Treatment of upper facial lines

Abstract

The present invention provides a modified BoNT / A for use in treating facial lines, wherein the modified BoNT / A is administered by intramuscular injection at multiple sites on a subject's face, wherein the modified BoNT / A is administered by a unit dose of greater than 1754 pg (e.g., greater than 1800 pg) of modified BoNT / A per site, wherein the multiple sites are selected from: up to two sites of the corrugator supercilii and one site of the procerus muscle to treat glabellar lines, up to five sites of the frontalis muscle to treat forehead lines, and up to three sites of the orbicularis oculi muscle to treat lateral canthal lines, wherein the total dose of modified BoNT / A administered during treatment is up to 88,000 pg, wherein the modified BoNT / A is a BoNT / A light chain and a translocation domain (H N domain) and the BoNT / B receptor binding domain (H C domain).

Description

[Technical field]

[0001] The present invention relates to cosmetic treatments (e.g., for treating hyperfunctional facial lines). More particularly, the present invention provides methods of cosmetic treatments that involve administration of a long-acting neurotoxin, and more particularly, methods of treating cosmetic irregularities using a long-acting botulinum neurotoxin. [Background technology]

[0002] Hyperfunctional facial lines are common aesthetic irregularities, including, but not limited to, glabellar lines, lateral canthal lines, forehead lines and wrinkles, crow's feet, brow lines, nasolabial folds, lip lines, and marionette lines. Upper facial lines can occur in the forehead, glabellar and lateral orbital regions. Wrinkles can also appear in the glabellar and forehead regions with a frowning expression, while wrinkles can appear in the lateral canthal region with a smiling expression. Overly pronounced lines in this region are often mistaken for fatigue, which can make the subject feel quite distressed about their appearance. Instead, overly pronounced facial lines appear as a result of the functional pulling of the underlying muscles, which ultimately creates wrinkles in the skin. In the glabellar complex, these muscles include the corrugator supercilii, procerus and depressor brow pencil, while the orbicularis oculi is involved in the generation of the lateral canthal crease.

[0003] Over the past few decades, as the aging population continues to grow, the demand for cosmetic procedures to reverse the signs of aging, especially in the facial area, has increased.This ever-increasing concern about physical appearance has led to the development of many different products and procedures, such as surgery, various types of resurfacing, and the use of fillers.Since none of these methods are completely risk-free, ongoing research is necessary to provide the safest and most effective methods for treating aging face.

[0004] Although there are five factors that interact in producing what is known as the aging face, there are two main factors (skin and underlying muscles) that interact to produce the more pronounced lines and folds as opposed to wrinkles. Numerous therapies have been developed to treat the skin factors of wrinkles and lines and folds, among them various types of resurfacing, dermatological products and injections for soft tissue augmentation.

[0005] An alternative cosmetic procedure for treating the appearance of facial wrinkles involves the administration of neurotoxins, specifically botulinum neurotoxins, to the muscles underlying the facial skin. Dysport® is a pharmaceutical product that contains the pharmaceutical ingredient BoNT / A hemagglutinin complex (BTX-A-HAC), isolated and purified from Clostridium botulinum type A strains. Several other medicinal BoNT / A products, naturally produced by Clostridium botulinum, are also commercially available.

[0006] BoNT / A selectively inhibits the release of acetylcholine from presynaptic nerve terminals, thus blocking cholinergic transmission at the neuromuscular junction, inducing muscle contraction and reduced muscle tone, and relaxing the injected muscle.

[0007] Dysport® is approved for the treatment of glabellar lines with a maximum total dose of up to 50 units to be administered over the corrugator supercilii and procerus muscles (see FIG. 1). The clinical effect of Dysport can last up to four months. Repeated dosing in clinical studies has demonstrated continued efficacy with up to four repeated doses. However, Dysport must be administered more frequently than every three months. Dysport has not been FDA approved to date for the treatment of forehead lines and lateral canthal lines.

[0008] To avoid systemic neurological effects, many clostridial toxin-based cosmetic treatments utilize direct administration of therapeutic clostridial toxins to a given target site (e.g., target tissue). A problem with administering clostridial toxin-based therapeutics in this manner is the spread of the toxin away from the administration site and into surrounding tissues or the systemic circulation. Diffusion of the toxin away from the target tissue is believed to be responsible for undesirable side effects, which in extreme cases can be life-threatening. This can be of particular concern when clostridial toxin therapeutics (e.g., BoNT therapeutics) are used at high doses, concentrations, and injection volumes. Adverse effects associated with this problem that have been reported for commercially available BoNT / A therapeutics include asthenia, generalized muscle weakness, diplopia, ptosis, dysphagia, dysphonia, dysarthria, urinary incontinence, and difficulty breathing. Difficulty swallowing and breathing can be life-threatening, and deaths have been reported that are associated with the spread of toxin effects.

