Modified BoNT / A for use in treating cervical dystonia

Abstract

The present invention is directed to the treatment of cervical dystonia with a modified botulinum neurotoxin A (BoNT / A), comprising a modified BoNT / A for use in treating cervical dystonia, the modified BoNT / A being administered by intramuscular injection to an affected neck muscle of an affected individual, the modified BoNT / A being administered in a unit dose of 750 pg to 17,000 pg of modified BoNT / A, at least a single unit dose being administered to the affected neck muscle, the total dose of modified BoNT / A administered during treatment being up to 170,000 pg, the modified BoNT / A comprising a BoNT / A light chain and translocation domain and a BoNT / B receptor binding domain (H C Also provided are related methods, uses, unit dosage forms and kits.

Description

[Technical field]

[0001] The present invention relates to the treatment of cervical dystonia. [Background technology]

[0002] Cervical dystonia (also known as spasmodic torticollis) is a chronic movement disorder of the nervous system that is typically extremely painful. It causes affected individuals to involuntarily bend their neck to the left, right, up, and / or down. Both agonist and antagonist muscles may contract simultaneously during dystonic movements.

[0003] The disorder typically presents with relatively mild symptoms, such as a non-visible head tremor, for several months after onset. Other early / progressive symptoms may include sudden head turning, pulling, and / or tilting movements. Additionally, early / progressive symptoms typically include persistent / prolonged involuntary head positioning. Involuntary spasms of the neck muscles tend to increase in frequency and intensity over time before reaching a plateau. Affected individuals with cervical dystonia may also experience muscle hypertrophy, neck pain, dysarthria, and / or tremor.

[0004] Symptoms of cervical dystonia may affect any of the affected neck muscles, and head postures may be diverse. Typically, the most common abnormal posture associated with cervical dystonia is a twisting of the chin toward the shoulder such that the head rotates to the side (torticollis). Other abnormal postures associated with cervical dystonia may include anterior neck flexion, where the head tilts forward, posterior neck flexion, where the head tilts back, or lateral neck flexion, where the head tilts to one side. The head may also shift over the shoulder in a sagittal anterior deviation (movement forward) or posterior sagittal deviation (movement backward). Most commonly, however, cervical dystonia presents as a complex condition in which affected individuals exhibit various angles of head movement.

[0005] Traditional treatment options include oral medications (such as dopamine blockers), deep brain stimulation, botulinum neurotoxin, and selective surgical denervation of the nerves that cause muscle contractions. Traditional oral medications are associated with many serious side effects, while deep brain stimulation and surgical denervation are invasive, carry risk of complications, and / or may be ineffective.

[0006] An example of a conventional botulinum neurotoxin serotype A (BoNT / A) treatment for cervical dystonia is Dysport®, a pharmaceutical product that contains the drug substance BoNT / A hemagglutinin complex (BTX-A-HAC), which is isolated and purified from a strain of Clostridium botulinum type A. Other medical BoNT / A products that are naturally produced by Clostridium botulinum are also commercially available (e.g., BOTOX® and XEOMIN®).

[0007] By paralyzing the dystonia antagonist muscles, BoNT / A may allow the agonist muscles to move freely. More specifically, BoNT / A selectively inhibits the release of acetylcholine from presynaptic nerve terminals, blocking cholinergic transmission at the neuromuscular junction, causing muscle contraction and decreased muscle tone, and relaxing the injected muscle. However, currently available BoNT / A products have a duration of action of approximately 12 to 14 weeks, during which time new nerve terminals sprout and nerve function returns to normal, and the original symptoms recur. As a result, injections must be repeated periodically to maintain efficacy. Thus, given the chronicity of the condition and the long-term nature of the required treatment, the frequency of BoNT / A injections is an important consideration for the treatment of cervical dystonia. Indeed, it impacts the direct and indirect health costs for the affected individual and caregivers, the logistics of injections in hospitals and clinics, and most importantly, the quality of life of the affected individual.

[0008] Dysport® is approved for the treatment of cervical dystonia with a maximum total dose of 1,000 units per treatment session (see FIG. 1). The clinician must administer Dysport® to the affected patient's neck muscles up to the upper limit of the standard total of 1,000 units per treatment session. The clinician is forced to make difficult choices while treating the affected patient. In other words, in conventional treatment plans, the clinician must find a balance between the relatively small total amount of BoNT / A that can be administered (1,000 units, required by the high toxicity of BoNT / A) and an amount that is effective against several different muscles. Thus, certain muscles are neglected while other muscles receive suboptimal amounts of BoNT / A, resulting in a suboptimal treatment.

[0009] Furthermore, conventional treatment regimens for cervical dystonia are complex and result in clinicians underdosing to avoid toxicity to the affected patient. Thus, there is a need for convenient, safe and effective single-dose units and corresponding guidance (including, for example, the number of injection sites per muscle) on the number of units that can be administered to the affected neck muscles during a treatment session without resulting in toxicity to the affected patient.

[0010] In conclusion, improved treatments for cervical dystonia are needed that allow for a personalized, patient-centered approach that avoids toxicity and provides long-lasting treatment (resulting in less frequent dosing), while tailoring treatment to a targeted clinical pattern that allows for different combinations of affected neck muscles to be injected depending on the distribution, degree, and severity of cervical dystonia.

[0011] The present invention overcomes one or more of the problems set forth above. Summary of the Invention

[0012] The present inventors have surprisingly found that modified BoNT / A is particularly useful for treating cervical dystonia. The modified BoNT / A comprises a BoNT / A light chain and translocation domain and a BoNT / B receptor binding domain (H CThe modified BoNT / A may comprise one or more modifications at surface-exposed amino acid residues that result in an increased net positive charge. The increased charge promotes electrostatic interactions between the polypeptide and anionic extracellular components, thereby promoting binding between the polypeptide and the cell surface. In turn, this also results in increased retention at the site of administration (reduced diffusion away) and / or increased duration of action (e.g., 6 to 9 months).

[0013] Advantageously, the modified BoNT / A has an improved safety profile when compared to an unmodified BoNT / A (e.g., Dysport®), which may be expressed by the increased safety margins described herein for the modified BoNT / A.

[0014] Based on the preclinical and clinical data herein (see Examples), it has been shown that a larger total dose of modified BoNT / A can be administered to an affected individual while achieving a similar safety profile to unmodified BoNT / A (e.g., Dysport®) at such a high dose. Thus, in treating cervical dystonia, more modified BoNT / A can be injected, and also into more neck muscles and locations, until a maximum total dose is reached. This is an important and advantageous discovery, improving cervical dystonia treatment and providing clinicians with a wider range of treatment options. The treatment may be improved in that it provides a more durable treatment (resulting in less frequent administration) and / or can be tailored to the affected individual, or results in an improved quality of life for the affected individual, when compared to treatment with unmodified BoNT / A (e.g., Dysport®). Thus, the treatment of the present invention is improved compared to conventional treatment regimes.

[0015] Furthermore, the present invention provides a convenient, safe and effective single unit dose as well as a total (maximum) dose that can be safely administered in a single treatment. The present invention also provides a guide to the number of times that the unit dose can be administered into the neck muscles (including, for example, the number of injection sites per muscle) without resulting in toxicity to the patient. Thus, the treatment of cervical dystonia according to the present invention is less complicated for the clinician, helping to avoid under-dosing and / or over-dosing. Furthermore, the treatment according to the present invention is much more satisfactory for the patient as it is better adapted to the needs of the patient when compared to conventional cervical dystonia treatments. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] In one aspect, the invention relates to a modified botulinum neurotoxin A (BoNT / A) for use in treating cervical dystonia, the modified BoNT / A being administered to an affected neck muscle of an affected individual by intramuscular injection, the modified BoNT / A being administered in a unit dose of between 750 pg and 17,000 pg of modified BoNT / A, at least a single unit dose being administered to the affected neck muscle, the total dose of modified BoNT / A administered during treatment being up to 170,000 pg, the modified BoNT / A comprising a BoNT / A light chain and translocation domain and a BoNT / B receptor binding domain (H C The present invention provides a modified BoNT / A comprising:

[0017] In a related aspect, the invention provides a modified BoNT / A for use in treating cervical dystonia in an affected individual for a longer duration than treatment with an unmodified BoNT / A (e.g., SEQ ID NO: 2), wherein the modified BoNT / A is administered by intramuscular injection to an affected neck muscle of the affected individual, the modified BoNT / A is administered in a unit dose of 750 pg to 17,000 pg of modified BoNT / A, at least a single unit dose is administered to the affected neck muscle, with a total dose administered during treatment of up to 170,000 pg of modified BoNT / A, and the modified BoNT / A comprises a BoNT / A light chain and translocation domain and a BoNT / B receptor binding domain (H C The present invention provides a modified BoNT / A comprising:

[0018] The term "treating the affected individual with cervical dystonia for a longer duration than when treated with unmodified BoNT / A" may mean that one or more symptoms of cervical dystonia in the affected individual are alleviated for a longer duration following administration of the modified BoNT / A of the present invention as compared to administration of unmodified BoNT / A. The duration of action may be at least 1.25-fold, 1.5-fold, 1.75-fold, 2.0-fold, or 2.25-fold. The duration of action of the modified BoNT / A may be between 6 and 9 months. For example, the duration of action may be at least: 4.5 months (from onset), 5.0 months, 5.5 months, 6 months, 6.5 months, 7.0 months, 7.5 months, 8.0 months, 8.5 months, or 9.0 months. In certain embodiments, the duration of action may be greater than 9.0 months. The alleviation may be determined by comparison with an equivalent control group with similar symptoms treated with unmodified BoNT / A. During a period in which the severity of one or more symptoms in the control group is substantially the same (e.g., the same) as before unmodified BoNT / A treatment, affected individuals treated with a modified BoNT / A according to the present invention may show at least a 5%, 10%, 25%, or 30% improvement in the equivalent one or more symptoms when compared to the severity of one or more symptoms before treatment with the modified BoNT / A. The unmodified BoNT / A is preferably SEQ ID NO:2, which exists in a two-chain form.

[0019] In one aspect, the invention provides a method for treating cervical dystonia, comprising administering a modified BoNT / A by intramuscular injection to an affected neck muscle of an affected individual, wherein the modified BoNT / A is administered in a unit dose of between 750 pg and 17,000 pg of modified BoNT / A, wherein at least a single unit dose is administered to the affected neck muscle, with a total dose administered during treatment of up to 170,000 pg of modified BoNT / A, wherein the modified BoNT / A comprises a BoNT / A light chain and translocation domain and a BoNT / B receptor binding domain (H C and a domain.

[0020] In a related aspect, the invention provides a method of treating cervical dystonia in an affected individual for a longer duration than treatment with unmodified BoNT / A (e.g., SEQ ID NO: 2), the method comprising administering modified BoNT / A by intramuscular injection to an affected neck muscle of the affected individual, the modified BoNT / A being administered in a unit dose of 750 pg to 17,000 pg of modified BoNT / A, at least a single unit dose being administered to the affected neck muscle, with a total dose administered during treatment of up to 170,000 pg of modified BoNT / A, the modified BoNT / A comprising a BoNT / A light chain and translocation domain and a BoNT / B receptor binding domain (H C and a domain.

[0021] In one aspect, the invention relates to the use of a modified botulinum neurotoxin A (BoNT / A) in the manufacture of a medicament for the treatment of cervical dystonia, wherein the modified BoNT / A is administered by intramuscular injection to an affected neck muscle of an affected individual, the modified BoNT / A being administered in a unit dose of 750 pg to 17,000 pg of modified BoNT / A, at least a single unit dose being administered to the affected neck muscle, with a total dose administered during treatment being up to 170,000 pg of modified BoNT / A, the modified BoNT / A comprising a BoNT / A light chain and translocation domain and a BoNT / B receptor binding domain (H C domain) and provide for use.

[0022] In a related aspect, the invention relates to the use of a modified BoNT / A (e.g., SEQ ID NO: 2) in the manufacture of a medicament for treating cervical dystonia in an affected individual for a longer duration than treatment with unmodified BoNT / A, wherein the modified BoNT / A is administered by intramuscular injection to an affected neck muscle of the affected individual, the modified BoNT / A is administered in a unit dose of 750 pg to 17,000 pg of modified BoNT / A, at least a single unit dose is administered to the affected neck muscle, with a total dose administered during treatment being up to 170,000 pg of modified BoNT / A, and the modified BoNT / A comprises a BoNT / A light chain and translocation domain and a BoNT / B receptor binding domain (H C domain) and provide for use.

[0023] The unit dose may be 750 pg to 17,000 pg of modified BoNT / A, the modified BoNT / A comprising a BoNT / A light chain and translocation domain and a BoNT / B receptor binding domain (H C The upper limit of the unit dose range can be 16,500, 15,500, 14,500, 13,500, 12,500, 11,500, 10,500, 9,500, 8,500, 7,500, 6,500, 5,500, 4,500, 3,500, 2,500, 2,250, 2,000, 1,500, 1,250, 1,000, 750, or 500 pg of modified BoNT / A, and preferably, the upper limit is 16,000 pg. The lower limit of the unit dose range may be 800 pg, 850 pg, 950 pg, 1,000 pg, 1,500 pg, 1,750 pg, 2,000 pg, 2,500 pg, 3,000 pg, 3,500 pg, 4,000 pg, 4,500 pg, or 5,000 pg of modified BoNT / A, preferably the lower limit is 1,000 pg. Preferably, the unit dose of modified BoNT / A is 1,000 pg to 16,000 pg of modified BoNT / A, for example 950 pg to 1,250 pg, 1,750 pg to 2,250 pg, or 8,000 pg to 12,000 pg. Preferably, the unit dose of the modified BoNT / A may be 1,000, 2,000, 3,000, 8,000, or 16,000 pg.

[0024] The total dose administered in practicing the therapeutic regimen of the present invention may be up to 170,000 pg of modified BoNT / A, which comprises the BoNT / A light chain and translocation domain, and the BoNT / B receptor binding domain (H CIn other words, the total amount of modified BoNT / A administered in a given treatment session may be up to 170,000 pg. The total dose may be up to 165,000, 160,000, 140,000, 110,000, 100,000, 90,000, 80,000, 70,000, 60,000, 50,000, 40,000, 30,000, 28,000, 25,000, 20,000, 15,000, 14,000, 10,000, 8,000, 7,000, or 5,000 pg. Preferably, the total dose is up to 160,000 pg of modified BoNT / A, for example, the total dose can be up to 7,000, 10,000, 14,000, 20,000, 28,000, 30,000, 80,000, or 160,000 pg. The total dose may be at least 900, 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 7,500, 10,000, 12,500, 13,000, 14,000, 15,000, 20,000, 25,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000, 100,000, 120,000, or 150,000 pg. Preferably, the total dose is at least 1,500 pg, more preferably at least 2,000 pg of modified BoNT / A, e.g., at least 5,000 pg. The total dose may be 5,250 pg to 170,000 pg, preferably 7,000 pg to 160,000 pg. For example, the total dose may be 6,000 pg to 30,000 pg, such as 6,000 to 15,000 pg, or 13,000 to 29,000 pg. More preferably, the total dose is 10,000 pg to 160,000 pg. Preferably, the total dose may be 7,000 pg, 10,000 pg, 14,000 pg, 20,000 pg, 28,000 pg, 30,000 pg, 80,000 pg, or 160,000 pg.

[0025] Thus, the unit dose may be between 750 pg and 17,000 pg of modified BoNT / A, the modified BoNT / A comprising a BoNT / A light chain and translocation domain and a BoNT / B receptor binding domain (H C domain), and the total administered dose in practicing the treatment regimen of the present invention may be up to 170,000 pg. The unit dose may be 1,000 pg and the total dose may be up to 7,000 pg. The unit dose may be 1,000 pg and the total dose may be up to 10,000 pg. The unit dose may be 1,000 pg and the total dose may be up to 14,000 pg. The unit dose may be 2,000 pg and the total dose may be up to 14,000 pg. The unit dose may be 2,000 pg and the total dose may be up to 20,000 pg. The unit dose may be 2,000 pg and the total dose may be up to 28,000 pg. The unit dose may be 3,000 pg and the total dose may be up to 30,000 pg. The unit dose may be 8,000 pg and the total dose may be up to 80,000 pg. The unit dose may be 16,000 pg and the total dose may be up to 160,000 pg.

[0026] Thus, the unit dose may be between 750 pg and 17,000 pg of modified BoNT / A, the modified BoNT / A comprising a BoNT / A light chain and translocation domain and a BoNT / B receptor binding domain (H Cdomain), and the total administered dose in practicing the treatment regimen of the present invention may be up to 170,000 pg. The unit dose may be 1,000 pg and the total dose may be 7,000 pg. The unit dose may be 1,000 pg and the total dose may be 10,000 pg. The unit dose may be 1,000 pg and the total dose may be 14,000 pg. The unit dose may be 2,000 pg and the total dose may be 14,000 pg. The unit dose may be 2,000 pg and the total dose may be 20,000 pg. The unit dose may be 2,000 pg and the total dose may be 28,000 pg. The unit dose may be 3,000 pg and the total dose may be 30,000 pg. The unit dose may be 8,000 pg and the total dose may be 80,000 pg. The unit dose may be 16,000 pg and the total dose may be 160,000 pg.

[0027] In one aspect, the invention relates to a modified botulinum neurotoxin A (BoNT / A) for use in treating cervical dystonia, the modified BoNT / A being administered to an affected neck muscle of an affected individual by intramuscular injection, the modified BoNT / A being administered as a unit dose of between 31 units and 707 units of modified BoNT / A, where 1 unit is the calculated median lethal dose (LD ) in mice. 50 ), wherein at least a single unit dose is administered to the affected neck muscle, with a total dose administered during treatment of up to 7,070 units of modified BoNT / A, and wherein the modified BoNT / A comprises a BoNT / A light chain and translocation domain and a BoNT / B receptor binding domain (H C The present invention provides a modified BoNT / A comprising:

[0028] In a related aspect, the invention provides a modified BoNT / A for use in treating cervical dystonia in an affected individual for a longer duration than treatment with an unmodified BoNT / A (e.g., SEQ ID NO: 2), wherein the modified BoNT / A is administered by intramuscular injection to an affected neck muscle of the affected individual; The modified BoNT / A is administered as a unit dose ranging from 31 units to 707 units of modified BoNT / A, where 1 unit is the calculated median lethal dose (LD 50 ), wherein at least a single unit dose is administered to the affected neck muscle, with a total dose administered during treatment of up to 7,070 units of modified BoNT / A, and wherein the modified BoNT / A comprises a BoNT / A light chain and translocation domain and a BoNT / B receptor binding domain (H C The present invention provides a modified BoNT / A comprising:

[0029] In one aspect, the invention provides a method for treating cervical dystonia, the method comprising administering a modified BoNT / A to an affected neck muscle of an affected individual by intramuscular injection, the modified BoNT / A being administered as a unit dose of between 31 units and 707 units of modified BoNT / A, where 1 unit is the calculated median lethal dose (LD ) in mice. 50 ), wherein at least a single unit dose is administered to the affected neck muscle, with a total dose administered during treatment of up to 7,070 units of modified BoNT / A, and wherein the modified BoNT / A comprises a BoNT / A light chain and translocation domain and a BoNT / B receptor binding domain (H C and a domain.

[0030] In a related aspect, the invention provides a method of treating cervical dystonia in an affected individual for a longer duration than treatment with an unmodified BoNT / A (e.g., SEQ ID NO:2), the method comprising administering modified BoNT / A by intramuscular injection to an affected neck muscle of the affected individual; The modified BoNT / A is administered as a unit dose of 31 units to 707 units of modified BoNT / A, where 1 unit is the calculated median lethal dose (LD) for mice. 50 ), wherein at least a single unit dose is administered to the affected neck muscle, with a total dose administered during treatment of up to 7,070 units of modified BoNT / A, and wherein the modified BoNT / A comprises a BoNT / A light chain and translocation domain and a BoNT / B receptor binding domain (H C and a domain.

[0031] In one aspect, the invention relates to the use of a modified botulinum neurotoxin A (BoNT / A) in the manufacture of a medicament for the treatment of cervical dystonia, wherein the modified BoNT / A is administered by intramuscular injection to an affected neck muscle of an affected individual, the modified BoNT / A being administered in a unit dose of between 31 units and 707 units of modified BoNT / A, where 1 unit is the calculated median lethal dose (LD ) in mice. 50 ), wherein at least a single unit dose is administered to the affected neck muscle, with a total dose administered during treatment of up to 7,070 units of modified BoNT / A, and wherein the modified BoNT / A comprises a BoNT / A light chain and translocation domain and a BoNT / B receptor binding domain (H C domain) and provide for use.

[0032] In a related aspect, the invention relates to the use of a modified BoNT / A in the manufacture of a medicament for treating cervical dystonia in an affected individual for a longer duration than treatment with an unmodified BoNT / A (e.g., SEQ ID NO: 2), wherein the modified BoNT / A is administered by intramuscular injection to an affected neck muscle of the affected individual, the modified BoNT / A being administered in a unit dose of between 31 units and 707 units of modified BoNT / A, where 1 unit is the calculated median lethal dose (LD ) in mice. 50 ), wherein at least a single unit dose is administered to the affected neck muscle, with a total dose administered during treatment of up to 7,070 units of modified BoNT / A, and wherein the modified BoNT / A comprises a BoNT / A light chain and translocation domain and a BoNT / B receptor binding domain (H C domain) and provide for use.

[0033] The unit dose may be 31 units to 707 units of modified BoNT / A, the modified BoNT / A comprising a BoNT / A light chain and translocation domain and a BoNT / B receptor binding domain (H CThe upper limit of the unit dose range may be 700, 650, 600, 550, 500, 450, 400, 350, 300, 250, 200, 150, 100, 95, 90, 85, 65, 60, 55, 50, or 31 units of modified BoNT / A, preferably the upper limit is 666 units. The lower limit of the unit dose range may be 35, 40, 45, 50, 60, 65, 70, 75, 80, 85, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, or 700 units of modified BoNT / A, preferably the lower limit is 42 units. Preferably, the unit dose of modified BoNT / A is between 42 and 666 units of modified BoNT / A, for example, between 40 and 50 units, between 70 and 95 units, or between 333 and 499 units. Preferably, the unit dose of modified BoNT / A may be 41.6 units, 83.2 units, 124.8 units, 332.8 units, or 665.6 units.

[0034] The total dose administered in practicing the treatment regimen of the present invention may be up to 7,070 units of modified BoNT / A, which comprises the BoNT / A light chain and translocation domain and the BoNT / B receptor (H CIn other words, the total amount of modified BoNT / A administered in a given treatment session may be up to 7,070 units. The total dose may be up to 7,000, 6,000, 5,000, 4,000, 3,500, 3,350, 3,000, 2,000, 1,500, 1,250, 1,175, 1,000, 900, 800, 700, 650, 625, 600, 575, 550, 500, 450, 425, 400, 350, 330, 300, 290, 250, or 200 units. Preferably, the total dose may be up to 6,660 units, for example the total dose may be up to 290, 425, 575, 600, 800, 1,000, 1,175, 1,250, 3,000, 3,350, 3,500, or 7,000 units. The total dose may be at least 35, 40, 50, 100, 150, 200, 250, 290, 300, 350, 400, 450, 500, 540, 550, 580, 600, 650, 700, 750, 800, 850, 900, 950, 1,000, 1,050, 1,500, 2,000, 3,000, 4,000, 5,000, 6,000, or 7,000 units. Preferably, the total dose may be at least 62 units, more preferably at least 83 units of modified BoNT / A, such as at least 208 units. The total dose may be 217 units to 7,070 units, preferably 294 units to 6,660 units. For example, the total dose administered may be 250 to 1,250 units or 540 units to 1200 units, such as 250 to 625 units. More preferably, the total dose administered is 420 units to 6,660 units. Preferably, the total dose administered may be 291 units, 416 units, 582 units, 832 units, 1,165 units, 1,248 units, 3,328 units, or 6,656 units.

[0035] Thus, the unit dose may be between 31 units and 707 units of modified BoNT / A, the modified BoNT / A comprising a BoNT / A light chain and translocation domain and a BoNT / B receptor binding domain (H Cdomain), and the total dose administered in practicing the treatment regimen of the invention may be up to 7,070 units. The unit dose may be 41.6 units and the total dose may be up to 291 units. The unit dose may be 41.6 units and the total dose may be up to 416 units. The unit dose may be 41.6 units and the total dose may be up to 582 units. The unit dose may be 83.2 units and the total dose may be up to 582 units. The unit dose may be 83.2 units and the total dose may be up to 832 units. The unit dose may be 83.2 units and the total dose may be up to 1,165 units. The unit dose may be 124.8 units and the total dose may be up to 1,248 units. The unit dose may be 332.8 units and the total dose may be up to 3,328 units. The unit dose may be 665.5 units and the total dose may be up to 6,656 units.

[0036] Thus, the unit dose may be between 31 units and 707 units of modified BoNT / A, the modified BoNT / A comprising a BoNT / A light chain and translocation domain and a BoNT / B receptor binding domain (H C domain), and the total dose administered in practicing the treatment regimen of the invention may be up to 7,070 units. The unit dose may be 41.6 units and the total dose may be 291 units. The unit dose may be 41.6 units and the total dose may be 416 units. The unit dose may be 41.6 units and the total dose may be 582 units. The unit dose may be 83.2 units and the total dose may be 582 units. The unit dose may be 83.2 units and the total dose may be 832 units. The unit dose may be 83.2 units and the total dose may be 1,165 units. The unit dose may be 124.8 units and the total dose may be 1,248 units. The unit dose may be 332.8 units and the total dose may be 3,328 units. The unit dose may be 665.5 units and the total dose may be 6,656 units.

[0037] In one aspect, the present invention provides a modified botulinum neurotoxin A (BoNT / A) for use in treating cervical dystonia, the modified BoNT / A being administered by intramuscular injection to an affected neck muscle of an affected individual, The modified BoNT / A is administered in a unit dose of 450 pg to 8,000 pg of modified BoNT / A; At least one unit dose is administered to the affected neck muscle; The total dose administered during treatment is up to 80,000 pg of modified BoNT / A, and The modified BoNT / A comprises a modification at one or more amino acid residues selected from: ASN886, ASN905, GLN915, ASN918, GLU920, ASN930, ASN954, SER955, GLN991, GLU992, GLN995, ASN1006, ASN1025, ASN1026, ASN1032, ASN1043, ASN1046, ASN1052, ASP1058, HIS1064, ASN1080, GLU1081, GLU1083, ASP1086, ASN1188, ASP1213, GLY1215, ASN1216, GLN1229, ASN1242, ASN1243, SER1274, and THR1277, the modification being selected from: (i) replacement of acidic surface-exposed amino acid residues with basic amino acid residues; (ii) replacement of acidic surface-exposed amino acid residues with uncharged amino acid residues; (iii) replacement of uncharged surface-exposed amino acid residues with basic amino acid residues; (iv) insertion of a basic amino acid residue; and (v) Deletion of acidic surface-exposed amino acid residues.

[0038] In a related aspect, the invention provides a modified BoNT / A for use in treating cervical dystonia in an affected individual for a longer duration than treatment with an unmodified BoNT / A (e.g., SEQ ID NO: 2), the modified BoNT / A being administered intramuscularly to an affected neck muscle of the affected individual. The modified BoNT / A is administered in a unit dose of 450 pg to 8,000 pg of modified BoNT / A; At least one unit dose is administered to the affected neck muscle; The total dose administered during treatment is up to 80,000 pg of modified BoNT / A. The modified BoNT / A comprises a modification at one or more amino acid residues selected from: ASN886, ASN905, GLN915, ASN918, GLU920, ASN930, ASN954, SER955, GLN991, GLU992, GLN995, ASN1006, ASN1025, ASN1026, ASN1032, ASN1043, ASN1046, ASN1052, ASP1058, HIS1064, ASN1080, GLU1081, GLU1083, ASP1086, ASN1188, ASP1213, GLY1215, ASN1216, GLN1229, ASN1242, ASN1243, SER1274, and THR1277, the modification being selected from: (i) replacement of acidic surface-exposed amino acid residues with basic amino acid residues; (ii) replacement of acidic surface-exposed amino acid residues with uncharged amino acid residues; (iii) replacement of uncharged surface-exposed amino acid residues with basic amino acid residues; (iv) insertion of a basic amino acid residue; and (v) Deletion of acidic surface-exposed amino acid residues.

[0039] In one aspect, the invention provides a method for treating cervical dystonia, the method comprising administering a modified BoNT / A by intramuscular injection to an affected neck muscle of an affected individual, The modified BoNT / A is administered in a unit dose of 450 pg to 8,000 pg of modified BoNT / A; At least one unit dose is administered to the affected neck muscle; The total dose of modified BoNT / A administered during treatment is up to 80,000 pg of modified BoNT / A; The modified BoNT / A comprises a modification at one or more amino acid residues selected from: ASN886, ASN905, GLN915, ASN918, GLU920, ASN930, ASN954, SER955, GLN991, GLU992, GLN 995, ASN1006, ASN1025, ASN1026, ASN1032, ASN1043, ASN1046, ASN1052, ASP1058, HIS1064, ASN1080, GLU1081, GLU1083, ASP1086, ASN1188, ASP1213, GLY1215, ASN1216, GLN1229, ASN1242, ASN1243, SER1274, and THR1277, the modification being selected from: (i) replacement of acidic surface-exposed amino acid residues with basic amino acid residues; (ii) replacement of acidic surface-exposed amino acid residues with uncharged amino acid residues; (iii) replacement of uncharged surface-exposed amino acid residues with basic amino acid residues; (iv) insertion of a basic amino acid residue; and (v) Deletion of acidic surface-exposed amino acid residues.

