.
[0033] . For use in the compositions of this invention, the limonoid plant alkaloids can be chemically synthesized. Alternatively, these plant alkaloids may be extracted from plants. When extracted, they may be in a pure form, a semi-purified form, or may be a component of an unpurified plant extract.
For example, toosendanin may be extracted from the plants Melia toosendan Sieb. et Zucc. and Me(ia azedarach t_. Azadirachtin may be extracted from the plant Melia azedarach L. As a result of some structural similarity, azadirachtin may mimic the function of toosendanin. Further, the compositions of the present invention can comprise a combination of plant alkaloids that are formulated to achieve the desired effects to combat wrinkles, and the tike, and hyperhidrosis. The alkaloids as active ingredients may exert their effects via multiple mechanisms and signal transduction pathways.
[0034] The limonoid plant alkaloids contained in the compositions of the present invention can be chemically synthesized at industrial scale in large amounts. Alternatively, the alkaloids may be extracted from natural raw materials from plants: Any and alt methods of preparation may be used, including- the use of-cultured plant seed cells, as disclosed in commonly owned patent apptidation U.S. publication ;number 2003-009T659.
The level of extraction and the degree of purity of the alkaloid may vary. For example, unpurifced plant ex>zacts may be employed in the present invention_ Depending on the solut~ility of the particular plant alkaloid in water or in organic solvent, the extraction process for each alkaloid may differ. Two methods for extracting the alkaloids from raw plant materials include organic solvent extraction, and aqueous-organic solvent extraction, as described in U.S. Patent No. 6,372,239 to Wu et at:
The organic extraction method involves a step of continuous washing and extracting the plant material against a stream of organic solvent. l=xamples of organic solvents include, but are not limited to methanol, ethanol, dichloromethane, chloroform, xylene, and petroleum ether. Alternatively, the alkaloid can be partially purified or completety purified. Chemical synthesis of the alkaloid obviates the need for extraction and purification.

[0035] In accordance with this invention, the limonoid constituents comprise compositions which include, without limitation, topically applied formulations, anti-oxidants, anti-inflammatories, sunscreens, cosmetics, including make-ups, anti-aging formulations, e.g., creams for fine lines and/or wrinkles, topicats, skin penetration enhancers, antiperspirants, deodorants, and the tike. Also in accordance with this invention, the timonoid constituents and additional ingredients cor»prising such compositions can be formulated in a variety of product formsf- Preferably, the compositions are prepared in targeted delivery systems, eg., transdermals, such as patches, and the like, particularly for topical administration.
j0036~ The present invention encompasses compositions"comprising one or more limonoid constituents; preferably in a pha'rtnaoeut~il~
acceptable ~sn~etic or dermatological formulation which is suitable for contact with living animal tissue, ~nduding~hutnan tissc~e, and fair topical administration, with virtually no adverse physiological effect, e;g:, irritation, to the user. Thus, the inventive oomposfions-are espeaatty sut~abte fot sensitive skin_ [0037 Compositions embraced by this invention-can be provided-in any cosrneticatlyandlor dermatotogicalty suitable fio; preferably as a - .
lotion, gel, cream,, or incorporated into a trapsdermai patch; .and also in an ointment or oil base, as weft as in a spcayabte liquid form. Other suitable cosmetic product forms for the compositions of this invention i~tcfude, for example, an emulsion, a mousse, a lip balm, ~alip gloss, a lotion, a mask, an ointment, a pomade, a solution, a serum, a spray; of a towelette. In addition, the compositions contemplated by this invenfiion. can include one.
or more compatible cosmeticatly acceptable adjuvarwts eoirtvrr~onty used and known by the skilled practfioner, such as colorants; fragrances; emzyilients, humectants, preservatives, vitamins, chetators, thickeners, and the tike, as well as other botanicals such as aloe, chamomile, and the tike, and as further described below. The compositions are preferably applied topically once or twice daily. The daily application can be for periods of up to two weeks, four weeks, or more.
[0038 In one embodiment encompassing sunscreen formulations, the compositions comprising timonoid constituents can further include, without limitation, ingredients such as avobenzone, cinnamic acid derivatives (e.g., octylmethoxycinnamate), octyl salicylate, oxybenzone, titanium oxide, zinc oxide and mixtures or combinations thereof. Such formulations can also preferably include an alpha hydroxy acid, an oxa acid, an oxa diacid, and mixtures or combinations thereof.
[0039 The compositions of this invention can also be formulated into liposomes which can comprise other additives or substances, and/or which can be modfied to more specifically reach or remain at a site following administration. Alternatively, the limonoid-containing compositions can be injected subcutaneously (s.c.) or intradermally (i.d.) at a site in need of wrinkle, Fine line, etc. reduction, improvement, prevention, andlor elimination, resulting from inhibition of acetylcholine release at the NMJ and concomitant relaxation of contracted muscle.
[00401 Pharmaceutical or physiologically acceptable compositions suitable for use in the present invention include compositions in which the active constituents, ingredients, or materials are contained in an amount effective to achieve the intended purpose. By way of example, in the present compositions, a limonoid constituent is present in an amount of from about 0.0001 wt% to about 10 wt%, based on the total weight of the composition. More preferably, the present compositions include one or more limonoid constituents in an amount from about 0.000 wt% to about 5 wt%. Most preferably, the present compositions include one or more limonoid constituents in an amount from about 0.001 wt% to about 1 wt% c~f ;

