Treatment of limb spasm

Abstract

The present invention relates to the treatment of limb spasm using a modified botulinum neurotoxin A (BoNT / A) comprising a BoNT / A light chain and a translocation domain, and a BoNT / B receptor binding domain (HC domain). Also provided are unit dosage forms of the modified BoNT / A and corresponding kits.

Description

Field of the Invention

[0001] The present invention relates to the use of modified botulinum neurotoxin A (BoNT / A) to treat limb spasticity. Background Art

[0002] Spasticity is a motor symptom characterized by a velocity-dependent increase in stretch reflexes with exaggerated tendon jerk caused by overexcitation of the stretch reflex and is a component of motor neuron syndrome. Spasticity is associated with various neurological disorders, such as multiple sclerosis, brain or spinal cord injury, traumatic brain injury (TBI) and cerebrovascular disorders (stroke). It is characterized by motor impairment (increased muscle tone, abnormal limb posture, excessive contraction of antagonist muscles and hyperreflexia), functional disability, pain and discomfort.

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

[0004] Management of upper limb spasticity after stroke is challenging due to the diversity of patient presentations and their goals. In these patients, muscle hypertonia often presents with several common clinical patterns that are determined by the muscles affected, which in turn correlate with the size, location, and age of the central nervous system lesion. These patterns were described in an international cross-sectional study of clinicians in 31 countries and subsequently used to establish a classification of 5 typical patterns of AUL spasticity. These patterns were defined relative to the location of the shoulder, elbow, forearm, and wrist joints. Current clinical consensus and existing guidelines recommend that the exact pattern of spasticity should be considered when selecting a treatment for the patient and that botulinum neurotoxin A (BoNT / A) therapy should be tailored accordingly by selecting the appropriate muscles for injection. However, no two patients are alike, and therefore a customized / personalized treatment plan is required.

[0005] These patterns were further studied by an expert panel to provide guidance on treatment parameters for upper limb spasticity (ULS) and target muscles for each clinical pattern (see Figure 1 ). Of note, the expert panel defined three overall posture combinations to target BoNT for treatment. These patterns are:

[0006] (1) Shoulder adduction, elbow flexion, forearm pronation, wrist flexion, and fist clenching;

[0007] (2) elbow flexion, forearm pronation, wrist flexion, and fist clenching; and

[0008] (3) Bend your wrist and make a fist.

[0009] Known Figure 1 The four upper limb joints shown in are involved in >90% of individuals (see Figure 1 ). Most spastic upper limb patterns (four of the five) are caused by internal rotation and adduction of the shoulder and flexion at the elbow, with postural differences caused by the position of the forearm and wrist.

[0010] In general, ULS treatment aims to relieve the signs and symptoms of spasticity - reduce muscle spasms and pain, improve posture, promote mobility and dexterity (voluntary motor functions to reach, grasp, move and release), minimize contractures and deformities and improve ease of patient care and hygiene / self-care and / or quality of life. Available pharmacological treatment options such as oral (benzodiazepines, baclofen, tizanidine and dantrolene) and intrathecal (baclofen) drugs 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.

[0011] The most effective treatment for spasticity is a combination of physical therapy and intramuscular (im) injections of BoNT / A, which has been recommended by several guidelines in recent years. BoNT / A has become the treatment of choice because it has minimal systemic side effects and is a local injection treatment that can be tailored to the individual's disease presentation by selectively targeting the affected muscles.

[0012] is a pharmaceutical product containing the drug substance BoNT / A hemagglutinin complex (BTX-A-HAC) isolated and purified from Clostridium botulinum type A strain. Several other pharmaceutical BoNT / A products naturally produced by Clostridium botulinum are also available on the market (e.g. and ).

[0013] BoNT / A selectively inhibits the release of acetylcholine from presynaptic nerve terminals, thereby blocking cholinergic transmission at the neuromuscular junction, thereby inducing muscle contraction and a decrease in muscle tone, resulting in relaxation of the injected muscle. This mechanism of action has been used therapeutically for more than two decades to treat a variety of clinical neurological disorders, including focal dystonia, focal muscle spasm, and aesthetic disorders.

[0014] However, the duration of action of currently available BoNT / A products is approximately 12 to 14 weeks, when new nerve endings sprout, allowing nerve function to return to normal and initial symptoms to reappear. Therefore, injections need to be repeated regularly to maintain the effect.

[0015] Therefore, the frequency of BoNT / A injections is an important consideration in the treatment of spasticity, given the chronic nature of the condition and the long-term treatment required. Indeed, it has an impact on the direct and indirect health costs involved for patients and caregivers, the organizational effort for injections within the hospital / clinic, and most importantly, the patient's quality of life.

[0016] Approved for the treatment of upper and lower limb spasticity, with a maximum total dose of 1,500 units per treatment period (see Figure 2 – The maximum dose for treating upper limb spasticity is 1,000 units). Clinicians need to administer to multiple muscles in the limbs. The total dose per treatment period is capped at 1,500 units. Clinicians are forced to make difficult choices during treatment of their patients. In other words, in conventional treatment regimens, clinicians must find a balance between the relatively low total amount of BoNT / A that can be administered (1,500 units or 1,000 units required for upper limb spasticity due to the highly toxic properties of BoNT / A) and the effective amount in multiple different muscles. As a result, certain muscles are neglected while other muscles receive suboptimal amounts of BoNT / A, resulting in suboptimal treatment. In addition, current treatment regimens exclude treatment of the shoulder, which requires multiple unit doses at multiple muscles thereof.

[0017] Furthermore, conventional treatment regimens are complex and result in under-dosing as clinicians strive to avoid toxicity to patients. Therefore, there is a need for a convenient, safe and effective single-dose unit that can be administered to a limb during treatment, along with corresponding guidance on the number of units (including the number of injection sites per muscle) without causing patient toxicity.

[0018] In summary, there is a need for an improved treatment of limb spasticity that allows for a patient-centered, individualized approach, tailoring treatment to the target clinical pattern, allowing injection of different combinations of limb muscles depending on the distribution, degree, and severity of spasticity, while avoiding toxicity and providing longer lasting treatment (resulting in less frequent administration). There is also a need for an improved treatment regimen that allows treatment of neglected muscles, such as those of the shoulder.

[0019] The present invention overcomes one or more of the above-identified problems. Summary of the invention

[0020] The present inventors surprisingly found that modified BoNT / A is particularly useful in treating 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 domain), which results in the modified BoNT / A exhibiting increased retention at the site of administration (reduced diffusion at 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 an unmodified BoNT / A (e.g., ). This improved safety profile can be represented by the high safety ratios of the modified BoNT / A described herein.

[0021] 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 an individual while achieving comparable efficacy to unmodified BoNT / A (e.g., ) with a similar safety profile. Thus, in the treatment of limb spasticity, the modified BoNT / A of the present invention can be injected and / or can be injected into more muscles / sites before the maximum total dose is reached. This is an important and advantageous discovery and results in improved treatment of limb spasticity while providing clinicians with a greater range of treatment options. It also provides for the first time the option of being able to treat other large muscles, such as those in the shoulder, while also treating the elbow, forearm, and / or wrist within the maximum dose range. The treatment may be improved because compared to unmodified BoNT / A (e.g. ) treatment, it can provide a more sustained treatment (thereby reducing the frequency of dosing) and / or can be customized to an individual and / or lead to an improved quality of life for the individual. Thus, the treatment of the present invention is improved over conventional treatment regimens.

[0022] In addition, the present invention provides a single unit dose and a total (maximum) dose that can be safely administered in a single treatment that is convenient, safe and effective. The present invention also provides corresponding guidance on the number of times the unit dose can be administered to a limb (e.g., including the number of injection sites per muscle) without causing patient toxicity. Therefore, treatment according to the present invention is much less complex for clinicians and helps avoid underdosing and / or overdosing. In addition, compared with traditional limb spasticity treatments, treatment according to the present invention is more satisfactory for patients because it is better tailored to the needs of the patient. DETAILED DESCRIPTION

[0023] In one aspect, the present invention provides a modified BoNT / A for use in treating limb spasticity (limb spasticity in adults), wherein the modified BoNT / A is administered by intramuscular injection to a plurality of affected muscles of a subject,

[0024] wherein the modified BoNT / A is administered to the plurality of affected muscles at a unit dose of greater than 17,000 pg of modified BoNT / A,

[0025] The plurality of affected muscles are selected from:

[0026] Group 1, including: 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 pollicis longus, adductor pollicis, flexor pollicis brevis, palmaris longus, lumbricals, opponens pollicis, adductor magnus, adductor longus, adductor brevis, gracilis, medial hamstring, lateral hamstring, tensor fasciae latae, rectus femoris, vastus lateralis, vastus medialis, vastus intermedius, gluteus maximus, tibialis anterior, flexor digitorum brevis, extensor pollicis longus, flexor hallucis brevis; and

[0027] The second group includes: 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; and

[0028] wherein a single unit dose is administered at a first group of affected muscles (preferably at a selected injection site) and / or a plurality of unit doses are administered at a second group of affected muscles (preferably at a different selected injection site), and wherein the total dose administered during the treatment period is at most 600,000 pg, and

[0029] The modified BoNT / A comprises a BoNT / A light chain and a translocation domain, and a BoNT / B receptor binding domain (H C domain).

[0030] In another aspect, the present invention provides a modified BoNT / A for use in treating limb spasticity (limb spasticity in adults) in an individual for a duration of treatment longer than the duration of treatment with an unmodified BoNT / A (e.g., SEQ ID NO: 2 [e.g., SEQ ID NO: 2 in di-chain form]), wherein the modified BoNT / A is administered by intramuscular injection to a plurality of affected muscles in the individual,

[0031] wherein the modified BoNT / A is administered to the plurality of affected muscles at a unit dose of greater than 17,000 pg of modified BoNT / A,

[0032] The plurality of affected muscles are selected from:

[0033] Group 1, including: 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 pollicis longus, adductor pollicis, flexor pollicis brevis, palmaris longus, lumbricals, opponens pollicis, adductor magnus, adductor longus, adductor brevis, gracilis, medial hamstring, lateral hamstring, tensor fasciae latae, rectus femoris, vastus lateralis, vastus medialis, vastus intermedius, gluteus maximus, tibialis anterior, flexor digitorum brevis, extensor pollicis longus, flexor hallucis brevis; and

[0034] The second group includes: 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; and

[0035] wherein a single unit dose is administered at a first group of affected muscles (preferably at a selected injection site) and / or a plurality of unit doses are administered at a second group of affected muscles (preferably at a different selected injection site), and wherein the total dose administered during the treatment period is at most 600,000 pg, and

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

[0037] In one aspect, the invention provides a method of treating limb spasticity, the method comprising administering a modified BoNT / A to a plurality of affected muscles of a subject by intramuscular injection,

[0038] wherein the modified BoNT / A is administered to the plurality of affected muscles at a unit dose of greater than 17,000 pg of modified BoNT / A,

[0039] The plurality of affected muscles are selected from:

[0040] Group 1, including: 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 pollicis longus, adductor pollicis, flexor pollicis brevis, palmaris longus, lumbricals, opponens pollicis, adductor magnus, adductor longus, adductor brevis, gracilis, medial hamstring, lateral hamstring, tensor fasciae latae, rectus femoris, vastus lateralis, vastus medialis, vastus intermedius, gluteus maximus, tibialis anterior, flexor digitorum brevis, extensor pollicis longus, flexor hallucis brevis; and

[0041] The second group includes: 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; and

[0042] wherein a single unit dose is administered at a first group of affected muscles (preferably at a selected injection site) and / or a plurality of unit doses are administered at a second group of affected muscles (preferably at a different selected injection site), and wherein the total dose administered during the treatment period is at most 600,000 pg, and

[0043] The modified BoNT / A comprises a BoNT / A light chain and a translocation domain, and a BoNT / B receptor binding domain (H C domain).

[0044] In a related aspect, the invention provides a method of treating limb spasticity (limb spasticity in adults) in an individual for a duration longer than that of treatment with an unmodified BoNT / A (e.g., SEQ ID NO: 2 [e.g., SEQ ID NO: 2 in di-chain form]), the method comprising administering the modified BoNT / A by intramuscular injection to a plurality of affected muscles of the individual,

[0045] wherein the modified BoNT / A is administered to the plurality of affected muscles at a unit dose of greater than 17,000 pg of modified BoNT / A,

[0046] The plurality of affected muscles are selected from:

[0047] Group 1, including: 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 pollicis longus, adductor pollicis, flexor pollicis brevis, palmaris longus, lumbricals, opponens pollicis, adductor magnus, adductor longus, adductor brevis, gracilis, medial hamstring, lateral hamstring, tensor fasciae latae, rectus femoris, vastus lateralis, vastus medialis, vastus intermedius, gluteus maximus, tibialis anterior, flexor digitorum brevis, extensor pollicis longus, flexor hallucis brevis; and

[0048] The second group includes: 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; and

[0049] wherein a single unit dose is administered at a first group of affected muscles (preferably at a selected injection site) and / or a plurality of unit doses are administered at a second group of affected muscles (preferably at a different selected injection site), and wherein the total dose administered during the treatment period is at most 600,000 pg, and

[0050] The modified BoNT / A comprises a BoNT / A light chain and a translocation domain, and a BoNT / B receptor binding domain (H C domain).

[0051] In another aspect, the present invention provides use of a modified BoNT / A in the manufacture of a medicament for treating limb spasticity (limb spasticity in adults), wherein the modified BoNT / A is administered by intramuscular injection to a plurality of affected muscles of a subject,

[0052] wherein the modified BoNT / A is administered to the plurality of affected muscles at a unit dose of greater than 17,000 pg of modified BoNT / A,

[0053] The plurality of affected muscles are selected from:

[0054] Group 1, including: 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 pollicis longus, adductor pollicis, flexor pollicis brevis, palmaris longus, lumbricals, opponens pollicis, adductor magnus, adductor longus, adductor brevis, gracilis, medial hamstring, lateral hamstring, tensor fasciae latae, rectus femoris, vastus lateralis, vastus medialis, vastus intermedius, gluteus maximus, tibialis anterior, flexor digitorum brevis, extensor pollicis longus, flexor hallucis brevis; and

[0055] The second group includes: 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; and

[0056] wherein a single unit dose is administered at a first group of affected muscles (preferably at a selected injection site) and / or a plurality of unit doses are administered at a second group of affected muscles (preferably at a different selected injection site), and wherein the total dose administered during the treatment period is at most 600,000 pg, and

[0057] The modified BoNT / A comprises a BoNT / A light chain and a translocation domain, and a BoNT / B receptor binding domain (H C domain).

[0058] In another aspect, the present invention provides a use of a modified BoNT / A in the manufacture of a medicament for treating limb spasticity (limb spasticity in adults) in a subject for a duration of treatment greater than the duration of treatment with an unmodified BoNT / A (e.g., SEQ ID NO: 2 [e.g., SEQ ID NO: 2 in di-chain form]), wherein the modified BoNT / A is administered by intramuscular injection to a plurality of affected muscles of the subject,

[0059] wherein the modified BoNT / A is administered to the plurality of affected muscles at a unit dose of greater than 17,000 pg of modified BoNT / A,

[0060] The plurality of affected muscles are selected from:

[0061] Group 1, including: 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 pollicis longus, adductor pollicis, flexor pollicis brevis, palmaris longus, lumbricals, opponens pollicis, adductor magnus, adductor longus, adductor brevis, gracilis, medial hamstring, lateral hamstring, tensor fasciae latae, rectus femoris, vastus lateralis, vastus medialis, vastus intermedius, gluteus maximus, tibialis anterior, flexor digitorum brevis, extensor pollicis longus, flexor hallucis brevis; and

[0062] The second group includes: 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; and

[0063] wherein a single unit dose is administered at a first group of affected muscles (preferably at a selected injection site) and / or a plurality of unit doses are administered at a second group of affected muscles (preferably at a different selected injection site), and wherein the total dose administered during the treatment period is at most 600,000 pg, and

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

[0065] The unit dose can be greater than 17,000 pg of modified BoNT / A. The upper limit of the unit dose range can be 40,000, 39,000, 38,000, 37,000, 36,000, 35,000, 30,000, 25,000, 24,000, 22,000, 20,000, or 18,000 pg of modified BoNT / A, with a preferred upper limit being 38,000 pg. The lower end of the unit dose range can be 17,500, 18,000, 20,000, 22,000, 24,000, 25,000, 26,000, 27,000, 28,000, 29,000, 30,000, 35,000, 36,000, 37,000, 38,000, or 39,000 pg of modified BoNT / A, with a preferred lower end being 17,500, 22,753, 22,790, or 33,181 pg. Preferably, the unit dose of modified BoNT / A is greater than 17,000 pg to up to 40,000 pg of modified BoNT / A, such as greater than 17,000 pg to up to 36,000 pg, or 20,000 pg to up to 39,000 pg. The unit dose of modified BoNT / A can be 22,000 to 38,000 pg, preferably 23,000 to 38,000 pg or 34,000 to 38,000 pg. The unit dose of modified BoNT / A can be 24,000 to 36,000 pg or 25,000 to 36,000 pg. The unit dose can be 25,000 pg to up to 40,000 pg of modified BoNT / A. In preferred embodiments, the unit dose of the modified BoNT / A is 24,000, 25,000, 30,000, or 36,000 pg. In more preferred embodiments, the unit dose of the modified BoNT / A is 30,000 or 36,000 pg (eg, 36,000 pg).

[0066] More preferably, the unit dose is 30,000 pg to 40,000 pg, such as 35,000 pg to 37,000 pg of modified BoNT / A. More preferably, the unit dose is 36,000 pg of modified BoNT / A.

[0067] Even more preferably, the unit dose is 20,000 pg to 30,000 pg, such as 24,000 pg to 26,000 pg of modified BoNT / A. Most preferably, the unit dose is 25,000 pg of modified BoNT / A.

[0068] When carrying out the treatment regimen of the invention, the total dose administered can be up to 600,000 pg. In other words, the total amount of modified BoNT / A administered in a given treatment period can be up to 600,000 pg. 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 200,000 pg. Preferably, the total dose may be at most 540,000 pg of modified BoNT / A, more preferably 375,000 pg. The total dose may be at least 17,500, 20,000, 22,500, 25,000, 27,500, 30,000, 35,000, 36,000, 37,000, 38,000, 39,000, 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 can be greater than 255,000 pg or greater than 341,849 pg, more preferably at least 350,000 pg of modified BoNT / A, such as at least 497,700 pg or 500,000 pg. The total dose may be 250,000-600,000 pg or 255,000-600,000 pg, preferably greater than 255,000 pg to a maximum of 600,000 pg, 350,000-600,000 or 500,000-600,000 pg. In a more preferred embodiment, the total dose administered is 520,000-600,000 pg, more preferably 350,000-400,000 pg. The total dose may be 360,000, 450,000 or 540,000 pg, preferably 450,000 or 540,000 pg (e.g., 540,000 pg).

[0069] More preferably, the total dose is 450,000 pg to 600,000 pg, such as 525,000 pg to 555,000 pg of modified BoNT / A. Most preferably, the total dose is at most 540,000 pg of modified BoNT / A (eg, the total dose can be 540,000 pg).

[0070] Even more preferably, the total dose is 300,000 pg to 450,000 pg, such as 360,000 pg to 390,000 pg of modified BoNT / A. Most preferably, the total dose is at most 375,000 pg of modified BoNT / A (eg, the total dose can be 375,000 pg).

[0071] Thus, the unit dose may be greater than 17,000 pg of modified BoNT / A, and the total dose administered when carrying out the treatment regimen of the 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.

[0072] In one aspect, the invention provides a modified BoNT / A for use in treating limb spasticity in a child, wherein the modified BoNT / A is administered by intramuscular injection to a plurality of affected muscles of the individual,

[0073] wherein the modified BoNT / A is administered to the plurality of affected muscles at a unit dose of greater than 8,500 pg of modified BoNT / A,

[0074] The plurality of affected muscles are selected from:

[0075] Group 1, including: 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 pollicis longus, adductor pollicis, flexor pollicis brevis, palmaris longus, lumbricals, opponens pollicis, adductor magnus, adductor longus, adductor brevis, gracilis, medial hamstring, lateral hamstring, tensor fasciae latae, rectus femoris, vastus lateralis, vastus medialis, vastus intermedius, gluteus maximus, tibialis anterior, flexor digitorum brevis, extensor pollicis longus, flexor hallucis brevis; and

[0076] The second group includes: 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; and

[0077] wherein a single unit dose is administered at a first group of affected muscles (preferably at a selected injection site) and / or a plurality of unit doses are administered at a second group of affected muscles (preferably at a different selected injection site), and wherein the total dose administered during the treatment period is at most 300,000 pg, and

[0078] The modified BoNT / A comprises a BoNT / A light chain and a translocation domain, and a BoNT / B receptor binding domain (H C domain).

[0079] In another aspect, the invention provides a modified BoNT / A for use in treating pediatric limb spasticity in an individual for a duration of treatment longer than the duration of treatment with an unmodified BoNT / A (e.g., SEQ ID NO: 2 [e.g., SEQ ID NO: 2 in di-chain form]), wherein the modified BoNT / A is administered by intramuscular injection to a plurality of affected muscles in the individual,

[0080] wherein the modified BoNT / A is administered to the plurality of affected muscles at a unit dose of greater than 8,500 pg of modified BoNT / A,

[0081] The plurality of affected muscles are selected from:

[0082] Group 1, including: 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 pollicis longus, adductor pollicis, flexor pollicis brevis, palmaris longus, lumbricals, opponens pollicis, adductor magnus, adductor longus, adductor brevis, gracilis, medial hamstring, lateral hamstring, tensor fasciae latae, rectus femoris, vastus lateralis, vastus medialis, vastus intermedius, gluteus maximus, tibialis anterior, flexor digitorum brevis, extensor pollicis longus, flexor hallucis brevis; and

[0083] The second group includes: 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; and

[0084] wherein a single unit dose is administered at a first group of affected muscles (preferably at a selected injection site) and / or a plurality of unit doses are administered at a second group of affected muscles (preferably at a different selected injection site), and wherein the total dose administered during the treatment period is at most 300,000 pg, and

[0085] The modified BoNT / A comprises a BoNT / A light chain and a translocation domain, and a BoNT / B receptor binding domain (H C domain).

[0086] In one aspect, the invention provides a method of treating limb spasticity in a child, the method comprising administering a modified BoNT / A to a plurality of affected muscles of a subject by intramuscular injection,

[0087] wherein the modified BoNT / A is administered to the plurality of affected muscles at a unit dose of greater than 8,500 pg of modified BoNT / A,

[0088] The plurality of affected muscles are selected from:

[0089] Group 1, including: 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 pollicis longus, adductor pollicis, flexor pollicis brevis, palmaris longus, lumbricals, opponens pollicis, adductor magnus, adductor longus, adductor brevis, gracilis, medial hamstring, lateral hamstring, tensor fasciae latae, rectus femoris, vastus lateralis, vastus medialis, vastus intermedius, gluteus maximus, tibialis anterior, flexor digitorum brevis, extensor pollicis longus, flexor hallucis brevis; and

[0090] The second group includes: 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; and

[0091] wherein a single unit dose is administered at a first group of affected muscles (preferably at a selected injection site) and / or a plurality of unit doses are administered at a second group of affected muscles (preferably at a different selected injection site), and wherein the total dose administered during the treatment period is at most 300,000 pg, and

[0092] The modified BoNT / A comprises a BoNT / A light chain and a translocation domain, and a BoNT / B receptor binding domain (H C domain).

[0093] In a related aspect, the invention provides a method of treating pediatric limb spasticity in an individual for a duration longer than that of treatment with an unmodified BoNT / A (e.g., SEQ ID NO: 2 [e.g., a di-chain form of SEQ ID NO: 2]), the method comprising administering the modified BoNT / A by intramuscular injection to a plurality of affected muscles of the individual,

[0094] wherein the modified BoNT / A is administered to the plurality of affected muscles at a unit dose of greater than 8,500 pg of modified BoNT / A,

[0095] The plurality of affected muscles are selected from:

[0096] Group 1, including: 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 pollicis longus, adductor pollicis, flexor pollicis brevis, palmaris longus, lumbricals, opponens pollicis, adductor magnus, adductor longus, adductor brevis, gracilis, medial hamstring, lateral hamstring, tensor fasciae latae, rectus femoris, vastus lateralis, vastus medialis, vastus intermedius, gluteus maximus, tibialis anterior, flexor digitorum brevis, extensor pollicis longus, flexor hallucis brevis; and

[0097] The second group includes: 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; and

[0098] wherein a single unit dose is administered at a first group of affected muscles (preferably at a selected injection site) and / or a plurality of unit doses are administered at a second group of affected muscles (preferably at a different selected injection site), and wherein the total dose administered during the treatment period is at most 300,000 pg, and

[0099] The modified BoNT / A comprises a BoNT / A light chain and a translocation domain, and a BoNT / B receptor binding domain (H C domain).

[0100] In another aspect, the present invention provides a use of a modified BoNT / A in the manufacture of a medicament for treating limb spasticity in a child, wherein the modified BoNT / A is administered by intramuscular injection to a plurality of affected muscles of a subject,

[0101] wherein the modified BoNT / A is administered to the plurality of affected muscles at a unit dose of greater than 8,500 pg of modified BoNT / A,

[0102] The plurality of affected muscles are selected from:

[0103] Group 1, including: 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 pollicis longus, adductor pollicis, flexor pollicis brevis, palmaris longus, lumbricals, opponens pollicis, adductor magnus, adductor longus, adductor brevis, gracilis, medial hamstring, lateral hamstring, tensor fasciae latae, rectus femoris, vastus lateralis, vastus medialis, vastus intermedius, gluteus maximus, tibialis anterior, flexor digitorum brevis, extensor pollicis longus, flexor hallucis brevis; and

[0104] The second group includes: 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; and

[0105] wherein a single unit dose is administered at a first group of affected muscles (preferably at a selected injection site) and / or a plurality of unit doses are administered at a second group of affected muscles (preferably at a different selected injection site), and wherein the total dose administered during the treatment period is at most 300,000 pg, and

[0106] The modified BoNT / A comprises a BoNT / A light chain and a translocation domain, and a BoNT / B receptor binding domain (H C domain).

[0107] In another aspect, the present invention provides a use of a modified BoNT / A in the manufacture of a medicament for treating pediatric limb spasticity in a subject for a duration of treatment greater than the duration of treatment with an unmodified BoNT / A (e.g., SEQ ID NO: 2 [e.g., SEQ ID NO: 2 in di-chain form]), wherein the modified BoNT / A is administered by intramuscular injection to a plurality of affected muscles of the subject,

[0108] wherein the modified BoNT / A is administered to the plurality of affected muscles at a unit dose of greater than 8,500 pg of modified BoNT / A,

[0109] The plurality of affected muscles are selected from:

[0110] Group 1, including: 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 pollicis longus, adductor pollicis, flexor pollicis brevis, palmaris longus, lumbricals, opponens pollicis, adductor magnus, adductor longus, adductor brevis, gracilis, medial hamstring, lateral hamstring, tensor fasciae latae, rectus femoris, vastus lateralis, vastus medialis, vastus intermedius, gluteus maximus, tibialis anterior, flexor digitorum brevis, extensor pollicis longus, flexor hallucis brevis; and

[0111] The second group includes: 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; and

[0112] wherein a single unit dose is administered at a first group of affected muscles (preferably at a selected injection site) and / or a plurality of unit doses are administered at a second group of affected muscles (preferably at a different selected injection site), and wherein the total dose administered during the treatment period is at most 300,000 pg, and

[0113] The modified BoNT / A comprises a BoNT / A light chain and a translocation domain, and a BoNT / B receptor binding domain (H C domain).

[0114] The terms "treating the duration of limb spasticity in a subject for longer than the duration of treatment with an unmodified BoNT / A" and "treating the duration of pediatric limb spasticity in a subject for longer than the duration of treatment with an unmodified BoNT / A" may mean that, following administration of a modified BoNT / A of the invention, the reduction in one or more symptoms of limb spasticity in the subject persists for a longer period of time than after administration of an unmodified BoNT / A. The duration of action may be at least 1.25x, 1.5x, 1.75x, 2.0x, or 2.25x or more. The duration of action of a modified BoNT / A may be between 6 and 9 months. For example, the duration of action may be at least: at least 4.5 months (from onset), 5.0 months, 5.5 months, 6 months, 6.5 months, 7.0 months, 7.5 months, 8.0 months, 8.5 months, or 9.0 months. In particular embodiments, the duration of action may be greater than 9.0 months. The reduction may be determined by comparison to an equivalent control subject exhibiting equivalent symptoms that has been treated with an unmodified BoNT / A. An individual treated with a modified BoNT / A according to the present invention can exhibit at least a 5%, 10%, 25%, or 50% improvement in the severity of the one or more symptoms compared to the severity of the one or more symptoms prior to treatment with the modified BoNT / A, over a period of time that is substantially the same (e.g., the same) as the severity of the one or more symptoms in the control individual prior to treatment with the unmodified BoNT / A. The unmodified BoNT / A is preferably SEQ ID NO: 2 in a double-stranded form.

[0115] A unit dose for treating limb spasticity in children can be greater than 8,500 pg of a 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 a modified BoNT / A, with a preferred upper limit being 19,000 pg. The lower end 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 a modified BoNT / A, with a preferred lower end being 8,750, 11,376, 11,377, 11,395, 11,400, or 16,591 pg. Preferably, the unit dose of modified BoNT / A is greater than 8,500 pg to up to 20,000 pg of modified BoNT / A, such as greater than 8,500 pg to up to 18,000 pg, 10,000 pg to up to 19,500 pg. The unit dose of modified BoNT / A can be 11,000 pg to 19,000 pg, preferably 11,500 to 19,000 pg or 17,000 to 19,000 pg. The unit dose of modified BoNT / A can be 12,000 pg to 18,000 pg or 12,500 pg to 18,000 pg. The unit dose can be 12,500 pg to 20,000 pg 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 (eg, 18,000 pg).

[0116] More preferably, the unit dose for treating limb spasticity in children is 15,000 pg to 20,000 pg, such as 17,500 to 18,500 pg of modified BoNT / A. More preferably, the unit dose for treating limb spasticity in children is 18,000 pg of modified BoNT / A.

[0117] Even more preferably, the unit dose for treating limb spasticity in children is 10,000 pg to 15,000 pg, such as 12,000 pg to 13,000 pg of modified BoNT / A. More preferably, the unit dose for treating limb spasticity in children is 12,500 pg of modified BoNT / A.

[0118] When carrying out the infantile spasms treatment regimen of the present invention, the total dose administered can be up to 300,000 pg. In other words, the total amount of modified BoNT / A administered in a given treatment period can be up to 300,000 pg. 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 100,000 pg. 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, such as at least 248,850 pg, 248,851 pg, or 250,000 pg. The total dose may be 125,000-300,000 pg or 127,500-300,000 pg, preferably greater than 127,500 pg to a maximum of 300,000 pg, 175,000-300,000 or 250,000-300,000 pg. In a more preferred embodiment, the total dose administered is 260,000-300,000 pg, more preferably 175,000-200,000 pg. The total dose may be 180,000, 225,000 or 270,000 pg, preferably 225,000 or 270,000 pg (e.g., 270,000 pg).

[0119] More preferably, the total dose when treating limb spasticity in children is 225,000 pg to 300,000 pg, such as 262,500 pg to 277,500 pg of modified BoNT / A. Most preferably, the total dose when treating limb spasticity in children is up to 270,000 pg of modified BoNT / A (e.g., the total dose can be 270,000 pg).

[0120] Even more preferably, the total dose when treating limb spasticity in children is 150,000 pg to 225,000 pg, such as 180,000 pg to 195,000 pg of modified BoNT / A. Most preferably, the total dose when treating limb spasticity in children is up to 187,500 pg of modified BoNT / A (e.g., the total dose can be 187,500 pg).

[0121] Thus, the unit dose for treating limb spasticity in children may be greater 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 limb spasticity in children 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 187,500 pg or may be up to 187,500 pg.

[0122] The unit dose can be expressed as an amount of a modified BoNT / A of the invention, a unit of the modified BoNT / A, or a combination thereof. Thus, the unit dose of a modified BoNT / A can also be expressed in both units and amounts (pg or ng, preferably pg).

[0123] In one aspect, the present invention provides a modified BoNT / A for use in treating limb spasticity (limb spasticity in adults), wherein the modified BoNT / A is administered by intramuscular injection to a plurality of affected muscles of a subject,

[0124] wherein the modified BoNT / A is administered to the affected muscles at a unit dose of greater than 707 units of the modified BoNT / A, and wherein 1 unit corresponds to a calculated median lethal dose (LD50) in mice 50 ),

[0125] The plurality of affected muscles are selected from:

[0126] Group 1, including: 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 pollicis longus, adductor pollicis, flexor pollicis brevis, palmaris longus, lumbricals, opponens pollicis, adductor magnus, adductor longus, adductor brevis, gracilis, medial hamstring, lateral hamstring, tensor fasciae latae, rectus femoris, vastus lateralis, vastus medialis, vastus intermedius, gluteus maximus, tibialis anterior, flexor digitorum brevis, extensor pollicis longus, flexor hallucis brevis; and

[0127] The second group includes: 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; and

[0128] wherein a single unit dose is administered at a first set of affected muscles (preferably at a selected injection site) and / or a plurality of unit doses are administered at a second set of affected muscles (preferably at a different selected injection site), and wherein the total dose administered during the treatment period is a maximum of 24,958 units, and

[0129] The modified BoNT / A comprises a BoNT / A light chain and a translocation domain, and a BoNT / B receptor binding domain (H C domain).

[0130] In a related aspect, the invention provides a modified BoNT / A for use in treating limb spasticity (limb spasticity in adults) in an individual for a duration of treatment longer than the duration of treatment with an unmodified BoNT / A (e.g., SEQ ID NO: 2 [e.g., SEQ ID NO: 2 in di-chain form]), wherein the modified BoNT / A is administered by intramuscular injection to a plurality of affected muscles of the individual,

[0131] wherein the modified BoNT / A is administered to the affected muscles at a unit dose of greater than 707 units of the modified BoNT / A, and wherein 1 unit corresponds to a calculated median lethal dose (LD50) in mice 50 ),

[0132] The plurality of affected muscles are selected from:

[0133] Group 1, including: 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 pollicis longus, adductor pollicis, flexor pollicis brevis, palmaris longus, lumbricals, opponens pollicis, adductor magnus, adductor longus, adductor brevis, gracilis, medial hamstring, lateral hamstring, tensor fasciae latae, rectus femoris, vastus lateralis, vastus medialis, vastus intermedius, gluteus maximus, tibialis anterior, flexor digitorum brevis, extensor pollicis longus, flexor hallucis brevis; and

[0134] The second group includes: 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; and

[0135] wherein a single unit dose is administered at a first set of affected muscles (preferably at a selected injection site) and / or a plurality of unit doses are administered at a second set of affected muscles (preferably at a different selected injection site), and wherein the total dose administered during the treatment period is a maximum of 24,958 units, and

[0136] The modified BoNT / A comprises a BoNT / A light chain and a translocation domain, and a BoNT / B receptor binding domain (H C domain).

