Modified BoNT / A for use in treatment of cervical muscular dystonia

Abstract

The present invention relates to the use of modified BoNT / A for the treatment of cervical dyskinesia, including modified botulinum neurotoxin A (BoNT / A) for use in the treatment of cervical dyskinesia wherein the modified BoNT / A is administered by intramuscular injection to affected neck muscles of a subject wherein the modified BoNT / A is administered in a unit dose of 750 pg to 17,000 pg of the modified BoNT / A, wherein at least a single unit dose is administered to the affected neck muscle wherein the total dose administered during treatment is at most 170,000 pg of the modified BoNT / A, and wherein the modified BoNT / A comprises a BoNT / A light chain and a translocation domain, and a BoNT / B receptor binding domain (HC domain). Related methods, uses, unit dosage forms, and kits are also provided.

Description

[0001] This application is a divisional application of China application 202280076093.9, filed on September 16, 2022, entitled “Modified BoNT / A for treatment of cervical dystonia”. TECHNICAL FIELD

[0002] The present invention relates to the treatment of cervical dystonia. BACKGROUND

[0003] Cervical dystonia, also known as spasmodic torticollis, is a chronic neuromotor disorder that is often associated with extreme pain. The condition causes the neck of an affected subject to involuntarily turn to the left, to the right, upwards and / or downwards. During a dystonia movement, both agonist and antagonist muscles can contract simultaneously.

[0004] The condition often presents with relatively mild symptoms, such as non-visual head tremors that last for several months at onset. Other early / progressive symptoms can include sudden movements of head turning, pulling and / or tilting. However, further early / progressive symptoms often include sustained / prolonged involuntary head positioning. The frequency and intensity of involuntary neck muscle spasms tend to increase over time before reaching a plateau. Subjects with cervical dystonia can also experience muscle hypertrophy, neck pain, dysarthria and / or tremor.

[0005] Symptoms of cervical dystonia can involve any of the subject’s neck muscles, and the head posture can vary. Typically, the most common abnormal posture associated with cervical dystonia is a twisted jaw towards the shoulder, causing the head to rotate sideways (torticollis). Other abnormal postures associated with cervical dystonia can include anterocollis (head tilted forward), retrocollis (head tilted backwards) or laterocollis (head tilted to one side). It can also occur that the head is displaced in a forward sagittal shift (forward displacement) or a posterior sagittal shift (backward displacement) on the shoulders. However, most commonly, cervical dystonia presents with complex symptoms, where the subject exhibits head movements in multiple angles.

[0006] Current treatment options include oral medications (such as dopamine blockers), deep brain stimulation, botulinum neurotoxin and selective denervation surgery, which removes the nerves that trigger muscle contractions. Traditional oral medications are associated with many serious side effects, while deep brain stimulation and denervation surgery are invasive, carry the risk of associated complications, and / or can be ineffective.

[0007] Dysport ®This is an example of a traditional serum type A botulinum toxin neurotoxin (BoNT / A) for the treatment of cervical dystonia, which contains botulinum toxin type A (BoNT / A). Clostridium botulinum Pharmaceutical products containing the drug substance BoNT / A hemagglutinin complex (BTX-A-HAC) isolated and purified from botulinum toxin. Several other products on the market are also made from botulinum toxin (Botox). Clostridium botulinum Naturally occurring medicinal BoNT / A products (e.g., BOTOX) ® and XEOMIN ® ).

[0008] By paralyzing the antagonist muscles of dystonia, BoNT / A allows the agonist muscles to move freely. More specifically, BoNT / A selectively inhibits the release of acetylcholine from presynaptic nerve endings, thereby blocking cholinergic transmission at the neuromuscular junction, inducing muscle contraction and a decrease in muscle tone, leading to relaxation of the injected muscle. However, the duration of action of currently available BoNT / A products is approximately 12 to 14 weeks, during which time new nerve endings sprout, nerve function returns to normal, and the original symptoms reappear. Therefore, to maintain the effect, regular repeat injections are necessary. Thus, considering the chronic nature of the condition and the need for long-term treatment, the frequency of BoNT / A injections is an important consideration in the treatment of cervical dystonia. In fact, this affects the direct and indirect health costs involved for patients and caregivers, the organizational workload of injections in hospitals / clinics, and, most importantly, the patient's quality of life.

[0009] Dysport ® Approved for the treatment of cervical dystonia, the maximum total dose per treatment course is 1,000 units (see [link to treatment]). Figure 1 Clinicians need to administer Dysport to the neck muscles of the test subjects. ® The maximum total dose for each treatment cycle is 1,000 units. Clinicians are forced to make difficult choices during patient treatment. In other words, in routine treatment regimens, 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 highly toxic nature of BoNT / A) and the effective dose at various different muscles. As a result, some muscles are neglected while others receive suboptimal doses of BoNT / A, leading to suboptimal treatment.

[0010] Furthermore, traditional treatment protocols for cervical dystonia are complex, leading to insufficient dosage by clinicians to minimize patient toxicity. Therefore, there is a need for a convenient, safe, and effective single-dose unit that can be administered to the affected neck muscles during treatment, along with corresponding guidelines for the number of units (e.g., including the number of injection sites per muscle), without causing patient toxicity.

[0011] In summary, there is a need for improved treatment of cervical dystonia that allows for a patient-centered, individualized approach, tailoring treatment to a target clinical model, allowing for different combinations of injections into the affected neck muscles based on the distribution, extent, and severity of the dystonia, while avoiding toxicity and providing more sustained treatment (leading to reduced administration frequency).

[0012] The present invention overcomes one or more of the above-mentioned problems. Summary of the Invention

[0013] The inventors have surprisingly discovered that modified BoNT / A is particularly useful in treating cervical dystonia. Modified BoNT / A may include a BoNT / A light chain and a translocation domain, as well as a BoNT / B receptor-binding domain (H). C This modification of BoNT / A (including its structural domain) leads to increased retention at the application site (reduced diffusion from the application site) and / or an increased duration of action (e.g., 6-9 months). Alternatively, the modified BoNT / A may contain one or more modifications of surface-exposed amino acid residues, resulting in an increased net positive charge. The increased charge promotes electrostatic interactions between the peptide and anionic extracellular components, thereby promoting peptide binding to the cell surface. This, in turn, also increases retention at the application site (reduced diffusion from the application site) and / or leads to an increased duration of action (e.g., 6-9 months).

[0014] Advantageously, with unmodified BoNT / A (e.g., Dysport) ® Compared to the original BoNT / A, the modified BoNT / A offers improved security. This improved security can be expressed by the high security ratio of the modified BoNT / A described herein.

[0015] Based on the preclinical and clinical data presented in this paper (see Examples), it has been shown that higher total amounts of modified BoNT / A can be administered to subjects while achieving the same results as unmodified BoNT / A (e.g., Dysport). ® Similar safety profile, despite such high doses. Therefore, in the treatment of cervical dystonia, more modified BoNT / A can be injected and / or injected at more neck muscles / sites before reaching the maximum total dose. This is an important and advantageous finding that provides improved treatment for cervical dystonia while offering clinicians a wider range of treatment options. Treatment can be improved because of the difference compared to using unmodified BoNT / A (e.g., Dysport). ® Compared to conventional treatments, it provides more durable treatment (leading to a reduced frequency of administration) and / or can be customized for the subject and / or results in an improvement in the subject's quality of life. Therefore, the treatment of the present invention is an improvement over conventional treatment regimens.

[0016] Furthermore, the present application provides a single unit dose and a total (maximum) dose which are convenient, safe and effective and can be safely administered in a single treatment. The present application also provides a corresponding guideline for the number of times the unit dose can be administered to the neck muscles (e.g. including the number of injection sites per muscle) without causing toxicity to the patient. Thus, the treatment of cervical dystonia according to the present application is much simpler for the clinician and helps to avoid under- and / or overdosing. Furthermore, the treatment according to the present application is more satisfactory for the patient compared to traditional treatments of cervical dystonia, as it is better tailored to the needs of the patient. DETAILED DESCRIPTION

[0017] In one aspect, the present application 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, wherein the modified BoNT / A is administered by means of a unit dose of 750 pg to 17,000 pg of the modified BoNT / A, wherein at least a single unit dose is administered to the affected neck muscle, wherein the total dose administered during the treatment is up to 170,000 pg of the modified BoNT / A, and wherein the modified BoNT / A comprises a BoNT / A light chain and translocation domain, and a BoNT / B receptor binding domain (H C domain).

[0018] In a related aspect, the present application provides a modified BoNT / A for use in the treatment of cervical dystonia in a subject for a longer duration than with an unmodified BoNT / A (e.g. SEQ ID NO: 2), wherein the modified BoNT / A is administered to an affected neck muscle of the subject by intramuscular injection, wherein the modified BoNT / A is administered by means of a unit dose of 750 pg to 17,000 pg of the modified BoNT / A, wherein at least a single unit dose is administered to the affected neck muscle, wherein the total dose administered during the treatment is up to 170,000 pg of the modified BoNT / A, wherein the modified BoNT / A comprises a BoNT / A light chain and translocation domain, and a BoNT / B receptor binding domain (H C domain).

[0019] The term "duration of treatment of cervical dystonia in a subject is longer than the duration of treatment with unmodified BoNT / A" can refer to the duration of relief of one or more symptoms of cervical dystonia in a subject after administration of a modified BoNT / A of the application is longer than when unmodified BoNT / A is administered. The duration of effect can be at least 1.25x, 1.5x, 1.75x, 2.0x, or 2.25x. The duration of effect of the modified BoNT / A can be between 6 and 9 months. For example, the duration of effect can be at least: 4.5 months (from onset), 5.0 months, 5.5 months, 6 months, 6.5 months, 7.0 months, 7.5 months, 8.0 months, 8.5 months, or 9.0 months. In particular embodiments, the duration of effect can be greater than 9.0 months. The relief can be determined by comparison to a comparable control subject exhibiting the same symptoms who has been treated with unmodified BoNT / A. A subject treated with a modified BoNT / A according to the application can exhibit at least a 5%, 10%, 25%, or 50% improvement in one or more comparable symptoms compared to the severity of the one or more symptoms prior to treatment with the modified BoNT / A, at a time period substantially the same (e.g., identical) to the severity of the one or more symptoms in the control subject prior to unmodified BoNT / A treatment. The unmodified BoNT / A is preferably SEQ ID NO: 2 in a di-chain form.

[0020] In one aspect, the application provides a method for treating cervical dystonia, the method comprising administering to a subject affected cervical muscle(s) a modified BoNT / A by intramuscular injection, wherein the modified BoNT / A is administered by way of a unit dose of 750 pg to 17,000 pg of the modified BoNT / A, wherein at least a single unit dose is administered to the affected cervical muscle, wherein the total dose administered during treatment is up to 170,000 pg of the modified BoNT / A, and wherein the modified BoNT / A comprises a BoNT / A light chain and translocation domain, and a BoNT / B receptor binding domain (H C domain).

[0021] In a related aspect, the application provides a method for treating cervical dystonia in a subject for a duration longer than the duration of treatment with unmodified BoNT / A (e.g., SEQ ID NO: 2), the method comprising administering to a subject affected cervical muscle(s) a modified BoNT / A by intramuscular injection, wherein the modified BoNT / A is administered by way of a unit dose of 750 pg to 17,000 pg of the modified BoNT / A, wherein at least a single unit dose is administered to the affected neck muscle, wherein the total dose administered during treatment is up to 170,000 pg of the modified BoNT / A, wherein the modified BoNT / A comprises a BoNT / A light chain and translocation domain, and a BoNT / B receptor binding domain (H C domain).

[0022] In one aspect, the present application provides 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, wherein the modified BoNT / A is administered by way of a unit dose of 750 pg to 17,000 pg of the modified BoNT / A, wherein at least a single unit dose is administered to the affected neck muscle, wherein the total dose administered during treatment is up to 170,000 pg of the modified BoNT / A, and wherein the modified BoNT / A comprises a BoNT / A light chain and translocation domain, and a BoNT / B receptor binding domain (H C domain).

[0023] In a related aspect, the present application provides use of a modified BoNT / A in the manufacture of a medicament for treating cervical dystonia in a subject for a duration that is longer than a duration of treatment with an unmodified BoNT / A (e.g., SEQ ID NO: 2), wherein the modified BoNT / A is administered to an affected neck muscle of the subject by intramuscular injection, wherein the modified BoNT / A is administered by way of a unit dose of 750 pg to 17,000 pg of the modified BoNT / A, wherein at least a single unit dose is administered to the affected neck muscle, wherein the total dose administered during treatment is up to 170,000 pg of the modified BoNT / A, wherein the modified BoNT / A comprises a BoNT / A light chain and translocation domain, and a BoNT / B receptor binding domain (H C domain).

[0024] A unit dose can be 750 pg to 17,000 pg of the modified BoNT / A, wherein the modified BoNT / A comprises a BoNT / A light chain and translocation domain, and a BoNT / B receptor binding domain (H C(Structural domain). The upper limit of the unit dose range can be 16,500, 15,500, 14,500, 13,500, 12,500, 11,500, 10,500, 9,500, 8,500, 7,500, 6,500, 5,500, 4,500, 3,500, 2,500, 2,250, 2,000, 1,500, 1,250, 1,000, 750 or 500 pg of modified BoNT / A, preferably with an upper limit of 16,000 pg. The lower limit of the unit dose range can be 800, 850, 950, 1,000, 1,500, 1,750, 2,000, 2,500, 3,000, 3,500, 4,000, 4,500, or 5,000 pg of modified BoNT / A, preferably 1,000 pg. Preferably, the unit dose of modified BoNT / A is from 1,000 pg to 16,000 pg, for example, 950 pg to 1,250 pg, 1,750 pg to 2,250 pg, or 8,000 pg to 12,000 pg. Preferably, the unit dose of modified BoNT / A can be 1,000, 2,000, 3,000, 8,000, or 16,000 pg.

[0025] When performing the treatment regimen of the present invention, the total dose administered can be up to 170,000 pg of modified BoNT / A, wherein the modified BoNT / A comprises a BoNT / A light chain and a translocation domain, and a BoNT / B receptor binding domain (H). C(Structural domain). In other words, the total amount of modified BoNT / A administered in a given treatment course can be up to 170,000 pg. The total dose can be up to 165,000, 160,000, 140,000, 110,000, 100,000, 90,000, 80,000, 70,000, 60,000, 50,000, 40,000, 30,000, 28,000, 25,000, 20,000, 15,000, 14,000, 10,000, 8,000, 7,000, or 5,000 pg. Preferably, the total dose can be up to 160,000 pg of modified BoNT / A, for example, the total dose can be up to 7,000, 10,000, 14,000, 20,000, 28,000, 30,000, 80,000 or 160,000 pg. The total dose can be at least 900, 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 7,500, 10,000, 12,500, 13,000, 14,000, 15,000, 20,000, 25,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000, 100,000, 120,000, or 150,000 pg. Preferably, the total dose can be at least 1,500 pg, more preferably at least 2,000 pg of modified BoNT / A, for example at least 5,000 pg. The total dose can be from 5,250 pg to 170,000 pg, preferably from 7,000 pg to 160,000 pg. For example, the total dose administered can be 6,000-30,000 pg, such as 6,000-15,000 pg or 13,000-29,000 pg. More preferably, the total dose administered is from 10,000 pg to 160,000 pg. Preferably, the total dose administered can be 7,000, 10,000, 14,000, 20,000, 28,000, 30,000, 80,000 or 160,000 pg.

[0026] Therefore, the unit dose can be from 750 pg to 17,000 pg of modified BoNT / A, wherein the modified BoNT / A contains a BoNT / A light chain and a translocation domain, as well as a BoNT / B receptor-binding domain (H). C(Structural domain), when performing the treatment regimen of the present invention, the total dose administered can be up to 170,000 pg. The unit dose can be 1,000 pg, and the total dose can be up to 7,000 pg. The unit dose can be 1,000 pg, and the total dose can be up to 10,000 pg. The unit dose can be 1,000 pg, and the total dose can be up to 14,000 pg. The unit dose can be 2,000 pg, and the total dose can be up to 14,000 pg. The unit dose can be 2,000 pg, and the total dose can be up to 20,000 pg. The unit dose can be 2,000 pg, and the total dose can be up to 28,000 pg. The unit dose can be 3,000 pg, and the total dose can be up to 30,000 pg. The unit dose can be 8,000 pg, and the total dose can be up to 80,000 pg. The unit dose can be 16,000 pg, and the total dose can be up to 160,000 pg.

[0027] Therefore, the unit dose can be from 750 pg to 17,000 pg of modified BoNT / A, wherein the modified BoNT / A contains a BoNT / A light chain and a translocation domain, as well as a BoNT / B receptor-binding domain (H). C (Structural domain), when performing the treatment regimen of the present invention, the total dose administered can be up to 170,000 pg. The unit dose can be 1,000 pg, and the total dose can be 7,000 pg. The unit dose can be 1,000 pg, and the total dose can be 10,000 pg. The unit dose can be 1,000 pg, and the total dose can be 14,000 pg. The unit dose can be 2,000 pg, and the total dose can be 14,000 pg. The unit dose can be 2,000 pg, and the total dose can be 20,000 pg. The unit dose can be 2,000 pg, and the total dose can be 28,000 pg. The unit dose can be 3,000 pg, and the total dose can be 30,000 pg. The unit dose can be 8,000 pg, and the total dose can be 80,000 pg. The unit dose can be 16,000 pg, and the total dose can be 160,000 pg.

[0028] On one hand, the present invention provides a modified botulinum neurotoxin A (BoNT / A) for treating cervical dystonia, wherein the modified BoNT / A is administered to the affected neck muscles of a subject via intramuscular injection. The modified BoNT / A was administered in unit doses ranging from 31 units (U) to 707 U, where 1 unit corresponds to the calculated median lethal dose (LD50) in mice. 50 The amount of BoNT / A modified by ) This involves administering at least a single unit dose to the affected neck muscles. The total dose of modified BoNT / A administered during treatment was as high as 7,070 U, and The modified BoNT / A includes the BoNT / A light chain and translocation domain, as well as the BoNT / B receptor-binding domain (H). C (structural domain).

[0029] In a related aspect, the present invention provides a modified BoNT / A for treating cervical dystonia in subjects, with a duration of treatment longer than that with unmodified BoNT / A (e.g., SEQ ID NO:2), wherein the modified BoNT / A is administered to the affected neck muscles of the subject via intramuscular injection. The modified BoNT / A was administered in unit doses ranging from 31 units (U) to 707 U, where 1 unit corresponds to the calculated median lethal dose (LD50) in mice. 50 The amount of BoNT / A modified by ) This involves administering at least a single unit dose to the affected neck muscles. The total dose of modified BoNT / A administered during treatment was as high as 7,070 U, and The modified BoNT / A includes the BoNT / A light chain and translocation domain, as well as the BoNT / B receptor-binding domain (H). C (structural domain).

[0030] On one hand, the present invention provides a method for treating cervical dystonia, the method comprising administering modified BoNT / A to the affected neck muscles of a subject via intramuscular injection. The modified BoNT / A was administered in unit doses ranging from 31 U to 707 U, where 1 unit corresponds to the calculated median lethal dose (LD50) in mice. 50 The amount of BoNT / A modified by ) This involves administering at least a single unit dose to the affected neck muscles. The total dose of modified BoNT / A administered during treatment was as high as 7,070 U, and The modified BoNT / A includes the BoNT / A light chain and translocation domain, as well as the BoNT / B receptor-binding domain (H). C (structural domain).

[0031] In a related aspect, the present invention provides a method for treating cervical dystonia in a subject, the duration of which is longer than the duration of treatment with unmodified BoNT / A (e.g., SEQ ID NO:2), the method comprising administering modified BoNT / A to the affected neck muscles of the subject via intramuscular injection. The modified BoNT / A was administered in unit doses ranging from 31 U to 707 U, where 1 unit corresponds to the calculated median lethal dose (LD50) in mice. 50 The amount of BoNT / A modified by ) This involves administering at least a single unit dose to the affected neck muscles. The total dose of modified BoNT / A administered during treatment was as high as 7,070 U, and The modified BoNT / A includes the BoNT / A light chain and translocation domain, as well as the BoNT / B receptor-binding domain (H). C (structural domain).

[0032] On one hand, the present invention provides the use of modified botulinum neurotoxin A (BoNT / A) in the preparation of a medicament for treating cervical dystonia, wherein the modified BoNT / A is administered to the affected neck muscles of a subject via intramuscular injection. The modified BoNT / A was administered in unit doses ranging from 31 U to 707 U, where 1 unit corresponds to the calculated median lethal dose (LD50) in mice. 50 The amount of BoNT / A modified by ) This involves administering at least a single unit dose to the affected neck muscles. The total dose of modified BoNT / A administered during treatment was as high as 7,070 U, and The modified BoNT / A includes the BoNT / A light chain and translocation domain, as well as the BoNT / B receptor-binding domain (H). C (structural domain).

[0033] In a related aspect, the present invention provides the use of modified BoNT / A in the preparation of a medicament for treating cervical dystonia in subjects, with a duration of effect longer than that of treatment with unmodified BoNT / A (e.g., SEQ ID NO:2), wherein the modified BoNT / A is administered to the affected neck muscles of the subject via intramuscular injection. The modified BoNT / A was administered in unit doses ranging from 31 U to 707 U, where 1 unit corresponds to the calculated median lethal dose (LD50) in mice. 50The amount of BoNT / A modified by ) This involves administering at least a single unit dose to the affected neck muscles. The total dose of modified BoNT / A administered during treatment was as high as 7,070 U, and The modified BoNT / A includes the BoNT / A light chain and translocation domain, as well as the BoNT / B receptor-binding domain (H). C (structural domain).

[0034] The unit dose can be 31 to 707 units of modified BoNT / A, wherein the modified BoNT / A comprises a BoNT / A light chain and a translocation domain, as well as a BoNT / B receptor-binding domain (H). C (Structural domain). The upper limit of the unit dose range can be 700, 650, 600, 550, 500, 450, 400, 350, 300, 250, 200, 150, 100, 95, 90, 85, 65, 60, 55, 50 or 31 units of modified BoNT / A, preferably 666 units. The lower limit of the unit dose range can be 35, 40, 45, 50, 60, 65, 70, 75, 80, 85, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650 or 700 units of modified BoNT / A, preferably 42 units. Preferably, the unit dose of the modified BoNT / A is 42 to 666 units, for example, 40 to 50 units, 70 to 95 units, or 333 to 499 units. Preferably, the unit dose of the modified BoNT / A can be 41.6 units, 83.2 units, 124.8 units, 332.8 units, or 665.6 units.

[0035] When performing the treatment regimen of the present invention, the total dose administered can be up to 7,070 units of modified BoNT / A, wherein the modified BoNT / A comprises a BoNT / A light chain and a translocation domain, and a BoNT / B receptor binding domain (H). C(Structural domain). In other words, the total amount of modified BoNT / A administered in a given treatment course can be up to 7,070 units. The total dose can be up to 7,000, 6,000, 5,000, 4,000, 3,500, 3,350, 3,000, 2,000, 1,500, 1,250, 1,175, 1,000, 900, 800, 700, 650, 625, 600, 575, 550, 500, 450, 425, 400, 350, 330, 300, 290, 250, or 200 units. Preferably, the total dose can be up to 6,660 units of modified BoNT / A, for example, the total dose can be up to 290, 425, 575, 600, 800, 1,000, 1,175, 1,250, 3,000, 3,350, 3,500, or 7,000 units. The total dose can be at least 35, 40, 50, 100, 150, 200, 250, 290, 300, 350, 400, 450, 500, 540, 550, 580, 600, 650, 700, 750, 800, 850, 900, 950, 1,000, 1,050, 1,500, 2,000, 3,000, 4,000, 5,000, 6,000, or 7,000 units. Preferably, the total dose may be at least 62 units, more preferably at least 83 units, of modified BoNT / A, for example at least 208 units. The total dose may be from 217 units to 7,070 units, preferably from 294 units to 6,660 units. For example, the total dose applied may be 250-1,250 units, for example 250-625 units, or 540-1,200 units. More preferably, the total dose applied is from 420 units to 6,660 units. Preferably, the total dose applied may be 291 units, 416 units, 582 units, 832 units, 1,165 units, 1,248 units, 3,328 units, or 6,656 units.

[0036] Therefore, the unit dose can be 31 to 707 units of modified BoNT / A, wherein the modified BoNT / A contains a BoNT / A light chain and a translocation domain, as well as a BoNT / B receptor-binding domain (H). C(Structural domain), when performing the treatment regimen of the present invention, the total dose administered can be up to 7,070 units. The unit dose can be 41.6 units and the total dose can be up to 291 units. The unit dose can be 41.6 units and the total dose can be up to 416 units. The unit dose can be 41.6 units and the total dose can be up to 582 units. The unit dose can be 83.2 units and the total dose can be up to 582 units. The unit dose can be 83.2 units and the total dose can be up to 832 units. The unit dose can be 83.2 units and the total dose can be up to 1,165 units. The unit dose can be 124.8 units and the total dose can be up to 1,248 units. The unit dose can be 332.8 units and the total dose can be up to 3,328 units. The unit dose can be 665.5 units and the total dose can be up to 6,656 units.

[0037] Therefore, the unit dose can be 31 to 707 units of modified BoNT / A, wherein the modified BoNT / A contains a BoNT / A light chain and a translocation domain, as well as a BoNT / B receptor-binding domain (H). C (Structural domain), when performing the treatment regimen of the present invention, the total dose administered can be up to 7,070 units. The unit dose can be 41.6 units and the total dose can be 291 units. The unit dose can be 41.6 units and the total dose can be 416 units. The unit dose can be 41.6 units and the total dose can be 582 units. The unit dose can be 83.2 units and the total dose can be 582 units. The unit dose can be 83.2 units and the total dose can be 832 units. The unit dose can be 83.2 units and the total dose can be 1,165 units. The unit dose can be 124.8 units and the total dose can be 1,248 units. The unit dose can be 332.8 units and the total dose can be 3,328 units. The unit dose can be 665.5 units and the total dose can be 6,656 units.

[0038] On one hand, the present invention provides a modified botulinum neurotoxin A (BoNT / A) for treating cervical dystonia, wherein the modified BoNT / A is administered to the affected neck muscles of a subject via intramuscular injection. The modified BoNT / A was administered at a unit dose of 450 pg to 8,000 pg. At least a single unit dose was administered to the affected neck muscles. The modified BoNT / A was administered at a total dose of up to 80,000 pg during treatment. The modified BoNT / A includes modifications at one or more amino acid residues selected from the following: ASN 886, ASN 905, GLN 915, ASN 918, GLU 920, ASN 930, ASN 954, SER 955, GLN 991, GLU 992, GLN995, ASN 1006, ASN 1025, ASN 1026, ASN 1032, ASN 1043, ASN 1046, ASN 1052, ASP 1058, HIS 1064, ASN 1080, GLU 1081, GLU 1083, ASP 1086, ASN 1188, ASP 1213, GLY 1215, ASN 1216, GLN 1229, ASN 1242, ASN 1243, SER 1274, and THR 1277, wherein the modifications are selected from: (i) Replace the acidic amino acid residues exposed on the surface with basic amino acid residues; (ii) Replace the surface-exposed acidic amino acid residues with uncharged amino acid residues; (iii) Replace the uncharged amino acid residues exposed on the surface with basic amino acid residues; (iv) Insertion of basic amino acid residues; and (v) Remove acidic amino acid residues exposed on the surface.

