HK1162925A - Injectable botulinum toxin formulations - Google Patents

Description

Injectable botulinum toxin formulations

Related patent application

The present application claims priority from U.S. provisional patent application No.61/142,063 filed 12/31/2008, 119, the contents of which are incorporated herein by reference in their entirety.

Technical Field

The present invention relates to novel injectable compositions comprising botulinum toxin that can be administered to a subject for a variety of therapeutic, cosmetic and/or cosmetic purposes.

Background

The skin protects the body organs from the external environment and acts as a thermostat to maintain body temperature. It consists of several different layers, each with a specific function. The major layers include the epidermis, dermis and subcutaneous tissue. The epidermis is a stratified layer of epithelial cells that covers the dermis, which is composed of connective tissue. Both the epidermis and dermis are further supported by the subcutaneous tissue, which is the inner layer of adipose tissue.

The epidermis, the uppermost layer of the Skin, is only 0.1 to 1.5 mm thick (Inlander, Skin, New York, n.y.: pearl's Medical Society, 1-7 (1998)). It consists of keratinocytes, which are divided into several layers according to their state of differentiation. The epidermis may be further divided into a stratum corneum and a viable epidermis consisting of grannulamelphigian and basal cells. The stratum corneum is hygroscopic and requires at least 10% by weight of water to retain its elasticity and softness. The hygroscopicity is due in part to the water holding capacity of keratin. When the stratum corneum loses its softness and elasticity, it becomes rough and brittle, resulting in dry skin.

The dermis, which is located immediately below the epidermis, is 1.5 to 4 mm thick. Which is the thickest of the three layers of skin. Most Skin structures, including sweat and sebaceous glands (which secrete material through pores in the Skin called pores or acne), hair follicles, nerve endings and blood and lymph vessels, are found in the dermis (Inlander, Skin, New York, n.y.: People's Medical Society, 1-7 (1998)). However, the main components of the dermis are collagen and elastin.

The subcutaneous tissue is the deepest layer of the skin. It acts as an insulator for body heat preservation and as a shock absorber for organ protection (Inlander, Skin, New York, n.y.: People's Medical Society, 1-7 (1998)). In addition, subcutaneous tissue also stores fat for energy storage. The pH of the skin is typically between 5 and 6. This acidity is due to the presence of amphoteric amino acids, lactic acid and fatty acids from sebaceous gland secretions. The term "acid mantle" refers to the presence of water-soluble substances in most areas of the skin. The buffering capacity of the skin is due in part to these secretions stored in the stratum corneum of the skin.

Wrinkles, one of the indicative characteristics of age, may be caused by biochemical, histological and physiological changes accumulated in the skin by environmental damage (Benedetto, "International journal of Dermatology", 38: 641-655 (1999)). In addition, there are other secondary factors that can cause wrinkles, creases and creases (fisses) in The Face (Stegman et al, The Skin of The Aging Face Cosmetic Dermatological Surgery, 2 nd edition, St. Louis eds., Mo.: Mosby Year Book: 5-15 (1990)). These secondary factors include constant traction by gravity, frequent and constant positional pressure on The Skin (e.g., during sleep), and repetitive facial movements due to contraction of facial muscles (Stegman et al, The Skin of The Aging Face Cosmetic facial surgery, 2 nd edition, St. Louis eds., Mo.: Mosby Yeast Book: 5-15 (1990)).

In order to possibly alleviate some signs of aging, different techniques have been used. These techniques range from facial moisturizers containing alpha hydroxy acids and retinol to surgical procedures and injections of neurotoxins. For example, In 1986, Jean and Alastair Carruther, a couple team consisting of plastic ophthalmic surgeons and dermatologists, studied a method of treating motion-related wrinkles In the glabellar region using Botulinum toxin type A (Schantz and Scott, In Lewis G.E, (Ed) biological assays of Botulinum, New York: Academic Press, 143-. In 1992, the method was first published by Carruthers for wrinkle treatment using Botulinum toxin type A (Schantz and Scott, In Lewis G.E (Ed) Biomedical accessories of Botulinum, New York: Academic Press, 143-150 (1981)). Until 1994, the same team reported experience with other motor-related wrinkles on the face (Scott, Ophthalmol, 87: 1044-1049 (1980)). This in turn led to the emergence of an era of cosmetic treatments using botulinum toxin type A.

Botulinum toxin type a is reported to be the most lethal natural biological agent known to man. Spores of clostridium botulinum (c. botulinum) can be found in soil and can grow in improperly sterilized and sealed food containers. Botulism (which may be fatal) can be caused by ingestion of the bacterium. Botulinum toxin causes muscle paralysis by preventing synaptic transmission (by inhibiting acetylcholine release across the neuromuscular junction), and it is believed to function in other ways as well. Its action is primarily to block the signals that normally cause muscle spasm or contraction, resulting in paralysis. The muscle-paralytic activity of botulinum toxin has been used during the last decade to obtain a variety of therapeutic effects. Controlled administration of botulinum toxin has been used to provide muscle paralysis to treat a variety of medical conditions, such as neuromuscular disorders characterized by hyperactive skeletal muscles. Conditions that have been treated with botulinum toxin include hemifacial spasm, adult onset spasmodic torticollis, anal fissure, blepharospasm, cerebral palsy, cervical dystonia, migraine headaches, strabismus, temporomandibular joint disorder, and various types of muscle cramps and spasms. Recently, the muscle-paralytic effect of botulinum toxin has been exploited in therapeutic and cosmetic facial applications, such as the treatment of wrinkles, frown lines (frown lines), and other consequences of facial muscle spasms or contractions.

In addition to botulinum toxin type A, there are seven other serologically distinct forms of botulinum toxin, which are also produced by the gram-positive bacterium Clostridium botulinum. Of these eight serologically distinct types of botulinum toxin, the seven that can cause paralysis are known as botulinum toxin serotypes A, B, C, D, E, F and G. Each of these toxins is characterized by neutralization with a specific type of antibody. Each botulinum toxin protein has a molecular weight of about 150 kD. Due to its molecular size and molecular structure, botulinum toxin cannot cross the stratum corneum and the multiple layers of the subcutaneous structure. The different serotypes of botulinum toxin vary in different animal species in the severity and duration of the paralysis they cause and in the effectiveness. For example, it has been determined that botulinum toxin type a has more than 500 times the efficacy of botulinum toxin type B in rats, as determined by the rate of paralysis. In addition, it has been determined that botulinum toxin type B is non-toxic in primates at a dose of 480U/kg, which is the primate LD for botulinum toxin type A₅₀About 12 times higher.

When released by a botulinum bacterium, the botulinum toxin is a component of a toxin complex comprising an approximately 150kD botulinum toxin protein molecule and an associated non-toxin protein. These endogenous non-toxin proteins are believed to include the hemagglutinin protein family and non-hemagglutinin proteins. It has been reported that the non-toxin protein stabilizes the botulinum toxin molecule in the toxin complex and protects it from denaturation by digestive acids (when the toxin complex is ingested). Thus, the non-toxin proteins in the toxin complexes preserve the activity of the botulinum toxin and thus increase systemic penetration when the toxin complexes are administered via the gastrointestinal tract. Furthermore, it is believed that certain non-toxin proteins specifically stabilize botulinum toxin molecules in blood.

The presence of non-toxin proteins in the toxin complex generally results in the toxin complex having a molecular weight greater than that of the botulinum toxin molecule alone, which is about 150kD, as previously stated. For example, a botulinum bacterium can produce a botulinum toxin type A complex having a molecular weight of about 900 kD, 500 kD or 300 kD. Botulinum toxin types B and C are produced as complexes having a molecular weight of about 700 kD or about 500 kD. Botulinum toxin type D is produced as a complex having a molecular weight of about 300 kD or about 500 kD. Botulinum toxin types E and F are produced only as complexes having a molecular weight of about 300 kD.

