Treatment of cervical dystonia

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 an affected neck muscle of a subject, wherein the modified BoNT / A is administered in a unit dose of greater than 17,000 pg, wherein at least a single unit dose is administered to the affected neck muscle, wherein the total dose of modified BoNT / A administered during treatment is up to 400,000 pg, wherein the modified BoNT / A comprises a BoNT / A light chain and translocation domain, and a BoNT / B receptor binding domain (H C The present invention also provides corresponding methods, uses, unit dosage forms, and kits.

Description

[Technical field]

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

[0002] Cervical dystonia (also known as spasmodic torticollis) is a chronic, usually severely painful neuromotor disorder that causes affected subjects to involuntarily rotate their neck to the left, right, upwards, and / or downwards. Both agonist and antagonist muscles may contract simultaneously during dystonic movements.

[0003] The disorder usually presents with relatively mild symptoms at onset, such as an unnoticeable head tremor that lasts for several months. Other early / progressive symptoms may include sudden head turns, lifts, and / or tilts. Still other early / progressive symptoms usually include persistent / prolonged involuntary head positioning. Involuntary neck muscle spasms tend to increase in frequency and intensity over time before reaching a plateau. Subjects with cervical dystonia may also experience muscle hypertrophy, neck pain, dysarthria, and / or tremor.

[0004] Symptoms of cervical dystonia may involve all of the subject's neck muscles and may result in changes in head posture. In general, the most common abnormal posture associated with cervical dystonia is a sideways rotation of the head with twisting of the chin towards the shoulder (torticollis). Other abnormal postures associated with cervical dystonia may include anterior neck flexion, where the head tilts forward, posterior neck flexion, where the head tilts backward, or lateral neck flexion, where the head tilts to one side. The head over the shoulder may also move in an anterior sagittal shift (forward translation) or posterior sagittal shift (backward translation). Most commonly, however, cervical dystonia presents with a complex symptom where the subject's head moves at several angles.

[0005] Current treatment options include oral drug therapy (e.g., dopamine blockers), deep brain stimulation, botulinum neurotoxin, and selective surgical denervation of nerves that induce muscle contractions. Traditional oral drug therapy is associated with many serious side effects, while deep brain stimulation and surgical denervation are invasive, carry risk of complications, and / or may be ineffective.

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

[0007] BoNT / A paralyzes the dystonic antagonist muscles, allowing the agonist muscles to move freely. More specifically, BoNT / A selectively inhibits the release of acetylcholine from presynaptic nerve terminals, thereby blocking cholinergic transmission at the neuromuscular junction, which induces muscle contraction and a decrease in muscle tone, and relaxes the injected muscle. However, the duration of action of currently available BoNT / A products is approximately 12-14 weeks, a period during which neurological function returns to normal through the sprouting of new nerve endings and the original symptoms reappear. Thus, to maintain efficacy, injections must be repeated periodically. Thus, given the chronicity of the condition and the need for a long treatment period, the frequency of BoNT / A injections is an important consideration in the treatment of cervical dystonia. Indeed, it affects the direct and indirect medical costs involved for the patient and caregiver, the logistics of the injected drug within the hospital / clinic, and most importantly, the quality of life of the patient.

[0008] Dysport (登録商標)Dysport is approved for the treatment of cervical dystonia with a maximum total dose of 1,000 units per treatment session (see Figure 1). Clinicians may administer up to a maximum upper threshold total of 1,000 units of Dysport per treatment session. (登録商標) In order to achieve this, a clinician must administer a dose of BoNT / A to the neck muscles of the subject. Clinicians are faced with difficult choices during the treatment of patients. In other words, in conventional treatment plans, clinicians must find a balance between the relatively low total amount of BoNT / A that can be administered (1,000 units - necessary due to the high toxicity of BoNT / A) and the effective amount in multiple different muscles. Thus, certain muscles are neglected while others receive a suboptimal amount of BoNT / A, resulting in a suboptimal treatment.

[0009] Furthermore, conventional cervical dystonia treatment regimens are complex, leading clinicians to under-dosage to avoid patient toxicity. Thus, there is a need for a simple, safe, and effective single-dose unit that does not cause patient toxicity during a treatment session, and a guide to the number of units that can be administered to the affected neck muscles (including, for example, the number of injection sites per muscle).

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

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

[0012] The present inventors have surprisingly found that modified BoNT / A is particularly useful for treating cervical dystonia. The modified BoNT / A 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 Cdomain), which may increase retention of the modified BoNT / A at the site of administration (by reducing diffusion) and / or increase duration of action (eg, 6-9 months).

[0013] Advantageously, the modified BoNT / A is a clonal antibody that is more effective than unmodified BoNT / A (e.g., Dysport (登録商標) ), which may be expressed by the high safety margins described herein for the modified BoNT / A.

[0014] Based on the preclinical and clinical data herein (see Examples), it is believed that higher total amounts of modified BoNT / A are more effective than unmodified BoNT / A (e.g., Dysport). (登録商標) Advantageously, the clinical data herein (see Example 9) demonstrates that a higher unit dose and total amount of modified BoNT / A can be administered to subjects while achieving a safety profile similar to that of unmodified BoNT / A (e.g., Dysport). (登録商標) It has been shown that such high doses can be administered to subjects while achieving a safety profile similar to that of unmodified BoNT / A. Thus, in treating cervical dystonia, more modified BoNT / A can be injected and / or a greater number of neck muscles / sites can be injected before the maximum total dose is reached. This is an important and advantageous finding, providing clinicians with a wider range of treatment options while resulting in improved treatment of cervical dystonia. The treatment may be improved in that it provides longer-lasting treatment (resulting in less frequent administration) and / or can be individualized to the subject and / or results in improved quality of life for the subject compared to treatment with unmodified BoNT / A (e.g., Dysport®). Thus, the treatment of the present invention is improved compared to conventional treatment regimes.

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

[0016] In one aspect, the invention provides a modified botulinum neurotoxin A (BoNT / A) for use in the treatment of cervical dystonia, wherein the modified BoNT / A is administered to an affected neck muscle of a subject by intramuscular injection; the modified BoNT / A is administered in a unit dose of more than 17,000 pg; at least a single unit dose is administered to the affected neck muscle; the total dose of modified BoNT / A administered during treatment is up to 400,000 pg; and the modified BoNT / A comprises a BoNT / A light chain and translocation domain, and a BoNT / B receptor binding domain (H C domain).

[0017] In a related aspect, the invention provides a modified BoNT / A for use in treating cervical dystonia in a subject for a longer period of time than a subject treated with an unmodified BoNT / A (e.g., SEQ ID NO:2 [e.g., a two-chain form of SEQ ID NO:2]), wherein the modified BoNT / A is administered by intramuscular injection into an affected neck muscle of the subject; The modified BoNT / A is administered in a unit dose of more than 17,000 pg; At least a single unit dose is administered to the affected neck muscle; The total dose of modified BoNT / A administered during treatment is up to 400,000 pg; The modified BoNT / A comprises the BoNT / A light chain and translocation domain, and the BoNT / B receptor binding domain (H Cdomain).

[0018] The term "treating a subject's cervical dystonia for a longer period of time than a subject treated with unmodified BoNT / A" may mean that one or more symptoms of the subject's cervical dystonia are alleviated for a longer period of time (e.g., 6 to 9 months) after administration of the modified BoNT / A of the present invention compared to administration of unmodified BoNT / A. The duration of action may be at least 1.25-fold, 1.5-fold, 1.75-fold, 2.0-fold, or 2.25-fold longer. The duration of action of the modified BoNT / A may be between 6 and 9 months. For example, the duration of action may be at least 4.5 months, 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 (from onset of action). In certain embodiments, the duration of action may be longer than 9.0 months. The reduction can be measured by comparison with a comparable control patient with comparable symptoms treated with unmodified BoNT / A. Subjects treated with modified BoNT / A according to the present invention for a period in which the severity of one or more symptoms of the control patient prior to treatment with unmodified BoNT / A is substantially the same (e.g., the same) can show at least a 5%, 10%, 25%, or 50% improvement in the comparable one or more symptoms compared to the severity of the one or more symptoms prior to treatment with modified BoNT / A. The unmodified BoNT / A is preferably SEQ ID NO: 2, which exists in a double-stranded form.

[0019] In one aspect, the invention provides a method of treating cervical dystonia comprising administering a modified BoNT / A to an affected neck muscle of a subject by intramuscular injection, wherein the modified BoNT / A is administered in a unit dose of greater than 17,000 pg, at least a single unit dose is administered to the affected neck muscle, the total dose of modified BoNT / A administered during treatment is up to 400,000 pg, and the modified BoNT / A comprises a BoNT / A light chain and translocation domain, and a BoNT / B receptor binding domain (H C domain).

[0020] In a related aspect, the invention provides a method for treating cervical dystonia in a subject for a longer period of time than subjects treated with unmodified BoNT / A (e.g., SEQ ID NO:2 [e.g., a di-stranded form of SEQ ID NO:2]). The method comprises administering modified BoNT / A to an affected neck muscle of the subject by intramuscular injection, wherein the modified BoNT / A is administered in a unit dose of greater than 17,000 pg, wherein at least a single unit dose is administered to the affected neck muscle, wherein the total dose of modified BoNT / A administered during treatment is up to 400,000 pg, and wherein the modified BoNT / A comprises a BoNT / A light chain and translocation domain, and a BoNT / B receptor binding domain (H C domain).

[0021] In one aspect, the invention provides the 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 a subject by intramuscular injection, the modified BoNT / A is administered in a unit dose of more than 17,000 pg, at least a single unit dose is administered to the affected neck muscle, the total dose of modified BoNT / A administered during treatment is up to 400,000 pg, and the modified BoNT / A comprises a BoNT / A light chain and translocation domain, and a BoNT / B receptor binding domain (H C domain).

[0022] In a related aspect, the invention provides for the use of a modified BoNT / A in the manufacture of a medicament for treating cervical dystonia in a subject for a longer period of time than a subject treated with an unmodified BoNT / A (e.g., SEQ ID NO:2 [e.g., a di-stranded form of SEQ ID NO:2]), wherein the modified BoNT / A is administered to the affected neck muscle of the subject by intramuscular injection; the modified BoNT / A is administered in a unit dose of greater than 17,000 pg; at least a single unit dose is administered to the affected neck muscle; the total dose of modified BoNT / A administered during treatment is up to 400,000 pg; and the modified BoNT / A comprises a BoNT / A light chain and translocation domain, and a BoNT / B receptor binding domain (H C domain).

[0023] The unit dose of the modified BoNT / A may be greater than 17,000 pg. The upper limit of the unit dose range of the modified BoNT / A may 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, preferably with an upper limit of 38,000 pg. The lower limit of the unit dose range of the modified BoNT / A may 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, preferably the lower limit is 17,500 pg or 25,000 pg. Preferably, the unit dose of the modified BoNT / A is greater than 17,000 pg and up to 40,000 pg, for example, greater than 17,000 pg and up to 36,000 pg, or between 20,000 pg and 39,000 pg. Most preferably, the unit dose of modified BoNT / A is 22,000 to 38,000 pg, for example, 24,000 to 36,000 pg, or 25,000 pg to 36,000 pg. The unit dose of modified BoNT / A may be 25,000 pg to a maximum of 40,000 pg. In a preferred embodiment, the unit dose of modified BoNT / A is 24,000, 25,000, 30,000 or 36,000 pg, for example, 25,000 pg or 36,000 pg. In a more preferred embodiment, the unit dose of modified BoNT / A is 30,000 or 36,000 pg (for example, 36,000 pg).

[0024] The unit dose of the modified BoNT / A can be between 20,000 pg and 30,000 pg, for example, between 24,000 pg and 26,000 pg. Most preferably, the unit dose of the modified BoNT / A is 25,000 pg.

[0025] The unit dose of the modified BoNT / A can be between 30,000 pg and 40,000 pg, for example, between 35,000 pg and 37,000 pg. Most preferably, the unit dose of the modified BoNT / A is 36,000 pg.

[0026] The total dose administered in practicing the treatment regimen of the present invention may be up to 400,000 pg. In other words, the total amount of modified BoNT / A administered in a given treatment session may be up to 400,000 pg. The total dose may 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 of modified BoNT / A may be up to 360,000 pg. 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, Preferably, the total dose of modified BoNT / may be greater than 170,000 pg, more preferably at least 240,000 pg or at least 250,000 pg, such as at least 300,000 pg. The total dose may be 160,000 to 400,000 pg, or 170,000 to 400,000 pg (e.g., greater than 170,000 to 400,000 pg), preferably 170,000 pg to up to 360,000 pg, or 200,000 to 370,000 pg. The total dose administered may be 250,000 pg to 400,000 pg. More preferably, the total dose administered is 250,000 to 360,000 pg. In a preferred embodiment, the total dose of modified BoNT / A is 240,000, 250,000, 300,000 or 360,000 pg (e.g., 250,000 or 360,000 pg).In more preferred embodiments, the total dose is 300,000 or 360,000 pg (e.g., 360,000 pg).

[0027] The total dose of the modified BoNT / A can be between 200,000 pg and 300,000 pg, for example, between 240,000 pg and 260,000 pg. Preferably, the total dose of the modified BoNT / A is up to 250,000 pg (for example, the total dose of the modified BoNT / A can be 250,000 pg).

[0028] The total dose of the modified BoNT / A can be between 300,000 pg and 400,000 pg, for example between 350,000 pg and 370,000 pg. Preferably, the total dose of the modified BoNT / A is up to 360,000 pg (for example, the total dose of the modified BoNT / A can be 360,000 pg).

[0029] Thus, the unit dose of modified BoNT / A may be greater than 17,000 pg, and the total dose administered in carrying out the treatment regimen of the present 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. In another preferred embodiment, the unit dose may be 36,000 pg, and the total dose may be 360,000 pg.

[0030] 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 to an affected neck muscle of a subject by intramuscular injection, and the modified BoNT / A is administered in a unit dose of greater than 707 units, where one unit is greater than or equal to the calculated median lethal dose (LD) in mice. 50), at least a single unit dose is administered to the affected neck muscle, the total dose of modified BoNT / A administered during treatment is up to 16,639 units, and the modified BoNT / A comprises a BoNT / A light chain and translocation domain, and a BoNT / B receptor binding domain (H C domain).

[0031] In a related aspect, the invention provides a modified BoNT / A for use in treating cervical dystonia in a subject for a longer period of time than a subject treated with an unmodified BoNT / A (e.g., SEQ ID NO:2 [e.g., a two-stranded form of SEQ ID NO:2]), wherein the modified BoNT / A is administered to an affected neck muscle of the subject by intramuscular injection, and wherein the modified BoNT / A is administered in a unit dose of greater than 707 units, one unit being greater than or equal to the calculated median lethal dose (LD) in mice. 50 ), at least a single unit dose is administered to the affected neck muscle, the total dose of modified BoNT / A administered during treatment is up to 16,639 units, and the modified BoNT / A comprises a BoNT / A light chain and translocation domain, and a BoNT / B receptor binding domain (H C domain).

[0032] In one aspect, the invention provides a method of treating cervical dystonia, comprising administering modified BoNT / A to an affected neck muscle of a subject by intramuscular injection, wherein the modified BoNT / A is administered in a unit dose of greater than 707 units, where 1 unit is greater than or equal to the calculated median lethal dose (LD) in mice. 50 ), at least a single unit dose is administered to the affected neck muscle, the total dose of modified BoNT / A administered during treatment is up to 16,639 units, and the modified BoNT / A comprises a BoNT / A light chain and translocation domain, and a BoNT / B receptor binding domain (H C domain).

[0033] In a related aspect, the invention provides a method of treating cervical dystonia in a subject for a longer period of time than subjects treated with unmodified BoNT / A (e.g., SEQ ID NO:2 [e.g., the double-stranded form of SEQ ID NO:2]), the method comprising administering modified BoNT / A by intramuscular injection to an affected neck muscle of the subject; Here, the modified BoNT / A was administered in a unit dose of more than 707 units, with one unit being the calculated median lethal dose (LD ) in mice. 50 ), at least a single unit dose is administered to the affected neck muscle, the total dose of modified BoNT / A administered during treatment is up to 16,639 units, and the modified BoNT / A comprises a BoNT / A light chain and translocation domain, and a BoNT / B receptor binding domain (H C domain).

[0034] In one aspect, the invention provides the 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 by intramuscular injection to an affected neck muscle of a subject, and the modified BoNT / A is administered in a unit dose of greater than 707 units, one unit being greater than or equal to the calculated median lethal dose (LD ) in mice. 50 ), at least a single unit dose is administered to the affected neck muscle, the total dose of modified BoNT / A administered during treatment is up to 16,639 units, and the modified BoNT / A comprises a BoNT / A light chain and translocation domain, and a BoNT / B receptor binding domain (H C domain).

[0035] In a related aspect, the invention provides for the use of a modified BoNT / A in the manufacture of a medicament for treating cervical dystonia in a subject for a longer period of time than a subject treated with an unmodified BoNT / A (e.g., SEQ ID NO:2 [e.g., a double-stranded form of SEQ ID NO:2]), wherein the modified BoNT / A is administered by intramuscular injection to an affected neck muscle of the subject, and wherein the modified BoNT / A is administered in a unit dose of greater than 707 units, one unit being greater than or equal to the calculated median lethal dose (LD) in mice. 50), at least a single unit dose is administered to the affected neck muscle, the total dose of modified BoNT / A administered during treatment is up to 16,639 units, and the modified BoNT / A comprises a BoNT / A light chain and translocation domain, and a BoNT / B receptor binding domain (H C domain).

[0036] The unit dose of the modified BoNT / A can be greater than 707 units. The upper limit of the unit dose range of the modified BoNT / A can be 1664, 1650, 1600, 1550, 1500, 1450, 1400, 1350, 1300, 1250, 1150, 1100, 1050, 1000, 950, 900, 850, 800 or 750 units, preferably the upper limit is 1500 units. The lower limit of the unit dose range of the modified BoNT / A may 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, preferably the lower limit is 728 units or 1,040 units. Preferably, the unit dose of the modified BoNT / A is greater than 707 units up to 1664 units, for example, greater than 707 units up to 1498 units, or 832 units to 1622 units. Most preferably, the unit dose of the modified BoNT / A is 915 to 1581 units, for example, 998 to 1498 units. The unit dose of modified BoNT / A can be from 1,040 units up to 1,664 units. In preferred embodiments, the unit dose of modified BoNT / A contains 998, 1,248, 1,040 or 1,498 units, e.g., 1,040 or 1,498 units, of modified BoNT / A. In more preferred embodiments, the unit dose contains 1,248 or 1,498 units, e.g., 1,248 units, of modified BoNT / A.

[0037] The unit dose of the modified BoNT / A can be between 832 and 1,248 units, for example, between 998 and 1,082 units. Most preferably, the unit dose of the modified BoNT / A is 1,040 units.

[0038] The unit dose of the modified BoNT / A can be between 1,248 and 1,664 units, for example, between 1,456 and 1,539 units. Most preferably, the unit dose of the modified BoNT / A is 1,498 units.

[0039] The total dose administered in carrying out the treatment regimen of the present invention may be up to 16,639 units. In other words, the total amount of modified BoNT / A administered in a given treatment session may be up to 16,639 units. The total dose may 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 of modified BoNT / A may be up to 14,975 units. 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 may 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 of modified BoNT / A is greater than 7,072 units, more preferably at least 9,983 units, or 10,399 units, for example at least 12,479 units. The total dose may be 6,656 to 16,639 units, or 7,072 to 16,639 units (e.g., greater than 7,072 units, up to 16,639 units), preferably 7,072 units up to 14,975 units, or 8,319 to 15,391 units. More preferably, the total dose administered is 9,983 to 14,975 units. The total dose administered may be 10,399 to 16,639 units. In a preferred embodiment, the total dose is 9,983, 10,399, 12,479 or 14,975 units (e.g., 10,399 or 14,975 units).In a more preferred embodiment, the total dose is 12,479 or 14,975 units pg (eg, 14,975 units).

[0040] The total dose of the modified BoNT / A can be between 8,319 and 12,479 units, for example, between 9,983 and 10,815 units. Preferably, the total dose is up to 103,999 units (for example, the total dose can be 103,999 units).

[0041] The total dose of modified BoNT / A can be between 12,479 units and 16,639 units, for example, between 14,559 units and 15,391 units. Preferably, the total dose is up to 14,975 units (for example, the total dose can be 14,975 units).

[0042] Thus, the unit dose of modified BoNT / A may be greater than 707 units, and the total dose administered in practicing the treatment regimen of the present invention may be up to 16,639 units. In a preferred embodiment, the unit dose of modified BoNT / A may be 998 units, and the total dose may be 9,983 units. In another preferred embodiment, the unit dose of modified BoNT / A may be 1,248 units, and the total dose may be 12,479 units. In another preferred embodiment, the unit dose of modified BoNT / A may be 1,498 units, and the total dose may be 14,975 units.

[0043] An "affected neck muscle" may be a neck muscle that contributes to (e.g., causes) the subject's cervical dystonia and / or symptoms thereof, or that contributes to (e.g., causes) the subject's cervical dystonia and / or symptoms thereof. It is not intended that an "affected neck muscle" necessarily must contribute to (e.g., causes) the subject's cervical dystonia and / or symptoms thereof when treated, although this is preferred. For example, the neck muscle may be one that has contributed to (e.g., caused) the subject's cervical dystonia and / or symptoms thereof in the past, or is expected to contribute to (e.g., cause) the subject's cervical dystonia and / or symptoms thereof in the future. In one embodiment, more than one neck muscle (e.g., a pair of agonist and antagonist neck muscles) may contribute to (e.g., cause) the subject's cervical dystonia and / or symptoms thereof. In such a case, modified BoNT / A may be administered to more than one neck muscle (e.g., a pair of agonist and antagonist neck muscles of a neck muscle).

