Treatment of limb spasticity

Abstract

The present invention relates to a BoNT / A light chain and translocation domain and a BoNT / B receptor binding domain (H C The present invention relates to the treatment of limb spasticity using a modified botulinum neurotoxin A (BoNT / A) containing a nucleotide sequence (SEQ ID NO: 1) and a corresponding kit.

Description

[Technical field]

[0001] The present invention relates to the treatment of limb spasticity using a modified botulinum neurotoxin A (BoNT / A). [Background technology]

[0002] Spasticity is a motor condition characterized by increased velocity-dependent stretch reflexes with exaggerated tendon reflexes due to the hyperexcitability of stretch reflexes as one component of motor neuron syndrome.Spasticity is associated with various neurological diseases, such as multiple sclerosis, brain or spinal cord injury, traumatic brain injury (TBI) and cerebrovascular accident (stroke).It is characterized by functional impairment, pain and discomfort due to motor disorders (increased muscle tone, abnormal limb posture, hypercontraction of antagonistic muscles and hyperreflexia).

[0003] Adult upper limb (AUL) spasticity is a common complication after stroke, is often painful (associated with spastic muscle contractions), and can cause significant functional disability by interfering with upper limb movement and limiting the use of the limb for active functional tasks. In severe cases, it can also interfere with "passive functions" such as washing, dressing, and caring for the affected limb, thereby increasing the burden on the caregiver. Secondary complications can include poor self-esteem and body image, impaired quality of life (reduced social and family interactions), and bedsores.

[0004] Upper limb post-stroke spasticity management is challenging due to the diversity of patients' symptoms and their goals. In these patients, muscle hypertonia usually manifests in several common clinical patterns determined by the affected muscles, which in turn relate to the size, location and age of the central nervous system lesion. These patterns were described in an international cross-sectional survey of clinicians from 31 countries and were subsequently used to develop a classification of five typical patterns of AUL spasticity. These are defined with respect to the joint locations of the shoulder, elbow, forearm and wrist. Current clinical consensus and existing guidelines recommend that the exact pattern of spasticity must be considered when selecting a patient's treatment, and that botulinum neurotoxin A (BoNT / A) therapy must be tailored according to the appropriate muscles selected for injection. However, no two patients are identical and a tailored / individualized treatment regimen is required.

[0005] An expert panel further explored these patterns to provide guidance on treatment parameters for upper limb spasticity (ULS) and the muscles targeted for each clinical pattern (see Figure 1). Importantly, the panel defined three collective posture combinations that would be targeted for treatment with BoNT. These were: (1) Adducted shoulders, flexed elbows, pronated forearms, flexed wrists, clenched fists, (2) flexed elbows, pronated forearms, flexed wrists, clenched fists, and (3) Bent wrist, clenched fist.

[0006] Four upper limb joints, shown in Figure 1, are known to be involved in >90% of patients (see Figure 1). The majority of spastic upper limb patterns (four out of five) are due to internal rotation and adduction of the shoulder and flexion at the elbow, with postural differences due to forearm and wrist posture.

[0007] Overall ULS treatment aims to alleviate the signs and symptoms of spasticity - reducing muscle spasms and pain, improving posture, facilitating mobility and dexterity (voluntary motor functions reaching grasping, moving and releasing), minimizing contractures and deformities and improving the ease of care and hygiene / self-care and / or quality of life of the patient.Available drug treatment options such as oral (benzodiazepines, baclofen, tizanidine and dantrolene) and intrathecal (baclofen) pharmacotherapy mostly cause non-selective muscle weakness with side effects such as generalized weakness and adverse effects on the central nervous system including ataxia, drowsiness, sedation and even withdrawal symptoms.

[0008] The most effective approach for the treatment of spasticity, recommended by several guidelines in recent years, is the combination of physical therapy and intramuscular (im) injection of BoNT / A. BoNT / A has emerged as the treatment of choice because it is a locally injectable treatment with minimal systemic side effects, and can be tailored to the individual's disease symptoms by selectively targeting the affected muscles.

[0009] Dysport® is a pharmaceutical product that contains the original BoNT / A hemagglutinin complex (BTX-A-HAC) isolated and purified from Clostridium botulinum type A strains. Several other pharmaceutical BoNT / A products, naturally produced by Clostridium botulinum, are also commercially available (e.g., BOTOX® and XEOMIN®).

[0010] BoNT / A selectively inhibits the release of acetylcholine from presynaptic nerve terminals, thus blocking cholinergic transmission at the neuromuscular junction, inducing muscle contraction and reduced muscle tone, and relaxing the injected muscle. This mechanism of action has been used therapeutically for more than 20 years to treat several clinical neurological conditions, including focal dystonia, focal muscle spasms, and cosmetic conditions.

[0011] However, currently available BoNT / A products have a duration of action of approximately 12-14 weeks, by which time new nerve endings have sprouted, allowing nerve function to return to normal and the original symptoms to reappear. As a result, repeated injections must be administered periodically to maintain efficacy.

[0012] Therefore, the frequency of BoNT / A injections is an important consideration for the treatment of spasticity, taking into account the chronicity of the condition and the long-term nature of the treatment required, with implications for direct and indirect medical costs associated with the patient and caregivers, the logistics of injections within the hospital / clinic, and most importantly, the quality of life of the patient.

[0013] Dysport® is approved for the treatment of upper and lower limb spasticity and has a maximum total dose per treatment session of 1,500 units (see FIG. 2 - up to 1,000 units for treating upper limb spasticity). Clinicians are required to administer Dysport® to multiple muscles on the limbs up to the upper threshold of a total of 1,500 units per treatment session. Clinicians are forced to make difficult choices during the treatment of patients. In other words, in conventional treatment regimens, clinicians must find a balance between the relatively small total amount of BoNT / A that can be administered (1,500 units or 1,000 units for upper limb spasticity - required by the high toxicity of BoNT / A) and the effective amount in multiple different muscles. Thus, certain muscles are neglected while others receive suboptimal amounts of BoNT / A, resulting in suboptimal therapy. Furthermore, current treatment regimens exclude treatment of the shoulder, which requires multiple unit doses in its multiple muscles.

[0014] Furthermore, conventional treatment regimens are complicated, resulting in clinicians underdosing to avoid toxicity to the patient.Therefore, there is a need for a convenient, safe, and effective single dose unit and a guide corresponding to the number of units (including the number of injection sites per muscle) that can be administered to a limb in a treatment session without resulting in patient toxicity.

[0015] In conclusion, there is a need for improved treatments for limb spasticity that allow for an individualized patient-centered approach to tailor treatment according to the targeted clinical pattern, and that allow injection into different combinations of limb muscles depending on the distribution, degree and severity of spasticity, while avoiding toxicity and providing long-lasting treatment (resulting in less frequent administration). There is also a need for improved treatment regimens that allow treatment of neglected muscles, such as those of the shoulder.

[0016] The present invention overcomes one or more of the problems set forth above. Summary of the Invention

[0017] The present inventors have surprisingly found that modified BoNT / A finds particular utility in the treatment of limb spasticity. The modified BoNT / A of the present invention comprises a BoNT / A light chain and a translocation domain (H N domain) and the BoNT / B receptor binding domain (H C The modified BoNT / A contains a 5'-terminal domain, which results in the modified BoNT / A exhibiting increased retention at (reduced diffusion from) the site of administration and / or increased duration of action (e.g., 6-9 months). Advantageously, the modified BoNT / A has an improved safety profile when compared to unmodified BoNT / A (e.g., Dysport®). This improved safety profile may be expressed by the increased safety margins described herein for the modified BoNT / A.

[0018] Based on the preclinical and clinical data herein, it has been shown that a higher total amount of the modified BoNT / A of the present invention can be administered to a subject, and such a high dose still achieves a similar safety profile as unmodified BoNT / A (e.g., Dysport®). Thus, the modified BoNT / A of the present invention can be injected, and / or can be injected into a larger number of muscles / sites in the treatment of limb spasticity before reaching the maximum total dose. This is an important, advantageous finding, and leads to an improvement in the treatment of limb spasticity, while providing clinicians with a greater range of treatment options. For the first time, it also provides the option to treat additional large muscles, such as those of the shoulder, while also adequately treating the elbow, forearm, and / or wrist within the maximum dose. The treatment can be improved in that it provides a longer-lasting treatment (resulting in less frequent administration) and / or can be tailored to the subject, and / or results in an improvement in the quality of life of the subject, when compared to treatment with unmodified BoNT / A (e.g., Dysport®). Thus, the treatment of the present invention is improved compared to conventional treatment regimes.

[0019] Furthermore, the present invention provides a convenient, safe, and effective single unit dose as well as a total (maximum) dose that can be safely administered in a single treatment. The present invention also provides a guide corresponding to the number of times (including, for example, the number of injection sites per muscle) that said unit dose can be administered to a limb without resulting patient toxicity. Treatment according to the present invention is therefore less complicated for clinicians and helps to avoid under-dosing and / or over-dosing. Furthermore, treatment according to the present invention is significantly more satisfying for patients, since it is better tailored to the needs of the patient compared to conventional limb spasticity treatments. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] In one aspect, the invention provides a modified BoNT / A for use in treating limb spasticity (adult limb spasticity), wherein the modified BoNT / A is administered by intramuscular injection to multiple affected muscles of a subject; wherein the modified BoNT / A is administered by a unit dose of greater than 17,000 pg of modified BoNT / A in multiple affected muscles; wherein the plurality of affected muscles is selected from: A first group including: flexor digitorum superficialis, flexor digitorum profundus, flexor carpi radialis, flexor carpi ulnaris, brachioradialis, pronator teres, biceps brachii, medial head of gastrocnemius (gastrocnemius medialis), lateral head of gastrocnemius (gastrocnemius lateralis), flexor digitorum longus, flexor hallucis longus, gastrocnemius, deltoid, levator scapulae, pronator quadratus, flexor hallucis longus, adductor pollicis brevis, palmaris longus, lumbricalis, opponens pollicis, adductor magnus, adductor longus, adductor brevis, gracilis, medial hamstrings, lateral hamstrings, tensor fasciae latae, rectus femoris, vastus lateralis, vastus medialis, vastus intermedius, gluteus maximus, tibialis anterior, flexor digitorum brevis, extensor hallucis longus, and flexor hallucis brevis, and A second group including: triceps brachii (long head), subscapularis, pectoralis major (e.g., pectoralis major), latissimus dorsi, biceps brachii, brachialis, soleus, tibialis posterior, brachioradialis, teres major, iliopsoas, adductor magnus, hamstrings (e.g., biceps femoris, semimembranosus, and / or semitendinosus), rectus femoris, and gastrocnemius; and wherein a single unit dose is administered in the affected muscle of a first group (preferably at a selected injection site) and / or multiple unit doses are administered in the affected muscle of a second group (preferably at different selected injection sites), wherein the total dose administered during treatment is up to 600,000 pg; and Here, 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 another aspect, the invention provides a modified BoNT / A for use in treating limb spasticity (adult limb spasticity) in a subject for a longer period of time than those treated with unmodified BoNT / A (e.g., SEQ ID NO:2 [e.g., a two-chain form of SEQ ID NO:2]), wherein the modified BoNT / A is administered by intramuscular injection into multiple affected muscles of the subject; wherein the modified BoNT / A is administered by a unit dose of greater than 17,000 pg of modified BoNT / A in multiple affected muscles; wherein the plurality of affected muscles is selected from: A first group including: flexor digitorum superficialis, flexor digitorum profundus, flexor carpi radialis, flexor carpi ulnaris, brachioradialis, pronator teres, biceps brachii, medial head of gastrocnemius (gastrocnemius medialis), lateral head of gastrocnemius (gastrocnemius lateralis), flexor digitorum longus, flexor hallucis longus, gastrocnemius, deltoid, levator scapulae, pronator quadratus, flexor hallucis longus, adductor pollicis brevis, palmaris longus, lumbricalis, opponens pollicis, adductor magnus, adductor longus, adductor brevis, gracilis, medial hamstrings, lateral hamstrings, tensor fasciae latae, rectus femoris, vastus lateralis, vastus medialis, vastus intermedius, gluteus maximus, tibialis anterior, flexor digitorum brevis, extensor hallucis longus, and flexor hallucis brevis, and A second group including: triceps brachii (long head), subscapularis, pectoralis major (e.g., pectoralis major), latissimus dorsi, biceps brachii, brachialis, soleus, tibialis posterior, brachioradialis, teres major, iliopsoas, adductor magnus, hamstrings (e.g., biceps femoris, semimembranosus, and / or semitendinosus), rectus femoris, and gastrocnemius; and wherein a single unit dose is administered in the affected muscle of a first group (preferably at a selected injection site) and / or multiple unit doses are administered in the affected muscle of a second group (preferably at different selected injection sites), wherein the total dose administered during treatment is up to 600,000 pg; and Here, 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 invention provides a method of treating limb spasticity, the method comprising administering a modified BoNT / A by intramuscular injection to multiple affected muscles of a subject; wherein the modified BoNT / A is administered by a unit dose of greater than 17,000 pg of modified BoNT / A in multiple affected muscles; wherein the plurality of affected muscles is selected from: A first group including: flexor digitorum superficialis, flexor digitorum profundus, flexor carpi radialis, flexor carpi ulnaris, brachioradialis, pronator teres, biceps brachii, medial head of gastrocnemius (gastrocnemius medialis), lateral head of gastrocnemius (gastrocnemius lateralis), flexor digitorum longus, flexor hallucis longus, gastrocnemius, deltoid, levator scapulae, pronator quadratus, flexor hallucis longus, adductor pollicis brevis, palmaris longus, lumbricalis, opponens pollicis, adductor magnus, adductor longus, adductor brevis, gracilis, medial hamstrings, lateral hamstrings, tensor fasciae latae, rectus femoris, vastus lateralis, vastus medialis, vastus intermedius, gluteus maximus, tibialis anterior, flexor digitorum brevis, extensor hallucis longus, and flexor hallucis brevis, and A second group including: triceps brachii (long head), subscapularis, pectoralis major (e.g., pectoralis major), latissimus dorsi, biceps brachii, brachialis, soleus, tibialis posterior, brachioradialis, teres major, iliopsoas, adductor magnus, hamstrings (e.g., biceps femoris, semimembranosus, and / or semitendinosus), rectus femoris, and gastrocnemius; and wherein a single unit dose is administered in the affected muscle of a first group (preferably at a selected injection site) and / or multiple unit doses are administered in the affected muscle of a second group (preferably at different selected injection sites), wherein the total dose administered during treatment is up to 600,000 pg; and Here, 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 invention provides a method of treating limb spasticity (adult limb spasticity) in a subject for a longer period of time than those treated with unmodified BoNT / A (e.g., SEQ ID NO:2 [e.g., a two-chain form of SEQ ID NO:2]), the method comprising administering modified BoNT / A by intramuscular injection to multiple affected muscles of the subject; wherein the modified BoNT / A is administered by a unit dose of greater than 17,000 pg of modified BoNT / A in multiple affected muscles; wherein the plurality of affected muscles is selected from: A first group including: flexor digitorum superficialis, flexor digitorum profundus, flexor carpi radialis, flexor carpi ulnaris, brachioradialis, pronator teres, biceps brachii, medial head of gastrocnemius (gastrocnemius medialis), lateral head of gastrocnemius (gastrocnemius lateralis), flexor digitorum longus, flexor hallucis longus, gastrocnemius, deltoid, levator scapulae, pronator quadratus, flexor hallucis longus, adductor pollicis brevis, palmaris longus, lumbricalis, opponens pollicis, adductor magnus, adductor longus, adductor brevis, gracilis, medial hamstrings, lateral hamstrings, tensor fasciae latae, rectus femoris, vastus lateralis, vastus medialis, vastus intermedius, gluteus maximus, tibialis anterior, flexor digitorum brevis, extensor hallucis longus, and flexor hallucis brevis, and A second group including: triceps brachii (long head), subscapularis, pectoralis major (e.g., pectoralis major), latissimus dorsi, biceps brachii, brachialis, soleus, tibialis posterior, brachioradialis, teres major, iliopsoas, adductor magnus, hamstrings (e.g., biceps femoris, semimembranosus, and / or semitendinosus), rectus femoris, and gastrocnemius; and wherein a single unit dose is administered in the affected muscle of a first group (preferably at a selected injection site) and / or multiple unit doses are administered in the affected muscle of a second group (preferably at different selected injection sites), wherein the total dose administered during treatment is up to 600,000 pg; and Here, the modified BoNT / A comprises a BoNT / A light chain and translocation domain and a BoNT / B receptor binding domain (H C domain).

[0024] In another aspect, the invention provides the use of a modified BoNT / A in the manufacture of a medicament for treating limb spasticity (adult limb spasticity), wherein the modified BoNT / A is administered by intramuscular injection to multiple affected muscles of a subject; wherein the modified BoNT / A is administered by a unit dose of greater than 17,000 pg of modified BoNT / A in multiple affected muscles; wherein the plurality of affected muscles is selected from: A first group including: flexor digitorum superficialis, flexor digitorum profundus, flexor carpi radialis, flexor carpi ulnaris, brachioradialis, pronator teres, biceps brachii, medial head of gastrocnemius (gastrocnemius medialis), lateral head of gastrocnemius (gastrocnemius lateralis), flexor digitorum longus, flexor hallucis longus, gastrocnemius, deltoid, levator scapulae, pronator quadratus, flexor hallucis longus, adductor pollicis brevis, palmaris longus, lumbricalis, opponens pollicis, adductor magnus, adductor longus, adductor brevis, gracilis, medial hamstrings, lateral hamstrings, tensor fasciae latae, rectus femoris, vastus lateralis, vastus medialis, vastus intermedius, gluteus maximus, tibialis anterior, flexor digitorum brevis, extensor hallucis longus, and flexor hallucis brevis, and A second group including: triceps brachii (long head), subscapularis, pectoralis major (e.g., pectoralis major), latissimus dorsi, biceps brachii, brachialis, soleus, tibialis posterior, brachioradialis, teres major, iliopsoas, adductor magnus, hamstrings (e.g., biceps femoris, semimembranosus, and / or semitendinosus), rectus femoris, and gastrocnemius; and wherein a single unit dose is administered in the affected muscle of a first group (preferably at a selected injection site) and / or multiple unit doses are administered in the affected muscle of a second group (preferably at different selected injection sites), wherein the total dose administered during treatment is up to 600,000 pg; and Here, the modified BoNT / A comprises a BoNT / A light chain and translocation domain and a BoNT / B receptor binding domain (H C domain).

[0025] In another aspect, the invention provides the use of a modified BoNT / A in the manufacture of a medicament for the treatment of limb spasticity (adult limb spasticity) in a subject for a longer period of time than that treated with an unmodified BoNT / A (e.g., SEQ ID NO:2 [e.g., a two-chain form of SEQ ID NO:2]), wherein the modified BoNT / A is administered by intramuscular injection into multiple affected muscles of the subject; wherein the modified BoNT / A is administered by a unit dose of greater than 17,000 pg of modified BoNT / A in multiple affected muscles; wherein the plurality of affected muscles is selected from: A first group including: flexor digitorum superficialis, flexor digitorum profundus, flexor carpi radialis, flexor carpi ulnaris, brachioradialis, pronator teres, biceps brachii, medial head of gastrocnemius (gastrocnemius medialis), lateral head of gastrocnemius (gastrocnemius lateralis), flexor digitorum longus, flexor hallucis longus, gastrocnemius, deltoid, levator scapulae, pronator quadratus, flexor hallucis longus, adductor pollicis brevis, palmaris longus, lumbricalis, opponens pollicis, adductor magnus, adductor longus, adductor brevis, gracilis, medial hamstrings, lateral hamstrings, tensor fasciae latae, rectus femoris, vastus lateralis, vastus medialis, vastus intermedius, gluteus maximus, tibialis anterior, flexor digitorum brevis, extensor hallucis longus, and flexor hallucis brevis, and A second group including: triceps brachii (long head), subscapularis, pectoralis major (e.g., pectoralis major), latissimus dorsi, biceps brachii, brachialis, soleus, tibialis posterior, brachioradialis, teres major, iliopsoas, adductor magnus, hamstrings (e.g., biceps femoris, semimembranosus, and / or semitendinosus), rectus femoris, and gastrocnemius; and wherein a single unit dose is administered in the affected muscle of a first group (preferably at a selected injection site) and / or multiple unit doses are administered in the affected muscle of a second group (preferably at different selected injection sites), wherein the total dose administered during treatment is up to 600,000 pg; Here, the modified BoNT / A comprises a BoNT / A light chain and translocation domain and a BoNT / B receptor binding domain (H C domain).

[0026] The unit dose can be greater than 17,000 pg of modified BoNT / A. The upper limit of the unit dose range can be 40,000, 39,000, 38,000, 37,000, 36,000, 35,000, 30,000, 25,000, 24,000, 22,000, 20,000 or 18,000 pg of modified BoNT / A, preferably the upper limit is 38,000 pg. The lower limit of the unit dose range can be 17,500, 18,000, 20,000, 22,000, 24,000, 25,000, 26,000, 27,000, 28,000, 29,000, 30,000, 35,000, 36,000, 37,000, 38,000 or 39,000 pg of modified BoNT / A, preferably the lower limit is 17,500 pg, 22,753 pg, 22,790 pg or 33,181 pg. Preferably, the unit dose of modified BoNT / A is more than 17,000pg and up to 40,000pg of modified BoNT / A, for example, more than 17,000pg and up to 36,000pg or 20,000pg to 39,000pg. The unit dose of modified BoNT / A can be 22,000 to 38,000pg, preferably 23,000 to 38,000pg or 34,000 to 38,000pg. The unit dose of modified BoNT / A can be 24,000 to 36,000pg or 25,000 to 36,000pg. The unit dose can be 25,000pg to up to 40,000pg of modified BoNT / A. In preferred embodiments, the unit dose of modified BoNT / A is 24,000, 25,000, 30,000 or 36,000 pg. In more preferred embodiments, the unit dose of modified BoNT / A is 30,000 or 36,000 pg (e.g., 36,000 pg).

[0027] More preferably, the unit dose is 30,000 pg to 40,000 pg, for example, 35,000 pg to 37,000 pg of the modified BoNT / A. More preferably, the unit dose is 36,000 pg of the modified BoNT / A.

[0028] Even more preferably, the unit dose is 20,000 pg to 30,000 pg, for example, 24,000 pg to 26,000 pg of the modified BoNT / A. Most preferably, the unit dose is 25,000 pg of the modified BoNT / A.

[0029] The total dose administered when carrying out the treatment regimen of the present invention can be up to 600,000pg. In other words, the total amount of modified BoNT / A administered in a given treatment session can be up to 600,000pg. The total dose can be up to 580,000, 560,000, 540,000, 520,000, 500,000, 480,000, 460,000, 450,000, 440,000, 420,000, 400,000, 380,000, 360,000, 340,000, 320,000, 300,000, 280,000, 260,000, 240,000, 220,000 or 200,000pg. Preferably, the total dose can be up to 540,000 pg of modified BoNT / A, more preferably 375,000 pg. The total dose can be at least 17,500, 20,000, 22,500, 25,000, 27,500, 30,000, 35,000, 36,000, 37,000, 38,000, 39,000 or 40,000 pg. The total dose can be at least 250,000, 255,000, 260,000, 270,000, 280,000, 290,000, 300,000, 320,000, 340,000, 360,000, 380,000, 400,000, 450,000, 500,000, 550,000 or 575,000 pg. Preferably, the total dose is greater than 255,000 pg or greater than 341,849 pg, more preferably at least 350,000 pg of modified BoNT / A, e.g., at least 497,700 pg or 500,000 pg. The total dose may be 250,000-600,000pg or 255,000-600,000pg, preferably more than 255,000pg, up to 600,000pg, 350,000-600,000 or 500,000-600,000pg. In a more preferred embodiment, the total dose administered is 520,000-600,000pg, more preferably 350,000-400,000pg. The total dose may be 360,000, 450,000 or 540,000pg, preferably 450,000 or 540,000pg (e.g., 540,000pg).

[0030] More preferably, the total dose is 450,000 pg to 600,000 pg, for example, 525,000 pg to 555,000 pg of modified BoNT / A. Most preferably, the total dose is up to 540,000 pg of modified BoNT / A (for example, the total dose can be 540,000 pg).

[0031] Even more preferably, the total dose is 300,000 pg to 450,000 pg, for example, 360,000 pg to 390,000 pg of modified BoNT / A. Most preferably, the total dose is up to 375,000 pg of modified BoNT / A (for example, the total dose can be 375,000 pg).

[0032] Thus, the unit dose may be more than 17,000 pg of modified BoNT / A, and the total dose administered when implementing the treatment regimen of the present invention may be up to 600,000 pg. In a preferred embodiment, the unit dose may be 24,000 pg, and the total dose may be 360,000 pg, or may be up to 360,000 pg. In another preferred embodiment, the unit dose may be 30,000 pg, and the total dose may be 450,000 pg, or may be up to 450,000 pg. In another preferred embodiment, the unit dose may be 36,000 pg, and the total dose may be 540,000 pg, or may be up to 540,000 pg. More preferably, the unit dose may be 25,000 pg, and the total dose may be 375,000 pg, or may be up to 375,000 pg.

[0033] In one aspect, the invention provides a modified BoNT / A for use in treating childhood limb spasticity, wherein the modified BoNT / A is administered by intramuscular injection to multiple affected muscles of a subject; wherein the modified BoNT / A is administered by a unit dose of greater than 8,500 pg of modified BoNT / A in multiple affected muscles; wherein the plurality of affected muscles is selected from: A first group including: flexor digitorum superficialis, flexor digitorum profundus, flexor carpi radialis, flexor carpi ulnaris, brachioradialis, pronator teres, biceps brachii, medial head of gastrocnemius (gastrocnemius medialis), lateral head of gastrocnemius (gastrocnemius lateralis), flexor digitorum longus, flexor hallucis longus, gastrocnemius, deltoid, levator scapulae, pronator quadratus, flexor hallucis longus, adductor pollicis brevis, palmaris longus, lumbricalis, opponens pollicis, adductor magnus, adductor longus, adductor brevis, gracilis, medial hamstrings, lateral hamstrings, tensor fasciae latae, rectus femoris, vastus lateralis, vastus medialis, vastus intermedius, gluteus maximus, tibialis anterior, flexor digitorum brevis, extensor hallucis longus, and flexor hallucis brevis, and A second group including: triceps brachii (long head), subscapularis, pectoralis major (e.g., pectoralis major), latissimus dorsi, biceps brachii, brachialis, soleus, tibialis posterior, brachioradialis, teres major, iliopsoas, adductor magnus, hamstrings (e.g., biceps femoris, semimembranosus, and / or semitendinosus), rectus femoris, and gastrocnemius; and wherein a single unit dose is administered in the affected muscle of a first group (preferably at a selected injection site) and / or multiple unit doses are administered in the affected muscle of a second group (preferably at different selected injection sites), wherein the total dose administered during treatment is up to 300,000 pg; and Here, the modified BoNT / A comprises a BoNT / A light chain and translocation domain and a BoNT / B receptor binding domain (H C domain).

[0034] In another aspect, the invention provides a modified BoNT / A for use in treating childhood limb spasticity in a subject for a longer period of time than those treated with an unmodified BoNT / A (e.g., SEQ ID NO:2 [e.g., a two-chain form of SEQ ID NO:2]), wherein the modified BoNT / A is administered by intramuscular injection into multiple affected muscles of the subject; wherein the modified BoNT / A is administered by a unit dose of greater than 8,500 pg of modified BoNT / A in multiple affected muscles; wherein the plurality of affected muscles is selected from: A first group including: flexor digitorum superficialis, flexor digitorum profundus, flexor carpi radialis, flexor carpi ulnaris, brachioradialis, pronator teres, biceps brachii, medial head of gastrocnemius (gastrocnemius medialis), lateral head of gastrocnemius (gastrocnemius lateralis), flexor digitorum longus, flexor hallucis longus, gastrocnemius, deltoid, levator scapulae, pronator quadratus, flexor hallucis longus, adductor pollicis brevis, palmaris longus, lumbricalis, opponens pollicis, adductor magnus, adductor longus, adductor brevis, gracilis, medial hamstrings, lateral hamstrings, tensor fasciae latae, rectus femoris, vastus lateralis, vastus medialis, vastus intermedius, gluteus maximus, tibialis anterior, flexor digitorum brevis, extensor hallucis longus, and flexor hallucis brevis, and A second group including: triceps brachii (long head), subscapularis, pectoralis major (e.g., pectoralis major), latissimus dorsi, biceps brachii, brachialis, soleus, tibialis posterior, brachioradialis, teres major, iliopsoas, adductor magnus, hamstrings (e.g., biceps femoris, semimembranosus, and / or semitendinosus), rectus femoris, and gastrocnemius; and wherein a single unit dose is administered in the affected muscle of a first group (preferably at a selected injection site) and / or multiple unit doses are administered in the affected muscle of a second group (preferably at different selected injection sites), wherein the total dose administered during treatment is up to 300,000 pg; and Here, the modified BoNT / A comprises a BoNT / A light chain and translocation domain and a BoNT / B receptor binding domain (H C domain).

[0035] In one aspect, the invention provides a method of treating infantile limb spasticity, the method comprising administering a modified BoNT / A by intramuscular injection to multiple affected muscles of a subject, wherein the modified BoNT / A is administered by a unit dose of greater than 8,500 pg of modified BoNT / A in multiple affected muscles; wherein the plurality of affected muscles is selected from: A first group including: flexor digitorum superficialis, flexor digitorum profundus, flexor carpi radialis, flexor carpi ulnaris, brachioradialis, pronator teres, biceps brachii, medial head of gastrocnemius (gastrocnemius medialis), lateral head of gastrocnemius (gastrocnemius lateralis), flexor digitorum longus, flexor hallucis longus, gastrocnemius, deltoid, levator scapulae, pronator quadratus, flexor hallucis longus, adductor pollicis brevis, palmaris longus, lumbricalis, opponens pollicis, adductor magnus, adductor longus, adductor brevis, gracilis, medial hamstrings, lateral hamstrings, tensor fasciae latae, rectus femoris, vastus lateralis, vastus medialis, vastus intermedius, gluteus maximus, tibialis anterior, flexor digitorum brevis, extensor hallucis longus, and flexor hallucis brevis, and A second group including: triceps brachii (long head), subscapularis, pectoralis major (e.g., pectoralis major), latissimus dorsi, biceps brachii, brachialis, soleus, tibialis posterior, brachioradialis, teres major, iliopsoas, adductor magnus, hamstrings (e.g., biceps femoris, semimembranosus, and / or semitendinosus), rectus femoris, and gastrocnemius; and wherein a single unit dose is administered in the affected muscle of a first group (preferably at a selected injection site) and / or multiple unit doses are administered in the affected muscle of a second group (preferably at different selected injection sites), wherein the total dose administered during treatment is up to 300,000 pg; and The modified BoNT / A comprises the BoNT / A light chain and translocation domain and the BoNT / B receptor binding domain (H C domain).

[0036] In a related aspect, the invention provides a method of treating infantile limb spasticity in a subject for a longer period of time than those treated with unmodified BoNT / A (e.g., SEQ ID NO:2 [e.g., a two-chain form of SEQ ID NO:2]), the method comprising administering modified BoNT / A by intramuscular injection to multiple affected muscles of the subject; wherein the modified BoNT / A is administered by a unit dose of greater than 8,500 pg of modified BoNT / A in multiple affected muscles; wherein the plurality of affected muscles is selected from: A first group including: flexor digitorum superficialis, flexor digitorum profundus, flexor carpi radialis, flexor carpi ulnaris, brachioradialis, pronator teres, biceps brachii, medial head of gastrocnemius (gastrocnemius medialis), lateral head of gastrocnemius (gastrocnemius lateralis), flexor digitorum longus, flexor hallucis longus, gastrocnemius, deltoid, levator scapulae, pronator quadratus, flexor hallucis longus, adductor pollicis brevis, palmaris longus, lumbricalis, opponens pollicis, adductor magnus, adductor longus, adductor brevis, gracilis, medial hamstrings, lateral hamstrings, tensor fasciae latae, rectus femoris, vastus lateralis, vastus medialis, vastus intermedius, gluteus maximus, tibialis anterior, flexor digitorum brevis, extensor hallucis longus, and flexor hallucis brevis, and A second group including: triceps brachii (long head), subscapularis, pectoralis major (e.g., pectoralis major), latissimus dorsi, biceps brachii, brachialis, soleus, tibialis posterior, brachioradialis, teres major, iliopsoas, adductor magnus, hamstrings (e.g., biceps femoris, semimembranosus, and / or semitendinosus), rectus femoris, and gastrocnemius; and wherein a single unit dose is administered in the affected muscle of a first group (preferably at a selected injection site) and / or multiple unit doses are administered in the affected muscle of a second group (preferably at different selected injection sites), wherein the total dose administered during treatment is up to 300,000 pg; and Here, the modified BoNT / A comprises a BoNT / A light chain and translocation domain and a BoNT / B receptor binding domain (H C domain).