[0009] The present invention overcomes one or more of the problems set forth above. Summary of the Invention

[0010] The present inventors have surprisingly found that modified BoNT / A finds particular utility in the cosmetic treatment of facial wrinkles. Modified BoNT / A contains the BoNT / A light chain and translocation domain and the BoNT / B receptor binding domain (H CThe modified BoNT / A may contain a modified amino acid residue (e.g., a carboxyl group) that results in a modified BoNT / A that exhibits increased retention at (reduced diffusion from) the site of administration and increased duration of action (e.g., 6-9 months). Advantageously, the modified BoNT / A has an improved safety profile when compared to unmodified BoNT / A (e.g., Dysport®). This improved safety profile may be expressed by the high safety factor described herein for the modified BoNT / A. The modified BoNT / A may contain one or more modifications of surface-exposed amino acid residues that result in an increased net positive charge. The increased charge promotes electrostatic interactions between the polypeptide and anionic extracellular components, thereby promoting binding between the polypeptide and the cell surface. In turn, this increases retention at (reduced diffusion from) the site of administration and results in an increased duration of action (e.g., 6-9 months).

[0011] Based on the preclinical data herein (see Example 11), it has been shown that a higher total amount of modified BoNT / A can be administered to a subject and achieve a similar safety profile as unmodified BoNT / A (e.g., Dysport®) at such high doses. Therefore, modified BoNT / A can be injected into a larger number of muscles / sites in the cosmetic treatment of facial wrinkles before reaching the maximum total dose. This is an important, advantageous finding, and provides clinicians with a greater range of treatment options while improving the cosmetic treatment of facial wrinkles. The treatment can be improved in that it provides a longer-lasting treatment (resulting in less frequent administration) and / or is tailorable to the subject, e.g., allows clinicians to administer to specific sites according to the aesthetic needs of the subject, when compared to treatment with unmodified BoNT / A (e.g., Dysport®). The treatment of the present invention is improved compared to conventional treatment regimens.

[0012] Furthermore, the present invention provides a convenient, safe, and effective single dose unit as well as the total (maximum) dose that can be safely administered in a single treatment. The present invention also provides a guide to the number of times that said dose unit can be administered into muscle (including the number of injection sites per muscle) without resulting patient toxicity. Treatments consistent with the present invention are therefore less complicated for clinicians and help to avoid under-dosing and / or over-dosing. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] In one aspect, the invention provides a modified BoNT / A for use in treating facial lines (e.g., for a longer period than that treated with an unmodified BoNT / A (e.g., SEQ ID NO: 2)), wherein the modified BoNT / A is administered by intramuscular injection at multiple sites on the face of a subject; Here, the modified BoNT / A is administered at a unit dose of greater than 73 units of modified BoNT / A per site, where 1 unit is the calculated median lethal dose in mice (LD 50 ) is the amount of modified BoNT / A corresponding to wherein the plurality of sites is selected from: up to two sites on the corrugator supercilii and one site on the procerus procerus to treat glabellar lines; Up to five sites on the frontalis muscle to treat forehead lines, as well as up to three sites of the orbicularis oculi muscle to treat lateral canthal lines (e.g., up to three sites of the outer portion of the orbicularis oculi muscle to treat lateral canthal lines); wherein the total dose of modified BoNT / A administered during treatment is up to 3,661 units; and Here, the modified BoNT / A comprises a BoNT / A light chain and translocation domain and a BoNT / B receptor binding domain (H C domain).

[0014] Corresponding uses (in the manufacture of a medicament) and methods of treatment of facial lines are also provided.