[0137] In a related aspect, the invention provides a method of treating limb spasticity (limb spasticity in adults), the method comprising administering a modified BoNT / A by intramuscular injection to a plurality of affected muscles of a subject,

[0138] wherein the modified BoNT / A is administered to the affected muscles at a unit dose of greater than 707 units of the modified BoNT / A, and wherein 1 unit corresponds to a calculated median lethal dose (LD50) in mice 50 ),

[0139] The plurality of affected muscles are selected from:

[0140] Group 1, including: 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 pollicis longus, adductor pollicis, flexor pollicis brevis, palmaris longus, lumbricals, opponens pollicis, adductor magnus, adductor longus, adductor brevis, gracilis, medial hamstring, lateral hamstring, tensor fasciae latae, rectus femoris, vastus lateralis, vastus medialis, vastus intermedius, gluteus maximus, tibialis anterior, flexor digitorum brevis, extensor pollicis longus, flexor hallucis brevis; and

[0141] The second group includes: 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; and

[0142] wherein a single unit dose is administered at a first set of affected muscles (preferably at a selected injection site) and / or a plurality of unit doses are administered at a second set of affected muscles (preferably at a different selected injection site), and wherein the total dose administered during the treatment period is a maximum of 24,958 units, and

[0143] The modified BoNT / A comprises a BoNT / A light chain and a translocation domain, and a BoNT / B receptor binding domain (H C domain).

[0144] In a related aspect, the invention provides a method of treating limb spasticity in an individual for a duration longer than that of treatment with an unmodified BoNT / A (e.g., SEQ ID NO: 2 [e.g., a di-chain form of SEQ ID NO: 2]), the method comprising administering the modified BoNT / A by intramuscular injection to a plurality of affected muscles of the individual,

[0145] wherein the modified BoNT / A is administered to the affected muscles at a unit dose of greater than 707 units of the modified BoNT / A, and wherein 1 unit corresponds to a calculated median lethal dose (LD50) in mice 50 ),

[0146] The plurality of affected muscles are selected from:

[0147] Group 1, including: 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 pollicis longus, adductor pollicis, flexor pollicis brevis, palmaris longus, lumbricals, opponens pollicis, adductor magnus, adductor longus, adductor brevis, gracilis, medial hamstring, lateral hamstring, tensor fasciae latae, rectus femoris, vastus lateralis, vastus medialis, vastus intermedius, gluteus maximus, tibialis anterior, flexor digitorum brevis, extensor pollicis longus, flexor hallucis brevis; and

[0148] The second group includes: 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; and

[0149] wherein a single unit dose is administered at a first set of affected muscles (preferably at a selected injection site) and / or a plurality of unit doses are administered at a second set of affected muscles (preferably at a different selected injection site), and wherein the total dose administered during the treatment period is a maximum of 24,958 units, and

[0150] The modified BoNT / A comprises a BoNT / A light chain and a translocation domain, and a BoNT / B receptor binding domain (H C domain).

[0151] In another related aspect, the present invention provides use of a modified BoNT / A in the manufacture of a medicament for treating limb spasticity (limb spasticity in adults), wherein the modified BoNT / A is administered by intramuscular injection to a plurality of affected muscles of a subject,

[0152] wherein the modified BoNT / A is administered to the affected muscles at a unit dose of greater than 707 units of the modified BoNT / A, and wherein 1 unit corresponds to a calculated median lethal dose (LD50) in mice 50 ),

[0153] The plurality of affected muscles are selected from:

[0154] Group 1, including: 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 pollicis longus, adductor pollicis, flexor pollicis brevis, palmaris longus, lumbricals, opponens pollicis, adductor magnus, adductor longus, adductor brevis, gracilis, medial hamstring, lateral hamstring, tensor fasciae latae, rectus femoris, vastus lateralis, vastus medialis, vastus intermedius, gluteus maximus, tibialis anterior, flexor digitorum brevis, extensor pollicis longus, flexor hallucis brevis; and

[0155] The second group includes: 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; and

[0156] wherein a single unit dose is administered at a first set of affected muscles (preferably at a selected injection site) and / or a plurality of unit doses are administered at a second set of affected muscles (preferably at a different selected injection site), and wherein the total dose administered during the treatment period is a maximum of 24,958 units, and

[0157] The modified BoNT / A comprises a BoNT / A light chain and a translocation domain, and a BoNT / B receptor binding domain (H C domain).

[0158] In another related aspect, the present invention provides use of a modified BoNT / A in the manufacture of a medicament for treating limb spasticity (limb spasticity in adults) in a subject for a duration of treatment greater than the duration of treatment with an unmodified BoNT / A (e.g., SEQ ID NO: 2 [e.g., SEQ ID NO: 2 in di-chain form]), wherein the modified BoNT / A is administered by intramuscular injection to a plurality of affected muscles of the subject,

[0159] wherein the modified BoNT / A is administered to the affected muscles at a unit dose of greater than 707 units of the modified BoNT / A, and wherein 1 unit corresponds to a calculated median lethal dose (LD50) in mice 50 ),

[0160] The plurality of affected muscles are selected from:

[0161] Group 1, including: 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 pollicis longus, adductor pollicis, flexor pollicis brevis, palmaris longus, lumbricals, opponens pollicis, adductor magnus, adductor longus, adductor brevis, gracilis, medial hamstring, lateral hamstring, tensor fasciae latae, rectus femoris, vastus lateralis, vastus medialis, vastus intermedius, gluteus maximus, tibialis anterior, flexor digitorum brevis, extensor pollicis longus, flexor hallucis brevis; and

[0162] The second group includes: 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; and

[0163] wherein a single unit dose is administered at a first set of affected muscles (preferably at a selected injection site) and / or a plurality of unit doses are administered at a second set of affected muscles (preferably at a different selected injection site), and wherein the total dose administered during the treatment period is a maximum of 24,958 units, and

[0164] The modified BoNT / A comprises a BoNT / A light chain and a translocation domain, and a BoNT / B receptor binding domain (H C domain).

[0165] The unit dose can be greater than 707 units of a modified BoNT / A. The upper limit of the unit dose range can be 1664, 1650, 1600, 1550, 1500, 1450, 1400, 1350, 1300, 1250, 1150, 1100, 1050, 1000, 950, 900, 850, 800, or 750 units of a modified BoNT / A, with a preferred upper limit being 1500 units. The lower end of the unit dose 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 a modified BoNT / A, with a preferred lower end being 725 units, 949 units, or 1381 units. Preferably, the unit dose of a modified BoNT / A is from greater than 707 units to up to 1664 units of a modified BoNT / A, e.g., from greater than 707 units to up to 1,498 units or from 832 units to 1622 units. The unit dose of the modified BoNT / A can be 915 to 1581 units, preferably 949 to 1581 units or 1414 to 1581 units. The unit dose of the modified BoNT / A can be 998 to 1,498 units or 1,040 to 1,498 units. In preferred embodiments, the unit dose of the modified BoNT / A is 998, 1,040, 1,248, or 1,498 units of the modified BoNT / A. In more preferred embodiments, the unit dose comprises 1,248 or 1,498 units (e.g., 1,498 units) of the modified BoNT / A.

[0166] More preferably, the unit dose is 1,248 units to 1,664 units, such as 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.

[0167] Even more preferably, the unit dose is 832 units to 1,248 units, such as 998 units to 1,082 units of the modified BoNT / A. Most preferably, the unit dose is 1,040 units of the modified BoNT / A.

[0168] When carrying out the treatment regimen of the present invention, the total dose administered can be up to 24,958 units. In other words, the total amount of modified BoNT / A administered in a given treatment period can be up to 24,958 units. The total dose may 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 can be greater than 10,607 units or greater than 14,220 units, more preferably at least 14,559 units of modified BoNT / A, such as at least 20,703 units or 20,799 units. The total dose can be 10,339-24,958 units, 10,399-24,958 units, or 10,607-24,958 units, preferably greater than 10,607 units up to 24,958 units, 14,559-24,958 units, or 20,799-24,958 units. More preferably, the total dose administered is 21,631-24,958 units, more preferably 14,559-16,639 units. The total dose may be 14,975 units, 18,719 units or 22,463 units, preferably 18,719 or 22,463 units (eg 22,463 units).

[0169] More preferably, the total dose is 18,719 units to 24,958 units, such as 21,839 units to 23,087 units of the modified BoNT / A. Most preferably, the total dose is at most 22,463 units of the modified BoNT / A (e.g., the total dose can be 22,463 units).

[0170] Even more preferably, the total dose is 12,479 units to 18,719 units, such as 14,975 units to 16,223 units of modified BoNT / A. Most preferably, the total dose is at most 15,599 units of modified BoNT / A (e.g., the total dose can be 15,599 units).

[0171] Thus, the unit dose may be greater than 707 units of modified BoNT / A, and the total dose administered when carrying out the treatment regimen of the 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 non-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 non-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.

[0172] In one aspect, the invention provides a modified BoNT / A for use in treating limb spasticity in a child, wherein the modified BoNT / A is administered by intramuscular injection to a plurality of affected muscles of the individual,

[0173] wherein the modified BoNT / A is administered to the affected muscles at a unit dose of greater than 353.5 units of the modified BoNT / A, wherein 1 unit is the median lethal dose (LD50) calculated in mice. 50 ) corresponds to the amount of modified BoNT / A,

[0174] The muscles affected are selected from:

[0175] Group 1, including: 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 pollicis longus, adductor pollicis, flexor pollicis brevis, palmaris longus, lumbricals, opponens pollicis, adductor magnus, adductor longus, adductor brevis, gracilis, medial hamstring, lateral hamstring, tensor fasciae latae, rectus femoris, vastus lateralis, vastus medialis, vastus intermedius, gluteus maximus, tibialis anterior, flexor digitorum brevis, extensor pollicis longus, flexor hallucis brevis; and

[0176] The second group includes: 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; and

[0177] wherein a single unit dose is administered at a first set of affected muscles (preferably at a selected injection site) and / or a plurality of unit doses are administered at a second set of affected muscles (preferably at a different selected injection site), and wherein the total dose administered during the treatment period is a maximum of 12,479 units, and

[0178] The modified BoNT / A comprises a BoNT / A light chain and a translocation domain, and a BoNT / B receptor binding domain (H C domain).

[0179] In another related aspect, the invention provides a modified BoNT / A for use in treating pediatric limb spasticity in an individual for a duration of treatment that is longer than the duration of treatment with an unmodified BoNT / A (e.g., SEQ ID NO: 2 [e.g., SEQ ID NO: 2 in di-chain form]), wherein the modified BoNT / A is administered by intramuscular injection to a plurality of affected muscles in the individual,

[0180] wherein the modified BoNT / A is administered to the affected muscles at a unit dose of greater than 353.5 units of the modified BoNT / A, and wherein 1 unit corresponds to a calculated median lethal dose (LD50) in mice. 50 ),

[0181] The plurality of affected muscles are selected from:

[0182] Group 1, including: 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 pollicis longus, adductor pollicis, flexor pollicis brevis, palmaris longus, lumbricals, opponens pollicis, adductor magnus, adductor longus, adductor brevis, gracilis, medial hamstring, lateral hamstring, tensor fasciae latae, rectus femoris, vastus lateralis, vastus medialis, vastus intermedius, gluteus maximus, tibialis anterior, flexor digitorum brevis, extensor pollicis longus, flexor hallucis brevis; and

[0183] The second group includes: 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; and

[0184] wherein a single unit dose is administered at a first set of affected muscles (preferably at a selected injection site) and / or a plurality of unit doses are administered at a second set of affected muscles (preferably at a different selected injection site), and wherein the total dose administered during the treatment period is a maximum of 12,479 units, and

[0185] The modified BoNT / A comprises a BoNT / A light chain and a translocation domain, and a BoNT / B receptor binding domain (H C domain).

[0186] In a related aspect, the invention provides a method of treating limb spasticity in a child, the method comprising administering a modified BoNT / A by intramuscular injection to a plurality of affected muscles of the individual,

[0187] wherein the modified BoNT / A is administered to the affected muscles at a unit dose of greater than 353.5 units of the modified BoNT / A, wherein 1 unit is the median lethal dose (LD50) calculated in mice. 50 ) corresponds to the amount of modified BoNT / A,

[0188] The muscles affected are selected from:

[0189] Group 1, including: 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 pollicis longus, adductor pollicis, flexor pollicis brevis, palmaris longus, lumbricals, opponens pollicis, adductor magnus, adductor longus, adductor brevis, gracilis, medial hamstring, lateral hamstring, tensor fasciae latae, rectus femoris, vastus lateralis, vastus medialis, vastus intermedius, gluteus maximus, tibialis anterior, flexor digitorum brevis, extensor pollicis longus, flexor hallucis brevis; and

[0190] The second group includes: 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; and

[0191] wherein a single unit dose is administered at a first set of affected muscles (preferably at a selected injection site) and / or a plurality of unit doses are administered at a second set of affected muscles (preferably at a different selected injection site), and wherein the total dose administered during the treatment period is a maximum of 12,479 units, and

[0192] The modified BoNT / A comprises a BoNT / A light chain and a translocation domain, and a BoNT / B receptor binding domain (H C domain).

[0193] In a related aspect, the invention provides a method of treating pediatric limb spasticity in an individual for a duration longer than that of treatment with an unmodified BoNT / A (e.g., SEQ ID NO: 2 [e.g., a di-chain form of SEQ ID NO: 2]), the method comprising administering the modified BoNT / A by intramuscular injection to a plurality of affected muscles of the individual,

[0194] wherein the modified BoNT / A is administered to the affected muscles at a unit dose of greater than 353.5 units of the modified BoNT / A, and wherein 1 unit corresponds to a calculated median lethal dose (LD50) in mice. 50 ),

[0195] The plurality of affected muscles are selected from:

[0196] Group 1, including: 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 pollicis longus, adductor pollicis, flexor pollicis brevis, palmaris longus, lumbricals, opponens pollicis, adductor magnus, adductor longus, adductor brevis, gracilis, medial hamstring, lateral hamstring, tensor fasciae latae, rectus femoris, vastus lateralis, vastus medialis, vastus intermedius, gluteus maximus, tibialis anterior, flexor digitorum brevis, extensor pollicis longus, flexor hallucis brevis; and

[0197] The second group includes: 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; and

[0198] wherein a single unit dose is administered at a first set of affected muscles (preferably at a selected injection site) and / or a plurality of unit doses are administered at a second set of affected muscles (preferably at a different selected injection site), and wherein the total dose administered during the treatment period is a maximum of 12,479 units, and

[0199] The modified BoNT / A comprises a BoNT / A light chain and a translocation domain, and a BoNT / B receptor binding domain (H C domain).

[0200] In another related aspect, the present invention provides a use of a modified BoNT / A in the manufacture of a medicament for treating limb spasticity in a child, wherein the modified BoNT / A is administered by intramuscular injection to a plurality of affected muscles of a subject,

[0201] wherein the modified BoNT / A is administered to the affected muscles at a unit dose of greater than 353.5 units of the modified BoNT / A, wherein 1 unit is the median lethal dose (LD50) calculated in mice. 50 ) corresponds to the amount of modified BoNT / A,

[0202] The muscles affected are selected from:

[0203] Group 1, including: 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 pollicis longus, adductor pollicis, flexor pollicis brevis, palmaris longus, lumbricals, opponens pollicis, adductor magnus, adductor longus, adductor brevis, gracilis, medial hamstring, lateral hamstring, tensor fasciae latae, rectus femoris, vastus lateralis, vastus medialis, vastus intermedius, gluteus maximus, tibialis anterior, flexor digitorum brevis, extensor pollicis longus, flexor hallucis brevis; and

[0204] The second group includes: 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; and

[0205] wherein a single unit dose is administered at a first set of affected muscles (preferably at a selected injection site) and / or a plurality of unit doses are administered at a second set of affected muscles (preferably at a different selected injection site), and wherein the total dose administered during the treatment period is a maximum of 12,479 units, and

[0206] The modified BoNT / A comprises a BoNT / A light chain and a translocation domain, and a BoNT / B receptor binding domain (H C domain).

[0207] In another related aspect, the invention provides use of a modified BoNT / A in the manufacture of a medicament for treating pediatric limb spasticity in a subject for a duration of treatment greater than the duration of treatment with an unmodified BoNT / A (e.g., SEQ ID NO: 2 [e.g., SEQ ID NO: 2 in di-chain form]), wherein the modified BoNT / A is administered by intramuscular injection to a plurality of affected muscles of the subject,

[0208] wherein the modified BoNT / A is administered to the affected muscles at a unit dose of greater than 353.5 units of the modified BoNT / A, and wherein 1 unit corresponds to a calculated median lethal dose (LD50) in mice. 50 ),

[0209] The plurality of affected muscles are selected from:

[0210] Group 1, including: 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 pollicis longus, adductor pollicis, flexor pollicis brevis, palmaris longus, lumbricals, opponens pollicis, adductor magnus, adductor longus, adductor brevis, gracilis, medial hamstring, lateral hamstring, tensor fasciae latae, rectus femoris, vastus lateralis, vastus medialis, vastus intermedius, gluteus maximus, tibialis anterior, flexor digitorum brevis, extensor pollicis longus, flexor hallucis brevis; and

[0211] The second group includes: 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; and

[0212] wherein a single unit dose is administered at a first set of affected muscles (preferably at a selected injection site) and / or a plurality of unit doses are administered at a second set of affected muscles (preferably at a different selected injection site), and wherein the total dose administered during the treatment period is a maximum of 12,479 units, and

[0213] The modified BoNT / A comprises a BoNT / A light chain and a translocation domain, and a BoNT / B receptor binding domain (H C domain).

[0214] A suitable unit dose for treating limb spasticity in children can be greater than 353.5 units of a modified BoNT / A. The upper limit of the unit dose range for treating limb spasticity in children can be 832, 825, 800, 775, 750, 725, 700, 675, 650, 625, 600, 575, 550, 525, 500, 475, 450, or 425 units of a modified BoNT / A, with a preferred upper limit being 790 units. The lower end of the unit dose range for treating limb spasticity in children 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 a modified BoNT / A, with a preferred lower end being 364 units, 475 units, or 690 units. Preferably, the unit dose of a modified BoNT / A for treating limb spasticity in children is from greater than 353.5 units to up to 832 units of a modified BoNT / A, such as from greater than 353.5 units to up to 749 units or from 416 units to 811 units. The unit dose of a modified BoNT / A for treating limb spasticity in children 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 a modified BoNT / A is 499, 520, 624, or 749 units. In more preferred embodiments, the unit dose of a modified BoNT / A is 624 or 749 units (e.g., 749 units).

[0215] More preferably, the unit dose for treating limb spasticity in children is 624 units to 832 units, such as 728 units to 770 units of the modified BoNT / A. More preferably, the unit dose for treating limb spasticity in children is 749 units of the modified BoNT / A.

[0216] Even more preferably, the unit dose for treating limb spasticity in children is 416 units to 624 units, such as 499 units to 541 units of the modified BoNT / A. Most preferably, the unit dose for treating limb spasticity in children is 520 units of the modified BoNT / A.

[0217] When carrying out the treatment regimen of the present invention for treating limb spasticity in children, the total dose administered can be up to 12,479 units. In other words, the total amount of modified BoNT / A administered in a given treatment period for treating limb spasticity in children can be up to 12,479 units. The total dose for treating limb spasticity in children 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 limb spasticity in children can be up to 11,250 units of a modified BoNT / A. Preferably, the total dose for treating limb spasticity in children can be up to 11,232 units of modified BoNT / A, more preferably 7,800 units. The total dose for treating limb spasticity in children 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 limb spasticity in children can be at least 5,304 units or greater than 7,110 units, more preferably at least 7,280 units of modified BoNT / A, such as at least 10,351 units or 10,400 units. The total dose for treating limb spasticity in children can be 5,200-12,479 units or 5,304-12,479 units, preferably greater than 5,200 units to a maximum of 12,479 units, greater than 5,301 units to a maximum of 12,479 units, 7,280-12,479 units, or 10,400-12,479 units. In a more preferred embodiment, the total dose administered is 10,815-12,479 units or 10,816-12,479 units, more preferably 7,280-8,320 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 (eg, 11,231 units).

[0218] More preferably, when treating limb spasticity in children, the total dose is 9,360 units to 12,479 units, such as 10,920 units to 11,544 units of the modified BoNT / A. Most preferably, when treating limb spasticity in children, the total dose is up to 11,232 units of the modified BoNT / A (e.g., the total dose can be 11,232 units).

[0219] Even more preferably, when treating limb spasticity in children, the total dose is 6,240 units to 9,360 units, such as 7,488 units to 8,112 units of the modified BoNT / A. Most preferably, when treating limb spasticity in children, the total dose is up to 7,800 units of the modified BoNT / A (e.g., the total dose can be 7,800 units).

[0220] Thus, the unit dose for treating limb spasticity in children can be greater 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 limb spasticity in children can be up to 12,479 units. In a preferred embodiment, the unit dose can be 499 units of modified BoNT / A, and the total dose can be 7,488 units or can be up to 7,488 units. In another preferred embodiment, the unit dose can be 624 units of modified BoNT / A, and the total dose can be 9,359 units or 9,360 units, or can be up to 9,359 units, or can be up to 9,360 units. In another preferred embodiment, the unit dose can be 749 units of modified BoNT / A, and the total dose can be 11,231 units or can be up to 11,231 units. In another preferred embodiment, the unit dose can be 749 units of modified BoNT / A, and the total dose can be 11,232 units or can 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.

[0221] An "affected muscle" is a muscle that exhibits symptoms of spasticity or causes spasticity in the affected limb. For example, the muscle may exhibit increased muscle tone or stiffness.

[0222] The plurality of affected muscles are selected from the first group and the second group as described herein. The selected plurality of affected muscles may be at least one muscle of the first group and / or at least one muscle of the second group. Alternatively, the plurality of affected muscles may be two or more muscles from the same group (e.g., two or more first group muscles or two or more second group muscles). Preferably, the plurality of affected muscles comprises at least one first group muscle and at least one second group muscle.

[0223] The multiple muscles can be muscles of the same limb or muscles of different limbs. However, preferably, the multiple muscles are muscles of the same limb. The present invention includes treating spasticity in one or more limbs simultaneously. For example, the modified BoNT / A can be administered to one or both upper limbs of an individual per treatment period, the modified BoNT / A can be administered to one or both lower limbs of an individual per treatment period, or the modified BoNT / A can be administered to a combination of lower and upper limbs per treatment period. Whether treating two or more limbs, preferably at least 2 muscles per limb are treated, such as at least 3, 4, or 5 muscles per limb.

[0224] The potency of the modified BoNT / A used in accordance with the present invention can be determined by mouse LD according to standard techniques. 50 In the test, 1 unit is defined as the amount corresponding to the median lethal dose (LD50) calculated in mice. 50 ) of the modified BoNT / A. Preferably, the calculated median lethal intraperitoneal dose in mice.

[0225] Specifically, in the test, the amount equivalent to 1 unit of modified BoNT / A is preferably 24.04 pg.

[0226] Preferably, the dose of the modified BoNT / A is administered to the affected muscle by intramuscular injection. More preferably, a single unit dose is administered per injection site. The term "administering a single unit dose" means administering substantially all of the single unit dose. For example, a residual 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 has been reconstituted. However, it is preferred that 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., administered to a muscle selected from the first group described herein) or multiple unit doses are administered (i.e., administered to 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), while some muscles are injected at two or more sites (i.e., muscles selected from the second group described herein).

[0227] In one embodiment, a single unit dose is administered at multiple injection sites in a first group of muscles affected and / or multiple unit doses are administered at multiple injection sites in a second group of muscles affected.

[0228] The term "up to" when used in reference to a value (e.g., up to 600,000 pg) means up to and including the referenced value. Thus, as an example, reference to administering "up to 600,000 pg" of a modified BoNT / A includes administering 600,000 pg of the modified BoNT / A as well as administering less than 600,000 pg of the modified BoNT / A.

[0229] When the first group of muscles is selected for treatment, a single unit dose of the modified BoNT / A is administered to a muscle selected from the first group described herein.

[0230] A plurality of unit doses of the modified BoNT / A is administered to a muscle selected from the second group (e.g., when the second group of muscles is selected for treatment). For example, at least 2×, 3×, or 4× unit doses can be administered. In some embodiments, 2-4× unit doses can be administered. Preferably, 2× unit doses of the modified BoNT / A are administered to a muscle selected from the second group.

[0231] Some muscles may be present in the first muscle group and the second muscle group. Thus, the clinician may decide whether to administer a single unit dose or multiple unit doses to said muscles.

[0232] Limb spasticity can be upper limb spasticity or lower limb spasticity.

[0233] When treating lower limb spasticity, the modified BoNT / A can be administered to a plurality of muscles selected from the group consisting of: a first group comprising (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 hamstring, lateral hamstring, tensor fasciae latae, rectus femoris, vastus lateralis, vastus medialis, vastus intermedius, gluteus maximus, tibialis anterior, flexor digitorum brevis, extensor hallucis longus, flexor hallucis brevis; and a second group comprising (preferably consisting of): soleus, tibialis posterior, iliopsoas, and gastrocnemius.

[0234] When administering to the flexor pollicis longus muscle, in some cases a single unit dose may be administered, while in other cases 2× unit doses may be administered.

[0235] Suitable doses for lower extremity muscles are as follows:

[0236] Injected muscle dose Gastrocnemius (medial head) 1×UD Gastrocnemius (lateral head) 1×UD Soleus 2×UD or 3×UD Tibialis posterior muscle 2×UD Flexor digitorum longus (FDL) 1×UD Flexor pollicis longus 1× or 2×UD

[0237] The modified BoNT / A can be administered to one or more muscles at the dosages shown in the table above.

[0238] The modified BoNT / A may be administered to one or more of the following muscles at the following doses:

[0239] Injected muscle dose Adductor magnus 1×UD adductor longus 1×UD Adductor brevis 1×UD Gracilis 1×UD Medial hamstrings 1×UD Lateral hamstrings 1×UD Tensor fasciae latae 1×UD Rectus femoris 1×UD Vastus lateralis 1×UD Vastus medialis 1×UD Vastus intermedius 1×UD gluteus maximus 1×UD Tibialis anterior muscle 1×UD FDL 1×UD Flexor digitorum brevis (FDB) 1×UD Flexor pollicis longus 1×UD Extensor pollicis longus 1×UD Flexor pollicis brevis 1×UD Iliopsoas 2×UD Gastrocnemius 2×UD Tibialis posterior muscle 2×UD Soleus 2×UD

[0240] Preferably, when treating lower limb spasticity, the modified BoNT / A can be administered to a plurality of muscles selected from the group consisting of: a first group comprising (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, gluteus maximus; and a second group comprising (preferably consisting of): soleus, tibialis posterior, rectus femoris, hamstrings (e.g., biceps femoris, semimembranosus and / or semitendinosus), and adductor magnus.

[0241] The hamstring muscle can be the biceps femoris, semimembranosus, and / or semitendinosus. For example, the modified BoNT / A can be administered to one or more (e.g., all) of the biceps femoris, semimembranosus, and / or semitendinosus.

[0242] Preferably, three unit doses are administered to the soleus muscle. Preferably, two unit doses are administered to the tibialis posterior muscle. Preferably, two unit doses are administered to the rectus femoris muscle. Preferably, two unit doses are administered to the hamstring muscle, for example, two units may be administered to the biceps femoris muscle, two units may be administered to the semimembranosus muscle, and / or two units may be administered to the semitendinosus muscle). Preferably, two unit doses are administered to the adductor magnus muscle.

[0243] The modified BoNT / A may be administered to one or more of the following muscles at the following doses:

[0244]

[0245]

[0246] Preferably, the limb spasm is upper limb spasm.

[0247] When treating upper limb spasticity, the modified BoNT / A can be administered to a plurality of affected muscles selected from: a first group comprising (preferably consisting of) 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, lumbricals, and opponens pollicis; and a second group comprising (preferably consisting of) triceps brachii (long head), subscapularis, pectoralis (e.g., pectoralis major), latissimus dorsi, biceps brachii, brachialis, brachioradialis, and teres major.

[0248] Preferably, when treating upper limb spasticity, the modified BoNT / A can be administered to a plurality of affected muscles selected from: a first group comprising (preferably consisting of): flexor digitorum superficialis, flexor digitorum profundus, flexor carpi radialis, flexor carpi ulnaris, brachioradialis, pronator teres, biceps brachii; and a second group comprising (preferably consisting of): triceps brachii (long head), subscapularis, pectoralis major, latissimus dorsi, biceps brachii, and brachialis.

[0249] For example, preferably, when treating upper limb spasticity, the modified BoNT / A can be administered to a plurality of affected muscles selected from: a first group comprising (preferably consisting of): flexor digitorum superficialis, flexor digitorum profundus, flexor carpi radialis, flexor carpi ulnaris, brachioradialis; and a second group comprising (preferably consisting of): brachialis, biceps brachii, triceps brachii (long head), subscapularis, pectoralis muscle (e.g., pectoralis major), and latissimus dorsi.

[0250] More preferably, the modified BoNT / A can be administered to muscles associated with shoulder spasm, such as shoulder adduction. The modified BoNT / A can be administered to at least one of the latissimus dorsi, subscapularis, pectoralis major, and triceps brachii (long head). For example, the modified BoNT / A can be administered to at least two or three (preferably all) of the latissimus dorsi, subscapularis, pectoralis major, or triceps brachii (long head).

[0251] When administered to the biceps brachii in some cases, a single unit dose may be administered, while in other cases 2× unit doses may be administered. Thus, the biceps brachii falls into the first and second groups of muscles. Preferably, the 2× unit dose is administered to the biceps brachii and this muscle is included in the second group of muscles.

[0252] Suitable doses for upper limb muscles are as follows:

[0253]

[0254] Preferably, the modified BoNT / A can be administered to one or more muscles at the dosages shown in the table above. In some cases, the pronator teres muscle can be omitted when treating limb spasticity.

[0255] Therefore, preferably, suitable doses for upper limb muscles are as follows:

[0256] Injected muscle dose Flexor digitorum superficialis (FDS) 1×UD Flexor digitorum profundus (FDP) 1×UD Flexor carpi radialis (FCR) 1×UD Flexor carpi ulnaris (FCU) 1×UD brachioradialis 1×UD Brachialis 2×UD Biceps 2×UD Triceps (long head) 2×UD Pectoralis major 2×UD Subscapularis 2×UD Latissimus dorsi 2×UD

[0257] The modified BoNT / A may be administered to one or more of the following muscles at the following doses:

[0258]

[0259]

[0260] To treat the upper limb spasticity clinical manifestation "clenching fist", the modified BoNT / A can be administered to the flexor digitorum superficialis and / or flexor digitorum profundus. To treat the upper limb spasticity clinical manifestation "wrist flexion", the modified BoNT / A can be administered to the flexor carpi radialis and / or flexor carpi ulnaris. To treat the upper limb spasticity clinical manifestation "elbow flexion", the modified BoNT / A can be administered to the brachioradialis, brachialis, and / or biceps brachii. To treat the upper limb spasticity clinical manifestation "shoulder adduction", the modified BoNT / A can be administered to the triceps brachii (long head), subscapularis, pectoralis major, and / or latissimus dorsi.

[0261] Particularly preferably, during the treatment of upper limb spasticity, at least the clinical manifestations "elbow flexion" and "shoulder adduction" are treated. More preferably, during the treatment of upper limb spasticity, the clinical manifestations "elbow flexion" and "shoulder adduction" are treated, and "fist clenching" or "wrist flexion" (preferably only one of them) are treated.

[0262] The total number of unit doses administered in a given treatment may be up to 15× unit doses. In other words, in one embodiment, 15× single unit doses may be administered to 15× injection sites. In other embodiments, 15× single unit doses may be administered to more than 15× injection sites. The total number of unit doses will be divided according to the muscles treated, for example, 2× unit doses may be administered to the latissimus dorsi, 2× to the subscapularis, 2× to the pectoralis major, and 1× to the ulnar flexor carpi, resulting in a total administration multiple of 7× unit doses. For example, the total number of unit doses administered may be up to 14×, 13×, 12×, 11×, 10×, 9×, 8×, or 7×. The total number of unit doses administered may be at least 2×, 3×, 4×, 5×, 6×, 7×, preferably at least 2× of the unit dose. The total number of unit doses administered may be from 2× to 15×, from 7× to 15×, or from 10× to 14×. Preferably, the number of unit doses administered is 15×.

[0263] One skilled in the art will consider whether the individual has recently received (or will subsequently receive) additional treatment with a Clostridial neurotoxin (e.g., an unmodified BoNT), e.g., as part of a cosmetic treatment or treatment for a different indication. Using routine techniques in the art, the skilled artisan will adjust the present treatment regimen accordingly.

[0264] Preferably, the limb spasticity treated according to the present invention is adult limb spasticity. Preferably, the dosage details provided above are for treating adult limb spasticity (unless the context indicates that the dosage details provided are for treating pediatric limb spasticity). However, the treatment of pediatric limb spasticity is also contemplated. When treating pediatric limb spasticity, the total combined dose is typically 50% or less of that used when treating adult limb spasticity. In some embodiments, the total combined dose is typically 70% or less (e.g. 67% or less) of that used when treating adult limb spasticity.

[0265] The total number of unit doses administered in a given treatment for treating limb spasticity in children may be up to 10× unit doses. In other words, in one embodiment, 10× single unit doses may be administered to 10× injection sites. However, the total number of unit doses administered in a given treatment for treating limb spasticity in children may be up to 15× unit doses. In another embodiment, 10× single unit doses may be administered to more than 10× injection sites. The total number of unit doses will be divided according to the muscles treated, for example, 2× unit doses may be administered to the latissimus dorsi, 2× to the subscapularis, 2× to the pectoralis major, and 1× to the ulnar flexor carpi, resulting in a total administration multiple of 7× unit doses. For example, the total number of unit doses administered may be up to 9×, 8×, 7×, 6×, 5×, 4×, or 3×. The total number of unit doses administered may be at least 2×, 3×, 4×, 5×, 6×, 7×, preferably at least 2×, of the unit dose. The total number of unit doses administered may 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.

[0266] When compared with unmodified BoNT / A (e.g. ), the modified BoNT / A of the invention preferably has a longer duration of action (e.g., at least 5%, 10%, 25%, or 50% improvement in one or more symptoms) compared to the control group. The duration of action can be at least 1.25×, 1.5×, 1.75×, 2.0×, or 2.25× or more. The duration of action of the modified BoNT / A can be between 6 and 9 months. For example, the duration of action can be at least: at least 4.5 months (from onset), 5.0 months, 5.5 months, 6 months, 6.5 months, 7.0 months, 7.5 months, 8.0 months, 8.5 months, or 9.0 months. In specific embodiments, the duration of action can be greater than 9.0 months.

[0267] Administration according to the invention to multiple muscles is preferably performed during the same treatment session.

[0268] Treatment is repeated at an appropriate time after administration of the modified BoNT / A. ) is approximately twice as high as that of the unmodified BoNT / A (e.g. ) for a longer period of time as appropriate when treating an individual. A modified BoNT / A according to the invention can be re-administered to an individual at least 18, 20, 25, or 30 weeks after a previous administration. For example, a modified BoNT / A according to the invention can be re-administered to an individual at least 18-45 weeks, preferably 20-35 weeks, after a previous administration.

