MX2008006328A - Compositions and methods of topical application and transdermal delivery of botulinum toxins with reduced non-toxin proteins - Google Patents

Abstract

This invention relates to novel compositions of botulinum toxin that can be applied topically for various therapeutic, aesthetic and/or cosmetic purposes. The compositions may include botulinum toxin complexes, wherein the amounts of hemagglutinin, non-toxin non-hemagglutinin and/or exogenous albumin are selectively and independently reduced compared to conventional commercially available botulinum toxin. The compositions may further contain molecules that are not native to botulinum toxin and that bind non-covalently to the botulinum toxin complexes, thereby acting as skin-tropic"adhesion molecules"to improve the ability of the toxin complexes to adhere to and to penetrate the skin epithelium. The compositions have an improved safety profile compared to existing botulinum-containing compositions that are injected subcutaneously. Methods for the use of such compositions are also contemplated by this invention.

Description

COMPOSITIONS AND METHODS OF TOPICAL APPLICATION AND TRANSDERMAL SUPPLY OF BOTULINIC TOXINS WITH NON-TOXIC PROTEINS FIELD OF THE INVENTION The present invention relates to novel botulinum toxin compositions which can be administered topically for various therapeutic, aesthetic and / or cosmetic purposes and which have an improved safety profile compared to existing botulin-containing compositions that are injected subcutaneously. BACKGROUND OF THE INVENTION The skin protects the organs of the body from environmental threats and acts as a thermostat to maintain body temperature. It consists of several different layers, each with its specialized functions. The major layers include the epidermis, the dermis and the hypodermis. The epidermis is a stratified layer of epithelial cells that are above the dermis, which consists of connective tissue. Both, the epidermis and the dermis are also supported by the hypodermis, an inner layer of adipose tissue. The epidermis, the highest layer of the skin, is only 0.1 to 1.5 millimeters thin (Inlander, Skin, New York, NY: People's Medical Society, 1-7 (1998)). It consists of keratinocytes and is divided into several layers based on their state of differentiation. The epidermis can also be classified into stratum corneum and the viable epidermis, which consists of melpgihian and granular base cells. The stratum corneo is hygroscopic and requires at least 10% wetting by weight to maintain its flexibility and softness.

The hygroscopicity is attributable in part to the ability to retain the water of keratin. When the cornea layer loses its softness and flexibility it becomes rough and fragile, resulting in dry skin. The dermis, which is placed just below the epidermis, is 1.5 to 4 millimeters thick. It is the thinnest layer of the three layers of the skin. In addition, the dermis is also home to most skin structures, including sweat and oil glands (which secrete substances through openings in the skin called pores, or comedos), hair follicles, endings of nerves, and vessels of blood and lymph (Inlander, Skin, New York, NY: People's Medical Society, 1-7 (1998)). However, the main component of the dermis are collagen and elastin. The hypodermis is the deepest layer of the skin. It acts as an insulator for the preservation of body heat and as a shock absorber for organ protection (Inlander, Skin, New York, NY: People's Medical Society, 1-7 (1998)). In addition, the hypodermis also stores fat for energy reserves. The pH of the skin is normally between 5 and 6. The acidity is due to the presence of amphoteric amino acids, lactic acid and fatty acids from the secretions of sebaceous glands. The term "acid blanket" refers to the presence of water-soluble substances in most skin reactions. The storage capacity of the skin is due in part to these secretions stored in the corneal layer of the skin. Wrinkles, one of the signs of age, can be caused by biochemical, histological and physiological changes that accumulate from the damage of the environment to the skin. (Benedetto, International Journal of Dermatology, 38: 641-655 (1999)). In addition, there are other secondary factors that can cause the characteristic folds, grooves and folds in facial wrinkles. (Stegman et al., The Skin of the Aging Face Cosmetic Dermatological Surgery, 2nd edition, ST.

Louis, MO: Mosby Year Book: 5-15 (1990)). These secondary factors include the constant shooting of gravity, frequent and constant positional pressure on the skin (for example during sleep), and repeated facial movements caused by the contraction of the facial muscles (Stegman et al.

The Skin of the Aging Face Cosmetic Dermatological Surgery, 2nd edition, St. Louis, MO: Mosby Year Book: 5-15 (1990)). Different techniques have been used to potentially attenuate some of the signs of aging. These facial humectant technique ranges contain alpha hydroxy acids and retinol for surgical procedures and injections of neurotoxins. For example, in 1986, Jean and Alastair Carruthers, a husband and wife team consisting of an ocuplastic and dermatologist, developed a method of using the type A form of botulinum toxin for the treatment of wrinkles related to movement in the area. between brow (Schantz and Scott, In Lewis GE (Ed) Biomedical Aspects of Botulinum, New York: Academic Press, 143-150 (1981)). By 1994, the same team reported experiences with other wrinkles related to movement in the face (Scott, Ophthalmol, 87: 1044-1049 (1980).

