WO2009139739A1 - Compositions comprising a combination of botulinum toxin a and botulinum toxin b for treating conditions characterized by unwanted or excessive presynaptic neuronal activity or secretion - Google Patents

Abstract

The instant invention provides methods and compositions for the treatment of diseases, disorders and conditions using combinations of botulinum toxins, e.g., botulinum toxin A and botulinum toxin B. The instant invention further provides an assay for determining the efficacy of neurotoxins, or combinations of neurotoxins.

Description

COMPOSITIONS COMPRISING A COMBINATION OF BOTULINUM TOXIN A AND BOTULINUM TOXIN B FOR TREATING CONDITIONS CHARACTERIZEDNWANTED OR EXCESSIVE PRESYNAPTIC NEURONAL ACTIVITY OR SECRETION

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. provisional application 60/773,412 filed February 14, 2006, which is incorporated by referenced herein it its entirety.

GOVERNMENT SUPPORT

Research supporting this application was carried out by the United States of America as represented by the Secretary, Department of Health and Human Services.

BACKGROUND OF THE INVENTION

Botulinum neurotoxins (BoNTs) cause the disease botulism, which is characterized by prolonged paralysis. In contrast, injections of low doses of purified BoNTs do not cause systemic illness but produce localized muscle paralysis that is beneficial for treating several human medical disorders involving excess presynaptic neuronal activity, e.g., uncontrollable muscle contraction.

Botulism is a neuromuscular disease characterized by progressive muscle weakness leading to paralysis, and possible death from respiratory failure. Of the seven known botulinum neurotoxins (BoNTs), only three routinely cause human disease, BoNT serotypes A, B or E. Since 1950, serotype F has been attributed to five cases, whereas, BoNT/C and /D have never been known to cause botulism in humans, yet these two serotypes cause significant disease among livestock and water fowl (Duncan and Jensen, 1976; Hubalek et al., 1991; Tucker, 2002). Poisoning by any one of these neurotoxins impairs communication between nerve and muscle. In humans, the resulting paralysis caused by BoNT/A or B tends to be protracted regardless of the route of entry. This makes treating the disease difficult but has led to using purified BoNT preparations to treat several human neuromuscular disorders (Hughes et al., 1981; Woodruff et al., 1992; Sloop et al., 1997; Brashear et al., 1999). In studies of healthy volunteers, local intramuscular (Lm.) injection of medical-grade BoNT/A had a prolonged effect, with muscle function gradually returning over 12 months. Paralysis from BoNT/B endured for up to four months (Sloop et al., 1997; Brin et al., 1999).

The therapeutic potential of individual botulinum toxins are well known in the art. However, improved therapeutic effects are desired to increase the efficacy of treatments of diseases, disorders and conditions using botulinum toxins.

SUMMARY OF THE INVENTION

The instant invention provides methods, pharmaceutical compositions, and kits for treating subjects using a combination of botulinum toxins, e.g., combination of botulinum toxin A and botulinum toxin B.

In one aspect, the instant invention provides a methods and compositions for the treatment of diseases, disorders or conditions characterized by unwanted or excessive presynaptic neuronal activity or secretion. The invention further provides an assay for monitoring the efficacy of one or more toxins, e.g., neurotoxins, on an animal.

In a further aspect, the instant invention provides methods and compositions for treating a subject having a disease, disorder or condition characterized by unwanted or excessive presynaptic neuronal activity or secretion by administering to the subject an effective amount of botulinum toxin A and botulinum toxin B, thereby treating the subject having a disease, disorder or condition characterized by unwanted or excessive presynaptic neuronal activity or secretion. In a related embodiment, the botulinum toxin A and botulinum toxin B are administered substantially simultaneously.

In another related embodiment, the botulinum toxin A and botulinum toxin B are in an admixture and administered to a subject in such manner. In a specific embodiment, approximately equal amounts of botulinum toxin A and botulinum toxin B are administered.

In a specific embodiment, the disease, disorder or condition is one or more of cervical dystonia, strabismus, blephrasopasm, VII nerve disorders, muscle spasticity, troticollois, hyperactive facial lines, Parkinson's disease, cosmetic wrinkles, hyperhidrosis, muscle spasms, migraines, tension headaches, anal fissures, glabeller furrows, hyperkinetic lines, hypertonia, thyroiditis, prostate enlargement, hyperparathyroidism, cardiovascular disease, neuromuscular disease, excessive sweating, juvenile cerebral palsy, epilepsy, sinus headache, fibromyalgia, urologic disorders, obsessive compulsive disorder, inner ear disorders, orofacial dyskinesia, athetosis, chorea, and/or diabetes.

In a further specific embodiment, the disease, disorder or condition is hyperactive facial lines, e.g., hyperactive facial lines of the upper face, forehead, periorbital, paranasal, perioral, lower facial, or nasolabial regions.

In one embodiment the botulinum toxin A and botulinum toxin B are administered intramuscularly, subcutaneously, or transdermally.

In one embodiment, the subject is human.

In one embodiment, the botulinum toxins are administered in an amount between about 1 unit and about 40,000 units each.

In another aspect, the instant invention provides methods of treating a subject having a neuromuscular disease or disorder comprising, administering to the subject an effective amount of botulinum toxin A and botulinum toxin B, thereby treating the subject having a neuromuscular disease or disorder.

In a related embodiment, the botulinum toxin A and botulinum toxin B are administered simultaneously. In another embodiment, the botulinum toxin A and botulinum toxin B are in an admixture. In one embodiment, approximately equal amounts of botulinum toxin A and botulinum toxin B are administered. In specific embodiments, the botulinum toxin A and botulinum toxin B are administered intramuscularly, subcutaneously, or transdermally.

In a another related embodiment, the botulinum toxins are administered in an amount between about 1 unit and about 40,000 units each.

In one embodiment, the subject is human.

In another aspect, the instant invention provides methods of treating a subject having cervical dystonia comprising, simultaneously administering to the subject an effective amount of botulinum toxin A and botulinum toxin B, thereby treating the subject having cervical dystonia.

In one embodiment, the botulinum toxin A and botulinum toxin B are in an admixture. In one embodiment, approximately equal amounts of botulinum toxin A and botulinum toxin B are administered. In specific embodiments, the botulinum toxin A and botulinum toxin B are administered intramuscularly, subcutaneously, or transdermally. In one embodiment, the botulinum toxins are administered in an amount between about 1 unit and about 40,000 units each.

In one embodiment, the subject is human.

In another aspect, the instant invention provides methods of treating a subject having cosmetic wrinkles comprising administering to the subject an effective amount of botulinum toxin A and botulinum toxin B, thereby treating the subject having cosmetic wrinkles.

In one embodiment, the botulinum toxin A and botulinum toxin B are administered substantially simultaneously. In another embodiment, the botulinum toxin A and botulinum toxin B are in an admixture and administered to a patient in such an admixture. In another embodiment, approximately equal amounts of botulinum toxin A and botulinum toxin B are administered. By indicating that botulinum toxin A and botulinum toxin B are administered substantially simultaneously to a patient, the distinct clinical agents are generally administered to the patient in the same treatment regime, e.g. where the clinical agents are administered in the same pharmaceutical composition or administered to a patient at least within 1 to 24 hours of each other, more typically within 0.25 to 6 hours, or 0.25 to 4 hours of each other.

In one embodiment, the botulinum toxins are administered in an amount between about 1 unit and about 40,000 units each.

In one embodiment, the subject or patient is human.