[0037] In another aspect, the invention provides the use of a modified BoNT / A in the manufacture of a medicament for treating childhood limb spasticity, wherein the modified BoNT / A is administered by intramuscular injection into multiple affected muscles of a subject; wherein the modified BoNT / A is administered by a unit dose of greater than 8,500 pg of modified BoNT / A in multiple affected muscles; wherein the plurality of affected muscles is selected from: A first group including: flexor digitorum superficialis, flexor digitorum profundus, flexor carpi radialis, flexor carpi ulnaris, brachioradialis, pronator teres, biceps brachii, medial head of gastrocnemius (gastrocnemius medialis), lateral head of gastrocnemius (gastrocnemius lateralis), flexor digitorum longus, flexor hallucis longus, gastrocnemius, deltoid, levator scapulae, pronator quadratus, flexor hallucis longus, adductor pollicis brevis, palmaris longus, lumbricalis, opponens pollicis, adductor magnus, adductor longus, adductor brevis, gracilis, medial hamstrings, lateral hamstrings, tensor fasciae latae, rectus femoris, vastus lateralis, vastus medialis, vastus intermedius, gluteus maximus, tibialis anterior, flexor digitorum brevis, extensor hallucis longus, and flexor hallucis brevis, and A second group including: triceps brachii (long head), subscapularis, pectoralis major (e.g., pectoralis major), latissimus dorsi, biceps brachii, brachialis, soleus, tibialis posterior, brachioradialis, teres major, iliopsoas, adductor magnus, hamstrings (e.g., biceps femoris, semimembranosus, and / or semitendinosus), rectus femoris, and gastrocnemius; and wherein a single unit dose is administered in the affected muscle of a first group (preferably at a selected injection site) and / or multiple unit doses are administered in the affected muscle of a second group (preferably at different selected injection sites), wherein the total dose administered during treatment is up to 300,000 pg; and Here, the modified BoNT / A comprises a BoNT / A light chain and translocation domain and a BoNT / B receptor binding domain (H C domain).

[0038] In another aspect, the invention provides the use of a modified BoNT / A in the manufacture of a medicament for the treatment of childhood limb spasticity in a subject for a longer period of time than that treated with an unmodified BoNT / A (e.g., SEQ ID NO:2 [e.g., a two-chain form of SEQ ID NO:2]), wherein the modified BoNT / A is administered by intramuscular injection into multiple affected muscles of the subject; wherein the modified BoNT / A is administered by a unit dose of greater than 8,500 pg of modified BoNT / A in multiple affected muscles; wherein the plurality of affected muscles is selected from: A first group including: flexor digitorum superficialis, flexor digitorum profundus, flexor carpi radialis, flexor carpi ulnaris, brachioradialis, pronator teres, biceps brachii, medial head of gastrocnemius (gastrocnemius medialis), lateral head of gastrocnemius (gastrocnemius lateralis), flexor digitorum longus, flexor hallucis longus, gastrocnemius, deltoid, levator scapulae, pronator quadratus, flexor hallucis longus, adductor pollicis brevis, palmaris longus, lumbricalis, opponens pollicis, adductor magnus, adductor longus, adductor brevis, gracilis, medial hamstrings, lateral hamstrings, tensor fasciae latae, rectus femoris, vastus lateralis, vastus medialis, vastus intermedius, gluteus maximus, tibialis anterior, flexor digitorum brevis, extensor hallucis longus, and flexor hallucis brevis, and A second group including: triceps brachii (long head), subscapularis, pectoralis major (e.g., pectoralis major), latissimus dorsi, biceps brachii, brachialis, soleus, tibialis posterior, brachioradialis, teres major, iliopsoas, adductor magnus, hamstrings (e.g., biceps femoris, semimembranosus, and / or semitendinosus), rectus femoris, and gastrocnemius; and wherein a single unit dose is administered in the affected muscle of a first group (preferably at a selected injection site) and / or multiple unit doses are administered in the affected muscle of a second group (preferably at different selected injection sites), wherein the total dose administered during treatment is up to 300,000 pg; Here, the modified BoNT / A comprises a BoNT / A light chain and translocation domain and a BoNT / B receptor binding domain (H C domain).

[0039] The terms "treating a subject's limb spasticity for a longer period than that treated by unmodified BoNT / A" and "treating a subject's pediatric limb spasticity for a longer period than that treated by unmodified BoNT / A" can mean that one or more symptoms of limb spasticity in a subject are reduced for a longer period after administration of the modified BoNT / A of the present invention compared to administration of unmodified BoNT / A. The duration of action can be at least 1.25x, 1.5x, 1.75x, 2.0x or 2.25x greater. The duration of action of modified BoNT / A can be between 6 and 9 months. For example, the duration of action can be at least 4.5 months (from onset), 5.0 months, 5.5 months, 6 months, 6.5 months, 7.0 months, 7.5 months, 8.0 months, 8.5 months or 9.0 months. In certain embodiments, the duration of action can be greater than 9.0 months. The reduction can be determined by comparison with a comparable control subject that is treated with unmodified BoNT / A and shows comparable symptoms. During the period in which the severity of one or more symptoms of the control subject is substantially the same (e.g., the same) as before unmodified BoNT / A treatment, the subject treated with modified BoNT / A according to the present invention can show at least 5%, 10%, 25% or 50% improvement in the comparable one or more symptoms when compared to the severity of one or more symptoms before treatment with modified BoNT / A. Unmodified BoNT / A is preferably SEQ ID NO: 2, which exists in a double-chain form.

[0040] The unit dose for treating pediatric limb spasticity can be more than 8,500 pg of modified BoNT / A. The upper limit of the unit dose range can be 20,000, 19,500, 19,000, 18,500, 18,000, 17,500, 15,000, 12,500, 12,000, 11,000, 10,000 or 9,000 pg of modified BoNT / A, preferably the upper limit is 19,000 pg. The lower limit of the unit dose range can be 8,750, 9,000, 10,000, 11,000, 12,000, 12,500, 13,000, 13,500, 14,000, 14,500, 15,000, 17,500, 18,000, 18,500, 19,000 or 19,500 pg of modified BoNT / A, preferably the lower limit is 8,750 pg, 11,376 pg, 11,377 pg, 11,395 pg, 11,400 pg or 16,591 pg. Preferably, the unit dose of modified BoNT / A is more than 8,500pg, up to 20,000pg of modified BoNT / A, for example, more than 8,500pg, up to 18,000pg, 10,000pg to 19,500pg. The unit dose of modified BoNT / A can be 11,000pg to 19,000pg, preferably 11,500 to 19,000pg or 17,000 to 19,000pg. The unit dose of modified BoNT / A can be 12,000pg to 18,000pg or 12,500pg to 18,000pg. The unit dose can be 12,500pg to 20,000pg of modified BoNT / A. The unit dose of the modified BoNT / A can be 12,000, 12,500, 15,000 or 18,000 pg. In a more preferred embodiment, the unit dose of the modified BoNT / A is 15,000 or 18,000 pg (e.g., 18,000 pg).

[0041] More preferably, the unit dose for treating childhood limb spasticity is 15,000 pg to 20,000 pg, for example, 17,500 to 18,500 pg of modified BoNT / A. More preferably, the unit dose for treating childhood limb spasticity is 18,000 pg of modified BoNT / A.

[0042] It is even more preferable that the unit dose for treating childhood limb spasticity is 10,000 pg to 15,000 pg, for example, 12,000 pg to 13,000 pg of modified BoNT / A. More preferably, the unit dose for treating childhood limb spasticity is 12,500 pg of modified BoNT / A.

[0043] The total dose administered when carrying out the pediatric spasticity treatment regimen of the present invention can be up to 300,000pg.In other words, the total amount of modified BoNT / A administered in a given treatment session can be up to 300,000pg.The total dose can be up to 290,000, 280,000, 270,000, 260,000, 250,000, 240,000, 230,000, 225,000, 220,000, 210,000, 200,000, 190,000, 180,000, 170,000, 160,000, 150,000, 140,000, 130,000, 120,000, 110,000 or 100,000pg. Preferably, the total dose can be up to 270,000 pg of modified BoNT / A. The total dose can be at least 8,750, 10,000, 11,250, 12,500, 13,750, 15,000, 17,500, 18,000, 18,500, 19,000, 19,500 or 20,000 pg. The total dose can be at least 125,000, 127,500, 130,000, 135,000, 140,000, 145,000, 150,000, 160,000, 170,000, 180,000, 190,000, 200,000, 225,000, 250,000, 275,000, 290,000 pg or 287,500 pg. Preferably, the total dose can be greater than 127,500 pg, or greater than 170,924 pg or 170,925 pg, more preferably at least 175,000 pg of modified BoNT / A, e.g., at least 248,850 pg, 248,851 pg or 250,000 pg. The total dose may be 125,000-300,000pg or 127,500-300,000pg, preferably more than 127,500pg, up to 300,000pg, 175,000-300,000pg or 250,000-300,000pg. In a more preferred embodiment, the total dose administered is 260,000-300,000pg, more preferably 175,000-200,000pg. The total dose may be 180,000, 225,000 or 270,000pg, preferably 225,000 or 270,000pg (e.g., 270,000pg).

[0044] More preferably, the total dose for treating pediatric limb spasticity is 225,000 pg to 300,000 pg, for example, 262,500 pg to 277,500 pg of modified BoNT / A. Most preferably, the total dose for treating pediatric limb spasticity is up to 270,000 pg of modified BoNT / A (for example, the total dose can be 270,000 pg).

[0045] More preferably, the total dose for treating pediatric limb spasticity is 150,000pg to 225,000pg, for example, 180,000pg to 195,000pg of modified BoNT / A. Most preferably, the total dose for treating pediatric limb spasticity is up to 187,500pg of modified BoNT / A (for example, the total dose can be 187,500pg).

[0046] Thus, the unit dose for treating pediatric limb spasticity may be more than 8,500 pg of modified BoNT / A, and the total dose administered when carrying out the treatment regimen of the present invention for treating pediatric limb spasticity may be up to 300,000 pg. In a preferred embodiment, the unit dose may be 12,000 pg, and the total dose may be 180,000 pg, or may be up to 180,000 pg. In another preferred embodiment, the unit dose may be 15,000 pg, and the total dose may be 225,000 pg, or may be up to 225,000 pg. In another preferred embodiment, the unit dose may be 18,000 pg, and the total dose may be 270,000 pg, or may be up to 270,000 pg. More preferably, the unit dose may be 12,500 pg and the total dose may be, or may be up to, 187,500 pg.

[0047] The unit dose may be expressed in terms of the amount of the modified BoNT / A of the present invention, in units of the modified BoNT / A, or a combination thereof. Thus, the unit dose of the modified BoNT / A may also be expressed simultaneously in both units and amount (pg or ng, preferably pg).

[0048] In one aspect, the invention provides a modified BoNT / A for use in treating limb spasticity (adult limb spasticity), wherein the modified BoNT / A is administered by intramuscular injection to multiple affected muscles of a subject; Here, the modified BoNT / A is administered in a unit dose of more than 707 units of modified BoNT / A in multiple affected muscles, where one unit is the calculated median lethal dose (LD ) in mice. 50 ) is the amount of modified BoNT / A corresponding to wherein the plurality of affected muscles is selected from: A first group including: flexor digitorum superficialis, flexor digitorum profundus, flexor carpi radialis, flexor carpi ulnaris, brachioradialis, pronator teres, biceps brachii, medial head of gastrocnemius (gastrocnemius medialis), lateral head of gastrocnemius (gastrocnemius lateralis), flexor digitorum longus, flexor hallucis longus, gastrocnemius, deltoid, levator scapulae, pronator quadratus, flexor hallucis longus, adductor pollicis brevis, palmaris longus, lumbricalis, opponens pollicis, adductor magnus, adductor longus, adductor brevis, gracilis, medial hamstrings, lateral hamstrings, tensor fasciae latae, rectus femoris, vastus lateralis, vastus medialis, vastus intermedius, gluteus maximus, tibialis anterior, flexor digitorum brevis, extensor hallucis longus, and flexor hallucis brevis, and A second group including: triceps brachii (long head), subscapularis, pectoralis major (e.g., pectoralis major), latissimus dorsi, biceps brachii, brachialis, soleus, tibialis posterior, brachioradialis, teres major, iliopsoas, adductor magnus, hamstrings (e.g., biceps femoris, semimembranosus, and / or semitendinosus), rectus femoris, and gastrocnemius; and wherein a single unit dose is administered in the affected muscle of a first group (preferably at a selected injection site) and / or multiple unit doses are administered in the affected muscle of a second group (preferably at different selected injection sites), wherein the total dose administered during treatment is up to 24,958 units; and Here, the modified BoNT / A comprises a BoNT / A light chain and translocation domain and a BoNT / B receptor binding domain (H C domain).

[0049] In a related aspect, the invention provides a modified BoNT / A for use in treating limb spasticity (adult limb spasticity) in a subject for a longer period of time than treated with an unmodified BoNT / A (e.g., SEQ ID NO:2 [e.g., a two-chain form of SEQ ID NO:2]), wherein the modified BoNT / A is administered by intramuscular injection into multiple affected muscles of the subject; Here, the modified BoNT / A is administered in a unit dose of more than 707 units of modified BoNT / A in multiple affected muscles, where one unit is the calculated median lethal dose (LD ) in mice. 50 ) is the amount of modified BoNT / A corresponding to wherein the plurality of affected muscles is selected from: A first group including: flexor digitorum superficialis, flexor digitorum profundus, flexor carpi radialis, flexor carpi ulnaris, brachioradialis, pronator teres, biceps brachii, medial head of gastrocnemius (gastrocnemius medialis), lateral head of gastrocnemius (gastrocnemius lateralis), flexor digitorum longus, flexor hallucis longus, gastrocnemius, deltoid, levator scapulae, pronator quadratus, flexor hallucis longus, adductor pollicis brevis, palmaris longus, lumbricalis, opponens pollicis, adductor magnus, adductor longus, adductor brevis, gracilis, medial hamstrings, lateral hamstrings, tensor fasciae latae, rectus femoris, vastus lateralis, vastus medialis, vastus intermedius, gluteus maximus, tibialis anterior, flexor digitorum brevis, extensor hallucis longus, and flexor hallucis brevis, and A second group including: triceps brachii (long head), subscapularis, pectoralis major (e.g., pectoralis major), latissimus dorsi, biceps brachii, brachialis, soleus, tibialis posterior, brachioradialis, teres major, iliopsoas, adductor magnus, hamstrings (e.g., biceps femoris, semimembranosus, and / or semitendinosus), rectus femoris, and gastrocnemius; and wherein a single unit dose is administered in the affected muscle of a first group (preferably at a selected injection site) and / or multiple unit doses are administered in the affected muscle of a second group (preferably at different selected injection sites), wherein the total dose administered during treatment is up to 24,958 units; Here, the modified BoNT / A comprises a BoNT / A light chain and translocation domain and a BoNT / B receptor binding domain (H C domain).

[0050] In a related aspect, the invention provides a method for treating limb spasticity (adult limb spasticity), the method comprising administering a modified BoNT / A by intramuscular injection to multiple affected muscles of a subject, Here, the modified BoNT / A is administered in a unit dose of more than 707 units of modified BoNT / A in multiple affected muscles, where one unit is the calculated median lethal dose (LD ) in mice. 50 ) is the amount of modified BoNT / A corresponding to wherein the plurality of affected muscles is selected from: A first group including: flexor digitorum superficialis, flexor digitorum profundus, flexor carpi radialis, flexor carpi ulnaris, brachioradialis, pronator teres, biceps brachii, medial head of gastrocnemius (gastrocnemius medialis), lateral head of gastrocnemius (gastrocnemius lateralis), flexor digitorum longus, flexor hallucis longus, gastrocnemius, deltoid, levator scapulae, pronator quadratus, flexor hallucis longus, adductor pollicis brevis, palmaris longus, lumbricalis, opponens pollicis, adductor magnus, adductor longus, adductor brevis, gracilis, medial hamstrings, lateral hamstrings, tensor fasciae latae, rectus femoris, vastus lateralis, vastus medialis, vastus intermedius, gluteus maximus, tibialis anterior, flexor digitorum brevis, extensor hallucis longus, and flexor hallucis brevis, and A second group including: triceps brachii (long head), subscapularis, pectoralis major (e.g., pectoralis major), latissimus dorsi, biceps brachii, brachialis, soleus, tibialis posterior, brachioradialis, teres major, iliopsoas, adductor magnus, hamstrings (e.g., biceps femoris, semimembranosus, and / or semitendinosus), rectus femoris, and gastrocnemius; and wherein a single unit dose is administered in the affected muscle of a first group (preferably at a selected injection site) and / or multiple unit doses are administered in the affected muscle of a second group (preferably at different selected injection sites), wherein the total dose administered during treatment is up to 24,958 units; and Here, the modified BoNT / A comprises a BoNT / A light chain and translocation domain and a BoNT / B receptor binding domain (H C domain).

[0051] In a related aspect, the invention provides a method for treating limb spasticity in a subject for a longer period of time than treated with an unmodified BoNT / A (e.g., SEQ ID NO:2 [e.g., a two-chain form of SEQ ID NO:2]), the method comprising administering a modified BoNT / A by intramuscular injection to multiple affected muscles of the subject; Here, the modified BoNT / A is administered in a unit dose of more than 707 units of modified BoNT / A in multiple affected muscles, where one unit is the calculated median lethal dose (LD ) in mice. 50 ) is the amount of modified BoNT / A corresponding to wherein the plurality of affected muscles is selected from: A first group including: flexor digitorum superficialis, flexor digitorum profundus, flexor carpi radialis, flexor carpi ulnaris, brachioradialis, pronator teres, biceps brachii, medial head of gastrocnemius (gastrocnemius medialis), lateral head of gastrocnemius (gastrocnemius lateralis), flexor digitorum longus, flexor hallucis longus, gastrocnemius, deltoid, levator scapulae, pronator quadratus, flexor hallucis longus, adductor pollicis brevis, palmaris longus, lumbricalis, opponens pollicis, adductor magnus, adductor longus, adductor brevis, gracilis, medial hamstrings, lateral hamstrings, tensor fasciae latae, rectus femoris, vastus lateralis, vastus medialis, vastus intermedius, gluteus maximus, tibialis anterior, flexor digitorum brevis, extensor hallucis longus, and flexor hallucis brevis, and A second group including: triceps brachii (long head), subscapularis, pectoralis major (e.g., pectoralis major), latissimus dorsi, biceps brachii, brachialis, soleus, tibialis posterior, brachioradialis, teres major, iliopsoas, adductor magnus, hamstrings (e.g., biceps femoris, semimembranosus, and / or semitendinosus), rectus femoris, and gastrocnemius; and wherein a single unit dose is administered in the affected muscle of a first group (preferably at a selected injection site) and / or multiple unit doses are administered in the affected muscle of a second group (preferably at different selected injection sites), wherein the total dose administered during treatment is up to 24,958 units; and Here, the modified BoNT / A comprises a BoNT / A light chain and translocation domain and a BoNT / B receptor binding domain (H C domain).

[0052] In another related aspect, the invention provides the use of a modified BoNT / A in the manufacture of a medicament for treating limb spasticity (adult limb spasticity), wherein the modified BoNT / A is administered by intramuscular injection into multiple affected muscles of a subject; Here, the modified BoNT / A is administered in a unit dose of more than 707 units of modified BoNT / A in multiple affected muscles, where one unit is the calculated median lethal dose (LD ) in mice. 50 ) is the amount of modified BoNT / A corresponding to wherein the plurality of affected muscles is selected from: A first group including: flexor digitorum superficialis, flexor digitorum profundus, flexor carpi radialis, flexor carpi ulnaris, brachioradialis, pronator teres, biceps brachii, medial head of gastrocnemius (gastrocnemius medialis), lateral head of gastrocnemius (gastrocnemius lateralis), flexor digitorum longus, flexor hallucis longus, gastrocnemius, deltoid, levator scapulae, pronator quadratus, flexor hallucis longus, adductor pollicis brevis, palmaris longus, lumbricalis, opponens pollicis, adductor magnus, adductor longus, adductor brevis, gracilis, medial hamstrings, lateral hamstrings, tensor fasciae latae, rectus femoris, vastus lateralis, vastus medialis, vastus intermedius, gluteus maximus, tibialis anterior, flexor digitorum brevis, extensor hallucis longus, and flexor hallucis brevis, and A second group including: triceps brachii (long head), subscapularis, pectoralis major (e.g., pectoralis major), latissimus dorsi, biceps brachii, brachialis, soleus, tibialis posterior, brachioradialis, teres major, iliopsoas, adductor magnus, hamstrings (e.g., biceps femoris, semimembranosus, and / or semitendinosus), rectus femoris, and gastrocnemius; and wherein a single unit dose is administered in the affected muscle of a first group (preferably at a selected injection site) and / or multiple unit doses are administered in the affected muscle of a second group (preferably at different selected injection sites), wherein the total dose administered during treatment is up to 24,958 units; and Here, the modified BoNT / A comprises a BoNT / A light chain and translocation domain and a BoNT / B receptor binding domain (H C domain).

[0053] In another related aspect, the invention provides the use of a modified BoNT / A in the manufacture of a medicament for treating limb spasticity (adult limb spasticity) in a subject for a longer period of time than treated with an unmodified BoNT / A (e.g., SEQ ID NO:2 [e.g., a two-chain form of SEQ ID NO:2]), wherein the modified BoNT / A is administered by intramuscular injection into multiple affected muscles of the subject; Here, the modified BoNT / A is administered in a unit dose of more than 707 units of modified BoNT / A in multiple affected muscles, where one unit is the calculated median lethal dose (LD ) in mice. 50 ) is the amount of modified BoNT / A corresponding to wherein the plurality of affected muscles is selected from: A first group including: flexor digitorum superficialis, flexor digitorum profundus, flexor carpi radialis, flexor carpi ulnaris, brachioradialis, pronator teres, biceps brachii, medial head of gastrocnemius (gastrocnemius medialis), lateral head of gastrocnemius (gastrocnemius lateralis), flexor digitorum longus, flexor hallucis longus, gastrocnemius, deltoid, levator scapulae, pronator quadratus, flexor hallucis longus, adductor pollicis brevis, palmaris longus, lumbricalis, opponens pollicis, adductor magnus, adductor longus, adductor brevis, gracilis, medial hamstrings, lateral hamstrings, tensor fasciae latae, rectus femoris, vastus lateralis, vastus medialis, vastus intermedius, gluteus maximus, tibialis anterior, flexor digitorum brevis, extensor hallucis longus, and flexor hallucis brevis, and A second group including: triceps brachii (long head), subscapularis, pectoralis major (e.g., pectoralis major), latissimus dorsi, biceps brachii, brachialis, soleus, tibialis posterior, brachioradialis, teres major, iliopsoas, adductor magnus, hamstrings (e.g., biceps femoris, semimembranosus, and / or semitendinosus), rectus femoris, and gastrocnemius; and wherein a single unit dose is administered in the affected muscle of a first group (preferably at a selected injection site) and / or multiple unit doses are administered in the affected muscle of a second group (preferably at different selected injection sites), wherein the total dose administered during treatment is up to 24,958 units; and Here, the modified BoNT / A comprises a BoNT / A light chain and translocation domain and a BoNT / B receptor binding domain (H C domain).

[0054] The unit dose can be greater than 707 units of modified BoNT / A. The upper limit of the unit dose range can be 1664, 1650, 1600, 1550, 1500, 1450, 1400, 1350, 1300, 1250, 1150, 1100, 1050, 1000, 950, 900, 850, 800 or 750 units of modified BoNT / A, preferably the upper limit is 1500 units. The lower limit of the unit dose range may be 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, 1000, 1025, 1050, 1075, 1100, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600 or 1650 units of modified BoNT / A, preferably the lower limit is 725 units, 949 units or 1381 units. Preferably the unit dose of modified BoNT / A is greater than 707 units up to 1664 units of modified BoNT / A, for example, greater than 707 units up to 1,498 units or 832 units to 1622 units. The unit dose of modified BoNT / A can be 915 to 1581 units, preferably 949 to 1581 units or 1414 to 1581 units. The unit dose of modified BoNT / A can be 998 to 1,498 units or 1,040 to 1,498 units. In a preferred embodiment, the unit dose of modified BoNT / A is 998, 1,040, 1,248 or 1,498 units of modified BoNT / A. In a more preferred embodiment, the unit dose is The modified BoNT / A comprises 1,248 or 1,498 units (e.g., 1,498 units). More preferably, the unit dose is 1,248 units to 1,664 units, for example, 1,456 units to 1,539 units of the modified BoNT / A. More preferably, the unit dose is 1,498 units of the modified BoNT / A.

[0055] Even more preferably, the unit dose is between 832 and 1,248 units, for example, between 998 and 1,082 units, of the modified BoNT / A. Most preferably, the unit dose is 1,040 units of the modified BoNT / A.

[0056] The total dose administered when practicing the treatment regimen of the present invention can be up to 24,958 units. In other words, the total amount of modified BoNT / A administered in a given treatment session can be up to 24,958 units. The total dose can be up to 24,500, 24,000, 23,500, 23,000, 22,500, 22,000, 21,500, 21,000, 20,500, 20,000, 19,500, 19,000, 18,500, 18,000, 17,500, 17,000, 16,500, 16,000, 15,500, 15,000, 14,500, 14,000, 13,500, 13,000, 12,500, 12,000, 11,500, 11,000, 10,500, 10,000, 9,500, 9,000 or 8,500 units. Preferably, the total dose can be up to 22,463 units of modified BoNT / A, more preferably 15,599 units. The total dose can be at least 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, 1000, 1,100, 1,200, 1,300, 1,400, 1,500 or 1,600 units. The total dose can be at least 10,000, 11,000, 12,000, 13,000, 14,000, 15,000, 16,000, 17,000, 18,000, 19,000, 20,000, 21,000, 22,000, 23,000, 24,000 or 24,500 units. Preferably, the total dose is more than 10,607 units, or more than 14,220 units, more preferably at least 14,559 units of modified BoNT / A, for example at least 20,703 units or 20,799 units. The total dose can be 10,339 to 24,958 units, 10,399 to 24,958 units, or 10,607 to 24,958 units, preferably more than 10,607 units, up to 24,958 units, 14,559 to 24,958 units, or 20,799 to 24,958 units. More preferably, the total dose administered is 21,631 to 24,958 units, more preferably 14,559 to 16,639 units. The total dose may be 14,975 units, 18,719 units or 22,463 units, preferably 18,719 or 22,463 units (e.g. 22,463 units).

[0057] More preferably, the total dose is between 18,719 and 24,958 units, for example, between 21,839 and 23,087 units, of the modified BoNT / A. Most preferably, the total dose is up to 22,463 units of the modified BoNT / A (for example, the total dose can be 22,463 units).

[0058] Even more preferably, the total dose is between 12,479 and 18,719 units, for example, between 14,975 and 16,223 units, of the modified BoNT / A. Most preferably, the total dose is up to 15,599 units of the modified BoNT / A (for example, the total dose can be 15,599 units).

[0059] Thus, the unit dose may be greater than 707 units of modified BoNT / A, and the total dose administered when implementing the treatment regimen of the present invention may be up to 24,958 units. In a preferred embodiment, the unit dose may be 998 units of modified BoNT / A, and the total dose may be 14,975 units, or may be up to 14,975 units. In another preferred embodiment, the unit dose may be 1248 units of modified BoNT / A, and the total dose may be 18,719 units, or may be up to 18,719 units. In another preferred embodiment, the unit dose may be 1498 units of modified BoNT / A, and the total dose may be 22,463 units, or may be up to 22,463 units. More preferably, the unit dose may be 1,040 units of modified BoNT / A, and the total dose may be 15,599 units, or may be up to 15,599 units.

[0060] In one aspect, the invention provides a modified BoNT / A for use in treating childhood limb spasticity, wherein the modified BoNT / A is administered by intramuscular injection to a plurality of affected muscles of a subject, wherein the modified BoNT / A is administered by a unit dose of greater than 353.5 units of modified BoNT / A in the plurality of affected muscles, wherein one unit is greater than or equal to the calculated median lethal dose in mice (LD 50 ) is the amount of modified BoNT / A corresponding to wherein the plurality of affected muscles is selected from: a first group including: flexor digitorum superficialis, flexor digitorum profundus, flexor carpi radialis, flexor carpi ulnaris, brachioradialis, pronator teres, biceps brachii, medial head of gastrocnemius (gastrocnemius medialis), lateral head of gastrocnemius (gastrocnemius lateralis), flexor digitorum longus, flexor hallucis longus, gastrocnemius, deltoid, levator scapulae, pronator quadratus, flexor hallucis longus, adductor pollicis brevis, palmaris longus, lumbricalis, opponens pollicis, adductor magnus, adductor longus, adductor brevis, gracilis, medial hamstrings, lateral hamstrings, tensor fasciae latae, rectus femoris, vastus lateralis, vastus medialis, vastus intermedius, gluteus maximus, tibialis anterior, flexor digitorum brevis, extensor hallucis longus, and flexor hallucis brevis; and A second group including: triceps brachii (long head), subscapularis, pectoralis major (e.g., pectoralis major), latissimus dorsi, biceps brachii, brachialis, soleus, tibialis posterior, brachioradialis, teres major, iliopsoas, adductor magnus, hamstrings (e.g., biceps femoris, semimembranosus, and / or semitendinosus), rectus femoris, and gastrocnemius; and wherein a single unit dose is administered in the affected muscle of a first group (preferably at a selected injection site) and / or multiple unit doses are administered in the affected muscle of a second group (preferably at different selected injection sites), wherein the total dose administered during treatment is up to 12,479 units; and Here, the modified BoNT / A comprises a BoNT / A light chain and translocation domain and a BoNT / B receptor binding domain (H C domain).

[0061] In another related aspect, the invention provides a modified BoNT / A for use in treating childhood limb spasticity in a subject for a longer period of time than treated with an unmodified BoNT / A (e.g., SEQ ID NO:2 [e.g., a two-chain form of SEQ ID NO:2]), wherein the modified BoNT / A is administered by intramuscular injection into multiple affected muscles of the subject; Here, the modified BoNT / A is administered in a unit dose of more than 353.5 units of modified BoNT / A in multiple affected muscles, where one unit is the calculated median lethal dose (LD ) in mice. 50 ) is the amount of modified BoNT / A corresponding to wherein the plurality of affected muscles is selected from: A first group including: flexor digitorum superficialis, flexor digitorum profundus, flexor carpi radialis, flexor carpi ulnaris, brachioradialis, pronator teres, biceps brachii, medial head of gastrocnemius (gastrocnemius medialis), lateral head of gastrocnemius (gastrocnemius lateralis), flexor digitorum longus, flexor hallucis longus, gastrocnemius, deltoid, levator scapulae, pronator quadratus, flexor hallucis longus, adductor pollicis brevis, palmaris longus, lumbricalis, opponens pollicis, adductor magnus, adductor longus, adductor brevis, gracilis, medial hamstrings, lateral hamstrings, tensor fasciae latae, rectus femoris, vastus lateralis, vastus medialis, vastus intermedius, gluteus maximus, tibialis anterior, flexor digitorum brevis, extensor hallucis longus, and flexor hallucis brevis, and A second group including: triceps brachii (long head), subscapularis, pectoralis major (e.g., pectoralis major), latissimus dorsi, biceps brachii, brachialis, soleus, tibialis posterior, brachioradialis, teres major, iliopsoas, adductor magnus, hamstrings (e.g., biceps femoris, semimembranosus, and / or semitendinosus), rectus femoris, and gastrocnemius; and wherein a single unit dose is administered in the affected muscle of a first group (preferably at a selected injection site) and / or multiple unit doses are administered in the affected muscle of a second group (preferably at different selected injection sites), wherein the total dose administered during treatment is up to 12,479 units; and Here, the modified BoNT / A comprises a BoNT / A light chain and translocation domain and a BoNT / B receptor binding domain (H C domain).

[0062] In a related aspect, the invention provides a method of treating infantile limb spasticity, the method comprising administering a modified BoNT / A by intramuscular injection to multiple affected muscles of a subject, Here, the modified BoNT / A is administered in a unit dose of more than 353.5 units of modified BoNT / A in multiple affected muscles, where one unit is the calculated median lethal dose (LD ) in mice. 50 ) is the amount of modified BoNT / A corresponding to wherein the plurality of affected muscles is selected from: A first group including: flexor digitorum superficialis, flexor digitorum profundus, flexor carpi radialis, flexor carpi ulnaris, brachioradialis, pronator teres, biceps brachii, medial head of gastrocnemius (gastrocnemius medialis), lateral head of gastrocnemius (gastrocnemius lateralis), flexor digitorum longus, flexor hallucis longus, gastrocnemius, deltoid, levator scapulae, pronator quadratus, flexor hallucis longus, adductor pollicis brevis, palmaris longus, lumbricalis, opponens pollicis, adductor magnus, adductor longus, adductor brevis, gracilis, medial hamstrings, lateral hamstrings, tensor fasciae latae, rectus femoris, vastus lateralis, vastus medialis, vastus intermedius, gluteus maximus, tibialis anterior, flexor digitorum brevis, extensor hallucis longus, and flexor hallucis brevis, and A second group including: triceps brachii (long head), subscapularis, pectoralis major (e.g., pectoralis major), latissimus dorsi, biceps brachii, brachialis, soleus, tibialis posterior, brachioradialis, teres major, iliopsoas, adductor magnus, hamstrings (e.g., biceps femoris, semimembranosus, and / or semitendinosus), rectus femoris, and gastrocnemius; and wherein a single unit dose is administered in the affected muscle of a first group (preferably at a selected injection site) and / or multiple unit doses are administered in the affected muscle of a second group (preferably at different selected injection sites), wherein the total dose administered during treatment is up to 12,479 units; and Here, the modified BoNT / A comprises a BoNT / A light chain and translocation domain and a BoNT / B receptor binding domain (H C domain).