the total composition.
[0041 ] The determination of an effective dose or amount is well within the capability of those skilled in the art. A therapeutically effective dose refers to that amount of active ingredient, for example, a limonoid constituent identified in accordance with the present invention, which, for instance, treats, prevents, ameliorates, reduces, or eliminates the condition, more specifically, wrinkles, fine lines, creases, and the like. The practitioner, who will consider the factors related to the individual requiring treatment, wilt determine the exact dosage. Dosage and administration are adjusted to provide sufficientJevels of the active constituent or to maintain the desinrd effect. Factors which are typically considered include the severity of the individual's particular need, general health of the patient, age, weight, and gender of the individual, diet, time and frequency of administration; drug combination(s), reaction sensitivities, and toierancelresponse to treatment.
As a general guide, long-acting pharmaceutical compositions can be administered once daily, every 2 to 4 days, every week, or once every two weeks, depending on half life and clearance rate of the particular formulation. Variations in these dosage levels can be adjusted using standard empirical routines for optimizafion, as is well understood in the art.
Guidance as to particular dosages and methods of delivery is provided in the literature and is generally available to practitioners in the art. Those skilled in the art will employ different formulations depending upon the nature, e.g., structure, composition, of the active limonoid constituent.
j0042] With particular regard to fimonoids as anti-hyperhidrosis agents in anti-perspirant or deodorant composifions and formulations for personal use, such products embrace a wide variety of dosage forms, such as stictcs, gels, roll-ons, aerosols, and creams. These dosage forms generally contain a solution of the active ingredient in a solvent, usually non-aqueous, or in a multiphasic dispersion or emulsion in which a solution of _ q9 _ the active ingredient is dispersed in some continuous phase or in which the solubilized active ingredient constitutes the continuous phase. To produce natural-looking, products that leave minimal visible residue on the skin;
clear and translucent antiperspirants, e.g., sticks or gels, can be utilized. Clear or translucent gelled antiperspirant sticks'which are substantially anhydrous, typically contain the active material, a stabilizer as an essential component, and are gelled in a gelling agent, such as dibenzylidene monosorbifot acetal (see, e.g., U.S. Patent No. 5,376,363 to A.J. Benfatto et al.). Other compositions provide antiperspirant active materials in geltants of different types, such as polyamide gelling agent (e.g., U.S. Patent No. 5,500,209 to M.S. Mendolia et at.).
[0043] The compositions of the present invention yield improvements to the aesthetic appearance of the skin by treating at least one of the following: signs of dermatologicat aging, especially chronological, actinic or hormonal aging, or signs of extrinsic aging, such as sun exposure. In particular, improvements to the aesthetic appearance of skin include at feast one of the following: makes facial lines appear tess noticeable, maker faaal lines and/or wrinkles feel plumped, improves appearance of suborbital tines andlor periorbital lines, improves appearance of crow's feet, reduces fcsr diminishes the appearance of wrinkles, particularly faaal wrinkles o~' cheeks, forehead (e.g., perpendicular wrinkles between eyes, horizontal wrinkles above the eyes), and/or around the mouth, (e.g., marionette tines), and particularly deep wrinkles, folds, or creases, improves skin suppleness;
reduces and/or eliminates fine andlor deep lines, folds and creases, and smoothes skin, e.g., to the extent that it reduces wrinkling/fines.
[0044] Embraced by the present invention are transdermal modes of delivery, such as patches and the like, with or without a suitable skin penetration enhancer. The methods and compositions embodied by the invention provide a means by which the one or more limonoid constituents can be effectively administered in a transdermal system. Frequently, compounds having poor topical absorption, or which are required at high dosage levels, are delivered transdermalty. Accordingly, a transdermal means of delivering a composition or formulation (often with a skin penetration, enhancer composition) to the skin is that of the transdermal patch or a similar device as known and described in the art Examples of such devices are disclosed in U.S. Patent Nos. 5,146,846, 5,223,262, 4,820,724, 4,379,454. and 4,956,'!71; such descriptions are not meant to be limiting. The transdermal mode of storing and delivering the compositions onto the skin and forming the active composition is convenient and well suited for the purposes of an embodiment of the present invention.
Preferably, when a topical patch is used, the patch is applied to the desired area for extended period of time. Preferably, the extended period of time is greater than one hour; more preferably, the extended period of time is overnight, e.g., when the user is sleeping.
[0045 A particular embodiment of the present invention is directed to the delivery of the described compositions by the use of targeted delivery systems, for example, tiposomes, microspheres (see, e.g-., U.S. Patent No.
5,770,222 to Unger et ai.); transdermat patches, and the like, so that the Iimonoid actives can more readily reach and affect the muscle layer of the area of application, e.g., face or neck, or the dermis layer of the skin, where sweat glands are located; e.g., underarm, foot; hand, etc. Compositions comprising limonoid constituents, including liposome formulations, can also be administered by direct injection subcutaneously or intradem~alty to more precisely deposit the active agents at sites which require muscle relaxation or decontraction via acetytcholine release inhibition.
[0046 Liposomes and delivery systems and vehicles involving liposomes are welt-known in the art. tn brief, liposomes are untlamellar or multifamellar lipid vesicles which entrap a significant fraction of aqueous solution. The vesicular microreservoirs of liposomes can contain a variety of water-soluble materials, which are suspended within the emulsion (e.g., reviewed in G. Gregorius (Ed.), 1991, Liposome Technology, Vols. 1, II, Ill, CRC Press, Boca Raton, Florida; Davis S.S. and Walker LM., 1987, Methods in Enzymoiogy, 149:51-64; Mayhew E. et al., 1987, Mefhods in Enzymoiogy, 149:64-77; and Shafer Korting M. et al., 1989, J. Am. Acad.
Dermatol_, 21:1271-1275). The preparation of liposomes and the variety of uses of fiposomes in biological systems have been described (e.g., in U.S.
Patent No. 4,708,861 to M.C. Popescu et al., U.S. Patent No. 4,224,179 to M. Schneider and U.S. Patent No. 4,235,871 to D.P. Papahadjopoulos et ai.). Accordingly, such liposomes can be formulated into any of the dermatological or cosmetic compositions as described herein.
[0047) In addition to the limonoid constituents as active agents, as described herein, the physiotogically acceptable and pharmaceutical compositions can contain suitable pharmaceutically acceptable carriers, diluents, or excipients comprising auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Further details on techniques for formulation and administration are provided in the latest edition of Remington's Pharmaceutical Sciences (Mack Publishing Co.; Easton, PA).
Pharmaceutical compositions containing the limonoid ingredients of the present invention can be manufactured in a manner that is known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, Ievigating, emulsifying, encapsulating, entrapping, or lyophilizing processes.
[0048 If applicable, the pharmaceutical composition can be provided as a salt and can be formed with many acids, including but not limited to, hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic, and the like.
Salts tend to be more soluble in aqueous solvents, or other protonic solvents, than are the corresponding free base forms. In other cases, the preparation can be a lyophilized powder.
[0049] A preferred embodiment of the topical compositions of the present invention also includes at feast one of the following: a surface smoother, a skin plumper, an optical d'~ffuser, a sunscreen, an exfoliation promoter, or an antioxidant.
[0050] A surface smoother provides the functional benefits of enhancing skin smoothness and reducing the appearance of fine lines and coarse wrinkles. F~camples include, without limitation, isopropyl myristate, petrolatum, isopropyl lanolate, silicones (e.g., methicone, dimethicone), or any mixtures thereof. The surface smoother is preferably present from about 0.1 wt% to about 50 wt% of the total weight of the composition. A skin plumper serves as a collagen enhancer to the skin. An example of a suitable and preferred skin plumper is patmitoyt otigopeptide. Other nontimiting examples of skirt piumpers include collagen andlor glycosaminogtycan (GAG) enhancing agents. The skin plurhper is preferably present from about 0.1 wt% to about 20 wt% of the total weight of the composition.
[005] An optical diffuser is a particle that changes the surFace optometrics of skin, resulting in a visual blurring and softening of, for example, lines and wrinkles. Examples of optical diffusers that can be used in the present invention include, but are not limited to, boron nitride, mica, nylon, potymethytmethacrytate (PNiMA), polyurethane powder, sericite, silica, silicone powder, talc, Tetfon~, titanium dioxide, zinc oxide, or any mixtures thereof. The optical diffuser is preferably present from about 0.01 wt% to about 20 wt% of the total weight of the composition.