[0269] As used herein, an "individual" may be a mammal, such as a human or other mammal. Preferably, an "individual" refers to a human individual. An "individual" is preferably an adult individual, i.e., an individual of at least 18 years old. The terms "individual" and "patient" are used synonymously herein. When treating limb spasticity in children, the individual is less than 18 years old, for example, less than 15, 10 or 5 years old, such as 1-17, 5-17 or 10-17 years old. Preferably, the individual has been diagnosed with limb spasticity. The individual may have been diagnosed with spastic hemiparesis. Limb spasticity may occur after a stroke or traumatic brain injury (e.g., caused by a stroke or traumatic brain injury).

[0270] One skilled in the art will understand that where the limb spasticity is an adult limb spasticity, the subject is an adult subject, and where the limb spasticity is a pediatric limb spasticity, the subject is a pediatric subject.

[0271] An individual treated in accordance with the present invention can be an individual who is not amenable to treatment with an unmodified BoNT / A and / or another clostridial neurotoxin. The individual can be an individual who is resistant to treatment with an unmodified BoNT / A and / or with another clostridial neurotoxin. Resistance can arise due to the development of an immune response in the individual to the clostridial neurotoxin, including the production of anti-clostridial neurotoxin antibodies.

[0272] Unless otherwise indicated, the dosages described herein (eg, unit dose and total dose) are for limb spasticity in adults.

[0273] The terms "treat" or "treating" as used herein include preventive treatment (e.g., to prevent the onset of a disorder) as well as corrective treatment (treating an individual already suffering from a disorder). Preferably, "treat" or "treating" as used herein means corrective treatment. The terms "treat" or "treating" as used herein refer to a disorder and / or its symptoms.

[0274] Suitable modified BoNT / A polypeptides (and nucleotide sequences encoding the same, if any) are described in WO 2015 / 004461A1 and WO 2017 / 191315, each of which is herein incorporated by reference in its entirety.

[0275] BoNT / A is an example of a clostridial neurotoxin produced by Clostridium bacteria. Other examples of such clostridial neurotoxins include those produced by C. Tetani (TeNT) and C. Botulinum (BoNT) serotypes BG and X (see WO 2018 / 009903 A2), as well as those produced by C. baratii and C. butyricum. The neurotoxins are highly potent and specific, capable of poisoning neurons and other cells to which they are delivered. Clostridial toxins are among the strongest toxins known. For example, botulinum neurotoxin has a median lethal dose (LD50) of 0.5-5 ng / kg for mice. 50 ), depending on the serotype. Both tetanus toxin and botulinum toxin act by inhibiting the function of the affected neurons, particularly the release of neurotransmitters. While botulinum toxin acts at the neuromuscular junction and inhibits cholinergic transmission in the peripheral nervous system, tetanus toxin acts on the central nervous system.

[0276] In nature, clostridial neurotoxins (including BoNT / A) are synthesized as single-chain polypeptides that are modified post-translationally by proteolytic cleavage events to form two polypeptide chains linked together by disulfide bonds. Cleavage occurs at a specific cleavage site, often referred to as the activation site (e.g., the activation loop), which is located between the cysteine ​​residues that provide the interchain disulfide bond. It is this two-chain form that is the active form of the toxin. The two chains are called 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 contains an N-terminal translocation component (H N domain) and the C-terminal targeting component (H C domain). The cleavage site is located between the L chain and the translocation domain component. C After the domain binds to its target neuron and the bound toxin enters the cell via endocytosis, H N The domain enables the translocation of the L chain across the endosomal membrane and into the cytosol, and the L chain provides the protease function (also known as a non-cytotoxic protease).

[0277] Non-cytotoxic proteases act by proteolytic cleavage of intracellular transporters 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 ( S oluble N SF A ttachment Re ceptor), where NSF refers to N-ethylmaleimide sensitive factor ( N -ethylmaleimide- S ensitive F actor). SNARE proteins are required for intracellular vesicle fusion and therefore for secretion of molecules from cells via vesicular transport. The protease function is a zinc-dependent endopeptidase activity and exhibits high substrate specificity for SNARE proteins. Therefore, once delivered to the desired target cells, the non-cytotoxic protease is able to inhibit cellular secretion from target cells. The L-chain protease of Clostridial neurotoxin is a non-cytotoxic protease that cleaves SNARE proteins.

[0278] The term "H" as used herein C The "domain" refers to the functionally distinct region of the neurotoxin heavy chain, which has a molecular weight of approximately 50 kDa and enables the neurotoxin to bind to receptors on the surface of target cells. C The domain consists of two structurally different subdomains, the "H CN Subdomain (H C N-terminal portion of the domain) and "H CC Subdomain (H C The C-terminal portion of the 24-mer domain), each of which has a molecular weight of approximately 25 kDa.

[0279] The term "LH N The "domain" refers to the lack of H C The neurotoxin consists of an endopeptidase domain (the "L chain" or "light chain") and a domain responsible for translocating the endopeptidase into the cytoplasm (the H chain of the heavy chain). N domain).

[0280] Given the ubiquitous nature of SNARE proteins, Clostridial neurotoxins such as botulinum toxin have been successfully used in a wide range of therapeutic applications.

[0281] For more details on the genetic basis of toxin production in Clostridium botulinum and C. tetani, see Henderson et al., (1997) The Clostridia: Molecular Biology and Pathogenesis, Academic press.

[0282] As described above, the clostridial neurotoxin is formed by two polypeptide chains: a heavy chain (H chain), which has a molecular weight of about 100 kDa; and a light chain (L chain), which has a molecular weight of about 50 kDa. The H chain contains a C-terminal targeting component (receptor binding domain or H C domain) and the N-terminal translocation component (H N domain).

[0283] Clostridial neurotoxin domains are described in more detail below.

[0284] Examples of light chain reference sequences include:

[0285] Botulinum neurotoxin type A: amino acid residues 1-448

[0286] Botulinum neurotoxin type B: amino acid residues 1-440

[0287] The reference sequences determined above should be considered as a guide, as slight variations may occur depending on the subserotype. For example, US2007 / 0166332 (incorporated herein in its entirety by reference) cites a slightly different Clostridium sequence:

[0288] Botulinum neurotoxin type A: amino acid residues M1-K448

[0289] Botulinum neurotoxin type B: amino acid residues M1-K441

[0290] The translocation domain is a fragment of the H chain of a clostridial neurotoxin corresponding approximately to the amino-terminal half of the H chain, or a domain corresponding to this fragment in an intact H chain.

[0291] Examples of reference translocation domains include:

[0292] Botulinum neurotoxin type A - amino acid residues (449-871)

[0293] Botulinum neurotoxin type B - amino acid residues (441-858)

[0294] The reference sequences determined above should be considered as a guide, as slight variations may occur depending on the subserotype. For example, US2007 / 0166332 (incorporated herein by reference) cites a slightly different Clostridium sequence:

[0295] Botulinum neurotoxin type A - amino acid residues (A449-K871)

[0296] Botulinum neurotoxin type B - amino acid residues (A442-S858)

[0297] In the context of the present invention, various BoNT / AH comprising a translocation domain N Regions may be used in aspects of the invention. H from a BoNT / A heavy chain N The region is approximately 410-430 amino acids long and contains the translocation domain. N The full length of the region is not necessary for the translocation activity of the translocation domain. Thus, aspects of this embodiment may include a BoNT / AH N A region comprising a translocation domain having a length of, for example, at least 350 amino acids, at least 375 amino acids, at least 400 amino acids, or at least 425 amino acids. Other aspects of this embodiment may include a BoNT / AH N A region comprising a translocation domain having a length of, for example, at most 350 amino acids, at most 375 amino acids, at most 400 amino acids, or at most 425 amino acids.

[0298] Terminology N Covers naturally occurring BoNT / AH N Portions, and modified BoNT / AHs having non-naturally occurring amino acid sequences and / or synthetic amino acid residues N Preferably, the modified BoNT / AH N Some still exhibit the above translocation function.

[0299] Clostridial neurotoxin receptor binding domain (H C ) Examples of reference sequences include:

[0300] BoNT / A-N872-L1296

[0301] BoNT / B-E859-E1291

[0302] Clostridial neurotoxins (e.g., BoNT) have a ~50 kDa H C The domain contains two distinct structural features, called H CC and H CNdomains, usually ~25 kDa each. The amino acid residues involved in receptor binding are thought to be located primarily in the H CC domain. H of natural Clostridial neurotoxin C The domain may contain about 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.

[0303] (Reference)H CN Examples of domains include:

[0304] Botulinum neurotoxin type A - amino acid residues (872-1110)

[0305] Botulinum neurotoxin type B - amino acid residues (859-1097)

[0306] The above sequence positions may vary slightly depending on the serotype / subtype, (reference) H CN Other examples of domains include:

[0307] Botulinum neurotoxin type A - amino acid residues (874-1110)

[0308] Botulinum neurotoxin type B - amino acid residues (861-1097)

[0309] (Reference)H CC Examples of domains include:

[0310] Botulinum neurotoxin type A - amino acid residues (Y1111-L1296)

[0311] Botulinum neurotoxin type B - amino acid residues (Y1098-E1291)

[0312] WO 2017 / 191315 A1 (which is incorporated herein by reference) teaches modified BoNT / As and methods of preparing and manufacturing them. Thus, the invention provides a method for preparing a modified BoNT / As comprising a botulinum neurotoxin A (BoNT / A) light chain and a translocation domain (BoNT / AH) N ) and BoNT / B receptor binding domain (H C The modified BoNT / A having a modified domain) can be the one taught in WO 2017 / 191315A1.

[0313] As used herein, the term "modified BoNT / A" or "chimeric neurotoxin" refers to a Clostridial neurotoxin comprising (preferably consisting of) a light chain and a translocation domain (HD) from a first Clostridial neurotoxin serotype. N domain) and the receptor binding domain from a second different Clostridial neurotoxin serotype (H C Specifically, the modified BoNT / A used in the present invention comprises a botulinum neurotoxin A (BoNT / A) light chain and a translocation domain (H N domain) and the BoNT / B receptor binding domain (H C Domain). BoNT / A LH of modified BoNT / A N Domains and BoNT / BH C The domains are covalently linked. The modified BoNT / A of the present invention may be referred to as a chimeric botulinum neurotoxin. The modified BoNT / A is also referred to herein as a "BoNT / AB," "mrBoNT / AB," or "BoNT / AB chimera."

[0314] L chain and H N The domains (optionally including a full or partial activation loop, e.g., a full activation loop when the modified BoNT / A is in single-chain form and a cleaved / partial activation loop when in two-chain form) may be collectively referred to as LH N domain. Therefore, LH N The domain does not further contain H C Structural domain.

[0315] The modified BoNT / A can consist essentially of a botulinum neurotoxin A (BoNT / A) light chain and a translocation domain (H N domain) and the BoNT / B receptor binding domain (H C domain).

[0316] As used herein, the term "consisting essentially of" means that, for example, when administered to an individual, the modified BoNT / A does not further comprise one or more amino acid residues that confer additional functionality to the polypeptide. In other words, "consisting essentially of" a botulinum neurotoxin A (BoNT / A) light chain and a translocation domain (H N domain) and the BoNT / B receptor binding domain (H C A polypeptide "composed of" a botulinum neurotoxin A (BoNT / A) light chain and a translocation domain (H N domain) and the BoNT / B receptor binding domain (H C The polypeptide may be similar to the amino acid residues of the polypeptide domain, but the one or more additional amino acid residues do not confer additional functions to the polypeptide, such as when administered to an individual. Additional functions may include enzymatic activity, binding activity and / or any physiological activity.

[0317] The modified BoNT / A may comprise a non-Clostridial neurotoxin sequence in addition to any Clostridial neurotoxin sequence, as long as the non-Clostridial neurotoxin sequence does not destroy the ability of the modified BoNT / A to achieve its therapeutic effect. Preferably, the non-Clostridial neurotoxin sequence is not a sequence with catalytic activity (e.g., enzymatic activity). In one embodiment, the modified BoNT / A of the present invention does not comprise a non-Clostridial catalytically active domain. In one embodiment, the modified BoNT / A does not comprise an additional catalytically active domain. In one embodiment, the non-Clostridial sequence is not a sequence that binds to a cell receptor. In other words, in one embodiment, the non-Clostridial sequence is not a ligand for a cell receptor. The cell receptor can be a protein cell receptor, such as an integral membrane protein. Examples of cell receptors can be found in the IUPHAR Guide to Pharmacology Database, version 2019.4, at https: / / www.guidetopharmacology.org / download.jsp#db_reports. The non-Clostridial neurotoxin sequence can include a tag that facilitates purification, such as a His tag. In one embodiment, a modified BoNT / A of the invention does not comprise a label or a site for adding a label, such as a sortase acceptor or donor site.

[0318] Preferably, the modified BoNT / A may be composed of a botulinum neurotoxin A (BoNT / A) light chain and a translocation domain (H N domain) and the BoNT / B receptor binding domain (H C domain).

[0319] The modified BoNT / A comprises a light chain that is capable of exhibiting non-cytotoxic protease activity and is capable of cleaving SNARE proteins in the cytosol of target neurons.

[0320] Cell-based and in vivo tests can be used to determine whether the clostridial neurotoxin comprising L chain and functional cell binding and translocation domain has non-cytotoxic protease activity. Tests such as Digit Abduction Score (DAS) test, dorsal root ganglion (DRG) test, spinal neuron (SCN) test and mouse phrenic nerve hemidiaphragm (PNHD) test are routine tests in the art. Suitable tests for determining non-cytotoxic protease activity can be Aoki KR, Toxicon 39: 1815-1820; 2001 or Donald et al. (2018), Pharmacol Res Perspect, e00446, 1-14 described in the test, which is incorporated herein by reference.

[0321] When administered to an individual, the modified BoNT / A is preferably in its active two-chain form, wherein the light chain and the heavy chain are linked together by a disulfide bond. When a BoNT / A (e.g., a modified BoNT / A) is defined herein by a polypeptide sequence (SEQ ID NO), the L chain portion of the sequence (SEQ ID NO) can constitute the first chain of the two-chain clostridial neurotoxin (e.g., a two-chain modified BoNT / A), and the H chain portion can constitute the first chain of the two-chain clostridial neurotoxin (e.g., a two-chain modified BoNT / A). N and H C The domains together can constitute the second chain of a two-chain clostridial neurotoxin (e.g., a two-chain modified BoNT / A), wherein the first chain and the second chain are linked together by a disulfide bond. It will be understood by those skilled in the art that the protease can cleave at one or more positions within the activation loop of the clostridial neurotoxin (e.g., a modified BoNT / A), preferably at two positions within the activation loop. When cleavage occurs at more than one position (preferably two positions) within the activation loop, a small fragment of the C-terminal L chain portion of the sequence may not be present in the two-chain clostridial neurotoxin sequence (e.g., a two-chain modified BoNT / A). In view of this, the sequence of a two-chain clostridial neurotoxin (e.g., a two-chain modified BoNT / A) may be slightly different from the sequence of the corresponding single-chain clostridial neurotoxin (e.g., a single-chain modified BoNT / A). The small fragment can be 1-15 amino acids. Specifically, in one embodiment, when Lys-C is used to convert a single-chain modified BoNT / A to a two-chain modified BoNT / A, the small fragment of the C-terminal L chain portion of the sequence that is absent can be SEQ ID NO: 9 or 10.

[0322] Most preferably, the modified BoNT / A for use in the present invention may comprise a BoNT / A light chain and a translocation domain (BoNT / ALHN domain) and BoNT / BH C Domain. BoNT / A LH N Domains and BoNT / BH C The modified BoNT / A is also referred to herein as a "BoNT / AB" or a "BoNT / AB chimera."

[0323] LH N The C-terminal amino acid residue of the domain may correspond to the LH that separates BoNT / A N and H C Domain 3 10 The first amino acid residue of the helix, and H C The N-terminal amino acid residues of the domain may correspond to the region separating LH N and H C Domain 3 10 The second amino acid residue of the helix.

[0324] An example of an (unmodified) BoNT / A polypeptide sequence is shown in SEQ ID NO:2.

[0325] An example of a BoNT / B polypeptide sequence is shown in SEQ ID NO:8 (UniProt Accession No. B1INP5).

[0326] The “LHN and H separation of BoNT / A” mentioned in this article C Domain 3 10 The first amino acid residue of the helix is ​​the N and H C Domain 3 10 The N-terminal residue of the helix.

[0327] The article mentions "LH that separates BoNT / B N and H C Domain 3 10 The second amino acid residue of the helix is ​​the N and H C Domain 3 10 The amino acid residue following the N-terminal residue of the helix.

[0328] "3 10 A "helix" is a type of secondary structure found in proteins and peptides, along with alpha helices, beta sheets, and reverse turns. 10The amino acids in the helix are arranged in a right-handed helical structure, in which each complete turn is realized by three residues and ten atoms separated by intramolecular hydrogen bonds between them. Each amino acid corresponds to a 120° turn in the helix (i.e., there are three residues per turn of the helix), and a translation along the helical axis. And there are 10 atoms in the ring formed by hydrogen bonding. Most importantly, the NH group of an amino acid forms a hydrogen bond with the C=O group of the amino acid three residues before it; this repeated i+3→i hydrogen bond is defined as 3 10 Spiral. 3 10 A helix is ​​a standard concept in structural biology with which those skilled in the art are familiar.

[0329] This 3 10 The helix corresponds to the four residues that form the actual helix and two cap (or transition) residues, one at each end of the four residues. N and H C Domain 3 10 The "helix" consists of those 6 residues.

[0330] By performing structural analysis and sequence alignment, the LH N and H C Domain 3 10 Spiral. This 3 10 The helix is ​​at its N-terminal end (i.e., at the end of LH N The C-terminal part of the domain is surrounded by an α-helix, and at its C-terminus (i.e., at the H C The N-terminal part of the domain) is surrounded by β-strands. 10 The first (N-terminal) residue of a helix (the cap or transition residue) also corresponds to the C-terminal residue of the alpha helix.

[0331] Separation of LH N and H C Domain 3 10 The helices can be determined, for example, from publicly available crystal structures of botulinum neurotoxins, such as 3BTA (http: / / www.rcsb.org / pdb / explore / explore.do?structureld=3BTA) and 1EPW (http: / / www.rcsb.org / pdb / explore / explore.do?structureld=1EPW) for botulinum neurotoxins A1 and B1, respectively.

[0332] Publicly available computer modeling and alignment tools can also be used to identify LH compartments in other neurotoxins. N and H C Domain 310 The positions of the helices are determined by, for example, the homology modeling servers LOOPP (Learning, Observing and 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 many other tools / services listed on the Internet Resources for Molecular and Cell Biologists (http: / / molbiol-tools.ca / ). In particular, “H N / H CN “The region surrounding the junction can be highly conserved in structure, making it an ideal region for superimposing different serotypes.

[0333] For example, the following method can be used to determine the presence of this 3 10 The sequence of the spiral:

[0334] 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);

[0335] 2. The structure (pdb) file thus obtained can be edited to include only H CN The N-terminus of the domain and the approximately 80 residues preceding it (which is the H N domain), thereby retaining the highly conserved "H N / H CN "area;

[0336] 3. The protein overlay server SuperPose (http: / / wishart.biology.ualberta.ca / superpose / ) can be used to overlay each serotype onto the 3BTA.pdb structure;

[0337] 4. Check the overlapping pdb files to locate the H of BoNT / A1C 3 at the beginning of the domain 10 helix, the corresponding residues in other serotypes could then be identified.

[0338] 5. Other BoNT serotype sequences can be aligned with Clustal Omega to check whether the corresponding residues are correct.

[0339] LH measured by this method N , H C and 3 10 An example of a helical domain is shown below:

[0340]

[0341]

[0342] Using structural analysis and sequence alignment, it was found that the LH N and H C Domain 3 10 The β-strand following the helix is ​​conserved in all botulinum and tetanus neurotoxins and is involved in the separation of LH N and H C Domain 3 10 The beta strand begins at the 8th residue starting with the first residue of the helix (eg, residue 879 of BoNT / A1).

[0343] A BoNT / AB chimera can include H from BoNT / B C Domain covalently linked to LH from BoNT / A N Domain,

[0344] ·LH N The C-terminal amino acid residue of the domain corresponds to the H C The eighth amino acid residue from the N-terminus of the β-strand at the start (N-terminus) of the domain, and

[0345] Wherein H C The N-terminal amino acid residue of the domain corresponds to the H C The domain starts at the seventh amino acid residue from the N-terminus of the β-strand.

[0346] A BoNT / AB chimera can include H from BoNT / B C Domain covalently linked to LH from BoNT / A N Domain,

[0347] ·LH NThe C-terminal amino acid residue of the domain corresponds to the LH N The C-terminal amino acid residue of the alpha helix at the end of the domain (C-terminus), and

[0348] Among them, H C The N-terminal amino acid residue of the domain corresponds to the LH N The amino acid residue immediately C-terminal to the C-terminal amino acid residue of the alpha helix at the domain termination end (C-terminus).

[0349] The rationale for the BoNT / AB chimera design process is to ensure that the secondary structure is as intact as possible, thereby minimizing any changes to the tertiary structure and the function of each domain. Without wishing to be bound by theory, it is hypothesized that by not disrupting the 3 domains in the BoNT / AB chimera, 10 The four central amino acid residues of the helix ensure the optimal conformation of the chimeric neurotoxins, thereby allowing the chimeric neurotoxins to exert their full functions.

[0351] In fact, surprisingly, only 3 10 The first amino acid residue of the helix and the second amino acid residue before the 310 helix of BoNT / B not only allow for the generation of soluble and functional BoNT / AB chimeras, but further result in improved properties relative to other BoNT / AB chimeras, particularly increased potency, increased safety ratio, and / or longer duration of action (as well as increased safety ratio and / or duration of action when compared to unmodified BoNT / A [e.g., SEQ ID NO: 2, e.g., SEQ ID NO: 2 in a two-chain form]).

[0352] BoNT / A light chain, BoNT / A translocation domain, and / or BoNT / BH C The domain can be a modified BoNT / A light chain, a BoNT / A translocation domain, and / or a BoNT / BH C Domains or derivatives thereof, including but not limited to those described below. Modified BoNT / A light chains, BoNT / A translocation domains, and / or BoNT / BH C The domain or derivative may contain a structure that is compatible with a BoNT / A light chain, a BoNT / A translocation domain, and / or a BoNT / BH C The native (unmodified) form of the BoNT / A light chain, BoNT / A translocation domain, and / or BoNT / BH chain may contain one or more amino acids that have been modified compared to the native (unmodified) form of the BoNT / A light chain, BoNT / A translocation domain, and / or BoNT / BH chain. CFor example, relative to a native (unmodified) BoNT / A light chain, a BoNT / A translocation domain, and / or a BoNT / BH C Domain sequences, modified BoNT / A light chain, BoNT / A translocation domain, and / or BoNT / BH C The domains may have modified amino acid sequences in one or more domains. Such modifications may modify aspects of their function, such as biological activity or persistence. Thus, in one embodiment, a BoNT / A light chain, a BoNT / A translocation domain, and / or a BoNT / BH C The domain is a modified BoNT / A light chain, BoNT / A translocation domain, and / or BoNT / BH C domain, or a modified BoNT / A light chain, BoNT / A translocation domain, and / or BoNT / BH C Derivatives of the domain.

[0353] Modified BoNT / BH C The domain may have one or more modifications that alter binding to target neural cells, such as when combined with native (unmodified) BoNT / BH C When the domains are compared, they provide higher or lower affinity binding. C Such modifications in the domain may include modifying H C The modification of a residue in a ganglioside binding site of a domain or a protein (e.g., synaptotagmin) binding site that alters binding to a ganglioside receptor and / or protein receptor of a target neural cell. Examples of such modified neurotoxins are described in WO 2006 / 027207 and WO 2006 / 114308, both of which are incorporated herein by reference in their entirety.

[0354] The modified light chain may have one or more modifications in its amino acid sequence, such as modifications in the substrate binding or catalytic domains, which may alter or modify the SNARE protein specificity of the modified light chain, preferably provided that the modification does not render the light chain catalytically inactive. Examples of such modified neurotoxins are described in WO 2010 / 120766 and US2011 / 0318385, both of which are incorporated herein by reference in their entirety.

[0355] LH from BoNT / A N The domain may correspond to amino acid residues 1 to 872 of SEQ ID NO: 2, or a polypeptide sequence having at least 70% sequence identity thereto. NThe domain may correspond to amino acid residues 1 to 872 of SEQ ID NO: 2, or a polypeptide sequence having at least 80%, 90%, or 95% sequence identity thereto. Preferably, the LH from a BoNT / A N The domain corresponds to amino acid residues 1 to 872 of SEQ ID NO:2.

[0356] H from BoNT / B C The domain may correspond to amino acid residues 860 to 1291 of SEQ ID NO:8, or a polypeptide sequence having at least 70% sequence identity thereto. C The domain may correspond to amino acid residues 860 to 1291 of SEQ ID NO:8, or a polypeptide sequence having at least 80%, 90%, or 95% sequence identity thereto. Preferably, the H from a BoNT / B C The domain corresponds to amino acid residues 860 to 1291 of SEQ ID NO:8.

[0357] Preferably, the BoNT / AB chimera comprises BoNT / A1 LH N Domains and BoNT / B1 H C More preferably, LH N The domain corresponds to amino acid residues 1 to 872 of BoNT / A1 (SEQ ID NO: 2), and H C The domain corresponds to amino acid residues 860 to 1291 of BoNT / B1 (SEQ ID NO: 8).

[0358] Most preferably, the BoNT / BH is C The domain also contains H CC At least one amino acid residue substitution, insertion, insertion / deletion (indel), or deletion in a subdomain that has the effect of increasing the binding affinity of the BoNT / B neurotoxin to human Syt II. CC Suitable amino acid residue substitutions, insertions, insertions / deletions (indels) or deletions in subdomains have been disclosed in WO 2013 / 180799 and WO 2016 / 154534 (both incorporated herein by reference).

[0359] BoNT / BH CCSuitable amino acid residue substitutions, insertions, insertions / deletions (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.

[0360] BoNT / BH CC Suitable amino acid residue substitutions, insertions, insertion / deletions (indels) or deletions in the subdomains may also include a combination of two substitution mutations selected from the group consisting of E1191M and S1199L, E1191M and S1199Y, E1191M and S1199F, E1191Q and S1199L, E1191Q and S1199Y, E1191Q and S1199F, E1191M and S1199W, E1191M and W1178Q, E1191C and S1199W, E1191C and S1199Y, E1191C and W1178Q, E1191Q and S1199W, E1191V and S1199W, E1191V and S1199Y, or E1191V and W1178Q.

[0361] BoNT / BH CC Suitable amino acid residue substitutions, insertions, insertion / deletions (indels) or deletions in the subdomains may also include a combination of three substitution mutations, which are E1191M, S1199W and W1178Q.

[0362] Preferably, BoNT / BH CC The amino acid residue substitutions, insertions / deletions, or deletions in the subdomain include a combination of two substitution mutations, which are E1191M and S1199Y. Such modifications are present in modified BoNT / A SEQ ID NO:5 and SEQ ID NO:6. E1191M can correspond to position 1204 of SEQ ID NO:6, and S1199Y can correspond to position 1212. Thus, SEQ ID NO:6 can include 1204M and 1212Y.

[0363] The modification can be a modification when compared to an unmodified BoNT / B as set forth in SEQ ID NO:8, wherein the amino acid residue numbering is determined by alignment with SEQ ID NO:8. Since the presence or absence of a methionine residue at position 1 of SEQ ID NO:8 (and SEQ ID NOs corresponding to the modified BoNT / A polypeptides described herein) is optional, one skilled in the art will take into account the presence / absence of the methionine residue when determining the amino acid residue numbering. For example, when SEQ ID NO:8 includes a methionine, the position numbering will be as defined above (e.g., E1191 will be E1191 of SEQ ID NO:8). Alternatively, when a methionine is absent in SEQ ID NO:8, the amino acid residue numbering should be modified to -1 (e.g., E1191 will be E1190 of SEQ ID NO:8). Thus, the initial methionine amino acid residue of the polypeptide sequence of the modified BoNT / A can be optional or absent. Similar considerations apply when a methionine is present / absent at position 1 of other polypeptide sequences described herein, and one skilled in the art can readily determine the correct amino acid residue numbering using routine techniques in the art.

[0364] The modified BoNT / A for use in the present invention can comprise a polypeptide sequence having at least 70% sequence identity to a polypeptide sequence selected from SEQ ID NOs: 3-7. For example, a polypeptide sequence having at least 80%, 90%, 95%, or 99.9% sequence identity to a polypeptide sequence selected from SEQ ID NOs: 3-7. Preferably, the modified BoNT / A for use in the present invention can comprise (more preferably consist of) a polypeptide sequence selected from SEQ ID NOs: 3-7.

[0365] Preferably, the modified BoNT / A comprises a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 6. For example, a polypeptide sequence having at least 80%, 90%, 95%, or 99.9% sequence identity to SEQ ID NO: 6. Most preferably, the modified BoNT / A for use in the present invention can comprise (more preferably consist of) SEQ ID NO: 6.

[0366] The term "deletion" as used herein refers to the removal of one or more amino acid residues of a polypeptide without replacing one or more amino acid residues at the site of the deletion. Thus, when one amino acid residue is deleted from a polypeptide sequence having x amino acid residues (for example), the resulting polypeptide has x-1 amino acid residues.

[0367] As used herein, the term "insertion / deletion (indel)" refers to the deletion of one or more amino acid residues of a polypeptide and the insertion of a different number of amino acid residues (more or fewer amino acid residues) than the number of amino acid residues deleted at the deletion site. Thus, for an insertion / deletion (indel) in which two amino acid residues are deleted from a polypeptide sequence having x amino acid residues (for example), the resulting polypeptide has x-1 amino acid residues or x+≥1 amino acid residues. Insertions and deletions can be performed in any order, sequentially or simultaneously.

[0368] As used herein, the term "substitution" 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 of a polypeptide sequence having x amino acid residues (for example), the resulting polypeptide also has x amino acid residues. Preferably, the substitution is at a single amino acid position.

[0369] The term "insertion" as used herein refers to the addition of one or more amino acid residues of a polypeptide at an insertion site without deleting one or more amino acid residues of the polypeptide. Thus, when an amino acid residue is inserted into a polypeptide sequence having x amino acid residues (for example), the resulting polypeptide has x+1 amino acid residues.

[0370] Methods for modifying proteins by substitution, insertion or deletion of amino acid residues are known in the art. For example, amino acid modifications can be introduced by modifying a DNA sequence encoding a BoNT / A (e.g., encoding an unmodified BoNT / A). This can be accomplished using standard molecular cloning techniques, such as by site-directed mutagenesis, where short strands of DNA (oligonucleotides) encoding the desired amino acids are used to replace the original coding sequence using a polymerase, or by inserting / deleting portions of a gene using various enzymes (e.g., ligases and restriction endonucleases). Alternatively, the modified gene sequence can be chemically synthesized. Generally, the modification can be made by modifying a nucleic acid encoding a native Clostridial neurotoxin (or a portion thereof) such that the modified BoNT / A (or a portion thereof) encoded by the nucleic acid comprises the modification. Alternatively, a nucleic acid encoding a modified BoNT / A (or a portion thereof) comprising the modification can be synthesized.

[0371] When the polypeptide sequence of the modified BoNT / A described herein comprises a tag (e.g., 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 of the invention.

[0372] As described above, the modified BoNT / A described herein have increased tissue retention properties, which also provide increased efficacy 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 ratio of the modified BoNT / A. In this regard, the undesirable effects of Clostridial toxins (caused by diffusion of the toxin from the site of administration) can be experimentally assessed by measuring the percent weight loss in a relevant animal model (e.g., mice, where weight loss is measured within seven days of administration). Conversely, the desired on-target effects of Clostridial toxins can be experimentally assessed by the toe abduction score (DAS) test (a measure of muscle paralysis). The DAS test can be performed by injecting 20 μl of neurotoxin formulated in gelatin phosphate buffer into the gastrocnemius / soleus complex of mice, followed by assessing the toe abduction score using the method of Aoki (Aoki KR, Toxicon 39: 1815-1820; 2001). In the DAS test, mice are suspended briefly by their tails to elicit a characteristic startle response in which the mice extend their hind limbs and abduct their hind toes. After injection of the neurotoxin, varying degrees of digital abduction are scored on a five-point scale (0 = normal to 4 = maximal reduction in digital abduction and leg extension).

[0373] The safety ratio of the neurotoxin can then be expressed as the ratio between the amount of toxin required to achieve a 10% decrease in body weight (measured at peak effect within the first seven days following administration to mice) and the amount of neurotoxin required to achieve a DAS score of 2. Thus, a high safety ratio score is desirable, indicating that the neurotoxin is able to effectively paralyze the target muscle with few undesirable off-target effects. The safety ratios of the modified BoNT / As of the present invention are higher than the safety ratios of the equivalent unmodified (natural) BoNT / As.

[0374] A high safety ratio is particularly advantageous in treatment because it represents an increase in the therapeutic index. In other words, this means that a reduced dose can be used and / or an increased dose can be used without any additional (e.g., deleterious) effects compared to alternative clostridial neurotoxin therapeutics. Detrimental effects may include systemic toxicity and / or undesirable spread to adjacent muscles. The possibility of using higher doses of neurotoxins without additional effects is particularly advantageous because higher doses generally result in a longer duration of action of the neurotoxin.

[0375] The potency of a modified BoNT / A can be expressed as the minimum dose of the neurotoxin that results in a given DAS score when administered to the gastrocnemius / soleus complex of mice, e.g., a DAS score of 2 (ED 50dose) or a DAS score of 4. The potency of the modified BoNT / A can also be expressed as the EC in a cellular assay measuring SNARE cleavage by neurotoxins. 50 Dose, e.g. EC in a cell assay measuring cleavage of SNAP25 by a modified BoNT / A 50 dose.

[0376] The duration of action of a modified BoNT / A can be expressed as the time required to restore a DAS score of 0 following administration of a given dose of neurotoxin (e.g., the minimum dose of neurotoxin that results in a DAS score of 4) to the gastrocnemius / soleus complex of mice.

[0377] Thus, in one embodiment, a modified BoNT / A of the invention has a safety ratio greater than 7 (e.g., at least 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50), wherein the safety ratio is calculated as follows: - the toxin dose required for a 10% change in body weight (pg / mouse) divided by the DAS ED 50 (pg / mouse) [ED 50 =dose required to produce a DAS score of 2]. For example, the modified BoNT / A can have a safety ratio of at least 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50.

[0378] In one embodiment, a modified BoNT / A of the invention has a safety ratio of at least 10. In one embodiment, a modified BoNT / A of the invention has a safety ratio of at least 15.

[0379] Preferably, the modified BoNT / A has a safety ratio of at least 10 (eg, a safety ratio of 10), more preferably at least 12 or 13 (eg, 14-15).

[0380] The modified BoNT / A can have a safety ratio of greater than 7 up to 50, such as 8-45, 10-20, or 12-15.

[0381] In use, the modified BoNT / A of the present invention is in a double-chain form.