To provide additional stability to botulinum toxin, the toxin complex is typically stabilized during the manufacturing process by combining the complex with albumin. For example,(Allergan, Inc., Irvine, CA) is a botulinum toxin containing formulation comprising 100 UA botulinum toxin, and an accessory protein, 0.5mg human albumin, and 0.9 mg sodium chloride. Albumin is used to bind and stabilize toxin complexes in different environments, including those associated with preparation, transport, storage, and administration.

Generally, botulinum toxin is administered to a patient by carefully controlled injection of a composition comprising botulinum toxin complexes and albumin. However, there are several problems associated with this approach. Not only is the pain of injection, but to achieve the desired therapeutic or cosmetic effect, large subcutaneous holes of toxin are generally created locally around the injection site. Botulinum toxin may migrate from these subcutaneous holes causing unwanted paralysis of adjacent areas of the body. This problem is exacerbated when the area to be treated is large and multiple injections of toxin are required to treat the area. Furthermore, because the injected toxin complexes comprise non-toxin proteins and albumin (which stabilizes botulinum toxin and increases the molecular weight of the toxin complex), the toxin complexes have a long half-life in vivo and may elicit an undesirable antigenic response in the patient. For example, some patients will develop an allergic reaction over time to albumin which is used as a stabilizer in current commercial formulations. Also, the toxin complex may induce the immune system of the patient to form neutralizing antibodies, such that a greater amount of toxin is required in subsequent administrations to achieve the same effect. When this occurs, subsequent injections must be carefully positioned so that they do not release large amounts of toxin into the patient's bloodstream, which can lead to fatal systemic toxicity, particularly because the non-toxin proteins and albumin stabilize botulinum toxin in the blood.

In view of the disadvantages associated with current botulinum toxin formulations, it would be highly desirable to have an injectable botulinum toxin formulation that is effective and stable, but exhibits reduced antigenicity and a lower propensity to spread locally upon injection. It would also be desirable to use such botulinum toxin formulations for various therapeutic, cosmetic and/or cosmetical purposes.

Summary of The Invention

The present invention provides injectable compositions comprising a botulinum toxin non-covalently bound to a positively charged carrier molecule. In a preferred embodiment, the compositions of the present invention are combined with conventional commercially available botulinum toxin formulations (e.g., botulinum toxin formulations)Or) With one or more advantages over other. For example, in certain embodiments, with conventional injectable meatsThe compositions may exhibit one or more advantages over a toxibacterium formulation, including reduced antigenicity, reduced propensity to diffuse into surrounding tissues following injection, increased duration or enhanced efficacy of clinical efficacy (relative to conventional botulinum toxin formulations), faster onset of clinical efficacy, and/or improved stability.

One aspect of the present invention is the recognition that certain non-native molecules (i.e., molecules not found in the botulinum toxin complexes obtained from botulinum bacteria) can be added to botulinum toxin, botulinum toxin complexes, particularly reduced botulinum toxin complexes (as defined herein) to enhance diffusion of the toxin through tissue. The non-native molecule is non-covalently bound to the toxin and acts as a penetration enhancer, which enhances the ability of the toxin to reach the target structure after injection. In addition, the non-native molecule may increase the stability of the toxin before and after injection. For example, the penetration enhancer may be a positively charged carrier, such as a cationic peptide, which has no inherent botulinum toxin-like activity and which further comprises one or more protein transduction domains, as described herein.

Another embodiment of the present invention is to provide a composition comprising a botulinum toxin, a botulinum toxin complex (or a reduced protein botulinum toxin complex, which includes only the 150kD neurotoxin itself or neurotoxin with some, but not all, native complex proteins), and a positively charged carrier.

The invention further relates to methods of producing a biological effect by injecting an effective amount of a composition of the invention into a subject or patient in need of such treatment. Biological effects may include, for example, muscle paralysis, reduction of hypersecretion or sweating, treatment of neuropathic pain or migraine, control of rhinitis or sinusitis, treatment of overactive bladder, reduction of muscle spasms, prevention or reduction of acne, reduction or enhancement of immune responses, reduction of wrinkles, or prevention or treatment of a variety of other conditions.

The invention also provides a kit for preparing a formulation comprising botulinum toxin, a botulinum toxin complex or a reduced protein botulinum toxin complex and a positively charged carrier or a premix which can be used to produce the formulation. Also provided are kits comprising means for sequentially administering a botulinum toxin complex (or a reduced botulinum toxin complex comprising only 150KD of the neurotoxin itself or the neurotoxin with some native complex proteins) and a positively charged carrier.

Brief description of the drawings

FIG. 1: bar graph showing repeated administration of RT003 orAnd then the time required to return to the baseline DAS value (0.4).

FIG. 2: figure 2A shows the hind limb of a mouse injected with dark dye indicating the gastrocnemius muscle portion of the mouse that received a midline lateral injection. Figure 2B shows the hind limb of a mouse injected with dark dye indicating the gastrocnemius muscle portion of the mouse that received a midline injection.

FIG. 3: toe abduction scores (Digital abduction scores) measured as RT003, RTT150 orTime after injection into the lateral or medial mid-line portion of the gastrocnemius muscle in mice.

Detailed Description

The present invention relates to novel injectable compositions comprising botulinum toxin, botulinum toxin complexes or reduced botulinum toxin complexes. In preferred embodiments, the composition stabilizes the toxin or enables transport or delivery of the toxin through tissue following injection, in order to interact with conventional commercially available and exogenous proteinsProtein-bound botulinum toxin complexes (e.g.Or) In contrast, the toxins have reduced antigenicity, better safety profile, enhanced efficacy, faster onset of clinical efficacy and/or longer duration of clinical efficacy. As described herein, the compositions of the present invention can be used as injectable administrations that provide botulinum toxin to a subject for various therapeutic, cosmetic and/or cosmetic purposes. The compositions of the present invention also have improved safety profiles as compared to other compositions and methods for delivering botulinum toxin. In addition, the composition may advantageously reduce the immune response to the toxin of the toxobacter.

The term "botulinum toxin" as used herein can refer to any known type of botulinum toxin (e.g., the 150kD botulinum toxin protein molecule associated with different serotypes of botulinum), whether produced by bacteria or by recombinant techniques, and any such type that may be subsequently discovered, including newly discovered serotypes and engineered variants or fusion proteins. As noted above, seven immunologically distinct botulinum neurotoxins have been characterized, namely botulinum neurotoxin serotypes A, B, C, D, E, F and G, each of which is characterized by neutralization with a specific type of antibody. The botulinum toxin serotypes are commercially available, for example, from Sigma-Aldrich (st. louis, Mo.) and Metabiologics, inc. (Madison, Wis), among other sources. The different serotypes of botulinum toxin vary with the species of animal, affecting the severity and duration of the paralysis they cause. At least two botulinum toxins, types a and B, are commercially available in formulations for the treatment of certain disorders. For example, type A is included under the trademarkIn Allergan formulations having the trademarkThe Ipsen preparation of (1), wherein form B is included under the trademark IpsenElan formulation of (1).

The term "botulinum toxin" as used in the compositions of the present invention may alternatively refer to botulinum toxin derivatives, i.e., compounds that possess botulinum toxin activity but contain one or more chemical or functional alterations at any portion or at any amino acid chain relative to naturally occurring or recombinant native botulinum toxin. For example, the botulinum toxin may be a modified neurotoxin, i.e. a neurotoxin having at least one amino acid deleted, modified or replaced compared to the native form, or the modified neurotoxin may be a recombinantly produced neurotoxin or a derivative or fragment thereof. For example, the botulinum toxin can be a toxin that has been modified in a manner that, for example, enhances its properties or reduces unwanted side effects, but still maintains the desired botulinum toxin activity. Alternatively, the botulinum toxin used in the present invention may be a toxin prepared using recombinant or synthetic chemical techniques, such as recombinant peptides, fusion proteins, or hybrid neurotoxins, for example prepared from subunits or domains of different botulinum toxin serotypes (see, e.g., U.S. Pat. No.6,444,209). The botulinum toxin can also be part of an entire molecule that has been shown to have the requisite botulinum toxin activity, and in this case can be used alone or as part of a combination or conjugate molecule such as a fusion protein. Alternatively, the botulinum toxin may be in the form of a botulinum toxin precursor, which itself may be non-toxic, e.g. a non-toxic zinc protease, which becomes toxic upon protease cleavage.