[0052 In another embodiment, the present invention embraces a sunscreen that protects skin from damaging ultraviolet rays. Illustratively, the sunscreen provides both UVA and UVB protection by using either a single sunscreen or a combination of sunscreens. Among the sunscreens that can be employed in the present compositions are avobenzone, cinnamic acid derivatives (such as octylmethoxy cinnamate), octyt salicylate, oxybenzone, ~tanium dioxide, zinc oxide, or any mixture or combination thereof. Preferably, the sunscreen is present from about 1 wt% to about 30 wt% of the total weight of the composfion. In particular, the addition of a sunscreen is preferred to prevent/reduce the photodegradation of the composition andlor ingredients therein white in the package andlor on the skin after application.
[0053 tt wilt be appreciated that the compositions of the present invention containing sunscreen bring about additional improvements to the aesthetic appearance of skin, including at least one of the following:
minimizes sunbuming, minimizes tanning, reduces redness, and reduces future wrinkle development. It will be appreciated that when the topical composition is intended to be applied prior to retiring for the evening, the addition of a sunscreen agent may not be preferred.
[0054] The present compositions may also have one or more exfoliation promoters. Suitable examples of an exfoliation promoter that can be used in the present compositions inGude keratolytic agents, i.e., an active agent having desquamating, exfotianf, or scrubbing properties; or an active agent which can soften the homy layer of the sidn; alpha (a) and/or beta (~i) hydroxy acids; benzoyl peroxide; keto acids, such as pyruvic acid, 2-oxopropanoic acid, 2-oxobutanoic acid, and 2-oxopentanoic acid; oxa acids, as disclosed in U.S. Patent Nos. 5,847,003 and 5,834,513;
urea; rettnoids, or any mixtures thereof. These anti-wrinkle or anti-fine line active agents can be formulated, for example, in amounts of from about 0.0001 % to 5% by weight relative to the total weight of the composition.
[0055] More specifically, examples of hydroxy acids include, but are not limited to, a-hydroxy acids or ~-hydroxy acids, either linear, branched, cyclic, saturated or unsaturated. The hydrogen atoms in the carbon-based backbone of these materials can be substituted with halogens, halogen-containing alkyl, acyl, acyloxy, alkoxycarbonyl, or alkoxy radicals having from 2 to 18 carbon atoms. Suitable hydroxy acids include, for example, glycolic acid, lactic acid, malic acid, tartaric acid, citric acid, 2-hydroxyalkanoic acid, mandelic acid, salicylic acid and alkyl derivatives thereof, including 5-n-octanoylsalicylic acid, 5-n-dodecanoytsalicyfic acid, 5-n-decanoylsalicylic acid, 5-n-octylsalicylic acid, 5-n-heptyloxysalicylic acid, 4-n-heptyloxysaiicylic acid and 2-hydroxy-3-methylbenzoic acid or atkoxy derivatives thereof, such as 2-hydroxy-3-methyoxybenzoic acid. Exemplary retinoids include, without limitation, retinoic acid (e.g., all-trans or 13-cis) and derivatives thereof, retinot (~tamin A) and esters thereof, such as retinol palmitate, retinol acetate and retinol propionate, and salts thereof.
Preferred exfoliation promoters are 3,6,9-trioxaundecanedioic aad, gtycotic acid, lactic acid, or any mixtures thereof Other acids, such as oxa acid (e.g., U.S.
Patent No. 6,069,169) and an oxa diacid (e.g., U.S. Patent No. 5,932,229) can be included in the compositions of this invention.
[0056] When the present invention includes an exfoliation promoter, the composition typically includes about 0.5 wt% to 30 wt°~, preferably about 1 wt% to about 15 wt%, more preferably about 2 wt% to about 10 wt%, and most preferably about 4 wt%, of the exfoliation promoter based on the total weight of the composition.
[007] An antioxidant functions, among other things, to scavenge free radicals from skin to protect the skin from environmental agressors.
Examples of antioxidants that can be used in the present compositions include compounds having phenalic hydroxy functions, such as ascorbic acid and its derivatives/esters; beta-carotene; catechins; curcumin; ferufic aad derivatives (e.g., ethyl fcrulate or sodium ferutate); Baltic acid derivatives (e.g., propyt gatlate); lycopene; reductic acid; rosmarinic acid;
tannic acid; tetrahydrocurcumin; tocopherol and its derivatives; uric acid; or any mixtures thereof. Other suitable antioxidants are those having one or more thiol (-SH) functions, in either reduced or non-reduced form, such as glutathione, lipoic acid, thioglycolic acid, and other sulfhydryl compounds.
The antioxidant may be inorganic, such as bisuffites, metabisutfites, sulfites, or.other inorganic salts and acids containing sulfur. Compositions of the present invention can include an antioxidant preferably from about 0.001 wt% to about 10 wt%, and more preferably from about 0.001 wt% to about 5 wt%, of the total weight of the composition.
[0058] The compositions of this invention can also include one or more of the following ingredients, additives, or adjuvants: anesthetics, anti-altergenics, anti-fungals, antiseptics, chelating agents, coEorants, dyestuffs, demulcents, emollients, emulsifiers, fragrances, fitters, humectants, lubricants, moisturizers, pH adjusters, pigment altering agents, preservatives, skin penetration enhancers, stabilizers, surfactants, thickeners, viscosity modifiers, hydrophilic or lipophilic getting agents, vitamins, or any mixtures thereof: The amounts of these various ingredients, additives, adjuuants, or active agents are those that are conventionally used in the cosmetic or pham~aceuticat fields, for example, they can constitute from about 0.01 % to 20% of the total weight of the composition. In addition, the adjuvants, ingredients, additives, or active agents can be introduced into the fatty phase, into the liquid phase, and/or into lipid vesicles, depending on their nature.
[0059] The components) of the present invention are preferably contained in a cosmetically acceptable medium (i.e., vehicle, diluent or carrier). In an embodiment embracing topical application, the compositions of this invention comprise a medium that is compatible with human skin.
The compositions can be formulated as aqueous, alcohol, or aqueous/alcohol-based solutions, ointments, lotions, gels, water-in-oil, oil-in-water, or water-oil-water triple emulsions having the appearance of a cream or gel, microemulsions, or aerosols. (n addition, the compositions can be in the form of vesicular dispersions containing ionic and/or nonionic lipids, as described above. Dosage units suitable for such pharmaceutical compositions are formulated according to the conventional knowledge and techniques used in the art.
j0060] More particularly, the compositions for topical application can be in the form of a protective care composition for the skin, preferably for the face, the neck, the hands, the feet, or other areas of the body. Nonlimiting examples indude day creams or lotions, night creams or lotions, sunscreen creams, lotions, or oils, body milks, makeup (a foundation), artfidal tanning compositions and depilatories.
[0061 ] If the composition of the invention is an emulsion, the proportion of fatty substances comprised therein can range from about 5%
to 80%, by weight; preferably from about 5% to 50% by weight, reia6ve to the total weight of the composition. The fatty substances and emulsifiers used in the composition in emulsion form are one or more of those conventionally employed in the cosmetic or pharmaceutical field.
Nontimiting examples of fatty substances include mineral oils (e.g., petroleum jelly), plant oils and hydrogenated derivatives thereof, animal oils, synthetic oils (e.g., perhydrosqualene), silicone oils (e.g., polydimethylsiloxane) and fluoro oils. Other exemplary fatty substances include fatty alcohols (e.g., stearyl or cetyl alcohol), fatty adds (e.g., steam acid} and waxes.
[0062] Emulsifiers are typically present in the compositions.of the invention in an amount of about 0.3% to 30%, by weight and preferably from about 0.5% to 30% by weight relative to the tots( weight of the composition.
X0063] In one embodiment, the present invention relates to the administration of an effective amount of at least one limonoid constituent or composition comprised thereof to inhibit or block acetylcholine release by nerve cells at the NMJ to relax and/or decontract subcutaneous muscles, particularly facial expression muscles, thereby smoothing out or unwrinkling skin, wherein the inhibition of acefylcholine release by nerve cells at the NMJ corresponds to muscle, preferably facial expression muscle relaxation or decontraction.
[00643 In another embodiment, the present invention encompasses a method of treating fine Lines, wrinkles, and/or other dermatological effects of . aging or photoexposure of skin, comprising applying to skin a composition containing a limonoid in a cosmetically and/or dermatofogically acceptable medium, and in an amount effective to treat, reduce, prevent and/or ameliorate fine lines, wrinkles andlor other dermatotogical effects of aging of skin. In the method the limonoid is preferably a member of the family Maliaecae, more preferably the limonoid is a plant alkaloid; most preferably toosendanin, azadirachtin, or a combination thereof. The application of. the limonoid containing composition is preferably topical. In addition, the composition is preferably applied via a directed mode of delivery, for example, by topical applicatiorf of an aqueous composition or transdermal patch.
[0065) In yet another embodiment, the present invention encompasses a method of treating hyperhidrosis or perspiration, comprising applying to skin a composition containing a iimonoid in a cosmetically and/or demzatologicaily acceptable medium, and in an amount effective to treat, reduce, prevent and/or ameliorate hyperhidrosis or perspiration. in the method the limonoid is preferably a member of the familyMaiiaecae, more preferably the limonoid is a plant alkaloid, most preferably toosendanin, azadirachtin, or a combination thereof. The application of the limonoid containing composition is preferably topical.