[0040] In a related aspect, the invention provides a method of treating cervical dystonia in an affected individual for a longer duration than treatment with an unmodified BoNT / A (e.g., SEQ ID NO:2), the method comprising administering modified BoNT / A by intramuscular injection to an affected neck muscle of the affected individual; wherein the modified BoNT / A is administered in a unit dose of between 450 pg and 8,000 pg of modified BoNT / A; At least one unit dose is administered to the affected neck muscle; the total dose administered during said treatment is up to 80,000 pg of modified BoNT / A; The modified BoNT / A includes modifications at one or more amino acid residues selected from the following: ASN886, ASN905, GLN915, ASN918, GLU920, ASN930, ASN954, STORAGE REGion955, GLN991, GLU992, GLN 995, ASN1006, ASN1025, ASN1026, ASN1032, ASN1043, ASN1046, ASN1052, ASP1058, HIS1064, ASN1080, GLU1081, GLU1083, ASP1086, ASN1188, ASP1213, GLY1215, ASN1216, GLN1229, ASN1242, ASN1243, SER1274, and THR1277, the modification being selected from: (i) replacement of acidic surface-exposed amino acid residues with basic amino acid residues; (ii) replacement of acidic surface-exposed amino acid residues with uncharged amino acid residues; (iii) replacement of uncharged surface-exposed amino acid residues with basic amino acid residues; (iv) insertion of a basic amino acid residue; and (v) Deletion of acidic surface-exposed amino acid residues.

[0041] In one aspect, the invention provides the use of a modified botulinum neurotoxin A (BoNT / A) in the manufacture of a medicament for the treatment of cervical dystonia, the modified BoNT / A being administered by intramuscular injection to an affected neck muscle of an affected individual; wherein the modified BoNT / A is administered in a unit dose of between 450 pg and 8,000 pg of modified BoNT / A; At least one unit dose is administered to the affected neck muscle; the total dose administered during said treatment is up to 80,000 pg of modified BoNT / A; The modified BoNT / A comprises a modification at one or more amino acid residues selected from: ASN886, ASN905, GLN915, ASN918, GLU920, ASN930, ASN954, SER955, GLN991, GLU992, GLN995, ASN1006, ASN1025, ASN1026, ASN1032, ASN1043, ASN1046, ASN1052, ASP1058, HIS1064, ASN1080, GLU1081, GLU1083, ASP1086, ASN1188, ASP1213, GLY1215, ASN1216, GLN1229, ASN1242, ASN1243, SER1274, and THR1277, the modification being selected from: (i) replacement of acidic surface-exposed amino acid residues with basic amino acid residues; (ii) replacement of acidic surface-exposed amino acid residues with uncharged amino acid residues; (iii) replacement of uncharged surface-exposed amino acid residues with basic amino acid residues; (iv) insertion of a basic amino acid residue; and (v) Deletion of acidic surface-exposed amino acid residues.

[0042] In a related aspect, the invention provides the use of a modified BoNT / A in the manufacture of a medicament for treating cervical dystonia in an affected individual for a longer duration than treatment with an unmodified BoNT / A (e.g., SEQ ID NO: 2), the modified BoNT / A being administered by intramuscular injection to an affected neck muscle of the affected individual; The modified BoNT / A is administered in a unit dose of 450 pg to 8,000 pg of modified BoNT / A; At least one unit dose is administered to the affected neck muscle; The total dose administered during treatment is up to 80,000 pg of modified BoNT / A. The modified BoNT / A comprises a modification at one or more amino acid residues selected from: ASN886, ASN905, GLN915, ASN918, GLU920, ASN930, ASN954, SER955, GLN991, GLU992, GLN 995, ASN1006, ASN1025, ASN1026, ASN1032, ASN1043, ASN1046, ASN1052, ASP1058, HIS1064, ASN1080, GLU1081, GLU1083, ASP1086, ASN1188, ASP1213, GLY1215, ASN1216, GLN1229, ASN1242, ASN1243, SER1274, and THR1277, the modification being selected from: (i) replacement of acidic surface-exposed amino acid residues with basic amino acid residues; (ii) replacement of acidic surface-exposed amino acid residues with uncharged amino acid residues; (iii) replacement of uncharged surface-exposed amino acid residues with basic amino acid residues; (iv) insertion of a basic amino acid residue; and (v) Deletion of acidic surface-exposed amino acid residues.

[0043] The unit dose can be between 450 pg and 8,000 pg of modified BoNT / A, where the modified BoNT / A includes a modification in one or more amino acid residues selected from the following: ASN886, ASN905, GLN915, ASN918, GLU920, ASN930, ASN954, SER955, GLN991, GLU992, GLN995, ASN1006, ASN1025, ASN1026, ASN1032, ASN1043, ASN1046, ASN1052, ASP1058, HIS1064, ASN1080, GLU1081, GLU1083, ASP1086, ASN1188, ASP1213, GLY1215, ASN1216, GLN1229, ASN1242, ASN1243, SER1274, and THR1277, the modification being selected from: (i) replacement of acidic surface-exposed amino acid residues with basic amino acid residues; (ii) replacement of acidic surface-exposed amino acid residues with uncharged amino acid residues; (iii) replacement of uncharged surface-exposed amino acid residues with basic amino acid residues; (iv) insertion of a basic amino acid residue; and (v) Deletion of acidic surface-exposed amino acid residues.

[0044] The upper limit of the unit dose range may be 7,750, 7,500, 7,000, 6,000, 5,000, 4,000, 3,000, 2,000, or 1,000 pg of modified BoNT / A, preferably the upper limit is 7,500 pg. The lower limit of the unit dose range may be 475, 500, 600, 700, 800, 900, 1,000, 1,500, 2,000, 3,000, 4,000, 5,000, 6,000, or 7,000 pg of modified BoNT / A, preferably the lower limit is 500 pg. Preferably, the unit dose of modified BoNT / A is 500 pg to 7,500 pg of modified BoNT / A, for example 4,000 pg to 6,000 pg. Most preferably, the unit dose of the modified BoNT / A is between 2,000 pg and 3,000 pg, such as between 2,400 pg and 2,600 pg.

[0045] The unit dose may be greater than 1,000 pg or 5,000 pg of modified BoNT / A, the modified BoNT / A comprising a modification at one or more amino acid residues selected from the following: ASN886, ASN905, GLN915, ASN918, GLU920, ASN930, ASN954, SER955, GLN991, GLU992, GLN 995, ASN1006, ASN1025, ASN1026, ASN1032, ASN1043, ASN1046, ASN1052, ASP1058, HIS1064, ASN1080, GLU1081, GLU1083, ASP1086, ASN1188, ASP1213, GLY1215, ASN1216, GLN1229, ASN1242, ASN1243, SER1274, and THR1277, the modification being selected from: (i) replacement of acidic surface-exposed amino acid residues with basic amino acid residues; (ii) replacement of acidic surface-exposed amino acid residues with uncharged amino acid residues; (iii) replacement of uncharged surface-exposed amino acid residues with basic amino acid residues; (iv) insertion of a basic amino acid residue; and (v) Deletion of acidic surface-exposed amino acid residues.

[0046] The unit dose of the modified BoNT / A can be greater than 1,000 pg up to 7,500 pg of the modified BoNT / A, for example, greater than 5,000 pg up to 7,500 pg of the modified BoNT / A.

[0047] The total dose administered in practicing the treatment regimens of the invention may be up to 80,000 pg of modified BoNT / A, where the modified BoNT / A contains a modification in one or more amino acid residues selected from the following: ASN886, ASN905, GLN915, ASN918, GLU920, ASN930, ASN954, SER955, GLN991, GLU992, GLN995, ASN1006, ASN1025, ASN1026, ASN1032, ASN1043, ASN1046, ASN1052, ASP1058, HIS1064, ASN1080, GLU1081, GLU1083, ASP1086, ASN1188, ASP1213, GLY1215, ASN1216, GLN1229, ASN1242, ASN1243, SER1274, and THR1277, the modification being selected from: (i) replacement of acidic surface-exposed amino acid residues with basic amino acid residues; (ii) replacement of acidic surface-exposed amino acid residues with uncharged amino acid residues; (iii) replacement of uncharged surface-exposed amino acid residues with basic amino acid residues; (iv) insertion of a basic amino acid residue; and (v) Deletion of acidic surface-exposed amino acid residues.

[0048] In other words, the total amount of modified BoNT / A administered in a given treatment session may be up to 80,000 pg. The total dose may be up to 75,000, 70,000, 60,000, 50,000, 40,000, 30,000, 20,000, 10,000, or 5,000 pg. Preferably, the total dose may be up to 75,000 pg of modified BoNT / A. The total dose may be at least 700, 800, 900, 1,000, 2,000, 3,000, 4,000, 5,000, 7,500, 10,000, 12,500, 15,000, 20,000, 30,000, 40,000, 50,000, 60,000, or 70,000 pg. Preferably, the total dose is at least 900 pg, more preferably at least 1,000 pg of modified BoNT / A, for example at least 3,000 pg. The total dose may be between 3,150 pg and 80,000 pg, preferably between 3,500 pg and 75,000 pg. More preferably, the total dose administered is between 7,500 and 75,000 pg.

[0049] The total administered dose may be greater than 1,000 pg or 5,000 pg of modified BoNT / A, the modified BoNT / A comprising a modification at one or more amino acid residues selected from the following: ASN886, ASN905, GLN915, ASN918, GLU920, ASN930, ASN954, SER955, GLN991, GLU992, GLN995, ASN1006, ASN1025, ASN1026, ASN1032, ASN1043, ASN1046, ASN1052, ASP1058, HIS1064, ASN1080, GLU1081, GLU1083, ASP1086, ASN1188, ASP1213, GLY1215, ASN1216, GLN1229, ASN1242, ASN1243, SER1274, and THR1277, the modification being selected from: (i) replacement of acidic surface-exposed amino acid residues with basic amino acid residues; (ii) replacement of acidic surface-exposed amino acid residues with uncharged amino acid residues; (iii) replacement of uncharged surface-exposed amino acid residues with basic amino acid residues; (iv) insertion of a basic amino acid residue; and (v) Deletion of acidic surface-exposed amino acid residues.

[0050] Preferably, the total dose is greater than 1,000 pg of modified BoNT / A, such as greater than 5,000 pg of modified BoNT / A.

[0051] In one aspect, the present invention provides a modified botulinum neurotoxin A (BoNT / A) for use in treating cervical dystonia, the modified BoNT / A being administered by intramuscular injection to an affected neck muscle of an affected individual, The modified BoNT / A is administered in unit doses ranging from 53 units to 948 units of modified BoNT / A, where 1 unit is the calculated median lethal dose (LD 50 ) is the amount of modified BoNT / A equivalent to At least one unit dose is administered to the affected neck muscle; The total dose administered during treatment is up to 9,480 units of modified BoNT / A. The modified BoNT / A comprises a modification at one or more amino acid residues selected from the following: ASN886, ASN905, GLN915, ASN918, GLU920, ASN930, ASN954, SER955, GLN991, GLU992, GLN995, ASN1006, ASN1025, ASN1026, ASN1032, ASN1043, ASN1046, ASN1052, ASP1058, HIS1064, ASN1080, GLU1081, GLU1083, ASP1086, ASN1188, ASP1213, GLY1215, ASN1216, GLN1229, ASN1242, ASN1243, SER1274, and THR1277, the modification being selected from: (i) replacement of acidic surface-exposed amino acid residues with basic amino acid residues; (ii) replacement of acidic surface-exposed amino acid residues with uncharged amino acid residues; (iii) replacement of uncharged surface-exposed amino acid residues with basic amino acid residues; (iv) insertion of a basic amino acid residue; and (v) Deletion of acidic surface-exposed amino acid residues.

[0052] In a related aspect, the invention provides a modified BoNT / A for use in treating cervical dystonia in an affected individual for a longer duration than treatment with an unmodified BoNT / A (e.g., SEQ ID NO: 2), wherein the modified BoNT / A is administered to an affected neck muscle of the affected individual by intramuscular injection; The modified BoNT / A is administered in unit doses ranging from 53 units to 948 units of modified BoNT / A, where 1 unit is the calculated median lethal dose (LD) for mice. 50 ) is the amount of modified BoNT / A equivalent to At least one unit dose is administered to the affected neck muscle; The total dose administered during treatment is up to 9,480 units of modified BoNT / A. The modified BoNT / A comprises a modification at one or more amino acid residues selected from: ASN 886, ASN905, GLN915, ASN918, GLU920, ASN930, ASN954, SER955, GLN991, GLU992, GLN995, ASN1006, ASN1025, ASN1026, ASN1032, ASN1043, ASN1046, ASN1052, ASP1058, HIS1064, ASN1080, GLU1081, GLU1083, ASP1086, ASN1188, ASP1213, GLY1215, ASN1216, GLN1229, ASN1242, ASN1243, SER1274, and THR1277, the modification being selected from: (i) replacement of acidic surface-exposed amino acid residues with basic amino acid residues; (ii) replacement of acidic surface-exposed amino acid residues with uncharged amino acid residues; (iii) replacement of uncharged surface-exposed amino acid residues with basic amino acid residues; (iv) insertion of a basic amino acid residue; and (v) Deletion of acidic surface-exposed amino acid residues.

[0053] In a related aspect, the invention provides a method for treating cervical dystonia, the method comprising administering a modified BoNT / A by intramuscular injection to an affected neck muscle of an affected individual, The modified BoNT / A is administered in unit doses ranging from 53 units to 948 units of modified BoNT / A, where 1 unit is the calculated median lethal dose (LD) for mice. 50 ) is the amount of modified BoNT / A equivalent to At least one unit dose is administered to the affected neck muscle; The total dose administered during said treatment is up to 9,480 units of modified BoNT / A; The modified BoNT / A comprises a modification at one or more amino acid residues selected from: ASN886, ASN905, GLN915, ASN918, GLU920, ASN930, ASN954, SER955, GLN991, GLU992, GLN995, ASN1006, ASN1025, ASN1026, ASN1032, ASN1043, ASN1046, ASN1052, ASP1058, HIS1064, ASN1080, GLU1081, GLU1083, ASP1086, ASN1188, ASP1213, GLY1215, ASN1216, GLN1229, ASN1242, ASN1243, SER1274, and THR1277, the modification being selected from: (i) replacement of acidic surface-exposed amino acid residues with basic amino acid residues; (ii) replacement of acidic surface-exposed amino acid residues with uncharged amino acid residues; (iii) replacement of uncharged surface-exposed amino acid residues with basic amino acid residues; (iv) insertion of a basic amino acid residue; and (v) Deletion of acidic surface-exposed amino acid residues.

[0054] In a related aspect, the invention provides a method of treating cervical dystonia in an affected individual for a longer duration than treatment with an unmodified BoNT / A (e.g., SEQ ID NO:2), the method comprising administering the modified BoNT / A to an affected neck muscle of the affected individual by intramuscular injection of the modified BoNT / A; The modified BoNT / A is administered in unit doses ranging from 53 units to 948 units of modified BoNT / A, where 1 unit is the calculated median lethal dose (LD) for mice. 50 ) is the amount of modified BoNT / A equivalent to At least one unit dose is administered to the affected neck muscle; The total dose administered during said treatment is up to 9,480 units of modified BoNT / A; The modified BoNT / A comprises a modification at one or more amino acid residues selected from: ASN886, ASN905, GLN915, ASN918, GLU920, ASN930, ASN954, SER955, GLN991, GLU992, GLN995, ASN1006, ASN1025, ASN1026, ASN1032, ASN1043, ASN1046, ASN1052, ASP1058, HIS1064, ASN1080, GLU1081, GLU1083, ASP1086, ASN1188, ASP1213, GLY1215, ASN1216, GLN1229, ASN1242, ASN1243, SER1274, and THR1277, the modification being selected from: (i) replacement of acidic surface-exposed amino acid residues with basic amino acid residues; (ii) replacement of acidic surface-exposed amino acid residues with uncharged amino acid residues; (iii) replacement of uncharged surface-exposed amino acid residues with basic amino acid residues; (iv) insertion of a basic amino acid residue; and (v) Deletion of acidic surface-exposed amino acid residues.

[0055] In another related aspect, the invention provides the use of a modified botulinum neurotoxin A (BoNT / A) in the manufacture of a medicament for the treatment of cervical dystonia, the modified BoNT / A being administered by intramuscular injection to an affected neck muscle; The modified BoNT / A is administered in unit doses ranging from 53 units to 948 units of modified BoNT / A, where 1 unit is the calculated median lethal dose (LD 50 ) is the amount of modified BoNT / A equivalent to At least one unit dose is administered to the affected neck muscle; The total dose administered during treatment is up to 9,480 units of modified BoNT / A. The modified BoNT / A comprises a modification at one or more amino acid residues selected from: ASN886, ASN905, GLN915, ASN918, GLU920, ASN930, ASN954, SER955, GLN991, GLU992, GLN995, ASN1006, ASN1025, ASN1026, ASN1032, ASN1043, ASN1046, ASN1052, ASP1058, HIS1064, ASN1080, GLU1081, GLU1083, ASP1086, ASN1188, ASP1213, GLY1215, ASN1216, GLN1229, ASN1242, ASN1243, SER1274, and THR1277, the modification being selected from: (i) replacement of acidic surface-exposed amino acid residues with basic amino acid residues; (ii) replacement of acidic surface-exposed amino acid residues with uncharged amino acid residues; (iii) replacement of uncharged surface-exposed amino acid residues with basic amino acid residues; (iv) insertion of a basic amino acid residue; and (v) Deletion of acidic surface-exposed amino acid residues.

[0056] In a related aspect, the invention provides the use of a modified BoNT / A in the manufacture of a medicament for treating cervical dystonia in an affected individual for a longer duration than treatment with an unmodified BoNT / A (e.g., SEQ ID NO: 2), the modified BoNT / A being administered by intramuscular injection to an affected neck muscle of the affected individual; The modified BoNT / A is administered in unit doses ranging from 53 units to 948 units of modified BoNT / A, where 1 unit is the calculated median lethal dose (LD 50 ) is the amount of modified BoNT / A equivalent to At least one unit dose is administered to the affected neck muscle; The total dose administered during treatment is up to 9,480 units of modified BoNT / A. The modified BoNT / A comprises a modification at one or more amino acid residues selected from: ASN886, ASN905, GLN915, ASN918, GLU920, ASN930, ASN954, SER955, GLN991, GLU992, GLN995, ASN1006, ASN1025, ASN1026, ASN1032, ASN1043, ASN1046, ASN1052, ASP1058, HIS1064, ASN1080, GLU1081, GLU1083, ASP1086, ASN1188, ASP1213, GLY1215, ASN1216, GLN1229, ASN1242, ASN1243, SER1274, and THR1277, the modification being selected from: (i) replacement of acidic surface-exposed amino acid residues with basic amino acid residues; (ii) replacement of acidic surface-exposed amino acid residues with uncharged amino acid residues; (iii) replacement of uncharged surface-exposed amino acid residues with basic amino acid residues; (iv) insertion of a basic amino acid residue; and (v) Deletion of acidic surface-exposed amino acid residues.

[0057] The unit dose can be between 53 units and 948 units of a modified BoNT / A, the modified BoNT / A comprising a modification in one or more amino acid residues selected from the following: ASN886, ASN905, GLN915, ASN918, GLU920, ASN930, ASN954, SER955, GLN991, GLU992, GLN995, ASN1006, ASN1025, ASN1026, ASN1032, ASN1043, ASN1046, ASN1052, ASP1058, HIS1064, ASN1080, GLU1081, GLU1083, ASP1086, ASN1188, ASP1213, GLY1215, ASN1216, GLN1229, ASN1242, ASN1243, SER1274, and THR1277, the modification being selected from: (i) replacement of acidic surface-exposed amino acid residues with basic amino acid residues; (ii) replacement of acidic surface-exposed amino acid residues with uncharged amino acid residues; (iii) replacement of uncharged surface-exposed amino acid residues with basic amino acid residues; (iv) insertion of a basic amino acid residue; and (v) Deletion of acidic surface-exposed amino acid residues.

[0058] The upper limit of the unit dose range can be 925, 900, 850, 800, 750, 700, 650, 600, 550, 50, 500, 450, 400, 350, 300, 250, 200, 150, or 100 units of modified BoNT / A, preferably the upper limit is 889 units. The lower limit of the unit dose range can be 55, 60, 65, 70, 75, 80, 85, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, or 900 units of modified BoNT / A, preferably the lower limit is 59 units. Preferably, the unit dose of modified BoNT / A is between 59 and 889 units of modified BoNT / A, such as between 200 and 600 units. Most preferably, the unit dose of modified BoNT / A is between 237 and 355 units, such as between 284 and 308 units.

[0059] The unit dose may be greater than 118.5 units, or greater than 592.5 units of a modified BoNT / A, the modified BoNT / A comprising a modification in one or more amino acid residues selected from the following: ASN886, ASN905, GLN915, ASN918, GLU920, ASN930, ASN954, SER955, GLN991, GLU992, GLN995, ASN1006, ASN1025, ASN1026, ASN1032, ASN1043, ASN1046, ASN1052, ASP1058, HIS1064, ASN1080, GLU1081, GLU1083, ASP1086, ASN1188, ASP1213, GLY1215, ASN1216, GLN1229, ASN1242, ASN1243, SER1274, and THR1277, the modification being selected from: (vi) replacement of acidic surface-exposed amino acid residues with basic amino acid residues; (vii) replacement of acidic surface-exposed amino acid residues with uncharged amino acid residues; (viii) replacement of uncharged surface-exposed amino acid residues with basic amino acid residues; (ix) insertion of a basic amino acid residue; and (x) Deletion of acidic surface-exposed amino acid residues.

[0060] The unit dose of modified BoNT / A can be greater than 118.5 units up to 888 units of modified BoNT / A, for example, greater than 592.5 units up to 888 units of modified BoNT / A.

[0061] The total dose administered in practicing the treatment regimen of the invention may be up to 9,480 units of a modified BoNT / A, the modified BoNT / A comprising a modification in one or more amino acid residues selected from the following: ASN886, ASN905, GLN915, ASN918, GLU920, ASN930, ASN954, SER955, GLN991, GLU992, GLN995, ASN1006, ASN1025, ASN1026, ASN1032, ASN1043, ASN1046, ASN1052, ASP1058, HIS1064, ASN1080, GLU1081, GLU1083, ASP1086, ASN1188, ASP1213, GLY1215, ASN1216, GLN1229, ASN1242, ASN1243, SER1274, and THR1277, the modification being selected from: (i) replacement of acidic surface-exposed amino acid residues with basic amino acid residues; (ii) replacement of acidic surface-exposed amino acid residues with uncharged amino acid residues; (iii) replacement of uncharged surface-exposed amino acid residues with basic amino acid residues; (iv) insertion of a basic amino acid residue; and (v) Deletion of acidic surface-exposed amino acid residues.

[0062] In other words, the total amount of modified BoNT / A administered in a given treatment session may be up to 9,480 units. The total dose may be up to 9,000, 8,000, 7,000, 6,000, 5,000, 4,000, 3,000, 2,000, or 1,000 units. Preferably, the total dose may be up to 8,890 units of modified BoNT / A. The total dose may be at least 83, 95, 106, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1,000, 1,500, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, or 9,000 units. Preferably, the total dose is at least 106 units, more preferably at least 118 units of modified BoNT / A, such as at least 355 units of modified BoNT / A. The total dose may be between 371 and 9,480 units, more preferably between 413 and 8,890 units. More preferably, the total administered dose is between 889 and 8,890 units.

[0063] The total administered dose can be greater than 118.5 units or greater than 592.5 units of a modified BoNT / A, the modified BoNT / A comprising a modification in one or more amino acid residues selected from the following: ASN886, ASN905, GLN915, ASN918, GLU920, ASN930, ASN954, SER955, GLN991, GLU992, GLN995, ASN1006, ASN1025, ASN1026, ASN1032, ASN1043, ASN1046, ASN1052, ASP1058, HIS1064, ASN1080, GLU1081, GLU1083, ASP1086, ASN1188, ASP1213, GLY1215, ASN1216, GLN1229, ASN1242, ASN1243, SER1274, and THR1277, the modification being selected from: (i) replacement of acidic surface-exposed amino acid residues with basic amino acid residues; (ii) replacement of acidic surface-exposed amino acid residues with uncharged amino acid residues; (iii) replacement of uncharged surface-exposed amino acid residues with basic amino acid residues; (iv) insertion of a basic amino acid residue; and (v) Deletion of acidic surface-exposed amino acid residues.

[0064] Preferably, the total dose is greater than 118.5 units of the modified BoNT / A, such as greater than 592.5 units of the modified BoNT / A.

[0065] An "affected neck muscle" may be a neck muscle that is involved (e.g., causes) the affected individual's cervical dystonia or symptoms. Although preferred, it is not intended that an "affected neck muscle" necessarily be involved (e.g., causes) the cervical dystonia or symptoms at the time of treatment. For example, the neck muscle may be one that has been involved (e.g., caused) the affected individual's cervical dystonia or symptoms in the past, or one that is expected to be involved (e.g., causes) the affected individual's cervical dystonia or symptoms in the future. In one embodiment, more than one neck muscle (e.g., a pair of agonist and antagonist neck muscles) may be involved (e.g., causes) the affected individual's cervical dystonia or symptoms. In such a case, the modified BoNT / A may be administered to more than one neck muscle (e.g., to the agonist and antagonist neck muscles of a pair of neck muscles).

[0066] The affected neck muscle is preferably involved in (e.g. causes) cervical dystonia and / or symptoms in the affected individual by contracting. Thus, preferably, the affected neck muscle of the affected individual is contracting or contracts resulting in cervical dystonia and / or symptoms in the affected individual. The neck muscle is preferably a neck muscle that contracts or has contracted involuntarily, e.g. during treatment. The neck muscle may be any muscle (e.g. skeletal muscle) that is operatively connected to the head and / or neck of the affected individual, e.g. any muscle that can change the position of the head of the affected individual (e.g. during contraction). The affected neck muscle may be a muscle that can: Causes the affected individual's jaw to bend toward the affected individual's shoulder, resulting in a rotation of the head to the side (torticollis), causes the affected individual's head to tilt forward (anteflexion), causes the affected individual's head to tilt backward (retroflexion), causes the affected individual's head to tilt to the side (lateral cervical bending), causes an anterior sagittal deviation (forward translation) of the affected individual's head, and / or causes a posterior sagittal deviation (backward translation) of the affected individual's head.

[0067] Affected neck muscles include the sternocleidomastoid / sternocleidomastoideus, splenius capitis, splenius cervix, scalene complex (e.g., anterior scalene and / or middle scalene), trapezius (e.g., upper trapezius and / or lower trapezius), levator scapulae, semispinalis capitis, or longissimus (e.g., longissimus capitis and / or longissimus cervix), posterior paravertebral (e.g., posterior scalene, middle scalene and / or anterior scalene, preferably posterior scalene), submandibular complex (e.g., digastric, geniohyoid, mylohyoid muscle, mylohyoid boutonniere and / or stylohyoid muscle), superior nuchal line-clavicle (lateral part), spinous process C3-Th3-mastoid process, spinous process Th3-Th5-transverse processes C1-C2, transverse process C3-Th6, spinous process C3-Th1-superior nuchal line, transverse process Th1-Th6-spinous processes C2-C7, transverse process C3-Th3-mastoid process, transverse process Th1-Th6-transverse processes C2-C6, oblique capitis inferior, spinous process C2-transverse process C1, suprasternal notch and clavicle (medial part)-mastoid process and superior nuchal line, transverse process C1-C4-scapula (superior angle), transverse process C2-C7-first rib, transverse process C3-C6-first rib, longus capitis muscle, transverse process C3-C6-occipital bone (base), longus colli muscle or transverse process C2-C5-atlas (anterior tubercle). The affected neck muscles may include the right levator scapulae, left levator scapulae, right trapezius, left trapezius, right sternocleidomastoid, left sternocleidomastoid, right splenius capitis, left splenius capitis, middle scalene, anterior scalene, right semispinalis capitis, left semispinalis capitis, right longissimus capitis, or left longissimus capitis.Affected neck muscles include the sternocleidomastoid (e.g., left or right sternocleidomastoid), left or right splenius capitis, anterior or middle scalene, left or right trapezius (e.g., left upper trapezius or right upper trapezius), left or right levator scapulae, left or right semispinalis capitis, longissimus (e.g., left or right longissimus capitis and / or left or right longissimus capitis), splenius cervicalis, scalene complex (e.g. anterior scalene and / or middle scalene), posterior paravertebral area (e.g. posterior scalene, middle scalene and / or anterior scalene, preferably posterior scalene), submandibular complex (e.g. digastric, geniohyoid, mylohyoid, mylohyoid boutonniere and / or stylohyoid), lower trapezius, superior nuchal line-clavicle (lateral area), spinous processes C3-Th3-mastoid, spinous processes Th3-Th5 - transverse processes C1-C2, transverse processes C3-Th6, spinous process C3-Th1 - superior nuchal line, transverse processes Th1-Th6 - spinous processes C2-C7, transverse processes C3-Th3 - mastoid process, transverse processes Th1-Th6 - transverse processes C2-C6, oblique capitis inferior, oblique capitis superior, spinous process C2 - transverse process C1, suprasternal notch and clavicle (medial part) - mastoid process and superior nuchal line, transverse processes C1-C4 - scapula (superior angle), transverse processes C2-C7 - first rib, transverse processes C3-C6 - first rib, longus capitis, transverse processes C3-C6 - occipital bone (base), longus colli, semispinalis cervi, rectus capitis major, rectus capitis minor, rectus capitis anterior, multiple tubes or transverse processes C2-C5 - atlas (anterior tubercle).

[0068] Affected neck muscles may include: semispinalis cervix, levator scapulae, splenius cervix, longissimus cervix, lower trapezius, sternocleidomastoid, semispinalis capitis, obliquus capitis inferior, longissimus capitis, splenius capitis, semispinalis cervix, scalene medialis, longissimus capitis, longus colli or longus capitis.

[0069] The multiple affected neck muscles treated in the present invention may include at least one (eg, at least two) of any of the muscles described herein.