The term "botulinum toxin complex" or "toxin complex" as used herein refers to an approximately 150kD botulinum toxin protein molecule (belonging to any of botulinum toxin serotypes a-G) along with associated endogenous non-toxin proteins (i.e., hemagglutinin protein and non-toxin non-hemagglutinin protein produced by botulinum bacteria). It should be noted, however, that the botulinum toxin complex need not originate from a botulinum bacterium as a single toxin complex. For example, a botulinum toxin or modified botulinum toxin can be first prepared recombinantly and then combined with non-toxin proteins. Recombinant botulinum toxin can also be purchased (e.g., from List Biological Laboratories, Campbell, Ca) and then combined with non-toxin proteins.

The present invention also relates to modulating the stability of a botulinum toxin molecule by the addition of one or more exogenous stabilizers, the removal of endogenous stabilizers, or a combination thereof. For example, the present invention relates to the use of "reduced botulinum toxin complexes" wherein the botulinum toxin complexes have a reduced amount of non-toxin proteins as compared to the amount naturally found in botulinum toxin complexes produced by a botulinum bacterium. In one embodiment, a reduced botulinum toxin complex is prepared by extracting a portion of the hemagglutinin protein or the non-toxin non-hemagglutinin protein from a botulinum toxin complex derived from a botulinum bacterium using any conventional protein isolation method. For example, reduced botulinum toxin complexes can be produced by exposing botulinum toxin complexes to red blood cells at pH 7.3 to isolate the botulinum toxin complexes (see, e.g., EP 1514556 a1, incorporated herein by reference). HPLC, dialysis, column, centrifugation, and other methods can be used to extract proteins from proteins. Alternatively, when reduced botulinum toxin complexes are produced by combining synthetically produced botulinum toxin with non-toxin proteins, one may simply add less hemagglutinin or non-toxin non-hemagglutinin protein to the mixture than is contained in naturally occurring botulinum toxin complexes. According to the present invention, any non-toxin protein (e.g., hemagglutinin protein or non-toxin non-hemagglutinin protein or both) in the reduced botulinum toxin complex can be reduced independently in any amount. In certain exemplary embodimentsThe one or more non-toxin proteins are reduced by at least about 0.5%, 1%, 3%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% relative to the amount typically found in a botulinum toxin complex. As described above, botulinum bacteria produce seven different serotypes of toxin and commercially available formulations (i.e., different amounts of toxin complexes) are prepared with different relative amounts of non-toxin proteins. For example, MYOBLOC^TMContains botulinum toxin type B/ml 5000U, human serum albumin 0.05%, sodium succinate 0.01M and sodium chloride 0.1M. DYSPORT^TMContains 500U of botulinum toxin type A-hemagglutinin complex with 125 mcg of albumin and 2.4 mg of lactose. In certain embodiments, substantially all of the non-toxin proteins typically found in botulinum toxin complexes derived from botulinum bacteria (e.g., greater than 95%, 96%, 97%, 98% or 99% of the hemagglutinin protein and the non-toxin non-hemagglutinin protein) are removed from the botulinum toxin complexes. Furthermore, while the amount of endogenous non-toxin proteins may be reduced in some instances by the same amount, the present invention also relates to reducing each endogenous non-toxin protein in a different amount, and reducing at least one endogenous non-toxin protein, but not the others.

As described above, exogenous stabilizers (e.g., albumin) are typically added to stabilize the botulinum toxin formulation. For example, it isIn particular, 0.5mg human albumin/botulinum toxin type 100U A complex was used to stabilize the complex. Generally, the amount of exogenous stabilizer that may be added to stabilize the composition according to the present invention is not particularly limited. In some embodiments, the amount of stabilizer added may be less than conventionally added due to the ability of the positively charged carriers of the present invention to act alone as a stabilizer. For example, the amount of exogenous albumin added may be any amount less than the conventional thousand-fold excess of exogenous albumin, and in certain exemplary embodiments of the invention, is only about 0.25, 0.20, 0.15, 0.10, 0.01, 0.005. 0.001, 0.0005, 0.00001, 0.000005, 0.000001, or 0.0000001 mg/100U botulinum toxin. In one embodiment, no exogenous albumin is added as a stabilizer to the composition of the invention.

In accordance with the present invention, as described herein, it has been found that positively charged carrier molecules having protein transduction domains or efficiency groups (efficiency groups) are suitable as transport systems for botulinum toxin, which enable the toxin to be injected with improved permeability into a target structure, such as muscle and/or other skin-related structures. The transport is performed without covalent modification of the botulinum toxin. In addition to enhancing the permeability of botulinum toxin, in certain preferred embodiments, the positively charged carrier of the present invention can also stabilize botulinum toxin against degradation. In such embodiments, the hemagglutinin protein and the non-toxin non-hemagglutinin protein that are typically present to stabilize the botulinum toxin may be reduced or omitted altogether. Similarly, exogenous albumin, which is typically added during the manufacturing process, may also be omitted.

The term "positively charged" or "cationic" as used in connection with the term "carrier" means that the carrier has a positive charge under at least some solution phase conditions, more preferably at least some physiologically compatible conditions. More specifically, "positively charged" and "cationic" as used herein means that the group of interest comprises a functional group (functional) that is charged under all pH conditions, such as a quaternary amine, or a functional group that can acquire a positive charge under certain solution phase conditions, such as in the case of primary amines, pH changes. More preferably, "positively charged" or "cationic" as used herein refers to a group that, under physiologically compatible conditions, behaves in conjunction with an anion. It will be apparent to those skilled in the art that the polymer having multiple positively charged moieties need not be a homopolymer. Other examples of positively charged moieties are well known in the art and can be readily used, as will be apparent to those skilled in the art.

Generally, a positively charged carrier (also referred to as a "positively charged backbone") is generally a chain of atoms, and groups in the chain either carry a positive charge at physiological pH, or the groups carrying a positive charge are attached to side chains extending from the backbone. In certain preferred embodiments, the positively charged backbone is a cationic peptide. As used herein, the term "peptide" refers to an amino acid sequence, but does not have a meaning with respect to the number of amino acid residues within the amino acid sequence. Thus, the term "peptide" may also include polypeptides and proteins. In certain preferred embodiments, the positively charged backbone itself does not have a defined enzymatic or therapeutic biological activity. In some embodiments, the backbone is a linear hydrocarbon backbone, which in some embodiments is interrupted by heteroatoms selected from nitrogen, oxygen, sulfur, silicon, and phosphorus. Most of the backbone atoms are typically carbon. The main chain is usually a polymer of a repeating unit (e.g., amino acid, poly (oxyethylene), poly (allylamine), polyalkyleneimine (polyalkyleneimine), or the like), but may be a heteropolymer. In one set of embodiments, the positively charged backbone is polypropyleneamine (polypropylenamine), in which a number of amine nitrogen atoms are present as positively charged ammonium groups (tetra-substituted). In another embodiment, the positively charged backbone is a non-peptidic polymer, which may be a heteropolymer or homopolymer, such as a polyalkyleneimine, for example, polyethyleneimine or polypropyleneimine, having a molecular weight of from about 10,000 to about 2,500,000, preferably from about 100,000 to about 1,800,000, and most preferably from about 500,000 to about 1,400,000. In another set of embodiments, the backbone has attached thereto a plurality of side chain moieties that include positively charged groups (e.g., ammonium, pyridyl, phosphonium, sulfonium, guanidinium, or amidinium groups). In this group of embodiments, the pendant moieties may be spaced along the backbone, with the spacing being uniform or variable. In addition, the length of the side chains may be the same or different. For example, in one set of embodiments, the side chain may be a straight or branched hydrocarbon chain having from one to twenty carbon atoms and terminating distally (away from the backbone) with one of the positively charged groups described above. The binding between the positively charged carrier and the botulinum toxin, reduced botulinum toxin complex is by non-covalent interactions, non-limiting examples of which include ionic interactions, hydrogen bonding, van der waals forces, or combinations thereof.