[0066] Another embodiment of the present invention relates to a method of improving the aesthetic appearance of skin and comprises applying to the skin, or introducing viv a directed mode of delivery, a composition including one or more limonoid constituents in an amount effective to improve the aesthetic appearance of the skin. According to this embodiment, the improvement in aesthetic appearance involves the treatment of at least one condition, such as signs of dermatological aging.
Dermatologicat aging can include chronological aging, actinic aging, hormonal aging, or any combination thereof.
[0067 As will be appreciated by the practitioner, cosmetic treatments comprising compositions containing the limonoid constituents of the invention can be carried out, for example, by topically applying the cosmetic composition as described herein according to the routine technique for administering such compositions. Routine and commonly practiced techniques encompass the application of creams, lotions, gels, sera, ointments, antiperspirants, or deodorants to the skin; spraying as a form of application is also envisioned.
EXAMPLES
[0068] The following examples describe specific aspects of the invention to illustrate the invention and provide a description of the present methods for those of skit) in the art. The examples should not be construed as limiting the invention, as the examples merely provide specific methodology useful in the understanding and practice of the invention and its 'various aspects.
Example 1 [00691 The plant alkaloids of the present invention can be extracted from natural raw materials by using the methods of organic solvent extraction or aqueous-organic solvent extraction, such as described in U.S.