[0382] The modified BoNT / A used in the present invention may comprise a polypeptide sequence having at least 70% sequence identity with a polypeptide sequence selected from SEQ ID NOs: 3-7. For example, a polypeptide sequence having at least 80%, 90%, 95%, or 99.9% sequence identity with a polypeptide sequence selected from SEQ ID NOs: 3-7. Preferably, the modified BoNT / A used in the present invention may comprise (more preferably consist of) a polypeptide sequence selected from SEQ ID NOs: 3-7. Among the modified BoNT / A, SEQ ID NO: 6 is preferred.

[0383] Thus, preferably, the modified BoNT / A comprises a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 6. More preferably, the polypeptide sequence has at least 80%, 90%, 95%, or 99.9% sequence identity to SEQ ID NO: 6. Most preferably, the modified BoNT / A for use in the present invention may comprise (more preferably consist of) SEQ ID NO: 6.

[0384] The double-chain modified BoNT / A of the present invention may include an L chain portion of a polypeptide sequence having at least 70%, 80%, 90%, 95%, 99.9% or 100% sequence identity to any one of SEQ ID NOs: 3-7, which constitutes the first chain of the double-chain modified BoNT / A, and may include an H chain portion of a polypeptide sequence having at least 70%, 80%, 90%, 95%, 99.9% or 100% sequence identity to any one of SEQ ID NOs: 3-7. N and H C The structural domains together constitute the second chain of the two-chain modified BoNT / A, wherein the first chain and the second chain are linked together by a disulfide bond.

[0385] Wherein cleavage occurs at more than one position (preferably two positions) within the activation loop of the 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, a small fragment of the C-terminal L chain portion of a sequence having at least 70%, 80%, 90%, 95%, 99.9%, or 100% sequence identity to any one of SEQ ID NOs: 3-7 may not be present in the two-chain modified BoNT / A. In view of this, the sequence of a two-chain modified BoNT / A (e.g., comprising a polypeptide sequence having at least 70%, 80%, 90%, 95%, 99.9%, or 100% sequence identity to any one of SEQ ID NOs: 3-7) can be slightly different from the corresponding single-chain modified BoNT / A, comprising a polypeptide sequence having at least 70%, 80%, 90%, 95%, 99.9%, or 100% sequence identity to any one of SEQ ID NOs: 3-7. The small fragment can be 1-15 amino acids. In particular, in one embodiment, when Lys-C is used to convert a single-chain modified BoNT / A into a two-chain clostridial neurotoxin, the small fragment of the C-terminal L chain portion of the absent sequence can be SEQ ID NO: 9 or 10.

[0386] Preferably, the double-chain modified BoNT / A of the present invention may include an L chain portion of a polypeptide sequence having at least 70%, 80%, 90%, 95%, 99.9% or 100% sequence identity to SEQ ID NO: 6, which constitutes the first chain of the double-chain modified BoNT / A, and may include an H chain portion of a polypeptide sequence having at least 70%, 80%, 90%, 95%, 99.9% or 100% sequence identity to SEQ ID NO: 6. N and H C The structural domains together constitute the second chain of the two-chain modified BoNT / A, wherein the first chain and the second chain are linked together by a disulfide bond.

[0387] Wherein cleavage occurs at more than one position (preferably two positions) within the activation loop of the modified BoNT / A comprising a polypeptide sequence having at least 70%, 80%, 90%, 95%, 99.9%, or 100% sequence identity to SEQ ID NO:6, a small fragment of the C-terminal L chain portion of a sequence having at least 70%, 80%, 90%, 95%, 99.9%, or 100% sequence identity to SEQ ID NO:6 may not be present in the two-chain modified BoNT / A. In view of this, the sequence of the two-chain modified BoNT / A (e.g., comprising a polypeptide sequence having at least 70%, 80%, 90%, 95%, 99.9%, or 100% sequence identity to SEQ ID NO: 6) can be slightly different from the corresponding single-chain chimeric clostridial neurotoxin, wherein the single-chain modified BoNT / A comprises a polypeptide sequence having at least 70%, 80%, 90%, 95%, 99.9%, or 100% sequence identity to SEQ ID NO: 6. The small fragment can be 1-15 amino acids. Specifically, in one embodiment, when Lys-C is used to convert a single-chain modified BoNT / A to a two-chain modified BoNT / A, the small fragment of the C-terminal L chain portion of the absent sequence can be SEQ ID NO: 9 or 10.

[0388] In particularly preferred embodiments, the two-chain modified BoNT / A comprises (or consists of) a light chain comprising a polypeptide sequence having at least 70%, 80%, 90%, 95%, or 99.9% sequence identity to SEQ ID NO: 11 or 12 (preferably SEQ ID NO: 11), and a heavy chain comprising a polypeptide sequence having at least 70%, 80%, 90%, 95%, or 99.9% sequence identity to SEQ ID NO: 13, wherein the light chain and heavy chain are linked together by a disulfide bond. More preferably, the two-chain modified BoNT / A comprises (or consists of) a light chain comprising SEQ ID NO: 11 or 12 (preferably SEQ ID NO: 11), and a heavy chain comprising SEQ ID NO: 13, wherein the light chain and heavy chain are linked together by a disulfide bond. Even more preferably, the two-chain modified BoNT / A comprises (or consists of) a light chain having SEQ ID NO: 11 and a heavy chain having SEQ ID NO: 13, wherein the light chain and the heavy chain are linked together by a disulfide bond. The disulfide bond is preferably formed by and / or between cysteine ​​residue 429 of SEQ ID NO: 11 or 12 and cysteine ​​residue 6 of SEQ ID NO: 13.

[0389] In preferred embodiments, the modified BoNT / A of the present invention does not comprise a therapeutic or diagnostic agent (e.g., a nucleic acid, protein, peptide, or small molecule therapeutic or diagnostic agent) other than the light and heavy chains. For example, in one embodiment, the modified BoNT / A may not comprise a covalently or non-covalently bound therapeutic or diagnostic agent. Thus, the modified BoNT / A of the present invention preferably does not serve as a delivery vehicle for other therapeutic or diagnostic agents.

[0390] In embodiments, the modified BoNT / A described herein has a tag (e.g., a His tag) and / or a linker for purification, which tag and / or linker is optional.

[0391] The modified BoNT / A may be free of complexing proteins present in the naturally occurring Clostridial neurotoxin complex. Examples of such complexing proteins include neurotoxin associated protein (NAP) and non-toxic non-hemagglutinin component (NTNH). However, it is preferred that the modified BoNT / A is a recombinant modified BoNT / A.

[0392] 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.

[0393] In one embodiment, a nucleic acid (e.g., DNA) comprising a nucleic acid sequence encoding a modified BoNT / A is provided. In one embodiment, the nucleic acid sequence is prepared as part of a DNA vector comprising a promoter and a terminator. The nucleic acid sequence can be selected from any of the nucleic acid sequences described herein.

[0394] In a preferred embodiment, the vector has a promoter selected from the group consisting of:

[0395] Typical induction conditions for promoter inducers

[0396] Tac (hybrid) IPTG 0.2mM (0.05-2.0mM)

[0397] AraBAD L-arabinose 0.2% (0.002-0.4%)

[0398] T7-lac operon IPTG 0.2mM (0.05-2.0mM)

[0399] In another preferred embodiment, the vector has a promoter selected from:

[0400] Typical induction conditions for promoter inducers

[0401] Tac (hybrid) IPTG 0.2mM (0.05-2.0mM)

[0402] AraBAD L-arabinose 0.2% (0.002-0.4%)

[0403] T7-lac operon IPTG 0.2mM (0.05-2.0mM)

[0404] T5-lac operon IPTG 0.2mM (0.05-2.0mM)

[0405] The nucleic acid molecules can be prepared using any suitable method known in the art. Therefore, chemical synthesis techniques can be used to prepare the nucleic acid molecules. Alternatively, molecular biology techniques can be used to prepare the nucleic acid molecules of the present invention.

[0406] The DNA constructs of the present invention are preferably designed in silico and then synthesized by conventional DNA synthesis techniques.

[0407] The above nucleic acid sequence information is optionally modified for codon preference depending on the final host cell (eg, E. coli) expression system to be employed.

[0408] The terms "nucleotide sequence" and "nucleic acid" are used synonymously herein. Preferably, the nucleotide sequence is a DNA sequence.

[0409] The modified BoNT / A of the present invention can exist as a single chain or as a double chain. However, it is preferred that the modified BoNT / A exists as a double chain, wherein the L chain is linked to the H chain (or its components, such as H) by disulfide bonds. N domain) connection.

[0410] The production of a single-chain modified BoNT / A having a light chain and a heavy chain can be achieved using a method comprising the steps of expressing a nucleic acid encoding the modified BoNT / A in an expression host, lysing the host cells to provide a host cell homogenate containing the single-chain modified BoNT / A, and isolating the single-chain modified BoNT / A. The single-chain modified BoNT / A described herein can be proteolytically processed using a method comprising contacting the single-chain modified BoNT / A with a protease (e.g., Lys-C) that hydrolyzes the peptide bond in the activation loop of the modified BoNT / A, thereby converting the single-chain modified BoNT / A to the corresponding double-chain modified BoNT / A (e.g., wherein the light chain and the heavy chain are linked together by a disulfide bond). The double-chain modified BoNT / A is preferably obtainable by this method.

[0411] Therefore, the modified BoNT / A used in the present invention is preferably a double-chain modified BoNT / A produced from a single-chain BoNT / A, wherein the single-chain BoNT / A comprises or consists of a polypeptide sequence described herein. For example, preferably, the modified BoNT / A used in the present invention is a double-chain modified BoNT / A produced from a polypeptide comprising a polypeptide sequence having at least 70% (e.g., at least 80%, 90%, 95% or 99.9%) sequence identity with SEQ ID NO: 6. Most preferably, the modified BoNT / A used in the present invention is a double-chain modified BoNT / A produced from a polypeptide comprising (even more preferably consisting of) SEQ ID NO: 6. Therefore, in some embodiments, the modified BoNT / A is a double-chain modified BoNT / A, wherein the light chain (L chain) is linked to the heavy chain (H chain) by a disulfide bond, and the modified BoNT / A can be obtained by the following method, which comprises 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 double-chain modified BoNT / A. Thus, in some embodiments, the modified BoNT / A is a two-chain modified BoNT / A, wherein the light chain (L chain) is linked to the heavy chain (H chain) by a disulfide bond, and the modified BoNT / A can be obtained by a method comprising contacting a modified BoNT / A comprising SEQ ID NO: 6 with a protease that hydrolyzes a peptide bond in its activation loop, thereby converting a 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, wherein the L chain is linked to the H chain by a disulfide bond, and the modified BoNT / A can be obtained by a method comprising contacting a modified BoNT / A consisting of SEQ ID NO: 6 with a protease that hydrolyzes a peptide bond in its activation loop, thereby converting a single-chain modified BoNT / A into the corresponding two-chain modified BoNT / A.

[0412] The protease used to cleave the activation loop is preferably Lys-C. Suitable proteases and methods for cleaving the activation loop to produce a two-chain clostridial neurotoxin are taught in WO 2014 / 080206, WO 2014 / 079495, and EP2677029A2, which are incorporated herein by reference. Lys-C may cleave the C-terminus of the activation loop at one or more lysine residues present therein. When Lys-C cleaves the activation loop more than once, the skilled artisan will understand that the small peptide of the activation loop of the two-chain modified BoNT / A may not be present when compared to the SEQ ID NOs shown herein.

[0413] As used herein, the term "obtainable" also encompasses the term "obtained." In one embodiment, the term "obtainable" refers to obtained.

[0414] As used herein, the term "one or more" may refer to at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20. In one embodiment, where "one or more" precedes a list, "one or more" may refer to all members of the list. Similarly, as used herein, the term "at least one" may refer to at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20. In one embodiment, where "at least one" precedes a list, "at least one" may refer to all members of the list.

[0415] As used herein, the term "disorder" also encompasses "disease." In one embodiment, the disorder is a disease.

[0416] The modified BoNT / A of the present invention can be formulated in any suitable manner for administration to an individual, such as as part of a pharmaceutical composition. Such a pharmaceutical composition can include a modified BoNT / A of the present invention and a pharmaceutically acceptable carrier, excipient, adjuvant, propellant, and / or salt.

[0417] Compositions suitable for injection may be in the form of solutions, suspensions or emulsions, or dry powders which are dissolved or suspended in a suitable vehicle before use.

[0418] Fluid dosage forms are typically prepared using a modified BoNT / A and a pyrogen-free sterile solvent. Depending on the solvent and concentration used, the modified BoNT / A can be dissolved or suspended in the solvent. In preparing a solution, the modified BoNT / A can be dissolved in the solvent, the solution can be made isotonic by the addition of sodium chloride if necessary, and sterilized by filtering through a sterile filter using aseptic techniques, and then filled into a suitable sterile vial or ampoule and sealed. Alternatively, if the solution stability is sufficient, the solution in the sealed container can be sterilized by autoclaving. Advantageously, additives such as buffers, solubilizers, stabilizers, preservatives or bactericides, suspending agents or emulsifiers, and / or local anesthetics can be dissolved in the solvent.

[0419] Dry powders can be prepared by filling pre-sterilized ingredients into sterile containers using aseptic techniques in a sterile area to be dissolved or suspended in a suitable solvent prior to use. Alternatively, the ingredients can be dissolved into a suitable container using aseptic techniques in a sterile area. The product is then freeze-dried and the container is aseptically sealed.

[0420] Parenteral suspensions suitable for the routes of administration described herein are prepared in substantially the same manner, except that the sterile components are suspended in a sterile vehicle instead of being dissolved, and sterilization cannot be accomplished by filtration. These components may be isolated in a sterile state, or may be sterilized after isolation, for example, by gamma irradiation.

[0421] Advantageously, a suspending agent such as polyvinylpyrrolidone is included in the composition to facilitate uniform distribution of the ingredients.

[0422] In one aspect, the invention provides a unit dosage form of a modified botulinum neurotoxin A (BoNT / A) (e.g., for treating limb spasticity in adults), the unit dosage form comprising:

[0423] a. Greater than 707 units of a modified BoNT / A, where 1 unit corresponds to the calculated median lethal dose (LD50) in mice. 50 ) of a modified BoNT / A; or

[0424] b. greater than 17,000 pg of modified BoNT / A; and

[0425] c. optionally a pharmaceutically acceptable carrier, excipient, adjuvant and / or salt,

[0426] The modified BoNT / A comprises a BoNT / A light chain and a translocation domain, and a BoNT / B receptor binding domain (H C domain).

[0427] Preferably, the modified BoNT / A of the unit dosage form comprises a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 6. For example, a polypeptide sequence having at least 80%, 90%, 95%, or 99.9% sequence identity to SEQ ID NO: 6. Most preferably, the modified BoNT / A can comprise (more preferably consist of) SEQ ID NO: 6.

[0428] The unit dosage form can comprise greater than 707 units of a modified BoNT / A. The upper limit of the range can be 1664, 1650, 1600, 1550, 1500, 1450, 1400, 1350, 1300, 1250, 1150, 1100, 1050, 1000, 950, 900, 850, 800, or 750 units of a modified BoNT / A, with a preferred upper limit being 1500 units. The lower end of the range can be 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 a modified BoNT / A, with a preferred lower end being 725 units, 949 units, or 1,381 units. Preferably, the unit dosage form comprises greater than 707 units to a maximum of 1664 units of a modified BoNT / A, e.g., greater than 707 units to a maximum of 1,498 units or 832 units to 1622 units. The unit dosage form may comprise 915 to 1581 units, preferably 949 to 1581 units or 1414 to 1581 units of a modified BoNT / A. The unit dosage form may comprise 998 to 1,498 units or 1,040 to 1,498 units of a modified BoNT / A. In preferred embodiments, the unit dosage form comprises 998, 1,040, 1,248, or 1,498 units of a modified BoNT / A. In more preferred embodiments, the unit dosage form comprises 1,248 or 1,498 units (e.g., 1,498 units) of a modified BoNT / A.

[0429] More preferably, the unit dosage form comprises 1,248 units to 1,664 units, such as 1,456 units to 1,539 units of the modified BoNT / A. More preferably, the unit dosage form comprises 1,498 units of the modified BoNT / A.

[0430] Even more preferably, the unit dosage form comprises 832 units to 1,248 units, such as 998 units to 1,082 units of the modified BoNT / A. Most preferably, the unit dosage form comprises 1,040 units of the modified BoNT / A.

[0431] The unit dosage form can comprise greater than 17,000 pg of a modified BoNT / A. The upper limit of the range can be 40,000, 39,000, 38,000, 37,000, 36,000, 35,000, 30,000, 25,000, 24,000, 22,000, 20,000, or 18,000 pg of a modified BoNT / A, with a preferred upper limit being 38,000 pg. The lower end of the range can be 17,500, 18,000, 20,000, 22,000, 24,000, 25,000, 26,000, 27,000, 28,000, 29,000, 30,000, 35,000, 36,000, 37,000, 38,000, or 39,000 pg of modified BoNT / A, with preferred lower ends being 17,500, 22,753, 22,790, or 33,1818 pg. Preferably, the unit dosage form comprises greater than 17,000 pg to up to 40,000 pg, e.g., greater than 17,000 pg to up to 36,000 pg, or 20,000 pg to 39,000 pg of modified BoNT / A. The unit dosage form may comprise 22,000 to 38,000 pg, preferably 23,000 to 38,000 pg, or 34,000 to 38,000 pg of the modified BoNT / A. The unit dosage form may comprise 24,000 to 36,000 pg or 25,000 to 36,000 pg of the modified BoNT / A. In preferred embodiments, the unit dosage form comprises 24,000, 25,000, 30,000, or 36,000 pg of the modified BoNT / A. In more preferred embodiments, the unit dosage form comprises 30,000 or 36,000 pg (e.g., 36,000 pg) of the modified BoNT / A.

[0432] More preferably, the unit dosage form comprises 30,000 pg to 40,000 pg, such as 35,000 pg to 37,000 pg of the modified BoNT / A. More preferably, the unit dosage form comprises 36,000 pg of the modified BoNT / A.

[0433] Even more preferably, the unit dosage form comprises 20,000 pg to 30,000 pg, such as 24,000 pg to 26,000 pg of the modified BoNT / A. Most preferably, the unit dosage form comprises 25,000 pg of the modified BoNT / A.

[0434] The above-described unit dosage forms may be suitable for treating adult subjects.

[0435] In one aspect, the present invention provides the above-mentioned unit dosage form for use in treating limb spasticity in adults. Also provided are corresponding methods for treating limb spasticity in adults, and uses thereof in the manufacture of a medicament for treating limb spasticity in adults. The treatment is preferably carried out according to the treatment described herein.

[0436] As previously mentioned, the unit dose for pediatric use may be 50% or less than that indicated above. In some embodiments, the unit dose is typically 70% or less (eg, 67% or less) of that used to treat limb spasticity in adults.

[0437] In another aspect, the present invention provides a unit dosage form of a modified botulinum neurotoxin A (BoNT / A) for use in treating a pediatric subject (e.g., for treating limb spasticity in a pediatric subject), the unit dosage form comprising:

[0438] a. Greater than 353.5 units of modified BoNT / A, where 1 unit corresponds to the calculated median lethal dose (LD50) in mice. 50 ) of a modified BoNT / A; or

[0439] b. greater than 8,500 pg of modified BoNT / A; and

[0440] c. optional pharmaceutically acceptable carriers, excipients, adjuvants and / or salts,

[0441] The modified BoNT / A comprises a BoNT / A light chain and a translocation domain, and a BoNT / B receptor binding domain (H C domain).

[0442] Preferably, the modified BoNT / A of the unit dosage form comprises a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 6. For example, a polypeptide sequence having at least 80%, 90%, 95%, or 99.9% sequence identity to SEQ ID NO: 6. Most preferably, the modified BoNT / A can comprise (more preferably consist of) SEQ ID NO: 6.

[0443] Unit dosage forms for treating pediatric subjects can comprise greater than 353.5 units of a modified BoNT / A. The upper limit of the range can be 832, 825, 800, 775, 750, 725, 700, 675, 650, 625, 600, 575, 550, 525, 500, 475, 450, or 425 units of a modified BoNT / A, with a preferred upper limit being 749, 750, or 790 units. The lower end 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 a modified BoNT / A, preferably the lower end is 364 units, 416 units, 474.5 units, 475 units, 690 units, or 690.5 units. Preferably, the unit dosage form comprises from 353.5 units to 832 units of a modified BoNT / A, e.g., from greater than 353.5 units to up to 749 units or from 416 units to 811 units. A unit dose of a modified BoNT / A for treating limb spasticity in children can 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 a modified BoNT / A. In more preferred embodiments, the unit dosage form comprises 624 or 749 units (e.g., 749 units) of a modified BoNT / A.

[0444] More preferably, the unit dosage form for treating limb spasticity in children comprises 624 units to 832 units, such as 728 units to 770 units of the modified BoNT / A. More preferably, the unit dosage form for treating limb spasticity in children comprises 749 units of the modified BoNT / A.

[0445] Even more preferably, the unit dosage form for treating limb spasticity in children comprises 416 units to 624 units, such as 499 units to 541 units of the modified BoNT / A. Most preferably, the unit dosage form for treating limb spasticity in children comprises 520 units of the modified BoNT / A.

[0446] Unit dosage forms for treating pediatric subjects can comprise greater than 8,500 pg of a modified BoNT / A. The upper limit of the 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 a modified BoNT / A, with a preferred upper limit being 19,000 pg. The lower end 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, with a preferred lower end being 8,750, 11,376, 11,377, 11,395, 11,400, 16,590, or 16,591 pg. Preferably, the unit dosage form comprises greater than 8,500 pg to up to 20,000 pg, such as greater than 8,500 pg to up to 18,000 pg, or 10,000 to 19,500 modified BoNT / A. The unit dosage form may comprise 11,000 to 19,000 pg, preferably 11,500 to 19,000 pg, or 17,000 to 19,000 pg modified BoNT / A. The unit dosage form may comprise 12,000 to 18,000 pg or 12,500 to 18,000 pg modified BoNT / A. The unit dosage form may comprise 12,500 to 20,000 pg modified BoNT / A. In preferred embodiments, the unit dosage form comprises 12,000, 12,500, 15,000, or 18,000 pg of a modified BoNT / A. In more preferred embodiments, the unit dosage form comprises 15,000 or 18,000 pg (e.g., 18,000 pg) of a modified BoNT / A.

[0447] More preferably, the unit dosage form for treating limb spasticity in children comprises 15,000 pg to 20,000 pg, such as 17,500 to 18,500 pg of the modified BoNT / A. More preferably, the unit dosage form for treating limb spasticity in children comprises 18,000 pg of the modified BoNT / A.

[0448] Even more preferably, a unit dosage form for treating limb spasticity in children comprises 10,000 pg to 15,000 pg, such as 12,000 pg to 13,000 pg of a modified BoNT / A. More preferably, a unit dosage form for treating limb spasticity in children comprises 12,500 pg of a modified BoNT / A.

[0449] In one aspect, the present invention provides the above-mentioned unit dosage form for treating limb spasticity in children. Also provided are corresponding methods for treating limb spasticity in children, and their use in the manufacture of a medicament for treating limb spasticity in children. The treatment is preferably carried out according to the treatment described herein.

[0450] 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).

[0451] The unit dosage form is preferably provided as a dry powder.

[0452] In another aspect, the present invention provides a kit comprising:

[0453] a. a unit dosage form according to the present invention; and

[0454] b. Instructions for use in the treatment of limb spasticity; and

[0455] c. Optional diluent.

[0456] In one aspect, the invention provides a modified botulinum neurotoxin A (BoNT / A) for use in treating limb spasticity in adults, wherein the modified BoNT / A is administered by intramuscular injection to a plurality of affected muscles of the individual,

[0457] wherein the modified BoNT / A is administered to the affected muscles 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 the modified BoNT / A,

[0458] The plurality of affected muscles are selected from:

[0459] Group 1, including: 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 pollicis longus, adductor pollicis, flexor pollicis brevis, palmaris longus, lumbricals, opponens pollicis, adductor magnus, adductor longus, adductor brevis, gracilis, medial hamstring, lateral hamstring, tensor fasciae latae, rectus femoris, vastus lateralis, vastus medialis, vastus intermedius, gluteus maximus, tibialis anterior, flexor digitorum brevis, extensor pollicis longus, flexor hallucis brevis; and

[0460] The second group includes: 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,

[0461] Preferably, at least one of the plurality of affected muscles is selected from the group consisting of: the adductor magnus, hamstring muscles (e.g., biceps femoris, semimembranosus and / or semitendinosus) and rectus femoris; and

[0462] wherein a single unit dose is administered at a first group of muscles affected and / or a plurality of unit doses are administered at a second group of muscles affected, and wherein the total dose administered during the treatment period is at most 300,000 pg (e.g. at most 240,000 pg, preferably at most 225,000 pg), and

[0463] The modified BoNT / A comprises a BoNT / A light chain and a translocation domain, and a BoNT / B receptor binding domain (H C domain).

[0464] In one aspect, the invention provides a method of treating limb spasticity in an adult, the method comprising administering a modified botulinum neurotoxin (BoNT / A) to a plurality of affected muscles of the individual by intramuscular injection,

[0465] wherein the modified BoNT / A is administered to the affected muscles 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 the modified BoNT / A,

[0466] The plurality of affected muscles are selected from:

[0467] Group 1, including: 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 pollicis longus, adductor pollicis, flexor pollicis brevis, palmaris longus, lumbricals, opponens pollicis, adductor magnus, adductor longus, adductor brevis, gracilis, medial hamstring, lateral hamstring, tensor fasciae latae, rectus femoris, vastus lateralis, vastus medialis, vastus intermedius, gluteus maximus, tibialis anterior, flexor digitorum brevis, extensor pollicis longus, flexor hallucis brevis; and

[0468] The second group includes: 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,

[0469] Preferably, at least one of the plurality of affected muscles is selected from the group consisting of: the adductor magnus, hamstring muscles (e.g., biceps femoris, semimembranosus and / or semitendinosus) and rectus femoris; and

[0470] wherein a single unit dose is administered at a first group of muscles affected and / or a plurality of unit doses are administered at a second group of muscles affected, and wherein the total dose administered during the treatment period is at most 300,000 pg (e.g. at most 240,000 pg, preferably at most 225,000 pg), and

[0471] The modified BoNT / A comprises a BoNT / A light chain and a translocation domain, and a BoNT / B receptor binding domain (H C domain).

[0472] In one aspect, the present invention provides a use of a modified botulinum neurotoxin (BoNT / A) in the manufacture of a medicament for treating limb spasticity in an adult, wherein the modified BoNT / A is administered by intramuscular injection to a plurality of affected muscles of an individual,

[0473] wherein the modified BoNT / A is administered to the affected muscles 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 the modified BoNT / A,

[0474] The plurality of affected muscles are selected from:

[0475] Group 1, including: 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 pollicis longus, adductor pollicis, flexor pollicis brevis, palmaris longus, lumbricals, opponens pollicis, adductor magnus, adductor longus, adductor brevis, gracilis, medial hamstring, lateral hamstring, tensor fasciae latae, rectus femoris, vastus lateralis, vastus medialis, vastus intermedius, gluteus maximus, tibialis anterior, flexor digitorum brevis, extensor pollicis longus, flexor hallucis brevis; and

[0476] The second group includes: 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,

[0477] Preferably, at least one of the plurality of affected muscles is selected from the group consisting of: the adductor magnus, hamstring muscles (e.g., biceps femoris, semimembranosus and / or semitendinosus) and rectus femoris; and

[0478] wherein a single unit dose is administered at a first group of muscles affected and / or a plurality of unit doses are administered at a second group of muscles affected, and wherein the total dose administered during the treatment period is at most 300,000 pg (e.g. at most 240,000 pg, preferably at most 225,000 pg), and

[0479] The modified BoNT / A comprises a BoNT / A light chain and a translocation domain, and a BoNT / B receptor binding domain (H C domain).

[0480] In the above aspects, preferably, the unit dose is 15,000 pg, and the total dose is at most 225,000 pg (eg, the total dose may be 225,000 pg).

[0481] In one aspect, the invention provides a modified botulinum neurotoxin A (BoNT / A) for use in treating limb spasticity in adults, wherein the modified BoNT / A is administered by intramuscular injection to a plurality of affected muscles of the individual,

[0482] wherein the modified BoNT / A is administered to the affected muscles in a unit dose of 416 units to 832 units (e.g., 582 units to 666 units, preferably 624 units) of the modified BoNT / A, and wherein 1 unit corresponds to a calculated median lethal dose (LD50) in mice. 50 ),

[0483] The plurality of affected muscles are selected from:

[0484] Group 1, including: 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 pollicis longus, adductor pollicis, flexor pollicis brevis, palmaris longus, lumbricals, opponens pollicis, adductor magnus, adductor longus, adductor brevis, gracilis, medial hamstring, lateral hamstring, tensor fasciae latae, rectus femoris, vastus lateralis, vastus medialis, vastus intermedius, gluteus maximus, tibialis anterior, flexor digitorum brevis, extensor pollicis longus, flexor hallucis brevis; and

[0485] The second group includes: 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,

[0486] Preferably, at least one of the plurality of affected muscles is selected from the group consisting of: the adductor magnus, hamstring muscles (e.g., biceps femoris, semimembranosus and / or semitendinosus) and rectus femoris; and

[0487] wherein a single unit dose is administered at the first group of muscles affected and / or a plurality of unit doses are administered at the second group of muscles affected, and wherein the total dose administered during the treatment period is at most 12,480 units (e.g. at most 9,990 units, preferably at most 9,360 units), and

[0488] The modified BoNT / A comprises a BoNT / A light chain and a translocation domain, and a BoNT / B receptor binding domain (H C domain).

[0489] In one aspect, the invention provides a method of treating limb spasticity in an adult, the method comprising administering a modified botulinum neurotoxin (BoNT / A) to a plurality of affected muscles of the individual by intramuscular injection,

[0490] wherein the modified BoNT / A is administered to the affected muscles in a unit dose of 416 units to 832 units (e.g., 582 units to 666 units, preferably 624 units) of the modified BoNT / A, and wherein 1 unit corresponds to a calculated median lethal dose (LD50) in mice. 50 ),

[0491] The plurality of affected muscles are selected from:

[0492] Group 1, including: 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 pollicis longus, adductor pollicis, flexor pollicis brevis, palmaris longus, lumbricals, opponens pollicis, adductor magnus, adductor longus, adductor brevis, gracilis, medial hamstring, lateral hamstring, tensor fasciae latae, rectus femoris, vastus lateralis, vastus medialis, vastus intermedius, gluteus maximus, tibialis anterior, flexor digitorum brevis, extensor pollicis longus, flexor hallucis brevis; and

[0493] The second group includes: 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,

[0494] Preferably, at least one of the plurality of affected muscles is selected from the group consisting of: the adductor magnus, hamstring muscles (e.g., biceps femoris, semimembranosus and / or semitendinosus) and rectus femoris; and

[0495] wherein a single unit dose is administered at the first group of muscles affected and / or a plurality of unit doses are administered at the second group of muscles affected, and wherein the total dose administered during the treatment period is at most 12,480 units (e.g. at most 9,990 units, preferably at most 9,360 units), and

[0496] The modified BoNT / A comprises a BoNT / A light chain and a translocation domain, and a BoNT / B receptor binding domain (HC domain).

[0497] In one aspect, the present invention provides a use of a modified botulinum neurotoxin (BoNT / A) in the manufacture of a medicament for treating limb spasticity in an adult, wherein the modified BoNT / A is administered by intramuscular injection to a plurality of affected muscles of an individual,

[0498] wherein the modified BoNT / A is administered to the affected muscles in a unit dose of 416 units to 832 units (e.g., 582 units to 666 units, preferably 624 units) of the modified BoNT / A, and wherein 1 unit corresponds to a calculated median lethal dose (LD50) in mice. 50 ),

[0499] The plurality of affected muscles are selected from:

[0500] Group 1, including: 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 pollicis longus, adductor pollicis, flexor pollicis brevis, palmaris longus, lumbricals, opponens pollicis, adductor magnus, adductor longus, adductor brevis, gracilis, medial hamstring, lateral hamstring, tensor fasciae latae, rectus femoris, vastus lateralis, vastus medialis, vastus intermedius, gluteus maximus, tibialis anterior, flexor digitorum brevis, extensor pollicis longus, flexor hallucis brevis; and

[0501] The second group includes: 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,

[0502] Preferably, at least one of the plurality of affected muscles is selected from the group consisting of: the adductor magnus, hamstring muscles (e.g., biceps femoris, semimembranosus and / or semitendinosus) and rectus femoris; and

[0503] wherein a single unit dose is administered at the first group of muscles affected and / or a plurality of unit doses are administered at the second group of muscles affected, and wherein the total dose administered during the treatment period is at most 12,480 units (e.g. at most 9,990 units, preferably at most 9,360 units), and

[0504] The modified BoNT / A comprises a BoNT / A light chain and a translocation domain, and a BoNT / B receptor binding domain (H C domain).

[0505] In the above aspects, preferably, the unit dose is 624 units and the total dose is at most 9,360 units (eg, the total dose may be 9,360 units).

[0506] In one aspect, the present invention provides a modified botulinum neurotoxin A (BoNT / A) for use in treating limb spasticity in children, wherein the modified BoNT / A is administered by intramuscular injection to a plurality of affected muscles of a subject,

[0507] wherein the modified BoNT / A is administered to the affected muscles 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 the modified BoNT / A,

[0508] The plurality of affected muscles are selected from:

[0509] Group 1, including: 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 pollicis longus, adductor pollicis, flexor pollicis brevis, palmaris longus, lumbricals, opponens pollicis, adductor magnus, adductor longus, adductor brevis, gracilis, medial hamstring, lateral hamstring, tensor fasciae latae, rectus femoris, vastus lateralis, vastus medialis, vastus intermedius, gluteus maximus, tibialis anterior, flexor digitorum brevis, extensor pollicis longus, flexor hallucis brevis; and

[0510] The second group includes: 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,

[0511] Preferably, at least one of the plurality of affected muscles is selected from the group consisting of: the adductor magnus, hamstring muscles (e.g., biceps femoris, semimembranosus and / or semitendinosus) and rectus femoris; and

[0512] wherein a single unit dose is administered at a first group of muscles affected and / or a plurality of unit doses are administered at a second group of muscles affected, and wherein the total dose administered during the treatment period is at most 150,000 pg (e.g. at most 120,000 pg, preferably at most 112,500 pg), and

[0513] The modified BoNT / A comprises a BoNT / A light chain and a translocation domain, and a BoNT / B receptor binding domain (H C domain).