[0063] In a related aspect, the invention provides a method of treating childhood limb spasticity in a subject for a longer period of time than treated with an unmodified BoNT / A (e.g., SEQ ID NO:2 [e.g., a two-chain form of SEQ ID NO:2]), the method comprising administering a modified BoNT / A by intramuscular injection to multiple affected muscles of the subject; Here, the modified BoNT / A is administered in a unit dose of more than 353.5 units of modified BoNT / A in multiple affected muscles, where one unit is the calculated median lethal dose (LD ) in mice. 50 ) is the amount of modified BoNT / A corresponding to wherein the plurality of affected muscles is selected from: A first group including: flexor digitorum superficialis, flexor digitorum profundus, flexor carpi radialis, flexor carpi ulnaris, brachioradialis, pronator teres, biceps brachii, medial head of gastrocnemius (gastrocnemius medialis), lateral head of gastrocnemius (gastrocnemius lateralis), flexor digitorum longus, flexor hallucis longus, gastrocnemius, deltoid, levator scapulae, pronator quadratus, flexor hallucis longus, adductor pollicis brevis, palmaris longus, lumbricalis, opponens pollicis, adductor magnus, adductor longus, adductor brevis, gracilis, medial hamstrings, lateral hamstrings, tensor fasciae latae, rectus femoris, vastus lateralis, vastus medialis, vastus intermedius, gluteus maximus, tibialis anterior, flexor digitorum brevis, extensor hallucis longus, and flexor hallucis brevis, and A second group including: triceps brachii (long head), subscapularis, pectoralis major (e.g., pectoralis major), latissimus dorsi, biceps brachii, brachialis, soleus, tibialis posterior, brachioradialis, teres major, iliopsoas, adductor magnus, hamstrings (e.g., biceps femoris, semimembranosus, and / or semitendinosus), rectus femoris, and gastrocnemius; and wherein a single unit dose is administered in the affected muscle of a first group (preferably at a selected injection site) and / or multiple unit doses are administered in the affected muscle of a second group (preferably at different selected injection sites), wherein the total dose administered during treatment is up to 12,479 units; and Here, the modified BoNT / A comprises a BoNT / A light chain and translocation domain and a BoNT / B receptor binding domain (H C domain).

[0064] In another related aspect, the invention provides the use of a modified BoNT / A in the manufacture of a medicament for treating infantile limb spasticity, wherein the modified BoNT / A is administered by intramuscular injection to multiple affected muscles of a subject; Here, the modified BoNT / A is administered in a unit dose of more than 353.5 units of modified BoNT / A in multiple affected muscles, where one unit is the calculated median lethal dose (LD ) in mice. 50 ) is the amount of modified BoNT / A corresponding to wherein the plurality of affected muscles is selected from: A first group including: flexor digitorum superficialis, flexor digitorum profundus, flexor carpi radialis, flexor carpi ulnaris, brachioradialis, pronator teres, biceps brachii, medial head of gastrocnemius (gastrocnemius medialis), lateral head of gastrocnemius (gastrocnemius lateralis), flexor digitorum longus, flexor hallucis longus, gastrocnemius, deltoid, levator scapulae, pronator quadratus, flexor hallucis longus, adductor pollicis brevis, palmaris longus, lumbricalis, opponens pollicis, adductor magnus, adductor longus, adductor brevis, gracilis, medial hamstrings, lateral hamstrings, tensor fasciae latae, rectus femoris, vastus lateralis, vastus medialis, vastus intermedius, gluteus maximus, tibialis anterior, flexor digitorum brevis, extensor hallucis longus, and flexor hallucis brevis, and A second group including: triceps brachii (long head), subscapularis, pectoralis major (e.g., pectoralis major), latissimus dorsi, biceps brachii, brachialis, soleus, tibialis posterior, brachioradialis, teres major, iliopsoas, adductor magnus, hamstrings (e.g., biceps femoris, semimembranosus, and / or semitendinosus), rectus femoris, and gastrocnemius; and wherein a single unit dose is administered in the affected muscle of a first group (preferably at a selected injection site) and / or multiple unit doses are administered in the affected muscle of a second group (preferably at different selected injection sites), wherein the total dose administered during treatment is up to 12,479 units; and Here, the modified BoNT / A comprises a BoNT / A light chain and translocation domain and a BoNT / B receptor binding domain (H C domain).

[0065] In another related aspect, the invention provides the use of a modified BoNT / A in the manufacture of a medicament for treating childhood limb spasticity in a subject for a longer period of time than treated with an unmodified BoNT / A (e.g., SEQ ID NO:2 [e.g., a two-chain form of SEQ ID NO:2]), wherein the modified BoNT / A is administered by intramuscular injection into a plurality of affected muscles of the subject; Here, the modified BoNT / A is administered in a unit dose of more than 353.5 units of modified BoNT / A in multiple affected muscles, where one unit is the calculated median lethal dose (LD ) in mice. 50 ) is the amount of modified BoNT / A corresponding to wherein the plurality of affected muscles is selected from: A first group including: flexor digitorum superficialis, flexor digitorum profundus, flexor carpi radialis, flexor carpi ulnaris, brachioradialis, pronator teres, biceps brachii, medial head of gastrocnemius (gastrocnemius medialis), lateral head of gastrocnemius (gastrocnemius lateralis), flexor digitorum longus, flexor hallucis longus, gastrocnemius, deltoid, levator scapulae, pronator quadratus, flexor hallucis longus, adductor pollicis brevis, palmaris longus, lumbricalis, opponens pollicis, adductor magnus, adductor longus, adductor brevis, gracilis, medial hamstrings, lateral hamstrings, tensor fasciae latae, rectus femoris, vastus lateralis, vastus medialis, vastus intermedius, gluteus maximus, tibialis anterior, flexor digitorum brevis, extensor hallucis longus, and flexor hallucis brevis, and A second group including: triceps brachii (long head), subscapularis, pectoralis major (e.g., pectoralis major), latissimus dorsi, biceps brachii, brachialis, soleus, tibialis posterior, brachioradialis, teres major, iliopsoas, adductor magnus, hamstrings (e.g., biceps femoris, semimembranosus, and / or semitendinosus), rectus femoris, and gastrocnemius; and wherein a single unit dose is administered in the affected muscle of a first group (preferably at a selected injection site) and / or multiple unit doses are administered in the affected muscle of a second group (preferably at different selected injection sites), wherein the total dose administered during treatment is up to 12,479 units; and Here, the modified BoNT / A comprises a BoNT / A light chain and translocation domain and a BoNT / B receptor binding domain (H C domain).

[0066] A suitable unit dose for treating pediatric limb spasticity may be greater than 353.5 units of modified BoNT / A. The upper end of the unit dose range for treating pediatric limb spasticity may be 832, 825, 800, 775, 750, 725, 700, 675, 650, 625, 600, 575, 550, 525, 500, 475, 450 or 425 units of modified BoNT / A, preferably the upper end is 790 units. The lower limit of the unit dose range for treating childhood limb spasticity may be 364, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825 or 830 units of modified BoNT / A, preferably the lower limit is 364 units, 475 units or 690 units. Preferably, the unit dose of modified BoNT / A for treating childhood limb spasticity is greater than 353.5 units, up to 832 units of modified BoNT / A, for example, greater than 353.5 units, up to 749 units or 416 units to 811 units. The unit dose of modified BoNT / A for treating childhood limb spasticity can be 458 units to 790 units or 458 units to 791 units, preferably 475 units to 790 units, 475 units to 791 units, 707 units to 790 units, or 707 units to 791 units. In preferred embodiments, the unit dose of modified BoNT / A is 499, 520, 624 or 749 units. In more preferred embodiments, the unit dose of modified BoNT / A is 624 or 749 units (e.g., 749 units).

[0067] More preferably, the unit dose for treating childhood spasticity is 624 units to 832 units, for example, 728 units to 770 units of modified BoNT / A. More preferably, the unit dose for treating childhood spasticity is 749 units of modified BoNT / A.

[0068] Even more preferably, the unit dose for treating childhood spasticity is 416 units to 624 units, for example, 499 units to 541 units, of modified BoNT / A. Most preferably, the unit dose for treating childhood spasticity is 520 units of modified BoNT / A.

[0069] The total dose administered when carrying out the treatment regimen of the present invention to treat pediatric limb spasticity can be up to 12,479 units. In other words, the total amount of modified BoNT / A administered in a given treatment session to treat pediatric limb spasticity can be up to 12,479 units. The total dose for treating pediatric limb spasticity can be up to 12,250, 12,000, 11,750, 11,500, 11,250, 11,000, 10,750, 10,500, 10,000, 9,750, 9,500, 9,250, 9,000, 8,750, 8,500, 8,250, 8,000, 7,750, 7,500, 7,250, 7,000, 6,750, 6,500, 6,250, 6,000, 5,750, 5,500, 5,250, 5,000, 4,750, 4,500 or 4,250 units. The total dose for treating pediatric limb spasticity can be up to 11,250 units of modified BoNT / A. Preferably, the total dose for treating pediatric limb spasticity can be up to 11,232 units of modified BoNT / A, more preferably 7,800 units. The total dose for treating pediatric limb spasticity can be at least 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825 or 850 units. The total dose can be at least 5,000, 5,500, 6,000, 6,500, 7,000, 7,500, 8,000, 8,500, 9,000, 9,500, 10,000, 10,500, 11,000 or 11,500 units. Preferably, the total dose for treating pediatric limb spasticity may be at least 5,304 units or more than 7,110 units, more preferably at least 7,280 units of modified BoNT / A, for example at least 10,351 units or 10,400 units. The total dose for treating pediatric limb spasticity may be 5,200 to 12,479 units or 5,304 to 12,479 units, preferably more than 5,200 units and up to 12,479 units, more than 5,301 units and up to 12,479 units, 7,280 to 12,479 units or 10,400 to 12,479 units.In a more preferred embodiment, the total dose administered is 10,815 to 12,479 units or 10,816 to 12,479 units, more preferably 7,280 to 8,320 units. The total dose may be 7,488, 9,360, 9,359, 11,231 or 11,232 units. In a more preferred embodiment, the total dose is 9,359 or 11,231 units (e.g., 11,231 units).

[0070] More preferably, the total dose for treating pediatric limb spasticity is 9,360 units to 12,479 units, for example, 10,920 units to 11,544 units of modified BoNT / A. Most preferably, the total dose for treating pediatric limb spasticity is up to 11,232 units of modified BoNT / A (for example, the total dose can be 11,232 units).

[0071] Even more preferably, the total dose for treating pediatric limb spasticity is 6,240 units to 9,360 units, for example, 7,488 units to 8,112 units of modified BoNT / A. Most preferably, the total dose for treating pediatric limb spasticity is up to 7,800 units of modified BoNT / A (for example, the total dose can be 7,800 units).

[0072] Thus, the unit dose for treating pediatric limb spasticity may be more than 353.5 units of modified BoNT / A, and the total dose administered when carrying out the treatment regimen of the present invention for treating pediatric limb spasticity may be up to 12,479 units. In a preferred embodiment, the unit dose may be 499 units of modified BoNT / A, and the total dose may be 7,488 units, or may be up to 7,488 units. In another preferred embodiment, the unit dose may be 624 units of modified BoNT / A, and the total dose may be 9,359 units or 9,360 units, or may be up to 9,359 units, or may be up to 9,360 units. In another preferred embodiment, the unit dose may be 749 units of modified BoNT / A, and the total dose may be 11,231 units, or may be up to 11,231 units. In another preferred embodiment, the unit dose may be 749 units of the modified BoNT / A and the total dose may be, or may be up to, 11,232 units. Most preferably, the unit dose may be 520 units and the total dose may be, or may be up to, 7,800 units.

[0073] An "affected muscle" is a muscle that exhibits symptoms of spasticity or contributes to spasticity of an affected limb. For example, the muscle may exhibit increased muscle tone or stiffness.

[0074] The multiple affected muscles are selected from the first and second groups as described herein.The multiple affected muscles selected can be at least one muscle of the first group and / or at least one muscle of the second group.Alternatively, the multiple affected muscles can be two or more muscles from the same group (e.g., two or more muscles of the first group or two or more muscles of the second group).Preferably, the multiple affected muscles include at least one muscle of the first group and at least one muscle of the second group.

[0075] The multiple muscles can be the muscles of the same limb or the muscles of different limbs.However, it is preferred that the multiple muscles are the muscles of the same limb.The present invention encompasses treating spasticity in one or more limbs simultaneously.For example, modified BoNT / A may be administered to one or both upper limbs of a subject per treatment session, one or both lower limbs of a subject per treatment session, or a combination of lower limbs and upper limbs per treatment session.Regardless of whether two or more limbs are treated, it is preferred that at least two muscles are treated per limb, for example, at least three, four or five muscles are treated per limb.

[0076] The potency of modified BoNT / A for use according to the present invention was determined using mouse LD 50 In the assay, one unit corresponds to a calculated median lethal dose (LD) in mice. 50 ) preferably the calculated median lethal intraperitoneal dose in mice.

[0077] In particular, the amount of modified BoNT / A corresponding to 1 unit in the assay is preferably 24.04 pg.

[0078] Preferably, the dose of modified BoNT / A is administered by intramuscular injection at the affected muscle. More preferably, a single unit dose is administered per injection site. The term "single unit dose is administered" means that substantially all of the single unit dose is administered. For example, a remaining amount of the unit dose (e.g., up to 1%, 0.1% or 0.01%) may remain in the vial in which the modified BoNT / A was reconstituted. However, preferably, all of the single unit dose is administered (e.g., at one or more injection sites). Depending on the nature of the muscle, a single unit dose is administered (i.e., at a muscle selected from the first group described herein) or multiple unit doses are administered (i.e., at a muscle selected from the second group described herein). The single unit dose or multiple unit doses may be administered at one or more injection sites (e.g., per muscle). For example, in some embodiments, less than a single unit dose may be administered per injection site. In preferred embodiments, some muscles are injected at only one site (i.e., muscles selected from the first group described herein) and some muscles are injected at two or more sites (i.e., muscles selected from the second group described herein).

[0079] In one embodiment, a single unit dose is administered at multiple injection sites in a first group of affected muscles and / or multiple unit doses are administered at multiple injection sites in a second group of affected muscles.

[0080] The term "up to" when used in reference to a value (e.g., up to 600,000 pg) means up to and including the recited value. Thus, as an example, reference to administering "up to 600,000 pg" of modified BoNT / A encompasses administration of 600,000 pg of modified BoNT / A as well as administration of less than 600,000 pg of modified BoNT / A.

[0081] A single unit dose of the modified BoNT / A is administered to a muscle selected from the first group described herein, when said first group muscle is selected for treatment.

[0082] Multiple unit doses of the modified BoNT / A are administered to the muscle selected from the second group (e.g., when the muscle of said second group is selected for treatment). For example, at least 2x, 3x or 4x unit doses may be administered. In some embodiments, 2-4x unit doses may be administered. Preferably, 2x unit doses of the modified BoNT / A are administered to the muscle selected from the second group.

[0083] Some muscles may be in a first muscle group and a second muscle group, and the clinician can then decide whether to administer a single unit dose to said muscles or multiple unit doses.

[0084] Limb spasticity can be upper limb spasticity or lower limb spasticity.

[0085] When treating lower limb spasticity, the modified BoNT / A can be administered to multiple muscles selected from the following: A first group including (preferably consisting of): medial head of gastrocnemius, lateral head of gastrocnemius, gastrocnemius, flexor digitorum longus, flexor hallucis longus, adductor magnus, adductor longus, adductor brevis, gracilis, medial hamstrings, lateral hamstrings, tensor fasciae latae, rectus femoris, vastus lateralis, vastus medialis, vastus intermedius, gluteus maximus, tibialis anterior, flexor digitorum brevis, extensor hallucis longus and flexor hallucis brevis; A second group including (preferably consisting of): soleus, tibialis posterior, iliopsoas and gastrocnemius.

[0086] When administered into the flexor hallucis longus, in some cases a single unit dose may be administered, while in other cases 2x the unit dose may be administered.

[0087] The appropriate dosages for different leg muscles are shown below: [Table A]

[0088] The modified BoNT / A can be administered into one or more of the muscles indicated in the doses indicated in the table above.

[0089] The modified BoNT / A may be administered in the following doses into one or more of the following muscles: [Table B]

[0090] Preferably, when treating lower limb spasticity, the modified BoNT / A can be administered to multiple muscles selected from the following: A first group including (preferably consisting of): medial head of gastrocnemius (also known as medial gastrocnemius), lateral head of gastrocnemius (also known as lateral gastrocnemius), tibialis anterior, flexor digitorum longus, flexor digitorum brevis, flexor hallucis longus, flexor hallucis brevis, gracilis, and gluteus maximus; and A second group including (preferably consisting of): soleus, tibialis posterior, rectus femoris, hamstring muscles (eg, biceps femoris, semimembranosus and / or semitendinosus) and adductor magnus.

[0091] The hamstring muscles can be the biceps femoris, semimembranosus, and / or semitendinosus. For example, modified BoNT / A can be administered to one or more (e.g., all) of the biceps femoris, semimembranosus, and / or semitendinosus.

[0092] Preferably, 3 unit doses are administered to the soleus muscle. Preferably, 2 unit doses are administered to the tibialis posterior muscle. Preferably, 2 unit doses are administered to the rectus femoris muscle. Preferably, 2 unit doses are administered to the hamstring muscle, e.g., 2 units may be administered to the biceps femoris muscle, 2 units may be administered to the semimembranosus muscle, and / or 2 units may be administered to the semitendinosus muscle). Preferably, 2 unit doses are administered to the adductor magnus muscle.

[0093] The modified BoNT / A may be administered in the following doses into one or more of the following muscles: [Table C]

[0094] Preferably, the limb spasticity is upper limb spasticity.

[0095] When treating upper limb spasticity, the modified BoNT / A can be administered to multiple affected muscles selected from the following: A first group including (preferably consisting of) the following: flexor digitorum superficialis, flexor digitorum profundus, flexor carpi radialis, flexor carpi ulnaris, brachioradialis, pronator teres, deltoid, levator scapulae, pronator quadratus, flexor pollicis longus, adductor pollicis, flexor pollicis brevis, palmaris longus, lumbricalis, and opponens pollicis; and A second group including (preferably consisting of): triceps brachii (long head), subscapularis, pectoral muscles (eg, pectoralis major), latissimus dorsi, biceps brachii, brachialis, brachioradialis and teres major.

[0096] Preferably, when treating upper limb spasticity, the modified BoNT / A can be administered to multiple affected muscles selected from the following: A first group including (preferably consisting of): flexor digitorum superficialis, flexor digitorum profundus, flexor carpi radialis, flexor carpi ulnaris, brachioradialis, pronator teres, and biceps brachii; A second group including (preferably consisting of): triceps brachii (long head), subscapularis, pectoralis major, latissimus dorsi, biceps brachii and brachialis.

[0097] For example, preferably, when treating upper limb spasticity, the modified BoNT / A can be administered to multiple affected muscles selected from the following: A first group including (preferably consisting of): flexor digitorum superficialis, flexor digitorum profundus, flexor carpi radialis, flexor carpi ulnaris and brachioradialis; and A second group including (preferably consisting of): brachialis, biceps, triceps (long head), subscapularis, chest muscles (eg, pectoralis major) and latissimus dorsi.

[0098] More preferably, modified BoNT / A can be administered to muscles associated with shoulder spasticity, for example, adducted shoulder.Modified BoNT / A can be administered to at least one of latissimus dorsi, subscapularis, pectoralis major and triceps brachii (long head).For example, modified BoNT / A can be administered to at least two or three (preferably all) of latissimus dorsi, subscapularis, pectoralis major or triceps brachii (long head).

[0099] When administering to the biceps, in some cases, a single unit dose may be administered, while in other cases, a 2x unit dose may be administered.Therefore, the biceps is included in both the first and second group of muscles.Preferably, the 2x unit dose is administered to the biceps, which is included in the second group of muscles.

[0100] The appropriate dosages for different upper extremity muscles are shown below: [Table D]

[0101] Preferably, the modified BoNT / A may be administered to one or more of the muscles indicated in the table above at the dosages indicated. In some cases, when treating limb spasticity, the pronator teres muscle may be omitted.

[0102] Therefore, preferably, suitable dosages by upper limb muscle are given below: [Table E]

[0103] The modified BoNT / A may be administered in the following doses into one or more of the following muscles: [Table F]

[0104] To treat "clenched fist", a clinical symptom of upper limb spasticity, modified BoNT / A can be administered to flexor digitorum superficialis and / or flexor digitorum profundus. To treat "flexed wrist", a clinical symptom of upper limb spasticity, modified BoNT / A can be administered to flexor carpi radialis and / or flexor carpi ulnaris. To treat "flexed elbow", a clinical symptom of upper limb spasticity, modified BoNT / A can be administered to brachioradialis, brachialis and / or biceps brachii. To treat "adducted shoulder", a clinical symptom of upper limb spasticity, modified BoNT / A can be administered to triceps brachii (long head), subscapularis, pectoralis major and / or latissimus dorsi.

[0105] Particularly preferably, in the treatment of upper limb spasticity, at least the clinical symptoms of "flexed elbow" and "adducted shoulder" are treated. More preferably, in the treatment of upper limb spasticity, the clinical symptoms of "flexed elbow" and "adducted shoulder" are treated, and either "clenched fist" or "flexed wrist" (preferably only one of them) is treated.

[0106] The total number of unit doses administered in a given treatment can be up to 15x unit doses. In other words, in one embodiment, 15x single unit doses can be administered at 15x injection sites. In another embodiment, 15x single unit doses can be administered at more than 15x injection sites. The total number of unit doses can be divided according to the muscle being treated, for example, 2x unit doses can be administered to the latissimus dorsi, 2x to the subscapularis, 2x to the pectoralis major, and 1x to the flexor carpi ulnaris, with the sum of the multiple administered unit doses being 7x. For example, the total number of unit doses administered can be up to 14x, 13x, 12x, 11x, 10x, 9x, 8x, or 7x. The total number of unit doses administered can be at least 2x, 3x, 4x, 5x, 6x, 7x unit doses, preferably at least 2x. The total number of unit doses administered can be 2x to 15x, 7x to 15x, or 10x to 14x. Preferably, the number of unit doses administered is 15x.

[0107] One of skill in the art will take into consideration cases where the subject has recently undergone (or has subsequently undergone) additional treatment with a Clostridial neurotoxin (e.g., unmodified BoNT), for example as part of a cosmetic treatment or treatment for a different indication, and will adapt the current treatment regimen accordingly, using routine techniques in the art.

[0108] The limb spasticity for treatment according to the present invention is preferably adult limb spasticity.Preferably, the dosage details provided above are for treating adult limb spasticity (unless the dosage details provided indicate that they are for treating child limb spasticity).However, treatment of child limb spasticity is also included.When treating child limb spasticity, the combined total dosage is usually 50% or less of that used when treating adult limb spasticity.In some embodiments, the combined total dosage is usually 70% or less (e.g., 67% or less) of that used when treating adult limb spasticity.

[0109] The total number of unit doses administered in a given treatment for treating pediatric limb spasticity can be up to 10x unit doses. In other words, in one embodiment, a single unit dose of 10x can be administered at 10x injection sites. However, the total number of unit doses administered in a given treatment for treating pediatric limb spasticity can be up to 15x unit doses. In another embodiment, a single unit dose of 10x can be administered at more than 10x injection sites. The total number of unit doses is divided according to the muscle being treated, for example, 2x unit doses are administered to the latissimus dorsi, 2x to the subscapularis, 2x to the pectoralis major, and 1x to the flexor carpi ulnaris, with the sum of the multiple administered unit doses being 7x. For example, the total number of unit doses administered can be up to 9x, 8x, 7x, 6x, 5x, 4x, or 3x. The total number of unit doses administered can be at least 2x, 3x, 4x, 5x, 6x, 7x unit doses, preferably at least 2x. The total number of unit doses administered can be 2x to 10x, 7x to 10x, or 4x to 8x. Preferably, the number of unit doses administered is 10x. Preferably, the number of unit doses administered is 15x.

[0110] The modified BoNT / A of the present invention preferably has a longer duration of action (e.g., at least 5%, 10%, 25% or 50% improvement in one or more symptoms) when compared to unmodified BoNT / A (e.g., Dysport®). The duration of action may be at least 1.25×, 1.5×, 1.75×, 2.0× or 2.25× greater. The duration of action of the modified BoNT / A may be between 6 and 9 months. For example, the duration of action may be at least 4.5 months (from onset), 5.0 months, 5.5 months, 6 months, 6.5 months, 7.0 months, 7.5 months, 8.0 months, 8.5 months or 9.0 months. In certain embodiments, the duration of action may be longer than 9.0 months.

[0111] Administration to multiple muscles according to the present invention preferably occurs in the same treatment session.

[0112] Treatment may be repeated at an appropriate time period after administration of modified BoNT / A. Given that the duration of action is approximately twice that of unmodified BoNT / A (e.g., Dysport®), it is appropriate to allow a longer period between subsequent administrations than when the subject is treated with unmodified BoNT / A (e.g., Dysport®). The subject may be re-administered modified BoNT / A according to the present invention at least 18, 20, 25 or 30 weeks after the previous administration. For example, the subject may be re-administered modified BoNT / A according to the present invention at least 18 to 45 weeks, preferably 20 to 35 weeks, after the previous administration.

[0113] A "subject" as used herein may be a mammal, for example, a human or other mammal. Preferably, a "subject" refers to a human subject. A "subject" is preferably an adult subject, i.e., a subject at least 18 years old. The terms "subject" and "patient" are used synonymously herein. When treating pediatric limb spasticity, the subject is less than 18 years old, for example, less than 15, 10 or 5 years old, for example, 1-17, 5-17 or 10-17 years old. Preferably, the subject has been diagnosed with limb spasticity. The subject may have been diagnosed with spastic hemiparesis. Limb spasticity may occur after (e.g., caused by) stroke or traumatic brain injury.

[0114] A person skilled in the art will understand that when the limb spasticity is adult limb spasticity, the subject is an adult subject, and when the limb spasticity is childhood limb spasticity, the subject is a childhood subject.

[0115] The subject of the treatment according to the present invention may be a subject that is unsuitable for treatment with unmodified BoNT / A and / or with another Clostridial neurotoxin. The subject may be a subject that is resistant to treatment with unmodified BoNT / A and / or with another Clostridial neurotoxin. Resistance may occur due to the development of an immune response against the Clostridial neurotoxin, including the production of anti-Clostridial neurotoxin antibodies by the subject.

[0116] Dosages (eg, unit doses and total doses) described herein are for adult limb spasticity unless otherwise specified.

[0117] The term "treat" or "treating" as used herein encompasses prophylactic treatment (e.g., to prevent the occurrence of a disorder) as well as corrective treatment (treatment of a subject already suffering from a disorder). Preferably, "treat" or "treating" as used herein means corrective treatment. The term "treat" or "treating" as used herein refers to a disorder and / or its symptoms.

[0118] Suitable modified BoNT / A polypeptides (and nucleotide sequences encoding them, if any) are described in WO2015 / 004461 A1 and WO2017 / 191315, both of which are incorporated by reference in their entireties.

[0119] BoNT / A is an example of a clostridial neurotoxin produced by bacteria of the genus Clostridium. Other examples of such clostridial neurotoxins include those produced by C. tetani (TeNT) and those produced by C. botulinum serotypes B-G and X (BoNT) (see WO2018 / 009903 A2) as well as those produced by C. baratii and C. butyricum. The neurotoxins are highly potent and specific, and can poison neurons and other cells to which they are delivered. Clostridial toxins are some of the most potent toxins known. By way of example, botulinum neurotoxins have a median lethal dose (LD) in mice ranging from 0.5 to 5 ng / kg depending on the serotype. 50 ) value. Both tetanus and botulinum toxins act by inhibiting the function of affected neurons, specifically, the release of neurotransmitters. Botulinum toxins act at the neuromuscular junction and inhibit cholinergic transmission in the peripheral nervous system, whereas tetanus toxins act in the central nervous system.

[0120] In nature, clostridial neurotoxins (including BoNT / A) are synthesized as single-chain polypeptides, which are post-translationally modified by a proteolytic cleavage event to form two polypeptide chains linked together by a disulfide bond. Cleavage occurs at a specific cleavage site, often referred to as the activation site (e.g., activation loop), located between cysteine ​​residues that provide the interchain disulfide bond. The active form of the toxin is this two-chain form. The two chains are referred to as the heavy chain (H chain), which has a molecular weight of approximately 100 kDa, and the light chain (L chain), which has a molecular weight of approximately 50 kDa. The H chain is linked to an N-terminal translocation component (H N domain) and the C-terminal targeting component (H C The cleavage site is located between the L chain and the translocation domain component. C After binding of the domain to its target neuron and internalization of the bound toxin into the cell by endosomes, H N The domain translocates the L chain across the endosomal membrane into the cytosol, where the L chain provides the protease function (also known as a non-cytotoxic protease).

[0121] Non-cytotoxic proteases act by proteolytically cleaving intracellular trafficking proteins known as SNARE proteins (e.g., SNAP-25, VAMP or syntaxin) - see Gerald K (2002) "Cell and Molecular Biology" (4th edition) John Wiley & Sons, Inc., preferably SNAP-25. The acronym SNARE is derived from the term Soluble NSF Attachment Receptor, where NSF stands for N-ethylmaleimide-Sensitive Factor. SNARE proteins are essential for intracellular vesicle fusion and thus for the secretion of molecules from cells via vesicle trafficking. The protease function is a zinc-dependent endopeptidase activity and shows high substrate specificity for SNARE proteins. Thus, once delivered to the desired target cell, non-cytotoxic proteases are capable of inhibiting cellular secretion from the target cell. The L-chain proteases of clostridial toxins are non-cytotoxic proteases that cleave SNARE proteins.

[0122] "H C The term "domain" as used herein means a functionally distinct region of a neurotoxin heavy chain having a molecular weight of approximately 50 kDa that enables the neurotoxin to bind to a receptor located on the surface of a target cell. C The domain is divided into two structurally distinct subdomains, “H CN Subdomains (H C N-terminal part of the domain) and "H CC Subdomains (H C The C-terminal portion of the domain), each of which has a molecular weight of approximately 25 kDa.

[0123] "L.H. N The term "domain" as used herein means CThe heavy chains lack the endopeptidase domain ("L" or "light chain") and the domain involved in the translocation of the endopeptidase to the cytoplasm (H N It refers to a neurotoxin consisting of the agonist (domain).

[0124] Given the ubiquity of SNARE proteins, clostridial neurotoxins, such as botulinum toxins, have been used successfully in a wide range of therapies.

[0125] For further details regarding the genetic basis of toxin production in C. botulinum and C. tetani, see Henderson et al (1997) in The Clostridia: Molecular Biology and Pathogenesis, Academic press.

[0126] As discussed above, clostridial neurotoxins are formed from two polypeptide chains, a heavy chain (H chain) having a molecular weight of approximately 100 kDa, and a light chain (L chain) having a molecular weight of approximately 50 kDa. The H chain contains a C-terminal targeting component (the receptor binding domain or H C domain) and N-terminal translocation component (H N domain).

[0127] Clostridial neurotoxin domains are described in more detail below.

[0128] Examples of light chain reference sequences include: Botulinum type A neurotoxin: amino acid residues 1-448 Botulinum type B neurotoxin: amino acid residues 1-440

[0129] The reference sequences identified above should be considered as a guide, as slight variations may occur according to subserotype. By way of example, US2007 / 0166332 (hereby incorporated by reference in its entirety) cites slightly different Clostridium sequences: Botulinum type A neurotoxin: Amino acid residues M1 to K448 Botulinum type B neurotoxin: Amino acid residues M1-K441

[0130] A translocation domain is a fragment of the H chain of a clostridial neurotoxin approximately equivalent to the amino-terminal half of the H chain, or the domain which corresponds to that fragment in an intact H chain.

[0131] Examples of reference translocation domains include: Botulinum type A neurotoxin - amino acid residues (449-871) Botulinum type B neurotoxin - amino acid residues (441-858)

[0132] The reference sequences identified above should be considered as a guide, as slight variations may occur according to subserotype. By way of example, US2007 / 0166332 (hereby incorporated by reference) cites slightly different Clostridium sequences: Botulinum type A neurotoxin - amino acid residues (A449 to K871) Botulinum type B neurotoxin - amino acid residues (A442 to S858)

[0133] In view of the present invention, various BoNT / AHs containing a translocation domain N Regions that may be useful in embodiments of the present invention include those derived from the heavy chain of BoNT / A. N The region is approximately 410-430 amino acids long and contains the translocation domain. N Studies have shown that the total length of the region is not essential for the translocation activity of the translocation domain. Thus, aspects of this embodiment include, for example, BoNT / AH polypeptides that include a translocation domain having a length of at least 350 amino acids, at least 375 amino acids, at least 400 amino acids, or at least 425 amino acids. NOther aspects of this embodiment include, for example, a BoNT / AH gene that includes a translocation domain having a length of at most 350 amino acids, at most 375 amino acids, at most 400 amino acids, or at most 425 amino acids. N It may include regions.