This, in turn, leads to the birth of the era of cosmetic treatment using the type A form of botulinum toxin. Interestingly, the type A form of botulinum toxin is said to be the most lethal natural biological agent known to man. C. Botulinum spores are found in the soil and can grow in improperly sterilized and sealed food containers. Ingestion of the bacteria can cause botulism, which can be fatal. Botulinum toxins act to produce muscle paralysis preventing synaptic transmission or release of acetylcholine through neuromuscular junction, and it is thought that it also acts in other pathways. Its action essentially blocks the signals that normally cause muscle spasms or contractions, which result in paralysis. However, the effects that paralyze the muscle of botulinum toxin have been used for therapeutic purposes. The controlled administration of botulinum toxin has been used for muscle paralysis to be brought to treatment conditions, for example, neuromuscular dysfunctions characterized by hyperactive skeletal muscles. Conditions that have been treated with botulinum toxin include hemifacial spasm, spasmodic torticollis in advanced age, anal fissures, blepharospasm, cerebral palsy, cervical dysphonia, migraine headaches, strabismus, temporomandibular dysfunction, and various types of cramps and muscle spasms. More recently the paralyzing effects of the botulinum toxin muscle have taken advantage in the applications-therapeutic and cosmetic facials such as wrinkle treatment, zone lines, and other results of spasms or contractions of facial muscles. In addition to the type A form of botulinum toxin, there are seven other distinct forms of botulinum toxin of the serological type that are also produced by the gram-positive bacterium Clostridium botulinum. Of these eight different types of botulinum toxin of serological type, the seven that can cause paralysis have been designated the name of toxin serotype A, B, C (also known as C, D, E, F and G. Each of it is distinguished by neutralization with antibodies of the specific type.The molecular weight of the botulinum toxin protein molecule for the seven of the botulinum toxin serotypes is approximately 150 kD The different serotypes of botulinum toxin vary in animal species which they affect and in the severity and duration of the paralysis that it evokes For example, it has been determined that botulinum toxin type A is 500 times more potent than botulinum toxin type B, measured by the range of paralysis produced in In addition, botulinum toxin type B has been determined to be non-toxic in primates at a dose of 480 U / kg, approximately 12 times of the primate LD50 for type A. Due to the medi Because of the molecule and molecular structure of botulinum toxin, the stratum corneum and the multiple layers of the underlying skin architecture can not be crossed. As it is released by the bacterium Clostridium botulinum, the botulinum toxin is a component of a toxin complex containing approximately 150 kD of botulinum toxin protein molecule together with the associated non-toxic proteins. These non-toxic endogenous proteins are believed to include a family of hemagglutinin proteins, as well as those that do not contain hemagglutinin. The non-toxic proteins are believed to be to establish the botulinum toxin molecule in the toxin complex and protect it against denaturation, for example, by digestive acids when the complex is ingested. In this way, the non-toxic proteins protect the activity of the botulinum toxin and carry out the systematic penetration, particularly when the toxin complex is administered by the gastrointestinal tract. More specifically, it is believed that some of the non-toxic proteins specifically improve penetration through the gastrointestinal epithelium while other non-toxic proteins stabilize the botulinum toxin molecule in the blood. further, the presence of non-toxic proteins in the toxin complexes typically cause the toxin complexes to have molecular weights that are greater than that of the botulinum toxin molecule, which is 150 kD, as has been previously noted. For example, the bacterium Clostridium botulinum can produce botulinum toxin type A complexes having molecular weights of approximately 900 kD, 500 kD or 300 kD. Interestingly, botulinum toxin type B and C are obviously produced with a complex of only 700 kD or 300 kD. Botulinum toxin type D is produced with both complexes of 300 kD and 500 kD. Botulinum toxin types E and F are produced with complexes of approximately 300 kD. To have additional stability of the botulinum toxin, the toxin complexes are often stabilized by combining them with endogenous stabilizers (eg gelatin, polysaccharides, or more common additional albumin) during processing. The stabilizers serve to bind and stabilize the toxin complexes in different environments, including those associated with processing, transportation, storage and administration.