_29_ Patent No. 6,372,239 to Wu et al., the contents of which are as set forth below.
[0070 Seed cell culture: Plant seed cells are extracted principally from the tissue of the embryo. The seed cell is then cultured. The total seed cell is then broken down through breaking or fracturing of cell watts to deliver the broad range of the plant cell constituents. Seed cell ccrltur~ are further disclosed in commonly owned patent application U.S. publication number 2003-0091659.
. [0071 J Oraanic solvent extraction The organic extraction method involves a step of continuous washing and extracting the plant material against a stream of organic solvent. F~camptes of organic solvents indude, but are not limited to methanol, ethanol, dichloromethane, chloroform, xylene, and petroleum ether. For example; organic solvent exbWon can be conducted in an extracting machine. Raw materials collected from the plant which contains the desired alkaloid(s), such as leaves, bark, seeds, fivits, and/or roots, are first ground to smaN particle sizes, and then put'into the extracting machine through an inlet for the raw materials by a measurable charging machine. The plant materials are pushed by a thruster in the extracting machine and move forward slowly. Organic solvent (e:g:, ethanol) is added into the machine through a solvent inlet at the top of a waste discharging outlet. Due to the difference of gravity and equilibrium, the solvent flows toward the raw material inlet, soaks the materials and flows out from the opposite side of the solvent inlet. Since the plant materials and the. solvent move in opposite directions against each other, the plant materials are constantly immersed in a solution that contains tov~
concentration of extracted alkaloid(s). As a result of equilibrium, high yield of plant alkatoid(s) can be achieved by continuously extracting the plant material against the tow-concentration solution.
E0072J The time of extraction is preferably between about 1.-8 hours, more preferably between about 2-6 hours, and most preferably between about 3-5 hours. The temperature of extraction is preferably between about 30-90°C, more preferably befinreen about 40-70°C, and most preferably between about 50-60°C. The collected extract is then fine-filtered to remove debris, and concentrated by distilling the solvent until the solid content reaches between about 25% and 45%. The distilled solvent can be reused for extraction.
[0073] Aqueous-organic solvent extraction Raw materials collected from a plant which contains the desired alkaloid(s), such as leaves, bark, seeds, and/or roots of the plant, are first ground to small particle sizes.
The ground plant material is soaked in aqueous solution that is acidic or alkaline, depending on the solubility and stability of the desire alkaloids) under acidic or alkaline (basic) conditions. For extraction under acidic conditions, an acid such as hydrochloric acid or sulfuric acid is added to water at concentration of about 3% (w/v). For extraction under alkaline conditions, an alkali such as sodium hydroxide or sodium carbonate is added to water.
[0074] The time of extraction is preferably between about 1-8 hours, more preferably between about 2-6 hours, and most preferably between about 3-5 hours. The temperature of extraction is preferably between about 30-90°C, more preferably between about 40-70°C, and most preferably befinreen about 50-60°C. The extract is then collected and fine-filtered to remove debris. Alkaline agents (e.g., ammonia) or acidifying agents (e.g., sulfuric acid) can be added to the extract to neutralize the solution by adjusting the pH, depending on the acidity or alkalinity of the collected extract. Organic solvent is then added to the neutralized solution to extract the alkaloid from aqueous phase to organic phase. F~camples of such organic solvents include, but are not limited to, butanot, pentanot, hexanol and xytene. The extracted alkaloids) dissolved in organic solvent is/are concentrated unfit the solid content reaches about 50-80%.