Methods of the invention include administering to a subject (e.g., a human or an animal) in need thereof an effective amount of botulinum toxin A and botulinum toxin B. The methods can also include the step of identifying that the subject is in need of treatment of diseases or disorders described herein. An effective amount of an effective amount of botulinum toxin A and botulinum toxin B can be administered to the identified subject. The identification can be in the judgment of a subject or a health professional and can be subjective (e.g., opinion) or objective (e.g., measurable by a test or a diagnostic method). The methods delineated herein can further include the step of assessing or identifying the effectiveness of the treatment or prevention regimen in the subject by assessing the presence, absence, increase, or decrease of a marker. Such assessment methodologies are know in the art and can be performed by commercial diagnostic or medical organizations, laboratories, clinics, hospitals and the like. The methods can further include the step of taking a sample from the subject and analyzing that sample. The sample can be a sampling of cells, genetic material, tissue, or fluid (e.g., blood, plasma, sputum, etc.) sample. The methods can further include the step of reporting the results of such analyzing to the subject or other health care professional.

In another aspect, the instant invention provides pharmaceutical composition comprising botulinum toxin A and botulinum toxin B optionally together with and a pharmaceutically acceptable carrier.

In a specific embodiment, the botulinum toxin A and botulinum toxin B are in approximately equal amounts, e.g. where botulinum toxin A and botulinum toxin B are present in a pharmaceutical composition in molar or weight amounts that are within about 2, 5, 10, 20, 25, 30 or 40 percent of each other.

In one embodiment, the botulinum toxin B is protease activated.

In another embodiment, the pharmaceutical composition comprising the botulinum toxin A and/or botulinum toxin B further comprising a stabilizing agent.

In another aspect, the instant invention provides a kit comprising one or more of the pharmaceutical compositions described herein and instructions for use. In another embodiment, the instant invention provides a kit comprising botulinum toxin A and botulinum toxin B and instructions for use.

In a related embodiment, the botulinum toxin A and botulinum toxin B are administered simultaneously.

In another embodiment, the instant invention provides a kit for the treatment of cosmetic wrinkles comprising botulinum toxin A and botulinum toxin B and instructions for use.

In a related embodiment, the kit contains a protease to activate the botulinum toxin B. In a specific embodiment, the protease is a serine protease, e.g., try spin.

In a related embodiment, the kit comprises one or more syringes.

In another aspect, the instant invention provides an assay for monitoring the efficacy of one or more neurotoxins on an animal comprising the steps of administering one or more neurotoxins to an animal, determining the distance the animal travels (e.g. runs) over a period of time, thereby monitoring the efficacy of a neurotoxin on the animal. In a specific embodiment, the animal is a rat or mouse. In another related embodiment, the neurotoxin is administered to the animal's hind legs. In one embodiment, the animal runs on a wheel. In another embodiment, the distance the animal runs is the distance the animal runs nocturnal Iy. Other aspects of the invention are disclosed infra.

DESCRIPTION OF THE DRAWINGS

Figures IA-C depict nightly running activity. Figure IA: Mice were housed in various sized groups with access to two exercise wheels in each cage. The average distance (km) per mouse was calculated from running values collected over three days. Data was compiled from at least four cages for each condition. Figure IB. The best paradigm required two mice co-housed with access to two running wheels. Mice that demonstrated at least 5 km per night were monitored and distances were recorded daily. Data from four cages (■, D, •, O) are shown with each symbol representing individual data points recorded at 24 hr intervals. Figure 1C: Two animals in each cage were treated as indicated and running activity was collected over three days. Injection of BoNT/A (0.2 LD₅₀ units) reduced running activity by about three-fold compared to untreated animals (n = 4). Animals injected with either saline or BoNT/A combined with antitoxin did not demonstrate reduced running over a period of three days.

Figures 2A-C depict dose-response of running distance versus BoNT dosage. Figure 2 A: Animals in groups of two were injected with BoNT/A (i.m. LD₅₀ units): 0.14 (D), 0.40 (♦), 0.6 (•), or 0.8 (■). One i.m. LD50 unit equals 0.06 mL of 1.25 pM BoNT/A. Error bars are averages of triplicate determinations except for 0.8 LD50 units (n=6). Non-linear regression analysis included data only after maximum paralysis subsided. The equation was y = (1 — exp(-k x t)) x M + c, as described in the materials and methods. Figure 2B: Dose-response of running distance versus BoNT/B (i.m. LDso units): 0.30 LD50 units (A), 0.60 (•), 0.90 (■). Regression analysis for BoNT/B data used the equation: y = B₀ + ((T_o-B₀)/l + 10^c) described in the materials and method section. Error bars are averages of triplicate determinations. Figure 2C. BoNT/E determinations were done in duplicate using 0.4 LD₅₀ units (D,O) or 0.90 (•,■). Each symbol from the BoNT/E trial represents a single determination. The arrow indicates the day of injection. Figures 3A-C depict recovery rates from BoNT/A paralysis. Data from Figure 2 A is graphed demonstrating two linear recovery phases for the return of running activity. Both phases intersect at 33 - 38% (dashed line) recovery (Figures 2A and 2B). The rapid and slow rates are depicted as linear lines. Figure 3C demonstrates the dose-dependent relationship of the rapid (•) and slow (■) phases.

Figures 4A-B depict a comparison of BoNT serotype duration. Figure 4A: Duplicate data for BoNT/A (■), BoNT/B (•), and BoNT/E (A.) was normalized to a percentage scale. BoNT/A and /B traces represent 0.9 LD50 units, BoNT/E trace is 0.8 LDso units. Figure 4B: The duration of total paralysis (zero running) was compiled and graphed relative to each toxin dose. BoNT/A (D) was fit using nonlinear regression analysis, whereas BoNT/B (O) and BoNT/E (Δ) were fit using linear regression analysis.

Figures 5A-B depict co-injection of toxin mixtures. Duplicate determinations were tested using 0.5 LD₅0 units of BoNT/ A mixed with either 0.3 LD₅₀ units of BoNT/B (Figure 5A) or 0.5 LD₅₀ units of BoNT/E (Figure 5B). In both circumstances the rate of recovery was reduced when two serotypes were present compared to the recovery rate for either serotype individually.

DETAILED DESCRIPTION

The anaerobic, gram positive bacterium Clostridium botulinum produces a potent polypeptide neurotoxin, botulinum toxin, which causes a neuroparalytic illness in humans and animals referred to as botulism. Clostridium botulinum and its spores are commonly found in soil and the bacterium can grow in improperly sterilized and sealed food containers of home based canneries, which are the cause of many of the cases of botulism. The effects of botulism typically appear 18 to 36 hours after eating the foodstuffs infected with a Clostridium botulinum culture or spores. The botulinum toxin can apparently pass unattenuated through the lining of the gut and attack peripheral motor neurons. Symptoms of botulinum toxin intoxication can progress from difficulty walking, swallowing, and speaking to paralysis of the respiratory muscles and death.

In contrast to the harmful effects of botulinum toxins detailed above, small amounts of botulinum toxins have been found to be effective in treating a number of diseases, disorders and conditions.

Optimizing the therapeutic efficacy while diminishing adverse reactions requires precise knowledge of toxin potency as well as a clear understanding of how each toxin causes disease.