[0044] The affected neck muscle is preferably a neck muscle that, upon contraction, contributes to (e.g., causes) the subject's cervical dystonia and / or symptoms thereof. Thus, the affected neck muscle is preferably a neck muscle of the subject that is contracted or that, upon contraction, results in cervical dystonia and / or symptoms thereof in the subject. Preferably, the neck muscle is a neck muscle that contracts or has contracted involuntarily, e.g., during treatment. The neck muscle may be any muscle (e.g., skeletal muscle) operably connected to the neck and / or head of the subject, e.g., any muscle that can alter the positioning of the subject's head (e.g., when contracted). The affected neck muscles may be capable of the following movements: twisting the subject's chin toward the subject's shoulder and rotating the head sideways (torticollis); tilting the subject's head forward (anteflexion); tilting the subject's head backward (retroflexion); tilting the subject's head sideways (lateral cervical flexion); performing an anterior sagittal shift (forward translation) of the subject's head; and / or performing a posterior sagittal shift (backward translation) of the subject's head.

[0045] Affected neck muscles may include the sternocleidomastoid, sternocleidomastoideus, 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, or longissimus (e.g., longissimus capitis and / or longissimus cervicis). Affected neck muscles may include: 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, longissimus (e.g., longissimus capitis and / or longissimus cervix), posterior paravertebral (e.g., posterior scalene, middle scalene, and / or anterior scalene, preferably posterior scalene), submandibular complex (e.g., digastric, geniohyoid, mylohyoid, mylohyoid boutonniere, and / or stylohyoid), superior nuchal line-clavicular (lateral portion), spinous processes C3-T h3-mastoid, spinous process Th3-Th5-transverse process C1-C2, transverse process C3-Th6, spinous process C3-Th1-superior nuchal line, transverse process Th1-Th6-spinous process C2-C7, transverse process C3-Th3-mastoid, transverse process Th1-Th6-transverse process C2-C6, inferior oblique muscle, spinous process C2- Transverse process C1, cervical notch and clavicle (medial part) - mastoid process and superior nuchal line, transverse process C1 - C4 - scapula (superior corner), transverse process C2 - C7 - 1st rib, transverse process C3 - C6 - 1st rib, longus capitis muscle, transverse process C3 - C6 - occipital bone (base), longus carpis muscle or transverse process C2 - C5 - atlas (anterior tuberosity). Affected neck muscles can 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: sternocleidomastoid (e.g., left or right sternocleidomastoid), left or right splenius capitis, anterior or middle scalene, left or right trapezius (e.g., left or right right trapezius), left or right levator scapulae, left or right semispinalis capitis, longissimus (e.g., left or right longissimus capitis and / or longissimus cervix), splenius cervix, scalene complex (e.g., anterior and / or middle scalene), posterior paravertebral (e.g., posterior scalene, middle scalene, and / or anterior scalene, preferably posterior scalene), submandibular complex (e.g., digastric, geniohyoid, mylohyoid, mylohyoid boutonniere, and / or stylohyoid), lower trapezius, superior nuchal line- Clavicle (lateral part), spinous process C3-Th3-mastoid process, spinous process Th3-Th5-transverse process C1-C2, transverse process C3-Th6, spinous process C3-Th1-superior nuchal line, transverse process Th1-Th6-spinous process C2-C7, transverse process C3-Th3-mastoid process, transverse process Th1-Th6-transverse process C2-C6, inferior oblique muscle, superior oblique muscle, spinous process C2-transverse process C1, cervical notch and clavicle (medial part)-mastoid process and superior nuchal line, transverse process C1-C4-scapula (superior angle), transverse process C2-C7-1st rib, transverse process C3-C6-1st rib, longus capitis, transverse process C3-C6-occipital bone (base), longus colli, semispinalis cervix, rectus capitis posterior greater, rectus capitis posterior less, rectus capitis anterior, multifidus, or transverse process C2-C5-atlas (anterior tubercle).

[0046] Affected neck muscles may include: sternocleidomastoid (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 right upper trapezius), levator scapulae (e.g., left or right levator scapulae), semispinalis capitis (e.g., left or right semispinalis capitis), semispinalis capitis intermediate, 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 paravertebral (e.g., posterior scalene, middle scalene, and / or anterior scalene, preferably posterior scalene), submandibular complex (e.g., digastric, geniohyoid, mylohyoid, mylohyoid boutonniere, and / or pedicle). prohyoid muscle), lower trapezius muscle, superior nuchal line - clavicle (lateral part), spinous process C3 - Th3 - mastoid process, spinous process Th3 - Th5 - transverse process C1 - C2, transverse process C3 - Th6, spinous process C3 - Th1-superior nuchal line, transverse process Th1-Th6-spinous process C2-C7, transverse process C3-Th3-mastoid process, transverse process Th1-Th6-transverse process C2-C6, inferior oblique muscle, superior oblique muscle, spinous process C2-transverse process C1, cervical notch and clavicle (medial part)-mastoid process and superior nuchal line, transverse process C1-C4-scapula (superior angle), transverse process C2-C7-1st rib, transverse process C3-C6-1st rib, longus capitis, transverse process C3-C6-occipital bone (base), longus colli, semispinalis cervix, spinalis capitis, rectus capitis posterior greater, rectus capitis posterior less, rectus capitis anterior, multifidus, or transverse process C2-C5-atlas (anterior tubercle).

[0047] Most preferably, the affected neck muscles may include: semispinalis cervix, levator scapulae, splenius cervix, longissimus cervix, trapezius (e.g., lower trapezius), sternocleidomastoid, semispinalis capitis, obliquus capitis inferior, longissimus capitis, splenius capitis, scalene (e.g., anterior, middle, and / or posterior scalene), longus colli, or longus capitis. For example, the affected neck muscles include or are one or more selected from the group consisting of: semispinalis cervix, levator scapulae, splenius cervix, longissimus cervix, trapezius (e.g., lower trapezius), sternocleidomastoid, semispinalis capitis, obliquus capitis inferior, longissimus capitis, splenius capitis, scalene (e.g., anterior, middle, and / or posterior scalene), longus colli, and longus capitis.

[0048] The multiple affected neck muscles treated in accordance with the present invention may include at least one (eg, at least two) of any of the muscles described herein.

[0049] In one embodiment, the modified BoNT / A may be administered to one or more affected neck muscles selected from the following: sternocleidomastoid, splenius capitis, splenius cervicis, 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 cervicis). Preferably, the modified BoNT / A is administered to multiple affected neck muscles. For example, the 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 following: 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, and longissimus (e.g., longissimus capitis and / or longissimus cervicis).

[0050] In one embodiment, the modified BoNT / A may be administered to one or more affected neck muscles selected from the following: right levator scapulae, left levator scapulae, right trapezius, left trapezius, right sternocleidomastoid, left sternocleidomastoid, right splenius capitis, left splenius capitis, middle scalene, anterior scalene, right semispinalis capitis, left semispinalis capitis, right longissimus capitis, and left longissimus capitis.

[0051] In one embodiment, the modified BoNT / A may be administered to one or more affected neck muscles selected from the following: sternocleidomastoid, sternocleidomastoideus, splenius capitis, splenius cervix, scalene complex (e.g., anterior scalene and / or middle scalene), trapezius (e.g., upper trapezius and / or lower trapezius), levator scapulae, semispinalis capitis, longissimus (e.g., longissimus capitis and / or longissimus cervix), posterior paravertebral (e.g., posterior scalene, middle scalene, and / or anterior scalene, preferably posterior scalene), submandibular complex (e.g., digastric, geniohyoid, mylohyoid, mylohyoid boutonniere, and / or stylohyoid), superior nuchal line-clavicular (lateral portion), spinous process C3- Th3-mastoid, spinous processes Th3-Th5-transverse processes C1-C2, transverse processes C3-Th6, spinous process C3-Th1-superior nuchal line, transverse processes Th1-Th6-spinous processes C2-C7, transverse processes C3-Th3-mastoid, transverse processes Th1-Th6-transverse processes C2-C6, oblique capitis inferior, spinous process C2-transverse process C1, cervical notch and clavicle (medial)-mastoid and superior nuchal line, transverse processes C1-C4-scapula (superior angle), transverse processes C2-C7-first rib, transverse processes C3-C6-first rib, longus capitis, transverse processes C3-C6-occipital bone (base), longus colli, and transverse processes C2-C5-atlas (anterior tubercle). Preferably, the modified BoNT / A is administered to multiple 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 following: sternocleidomastoid, sternocleidomastoideus), splenius capitis, splenius cervix, scalene complex (e.g., anterior scalene and / or middle scalene), trapezius (e.g., upper trapezius and / or lower trapezius), levator scapulae, semispinalis capitis, longissimus (e.g., longissimus capitis and / or longissimus cervix), posterior paravertebral area (e.g., posterior scalene, middle scalene, and / or anterior scalene, preferably posterior scalene), submandibular complex (e.g., digastric, geniohyoid, mylohyoid, mylohyoid boutonniere, and / or stylohyoid), superior nuchal line-clavicle (lateral area), spinous process C3- Th3-mastoid, spinous process Th3-Th5-transverse process C1-C2, transverse process C3-Th6, spinous process C3-Th1-superior nuchal line, transverse process Th1-Th6-spinous process C2-C7, transverse process C3-Th3-mastoid, transverse process Th1-Th6-transverse process C2-C6, inferior oblique muscle, spinous process C2- Transverse process C1, cervical notch and clavicle (medial part) - mastoid process and superior nuchal line, transverse process C1 - C4 - scapula (superior angle), transverse process C2 - C7 - 1st rib, transverse process C3 - C6 - 1st rib, longus capitis muscle, transverse process C3 - C6 - occipital bone (bottom), longus neck muscle, and transverse process C2 - C5 - atlas (anterior tubercle).

[0052] In one embodiment, the modified BoNT / A may be administered to one or more affected muscles of the neck, 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 right upper trapezius), levator scapulae (e.g., left or right levator scapulae), semispinalis capitis (e.g., left or right semispinalis capitis), semispinalis capitis intermediate, 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 paravertebral (e.g., posterior scalene, middle scalene, and / or anterior scalene, preferably posterior scalene), submandibular complex (e.g., digastric, geniohyoid, mylohyoid, mylohyoid boot ... Nierere and / or stylohyoid muscle), lower trapezius muscle, superior nuchal line-clavicle (lateral part), spinous process C3-Th3-mastoid process, spinous process Th3-Th5-transverse process C1-C2, transverse process C3-T h6, spinous process C3-Th1-superior nuchal line, transverse process Th1-Th6-spinous process C2-C7, transverse process C3-Th3-mastoid, transverse process Th1-Th6-transverse process C2-C6, inferior oblique muscle, superior oblique muscles, spinous process C2-transverse process C1, cervical notch and clavicle (medial)-mastoid and superior nuchal line, transverse process C1-C4-scapula (superior angle), transverse process C2-C7-first rib, transverse process C3-C6-first rib, longus capitis, transverse process C3-C6-occipital bone (base), longus colli, semispinalis cervix, spinalis capitis, rectus capitis posterior greater, rectus capitis posterior less, rectus capitis anterior, multifidus, and / or transverse process C2-C5-atlas (anterior tubercle). Preferably, the modified BoNT / A is administered to multiple affected neck muscles.For example, a modified BoNT / A 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, including: sternocleidomastoid (e.g., left or right sternocleidomastoid), splenius capitis (e.g., left or right splenius capitis), or sternocleidomastoid (e.g., right or left splenius capitis). scapulae (e.g., left or right semispinalis capitis), semispinalis capitis middle, longissimus capitis (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 (e.g., posterior scalene, middle scalene, and / or anterior scalene, preferably posterior scalene), submandibular complex (e.g., digastric, geniohyoid, mylohyoid, mylohyoid boutonniere, and / or stylohyoid), lower trapezius, superior nuchal line-clavicle (lateral portion), spinous process C3-Th3-mastoid, spinous process Th3-Th5-transverse process C1-C2, transverse process C3-Th6, spinous process C3-Th1-superior nuchal line, transverse process Th1-Th6-spinous process C2-C7, transverse process C3-Th3-mastoid, transverse process Th1-Th6-transverse process C2-C6, inferior oblique muscle, superior oblique muscle, spinous process C2-transverse process C1, cervical notch and clavicle (medial part)-mastoid process and superior nuchal line, transverse process C1-C4-scapula (superior angle), transverse process C2-C7-1st rib , transverse process C3-C6 - first rib, longus capitis, transverse process C3-C6 - occipital (bottom), longus cervix, semispinalis cervicis, spinae capitis, rectus capitis major, rectus occipital minor, rectus capitis frontalis, multifidus, and / or transverse process C2-C5 - atlas (anterior tuberosity).

[0053] In one embodiment, the modified BoNT / A may be administered to one or more affected neck muscles selected from the following: sternocleidomastoid (e.g., left 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 right upper trapezius), levator scapulae (e.g., left or right levator scapulae), semispinalis capitis (e.g., left or right semispinalis capitis), semispinalis capitis intermediate, 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 paravertebral (e.g., posterior scalene, middle scalene, and / or anterior scalene, preferably posterior scalene), submandibular complex (e.g., digastric, geniohyoid, mylohyoid, geniohyoid, hyoglossus ... bone boutonniere and / or stylohyoid muscle), lower trapezius muscle, superior nuchal line-clavicle (lateral part), spinous process C3-Th3-mastoid process, spinous process Th3-Th5-transverse process C1-C2, transverse process C3-Th6, spinous process C3-Th1-superior nuchal line, transverse process Th1-Th6-spinous process C2-C7, transverse process C3-Th3-mastoid process, transverse process Th1-Th6-transverse process C2-C6, oblique head inferior muscle, oblique capitis superior, spinous process C2-transverse process C1, cervical notch and clavicle (medial)-mastoid and superior nuchal line, transverse process C1-C4-scapula (superior angle), transverse process C2-C7-first rib, transverse process C3-C6-first rib, longus capitis, transverse process C3-C6-occipital bone (base), longus colli, semispinalis cervix, spinalis capitis, rectus capitis posterior greater, rectus capitis posterior less, rectus capitis anterior, multifidus, and transverse process C2-C5-atlas (anterior tubercle). Preferably, the modified BoNT / A is administered to multiple 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 following: 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 the 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 intermediate, 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 (e.g., posterior scalene, middle scalene, and / or or anterior scalene, preferably posterior scalene), submandibular complex (e.g., digastric, geniohyoid, mylohyoid, mylohyoid boutonniere, and / or stylohyoid), lower trapezius, superior nuchal line-clavicle (lateral portion), spinous processes C3-Th3-mastoid, 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, Transverse process Th1-Th6-transverse process C2-C6, inferior oblique muscle, superior oblique muscle, spinous process C2-transverse process C1, cervical notch and clavicle (medial part) - mastoid process and superior nuchal line, transverse process C1-C4-scapula (superior angle), transverse process C2-C7-th 1st rib, transverse process C3-C6 - 1st rib, longus capitis muscle, transverse process C3-C6 - occipital bone (bottom), longus cervix muscle, semispinalis cervicis, spinae capitis muscle, rectus capitis major, rectus occipitalis minor, rectus capitis frontalis, multifidus muscle, and transverse process C2-C5 - atlas (anterior tuberosity).

[0054] Most preferably, the modified BoNT / A can be administered to one or more affected neck muscles, including: semispinalis cervix, levator scapulae, splenius cervix, longissimus cervix, trapezius (e.g., lower trapezius), sternocleidomastoid, semispinalis capitis, obliquus capitis inferior, longissimus capitis, splenius capitis, scalene (e.g., anterior, middle, 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 cervix, levator scapulae, splenius cervix, longissimus cervix, trapezius (e.g., lower trapezius), sternocleidomastoid, semispinalis capitis, obliquus capitis inferior, longissimus capitis, splenius capitis, scalene (e.g., anterior, middle, and / or posterior scalene), longus colli, and longus capitis. For example, the modified BoNT / A can be administered to at least two (eg, at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15) of said affected neck muscles.

[0055] In one embodiment, the modified BoNT / A may be administered to one or more affected neck muscles, including: 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. In one embodiment, the modified BoNT / A may be administered to one or more affected neck muscles selected from: right levator scapulae, left levator scapulae, right trapezius, left trapezius, right sternocleidomastoid, left sternocleidomastoid, right splenius capitis, left splenius capitis, middle scalene, anterior scalene, right semispinalis capitis, left semispinalis capitis, right longissimus capitis, and left longissimus capitis.

[0056] In cases where there are two corresponding cervical muscles on either side of the neck (e.g., sternocleidomastoid muscles, such as the left and right sternocleidomastoid muscles), the modified BoNT / A can be administered unilaterally (e.g., to one muscle, if only one is contracting) or bilaterally (e.g., to both muscles, if both muscles are contracting). When treating multiple affected muscles, it is preferred that there are multiple, different types of affected muscles. For example, when bilateral administration is performed to two affected sternocleidomastoid muscles, it is preferred that additional muscles are treated when treating multiple affected muscles as described herein.

[0057] In the case where there are two corresponding neck muscles on either side of the neck, unilateral administration can be to either of the corresponding neck muscles. For example, administration can be to a corresponding muscle that is contracted or not contracted. In one embodiment, unilateral administration is to a side of the subject's neck, where the side of the subject's neck exhibits symptoms of cervical dystonia, or to a corresponding muscle on the opposite side of the subject's neck, where the opposite side of the subject's neck does not exhibit symptoms of cervical dystonia.

[0058] The affected neck muscles selected for treatment according to the invention may vary depending on the specific symptoms of cervical dystonia in the subject to be treated (e.g., torticollis, lateral neck flexion, anterior neck flexion, posterior neck flexion, lateral head flexion, head tilt, anterior head flexion, posterior head flexion, lateral deviation, sagittal shift, or a combination thereof). 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 typical symptoms of cervical dystonia and the associated muscles involved. Thus, the muscle or set of muscles treated according to the invention may be the muscle or set of muscles described in the aforementioned reference Jost & Tatu (2015), which is incorporated herein by reference.

[0059] In one embodiment, when treating torticollis, modified BoNT / A may be administered to one or more affected neck muscles selected from the following: sternocleidomastoid, trapezius (e.g., upper trapezius), scalene anterior, splenius capitis, splenius cervicis, levator scapulae, and longissimus (e.g., longissimus capitis and / or longissimus cervicis). In one embodiment, when treating torticollis, modified BoNT / A may be administered to the contralateral side of one or more affected neck muscles selected from the following: sternocleidomastoid, trapezius (e.g., upper trapezius), and scalene anterior; and / or to the ipsilateral side of one or more affected neck muscles selected from the following: splenius capitis, splenius cervicis, levator scapulae, and longissimus (e.g., longissimus capitis and / or longissimus cervicis).

[0060] In one embodiment, when treating torticollis, modified BoNT / A may be administered to one or more affected neck muscles selected from the following: sternocleidomastoid, trapezius (e.g., upper trapezius), scalene anterior, splenius capitis, splenius cervicis, levator scapulae, longissimus (e.g., longissimus capitis and / or longissimus cervicis), and semispinalis cervicis. In one embodiment, when treating torticollis, modified BoNT / A may be administered to the contralateral side of one or more affected neck muscles selected from the following: sternocleidomastoid, trapezius (e.g., upper trapezius), and scalene anterior; and / or to the ipsilateral side of one or more affected neck muscles selected from the following: splenius capitis, splenius cervicis, levator scapulae, longissimus (e.g., longissimus capitis and / or longissimus cervicis), and semispinalis cervicis.

[0061] In one embodiment, when treating torticollis, modified BoNT / A may be administered to one or more affected neck muscles selected from the following: sternocleidomastoid, trapezius (e.g., upper trapezius), scalene anterior, splenius capitis, splenius cervicis, levator scapulae, longissimus (e.g., longissimus capitis and / or longissimus cervicis), semispinalis cervicis, rectus capitis posterior greater, multifidus, and obliquus capitis inferior. In one embodiment, when treating torticollis, modified BoNT / A may be administered to the contralateral side of one or more affected neck muscles selected from the following: sternocleidomastoid, trapezius (e.g., upper trapezius), scalene anterior, semispinalis cervicis, and multifidus; and / or to the ipsilateral side of one or more affected neck muscles selected from the following: splenius capitis, splenius cervicis, levator scapulae, longissimus (e.g., longissimus capitis and / or longissimus cervicis), rectus capitis posterior greater, and obliquus capitis inferior. In one embodiment, when treating torticollis, modified BoNT / A may be administered to one or more affected neck muscles, including: sternocleidomastoid, trapezius (e.g., upper trapezius), scalene anterior, splenius capitis, splenius cervicis, levator scapulae, longissimus (e.g., longissimus capitis and / or longissimus cervicis), semispinalis cervicis, rectus capitis posterior greater, multifidus, and / or obliquus capitis inferior. In one embodiment, when treating torticollis, modified BoNT / A may be administered contralaterally to one or more affected neck muscles, including: sternocleidomastoid, trapezius (e.g., upper trapezius), scalene anterior, semispinalis cervicis, and / or multifidus; and / or ipsilaterally to one or more affected neck muscles, including: splenius capitis, splenius cervicis, levator scapulae, longissimus (e.g., longissimus capitis and / or longissimus cervicis), rectus capitis posterior greater, and / or obliquus capitis inferior.

[0062] In one embodiment, when treating torticollis, the modified BoNT / A is administered to one or more affected neck muscles selected from or including: semispinalis cervix, levator scapulae, splenius cervix, and / or longissimus cervix. The modified BoNT / A may also be administered ipsilaterally.

[0063] In one embodiment, when treating cervical lateral bending, the modified BoNT / A may be administered to one or more affected neck muscles selected from the following: levator scapulae, trapezius (e.g., upper trapezius), scalene complex (e.g., anterior scalene and / or middle scalene), sternocleidomastoid, splenius capitis, splenius cervix, and longissimus (e.g., longissimus capitis and / or longissimus cervix).

[0064] In one embodiment, when treating cervical lateral bending, the modified BoNT / A may be administered to one or more affected neck muscles selected from the following: levator scapulae, trapezius (e.g., upper trapezius), scalene complex (e.g., anterior scalene and / or middle scalene), sternocleidomastoid, splenius capitis, splenius cervix, longissimus (e.g., longissimus capitis and / or longissimus cervix), and semispinalis cervix.