[0070] In one embodiment, the modified BoNT / A may be administered to one or more affected neck muscles selected from the following: sternocleidomastoid, splenius capitis, splenius cervix, scalene complex (e.g., anterior scalene and / or middle scalene), trapezius (e.g., upper trapezius and / or lower trapezius), levator scapulae, semispinalis capitis and longissimus (e.g., longissimus capitis and / or longissimus cervix). Preferably, the modified BoNT / A is administered to multiple affected neck muscles. For example, the modified BoNT / A may be administered to at least two (e.g., at least three, four, five, six, or seven, preferably eight) affected neck muscles selected from the following: sternocleidomastoid, splenius capitis, splenius cervix, scalene complex (e.g., anterior scalene and / or middle scalene), trapezius (e.g., upper trapezius and / or lower trapezius), levator scapulae, semispinalis capitis and longissimus (e.g., longissimus capitis and / or longissimus cervix).

[0071] In one embodiment, the modified BoNT / A may be administered to one or more affected neck muscles selected from the following: right levator scapulae, left levator scapulae, right trapezius, left trapezius, right sternocleidomastoid, left sternocleidomastoid, right splenius capitis, left splenius capitis, middle scalene, anterior scalene, right semispinalis capitis, left semispinalis capitis, right longissimus capitis, and left longissimus capitis.

[0072] In one embodiment, the modified BoNT / A may be administered to one or more affected neck muscles selected from the following: sternocleidomastoid / sternocleidomastoideus, splenius capitis, splenius cervix, scalene complex (e.g., anterior scalene and / or middle scalene), trapezius (e.g., upper trapezius and / or lower trapezius), levator scapulae, semispinalis capitis, longissimus (e.g., longissimus capitis and / or longissimus cervix), posterior paravertebral (e.g., posterior scalene, middle scalene and / or anterior scalene, preferably posterior scalene), submandibular complex (e.g., digastric, geniohyoid, mylohyoid, mylohyoid boutonniere and / or stylohyoid), superior nuchal line-clavicular (lateral portion), spine (lateral portion), spinous ... C3-Th3-mastoid process, Th3-Th5-transverse processes C1-C2, C3-Th6-transverse processes C3-Th1-superior nuchal line, Th1-Th6-C2-C7-C7, C3-Th3-mastoid process, Th1-Th6-C2-C6-C6-C6-C6-C6-C7-C4-Scapula (superior angle), C2-C7-C7-1st rib, C3-C6-C6-C7-C6-C6-C7-C6-C6-C6-C7-C6-C6-C6-C4-C7-C6 ...For example, a modified BoNT / A may be administered to at least two (e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35) affected neck muscles selected from the following: Sternocleidomastoid / sternocleidomastoideus, splenius capitis, splenius cervix, scalene complex (e.g. anterior scalene and / or middle scalene), trapezius (e.g. upper trapezius and / or lower trapezius), levator scapulae, semispinalis capitis, longissimus (e.g. longissimus capitis and / or longissimus cervix), posterior paravertebral (e.g. posterior scalene, middle scalene and / or anterior scalene, preferably posterior scalene), submandibular complex (e.g. digastric, geniohyoid, mylohyoid, mylohyoid boutonniere and / or stylohyoid), superior nuchal line-clavicle (lateral portion), spinous processes C3-Th3-mastoid, spinous processes Th3-Th5-transverse processes C1-C2, transverse processes C3-Th6, spinous processes C3-Th1 - superior nuchal line, transverse process Th1-Th6 - spinous process C2-C7, transverse process C3-Th3 - mastoid process, transverse process Th1-Th6 - transverse process C2-C6, inferior oblique muscle, spinous process C2-transverse process C1, suprasternal notch and clavicle (medial part) - mastoid process and superior nuchal line, transverse process C1-C4 - scapula (superior angle), transverse process C2-C7 - first rib, longus capitis muscle, transverse process C3-C6 - occipital bone (bottom), longus maxilla muscle and transverse process C2-C5 - atlas (anterior tuberosity).

[0073] In one embodiment, the modified BoNT / A may be administered to one or more affected neck muscles, including the following: sternocleidomastoid muscle (e.g., left sternocleidomastoid or right sternocleidomastoid), left splenius capitis or right splenius capitis, anterior scalene or middle scalene, left trapezius or right trapezius (e.g., left upper trapezius or right upper trapezius), left levator scapulae or right levator scapulae, left semispinalis capitis or right semispinalis capitis, middle semispinalis capitis, longissimus muscle (e.g., left longissimus capitis or right longissimus capitis and / or left longissimus capitis or right longissimus capitis), cervical latissimus posterior paravertebral area (e.g. posterior scalene, middle scalene and / or anterior scalene, preferably posterior scalene), submandibular complex (e.g. digastric, geniohyoid, mylohyoid, mylohyoid boutonniere and / or stylohyoid), lower trapezius, superior nuchal line - clavicle (lateral area), spinous processes C3 - Th3 - mastoid, spinous processes Th3 - Th5 - transverse processes C1-C2, transverse process C3-Th6, spinous process C3-Th1-superior nuchal line, transverse process Th1-Th6-spinous process C2-C7, transverse process C3-Th3-mastoid process, transverse process Th1-Th6-transverse process C2-C6, oblique capitis inferior, oblique capitis superior, spinous process C2-transverse process C1, suprasternal notch and clavicle (medial part)-mastoid process and superior nuchal line, transverse process C1-C4-scapula (superior angle), transverse process C2-C7-first rib, transverse process C3-C6-first rib, longus capitis, transverse process C3-C6-occipital bone (base), longus colli, semispinous cervix, rectus capitis posterior greater, rectus capitis posterior less, rectus capitis anterior, multifidus or transverse process C2-C5-atlas (anterior tubercle). Preferably, the modified BoNT / A is administered to multiple affected neck muscles.For example, a modified BoNT / A may be administered to at least two (e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35) affected neck muscles selected from the following: Sternocleidomastoid (e.g. left sternocleidomastoid or right sternocleidomastoid), splenius capitis or right splenius capitis, anterior or middle scalene, left or right trapezius (e.g. upper trapezius or right upper trapezius), levator scapulae or right levator scapulae, semispinalis capitis or right semispinalis capitis, middle semispinalis capitis, longissimus (e.g. longus capitis or right longus capitis and / or longissimus capitis or right longissimus capitis), splenius cervix, scalene complex (e.g. anterior scalene and / or middle scalene), posterior paravertebral (e.g. posterior scalene, middle scalene and / or anterior scalene, preferably posterior scalene), submandibular complex (e.g. digastric, geniohyoid, mylohyoid, mylohyoid boutonniere and / or stylohyoid), scapulae, Lower caps muscle, superior nuchal line - clavicle (lateral part), spinous process C3 - Th3 - mastoid process, spinous process Th3 - Th5 - transverse process C1 - C2, transverse process C3 - Th6, spinous process C3 - Th1 - superior nuchal line, transverse process Th1 - Th6 - spinous process C2 - C7, transverse process C3 - Th3 - mastoid process, transverse process Th1 - Th6 - transverse process C2 - C6, inferior oblique muscle, superior head Oblique muscle, spinous process C2-transverse process C1, suprasternal notch and clavicle (medial part) - mastoid process and superior nuchal line, transverse process C1-C4 - scapula (superior corner), transverse process C2-C7 - first rib Bones, transverse processes C3-C6 - occipital bone (bottom), longus cervix, semispinalis cervicis, rectus occipitalis major, rectus occipital minor, rectus frontalis, multifidus canal and or transverse processes C2-C5 - atlas (anterior tuberosity).

[0074] In one embodiment, the modified BoNT / A may be administered to one or more affected neck muscles selected from the following: sternocleidomastoid (e.g., left sternocleidomastoid or right sternocleidomastoid), splenius capitis or right splenius capitis, anterior or middle scalene, left or right trapezius (e.g., upper left trapezius or right upper trapezius), levator scapulae or right levator scapulae, left or right semispinalis capitis, longissimus (e.g., longus capitis or right longus capitis and / or left or right longissimus capitis), splenius cervix, scalene complex (e.g., anterior scalene and / or middle scalene), posterior paraspinal (e.g., posterior scalene, middle scalene and / or anterior scalene, preferably posterior scalene), submandibular complex (e.g., digastric, geniohyoid, mylohyoid, mylohyoid boutonniere and / or stylohyoid), lower trapezius, superior nuchal line-clavicular (lateral part), spinous process C3-Th3-mastoid, spinous process Th3-Th5-transverse process C1-C2, transverse process C3-Th6, spinous process C3-Th1-superior nuchal line, transverse process Th1-Th6-spinous process C2-C7, transverse process C3-Th3-mastoid, transverse process Th1-Th6-transverse process C2-C6, inferior oblique muscle, superior capitis oblique muscle, spinous process C2-transverse process C 1. Suprasternal notch and clavicle (medial part) - mastoid process and superior nuchal line, transverse process C1-C4 - scapula (superior angle), transverse process C2-C7 - first rib, transverse process C3-C6 - first rib Bones, longus capitis, transverse processes C3-C6 - occipital bone (bottom), longus capitis, semispinalis cervicis, rectus occipitalis major, rectus occipital minor, rectus capitis frontalis, multifidus and transverse processes C2-C5 - atlas (anterior tuberosity). Preferably, the modified BoNT / A is administered to multiple affected neck muscles.For example, a modified BoNT / A may be administered to at least two (e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35) affected neck muscles selected from the following: Sternocleidomastoid (e.g. left sternocleidomastoid or right sternocleidomastoid), splenius capitis or right splenius capitis, anterior or middle scalene, left or right trapezius (e.g. upper left or right trapezius), levator scapulae or right levator scapulae, left or right semispinalis capitis, longissimus (e.g. longus capitis or right longus capitis and / or left or right longissimus capitis), splenius cervix, scalene complex (e.g. anterior scalene and / or middle scalene), posterior paraspinal (e.g. posterior scalene, middle scalene and / or anterior scalene, preferably posterior scalene), submandibular complex (e.g. digastric muscle, geniohyoid muscle, mylohyoid muscle, mylohyoid boutonniere and / or stylohyoid muscle), lower trapezius muscle, superior nuchal line-clavicle (lateral part), spinous process C3-Th3-mastoid process, spinous process Th3-Th5-transverse process C1-C2, transverse process C3-Th6, spinous process C3-Th1-superior nuchal line, transverse process Th1-Th6-spinous process C2-C7, transverse process C3-Th3-mastoid process, transverse process Th1-Th6-transverse process C2-C6, obliquus capitis inferior, obliquus capitis superior, spinous process C2-transverse process C1, suprasternal notch and clavicle (medial part)-mastoid process and superior nuchal line, transverse process C1-C4- Scapula (superior angle), transverse process C2-C7 – first rib, transverse processes C3-C6 – first rib, longus capitis, transverse processes C3-C6 – occipital bone (base), longus colli, semispinalis cervi, rectus capitis posterior greater, rectus capitis posterior less, rectus capitis anterior, multifidus and transverse processes C2-C5 – atlas (anterior tubercle).

[0075] In one embodiment, the modified BoNT / A may be administered to one or more affected neck muscles including: semispinalis cervix, levator scapulae, splenius cervix, longissimus cervix, lower trapezius, sternocleidomastoid, semispinalis capitis, oblique capitis inferior, longissimus capitis, splenius capitis, semispinalis cervix, scalene medial, longissimus capitis, longissimus capitis, or longus capitis. In one embodiment, the modified BoNT / A may be administered to one or more affected neck muscles selected from: semispinalis cervix, levator scapulae, splenius cervix, longissimus cervix, lower trapezius, sternocleidomastoid, semispinalis capitis, oblique capitis inferior, longissimus capitis, splenius capitis, semispinalis cervix, scalene medial, longissimus capitis, longissimus capitis, or longus capitis. For example, the modified BoNT / A may be administered to at least two (e.g., at least three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or fifteen) of the affected neck muscles.

[0076] In the case of two equivalent neck muscles on either side of the neck (e.g., sternocleidomastoid muscles, such as the left and right sternocleidomastoid muscles), the modified BoNT / A may be administered unilaterally (e.g., to one of the muscles if only one muscle is contracted) or bilaterally (e.g., to both muscles if both muscles are contracted). When treating multiple affected muscles, it is preferred that the affected muscles are of multiple different types. For example, when bilateral administration is performed to two affected sternocleidomastoid muscles, it is preferred to treat additional muscles when treating multiple affected muscles as described herein.

[0077] In the case where there are two equivalent neck muscles on either side of the neck, unilateral administration may be performed to either of said equivalent neck muscles. For example, administration may be performed to a constrictor muscle or an equivalent non-contractor muscle. In one embodiment, unilateral administration is performed to a muscle located on one side of the neck of an affected individual, where said side of the neck of the affected individual exhibits symptoms of cervical dystonia, or to an equivalent muscle located on the opposite side of the neck of an affected individual, where said side of the neck of the affected individual does not exhibit symptoms of cervical dystonia.

[0078] The affected neck muscles selected for treatment according to the present invention may be determined by the specific symptoms of cervical dystonia in the patient to be treated (e.g., torticollis, lateral cervical flexion, anterior cervical flexion, posterior cervical flexion or a combination thereof).

[0079] The affected neck muscles selected for treatment according to the present invention may be determined by the specific symptoms of cervical dystonia in the patient to be treated (e.g., torticollis, lateral neck flexion, forward neck flexion, backward neck flexion, lateral head flexion, head rotation, forward head flexion, backward head flexion, lateral deviation, sagittal deviation, or any combination thereof).

[0080] In one embodiment, when treating torticollis, the modified BoNT / A may be administered to one or more affected neck muscles selected from the following: sternocleidomastoid, trapezius (e.g., upper trapezius), scalene anterior, splenius capitis, splenius cervicis, levator scapulae, and longissimus (e.g., longissimus capitis and / or longissimus cervicis). Preferably, when treating torticollis, the modified BoNT / A may be administered contralaterally to one or more affected neck muscles selected from the following: sternocleidomastoid, trapezius (e.g., upper trapezius), and scalene anterior, and / or ipsilaterally to one or more affected neck muscles selected from the following: splenius capitis, splenius cervicis, levator scapulae, and longissimus (e.g., longissimus capitis and / or longissimus cervicis).

[0081] In one embodiment, when treating torticollis, the modified BoNT / A may be administered to one or more neck muscles selected from the following: sternocleidomastoid, trapezius (e.g., upper trapezius), scalene anterior, splenius capitis, splenius cervicis, levator scapulae, longissimus (e.g., longissimus capitis and / or longissimus cervicis), and semispinalis cervicis. In one embodiment, when treating torticollis, the modified BoNT / A may be administered contralaterally to one or more affected neck muscles selected from the following: sternocleidomastoid, trapezius (e.g., upper trapezius), and scalene anterior, and / or ipsilaterally to one or more affected neck muscles selected from the following: splenius capitis, splenius cervicis, levator scapulae, longissimus (e.g., longissimus capitis and / or longissimus cervicis), and semispinalis cervicis.

[0082] In one embodiment, when treating torticollis, the modified BoNT / A may be administered to one or more affected neck muscles selected from the following: sternocleidomastoid, trapezius (e.g., upper trapezius), scalene anterior, splenius capitis, splenius cervicis, levator scapulae, longissimus (e.g., longissimus capitis and / or longissimus cervicis), semispinalis cervicis, rectus capitis posterior greater, multifidus, and obliquus capitis occipitalis. In one embodiment, when treating torticollis, the modified BoNT / A may be administered contralaterally to one or more affected neck muscles selected from the following: sternocleidomastoid, trapezius (e.g., upper trapezius), scalene anterior, semispinalis cervicis, and multifidus, and / or ipsilaterally to one or more affected neck muscles selected from the following: splenius capitis, splenius cervicis, levator scapulae, longissimus (e.g., longissimus capitis and / or longissimus cervicis), rectus capitis posterior greater, and obliquus capitis inferior.

[0083] In one embodiment, when treating torticollis, the modified BoNT / A may be administered to affected neck muscles, including: sternocleidomastoid, trapezius (e.g., upper trapezius), scalene anterior, splenius capitis, splenius cervicis, levator scapulae, longissimus (e.g., longissimus capitis and / or longissimus cervicis), semispinalis cervicis, rectus capitis posterior greater, multifidus, or obliquus capitis inferior. In one embodiment, when treating torticollis, the modified BoNT / A may be administered contralaterally to one or more affected neck muscles, including: sternocleidomastoid, trapezius (e.g., upper trapezius), scalene anterior, semispinalis cervicis, or multifidus, and / or ipsilaterally to one or more affected neck muscles, including: splenius capitis, splenius cervicis, levator scapulae, longissimus (e.g., longissimus capitis and / or longissimus cervicis), rectus capitis posterior greater, or obliquus capitis inferior.

[0084] In one embodiment, when treating cervical lateral curvature (later collis), the modified BoNT / A may be administered to one or more affected neck muscles selected from the following: levator scapulae, trapezius (e.g., upper trapezius), scalene complex (anterior scalene and / or middle scalene), sternocleidomastoid, splenius capitis, splenius cervix and longissimus (e.g., longissimus capitis and / or longissimus cervix). Preferably, when treating cervical lateral curvature, the modified BoNT / A may be administered ipsilaterally to one or more of said affected neck muscles.

[0085] In one embodiment, when treating cervical lateral bending, the modified BoNT / A may be administered to one or more affected neck muscles selected from the following: levator scapulae, trapezius (e.g., upper trapezius), scalene complex (anterior scalene and / or middle scalene), sternocleidomastoid, splenius capitis, splenius cervix, longissimus (e.g., longissimus capitis and / or longissimus cervix), and semispinalis cervix. In one embodiment, when treating cervical lateral bending, the modified BoNT / A may be administered ipsilaterally to one or more of said affected neck muscles.

[0086] In one embodiment, when treating cervical lateral bending, the modified BoNT / A may be administered to one or more affected neck muscles selected from the following: levator scapulae, trapezius (e.g., upper trapezius), scalene complex (anterior scalene and / or middle scalene), sternocleidomastoid, splenius capitis, splenius cervicis, longissimus (e.g., longissimus capitis and / or longissimus cervicis), and multifidus. In one embodiment, when treating cervical lateral bending, the modified BoNT / A may be administered ipsilaterally to one or more of said affected neck muscles.

[0087] In one embodiment, when treating cervical lateral bending, the modified BoNT / A may be administered to one or more affected neck muscles selected from the following: levator scapulae, trapezius (e.g., upper trapezius), scalene complex (e.g., anterior scalene and / or middle scalene), sternocleidomastoid, splenius capitis, splenius cervix, longissimus (e.g., longissimus capitis and / or longissimus cervix), semispinalis cervix, and multifidus. In one embodiment, when treating cervical lateral bending, the modified BoNT / A may be administered ipsilaterally to one or more of said affected neck muscles.

[0088] In one embodiment, when treating cervical lateral bending, the modified BoNT / A may be administered to one or more affected neck muscles, including: levator scapulae, trapezius (e.g., upper trapezius), scalene complex (e.g., anterior scalene and / or middle scalene), sternocleidomastoid, splenius capitis, splenius cervix, longissimus (e.g., longissimus capitis and / or longissimus cervix), semispinalis cervix, or multifidus. In one embodiment, when treating cervical lateral bending, the modified BoNT / A may be administered ipsilaterally to one or more of said affected neck muscles.

[0089] In one embodiment, when treating anterocollis, the modified BoNT / A may be administered to one or more affected neck muscles selected from the following: sternocleidomastoid, anterior scalene, and middle scalene. Preferably, when treating anterocollis, the modified BoNT / A may be administered bilaterally.

[0090] In one embodiment, when treating neck flexion, the modified BoNT / A may be administered to one or more affected neck muscles selected from the following: sternocleidomastoid, anterior scalene, middle scalene, levator scapulae, longus colli and submandibular complex (e.g., digastric, geniohyoid, mylohyoid, mylohyoid boutonniere and / or stylohyoid). Preferably, when treating neck flexion, the modified BoNT / A may be administered bilaterally.

[0091] In one embodiment, when treating neck flexion, the modified BoNT / A may be administered to one or more affected neck muscles selected from the following: sternocleidomastoid, anterior scalene, middle scalene, longus capitis, longus colli, and rectus capitis anterior. In one embodiment, when treating neck flexion, the modified BoNT / A may be administered bilaterally.

[0092] In one embodiment, when treating neck flexion, the modified BoNT / A may be administered to one or more affected neck muscles selected from the following: sternocleidomastoid, scalene anterior, scalene middle, levator scapulae, longus colli, submandibular complex (e.g., digastric, geniohyoid, mylohyoid, mylohyoid boutonniere, and / or stylohyoid), longus capitis, and rectus capitis anterior. In one embodiment, when treating neck flexion, the modified BoNT / A may be administered bilaterally.

[0093] In one embodiment, when treating neck flexion, the modified BoNT / A may be administered to one or more affected neck muscles, including: sternocleidomastoid, scalene anterior, scalene middle, levator scapulae, longus colli, submandibular complex (e.g., digastric cervicalis, geniohyoid, mylohyoid, mylohyoid boutonniere, and / or stylohyoid), longus capitis, or rectus capitis anterior. In one embodiment, when treating neck flexion, the modified BoNT / A may be administered bilaterally.

[0094] In one embodiment, when treating retrocollis, the modified BoNT / A may be administered to one or more affected neck muscles selected from the following: levator scapulae, trapezius (e.g., upper trapezius), longissimus (e.g., longissimus capitis and / or longissimus cervix), splenius capitis, splenius cervix, and semispinalis capitis. Preferably, when treating retrocollis, the modified BoNT / A may be administered bilaterally.

[0095] In one embodiment, when treating cervical retroversion, the modified BoNT / A may be administered to one or more affected neck muscles selected from the following: levator scapulae, trapezius (e.g., upper trapezius), longissimus (e.g., longissimus capitis and / or longissimus cervix), splenius capitis, splenius cervix, semispinalis capitis, semispinalis cervix, and the posterior paraspinal (e.g., posterior scalene, middle scalene, and / or anterior scalene, preferably posterior scalene). Preferably, when treating cervical retroversion, the modified BoNT / A may be administered bilaterally.

[0096] In one embodiment, when treating cervical retroversion, the modified BoNT / A may be administered to one or more affected neck muscles selected from the following: levator scapulae, trapezius (e.g., upper trapezius), longissimus (e.g., longissimus capitis and / or longissimus cervix), splenius capitis, splenius cervix, semispinalis capitis, semispinalis cervix, spinalis capitis, rectus capitis posterior greater, rectus capitis posterior less, and obliquus capitis superior. Preferably, when treating cervical retroversion, the modified BoNT / A may be administered bilaterally.

[0097] In one embodiment, when treating cervical retroversion, the modified BoNT / A may be administered to one or more affected neck muscles selected from the following: levator scapulae, trapezius (e.g., upper trapezius), longissimus (e.g., longissimus capitis and / or longissimus cervix), splenius capitis, splenius cervix, semispinalis capitis, semispinalis cervix, posterior paraspinal (e.g., posterior scalene, middle scalene and / or anterior scalene, preferably posterior scalene), spinalis capitis, rectus capitis posterior greater, rectus capitis posterior less and obliquus capitis superior. Preferably, when treating cervical retroversion, the modified BoNT / A may be administered bilaterally.

[0098] In one embodiment, when treating cervical retroversion, the modified BoNT / A may be administered to one or more affected neck muscles, including: levator scapulae, trapezius (e.g., upper trapezius), longissimus (e.g., longissimus capitis and / or longissimus cervix), splenius capitis, splenius cervix, semispinalis capitis, semispinalis cervix, posterior paraspinal (e.g., posterior scalene, middle scalene and / or anterior scalene, preferably posterior scalene), spinalis capitis, rectus capitis posterior greater, rectus capitis posterior less or obliquus capitis superior. Preferably, when treating cervical retroversion, the modified BoNT / A may be administered bilaterally.

[0099] In one embodiment, when treating a lateral shift, the modified BoNT / A may be administered to one or more affected neck muscles selected from the following: levator scapulae, trapezius (e.g., upper trapezius or lower trapezius), scalene complex (e.g., anterior scalene and / or middle scalene), sternocleidomastoid, splenius capitis, splenius cervix, longissimus (e.g., longissimus capitis and / or longissimus cervix), and semispinalis cervix. Preferably, when treating a lateral shift, the modified BoNT / A may be administered to affected neck muscles selected from the following: levator scapulae, semispinalis cervix, scalene medial, and longissimus cervix on a first side (e.g., left side) of the neck, and the modified BoNT / A may be administered to affected neck muscles selected from the following: sternocleidomastoid, lower trapezius, splenius capitis, semispinalis capitis, longissimus capitis, and levator scapulae on a second side (e.g., right side) of the neck.

[0100] In one embodiment, when treating lateral deviation, the modified BoNT / A may be administered to one or more affected neck muscles, including: levator scapulae, trapezius (e.g., upper trapezius or lower trapezius), scalene complex (e.g., anterior scalene and / or middle scalene), sternocleidomastoid, splenius capitis, splenius cervix, longissimus (e.g., longissimus capitis and / or longissimus cervix), or semispinalis cervix. Preferably, when treating lateral deviation, the modified BoNT / A may be administered to affected neck muscles, including: levator scapulae, semispinalis cervix, middle scalene or longissimus cervix on a first side (e.g., left side) of the neck, and the modified BoNT / A may be administered to affected neck muscles, including: sternocleidomastoid, lower trapezius, splenius capitis, semispinalis capitis, longissimus capitis, or levator scapulae on a second side (e.g., right side) of the neck.

[0101] In one embodiment, when treating laterocaput, the modified BoNT / A may be administered to one or more affected neck muscles selected from the following: lower trapezius, sternocleidomastoid, longissimus capitis, splenius capitis, semispinalis capitis, levator scapulae, and the posterior paraspinal muscles (e.g., posterior scalene, middle scalene, and / or anterior scalene, preferably posterior scalene). Preferably, when treating laterocaput, the modified BoNT / A may be administered ipsilaterally.

[0102] In one embodiment, when treating lateral head bending, the modified BoNT / A may be administered to one or more affected neck muscles, including the lower trapezius, sternocleidomastoid, longissimus capitis, splenius capitis, semispinalis capitis, levator scapulae, or posterior paraspinal muscles (e.g., posterior scalene, middle scalene, and / or anterior scalene, preferably posterior scalene). Preferably, when treating lateral head bending, the modified BoNT / A may be administered ipsilaterally.

[0103] In one embodiment, when treating torticaput, the modified BoNT / A may be administered to one or more affected neck muscles selected from the following: lower trapezius, sternocleidomastoid, longissimus capitis, splenius capitis, semispinalis capitis middle, and obliquus capitis inferior. Preferably, when treating torticaput, the modified BoNT / A may be administered ipsilaterally or contralaterally. For example, the modified BoNT / A may be administered contralaterally to one or more affected neck muscles selected from the following: lower trapezius, sternocleidomastoid, and semispinalis capitis middle, and / or the modified BoNT / A may be administered ipsilaterally to one or more affected neck muscles selected from the following: obliquus capitis inferior, longissimus capitis, and splenius capitis.

[0104] In one embodiment, when treating head rotation, the modified BoNT / A may be administered to one or more affected neck muscles, including the lower trapezius, sternocleidomastoid, longissimus capitis, splenius capitis, semispinalis capitis middle, or obliquus capitis inferior. Preferably, when treating head rotation, the modified BoNT / A may be administered ipsilaterally or contralaterally. For example, the modified BoNT / A may be administered contralaterally to one or more affected neck muscles, including the lower trapezius, sternocleidomastoid, or semispinalis capitis middle, and / or the modified BoNT / A may be administered ipsilaterally to one or more affected neck muscles, including the obliquus capitis inferior, longissimus capitis, or splenius capitis.

[0105] In one embodiment, when treating head antecaput, the modified BoNT / A may be administered to one or more affected neck muscles, including: longus capitis, levator scapulae, and sternocleidomastoid. Preferably, when treating head antecaput, the modified BoNT / A may be administered bilaterally.

[0106] In one embodiment, when treating head forward, the modified BoNT / A may be administered to one or more affected neck muscles, including: longus capitis, levator scapulae, or sternocleidomastoid. Preferably, when treating head forward, the modified BoNT / A may be administered bilaterally.

[0107] In one embodiment, when treating retrocaput, the modified BoNT / A may be administered to one or more affected neck muscles selected from: obliquus capitis inferior, semispinalis capitis, inferior trapezius, and splenius capitis. Preferably, when treating retrocaput, the modified BoNT / A may be administered bilaterally.

[0108] In one embodiment, when treating head tilt, the modified BoNT / A may be administered to one or more affected neck muscles, including: obliquus capitis inferior, semispinalis capitis, inferior trapezius, or splenius capitis. Preferably, when treating head tilt, the modified BoNT / A may be administered bilaterally.

[0109] In one embodiment, when treating sagittal shift, the modified BoNT / A may be administered to one or more affected neck muscles selected from the following: sternocleidomastoid, scalene anterior, scalene middle, levator scapulae, longus colli, submandibular complex (e.g., digastric, geniohyoid, mylohyoid, mylohyoid boutonniere, and / or stylohyoid), obliquus capitis inferior, semispinalis capitis, lower trapezius, and splenius capitis. Preferably, when treating sagittal deviation, the modified BoNT / A may be administered to affected neck muscles selected from: the sternocleidomastoid, anterior scalene, middle scalene, levator scapulae, longus colli and submandibular complex (e.g., digastric cervicalis, geniohyoid, mylohyoid, mylohyoid boutonniere and / or stylohyoid) on a first side (e.g., left side) of the neck, and the modified BoNT / A may be administered to affected neck muscles selected from: the inferior obliquus capitis, semispinalis capitis, inferior trapezius and splenius capitis on a second side (e.g., right side) of the neck.