[0015] In a related aspect, the invention provides a method of treating facial wrinkles (e.g., for a longer period of time than treatment with unmodified BoNT / A (e.g., SEQ ID NO:2)), the method comprising administering modified botulinum neurotoxin A (BoNT / A) by intramuscular injection at multiple sites on the face of a subject; Here, the modified BoNT / A is administered at a unit dose of greater than 73 units of modified BoNT / A per site, where 1 unit is the calculated median lethal dose in mice (LD 50 ) is the amount of modified BoNT / A corresponding to wherein the plurality of sites is selected from: up to two sites on the corrugator supercilii and one site on the procerus procerus to treat glabellar lines; Up to five sites on the frontalis muscle to treat forehead lines, as well as up to three sites of the orbicularis oculi muscle to treat lateral canthal lines (e.g., up to three sites of the outer portion of the orbicularis oculi muscle to treat lateral canthal lines); wherein the total dose of modified BoNT / A administered during treatment is up to 3,661 units; and Here, the modified BoNT / A comprises a BoNT / A light chain and translocation domain and a BoNT / B receptor binding domain (H C domain).

[0016] In another related aspect, the invention provides for the use of a modified botulinum neurotoxin A (BoNT / A) in the manufacture of a medicament for treating facial lines (e.g., for a longer period of time than would be treated with an unmodified BoNT / A (e.g., SEQ ID NO: 2)), wherein the modified BoNT / A is administered by intramuscular injection at multiple sites on the face of a subject; Here, the modified BoNT / A is administered at a unit dose of greater than 73 units of modified BoNT / A per site, where 1 unit is the calculated median lethal dose in mice (LD 50 ) is the amount of modified BoNT / A corresponding to wherein the plurality of sites is selected from: up to two sites on the corrugator supercilii and one site on the procerus procerus to treat glabellar lines; Up to five sites on the frontalis muscle to treat forehead lines, and up to three sites of the orbicularis oculi muscle to treat lateral canthal lines (e.g., up to three sites of the outer portion of the orbicularis oculi muscle to treat lateral canthal lines); wherein the total dose of modified BoNT / A administered during treatment is up to 3,661 units; and Here, the modified BoNT / A comprises a BoNT / A light chain and translocation domain and a BoNT / B receptor binding domain (H C domain).

[0017] In one aspect, the invention provides a modified BoNT / A for use in treating facial lines (e.g., for a longer period than that treated with an unmodified BoNT / A (e.g., SEQ ID NO: 2)), wherein the modified BoNT / A is administered by intramuscular injection at multiple sites on the face of a subject; Here, the modified BoNT / A is administered at a unit dose of greater than 73 units of modified BoNT / A per site, where 1 unit is the calculated median lethal dose in mice (LD 50 ) is the amount of modified BoNT / A corresponding to wherein the plurality of sites is selected from: up to two sites on the corrugator supercilii and one site on the procerus procerus to treat glabellar lines; Up to five sites on the frontalis muscle to treat forehead lines, as well as up to three sites of the orbicularis oculi muscle to treat lateral canthal lines (e.g., up to three sites of the outer portion of the orbicularis oculi muscle to treat lateral canthal lines); Preferably, wherein the multiple sites include (or consist of) up to two sites of the corrugator supercilii and one site of the procerus for treating glabellar lines; wherein the total dose of modified BoNT / A administered during treatment is up to 1144 units (e.g., up to 1019 units); and Here, the modified BoNT / A comprises a BoNT / A light chain and translocation domain and a BoNT / B receptor binding domain (H C domain).

[0018] In a related aspect, the invention provides a method of treating facial wrinkles (e.g., for a longer period than treatment with unmodified BoNT / A (e.g., SEQ ID NO:2)), the method comprising administering modified botulinum neurotoxin A (BoNT / A) by intramuscular injection at multiple sites on the face of a subject; Here, the modified BoNT / A is administered at a unit dose of greater than 73 units of modified BoNT / A per site, where 1 unit is the calculated median lethal dose in mice (LD 50 ) is the amount of modified BoNT / A corresponding to wherein the plurality of sites is selected from: up to two sites on the corrugator supercilii and one site on the procerus procerus to treat glabellar lines; Up to five sites on the frontalis muscle to treat forehead lines, as well as up to three sites of the orbicularis oculi muscle to treat lateral canthal lines (e.g., up to three sites of the outer portion of the orbicularis oculi muscle to treat lateral canthal lines); Preferably, wherein the multiple sites include (or consist of) up to two sites of the corrugator supercilii and one site of the procerus for treating glabellar lines; wherein the total dose of modified BoNT / A administered during treatment is up to 1144 units (e.g., up to 1019 units); and Here, the modified BoNT / A comprises a BoNT / A light chain and translocation domain and a BoNT / B receptor binding domain (H C domain).

