Treatment of cervical muscular dynia

Abstract

The present invention relates, inter alia, to a modified botulinum neurotoxin A (BoNT / A) for use in the treatment of cervical dystonia wherein the modified BoNT / A is administered by intramuscular injection to the affected neck muscle of an individual wherein the modified BoNT / A is administered at a unit dose greater than 17,000 pg of the modified BoNT / A wherein at least one unit dose is administered to the affected neck muscle, and wherein the modified BoNT / A is administered by intramuscular injection to the affected neck muscle. Wherein the total dose administered during treatment is at most 400,000 pg of the modified BoNT / A, and wherein the modified BoNT / A comprises a BoNT / A light chain and a translocation domain, and a BoNT / B receptor binding domain (HC domain). Corresponding methods, uses, unit dosage forms and kits are also provided.

Description

Technical Field

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

[0002] Cervical dystonia (also known as spasmodic torticollis) is a chronic neurological movement disorder that is often associated with extreme pain. The condition causes the affected individual's neck to involuntarily turn left, right, up, and / or down. During dystonic movements, both the agonist and antagonist muscles may contract simultaneously.

[0003] This disorder usually presents relatively mild symptoms, such as non-visual head tremors that last for several months at the onset of the disease. Other early / progressive symptoms may include sudden movements of the head turning, pulling and / or tilting. However, further early / progressive symptoms usually include persistent / long-term involuntary head positioning. Before reaching a plateau, the frequency and intensity of involuntary neck muscle spasms tend to increase over time. Individuals suffering from cervical dystonia may also experience muscle hypertrophy, neck pain, dysarthria and / or tremor.

[0004] Symptoms of cervical dystonia may involve any of the neck muscles of an individual, and the head posture may vary. Typically, the most common abnormal posture associated with cervical dystonia is a twisting of the chin toward the shoulder, causing the head to rotate to the side (torticollis). Other abnormal postures associated with cervical dystonia may include anterocollis (head tilted forward), retrocollis (head tilted backward), or laterocollis (head tilted to one side). Anterior sagittal shift (forward displacement) or posterior sagittal shift (backward displacement) of the head on the shoulders may also occur. However, most commonly, cervical dystonia presents complex symptoms in which individuals exhibit multiple angles of head movement.

[0005] Current treatment options include oral medications (e.g., dopamine blockers), deep brain stimulation, botulinum neurotoxin, and selective denervation surgery, which removes the nerves that trigger muscle contraction. Traditional oral medications are associated with a number of serious side effects, while deep brain stimulation and denervation surgery are invasive, have associated risks of complications, and / or may not be effective.

[0006] is an example of a traditional serotype A botulinum neurotoxin (BoNT / A) for the treatment of cervical dystonia, which is a pharmaceutical product containing the drug substance BoNT / A hemagglutinin complex (BTX-A-HAC) isolated and purified from Clostridium botulinum. There are several other pharmaceutical BoNT / A products on the market that are naturally produced by Clostridium botulinum (e.g. and ).

[0007] By paralyzing the antagonist muscles of dystonia, BoNT / A can allow the agonist muscles to move freely. In more detail, BoNT / A selectively inhibits the release of acetylcholine from presynaptic nerve endings, thereby blocking cholinergic transmission at the neuromuscular junction, thereby inducing muscle contraction and a decrease in muscle tone, leading to relaxation of the injected muscle. However, the duration of action of currently available BoNT / A products is about 12 to 14 weeks, at which time new nerve endings sprout, nerve function returns to normal, and the original symptoms reappear. Therefore, in order to maintain the effect, injections need to be repeated regularly. Therefore, considering the chronic nature of the condition and the need for long-term treatment, the frequency of BoNT / A injections is an important consideration in the treatment of cervical dystonia. In fact, this affects the direct and indirect health costs involved for patients and caregivers, the organization of injections in hospitals / clinics, and most importantly, the quality of life of patients.

[0008] Approved for the treatment of cervical dystonia, the maximum total dose per treatment course is 1,000 units (see Figure 1 ). The clinician needs to apply a The total dose per treatment course is capped at 1,000 units. Clinicians are forced to make difficult choices when treating their patients. In other words, in conventional treatment regimens, clinicians must find a balance between the relatively low total amount of BoNT / A that can be administered (1,000 units - necessitated by the highly toxic nature of BoNT / A) and the effective amount in multiple different muscles. As a result, certain muscles are neglected, while other muscles receive suboptimal amounts of BoNT / A, resulting in suboptimal treatment.

[0009] Furthermore, conventional cervical dystonia treatment regimens are complex, leading clinicians to underdose in order to minimize patient toxicity. Therefore, there is a need for a convenient, safe, and effective single-dose unit that can be administered to the affected neck muscles during a treatment session and corresponding guidance for the number of units (e.g., including the number of injection sites per muscle) without resulting in patient toxicity.

[0010] In conclusion, there is a need for improved treatments for cervical dystonia that allow for an individualized, patient-centered approach, tailoring treatment to the targeted clinical pattern, allowing injections into different combinations of affected neck muscles depending on the distribution, extent, and severity of cervical dystonia, while avoiding toxicity and providing more durable treatment (resulting in less frequent administration).

[0011] The present invention overcomes one or more of the above-identified problems. Summary of the invention

[0012] The present inventors surprisingly found that modified BoNT / A is particularly useful in treating cervical dystonia. The modified BoNT / A of the present invention comprises a BoNT / A light chain and a translocation domain (H N domain) and the BoNT / B receptor binding domain (H C domain), which can result in the modified BoNT / A exhibiting increased retention at the administration site (reduced diffusion from the administration site) and / or increased duration of action (e.g., 6-9 months).

[0013] Advantageously, compared to unmodified BoNT / A (e.g. ) have an improved safety profile. This improved safety profile can be expressed by the high safety ratio of the modified BoNT / A described herein.

[0014] Based on the preclinical and clinical data herein (see Examples), it has been shown that a higher total amount of modified BoNT / A can be administered to an individual while achieving comparable results to unmodified BoNT / A (e.g. ) similar safety profile, albeit at such high doses. Advantageously, the clinical data herein (see Example 9) indicate that higher unit doses and total amounts of modified BoNT / A can be administered to an individual while achieving comparable efficacy to unmodified BoNT / A (e.g., ) with a similar safety profile. Thus, in treating cervical dystonia, more modified BoNT / A can be injected and / or injections can be made at a greater number of cervical muscles / sites before a maximum total dose is reached. This is an important and advantageous discovery that provides improved treatment of cervical dystonia while providing clinicians with a wider range of treatment options. Treatment can be improved because compared to unmodified BoNT / A (e.g. ) treatment, it provides a more durable treatment (resulting in a reduced frequency of administration) and / or can be tailored to an individual and / or results in an improved quality of life for the individual. Thus, the treatment of the present invention is improved over conventional treatment regimens.

[0015] In addition, the present invention provides a single unit dose and a total (maximum) dose that can be safely administered in a single treatment that is convenient, safe and effective. The present invention also provides corresponding guidelines for the number of times the unit dose can be administered to the neck muscles (e.g., including the number of injection sites for each muscle) without causing patient toxicity. Therefore, the treatment of cervical dystonia according to the present invention is much simpler for clinicians and helps to avoid insufficient and / or overdosage. In addition, compared with traditional cervical dystonia treatments, the treatment according to the present invention is more satisfactory for patients because it is better customized to the needs of the patient. DETAILED DESCRIPTION

[0016] In one aspect, the invention provides a modified botulinum neurotoxin A (BoNT / A) for use in treating cervical dystonia, wherein the modified BoNT / A is administered by intramuscular injection to an affected neck muscle of an individual,

[0017] wherein the modified BoNT / A is administered in a unit dose of greater than 17,000 pg of the modified BoNT / A,

[0018] wherein at least a single unit dose is administered to the affected neck muscle,

[0019] wherein the total dose administered during the treatment period is at most 400,000 pg of the modified BoNT / A, and

[0020] The modified BoNT / A comprises a BoNT / A light chain and a translocation domain, and a BoNT / B receptor binding domain (H C domain).

[0021] In a related aspect, the invention provides a modified BoNT / A for use in treating cervical dystonia in an individual for a duration longer than treatment with an unmodified BoNT / A (e.g., SEQ ID NO: 2 [e.g., SEQ ID NO: 2 in di-chain form]), wherein the modified BoNT / A is administered by intramuscular injection to the affected neck muscles of the individual,

[0022] wherein the modified BoNT / A is administered in a unit dose of greater than 17,000 pg of the modified BoNT / A,

[0023] wherein at least a single unit dose is administered to the affected neck muscle,

[0024] wherein the total dose administered during the treatment period is at most 400,000 pg of the modified BoNT / A,

[0025] The modified BoNT / A comprises a BoNT / A light chain and a translocation domain, and a BoNT / B receptor binding domain (H C domain).

[0026] The term "treating a subject's cervical dystonia for a duration longer than treatment with an unmodified BoNT / A" may refer to a subject's reduction in one or more symptoms of cervical dystonia lasting longer (e.g., 6-9 months) after administration of a modified BoNT / A of the invention compared to administration of an unmodified BoNT / A. The duration of action may be at least 1.25×, 1.5×, 1.75×, 2.0×, or 2.25×. The duration of action of a 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 specific embodiments, the duration of action may be greater than 9.0 months. The reduction may be determined by comparison to an equivalent control subject exhibiting the same symptoms who has been treated with an unmodified BoNT / A. An individual treated with a modified BoNT / A according to the present invention can exhibit at least a 5%, 10%, 25%, or 50% improvement in the severity of one or more equivalent symptoms compared to the severity of one or more equivalent symptoms prior to treatment with the modified BoNT / A, for a period of time that is substantially the same (e.g., the same) as the severity of one or more equivalent symptoms in the control individual prior to treatment with the unmodified BoNT / A. The unmodified BoNT / A is preferably SEQ ID NO: 2 in a double-stranded form.

[0027] 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 individual by intramuscular injection,

[0028] wherein the modified BoNT / A is administered in a unit dose of greater than 17,000 pg of the modified BoNT / A,

[0029] wherein at least a single unit dose is administered to the affected neck muscle,

[0030] wherein the total dose administered during the treatment period is at most 400,000 pg of the modified BoNT / A, and

[0031] The modified BoNT / A comprises a BoNT / A light chain and a translocation domain, and a BoNT / B receptor binding domain (H C domain).

[0032] In a related aspect, the invention provides a method for treating cervical dystonia in a subject for a longer duration than treatment with an unmodified BoNT / A (e.g., SEQ ID NO: 2 [e.g., a di-chain form of SEQ ID NO: 2]), the method comprising administering a modified BoNT / A to an affected neck muscle of the subject by intramuscular injection,

[0033] wherein the modified BoNT / A is administered in a unit dose of greater than 17,000 pg of the modified BoNT / A,

[0034] wherein at least a single unit dose is administered to the affected neck muscle,

[0035] wherein the total dose administered during the treatment period is at most 400,000 pg of the modified BoNT / A,

[0036] The modified BoNT / A comprises a BoNT / A light chain and a translocation domain, and a BoNT / B receptor binding domain (H C domain).

[0037] In one aspect, the present invention provides a use of a modified botulinum neurotoxin A (BoNT / A) in the preparation of a medicament for treating cervical dystonia, wherein the modified BoNT / A is administered by intramuscular injection to an affected neck muscle of an individual,

[0038] wherein the modified BoNT / A is administered in a unit dose of greater than 17,000 pg of the modified BoNT / A,

[0039] wherein at least a single unit dose is administered to the affected neck muscle,

[0040] wherein the total dose administered during the treatment period is at most 400,000 pg of the modified BoNT / A, and

[0041] The modified BoNT / A comprises a BoNT / A light chain and a translocation domain, and a BoNT / B receptor binding domain (H C domain).

[0042] In a related aspect, the invention provides use of a modified BoNT / A in the preparation of a medicament for treating cervical dystonia in an individual for a longer duration than treatment with an unmodified BoNT / A (e.g., SEQ ID NO: 2 [e.g., SEQ ID NO: 2 in di-chain form]), wherein the modified BoNT / A is administered to the affected neck muscles of the individual by intramuscular injection,

[0043] wherein the modified BoNT / A is administered in a unit dose of greater than 17,000 pg of the modified BoNT / A,

[0044] wherein at least a single unit dose is administered to the affected neck muscle,

[0045] wherein the total dose administered during the treatment period is at most 400,000 pg of the modified BoNT / A,

[0046] The modified BoNT / A comprises a BoNT / A light chain and a translocation domain, and a BoNT / B receptor binding domain (H C domain).

[0047] The unit dose can be greater than 17,000 pg of modified BoNT / A. The upper limit of the unit dose range can be 40,000, 39,000, 38,000, 37,000, 36,000, 35,000, 30,000, 25,000, 24,000, 22,000, 20,000, or 18,000 pg of modified BoNT / A, with a preferred upper limit of 38,000 pg. The lower end of the unit dose range can be 17,500, 18,000, 20,000, 22,000, 24,000, 25,000, 26,000, 27,000, 28,000, 29,000, 30,000, 35,000, 36,000, 37,000, 38,000, or 39,000 pg of modified BoNT / A, with a preferred lower end being 17,500 pg or 25,000 pg. Preferably, the unit dose of modified BoNT / A is greater than 17,000 pg to up to 4,000 pg of modified BoNT / A, such as greater than 17,000 pg to up to 36,000 pg, or 20,000 pg to 39,000 pg. Most preferably, the unit dose of the modified BoNT / A is 22,000 to 38,000 pg, such as 24,000 to 36,000 pg or 25,000 to 36,000 pg. The unit dose can be 25,000 pg up to 40,000 pg of the modified BoNT / A, and in preferred embodiments, the unit dose of the modified BoNT / A is 24,000, 25,000, 30,000, or 36,000 pg, such as 25,000 or 36,000 pg. In more preferred embodiments, the unit dose of the modified BoNT / A is 30,000 or 36,000 pg (e.g., 36,000 pg).

[0048] The unit dose can be 20,000 pg to 30,000 pg, such as 24,000 pg to 26,000 pg of modified BoNT / A, and most preferably, the unit dose is 25,000 pg of modified BoNT / A.

[0049] The unit dose can be 30,000 pg to 40,000 pg, such as 35,000 pg to 37,000 pg of modified BoNT / A, and most preferably, the unit dose is 36,000 pg of modified BoNT / A.

[0050] The total dose administered when carrying out the treatment regimen of the invention can be up to 400,000 pg. In other words, the total amount of modified BoNT / A administered in a given treatment course can be up to 400,000 pg. The total dose can be up to 380,000, 360,000, 340,000, 320,000, 300,000, 280,000, 260,000, 250,000, 240,000, 220,000, 200,000, 180,000, 160,000, 140,000, 120,000, 100,000, 80,000, 60,000, 40,000, or 20,000 pg. Preferably, the total dose can be up to 360,000 pg of modified BoNT / A. The total dose may be at least 17,500, 20,000, 22,500, 25,000, 27,500, 30,000, 35,000, 36,000, 37,000, 38,000, 39,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000, 100,000, 150,000, 160,000, 170,000, 180,000, 190,000, 200,000, 210,000, 220,000, 250,000, 275,000, 30,000, 35,0 Preferably, the total dose can be greater than 170,000 pg, more preferably at least 240,000 pg or at least 250,000 pg of modified BoNT / A, such as at least 300,000 pg. The total dose can be 160,000-400,000 pg, or 170,000-400,000 pg (e.g., greater than 170,000-400,000 pg), preferably 170,000 pg up to 360,000 pg or 200,000-370,000 pg. The total dose administered can be 250,000 pg to 400,000 pg. More preferably, the total dose administered is 250,000-360,000 pg. In preferred embodiments, the total dose is 240,000, 250,000, 300,000, or 360,000 pg (e.g., 250,000 or 360,000 pg) of modified BoNT / A. In a more preferred embodiment, the total dose is 300,000 or 360,000 pg (eg, 360,000 pg).

[0051] The total dose can be 200,000 pg to 300,000 pg, such as 240,000 pg to 260,000 pg modified BoNT / A. Preferably, the total dose is at most 250,000 pg modified BoNT / A (eg, the total dose can be 250,000 pg modified BoNT / A).

[0052] The total dose can be 300,000 pg to 400,000 pg, such as 350,000 pg to 370,000 pg modified BoNT / A. Preferably, the total dose is at most 360,000 pg modified BoNT / A (eg, the total dose can be 360,000 pg modified BoNT / A).

[0053] Thus, the unit dose may be greater than 17,000 pg of modified BoNT / A, and the total dose administered when carrying out the treatment regimen of the invention may be up to 400,000 pg. In a preferred embodiment, the unit dose may be 24,000 pg, and the total dose may be 240,000 pg. In another preferred embodiment, the unit dose may be 25,000 pg, and the total dose may be 250,000 pg. In another preferred embodiment, the unit dose may be 30,000 pg, and the total dose may be 300,000 pg.

[0054] In another preferred embodiment, the unit dose may be 36,000 pg and the total dose may be 360,000 pg.

[0055] In one aspect, the invention provides a modified botulinum neurotoxin A (BoNT / A) for use in treating cervical dystonia, wherein the modified BoNT / A is administered by intramuscular injection to an affected neck muscle of an individual,

[0056] wherein the modified BoNT / A is administered in a unit dose of greater than 707 units of the modified BoNT / A, wherein 1 unit corresponds to a calculated median lethal dose (LD50) in mice. 50 ),

[0057] wherein at least a single unit dose is administered to the affected neck muscles,

[0058] wherein the total dose administered during the treatment period is at most 16,639 U of modified BoNT / A, and

[0059] The modified BoNT / A comprises a BoNT / A light chain and a translocation domain, and a BoNT / B receptor binding domain (H C domain).

[0060] In a related aspect, the invention provides a modified BoNT / A for use in treating cervical dystonia in an individual for a duration longer than treatment with an unmodified BoNT / A (e.g., SEQ ID NO: 2 [e.g., SEQ ID NO: 2 in di-chain form]), wherein the modified BoNT / A is administered by intramuscular injection to the affected neck muscles of the individual,

[0061] wherein the modified BoNT / A is administered in a unit dose of greater than 707 units of the modified BoNT / A, wherein 1 unit corresponds to a calculated median lethal dose (LD50) in mice. 50 ),

[0062] wherein at least a single unit dose is administered to the affected neck muscles,

[0063] wherein the total dose administered during the treatment period is at most 16,639 U of modified BoNT / A, and

[0064] The modified BoNT / A comprises a BoNT / A light chain and a translocation domain, and a BoNT / B receptor binding domain (H C domain).

[0065] 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 individual by intramuscular injection,

[0066] The modified BoNT / A is administered in a unit dose of greater than 707 U of the modified BoNT / A, wherein 1 unit corresponds to a calculated median lethal dose (LD50) in mice. 50 ),

[0067] wherein at least a single unit dose is administered to the affected neck muscles,

[0068] wherein the total dose administered during the treatment period is at most 16,639 U of modified BoNT / A, and

[0069] The modified BoNT / A comprises a BoNT / A light chain and a translocation domain, and a BoNT / B receptor binding domain (H C domain).

[0070] In a related aspect, the invention provides a method for treating cervical dystonia in an individual for a longer duration than treatment with an unmodified BoNT / A (e.g., SEQ ID NO: 2 [e.g., SEQ ID NO: 2 in di-chain form]), the method comprising administering a modified BoNT / A to an affected neck muscle of the individual by intramuscular injection,

[0071] wherein the modified BoNT / A is administered in a unit dose of greater than 707 units of the modified BoNT / A, wherein 1 unit corresponds to a calculated median lethal dose (LD50) in mice. 50 ),

[0072] wherein at least a single unit dose is administered to the affected neck muscles,

[0073] wherein the total dose administered during the treatment period is at most 16,639 U of modified BoNT / A, and

[0074] The modified BoNT / A comprises a BoNT / A light chain and a translocation domain, and a BoNT / B receptor binding domain (H C domain).

[0075] In one aspect, the present invention provides a use of a modified botulinum neurotoxin A (BoNT / A) in the preparation of a medicament for treating cervical dystonia, wherein the modified BoNT / A is administered by intramuscular injection to an affected neck muscle of an individual,

[0076] wherein the modified BoNT / A is administered in a unit dose of greater than 707 units of the modified BoNT / A, wherein 1 unit corresponds to a calculated median lethal dose (LD50) in mice. 50 ),

[0077] wherein at least a single unit dose is administered to the affected neck muscles,

[0078] wherein the total dose administered during the treatment period is at most 16,639 U of modified BoNT / A, and

[0079] The modified BoNT / A comprises a BoNT / A light chain and a translocation domain, and a BoNT / B receptor binding domain (H C domain).

[0080] In a related aspect, the invention provides use of a modified BoNT / A in the manufacture of a medicament for treating cervical dystonia in an individual for a longer duration than treatment with an unmodified BoNT / A (e.g., SEQ ID NO: 2 [e.g., SEQ ID NO: 2 in di-chain form]), wherein the modified BoNT / A is administered to the affected neck muscles of the individual by intramuscular injection,

[0081] wherein the modified BoNT / A is administered in a unit dose of greater than 707 units of the modified BoNT / A, wherein 1 unit corresponds to a calculated median lethal dose (LD50) in mice.50 ),

[0082] wherein at least a single unit dose is administered to the affected neck muscles,

[0083] wherein the total dose administered during the treatment period is at most 16,639 U of modified BoNT / A, and

[0084] The modified BoNT / A comprises a BoNT / A light chain and a translocation domain, and a BoNT / B receptor binding domain (H C domain).

[0085] The unit dose can be greater than 707 units of a modified BoNT / A. The upper limit of the unit dose range can be 1664, 1650, 1600, 1550, 1500, 1450, 1400, 1350, 1300, 1250, 1150, 1100, 1050, 1000, 950, 900, 850, 800, or 750 units of a modified BoNT / A, with a preferred upper limit of 1500 units. The lower end of the unit dose range can be 728, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, 1000, 1025, 1050, 1075, 1100, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, or 1650 units of a modified BoNT / A, with a preferred lower end being 728 units or 1,040 units. Preferably, the unit dose of a modified BoNT / A is from greater than 707 units to up to 1664 units of a modified BoNT / A, such as from greater than 707 units to up to 1498 units or 832 units to 1622 units, and most preferably, the unit dose of a modified BoNT / A is from 915 to 1518 units, such as from 998 to 1498 units. The unit dose of the modified BoNT / A can be from 1,040 units to a maximum of 1,664 units. In preferred embodiments, the unit dose of the modified BoNT / A comprises 998, 1,248, 1,040, or 1,498 units, e.g., 1,040 or 1,498 units of the modified BoNT / A. In more preferred embodiments, the unit dose comprises 1,248 or 1,498 units (e.g., 1,248 units) of the modified BoNT / A.

[0086] The unit dose can be 832 units to 1,248 units, such as 998 units to 1,082 units of the modified BoNT / A. Most preferably, the unit dose is 1,040 units of the modified BoNT / A.

[0087] The unit dose can be 1,248 units to 1,664 units, such as 1,456 units to 1,539 units of the modified BoNT / A. Most preferably, the unit dose is 1,498 units of the modified BoNT / A.