[0110] In one embodiment, when treating sagittal deviation, the modified BoNT / A may be administered to one or more affected neck muscles, including: the sternocleidomastoid, anterior scalene, middle scalene, levator scapulae, longus colli, submandibular complex (e.g., digastric, geniohyoid, mylohyoid, mylohyoid boutonniere, and / or stylohyoid), inferior obliquus capitis, semispinalis capitis, inferior trapezius, or splenius capitis on a first side (e.g., the left side) of the neck. Preferably, when treating sagittal deviation, the modified BoNT / A may be administered to affected neck muscles including: the sternocleidomastoid, anterior scalene, middle scalene, levator scapulae, longus colli, or submandibular complex (e.g., digastric, geniohyoid, mylohyoid, mylohyoid boutonniere, and / or stylohyoid) on a first side (e.g., left side) of the neck, and the modified BoNT / A may be administered to affected neck muscles including: the inferior obliquus capitis, semispinalis capitis, inferior trapezius, or splenius capitis on a second side (e.g., right side) of the neck.

[0111] The modified BoNT / A is administered to the affected neck muscle by intramuscular injection. One or more unit doses (e.g., at least two unit doses) of the modified BoNT / A may be administered to the affected neck muscle. However, it is preferred to administer only a single unit dose per affected neck muscle. If the neck muscle is the sternocleidomastoid muscle, two unit doses may be administered. For example, two unit doses may be administered to the left sternocleidomastoid muscle and / or two unit doses may be administered to the right sternocleidomastoid muscle. If the neck muscle is the trapezius muscle (e.g., lower trapezius muscle), two unit doses may be administered. For example, two unit doses may be administered to the left trapezius muscle (e.g., upper left trapezius muscle) and / or two unit doses may be administered to the right trapezius muscle (e.g., upper right trapezius muscle).

[0112] A unit dose may be administered to the affected neck muscle at a single injection site. Thus, in some embodiments, the modified BoNT / A is administered at a unit dose per injection site in the affected neck muscle. However, it may be desirable to administer less than a unit dose at a single injection site, in which case the unit dose may be split (evenly or unevenly) between two or more injection sites in the affected neck muscle. Thus, the modified BoNT / A may be administered at two or more injection sites in the affected neck muscle. In preferred embodiments, the modified BoNT / A is administered at a less than unit dose per injection site in the affected neck muscle. Advantageously, administering more of the modified BoNT / A to the site may allow the clinician to clearly distinguish the muscle from other muscles and / or treat the particularly affected areas of the muscle when compared to less affected sites.

[0113] Most preferably, a unit dose is administered per injection site.

[0114] The modified BoNT / A may be administered at a dose of 750 pg to 4,000 pg per injection site, where the modified BoNT / A comprises a BoNT / A light chain and translocation domain and a BoNT / B receptor binding domain (H CPreferably, the modified BoNT / A is administered at a dose of 1,000 pg or 2,000 pg per injection site, where the modified BoNT / A consists of the BoNT / A light chain and translocation domain and the BoNT / B receptor binding domain (H C domain).

[0115] The modified BoNT / A may be administered at a dose of 31 units to 166.4 units per injection site, wherein the modified BoNT / A comprises a BoNT / A light chain and translocation domain and a BoNT / B receptor binding domain (H C Preferably, the modified BoNT / A is administered at a dose of 41.6 or 83.2 units per injection site, wherein the modified BoNT / A comprises a BoNT / A light chain and translocation domain and a BoNT / B receptor binding domain (H domain). C domain).

[0116] The phrase "at least a single unit dose is administered" means that at least substantially all of the single unit dose is administered. For example, a residual amount of the unit dose (e.g., up to 1%, up to 0.1%, or up to 0.01%) may remain in the vial in which the modified BoNT / A was reconstituted. However, preferably, at least all of the single unit dose is administered (e.g., to one or more injection sites).

[0117] The efficacy of modified BoNT / A for use in the present invention can be determined using mouse LD assays according to standard techniques. 50 In said assay, one unit is calculated as the median lethal dose (LD ) in mice. 50 ) is defined as the amount of modified BoNT / A equivalent to the calculated median intraperitoneal lethal dose in mice.

[0118] The modified BoNT / A for use in the present invention comprises a BoNT / A light chain and translocation domain and a BoNT / B receptor binding domain (H CIn the case of a modified BoNT / A consisting of the nucleotide sequence (nucleotide sequence) and the nucleotide sequence (domain), the amount of modified BoNT / A corresponding to 1 unit in the assay is preferably 24.04 pg.

[0119] When the modified BoNT / A for use in the present invention is a modified BoNT / A that contains one or more modifications to amino acid residues selected from the following: ASN886, ASN905, GLN915, ASN918, GLU920, ASN930, ASN954, SER955, GLN991, GLU992, GLN995, ASN1006, ASN1025, ASN1026, ASN1032, ASN1043, ASN1046, ASN1052, ASP1058, HIS1064, ASN1080, GLU1081, GLU1083, ASP1086, ASN1188, ASP1213, GLY1215, ASN1216, GLN1229, ASN1242, ASN1243, SER1274, and THR1277, wherein the modification is selected from: replacement of an acidic surface-exposed amino acid residue with a basic amino acid residue, replacement of an acidic surface-exposed amino acid residue with an uncharged amino acid residue, replacement of an uncharged surface-exposed amino acid residue with a basic amino acid residue, insertion of a basic amino acid residue and deletion of an acidic surface-exposed amino acid residue; The amount of modified BoNT / A corresponding to one unit in the assay is preferably 8.44 pg.

[0120] The term "up to" when used in reference to a numerical value (e.g., up to 170,000 pg) means up to and including the recited numerical value. Thus, by way of example, a reference to an "up to 170,000 pg" administration of a modified BoNT / A includes administration of 170,000 pg of modified BoNT / A as well as administration of less than 170,000 pg.

[0121] The unit dose may be expressed in terms of an amount of modified BoNT / A, in units of modified BoNT / A, or a combination thereof.

[0122] In one embodiment, the modified BoNT / A may be administered in the doses set forth below, as follows, into one or more of the following neck muscles: [Table A]

[0123] In one embodiment, the modified BoNT / A may be administered in the doses set forth below into one or more of the following neck muscles as follows: [Table B]

[0124] (The modified BoNT / A comprises a modified BoNT / A light chain and a translocation domain, and a BoNT / B receptor binding domain (H C domain), so long as the total dose administered during the treatment does not exceed an upper limit of 170,000 pg or 7,070 units, or the modified BoNT / A is ASN886, ASN905, GLN915, ASN918, GLU920, ASN930, ASN954, SER955, GLN991, GLU992, GLN995, ASN1006, ASN1025, ASN1026, ASN1032, ASN1043, ASN1046, ASN1052, ASP1058, HIS1064, ASN1080, GLU1081, GLU1083, ASP1086, ASN1188, ASP1213, GLY1215, ASN1216, GLN12 29, ASN1242, ASN1243, SER1274, and THR1277, where the modification is selected from substitution of an acidic surface-exposed amino acid residue with a basic amino acid residue, substitution of an acidic surface-exposed amino acid residue with an uncharged amino acid residue, substitution of an uncharged surface-exposed amino acid residue with a basic amino acid residue, insertion of a basic amino acid residue, and deletion of an acidic surface-exposed amino acid residue, so long as the total dose administered during treatment does not exceed an upper limit of 80,000 pg or 9,480 units, in one embodiment, the modified BoNT / A may be administered at the following doses, as follows, into one or more of the following neck muscles: [Table C]

[0125] (The modified BoNT / A comprises a modified BoNT / A light chain and a translocation domain, and a BoNT / B receptor binding domain (H C domain), as long as the total dose administered during treatment does not exceed an upper limit of 170,000 pg or 7,070 units, or the modified BoNT / A is ASN886, ASN905, GLN915, ASN918, GLU920, ASN930, ASN954, SER955, GLN991, GLU992, GLN995, ASN1006, ASN1025, ASN1026, ASN1032, ASN1043, ASN1046, ASN1052, ASP1058, HIS1064, ASN1080, GLU1081, GLU1083, ASP1086, ASN1188, ASP1213, GLY1215, AS When the modified BoNT / A comprises a modification at one or more amino acid residues selected from N1216, GLN1229, ASN1242, ASN1243, SER1274, and THR1277, where the modification is selected from replacement of an acidic surface-exposed amino acid residue with a basic amino acid residue, replacement of an acidic surface-exposed amino acid residue with an uncharged amino acid residue, replacement of an uncharged surface-exposed amino acid residue with a basic amino acid residue, insertion of a basic amino acid residue, and deletion of an acidic surface-exposed amino acid residue, the modified BoNT / A may be administered at the following doses and into the following neck muscles as follows (unless an upper limit of 80,000 pg or 9,480 units is exceeded): [Table D]

[0126] Preferably, the modified BoNT / A may be administered in the doses set forth below, as follows, into one or more of the following neck muscles: [Table E]

[0127] Preferably, the modified BoNT / A may be administered in the following doses, as follows, into the following neck muscles: [Table F]

[0128] As used herein, the terms "right" and "left" have their ordinary meanings, e.g., an affected individual's right levator scapulae muscle is the levator scapulae muscle on the affected individual's right side, while an affected individual's left levator scapulae muscle is the levator scapulae muscle on the affected individual's left side.

[0129] The total number of unit doses administered in a given treatment may be up to 10 times or up to 7 times the unit dose. The total number of unit doses may be divided according to the affected neck muscles to be treated, for example, in one embodiment, if the dose delivered during a treatment is 1 unit dose, only one affected neck muscle may be treated. However, if the total number is 2 unit doses, two affected neck muscles may be treated. The total number of unit doses administered may be up to 9 times, up to 8 times, up to 7 times, or up to 6 times. The total number of unit doses may be at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, preferably at least 2 times, the unit dose. The total number of unit doses administered may be 1 to 10 times, or 5 to 10 times, preferably 7 to 10 times.

[0130] The modified BoNT / A comprises a modified BoNT / A light chain and translocation domain, and a BoNT / B receptor binding domain (H Cdomain), as long as the total dose administered in the treatment does not exceed the upper limit of 170,000 pg or 7,070 units, or the modified BoNT / A is ASN886, ASN905, GLN915, ASN918, GLU920, ASN930, ASN954, SER955, GLN991, GLU992, GLN995, ASN1006, ASN1025, ASN1026, ASN1032, ASN1043, ASN1046, ASN1052, ASP1058, HIS1064, ASN1080, GLU1081, GLU1083, ASP1086, ASN1188, ASP1213, GLY1215, ASN1216, GLN1229, ASN12 42, ASN1243, SER1274, and THR1277, where the modification is selected from substitution of an acidic surface-exposed amino acid residue with a basic amino acid residue, substitution of an acidic surface-exposed amino acid residue with an uncharged amino acid residue, substitution of an uncharged surface-exposed amino acid residue with a basic amino acid residue, insertion of a basic amino acid residue, and deletion of an acidic surface-exposed amino acid residue, the total number of unit doses administered in the treatment may be up to 20-fold or up to 15-fold (preferably up to 14-fold or up to 7-fold) the unit dose, so long as the total dose administered in the treatment does not exceed the upper limit of 80,000 pg or 9,480 units.

[0131] Therefore, the modified BoNT / A comprises a modified BoNT / A light chain and translocation domain, and a BoNT / B receptor binding domain (H Cdomain), as long as the total dose administered during treatment does not exceed the upper limit of 170,000 pg or 7,070 units, or the modified BoNT / A is ASN886, ASN905, GLN915, ASN918, GLU920, ASN930, ASN954, SER955, GLN991, GLU992, GLN995, ASN1006, ASN1025, ASN1026, ASN1032, ASN1043, ASN1046, ASN1052, ASP1058, HIS1064, ASN1080, GLU1081, GLU1083, ASP1086, ASN1188, ASP1213, GLY1215, ASN1216, GLN1229, A When the compound comprises a modification at one or more amino acid residues selected from SN1242, ASN1243, SER1274, and THR1277, where the modification is selected from substitution of an acidic surface-exposed amino acid residue with a basic amino acid residue, substitution of an acidic surface-exposed amino acid residue with an uncharged amino acid residue, substitution of an uncharged surface-exposed amino acid residue with a basic amino acid residue, insertion of a basic amino acid residue, and deletion of an acidic surface-exposed amino acid residue, the total number of unit doses administered may be up to 13-fold, up to 12-fold, up to 10-fold, up to 9-fold, up to 8-fold, up to 7-fold, or up to 6-fold, so long as the total dose administered during treatment does not exceed the upper limit of 80,000 pg or 9,480 units.

[0132] The modified BoNT / A comprises a modified BoNT / A light chain and translocation domain, and a BoNT / B receptor binding domain (H Cdomain), as long as the total dose administered during treatment does not exceed the upper limit of 170,000 pg or 7,070 units, or the modified BoNT / A is ASN886, ASN905, GLN915, ASN918, GLU920, ASN930, ASN954, SER955, GLN991, GLU992, GLN995, ASN1006, ASN1025 , ASN1026, ASN1032, ASN1043, ASN1046, ASN1052, ASP1058, HIS1064, ASN1080, GLU1081, GLU1083, ASP1086, ASN1188, ASP1213, GLY1215, ASN1216, GLN1229, ASN1242, ASN1243, SER1274, and THR1277. In the case of a compound comprising a modification at one or more selected amino acid residues, wherein the modification is selected from substitution of an acidic surface-exposed amino acid residue with a basic amino acid residue, substitution of an acidic surface-exposed amino acid residue with an uncharged amino acid residue, substitution of an uncharged surface-exposed amino acid residue with a basic amino acid residue, insertion of a basic amino acid residue, and deletion of an acidic surface-exposed amino acid residue, the total number of unit doses administered may be at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, and at least 10-fold the unit dose, preferably at least 2-fold (e.g., 7-fold or 14-fold), so long as the total dose administered during the treatment does not exceed the upper limit of 80,000 pg or 9,480 units.

[0133] The modified BoNT / A comprises a modified BoNT / A light chain and translocation domain, and a BoNT / B receptor binding domain (H Cdomain), as long as the total dose administered during treatment does not exceed the upper limit of 170,000 pg or 7,070 units, or the modified BoNT / A is ASN886, ASN905, GLN915, ASN918, GLU920, ASN930, ASN954, SER955, GLN991, GLU992, GLN995, ASN1 006, ASN1025, ASN1026, ASN1032, ASN1043, ASN1046, ASN1052, ASP1058, HIS1064, ASN1080, G LU1081, GLU1083, ASP1086, ASN1188, ASP1213, GLY1215, ASN1216, GLN1229, ASN1242, ASN124 3, SER1274, and THR1277, where the modification is selected from substitution of an acidic surface-exposed amino acid residue with a basic amino acid residue, substitution of an acidic surface-exposed amino acid residue with an uncharged amino acid residue, substitution of an uncharged surface-exposed amino acid residue with a basic amino acid residue, insertion of a basic amino acid residue, and deletion of an acidic surface-exposed amino acid residue, the total number of unit doses administered may be 1-20 times (e.g., 12-16 times), 1-15 times, 1-14 times, or 5-14 times, preferably 7-14 times, so long as the total dose administered during treatment does not exceed the upper limit of 80,000 pg or 9,480 units.

[0134] One of skill in the art will take into consideration cases where the affected individual has recently undergone (or will subsequently undergo) additional treatment with a Clostridial neurotoxin (e.g., BoNT), for example as part of cosmetic surgery or treatment for another condition, and using routine techniques in the art, one of skill in the art will be able to adapt the treatment regimen appropriately.

[0135] The modified BoNT / A of the present invention preferably has a longer duration of action (e.g., at least 5%, 10%, 25%, or 50% improvement in one or more symptoms) when compared to unmodified BoNT / A (e.g., Dysport®). The duration of action can be at least 1.25-fold, 1.5-fold, 1.75-fold, 2.0-fold, or 2.25-fold. The duration of action of the modified BoNT / A can be between 6 and 9 months. For example, the duration of action can be at least: 4.5 months, 5.0 months, 5.5 months, 6 months, 6.5 months, 7.0 months, 7.5 months, 8 months, 8.5 months, or 9.0 months (from onset). In certain embodiments, the duration of action can be longer than 9.0 months.

[0136] When multiple affected neck muscles are administered, said administrations are preferably performed in the same treatment session.

[0137] Treatment may be repeated at an appropriate time period after administration of modified BoNT / A. Given that the duration of action is twice that of unmodified BoNT / A (e.g., Dysport®), there is an appropriately longer period between subsequent administrations than if the affected individual were to undergo treatment with unmodified BoNT / A (e.g., Dysport®). In the present invention, the affected individual may be re-administered BoNT / A at least 18 weeks, 20 weeks, 25 weeks, or 30 weeks after the previous administration. For example, in the present invention, the affected individual may be re-administered modified BoNT / A at least 18 weeks to 45 weeks, preferably 20 weeks to 35 weeks, after the previous administration.

[0138] The effectiveness of the treatment (including the severity of the affected person's symptoms) may be assessed using the Toronto Western Spasmodic Torticollis Rating Scale (TWSTRS) reviewed by Jost et al. 2013 J Neural Transm (Vienna) 120(3):487-496. The TWSTRS is a composite scale consisting of the TWSTRS Severity Scale, the TWSTRS Disability Scale, and the TWSTRS Pain Scale. Higher TWSTRS scores indicate more severe disease. The TWSTRS Pain Scale includes the following items: A. Maximum range of motion (rotation, tilt, forward or backward bending, lateral deviation, sagittal deviation), B. Duration factor, C. Effect of sensory tricks, D. Shoulder elevation / forward displacement, E. Range of motion (without the aid of sensory illusions), F. Time (maximum 60 seconds that the affected person can maintain the head within 10 degrees of neutral position without the aid of sensory tricks). The duration factor is weighted twice, and the sum of A to F can be a maximum score of 35. The TWSTRS disability scale is a six-item scale that includes assessments of performance in daily activities that may be affected by cervical dystonia: occupational performance (work or housework), activities of daily living (eating, dressing, hygiene), driving, reading, watching television and leisure activities outside the home. Each item is rated on a six-point scale (0=no difficulty, 5=highest degree of disability) with the sum of the items giving a maximum score of 30. The TWSTRS pain scale consists of severity ratings for the patient's most common, worst and best pain in the past week, as well as a duration component and an assessment of the contribution of pain to disability. The scale ranges between 0 and 20, with 20 being assigned to the most severe pain that can be experienced.

[0139] As used herein, a "subject" may be a mammal, such as a human or other mammal. Preferably, a "subject" refers to a human subject. A "subject" is preferably an adult subject, i.e., a subject who is at least 18 years of age. The terms "subject" and "patient" are used synonymously herein.

[0140] As used herein, the term "treatment" or "treating" encompasses corrective treatment (treatment of an affected individual who already has a disease) as well as preventative treatment (e.g., to prevent the onset of a disease). Preferably, as used herein, "treatment" or "treating" refers to corrective treatment. As used herein, the term "treatment" or "treating" refers to those diseases and / or symptoms.

[0141] Suitable modified BoNT / A polypeptides (and nucleotide sequences encoding the same, if present) are described in WO2015 / 004461A1 and WO2017 / 191315, both of which are incorporated by reference in their entireties herein.

[0142] An example of a clostridial neurotoxin produced by bacteria of the genus Clostridium is BoNT / A. Other examples of such clostridial neurotoxins include those produced by C. baratii and C. butyricum, as well as those produced by Clostridium tetani (TeNT) and Clostridium botulinum (BoNT) serotypes B to G. The neurotoxins are highly potent and specific, and can poison nerves and other cells to which they are delivered. The clostridial toxins are some of the most potent toxins known. For example, botulinum neurotoxins have a median lethal dose (LD ) in mice ranging from 0.5 to 5 ng / kg, depending on the serotype. 50 ) value. Both tetanus toxin and botulinum toxin act by inhibiting the function of affected neurons, specifically the release of neurotransmitters. Tetanus toxin acts in the central nervous system, while botulinum toxin acts at the neuromuscular junction and inhibits cholinergic transmission in the peripheral nervous system.

[0143] In nature, clostridial neurotoxins (including BoNT / A) are synthesized as single-chain polypeptides by a proteolytic cleavage event that results in the formation of two polypeptide chains linked by a disulfide bond. Cleavage occurs at a specific cleavage site, often referred to as the active site, located between the cysteine ​​residues that provide the interchain disulfide bond. It is this two-chain form that is the active form of the toxin. The two chains are referred to as the heavy chain (H chain), with a molecular weight of approximately 100 kDa, and the light chain (L chain), with a molecular weight of approximately 50 kDa. The H chain is composed of an N-terminal cartwheel component (H N domain) and C-terminal targeting component (H C The cleavage site is located between the L chain and the translocation domain component. C After the H domain binds to the target neuron and the bound toxin is internalized into the cell by endosomes, N The domain translocates the L chain across the endosomal membrane into the cytosol, where it provides a protease function (also known as a non-cytotoxic protease).

[0144] Non-cytotoxic proteases act by proteolytic cleavage of intracellular transport proteins known as SNARE proteins (e.g., SNAP-25, VAMP, or syntaxin) - Gerald K (2002) "Cell and Molecular Biology" (4 th See the American Journal of Clinical Toxins (2011) (edition 11, John Wiley & Sons, Inc.). The acronym SNARE is derived from Solute NSF Attachment Receptor, where NSF stands for N-ethylmaleimide-sensitive factor. SNARE proteins are essential for intracellular vesicle fusion and thus for the secretion of molecules from cells via vesicular trafficking. The protease function is a zinc-dependent endopeptidase activity that shows high substrate specificity for SNARE proteins. Thus, once delivered to the desired target cells, the non-cytotoxic proteases can inhibit cellular secretion from the target cells. The L-chain proteases of clostridial toxins are non-cytotoxic proteases that cleave SNARE proteins.

[0145] Given the ubiquitous nature of SNARE proteins, clostridial neurotoxins, such as botulinum toxin, have been used successfully in a wide range of therapeutic approaches.

[0146] For further details regarding the genetic basis of toxin production in C. botulinum and C. tetani, see Henderson et al (1997), The Clostridia: Molecular Biology and Pathogenesis, Academic press.

[0147] As mentioned above, clostridial neurotoxins are composed of two polypeptide chains, a heavy chain (H chain) with a molecular weight of about 100 kDa and a light chain (L chain) with a molecular weight of about 50 kDa. The H chain contains a C-terminal targeting component (receptor binding domain or H C domain) and the N-terminal cartwheel component (H N domain).

[0148] Clostridial neurotoxin domains are described in more detail below.

[0149] Examples of light chain reference sequences include: Botulinum neurotoxin type A: amino acid residues 1-448 Botulinum neurotoxin type B: amino acid residues 1-440

[0150] The above specific reference sequences should be considered as a guideline, as slight variations may occur depending on the subserotype. For example, US2007 / 0166332 (hereby incorporated by reference in its entirety) cites the following slightly different Clostridium sequences: Botulinum neurotoxin type A: amino acid residues M1 to K448 Botulinum neurotoxin type B: amino acid residues M1 to K441

[0151] A translocation domain is a fragment of the heavy chain of a clostridial neurotoxin approximately equal to the amino-terminal half of the heavy chain, or the domain corresponding to that fragment in an intact heavy chain.

[0152] Examples of translocation domain references include: Botulinum neurotoxin type A - amino acid residues (449-871) Botulinum neurotoxin type B - amino acid residues (441-858)

[0153] The reference sequences identified above should be considered as a guideline, as slight variations may occur depending on the subserotype. By way of example, US2007 / 0166332 (hereby incorporated by reference) cites slightly different Clostridium sequences: Botulinum neurotoxin type A - amino acid residues (A449~K871) Botulinum neurotoxin type B - amino acid residues (A442-S858)

[0154] In the context of the present invention, various BoNT / AHs containing a translocation domain N The regions may be useful in embodiments of the present invention. N The region is approximately 410 to 430 amino acids in length and contains the translocation domain. N Studies have shown that the full length of the region is not necessary for the translocation activity of the translocation domain. Thus, aspects of this embodiment include BoNT / AH polypeptides that include a translocation domain having a length of, for example, at least 350 amino acids, at least 375 amino acids, at least 400 amino acids, or at least 425 amino acids. N Other aspects of this embodiment may include a BoNT / AH1 gene that includes a translocation domain having a length of, for example, at most 350 amino acids, at most 375 amino acids, at most 400 amino acids, or at most 425 amino acids. N It may contain areas.

[0155] H N The term refers to naturally occurring BoNT / AH.N and modified BoNT / AH having non-naturally occurring amino acid sequences and / or artificial amino acid residues. N Preferably, the modified BoNT / AH N The moiety still exhibits the translocation function described above.

[0156] Clostridial neurotoxin receptor binding domain (H C ) Examples of reference sequences include: BoNT / A‐N872-L1296 BoNT / B‐E859-E1291

[0157] The Hc domain of clostridial neurotoxins (such as BoNT / A) is approximately 50 kDa. CC Domain and H CN It contains two distinct structural features called domains, both typically around 25 kDa. The amino acid residues involved in receptor binding are mainly H CC The H domain of natural Clostridial neurotoxins is thought to be located in C A domain may comprise between about 400 and 440 amino acid residues. This fact is confirmed by the following publications, each of which is incorporated herein by reference in its entirety: Umland TC (1997) Nat. Struct. Biol. 4: 788-792; Herreros J (2000) Biochem. J. 347: 199-204; Halpern J (1993) J. Biol. Chem. 268: 15, pp. 11188-11192; Rummel A (2007) PNAS 104: 359-364; Lacey DB (1998) Nat. Struct. Biol. 5: 898-902; Knapp (1998) Am. Cryst. Assoc. Abstract Papers 25: 90; Swaminathan and Eswaramoorthy (2000) Nat. Struct. Biol. 7: 1751-1759; and Rummel A (2004) Mol. Microbiol. 51(3), 631-643.

[0158] H CN Example domains (references) include: Botulinum neurotoxin type A - amino acid residues (872-1110) Botulinum neurotoxin type B - amino acid residues (859-1097)

[0159] The above sequence positions may vary slightly depending on the serotype / subserotype, and further H CN Example domains (references) include: Botulinum neurotoxin type A - amino acid residues (874-1110) Botulinum neurotoxin type B - amino acid residues (861-1097)

[0160] H CC Examples of domains include: Botulinum neurotoxin type A - amino acid residues (Y1111 to L1296) Botulinum neurotoxin type B - amino acid residues (Y1098-E1291)

[0161] L chain and H N The domains (optionally complete or partial activation loops, e.g., including the complete activation loop if the modified BoNT / A is in single-chain form, or including a truncated / partial activation loop if the modified BoNT / A is in two-chain form) are collectively referred to as the LH domain. N This LH may be called a domain. N The domain is therefore further C It does not include the domain.

[0162] A modified BoNT / A for use in the present invention may contain a modification at one or more amino acid residues selected from the following: ASN886, ASN905, GLN915, ASN918, GLU920, ASN930, ASN954, SER955, GLN991, GLU992, GLN995, ASN1006, ASN1025, ASN1026, ASN1032, ASN1043, ASN1046, ASN1052, ASP1058, HIS1064, ASN1080, GLU1081, GLU1083, ASP1086, ASN1188, ASP1213, GLY1215, ASN1216, GLN1229, ASN1242, ASN1243, SER1274, and THR1277. Such modified BoNT / As exhibit reduced or absent side effects as compared to the use of known BoNT / As. The enhanced tissue retention properties of the modified BoNT / As of the present invention may also provide increased utility and / or duration of action, allowing for reduced dosages (or increased dosages without additional side effects) to be used as compared to known Clostridial neurotoxin therapies, thus providing further advantages.

[0163] The modification may be a modification relative to the unmodified BoNT / A shown as SEQ ID NO:2, where the numbering of the amino acid residues is determined by alignment with SEQ ID NO:2. Since the presence of a methionine residue at position 1 of SEQ ID NO:2 is optional (as are the SEQ ID NOs corresponding to the modified BoNT / A polypeptides described herein), one of skill in the art would take into account the presence / absence of the methionine residue when determining the numbering of the amino acid residues. For example, if SEQ ID NO:2 contains a methionine, the position numbering is as defined above (e.g., ASN886 becomes ASN886 in SEQ ID NO:2). Alternatively, if a methionine is not present in SEQ ID NO:2, the numbering of that amino acid residue should be corrected by -1 (e.g., ASN886 becomes ASN885 in SEQ ID NO:2). Similar considerations apply to the presence / absence of a methionine at position 1 of other polypeptide sequences described herein, and one of skill in the art would easily determine the correct numbering of the amino acid residues using routine techniques in the art.

[0164] Alignment to determine the numbering of amino acid residues described herein may be performed using any of the methods described herein for determining sequence matches and / or % sequence identity.

[0165] The amino acid residues designated for modification are surface exposed amino acid residues.

[0166] The modified BoNT / A comprises a modification at one or more amino acid residues selected from the following: ASN886, ASN930, ASN954, SER955, GLN991, ASN1025, ASN1026, ASN1052, ASN1188, ASP1213, GLY1215, ASN1216, GLN1229, ASN1242, ASN1243, SER1274, and THR1277. The modified BoNT / A may be encoded by a nucleic acid sequence having at least 70% sequence identity to a nucleic acid selected from SEQ ID NOs: 3, 5, 7, and 9. For example, a nucleic acid sequence having at least 80%, 90%, 95%, or 99.9% sequence identity to a nucleic acid sequence selected from SEQ ID NOs: 3, 5, 7, and 9. Preferably, the modified BoNT / A used in the present invention may be encoded by a nucleic acid comprising (or consisting of) SEQ ID NOs: 3, 5, 7, or 9. The modified BoNT / A may comprise a polypeptide sequence having at least 70% sequence identity to a polypeptide sequence selected from SEQ ID NOs: 4, 6, 8, and 10. For example, a polypeptide sequence having at least 80%, 90%, 95%, or 99.9% sequence identity to a polypeptide sequence selected from SEQ ID NOs: 4, 6, 8, and 10. Preferably, the modified BoNT / A used in the present invention may comprise (and more preferably consists of) a polypeptide sequence selected from SEQ ID NOs: 4, 6, 8, and 10.

[0167] The term "one or more amino acid residues" when used in the context of a modified BoNT / A preferably means at least 2, 3, 4, 5, 6, or 7 of the displayed amino acid residues. Thus, a modified BoNT / A may contain at least 2, 3, 4, 5, 6, or 7 (preferably 7) modifications in the displayed amino acid residues. The modified BoNT / A may contain 1 to 30, 3 to 20, or 5 to 10 amino acid modifications. More preferably, the term "one or more amino acid residues" when used in the context of a modified BoNT / A means all of the displayed amino acid residues.