Typically, botulinum toxin is administered to patients by carefully controlling injections of compositions containing botulinum toxin complex and albumin, but there are several problems related to that scope. Not only are the injections painful, but they must often supply too many toxins to create large subdermal wells of local toxin around the site of the injection, in order to achieve the desired therapeutic and aesthetic effects. Worse yet, many injections may be required when the area to be treated is large. In addition, because the injected toxin complexes contain non-toxic proteins and albumin that stabilizes botulinum toxin-and increase the molecular weight of the toxic complex, the toxin complexes have a long half-life in the body, are slow to disperse in the tissue and can cause an undesirable antigenic response in the patient. Also, since non-toxic proteins and albumin stabilize botulinum toxin in the blood, the injections can be carefully placed so that they do not release a large amount of toxin into the patient's bloodstream, which can lead to systemic poisoning. fatal. In this way, injections can typically be carried out precisely by highly trained medical personnel with a great understanding of the human anatomy. In view of all the problems discussed above, it would be highly desirable to have a method for administering botulinum toxin that would be painless and requires fewer toxins than conventional injection-based methods. Furthermore, it would be highly desirable if this method would reduce the antigenicity and blood stability of botuinic toxin, while increasing the diffusion range of botulinum toxin complexes within the body, and thus it is safer to use botulinum toxin for several. therapeutic, aesthetic and / or cosmetic purposes. It is also desirable to have a method of administration that does not critically depend on the precise injection of the botulinum toxin by a medical professional in order to have a safe administration of the toxin. Brief Description of the Invention The present invention provides a solution to the aforementioned problems by providing a botulinum toxin composition that can be administered topically to the epithelium of the skin easily and painlessly. The botulinum toxin complexes in the topical compositions of this invention have reduced the antigenicity, the lower blood stability, a better safety profile, and higher diffusion ranges through the epithelium of the skin compared to the complexes of Conventional commercial botulinum toxin that are bound to exogenous albumin (for example BOTOX ® or MYOBLOC ®). In addition, using the compositions and related methods of the present invention, less botulinum toxin is required to achieve the same clinical result compared to methods based on administration injections. One aspect of the present invention is the recognition that the non-toxic endogenous proteins in a botulinum toxin complex obtained from the Clostridium botulinum bacterium (viz.

Non-toxic hemagglutinin and proteins without hemagglutinin) undesirably increase the stability and toxicity of the toxin complex, while undesirably decreasing the ability of the toxin to disperse through the epithelium of the skin. The present invention recognizes that these effects are exacerbated when an exogenous stabilizer, such as albumin, binds to the botulinum toxin during the manufacturing process. Thus, one aspect of the invention is to provide botulinum toxin complexes wherein the amounts of hemagglutinin, without non-toxic hemagglutinin, and / or exogenous albumin are selectively and independently reduced compared to the commercially available conventional botulinum toxin (e.g. BOTOX® or MYOBLOC® Another aspect of the present invention is the recognition that some non-native molecules (ie molecules not found in the botulinum toxin complexes obtained from the bacterium Clostridium botulinum can be added to the botulinum toxin complexes)., and in particular reduced botulinum toxin complexes, to increase the ability of the botulinum toxin complex to disperse through the epithelium of the skin. In a particular preferred embodiment, the non-native molecules bind non-covalently to botulinum toxin complexes to adhere to and penetrate the epithelium of the skin, and further reduce the stability of the botulinum complex in the blood. By way of example, the adhesion molecules may be some proteins, such as sialoproteins. Accordingly, an object of this invention is to provide a composition comprising a botulinum toxin complex (or a reduced botulinum toxin complex) and non-native target adhesion molecules in the skin that improve the transdermal penetration of the composition for cosmetic treatments and therapeutic The composition may optionally contain aggregated exogenous stabilizers, such as albumin. The invention further relates to a method of producing a biological effect by topically administering an effective amount of the compositions within the invention, preferably to the skin, or to a subject or a patient in need of this treatment. The biological effect may include, for example, muscle paralysis, reduction of hypersecretion or sweating, treatment of neurological pain or migraine, reduction of muscle spasms, prevention and reduction of acne, reduction and improvement of immune response, reduction of wrinkles, or prevention of treatment of several other dysfunctions. The invention also provides kits for preparing formulations containing a botulinum toxin complex (or a reduced botulinum toxin complex) and adhesion molecules that can in turn be used to produce this formulation. Kits are also provided which contain means for sequentially administering a complex of botulinum toxin (or a reduced botulinum toxin complex) and adhesion molecules to a subject. Detailed Description of the Invention The present invention relates to novel compositions comprising a botulinum toxin, more specifically to compositions that improve the transport or delivery of botulinum toxin through the epithelium of the skin (also referred to as "transdermal delivery") with Improved adhesion to the skin and penetration, reducing the antigenicity and stability of the blood. The compositions of the invention can be used as topical applications to provide a botulinum toxin to a subject, for various purposes, such as therapeutic, aesthetic and / or cosmetic. The compositions of the invention also have an improved safety profile compared to the compositions and methods of botulinum toxin delivery. In addition, these compositions may offer beneficial reductions in the immune response of botulinum toxin.