[0088] The total dose administered when carrying out the treatment regimen of the invention can be up to 16,639 units. In other words, the total amount of modified BoNT / A administered in a given treatment course can be up to 16,639 units. The total dose can be up to 16,000, 15,000, 14,000, 13,000, 12,000, 11,000, 10,000, 9,000, 8,000, 7,000, 6,000, 5,000, 4,000, 3,000, 2,000, 1,000, or 832 units. Preferably, the total dose can be up to 14,975 units of modified BoNT / A. The total dose may be at least 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, 1000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 2,000, 2,500, 3,000, 3,500, 4,000, 4,500, 5,000, 5,500, 6,000, 7,000, 8,000, 9,000, 10,000, 11,000, 12,000, 13,000, 14,000, 15,000, or 16,000 units. The total dose can be at least 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 11,000, 12,000, 13,000, 14,000, 15,000, 16,000, or 16,300 units. Preferably, the total dose can be greater than 7,072 units, more preferably at least 9,983 units or 10,399 units of modified BoNT / A, such as at least 12,479 units. The total dose can be 6,656-16,639 units or 7,072-16,639 units (e.g., greater than 7,072 units to up to 16,639 units), preferably 7,072 to up to 14,975 units or 8,319-15,391 units. More preferably, the total dose administered is 9,983-14,975 units. The total dose administered can be 10,399 units to 16,639 units of modified BoNT / A. In preferred embodiments, the total dose is 9,983, 10,399, 12,479 or 14,975 units (e.g., 10,399 or 14,975 units). In preferred embodiments, the total dose is 12,479 or 14,975 pg (e.g., 14,975 units).

[0089] The total dose can be 8,319 units to 12,479 units, such as 9,983 units to 10,815 units of modified BoNT / A. Preferably, the total dose is at most 103,999 units (eg, the total dose can be 103,999 units).

[0090] The total dose can be 12,479 units to 16,639 units, such as 14,559 units to 15,391 units of modified BoNT / A. Preferably, the total dose is at most 14,975 units (e.g., the total dose can be 14,975 units).

[0091] Thus, the unit dose can be greater than 707 units of modified BoNT / A and the total dose administered when carrying out the treatment regimen of the invention can be up to 16,639 units. In a preferred embodiment, the unit dose can be 998 units of modified BoNT / A and the total dose can be 9,983 units. In another preferred embodiment, the unit dose can be 1,248 units of modified BoNT / A and the total dose can be 12,479 units.

[0092] In another preferred embodiment, the unit dose can be 1,498 units of modified BoNT / A, and the total dose can be 14,975 units.

[0093] An "affected neck muscle" can be a neck muscle that contributes to (e.g., causes) cervical dystonia and / or its symptoms in an individual, or a neck muscle that contributes to (e.g., causes) cervical dystonia and / or its symptoms in an individual. This does not mean that an "affected neck muscle" must necessarily contribute to (e.g., cause) cervical dystonia and / or its symptoms at the time of treatment, although this is preferred. For example, a neck muscle can be a muscle that contributed to (e.g., caused) cervical dystonia and / or its symptoms in an individual in the past, or a muscle that is expected to contribute to (e.g., cause) cervical dystonia and / or its symptoms in an individual in the future. In one embodiment, two or more neck muscles (e.g., an agonist pair and an antagonist pair of neck muscles) can contribute to (e.g., cause) cervical dystonia and / or its symptoms in an individual. In this case, the modified BoNT / A can be administered to two or more neck muscles (e.g., to the agonist neck muscle and the antagonist neck muscle of the neck muscle pair).

[0094] The affected neck muscles preferably contribute to (e.g., cause) individual cervical dystonia and / or its symptoms by contraction. Therefore, the affected neck muscles are preferably individual neck muscles, and their contraction or its contraction causes individual cervical dystonia and / or its symptoms. The neck muscles are preferably neck muscles that, for example, contract involuntarily or have contracted involuntarily during treatment. The neck muscles can be any muscles (e.g., skeletal muscles) that are effectively connected to the neck and / or head of an individual, such as any muscles that can change the head positioning of an individual (e.g., when contracted). The affected neck muscles can be capable of: causing the individual's chin to twist in the direction of the individual's shoulders, thereby causing the head to rotate laterally (torticollis); causing the individual's head to tilt forward (flexed neck); causing the individual's head to tilt backward (extended neck); causing the individual's head to tilt laterally (lateral neck); causing the individual's head to shift anteriorly (displace forward); and / or causing the individual's head to shift posteriorly (displace backward).

[0095] Affected neck muscles may include the sternocleidomastoid, sternocleidomastoideus, splenius capitis, splenius cervicalis, 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 cervicalis). Affected neck muscles may include the sternocleidomastoid, splenius capitis, splenius cervicalis, 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 cervi), posterior paraspinal muscles (e.g., posterior scalene, middle scalene and / or anterior scalene, preferably posterior scalene), submental complex (e.g., digastric, geniohyoid, mylohyoid, mylohyoid buttonhole and / or stylohyoid), superior nuchal line-clavicle (lateral portion), spinous process C3-3, Th3-3, and Th3-4. 5—transverse processes C1-C2, transverse processes C3-Th6, spinous processes 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, inferior oblique muscle of the capitis, 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 of scapula), transverse processes C2-C7—first rib, transverse processes C3-C6—first rib, longus capitis, transverse processes C3-C6—occipital bone (basal part), longus colli or transverse processes 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 may include the sternocleidomastoid (e.g., left or right sternocleidomastoid), left or right splenius capitis, anterior or middle scalene muscles, left or right trapezius (e.g., left or right upper trapezius), left or right levator scapulae, left or right semispinalis capitis, longissimus (e.g., left or right longissimus capitis and / or longissimus cervicis), splenius cervicis, scalene complex (e.g., anterior scalene and / or middle scalene), posterior paraspinal muscles (e.g., posterior scalene, middle scalene and / or anterior scalene, preferably posterior scalene), submental complex (e.g., digastric, geniohyoid, mylohyoid, mylohyoid buttonhole and / or stylohyoid), lower trapezius, superior nuchal line-clavicle (lateral portion), spinous process C3-Th3-mastoid, spinous process C3-Th3-mastoid. -Th5—transverse processes C1-C2, transverse processes C3-Th6, spinous processes 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 scapular angle), transverse processes C2-C7—first rib, transverse processes C3-C6—first rib, longus capitis, transverse processes C3-C6—occipital bone (basal part), longus colli, semispinalis cervicis, rectus capitis posterior major, rectus capitis posterior minor, rectus capitis anterior, multifidus or transverse processes C2-C5—atlas (anterior tubercle).

[0096] The affected neck muscles may include the sternocleidomastoid (e.g., left or right sternocleidomastoid), the splenius capitis (e.g., left or right splenius capitis), the anterior scalene, the middle scalene, the trapezius (e.g., left or right trapezius, such as the left or right upper trapezius), the levator scapulae (e.g., left or right levator scapulae), the semispinalis capitis (e.g., left or right semispinalis capitis), the middle part of the semispinalis capitis, the longissimus (e.g., left or right longissimus capitis and / or longissimus cervicis), the splenius cervicis, the scalene complex (e.g., anterior scalene and / or middle scalene), the posterior paraspinal muscles (e.g., posterior scalene, middle scalene and / or anterior scalene, preferably posterior scalene), the submental complex (e.g., digastric, geniohyoid, mylohyoid, mylohyoid buttonhole and / or stylohyoid), the lower trapezius, Superior nuchal line—clavicle (lateral part), spinous process C3-Th3-mastoid, spinous process Th3-Th5—transverse processes C1-C2, transverse processes C3-Th6, spinous process C3-Th1—superior nuchal line, transverse processes Th1-Th6—spinous process C2-C7, transverse process C3-Th3—mastoid, transverse processes Th1-Th6—transverse processes C2-C6, inferior oblique muscle of the capitis, superior oblique muscle of the capitis, spinous process C2—transverse process C1, suprasternal notch and clavicle (medial part)—mastoid and superior nuchal line, transverse processes C1-C4—scapula (superior angle of scapula), transverse processes C2-C7—first rib, transverse processes C3-C6—first rib, longus capitis, transverse processes C3-C6—occipital bone (basal part), longus colli, semispinalis cervicis, spinalis capitis (spinalis capitis) capitis), rectus capitis major, rectus capitis minor, rectus capitis anterior, multifidus or transverse processes C2-C5—atlas (anterior tubercle).

[0097] Most preferably, the affected neck muscles may include: semispinalis cervicis, levator scapulae, splenius cervicis, longissimus cervicis, trapezius (e.g., lower trapezius), sternocleidomastoid, semispinalis capitis, inferior oblique capitis, longissimus capitis, splenius capitis, or scalene muscles (e.g., anterior scalene muscles, middle scalene muscles, and / or posterior scalene muscles), longus colli, or longus capitis. For example, the affected neck muscles are one or more selected from the group comprising or consisting of: semispinalis cervicis, levator scapulae, splenius cervicis, longissimus cervicis, trapezius (e.g., lower trapezius), sternocleidomastoid, semispinalis capitis, inferior oblique capitis, longissimus capitis, splenius capitis, or scalene muscles (e.g., anterior scalene muscles, middle scalene muscles, and / or posterior scalene muscles), longus colli, and longus capitis.

[0098] The plurality of affected neck muscles treated according to the present invention may include at least one (eg, at least two) of any of the muscles described herein.

[0099] In one embodiment, a modified BoNT / A can be administered to one or more affected neck muscles selected from the group consisting of: 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 a maximum number of affected neck muscles. For example, a modified BoNT / A can be administered to at least two (e.g., at least three, four, five, six, or seven, preferably eight) affected neck muscles selected from the group consisting of: 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).

[0100] In one embodiment, a modified BoNT / A can be administered to one or more affected neck muscles selected from the group consisting of right levator scapulae, left levator scapulae, right trapezius, left trapezius, right sternocleidomastoid, left sternocleidomastoid, right splenius capitis, left splenius capitis, middle scalene muscles, anterior scalene muscles, right semispinalis capitis, left semispinalis capitis, right longissimus capitis, and left longissimus capitis.

[0101] In one embodiment, the modified BoNT / A can be administered to one or more affected neck muscles selected from the group consisting of: sternocleidomastoid, sternocleidomastoid, splenius capitis, splenius cervicalis, 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 cervi), posterior paraspinal muscles (e.g., posterior scalene, middle scalene and / or anterior scalene, preferably posterior scalene), submental complex (e.g., digastric, geniohyoid, mylohyoid, mylohyoid buttonhole and / or stylohyoid), superior nuchal-clavicle (lateral portion ), spinous process C3-Th3-mastoid, spinous process 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, transverse processes Th1-Th6-transverse processes C2-C6, inferior oblique capitis, spinous process C2-transverse process C1, suprasternal notch and clavicle (medial portion)-mastoid and superior nuchal line, transverse processes C1-C4-scapula (superior scapular angle), transverse processes C2-C7-first rib, transverse processes C3-C6-first rib, longus capitis, transverse processes C3-C6-occipital (basal portion), longus colli, and transverse processes C2-C5-atlas (anterior tubercle). Preferably, the modified BoNT / A is administered to the maximum number of affected neck muscles.For example, a modified BoNT / A can 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 group consisting of: sternocleidomastoid, sternocleidomastoid, splenius capitis, splenius cervicis, 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 cervicis), posterior paraspinal muscles (e.g., posterior scalene, middle scalene and / or anterior scalene, preferably posterior scalene), submental complex. (e.g. digastric, geniohyoid, mylohyoid, mylohyoid buttonhole and / or stylohyoid), superior nuchal line—clavicle (lateral part), spinous process C3-Th3-mastoid, spinous process 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, transverse processes Th1 -Th6—transverse processes C2-C6, inferior oblique capitis, spinous process C2—transverse process C1, suprasternal notch and clavicle (medial part)—mastoid process and superior nuchal line, transverse processes C1-C4—scapula (superior scapular angle), transverse processes C2-C7—first rib, transverse processes C3-C6—first rib, longus capitis, transverse processes C3-C6—occipital bone (basal part), longus colli and transverse processes C2-C5—atlas (anterior tubercle).

[0102] In one embodiment, a modified BoNT / A can be administered to one or more affected neck muscles, including: sternocleidomastoid (e.g., left or right sternocleidomastoid), splenius capitis (e.g., left or right splenius capitis), anterior scalene, middle scalene, trapezius (e.g., left or right trapezius, such as left or right upper trapezius), levator scapulae (e.g., left or right levator scapulae), semispinalis capitis (e.g., left or right semispinalis capitis), middle semispinalis capitis, longissimus (e.g., left or right longissimus capitis and / or longissimus cervicis), splenius cervicis, scalene complex (e.g., anterior scalene and / or middle scalene), posterior paraspinal muscles (e.g., posterior scalene, middle scalene and / or anterior scalene, preferably posterior scalene), submental complex (e.g., digastric, geniohyoid, mylohyoid, mylohyoid buttonhole, and / or stylohyoid), trapezius Lower part of the muscle, upper 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 - upper 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 of the head, superior oblique muscle of the head, spinous process C2 - transverse process C1, thoracic Suprascapular notch and clavicle (medial portion)—mastoid process and superior nuchal line, transverse processes C1-C4—scapula (superior scapular angle), transverse processes C2-C7—first rib, transverse processes C3-C6—first rib, longus capitis, transverse processes C3-C6—occipital bone (basal portion), longus colli, semispinalis cervicis, spinalis capitis, rectus capitis posterior major, rectus capitis posterior minor, rectus capitis anterior, multifidus and / or transverse processes C2-C5—atlas (anterior tubercle).

[0103] Preferably, the modified BoNT / A is administered to a maximum number of affected neck muscles. For example, a modified BoNT / A can 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 comprising: the sternocleidomastoid muscle (e.g., left or right sternocleidomastoid muscle), the cranial nerve, the anterior cruciate ligament, the posterior cruciate ligament, the posterior cruciate ligament, the posterior cruciate ligament, the posterior cruciate ligament splenius (e.g., left or right splenius capitis), anterior scalene muscles, middle scalene muscles, trapezius muscles (e.g., left or right trapezius muscles, such as left or right upper trapezius muscles), levator scapulae (e.g., left or right levator scapulae), semispinalis capitis (e.g., left or right semispinalis capitis), middle semispinalis capitis, longissimus (e.g., left or right longissimus capitis and / or longissimus cervicis), splenius cervicis, scalene muscle complex (e.g., anterior scalene muscles and / or middle scalene muscles), posterior paraspinal muscles (e.g., posterior scalene muscles, middle scalene muscles and / or anterior scalene muscles, preferably posterior scalene muscles horn), submental complex (e.g., digastric, geniohyoid, mylohyoid, mylohyoid buttonhole, and / or stylohyoid), lower trapezius, superior nuchal line—clavicle (lateral portion), spinous process C3-Th3-mastoid, spinous process 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, transverse processes Th1-Th6—transverse processes C2-C 6. 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 scapular angle), transverse processes C2-C7—first rib, transverse processes C3-C6—first rib, longus capitis, transverse processes C3-C6—occipital bone (basal part), longus colli, semispinalis cervicis, spinalis capitis, rectus capitis posterior major, rectus capitis posterior minor, rectus capitis anterior, multifidus and / or transverse processes C2-C5—atlas (anterior tubercle).

[0104] In one embodiment, the modified BoNT / A can be administered to one or more affected neck muscles selected from the group consisting of: sternocleidomastoid (e.g., left or right sternocleidomastoid), splenius capitis (e.g., left or right splenius capitis), anterior scalene, middle scalene, trapezius (e.g., left or right trapezius, such as left or right upper trapezius), levator scapulae (e.g., left or right levator scapulae), semispinalis capitis (e.g., left or right semispinalis capitis), middle semispinalis capitis, longissimus (e.g., left or right longissimus capitis and / or longissimus cervicis), splenius cervicis, scalene complex (e.g., anterior scalene and / or middle scalene), posterior paraspinal muscles (e.g., posterior scalene, middle scalene and / or anterior scalene, preferably posterior scalene), submental complex (e.g., digastric, geniohyoid, mylohyoid, mylohyoid buttonhole, and / or stylohyoid), Lower part of quadratus 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 of capitis, superior oblique muscle of capitis, spinous process C2—transverse process C1 , suprasternal notch and clavicle (medial portion) - mastoid process and superior nuchal line, transverse processes C1-C4 - scapula (superior scapular angle), transverse processes C2-C7 - first rib, transverse processes C3-C6 - first rib, longus capitis, transverse processes C3-C6 - occipital (basal portion), longus colli, semispinalis cervicis, spinalis capitis, rectus capitis posterior major, rectus capitis posterior minor, rectus capitis anterior, multifidus, and transverse processes C2-C5 - atlas (anterior tubercle). Preferably, the modified BoNT / A is administered to the most affected neck muscles.For example, the modified BoNT / A can 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 group consisting of: sternocleidomastoid muscle (e.g., left or right sternocleidomastoid muscle), splenius capitis (e.g., left or right splenius capitis), anterior scalene muscles, middle scalene muscles, trapezius muscles (e.g., left or right trapezius muscles, such as left or right upper trapezius muscles), levator scapulae (e.g., left or right levator scapulae), semispinalis capitis (e.g., left or right semispinalis capitis), middle semispinalis capitis, longissimus (e.g., left or right longissimus capitis and / or longissimus cervicis), splenius cervicis, scalene muscle complex (e.g., anterior scalene muscles and / or middle scalene muscles), posterior paraspinal muscles (e.g., posterior scalene muscles, middle scalene muscles and / or anterior scalene muscles, preferably posterior scalene), submental complex (e.g., digastric, geniohyoid, mylohyoid, mylohyoid buttonhole, and / or stylohyoid), lower trapezius, superior nuchal line—clavicle (lateral portion), spinous process C3-Th3-mastoid, spinous process 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, 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 process C1-C4 — scapula (superior scapular angle), transverse process C2-C7 — first rib, transverse process C3-C6 — first rib, longus capitis, transverse process C3-C6 — occipital bone (basal part), longus colli, semispinalis cervicis, spinalis capitis, rectus capitis posterior major, rectus capitis posterior minor, rectus capitis anterior, multifidus and transverse process C2-C5 — atlas (anterior tubercle).

[0105] Most preferably, the modified BoNT / A can be administered to one or more affected neck muscles comprising: semispinalis cervicis, levator scapulae, splenius cervicis, longissimus cervicis, trapezius (e.g., lower trapezius), sternocleidomastoid, semispinalis capitis, inferior oblique capitis, longissimus capitis, splenius capitis, scalene muscles (e.g., anterior scalene, middle scalene, and / or posterior scalene), longus colli, and / or longus capitis. For example, the modified BoNT / A can be administered to one or more affected neck muscles selected from: semispinalis cervicis, levator scapulae, splenius cervicis, longissimus cervicis, trapezius (e.g., lower trapezius), sternocleidomastoid, semispinalis capitis, inferior oblique capitis, longissimus capitis, splenius capitis, scalene muscles (e.g., anterior scalene, middle scalene, and / or posterior scalene), longus colli, and longus capitis. For example, a modified BoNT / A can be administered to at least two (e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15) of the affected neck muscles.

[0106] In one embodiment, a modified BoNT / A can be administered to one or more affected neck muscles comprising: 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 / or left longissimus capitis.

[0107] In one embodiment, a modified BoNT / A may be administered to one or more affected neck muscles selected from the group consisting of right levator scapulae, left levator scapulae, right trapezius, left trapezius, right sternocleidomastoid, left sternocleidomastoid, right splenius capitis, left splenius capitis, middle scalene muscles, anterior scalene muscles, right semispinalis capitis, left semispinalis capitis, right longissimus capitis, and left longissimus capitis.

[0108] When there are two equal neck muscles on either side of the neck (e.g., sternocleidomastoid muscles, e.g., left and right sternocleidomastoid muscles), the modified BoNT / A can be administered unilaterally (e.g., to one of the muscles where only one muscle is contracted) or bilaterally (e.g., to both muscles where both muscles are contracted). When treating multiple affected muscles, it is preferred that multiple affected muscles of different types are treated. For example, when administering bilaterally to both affected sternocleidomastoid muscles, it is preferred that additional muscles are treated when treating multiple affected muscles as described herein.

[0109] When there are two equivalent neck muscles on either side of the neck, unilateral administration may be performed to either of the equivalent neck muscles. For example, administration may be performed to a contracted muscle or to a non-contracted equivalent muscle. In one embodiment, unilateral administration is performed to a muscle on one side of the neck of an individual, where one side of the individual's neck exhibits symptoms of cervical dystonia, or unilateral administration is performed to an equivalent muscle on the contralateral side of the neck of an individual, where the contralateral side of the individual's neck does not exhibit symptoms of cervical dystonia.

[0110] The affected neck muscles selected for treatment according to the present invention can depend on the specific manifestations of cervical dystonia (e.g., torticollis, lateral neck, flexion neck, extension neck, lateral head, oblique head, flexion head, extension head, lateral displacement, sagittal displacement or a combination thereof) of the individual to be treated. Jost, WH, & Tatu, L. (2015). Selection of Muscles for Botulinum Toxin Injections in Cervical Dystonia. Movement disorders clinical practice, 2 (3), 224–226. https: / / doi.org / 10.1002 / mdc3.12172 describes the typical manifestations and related muscles of cervical dystonia. Therefore, the muscle or muscle group treated according to the present invention can be the muscle or muscle group described in the above-mentioned reference Jost & Tatu (2015), which is incorporated herein by reference.

[0111] In one embodiment, when treating torticollis, the modified BoNT / A can be administered to one or more affected neck muscles selected from the group consisting of sternocleidomastoid, trapezius (e.g., upper trapezius), scalene anterior, splenius capitis, splenius cervix, levator scapulae, and longissimus (e.g., longissimus capitis and / or longissimus cervix). In one embodiment, when treating torticollis, the modified BoNT / A can be administered contralaterally to one or more affected neck muscles selected from the group consisting of sternocleidomastoid, trapezius (e.g., upper trapezius), and scalene anterior; and / or ipsilaterally to one or more affected neck muscles selected from the group consisting of splenius capitis, splenius cervix, levator scapulae, and longissimus (e.g., longissimus capitis and / or longissimus cervix).

[0112] In one embodiment, when treating torticollis, the modified BoNT / A can be administered to one or more affected neck muscles selected from the group consisting of sternocleidomastoid, trapezius (e.g., upper trapezius), anterior scalene, splenius capitis, splenius cervi, levator scapulae, and longissimus (e.g., longissimus capitis and / or longissimus cervi), and semispinalis cervi. In one embodiment, when treating torticollis, the modified BoNT / A can be administered contralaterally to one or more affected neck muscles selected from the group consisting of sternocleidomastoid, trapezius (e.g., upper trapezius), and anterior scalene; and / or ipsilaterally to one or more affected neck muscles selected from the group consisting of splenius capitis, splenius cervi, levator scapulae, and longissimus (e.g., longissimus capitis and / or longissimus cervi), and semispinalis.

[0113] In one embodiment, when treating torticollis, the modified BoNT / A can be administered to one or more affected neck muscles selected from the group consisting of sternocleidomastoid, trapezius (e.g., upper trapezius), anterior scalene, splenius capitis, splenius cervi, levator scapulae, longissimus (e.g., longissimus capitis and / or longissimus cervi), semispinalis cervi, rectus capitis posterior major, multifidus, and inferior oblique capitis. In one embodiment, when treating torticollis, the modified BoNT / A can be administered contralaterally to one or more affected neck muscles selected from the group consisting of sternocleidomastoid, trapezius (e.g., upper trapezius), anterior scalene, semispinalis cervi, and multifidus; and / or ipsilaterally to one or more affected neck muscles selected from the group consisting of splenius capitis, splenius cervi, levator scapulae, longissimus (e.g., longissimus capitis and / or longissimus cervi), rectus capitis posterior major, and inferior oblique capitis.