[0065] In one embodiment, when treating cervical lateral flexion, modified BoNT / A may be administered to one or more affected neck muscles selected from the following: levator scapulae, trapezius (e.g., upper trapezius), scalene complex (e.g., anterior scalene and / or middle scalene), sternocleidomastoid, splenius capitis, splenius cervicis, longissimus (e.g., longissimus capitis and / or longissimus cervicis), and multifidus. In one embodiment, when treating cervical lateral flexion, modified BoNT / A may be administered to one or more affected neck muscles including the following: levator scapulae, trapezius (e.g., upper trapezius), scalene complex (e.g., anterior scalene and / or middle scalene), sternocleidomastoid, splenius capitis, splenius cervicis, longissimus (e.g., longissimus capitis and / or longissimus cervicis), and / or multifidus.

[0066] In one embodiment, when treating cervical lateral bending, the modified BoNT / A may be administered to one or more affected neck muscles selected from the following: levator scapulae, trapezius (e.g., upper trapezius), scalene complex (e.g., anterior scalene and / or middle scalene), sternocleidomastoid, splenius capitis, splenius cervix, longissimus (e.g., longissimus capitis and / or longissimus cervix), semispinalis cervix, and multifidus.

[0067] In one embodiment, when treating cervical lateral bending, the modified BoNT / A may be administered to one or more affected neck muscles, including: levator scapulae, trapezius (e.g., upper trapezius), scalene complex (e.g., anterior scalene and / or middle scalene), sternocleidomastoid, splenius capitis, splenius cervix, longissimus (e.g., longissimus capitis and / or longissimus cervix), semispinalis cervix, and / or multifidus.

[0068] In one embodiment, when treating cervical lateral bending, the modified BoNT / A is administered to one or more affected neck muscles selected from or including: levator scapulae, semispinalis cervix, scalene medius, and / or longissimus cervix.

[0069] Preferably, when treating cervical lateral curvature, the modified BoNT / A can be administered ipsilateral to one or more of the affected neck muscles.

[0070] In one embodiment, when treating cervical flexion, the modified BoNT / A can be administered to one or more affected neck muscles selected from the following: sternocleidomastoid, anterior scalene, and middle scalene.

[0071] In one embodiment, when treating cervical flexion, the modified BoNT / A may be administered to one or more affected neck muscles selected from the following: sternocleidomastoid, anterior scalene, middle scalene, levator scapulae, longus colli, and the submandibular complex (e.g., digastric, geniohyoid, mylohyoid, mylohyoid boutonniere, and / or stylohyoid).

[0072] In one embodiment, when treating neck flexion, the modified BoNT / A may be administered to one or more affected neck muscles selected from the following: sternocleidomastoid, anterior scalene, middle scalene, longus capitis, longus colli, and rectus capitis anterior. In one embodiment, when treating neck flexion, the modified BoNT / A may be administered to one or more affected neck muscles including the following: sternocleidomastoid, anterior scalene, middle scalene, longus capitis, longus colli, and / or rectus capitis anterior.

[0073] In one embodiment, when treating neck flexion, the modified BoNT / A may be administered to one or more affected neck muscles selected from the following: sternocleidomastoid, anterior scalene, middle scalene, levator scapulae, longus colli, submandibular complex (e.g., digastric, geniohyoid, mylohyoid, mylohyoid boutonniere, and / or stylohyoid), longus capitis, and rectus capitis anterior. In one embodiment, when treating neck flexion, the modified BoNT / A may be administered to one or more affected neck muscles selected from the following: sternocleidomastoid, anterior scalene, middle scalene, levator scapulae, longus colli, submandibular complex (e.g., digastric, geniohyoid, mylohyoid, mylohyoid boutonniere, and / or stylohyoid), longus capitis, and / or rectus capitis anterior.

[0074] In one embodiment, when treating cervical flexion, the modified BoNT / A is administered to one or more affected neck muscles selected from or including: scalene middle, levator scapulae, and / or longus colli.

[0075] Preferably, when treating cervical flexion, the modified BoNT / A may be administered bilaterally.

[0076] In one embodiment, when treating cervical retroflexion, the modified BoNT / A may be administered to one or more affected neck muscles selected from the following: levator scapulae, trapezius (e.g., upper trapezius), longissimus (e.g., longissimus capitis and / or longissimus cervix), splenius capitis, splenius cervix, and semispinalis capitis.

[0077] In one embodiment, when treating cervical retroflexion, the modified BoNT / A may be administered to one or more affected neck muscles selected from the following: levator scapulae, trapezius (e.g., upper trapezius), longissimus (e.g., longissimus capitis and / or longissimus cervix), splenius capitis, splenius cervix, semispinalis capitis, semispinalis cervix, and posterior paraspinal (e.g., posterior scalene, middle scalene, and / or anterior scalene, preferably posterior scalene).

[0078] In one embodiment, when treating cervical retroflexion, modified BoNT / A may be administered to one or more affected neck muscles selected from the following: levator scapulae, trapezius (e.g., upper trapezius), longissimus (e.g., longissimus capitis and / or longissimus cervix), splenius capitis, splenius cervix, semispinalis capitis, semispinalis cervix, spinalis capitis, rectus capitis posterior greater, rectus capitis posterior less, and obliquus capitis superior. In one embodiment, when treating cervical retroflexion, modified BoNT / A may be administered to one or more affected neck muscles selected from the following: levator scapulae, trapezius (e.g., upper trapezius), longissimus (e.g., longissimus capitis and / or longissimus cervix), splenius capitis, splenius cervix, semispinalis capitis, semispinalis cervix, spinalis capitis, rectus capitis posterior greater, rectus capitis posterior less, and / or obliquus capitis superior.

[0079] In one embodiment, when treating cervical retroflexion, the modified BoNT / A may be administered to one or more affected neck muscles selected from the following: levator scapulae, trapezius (e.g., upper trapezius), longissimus (e.g., longissimus capitis and / or longissimus cervix), splenius capitis, splenius cervix, semispinalis capitis, semispinalis cervix, posterior paraspinal (e.g., posterior scalene, middle scalene, and / or anterior scalene, preferably posterior scalene), spinalis capitis, rectus capitis posterior greater, rectus capitis posterior less, and obliquus capitis superior. In one embodiment, when treating cervical retroflexion, the modified BoNT / A may be administered to one or more affected neck muscles, including: levator scapulae, trapezius (e.g., upper trapezius), longissimus (e.g., longissimus capitis and / or longissimus cervix), splenius capitis, splenius cervix, semispinalis capitis, semispinalis cervix, posterior paraspinal (e.g., posterior scalene, middle scalene, and / or anterior scalene, preferably posterior scalene), spinalis capitis, rectus capitis posterior greater, rectus capitis posterior less, and / or obliquus capitis superior.

[0080] In one embodiment, when treating cervical retroflexion, the modified BoNT / A is administered to at least the semispinalis cervix muscle.

[0081] Preferably, when treating cervical retroversion, the modified BoNT / A may be administered bilaterally.

[0082] When treating the lateral deviation symptoms of cervical dystonia, the muscles selected for administration may be a combination of the muscles targeted for administration when treating cervical lateral flexion (e.g., of a first side) and head lateral flexion (e.g., of a second side).

[0083] In one embodiment, when treating lateral deviation, modified BoNT / A may be administered to one or more affected neck muscles selected from the following: levator scapulae, trapezius (e.g., upper trapezius or lower trapezius), scalene complex (e.g., anterior scalene and / or middle scalene), sternocleidomastoid, splenius capitis, splenius cervix, longissimus (e.g., longissimus capitis and / or longissimus cervix), and semispinalis cervix. Preferably, when treating lateral deviation, modified BoNT / A may be administered to affected neck muscles selected from the following: levator scapulae, semispinalis cervix, middle scalene, and longissimus cervix of a first neck side (e.g., left side). Modified BoNT / A may also be administered to affected muscles selected from sternocleidomastoid, lower trapezius, splenius capitis, semispinalis capitis, longissimus capitis, and levator scapulae of a second neck side (e.g., right side).

[0084] In one embodiment, when treating a lateral deviation, the modified BoNT / A may be administered to one or more affected neck muscles, including: levator scapulae, trapezius (e.g., upper trapezius or lower trapezius), scalene complex (e.g., anterior scalene and / or middle scalene), sternocleidomastoid, splenius capitis, splenius cervix, longissimus (e.g., longissimus capitis and / or longissimus cervix), and / or semispinalis cervix. Preferably, when treating a lateral deviation, the modified BoNT / A may be administered to one or more affected neck muscles, including: levator scapulae, semispinalis cervix, scalene middle, and / or longissimus cervix (e.g., on the first neck side (e.g., left side)). The modified BoNT / A may also be administered to one or more affected muscles, including: sternocleidomastoid, lower trapezius, splenius capitis, semispinalis capitis, longissimus capitis, and / or levator scapulae (on the second side of the neck (e.g., the right side)).

[0085] In one embodiment, when treating lateral head bending, modified BoNT / A may be administered to one or more affected neck muscles selected from the following: lower trapezius, sternocleidomastoid, longissimus capitis, splenius capitis, semispinalis capitis, levator scapulae, and posterior paravertebral (e.g., posterior scalene, middle scalene, and / or anterior scalene, preferably posterior scalene). In one embodiment, when treating lateral head bending, modified BoNT / A may be administered to one or more affected neck muscles selected from the following: lower trapezius, sternocleidomastoid, longissimus capitis, splenius capitis, semispinalis capitis, levator scapulae, and / or posterior paravertebral (e.g., posterior scalene, middle scalene, and / or anterior scalene, preferably posterior scalene).

[0086] In one embodiment, when treating lateral head bending, the modified BoNT / A is administered to one or more affected neck muscles selected from or including: sternocleidomastoid, lower trapezius, splenius capitis, semispinalis capitis, longissimus capitis, and / or levator scapulae.

[0087] Preferably, when treating head tilt, the modified BoNT / A can be administered ipsilaterally.

[0088] In one embodiment, when treating head tilt, the modified BoNT / A may be administered to one or more affected neck muscles selected from the following: lower trapezius, sternocleidomastoid, longissimus capitis, splenius capitis, semispinalis capitis intermediate, and obliquus capitis inferior. For example, the modified BoNT / A may be administered contralaterally to one or more affected neck muscles selected from the following: lower trapezius, sternocleidomastoid, and semispinalis capitis intermediate; and / or the modified BoNT / A may be administered ipsilaterally to one or more neck muscles selected from the following: obliquus capitis inferior, longissimus capitis, and splenius capitis.

[0089] In one embodiment, when treating head tilt, the modified BoNT / A may be administered to one or more affected neck muscles, including: lower trapezius, sternocleidomastoid, longissimus capitis, splenius capitis, semispinalis capitis middle, and / or obliquus capitis inferior. For example, the modified BoNT / A may be administered contralaterally to one or more affected neck muscles, including: lower trapezius, sternocleidomastoid, and / or semispinalis capitis middle; and / or the modified BoNT / A may be administered ipsilaterally to one or more neck muscles, including: obliquus capitis inferior, longissimus capitis, and / or splenius capitis.

[0090] Preferably, when treating head tilt, the modified BoNT / A may be administered ipsilaterally or contralaterally.

[0091] In one embodiment, when treating head forward bending (also known as head and neck forward bending), modified BoNT / A may be administered to one or more affected neck muscles selected from the following: longus capitis, levator scapulae, and sternocleidomastoid. In one embodiment, when treating head forward bending, modified BoNT / A may be administered to one or more affected neck muscles including the following: longus capitis, levator scapulae, and / or sternocleidomastoid.

[0092] Preferably, when treating head forward, the modified BoNT / A may be administered bilaterally.

[0093] In one embodiment, when treating head tilt, modified BoNT / A may be administered to one or more affected neck muscles selected from the following: oblique capitis inferior, semispinalis capitis, lower trapezius, and splenius capitis. In one embodiment, when treating head tilt, modified BoNT / A may be administered to one or more affected neck muscles including the following: oblique capitis inferior, semispinalis capitis, lower trapezius, and / or splenius capitis.

[0094] Preferably, when treating head tilt, the modified BoNT / A may be administered bilaterally.

[0095] When treating the sagittal shift symptom of cervical dystonia, the muscles selected for administration may be a combination of the muscles targeted for administration when treating neck flexion (e.g., in a first aspect) and head flexion (e.g., in a second aspect).

[0096] In one embodiment, when treating sagittal shift, modified BoNT / A may be administered to one or more affected neck muscles selected from the following: sternocleidomastoid, anterior scalene, middle scalene, levator scapulae, longus colli, submandibular complex (e.g., digastric, geniohyoid, mylohyoid, mylohyoid boutonniere, and / or stylohyoid), oblique capitis inferior, semispinalis capitis, inferior trapezius, and splenius capitis. Preferably, when treating sagittal shift, modified BoNT / A may be administered to affected neck muscles selected from the following: sternocleidomastoid, anterior scalene, middle scalene, levator scapulae, longus colli, and submandibular complex (e.g., digastric, geniohyoid, mylohyoid, mylohyoid boutonniere, and / or stylohyoid) on a first neck side (e.g., left side). The modified BoNT / A can also be administered to an affected neck muscle selected from the following: obliquus capitis inferior, semispinalis capitis, inferior trapezius, and splenius capitis on the second side of the neck (eg, the right side).

[0097] In one embodiment, when treating sagittal shift, modified BoNT / A may be administered to one or more affected neck muscles, including: sternocleidomastoid, anterior scalene, middle scalene, levator scapulae, longus colli, submandibular complex (e.g., digastric, geniohyoid, mylohyoid, mylohyoid boutonniere, and / or stylohyoid), oblique capitis inferior, semispinalis capitis, lower trapezius, and / or splenius capitis. Preferably, when treating sagittal shift, modified BoNT / A may be administered to one or more affected neck muscles, including: sternocleidomastoid, anterior scalene, middle scalene, levator scapulae, longus colli, and / or submandibular complex (e.g., digastric, geniohyoid, mylohyoid, mylohyoid boutonniere, and / or stylohyoid) (e.g., on a first neck side (e.g., left side)). The modified BoNT / A may also be administered to one or more affected neck muscles, including: the oblique capitis inferior, semispinalis capitis, lower trapezius, and / or splenius capitis (e.g., on the second side of the neck (e.g., the right side)).

[0098] In one embodiment, when treating sagittal shift, the modified BoNT / A is administered to one or more affected neck muscles selected from or including: middle scalene, 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: middle scalene, levator scapulae, and / or longus colli (e.g., of a first neck side); and one or more affected neck muscles selected from or including: oblique capitis inferior, semispinalis capitis, lower trapezius, and / or splenius capitis (e.g., of a second neck side).

[0099] When a muscle is recited for administration by a treatment of the invention, the invention can further include administering the modified BoNT / A to other muscles not recited.

[0100] The modified BoNT / A is administered to the affected neck muscle by intramuscular injection. One or more unit doses (e.g., at least two unit doses) of the modified BoNT / A may be administered to the affected neck muscle. However, it is preferred that only a single unit dose is administered per affected neck muscle. If the neck muscle is the splenius capitis, longissimus cervix, trapezius, sternocleidomastoid, semispinalis capitis, or levator scapulae, two unit doses may be administered. In the case of bilateral administration, two unit doses may be administered to the first neck side and two unit doses may be administered to the corresponding muscle of the second neck side. Thus, if the neck muscle is the sternocleidomastoid, two unit doses may be administered. For example, two unit doses may be administered to the left sternocleidomastoid and / or two unit doses may be administered to the right sternocleidomastoid. If the neck muscle is the trapezius (e.g., lower trapezius), two unit doses may be administered. For example, two unit doses may be administered to the left trapezius muscle (eg, left upper trapezius muscle) and / or two unit doses may be administered to the right trapezius muscle (eg, right upper trapezius muscle).

[0101] Thus, a single unit dose of modified BoNT / A may be administered to one or more affected neck muscles selected from a first group including: splenius capitis, oblique capitis inferior, semispinalis cervix, scalene muscles (e.g., anterior, middle, and / or posterior scalene), longissimus capitis, longus colli, and / or longus capitis; and / or a single or multiple unit doses (preferably multiple unit doses) of modified BoNT / A may be administered to one or more affected neck muscles selected from a second group including: splenius capitis, longissimus cervix, trapezius (e.g., lower trapezius), sternocleidomastoid, semispinalis capitis, and / or levator scapulae.

[0102] A single unit dose of the modified BoNT / A may be administered to one or more affected neck muscles selected from a first group including: splenius capitis, obliquus capitis inferior, semispinalis cervix, scalene muscles (e.g., anterior, middle, and / or posterior scalene muscles), longissimus capitis, longus colli, and / or longus capitis; and / or multiple unit doses (preferably two unit doses) of the modified BoNT / A may be administered to one or more affected neck muscles selected from a second group including: splenius capitis, longissimus cervix, trapezius (e.g., lower trapezius), sternocleidomastoid, semispinalis capitis, and / or levator scapulae.

[0103] A unit dose may be administered to one injection site of the affected neck muscle. Thus, in some embodiments, the modified BoNT / A is administered in a unit dose per injection site of the affected neck muscle. However, less than a unit dose may be administered to one injection site, in which case the unit dose may be split (evenly or unevenly) between two or more injection sites of the affected neck muscle. Thus, the modified BoNT / A may be administered to two or more injection sites of the affected neck muscle. In some instances, the modified BoNT / A may be administered in less than a unit dose per injection site of the affected neck muscle. Advantageously, this allows the clinician to delineate the muscle and / or administer more modified BoNT / A to a particularly symptomatic muscle area, as compared to a less symptomatic area, to perform the treatment.

[0104] 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.

[0105] The modified BoNT / A may be administered at a dose of more than 17,000 pg per injection site. Preferably, the modified BoNT / A is administered at a dose of 25,000 pg or 36,000 pg 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, for example 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, for example 36,000 pg.

[0106] The modified BoNT / A may be administered at a dose of greater than 707 units per injection site. Preferably, the modified BoNT / A is administered at a dose of 1,040 or 1,498 units per injection site. The modified BoNT / A may be administered at a dose of 998 units to 1,082 units, e.g., 1,040 units, 832 units to 1,248 units. The modified BoNT / A may be administered at a dose of 1,456 units to 1,539 units, e.g., 1,498 units, 1,248 units to 1,664 units.

[0107] The term "at least a single unit dose is administered" means that at least substantially all of the single unit dose is administered. For example, a vial into which modified BoNT / A has been reconstituted may have a residual amount of a unit dose remaining (e.g., up to 1%, 0.1%, or 0.01%). However, preferably, at least all of the single unit dose is administered (e.g., to one or more injection sites).

[0108] Similarly, administering a portion of a unit dose or multiple unit doses can mean administering substantially a portion of the unit dose or all of the multiple unit doses. For example, the vial from which the modified BoNT / A was removed (e.g., the vial into which the modified BoNT / A was reconstituted) may contain a portion of the unit dose or a remainder of multiple unit doses (e.g., up to 1%, 0.1%, or 0.01%). However, preferably, the portion of the unit dose or all of the multiple unit doses is administered (e.g., to one or more injection sites).

[0109] The potency of modified BoNT / A for use according to the invention was determined using mouse LD 50 In the assay, one unit corresponds to the calculated median lethal dose (LD 50 ) is defined as the amount of modified BoNT / A equivalent to the median intraperitoneal lethal dose calculated in mice.

[0110] The modified BoNT / A used in the present invention comprises a BoNT / A light chain and translocation domain, and a BoNT / B receptor binding domain (H C In the case of a modified BoNT / A containing the IgG1 domain, the amount of modified BoNT / A corresponding to 1 unit in the assay is preferably 24.04 pg.

[0111] The term "up to," when used in reference to a value (e.g., up to 400,000 pg), means up to and including the recited value. Thus, by way of example, reference to administering "up to 400,000 pg" of modified BoNT / A encompasses administration of 400,000 pg of modified BoNT / A as well as administration of less than 400,000 pg of modified BoNT / A.

[0112] The unit dose can be expressed in terms of an amount of modified BoNT / A, in terms of units of modified BoNT / A, or a combination thereof.

[0113] In one embodiment, the modified BoNT / A may be administered in the following dosages into one or more of the following neck muscles as follows: [Table A]

[0114] In one embodiment, the modified BoNT / A may be administered in the following dosages into one or more of the following neck muscles as follows: [Table B]

[0115] The modified BoNT / A may be administered in the following dosages into one or more of the following neck muscles as follows: [Table C]

[0116] The modified BoNT / A may be administered in the following doses into the following neck muscles as follows: [Table D]

[0117] As used herein, the terms "right" and "left" have their ordinary meanings, e.g., a subject's right levator scapulae muscle is the levator scapulae muscle on the subject's right hand side, and a subject's left levator scapulae muscle is the levator scapulae muscle on the subject's left hand side.

[0118] The total number of unit doses administered in a given treatment may be up to 10 times the unit dose, or up to 7 times the unit dose. The total number of total unit doses may be divided according to the affected neck muscles to be treated. For example, in one embodiment, if the number of doses delivered during a treatment is 1 time the unit dose, only one affected neck muscle may be treated, but if the total is 2 times the unit dose, two affected neck muscles may be treated. The total number of unit doses administered may be up to 9 times, 8 times, 7 times, or 6 times. The total number of unit doses administered may be at least 2 times, 3 times, 4 times, 5 times, 6 times, 7 times the unit dose, and preferably at least 2 times. The total number of unit doses administered may be 1 times to 10 times, or 5 times to 10 times, preferably 7 times to 10 times.

[0119] One of skill in the art would take into consideration if the subject had recently undergone (or continues to undergo) additional treatment with a Clostridial neurotoxin (e.g., unmodified BoNT), e.g., as part of a cosmetic procedure or for the treatment of a different indication, and would adapt the treatment regimen accordingly, using techniques routine in the art.

[0120] The modified BoNT / A of the present invention preferably has a longer duration of action (e.g., at least 5%, 10%, 25%, or 50% improvement in one or more symptoms) compared to unmodified BoNT / A (e.g., Dysport®). The duration of action may be at least 1.25-fold, 1.5-fold, 1.75-fold, 2.0-fold, or 2.25-fold longer. The duration of action of the modified BoNT / A may be between 6 and 9 months. For example, the duration of action may be at least 4.5 months, 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 (from onset of action). In certain embodiments, the duration of action may be longer than 9.0 months.

[0121] When administration is performed to multiple affected neck muscles, preferably said administrations are performed during the same treatment session.