The term "botulinum toxin" as used herein, refers to any of the known types of botulinum toxin (ie approximately 150 kD of botulinum toxin protein molecule), whether produced by the bacterium or by recombinant techniques, as well as any type that can subsequently be discovered including newly discovered serotypes and energized variants or fusion proteins. As mentioned above, currently seven immunologically distinct botulinum neurotoxins have been characterized, precisely serotypes of botulinum neurotoxin A, B, C, D, E, F and G, each being distinguished by neutralization with specific type antibodies. Botulinum toxin serotypes are commercially available, for example, from Sigma-Aldrich (St. Louis, MO) and Metabiologics, Inc. (Madison, Wisconsin) as well as from other sources. The different serotypes of botulinum toxin vary in the species of animals that affect them and in the severity and duration of paralysis they evoke. At least two types of botulinum toxin, types A and B, are commercially available in formulations for the treatment of some conditions. Type A, for example, is contained in preparations of Allergan that has the trademark BOTOX® and of Ipsen that has the trademark DYSPORT®, and of type B is contained in preparations of Elan that has the trademark MYOBLOC®. The term "botulinum toxin" used in the compositions of the present invention can alternatively be referred to as a botulinum toxin derivative, that is, a compound having botulinum toxin activity but containing one or more chemical or functional alterations anywhere or in any chain relative to native or recombinant botulinum toxins that occur naturally. For example, botulinum toxin can be a modified neurotoxin which is a neurotoxin which has at least one of its amino acids removed, modified or replaced, comparing it to a native, or the modified neurotoxin can be a neurotoxin produced from recombinant handle or a derivative or fragment of it. For example, the botulinum toxin may be one that has been modified so that, for example, it improves its properties or diminished side effects, but this still retains the desired botulinum toxin activity. The botulinum toxin may be one of the botulinum toxin complexes produced by the bacterium as described above. Alternatively, the botulinum toxin used in this invention can be a toxin prepared using synthetic or recombinant chemical techniques, for example, recombinant peptide, fusion protein and hybrid neurotoxin, for example prepared from subunits or serotype domains of botulinum toxin (see U.S. Patent 6,444,209 , for example). The botulinum toxin may also be a portion of the total molecule that has been shown to have the necessary botulinum toxin activity, and in this case it may be used by itself or as part of a combination or conjugated molecule, for example, a fusion protein. . Alternatively, the botulinum toxin may be in the form of a botulinum toxin precursor, which may be non-toxic, for example a non-toxic zinc protease that becomes toxic in proteolytic cleavage. The term "botulinum toxin complex" or "toxin complex" as used herein refers to approximately 150kD of botulinum toxin protein molecule (belonging to any serotype of botulinum toxin AG), together with endogenous non-toxic proteins (ie hemagglutinin protein and protein without hemagglutinin and without toxin produced by the bacterium Clostridium botulinum). However, it should be noted that the botulinum toxin complex must be derived from the bacterium Clostridium botulinum as a unit toxin complex. For example, botulinum toxin or modified botulinum toxin can be first prepared recombinantly and subsequently combined with non-toxic proteins. The recombinant botulinum toxin can also be acquired (for example from List Biological Laboratories, Campbell, CA) and then combined with non-toxic proteins. The present invention further contemplates "reduced botulinum toxin complexes", in which the botulinum toxin complexes have reduced the amounts produced by the bacterium Clostridium botulinum. In one embodiment, reduced botulinum toxin complexes are prepared using any conventional protein separation method to extract a hemagglutinin or protein fraction without non-toxic hemagglutinin from the botulinum toxin complexes derived from the bacterium Clostridium botulinum. For example, reduced botulinum toxin complexes can be produced by dissociating the botulinum toxin complexes through exposure to red blood cells at pH 7.3 (for example, see European patent 1514556 A1, and thus incorporated herein by reference). ). HPLC, dialysis, columns, centrifugation and other methods to extract protein proteins, can be used. Alternatively, when the botulinum toxin complexes are to be produced by synthetically combining the botulinum toxin produced with non-toxic proteins, only less haemagglutinin or non-toxic hemagglutinin protein can be added to the mixture, which would be present for toxin complexes. botuiinica that occur naturally. Any of the non-toxic proteins (e.g., hemagglutinin protein or protein without non-toxic hemagglutinin, or both) in the botulinum toxin complexes according to the invention can be independently reduced by any amount. In any exemplary embodiment, one or more non-toxic proteins are reduced by at least about 0.5%, 1%, 3%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% compared to the amounts normally found in botulinum toxin complexes. The bacterium Clostridium botulinum produces seven different types of toxin and commercial preparations are made with different relative amounts of non-toxic proteins (i.e. different amounts of toxin complexes). For example, Myobloc has 5000 U of botulinum toxin type B by me with 0.05% human serum albumin, 0.01 M sodium succinate, and 0.1 M sodium chloride. Dysport has 500U of botulinum toxin type A of hemagglutinin complex with 125 mg of albumin and 2.4 mg of lactose. In a particularly interesting embodiment, substantially all non-toxic proteins (eg, 95% hemagglutinin protein and protein without non-toxic hemagglutinin) that would normally be found in the botulinum toxin complex derived from the bacterium Clsotridium botulinum would be removed from the toxin complex botulinum In addition, although the non-toxic proteins of endogenous amount can be reduced by the same amount in some cases, this invention also contemplates reducing each of the non-toxic proteins by different amounts, as well as reducing at least one of the non-toxic endogenous proteins, but not others. In addition to (instead of) reducing the amount of endogenous non-toxic protein to destabilize the botulinum toxin complex, this invention also contemplates reducing the amount of exogenous stabilizers that are normally added during processing. An example of such an exogenous stabilizer is albumin which is normally added during the elaboration of botulinum toxin complexes in amount equal to 1000 times the amount of albumin found in the nontoxic component without hemagglutinin of a naturally occurring botulinum toxin complex. In accordance with this invention, the amount of exogenous albumin added can be any amount less than the 1000-fold conventional excess of