[0114] In one embodiment, when treating torticollis, a modified BoNT / A can be administered to one or more affected neck muscles, including: sternocleidomastoid, trapezius (e.g., upper trapezius), anterior scalene, splenius capitis, splenius cervi, levator scapulae, longissimus (e.g., longissimus capitis and / or longissimus cervi), semispinalis cervi, rectus capitis posterior major, multifidus, and / or inferior oblique capitis. In one embodiment, when treating torticollis, a modified BoNT / A can be administered contralaterally to one or more affected neck muscles, including: sternocleidomastoid, trapezius (e.g., upper trapezius), anterior scalene, semispinalis cervi, and / or multifidus; and / or ipsilaterally to one or more affected neck muscles, including: splenius capitis, splenius cervi, levator scapulae, longissimus (e.g., longissimus capitis and / or longissimus cervi), rectus capitis posterior major, and / or inferior oblique capitis.

[0115] In one embodiment, when treating torticollis, the modified BoNT / A is administered to one or more affected neck muscles selected from or including: the semispinalis cervicis, levator scapulae, splenius cervicis, and / or the longissimus cervicis. The modified BoNT / A can be administered to the same side.

[0116] In one embodiment, when treating the laterocollis, the modified BoNT / A can be administered to one or more affected neck muscles selected from the group consisting of the levator scapulae, trapezius (e.g., upper trapezius), scalene complex (e.g., anterior scalene and / or middle scalene), sternocleidomastoid, splenius capitis, splenius cervicis, and longissimus (e.g., longissimus capitis and / or longissimus cervicis).

[0117] In one embodiment, when treating the lateral neck, the modified BoNT / A can be administered to one or more affected neck muscles selected from the group consisting of the levator scapulae, trapezius (e.g., upper trapezius), scalene complex (e.g., anterior scalene and / or middle scalene), sternocleidomastoid, splenius capitis, splenius cervi, and longissimus (e.g., longissimus capitis and / or longissimus cervi), and semispinalis cervi.

[0118] In one embodiment, when treating the lateral neck, the modified BoNT / A can be administered to one or more affected neck muscles selected from the group consisting of the 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), and multifidus. In one embodiment, when treating the lateral neck, the modified BoNT / A can be administered to one or more affected neck muscles, the affected neck muscles comprising the 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), and / or multifidus.

[0119] In one embodiment, when treating the lateral neck, the modified BoNT / A can be administered to one or more affected neck muscles selected from the group consisting of the levator scapulae, trapezius (e.g., upper trapezius), scalene complex (e.g., anterior scalene and / or middle scalene), sternocleidomastoid, splenius capitis, splenius cervi, longissimus (e.g., longissimus capitis and / or longissimus cervi), semispinalis cervi, and multifidus.

[0120] In one embodiment, when treating the lateral neck, the modified BoNT / A can be administered to one or more of the 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 cervi, longissimus (e.g., longissimus capitis and / or longissimus cervi), semispinalis cervi, and / or multifidus.

[0121] In one embodiment, when treating the lateral neck, the modified BoNT / A is administered to one or more affected neck muscles selected from or including the levator scapulae, semispinalis cervicis, scalene medius, and / or longissimus cervicis.

[0122] Preferably, when treating the side of the neck, the modified BoNT / A may be administered ipsilaterally to one or more of the affected neck muscles.

[0123] In one embodiment, when treating anterocollis, the modified BoNT / A may be administered to one or more affected neck muscles selected from the group consisting of the sternocleidomastoid, anterior scalene, and middle scalene.

[0124] In one embodiment, when treating neck flexion, the modified BoNT / A can be administered to one or more affected neck muscles selected from the group consisting of the sternocleidomastoid, scalene anterior, scalene medius, levator scapulae, longus colli, and submental complex (e.g., digastric, geniohyoid, mylohyoid, mylohyoid buttonhole, and / or stylohyoid).

[0125] In one embodiment, when treating neck flexion, the modified BoNT / A can be administered to one or more affected neck muscles selected from the group consisting of the sternocleidomastoid, anterior scalene, medius scalene, longus capitis, longus colli, and rectus capitis anterior.

[0126] In one embodiment, when treating the neck flexor muscles, the modified BoNT / A can be administered to one or more of the affected neck muscles including: the sternocleidomastoid, the anterior scalene, the medius scalene, the longus capitis, the longus colli, and / or the rectus capitis anterior.

[0127] In one embodiment, when treating neck flexion, a modified BoNT / A can be administered to one or more affected neck muscles selected from the group consisting of: sternocleidomastoid, scalene anterior, scalene middle, levator scapulae, longus colli, submental complex (e.g., digastric, geniohyoid, mylohyoid, mylohyoid buttonhole, and / or stylohyoid), longus capitis, and rectus capitis anterior. In one embodiment, when treating neck flexion, a modified BoNT / A can be administered to one or more affected neck muscles comprising: sternocleidomastoid, scalene anterior, scalene middle, levator scapulae, longus colli, submental complex (e.g., digastric, geniohyoid, mylohyoid, mylohyoid buttonhole, and / or stylohyoid), longus capitis, and / or rectus capitis anterior.

[0128] In one embodiment, when treating neck flexion, the modified BoNT / A is administered to one or more affected neck muscles selected from or including the following: scalene medius, levator scapulae, and / or longus colli. Preferably, when treating neck flexion, the modified BoNT / A may be administered bilaterally.

[0129] In one embodiment, when treating retrocollis, the modified BoNT / A can be administered to one or more affected neck muscles selected from the group consisting of the levator scapulae, the trapezius (e.g., the upper trapezius), the longissimus (e.g., the longissimus capitis and / or the longissimus cervix), the splenius capitis, the splenius cervix, and the semispinalis capitis.

[0130] In one embodiment, when treating neck extension, the modified BoNT / A can be administered to one or more affected neck muscles selected from the group consisting of the 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 posterior paraspinal muscles (e.g., posterior scalene, middle scalene, and / or anterior scalene, preferably posterior scalene).

[0131] In one embodiment, when treating neck extension, the modified BoNT / A can be administered to one or more affected neck muscles selected from the group consisting of the levator scapulae, trapezius (e.g., upper trapezius), longissimus (e.g., longissimus capitis and / or longissimus cervix), splenius capitis, splenius cervi, semispinalis capitis, semispinalis cervi, spinalis capitis, rectus capitis posterior major, rectus capitis posterior minor, and oblique capitis superior.

[0132] In one embodiment, when treating neck extension, a modified BoNT / A can 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 cervi, spinalis capitis, rectus capitis posterior major, rectus capitis posterior minor, and / or oblique capitis superior.

[0133] In one embodiment, when treating neck extension, the modified BoNT / A can be administered to one or more affected neck muscles selected from the group consisting of the levator scapulae, trapezius (e.g., upper trapezius), longissimus (e.g., longissimus capitis and / or longissimus cervicis), splenius capitis, splenius cervicis, semispinalis capitis, semispinalis cervicis, posterior paraspinal muscles (e.g., posterior scalene, middle scalene, and / or anterior scalene, preferably posterior scalene), spinalis capitis, rectus capitis posterior major, rectus capitis posterior minor, and oblique capitis superior. In one embodiment, when treating neck extension, the modified BoNT / A can 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 muscles (e.g., posterior scalene, middle scalene, and / or anterior scalene, preferably posterior scalene), spinalis capitis, rectus capitis posterior major, rectus capitis posterior minor, and / or oblique capitis superior.

[0134] In one embodiment, when treating neck extensor, the modified BoNT / A is administered to at least the semispinalis cervicis muscle.

[0135] Preferably, when treating neck extension, the modified BoNT / A may be administered bilaterally.

[0136] When treating a lateral shift manifestation of cervical dystonia, the muscles selected for administration may be a combination of muscles administered when treating the lateral neck (eg, on a first side) and lateral head (eg, on a second side).

[0137] In one embodiment, when treating lateral displacement, the modified BoNT / A can be administered to one or more affected neck muscles selected from the group consisting of the 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 lateral displacement, the modified BoNT / A can be administered to the affected neck muscles selected from the group consisting of the levator scapulae, semispinalis cervix, middle scalene, and longissimus cervix on a first side of the neck (e.g., the left side), and the modified BoNT / A can be administered to the affected neck muscles selected from the group consisting of the sternocleidomastoid, lower trapezius, splenius capitis, semispinalis capitis, longissimus capitis, and levator scapulae on a second side of the neck (e.g., the right side).

[0138] In one embodiment, when treating lateral displacement, the modified BoNT / A can 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), and / or semispinalis cervix. Preferably, when treating lateral displacement, the modified BoNT / A can be administered to one or more affected neck muscles including: levator scapulae, semispinalis cervix, middle scalene, and / or longissimus cervix (e.g., on a first side of the neck (e.g., left side)), and the modified BoNT / A can be administered to one or more affected muscles including: sternocleidomastoid, lower trapezius, splenius capitis, semispinalis capitis, longissimus capitis, and / or levator scapulae (e.g., on a second side of the neck (e.g., right side)).

[0139] In one embodiment, when treating the side of the head, the modified BoNT / A can be administered to one or more affected neck muscles selected from the group consisting of the lower trapezius, sternocleidomastoid, longissimus capitis, splenius capitis, semispinalis capitis, levator scapulae, and posterior paraspinal muscles (e.g., posterior scalene, middle scalene, and / or anterior scalene, preferably the posterior scalene).

[0140] In one embodiment, when treating the side of the head, the modified BoNT / A can be administered to one or more affected neck muscles including: the lower trapezius, sternocleidomastoid, longissimus capitis, splenius capitis, semispinalis capitis, levator scapulae, and / or posterior paraspinal muscles (e.g., posterior scalene, middle scalene, and / or anterior scalene, preferably the posterior scalene).

[0141] In one embodiment, when treating laterocaput, the modified BoNT / A is administered to one or more affected neck muscles selected from or including the sternocleidomastoid, lower trapezius, splenius capitis, semispinalis capitis, longissimus capitis, and / or levator scapulae.

[0142] Preferably, when treating the side of the head, the modified BoNT / A can be administered ipsilaterally.

[0143] In one embodiment, when treating torticaput, the modified BoNT / A can be administered to one or more affected neck muscles selected from the group consisting of: the lower trapezius, sternocleidomastoid, longissimus capitis, splenius capitis, semispinalis capitis middle, and oblique capitis inferior. For example, the modified BoNT / A can be administered contralaterally to one or more affected neck muscles selected from the group consisting of: the lower trapezius, sternocleidomastoid, and semispinalis capitis middle; and / or the modified BoNT / A can be administered ipsilaterally to one or more neck muscles selected from the group consisting of: oblique capitis inferior, longissimus capitis, and splenius capitis.

[0144] In one embodiment, when treating plagiocephaly, the modified BoNT / A can be administered to one or more affected neck muscles including: the lower trapezius, sternocleidomastoid, longissimus capitis, splenius capitis, semispinalis capitis middle, and / or oblique capitis inferior. For example, the modified BoNT / A can be administered contralaterally to one or more affected neck muscles including: the lower trapezius, sternocleidomastoid, and / or semispinalis capitis middle; and / or the modified BoNT / A can be administered ipsilaterally to one or more neck muscles including: oblique capitis inferior, longissimus capitis, and / or splenius capitis.

[0145] Preferably, when treating plagiocephaly, the modified BoNT / A can be administered ipsilaterally or contralaterally.

[0146] In one embodiment, when treating antecaput (also known as anterocaput), the modified BoNT / A can be administered to one or more affected neck muscles selected from the group consisting of: longus capitis, levator scapulae, and sternocleidomastoid.

[0147] In one embodiment, when treating head flexion, the modified BoNT / A can be administered to one or more affected neck muscles including: the longus capitis, levator scapulae, and / or sternocleidomastoid.

[0148] Preferably, when treating dactyly, the modified BoNT / A may be administered bilaterally.

[0149] In one embodiment, when treating retrocaput, the modified BoNT / A can be administered to one or more affected neck muscles selected from the group consisting of: oblique capitis inferior, semispinalis capitis, trapezius inferior, and splenius capitis. In one embodiment, when treating retrocaput, the modified BoNT / A can be administered to one or more affected neck muscles selected from the group consisting of: oblique capitis inferior, semispinalis capitis, trapezius inferior, and / or splenius capitis.

[0150] Preferably, when treating the head, the modified BoNT / A may be administered bilaterally.

[0151] When treating a sagittal shift presentation of cervical dystonia, the muscles selected for administration may be a combination of muscles administered when treating neck flexion (eg, on a first side) and head extension (eg, on a second side).

[0152] In one embodiment, when treating sagittal shift, the modified BoNT / A can be administered to one or more affected neck muscles selected from the group consisting of the sternocleidomastoid, scalene anterior, scalene medius, levator scapulae, longus colli, submental complex (e.g., digastric, geniohyoid, mylohyoid, mylohyoid buttonhole, and / or stylohyoid), oblique capitis inferior, semispinalis capitis, lower trapezius, and splenius capitis. Preferably, when treating sagittal shift, the modified BoNT / A can be administered to an affected neck muscle selected from the group consisting of the sternocleidomastoid, anterior scalene, middle scalene, levator scapulae, longus colli, and submental complex (e.g., digastric, geniohyoid, mylohyoid, mylohyoid buttonhole, and / or stylohyoid) on a first side of the neck (e.g., the left side), and the modified BoNT / A can be administered to an affected neck muscle selected from the group consisting of the inferior oblique capitis, semispinalis capitis, lower trapezius, and splenius capitis on a second side of the neck (e.g., the right side).

[0153] In one embodiment, when treating sagittal shift, a modified BoNT / A can be administered to one or more affected neck muscles including: sternocleidomastoid, scalene anterior, scalene medius, levator scapulae, longus colli, submental complex (e.g., digastric, geniohyoid, mylohyoid, mylohyoid buttonhole, and / or stylohyoid), oblique capitis inferior, semispinalis capitis, lower trapezius, and / or splenius capitis. Preferably, when treating sagittal shift, the modified BoNT / A can be administered to one or more affected neck muscles including: sternocleidomastoid, scalene anterior, scalene middle, levator scapulae, longus colli, and / or submental complex (e.g., digastric, geniohyoid, mylohyoid, mylohyoid buttonhole, and / or stylohyoid) (e.g., on a first side of the neck (e.g., left side)), and the modified BoNT / A can be administered to one or more affected neck muscles including: oblique capitis inferior, semispinalis capitis, lower trapezius, and / or splenius capitis (e.g., on a second side of the neck (e.g., right side)).

[0154] In one embodiment, when treating sagittal shift, the modified BoNT / A can be administered to one or more affected neck muscles selected from or including the scalene medius, levator scapulae, longus colli, oblique capitis inferior, semispinalis capitis, lower trapezius, and / or splenius capitis. For example, the modified BoNT / A can be administered to one or more affected neck muscles selected from or including the scalene medius, levator scapulae, and / or longus colli (e.g., on a first side of the neck), and one or more affected neck muscles selected from or including the oblique capitis inferior, semispinalis capitis, lower trapezius, and / or splenius capitis (e.g., on a second side of the neck).

[0155] When muscles are listed for administration according to the methods of treatment of the present invention, the present invention may also include administering a modified BoNT / A to additional, unlisted muscles.

[0156] The modified BoNT / A is administered by intramuscular injection into the affected neck muscle. One or more unit doses (e.g., at least two unit doses) of the modified BoNT / A can be administered to the affected neck muscle. However, it is preferred that only a single unit dose is administered to each affected neck muscle. When the neck muscle is the splenius capitis, the longissimus cervicis, the trapezius, the sternocleidomastoid, the semispinalis capitis, or the levator scapulae, two unit doses can be administered. In the case of bilateral administration, two unit doses can be administered on a first side of the neck and two unit doses can be administered to the corresponding muscle on a second side of the neck. Thus, when the neck muscle is the sternocleidomastoid, two unit doses can be administered. For example, two unit doses can be administered to the left sternocleidomastoid and / or two unit doses can be administered to the right sternocleidomastoid. When the neck muscle is the trapezius (e.g., the lower trapezius), two unit doses can be administered. For example, two unit doses can be administered to the left trapezius (e.g., the left upper trapezius) and / or two unit doses can be administered to the right trapezius (e.g., the right upper trapezius).

[0157] Thus, a unit dose of a modified BoNT / A can be administered to one or more affected neck muscles selected from a first group comprising: splenius cervicis, oblique capitis inferior, semispinalis cervicis, scalene muscles (e.g., anterior scalene, middle scalene, and / or posterior scalene), longissimus capitis, longus colli, and / or longus capitis; and / or a single unit dose or multiple unit doses (preferably multiple unit doses) of a modified BoNT / A can be administered to one or more affected neck muscles selected from a second group comprising: splenius capitis, longissimus cervicis, trapezius muscles (e.g., lower trapezius), sternocleidomastoid, semispinalis capitis, and / or levator scapulae.

[0158] A single unit dose of the modified BoNT / A can be administered to one or more affected neck muscles selected from the first group comprising: the splenius cervii, the inferior oblique capitis, the semispinalis cervii, the scalene muscles (e.g., the anterior scalene, the middle scalene, and / or the posterior scalene), the longissimus capitis, the longus colli, and / or the longus capitis; and / or multiple unit doses (preferably two unit doses) of the modified BoNT / A can be administered to one or more affected neck muscles selected from the second group comprising: the splenius capitis, the longissimus cervii, the trapezius muscles (e.g., the lower trapezius), the sternocleidomastoid, the semispinalis capitis, and / or the levator scapulae.

[0159] 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 the affected neck muscle in a unit dose per injection site. However, less than a unit dose may be administered at a single injection site, in which case the unit dose may be divided (equally or unequally) between two or more injection sites at the affected neck muscle. Thus, the modified BoNT / A may be administered to the affected neck muscle at two or more injection sites. In some cases, the modified BoNT / A may be administered at the affected neck muscle in a less than a unit dose per injection site. Advantageously, this may allow the clinician to contour the muscle and / or treat a particularly affected muscle area by administering more of the modified BoNT / A to that area when compared to less affected areas.

[0160] In one embodiment, at least 0.25, 0.5, 1 or 2 unit doses may be administered per injection (e.g., per injection site). For example, 0.25, 0.5, 1 or 2 unit doses may be administered per injection (e.g., per injection site). Most preferably, a unit dose is administered per injection site.

[0161] A dose of greater than 17,000 pg may be administered per injection site. Preferably, a dose of 25,000 pg or 36,000 pg of the modified BoNT / A is administered per injection site. The modified BoNT / A may be administered at a dose of 20,000 pg to 30,000 pg, such as 24,000 pg to 26,000 pg, such as 25,000 pg. The modified BoNT / A may be administered at a dose of 30,000 pg to 40,000 pg, such as 35,000 pg to 37,000, such as 36,000 pg.

[0162] A dose of greater than 707 units of the modified BoNT / A can be administered per injection site. Preferably, a dose of 1,040 units or 1,498 units of the modified BoNT / A can be administered per injection site. The modified BoNT / A can be administered at a dose of 832 units to 1,248 units, such as 998 units to 1,082 units, such as 1,040 units. The modified BoNT / A can be administered at a dose of 1,248 units to 1,664 units, such as 1,456 units to 1,539 units, such as 1,498 units.

[0163] The term "administering at least a single unit dose" means administering at least substantially all of a single unit dose. For example, in a vial in which the modified BoNT / A has been reconstituted, a residual amount of a unit dose may remain (e.g., up to 1%, 0.1%, or 0.01%). However, it is preferred that at least all of a single unit dose is administered (e.g., at one or more injection sites).

[0164] Likewise, when a portion or multiples of a unit dose are administered, this may mean that substantially all of the portion or multiples of the unit dose are administered. For example, a residual amount (e.g., up to 1%, 0.1%, or 0.01%) of a portion or multiples of the unit dose may remain in the vial from which the modified BoNT / A was removed (e.g., in which the modified BoNT / A was reconstituted). However, it is preferred that the entire portion or multiples of the unit dose be administered (e.g., at one or more injection sites).

[0165] The potency of the modified BoNT / A used in accordance with the present invention can be determined by mouse LD according to standard techniques. 50 In the test, 1 unit is defined as the amount corresponding to the calculated median lethal dose (LD50) in mice. 50 ). Preferably, the calculated mouse intraperitoneal median lethal dose is.

[0166] The modified BoNT / A used in the present invention is a BoNT / A light chain and translocation domain, and a BoNT / B receptor binding domain (H C When the modified BoNT / A contains a modified BoNT / A having a glycosylated domain, the amount corresponding to 1 unit of the modified BoNT / A in the assay is preferably 24.04 pg.

[0167] When used in reference to a numerical value, the term "up to" (e.g., up to 400,000 pg) means up to and including the recited value. Thus, as an example, reference to administering "up to 400,000 pg" of a modified BoNT / A encompasses administering 400,000 pg of a modified BoNT / A as well as administering less than 400,000 pg of a modified BoNT / A.

[0168] A unit dose can be expressed in terms of the amount of a modified BoNT / A, units of a modified BoNT / A, or a combination thereof.

[0169] In one embodiment, the modified BoNT / A may be administered to one or more of the following neck muscles at the following dosages:

[0170] Neck muscles Dosage (unit dose) Sternocleidomastoid muscle 1x UD Splenius capitis 1x UD Splenius cervicis 1x UD Trapezius 1x UD Levator scapulae 1x UD Scalene medius 1x UD scalene anterior muscle 1x UD Semispinalis capitis 1x UD Longissimus 1x UD Posterior paraspinal muscles 1x UD Submental complex 1x UD

[0171] In one embodiment, the modified BoNT / A may be administered to one or more of the following neck muscles at the following dosages:

[0172]

[0173] The modified BoNT / A may be administered to one or more of the following neck muscles at the following dosages:

[0174]

[0175]

[0176] The modified BoNT / A can be administered to the following neck muscles at the following dosages:

[0177] Neck muscles Dosage (unit dose) Right levator scapulae or left levator scapulae 1x UD Right or left trapezius 1x UD Right sternocleidomastoid muscle or left sternocleidomastoid muscle 1x UD Right or left splenius capitis 1x UD scalene middle or anterior 1x UD Right or left semispinalis capitis 1x UD Longissimus capitis right or longissimus capitis left 1x UD

[0178] As used herein, the terms "right" and "left" have their normal meanings. For example, an individual's right levator scapulae would be the levator scapulae on the individual's right hand side, and an individual's left levator scapulae would be the levator scapulae on the individual's left hand side.

[0179] The total number of unit doses administered in a given treatment may be up to 10× unit doses or up to 7× unit doses. The total number of unit doses may be divided according to the affected neck muscles treated, for example, in one embodiment, when the number of doses to be delivered during treatment is 1× unit dose, then only one affected neck muscle may be treated, however, if the total number is 2× unit doses, then two affected neck muscles may be treated. The total number of unit doses administered may be up to 9×, 8×, 7× or 6×. The total number of unit doses administered may be at least 2×, 3×, 4×, 5×, 6×, 7× unit doses, preferably at least 2×. The total number of unit doses administered may be from 1× to 10×, or from 5× to 10×, preferably from 7× to 10×.

[0180] The skilled artisan will take into account whether the individual has recently (or subsequently is) undergoing additional treatment with a Clostridial neurotoxin (e.g., an unmodified BoNT), e.g., as part of a cosmetic treatment or treatment for a different indication. Using routine skills in the art, the skilled artisan will adjust the present treatment regimen accordingly.