[0122] Treatment may be repeated at an appropriate time period after administration of the modified BoNT / A. Assuming that the duration of action is approximately twice that of unmodified BoNT / A (e.g., Dysport®), there is preferably a longer period between subsequent administrations compared to when the subject is treated with unmodified BoNT / A (e.g., Dysport®). The subject may be re-administered with the modified BoNT / A of the present invention at least 18, 20, 25, or 30 weeks after the previous administration. For example, the subject may be re-administered with the modified BoNT / A of the present invention at least 18 to 45 weeks, preferably 20 to 35 weeks, after the previous administration.

[0123] Treatment efficacy (including the severity of the subject's symptoms) can be assessed using the Toronto Western Spasmodic Torticollis Rating Scale (TWSTRS), as outlined in 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-Impairment Scale, and the TWSTRS-Pain Scale. Higher scores on the TWSTRS indicate more severe disease. The TWSTRS-Severity Scale includes the following items: A. Maximum range of motion (rotation, tilt, forward or backward neck flexion, lateral deviation, sagittal shift), B. Duration factor, C. Effect of sensory tricks, D. Shoulder elevation / forward displacement, E. Range of motion (without the aid of sensory tricks), F. Time (time the patient can maintain the head within 10° of neutral position without using sensory tricks, up to 60 seconds). A through F are summed, with the duration factor weighted twice, for a maximum score of 35. The TWSTRS-Impairment Scale is a 6-item scale that includes an assessment of the ability to perform daily activities that may be affected by cervical dystonia, including the ability to perform duties (work or housework), activities of daily living (eating, dressing, hygiene), driving, reading, watching television, and leisure activities outside the home. Each item is rated on a 6-point scale (0=no difficulty, 5=highest impairment), with each item summed for a maximum score of 30. The TWSTRS-Pain Scale consists of severity scores for usual pain, worst pain, and best pain in the patient's past week, a duration factor, and an assessment of the impact of pain on impairment. Scores range from 0 to 20, with 20 being assigned to the most intense pain that can be experienced.

[0124] A "subject" as used herein may be a mammal, such as a human or other mammal. Preferably, a "subject" refers to a human subject. Preferably, a "subject" is an adult subject, i.e., a subject aged 18 years or older. As used herein, the terms "subject" and "patient" are used synonymously. Preferably, the subject has been diagnosed with cervical dystonia.

[0125] The subject of treatment according to the present invention may be a subject for whom treatment with unmodified BoNT / A and / or other Clostridial neurotoxins is unsuitable. The subject may be a subject who is resistant to treatment with unmodified BoNT / A and / or other Clostridial neurotoxins. Resistance may occur due to the subject's development of an immune response against the Clostridial neurotoxin, including production of anti-Clostridial neurotoxin antibodies.

[0126] The term "treat" or "treatment" as used herein encompasses preventative treatment (e.g., preventing the onset of pain) as well as corrective treatment (e.g., treatment of a subject already suffering from a disorder). Preferably, "treat" or "treatment" as used herein means corrective treatment. The term "treat" or "treatment" as used herein refers to a disorder and / or its symptoms.

[0127] Suitable modified BoNT / A polypeptides (and nucleotide sequences encoding the modified BoNT / A polypeptides, if any) are described in WO 2015 / 004461 A1 and WO 2017 / 191315, both of which are incorporated by reference in their entireties herein.

[0128] BoNT / A is an example of a clostridial neurotoxin produced by bacteria of the genus Clostridium. Other examples of such clostridial neurotoxins include those produced by Clostridium tetani (TeNT) and Clostridium botulinum (BoNT) serotypes B to G and X (see WO 2018 / 009903 A2), along with those produced by Clostridium baratii and Clostridium butyricum. The neurotoxins are highly potent and specific, and can harm neurons and other cells to which they are delivered. Clostridial toxins are some of the most potent toxins known. For example, botulinum neurotoxins have a median lethal dose (LD ) in mice of 0.5 to 5 ng / kg, depending on the serotype. 50) value. Both tetanus and botulinum toxins act by inhibiting the function of affected neurons, specifically by inhibiting the release of neurotransmitters. Botulinum toxins act at the neuromuscular junction and inhibit cholinergic transmission in the peripheral nervous system, whereas tetanus toxins act in the central nervous system.

[0129] In nature, clostridial neurotoxins (including BoNT / A) are synthesized as single-chain polypeptides that are post-translationally modified by a proteolytic cleavage event to form two polypeptide chains linked together by a disulfide bond. Cleavage occurs at a specific cleavage site, often referred to as the activation site (e.g., activation loop), located between cysteine ​​residues that provide an interchain disulfide bond. This two-chain form is the active form of the toxin. The two chains are termed heavy chains (H chains) with a molecular weight of approximately 100 kDa, and light chains (L chains) with a molecular weight of approximately 50 kDa. The H chain is linked to an N-terminal translocation component (H N domain) and the C-terminal targeting moiety (H C The cleavage site is located between the L chain and the translocation domain component. C Following binding of the H domain to its target neurons and internalization of the bound toxin into the cell via endosomes, N The domain translocates the L chain across the endosomal membrane into the cytosol, where the L chain provides the protease function (also known as a non-cytotoxic protease).

[0130] The non-cytotoxic protease acts by proteolytically cleaving intracellular trafficking proteins known as SNARE proteins (e.g., SNAP-25, VAMP, or Syntaxin), preferably SNAP-25 - see Gerald K (2002) "Cell and Molecular Biology" (4th edition) John Wiley & Sons, Inc. The acronym SNARE is derived from Soluble NSF Attachment Receptor, where NSF stands for N-ethylmaleimide-Sensitive Factor. SNARE proteins are essential for intracellular vesicle fusion and therefore secretion of molecules from cells via vesicle trafficking. The protease function is a zinc-dependent endopeptidase activity and shows high substrate specificity for SNARE proteins. Thus, when delivered to the desired target cells, the non-cytotoxic protease is capable of inhibiting cellular secretion from the target cells. The light chain proteases of clostridial neurotoxins are non-cytotoxic proteases that cleave SNARE proteins.

[0131] Because of the ubiquitous nature of SNARE proteins, clostridial neurotoxins, such as botulinum toxins, have been used successfully in a wide range of therapeutic approaches.

[0132] For further details on the genetic basis of toxin production in Clostridium botulinum and Clostridium tetani, see The Clostridia: Molecular Biology and Pathogenesis by Henderson et al (1997), Academic press.

[0133] As described above, Clostridial neurotoxins are formed from two polypeptide chains, a heavy chain (H chain) with a molecular weight of approximately 100 kDa and a light chain (L chain) with a molecular weight of approximately 50 kDa. The H chain contains a C-terminal targeting component (receptor binding domain or H C domain) and N-terminal translocation component (HN domain).

[0134] Clostridial neurotoxin domains are described in further detail below.

[0135] Examples of L chain reference sequences include the following: Botulinum type A neurotoxin: amino acid residues 1 to 448 Botulinum type B neurotoxin: amino acid residues 1 to 440

[0136] The reference sequences given above should be considered as a guideline and slight differences may occur depending on the subserotype. For example, US 2007 / 0166332 (hereby incorporated by reference in its entirety) lists slightly different Clostridium sequences. Botulinum type A neurotoxin: amino acid residues M1 to K448 Botulinum type B neurotoxin: amino acid residues M1 to K441

[0137] The translocation domain is a fragment of the H chain of a clostridial neurotoxin that is approximately equivalent to the amino-terminal half of the H chain, or to the domain that corresponds to that fragment in an intact H chain.

[0138] Examples of reference translocation domains include the following: Botulinum type A neurotoxin: amino acid residues (449 to 871) Botulinum type B neurotoxin: amino acid residues (441 to 858)

[0139] The reference sequences given above should be considered as a guideline and slight variations may occur depending on the subserotype. For example, US 2007 / 0166332 (hereby incorporated by reference) lists slightly different Clostridium sequences. Botulinum type A neurotoxin: amino acid residues (A449 to K871) Botulinum type B neurotoxin: amino acid residues (A442 to S858)

[0140] In the context of the present invention, various BoNT / AHs containing a translocation domain N The H region of the BoNT / A heavy chain may be useful in embodiments of the present invention. N The region is approximately 410-430 amino acids long and contains a translocation domain. Studies have shown that the translocation domain translocates to the H of the clostridial neurotoxin heavy chain due to its translocation activity. N It has been shown that the full length of the region is not necessary. Thus, aspects of this embodiment include, for example, a BoNT / AH gene that includes a translocation domain that is at least 350 amino acids, at least 375 amino acids, at least 400 amino acids, or at least 425 amino acids in length. N Other aspects of this embodiment include a BoNT / AH gene that includes a translocation domain that is, 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. N It may include regions.

[0141] Term H N BoNT / AH of natural origin N Modified BoNT / AHs having portions, and non-naturally occurring amino acid sequences and / or synthetic amino acid residues. N Preferably, the modified BoNT / AH N The moiety still exhibits said translocation function.

[0142] Clostridial neurotoxin receptor binding domain (H C ) Examples of reference sequences include the following: BoNT / A-N872 to L1296 BoNT / B-E859 to E1291

[0143] Clostridial neurotoxins (BoNT, etc.) have a molecular weight of about 50 kDa C The domains are H, which are usually about 25 kDa each. CC Domain and H CN It has two distinct structural features called domains. The amino acid residues involved in receptor binding are mainly H CC It is believed to be located in the H domain of natural clostridial neurotoxins.C A domain may contain approximately 400 to 440 amino acid residues. This reality is confirmed by the following publications, each of which is incorporated herein by reference in its entirety: Umland TC (1997) Nat. Struct. Biol. 4: 788-792; Herreros J (2000) Biochem. J. 347: 199-204; Halpern J (1993) J. Biol. 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.

[0144] (Reference)H CN Examples of domains include: Botulinum type A neurotoxin: amino acid residues (872 to 1110) Botulinum type B neurotoxin: amino acid residues (859 to 1097)

[0145] The above sequence positions may vary slightly depending on the serotype / subtype. CN Further examples of domains include: Botulinum type A neurotoxin: amino acid residues (874 to 1110) Botulinum type B neurotoxin: amino acid residues (861 to 1097)

[0146] (Reference)H CC Examples of domains include: Botulinum type A neurotoxin: amino acid residues (Y1111 to L1296) Botulinum type B neurotoxin: amino acid residues (Y1098 to E1291)

[0147] L chain and H N The domains (optionally including a complete or partial activation loop, e.g., a complete activation loop if the modified BoNT / A is in single-chain form, or a truncated / partial activation loop if the modified BoNT / A is in two-chain form) are collectively referred to as the LH N It is sometimes called a domain. Therefore, LH N The domain is further H C Does not include the domain.

[0148] WO 2017 / 191315 A1 (hereby incorporated by reference) teaches modified BoNT / A and methods for its preparation and manufacture. Thus, the botulinum neurotoxin A (BoNT / A) light chain and translocation domain (BoNT / AH) used in the present invention are N domain), and the BoNT / B receptor binding domain (H C The modified BoNT / A including the IL-1 domain may be one taught in WO 2017 / 191315 A1.

[0149] The term "modified BoNT / A" or "chimeric neurotoxin" as used herein refers to a chimeric neurotoxin that is a clostridial ... N domain), as well as a receptor-binding domain (H C Specifically, the modified BoNT / A used in the present invention refers to a neurotoxin comprising (preferably consisting of) a botulinum neurotoxin A (BoNT / A) light chain and a translocation domain (H domain). N domain), and the BoNT / B receptor binding domain (H C The modified BoNT / A contains the BoNT / A LH domain. N The domain is BoNT / BH CThe modified BoNT / A of the present invention may also 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".

[0150] L chain and H N The domains (optionally including a complete or partial activation loop, e.g., a complete activation loop if the modified BoNT / A is in single-chain form, or a truncated / partial activation loop if the modified BoNT / A is in two-chain form) are collectively referred to as the LH N It is sometimes called a domain. Therefore, LH N The domain is further H C Does not include the domain.

[0151] The modified BoNT / A is a botulinum neurotoxin A (BoNT / A) light chain and translocation domain (H N domain), and the BoNT / B receptor binding domain (H C The scalable domain may consist essentially of:

[0152] The term "consisting essentially of" as used in this context means that the modified BoNT / A does not further comprise one or more amino acid residues that confer additional functionality to the polypeptide, for example, when administered to a subject. In other words, the modified BoNT / A comprises a botulinum neurotoxin A (BoNT / A) light chain and translocation domain (H N domain), and the BoNT / B receptor binding domain (H C A polypeptide "consisting essentially of" a botulinum neurotoxin A (BoNT / A) light chain and translocation domain (H domain) can further include a polypeptide "consisting essentially of" a botulinum neurotoxin A (BoNT / A) light chain and translocation domain (H domain). N domain), and the BoNT / B receptor binding domain (H C A polypeptide may contain one or more amino acid residues (in addition to those of the corresponding domain), but said one or more additional amino acid residues do not confer additional functionality to the polypeptide, e.g., when administered to a subject. Additional functionality may include enzymatic activity, binding activity, and / or any other physiological activity.

[0153] The modified BoNT / A may contain non-clostridial neurotoxin sequences in addition to any clostridial neurotoxin sequences, so long as it does not interfere with the ability of the modified BoNT / A to achieve its therapeutic effect. The non-clostridial neurotoxin sequence is preferably one that does not have catalytic activity, such as enzymatic activity. In one embodiment, the modified BoNT / A of the present invention does not contain a non-clostridial catalytic activity domain. In one embodiment, the modified BoNT / A does not contain an additional catalytic activity domain. In one embodiment, the non-clostridial sequence is not a sequence that binds to a cellular receptor. In other words, in one embodiment, the non-clostridial sequence is not a ligand for a cellular receptor. The cellular receptor may be a proteinaceous cellular receptor, such as an integral membrane protein. Examples of cellular receptors are listed in the IUPHAR Guide to Pharmacology Database, version 2019.4, available at https: / / www.guidetopharmacology.org / download.jsp#db_reports. The non-clostridial neurotoxin sequence may contain a tag that aids in purification, such as a histidine tag. In one embodiment, a modified BoNT / A of the invention does not contain a label or a site for adding a label, such as a sortase acceptor site or donor site.

[0154] Preferably, the modified BoNT / A 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 The domain may consist of:

[0155] The modified BoNT / A contains a light chain that exhibits non-cytotoxic protease activity and is capable of cleaving SNARE proteins in the cytosol of target neurons.

[0156] By using cell assays and in vivo assays, it can be determined whether a Clostridial neurotoxin containing an L chain, a functional cell binding, and a translocation domain has a non-cytotoxic protease activity. Assays such as digit abduction score (DAS) assay, dorsal root ganglion (DRG) assay, spinal cord neuron (SCN) assay, and mouse phrenic nerve hemidiaphragm (PNHD) assay are common in the art. Suitable assays for measuring non-cytotoxic protease activity may be those described in Aoki KR, Toxicon 39: 1815-1820; 2001 or Donald et al (2018), Pharmacol Res Perspect, e00446, 1-14, which are incorporated herein by reference.

[0157] When administered to a subject, the modified BoNT / A is preferably in an active two-chain form, with the light and heavy chains linked together by a disulfide bond. When BoNT / A (e.g., modified BoNT / A) is defined herein by a polypeptide sequence (SEQ ID NO:), the L chain portion of the sequence (SEQ ID NO:) may constitute the first chain of a two-chain Clostridial neurotoxin (e.g., a two-chain modified BoNT / A), and the H chain portion of the sequence (SEQ ID NO:) may constitute the first chain of a two-chain Clostridial neurotoxin (e.g., a two-chain modified BoNT / A). N Domain and H CThe domains together may constitute the second chain of a di-chain clostridial neurotoxin (e.g., di-chain modified BoNT / A), where the first and second chains are linked to each other by disulfide bonds. Those skilled in the art will recognize that the protease may cleave at one or more positions in the activation loop of a clostridial neurotoxin (e.g., modified BoNT / A), preferably at two positions in the activation loop. If cleavage occurs at more than one position (preferably at two positions) in the activation loop, a small fragment of the C-terminal L chain portion of the sequence may not be present in the di-chain clostridial neurotoxin sequence (e.g., di-chain modified BoNT / A). For this reason, the sequence of a di-chain clostridial neurotoxin (e.g., di-chain modified BoNT / A) may differ slightly from the sequence of the corresponding single-chain clostridial neurotoxin (e.g., single-chain modified BoNT / A). The small fragment may be 1 to 15 amino acids. In particular, in one embodiment, when Lys-C is used to convert a single-chain modified BoNT / A to a two-chain modified BoNT / A, a subfragment of the C-terminal L chain portion of the absent sequence may be SEQ ID NO: 9 or 10.

[0158] Most preferably, the modified BoNT / A used in the present invention comprises a BoNT / A light chain and a translocation domain (BoNT / A LH N domain), and BoNT / BH C BoNT / A LH domain. N The domain is BoNT / BH C The modified BoNT / A is also referred to herein as a "BoNT / AB" or a "BoNT / AB chimera."

[0159] LH N The C-terminal amino acid residues of the domain are the same as those of the LH domain of BoNT / A. N Domain and H C Separate domains 3 10 may correspond to the first amino acid residue of the helix, H C The N-terminal amino acid residues of the domain are the LH N Domain and H C Separate domains 3 10It may correspond to the second amino acid residue of the helix.

[0160] An example of an (unmodified) BoNT / A polypeptide sequence is set forth in SEQ ID NO:2.

[0161] An example of a BoNT / B polypeptide sequence is set forth in SEQ ID NO:8 (UniProt Accession No. B1INP5).

[0162] As used herein, the term "BoNT / A LH N Domain and H C Separate domains 3 10 When referring to "the first amino acid residue of the helix," N Domain and H C Separate domains 3 10 It refers to the N-terminal residue of the helix.

[0163] As used herein, the term "BoNT / B LH N Domain and H C Separate domains 3 10 When referring to "the second amino acid residue of the helix," N Domain and H C Separate domains 3 10 It refers to the amino acid residue following the N-terminal residue of the helix.

[0164] "3 10 A "helix" is a type of secondary structure found in proteins and polypeptides, along with α-helices, β-sheets, and reverse turns. 3 10The amino acids in the helix are arranged in a right-handed helical structure, with each full turn completed by 10 atoms separating the three residues and the intramolecular hydrogen bonds between them. Each amino acid has 10 atoms in the ring formed by the formation of hydrogen bonds, corresponding to a 120° turn in the helix (i.e., the helix has three residues per turn) and a translational movement of 2.0 Å (=0.2 nm) along the helix axis. Most importantly, the NH group of an amino acid forms a hydrogen bond with the C=O group of the amino acid three residues earlier, and this repeated i+3→i hydrogen bond is repeated three times. 10 Define a helix. 3 10 A helix is ​​a standard concept in structural biology with which those skilled in the art are familiar.

[0165] These three 10 The helix corresponds to the four residues that form the actual helix plus two cap (or transition) residues, one at each end of these four residues. N Domain and H C Separate domains 3 10 A "helix," as used herein, consists of those six residues.

[0166] By performing structural analysis and sequence alignment, LH N Domain and H C Separate domains 3 10 The three helices were identified. 10 The helix is ​​located at its N-terminus (i.e., LH N at the C-terminal part of the domain) by an α-helix and at its C-terminus (i.e. 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 this α-helix.

[0167] LH N Domain and H C Separate domains 3 10The helices can be determined, for example, from the publicly available crystal structures of botulinum neurotoxins, such as 3BTA (http: / / www.rcsb.org / pdb / explore / explore.do?structureId=3BTA) and 1EPW (http: / / www.rcsb.org / pdb / explore / explore.do?structureId=1EPW) for botulinum neurotoxins A1 and B1, respectively.

[0168] LH in other neurotoxins N Domain and H C Separate domains 3 10 To determine the position of the helices, publicly available computational modeling and alignment tools can also be used, such as the homology modeling servers LOOPP (Learning, Observing and Outputting Protein Patterns, http: / / loopp.org), PHYRE (Protein Homology / analogY Recognition Engine, http: / / www.sbg.bio.ic.ac.uk / phyre2 / ), and Rosetta (https: / / www.rosettacommons.org / ), the protein superposition server SuperPose (http: / / wishart.biology.ualberta.ca / superpose / ), the alignment program Clustal Omega (http: / / www.clustal.org / omega / ), as well as many other tools / services listed in Internet Resources for Molecular and Cell Biologists (http: / / molbiol-tools.ca / ). In particular, N / H CN "The perijunctional region can be highly structurally conserved, making it an ideal region for overlapping different serotypes.

[0169] For example, this 3 10To determine the sequence of the helix, the following methodology may be used. 1. Based on the BoNT / A1 crystal structure (3BTA.pdb), the structural homology modeling tool LOOP (http: / / loopp.org) may be used to obtain predicted structures of other BoNT serotypes. 2. Copy the resulting structure (pdb) file to H CN The N-terminal part of the domain, and (H N This contains only about 80 residues preceding the α-terminal domain (which is part of the α-terminal domain), thereby forming a highly structurally conserved "H N / H CN " area can be edited to preserve it. 3. The protein superposition server SuperPose (http: / / wishart.biology.ualberta.ca / superpose / ) may be used to superimpose each serotype onto the 3BTA.pdb structure. 4. Examine the overlapping pdb files to find the H of BoNT / A1. C 3 at the start of the domain 10 The helices may be located and then the corresponding residues in other serotypes identified. 5. Other BoNT serotype sequences may be aligned using Clustal Omega to see if corresponding residues were correct.

[0170] LH determined by this method N , H C and 3 10 Examples of helical domains are shown below. [Table E]

[0171] Using structural analysis and sequence alignment, LH N Domain and H C Separate domains 3 10 The β-strand following the helix is ​​a conserved structure in all botulinum and tetanus neurotoxins and is the LHN Domain and H C Separate domains 3 10 It was found to start at the eighth residue (eg, residue 879 of BoNT / A1) when starting from the first residue of the helix.

[0172] BoNT / AB chimera is a chimera that binds the H C BoNT / A LH covalently bound to the domain N domain, where LH N The C-terminal amino acid residue of the domain is H C corresponds to the eighth amino acid residue at the N-terminus of the β-strand located at the beginning (N-terminus) of the domain, where H C The N-terminal amino acid residue of the domain is H C It corresponds to the seventh amino acid residue at the N-terminus of the β-strand located at the beginning (N-terminus) of the domain.