[0039] In a related aspect, the present invention provides a modified BoNT / A for treating cervical dystonia in subjects, with a duration of treatment longer than that with unmodified BoNT / A (e.g., SEQ ID NO:2), wherein the modified BoNT / A is administered to the affected neck muscles of the subject via intramuscular injection. The modified BoNT / A was administered at a unit dose of 450 pg to 8,000 pg. At least a single unit dose was administered to the affected neck muscles. The modified BoNT / A was administered at a total dose of up to 80,000 pg during treatment. The modified BoNT / A contains modifications at one or more amino acid residues selected from the following: , SER 1274 and THR 1277, wherein the modification is selected from: (i) Replace the acidic amino acid residues exposed on the surface with basic amino acid residues; (ii) Replace the surface-exposed acidic amino acid residues with uncharged amino acid residues; (iii) Replace the uncharged amino acid residues exposed on the surface with basic amino acid residues; (iv) Insertion of basic amino acid residues; and (v) Remove acidic amino acid residues exposed on the surface.

[0040] On one hand, the present invention provides a method for treating cervical dystonia, the method comprising administering modified BoNT / A to the affected neck muscles of a subject via intramuscular injection. The modified BoNT / A was administered at a unit dose of 450 pg to 8,000 pg. At least a single unit dose was administered to the affected neck muscles. The modified BoNT / A was administered at a total dose of up to 80,000 pg during treatment. The modified BoNT / A contains modifications at one or more amino acid residues selected from the following: , SER 1274 and THR 1277, wherein the modification is selected from: (i) Replace the acidic amino acid residues exposed on the surface with basic amino acid residues; (ii) Replace the surface-exposed acidic amino acid residues with uncharged amino acid residues; (iii) Replace the uncharged amino acid residues exposed on the surface with basic amino acid residues; (iv) Insertion of basic amino acid residues; and (v) Remove acidic amino acid residues exposed on the surface.

[0041] In a related aspect, the present invention provides a method for treating cervical dystonia in a subject, the duration of which is longer than the duration of treatment with unmodified BoNT / A (e.g., SEQ ID NO:2), the method comprising administering modified BoNT / A to the affected neck muscles of the subject via intramuscular injection. The modified BoNT / A was administered at a unit dose of 450 pg to 8,000 pg. At least a single unit dose was administered to the affected neck muscles. The modified BoNT / A was administered at a total dose of up to 80,000 pg during treatment. The modified BoNT / A contains modifications at one or more amino acid residues selected from the following: , SER 1274 and THR 1277, wherein the modifications are selected from: (i) Replace the acidic amino acid residues exposed on the surface with basic amino acid residues; (ii) Replace the surface-exposed acidic amino acid residues with uncharged amino acid residues; (iii) Replace the uncharged amino acid residues exposed on the surface with basic amino acid residues; (iv) Insertion of basic amino acid residues; and (v) Remove acidic amino acid residues exposed on the surface.

[0042] On one hand, the present invention provides the use of modified botulinum neurotoxin A (BoNT / A) in the preparation of a medicament for treating cervical dystonia, wherein the modified BoNT / A is administered to the affected neck muscles of a subject via intramuscular injection. The modified BoNT / A was administered at a unit dose of 450 pg to 8,000 pg. At least a single unit dose was administered to the affected neck muscles. The modified BoNT / A was administered at a total dose of up to 80,000 pg during treatment. The modified BoNT / A contains modifications at one or more amino acid residues selected from the following: , SER 1274 and THR 1277, wherein the modifications are selected from: (i) Replace the acidic amino acid residues exposed on the surface with basic amino acid residues; (ii) Replace the surface-exposed acidic amino acid residues with uncharged amino acid residues; (iii) Replace the uncharged amino acid residues exposed on the surface with basic amino acid residues; (iv) Insertion of basic amino acid residues; and (v) Remove acidic amino acid residues exposed on the surface.

[0043] In a related aspect, the present invention provides the use of modified BoNT / A in the preparation of a medicament for treating cervical dystonia in subjects, with a duration of effect longer than that of treatment with unmodified BoNT / A (e.g., SEQ ID NO:2), wherein the modified BoNT / A is administered to the affected neck muscles of the subject via intramuscular injection. The modified BoNT / A was administered at a unit dose of 450 pg to 8,000 pg. At least a single unit dose was administered to the affected neck muscles. The modified BoNT / A was administered at a total dose of up to 80,000 pg during treatment. The modified BoNT / A contains modifications at one or more amino acid residues selected from the following: , SER 1274 and THR 1277, wherein the modifications are selected from: (i) Replace the acidic amino acid residues exposed on the surface with basic amino acid residues; (ii) Replace the surface-exposed acidic amino acid residues with uncharged amino acid residues; (iii) Replace the uncharged amino acid residues exposed on the surface with basic amino acid residues; (iv) Insertion of basic amino acid residues; and (v) Remove acidic amino acid residues exposed on the surface.

[0044] The unit dose can be from 450 pg to 8,000 pg of modified BoNT / A, wherein the modified BoNT / A contains a modification at one or more amino acid residues selected from the following: ASN 886, ASN , And THR 1277, wherein the modification is selected from: (i) replacing surface-exposed acidic amino acid residues with basic amino acid residues; (ii) replacing surface-exposed acidic amino acid residues with uncharged amino acid residues; (iii) replacing surface-exposed uncharged amino acid residues with basic amino acid residues; (iv) inserting basic amino acid residues; (v) deleting surface-exposed acidic amino acid residues. The upper limit of the unit dose range may be 7,750, 7,500, 7,000, 6,000, 5,000, 4,000, 3,000, 2,000 or 1,000 pg of modified BoNT / A, preferably with an upper limit of 7,500 pg. The lower limit of the unit dose range can be 475, 500, 600, 700, 800, 900, 1,000, 1,500, 2,000, 3,000, 4,000, 5,000, 6,000, or 7,000 pg of modified BoNT / A, preferably 500 pg. Preferably, the unit dose of modified BoNT / A is from 500 pg to 7,500 pg, for example, from 4,000 pg to 6,000 pg. Most preferably, the unit dose of modified BoNT / A is from 2,000 pg to 3,000 pg, for example, from 2,400 pg to 2,600 pg.

[0045] The unit dose can be greater than 1,000 or greater than 5,000 pg of modified BoNT / A, wherein the modified BoNT / A contains modifications at one or more amino acid residues selected from the following: ASN 886, ASN 905, GLN 915, ASN 918, GLU 920, ASN 930, ASN 954, SER 955, GLN 991, GLU 992, GLN 995, ASN 1006, ASN 1025, ASN 1026, ASN 1032, ASN 1043, ASN 1046, ASN 1052, ASP 1058, HIS 1064, ASN 1080, GLU 1081, GLU 1083, ASP 1086, ASN 1188, ASP 1213, GLY 1215, ASN 1216, GLN 1229, ASN 1242, ASN 1243, SER 1274, and THR 1277, wherein the modification is selected from: (i) replacing surface-exposed acidic amino acid residues with basic amino acid residues; (ii) replacing surface-exposed acidic amino acid residues with uncharged amino acid residues; (iii) replacing surface-exposed uncharged amino acid residues with basic amino acid residues; (iv) inserting basic amino acid residues; and (v) deleting surface-exposed acidic amino acid residues. The unit dose of modified BoNT / A can be greater than 1,000 pg to up to 7,500 pg of modified BoNT / A, for example, greater than 5,000 pg to up to 7,500 pg of modified BoNT / A.

[0046] The total dose administered during the treatment regimen of the present invention can be up to 80,000 pg of modified BoNT / A, wherein the modified BoNT / A contains modifications at one or more amino acid residues selected from the following: , And THR 1277, wherein the modification is selected from: (i) replacing surface-exposed acidic amino acid residues with basic amino acid residues; (ii) replacing surface-exposed acidic amino acid residues with uncharged amino acid residues; (iii) replacing surface-exposed uncharged amino acid residues with basic amino acid residues; (iv) inserting basic amino acid residues; (v) deleting surface-exposed acidic amino acid residues. In other words, the total amount of modified BoNT / A administered in a given treatment course can be up to 80,000 pg. The total dose can be up to 75,000, 70,000, 60,000, 50,000, 40,000, 30,000, 20,000, 10,000, or 5,000 pg. Preferably, the total dose of the modified BoNT / A can be up to 75,000 pg. The total dose can be at least 700, 800, 900, 1,000, 2,000, 3,000, 4,000, 5,000, 7,500, 10,000, 12,500, 15,000, 20,000, 30,000, 40,000, 50,000, 60,000, or 70,000 pg. Preferably, the total dose can be at least 900 pg, more preferably at least 1,000 pg of modified BoNT / A, for example at least 3,000 pg. The total dose can be from 3,150 pg to 80,000 pg, preferably from 3,500 pg to 75,000 pg. More preferably, the total dose administered is 7,500-75,000 pg.

[0047] The total dose administered may be greater than 1,000 or greater than 5,000 pg of modified BoNT / A, wherein the modified BoNT / A contains modifications at one or more amino acid residues selected from the following: , SER 1274 and THR 1277, wherein the modification is selected from: (i) replacing surface-exposed acidic amino acid residues with basic amino acid residues; (ii) replacing surface-exposed acidic amino acid residues with uncharged amino acid residues; (iii) replacing surface-exposed uncharged amino acid residues with basic amino acid residues; (iv) inserting basic amino acid residues; and (v) deleting surface-exposed acidic amino acid residues. Preferably, the total dose may be greater than 1,000 pg of modified BoNT / A, for example, greater than 5,000 pg of modified BoNT / A.

[0048] On one hand, the present invention provides a modified botulinum neurotoxin A (BoNT / A) for treating cervical dystonia, wherein the modified BoNT / A is administered to the affected neck muscles of a subject via intramuscular injection. The modified BoNT / A was administered in unit doses ranging from 53 U to 948 U, where 1 unit corresponds to the median lethal dose (LD50) calculated in mice. 50 The amount of BoNT / A modified by ) At least a single unit dose was administered to the affected neck muscles. The total dose of modified BoNT / A administered during treatment was as high as 9,480 U, and The modified BoNT / A contains modifications at one or more amino acid residues selected from the following: , SER 1274 and THR 1277, wherein the modifications are selected from: (i) Replace the acidic amino acid residues exposed on the surface with basic amino acid residues; (ii) Replace the surface-exposed acidic amino acid residues with uncharged amino acid residues; (iii) Replace the uncharged amino acid residues exposed on the surface with basic amino acid residues; (iv) Insertion of basic amino acid residues; and (v) Remove acidic amino acid residues exposed on the surface.

[0049] In a related aspect, the present invention provides a modified BoNT / A for treating cervical dystonia in subjects, with a duration of treatment longer than that with unmodified BoNT / A (e.g., SEQ ID NO:2), wherein the modified BoNT / A is administered to the affected neck muscles of the subject via intramuscular injection. The modified BoNT / A was administered in unit doses ranging from 53 U to 948 U, where 1 unit corresponds to the median lethal dose (LD50) calculated in mice. 50 The amount of BoNT / A modified by ) At least a single unit dose was administered to the affected neck muscles. The total dose of modified BoNT / A administered during treatment was as high as 9,480 U, and The modified BoNT / A includes modifications at one or more amino acid residues selected from the following: ASN 886, ASN 905, GLN 915, ASN 918, GLU 920, ASN 930, ASN 954, SER 955, GLN 991, GLU 992, GLN995, ASN 1006, ASN 1025, ASN 1026, ASN 1032, ASN 1043, ASN 1046, ASN 1052, ASP 1058, HIS 1064, ASN 1080, GLU 1081, GLU 1083, ASP 1086, ASN 1188, ASP 1213, GLY 1215, ASN 1216, GLN 1229, ASN 1242, ASN 1243, SER 1274, and THR 1277, wherein the modifications are selected from: (i) Replace the acidic amino acid residues exposed on the surface with basic amino acid residues; (ii) Replace the surface-exposed acidic amino acid residues with uncharged amino acid residues; (iii) Replace the uncharged amino acid residues exposed on the surface with basic amino acid residues; (iv) Insertion of basic amino acid residues; and (v) Remove acidic amino acid residues exposed on the surface.

[0050] In a related aspect, the present invention provides a method for treating cervical dystonia, the method comprising administering modified BoNT / A to the affected neck muscles of a subject via intramuscular injection. The modified BoNT / A was administered in unit doses ranging from 53 U to 948 U, where 1 unit corresponds to the median lethal dose (LD50) calculated in mice. 50 The amount of BoNT / A modified by ) At least a single unit dose was administered to the affected neck muscles. The total dose of modified BoNT / A administered during treatment was as high as 9,480 U, and The modified BoNT / A includes modifications at one or more amino acid residues selected from the following: ASN 886, ASN 905, GLN 915, ASN 918, GLU 920, ASN 930, ASN 954, SER 955, GLN 991, GLU 992, GLN995, ASN 1006, ASN 1025, ASN 1026, ASN 1032, ASN 1043, ASN 1046, ASN 1052, ASP 1058, HIS 1064, ASN 1080, GLU 1081, GLU 1083, ASP 1086, ASN 1188, ASP 1213, GLY 1215, ASN 1216, GLN 1229, ASN 1242, ASN 1243, SER 1274, and THR 1277, wherein the modifications are selected from: (i) Replace the acidic amino acid residues exposed on the surface with basic amino acid residues; (ii) Replace the surface-exposed acidic amino acid residues with uncharged amino acid residues; (iii) Replace the uncharged amino acid residues exposed on the surface with basic amino acid residues; (iv) Insertion of basic amino acid residues; and (v) Remove acidic amino acid residues exposed on the surface.

[0051] In a related aspect, the present invention provides a method for treating cervical dystonia in a subject, the duration of which is longer than the duration of treatment with unmodified BoNT / A (e.g., SEQ ID NO:2), the method comprising administering modified BoNT / A to the affected neck muscles of the subject via intramuscular injection. The modified BoNT / A was administered in unit doses ranging from 53 U to 948 U, where 1 unit corresponds to the median lethal dose (LD50) calculated in mice. 50 The amount of BoNT / A modified by ) At least a single unit dose was administered to the affected neck muscles. The total dose of modified BoNT / A administered during treatment was as high as 9,480 U, and The modified BoNT / A includes modifications at one or more amino acid residues selected from the following: ASN 886, ASN 905, GLN 915, ASN 918, GLU 920, ASN 930, ASN 954, SER 955, GLN 991, GLU 992, GLN995, ASN 1006, ASN 1025, ASN 1026, ASN 1032, ASN 1043, ASN 1046, ASN 1052, ASP 1058, HIS 1064, ASN 1080, GLU 1081, GLU 1083, ASP 1086, ASN 1188, ASP 1213, GLY 1215, ASN 1216, GLN 1229, ASN 1242, ASN 1243, SER 1274, and THR 1277, wherein the modifications are selected from: (i) Replace the acidic amino acid residues exposed on the surface with basic amino acid residues; (ii) Replace the surface-exposed acidic amino acid residues with uncharged amino acid residues; (iii) Replace the uncharged amino acid residues exposed on the surface with basic amino acid residues; (iv) Insertion of basic amino acid residues; and (v) Remove acidic amino acid residues exposed on the surface.

[0052] In another related aspect, the present invention provides the use of modified botulinum neurotoxin A (BoNT / A) in the preparation of a medicament for treating cervical dystonia, wherein the modified BoNT / A is administered to the affected neck muscles of a subject via intramuscular injection. The modified BoNT / A was administered in unit doses ranging from 53 U to 948 U, where 1 unit corresponds to the median lethal dose (LD50) calculated in mice. 50 The amount of BoNT / A modified by ) At least a single unit dose was administered to the affected neck muscles. The total dose of modified BoNT / A administered during treatment was as high as 9,480 U, and The modified BoNT / A includes modifications at one or more amino acid residues selected from the following: ASN 886, ASN 905, GLN 915, ASN 918, GLU 920, ASN 930, ASN 954, SER 955, GLN 991, GLU 992, GLN995, ASN 1006, ASN 1025, ASN 1026, ASN 1032, ASN 1043, ASN 1046, ASN 1052, ASP 1058, HIS 1064, ASN 1080, GLU 1081, GLU 1083, ASP 1086, ASN 1188, ASP 1213, GLY 1215, ASN 1216, GLN 1229, ASN 1242, ASN 1243, SER 1274, and THR 1277, wherein the modifications are selected from: (i) Replace the acidic amino acid residues exposed on the surface with basic amino acid residues; (ii) Replace the surface-exposed acidic amino acid residues with uncharged amino acid residues; (iii) Replace the uncharged amino acid residues exposed on the surface with basic amino acid residues; (iv) Insertion of basic amino acid residues; and (v) Remove acidic amino acid residues exposed on the surface.

[0053] In a related aspect, the present invention provides the use of modified BoNT / A in the preparation of a medicament for treating cervical dystonia in subjects, with a duration of effect longer than that of treatment with unmodified BoNT / A (e.g., SEQ ID NO:2), wherein the modified BoNT / A is administered to the affected neck muscles of the subject via intramuscular injection. The modified BoNT / A was administered in unit doses ranging from 53 U to 948 U, where 1 unit corresponds to the median lethal dose (LD50) calculated in mice. 50 The amount of BoNT / A modified by ) At least a single unit dose was administered to the affected neck muscles. The total dose of modified BoNT / A administered during treatment was as high as 9,480 U, and The modified BoNT / A includes modifications at one or more amino acid residues selected from the following: ASN 886, ASN 905, GLN 915, ASN 918, GLU 920, ASN 930, ASN 954, SER 955, GLN 991, GLU 992, GLN995, ASN 1006, ASN 1025, ASN 1026, ASN 1032, ASN 1043, ASN 1046, ASN 1052, ASP 1058, HIS 1064, ASN 1080, GLU 1081, GLU 1083, ASP 1086, ASN 1188, ASP 1213, GLY 1215, ASN 1216, GLN 1229, ASN 1242, ASN 1243, SER 1274, and THR 1277, wherein the modifications are selected from: (i) Replace the acidic amino acid residues exposed on the surface with basic amino acid residues; (ii) Replace the surface-exposed acidic amino acid residues with uncharged amino acid residues; (iii) Replace the uncharged amino acid residues exposed on the surface with basic amino acid residues; (iv) Insertion of basic amino acid residues; and (v) Remove acidic amino acid residues exposed on the surface.

[0054] The unit dose can be 53 to 948 units of modified BoNT / A, wherein the modified BoNT / A contains modifications at one or more amino acid residues selected from the following: ASN 886, ASN 905, GLN 915, ASN 918, GLU 920, ASN 930, ASN 954, SER 955, GLN 991, GLU 992, GLN 995, ASN 1006, ASN 1025, ASN 1026, ASN 1032, ASN 1043, ASN 1046, ASN 1052, ASP 1058, HIS 1064, ASN 1080, GLU 1081, GLU 1083, ASP 1086, ASN 1188, ASP 1213, GLY 1215, ASN 1216, GLN 1229, ASN 1242, ASN 1243, SER 1274, and THR 1277, wherein the modification is selected from: (i) replacing surface-exposed acidic amino acid residues with basic amino acid residues; (ii) replacing surface-exposed acidic amino acid residues with uncharged amino acid residues; (iii) replacing surface-exposed uncharged amino acid residues with basic amino acid residues; (iv) inserting basic amino acid residues; and (v) deleting surface-exposed acidic amino acid residues. The upper limit of the unit dose range may be 925, 900, 850, 800, 750, 700, 650, 600, 550, 500, 450, 400, 350, 300, 250, 200, 150, or 100 units of modified BoNT / A, preferably an upper limit of 889 units. The lower limit of the unit dose range can be 55, 60, 65, 70, 75, 80, 85, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, or 900 units of modified BoNT / A, preferably 59 units. Preferably, the unit dose of modified BoNT / A is from 59 units to 889 units, for example, from 200 units to 600 units. Most preferably, the unit dose of modified BoNT / A is from 237 units to 355 units, for example, from 284 units to 308 units.

[0055] The unit dose can be greater than 118.5 units or greater than 592.5 units of modified BoNT / A, wherein the modified BoNT / A contains modifications at one or more amino acid residues selected from the following: ASN 886, ASN 905, GLN 915, ASN 918, GLU 920, ASN 930, ASN 954, SER 955, GLN 991, GLU 992, GLN 995, ASN 1006, ASN 1025, ASN 1026, ASN 1032, ASN 1043, ASN 1046, ASN 1052, ASP 1058, HIS 1064, ASN 1080, GLU 1081, GLU 1083, ASP 1086, ASN 1188, ASP 1213, GLY 1215, ASN 1216, GLN 1229, ASN 1242, ASN 1243, SER 1274, and THR 1277, wherein the modification is selected from: (i) replacing surface-exposed acidic amino acid residues with basic amino acid residues; (ii) replacing surface-exposed acidic amino acid residues with uncharged amino acid residues; (iii) replacing surface-exposed uncharged amino acid residues with basic amino acid residues; (iv) inserting basic amino acid residues; and (v) deleting surface-exposed acidic amino acid residues. The modified BoNT / A per unit dose can be a modified BoNT / A greater than 118.5 units up to 888 units, for example, a modified BoNT / A greater than 592.5 units up to 888 units.

[0056] The total dose administered during the treatment regimen of this invention can be up to 9,480 units of modified BoNT / A, wherein the modified BoNT / A comprises modifications at one or more amino acid residues selected from the following: ASN 886, ASN 905, GLN 915, ASN 918, GLU 920, ASN 930, ASN 954, SER 955, GLN 991, GLU 992, GLN 995, ASN 1006, ASN 1025, ASN 1026, ASN 1032, ASN 1043, ASN 1046, ASN 1052, ASP 1058, HIS 1064, ASN 1080, GLU 1081, GLU 1083, ASP 1086, ASN 1188, ASP 1213, GLY 1215, ASN 886, 9480 ... 1216, GLN1229, ASN 1242, ASN 1243, SER 1274, and THR 1277, wherein the modification is selected from: (i) replacing surface-exposed acidic amino acid residues with basic amino acid residues; (ii) replacing surface-exposed acidic amino acid residues with uncharged amino acid residues; (iii) replacing surface-exposed uncharged amino acid residues with basic amino acid residues; (iv) inserting basic amino acid residues; and (v) deleting surface-exposed acidic amino acid residues. In other words, the total amount of modified BoNT / A administered in a given treatment course can be up to 9,480 units. The total dose can be up to 9,000, 8,000, 7,000, 6,000, 5,000, 4,000, 3,000, 2,000, or 1,000 units. Preferably, the total dose can be up to 8,890 units of modified BoNT / A. The total dose can be at least 83, 95, 106, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1,000, 1,500, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, or 9,000 units. Preferably, the total dose can be at least 106 units, more preferably at least 118 units of modified BoNT / A, for example, at least 355 units. The total dose can be from 371 units to 9,480 units, preferably from 413 units to 8,890 units. More preferably, the total dose administered is from 889 units to 8,890 units.

[0057] The total dose administered may be greater than 118.5 units or greater than 592.5 units of modified BoNT / A, wherein the modified BoNT / A contains modifications at one or more amino acid residues selected from the following: ASN 886, ASN 905, GLN 915, ASN 918, GLU 920, ASN 930, ASN 954, SER 955, GLN 991, GLU 992, GLN 995, ASN 1006, ASN 1025, ASN 1026, ASN 1032, ASN 1043, ASN 1046, ASN 1052, ASP 1058, HIS 1064, ASN 1080, GLU 1081, GLU 1083, ASP 1086, ASN 1188, ASP 1213, GLY 1215, ASN 1216, GLN 1229, ASN 1242, ASN 1243, SER 1274, and THR 1277, wherein the modification is selected from: (i) replacing surface-exposed acidic amino acid residues with basic amino acid residues; (ii) replacing surface-exposed acidic amino acid residues with uncharged amino acid residues; (iii) replacing surface-exposed uncharged amino acid residues with basic amino acid residues; (iv) inserting basic amino acid residues; and (v) deleting surface-exposed acidic amino acid residues. Preferably, the total dose may be greater than 118.5 units of modified BoNT / A, for example, greater than 592.5 units of modified BoNT / A.

[0058] "Affected neck muscles" can be neck muscles that contribute to (e.g., cause) neck dystonia and / or symptoms in the subject, or neck muscles that contribute to (e.g., cause) neck dystonia and / or symptoms in the subject. This does not mean that "affected neck muscles" must necessarily contribute to (e.g., cause) neck dystonia and / or symptoms at the time of treatment, although this is preferred. For example, neck muscles can be muscles that have contributed to (e.g., caused) neck dystonia and / or symptoms in the subject in the past, or muscles that are expected to contribute to (e.g., cause) neck dystonia and / or symptoms in the subject in the future. In one embodiment, two or more neck muscles (e.g., an agonist pair and an antagonist pair of neck muscles) can contribute to (e.g., cause) neck dystonia and / or symptoms in the subject. In this case, modified BoNT / A can be applied to two or more neck muscles (e.g., to the agonist and antagonist neck muscles of the neck muscle pair).

[0059] The affected neck muscles preferably contribute to (e.g., cause) neck dystonia and / or its symptoms in the subject through contraction. Therefore, the affected neck muscles are preferably the subject's neck muscles whose contraction or contraction leads to neck dystonia and / or its symptoms. The neck muscles are preferably, for example, neck muscles that contract involuntarily during treatment or have already contracted involuntarily. Neck muscles can be any muscle (e.g., skeletal muscle) effectively connected to the subject's neck and / or head, such as any muscle capable of altering the subject's head position (e.g., when contracting). The affected neck muscles can be capable of: causing the subject's chin to twist toward the subject's shoulder, resulting in lateral head rotation (torticollis); causing the subject's head to tilt forward (neck flexion); causing the subject's head to tilt backward (neck extension); causing the subject's head to tilt laterally (lateral neck); causing the subject's head to shift anteriorly (forward displacement); and / or causing the subject's head to shift posteriorly (backward displacement).

[0060] Affected neck muscles may include the sternocleidomastoid, sternocleidomastoid, splenius capitis, splenius cervicis, scalene complex (e.g., anterior and / or middle scalene), trapezius (e.g., upper and / or lower trapezius), levator scapulae, semispinalis capitis, or longissimus capitis (e.g., longissimus capitis and / or longissimus cervicis). Affected neck muscles may include the sternocleidomastoid, splenius capitis, splenius cervicis, scalene complex (e.g., anterior and / or middle scalene), trapezius (e.g., upper and / or lower trapezius), levator scapulae, semispinalis capitis, longissimus (e.g., longissimus capitis and / or longissimus cervicis), posterior paravertebral muscles (e.g., posterior, middle, and / or anterior scalene, preferably posterior scalene), submental complex (e.g., digastric, geniohyoid, mylohyoid, mylohyoid, button eye, and / or stylohyoid), upper cervical line—clavicle (lateral portion), spinous processes C3-Th3-mastoid, spinous processes Th3-Th 5—Transverse processes C1-C2, transverse processes C3-Th6, spinous processes C3-Th1—upper cervical line, transverse processes Th1-Th6—spinous processes C2-C7, transverse processes C3-Th3—mastoid process, transverse processes Th1-Th6—transverse processes C2-C6, inferior oblique capitis muscle, spinous process C2—transverse process C1, suprasternal notch and clavicle (medial part)—mastoid process and upper cervical line, transverse processes C1-C4—scapula (superior angle of scapula), transverse processes C2-C7—first rib, transverse processes C3-C6—first rib, longus capitis muscle, transverse processes C3-C6—occipital bone (basal part), longus colli muscle or transverse processes C2-C5—atlas (anterior tubercle). The affected neck muscles may include the right levator scapulae, left levator scapulae, right trapezius, left trapezius, right sternocleidomastoid, left sternocleidomastoid, right splenius capitis, left splenius capitis, middle scalene, anterior scalene, right semispinalis capitis, left semispinalis capitis, right longissimus capitis, or left longissimus capitis.Affected neck muscles may include the sternocleidomastoid (e.g., left or right sternocleidomastoid), splenius capitis (left or right), anterior or middle scalene, left or right trapezius (e.g., left or right upper trapezius), levator scapulae (left or right), semispinalis capitis (left or right), longissimus (e.g., left or right longissimus capitis and / or longissimus cervicis), splenius cervicis, scalene muscle complex (e.g., anterior and / or middle scalene), posterior paravertebral muscles (e.g., posterior, middle, and / or anterior scalene, preferably posterior scalene), submental complex (e.g., digastric, geniohyoid, mylohyoid, mylohyoid, button eye, and / or stylohyoid), lower trapezius, superior nuchal line—clavicle (lateral portion), spinous processes C3-Th3-mastoid, spinous process Th3 -Th5—Transverse processes C1-C2, Transverse processes C3-Th6, Spinous processes C3-Th1—Superior nuchal line, Transverse processes Th1-Th6—Spinous processes C2-C7, Transverse processes C3-Th3—Mastoid process, Transverse processes Th1-Th6—Transverse processes C2-C6, Inferior oblique capitis, Superior oblique capitis, Spinous process C2—Transverse process C1, Suprasternal notch and clavicle (medial part)—Mastoid process and superior nuchal line, Transverse processes C1-C4—Scapula (superior angle of scapula), Transverse processes C2-C7—First rib, Transverse processes C3-C6—First rib, Longus capitis, Transverse processes C3-C6—Occipital bone (basal part), Longus colli, Semispinalis cervicis, Rectus capitis posterior major, Rectus capitis posterior minor, Rectus capitis anterior, Multifidus or Transverse processes C2-C5—Atlanta (anterior tubercle).