[0181] When compared with unmodified BoNT / A (e.g. ), the modified BoNT / A of the invention preferably has a longer duration of action (e.g., at least 5%, 10%, 25%, or 50% improvement in one or more symptoms) compared to the control group. The duration of action can be at least 1.25×, 1.5×, 1.75×, 2.0×, or 2.25×. 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 (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 specific embodiments, the duration of action can be greater than 9.0 months.

[0182] When administering to the maximum number of affected neck muscles, preferably the administration occurs in the same treatment session.

[0183] Treatment can be repeated at appropriate time intervals after administration of the modified BoNT / A. Considering that the duration of action is approximately the same as that of unmodified BoNT / A (e.g. ), the time period between subsequent administrations was twice as long as with unmodified BoNT / A (e.g. ) is appropriately extended to the individual treated with a modified BoNT / A according to the present invention. The modified BoNT / A according to the present invention can be re-administered to the individual at least 18, 20, 25, or 30 weeks after the previous administration. For example, the modified BoNT / A according to the present invention can be re-administered to the individual at least 18-45 weeks, preferably 20-35 weeks, after the previous administration.

[0184] The efficacy of treatment (including the severity of individual symptoms) can be assessed using the Toronto Western Spasmodic Torticollis Rating Scale (TWSTRS), as 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-Severity Scale includes the following items: A. Maximum excursion (rotation, tilt, flexion or extension of the neck, lateral displacement, sagittal displacement), B. Duration factor, C. Effect of sensory techniques, D. Shoulder elevation / anterior displacement, E. Range of motion (without the aid of sensory techniques), and F. Time (patients are able to hold their head within 10° of a neutral position for up to 60 seconds without the use of sensory techniques). The sum of A to F is up to 35 points, with the duration factor weighted twice. The TWSTRS-Disability Scale is a six-item scale that includes assessments of performance in daily activities that may be affected by CD: work performance (work or housework), activities of daily living (eating, dressing, hygiene), driving, reading, watching television, and outdoor leisure activities. Each item is scored on a 6-point scale (0 = no difficulty, 5 = highest degree of disability), with a maximum total of 30 points. The TWSTRS-Pain Scale consists of the patient's average, worst, and best pain severity ratings over the past week, as well as a duration component and an assessment of the impact of pain on disability. Scores range from 0 to 20, with 20 assigned to the highest possible pain experienced.

[0185] As used herein, an "individual" may be a mammal, such as a human or other mammal. Preferably, an "individual" refers to a human individual. An "individual" is preferably an adult individual, i.e., an individual of at least 18 years old. The terms "individual" and "patient" are used synonymously herein. Preferably, the individual has been diagnosed with cervical dystonia.

[0186] An individual treated in accordance with the present invention may be an individual who is not suitable for treatment with an unmodified BoNT / A and / or with another clostridial neurotoxin. The individual may be an individual who is resistant to treatment with an unmodified BoNT / A and / or with another clostridial neurotoxin. Resistance may result from an immune response in the individual to the clostridial neurotoxin, including the production of anti-clostridial neurotoxin antibodies.

[0187] As used herein, the terms "treat" or "treatment" include prophylactic treatment (e.g., preventing the onset of a disorder) as well as corrective treatment (treating an individual who already has a disorder). As used herein, preferably "treat" or "treatment" refers to corrective treatment. As used herein, the terms "treat" or "treatment" refer to a disorder and / or its symptoms.

[0188] Suitable modified BoNT / A polypeptides (and nucleotide sequences encoding the same, if any) are described in WO2015 / 004461A1 and WO2017 / 191315, both of which are incorporated herein by reference in their entireties.

[0189] BoNT / A is an example of a clostridial neurotoxin produced by Clostridium bacteria. Other examples of such clostridial neurotoxins include those produced by Clostridium tetani (C. Tetani, TeNT) and Clostridium botulinum (C. botulinum, BoNT) serotypes BG, and X (see WO 2018 / 009903 A2), as well as C. Baratii and C. Butyricum. The neurotoxins are highly efficient and specific, and are able to poison neurons and other cells to which they are delivered. Clostridial toxins are the most potent toxins known. For example, botulinum neurotoxins have a median lethal dose (LD50) of 0.5 to 5 ng / kg for mice, depending on the serotype. 50 ) values. Both tetanus toxin and botulinum toxin act by inhibiting the function of affected neurons, particularly by inhibiting the release of neurotransmitters. Botulinum toxin acts at the neuromuscular junction and inhibits cholinergic transmission in the peripheral nervous system, while tetanus toxin acts on the central nervous system.

[0190] In nature, clostridial neurotoxins (including BoNT / A) are synthesized as single-chain polypeptides that are modified post-translationally by proteolytic cleavage events to form two polypeptide chains linked together by disulfide bonds. Cleavage occurs at a specific cleavage site, often referred to as the activation site (e.g., the activation loop), i.e., located between the cysteine ​​residues that provide the interchain disulfide bond. It is this two-chain form that serves as the active form of the toxin. The two chains are called heavy chains (H chains) and light chains (L chains), where the heavy chain (H chain) has a molecular weight of approximately 100 kDa and the light chain (L chain) has a molecular weight of approximately 50 kDa. The H chain contains an N-terminal translocation component (H N domain) and the C-terminal targeting component (H C domain). The cleavage site is located between the L chain and the translocation domain component. c After the domain binds to its target neuron and internalizes the bound toxin into the cell via endosomes, H NThe domain enables the translocation of the L chain across the endosomal membrane and into the cytoplasm, where the L chain provides the protease function (also known as a non-cytotoxic protease).

[0191] Non-cytotoxic proteases act by proteolytic cleavage of intracellular transporters called SNARE proteins (e.g. SNAP-25, VAMP, or syntaxin) - see Gerald K (2002) "Cell and Molecular Biology" (4th edition) John Wiley & Sons, Inc., preferably SNAP-25. The acronym SNARE is derived from the term Soluble NSF Attachment Receptor. Re ceptor), where NSF stands for N-ethylmaleimide sensitivity factor ( N -ethylmaleimide- S ensitive F SNARE proteins are essential for intracellular vesicle fusion and therefore for the secretion of molecules from cells via vesicle transport. The protease function is a zinc-dependent endopeptidase activity and exhibits high substrate specificity for SNARE proteins. Therefore, once delivered to the target cell of interest, the non-cytotoxic protease is able to inhibit cellular secretion of the target cell. The L-chain protease of Clostridial neurotoxin is a non-cytotoxic protease that cleaves SNARE proteins.

[0192] Given the ubiquitous nature of SNARE proteins, fusiform neurotoxins (such as botulinum toxin) have been successfully used in a variety of therapeutic applications.

[0193] For more details on the genetic basis of toxin production by Clostridium botulinum and C. tetani, see Henderson et al. (1997), Thelostridia: Molecular Biology and Pathogenesis, Academic press.

[0194] As mentioned above, the clostridial neurotoxin is composed of two polypeptide chains, a heavy chain and a light chain, the heavy chain (H chain) having a molecular weight of about 100 kDa and the light chain (L chain) having 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 translocation component (H N domain).

[0195] Clostridial neurotoxin domains are described in more detail below.

[0196] Examples of L chain reference sequences include:

[0197] Botulinum neurotoxin type A: amino acid residues 1-448

[0198] Botulinum neurotoxin type B: amino acid residues 1-440

[0199] The reference sequences identified above should be considered as a guide, as slight variations may occur depending on serosubtype. For example, US2007 / 0166332 (incorporated herein by reference in its entirety) cites a slightly different Clostridium sequence:

[0200] Botulinum neurotoxin type A: amino acid residues M1-K448

[0201] Botulinum neurotoxin type B: amino acid residues M1-K441

[0202] The translocation domain is a fragment of the H chain of a clostridial neurotoxin (equivalent approximately to the amino-terminal half of the H chain), or a domain corresponding to such a fragment in an intact H chain.

[0203] Examples of reference translocation domains include:

[0204] Botulinum neurotoxin type A - amino acid residues (449-871)

[0205] Botulinum neurotoxin type B - amino acid residues (441-858)

[0206] The reference sequences identified above should be considered as a guide, as slight variations may occur depending on serosubtype. For example, US2007 / 0166332 (incorporated herein by reference in its entirety) cites a slightly different Clostridium sequence:

[0207] Botulinum neurotoxin type A - amino acid residues (A449-K871)

[0208] Botulinum neurotoxin type B - amino acid residues (A442-S858)

[0209] In the context of the present invention, various BoNT / AH comprising a translocation domain N The H region of the BoNT / A heavy chain can be used in aspects of the present invention. N The region is approximately 410-430 amino acids long and contains the translocation domain. N The full length of the region is not necessary for the translocation activity of the translocation domain. Thus, aspects of this embodiment may include a BoNT / AH NA region comprising 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. Other aspects of this embodiment may include a BoNT / AH N Region comprising a translocation domain of, for example, up to 350 amino acids, up to 375 amino acids, up to 400 amino acids, or up to 425 amino acids in length.

[0210] Terminology N Including naturally occurring BoNT / AH N Partial and modified BoNT / AH N part, the modified BoNT / AH N The modified BoNT / AH N Some still exhibit the above translocation function.

[0211] Clostridial neurotoxin receptor binding domain (H C ) Examples of reference sequences include:

[0212] BoNT / A-N872-L1296

[0213] BoNT / B-E859-E1291

[0214] The approximately 50 kDa H of Clostridial neurotoxins (e.g., BoNT) C The domain contains two distinct structural features, called H CC and H CN The amino acid residues believed to be involved in receptor binding are mainly located in the H CC Domain. H of natural Clostridial neurotoxin CA domain may contain about 400-440 amino acid residues. The following publications confirm this fact, 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.

[0215] (Reference)H CN Examples of domains include:

[0216] Botulinum neurotoxin type A - amino acid residues (872-1110)

[0217] Botulinum neurotoxin type B - amino acid residues (859-1097)

[0218] The above sequence positions may vary slightly depending on the serotype / subtype, (reference) H CN Other examples of domains include:

[0219] Botulinum neurotoxin type A - amino acid residues (874-1110)

[0220] Botulinum neurotoxin type B - amino acid residues (861-1097)

[0221] (Reference)H CC Examples of domains include:

[0222] Botulinum neurotoxin type A - amino acid residues (Y1111-L1296)

[0223] Botulinum neurotoxin type B - amino acid residues (Y1098-E1291)

[0224] L chain and H NThe domains (optionally including a complete or partial activation loop, e.g., a complete activation loop when the modified BoNT / A is in single-chain form and a cleavage / partial activation loop when in two-chain form) can be collectively referred to as LH N domain. Therefore, LH N The domain may not contain H C Structural domain.

[0225] WO2017 / 191315A1 (which is incorporated herein by reference) teaches modified BoNT / A and methods for preparing and manufacturing the same. N ) and the BoNT / B receptor binding domain (H C The modified BoNT / A having a structural domain) may be one taught in WO2017 / 191315A1.

[0226] As used herein, the term "modified BoNT / A" or "chimeric neurotoxin" refers to a Clostridial neurotoxin comprising (preferably consisting of) a light chain and a translocation domain (H domain) from a first Clostridial neurotoxin serotype. N domain) and a receptor binding domain (H C In particular, the modified BoNT / A for use in the present invention comprises a botulinum neurotoxin A (BoNT / A) light chain and a translocation domain (H N domain), and the BoNT / B receptor binding domain (H C Domain). BoNT / A LH of modified BoNT / A N Domains and BoNT / BH C The domains are covalently linked. 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."

[0227] L chain and H N The domains (optionally including a complete or partial activation loop, e.g., a complete activation loop when the modified BoNT / A is in single-chain form, and a cleavage / partial activation loop when the modified BoNT / A is in two-chain form) may be collectively referred to as LH N domain. Therefore, LH N The domain does not contain H C Structural domain.

[0228] The modified BoNT / A can consist essentially of a botulinum neurotoxin A (BoNT / A) light chain and a translocation domain (H Ndomain) and the BoNT / B receptor binding domain (H C domain).

[0229] As used herein, the term "consisting essentially of" means that the modified BoNT / A also does not include one or more amino acid residues that confer additional functionality to the polypeptide, such as when administered to an individual. In other words, "consisting essentially of" a botulinum neurotoxin A (BoNT / A) light chain and a translocation domain (H N domain) and the BoNT / B receptor binding domain (H C The polypeptide composed of a botulinum neurotoxin A (BoNT / A) light chain and a translocation domain (H N domain) and the BoNT / B receptor binding domain (H C The amino acid residues of the polypeptide may be similar to those of the amino acid residues of the polypeptide (i.e., ...

[0230] The modified BoNT / A may comprise a non-Clostridial neurotoxin sequence in addition to any Clostridial neurotoxin sequence, as long as the non-Clostridial neurotoxin sequence does not destroy the ability of the modified BoNT / A to achieve its therapeutic effect. Preferably, the non-Clostridial neurotoxin sequence is not a sequence with catalytic activity (e.g., enzymatic activity). In one embodiment, the modified BoNT / A of the present invention does not comprise a non-Clostridial catalytically active domain. In one embodiment, the modified BoNT / A does not comprise an additional catalytically active domain. In one embodiment, the non-Clostridial sequence is not a sequence that binds to a cell receptor. In other words, in one embodiment, the non-Clostridial sequence is not a ligand for a cell receptor. The cell receptor can be a protein cell receptor, such as an integral membrane protein. Examples of cell receptors can be found in the IUPHAR Guide to Pharmacology Database, version 2019.4, at https: / / www.guidetopharmacology.org / download.jsp#db_reports. The non-Clostridial neurotoxin sequence can include a tag that facilitates purification, such as a His tag. In one embodiment, a modified BoNT / A of the invention does not comprise a label or a site for adding a label, such as a sortase acceptor or donor site.

[0231] Preferably, the modified BoNT / A may be composed of a botulinum neurotoxin A (BoNT / A) light chain and a translocation domain (H N domain) and the BoNT / B receptor binding domain (H Cdomain).

[0232] The modified BoNT / A comprises a light chain that is capable of exhibiting non-cytotoxic protease activity and is capable of cleaving SNARE proteins in the cytosol of target neurons.

[0233] Cell-based and in vivo tests can be used to determine whether the clostridial neurotoxin comprising L chain and functional cell binding and translocation domain has non-cytotoxic protease activity. Tests such as toe abduction score (DAS) test, dorsal root ganglion (DRG) test, spinal neuron (SCN) test and mouse phrenic nerve hemidiaphragm (PNHD) test are routine tests in the art. Suitable tests for determining non-cytotoxic protease activity can be Aoki KR, Toxicon 39: 1815-1820; 2001 or Donald et al. (2018), Pharmacol Res Perspect, e00446, 1-14 described in the test, which is incorporated herein by reference.

[0234] When administered to an individual, the modified BoNT / A is preferably in its active two-chain form, wherein the light chain and the heavy chain are linked together by a disulfide bond. When a BoNT / A (e.g., a modified BoNT / A) is defined herein by means of a polypeptide sequence (SEQ ID NO), the L chain portion of the sequence (SEQ ID NO) can constitute the first chain of the two-chain clostridial neurotoxin (e.g., a two-chain modified BoNT / A), and the H chain portion can constitute the first chain of the two-chain clostridial neurotoxin (e.g., a two-chain modified BoNT / A). N and H C The domains together may constitute the second chain of a two-chain clostridial neurotoxin (e.g., a two-chain modified BoNT / A), wherein the first chain and the second chain are linked together by a disulfide bond. It will be appreciated by those skilled in the art that a protease may cleave at one or more positions within the activation loop of a clostridial neurotoxin (e.g., a modified BoNT / A), preferably at two positions within the activation loop. When cleavage occurs at more than one position (preferably two positions) within the activation loop, a small fragment of the C-terminal L chain portion of the sequence may not be present in the two-chain clostridial neurotoxin sequence (e.g., a two-chain modified BoNT / A). In view of this, the sequence of a two-chain clostridial neurotoxin (e.g., a two-chain modified BoNT / A) may differ slightly from the sequence of a corresponding single-chain clostridial neurotoxin (e.g., a single-chain modified BoNT / A). The small fragment may be 1-15 amino acids. Specifically, in one embodiment, when a single-chain modified BoNT / A is converted to a two-chain modified BoNT / A using Lys-C, the small fragment of the C-terminal L chain portion of the sequence that is absent may be SEQ ID NO: 9 or 10.

[0235] Most preferably, the modified BoNT / A for use in the present invention may comprise a BoNT / A light chain and a translocation domain (BoNT / A LH N domain) and BoNT / BH C Domain. BoNT / A LH N Domains and BoNT / BH C The modified BoNT / A is also referred to herein as a "BoNT / AB," or a "BoNT / AB chimera."

[0236] LH N The C-terminal amino acid residue of the domain may correspond to the LH that separates BoNT / A N and H C Domain 3 10 The first amino acid residue of the helix, and H C The N-terminal amino acid residue of the domain may correspond to the LH in the BoNT / B N and H C Domain 3 10 The second amino acid residue of the helix.

[0237] An example of an (unmodified) BoNT / A polypeptide sequence is shown in SEQ ID NO:2.

[0238] An example of a BoNT / B polypeptide sequence is shown in SEQ ID NO:8 (UniProt Accession No. B1INP5).

[0239] The article refers to "LH that separates BoNT / A N and H C Domain 3 10 The first amino acid residue of the helix is ​​the N and H C Domain 3 10 The N-terminal residue of the helix.

[0240] The article mentions "LH that separates BoNT / B N and H C Domain 3 10 The second amino acid residue of the helix is ​​the N and H C Domain 3 10 The amino acid residue following the N-terminal residue of the helix.

[0241] "3 10 A helix is ​​a type of secondary structure found in proteins and peptides, along with alpha helices, beta sheets, and inversions. 10The amino acids in the helix are arranged in a right-handed helical structure, in which each complete turn is made by three residues and ten atoms separated by intramolecular hydrogen bonds between them. Each amino acid corresponds to a 120° turn in the helix (i.e., the helix has 3 residues per turn), with a translation along the helical axis of (=0.2 nm) and has 10 atoms in the ring formed by hydrogen bonding. Most importantly, the NH 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 defined as 3 10 Spiral. 3 10 The helix is ​​a standard concept familiar to those skilled in structural biology.

[0242] The 3 10 The helix corresponds to the four residues that form the actual helix and two cap (or transition) residues, one at each end of the four residues. N and H C Domain 3 10 The "helix" consists of those 6 residues.

[0243] By performing structural analysis and sequence alignment, it was identified that the LH N and H C Domain 3 10 Spiral. The 3 10 The helix at its N-terminus (i.e., LH N The C-terminal part of the domain is surrounded by an α-helix, and at its C-terminus (i.e., H c The N-terminal part of the domain) is surrounded by β-strands. 10 The first (N-terminal) residue of a helix (the cap or transition residue) also corresponds to the C-terminal residue of that alpha helix.

[0244] Separation of LH N and H C Domain 3 10 Helices can be determined, for example, from publicly available crystal structures of botulinum neurotoxins, such as 3BTA (http: / / www.rcsb.org / pdb / explore / explore.do?structureld=3BTA) and 1EPW (http: / / www.rcsb.org / pdb / explore / explore.do?structureld=1EPW) for botulinum neurotoxins A1 and B1, respectively.

[0245] Publicly available computer modeling and alignment tools can also be used to identify LH compartments in other neurotoxins. N and H C Domain 310 The positions of the helices are determined by, for example, the homology modeling servers LOOPP (Learning, Observing and Exporting Protein Patterns, http: / / loopp.org), PHYRE (Protein Homology / Mock Recognition Engine, http: / / www.sbg.bio.ic.ac.uk / phyre2 / ) and Rosetta (http: / / www.rosettacommons.org / ), the protein superposition server SuperPose (http: / / wishart.biology.ualberta.ca / superpose / ), the alignment program Clustal Omega (http: / / www.clustal.org / omega / ), and many other tools / services listed in Internet Resources for Molecular and Cell Biologists (http: / / molbiol-tools.ca / ). In particular, “H N / H CN “The region surrounding the junction can be highly conserved in structure, making it an ideal region for superimposing different serotypes.

[0246] For example, the following method can be used to determine the presence of this 3 10 The sequence of the spiral:

[0247] 1. The structural homology modeling tool LOOP (http: / / loopp.org) can be used to obtain predicted structures of other BoNT serotypes based on the BoNT / A1 crystal structure (3BTA.pdb);

[0248] 2. The structure (pdb) file thus obtained can be edited to contain only H CN The N-terminus of the domain and the approximately 80 residues preceding it (which are H N domain), thereby retaining the highly conserved "H N / H CN "area;

[0249] 3. The protein overlay server SuperPose (http: / / wishart.biology.ualberta.ca / superpose / ) can be used to overlay each serotype onto the 3BTA.pdb structure;

[0250] 4. You can check the superimposed pdb files to locate the H C 3 at the beginning of the domain 10 helix, the corresponding residues in other serotypes could then be identified.

[0251] 5. Other BoNT serotype sequences can be aligned with Clustal Omega to check if the corresponding residues are correct.

[0252] LH measured by this method N , H C and 3 10 An example of a helical domain is shown below:

[0253]

[0254]

[0255] Using structural analysis and sequence alignment, it was found that the LH N and H C Domain 3 10 The β-strand following the helix is ​​conserved in all botulinum and tetanus neurotoxins and separates LH from N and H C Domain 3 10 When the first residue of the helix begins, the beta strand begins at residue 8 (eg, residue 879 of BoNT / A1).

[0256] The BoNT / AB chimera can contain H from BoNT / B C Domain covalently linked to LH from BoNT / A N domain, in which LH N The C-terminal amino acid residue of the domain corresponds to the H C The eighth amino acid residue from the N-terminus of the β-chain of the starting end (N-terminus) of the domain, and wherein H C The N-terminal amino acid residue of the domain corresponds to the H C The seventh amino acid residue from the N-terminus of the β-chain at the start (N-terminus) of the domain.

[0257] The BoNT / AB chimera can contain H from BoNT / B C Domain covalently linked to LH from BoNT / A N domain, in which LH N The C-terminal amino acid residue of the domain corresponds to the LH N The C-terminal amino acid residue of the alpha helix at the terminal end (C-terminus) of the domain, and wherein H C The N-terminal amino acid residue of the domain corresponds to the LH N The terminal end (C-terminus) of the domain is adjacent to the amino acid residue C-terminal to the C-terminal amino acid residue of the alpha helix.

[0258] The rationale for the BoNT / AB chimera design process is to ensure that the secondary structure is as intact as possible, thereby minimizing any changes to the tertiary structure and the function of each domain. 10 The four central amino acid residues of the helix ensure the optimal conformation of the chimeric neurotoxin, thereby allowing the chimeric neurotoxin to exert its effect and fully exert its function. In fact, surprisingly, only the 3 10 The first amino acid residue of the helix and the 3 10 The second amino acid residue further along the helix not only allows for the generation of soluble and functional BoNT / AB chimeras, but further results in improved properties over other BoNT / AB chimeras, particularly increased potency, increased safety ratio, and / or longer duration of action (as well as increased safety ratio and / or duration of action compared to an unmodified BoNT / A [e.g., SEQ ID NO: 2, e.g., SEQ ID NO: 2 in a two-chain form]).