[0019] In another related aspect, the invention provides the use of a modified botulinum neurotoxin A (BoNT / A) in the manufacture of a medicament for treating facial lines (e.g., for a longer period than that treated with an unmodified BoNT / A (e.g., SEQ ID NO: 2)), wherein the modified BoNT / A is administered by intramuscular injection at multiple sites on the face of a subject; Here, the modified BoNT / A is administered at a unit dose of greater than 73 units of modified BoNT / A per site, where 1 unit is the calculated median lethal dose in mice (LD 50 ) is the amount of modified BoNT / A corresponding to wherein the plurality of sites is selected from: up to two sites on the corrugator supercilii and one site on the procerus procerus to treat glabellar lines; Up to five sites on the frontalis muscle to treat forehead lines, as well as up to three sites of the orbicularis oculi muscle to treat lateral canthal lines (e.g., up to three sites of the outer portion of the orbicularis oculi muscle to treat lateral canthal lines); Preferably, wherein the multiple sites include (or consist of) up to two sites of the corrugator supercilii and one site of the procerus for treating glabellar lines; wherein the total dose of modified BoNT / A administered during treatment is up to 1144 units (e.g., up to 1019 units); and Here, the modified BoNT / A comprises a BoNT / A light chain and translocation domain and a BoNT / B receptor binding domain (H C domain).

[0020] In another aspect, the invention provides a modified BoNT / A for use in treating facial lines (e.g., for a longer period than that treated with an unmodified BoNT / A (e.g., SEQ ID NO: 2)), wherein the modified BoNT / A is administered by intramuscular injection at multiple sites on the face of a subject; wherein the modified BoNT / A is administered in a unit dose of greater than 1754 pg (e.g., greater than 1800 pg) of modified BoNT / A per site; wherein the plurality of sites is selected from: up to two sites on the corrugator supercilii and one site on the procerus procerus to treat glabellar lines; Up to five sites on the frontalis muscle to treat forehead lines, as well as up to three sites of the orbicularis oculi muscle to treat lateral canthal lines (e.g., up to three sites of the outer portion of the orbicularis oculi muscle to treat lateral canthal lines); wherein the total dose of modified BoNT / A administered during treatment is up to 88,000 (e.g., up to 80,000 pg); and Here, the modified BoNT / A comprises a BoNT / A light chain and translocation domain and a BoNT / B receptor binding domain (H C domain).

[0021] In a related aspect, the invention provides a method of treating facial wrinkles (e.g., for a longer period than treatment with unmodified BoNT / A (e.g., SEQ ID NO:2)), the method comprising administering modified botulinum neurotoxin A (BoNT / A) by intramuscular injection at multiple sites on the face of a subject; wherein the modified BoNT / A is administered in a unit dose of greater than 1754 pg (e.g., greater than 1800 pg) of modified BoNT / A per site; wherein the plurality of sites is selected from: up to two sites on the corrugator supercilii and one site on the procerus procerus to treat glabellar lines; Up to five sites on the frontalis muscle to treat forehead lines, as well as up to three sites of the orbicularis oculi muscle to treat lateral canthal lines (e.g., up to three sites of the outer portion of the orbicularis oculi muscle to treat lateral canthal lines); wherein the total dose of modified BoNT / A administered during treatment is up to 88,000 (e.g., up to 80,000 pg); and Here, the modified BoNT / A comprises a BoNT / A light chain and translocation domain and a BoNT / B receptor binding domain (H C domain).

[0022] In another related aspect, the invention provides the use of a modified botulinum neurotoxin A (BoNT / A) in the manufacture of a medicament for treating facial lines (e.g., for a longer period of time than that treated with an unmodified BoNT / A (e.g., SEQ ID NO: 2)), wherein the modified BoNT / A is administered by intramuscular injection at multiple sites on the face of a subject; wherein the modified BoNT / A is administered in a unit dose of greater than 1754 pg (preferably greater than 1800 pg, more preferably greater than 2555 pg) of modified BoNT / A per site; wherein the plurality of sites is selected from: up to two sites on the corrugator supercilii and one site on the procerus procerus to treat glabellar lines; Up to five sites on the frontalis muscle to treat forehead lines, as well as up to three sites of the orbicularis oculi muscle to treat lateral canthal lines (e.g., up to three sites of the outer portion of the orbicularis oculi muscle to treat lateral canthal lines); wherein the total dose of modified BoNT / A administered during treatment is up to 88,000 (e.g., up to 80,000 pg); and Here, the modified BoNT / A comprises a BoNT / A light chain and translocation domain and a BoNT / B receptor binding domain (H C domain).