[0134] H N The term refers to naturally occurring BoNT / AH N Modified BoNT / AHs having portions and non-naturally occurring amino acid sequences and / or synthetic amino acid residues N Preferably, the modified BoNT / AH N The moiety still demonstrates the translocation function described above.

[0135] Clostridial neurotoxin receptor binding domain (H C ) Examples of reference sequences include: BoNT / A-N872~L1296 BoNT / B-E859~E1291

[0136] The approximately 50 kDa H of clostridial neurotoxins (e.g., BoNT) C The domain is H CC and H CN It contains two distinct structural features, called domains, each usually of about 25 kDa. The amino acid residues involved in receptor binding are mainly H CC It is believed to be located in the H domain of natural clostridial neurotoxins. CThe domain may comprise approximately 400-440 amino acid residues. This fact is confirmed by the following publications, each of which is incorporated herein by reference in its entirety: Umland TC (1997) Nat. Struct. Biol. 4: 788-792; Herreros J (2000) Biochem. J. 347: 199-204; Halpern J (1993) J. Biol. Chem. 268: 15, pp. 11188-11192; Rummel A (2007) PNAS 104: 359-364; Lacey DB (1998) Nat. Struct. Biol. 5: 898-902; Knapp (1998) Am. Cryst. Assoc. Abstract Papers 25: 90; Swaminathan and Eswaramoorthy (2000) Nat. Struct. Biol. 7: 1751-1759; and Rummel A (2004) Mol. Microbiol. 51(3), 631-643.

[0137] (Reference)H CN Examples of domains include: Botulinum type A neurotoxin - amino acid residues (872-1110) Botulinum type B neurotoxin - amino acid residues (859-1097)

[0138] The above sequence positions may vary slightly according to serotype / subtype (see CN Further examples of domains include: Botulinum type A neurotoxin - amino acid residues (874-1110) Botulinum type B neurotoxin - amino acid residues (861-1097)

[0139] (Reference)H CC Examples of domains include: Botulinum type A neurotoxin - amino acid residues (Y1111 to L1296) Botulinum type B neurotoxin - amino acid residues (Y1098 to E1291)

[0140] WO2017 / 191315 A1 (herein incorporated by reference) teaches modified BoNT / A and methods for preparing and producing same. Thus, botulinum neurotoxin A (BoNT / A) light chain and translocation domain (BoNT / AH) for use in the present invention are N ) and the BoNT / B receptor binding domain (H C The modified BoNT / A including the ribozyme domain can be that taught in WO2017 / 191315 A1.

[0141] The term "modified BoNT / A" or "chimeric neurotoxin" as used herein refers to a clostridial neurotoxin comprising a clostridial neurotoxin light chain and a translocation domain (H) derived from a first clostridial neurotoxin serotype. N domain) and a receptor-binding domain (H) attributed to a second distinct Clostridial neurotoxin serotype. C Specifically, the modified BoNT / A for use in the present invention refers to a neurotoxin comprising (preferably consisting of) a botulinum neurotoxin A (BoNT / A) light chain and a translocation domain (H domain). N domain) and the BoNT / B receptor binding domain (H C The modified BoNT / A contains the BoNT / A LH domain. N The domain is BoNT / BH C The modified BoNT / A of the present invention may be referred to as a chimeric botulinum neurotoxin. The modified BoNT / A is also referred to herein as "BoNT / AB", "mrBoNT / AB" or "BoNT / AB chimera".

[0142] Light chains and heavy chains N The domains (optionally including a complete or partial activation loop, e.g., a complete activation loop if the modified BoNT / A is in single-chain form and a truncated / partial activation loop if the modified BoNT / A is in two-chain form) are collectively referred to as LH N Sometimes called a domain. NThe domain is therefore H C Does not further include the domain.

[0143] The modified BoNT / A is essentially a botulinum neurotoxin A (BoNT / A) light chain and translocation domain (H N domain) and the BoNT / B receptor binding domain (H C domain).

[0144] The term "consists essentially of" as used in this context means that the modified BoNT / A does not further comprise one or more amino acid residues that impart additional functionality to the polypeptide when, for example, administered to a subject. In other words, the modified BoNT / A comprises a botulinum neurotoxin A (BoNT / A) light chain and a translocation domain (H N domain) and the BoNT / B receptor binding domain (H C A polypeptide "consisting essentially of" a botulinum neurotoxin A (BoNT / A) light chain and a translocation domain (H domain) is N domain) and the BoNT / B receptor binding domain (H C The polypeptide may further comprise one or more amino acid residues (such as those of the nucleotide sequence of the polypeptide of the present invention) which do not confer an additional function to the polypeptide when, for example, administered to a subject. The additional function may include an enzymatic activity, a binding activity, and / or any physiological activity whatsoever.

[0145] The modified BoNT / A may include a non-clostridial neurotoxin sequence in addition to any clostridial neurotoxin sequence, so long as the non-clostridial neurotoxin sequence does not disrupt the ability of the modified BoNT / A to achieve its therapeutic effect. Preferably, the non-clostridial neurotoxin sequence does not have catalytic activity, e.g., enzymatic activity. In one embodiment, the modified BoNT / A of the present invention does not include a non-clostridial catalytic activity domain. In one embodiment, the modified BoNT / A does not include an additional catalytic activity domain. In one embodiment, the non-clostridial sequence does not bind to a cellular receptor. In other words, in one embodiment, the non-clostridial sequence is not a ligand of the cellular receptor. The cellular receptor may be a proteinaceous cellular receptor, e.g., an integral membrane protein. Examples of cellular receptors can be found in the IUPHAR Guide to Pharmacology Database, April 2019 edition, available at https: / / www.guidetopharmacology.org / download.jsp#db_reports. The non-clostridial neurotoxin sequence may include a tag, e.g., a His-tag, that aids in purification. In one embodiment, the modified BoNT / A of the invention does not include a label or a site for adding a label, e.g., a sortase acceptor or donor site.

[0146] Preferably, the modified BoNT / A comprises a botulinum neurotoxin A (BoNT / A) light chain and a translocation domain (H N domain) and the BoNT / B receptor binding domain (H C domain).

[0147] The modified BoNT / A contains a light chain that is capable of exhibiting non-cytotoxic protease activity and is capable of cleaving SNARE proteins in the cytosol of target neurons.

[0148] Cell-based, in vivo assays can be used to determine whether the Clostridial neurotoxin containing L chain and functional cell binding and translocation domain has non-cytotoxic protease activity.Assays such as Digit Abduction Score (DAS) assay, Dorsal Root Ganglion (DRG) assay, Spinal Cord Neuron (SCN) assay and Mouse Phrenic Nerve Hemidiaphragm (PNHD) assay are routine in the art.Suitable assays for determining non-cytotoxic protease activity can be those described in Aoki KR, Toxicon 39: 1815-1820; 2001 or Donald et al (2018), Pharmacol Res Perspect, e00446, 1-14, which are incorporated herein by reference.

[0149] BoNT / A (e.g., modified BoNT / A) is defined herein by a polypeptide sequence (SEQ ID NO:), the L chain portion of the sequence (SEQ ID NO:) may constitute the first chain of a dichain Clostridial neurotoxin (e.g., a dichain modified BoNT / A), and the H chain portion of the sequence (SEQ ID NO:) may constitute the first chain of a dichain Clostridial neurotoxin (e.g., a dichain modified BoNT / A). N and H CThe domains together may constitute the second chain of a di-chain clostridial neurotoxin (e.g., di-chain modified BoNT / A), where the first and second chains are linked together by a disulfide bond. Those skilled in the art will understand that the protease may cleave at one or more positions in the activation loop of a clostridial neurotoxin (e.g., modified BoNT / A), preferably at two positions in the activation loop. If cleavage occurs at more than one position (preferably at two positions) in the activation loop, a small fragment of the C-terminal L chain portion of the sequence may not be present in the di-chain clostridial neurotoxin sequence (e.g., di-chain modified BoNT / A). In view of this, the sequence of a di-chain clostridial neurotoxin (e.g., di-chain modified BoNT / A) may differ slightly from that of the corresponding single-chain clostridial neurotoxin (e.g., single-chain modified BoNT / A). The small fragment may be 1 to 15 amino acids. In particular, in one embodiment, when Lys-C is used to convert a single-chain modified BoNT / A to a two-chain modified BoNT / A, the small fragment of the C-terminal L chain portion of the absent sequence may be SEQ ID NO: 9 or 10.

[0150] Most preferably, the modified BoNT / A for use in the present invention comprises a BoNT / A light chain and a translocation domain (BoNT / A LH N domain) and BoNT / BH C BoNT / A LH domain. N The domain is BoNT / BH C The modified BoNT / A is also referred to herein as a "BoNT / AB" or a "BoNT / AB chimera."

[0151] LH N The C-terminal amino acid residues of the domain are the same as those of the LH domain of BoNT / A. N and H C Separate domains 3 10 may correspond to the first amino acid residue of the helix, H C The N-terminal amino acid residues of the domain are the same as those of the LH domain in BoNT / B. N and HC Separate domains 3 10 It may correspond to the second amino acid residue of the helix.

[0152] An example of an (unmodified) BoNT / A polypeptide sequence is provided as SEQ ID NO:2.

[0153] An example of a BoNT / B polypeptide sequence is provided as SEQ ID NO:8 (UniProt Accession No. B1INP5).

[0154] "BoNT / A LH N and H C Separate domains 3 10 References herein to "the first amino acid residue of the helix" include N and H C Separate domains 3 10 It refers to the N-terminal residue of the helix.

[0155] "BoNT / B LH N and H C Separate domains 3 10 References herein to "the second amino acid residue of the helix" include N and H C Separate domains 3 10 It refers to the amino acid residue following the N-terminal residue of the helix.

[0156] "3 10 A "helix" is a type of secondary structure found in proteins and polypeptides, along with α-helices, β-sheets, and reverse turns. 3 10The amino acids in the helix are arranged in a right-handed helical structure, with each full turn completed by three residues and 10 atoms separating the intramolecular hydrogen bonds between them. Each amino acid has 10 atoms in a ring formed by forming hydrogen bonds, corresponding to a rotation of 120° in the helix (i.e., the helix has 3 residues per turn), a movement of 2.0 Å (=0.2 nm) along the helix axis. Most importantly, the NH group of an amino acid forms a hydrogen bond with the C=O group of the amino acid three residues before it, and this repeated i+3→i hydrogen bond is repeated 3 times. 10 Define the helix. 3 10 A helix is ​​a standard concept in structural biology and is familiar to those of skill in the art.

[0157] These three 10 The helix corresponds to the four residues that form the actual helix and two cap (or transition) residues, one at each end of these four residues. N and H C Separate domains 3 10 The term "helix" as used herein consists of those six residues.

[0158] By performing structural analysis and sequence alignment, LH N and H C Separate domains 3 10 The three helices were identified. 10 The helix is ​​located at its N-terminus (i.e., LH N at the C-terminal part of the domain) by an α-helix and at its C-terminus (i.e. C The N-terminal part of the domain is surrounded by β-strands. 10 The first (N-terminal) residue of a helix (the cap or transition residue) also corresponds to the C-terminal residue of this α-helix.

[0159] LH N and H C Separate domains 3 10The helices can be determined, for example, from the publicly available crystal structures of botulinum neurotoxins, such as 3BTA (http: / / www.rcsb.org / pdb / explore / explore.do?structureId=3BTA) and 1EPW (http: / / www.rcsb.org / pdb / explore / explore.do?structureId=1EPW) for botulinum neurotoxins A1 and B1, respectively.

[0160] We also used publicly available in silico modeling and alignment tools to characterize LH in other neurotoxins. N and H C Separate domains 3 10 The position of the helices can be determined, for example, by the homology modeling servers LOOPP (Learning, Observing and Outputting Protein Patterns, http: / / loopp.org), PHYRE (Protein Homology / analogY Recognition Engine, http: / / www.sbg.bio.ic.ac.uk / phyre2 / ) and Rosetta (https: / / www.rosettacommons.org / ), the protein superposition server SuperPose (http: / / wishart.biology.ualberta.ca / superpose / ), the alignment program Clustal Omega (http: / / www.clustal.org / omega / ) and several other tools / services listed in Internet Resources for Molecular and Cell Biologists (http: / / molbiol-tools.ca / ). N / H CN " The regions surrounding the junctions are highly structurally conserved, making them ideal regions for overlapping of different serotypes.

[0161] For example, the following methodology can be used to investigate the effect of these three 10 The sequence of the helix can be determined: 1. The structural homology modeling tool LOOP (http: / / loopp.org) can be used to obtain predicted structures of other BoNT serotypes based on the BoNT / A1 crystal structure (3BTA.pdb); 2. Copy the structure (pdb) file obtained in this way to H CN The N-terminus of the domain and the approximately 80 residues preceding it (H N This results in a highly structurally conserved "H N / H CN " can be edited to preserve the area; 3. Each serotype can be superimposed onto the 3BTA.pdb structure using the protein superposition server SuperPose (http: / / wishart.biology.ualberta.ca / superpose / ); 4. Investigate the overlapping pdb files and find 3 10 The helix is ​​the H C It is possible to map to the start of the domain and then identify the corresponding residues in other serotypes; 5. Other BoNT serotype sequences can be aligned using Clustal Omega to check that corresponding residues are correct.

[0162] LH determined by this method N , H C and 3 10 An example of a helical domain is shown below: [Table G]

[0163] Using structural analysis and sequence alignment, LH N and H C Separate domains 3 10 The posthelical β-strand is a conserved structure in all botulinum and tetanus neurotoxins and is involved in the formation of LH N and H C Separate domains 3 10It was found that if one starts at the first residue of the helix, it begins at the eighth residue (eg, at residue 879 in BoNT / A1).

[0164] BoNT / AB chimera is a BoNT / B derived H C LH from BoNT / A covalently linked to the domain N May include the domain, Where LH N The C-terminal amino acid residue of the domain is H C corresponds to the 8th amino acid residue at the N-terminus of the β-strand located at the beginning (N-terminus) of the domain, Where H C The N-terminal amino acid residue of the domain is H C It corresponds to the seventh amino acid residue at the N-terminus of the β-strand located at the beginning (N-terminus) of the domain.

[0165] BoNT / AB chimera is a BoNT / B derived H C LH from BoNT / A covalently linked to the domain N May include the domain, Where LH N The C-terminal amino acid residues of the domain are the same as those of the LH domain of BoNT / A. N corresponds to the C-terminal amino acid residue of the α-helix located at the end (C-terminus) of the domain, Where H C The N-terminal amino acid residues of the domain are the LH N It corresponds to the amino acid residue immediately C-terminal to the C-terminal amino acid residue of the α-helix located at the end (C-terminus) of the domain.

[0166] The rationale for the design process of the BoNT / AB chimera was to ensure that the secondary structure was intact, thereby minimizing any changes to the tertiary structure and to the function of each domain. Without wishing to be bound by theory, 10It is hypothesized that not disrupting the four central amino acid residues of the helix ensures an optimal conformation of the chimeric neurotoxin, thereby allowing the chimeric neurotoxin to perform its function to its full potential.

[0167] In fact, surprisingly, 3 10 The first amino acid residue of helix 1 and the third amino acid residue of BoNT / B 10 Retaining only the second amino acid residue after the helix not only allows for the production of a soluble, functional BoNT / AB chimera, but also leads to improved properties over other BoNT / AB chimeras, particularly increased potency, increased safety factor and / or longer duration of action (as well as increased safety factor and / or increased duration of action when compared to unmodified BoNT / A [e.g., SEQ ID NO:2, e.g., SEQ ID NO:2 in its dichain form]).

[0168] BoNT / A light chain, BoNT / A translocation domain and / or BoNT / BH C The domains may include modified BoNT / A light chains, BoNT / A translocation domains and / or BoNT / BH C The BoNT / A light chain may be a modified BoNT / A light chain, a BoNT / A translocation domain, and / or a BoNT / BH domain or a derivative thereof, including, but not limited to, those described below. C The domain or derivative may be a BoNT / A light chain, a BoNT / A translocation domain, and / or a BoNT / BH C The BoNT / A light chain, the BoNT / A translocation domain and / or the BoNT / BH light chain may contain one or more amino acids that are modified compared to the native (unmodified) form of the domain, or may contain one or more amino acids that are modified compared to the native (unmodified) form of the domain. C It may contain one or more inserted amino acids not present in the native (unmodified) form of the domain. Examples include modified BoNT / A light chains, BoNT / A translocation domains and / or BoNT / BH domains. C The domains may be any of the native (unmodified) BoNT / A light chain, the BoNT / A translocation domain and / or the BoNT / BH domain. CThe domain sequence may have an altered amino acid sequence in one or more of the domains. Such alterations may alter its functional aspects, such as biological activity or persistence. Thus, in one embodiment, the BoNT / A light chain, the BoNT / A translocation domain and / or the BoNT / BH C The domains may include modified BoNT / A light chains, BoNT / A translocation domains and / or BoNT / BH C Domain or modified BoNT / A light chain, BoNT / A translocation domain and / or BoNT / BH C It is a domain derivative.

[0169] Modified BoNT / BH C The domain modifies binding to a target neuron, e.g., the native (unmodified) BoNT / BH C The BoNT / BH domain may have one or more modifications that provide higher or lower affinity binding when compared to the BoNT / BH domain. C Such modifications in the H domain alter binding to ganglioside and / or protein receptors on target neurons. C This may involve modifying residues in the ganglioside binding site or in the protein (e.g., synaptotagmin) binding site of the domain. Examples of such modified neurotoxins are described in WO2006 / 027207 and WO2006 / 114308, both of which are incorporated herein by reference in their entirety.

[0170] The modified light chain may have one or more modifications in its amino acid sequence, for example, modifications in the substrate binding or catalytic domains that may alter or modify the SNARE protein specificity of the modified light chain, preferably provided that said modifications do not catalytically inactivate the light chain. Examples of such modified neurotoxins are described in WO2010 / 120766 and US2011 / 0318385, both of which are incorporated herein by reference in their entirety.

[0171] BoNT / A-derived LH NThe domain may correspond to amino acid residues 1-872 of SEQ ID NO:2, or a polypeptide sequence having at least 70% sequence identity thereto. N The domain may correspond to amino acid residues 1 to 872 of SEQ ID NO:2, or a polypeptide sequence having at least 80%, 90% or 95% sequence identity thereto. N The domain corresponds to amino acid residues 1-872 of SEQ ID NO:2.

[0172] BoNT / B-derived H C The domain may correspond to amino acid residues 860 to 1291 of SEQ ID NO:8, or a polypeptide sequence having at least 70% sequence identity thereto. C The domain may correspond to amino acid residues 860 to 1291 of SEQ ID NO:8, or a polypeptide sequence having at least 80%, 90% or 95% sequence identity thereto. C The domain corresponds to amino acid residues 860 to 1291 of SEQ ID NO:8.

[0173] Preferably, the BoNT / AB chimera is BoNT / A1 LH N Domain and BoNT / B1 H C More preferably, the LH domain is N The domain corresponds to amino acid residues 1 to 872 of BoNT / A1 (SEQ ID NO: 2), C The domain corresponds to amino acid residues 860 to 1291 of BoNT / B1 (SEQ ID NO:8).

[0174] Most preferably, BoNT / BH C The domain is H CC The BoNT / BH further comprises at least one amino acid residue substitution, insertion, indel or deletion in the subdomain, which has the effect of increasing the binding affinity of the BoNT / B neurotoxin for human Syt II, as compared to the native BoNT / B sequence. CCSuitable amino acid residue substitutions, insertions, indels or deletions in the subdomains are disclosed in WO2013 / 180799, and in WO2016 / 154534 (both of which are incorporated herein by reference).

[0175] BoNT / BH CC Suitable amino acid residue substitutions, insertions, indels or deletions in the subdomains may include substitution mutations selected from the group consisting of V1118M, Y1183M, E1191M, E1191I, E1191Q, E1191T, S1199Y, S1199F, S1199L, S1201V, E1191C, E1191V, E1191L, E1191Y, S1199W, S1199E, S1199H, W1178Y, W1178Q, W1178A, W1178S, Y1183C, Y1183P and combinations thereof.

[0176] BoNT / BH CC Suitable amino acid residue substitutions, insertions, indels or deletions in the subdomain may further include a combination of two substitution mutations selected from the group consisting of E1191M and S1199L, E1191M and S1199Y, E1191M and S1199F, E1191Q and S1199L, E1191Q and S1199Y, E1191Q and S1199F, E1191M and S1199W, E1191M and W1178Q, E1191C and S1199W, E1191C and S1199Y, E1191C and W1178Q, E1191Q and S1199W, E1191V and S1199W, E1191V and S1199Y or E1191V and W1178Q.

[0177] BoNT / BH CC Suitable amino acid residue substitutions, insertions, indels or deletions in the subdomains may also include a combination of the three substitution mutations: E1191M, S1199W and W1178Q.

[0178] Preferably, BoNT / BH CCThe amino acid residue substitution, insertion, indel or deletion in the subdomain includes a combination of two substitution mutations, E1191M and S1199Y. Such modifications are present in modified BoNT / A of SEQ ID NO:5 and SEQ ID NO:6. E1191M may correspond to position 1204 and S1199Y may correspond to position 1212 of SEQ ID NO:6. Thus, SEQ ID NO:6 may include 1204M and 1212Y.

[0179] The modification may be a modification in comparison with unmodified BoNT / B shown as SEQ ID NO:8, where the amino acid residue numbering is determined by alignment with SEQ ID NO:8. Since the presence of a methionine residue at position 1 of SEQ ID NO:8 (as well as the SEQ ID NOs corresponding to the modified BoNT / A polypeptides described herein) is optional, a person skilled in the art will take into account the presence / absence of the methionine residue when determining the amino acid residue numbering. For example, if SEQ ID NO:8 contains a methionine, the position numbering will be as defined above (e.g., E1191 will be E1191 in SEQ ID NO:8). Alternatively, if a methionine is not present in SEQ ID NO:8, the amino acid residue numbering must be modified by -1 (e.g., E1191 will be E1190 in SEQ ID NO:8). Thus, the first methionine amino acid residue of the polypeptide sequence of modified BoNT / A is optional and may not be present. Similar considerations apply to the presence / absence of a methionine at position 1 of other polypeptide sequences described herein, and a person skilled in the art will easily determine the correct amino acid residue numbering using routine techniques in the art.

[0180] A modified BoNT / A for use in the present invention may comprise a polypeptide sequence having at least 70% sequence identity to a polypeptide sequence selected from SEQ ID NOs: 3 to 7. For example, a polypeptide sequence having at least 80%, 90%, 95% or 99.9% sequence identity to a polypeptide sequence selected from SEQ ID NOs: 3 to 7. Preferably, a modified BoNT / A for use in the present invention may comprise (and more preferably consist of) a polypeptide sequence selected from SEQ ID NOs: 3 to 7.

[0181] It is preferred that the modified BoNT / A comprises a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 6. For example, a polypeptide sequence having at least 80%, 90%, 95 or 99.9% sequence identity to SEQ ID NO: 6. Most preferably, a modified BoNT / A for use in the present invention may comprise (and more preferably consist of) SEQ ID NO: 6.

[0182] The term "deletion," as used herein, refers to the removal of one or more amino acid residues of a polypeptide without replacement of one or more amino acid residues at the site of the deletion. Thus, (for example), if one amino acid residue is deleted from a polypeptide sequence having x number of amino acid residues, the resulting polypeptide will have x-1 amino acid residues.

[0183] The term "insertion / deletion" as used herein refers to the deletion of one or more amino acid residues of a polypeptide and the insertion at the deletion site of a different number of amino acid residues (more or fewer amino acid residues) as compared to the number of deleted amino acid residues. Thus, for an insertion / deletion in which two amino acid residues are deleted from a polypeptide sequence having (for example) x number of amino acid residues, the resulting polypeptide has x-1 amino acid residues or x+≧1 amino acid residues. Insertions and deletions can be made in any order, sequentially or simultaneously.

[0184] The term "substitution" as used herein refers to the replacement of one or more amino acid residues with the same number of amino acid residues at the same site. Thus, for a substitution in a polypeptide sequence having (for example) x number of amino acid residues, the resulting polypeptide also has x number of amino acid residues. Preferably, the substitution is at a single amino acid position.

[0185] The term "insertion," as used herein, refers to the addition of one or more amino acid residues to a polypeptide without deletion of one or more amino acid residues of the polypeptide at the site of the insertion. Thus, (for example), if a single amino acid residue is inserted into a polypeptide sequence having x number of amino acid residues, the resulting polypeptide will have x+1 amino acid residues.

[0186] Methods for modifying proteins by substitution, insertion or deletion of amino acid residues are known in the art. By way of example, amino acid modifications can be introduced by modification of the DNA sequence encoding BoNT / A (e.g., encoding unmodified BoNT / A). This can be accomplished using standard molecular cloning techniques, for example, by site-directed mutagenesis, in which a short strand of DNA (oligonucleotide) encoding the desired amino acid(s) is used to replace the original coding sequence using a polymerase enzyme, or by inserting / deleting portions of the gene using various enzymes (e.g., ligases and restriction endonucleases). Alternatively, modified gene sequences can be chemically synthesized. Typically, modifications can be performed by modifying a nucleic acid encoding a native Clostridial neurotoxin (or a portion thereof), such that the modified BoNT / A (or a portion thereof) encoded by the nucleic acid contains the modification(s). Alternatively, a nucleic acid encoding a modified BoNT / A (or a portion thereof) containing the modification(s) can be synthesized.

[0187] If the polypeptide sequence of the modified BoNT / A described herein includes, for example, a tag for purification, such as a His-tag, the tag is optional. Preferably, the tag is removed prior to use of the modified BoNT / A according to the present invention.

[0188] As discussed above, the modified BoNT / A described herein has increased tissue retention properties, which also provide increased potency and / or duration of action, and can allow for increased dosage without any additional negative effects. One way these advantageous properties can be defined is in terms of the safety factor of modified BoNT / A. In this regard, the undesired effects of clostridial toxins (caused by toxin diffusion away from the site of administration) can be experimentally evaluated by measuring the percentage of body weight loss in relevant animal models (e.g., mice, where body weight loss is detected within 7 days of administration). Conversely, the desired on-target effects of clostridial toxins can be experimentally evaluated by the digit abduction score (DAS) assay, which is a measurement of muscle paralysis. The DAS assay can be performed by injection of 20 μl of neurotoxin formulated in gelatin phosphate buffer into the mouse gastrocnemius / soleus muscle complex, followed by assessment of digit abduction scores using the method of Aoki (Aoki KR, Toxicon 39: 1815-1820; 2001). In the DAS assay, mice are briefly suspended by their tails to elicit a characteristic startle response in which the mouse extends its hind limbs and abducts its hind digits. After neurotoxin injection, various degrees of digit abduction are scored on a 5-point scale (0=normal to 4=maximal reduction in digit abduction and leg extension).

[0189] The safety factor of the neurotoxin can then be expressed as the ratio between the amount of toxin required for a 10% reduction in body weight (measured at peak effect within the first 7 days after dosing in mice) and the amount of toxin required for a DAS score of 2. Thus, a high safety factor score is desirable and indicates a neurotoxin that can effectively paralyze the target muscle with few undesired off-target effects. The modified BoNT / A of the present invention has a higher safety factor than the safety factor of the equivalent unmodified (natural) BoNT / A.

[0190] A high safety margin is particularly advantageous in therapy, since it represents an increased therapeutic index. In other words, this means that a reduced dosage can be used compared to alternative Clostridial neurotoxin therapeutics, and / or an increased dosage can be used without any additional (e.g., adverse) effects. Adverse effects can include systemic toxicity and / or undesirable spread to adjacent muscles. The possibility of using higher doses of neurotoxin without additional effects is particularly advantageous, since higher doses usually lead to a longer duration of action of the neurotoxin.

[0191] The efficacy of the modified BoNT / A is determined by the expression of a given DAS score, e.g., a DAS score of 2 (ED 50 The potency of modified BoNT / A can also be expressed as the EC 50 Dose, e.g., EC in a cellular assay measuring SNAP25 cleavage by modified BoNT / A 50 It can also be expressed as a dose.

[0192] The duration of action of the modified BoNT / A can be expressed as the time required to recover a DAS score of 0 following administration of a given dose of neurotoxin to the mouse gastrocnemius / soleus muscle complex, for example, the minimum dose of neurotoxin that leads to a DAS score of 4.

[0193] Thus, in one embodiment, a modified BoNT / A of the invention has a safety factor of greater than 7 (e.g., at least 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50), and the safety factor is greater than the DAS ED 50 Dose of toxin required for -10% body weight change (pg / mouse) divided by (pg / mouse) [ED 50 Dose required to produce a DAS score of 0.05 to 0.25, for example, ...

[0194] In one embodiment, the modified BoNT / A of the present invention has a safety factor of at least 10. In one embodiment, the modified BoNT / A of the present invention has a safety factor of at least 15.

[0195] Preferably, the modified BoNT / A has a safety factor of at least 10 (eg, a safety factor of 10), more preferably at least 12 or 13 (eg, 14-15).

[0196] The modified BoNT / A may have a safety factor of greater than 7, up to 50, for example, 8-45, 10-20, or 12-15.

[0197] During use, the modified BoNT / A of the invention is in a two-chain form.

[0198] The modified BoNT / A for use in the present invention may comprise a polypeptide sequence having at least 70% sequence identity to a polypeptide sequence selected from SEQ ID NOs: 3 to 7. For example, a polypeptide sequence having at least 80%, 90%, 95% or 99.9% sequence identity to a polypeptide sequence selected from SEQ ID NOs: 3 to 7. Preferably, the modified BoNT / A for use in the present invention may comprise (and more preferably consist of) a polypeptide sequence selected from SEQ ID NOs: 3 to 7. Of the modified BoNT / As, SEQ ID NO: 6 is preferred.

[0199] Thus, it is preferred that the modified BoNT / A comprises a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 6. More preferably, a polypeptide sequence having at least 80%, 90%, 95% or 99.9% sequence identity to SEQ ID NO: 6. Most preferably, a modified BoNT / A for use in the present invention may comprise (and more preferably consist of) SEQ ID NO: 6.

[0200] The two-chain modified BoNT / A of the present invention may comprise a light chain portion of a polypeptide sequence having at least 70%, 80%, 90%, 95%, 99.9% or 100% sequence identity to any one of SEQ ID NOs: 3-7 constituting the first chain of the two-chain modified BoNT / A, and a heavy chain portion of a polypeptide sequence having at least 70%, 80%, 90%, 95%, 99.9% or 100% sequence identity to any one of SEQ ID NOs: 3-7 constituting together the second chain of the two-chain modified BoNT / A. N and H C domain, and the first and second chains are linked together by disulfide bonds.

[0201] When cleavage occurs at more than one position (preferably at two positions) within the activation loop of a modified BoNT / A that contains a polypeptide sequence having at least 70%, 80%, 90%, 95%, 99.9% or 100% sequence identity to any one of SEQ ID NOs: 3-7, a small fragment of the C-terminal L-chain portion of the sequence having at least 70%, 80%, 90%, 95%, 99.9% or 100% sequence identity to any one of SEQ ID NOs: 3-7 may not be present in the two-chain modified BoNT / A. In view of this, the sequence of a two-chain modified BoNT / A (e.g., comprising a polypeptide sequence having at least 70%, 80%, 90%, 95%, 99.9% or 100% sequence identity to any one of SEQ ID NOs: 3-7) may differ slightly from that of a corresponding single-chain modified BoNT / A comprising a polypeptide sequence having at least 70%, 80%, 90%, 95%, 99.9% or 100% sequence identity to any one of SEQ ID NOs: 3-7. The subfragment may be 1 to 15 amino acids. In particular, in one embodiment, when Lys-C is used to convert a single-chain modified BoNT / A to a two-chain Clostridial neurotoxin, the subfragment of the C-terminal L-chain portion of the absent sequence may be SEQ ID NO: 9 or 10.

[0202] Preferably, the two-chain modified BoNT / A of the present invention may comprise a light chain portion of a polypeptide sequence having at least 70%, 80%, 90%, 95%, 99.9% or 100% sequence identity to SEQ ID NO: 6 constituting the first chain of the two-chain modified BoNT / A, and a heavy chain portion of a polypeptide sequence having at least 70%, 80%, 90%, 95%, 99.9% or 100% sequence identity to SEQ ID NO: 6 constituting together the second chain of the two-chain modified BoNT / A. N and H C domain, the first and second chains being linked together by disulfide bonds.