[0259] BoNT / A light chain, BoNT / A translocation domain, and / or BoNT / BH C The domain can be a modified BoNT / A light chain, a BoNT / A translocation domain, and / or a BoNT / BH C Domains or derivatives thereof, including but not limited to those described below. Modified BoNT / A light chains, BoNT / A translocation domains, and / or BoNT / BH C The domain or derivative can contain one or more amino acids that have been modified compared to the native (unmodified) form of a BoNT / A light chain, a BoNT / A translocation domain, and / or a BoNT / B Hc domain, or can contain one or more inserted amino acids that are not present in the native (unmodified) form of a BoNT / A light chain, a BoNT / A translocation domain, and / or a BoNT / B Hc domain. For example, a modified BoNT / A light chain, a BoNT / A translocation domain, and / or a BoNT / B Hc domain C The domain can have a modified amino acid sequence in one or more domains relative to a native (unmodified) BoNT / A light chain, BoNT / A translocation domain, and / or BoNT / BHc domain. Such modifications can alter aspects of their function, such as biological activity or persistence. Thus, in one embodiment, a BoNT / A light chain, a BoNT / A translocation domain, and / or a BoNT / BHc domain can have a modified amino acid sequence in one or more domains relative to a native (unmodified) BoNT / A light chain, a BoNT / A translocation domain, and / or a BoNT / BHc domain. Such modifications can alter aspects of their function, such as biological activity or persistence. C The domain is a modified BoNT / A light chain, BoNT / A translocation domain, and / or BoNT / BH Cdomain, or a modified BoNT / A light chain, BoNT / A translocation domain, and / or BoNT / BH C Domain derivatives.

[0260] The modified BoNT / B Hc domain can have one or more modifications that alter binding to target neural cells, for example, when compared to native (unmodified) BoNT / B Hc domains. C When the domains are compared, they provide higher or lower affinity binding. C Such modifications in the domain may include modifying H C The modification of a residue in a ganglioside binding site of a domain or a residue in a protein (e.g., synaptotagmin) binding site that alters binding to a ganglioside receptor and / or protein receptor of a target neural cell. Examples of such modified neurotoxins are described in WO 2006 / 027207 and WO 2006 / 114308, both of which are hereby incorporated by reference in their entirety.

[0261] The modified light chain may have one or more modifications in its amino acid sequence, such as modifications in the substrate binding or catalytic domains, which may alter or modify the SNARE protein specificity of the modified light chain, preferably provided that the modification does not catalytically inactivate the light chain. Examples of such modified neurotoxins are described in WO 2010 / 120766 and US2011 / 0318385, both of which are hereby incorporated by reference in their entirety.

[0262] LH from BoNT / A 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. Preferably, the LH from a BoNT / A N The domain corresponds to amino acid residues 1 to 872 of SEQ ID NO:2.

[0263] H from BoNT / B C The domain may correspond to amino acid residues 860 to 1291 of SEQ ID NO:8, 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:8, or a polypeptide sequence having at least 80%, 90%, or 95% sequence identity thereto. Preferably, the H from a BoNT / B CThe domain corresponds to amino acid residues 860 to 1291 of SEQ ID NO:8.

[0264] Preferably, the BoNT / AB chimera comprises BoNT / A1 LH N Domains and BoNT / B1 H C More preferably, LH N The domain corresponds to amino acid residues 1 to 872 of BoNT / A1 (SEQ ID NO: 2), and H C The domain corresponds to amino acid residues 860 to 1291 of BoNT / B1 (SEQ ID NO: 8).

[0265] Most preferably, the BoNT / BH is C The domain is still in H CC The subdomain comprises at least one amino acid residue substitution, insertion, insertion / deletion (indel), or deletion that has the effect of increasing the binding affinity of the BoNT / B neurotoxin to human Syt II. CC Suitable amino acid residue substitutions, insertions, insertions / deletions or deletions in subdomains have been disclosed in WO 2013 / 180799 and WO 2016 / 154534 (both incorporated herein by reference).

[0266] BoNT / BH CC Suitable amino acid residue substitutions, insertions, insertions / deletions or deletions in the subdomains may include substitution mutations selected from the group consisting of: V1118M; Y1183M; E1191M; E1191I; E1191Q; E1191T; S1199Y; S1199F; S1199L; S1201V; E1191C, E1191V, E1191L, E1191Y, S1199W, S1199E, S1199H, W1178Y, W1178Q, W1178A, W1178S, Y1183C, Y1183P, and combinations thereof.

[0267] BoNT / BH CCSuitable amino acid residue substitutions, insertions, insertions / deletions or deletions in the subdomains may also include a combination of two substitution mutations 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.

[0268] BoNT / BH CC Suitable amino acid residue substitutions, insertions, insertions / deletions or deletions in the subdomains may also include a combination of three substitution mutations, namely E1191M, S1199W and W1178Q.

[0269] Preferably, BoNT / BH CC Amino acid residue substitutions, insertions, insertions / deletions, or deletions in the subdomain include a combination of two substitution mutations, namely E1191M and S1199Y. Such modifications are present, for example, in BoNT / AB chimeras SEQ ID NO:5 and SEQ ID NO:6. E1191M may correspond to position 1204 of SEQ ID NO:6, and S1199Y may correspond to position 1212. Thus, SEQ ID NO:6 may include 1204M and 1212Y.

[0270] The modification can be a modification when compared to a BoNT / B as set forth in SEQ ID NO:8, wherein the amino acid residue numbering is determined by alignment with SEQ ID NO:8. Since the presence of a methionine residue at position 1 of SEQ ID NO:8 (and SEQ ID NOs corresponding to the modified BoNT / A polypeptides described herein) is optional, one skilled in the art will take into account the presence / absence of the methionine residue when determining the amino acid residue numbering. For example, where SEQ ID NO:8 comprises a methionine, the position numbering will be as defined above (e.g., E1191 would be E1191 of SEQ ID NO:8). Alternatively, when a methionine is not present in SEQ ID NO:8, the amino acid residue numbering should be modified to -1 (e.g., E1191 would be E1190 of SEQ ID NO:8). Thus, the initial methionine amino acid residue of the polypeptide sequence of the modified BoNT / A can be optional or absent. Similar considerations apply when a methionine is present / absent at position 1 of other polypeptide sequences described herein, and the skilled artisan will readily determine the correct amino acid residue numbering using routine techniques in the art.

[0271] The modified BoNT / A for use in the present invention can comprise a polypeptide sequence having at least 70% sequence identity to a polypeptide sequence selected from SEQ ID NOs: 3-7. 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: 3-7. Preferably, the modified BoNT / A for use in the present invention can comprise (more preferably consist of) a polypeptide sequence selected from SEQ ID NOs: 3-7.

[0272] Preferably, the modified BoNT / A comprises a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 6. For example, a polypeptide sequence having at least 80%, 90%, 95%, or 99.9% sequence identity to SEQ ID NO: 6. Most preferably, the modified BoNT / A for use in the present invention may comprise (more preferably consist of) SEQ ID NO: 6.

[0273] The term "deletion" as used herein refers to the removal of one or more amino acid residues of a polypeptide without replacing one or more amino acid residues at the site of the deletion. Thus, when one amino acid residue is deleted from a polypeptide sequence having x amino acid residues (for example), the resulting polypeptide has x-1 amino acid residues.

[0274] As used herein, the term "insertion / deletion (indel)" refers to the deletion of one or more amino acid residues of a polypeptide and the insertion of a different number of amino acid residues (more or fewer amino acid residues) than the number of amino acid residues deleted at the deletion site. Thus, for an indel (for example) in which two amino acid residues are deleted from a polypeptide sequence having x amino acid residues, the resulting polypeptide has x-1 amino acid residues or x+≥1 amino acid residues. Insertions and deletions can be performed in any order, sequentially or simultaneously.

[0275] The term "substitution" as used herein refers to the replacement of one or more amino acid residues with the same number of amino acid residues at the same position. Thus, for a substitution of a polypeptide sequence having x amino acid residues (for example), the resulting polypeptide also has x amino acid residues. Preferably, the substitution is at a single amino acid position.

[0276] The term "insertion" as used herein refers to the addition of one or more amino acid residues of a polypeptide at an insertion site without deleting one or more amino acid residues of the polypeptide. Thus, when an amino acid residue is inserted into a polypeptide sequence having x amino acid residues (for example), the resulting polypeptide has x+1 amino acid residues.

[0277] Methods for modifying proteins by substitution, insertion or deletion of amino acid residues are known in the art. For example, amino acid modifications can be introduced by modification of a DNA sequence encoding a BoNT / A (e.g., encoding an unmodified BoNT / A). This can be accomplished using standard molecular cloning techniques, such as by site-directed mutagenesis, by using a polymerase, where short strands of DNA (oligonucleotides) encoding the desired amino acids are used to replace the original coding sequence, or by inserting / deleting a portion of a gene with various enzymes (e.g., ligases and restriction endonucleases). Alternatively, the modified gene sequence can be chemically synthesized. Generally, the modification can be made by modifying a nucleic acid encoding a native clostridial neurotoxin (or a portion thereof) such that the modified BoNT / A (or a portion thereof) encoded by the nucleic acid comprises the modification. Alternatively, a nucleic acid encoding a modified clostridial neurotoxin (or a portion thereof) comprising the modification can be synthesized.

[0278] When the polypeptide sequence of the modified BoNT / A described herein comprises a tag, such as a His tag, for example, for purification, the tag is optional. Preferably, the tag is removed prior to use of the modified BoNT / A according to the present invention.

[0279] As discussed above, the modified BoNT / A described herein have increased tissue retention properties, which also provide increased potency and / or duration of action, and can allow for increased dosage without any additional negative effects. One way to define these favorable properties is in terms of the safety ratio of the modified BoNT / A. In this regard, the undesirable effects of Clostridial toxins (caused by diffusion of the toxin from the site of administration) can be experimentally assessed by measuring the percentage of weight loss in relevant animal models (e.g., mice, where weight loss is measured within 7 days of administration). Conversely, the intended target effects of Clostridial toxins can be experimentally assessed by the Digital Abduction Score (DAS) test (a measure of muscle paralysis). The DAS test can be performed by injecting 20 μl of the neurotoxin formulated in phosphate gelatin buffer into the gastrocnemius / soleus complex of mice, followed by assessment of the digital abduction score using the Aoki method (Aoki KR, Toxicon 39: 1815-1820; 2001). In the DAS test, mice are suspended briefly by their tails to elicit a characteristic startle response in which the mice extend their hind limbs and abduct their hind toes. After neurotoxin injection, varying degrees of digital abduction are scored on a five-point scale (0 = normal, 4 = maximal reduction in digital abduction and leg extension).

[0280] The safety ratio of the modified BoNT / A of the present invention (or unmodified BoNT / A for comparison) can then be expressed as the ratio between the amount of toxin required to reduce body weight by 10% (measured at peak effect within the first 7 days after dosing in mice) and the amount of neurotoxin required to achieve a DAS score of 2. Thus, a high safety ratio score is desirable, indicating that the neurotoxin is able to effectively paralyze the target muscle with few undesirable off-target effects. The safety ratio of the modified BoNT / A of the present invention is higher than the safety ratio of the equivalent unmodified (natural) BoNT / A.

[0281] A high safety ratio is particularly advantageous in treatment because it represents an increase in the therapeutic index. In other words, this means that a reduced dose can be used compared to alternative clostridial neurotoxin therapeutics and / or an increased dose can be used without any additional (e.g., deleterious) effects. Detrimental effects include systemic toxicity and undesirable spread to adjacent muscles. The possibility of using higher doses of neurotoxins without additional effects is particularly advantageous because higher doses generally result in a longer duration of action of the neurotoxin.

[0282] The potency of a modified BoNT / A can be expressed as the minimum dose of the neurotoxin that results in a given DAS score when administered to the gastrocnemius / soleus complex of mice, e.g., a DAS score of 2 (ED 50The potency of the modified BoNT / A can also be expressed as the EC in a cellular assay measuring SNARE cleavage by neurotoxins. 50 Dose, e.g. EC in a cell-based assay measuring SNAP25 cleavage by modified BoNT / A 50 dose.

[0283] The duration of action of a modified BoNT / A can be expressed as the time required to restore a DAS score of 0 following administration of a given dose of the neurotoxin to the gastrocnemius / soleus complex of mice, wherein the given dose is, for example, the minimum dose of the neurotoxin that results in a DAS score of 4.

[0284] Thus, in one embodiment, a modified BoNT / A of the invention has a safety ratio greater than 7 (e.g., at least 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50), wherein the safety ratio is calculated as: -10% body weight change required toxin dose (pg / mouse) divided by DAS ED 50 (pg / mouse) [ED 50 =dose required to produce a DAS score of 2]. For example, the modified BoNT / A can have a safety ratio of at least 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50.

[0285] In one embodiment, a modified BoNT / A of the invention has a safety ratio of at least 10. In one embodiment, a modified BoNT / A of the invention has a safety ratio of at least 15.

[0286] Preferably, the modified BoNT / A has a safety ratio of at least 10 (e.g., a safety ratio of 10), more preferably at least 12 or 13 (e.g., 14-15). The chimeric Clostridial neurotoxin can have a safety ratio of greater than 7 up to 50, e.g., 8-45, 10-20, or 12-15.

[0287] The modified BoNT / A for use in the present invention may comprise a polypeptide sequence having at least 70% sequence identity with a polypeptide sequence selected from SEQ ID NOs: 3-7. For example, a polypeptide sequence having at least 80%, 90%, 95%, or 99.9% sequence identity with a polypeptide sequence selected from SEQ ID NOs: 3-7. Preferably, the modified BoNT / A for use in the present invention may comprise (more preferably consist of) a polypeptide sequence selected from SEQ ID NOs: 3-7. Among the modified BoNT / A, SEQ ID NO: 6 is preferred.

[0288] Thus, preferably, the modified BoNT / A comprises a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 6. More preferably, the polypeptide sequence has at least 80%, 90%, 95%, or 99.9% sequence identity to SEQ ID NO: 6. Most preferably, the modified BoNT / A for use in the present invention may comprise (more preferably consist of) SEQ ID NO: 6.

[0289] The double-chain modified BoNT / A of the present invention can include an L chain portion of a polypeptide sequence having at least 70%, 80%, 90%, 95%, 99.9% or 100% sequence identity to any one of SEQ ID NOs: 3-7, which constitutes the first chain of the double-chain modified BoNT / A, and can include an H chain portion of a polypeptide sequence having at least 70%, 80%, 90%, 95%, 99.9% or 100% sequence identity to any one of SEQ ID NOs: 3-7. N and H C The domains together constitute the second chain of the two-chain modified BoNT / A, wherein the first chain and the second chain are linked together by a disulfide bond.

[0290] Wherein cleavage occurs at more than one position (preferably two positions) within the activation loop of a modified BoNT / A comprising a polypeptide sequence having at least 70%, 80%, 90%, 95%, 99.9%, or 100% sequence identity to any one of SEQ ID NOs: 3-7, then a small fragment of the C-terminal L chain portion of a sequence having at least 70%, 80%, 90%, 95%, 99.9%, or 100% sequence identity to any one of SEQ ID NOs: 3-7 may not be present in the two-chain modified BoNT / A. In view of this, the sequence of a two-chain modified BoNT / A (e.g., comprising a polypeptide sequence having at least 70%, 80%, 90%, 95%, 99.9%, or 100% sequence identity to any one of SEQ ID NOs: 3-7) may differ slightly from the sequence of a corresponding single-chain modified BoNT / A comprising a polypeptide sequence having at least 70%, 80%, 90%, 95%, 99.9%, or 100% sequence identity to any one of SEQ ID NOs: 3-7. The small fragment can be 1-15 amino acids. Specifically, in one embodiment, when Lys-C is used to convert a single-chain modified BoNT / A into a two-chain clostridial neurotoxin, the small fragment of the C-terminal L chain portion of the sequence that is absent can be SEQ ID NO: 9 or 10.

[0291] Preferably, the double-chain modified BoNT / A of the present invention may include an L chain portion of a polypeptide sequence having at least 70%, 80%, 90%, 95%, 99.9% or 100% sequence identity to SEQ ID NO: 6, which constitutes the first chain of the double-chain modified BoNT / A, and may include an H chain portion of a polypeptide sequence having at least 70%, 80%, 90%, 95%, 99.9% or 100% sequence identity to SEQ ID NO: 6. N and H C domains, which together constitute the second chain of the two-chain modified BoNT / A, wherein the first chain and the second chain are connected by a disulfide bond.

[0292] Wherein cleavage occurs at more than one position (preferably two positions) within the activation loop of a modified BoNT / A comprising a polypeptide sequence having at least 70%, 80%, 90%, 95%, 99.9%, or 100% sequence identity to SEQ ID NO:6, then a small fragment of the C-terminal L chain portion of a sequence having at least 70%, 80%, 90%, 95%, 99.9%, or 100% sequence identity to SEQ ID NO:6 may not be present in the two-chain modified BoNT / A. In view of this, the sequence of a two-chain modified BoNT / A (e.g., comprising a polypeptide sequence having at least 70%, 80%, 90%, 95%, 99.9%, or 100% sequence identity to SEQ ID NO: 6) may be slightly different from the sequence of the corresponding single-chain modified BoNT / A, which comprises a polypeptide sequence having at least 70%, 80%, 90%, 95%, 99.9%, or 100% sequence identity to SEQ ID NO: 6. The small fragment can be 1-15 amino acids. Specifically, in one embodiment, when Lys-C is used to convert a single-chain modified BoNT / A to a two-chain modified BoNT / A, the small fragment of the C-terminal L chain portion of the absent sequence can be SEQ ID NO: 9 or 10.

[0293] In particularly preferred embodiments, the two-chain modified BoNT / A comprises (or consists of) a light chain comprising a polypeptide sequence having at least 70%, 80%, 90%, 95%, or 99.9% sequence identity to SEQ ID NO: 11 or 12 (preferably SEQ ID NO: 11), and a heavy chain comprising a polypeptide sequence having at least 70%, 80%, 90%, 95%, or 99.9% sequence identity to SEQ ID NO: 13, wherein the light chain and the heavy chain are linked together by a disulfide bond. More preferably, the two-chain modified BoNT / A comprises (or consists of) a light chain comprising SEQ ID NO: 11 or 12 (preferably SEQ ID NO: 11), and a heavy chain comprising SEQ ID NO: 13, wherein the light chain and the heavy chain are linked together by a disulfide bond. Even more preferably, the two-chain modified BoNT / A comprises (or consists of) a light chain having SEQ ID NO: 11 and a heavy chain having SEQ ID NO: 13, wherein the light chain and the heavy chain are linked together by a disulfide bond. The disulfide bond is preferably formed by and / or between cysteine ​​residue 429 of SEQ ID NO: 11 or 12 and cysteine ​​residue 6 of SEQ ID NO: 13.

[0294] In preferred embodiments, the modified BoNT / A of the present invention does not comprise therapeutic or diagnostic agents (e.g., nucleic acid, protein, peptide, or small molecule therapeutic or diagnostic agents) other than the light and heavy chains. For example, in one embodiment, the modified BoNT / A may not comprise a covalently or non-covalently bound therapeutic or diagnostic agent. Thus, the modified BoNT / A of the present invention preferably does not serve as a delivery vehicle for other therapeutic or diagnostic agents.

[0295] In embodiments, the modified BoNT / A described herein has a tag (eg, a His tag) and / or a linker for purification, which tag and / or linker is optional.

[0296] The modified BoNT / A is preferably in a non-complexed form (i.e., does not contain complexing proteins naturally present in BoNT / A). Examples of such complexing proteins include neurotoxin associated protein (NAP) and non-toxic non-hemagglutinin component (NTNH). However, it is preferred that the modified BoNT / A is a recombinant modified BoNT / A.

[0297] The modified BoNT / A of the present invention can be produced using recombinant nucleic acid technology. Thus, in one embodiment, the modified BoNT / A (as described herein) is a recombinant modified BoNT / A.

[0298] In one embodiment, a nucleic acid (e.g., DNA) comprising a nucleic acid sequence encoding a modified BoNT / A is provided. In one embodiment, the nucleic acid sequence is prepared as part of a DNA vector comprising a promoter and a terminator. The nucleic acid sequence can be selected from any nucleic acid sequence described herein.

[0299] In a preferred embodiment, the vector has a promoter selected from:

[0300]

[0301] In another preferred embodiment, the vector has a promoter selected from:

[0302]

[0303] Any suitable method known in the art can be used to prepare nucleic acid molecules. Therefore, chemical synthesis techniques can be used to prepare nucleic acid molecules. Alternatively, molecular biology techniques can be used to prepare nucleic acid molecules of the present invention.

[0304] The DNA constructs of the present invention are preferably designed in silico and then synthesized by conventional DNA synthesis techniques.

[0305] The above nucleic acid sequence information is optionally modified for codon preference depending on the final host cell (eg, E. coli) expression system to be used.

[0306] The terms "nucleotide sequence" and "nucleic acid" are used synonymously herein. Preferably, the nucleotide sequence is a DNA sequence.

[0307] The modified BoNT / A of the present invention can exist as a single chain or as a double chain. However, it is preferred that the modified BoNT / A exists as a double chain, wherein the L chain is linked to the H chain (or its component, such as H) by a disulfide bond. N domain).

[0308] The production of a single-chain modified BoNT / A having a light chain and a heavy chain can be achieved using a method comprising expressing a nucleic acid encoding the modified BoNT / A in an expression host, lysing 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 can be proteolytically processed using a method comprising contacting the single-chain modified BoNT / A with a protease (e.g., Lys-C) that hydrolyzes a peptide bond in the activation loop of the modified BoNT / A, thereby converting the single-chain modified BoNT / A to a corresponding double-chain modified BoNT / A (e.g., wherein the light chain and the heavy chain are linked together by a disulfide bond). Double-chain modified BoNT / A is preferably obtainable by such a method.

[0309] Thus, the modified BoNT / A used in the present invention is preferably a double-chain modified BoNT / A produced from a single-chain BoNT / A, wherein the single-chain BoNT / A comprises or consists of a polypeptide sequence described herein. For example, preferably, the modified BoNT / A used in the present invention is a double-chain modified BoNT / A produced 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: 6. Most preferably, the modified BoNT / A used in the present invention is a double-chain modified BoNT / A produced from a polypeptide comprising (even more preferably consisting of) SEQ ID NO: 6. Thus, in some embodiments, the modified BoNT / A is a two-chain modified BoNT / A, wherein the light chain (L chain) is linked to the heavy chain (H chain) by a disulfide bond, and the modified BoNT / A can be obtained by a method comprising contacting a single-chain modified BoNT / A comprising SEQ ID NO: 6 with a protease that hydrolyzes a peptide bond in an activation loop thereof, thereby converting the single-chain modified BoNT / A to a corresponding two-chain modified BoNT / A. In some embodiments, the modified BoNT / A is a two-chain modified BoNT / A, wherein the L chain is linked to the H chain by a disulfide bond, and the modified BoNT / A can be obtained by a method comprising contacting a single-chain modified BoNT / A consisting of SEQ ID NO: 6 with a protease that hydrolyzes a peptide bond in an activation loop thereof, thereby converting the single-chain modified BoNT / A to a corresponding two-chain modified BoNT / A.

[0310] The protease used to cleave the activation loop is preferably Lys-C. Suitable proteases and methods for cleaving the activation loop to produce a two-chain clostridial neurotoxin are taught in WO 2014 / 080206, WO 2014 / 079495, and EP2677029A2, which are incorporated herein by reference. Lys-C can cleave the activation loop C-terminal to one or more lysine residues present therein. When Lys-C cleaves the activation loop multiple times, the skilled artisan will understand that the small peptide of the activation loop of the two-chain modified BoNT / A may not be present when compared to the SEQ ID NO shown herein.