A novel in vivo mouse assay has been used to correlate toxin dosage with the duration of muscle paralysis. Voluntary running activity performed by mice was proportional to the amount of toxin injected into the hind limbs and the subsequent rate of recovery over the ensuing days or weeks was a function of neurotoxin type, amount and/or combination of toxins, e.g., serotypes of BoNTs. BoNT/A produced longer paralysis than BoNTTB consistent with human observations. BoNT/A recovery appeared biphasic with the initial phase about two-fold faster than the final phase. Over four weeks, muscle activity had gradually improved following the highest BoNT/A dose, reaching about half of the normal running activity. Lower BoNT/A doses led to incrementally faster and complete recovery. Persistence of maximum paralysis was exponentially related to BoNT/A dosage, with a doubling of the paralysis time occurring with every 25% increase of the toxin concentration. The rate of recovery from BoNT/B was monophasic relative to toxin dosage and the duration of maximum paralysis was linear relative to dosage. Combinations of BoNT/A & B and BoNT/A & E were tested and shown to exacerbate paralysis compared to individually administered serotypes. The results detailed in the experiments demonstrate that a combination of botulinum toxin A and botulinum toxin B results in faster onset of the biological effect of the toxins and a longer duration of action.

The instant invention thus provides methods, pharmaceutical compositions, and kits for treating subjects using a combination of botulinum toxins, e.g., combination of botulinum toxin A and botulinum toxin B.

The instant invention provides methods and compositions for the treatment of subjects having diseases, disorders and conditions characterized by unwanted or excessive presynaptic neuronal activity or secretion, or subjects having diseases, disorders and conditions known to be treatable with botulinum toxin. Botulinum Toxins

"Botulinum toxin" (also identified as "BoNT" herein) means a neurotoxin produced by Clostridium botulinum, as well as a botulinum toxin (or the light chain or the heavy chain thereof) made recombinantly by a non-Clostridial species. The phrase "botulinum toxin", as used herein, encompasses the botulinum toxin serotypes A, B, C, D, E, F and G. Botulinum toxin, as used herein, also encompasses both a botulinum toxin complex (i.e. the 300, 600 and 900 kDa complexes) as well as the purified botulinum toxin (i.e. about 150 kDa). "Purified botulinum toxin" is defined as a botulinum toxin that is isolated, or substantially isolated, from other proteins, including proteins that form a botulinum toxin complex. A purified botulinum toxin may be greater than 95% pure, and preferably is greater than 99% pure. Botulinum toxin A, as used herein, refers to botulinum toxin type A as further described herein and as known in the art. Botulinum toxin B, as used herein, refers to botulinum toxin type B as further described herein and as known in the art. Botulinum toxin A has been previously described by, for example, Thompson, D.E. et al. (1990) Eur. J. Biochem. 189:73-81 and Binz,T., et al. (1990) J. Biol. Chem. 265:9153-9158. The sequence of botulinum toxin A can be found, for example, as Genbank Accession Number Q45894. Botulinum toxin B has been previously described by, for example, Whelan, S.M. et al. (1992) Appl. Environ. Microbiol. 58:2345-2354. The sequence of botulinum toxin B can be found, for example, as Genbank Accession Number: P 10844.

"Clostridial neurotoxin" means a neurotoxin produced from, or native to, a Clostridial bacterium, such as Clostridium botulinum, Clostridium butyricum or Clostridium beratti, as well as a Clostridial neurotoxin made recombinantly by a non- Clostridial species.

Botulinum toxin type A is the most lethal natural biological agent known to man. About 50 picograms of botulinum toxin (purified neurotoxin complex) type A is a LD₅₀ in mice. On a molar basis, botulinum toxin type A is 1.8 billion times more lethal than diphtheria, 600 million times more lethal than sodium cyanide, 30 million times more lethal than cobrotoxin and 12 million times more lethal than cholera. Singh, Critical Aspects of Bacterial Protein Toxins, pages 63-84 (chapter 4) of Natural Toxins II, edited by B. R. Singh et al., Plenum Press, New York (1976) (where the stated LD50 of botulinum toxin type A of 0.3 ng equals 1 U is corrected for the fact that about 0.05 ng of BOTOX equals 1 unit). One unit (U) of botulinum toxin is defined as the LD₅₀ upon intraperitoneal injection into female Swiss Webster mice weighing 18-20 grams each. In other words, one unit of botulinum toxin is the amount of botulinum toxin that kills 50% of a group of female Swiss Webster mice. Seven generally immunologically distinct botulinum neurotoxins have been characterized, these being respectively botulinum neurotoxin serotypes A, B, Ci, D, E, F, and G, each of which is distinguished by neutralization with type-specific antibodies. The different serotypes of botulinum toxin vary in the animal species that they affect and in the severity and duration of the paralysis they evoke. For example, it has been determined that botulinum toxin type A is 500 times more potent, as measured by the rate of paralysis produced in the rat, than is botulinum toxin type B. The botulinum toxins apparently bind with high affinity to cholinergic motor neurons, are translocated into the neuron and block the presynaptic release of acetylcholine. In preferred embodiments, the instant invention uses combinations of botulinum toxin A and botulinum toxin B to treat a mammal, e.g., a human, for any one or more of the diseases, disorders or conditions described herein.

Botulinum toxins can be obtained from commercial sources, e.g., List Biological Laboratories, Inc., Campbell, Calif.; the Centre for Applied Microbiology and Research, Porton Down, U.K.; Wako (Osaka, Japan), as well as from Sigma Chemicals of St Louis, Mo. In an alternative embodiment, botulinum toxins can be produced recombinantly or isolated from the natural source, see, for example, Animal product free system and process for purifying a botulinum toxin (US Patent Appln No. 20050238669) and Media for Clostridium bacterium and processes for obtaining a clostridial toxin (US Patent Appln No. 20050238668).

Botulinum toxins have been used in clinical settings for the treatment of neuromuscular disorders characterized by hyperactive skeletal muscles. Botulinum toxin type A was approved by the U.S. Food and Drug Administration in 1989 for the treatment of essential blepharospasm, strabismus and hemifacial spasm in patients over the age of twelve. Clinical effects of peripheral injection (i.e. intramuscular or subcutaneous) botulinum toxin type A are usually seen within one week of injection, and often within a few hours after injection. The typical duration of symptomatic relief (i.e. flaccid muscle paralysis) from a single intramuscular injection of botulinum toxin type A can be about three months to about six months. Pharmaceutical Compositions

"Pharmaceutical composition" means a formulation in which an active ingredient can be a neurotoxin, such as a one or more botulinum toxins. The word "formulation" means that there is at least one additional ingredient in the pharmaceutical composition besides the neurotoxin(s). A pharmaceutical composition is therefore a formulation which is suitable for diagnostic or therapeutic administration (i.e. by intramuscular or subcutaneous injection or by insertion of a depot or implant) to a subject, such as a human patient. The pharmaceutical composition can be: in a lyophilized or vacuum dried condition; a solution formed after reconstitution of the lyophilized or vacuum dried pharmaceutical composition with saline or water, or; as a solution which does not require reconstitution. The neurotoxin active ingredient can be one or more of the botulinum toxin serotypes A, B, Ci, D, E, F or G, all of which can be made natively by Clostridial bacteria or obtained commercially. In preferred embodiments the pharmaceutical compositions comprise botulinum toxin A and/or B. As stated, a pharmaceutical composition can be liquid or solid, for example vacuum- dried. The constituent ingredients of a pharmaceutical composition can be included in a single composition (that is all the constituent ingredients, except for any required reconstitution fluid, are present at the time of initial compounding of the pharmaceutical composition) or as a two-component system, for example a vacuum- dried composition reconstituted with a diluent such as saline which diluent contains an ingredient not present in the initial compounding of the pharmaceutical composition. A two-component system provides the benefit of allowing incorporation of ingredients which are not sufficiently compatible for long-term shelf storage with the first component of the two component system. For example, the reconstitution vehicle or diluent may include a preservative which provides sufficient protection against microbial growth for the use period, for example one-week of refrigerated storage, but is not present during the two-year freezer storage period during which time it might degrade the toxin. Other ingredients, which may not be compatible with a Clostridial toxin or other ingredients for long periods of time, may be incorporated in this manner; that is, added in a second vehicle (i.e. in the reconstitution fluid) at the approximate time of use.