[0203] In the case where cleavage occurs at more than one position (preferably at two positions) within the activation loop of a modified BoNT / A that includes a polypeptide sequence having at least 70%, 80%, 90%, 95%, 99.9% or 100% sequence identity to SEQ ID NO: 6, a small fragment of the C-terminal L chain portion of the sequence having at least 70%, 80%, 90%, 95%, 99.9% or 100% sequence identity to SEQ ID NO: 6 may not be present in the two-chain modified BoNT / A. In light of this, the sequence of a two-chain modified BoNT / A (e.g., including a polypeptide sequence having at least 70%, 80%, 90%, 95%, 99.9% or 100% sequence identity to SEQ ID NO: 6) may differ slightly from that of a corresponding single-chain modified BoNT / A that includes a polypeptide sequence having at least 70%, 80%, 90%, 95%, 99.9% or 100% sequence identity to SEQ ID NO: 6. The subfragment can be 1 to 15 amino acids. In particular, in one embodiment, when Lys-C is used to convert a single-chain modified BoNT / A to a two-chain modified BoNT / A, the subfragment of the C-terminal L-chain portion of the absent sequence can be SEQ ID NO: 9 or 10.

[0204] In a particularly preferred embodiment, the two-chain modified BoNT / A comprises (or consists of) a light chain comprising a polypeptide sequence having at least 70%, 80%, 90%, 95% or 99.9% sequence identity to SEQ ID NO: 11 or 12 (preferably SEQ ID NO: 11) and a heavy chain comprising a polypeptide sequence having at least 70%, 80%, 90%, 95% or 99.9% sequence identity to SEQ ID NO: 13, where the light chain and the heavy chain are linked together by a disulfide bond. More preferably, the two-chain modified BoNT / A comprises (or consists of) a light chain comprising SEQ ID NO: 11 or 12 (preferably SEQ ID NO: 11) and a heavy chain comprising SEQ ID NO: 13, where the light chain and the heavy chain are linked together by a disulfide bond. Even more preferably, the two-chain modified BoNT / A comprises (or consists of) a light chain having SEQ ID NO: 11 and a heavy chain having SEQ ID NO: 13, where the light chain and the heavy chain are linked together by a disulfide bond. The disulfide bond is preferably formed by and / or between cysteine ​​residue 429 of SEQ ID NO:11 or 12 and cysteine ​​residue 6 of SEQ ID NO:13.

[0205] In a preferred embodiment, the modified BoNT / A of the present invention does not include a therapeutic or diagnostic agent (e.g., a nucleic acid, protein, peptide, or small molecule therapeutic or diagnostic agent) in addition to the light and heavy chains. For example, in one embodiment, the modified BoNT / A may not include a therapeutic or diagnostic agent that is covalently or non-covalently associated therewith. Thus, the modified BoNT / A of the present invention preferably does not function as a delivery vehicle for additional therapeutic or diagnostic agents.

[0206] In embodiments in which the modified BoNT / A described herein has a tag for purification (eg, a His-tag) and / or a linker, the tag and / or linker are optional.

[0207] The modified BoNT / A may not contain complexing proteins present in the naturally occurring Clostridial neurotoxin complex. Examples of such complexing proteins include neurotoxin-associated proteins (NAPs) and non-toxic non-hemagglutinin components (NTNHs). However, the modified BoNT / A is preferably a recombinant modified BoNT / A.

[0208] The modified BoNT / A of the present invention can be produced using recombinant nucleic acid technology. Thus, in one embodiment, the modified BoNT / A (as described herein) is a recombinant modified BoNT / A.

[0209] In one embodiment, a nucleic acid (e.g., DNA) is provided that comprises a nucleic acid sequence that encodes a modified BoNT / A. In one embodiment, the nucleic acid sequence is prepared as part of a DNA vector that includes a promoter and a terminator. The nucleic acid sequence can be selected from any of the nucleic acid sequences described herein.

[0210] In a preferred embodiment, the vector has a promoter selected from the following: Promoter / Inducer / Normal induction conditions Tac (hybrid) / IPTG / 0.2mM (0.05~2.0mM) AraBAD / L-arabinose / 0.2% (0.002~0.4%) T7-lac operator / IPTG / 0.2mM (0.05~2.0mM)

[0211] In another preferred embodiment, the vector has a promoter selected from: Promoter / Inducer / Normal induction conditions Tac (hybrid) / IPTG / 0.2mM (0.05~2.0mM) AraBAD / L-arabinose / 0.2% (0.002~0.4%) T7-lac operator / IPTG / 0.2mM (0.05~2.0mM) T5-lac operator / IPTG / 0.2mM (0.05~2.0mM)

[0212] The nucleic acid molecule can be produced using any suitable process known in the art. Thus, the nucleic acid molecule can be produced using chemical synthesis techniques. Alternatively, the nucleic acid molecule of the present invention can be produced using molecular biology techniques.

[0213] The DNA constructs of the present invention are preferably designed on a computer and then synthesized by conventional DNA synthesis techniques.

[0214] The above nucleic acid sequence information is optionally modified for codon bias according to the final host cell (eg, E. coli) expression system to be utilized.

[0215] The terms "nucleotide sequence" and "nucleic acid" are used synonymously herein. Preferably, the nucleotide sequence is a DNA sequence.

[0216] The modified BoNT / A of the present invention can exist as a single chain or as a two-chain. However, the modified BoNT / A can be a single chain in which the L chain is linked to the H chain (or a component thereof, e.g., H) via a disulfide bond. N It is preferred that the nucleic acid be present as a duplex linked to a single nucleic acid domain.

[0217] The production of a single-chain modified BoNT / A having a light chain and a heavy chain can be achieved using a method that includes expressing a nucleic acid encoding the modified BoNT / A in an expression host, lysing the host cell to provide a host cell homogenate containing the single-chain modified BoNT / A, and isolating the single-chain modified BoNT / A. The single-chain modified BoNT / A can be proteolytically processed using a method that includes contacting the single-chain modified BoNT / A protein with a protease (e.g., Lys-C) that hydrolyzes the peptide bond in the activation loop of the modified BoNT / A, thereby converting the single-chain modified BoNT / A into the corresponding two-chain modified BoNT / A (e.g., the light chain and the heavy chain are linked together by a disulfide bond). The two-chain modified BoNT / A can preferably be obtained by such a method.

[0218] Therefore, the modified BoNT / A used in the present invention is preferably a two-chain modified BoNT / A generated from a single-chain BoNT / A, and the single-chain BoNT / A comprises or consists of the polypeptide sequence described herein.For example, it is preferred that the modified BoNT / A used in the present invention is a two-chain modified BoNT / A generated from a polypeptide comprising a polypeptide sequence having at least 70% (e.g., at least 80%, 90%, 95% or 99.9%) sequence identity to SEQ ID NO:6.Most preferably, the modified BoNT / A used in the present invention is a two-chain modified BoNT / A generated from a polypeptide comprising (and even more preferably consists of) SEQ ID NO:6. Thus, in some embodiments, the modified BoNT / A is a two-chain modified BoNT / A in which the light chain (L chain) is linked to the heavy chain (H chain) via a disulfide bond, and can be obtained by a method comprising contacting the single-chain modified BoNT / A with a protease that hydrolyzes the peptide bond in its activation loop, thereby converting the single-chain modified BoNT / A into the corresponding two-chain modified BoNT / A. Thus, in some embodiments, the modified BoNT / A is a two-chain modified BoNT / A in which the light chain (L chain) is linked to the heavy chain (H chain) via a disulfide bond, and can be obtained by a method comprising contacting the single-chain modified BoNT / A comprising SEQ ID NO: 6 with a protease that hydrolyzes the peptide bond in its activation loop, thereby converting the single-chain modified BoNT / A into the corresponding two-chain modified BoNT / A. In some embodiments, the modified BoNT / A is a two-chain modified BoNT / A that can be obtained by a method comprising contacting a single-chain modified BoNT / A consisting of SEQ ID NO:6, in which the L chain is linked to the H chain via a disulfide bond, with a protease that hydrolyzes the peptide bond in its activation loop, thereby converting the single-chain modified BoNT / A into the corresponding two-chain modified BoNT / A.

[0219] The protease used to cleave activation loop is preferably Lys-C. Suitable proteases and methods for cleaving activation loop to generate two-chain clostridial neurotoxins are taught in WO2014 / 080206, WO2014 / 079495 and EP2677029A2, which are incorporated herein by reference. Lys-C can cleave the activation loop at the C-terminus of one or more lysine residues present therein. If Lys-C cleaves activation loop more than once, those skilled in the art will understand that the small peptide of the activation loop of two-chain modified BoNT / A may not exist when compared with the sequence number shown herein.

[0220] The term "obtainable" as used herein also encompasses the term "obtained." In one embodiment, the term "obtainable" means obtained.

[0221] The term "one or more," as used herein, may mean at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20. In one embodiment, when "one or more" precedes a list, "one or more" may mean all of the members of the list. Similarly, the term "at least one," as used herein, may mean at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20. In one embodiment, when "at least one" precedes a list, "at least one" may mean all of the members of the list.

[0222] The term "disorder" as used herein also encompasses "disease." In one embodiment, the disorder is a disease.

[0223] The modified BoNT / A of the present invention can be formulated in any suitable manner for administration to a subject, for example, as part of a pharmaceutical composition. Such a pharmaceutical composition can include the modified BoNT / A of the present invention and a pharma- ceutically acceptable carrier, excipient, adjuvant, propellant, and / or salt.

[0224] Compositions suitable for injection can be in the form of solutions, suspensions or emulsions, or dry powders that are dissolved or suspended in a suitable vehicle prior to use.

[0225] Liquid dosage forms are usually prepared using modified BoNT / A and a pyrogen-free sterile medium. Depending on the vehicle and concentration used, modified BoNT / A may be dissolved or suspended in the vehicle. In preparing a solution, modified BoNT / A can be dissolved in the vehicle, the solution is made isotonic by adding sodium chloride as necessary, sterilized by filtration through a sterile filter using aseptic techniques, and then filled into suitable sterile vials or ampoules and sealed. Alternatively, if the solution stability is sufficient, the solution in the sealed container may be sterilized by autoclaving. Advantageously, additives such as buffers, solubilizers, stabilizers, preservatives or bactericides, suspending or emulsifying agents, and or local anesthetics can be dissolved in the vehicle.

[0226] By filling pre-sterilized ingredients into sterile containers using aseptic technique in a sterile area, dry powders can be prepared that are dissolved or suspended in a suitable medium prior to use. Alternatively, the ingredients can be dissolved in a suitable container using aseptic technique in a sterile area. The product is then lyophilized and the container is aseptically sealed.

[0227] Parenteral suspensions suitable for the routes of administration described herein are prepared in substantially the same manner, except that the sterile components are suspended in the sterile vehicle instead of dissolved, and sterilization cannot be accomplished by filtration. The components may be isolated in a sterile state or may be sterilized after isolation, for example, by gamma irradiation.

[0228] Advantageously, a suspending agent, such as polyvinylpyrrolidone, can be included in the composition(s) to facilitate uniform distribution of the components.

[0229] In one aspect, the invention provides a unit dosage form of modified botulinum neurotoxin A (BoNT / A) (e.g., for treating adult quadriplegia), the unit dosage form comprising: a. greater than 707 units of modified BoNT / A, where one unit is the amount of modified BoNT / A that corresponds to the calculated median lethal dose (LD50) in mice; or b. greater than 17,000 pg of modified BoNT / A; and c. optionally, pharma- ceutically acceptable carriers, excipients, adjuvants and / or salts; Here, the modified BoNT / A comprises a BoNT / A light chain and translocation domain and a BoNT / B receptor binding domain (H C domain).

[0230] The modified BoNT / A in unit dosage form preferably comprises a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 6. For example, a polypeptide sequence having at least 80%, 90%, 95% or 99.9% sequence identity to SEQ ID NO: 6. Most preferably, the modified BoNT / A may comprise (and more preferably consist of) SEQ ID NO: 6.

[0231] The unit dosage form may contain more than 707 units of modified BoNT / A. The upper limit of the range may be 1664, 1650, 1600, 1550, 1500, 1450, 1400, 1350, 1300, 1250, 1150, 1100, 1050, 1000, 950, 900, 850, 800 or 750 units of modified BoNT / A, preferably the upper limit is 1500 units. The lower limit of the range can be 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, 1000, 1025, 1050, 1075, 1100, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600 or 1650 Units of modified BoNT / A, preferably the lower limit is 725 Units, 949 Units or 1,381 Units. Preferably the unit dosage form contains more than 707 Units up to 1664 Units of modified BoNT / A, for example more than 707 Units up to 1,498 Units or 832 Units to 1622 Units. The unit dosage form may contain 915 to 1581 units, preferably 949 to 1581 units or 1414 to 1581 units of modified BoNT / A. The unit dosage form may contain 998 to 1,498 units or 1,040 to 1,498 units of modified BoNT / A. In preferred embodiments, the unit dosage form contains 998, 1,040, 1,248 or 1,498 units of modified BoNT / A. In more preferred embodiments, the unit dosage form contains 1,248 or 1,498 units (e.g., 1,498 units) of modified BoNT / A.

[0232] More preferably, the unit dosage form contains 1,248 units to 1,664 units, for example, 1,456 units to 1,539 units, of the modified BoNT / A. More preferably, the unit dosage form contains 1,498 units of the modified BoNT / A.

[0233] Even more preferably, the unit dosage form contains between 832 and 1,248 units of modified BoNT / A, for example between 998 and 1,082 units. Most preferably, the unit dosage form contains 1,040 units of modified BoNT / A.

[0234] The unit dosage form may contain more than 17,000 pg of modified BoNT / A. The upper end of the range may be 40,000, 39,000, 38,000, 37,000, 36,000, 35,000, 30,000, 25,000, 24,000, 22,000, 20,000 or 18,000 pg of modified BoNT / A, preferably the upper end is 38,000 pg. The lower limit of the range can be 17,500, 18,000, 20,000, 22,000, 24,000, 25,000, 26,000, 27,000, 28,000, 29,000, 30,000, 35,000, 36,000, 37,000, 38,000 or 39,000 pg of modified BoNT / A, preferably the lower limit is 17,500 pg, 22,753 pg, 22,790 pg or 33,1818 pg. Preferably, the unit dosage form contains more than 17,000 pg, up to 40,000 pg, for example, more than 17,000 pg, up to 36,000 pg or 20,000 pg to 39,000 pg of modified BoNT / A. The unit dosage form may contain 22,000 to 38,000 pg, preferably 23,000 to 38,000 pg or 34,000 to 38,000 pg of modified BoNT / A. The unit dosage form may contain 24,000 to 36,000 pg or 25,000 to 36,000 pg of modified BoNT / A. In a preferred embodiment, the unit dosage form contains 24,000, 25,000, 30,000 or 36,000 pg of modified BoNT / A. In a more preferred embodiment, the unit dosage form contains 30,000 or 36,000 pg (eg, 36,000 pg) of the modified BoNT / A.

[0235] More preferably, the unit dosage form contains 30,000 pg to 40,000 pg, for example 35,000 pg to 37,000 pg of the modified BoNT / A. More preferably, the unit dosage form contains 36,000 pg of the modified BoNT / A.

[0236] Even more preferably, the unit dosage form contains 20,000 pg to 30,000 pg, for example 24,000 pg to 26,000 pg of the modified BoNT / A. Most preferably, the unit dosage form contains 25,000 pg of the modified BoNT / A.

[0237] The above unit dosage forms may be suitable for treating adult subjects.

[0238] In one aspect, the present invention provides the above-mentioned unit dosage form for use in treating adult limb spasticity.Also provided is the corresponding method for treating adult limb spasticity and its use in manufacturing medicament for treating adult limb spasticity.The treatment is preferably consistent with the treatment described herein.

[0239] As discussed above, the unit dose for pediatric applications may be 50% or less of those set forth above. In some embodiments, the unit dose is 70% or less (e.g., 67% or less) of that typically used when treating adult limb spasticity.

[0240] In another aspect, the invention provides a unit dosage form of modified botulinum neurotoxin A (BoNT / A) for treating a pediatric subject (e.g., for treating childhood limb spasticity), the unit dosage form comprising: a. More than 353.5 units of modified BoNT / A, where 1 unit is the calculated median lethal dose (LD) in mice. 50 ), or b. greater than 8,500 pg of modified BoNT / A; and c. optionally, pharma- ceutically acceptable carriers, excipients, adjuvants and / or salts; Here, the modified BoNT / A comprises a BoNT / A light chain and translocation domain and a BoNT / B receptor binding domain (H C domain).

[0241] It is preferred that the modified BoNT / A in unit dosage form comprises a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 6. For example, a polypeptide sequence having at least 80%, 90%, 95% or 99.9% sequence identity to SEQ ID NO: 6. Most preferably, the modified BoNT / A can comprise (and more preferably consist of) SEQ ID NO: 6.

[0242] A unit dosage form for treating a pediatric subject may contain more than 353.5 units of modified BoNT / A. The upper limit of the range may be 832, 825, 800, 775, 750, 725, 700, 675, 650, 625, 600, 575, 550, 525, 500, 475, 450 or 425 units of modified BoNT / A, preferably the upper limit is 749, 750 or 790 units. The lower limit of the range can be 364, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825 or 830 Units of modified BoNT / A, preferably the lower limit is 364 Units, 416 Units, 474.5 Units, 475 Units, 690 Units or 690.5 Units. Preferably the unit dosage form contains between 353.5 Units and 832 Units of modified BoNT / A, for example, greater than 353.5 Units, up to 749 Units or 416 Units to 811 Units. A unit dose of modified BoNT / A for treating infantile limb spasticity may comprise 457.5 Units to 749 Units, 458 Units to 790 Units or 458 Units to 791 Units, preferably 474.5 Units to 749 Units, 707 Units to 749 Units, 475 Units to 790 Units, 475 Units to 791 Units, 707 Units to 790 Units or 707 Units to 791 Units. In preferred embodiments, the unit dosage form comprises 499, 520, 624 or 749 Units of modified BoNT / A. In more preferred embodiments, the unit dosage form comprises 624 or 749 Units (e.g., 749 Units) of modified BoNT / A.

[0243] More preferably, the unit dosage form for treating childhood spasticity contains 624 units to 832 units, for example, 728 units to 770 units, of modified BoNT / A. More preferably, the unit dosage form for treating childhood spasticity contains 749 units of modified BoNT / A.

[0244] Even more preferably, the unit dosage form for treating childhood spasticity contains 416 units to 624 units, for example, 499 units to 541 units, of modified BoNT / A. Most preferably, the unit dosage form for treating childhood spasticity contains 520 units of modified BoNT / A.

[0245] A unit dosage form for treating a pediatric subject may contain more than 8,500 pg of modified BoNT / A. The upper end of the range may be 20,000, 19,500, 19,000, 18,500, 18,000, 17,500, 15,000, 12,500, 12,000, 11,000, 10,000 or 9,000 pg of modified BoNT / A, preferably the upper end is 19,000 pg. The lower limit of the range can be 8,750, 9,000, 10,000, 11,000, 12,000, 12,500, 13,000, 13,500, 14,000, 14,500, 15,000, 17,500, 18,000, 18,500, 19,000 or 19,500 pg of modified BoNT / A, preferably the lower limit is 8,750 pg, 11,376 pg, 11,377 pg, 11,395 pg, 11,400 pg, 16,590 pg or 16,591 pg. Preferably, the unit dosage form contains more than 8,500 pg and up to 20,000 pg, for example, more than 8,500 pg and up to 18,000 pg or 10,000 to 19,500 modified BoNT / A. The unit dosage form may contain 11,000 to 19,000 pg, preferably 11,500 pg to 19,000 pg or 17,000 to 19,000 pg modified BoNT / A. The unit dosage form may contain 12,000 pg to 18,000 pg or 12,500 pg to 18,000 pg modified BoNT / A. The unit dosage form may contain 12,500 pg to 20,000 pg modified BoNT / A. In preferred embodiments, the unit dosage form contains 12,000, 12,500, 15,000 or 18,000 pg of modified BoNT / A. In more preferred embodiments, the unit dosage form contains 15,000 or 18,000 pg (e.g., 18,000 pg) of modified BoNT / A.

[0246] More preferably, the unit dosage form for treating childhood limb spasticity contains 15,000 pg to 20,000 pg, for example, 17,500 to 18,500 pg of modified BoNT / A. More preferably, the unit dosage form for treating childhood limb spasticity contains 18,000 pg of modified BoNT / A.

[0247] It is even more preferred that the unit dosage form for treating childhood limb spasticity contains 10,000 pg to 15,000 pg, for example, 12,000 pg to 13,000 pg of modified BoNT / A. More preferably, the unit dosage form for treating childhood limb spasticity contains 12,500 pg of modified BoNT / A.

[0248] The present invention provides, in one aspect, the above-mentioned unit dosage form for use in treating pediatric limb spasticity.Also provides the corresponding method for treating pediatric limb spasticity and its use in manufacturing the medicament for treating pediatric limb spasticity.The treatment is preferably consistent with the treatment described herein.

[0249] The modified BoNT / A present in the unit dosage form is preferably in a two-chain form. Thus, the modified BoNT / A is preferably a two-chain modified BoNT / A as described herein (and / or produced as described herein).

[0250] The unit dosage form is preferably provided as a dry powder.

[0251] In another aspect, the present invention provides a kit comprising: a. a unit dosage form according to the present invention, and b. instructions for its use in treating limb spasticity, and c. Optionally, a diluent.

[0252] In one aspect, the invention provides a modified botulinum neurotoxin A (BoNT / A) for use in treating adult limb spasticity, wherein the modified BoNT / A is administered by intramuscular injection into multiple affected muscles of a subject; wherein the modified BoNT / A is administered in a unit dose of 10,000 pg to 20,000 pg (e.g., 14,000 pg to 16,000 pg, preferably 15,000 pg) of modified BoNT / A in multiple affected muscles; wherein the plurality of affected muscles is selected from: A first group including: flexor digitorum superficialis, flexor digitorum profundus, flexor carpi radialis, flexor carpi ulnaris, brachioradialis, pronator teres, biceps brachii, medial head of gastrocnemius (gastrocnemius medialis), lateral head of gastrocnemius (gastrocnemius lateralis), flexor digitorum longus, flexor hallucis longus, gastrocnemius, deltoid, levator scapulae, pronator quadratus, flexor hallucis longus, adductor pollicis brevis, palmaris longus, lumbricalis, opponens pollicis, adductor magnus, adductor longus, adductor brevis, gracilis, medial hamstrings, lateral hamstrings, tensor fasciae latae, rectus femoris, vastus lateralis, vastus medialis, vastus intermedius, gluteus maximus, tibialis anterior, flexor digitorum brevis, extensor hallucis longus, and flexor hallucis brevis, and A second group including: triceps brachii (long head), subscapularis, pectoralis muscles (e.g., pectoralis major), latissimus dorsi, biceps brachii, brachialis, soleus, tibialis posterior, brachioradialis, teres major, iliopsoas, adductor magnus, hamstring muscles (e.g., biceps femoris, semimembranosus, and / or semitendinosus), rectus femoris, and gastrocnemius; Preferably, wherein at least one of the multiple affected muscles is selected from the following: adductor magnus, hamstring muscles (e.g., biceps femoris, semimembranosus and / or semitendinosus) and rectus femoris, and wherein a single unit dose is administered in the affected muscle of a first group and / or multiple unit doses are administered in the affected muscle of a second group, wherein the total dose administered during treatment is up to 300,000 pg (e.g., up to 240,000 pg, preferably up to 225,000 pg); and Here, the modified BoNT / A is a BoNT / A comprising a light chain and a translocation domain and a BoNT / B receptor binding domain (H C domain).

[0253] In one aspect, the invention provides a method of treating adult limb spasticity, the method comprising administering a modified botulinum neurotoxin A (BoNT / A) by intramuscular injection to multiple affected muscles of a subject; wherein the modified BoNT / A is administered in a unit dose of 10,000 pg to 20,000 pg (e.g., 14,000 pg to 16,000 pg, preferably 15,000 pg) of modified BoNT / A in multiple affected muscles; wherein the plurality of affected muscles is selected from: A first group including: flexor digitorum superficialis, flexor digitorum profundus, flexor carpi radialis, flexor carpi ulnaris, brachioradialis, pronator teres, biceps brachii, medial head of gastrocnemius (gastrocnemius medialis), lateral head of gastrocnemius (gastrocnemius lateralis), flexor digitorum longus, flexor hallucis longus, gastrocnemius, deltoid, levator scapulae, pronator quadratus, flexor hallucis longus, adductor pollicis brevis, palmaris longus, lumbricalis, opponens pollicis, adductor magnus, adductor longus, adductor brevis, gracilis, medial hamstrings, lateral hamstrings, tensor fasciae latae, rectus femoris, vastus lateralis, vastus medialis, vastus intermedius, gluteus maximus, tibialis anterior, flexor digitorum brevis, extensor hallucis longus, and flexor hallucis brevis, and A second group including: triceps brachii (long head), subscapularis, pectoralis muscles (e.g., pectoralis major), latissimus dorsi, biceps brachii, brachialis, soleus, tibialis posterior, brachioradialis, teres major, iliopsoas, adductor magnus, hamstring muscles (e.g., biceps femoris, semimembranosus, and / or semitendinosus), rectus femoris, and gastrocnemius; Preferably, wherein at least one of the plurality of affected muscles is selected from the adductor magnus, the hamstring muscles (e.g., the biceps femoris, the semimembranosus and / or the semitendinosus) and the rectus femoris; and wherein a single unit dose is administered in the affected muscle of a first group and / or multiple unit doses are administered in the affected muscle of a second group, wherein the total dose administered during treatment is up to 300,000 pg (e.g., up to 240,000 pg, preferably up to 225,000 pg); and Here, the modified BoNT / A comprises a BoNT / A light chain and translocation domain and a BoNT / B receptor binding domain (H C domain).

[0254] In one aspect, the invention provides the use of a modified botulinum neurotoxin A (BoNT / A) in the manufacture of a medicament for treating adult limb spasticity, wherein the modified BoNT / A is administered by intramuscular injection into multiple affected muscles of a subject; wherein the modified BoNT / A is administered in a unit dose of 10,000 pg to 20,000 pg (e.g., 14,000 pg to 16,000 pg, preferably 15,000 pg) of modified BoNT / A in multiple affected muscles; wherein the plurality of affected muscles is selected from: A first group including: flexor digitorum superficialis, flexor digitorum profundus, flexor carpi radialis, flexor carpi ulnaris, brachioradialis, pronator teres, biceps brachii, medial head of gastrocnemius (gastrocnemius medialis), lateral head of gastrocnemius (gastrocnemius lateralis), flexor digitorum longus, flexor hallucis longus, gastrocnemius, deltoid, levator scapulae, pronator quadratus, flexor hallucis longus, adductor pollicis brevis, palmaris longus, lumbricalis, opponens pollicis, adductor magnus, adductor longus, adductor brevis, gracilis, medial hamstrings, lateral hamstrings, tensor fasciae latae, rectus femoris, vastus lateralis, vastus medialis, vastus intermedius, gluteus maximus, tibialis anterior, flexor digitorum brevis, extensor hallucis longus, and flexor hallucis brevis, and A second group including: triceps brachii (long head), subscapularis, pectoralis muscles (e.g., pectoralis major), latissimus dorsi, biceps brachii, brachialis, soleus, tibialis posterior, brachioradialis, teres major, iliopsoas, adductor magnus, hamstring muscles (e.g., biceps femoris, semimembranosus, and / or semitendinosus), rectus femoris, and gastrocnemius; Preferably, wherein at least one of the plurality of affected muscles is selected from the adductor magnus, the hamstring muscles (e.g., the biceps femoris, the semimembranosus and / or the semitendinosus) and the rectus femoris; and wherein a single unit dose is administered in the affected muscle of a first group and / or multiple unit doses are administered in the affected muscle of a second group, wherein the total dose administered during treatment is up to 300,000 pg (e.g., up to 240,000 pg, preferably up to 225,000 pg); and Here, the modified BoNT / A comprises a BoNT / A light chain and translocation domain and a BoNT / B receptor binding domain (H C domain).

[0255] In the above embodiments, preferably the unit dose is 15,000 pg and the total dose is up to 225,000 pg (eg, the total dose can be 225,000 pg).

[0256] In one aspect, the invention provides a modified botulinum neurotoxin A (BoNT / A) for use in treating adult limb spasticity, wherein the modified BoNT / A is administered by intramuscular injection into multiple affected muscles of a subject; Here, the modified BoNT / A is administered in a unit dose of 416 units to 832 units (e.g., 582 units to 666 units, preferably 624 units) of modified BoNT / A in multiple affected muscles, where one unit is the calculated median lethal dose in mice (LD 50 ) is the amount of modified BoNT / A corresponding to wherein the plurality of affected muscles is selected from: A first group including: flexor digitorum superficialis, flexor digitorum profundus, flexor carpi radialis, flexor carpi ulnaris, brachioradialis, pronator teres, biceps brachii, medial head of gastrocnemius (gastrocnemius medialis), lateral head of gastrocnemius (gastrocnemius lateralis), flexor digitorum longus, flexor hallucis longus, gastrocnemius, deltoid, levator scapulae, pronator quadratus, flexor hallucis longus, adductor pollicis brevis, palmaris longus, lumbricalis, opponens pollicis, adductor magnus, adductor longus, adductor brevis, gracilis, medial hamstrings, lateral hamstrings, tensor fasciae latae, rectus femoris, vastus lateralis, vastus medialis, vastus intermedius, gluteus maximus, tibialis anterior, flexor digitorum brevis, extensor hallucis longus, and flexor hallucis brevis, and A second group including: triceps brachii (long head), subscapularis, pectoralis muscles (e.g., pectoralis major), latissimus dorsi, biceps brachii, brachialis, soleus, tibialis posterior, brachioradialis, teres major, iliopsoas, adductor magnus, hamstring muscles (e.g., biceps femoris, semimembranosus, and / or semitendinosus), rectus femoris, and gastrocnemius; Preferably, wherein at least one of the plurality of affected muscles is selected from the adductor magnus, the hamstring muscles (e.g., the biceps femoris, the semimembranosus and / or the semitendinosus) and the rectus femoris; and wherein a single unit dose is administered in the affected muscle of a first group and / or multiple unit doses are administered in the affected muscle of a second group, wherein the total dose administered during treatment is up to 12,480 units (e.g., up to 9,990 units, preferably up to 9,360 units); and Here, the modified BoNT / A comprises a BoNT / A light chain and translocation domain and a BoNT / B receptor binding domain (H C domain).

[0257] In one aspect, the invention provides a method of treating adult limb spasticity, the method comprising administering modified botulinum neurotoxin A (BoNT / A) by intramuscular injection to a plurality of affected muscles in a subject; Here, the modified BoNT / A is administered in a unit dose of 416 units to 832 units (e.g., 582 units to 666 units, preferably 624 units) of modified BoNT / A in multiple affected muscles, where one unit is the calculated median lethal dose in mice (LD 50 ) is the amount of modified BoNT / A corresponding to wherein the plurality of affected muscles is selected from: A first group including: flexor digitorum superficialis, flexor digitorum profundus, flexor carpi radialis, flexor carpi ulnaris, brachioradialis, pronator teres, biceps brachii, medial head of gastrocnemius (gastrocnemius medialis), lateral head of gastrocnemius (gastrocnemius lateralis), flexor digitorum longus, flexor hallucis longus, gastrocnemius, deltoid, levator scapulae, pronator quadratus, flexor hallucis longus, adductor pollicis brevis, palmaris longus, lumbricalis, opponens pollicis, adductor magnus, adductor longus, adductor brevis, gracilis, medial hamstrings, lateral hamstrings, tensor fasciae latae, rectus femoris, vastus lateralis, vastus medialis, vastus intermedius, gluteus maximus, tibialis anterior, flexor digitorum brevis, extensor hallucis longus, and flexor hallucis brevis, and A second group including: triceps brachii (long head), subscapularis, pectoralis muscles (e.g., pectoralis major), latissimus dorsi, biceps brachii, brachialis, soleus, tibialis posterior, brachioradialis, teres major, iliopsoas, adductor magnus, hamstring muscles (e.g., biceps femoris, semimembranosus, and / or semitendinosus), rectus femoris, and gastrocnemius; Preferably, wherein at least one of the plurality of affected muscles is selected from the adductor magnus, the hamstring muscles (e.g., the biceps femoris, the semimembranosus and / or the semitendinosus) and the rectus femoris; and wherein a single unit dose is administered in the affected muscle of a first group and / or multiple unit doses are administered in the affected muscle of a second group, wherein the total dose administered during treatment is up to 12,480 units (e.g., up to 9,990 units, preferably up to 9,360 units); Here, the modified BoNT / A comprises a BoNT / A light chain and translocation domain and a BoNT / B receptor binding domain (H C domain).