[0168] Preferably, other than one or more modifications at the indicated amino acid residues, when compared to SEQ ID NO:2, the modified BoNT / A contains no further amino acid modifications.

[0169] Most preferably, the modified BoNT / A comprises (more preferably consists of) a modification in one or more amino acid residues selected from the following: ASN886, ASN930, SER955, GLN991, ASN1026, ASN1052, and GLN1229. The modified BoNT / A may be encoded by a nucleic acid sequence having at least 70% sequence identity to SEQ ID NO:3. For example, a nucleic acid sequence having at least 80%, 90%, 95%, or 99.9% sequence identity to SEQ ID NO:3. Preferably, the modified BoNT / A used in the present invention may be encoded by a nucleic acid comprising (or consisting of) SEQ ID NO:3. The modified BoNT / A may comprise a polypeptide sequence having at least 70% sequence identity to SEQ ID NO:4. For example, a polypeptide sequence having at least 80%, 90%, 95%, or 99.9% sequence identity to SEQ ID NO:4. Preferably, the modified BoNT / A used in the present invention may comprise (more preferably consists of) SEQ ID NO:4.

[0170] The modifications may be selected from the following: (i) replacement of acidic surface-exposed amino acid residues with basic amino acid residues; (ii) replacement of acidic surface-exposed amino acid residues with uncharged amino acid residues; (iii) replacement of uncharged surface-exposed amino acid residues with basic amino acid residues; (iv) insertion of a basic amino acid residue; and (v) Deletion of acidic surface-exposed amino acid residues.

[0171] The modifications as described above result in a modified BoNT / A that has an increased positive surface charge and an increased isoelectric point when compared to the corresponding unmodified BoNT / A.

[0172] The isoelectric point (pI) is a characteristic property that is unique to a given protein. As is well known in the art, proteins are made up of specific sequences of amino acids (also called amino acid residues in the case of proteins). Each of the 20 standard amino acids has a different side chain (or R group), which means that each amino acid residue in a protein exhibits different chemical properties, such as charge and hydrophobicity. These properties can be influenced by the surrounding chemical environment, such as temperature and pH. The overall chemical properties of a protein depend on the sum of these various factors.

[0173] Certain amino acid residues (discussed in more detail below) have ionizable side chains that can exhibit a charge depending on the pH of their surroundings. Whether such a side chain is charged at a given pH depends on the pKa of the associated ionizable moiety, where pKa is the negative logarithm of the acid dissociation constant (Ka) for a particular proton from the conjugate base. For example, acidic residues such as aspartic acid and glutamic acid have side chain carboxylic acid groups with pKa values ​​of approximately 4.1 (the exact pKa value can depend on temperature, ionic strength, and the microenvironment of the ionizable group). These side chains therefore exhibit a negative charge at pH 7.4 (often referred to as "physiological pH"). At lower pH values, these side chains become protonated and lose their charge.

[0174] Conversely, basic residues such as lysine and arginine have nitrogen-containing side groups with pKa values ​​of approximately 10 to 12. These side chains therefore exhibit a positive charge at pH 7.4. These side chains become deprotonated and lose their charge at higher pH values.

[0175] Thus, the overall (net) charge of a protein molecule depends on the number of acidic and basic residues present in the protein (and their degree of surface exposure) and on the surrounding pH. Changing the surrounding pH changes the overall charge of the protein. Thus, for any protein, there exists a given pH at which the number of positive and negative charges is equal and the protein exhibits no overall net charge. This point is known as the isoelectric point (pI). Isoelectric point is a standard concept in protein biochemistry with which those skilled in the art are familiar.

[0176] The isoelectric point (pI) is therefore defined as the pH value at which a protein exhibits a net charge of zero. An increase in pI means that a higher pH value is required for the protein to exhibit a net charge of zero. Hence, an increase in pI represents an increase in the net positive charge of a protein at a given pH. Conversely, a decrease in pI means that a lower pH value is required for the protein to exhibit a net charge of zero. Thus, a decrease in pI represents a decrease in the net positive charge of the protein at a given pH.

[0177] Methods for determining the pI of a protein are known in the art and will be familiar to those skilled in the art. As an example, the pI of a protein can be calculated from the average pKa value of each amino acid present in the protein ("calculated pI"). Such calculations can be performed using computer programs known in the art, such as ExPASy's Compute pI / MW Tool (.expasy.org / compute_pi / ), which is the preferred method for calculating pI in the present invention. Comparison of pI values ​​between different molecules should be performed using the same calculation method / program.

[0178] If desired, the calculated pI of a protein can be experimentally confirmed using the technique of isoelectric focusing ("actual pI"). In this technique, electrophoresis is used to separate proteins according to their pI. Isoelectric focusing is typically performed using a gel with an immobilized pH gradient. When an electric field is applied, the protein migrates through the pH gradient until it reaches a pH at which the net charge is zero (this point is the pI of the protein). Since the results provided by isoelectric focusing are typically relatively low resolution in nature, the inventors believe that the results obtained by calculated pI (as described above) are more suitable for use.

[0179] Throughout this specification, unless otherwise indicated, "pI" means "calculated pI."

[0180] The pI of a protein can be increased or decreased by altering the number of basic and / or acidic groups displayed on its surface. This can be achieved by modifying one or more amino acids of the protein. For example, an increase in pI can be brought about by decreasing the number of acidic residues or by increasing the number of basic residues.

[0181] The modified BoNT / A of the present invention may have a pI value that is at least 0.2, 0.4, 0.5, or 1 pI unit higher than the pI value of the unmodified BoNT / A (e.g., SEQ ID NO: 2). Preferably, the modified BoNT / A may have a pI of at least 6.6, such as at least 6.8.

[0182] The properties of the 20 common amino acids are shown in the table below: [Table G]

[0183] The following amino acids are considered charged amino acids: aspartic acid (negative), glutamic acid (negative), arginine (positive) and lysine (positive).

[0184] At pH 7.4, the side chains of aspartic acid (pKa 3.1) and glutamic acid (pKa 4.1) are negatively charged, while the side chains of arginine (pKa 12.5) and lysine (pKa 10.8) are positively charged. Aspartic acid and glutamic acid are called acidic amino acid residues. Arginine and lysine are called basic amino acid residues.

[0185] The following amino acids are considered to be uncharged polar (meaning they can participate in hydrogen bonds) amino acids: asparagine, glutamine, histidine, serine, threonine, tyrosine, cysteine, methionine, and tryptophan.

[0186] The following amino acids are considered to be uncharged hydrophobic amino acids: alanine, valine, leucine, isoleucine, phenylalanine, proline and glycine.

[0187] In an amino acid insertion, an additional amino acid residue (one that is not normally present) is incorporated into the BoNT / A polypeptide sequence, thereby increasing the total number of amino acid residues in the sequence. In an amino acid deletion, an amino acid residue is removed from the amino acid sequence of a Clostridial toxin, thereby decreasing the total number of amino acid residues in the sequence.

[0188] Preferably, the modification is a substitution, advantageously retaining the same number of amino acid residues in the modified BoNT / A. In an amino acid substitution, an amino acid residue that forms part of the BoNT / A polypeptide sequence is replaced with a different amino acid residue. As mentioned above, the substituted amino acid residue may be one of the 20 standard amino acids. Alternatively, the substituted amino acid in an amino acid substitution may be a non-standard amino acid (an amino acid that is not part of the above 20 standard set). By way of example, the substituted amino acid may be a basic non-standard amino acid, such as, for example, L-ornithine, L-2-amino-3-guanidinopropionic acid or the D-isomers of lysine, arginine and ornithine. Methods for introducing non-standard amino acids into proteins are known in the art and include recombinant protein synthesis using an E. coli auxotrophic expression host.

[0189] In one embodiment, the substitution is selected from: replacement of an acidic amino acid residue with a basic amino acid residue, replacement of an acidic amino acid residue with an uncharged amino acid residue, and replacement of an uncharged amino acid residue with a basic amino acid residue. In one embodiment, when the substitution is a replacement of an acidic amino acid residue with an uncharged amino acid residue, the acidic amino acid residue is replaced with the corresponding uncharged amide amino acid residue (i.e. aspartic acid is replaced with asparagine, glutamic acid with glutamic acid).

[0190] Preferably, the basic amino acid residue is a lysine or arginine residue. In other words, the substitution is with lysine or arginine. Most preferably, the modification is with lysine.

[0191] After modification in accordance with the present invention, the modified BoNT / A is capable of binding to a target cell receptor to which unmodified BoNT / A (eg, SEQ ID NO:2) binds.

[0192] The modified BoNT / A used in the present invention is Clostridial toxin H CNThe modified BoNT / A may include between 4 and 40 amino acid modifications located in the domain. The modified BoNT / A also preferably has a pI of at least 6.6. The modified BoNT / A preferably includes modifications of at least four amino acids selected from the following: ASN886, ASN930, ASN954, SER955, GLN991, ASN1025, ASN1026, and ASN1052, where the modifications include substitution of the amino acid with a lysine or arginine residue. For example, the modified BoNT / A or fragment thereof may include modifications in at least five amino acids selected from the following: ASN886, ASN930, ASN954, SER955, GLN991, ASN1025, ASN1026, ASN1052, and GLN1229, where the modifications include substitution of the amino acid with a lysine or arginine residue.

[0193] As used herein, the term "modified BoNT / A" or "chimeric neurotoxin" preferably refers to a clostridial neurotoxin comprising a clostridial neurotoxin light chain and a translocation domain (H) from a first clostridial neurotoxin serotype. N domain) and a receptor-binding domain (H) derived from a second different Clostridial neurotoxin serum. C Specifically, the modified BoNT / A used in the present invention refers to a neurotoxin comprising (preferably consisting of) a botulinum neurotoxin A (BoNT / A) light chain and a translocation domain (H domain). N domain) and the BoNT / B receptor binding domain (H C The modified BoNT / A includes the BoNT / ALH domain. N The domain is BoNT / BH C The modified BoNT / A of the present invention may be referred to as a chimeric botulinum neurotoxin. The modified BoNT / A is also referred to herein as a "BoNT / AB", "mrBoNT / AB" or "BoNT / AB chimera".

[0194] Most preferably, the modified BoNT / A used in the present invention comprises a BoNT / A light chain and a translocation domain (BoNT / ALH Ndomain) and BoNT / BH C The BoNT / ALH may include a domain. N The domain is the BoNT / BH C The modified BoNT / A is also referred to herein as a "BoNT / AB" or a "BoNT / AB chimera."

[0195] LH N The C-terminal amino acid residues of the domain are the same as those of the LH domain of BoNT / A. N Domain and H C Separate domains 3 10 It may correspond to the first amino acid residue of the helix. C The N-terminal amino acid residues of the domain are the LH N Domain and H C Separate domains 3 10 It may correspond to the second amino acid residue of the helix.

[0196] An example of a BoNT / B polypeptide sequence is provided as SEQ ID NO: 16 (UniProt Accession No. B1INP5).

[0197] As used herein, the term "BoNT / A LH N Domain and H C Separate domains 3 10 A reference to "the first amino acid residue of a helix" refers to the LH N Domain and H C Separate domains 3 10 It refers to the N-terminal residue of the helix.

[0198] As used herein, the term "BoNT / B LH N Domain and H C Separate domains 3 10 A reference to "the second amino acid residue of the helix" refers to the LH N Domain and H C Separate domains 3 10 It refers to the amino acid residue following the N-terminal residue of the helix.

[0199] "310 A "helix" is a type of secondary structure found in proteins and polypeptides, along with alpha helices, beta sheets, and reverse turns. 3 10 The amino acids in the helix are arranged in a right-handed helical structure where each full turn is completed by three residues and 10 atoms separating the intramolecular hydrogen bonds between the turns. Each amino acid has 10 atoms in a ring formed by hydrogen bonds, corresponding to a 120° rotation in the helix (i.e. the helix has 3 residues per turn) and a translation of 2.0 Å (= 0.2 nm) along the helical axis. Most importantly, the N-H group of an amino acid forms a hydrogen bond with the C=O group of the amino acid three residues before it. This repeated i+3→i hydrogen bond is 3 10 Define a helix. 3 10 A helix is ​​a standard concept in structural biology with which those skilled in the art are familiar.

[0200] These three 10 A helix corresponds to the four residues that form the actual helix plus two cap (or transition) residues, one at each end of these four residues. N Domain and H C Separate domains 3 10 The term "helix" consists of those six residues.

[0201] Through structural analysis and sequence alignment, LH N Domain and H C Separate domains 3 10 The helix was identified. 10 The helix is ​​located at its N-terminus (i.e., the LH N The C-terminus of the domain is surrounded by α-helices, and the C-terminus (i.e., the H C The N-terminal part of the domain is surrounded by β-strands. 10 The first (N-terminal) residue of the helix (the cap or transition residue) also corresponds to the C-terminal residue of this α-helix.

[0202] LH N Domain and HC Separate domains 3 10 The helices can be determined, for example, from published crystal structures of botulinum neurotoxins, such as 3BTA (w.rcsb.org / pdb / explore / explore.do?structureId=3BTA) and 1EPW (w.rcsb.org / pdb / explore / explore.do?structureId=1EPW) for botulinum neurotoxins A1 and B1, respectively.

[0203] The LH sequences of other neurotoxins were identified using publicly available in silico modeling and alignment tools, such as the homology modeling servers LOOPP (Learning, Observing and Outputting Protein Patterns, p.org), PHYRE (Protein Homology / Similarity Recognition Engine, w.sbg.bio.ic.ac.uk / phyre2 / ) and Rosetta (w.rosettacommons.org / ), the protein superposition server SuperPose (t.biology.ualberta .ca / superpose / ), the alignment program ClustalOmega (w.clustal.org / omega / ) and many other tools / services listed in the Internet resource for molecular and cell biologists l-tools.ca / . N Domain and H C Separate domains 3 10 It is also possible to determine the position of the helix. N / H CN "The perijunctional regions may be highly structurally conserved, making them ideal regions for overlapping of different serotypes.

[0204] For example, the following methodology can be used to investigate this effect in other neurotoxins: 10 The sequence of the helix may be determined: 1. The structural homology modeling tool LOOP (p.org) may be used to obtain predicted structures of other BoNT serotypes based on the BoNT / A1 crystal structure (3BTA.pdb). 2. Copy the structural (pdb) file obtained in this way to H CN The N-terminus of the domain and the preceding (H N By editing the protein to contain only about 80 residues (which are part of the H domain), the highly structurally conserved H N / H CN " area. 3. The protein superposition server SuperPose (t.biology.ualberta.ca / superpose / ) may be used to superimpose each serotype onto the corresponding 3BTA.pdb structure. 4. By examining the overlapping pdb files, the H of the BoNT / A1 N The domain begins with the 3 10 The helices may be mapped and corresponding residues in other serotypes may be identified. 5. Other BoNT serum sequences may be aligned with ClustalOmega to ensure that corresponding residues are correct.

[0205] LH determined by this method N , H C and 3 10 Examples of helical domains are shown below. [Table H]

[0206] Using structural analysis and sequence alignment, LH N Domain and H C Separate domains 3 10 The helix followed by a β-strand is a conserved structure in all botulinum and tetanus neurotoxins and is the LH N Domain and H C Separate domains 3 10 It was found to begin at the eighth residue when starting from the first residue of the helix (eg, BoNT / A1 residue 879).

[0207] BoNT / AB chimera is a BoNT / B-derived HC LH from BoNT / A covalently bound to the domain N domain, where the LH N The C-terminal amino acid residue of the domain is the H C It corresponds to the 8th amino acid residue from the N-terminus of the β chain located at the beginning (N-terminus) of the domain, and C The N-terminal amino acid residue of the domain is the H of BoNT / B. C It corresponds to the seventh amino acid residue from the N-terminus of the β chain located at the beginning (N-terminus) of the domain.

[0208] BoNT / AB chimera is a BoNT / B-derived H C LH from BoNT / A covalently bound to the domain N domain, where the LH N The C-terminal amino acid residue of the domain is the same as that of the LH of BoNT / A. N The H corresponds to the C-terminal amino acid residue of the α-helix located at the end (C-terminus) of the domain. C The N-terminal amino acid residue of the domain is the same as that of the LH of BoNT / B. N It corresponds to the amino acid residue adjacent to the C-terminal amino acid residue of the α-helix located at the end (C-terminus) of the domain.

[0209] The rationale for the design process of the BoNT / AB chimera is to ensure that the secondary structure is intact, thereby attempting to minimize any changes to the tertiary structure and function of each domain. 10 It is hypothesized that not disrupting the four central amino acid residues of the helix would ensure an optimal conformation for the chimeric neurotoxin, thereby allowing it to maximize its efficacy. Indeed, surprisingly, the 3 10 The first amino acid residue of the helix and the 3 10Retaining only the second amino acid residue after the helix not only allows for the production of a soluble and functional BoNT / AB, but also leads to superior properties over other BoNT / AB chimeras, particularly increased potency, increased safety factor and / or longer duration of action (as well as increased safety factor and / or duration of action compared to unmodified BoNT / A).

[0210] The BoNT / A light chain, BoNT / A translocation domain, and / or BoNT / BHc domain may be a modified BoNT / A light chain, a modified BoNT / A translocation domain, and / or a modified BoNT / BHc domain, or a derivative thereof, including, but not limited to, those described below. C The domain or derivative may be the BoNT / A light chain, the BoNT / A translocation domain, and / or the BoNT / BH. C The BoNT / A light chain, the BoNT / A translocation domain, and / or the BoNT / BH domain may contain one or more amino acids that are modified as compared to the naturally occurring (unmodified) form of the domain. C The modified BoNT / A light chain, BoNT / A translocation domain, and / or BoNT / BHc domain may contain one or more inserted amino acids that are not present in the naturally occurring (unmodified) form of the domain. By way of example, a modified BoNT / A light chain, BoNT / A translocation domain, and / or BoNT / BHc domain may be a BoNT / A light chain, BoNT / A translocation domain, and / or BoNT / BHc domain that is similar to a naturally occurring (unmodified) BoNT / A light chain, BoNT / A translocation domain, and / or BoNT / BHc domain. C The BoNT / A light chain, the BoNT / A translocation domain, and / or the BoNT / BH domain may have a modified amino acid sequence in one or more of the domains compared to the domain sequence. Such modifications may alter its functional aspects, such as its biological activity or persistence. Thus, in one embodiment, the BoNT / A light chain, the BoNT / A translocation domain, and / or the BoNT / BH domain may have a modified amino acid sequence in one or more of the domains compared to the domain sequence. C The domains may be modified BoNT / A light chains, BoNT / A translocation domains, and / or BoNT / BH domains. C domain, or modified BoNT / A light chain, BoNT / A translocation domain, and / or BoNT / BH C It is a derivative of the domain.

[0211] Modifications that alter binding to target neurons, such as native (unmodified) BoNT / BH C The modified BoNT / BH domain exhibits higher or lower binding affinity compared to the C The BoNT / BH may have one or more domains. C Such modifications in the H domain include those that alter the binding of the H domain to ganglioside receptors and / or protein receptors of the target neuron. C Modifications of residues in the ganglioside binding site or protein (e.g., synaptotagmin) binding site of the domain may be included. Examples of such modified neurotoxins are described in WO2006 / 027207 and WO2006 / 114308, both of which are incorporated herein by reference in their entirety.

[0212] The modified light chain may have one or more modifications in its amino acid sequence, such as modifications in the substrate binding domain or catalytic domain that may alter or modify the SNARE protein specificity of the modified light chain, preferably with the proviso that said modifications do not catalytically inactivate the light chain. Examples of such modified neurotoxins are described in WO2010 / 120766 and US2011 / 0318385, both of which are incorporated by reference in their entirety.

[0213] BoNT / A-derived LH N The domain may correspond to amino acid residues 1 to 872 of SEQ ID NO:2, or a polypeptide sequence having at least 70% sequence identity thereto. N The domain may correspond to amino acid residues 1 to 872 of SEQ ID NO:2, or a polypeptide sequence having at least 80%, 90%, or 95% sequence identity thereto. N The domain corresponds to amino acid residues 1 to 872 of SEQ ID NO:2.

[0214] BoNT / B-derived H CThe domain may correspond to amino acid residues 860 to 1291 of SEQ ID NO: 16, or a polypeptide sequence having at least 70% sequence identity thereto. C The domain may correspond to amino acid residues 860 to 1291 of SEQ ID NO: 16, or a polypeptide sequence having at least 80%, 90%, or 95% sequence identity thereto. C The domain corresponds to amino acid residues 860 to 1291 of SEQ ID NO:16.

[0215] Preferably, the BoNT / AB chimera is BoNT / A1LH N Domains and BoNT / B1H C More preferably, the LH N The domain corresponds to amino acid residues 1 to 872 of BoNT / A1 (SEQ ID NO: 2), and the H C The domain corresponds to amino acid residues 860 to 1291 of BoNT / B1 (SEQ ID NO: 16).

[0216] Preferably, BoNT / BH C The H domain further has the effect of increasing the binding affinity of the BoNT / B neurotoxin to human Syt2 as compared to the native BoNT / B sequence. CC The subdomain comprises at least one substitution, addition, or deletion of an amino acid residue. Suitable substitutions, additions, or deletions of amino acid residues in the BoNT / BHcc subdomain are disclosed in WO2013 / 180799 and WO2016 / 154534, both of which are incorporated herein by reference.

[0217] Suitable amino acid residue substitutions, additions, or deletions in the BoNT / BHcc subdomain can include substitution modifications selected from the group consisting of V1118M, V1183M, E1191M, E1191I, E1191Q, E1191T, S1199Y, S1199F, S1199L, S1201V, E1191C, E1191V, E1191L, E1191Y, S1199W, S1199E, S1199H, W1178Y, W1178Q, W1178A, W1178S, Y1183C, Y1183P, and combinations thereof.

[0218] The substitution, addition, or deletion of suitable amino acid residues in the BoNT / BHcc subdomain may further include a combination of two substitution modifications selected from the group consisting of: E1191M and S1199L, E1191M and S1199Y, E1191M and S1199F, E1191Q and S1199L, E1191Q and S1199Y, E1191Q and S1199F, E1191M and S1199W, E1191M and W1178Q, E1191C and S1199W, E1191C and S1199Y, E1191C and W1178Q, E1191Q and S1199W, E1191V and S1199W, E1191V and S1199Y, or E1191V and W1178Q.

[0219] Substitution, addition, or deletion of appropriate amino acid residues in the BoNT / BHcc subdomain can also include a combination of the three substitution modifications, E1191M, S1199W, and W1178Q.

[0220] Most preferably, the substitution, addition, or deletion of appropriate amino acid residues in the BoNT / B Hcc subdomain includes a combination of the two substitution modifications, E1191M and S1199Y, such as those present in the BoNT / AB chimeras SEQ ID NO:13 and SEQ ID NO:14.

[0221] The modification may be a modification relative to unmodified BoNT / B shown as SEQ ID NO: 16, where the numbering of the amino acid residues is determined by alignment with SEQ ID NO: 16. Since the presence of a methionine residue at position 1 of SEQ ID NO: 16 is optional (as are the SEQ ID NOs corresponding to the modified BoNT / A polypeptides described herein), the skilled artisan will take into account the presence / absence of the methionine residue when determining the numbering of the amino acid residues. For example, if SEQ ID NO: 16 contains a methionine, the numbering of the position will be as defined above (e.g., E1191 becomes E1191 in SEQ ID NO: 16). Alternatively, if a methionine is not present in SEQ ID NO: 16, the numbering of the amino acid residue should be corrected by -1 (e.g., E1191 becomes E1190 in SEQ ID NO: 16). Similar considerations apply to the presence / absence of a methionine at position 1 of other polypeptide sequences described herein, and the skilled artisan will easily determine the correct numbering of the amino acid residues using routine techniques in the art.

[0222] A modified BoNT / A used in the present invention may comprise a polypeptide sequence having at least 70% sequence identity to a polypeptide sequence selected from SEQ ID NOs: 11 to 15. For example, a polypeptide sequence having at least 80%, 90%, 95%, or 99.9% sequence identity to a polypeptide sequence selected from SEQ ID NOs: 11 to 15. Preferably, a modified BoNT / A used in the present invention may comprise a polypeptide sequence selected from SEQ ID NOs: 11 to 15 (and more preferably, consists of said polypeptide sequence).

[0223] When the modified BoNT / A is a BoNT / AB chimera, it is preferred that the modified BoNT / A comprises a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 14. For example, a polypeptide sequence having at least 80%, 90%, 95%, or 99.9% sequence identity to SEQ ID NO: 14. Most preferably, the modified BoNT / A used in the present invention may comprise (and more preferably consist of) SEQ ID NO: 14.

[0224] Methods for modifying proteins by substitution, insertion or deletion of amino acid residues are known in the art. By way of example, amino acid modifications may be introduced by modification of the DNA sequence encoding BoNT / A (e.g., encoding unmodified BoNT / A). This can be achieved, for example, by using standard molecular cloning techniques such as site-directed mutagenesis, in which short strands of DNA (oligonucleotides) encoding the desired amino acids are used to replace the original coding sequence using a polymerase enzyme, or by inserting / deleting parts of a gene using various enzymes (e.g., ligases and restriction endonucleases). Alternatively, modified gene sequences can be chemically synthesized.

[0225] Where the polypeptide sequence of a modified BoNT / A described herein includes a tag, such as a His tag, for example for purification purposes, the tag is optional. Preferably, the tag is removed prior to use of the modified BoNT / A in the present invention.

[0226] As mentioned above, the modified BoNT / A described herein has increased tissue retention properties that also result in increased efficacy and / or duration of action, allowing for increased dosage without additional negative effects. One way in which these advantageous properties may be defined is in terms of the safety factor of the modified BoNT / A. In this regard, the undesirable effects of a clostridial toxin (caused by diffusion of the toxin from the site of administration) can be experimentally evaluated by measuring the rate of weight loss in a suitable animal model (e.g., mice in which weight loss is detected within 7 days of administration). Conversely, the desirable on-target effects of a clostridial toxin can be experimentally evaluated by the digit abduction score (DAS) assay, which is an index of muscle paralysis. The DAS assay may be performed by injecting 20 μl of clostridial toxin formulated in gelatin phosphate buffer into the gastrocnemius / soleus muscle complex of mice, followed by evaluation of the digit abduction score using the method of Aoki (Aoki KR, Toxicon 39: 1815-1820;2001). In the DAS assay, mice are briefly suspended by their tails to elicit a characteristic startle response in which the mice extend their hind limbs and abduct their hind digits. After clostridial toxin injection, the varying degrees of digit abduction are scored on a 5-point scale (0=normal, 4=maximal reduction in digit abduction and limb extension).

[0227] The safety factor of a modified BoNT / A of the invention (or unmodified BoNT / A for comparison) may then be expressed as the ratio of the amount of toxin required to achieve a 10% reduction in body weight (measured at peak effect in the first 7 days after administration to mice) to the amount of toxin required for a DAS score of 2. Thus, a high safety factor score is desirable and indicates a toxin that can effectively paralyze a target muscle with few undesirable off-target effects. The modified BoNT / A of the invention has a higher safety factor than the safety factor of a comparable unmodified (native) BoNT / A.

[0228] A high safety margin is particularly advantageous in therapy because it means an increased therapeutic index. In other words, this means that lower doses can be used and / or higher doses can be used without additional (e.g., harmful) effects compared to alternative Clostridial neurotoxin therapies. Harmful effects include systemic toxicity and undesirable diffusion to adjacent muscles. The possibility of using high doses of a neurotoxin without additional effects is particularly advantageous, since high doses usually increase the duration of action of the neurotoxin in question.

[0229] The efficacy of modified BoNT / A is evaluated by measuring the efficacy of a given DAS score, e.g., DAS score 2 (ED 50 The efficacy of modified BoNT / A may be expressed as the EC value (EC20) in a cellular assay measuring SNARE cleavage by the neurotoxin. 50 Dose, e.g., EC in a cellular assay measuring SNAP25 cleavage by modified BoNT / A 50 It may also be expressed as dose.

[0230] The duration of action of a modified BoNT / A may be expressed as the time required to restore a DAS score to 0 after administration of a given dose of neurotoxin, e.g., the minimum dose of neurotoxin that results in a DAS score of 4, to the gastrocnemius / soleus muscle complex of a mouse.

[0231] Thus, in one embodiment, a modified BoNT / A of the invention has a safety factor of greater than 7 (e.g., at least 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50), where the safety factor is calculated as: DASED 50 Dose of toxin required for 10% body weight change (pg / mouse) divided by ED 50 = dose required to produce a DAS score of 2].

[0232] In one embodiment, the modified BoNT / A of the invention has a safety factor of at least 10. In one embodiment, the modified BoNT / A of the invention has a safety factor of at least 15.

[0233] Preferably, as described herein, the modified BoNT / A has a safety factor of at least 20, and more preferably at least 22 (e.g., 23 to 25), when the modified BoNT / A comprises one or more amino acid residues selected from the following: ASN886, ASN930, ASN954, SER955, GLN991, ASN1025, ASN1026, ASN1052, ASN1188, ASP1213, GLY1215, ASN1216, GLN1229, ASN1242, ASN1243, SER1274, and THR1277.

[0234] Preferably, the modified BoNT / A comprises a BoNT / A light chain and translocation domain and a BoNT / BH C domain, the modified BoNT / A has a safety factor of at least 10, more preferably at least 12 (eg, 14-15).

[0235] The modified BoNT / A is preferably in an uncomplexed form (i.e., does not contain the complexing proteins present in naturally occurring BoNT / A). Examples of such complexing proteins include neurotoxin binding proteins (NAPs) and non-toxic components lacking hemagglutination activity (NTNHs). However, it is preferred that the modified BoNT / A is a recombinant modified BoNT / A. Such modified BoNT / As of the invention can be produced using recombinant nucleic acid technology.