The instant invention provides pharmaceutical compositions comprising botulinum toxin A and/or B. In one embodiment, the pharmaceutical composition is a formulation which contains at least one active ingredient, i.e., botulinum toxin A and/or B as well as, for example, one or more excipients, buffers, carriers, stabilizers, preservatives and/or bulking agents, and is suitable for administration to a patient to achieve a desired diagnostic result or therapeutic effect.

"Protein stabilizer" (or "primary stabilizer") is a chemical agent that assists to preserve or maintain the biological structure (i.e., the three dimensional conformation) and/or biological activity of a protein (such as a botulinum toxin). Stabilizers can be proteins or polysaccharides. Examples of protein stabilizers include hydroxyethyl starch (hetastarch), serum albumin, gelatin, collagen, as well as a recombinant albumin, gelatin or collagen. As disclosed herein, the primary stabilizer can be a synthetic agent that would not produce an immunogenic response (or produces an attenuated immune response) in a subject receiving a composition containing the primary stabilizer. In other embodiments of the invention, the protein stabilizers may be proteins from the same species of animal that is being administered the protein. Additional stabilizers may also be included in a pharmaceutical composition. These additional or secondary stabilizers may be used alone or in combination with primary stabilizers, such as proteins and polysaccharides. Exemplary secondary stabilizers include, but are not limited to non-oxidizing amino acid derivatives (such as a tryptophan derivate, such as N-acetyl-tryptophan ("NAT")), caprylate (i.e. sodium caprylate), a polysorbate (e.g., P80), amino acids, and divalent metal cations such as zinc. A pharmaceutical composition can also include preservative agents such as benzyl alcohol, benzoic acid, phenol, parabens and sorbic acid. A "recombinant stabilizer" is a "primary stabilizer" made by recombinant means, such as for example, a recombinantly made albumin (such as a recombinantly made human serum albumin), collagen, gelatin or a cresol, such as an M-cresol.

The pharmaceutical compositions disclosed herein have diagnostic, therapeutic and/or research utility in patients such as humans, as well in, for example, canine, equine, bovine and porcine mammalian species patients, and in non-mammalian avian species patients.

Pharmaceutical compositions comprising either botulinum toxin A or botulinum toxin B have been described previously. For example, the following U.S. patents and patent applications describe pharmaceutical compositions comprising botulinum toxins: Pharmaceutical composition of botulinum neurotoxin and method of preparation (U.S. Pat. No. 5,512,547); Botulinum toxin pharmaceutical compositions with multiple stabilizers (U.S. Pat. Appln. No. 20050238664); Recombinant stabilizer botulinum toxin pharmaceutical compositions (U.S. Pat. Appln. No. 20050238663); Botulinum toxin pharmaceutical composition with enhanced potency (U.S. Pat. Appln. No. 20050208076); Compositions and methods for topical application and transdermal delivery of botulinum toxins (U.S. Pat. Appln. No. 20050196414); Clostridial neurotoxin compositions and modified clostridial neurotoxins (U.S. Pat. Appln. No. 20040220386); Solid dose delivery vehicle and methods of making same (U.S. Pat. Appln. No. 20040219206); and Controlled release botulinum toxin system (U.S. Pat. Appln. No. 2004003324). The teachings of these patents and patent applications can be used to administer botulinum toxin A and botulinum toxin B, or can be adapted to formulate a composition that comprises both botulinum toxin A and botulinum toxin B.

The invention also provides kits comprising botulinum toxin A and botulinum toxin B and instructions for use. In further embodiments, botulinum toxin A and botulinum toxin B are formulated as pharmaceutical compositions for inclusion in the kits. Kits of the invention may also provide one or more syringes for administration of botulinum toxin A and botulinum toxin B to the subject.

Methods of Treatment

The instant invention provides a methods and compositions for the treatment of diseases, disorders and/or conditions characterized by unwanted or excessive presynaptic neuronal activity or secretion. In a specific embodiments, the disease, disorder or condition is one or more of cervical dystonia, strabismus, blephrasopasm, VII nerve disorders, muscle spasticity, troticollois, hyperactive facial lines, Parkinson's disease, cosmetic wrinkles, hyperhidrosis, muscle spasms, migraines, tension headaches, anal fissures, glabeller furrows, hyperkinetic lines, hypertonia, thyroiditis, prostate enlargement, hyperparathyroidism, cardiovascular disease, neuromuscular disease, excessive sweating, juvenile cerebral palsy, epilepsy, sinus headache, fibromyalgia, urologic disorders, obsessive compulsive disorder, inner ear disorders, orofacial dyskinesia, athetosis, chorea, or diabetes.

In addition to the diseases, disorders and conditions described above, botulinum toxins have been previously described to be beneficial in the treatment of: intrathecal pain (see e.g. U.S. Pat. No. 6,113,915); paragangliomas (see e.g. U.S. Pat. No. 6,139,845); otic disorders (see e.g. U.S. Pat. No. 6,265,379); pancreatic disorders (see e.g. U.S. Pat. Nos. 6,143,306 and 6,261,572); migraine (see e.g. U.S. Pat. No. 5,714,468); smooth muscle disorders (see e.g. U.S. Pat. No. 5,437,291); prostate disorders, including prostatic hyperplasia (see e.g. WO 99/03483 and Doggweiler R., et al Botulinum toxin type A causes diffuse and highly selective atrophy of rat prostate, Neurourol Urodyn 1998; 17(4): 363); autonomic nerve disorders, including hyperplasic sweat glands (see e.g. U.S. Pat. No. 5,766,606); wound healing (see e.g. WO 00/24419); reduced hair loss (see e.g. WO 00/62746); skin lesions (see e.g. U.S. Pat. No. 5,670,484), neurogenic inflammatory disorders (see e.g. U.S. Pat. No. 6,063,768) bradycardia (see e.g. U.S. Pat. No.6,977,080); hyperparathyroid disorders (see e.g. U.S. Pat. No.6,974,793); vascular disorders (see e.g. U.S. Pat. No.6,974,579); secretions and glands (see e.g. U.S. Pat. No.6,974,578); neuropsychiatric disorders (see e.g. U.S. Pat. No.6,921,538); pain (see e.g. U.S. Pat. No.6,887,476); muscle spasm (see e.g. U.S. Pat. No.6,841,156); sinus headache (see e.g. U.S. PaL No.6,838,434); Hashimoto's thyroiditis (see e.g. U.S. Pat. No.6,821,520); pain (see e.g. U.S. Pat. No.6,806,251); pain (see e.g. U.S. Pat. No.6,787,517); tension headache (see e.g. U.S. Pat. No.6,776,992); priapism (see e.g. U.S. Pat. No.6,776,991); Hashimoto's thyroiditis (see e.g. U.S. Pat. No.6,773,711); hypocalcemia (see e.g. U.S. Pat. No.6,716,427); Hypoparathyroid (see e.g. U.S. Pat. No.6,635,247); Parkinson's disease (see e.g. U.S. Pat. No.6,620,415); bone tumors (see e.g. U.S. Pat. No.6,565,870); thyroid disorders (see e.g. U.S. Pat. No.6,524,580); headache (see e.g. U.S. Pat. No.6,458,365); cerebral palsy (see e.g. U.S. Pat. No.6,448,231); wound healing (see e.g. U.S. Pat. No.6,447,787); hypercalcemia (see e.g. U.S. Pat. No.6,447,785); pancreatic disorders (see e.g. U.S. Pat. No.6,143,306); cancer (see e.g. U.S. Pat. No.6, 139,845); neurogenic inflammatory disorders (see e.g. U.S. Pat. No.6,063, 768); gastrointestinal muscle disorders and other smooth muscle dysfunction (see e.g. U.S. Pat. No.5,437,291); neoplasms (see e.g. U.S. Pat. App. No.20050260231); neurological and neuropsychiatric disorders (see e.g. U.S. Pat. App. No. 20050147626); kinesia (see e.g. U.S. Pat. App. No. 20050147625); skin disorders (see e.g. U.S. Pat. App. No. 20050123567); cancers (see e.g. U.S. Pat. App. No. 20050031648); eye disorders (see e.g. U.S. Pat. App. No. 20040234532); cardiovascular diseases (see e.g. U.S. Pat. App. No. 20040223975); cervical dystonia (see e.g. U.S. Pat. No.6,632,433); and dystonia (see e.g. U.S. Pat. No.6,290,961), and the treatment methods, pharmaceutical compositions and kits of the invention can be employed to treat subjects (particularly humans) suffering from or susceptible to one or more of these disorders and/or diseases.