[0023] In another aspect, the invention provides a modified BoNT / A for use in treating facial lines (e.g., for a longer period than that treated with an unmodified BoNT / A (e.g., SEQ ID NO: 2)), wherein the modified BoNT / A is administered by intramuscular injection at multiple sites on the face of a subject; wherein the modified BoNT / A is administered in a unit dose of greater than 1754 pg (e.g., greater than 1800 pg) of modified BoNT / A per site; wherein the plurality of sites is selected from: up to two sites on the corrugator supercilii and one site on the procerus procerus to treat glabellar lines; Up to five sites on the frontalis muscle to treat forehead lines, as well as up to three sites of the orbicularis oculi muscle to treat lateral canthal lines (e.g., up to three sites of the outer portion of the orbicularis oculi muscle to treat lateral canthal lines); Preferably, wherein the multiple sites include (or consist of) up to two sites of the corrugator supercilii and one site of the procerus for treating glabellar lines; wherein the total dose of modified BoNT / A administered during treatment is up to 27,500 (e.g., up to 25,000 pg); and Here, the modified BoNT / A comprises a BoNT / A light chain and translocation domain and a BoNT / B receptor binding domain (H C domain).

[0024] In a related aspect, the invention provides a method of treating facial wrinkles (e.g., for a longer period than treatment with unmodified BoNT / A (e.g., SEQ ID NO:2)), the method comprising administering modified botulinum neurotoxin A (BoNT / A) by intramuscular injection at multiple sites on the face of a subject; wherein the modified BoNT / A is administered in a unit dose of greater than 1754 pg (e.g., greater than 1800 pg) of modified BoNT / A per site; wherein the plurality of sites is selected from: up to two sites on the corrugator supercilii and one site on the procerus procerus to treat glabellar lines; Up to five sites on the frontalis muscle to treat forehead lines, as well as up to three sites of the orbicularis oculi muscle to treat lateral canthal lines (e.g., up to three sites of the outer portion of the orbicularis oculi muscle to treat lateral canthal lines); Preferably, wherein the multiple sites include (or consist of) up to two sites of the corrugator supercilii and one site of the procerus for treating glabellar lines; wherein the total dose of modified BoNT / A administered during treatment is up to 27,500 (e.g., up to 25,000 pg); and Here, the modified BoNT / A comprises a BoNT / A light chain and translocation domain and a BoNT / B receptor binding domain (H Cdomain).

[0025] In another related aspect, the invention provides the use of a modified botulinum neurotoxin A (BoNT / A) in the manufacture of a medicament for treating facial lines (e.g., for a longer period of time than that treated with an unmodified BoNT / A (e.g., SEQ ID NO: 2)), wherein the modified BoNT / A is administered by intramuscular injection at multiple sites on the face of a subject; wherein the modified BoNT / A is administered via a unit dose of greater than 1754 pg (preferably, greater than 1800 pg, more preferably, greater than 2555 pg) of modified BoNT / A per site; wherein the plurality of sites is selected from: up to two sites on the corrugator supercilii and one site on the procerus procerus to treat glabellar lines; Up to five sites on the frontalis muscle to treat forehead lines, as well as up to three sites of the orbicularis oculi muscle to treat lateral canthal lines (e.g., up to three sites of the outer portion of the orbicularis oculi muscle to treat lateral canthal lines); Preferably, the multiple sites include (or consist of) up to two sites of the corrugator supercilii and one site of the procerus for treating glabellar lines; wherein the total dose of modified BoNT / A administered during treatment is up to 27,500 pg (e.g., up to 25,000 pg); and Here, the modified BoNT / A comprises a BoNT / A light chain and translocation domain and a BoNT / B receptor binding domain (H C domain).

[0026] Said facial lines may most preferably be referred to as "upper facial lines" and include glabellar lines, forehead lines and lateral canthus lines (as reflected by the above embodiment). The present invention may include treating any one of said facial lines (e.g., upper facial lines) or any combination thereof. For example, all three of glabellar lines, forehead lines and lateral canthus lines may be treated by the method of the present invention. As such, a particular type of facial lines may be targeted. For example, the facial lines to be treated may be glabellar lines. Additionally or alternatively, the facial lines to be treated may be forehead lines. Additionally or alternatively, the facial lines to be treated may be lateral canthus lines.

[0027] Certain combinations of facial lines may be treated by the present invention. Examples of facial line combinations include: Frowning lines and forehead lines, Glabbear and outer canthal lines, Forehead and outer canthal lines.