exogenous albumin. In some exemplary embodiments of the invention, only approximately 500x, 400x, 300x, 200x, 100x, 50x, 10x, 1x, 0.5x or 0.01x of albumin amount is added in naturally occurring botulinum toxin complexes. In a modality, the non-exogenous albumin is added as a stabilizer to the compositions of the invention. In other embodiments the exogenous stabilizers in addition to (instead of) albumin are added to the therapeutic topical compositions of the invention. For example, other stabilizers contemplated by the invention include lactose, gelatin and polysaccharides. An "adhesion molecule" according to the invention may be a protein or other molecule having at least the following properties: (1) it is not found in botulinum toxin complexes that occur naturally (ie non-native), (2) ) serves to stabilize botulinum toxin complexes or reduced botulinum toxin complexes, especially those that have been combined with little or no excess of exogenous albumin and other stabilizers and (3) when mixed with botulinum toxin complex or reduced botulinum toxin, the transdermal penetration of botulinum toxin is promoted, enabling the toxin to be administered to the muscles and / or other structures related to the skin in amounts that are sufficient to produce a desired therapeutic or cosmetic effect. Generally speaking, it is preferable if transport can occur without covalent modification of botulinum toxin. In some preferred embodiments, the adhesion molecules are capable of binding to specific components of the skin, unlimited examples of keratinocytes, epidermal cells and hair follicles. By way of example, the adhesion molecules according to the invention can be proteins capable of binding to the keratinocyte growth factor, keratinocyte binding proteins, epidermal growth factor (EGF), proteins similar to EGF and neurotrophins such as nerve growth factor, neurotrophic factor derived from the brain, neurotrophin-3 and neurotrophin 4/5. In some embodiments of the invention, the topical therapeutic composition includes more than one different type of non-native adhesion molecule. In a particularly interesting embodiment, non-native adhesion molecules are sialoproteins. Without wishing to unite them by any particular scientific theory, it is believed that sialoproteins promote skin adhesion and transdermal penetration of botulinum toxin, while improving the stabilization of botulinum toxin in the skin and in vitro and reducing blood and systemic activity for an improved security profile. Non-limited examples of sialoproteins contemplated by that invention include bone sialoproteins I (also known as BSPII, bone sialoprotein, osteopontin, OPN, secreted phosphoprotein 1, Spp 1, early T lymphocyte activation protein-1, ETA-1, bone protein urinary, nephropontine and bone sialoprotein II (also known as BSPII, integrin-binding sialoprotein, cell-binding sialoprotein, BNSP.) Sialoproteins are commercially available, for example, from Chemicon International. epithelial cells especially skin and biliary epithelial cells can be used.The family of adhesion molecules such as "cadherins" or "integrins", the immunoglobulin family, selectins and other transmembrane sialoproteins such as podocalixin, can be added. , the concentration of adhesion molecules in the compositions according to the invention should be sufficient to allow the botulinum toxin to be delivered transdermally. Furthermore, without wishing to be bound by theory, it is believed that the range of transdermal transport follows the kinetics mediated by the receptor, as transdermal transport increases with increased amounts of adhesion molecules to a point of saturation, under which the Transport range becomes constant. Thus, in a preferred embodiment, the amount of aggregate adhesion molecules is equal to the amount that maximizes the transdermal penetration range just before saturation. A useful concentration range for the adhesion molecules in the topical compositions of this invention is from 0.1 to about 1.0 mg per unit of botulinum toxin composition as described above. More preferably, the adhesion molecules in the topical compositions of the invention are in a range of 0.1 mg to approximately 0.5 mg per unit of botulinum toxin. For example, in case of bone sialoprotein I, which is an example of a sialoprotein contemplated by the invention, a useful range is between 0.1 mg to about 1.0 mg, more preferably between 0.1 mg to about 0.5 mg. The compositions of this invention are preferably in the form of products to be applied to the skin or epithelium of subjects or patients, i.e. humans or other mammals in need of the particular treatment. The term "in need" is intended to include both needs; pharmaceutical and health related, for example, treatment conditions involving undesirable facial muscle spasms, as well as cosmetic and subjective needs, for example, altering or improving the appearance of facial tissue. In general, the compositions are prepared by mixing the botulinum toxin (containing related non-toxic proteins or reduced non-toxic related proteins) with non-native adhesion molecules and usually with one or more additional pharmaceutically acceptable carriers or excipients. In their simplest form they may contain a pharmaceutically acceptable carrier or diluent such as neutral saline. However, the compositions may contain other typical ingredients in pharmaceutical or cosmetic compositions, that is, a pharmaceutically or dermatologically acceptable carrier, vehicle or medium, i.e. a carrier, vehicle or medium that is compatible with the tissues to which they will be applied. The term "dermatologically or pharmaceutically acceptable", as used herein, means that the compositions or components thereof described above are suitable for use in contact with these tissues or for use in general patients without undue toxicity, incompatibility, instability, allergic response and the like. As is appropriate, the compositions of the invention may comprise any ingredient in a conventional manner used in the fields under consideration, and particularly in cosmetics and dermatology. In terms of their form, the compositions of this invention can include solutions, emulsions (including microemulsions), suspensions, creams, lotions, gels, powders or other typical solids or liquid compositions used for application to the skin and other tissues wherein the compositions can be used. Such compositions may contain, in addition to the botulinum toxin and non-native adhesion molecules, other ingredients typically used in these products, such as antimicrobial humectants and hydrating agents, penetrating agents, preservatives, emulsifiers, natural or synthetic oils, solvents , surfactants, detergents, gelling agents, emollients, antioxidants, fragrances, fillers, slimming, waxes, absorbs odors, driers, coloring agents, powders, viscosity control agents and water, and optionally including anesthetics, active for itching, botanical extracts, softening agents for darkening or whitening, gloss, moisturizers, mica, minerals, polyphenols, silicones or derivatives thereof, sunblocks, vitamins and phytomedicinals.