In one set of embodiments, the positively charged backbone is a polypeptide having a plurality of positively charged side chain groups (e.g., lysine, arginine, ornithine, homoarginine, etc.). Preferably, the polypeptide has a molecular weight of about 100 to about 1,500,000, more preferably about 500 to about 1,200,000, most preferably about 1000 to about 1,000,000. It will be appreciated by those skilled in the art that when amino acids are used in this part of the invention, the side chains may have a D-or L-form (R or S configuration) at the center of attachment. In certain preferred embodiments, the polypeptide has a molecular weight of about 500 to about 5000, more preferably about 1000 to about 4000, more preferably 2000 to about 3000.

Alternatively, the backbone may comprise amino acid analogs and/or synthetic amino acids. The backbone may also be a polypeptide analog, such as a peptoid. See, e.g., Kessler, angelw.chem.int.ed.engl.32: 543 (1993); zuckermann et al, Chemtracts-Macromol. chem.4: 80 (1992); and Simon et al, proc.nat' 1.acad.sci.usa 89: 9367(1992). Briefly, a peptoid is a polyglycine in which the side chains are attached to the backbone nitrogen atoms instead of the α -carbon atoms. As noted above, a portion of the side chains typically terminate in positively charged groups to provide a positively charged backbone component. The synthesis of peptoids is described, for example, in U.S. patent 5,877,278, which is incorporated herein by reference in its entirety. As the term is used herein, positively charged backbones having a peptoid backbone structure are considered "non-peptidic" in that they do not consist of amino acids having side chains naturally occurring at the α -carbon position.

A variety of other backbones can be used, for example, steric or electronic mimetics of polypeptides in which the amide bond of the peptide is replaced by a surrogate such as an ester bond, thioamide (- -CSNH- -), retro thioamide (- -NHCS- -), aminomethylene (- -NHCH)₂- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -₂NH- -) group, keto-methylene (- -COCH)₂- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -₂RCH₂- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -₂RNH- -), reverse peptide (- -NHCO- -), trans olefin (- -CR ═ CH- -), fluoroolefin (- -CF ═ CH- -), dimethylene (- -CH- -)₂CH₂- - - - - -, thioether (- -CH)₂S- -), hydroxyethylidene (- -CH (OH) CH₂- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -₂O- -), tetrazole (CN)₄) Sulfonamido (- -SO)₂NH- -), methylenesulfonamido (- -CHRSO)₂NH- -), reverse sulfonamido (- -NHSO)₂- -) and a backbone with malonate and/or gem-diaminoalkyl subunits, e.g. as described in Fletcher et al ((1998) chem. rev.98: 763) as discussed and described in the documents cited therein. Many of the prior substitutions result in a (isostatic) polymer backbone that is approximately equielectronically aligned relative to the backbone formed by the alpha amino acids.

In each of the backbones provided above, a pendant group may be attached, the pendant group carrying a positively charged group. For example, a sulfonamido-linked backbone (- -SO)₂NH- -and- -NHSO₂- -) may have a side chain group attached to the nitrogen atom. Similarly, hydroxyethylidene (- -CH (OH) CH₂- - - -) linkages may have a side chain group attached to a hydroxyl substituent. One skilled in the art can readily employ other chemical linkages to provide positively charged side chain groups using standard synthetic methods.

In one embodiment, the positively charged backbone is a polypeptide having a protein transduction domain (also referred to as a efficiency group). As used herein, an "efficiency group" is any agent that has the effect of promoting transfer of a positively charged backbone through a tissue or cell membrane. Non-limiting examples of protein transduction domains or efficiency groups include- (gly)_n1-(arg)_n2HIV-TAT or fragments thereof, or a protein transduction domain of Antennapedia (Antennapedia), or fragments thereof, wherein subscript n1 is an integer from 0 to 20, more preferably from 0 to 8, more preferably from 2 to 5, and subscript n2 is individually an odd integer from about 5 to about 25, more preferably from about 7 to about 17, most preferably from about 7 to about 13. In some embodiments, the HIV-TAT fragment does not comprise an HIV-TAT moleculeCysteine-rich regions to minimize problems associated with disulfide aggregation. Further preferred are embodiments wherein the HIV-TAT fragment has the formula (gly)_p-RGRDDRRQRRR-(gly)_q、(gly)_p-YGRKKRRQRRR-(gly)_qOr (gly)_p-RKKRRQRRR-(gly)_qWherein subscripts p and q are each independently integers of 0 to 20 and the fragments are linked to the backbone via the C-terminus or N-terminus of the fragment. In certain preferred embodiments, p is 1 and q is 0 or p is 0 and q is 1. Preferred HIV-TAT fragments are those wherein subscripts p and q are each independently an integer from 0 to 8, more preferably from 0 to 5. In another preferred embodiment, the positively charged side chain or branching group is the antennapedia (Antp) Protein Transduction Domain (PTD), or a fragment thereof that retains activity. These are known in the art, see, for example, Console et al, j.biol.chem.278: 35109(2003), and a non-limiting example of an Antp PTD to which the present invention relates is SGRQIKIWFQNRRMKWKKC.

Preferably, the positively charged carrier comprises the protein transduction domain with a positively charged side chain in an amount of at least about 0.01% (percentage of the total carrier weight), preferably from about 0.01 to about 50 wt.%, more preferably from about 0.05 to about 45 wt.%, and most preferably from about 0.1 to about 30 wt.%. For having the formula- (gly)_n1-(arg)_n2The preferred range of (a) is from about 0.1 to about 25%.

In another embodiment, the backbone moiety is polylysine and the positively charged protein transduction domain is attached to the amino groups of the lysine side chains or to the C-or N-terminus. In some preferred embodiments, polylysine may have a molecular weight of at least 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, or 6000D, and less than about 2,000,000, 1,000,000, 500,000, 250,000, 100,000, 75,000, 50,000, and 25,000D. Within the range of 100 to 2,000,000D, it is contemplated that the lower and/or higher ranges may be increased or decreased by 100, respectively, with each resulting sub-range being an embodiment specifically contemplated by the present invention. In thatIn some exemplary embodiments, polylysine has a molecular weight of from about 1,000 to about 1,500,000D, from about 2,000 to about 800,000D, or from about 3,000 to about 200,000D. In other exemplary embodiments, polylysine has a molecular weight of from about 100 to about 10,000D, from about 500 to about 5,000D, from about 1,000 to about 4,000D, from about 1,500 to about 3,500D, or from about 2,000 to about 3,000D. In some embodiments, polylysines contemplated by the present invention may be any commercially available (Sigma Chemical Company, st. louis, mo., USA) polylysine, such as, for example, polylysine with MW > 70,000, polylysine with MW of 70,000 to 150,000, polylysine with MW of 150,000 to 300,000, and polylysine with MW > 300,000. The selection of a suitable polylysine depends on the other components of the composition, and will be sufficient to provide a total net positive charge to the composition and a length preferably one to four times the combined length of the negatively charged components. Preferred positively charged protein transduction domains or efficiency groups include, for example, -Gly-Gly-Gly-arg-arg-arg-arg-arg-arg-arg (-Gly)₃Arg₇) Or HIV-TAT.

In another preferred embodiment, the positively charged backbone is a polyalkyleneimine, non-limiting examples of which include polyethyleneimine, polypropyleneimine, and polybutyleneimine. In certain preferred embodiments, the polyalkyleneimines have a molecular weight of at least 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, or 6000D and less than about 2,000,000, 1,000,000, 500,000, 250,000, 100,000, 75,000, 50,000, and 25,000D. Within the range of 100 to 2,000,000D, it is contemplated that the lower and/or higher ranges may be increased or decreased by 100, respectively, with each resulting sub-range being an embodiment specifically contemplated by the present invention.