[0311] The term "obtainable" as used herein also includes the term "obtained." In one embodiment, the term "obtainable" refers to obtained.

[0312] As used herein, the term "one or more" may mean at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20. In one embodiment, where "one or more" precedes a list, "one or more" may mean all members of the list. Similarly, as used herein, the term "at least one" may mean at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20. In one embodiment, where "at least one" precedes a list, "at least one" may mean all members of the list.

[0313] As used herein, the term "disorder" also encompasses "disease." In one embodiment, the disorder is a disease.

[0314] The modified BoNT / A of the present invention can be formulated in any suitable manner for administration to an individual, such as as part of a pharmaceutical composition. Such pharmaceutical compositions can include a modified BoNT / A of the present invention and a pharmaceutically acceptable carrier, excipient, adjuvant, propellant, and / or salt.

[0315] The modified BoNT / A of the present invention can be formulated for oral, parenteral, continuous infusion, inhalation or topical application. Compositions suitable for injection can be in the form of solutions, suspensions or emulsions, or dry powders that are dissolved or suspended in a suitable solvent before use.

[0316] For modified BoNT / A to be delivered topically, the modified BoNT / A can be formulated as a cream (eg, for topical application) or for subcutaneous injection.

[0317] Topical delivery methods may include aerosols or other sprays (eg, nebulizers). In this regard, aerosol formulations of the modified BoNT / A can be delivered to the lungs and / or other nasal cavities and / or bronchial or airway passages.

[0318] Liquid dosage forms are typically prepared using the modified BoNT / A and a pyrogen-free, sterile carrier. Depending on the carrier and concentration used, the modified BoNT / A can be dissolved or suspended in the carrier. In preparing a solution, the modified BoNT / A can be dissolved in the carrier, the solution can be made isotonic by the addition of sodium chloride, if necessary, and sterilized by filtration through a sterile filter using aseptic technique, and then filled into suitable sterile vials or ampoules and sealed. Alternatively, if the stability of the solution is sufficient, the solution in the sealed container can be sterilized by autoclaving. Advantageously, additives can be dissolved in the carrier, such as buffers, solubilizers, stabilizers, preservatives or bactericides, suspending agents or emulsifiers, and / or local anesthetics.

[0319] Dry powders can be prepared by filling pre-sterilized ingredients into sterile containers using aseptic techniques in a sterile area to be dissolved or suspended in a suitable vehicle prior to use. Alternatively, the ingredients can be dissolved into suitable containers using aseptic techniques in a sterile area. The product is then freeze-dried and the container sealed aseptically.

[0320] Parenteral 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, rather than dissolved, and sterilization cannot be achieved by filtration. The components may be isolated in an aseptic state, or alternatively, they may be sterilized after isolation, for example by gamma irradiation.

[0321] Advantageously, a suspending agent, such as polyvinylpyrrolidone, is included in one or more of the compositions to facilitate uniform distribution of the ingredients.

[0322] Administration according to the present invention may utilize a variety of delivery techniques including microparticle encapsulation or high pressure aerosol bombardment.

[0323] In one aspect, the invention provides a unit dosage form of a modified BoNT / A (e.g., for treating cervical dystonia) comprising:

[0324] a. Greater than 707 units of modified BoNT / A, where 1 unit corresponds to the calculated median lethal dose (LD50) in mice 50 ) of a modified BoNT / A; or

[0325] b. Greater than 17,000 pg modified BoNT / A; and

[0326] c. optionally a pharmaceutically acceptable carrier, excipient, adjuvant and / or salt,

[0327] The modified BoNT / A comprises a BoNT / A light chain and a translocation domain, and a BoNT / B receptor binding domain (H C domain).

[0328] Preferably, the modified BoNT / A of the unit dosage form comprises a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 6. For example, a polypeptide sequence having at least 80%, 90%, 95%, or 99.9% sequence identity to SEQ ID NO: 6. Most preferably, the modified BoNT / A may comprise (more preferably consist of) SEQ ID NO: 6.

[0329] The unit dosage form can comprise greater than 707 units of a modified BoNT / A. The upper limit of the range can be 1664, 1650, 1600, 1550, 1500, 1450, 1400, 1350, 1300, 1250, 1150, 1100, 1050, 1000, 950, 900, 850, 800, or 750 units of a modified BoNT / A, with a preferred upper limit being 1500 units. The lower end of the range can be 728, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, 1000, 1025, 1050, 1075, 1100, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, or 1650 units of modified BoNT / A, with a preferred lower end being 728 units or 1,040 units. Preferably,

[0330] The unit dosage form comprises greater than 707 units to a maximum of 1664 units of a modified BoNT / A, e.g., greater than 707 units to a maximum of 1498 units, 832 units to 1622 units. Most preferably, the unit dosage form comprises 915 to 1581 units of a modified BoNT / A, e.g., 998 to 1498 units.

[0331] The unit dosage form may comprise 10,399 units to 16,639 units of a modified BoNT / A. The unit dosage form may comprise 1,040 units to 1,664 units of a modified BoNT / A. In preferred embodiments, the unit dosage form of a modified BoNT / A comprises 998, 1,248, 1,040, or 1,498 units, e.g., 1,040 or 1,498 units of a modified BoNT / A. In more preferred embodiments, the unit dosage form comprises 1,248 or 1,498 units (e.g., 1,248 units) of a modified BoNT / A.

[0332] The unit dosage form can contain 832 units to 1,248 units, such as 998 units to 1,082 units of the modified BoNT / A. Most preferably, the unit dosage form can contain 1,040 units of the modified BoNT / A.

[0333] The unit dosage form can include 1,248 units to 1,664 units, such as 1,456 units to 1,539 units of the modified BoNT / A. Most preferably, the unit dosage form can include 1,498 units of the modified BoNT / A.

[0334] The unit dosage form can comprise greater than 17,000 pg of a modified BoNT / A. The upper limit of the range can be 40,000, 39,000, 38,000, 37,000, 36,000, 35,000, 30,000, 25,000, 24,000, 22,000, 20,000, or 18,000 pg of a modified BoNT / A, preferably the upper limit is 38,000 pg. The lower end of the range can be 17,500, 18,000, 20,000, 22,000, 24,000, 25,000, 26,000, 27,000, 28,000, 29,000, 30,000, 35,000, 36,000, 37,000, 38,000, or 39,000 pg of a modified BoNT / A, preferably, the lower end is 17,500 pg or 25,000 pg. Preferably, the unit dosage form comprises greater than 17,000 pg to up to 40,000 pg, e.g., greater than 17,000 pg to up to 36,000 pg, 20,000 pg to up to 39,000 pg of a modified BoNT / A. Most preferably, the unit dosage form comprises 22,000 to 38,000 pg, e.g., 24,000 to 36,000 pg or 25,000 to 36,000 pg of the modified BoNT / A. The unit dosage form may comprise 25,000 pg to 40,000 pg of the modified BoNT / A. In preferred embodiments, the unit dosage form comprises 24,000, 25,000, 30,000, or 36,000 pg, e.g., 25,000 or 36,000 pg of the modified BoNT / A. In more preferred embodiments, the unit dosage form comprises 30,000 or 36,000 ng (e.g., 36,000 pg) of the modified BoNT / A.

[0335] The unit dosage form can contain 20,000 pg to 30,000 pg, e.g., 24,000 pg to 26,000 pg of the modified BoNT / A. Most preferably, the unit dosage form can contain 25,000 pg of the modified BoNT / A.

[0336] The unit dosage form can comprise 30,000 pg to 40,000 pg, e.g., 35,000 pg to 37,000 pg of the modified BoNT / A. Most preferably, the unit dosage form can comprise 36,000 pg of the modified BoNT / A.

[0337] The unit dosage form is preferably provided as a dry powder.

[0338] In another aspect, the present invention provides a kit comprising:

[0339] a. a unit dosage form according to the present invention; and

[0340] b. Instructions for use in the treatment of cervical dystonia; and

[0341] c. Optionally, a diluent.

[0342] Embodiments relating to the various therapeutic uses of the present invention may be applied to the methods, unit dosage forms and kits of the present invention, and vice versa.

[0343] Sequence homology

[0344] Any of a variety of sequence alignment methods can be used to determine percent identity, including but not limited to global methods, local methods, and hybrid methods, such as segment methods. Protocols for determining percent identity are routine procedures within the scope of those skilled in the art. Global methods align sequences from the beginning to the end of the molecule and determine the best alignment by accumulating the scores for each residue pair and by applying gap penalties. Non-limiting methods include, for example, CLUSTAL W, 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, 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 common to all input sequences.Non-limiting methods include, for example, Match-box, see, for example, 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); Gibbs sampling, see, for example, CE Lawrence et al., Detecting Subtle Sequence Signals: A Gibbs Sampling Strategy for Multiple Alignment, 262(5131) Science 208-214 (1993); Align-M, see, for example, Ivo Van WaIIe et al., Align-MA New Algorithm for Multiple Alignment of Highly Divergent Sequences, 20(9) Bioinformatics: 1428-1435 (2004).

[0345] Therefore, the percent sequence identity is determined by conventional methods. See, e.g., 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, two amino acid sequences are aligned using a gap opening penalty of 10, a gap extension penalty of 1, and the "blosum 62" scoring matrix of Henikoff and Henikoff (supra) to optimize the alignment score (amino acids are represented by standard single-letter codes); preferably, this method is used to align sequences with the subject sequences of this article (e.g., SEQ ID NO: 2) to define the amino acid position numbers as described herein.

[0346] The "percentage of 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, % identity can 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 % sequence identity can also take into account the number of spaces that need to be introduced to optimize the alignment of two or more sequences, as well as the length of each space. Specific mathematical algorithms familiar to those skilled in the art (e.g., BLAST) can be used to compare sequences and determine the percent identity between two or more sequences.

[0347] Determining alignment scores for sequence identity

[0348]

[0349] The percent identity is then calculated as:

[0350]

[0351] Substantially homologous polypeptides are characterized by having one or more amino acid substitutions, deletions or additions. These changes are preferably insignificant, i.e., conservative amino acid substitutions (see below) and other substitutions that do not significantly affect polypeptide folding or activity; small deletions, typically 1 to about 30 amino acids; and small amino or carboxyl terminal extensions, such as an amino terminal methionine residue, a small linker peptide of up to about 20-25 residues or an affinity tag.

[0352] Conservative amino acid substitution

[0353] Alkaline: Arginine

[0354] Lysine

[0355] Histidine

[0356] Acid: Glutamic acid

[0357] Aspartic acid

[0358] Polarity: Glutamine

[0359] Asparagine

[0360] Hydrophobicity: Leucine

[0361] Isoleucine

[0362] Valine

[0363] Aromatic: Phenylalanine

[0364] Tryptophan

[0365] Tyrosine

[0366] Small: Glycine

[0367] Alanine

[0368] Serine

[0369] Threonine

[0370] Methionine

[0371] In addition to the 20 standard amino acids, non-standard amino acids (e.g., 4-hydroxyproline, 6-N-methyllysine, 2-aminoisobutyric acid, isovaline, and α-methylserine) can replace the amino acid residues of the polypeptides of the present invention. A limited number of non-conservative amino acids, amino acids not encoded by the genetic code, and non-natural amino acids can replace polypeptide amino acid residues. The polypeptides of the present invention can also contain non-naturally occurring amino acid residues.

[0372] Non-naturally occurring amino acids include, but are not limited to, trans-3-methylproline, 2,4-methanol-proline, cis-4-hydroxyproline, trans-4-hydroxyproline, N-methylglycine, allo-threonine, methylthreonine, hydroxyethylcysteine, hydroxyethylhomocysteine, nitroglutamine, homoglutamine, pipecolic acid, tert-leucine, norvaline, 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine and 4-fluorophenylalanine. Several methods for incorporating non-naturally occurring amino acid residues into proteins are known in the art. 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 aminoacylated tRNAs are known in the art. Transcription and translation of plasmids containing nonsense mutations are performed in a cell-free system comprising 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 a second approach, translation is performed in Xenopus oocytes by microinjection of mutant mRNA and chemically aminoacylated suppressor tRNA (Turcatti et al., J. Biol. Chem. 271:19991-8, 1996). In the third method, E. coli cells are cultured in the absence of the natural amino acid to be replaced (e.g., phenylalanine) and in the presence of the desired non-natural amino acid (e.g., 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine or 4-fluorophenylalanine). Non-natural amino acids are incorporated into polypeptides in place of their natural counterparts. See, Koide et al., Biochem. 33: 7470-6, 1994. Naturally occurring amino acid residues can be converted to non-naturally occurring substances by in vitro chemical modification. Chemical modification can be used in combination with site-directed mutagenesis to further expand the scope of substitution (Wynn and Richards, Protein Sci. 2: 395-403, 1993).

[0373] A limited number of non-conservative amino acids, amino acids not encoded by the genetic code, non-naturally occurring amino acids and unnatural amino acids can be substituted for amino acid residues in the polypeptides of the invention.

[0374] 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). The sites of biological interaction can also be determined by physical analysis of the structure, such as by techniques such as nuclear magnetic resonance, crystallography, electron diffraction or photoaffinity labeling, combined 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 homology analysis with related components of the polypeptides of the invention (e.g., translocation or protease components).

[0375] A variety of amino acid substitutions can be made and tested using known methods of mutagenesis and screening, such as those disclosed in Reidhaar-Olson and Sauer (Science 241:53-7, 1988) or Bowie and Sauer (Proc. Natl. Acad. Sci. USA 86:2152-6, 1989). In short, these authors disclose a method of simultaneously randomizing two or more positions in a polypeptide, selecting functional polypeptides, and then sequencing the mutagenized polypeptides to determine the spectrum of substitutions allowed 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., U.S. Pat. No. 5,223,409; Huse, WIPO Publication No. WO 92 / 06204) and region-directed mutagenesis (Derbyshire et al., Gene 46:145, 1986; Ner et al., DNA 7:127, 1988).

[0376] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present disclosure belongs. Singleton et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 20th Edition, John Wiley and Sons, New York (1994), and Hale & Marham, THE HARPER COLLINS DICTIONARY OF BIOLOGY, Harper Perennial, NY (1991), provide the skilled artisan with a general dictionary of many of the terms used in the present disclosure.

[0377] The present 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 the embodiments of the present disclosure. Numerical ranges include the numbers defining the ranges. Unless otherwise indicated, any nucleic acid sequence is written from left to right in a 5' to 3' direction; amino acid sequences are written from left to right in an amino to carboxyl direction, respectively.

[0378] The headings provided herein are not limitations of the various aspects or embodiments of the disclosure.

[0379] In this article, amino acid names, three-letter abbreviations or single-letter abbreviations are used to refer to amino acids. As used herein, the term "protein" includes protein, polypeptide and peptide. 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 of amino acid residues can be used. The 3-letter code of amino acids is defined according to the Joint Committee on Biochemical Nomenclature (JCBN) of IUPAC IUB. It should also be understood that due to the degeneracy of the genetic code, a polypeptide can be encoded by more than one nucleotide sequence.

[0380] Other definitions of terms may appear throughout the specification. Before describing the exemplary embodiments in more detail, it should be understood that the present disclosure is not limited to the specific embodiments described and may vary accordingly. It should also be understood that the terms used herein are only for the purpose of describing specific embodiments and are not intended to be limiting, as the scope of the present disclosure is limited only by the appended claims.

[0381] In the case of providing a range of values, it is understood that, unless the context clearly indicates otherwise, between the upper and lower limits of the range, each intermediate value, one-tenth of the lower limit unit, is specifically included in the present disclosure. Each smaller range between any specified value or intermediate value in a specified range and any other specified value or intermediate value in the specified range is included in the present disclosure. The upper and lower limits of these smaller ranges may be independently included in the range or excluded in the range, and each range is also included in the present disclosure, including one of the upper and lower limits in the smaller range, excluding both, or including both, subject to any explicitly excluded limit in the range. In the case where the range includes one or two limits, the range excluding any one or two included in these limits is also included in the present disclosure.

[0382] It must 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 and reference to "a modified botulinum neurotoxin A" includes reference to one or more modified botulinum neurotoxin A and equivalents thereof known to those skilled in the art, and so forth.

[0383] The publications discussed herein are provided only 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 appended claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0384] Embodiments of the invention will now be described, by way of example only, with reference to the following Figures and Examples.

[0385] Figure 1 FDA-approved drug for cervical dystonia dose.

[0386] Figure 2 Shown is an SDS-PAGE of purified recombinant BoNT / AB chimeras 1, 2, and 3A (SEQ ID NOs: 3, 4, and 5, respectively). Lanes are labeled "Marker" (molecular weight marker), "-DTT" (oxidized BoNT / AB chimera sample), and "+DTT" (reduced BoNT / AB chimera sample).

[0387] Figure 3SNAP-25 cleavage in rat spinal cord neurons by recombinant BoNT / AB chimeras 1, 2, and 3A (SEQ ID NOs: 3, 4, and 5, respectively) is shown. Cultured rat primary spinal cord neurons (SCN) were exposed to different concentrations of recombinant BoNT / AB chimeras 1, 2, or 3A for 24 hours at 37°C in a humidified atmosphere containing 10% CO2. The cells were then lysed with 1× 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 (Sigma #S9684) that detects both full-length and cleaved forms of SNAP-25. Anti-rabbit HRP (Sigma #A6154) was used as a secondary antibody.

[0388] Figure 4 The mouse toe abduction score test is shown. Under brief general anesthesia, the gastrocnemius-soleus complex muscle of one hind limb of the mouse was injected; muscle weakness was measured using the toe abduction score (DAS) on a scale of 0-4. The maximum DAS value for each dose was determined and plotted against the dose, and the data were fitted to a 4-parameter logistic equation to determine the ED50 and dose (DAS4 dose) value that resulted in DAS 4.

[0389] Figure 5 Shown is an SDS-PAGE of purified recombinant BoNT / AB chimeras 3B and 3C (SEQ ID NOs: 6 and 7, respectively). Lanes are labeled "Marker" (molecular weight marker), "-DTT" (oxidized BoNT / AB chimera sample), and "+DTT" (reduced BoNT / AB chimera sample).

[0390] Figure 6Cleavage of SNAP-25 by BoNT / A and BoNT / AB chimeras 3B and 3C (SEQ ID NOs: 2, 6, and 7, respectively) in human induced pluripotent stem cell-derived peripheral neurons (PERI.4U-Axiogenesis, Germany) is shown. PERI.4U cells were exposed to different 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 with 1× NuPAGE buffer supplemented with DTT and Benzonase. Samples were transferred to microcentrifuge tubes, heated at 90°C for 5 minutes on a heat block, and stored at -20°C before analysis of SNAP-25 cleavage by Western blot. SNAP-25 was detected using a polyclonal antibody (Sigma #S9684) that detects both full-length and cleaved forms of SNAP-25. Anti-rabbit HRP (Sigma #A6154) was used as a secondary antibody.

[0391] Figure 7 The duration of muscle weakness over time in the mouse digital abduction score test is shown. Under brief general anesthesia, mice were injected into the gastrocnemius-soleus complex muscle of one hind limb; muscle weakness was measured using the digital abduction score (DAS) on a scale of 0-4. Animals in the group injected with the lowest dose, which induced a DAS of 4 during the first four days of injection, were monitored until muscle weakness fully recovered to a DAS of 0 (no muscle weakness observed).

[0392] Sequence Listing

[0393] Where an initiating Met amino acid residue or a corresponding initiation codon is indicated in any of the following SEQ ID NOs, said residue / codon is optional.

[0394] SEQ ID NO: 1 (nucleotide sequence of unmodified BoNT / A)

[0395]

[0396] SEQ ID NO: 2 (polypeptide sequence of unmodified BoNT / A)

[0397]

[0398] SEQ ID NO: 3 (Polypeptide sequence of modified BoNT / A "Chimera 1")

[0399]

[0400] SEQ ID NO:4 (Polypeptide sequence of modified BoNT / A "Chimera 2")

[0401]

[0402] SEQ ID NO:5 (Polypeptide sequence of modified BoNT / A "Chimera 3A")

[0403]

[0404] SEQ ID NO:6 (Polypeptide sequence of modified BoNT / A "Chimera 3B")

[0405] SEQ ID NO:7 (Polypeptide sequence of modified BoNT / A "Chimera 3C")

[0406]

[0407] SEQ ID NO: 8 (BoNT / B polypeptide sequence)

[0408]

[0409] SEQ ID NO:9 C-terminal L chain fragment

[0410] TKSLDKGYNK

[0411] SEQ ID NO:10—C-terminal L chain fragment 2

[0412] SLDKGYNK

[0413] SEQ ID NO:11-Double-stranded L chain 1

[0414] PFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGGSTIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNGYGSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPNRVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKAKSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIYTEDNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTKLKNFTGLFEFYKLLCVRGIITSK

[0415] SEQ ID NO:12-Double-stranded L chain 2

[0416] PFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGGSTIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNGYGSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPNRVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKAKSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIYTEDNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTKLKNFTGLFEFYKLLCVRGIITSKTK

[0417] SEQ ID NO:13-Double-stranded H chain

[0418] ALNDLCIKVNNWDLFFSPSEDNFTNDLNKGEEITSDTNIEAAEENISLDLIQQYYLTFNFDNEPENISIENLSSDIIGQLELMPNIERFPNGKKYELDKYTMFHYLRAQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSDYVKKVNKATEAAMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYKDDFVGALIFSGAVILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALSKRNEKWDEVYKYIVTNWLAKVNTQIDLIRKKMKEALENQAEATKAIINYQYNQYTEEEKNNINFNIDDLSSKLNESINKAMININKFLNQCSVSYLMNSMIPYGVKRLEDFDASLKDALLKYIYDNRGTLIGQVDRLKDKVNNTLSTDIPFQLSKYVDNQRLLSTFTEYIKNILNNIILNLRYKDNNLIDLSGYGAKVEVYDGVELNDKNQFKLTSSANSKIRVTQNQNIIFNSVFLDFSVSFWIRIPKYKNDGIQNYIHNEYTIINCMKNNSGWKISIRGNRIIWTLIDINGKTKSVFFEYNIREDISEYINRWFFVTITNNLNNAKIYINGKLESNTDIKDIREVIANGEIIFKLDGDIDRTQFIWMKYFSIFNTELSQSNIEERYKIQSYSEYLKDFWGNPLMYNKEYYMFNAGNKNSYIKLKKDSPVGEILTRSKYNQNSKYINYRDLYIGEKFIIRRKSNSQSINDDIVRKEDYIYLDFFNLNQEWRVYTYKYFKKEEMKLFLAPIYDSDEFYNTIQIKEYDEQPTYSCQLLFKKDEESTDEIGLIGIHRFYESGIVFEEYKDYFCISKWYLKEVKRKPYNLKLGCNWQFIPKDEGWTE

[0419] Example

[0420] Example 1

[0421] Cloning, expression and purification of modified BoNT / A (BoNT / AB chimera)

[0422] Using standard molecular biology techniques, BoNT / AB chimeric constructs 1, 2, 3A, 3B, and 3C (SEQ ID NOs: 3 to 7, respectively) were constructed from DNA encoding the parental serotype molecules and appropriate oligonucleotides. They were then cloned into 10 The pJ401 expression vector with the A-tag was transformed into BLR(DE3) E. coli cells for overexpression. These cells were grown in 2L baffled Erlenmeyer flasks at 37°C and 225RPM shaking containing 1L of modified Terrific broth (mTB) supplemented with appropriate antibiotics. Once A 600 Once >0.5 was reached, the incubator temperature was lowered to 16°C and then one hour later, induced with 1 mM IPTG at 225 RPM shaking for 20 hours to express the recombinant BoNT / AB construct.