[0173] BoNT / AB chimera is a chimera that binds the H C BoNT / A LH covalently bound to the domain N domain, where LH N The C-terminal amino acid residues of the domain are the same as those of the LH domain of BoNT / A. N corresponds to the C-terminal amino acid residue of the α-helix located at the end (C-terminus) of the domain, where H C The N-terminal amino acid residues of the domain are the LH N It corresponds to the amino acid residue immediately C-terminal to the C-terminal amino acid residue of the α-helix located at the end (C-terminus) of the domain.

[0174] The principle in the design process of the BoNT / AB chimera was to ensure that the secondary structure was intact, thereby minimizing any changes in the tertiary structure and function of each domain. 10It is hypothesized that not disrupting the four central amino acid residues of the helix will ensure an optimal conformation of the chimeric neurotoxin, thereby allowing it to maximize its functionality. Indeed, surprisingly, the 3 10 The first amino acid residue of the helix and the first three amino acids of BoNT / B 10 Retaining only the second amino acid residue of the helix not only allows for the production of a soluble and functional BoNT / AB chimera, but also provides improved properties over other BoNT / AB chimeras, in particular enhanced potency, improved safety factor and / or longer duration of action (and improved safety factor and / or longer duration of action compared to unmodified BoNT / A [e.g., SEQ ID NO:2 in its dichain form]).

[0175] BoNT / A light chain, BoNT / A translocation domain, and / or BoNT / BH C The domains include, but are not limited to, modified BoNT / A light chains, BoNT / A translocation domains, and / or BoNT / BH domains, including those described below. C The modified BoNT / A light chain, the BoNT / A translocation domain, and / or the BoNT / BH domain, or a derivative thereof. C The domains or derivatives may be the naturally occurring (unmodified) forms of the BoNT / A light chain, the BoNT / A translocation domain, and / or the BoNT / BH domain. C The BoNT / A light chain, the BoNT / A translocation domain, and / or the BoNT / BH domain may contain one or more amino acids that are modified compared to the naturally occurring (unmodified) form of the BoNT / A light chain, the BoNT / A translocation domain, and / or the BoNT / BH domain. C The modified BoNT / A light chain, the BoNT / A translocation domain, and / or the BoNT / BH domain may include one or more inserted amino acids that are not present in the modified BoNT / A light chain, the BoNT / A translocation domain, and / or the BoNT / BH domain. C The domains may be native (unmodified) BoNT / A light chain, BoNT / A translocation domain, and / or BoNT / BH. CFor domain sequences, one or more domains may contain modified amino acid sequences. Such modifications may, for example, modify their functional aspects, such as biological activity or biological persistence. Thus, in one embodiment, the BoNT / A light chain, the BoNT / A translocation domain, and / or the BoNT / BH C The domain may be a modified BoNT / A light chain, a BoNT / A translocation domain, and / or a BoNT / BH domain. C domain, or modified BoNT / A light chain, BoNT / A translocation domain, and / or BoNT / BH C It may be a derivative of a domain.

[0176] Modified BoNT / BH C The domain may have one or more modifications that modify binding to a target neuronal cell, for example, a natural (unmodified) BoNT / BH domain. C The BoNT / BH domain provides higher or lower affinity binding. C Such modifications in the H domain alter binding to ganglioside receptors and / or protein receptors on target neurons. C This may involve modifying residues in the ganglioside binding site or in the protein (e.g., synaptotagmin) binding site of the domain. Examples of such modified neurotoxins are described in WO 2006 / 027207 and WO 2006 / 114308, both of which are incorporated by reference in their entireties.

[0177] 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 that may alter or modify the SNARE protein specificity of the modified light chain, but preferably do not catalytically inactivate the light chain. Examples of such modified neurotoxins are described in WO 2010 / 120766 and US 2011 / 0318385, both of which are incorporated by reference in their entireties.

[0178] BoNT / A LH NThe domain may correspond to amino acid residues 1 to 872 of SEQ ID NO:2, or a polypeptide sequence having at least 70% sequence identity thereto. N The domain may correspond to amino acid residues 1 to 872 of SEQ ID NO:2, or a polypeptide sequence having at least 80%, 90% or 95% sequence identity thereto. N The domain corresponds to amino acid residues 1 to 872 of SEQ ID NO:2.

[0179] BoNT / B H 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. C The domain corresponds to amino acid residues 860 to 1291 of SEQ ID NO:8.

[0180] Preferably, the BoNT / AB chimera is BoNT / A1 LH N Domain and BoNT / B1 H C More preferably, the LH domain is N The domain corresponds to amino acid residues 1 to 872 of BoNT / A1 (SEQ ID NO: 2), C The domain corresponds to amino acid residues 860 to 1291 of BoNT / B1 (SEQ ID NO:8).

[0181] Most preferably, BoNT / BH C The H domain has the effect of increasing the binding affinity of BoNT / B neurotoxin to human Syt II as compared to the native BoNT / B sequence. CC The BoNT / BH subdomain further comprises a substitution, insertion, indel, or deletion of at least one amino acid residue in the subdomain. CCSuitable amino acid residue substitutions, insertions, indels, or deletions in the subdomains are disclosed in WO 2013 / 180799 and WO 2016 / 154534 (both of which are incorporated herein by reference).

[0182] BoNT / BH CC Suitable amino acid residue substitutions, insertions, indels, or deletions in the subdomains may comprise 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.

[0183] BoNT / BH CC The substitution, insertion, indel, or deletion of a suitable amino acid residue in the subdomain may further comprise 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, E11 91Q 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.

[0184] BoNT / BH CC Suitable amino acid residue substitutions, insertions, indels, or deletions in the subdomains may also include a combination of the three substitution mutations E1191M, S1199W, and W1178Q.

[0185] Preferably, BoNT / BH CCSuitable amino acid residue substitutions, insertions, indels, or deletions in the subdomain include a combination of two substitution mutations, E1191M and S1199Y. Such modifications are present, for example, in SEQ ID NO:5 and SEQ ID NO:6 of the BoNT / AB chimera. E1191M can correspond to position 1204 and S1199Y can correspond to position 1212 of SEQ ID NO:6. Thus, SEQ ID NO:6 can include 1204M and 1212Y.

[0186] The modification may be a modification relative to BoNT / B shown as SEQ ID NO:8, where the numbering of the amino acid residues is determined by alignment with SEQ ID NO:8. Since the presence of a methionine residue at position 1 of SEQ ID NO:8 is optional (as are the SEQ ID NOs corresponding to the modified BoNT / A polypeptides described herein), the skilled artisan will take into account the presence / absence of the methionine residue when determining the amino acid residue numbering. For example, if SEQ ID NO:8 contains a methionine, the position numbering will be as defined above (e.g., E1191 becomes E1191 in SEQ ID NO:8). Alternatively, if a methionine is not present in SEQ ID NO:8, the amino acid residue numbering must be modified by -1 (e.g., E1191 becomes E1190 in SEQ ID NO:8). Thus, the first methionine amino acid residue in the polypeptide sequence of the modified BoNT / A may be optional or absent. Similar considerations apply to the presence / absence of a methionine at position 1 of other polypeptide sequences described herein, and the skilled artisan will readily determine the correct amino acid residue numbering using techniques routine in the art.

[0187] A modified BoNT / A for use in the present invention may comprise a polypeptide sequence having at least 70% sequence identity to a polypeptide sequence selected from SEQ ID NOs: 3 to 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 to 7. Preferably, a modified BoNT / A for use in the present invention may comprise (and more preferably consists of) a polypeptide sequence selected from SEQ ID NOs: 3 to 7.

[0188] Thus, it is preferred that 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 used in the present invention may comprise (and more preferably consist of) SEQ ID NO: 6.

[0189] The term "deletion," as used herein, refers to the removal of one or more amino acid residues of a polypeptide without replacing the amino acid residue or residues at the deletion site. Thus, (for example), if one amino acid residue is deleted from a polypeptide sequence having x amino acid residues, the resulting polypeptide has x-1 amino acid residues.

[0190] The term "indel" as used herein refers to the deletion of one or more amino acid residues of a polypeptide and the insertion at the deletion site of a different number of amino acid residues (more or fewer amino acid residues) compared to the number of deleted amino acid residues. Thus, in the case of an indel in which two amino acid residues are deleted from a polypeptide sequence having (for example) 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.

[0191] 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 positions. Thus, in the case of a substitution of a polypeptide sequence having (for example) x amino acid residues, the resulting polypeptide also has x amino acid residues. Preferably, the substitution is at a single amino acid position.

[0192] The term "insertion," as used herein, refers to the addition of one or more amino acid residues to a polypeptide without deletion of one or more amino acid residues of the polypeptide at the site of insertion. Thus, (for example), if one amino acid residue is inserted into a polypeptide sequence having x amino acid residues, the resulting polypeptide will have x+1 amino acid residues.

[0193] Methods for modifying proteins by substitution, insertion, or deletion of amino acid residues are known in the art. By way of example, amino acid modifications can be introduced by modification of the DNA sequence encoding BoNT / A (e.g., encoding unmodified BoNT / A). This can be achieved by standard molecular cloning techniques, for example, by site-directed mutagenesis using a short strand of DNA (oligonucleotide) encoding the desired amino acid(s) and replacing the original coding sequence using a polymerase enzyme, or by inserting / deleting parts of a gene with various enzymes (e.g., ligase and restriction enzymes). Alternatively, modified gene sequences can be chemically synthesized. Typically, modifications are made by modifying a nucleic acid encoding a naturally occurring Clostridial neurotoxin (or a portion thereof), such that the modified BoNT / A (or a portion thereof) encoded by the nucleic acid contains the modification(s). Alternatively, a nucleic acid encoding a modified Clostridial neurotoxin (or a portion thereof) containing the modification(s) can be synthesized.

[0194] Where the polypeptide sequence of a modified BoNT / A described herein includes a tag, such as a histidine 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.

[0195] As mentioned above, the modified BoNT / A described herein has improved tissue retention properties that also provide increased potency and / or duration of action, allowing for increased dosage without any additional negative effects. One way these beneficial properties can be defined is in terms of the safety factor of the modified BoNT / A. In this regard, the undesirable effects of clostridial neurotoxins (caused by toxin diffusion away from the site of administration) can be experimentally evaluated by measuring the rate of weight loss in relevant animal models (e.g., mice, where weight loss is detected within 7 days of administration). Conversely, the desired on-target effects of clostridial toxins can be experimentally evaluated by the digit abduction score (DAS) assay, which measures muscle paralysis. The DAS assay can be performed by injecting 20 μL of neurotoxin formulated in gelatin phosphate buffer into the mouse gastrocnemius / soleus muscle complex, followed by evaluating the digit abduction score using the method of Aoki (Aoki KR, Toxicon 39: 1815-1820; 2001). In the DAS assay, mice are briefly suspended by their tails to elicit a characteristic startle response in which the mice extend their hind limbs and abduct their hind digits. After injection of a neurotoxin, the degree of change in digit abduction is scored on a 5-point scale (0=normal to 4=maximal reduction in digit abduction and leg extension).

[0196] The safety factor of the modified BoNT / A of the present invention (or a comparable unmodified BoNT / A) can then be expressed as the ratio of the amount of toxin required for 10% weight loss (measured at peak effect within the first 7 days after dosing in mice) to the amount of neurotoxin required for a DAS score of 2. Thus, a high safety factor score is desired, and a neurotoxin capable of effectively paralyzing target muscles with few undesirable off-target effects is required. The modified BoNT / A of the present invention has a higher safety factor than the safety factor of the equivalent unmodified (natural) BoNT / A.

[0197] A high safety margin is particularly advantageous in a therapeutic method since it represents an increased therapeutic index. In other words, this means that the dosage can be reduced and / or increased dosage can be used without any additional (e.g., adverse) effects compared to alternative Clostridial neurotoxin therapeutics. Adverse effects include systemic toxicity and undesirable diffusion to adjacent muscles. The possibility of using higher doses of neurotoxin without additional effects is particularly advantageous since higher doses usually result in a longer duration of action of the neurotoxin.

[0198] The efficacy of the modified BoNT / A is determined by the efficacy of the modified BoNT / A when administered to the mouse gastrocnemius / soleus muscle complex, based on a given DAS score, e.g., DAS score 2 (ED 50 The potency of modified BoNT / A can also be expressed as the EC 50 Dose, e.g., EC in a cellular assay measuring SNAP25 cleavage by modified BoNT / A 50 It can also be expressed as a dose.

[0199] The duration of action of a modified BoNT / A can be expressed as the time required to restore the DAS score to 0 after administration of a given dose of neurotoxin, e.g., the minimum dose of neurotoxin that results in a DAS score of 4, to the mouse gastrocnemius / soleus muscle complex.

[0200] Thus, in one embodiment, a modified BoNT / A of the invention has a safety factor of greater than 7 (e.g., at least 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50), where the safety factor is the dose of toxin (pg / mouse) required for a -10% change in body weight relative to the DAS ED 50 Calculate [ED 50 = dose required to obtain a DAS score of 2. For example, the modified BoNT / A can have a safety factor of at least 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50.

[0201] In one embodiment, the modified BoNT / A of the invention has a safety factor of at least 10. In one embodiment, the modified BoNT / A of the invention has a safety factor of at least 15. Preferably, the modified BoNT / A has a safety factor of at least 10 (e.g., a safety factor of 10), more preferably at least 12 or 13 (e.g., 14-15). The chimeric Clostridial neurotoxin can have a safety factor of greater than 7, up to 50, e.g., 8-45, 10-20, or 12-15.

[0202] The modified BoNT / A used in the present invention may comprise a polypeptide sequence having at least 70% sequence identity to a polypeptide sequence selected from SEQ ID NOs: 3 to 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 to 7. Preferably, the modified BoNT / A used in the present invention may comprise (and more preferably consists of) a polypeptide sequence selected from SEQ ID NOs: 3 to 7. Of the modified BoNT / As, SEQ ID NO: 6 is preferred.

[0203] Thus, it is preferred that the modified BoNT / A comprises a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 6. More preferably, 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 used in the present invention may comprise (and more preferably consist of) SEQ ID NO: 6.

[0204] The two-stranded modified BoNT / A of the present invention may comprise a light 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 to 7 which together constitute the first strand of the two-stranded modified BoNT / A, and a heavy 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 to 7 which together constitute the second strand of the two-stranded modified BoNT / A. N Domain and H Cdomain, where the first and second chains are linked to each other by disulfide bonds.

[0205] When cleavage occurs at two or more positions (preferably two positions) within the activation loop of a modified BoNT / A that contains 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, a small fragment of the C-terminal L chain portion of the 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 this respect, 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 subfragment may be 1 to 15 amino acids. In particular, in one embodiment, when Lys-C is used to convert a single-chain modified BoNT / A to a two-chain Clostridial neurotoxin, the subfragment of the C-terminal L chain portion of the absent sequence may be SEQ ID NO: 9 or 10.

[0206] Preferably, the two-chain modified BoNT / A of the present invention may comprise a light 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 two-chain modified BoNT / A, and a heavy 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 together constitute the second chain of the two-chain modified BoNT / A. N Domain and H C domain, where the first and second chains are linked to each other by disulfide bonds.

[0207] If truncations occur at more than one position (preferably two positions) within the activation loop of a modified BoNT / A that contains a polypeptide sequence that has at least 70%, 80%, 90%, 95%, 99.9% or 100% sequence identity to SEQ ID NO: 6, a subfragment of the C-terminal L chain portion of the sequence that has 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 this regard, the sequence of a two-chain modified BoNT / A (e.g., containing a polypeptide sequence that has at least 70%, 80%, 90%, 95%, 99.9% or 100% sequence identity to SEQ ID NO: 6) may differ slightly from the sequence of a corresponding single-chain modified BoNT / A that contains a polypeptide sequence that has at least 70%, 80%, 90%, 95%, 99.9% or 100% sequence identity to SEQ ID NO: 6. The subfragment may be 1 to 15 amino acids. In particular, in one embodiment, when Lys-C is used to convert a single-chain modified BoNT / A to a two-chain modified BoNT / A, a subfragment of the C-terminal L chain portion of the absent sequence may be SEQ ID NO: 9 or 10.

[0208] In a particularly preferred embodiment, 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, where the light chain and the heavy chain are linked to each other 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, where the light chain and the heavy chain are linked to each other 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, where the light chain and the heavy chain are linked to each other 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.

[0209] In a preferred embodiment, the modified BoNT / A of the present invention does not include a therapeutic or diagnostic agent (e.g., a nucleic acid, protein, peptide, or small molecule therapeutic, or diagnostic agent) in addition to the light and heavy chains. For example, in one embodiment, the modified BoNT / A may not include a covalently or non-covalently bound therapeutic or diagnostic agent. Thus, the modified BoNT / A of the present invention preferably does not function as a delivery vehicle for additional therapeutic or diagnostic agents.

[0210] In embodiments in which the modified BoNT / A described herein has a tag for purification (eg, a histidine tag) and / or a linker, the tag and / or linker are optional.

[0211] The modified BoNT / A is preferably uncomplexed (i.e., does not contain the complexing proteins present in naturally occurring BoNT / A). Examples of such complexing proteins include neurotoxin-associated proteins (NAPs) and non-toxic non-hemagglutinin components (NTNHs). However, it is preferred that the modified BoNT / A is a recombinant modified BoNT / A.

[0212] 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.

[0213] In one embodiment, a nucleic acid (e.g., DNA) is provided that comprises a nucleic acid sequence encoding a modified BoNT / A. In one embodiment, the nucleic acid sequence is prepared as part of a DNA vector that includes a promoter and a terminator. The nucleic acid sequence can be selected from any of the nucleic acid sequences described herein.

[0214] In a preferred embodiment, the vector has a promoter selected from the following: Promoter / Inducer / General induction conditions Tac (hybrid) / IPTG / 0.2mM (0.05 to 2.0mM) AraBAD / L-arabinose / 0.2% (0.002 to 0.4%) T7-lac operator / IPTG / 0.2mM (0.05 to 2.0mM)

[0215] In another preferred embodiment, the vector has a promoter selected from: Promoter / Inducer / General induction conditions Tac (hybrid) / IPTG / 0.2mM (0.05 to 2.0mM) AraBAD / L-arabinose / 0.2% (0.002 to 0.4%) T7-lac operator / IPTG / 0.2mM (0.05 to 2.0mM) T5-lac operator / IPTG / 0.2mM (0.05 to 2.0mM)

[0216] The nucleic acid molecules can be produced using any suitable process known in the art. Thus, the nucleic acid molecules can be produced using chemical synthesis techniques. Alternatively, the nucleic acid molecules of the present invention can be produced using molecular biology techniques.

[0217] The DNA constructs of the present invention are preferably designed on a computer and then synthesized by conventional DNA synthesis techniques.

[0218] The above nucleic acid sequence information is optionally modified for codon bias depending on the final host cell (eg, E. coli) expression system used.

[0219] As used herein, the terms "nucleotide sequence" and "nucleic acid" are used synonymously. Preferably, the nucleotide sequence is a DNA sequence.

[0220] The modified BoNT / A of the present invention may exist as a single chain or as a two-chain. However, the modified BoNT / A has an L chain connected to an H chain (or a component thereof, such as an H chain) via a disulfide bond. N It is preferred that the nucleic acid be present as a double strand linked to a single nucleic acid domain.

[0221] 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 obtain 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 into the corresponding two-chain modified BoNT / A (e.g., the light chain and the heavy chain are linked together by a disulfide bond). The two-chain modified BoNT / A is preferably obtained by such a method.

[0222] Thus, the modified BoNT / A used in the present invention is preferably a two-chain modified BoNT / A generated from a single-chain BoNT / A, where the single-chain BoNT / A comprises or consists of a polypeptide sequence described herein. For example, the modified BoNT / A used in the present invention is preferably a two-chain modified BoNT / A generated from a polypeptide comprising a polypeptide sequence having at least 70% (e.g., at least 80%, 90%, 95% or 99.9%) sequence identity to SEQ ID NO:6. Most preferably, the modified BoNT / A used in the present invention is a two-chain modified BoNT / A generated from a polypeptide comprising (more preferably consisting of) SEQ ID NO:6. Thus, in some embodiments, the modified BoNT / A is a two-chain modified BoNT / A in which the light chain (L chain) is linked to the heavy chain (H chain) via a disulfide bond, obtained by a method comprising contacting a single-chain modified BoNT / A comprising SEQ ID NO:6 with a protease that hydrolyzes a peptide bond in its activation loop, thereby converting the single-chain modified BoNT / A to the corresponding two-chain modified BoNT / A. In some embodiments, the modified BoNT / A is a two-chain modified BoNT / A in which the L chain is linked to the H chain via a disulfide bond, obtained by a method comprising contacting a single-chain modified BoNT / A consisting of SEQ ID NO:6 with a protease that hydrolyzes a peptide bond in its activation loop, thereby converting the single-chain modified BoNT / A to the corresponding two-chain modified BoNT / A.

[0223] The protease used to cleave the activation loop is preferably Lys-C. Suitable proteases and methods for cleaving the activation loop to generate a di-chain clostridial neurotoxin are taught in WO 2014 / 080206, WO2014 / 079495 and EP2677029A2, which are incorporated herein by reference. Lys-C can cleave the C-terminus of the activation loop to one or more lysine residues present therein. If Lys-C cleaves the activation loop more than once, those skilled in the art will understand that the small peptide of the activation loop of the di-chain modified BoNT / A may not be present, compared to the sequence number shown herein.

[0224] The term "obtained" as used herein also encompasses the term "obtained." In one embodiment, the term "obtained" means obtained.

[0225] The term "one or more," as used herein, may mean at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20. In one embodiment, when "one or more" precedes a list, "one or more" may mean every element of the list. Similarly, the term "at least one," as used herein, may mean at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20. In one embodiment, when "at least one" precedes a list, "at least one" may mean every element of the list.

[0226] The term "disorder" as used herein also encompasses "disease." In one embodiment, the disorder is a disease.

[0227] The modified BoNT / A of the present invention can be formulated in any suitable manner for administration to a subject, for example, as part of a pharmaceutical composition. Such a pharmaceutical composition can include the modified BoNT / A of the present invention, a pharma- ceutically acceptable carrier, excipient, adjuvant, propellant, and / or salt.