In other embodiments of the invention, the carrier is a relatively short polylysine or Polyethyleneimine (PEI) backbone (which may be linear or branched) and which has positively charged branching groups. Without wishing to be bound by theory, it is believed that such carriers can be used to minimize uncontrolled aggregation of botulinum toxin in the backbone and therapeutic composition, which results in a significant decrease in transport efficiency. When the carrier is a relatively short linear polylysine or PEI backbone, the backbone has a molecular weight of less than 75,000D, more preferably less than 30,000D, and most preferably less than 25,000D. However when the carrier is a relatively short branched polylysine or PEI backbone, the backbone has a molecular weight of less than 60,000D, more preferably less than 55,000D, and most preferably less than 50,000D.

In a particularly interesting embodiment, the non-native molecule is a cationic peptide that has no inherent botulinum toxin-like activity and further comprises one or more protein transduction domains, as described herein. Without wishing to be bound by any particular scientific theory, it is believed that the peptides enhance tissue permeability of the molecules bound in the complex after injection, while enhancing stability of the botulinum toxin in the skin and in vitro. It is believed that the enhanced tissue permeability conferred by these peptides provides, inter alia, reduced antigenicity, better safety profile, enhanced efficacy, faster onset of clinical efficacy or longer duration of clinical efficacy, and conventional commercially available botulinum toxin complexes bound to exogenous albumin (e.g., botulinum toxin complexes with exogenous albumin)Or) And (4) comparing.

In a preferred embodiment, the concentration of the positively charged carrier in the compositions of the present invention is sufficient to enhance the delivery of the botulinum toxin to a molecular target, such as, for example, a motor nerve plate. Furthermore, without wishing to be bound by theory, it is believed that the rate of penetration follows receptor-mediated kinetics, whereby tissue permeability increases with increasing amount of penetration enhancing molecules up to the saturation point, after which the rate of transport becomes constant. Thus, in a preferred embodiment, the amount of permeation enhancing molecules added is equal to the amount that maximizes permeation rate just prior to saturation. As described herein, useful concentrations of the positively charged carrier in the injectable compositions of the present invention range from about 0.1pg to about 1.0mg per unit of botulinum toxin composition. More preferably, the positively charged carrier in the topical composition of the present invention is present at about 1.0pg to 0.5mg per unit of botulinum toxin.

The compositions of the invention are preferably in a form that allows injection into the skin or epithelium of a subject or patient (i.e., a human or other mammal in need of specific treatment). The term "need" is intended to include pharmaceutical or health related needs (e.g., treatment of conditions, including undesirable facial muscle spasms), as well as cosmetic and subjective needs (e.g., altering or improving the appearance of facial tissue). In a preferred embodiment, the composition is prepared by mixing the botulinum toxin (comprising or reduced in the relevant non-toxin protein) and the positively charged carrier, and typically one or more additional pharmaceutically acceptable carriers or excipients. In their simplest form, they may comprise an aqueous pharmaceutically acceptable diluent, such as buffered saline (e.g. phosphate buffered saline). However, the composition may contain other ingredients typically found in injectable pharmaceutical or cosmeceutical compositions, including dermatologically or pharmaceutically acceptable carriers, vehicles, or vehicles, which are compatible with the tissue to which it is to be administered. As used herein, the term "dermatologically or pharmaceutically acceptable" means that the composition or components thereof are suitable for use in contact with such tissues or in a patient, generally without undue toxicity, incompatibility, instability, allergic response, and the like. The compositions of the invention may, if desired, comprise any ingredient normally used in the fields concerned, in particular in cosmetics and dermatology.

Depending on their form, the compositions of the present invention may include solutions, emulsions (including microemulsions), suspensions, gels, powders or other generally solid or liquid compositions for injection into muscle and other tissues where the composition may be used. In a preferred embodiment, the composition of the invention is present in a low viscosity, sterile formulation suitable for injection by syringe. The compositions of the invention may be in the form of a lyophilized powder for reconstitution with a pharmaceutically acceptable liquid diluent prior to injection. In certain embodiments, the lyophilized powder is reconstituted with a liquid diluent to form an injectable formulation having a viscosity of about 0.1 to about 2000cP, more preferably about 0.2 to about 500cP, more preferably about 0.3 to about 50cP, more preferably about 0.4 to about 2.0 cP. The compositions of the present invention may contain, in addition to the botulinum toxin and positively charged carrier, other ingredients commonly used in such products, such as antimicrobial agents, hydrating agents, tissue bulking agents (tissue bulking agents) or tissue bulking agents, preservatives, emulsifiers, natural or synthetic oils, solvents, surfactants, detergents, gelling agents, antioxidants, fillers, thickeners, powders, viscosity control agents and water, and optionally including anesthetics, anti-itch actives, botanical extracts, conditioning agents, minerals, polyphenols, silicones or derivatives thereof, vitamins and botanicals (phytomedicinals).

The injectable compositions of the present invention may be in the form of controlled or sustained release compositions comprising a botulinum toxin and a positively charged carrier encapsulated or otherwise contained within a material such that they are released in a controlled manner over time within a tissue. The composition comprising the botulinum toxin and the positively charged carrier may be contained within a matrix, liposome, vesicle, microcapsule, microsphere, or the like, or within a solid particulate material, all selected and/or configured to cause release of the botulinum toxin over time. The botulinum toxin and the positively charged carrier may be encapsulated together (i.e., in the same capsule) or separately (i.e., in separate capsules).

The botulinum toxin formulations of the invention can be delivered by injection (typically using a syringe) to muscle under the skin, or to glandular structures within the skin, in an amount effective to produce paralysis, produce relaxation, reduce contractions, prevent or reduce spasticity, reduce glandular output (glandular output), or produce other desired effects. Local delivery of botulinum toxin in this manner can reduce dosage, reduce toxicity and allow more precise dosage optimization (as compared to injectable or implantable materials) for the desired effect.

The compositions of the present invention are applied to deliver an effective amount of botulinum toxin. As used herein, the term "effective amount" refers to an amount of botulinum toxin, as defined above, that is sufficient to produce the desired muscle paralysis or other biological or cosmetic effect, and is specifically a safe amount, i.e., an amount low enough to avoid serious side effects. Desirable effects include relaxation of certain muscles, for example, to reduce the appearance of fine lines and/or wrinkles (particularly in the face), or to otherwise modify the appearance of the face, for example, to enlarge the eyes, to raise the corners of the mouth, or to smooth fine lines that fan out from the upper lip, or to generally relieve muscle tension. In the last effect, general relief of muscle tension can be achieved on the face or other parts. The compositions of the present invention may comprise a suitable effective amount of a botulinum toxin for administration in a single dose therapeutic form, or may be more concentrated for dilution at the time of administration or for multiple administrations. By using the positively charged carriers of the present invention, botulinum toxin can be administered by injection to a subject to treat a condition, such as wrinkles, undesirable facial or other muscle spasms, hyperhidrosis, acne, or a condition elsewhere in the body where relief of muscle soreness or spasms is desired. Botulinum toxin is administered by injection into muscle or other skin-related structures or other target tissue structures. Can be applied, for example, to the legs, shoulders, back (including the waist), armpits, palms, feet, neck, face, groin, back of the hands or feet, elbows, upper arms, knees, thighs, buttocks, torso, pelvis, or any other part of the body to which botulinum toxin is desired to be applied.

The injectable compositions of the invention comprising botulinum toxin may also be administered to treat other conditions, including any condition for which prevention of synaptic transmission or release of acetylcholine may confer a therapeutic benefit. For example, conditions that may be treated by the compositions of the present invention include, but are not limited to, neuropathic pain, migraine or other headaches, overactive bladder, rhinitis, sinusitis, acne, dystonia, dystonic contractions (whether subjective or clinical), hyperhidrosis (whether subjective or clinical), and hypersecretion of one or more glands controlled by the cholinergic nervous system. The compositions of the present invention may also be used to reduce or enhance an immune response, or to treat other conditions for which administration of botulinum toxin by injection has been proposed or carried out.