The instant invention is based, at least in part, on the discovery that the administration of two or more botulinum toxins results in improved therapeutic results when compared to the administration of either toxin alone. Specifically, the examples show that administration of botulinum toxin A and botulinum toxin B to an animal results in improved muscle paralysis characteristics when compared to the administration of botulinum toxin A or botulinum toxin B alone. Accordingly, the administration of botulinum toxin A and botulinum toxin B will lead to improved therapeutic results as compared to the administration of either of botulinum toxin alone. In preferred embodiments, botulinum toxin A and boulinum toxin B are administered to a subject simultaneously. As used herein "simultaneously" means that the two toxins are administered at approximately the same time, e.g., in a mixture or as multiple injections. Alternatively, simultaneously can mean the administration of one botulinum toxin prior to the administration of the second botulinum toxin such that the second botulinum toxin is administered prior to the first botulinum toxin achieving full effect, e.g., preferably long before the first botulinum toxin achieving full effect. In preferred embodiments, the second botulinum toxin is administered within 72 hours, 48 hours, 24 hours, 12, hours, 6 hours, 3 hours, 1 hour, 30 minutes, 20 minutes, 15 minutes, 10 minutes, 5 minutes, 4 minutes, 3 minutes, 2 minutes, or 1 minute of the first botulinum toxin. In the methods of the invention, botulinum toxin A or botulinum toxin B can be administered first.

Due to the toxic nature of botulinum toxins they are often administered locally and proximal to the site of desired therapeutic action. For example, for treatment of tumors botulinum toxin A and botulinum toxin B are administered locally to the site of the tumor, or targeted to that location. For treatment of muscular conditions, the toxins are administered to the affected muscle. For cosmetic treatments, the toxins are administered locally to the site of the desired cosmetic effect, e.g., by direct injection of such toxin into the facial muscles. For many treatments intramuscular injection is the preferred route of administration, but other routes of local administration are available, such as subcutaneous administration. "Local administration" means direct administration of a pharmaceutical at or to the vicinity of a site on or within an animal body, at which site a biological effect of the pharmaceutical is desired. Local administration excludes systemic routes of administration, such as intravenous or oral administration.

The methods described herein use two or more botulinum toxins, e.g., botulinum toxin A and botulinum toxin B. Depending on the disease, disorder or condition being treated, a medical professional may administer from 1 -40,000 units of botulinum toxin and botulinum toxin B to a subject. In specific embodiments a medical professional may administer, 5-20,000 units, 10-15,000 units, 20-10,000 units, 50-1000 units, 1-3000 units, 100-500 units, of 300-1000 units of each botulinum toxin, e.g., botulinum A and botulinum toxin B to a subject depending on the specific course of treatment and condition being treated.

In certain embodiments, botulinum toxin A and botulinum toxin B are administered in approximately equal amounts, i.e., in approximately a 1:1 ratio, based on weight or molar amounts. In other embodiments, the ratio of botulinum toxin A and botulinum toxin B administered is approximately 0.001: 1, 0.005:1, 0.01:1, 0.05:1, 0.1:1, 0.2: 1, 0.3:1, 0.4:1, 0.5: 1, 0.6:1, 0.7:1, 0.8:1, 0.9:1, 1.1:1, 1.2:1, 1.3:1, 1.4:1, 1.5: 1 , 1.6:1, 1.7:1, 1.8:1, 1.9: 1, 2:1, 3:1, 4:1, 5:1, 10:1, 100:1, 300:1, 500:1, 1000:1 or 10,000: 1 , based on weight or molar amounts.

A medical professional having ordinary skill in the art can determine the amount, ratio, and method of administration for botulinum toxins to be administered to a subject. In making these determinations, the medical provider will take into account the disease, disorder or condition being treated; the progression of the particular disease, disorder or condition; and characteristics of the individual being treated, e.g., size, previous treatments, etc.

In specific embodiments, the botulinum toxins used in the methods of the invention can be administered with other therapeutic agents that aid in treating the particular disease, disorder or condition that the subject is being treated for.

Assays

The instant invention also provides assays to determine the efficacy of toxins, e.g., neurotoxins such as botulinum toxins, on an animal. The assays are based on the observation that neuroxotins such as botulinum toxins when injected into the muscles of an animal, result in a decrease in the amount of running the animal does. The more effective the toxin, or combination of toxins, the larger the decrease in the running activity of the animal as compared to an untreated animal. Specifically, the assay comprises injecting one or more toxins into the gastrocnemius (calf) muscle of a mouse and measuring the running activity of the animal over a number of days or weeks. In particular embodiments, the nocturnal running activity is measured. In one embodiment, the running distances are measured for 8 hours, 12 hours, 24 hours, 2 days, 7 days, 14 days, 30 days or more after the administration of the toxins. Running distances may be reduced up to 99%, 90%, 85%, 80%, or less. In one embodiment, recovery may also be measured. As used herein, recovery is a measure of the percent of running distance recovered over time as compared to a pre-treatment level of running distance. In one embodiment, a toxin is efficacious if it reduces normal running distance by up to 90% for over 7 days. In another embodiment, a toxin is efficacious if it reduces running distance from between about 50% to about 99% for from between 1 — 30 days.

EXAMPLES

It should be appreciated that the invention should not be construed to be limited to the examples that are now described; rather, the invention should be construed to include any and all applications provided herein and all equivalent variations within the skill of the ordinary artisan.

MATERIALS AND METHODS

Animals. BALB/cJ male mice at five weeks old (18 — 20 g) were obtained from NCT (Frederick, Maryland). All aspects of the experiments were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The research had prior approval from the CBER Animal Care- and-Use Committee. Groups of eight animals were housed with free access to exercise wheels (Penn-Plax, Inc., NY) according to previous descriptions (Allen et al., 2001). Each wheel with a diameter of 11.5 cm was fitted with a magnet and microcomputer that recorded the number of revolutions, speed and total distance (Cat Eye, Co, LTD, Osaka, Japan). At three-day increments animals were divided into groups or four then two per cage. Rooms were maintained with a 12 : 12 hr light/dark cycle, 20 — 22 ⁰C. Use of the wheels, feeding and drinking were ad libitum. Distance data was collected at 24 hr intervals.