[0423] The harvested cells were lysed by sonication and clarified by centrifugation at 4500 RPM for 1 hour at 4°C. The recombinant BoNT / AB chimeric molecules were then extracted with ammonium sulfate and purified by standard fast protein liquid chromatography (FPLC) techniques. This involved the use of a hydrophobic interaction resin for capture and an anion exchange resin for an intermediate purification step. The partially purified molecules were then proteolytically cleaved with the endoproteinase Lys-C to generate an active duplex. Further purification was performed with a second hydrophobic interaction resin to obtain the final BoNT / AB chimera.

[0424] For the case with a ten-histidine tag (H 10 ) of the BoNT / AB chimeric molecules (chimeras 1, 2, and 3A), and the capture step used immobilized nickel resin instead of hydrophobic interaction resin.

[0425] The sequence of each chimera is shown in Table 1.

[0426]

[0427] Table 1 - Chimeric BoNT / AB Constructs

[0428] Example 2

[0429] Comparison of BoNT / AB chimeras 1, 2, and 3A

[0430] As in Example 1 ( Figure 2 ) as described in the purification of 10 BoNT / AB chimeras 1, 2, and 3A with E1191M / S1199Y double mutations were tagged and tested for functional activity.

[0431] SNAP-25 cleavage assay in rat spinal cord neurons

[0432] Primary cultures of rat spinal cord neurons (SCN) were prepared and cultured in 96-well tissue culture plates for 3 weeks (as described in Masuyer et al., 2011, J. Struct. Biol. Structure and activity of a functional derivative of Clostridium botulinum neurotoxin B; and Chaddock et al., 2002, Protein Expr. Purif. Expression and purification of catalytically active, non-toxic endopeptidase derivatives of Clostridium botulinum toxin type A). Serial dilutions of BoNT / AB were prepared in SCN feed medium. Growth medium from the wells to be treated was collected and filtered (0.2 μm filter). 125 μL of filtered medium was added back to each test well. 125 μL of diluted toxin was then added to the plate (in triplicate). Treated cells were incubated at 37°C, 10% CO2 for 24 ± 1 h.

[0433] Analysis of BoNT activity using SNAP-25 cleavage assay

[0434] After treatment, BoNT was removed and cells were washed once in PBS (Gibco, UK). Cells were lysed in 1× NuPAGE lysis buffer (Life Technologies) supplemented with 0.1 M dithiothreitol (DTT) and 250 units / mL benzoase (Sigma). Lysate proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes. The membrane was probed with a SNAP-25-specific primary antibody (Sigma # S9684), which recognizes uncut SNAP-25 as well as SNAP-25 cleaved 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 SNAP-25 cleaved at each BoNT concentration was calculated. Data were fitted to a 4-parameter logistic equation and pEC was calculated using GraphPad Prism version 6 (GraphPad) 50 .

[0435] Table 2 below provides the pEC values ​​determined for chimeras 1, 2, and 3A in the rat SCN SNAP-25 cleavage assay.50 These results indicate that the three BoNT / AB chimeras retain the ability to enter rat spinal cord neurons and cleave their target substrates. However, chimera 3A was more potent than chimeras 1 and 2 in this assay (see also Figure 3 ).

[0436]

[0437]

[0438] Table 2. pEC 50 value.

[0439] Digital abduction score (DAS) test

[0440] The method for measuring the activity of BoNT / AB chimeras 1, 2, and 3A in the DAS assay is based on the startle toe splay reflex of mice when they are briefly suspended by their tails. This reflex is scored as the digital abduction score (DAS) and is inhibited after BoNT is applied to the gastrocnemius-soleus muscles of the hind paw. Mice are briefly suspended by their tails to elicit a characteristic startle response in which the animal extends its hind limbs and abducts its hind toes. (Aoki et al., 1999, Eur. J. Neurol.; 6(suppl. 4) S3-S10).

[0441] On the day of injection, mice were anesthetized in an induction chamber receiving 3% isoflurane in oxygen.Each mouse received an intramuscular injection of BoNT / AB chimeras or vehicle (phosphate buffered saline containing 0.2% gelatin) in the gastrocnemius-soleus muscle of the right hind paw.

[0442] After injection of the neurotoxin, the degree of toe abduction was scored on a scale from 0 to 4, where 0 = normal and 4 = maximal reduction in toe abduction and leg extension. The ED was determined by nonlinear adjustment analysis using the mean of the maximal effect of each dose. 50 The mathematical model used was a 4-parameter logistic model.

[0443] DAS was performed every 2 hours on the first day after drug administration and 3 times a day thereafter for 4 days.

[0444] Figure 4 The fitted curves for chimeras 1, 2, and 3A (SEQ ID NOs: 3, 4, and 5 converted to double-stranded form, respectively) are shown. The curve for chimera 3A is shifted to the left, meaning 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 other chimeras in the mouse DAS assay; see also the table below (Table 3), which provides the calculated ED for each chimera 50 values ​​and the dose that resulted in DAS 4 (the highest score).

[0445] Table 3 below provides the ED values ​​determined in the mouse DAS assay for recombinant BoNT / A1 (rBoNT / A1-converted to double-chain form in SEQ ID NO: 2) and Chimeras 1, 2, and 3A. 50 and DAS 4 doses. These results indicate that among the three chimeras, chimera 3A has the highest in vivo efficacy in inducing muscle weakness. Figure 4 The studies shown in Table 3 were performed on mice obtained from Charles River Laboratories.

[0446]

[0447]

[0448] Table 3ED 50 value.

[0449] Example 3

[0450] Comparison of BoNT / AB chimeras 3B, 3C and unmodified BoNT / A1

[0451] Untagged BoNT / AB chimeras 3B and 3C, with or without the E1191M / S1199Y double mutation (SEQ ID NOs: 6 and 7), respectively, were purified as described in Example 1 ( Figure 2 ), and the functional activity was tested using unmodified BoNT / A (SEQ ID NO: 2 converted into a double-chain form) as a reference.

[0452] Human pluripotent stem cell SNAP-25 cleavage assay

[0453] Cryopreserved PERI.4U cells were purchased from Axiogenesis (Cologne, Germany). Thawing and plating of cells were performed according to the manufacturer's recommendations. Briefly, cryovials containing cells were thawed in a 37°C water bath for 2 min. After gentle resuspension, cells were transferred to 50 mL tubes. 1 mL of Wash the cryovials with thawed medium and transfer the medium dropwise to the cell suspension in the 50 mL tube, then add an additional 2 mL Thaw the culture medium and add it dropwise to a 50 mL tube. Then count the cells using a hemocytometer. Afterwards, add another 6 mL Thaw medium is added to the cell suspension. Cell pellets are obtained by centrifugation at 260 x g (e.g., 1,100 RPM) for 6 minutes at room temperature. Cells are then resuspended in the manufacturer's complete The cells were plated at 50,000 to 150,000 cells / cm 2The cells were plated at a density of 100 μg / mL on cell culture plates coated with poly-L-ornithine and laminin. The cells were cultured at 37°C in a humidified CO2 atmosphere, and the culture medium was completely replaced every 2-3 days during the culture period.

[0454] For toxin treatment, Prepare serial dilutions of BoNT in culture medium. Collect and filter the culture medium from the wells to be treated (0.2 μm filter). Add 125 μL of filtered culture medium back to each test well. Then add 125 μL of diluted toxin to the plate (in triplicate). Incubate the treated cells at 37°C, 10% CO2 for 48 ± 1 h.

[0455] Analysis of BoNT activity using SNAP-25 cleavage assay

[0456] After treatment, BoNT was removed and cells were washed once in PBS (Gibco, UK). Cells were lysed in 1× NuPAGE lysis buffer (Life Technologies) supplemented with 0.1 M dithiothreitol (DTT) and 250 units / mL benzoase (Sigma). Lysate proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes. The membrane was probed with a SNAP-25-specific primary antibody (Sigma # S9684), which recognizes uncut SNAP-25 as well as SNAP-25 cleaved 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 SNAP-25 cleaved at each BoNT concentration was calculated. Data were fitted to a 4-parameter logistic equation and pEC was calculated using GraphPad Prism version 6 (GraphPad) 50 .

[0457] Figure 6 The chimeras 3B and 3C were shown to exhibit greater potency than rBoNT / A1 in cleaving SNAP-25 in induced human pluripotent stem cells, but the former was significantly more effective. This could be explained by the double mutation, which increases the affinity of chimera 3B for the human synaptotagmin II protein receptor present in these cells ( Figure 6 , Table 4).

[0458] <![CDATA[pEC 50 ±SEM]]> rBoNT / A1 10.21±0.05 Chimera 3B 12.38±0.06 Chimera 3C 10.72±0.08

[0459] Table 4. pEC 50 value.

[0460] Digital Abduction Score (DAS) Test – Safety Ratio

[0461] The method for measuring BoNT activity in the DAS assay is based on the startle toe splay reflex of mice when they are briefly suspended by their tails. This reflex is scored as the digital abduction score (DAS) and is inhibited after BoNT is applied to the gastrocnemius-soleus muscles of the hind paw. Mice are briefly suspended by their tails to elicit a characteristic startle response in which the animal extends its hind limbs and abducts its hind toes. (Aoki et al., 1999, Eur. J. Neurol.; 6(suppl. 4) S3-S10).

[0462] On the day of injection, mice were anesthetized in an induction chamber receiving 3% isoflurane in oxygen.Each mouse received an intramuscular injection of BoNT or vehicle (phosphate buffered saline containing 0.2% gelatin) in the gastrocnemius-soleus muscle of the right hind paw.

[0463] After injection of the neurotoxin, the degree of toe abduction was scored on a scale from 0 to 4, where 0 = normal and 4 = maximal reduction in toe abduction and leg extension. The ED was determined by nonlinear adjustment analysis using the mean of the maximal effect of each dose. 50 The mathematical model used was a 4-parameter logistic model.

[0464] DAS was performed every 2 hours on the first day after dosing; thereafter, it was performed 3 times a day for 4 days for all doses. Thereafter, animals in the vehicle-injected and lowest dose groups, which induced a DAS of 4 during the first four days of injection, were monitored until muscle weakness fully recovered to a DAS of 0 (no muscle weakness was observed).

[0465] To calculate the safety ratio, all animals were weighed one day before toxin injection (D0) and thereafter once daily throughout the study. The mean body weight, its standard deviation, and standard error mean were calculated for each dose group daily. To obtain the safety ratio (-10% ΔBW / ED 50 ) the dose that causes the mean body weight of the dose group at any time during the study to be less than 10% of the mean body weight of the same dose group at D0 divided by the ED value of the BoNT being studied 50 The lethal dose is defined as the dose at which one or more animals in the group die.

[0466] Figure 7 The duration of muscle weakness over time in the mouse digital abduction score assay is shown for unmodified BoNT / A, chimera 3B, and chimera 3C (SEQ ID NOs: 2, 6, and 7 converted to double-chain form), indicating that the chimeras have a longer duration of action.

[0467] Table 5 below provides the ED values ​​determined for rBoNT / A1 and chimeras 3B and 3C in the mouse DAS assay. 50 The table also provides the total duration of action of the DAS 4 dose until muscle weakness is fully restored to DAS 0 (no muscle weakness is observed). In addition, the table also shows the mouse lethal dose and safety ratio (-10% ΔBW / ED 50 ), as defined above. Compared to rBoNT / A1, chimeras 3B and 3C had longer duration of action, better safety ratio, and higher lethal dose.

[0468] Figure 7 The studies shown in and Table 5 were performed on mice obtained from the Janvier laboratory.

[0469]

[0470] Table 5. DAS and safety ratios of BoNT / AB chimeras.

[0471] Example 4

[0472] Preclinical testing of modified BoNT / A (BoNT / AB chimera [SEQ ID NO: 6 converted to two-chain form])

[0473] The BoNT / AB chimera SEQ ID NO:6 converted into a double-chain form was expressed in mouse LD 50 The result was 1.202 ng / kg. Therefore, 1 unit of SEQ ID NO: 6 is equivalent to 24.04 pg in this test.

[0474] Additionally, the BoNT / AB chimeras were tested in the rat DAS assay to determine The results are shown in Table 6 below:

[0475]

[0476] Table 6. Duration of action.

[0477] In conclusion, the duration of action of BoNT / AB is much longer than

[0478] Example 5

[0479] Determination of the modified BoNT / A (SEQ ID NO: 6 converted into a two-chain form) for the treatment of cervical dystonia Unit dose

[0480] Based on preclinical pharmacology data, a suitable unit dose range (UD) for administration of modified BoNT / A in humans has been determined.

[0481] Calculated DAS ED of SEQ ID NO: 6 50 It is 13pg / kg. 50 It is considered to be the minimum pharmacologically active dose, which is approximately 300 times lower than the no observed adverse effect level (NOAEL) of 4 ng / kg in the same animal species. The ED of 13 pg / kg of SEQ ID NO: 6 in rats 50 This is equivalent to a dose of 0.8 ng for a person weighing 60 kg.

[0482] Therefore, the lower limit of the unit dose is selected to be 1,000 pg. The upper limit of the unit dose is selected to be 16,000 pg, which is lower than the NOAEL of 4 ng / kg in two non-clinical safety species (rats and monkeys) converted to a human dose of 60 kg body weight. Therefore, the unit dose is determined to be 1,000 pg to 16,000 pg (about 42 units to about 666 units).

[0483] In view of the improved safety, the maximum total dose for the treatment of cervical dystonia was set at 160,000 pg (approximately 7,070 units), which was derived from the NOAEL of 4 ng / kg in two nonclinical safety species (rat and monkey) converted to a 60 kg body weight human dose.

[0484] In view of the preclinical data as determined in Example 4 Compared with the improved safety profile, the total dose (in units) expected to be administered in cervical dystonia is almost 7 times. The maximum total dose for cervical dystonia is 1,000 units (see Figure 1 ).

[0485] Advantageously, in treating cervical dystonia, more modified BoNT / A (SEQ ID NO: 6) can be injected and / or can be injected at a greater number of cervical muscles / sites before a maximum dose is reached. This is an important and advantageous discovery that results in improved treatment of cervical dystonia while providing clinicians with a wider range of treatment options.

[0486] Example 6

[0487] Methods of administration of modified BoNT / A (SEQ ID NO: 6 converted into a double-chain form) for treating cervical dystonia case

[0488] The modified BoNT / A (eg, SEQ ID NO: 6 converted to a double-chain form) is provided as a lyophilized powder in vials containing 36 ng of modified BoNT / A per vial. The lyophilized powder is reconstituted.

[0489] The unit dose (UD) is 1,000-16,000 pg (approximately 42-666 units [LD 50 Measurement]).

[0490] Cervical dystonia is treated by intramuscular injection according to the following dosing schedule (Table 7):

[0491] Neck muscles Dosage (unit dose) Sternocleidomastoid muscle 1x UD Splenius capitis 1x UD Splenius cervicis 1x UD Trapezius 1x UD Levator scapulae 1x UD Scalene medius 1x UD scalene anterior muscle 1x UD Semispinalis capitis 1x UD Longissimus 1x UD Posterior paraspinal muscles 1x UD Submental complex 1x UD

[0492] Table 7. Dosing regimen.

[0493] Administration can be unilateral or bilateral, as required by the particular presentation.

[0494] The maximum total dose administered is 10×UD (e.g., in some cases, 2×UD is administered to a given neck muscle or muscles). This corresponds to 160,000 pg / about 6,660 units. This is about as much as can be administered during treatment of cervical dystonia without approaching the limits of toxicity, a concern with conventional treatment regimens. Thus, clinicians are able to tailor treatment for their patients knowing that 10× UD can be administered without concern for toxicity, thereby allowing for treatment of additional neck muscles in an individual and / or ensuring that each neck muscle receives a pharmaceutically effective dose.

[0495] Example 7

[0496] Treatment of patients with cervical dystonia (lateral neck)

[0497] Jane, 65 years old, was diagnosed with cervical dystonia by her general practitioner. The lateral neck was specifically manifested by a single unit dose (3,000 pg) of modified BoNT / A (SEQ ID NO: 6 converted to a two-chain form) was administered ipsilaterally to Jane's levator scapulae muscle, and a single unit dose was also administered ipsilaterally to Jane's sternocleidomastoid muscle (resulting in a total dose of 6,000 pg during treatment). The lateral neck was relieved, and due to the longer duration of the modified BoNT / A, Jane did not require further treatment for more than 9 months. As a result, Jane received less frequent injections (e.g., annually) compared to an equivalent individual administered unmodified BoNT / A. In addition, due to the improved safety profile of the modified BoNT / A, Jane did not exhibit any side effects.

[0498] Example 8

[0499] Treatment of patients with cervical dystonia (neck extension)

[0500] Brian, 48 years old, was diagnosed with cervical dystonia by his general practitioner. The symptoms were neck extension. The modified BoNT / A (SEQ ID NO: 6 converted to a double-chain form) was administered to each of Brian's following muscles:

[0501] · 1× unit dose (UD) of 10,000 pg per levator scapulae muscle;

[0502] 10,000 pg of 1×UD per trapezius muscle;

[0503] 10,000 pg of 1×UD was administered to each longissimus muscle;

[0504] 10,000 pg of 1×UD per splenius capitis muscle; and

[0505] · 10,000 pg of 1×UD was administered to each cervical splenius muscle.

[0506] The total dose administered was 10×UD (100,000 pg), which is well within the upper limit of 160,000 pg and is feasible given the improved safety profile of modified BoNT / A compared to unmodified BoNT / A. Neck extension was alleviated, and due to the longer duration of the modified BoNT / A, Brian did not require further treatment for 12 months. Therefore, Brian received less frequent injections than an equivalent individual administered unmodified BoNT / A.

[0507] Example 9

[0508] Safety and efficacy of modified BoNT / A (SEQ ID NO: 6 converted to a double-chain form) in humans

[0509] SEQ ID NO: 6 (converted into a two-chain form) was administered to a human subject by way of a single unit dose of modified BoNT / A. Different (increasing) amounts of modified BoNT / A (SEQ ID NO: 6 converted into a two-chain form) were administered to 5 cohorts. Cohort 1 was administered 2 x 1,000 pg unit doses of modified BoNT / A (i.e., a maximum of 2,000 pg), while Cohort 5 was administered 2 x 16,000 pg unit doses of modified BoNT / A (i.e., a maximum of 32,000 pg).

[0510] The results showed that all unit doses of modified BoNT / A tested (i.e., up to 16,000 pg unit dose) were effective against muscle paralysis, were safely tolerated, and no adverse effects were observed despite the unusually high dose per muscle. This suggests that the modified BoNT / A does not spread from the injection site and highlights the unusual safety of the modified BoNT / A (SEQ ID NO: 6 converted to a two-chain form).

[0511] Example 10

[0512] Methods of administration of modified BoNT / A (SEQ ID NO: 6 converted into a double-chain form) for treating cervical dystonia case

[0513] The modified BoNT / A (e.g., SEQ ID NO: 6 converted to a double-chain form) is provided as a lyophilized powder in vials, each containing 36 ng of the modified BoNT / A. The lyophilized powder is reconstituted.

[0514] The unit dose (UD) is 17,000-36,000 pg (~707-1,498 units [based on mouse LD 50 Measurement]).

[0515] Cervical dystonia is treated by intramuscular injection according to the following dosing schedule (Table 8):

[0516] Neck muscles Dosage (unit dose) Sternocleidomastoid muscle 1x UD Splenius capitis 1x UD Splenius cervicis 1x UD Trapezius 1x UD Levator scapulae 1x UD Scalene medius 1x UD scalene anterior muscle 1x UD Semispinalis capitis 1x UD Longissimus 1x UD Posterior paraspinal muscles 1x UD Submental complex 1x UD

[0517] Table 8 Dosage regimen

[0518] Administration may be unilateral or bilateral, depending on the circumstances.

[0519] The maximum total dose administered was 10×UD (e.g., in some cases, 2×UD was administered to one or more neck muscles). This is equivalent to 360,000 pg / ~14,975 units. The maximum total dose that can be administered during treatment of cervical dystonia is more than 20 times higher without approaching the toxicity limit (a concern with traditional treatment regimens). Therefore, clinicians are able to tailor treatment to their patients knowing that 10× UD can be administered without concern for toxicity, thereby allowing treatment of additional neck muscles in an individual and / or ensuring that each neck muscle receives a pharmaceutically effective dose.

[0520] Embodiment 11

[0521] Treatment of patients with cervical dystonia (lateral neck)

[0522] Elizabeth, 62 years old, was diagnosed with cervical dystonia by her general practitioner. The lateral neck was specifically manifested. A single unit dose (36,000 pg) of modified BoNT / A (SEQ ID NO:6 converted to a double-chain form) was administered to Elizabeth's levator scapulae muscle ipsilateral to the sternocleidomastoid muscle, while a single unit dose was administered to Elizabeth's sternocleidomastoid muscle ipsilateral to the sternocleidomastoid muscle (total dose during treatment was 72,000 pg). The lateral neck was relieved, and due to the long duration of the modified BoNT / A, Elizabeth did not require further treatment for more than 9 months. Therefore, Elizabeth received less frequent injections (e.g., annually) than an equivalent individual injected with unmodified BoNT / A. In addition, due to the improved safety of the modified BoNT / A, Elizabeth did not exhibit any side effects.

[0523] Example 12

[0524] Treatment of patients with cervical dystonia (neck extension)

[0525] Donald, 54 years old, was diagnosed with cervical dystonia by his general practitioner. The symptoms were neck extension. Modified BoNT / A (SEQ ID NO: 6 converted to a two-chain form) was applied bilaterally to each of Donald's following muscles:

[0526] · 1× unit dose (UD) of 36,000 pg per levator scapulae muscle;

[0527] · 1×UD 36,000pg per trapezius muscle;

[0528] · 1×UD 36,000pg per longissimus muscle;

[0529] · 1×UD 36,000pg per splenius capitis muscle;

[0530] · 1×UD 36,000 pg was administered to each cervical splenius muscle.

[0531] The total dose administered was 10×UD (360,000 pg), which was possible due to the improved safety profile of the modified BoNT / A compared to unmodified BoNT / A. Neck extension symptoms were alleviated, and due to the long-lasting effect of the modified BoNT / A, Donald did not require further treatment for 12 months. Therefore, Donald received less frequent injections than an equivalent individual administered unmodified BoNT / A. In addition, Donald did not experience any side effects due to the improved safety profile of the modified BoNT / A.

[0532] Example 13

[0533] Safety and efficacy of modified BoNT / A (SEQ ID NO: 6 converted to double-chain form) in humans

[0534] SEQ ID NO: 6 (converted to a double-stranded form) was administered to human subjects by intramuscular injection. The subjects were administered 2×15,000 pg unit doses (i.e., 30,000 pg in total), 2×25,000 pg unit doses (i.e., 50,000 pg in total), or 2×36,000 pg unit doses (i.e., 72,000 pg in total) of the modified BoNT / A (SEQ ID NO: 6 converted to a double-stranded form).

[0535] The results showed that all modified BoNT / A unit doses tested were effective in paralyzing muscles, safely tolerated, and no adverse reactions were observed, despite extremely high doses per muscle (e.g., 25,000 pg and 36,000 pg unit doses). This indicates that the modified BoNT / A does not spread from the injection site and highlights the excellent safety of the modified BoNT / A (SEQ ID NO: 6 converted to a two-chain form).