[0061] The neck muscles that may be affected include: semispinalis cervicis, levator scapulae, splenius cervicis, longissimus cervicis, lower trapezius, sternocleidomastoid, semispinalis capitis, inferior oblique capitis, longissimus capitis, splenius capitis, semispinalis cervicis, middle scalene, longissimus capitis, longus cervicis, or longus capitis.

[0062] The multiple affected neck muscles treated according to the present invention may include at least one (e.g., at least two) of any of the muscles described herein.

[0063] In one embodiment, the modified BoNT / A can be applied to one or more affected neck muscles selected from: sternocleidomastoid, splenius capitis, splenius cervicis, scalene complex (e.g., anterior and / or middle scalene), trapezius (e.g., upper and / or lower trapezius), levator scapulae, semispinalis capitis, and longissimus (e.g., longissimus capitis and / or longissimus cervicis). Preferably, the modified BoNT / A is applied to multiple affected neck muscles. For example, the modified BoNT / A can be applied to at least two (e.g., at least three, four, five, six, or seven, preferably eight) affected neck muscles selected from: sternocleidomastoid, splenius capitis, splenius cervicis, scalene complex (e.g., anterior and / or middle scalene), trapezius (e.g., upper and / or lower trapezius), levator scapulae, semispinalis capitis, and longissimus (e.g., longissimus capitis and / or longissimus cervicis).

[0064] In one embodiment, the modified BoNT / A can be applied to one or more affected neck muscles selected from 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, and left longissimus capitis.

[0065] In one embodiment, the modified BoNT / A can be applied to one or more affected neck muscles selected from: sternocleidomastoid, splenius capitis, splenius cervicis, scalene muscle complex (e.g., anterior and / or middle scalene), trapezius (e.g., upper and / or lower trapezius), levator scapulae, semispinalis capitis, longissimus (e.g., longissimus capitis and / or longissimus cervicis), posterior paravertebral muscles (e.g., posterior, middle, and / or anterior scalene, preferably posterior scalene), submental complex (e.g., digastric, geniohyoid, mylohyoid, mylohyoid oculi, and / or stylohyoid), upper cervical line—clavicle (lateral portion) The following are the muscle groups involved: spinous processes C3-Th3-mastoid process, spinous processes Th3-Th5-transverse processes C1-C2, transverse processes C3-Th6, spinous processes C3-Th1-upper cervical line, transverse processes Th1-Th6-spinous processes C2-C7, transverse processes C3-Th3-mastoid process, transverse processes Th1-Th6-transverse processes C2-C6, inferior oblique capitis muscle, spinous process C2-transverse process C1, suprasternal notch and clavicle (medial portion)-mastoid process and upper cervical line, transverse processes C1-C4-scapula (superior angle of scapula), transverse processes C2-C7-first rib, transverse processes C3-C6-first rib, longus capitis muscle, transverse processes C3-C6-occipital bone (basal portion), longus colli muscle and transverse processes C2-C5-atlas (anterior tubercle). Preferably, the modified BoNT / A is applied to multiple affected neck muscles.For example, the modified BoNT / A can be applied 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, said affected neck muscles being selected from: sternocleidomastoid, sternocleidomastoid, splenius capitis, splenius cervicis, scalene muscle complex (e.g., anterior scalene and / or middle scalene), trapezius (e.g., upper trapezius and / or lower trapezius), levator scapulae, semispinalis capitis, longissimus (e.g., longissimus capitis and / or longissimus cervicis), posterior paravertebral muscles (e.g., posterior scalene, middle scalene, and / or anterior scalene, preferably posterior scalene), submental complex (e.g., digastric muscle, geniohyoid muscle, mylohyoid muscle, mylohyoid muscle buttonhole and / or stylohyoid muscle), upper cervical line—clavicle (lateral part), spinous process C3-Th3-mastoid, spinous process Th3-Th5—transverse process C1-C2, transverse process C3-Th6, spinous process C3-Th1—upper cervical line, transverse process Th1-Th6—spinous process C2-C7, transverse process C3-Th3—mastoid, transverse process Th1 -Th6—transverse processes C2-C6, inferior oblique capitis muscle, spinous process C2—transverse process C1, suprasternal notch and clavicle (medial part)—mastoid process and upper cervical line, transverse processes C1-C4—scapula (superior angle of scapula), transverse processes C2-C7—first rib, transverse processes C3-C6—first rib, longus capitis muscle, transverse processes C3-C6—occipital bone (basal part), longus colli muscle and transverse processes C2-C5—atlas (anterior tubercle).

[0066] In one embodiment, the modified BoNT / A can be applied to one or more affected neck muscles, including: 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 upper trapezius), left or right levator scapulae, left or right semispinalis capitis, middle semispinalis capitis, longissimus (e.g., left or right longissimus capitis and / or longissimus cervicis), splenius cervicis, scalene muscle complex (e.g., anterior and / or middle scalene), posterior paravertebral muscles (e.g., posterior, middle, and / or anterior scalene, preferably posterior scalene), submental complex (e.g., digastric, geniohyoid, mylohyoid, mylohyoid, button eye, 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 capitis muscle, superior oblique capitis muscle, spinous process C2-transverse process C1, suprasternal notch and clavicle (medial part)-mastoid process and superior nuchal line, transverse process C1-C4-scapula (superior angle of scapula), transverse process C2-C7-first rib, transverse process C3-C6-first rib, longus capitis muscle, transverse process C3-C6-occipital bone (basal part), longus colli muscle, semispinalis cervicis muscle, rectus capitis posterior major muscle, rectus capitis posterior minor muscle, rectus capitis anterior muscle, multifidus muscle or transverse process C2-C5-atlas (anterior tubercle). Preferably, the modified BoNT / A is applied to multiple affected neck muscles.For example, the modified BoNT / A can be applied 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, said affected neck muscles being selected from: the sternocleidomastoid muscle (e.g., left or right). Sternocleidomastoid muscle), left or right splenius capitis, anterior or middle scalene muscles, left or right trapezius (e.g., left or right upper trapezius), left or right levator scapulae, left or right semispinalis capitis, middle semispinalis capitis, longissimus (e.g., left or right longissimus capitis and / or longissimus cervicis), splenius cervicis, scalene muscle complex (e.g., anterior and / or middle scalene muscles), posterior paravertebral muscles (e.g., posterior scalene, middle scalene and / or anterior scalene, preferably posterior scalene), submental complex (e.g. Digastric muscle, geniohyoid muscle, mylohyoid muscle, mylohyoid muscle (buttock 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, subcephalic region Oblique muscles, oblique superior, spinous process C2-transverse process C1, suprasternal notch and clavicle (medial part)-mastoid process and superior nuchal line, transverse processes C1-C4-scapula (superior angle of scapula), transverse processes C2-C7-first rib, transverse processes C3-C6-first rib, longus capitis, transverse processes C3-C6-occipital bone (basal part), longus colli, semispinalis cervicis, rectus capitis posterior major, rectus capitis posterior minor, rectus capitis anterior, multifidus and / or transverse processes C2-C5-atlas (anterior tubercle).

[0067] In one embodiment, the modified BoNT / A can be applied to one or more affected neck muscles selected from: 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 upper trapezius), left or right levator scapulae, left or right semispinalis capitis, longissimus (e.g., left or right longissimus capitis and / or longissimus cervicis), splenius cervicis, scalene muscle complex (e.g., anterior and / or middle scalene), posterior paravertebral muscles (e.g., posterior, middle, and / or anterior scalene, preferably posterior scalene), submental complex (e.g., digastric, geniohyoid, mylohyoid, mylohyoid, buttonhole, and / or stylohyoid), lower trapezius, superior nuchal line—clavicle (lateral portion) (Divided into), 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 processes Th1-Th6-transverse processes C2-C6, inferior oblique capitis, superior oblique capitis, spinous process C2-transverse process C1, suprasternal notch and clavicle (medial part)-mastoid and superior nuchal line, transverse processes C1-C4-scapula (superior angle of scapula), transverse processes C2-C7-first rib, transverse processes C3-C6-first rib, longus capitis, transverse processes C3-C6-occipital bone (basal part), longus colli, semispinalis cervicis, rectus capitis posterior major, rectus capitis posterior minor, rectus capitis anterior, multifidus and transverse processes C2-C5-atlas (anterior tubercle). Preferably, the modified BoNT / A is applied to multiple affected neck muscles.For example, the modified BoNT / A can be applied 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, said affected neck muscles being selected from: the sternocleidomastoid muscle (e.g., Left or right sternocleidomastoid muscle), left or right splenius capitis, anterior or middle scalene muscles, left or right trapezius (e.g., left or right upper trapezius), left or right levator scapulae, left or right semispinalis capitis, longissimus (e.g., left or right longissimus capitis and / or longissimus cervicis), splenius cervicis, scalene muscle complex (e.g., anterior and / or middle scalene muscles), posterior paravertebral muscles (e.g., posterior, middle, and / or anterior scalene muscles, preferably posterior scalene), submental complex (e.g., digastric... Muscles, including the geniohyoid, mylohyoid, mylohyoid, button eye and / or stylohyoid muscles, lower part of the trapezius, superior nuchal line—clavicle (lateral part), 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 processes Th1-Th6—transverse processes C2-C6, subcephalic region Oblique muscles, oblique superior, spinous process C2—transverse process C1, suprasternal notch and clavicle (medial part)—mastoid process and superior nuchal line, transverse processes C1-C4—scapula (superior angle of scapula), transverse processes C2-C7—first rib, transverse processes C3-C6—first rib, longus capitis, transverse processes C3-C6—occipital bone (basal part), longus colli, semispinalis capitis, rectus capitis posterior major, rectus capitis posterior minor, rectus capitis anterior, multifidus and transverse processes C2-C5—atlas (anterior tubercle).

[0068] In one embodiment, the modified BoNT / A can be applied to one or more affected neck muscles, including: semispinalis cervicis, levator scapulae, splenius cervicis, longissimus cervicis, lower trapezius, sternocleidomastoid, semispinalis capitis, inferior oblique capitis, longissimus capitis, splenius capitis, semispinalis cervicis, middle scalene, longissimus capitis, longus colli, or longus capitis. In another embodiment, the modified BoNT / A can be applied to one or more affected neck muscles selected from: semispinalis cervicis, levator scapulae, splenius cervicis, longissimus cervicis, lower trapezius, sternocleidomastoid, semispinalis capitis, inferior oblique capitis, longissimus capitis, splenius capitis, semispinalis cervicis, middle scalene, longus capitis, longus colli, and longus capitis. For example, the modified BoNT / A can be applied to at least two (e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15) of the affected neck muscles.

[0069] When there are two equal neck muscles on both sides of the neck (e.g., sternocleidomastoid muscles, such as the left and right sternocleidomastoid muscles), modified BoNT / A can be applied unilaterally (e.g., to one of the muscles where only one muscle contracts) or bilaterally (e.g., to both muscles where both muscles contract). When treating multiple affected muscles, it is preferable to treat multiple different types of affected muscles. For example, when applying bilaterally to two affected sternocleidomastoid muscles, it is preferable to treat additional muscles while treating multiple affected muscles as described herein.

[0070] When there are two equal neck muscles on both sides of the neck, one of the equal neck muscles can be applied unilaterally. For example, the contracting muscle or the non-contracting equal muscle can be applied. In one embodiment, the muscle on one side of the subject's neck is applied unilaterally, where the subject's neck exhibits symptoms of dystonia, or the equal muscle on the opposite side of the subject's neck is applied unilaterally, where the subject's neck does not exhibit symptoms of dystonia.

[0071] The affected neck muscles selected for treatment according to the invention may depend on the specific manifestation of neck dystonia in the subject to be treated (e.g., torticollis, lateral neck, flexion, extension, or a combination thereof).

[0072] The affected neck muscles selected for treatment according to the invention may depend on the specific manifestation of neck dystonia in the subject to be treated (e.g., torticollis, lateral neck, flexion of the neck, extension of the neck, lateral head tilt, torticollis, flexion of the head, extension of the head, lateral displacement, sagittal displacement or a combination thereof).

[0073] In one embodiment, when treating torticollis, the modified BoNT / A can be applied to one or more affected neck muscles selected from the following: sternocleidomastoid, trapezius (e.g., upper trapezius), anterior scalene, splenius capitis, splenius cervicis, levator scapulae, and longissimus (e.g., longissimus capitis and / or longissimus cervicis). Preferably, when treating torticollis, the modified BoNT / A can be applied contralaterally to one or more affected neck muscles selected from the following: sternocleidomastoid, trapezius (e.g., upper trapezius), and anterior scalene; and / or ipsilaterally to one or more affected neck muscles selected from the following: splenius capitis, splenius cervicis, levator scapulae, and longissimus (e.g., longissimus capitis and / or longissimus cervicis).

[0074] In one embodiment, when treating torticollis, the modified BoNT / A may be applied to one or more affected neck muscles selected from the following: sternocleidomastoid, trapezius (e.g., upper trapezius), anterior scalene, splenius capitis, splenius cervicis, levator scapulae, and longissimus (e.g., longissimus capitis and / or longissimus cervicis) and semispinalis cervicis. In one embodiment, when treating torticollis, the modified BoNT / A may be applied contralaterally to one or more affected neck muscles selected from the following: sternocleidomastoid, trapezius (e.g., upper trapezius), and anterior scalene; and / or ipsilaterally to one or more affected neck muscles selected from the following: splenius capitis, splenius cervicis, levator scapulae, and longissimus (e.g., longissimus capitis and / or longissimus cervicis) and semispinalis cervicis.

[0075] In one embodiment, when treating torticollis, the modified BoNT / A may be applied to one or more affected neck muscles selected from the following: sternocleidomastoid, trapezius (e.g., upper trapezius), anterior scalene, splenius capitis, splenius cervicis, levator scapulae, longissimus (e.g., longissimus capitis and / or longissimus cervicis), semispinalis capitis, rectus capitis posterior major, multifidus, and inferior oblique capitis. In one embodiment, when treating torticollis, the modified BoNT / A may be applied contralaterally to one or more affected neck muscles selected from the following: sternocleidomastoid, trapezius (e.g., upper trapezius), anterior scalene, semispinalis capitis, and multifidus; and / or ipsilaterally to one or more affected neck muscles selected from the following: splenius capitis, splenius cervicis, levator scapulae, longissimus (e.g., longissimus capitis and / or longissimus cervicis), rectus capitis posterior major, and inferior oblique capitis.

[0076] In one embodiment, when treating torticollis, the modified BoNT / A can be applied to one or more affected neck muscles, including: sternocleidomastoid, trapezius (e.g., upper trapezius), anterior scalene, splenius capitis, splenius cervicis, levator scapulae, longissimus (e.g., longissimus capitis and / or longissimus cervicis), semispinalis capitis, rectus capitis posterior major, multifidus, or inferior oblique capitis. In one embodiment, when treating torticollis, the modified BoNT / A can be applied contralaterally to one or more affected neck muscles, including: sternocleidomastoid, trapezius (e.g., upper trapezius), anterior scalene, semispinalis capitis, 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 major, or inferior oblique capitis.

[0077] In one embodiment, when treating lateral neck, the modified BoNT / A can be applied to one or more affected neck muscles selected from the following: levator scapulae, trapezius (e.g., upper trapezius), scalene complex (e.g., anterior and / or middle scalene), sternocleidomastoid, splenius capitis, splenius cervicis, and longissimus capitis (e.g., longissimus capitis and / or longissimus cervicis). Preferably, when treating lateral neck, the modified BoNT / A can be applied ipsilaterally to one or more of the affected neck muscles.

[0078] In one embodiment, when treating lateral neck, the modified BoNT / A can be applied to one or more affected neck muscles selected from the following: levator scapulae, trapezius (e.g., upper trapezius), scalene complex (e.g., anterior and / or middle scalene), sternocleidomastoid, splenius capitis, splenius cervicis, and longissimus cervicis (e.g., longissimus capitis and / or longissimus cervicis), and semispinalis cervicis. In one embodiment, when treating lateral neck, the modified BoNT / A can be applied ipsilaterally to one or more of the said affected neck muscles.

[0079] In one embodiment, when treating lateral neck, the modified BoNT / A can be applied to one or more affected neck muscles selected from the following: levator scapulae, trapezius (e.g., upper trapezius), scalene complex (e.g., anterior 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 lateral neck, the modified BoNT / A can be applied ipsilaterally to one or more of the said affected neck muscles.

[0080] In one embodiment, when treating lateral neck, the modified BoNT / A can be applied to one or more affected neck muscles selected from the following: levator scapulae, trapezius (e.g., upper trapezius), scalene complex (e.g., anterior and / or middle scalene), sternocleidomastoid, splenius capitis, splenius cervicis, and longissimus (e.g., longissimus capitis and / or longissimus cervicis), semispinalis cervicis, and multifidus. In one embodiment, when treating lateral neck, the modified BoNT / A can be applied ipsilaterally to one or more of the said affected neck muscles.

[0081] In one embodiment, when treating lateral neck, the modified BoNT / A can be applied to one or more affected neck muscles, including: levator scapulae, trapezius (e.g., upper trapezius), scalene complex (e.g., anterior and / or middle scalene), sternocleidomastoid, splenius capitis, splenius cervicis, longissimus (e.g., longissimus capitis and / or longissimus cervicis), semispinalis cervicis, or multifidus. In one embodiment, when treating lateral neck, the modified BoNT / A can be applied ipsilaterally to one or more of the affected neck muscles.

[0082] In one embodiment, when treating neck flexion, the modified BoNT / A can be applied to one or more affected neck muscles selected from the following: sternocleidomastoid, anterior scalene, and middle scalene. Preferably, when treating neck flexion, the modified BoNT / A can be applied bilaterally.

[0083] In one embodiment, when treating neck flexors, the modified BoNT / A can be applied to one or more affected neck muscles selected from: sternocleidomastoid, anterior scalene, middle scalene, levator scapulae, longus colli, and submental complex (e.g., digastric, geniohyoid, mylohyoid, mylohyoid nodule, and / or stylohyoid). Preferably, when treating neck flexion, the modified BoNT / A can be applied bilaterally.

[0084] In one embodiment, when treating neck flexors, the modified BoNT / A may be applied 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 applied bilaterally.

[0085] In one embodiment, when treating neck flexors, the modified BoNT / A may be applied to one or more affected neck muscles selected from the following: sternocleidomastoid, anterior scalene, middle scalene, levator scapulae, longus colli, submental complex (e.g., digastric, geniohyoid, mylohyoid, mylohyoid oculi, and / or stylohyoid), longus capitis, and rectus capitis anterior. In one embodiment, when treating neck flexion, the modified BoNT / A may be applied bilaterally.

[0086] In one implementation, when treating neck flexors, the modified BoNT / A can be applied to one or more affected neck muscles, including: sternocleidomastoid, anterior scalene, middle scalene, levator scapulae, longus colli, submental complex (e.g., digastric, geniohyoid, mylohyoid, mylohyoid nodule, and / or stylohyoid), longus capitis, or rectus capitis anterior. In one implementation, when treating neck flexion, the modified BoNT / A can be applied bilaterally.

[0087] In one embodiment, when treating neck extension, the modified BoNT / A can be applied to one or more affected neck muscles selected from: levator scapulae, trapezius (e.g., upper trapezius), longissimus (e.g., longissimus capitis and / or longissimus cervicis)), splenius capitis, splenius cervicis, and semispinalis capitis. Preferably, when treating neck extension, the modified BoNT / A can be applied bilaterally.

[0088] In one embodiment, when treating neck extension, the modified BoNT / A can be applied to one or more affected neck muscles selected from: levator scapulae, trapezius (e.g., upper trapezius), longissimus (e.g., longissimus capitis and / or longissimus cervicis), splenius capitis, splenius cervicis, semispinalis capitis, semispinalis cervicis, and posterior paraspinal muscles (e.g., posterior scalene, middle scalene, and / or anterior scalene, preferably posterior scalene). Preferably, when treating neck extension, the modified BoNT / A can be applied bilaterally.

[0089] In one embodiment, when treating neck extension, the modified BoNT / A can be applied 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 cervicis), splenius capitis, splenius cervicis, semispinalis capitis, semispinalis cervicis, spinae capitis, rectus capitis posterior major, rectus capitis posterior minor, and oblique capitis superior. Preferably, when treating neck extension, the modified BoNT / A can be applied bilaterally.

[0090] In one embodiment, when treating neck extension, the modified BoNT / A can be applied to one or more affected neck muscles selected from: levator scapulae, trapezius (e.g., upper trapezius), longissimus (e.g., longissimus capitis and / or longissimus cervicis), splenius capitis, splenius cervicis, semispinalis capitis, semispinalis cervicis, posterior paravertebral muscles (e.g., posterior scalene, middle scalene and / or anterior scalene, preferably posterior scalene), spinae capitis, rectus capitis posterior major, rectus capitis posterior minor, and oblique capitis superior. Preferably, the modified BoNT / A can be applied bilaterally when treating neck extension.

[0091] In one embodiment, when treating neck extension, the modified BoNT / A can be applied to one or more affected neck muscles, including: levator scapulae, trapezius (e.g., upper trapezius), longissimus (e.g., longissimus capitis and / or longissimus cervicis), splenius capitis, splenius cervicis, semispinalis capitis, semispinalis cervicis, posterior paravertebral muscles (e.g., posterior scalene, middle scalene and / or anterior scalene, preferably posterior scalene), spinae capitis, rectus capitis posterior major, rectus capitis posterior minor, or oblique capitis superior. Preferably, when treating neck extension, the modified BoNT / A can be applied bilaterally.

[0092] In one embodiment, when treating lateral displacement, the modified BoNT / A can be applied 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 cervicis, longissimus (e.g., longissimus capitis and / or longissimus cervicis), and semispinalis cervicis. Preferably, when treating lateral displacement, the modified BoNT / A can be applied to the affected neck muscles on a first side of the neck (e.g., left side), the affected neck muscles being selected from the following: levator scapulae, semispinalis cervicis, middle scalene, and longissimus cervicis, and the modified BoNT / A can be applied to the affected muscles on a second side of the neck (e.g., right side), the affected muscles being selected from the following: sternocleidomastoid, lower trapezius, splenius capitis, semispinalis capitis, longissimus capitis, and levator scapulae.

[0093] In one embodiment, when treating lateral displacement, the modified BoNT / A can be applied 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 cervicis, longissimus (e.g., longissimus capitis and / or longissimus cervicis), or semispinalis cervicis. Preferably, when treating lateral displacement, the modified BoNT / A can be applied to the affected neck muscles on a first side of the neck (e.g., left side), including: levator scapulae, semispinalis cervicis, middle scalene, or longissimus cervicis, and the modified BoNT / A can be applied to the affected muscles on a second side of the neck (e.g., right side), including: sternocleidomastoid, lower trapezius, splenius capitis, semispinalis capitis, longissimus capitis, or levator scapulae.

[0094] In one embodiment, when treating lateral head tilt, the modified BoNT / A can be applied to one or more affected neck muscles selected from: the lower trapezius, sternocleidomastoid, longissimus capitis, splenius capitis, semispinalis capitis, levator scapulae, and posterior paravertebral muscles (e.g., posterior scalene, middle scalene, and / or anterior scalene, preferably posterior scalene). Preferably, when treating lateral head tilt, the modified BoNT / A can be applied ipsilaterally.

[0095] In one embodiment, when treating lateral head tilt, the modified BoNT / A can be applied to one or more affected neck muscles, including: lower trapezius, sternocleidomastoid, longissimus capitis, splenius capitis, semispinalis capitis, levator scapulae, or posterior paravertebral muscles (e.g., posterior scalene, middle scalene, and / or anterior scalene, preferably posterior scalene). Preferably, when treating lateral head tilt, the modified BoNT / A can be applied ipsilaterally.

[0096] In one embodiment, when treating plagiocephaly, the modified BoNT / A can be applied to one or more affected neck muscles selected from the following: lower trapezius, sternocleidomastoid, longissimus capitis, splenius capitis, middle semispinalis capitis, and inferior oblique capitis. Preferably, when treating plagiocephaly, the modified BoNT / A can be applied ipsilaterally or contralaterally. For example, the modified BoNT / A can be applied contralaterally to one or more affected neck muscles selected from the following: lower trapezius, sternocleidomastoid, and middle semispinalis capitis; and / or the modified BoNT / A can be applied ipsilaterally to one or more neck muscles selected from the following: inferior oblique capitis, longissimus capitis, and splenius capitis.

[0097] In one embodiment, when treating plagiocephaly, the modified BoNT / A can be applied to one or more affected neck muscles, including the lower trapezius, sternocleidomastoid, longissimus capitis, splenius capitis, middle semispinalis capitis, or inferior oblique capitis. Preferably, when treating plagiocephaly, the modified BoNT / A can be applied ipsilaterally or contralaterally. For example, the modified BoNT / A can be applied contralaterally to one or more affected neck muscles, including the lower trapezius, sternocleidomastoid, or middle semispinalis capitis; and / or the modified BoNT / A can be applied ipsilaterally to one or more neck muscles, including the inferior oblique capitis, longissimus capitis, or splenius capitis.

[0098] In one embodiment, when treating head flexion, the modified BoNT / A can be applied to one or more affected neck muscles selected from the longus capitis, levator scapulae, and sternocleidomastoid muscles. Preferably, when treating head flexion, the modified BoNT / A can be applied bilaterally.

[0099] In one embodiment, when treating head flexion, the modified BoNT / A can be applied to one or more affected neck muscles, including the longus capitis, levator scapulae, or sternocleidomastoid muscles. Preferably, when treating head flexion, the modified BoNT / A can be applied bilaterally.

[0100] In one embodiment, when treating head extension, the modified BoNT / A can be applied to one or more affected neck muscles selected from the inferior oblique capitis, semispinalis capitis, lower trapezius, and splenius capitis. Preferably, when treating head extension, the modified BoNT / A can be applied bilaterally.

[0101] In one embodiment, when treating head extension, the modified BoNT / A can be applied to one or more affected neck muscles, including the inferior oblique capitis, semispinalis capitis, lower trapezius, or splenius capitis. Preferably, when treating head extension, the modified BoNT / A can be applied bilaterally.