[0514] In one aspect, the present invention provides a method of treating limb spasticity in a child, the method comprising administering a modified botulinum neurotoxin (BoNT / A) to a plurality of affected muscles of a subject by intramuscular injection,

[0515] wherein the modified BoNT / A is administered to the affected muscles 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 the modified BoNT / A,

[0516] The plurality of affected muscles are selected from:

[0517] Group 1, including: 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 pollicis longus, adductor pollicis, flexor pollicis brevis, palmaris longus, lumbricals, opponens pollicis, adductor magnus, adductor longus, adductor brevis, gracilis, medial hamstring, lateral hamstring, tensor fasciae latae, rectus femoris, vastus lateralis, vastus medialis, vastus intermedius, gluteus maximus, tibialis anterior, flexor digitorum brevis, extensor pollicis longus, flexor hallucis brevis; and

[0518] The second group includes: 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,

[0519] Preferably, at least one of the plurality of affected muscles is selected from the group consisting of: the adductor magnus, hamstring muscles (e.g., biceps femoris, semimembranosus and / or semitendinosus) and rectus femoris; and

[0520] wherein a single unit dose is administered to a first group of affected muscles and / or multiple unit doses are administered to a second group of affected muscles, and wherein the total dose administered during the treatment period is at most 150,000 pg (e.g., at most 120,000 pg, preferably at most 112,500 pg), and wherein the modified BoNT / A comprises a BoNT / A light chain and a translocation domain, and a BoNT / B receptor binding domain (H C domain).

[0521] In one aspect, the present invention provides a use of a modified botulinum neurotoxin (BoNT / A) in the manufacture of a medicament for treating limb spasticity in a child, wherein the modified BoNT / A is administered by intramuscular injection to a plurality of affected muscles of a subject,

[0522] wherein the modified BoNT / A is administered to the affected muscles 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 the modified BoNT / A,

[0523] The plurality of affected muscles are selected from:

[0524] Group 1, including: 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 pollicis longus, adductor pollicis, flexor pollicis brevis, palmaris longus, lumbricals, opponens pollicis, adductor magnus, adductor longus, adductor brevis, gracilis, medial hamstring, lateral hamstring, tensor fasciae latae, rectus femoris, vastus lateralis, vastus medialis, vastus intermedius, gluteus maximus, tibialis anterior, flexor digitorum brevis, extensor pollicis longus, flexor hallucis brevis; and

[0525] The second group includes: 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,

[0526] Preferably, at least one of the plurality of affected muscles is selected from the group consisting of: the adductor magnus, hamstring muscles (e.g., biceps femoris, semimembranosus and / or semitendinosus) and rectus femoris; and

[0527] wherein a single unit dose is administered at a first group of muscles affected and / or a plurality of unit doses are administered at a second group of muscles affected, and wherein the total dose administered during the treatment period is at most 150,000 pg (e.g. at most 120,000 pg, preferably at most 112,500 pg), and

[0528] The modified BoNT / A comprises a BoNT / A light chain and a translocation domain, and a BoNT / B receptor binding domain (H C domain).

[0529] In the above aspects, preferably, the unit dose is 7,500 pg, and the total dose is at most 112,500 pg (eg, the total dose may be 112,500 pg).

[0530] In one aspect, the present invention provides a modified botulinum neurotoxin A (BoNT / A) for use in treating limb spasticity in children, wherein the modified BoNT / A is administered by intramuscular injection to a plurality of affected muscles of a subject,

[0531] wherein the modified BoNT / A is administered to the affected muscles in a unit dose of 208 units to 416 units (e.g., 291 units to 333 units, preferably 312 units) of the modified BoNT / A, and wherein 1 unit corresponds to a calculated median lethal dose (LD50) in mice. 50 ),

[0532] The plurality of affected muscles are selected from:

[0533] Group 1, including: 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 pollicis longus, adductor pollicis, flexor pollicis brevis, palmaris longus, lumbricals, opponens pollicis, adductor magnus, adductor longus, adductor brevis, gracilis, medial hamstring, lateral hamstring, tensor fasciae latae, rectus femoris, vastus lateralis, vastus medialis, vastus intermedius, gluteus maximus, tibialis anterior, flexor digitorum brevis, extensor pollicis longus, flexor hallucis brevis; and

[0534] The second group includes: 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,

[0535] Preferably, at least one of the plurality of affected muscles is selected from the group consisting of: the adductor magnus, hamstring muscles (e.g., biceps femoris, semimembranosus and / or semitendinosus) and rectus femoris; and

[0536] wherein a single unit dose is administered at the first group of muscles affected and / or a plurality of unit doses are administered at the second group of muscles affected, and wherein the total dose administered during the treatment period is at most 6,240 units (e.g. at most 4,995 units, preferably at most 4,680 units), and

[0537] The modified BoNT / A comprises a BoNT / A light chain and a translocation domain, and a BoNT / B receptor binding domain (H C domain).

[0538] In one aspect, the present invention provides a method of treating limb spasticity in a child, the method comprising administering a modified botulinum neurotoxin (BoNT / A) to a plurality of affected muscles of a subject by intramuscular injection,

[0539] wherein the modified BoNT / A is administered to the affected muscles in a unit dose of 208 units to 416 units (e.g., 291 units to 333 units, preferably 312 units) of the modified BoNT / A, and wherein 1 unit corresponds to a calculated median lethal dose (LD50) in mice. 50 ),

[0540] The plurality of affected muscles are selected from:

[0541] Group 1, including: 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 pollicis longus, adductor pollicis, flexor pollicis brevis, palmaris longus, lumbricals, opponens pollicis, adductor magnus, adductor longus, adductor brevis, gracilis, medial hamstring, lateral hamstring, tensor fasciae latae, rectus femoris, vastus lateralis, vastus medialis, vastus intermedius, gluteus maximus, tibialis anterior, flexor digitorum brevis, extensor pollicis longus, flexor hallucis brevis; and

[0542] The second group includes: 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,

[0543] Preferably, at least one of the plurality of affected muscles is selected from the group consisting of: the adductor magnus, hamstring muscles (e.g., biceps femoris, semimembranosus and / or semitendinosus) and rectus femoris; and

[0544] wherein a single unit dose is administered at the first group of muscles affected and / or a plurality of unit doses are administered at the second group of muscles affected, and wherein the total dose administered during the treatment period is at most 6,240 units (e.g. at most 4,995 units, preferably at most 4,680 units), and

[0545] The modified BoNT / A comprises a BoNT / A light chain and a translocation domain, and a BoNT / B receptor binding domain (H C domain).

[0546] In one aspect, the present invention provides a use of a modified botulinum neurotoxin (BoNT / A) in the manufacture of a medicament for treating limb spasticity in a child, wherein the modified BoNT / A is administered by intramuscular injection to a plurality of affected muscles of a subject,

[0547] wherein the modified BoNT / A is administered to the affected muscles in a unit dose of 208 units to 416 units (e.g., 291 units to 333 units, preferably 312 units) of the modified BoNT / A, and wherein 1 unit corresponds to a calculated median lethal dose (LD50) in mice. 50 ),

[0548] The plurality of affected muscles are selected from:

[0549] Group 1, including: 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 pollicis longus, adductor pollicis, flexor pollicis brevis, palmaris longus, lumbricals, opponens pollicis, adductor magnus, adductor longus, adductor brevis, gracilis, medial hamstring, lateral hamstring, tensor fasciae latae, rectus femoris, vastus lateralis, vastus medialis, vastus intermedius, gluteus maximus, tibialis anterior, flexor digitorum brevis, extensor pollicis longus, flexor hallucis brevis; and

[0550] The second group includes: 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,

[0551] Preferably, at least one of the plurality of affected muscles is selected from the group consisting of: the adductor magnus, hamstring muscles (e.g., biceps femoris, semimembranosus and / or semitendinosus) and rectus femoris; and

[0552] wherein a single unit dose is administered at the first group of muscles affected and / or a plurality of unit doses are administered at the second group of muscles affected, and wherein the total dose administered during the treatment period is at most 6,240 units (e.g. at most 4,995 units, preferably at most 4,680 units), and

[0553] The modified BoNT / A comprises a BoNT / A light chain and a translocation domain, and a BoNT / B receptor binding domain (H C domain).

[0554] In the above aspects, preferably, the unit dose is 312 units and the total dose is at most 4,680 units (eg, the total dose may be 4,680 units).

[0555] In any of the above aspects, the duration of treatment of limb spasticity can be longer than the duration of treatment with an unmodified BoNT / A (eg, SEQ ID NO: 2 [eg, SEQ ID NO: 2 in a di-chain form]).

[0556] The total number of unit doses administered in a given treatment may be up to 15× unit doses. In other words, in one embodiment, 15× single unit doses may be administered to 15× injection sites. In other embodiments, 15× single unit doses may be administered to more than 15× injection sites. The total number of unit doses will be divided according to the muscles treated, for example, 2× unit doses may be administered to the latissimus dorsi, 2× to the subscapularis, 2× to the pectoralis major, and 1× to the ulnar flexor carpi, resulting in a total administration multiple of 7× unit doses. For example, the total number of unit doses administered may be up to 14×, 13×, 12×, 11×, 10×, 9×, 8×, or 7×. The total number of unit doses administered may be at least 2×, 3×, 4×, 5×, 6×, 7×, preferably at least 2× of the unit dose. The total number of unit doses administered may be from 2× to 15×, from 7× to 15×, or from 10× to 14×. Preferably, the number of unit doses administered is 15×.

[0557] Embodiments relating to the various therapeutic uses of the invention are intended to apply equally to the methods, compositions (eg, unit dosage forms), and kits of the invention, and vice versa.

[0558] Sequence homology

[0559] Percent identity can be determined using any of a variety of sequence alignment methods, including but not limited to global methods, local methods, and hybrid methods, such as segment methods. The scheme for determining percent identity is a routine procedure within the capabilities of those skilled in the art. The global method aligns the sequence from the beginning to the end of the molecule and determines the optimal alignment by adding the scores of single residue pairs and applying gap penalties. Non-limiting methods include, for example, CLUSTAL W, see, for example, Julie D. Thompson et al., CLUSTAL W: Improving the Sensitivity of Progressive Multiple Sequence Alignment Through Sequence Weighting, Position-Specific Gap Penalties and Weight Matrix Choice, 22 (22) Nucleic Acids Research 4673-4680 (1994); and iterative refinement, see, for example, Osamu Gotoh, Significant Improvement in Accuracy of Multiple Protein. Sequence Alignments by Iterative Refinement as Assessed by Reference to Structural Alignments, 264 (4) J. MoI. Biol. 823-838 (1996). Local methods align sequences by identifying one or more conserved motifs common to all input sequences.Non-limiting methods include, for example, Match-box, see, for example, Eric Depiereux and Ernest Feytmans, Match-Box: A Fundamentally New Algorithm for the Simultaneous Alignment of Several Protein Sequences, 8(5) CABIOS 501-509 (1992); Gibbs sampling, see, for example, CE Lawrence et al., Detecting Subtle Sequence Signals: A Gibbs Sampling Strategy for Multiple Alignment, 262(5131) Science 208-214 (1993); Align-M, see, for example, Ivo Van WaIIe et al., Align-MA New Algorithm for Multiple Alignment of Highly Divergent Sequences, 20(9) Bioinformatics: 1428-1435 (2004).

[0560] Therefore, the 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, as shown below, two amino acid sequences are aligned using a gap opening penalty of 10, a gap extension penalty of 1, and the "blosum 62" scoring matrix of Henikoff and Henikoff (supra) to optimize the alignment score (amino acids are represented by standard single letter codes); preferably, the method is used to align the sequence with the subject sequence of this article (e.g., SEQ ID NO: 2) to define the amino acid position numbering described herein.

[0561] "Percentage of sequence identity" between two or more nucleic acid or amino acid sequences is a function of the number of identical positions shared by the sequences. Therefore, % identity can be calculated as the number of identical nucleotides / amino acids divided by the total number of nucleotides / amino acids, multiplied by 100. The calculation of % sequence identity can also take into account the number of spaces that need to be introduced to optimize the comparison of two or more sequences, and the length of each space. Specific mathematical algorithms such as BLAST that are familiar to those skilled in the art can be used to compare sequences and determine the percentage identity between two or more sequences.

[0562] Alignment score used to determine sequence identity

[0563]

[0564] The percent identity is then calculated as follows:

[0565]

[0566] Substantially homologous polypeptides are characterized by having one or more amino acid substitutions, deletions or additions. These changes are preferably insignificant, i.e., conservative amino acid substitutions (see below) and other substitutions that do not significantly affect the folding or activity of the polypeptide; small deletions, typically 1 to about 30 amino acids; and small amino-terminal or carboxyl-terminal extensions, such as an amino-terminal methionine residue, a small linker peptide of up to about 20-25 residues, or an affinity tag.

[0567] Conservative amino acid substitution

[0568] Alkaline: Arginine

[0569] Lysine

[0570] Histidine

[0571] Acid: Glutamic acid

[0572] Aspartic acid

[0573] Polarity: Glutamine

[0574] Asparagine

[0575] Hydrophobicity: Leucine

[0576] Isoleucine

[0577] Valine

[0578] Aromatic: Phenylalanine

[0579] Tryptophan

[0580] Tyrosine

[0581] Small: Glycine

[0582] Alanine

[0583] Serine

[0584] Threonine

[0585] Methionine

[0586] In addition to the 20 standard amino acids, non-standard amino acids (such as 4-hydroxyproline, 6-N-methyllysine, 2-aminoisobutyric acid, isovaline and α-methylserine) can replace the amino acid residues of the polypeptides of the present invention. A limited number of non-conservative amino acids, amino acids not encoded by the genetic code and unnatural amino acids can replace polypeptide amino acid residues. The polypeptides of the present invention can also contain non-naturally occurring amino acid residues.

[0587] Non-naturally occurring amino acids include, but are not limited to, trans-3-methylproline, 2,4-methano-proline, cis-4-hydroxyproline, trans-4-hydroxyproline, N-methylglycine, allo-threonine, methylthreonine, hydroxyethylcysteine, hydroxyethylhomocysteine, nitroglutamine, homoglutamine, pipecolic acid, tert-leucine, norvaline, 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, and 4-fluorophenylalanine. Several methods for incorporating non-naturally occurring amino acid residues into proteins are known in the art. For example, an in vitro system can be used in which a chemically aminoacylated suppressor tRNA is used to suppress nonsense mutations. Methods for synthesizing amino acids and aminoacylated tRNAs are known in the art. Transcription and translation of plasmids containing nonsense mutations are performed in a cell-free system comprising an E. coli S30 extract and commercially available enzymes and other reagents. The protein is purified by chromatography. See, e.g., Robertson et al., J. Am. Chem. Soc. 113:2722, 1991; Ellman et al., Methods Enzymol. 202:301, 1991; Chung et al., Science 259:806-9, 1993; and Chung et al., Proc. Natl. Acad. Sci. USA 90:10145-9, 1993). In a second approach, translation is performed in Xenopus oocytes by microinjection of mutant mRNA and chemically aminoacylated suppressor tRNA (Turcatti et al., J. Biol. Chem. 271:19991-8, 1996). In the third method, E. coli cells are cultured in the absence of a natural amino acid to be replaced (e.g., phenylalanine) and in the presence of a desired non-natural amino acid (e.g., 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, or 4-fluorophenylalanine). Non-naturally occurring amino acids are incorporated into polypeptides in place of their natural counterparts. See Koide et al., Biochem. 33: 7470-6, 1994. Naturally occurring amino acid residues can be converted to non-naturally occurring species by in vitro chemical modification. Chemical modification can be combined with site-directed mutagenesis to further expand the scope of substitution (Wynn and Richards, Protein Sci. 2: 395-403, 1993).

[0588] A limited number of non-conservative amino acids, amino acids not encoded by the genetic code, non-naturally occurring amino acids and unnatural amino acids can be substituted for amino acid residues in the polypeptides of the invention.

[0589] Essential amino acids in the polypeptides of the invention can be identified according to methods known in the art, such as site-directed mutagenesis or alanine scanning mutagenesis (Cunningham and Wells, Science 244: 1081-5, 1989). The site of biological interaction can also be determined by physical analysis of the structure, such as by techniques such as nuclear magnetic resonance, crystallography, electron diffraction or photoaffinity labeling, combined with mutations of putative contact site amino acids. See, for example, de Vos et al., Science 255: 306-12, 1992; Smith et al., J. Mol. Biol. 224: 899-904, 1992; Wlodaver et al., FEBS Lett. 309: 59-64, 1992. The identity of essential amino acids can also be inferred by homology analysis with related components of the polypeptides of the invention (e.g., translocation or protease components).

[0590] A variety of amino acid substitutions can be made and tested using known mutagenesis and screening methods, 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). In short, these authors disclose methods for simultaneously randomizing two or more positions in a polypeptide, selecting functional polypeptides, and then sequencing the mutagenized polypeptides to determine the substitution spectrum allowed at each position. Other methods that can be used include phage display (e.g., Lowman et al., Biochem. 30:10832-7, 1991; Ladner et al., US Patent No. 5,223,409; Huse, WIPO Publication WO 92 / 06204) and regional site-directed mutagenesis (Derbyshire et al., Gene 46:145, 1986; Ner et al., DNA 7:127, 1988).

[0591] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Singleton et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 20th Edition, John Wiley and Sons, New York (1994); and Hale & Marham, THE HARPER COLLINSDICTIONARY 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.

[0592] The present disclosure is not limited to the exemplary methods and materials disclosed herein, and any methods and materials similar or equivalent to those described herein can be used to practice or test the embodiments of the present disclosure. Numerical ranges include the numbers defining the ranges. Unless otherwise indicated, any nucleic acid sequence is written from left to right in the 5' to 3' direction; amino acid sequences are written from left to right in the direction of amino to carboxyl, respectively.

[0593] The headings provided herein are not limitations of the various aspects or embodiments of the invention.

[0594] The name of amino acid, three-letter abbreviation or single-letter abbreviation is used herein to refer to amino acid. The term "protein" used herein includes protein, polypeptide and peptide. As used herein, the term "amino acid sequence" is synonymous with the term "polypeptide" and / or the term "protein". In some cases, the term "amino acid sequence" is synonymous with the term "peptide". In some cases, the term "amino acid sequence" is synonymous with the term "enzyme". The terms "protein" and "polypeptide" are used interchangeably herein. In the present disclosure and claims, conventional single-letter and three-letter codes of amino acid residues can be used. The 3-letter code of amino acid defined by the Joint Commission on Biochemical Nomenclature (JCBN) of IUPACIUB is met. It should also be understood that due to the degeneracy of the genetic code, a polypeptide can be encoded by more than one nucleotide sequence.

[0595] Other definitions of terms may appear throughout the specification. Before describing the exemplary embodiments in more detail, it should be understood that the present invention is not limited to the specific embodiments described and may vary accordingly. It should also be understood that the terminology used herein is for the purpose of describing specific embodiments only and is not intended to be limiting, as the scope of the present invention will be defined only by the appended claims.

[0596] Where a numerical range is provided, it is to be understood that, unless the context clearly indicates otherwise, each intermediate value (to one tenth of the lower limit unit) between the upper and lower limits of the range is also specifically disclosed. Each smaller range between any specified value or intermediate value within a specified range and any other specified value or intermediate value within the specified range is included in the present disclosure. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope, and each range is also included in the present disclosure, including one of the upper and lower limits within the smaller range, excluding none, or including both, subject to any explicitly excluded limit within the scope. When the range includes one or two limits, the range excluding one or two of these included limits is also included in the present disclosure.

[0597] It must be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "botulinum neurotoxin A" includes a plurality of such candidate agents and reference to "botulinum neurotoxin A" includes reference to one or more clostridial neurotoxins and equivalents thereof known to those skilled in the art, and so forth.

[0598] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein should be construed as an admission that such publications constitute prior art to the appended claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0599] Embodiments of the invention will now be described, by way of example only, with reference to the following Figures and Examples.

[0600] Figure 1 The five most common clinical patterns of upper limb spasticity involving the upper arm joints are shown.

[0601] Figure 2 Showing FDA approval for the treatment of spasticity in adults dosage.

[0602] Figure 3 Shown is an SDS-PAGE of purified recombinant BoNT / AB chimeras 1, 2, and 3A (SEQ ID NOs: 3, 4, and 5, respectively). Lanes are labeled "Maker" (molecular weight marker), "-DTT" (oxidized BoNT / AB chimera sample), and "+DTT" (reduced BoNT / AB chimera sample).

[0603] Figure 4Cleavage of SNAP-25 in rat spinal cord neurons by recombinant BoNT / AB chimeras 1, 2, and 3A (SEQ ID NOs: 3, 4, and 5 converted to double-chain form, respectively) is shown. 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 with 10% CO2. The cells were then lysed with 1× NuPAGE buffer supplemented with DTT and Benzonase. The samples were transferred to microcentrifuge tubes, heated at 90°C for 5 minutes on a heating block, and stored at -20°C before analysis of SNAP-25 cleavage by Western blot. SNAP-25 was detected using a polyclonal antibody (Sigma #S9684) that detects the full length and cleaved forms of SNAP-25. Anti-rabbit HRP (Sigma #A6154) was used as a secondary antibody.

[0604] Figure 5 The mouse digital abduction score test is shown. Under brief general anesthesia, mice were injected into the gastrocnemius-soleus complex muscle of one hind limb; the degree of muscle weakness was measured using the digital abduction score (DAS) on a scale of 0-4. The maximum DAS for each dose was determined and plotted against the doses, and the data were fitted to a 4-parameter logistic equation to determine the ED that resulted in a DAS of 4. 50 and dose (DAS 4 dose) values.

[0605] Figure 6 Shown is an SDS-PAGE of purified recombinant BoNT / AB chimeras 3B and 3C (SEQ ID NOs: 6 and 7, respectively). Lanes are labeled "Maker" (molecular weight marker), "-DTT" (oxidized BoNT / AB chimera sample), and "+DTT" (reduced BoNT / AB chimera sample).

[0606] Figure 7Cleavage 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) is shown. 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. The cells were then lysed with 1× NuPAGE buffer supplemented with DTT and Benzonase. The samples were transferred to microcentrifuge tubes, heated at 90°C for 5 minutes on a heating block, and stored at -20°C before analysis of SNAP-25 cleavage by Western blot. SNAP-25 was detected using a polyclonal antibody (Sigma #S9684) that detects both the full-length and cleaved forms of SNAP-25. Anti-rabbit HRP (Sigma #A6154) was used as secondary antibody.

[0607] Figure 8 The duration of muscle weakness over time in the mouse digital abduction score test is shown. Under brief general anesthesia, the gastrocnemius-soleus complex muscle of one hind limb of the mouse was injected; the degree of muscle weakness was measured using the digital abduction score (DAS) on a scale of 0-4. Animals in the group injected with the lowest dose (which caused a DAS of 4 in the first four days of injection) were monitored until muscle weakness fully recovered to DAS 0 (no muscle weakness was observed).

[0608] Sequence Listing

[0609] When an initiating Met amino acid residue or the corresponding initiation codon is indicated in any of the following SEQ ID NOs, said residue / codon is optional.

[0610] SEQ ID NO: 1 (nucleotide sequence of unmodified BoNT / A)

[0611] ATGCCATTCGTCAACAAGCAATTCAACTACAAAGACCCAGTCAACGGCGTCGACATCGCATACATCAAGATTCCGAACGC

[0612] CGGTCAAATGCAGCCGGTTAAGGCTTTTAAGATCCACAACAAGATTTGGGTTATCCCGGAGCGTGACACCTTCACGAACC

[0613] CGGAAGAAGGCGATCTGAACCCGCCACCGGAAGCGAAGCAAGTCCCTGTCAGCTACTACGATTCGACGTACCTGAGCACG

[0614] GATAACGAAAAAGATAACTACCTGAAAGGTGTGACCAAGCTGTTCGAACGTATCTACAGCACGGATCTGGGTCGCATGCT

[0615] GCTGACTAGCATTGTTCGCGGTATCCCGTTCTGGGGTGGTAGCACGATTGACACCGAACTGAAGGTTATCGACACTAACT

[0616] GCATTAACGTTATTCAACCGGATGGTAGCTATCGTAGCGAAGAGCTGAATCTGGTCATCATTGGCCCGAGCGCAGACATT

[0617] ATCCAATTCGAGTGCAAGAGCTTTGGTCACGAGGTTCTGAATCTGACCCGCAATGGCTATGGTAGCACCCAGTACATTCG

[0618] TTTTTCGCCGGATTTTACCTTCGGCTTTGAAGAGAGCCTGGAGGTTGATACCAATCCGTTGCTGGGTGCGGGCAAATTCG

[0619] CTACCGATCCGGCTGTCACGCTGGCCCATGAACTGATCCACGCAGGCCACCGCCTGTACGGCATTGCCATCAACCCAAAC

[0620] CGTGTGTTCAAGGTTAATACGAATGCATACTACGAGATGAGCGGCCTGGAAGTCAGCTTCGAAGAACTGCGCACCTTCGG

[0621] TGGCCATGACGCTAAATTCATTGACAGCTTGCAAGAGAATGAGTTCCGTCTGTACTACTATAACAAATTCAAAGACATTG

[0622] CAAGCACGTTGAACAAGGCCAAAAGCATCGTTGGTACTACCGCGTCGTTGCAGTATATGAAGAATGTGTTTAAAGAGAAG

[0623] TACCTGCTGTCCGAGGATACCTCCGGCAAGTTTAGCGTTGATAAGCTGAAGTTTGACAAACTGTACAAGATGCTGACCGA

[0624] GATTTACACCGAGGACAACTTTGTGAAATTCTTCAAAGTGTTGAATCGTAAAACCTATCTGAATTTTGACAAAGCGGTTT

[0625] TCAAGATTAACATCGTGCCGAAGGTGAACTACACCATCTATGACGGTTTTAACCTGCGTAACACCAACCTGGCGGCGAAC

[0626] TTTAACGGTCAGAATACGGAAATCAACAACATGAATTTCACGAAGTTGAAGAACTTCACGGGTCTGTTCGAGTTCTATAA

[0627] GCTGCTGTGCGTGCGCGGTATCATCACCAGCAAAACCAAAAGCCTGGACAAAGGCTACAACAAGGCGCTGAATGACCTGT

[0628] GCATTAAGGTAAACAATTGGGATCTGTTCTTTTCGCCATCCGAAGATAATTTTACCAACGACCTGAACAAGGGTGAAGAA

[0629] ATCACCAGCGATACGAATATTGAAGCAGCGGAAGAGAATATCAGCCTGGATCTGATCCAGCAGTACTATCTGACCTTTAA

[0630] CTTCGACAATGAACCGGAGAACATTAGCATTGAGAATCTGAGCAGCGACATTATCGGTCAGCTGGAACTGATGCCGAATA

[0631] TCGAACGTTTCCCGAACGGCAAAAAGTACGAGCTGGACAAGTACACTATGTTCCATTACCTGCGTGCACAGGAGTTTGAA

[0632] CACGGTAAAAGCCGTATCGCGCTGACCAACAGCGTTAACGAGGCCCTGCTGAACCCGAGCCGTGTCTATACCTTCTTCAG

[0633] CAGCGACTATGTTAAGAAAGTGAACAAAGCCACTGAGGCCGCGATGTTCCTGGGCTGGGTGGAACAGCTGGTATATGACT

[0634] TCACGGACGAGACGAGCGAAGTGAGCACTACCGACAAAATTGCTGATATTACCATCATTATCCCGTATATTGGTCCGGCA

[0635] CTGAACATTGGCAACATGCTGTACAAAGACGATTTTGTGGGTGCCCTGATCTTCTCCGGTGCCGTGATTCTGCTGGAGTT

[0636] CATTCCGGAGATTGCGATCCCGGTGTTGGGTACCTTCGCGCTGGTGTCCTACATCGCGAATAAGGTTCTGACGGTTCAGA

[0637] CCATCGATAACGCGCTGTCGAAACGTAATGAAAAATGGGACGAGGTTTACAAATACATTGTTACGAATTGGCTGGCGAAA

[0638] GTCAATACCCAGATCGACCTGATCCGTAAGAAAATGAAAGAGGCGCTGGAGAATCAGGCGGAGGCCACCAAAGCAATTAT

[0639] CAACTACCAATACAACCAGTACACGGAAGAAGAGAAGAATAACATTAACTTCAATATCGATGATTTGAGCAGCAAGCTGA

[0640] ATGAATCTATCAACAAAGCGATGATCAATATCAACAAGTTTTTGAATCAGTGTAGCGTTTCGTACCTGATGAATAGCATG

[0641] ATTCCGTATGGCGTCAAACGTCTGGAGGACTTCGACGCCAGCCTGAAAGATGCGTTGCTGAAATACATTTACGACAATCG

[0642] TGGTACGCTGATTGGCCAAGTTGACCGCTTGAAAGACAAAGTTAACAATACCCTGAGCACCGACATCCCATTTCAACTGA

[0643] GCAAGTATGTTGATAATCAACGTCTGTTGAGCACTTTCACCGAGTATATCAAAAACATCATCAATACTAGCATTCTGAAC

[0644] CTGCGTTACGAGAGCAATCATCTGATTGATCTGAGCCGTTATGCAAGCAAGATCAACATCGGTAGCAAGGTCAATTTTGA

[0645] CCCGATCGATAAGAACCAGATCCAGCTGTTTAATCTGGAATCGAGCAAAATTGAGGTTATCCTGAAAAACGCCATTGTCT

[0646] ACAACTCCATGTACGAGAATTTCTCCACCAGCTTCTGGATTCGCATCCCGAAATACTTCAACAGCATTAGCCTGAACAAC

[0647] GAGTATACTATCATCAACTGTATGGAGAACAACAGCGGTTGGAAGGTGTCTCTGAACTATGGTGAGATCATTTGGACCTT

[0648] GCAGGACACCCAAGAGATCAAGCAGCGCGTCGTGTTCAAGTACTCTCAAATGATCAACATTTCCGATTACATTAATCGTT

[0649] GGATCTTCGTGACCATTACGAATAACCGTCTGAATAACAGCAAGATTTACATCAATGGTCGCTTGATCGATCAGAAACCG

[0650] ATTAGCAACCTGGGTAATATCCACGCAAGCAACAACATTATGTTCAAATTGGACGGTTGCCGCGATACCCATCGTTATAT

[0651] CTGGATCAAGTATTTCAACCTGTTTGATAAAGAACTGAATGAGAAGGAGATCAAAGATTTGTATGACAACCAATCTAACA

[0652] GCGGCATTTTGAAGGACTTCTGGGGCGATTATCTGCAATACGATAAGCCGTACTATATGCTGAACCTGTATGATCCGAAC

[0653] AAATATGTGGATGTCAATAATGTGGGTATTCGTGGTTACATGTATTTGAAGGGTCCGCGTGGCAGCGTTATGACGACCAA

[0654] CATTTACCTGAACTCTAGCCTGTACCGTGGTACGAAATTCATCATTAAGAAATATGCCAGCGGCAACAAAGATAACATTG

[0655] TGCGTAATAACGATCGTGTCTACATCAACGTGGTCGTGAAGAATAAAGAGTACCGTCTGGCGACCAACGCTTCGCAGGCG

[0656] GGTGTTGAGAAAATTCTGAGCGCGTTGGAGATCCCTGATGTCGGTAATCTGAGCCAAGTCGTGGTTATGAAGAGCAAGAA

[0657] CGACCAGGGTATCACTAACAAGTGCAAGATGAACCTGCAAGACAACAATGGTAACGACATCGGCTTTATTGGTTTCCACC

[0658] AGTTCAACAATATTGCTAAACTGGTAGCGAGCAATTGGTACAATCGTCAGATTGAGCGCAGCAGCCGTACTTTGGGCTGT

[0659] AGCTGGGAGTTTATCCCGGTCGATGATGGTTGGGGCGAACGTCCGCTG

[0660] SEQ ID NO: 2 (polypeptide sequence of unmodified BoNT / A)

[0661] MPFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLNPPPEAKQVPVSYYDSTYLST

[0662] DNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGGSTIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADI

[0663] IQFECKSFGHEVLNLTRNGYGSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPN

[0664] RVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKAKSIVGTTASLQYMKNVFKEK

[0665] YLLSEDTSGKFSVDKLKFDKLYKMLTEIYTEDNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAAN

[0666] FNGQNTEINNMNFTKLKNFTGLFEFYKLLCVRGIITSKTKSLDKGYNKALNDLCIKVNNWDLFFSPSEDNFTNDLNKGEE

[0667] ITSDTNIEAAEENISLDLIQQYYLTFNFDNEPENISIENLSSDIIGQLELMPNIERFPNGKKYELDKYTMFHYLRAQEFE

[0668] HGKSRIALTNSVNEALLNPSRVYTFFSSDYVKKVNKATEAAMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPA

[0669] LNIGNMLYKDDFVGALIFSGAVILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALSKRNEKWDEVYKYIVTNWLAK

[0670] VNTQIDLIRKKMKEALENQAEATKAIINYQYNQYTEEEKNNINFNIDDLSSKLNESINKAMININKFLNQCSVSYLMNSM

[0671] IPYGVKRLEDFDASLKDALLKYIYDNRGTLIGQVDRLKDKVNNTLSTDIPFQLSKYVDNQRLLSTFTEYIKNIINTSILN

[0672] LRYESNHLIDLSRYASKINIGSKVNFDPIDKNQIQLFNLESSKIEVILKNAIVYNSMYENFSTSFWIRIPKYFNSISLNN

[0673] EYTIINCMENNSGWKVSLNYGEIIWTLQDTQEIKQRVVFKYSQMINISDYINRWIFVTITNNRLNNSKIYINGRLIDQKP

[0674] ISNLGNIHASNNIMFKLDGCRDTHRYIWIKYFNLFDKELNEKEIKDLYDNQSNSGILKDFWGDYLQYDKPYYMLNLYDPN

[0675] KYVDVNNVGIRGYMYLKGPRGSVMTTNIYLNSSLYRGTKFIIKKYASGNKDNIVRNNDRVYINVVVKNKEYRLATNASQA

[0676] GVEKILSALEIPDVGNLSQVVVMKSKNDQGITNKCKMNLQDNNGNDIGFIGFHQFNNIAKLVASNWYNRQIERSSRTLGC

[0677] SWEFIPVDDGWGERPL

[0678] SEQ ID NO: 3 (Polypeptide sequence of modified BoNT / A "Chimera 1")

[0679] MPFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLN

[0680] PPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGG

[0681] STIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNGY

[0682] GSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPN

[0683] RVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKA

[0684] KSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIYTEDNFVKFFKV

[0685] LNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTKLKNFT

[0686] GLFEFYKLLCVRGIITSKTKSLDKGYNKALNDLCIKVNNWDLFFSPSEDNFTNDLNKGEE

[0687] ITSDTNIEAAEENISLDLIQQYYLTFNFDNEPENISIENLSSDIIGQLELMPNIERFPNG

[0688] KKYELDKYTMFHYLRAQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSDYVKKVNKATEA

[0689] AMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYKDDFVGALIFSG

[0690] AVILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALSKRNEKWDEVYKYIVTNWLAK