Most preferably, the composition is administered by or under the direction of a physician or other health care professional. They may be administered in a single treatment or over a period of time in a series of treatments. In a preferred embodiment, the compositions of the present invention are injected at a site where the effect associated with botulinum toxin is desired. Because of its nature, it is preferred to administer the botulinum toxin in an amount, at an administration rate, and at a frequency that produces the desired results without producing any adverse or undesirable results. For example, in certain embodiments, the compositions of the present invention are applied at a rate of from about 1U to about 20,000U, preferably from about 1U to about 10,000U botulinum toxin per square centimeter of skin surface. Higher doses within these ranges may be used, for example, where the botulinum toxin and the controlled release material are administered together, as described herein. In certain embodiments, the botulinum toxin formulation of the present invention is administered to provide a botulinum toxin/injection of 1 to 400U, more preferably 10 to 350U, more preferably 30 to 250U and most preferably 50 to 200U.

The present invention also relates to the use of various delivery devices for injecting a botulinum toxin containing composition described herein through the skin. The device may include, but is not limited to, a needle and syringe, or may include a more advanced device capable of dispensing the composition and monitoring the dispensing of the composition, and optionally monitoring the condition of the subject in one or more aspects (e.g., monitoring the subject's response to the substance being dispensed).

In some embodiments, the composition may be pre-formulated and/or pre-assembled in such delivery devices. The invention also relates to embodiments wherein the composition is provided in a kit storing one or more components separate from other components. For example, in certain embodiments, the present invention provides a kit that stores botulinum toxin and a positively charged carrier separately, combined at or prior to administration. The amount of positively charged carrier or the concentration ratio of these molecules to botulinum toxin depends on the carrier selected for the composition in question. Suitable amounts or ratios of carrier molecules in a given situation can be readily determined, for example by performing one or more of the experiments described below.

Generally, the present invention also relates to a method for administering a botulinum toxin (optionally, a botulinum toxin complex or a reduced botulinum toxin complex) to a subject or patient in need thereof, wherein an effective amount of the botulinum toxin is administered with a positively charged carrier, as described herein. Together "means that the two components (botulinum toxin and positively charged carrier) are administered in a combined process, which may include combining them prior to administration to a subject, or administering them separately, but in a manner such that they work together to provide the necessary delivery of an effective amount of the therapeutic protein. For example, a composition comprising a positively charged carrier can be first applied to the skin of a subject, followed by application of a skin patch, syringe, or other device comprising a botulinum toxin. The botulinum toxin can be stored in dry form in a syringe or other dispensing device, and the positively charged carrier can be injected prior to administration of the toxin so that the two act together to produce the desired enhancement of tissue penetration. Thus, in this case, the two substances (positively charged carrier and botulinum toxin) act in combination or possibly interact to form an in situ composition or combination. Thus, the invention also includes kits having a means for dispensing botulinum toxin and a liquid, gel, or the like containing a positively charged carrier, and which are suitable for injection into the skin or target tissue of a subject. Kits for administering the compositions of the invention under the direction of a health care professional or by a patient or subject may also include conventional applicators suitable for the purpose.

The compositions of the present invention are suitable for use in physiological environments having a pH of about 4.5 to about 6.3, and thus may have such a pH. The compositions of the present invention may be stored at room temperature or under refrigerated conditions.

It is understood that the following examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are incorporated by reference in their entirety for all purposes.

Example 1

Duration of local muscle paralysis in mouse model

RT003 or RT003 for comparison in the present exampleDuration of local muscle paralysis in the injected mice. RT003 is an exemplary injectable formulation of the present invention comprising botulinum toxin type A (purified to remove all endogenous non-toxin proteins) and having the sequence RKKRRQRRRG- (K)₁₅-a positively charged carrier of GRKKRRQRRR.Also included is botulinum toxin type a, but exogenous albumin is added to stabilize the botulinum toxin type a molecules.

Muscle paralysis is measured using a toe abduction score (DAS) analysis, such as Aoki, k.r. in "acoustparison of the safety markers of botulin neuroleptospermypes a, B and F in mice", Toxicon 2001; 39(12): 1815 and 1820. In the DAS analysis, the mouse is simply suspended by its tail to elicit a characteristic startle response in which the mouse stretches its hind limb and abducts its hind toe. Mice were scored on five points (from 0-4) for the extent to which they were able to exhibit a startle response, with 0 indicating a normal startle response and 4 indicating the greatest reduction in toe abduction and leg extension. Scoring is performed by an observer who is unaware of the extent to which the subject mouse is treated with neurotoxin. The baseline score using DAS analysis was determined to be 0.4 for the untreated animal population.

The study reported in this example included 10 animals (5 mice in the RT003 group and5 mice of the group). With separate botulinum toxin formulations (i.e. RT003 or) Each animal was injected three times with 40 days between each dose. After injection, all animals in each experimental group were counted for days above 0.4 baseline of DAS analysis. The results shown in figure 1 indicate that the DAS analysis score of the RT 003-treated group remained above the 0.4 baseline value for 25, 22 and 21 days, respectively, after the first, second and third treatments. In contrast, after the first, second and third treatments,the days in which the DAS analysis score of the treatment group remained above the 0.4 baseline value were 11, 8 and 11 days, respectively.

The data from DAS analysis indicated that the local muscle paralysis caused by the RT003 preparation lasted approximately fromTwice as long as the local muscle paralysis caused. This result is of great significance for the therapeutic use of RT003 and other injectable botulinum toxin containing compounds of the present invention. In particular, by using the injectable composition of the present invention, the frequency of subsequent injections required to maintain a particular cosmetic or therapeutic effect induced by botulinum toxin can be significantly reduced. Further, reduced frequency of administration may result in better long term efficacy, as subjects are less likely to be treatedTo generate antibodies against botulinum toxin.

Example 2

Injectable botulinum toxin formulations with improved safety profile

Botulinum toxin has been used as a therapeutic agent for the treatment of a variety of conditions over the last several decades, including wrinkles, hyperhidrosis, and muscle spasms. However, because botulinum toxin is the most potent naturally occurring toxin known to man, improper administration of the toxin can be very dangerous. For example, accidental systemic delivery of botulinum toxin can lead to paralysis, dyspnea, and even death. Furthermore, even if botulinum toxin is properly delivered to a localized area of the body as part of a treatment, the toxin has a natural tendency to spread over time, thereby increasing the risk of unwanted paralysis in other parts of the body. For example, when botulinum toxin is injected around the eye to treat wrinkles, it may spread to muscles that control eyelid movement. If this occurs, the eyelid muscles may become partially paralyzed, resulting in a condition known as "eyelid droop" in which the eyelids are partially closed and interfere with normal vision.

It is an aspect of the present invention to provide injectable botulinum toxin formulations having improved safety profiles compared to currently commercially available botulinum toxin formulations. In a preferred embodiment, the injectable botulinum toxin formulation has a reduced tendency to diffuse upon injection. Thus, certain preferred formulations of the present invention allow for more precise delivery of botulinum toxin, significantly reducing unwanted side effects associated with uncontrolled local diffusion of botulinum toxin.

This example reports a comparative study of the diffusion tendencies of botulinum toxin in various formulations after injection. The study included three botulinum toxin formulations: (1)(2) RT003, comprising and having the formula RKKRRQRRRG- (K)₁₅-a buffered and stabilized solution of a positively charged carrier non-covalently bound 150kD type a botulinum toxin molecule of GRKKRRQRRR; and (3) RTT150, which is identical to the RT003 formulation except that it does not contain positively charged carriers present in RT 003.