[0102] In one embodiment, when treating sagittal displacement, the modified BoNT / A can be applied to one or more affected neck muscles selected from: sternocleidomastoid, anterior scalene, middle scalene, levator scapulae, longus colli, submental complex (e.g., digastric, geniohyoid, mylohyoid, mylohyoid eye and / or stylohyoid), inferior oblique capitis, semispinalis capitis, lower trapezius and splenius capitis. Preferably, when treating sagittal displacement, the modified BoNT / A can be applied to the affected neck muscles on the first side of the neck (e.g., the left side), the affected neck muscles being selected from: sternocleidomastoid, anterior scalene, middle scalene, levator scapulae, longus colli, and submental complex (e.g., digastric, geniohyoid, mylohyoid, mylohyoid eye, and / or stylohyoid), and the modified BoNT / A can be applied to the affected neck muscles on the second side of the neck (e.g., the right side), the affected neck muscles being selected from: inferior oblique capitis, semispinalis capitis, lower trapezius, and splenius capitis.

[0103] In one implementation, when treating sagittal displacement, the modified BoNT / A can be applied to one or more affected neck muscles, including: sternocleidomastoid, anterior scalene, middle scalene, levator scapulae, longus colli, submental complex (e.g., digastric, geniohyoid, mylohyoid, mylohyoid eye and / or stylohyoid), inferior oblique capitis, semispinalis capitis, lower trapezius or splenius capitis. Preferably, when treating sagittal displacement, the modified BoNT / A can be applied to the affected neck muscles on the first side of the neck (e.g., the left side), the affected neck muscles including: sternocleidomastoid, anterior scalene, middle scalene, levator scapulae, longus colli, or submental complex (e.g., digastric, geniohyoid, mylohyoid, mylohyoid eye and / or stylohyoid), and the modified BoNT / A can be applied to the affected neck muscles on the second side of the neck (e.g., the right side), the affected neck muscles including: inferior oblique capitis, semispinalis capitis, lower trapezius or splenius capitis.

[0104] The modified BoNT / A is administered via intramuscular injection into the affected neck muscles. One or more unit doses (e.g., at least two unit doses) of the modified BoNT / A may be administered to the affected neck muscles. However, it is preferable to administer only a single unit dose to each affected neck muscle. When the neck muscle is the sternocleidomastoid muscle, two unit doses may be administered. For example, two unit doses may be administered to the left sternocleidomastoid muscle and / or two unit doses may be administered to the right sternocleidomastoid muscle. When the neck muscle is the trapezius muscle (e.g., the lower trapezius), two unit doses may be administered. For example, two unit doses may be administered to the left trapezius muscle (e.g., the upper left trapezius) and / or two unit doses may be administered to the right trapezius muscle (e.g., the upper right trapezius).

[0105] A unit dose can be administered to the affected neck muscle at a single injection site. Therefore, in some embodiments, the modified BoNT / A is administered at each injection site in a unit dose manner at the affected neck muscle. However, it is preferable to administer less than a unit dose at a single injection site, in which case the unit dose may be distributed (equal or unequal) among two or more injection sites of the affected neck muscle. Thus, the modified BoNT / A can be administered to the affected neck muscle at two or more injection sites. In a preferred embodiment, the modified BoNT / A is administered at each injection site of the affected neck muscle in a less than unit dose manner. Advantageously, this allows clinicians to delineate the muscle contour and / or treat the affected muscle area by applying more modified BoNT / A to the area that is less affected compared to the less affected area.

[0106] Preferably, a unit dose is applied to each injection site.

[0107] Each injection site can be treated with 750-4,000 pg of modified BoNT / A, wherein the modified BoNT / A comprises a BoNT / A light chain and a translocation domain, as well as a BoNT / B receptor-binding domain (H). C (Structural domain). Preferably, 1,000 pg or 2,000 pg of modified BoNT / A is administered at each injection site, wherein the modified BoNT / A comprises a BoNT / A light chain and a translocation domain, as well as a BoNT / B receptor-binding domain (H). C (structural domain).

[0108] Each injection site may administer a dose of 31-166.4 units of modified BoNT / A, wherein the modified BoNT / A comprises a BoNT / A light chain and a translocation domain, as well as a BoNT / B receptor-binding domain (H). C(Structural domain). Preferably, 41.6 units or 83.2 units of the modified BoNT / A are administered at each injection site, wherein the modified BoNT / A comprises a BoNT / A light chain and a translocation domain, as well as a BoNT / B receptor-binding domain (H). C (structural domain).

[0109] The term "administer at least a single unit dose" means that at least substantially all of the single unit dose is administered. For example, in a modified BoNT / A reconstructed vial, a residual amount of the unit dose (e.g., up to 1%, 0.1%, or 0.01%) may be retained. However, it is preferable to administer the entirety of at least a single unit dose (e.g., at one or more injection sites).

[0110] The potency of the modified BoNT / A used according to the present invention can be measured by mouse LD50 using standard techniques. 50 The determination is made by a assay. In this assay, one unit is defined as the calculated median lethal dose (LD50) in mice. 50 The amount of modified BoNT / A. Preferably, the calculated intraperitoneal median lethal dose for mice.

[0111] When the modified BoNT / A used in this invention comprises a BoNT / A light chain and a translocation domain, as well as a BoNT / B receptor-binding domain (H... C When BoNT / A is modified with a structural domain, the amount of modified BoNT / A corresponding to 1 unit in the determination method is preferably 24.04 pg.

[0112] When the modified BoNT / A used in this invention is a modified BoNT / A containing modifications at one or more amino acid residues selected from the following: ASN 886, ASN 905, GLN 915, ASN 918, GLU 920, ASN 930, ASN 954, SER 955, GLN 991, GLU 992, GLN 995, ASN 1006, ASN 1025, ASN 1026, ASN 1032, ASN 1043, ASN 1046, ASN 1052, ASP 1058, HIS 1064, ASN 1080, GLU 1081, GLU 1083, ASP 1086, ASN 1188, ASP 1213, GLY 1215, ASN 1216, GLN 1229, ASN 1242, ASN 1243, SER 1274 and THR1277, wherein the modification is selected from: replacing surface-exposed acidic amino acid residues with basic amino acid residues; replacing surface-exposed acidic amino acid residues with uncharged amino acid residues; replacing surface-exposed uncharged amino acid residues with basic amino acid residues; inserting basic amino acid residues; and deleting surface-exposed acidic amino acid residues, wherein the amount of BoNT / A corresponding to 1 unit of modification in the assay is preferably 8.44 pg.

[0113] When numerical values ​​are involved, the term "at most" (e.g., at most 170,000 pg) means at most and includes the listed values. Thus, as an example, references to BoNT / A modified with "at most 170,000 pg" cover BoNT / A modified with 170,000 pg as well as BoNT / A modified with less than 170,000 pg.

[0114] The unit dose can be expressed as the amount of modified BoNT / A, the unit of modified BoNT / A, or a combination thereof.

[0115] In one embodiment, the modified BoNT / A can be applied to one or more of the following neck muscles at the following dosages:

[0116] In one embodiment, the modified BoNT / A can be applied to one or more of the following neck muscles at the following dosages:

[0117] In one embodiment, the modified BoNT / A can be administered to one or more of the following neck muscles at the following dosages (provided that the modified BoNT / A contains a BoNT / A light chain and a translocation domain, as well as a BoNT / B receptor-binding domain (H).C When the modified BoNT / A contains a domain, the total dose administered during treatment shall not exceed the upper limit of 170,000 pg or 7,070 units; or when the modified BoNT / A contains one or more modifications at amino acid residues selected from the following, the dose shall not exceed 80,000 pg or 9,480 units: ASN 886, ASN 905, GLN 915, ASN 918, GLU 920, ASN 930, ASN 954, SER 955, GLN 991, GLU 992, GLN 995, ASN 1006, ASN 1025, ASN 1026, ASN 1032, ASN 1043, ASN 1046, ASN 1052, ASP 1058, HIS 1064, ASN 1080, GLU 1081, GLU 1083, ASP 1086, ASN 1188, ASP 1213, GLY 1215, ASN 1216, GLN 1229, ASN 1242, ASN 1243, SER 1274, and THR 1277, wherein the modification is selected from: replacing surface-exposed acidic amino acid residues with basic amino acid residues; replacing surface-exposed acidic amino acid residues with uncharged amino acid residues; replacing surface-exposed uncharged amino acid residues with basic amino acid residues; inserting basic amino acid residues; and deleting surface-exposed acidic amino acid residues.

[0118] Preferably, the modified BoNT / A can be applied to the following neck muscles at the following dosages (provided that the modified BoNT / A contains a BoNT / A light chain and a translocation domain, as well as a BoNT / B receptor-binding domain (H). CWhen the modified BoNT / A contains a domain, the total dose administered during treatment shall not exceed the upper limit of 170,000 pg or 7,070 units; or when the modified BoNT / A contains one or more modifications at amino acid residues selected from the following, the dose shall not exceed 80,000 pg or 9,480 units: ASN 886, ASN 905, GLN915, ASN 918, GLU 920, ASN 930, ASN 954, SER 955, GLN 991, GLU 992, GLN 995, ASN 1006, ASN 1025, ASN 1026, ASN 1032, ASN 1043, ASN 1046, ASN 1052, ASP 1058, HIS 1064, ASN 1080, GLU 1081, GLU 1083, ASP 1086, ASN 1188, ASP 1213, GLY 1215, ASN 1216, GLN 1229, ASN 1242, ASN 1243, SER 1274, and THR 1277, wherein the modification is selected from: replacing surface-exposed acidic amino acid residues with basic amino acid residues; replacing surface-exposed acidic amino acid residues with uncharged amino acid residues; replacing surface-exposed uncharged amino acid residues with basic amino acid residues; inserting basic amino acid residues; and deleting surface-exposed acidic amino acid residues.

[0119]

[0120] Preferably, the modified BoNT / A can be applied to one or more of the following neck muscles at the following dosages:

[0121] Preferably, the modified BoNT / A can be applied to the following neck muscles at the following dosages:

[0122] As used herein, the terms “right” and “left” have their normal meanings. For example, the subject’s right levator scapulae would be the levator scapulae on the subject’s right hand side, while the subject’s left levator scapulae would be the levator scapulae on the subject’s left hand side.

[0123] The total number of unit doses administered in a given treatment can be up to 10x unit doses or up to 7x unit doses. The total number of unit doses can be determined based on the affected neck muscles being treated; for example, in one embodiment, when the number of doses to be delivered during treatment is 1x unit dose, only one affected neck muscle can be treated; however, if the total is 2x unit doses, two affected neck muscles can be treated. The total number of unit doses administered can be up to 9x, 8x, 7x, or 6x. The total number of unit doses administered can be at least 2x, 3x, 4x, 5x, 6x, or 7x unit doses, preferably at least 2x. The total number of unit doses administered can be from 1x to 10x, or from 5x to 10x, preferably from 7x to 10x.

[0124] The total number of unit doses administered in a given treatment may be up to 20x or 15x (preferably up to 14x or up to 7x) unit doses, provided that the modified BoNT / A contains a BoNT / A light chain and a translocation domain, as well as a BoNT / B receptor-binding domain (H). C When the modified BoNT / A contains a domain, the total dose administered during treatment shall not exceed the upper limit of 170,000 pg or 7,070 units; or when the modified BoNT / A contains one or more modifications at amino acid residues selected from the following, the dose shall not exceed 80,000 pg or 9,480 units: ASN 886, ASN 905, GLN 915, ASN 918, GLU 920, ASN 930, ASN 954, SER 955, GLN 991, GLU 992, GLN 995, ASN 1006, ASN 1025, ASN 1026, ASN 1032, ASN 1043, ASN 1046, ASN 1052, ASP 1058, HIS 1064, ASN 1080, GLU 1081, GLU 1083, ASP 1086, ASN 1188, ASP 1213, GLY 1215, ASN 1216, GLN 1229, ASN 1242, ASN 1243, SER 1274, and THR 1277, wherein the modification is selected from: replacing surface-exposed acidic amino acid residues with basic amino acid residues; replacing surface-exposed acidic amino acid residues with uncharged amino acid residues; replacing surface-exposed uncharged amino acid residues with basic amino acid residues; inserting basic amino acid residues; and deleting surface-exposed acidic amino acid residues. Therefore, the total number of unit doses administered can be up to 13x, 12x, 10x, 9x, 8x, 7x, or 6x, provided that the modified BoNT / A contains a BoNT / A light chain and a translocation domain, and a BoNT / B receptor-binding domain (H). CWhen the modified BoNT / A contains a domain, the total dose administered during treatment shall not exceed the upper limit of 170,000 pg or 7,070 units; or when the modified BoNT / A contains one or more modifications at amino acid residues selected from the following, the dose shall not exceed 80,000 pg or 9,480 units: ASN 886, ASN 905, GLN 915, ASN 918, GLU 920, ASN 930, ASN 954, SER 955, GLN 991, GLU 992, GLN 995, ASN 1006, ASN 1025, ASN 1026, ASN 1032, ASN 1043, ASN 1046, ASN 1052, ASP 1058, HIS 1064, ASN 1080, GLU 1081, GLU 1083, ASP1086, ASN 1188, ASP 1213, GLY 1215, ASN 1216, GLN 1229, ASN 1242, ASN 1243, SER 1274, and THR 1277, wherein the modification is selected from: replacing surface-exposed acidic amino acid residues with basic amino acid residues; replacing surface-exposed acidic amino acid residues with uncharged amino acid residues; replacing surface-exposed uncharged amino acid residues with basic amino acid residues; inserting basic amino acid residues; and deleting surface-exposed acidic amino acid residues. The total number of unit doses administered can be at least 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, or 10x unit doses, preferably at least 2x (e.g., 7x or 14x), provided that the modified BoNT / A contains a BoNT / A light chain and a translocation domain, and a BoNT / B receptor-binding domain (H). CWhen the modified BoNT / A contains a domain, the total dose administered during treatment shall not exceed the upper limit of 170,000 pg or 7,070 units; or when the modified BoNT / A contains one or more modifications at amino acid residues selected from the following, the dose shall not exceed 80,000 pg or 9,480 units: ASN 886, ASN 905, GLN 915, ASN 918, GLU 920, ASN 930, ASN 954, SER 955, GLN 991, GLU 992, GLN 995, ASN 1006, ASN 1025, ASN 1026, ASN 1032, ASN 1043, ASN 1046, ASN 1052, ASP 1058, HIS 1064, ASN 1080, GLU 1081, GLU 1083, ASP 1086, ASN 1188, ASP 1213, GLY 1215, ASN 1216, GLN 1229, ASN 1242, ASN 1243, SER 1274, and THR 1277, wherein the modification is selected from: replacing surface-exposed acidic amino acid residues with basic amino acid residues; replacing surface-exposed acidic amino acid residues with uncharged amino acid residues; replacing surface-exposed uncharged amino acid residues with basic amino acid residues; inserting basic amino acid residues; and deleting surface-exposed acidic amino acid residues. The total number of unit doses administered can be 1x to 20x (e.g., 12x to 16x), 1x to 15x, 1x to 14x, or 5x to 14x, preferably 7x to 14x, provided that the modified BoNT / A contains a BoNT / A light chain and a translocation domain, and a BoNT / B receptor-binding domain (H). CWhen the modified BoNT / A contains a domain, the total dose administered during treatment shall not exceed the upper limit of 170,000 pg or 7,070 units; or when the modified BoNT / A contains one or more modifications at amino acid residues selected from the following, the dose shall not exceed 80,000 pg or 9,480 units: ASN 886, ASN 905, GLN 915, ASN 918, GLU 920, ASN 930, ASN 954, SER 955, GLN 991, GLU 992, GLN 995, ASN1006, ASN 1025, ASN 1026, ASN 1032, ASN 1043, ASN 1046, ASN 1052, ASP 1058, HIS 1064, ASN 1080, GLU 1081, GLU 1083, ASP 1086, ASN 1188, ASP 1213, GLY 1215, ASN 1216, GLN1229, ASN 1242, ASN 1243, SER 1274, and THR 1277, wherein the modification is selected from: replacing surface-exposed acidic amino acid residues with basic amino acid residues; replacing surface-exposed acidic amino acid residues with uncharged amino acid residues; replacing surface-exposed uncharged amino acid residues with basic amino acid residues; inserting basic amino acid residues; and deleting surface-exposed acidic amino acid residues.

[0125] The technician will consider that the subject has recently (or is subsequently receiving) additional treatment with clostridial neurotoxins (such as BoNT), for example, as part of a cosmetic treatment or treatment for a different indication. Using techniques conventional in the art, the technician will adjust the treatment regimen accordingly.

[0126] When used with unmodified Bont / A (e.g., Dysport) ® Compared to [previous formulation], the modified BoNT / A of the present invention preferably has a longer duration of action (e.g., improvement of at least 5%, 10%, 25%, or 50% in one or more symptoms). The duration of action can be at least 1.25x, 1.5x, 1.75x, 2.0x, or 2.25x. The duration of action of the modified BoNT / A can be between 6 and 9 months. For example, the duration of action can be at least: 4.5 months (from the onset of symptoms), 5.0 months, 5.5 months, 6 months, 6.5 months, 7.0 months, 7.5 months, 8.0 months, 8.5 months, or 9.0 months. In a specific embodiment, the duration of action can be greater than 9.0 months.

[0127] When applied to multiple affected neck muscles, the application preferably occurs within the same treatment course.

[0128] Treatment can be repeated at an appropriate time after application of the modified BoNT / A. This is because the duration of action is approximately the same as that of unmodified BoNT / A (e.g., Dysport). ® The time interval between subsequent applications is twice that of using unmodified BoNT / A (e.g., Dysport). ® The treatment period for the subjects may be appropriately extended. The modified BoNT / A according to the invention may be re-administered to the subjects at least 18, 20, 25, or 30 weeks after the previous administration. For example, the modified BoNT / A according to the invention may be re-administered to the subjects at least 18-45 weeks, preferably 20-35 weeks, after the previous administration.

[0129] The efficacy of treatment (including the severity of symptoms in the subjects) could be assessed using the Toronto Western Spasmodic Torticollis Rating Scale (TWSTRS), as reviewed by Jost et al., 2013 J Neural Transm (Vienna) 120(3):487-496. The TWSTRS is a composite scale consisting of the TWSTRS Severity Scale, the TWSTRS Disability Scale, and the TWSTRS Pain Scale. A higher TWSTRS score indicates a more severe condition. The TWSTRS Severity Scale includes the following items: A. Maximum displacement (rotation, tilt, flexion or extension, lateral displacement, sagittal displacement), B. Duration factor, C. Effect of sensory skills, D. Shoulder elevation / forward movement, E. Range of motion (without sensory skills), F. Time (the patient is able to keep their head in a neutral position within 10° for up to 60 seconds without using sensory skills). The total score from A to F is capped at 35, with the duration factor weighted twice. The TWSTRS-Disability Scale is a six-item scale that assesses performance in daily activities that may be affected by CD: work performance (work or housework), activities of daily living (eating, dressing, hygiene), driving, reading, watching television, and outdoor recreation. Each item is scored on a 6-point scale (0 = no difficulty, 5 = highest degree of disability), with a maximum total score of 30. The TWSTRS-Pain Scale consists of the patient's past week's scores for general, most severe, and best pain severity, along with a duration component and an assessment of the impact of pain on disability. Scores range from 0 to 20, with 20 assigned to the highest level of pain likely experienced.

[0130] As used herein, “subject” can refer to a mammal, such as a human or other mammal. Preferably, “subject” refers to a human subject. A “subject” is preferably an adult subject, i.e., a subject at least 18 years of age. The terms “subject” and “patient” are used as synonyms herein.

[0131] As used herein, the term "treatment" or "treatment" includes preventative treatment (e.g., preventing a patient's onset) and corrective treatment (treating a subject who already has the disease). As used herein, preferably "treatment" or "treatment" refers to corrective treatment. As used herein, the term "treatment" or "treatment" refers to a patient and / or their symptoms.

[0132] The appropriate modified BoNT / A peptide (and the nucleotide sequence encoding it, if present) is described in WO2015 / 004461 A1 and WO2017 / 191315, both of which are incorporated herein by reference in their entirety.

[0133] BoNT / A is an example of a clostridium neurotoxin produced by Clostridium bacteria. Other examples of such clostridium neurotoxins include those produced by Clostridium tetani (…). C. Tetani TeNT) and Clostridium botulinum ( C. botulinum BoNT serotype BG, as well as Clostridium baratii and Clostridium butyricum (BG). C. Butyricum The neurotoxins produced by botulinum toxin are highly potent and specific, capable of poisoning the neurons and other cells they are delivered to. Clostridium toxins are among the most toxic toxins known. For example, depending on the serotype, botulinum neurotoxin has a median lethal dose (LD50) of 0.5 to 5 ng / kg in mice. 50 The values ​​are as follows. Both tetanus toxin and botulinum toxin work by inhibiting the function of affected neurons, particularly by inhibiting the release of neurotransmitters. Botulinum toxin acts on the neuromuscular junction and inhibits cholinergic transmission in the peripheral nervous system, while tetanus toxin acts on the central nervous system.

[0134] In nature, clostridial neurotoxins (including BoNT / A) are synthesized as single-chain polypeptides, which are modified post-translational by proteolytic cleavage events to form two polypeptide chains linked together by disulfide bonds. Cleavage occurs at specific cleavage sites, often called activation sites, located between cysteine ​​residues that provide the interchain disulfide bonds. This double-chain form is the active form of the toxin. These two chains are called the heavy chain (H chain) and the light chain (L chain), with the heavy chain (H chain) having a molecular weight of approximately 100 kDa and the light chain (L chain) having a molecular weight of approximately 50 kDa. The H chain contains an N-terminal translocation component (H... N (structural domain) and C-terminal targeting component (H) C (Structural domain). The cleavage site is located between the L-chain and the translocation structural domain components. In H c After the domain binds to its target neuron and internalizes the bound toxin into the cell via endosomes, H... NThe domain allows the L chain to translocate across the endosome membrane and into the cytoplasm, where it provides protease function (also known as non-cytotoxic protease).

[0135] Non-cytotoxic proteases function by proteolytically cleaving intracellular transport proteins called SNARE proteins (such as SNAP-25, VAMP, or syntaxin) – see Gerald K (2002) “Cell and Molecular Biology” (4th edition). John Wiley & Sons, Inc. The acronym SNARE originates from the term soluble NSF attachment receptor (SNARE). S oluble N SF A ttachment Re ceptor), where NSF represents N-ethylmaleimide sensitivity factor ( N -ethylmaleimide- S ensitive F SNARE proteins are essential for intracellular vesicle fusion and therefore for the transport of secreted molecules from cells via vesicles. The protease function is a zinc-dependent endopeptidase activity and exhibits high substrate specificity for SNARE proteins. Therefore, once delivered to the target cell, the non-cytotoxic protease can inhibit the cellular secretion of the target cell. Clostridium toxin's L-chain protease is a non-cytotoxic protease that cleaves SNARE proteins.

[0136] Given the ubiquitous nature of SNARE proteins, fusiform neurotoxins (such as botulinum toxin) have been successfully used in a variety of treatments.

[0137] Clostridium botulinum (related to Clostridium botulinum) Clostridium botulinum ) and Clostridium tetani ( C. tetani For more detailed information on the genetic basis of toxin production, see Henderson et al. (1997). The lostridia: Molecular Biology and Pathogenesis, Academic press.

[0138] As described above, clostridial neurotoxin consists of two polypeptide chains, a heavy chain and a light chain. The heavy chain (H chain) has a molecular weight of approximately 100 kDa, and the light chain (L chain) has a molecular weight of approximately 50 kDa. The H chain contains a C-terminal targeting component (receptor-binding domain or H+). C (structural domain) and N-terminal translocation component (H) N (structural domain).

[0139] The clostridial neurotoxin domain is described in more detail below.

[0140] Examples of L-chain reference sequences include: Botulinum toxin type A neurotoxin: amino acid residues 1-448 Botulinum toxin type B neurotoxin: amino acid residues 1-440 The reference sequences used for identification above should be considered as guidelines, as subtle variations may occur depending on the serotype. For example, US 2007 / 0166332 (incorporated hereinth by reference) cites a slightly different Clostridium sequence: Botulinum toxin type A neurotoxin: amino acid residue M1-K448 Botulinum toxin type B neurotoxin: amino acid residue M1-K441 The translocation domain is a fragment of the H chain of clostridial neurotoxin (approximately half of the amino terminus of the H chain), or a domain corresponding to that fragment in the intact H chain.

[0141] Examples of reference translocation domains include: Botulinum toxin type A neurotoxin - amino acid residues (449-871) Botulinum toxin type B neurotoxin - amino acid residues (441-858) The reference sequences used for identification above should be considered as guidelines, as subtle variations may occur depending on the serotype. For example, US 2007 / 0166332 (incorporated hereinth by reference) cites a slightly different Clostridium sequence: Botulinum toxin type A neurotoxin - amino acid residues (A449-K871) Botulinum toxin type B neurotoxin - amino acid residues (A442-S858) In the context of this invention, various BoNT / AHs containing translocation structural domains N The region can be used in aspects of this invention. H of the BoNT / A heavy chain N The region is approximately 410-430 amino acids long and contains a translocation domain. Studies have shown that the H from the clostridium neurotoxin heavy chain... N The full length of the region is not necessary for the translocation activity of the translocation domain. Therefore, aspects of this embodiment may include BoNT / AH. N The region comprises a translocation domain having a length of, for example, at least 350 amino acids, at least 375 amino acids, at least 400 amino acids, or at least 425 amino acids. Other aspects of this embodiment may include BoNT / AH. N The region contains translocation domains with lengths of, for example, up to 350 amino acids, up to 375 amino acids, up to 400 amino acids, or up to 425 amino acids.

[0142] Term H N Including naturally occurring BoNT / AH N Partial and modified BoNT / AH N Partially, the modified BoNT / AH N Some of them have amino acid sequences that do not exist in nature and / or synthetic amino acid residues. Preferably, the modified BoNT / AH N Some still exhibit the aforementioned translocation function.

[0143] Clostridium neurotoxin receptor binding domain (H C Examples of reference sequences include: BoNT / A - N872-L1296 BoNT / B - E859-E1291 Clostridium neurotoxins (e.g., BoNT) have approximately 50 kDa of H+. C The domain contains two distinct structural features, called H CC and H CN Each structural feature typically comprises a domain of approximately 25 kDa. The amino acid residues believed to be involved in receptor binding are primarily located in the H+ region. CC Within the structural domain. H of the natural clostridium neurotoxin. C The domain can contain approximately 400-440 amino acid residues. The following publications corroborate this fact, each of which is incorporated herein by reference in its entirety: Umland TC (1997) Nat. Struct. Biol. 4:788-792; Herreros J (2000) Biochem. J. 347:199-204; Halpern J (1993) J. Biol. Chem. 268: 15, pp. 11188-11192; Rummel A (2007) PNAS 104: 359-364; Lacey DB (1998) Nat. Struct. Biol. 5:898-902; Knapp (1998) Am. Cryst. Assoc. Abstract Papers 25:90; Swaminathan and Eswaramoorthy (2000) Nat. Struct. Biol. 7:1751-1759; and Rummel A (2004) Mol. Microbiol. 51(3), 631-643. (Reference) H CN Examples of structural domains include: Botulinum toxin type A neurotoxin - amino acid residues (872-1110) Botulinum toxin type B neurotoxin - amino acid residues (859-1097) The above sequence positions may vary slightly depending on the serotype / subtype. (Reference) H CN Other examples of structural domains include: Botulinum toxin type A neurotoxin - amino acid residues (874-1110) Botulinum toxin type B neurotoxin - amino acid residues (861-1097) (Reference) H CC Examples of structural domains include: Botulinum toxin type A neurotoxin - amino acid residues (Y1111-L1296) Botulinum toxin type B neurotoxin - amino acid residues (Y1098-E1291) L-chain and H N The structural domain (optionally including a complete or partial activation loop, e.g., a complete activation loop when the modified BoNT / A is in single-stranded form and a cleaved / partial activation loop when it is in double-stranded form) can be collectively referred to as LH. N Structural domain. Therefore, LH N The structure does not further include H C Structural domain.