[0028] A preferred combination of facial lines to be treated by the present invention may be glabellar lines and lateral canthal lines.

[0029] Reference to "muscle" means a muscle that is adversely affected (eg, by excessive stiffness, tension or strain) that contributes to the underlying aesthetic condition that the present invention addresses.

[0030] The first group of muscles is defined by the procerus muscle, the second group of muscles is defined by the corrugator supercilii muscle, the third group of muscles is defined by the orbicularis oculi muscle, and the fourth group of muscles is defined by the frontalis muscle.

[0031] Throughout this disclosure, the term "up to two sites in the corrugator supercilii" may be used interchangeably with the term "up to two sites per corrugator supercilii," noting that there are two corrugator supercilii. As such, for example, modified BoNT / A may be administered at up to four sites in the corrugator supercilii, provided that modified BoNT / A is administered at up to two sites per corrugator supercilii.

[0032] The corrugator is also known as the corrugator supercilii.

[0033] The site of the orbicularis occuli muscle (where the unit dose of modified BoNT / A is administered) is preferably in the outer portion of the orbicularis oculi muscle. The term "outer portion of the orbicularis oculi muscle" may be used synonymously with the term "outer upper orbicularis oculi muscle."

[0034] Administration to "up to three sites of the orbicularis oculi" may include: a) administering a unit dose of a modified BoNT / A to the outer superior orbicularis oculi muscle (e.g., proximal to a first eye of a subject); b) administering a unit dose of the modified BoNT / A to the medial superior orbicularis oculi muscle (e.g., proximal to the first eye of the subject); and c) Administering a unit dose of the modified BoNT / A into the outer inferior orbicularis oculi muscle (e.g., proximal to the first eye of the subject).

[0035] Administration to "up to three sites of the orbicularis oculi" may include: a) administering a unit dose of a modified BoNT / A to the outer superior orbicularis oculi muscle (e.g., proximal to a first eye of a subject); b) administering a unit dose of the modified BoNT / A to a first site in the outer inferior orbicularis oculi muscle (e.g., proximal to a first eye of the subject); and c) administering a unit dose of the modified BoNT / A to a second (different) site in the outer inferior orbicularis oculi muscle (e.g., proximal to the first eye of the subject).

[0036] Throughout this disclosure, the term "up to three sites in the orbicularis oculi muscle" may be used synonymously with the term "up to three sites per orbicularis oculi muscle," noting that there are two orbicularis oculi muscles. Similarly, throughout this disclosure, the term "up to three sites in the outer portion of the orbicularis oculi muscle" may be used synonymously with the term "up to three sites per orbicularis oculi muscle in the outer portion of the orbicularis oculi muscle," noting that there are two orbicularis oculi muscles. As such, for example, modified BoNT / A may be administered at up to six sites in the orbicularis oculi muscle, provided that modified BoNT / A is administered at up to three sites per orbicularis oculi muscle.

[0037] Administration to up to three sites in the orbicularis oculi may include: a) administering a unit dose of modified BoNT / A to the lateral superior orbicularis oculi muscle; b) administering a unit dose of modified BoNT / A to the medial superior orbicularis oculi muscle; and c) Administering a unit dose of modified BoNT / A to the inferior lateral orbicularis oculi muscle. Administration to "up to three sites of the orbicularis oculi" may include: a) administering a unit dose of modified BoNT / A to the outer superior orbicularis oculi muscle (e.g., proximal to the first eye of the subject); b) administering a unit dose of the modified BoNT / A to a first site in the outer inferior orbicularis oculi muscle (e.g., proximal to a first eye of the subject); and c) administering a unit dose of the modified BoNT / A to a second (different) site in the outer inferior orbicularis oculi muscle (e.g., proximal to the subject's first eye).

[0038] References to the "upper" orbicularis oculi muscle refer to the orbicularis oculi muscle of the upper eyelid. Similarly, references to the "lower" orbicularis oculi muscle refer to the orbicularis oculi muscle of the lower eyelid.

[0039] Those of skill in the art will understand that the term "medial" (e.g., from an anatomical perspective) means toward the midline of the body. Similarly, those of skill in the art will understand that "lateral" (e.g., from an anatomical perspective) means away from the midline of the body. Thus: - "Outer" upper orbicularis oculi refers to the portion of the upper eyelid orbicularis oculi that lies away from the midline of the body. - "Medial" upper orbicularis oculi refers to the portion of the upper eyelid orbicularis oculi that lies toward the midline of the body; - "The lateral inferior orbicularis oculi refers to the portion of the orbicularis oculi muscle of the lower eyelid that lies away from the midline of the body.