[0236] In one embodiment, a nucleic acid (e.g., DNA) is provided that includes a nucleic acid sequence that encodes a modified BoNT / A. In one embodiment, the nucleic acid sequence is generated as part of a DNA vector that includes a promoter and a terminator. The nucleic acid sequence may be selected from any of the nucleic acid sequences described herein.

[0237] In a preferred embodiment, the vector comprises a promoter selected from: Promoter / Inducer / Typical induction conditions Tac (hybrid) / IPTG / 0.2mM (0.05-2.0mM) AraBAD / L-arabinose / 0.2% (0.002-0.4%) T7-lac operator / IPTG / 0.2 mM (0.05-2.0mM)

[0238] In another preferred embodiment, the vector comprises a promoter selected from: Promoter / Inducer / Typical induction conditions Tac (hybrid) / IPTG / 0.2mM (0.05-2.0mM) AraBAD / L-arabinose / 0.2% (0.002-0.4%) T7-lac operator / IPTG / 0.2 mM (0.05-2.0mM) T5-lac operator / IPTG / 0.2 mM (0.05-2.0mM)

[0239] The nucleic acid molecules may be produced using any suitable procedure known in the art. Thus, the nucleic acid molecules may be produced using chemical synthesis techniques. Alternatively, the nucleic acid molecules of the present invention may be produced using molecular biology techniques.

[0240] The DNA constructs of the present invention are preferably designed in silico and then synthesized by conventional DNA synthesis techniques.

[0241] The above-mentioned nucleic acid sequence information is optionally modified for codon bias in the expression system of the final host cell (eg, E. coli) used.

[0242] The terms "nucleotide sequence" and "nucleic acid" are used interchangeably herein. Preferably, the nucleotide sequence is a DNA sequence.

[0243] The modified BoNT / A of the present invention may exist as a single chain or two chains. However, the modified BoNT / A may have an L chain linked to an H chain (or a component thereof, such as an H chain) via a disulfide bond. N It is preferred that the polypeptide exists as two chains linked together (domain).

[0244] Production of a single-chain modified BoNT / A having a light chain and a heavy chain may be achieved using a method comprising expressing a nucleic acid encoding the modified BoNT / A in an expression host, disrupting the host cells to provide a host cell homogenate containing the single-chain modified BoNT / A, and isolating the single-chain modified BoNT / A. The single-chain modified BoNT / A described herein may be proteolytically processed using a method comprising contacting the single-chain modified BoNT / A with a protease that hydrolyzes a peptide bond in the activation loop of the modified BoNT / A to convert the single-chain modified BoNT / A into the corresponding two-chain modified BoNT / A (e.g., where the light and heavy chains are linked by a disulfide bond). The two-chain modified BoNT / A is preferably obtained by such a method.

[0245] Thus, the modified BoNT / A used in the present invention is preferably a two-chain modified BoNT / A generated from a single-chain BoNT / A, where the single-chain BoNT / A comprises or consists of a polypeptide sequence described herein. For example, the modified BoNT / A used in the present invention is preferably a two-chain modified BoNT / A generated from a polypeptide comprising a polypeptide sequence having at least 70% (e.g., at least 80%, 90%, 95%, or 99.9%) sequence identity to SEQ ID NO: 14. Most preferably, the modified BoNT / A used in the present invention is a two-chain modified BoNT / A generated from a polypeptide comprising SEQ ID NO: 14 (and even more preferably consisting of SEQ ID NO: 14). Thus, in some embodiments, the modified BoNT / A is a two-chain modified BoNT / A in which the light chain (L chain) is linked to the heavy chain (H chain) via a disulfide bond, obtained by a method comprising contacting a single-chain modified BoNT / A comprising SEQ ID NO: 14 with a protease that hydrolyzes a peptide bond in the activation loop to convert the single-chain modified BoNT / A into a corresponding two-chain modified BoNT / A. In some embodiments, the modified BoNT / A is a two-chain modified BoNT / A in which the L chain is linked to the H chain via a disulfide bond, obtained by a method comprising contacting a single-chain modified BoNT / A consisting of SEQ ID NO: 14 with a protease that hydrolyzes a peptide bond in the activation loop to convert the single-chain modified BoNT / A into a corresponding two-chain modified BoNT / A.

[0246] In one embodiment, the modified BoNT / A used in the present invention is a two-chain modified BoNT / A generated from a polypeptide comprising a polypeptide sequence having at least 70% (e.g., at least 80%, 90%, 95%, or 99.9%) sequence identity to SEQ ID NO: 4. Preferably, the modified BoNT / A used in the present invention is a two-chain modified BoNT / A generated from a polypeptide comprising SEQ ID NO: 4 (more preferably consisting of SEQ ID NO: 4). Thus, in some embodiments, the modified BoNT / A is a two-chain modified BoNT / A in which a light chain (L chain) is linked to a heavy chain (H chain) via a disulfide bond, obtained by a method comprising contacting a single-chain modified BoNT / A comprising SEQ ID NO: 4 with a protease that hydrolyzes a peptide bond in its activation loop to convert the single-chain modified BoNT / A into the corresponding two-chain modified BoNT / A. In some embodiments, the modified BoNT / A is a two-chain modified BoNT / A in which the L chain is linked to the H chain via a disulfide bond, obtained by a method comprising converting a single-chain modified BoNT / A consisting of SEQ ID NO: 4 into the corresponding two-chain modified BoNT / A by contacting the single-chain modified BoNT / A with a protease that hydrolyzes the peptide bond in its active loop.

[0247] As used herein, the term "obtained" also encompasses the term "obtained." In one embodiment, the term "obtained" means obtained.

[0248] The protease used to cleave the activation loop is preferably Lys-C. Suitable proteases and methods for cleaving the activation loop to generate a dichain Clostridial neurotoxin are taught in WO2014 / 080206, WO2014 / 079495, and EP2677029A2, which are incorporated herein by reference. Lys-C may cleave the activation loop C-terminal to one or more lysine residues present at the activation loop C-terminus. It will be understood by those skilled in the art that if Lys-C cleaves the activation loop multiple times, small peptides of the activation loop of the dichain modified BoNT / A may not be present when compared to the SEQ ID NOs shown herein.

[0249] The modified BoNT / A of the invention may be formulated in any suitable manner for administration to an affected individual, for example as part of a pharmaceutical composition. Thus, in one embodiment, the invention provides a pharmaceutical composition comprising a modified BoNT / A of the invention and a pharma- ceutically acceptable carrier, excipient, adjuvant, and / or salt.

[0250] Liquid dosage forms are typically prepared using the modified BoNT / A and a pyrogen-free sterile solvent. Depending on the solvent and concentration used, the modified BoNT / A may be dissolved or suspended in the solvent. In preparing a solution, the modified BoNT / A may be dissolved in the solvent, the solution may be made isotonic by adding sodium chloride as necessary, sterilized by filtration through a sterile filter using aseptic techniques, and then filled into a suitable sterile bottle or ampoule and sealed. Alternatively, if the stability of the solution is sufficient, the solution in a sealed container may be sterilized by autoclaving. Advantageously, additives such as buffers, solubilizers, stabilizers, preservatives or bactericides, suspending or emulsifying agents, and / or local anesthetics may be dissolved in the solvent.

[0251] Dry powders that are dissolved or suspended in a suitable solvent prior to use may be prepared by filling sterile containers with pre-sterilized ingredients using aseptic techniques in a sterile area. Alternatively, the ingredients may be dissolved in suitable containers using aseptic techniques in a sterile area. The product is then lyophilized and the containers are aseptically sealed.

[0252] Parenterally administered suspensions suitable for the routes of administration described herein are prepared in substantially the same manner, except that the sterile components are suspended in a sterile vehicle instead of being dissolved, and sterilization cannot be achieved by filtration. The components may be isolated under aseptic conditions or may be sterilized after isolation, for example by gamma irradiation.

[0253] Advantageously, a suspending agent, such as polyvinylpyrrolidone, is included in the composition to facilitate uniform distribution of the ingredients.

[0254] In another aspect, the present invention provides a unit dosage form of modified botulinum neurotoxin A (BoNT / A) for the treatment of cervical dystonia, the unit dosage form comprising: a. 31 to 707 units of modified BoNT / A, where 1 unit is the calculated median lethal dose (LD 50 or b. 750 pg to 17,000 pg of modified BoNT / A, and c. any pharma- ceutically acceptable carriers, excipients, adjuvants, and / or salts; The modified BoNT / A contains a BoNT / A light chain and translocation domain, and a BoNT / B receptor binding domain (Hc domain).

[0255] The modified BoNT / A in the unit dosage form preferably comprises a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 14. For example, a polypeptide sequence having at least 80%, 90%, 95%, or 99.9% sequence identity. Most preferably, the modified BoNT / A may comprise (and more preferably consist of) SEQ ID NO: 14.

[0256] A unit dosage form for treating cervical dystonia may contain between 750 pg and 17,000 pg of modified BoNT / A, the modified BoNT / A comprising a BoNT / A light chain and translocation domain, and a BoNT / B receptor binding domain (Hc domain). The upper limit of the unit dose range can be 16,500, 15,500, 14,500, 13,500, 12,500, 11,500, 10,500, 9,500, 8,500, 7,500, 6,500, 5,500, 4,500, 3,500, 2,500, 2,250, 2,000, 1,500, 1,250, 1,000, or 750 pg of modified BoNT / A, preferably, the upper limit is 16,000 pg. The lower limit of the unit dose range may be 800, 850, 950, 1,000, 1,500, 1,750, 2,000, 2,500, 3,000, 3,500, 4,000, 4,500, or 5,000 pg of modified BoNT / A, preferably the lower limit is 1,000 pg. Preferably, the unit dose of modified BoNT / A is 1,000 pg to 16,000 pg of modified BoNT / A, for example 950 pg to 1,250 pg, 1,750 pg to 2,250 pg, or 8,000 pg to 12,000 pg. Preferably, the unit dose of modified BoNT / A may be 1,000, 2,000, 3,000, 8,000, or 16,000 pg.

[0257] A unit dosage form for treating cervical dystonia may contain 31 units to 707 units of a modified BoNT / A, wherein the modified BoNT / A comprises a BoNT / A light chain and translocation domain and a BoNT / B receptor binding domain (H CThe upper limit of the unit dose range may be 700, 650, 600, 550, 500, 450, 400, 350, 300, 250, 200, 150, 100, 95, 90, 85, 65, 60, 55, 50, or 31 units of modified BoNT / A, preferably the upper limit is 666 units. The lower limit of the unit dose range may be 35, 40, 45, 50, 60, 65, 70, 75, 80, 85, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, or 700 units of modified BoNT / A, preferably the lower limit is 42 units. Preferably, the unit dose of modified BoNT / A is between 42 and 666 units of modified BoNT / A, such as between 40 and 50 units, between 70 and 95 units, or between 333 and 499 units. Preferably, the unit dose of modified BoNT / A can be 41.6 units, 83.2 units, 124.8 units, 332.8 units, or 665.6 units.

[0258] In another aspect, the present invention provides a unit dosage form of modified botulinum neurotoxin A (BoNT / A) for treating cervical dystonia, the unit dosage form comprising: a. 53 units to 948 units of modified BoNT / A, where 1 unit is the calculated median lethal dose (LD ) in mice. 50 ), or b. 450 pg to 8,000 pg of modified BoNT / A; c. optionally, pharma- ceutically acceptable carriers, excipients, adjuvants and / or salts; The modified BoNT / A includes a modification at one or more amino acid residues selected from the following: ASN886, ASN905, GLN915, ASN918, GLU920, ASN930, ASN954, SER955, GLN991, GLU992, GLN995, ASN1006, ASN1025, ASN1026, ASN1032, ASN1043, ASN1046, ASN1052, ASP1058, HIS1064, ASN1080, GLU1081, GLU1083, ASP1086, ASN1188, ASP1213, GLY1215, ASN1216, GLN1229, ASN1242, ASN1243, SER1274, and THR1277, the modification being selected from the following: i. replacement of acidic surface-exposed amino acid residues with basic amino acid residues; ii. replacement of acidic surface-exposed amino acid residues with uncharged amino acid residues; iii. Substitution of uncharged surface-exposed amino acid residues with basic amino acid residues; iv. insertion of a basic amino acid residue; v. Deletion of acidic surface exposed amino acid residues.

[0259] A unit dosage form for treating cervical dystonia may comprise between 450 pg and 8,000 pg of a modified BoNT / A, the modified BoNT / A comprising a modification in one or more amino acid residues selected from the following: ASN886, ASN905, GLN915, ASN918, GLU920, ASN930, ASN954, SER955, GLN991, GLU992, GLN995, ASN1006, ASN1025, ASN1026, ASN1032, ASN1043, ASN1046, ASN1052, ASP1058, HIS1064, ASN1080, GLU1081, GLU1083, ASP1086, ASN1188, ASP1213, GLY1215, ASN1216, GLN1229, ASN1242, ASN1243, SER1274, and THR1277, the modification is selected from: (i) replacement of a surface-exposed acidic amino acid residue with a basic amino acid residue; (ii) substitution of an acidic surface-exposed amino acid residue with an uncharged amino acid residue; (iii) substitution of an uncharged surface-exposed amino acid residue with a basic amino acid residue; (iv) insertion of a basic amino acid residue; and (v) deletion of an acidic surface-exposed amino acid residue. The upper end of the range can be 7,750, 7,500, 7,000, 6,000, 5,000, 4,000, 3,000, 2,000, or 1,000 pg of modified BoNT / A, preferably the upper end is 7,500 pg. The lower end of the range can be 475, 500, 600, 700, 800, 900, 1,000, 1,500, 2,000, 3,000, 4,000, 5,000, 6,000, or 7,000 pg of modified BoNT / A, preferably the lower end is 500 pg. Preferably, the unit dosage form contains 500 pg to 7,500 pg of modified BoNT / A, such as 4,000 pg to 6,000 pg. Most preferably, the unit dosage form contains 2,000 pg to 3,000 pg of modified BoNT / A, such as 2,400 pg to 2,600 pg.

[0260] A unit dosage form for treating cervical dystonia may comprise 53 units to 948 units of a modified BoNT / A, the modified BoNT / A comprising a modification in one or more amino acid residues selected from the following: ASN886, ASN905, GLN915, ASN918, GLU920, ASN930, ASN954, SER955, GLN991, GLU992, GLN995, ASN1006, ASN1025, ASN1026, ASN1032, ASN1043, ASN1046, ASN1052, ASP1058, HIS1064, ASN1080, GLU1081, GLU1083, ASP1086, ASN1188, ASP1213, GLY1215, ASN1216, GLN1229, ASN1242, ASN1243, SER1274 and THR1277, wherein the modification is selected from: (i) replacement of a surface-exposed acidic amino acid residue with a basic amino acid residue; (ii) substitution of an acidic surface-exposed amino acid residue with an uncharged amino acid residue; (iii) substitution of an uncharged surface-exposed amino acid residue with a basic amino acid residue; (iv) insertion of a basic amino acid residue; and (v) deletion of an acidic surface-exposed amino acid residue. The upper end of the range can be 925, 900, 850, 800, 750, 700, 650, 600, 550, 500, 450, 400, 350, 300, 250, 200, 150, or 100 units of the modified BoNT / A, preferably, the upper end is 889 units. The lower limit of the range may be 55, 60, 65, 70, 75, 80, 85, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, or 900 units of modified BoNT / A, preferably the lower limit is 59 units. Preferably, the unit dosage form contains between 59 and 889 units, e.g., between 200 and 600 units of modified BoNT / A. Most preferably, the unit dosage form contains between 237 and 355 units, e.g., between 284 and 308 units of modified BoNT / A.

[0261] In another aspect, the present invention provides a kit comprising: a. the unit dosage form according to the present invention; b. Instructions for use of said unit dosage form in the treatment of cervical dystonia; c. Optionally, a diluent.

[0262] The various therapeutic application embodiments of the invention can be applied to the methods, compositions (eg, unit dosage forms), and kits of the invention, and vice versa.

[0263] sequence homology

[0264] To determine percent identity, any of a variety of sequence alignment methods can be used, including but not limited to global methods, local methods, and hybrid methods such as segment approach methods. Protocols for determining percent identity are routine procedures within the skill of the art. In global methods, the molecular sequences are aligned from beginning to end, and the optimal alignment is determined by summing the scores of individual residue pairs and applying gap penalties. Non-limiting methods include, for example, CLUSTALW, see, for example, Julie D. Thompson et al., CLUSTAL W: Improving the Sensitivity of Progressive Multiple Sequence Alignment Through Sequence Weighting, Position- Specific Gap Penalties and Weight Matrix Choice, 22(22) Nucleic Acids Research 4673-4680 (1994), and iterative improvement methods, see, for example, Osamu Gotoh, Significant Improvement in Accuracy of Multiple Protein. Sequence Alignments by Iterative Refinement as Assessed by Reference to Structural Alignments,264(4) J. MoI. Biol. 823-838 (1996). Local methods align sequences by identifying one or more conserved motifs shared by all input sequences. Non-limiting methods include Matchbox, see, e.g., Eric Depiereux and Ernest Feytmans, Match-Box: A Fundamentally New Algorithm for the Simultaneous Alignment of Several Protein Sequences, 8(5) CABIOS 501 -509 (1992).For Gibbs sampling, see, e.g., CE Lawrence et al., Detecting Subtle Sequence Signals: A Gibbs Sampling Strategy for Multiple Alignment, 262(5131) Science 208-214 (1993). For Align-M, see, e.g., Ivo Van WaIIe et al., Align-M - A New Algorithm for Multiple Alignment of Highly Divergent Sequences, 20(9) Bioinformatics:1428-1435 (2004).

[0265] Thus, the percent sequence identity is determined by conventional methods. See, for example, Altschul et al, Bull. Math. Bio. 48: 603-16, 1986 and Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915-19, 1992. Briefly, as shown below (amino acids are indicated by standard single letter code), two amino acid sequences are aligned using a gap open penalty of 10, a gap extension penalty of 1, and the "blosum62" score matrix of Henikoff and Henikoff (ibid.) to optimize the alignment score. Preferably, this method is used to align a sequence with the subject sequence herein (e.g., SEQ ID NO:2) and define the numbering of amino acid positions, as described herein.

[0266] The "percent sequence identity" between two or more nucleic acid or amino acid sequences is a function of the number of identical positions shared by the sequences. Thus, the percent identity may be calculated as the number of identical nucleotides / amino acids divided by the total number of nucleotides / amino acids multiplied by 100. The calculation of percent sequence identity may also take into account the number of gaps and the length of each gap that needs to be inserted to optimize the alignment of two or more sequences. The comparison of sequences and the determination of percent identity between two or more sequences can be performed using certain mathematical algorithms, such as BLAST, which are well known to those skilled in the art.

[0267] Alignment scores for determining sequence identity

number

[0268] The percent identity is calculated as follows: [total number of perfect matches × 100] / [length of the longer sequence + number of gaps inserted into the longer sequence to align the two sequences]

[0269] Substantially homologous polypeptides are considered to have one or more amino acid substitutions, deletions or additions. These changes are preferably of a relatively minor nature, i.e., conservative amino acid substitutions (see below) and other substitutions that do not significantly affect the folding or activity of the polypeptide: typically small deletions of from one to about 30 amino acids, and small amino- or carboxyl-terminal extensions such as an amino-terminal methionine residue, small linker peptides of up to about 20-25 residues, or affinity tags.

[0270] Conservative Amino Acid Substitutions Basicity: Arginine lysine Histidine Acidic: Glutamic acid Aspartic acid polarity: glutamine Asparagine Hydrophobicity: Leucine Isoleucine Ballin Aromatic: Phenylalanine Tryptophan Tyrosine small: glycine Alanine Serine Threonine Methionine

[0271] In addition to the 20 standard amino acids, non-standard amino acids (such as 4-hydroxyproline, 6-N-methyllysine, 2-aminoisobutyric acid, isovaline, and α-methylserine) may be substituted for amino acid residues in the polypeptides of the invention. A limited number of non-conserved amino acids, as well as amino acids that are not encoded by the genetic code and that do not occur naturally, may be substituted for polypeptide amino acid residues. The polypeptides of the invention may also include non-naturally occurring amino acid residues.

[0272] Non-naturally occurring amino acids include, but are not limited to, trans-3-methylproline, 2,4-methanoproline, cis-4-hydroxyproline, trans-4-hydroxyproline, N-methylglycine, allothreonine, methylthreonine, hydroxyethylcysteine, hydroxyethylhomocysteine, nitroglutamine, homoglutamine, pipecolic acid, tertroucine, norvaline, 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, and 4-fluorophenylalanine. Various methods are known in the art for incorporating non-naturally occurring amino acid residues into proteins. For example, an in vitro system can be used in which nonsense mutations are suppressed using chemically aminoacylated suppressor tRNAs. Methods for synthesizing amino acids and aminoacylating tRNAs are known in the art. Transcription and translation of the plasmid containing the nonsense mutation is carried out in a cell-free system containing an E. coli S30 extract and commercially available enzymes and other reagents. The protein is purified by chromatography. See, e.g., Robertson et al., J. Am. Chem. Soc. 113:2722, 1991; Ellman et al., Methods Enzymol. 202:301, 1991; Chung et al., Science 259:806-9, 1993; and Chung et al., Proc. Natl. Acad. Sci. USA 90:10145-9, 1993. In the second method, translation is driven in Xenopus oocytes by microinjection of mutant mRNA and chemically aminoacylated suppressor tRNA (Turcatti et al., J. Biol. Chem. 271:1991-8, 1996). In the third method, E. coli cells are cultured in the absence of the natural amino acid to be substituted (e.g., phenylalanine) and in the presence of the desired non-naturally occurring amino acid (e.g., 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, or 4-fluorophenylalanine) that is incorporated into the polypeptide in place of the natural amino acid.See Koide et al., Biochem. 33:7470-6, 1994. Naturally occurring amino acid residues can be converted to non-naturally occurring species by in vitro chemical modification. Chemical modification can be combined with site-directed mutagenesis to further expand the range of such substitutions (Wynn and Richards, Protein Sci. 2:395-403, 1993).

[0273] A limited number of non-conservative amino acids, amino acids that are not encoded by the genetic code, non-naturally occurring amino acids, and unnatural amino acids may be substituted for amino acid residues in the polypeptides of the invention.

[0274] Essential amino acids in the polypeptides of the invention can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine scanning mutagenesis (Cunningham and Wells, Science 244: 1081-5, 1989). Biological interaction sites can also be determined by physical analysis of structures determined by techniques such as nuclear magnetic resonance, crystallography, electron diffraction or photoaffinity labeling, in combination with mutations of putative contact site amino acids. See, for example, de Vos et al., Science 255:306-12, 1992; Smith et al., J. Mol. Biol. 224:899-904, 1992; Wlodaver et al., FEBS Lett. 309:59-64, 1992. The identity of essential amino acids can also be inferred from analysis of homology with related components of the polypeptides of the invention, such as translocation components or protease components.

[0275] Multiple amino acid substitutions can be made and tested using known methods for mutagenesis and screening, such as those disclosed by Reidhaar-Olson and Sauer (Science 241:53-7, 1988) or Bowie and Sauer (Proc. Natl. Acad. Sci. USA 86:2152-6, 1989). Briefly, these authors disclose methods for simultaneously randomizing two or more positions within a polypeptide, selecting functional polypeptides, and then sequencing the mutagenized polypeptides to determine the spectrum of substitutions tolerated at each position. Other methods that can be used include phage display (e.g., Lowman et al., Biochem. 30:10832-7, 1991; Ladner et al., US Patent No. 5,223,409; Huse, WIPO Publication WO 92 / 06204) and site-directed mutagenesis (Derbyshire et al., Gene 46:145, 1986; Ner et al., DNA 7:127, 1988).

[0276] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Singleton, et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 20 ED., John Wiley and Sons, New York (1994), and Hale & Marham, THE HARPER COLLINS DICTIONARY OF BIOLOGY, Harper Perennial, NY (1991) provide those of ordinary skill in the art with a general dictionary of many of the terms used in this disclosure.

[0277] The disclosure is not limited by the exemplary methods and materials disclosed herein, and any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the disclosure. Numeric ranges include the numbers defining the range. Unless otherwise specified, any nucleic acid sequence is written left to right in a 5' to 3' orientation, and any amino acid sequence is written left to right in an amino to carboxy orientation, respectively.

[0278] The headings provided herein are not intended to limit the various aspects or embodiments of the disclosure. Amino acids are referred to herein using the amino acid name, three letter abbreviation, or one letter abbreviation. As used herein, the term "protein" includes proteins, polypeptides, and peptides. As used herein, the term "amino acid sequence" is synonymous with the term "polypeptide" and / or the term "protein." In some cases, the term "amino acid sequence" is synonymous with the term "peptide." In some cases, the term "amino acid sequence" is synonymous with the term "enzyme." The terms "protein" and "polypeptide" are used interchangeably herein. In the present disclosure and claims, conventional single-letter and three-letter codes for amino acid residues may be used. The three-letter codes for amino acids are as defined in accordance with the IUPACIUB Joint Commission on Biochemical Nomenclature (JCBN). It is also understood that due to the degeneracy of the genetic code, a polypeptide may be encoded by one or more nucleotide sequences.

[0279] Different definitions of terms may appear throughout the specification. Before a more detailed description of the exemplary embodiments, it is to be understood that the present disclosure is not limited to the particular embodiments described, as such may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure is defined only by the appended claims.

[0280] Where a range of values ​​is given, it is understood that each intervening value between the upper and lower limit of that range is also specifically disclosed, to the tenth of the unit of the lower limit, unless the context clearly dictates otherwise. Each smaller range between any stated or intervening value in a stated range and any other stated or intervening value in that stated range is included within the disclosure.

[0281] The upper and lower limits of these smaller ranges may each independently be included or excluded in the range, and each of the smaller ranges including either, neither, or both of those limits is also included in the disclosure, subject to any specifically excluded limit in the stated range. If the stated range includes one or both of those limits, then ranges excluding either or both of those included limits are also included in the disclosure.

[0282] It should be noted that, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a modified botulinum neurotoxin A" includes a plurality of such candidate agents, reference to "the modified botulinum neurotoxin A" includes reference to one or more modified botulinum neurotoxins A and equivalents thereof known to those skilled in the art, and so forth.

[0283] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein should be construed as an admission that such publications constitute prior art to the claims appended hereto.

[0284] Embodiments of the invention will now be described, by way of example only, with reference to the following figures and examples. [Brief description of the drawings]

[0285] [Figure 1]FIG. 1 shows the FDA-approved dosages of Dysport® for the treatment of cervical dystonia. [Diagram 2] FIG. 2 shows an isoelectric focusing (IEF) gel of the cationic constructs. [Diagram 3] Figure 3 shows a summary of the percentage of SNAP-25 cleavage in rat embryonic spinal cord neurons (eSCN) and the pEC50 for nBoNT / A1 for Cat5v2(K1064H / N954K) (A), Cat5v2(K1064H / N886K) (B), and Cat5v2(K1064H / N1025K) (C). (A, B, C) Rat embryonic spinal cord neurons were cultured for 3 weeks and treated with Cat5v4 for 24 hours, followed by Western blotting with a SNAP-25 specific antibody. Data are the mean ± SEM of triplicate independent experiments. (D) Relative potency of Cat5v2(K1064H / N886K), Cat5v2(K1064H / N954K) and Cat5v2(K1064H / N1025K) versus nBoNT / A1 (List Biological Laboratories) in the rat eSCN SNAP-25 cleavage potency assay. Each point corresponds to an individual batch and is the mean of three independent pEC50 determinations based on an 8-point concentration-response curve (CRC). Each concentration in the CRC was evaluated in triplicate. Potency comparisons are made against the mean of List batches with pooled data n=24. Data are the mean ± SEM of n=3 batches per Cat5v4. [Figure 4] FIG. 4 shows the potency (t50) of nBoNT / A1 and Cat5v4 in the mouse phrenic nerve hemidiaphragm assay (mPNHD). Mouse phrenic nerve hemidiaphragm tissue was incubated with Cat5v4 or native BoNT / A1 as shown. Diaphragm contraction force was recorded until no contraction was detectable or after 140 min. Each point corresponds to an independent measurement. The t50 value is the time required to inhibit mouse hemidiaphragm contraction force by 50%. [Diagram 5]5 shows SDS-PAGE of purified recombinant BoNT / AB chimeras 1, 2, and 3A (SEQ ID NOs: 11, 12, and 13, respectively). Lanes are labeled with "Marker" (molecular weight marker), "-DTT" (oxidized BoNT / AB chimera sample), and "+DTT" (reduced BoNT / AB chimera sample). [Figure 6] Figure 6 shows cleavage of SNAP-25 by recombinant BoNT / AB chimeras 1, 2, and 3A (converted to dichain form with SEQ ID NO: 11, 12, and 13, respectively) in rat spinal cord neurons. Primary cultured rat spinal cord neurons (SCN) were exposed to various concentrations of recombinant BoNT / AB chimeras 1, 2, or 3A for 24 hours at 37°C in a humidified atmosphere containing 10% CO2. Cells were then lysed in 1x NuPAGE buffer supplemented with DTT and benzonase. The samples were transferred to microcentrifuge tubes, heated at 90°C on a heat block for 5 minutes, and stored at -20°C before analyzing SNAP-25 cleavage by Western blot. SNAP-25 was detected using a polyclonal antibody that detects both full-length and cleaved forms of SNAP-25 (Sigma #S9684). Anti-rabbit HRP (Sigma #A6154) was used as the secondary antibody. [Figure 7] Figure 7 shows the mouse digit abduction scoring assay. Under brief general anesthesia, mice were injected into the gastrocnemius-soleus compound muscle of one hind limb, and muscle weakness was measured using the digit abduction score (DAS) on a scale of 0 to 4. Maximum DAS values ​​for each dose were determined and plotted against dose, and the data were fitted to a four-parameter logistic equation to determine the ED50 and the dose value that induces DAS4 (DAS4 dose). [Figure 8] 8 shows SDS-PAGE of purified recombinant BoNT / AB chimeras 3B and 3C (SEQ ID NOs: 14 and 15, respectively). Lanes are labeled "Marker" (molecular weight marker), "-DTT" (oxidized BoNT / AB chimera sample), and "+DTT" (reduced BoNT / AB chimera sample). [Figure 9]Figure 9 shows the cleavage of SNAP-25 by unmodified BoNT / A and BoNT / AB chimeras 3B and 3C (converted to the two-chain form with SEQ ID NOs: 2, 14, and 15, respectively) in human induced pluripotent stem cell-derived peripheral neurons (PERI.4U-Axiogenesis, Germany). PEDRI.4U cells were exposed to various concentrations of recombinant BoNT / A, or BoNT / AB chimeras 3B or 3C for 24 hours at 37°C in a humidified CO2 atmosphere containing 5% CO2. Cells were then lysed in 1x NuPAGE buffer supplemented with DTT and benzonase. The samples were transferred to microcentrifuge tubes, heated at 90°C for 5 minutes on a heat block, and stored at -20°C before analyzing SNAP-25 cleavage by Western blot. SNAP-25 was detected using a polyclonal antibody that detects both full-length and cleaved forms of SNAP-25 (Sigma #S9684). Anti-rabbit HRP (Sigma #A6154) was used as the secondary antibody. [Figure 10] Figure 10 shows the duration of muscle weakness over time in the mouse digit abduction scoring assay. Under brief general anesthesia, mice were injected into the gastrocnemius-soleus compound muscle of one hind limb and muscle weakness was measured using the digit abduction score (DAS) on a scale of 0 to 4. Animals in the group injected with the lowest dose inducing DAS4 during the first 4 days after injection were monitored until complete recovery of muscle weakness and DAS0 (no observed muscle weakness).