[0228] 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 a solution, suspension, or emulsion, or a dry powder that is dissolved or suspended in a suitable solvent before use.

[0229] In the case of a modified BoNT / A delivered locally, the modified BoNT / A can be formulated as a cream (eg, for topical application) or for subcutaneous injection.

[0230] Local delivery means can include aerosols or other sprays (e.g., nebulizers). In this regard, aerosol formulations of modified BoNT / A allow for delivery to the lungs and / or other nasal and / or bronchi or airways.

[0231] Liquid dosage forms are usually prepared using modified BoNT / A and a sterile pyrogen-free solvent. Depending on the solvent and concentration used, modified BoNT / A may be dissolved or suspended in the solvent. In preparing a solution, modified BoNT / A may be dissolved in the solvent, sodium chloride may be added if necessary to make the solution isotonic, sterilized by filtration through a sterile filter using aseptic techniques, 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 may be sterilized by autoclaving. Advantageously, additives such as buffers, solubilizers, stabilizers, preservatives or bactericides, suspending or emulsifying agents, and / or local anesthetics may be dissolved in the solvent.

[0232] The pre-sterilized ingredients may be filled into a sterile container using aseptic techniques in a sterile area to prepare a dry powder that is dissolved or suspended in a suitable solvent prior to use. Alternatively, the ingredients may be dissolved in a suitable container using aseptic techniques in a sterile area. The product is then lyophilized and the container is aseptically sealed.

[0233] Parenteral suspensions suitable for the routes of administration described herein are prepared in substantially the same manner, except that the sterile ingredients are suspended in a sterile solvent rather than dissolved, and sterilization cannot be achieved by filtration. The ingredients may be separated under sterile conditions or may be sterilized after separation, for example, by gamma irradiation.

[0234] Advantageously, the composition contains a suspending agent, such as polyvinylpyrrolidone, to promote uniform distribution of the ingredients.

[0235] Administration according to the present invention may utilize a variety of delivery techniques, including microparticle encapsulation or high pressure aerosol impaction.

[0236] In one aspect, the invention provides a unit dosage form of a modified BoNT / A (eg, for treating cervical dystonia), the unit dosage form comprising: a. More than 707 units of modified BoNT / A, where 1 unit is the calculated median lethal dose (LD 50 or b. greater than 17,000 pg of modified BoNT / A; and c. optionally, pharma- ceutically acceptable carriers, excipients, adjuvants, and / or salts; Here, the modified BoNT / A comprises a BoNT / A light chain and translocation domain, and a BoNT / B receptor binding domain (H C domain).

[0237] The modified BoNT / A in unit dosage form preferably 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 (and more preferably consist of) SEQ ID NO: 6.

[0238] The unit dosage form may contain more than 707 units of modified BoNT / A. The upper end of the range of modified BoNT / A may be 1664, 1650, 1600, 1550, 1500, 1450, 1400, 1350, 1300, 1250, 1150, 1100, 1050, 1000, 950, 900, 850, 800 or 750 units, preferably the upper end is 1500 units. The lower limit of the range of modified BoNT / A may 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, preferably the lower limit is 728 units or 1,040 units. Preferably the unit dosage form contains more than 707 units up to 1664 units of modified BoNT / A, for example, more than 707 units up to 1498 units, 832 units to 1622 units. Most preferably the unit dosage form contains 915 to 1581 units of modified BoNT / A, for example, 998 to 1498 units. The unit dosage form may contain 10,399 units to 16,639 units of modified BoNT / A. The unit dosage form may contain 1,040 units up to 1,664 units of modified BoNT / A. In preferred embodiments, the unit dosage form contains 998, 1,248, 1,040 or 1,498 units, e.g., 1,040 or 1,498 units of modified BoNT / A. In more preferred embodiments, the unit dosage form contains 1,248 or 1,498 units (e.g., 1,248 units) of modified BoNT / A.

[0239] The unit dosage form may contain between 832 and 1,248 units of modified BoNT / A, for example between 998 and 1,082 units. Most preferably, the unit dosage form may contain 1,040 units of modified BoNT / A.

[0240] The unit dosage form may contain between 1,248 and 1,664 units of the modified BoNT / A, for example between 1,456 and 1,539 units. Most preferably, the unit dosage form may contain 1,498 units of the modified BoNT / A.

[0241] The unit dosage form may contain more than 17,000 pg of modified BoNT / A. The upper end of the range of modified BoNT / A may 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, preferably the upper end is 38,000 pg. The lower limit of the range of modified BoNT / A may 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, preferably the lower limit is 17,500 pg or 25,000 pg. Preferably, the unit dosage form contains more than 17,000 pg, up to 40,000 pg, e.g., more than 17,000 pg, up to 36,000 pg, 20,000 pg to 39,000 pg of modified BoNT / A. Most preferably, the unit dosage form contains 22,000 to 38,000 pg, e.g., 24,000 to 36,000 pg, or 25,000 to 36,000 pg of modified BoNT / A. The unit dosage form may contain 25,000 pg up to 40,000 pg of modified BoNT / A. In preferred embodiments, the unit dosage form contains 24,000, 25,000, 30,000 or 36,000 pg, e.g., 25,000 or 36,000 pg of modified BoNT / A. In more preferred embodiments, the unit dosage form contains 30,000 or 36,000 ng (e.g., 36,000 pg) of modified BoNT / A.

[0242] A unit dosage form may contain between 20,000 pg and 30,000 pg, for example between 24,000 pg and 26,000 pg, of the modified BoNT / A. Most preferably, the unit dosage form may contain 25,000 pg of the modified BoNT / A.

[0243] The unit dosage form may contain between 30,000 pg and 40,000 pg, for example between 35,000 pg and 37,000 pg, of the modified BoNT / A. Most preferably, the unit dosage form may contain 36,000 pg of the modified BoNT / A.

[0244] The unit dosage form is preferably provided as a dry powder.

[0245] In another aspect, the present invention provides a kit comprising: a. a unit dosage form according to the present invention; b. instructions for use of the kit in treating cervical dystonia; and c. Optional diluent.

[0246] The embodiments of the present invention relating to the various therapeutic applications can be applied to the methods, unit dosage forms, and kits of the present invention, and vice versa.

[0247] sequence homology

[0248] Any of a variety of sequence alignment methods may be used to determine percent identity, including, but not limited to, global, local, and hybrid methods, such as segmental approaches. Protocols for determining percent identity are routine procedures within the skill of the art. Global methods align the sequence from beginning to end of the molecule, summing the scores of individual residue pairs, and determine optimal alignment by imposing gap penalties. Non-limiting methods include, for example, CLUSTAL W (see, e.g., 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 refinement (see, e.g., Osamu Gotoh, Significant Improvement in Accuracy of Multiple Protein. Sequence Alignments by Iterative Refinement as Assessed by Reference to Structural Alignments, 264(4) J. MoI. Biol. 823-838 (1996)). Local methods align sequences by identifying one or more conserved motifs shared by all input sequences.Non-limiting methods include, for example, Match-box (see, e.g., Eric Depiereux and Ernest Feytmans, Match-Box: A Fundamentally New Algorithm for the Simultaneous Alignment of Several Protein Sequences, 8(5) CABIOS 501 -509 (1992)); Gibbs sampling (see, e.g., CE Lawrence et al., Detecting Subtle Sequence Signals: A Gibbs Sampling Strategy for Multiple Alignment, 262(5131) Science 208-214 (1993)); Align-M (see, e.g., Ivo Van Walle et al., Align-M - A New Algorithm for Multiple Alignment of Highly Divergent Sequences, 20(9) Bioinformatics:1428-1435 (2004)).

[0249] Thus, the percent sequence identity is determined by conventional methods. See, for example, Altschul et al., Bull. Math. Bio. 48: 603-16, 1986 and Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915-19, 1992. Briefly, the two amino acid sequences are aligned to optimize the alignment score using a gap opening penalty of 10, a gap extension penalty of 1, and the "blosum 62" scoring matrix of Henikoff and Henikoff (ibid.) (amino acids are represented by standard single letter codes) as described below. Preferably, this method is used to align the sequence with the subject sequence herein (e.g., SEQ ID NO: 2) and define the amino acid position numbering as described herein.

[0250] The "percent sequence identity" between two or more nucleic acid or amino acid sequences is a function of the number of identical positions shared by the sequences. Thus, the percent identity can be calculated by dividing the number of identical nucleotides / amino acids by the total number of nucleotides / amino acids, and multiplying it by 100. The calculation of percent sequence identity may also take into account the number of gaps that need to be introduced to optimize the alignment of two or more sequences, and the length of each gap. The sequence comparison and the determination of percent identity between two or more sequences can be performed using a specific mathematical algorithm, such as BLAST, which is well known to those skilled in the art.

[0251] Alignment scores for determining sequence identity

number

[0252] The percent identity is then calculated as follows: [total number of identical matches × 100] / [length of the longer sequence + number of gaps introduced into the longer sequence to align the two sequences]

[0253] Substantially homologous polypeptides are characterized as having one or more amino acid substitutions, deletions, or additions. These changes are preferably of a minor nature, i.e., conservative amino acid substitutions (see below) and other substitutions that do not significantly affect the folding or activity of the polypeptide; small deletions, usually from one to about 30 amino acids; and small amino- or carboxyl-terminal extensions, such as, for example, an amino-terminal methionine residue, a small linker peptide of up to about 20-25 residues, or an affinity tag.

[0254] Conservative Amino Acid Substitutions

[0255] Basicity: Arginine lysine Histidine Acidic: Glutamic acid Aspartic acid polarity: glutamine Asparagine Hydrophobicity: Leucine Isoleucine Ballin Aromatic: Phenylalanine Tryptophan Tyrosine Small size: glycine Alanine Serine Threonine Methionine

[0256] In addition to the 20 standard amino acids, non-standard amino acids (such as 4-hydroxyproline, 6-N-methyllysine, 2-aminoisobutyric acid, isovaline, and α-methylserine) may be substituted for amino acid residues in the polypeptides of the invention. A limited number of non-conservative amino acids, amino acids that are not encoded by the genetic code, and unnatural amino acids may be substituted for polypeptide amino acid residues. The polypeptides of the invention may also include amino acid residues of non-natural origin.

[0257] Non-naturally occurring amino acids include, but are not limited to, trans-3-methylproline, 2,4-methano-proline, cis-4-hydroxyproline, trans-4-hydroxy-proline, N-methylglycine, allo-threonine, methyl-threonine, hydroxy-ethylcysteine, hydroxyethylhomo-cysteine, nitro-glutamine, homoglutamine, pipecolic acid, tert-leucine, norvaline, 2-azaphenylalanine, 3-azaphenyl-alanine, 4-azaphenyl-alanine, and 4-fluorophenylalanine. Several methods are known in the art for incorporating non-naturally occurring amino acid residues into proteins. For example, an in vitro system can be used in which chemically aminoacylated suppressor tRNAs are used to suppress nonsense mutations. Methods for synthesizing amino acids and aminoacylating tRNAs are known in the art. Transcription and translation of plasmids containing nonsense mutations are carried out in a cell-free system containing E. coli S30 extracts, commercially available enzymes, and other reagents. Proteins are 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 method, translation is carried out in Xenopus oocytes by microinjecting mutant mRNA and chemically aminoacylated suppressor tRNAs (Turcatti et al., J. Biol. Chem. 271:19991-8, 1996). In a third method, E. coli cells are cultured in the absence of the natural amino acid to be substituted (e.g., phenylalanine) and in the presence of the desired non-naturally occurring amino acid(s) (e.g., 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, or 4-fluorophenylalanine).Non-naturally occurring 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 species by in vitro chemical modification. Chemical modification can be combined with site-directed mutagenesis to further expand the scope of substitutions (Wynn and Richards, Protein Sci. 2:395-403, 1993).

[0258] A limited number of non-conservative amino acids, amino acids that are not encoded by the genetic code, non-naturally occurring amino acids, and unnatural amino acids may be substituted for amino acid residues in the polypeptides of the invention.

[0259] Essential amino acids in the polypeptides of the invention can be identified by procedures known in the art, such as site-directed mutagenesis or alanine scanning mutagenesis (Cunningham and Wells, Science 244: 1081-5, 1989). Sites of biological interaction can also be determined by physical analysis of the structure, as determined by techniques such as nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, in combination with mutations of putative contact site amino acids. See, e.g., de Vos et al., Science 255:306-12, 1992; Smith et al., J. Mol. Biol. 224:899-904, 1992; Wlodaver et al., FEBS Lett. 309:59-64, 1992. The identity of essential amino acids can also be inferred from analysis of homology with related components of the polypeptides of the invention (e.g., translocation or protease components).

[0260] Multiple amino acid substitutions can be made and tested using known methods of mutagenesis and screening, such as those disclosed by Reidhaar-Olson and Sauer (Science 241:53-7, 1988) or Bowie and Sauer (Proc. Natl. Acad. Sci. USA 86:2152-6, 1989). Briefly, these authors disclose methods for simultaneously randomizing two or more positions in a polypeptide, selecting functional polypeptides, and then sequencing the mutagenized polypeptides to determine the spectrum of allowed substitutions at each position. Other methods that can be used include phage display (e.g., Lowman et al., Biochem. 30:10832-7, 1991; Ladner et al., US Patent No. 5,223,409; Huse, WIPO Publication WO 92 / 06204), and region-directed mutagenesis (Derbyshire et al., Gene 46:145, 1986; Ner et al., DNA 7:127, 1988).

[0261] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Singleton, et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 20 ED., John Wiley and Sons, New York (1994), and Hale & Marham, THE HARPER COLLINS DICTIONARY OF BIOLOGY, Harper Perennial, NY (1991) provide those of ordinary skill in the art with a general dictionary of many of the terms used in this disclosure.

[0262] 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 embodiments of the present disclosure. Numeric ranges include the numbers defining the range. Unless otherwise specified, each nucleic acid sequence is written left to right in 5' to 3' orientation, and amino acid sequences are written left to right in amino to carboxy orientation, respectively.

[0263] The headings provided herein do not limit the various aspects or embodiments of the disclosure.

[0264] Amino acids are described herein using the name, three-letter abbreviation, or one-letter abbreviation of the amino acid. The term "protein" as used herein includes proteins, polypeptides, and peptides. The term "amino acid sequence" as used herein is synonymous with the term "polypeptide" and / or the term "protein". In some instances, the term "amino acid sequence" is synonymous with the term "peptide". In some instances, 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 one-letter and three-letter codes for amino acid residues may be used. The three-letter codes for amino acids are defined in accordance with the Joint Commission on Biochemical Nomenclature (JCBN) of the IUPACIUB. It is also understood that due to the degeneracy of the genetic code, a polypeptide may be encoded by more than one nucleotide sequence.

[0265] Other definitions of terms may appear throughout the specification. Before describing the exemplary embodiments in more detail, it is to be understood that the present disclosure is not limited to the specific embodiments described, as such may vary. It is also to be understood that the technical terms used herein are for the purpose of describing specific embodiments only, and are not intended to be limiting, since the scope of the present disclosure is defined only by the appended claims.

[0266] Where a range of values ​​is presented, each intervening value between the upper and lower limit of that range is also understood to be specifically disclosed, to the tenth of the unit of the lower limit, unless the context clearly dictates otherwise. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the disclosure. The upper and lower limits of these smaller ranges may be independently included or excluded in the range, and each range where either limit, neither limit, or both limits are included in the smaller range is also encompassed within the disclosure and follows where a stated range specifically excludes a limit. Where a stated range includes either or both limits, ranges excluding either or both of those included limits are also included within the disclosure.

[0267] It should be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "modified botulinum neurotoxin A" includes a plurality of such candidate agents, reference to "the modified botulinum neurotoxin A" includes reference to one or more modified botulinum neurotoxins A and equivalents thereof known to those skilled in the art, and so forth.

[0268] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein should be construed as an admission that such publications constitute prior art to the claims appended hereto.

[0269] Embodiments of the invention will now be described, by way of example only, with reference to the following Figures and Examples. [Brief description of the drawings]

[0270] [Figure 1]FIG. 1 shows the U.S. Food and Drug Administration (FDA)-approved dosages of Dysport® for treating cervical dystonia. [Diagram 2] 2 shows SDS-polyacrylamide gel electrophoresis 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). [Diagram 3] FIG. 3 is a graph showing cleavage of SNAP-25 in rat spinal cord neurons by recombinant BoNT / AB chimeras 1, 2, and 3A (SEQ ID NOs: 3, 4, and 5, respectively). Cultured rat primary spinal cord neurons (SCN) were exposed to various concentrations of recombinant BoNT / AB chimeras 1, 2, or 3A for 24 hours at 37°C in a humidified atmosphere containing 10% CO2. Cells were then lysed in 1xNuPAGE buffer supplemented with DTT and benzonase. Samples were transferred to microcentrifuge tubes, heated at 90°C in a heat block for 5 minutes, and stored at -20°C. SNAP-25 cleavage was then analyzed by Western blot. SNAP-25 was detected using a polyclonal antibody that detects both full-length and cleaved forms of SNAP-25 (Sigma #S9684). Anti-rabbit HRP (Sigma #A6154) was used as the secondary antibody. [Figure 4] Figure 4 is a graph showing the mouse digit abduction scoring assay. Mice were injected into the gastrocnemius-soleus muscle complex of one hind limb under brief general anesthesia. The digit abduction score (DAS) was used to measure muscle weakness on a scale of 0 to 4. Maximum DAS values ​​were measured for each dose and plotted against dose, and the data were fitted to a 4-parameter logistic equation to determine the ED50 and the dose producing DAS4 (DAS4 dose). [Diagram 5]5 shows SDS-polyacrylamide gel electrophoresis 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). [Figure 6] FIG. 6 is a graph showing the cleavage of SNAP-25 in human induced pluripotent stem cell-derived peripheral neurons (PERI.4U, Axiogenesis, Germany) by BoNT / A and BoNT / AB chimeras 3B and 3C (SEQ ID NOs: 2, 6 and 7, respectively). PERI.4U cells were exposed to various concentrations of recombinant BoNT / A or BoNT / AB chimeras 3B or 3C for 24 hours at 37°C in a humidified CO2 atmosphere containing 5% CO2. Cells were then lysed with 1xNuPAGE buffer supplemented with DTT and benzonase. Samples were transferred to microcentrifuge tubes, heated at 90°C for 5 minutes in a heat block and stored at -20°C. SNAP-25 cleavage was then analyzed by Western blot. SNAP-25 was detected using a polyclonal antibody that detects both full-length and cleaved forms of SNAP-25 (Sigma #S9684). Anti-rabbit HRP (Sigma #A6154) was used as the secondary antibody. [Figure 7] Figure 7 is a graph showing the duration of muscle weakness in the mouse digit abduction scoring assay. Mice were injected into the gastrocnemius-soleus muscle complex of one hind limb under brief general anesthesia. The digit abduction score (DAS) was used to measure muscle weakness on a scale of 0 to 4. The group of animals injected with the lowest dose, which induced DAS4 during the first 4 days after injection, was monitored until muscle weakness fully recovered to DAS0 (no muscle weakness).

[0271] Sequence Listing

[0272] Where the first Met amino acid residue or the corresponding first codon is shown in any of the SEQ ID NOs below, said residue / codon is optional.