[0040] The term "lateral upper orbicularis oculi muscle" may be used synonymously with the term "external portion of the orbicularis oculi muscle of the upper eyelid." The term "medial upper orbicularis oculi muscle" may be used synonymously with the term "internal portion of the orbicularis oculi muscle of the upper eyelid." The term "lateral lower orbicularis oculi muscle" may be used synonymously with the term "external portion of the orbicularis oculi muscle of the lower eyelid."

[0041] The orbicularis oculi muscle includes a "pretarsal portion" and a "preseptal portion," either of which can be injected. The orbicularis oculi muscle includes an "orbital portion."

[0042] Preferably, the unit dose administered to the orbital muscle may be administered to the orbital portion of said muscle, more preferably to the orbital portion of the external (e.g., lateral) portion of the orbital muscle. Thus, the multiple sites include (or consist of) up to three sites of the orbital portion of the external (e.g., lateral) portion of the orbital muscle to treat lateral canthal lines.

[0043] Administration to the lateral superior orbicularis oculi muscle can refer to administration to the lateral "pretarsal" superior orbicularis oculi muscle or to the lateral "preseptal" superior orbicularis oculi muscle.

[0044] Administration to the medial superior orbicularis oculi muscle can refer to administration to the medial superior "pretarsal" orbicularis oculi muscle or to the medial superior "preseptal" orbicularis oculi muscle.

[0045] Administration to the outer lower orbicularis oculi muscle can refer to administration to the outer lower "pretarsal" orbicularis oculi muscle or to the outer lower "preseptal" orbicularis oculi muscle.

[0046] When administered to the lateral superior orbicularis oculi muscle, the unit dose may be administered to the anterior portion of the diaphragm (in other words, to the lateral "pre-diaphragm" superior orbicularis oculi muscle). When administered to the medial superior orbicularis oculi muscle, the unit dose may be administered to the anterior portion of the diaphragm (in other words, to the medial superior "pre-diaphragm" orbicularis oculi muscle). When administered to the lateral inferior orbicularis oculi muscle, the unit dose may be administered to the anterior portion of the diaphragm (in other words, to the lateral inferior "pre-diaphragm" orbicularis oculi muscle).

[0047] The number of sites to which the modified BoNT / A is administered can vary depending on the facial lines to be treated.

[0048] For example, the facial wrinkle to be treated may be a glabellar wrinkle, and the term "multiple sites" may mean up to three sites, where the multiple sites include (or consist of) up to two sites of the corrugator supercilii and one site of the procerus muscle for treating the glabellar wrinkle.To treat the glabellar wrinkle, it is preferred that two unit doses of modified BoNT / A are administered to two different sites of the corrugator supercilii and one unit dose to one site of the procerus muscle (e.g., so that three unit doses are administered at three different sites).Glabellar wrinkles on one side of the face may be treated, and the term "multiple sites" may mean up to three sites as defined above, so that the subject receives up to three unit doses of modified BoNT / A.

[0049] The facial wrinkles to be treated may be glabellar wrinkles, and the term "multiple sites" may mean up to six sites, where the unit dose of modified BoNT / A is administered at up to two sites per corrugator supercilii and one site per procerus muscle to treat glabellar wrinkles.To treat glabellar wrinkles, it is preferred that two unit doses of modified BoNT / A are administered at two different sites per corrugator supercilii and one unit dose is administered at one site per procerus muscle (e.g., so that five unit doses are administered at five different sites).Glabellar wrinkles on both sides of the face may be treated, and the term "multiple sites" may mean up to six sites as defined above, so that the subject receives up to six unit doses of modified BoNT / A.

[0050] The facial lines to be treated may be forehead lines, and the term "multiple sites" may refer to up to 2, 3, 4 or 5 sites on the frontalis muscle for treating forehead lines. Preferably, five unit doses are administered to five different sites on the frontalis (e.g., such that five unit doses are administered to five different sites) for treating forehead lines.