j0075J It should be noted that different plants containing different kinds of alkaloids can be mixed and extracted together. This process of mixed extraction can preferably be used for extracting those plants containing alkaloids with similar solubility in the solvent used for extraction, such as ethanol. The mixture of alkaloids extracted can be concentrated and stored in an appropriate solvent.
j0076j Toosendanin has a molea~tar weight of 574.60, is colorless crystal, dissolves in methanol, chloroform and petroleum ether, and has a melting. point of 244-245°C. For the extraction of toosendanin from Melia toosendan Sieb. et Zucc., dried seeds from Melia toosendan Sieb. et Zucc.
can be ground and extracted against methanot_ After fine-filtrafion and concentration, the extract containing toosendanin is transferred into turpentine and stored in a coot, dark place. The content of foosendanin is fypicatty on the order of 20% by this procedure.
j007Tj Methods for extracting azadirachtin from plant materials, e.g., neem seeds, are described in H1, 541 to K.S. Holta.
j0078j Plant extracts for obtaining limonoids for use acco~ng to the present invention are commerciaNy available. For example; Ku taan Pi extract (containing toosendanin) is available from Premier Speddltres tnc., Middlesex, NJ. Ku Lian Pi is the Chinese name of the bark and root cortex of MeGa toosendan Sieb. et Zucc., or M. azedarach L.; this material can also be called Cortex Meliae. In addition; neem seed cell broth is available from Phyton, tnc. Ithaca, NY. Neem extract is available from Carlisle International Corp. NY.
Example 2 Clinic~evaluation of cosmefic~contraining limonoids j0079] The safety arid efficacy of a topically administered composition containing one or more limonoid constituents, e.g., toosendanin or azadirachtin, is studied in human subjects after a single-dose administration of a cosmetic formulation comprising the composition. Ten to fifteen panelists (preferably between 35 and 50 years old) with mild to moderate forehead lines and wrinkles are selected. The test subjects initially have a skin replicate made to establish a baseline depth and severity of wrinkles.
[0080] Dermatotogical tests are known in the art for testing the efficacy of an anti-wrinkling imposition. Any such test would be useful for the present invention. For example, a layer of a flexible "non-cured"
material, such as latex, may be applied to the skin. Upon removal of the "~~u material from the skin, the side that was adjacent to the skin can be assessed, either visually or via instrumentation, to determine the extent of wrinkling before treatment The same area of skin may then be assessed after treatment to determine if there has been a change in number or depth of wrinkles.
[008'1 ] On the following day, study ted~nicians apply 0:6 grams of product (at a concentration of timonoid constituent of 140 mg/g) over an approximate 6 cm2 semi-occlusive patch area on the subjects' foreheads.
The product formulation preferably consists of a formulation designed to increase the penetrafion of water-soluble materials. The product remains on the subjects' skin for one hour. After the one hour exposure, a repeat stun replicate is taken to assess changes from the previous day. In addition, the subjects are asked to actively contract the forehead skin and brow to determine the overall efficacy and potency of the formulation.
am le 3 [0082] A composition comprising limonoid constituents according to the present invention is prepared to assess its effects on one or more derrnatofogical related skin conditions. The composition is in the form of an oil-and=water emulsion and comprises the following ingredients:

Table 1 Oil-in-Water Emulsion In redient Amount Humectant e. . I cots, 1 0.5-15%
cerols Thickeners (e.g. gums, starches,0.1-4%

t mers Chelants t 0.001-0.5%

(e.g. disodium EDTA; tetrasodium EDTA

Preservatives 0.01-2%

Sunscreen (e.g., benzophenone,0.1-50%

eth the Imetho cinnamate Silicone 0.1-15%

Silica 0.01-1 p%

Fatty Alcohol/ Emulsifers 0.5-15%
/Wax/Fatty Aad Emoilienfs 0.1-20%

Extracts for limonoid constituents0.0001-50%

(e.g., one or more of Neem seed cell broth, Ku Lian Pi extract, Neem extract or an combination Demineralized water ' Q.S.

Example 4 [0083] A composition comprising limonoid constituents according to the presenfi invention is prepared to assess its effects on one or more dermatological related skin conditions. The composition is in the form of an oil-and-water emulsion and comprises the following ingredients:
Table 2 Oit-in-Water Emulsion In redient Amount Humectant e. ., I cots, 0.5-15%
( cerols Thickeners (e.g. gums, starches, 0.1-4%

of mers Chelants I 0.001-0.5%

e. . disodium EDTA~ tetrasodium EDTA

Preservatives 0.01-2%

Sunscreen (e.g. Parsol~ 1789;0.1-50%

ethylhexylmethoxycinnamate;

benzo henone-3 Silicone 0.1-15%

Silica 0.01-10%

Fatty Alcohol/ Emulsifers 0.5-15%
/Vllax/Fatty Acid (e.g., ceteth-20 phosphate/cetearyl alcohol/dicetyl phosphate, Tribehenin PEG-20 Ester, Sodium Dihydroxycethyl phosphate, cetearyl glucoside, co ceride Emulsion Stabilizers~scosityO.t-20%
Modfiers (e.g., acryiateslC~o~o altcyl acrylate crosspolymer, AcrylatelAminoacrylates/C~o-~
alkyl PEG-20 ttaconate, Sodium AcrylatelAcryloytdimethyt Taurate Coploymer, Acrylates/Ga3o attcyl a to cross mer Film Formers (e.g. decene/butene0.001-2%

copolymer, acrytates/octylaaytamide copolymer, adipic acidldiethylene cot/ cerin cross 1 mer Emollients 0.1-20%

Neem seed cell broth 0.01-10%

Neem extract U_01-10~

Ku Lian Pi exfract 0.01-10~

Demineralized water Q.S.

Example 5 [0084] A composition comprising limonoid constituents according to the present invention is prepared to assess its effects on one or more dermatological related skin conditions. 'Fhe composition is in the fom~ of a water/silicone emulsion and comprises the following ingredients:

Tab 3 Water/Siticone Emulsion M radiant Amount Sodium PCA 50% 0.1-4%

Sodium Lactate 60% 0.01-10%

Sodium Chloride 0.1-10%

Humectant (e.g., glycerin, 0.5-10%
glyools, 1 carols Ammonium h droxide 0.01-10%

C clomethicon 0.1-20%

C ctomethicone/Dimethicone 0.1-20%
Co ! Col Emollients e. ., ce octanoate0.1 20%

Dimethicone Copolyoll 0.1-10%
C clo entasitoxane Neem seed cell broth 0.01-10%

Neem extract 0_01-10%

Ku Lian Pi extract 0.01-1090 Demineralized water Q.S.

Example 6 j0085~ A composition comprising limonoid constituents according_to the present invention is prepared to assess, its effects on one or more dennatological related skin conditions. The composition is in the form of a gel and comprises the following ingredients.

Tabte 4 el ~edien Amount .
t in _ 0.01-3%
_ Carbo of GI cerin 0.1-30%

B ene col 0.1-30%

Disodium EDTA 0.01-2%

Meth t araben 0.01 2%

dro eth ! ce!!ulose 0.01-2%

Com Zea ma s starch 0.01-10%

C.S. D&C Yetfow No.10 0.001-1 ~o C.S. FD&C Blue No. 1 0.001-1 POE 20M meth i lucose ether0.01-10%

Dimeth I itoxane 0.01-10%

PEG 50 Shea butter 0.01-10%

Sodium h roxide solution 0.01-5~6 Ben alcohol 0.01-5~

Neem seed cell broth 0.01-10~

Neem extract 0.01-10%

Ku Van Pi extract 0.01-10%

Demineratized water [ Q.S.