The compositions according to this invention may be in the form of controlled release or substituted release compositions, wherein the botulinum toxin and the non-native adhesion molecules are encapsulated and otherwise contained within a material so that they are released in the skin in a controlled manner over time. The composition comprising botulinum toxin and non-native adhesion molecules can be contained within the matrices, liposomes, vesicles, microcapsules, micro spheres and the like or within a particular solid material, all are chosen and / or constructed to provide toxin release botulinum through time. Botulinum toxin and non-native adhesion molecules can be encapsulated together (for example in the same capsule) or separately (in separate capsules). Botulinum toxin can be delivered to the muscles under the skin, or to glandular structures within the skin in an amount effective to produce paralysis, produce relaxation, alleviate contractions, prevent or relieve spasms, reduce glandular output or other desired effects. The local supply of botulinum toxin in this manner could allow dosage reduction "reduces toxicity" and allows more accurate dosage optimization for the desired effects in relation to injectable or implantable materials. The compositions of the invention are applied so as to administer an effective amount of botulinum toxin. He term "effective amount" as used herein, means an amount of botulinum toxin as defined above that is sufficient to produce the desired muscle paralysis or other biological or aesthetic effect, but this is implicitly a safe quantity, i.e. one that is too low to avoid serious side effects. The desired effects include the relaxation of some muscles in order, for example, to diminish the appearance of fine lines and / or wrinkles, especially on the face, or to adjust the facial appearance in other ways so that the eyes are dilated, the corners of the mouth, or soften lines that extend from the upper lip or the general relief of muscle tension. The last mentioned effect, the general relief of muscular tension can be carried out on the face or on any other side. The compositions of the invention may contain an appropriate effective amount of botulinum toxin for application as a single dose treatment or may be concentrated, either for dilution at the site of administration or for use in multiple applications. Through the use of non-native adhesion molecules for the skin of this invention, a botulinum toxin can be administered transdermally to a subject to treat conditions such as undesirable facial muscles or other muscle spasms, hyperhidrosis, acne conditions or in some other side of the body in which the relief of muscular pain or spasms is desired. Botulinum toxin is administered topically for transdermal delivery to muscles or other structures associated with the skin. The administration can be done, for example, to the legs, shoulders, back (including lower back), armpits, palms, feet, neck, groin, the back of the hands or feet, elbows, upper arm, knees, upper part of the legs, buttocks, torso, pelvis, or any other part of the body where the administration of botulinum toxin is desired. The administration of botulinum toxin can also be carried out to treat other conditions, including but not limited to treating neurological pain, prevention or reduction of migraines. or other headaches, prevention or reduction of acne, prevention or reduction of distomy or dystonic contractions (whether subjective or clinical), prevention or reduction of symptoms related to subjective or clinical hyperhidrosis, reducing hypersecretion or sweating, reducing or increasing the immune response, or treatment of other conditions for which the administration of botulinum toxin by injection has been suggested or improved. More preferably, the compositions are administered by or under the direction of a physician or other health care professional. They can be administered in a single treatment or in series of periodic treatments over time. For transdermal delivery of botulinum toxin for the aforementioned purposes, a composition described above is applied topically to the skin or to some location where the effect is desired. Because of its nature, more preferable is the amount of botulinum toxin that would be administered with care, in a range of application and frequency of application that will produce the desired result without producing any adverse or undesired result. Accordingly, for example, the topical compositions of the invention should be applied in a range from 1U to about 20,000U, preferably from 1U to about 10,000U per cm2 of skin surface. Higher dosages within those ranges could preferably be used in conjunction with controlled release materials, for example, or allow a shorter residence time before disposal. This invention also includes transdermal delivery devices for transmitting botulinum toxin-containing compositions described herein through the skin. These devices may be as simple in construction as a skin patch, or may be a more complicated device that includes means for dispensing and monitoring the supply of the composition, and optionally means for monitoring the condition of the subject in one or more aspects, including monitoring the subject's reaction to the substances that are supplied. The compositions, both in general, and in these devices, can be pre-formulated or pre-installed in the device, or prepared afterwards, for example using a kit containing the two ingredients (botulinum toxin and non-native adhesion molecules) to combine or before the application time. The amount of non-native adhesion molecules or the range of botulinum toxin will depend on which carrier is chosen for use in the composition in question. The appropriate amount or range of carrier molecule in a given case can be determined, for example, by carrying out one or more experiments such as those described above. In general, the invention also contemplates a method for administering botulinum toxin (preferably as reduced botulinum toxin complexes) to a subject or patient in need thereof, in