[0536] All publications mentioned in the above specification are incorporated herein by reference. Without departing from the scope and spirit of the present invention, various modifications and variations of the method and system described in the present invention will be apparent to those skilled in the art. Although the present invention has been described in conjunction with specific preferred embodiments, it should be understood that the claimed invention should not be unduly limited to such specific embodiments. In fact, for those skilled in the art of biochemistry and biotechnology or related fields, it is apparent that various modifications are made to the described modes for performing the present invention, and fall within the scope of the following claims.

Claims

1. A modified botulinum neurotoxin A (BoNT / A) for use in treating cervical dystonia, wherein the modified BoNT / A is administered by intramuscular injection into the affected neck muscles of a subject, wherein the modified BoNT / A is administered at a unit dose of greater than 17,000 pg of modified BoNT / A, wherein at least a single unit dose is administered to the affected neck muscle, wherein the total dose administered during the treatment period is up to 400,000 pg of the modified BoNT / A, and wherein the modified BoNT / A comprises a BoNT / A light chain and a translocation domain, and a BoNT / B receptor binding domain (H C domain).

2. A method of treating cervical dystonia, the method comprising administering a modified BoNT / A to an affected neck muscle of an individual by intramuscular injection, wherein the modified BoNT / A is administered at a unit dose of greater than 17,000 pg of modified BoNT / A, wherein at least a single unit dose is administered to the affected neck muscles, wherein the total dose administered during the treatment period is up to 400,000 pg of the modified BoNT / A, and wherein the modified BoNT / A comprises a BoNT / A light chain and a translocation domain, and a BoNT / B receptor binding domain (H C domain).

3. Use of a modified botulinum neurotoxin A (BoNT / A) in the manufacture of a medicament for treating cervical dystonia, wherein the modified BoNT / A is administered to the affected neck muscles of an individual by intramuscular injection, wherein the modified BoNT / A is administered at a unit dose of greater than 17,000 pg of the modified BoNT / A, wherein at least a single unit dose is administered to an affected neck muscle, wherein the total dose administered during the treatment period is up to 400,000 pg of the modified BoNT / A, and wherein the modified BoNT / A comprises a BoNT / A light chain and a translocation domain, and a BoNT / B receptor binding domain (H C domain).

4. The modified BoNT / A for use, method, or use according to any one of the preceding claims, wherein the total dose administered is 170,000 pg to 400,000 pg (e.g., greater than 170,000 pg to at most 400,000 pg) of the modified BoNT / A.

5. The modified BoNT / A for use, method, or use according to any one of claims 1 to 4, wherein the total dose administered is 170,000 pg to 360,000 pg (e.g., greater than 170,000 pg to a maximum of 360,000 pg) of the modified BoNT / A 6. The modified BoNT / A for use, method or use according to any one of claims 1-4, wherein the total dose administered is 250,000 pg to 400,000 pg (e.g., 250,000 pg to a maximum of 360,000 pg) of the modified BoNT / A.

7. The modified BoNT / A for use, method or use according to any one of claims 1 to 5, wherein the total dose administered is 200,000 pg to 300,000 pg, such as 240,000 to 260,000 pg (e.g., 250,000 pg) of the modified BoNT / A.

8. The modified BoNT / A for use, method or use according to any one of claims 1-4 or 6, wherein the total dose administered is 300,000 pg to a maximum of 400,000 pg, such as 350,000 pg to 370,000 pg, (e.g. 360,000 pg) of the modified BoNT / A.

9. The modified BoNT / A for use, method or use according to any one of the preceding claims, wherein the total dose administered during treatment is at most 250,000 pg of modified BoNT / A.

10. The modified BoNT / A for use, method or use according to any one of the preceding claims, wherein the total dose administered during treatment is at most 360,000 pg of modified BoNT / A.

11. The modified BoNT / A for use, method or use according to any one of the preceding claims, wherein the total dose administered during treatment is at least 250,000 pg of the modified BoNT / A.

12. The modified BoNT / A for use, method, or use according to any of the preceding claims, wherein the unit dose is greater than 17,000 pg to up to 40,000 pg (e.g., 17,001 pg to up to 40,000 pg) of the modified BoNT / A.

13. The modified BoNT / A for use, method, or use according to any of the preceding claims, wherein the unit dose is greater than 17,000 pg to up to 36,000 pg (e.g., 17,001 pg to up to 36,000 pg) of the modified BoNT / A.

14. The modified BoNT / A for use, method, or use according to any one of claims 1-2, wherein the unit dose is 25,000 pg to a maximum of 40,000 pg (e.g., 25,000 pg to 36,000 pg) of the modified BoNT / A.

15. The modified BoNT / A for use, method or use according to any one of claims 1-12 or 14, wherein the total dose administered is 30,000 pg to a maximum of 40,000 pg, such as 35,000 pg to 37,000 pg (preferably 36,000 pg) of the modified BoNT / A.

16. The modified BoNT / A for use, method or use according to any one of claims 1-13, wherein the unit dose is 20,000 pg to 30,000 pg, such as 24,000 to 26,000 pg (preferably 25,000 pg) of the modified BoNT / A.

17. A modified botulinum neurotoxin A (BoNT / A) for use in treating cervical dystonia, wherein the modified BoNT / A is administered by intramuscular injection into the affected neck muscles of a subject, wherein the modified BoNT / A is administered at a unit dose of greater than 707 U of the modified BoNT / A, wherein 1 unit corresponds to a calculated median lethal dose (LD50) in mice 50 ), wherein at least a single unit dose is administered to the affected neck muscles, wherein the total dose administered during the treatment period is up to 16,639 U of the modified BoNT / A, and wherein the modified BoNT / A comprises a BoNT / A light chain and a translocation domain, and a BoNT / B receptor binding domain (H C domain).

18. A method of treating cervical dystonia, the method comprising administering a modified BoNT / A to an affected neck muscle of an individual by intramuscular injection, wherein the modified BoNT / A is administered at a unit dose of greater than 707 U of the modified BoNT / A, wherein 1 unit corresponds to a calculated median lethal dose (LD50) in mice. 50 ), wherein at least a single unit dose is administered to the affected neck muscles, wherein the total dose administered during the treatment period is up to 16,639 U of the modified BoNT / A, and wherein the modified BoNT / A comprises a BoNT / A light chain and a translocation domain, and a BoNT / B receptor binding domain (H C domain).

19. Use of a modified botulinum neurotoxin A (BoNT / A) in the manufacture of a medicament for treating cervical dystonia, wherein the modified BoNT / A is administered to an affected neck muscle of an individual by intramuscular injection, wherein the modified BoNT / A is administered at a unit dose of greater than 707 units of the modified BoNT / A, wherein 1 unit corresponds to a calculated median lethal dose (LD50) in mice. 50 ), wherein at least a single unit dose is administered to the affected neck muscle, wherein the total dose administered during the treatment period is up to 16,639 U of the modified BoNT / A, and wherein the modified BoNT / A comprises a BoNT / A light chain and a translocation domain, and a BoNT / B receptor binding domain (H C domain).

20. The modified BoNT / A for use, method, or use according to any one of claims 17-19, wherein the total dose administered is 7,072 units to a maximum of 16,639 units (e.g., greater than 7,072 units to a maximum of 16,639 units) of the modified BoNT / A.

21. The modified BoNT / A for use, method or use according to any one of claims 17-20, wherein the total dose administered is 10,399 units to 16,639 units (eg, 10,399 units to a maximum of 14,975 units) of the modified BoNT / A.

22. The modified BoNT / A for use, method or use according to any one of claims 17-20, wherein the total dose administered is 8,319 units to 12,479 units, such as 9,983 units to 10,815 units (e.g., 10,399 units) of the modified BoNT / A.

23. The modified BoNT / A for use, method or use according to any one of claims 17-21, wherein the total dose administered is 12,479 units to 16,639 units, such as 14,559 units to 15,391 units (e.g., 14,975 units) of the modified BoNT / A.

24. The modified BoNT / A for use, method or use according to any one of claims 17-23, wherein the total dose administered during the treatment period is a maximum of 14,975 U of the modified BoNT / A.

25. The modified BoNT / A for use, method or use according to any one of claims 17-22, wherein the total dose administered during treatment is at most 10,399 units.

26. The modified BoNT / A for use, method, or use according to any one of claims 17-25, wherein the unit dose is greater than 707 units to a maximum of 1,664 units (e.g., 707.2 or 708 units to a maximum of 1,664 units) of the modified BoNT / A.

27. The modified BoNT / A for use, method, or use according to any one of claims 17-26, wherein the unit dose is greater than 707 units to a maximum of 1,498 units (e.g., 707.2 or 708 units to a maximum of 1,498 units) of the modified BoNT / A.

28. The modified BoNT / A for use, method, or use according to any one of claims 17-26, wherein the unit dose is 1,040 units to a maximum of 1,664 units (eg, 1,040 units to 1,498 units) of the modified BoNT / A.

29. The modified BoNT / A for use, method or use according to any one of claims 17-27, wherein the unit dose is 832 units to 1,248 units, such as 998 units to 1,082 units (preferably 1,040 units) of the modified BoNT / A.

30. The modified BoNT / A for use, method or use according to any one of claims 17-26 or 28, wherein the unit dose is 1,248 units to 1,664 units, such as 1,456 units to 1,539 units, (preferably 1,498 units) of the modified BoNT / A.

31. The modified BoNT / A for use, method, or use according to any one of the preceding claims, wherein the modified BoNT / A comprises, comprises, or consists of a polypeptide sequence having at least 70% sequence identity to SEQ ID NO:

6.

32. The modified BoNT / A for use, method or use according to any one of the preceding claims, wherein the modified BoNT / A is a two-chain modified BoNT / A wherein the L chain is linked to the H chain by a disulfide bond, the modified BoNT / A being obtainable by a method comprising contacting a single-chain modified BoNT / A consisting of SEQ ID NO: 6 with a protease that hydrolyzes a peptide bond in an activation loop thereof, thereby converting the single-chain modified BoNT / A to the corresponding two-chain modified BoNT / A.

33. The modified BoNT / A for use, method, or use according to any one of the preceding claims, wherein the initial methionine amino acid residue of the polypeptide sequence of the modified BoNT / A is optional.

34. The modified BoNT / A for use, method, or use according to any one of the preceding claims, wherein the initial methionine amino acid residue of the polypeptide sequence of the modified BoNT / A is absent.

35. The modified BoNT / A for use, method, or use according to any one of the preceding claims, wherein the modified BoNT / A is a two-chain modified BoNT / A comprising (or consisting of) a light chain comprising SEQ ID NO: 11 or 12 (preferably SEQ ID NO: 11) and a heavy chain comprising SEQ ID NO: 13, wherein the light chain and heavy chain are linked together by a disulfide bond.

36. The modified BoNT / A for use, method, or use of any of the preceding claims, wherein the modified BoNT / A has a safety ratio greater than 7, wherein the safety ratio is calculated as: toxin dose required for -10% body weight change measured in pg / mouse divided by DAS ED measured in pg / mouse 50 , where ED 50 = the dose required to produce a DAS score of 2.

37. The modified BoNT / A for use, method, or use of any preceding claim, wherein the affected neck muscle is selected from the group consisting of: sternocleidomastoid, sternocleidomastoideus, splenius capitis, splenius cervicalis, scalene complex (e.g., anterior scalene and / or middle scalene), trapezius (e.g., superior trapezius), levator scapulae, semispinalis capitis, longissimus (e.g., longissimus capitis and / or longissimus cervicalis), posterior paraspinal muscles and submental complex (e.g., digastric, geniohyoid, mylohyoid, mylohyoid buttonhole, and / or stylohyoid), lower trapezius, superior nuchal-clavicularis Bone (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 of the capitis, spinous process C2—transverse process C1, suprasternal notch and clavicle (medial part)—mastoid process and superior nuchal line, transverse process C1-C4—scapula (superior scapular angle), transverse process C2-C7—first rib, transverse process C3-C6—first rib, longus capitis, transverse process C3-C6—occipital bone (basal part), longus colli, transverse process C2-C5—atlas (anterior tubercle).

38. The modified BoNT / A for use, method, or use of any preceding claim, wherein the affected neck muscle is selected from the group consisting of: sternocleidomastoid (e.g., left or right sternocleidomastoid), splenius capitis (e.g., left or right splenius capitis), anterior scalene, middle scalene, trapezius (e.g., left or right trapezius, such as left or right upper trapezius), levator scapulae (e.g., left or right levator scapulae), semispinalis capitis (e.g., left or right semispinalis capitis), middle semispinalis capitis, longissimus (e.g., left or right longissimus capitis and / or longissimus cervicis), splenius cervicis, scalene complex (e.g., anterior scalene and / or middle scalene), posterior paraspinal muscles (e.g., posterior scalene, middle scalene and / or anterior scalene, preferably posterior scalene), submental complex (e.g., digastric, geniohyoid, mylohyoid, mylohyoid buttonhole, and / or stylohyoid), trapezius Lower part, upper nuchal line - clavicle (outer part), spinous process C3-Th3-mastoid process, spinous process Th3-Th5-transverse process C1-C2, transverse process C3-Th6, spinous process C3-Th1-upper 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 of the head, superior oblique muscle of the head, spinous process C2-transverse process C1, upper sternum Notch and clavicle (medial part)—mastoid process and superior nuchal line, transverse processes C1-C4—scapula (superior scapular angle), transverse processes C2-C7—first rib, transverse processes C3-C6—first rib, longus capitis, transverse processes C3-C6—occipital bone (basal part), longus colli, semispinalis cervicis, spinalis capitis, rectus capitis posterior major, rectus capitis posterior minor, rectus capitis anterior, multifidus, transverse processes C2-C5—atlas (anterior tubercle).

39. The modified BoNT / A for use, method, or use of any preceding claim, wherein the affected neck muscles comprise sternocleidomastoid (e.g., left or right sternocleidomastoid), splenius capitis (e.g., left or right splenius capitis), anterior scalene, middle scalene, trapezius (e.g., left or right trapezius, such as left or right upper trapezius), levator scapulae (e.g., left or right levator scapulae), semispinalis capitis (e.g., left or right semispinalis capitis), semispinalis capitis internalis, longissimus (e.g., left or right longissimus capitis and / or longissimus cervix), splenius cervix, scalene complex (e.g., anterior scalene and / or middle scalene), posterior paraspinal muscles (e.g., posterior scalene, middle scalene and / or anterior scalene, preferably posterior scalene), submental complex (e.g., digastric, geniohyoid, mylohyoid, mylohyoid buttonhole, and / or stylohyoid), Quadratus inferior, superior nuchal line—clavicle (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, 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 of C1-C4 - scapula (superior scapular angle), transverse processes of C2-C7 - first rib, transverse processes of C3-C6 - first rib, longus capitis, transverse processes of C3-C6 - occipital bone (basal part), longus colli, semispinalis cervicis, spinalis capitis, rectus capitis posterior major, rectus capitis posterior minor, rectus capitis anterior, multifidus or transverse processes of C2-C5 - atlas (anterior tubercle).

40. The modified BoNT / A for use, method, or use of any preceding claim, wherein the modified BoNT / A is administered to one or more affected neck muscles selected from or comprising the semispinalis cervicis, levator scapulae, splenius cervicis, longissimus cervicis, trapezius (e.g., lower trapezius), sternocleidomastoid, semispinalis capitis, oblique capitis inferior, longissimus capitis, splenius capitis, scalene muscles (e.g., anterior, intermediate, and / or posterior scalene muscles), longus colli, and / or longus capitis.

41. The modified BoNT / A for use, method, or use according to any one of the preceding claims, wherein the modified BoNT / A is administered to a plurality of affected neck muscles of an individual by intramuscular injection, and wherein at least a single unit dose is administered to each affected neck muscle, Preferably, the plurality of affected neck muscles are selected from the group consisting of: sternocleidomastoid, sternocleidomastoid, splenius capitis, splenius cervicalis, scalene complex (e.g., anterior scalene and / or middle scalene), trapezius (e.g., superior trapezius), levator scapulae, semispinalis capitis, longissimus (e.g., longissimus capitis and / or longissimus cervicalis), posterior paraspinal muscles and submental complex (e.g., digastric, geniohyoid, mylohyoid, mylohyoid buttonhole and / or stylohyoid), lower trapezius, superior nuchal line-clavicle (lateral portion), 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 process, transverse process Th1-Th6—transverse process C2-C6, inferior oblique muscle of the capitis, spinous process C2—transverse process C1, suprasternal notch and clavicle (medial part)—mastoid process and superior nuchal line, transverse process C1-C4—scapula (superior scapular angle), transverse process C2-C7—first rib, transverse process C3-C6—first rib, longus capitis, transverse process C3-C6—occipital bone (basal part), longus colli, transverse process C2-C5—atlas (anterior tubercle).

42. The modified BoNT / A for use, method, or use of any preceding claim, wherein the modified BoNT / A is administered to a plurality of affected neck muscles of a subject by intramuscular injection, and wherein at least a single unit dose is administered to each affected neck muscle, wherein the plurality of affected neck muscles is selected from the group consisting of: sternocleidomastoid (e.g., left or right sternocleidomastoid), splenius capitis (e.g., left or right splenius capitis), scalene anterior, scalene middle, trapezius (e.g., left or right trapezius, such as left or right upper trapezius), levator scapulae (e.g., left or right levator scapulae), semispinalis capitis (e.g., left or right semispinalis capitis), semispinalis capitis middle, longissimus (e.g., left or right longissimus capitis and / or longissimus cervicis), splenius cervicis, scalene complex (e.g., anterior scalene and / or middle scalene), posterior paraspinal muscles (e.g., posterior scalene, middle scalene and / or anterior scalene, preferably posterior scalene), submental complex (e.g., digastric, Geniohyoid, mylohyoid, mylohyoid buttonhole and / or stylohyoid), lower part of trapezius, superior nuchal line-clavicle (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 capitis, head Oblique muscles, spinous process C2—transverse process C1, suprasternal notch and clavicle (medial part)—mastoid process and superior nuchal line, transverse processes C1-C4—scapula (superior scapular angle), transverse processes C2-C7—first rib, transverse processes C3-C6—first rib, longus capitis, transverse processes C3-C6—occipital bone (basal part), longus colli, semispinalis cervicis, spinalis capitis, rectus capitis posterior major, rectus capitis posterior minor, rectus capitis anterior, multifidus, transverse processes C2-C5—atlas (anterior tubercle).

43. The modified BoNT / A for use, method, or use of any preceding claim, wherein the modified BoNT / A is administered to a plurality of affected neck muscles of a subject by intramuscular injection, and wherein at least a single unit dose is administered to each affected neck muscle, wherein the plurality of affected neck muscles comprises: Sternocleidomastoid (e.g., left or right sternocleidomastoid), splenius capitis (e.g., left or right splenius capitis), anterior scalene, middle scalene, trapezius (e.g., left or right trapezius, such as left or right upper trapezius), levator scapulae (e.g., left or right levator scapulae), semispinalis capitis (e.g., left or right semispinalis capitis), middle part of semispinalis capitis, longissimus (e.g., left or right longissimus capitis and / or longissimus cervicis), splenius cervicis, scalene complex (e.g., anterior scalene and / or middle scalene), posterior paraspinal muscles (e.g., posterior scalene, middle scalene and / or anterior scalene, preferably posterior scalene), submental complex (e.g., digastric, geniohyoid, mylohyoid, mylohyoid buttonhole and / or stylohyoid), lower part of trapezius, superior nuchal line-clavicle (lateral part), spinous process C3-Th3- Mastoid process, spinous processes Th3-Th5—transverse processes C1-C2, transverse processes C3-Th6, spinous processes 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 (basal part), longus colli, semispinalis cervicis, spinalis capitis, rectus capitis posterior major, rectus capitis posterior minor, rectus capitis anterior, multifidus and / or transverse processes C2-C5—atlas (anterior tubercle).

44. The modified BoNT / A for use, method, or use according to any preceding claim, wherein a single unit dose of the modified BoNT / A is administered to one or more affected neck muscles selected from a first group comprising: the splenius cervicis, the inferior oblique capitis, the semispinalis cervicis, the scalene muscles (e.g., the anterior scalene, the middle scalene, and / or the posterior scalene), the longissimus capitis, the longus colli, and / or the longus capitis; and / or a single or multiple unit doses of the modified BoNT / A are administered to one or more affected neck muscles selected from a second group comprising: the splenius capitis, the longissimus cervicis, the trapezius muscles (e.g., the descending trapezius), the sternocleidomastoid, the semispinalis capitis, and / or the levator scapulae.

45. The modified BoNT / A for use, method, or use according to any preceding claim, wherein a single unit dose of the modified BoNT / A is administered to one or more affected neck muscles selected from a first group comprising: the splenius cervicis muscle, the oblique capitis inferior muscle, the semispinalis cervicis muscle, the scalene muscles (e.g., the anterior scalene, the middle scalene, and / or the posterior scalene), the longissimus capitis muscle, the longus colli muscle, and / or the longus capitis muscle; and / or multiple unit doses (preferably two unit doses) of the modified BoNT / A are administered to one or more affected neck muscles selected from a second group comprising: the splenius capitis muscle, the longissimus cervicis muscle, the trapezius muscles (e.g., the lower trapezius muscle), the sternocleidomastoid muscle, the semispinalis capitis muscle, and / or the levator scapulae muscle.

46. ​​The modified BoNT / A for use, method or use according to claim 44 or 45, wherein the unit dose is: (a) 20,000 pg to 30,000 pg, e.g., 24,000 to 26,000 pg (preferably 25,000 pg) of a modified BoNT / A; or (b) 30,000 pg to a maximum of 40,000 pg, such as 35,000 pg to 37,000 pg, (preferably 36,000 pg) of the modified BoNT / A.

47. The modified BoNT / A for use, method or use according to any one of claims 44-46, wherein the total number of unit doses administered during the treatment period is at most 10 unit doses or is at most 7 unit doses.

48. The modified BoNT / A for use, method, or use according to any one of the preceding claims, wherein the modified BoNT / A is administered at the affected neck muscle in a unit dose per injection site, or wherein the modified BoNT / A is administered in less than a unit dose per injection site.

49. The modified BoNT / A for use, method or use according to any one of the preceding claims, wherein a single unit dose is administered at multiple injection sites in the affected neck muscles and / or two or more unit doses are administered at multiple injection sites in the affected neck muscles.

50. The modified BoNT / A for use, method, or use according to any one of the preceding claims, wherein a single unit dose is administered at the affected neck muscle (eg, a single unit dose is administered at each affected neck muscle).

51. The modified BoNT / A for use, method, or use according to any one of the preceding claims, wherein the duration of treatment of cervical dystonia is longer than the duration of treatment of an unmodified BoNT / A (e.g., SEQ ID NO: 2, e.g., SEQ ID NO: 2 in di-chain form).

52. A unit dosage form of a modified BoNT / A (e.g., for treating cervical dystonia), comprising: (a) greater than 707 units of a modified BoNT / A, wherein 1 unit corresponds to the calculated median lethal dose (LD50) in mice. 50 ) of a modified BoNT / A; or (b) greater than 17,000 pg of modified BoNT / A; and (c) optionally a pharmaceutically acceptable carrier, excipient, adjuvant and / or salt, The modified BoNT / A comprises a BoNT / A light chain and a translocation domain, and a BoNT / B receptor binding domain (H C domain).

53. A kit comprising: (a) a unit dosage form according to claim 52; and (b) instructions for use of the kit for treating cervical dystonia; and (c) Optional diluent.

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