Example 7 [0086] A composition comprising limonoid constituents according to the present invention is prepared to assess its effects on one or more dermatological related skin conditions. The composition is in the form of a cleansing foam and comprises the following ingredients:

Table 5 ~leansin. Fg oam In redient Amount Humectant (e.g., glycerin, butylene5-25%
I cot Pot eth lene l cot 0.1-20%

Bentonite 0.1-20%

Stearic acid 0.1-30%

_ _ 0.1-20%
M risitc acid Cetea ! AlcohotlCeteareth-20 1.00000%

Potassium h droxide 45% 0.1 20%

Preservatives (e.g., Benzyl 0.1-10%
alcohol, 2- _ Pheno ethanol Neem seed cell broth 0.01-10%

Neem extract 0.01-10~

Ku dart Pi extract 0:09-10%

Demi~eratized water Q.S.

Example 8 [0087] A iiposome composition comprising tirt~onoid constituents according to the present invention is prepared as fotiows:
abe6 i-i osome In ~edierEt Amount Lecicthir~ 5-25~

Ct _,s a# i benzoate '!-95%

Quatemium 15 Q_01:-2%

Parabens 0.01-2%

Neem seed ceii broth 0.09-10~

teem extract 0.01-10%

' Ku Lian Pi extract 0.01-10~

[0 0 8 8j The contents of all patents, patent applications, published articles, at~stract's, boots, rei'erence manuals and abstracts, as cited herein are to more fully describe the state of the art to which the invention pertains.
[0089j As various changes can be made in the above-described subject matter without departing from the scope and spirit of the present invention, it is intended that all subject matter contained in the above description, or defined in the appended claims, be interpreted as descriptive and illustrative of the present invention. Many modifications and variations of the present invention are possible in light of the above teachings.

Claims (14)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE PROPERTY
OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. Use of an effective amount of a composition comprising one or more limonoid constituents and an acceptable vechicle, carrier or diluent for treatment, prevention, reduction and/or amelioration of hyperhidrosis.

2. Use of one or more limonoid constituents in the manufacture of a composition for treatment, prevention, reduction and/or amelioration of hyperhidrosis.

3. The use according to claim 1 or 2, wherein the one or more limonoid constituents is toosendanin, azadirachtin, or a combination thereof.

4. The use according to claim 1 or 2, wherein the one or more limonoid constituents is present in an amount of from about 0.0001 wt% to about 10 wt%
of the total weight of the composition.

5. The use according to claim 1 or 2, wherein the one or more limonoid constituents is present in an amount of from about 0.0005 wt% to about 5 wt%
of the total weight of the composition.

6. The use according to claim 1 or 2, wherein the one or more limonoid constituents is present in an amount of from about 0.001 wt% to about 1 wt% of the total weight of the composition.

7. The use according to claim 1 or 2, wherein the composition is for topical application to the skin to treat, prevent, reduce, ameliorate and/or eliminate excessive sweating of underarm, face, feet and hands.

8. Use of an effective amount of a composition comprising one or more limonoid constituents and an acceptable vehicle, carrier or diluent, for inhibition or reduction of acetylcholine release at a neuromuscular junction (NMJ) in the fibers of sweat glands, wherein said inhibition or reduction of acetylcholine release at the neuromuscular junction (NMJ) concomitantly inhibits or reduces contraction of muscle tissue of the fibers of sweat glands, thereby treating, preventing, reducing, ameliorating and/or eliminating hyperhidrosis.

9. Use of one or more limonoid constituent in the manufacture of a composition for inhibition or reduction of acetylcholine release at a neuromuscular junction (NMJ) in the fibers of sweat glands, wherein said inhibition or reduction of acetylcholine release at the neuromuscular junction (NMJ) concomitantly inhibits or reduces contraction of muscle tissue of the fibers of sweat glands, thereby treating, preventing, reducing, ameliorating and/or eliminating hyperhidrosis.

10. The use according to claim 8 or 9, wherein the one or more limonoid constituents is toosendanin, azadirachtin, or a combination thereof.

11. The use according to claim 8 or 9, wherein the one or more limonoid constituents is present in an amount of from about 0.0001 wt% to about 10 wt%
of the total weight of the composition.

12. The use according to claim 8 or 9, wherein the one or more limonoid constituents is present in an amount of from about 0.0005 wt% to about 5 wt%
of the total weight of the composition.

13. The use according to claim 8 or 9, wherein the one or more limonoid constituents is present in an amount of from about 0.001 wt% to about 1 wt% of the total weight of the composition.

14. The use according to claim 8 or 9, wherein the composition is for topical application to the skin to treat, prevent, reduce, ameliorate and/or eliminate excessive sweating of underarm, face, feet and hands.