[0273] SEQ ID NO:1 (Nucleotide sequence of unmodified BoNT / A)

[0274] SEQ ID NO:2 (polypeptide sequence of unmodified BoNT / A)

[0275] SEQ ID NO:3 (polypeptide sequence of modified BoNT / A "chimera 1") MPFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLN PPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGG STIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNGY GSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPN RVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKA KSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIYTEDNFVKFFKV LNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTKLKNFT GLFEFYKLLCVRGIITSKTKSLDKGYNKALNDLCIKVNNWDLFFSPSEDNFTNDLNKGEE ITSDTNIEAAEENISLDLIQQYYLTFNFDNEPENISIENLSSDIIGQLELMPNIERFPNG KKYELDKYTMFHYLRAQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSDYVKKVNKATEA AMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYKDDFVGALIFSG AVILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALSKRNEKWDEVYKYIVTNWLAK VNTQIDLIRKKMKEALENQAEATKAIINYQYNQYTEEEKNNINFNIDDLSSKLNESINKA MININKFLNQCSVSYLMNSMIPYGVKRLEDFDASLKDALLKYIYDNRGTLIGQVDRLKDK VNNTLSTDIPFQLSKYVDNQRLLSTFTEYIKSEILNNIILNLRYKDNNLIDLSGYGAKVE VYDGVELNDKNQFKLTSSANSKIRVTQNQNIIFNSVFLDFSVSFFWIRIPKYKNDGIQNYI HNEYTIINCMKNNSGWKISIRGNRIIWTLIDINGKTKSVFFEYNIREDISEYINRWFFVT ITNNLNNAKIYINGKLESNTDIKDIREVIANGEIIFKLDGDIDRTQFIWMKYFSIFNTEL SQSNIEERYKIQSYSEYLKDFWGNPLMYNKEYYMFNAGNKNSYIKLKKDSPVGEILTRSK YNQNSKYINYRDLYIGEKFIIRRKSNSQSINDDIVRKEDYIYLDFFNLNQEWRVYTYKYF KKEEMKLFLAPIYDSDEFYNTIQIKEYDEQPTYSCQLLFKKDEESTDEIGLIGIHRFYES GIVFEEYKDYFCISKWYLKEVKRKPYNLKLGCNWQFIPKDEGWTEHHHHHHHHHH

[0276] SEQ ID NO: 4 (polypeptide sequence of modified BoNT / A "chimera 2") MPFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLN PPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGG STIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNGY GSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPN RVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKA KSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIYTEDNFVKFFKV LNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTKLKNFT GLFEFYKLLCVRGIITSKTKSLDKGYNKALNDLCIKVNNWDLFFSPSEDNFTNDLNKGEE ITSDTNIEAAEENISLDLIQQYYLTFNFDNEPENISIENLSSDIIGQLELMPNIERFPNG KKYELDKYTMFHYLRAQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSDYVKKVNKATEA AMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYKDDFVGALIFSG AVILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALSKRNEKWDEVYKYIVTNWLAK VNTQIDLIRKKMKEALENQAEATKAIINYQYNQYTEEEKNNINFNIDDLSSKLNESINKA MININKFLNQCSVSYLMNSMIPYGVKRLEDFDASLKDALLKYIYDNRGTLIGQVDRLKDK VNNTLSTDIPFQLSKYVDNQRLLSTFTEYIKNIIELGGGGSELSEILNNIILNLRYKDNN LIDLSGYGAKVEVYDGVELNDKNQFKLTSSANSKIRVTQNQNIIFNSVFLDFSVSFWIRI PKYKNDGIQNYIHNEYTIINCMKNNSGWKISIRGNRIIWTLIDINGKTKSVFFEYNIRED ISEYINRWFFVTITNNNLNNAKIYINGKLESNTDIKDIREVIANGEIIFKLDGDIDRTQFI WMKYFSIFNTELSQSNIEERYKIQSYSEYLKDFWGNPLMYNKEYYMFNAGNKNSYIKLKK DSPVGEILTRSKYNQNSKYINYRDLYIGEKFIIRRKSNSQSINDDIVRKEDYIYLDFFNL NQEWRVYTYKYFKKEEMKLFLAPIYDSDEFYNTIQIKEYDEQPTYSCQLLFKKDEESTDE IGLIGIHRFYESGIVFEEYKDYFCISKWYLKEVKRKPYNLKLGCNWQFIPKDEGWTEHHH HHHHHHH

[0277] SEQ ID NO:5 (polypeptide sequence of modified BoNT / A "chimera 3A") MPFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLN PPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGG STIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNGY GSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPN RVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKA KSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIYTEDNFVKFFKV LNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTKLKNFT GLFEFYKLLCVRGIITSKTKSLDKGYNKALNDLCIKVNNWDLFFSPSEDNFTNDLNKGEE ITSDTNIEAAEENISLDLIQQYYLTFNFDNEPENISIENLSSDIIGQLELMPNIERFPNG KKYELDKYTMFHYLRAQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSDYVKKVNKATEA AMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYKDDFVGALIFSG AVILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALSKRNEKWDEVYKYIVTNWLAK VNTQIDLIRKKMKEALENQAEATKAIINYQYNQYTEEEKNNINFNIDDLSSKLNESINKA MININKFLNQCSVSYLMNSMIPYGVKRLEDFDASLKDALLKYIYDNRGTLIGQVDRLKDK VNNTLSTDIPFQLSKYVDNQRLLSTFTEYIKNILNNIILNLRYKDNNLIDLSGYGAKVEV YDGVELNDKNQFKLTSSANSKIRVTQNQNIIFNSVFLDFSVSFWIRIPKYKNDGIQNYIH NEYTIINCMKNNSGWKISIRGNRIIWTLIDINGKTKSVFFEYNIREDISEYINRWFFVTI TNNLNNAKIYINGKLESNTDIKDIREVIANGEIIFKLDGDIDRTQFIWMKYFSIFNTELS QSNIEERYKIQSYSEYLKDFWGNPLMYNKEYYMFNAGNKNSYIKLKKDSPVGEILTRSKY NQNSKYINYRDLYIGEKFIIRRKSNSQSINDDIVRKEDYIYLDFFNLNQEWRVYTYKYFK KEEMKLFLAPIYDSDEFYNTIQIKEYDEQPTYSCQLLFKKDEESTDEIGLIGIHRFYESG IVFEEYKDYFCISKWYLKEVKRKPYNLKLGCNWQFIPKDEGWTEHHHHHHHHHH

[0278] SEQ ID NO:6 (Polypeptide sequence of modified BoNT / A "chimera 3B")

[0279] SEQ ID NO: 7 (Polypeptide sequence of modified BoNT / A "chimera 3C") MPFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLN PPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGG STIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNGY GSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPN RVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKA KSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIYTEDNFVKFFKV LNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTKLKNFT GLFEFYKLLCVRGIITSKTKSLDKGYNKALNDLCIKVNNWDLFFSPSEDNFTNDLNKGEE ITSDTNIEAAEENISLDLIQQYYLTFNFDNEPENISIENLSSDIIGQLELMPNIERFPNG KKYELDKYTMFHYLRAQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSDYVKKVNKATEA AMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYKDDFVGALIFSG AVILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALSKRNEKWDEVYKYIVTNWLAK VNTQIDLIRKKMKEALENQAEATKAIINYQYNQYTEEEKNNINFNIDDLSSKLNESINKA MININKFLNQCSVSYLMNSMIPYGVKRLEDFDASLKDALLKYIYDNRGTLIGQVDRLKDK VNNTLSTDIPFQLSKYVDNQRLLSTFTEYIKNILNNIILNLRYKDNNLIDLSGYGAKVEV YDGVELNDKNQFKLTSSANSKIRVTQNQNIIFNSVFLDFSVSFFWIRIPKYKNDGIQNYIH NEYTIINCMKNNSGWKISIRGNRIIWTLIDINGKTKSVFFEYNIREDISEYINRWFFVTI TNNLNNNAKIYINGKLESNTDIKDIREVIANGEIIFKLDGDIDRTQFIWMKYFSIFTELS QSNIEERYKIQSYSEYLKDFWGNPLMYNKEYYMFNAGNKNSYIKLKKDSPVGEILTRSKY NQNSKYINYRDLYIGEKFIIRRKSNSQSINDDIVRKEDYIYLDFFNLNQEWRVYTYKYFK KEEEKLFLAPISDSDEFYNTIQIKEYDEQPTYSCQLLFKKDEESTDEIGLIGIHRFYESG IVFEEYKDYFCISKWYLKEVKRKPYNLKLGCNWQFIPKDEGWTE

[0280] SEQ ID NO:8 (Polypeptide sequence of BoNT / B) MPVTINNFNYNDPIDNNNIIMMEPPFARGTGRYYKAFKITDRIWIIPERYTFGYKPEDFN KSSGIFNRDVCEYYDPDYLNTNDKKNIFLQTMIKLFNRIKSKPLGEKLLEMIINGIPYLG DRRVPLEEFNTNIASVTVNKLISNPGEVERKKGIFANLIIFGPGPVLNENETIDIGIQNH FASREGFGGIMQMKFCPEYVSVFNNVQENKGASIFNRRGYFSDPALILMHELIHVLHGLY GIKVDDLPIVPNEKKFFMQSTDAIQAEELYTFGGQDPSIITPSTDKSIYDKVLQNFRGIV DRNLKVLVCISDPNININIYKNKFKDKYKFVEDSEGKYSIDVESFDKLYKSLMFGFTETN IAENYKIKTRASYFSDSLPPVKIKNLLDNEIYTIEEGFNISDKDMEKEYRGQNKAINKQA YEEISKEHLAVYKIQMCKSVKAPGICIDVDNEDLFFIADKNSFSDDLSKNERIEYNTQSN YIENDFPINELILDTDLISKIELPSENTESLTDFNVDVPVYEKQPAIKKIFTDENTIFQY LYSQTFPLDIRDISLTSSFDDALLFSNKVYSFFSMDYIKTANKVVEAGLFAGWVKQIVND FVIEANKSNTMDKIADISLIVPYIGLALNVGNETAKGNFENAFEIAGASILLEFIPELLI PVVGAFLLESYIDNKNKIIKTIDNALTKRNEKWSDMYGLIVAQWLSTVNTQFYTIKEGMY KALNYQAQALEEIIKYRYNIYSEKEKSNINIDFNDINSKLNEGINQAIDNINNFINGCSV SYLMKKMIPLAVEKLLDFDNTLKKNLLNYIDENKLYLIGSAEYEKSKVNKYLKTIMPFDL SIYTNTILIEMFNKYNSEILNNIILNLRYKDNNLIDLSGYGAKVYDGVELNDKNQFK LTSSANSKIRVTQNQNIIFNSVFLDFSVSFWIRIPKYKNDGIQNYIHNEYTIINCMKNNS GWKISIRGNRIIWTLIDINGKTKSVFFEYNIREDISEYINRWFFVTITNNNLNNNAKIYING KLESNTDIKDIREVIANGEIIFKLDGDIDRTQFIWMKYFSIFTELSQSNIEERYKIQSY SEYLKDFWGNPLMYNKEYYMFNAGNKNSYIKLKKDSPVGEILTRSKYNQNSKYINYRDLY IGEKFIIRRKSNSQSINDDIVRKEDYIYLDFFNLNQEWRVYTYKYFKKEEEKLFLAPISD SDEFYNTIQIKEYDEQPTYSCQLLFKKDEESTDEIGLIGIHRFYESGIVFEEYKDYFCIS KWYLKEVKRKPYNLKLGCNWQFIPKDEGWTE

[0281] SEQ ID NO:9 - C-terminal light chain fragment TKSLDKGYNK

[0282] SEQ ID NO:10 - C-terminal light chain fragment 2 SLDKGYNK

[0283] SEQ ID NO:11 - double light chain 1 PFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGGSTIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNGYGSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPNRVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKAKSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIYTEDNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTKLKNFTGLFEFYKLLCVRGIITSK

[0284] Sequence number 12 - double-stranded L chain 2 PFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGGSTIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNGYGSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPNRVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKAKSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIYTEDNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTKLKNFTGLFEFYKLLCVRGIITSKTK

[0285] Sequence number 13 - double-stranded H chain ALNDLCIKVNNWDLFFSPSEDNFTNDLNKGEEITSDTNIEAAEENISLDLIQQYYLTFNFDNEPENISIENLSSDIIGQLELMPNIERFPNGKKYELDKYTMFHYLRAQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSDYVKKVNKATEAAMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPANLIGNMLYKDDFVGALIFSGAV ILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALSKRNEKWDEVYKYIVTNWLAKVNTQIDLIRKKMKEALENQAEEATKAIINYQYNQYTEEEKNNINFNIDDLSSKLNESINKAMININKFLNQCSVSYLMNSMIPYGVKRLEDFDASLKDALLKYIYDNRGTLIGQVDRLKDKVNNTLSTDIPFQLSKYVDNQRLLSTFTEYIKNILNN IILNLRYKDNNLIDLSGYGAKVEVYDGVELNDKNQFKLTSSANSKIRVTQNQNIIFNSVFLDFSVSFWIRIPKYKNDGIQNYIHNEYTIINCMKNNSGWKISIRGNRIIWTLIDINGKTKSVFFEYNIREDISEYINRWFFVTITNNLNNAKIYINGKLESNTDIKDIREVIANGEIIFKLDGDIDRTQFIWMKYFSIFNTELSQSNIEERYKI QSYSEYLKDFWGNPLMYNKEYYMFNAGNKNSYIKLKDSPVGEILTRSKYNQNSKYINYRDLYIGEKFIIRRKSNSQSINDDIVRKEDYIYLDFFNNLNQEWRWYTYKYFKKEEMKLFLAPIYDSDEFYNTIQIKEYDEQPTYSCQLLFKKDEESTDEIGLIGIHRFYESGIVFEEYKDYFCISKWYLKEVKRKPYNLKGCNWQFIPKDEGWTE

example

[0286] Example 1

[0287] Cloning, expression, and purification of modified BoNT / A (BoNT / AB chimera)

[0288] BoNT / A chimeric constructs 1, 2, 3A, 3B and 3C (SEQ ID NOs: 3-7, respectively) were constructed from DNA encoding the parent serotype molecules and appropriate oligonucleotides using standard molecular biology techniques. These constructs were then modified to include a C-terminal His 10 The vectors were cloned with or without tags into the pJ401 expression vector and transformed into BLR(DE3) E. coli cells for overexpression. The cells were grown in 2 L baffled conical flasks containing 1 L of modified terrific broth (mTB) supplemented with the appropriate antibiotics at 37°C with shaking at 225 RPM. 600 After reaching >0.5, the incubator temperature was lowered to 16° C. and 1 hour later induced with 1 mM IPTG for 20 hours with shaking at 225 RPM to allow expression of the recombinant BoNT / AB construct.

[0289] Harvested cells were lysed by sonication and clarified by centrifugation at 4500 RPM for 1 hour at 4°C. The recombinant BoNT / AB chimera molecule was then extracted with ammonium sulfate and purified by standard fast protein liquid chromatography (FPLC) techniques, including capture using a hydrophobic interaction resin and intermediate purification steps using an anion exchange resin. The partially purified molecule was then proteolytically cleaved with endoprotease Lys-C to yield the active duplex, which was further purified by a second hydrophobic interaction resin to yield the final BoNT / AB chimera.

[0290] Decahistidine tag (H 10 For the BoNT / AB chimera molecules (chimeras 1, 2, and 3A) having the above structure, immobilized nickel resin was used in place of the hydrophobic interaction resin in the capture step.

[0291] The sequences of each chimera are shown in Table 1. [Table 1]

[0292] Example 2

[0293] Comparison of BoNT / AB chimeras 1, 2, and 3A

[0294] C-terminal His 10 BoNT / AB chimeras 1, 2 and 3A carrying the tag and the E1191M / S1199Y double mutation were purified as described in Example 1 (FIG. 2) and tested for functional activity.

[0295] Rat spinal cord neuron SNAP-25 cleavage assay

[0296] Primary cultures of rat spinal cord neurons (SCN) were prepared in 96-well tissue culture plates and grown for 3 weeks (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-fed medium. Growth medium was removed from treatment wells and filtered (0.2 μm filter). 125 μL of filtered medium was added back to each test well. Then, 125 μL of diluted toxin was added to the plate (triplicate wells). Treated cells were incubated at 37°C in 10% CO2 for 24 ± 1 h.

[0297] Analysis of BoNT activity using the SNAP-25 cleavage assay

[0298] After treatment, BoNT was removed and cells were washed once with PBS (Gibco, UK). Cells were lysed in 1x NuPAGE lysis buffer (Life Technologies) supplemented with 0.1M dithiothreitol (DTT) and 250 units / mL benzonase (Sigma). Lysate proteins were separated by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. Membranes were probed with a primary antibody specific for SNAP-25 (Sigma #S9684), which recognizes uncleaved SNAP-25 as well as BoNT / A endopeptidase-cleaved SNAP-25. HRP-conjugated anti-rabbit IgG (Sigma #A6154) was used as the secondary antibody. Bands were detected by enhanced chemiluminescence and imaged using pXi6 Access (Synoptics, UK). Band intensities were quantified using GeneTools software (Syngene, Cambridge, UK) and the percentage of SNAP-25 cleaved by each concentration of BoNT was calculated. Data were fitted to a 4-parameter logistic equation to obtain pEC 50 was calculated using GraphPad Prism version 6 (GraphPad Inc.).

[0299] Table 2 below shows the pECs of chimeras 1, 2 and 3A measured in the rat SCN SNAP-25 cleavage assay. 50 These results indicate that the three BoNT / AB chimeras retained 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 FIG. 3). [Table 2]

[0300] Digital Abduction Scoring (DAS) Assay

[0301] The method to measure the activity of BoNT / AB chimeras 1, 2, and 3A in the DAS assay is based on the toe extension reflex, a startle response when mice are briefly suspended by their tail. This reflex, scored as the digit abduction score (DAS), is suppressed after BoNT is administered into the gastrocnemius-soleus muscles of the hind limb. Mice are briefly suspended by their tail to elicit a characteristic startle response in which the animals extend their hind limbs and abduct their hind digits (Aoki et al. 1999, Eur. J. Neurol.; 6 (suppl. 4) S3-S10).

[0302] On the day of injection, mice were anesthetized with 3% isoflurane in oxygen in an induction chamber. Each mouse received an intramuscular injection of the BoNT / AB chimera or vehicle (phosphate buffer containing 0.2% gelatin) into the gastrocnemius and soleus muscles of the right hind limb.

[0303] After neurotoxin injection, the change in the degree of finger abduction was scored on a scale of 0 to 4 (0=normal and 4=maximal reduction in finger abduction and leg extension). 50 was determined by nonlinear adjustment analysis using the mean maximum effect at each dose. The mathematical model used was a four-parameter logistic model.

[0304] The DAS was performed every 2 hours on the day after dosing, and then three times a day for four days.

[0305] Figure 4 shows the fitted curves for chimeras 1, 2 and 3A (SEQ ID NOs: 3, 4 and 5, respectively, converted to double-stranded form). The curve for chimera 3A is shifted to the left, implying that lower doses of chimera 3A produced DAS responses comparable to chimeras 1 and 2, and therefore chimera 3A is more potent in the mouse DAS assay than the other two chimeras. Calculated ED 50 See also the table below (Table 3) showing the dose values ​​that resulted in DAS4 (maximum value) in each chimera.

[0306] Table 3 below shows the ED of recombinant BoNT / A1 (rBoNT / A1 - SEQ ID NO: 2 converted to a two-chain form) and chimeras 1, 2 and 3A as measured in the mouse DAS assay. 50 and DAS4 doses are shown. These results show that of the three chimeras, chimera 3A has the highest in vivo potency to induce muscle weakness. The study shown in Figure 4 and Table 3 was performed in mice obtained from Charles River laboratories. [Table 3]

[0307] Example 3

[0308] Comparison of BoNT / AB chimeras 3B, 3C and unmodified BoNT / A

[0309] The untagged BoNT / AB chimeras 3B and 3C (sequence numbers 6 and 7), respectively, with and without the E1191M / S1199Y double mutation, were purified as described in Example 1 (Figure 2) and tested for functional activity using unmodified BoNT / A (sequence number 2 converted to a two-chain form) as a reference.

[0310] Human pluripotent stem cell SNAP-25 cleavage assay

[0311] Cryopreserved PERI.4U cells were purchased from Axiogenesis (Cologne, Germany). Thawing and plating of cells was performed as recommended by the manufacturer. Briefly, a cryopreservation vial containing cells was thawed in a water bath at 37°C for 2 minutes. After gentle resuspension, the cells were transferred to a 50 mL tube. The cryopreservation vial was washed with 1 mL of Peri.4U® Thawing Medium provided by the manufacturer, and the medium was added dropwise to the cell suspension in the 50 mL tube, followed by an additional 2 mL of Peri.4U® Thawing Medium added dropwise to the 50 mL tube. The cells were then counted using a hemocytometer. After this, an additional 6 mL of Peri.4U® Thawing Medium was added to the cell suspension. A cell pellet was obtained by centrifugation at 260xg (e.g., 1,100 RPM) for 6 minutes at room temperature. The cells were then resuspended in complete Peri.4U® Medium provided by the manufacturer. Cells were plated on cell culture plates coated with poly-L-ornithine and laminin at 37 °C. 2 Cells were cultured at 37°C in a humidified CO2 atmosphere, and the medium was completely changed every 2-3 days during culture.

[0312] For toxin treatment, serial dilutions of BoNT were prepared in Peri.4U® medium. From treatment wells, medium was collected and filtered (0.2 μm filter). 125 μL of filtered medium was added back to each test well. Then, 125 μL of diluted toxin was added to the plate (triplicate wells). Treated cells were incubated at 37° C., 10% CO2 for 48±1 h.

[0313] Analysis of BoNT activity using the SNAP-25 cleavage assay

[0314] After treatment, BoNT was removed and cells were washed once with PBS (Gibco, UK). Cells were lysed in 1x NuPAGE lysis buffer (Life Technologies) supplemented with 0.1M dithiothreitol (DTT) and 250 units / mL benzonase (Sigma). Lysate proteins were separated by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. Membranes were probed with a primary antibody specific for SNAP-25 (Sigma #S9684), which recognizes uncleaved SNAP-25 as well as BoNT / A endopeptidase-cleaved SNAP-25. HRP-conjugated anti-rabbit IgG (Sigma #A6154) was used as the secondary antibody. Bands were detected by enhanced chemiluminescence and imaged using pXi6 Access (Synoptics, UK). Band intensities were quantified using GeneTools software (Syngene, Cambridge, UK) and the percentage of SNAP-25 cleaved by each concentration of BoNT was calculated. Data were fitted to a 4-parameter logistic equation to obtain pEC 50 was calculated using GraphPad Prism version 6 (GraphPad Inc.).

[0315] Figure 6 shows that chimeras 3B and 3C exhibited stronger potency than rBoNT / A1 in cleaving SNAP-25 in human induced pluripotent stem cells, but the potency was more pronounced for chimera 3B, which can be explained by a double mutation that increases the affinity of chimera 3B for the human synaptotagmin II protein receptor present in these cells (Figure 6, Table 4). [Table 4]

[0316] Digital Abduction Scoring (DAS) Assay - Safety Factor

[0317] The method to measure the activity of BoNT in the DAS assay is based on the toe extension reflex, a startle response when mice are briefly suspended by their tail. This reflex, scored as the digit abduction score (DAS), is suppressed after BoNT is administered into the gastrocnemius-soleus muscles of the hind limb. Mice are briefly suspended by their tail to elicit a characteristic startle response in which the animals extend their hind limbs and abduct their hind digits (Aoki et al. 1999, Eur. J. Neurol.; 6 (suppl. 4) S3-S10).

[0318] On the day of injection, mice were anesthetized with 3% isoflurane in oxygen in an induction chamber. Each mouse received BoNT or vehicle (phosphate buffer containing 0.2% gelatin) via intramuscular injection into the gastrocnemius and soleus muscles of the right hind limb.

[0319] After neurotoxin injection, the change in the degree of finger abduction was scored on a scale of 0 to 4 (0=normal and 4=maximal reduction in finger abduction and leg extension). 50 was determined by nonlinear adjustment analysis using the mean maximum effect at each dose. The mathematical model used was a four-parameter logistic model.

[0320] The day after dosing, DAS was performed every 2 hours. Thereafter, DAS was performed three times a day for 4 days for all doses. Animals injected with vehicle and the lowest dose that induced DAS4 during the first 4 days after injection were then monitored until muscle weakness was fully restored to DAS0 (no muscle weakness).

[0321] To calculate the safety factor, all animals were weighed the day before toxin injection (D0) and once a day thereafter throughout the duration of the study. The mean body weight, its standard deviation, and standard error of the mean were calculated for each dose group on a daily basis. The safety factor of BoNT (-10%ΔBW / ED 50 ) was defined as the dose at which the mean weight of a dose group was 10% lower than the mean weight of the same dose group at D0 at any time during the study. 50A lethal dose was defined as the dose at which one or more animals died within that dose group.