[0144] The modified BoNT / A used in this invention can be a modified BoNT / A containing modifications at one or more amino acid residues selected from the following: ASN 886, ASN 905, GLN 915, ASN 918, GLU 920, ASN 930, ASN 954, SER 955, GLN 991, GLU 992, GLN 995, ASN 1006, ASN 1025, ASN 1026, ASN 1032, ASN 1043, ASN 1046, ASN 1052, ASP 1058, HIS 1064, ASN 1080, GLU 1081, GLU 1083, ASP 1086, ASN 1188, ASP 1213, GLY 1215, ASN 1216, GLN 1229, ASN 1242, ASN 1243, SER 1274, and THR1277. Compared to known BoNT / A, this modified BoNT / A exhibits reduced or absent side effects. The increased tissue retention properties of the modified BoNT / A of the present invention, compared to known clostoxin therapeutics, also provide increased potency and / or duration of action, and can allow for the use of reduced doses (or increased doses without any additional side effects), thus providing further advantages.

[0145] This modification can be a comparison with the unmodified BoNT / A as shown in SEQ ID NO:2, where the amino acid residue numbering is determined by comparison with SEQ ID NO:2. Since the presence of a methionine residue at position 1 of SEQ ID NO:2 (and SEQ ID NO corresponding to the modified BoNT / A polypeptide described herein) is optional, those skilled in the art will consider the presence / absence of the methionine residue when determining the amino acid residue numbering. For example, where SEQ ID NO:2 contains methionine, the position numbering will be as defined above (e.g., ASN 886 would be ASN 886 of SEQ ID NO:2). Alternatively, when methionine is absent in SEQ ID NO:2, the amino acid residue numbering should be modified to -1 (e.g., ASN 886 would be ASN 885 of SEQ ID NO:2). Similar considerations apply when methionine is present / absent at position 1 of other polypeptide sequences described herein, and those skilled in the art will readily determine the correct amino acid residue numbering using techniques conventional in the art.

[0146] The alignments described herein for determining amino acid residue numbers can be performed using any of the methods described herein for determining sequence homology and / or % sequence identity.

[0147] The amino acid residues specified for modification are those exposed on the surface.

[0148] The modified BoNT / A may include modifications at one or more amino acid residues selected from the following: ASN 886, ASN 930, ASN 954, SER 955, GLN 991, ASN 1025, ASN 1026, ASN 1052, ASN 1188, ASP 1213, GLY 1215, ASN 1216, GLN 1229, ASN 1242, ASN 1243, SER 1274, and THR 1277. The modified BoNT / A may be encoded by a nucleic acid sequence having at least 70% sequence identity with a nucleic acid sequence selected from SEQ ID NO: 3, 5, 7, and 9. For example, a nucleic acid sequence having at least 80%, 90%, 95%, or 99.9% sequence identity with a nucleic acid sequence selected from SEQ ID NO: 3, 5, 7, and 9. Preferably, the modified BoNT / A used in this invention may be encoded by a nucleic acid comprising (or composed of) SEQ ID NO: 3, 5, 7 or 9. The modified BoNT / A may comprise a polypeptide sequence having at least 70% sequence identity with a polypeptide sequence selected from SEQ ID NO: 4, 6, 8 and 10. For example, a polypeptide sequence having at least 80%, 90%, 95% or 99.9% sequence identity with a polypeptide sequence selected from SEQ ID NO: 4, 6, 8 and 10. Preferably, the modified BoNT / A used in this invention may comprise a polypeptide sequence selected from SEQ ID NO: 4, 6, 8 and 10 (more preferably composed of).

[0149] When used in the context of a modified BoNT / A, the term "one or more amino acid residues" preferably means at least 2, 3, 4, 5, 6, or 7 specified amino acid residues. Therefore, a modified BoNT / A may contain at least 2, 3, 4, 5, 6, or 7 (preferably 7) modifications at specified amino acid residues. A modified BoNT / A may contain 1-30, 3-20, or 5-10 amino acid modifications. More preferably, when used in the context of a modified BoNT / A, the term "one or more amino acid residues" means all specified amino acid residues.

[0150] Preferably, when compared with SEQ ID NO:2, the modified BoNT / A does not contain any further amino acid modifications other than one or more amino acid modifications at the specified amino acid residues.

[0151] Most preferably, the modified BoNT / A comprises modifications (more preferably consisting of) one or more amino acid residues selected from the following: ASN 886, ASN 930, SER 955, GLN 991, ASN 1026, ASN 1052, and GLN 1229. The modified BoNT / A may be encoded by a nucleic acid sequence having at least 70% sequence identity with SEQ ID NO:3. For example, a nucleic acid sequence having at least 80%, 90%, 95%, or 99.9% sequence identity with SEQ ID NO:3. Preferably, the modified BoNT / A used in this invention may be encoded by a nucleic acid comprising SEQ ID NO:3 (or consisting of). The modified BoNT / A may comprise a polypeptide sequence having at least 70% sequence identity with SEQ ID NO:4. For example, a polypeptide sequence having at least 80%, 90%, 95%, or 99.9% sequence identity with SEQ ID NO:4. Preferably, the modified BoNT / A used in this invention may comprise SEQ ID NO:4 (more preferably consisting of).

[0152] Modifications can be selected from: i. Replace the acidic amino acid residues exposed on the surface with basic amino acid residues; ii. Replace the surface-exposed acidic amino acid residues with uncharged amino acid residues; iii. Replace the uncharged amino acid residues exposed on the surface with basic amino acid residues; iv. Insertion of basic amino acid residues; and v. To remove acidic amino acid residues that are exposed on the surface.

[0153] The modifications described above produce modified BoNT / A, which, compared to the corresponding unmodified BoNT / A, has an increased positive surface charge and an increased isoelectric point.

[0154] The isoelectric point (pI) is a specific property of a given protein. As is well known in the art, proteins are composed of a specific sequence of amino acids (also called amino acid residues when referring to proteins). Each amino acid in the twenty standard groups has a different side chain (or R group), meaning that each amino acid residue in a protein exhibits different chemical properties, such as charge and hydrophobicity. These properties can be influenced by the surrounding chemical environment, such as temperature and pH. The overall chemical properties of a protein depend on the sum of these different factors.

[0155] Some amino acid residues (described in detail below) have ionizable side chains that can display a charge depending on the surrounding pH. Whether such a side chain is charged at a given pH depends on the pKa of the relevant ionizable portion, where pKa is the negative logarithm of the acid dissociation constant (Ka) of the specified proton from the conjugate base.

[0156] For example, acidic residues such as aspartic acid and glutamic acid have side-chain carboxylic acid groups with pKa values ​​of approximately 4.1 (the exact pKa value can depend on temperature, ionic strength, and the microenvironment of the ionizable group). Therefore, these side chains exhibit a negative charge at pH 7.4 (often referred to as the "physiological pH"). At lower pH values, these side chains protonate and lose their charge.

[0157] Conversely, basic residues such as lysine and arginine have nitrogen-containing side chains with pKa values ​​of approximately 10⁻¹². Therefore, these side chains exhibit a positive charge at pH 7.4. These side chains will deprotonate and lose their charge at higher pH values.

[0158] Therefore, the total (net) charge of a protein molecule depends on the number of acidic and basic residues present in the protein (and their surface exposure) as well as the surrounding pH value. Changing the surrounding pH value changes the total charge of the protein. Thus, for each protein, there is a given pH value at which the amount of positive and negative charges is equal, and the protein as a whole does not exhibit a net charge. This point is called the isoelectric point (pI). The isoelectric point is a standard concept in protein biochemistry familiar to those skilled in the art.

[0159] Therefore, the isoelectric point (pI) is defined as the pH at which a protein exhibits a net charge of zero. An increase in pI means that the protein requires a higher pH to exhibit a net charge of zero. Thus, an increase in pI represents an increase in the net positive charge of the protein at a given pH. Conversely, a decrease in pI means that the protein requires a lower pH to exhibit a net charge of zero. Thus, a decrease in pI represents a decrease in the net positive charge of the protein at a given pH.

[0160] Methods for determining the pI of a protein are known in the art and familiar to those skilled in the art. For example, the pI of a protein can be calculated based on the average pKa value of each amino acid present in the protein (“calculated pI”). This calculation can be performed using computer programs known in the art, such as the pI / MW calculation tool from ExPASy (.expasy.org / compute_pi / ), which is a preferred method for calculating pI according to the present invention. Comparisons of pI values ​​between different molecules should be performed using the same calculation technique / program.

[0161] Where appropriate, the calculated protein pI can be experimentally confirmed using isoelectric focusing (“observed pI”). This technique uses electrophoresis to separate proteins based on their pI. Isoelectric focusing is typically performed using a gel with a fixed pH gradient. When an electric field is applied, the protein migrates along the pH gradient until it reaches a pH value where the net charge is zero; this is the protein's pI. The results provided by isoelectric focusing are inherently relatively low-resolution, therefore the inventors believe that results provided by calculated pI (as described above) are more suitable for use.

[0162] Throughout this specification, unless otherwise stated, "pI" means "calculated pI".

[0163] The pI of a protein can be increased or decreased by altering the number of basic and / or acidic groups displayed on its surface. This can be achieved by modifying one or more amino acids of the protein. For example, an increase in pI can be provided by decreasing the number of acidic residues or by increasing the number of basic residues.

[0164] The modified BoNT / A of the present invention can have a pI value that is at least 0.2, 0.4, 0.5, or 1 pI unit higher than that of the unmodified BoNT / A (e.g., SEQ ID NO: 2). Preferably, the modified BoNT / A can have a pI of at least 6.6, for example, at least 6.8.

[0165] The properties of 20 standard amino acids are shown in the table below:

[0166] The following amino acids are considered to be charged amino acids: aspartic acid (negative charge), glutamic acid (negative charge), arginine (positive charge), and lysine (positive charge).

[0167] At pH 7.4, the side chains of aspartic acid (pKa 3.1) and glutamic acid (pKa 4.1) carry a negative charge, while the side chains of arginine (pKa 12.5) and lysine (pKa 10.8) carry a positive charge. Aspartic acid and glutamic acid are referred to as acidic amino acid residues. Arginine and lysine are referred to as basic amino acid residues.

[0168] The following amino acids are considered to be uncharged polar amino acids (meaning they can participate in hydrogen bonding): asparagine, glutamine, histidine, serine, threonine, tyrosine, cysteine, methionine, and tryptophan.

[0169] The following amino acids are considered to be uncharged hydrophobic amino acids: alanine, valine, leucine, isoleucine, phenylalanine, proline, and glycine.

[0170] In amino acid insertion, additional amino acid residues (normally absent amino acid residues) are incorporated into the BoNT / A polypeptide sequence, thereby increasing the total number of amino acid residues in the sequence. In amino acid deletion, amino acid residues are removed from the clostoxin amino acid sequence, thereby reducing the total number of amino acid residues in the sequence.

[0171] Preferably, the modification is a substitution, which advantageously maintains the same number of amino acid residues in the modified BoNT / A. In amino acid substitution, the amino acid residues forming part of the BoNT / A polypeptide sequence are replaced by different amino acid residues. The substituted amino acid residues can be one of 20 standard amino acids, as described above. Alternatively, the substituted amino acid in amino acid substitution can be a non-standard amino acid (an amino acid not belonging to the aforementioned 20 standard groups). For example, the substituted amino acid can be a basic non-standard amino acid, such as L-ornithine, L-2-amino-3-guanidinopropionic acid, or the D-isomers of lysine, arginine, and ornithine. Methods for introducing non-standard amino acids into proteins are known in the art and include the synthesis of recombinant proteins using E. coli auxotrophic expression hosts.

[0172] In one embodiment, the substitution is selected from: replacing an acidic amino acid residue with a basic amino acid residue, replacing an acidic amino acid residue with a non-charged amino acid residue, and replacing a non-charged amino acid residue with a basic amino acid. In one embodiment, the substitution is replacing an acidic amino acid residue with a non-charged amino acid residue, wherein the acidic amino acid residue is replaced by its corresponding non-charged amide amino acid residue (i.e., aspartic acid is replaced by asparagine, and glutamic acid is replaced by glutamine).

[0173] Preferably, the basic amino acid residue is a lysine residue or an arginine residue. In other words, the substitution is with lysine or arginine. Most preferably, the modification is with lysine substitution.

[0174] After modification according to the present invention, the modified BoNT / A is able to bind to the target cell receptor bound by the unmodified BoNT / A (e.g., SEQ ID NO: 2).

[0175] The modified BoNT / A used in this invention may contain components located at clostoxin H. CNThe modified BoNT / A contains 4 to 40 amino acid modifications within its domain. The modified BoNT / A preferably also has a pI of at least 6.6. The modified BoNT / A preferably comprises modifications of at least four amino acids selected from the following: ASN 886, ASN 930, ASN 954, SER 955, GLN 991, ASN 1025, ASN 1026, and ASN 1052, wherein the modification includes substituting an amino acid with a lysine or arginine residue. For example, the modified BoNT / A or a fragment thereof may comprise modifications of at least five amino acids selected from the following: ASN 886, ASN 930, ASN 954, SER 955, GLN 991, ASN 1025, ASN 1026, ASN 1052, and GLN 1229, wherein the modification includes substituting an amino acid with a lysine or arginine residue.

[0176] As used herein, the terms "modified BoNT / A" or "chimeric neurotoxin" preferably refer to a clostridium neurotoxin light chain and translocation domain (H2O) derived from a first clostridium neurotoxin serotype. N (H) and receptor-binding domains derived from different Clostridium difficile neurotoxin serotypes (H) C (H domain). Specifically, the modified BoNT / A used in this invention comprises a botulinum neurotoxin A (BoNT / A) light chain and a translocation domain (H). N The structure domain), and the BoNT / B receptor binding domain (H C (Structure domain). Modified BoNT / A of BoNT / A LH N Structural domains and BoNT / BH C The structural domains are covalently connected. The modified BoNT / A of this invention may be referred to as chimeric botulinum neurotoxin. The modified BoNT / A is also referred to herein as "BoNT / AB", "mrBoNT / AB", or "BoNT / AB chimera".

[0177] Most preferably, the modified BoNT / A used in this invention may comprise a BoNT / A light chain and a translocation domain (BoNT / A LH). N (structural domain) and BoNT / BH C Domain. BoNT / A LH N Structural domains and BoNT / BH C The structural domains are covalently connected. The modified BoNT / A is also referred to herein as "BoNT / AB" or "BoNT / AB chimera".

[0178] LH N The C-terminal amino acid residue of the domain can correspond to the LH that separates BoNT / A.N and H C 3 of the structural domain 10 The first amino acid residue of the helix, and H C The N-terminal amino acid residue of the domain can correspond to the LH in the BoNT / B separator. N and H C 3 of the structural domain 10 The second amino acid residue of the helix.

[0179] An example of the BoNT / B polypeptide sequence is provided as SEQ ID NO:16 (UniProt accession number B1INP5).

[0180] The "LH separating BoNT / A" mentioned in this article N and H C 3 of the structural domain 10 The first amino acid residue of the helix refers to the LH... N and H C 3 of the structural domain 10 The N-terminal residue of the helix.

[0181] The "LH separating BoNT / B" mentioned in this article N and H C 3 of the structural domain 10 The "second amino acid residue of the helix" refers to the part that separates LH. N and H C 3 of the structural domain 10 The amino acid residue following the N-terminal residue of the helix.

[0182] 3 10 "Helix" is a type of secondary structure found in proteins and polypeptides; other types include α-helix, β-sheet, and inversion. 10 The amino acids in the helix are arranged in a right-handed helical structure, where each complete turn is achieved by three residues and ten atoms, which separate the intramolecular hydrogen bonds between them. Each amino acid corresponds to a 120° turn in the helix (i.e., the helix has 3 residues per turn), translates 2.0 Å (= 0.2 nm) along the helical axis, and has 10 atoms in the ring formed by the hydrogen bonds. Crucially, the NH group of the amino acid forms a hydrogen bond with the C=O group of the amino acid three residues preceding it; this repeating i + 3 → i hydrogen bond is defined as 3 10 Spiral. 3 10 Helices are a standard concept familiar to technicians in structural biology.

[0183] The 3 10The helix corresponds to the four residues that form the actual helix and two cap (or transition) residues, with a cap residue at each end of the four residues. The term "separated LH" is used in this paper. N and H C 3 of the structural domain 10 The "helix" is composed of those 6 residues.

[0184] Structural analysis and sequence alignment were performed to identify the LH separator. N and H C 3 of the structural domain 10 Spiral. The 3 10 The helix at its N-terminus (i.e., LH) N The C-terminal portion of the structural domain is surrounded by an α-helix, and at its C-terminus (i.e., H... c The N-terminal portion of the structural domain is surrounded by a β chain. 3 10 The first (N-terminal) residue (cap or transition residue) of the helix also corresponds to the C-terminal residue of the α-helix.

[0185] Separating LH N and H C 3 of the structural domain 10 The helix can be determined, for example, from publicly available crystal structures of botulinum neurotoxins, such as 3BTA (w.rcsb.org / pdb / explore / explore.do?structureId=3BTA) and 1EPW (w.rcsb.org / pdb / explore / explore.do?structureId=1EPW) for botulinum neurotoxins A1 and B1, respectively.

[0186] Publicly available computer modeling and comparison tools can also be used to identify septated LH in other neurotoxins. N and H C 3 of the structural domain 10 The location of helices is listed in many tools / services, such as the homology modeling server LOOPP (learns, observes, and outputs protein patterns, p.org), PHYRE (protein homology / simulation recognition engine, w.sbg.bio.ic.ac.uk / phyre2 / ) and Rosettaw.rosettacommons.org / ), the protein overlay server SuperPose t.biology.ualberta.ca / superpose / , the alignment program Clustal Omega w.clustal.org / omega / , and many other tools / services listed in the Internet resource for molecular and cell biologists l-tools.ca / . In particular, "H N / H CN"The area around the junction can be structurally highly conservative, making it an ideal region for superimposing different serotypes."

[0187] For example, the following methods can be used to determine the presence of this 3 in other neurotoxins. 10 Spiral sequence: 1. The structural homology modeling tool LOOP (p.org) can be used to obtain the predicted structure of other BoNT serotypes based on the BoNT / A1 crystal structure (3BTA.pdb); 2. The resulting structure (pdb) file can be edited to contain only H... CN The N-terminus of the domain and the approximately 80 residues preceding it (these are H) N (part of the structural domain), thus preserving the highly conservative "H" structure. N / H CN "area; 3. The protein stacking server SuperPose (t.biology.ualberta.ca / superpose / ) can be used to stack each serotype onto a 3BTA.pdb structure; 4. The superimposed PDB file can be examined to locate the H in BoNT / A1. C 3 at the beginning of the structural domain 10 The helix can then be used to identify the corresponding residues in other serotypes.

[0188] 5. Other BoNT serotype sequences can be compared with Clustal Omega to check if the corresponding residues are correct.

[0189] LH measured by this method N H C and 3 10 An example of a spiral structure domain is shown below:

[0190] Using structural analysis and sequence alignment, the LH separator was found. N and H C 3 of the structural domain 10 The β-chain following the helix is ​​a conserved structure in all botulinum toxins and tetanus neurotoxins, and it separates from the LH... N and H C 3 of the structural domain 10 When the first residue of the helix begins, the β chain starts from residue 8 (e.g., residue 879 of BoNT / A1).

[0191] BoNT / AB chimeras can contain H from BoNT / B C LH from BoNT / A are covalently connected to the structural domain.N Structural domain, where LH N The C-terminal amino acid residue of the domain corresponds to the H located at BoNT / A. C The 8th amino acid residue at the N-terminus of the β-chain at the start (N-terminus) of the domain, and where H C The N-terminal amino acid residue of the domain corresponds to the H located in BoNT / B. C The 7th amino acid residue at the N-terminus of the β-chain at the start (N-terminus) of the domain.

[0192] BoNT / AB chimeras can contain H from BoNT / B C LH from BoNT / A are covalently connected to the structural domain. N Structural domain, where LH N The C-terminal amino acid residue of the domain corresponds to the LH located at BoNT / A. N The C-terminal amino acid residue of the α-helix at the termination end (C-terminus) of the structural domain, and wherein H C The N-terminal amino acid residue of the domain corresponds to the LH located in BoNT / B. N The C-terminal amino acid residue of the α-helix at the termination end (C-terminus) of the domain is adjacent to the C-terminal amino acid residue.

[0193] The fundamental principle of the BoNT / AB chimera design process is to ensure that the secondary structure remains largely unaffected, thereby minimizing any alterations to the tertiary structure and the function of each structural domain. Not wishing to be bound by theory, it is assumed that by not disrupting the tertiary structure within the BoNT / AB chimera... 10 The four central amino acid residues of the helix ensure the optimal conformation of the chimeric neurotoxin, thus allowing it to exert its effects and fully realize its function. In fact, surprisingly, only the three amino acid residues of BoNT / A are retained. 10 The first amino acid residue of the helix and the 3rd amino acid of BoNT / B 10 The forward-helical second amino acid residue not only allows for the creation of soluble and functional BoNT / AB chimeras, but also leads to improved properties compared to other BoNT / AB chimeras, particularly increased potency, increased safety ratio, and / or longer duration of action (as well as increased safety ratio and / or duration of action compared to unmodified BoNT / A).

[0194] BoNT / A light chain, BoNT / A translocation domain and / or BoNT / BH C The structural domain can be a modified BoNT / A light chain, a BoNT / A translocation structural domain, and / or a BoNT / BH domain. CDomains or their derivatives, including but not limited to those described below. Modified BoNT / A light chains, BoNT / A translocation domains, and / or BoNT / BH domains. C The domains or derivatives may contain one or more modified amino acids compared to the native (unmodified) forms of the BoNT / A light chain, BoNT / A translocation domain, and / or BoNT / B Hc domain, or may contain one or more inserted amino acids not present in the native (unmodified) forms of the BoNT / A light chain, BoNT / A translocation domain, and / or BoNT / B Hc domain. For example, modified BoNT / A light chains, BoNT / A translocation domains, and / or BoNT / B Hc domains... C The domains may have modified amino acid sequences in one or more of the natural (unmodified) BoNT / A light chain, BoNT / A translocation domain, and / or BoNT / B Hc domain. Such modifications can alter functional aspects, such as biological activity or persistence. Therefore, in one embodiment, the BoNT / A light chain, BoNT / A translocation domain, and / or BoNT / B Hc domain... C The structural domain is a modified BoNT / A light chain, a BoNT / A translocation structural domain, and / or a BoNT / BH domain. C Structural domains, or modified BoNT / A light chains, BoNT / A translocation structural domains, and / or BoNT / BH C Domain derivatives.

[0195] The modified BoNT / BHc domain can have one or more modifications that alter its binding to target neurons, for example, when combined with the native (unmodified) BoNT / BHc domain. C Compared to other domains, it provides either higher or lower affinity binding. In BoNT / BH C Such modifications in a structural domain may include modifications in H C Residues in the ganglioside binding site of the domain, or residues modified in the binding site of a protein (e.g., a synaptic binding protein) that alters the binding to ganglioside receptors and / or protein receptors of target nerve cells. Examples of such modified neurotoxins are described in WO 2006 / 027207 and WO 2006 / 114308, both of which are incorporated herein by reference in their entirety.

[0196] The modified light chain may have one or more modifications in its amino acid sequence, such as modifications in the substrate-binding or catalytic domains, which may alter or modify the SNARE protein specificity of the modified light chain, preferably provided that the modification does not catalytically inactivate the light chain. Examples of such modified neurotoxins are described in WO 2010 / 120766 and US 2011 / 0318385, both of which are incorporated herein by reference in their entirety.

[0197] LH from BoNT / A N The domain may correspond to amino acid residues 1 to 872 of SEQ ID NO:2, or a polypeptide sequence having at least 70% sequence identity with it. LH from BoNT / A N The domain may correspond to amino acid residues 1 to 872 of SEQ ID NO:2, or a polypeptide sequence having at least 80%, 90%, or 95% sequence identity with it. Preferably, it is an LH from BoNT / A. N The domains correspond to amino acid residues 1 to 872 of SEQ ID NO:2.

[0198] H from BoNT / B C The domain may correspond to amino acid residues 860 to 1291 of SEQ ID NO:16, or a polypeptide sequence having at least 70% sequence identity with it. (H from BoNT / B) C The domain may correspond to amino acid residues 860 to 1291 of SEQ ID NO:16, or a polypeptide sequence having at least 80%, 90%, or 95% sequence identity with it. Preferably, the H domain is derived from BoNT / B. C The domain corresponds to amino acid residues 860 to 1291 of SEQ ID NO:16.

[0199] Preferably, the BoNT / AB chimera comprises BoNT / A1 LH N Structural domains and BoNT / B1 H C Structural domain. More preferably, LH N The structural domain corresponds to amino acid residues 1 to 872 of BoNT / A1 (SEQ ID NO: 2), and H C The domain corresponds to amino acid residues 860 to 1291 of BoNT / B1 (SEQ ID NO: 16).

[0200] Preferably, compared to the natural BoNT / B sequence, BoNT / BH C The structural domain is still in H CCThe subdomain contains at least one substituted, added, or deleted amino acid residue, which increases the binding affinity of BoNT / B neurotoxin to human Syt II. BoNT / BH CC Suitable amino acid residue substitutions, additions, or deletions in the subdomains have been disclosed in WO 2013 / 180799 and WO 2016 / 154534 (both incorporated herein by reference).

[0201] BoNT / BH CC Suitable amino acid residue substitutions, additions, or deletions in the subdomain may include substitution mutations selected from the following: V1118M; Y1183M; , Y1183C, Y1183P, and combinations thereof.

[0202] BoNT / BH CC Suitable amino acid residue substitutions, additions, or deletions in the subdomain may also include combinations of two substitution mutations selected from the following: E1191M and S1199L, E1191M and S1199Y, E1191M and S1199F, E1191Q and S1199L, E1191Q and S1199Y, E1191Q and S1199F, E1191M and S1199W, E1191M and W1178Q, E1191C and S1199W, E1191C and S1199Y, E1191C and W1178Q, E1191Q and S1199W, E1191V and S1199W, E1191V and S1199Y, or E1191V and W1178Q.

[0203] BoNT / BH CC Suitable amino acid residue substitutions, additions, or deletions in the subdomain can also include combinations of three substitution mutations, namely E1191M, S1199W, and W1178Q.

[0204] The preferred option is BoNT / BH. CC Suitable amino acid residue substitutions, additions, or deletions in the subdomain include combinations of two substitution mutations, namely E1191M and S1199Y. Such modifications are present, for example, in the BoNT / AB chimeras SEQ ID NO:13 and SEQ ID NO:14.

[0205] This modification can be a comparison with the unmodified BoNT / B as shown in SEQ ID NO:16, where the amino acid residue numbering is determined by comparison with SEQ ID NO:16. Since the presence of a methionine residue at position 1 of SEQ ID NO:16 (and SEQ ID NO corresponding to the modified BoNT / A polypeptide described herein) is optional, those skilled in the art will consider the presence / absence of the methionine residue when determining the amino acid residue numbering. For example, where SEQ ID NO:16 contains methionine, the position numbering will be as defined above (e.g., E1191 would be E1191 of SEQ ID NO:16). Alternatively, when methionine is absent in SEQ ID NO:16, the amino acid residue numbering should be modified to -1 (e.g., E1191 would be E1190 of SEQ ID NO:16). Similar considerations apply when methionine is present / absent at position 1 of other polypeptide sequences described herein, and those skilled in the art will readily determine the correct amino acid residue numbering using techniques conventional in the art.