The gastrocnemius muscle of each mouse used in this study was injected with one of the botulinum toxin formulations described above, either in the paramidline part of the muscle (fig. 2A) or in the midline part of the muscle (fig. 2B). After botulinum toxin injection, each mouse was subjected to a DAS analysis for 4 days to determine whether each formulation of botulinum toxin exhibited any tendency to diffuse from the gastrocnemius muscle to the hind paw of the mouse. According to the DAS analysis, any reduction in the ability of the test animal to abduct its hind toe is interpreted as an indication of botulinum toxin diffusion.

FIG. 3 shows the results of DAS analysis performed after injection of test animals with different botulinum toxin formulations as described above. Note that the toe abduction scores were divided into two groups, corresponding to whether the injection was in the midline or the paramidline portion of the gastrocnemius muscle. The generally lower DAS score for the midline injection compared to the DAS score for the midline lateral injection indicated that the degree of paralysis of the hind paws of the test animals was generally less after the midline injection. Without wishing to be bound by theory, it is believed that this result is due to the fact that botulinum toxin must travel a longer distance to reach the hind toe of the test animal following a midline injection than a midline, lateral injection. It is believed that the longer distance required to transport the botulinum toxin reduces the likelihood of posterior toe paralysis.

Figure 3 shows that all of the toe abduction scores were zero 4 days after the midline injection of the RT003 formulation. This result indicates that botulinum toxin in the RT003 formulation remained localized in the midline portion of the gastrocnemius muscle after injection and that no diffusion causing paralysis occurred over the period of the experiment. In contrast, at injection RTT150 andafter formulation, a toe abduction score above 0.4DAS baseline was observed, andthe mean DAS score of the formulation was higher. RTT150 andDAS results of the formulations indicated that after midline injection of these formulations, hind toe paralysis in the test animals was observed, and after injectionA higher degree of paralysis was observed after the formulation. These data indicate that RTT150 andthe botulinum toxin molecules in the formulation are capable of local diffusion upon injection, andthe local diffusion of the botulinum toxin molecules in the formulation is higher.

Figure 3 also shows that posterior toe paralysis was observed in all tested animals after a lateral midline injection, regardless of the particular botulinum toxin formulation. As noted above, the higher degree of paralysis following a lateral midline injection is believed to be associated with a shorter transport distance of botulinum toxin to the hind paws of the test animals as compared to a midline injection. However, while all three botulinum toxin formulations showed a spread causing paralysis after midline lateral injection, on average, the time period of the experiment compared to that for RTT150 and RTT150The degree of paralysis observed with the formulation was less in the test animals injected with RT 003. Thus, DAS analysis data corresponding to a midline lateral injection is qualitatively similar to that of a midline injection, which shows RTT150 andin contrast, the RT003 formulations had a reduced tendency to local spread of botulinum toxin.

Comparison of local diffusion rates after midline injection and mid-lateral injection can be made by considering a parameter known as the "diffusion index", which is defined by the following formula (1):

since the toe abduction score may be 0 to 4, and the toe abduction score lateral to the midline is expected to be higher than the midline toe abduction score (as described above), the diffusion index value is typically 0 to 100. A diffusion index value near 100 indicates that the ratio of the midline and the toe abduction score lateral to the midline is nearly uniform. This can occur if the post-injection diffusion rate is sufficiently high that the diffusion time for botulinum toxin to reach and paralyze the hind toe of the test animal after the midline and midline paralateral injections is comparable or nearly the same. In the other extreme, a diffusion index value close to zero indicates that the ratio of the midline and the toe abduction score lateral to the midline is close to zero. This can occur if the spread of botulinum toxin following midline injection is too low to cause posterior toe paralysis in the test animals (even if paralysis is observed following midline lateral injection).

Table 1 shows the mid-line or midline side injectionToe abduction scores after RT003 and RTT150, as reported in the experiments corresponding to fig. 3. During the period of the experiment, corresponding toThe diffusion index values for the formulation injections were higher than those observed for RTT150 and RT003 formulations. This indicates a correlation with the RTT150 compared to the ratio observed for the RT003 formulation, for injectionThe ratio of the formulation, midline and abduction score on the side of the midline was more nearly uniform. Because botulinum toxin must diffuse farther after a midline injection to cause hind toe paralysis in test animals,observations that the ratio of the midline post-injection and midline lateral toe abduction scores were more nearly identical showed that midline injection relative to the rate post midline lateral injectionThe diffusion rate of the latter botulinum toxin is quite significant. In other words, the increased length of the diffusion pathway associated with the midline injection is less obstructive to causing posterior toe paralysis.

In contrast, the diffusion index values of RT003 were all zero over the 4 day experimental period. The results indicate that no diffusion causing paralysis was observed after midline injection of RT 003. In other words, an RT003 formulation comprising a botulinum toxin type a molecule non-covalently bound to a positively charged carrier allows for enhanced local localization of an injected botulinum toxin type a. Thus, RT003 formulations conferImproved safety profile compared to the formulation and minimized unwanted paralysis.

The diffusion index value of observed RTT150, although not zero like RT003, is still better than forThe observed values for the formulations are small [ see Table 1]. This result indicates that, over the 4 day experimental period, diffusion of botulinum toxin sufficient to produce observable post-paresis occurred, but that paralytic expansion of botulinum toxin following midline injection resultedThe time required for dispersion is relatively longer.

Table 1: RTT150,And measurement of botulinum toxin spreading index at RT 003.

Example 3

Injectable botulinum toxin formulations with reduced propensity to produce antibodies

When botulinum toxin is periodically injected into a patient to treat an undesirable condition (e.g., wrinkles), it is often observed that the efficacy of botulinum toxin decreases with continued injection, even though the duration of the effect of botulinum toxin remains the same. This phenomenon is believed to be a result of the patient's immune system developing antibodies against botulinum toxin. From a therapeutic point of view, it is undesirable for the patient to develop antibodies against botulinum toxin, since then progressively larger doses of botulinum toxin are required to achieve the same effect, which leads to serious problems related to safety and cost.

In certain embodiments, the present invention provides injectable botulinum toxin formulations having a reduced tendency to induce antibody formation as compared to currently commercially available injectable botulinum toxin formulations. Thus, in these embodiments, the botulinum toxin formulation helps minimize the risks associated with botulinum toxin injections by allowing less toxin to be used over a period of time to achieve the same effect.

In this example, RT003 andthe DAS analysis data (as described in example 2) obtained thereafter was analyzed as a function of time to determine changes in efficacy of both formulations upon repeated administration to the same test animal. Generally, the duration of the effect associated with botulinum toxin is the same after repeated applications of each formulation. However, the degree of muscle paralysis after repeated administration varies depending on the formulation. To quantify the change in the degree of muscle paralysis, the injection of RT003 or RT003 is determined according to equation (2)Percent change in hind toe abduction score:

since the numerator of equation (2) is the difference of the measured toe abduction score for the nth treatment and the first treatment, the% change in DAS will be negative if the measured toe abduction score for the nth treatment is less than the measured toe abduction score for the first treatment. In other words, the% change in DAS is negative when less paralysis is observed after the nth treatment compared to the first treatment. Table 2 shows the procedure described in example 2, with repeated administration of RT003 andafter formulation, change% in measured DAS value.

Table 2: repeated administration of RT003 andchange in post DAS value%

As shown in Table 2After the first reprocessing,the% change in toe extension score of the formulation was-44%, indicating a significant decrease in efficacy. In contrast, the% change in toe extension score for the RT003 formulation was zero, indicating that the DAS score after the second retreatment was the same as after the first application and the first retreatment. The results indicate that the degree of paralysis observed after the first re-treatment of RT003 was the same as after the first treatment, and that negligible formation of neutralizing antibodies occurred in the test animals even after the first re-treatment. At RT003 andthe calculated% change in DAS values for both formulations was negative after the second reprocessing, although the magnitude of the% change in DAS value for the RT003 formulation was negativeHalf the value of (c). For theThe% change in DAS observed was greater and negative, indicating that the test animal pairs compared to RT003Has a higher antibody production rate. Thus, these data indicate that formulations related to the present invention, such as RT003, may have a lower propensity to induce the formation of antibodies that neutralize the effects of botulinum toxin. Thus, the results show that by using the formulations of the present invention, less botulinum toxin can be used over a period of time to achieve the same therapeutic effect.