[0258] In one aspect, the invention provides the use of a modified botulinum neurotoxin A (BoNT / A) in the manufacture of a medicament for treating adult limb spasticity, wherein the modified BoNT / A is administered by intramuscular injection into multiple affected muscles of a subject; Here, the modified BoNT / A is administered in a unit dose of 416 units to 832 units (e.g., 582 units to 666 units, preferably 624 units) of modified BoNT / A in multiple affected muscles, where one unit is the calculated median lethal dose in mice (LD 50 ) is the amount of modified BoNT / A corresponding to wherein the plurality of affected muscles is selected from: A first group including: flexor digitorum superficialis, flexor digitorum profundus, flexor carpi radialis, flexor carpi ulnaris, brachioradialis, pronator teres, biceps brachii, medial head of gastrocnemius (gastrocnemius medialis), lateral head of gastrocnemius (gastrocnemius lateralis), flexor digitorum longus, flexor hallucis longus, gastrocnemius, deltoid, levator scapulae, pronator quadratus, flexor hallucis longus, adductor pollicis brevis, palmaris longus, lumbricalis, opponens pollicis, adductor magnus, adductor longus, adductor brevis, gracilis, medial hamstrings, lateral hamstrings, tensor fasciae latae, rectus femoris, vastus lateralis, vastus medialis, vastus intermedius, gluteus maximus, tibialis anterior, flexor digitorum brevis, extensor hallucis longus, and flexor hallucis brevis, and A second group including: triceps brachii (long head), subscapularis, pectoralis muscles (e.g., pectoralis major), latissimus dorsi, biceps brachii, brachialis, soleus, tibialis posterior, brachioradialis, teres major, iliopsoas, adductor magnus, hamstring muscles (e.g., biceps femoris, semimembranosus, and / or semitendinosus), rectus femoris, and gastrocnemius; Preferably, wherein at least one of the plurality of affected muscles is selected from the adductor magnus, the hamstring muscles (e.g., the biceps femoris, the semimembranosus and / or the semitendinosus) and the rectus femoris; and wherein a single unit dose is administered in the affected muscle of a first group and / or multiple unit doses are administered in the affected muscle of a second group, wherein the total dose administered during treatment is up to 12,480 units (e.g., up to 9,990 units, preferably up to 9,360 units); and Here, the modified BoNT / A comprises a BoNT / A light chain and translocation domain and a BoNT / B receptor binding domain (H C domain).

[0259] In the above embodiment, preferably the unit dose is 624 units and the total dose is up to 9,360 units (eg, the total dose can be 9,360 units).

[0260] In one aspect, the invention provides a modified botulinum neurotoxin A (BoNT / A) for use in treating childhood limb spasticity, wherein the modified BoNT / A is administered by intramuscular injection into multiple affected muscles of a subject; wherein the modified BoNT / A is administered in a unit dose of 5,000 pg to 10,000 pg (e.g., 7,000 pg to 8,000 pg, preferably 7,500 pg) of modified BoNT / A in multiple affected muscles; wherein the plurality of affected muscles is selected from: A first group including: flexor digitorum superficialis, flexor digitorum profundus, flexor carpi radialis, flexor carpi ulnaris, brachioradialis, pronator teres, biceps brachii, medial head of gastrocnemius (gastrocnemius medialis), lateral head of gastrocnemius (gastrocnemius lateralis), flexor digitorum longus, flexor hallucis longus, gastrocnemius, deltoid, levator scapulae, pronator quadratus, flexor hallucis longus, adductor pollicis brevis, palmaris longus, lumbricalis, opponens pollicis, adductor magnus, adductor longus, adductor brevis, gracilis, medial hamstrings, lateral hamstrings, tensor fasciae latae, rectus femoris, vastus lateralis, vastus medialis, vastus intermedius, gluteus maximus, tibialis anterior, flexor digitorum brevis, extensor hallucis longus, and flexor hallucis brevis, and A second group including: triceps brachii (long head), subscapularis, pectoralis muscles (e.g., pectoralis major), latissimus dorsi, biceps brachii, brachialis, soleus, tibialis posterior, brachioradialis, teres major, iliopsoas, adductor magnus, hamstring muscles (e.g., biceps femoris, semimembranosus, and / or semitendinosus), rectus femoris, and gastrocnemius; Preferably, wherein at least one of the plurality of affected muscles is selected from the adductor magnus, the hamstring muscles (e.g., the biceps femoris, the semimembranosus and / or the semitendinosus) and the rectus femoris; and wherein a single unit dose is administered in the affected muscle of a first group and / or multiple unit doses are administered in the affected muscle of a second group, wherein the total dose administered during treatment is up to 150,000 pg (e.g., up to 120,000 pg, preferably up to 112,500 pg); and Here, the modified BoNT / A comprises a BoNT / A light chain and translocation domain and a BoNT / B receptor binding domain (H C domain).

[0261] In one aspect, the invention provides a method of treating childhood limb spasticity, the method comprising administering modified botulinum neurotoxin A (BoNT / A) by intramuscular injection to a plurality of affected muscles of a subject; wherein the modified BoNT / A is administered in a unit dose of 5,000 pg to 10,000 pg (e.g., 7,000 pg to 8,000 pg, preferably 7,500 pg) of modified BoNT / A in multiple affected muscles; wherein the plurality of affected muscles is selected from: A first group including: flexor digitorum superficialis, flexor digitorum profundus, flexor carpi radialis, flexor carpi ulnaris, brachioradialis, pronator teres, biceps brachii, medial head of gastrocnemius (gastrocnemius medialis), lateral head of gastrocnemius (gastrocnemius lateralis), flexor digitorum longus, flexor hallucis longus, gastrocnemius, deltoid, levator scapulae, pronator quadratus, flexor hallucis longus, adductor pollicis brevis, palmaris longus, lumbricalis, opponens pollicis, adductor magnus, adductor longus, adductor brevis, gracilis, medial hamstrings, lateral hamstrings, tensor fasciae latae, rectus femoris, vastus lateralis, vastus medialis, vastus intermedius, gluteus maximus, tibialis anterior, flexor digitorum brevis, extensor hallucis longus, and flexor hallucis brevis, and A second group including: triceps brachii (long head), subscapularis, pectoralis muscles (e.g., pectoralis major), latissimus dorsi, biceps brachii, brachialis, soleus, tibialis posterior, brachioradialis, teres major, iliopsoas, adductor magnus, hamstring muscles (e.g., biceps femoris, semimembranosus, and / or semitendinosus), rectus femoris, and gastrocnemius; Preferably, wherein at least one of the plurality of affected muscles is selected from the adductor magnus, the hamstring muscles (e.g., the biceps femoris, the semimembranosus and / or the semitendinosus) and the rectus femoris; and wherein a single unit dose is administered in the affected muscle of a first group and / or multiple unit doses are administered in the affected muscle of a second group, wherein the total dose administered during treatment is up to 150,000 pg (e.g., up to 120,000 pg, preferably up to 112,500 pg); and Here, the modified BoNT / A comprises a BoNT / A light chain and translocation domain and a BoNT / B receptor binding domain (H C domain).

[0262] In one aspect, the invention provides the use of a modified botulinum neurotoxin A (BoNT / A) in the manufacture of a medicament for treating childhood limb spasticity, wherein the modified BoNT / A is administered by intramuscular injection into multiple affected muscles of a subject; wherein the modified BoNT / A is administered in a unit dose of 5,000 pg to 10,000 pg (e.g., 7,000 pg to 8,000 pg, preferably 7,500 pg) of modified BoNT / A in multiple affected muscles; wherein the plurality of affected muscles is selected from: A first group including: flexor digitorum superficialis, flexor digitorum profundus, flexor carpi radialis, flexor carpi ulnaris, brachioradialis, pronator teres, biceps brachii, medial head of gastrocnemius (gastrocnemius medialis), lateral head of gastrocnemius (gastrocnemius lateralis), flexor digitorum longus, flexor hallucis longus, gastrocnemius, deltoid, levator scapulae, pronator quadratus, flexor hallucis longus, adductor pollicis brevis, palmaris longus, lumbricalis, opponens pollicis, adductor magnus, adductor longus, adductor brevis, gracilis, medial hamstrings, lateral hamstrings, tensor fasciae latae, rectus femoris, vastus lateralis, vastus medialis, vastus intermedius, gluteus maximus, tibialis anterior, flexor digitorum brevis, extensor hallucis longus, and flexor hallucis brevis, and A second group including: triceps brachii (long head), subscapularis, pectoralis muscles (e.g., pectoralis major), latissimus dorsi, biceps brachii, brachialis, soleus, tibialis posterior, brachioradialis, teres major, iliopsoas, adductor magnus, hamstring muscles (e.g., biceps femoris, semimembranosus, and / or semitendinosus), rectus femoris, and gastrocnemius; Preferably, wherein at least one of the plurality of affected muscles is selected from the adductor magnus, the hamstring muscles (e.g., the biceps femoris, the semimembranosus and / or the semitendinosus) and the rectus femoris; and wherein a single unit dose is administered in the affected muscle of a first group and / or multiple unit doses are administered in the affected muscle of a second group, wherein the total dose administered during treatment is up to 150,000 pg (e.g., up to 120,000 pg, preferably up to 112,500 pg); and Here, the modified BoNT / A comprises a BoNT / A light chain and translocation domain and a BoNT / B receptor binding domain (H C domain).

[0263] In the foregoing embodiment, preferably the unit dose is 7,500 pg and the total dose is up to 112,500 pg (eg, the total dose can be 112,500 pg).

[0264] In one aspect, the invention provides a modified botulinum neurotoxin A (BoNT / A) for use in treating childhood limb spasticity, wherein the modified BoNT / A is administered by intramuscular injection into multiple affected muscles of a subject; Here, the modified BoNT / A is administered in a unit dose of 208 units to 416 units (e.g., 291 units to 333 units, preferably 312 units) of modified BoNT / A in multiple affected muscles, where one unit is the calculated median lethal dose in mice (LD 50 ) is the amount of modified BoNT / A corresponding to wherein the plurality of affected muscles is selected from: A first group including: flexor digitorum superficialis, flexor digitorum profundus, flexor carpi radialis, flexor carpi ulnaris, brachioradialis, pronator teres, biceps brachii, medial head of gastrocnemius (gastrocnemius medialis), lateral head of gastrocnemius (gastrocnemius lateralis), flexor digitorum longus, flexor hallucis longus, gastrocnemius, deltoid, levator scapulae, pronator quadratus, flexor hallucis longus, adductor pollicis brevis, palmaris longus, lumbricalis, opponens pollicis, adductor magnus, adductor longus, adductor brevis, gracilis, medial hamstrings, lateral hamstrings, tensor fasciae latae, rectus femoris, vastus lateralis, vastus medialis, vastus intermedius, gluteus maximus, tibialis anterior, flexor digitorum brevis, extensor hallucis longus, and flexor hallucis brevis, and A second group including: triceps brachii (long head), subscapularis, pectoralis muscles (e.g., pectoralis major), latissimus dorsi, biceps brachii, brachialis, soleus, tibialis posterior, brachioradialis, teres major, iliopsoas, adductor magnus, hamstring muscles (e.g., biceps femoris, semimembranosus, and / or semitendinosus), rectus femoris, and gastrocnemius; Preferably, wherein at least one of the plurality of affected muscles is selected from the adductor magnus, the hamstring muscles (e.g., the biceps femoris, the semimembranosus and / or the semitendinosus) and the rectus femoris; and wherein a single unit dose is administered in the affected muscle of a first group and / or multiple unit doses are administered in the affected muscle of a second group, wherein the total dose administered during treatment is up to 6,240 units (e.g., up to 4,995 units, preferably up to 4,680 units); and Here, the modified BoNT / A comprises a BoNT / A light chain and translocation domain and a BoNT / B receptor binding domain (H C domain).

[0265] In one aspect, the invention provides a method of treating childhood limb spasticity, the method comprising administering modified botulinum neurotoxin A (BoNT / A) by intramuscular injection to a plurality of affected muscles of a subject; Here, the modified BoNT / A is administered in a unit dose of 208 units to 416 units (e.g., 291 units to 333 units, preferably 312 units) of modified BoNT / A in multiple affected muscles, where one unit is the calculated median lethal dose in mice (LD 50 ) is the amount of modified BoNT / A corresponding to wherein the plurality of affected muscles is selected from: A first group including: flexor digitorum superficialis, flexor digitorum profundus, flexor carpi radialis, flexor carpi ulnaris, brachioradialis, pronator teres, biceps brachii, medial head of gastrocnemius (gastrocnemius medialis), lateral head of gastrocnemius (gastrocnemius lateralis), flexor digitorum longus, flexor hallucis longus, gastrocnemius, deltoid, levator scapulae, pronator quadratus, flexor hallucis longus, adductor pollicis brevis, palmaris longus, lumbricalis, opponens pollicis, adductor magnus, adductor longus, adductor brevis, gracilis, medial hamstrings, lateral hamstrings, tensor fasciae latae, rectus femoris, vastus lateralis, vastus medialis, vastus intermedius, gluteus maximus, tibialis anterior, flexor digitorum brevis, extensor hallucis longus, and flexor hallucis brevis, and A second group including: triceps brachii (long head), subscapularis, pectoralis muscles (e.g., pectoralis major), latissimus dorsi, biceps brachii, brachialis, soleus, tibialis posterior, brachioradialis, teres major, iliopsoas, adductor magnus, hamstring muscles (e.g., biceps femoris, semimembranosus, and / or semitendinosus), rectus femoris, and gastrocnemius; Preferably, wherein at least one of the plurality of affected muscles is selected from the adductor magnus, the hamstring muscles (e.g., the biceps femoris, the semimembranosus and / or the semitendinosus) and the rectus femoris; and wherein a single unit dose is administered in the affected muscle of a first group and / or multiple unit doses are administered in the affected muscle of a second group, wherein the total dose administered during treatment is up to 6,240 units (e.g., up to 4,995 units, preferably up to 4,680 units); and Here, the modified BoNT / A comprises a BoNT / A light chain and translocation domain and a BoNT / B receptor binding domain (H C domain).

[0266] In one aspect, the invention provides the use of a modified botulinum neurotoxin A (BoNT / A) in the manufacture of a medicament for treating childhood limb spasticity, wherein the modified BoNT / A is administered by intramuscular injection into multiple affected muscles of a subject; Here, the modified BoNT / A is administered in a unit dose of 208 units to 416 units (e.g., 291 units to 333 units, preferably 312 units) of modified BoNT / A in multiple affected muscles, where one unit is the calculated median lethal dose in mice (LD 50 ) is the amount of modified BoNT / A corresponding to wherein the plurality of affected muscles is selected from: A first group including: flexor digitorum superficialis, flexor digitorum profundus, flexor carpi radialis, flexor carpi ulnaris, brachioradialis, pronator teres, biceps brachii, medial head of gastrocnemius (gastrocnemius medialis), lateral head of gastrocnemius (gastrocnemius lateralis), flexor digitorum longus, flexor hallucis longus, gastrocnemius, deltoid, levator scapulae, pronator quadratus, flexor hallucis longus, adductor pollicis brevis, palmaris longus, lumbricalis, opponens pollicis, adductor magnus, adductor longus, adductor brevis, gracilis, medial hamstrings, lateral hamstrings, tensor fasciae latae, rectus femoris, vastus lateralis, vastus medialis, vastus intermedius, gluteus maximus, tibialis anterior, flexor digitorum brevis, extensor hallucis longus, and flexor hallucis brevis, and A second group including: triceps brachii (long head), subscapularis, pectoralis muscles (e.g., pectoralis major), latissimus dorsi, biceps brachii, brachialis, soleus, tibialis posterior, brachioradialis, teres major, iliopsoas, adductor magnus, hamstring muscles (e.g., biceps femoris, semimembranosus, and / or semitendinosus), rectus femoris, and gastrocnemius; Preferably, wherein at least one of the plurality of affected muscles is selected from the adductor magnus, the hamstring muscles (e.g., the biceps femoris, the semimembranosus and / or the semitendinosus) and the rectus femoris; and wherein a single unit dose is administered in the affected muscle of a first group and / or multiple unit doses are administered in the affected muscle of a second group, wherein the total dose administered during treatment is up to 6,240 units (e.g., up to 4,995 units, preferably up to 4,680 units); and Here, the modified BoNT / A comprises a BoNT / A light chain and translocation domain and a BoNT / B receptor binding domain (H C domain).

[0267] In the above embodiment, preferably the unit dose is 312 units and the total dose is up to 4,680 units (eg, the total dose can be 4,680 units).

[0268] In any of the above embodiments, limb spasticity can be treated for a longer period of time than that treated with unmodified BoNT / A (eg, SEQ ID NO:2 [eg, the two-chain form of SEQ ID NO:2]).

[0269] The total number of unit doses administered in a given treatment can be up to 15x unit doses. In other words, in one embodiment, 15x single unit doses can be administered at 15x injection sites. In another embodiment, 15x single unit doses can be administered at more than 15x injection sites. The total number of unit doses can be divided according to the muscle being treated, for example, 2x unit doses can be administered to the latissimus dorsi, 2x to the subscapularis, 2x to the pectoralis major, and 1x to the flexor carpi ulnaris, with the sum of the administered multiple unit doses being 7x. For example, the total number of unit doses administered can be up to 14x, 13x, 12x, 11x, 10x, 9x, 8x, or 7x. The total number of unit doses administered can be at least 2x, 3x, 4x, 5x, 6x, 7x unit doses, preferably at least 2x. The total number of unit doses administered can be 2x to 15x, 7x to 15x, or 10x to 14x. Preferably, the number of unit doses administered is 15x.

[0270] The embodiments of the invention relating to the various therapeutic uses are intended to apply equally to the methods, compositions (eg, unit dosage forms) and kits of the invention, and vice versa.

[0271] sequence homology

[0272] Any of a variety of sequence alignment methods can be used to determine percent identity, including, but not limited to, global, local and hybrid methods, such as segment approach methods. Protocols for determining percent identity are routine within the skill of the art. Global methods align sequences from the beginning to the end of the molecule and determine the best alignment by summing the scores of individual residue pairs and by imposing gap penalties. Non-limiting methods include, for example, CLUSTAL W, see, e.g., Julie D. Thompson et al., CLUSTAL W: Improving the Sensitivity of Progressive Multiple Sequence Alignment Through Sequence Weighting, Position- Specific Gap Penalties and Weight Matrix Choice, 22(22) Nucleic Acids Research 4673-4680 (1994), and iterative refinement, see, e.g., Osamu Gotoh, Significant Improvement in Accuracy of Multiple Protein. Sequence Alignments by Iterative Refinement as Assessed by Reference to Structural Alignments, 264(4) J. MoI. Biol. 823-838 (1996). Local methods align sequences by identifying one or more conserved motifs shared by all of the input sequences.Non-limiting methods include, for example, Match-box, see e.g., Eric Depiereux and Ernest Feytmans, Match-Box: A Fundamentally New Algorithm for the Simultaneous Alignment of Several Protein Sequences, 8(5) CABIOS 501 -509 (1992), Gibbs sampling, see e.g., CE Lawrence et al., Detecting Subtle Sequence Signals: A Gibbs Sampling Strategy for Multiple Alignment, 262(5131 ) Science 208-214 (1993), Align-M, see e.g., Ivo Van WaIIe et al., Align-MA New Algorithm for Multiple Alignment of Highly Divergent Sequences, 20(9) Bioinformatics:1428-1435 (2004).

[0273] Thus, sequence identity percentage is determined by conventional methods.See, for example, Altschul et al., Bull. Math. Bio. 48: 603-16, 1986 and Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915-19, 1992.In brief, two amino acid sequences are aligned to optimize alignment score using a gap opening penalty of 10, a gap extension penalty of 1, and the "blosum 62" scoring matrix of Henikoff and Henikoff (ibid.) as shown below (amino acids are indicated by standard single letter code), and preferably, this method is used to align the sequence with the subject sequence herein (e.g., SEQ ID NO: 2) to define the amino acid position numbering as described herein.

[0274] The "percent sequence identity" between two or more nucleic acid or amino acid sequences is a function of the number of identical positions shared by the sequences.Thus, percent identity can be calculated as the number of identical nucleotides / amino acids divided by the total number of nucleotides / amino acids and multiplied by 100.The calculation of percent sequence identity can also take into account the number of gaps and the length of each gap that need to be introduced to optimize the alignment of two or more sequences.The sequence comparison and percent identity determination between two or more sequences can be carried out using a certain mathematical algorithm, such as BLAST, that will be familiar to those skilled in the art.

[0275] Alignment scores for determining sequence identity

number

[0276] The percent identity is then calculated as: [total number of identical matches × 100] / [length of the longer sequence and number of gaps introduced in the longer sequence to align the two sequences]

[0277] Substantially homologous polypeptides are characterized as having one or more amino acid substitutions, deletions, or additions. These changes are preferably of a minor nature, such as conservative amino acid substitutions (see below) and other substitutions that do not significantly affect the folding or activity of the polypeptide, usually small deletions of from one to about 30 amino acids, and small amino- or carboxyl-terminal extensions, such as an amino-terminal methionine residue, small linker peptides of up to about 20-25 residues, or affinity tags.

[0278] Conservative Amino Acid Substitutions Basicity: Arginine Lysine Histidine Acidic: Glutamic acid Aspartic acid polarity: glutamine Asparagine Hydrophobicity: Leucine Isoleucine Ballin Aromatic: Phenylalanine Tryptophan Tyrosine small: glycine Alanine Serine Threonine Methionine

[0279] In addition to the 20 standard amino acids, non-standard amino acids (e.g., 4-hydroxyproline, 6-N-methyllysine, 2-aminoisobutyric acid, isovaline and α-methylserine) can be substituted for the amino acid residues of the polypeptides of the present invention. A limited number of non-conservative amino acids, amino acids that are not encoded by the genetic code and unnatural amino acids can be substituted for the polypeptide amino acid residues. The polypeptides of the present invention can also include non-naturally occurring amino acid residues.

[0280] Non-naturally occurring amino acids include, but are not limited to, trans-3-methylproline, 2,4-methano-proline, cis-4-hydroxyproline, trans-4-hydroxy-proline, N-methylglycine, allo-threonine, methyl-threonine, hydroxy-ethylcysteine, hydroxyethylhomo-cysteine, nitro-glutamine, homoglutamine, pipecolic acid, t-leucine, norvaline, 2-azaphenylalanine, 3-azaphenyl-alanine, 4-azaphenyl-alanine and 4-fluorophenylalanine. Several methods are known in the art for incorporating non-naturally occurring amino acid residues into proteins. For example, an in vitro system can be used in which nonsense mutations are suppressed using chemically aminoacylated suppressor tRNAs. Methods for synthesizing amino acids and aminoacylating tRNAs are known in the art. Transcription and translation of plasmids containing nonsense mutations are carried out in a cell-free system that includes E. coli S30 extracts and commercially available enzymes and other reagents. Proteins are 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 carried out in Xenopus oocytes by microinjection of mutated mRNA and chemically aminoacylated suppressor tRNA (Turcatti et al., J. Biol. Chem. 271:19991-8, 1996). Within the third method, E. coli cells are cultured in the absence of the natural amino acid to be replaced (e.g., phenylalanine) and in the presence of the desired non-naturally occurring amino acid(s) (e.g., 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, or 4-fluorophenylalanine).Non-naturally occurring amino acids are incorporated into polypeptides in place of their natural counterparts. See Koide et al., Biochem. 33:7470-6, 1994. Naturally occurring amino acid residues can be converted to non-naturally occurring species by in vitro chemical modification. To further expand the scope of substitutions, chemical modification can also be combined with site-directed mutagenesis (Wynn and Richards, Protein Sci. 2:395-403, 1993).

[0281] A limited number of non-conservative amino acids, amino acids that are not encoded by the genetic code, non-naturally occurring amino acids, and unnatural amino acids may be substituted for amino acid residues in the polypeptides of the invention.

[0282] Essential amino acids in the polypeptides of the present invention can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine scanning mutagenesis (Cunningham and Wells, Science 244: 1081-5, 1989). Along with mutations of putative contact site amino acids, the sites of biological interaction can also be determined by physical analysis of structures, such as those determined by techniques such as nuclear magnetic resonance, crystallography, electron diffraction or photoaffinity labeling. See, for example, de Vos et al., Science 255:306-12, 1992, Smith et al., J. Mol. Biol. 224:899-904, 1992, Wlodaver et al., FEBS Lett. 309:59-64, 1992. The identity of essential amino acids can also be inferred from the analysis of homology with related components (e.g., translocation or protease components) of the polypeptides of the present invention.

[0283] Multiple amino acid substitutions can be made and tested using known methods of mutagenesis and screening, such as those disclosed by Reidhaar-Olson and Sauer (Science 241:53-7, 1988) or Bowie and Sauer (Proc. Natl. Acad. Sci. USA 86:2152-6, 1989). Briefly, these authors disclose methods in which two or more positions in a polypeptide are simultaneously randomized, functional polypeptides are selected, and the mutagenized polypeptides are then sequenced to determine the range 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. Pat. 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).

[0284] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs. Singleton, et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 20 ED., John Wiley and Sons, New York (1994) and Hale & Marham, THE HARPER COLLINS DICTIONARY OF BIOLOGY, Harper Perennial, NY (1991) provide those skilled in the art with a general dictionary of many of the terms used in this disclosure.

[0285] The present disclosure is not limited by the exemplary methods and materials disclosed herein, and any methods and materials similar or equivalent to those described herein can be used in carrying out or testing the embodiments of the present disclosure.Numerical ranges include the numerical values ​​that define the range.Unless otherwise specified, each nucleic acid sequence is written from left to right in 5' to 3' orientation, and amino acid sequence is written from left to right in amino to carboxy orientation.

[0286] The headings provided herein do not limit the various aspects or embodiments of the disclosure.

[0287] Amino acids are referred to herein using the name, three-letter abbreviation or single-letter abbreviation of the amino acid. The term "protein" as used herein includes proteins, polypeptides and peptides. As used herein, the term "amino acid sequence" is synonymous with the term "polypeptide" and / or the term "protein". In some instances, the term "amino acid sequence" is synonymous with the term "peptide". In some instances, the term "amino acid sequence" is synonymous with the term "enzyme". The terms "protein" and "polypeptide" are used synonymously herein. In the present disclosure and claims, conventional single-letter and three-letter codes for amino acid residues may be used. The three-letter codes for amino acids defined in accordance with the IUPACIUB Joint Commission on Biochemical Nomenclature (JCBN). It is also understood that a polypeptide may be encoded by more than one nucleotide sequence due to the degeneracy of the genetic code.

[0288] Other definitions of terms may appear throughout this specification. Before exemplary embodiments are described in more detail, it should be understood that the present disclosure is not limited to the specific embodiments described, and may vary as such. It should also be understood that the technical terms used in this specification are merely for the purpose of describing specific embodiments, and are not intended to be limiting, since the scope of the present disclosure is defined only by the appended claims.

[0289] Where a range of values ​​is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit, between the upper and lower limits of that range is also specifically disclosed, unless the context clearly dictates otherwise. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the disclosure. The upper and lower limits of these smaller ranges may be independently included or excluded in the range, and each range where either, neither, or both limits are included in the smaller range is also encompassed within the disclosure, subject to any specifically excluded limits in the stated range. Where a stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0290] It must be noted that, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a botulinum neurotoxin A" includes a plurality of such candidate agents, reference to "the botulinum neurotoxin A" includes reference to one or more Clostridial neurotoxins and equivalents thereof known to those skilled in the art, and so forth.

[0291] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application, and nothing herein should be construed as an admission that such publications constitute prior art to the claims appended hereto.

[0292] Embodiments of the present invention will now be described, by way of example only, with reference to the following figures and examples. [Brief description of the drawings]

[0293] [Figure 1] FIG. 1 illustrates the five most common clinical patterns of upper limb spasticity involving the glenohumeral joint. [Diagram 2] FIG. 2 shows the FDA approved dosages of Dysport® for treating spasticity in adults. [Diagram 3] 3 shows an SDS-PAGE of purified recombinant BoNT / AB chimeras 1, 2 and 3A (SEQ ID NOs: 3, 4 and 5, respectively). Lanes are labeled "Marker" (molecular weight marker), "-DTT" (oxidized BoNT / AB chimera sample) and "+DTT" (reduced BoNT / AB chimera sample). [Figure 4]FIG. 4 shows the cleavage of SNAP-25 in rat spinal cord neurons by recombinant BoNT / AB chimeras 1, 2 and 3A (SEQ ID NOs: 3, 4 and 5, respectively, converted to a two-chain form). Cultured rat primary spinal cord neurons (SCN) were exposed to various concentrations of recombinant BoNT / AB chimeras 1, 2 or 3A for 24 hours at 37° C. in a humidified atmosphere containing 10% CO2. Cells were then lysed using 1× NuPAGE buffer supplemented with DTT and benzonase. Samples were transferred to microcentrifuge tubes, heated at 90° C. for 5 minutes on a heat block, stored at −20° C., and then analyzed for SNAP-25 cleavage by Western blot. SNAP-25 was detected using a polyclonal antibody (Sigma #S9684) that detects both full-length and cleaved forms of SNAP-25. Anti-rabbit HRP (Sigma #A6154) was used as the secondary antibody. [Diagram 5] Figure 5 shows the mouse digit abduction scoring assay. Mice were injected into the gastrocnemius-soleus complex muscle of one hind limb under brief general anesthesia, and muscle weakness was measured using the digit abduction score (DAS) on a 0-4 scale. The highest DAS value for each dose was determined and plotted against dose, and the data was fitted to a 4-parameter logistic equation to determine the ED50 and the dose leading to DAS 4 (DAS 4 dose) values. [Figure 6] 6 shows an SDS-PAGE of purified recombinant BoNT / AB chimeras 3B and 3C (SEQ ID NOs: 6 and 7, respectively). Lanes are labeled "Marker" (molecular weight marker), "-DTT" (oxidized BoNT / AB chimera sample), and "+DTT" (reduced BoNT / AB chimera sample). [Figure 7]FIG. 7 shows cleavage of SNAP-25 by unmodified BoNT / A and BoNT / AB chimeras 3B and 3C (SEQ ID NOs: 2, 6 and 7, respectively) in human induced pluripotent stem cell-derived peripheral neurons (PERI.4U-Axiogenesis, Germany). PERI.4U cells were exposed to various concentrations of recombinant BoNT / A or BoNT / AB chimeras 3B or 3C for 24 hours at 37° C. in a humidified CO2 atmosphere containing 5% CO2. Cells were then lysed using 1× NuPAGE buffer supplemented with DTT and benzonase. Samples were transferred to microcentrifuge tubes, heated at 90° C. for 5 minutes on a heat block, stored at −20° C., and then analyzed for SNAP-25 cleavage by Western blot. SNAP-25 was detected using a polyclonal antibody (Sigma #S9684) that detects both full-length and cleaved forms of SNAP-25. Anti-rabbit HRP (Sigma #A6154) was used as the secondary antibody. [Figure 8] Figure 8 shows the duration of muscle weakness over time in a mouse digit abduction scoring assay. Mice were injected into the gastrocnemius-soleus complex muscle of one hind limb under brief general anesthesia, and muscle weakness was measured using the digit abduction score (DAS) on a 0-4 scale. Animals from the group injected with the lowest dose that induced a DAS of 4 during the first 4 days of injection were monitored until complete recovery of muscle weakness to a DAS of 0 (no observed muscle weakness).

[0294] Sequence Listing

[0295] In any of the following SEQ ID NOs, where the first Met amino acid residue or the corresponding first codon is shown, said residue / codon is optional.