[0206] The modified BoNT / A used in this invention may comprise a polypeptide sequence having at least 70% sequence identity with a polypeptide sequence selected from SEQ ID NO:11-15. For example, a polypeptide sequence having at least 80%, 90%, 95%, or 99.9% sequence identity with a polypeptide sequence selected from SEQ ID NO:11-15. Preferably, the modified BoNT / A used in this invention may comprise a polypeptide sequence selected from SEQ ID NO:11-15 (more preferably composed of therefrom).

[0207] When the modified BoNT / A is a BoNT / AB chimera, the modified BoNT / A preferably comprises a polypeptide sequence having at least 70% sequence identity with SEQ ID NO:14. For example, a polypeptide sequence having at least 80%, 90%, 95%, or 99.9% sequence identity with SEQ ID NO:14. Most preferably, the modified BoNT / A used in this invention may comprise SEQ ID NO:14 (more preferably composed of it).

[0208] Methods for modifying proteins by substituting, inserting, or deleting amino acid residues are known in the art. For example, amino acid modifications can be introduced by modifying the DNA sequence encoding BoNT / A (e.g., encoding unmodified BoNT / A). This can be achieved using standard molecular cloning techniques, such as site-directed mutagenesis, by using polymerases in which short-chain DNA (oligonucleotides) encoding the desired amino acids are used in place of the original coding sequence, or by inserting / deleting portions of the gene using various enzymes such as ligases and restriction endonucleases. Alternatively, the modified gene sequence can be chemically synthesized.

[0209] When the polypeptide sequence of the modified BoNT / A described herein contains, for example, a tag for purification, such as a His tag, the tag is optional. Preferably, the tag is removed before using the modified BoNT / A according to the invention.

[0210] As discussed above, the modified BoNT / A described herein possesses increased tissue retention properties, which also provide increased potency and / or duration of action, and allow for increased doses without any additional negative effects. One way to define these advantageous properties is based on the safety ratio of the modified BoNT / A. In this regard, the undesirable effects of clostridial toxins (caused by toxin diffusion from the application site) can be experimentally assessed by measuring the percentage of weight loss in relevant animal models (e.g., mice, where weight loss was detected within 7 days of administration). Conversely, the intended target effect of clostridial toxins can be experimentally assessed by the Digital Abduction Score (DAS) assay (a measure of muscle paralysis). The DAS assay is performed by injecting 20 μl of clostridial toxin prepared in gelatin phosphate buffer into the gastrocnemius / soleus muscle complex of mice, followed by assessment of the abduction score using the Aoki method (Aoki KR, Toxicon 39:1815-1820; 2001). In the DAS assay, mice are briefly suspended by their tails to elicit a specific startle response, in which the mice extend their hind limbs and abduct their hind toes. After clostridium toxin injection, different degrees of toe abduction are scored using a five-point scale (0 = normal, 4 = reduced maximum abduction and leg extension).

[0211] The safety ratio of the modified BoNT / A of the present invention (or the unmodified BoNT / A used for comparison) can then be expressed as the ratio between the amount of toxin required for a 10% weight loss (measured at peak effect within the first 7 days after administration in mice) and the amount of toxin required for a DAS score of 2. Therefore, a high safety ratio score is desirable, indicating that the toxin effectively paralyzes the target muscle with minimal undesirable off-target effects. The safety ratio of the modified BoNT / A of the present invention is higher than that of an equivalent unmodified (natural) BoNT / A.

[0212] A high safety ratio is particularly advantageous in treatment because it represents an increase in the therapeutic index. In other words, this means that a reduced dose can be used and / or an increased dose can be used without any additional (e.g., harmful) effects compared to alternative clostridial neurotoxin treatments. Harmful effects include systemic toxicity and undesirable diffusion to adjacent muscles. The possibility of using higher doses of neurotoxin without additional effects is particularly advantageous because higher doses generally result in a longer duration of action of the neurotoxin.

[0213] The potency of modified BoNT / A can be expressed as the minimum dose of neurotoxin that results in a given DAS fraction when administered to the gastrocnemius / soleus muscle complex in mice, for example, a DAS fraction of 2 (ED). 50 The dose) or DAS score is 4. The potency of modified BoNT / A can also be expressed as EC in a cellular assay measuring the neurotoxin's effect on SNARE cleavage. 50 Dosage, for example, in the assay of modified BoNT / A against SNAP25-digested cells, EC 50 dose.

[0214] The duration of action of modified BoNT / A can be expressed as the time required to restore a DAS score of 0 after administration of a given dose of neurotoxin to the gastrocnemius / soleus muscle complex of a mouse, said given dose being, for example, the minimum dose of neurotoxin that results in a DAS score of 4.

[0215] Therefore, in one embodiment, the modified BoNT / A of the present invention has a safety ratio greater than 7 (e.g., at least 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50), wherein the safety ratio is calculated as: the toxin dose (pg / mouse) required for -10% body weight change divided by DAS ED. 50 (pg / mouse) [ED] 50 =The dose required to produce a DAS score of 2].

[0216] In one embodiment, the modified BoNT / A of the present invention has a safety ratio of at least 10. In another embodiment, the modified BoNT / A of the present invention has a safety ratio of at least 15.

[0217] Preferably, the modified BoNT / A is a BoNT / A containing one or more amino acid residues selected from the following: ASN 886, ASN 930, ASN 954, SER 955, GLN 991, ASN 1025, ASN 1026, ASN 1052, ASN 1188, ASP 1213, GLY 1215, ASN 1216, GLN 1229, ASN 1242, ASN 1243, SER 1274, and THR 1277, and as described herein, the modified BoNT / A has a safety ratio of at least 20, more preferably at least 22 (e.g., 23-25).

[0218] Preferably, when the modified BoNT / A is a light chain containing BoNT / A and a translocation domain, as well as BoNT / BH C When the BoNT / A of the structural domain is modified, the modified BoNT / A has a safety ratio of at least 10, more preferably at least 12 (e.g., 14-15).

[0219] The modified BoNT / A is preferably in a non-complex form (i.e., without the complex protein naturally present in BoNT / A). Examples of such complex proteins include neurotoxin-associated protein (NAP) and non-toxic non-hemagglutinin component (NTNH). However, the preferred modified BoNT / A is recombinant modified BoNT / A. This modified BoNT / A of the present invention can be produced using recombinant nucleic acid technology.

[0220] In one embodiment, a nucleic acid (e.g., DNA) comprising a nucleic acid sequence encoding a modified BoNT / A is provided. In one embodiment, the nucleic acid sequence is prepared as part of a DNA vector comprising a promoter and a terminator. The nucleic acid sequence may be selected from any nucleic acid sequence described herein.

[0221] In a preferred embodiment, the vector has a promoter selected from the following: Typical induction conditions for promoter inducers Tac (hybrid) IPTG 0.2 mM (0.05-2.0 mM) AraBAD L-arabinose 0.2% (0.002-0.4%) T7-lac operator IPTG 0.2 mM (0.05-2.0 mM) In another preferred embodiment, the vector has a promoter selected from the following: Typical induction conditions for promoter inducers Tac (hybrid) IPTG 0.2 mM (0.05-2.0 mM) AraBAD L-arabinose 0.2% (0.002-0.4%) T7-lac operator IPTG 0.2 mM (0.05-2.0 mM) T5-lac operator IPTG 0.2 mM (0.05-2.0 mM) Nucleic acid molecules can be prepared using any suitable method known in the art. Therefore, chemical synthesis techniques can be used to prepare nucleic acid molecules. Alternatively, molecular biology techniques can be used to prepare the nucleic acid molecules of this invention.

[0222] The DNA constructs of the present invention are preferably designed on a computer and then synthesized using conventional DNA synthesis techniques.

[0223] Depending on the final host cell (e.g., E. coli) expression system to be used, the above nucleic acid sequence information may be optionally modified for codon preference.

[0224] The terms "nucleotide sequence" and "nucleic acid" are used synonymously herein. Preferably, the nucleotide sequence is a DNA sequence.

[0225] The modified BoNT / A of the present invention can exist as a single chain or as a double chain. However, it is preferred that the modified BoNT / A exist as a double chain, wherein the L chain is connected to the H chain (or its components, such as H+) via disulfide bonds. N (structural domain).

[0226] The production of single-chain modified BoNT / A with light and heavy chains can be achieved by expressing nucleic acids encoding the modified BoNT / A in an expression host, lysing host cells to provide host cell homogenates 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 by contacting the single-chain modified BoNT / A with a protease, which hydrolyzes the peptide bonds in the activated ring of the modified BoNT / A, thereby converting the single-chain modified BoNT / A into the corresponding double-chain modified BoNT / A (e.g., where the light and heavy chains are linked together by disulfide bonds). Double-chain modified BoNT / A is preferably obtained by such a method.

[0227] Therefore, the modified BoNT / A used in this invention is preferably a double-stranded modified BoNT / A derived from a single-stranded BoNT / A, wherein the single-stranded BoNT / A comprises or is composed of the polypeptide sequence described herein. For example, preferably, the modified BoNT / A used in this invention is a double-stranded modified BoNT / A derived from a polypeptide that comprises a polypeptide sequence having at least 70% (e.g., at least 80%, 90%, 95%, or 99.9%) sequence identity with SEQ ID NO:14. Most preferably, the modified BoNT / A used in this invention is a double-stranded modified BoNT / A derived from a polypeptide comprising SEQ ID NO:14 (or even more preferably, composed of). Therefore, in some embodiments, the modified BoNT / A is a double-stranded modified BoNT / A, wherein the light chain (L chain) is linked to the heavy chain (H chain) via disulfide bonds. The modified BoNT / A can be obtained by contacting a single-stranded modified BoNT / A comprising SEQ ID NO:14 with a protease, the protease hydrolyzing the peptide bonds in its activated ring, thereby converting the single-stranded modified BoNT / A into the corresponding double-stranded modified BoNT / A. In some embodiments, the modified BoNT / A is a double-stranded modified BoNT / A, wherein the L chain is linked to the H chain via disulfide bonds. The modified BoNT / A can be obtained by contacting a single-stranded modified BoNT / A comprising SEQ ID NO:14 with a protease, the protease hydrolyzing the peptide bonds in its activated ring, thereby converting the single-stranded modified BoNT / A into the corresponding double-stranded modified BoNT / A.

[0228] In one embodiment, the modified BoNT / A used in this invention is a double-stranded modified BoNT / A derived from a polypeptide comprising a polypeptide sequence having at least 70% (e.g., at least 80%, 90%, 95%, or 99.9%) sequence identity with SEQ ID NO:4. Preferably, the modified BoNT / A used in this invention is a double-stranded modified BoNT / A derived from a polypeptide comprising SEQ ID NO:4 (more preferably composed of therein). Thus, in some embodiments, the modified BoNT / A is a double-stranded modified BoNT / A wherein the light chain (L chain) is linked to the heavy chain (H chain) via a disulfide bond, the modified BoNT / A being obtained by a method comprising contacting a single-stranded modified BoNT / A comprising SEQ ID NO:4 with a protease, the protease hydrolyzing the peptide bonds in its activated ring, thereby converting the single-stranded modified BoNT / A into the corresponding double-stranded modified BoNT / A. In some embodiments, the modified BoNT / A is a double-stranded modified BoNT / A, wherein the L chain is linked to the H chain via a disulfide bond. The modified BoNT / A can be obtained by contacting a single-stranded modified BoNT / A consisting of SEQ ID NO:4 with a protease, the protease hydrolyzing the peptide bonds in its activation ring, thereby converting the single-stranded modified BoNT / A into the corresponding double-stranded modified BoNT / A.

[0229] As used herein, the term "available" also includes the term "obtained." In one implementation, the term "available" means "obtained."

[0230] Lys-C is the preferred protease for cleaving the activation loop. Suitable proteases and methods for cleaving the activation loop to produce double-stranded Clostridium 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 containing one or more lysine residues. When Lys-C cleaves the activation loop multiple times, those skilled in the art will understand that the small peptide of the activation loop of the double-stranded modified BoNT / A may not be present when compared with SEQ ID NO shown herein.

[0231] 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. Therefore, in one aspect, the present invention provides a pharmaceutical composition comprising the modified BoNT / A of the present invention and a pharmaceutically acceptable carrier, excipient, adjuvant, and / or salt.

[0232] Liquid dosage forms are typically prepared using modified BoNT / A and a pyrogen-free sterile carrier. Depending on the carrier used and the concentration, the modified BoNT / A can be dissolved or suspended in the carrier. In preparing the solution, the modified BoNT / A can be dissolved in the carrier, and if necessary, the solution can be made isotonic by adding sodium chloride. The solution is then sterilized by aseptic filtration through a sterile filter using aseptic techniques, and subsequently filled into suitable sterile vials or ampoules and sealed. Alternatively, if the solution is sufficiently stable, the solution in the sealed container can be sterilized by autoclaving. Advantageously, additives can be dissolved in the carrier; these additives may be, for example, buffers, solubilizers, stabilizers, preservatives or bactericides, suspending agents or emulsifiers, and / or local anesthetics.

[0233] A dry powder can be prepared by filling pre-sterilized ingredients into sterile containers in a sterile area using aseptic techniques, so that it can be dissolved or suspended in a suitable carrier before use. Alternatively, the ingredients can be dissolved into a suitable container in a sterile area using aseptic techniques. The product is then freeze-dried and the container is aseptically sealed.

[0234] The parenteral suspension suitable for the route of administration described herein is prepared in essentially the same manner, except that the sterile components are suspended in a sterile carrier rather than dissolved, and sterilization is not achieved by filtration. The components can be separated in a sterile state, or alternatively, they can be sterilized after separation, for example, by gamma radiation.

[0235] Advantageously, one or more compositions may include a suspending agent, such as polyvinylpyrrolidone, to promote uniform distribution of the components.

[0236] On the other hand, the present invention provides a unit dosage form of modified botulinum neurotoxin A (BoNT / A) for treating cervical dystonia, the unit dosage form comprising: a. 31-707 units of modified BoNT / A, where 1 unit corresponds to the calculated median lethal dose (LD50) in mice. 50 The amount of BoNT / A modified by ); or b. BoNT / A modified to 750 pg to 17,000 pg; and c. Optional pharmaceutically acceptable transporters, excipients, adjuvants, and / or salts. The modified BoNT / A includes the BoNT / A light chain and translocation domain, as well as the BoNT / B receptor-binding domain (H). C (structural domain).

[0237] Preferably, the modified BoNT / A in unit dosage form comprises a polypeptide sequence having at least 70% sequence identity with SEQ ID NO: 14. For example, a polypeptide sequence having at least 80%, 90%, 95%, or 99.9% sequence identity with SEQ ID NO: 14. Most preferably, the modified BoNT / A may comprise SEQ ID NO: 14 (more preferably composed of it).

[0238] Unit dosage forms for treating cervical dystonia may comprise 750 pg to 17,000 pg of modified BoNT / A, wherein the modified BoNT / A comprises a BoNT / A light chain and translocation domain and a BoNT / B receptor-binding domain (H). C (Structural domain). The upper limit of the unit dose range can be 16,500, 15,500, 14,500, 13,500, 12,500, 11,500, 10,500, 9,500, 8,500, 7,500, 6,500, 5,500, 4,500, 3,500, 2,500, 2,250, 2,000, 1,500, 1,250, 1,000 or 750 pg of modified BoNT / A, preferably with an upper limit of 16,000 pg. The lower limit of the unit dose range can be 800, 850, 950, 1,000, 1,500, 1,750, 2,000, 2,500, 3,000, 3,500, 4,000, 4,500, or 5,000 pg of modified BoNT / A, preferably 1,000 pg. Preferably, the unit dose of modified BoNT / A is from 1,000 pg to 16,000 pg, for example, 950 pg to 1,250 pg, 1,750 pg to 2,250 pg, or 8,000 pg to 12,000 pg. Preferably, the unit dose of modified BoNT / A can be 1,000, 2,000, 3,000, 8,000, or 16,000 pg.

[0239] Unit formulations for treating cervical dystonia may contain 31 to 707 units of modified BoNT / A, wherein the modified BoNT / A comprises a BoNT / A light chain and a translocation domain, and a BoNT / B receptor-binding domain (H). C(Structural domain). The upper limit of the unit dose range can be 700, 650, 600, 550, 500, 450, 400, 350, 300, 250, 200, 150, 100, 95, 90, 85, 65, 60, 55, 50, or 31 units of modified BoNT / A, preferably 666 units. The lower limit of the unit dose range can be 35, 40, 45, 50, 60, 65, 70, 75, 80, 85, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, or 700 units of modified BoNT / A, preferably 42 units. Preferably, the unit dose of the modified BoNT / A is 42 to 666 units, for example, 40 to 50 units, 70 to 95 units, or 333 to 499 units. Preferably, the unit dose of the modified BoNT / A can be 41.6 units, 83.2 units, 124.8 units, 332.8 units, or 665.6 units.

[0240] On the other hand, the present invention provides a unit dosage form of modified botulinum neurotoxin A (BoNT / A) for treating cervical dystonia, the unit dosage form comprising: a. Modified BoNT / A units ranging from 53 U to 948 U, where 1 unit corresponds to the calculated median lethal dose (LD50) in mice. 50 The amount of BoNT / A modified by ); or b. BoNT / A modified to 450 pg to 8,000 pg; and c. Optional pharmaceutically acceptable transporters, excipients, adjuvants, and / or salts. The modified BoNT / A includes modifications at one or more amino acid residues selected from the following: ASN 886, ASN 905, GLN 915, ASN 918, GLU 920, ASN 930, ASN 954, SER 955, GLN 991, GLU 992, GLN995, ASN 1006, ASN 1025, ASN 1026, ASN 1032, ASN 1043, ASN 1046, ASN 1052, ASP 1058, HIS 1064, ASN 1080, GLU 1081, GLU 1083, ASP 1086, ASN 1188, ASP 1213, GLY 1215, ASN 1216, GLN 1229, ASN 1242, ASN 1243, SER 1274, and THR 1277, wherein the modifications are selected from: i. Replace the acidic amino acid residues exposed on the surface with basic amino acid residues; ii. Replace the surface-exposed acidic amino acid residues with uncharged amino acid residues; iii. Replace the uncharged amino acid residues exposed on the surface with basic amino acid residues; iv. Insertion of basic amino acid residues; and v. To remove acidic amino acid residues that are exposed on the surface.

[0241] Unit dosage forms for the treatment of cervical dystonia may comprise 450 pg to 8,000 pg of modified BoNT / A, wherein the modified BoNT / A contains modifications at one or more amino acid residues selected from the following: ASN 886, ASN 905, GLN915, ASN 918, GLU 920, ASN 930, ASN 954, SER 955, GLN 991, GLU 992, GLN 995, ASN 1006, ASN 1025, ASN 1026, ASN 1032, ASN 1043, ASN 1046, ASN 1052, ASP 1058, HIS 1064, ASN 1080, GLU 1081, GLU 1083, ASP 1086, ASN 1188, ASP 1213, GLY 1215, ASN 1216, GLN 1229, ASN 1242, ASN 1243, SER 1274, and THR 1277, wherein the modification is selected from: (i) replacing surface-exposed acidic amino acid residues with basic amino acid residues; (ii) replacing surface-exposed acidic amino acid residues with uncharged amino acid residues; (iii) replacing surface-exposed uncharged amino acid residues with basic amino acid residues; (iv) inserting basic amino acid residues; and (v) deleting surface-exposed acidic amino acid residues. The upper limit of this range may be 7,750, 7,500, 7,000, 6,000, 5,000, 4,000, 3,000, 2,000, or 1,000 pg of modified BoNT / A, preferably 7,500 pg. The lower limit of this range can be 475, 500, 600, 700, 800, 900, 1,000, 1,500, 2,000, 3,000, 4,000, 5,000, 6,000, or 7,000 pg of modified BoNT / A, with a preferred lower limit of 500 pg. Preferably, the unit dosage form contains 500 pg to 7,500 pg of modified BoNT / A, for example, 4,000 pg to 6,000 pg. Most preferably, the unit dosage form contains 2,000 pg to 3,000 pg, for example, 2,400 pg to 2,600 pg.

[0242] Unit formulations for treating cervical dystonia may contain 53 to 948 units of modified BoNT / A, wherein the modified BoNT / A comprises modifications at one or more amino acid residues selected from the following: ASN 886, ASN 905, GLN 915, ASN 918, GLU 920, ASN 930, ASN 954, SER 955, GLN 991, GLU 992, GLN 995, ASN 1006, ASN 1025, ASN 1026, ASN 1032, ASN 1043, ASN 1046, ASN 1052, ASP 1058, HIS 1064, ASN 1080, GLU 1081, GLU 1083, ASP 1086, ASN 1188, ASP 1213, GLY 1215, ASN 1216, GLN1229, ASN 1242, ASN 1243, SER 1274, and THR 1277, wherein the modification is selected from: (i) replacing surface-exposed acidic amino acid residues with basic amino acid residues; (ii) replacing surface-exposed acidic amino acid residues with uncharged amino acid residues; (iii) replacing surface-exposed uncharged amino acid residues with basic amino acid residues; (iv) inserting basic amino acid residues; and (v) deleting surface-exposed acidic amino acid residues. The upper limit of this range may be 925, 900, 850, 800, 750, 700, 650, 600, 550, 500, 450, 400, 350, 300, 250, 200, 150, or 100 units of modified BoNT / A, preferably, the upper limit is 889 units. The lower limit of this range can be 55, 60, 65, 70, 75, 80, 85, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, or 900 units of modified BoNT / A, preferably 59 units. Preferably, the unit dosage form contains 59 to 889 units of modified BoNT / A, for example, 200 to 600 units. Most preferably, the unit dosage form contains 237 to 355 units, for example, 284 to 308 units.

[0243] In another aspect, the present invention provides a kit comprising: a. The unit dosage form according to the invention; and b. Instructions for use in the treatment of cervical dystonia; and c. Optional, diluent.

[0244] Embodiments relating to the various therapeutic uses of the present invention can be applied to the methods, compositions (e.g., unit dosage forms) and kits of the present invention, and vice versa.

[0245] Sequence homology

[0246] Any of a variety of sequence alignment methods can be used to determine the percentage of identity, including but not limited to global methods, local methods, and hybrid methods such as segmentation methods. Schemes for determining the percentage of identity are standard procedures within the scope of those skilled in the art. Global methods align sequences from the beginning to the end of the molecule and determine the optimal alignment by accumulating the scores of individual residue pairs and by applying a vacancy penalty. Non-restrictive methods include, for example, CLUSTAL W, see, for example, Julie D. Thompson et al., CLUSTAL W: Improving the Sensitivity of Progressive Multiple Sequence Alignment Through Sequence Weighting, Position-Specific GapPenalties and Weight Matrix Choice, 22(22) Nucleic Acids Research 4673-4680 (1994); and iterative improvements, see, for example, Osamu Gotoh, Significant Improvement in Accuracy of Multiple Protein Sequence Alignments by Iterative Refinement as Assessed by Reference to Structural Alignments, 264(4) J. MoI. Biol. 823-838 (1996). Local methods align sequences by identifying one or more conserved motifs common to all input sequences.Non-restrictive methods include, for example, match-box, see, for example, Eric Depiereux and Ernest Feytmans, Match-Box: A Fundamentally New Algorithm for the Simultaneous Alignment of Several Protein Sequences, 8(5) CABIOS 501-509 (1992); Gibbs sampling, see, for example, C.E. Lawrence et al., Detecting Subtle Sequence Signals: A Gibbs Sampling Strategy for Multiple Alignment, 262(5131) Science 208-214 (1993); Align-M, see, for example, Ivo Van Waille et al., Align-M - A New Algorithm for Multiple Alignment of Highly Divergent Sequences, 20(9) Bioinformatics:1428-1435 (2004).

[0247] Therefore, the percentage of sequence identity is determined using conventional methods. See, for example, Altschul et al., Bull. Math. Bio. 48: 603-16, 1986 and Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915-19, 1992. Briefly, as shown below, two amino acid sequences are aligned using a vacancy opening penalty of 10, a vacancy extension penalty of 1, and the Henikoff and Henikoff “blosum 62” scoring matrix (ibid.) to optimize the alignment score (amino acids are represented by standard single-letter codes); preferably, this method is used to align sequences with the subject sequence of this paper (e.g., SEQ ID NO: 2) to define amino acid position numbers as described herein.

[0248] The "percentage of sequence identity" between two or more nucleic acid or amino acid sequences is a function of the number of common positions in the sequences. Therefore, identity % can be calculated as the number of identical nucleotides / amino acids divided by the total number of nucleotides / amino acids, then multiplied by 100. The calculation of % sequence identity can also take into account the number of vacancies that need to be introduced to optimize the alignment of two or more sequences, as well as the length of each vacancy. Specific mathematical algorithms familiar to those skilled in the art (e.g., BLAST) can be used for sequence comparison and to determine the percentage of identity between two or more sequences.

[0249] Determination of alignment score for sequence identity

[0250] ARNDCQEGHILKMFPSTWYV

[0251] A 4

[0252] R -1 5

[0253] N -2 0 6

[0254] D -2 -2 1 6

[0255] C 0 -3 -3 -3 9

[0256] Q -1 1 0 0 -3 5

[0257] E -1 0 0 2 -4 2 5

[0258] G 0 -2 0 -1 -3 -2 -2 6

[0259] H -2 0 1 -1 -3 0 0 -2 8

[0260] I -1 -3 -3 -3 -1 -3 -3 -4 -3 4

[0261] L -1 -2 -3 -4 -1 -2 -3 -4 -3 2 4

[0262] K -1 2 0 -1 -3 1 1 -2 -1 -3 -2 5

[0263] M -1 -1 -2 -3 -1 0 -2 -3 -2 1 2 -1 5

[0264] F -2 -3 -3 -3 -2 -3 -3 -3 -1 0 0 -3 0 6

[0265] P -1 -2 -2 -1 -3 -1 -1 -2 -2 -3 -3 -1 -2 -4 7

[0266] S 1 -1 1 0 -1 0 0 0 -1 -2 -2 0 -1 -2 -1 4

[0267] T 0 -1 0 -1 -1 -1 -1 -2 -2 -1 -1 -1 -1 -2 -1 1 5

[0268] W -3 -3 -4 -4 -2 -2 -3 -2 -2 -3 -2 -3 -1 1 -4 -3 -2 11

[0269] Y -2 -2 -2 -3 -2 -1 -2 -3 2 -1 -1 -2 -1 3 -3 -2 -2 2 7

[0270] V 0 -3 -3 -3 -1 -2 -2 -3 -3 3 1 -2 1 -1 -2 -2 0 -3 -1 4

[0271] Then, the percentage identity is calculated as follows: Total number of identical matches ________________________________________ x 100 [Length of the longer sequence plus] Introduce a longer sequence to compare the number of gaps between the two sequences. Essentially homologous peptides are characterized by having one or more amino acid substitutions, deletions, or additions. These changes are preferably insignificant, i.e., conserved amino acid substitutions (see below) and other substitutions that do not significantly affect peptide folding or activity; small deletions, typically from 1 to about 30 amino acids; and small amino or carboxyl terminal extensions, such as N-terminal methionine residues, or small linker peptides or affinity tags of up to about 20-25 residues.