Toxin injection procedure. Purified BoNT complexes were used in this study. BoNT/A (WAKO Chemicals, VA) was stored as a 1 μg / mL stock solution (2.0 x 10⁷ ipLD₅₀ / mg). BoNT/B and E (WAKO Chemicals, VA) were activated with trypsin and aliquoted as described previously (Ohishi and Sakaguchi, 1977; Keller et al., 1999). Potency of each was reported by the manufacturer to be 3.0 x 10⁷ and 1.0 x 10⁷ ipLD₅₀ per mg of BoNT/B and E, respectively. Actual potency after trypsin treatment was 2.7 x 10⁷ and 1.0 x 10⁷ LD₅₀ per mg of BoNT/B and BoNTVE. Aliquots of each toxin preparation were stored at— 80 ⁰C. Dilution of each toxin was performed on the day of the experiment using sterile physiological saline containing 0.5 % bovine serum albumin. Animals were lightly sedated with 2% halothane and the injection area was moistened with 70% isopropanol prior to administering toxin. Intramuscular injections of 30 μL were given directly into the largest, central region of the gastrocnemius (calf) muscle mass. Since the animals had been running for 10 — 14 days prior to injection, this injection volume did not cause visible swelling of the leg, as happened with non-running animals (Meunier et al., 2003). Because physical tissue trauma enhances the recovery rate from BoNT/ A, syringes with a 32-gauge diameter were used to reduce the likelihood of injury.

Statistical analysis. Compiled distances from three or more cages are represented as average determinations regardless of the number of animals within each cage. Error bars represent one standard deviation unit. Linear and non-linear regression analyses were performed using Prism 3.0 (Graphpad, San Diego, CA). In the case of recovery times, data during paralysis onset were excluded from regression analysis. Linear regression analysis of BoNT/A used the standard equation: y = mx + b. Nonlinear analysis used the following equations: (BoNT/A) y = (1 — exp(— k x t)) x M + c. Y is the distance (km); M, the plateau maximum was predicted at time (t), with a first-order rate constant, k or for (BoNT/B): y = B₀ + ((T₀-B₀)/l + 10^c). B₀ and T₀ represented the lower and upper limit values of the curve. The constant, C, was log (EC50 - 1) x H. Where EC₅₀, t and H represent the 50% recovery point at time, t, which was influenced by a cooperativity coefficient, H. RESULTS

Gastrointestinal symptoms were observed in response to i.m. injection of BoNT/A and B when the dose exceeded 0.7 LD₅₀. BoNT/A i.m. injection into the hind limb elicited diarrhea, whereas, BoNT/B reduced fecal accumulation within the cages, suggesting sub-lethal i.m. administered BoNT/A or B can selectively poison different autonomic neurons of the GI tract. Interestingly, BoNT/E toxicity was very sharp; animals did not display systemic signs of illness even at 0.9 LD50 units but all died within 18 hr if given 1.0 LD50. At all doses of BoNT/A, B or E tested, animals demonstrated an ability to move, eat, drink & groom regardless of the toxin type.

Animal housing was examined to optimize the distance and consistency of voluntary running activity. Mice were active almost exclusively during the 12 hr dark cycle (>95%) with only slight running displayed during the daytime. Initially eight animals, then subsequently smaller groups were tested. Housing three or more animals within a cage was counter-productive since individually each animal ran approximately five hours per night (Konhilas et al., 2004; Lightfoot et al., 2004; Irani et al., 2005), thus limiting wheel access during the 12 hr active period (Figure IA). This was resolved by placing two animals together, which eliminated aggressive competition that often developed within larger groups. When single animals were tested, running became sporadic with increasing variation (Figure IA). Based on these data, the experimental design consisted of two mice in a single cage; running distances were reported as the average between the two animals. Pairs of animals that ran consistently near six km per night were used for further experimentation (Figure IB) (Lightfoot et al., 2004; Irani et al., 2005). The running distances remained generally constant over a period of several days and were not affected by injection of saline or BoNT/A premixed with mouse antitoxin (Figure 1C). Injection of BoNTs into the hind limb reduced running distances when toxin was administered over a four- to fivefold range of sub-lethal doses. The effects caused by each toxin are described below in terms of onset of paralysis, duration of total paralysis (zero running) and the rate of recovery after total paralysis subsided.

Running distances were recorded for up to four weeks following injection of BoNT/A (Figure 2A). Paralysis on-set was similar for all doses tested with the maximum effect occurring within 48 hr of injection. At the lowest dose, 0.14 LD5₀ unit, the total running distance was reduced by 85%. Recovery began on the third day with gradual improvement leading to normal physical activity about seven days after injection. Higher doses of BoNIVA led to complete cessation of running that persisted for many days relative to toxin dosage. The rate of recovery following total paralysis was similarly influenced by higher amounts of toxin. The highest dose tested, 0.8 LD₅₀ units, produced eight to nine days of total inactivity followed by about 50% recovery (~3 km) 28 days later. Reducing BoNT/A by two-fold to 0.4 LD₅₀ units resulted in only one day of about 95% inhibition of running and recovery began the following day. Full recovery was achieved within 15 days of the injection of 0.4 LD50 units.

Regression analysis using a single exponential rate constant predicted that 90% recovery required 11, 24.1, 45.3 or 61 days post-injection for the four doses of toxin tested, which overestimated the actual recovery times observed for the two lowest BoNT/A doses. Since recovery from the highest dose of BoNT/ A appeared to follow a nearly linear path from day 12 to day 30, the recovery profile for the BoNT/A poisoning was evaluated using two linear equations overlapping at day 19. Although BoNT/A dosage clearly influences the duration of total paralysis, the ensuing recovery may entail a two-part process. Recovery proceeded with a faster, initial phase until 33 - 38% of normal running was achieved after three to seven days, depending on the dose of toxin. A second recovery phase was 2.4-fold slower than the initial phase (Figure 3C), which was too slow to allow full recovery from the highest dose of BoNT/A during the time of the experiment. However, extrapolating from the lower doses, full recovery from 0.8 LD₅₀ units would require 48 ± 8 days. Both linear rates of recovery were directly related to toxin dosage, and the analysis predicts that each rate becomes zero if a lethal amount of toxin were injected (Figure 3C).

Similar experiments using BoNT/B and E yielded results that were generally distinct from BoNT/A (Figure 2B and 2C). BoNT/B, like BoNT/A, produced a dose- dependent duration of total paralysis. However, once total paralysis had subsided, the recovery phase proceeded at approximately the same rate for all BoNT/B concentrations. Unlike BoNT/A, recovery from BoNT/B was best represented by a sigmoidal curve representing early, middle and late phases of recovery that begin within several days of disease on-set. Full recovery to 100% normal running activity occurred eight to nine days after total paralysis regardless of toxin concentration. The lowest concentration of BoNT/B tested (0.3 LD50 units) demonstrated a delayed onset time, which peaked four to five days post-injection with about 90% blockade in running; the higher doses of BoNT/B completely inhibited running two to three days after injection. As with BoNT/A, the duration of maximum paralysis from BoNT/B was dose-dependent but unlike BoNT/A, the recovery rate was influenced to a lesser extent by BoNT/B dose (Figure 2B).