which an effective amount of botulinum toxin is administered topically in conjunction with the adhesion molecules as described above. By "in conjunction with" it is meant that the two components (botulinum toxin and adhesion molecules) are administered in a combination procedure, which may involve combining them before topical administration to a subject, or administering them separately, but a way that they act together to provide the required supply of an effective amount of therapeutic protein. For example, a composition containing adhesion molecules can first be applied to the skin of a subject, followed by applying a patch or other device containing botulinum toxin. Botulinum toxin can be incorporated in a dry form in a skin patch or other delivery device and adhesion molecules can be applied to the surface of the skin before application of the patch so that they act together, resulting in the delivery desired transdermal In this sense, in this way, the two substances (adhesion molecule and botulinum toxin) act in combination or probably interact to form a composition or combination in situ. Consequently so, the invention includes a kit with a device for delivering botulinum toxin through the skin and a liquid, gel, cream or the like containing adhesion molecules, and which is suitable for application to the skin or epithelium of a subject. Kits for administering the compositions of the invention, under the supervision of a health care professional or by a patient or subject, may include an appropriate custom applicator for this purpose. The compositions of this invention are suitable for use in physiological environments with the pH range from 4.5 to about 6.3 and thus may have such a pH. The compositions according to the present invention can be stored at room temperature or under refrigeration conditions. It should be understood that the following examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in view thereof would be suggested to persons skilled in the art and should be included within the spirit and scope of the application and the scope of the appended claims. All publications, patents and patent applications cited are here incorporated by reference in your entity for all purposes. Example 1. Transport of botulinum toxin in vivo using sialoproteins. This experiment demonstrates the use of sialoproteins to transport a large complex containing botulinum toxin from intact protein through the intact skin after a single-time administration. The Botox® brand of botulinum toxin type A (Allergan, Irving, CA) is selected for this experiment. Botulinum toxin is reconstituted according to the manufacturer's instructions. An aliquot of the protein is biotinylated with a molar excess of 12 calculated folds of biotin sulfa-NHS-LC (Pierce Chemical, Rockford, IL). 2.0 units of botulinum toxin per aliquot (i.e., 20 U in total) and sialoprotein in a calculated MW range of 4: 1 is mixed until homogenous and diluted to 200 microliters with phosphate saline. The resulting composition is mixed until homogeneous with 1.8 ml of Cetaphil® lotion and aliquoted into 200 microlitre portions. Animal experiment to determine transdermal delivery efficiencies after single time treatment with botulinum toxin composition containing sialoproteins Animals are anesthetized during the inhalation of isoflurane during the application of treatments. After they have been anesthetized, C57BLK / 6 rats (n = 10) is subjected to topical application of 200 microliter dose appropriate treatment measure applied to the cranial portion of the skin of the back (selected because the mouse can not reach this region with your mouth or extremities). The animals do not undergo hair removal. In 30 minutes after the initial treatment, the mice are euthanized via CO2 inhalation and treated with skin segments are collected at full thickness by hidden observers. The treated segments are divided into three equal portions; the cranial portion was fixed in 10% neutral diluted formalin for 12-16 hours and then stored in 70% ethanol before paraffin bonding. The central portion is frozen and used directly for visualization of biotin by hidden observers as has been previously summarized. The treated caudal segment is frozen by solubilization studies. The visualization is carried out as follows. In brief, each section is subjected to NeutrAvidin® (Pierce Biotechnology, Rockford, IL) neutral solution at room temperature. To visualize alkaline phosphatose activity, cross sections are washed in saline four times and then subjected to NBT BCIP (Pierce Biotechnology) for approximately 1 hour. The sections are then washed and then distilled and photographed in their entirety in a Nikon E600 microscope with flat apochromate lenses. The total positive coloration is determined by the hidden observer via the analysis using Image Pro Plus software (Media Cybernetics, Silver pring, MD) and is normalized to the cross sectional area and to determine the positive coloration of percentage for each one. The mean and the standard error are then determined by each group with the analysis with 95% confidence significance in a way of repeated ANOVA measures using the Statview program (Abacus, Berkeley, CA). The results demonstrate that sialoproteins allow efficient transfer of botulinum toxin after topical administration in a murine model of intact skin. Example 2. Botulinum toxin administered transdermally to treat facial wrinkles. A woman wants to reduce the fine lines that come out of the side of her upper lip. A transdermal patch contains a composition containing 1 unit of botulinum toxin type A, 0.01 mg of sialoprotein and is essentially free of non-toxic proteins and albumin is applied to the area on its face where there are fine lines. The patch is applied only at night when the subject is asleep. Within 1-7 days the appearance of fine lines is greatly reduced. The benefit effect persists with continuous application of the patch. The reduced antigenicity is a result of lack of albumin derived from animals or gelatin allows repeated use of the botulinum toxin composition.