[0272] Conservative amino acid substitution

[0273] Alkaline: Arginine

[0274] Lysine

[0275] Histidine

[0276] Acidity: Glutamic acid

[0277] Aspartic acid

[0278] Polarity: Glutamine

[0279] Asparagine

[0280] Hydrophobicity: Leucine

[0281] Isoleucine

[0282] Valine

[0283] Aromatic compounds: Phenylalanine

[0284] Tryptophan

[0285] Tyrosine

[0286] Small: Glycine

[0287] alanine

[0288] Serine

[0289] threonine

[0290] Methionine

[0291] In addition to the 20 standard amino acids, non-standard amino acids (e.g., 4-hydroxyproline, 6-N-methyllysine, 2-aminoisobutyric acid, isovaleine, and α-methylserine) may replace amino acid residues in the polypeptides of the present invention. A limited number of non-conserved amino acids, amino acids not encoded by the genetic code, and non-natural amino acids may replace amino acid residues in the polypeptides. The polypeptides of the present invention may also contain non-naturally occurring amino acid residues.

[0292] Non-naturally occurring amino acids include, but are not limited to, trans-3-methylproline, 2,4-methylproline, cis-4-hydroxyproline, trans-4-hydroxyproline, N-methylglycine, allothreonine, methylthreonine, hydroxyethylcysteine, hydroxyethylhomocysteine, nitroglutamine, homoglutamine, piperidinic acid, tert-leucine, valine, 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, and 4-fluorophenylalanine. Several methods for incorporating non-naturally occurring amino acid residues into proteins are known in the art. For example, an in vitro system can be used in which nonsense mutations are suppressed using chemically aminoacylated repressor tRNAs. Methods for synthesizing amino acids and aminoacylated tRNAs are known in the art. Transcription and translation of plasmids containing nonsense mutations are performed in a cell-free system containing *E. coli* S30 extract and commercially available enzymes and other reagents. The protein is purified by chromatography. See, for example, Robertson et al., J. Am. Chem. Soc. 113:2722, 1991; Ellman et al., Methods Enzymol. 202:301, 1991; Chung et al., Science 259:806-9, 1993; and Chung et al., Proc. Natl. Acad. Sci. USA 90:10145-9, 1993). In the second method, translation is performed in Xenopus oocytes by microinjection of mutated mRNA and chemically aminoacylated repressor tRNA (Turcatti et al., J. Biol. Chem. 271:19991-8, 1996). In the third method, *E. coli* cells are cultured in the absence of the natural amino acid to be substituted (e.g., phenylalanine) and in the presence of the desired non-natural amino acid (e.g., 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, or 4-fluorophenylalanine). The non-natural amino acid is incorporated into the polypeptide, replacing its natural counterpart. See Koide et al., *Biochem.* 33:7470-6, 1994. Naturally occurring amino acid residues can be converted into non-natural substances through in vitro chemical modification. Chemical modification can be combined with site-directed mutagenesis to further expand the range of substitutions (Wynn and Richards, *Protein Sci.* 2:395-403, 1993).

[0293] A limited number of non-conserved amino acids, amino acids not encoded by the genetic code, non-naturally occurring amino acids, and non-natural amino acids can replace the amino acid residues of the polypeptides of the present invention.

[0294] Essential amino acids in the polypeptides of the present invention can be identified using 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 through physical structural analysis, such as by techniques like nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, combined with assumed contact site amino acid mutations. See, for example, de Vos et al., Science 255:306-12, 1992; Smith et al., J. Mol. Biol. 224:899-904, 1992; Wlodaver et al., FEBS Lett. 309:59-64, 1992. The identification of essential amino acids can also be inferred from homology analysis of related components (e.g., translocation or protease components) of the polypeptides of the present invention.

[0295] Multiple amino acid substitutions can be performed and tested using known methods of mutagenesis and screening, such as those disclosed in Reidhaar-Olson and Sauer (Science 241:53-7, 1988) or Bowie and Sauer (Proc.Natl. Acad. Sci. USA 86:2152-6, 1989). In short, these authors disclosed methods for simultaneously randomizing two or more positions in a polypeptide, selecting functional polypeptides, and then sequencing the mutagenized polypeptide to determine the profile of permissible 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., U.S. 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).

[0296] 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 pertains. Singleton et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 20th edition, John Wiley and Sons, New York (1994), and Hale & Marham, THE HARPER COLLINS DICTIONARY OF BIOLOGY, Harper Perennial, NY (1991), provide a general dictionary for skilled persons of the art of the use of many terms in this disclosure.

[0297] This disclosure is not limited to the exemplary methods and materials disclosed herein, and any methods and materials similar to or equivalent to those described herein may be used in the practice or testing of embodiments of this disclosure. Numerical ranges include the numbers defining the range. Unless otherwise stated, any nucleic acid sequence is written from left to right in a 5' to 3' orientation; amino acid sequences are written from left to right, respectively, from amino to carboxyl.

[0298] The headings provided herein are not intended to limit any aspect or implementation of this disclosure.

[0299] In this document, amino acid names, three-letter abbreviations, or single-letter abbreviations are used to refer to amino acids. As used herein, the term "protein" includes proteins, polypeptides, and peptides. As used herein, the term "amino acid sequence" is synonymous with the terms "polypeptide" and / or "protein." In some cases, the term "amino acid sequence" is synonymous with the term "peptide." In some cases, the term "amino acid sequence" is synonymous with the term "enzyme." The terms "protein" and "polypeptide" are used interchangeably herein. In this disclosure and claims, conventional single-letter and three-letter codons for amino acid residues may be used. The three-letter codon for amino acids is defined according to the Joint Committee on Biochemical Nomenclature of IUPACIUB (JCBN). It should also be understood that, due to the degeneracy of the genetic codon, a polypeptide can be encoded by more than one nucleotide sequence.

[0300] Other definitions of terms may appear throughout the specification. Before describing the exemplary embodiments in more detail, it should be understood that this disclosure is not limited to the specific embodiments described and therefore can vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting, as the scope of this disclosure is defined only by the appended claims.

[0301] In the case of a range of values ​​provided, it should be understood that, unless the context explicitly indicates otherwise, each midpoint value between the upper and lower limits of the range, expressed as one-tenth of the lower limit unit, is specifically included in this disclosure. Each smaller range between any specified value or midpoint within the specified range and any other specified value or midpoint within that specified range is included in this disclosure. The upper and lower limits of these smaller ranges may be independently included in or excluded from the range, and each range is also included in this disclosure, including, excluding, or including both of the upper and lower limits within the smaller range, as specified in any explicitly excluded limit within the range. Where the range includes one or two limits, ranges excluding any one or both of those limits are also included in this disclosure.

[0302] It is important to note that, as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural indicators unless the context clearly indicates otherwise. Thus, for example, reference to “modified botulinum neurotoxin A” includes a variety of such candidate agents, and reference to “modified botulinum neurotoxin A” includes reference to one or more modified botulinum neurotoxin A and its equivalents known to those skilled in the art, etc.

[0303] The publications discussed herein have only provided their public information prior to the filing date of this application. Nothing herein should be construed as an admission that such publications constitute prior art as described in the appended claims. Attached Figure Description

[0304] Schemes of the present invention will now be described by way of example only with reference to the following figures and embodiments.

[0305] Figure 1 This indicates that Dysport is FDA-approved for the treatment of cervical dystonia. ® dose.

[0306] Figure 2 An isoelectric focusing (IEF) gel of a cationic construct is shown.

[0307] Figure 3The percentage of SNAP-25 cleavage of Cat5v2(K1064H / N954K) (A), Cat5v2(K1064H / N886K) (B), and Cat5v2(K1064H / N1025K) (C) in rat embryonic spinal cord neurons (eSCN) is shown, along with a summary of pEC50 relative to nBoNT / A1. (A, B, C) Rat embryonic spinal cord neurons were cultured for three weeks, treated with Cat5v4 for 24 hours, and then blotted with a SNAP-25-specific antibody. Data are the mean ± SEM values ​​from three independent experiments, triplicate. (D) Relative potency of Cat5v2 (K1064H / N886K), Cat5v2 (K1064H / N954K), and Cat5v2 (K1064H / N1025K) relative to nBoNT / A1 (List Biological Laboratories) in the rat eSCN SNAP-25 cleavage potency assay. Each point corresponds to a separate batch and is the average of three independent pEC50 measurements based on an 8-point concentration response curve (CRC). Each concentration in the CRC was evaluated in triplicate. Potency was compared with the average of the List batches, with data pooled (n=24). Data are the average ± SEM of each Cat5v4 (n=3) batch.

[0308] Figure 4 This demonstrates the potency of nBoNT / A1 and Cat5v4 in the mouse phrenic hemidiaphragmatic assay (mPNHD). 50 As shown, mouse phrenic hemidiaphragmatic tissue was incubated with Cat5v4 or native BoNT / A1. Diaphragmatic contractility was recorded until no contraction was detected or after 140 minutes. Each point corresponds to an independent measurement. 50 The value represents the time required to inhibit 50% of the contractile force of the mouse hemidiaphragm.

[0309] Figure 5 The images show protein imprints of purified recombinant BoNT / AB chimeras 1, 2, and 3A (SEQ ID NO: 11, 12, and 13, respectively). Lanes are labeled “Marker” (molecular weight marker), “-DTT” (oxidized BoNT / AB chimera sample), and “+DTT” (reduced BoNT / AB chimera sample).

[0310] Figure 6This demonstrates the SNAP-25 cleavage of recombinant BoNT / AB chimeras 1, 2, and 3A (SEQ ID NO: 11, 12, and 13, respectively, converted to double-stranded form) in rat spinal cord neurons. Cultured primary rat spinal cord neurons (SCNs) were exposed to different concentrations of recombinant BoNT / AB chimeras 1, 2, or 3A for 24 hours at 37°C in a humid atmosphere containing 10% CO2. Cells were then lysed with 1x NuPAGE buffer supplemented with DTT and benzoxoside. Samples were transferred to microcentrifuge tubes, heated on a heat block at 90°C for 5 minutes, and stored at -20°C, followed by Western blot analysis of SNAP-25 cleavage. 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 a secondary antibody.

[0311] Figure 7 The toe abduction score assay for mice is shown. Under brief general anesthesia, an injection was administered into the gastrocnemius-soleus complex muscle of one hind limb of mice; muscle weakness was measured using the toe abduction score (DAS) on a 0-4 scale. The maximum DAS value for each dose was determined and plotted against dose. The data were fitted to a 4-parameter logistic equation to determine the ED50 and dose (DAS4 dose) value leading to DAS 4.

[0312] Figure 8 The images show protein imprints of purified recombinant BoNT / AB chimeras 3B and 3C (SEQ ID NO: 14 and 15, respectively). Lanes are labeled “Marker” (molecular weight marker), “-DTT” (oxidized BoNT / AB chimera sample), and “+DTT” (reduced BoNT / AB chimera sample).

[0313] Figure 9The SNAP-25 cleavage is shown in unmodified BoNT / A and BoNT / AB chimeras 3B and 3C (SEQ ID Nos. 2, 14, and 15, respectively, converted to double-stranded form) in human induced pluripotent stem cell-derived peripheral neurons (PERI.4U – Axiogenesis, Germany). PERI.4U cells were exposed to different concentrations of recombinant BoNT / A or BoNT / AB chimeras 3B or 3C for 24 hours at 37°C in a humidified CO2 atmosphere containing 5% CO2. Cells were then lysed with 1x NuPAGE buffer supplemented with DTT and benzoxoside. Samples were transferred to microcentrifuge tubes, heated on a hot block at 90°C for 5 minutes, and stored at -20°C, followed by Western blot analysis of SNAP-25 cleavage. SNAP-25 was detected using a polyclonal antibody that can detect both full-length and cleaved forms of SNAP-25 (Sigma #S9684). Anti-rabbit HRP (Sigma #A6154) was used as a secondary antibody.

[0314] Figure 10 The duration of muscle weakness over time is shown in the mouse toe abduction score assay. Under brief general anesthesia, the muscle was injected into the gastrocnemius-soleus complex of one hind limb of mice; muscle weakness was measured using the toe abduction score (DAS) on a 0-4 scale. Animals in the group receiving the lowest dose, which induced a DAS of 4 for the first four days after injection, were monitored until the muscle weakness fully recovered to a DAS of 0 (no muscle weakness observed).

[0315] Sequence Listing

[0316] The residue / codon is optional when the initial Met amino acid residue or the corresponding initial codon is indicated in any of the following SEQ ID NO.

[0317] SEQ ID NO: 1 (nucleotide sequence of unmodified BoNT / A)

[0318]

[0319] SEQ ID NO: 2 (polypeptide sequence of unmodified BoNT / A)

[0320]

[0321] SEQ ID NO: 3 (nucleotide sequence of modified BoNT / A "Cat-A")

[0322]

[0323] SEQ ID NO: 4 (polypeptide sequence of modified BoNT / A "Cat-A")

[0324]

[0325] SEQ ID NO: 5 (nucleotide sequence of modified BoNT / A "Cat-B")

[0326]

[0327] SEQ ID NO: 6 (polypeptide sequence of modified BoNT / A "Cat-B")

[0328]

[0329] SEQ ID NO: 7 (nucleotide sequence of modified BoNT / A "Cat-C")

[0330]

[0331] SEQ ID NO: 8 (polypeptide sequence of modified BoNT / A "Cat-C")

[0332]

[0333] SEQ ID NO: 9 (nucleotide sequence of modified BoNT / A "Cat-D")

[0334]

[0335] SEQ ID NO: 10 (polypeptide sequence of modified BoNT / A "Cat-D")

[0336]

[0337] SEQ ID NO: 11 (polypeptide sequence of modified BoNT / A "Chimera 1")

[0338]

[0339] SEQ ID NO: 12 (polypeptide sequence of modified BoNT / A "Chimera 2")

[0340]

[0341] SEQ ID NO: 13 (polypeptide sequence of modified BoNT / A "Chimera 3A")

[0342]

[0343] SEQ ID NO: 14 (polypeptide sequence of modified BoNT / A "Chimera 3B")

[0344]

[0345] SEQ ID NO: 15 (polypeptide sequence of modified BoNT / A "Chimera 3C")

[0346]

[0347] SEQ ID NO: 16 (polypeptide sequence of BoNT / B)

[0348]

[0349] Example

[0350] Example 1

[0351] Cloning, expression, and purification

[0352] The nucleotide sequence SEQ ID NO: 1 encodes wild-type BoNT / A (SEQ ID NO: 2), which is mutated to introduce the following substitutions, forming the four constructs shown in Table 1 below:

[0353] Table 1 Constructs The calculated pI of Cat-D is 7.45, and the molecular weight is 149,859.

[0354] A DNA construct encoding the modified BoNT / A molecule was synthesized, cloned into the pJ401 expression vector, and then transformed into BL21(DE3) Escherichia coli. This enabled soluble overexpression of recombinant Cat-A, Cat-B, Cat-C, and Cat-D proteins in BL21(DE3) Escherichia coli.

[0355] Recombinant-modified BoNTs were purified from *E. coli* lysates using classical chromatographic techniques. An initial purification step using a cation exchange resin was employed, followed by an intermediate purification step using a hydrophobic interaction resin. The recombinant-modified BoNT single strands were then cleaved by proteolytic hydrolysis to generate activated double-stranded modified BoNTs. A final purification step was then performed to remove residual contaminants. Suitable techniques are taught in WO2015 / 166242, WO2017055274A1, EP2524963B1, EP2677029B1, and US10087432B2.

[0356] Example 2

[0357] Characterization of purified modified BoNT / A

[0358] The modified BoNT described in Example 1 above was characterized experimentally as follows.

[0359] pI measurements show that the isoelectric point of modified BoNT is higher than that of unmodified (natural) BoNT / A1 - see [link to pI measurement] Figure 2 and Table 2 below.

[0360]

[0361] Table 2. Modified BoNT / A pI values.

[0362] The ability of modified BoNTs to enter neurons and cleave SNAP-25 (a target of BoNT / A1) was assessed using rat embryonic spinal cord neurons (eSCN). Figure 3 The results showed that the modified BoNT retained the same ability to enter neurons and cleave SNAP-25 as the natural BoNT / A1.

[0363] The potency of the modified BoNT was further evaluated using the mouse phrenic hemi-septum assay (mPNHD). Figure 4 The modified BoNT showed that it retained the same ability as the natural BoNT / A1 to inhibit mouse hemidiaphragmatic contraction.

[0364] In vivo mouse toe abduction score (DAS) assays were used to assess potency and safety relative to native BoNT / A1. Both molecules (converted to the double-stranded form Cat-A [SEQ ID NO: 4] and Cat-B [converted to the double-stranded form SEQ ID NO: 6]) showed a higher safety ratio and slightly stronger potency compared to native BoNT / A1. These data are shown in Table 3 below:

[0365] Table 3. DAS determination method and safety ratio.

[0366] -DAS ED 50 Calculated dose of induced DAS 2

[0367] - Dosage of DAS 4: Experimental dose for inducing DAS 4

[0368] -BW: body weight

[0369] -10% ΔBW dose: The calculated induced BW is reduced by 10% compared to the BW at D0.

[0370] - Safety ratio: -10% ΔBW dose / DAS ED 50

[0371] The safety ratio is a measure of the negative impact of BoNT treatment (weight loss) relative to its efficacy (half-maximum abduction score (DAS)). It is calculated as -10% body weight (BW) versus DAS ED. 50 The ratio between them, where -10% BW refers to the amount of BoNT (pg / animal) required to reduce body weight by 10%, ED 50 This refers to the amount (pg / animal) of BoNT that produces a DAS of 2.

[0372] The DAS assay was performed by injecting 20 μl of modified BoNT / A prepared in gelatin phosphate buffer into the gastrocnemius / soleus muscle complex of mice, and then assessing toe abduction as previously reported by Aoki (Aoki KR, Toxicon 39:1815-1820;2001).

[0373] Example 3

[0374] Cloning, expression, and purification of modified BoNT / A (BoNT / AB chimeras)

[0375] Using standard molecular biology techniques, BoNT / AB chimeric constructs 1, 2, 3A, 3B, and 3C (SEQ ID NO: 11 to 15, respectively) were constructed using DNA encoding the parental serotype molecule and appropriate oligonucleotides. They were then cloned into DNA with or without a C-terminal His... 10 The pJ401 expression vector was tagged and transformed into BLR(DE3) E. coli cells for overexpression. These cells were grown in 2 L baffled Erlenmeyer flasks containing 1 L of modified Terrific broth (mTB) supplemented with appropriate antibiotics at 37°C and 225 RPM with shaking. 600 Once the value reached >0.5, the incubator temperature was lowered to 16°C, and then after one hour, the incubator was induced with 1 mM IPTG at 225 RPM for 20 hours to express the recombinant BoNT / AB construct.

[0376] Harvested cells were lysed by sonication and clarified by centrifugation at 4500 RPM for 1 hour at 4°C. The recombinant BoNT / AB chimeric molecules were then extracted with ammonium sulfate and purified by standard rapid protein liquid chromatography (FPLC). This involved capture using a hydrophobic interaction resin and intermediate purification using an anion exchange resin. The partially purified molecules were then proteolytically cleaved with the endopeptide Lys-C to produce active double strands. Further purification with a second hydrophobic interaction resin yielded the final BoNT / AB chimeric molecules.

[0377] For those with decahistidine tag (H 10 The BoNT / AB chimeric molecules (chimers 1, 2, 3A) were captured using immobilized nickel resin instead of hydrophobic interaction resin.

[0378] The sequence of each chimera is shown in Table 4.

[0379]

[0380] Table 4 – Chimeric BoNT / AB Constructs

[0381] Example 4

[0382] Comparison of BoNT / AB chimeras 1, 2, and 3A

[0383] As in Example 3 ( Figure 5 The purification of C-terminal His described in the article) 10 We tagged and tested the functional activity of BoNT / AB chimeras 1, 2 and 3A with the E1191M / S1199Y double mutation.

[0384] Rat spinal cord neuron SNAP-25 cleavage assay

[0385] Primary cultures of rat spinal cord neurons (SCN) were prepared and cultured in 96-well tissue culture plates for 3 weeks (as described below: Masuyer et al., 2011, J. Struct. Biol. Structure and activity of afunctional derivative of Clostridium botulinum neurotoxin B; and Chaddock et al., 2002, Protein Expr. Purif. Expression and purification of catalyticallyactive, non-toxic endopeptidase derivatives of Clostridium botulinum toxintype A). Serial dilutions of BoNT / AB were prepared in SCN fed medium. Growth medium (0.2 g / mL) from the wells to be treated was collected and filtered. m filter). 125 The filtered culture medium was added back into each test well. Then 125 Add L-diluted toxin to the plate (in triplicate). Incubate the treated cells at 37°C and 10% CO2 for 24 ± 1 hours.

[0386] Analysis of BoNT activity using SNAP-25 cleavage assay

[0387] Following treatment, BoNTs were removed and cells were washed once in PBS (Gibco, UK). Cells were lysed in 1x NuPAGE lysis buffer (Life Technologies) supplemented with 0.1M dithiothreitol (DTT) and 250 units / mL benzoxoside (Sigma). Lysate proteins were separated by SDS-PAGE and transferred to a nitrocellulose membrane. The membrane was detected using a SNAP-25-specific primary antibody probe (Sigma #S9684), which recognizes both uncleaved and BoNT / A endopeptidase-cleaved SNAP-25. The secondary antibody used was HRP-conjugated anti-rabbit IgG (Sigma #A6154). Bands were detected by enhanced chemiluminescence and imaged using pXi6 Access (Synoptics, UK). Band intensity was determined using GeneTools software (Syngene, Cambridge, UK), and the percentage of SNAP-25 cleaved at each BoNT concentration was calculated. The data was fitted to a 4-parameter logistic equation, and pEC was calculated using GraphPad Prism version 6 (GraphPad). 50 .

[0388] Table 5 below provides the pEC values ​​for chimeras 1, 2, and 3A as determined by the rat SCN SNAP-25 cleavage assay. 50 These results indicate that the three BoNT / AB chimeras retained their ability to enter rat spinal cord neurons and cleave their target substrates. However, in this assay, chimera 3A was more effective than chimeras 1 and 2 (see also...). Figure 6 ).

[0389]

[0390] Table 5. pEC 50 value.

[0391] Digiugus angle score (DAS) assay

[0392] The method for measuring BoNT / AB chimera 1, 2, and 3A activity in the DAS assay is based on the startle response to the toe-spreading reflex in mice when briefly suspended by their tails. This reflex is scored as the toe-spreading score (DAS) and is inhibited after BoNT is applied to the gastrocnemius-soleus muscles of the hind paw. The mouse's tail is briefly suspended to elicit the characteristic startle response, in which the animal extends its hind limb and abducts its hind toes. (Aoki et al., 1999, Eur. J. Neurol.; 6 (suppl. 4) S3–S10).

[0393] On the day of injection, mice were anesthetized in an induction chamber containing oxygen with 3% isoflurane. Each mouse received an intramuscular injection of BoNT / AB chimera or a carrier (phosphate buffered saline containing 0.2% gelatin) into the gastrocnemius-soleus muscle of the right hind paw.

[0394] Following neurotoxin injection, different degrees of toe abduction were scored on a scale from 0 to 4, where 0 = normal and 4 = reduced maximum toe abduction and leg extension. The effect-limiting factor (ED) was determined using the mean of the maximum effect for each dose via nonlinear adjustment analysis. 50 The mathematical model used is a 4-parameter logic model.

[0395] DAS was performed every 2 hours on the first day after administration; thereafter, it was performed 3 times a day for 4 days.

[0396] Figure 7 Fitting curves for chimeras 1, 2, and 3A (SEQ ID NO: 11, 12, and 13, respectively, converted to double-stranded forms) are shown. The curve for chimera 3A shifts to the left, indicating that a lower dose of chimera 3A achieved a similar DAS response compared to chimeras 1 and 2, thus suggesting that chimera 3A is more effective than other chimeras in mouse DAS assays; see also the table below (Table 6), which provides the calculated ED for each chimera. 50 Value and the dose that resulted in DAS 4 (highest score).

[0397] Table 6 below provides the ED determined by the mouse DAS assay for unmodified recombinant BoNT / A1 (rBoNT / A1-converted to double-stranded form SEQ ID NO: 2) and chimeras 1, 2 and 3A. 50 And DAS 4 dose. These results indicate that, among the three chimeras, chimera 3A has the highest in vivo potency in inducing muscle weakness. Figure 7 The studies shown in Table 6 were conducted on mice obtained from the Charles River laboratory.

[0398]

[0399] Table 6 ED 50 value.

[0400] Example 5

[0401] Comparison of BoNT / AB chimeras 3B, 3C, and unmodified BoNT / A 1

[0402] As described in Example 3, unlabeled BoNT / AB chimeras 3B and 3C were purified, with or without the E1191M / S1199Y double mutation (SEQ ID NO: 14 and 15), respectively. Figure 8Unmodified BoNT / A (converted to double-stranded form SEQ ID NO: 2) was used as a reference to test functional activity.

[0403] Human pluripotent stem cell SNAP-25 cleavage assay

[0404] Cryopreserved PERI.4U cells were purchased from Axiogenesis (Cologne, Germany). Cell thawing and plating were performed according to the manufacturer's recommendations. In short, the frozen tubes containing cells were thawed in a 37°C water bath for 2 minutes. After gentle resuspending, the cells were transferred to 50 mL tubes. The frozen tubes were washed with 1 mL of the manufacturer's provided Peri.4U® thawing medium, and the medium was added dropwise to the cell suspension in the 50 mL tubes. Then, another 2 mL of Peri.4U® thawing medium was added dropwise to the 50 mL tubes. Cells were then counted using a hemocytometer. Subsequently, another 6 mL of Peri.4U® thawing medium was added to the cell suspension. Cell pellet was obtained by centrifugation at 260 xg (e.g., 1,100 RPM) for 6 minutes at room temperature. The cells were then resuspended in the manufacturer's provided complete Peri.4U® medium. Cells were plated at concentrations ranging from 50,000 to 150,000 cells / cm². 2 Cells were seeded at a density on cell culture plates coated with poly-L-ornithine and laminin. The cells were cultured at 37°C in a humidified CO2 atmosphere, with the culture medium completely replaced every 2-3 days during the culture period.

[0405] For toxin treatment, serially diluted BoNTs were prepared in Peri.4U® medium. The medium from the wells to be treated (0.2 g / L) was collected and filtered. m filter). 125 The filtered culture medium was added back into each test well. Then 125 Add L-diluted toxin to the plate (in triplicate). Incubate the treated cells at 37°C and 10% CO2 for 48 ± 1 hours.

[0406] Analysis of BoNT activity using SNAP-25 cleavage assay

[0407] Following treatment, BoNTs were removed and cells were washed once in PBS (Gibco, UK). Cells were lysed in 1x NuPAGE lysis buffer (Life Technologies) supplemented with 0.1M dithiothreitol (DTT) and 250 units / mL benzoxoside (Sigma). Lysate proteins were separated by SDS-PAGE and transferred to a nitrocellulose membrane. The membrane was detected using a SNAP-25-specific primary antibody probe (Sigma #S9684), which recognizes both uncleaved and BoNT / A endopeptidase-cleaved SNAP-25. The secondary antibody used was HRP-conjugated anti-rabbit IgG (Sigma #A6154). Bands were detected by enhanced chemiluminescence and imaged using pXi6 Access (Synoptics, UK). Band intensity was determined using GeneTools software (Syngene, Cambridge, UK), and the percentage of SNAP-25 cleaved at each BoNT concentration was calculated. The data was fitted to a 4-parameter logistic equation, and pEC was calculated using GraphPad Prism version 6 (GraphPad). 50 .