Example 4

Injectable botulinum toxin formulations with improved stability

This example demonstrates that positively charged carrier molecules used in the injectable botulinum toxin formulations of the present invention not only enhance the safety of the formulations (example 2), but also improve their stability. Table 3 shows the results of the aging experiment in which RT003 and RTT150 formulations were aged at 4 ℃ (RT003 only) and at 40 ℃ (RT003 and RTT150) for various time intervals. The potency of RT003 and RTT150 formulations was measured via a series of mouse IP LD50 assays after aging at the indicated temperatures for the indicated times. The results are summarized in table 3, which shows that the potency of RT003 is essentially unchanged after aging at 4 ℃ (even after 6 months). Furthermore, even though RT003 formulations aged for 6 months at elevated temperatures (40 ℃), the efficacy of RT003 formulations (as measured by the ability of the formulations to kill target animals in the mouse IP LD50 assay) decreased only slightly. In contrast, RTT150 formulations show a significant drop in efficacy after only one month of aging at 40 ℃. Since formulations other than RT003 also comprise compounds having the formula RKKRRQRRRG- (K)₁₅The RT003 and RTT150 formulations are identical except for the positively charged carrier molecule of GRKKRRQRRR, so these data indicate that the positively charged carrier molecule improves the stability of botulinum toxin in RT003 formulations.

Table 3: results of mouse IP LD50 analysis after aging of RT003 and RTT150 under various conditions

Claims (23)

1.A method of administering a botulinum toxin to an individual in need thereof to achieve a therapeutic or cosmetic effect, the method comprising

Injecting the subject with an effective amount of the composition to achieve a therapeutic or cosmetic effect,

wherein the composition comprises

A positively charged carrier comprising a positively charged backbone having a plurality of efficiency groups attached thereto,

a botulinum toxin complex, a reduced botulinum toxin complex, or a botulinum toxin, and

wherein the positively charged carrier is non-covalently associated with the botulinum toxin complex, reduced botulinum toxin complex or botulinum toxin.

2. The method of claim 1, wherein the botulinum toxin complex, reduced botulinum toxin complex, or botulinum toxin is obtained from botulinum serotype A, B, C, D, E, F or G.

3. The method of claim 1, wherein the positively charged backbone comprises a polyamino acid.

4. The method of claim 3, wherein the polyamino acid is selected from the group consisting of polylysine, polyarginine, polyhistidine, and polyornithine.

5. The method of claim 1, wherein the efficiency group comprises an amino acid sequence selected from the group consisting of: (gly)_n1-(arg)_n2HIV-TAT or fragments thereof, protein transduction domains of antennapedia proteins or fragments thereof, (gly)_p-RGRDDRRQRRR-(gly)_q，(gly)_p-YGRKKRRQRRR-(gly)_qOr (gly)_p-RKKRRQRRR-(gly)_qAnd SGRQIKIWFQNRRMKWKKC;

wherein subscript n1 is an integer of from 0 to 20, subscript n2 is independently an odd integer of from about 5 to about 25, more preferably from about 7 to about 17, most preferably from about 7 to about 13; and

wherein subscripts p and q are each independently an integer of 0 to 8.

6. The method of claim 1, wherein the positively charged carrier stabilizes botulinum toxin against degradation.

7. The method of claim 1, wherein the positively charged carrier reduces local diffusion of botulinum toxin upon injection.

8. The method of claim 1, wherein the positively charged carrier has the amino acid sequence RKKRRQRRRG- (K)₁₅-GRKKRRQRRR。

9. The method of claim 1, wherein the composition is administered to the subject after injection into the subject's bodyIn contrast, there is a reduced tendency to induce antibody production.

10. The method of claim 1, wherein the cosmetic effect is the treatment of wrinkles.

11. The method of claim 1, wherein the therapeutic effect is a reduction in symptoms associated with a condition selected from the group consisting of: hemifacial spasm, adult onset spasmodic torticollis, anal fissure, blepharospasm, cerebral palsy, headache, strabismus, temporomandibular joint disorder, neuropathic pain, overactive bladder, rhinitis, sinusitis, acne, dystonia, dystonic contractions, hyperhidrosis, and hypersecretion of glands controlled by the cholinergic nervous system.

12. A method of making an injectable botulinum toxin formulation, the method comprising

Providing an effective amount of a botulinum toxin complex, a reduced botulinum toxin complex, or a botulinum toxin;

providing a positively charged carrier comprising a positively charged backbone having a plurality of efficiency groups attached thereto;

combining the botulinum toxin complex, reduced botulinum toxin complex or botulinum toxin with the positively charged carrier and a pharmaceutically acceptable diluent.

13. The method of claim 11, wherein the botulinum toxin complex, reduced botulinum toxin complex, or botulinum toxin is obtained from botulinum serotype A, B, C, D, E, F or G.

14. The method of claim 11, wherein the positively charged backbone comprises a polyamino acid.

15. The method of claim 11, wherein the polyamino acid is selected from the group consisting of polylysine, polyarginine, polyhistidine, and polyornithine.

16. The method of claim 9, wherein the efficiency group is an amino acid sequence selected from the group consisting of: (gly)_n1-(arg)_n2HIV-TAT or fragments thereof, protein transduction domains of antennapedia proteins or fragments thereof, (gly)_p-RGRDDRRQRRR-(gly)_q，(gly)_p-YGRKKRRQRRR-(gly)_qOr (gly)_p-RKKRRQRRR-(gly)_qAnd SGRQIKIWFQNRRMKWKKC;

wherein subscript n1 is an integer of from 0 to 20, subscript n2 is independently an odd integer of from about 5 to about 25, more preferably from about 7 to about 17, most preferably from about 7 to about 13; and

wherein subscripts p and q are each independently an integer of 0 to 8.

17. The method of claim 1, wherein the positively charged carrier stabilizes botulinum toxin against degradation.

18. The method of claim 1, wherein the positively charged carrier reduces local diffusion of botulinum toxin upon injection.

19. The method of claim 1The method wherein the positively charged carrier has the amino acid sequence RKKRRQRRRG- (K)₁₅-GRKKRRQRRR。

20. The method of claim 1, wherein the formulation is administered to the subject after injection into the subject's bodyIn contrast, there is a reduced tendency to induce antibody production.

21. A sterile injectable composition comprising

An effective amount of a botulinum toxin complex, a reduced botulinum toxin complex, or a botulinum toxin; and

a positively charged carrier comprising a positively charged backbone having a plurality of efficiency groups attached thereto;

wherein the botulinum toxin complex, reduced botulinum toxin complex, or botulinum toxin is non-covalently associated with the positively charged carrier.

22. A syringe comprising

An effective amount of a botulinum toxin complex, a reduced botulinum toxin complex, or a botulinum toxin; and

a positively charged carrier comprising a positively charged backbone having a plurality of efficiency groups attached thereto;

wherein the botulinum toxin complex, reduced botulinum toxin complex, or botulinum toxin is non-covalently associated with the positively charged carrier.

23. Reconstituted botulinum toxin composition comprising

An effective amount of a botulinum toxin complex, a reduced botulinum toxin complex, or a botulinum toxin; and

a positively charged carrier comprising a positively charged backbone having a plurality of efficiency groups attached thereto; and

a pharmaceutically acceptable aqueous liquid diluent in a pharmaceutically acceptable carrier,

wherein the botulinum toxin complex, reduced botulinum toxin complex or botulinum toxin is non-covalently bound to the positively charged carrier,

and wherein the reconstituted botulinum toxin composition has a viscosity of about 0.4 to about 2.0 cP.