[0286] Sequence Listing

[0287] Where the first Met amino acid residue, or the corresponding first codon, is shown in any of the following SEQ ID NOs, said residue / codon is optional.

[0288] SEQ ID NO:1 (nucleotide sequence, unmodified BoNT / A)

[0289] SEQ ID NO:2 (polypeptide sequence, unmodified BoNT / A)

[0290] SEQ ID NO:3 (Nucleotide sequence, modified BoNT / A “Cat-A”)

[0291] SEQ ID NO:4 (polypeptide sequence, modified BoNT / A “Cat-A”)

[0292] SEQ ID NO:5 (Nucleotide sequence, modified BoNT / A “Cat-B”)

[0293] SEQ ID NO:6 (polypeptide sequence, modified BoNT / A “Cat-B”)

[0294] SEQ ID NO:7 (Nucleotide sequence, modified BoNT / A “Cat-C”) SEQ ID NO:8 (polypeptide sequence, modified BoNT / A “Cat-C”)

[0295] SEQ ID NO:9 (Nucleotide sequence, modified BoNT / A “Cat-D”)

[0296] SEQ ID NO:10 (polypeptide sequence, modified BoNT / A “Cat-D”)

[0297] SEQ ID NO:11 (polypeptide sequence, modified BoNT / A "chimera 1")

[0298] SEQ ID NO:12 (polypeptide sequence, modified BoNT / A "chimera 2")

[0299] SEQ ID NO:13 (polypeptide sequence, modified BoNT / A "chimera 3A")

[0300] SEQ ID NO:14 (polypeptide sequence, modified BoNT / A "chimera 3B")

[0301] SEQ ID NO:15 (polypeptide sequence, modified BoNT / A "chimera 3C")

[0302] SEQ ID NO: 16 (polypeptide sequence, BoNT / B) EXAMPLES

[0303] [Example 1]

[0304] Cloning, expression and purification

[0305] The nucleotide sequence of SEQ ID NO:1, which encodes wild-type BoNT / A (SEQ ID NO:2), was mutated to introduce the following substitutions to form the four constructs shown in Table 1 below. [Table 1]

[0306] DNA constructs encoding the modified BoNT / A molecules were synthesized, cloned into the pJ401 expression vector, and then transformed into BL21(DE3) E. coli, allowing for the soluble overexpression of recombinant Cat-A, Cat-B, Cat-C, and Cat-D proteins in BL21(DE3) E. coli.

[0307] The recombinant modified BoNT was purified from E. coli lysate using classical chromatographic techniques. An initial purification step using a cation exchange resin was used, followed by an intermediate purification step using a hydrophobic interaction resin. The recombinant modified BoNT single chain was then proteolytically cleaved, resulting in an activated two-chain modified BoNT. A final purification step was then used to remove remaining contaminants. Suitable techniques are taught in WO2015 / 166242, WO2017055274A1, EP2524963B1, EP2677029B1, and US10087432B2.

[0308] [Example 2]

[0309] Characteristics of purified and modified BoNT / A

[0310] The modified BoNTs described in Example 1 above were experimentally characterized as follows.

[0311] pI measurements showed that the isoelectric point of the modified BoNT was greater than that of unmodified (native) BoNT / A1 (see FIG. 2 and Table 2 below). [Table 2]

[0312] The ability of the modified BoNT to enter neurons and cleave SNAP-25 (the target of BoNT / A1) was assessed using rat embryonic spinal cord neurons (eSCN). Figure 3 shows that the modified BoNT retains the same ability as native BoNT / A1 to enter neurons and cleave SNAP-25.

[0313] The potency of the modified BoNT was further evaluated using the mouse phrenic nerve hemidiaphragm assay (mPNHD), and Figure 4 shows that the modified BoNT retains the same ability as native BoNT / A1 to inhibit the contraction ability of the mouse hemidiaphragm.

[0314] An in vivo mouse digit abduction score (DAS) assay was used to evaluate efficacy and safety compared to native BoNT / A1. Both molecules (Cat-A [SEQ ID NO: 4 converted to a bichain form] and Cat-B [SEQ ID NO: 6 converted to a bichain form]) showed a higher safety rate and were slightly more potent compared to native BoNT / A1. These data are shown in Table 3 below. [Table 3]

[0315] The safety factor is a measure of the negative effect (weight loss) of BoNT treatment on efficacy (half maximum digital abduction score (DAS)). It ... 50 -10%BW refers to the amount of BoNT (pg / animal) required to reduce body weight by 10%, and ED 50 refers to the amount of BoNT (pg / animal) that produces DAS2.

[0316] 20 μl of modified BoNT / A formulated in gelatin phosphate buffer is injected into the gastrocnemius / soleus muscle complex of mice, after which the DAS assay is performed by assessing digit abduction as previously reported by Aoki (Aoki KR, Toxicon 39: 1815-1820; 2001).

[0317] [Example 3]

[0318] Cloning, expression, and purification of modified BoNT / A (BoNT / AB chimera)

[0319] BoNT / AB chimeric constructs 1, 2, 3A, 3B and 3C (SEQ ID NOs: 11-15, respectively) were constructed from DNA encoding the parent serotype molecules and appropriate oligonucleotides using standard molecular biology techniques. 10 The constructs were cloned into the pJ401 expression vector with or without tags and overexpressed by transformation into BLR(DE3) E. coli cells. The cells were grown in baffled Erlenmeyer flasks containing 1 L of modified Terrific Broth (mTB) supplemented with the appropriate antibiotics at 37°C and 225 RPM shaking. 600 As soon as the β-actin reached >0.5, the incubator temperature was reduced to 16° C. and 1 hour later the culture was induced with 1 mM IPTG for 20 hours with shaking at 225 RPM to allow expression of the recombinant BoNT / AB construct.

[0320] Harvested cells were lysed by sonication and clarified by centrifugation at 4500 RPM for 1 hour at 4°C. The recombinant BoNT / AB chimera molecule was then extracted with ammonium sulfate and purified by standard fast protein liquid chromatography (FPLC) techniques, involving the use of a hydrophobic interaction resin for capture and an anion exchange resin for intermediate purification steps. The partially purified molecule was then proteolytically cleaved with endoproteinase Lys-C to generate the active dichain, which was further purified on a second hydrophobic interaction resin to yield the final BoNT / AB chimera.

[0321] Decahistidine (H 10 For BoNT / AB chimeric molecules bearing the .DELTA.) tag (chimeras 1, 2, 3A), immobilized nickel resin was used in place of the hydrophobic interaction resin in the capture step.

[0322] The sequences of each chimera are shown in Table 4. [Table 4]

[0323] [Example 4]

[0324] Comparison of BoNT / AB chimeras 1, 2, and 3A

[0325] C-terminal His 10 BoNT / AB chimeras 1, 2, and 3A carrying the tag and the E1191M / S1199Y double mutation were purified and tested for functional activity as described in Example 3 (FIG. 5).

[0326] Rat spinal cord neuron SNAP-25 cleavage assay

[0327] Primary cultures of rat spinal cord neurons (SCN) were generated and grown for 3 weeks in 96-well tissue culture plates (as described in Masuyer et al., 2011, J. Struct. Biol. Structure and activity of a functional derivative of Clostridium botulinum neurotoxin B; and in: Chaddock et al., 2002, Protein Expr. Purif. Expression and purification of catalytically active, non-toxic endopeptidase derivatives of Clostridium botulinum toxin type A). A dilution series of BoNT / AB was prepared in SCN feeding medium. Growth medium from treated wells was collected and filtered (0.2 μm filter). 125 μL of filtered medium was returned to each test well. 125 μL of diluted toxin was then added to the plate (triplicate wells). The treated cells were incubated at 37°C and 10% CO2 for 24 ± 1 h.

[0328] Analysis of BoNT activity using the SNAP-25 cleavage assay

[0329] After treatment, BoNT was removed and cells were washed once with PBS (Gibco, UK). Cells were lysed in 1xNuPAGE lysis buffer (Life Technologies) supplemented with 0.1M dithiothreitol (DTT) and 250 units / mL benzonase (Sigma). Protein lysates were separated by SDS-PAGE and transferred to nitrocellulose membranes. Membranes were probed with a primary antibody specific for SNAP-25 (Sigma #S9684), which recognizes uncleaved SNAP-25 as well as cleaved SNAP-25 by BoNT / A endopeptidase. The secondary antibody used was HRP-conjugated anti-rabbit IgG (Sigma #A6154). Bands were detected by enhanced chemiluminescence and imaged using pXi6 Access (Synoptics, UK). Band intensities were determined using GeneTools software (Syngene, Cambridge, UK) and the percentage of cleaved SNAP-25 at each concentration of BoNT was calculated. Data were fitted to a four-parameter logistic equation and pEC was calculated using GraphPad Prism version 6 (Graphpad). 50 was calculated.

[0330] Table 5 below shows the pEC5A determined for chimeras 1, 2 and 3A in the rat SCN SNAP-25 cleavage assay. 50 Values ​​are shown in Table 6. These results demonstrated that the three BoNT / A chimeras retained the ability to enter rat spinal cord neurons and cleave target substrates, although chimera 3A was more potent than chimeras 1 and 2 in this assay (see also FIG. 6). [Table 5]

[0331] Digital Abduction Score (DAS) Assay

[0332] The method to measure the activity of BoNT / AB chimeras 1, 2 and 3A in the DAS assay is based on the startle response digit extension reflex when mice are temporarily suspended by their tail. This reflex is recorded as the digit abduction score (DAS) and is suppressed after BoNT administration into the gastrocnemius-soleus muscles of the hind limb. Brief suspension of mice by their tail induces a characteristic startle response in which the animals extend their hind limbs and abduct their hind digits (Aoki et al. 1999, Eur. J. Neurol.; 6 (suppl. 4) S3-S10).

[0333] On the day of injection, mice were anesthetized in an induction chamber with 3% isoflurane in oxygen and received an intramuscular injection of the BoNT / AB chimera or vehicle (0.2% gelatin in phosphate buffer) in the gastrocnemius and soleus muscles of the right hind limb.

[0334] After neurotoxin injection, the various degrees of finger abduction were scored on a scale of 0 to 4, where 0 = normal and 4 = maximal reduction in finger abduction and limb extension. 50 was determined by nonlinear adjustment analysis using the mean of the maximum effect at each dose. The mathematical model used was a four-parameter logistic model.

[0335] On the first day of treatment, DAS was performed every 2 hours, then three times daily for 4 days.

[0336] Figure 7 shows the fitted curves of chimeras 1, 2 and 3A (SEQ ID NOs: 11, 12 and 13, respectively, converted to two-chain form). The chimera 3A curve is shifted to the left, which means that a lower dose of chimera 3A achieved a similar DAS response compared to chimeras 1 and 2, thus indicating that chimera 3A is more effective than the others in the mouse DAS assay. The calculated ED of each chimera 50 See also the table below (Table 6) showing the doses that lead to a DAS4 (maximum score) for each chimera.

[0337] Table 6 below shows the EDs determined for unmodified recombinant BoNT / A1 (rBoNT / A1-converted to two-chain form SEQ ID NO: 2) and chimeras 1, 2 and 3A in the mouse DAS assay. 50 and DAS4 doses are shown. These results indicate that of the three chimeras, chimera 3A is the most potent in inducing muscle weakness in vivo. The studies shown in Figure 7 and Table 6 were performed in mice obtained from Charles River Laboratories. [Table 6]

[0338] [Example 5]

[0339] Comparison of BoNT / AB chimeras 3B and 3C with unmodified BoNT / A1

[0340] Untagged BoNT / AB chimeras 3B and 3C, both with and without the E1191M / S1199Y double modification (SEQ ID NO: 14 and 15), respectively, were purified as described in Example 3 (Figure 8) and tested for functional activity using unmodified BoNT / A (SEQ ID NO: 2 converted to a two-chain form) as a reference.

[0341] Human pluripotent stem cell SNAP-25 cleavage assay

[0342] Cryopreserved PERI.4U cells were purchased from Axiogenesis (Cologne, Germany). Thawing and plating of the cells were performed according to the manufacturer's recommendations. Briefly, the cryovial containing the cells was thawed in a 37° C. water bath for 2 minutes. After gentle resuspension, the cells were transferred to a 50 mL tube. The cryovial was washed with 1 mL of Peri.4U® Thawing Medium provided by the manufacturer, which was then transferred dropwise to the cell suspension before transferring an additional 2 mL of Peri.4U® Thawing Medium to the 50 mL tube. Cells were then counted using a hemocytometer. After this, an additional 6 mL of Peri.4U® Thawing Medium was added to the cell suspension. A cell pellet was obtained by centrifugation at 260×g (e.g., 1,100 RPM) for 6 minutes at room temperature. Cells were then resuspended in Peri.4U® complete Medium provided by the manufacturer. The cells were plated on cell culture plates coated with poly-L-ornithine and laminin at 1 cm 2 Cells were plated at a density of 50,000-150,000 cells per well. Cells were cultured at 37°C in a humidified CO2 atmosphere, and the medium was completely changed every 2-3 days during culture.

[0343] For toxin treatment, a dilution series of BoNT was prepared in Peri.4U® medium. The medium from the wells to be treated was collected and filtered (0.2 μm filter). 125 μL of the filtered medium was returned to each test well. 125 μL of diluted toxin was then added to the plate (triplicate wells). Treated cells were incubated at 37° C., 10% CO2 for 48±1 h.

[0344] Analysis of BoNT activity using the SNAP-25 cleavage assay

[0345] After treatment, BoNT was removed and cells were washed once with PBS (Gibco, UK). Cells were lysed in 1x NuPAGE lysis buffer (Life Technologies) supplemented with 0.1 M dithiothreitol (DTT) and 250 units / mL benzonase (Sigma). Protein lysates were separated by SDS-PAGE and transferred to nitrocellulose membranes. Membranes were probed with a primary antibody specific for SNAP-25 (Sigma #S9684), which recognizes uncleaved SNAP-25 as well as cleaved SNAP-25 by BoNT / A endopeptidase. The secondary antibody used was HRP-conjugated anti-rabbit IgG (Sigma #A6154). Bands were detected by enhanced chemiluminescence and imaged using pXi6Access (Synoptics, UK). Band intensities were determined using GeneTools software (Syngene, Cambridge, UK) and the percentage of cleaved SNAP-25 at each concentration of BoNT was calculated. Data were fitted to a four-parameter logistic equation and pEC was calculated using GraphPad Prism version (GraphPad). 50 was calculated.

[0346] Figure 9 shows that chimeras 3B and 3C exhibited greater potency than rBoNT / A1 in cleaving SNAP-25 in human induced pluripotent stem cells, with the former possessing a significantly greater potency, which can be explained by a dual modification that increases the affinity of chimera 3B for the human synaptotagmin II protein receptor present in these cells (Figure 9, Table 7). [Table 7]

[0347] Digital Abduction Scoring (DAS) Assay - Safety Factor

[0348] The method to measure the activity of BoNT in the DAS assay is based on the startle digit extension reflex of mice when briefly suspended by their tail. This reflex is scored as the digit abduction score (DAS) and is inhibited after BoNT administration into the gastrocnemius-soleus muscle of the hind limb. Briefly suspending a mouse by its tail induces a characteristic startle response in which the animal extends its hind limb and abducts its hind digits (Aoki et al. 1999, Eur. J. Neurol.; 6 (suppl. 4) S3-S10).

[0349] On the day of injection, mice were anesthetized in an induction chamber with 3% isoflurane in oxygen and each mouse was injected intramuscularly with BoNT or vehicle (0.2% gelatin in phosphate buffer) into the gastrocnemius and soleus muscles of the right hind leg.

[0350] After neurotoxin injection, the various degrees of finger abduction were scored on a scale of 0 to 4, where 0 = normal and 4 = maximal reduction in finger abduction and leg extension. ED was determined by nonlinear adjustment analysis using the mean of the maximum effect at each dose. 50 The mathematical model used was a four-parameter logistic model.

[0351] DAS were performed every 2 hours on the first day after dosing, then 3 times daily for 4 days for all doses. Animals in the vehicle and the lowest dose that induced DAS4 during the first 4 days of injection were then monitored until muscle weakness reached DAS0 (no observed muscle weakness) and full recovery.

[0352] To calculate the safety factor, all animals were weighed the day before toxin injection (D0) and then once a day for the duration of the study. The mean body weight, its standard deviation, and the standard error of the sample mean were calculated for each dose group on each day. The safety factor of BoNT (-10%ΔBW / ED 50 ), the dose at which the mean body weight of a treatment group at any time during the study is less than 10% of the mean body weight of that treatment group at D0 is defined as the ED for the BoNT under study. 50The lethal dose was defined as the dose at which one or more animals in the treatment group died.

[0353] Figure 10 shows the duration of muscle weakness over time in the mouse digit abduction scoring assay for unmodified BoNT / A, chimera 3B, and chimera 3C (sequence numbers 2, 14, and 15 converted to two-chain forms), indicating that the chimeras have a longer duration of action.

[0354] Table 8 below shows the EDs determined for rBoNT / A1 and chimeras 3B and 3C in the mouse DAS assay. 50 The table also shows the total duration of effect of the DAS4 dose, which is the time from complete recovery from muscle weakness to DAS0 (no muscle weakness observed). In addition, the table shows the lethal dose for mice and the safety factor (-10%ΔBW / ED 50 ) and . Compared to rBoNT / A1, chimeras 3B and 3C have a longer duration of action, a better safety margin, and a higher lethal dose. The studies shown in Figure 10 and Table 8 were performed in mice obtained from Janvier Laboratories. [Table 8]

[0355] [Example 6]

[0356] Preclinical testing of modified BoNT / A (SEQ ID NO: 4 converted to a two-chain form)

[0357] The modified BoNT / A "Cat-A" (SEQ ID NO: 4 converted to a two-chain form) was subjected to additional preclinical trials.

[0358] Materials and Methods

[0359] Rat Digital Abduction Score (DAS) Assay

[0360] To evaluate the effect of modified BoNT / A (SEQ ID NO: 4 converted to a two-chain form) on muscle activity in vivo, a dose-response study was performed using the rat DAS assay. The rat DAS assay is based on the characteristic digit extension reflex, a startle response when the animal is briefly grasped. After a single injection of neurotoxin into the left peroneal muscle complex, muscle weakness results in reduced digit abduction. The various degrees of digit abduction are scored on a 5-point scale: 0 = normal to 4 = maximal reduction in digit abduction and limb extension (Broide RS, Rubino J, Nicholson GS, et al. The rat Digit Abduction Score (DAS) assay: A physiological model for assessing botulinum neurotoxin-induced skeletal muscle paralysis. Toxicon 2013;71:18-24). DAS values ​​were measured for the first 5 consecutive days after toxin injection, and then at 2-3 day intervals until the effect of low doses of modified BoNT / A (SEQ ID NO: 4 converted to the two-chain form) on the digit extension reflex was completely abolished, and doses resulting in DAS4 restored DAS2. A transient BoNT-induced dose-dependent effect on weight gain was considered evidence of a systemic toxin effect (Torii Y, Goto Y, Nakahira S, et al. Comparison of Systemic Toxicity between Botulinum Toxin Subtypes A1 and A2 in Mice and Rats. Basic Clin. Pharmacol. Toxicol. 2015;116:524-528.). Rats were therefore weighed at each evaluation time point, and side effects were recorded. The dosing solution of BoNT was masked (assigned random letters) before injection and until the end of the study. Efficacy was determined as the dose required to induce 50% of the effect (ED 50 : the dose that produces a DAS value of 2. 50Doses ranging from 2.5 to 750 pg / kg were tested to determine the mean and 95% confidence intervals (CIs). Higher doses of 1, 1.5, 2, 2.4, 3, 4, and 5 ng / kg were also administered to assess possible side effects.

[0361] To evaluate the duration of action of modified BoNT / A (sequence number 4 converted to a two-chain form) and compare it to the duration of action of unmodified BoNT / A (sequence number 2 converted to a two-chain form), the median time required for a DAS2 reading to return to 2 was evaluated for maximally tolerated doses of both toxins (which had no effect on body weight movement compared to untreated rats) in two independent head-to-head studies.

[0362] Single-dose study in rats

[0363] Intramuscular injections of modified BoNT / A (SEQ ID NO: 4 converted to the two-chain form) at doses of 0, 0.1, 1 and 3 ng / kg were injected into the right gastrocnemius muscle of rats. Control animals received a diluent of SEQ ID NO: 4 in the right gastrocnemius muscle. Animals were euthanized after 7 days of treatment (10 males and 10 females per group) or after an observation period of 13 or 26 weeks (5 males and 5 females per dose). Irwin test observations to evaluate central nervous system function were performed pretest (day -1), on day 8, and between weeks 13 and 27. Other clinical (adverse) signs were evaluated by limp, reduced size of the toxin-injected muscle and soft, distended abdomen.

[0364] Monkey research

[0365] A single intramuscular injection of modified BoNT / A (SEQ ID NO: 4 converted to a two-chain form) at doses of 0, 0.1, 0.25 and 0.75 ng / kg was injected into the right gastrocnemius muscle of monkeys. Animals were euthanized after 7 days of treatment (3 males and 3 females per group) or after an observation period of 13 or 26 weeks (2 males and 2 females per dose). Cardiovascular examinations including hemodynamics, electrocardiograms and respiratory parameters were performed by external telemetry pretest on days 8 and 15.

[0366] Preliminary enriched EFD in pregnant rats

[0367] The purpose of the study was to provide initial information on the effects of modified BoNT / A (SEQ ID NO: 4 converted to a two-chain form) on rat embryonic and fetal development when administered by intramuscular injection route throughout the organogenesis period. A group of nine mated female Sprague-Dawley rats from gestation day 6 (G6) to gestation day 17 (G17) (inclusive) were administered daily intramuscular injections (gastrocnemius) of modified BoNT / A (SEQ ID NO: 4 converted to a two-chain form) at dose levels of 0.02, 0.05 and 0.1 ng / kg / day. Clinical status, body weight and food intake were monitored throughout the study. The females underwent Caesarean section examination on G21 and fetal parameters were recorded. At necropsy, the females were examined grossly, the pregnant uterus was weighed, and in females with a small injected gastrocnemius muscle, the muscle and the contralateral muscle were weighed. All fetuses were weighed. The fetuses were then examined for external and visceral abnormalities and sexed. The heads of approximately half of the fetuses were fixed for internal examination by serial sectioning. All fetal eviscerated carcasses were processed for skeletal examination.

[0368] Preliminary enriched EFD in pregnant rabbits

[0369] The purpose of the study was to provide initial information on the effect of modified BoNT / A (SEQ ID NO: 4 converted to the two-chain form) on rabbit embryo and fetal development when administered by intramuscular injection route throughout the organogenesis period. A group of nine mated female New Zealand White rabbits from gestation day 6 (G6) to gestation day 19 (G19) (inclusive) were administered daily intramuscular injections (gastrocnemius) of modified BoNT / A (SEQ ID NO: 4 converted to the two-chain form) at dose levels of 0.02, 0.05 and 0.1 ng / kg / day. Clinical condition, body weight and food intake were monitored throughout the study. The females underwent Caesarean section examination on G29 and fetal parameters were recorded. At necropsy, the females were grossly examined, the pregnant uterus was weighed, and in females with a small injected gastrocnemius muscle, the muscle and the contralateral muscle were weighed. All fetuses were weighed. They were then examined for external and visceral abnormalities and sexed. Approximately half of the fetuses had their heads fixed for internal examination by serial sectioning.

[0370] result

[0371] By conducting studies such as those described above, the following pharmacological data (shown in Table 9 below) was obtained for a number of different species administered modified BoNT / A. [Table 9]

[0372] Additionally, modified BoNT / A (SEQ ID NO: 4 converted to a two-chain form) was tested in a rat DAS assay to determine its duration of action compared to Dysport®. The results are shown in Table 10 below. [Table 10]

[0373] These data indicate that the modified BoNT / A has more than twice the duration of action of Dysport®.

[0374] [Example 7]

[0375] Determination of the unit dose of modified BoNT / A (SEQ ID NO: 4 converted to a two-chain form) for the treatment of cervical dystonia

[0376] Considering the preclinical pharmacology data obtained in Example 6 above, a suitable unit dose range (UD) for administration of modified BoNT / A in humans was determined. The study revealed that modified BoNT / A provides a longer duration of action than unmodified BoNT / A while at the same time exhibiting an improved safety profile. This improved safety profile may be expressed by the higher safety margins described herein for the modified BoNT / A.

[0377] Because modified BoNT / A shares the same mechanism of action as Dysport® (albeit with an increased safety margin due to its modified properties), the minimum dose of modified BoNT / A to treat affected individuals was placed relative to the labeled dose of Dysport® in the same muscle groups: ED of modified BoNT / A in the Digit Abduction Score rat model 50 The ED of Dysport® in the same rat model was 13 pg / kg, more than 100-fold lower than the estimated no observed adverse effect level (NOAEL) of 1500 pg / kg in the same species. 50 Based on these animal data, a 2.6 ng dose of modified BoNT / A is estimated to be a 100 unit dose of Dysport®. Mouse intraperitoneal LD 50 was set at 8.44 pg. Under these conditions, a dose of 0.84 ng of modified BoNT / A corresponds to a dose of 100 units of Dysport®.

[0378] Therefore, the calculated minimum dose is 500 pg (0.5 ng). 50Using this data, 0.5 ng of modified BoNT / A is equivalent to approximately 60 units of Dysport® and therefore may be efficacious when administered intramuscularly for the treatment of cervical dystonia.

[0379] The estimated NOAEL of 1.5 ng / kg for modified BoNT / A in rats is equivalent to a 90 ng dose in a 60 kg human. In monkeys, the more sensitive of the two nonclinical species, the estimated NOAEL of 0.125 ng / kg for modified BoNT / A is equivalent to a 7.5 ng dose in a 60 kg human.

[0380] Therefore, the upper limit of the unit dose is determined to be 7,500 pg (7.5 ng), which is below the NOAEL in rats converted to a human dose.

[0381] Therefore, the unit dose suitable for treating cervical dystonia with modified BoNT / A was determined to be 500-7,500 pg. Based on available preclinical data, this is within the range of the calculated intraperitoneal median lethal dose (LD ) in mice, as determined using the mouse intraperitoneal lethal dose assay. 50 ) based on about 59 to 889 units of modified BoNT / A (also corresponding to about 59 to 889 units of Dysport®).

[0382] Given the improved safety profile compared to Dysport®, as determined by the preclinical data in Example 6, the total dose (units) administered for cervical dystonia is expected to be nearly nine times greater than that of Dysport®. The maximum total dose of Dysport® for the treatment of cervical dystonia is 1,000 units (see FIG. 1).

[0383] Advantageously, more modified BoNT / A can be injected and / or more neck muscles / areas can be injected in the treatment of cervical dystonia until that maximum dose is reached. This is an important and advantageous discovery that will provide clinicians with a wider range of treatment options while at the same time leading to improved treatment of cervical dystonia.

[0384] [Example 8]

[0385] Dosage regimen for the treatment of cervical dystonia

[0386] Modified BoNT / A (SEQ ID NO: 4 converted to a two-chain form) is provided as a lyophilized powder in 2 mL clear glass vials containing 15 ng of modified BoNT / A per vial. The lyophilized powder is reconstituted with a mixture of sterile preservative-free 0.9% v / w sodium chloride solution and diluent (a buffer formulation containing only the modified BoNT / A excipients). After reconstitution, the solution is further diluted as necessary.

[0387] The unit dose (UD) is 500 to 7,500 pg (about 59 to 889 units).

[0388] Cervical dystonia is treated by intramuscular injection according to the following dosing regimen (Table 11): [Table 11]

[0389] Such administration may be unilateral or bilateral, as necessary, based on the particular condition.

[0390] The maximum total dose administered is 10 times the unit dose (e.g., in some cases, 2 times the unit dose is administered to the indicated neck muscle or muscles). This corresponds to 75,000 pg / approximately 8,890 units. This is approximately 9 times the maximum total dose of Dysport® that can be administered during treatment of cervical dystonia without approaching toxicity limits (a concern in conventional treatment regimens). Thus, clinicians can tailor treatment to affected individuals with the knowledge that 10 times the unit dose can be administered without toxicity concerns, thereby allowing treatment to additional neck muscles of the affected individual and / or ensuring that each neck muscle receives a pharmacologically effective dose.