[0296] SEQ ID NO:1 (Nucleotide sequence of unmodified BoNT / A)

[0297] SEQ ID NO:2 (polypeptide sequence of unmodified BoNT / A)

[0298] SEQ ID NO:3 (polypeptide sequence of modified BoNT / A "chimera 1") MPFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLN PPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGG STIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNGY GSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPN RVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKA KSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIYTEDNFVKFFKV LNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTKLKNFT GLFEFYKLLCVRGIITSKTKSLDKGYNKALNDLCIKVNNWDLFFSPSEDNFTNDLNKGEE ITSDTNIEAAEENISLDLIQQYYLTFNFDNEPENISIENLSSDIIGQLELMPNIERFPNG KKYELDKYTMFHYLRAQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSDYVKKVNKATEA AMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYKDDFVGALIFSG AVILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALSKRNEKWDEVYKYIVTNWLAK VNTQIDLIRKKMKEALENQAEATKAIINYQYNQYTEEEKNNINFNIDDLSSKLNESINKA MININKFLNQCSVSYLMNSMIPYGVKRLEDFDASLKDALLKYIYDNRGTLIGQVDRLKDK VNNTLSTDIPFQLSKYVDNQRLLSTFTEYIKSEILNNIILNLRYKDNNLIDLSGYGAKVE VYDGVELNDKNQFKLTSSANSKIRVTQNQNIIFNSVFLDFSVSFFWIRIPKYKNDGIQNYI HNEYTIINCMKNNSGWKISIRGNRIIWTLIDINGKTKSVFFEYNIREDISEYINRWFFVT ITNNLNNAKIYINGKLESNTDIKDIREVIANGEIIFKLDGDIDRTQFIWMKYFSIFNTEL SQSNIEERYKIQSYSEYLKDFWGNPLMYNKEYYMFNAGNKNSYIKLKKDSPVGEILTRSK YNQNSKYINYRDLYIGEKFIIRRKSNSQSINDDIVRKEDYIYLDFFNLNQEWRVYTYKYF KKEEMKLFLAPIYDSDEFYNTIQIKEYDEQPTYSCQLLFKKDEESTDEIGLIGIHRFYES GIVFEEYKDYFCISKWYLKEVKRKPYNLKLGCNWQFIPKDEGWTEHHHHHHHHHH

[0299] SEQ ID NO: 4 (polypeptide sequence of modified BoNT / A "chimera 2") MPFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLN PPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGG STIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNGY GSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPN RVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKA KSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIYTEDNFVKFFKV LNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTKLKNFT GLFEFYKLLCVRGIITSKTKSLDKGYNKALNDLCIKVNNWDLFFSPSEDNFTNDLNKGEE ITSDTNIEAAEENISLDLIQQYYLTFNFDNEPENISIENLSSDIIGQLELMPNIERFPNG KKYELDKYTMFHYLRAQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSDYVKKVNKATEA AMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYKDDFVGALIFSG AVILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALSKRNEKWDEVYKYIVTNWLAK VNTQIDLIRKKMKEALENQAEATKAIINYQYNQYTEEEKNNINFNIDDLSSKLNESINKA MININKFLNQCSVSYLMNSMIPYGVKRLEDFDASLKDALLKYIYDNRGTLIGQVDRLKDK VNNTLSTDIPFQLSKYVDNQRLLSTFTEYIKNIIELGGGGSELSEILNNIILNLRYKDNN LIDLSGYGAKVEVYDGVELNDKNQFKLTSSANSKIRVTQNQNIIFNSVFLDFSVSFWIRI PKYKNDGIQNYIHNEYTIINCMKNNSGWKISIRGNRIIWTLIDINGKTKSVFFEYNIRED ISEYINRWFFVTITNNNLNNAKIYINGKLESNTDIKDIREVIANGEIIFKLDGDIDRTQFI WMKYFSIFNTELSQSNIEERYKIQSYSEYLKDFWGNPLMYNKEYYMFNAGNKNSYIKLKK DSPVGEILTRSKYNQNSKYINYRDLYIGEKFIIRRKSNSQSINDDIVRKEDYIYLDFFNL NQEWRVYTYKYFKKEEMKLFLAPIYDSDEFYNTIQIKEYDEQPTYSCQLLFKKDEESTDE IGLIGIHRFYESGIVFEEYKDYFCISKWYLKEVKRKPYNLKLGCNWQFIPKDEGWTEHHH HHHHHHH

[0300] SEQ ID NO:5 (polypeptide sequence of modified BoNT / A "chimera 3A") MPFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLN PPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGG STIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNGY GSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPN RVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKA KSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIYTEDNFVKFFKV LNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTKLKNFT GLFEFYKLLCVRGIITSKTKSLDKGYNKALNDLCIKVNNWDLFFSPSEDNFTNDLNKGEE ITSDTNIEAAEENISLDLIQQYYLTFNFDNEPENISIENLSSDIIGQLELMPNIERFPNG KKYELDKYTMFHYLRAQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSDYVKKVNKATEA AMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYKDDFVGALIFSG AVILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALSKRNEKWDEVYKYIVTNWLAK VNTQIDLIRKKMKEALENQAEATKAIINYQYNQYTEEEKNNINFNIDDLSSKLNESINKA MININKFLNQCSVSYLMNSMIPYGVKRLEDFDASLKDALLKYIYDNRGTLIGQVDRLKDK VNNTLSTDIPFQLSKYVDNQRLLSTFTEYIKNILNNIILNLRYKDNNLIDLSGYGAKVEV YDGVELNDKNQFKLTSSANSKIRVTQNQNIIFNSVFLDFSVSFWIRIPKYKNDGIQNYIH NEYTIINCMKNNSGWKISIRGNRIIWTLIDINGKTKSVFFEYNIREDISEYINRWFFVTI TNNLNNAKIYINGKLESNTDIKDIREVIANGEIIFKLDGDIDRTQFIWMKYFSIFNTELS QSNIEERYKIQSYSEYLKDFWGNPLMYNKEYYMFNAGNKNSYIKLKKDSPVGEILTRSKY NQNSKYINYRDLYIGEKFIIRRKSNSQSINDDIVRKEDYIYLDFFNLNQEWRVYTYKYFK KEEMKLFLAPIYDSDEFYNTIQIKEYDEQPTYSCQLLFKKDEESTDEIGLIGIHRFYESG IVFEEYKDYFCISKWYLKEVKRKPYNLKLGCNWQFIPKDEGWTEHHHHHHHHHH

[0301] SEQ ID NO:6 (Polypeptide sequence of modified BoNT / A "chimera 3B")

[0302] SEQ ID NO: 7 (Polypeptide sequence of modified BoNT / A "chimera 3C") MPFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLN PPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGG STIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNGY GSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPN RVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKA KSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIYTEDNFVKFFKV LNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTKLKNFT GLFEFYKLLCVRGIITSKTKSLDKGYNKALNDLCIKVNNWDLFFSPSEDNFTNDLNKGEE ITSDTNIEAAEENISLDLIQQYYLTFNFDNEPENISIENLSSDIIGQLELMPNIERFPNG KKYELDKYTMFHYLRAQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSDYVKKVNKATEA AMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYKDDFVGALIFSG AVILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALSKRNEKWDEVYKYIVTNWLAK VNTQIDLIRKKMKEALENQAEATKAIINYQYNQYTEEEKNNINFNIDDLSSKLNESINKA MININKFLNQCSVSYLMNSMIPYGVKRLEDFDASLKDALLKYIYDNRGTLIGQVDRLKDK VNNTLSTDIPFQLSKYVDNQRLLSTFTEYIKNILNNIILNLRYKDNNLIDLSGYGAKVEV YDGVELNDKNQFKLTSSANSKIRVTQNQNIIFNSVFLDFSVSFFWIRIPKYKNDGIQNYIH NEYTIINCMKNNSGWKISIRGNRIIWTLIDINGKTKSVFFEYNIREDISEYINRWFFVTI TNNLNNNAKIYINGKLESNTDIKDIREVIANGEIIFKLDGDIDRTQFIWMKYFSIFTELS QSNIEERYKIQSYSEYLKDFWGNPLMYNKEYYMFNAGNKNSYIKLKKDSPVGEILTRSKY NQNSKYINYRDLYIGEKFIIRRKSNSQSINDDIVRKEDYIYLDFFNLNQEWRVYTYKYFK KEEEKLFLAPISDSDEFYNTIQIKEYDEQPTYSCQLLFKKDEESTDEIGLIGIHRFYESG IVFEEYKDYFCISKWYLKEVKRKPYNLKLGCNWQFIPKDEGWTE

[0303] SEQ ID NO:8 (Polypeptide sequence of BoNT / B) MPVTINNFNYNDPIDNNNIIMMEPPFARGTGRYYKAFKITDRIWIIPERYTFGYKPEDFN KSSGIFNRDVCEYYDPDYLNTNDKKNIFLQTMIKLFNRIKSKPLGEKLLEMIINGIPYLG DRRVPLEEFNTNIASVTVNKLISNPGEVERKKGIFANLIIFGPGPVLNENETIDIGIQNH FASREGFGGIMQMKFCPEYVSVFNNVQENKGASIFNRRGYFSDPALILMHELIHVLHGLY GIKVDDLPIVPNEKKFFMQSTDAIQAEELYTFGGQDPSIITPSTDKSIYDKVLQNFRGIV DRNLKVLVCISDPNININIYKNKFKDKYKFVEDSEGKYSIDVESFDKLYKSLMFGFTETN IAENYKIKTRASYFSDSLPPVKIKNLLDNEIYTIEEGFNISDKDMEKEYRGQNKAINKQA YEEISKEHLAVYKIQMCKSVKAPGICIDVDNEDLFFIADKNSFSDDLSKNERIEYNTQSN YIENDFPINELILDTDLISKIELPSENTESLTDFNVDVPVYEKQPAIKKIFTDENTIFQY LYSQTFPLDIRDISLTSSFDDALLFSNKVYSFFSMDYIKTANKVVEAGLFAGWVKQIVND FVIEANKSNTMDKIADISLIVPYIGLALNVGNETAKGNFENAFEIAGASILLEFIPELLI PVVGAFLLESYIDNKNKIIKTIDNALTKRNEKWSDMYGLIVAQWLSTVNTQFYTIKEGMY KALNYQAQALEEIIKYRYNIYSEKEKSNINIDFNDINSKLNEGINQAIDNINNFINGCSV SYLMKKMIPLAVEKLLDFDNTLKKNLLNYIDENKLYLIGSAEYEKSKVNKYLKTIMPFDL SIYTNTILIEMFNKYNSEILNNIILNLRYKDNNLIDLSGYGAKVYDGVELNDKNQFK LTSSANSKIRVTQNQNIIFNSVFLDFSVSFWIRIPKYKNDGIQNYIHNEYTIINCMKNNS GWKISIRGNRIIWTLIDINGKTKSVFFEYNIREDISEYINRWFFVTITNNNLNNAKIYING KLESNTDIKDIREVIANGEIIFKLDGDIDRTQFIWMKYFSIFTELSQSNIEERYKIQSY SEYLKDFWGNPLMYNKEYYMFNAGNKNSYIKLKKDSPVGEILTRSKYNQNSKYINYRDLY IGEKFIIRRKSNSQSINDDIVRKEDYIYLDFFNLNQEWRVYTYKYFKKEEEKLFLAPISD SDEFYNTIQIKEYDEQPTYSCQLLFKKDEESTDEIGLIGIHRFYESGIVFEEYKDYFCIS KWYLKEVKRKPYNLKLGCNWQFIPKDEGWTE

[0304] SEQ ID NO:9 - C-terminal light chain fragment TKSLDKGYNK

[0305] SEQ ID NO:10 - C-terminal light chain fragment 2 SLDKGYNK

[0306] SEQ ID NO:11 - double light chain 1 PFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGGSTIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNGYGSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPNRVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKAKSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIYTEDNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTKLKNFTGLFEFYKLLCVRGIITSK

[0307] Sequence number 12 - double-stranded L chain 2 PFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGGSTIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNGYGSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPNRVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKAKSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIYTEDNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTKLKNFTGLFEFYKLLCVRGIITSKTK

[0308] Sequence number 13 - double-stranded H chain ALNDLCIKVNNWDLFFSPSEDNFTNDLNKGEEITSDTNIEAAENISLDLIQQYYLTFNFDNEPENISIENLSSDIIGQLELMPNIERFPNGKKYELDKYTMFHYLRAQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSDYVKKVNKATEAAMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPANLIGNMLYKDDFVGALIFSGAV ILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALSKRNEKWDEVYKYIVTNWLAKVNTQIDLIRKKMKEALENQAEEATKAIINYQYNQYTEEEKNNINFNIDDLSSKLNESINKAMININKFLNQCSVSYLMNSMIPYGVKRLEDFDASLKDALLKYIYDNRGTLIGQVDRLKDKVNNTLSTDIPFQLSKYVDNQRLLSTFTEYIKNILNN IILNLRYKDNNLIDLSGYGAKVEVYDGVELNDKNQFKLTSSANSKIRVTQNQNIIFNSVFLDFSVSFWIRIPKYKNDGIQNYIHNEYTIINCMKNNSGWKISIRGNRIIWTLIDINGKTKSVFFEYNIREDISEYINRWFFVTITNNLNNAKIYINGKLESNTDIKDIREVIANGEIIFKLDGDIDRTQFIWMKYFSIFNTELSQSNIEERYKI QSYSEYLKDFWGNPLMYNKEYYMFNAGNKNSYIKLKDSPVGEILTRSKYNQNSKYINYRDLYIGEKFIIRRKSNSQSINDDIVRKEDYIYLDFFNNLNQEWRWYTYKYFKKEEMKLFLAPIYDSDEFYNTIQIKEYDEQPTYSCQLLFKKDEESTDEIGLIGIHRFYESGIVFEEYKDYFCISKWYLKEVKRKPYNLKGCNWQFIPKDEGWTE

example

[0309] [example1]

[0310] Cloning, expression and purification of BoNT / AB chimeras

[0311] BoNT / AB chimeric constructs 1, 2, 3A, 3B and 3C (SEQ ID NOs: 3-7, respectively) were constructed using standard molecular biology techniques from DNA encoding the parent serotype molecules and appropriate oligonucleotides. These were then modified to include a C-terminal His 10 The A-tags were cloned into the pJ401 expression vector and transformed into BLR(DE3) E. coli cells for overexpression. The cells were grown in 2L baffled Erlenmeyer flasks containing 1L of modified Terrific Broth (mTB) supplemented with the appropriate antibiotics at 37°C and 225 RPM shaking. 600 When the β reached >0.5, the incubator temperature was reduced to 16° C. and then induced 1 hour later with 1 mM IPTG for 20 hours at 225 RPM shaking to allow expression of the recombinant BoNT / AB constructs.

[0312] Harvested cells were lysed by sonication and clarified by centrifugation at 4500 RPM for 1 hour at 4°C. The recombinant BoNT / AB chimera molecule was then extracted in ammonium sulfate and purified by standard fast protein liquid chromatography (FPLC) techniques, which involved the use of a hydrophobic interaction resin for capture and an anion exchange resin for intermediate purification steps. The partially purified molecule was then proteolytically cleaved with endoproteinase Lys-C to obtain the active duplex, which was further purified using a second hydrophobic interaction resin to obtain the final BoNT / AB chimera.

[0313] Decahistadine Tag (H 10 For the BoNT / AB chimera molecules (chimeras 1, 2, 3A) having the .sup.-hydroxypropyl ester group, the capture step employed was the use of immobilized nickel resin instead of the hydrophobic interaction resin.

[0314] The sequences of each chimera are shown in Table 1. [Table 1]

[0315] [Example 2]

[0316] Comparison of BoNT / AB chimeras 1, 2, and 3A

[0317] C-terminal His 10 BoNT / AB chimeras 1, 2 and 3A carrying the tag and the E1191M / S1199Y double mutation were purified and tested for functional activity as described in Example 1 (FIG. 3).

[0318] Rat spinal cord neuron SNAP-25 cleavage assay

[0319] Primary cultures of rat spinal cord neurons (SCN) were prepared and grown for 3 weeks in 96-well tissue culture plates (as described in Masuyer et al., 2011, J. Struct. Biol. Structure and activity of a functional derivative of Clostridium botulinum neurotoxin B, and Chaddock et al., 2002, Protein Expr. Purif. Expression and purification of catalytically active, non-toxic endopeptidase derivatives of Clostridium botulinum toxin type A). Serial dilutions of BoNT / AB were prepared in SCN feeding medium. Growth medium from the wells to be treated was collected and filtered (0.2 μm filter). 125 μL of filtered medium was added back to each test well. Then 125 μL of diluted toxin was added to the plate (triplicate wells). Treated cells were incubated at 37° C., 10% CO2 for 24±1 h.

[0320] Analysis of BoNT activity using a SNAP-25 cleavage assay

[0321] After treatment, BoNT was removed and cells were washed once with PBS (Gibco, UK). Cells were lysed in 1x NuPAGE lysis buffer (Life Technologies) supplemented with 0.1 M dithiothreitol (DTT) and 250 units / mL benzonase (Sigma). Lysate proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes. Membranes were probed with a primary antibody specific for SNAP-25 (Sigma #S9684), which recognizes uncleaved SNAP-25 as well as SNAP-25 cleaved by BoNT / A endopeptidase. The secondary antibody used was anti-rabbit IgG conjugated with HRP (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 calculated percentage of cleaved SNAP-25 for each concentration of BoNT. Data were fitted to a four-parameter logistic equation using GraphPad Prism version 6 (GraphPad) and pEC 50 It was fitted to.

[0322] Table 2 below shows the pECs determined for chimeras 1, 2 and 3A in the rat SCN SNAP-25 cleavage assay. 50 These results indicate that the three BoNT / AB chimeras retained the ability to enter rat spinal cord neurons and cleave their target substrates. However, chimera 3A was more potent in this assay than chimeras 1 and 2 (see also FIG. 4). [Table 2]

[0323] Digital Abduction Scoring (DAS) Assay

[0324] The method to measure the activity of BoNT / AB chimeras 1, 2 and 3A in the DAS assay is based on the toe-spreading reflex, a startle response when briefly suspended by the tail. This reflex is scored as the digit abduction score (DAS) and is inhibited after administration of BoNT into the gastrocnemius-soleus muscle of the hind limb. Mice are briefly suspended by the tail to elicit a characteristic startle response in which the animal extends its hind limbs and then abducts the digits. (Aoki et al. 1999, Eur. J. Neurol.; 6 (suppl. 4) S3-S10).

[0325] On the day of injection, mice were anesthetized with 3% isoflurane in oxygen in an induction chamber. Each mouse received an intramuscular injection of the BoNT / AB chimera or vehicle (phosphate buffer containing 0.2% gelatin) in the gastrocnemius-soleus muscle of the right hind leg.

[0326] After neurotoxin injection, the various degrees of finger abduction were scored on a scale of 0 to 4, where 0 = normal and 4 = maximal reduction in finger abduction and leg extension. The ED50 was determined by nonlinear adjustment analysis using the mean of the maximal effect at each dose. The mathematical model used was a four-parameter logistic model.

[0327] The DAS was performed every 2 hours during the first day after dosing, then three times per day for four days.

[0328] Figure 5 shows the fitted curves of chimeras 1, 2 and 3A (SEQ ID NOs: 3, 4 and 5, respectively, converted to double-stranded form). The chimera 3A curve is shifted to the left, which means that a lower dose of chimera 3A achieved a similar DAS response compared to chimeras 1 and 2, thus indicating that chimera 3A is more potent than the others in the mouse DAS assay; see also the table below (Table 3), which provides the calculated ED50 of each chimera and the value of the dose leading to DAS 4 (highest score).

[0329] Table 3 below shows the EDs determined for unmodified recombinant BoNT / A1 (rBoNT / A1 - SEQ ID NO: 2 converted to a two-chain form) and chimeras 1, 2 and 3A in the mouse DAS assay. 50 and DAS 4 doses. These results indicate that of the three chimeras, chimera 3A has the highest in vivo efficacy in inducing muscle weakness. The studies shown in Figure 5 and Table 3 were performed in mice obtained from Charles River Laboratories. [Table 3]

[0330] [Example 3]

[0331] Comparison of BoNT / AB chimeras 3B, 3C and unmodified BoNT / A1

[0332] The untagged BoNT / AB chimeras 3B and 3C (sequence numbers 6 and 7), with and without the E1191M / S1199Y double mutation, respectively, were purified as described in Example 1 (Figure 6) and tested for functional activity using unmodified BoNT / A (sequence number 2 converted to a two-chain form) as a reference.

[0333] Human pluripotent stem cell SNAP-25 cleavage assay

[0334] Cryopreserved PERI.4U-cells were purchased from Axiogenesis (Cologne, Germany). Thawing and plating of cells was performed as recommended by the manufacturer. Briefly, cryovials containing cells were thawed for 2 minutes in a water bath at 37°C. After gentle resuspension, cells were transferred to a 50 mL tube. The cryovial was washed with 1 mL of Peri.4U® Thawing Medium supplied by the manufacturer, and the medium was transferred dropwise to the cell suspension into the 50 mL tube, after which another 2 mL of Peri.4U® Thawing Medium was added dropwise to the 50 mL tube. Cells were then counted using a hemocytometer. After this, another 6 mL of Peri.4U® Thawing Medium was added to the cell suspension. Cell pellets were obtained by centrifugation at 260 x g (e.g., 1,100 RPM) at room temperature for 6 minutes. Cells were then resuspended in the complete Peri.4U® culture medium supplied by the manufacturer. Cells were plated on cell culture plates coated with poly-L-ornithine and laminin at 1 cm 2 Cells were plated at a density of 50,000–150,000 cells per well. Cells were cultured at 37°C in a humidified CO2 atmosphere, with complete medium changes every 2–3 days during culture.

[0335] For toxin treatment, serial dilutions of BoNT were prepared in Peri.4U® culture medium. The medium from the wells to be treated was collected and filtered (0.2 μm filter). 125 μL of filtered medium was added back to each test well. Then, 125 μL of diluted toxin was added to the plate (triplicate wells). Treated cells were incubated at 37° C., 10% CO2 for 48±1 h.

[0336] Analysis of BoNT activity using a SNAP-25 cleavage assay

[0337] After treatment, BoNT was removed and cells were washed once with PBS (Gibco, UK). Cells were lysed in 1x NuPAGE lysis buffer (Life Technologies) supplemented with 0.1 M dithiothreitol (DTT) and 250 units / mL benzonase (Sigma). Lysate proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes. Membranes were probed with a primary antibody specific for SNAP-25 (Sigma #S9684), which recognizes uncleaved SNAP-25 as well as SNAP-25 cleaved by BoNT / A endopeptidase. The secondary antibody used was anti-rabbit IgG conjugated with HRP (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 calculated percentage of cleaved SNAP-25 for each concentration of BoNT. Data were fitted to a four-parameter logistic equation using GraphPad Prism version 6 (GraphPad) and pEC 50 It was fitted to.

[0338] Figure 7 shows that chimeras 3B and 3C were more potent than rBoNT / A1 in cleaving SNAP-25 in induced human pluripotent stem cells, with the former being significantly more potent, which can be explained by a double mutation that increases the affinity of chimera 3B for the human synaptotagmin II protein receptor present in these cells (Figure 7, Table 4). [Table 4]

[0339] Digital Abduction Scoring (DAS) Assay – Safety Factor

[0340] The method for measuring the activity of BoNT in the DAS assay is based on the startle response of mice when briefly suspended by their tails, the toe-spreading reflex. This reflex is scored as the digit abduction score (DAS) and is inhibited after administration of BoNT into the gastrocnemius-soleus muscle of the hind limb. Mice are briefly suspended by their tails to elicit a characteristic startle response in which the animals extend their hind limbs and then abduct their toes. (Aoki et al. 1999, Eur. J. Neurol.; 6 (suppl. 4) S3-S10).

[0341] On the day of injection, mice were anesthetized with 3% isoflurane in oxygen in an induction chamber. Each mouse received an intramuscular injection of BoNT or vehicle (phosphate buffer containing 0.2% gelatin) in the gastrocnemius-soleus muscle of the right hind leg.

[0342] After neurotoxin injection, the various degrees of finger abduction were scored on a scale of 0 to 4, where 0 = normal and 4 = maximal reduction in finger abduction and leg extension. The ED50 was determined by nonlinear adjustment analysis using the mean of the maximal effect at each dose. The mathematical model used was a four-parameter logistic model.

[0343] DAS were performed every 2 hours for all doses during the first day post-dosing, then 3 times per day for 4 days. Animals in the vehicle and lowest dose injected groups that induced a DAS of 4 during the first 4 days of injection were then monitored until complete recovery of muscle weakness to a DAS of 0 (no observed muscle weakness).

[0344] To calculate the safety rate, all animals were weighed the day before toxin injection (D0) and then once a day throughout the study period. The mean body weight, its standard deviation and standard error of the mean were calculated daily for each dose group. The safety rate of BoNT (-10%ΔBW / ED 50 To obtain the ED of the BoNT being studied, doses were administered at any time during the study where the mean body weight of a dose group was 10% lower than the mean body weight of that same dose group at D0. 50A lethal dose was defined as the dose at which at least one animal in that dose group died.

[0345] Figure 8 shows the duration of muscle weakness over time in a mouse digit abduction scoring assay for unmodified BoNT / A, chimera 3B and chimera 3C (sequence numbers 2, 6 and 7 converted to two-chain forms), indicating that the chimeras have a longer duration of action.

[0346] Table 5 below shows the EDs determined for rBoNT / A1 and chimeras 3B and 3C in the mouse DAS assay. 50 and DAS 4 doses. The table also provides the total duration of action of the DAS 4 doses until full recovery of muscle weakness to a DAS of 0 (no muscle weakness observed). Additionally, the table provides the mouse lethal dose and safety factor (-10%ΔBW / ED 50 ) are shown. In comparison to rBoNT / A1, chimeras 3B and 3C have a longer duration of action, a better safety rate and a higher lethal dose. The studies shown in FIG. 8 and Table 5 were performed in mice obtained from Janvier Laboratories. [Table 5]

[0347] [Example 4]

[0348] Further characterization of the BoNT / AB chimera (SEQ ID NO:6 converted to a two-stranded form)

[0349] SEQ ID NO:6, converted to a two-chain form that is a BoNT / AB chimera, was used in mouse LD 50 It was tested in the assay with a result of 1.202 ng / kg. Therefore, 1 unit of SEQ ID NO:6 is equivalent to 24.04 pg in this assay.

[0350] Additionally, the BoNT / AB chimera was tested in a rat DAS assay to determine duration of action as compared to Dysport®. The results are shown in Table 6 below: [Table 6]

[0351] In conclusion, the duration of action of BoNT / AB was significantly greater than that of Dysport®.

[0352] [Example 5]

[0353] Calculation of unit dose of modified BoNT / A (SEQ ID NO: 6 converted to two-chain form) for the treatment of limb spasticity

[0354] Taking into account the preclinical pharmacology data, a suitable unit dose range (UD) for administration of modified BoNT / A in humans was calculated.

[0355] For SEQ ID NO: 6, DAS ED of 13 pg / kg 50 was calculated. ED 50 was considered to be the minimum pharmacologically active dose, which was approximately 300-fold lower than the no observed adverse effect level (NOAEL) of 4 ng / kg in the same animal species. 50 corresponds to a 0.8 ng dose for a human weighing 60 kg.

[0356] Therefore, a lower unit dose limit of 1000 pg was selected. An upper unit dose limit of 16,000 pg was selected, which is lower than the NOAEL of 4 ng / kg obtained from both non-clinical safety species (rat and monkey) converted to a human dose of 60 kg body weight.

[0357] Considering the improved safety profile, the maximum total dose for the treatment of limb spasticity was set at 240,000 pg, derived from the NOAEL of 4 ng / kg from both non-clinical safety species (rat and monkey) converted to a human dose of 60 kg body weight.

[0358] Advantageously, modified BoNT / A (SEQ ID NO: 6) can be injected into more muscles in the treatment of limb spasticity before reaching maximum dose.This is a significant and advantageous finding that leads to improved treatment of limb spasticity, while providing clinicians with a greater range of treatment options.For the first time, it also provides the option of treating additional larger muscles, such as those of the shoulder, while still being able to treat elbow, forearm, and / or wrist well within maximum dose.

[0359] [Example 6]

[0360] Dosing regimen for treating adult upper limb spasticity using modified BoNT / A (SEQ ID NO: 6 converted to a two-chain form)

[0361] The modified BoNT / A is provided as a lyophilized powder in vials containing 36 ng of modified BoNT / A per vial. The lyophilized powder is reconstituted. The unit dose (UD) is 1000-16,000 pg (42-666 units [measured by mouse LD50]).

[0362] Adult upper limb spasticity is treated by intramuscular injection according to the following dosing regimen (Table 7): [Table 7]

[0363] The maximum total dose administered is 15xUD, which is equivalent to 240,000pg / 9,990 units. Thus, clinicians can tailor treatment to patients with the knowledge that 15xUD can be administered without any concerns of toxicity, thereby allowing treatment of additional muscles of interest, including the shoulder, and / or ensuring that each muscle receives a pharmacologic effective dose.

[0364] [Example 7]

[0365] Dosing regimen for treating adult leg spasticity using modified BoNT / A (SEQ ID NO: 6 converted to a two-chain form)

[0366] The modified BoNT / A is provided as a lyophilized powder in vials containing 36 ng of modified BoNT / A per vial. The lyophilized powder is reconstituted.

[0367] The unit dose (UD) is 1000-16,000 pg (42-666 units).

[0368] Adult lower limb spasticity is treated by intramuscular injection according to the following dosing regimen (Table 8): [Table 8]

[0369] The maximum total dose administered is 15xUD, which is equivalent to 240,000pg / 9,990 units. Thus, clinicians can tailor treatment to patients with the knowledge that 15xUD can be administered without any concerns of toxicity, thereby allowing treatment of additional muscles of interest and / or ensuring that each muscle receives a pharmacologic effective dose.

[0370] [Example 8]

[0371] Safety & Efficacy of Modified BoNT / A (SEQ ID NO:6 Converted to Two-Stranded Form) in Humans

[0372] An integrated Phase I / II multicenter, double-blind, randomized, Dysport and placebo-controlled, dose-escalation and dose-finding study was conducted to evaluate the safety and efficacy of modified BoNT / A (SEQ ID NO: 6 converted to the two-stranded form) in the treatment of adult upper limb spasticity in subjects aged 18-70 years with spastic hemiparesis following stroke or traumatic brain injury (TBI).

[0373] The study involved human sequential dose escalation in a unique cohort of patients with adult upper limb spasticity with a fist pattern. All participants were injected into the flexor digitorum profundus (FDP) and flexor digitorum superficialis (FDS). Each muscle was injected with a single unit dose of modified BoNT / A, Dysport, or placebo. Five cohorts were administered different (escalating) amounts of modified BoNT / A (SEQ ID NO: 6 converted to a two-chain form). Cohort 1 was administered 2 x 1,000 pg unit doses of modified BoNT / A (i.e., up to 2,000 pg), while cohort 5 was administered 2 x 16,000 pg unit doses of modified BoNT / A (i.e., up to 32,000 pg).

[0374] The results showed that, despite the exceptionally high doses per muscle, all unit doses of modified BoNT / A tested (i.e., unit doses up to 16,000 pg) were effective in muscle paralysis, were safely tolerated, and no adverse effects were observed. This indicates that modified BoNT / A does not diffuse away from the injection site, highlighting the exceptional safety profile of modified BoNT / A (SEQ ID NO: 6 converted to a two-stranded form). Based on these findings, it is considered reliable that higher unit doses per muscle can be administered without resulting adverse effects. Furthermore, considering the lack of systemic diffusion of the toxin, it is considered reliable that up to 15x higher unit doses can be administered without safety concerns.

[0375] Therefore, unit doses of 30,000 pg and 36,000 pg (each administered 15x maximum) were selected for therapeutic treatment of adult limb spasticity, resulting in total doses administered during a treatment session of 450,000 pg and 540,000 pg, respectively.

[0376] [Example 9]

[0377] Dosing regimen for treating adult upper limb spasticity using modified BoNT / A (SEQ ID NO: 6 converted to a two-chain form)

[0378] The modified BoNT / A is provided as a lyophilized powder in multiple vials, each containing 36 ng of the modified BoNT / A. The lyophilized powder is reconstituted.

[0379] The unit dose (UD) is 30,000 pg (1,248 units [mouse LD 50 ) or 36,000 pg (1,498 units [mouse LD 50 [measured by].

[0380] Adult upper limb spasticity is treated by intramuscular injection according to the following dosing regimen (Table 9): [Table 9]

[0381] The maximum total dose administered is 15xUD, which is equivalent to 450,000pg / 18,719 units or 540,000pg / 22,463 units. Thus, clinicians can tailor treatment to patients with the knowledge that 15xUD can be administered without any concerns of toxicity, thereby allowing treatment of additional muscles of interest, including the shoulder, and / or ensuring that each muscle receives a pharmacologic effective dose.

[0382] [Example 10]

[0383] Dosing regimen for treating adult leg spasticity using modified BoNT / A (SEQ ID NO: 6 converted to a two-chain form)

[0384] The modified BoNT / A is provided as a lyophilized powder in multiple vials, each containing 36 ng of the modified BoNT / A. The lyophilized powder is reconstituted.

[0385] The unit dose (UD) is 30,000 pg (1,248 units [mouse LD 50 ) or 36,000 pg (1,498 units [mouse LD 50 [measured by].

[0386] Adult lower limb spasticity is treated by intramuscular injection according to the following dosing regimen (Table 10): [Table 10]

[0387] The maximum total dose administered is 15xUD, which is equivalent to 450,000pg / 18,719 units or 540,000pg / 22,463 units. Thus, clinicians can tailor treatment to patients with the knowledge that 15xUD can be administered without any concerns of toxicity, thereby allowing treatment to additional muscles of interest and / or ensuring that each muscle receives a pharmacologic effective dose.

[0388] [Example 11]

[0389] Treatment of Adult Patients with Upper Limb Spasticity

[0390] Bill, age 53, has been diagnosed by his GP with adult upper limb spasticity. Modified BoNT / A (SEQ ID NO:6 converted to a two-chain form) is administered by one or more unit doses (1 x unit dose = 36,000 pg) into the following muscles as shown below: [Table H]

[0391] The total dose administered is 540,000 pg of modified BoNT / A (SEQ ID NO:6 converted to the two-chain form).

[0392] Adult upper limb spasticity is alleviated, and Bill does not require further treatment for longer than 9 months due to the long duration of modified BoNT / A. Therefore, Bill receives less frequent injections (e.g., per year) when compared with a comparable subject receiving unmodified BoNT / A. Furthermore, Bill does not show any side effects due to the improved safety profile of modified BoNT / A.

[0393] [Example 12]

[0394] Safety and Efficacy of Modified BoNT / A (SEQ ID NO:6 Converted to a Two-Stranded Form) in Humans

[0395] An integrated Phase I / II multicenter, double-blind, randomized, Dysport and placebo-controlled, dose-escalation and dose-finding study was conducted to evaluate the safety and efficacy of modified BoNT / A (SEQ ID NO: 6 converted to the two-stranded form) in the treatment of adult upper limb spasticity in subjects aged 18-70 years with spastic hemiparesis following stroke or traumatic brain injury (TBI).