[0322] Figure 7 is a graph showing the duration of muscle weakness in a mouse digit abduction scoring assay for unmodified BoNT / A, chimera 3B, and chimera 3C (sequence numbers 2, 6, and 7 converted to two-chain forms), indicating that the chimeras have a longer duration of action.

[0323] Table 5 below shows the EDs measured for rBoNT / A1 and chimeras 3B and 3C in the mouse DAS assay. 50 The table also shows the total duration of action of the DAS4 dose until muscle weakness was fully restored to DAS0 (no muscle weakness). Additionally, the table shows the lethal dose and safety factor (-10%ΔBW / ED 50 ) are shown. Compared to rBoNT / A1, chimeras 3B and 3C have a longer duration of action, a higher safety margin, and a higher lethal dose. The study shown in Figure 7 and Table 5 was performed in mice obtained from Janvier laboratories. [Table 5]

[0324] Example 4

[0325] Preclinical testing of modified BoNT / A (BoNT / AB chimera [SEQ ID NO: 6 converted to two-chain form])

[0326] The BoNT / AB chimera SEQ ID NO:6 converted to a two-chain form was incubated with mouse LD 50 When tested in the assay, a result of 1.202 ng / kg was obtained. Therefore, 1 unit of SEQ ID NO:6 corresponds to 24.04 pg in this assay.

[0327] Additionally, the BoNT / AB chimera was tested in a rat DAS assay to determine duration of action compared to Dysport®, and the results are shown in Table 6 below. [Table 6]

[0328] In conclusion, the duration of action of BoNT / AB was much longer than that of Dysport®.

[0329] Example 5

[0330] Determination of the unit dose of modified BoNT / A (SEQ ID NO: 6 converted to a two-chain form) for treating cervical dystonia

[0331] Taking into account the preclinical pharmacology data, a suitable unit dose range (UD) for administration of modified BoNT / A in humans was determined.

[0332] DAS ED of SEQ ID NO:6 50 The calculated ED was 13 pg / kg. 50 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 corresponds to a dose of 0.8 ng for a 60 kg human.

[0333] Therefore, 1,000 pg was selected as the lower limit of the unit dose. 16,000 pg was selected as the upper limit of the unit dose, which is lower than the NOAEL of 4 ng / kg obtained from both non-clinical safety species (rats and monkeys) converted to a human dose of 60 kg body weight. Therefore, the unit dose was determined to be 1,000 pg to 16,000 pg (approximately 42 units to approximately 666 units).

[0334] Considering the improved safety profile, the maximum total dose for the treatment of cervical dystonia was set at 160,000 pg (approximately 7,070 units), derived by converting the NOAEL of 4 ng / kg obtained from both non-clinical safety species (rat and monkey) to a human dose of 60 kg body weight.

[0335] Given the improved safety profile compared to Dysport®, as determined by the preclinical data in Example 4, the total dose (units) administered for cervical dystonia is expected to be approximately 7-fold higher than Dysport®. (登録商標) The maximum total dose is 1,000 units (see Figure 1).

[0336] Advantageously, in treating cervical dystonia, more modified BoNT / A (SEQ ID NO: 6) can be injected and / or more neck muscles / sites can be injected before a maximum dose is reached, an important and advantageous discovery that could lead to improved treatment of cervical dystonia, providing clinicians with a wider range of treatment options.

[0337] Example 6

[0338] Dosage regimen for treating cervical dystonia with modified BoNT / A (SEQ ID NO: 6 converted to a two-stranded form)

[0339] The modified BoNT / A (eg, SEQ ID NO:6 converted to a di-stranded form) is provided as a lyophilized powder in vials containing 36 ng of modified BoNT / A per vial. The lyophilized powder is reconstituted.

[0340] The unit dose (UD) is 1,000 to 16,000 pg (approximately 42 to 666 units [mouse LD 50 [Measured by].

[0341] Cervical dystonia is treated by intramuscular injection according to the following dosing regimen (Table 7). [Table 7]

[0342] Administration may be unilateral or bilateral, as necessary, based on the particular condition.

[0343] The maximum total dose administered is 10 unit doses (e.g., in some cases, 2 unit doses are administered to the indicated neck muscle or muscles). This corresponds to 160,000 pg / approximately 6,660 units. This is nearly 7 times the maximum total dose of Dysport® that can be administered during the treatment of cervical dystonia without reaching toxicity limits (a concern in conventional treatment regimens). Thus, clinicians can tailor treatment to their patients with the knowledge that a 10 unit dose can be administered without toxicity concerns, thereby treating additional neck muscles of a subject and / or ensuring that each neck muscle receives a pharmacologic effective dose.

[0344] Example 7

[0345] Treatment of patients with cervical dystonia (lateral cervical flexion)

[0346] Jane, 65, is diagnosed with cervical dystonia by her family doctor. The specific symptom is cervical lateral flexion. A single unit dose (3,000 pg) of modified BoNT / A (SEQ ID NO: 6 converted to a two-stranded form) is administered ipsilaterally to Jane's levator scapulae muscle and a single unit dose is administered ipsilaterally to Jane's sternocleidomastoid muscle (total dose for treatment session is 6,000 pg). The cervical lateral flexion is relieved and due to the long duration of modified BoNT / A, Jane does not require additional treatment for more than 9 months. Thus, Jane receives injections less frequently (e.g., once a year) compared to comparable subjects administered unmodified BoNT / A. Furthermore, Jane does not show any side effects due to the improved safety profile of modified BoNT / A.

[0347] Example 8

[0348] Treatment for patients with cervical dystonia (neck retroflexion)

[0349] Brian, age 48, is diagnosed with cervical dystonia by his family doctor. The specific symptom is neck retroflexion. Modified BoNT / A (SEQ ID NO: 6 converted to a two-stranded form) is administered to Brian in each of the following muscles: ·One unit dose of 10,000 pg into each levator scapulae; ·One unit dose of 10,000 pg in each trapezius muscle; ·One unit dose of 10,000 pg for each longissimus muscle; One unit dose of 10,000 pg into each splenius capitis muscle; and · One unit dose of 10,000 pg into each splenius cervix.

[0350] The total dose was 10 unit doses (100,000 pg), which is well below the upper limit of 160,000 pg and is possible given the higher safety profile of modified BoNT / A compared to unmodified BoNT / A. The neck flexion was relieved and due to the long duration of modified BoNT / A, Brian has not required additional treatment for 12 months. Thus, Brian receives injections less frequently compared to comparable subjects receiving unmodified BoNT / A.

[0351] Example 9

[0352] Safety and efficacy of modified BoNT / A (SEQ ID NO: 6 converted to a two-chain form) in humans

[0353] SEQ ID NO:6 (converted to a two-chain form) was administered to human subjects in a single unit dose of modified BoNT / A. Five cohorts were administered different (increasing) amounts of modified BoNT / A (SEQ ID NO:6 converted to a two-chain form). Cohort 1 received two 1,000 pg unit doses of modified BoNT / A (i.e., up to 2,000 pg), and cohort 5 received two 16,000 pg unit doses of modified BoNT / A (i.e., up to 32,000 pg).

[0354] The results showed that all unit doses of modified BoNT / A tested (i.e., unit doses up to 16,000 pg) were effective in muscle paralysis and were safely tolerated, with no adverse effects observed despite the exceptionally high doses administered per muscle, indicating that modified BoNT / A does not diffuse from the injection site and highlighting the exceptional safety profile of modified BoNT / A (SEQ ID NO: 6 converted to a two-stranded form).

[0355] Example 10

[0356] Dosage regimen for treating cervical dystonia with modified BoNT / A (SEQ ID NO: 6 converted to a two-stranded form)

[0357] The modified BoNT / A (eg, SEQ ID NO:6 converted to a di-stranded form) is provided as a lyophilized powder in vials containing 36 ng of modified BoNT / A per vial. The lyophilized powder is reconstituted.

[0358] The unit dose (UD) is 17,000 to 36,000 pg (approximately 707 to 1,498 units [mouse LD 50 [Measured by].

[0359] Cervical dystonia is treated by intramuscular injection according to the following dosing regimen (Table 8). [Table 8]

[0360] Administration may be unilateral or bilateral, as necessary, based on the particular condition.

[0361] The maximum total dose administered is 10 unit doses (e.g., in some cases, 2 unit doses are administered to the indicated neck muscle or muscles). This corresponds to 360,000 pg / approximately 14,975 units. This is more than 20 times the maximum total dose of Dysport® that can be administered during the treatment of cervical dystonia without reaching toxicity limits (a concern in conventional treatment regimens). Thus, clinicians can tailor treatment to patients with the knowledge that 10 unit doses can be administered without toxicity concerns, thereby allowing treatment of additional neck muscles of a subject and / or ensuring that each neck muscle receives a pharmacologic effective dose.

[0362] Example 11

[0363] Treatment of patients with cervical dystonia (lateral cervical flexion)

[0364] Elizabeth, age 62, is diagnosed with cervical dystonia by her family doctor. The specific symptom is cervical lateral bending. A single unit dose (36,000 pg) of modified BoNT / A (SEQ ID NO: 6 converted to a two-stranded form) is administered ipsilaterally to Elizabeth's levator scapulae muscle and a single unit dose is administered ipsilaterally to Elizabeth's sternocleidomastoid muscle (total dose for treatment session is 72,000 pg). The cervical lateral bending is relieved and due to the long duration of modified BoNT / A, Elizabeth does not require additional treatment for more than 9 months. Thus, Elizabeth receives injections less frequently (e.g., once a year) compared to comparable subjects administered unmodified BoNT / A. Furthermore, Elizabeth does not show any side effects due to the improved safety profile of modified BoNT / A.

[0365] Example 12

[0366] Treatment for patients with cervical dystonia (neck retroflexion)

[0367] Donald, age 54, is diagnosed with cervical dystonia by his family doctor. The specific symptom is neck retroflexion. Modified BoNT / A (SEQ ID NO: 6 converted to a two-stranded form) is administered bilaterally to each of Donald's muscles as follows: ·One unit dose of 36,000 pg into each levator scapulae; ·One unit dose of 36,000 pg in each trapezius muscle; ·One unit dose of 36,000 pg in each longissimus muscle; One unit dose of 36,000 pg into each splenius capitis muscle; and · One unit dose of 36,000 pg into each splenius cervix.

[0368] The total dose administered is a 10 unit dose (360,000 pg), which is possible given the higher safety profile of modified BoNT / A compared to unmodified BoNT / A. The neck flexion is relieved and Donald does not require additional treatment for 12 months due to the long duration of modified BoNT / A. Thus, Donald receives injections less frequently compared to comparable subjects administered unmodified BoNT / A. Furthermore, Donald does not show any side effects due to the improved safety profile of modified BoNT / A.

[0369] Example 13

[0370] Safety and efficacy of modified BoNT / A (SEQ ID NO: 6 converted to a two-chain form) in humans

[0371] Human subjects were administered SEQ ID NO:6 (converted to a two-chain form) by intramuscular injection. Subjects were administered two 15,000 pg unit doses (i.e., 30,000 pg total), two 25,000 pg unit doses (i.e., 50,000 pg total), or two 36,000 pg unit doses (i.e., 72,000 pg total) of modified BoNT / A (SEQ ID NO:6 converted to a two-chain form).

[0372] The results showed that all unit doses of modified BoNT / A tested were effective in muscle paralysis and were safely tolerated, with no adverse effects observed despite exceptionally high doses administered per muscle (e.g., unit doses of 25,000 pg and 36,000 pg), indicating that modified BoNT / A does not diffuse from the injection site and highlighting the exceptional safety profile of modified BoNT / A (SEQ ID NO: 6 converted to a two-stranded form).

[0373] All publications mentioned in the above specification are incorporated herein by reference. Various modifications and variations of the described methods and systems of the invention will be apparent to those skilled in the art that do not depart from the scope and spirit of the invention. Although the invention has been described in connection with certain preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in biochemistry and biotechnology or related fields are intended to be within the scope of the following claims. [Explanation of symbols]

[0374] [Figure 1] Indication: Cervical Dystonia, Adults: Cervical dystonia in adults Recommended Concentration: 50 Units / 0.1 mL or 25 Units / 0.1 mL: 50 Units / 0.1 mL or 25 Units / 0.1 mL Recommended DYSPORT Dose: Recommended DYSPORT Dose 500 Units to 1000 Units: 500 units to 1000 units [Figures 2 and 5] Marker: Marker [Figures 2-7] SEQ ID NO: Sequence number [Figure 3] % SNAP-25 cleavage (in rat spinal cord neurons): Percentage of SNAP-25 cleavage (in rat spinal cord neurons) [Figures 3 and 6] Un-Tr: Untreated [Figure 4] Mean DAS: Average DAS Dose (pg / mouse): Dose (pg / mouse) [Figure 6] % SNAP-25 cleavage (in human pluripotent stem cells): SNAP-25 cleavage percentage (in human pluripotent stem cells) [Figure 7] Time (day): Time (days)

Claims

1. A pharmaceutical product for use in the treatment of cervical dystonia, comprising modified botulinum neurotoxin A (BoNT / A). Herein, the modified BoNT / A is administered to the affected cervical muscle of the subject by intramuscular injection. The modified BoNT / A described above is administered in a unit dose exceeding 17,000 pg. At least a single dose is administered to the affected neck muscle. The total dose of the modified BoNT / A administered during the aforementioned treatment is a maximum of 400,000 pg, and The modified BoNT / A comprises the BoNT / A light chain and transposition domain, and the BoNT / B receptor binding domain (H C (including domain)

2. The pharmaceutical product according to Claim 1. Here, (a) The total dose of the modified BoNT / A administered is between 170,000 pg and 400,000 pg; (b) The total dose of the modified BoNT / A administered is 170,000 pg to a maximum of 360,000 pg; (c) The total dose of the modified BoNT / A administered is between 250,000 pg and 400,000 pg; (d) The total dose of the modified BoNT / A administered is between 200,000 pg and 300,000 pg; (e) The total dose of the modified BoNT / A administered is 300,000 pg to a maximum of 400,000 pg; (f) The total dose of the modified BoNT / A administered during the treatment is a maximum of 250,000 pg; (g) The total dose of the modified BoNT / A administered during the treatment is a maximum of 360,000 pg; and / or (h) The total dose of the modified BoNT / A administered during the treatment is at least 250,000 pg.

3. The pharmaceutical product according to claim 1 or 2. Here, (a) The unit dose of the modified BoNT / A is greater than 17,000 pg and up to 40,000 pg; (b) The unit dose of the modified BoNT / A is 25,000 pg to a maximum of 40,000 pg; and / or (c) The unit dose of the modified BoNT / A is 30,000 pg to a maximum of 40,000 pg.

4. The pharmaceutical product according to claim 1 or 2. Here, The unit dose of the modified BoNT / A is greater than 17,000 pg and a maximum of 36,000 pg; and / or The unit dose of the modified BoNT / A is 20,000 pg to 30,000 pg.

5. The pharmaceutical product according to claim 1 or 2. Here, (a) The unit dose is greater than 17,000 pg and a maximum of 25,000 pg; (b) The unit dose is 25,000 pg; (c) The unit dose is 25,000 pg and the total dose is a maximum of 250,000 pg; and / or (d) The unit dose is 25,000 pg and the total dose is a maximum of 180,000 pg.

6. The pharmaceutical product according to claim 1 or 2. Here, (a) The modified BoNT / A comprises a polypeptide sequence having at least 70% sequence identity with SEQ ID NO: 6, or comprises a polypeptide sequence of SEQ ID NO: 6, or consists of a polypeptide sequence of SEQ ID NO: 6; (b) The modified BoNT / A is a double-stranded modified BoNT / A in which the L chain is linked to the H chain via a disulfide bond, obtained by a method comprising contacting the single-strand modified BoNT / A of Sequence ID No. 6 with a protease that hydrolyzes the peptide bond in its activation loop, thereby converting the single-strand modified BoNT / A to the corresponding double-stranded modified BoNT / A; (c) The modified BoNT / A is a double-stranded modified BoNT / A comprising (or consisting of) a light chain containing SEQ ID NO: 11 or 12 and a heavy chain containing SEQ ID NO: 13, wherein the light chain and the heavy chain are linked to each other by disulfide bonds; and / or (d) The modified BoNT / A has a safety factor of greater than 7, which is calculated by dividing the dose of the toxin measured as pg / mouse required for a -10% change in body weight by the DAS ED 50 measured as pg / mouse, where ED 50 is the dose required to obtain a DAS score of 2.

7. The pharmaceutical product according to claim 1 or 2. Here, (a) The first methionine amino acid residue of the polypeptide sequence of the modified BoNT / A is optional; or (b) The first methionine amino acid residue in the polypeptide sequence of the modified BoNT / A is absent.

8. The pharmaceutical product according to claim 1 or 2. Herein, the affected cervical muscles are selected from the following: sternocleidomastoid muscle, splenius capitis muscle, splenius cervicis muscle, scalene complex, anterior scalene muscle, middle scalene muscle, trapezius muscle, upper trapezius muscle, levator scapulae muscle, semispinalis capitis muscle, longissimus muscle, longissimus capitis muscle, longissimus cervicis muscle, paravertebral posterior and submandibular complex, digastric muscle, geniohyoid muscle, mylohyoid muscle, mylohyoid boutonniere, stylohyoid muscle, lower trapezius muscle, superior nuchal line-clavicular (lateral part), spinous process C3-Th3-mastoid process, spinous process Th3-Th5-transverse process C1-C2, transverse process C3-Th6, spines Process C3-Th1-superior nuchal line, transverse process Th1-Th6-spinous process C2-C7, transverse process C3-Th3-mastoid process, transverse process Th1-Th6-transverse process C2-C6, inferior oblique muscle, spinous process C2-transverse process C1, cervical notch and clavicle (medial part)- Mastoid process and superior nuchal line, transverse process C1-C4 - scapula (superior corner), transverse process C2-C7 - 1st rib, transverse process C3-C6 - 1st rib, longus capitis muscle, transverse process C3-C6 - occipital bone (base), longus neck muscle, and transverse process C2-C5 - atlas (anterior tubercle)

9. The pharmaceutical product according to claim 1 or 2, wherein the modified BoNT / A is administered by intramuscular injection to a plurality of affected cervical muscles of the subject, and at least a single dose is administered to each affected cervical muscle.

10. The pharmaceutical product according to claim 1 or 2. Here, The aforementioned multiple affected cervical muscles are selected from the following: sternocleidomastoid, splenius capitis, splenius cervicis, scalene complex, anterior scalene, middle scalene, trapezius, upper trapezius, levator scapulae, semispinalis capitis, longissimus, longissimus capitis, longissimus cervicis, paravertebral posterior and submandibular complex, digastric, geniohyoid, mylohyoid, mylohyoid boutonniere, stylohyoid, lower trapezius, superior nuchal line-clavicle (lateral part), spinous process C3-Th3-mastoid, spinous process Th3-Th5-transverse process C1-C2, transverse process C3-Th6, spines Process C3-Th1-superior nuchal line, transverse process Th1-Th6-spinous process C2-C7, transverse process C3-Th3-mastoid process, transverse process Th1-Th6-transverse process C2-C6, inferior oblique muscle, spinous process C2-transverse process C1, cervical notch and clavicle (medial part)- Mastoid process and superior nuchal line, transverse process C1-C4 - scapula (superior corner), transverse process C2-C7 - 1st rib, transverse process C3-C6 - 1st rib, longus capitis muscle, transverse process C3-C6 - occipital bone (base), longus neck muscle, and transverse process C2-C5 - atlas (anterior tubercle)

11. The pharmaceutical product according to claim 1 or 2. Here, (a) A single unit dose of the modified BoNT / A is administered to one or more affected cervical muscles selected from the first group, which includes: splenius cervicis, oblique capitis inferior, semispinalis cervicis, scalene muscles, anterior, middle, and / or posterior scalene muscles, longissimus capitis, longus colli, and / or longus capitis; and / or a single or multiple unit doses of the modified BoNT / A are administered to one or more affected cervical muscles selected from the second group, which includes: splenius capitis, longissimus cervicis, trapezius, lower trapezius, sternocleidomastoid, semispinalis capitis, and / or levator scapulae; and / or (b) A single unit dose of the modified BoNT / A is administered to one or more affected cervical muscles selected from the first group, which includes: the splenius cervicis, oblique capitis inferior, semispinalis cervicis, scalene muscles, anterior, middle, and / or posterior scalene muscles, longissimus capitis, longus colli, and / or longus capitis; and / or multiple unit doses of the modified BoNT / A are administered to one or more affected cervical muscles selected from the second group, which includes: the splenius capitis, longissimus cervicis, trapezius, subtrapezius, sternocleidomastoid, semispinalis capitis, and / or levator scapulae.

12. The pharmaceutical product according to claim 11. Herein, the unit dose is (a) the modified BoNT / A in amounts of 20,000 pg to 30,000 pg; or (b) The modified BoNT / A of 30,000 pg to a maximum of 40,000 pg; At the discretion of the patient, the total unit dose administered during the aforementioned treatment may be a maximum of 10 units or a maximum of 7 units.

13. The pharmaceutical product according to claim 1 or 2. Here, (a) The modified BoNT / A is administered in a unit dose for each injection site in the affected cervical muscle, or the modified BoNT / A is administered in a dose less than a unit dose for each injection site; (b) A single unit dose is administered to multiple injection sites in the affected cervical muscle, and / or two or more unit doses are administered to multiple injection sites in the affected cervical muscle; (c) A single dose is administered to the affected neck muscle; and / or (d) Cervical dystonia is treated for a longer period than when treated with unmodified BoNT / A (Sequence ID 2).

14. Unit dosage forms of modified BoNT / A for use in the treatment of cervical dystonia, including the following: (a) the aforementioned modified BoNT / A exceeding 17,000 pg; and (b) Pharmacologically acceptable carriers, excipients, adjuvants, and / or salts, Here, the modified BoNT / A comprises the BoNT / A light chain and transposition domain, and the BoNT / B receptor binding domain (H C (including domain)

15. The kit includes the following: (a) The unit dosage form according to claim 14; (b) Instructions for use of the kit in the treatment of cervical dystonia; and (c) Diluent of your choice

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