[0408] Figure 9 Chimeric 3B and 3C showed greater potency than rBoNT / A1 in induced pluripotent stem cell cleavage of SNAP-25 in induced pluripotent stem cells, but the former was significantly more effective. This can be explained by a double mutation, which increases the affinity of chimeric 3B for the human synaptic binding protein II receptor present in these cells. Figure 9 (Table 7).

[0409]

[0410] Table 7. pEC 50 value.

[0411] Digiugus angle score (DAS) assay - safety ratio

[0412] The method for measuring BoNT activity in the DAS assay is based on the startle response of a mouse's toe-spreading reflex during brief tail suspension. This reflex is scored as the toe-spreading score (DAS) and is inhibited after BoNT is applied to the gastrocnemius-soleus muscles of the hind paw. The mouse's tail is briefly suspended to elicit the characteristic startle response, in which the animal extends its hind limb and abducts its hind toes. (Aoki et al., 1999, Eur. J. Neurol.; 6 (suppl. 4) S3–S10).

[0413] On the day of injection, mice were anesthetized in an induction chamber containing oxygen with 3% isoflurane. Each mouse received an intramuscular injection of BoNT or a carrier (phosphate buffered saline containing 0.2% gelatin) into the gastrocnemius-soleus muscle of the right hind paw.

[0414] Following neurotoxin injection, different degrees of toe abduction were scored on a scale from 0 to 4, where 0 = normal and 4 = reduced maximum toe abduction and leg extension. The effect-limiting factor (ED) was determined using the mean of the maximum effect for each dose via nonlinear adjustment analysis. 50 The mathematical model used is a 4-parameter logic model.

[0415] DAS was performed every 2 hours on the first day after administration; thereafter, it was performed 3 times daily for 4 days for all doses. Animals in the carrier group and the lowest dose group were then monitored, which induced a DAS of 4 during the first four days after administration, until muscle weakness fully recovered to a DAS of 0 (no muscle weakness was observed).

[0416] To calculate the safety ratio, all animals were weighed one day prior to toxin injection (D0) and subsequently daily throughout the study period. The mean body weight, standard deviation, and mean standard error were calculated daily for each dose group. To obtain the safety ratio of BoNT (-10% ΔBW / ED), 50 The dose that causes the mean body weight of the dose group at any time during the study period to be less than 10% of the mean body weight of the same dose group at D0 is divided by the ED of the studied BoNT. 50 The lethal dose is defined as the dose that would cause one or more animals in that dose group to die.

[0417] Figure 10 The duration of muscle attenuation over time for unmodified BoNT / A, chimera 3B, and chimera 3C (converted to double-stranded forms SEQ ID NO:2, 14, and 15) in the mouse toe abduction scoring assay is shown, indicating that the chimeras have a longer duration of action.

[0418] Table 8 below provides the ED determined for rBoNT / A1 and chimeric 3B and 3C using the mouse DAS assay. 50 The table also provides the total duration of action of the DAS 4 dose until muscle weakness fully recovers to DAS 0 (no muscle weakness observed). Furthermore, the table shows the lethal dose and safety ratio in mice (-10% ΔBW / ED). 50 As defined above, chimeric compounds 3B and 3C have a longer duration of action, a better safety ratio, and a higher lethal dose compared to rBoNT / A1. Figure 10 The studies shown in Table 8 were conducted on mice obtained from Janvier's laboratory.

[0419]

[0420] Table 8. DAS and safety ratio of BoNT / AB chimeras.

[0421] Example 6

[0422] Preclinical testing of modified BoNT / A (SEQ ID NO: 4 converted to a double chain form)

[0423] Further preclinical testing was conducted on the modified BoNT / A “Cat-A” (converted to the double-stranded form SEQ ID NO: 4).

[0424] Materials and Methods

[0425] Rat Digit Abduction Score (DAS) Assay

[0426] To assess the effects of modified BoNT / A (converted to the double-stranded form SEQ ID NO:4) on in vivo muscle activity, a dose-response study was conducted using the rat Digit Abduction Score (DAS) assay. The rat DAS assay is based on the toe spread reflex, a characteristic startle response of animals when briefly grasped. Following a single injection of neurotoxin into the left peroneal muscle complex, muscle weakness resulted in reduced toe abduction. Different degrees of toe abduction were scored using a 5-point scale: 0 = normal to 4 = maximum reduction in toe abduction and leg extension (Broide RS, Rubino J, Nicholson GS, et al., The rat Digit Abduction Score (DAS) assay: A physiological model for assessing botulinum neurotoxin-induced skeletal muscle paralysis. Toxicon 013;71:18-24). DAS values ​​were measured consecutively for the first five days following toxin injection, and then every two to three days thereafter until the effect of a lower dose of modified BoNT / A (converted to the double-stranded form SEQ ID NO: 4) on the toe extension reflex completely disappeared, and the effect of a dose leading to DAS4 on the toe extension reflex returned to DAS2. A transient BoNT-induced dose-dependent weight gain was considered evidence of a generalized toxin effect (Torii Y, Goto Y, Nakahira S, et al., Comparison of Systemic Toxicity between Botulinum Toxin Subtypes A1 and A2 in Mice and Rats. BasicClin. Pharmacol. Toxicol. 2015;116:524-528). Rats were weighed and side effects were recorded at each assessment time point. The BoNT administration solution was masked before injection until the end of the study (assigned random letters). Potency was determined as the dose required to induce 50% effect (ED). 50 (The dose that results in a DAS value of 2). To determine ED 50 The dose range of 2.5 to 750 pg / kg was tested with a 95% confidence interval (CI). Higher doses of 1, 1.5, 2, 2.4, 3, 4, and 5 ng / kg were also administered to assess potential side effects.

[0427] To assess the duration of action of modified BoNT / A (converted to the double-stranded form of SEQ ID NO:4) and compare it with that of unmodified BoNT / A (converted to the double-stranded form of SEQ ID NO:2), the median time required to return to DAS2 reading 2 at the highest tolerated dose of both toxins (with no effect on body weight change compared to untreated rats) was evaluated in two independent, direct head-to-head studies.

[0428] Rat Single Dose Study

[0429] Rats received a single intramuscular (im) injection of modified BoNT / A (converted to the double-stranded form of SEQ ID NO:4) into the right gastrocnemius muscle at doses of 0, 0.1, 1, and 3 ng / kg. Control animals received a dilution of SEQ ID NO:4 into the right gastrocnemius muscle. Animals were euthanized after 7 days post-treatment (n=10 males and 10 females per group) or after a 13 or 26-week observation period (n=5 males and 5 females per dose). Irvine tests for assessing central nervous system function were performed during the pre-test (day-1), day 8, and weeks 13 and 27. Other clinical (adverse) signs assessed were lameness, muscle size at small injections of toxin, and abdominal distension.

[0430] Monkey Study

[0431] Monkeys received a single intramuscular injection of modified BoNT / A (converted to the double-stranded form SEQ ID NO: 4) into the right gastrocnemius muscle at doses of 0, 0.1, 0.25, and 0.75 ng / kg. Animals were euthanized after 7 days post-treatment (3 males and 3 females per group) or a 13 or 26-week observation period (2 males and 2 females per dose). Cardiovascular examinations, including hemodynamics, electrocardiogram, and respiratory parameters, were performed on days 8 and 15 via external telemetry pre-testing.

[0432] Preliminary enhanced EFD in pregnant rats

[0433] The aim of this study was to provide preliminary information on the effects of modified BoNT / A (converted to the double-stranded form SEQ ID NO: 4) administered intramuscularly throughout organogenesis on rat embryonic and fetal development. Modified BoNT / A (converted to the double-stranded form SEQ ID NO: 4) was administered daily via intramuscular injection (gastrocnemius muscle) to nine mated female Sprague-Dawley rats from day 6 (G6) to day 17 (G17) (inclusive of G6 and G17) at dose levels of 0.02, 0.05, and 0.1 ng / kg / day. Clinical status, body weight, and food consumption were monitored throughout the study. Females underwent cesarean section at G21, and litter parameters were recorded. At necropsy, females underwent gross examination, and the pregnant uterus was weighed. For females exhibiting small injected gastrocnemius muscles, both the injected and contralateral muscles were weighed. All fetuses were weighed. External and visceral abnormalities of the fetuses were then examined, and sex was determined. Approximately half of the fetuses' heads were fixed for internal examination via serial slicing. All fetal eviscerated remains were processed for skeletal examination.

[0434] Preliminary extended EFD in pregnant rabbits

[0435] The aim of this study was to provide preliminary information on the effects of modified BoNT / A (SEQ ID NO: 4 in double-stranded form) administered intramuscularly throughout organogenesis on rabbit embryonic and fetal development. Modified BoNT / A (SEQ ID NO: 4 in double-stranded form) was administered daily via intramuscular injection (gastrocnemius muscle) to nine mated female New Zealand white rabbits from day 6 of gestation (G6) to day 19 of gestation (G19) (inclusive of G6 and G19) at dose levels of 0.002, 0.005, and 0.01 ng / kg / day. Clinical condition, body weight, and food consumption were monitored throughout the study. Females underwent cesarean section at G29, and litter parameters were recorded. At necropsy, females underwent gross examination, and the pregnant uterus was weighed. For females exhibiting small injected gastrocnemius muscles, both the injected and contralateral muscles were weighed. All fetuses were weighed. External and visceral abnormalities of the fetuses were then examined, and sex was determined. Approximately half of the fetuses' heads were fixed in place for internal examination via sequential slices.

[0436] Results

[0437] Through the studies described above, the following pharmacological data were obtained for many different species that were given modified BoNT / A (shown in Table 9 below).

[0438]

[0439] Table 9. Preclinical results.

[0440] In addition, the modified BoNT / A (converted to the double-stranded form of SEQ ID NO:4) was tested in a rat DAS assay to determine the duration of action compared to Dysport®. The results are shown in Table 10 below:

[0441] Table 10. Duration of action.

[0442] These data indicate that the duration of the effect of the modified BoNT / A is Dysport ® More than twice as much.

[0443] Example 7

[0444] Determination of unit dose of modified BoNT / A (SEQ ID NO: 4 converted to a double chain form) for treatment of cervical dystonia Unit Dose

[0445] Based on the preclinical pharmacological data obtained in Example 6 above, an appropriate unit dose range (UD) for the administration of modified BoNT / A in humans has been determined. Studies have shown that modified BoNT / A has a longer duration of action and exhibits improved safety compared to unmodified BoNT / A. This improved safety is reflected in the high safety ratio of modified BoNT / A described herein.

[0446] Due to the modification of BoNT / A and Dysport ® Having the same mechanism of action (although with an increased safety ratio due to its modified properties), the minimum dose of modified BoNT / A used to treat subjects has been determined relative to Dysport in the context of the same muscle group. ® The labeled dosage was located: In a rat model of toe abduction fraction, the ED of modified BoNT / A 50 The level was 13 pg / kg, more than 100-fold lower than the estimated no-observed adverse effect level (NOAEL) of 1500 pg / kg in the same animal species. In the same rat model, Dysport... ® ED 50 The dose was 0.5 U / kg. Based on these animal data, the dose of 2.6 ng modified BoNT / A could be estimated at 100 U / kg. ® The dosage.

[0447] Intraperitoneal LD ​​in mice 50The dose was determined to be 8.44 pg. Under these conditions, 0.84 ng of modified BoNT / A is equivalent to 100 U Dysport. ® The dosage.

[0448] Therefore, the calculated minimum dose was 500 pg (0.5 ng). To provide some background information, the above intraperitoneal mouse LD50 was used. 50 Data shows that 0.5 ng of modified BoNT / A is equivalent to approximately 60 U Dysport. ® Therefore, it will be active when used for intramuscular injection to treat cervical dystonia.

[0449] In rats, the estimated NOAEL of 1.5 ng / kg modified BoNT / A is equivalent to a dose of 90 ng in a 60 kg human. In monkeys (the more sensitive of the two non-clinical species tested), the estimated NOAEL of 0.125 ng / kg modified BoNT / A is equivalent to a dose of 7.5 ng in a 60 kg human.

[0450] Therefore, the upper limit of the unit dose was set at 7,500 pg (7.5 ng), as this is still below the rat NOAEL converted to human dose.

[0451] Therefore, the appropriate unit dose for treating cervical dystonia with modified BoNT / A has been determined to be 500-7,500 pg. Based on obtained preclinical data, such as those determined using mouse intraperitoneal lethal dose assays, the calculated mouse intraperitoneal median lethal dose (LD50) is used. 50 This dose is approximately 59-889 units of modified BoNT / A (which also corresponds to approximately 59-889 units of Dysport). ® ).

[0452] Based on the preclinical data identified in Example 6, and Dysport ® Compared to the improved safety profile, the total dose (in units) expected to be administered in cervical dystonia is almost the same as that of Dysport. ® Nine times that of Dysport ® The maximum total dose for treating cervical dystonia is 1,000 units (see [link to relevant treatment]). Figure 1 ).

[0453] Advantageously, in the treatment of cervical dystonia, more modified BoNT / A can be injected and / or injected into a greater number of neck muscles / sites before reaching the maximum dose. This is an important and advantageous finding that leads to improved treatment of cervical dystonia while providing clinicians with a wider range of treatment options.

[0454] Example 8

[0455] Dose regimen for treatment of cervical dystonia

[0456] Modified BoNT / A (e.g., converted to the double-stranded form SEQ ID NO: 4) is provided as a lyophilized powder in 2 mL clear glass vials, each containing 15 ng of modified BoNT / A. The lyophilized powder is reconstituted with a mixture of sterile 0.9% v / w preservative-free sodium chloride solution and a diluent (a formulation buffer containing only the excipients of modified BoNT / A). After reconstitution, the solution is further diluted as needed.

[0457] The unit dose (UD) is 500-7,500 pg (approximately 59-889 units).

[0458] Neck dystonia can be treated by intramuscular injection according to the following dosage regimen (Table 11):

[0459] Table 11. Dosage regimen.

[0460] Depending on the specific needs of the patient, the application can be unilateral or bilateral.

[0461] The maximum total dose administered is 10 x UD (e.g., in some cases, 2 x UD may be administered to one or more designated neck muscles). This corresponds to 75,000 pg / approximately 8,890 units. This is almost certainly the dose of Dysport that can be administered during the treatment of cervical dystonia without approaching the limits of toxicity (a concern in conventional treatment regimens). ® This is nine times the maximum total dose. Therefore, clinicians are able to tailor treatment to patients knowing that 10x UD can be administered without concern for toxicity, thus allowing for treatment of additional neck muscles and / or ensuring that each neck muscle receives a pharmaceutically effective dose.

[0462] Example 9

[0463] Preclinical testing of modified BoNT / A (BoNT / AB chimera [SEQ ID NO: 14 converted to a double chain form])

[0464] In mouse LD 50 The assay tested BoNT / AB chimera SEQ ID NO:14 (converted to double-stranded form), yielding a result of 1.202 ng / kg. Therefore, in this assay, 1 unit of SEQ ID NO:14 (converted to double-stranded form) corresponds to 24.04 pg.

[0465] In addition, the BoNT / AB chimera was tested in a rat DAS assay to determine its compatibility with Dysport. ®The duration of action was compared (according to Example 6). The results are shown in Table 12 below:

[0466] Table 12. Duration of action.

[0467] In summary, the duration of BoNT / AB's effect is much longer than that of Dysport. ® Furthermore, it is similar to SEQ ID NO:4 (converted to double-stranded form). Therefore, it is expected that the unit dose and dosing regimen of SEQ ID NO:4 (converted to double-stranded form) can also be applied to BoNT / AB to provide improved treatment for cervical dystonia.

[0468] Example 10

[0469] Determination of unit dose of modified BoNT / A (SEQ ID NO: 14 converted to a double chain form) for treatment of cervical dystonia Unit Dose

[0470] Based on preclinical pharmacological data, an appropriate unit dose range (UD) for the administration of modified BoNT / A in humans has been determined.

[0471] Calculated DAS ED of SEQ ID NO: 14 (converted to double-stranded form) 50 It is 13 pg / kg. ED 50 It is considered to be the minimum pharmacologically active dose, approximately 300 times lower than the no-observed adverse reaction level (NOAEL) of 4 ng / kg in the same animal species. In rats, the ED at 13 pg / kg of SEQ ID NO: 14 (converted to double-stranded form) is... 50 This is equivalent to a dose of 0.8 ng for a person weighing 60 kg.

[0472] Therefore, a lower limit for the unit dose was chosen as 1,000 pg. An upper limit for the unit dose was chosen as 16,000 pg, which is lower than the NOAEL of 4 ng / kg for two non-clinically safe species (rats and monkeys) converted to a dose for a 60 kg human body. Therefore, the unit dose was determined to be from 1,000 pg to 16,000 pg (approximately 42 units to approximately 666 units).

[0473] Given the improved safety profile, the maximum total dose for treating cervical dystonia was set at 160,000 pg (approximately 7,070 units), derived from a NOAEL of 4 ng / kg in two non-clinically safe species (rats and monkeys) converted to a dose for a 60 kg human.

[0474] Based on the preclinical data identified in Example 9, and Dysport ®Compared to the improved safety profile, the total dose (in units) expected to be administered in cervical dystonia is almost the same as that of Dysport. ® Seven times that of the previous dose. The maximum total dose of Dysport® for the treatment of cervical dystonia is 1,000 units (see [link to relevant information]). Figure 1 ).

[0475] Advantageously, in the treatment of cervical dystonia, more modified BoNT / A (converted to the double-stranded form of SEQ ID NO:14) can be injected before reaching the maximum dose and / or injected at a greater number of neck muscles / sites. This is an important and advantageous finding that leads to improved treatment of cervical dystonia while providing clinicians with a wider range of treatment options.

[0476] Example 11

[0477] Dose regimen for treatment of cervical dystonia using modified BoNT / A (SEQ ID NO: 14 converted to a double chain form) Example 12

[0478] The modified BoNT / A (e.g., converted to the double-stranded form SEQ ID NO: 14) is provided as a lyophilized powder in vials containing 36 ng of the modified BoNT / A per vial. The lyophilized powder is then reconstituted.

[0479] The unit dose (UD) is 1,000-16,000 pg (approximately 42-666 units [based on mouse LD50]). 50 Measurement]).

[0480] Neck dystonia can be treated by intramuscular injection according to the following dosage regimen (Table 13):

[0481] Table 13. Dosage regimen.

[0482] Depending on the specific needs of the patient, the application can be unilateral or bilateral.

[0483] The maximum total dose administered is 10 x UD (e.g., in some cases, 2 x UD may be administered to one or more designated neck muscles). This corresponds to 160,000 pg / approximately 6,660 units. This is almost certainly the dose of Dysport that can be administered during the treatment of cervical dystonia without approaching the limits of toxicity (a concern in conventional treatment regimens). ® This is up to 7 times the maximum total dose. Therefore, clinicians are able to tailor treatment to patients knowing that 10x UD can be administered without concern for toxicity, thus allowing for treatment of additional neck muscles and / or ensuring that each neck muscle receives a pharmaceutically effective dose.

[0484] Treatment of cervical dystonia (lateral neck) patients

[0485] Example 13

[0486] Jane, 65, was diagnosed with cervical dystonia by a general practitioner, specifically lateral cervical dystonia. A single unit dose (3,000 pg) of modified BoNT / A (converted to the double-chain form SEQ ID NO: 4) was administered ipsilaterally to Jane's levator scapulae muscle, and a single unit dose was also administered ipsilaterally to Jane's sternocleidomastoid muscle (resulting in a total dose of 6,000 pg during treatment). Lateral cervical dystonia was relieved, and due to the longer duration of modified BoNT / A administration, Jane did not require further treatment for over 9 months. Therefore, Jane received fewer injections (e.g., annually) compared to equivalent subjects receiving unmodified BoNT / A. Furthermore, due to the improved safety profile of modified BoNT / A, Jane did not experience any side effects.

[0487] Treatment of cervical dystonia (torticollis) patients

[0488] Example 14

[0489] Brian, 48, was diagnosed with cervical dystonia by a general practitioner. His specific symptom was neck extension. Modified BoNT / A (converted to the double-stranded form of SEQ ID NO:14) was applied bilaterally to each of Brian's following muscles: Administer a 1x unit dose (UD) of 10,000 pg to each levator scapulae muscle; Administer 10,000 pg of 1xUD to each trapezius muscle; Administer 10,000 pg of 1xUD to each longissimus muscle; Administer 10,000 pg of 1xUD to each splenius capitis muscle; and Administer 10,000 pg of 1xUD to each splenius cervicis muscle.

[0490] The total dose administered was 10 x UD (100,000 pg), well within the upper limit of 160,000 pg, and feasible given the superior safety profile of modified BoNT / A compared to unmodified BoNT / A. Neck extension was relieved, and due to the longer duration of modified BoNT / A administration, Brian required no further treatment for 12 months. Therefore, Brian received fewer injections compared to equivalent subjects receiving unmodified BoNT / A.

[0491] Safety and efficacy of modified BoNT / A (SEQ ID NO: 14 converted to a double chain form) in humans

[0492] Example 15

[0493] SEQ ID NO:14 (converted to double-stranded form) was administered to human subjects via a single unit dose of modified BoNT / A. Different (increased) amounts of modified BoNT / A (converted to double-stranded form of SEQ ID NO:14) were administered to five cohorts. Cohort 1 received 2 x 1,000 pg units of modified BoNT / A (i.e., a maximum of 2,000 pg), while Cohort 5 received 2 x 16,000 pg units of modified BoNT / A (i.e., a maximum of 32,000 pg).

[0494] The results showed that all tested unit doses of modified BoNT / A (i.e., up to 16,000 pg unit doses) were effective and well-tolerated for muscle paralysis, and no adverse reactions were observed despite the unusually high doses per muscle. This indicates that the modified BoNT / A does not diffuse from the injection site and highlights the exceptional safety profile of the modified BoNT / A (converted to the double-stranded form SEQ ID NO:14).

[0495] Treatment of cervical dystonia patients

[0496] Example 16

[0497] Sally, 64, was diagnosed with cervical dystonia by her general practitioner. She was treated with a 1000 pg unit dose (UD) of SEQ ID NO:14 (converted to double-stranded form), administered as follows:

[0498] She received a total dose of 14,000 pg of SEQ ID NO:14 (converted to double-stranded form). The treatment was successful, and her symptoms were relieved. She did not require treatment for more than 9 months.

[0499] Treatment of cervical dystonia patients

[0500] ​

[0501] Francesco, 43, was diagnosed with cervical dystonia by a general practitioner. He was treated with a 2000 pg unit dose (UD) of SEQ ID NO:14 (converted to double-stranded form), administered as follows:

[0502] He received a total dose of 28,000 pg of SEQ ID NO:14 (converted to double-stranded form). The treatment was successful, and his symptoms were relieved. She did not require treatment for over 10 months.

[0503] All publications mentioned in the foregoing specification are incorporated herein by reference. Various modifications and variations to the methods and systems described herein will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in conjunction with specific preferred embodiments, it should be understood that the claimed invention should not be unduly limited to such specific embodiments. Indeed, various modifications to the described methods for carrying out the invention will be apparent to those skilled in the art of biochemistry and biotechnology or related fields, and fall within the scope of the following claims.

Claims

1. The use of a modified botulinum neurotoxin A (BoNT / A) for the treatment of cervical dystonia, wherein the modified BoNT / A is administered to the affected neck muscles of a subject via intramuscular injection. The modified BoNT / A is administered at a unit dose of 750 pg to 17,000 pg of modified BoNT / A. This involves applying at least a single unit dose to the affected neck muscles. The total dose administered during treatment was as high as 170,000 pg of modified BoNT / A, and The modified BoNT / A comprises a BoNT / A light chain and a translocation domain, as well as a BoNT / B receptor-binding domain (H). C (structural domain).

2. The modified BoNT / A according to claim 1, wherein: (a) The total dose administered is from 5,250 pg to 170,000 pg, preferably from 7,000 pg to 160,000 pg; (b) The unit dose is from 1,000 pg to 16,000 pg; and / or (c) Modified BoNT / A administered at a total dose of up to 160,000 pg during treatment.

3. The modified BoNT / A according to claim 1 or claim 2, wherein the modified BoNT / A is applied to the affected neck muscles at a single injection site or at two or more injection sites (e.g., two injection sites).

4. The modified BoNT / A according to any one of the preceding claims, wherein the duration of treatment for cervical dystonia is longer than the duration of treatment with unmodified BoNT / A (e.g., SEQ ID NO:2 (e.g., SEQ ID NO:2 in double strand form)).

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

6. A modified BoNT / A according to the use described in any one of the preceding claims, wherein: The affected neck muscles are selected from: sternocleidomastoid, splenius capitis, splenius cervicis, scalene complex (e.g., anterior scalene and / or middle scalene), trapezius (e.g., upper trapezius), levator scapulae, semispinalis capitis, and longissimus (e.g., longissimus capitis and / or longissimus cervicis).

7. A modified BoNT / A according to any one of the preceding claims, wherein the modified BoNT / A is administered to a plurality of affected neck muscles of a subject by intramuscular injection, and wherein at least a single unit dose is administered to each affected neck muscle. Preferably, wherein: Multiple affected neck muscles are selected from: sternocleidomastoid, splenius capitis, splenius cervicis, scalene complex (e.g., anterior and / or middle scalene), trapezius (e.g., upper trapezius), levator scapulae, semispinalis capitis, and longissimus (e.g., longissimus capitis and / or longissimus cervicis).

8. A modified BoNT / A for the use described in any of the preceding claims, wherein: (a) The modified BoNT / A is applied to the affected neck muscles in a unit dose per injection site, or the modified BoNT / A is applied in a less than unit dose per injection site. (b) Administer a single unit dose at multiple injection sites in the affected neck muscles, and / or administer two or more unit doses at multiple injection sites in the affected neck muscles; (c) Administer a single unit dose to the affected neck muscles (e.g., administer a single unit dose to each affected neck muscle).

9. The modified BoNT / A according to any one of the preceding claims, wherein the subject is a human subject.

10. A modified BoNT / A unit dosage form for treating cervical dystonia, said unit dosage form comprising: (a) 750 pg to 17,000 pg modified BoNT / A; and (b) Optional pharmaceutically acceptable carriers, excipients, adjuvants and / or salts The modified BoNT / A includes the BoNT / A light chain and translocation domain, as well as the BoNT / B receptor-binding domain (H). C (structural domain).

11. A modified BoNT / A for use according to any one of claims 1-9, or a unit dosage form for use according to claim 10, wherein: (a) The modified BoNT / A comprises a combination of two substitution mutations, namely E1191M and S1199Y; and / or (b) The modified BoNT / A contains a polypeptide sequence that has at least 70% sequence identity with SEQ ID NO:

14.

12. A modified BoNT / A for use according to any one of claims 1-9 or 11, or a unit dosage form for use according to claim 10 or 11, wherein the modified BoNT / A is a double-stranded modified BoNT / A, wherein the light chain (L chain) is linked to the heavy chain (H chain) by a disulfide bond, the modified BoNT / A being obtained by means of a method comprising contacting a single-stranded modified BoNT / A comprising a polypeptide sequence having at least 70%, 80%, 90%, 95%, or 99.9% sequence identity with SEQ ID NO:14 with a protease, the protease hydrolyzing the peptide bonds in its activated ring, thereby converting the single-stranded modified BoNT / A into the corresponding double-stranded modified BoNT / A.

13. A modified BoNT / A for use according to any one of claims 1-9 or 11-12, or a unit dosage form for use according to any one of claims 10-12, wherein the C-terminal amino acid residue of the translocation domain corresponds to the LH separating the BoNT / A. N and H C 3 of the structural domain 10 The first amino acid residue of the helix, and H C The N-terminal amino acid residue of the domain corresponds to the LH in the BoNT / B separator. N and H C 3 of the structural domain 10 The second amino acid residue of the helix.

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