Injection of BoNTVE produced short-term paralysis relative to either BoNT/A or B (Figure 2C). The concentration of BoNT/E did not influence the onset of muscle weakness nor did it alter the duration of paralysis. Toxin action persisted for up to seven days after injection regardless of the BoNT/E dose. This is similar to other studies that have examined BoNT/E in cultured neurons (Keller et al., 1999) and in rat muscle tissue (Sellin et al., 1983a; Adler et al., 2001), where recovery was complete within two weeks. In addition to confirming the brevity of duration, mouse running demonstrated that paralysis on-set, duration of maximal blockade and the rate of recovery were generally independent of BoNT/E dose. The degree of paralysis observed on the second day after injection was the only symptom clearly influenced by the amount of toxin.

Graphical depiction of the duration of total paralysis versus toxin concentration shows an exponential relationship between BoNT/A concentration and muscle inactivity (Figure 4). In comparison, the duration of paralysis caused by BoNT/B is linear with toxin concentration producing similar paralysis to BoNT/A when both toxins were at relatively low levels but at higher BoNT/B concentrations, paralysis was brief compared to BoNT/A. BoNT/E, unlike BoNT/A and B, had practically no effect on the time of muscle inactivity. These observations are consistent with clinical observations of pharmaceutical-grade BoNT injections used to treat several medical disorders in human patients (Sloop et al., 1997; Eleopra et al., 1998).

To examine if BoNTs can influence each other when simultaneously injected, combinations were tested and the results compared to previously reported observations (Sloop et al., 1997; Eleopra et al., 1998)BoNT/A (0.5 LD₅₀ units) was combined with either BoNT/B or E. This dose of BoNT/A, as suggested by Figure 2 and Figure 4, produced maximum paralysis for two to three days (Figure 5). Injection of BoNT/B at 0.3 LD50 units produced paralysis developing over several days leading to about a 90% reduction of running four days after injection but did not cause complete paralysis. Animals recovered fully within two to three weeks from the individual injections of BoNTYB and /A, respectively. In contrast, the AB mixture caused complete paralysis in less than 12 hr compared to the slower on-set times observed for the separate toxins (Figure 2 and 5). Running activity from the AB combination remained suppressed by more than 90% for seven days followed by biphasic recovery over several weeks. Running activity returned to about half of the normal distance at the end of four weeks. A similar experiment was performed by mixing BoNT/A and E (Figure 5). In this situation the on-set of paralysis was not affected by the combination. Recovery from BoNT/E was complete within seven days but the extent of the recovery from the AE combination was incomplete compared to BoNTYA alone, and reached a plateau where running distances remained depressed one month later. Paralysis caused by BoNTYA alone had subsided completely in three weeks.

DISCUSSION

Botulism has long been associated with food poisoning where the primary symptom, muscle paralysis, develops slowly over several days and may persist for many weeks or months. The disease is derived from the ingestion of BoNT, which eventually enters nerve terminals where it proteolytically cuts certain nerve terminal proteins controlling synaptic vesicle function (Sloop et al., 1997; Eleopra et al., 1998). This, in turn, causes a gradual blockade of neurotransmission, which stops muscle function. Death may result depending on the severity of symptoms and the timing of medical intervention, however, should a patient survive beyond the initial onset, recovery is very likely. The duration of the recovery phase in human cases can range from several days to many months (Ball et al., 1979; Colebatch et al., 1989; Shapiro et al., 1998). The precise reason(s) for the variable recovery times are not clearly known because natural occurrences of botulism rarely reveal when the poisoning occurred or how much toxin was consumed.

In the current study, running activity from mice injected with BoNT/A, B or E has provided useful data for understanding how each neurotoxin causes botulism and how recovery proceeds after poisoning. Complete and partial paralysis produced by BoNT/A, B and E persisted in the order of A>B>E (Figure 4) in agreement with results compiled from previous studies (Ball et al., 1979; Colebatch et al., 1989; Shapiro et al., 1998). Previously, ex vivo or in vivo nerve-muscle analysis has been used to examine the duration of BoNT symptoms but these studies tended to produce minimal data because of the inherent difficulties of the experimental approach. Generally, this required large numbers of animals which were monitored at broad time intervals after poisoning (Simpson, 1973; Simpson, 1978; Polak et al., 1981 ; Sellin, 1981; Sellin and Thesleff, 1981; Adler et ah, 2000; Adler et al., 2001), thus greatly limiting the conditions that have been examined. Those methods have not been used to assess variable BoNT/A, B or E dosages on muscle paralysis, for example, or to measure the ensuing recovery on a daily basis. Running activity in response to i.m. injection allowed simultaneous testing of many dosages from the three BoNTs and demonstrated that each toxin has distinct characteristics that differentially influence the duration of paralysis and the rate of recovery. The persistence of symptoms from BoNT/A poisoning is exacerbated with small increases in toxin exposure, whereas paralysis caused by BoNT/B and /E is influenced relatively less by toxin dosage. Whether BoNT/A recovery encompasses a single exponential rate or a two-part linear process is unclear at this time, however, full recovery from BoNT/A poisoning is considered to entail several steps, each proceeding with a separate rate. Some of these factors are: the rate of toxin degradation within poisoned nerve terminals, the rate of repair of the poisoned nerve terminals, the rate of new (non-poisoned) nerve terminal growth, recruitment of resting nerve terminals that escaped poisoning and a return of normal muscle mass. In this study the multifunctional requirements for recovery from BoNT/A seem to be distinguished from the simpler recovery profiles for BoNT/B and /E but the individual processes are not clearly depicted in the data.

The running assay has revealed several unknown characteristics of BoNT/A, B and E while confirming several previously known properties. The similarities to human cases of botulism provide continued support for the mouse model as a good predictor of toxin action in human tissue. Because this approach can quantitatively monitor recovery, the running assay can benefit in vivo research that is aimed at developing or screening BoNT antagonists to stimulate the return of normal muscle function after poisoning. Although this assay relies upon mice, the separate findings for recovery from BoNT/A and B are consistent with the duration of paralysis produced with controlled, low-dose injections used to treat human muscle disorders. In this sense, the different recovery phases observed for the individual toxins in the present study have the potential to improve our understanding of how each BoNT acts in human tissue, which consequently may help develop alternative or improved BoNT regimens.

This research was extended to examine the effect of BoNT combinations. Two previous in vivo studies using rodents have reported opposite findings for BoNT/A & E mixtures (Adler et al., 2001 ; Meunier et al., 2003). To attempt to provide additional data, running distances were measured after injecting combinations of BoNT/ A & B or BoNT/A & E. When BoNT/A was combined with BoNT/B the resulting paralysis developed more rapidly and persisted much longer than the individually injected BoNTs (Figure 5A). Combination of BoNT/ A and E in similar ratios prolonged paralysis compared to BoNT/A alone. This final observation is in agreement with Adler et. al. (Adler et al., 2001) where BoNT/ A and E were found to act independently following injection of A, then E into rat extensor digitorum longus (EDL) muscle. In that study, muscle contractions were elicited by electrical stimulation of the poisoned nerve - muscle group. Muscle force had returned to normal within one month after injection of BoNT/E but was 70% depressed following BoNT/A injection, and about 80% depressed by the AE combination. This trend is identical to the running data presented here. The in vivo results from both studies support data obtained from cultured neurons showing that BoNT/A and E do not influence each other at the molecular level within nerve terminals (Keller et al., 1999).