Claims (10)

1. A composition for transdermal botulinum toxin delivery comprises: a botulinum toxin complex or a reduced botulinum toxin complex; and an adhesion molecule; wherein the adhesion molecule forms a complex with the botulinum toxin complex or reduced botulinum toxin complex.

2. The composition according to claim 1, wherein the composition further comprises an exogenous stabilizer.

3. The composition according to claim 1, wherein the complex that is formed between the adhesion molecule and the botulinum toxin complex or reduced botulinum toxin complex is not covalent.

4. The composition according to any of claims 1-3, wherein the adhesion molecule is a sialoprotein.

The composition according to claim 4, wherein the reduced botulinum toxin complex contains a reduced amount of hemagglutinin or non-toxic protein, protein without haemagglutinin or both compared to the amount that naturally occurs in the botulinum toxin complexes. extracted from Clostridium botulinum.

6. The composition according to claim 2, wherein the exogenous stabilizer is albumin. The composition according to claim 6, wherein the albumin is present in an equal amount of about 500, 400, 300, 200, 100, 50, 10, 5, 0.5, 0.1 or

0. 01 times the amount of albumin from botulinum toxin complexes. they occur naturally. The composition according to claim 1, wherein the botulinum toxin complex or the reduced botulinum toxin complex contains botulinum toxin selected from the group consisting of a botulinum toxin derivative, a recombinant botulinum toxin, a modified botulinum toxin, botulinum toxin type A, botulinum toxin type B, botulinum toxin type C, botulinum toxin type E, botulinum toxin type F and botulinum toxin type G. 9. A method for administering a botulinum toxin to a subject comprising topically applying the composition according to claim 1. The method according to claim 9, wherein the composition is applied to an area of the subject's body selected from the group consisting of face, armpits, palm of the hands, hands, feet, lower back, neck, leg, groin, arm, elbow, knee, pelvis, buttocks and torso.