[0396] The study included a human sequential dose escalation in a unique cohort of patients with adult upper limb spasticity with fist pattern. All participants were injected into the flexor digitorum profundus (FDP) and flexor digitorum superficialis (FDS). Each muscle was injected with a single unit dose of modified BoNT / A, Dysport or placebo. Subjects were administered modified BoNT / A (SEQ ID NO: 6 converted to a two-stranded form) as follows: by a unit dose of 15,000pg to FDP and a unit dose of 15,000pg to FDS (i.e., a total dose of 30,000pg), by a unit dose of 25,000pg to FDP and a unit dose of 25,000pg to FDS (i.e., a total dose of 50,000pg) or by a unit dose of 36,000pg to FDP and a unit dose of 36,000pg to FDS (i.e., a total dose of 72,000pg).

[0397] The results showed that all unit doses of modified BoNT / A tested were effective in muscle paralysis, were safely tolerated, and no adverse effects were observed, despite exceptionally high doses per muscle (e.g., for unit doses of 25,000 pg and 36,000 pg), indicating that modified BoNT / A does not diffuse away from the injection site and highlighting the exceptional safety profile of modified BoNT / A (SEQ ID NO: 6 converted to a two-stranded form).

[0398] Considering the lack of systemic diffusion of the toxin, it is reliable that higher unit doses up to 15x can be administered without safety concerns. Therefore, unit doses of 25,000pg and 36,000pg (each administered up to 15x) were selected for therapeutic treatment of adult limb spasticity. Thus, the total doses administered during a treatment session are 375,000pg and 540,000pg, respectively.

[0399] [Example 13]

[0400] Treatment of Adult Patients with Lower Limb Spasticity

[0401] Ryan, age 63, has been diagnosed by his GP with adult leg spasticity. Modified BoNT / A (SEQ ID NO:6 [converted to the two-chain form]) is administered in one or more unit doses (1 x unit dose = 25,000 pg) to multiple of the following muscles as shown below: [Table I]

[0402] The maximum dose is 15×UD (ie, 375,000 pg) of the modified BoNT / A.

[0403] Spasticity is reduced, and due to the long duration of modified BoNT / A, Ryan does not require further treatment for longer than 8 months. Thus, Ryan receives less frequent injections (e.g., per year) when compared to comparable subjects receiving unmodified BoNT / A. Furthermore, Ryan does not show any side effects due to the improved safety profile of modified BoNT / A.

[0404] [Example 14]

[0405] Treatment of patients with pediatric lower limb spasticity

[0406] Sharon, age 7, has been diagnosed by her GP with infantile leg spasticity. Modified BoNT / A (SEQ ID NO:6 [converted to the two-chain form]) is administered in one or more unit doses (1 x unit dose = 12,500 pg) to multiple of the following muscles as shown below: [Table J]

[0407] The maximum dose is 15×UD (ie, 187,500 pg) of modified BoNT / A.

[0408] Spasticity is reduced, and due to the long duration of modified BoNT / A, Sharon does not require further treatment for longer than 8 months. Thus, Sharon receives less frequent injections (e.g., per year) when compared to comparable subjects receiving unmodified BoNT / A. Furthermore, Sharon does not show any side effects due to the improved safety profile of modified BoNT / A.

[0409] [Example 15]

[0410] Treatment of patients with pediatric lower limb spasticity

[0411] Timmy, age 8, has been diagnosed by his GP with childhood leg spasticity. Modified BoNT / A (SEQ ID NO:6 [converted to the two-chain form]) is administered by one or more unit doses (1 x unit dose = 18,000 pg) to multiple of the following muscles as shown below: [Table K]

[0412] The maximum dose is 15×UD (ie, 270,000 pg) of the modified BoNT / A.

[0413] Spasticity is reduced, and Timmy does not require further treatment for more than 9 months due to the long duration of modified BoNT / A.Thus, Timmy receives less frequent injections (e.g., per year) when compared to comparable subjects receiving unmodified BoNT / A.Furthermore, Timmy does not show any side effects due to the improved safety profile of modified BoNT / A.

[0414] [Example 16]

[0415] Treatment of Adult Patients with Upper Limb Spasticity

[0416] Larry, age 21, has been diagnosed by his GP with upper limb spasticity. Modified BoNT / A (SEQ ID NO:6 [converted to the two-chain form]) is administered in one or more unit doses (1 x unit dose = 25,000 pg) to multiple of the following muscles as shown below: [Table L]

[0417] The maximum dose is 15×UD (ie, 375,000 pg) of the modified BoNT / A.

[0418] Spasticity is reduced, and due to the long duration of modified BoNT / A, Larry does not require further treatment for longer than 9 months. Thus, Larry receives less frequent injections (e.g., per year) when compared to comparable subjects receiving unmodified BoNT / A. Furthermore, Larry does not show any side effects due to the improved safety profile of modified BoNT / A.

[0419] [Example 17]

[0420] Treatment of Adult Patients with Upper Limb Spasticity

[0421] Loretta, age 39, has been diagnosed by her GP with upper limb spasticity. Modified BoNT / A (SEQ ID NO:6 [converted to the two-chain form]) is administered in one or more unit doses (1 x unit dose = 36,000 pg) to multiple of the following muscles as shown below: [Table M]

[0422] The maximum dose is 15×UD (ie, 540,000 pg) of the modified BoNT / A.

[0423] Spasticity is reduced, and due to the long duration of modified BoNT / A, Loretta does not require further treatment for longer than 9 months. Thus, Loretta receives less frequent injections (e.g., per year) when compared to comparable subjects receiving unmodified BoNT / A. Furthermore, Loretta does not show any side effects due to the improved safety profile of modified BoNT / A.

[0424] [Example 18]

[0425] Treatment of patients with pediatric upper limb spasticity

[0426] Jack, age 13, has been diagnosed by his GP with upper limb spasticity. Modified BoNT / A (SEQ ID NO:6 [converted to the two-chain form]) is administered in one or more unit doses (1 x unit dose = 12,500 pg) to multiple of the following muscles as shown below: [Table N]

[0427] The maximum dose is 15×UD (ie, 187,500 pg) of modified BoNT / A.

[0428] Spasticity is reduced, and due to the long duration of modified BoNT / A, Jack does not require further treatment for longer than 9 months. Thus, Jack receives less frequent injections (e.g., per year) when compared to a comparable subject receiving unmodified BoNT / A. Furthermore, Jack does not show any side effects due to the improved safety profile of modified BoNT / A.

[0429] [Example 19]

[0430] Treatment of patients with pediatric upper limb spasticity

[0431] Julia, age 11, has been diagnosed by her GP with upper limb spasticity. Modified BoNT / A (SEQ ID NO:6 [converted to the two-chain form]) is administered in one or more unit doses (1 x unit dose = 18,000 pg) to multiple of the following muscles as shown below: [Table O]

[0432] The maximum dose is 15×UD (ie, 270,000 pg) of the modified BoNT / A.

[0433] Spasticity is reduced, and due to the long duration of modified BoNT / A, Julia does not require further treatment for longer than 9 months.Thus, Julia receives less frequent injections (e.g., per year) when compared to a comparable subject receiving unmodified BoNT / A.Furthermore, Julia does not show any side effects due to the improved safety profile of modified BoNT / A.

[0434] [Example 20]

[0435] Treatment of Adult Patients with Lower Limb Spasticity

[0436] Claire, age 57, has been diagnosed by her GP with adult leg spasticity. Modified BoNT / A (SEQ ID NO:6 [converted to the two-chain form]) is administered by one or more unit doses (1 x unit dose = 36,000 pg) to multiple of the following muscles as shown below: [Table P]

[0437] The maximum dose is 15×UD (ie, 540,000 pg) of the modified BoNT / A.

[0438] Spasticity is reduced, and due to the long duration of modified BoNT / A, Claire does not require further treatment for longer than 8 months. Therefore, Claire receives less frequent injections (e.g., per year) when compared with a comparable subject receiving unmodified BoNT / A. Furthermore, Claire does not show any side effects due to the improved safety profile of modified BoNT / A.

[0439] Terms

[0440] 1. A modified BoNT / A for use in treating limb spasticity (adult limb spasticity), wherein the modified BoNT / A is administered by intramuscular injection into multiple affected muscles of a subject; wherein the modified BoNT / A is administered by a unit dose of greater than 17,000 pg of modified BoNT / A in multiple affected muscles; wherein the plurality of affected muscles is selected from: a first group including: flexor digitorum superficialis, flexor digitorum profundus, flexor carpi radialis, flexor carpi ulnaris, brachioradialis, pronator teres, biceps brachii, medial head of gastrocnemius, lateral head of gastrocnemius, flexor digitorum longus, flexor hallucis longus, gastrocnemius, deltoid, levator scapulae, pronator quadratus, flexor hallucis longus, adductor pollicis brevis, palmaris longus, lumbricalis, opponens pollicis, adductor magnus, adductor longus, adductor brevis, gracilis, medial hamstrings, lateral hamstrings, tensor fasciae latae, rectus femoris, vastus lateralis, vastus medialis, vastus intermedius, gluteus maximus, tibialis anterior, flexor digitorum brevis, extensor hallucis longus, and flexor hallucis brevis; A second group including: triceps brachii (long head), subscapularis, pectoralis major (e.g., pectoralis major), latissimus dorsi, biceps brachii, brachialis, soleus, tibialis posterior, brachioradialis, teres major, iliopsoas, and gastrocnemius; and wherein a single unit dose is administered in the affected muscle of a first group and / or multiple unit doses are administered in the affected muscle of a second group, wherein the total dose administered during treatment is up to 600,000 pg; and Here, the modified BoNT / A comprises a BoNT / A light chain and translocation domain and a BoNT / B receptor binding domain (H C domain). 2. A modified BoNT / A for use according to clause 1, wherein the limb spasticity is upper limb spasticity. 3. A modified BoNT / A for use according to clause 1 or 2, wherein the unit dose is greater than 17,000 pg and up to 40,000 pg (eg, 17,001 pg and up to 40,000 pg) of modified BoNT / A. 4. A modified BoNT / A for use according to any one of clauses 1 to 3, wherein the unit dose is more than 23,000 pg and up to 38,000 pg (eg, 23,001 pg to up to 38,000 pg) of modified BoNT / A. 5. A modified BoNT / A for use according to any one of clauses 1 to 4, wherein the unit dose is more than 34,000 pg and up to 38,000 pg (eg, 34,001 pg to up to 38,000 pg) of modified BoNT / A. 6. A modified BoNT / A for use according to any one of clauses 1 to 5, wherein the total dose administered is between 250,000 pg and 600,000 pg of modified BoNT / A (e.g., between 255,000 pg and 600,000 pg). 7. A modified BoNT / A for use according to any one of clauses 1 to 6, wherein the total dose administered is greater than 255,000 pg to 600,000 pg of modified BoNT / A. 8. The modified BoNT / A for use according to any one of clauses 1 to 7, wherein the total dose administered is between 350,000 and 600,000 pg of modified BoNT / A. 9. The modified BoNT / A for use according to any one of clauses 1 to 8, wherein the total dose administered is between 500,000 and 600,000 pg of modified BoNT / A. 10. A modified BoNT / A for use in treating childhood limb spasticity, wherein the modified BoNT / A is administered by intramuscular injection into multiple affected muscles of a subject; wherein the modified BoNT / A is administered by a unit dose of greater than 8,500 pg of modified BoNT / A in multiple affected muscles; wherein the plurality of affected muscles is selected from: a first group including: flexor digitorum superficialis, flexor digitorum profundus, flexor carpi radialis, flexor carpi ulnaris, brachioradialis, pronator teres, biceps brachii, medial head of gastrocnemius, lateral head of gastrocnemius, flexor digitorum longus, flexor hallucis longus, gastrocnemius, deltoid, levator scapulae, pronator quadratus, flexor hallucis longus, adductor pollicis brevis, palmaris longus, lumbricalis, opponens pollicis, adductor magnus, adductor longus, adductor brevis, gracilis, medial hamstrings, lateral hamstrings, tensor fasciae latae, rectus femoris, vastus lateralis, vastus medialis, vastus intermedius, gluteus maximus, tibialis anterior, flexor digitorum brevis, extensor hallucis longus, and flexor hallucis brevis; A second group including: triceps brachii (long head), subscapularis, pectoralis major (e.g., pectoralis major), latissimus dorsi, biceps brachii, brachialis, soleus, tibialis posterior, brachioradialis, teres major, iliopsoas, and gastrocnemius; and wherein a single unit dose is administered in the affected muscle of a first group and / or multiple unit doses are administered in the affected muscle of a second group, wherein the total dose administered during treatment is up to 300,000 pg; and Here, the modified BoNT / A comprises a BoNT / A light chain and translocation domain and a BoNT / B receptor binding domain (H C domain). 11. A modified BoNT / A for use according to clause 10, wherein the infantile limb spasticity is infantile upper limb spasticity. 12. A modified BoNT / A for use according to clause 10 or clause 11, wherein the unit dose is greater than 8,500 pg and up to 20,000 pg (e.g., 8,501 pg and up to 20,000 pg) of modified BoNT / A, preferably, the unit dose is greater than 8,500 pg and up to 18,000 pg. 13. A modified BoNT / A for use according to any one of clauses 10 to 12, wherein the unit dose is more than 11,500 pg and up to 19,000 pg (eg, 11,501 pg to up to 19,000 pg) of modified BoNT / A. 14. A modified BoNT / A for use according to any one of clauses 10 to 13, wherein the unit dose is more than 17,000 pg and up to 19,000 pg (eg, 17,001 pg to up to 19,000 pg) of modified BoNT / A. 15. The modified BoNT / A for use according to any one of clauses 10 to 14, wherein the total dose administered is greater than 125,000 pg to 300,000 pg (e.g., 127,500 pg to 300,000 pg) of modified BoNT / A. 16. The modified BoNT / A for use according to any one of clauses 10 to 15, wherein the total dose administered is greater than 127,500 pg to 300,000 pg of the modified BoNT / A. 17. The modified BoNT / A for use according to any one of clauses 10 to 16, wherein the total dose administered is between 175,000 and 300,000 pg of modified BoNT / A. 18. The modified BoNT / A for use according to any one of clauses 10 to 17, wherein the total dose administered is between 250,000 and 300,000 pg of the modified BoNT / A. 19. A modified BoNT / A for use in treating limb spasticity (adult limb spasticity), wherein the modified BoNT / A is administered by intramuscular injection into multiple affected muscles of a subject; wherein the modified BoNT / A is administered in a unit dose of greater than 707 units of modified BoNT / A in multiple affected muscles; Here, 1 unit is the calculated median lethal dose (LD ) in mice. 50 ) is the amount of modified BoNT / A corresponding to wherein the plurality of affected muscles is selected from: a first group including: flexor digitorum superficialis, flexor digitorum profundus, flexor carpi radialis, flexor carpi ulnaris, brachioradialis, pronator teres, biceps brachii, medial head of gastrocnemius, lateral head of gastrocnemius, flexor digitorum longus, flexor hallucis longus, gastrocnemius, deltoid, levator scapulae, pronator quadratus, flexor hallucis longus, adductor pollicis brevis, palmaris longus, lumbricalis, opponens pollicis, adductor magnus, adductor longus, adductor brevis, gracilis, medial hamstrings, lateral hamstrings, tensor fasciae latae, rectus femoris, vastus lateralis, vastus medialis, vastus intermedius, gluteus maximus, tibialis anterior, flexor digitorum brevis, extensor hallucis longus, and flexor hallucis brevis; A second group including: triceps brachii (long head), subscapularis, pectoralis major (e.g., pectoralis major), latissimus dorsi, biceps brachii, brachialis, soleus, tibialis posterior, brachioradialis, teres major, iliopsoas, and gastrocnemius; and wherein a single unit dose is administered in the affected muscle of a first group and / or multiple unit doses are administered in the affected muscle of a second group, wherein the total dose administered during treatment is up to 24,958 units; and Here, the modified BoNT / A comprises a BoNT / A light chain and translocation domain and a BoNT / B receptor binding domain (H C domain). 20. A modified BoNT / A for use according to clause 19, wherein the limb spasticity is upper limb spasticity. 21. A modified BoNT / A for use according to clause 19 or clause 20, wherein the unit dose is more than 707 units and up to 1,664 units (eg, 708 units and up to 1,664 units) of modified BoNT / A. 22. A modified BoNT / A for use according to any one of clauses 19 to 21, wherein the unit dose is more than 949 units and up to 1,581 units (e.g., from 950 units to up to 1,581 units) of modified BoNT / A. 23. A modified BoNT / A for use according to any one of clauses 19 to 22, wherein the unit dose is more than 1,414 units and up to 1,581 units (e.g., 1,415 units and up to 1,581 units) of modified BoNT / A. 24. The modified BoNT / A for use according to any one of clauses 19 to 23, wherein the total dose administered is greater than 10,399 Units to 24,958 Units (e.g., 10,607 Units to 24,958 Units) of modified BoNT / A. 25. The modified BoNT / A for use according to any one of clauses 19 to 24, wherein the total dose administered is greater than 10,607 units and greater than 24,958 units of the modified BoNT / A. 26. The modified BoNT / A for use according to any one of claims 19 to 25, wherein the total dose administered is between 14,559 units and 24,958 units of the modified BoNT / A. 27. The modified BoNT / A for use according to any one of clauses 19 to 26, wherein the total dose administered is between 20,799 units and 24,958 units of the modified BoNT / A. 28. A modified BoNT / A for use in treating childhood limb spasticity, wherein the modified BoNT / A is administered by intramuscular injection into multiple affected muscles of a subject; wherein the modified BoNT / A is administered in a unit dose of greater than 353.5 units of modified BoNT / A in multiple affected muscles; Here, 1 unit is the calculated median lethal dose (LD ) in mice. 50 ) is the amount of modified BoNT / A corresponding to wherein the plurality of affected muscles is selected from: a first group including: flexor digitorum superficialis, flexor digitorum profundus, flexor carpi radialis, flexor carpi ulnaris, brachioradialis, pronator teres, biceps brachii, medial head of gastrocnemius, lateral head of gastrocnemius, flexor digitorum longus, flexor hallucis longus, gastrocnemius, deltoid, levator scapulae, pronator quadratus, flexor hallucis longus, adductor pollicis brevis, palmaris longus, lumbricalis, opponens pollicis, adductor magnus, adductor longus, adductor brevis, gracilis, medial hamstrings, lateral hamstrings, tensor fasciae latae, rectus femoris, vastus lateralis, vastus medialis, vastus intermedius, gluteus maximus, tibialis anterior, flexor digitorum brevis, extensor hallucis longus, and flexor hallucis brevis; A second group including: triceps brachii (long head), subscapularis, pectoralis major (e.g., pectoralis major), latissimus dorsi, biceps brachii, brachialis, soleus, tibialis posterior, brachioradialis, teres major, iliopsoas, and gastrocnemius; and wherein a single unit dose is administered in the affected muscle of a first group and / or multiple unit doses are administered in the affected muscle of a second group, wherein the total dose administered during treatment is up to 12,479 units; and Here, the modified BoNT / A comprises a BoNT / A light chain and translocation domain and a BoNT / B receptor binding domain (H C domain). 29. A modified BoNT / A for use according to article 28, wherein the childhood limb spasticity is childhood upper limb spasticity. 30. A modified BoNT / A for use according to clause 28 or clause 29, wherein the unit dose is greater than 353.5 units and up to 832 units (e.g., 354 units and up to 832 units), preferably greater than 353.5 units and up to 749 units (e.g., 354 units and up to 749 units). 31. A modified BoNT / A for use according to any one of clauses 28 to 30, wherein the unit dose is more than 478 units and up to 749 units (eg, from 479 units to up to 749 units) of modified BoNT / A. 32. A modified BoNT / A for use according to any one of clauses 28 to 31, wherein the unit dose is more than 707 units and up to 749 units (eg, 708 units to up to 749 units) of modified BoNT / A. 33. The modified BoNT / A for use according to any one of claims 28 to 32, wherein the total dose administered is greater than 5,200 units to 12,479 units (e.g., 5,304 units to 12,479 units) of modified BoNT / A. 34. The modified BoNT / A for use according to any one of clauses 28 to 33, wherein the total dose administered is greater than 5,304 units to 12,479 units of the modified BoNT / A. 35. The modified BoNT / A for use according to any one of claims 28 to 34, wherein the total dose administered is between 7,280 units and 12,479 units of the modified BoNT / A. 36. The modified BoNT / A for use according to any one of clauses 28 to 35, wherein the total dose administered is from 10,400 units to a maximum of 12,479 units of the modified BoNT / A. 37. A modified BoNT / A for use according to any one of the preceding clauses, wherein the modified BoNT / A comprises a polypeptide sequence having at least 70% sequence identity to SEQ ID NO:6. 38. A modified BoNT / A for use according to any one of the preceding clauses, wherein the modified BoNT / A comprises the polypeptide sequence of SEQ ID NO:6. 39. A modified BoNT / A for use according to any one of the preceding clauses, wherein the modified BoNT / A consists of the polypeptide sequence of SEQ ID NO:6. 40. The modified BoNT / A has a safety margin of greater than 7, the safety margin being a DAS ED measured in pg / mouse. 50 Calculated as the dose of toxin required for a -10% body weight change measured in pg / mouse divided by ED 50 The modified BoNT / A for use according to any one of the preceding clauses, wherein the dose is necessary to result in a DAS score of =2. 41. The modified BoNT / A for use according to any one of the preceding clauses, wherein the plurality of muscles is selected from the following: A first group including: flexor digitorum superficialis, flexor digitorum profundus, flexor carpi radialis, flexor carpi ulnaris, brachioradialis, pronator teres, deltoid, levator scapulae, pronator quadratus, flexor pollicis longus, adductor pollicis, flexor pollicis brevis, palmaris longus, lumbricalis, and opponens pollicis; and A second group includes: triceps brachii (long head), subscapularis, pectoral muscles (e.g., pectoralis major), latissimus dorsi, biceps brachii, brachialis, brachioradialis, and teres major. 42. A unit dosage form of a modified BoNT / A comprising: (a) More than 707 units of modified BoNT / A, where 1 unit is the calculated median lethal dose (LD) in mice. 50 ), or (b) greater than 17,000 pg of modified BoNT / A; and (c) optionally, pharma- ceutically acceptable carriers, excipients, adjuvants and / or salts; Here, the modified BoNT / A comprises a BoNT / A light chain and translocation domain and a BoNT / B receptor binding domain (H C domain). 43. A unit dosage form according to clause 42, comprising: (a) a modified BoNT / A having more than 707 units, up to 1,664 units (e.g., between 708 units and 1,664 units); or (b) A modified BoNT / A of more than 17,000 pg up to 40,000 pg (e.g., 17,001 pg up to 40,000 pg). 44. A kit comprising: (a) unit dosage forms in accordance with article 42 or 43; and (b) instructions for its use in treating limb spasticity, and (c) Optionally, a diluent. 45. A unit dosage form of modified BoNT / A (e.g., for treating adult limb spasticity), comprising: (a) More than 353.5 units of modified BoNT / A, where 1 unit is the calculated median lethal dose (LD) in mice. 50 ), or (b) greater than 8,500 pg of modified BoNT / A; and (c) optionally, pharma- ceutically acceptable carriers, excipients, adjuvants and / or salts; Here, the modified BoNT / A comprises a BoNT / A light chain and translocation domain and a BoNT / B receptor binding domain (H C domain). 46. ​​Unit dosage forms according to Article 45, including ranks: (a) a modified BoNT / A having more than 353.5 units and up to 832 units (e.g., 354 units and up to 832 units); or (b) A modified BoNT / A of more than 8,500 pg up to 20,000 pg (e.g., 8,501 pg up to 20,000 pg). 47. A kit containing: (a) unit dosage forms in accordance with article 45 or 46; and (b) instructions for its use in the treatment of childhood limb spasticity; and (c) Optionally, a diluent.

[0441] All publications mentioned in the above specification are incorporated herein by reference. Various modifications and variations of the described methods and systems of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with certain preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in the art of biochemistry and biotechnology or related fields are intended to be within the scope of the following claims. [Explanation of symbols]

[0442] [Figure 1] Shoulder: Elbow: Forearm: Wrist: Internal rotation / adduction: Flexion: Flexion Supination: Extension: Neutral: Neutral Pronation: Internal rotation / retroversion: Upper limb spasticity patterns. Note: All five upper limb patterns could be combined with any spastic hand and finger position (e.g. claw, spastic flexed, intrinsic lumbrical).: Upper limb spasticity patterns. Note: All five upper limb patterns could be combined with any spastic hand and finger position (e.g. claw, spastic flexed, intrinsic lumbrical). [Figure 2] Indication: Recommended Concentration: Recommended DYSPORT Doase: Recommended Dysport Dose Spasticity, Adults: Spasticity, Adults 10 Units / 0.1 mL or 20 Units / 0.1 mL: 10 Units / 0.1 mL or 20 Units / 0.1 mL Upper Limb: 500 Units to 1000 Units: Lower Limb: 1000 Units to 1500 Units: Maximum total dose per treatment session = 1500 Units: [Figure 3, 6] Marker: Marker SEQ ID NO: Sequence number [Figure 4, 7] % SNPA-25 cleavage: SNAP-25 cleavage percentage in rat spinal cord neurones: in human pluripotent stem cells Un-Tr: Untreated SEQ ID NO: Sequence number [Figure 5] Mean DAS: Average DAS Dose (pg / mouse): Dose (pg / mouse) SEQ ID NO: Sequence number [Figure 8] Time (day): Time (day) SEQ ID NO: Sequence number

Claims

1. A pharmaceutical product comprising modified botulinum neurotoxin A (BoNT / A) for use in the treatment of limb spasticity. Here, Modified BoNT / A is administered by intramuscular injection to one or more affected muscles of the subject. Modified BoNT / A is administered in unit doses ranging from 17,000 pg to 40,000 pg, and Modified BoNT / A includes the light chain and transposition domain of BoNT / A, as well as the receptor-binding domain (HC domain) of BoNT / B.

2. The affected muscle is selected from the following, according to Claim 1. The first group includes the following: flexor digitorum superficialis, flexor digitorum profundus, flexor carpi radialis, flexor carpi ulnaris, brachioradialis, pronator teres, biceps brachii, medial head of gastrocnemius (medial gastrocnemius), lateral head of gastrocnemius (lateral gastrocnemius), flexor digitorum longus, flexor hallucis longus, gastrocnemius, deltoid, levator scapulae, pronator quadratus, flexor hallucis longus, adductor pollicis, flexor pollicis brevis, palmaris longus, lumbricals, opponens pollicis, adductor magnus, adductor longus, adductor brevis, gracilis, medial hamstrings, lateral hamstrings, tensor fasciae latae, rectus femoris, vastus lateralis, vastus medialis, vastus intermedius, gluteus maximus, tibialis anterior, flexor digitorum brevis, extensor hallucis longus, and flexor hallucis brevis, and The second group includes the following: triceps brachii (long head), subscapularis, pectoralis (e.g., pectoralis major), latissimus dorsi, biceps brachii, brachialis, soleus, tibialis posterior, brachioradialis, teres major, iliopsoas, adductor magnus, hamstrings (e.g., biceps femoris, semimembranosus, and / or semitendinosus), rectus femoris, and gastrocnemius, and Here, a single unit dose is administered to the muscles of the first affected group, and / or multiple unit doses are administered to the muscles of the second affected group.

3. The pharmaceutical product according to claim 1, wherein the unit dose is 25,000 pg or 35,000 pg.

4. The pharmaceutical product according to claim 1, wherein the total dose administered is 250,000 pg to 600,000 pg.

5. The pharmaceutical product according to claim 1, wherein the total dose administered is a maximum of 375,000 pg or a maximum of 525,000 pg.

6. The pharmaceutical product according to claim 1, wherein the limb spasticity is upper limb spasticity.

7. A pharmaceutical product comprising modified botulinum neurotoxin A (BoNT / A) for use in the treatment of spasticity of the limbs in children. Here, the modified BoNT / A is administered by intramuscular injection to one or more affected muscles of the subject. Here, the modified BoNT / A was administered in unit doses ranging from 8,500 pg to 20,000 pg. Here, the affected muscles are selected from the following: The first group includes the following: flexor digitorum superficialis, flexor digitorum profundus, flexor carpi radialis, flexor carpi ulnaris, brachioradialis, pronator teres, biceps brachii, medial head of gastrocnemius (medial gastrocnemius), lateral head of gastrocnemius (lateral gastrocnemius), flexor digitorum longus, flexor hallucis longus, gastrocnemius, deltoid, levator scapulae, pronator quadratus, flexor hallucis longus, adductor pollicis, flexor pollicis brevis, palmaris longus, lumbricals, opponens pollicis, adductor magnus, adductor longus, adductor brevis, gracilis, medial hamstrings, lateral hamstrings, tensor fasciae latae, rectus femoris, vastus lateralis, vastus medialis, vastus intermedius, gluteus maximus, tibialis anterior, flexor digitorum brevis, extensor hallucis longus, and flexor hallucis brevis, and The second group includes the following: triceps brachii (long head), subscapularis, pectoralis (e.g., pectoralis major), latissimus dorsi, biceps brachii, brachialis, soleus, tibialis posterior, brachioradialis, teres major, iliopsoas, adductor magnus, hamstrings (e.g., biceps femoris, semimembranosus, and / or semitendinosus), rectus femoris, and gastrocnemius, and Here, modified BoNT / A includes the light chain and transposition domain of BoNT / A and the receptor-binding domain (HC domain) of BoNT / B.

8. The pharmaceutical product according to claim 7, wherein the total dose administered is a modified BoNT / A of more than 127,500 pg to 300,000 pg.

9. The pharmaceutical product according to claim 7, wherein the spasticity of the limbs in children is spasticity of the upper limbs in children.

10. The pharmaceutical product according to claim 2 or 7. Here, The spasticity is upper limb spasticity, The affected muscle is selected from the following: The first group includes: the superficial flexor muscles of the fingers, the deep flexor muscles of the fingers, the radial carpal flexor muscle, the ulnar carpal flexor muscle, and the brachioradialis muscle; and The second group includes: the brachialis muscle, biceps brachii muscle, triceps brachii (long head), subscapularis muscle, pectoralis muscle (e.g., pectoralis major), and latissimus dorsi muscle.

11. The pharmaceutical product according to claim 10, wherein up to two units are administered to the muscles of the second group affected.

12. The pharmaceutical product according to claim 2 or 7. Here, The spasticity is lower limb spasticity, The affected muscle is selected from the following: A first group including (preferably consisting of): the medial head of the gastrocnemius muscle (medial gastrocnemius), the lateral head of the gastrocnemius muscle (lateral gastrocnemius), the tibialis anterior muscle, the flexor digitorum longus, the flexor digitorum brevis, the flexor hallucis longus, the flexor hallucis brevis, the gracilis muscle, and the gluteus maximus; and A second group including (preferably consisting of): soleus, tibialis posterior, rectus femoris, hamstrings (e.g., biceps femoris, semimembranosus, and / or semitendinosus), and adductor magnus.

13. The pharmaceutical product according to claim 12. Here, The muscles of the second group: (a) In the case of the soleus muscle, a dose of 3 units is administered to it; or (b) In the case of the tibialis posterior, rectus femoris, hamstring muscles (e.g., biceps femoris, semimembranosus, and / or semitendinosus), or adductor magnus, a dose of 2 units is administered.

14. The pharmaceutical product according to claim 1 or 7, wherein the total number of unit doses administered during the treatment is a maximum of 15.

15. The pharmaceutical product according to claim 1 or 7, wherein the modified BoNT / A comprises a polypeptide sequence having at least 70% sequence identity with respect to SEQ ID NO:

6.

16. The pharmaceutical product according to claim 1 or 7, wherein the modified BoNT / A comprises the polypeptide sequence of SEQ ID NO:

6.

17. A unit dosage form of modified botulinum neurotoxin A (BoNT / A), comprising the following: (a) Modified BoNT / A from 17,000pg to 40,000pg, and (b) pharmaceutically acceptable carriers, excipients, adjuvants, and / or salts Here, modified BoNT / A includes the light chain and transposition domain of BoNT / A and the receptor-binding domain (HC domain) of BoNT / B.

18. The pharmaceutical product according to claim 1 or 7. Here, the HC domain of BoNT / B contains one or more substitution mutations selected from the group consisting of E1191M, S1199Y, V1118M, Y1183M, E1191I, E1191Q, E1191T, S1199F, S1199L, S1201V and combinations thereof.

19. The pharmaceutical product according to claim 1 or 7. Here, the modified BoNT / A is a double-stranded modified BoNT / A that includes a light chain containing SEQ ID NO: 11 or 12 and a heavy chain containing SEQ ID NO:

13. The light and heavy chains are joined together by disulfide bonds.

20. A kit comprising the following: (a) The unit dosage form according to claim 17, and (b) Instructions for use in the treatment of limb spasticity, (c) Diluent

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