Results from human volunteers (Eleopra et al., 1998) suggest that co-injection of BoNT/A & E into the extensor digitorum brevis (EDB) muscle resulted in a shortened duration of paralysis. The EDB is a slow-twitch muscle group, whereas, the EDL tested in rats and the gastrocnemius group tested here are fast-twitch muscle groups. The duration of paralysis in each type of muscle is strongly influenced by innervation properties. Comparison studies concluded that the slow-twitch soleus muscle in mice regains normal contractile properties within several days after injection of BoNT/A but the fast-twitch gastrocnemius muscle group recovered partially over several weeks (Keller et al., 1999). The present study targeted the fast-twitch gastrocnemius mouse muscle, and produced results similar the rat EDL muscle study, both results are consistent with these muscle groups having fast-twitch electrical properties. The contrasting results between human and rodent studies may entail species variation, tissue types, size of muscle groups and experimental designs. Rats are naturally resistant to BoNT/B so BoNT/A & B combinations were not attempted in the rat EDL study, and were not mentioned in the human EDB study. The apparent additive effect observed in mice by mixing sub-optimal doses of BoNT/A and BoNT/B has not been reported elsewhere and may represent compounded disruption of synaptic vesicle trafficking due to each toxin cleaving different synaptic proteins (Keller et al., 1999).

Comparison of the three BoNT serotypes commonly associated with causing human disease, shows that running activity decreases in direct proportion to the amount of toxin injected. Furthermore, each of the three BoNTs demonstrates distinct biological effects on both the duration of paralysis and the manner in which recovery proceeds. BoNT/A concentration affects at least two aspects of muscle function: duration of maximum paralysis followed by a prolonged and relatively slow rate of recovery. In contrast, BoNT/B concentration primarily influences the duration of maximum paralysis while slightly prolonging the subsequent return of normal muscle action. BoNT/E concentration does not greatly affect the duration of maximum paralysis or the time to recovery but reduces only the extent of muscle activity after the initial poisoning. These distinct variations represent biochemical properties unique to each BoNT, and suggest that each toxin influences different biological process(es) required for a return of normal synaptic function.

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Incorporation by Reference

The contents of all references, patents, pending patent applications and published patents, cited throughout this application are hereby expressly incorporated by reference.

Equivalents

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

What is claimed is:

1. A method for treating a subject having a disease, disorder or condition characterized by unwanted or excessive presynaptic neuronal activity or secretion, comprising: administering to the subject an effective amount of botulinum toxin A and botulinum toxin B; thereby treating the subject.

2. The method of claim 1 , wherein the botulinum toxin A and botulinum toxin B are administered simultaneously.

3. The method of claim 2, wherein the botulinum toxin A and botulinum toxin B are in an admixture.

4. The method of claim 1 , wherein approximately equal amounts of botulinum toxin A and botulinum toxin B are administered.

5. The method of claim 1, wherein the disease, disorder or condition is selected from the group consisting of cervical dystonia, strabismus, blephrasopasm, VII nerve disorders, muscle spasticity, troticollois, hyperactive facial lines, Parkinson's disease, cosmetic wrinkles, hyperhidrosis, muscle spasms, migraines, tension headaches, anal fissures, glabeller furrows, hyperkinetic lines, hypertonia, thyroiditis, prostate enlargement, hyperparathyroidism, cardiovascular disease, neuromuscular disease, excessive sweating, juvenile cerebral palsy, epilepsy, sinus headache, fibromyalgia, urologic disorders, obsessive compulsive disorder, inner ear disorders, orofacial dyskinesia, athetosis, chorea, and diabetes.

6. The method of claim 5, wherein the disease, disorder or condition is hyperactive facial lines.

7. The method of claim 6, wherein the hyperactive facial lines are of the upper face, forehead, periorbital, paranasal, perioral, lower facial, or nasolabial regions.

8. The method of claim 1 , wherein the botulinum toxin A and botulinum toxin B are administered intramuscularly, subcutaneously, or transdermally.

9. The method of claim 1, wherein the subject is human.

10. The method of claim 1, wherein the botulinum toxins are administered in an amount between about 1 unit and about 40,000 units each.

11. A method of treating a subject having a neuromuscular disease or disorder, comprising: administering to the subject an effective amount of botulinum toxin A and botulinum toxin B; thereby treating the subject.

12. The method of claim 11, wherein the botulinum toxin A and botulinum toxin B are administered simultaneously.

13. The method of claim 12, wherein the botulinum toxin A and botulinum toxin B are in an admixture.

14. The method of claim 11, wherein approximately equal amounts of botulinum toxin A and botulinum toxin B are administered.

15. The method of claim 11, wherein the botulinum toxin A and botulinum toxin B are administered intramuscularly, subcutaneously, or transdermally.

16. The method of claim 1, wherein the botulinum toxins are administered in an amount between about 1 unit and about 40,000 units each.

17. The method of claim 11, wherein the subject is human.

18. A method of treating a subject having a cervical dystonia comprising: simultaneously administering to the subject an effective amount of botulinum toxin A and botulinum toxin B; thereby treating the subject.

19. The method of claim 18, wherein the botulinum toxin A and botulinum toxin B are in an admixture.

20. The method of claim 18, wherein approximately equal amounts of botulinum toxin A and botulinum toxin B are administered.

21. The method of claim 18, wherein the botulinum toxin A and botulinum toxin B are administered intramuscularly, subcutaneously, or transdermally.

22. The method of claim 18, wherein the botulinum toxins are administered in an amount between about 1 unit and about 40,000 units each.

23. The method of claim 18, wherein the subject is human.

24. A method of treating a subject having cosmetic wrinkles comprising: administering to the subject an effective amount of botulinum toxin A and botulinum toxin B; thereby treating the subject.

25. The method of claim 24, wherein the botulinum toxin A and botulinum toxin B are administered simultaneously.

26. The method of claim 25, wherein the botulinum toxin A and botulinum toxin B are in an admixture.

27. The method of claim 26, wherein approximately equal amounts of botulinum toxin A and botulinum toxin B are administered.

28. The method of claim 24, wherein the botulinwn toxins are administered in an amount between about 1 unit and about 40,000 units each.

29. The method of claim 24, wherein the subject is human.

30. A pharmaceutical composition comprising botulinum toxin A and botulinum toxin B and a pharmaceutically acceptable carrier.

31. The pharmaceutical composition of claim 30, wherein the botulinum toxin A and botulinum toxin B are in approximately equal amounts.

32. The pharmaceutical composition of claim 30, wherein the botulinum toxin B is protease activated.

33. The pharmaceutical composition of claim 30, further comprising a stabilizing agent.

34. A kit comprising the pharmaceutical composition of claim 30, 31, or 32 and instructions for use.

35. A kit comprising botulinum toxin A and botulinum toxin B and instructions for use.

36. The kit of claim 35, wherein the botulinum toxin A and botulinum toxin B are administered simultaneously.

37. A kit for the treatment of cosmetic wrinkles comprising botulinum toxin A and botulinum toxin B and instructions for use.

38. The kit of claim 37, further comprising a protease to activate the botulinum toxin B.

39. The kit of claim 38, wherein the protease is tryspin.

40. The kit of any one of claims 34-39, further comprising one or more syringes.

41. An assay for monitoring the efficacy of one or more neurotoxin on an animal comprising the steps of: administering one or more neurotoxins to an animal; determining the distance the animal travels over a period of time; thereby monitoring the efficacy of a neurotoxin on the animal.

42. The assay of claim 41 , wherein the animal is a rat or mouse.

43. The assay of claim 41 , wherein the neurotoxin is administered to the animals hind legs.

44. The assay of claim 41, wherein the animal runs on a wheel.

45. The assay of claim 41, wherein the distance the animal travels is the distance the animal travels at night.