[0691] VNTQIDLIRKKMKEALENQAEATKAIINYQYNQYTEEEKNNINFNIDDLSSKLNESINKA

[0692] MININKFLNQCSVSYLMNSMIPYGVKRLEDFDASLKDALLKYIYDNRGTLIGQVDRLKDK

[0693] VNNTLSTDIPFQLSKYVDNQRLLSTFTEYIKSEILNNIILNLRYKDNNLIDLSGYGAKVE

[0694] VYDGVELNDKNQFKLTSSANSKIRVTQNQNIIFNSVFLDFSVSFWIRIPKYKNDGIQNYI

[0695] HNEYTIINCMKNNSGWKISIRGNRIIWTLIDINGKTKSVFFEYNIREDISEYINRWFFVT

[0696] ITNNLNNAKIYINGKLESNTDIKDIREVIANGEIIFKLDGDIDRTQFIWMKYFSIFNTEL

[0697] SQSNIEERYKIQSYSEYLKDFWGNPLMYNKEYYMFNAGNKNSYIKLKKDSPVGEILTRSK

[0698] YNQNSKYINYRDLYIGEKFIIRRKSNSQSINDDIVRKEDYIYLDFFNLNQEWRVYTYKYF

[0699] KKEEMKLFLAPIYDSDEFYNTIQIKEYDEQPTYSCQLLFKKDEESTDEIGLIGIHRFYES

[0700] GIVFEEYKDYFCISKWYLKEVKRKPYNLKLGCNWQFIPKDEGWTEHHHHHHHHHH

[0701] SEQ ID NO: 4 (Polypeptide sequence of modified BoNT / A "Chimera 2")

[0702] MPFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLN

[0703] PPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGG

[0704] STIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNGY

[0705] GSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPN

[0706] RVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKA

[0707] KSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIYTEDNFVKFFKV

[0708] LNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTKLKNFT

[0709] GLFEFYKLLCVRGIITSKTKSLDKGYNKALNDLCIKVNNWDLFFSPSEDNFTNDLNKGEE

[0710] ITSDTNIEAAEENISLDLIQQYYLTFNFDNEPENISIENLSSDIIGQLELMPNIERFPNG

[0711] KKYELDKYTMFHYLRAQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSDYVKKVNKATEA

[0712] AMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYKDDFVGALIFSG

[0713] AVILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALSKRNEKWDEVYKYIVTNWLAK

[0714] VNTQIDLIRKKMKEALENQAEATKAIINYQYNQYTEEEKNNINFNIDDLSSKLNESINKA

[0715] MININKFLNQCSVSYLMNSMIPYGVKRLEDFDASLKDALLKYIYDNRGTLIGQVDRLKDK

[0716] VNNTLSTDIPFQLSKYVDNQRLLSTFTEYIKNIIELGGGGSELSEILNNIILNLRYKDNN

[0717] LIDLSGYGAKVEVYDGVELNDKNQFKLTSSANSKIRVTQNQNIIFNSVFLDFSVSFWIRI

[0718] PKYKNDGIQNYIHNEYTIINCMKNNSGWKISIRGNRIIWTLIDINGKTKSVFFEYNIRED

[0719] ISEYINRWFFVTITNNLNNAKIYINGKLESNTDIKDIREVIANGEIIFKLDGDIDRTQFI

[0720] WMKYFSIFNTELSQSNIEERYKIQSYSEYLKDFWGNPLMYNKEYYMFNAGNKNSYIKLKK

[0721] DSPVGEILTRSKYNQNSKYINYRDLYIGEKFIIRRKSNSQSINDDIVRKEDYIYLDFFNL

[0722] NQEWRVYTYKYFKKEEMKLFLAPIYDSDEFYNTIQIKEYDEQPTYSCQLLFKKDEESTDE

[0723] IGLIGIHRFYESGIVFEEYKDYFCISKWYLKEVKRKPYNLKLGCNWQFIPKDEGWTEHHH

[0724] HHHHHHH

[0725] SEQ ID NO:5 (Polypeptide sequence of modified BoNT / A "Chimera 3A")

[0726] MPFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLN

[0727] PPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGG

[0728] STIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNGY

[0729] GSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPN

[0730] RVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKA

[0731] KSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIYTEDNFVKFFKV

[0732] LNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTKLKNFT

[0733] GLFEFYKLLCVRGIITSKTKSLDKGYNKALNDLCIKVNNWDLFFSPSEDNFTNDLNKGEE

[0734] ITSDTNIEAAEENISLDLIQQYYLTFNFDNEPENISIENLSSDIIGQLELMPNIERFPNG

[0735] KKYELDKYTMFHYLRAQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSDYVKKVNKATEA

[0736] AMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYKDDFVGALIFSG

[0737] AVILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALSKRNEKWDEVYKYIVTNWLAK

[0738] VNTQIDLIRKKMKEALENQAEATKAIINYQYNQYTEEEKNNINFNIDDLSSKLNESINKA

[0739] MININKFLNQCSVSYLMNSMIPYGVKRLEDFDASLKDALLKYIYDNRGTLIGQVDRLKDK

[0740] VNNTLSTDIPFQLSKYVDNQRLLSTFTEYIKNILNNIILNLRYKDNNLIDLSGYGAKVEV

[0741] YDGVELNDKNQFKLTSSANSKIRVTQNQNIIFNSVFLDFSVSFWIRIPKYKNDGIQNYIH

[0742] NEYTIINCMKNNSGWKISIRGNRIIWTLIDINGKTKSVFFEYNIREDISEYINRWFFVTI

[0743] TNNLNNAKIYINGKLESNTDIKDIREVIANGEIIFKLDGDIDRTQFIWMKYFSIFNTELS

[0744] QSNIEERYKIQSYSEYLKDFWGNPLMYNKEYYMFNAGNKNSYIKLKKDSPVGEILTRSKY

[0745] NQNSKYINYRDLYIGEKFIIRRKSNSQSINDDIVRKEDYIYLDFFNLNQEWRVYTYKYFK

[0746] KEEMKLFLAPIYDSDEFYNTIQIKEYDEQPTYSCQLLFKKDEESTDEIGLIGIHRFYESG

[0747] IVFEEYKDYFCISKWYLKEVKRKPYNLKLGCNWQFIPKDEGWTEHHHHHHHHHH

[0748] SEQ ID NO:6 (Polypeptide sequence of modified BoNT / A "Chimera 3B")

[0749] MPFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLNPPPEAKQVPVSYYDSTYLST

[0750] DNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGGSTIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADI

[0751] IQFECKSFGHEVLNLTRNGYGSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPN

[0752] RVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKAKSIVGTTASLQYMKNVFKEK

[0753] YLLSEDTSGKFSVDKLKFDKLYKMLTEIYTEDNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAAN

[0754] FNGQNTEINNMNFTKLKNFTGLFEFYKLLCVRGIITSKTKSLDKGYNKALNDLCIKVNNWDLFFSPSEDNFTNDLNKGEE

[0755] ITSDTNIEAAEENISLDLIQQYYLTFNFDNEPENISIENLSSDIIGQLELMPNIERFPNGKKYELDKYTMFHYLRAQEFE

[0756] HGKSRIALTNSVNEALLNPSRVYTFFSSDYVKKVNKATEAAMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPA

[0757] LNIGNMLYKDDFVGALIFSGAVILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALSKRNEKWDEVYKYIVTNWLAK

[0758] VNTQIDLIRKKMKEALENQAEATKAIINYQYNQYTEEEKNNINFNIDDLSSKLNESINKAMININKFLNQCSVSYLMNSM

[0759] IPYGVKRLEDFDASLKDALLKYIYDNRGTLIGQVDRLKDKVNNTLSTDIPFQLSKYVDNQRLLSTFTEYIKNILNNIILN

[0760] LRYKDNNLIDLSGYGAKVEVYDGVELNDKNQFKLTSSANSKIRVTQNQNIIFNSVFLDFSVSFWIRIPKYKNDGIQNYIH

[0761] NEYTIINCMKNNSGWKISIRGNRIIWTLIDINGKTKSVFFEYNIREDISEYINRWFFVTITNNLNNAKIYINGKLESNTD

[0762] IKDIREVIANGEIIFKLDGDIDRTQFIWMKYFSIFNTELSQSNIEERYKIQSYSEYLKDFWGNPLMYNKEYYMFNAGNKN

[0763] SYIKLKKDSPVGEILTRSKYNQNSKYINYRDLYIGEKFIIRRKSNSQSINDDIVRKEDYIYLDFFNLNQEWRVYTYKYFK

[0764] KEEMKLFLAPIYDSDEFYNTIQIKEYDEQPTYSCQLLFKKDEESTDEIGLIGIHRFYESGIVFEEYKDYFCISKWYLKEV

[0765] KRKPYNLKLGCNWQFIPKDEGWTE

[0766] SEQ ID NO:7 (Polypeptide sequence of modified BoNT / A "Chimera 3C")

[0767] MPFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLN

[0768] PPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGG

[0769] STIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNGY

[0770] GSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPN

[0771] RVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKA

[0772] KSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIYTEDNFVKFFKV

[0773] LNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTKLKNFT

[0774] GLFEFYKLLCVRGIITSKTKSLDKGYNKALNDLCIKVNNWDLFFSPSEDNFTNDLNKGEE

[0775] ITSDTNIEAAEENISLDLIQQYYLTFNFDNEPENISIENLSSDIIGQLELMPNIERFPNG

[0776] KKYELDKYTMFHYLRAQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSDYVKKVNKATEA

[0777] AMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYKDDFVGALIFSG

[0778] AVILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALSKRNEKWDEVYKYIVTNWLAK

[0779] VNTQIDLIRKKMKEALENQAEATKAIINYQYNQYTEEEKNNINFNIDDLSSKLNESINKA

[0780] MININKFLNQCSVSYLMNSMIPYGVKRLEDFDASLKDALLKYIYDNRGTLIGQVDRLKDK

[0781] VNNTLSTDIPFQLSKYVDNQRLLSTFTEYIKNILNNIILNLRYKDNNLIDLSGYGAKVEV

[0782] YDGVELNDKNQFKLTSSANSKIRVTQNQNIIFNSVFLDFSVSFWIRIPKYKNDGIQNYIH

[0783] NEYTIINCMKNNSGWKISIRGNRIIWTLIDINGKTKSVFFEYNIREDISEYINRWFFVTI

[0784] TNNLNNAKIYINGKLESNTDIKDIREVIANGEIIFKLDGDIDRTQFIWMKYFSIFNTELS

[0785] QSNIEERYKIQSYSEYLKDFWGNPLMYNKEYYMFNAGNKNSYIKLKKDSPVGEILTRSKY

[0786] NQNSKYINYRDLYIGEKFIIRRKSNSQSINDDIVRKEDYIYLDFFNLNQEWRVYTYKYFK

[0787] KEEEKLFLAPISDSDEFYNTIQIKEYDEQPTYSCQLLFKKDEESTDEIGLIGIHRFYESG

[0788] IVFEEYKDYFCISKWYLKEVKRKPYNLKLGCNWQFIPKDEGWTE

[0789] SEQ ID NO: 8 (BoNT / B polypeptide sequence)

[0790] MPVTINNFNYNDPIDNNNIIMMEPPFARGTGRYYKAFKITDRIWIIPERYTFGYKPEDFN

[0791] KSSGIFNRDVCEYYDPDYLNTNDKKNIFLQTMIKLFNRIKSKPLGEKLLEMIINGIPYLG

[0792] DRRVPLEEFNTNIASVTVNKLISNPGEVERKKGIFANLIIFGPGPVLNENETIDIGIQNH

[0793] FASREGFGGIMQMKFCPEYVSVFNNVQENKGASIFNRRGYFSDPALILMHELIHVLHGLY

[0794] GIKVDDLPIVPNEKKFFMQSTDAIQAEELYTFGGQDPSIITPSTDKSIYDKVLQNFRGIV

[0795] DRNLKVLVCISDPNININIYKNKFKDKYKFVEDSEGKYSIDVESFDKLYKSLMFGFTETN

[0796] IAENYKIKTRASYFSDSLPPVKIKNLLDNEIYTIEEGFNISDKDMEKEYRGQNKAINKQA

[0797] YEEISKEHLAVYKIQMCKSVKAPGICIDVDNEDLFFIADKNSFSDDLSKNERIEYNTQSN

[0798] YIENDFPINELILDTDLISKIELPSENTESLTDFNVDVPVYEKQPAIKKIFTDENTIFQY

[0799] LYSQTFPLDIRDISLTSSFDDALLFSNKVYSFFSMDYIKTANKVVEAGLFAGWVKQIVND

[0800] FVIEANKSNTMDKIADISLIVPYIGLALNVGNETAKGNFENAFEIAGASILLEFIPELLI

[0801] PVVGAFLLESYIDNKNKIIKTIDNALTKRNEKWSDMYGLIVAQWLSTVNTQFYTIKEGMY

[0802] KALNYQAQALEEIIKYRYNIYSEKEKSNINIDFNDINSKLNEGINQAIDNINNFINGCSV

[0803] SYLMKKMIPLAVEKLLDFDNTLKKNLLNYIDENKLYLIGSAEYEKSKVNKYLKTIMPFDL

[0804] SIYTNDTILIEMFNKYNSEILNNIILNLRYKDNNLIDLSGYGAKVEVYDGVELNDKNQFK

[0805] LTSSANSKIRVTQNQNIIFNSVFLDFSVSFWIRIPKYKNDGIQNYIHNEYTIINCMKNNS

[0806] GWKISIRGNRIIWTLIDINGKTKSVFFEYNIREDISEYINRWFFVTITNNLNNAKIYING

[0807] KLESNTDIKDIREVIANGEIIFKLDGDIDRTQFIWMKYFSIFNTELSQSNIEERYKIQSY

[0808] SEYLKDFWGNPLMYNKEYYMFNAGNKNSYIKLKKDSPVGEILTRSKYNQNSKYINYRDLY

[0809] IGEKFIIRRKSNSQSINDDIVRKEDYIYLDFFNLNQEWRVYTYKYFKKEEEKLFLAPISD

[0810] SDEFYNTIQIKEYDEQPTYSCQLLFKKDEESTDEIGLIGIHRFYESGIVFEEYKDYFCIS

[0811] KWYLKEVKRKPYNLKLGCNWQFIPKDEGWTE

[0812] SEQ ID NO:9—C-terminal L chain fragment

[0813] TKSLDKGYNK

[0814] SEQ ID NO:10—C-terminal L chain fragment 2

[0815] SLDKGYNK

[0816] SEQ ID NO:11-Double-stranded L chain 1

[0817] PFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLNPPPEAKQVPVSYYDSTYLSTD

[0818] NEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGGSTIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADII

[0819] QFECKSFGHEVLNLTRNGYGSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPNR

[0820] VFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKAKSIVGTTASLQYMKNVFKEKY

[0821] LLSEDTSGKFSVDKLKFDKLYKMLTEIYTEDNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANF

[0822] NGQNTEINNMNFTKLKNFTGLFEFYKLLCVRGIITSK

[0823] SEQ ID NO:12-Double-stranded L chain 2

[0824] PFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLNPPPEAKQVPVSYYDSTYLSTD

[0825] NEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGGSTIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADII

[0826] QFECKSFGHEVLNLTRNGYGSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPNR

[0827] VFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKAKSIVGTTASLQYMKNVFKEKY

[0828] LLSEDTSGKFSVDKLKFDKLYKMLTEIYTEDNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANF

[0829] NGQNTEINNMNFTKLKNFTGLFEFYKLLCVRGIITSKTK

[0830] SEQ ID NO:13-Double-stranded H chain

[0831] ALNDLCIKVNNWDLFFSPSEDNFTNDLNKGEEITSDTNIEAAEENISLDLIQQYYLTFNFDNEPENISIENLSSDIIGQL

[0832] ELMPNIERFPNGKKYELDKYTMFHYLRAQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSDYVKKVNKATEAAMFLGWVE

[0833] QLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYKDDFVGALIFSGAVILLEFIPEIAIPVLGTFALVSYIANK

[0834] VLTVQTIDNALSKRNEKWDEVYKYIVTNWLAKVNTQIDLIRKKMKEALENQAEATKAIINYQYNQYTEEEKNNINFNIDD

[0835] LSSKLNESINKAMININKFLNQCSVSYLMNSMIPYGVKRLEDFDASLKDALLKYIYDNRGTLIGQVDRLKDKVNNTLSTD

[0836] IPFQLSKYVDNQRLLSTFTEYIKNILNNIILNLRYKDNNLIDLSGYGAKVEVYDGVELNDKNQFKLTSSANSKIRVTQNQ

[0837] NIIFNSSVFLDFSVSFWIRIPKYKNDGIQNYIHNEYTIINCMKNNSGWKISIRGNRIIWTLIDINGKTKSVFFEYNIREDI

[0838] SEYINRWFFVTITNNLNNAKIYINGKLESNTDIKDIREVIANGEIIFKLDGDIDRTQFIWMKYFSIFNTELSQSNIEERY

[0839] KIQSYSEYLKDFWGNPLMYNKEYYMFNAGNKNSYIKLKKDSPVGEILTRSKYNQNSKYINYRDLYIGEKFIIRRKSNSQS

[0840] INDDIVRKEDYIYLDFFNLNQEWRVYTYKYFKKEEMKLFLAPIYDSDEFYNTIQIKEYDEQPTYSCQLLFKKDEESTDEI

[0841] GLIGIHRFYESGIVFEEYKDYFCISKWYLKEVKRKPYNLKLGCNWQFIPKDEGWTE

[0842] Example

[0843] Example 1

[0844] Cloning, expression and purification of BoNT / AB chimeras

[0845] The nucleotide sequences of BoNT / AB chimeric constructs 1, 2, 3A, 3B, and 3C (SEQ ID NOs: 3 to 7, respectively) were constructed from DNA encoding the parental serotype molecules and appropriate oligonucleotides using standard molecular biology techniques. They were then cloned into 10 -tagged pJ401 expression vector and transformed into BLR(DE3) E. coli cells for overexpression. These cells were grown at 37°C and 225 RPM shaking in 2L baffled Erlenmeyer flasks containing 1L of modified Terrific Broth (mTB) supplemented with appropriate antibiotics. Once A 600 Once >0.5 was reached, the incubator temperature was lowered to 16°C and then one hour later, induced with 1 mM IPTG at 225 RPM shaking for 20 hours to express the recombinant BoNT / AB construct.

[0846] The harvested cells were lysed by sonication and clarified by centrifugation at 4500 RPM for 1 hour at 4°C, and the recombinant BoNT / AB chimeric molecules were then extracted in ammonium sulfate and purified by standard fast protein liquid chromatography (FPLC) techniques. This included the use of a hydrophobic interaction resin for capture and an anion exchange resin for an intermediate purification step. The partially purified molecules were then proteolytically cleaved with endoproteinase Lys-C to generate an active duplex. Further purification was performed with a second hydrophobic interaction resin to obtain the final BoNT / AB chimera.

[0847] For the case with a ten-histidine tag (H 10 ) of the BoNT / AB chimeric molecules (chimeras 1, 2, and 3A), and the capture step used immobilized nickel resin instead of hydrophobic interaction resin.

[0848] The sequence of each chimera is shown in Table 1.

[0849]

[0850] Table 1 - Chimeric BoNT / AB Constructs

[0851] Example 2

[0852] Comparison of BoNT / AB chimeras 1, 2, and 3A

[0853] The C-terminal His 10 Tag and E1191M / S1199Y double mutant BoNT / AB chimeras 1, 2, and 3A ( Figure 3 ), and tested for functional activity.

[0854] SNAP-25 cleavage assay in rat spinal cord neurons

[0855] Primary cultures of rat spinal cord neurons (SCN) were prepared and cultured in 96-well tissue culture plates for 3 weeks (as described in Masuyer et al., 2011, J. Struct. Biol. Structure and activity of a functional derivative of Clostridium botulinum neurotoxin B; and in Chaddock et al., 2002, Protein Expr. Purif. Expression and purification of catalytically active, non-toxic endopeptidase derivatives of Clostridiumbotulinum toxin type A). Serial dilutions of BoNT / AB were prepared in SCN feed medium. Growth medium from wells to be treated was collected and filtered (0.2 μm filter). 125 μL of filtered medium was added back to each test well. 125 μL of diluted toxin was then added to the plate (in triplicate). Treated cells were incubated at 37°C, 10% CO2 for 24 ± 1 h).

[0856] Analysis of BoNT activity using SNAP-25 cleavage assay

[0857] After treatment, BoNT was removed and the cells were washed once with PBS (Gibco, UK). The cells were lysed in 1×NuPAGE lysis buffer (Life Technologies) supplemented with 0.1 M dithiothreitol (DTT) and 250 units / mL benzonase (Sigma). The lysate proteins were separated by SDS-PAGE and transferred to a nitrocellulose membrane. The membrane was probed with a SNAP-25-specific primary antibody (Sigma # S9684), which recognizes uncut SNAP-25 as well as SNAP-25 cleaved by BoNT / A endopeptidase. The secondary antibody used was HRP-conjugated anti-rabbit IgG (Sigma # A6154). The bands were detected by enhanced chemiluminescence and imaged using pXi6 Access (Synoptics, UK). The band intensity was determined using GeneTools software (Syngene, Cambridge, UK), and the percentage of SNAP-25 cleavage at each BoNT concentration was calculated. Data were fitted to a 4-parameter logistic equation and pEC was calculated using GraphPad Prism version 6 (GraphPad) 50 .

[0858] Table 2 below provides the pEC values ​​of chimeras 1, 2, and 3A as determined in the rat SCN SNAP-25 cleavage assay. 50 These results indicate that the three BoNT / AB chimeras retain the ability to enter rat spinal cord neurons and cleave their target substrates. However, chimera 3A was more potent than chimeras 1 and 2 in this assay (see also Figure 4 ).

[0859] <![CDATA[pEC 50 ±SEM]]> Chimera 1 12.42±0.04 Chimera 2 12.57±0.01 Chimera 3A 12.89±0.04

[0860] Table 2. pEC 50 value.

[0861] Digital abduction score (DAS) test

[0862] The method for measuring the activity of BoNT / AB chimeras 1, 2, and 3A in the DAS assay is based on the startle toe extension reflex of mice when they are briefly suspended by their tails. This reflex, scored as the digital abduction score (DAS), is inhibited following injection of BoNT into the gastrocnemius-soleus muscles of the hind paw. The tails of mice are briefly suspended to elicit a characteristic startle response in which the animals extend their hind limbs and abduct their hind toes. (Aoki et al., 1999, Eur. J. Neurol.; 6(Suppl. 4) S3-S10).

[0863] On the day of injection, mice received anesthesia with 3% isoflurane in oxygen in an induction chamber.Each mouse received an intramuscular injection of BoNT / AB chimeras or vehicle (phosphate buffered saline containing 0.2% gelatin) in the gastrocnemius-soleus muscle of the right hind paw.

[0864] After injection of the neurotoxin, the degree of toe abduction was scored on a scale of 0 to 4, where 0 = normal and 4 = maximal reduction in toe abduction and leg extension. The ED was determined using the mean of the maximal effect of each dose in a nonlinearly adjusted analysis. 50 The mathematical model used was a 4-parameter logistic model.

[0865] DAS was performed every 2 hours on the first day after drug administration and 3 times a day thereafter for 4 days.

[0866] Figure 5 Fitted curves for chimeras 1, 2, and 3A (SEQ ID NOs: 3, 4, and 5 converted to double-stranded form, respectively) are shown. The chimera 3A curve is shifted to the left, meaning that a lower dose of chimera 3A achieved a similar DAS response compared to chimeras 1 and 2, thus indicating that chimera 3A is more effective than the other chimeras in the mouse DAS assay; see also the table below (Table 3), which provides the calculated ED for each chimera 50 values ​​and the dose that resulted in DAS 4 (the highest score).

[0867] Table 3 below provides the ED values ​​of unmodified recombinant BoNT / A1 (rBoNT / A1 - SEQ ID NO: 2 converted to a double-chain form) and chimeras 1, 2, and 3A as determined in the mouse DAS assay. 50 and DAS 4 doses. These results indicate that among the three chimeras, chimera 3A has the highest in vivo efficacy in inducing muscle weakness. Figure 5 The studies shown in and Table 3 were performed in mice obtained from Charles River Laboratories.

[0868]

[0869] Table 3. ED 50 value.

[0870] Example 3

[0871] Comparison of BoNT / AB chimeras 3B and 3C with unmodified BoNT / A1

[0872] Untagged BoNT / AB chimeras 3B and 3C (with and without the E1191M / S1199Y double mutation, respectively) (SEQ ID NOs: 6 and 7) were purified as described in Example 1 ( Figure 6 ), and the functional activity was tested using unmodified BoNT / A (SEQ ID NO: 2 converted into a double-chain form) as a reference.

[0873] Human pluripotent stem cell SNAP-25 cleavage assay

[0874] Cryopreserved PERI.4U cells were purchased from Axiogenesis (Cologne, Germany). Thawing and plating of cells were performed according to the manufacturer's recommendations. Briefly, cryovials containing cells were thawed in a 37°C water bath for 2 min. After gentle resuspension, cells were transferred to 50 mL tubes. 1 mL of 4% paraformaldehyde was used as provided by the manufacturer. Thaw medium to wash the cryovials, then transfer the medium dropwise to the cell suspension in the 50 mL tube, and then transfer 2 mL Thaw medium was added dropwise to the 50 mL tube. The cells were then counted using a hemocytometer. Afterwards, another 6 mL Add thawing medium to the cell suspension. Centrifuge at 260 × g (e.g., 1,100 RPM) for 6 minutes at room temperature to obtain a cell pellet. Then resuspend the cells in the manufacturer's complete The cells were cultured at 50,000 to 150,000 cells / cm 2The cells were seeded at a density of 1.5 ng / mL on cell culture plates coated with poly-L-ornithine and laminin. The cells were cultured at 37°C in a humidified CO2 atmosphere, and the culture medium was completely replaced every 2-3 days during the culture period.

[0875] For toxin treatment, Prepare serial dilutions of BoNT in culture medium. Collect the culture medium from the wells to be treated and filter (0.2 μm filter). Add 125 μL of filtered culture medium back to each test well. Then add 125 μL of diluted toxin to the plate (in triplicate). Incubate the treated cells at 37°C, 10% CO2 for 48 ± 1 h).

[0876] Analysis of BoNT activity using SNAP-25 cleavage assay

[0877] After treatment, BoNT was removed and the cells were washed once with PBS (Gibco, UK). The cells were lysed in 1×NuPAGE lysis buffer (Life Technologies) supplemented with 0.1 M dithiothreitol (DTT) and 250 units / mL benzonase (Sigma). The lysate proteins were separated by SDS-PAGE and transferred to a nitrocellulose membrane. The membrane was probed with a SNAP-25-specific primary antibody (Sigma # S9684), which recognizes uncut SNAP-25 as well as SNAP-25 cleaved by BoNT / A endopeptidase. The secondary antibody used was HRP-conjugated anti-rabbit IgG (Sigma # A6154). The bands were detected by enhanced chemiluminescence and imaged using pXi6 Access (Synoptics, UK). The band intensity was determined using GeneTools software (Syngene, Cambridge, UK), and the percentage of SNAP-25 cleavage at each BoNT concentration was calculated. Data were fitted to a 4-parameter logistic equation and pEC was calculated using GraphPad Prism version 6 (GraphPad) 50 .

[0878] Figure 7 The chimeras 3B and 3C showed a stronger potency than rBoNT / A1 in cleaving SNAP-25 in induced human pluripotent stem cells, but the former was significantly more potent. This could be explained by the double mutation, which increases the affinity of chimera 3B for the human synaptotagmin II protein receptor present in these cells ( Figure 7 , Table 4).

[0879] <![CDATA[pEC 50 ±SEM]]> rBoNT / A1 10.21±0.05 Chimera 3B 12.38±0.06 Chimera 3C 10.72±0.08

[0880] Table 4. pEC 50 value.

[0881] Digital Abduction Score (DAS) Test – Safety Ratio

[0882] The method for measuring BoNT activity in the DAS assay is based on the startle toe extension reflex of mice when they are briefly suspended by their tails. This reflex is scored as the digital abduction score (DAS) and is inhibited after BoNT is applied to the gastrocnemius-soleus muscles of the hind paw. The tails of mice are briefly suspended to elicit a characteristic startle response in which the animals extend their hind limbs and abduct their hind toes. (Aoki et al., 1999, Eur. J. Neurol.; 6(Suppl. 4) S3-S10).

[0883] On the day of injection, mice received anesthesia with 3% isoflurane in oxygen in an induction chamber.Each mouse received an intramuscular injection of BoNT or vehicle (phosphate buffered saline containing 0.2% gelatin) in the gastrocnemius-soleus muscle of the right hind paw.

[0884] After injection of the neurotoxin, the degree of toe abduction was scored on a scale of 0 to 4, where 0 = normal and 4 = maximal reduction in toe abduction and leg extension. The ED was determined using the mean of the maximal effect of each dose in a nonlinearly adjusted analysis. 50 The mathematical model used was a 4-parameter logistic model.

[0885] DAS was performed every 2 hours on the first day after dosing; thereafter, DAS was performed 3 times a day for 4 days for all doses. Animals injected with vehicle and the lowest dose group that induced a DAS of 4 four days before injection were subsequently monitored until muscle weakness fully recovered to a DAS of 0 (no muscle weakness was observed).

[0886] To calculate the safety ratio, all animals were weighed one day before toxin injection (D0) and thereafter once daily throughout the study. The mean body weight, standard deviation, and standard error mean were calculated for each dose group daily. To obtain the safety ratio (-10% ΔBW / ED 50 ), the dose at which the mean body weight of a dose group at any time during the study was less than 10% of the mean body weight of the same dose group at D0 divided by the ED value of the BoNT being studied 50 The lethal dose was defined as the dose at which one or more animals in the dose group died.

[0887] Figure 8 The duration of muscle weakness in the mouse toe abduction score test is shown over time for unmodified BoNT / A, chimera 3B, and chimera 3C (SEQ ID NOs: 2, 6, and 7 converted to double-chain form), indicating that the chimera has a longer duration of action.

[0888] Table 5 below provides the ED values ​​of rBoNT / A1 and chimeras 3B and 3C as determined in the mouse DAS assay.50 The table also provides the total duration of action of the DAS 4 dose until muscle weakness is completely restored to DAS 0 (no muscle weakness is observed). In addition, the table also shows the mouse lethal dose and safety ratio (-10% ΔBW / ED 50 ), as defined above. Compared with rBoNT / A1, chimeras 3B and 3C had a longer duration of action, a better safety profile, and a higher lethal dose. Figure 8 The studies shown in and Table 5 were performed in mice obtained from the Janvier laboratory.

[0889]

[0890] Table 5. DAS and safety ratios of BoNT / AB chimeras.

[0891] Example 4

[0892] Further characterization of the BoNT / AB chimera (SEQ ID NO: 6 converted to double-chain form)

[0893] The BoNT / AB chimera SEQ ID NO:6 converted into a double-chain form was expressed in mouse LD 50 The result of the test in the experiment was 1.202 ng / kg. Therefore, in this test, 1 unit of SEQ ID NO: 6 corresponds to 24.04 pg.

[0894] Additionally, the BoNT / AB chimeras were tested in the rat DAS assay to determine The results are listed in the following Table 6:

[0895]

[0896] Table 6. Duration of action.

[0897] In conclusion, the duration of action of BoNT / AB is Much higher.

[0898] Example 5

[0899] Unit dosage of modified BoNT / A (SEQ ID NO: 6 converted into a two-chain form) for treating limb spasticity calculate

[0900] Taking into account preclinical pharmacology data, a suitable unit dose range (UD) for administration of modified BoNT / A in humans has been calculated.

[0901] For SEQ ID NO:6, the calculated DAS ED 50 It is 13pg / kg. 50It is considered to be the minimum pharmacologically active dose, which is about 300 times lower than the no observed adverse effect level (NOAEL) of 4 ng / kg in the same animal species. The ED of SEQ ID NO: 6 at 13 pg / kg in rats 50 This corresponds to a dose of 0.8 ng for a human of 60 kg body weight.

[0902] Therefore, the lower limit of the unit dose was selected as 1000 pg. The upper limit of the unit dose was selected as 16,000 pg, which is lower than the NOAEL of 4 ng / kg from two non-clinical safety species (rat and monkey) converted to a human dose of 60 kg body weight.

[0903] In view of the improved safety, the maximum total dose for treating limb spasticity was set at 240,000 pg, which was derived from the NOAEL of 4 ng / kg in two nonclinical safety species (rat and monkey) which translates to a human dose of 60 kg body weight.

[0904] Advantageously, the modified BoNT / A (SEQ ID NO: 6) can be injected into more muscles when treating limb spasticity before the maximum dose is reached. This is a significant and advantageous discovery that improves the treatment of limb spasticity while providing clinicians with a wider range of treatment options. For the first time, it also provides the option of being able to treat additional large muscles, such as those in the shoulder, while also treating the elbow, forearm and / or wrist within the maximum dose.

[0905] Example 6

[0906] Methods of administering modified BoNT / A (SEQ ID NO: 6 converted into a double-chain form) to treat upper limb spasticity in adults case

[0907] The modified BoNT / A is supplied as a lyophilized powder in vials, each containing 36 ng of modified BoNT / A. The lyophilized powder is reconstituted. The unit dose (UD) is 1000-16,000 pg (42-666 units [measured by mouse LD 50 Measurement]).

[0908] For the treatment of upper limb spasticity in adults, the following dosing schedule is given by intramuscular injection (Table 7):

[0909]

[0910] Table 7. Dosing regimen.

[0911] The maximum total dose administered is 15×UD. This corresponds to 240,000 pg / 9,990 units. Therefore, clinicians are able to tailor treatment to their patients, knowing that 15×UD can be administered without concern for toxicity, allowing them to treat other muscles in their patients (including the shoulder), and / or ensuring that each muscle receives an effective dose of the drug.

[0912] Example 7

[0913] Methods of administering modified BoNT / A (SEQ ID NO: 6 converted into a double-chain form) to treat lower limb spasticity in adults case

[0914] The modified BoNT / A is supplied as a lyophilized powder in vials, each containing 36 ng of modified BoNT / A. The lyophilized powder is reconstituted.

[0915] The unit dose (UD) is 1000-16,000 pg (42-666 units).

[0916] For the treatment of lower limb spasticity in adults, the following dosing regimen is given by intramuscular injection (Table 8):

[0917] Injected muscle dose Gastrocnemius (medial head) 1×UD Gastrocnemius (lateral head) 1×UD Soleus 3×UD Tibialis posterior muscle 2×UD Flexor digitorum longus 1×UD Flexor pollicis longus 1× or 2×UD

[0918] Table 8. Dosing regimen.

[0919] The maximum total dose administered is 15x UD. This corresponds to 240,000 pg / 9,990 units. Therefore, clinicians are able to tailor treatment to their patients, knowing that 15x UD can be administered without concern for toxicity, allowing them to treat other muscles in their patients and / or ensure that every muscle receives an effective dose of the drug.

[0920] Example 8

[0921] Safety and efficacy of modified BoNT / A (SEQ ID NO: 6 converted to double-chain form) in humans

[0922] A comprehensive Phase I / II multicenter, double-blind, randomized, Dysport- and placebo-controlled, dose-escalation and dose-finding study was conducted in individuals aged 18 to 70 years with spastic hemiparesis following stroke or traumatic brain injury (TBI) to evaluate the safety and efficacy of modified BoNT / A (SEQ ID NO:6 converted to a two-chain form) in the treatment of upper limb spasticity in adults.