[0391] [Example 9]

[0392] Preclinical testing of modified BoNT / AB (BoNT / AB chimera [SEQ ID NO: 14 converted to two-chain form])

[0393] The BoNT / AB chimera of SEQ ID NO:14 (converted to its two-chain form) was incubated with mouse LD 50 When tested in the assay, a result of 1.202 ng / kg was obtained. Thus, 1 unit of SEQ ID NO: 14 (converted to the two-chain form) corresponds to 24.04 pg in this assay.

[0394] The BoNT / AB chimera was further tested in the rat DAS assay to determine duration of action compared to Dysport® (according to Example 6). The results are shown in Table 12 below. [Table 12]

[0395] In conclusion, the duration of action of BoNT / AB was much longer than Dysport® and similar to that of SEQ ID NO:4 (converted to the two-chain form). It is therefore expected that the unit dose and dosing regimen for SEQ ID NO:4 (converted to the two-chain form) can be similarly applied to BoNT / AB to provide an improved treatment for cervical dystonia.

[0396] [Example 10]

[0397] Determination of the unit dose of modified BoNT / A (SEQ ID NO: 14 converted to a two-chain form) for the treatment of cervical dystonia

[0398] Taking into account the preclinical pharmacology data, a unit dose range (UD) suitable for administration of modified BoNT / A in humans was determined.

[0399] For SEQ ID NO: 14 (converted to the two-chain form), the DAS ED of 13 pg / kg 50 was calculated. ED 50 is considered to be the minimum pharmacologically effective dose, approximately 300 times lower than the no observed adverse effect level (NOAEL) of 4 ng / kg in the same animal species. 50 13 pg / kg corresponds to a 0.8 ng dose in a 60 kg human.

[0400] Therefore, 1,000 pg was selected as the lower limit of the unit dose. The upper limit of the unit dose was selected as 16,000 pg, which is lower than the NOAEL of 4 ng / kg, calculated as a human dose of 60 kg body weight, from both non-clinical safety species (rats and monkeys). Therefore, the unit dose was determined to be 1,000 pg to 16,000 pg (about 42 units to about 666 units).

[0401] Taking into account the improved safety profile, the maximum total dose for the treatment of cervical dystonia has been set at 160,000 pg (approximately 7,070 units), derived from a NOAEL of 4 ng / kg from both non-clinical safety species (rat and monkey) converted to a human dose of 60 kg body weight.

[0402] Given the improved safety profile compared to Dysport®, as determined by the preclinical data in Example 9, the total dosage (units) administered in cervical dystonia is expected to be approximately seven times that of Dysport®. The maximum total dose of Dysport® for treating cervical dystonia is 1,000 units (see FIG. 1).

[0403] Advantageously, in the treatment of cervical dystonia, more modified BoNT / A (SEQ ID NO: 14 converted to the two-chain form) can be injected and / or more neck muscles / necks can be injected until the maximum dose is reached, which is an important and advantageous discovery that could lead to improved treatment of cervical dystonia while offering clinicians a wider range of treatment options.

[0404] [Example 11]

[0405] Dosage regimen for treating cervical dystonia with modified BoNT / A (SEQ ID NO: 14 converted to a two-chain form)

[0406] The modified BoNT / A (SEQ ID NO: 14 converted to its dichain form) is provided as a lyophilized powder in vials containing 36 ng of modified BoNT / A per vial. The lyophilized powder is reconstituted.

[0407] The unit dose (UD) is between 1,000 and 16,000 pg (approximately 42 to 666 units [mouse LD 50 [Measurement by].

[0408] Cervical dystonia is treated by intramuscular injection according to the following dosing schedule (Table 13): [Table 13]

[0409] Such administration may be unilateral or bilateral, as appropriate, based on the particular condition.

[0410] The maximum total dose administered is 10 times the unit dose (e.g., in some cases, 2 times the unit dose is administered to the indicated neck muscle or muscles). This corresponds to 160,000 pg / approximately 6,660 units. This is approximately 7 times the maximum total dose of Dysport® that can be administered during treatment of cervical dystonia without approaching toxicity limits (a concern in conventional treatment regimens). Thus, clinicians can tailor treatment to affected individuals with the knowledge that 10 times the unit dose can be administered without toxicity concerns, thereby allowing treatment of additional neck muscles of that affected individual and / or ensuring that each neck muscle receives a pharmacologically effective dose.

[0411] [Example 12]

[0412] Treatment for patients with cervical dystonia (lateral neck flexion)

[0413] Jane, 65, is diagnosed with cervical dystonia by her general practitioner. The specific symptoms are as follows: neck flexion. A single unit dose (3,000 pg) of modified BoNT / A (SEQ ID NO: 4 converted to a two-chain form) is administered ipsilaterally to Jane's levator scapulae muscle, and a single unit dose is also administered ipsilaterally to Jane's sternocleidomastoid muscle (resulting in a total dose of 6,000 pg in the treatment session). The neck flexion is alleviated, and due to the long duration of the modified BoNT / A, Jane does not require further treatment for more than 9 months. Thus, Jane receives less frequent injections (e.g., per year) when compared to a comparable affected individual administered unmodified BoNT / A. Furthermore, due to the improved safety profile of the modified BoNT / A, Jane does not show any side effects.

[0414] [Example 13]

[0415] Treatment for patients with cervical dystonia (neck retroflexion)

[0416] Brian, age 48, has been diagnosed with cervical dystonia by his general practitioner. Specific symptoms include neck tilt. Modified BoNT / A (SEQ ID NO: 14 converted to a two-chain form) is administered bilaterally to Brian's muscles: · 1x unit dose of 10,000 pg to each levator scapulae; · 1x unit dose of 10,000 pg to each trapezius muscle; · 1x unit dose of 10,000 pg for each longissimus muscle; 1x unit dose of 10,000 pg into each splenius capitis muscle; and · 1x unit dose of 10,000 pg into each splenius cervix muscle.

[0417] The total dose administered is 10 times the unit dose (100,000 pg), well within the upper limit of 160,000 pg, and is possible given the higher safety profile of the modified BoNT / A compared to unmodified BoNT / A. Due to the reduction in the neck tilt and the longer duration of the modified BoNT / A, Brian has not required further treatment for 12 months. Thus, Brian will receive injections less frequently compared to comparable affected individuals receiving unmodified BoNT / A.

[0418] [Example 14]

[0419] Safety and efficacy of modified BoNT / A (SEQ ID NO: 14 converted to a two-chain form) in humans

[0420] SEQ ID NO:14 (converted to the dichain form) was administered to human patients as a single unit dose of modified BoNT / A. Five cohorts were administered different (increasing) amounts of modified BoNT / A (SEQ ID NO:14 converted to the dichain form). Cohort 1 received two 1,000 pg unit doses of modified BoNT / A (i.e., up to 2,000 pg), while cohort 5 received two 16,000 pg unit doses of modified BoNT / A (i.e., up to 32,000 pg).

[0421] The results showed that all unit doses of modified BoNT / A tested (i.e., up to the 16,000 pg unit dose) were effective in muscle paralysis, were well tolerated, and no side effects were observed despite the exceptionally high doses per muscle, indicating that the modified BoNT / A does not diffuse from the injection site and highlighting the exceptional safety profile of modified BoNT / A (SEQ ID NO: 14 converted to a two-chain form).

[0422] [Example 15]

[0423] Treatment for Patients with Cervical Dystonia

[0424] Sally, age 64, is diagnosed with cervical dystonia by her general practitioner. She is treated with a unit dose (UD) of 1000 pg of SEQ ID NO: 14 (converted to the two-chain form) administered as follows: [Table I]

[0425] She receives a total dose of 14,000 pg of SEQ ID NO: 14 (converted to the two-chain form). The treatment is successful and her symptoms are alleviated. She has not required treatment for more than 9 months.

[0426] [Example 16]

[0427] Treatment for Patients with Cervical Dystonia

[0428] Francesco, age 43, is diagnosed with cervical dystonia by his general practitioner. He is treated with a unit dose (UD) of 2000 pg of SEQ ID NO: 14 (converted to the two-chain form) administered as follows: [Table J]

[0429] He receives a total dose of 28,000 pg of SEQ ID NO: 14 (converted to the two-chain form). The treatment is successful and the symptoms are alleviated. He has not required treatment for more than 10 months.

[0430] All publications mentioned in the above specification are incorporated herein by reference. Various modifications and variations of the described methods and systems of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in biochemistry and biotechnology or related fields are intended to be within the scope of the claims. [Explanation of symbols]

[0431] [Figure 1] Indication: Recommended Concentration: Recommended DYSPORT Dose: Recommended Dysport Dose Cervical Dystonia: Cervical dystonia Adults: Adults 50 units / 0.1ml or 25 units / 0.1ml 500 to 1000 units [Figures 2, 5, and 8] Marker: Marker [Figures 3A, 3B, 3C, 6, 9] % SNAP-25 cleavage: Percentage of SNAP-25 cleavage Basal: Basal [Figure 3D] mean ΔpEC50 v nBoNT / A1: Mean ΔpEC50 vs nBoNT / A1 log units: log / unit 10x less poteint: 10 times less potent [Figure 4] min: minutes mean min ± sem: mean min ± standard error of the mean [Figures 5 to 10] SEQ ID NO: Sequence number [Figure 6] in rat spinal cord neurons [Figure 7] Mean DAS: Average DAS Dose (pg / mouse): Dose (pg / mouse) [Figure 9] in human pluripotent stem cells [Figure 10] Time (day): Time (day)

Claims

1. A pharmaceutical for treating cervical dystonia, comprising modified botulinum neurotoxin A (BoNT / A), wherein: the modified BoNT / A is administered intramuscularly to an affected neck muscle of the affected individual; the modified BoNT / A is administered in a unit dose of 750 pg to 17,000 pg of modified BoNT / A; administering at least a single unit dose to said affected neck muscle; The total dose of modified BoNT / A administered during said treatment is up to 170,000 pg; and The modified BoNT / A comprises a BoNT / A light chain and translocation domain and a BoNT / B receptor binding domain (H C domain) and includes

2. The pharmaceutical composition according to claim 1, wherein: (a) the total dose administered is between 5,250 pg and 170,000 pg, preferably between 7,000 pg and 160,000 pg; (b) the unit dose is between 1,000 pg and 6,000 pg; (c) the total dose of modified BoNT / A administered during said treatment is up to 160,000 pg; and / or (d) The modified BoNT / A is administered to the affected neck muscle at a single injection site or at two or more injection sites (e.g., two injection sites). Optionally, the modified BoNT / A is administered at a unit dose of 750 pg to 4,000 pg (preferably 1,000 pg or 2,000 pg) per injection site.

3. A pharmaceutical for treating cervical dystonia, comprising modified botulinum neurotoxin A (BoNT / A), wherein: the modified BoNT / A is administered intramuscularly to an affected neck muscle of the affected individual; the modified BoNT / A is administered in a unit dose of between 31 units and 707 units of modified BoNT / A; One unit is the calculated median lethal dose (LD) for mice. 50 ) is the amount of modified BoNT / A equivalent to; administering at least a single unit dose to said affected neck muscle; The total dose of modified BoNT / A administered during said treatment is up to 7,070 units; and The modified BoNT / A comprises a BoNT / A light chain and translocation domain, and a BoNT / B receptor binding domain (Hc domain).

4. The pharmaceutical composition according to claim 3, wherein: (a) the total dose administered is between 217 and 7,070 units, preferably between 294 and 6,660 units; (b) the unit dose is between 42 units and 666 units; (c) the total dose of modified BoNT / A administered during said treatment is up to 6,660 units; and / or (d) the modified BoNT / A is administered to the affected neck muscle at a single injection site or at two or more injection sites (e.g., two injection sites); optionally, the modified BoNT / A is administered at a unit dose of between 31 units and 166.4 units (preferably 41.6 units or 83.2 units) per injection site.

5. A medicament for treating cervical dystonia, comprising modified botulinum neurotoxin A (BoNT / A), wherein: the modified BoNT / A is administered intramuscularly to an affected neck muscle of the affected individual; the modified BoNT / A is administered in a unit dose of 450 pg to 8,000 pg of modified BoNT / A; administering at least a single unit dose to said affected neck muscle; the total dose of modified BoNT / A administered during said treatment is up to 80,000 pg; The modified BoNT / A comprises a modification at one or more amino acid residues selected from the following: ASN886, ASN905, GLN915, ASN918, GLU920, ASN930, ASN954, SER955, GLN991, GLU992, GLN995, ASN1006, ASN1025, ASN1026, ASN1032, ASN1043, ASN1046, ASN1052, ASP1058, HIS1064, ASN1080, GLU1081, GLU1083, ASP1086, ASN1188, ASP1213, Gly1215, ASN1216, GLN1229, ASN1242, ASN1243, SER1274, and THR1277; and The modification is selected from the following: (i) replacement of acidic surface-exposed amino acid residues with basic amino acid residues; (ii) replacement of acidic surface-exposed amino acid residues with uncharged amino acid residues; (iii) substitution of uncharged surface-exposed amino acid residues with basic amino acid residues; (iv) insertion of a basic amino acid residue; and (v) Deletion of acidic surface-exposed amino acid residues

6. The pharmaceutical composition according to claim 5, wherein: (a) the total dose administered is between 3,150 pg and 80,000 pg, preferably between 3,500 pg and 75,000 pg; (b) the unit dose is between 500 pg and 75,00 pg; and / or (c) the total dose of modified BoNT / A administered during said treatment is up to 75,000 pg; 7. A pharmaceutical composition for treating cervical dystonia, comprising modified botulinum neurotoxin A (BoNT / A), wherein: the modified BoNT / A is administered intramuscularly to an affected neck muscle of the affected individual; the modified BoNT / A is administered in a unit dose of between 53 units and 948 units of modified BoNT / A; One unit is the calculated median lethal dose (LD) for mice. 50 ) is the amount of modified BoNT / A equivalent to; administering at least a single unit dose to said affected modified neck muscle; The total dose of modified BoNT / A administered during treatment is up to 9,480 units; The modified BoNT / A comprises a modification at one or more amino acid residues selected from the following: ASN886, ASN905, GLN915, ASN918, GLU920, ASN930, ASN954, SER955, GLN991, GLU992, GLN995, ASN1006, ASN1025, ASN1026, ASN1032, ASN1043, ASN1046, ASN1052, ASP1058, HIS1064, ASN1080, GLU1081, GLU1083, ASP1086, ASN1188, ASP1213, Gly1215, ASN1216, GLN1229, ASN1242, ASN1243, SER1274, and THR1277; and The modification is selected from the following: (i) replacement of acidic surface-exposed amino acid residues with basic amino acid residues; (ii) replacement of acidic surface-exposed amino acid residues with uncharged amino acid residues; (iii) substitution of uncharged surface-exposed amino acid residues with basic amino acid residues; (iv) insertion of a basic amino acid residue; and (v) Deletion of acidic surface-exposed amino acid residues

8. The pharmaceutical composition according to claim 7, wherein: (a) The total dose administered is between 371 and 9,480 units, preferably between 413 and 8,890 units. (b) the unit dose is between 59 and 889 units; and / or (c) The total dose of modified BoNT / A administered during treatment is a maximum of 8,890 units.

9. The pharmaceutical composition according to claim 1, 3, 5, or 7, wherein: The affected neck muscle is selected from: sternocleidomastoid, splenius capitis, splenius cervicis, scalene complex (e.g., anterior scalene and / or middle scalene), trapezius (e.g., upper trapezius), levator scapulae, semispinalis capitis, and longissimus (e.g., longissimus capitis and / or longissimus cervicis); The affected neck muscle is selected from: Right levator scapulae, left levator scapulae, right trapezius, left trapezius, right sternocleidomastoid, left sternocleidomastoid, right splenius capitis, left splenius capitis, middle scalene, anterior scalene, right semispinalis capitis, left semispinalis capitis, right longissimus capitis, left longissimus capitis; The affected neck muscle is selected from: Sternocleidomastoid muscle (sternocleidomastoid / sternocleidomastoideus), splenius capitis, splenius cervix, scalene complex (e.g., anterior scalene and / or middle scalene), trapezius (e.g., upper trapezius), levator scapulae, semispinalis capitis, longissimus (e.g., longissimus capitis and / or longissimus cervix), posterior paravertebral region (e.g., posterior scalene, middle scalene and / or anterior scalene, preferably posterior scalene), submandibular complex (e.g., digastric, geniohyoid, mylohyoid, mylohyoid boutonniere and / or stylohyoid), lower trapezius, superior nuchal line - clavicle (lateral portion), spinous Origin C3-Th3-mastoid, spinous process Th3-Th5-transverse process C1-C2, transverse process C3-Th6, spinous process C3-Th1-superior nuchal line, transverse process Th1-Th6-spinous process C2-C7, transverse process C3-Th3-mastoid, transverse process Th1-Th6-transverse process C2-C6, inferior oblique muscle, spinous process C 2 - transverse process C1, suprasternal notch and clavicle (medial part) - mastoid process and superior nuchal line, transverse process C1 - C4 - scapula (superior corner), transverse process C2 - C7 - first rib, transverse process C3 - C6 - first rib, longus capitis muscle, transverse process C3 - C6 - occipital bone (base), longus carpis muscle, transverse process C2 - C5 - atlas (anterior tubercle); or, The affected neck muscle is selected from: Sternocleidomastoid muscle (e.g., left sternocleidomastoid or right sternocleidomastoid), left splenius capitis or right splenius capitis, anterior scalene or middle scalene, left or right trapezius muscle (e.g., left upper trapezius or right upper trapezius), left or right levator scapulae, left or right semispinalis capitis, longissimus muscle (e.g., left and right longissimus capitis and / or left and right longissimus capitis), splenius cervix, scalene complex (e.g., anterior scalene and / or middle scalene), posterior paraspinal region (e.g., posterior scalene, middle scalene and / or anterior scalene, preferably posterior scalene), submandibular complex (e.g., digastric, geniohyoid, mylohyoid, mylohyoid boutonniere and / or stylohyoid), lower trapezius, superior nuchal line Clavicle (lateral part), spinous process C3-Th3 - mastoid process, spinous process Th3-Th5 - transverse process C1-C2, transverse process C3-Th6, spinous process C3-Th1 - superior nuchal line, transverse process Th1-Th6 - spinous process C2-C7, transverse process C3-Th3 - Mastoid process, transverse process Th1-Th6 - transverse process C2-C6, inferior oblique muscle, superior oblique muscle, spinous process C2 - transverse process C1, suprasternal notch and clavicle (medial part) - mastoid process and superior nuchal line, transverse process C1-C4 - scapula (superior corner), transverse process C 2-C7 - first rib, transverse process C3-C6 - first rib, longus capitis muscle, transverse process C3-C6 - occipital bone (bottom), longus neck muscle, semispinalis cervicis, rectus occipitalis major, rectus occipitalis minor, rectus capitis frontalis, multifidus canal, and transverse process C2-C5 - atlas (anterior tuberosity) 10. The pharmaceutical composition according to any one of claims 1, 3, 5, or 7, wherein: the modified BoNT / A is administered by intramuscular injection to multiple affected neck muscles of the affected individual; At least one unit dose is administered to each affected nape of the neck; Preferably, The plurality of affected neck muscles are selected from: Sternocleidomastoid, splenius capitis, splenius cervicis, scalene complex (e.g., anterior scalene and / or middle scalene), trapezius (e.g., upper trapezius), levator scapulae, semispinalis capitis and longissimus (e.g., longissimus capitis and / or longissimus cervicis); The plurality of affected neck muscles are selected from: Right levator scapulae, left levator scapulae, right trapezius, left trapezius, right sternocleidomastoid, left sternocleidomastoid, right splenius capitis, left splenius capitis, middle scalene, anterior scalene, right semispinalis capitis, left semispinalis capitis, right longissimus capitis, left longissimus capitis; The plurality of affected neck muscles are selected from: Sternocleidomastoid muscle (sternocleidomastoid / sternocleidomastoideus), splenius capitis muscle, splenius cervix muscle, scalene complex (e.g., anterior scalene and / or middle scalene), trapezius muscle (e.g., upper trapezius), levator scapulae muscle, semispinalis capitis muscle, longissimus muscle (e.g., longissimus capitis and / or longissimus cervix), posterior paravertebral region (e.g., posterior scalene, middle scalene and / or anterior scalene, preferably posterior scalene), submandibular complex (e.g., digastric muscle, geniohyoid muscle, mylohyoid muscle, mylohyoid boutonniere and / or stylohyoid muscle), lower trapezius muscle, superior nuchal line - clavicle (lateral part), spinous processes C3-Th3 - mastoid process, spinous processes Th3-Th5 - transverse processes C1-C2, transverse processes C3-Th6, spinous process C3-Th1 - superior nuchal line, transverse process Th1-Th6 - spinous process C2-C7, transverse process C3-Th3 - mastoid process, transverse process Th1-Th6 - transverse process C2-C6, inferior oblique muscle, spinous process C2 - transverse process C1, suprasternal notch and clavicle (medial part) - mastoid process and superior nuchal line, transverse process C1-C4 - scapula (superior angle), transverse process C2-C7 - first rib, transverse process C3-C6 - first rib, longus capitis, transverse process C3-C6 - occipital (base), longus neck muscle, transverse process C2-C5 - atlas (anterior tubercle); or The plurality of affected neck muscles are selected from: Sternocleidomastoid muscle (e.g., left sternocleidomastoid muscle or right sternocleidomastoid muscle), left splenius capitis muscle or right splenius capitis muscle, anterior scalene muscle or middle scalene muscle, left or right trapezius muscle (e.g., upper left trapezius muscle or upper right trapezius muscle), left or right levator scapulae muscle, left or right semispinalis capitis muscle, middle part of semispinalis capitis muscle, longissimus muscle (e.g., left and right longissimus capitis muscles and / or left and right longissimus capitis muscles), splenius cervix muscle, scalene muscle complex (e.g., anterior scalene muscle and / or middle scalene muscle), posterior paravertebral region (e.g., posterior scalene muscle, middle scalene muscle and / or anterior scalene muscle, preferably posterior scalene muscle), submandibular complex (e.g., digastric muscle, geniohyoid muscle, mylohyoid muscle, mylohyoid boutonniere muscle and / or stylohyoid muscle), lower trapezius muscle, superior nuchal line - clavicle (lateral part), spinous processes C3-Th3 - Mastoid, spinous process Th3-Th5 - transverse process C1-C2, transverse process C3-Th6, spinous process C3-Th1 - superior nuchal line, transverse process Th1-Th6 - spinous process C2-C7, transverse process C3-Th3 - mastoid, transverse process Th1-Th6 - transverse process C2-C6, inferior oblique muscle, superior capitis oblique muscle, spinous process C2 - transverse process C1, suprasternal notch and clavicle (inner part) - Mastoid and superior nuchal line, transverse process C1-C4 - Scapula (superior angle), transverse process C2-C7 - First rib, transverse process C3-C6 - First rib, longus capitis, transverse process C3-C6 - Occipital bone (bottom), longus cervix, semispinalis cervicis, rectus occipital major, rectus occipital minor, rectus frontalis, multifidus canal, and transverse process C2-C5 - atlas (anterior tuberosity) 11. The pharmaceutical composition according to any one of claims 1, 3, 5, or 7, wherein: (a) the modified BoNT / A is administered to the affected neck muscle at a unit dose per injection site, or the modified BoNT / A is administered at less than a unit dose per injection site; (b) a single unit dose is administered to multiple injection sites in the affected neck muscle, and / or two or more unit doses are administered to multiple injection sites in the affected neck muscle; (c) a single unit dose is administered to the affected neck muscles (e.g., a single unit dose is administered to each affected neck muscle); (d) the affected subject is a human; (e) the modified BoNT / A treats cervical dystonia in an affected individual for a longer duration than treatment with an unmodified BoNT / A (e.g., SEQ ID NO: 2); and / or (f) the modified BoNT / A has a safety factor of greater than 7, the safety factor being calculated by dividing the dose of toxin required to produce a -10% change in body weight measured in pg / mouse by the DAS ED 50 measured in pg / mouse, where ED 50 is the dose required to obtain a DAS score of 2.

12. A unit dosage form of modified BoNT / A (e.g., for treating cervical dystonia), wherein: The unit dosage form includes: (a) 750 pg to 17,000 pg of modified BoNT / A; or (b) 31 units to 707 units of modified BoNT / A, where 1 unit is the median lethal dose (LD ) for mice. 50 ) is the amount of modified BoNT / A equivalent to; and (c) optionally pharmaceutically acceptable carriers, excipients, adjuvants and / or salts; wherein the modified BoNT / A comprises a BoNT / A light chain and translocation domain, and a BoNT / B receptor binding domain (Hc domain).

13. A pharmaceutical composition according to claim 1 or 3 or a unit dosage form according to claim 12, wherein: (a) the modified BoNT / A contains a combination of two substitution mutations, E1191M and S1199Y; (b) the modified BoNT / A comprises a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 14; (c) the modified BoNT / A is a two-chain modified BoNT / A in which the light chain is linked to the heavy chain via a disulfide bond, obtained by a method of converting a single-chain modified BoNT / A containing a polypeptide sequence having at least 70%, 80%, 90%, 95%, or 99.9% sequence identity to SEQ ID NO: 14 or SEQ ID NO: 14 into the corresponding two-chain modified BoNT / A by contacting the single-chain modified BoNT / A with a protease that hydrolyzes the peptide bond in the active loop; (d) the modified BoNT / A is a two-chain modified BoNT / A in which the light chain is linked to the heavy chain via a disulfide bond, obtained by a method of converting a single-chain modified BoNT / A consisting of SEQ ID NO: 14 into the corresponding two-chain modified BoNT / A by contacting the single-chain modified BoNT / A with a protease that hydrolyzes the peptide bond in the activation loop; and / or (e) the C-terminal amino acid residue of the LH N domain of the modified BoNT / A corresponds to the first amino acid residue of the 3 10 helix separating the LH N domain and the HC domain of BoNT / A, and the N-terminal amino acid residue of the HC domain of the modified BoNT / A corresponds to the second amino acid residue of the 3 10 helix separating the LH N domain and the HC domain of BoNT / B.

14. A unit dosage form of modified BoNT / A (e.g., for treating cervical dystonia), wherein: The unit dosage form comprises: (a) 450 pg to 8,000 pg of modified BoNT / A; or (b) 53 units to 948 units of modified BoNT / A, where 1 unit is the median lethal dose (LD 50 ) is the amount of modified BoNT / A equivalent to; and (c) optionally pharmaceutically acceptable carriers, excipients, adjuvants and / or salts; The modified BoNT / A comprises a modification at one or more amino acid residues selected from the following: ASN886, ASN905, GLN915, ASN918, GLU920, ASN930, ASN954, SER955, GLN991, GLU992, GLN995, ASN1006, ASN1025, ASN1026, ASN1032, ASN1043, ASN1046, ASN1052, ASP1058, HIS1064, ASN1080, GLU1081, GLU1083, ASP1086, ASN1188, ASP1213, Gly1215, ASN1216, GLN1229, ASN1242, ASN1243, SER1274, and THR1277; The modification is selected from the following: (i) replacement of acidic surface-exposed amino acid residues with basic amino acid residues; (ii) replacement of acidic surface-exposed amino acid residues with uncharged amino acid residues; (iii) substitution of uncharged surface-exposed amino acid residues with basic amino acid residues; (iv) insertion of a basic amino acid residue; and (v) Deletion of acidic surface-exposed amino acid residues 15. The pharmaceutical composition of claim 5 or 7 or the unit dosage form of claim 14, wherein: (a) the modification comprises (and preferably consists of) a modification in one or more amino acid residues selected from the following: ASN886, ASN930, ASN954, SER955, GLN991, ASN1025, ASN1026, ASN1052, ASN1188, ASP1213, GLY1215, ASN1216, GLN1229, ASN1242, ASN1243, SER1274, or THR1277; The modified BoNT / A is encoded by a nucleic acid sequence having at least 70% sequence identity to a nucleic acid sequence selected from SEQ ID NOs: 3, 5, 7, and 9, and / or comprises a polypeptide sequence having at least 70% sequence identity to a polypeptide sequence selected from SEQ ID NOs: 4, 6, 8, and 10; Preferably, the modification comprises (and preferably consists of) a modification in one or more amino acid residues selected from the following: ASN886, ASN930, SER955, GLN991, ASN1026, ASN1052, and GLN1229; the modified BoNT / A is encoded by a nucleic acid sequence having at least 70% sequence identity to SEQ ID NO:3 and / or comprises a polypeptide sequence having at least 70% sequence identity to an amino acid sequence selected from SEQ ID NO:4; (b) the modification is a substitution, preferably a substitution with lysine or arginine; (c) the modified BoNT / A is a two-chain modified BoNT / A in which the light chain (L chain) is linked to the heavy chain (H chain) via a disulfide bond, obtained by a method of converting a single-chain modified BoNT / A comprising a polypeptide sequence having at least 70%, 80%, 90%, 95%, or 99.9% sequence identity to SEQ ID NO: 4 into the corresponding two-chain modified BoNT / A by contacting the single-chain modified BoNT / A with a protease that hydrolyzes the peptide bond in the active loop; and / or (d) The modified BoNT / A is a two-chain modified BoNT / A in which the light chain is linked to the heavy chain via a disulfide bond, obtained by a method of converting a single-chain modified BoNT / A consisting of SEQ ID NO: 4 into the corresponding two-chain modified BoNT / A by contacting the single-chain modified BoNT / A with a protease that hydrolyzes the peptide bond in the active loop.

16. A pharmaceutical for treating cervical dystonia, comprising a unit dosage form according to claim 12 or 14.

17. Kit including: (a) a unit dosage form according to claim 12 or 14; (b) instructions for use of the unit dosage form in the treatment of cervical dystonia; (c) optionally, a diluent;

18. A pharmaceutical kit for treating cervical dystonia, comprising: (a) a unit dosage form according to claim 12 or 14; (b) instructions for use of the unit dosage form in the treatment of cervical dystonia; (c) optionally, a diluent;

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