[0923] This study involved human serial dose escalation in a unique patient cohort with adult upper limb spasticity with a fisting pattern. All participants were injected in the flexor digitorum profundus (FDP) and flexor digitorum superficialis (FDS). A single unit dose of modified BoNT / A, Dysport, or placebo was injected into each muscle. Different (increasing) amounts of modified BoNT / A (SEQ ID NO:6 converted to a two-chain form) were administered to the five cohorts. Cohort 1 was administered 2×1,000 pg unit doses of modified BoNT / A (i.e., 2,000 pg maximum), while cohort 5 was administered 2×16,000 pg unit doses of modified BoNT / A (i.e., 32,000 pg maximum).

[0924] The results showed that despite the extremely high dose per muscle, all modified BoNT / A unit doses tested (i.e., up to 16,000 pg unit dose) were effective for muscle paralysis, safely tolerated, and no side effects were observed. This indicates that the modified BoNT / AB does not diffuse from the injection site and highlights the excellent safety profile of the modified BoNT / AB (SEQ ID NO: 6 converted to a two-chain form). Based on these findings, it is believed that higher unit doses per muscle can be administered without side effects. In addition, due to the lack of systemic spread of the toxin, it is believed that up to 15-fold higher unit doses can be administered without safety issues.

[0925] Therefore, unit doses of 30,000 pg and 36,000 pg (each administered up to 15x) have been selected for therapeutic treatment of limb spasticity in adults. Thus, the total dose administered during the treatment period would be 450,000 pg and 540,000 pg, respectively.

[0926] Example 9

[0927] Methods of administering modified BoNT / A (SEQ ID NO: 6 converted into a double-chain form) to treat upper limb spasticity in adults case

[0928] The modified BoNT / A is supplied as a lyophilized powder in vials, each containing 36 ng of modified BoNT / A. The lyophilized powder is reconstituted.

[0929] The unit dose (UD) was 30,000 pg (1,248 units [LD 50 measured]) or 36,000 pg (1,498 units [measured by mouse LD 50 Measurement]).

[0930] For the treatment of upper limb spasticity in adults, inject intramuscularly according to the following dosing schedule (Table 9):

[0931]

[0932] Table 9. Dosing regimen.

[0933] The maximum total dose administered is 15×UD. This corresponds to 450,000 pg / 18,719 units or 540,000 pg / 22,463 units. Therefore, clinicians are able to tailor treatment to their patients, knowing that 15×UD can be administered without concern for toxicity, allowing them to treat other muscles in their patients (including the shoulder) and / or ensure that every muscle receives an effective dose of the drug.

[0934] Example 10

[0935] Methods of administering modified BoNT / A (SEQ ID NO: 6 converted into a double-chain form) to treat lower limb spasticity in adults case

[0936] The modified BoNT / A is supplied as a lyophilized powder in vials, each containing 36 ng of modified BoNT / A. The lyophilized powder is reconstituted.

[0937] The unit dose (UD) was 30,000 pg (1,248 units [LD 50 measured]) or 36,000 pg (1,498 units [measured by mouse LD 50 Measurement]).

[0938] For the treatment of lower limb spasticity in adults, the following dosing regimen is given by intramuscular injection (Table 10):

[0939] Injected muscle dose Gastrocnemius (medial head) 1×UD Gastrocnemius (lateral head) 1×UD Soleus 3×UD Tibialis posterior muscle 2×UD Flexor digitorum longus 1×UD Flexor pollicis longus 1× or 2×UD

[0940] Table 10. Dosing regimen.

[0941] The maximum total dose administered is 15x UD. This corresponds to 450,000 pg / 18,719 units or 540,000 pg / 22,463 units. Therefore, clinicians are able to tailor treatment to their patients, knowing that 15x UD can be administered without concern for toxicity, allowing them to treat other muscles in their patients and / or ensure that every muscle receives an effective dose of the drug.

[0942] Embodiment 11

[0943] Treatment of patients with upper limb spasticity in adults

[0944] Bill, 53 years old, was diagnosed with adult upper limb spasticity by his GP. Modified BoNT / A (converted to the di-chain form SEQ ID NO: 6) was administered in one or more unit doses (1 x unit dose = 36,000 pg) to the following muscles as shown below:

[0945]

[0946] The total dose administered was 540,000 pg of modified BoNT / A (SEQ ID NO: 6 converted to double-chain form).

[0947] The adult upper limb spasticity was alleviated, and due to the long-lasting effect of the modified BoNT / A, Bill did not require further treatment for more than 9 months. Thus, Bill received less frequent injections (e.g., annually) than an equivalent individual administered unmodified BoNT / A. Additionally, due to the improved safety profile of the modified BoNT / A, Bill did not experience any side effects.

[0948] Example 12

[0949] Safety and efficacy of modified BoNT / A (SEQ ID NO: 6 converted to double-chain form) in humans

[0950] A comprehensive 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 di-chain form]) in the treatment of upper limb spasticity in individuals aged 18 to 70 years with spastic hemiparesis following stroke or traumatic brain injury (TBI).

[0951] This study involved human serial dose escalation in a unique patient cohort with adult upper limb spasticity with a fisting pattern. All participants were injected in the flexor digitorum profundus (FDP) and flexor digitorum superficialis (FDS). A single unit dose of modified BoNT / A, Dysport, or placebo was injected into each muscle. Modified BoNT / A (SEQ ID NO: 6 converted to a two-chain form) was administered to individuals as follows: 15,000 pg unit dose to FDP and 15,000 pg unit dose to FDS (i.e., a total dose of 30,000 pg), 25,000 pg unit dose to FDP and 25,000 pg unit dose to FDS (i.e., a total dose of 50,000 pg), or 36,000 pg unit dose to FDP and 36,000 pg unit dose to FDS (i.e., a total dose of 72,000 pg).

[0952] The results showed that despite the extremely high dose per muscle, all modified BoNT / A unit doses tested were effective for muscle paralysis, safely tolerated, and no side effects were observed (e.g., 25,000 pg and 36,000 pg unit doses). This indicates that the modified BoNT / AB does not spread from the injection site and highlights the excellent safety profile of the modified BoNT / AB (SEQ ID NO: 6 converted to a double-chain form).

[0953] Due to the lack of systemic spread of the toxin, it is believed that up to 15 times higher unit doses can be administered without safety issues. Therefore, unit doses of 25,000 pg and 36,000 pg (each administered up to 15×) have been selected for therapeutic treatment of limb spasticity in adults. Therefore, the total dose administered during the treatment period would be 375,000 pg and 540,000 pg, respectively.

[0954] Embodiment 13

[0955] Management of patients with lower limb spasticity in adults

[0956] Ryan, 63 years old, was diagnosed with adult lower limb spasticity by his GP. Modified BoNT / A (SEQ ID NO:6 [converted to di-chain form]) was administered in one or more unit doses (1 x unit dose = 25,000 pg) to multiple of the following muscles as shown below:

[0957]

[0958] The maximum dose was 15 x UD (ie, 375,000 pg) of modified BoNT / A.

[0959] The spasticity was relieved, and due to the long-lasting nature of the modified BoNT / A, Ryan did not require further treatment for more than 8 months. As a result, Ryan received less frequent injections (e.g., annually) than an equivalent individual administered unmodified BoNT / A. Additionally, due to the improved safety profile of the modified BoNT / A, Ryan did not experience any side effects.

[0960] Embodiment 14

[0961] Treating patients with pediatric lower limb spasticity

[0962] Sharon, 7 years old, was diagnosed with pediatric leg spasticity by her GP. The modified BoNT / A (SEQ ID NO:6 [converted to di-chain form]) was administered in one or more unit doses (1 x unit dose = 12,500 pg) to the following muscles as shown below:

[0963]

[0964] The maximum dose was 15 x UD (ie, 187,500 pg) of modified BoNT / A.

[0965] The spasticity was relieved, and due to the long-lasting effect of the modified BoNT / A, Sharon did not require further treatment for more than 8 months. As a result, Sharon received less frequent injections (e.g., annually) than an equivalent individual administered unmodified BoNT / A. Additionally, due to the improved safety profile of the modified BoNT / A, Sharon did not experience any side effects.

[0966] Embodiment 15

[0967] Treating patients with pediatric lower limb spasticity

[0968] Timmy, 8 years old, was diagnosed with pediatric leg spasticity by his GP. Modified BoNT / A (SEQ ID NO:6 [converted to di-chain form]) was administered in one or more unit doses (1 x unit dose = 18,000 pg) to the following muscles as shown below:

[0969]

[0970] The maximum dose was 15 x UD (ie, 270,000 pg) of modified BoNT / A.

[0971] The spasticity was relieved, and due to the long-lasting effect of the modified BoNT / A, Timmy did not require further treatment for more than 9 months. Thus, Timmy received less frequent injections (e.g., annually) than an equivalent individual administered unmodified BoNT / A. Additionally, due to the improved safety profile of the modified BoNT / A, Timmy did not experience any side effects.

[0972] Example 16

[0973] Treatment of patients with upper limb spasticity in adults

[0974] Larry, 21 years old, was diagnosed with upper limb spasticity by his GP. Modified BoNT / A (SEQ ID NO:6 [converted to di-chain form]) was administered in one or more unit doses (1 x unit dose = 25,000 pg) to the following muscles as shown below:

[0975]

[0976]

[0977] The maximum dose was 15 x UD (ie, 375,000 pg) of modified BoNT / A.

[0978] The spasticity was relieved, and due to the long-lasting effect of the modified BoNT / A, Larry did not require further treatment for more than 9 months. Thus, Larry received less frequent injections (e.g., annually) than an equivalent individual administered unmodified BoNT / A. Additionally, due to the improved safety profile of the modified BoNT / A, Larry did not experience any side effects.

[0979] Embodiment 17

[0980] Treatment of patients with upper limb spasticity in adults

[0981] Loretta, 39 years old, was diagnosed with upper limb spasticity by her GP. Modified BoNT / A (SEQ ID NO:6 [converted to di-chain form]) was administered in one or more unit doses (1 x unit dose = 36,000 pg) to the following muscles as shown below:

[0982] Injected muscle dose Flexor digitorum superficialis (FDS) 1×UD Flexor digitorum profundus (FDP) 1×UD Flexor carpi radialis (FCR) 1×UD Flexor carpi ulnaris (FCU) 1×UD brachioradialis 1×UD Brachialis 2×UD Biceps 2×UD Triceps (long head) 2×UD Pectoralis major 2×UD Subscapularis 2×UD Latissimus dorsi 2×UD

[0983] The maximum dose was 15 x UD (ie, 540,000 pg) of modified BoNT / A.

[0984] The spasticity was relieved, and due to the long-lasting effect of the modified BoNT / A, Loretta did not require further treatment for more than 9 months. As a result, Loretta received less frequent injections (e.g., annually) than an equivalent individual administered unmodified BoNT / A. Additionally, due to the improved safety profile of the modified BoNT / A, Loretta did not experience any side effects.

[0985] Embodiment 18

[0986] Treatment of patients with pediatric upper limb spasticity

[0987] Jack, 13 years old, was diagnosed with upper limb spasticity by his GP. Modified BoNT / A (SEQ ID NO:6 [converted to di-chain form]) was administered in one or more unit doses (1 x unit dose = 12,500 pg) to the following muscles as shown below:

[0988] Injected muscle dose Flexor digitorum superficialis (FDS) 1×UD Flexor digitorum profundus (FDP) 1×UD Flexor carpi radialis (FCR) 1×UD Flexor carpi ulnaris (FCU) 1×UD brachioradialis 1×UD Brachialis 2×UD Biceps 2×UD Triceps (long head) 2×UD Pectoralis major 2×UD Subscapularis 2×UD Latissimus dorsi 2×UD

[0989] The maximum dose was 15 x UD (ie, 187,500 pg) of modified BoNT / A.

[0990] The spasticity was relieved, and due to the long-lasting effect of the modified BoNT / A, Jack did not require further treatment for more than 9 months. As a result, Jack received less frequent injections (e.g., annually) than an equivalent individual administered unmodified BoNT / A. Additionally, due to the improved safety profile of the modified BoNT / A, Jack did not exhibit any side effects.

[0991] Embodiment 19

[0992] Treatment of patients with pediatric upper limb spasticity

[0993] Julia, 11 years old, was diagnosed with upper limb spasticity by her GP. Modified BoNT / A (SEQ ID NO:6 [converted to di-chain form]) was administered in one or more unit doses (1 x unit dose = 18,000 pg) to the following muscles as shown below:

[0994] Injected muscle dose Flexor digitorum superficialis (FDS) 1×UD Flexor digitorum profundus (FDP) 1×UD Flexor carpi radialis (FCR) 1×UD Flexor carpi ulnaris (FCU) 1×UD brachioradialis 1×UD Brachialis 2×UD Biceps 2×UD Triceps (long head) 2×UD Pectoralis major 2×UD Subscapularis 2×UD Latissimus dorsi 2×UD

[0995] The maximum dose was 15 x UD (ie, 270,000 pg) of modified BoNT / A.

[0996] The spasticity was relieved, and due to the long-lasting effect of the modified BoNT / A, Julia did not require further treatment for more than 9 months. As a result, Julia received less frequent injections (e.g., annually) than an equivalent individual administered unmodified BoNT / A. Additionally, due to the improved safety profile of the modified BoNT / A, Julia did not experience any side effects.

[0997] Embodiment 20

[0998] Management of patients with lower limb spasticity in adults

[0999] Clare, 57 years old, was diagnosed with adult lower limb spasticity by her GP. Modified BoNT / A (SEQ ID NO:6 [converted to di-chain form]) was administered in one or more unit doses (1 x unit dose = 36,000 pg) to the following muscles as shown below:

[1000]

[1001] The maximum dose was 15 x UD (ie, 540,000 pg) of modified BoNT / A.

[1002] The spasticity was relieved, and due to the long-lasting nature of the modified BoNT / A, Clare did not require further treatment for more than 8 months. Thus, Clare received less frequent injections (e.g., annually) than an equivalent individual administered unmodified BoNT / A. Additionally, due to the improved safety profile of the modified BoNT / A, Clare did not experience any side effects.

[1003] Terms

[1004] 1. A modified BoNT / A for use in treating limb spasticity (limb spasticity in adults), wherein the modified BoNT / A is administered by intramuscular injection to a plurality of affected muscles of a subject,

[1005] wherein the modified BoNT / A is administered to the plurality of affected muscles at a unit dose of greater than 17,000 pg of modified BoNT / A,

[1006] The plurality of affected muscles are selected from:

[1007] Group 1, 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 policis longus, adductor pollicis, flexor policis brevis, palmaris longus, lumbricals, opponens pollicis, adductor magnus, adductor longus, adductor brevis, gracilis, medial hamstring, lateral hamstring, tensor fasciae latae, rectus femoris, vastus lateralis, vastus medialis, vastus intermedius, gluteus maximus, tibialis anterior, flexor digitorum brevis, extensor pollicis longus, flexor hallucis brevis; and

[1008] A second group, comprising: triceps brachii (long head), subscapularis, pectoralis (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 to the affected first group of muscles and / or multiple unit doses are administered to the affected second group of muscles, and wherein the total dose administered during the treatment period is up to 600,000 pg, and wherein the modified BoNT / A comprises a BoNT / A light chain and a translocation domain, and a BoNT / B receptor binding domain (H C domain).

[1009] 2. The modified BoNT / A for use according to clause 1, wherein the limb spasticity is upper limb spasticity.

[1010] 3. A modified BoNT / A for use according to clause 1 or clause 2, wherein the unit dose is greater than 17,000 pg to up to 40,000 pg (e.g., 17,001 pg to up to 40,000 pg) of the modified BoNT / A.

[1011] 4. A modified BoNT / A for use according to any one of clauses 1-3, wherein the unit dose is greater than 23,000 pg to up to 38,000 pg (eg, 23,001 pg to up to 38,000 pg) of the modified BoNT / A.

[1012] 5. A modified BoNT / A for use according to any one of clauses 1-4, wherein the unit dose is greater than 34,000 pg to up to 38,000 pg (eg, 34,001 pg to up to 38,000 pg) of the modified BoNT / A.

[1013] 6. A modified BoNT / A for use according to any one of clauses 1-5, wherein the total dose administered is 250,000 pg to 600,000 pg of modified BoNT / A (eg, 255,000 pg to 600,000 pg).

[1014] 7. A modified BoNT / A for use according to any one of clauses 1-6, wherein the total dose administered is greater than 255,000 pg to 600,000 pg of modified BoNT / A.

[1015] 8. A modified BoNT / A for use according to any one of clauses 1-7, wherein the total dose administered is 350,000 to 600,000 pg of modified BoNT / A.

[1016] 9. A modified BoNT / A for use according to any one of clauses 1 to 8, wherein the total dose administered is 500,000 to 600,000 pg of modified BoNT / A.

[1017] 10. A modified BoNT / A for use in treating limb spasticity in a child, wherein the modified BoNT / A is administered to a plurality of affected muscles of a subject by intramuscular injection,

[1018] wherein the modified BoNT / A is administered to the plurality of affected muscles at a unit dose of greater than 8,500 pg of modified BoNT / A,

[1019] The plurality of affected muscles are selected from:

[1020] Group 1, 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 policis longus, adductor pollicis, flexor policis brevis, palmaris longus, lumbricals, opponens pollicis, adductor magnus, adductor longus, adductor brevis, gracilis, medial hamstring, lateral hamstring, tensor fasciae latae, rectus femoris, vastus lateralis, vastus medialis, vastus intermedius, gluteus maximus, tibialis anterior, flexor digitorum brevis, extensor pollicis longus, flexor hallucis brevis; and

[1021] A second group, comprising: triceps brachii (long head), subscapularis, pectoralis (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 to the affected first group of muscles and / or multiple unit doses are administered to the affected second group of muscles, and wherein the total dose administered during the treatment period is up to 300,000 pg, and wherein the modified BoNT / A comprises a BoNT / A light chain and a translocation domain, and a BoNT / B receptor binding domain (H C domain).

[1022] 11. The modified BoNT / A for use according to clause 10, wherein the pediatric limb spasticity is pediatric upper limb spasticity.

[1023] 12. A modified BoNT / A for use according to clause 10 or clause 11, wherein the unit dose is greater than 8,500 pg to up to 20,000 pg (e.g., 8,501 pg to up to 20,000 pg) of the modified BoNT / A, preferably wherein the unit dose is greater than 8,500 pg to up to 18,000 pg.

[1024] 13. A modified BoNT / A for use according to any one of clauses 10-12, wherein the unit dose is greater than 11,500 pg to up to 19,000 pg (eg, 11,501 pg to up to 19,000 pg) of the modified BoNT / A.

[1025] 14. A modified BoNT / A for use according to any one of clauses 10-13, wherein the unit dose is greater than 17,000 pg to up to 19,000 pg (eg, 17,001 pg to up to 19,000 pg) of the modified BoNT / A.

[1026] 15. A modified BoNT / A for use according to any one of clauses 10-14, wherein the total dose administered is greater than 125,000 pg to 300,000 pg (eg, 127,500 pg to 300,000 pg) of the modified BoNT / A.

[1027] 16. A modified BoNT / A for use according to any one of clauses 10-15, wherein the total dose administered is greater than 127,500 pg to 300,000 pg of modified BoNT / A.

[1028] 17. A modified BoNT / A for use according to any one of clauses 10-16, wherein the total dose administered is 175,000 to 300,000 pg of modified BoNT / A.

[1029] 18. A modified BoNT / A for use according to any one of clauses 10-17, wherein the total dose administered is 250,000 to 300,000 pg of modified BoNT / A.

[1030] 19. A modified BoNT / A for use in treating limb spasticity (limb spasticity in adults), wherein the modified BoNT / A is administered to a plurality of affected muscles of a subject by intramuscular injection,

[1031] wherein the modified BoNT / A is administered to the plurality of affected muscles at a unit dose of greater than 707 units of the modified BoNT / A, and wherein 1 unit is the median lethal dose (LD50) calculated in mice. 50 ) corresponds to the amount of modified BoNT / A,

[1032] The muscles affected are selected from:

[1033] Group 1, 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 policis longus, adductor pollicis, flexor policis brevis, palmaris longus, lumbricals, opponens pollicis, adductor magnus, adductor longus, adductor brevis, gracilis, medial hamstring, lateral hamstring, tensor fasciae latae, rectus femoris, vastus lateralis, vastus medialis, vastus intermedius, gluteus maximus, tibialis anterior, flexor digitorum brevis, extensor pollicis longus, flexor hallucis brevis; and

[1034] A second group, comprising: triceps brachii (long head), subscapularis, pectoralis (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 to the affected first group of muscles and / or multiple unit doses are administered to the affected second group of muscles, and wherein the total dose administered during the treatment period is a maximum of 24,958 units, and wherein the modified BoNT / A comprises a BoNT / A light chain and a translocation domain, and a BoNT / B receptor binding domain (H C domain).

[1035] 20. The modified BoNT / A for use according to clause 19, wherein the limb spasticity is upper limb spasticity.

[1036] 21. A modified BoNT / A for use according to clause 19 or clause 20, wherein the unit dose is greater than 707 units to a maximum of 1,664 units (e.g., 708 units to a maximum of 1,664 units) of the modified BoNT / A. 22. A modified BoNT / A for use according to any of clauses 19-21, wherein the unit dose is greater than 949 units to a maximum of 1,581 units (e.g., 950 units to a maximum of 1,581 units) of the modified BoNT / A.

[1037] 23. A modified BoNT / A for use according to any one of clauses 19-22, wherein the unit dose is greater than 1,414 units to up to 1,581 units (eg, 1,415 units to up to 1,581 units) of the modified BoNT / A.

[1038] 24. A modified BoNT / A for use according to any one of clauses 19-23, wherein the total dose administered is greater than 10,399 Units to 24,958 Units (eg, 10,607 Units to 24,958 Units) of the modified BoNT / A.

[1039] 25. A modified BoNT / A for use according to any one of clauses 19-24, wherein the total dose administered is greater than 10,607 units to 24,958 units of the modified BoNT / A.

[1040] 26. A modified BoNT / A for use according to any one of clauses 19-25, wherein the total dose administered is between 14,559 units and 24,958 units of the modified BoNT / A.

[1041] 27. A modified BoNT / A for use according to any one of clauses 19-26, wherein the total dose administered is 20,799 units to 24,958 units of the modified BoNT / A.

[1042] 28. A modified BoNT / A for use in treating limb spasticity in a child, wherein the modified BoNT / A is administered to a plurality of affected muscles of a subject by intramuscular injection,

[1043] wherein the modified BoNT / A is administered to the plurality of affected muscles at a unit dose of greater than 353.5 units of the modified BoNT / A, and wherein 1 unit is the median lethal dose (LD50) calculated in mice. 50 ) corresponds to the amount of modified BoNT / A,

[1044] The muscles affected are selected from:

[1045] Group 1, 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 policis longus, adductor pollicis, flexor policis brevis, palmaris longus, lumbricals, opponens pollicis, adductor magnus, adductor longus, adductor brevis, gracilis, medial hamstring, lateral hamstring, tensor fasciae latae, rectus femoris, vastus lateralis, vastus medialis, vastus intermedius, gluteus maximus, tibialis anterior, flexor digitorum brevis, extensor pollicis longus, flexor hallucis brevis; and

[1046] The second group includes: 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

[1047] wherein a single unit dose is administered at a first group of affected muscles and / or a plurality of unit doses are administered at a second group of affected muscles, and wherein the total dose administered during the treatment period is at most 12,479 units, and wherein the modified BoNT / A comprises a BoNT / A light chain and a translocation domain, and a BoNT / B receptor binding domain (H C domain).

[1048] 29. The modified BoNT / A for use according to clause 28, wherein the pediatric limb spasticity is pediatric upper limb spasticity.

[1049] 30. A modified BoNT / A for use according to clause 28 or clause 29, wherein the unit dose is greater than 353.5 units to a maximum of 832 units (e.g., 354 units to a maximum of 832 units) of the modified BoNT / A, preferably wherein the unit dose is greater than 353.5 units to a maximum of 749 units (e.g., 354 units to a maximum of 749 units).

[1050] 31. A modified BoNT / A for use according to any one of clauses 28-30, wherein the unit dose is greater than 478 units to up to 749 units (eg, 479 units to up to 749 units) of the modified BoNT / A.

[1051] 32. A modified BoNT / A for use according to any one of clauses 28-31, wherein the unit dose is greater than 707 units to up to 749 units (eg, 708 units to up to 749 units) of the modified BoNT / A.

[1052] 33. A modified BoNT / A for use according to any one of clauses 28-32, wherein the total dose administered is greater than 5,200 units to 12,479 units (eg, 5,304 units to 12,479 units) of the modified BoNT / A.

[1053] 34. A modified BoNT / A for use according to any one of clauses 28-33, wherein the total dose administered is greater than 5,304 units to 12,479 units of the modified BoNT / A.

[1054] 35. A modified BoNT / A for use according to any one of clauses 28-34, wherein the total dose administered is 7,280 units to 12,479 units of the modified BoNT / A.

[1055] 36. A modified BoNT / A for use according to any one of clauses 28-35, wherein the total dose administered is 10,400 units to a maximum of 12,479 units of the modified BoNT / A.

[1056] 37. A modified BoNT / A for use according to any of the preceding clauses, wherein the modified BoNT / A comprises a polypeptide sequence having at least 70% sequence identity to SEQ ID NO:6.

[1057] 38. A modified BoNT / A for use according to any of the preceding clauses, wherein the modified BoNT / A comprises the polypeptide sequence of SEQ ID NO: 6.

[1058] 39. The modified BoNT / A for use according to any of the preceding clauses, wherein the modified BoNT / A consists of the polypeptide sequence of SEQ ID NO: 6.

[1059] 40. A modified BoNT / A for use according to any of the preceding clauses, wherein the modified BoNT / A has a safety ratio greater than 7, wherein the safety ratio is calculated as: the toxin dose required for -10% body weight change measured in pg / mouse divided by the DAS EC measured in pg / mouse 50 , where EC 50 = the dose required to produce a DAS score of 2.

[1060] 41. A modified BoNT / A for use according to any of the preceding clauses, wherein the plurality of muscles are selected from:

[1061] The first group includes: 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, lumbricals, and opponens pollicis; and

[1062] The second group, which includes: triceps brachii (long head), subscapularis, pectoralis muscles (eg, pectoralis major), latissimus dorsi, biceps brachii, brachialis, brachioradialis, and teres major.

[1063] 42. A unit dosage form of a modified BoNT / A, the unit dosage form comprising:

[1064] (a) greater than 707 units of a modified BoNT / A, wherein 1 unit corresponds to the calculated median lethal dose (LD50) in mice. 50 ) of a modified BoNT / A; or

[1065] (b) greater than 17,000 pg of modified BoNT / A; and

[1066] (c) optionally pharmaceutically acceptable carriers, excipients, adjuvants and / or salts,

[1067] The modified BoNT / A comprises a BoNT / A light chain and a translocation domain, and a BoNT / B receptor binding domain (H C domain).

[1068] 43. The unit dosage form according to clause 42, comprising:

[1069] (a) a modified BoNT / A of greater than 707 units to up to 1,664 units (e.g., 708 units to up to 1,664 units); or

[1070] (b) greater than 17,000 pg to at most 40,000 pg (e.g., 17,001 pg to at most 40,000 pg) of a modified BoNT / A.

[1071] 44. A kit comprising:

[1072] (a) a unit dosage form according to clause 42 or 43; and

[1073] (b) instructions for use in the treatment of limb spasticity; and

[1074] (c) optional diluent.

[1075] 45. A unit dosage form of a modified BoNT / A (e.g., for treating limb spasticity in adults), comprising:

[1076] (a) greater than 353.5 units of a modified BoNT / A, wherein 1 unit corresponds to the calculated median lethal dose (LD50) in mice. 50 ) of a modified BoNT / A; or

[1077] (b) greater than 8,500 pg of modified BoNT / A; and

[1078] (c) optionally pharmaceutically acceptable carriers, excipients, adjuvants and / or salts,

[1079] The modified BoNT / A comprises a BoNT / A light chain and a translocation domain, and a BoNT / B receptor binding domain (H C domain).

[1080] 46. ​​The unit dosage form according to clause 45, comprising:

[1081] (a) greater than 353.5 units to up to 832 units (e.g., 354 units to up to 832 units) of a modified BoNT / A; or

[1082] (b) greater than 8,500 pg to at most 20,000 pg (e.g., 8,501 pg to at most 20,000 pg) of a modified BoNT / A.

[1083] 47. A kit comprising:

[1084] (a) a unit dosage form according to clause 45 or 46; and

[1085] (b) instructions for use in the treatment of limb spasticity in children; and

[1086] (c) optional diluent.

[1087] All publications mentioned in the above description are incorporated herein by reference. Various modifications and variations of the method and system of the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been described in conjunction with specific preferred embodiments, it should be understood that the claimed invention should not be unduly limited to these specific embodiments. In fact, various modifications of the modes for implementing the present invention that are apparent to those skilled in the art of biochemistry and biotechnology or related fields are within the scope of the appended claims.

Claims

1. A modified botulinum neurotoxin A (BoNT / A) for use in treating 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 to the plurality of affected muscles at a unit dose of greater than 17,000 pg of the modified BoNT / A, wherein the plurality of affected muscles are selected from the group consisting of: The first group, include: 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 hamstring, lateral hamstring, tensor fasciae latae, rectus femoris, vastus lateralis, vastus medialis, vastus intermedius, gluteus maximus, tibialis anterior, flexor digitorum brevis, extensor pollicis longus, flexor hallucis brevis; as well as The second group includes: 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; and wherein a single unit dose is administered at a first group of affected muscles and / or a plurality of unit doses are administered at a second group of affected muscles, and wherein the total dose administered during the treatment period is up to 600,000 pg, and wherein the modified BoNT / A comprises a BoNT / A light chain and a translocation domain, and a BoNT / B receptor binding domain (H C domain).

2. A method for treating limb spasticity, the method comprising administering a modified botulinum neurotoxin A (BoNT / A) to a plurality of affected muscles of an individual by intramuscular injection, wherein the modified BoNT / A is administered to the plurality of affected muscles at a unit dose of greater than 17,000 pg of the modified BoNT / A, wherein the plurality of affected muscles are selected from: The first group, include: 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 hamstring, lateral hamstring, tensor fasciae latae, rectus femoris, vastus lateralis, vastus medialis, vastus intermedius, gluteus maximus, tibialis anterior, flexor digitorum brevis, extensor pollicis longus, flexor hallucis brevis; as well as The second group includes: 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; and wherein a single unit dose is administered at a first group of affected muscles and / or a plurality of unit doses are administered at a second group of affected muscles, and wherein the total dose administered during the treatment period is up to 600,000 pg, and wherein the modified BoNT / A comprises a BoNT / A light chain and a translocation domain, and a BoNT / B receptor binding domain (H C domain).

3. Use of a modified botulinum neurotoxin A (BoNT / A) in the manufacture of a medicament for treating limb spasticity, wherein the modified BoNT / A is administered to a plurality of affected muscles of a subject by intramuscular injection, wherein the modified BoNT / A is administered to the plurality of affected muscles at a unit dose of greater than 17,000 pg of the modified BoNT / A, The muscles affected are selected from: The first group, include: 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 hamstring, lateral hamstring, tensor fasciae latae, rectus femoris, vastus lateralis, vastus medialis, vastus intermedius, gluteus maximus, tibialis anterior, flexor digitorum brevis, extensor pollicis longus, flexor hallucis brevis; as well as The second group includes: 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; and wherein a single unit dose is administered at a first group of affected muscles and / or a plurality of unit doses are administered at a second group of affected muscles, and wherein the total dose administered during the treatment period is up to 600,000 pg, and wherein the modified BoNT / A comprises a BoNT / A light chain and a translocation domain, and a BoNT / B receptor binding domain (H C domain).

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

5. The modified BoNT / A for use, method or use according to any one of the preceding claims, wherein the unit dose is 20,000 pg to 30,000 pg, preferably 24,000 pg to 26,000 pg, more preferably 25,000 pg of modified BoNT / A.

6. The modified BoNT / A for use, method or use according to any one of claims 1 to 4, wherein the unit dose is 30,000 pg to 40,000 pg, preferably 35,000 pg to 37,000 pg, more preferably 36,000 pg of modified BoNT / A.

7. The modified BoNT / A for use, method or use according to any one of claims 1-4, wherein the unit dose is greater than 23,000 pg to up to 38,000 pg (e.g., 23,001 pg to up to 38,000 pg) of the modified BoNT / A.

8. The modified BoNT / A for use, method, or use according to any one of claims 1-4 or 6-7, wherein the unit dose is greater than 34,000 pg to up to 38,000 pg (e.g., 34,001 pg to up to 38,000 pg) of the modified BoNT / A.

9. The modified BoNT / A for use, method or use according to any one of the preceding claims, wherein the total dose administered is 250,000 pg to 600,000 pg (e.g., 255,000 pg to 600,000 pg) of the modified BoNT / A.

10. The modified BoNT / A for use, method or use according to any of the preceding claims, wherein the total dose administered is greater than 255,000 pg to 600,000 pg of the modified BoNT / A.

11. The modified BoNT / A for use, method or use according to any one of the preceding claims, wherein the total dose administered is 350,000 pg to 600,000 pg of modified BoNT / A.

12. The modified BoNT / A for use, method or use according to any one of claims 1-10, wherein the total dose administered is at most 300,000 pg to 450,000 pg, preferably at most 360,000 pg to 390,000 pg, more preferably at most 375,000 pg of modified BoNT / A.

13. The modified BoNT / A for use, method or use according to any one of claims 1-11, wherein the total dose administered is at most 450,000 pg to 600,000 pg, preferably at most 525,000 pg to 555,000 pg, more preferably at most 540,000 pg of modified BoNT / A.

14. The modified BoNT / A for use, method or use according to any one of claims 1-11 or 13, wherein the total dose administered is 500,000 to 600,000 pg of the modified BoNT / A.

15. A modified botulinum neurotoxin A (BoNT / A) for use in treating 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 to the affected muscles at a unit dose of greater than 707 units of the modified BoNT / A, and wherein 1 unit is the median lethal dose (LD50) calculated in mice. 50 ) corresponds to the amount of modified BoNT / A, The muscles affected are selected from: The first group, include: 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 hamstring, lateral hamstring, tensor fasciae latae, rectus femoris, vastus lateralis, vastus medialis, vastus intermedius, gluteus maximus, tibialis anterior, flexor digitorum brevis, extensor pollicis longus, flexor hallucis brevis; as well as The second group includes: 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; and wherein a single unit dose is administered at the first group of affected muscles and / or a plurality of unit doses are administered at the second group of affected muscles, and wherein the total dose administered during the treatment period is at most 24,958 units, and wherein the modified BoNT / A comprises a BoNT / A light chain and a translocation domain, and a BoNT / B receptor binding domain (H C domain).

16. A method for treating limb spasticity, the method comprising administering a 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 to the affected muscles at a unit dose of greater than 707 units of the modified BoNT / A, wherein 1 unit is the median lethal dose (LD50) calculated in mice. 50 ) corresponds to the amount of modified BoNT / A, The muscles affected are selected from: The first group, include: 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 hamstring, lateral hamstring, tensor fasciae latae, rectus femoris, vastus lateralis, vastus medialis, vastus intermedius, gluteus maximus, tibialis anterior, flexor digitorum brevis, extensor pollicis longus, flexor hallucis brevis; as well as The second group includes: 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; and wherein a single unit dose is administered at the first group of affected muscles and / or a plurality of unit doses are administered at the second group of affected muscles, and wherein the total dose administered during the treatment period is at most 24,958 units, and wherein the modified BoNT / A comprises a BoNT / A light chain and a translocation domain, and a BoNT / B receptor binding domain (H C domain).

17. Use of a modified botulinum neurotoxin A (BoNT / A) in the manufacture of a medicament for treating limb spasticity, wherein the modified BoNT / A is administered to a plurality of affected muscles of an individual by intramuscular injection, wherein the modified BoNT / A is administered to the plurality of affected muscles at a unit dose of greater than 707 units of the modified BoNT / A, and wherein 1 unit is the amount of the modified BoNT / A corresponding to a calculated median lethal dose (LD50) in mice, The muscles affected are selected from: The first group, include: 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 hamstring, lateral hamstring, tensor fasciae latae, rectus femoris, vastus lateralis, vastus medialis, vastus intermedius, gluteus maximus, tibialis anterior, flexor digitorum brevis, extensor pollicis longus, flexor hallucis brevis; as well as The second group includes: 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; and wherein a single unit dose is administered at the first group of affected muscles and / or a plurality of unit doses are administered at the second group of affected muscles, and wherein the total dose administered during the treatment period is at most 24,958 units, and wherein the modified BoNT / A comprises a BoNT / A light chain and a translocation domain, and a BoNT / B receptor binding domain (H C domain).

18. The modified BoNT / A for use, method or use according to any one of claims 15-17, wherein the unit dose is greater than 707 units to up to 1,664 units (e.g., 708 units to up to 1,664 units) of the modified BoNT / A.

19. The modified BoNT / A for use, method or use according to any one of claims 15-18, wherein the unit dose is 832 units to 1,248 units, preferably 998 units to 1,082 units, more preferably 1,040 units of the modified BoNT / A.

20. The modified BoNT / A for use, method or use according to any one of claims 15-18, wherein the unit dose is 1,248 units to 1,664 units, preferably 1,456 units to 1,539 units, more preferably 1,498 units of the modified BoNT / A.

21. The modified BoNT / A for use, method, or use according to any one of claims 15-18, wherein the unit dose is greater than 949 units to up to 1,581 units or greater than 957 units to up to 1,581 units (e.g., 950 units to up to 1,581 units or 958 units to up to 1,581 units) of the modified BoNT / A.

22. The modified BoNT / A for use, method, or use according to any of claims 15-18 or 20-21, wherein the unit dose is greater than 1,414 units to up to 1,581 units (e.g., 1,415 units to up to 1,581 units) of the modified BoNT / A.

23. The modified BoNT / A for use, method or use according to any one of claims 15-22, wherein the total dose administered is 10,399 units to 24,958 units (eg, 10,607 units to 24,958 units) of the modified BoNT / A.

24. The modified BoNT / A for use, method or use according to any one of claims 15-23, wherein the total dose administered is greater than 10,607 units to 24,958 units of the modified BoNT / A.

25. The modified BoNT / A for use, method or use according to any one of claims 15-24, wherein the total dose administered is at most 12,479 units to 18,719 units, preferably at most 14,975 units to 16,223 units, more preferably at most 15,599 units of the modified BoNT / A.

26. The modified BoNT / A for use, method or use according to any one of claims 12-20, 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, method or use according to any one of claims 15-24 or 26, wherein the total dose administered is at most 18,719 units to 24,958 units, preferably at most 21,839 units to 23,087 units, more preferably at most 22,463 units of the modified BoNT / A.

28. The modified BoNT / A for use, method or use according to any one of claims 15-24 or 26-27, wherein the total dose administered is 20,799 units to 24,958 units of the modified BoNT / A.

29. The modified BoNT / A for use, method, or use according to any one of the preceding claims, wherein the spasticity is an adult limb spasticity and the individual is an adult individual.

30. The modified BoNT / A for use, method, or use according to any one of the preceding claims, wherein the limb spasticity is upper limb spasticity.

31. A modified botulinum neurotoxin A (BoNT / A) for use in treating limb spasticity in children, 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 to the plurality of affected muscles at a unit dose of greater than 8,500 pg of the modified BoNT / A, The muscles affected are selected from: The first group, include: 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 hamstring, lateral hamstring, tensor fasciae latae, rectus femoris, vastus lateralis, vastus medialis, vastus intermedius, gluteus maximus, tibialis anterior, flexor digitorum brevis, extensor pollicis longus, flexor hallucis brevis; as well as The second group includes: 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; and wherein a single unit dose is administered at a first group of affected muscles and / or a plurality of unit doses are administered at a second group of affected muscles, and wherein the total dose administered during the treatment period is at most 300,000 pg, and wherein the modified BoNT / A comprises a BoNT / A light chain and a translocation domain, and a BoNT / B receptor binding domain (H C domain).

32. A method for treating limb spasticity in a child, the method comprising administering a modified botulinum neurotoxin A (BoNT / A) to a plurality of affected muscles of a subject by intramuscular injection, wherein the modified BoNT / A is administered to the plurality of affected muscles at a unit dose of greater than 8,500 pg of the modified BoNT / A, The muscles affected are selected from: The first group, include: 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 hamstring, lateral hamstring, tensor fasciae latae, rectus femoris, vastus lateralis, vastus medialis, vastus intermedius, gluteus maximus, tibialis anterior, flexor digitorum brevis, extensor pollicis longus, flexor hallucis brevis; as well as The second group includes: 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; and wherein a single unit dose is administered at a first group of affected muscles and / or a plurality of unit doses are administered at a second group of affected muscles, and wherein the total dose administered during the treatment period is at most 300,000 pg, and wherein the modified BoNT / A comprises a BoNT / A light chain and a translocation domain, and a BoNT / B receptor binding domain (H C domain).

33. Use of a modified botulinum neurotoxin A (BoNT / A) in the manufacture of a medicament for treating limb spasticity in children, wherein the modified BoNT / A is administered to a plurality of affected muscles of a subject by intramuscular injection, wherein the modified BoNT / A is administered to the plurality of affected muscles at a unit dose of greater than 8,500 pg of the modified BoNT / A, The muscles affected are selected from: The first group, include: 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 hamstring, lateral hamstring, tensor fasciae latae, rectus femoris, vastus lateralis, vastus medialis, vastus intermedius, gluteus maximus, tibialis anterior, flexor digitorum brevis, extensor pollicis longus, flexor hallucis brevis; as well as The second group includes: 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; and wherein a single unit dose is administered at a first group of affected muscles and / or a plurality of unit doses are administered at a second group of affected muscles, and wherein the total dose administered during the treatment period is at most 300,000 pg, and wherein the modified BoNT / A comprises a BoNT / A light chain and a translocation domain, and a BoNT / B receptor binding domain (H C domain).

34. The modified BoNT / A for use, method, or use according to any one of claims 31-33, wherein the unit dose is greater than 8,500 pg to up to 20,000 pg (e.g., 8,501 pg to up to 20,000 pg) of the modified BoNT / A, preferably wherein the unit dose is greater than 8,500 pg to up to 18,000 pg.

35. The modified BoNT / A for use, method or use according to any one of claims 31-34, wherein the unit dose is 10,000 pg to 15,000, preferably 12,000 pg to 13,000 pg, more preferably 12,500 pg of modified BoNT / A.

36. The modified BoNT / A for use, method or use according to any one of claims 31-34, wherein the unit dose is 15,000 pg to 20,000, preferably 17,500 pg to 18,500 pg, more preferably 18,000 pg of modified BoNT / A.

37. The modified BoNT / A for use, method, or use according to any one of claims 31-34, wherein the unit dose is greater than 11,500 pg to up to 19,000 pg (e.g., 11,501 pg to up to 19,000 pg) of the modified BoNT / A.

38. The modified BoNT / A for use, method, or use according to any one of claims 31-34 or 36-37, wherein the unit dose is greater than 17,000 pg to up to 19,000 pg (e.g., 17,001 pg to up to 19,000 pg) of the modified BoNT / A.

39. The modified BoNT / A for use, method, or use according to any one of claims 31-38, wherein the total dose administered is 125,000 pg to 300,000 pg (e.g., 127,500 pg to 300,000 pg) of the modified BoNT / A.

40. The modified BoNT / A for use, method, or use according to any one of claims 31-39, wherein the total dose administered is greater than 127,500 pg to 300,000 pg of the modified BoNT / A.

41. The modified BoNT / A for use, method or use according to any one of claims 31-40, wherein the total dose administered is 175,000 to 300,000 pg of the modified BoNT / A.

42. The modified BoNT / A for use, method or use according to any one of claims 31-40, wherein the total dose administered is at most 150,000 pg to 225,000 pg, preferably at most 180,000 pg to 195,000 pg, more preferably at most 187,500 pg of modified BoNT / A.

43. The modified BoNT / A for use, method or use according to any one of claims 31-41, wherein the total dose administered is at most 225,000 pg to 300,000 pg, preferably at most 262,500 pg to 277,500 pg, more preferably at most 270,000 pg of modified BoNT / A.

44. The modified BoNT / A for use, method or use according to any one of claims 31-41 or 43, wherein the total dose administered is 250,000 to 300,000 pg of the modified BoNT / A.

45. A modified BoNT / A for use in treating limb spasticity in a child, wherein the modified BoNT / A is administered to a plurality of affected muscles of a subject by intramuscular injection, wherein the modified BoNT / A is administered to the plurality of affected muscles at a unit dose of greater than 353.5 units of the modified BoNT / A, and wherein 1 unit is the median lethal dose (LD50) calculated in mice. 50 ) corresponds to the amount of modified BoNT / A, The muscles affected are selected from: The first group, include: 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 hamstring, lateral hamstring, tensor fasciae latae, rectus femoris, vastus lateralis, vastus medialis, vastus intermedius, gluteus maximus, tibialis anterior, flexor digitorum brevis, extensor pollicis longus, flexor hallucis brevis; as well as The second group includes: 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; and wherein a single unit dose is administered at the first group of affected muscles and / or a plurality of unit doses are administered at the second group of affected muscles, and wherein the total dose administered during the treatment period is at most 12,479 units, and wherein the modified BoNT / A comprises a BoNT / A light chain and a translocation domain, and a BoNT / B receptor binding domain (H C domain).

46. ​​A method for treating limb spasticity in a child, the method comprising administering a 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 to the affected muscles at a unit dose of greater than 353.5 units of the modified BoNT / A, wherein 1 unit is the median lethal dose (LD50) calculated in mice. 50 ) corresponds to the amount of modified BoNT / A, The muscles affected are selected from: The first group, include: 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 hamstring, lateral hamstring, tensor fasciae latae, rectus femoris, vastus lateralis, vastus medialis, vastus intermedius, gluteus maximus, tibialis anterior, flexor digitorum brevis, extensor pollicis longus, flexor hallucis brevis; as well as The second group includes: 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; and wherein a single unit dose is administered at the first group of affected muscles and / or a plurality of unit doses are administered at the second group of affected muscles, and wherein the total dose administered during the treatment period is at most 12,479 units, and wherein the modified BoNT / A comprises a BoNT / A light chain and a translocation domain, and a BoNT / B receptor binding domain (H C domain).

47. Use of a modified BoNT / A in the manufacture of a medicament for treating limb spasticity in a child, wherein the modified BoNT / A is administered to a plurality of affected muscles of a subject by intramuscular injection, wherein the modified BoNT / A is administered to the plurality of affected muscles at a unit dose of greater than 353.5 units of the modified BoNT / A, and wherein 1 unit is the amount of the modified BoNT / A corresponding to a calculated median lethal dose (LD50) in mice, The muscles affected are selected from: The first group, include: 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 hamstring, lateral hamstring, tensor fasciae latae, rectus femoris, vastus lateralis, vastus medialis, vastus intermedius, gluteus maximus, tibialis anterior, flexor digitorum brevis, extensor pollicis longus, flexor hallucis brevis; as well as The second group includes: 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; and wherein a single unit dose is administered at the first group of affected muscles and / or a plurality of unit doses are administered at the second group of affected muscles, and wherein the total dose administered during the treatment period is at most 12,479 units, and wherein the modified BoNT / A comprises a BoNT / A light chain and a translocation domain, and a BoNT / B receptor binding domain (H C domain).

48. The modified BoNT / A for use, method, or use according to any one of claims 45-47, wherein the unit dose is greater than 353.5 Units to a maximum of 832 Units (e.g., 354 Units to a maximum of 832 Units) of the modified BoNT / A, preferably wherein the unit dose is greater than 353.5 Units to a maximum of 749 Units (e.g., 354 Units to a maximum of 749 Units).

49. The modified BoNT / A for use, method or use according to any one of claims 45-48, wherein the unit dose is 416 units to 624 units, preferably 499 units to 541 units, more preferably 520 units of the modified BoNT / A.

50. The modified BoNT / A for use, method or use according to any one of claims 45-48, wherein the unit dose is 624 units to 832 units, preferably 728 units to 770 units, more preferably 749 units of the modified BoNT / A.

51. The modified BoNT / A for use, method, or use according to any one of claims 45-48, wherein the unit dose is greater than 478 units to up to 790 units (e.g., 479 units to up to 790 units) of the modified BoNT / A.

52. The modified BoNT / A for use, method, or use according to any one of claims 45-48, wherein the unit dose is greater than 707 units to up to 790 units (e.g., 708 units to up to 790 units) of the modified BoNT / A.

53. The modified BoNT / A for use, method, or use according to any one of claims 45-52, wherein the total dose administered is 5,200 units to 12,479 units (eg, 5,304 units to 12,479 units) of the modified BoNT / A.

54. The modified BoNT / A for use, method, or use according to any one of claims 45-53, wherein the total dose administered is greater than 5,304 units to 12,479 units of the modified BoNT / A.

55. The modified BoNT / A for use, method or use according to any one of claims 45-54, wherein the total dose administered is at most 6,240 units to 9,360 units, preferably at most 7,488 units to 8,112 units, more preferably at most 7,800 units of the modified BoNT / A.

56. The modified BoNT / A for use, method or use according to any one of claims 45-54, wherein the total dose administered is 7,280 units to 12,479 units of the modified BoNT / A.

57. The modified BoNT / A for use, method or use according to any one of claims 45-54 or 56, wherein the total dose administered is at most 9,360 Units to 12,479 Units, preferably at most 10,920 Units to 11,544 Units, more preferably at most 11,232 Units of the modified BoNT / A.

58. The modified BoNT / A for use, method, or use according to any one of claims 45-54 or 56-57, wherein the total dose administered is between 10,399 units and 12,479 units of the modified BoNT / A.

59. The modified BoNT / A for use, method, or use according to any one of claims 31-58, wherein the pediatric limb spasticity is pediatric upper limb spasticity.

60. The modified BoNT / A for use, method or use according to any one of the preceding claims, in: The first group of affected muscles includes: 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 policis longus, adductor pollicis, flexor policis brevis, palmaris longus, lumbricals, opponens pollicis, adductor magnus, adductor longus, adductor brevis, gracilis, medial hamstring, lateral hamstring, tensor fasciae latae, rectus femoris, vastus lateralis, vastus medialis, vastus intermedius, gluteus maximus, tibialis anterior, flexor digitorum brevis, extensor hallucis longus, flexor hallucis brevis; and The second group of muscles affected include: triceps brachii (long head), subscapularis, pectoralis muscles (eg, pectoralis major), latissimus dorsi, biceps brachii, brachialis, soleus, tibialis posterior, brachioradialis, teres major, iliopsoas, and gastrocnemius.

61. A modified botulinum neurotoxin A (BoNT / A) for use in treating limb spasticity, preferably limb spasticity in adults, 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 to the plurality of affected muscles at 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 the modified BoNT / A, The muscles affected are selected from: The first group, include: 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 hamstring, lateral hamstring, tensor fasciae latae, rectus femoris, vastus lateralis, vastus medialis, vastus intermedius, gluteus maximus, tibialis anterior, flexor digitorum brevis, extensor pollicis longus, flexor hallucis brevis; as well as The second group includes: 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, and wherein a single unit dose is administered at the affected first group of muscles and / or multiple unit doses are administered at the affected second group of muscles, and wherein the total dose administered during the treatment period is at most 300,000 pg (e.g., at most 240,000 pg, preferably at most 225,000 pg), and The modified BoNT / A comprises a BoNT / A light chain and a translocation domain, and a BoNT / B receptor binding domain (H C domain).

62. A method for treating limb spasticity, preferably limb spasticity in adults, the method comprising administering a modified botulinum neurotoxin A (BoNT / A) to a plurality of affected muscles of an individual by intramuscular injection, wherein the modified BoNT / A is administered to the plurality of affected muscles at 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 the modified BoNT / A, The muscles affected are selected from: The first group, include: 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 hamstring, lateral hamstring, tensor fasciae latae, rectus femoris, vastus lateralis, vastus medialis, vastus intermedius, gluteus maximus, tibialis anterior, flexor digitorum brevis, extensor pollicis longus, flexor hallucis brevis; as well as The second group includes: 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, wherein a single unit dose is administered at a first group of muscles affected and / or a plurality of unit doses are administered at a second group of muscles affected, and wherein the total dose administered during the treatment period is at most 300,000 pg (e.g. at most 240,000 pg, preferably at most 225,000 pg), and The modified BoNT / A comprises a BoNT / A light chain and a translocation domain, and a BoNT / B receptor binding domain (H C domain).

63. Use of a modified botulinum neurotoxin A (BoNT / A) in the manufacture of a medicament for treating limb spasticity, preferably limb spasticity in adults, wherein the modified BoNT / A is administered to a plurality of affected muscles of a subject by intramuscular injection, wherein the modified BoNT / A is administered to the plurality of affected muscles at 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 the modified BoNT / A, The muscles affected are selected from: The first group, include: 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 hamstring, lateral hamstring, tensor fasciae latae, rectus femoris, vastus lateralis, vastus medialis, vastus intermedius, gluteus maximus, tibialis anterior, flexor digitorum brevis, extensor pollicis longus, flexor hallucis brevis; as well as The second group includes: 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, wherein a single unit dose is administered at a first group of muscles affected and / or a plurality of unit doses are administered at a second group of muscles affected, and wherein the total dose administered during the treatment period is at most 300,000 pg (e.g. at most 240,000 pg, preferably at most 225,000 pg), and The modified BoNT / A comprises a BoNT / A light chain and a translocation domain, and a BoNT / B receptor binding domain (H C domain).

64. A modified botulinum neurotoxin A (BoNT / A) for use in treating limb spasticity, preferably limb spasticity in adults, wherein the modified BoNT / A is administered to a plurality of affected muscles of an individual by intramuscular injection, wherein the modified BoNT / A is administered to the plurality of affected muscles at a unit dose of 416 units to 832 units (e.g., 582 units to 666 units, preferably 624 units) of the modified BoNT / A, and wherein 1 unit is the amount of the modified BoNT / A corresponding to a calculated median lethal dose (LD50) in mice, The muscles affected are selected from: The first group, include: 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 hamstring, lateral hamstring, tensor fasciae latae, rectus femoris, vastus lateralis, vastus medialis, vastus intermedius, gluteus maximus, tibialis anterior, flexor digitorum brevis, extensor pollicis longus, flexor hallucis brevis; as well as The second group includes: 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, wherein a single unit dose is administered at the first group of muscles affected and / or a plurality of unit doses are administered at the second group of muscles affected, and wherein the total dose administered during the treatment period is at most 12,480 units (e.g. at most 9,990 units, preferably at most 9,360 units), and The modified BoNT / A comprises a BoNT / A light chain and a translocation domain, and a BoNT / B receptor binding domain (H C domain).

65. A method for treating limb spasticity, preferably limb spasticity in adults, the method comprising administering a modified botulinum neurotoxin A (BoNT / A) to a plurality of affected muscles of an individual by intramuscular injection, wherein the modified BoNT / A is administered to the plurality of affected muscles at a unit dose of 416 units to 832 units (e.g., 582 units to 666 units, preferably 624 units) of the modified BoNT / A, wherein 1 unit is the median lethal dose (LD50) calculated in mice. 50 ) corresponds to the amount of modified BoNT / A, The muscles affected are selected from: The first group, include: 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 hamstring, lateral hamstring, tensor fasciae latae, rectus femoris, vastus lateralis, vastus medialis, vastus intermedius, gluteus maximus, tibialis anterior, flexor digitorum brevis, extensor pollicis longus, flexor hallucis brevis; as well as The second group includes: 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, and wherein a single unit dose is administered at the affected first group of muscles and / or multiple unit doses are administered at the affected second group of muscles, and wherein the total dose administered during the treatment period is at most 12,480 units (e.g., at most 9,990 units, preferably at most 9,360 units), and The modified BoNT / A comprises a BoNT / A light chain and a translocation domain, and a BoNT / B receptor binding domain (H C domain).

66. Use of a modified botulinum neurotoxin A (BoNT / A) in the manufacture of a medicament for treating limb spasticity, preferably limb spasticity in adults, 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 to the plurality of affected muscles at a unit dose of 416 units to 832 units (e.g., 582 units to 666 units, preferably 624 units) of the modified BoNT / A, and wherein 1 unit is the median lethal dose (LD50) calculated in mice. 50 ) corresponds to the amount of modified BoNT / A, The muscles affected are selected from: The first group, include: 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 hamstring, lateral hamstring, tensor fasciae latae, rectus femoris, vastus lateralis, vastus medialis, vastus intermedius, gluteus maximus, tibialis anterior, flexor digitorum brevis, extensor pollicis longus, flexor hallucis brevis; as well as The second group includes: 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, wherein a single unit dose is administered at the first group of muscles affected and / or a plurality of unit doses are administered at the second group of muscles affected, and wherein the total dose administered during the treatment period is at most 12,480 units (e.g. at most 9,990 units, preferably at most 9,360 units), and The modified BoNT / A comprises a BoNT / A light chain and a translocation domain, and a BoNT / B receptor binding domain (H C domain).

67. A modified botulinum neurotoxin A (BoNT / A) for use in treating limb spasticity in children, 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 to the plurality of affected muscles at 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 the modified BoNT / A, The muscles affected are selected from: The first group, include: 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 hamstring, lateral hamstring, tensor fasciae latae, rectus femoris, vastus lateralis, vastus medialis, vastus intermedius, gluteus maximus, tibialis anterior, flexor digitorum brevis, extensor pollicis longus, flexor hallucis brevis; as well as The second group includes: 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, wherein a single unit dose is administered at a first group of muscles affected and / or a plurality of unit doses are administered at a second group of muscles affected, and wherein the total dose administered during the treatment period is at most 150,000 pg (e.g. at most 120,000 pg, preferably at most 112,500 pg), and The modified BoNT / A comprises a BoNT / A light chain and a translocation domain, and a BoNT / B receptor binding domain (H C domain).

68. A method for treating limb spasticity in a child, the method comprising administering a 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 to a plurality of affected muscles at 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 the modified BoNT / A, wherein the plurality of affected muscles are selected from: The first group, include: 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 hamstring, lateral hamstring, tensor fasciae latae, rectus femoris, vastus lateralis, vastus medialis, vastus intermedius, gluteus maximus, tibialis anterior, flexor digitorum brevis, extensor pollicis longus, flexor hallucis brevis; as well as The second group includes: 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, wherein a single unit dose is administered at a first group of muscles affected and / or a plurality of unit doses are administered at a second group of muscles affected, and wherein the total dose administered during the treatment period is at most 150,000 pg (e.g. at most 120,000 pg, preferably at most 112,500 pg), and The modified BoNT / A comprises a BoNT / A light chain and a translocation domain, and a BoNT / B receptor binding domain (H C domain).

69. Use of a modified botulinum neurotoxin A (BoNT / A) in the manufacture of a medicament for treating limb spasticity in children, 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 to the plurality of affected muscles at 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 the modified BoNT / A, The muscles affected are selected from: The first group, include: 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 hamstring, lateral hamstring, tensor fasciae latae, rectus femoris, vastus lateralis, vastus medialis, vastus intermedius, gluteus maximus, tibialis anterior, flexor digitorum brevis, extensor pollicis longus, flexor hallucis brevis; as well as The second group includes: 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, wherein a single unit dose is administered at a first group of muscles affected and / or a plurality of unit doses are administered at a second group of muscles affected, and wherein the total dose administered during the treatment period is at most 150,000 pg (e.g. at most 120,000 pg, preferably at most 112,500 pg), and The modified BoNT / A comprises a BoNT / A light chain and a translocation domain, and a BoNT / B receptor binding domain (H C domain).

70. A modified botulinum neurotoxin A (BoNT / A) for use in treating limb spasticity in a child, wherein the modified BoNT / A is administered by intramuscular injection to a plurality of affected muscles of the subject, wherein the modified BoNT / A is administered to the plurality of affected muscles at a unit dose of 208 units to 416 units (e.g., 291 units to 333 units, preferably 312 units) of the modified BoNT / A, and wherein 1 unit is the median lethal dose (LD50) calculated in mice. 50 ) corresponds to the amount of modified BoNT / A, The muscles affected are selected from: The first group, include: 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 hamstring, lateral hamstring, tensor fasciae latae, rectus femoris, vastus lateralis, vastus medialis, vastus intermedius, gluteus maximus, tibialis anterior, flexor digitorum brevis, extensor pollicis longus, flexor hallucis brevis; as well as The second group includes: 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, wherein a single unit dose is administered at the first group of muscles affected and / or a plurality of unit doses are administered at the second group of muscles affected, and wherein the total dose administered during the treatment period is at most 6,240 units (e.g. at most 4,995 units, preferably at most 4,680 units), and The modified BoNT / A comprises a BoNT / A light chain and a translocation domain, and a BoNT / B receptor binding domain (H C domain).

71. A method for treating limb spasticity in children, the method comprising administering a modified botulinum neurotoxin A (BoNT / A) to a plurality of affected muscles of a subject by intramuscular injection, wherein the modified BoNT / A is administered to the plurality of affected muscles at a unit dose of 208 units to 416 units (e.g., 291 units to 333 units, preferably 312 units) of the modified BoNT / A, wherein 1 unit is the median lethal dose (LD50) calculated in mice. 50 ) corresponds to the amount of modified BoNT / A, The muscles affected are selected from: The first group, include: 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 hamstring, lateral hamstring, tensor fasciae latae, rectus femoris, vastus lateralis, vastus medialis, vastus intermedius, gluteus maximus, tibialis anterior, flexor digitorum brevis, extensor pollicis longus, flexor hallucis brevis; as well as The second group includes: 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, wherein a single unit dose is administered at the first group of muscles affected and / or a plurality of unit doses are administered at the second group of muscles affected, and wherein the total dose administered during the treatment period is at most 6,240 units (e.g. at most 4,995 units, preferably at most 4,680 units), and The modified BoNT / A comprises a BoNT / A light chain and a translocation domain, and a BoNT / B receptor binding domain (H C domain).

72. Use of a modified botulinum neurotoxin A (BoNT / A) in the manufacture of a medicament for treating limb spasticity in a child, 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 to the plurality of affected muscles at a unit dose of 208 units to 416 units (e.g., 291 units to 333 units, preferably 312 units) of the modified BoNT / A, and wherein 1 unit is the median lethal dose (LD50) calculated in mice. 50 ) corresponds to the amount of modified BoNT / A, The muscles affected are selected from: The first group, include: 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 hamstring, lateral hamstring, tensor fasciae latae, rectus femoris, vastus lateralis, vastus medialis, vastus intermedius, gluteus maximus, tibialis anterior, flexor digitorum brevis, extensor pollicis longus, flexor hallucis brevis; as well as The second group includes: 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, wherein a single unit dose is administered at the first group of muscles affected and / or a plurality of unit doses are administered at the second group of muscles affected, and wherein the total dose administered during the treatment period is at most 6,240 units (e.g. at most 4,995 units, preferably at most 4,680 units), and The modified BoNT / A comprises a BoNT / A light chain and a translocation domain, and a BoNT / B receptor binding domain (H C domain).

73. The modified BoNT / A for the use, method, or use of any preceding claim, wherein the spasticity is upper limb spasticity, and wherein the plurality of affected muscles are selected from: A first group, which comprises (preferably consists of): flexor digitorum superficialis, flexor digitorum profundus, flexor carpi radialis, flexor carpi ulnaris, brachioradialis; and The second group comprises (preferably consists of) the brachialis, biceps brachii, triceps brachii (long head), subscapularis, pectoralis muscles (eg, pectoralis major) and latissimus dorsi.

74. The modified BoNT / A for use, method or use of claim 73, wherein two unit doses are administered to a second group of affected muscles.

75. The modified BoNT / A for use, method, or use according to any one of claims 1-29, 31-58, or 60-72, wherein at least one of the plurality of affected muscles is selected from the group consisting of: the adductor magnus, hamstring muscles (e.g., biceps femoris, semimembranosus, and / or semitendinosus), and rectus femoris.

76. The modified BoNT / A for use, method, or use according to any one of claims 1-29, 31-58, 60-72, or 75, wherein the spasticity is lower limb spasticity, and wherein the plurality of affected muscles are selected from: A first group, which comprises (preferably consists of): the medial head of the gastrocnemius (medial gastrocnemius), the lateral head of the gastrocnemius (lateral gastrocnemius), the tibialis anterior, the flexor digitorum longus, the flexor digitorum brevis, the flexor hallucis longus, the flexor hallucis brevis, the gracilis, and the gluteus maximus; and The second group comprises (preferably consists of): the soleus, tibialis posterior, rectus femoris, hamstrings (eg, biceps femoris, semimembranosus and / or semitendinosus) and adductor magnus.

77. The modified BoNT / A for use, method or use of claim 76, wherein when the second group of muscles is: (a) the soleus muscle, to which three unit doses are administered; or (b) the tibialis posterior, rectus femoris, hamstring (eg, biceps femoris, semimembranosus and / or semitendinosus), or adductor magnus, to which two unit doses are administered.

78. The modified BoNT / A for use, method or use according to any preceding claim, wherein the total number of unit doses administered during the treatment period is at most 15 unit doses.

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

6.

80. The modified BoNT / A for use, method or use according to any preceding claim, wherein the modified BoNT / A comprises the polypeptide sequence of SEQ ID NO:

6.

81. The modified BoNT / A for use, method or use according to any preceding claim, wherein the modified BoNT / A consists of the polypeptide sequence of SEQ ID NO:

6.

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

83. The modified BoNT / A for use, method, or use of any preceding claim, wherein the duration of treatment of limb spasticity is longer than the duration of treatment of an unmodified BoNT / A (e.g., SEQ ID NO: 2 [e.g., SEQ ID NO: 2 in di-chain form]).

84. A unit dosage form of a modified botulinum neurotoxin A (BoNT / A) (e.g., for treating limb spasticity in adults), the unit dosage form comprising: (a) greater than 17,000 pg of modified BoNT / A; or (b) greater than 707 units of a modified BoNT / A, wherein 1 unit corresponds to the calculated median lethal dose (LD50) in mice. 50 ) of the modified BoNT / A; and (c) optionally pharmaceutically acceptable carriers, excipients, adjuvants and / or salts, The modified BoNT / A comprises a BoNT / A light chain and a translocation domain, and a BoNT / B receptor binding domain (H C domain).

85. The unit dosage form of claim 84, wherein the unit dosage form comprises 20,000 pg to 30,000 pg, preferably 24,000 pg to 26,000 pg, more preferably 25,000 pg of modified BoNT / A.

86. The unit dosage form of claim 84, wherein the unit dosage form comprises 832 units to 1,248 units, preferably 998 units to 1,082 units, more preferably 1,040 units of modified BoNT / A.

87. The unit dosage form of claim 84, wherein the unit dosage form comprises 30,000 pg to 40,000 pg, preferably 35,000 pg to 37,000 pg, more preferably 36,000 pg of modified BoNT / A.

88. The unit dosage form of claim 84, wherein the unit dosage form comprises 1,248 units to 1,664 units, preferably 1,456 units to 1,539 units, more preferably 1,498 units of modified BoNT / A.

89. A unit dosage form of a modified botulinum neurotoxin A (BoNT / A) for use in treating a pediatric subject (e.g., for treating pediatric limb spasticity), the unit dosage form comprising: (a) greater than 8,500 pg of modified BoNT / A; or (b) greater than 353.5 units of a modified BoNT / A, wherein 1 unit corresponds to the calculated median lethal dose (LD50) in mice. 50 ) of the modified BoNT / A; and (c) optionally pharmaceutically acceptable carriers, excipients, adjuvants and / or salts, The modified BoNT / A comprises a BoNT / A light chain and a translocation domain, and a BoNT / B receptor binding domain (H C domain).

90. The unit dosage form of claim 89, wherein the unit dosage form comprises 10,000 pg to 15,000 pg, preferably 12,000 pg to 13,000 pg, more preferably 12,500 pg of modified BoNT / A.

91. The unit dosage form of claim 89, wherein the unit dosage form comprises 416 units to 624 units, preferably 499 units to 541 units, more preferably 520 units of modified BoNT / A.

92. The unit dosage form of claim 89, wherein the unit dosage form comprises 15,000 pg to 20,000 pg, preferably 17,500 pg to 18,500 pg, more preferably 18,000 pg of modified BoNT / A.

93. The unit dosage form of claim 89, wherein said unit dosage form comprises 624 units to 832 units, preferably 728 units to 770 units, more preferably 749 units of modified BoNT / A.

94. The modified BoNT / A for use, method, use or unit dosage form according to any preceding claim, wherein the modified BoNT / A is a two-chain modified BoNT / A wherein the L chain is linked to the H chain by a disulfide bond, the modified BoNT / A being obtainable by a method comprising contacting a single-chain modified BoNT / A comprising (or consisting of) SEQ ID NO: 6 with a protease that hydrolyzes a peptide bond in its activation loop, thereby converting the single-chain modified BoNT / A into the corresponding two-chain modified BoNT / A.

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

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

97. The modified BoNT / A for use, method, use or unit dosage form according to any one of the preceding claims, wherein the BoNT / BH C The domain comprises one or more substitution mutations selected from the group consisting of: E1191M; S1199Y; V1118M; Y1183M; E1191I; E1191Q; E1191T; S1199F; S1199L; S1201V and combinations thereof, preferably wherein the BoNT / BH C The domain contains the substitution mutations E1191M and S1199Y.

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

99. A kit comprising: (a) a unit dosage form according to any one of claims 84-98; and (b) instructions for use in the treatment of limb spasticity; and (c) optional diluent.

100. The unit dosage form according to any one of claims 84-88 or 94-98 for use in treating limb spasticity in adults.

101. A method for treating limb spasticity in an adult, the method comprising administering to the individual a dosage form according to any one of claims 84-88 or 94-98.

102. Use of a unit dosage form according to any one of claims 84-88 or 94-98 in the manufacture of a medicament for treating limb spasticity in adults.

103. The unit dosage form according to any one of claims 89-98, for use in the treatment of limb spasticity in children.

104. A method for treating limb spasticity in a child, the method comprising administering to a subject a dosage form according to any one of claims 89-98.

105. Use of the unit dosage form according to any one of claims 89-98 in the manufacture of a medicament for treating limb spasticity in children.

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