[0051] The facial wrinkles to be treated may be lateral canthus wrinkles, and the term "multiple sites" may mean up to two or three sites of the orbicularis oculi muscle (e.g., up to two or three sites of the outer part of the orbicularis oculi muscle). To treat lateral canthus wrinkles, three unit doses of modified BoNT / A are preferably administered to three different sites of the orbicularis oculi muscle (e.g., so that three unit doses are administered at three different sites). The lateral canthus wrinkles on one side of the face may be treated, and "multiple sites" may mean up to three sites as defined above, so that the subject receives up to three unit doses of modified BoNT / A.

[0052] The facial wrinkles to be treated may be lateral canthus wrinkles, and the term "multiple sites" may mean up to two or three sites per orbicularis oculi muscle (e.g., up to two or three sites per orbicularis oculi muscle in the outer part of the orbicularis oculi muscle). To treat lateral canthus wrinkles, it is preferred that three unit doses of modified BoNT / A are administered to three different sites per orbicularis oculi muscle (e.g., so that six unit doses are administered to six different sites). The lateral canthus wrinkles on both sides of the face may be treated, and "multiple sites" may mean up to six sites as defined above, so that the subject receives up to six unit doses of modified BoNT / A.

[0053] The facial lines to be treated may be glabellar lines and forehead lines, and the term "multiple sites" may mean up to eight sites, where multiple sites include (or consist of) up to two sites of the corrugator supercilii and one site of the procerus muscle for treating glabellar lines and up to five sites of the frontalis muscle for treating forehead lines. It is preferred that two unit doses of modified BoNT / A are administered at two different sites of the corrugator supercilii to treat glabellar lines, one unit dose is administered at one site of the procerus muscle, and five unit doses are administered at five different sites of the frontalis muscle to treat forehead lines (e.g., so that eight unit doses are administered at eight different sites).

[0054] The facial lines to be treated may be glabellar lines and forehead lines, and the term "multiple sites" may mean up to 10 sites, where multiple sites include (or consist of) up to 2 sites per corrugator supercilii and one site per procerus muscle for treating glabellar lines, and up to 5 sites per frontalis muscle for treating forehead lines. It is preferred that 2 unit doses of modified BoNT / A are administered at 2 different sites per corrugator supercilii, 1 unit dose is administered at 1 site per procerus muscle, and 5 unit doses are administered at 5 different sites per frontalis muscle for treating forehead lines (e.g., 10 unit doses are administered at 10 different sites).

[0055] The facial wrinkles to be treated may be glabellar and lateral canthus wrinkles, and the term "multiple sites" may mean up to 3, 4, 5 or 6 sites, where multiple sites include (or consist of) up to 2 sites of the corrugator supercilii and 1 site of the procerus muscle for treating glabellar wrinkles, and up to 2 or 3 sites of the orbicularis oculi (e.g., up to 2 or 3 sites of the outer part of the orbicularis oculi) for treating lateral canthus wrinkles. It is preferred that two unit doses of modified BoNT / A are administered to two different sites of the corrugator supercilii and one unit dose is administered to one site of the procerus muscle for treating glabellar wrinkles, and three unit doses of modified BoNT / A are administered to three different sites of the orbicularis oculi (e.g., six unit doses are administered at six different sites) for treating lateral canthus wrinkles.

[0056] The facial lines to be treated may be glabellar lines and lateral canthal lines, and the term "multiple sites" may mean up to 3, 4, 5, 6, 7, 8, 9, 10, or 11 sites, where multiple sites include (or consist of) up to two sites per corrugator supercilii and one site per procerus muscle for treating glabellar lines, and up to two or three sites per orbicularis oculi (e.g., up to two or three sites per orbicularis oculi in the outer portion of the orbicularis oculi) for treating lateral canthal lines. Preferably, to treat glabellar lines, two unit doses of the modified BoNT / A are administered to two different sites for the corrugator supercilii muscle, one unit dose is administered to one site for the procerus muscle, and to treat lateral canthal lines, three unit doses of the modified BoNT / A are administered to three different sites for the orbicularis oculi muscle (e.g., resulting in 11 unit doses administered to 11 different sites).

[0057] The facial lines to be treated may be brow lines and lateral canthus lines, and the term "multiple sites" may mean up to 4, 5, 6, 7 or 8 sites, where multiple sites include (or consist of) up to 2, 3, 4 or 5 sites of the frontalis muscle to treat brow lines and up to 2 or 3 sites of the orbicularis oculi muscle (e.g., up to 2 or 3 sites of the outer part of the orbicularis oculi muscle) to treat lateral canthus lines. It is preferred that 5 unit doses are administered to 5 different sites of the frontalis muscle to treat brow lines, and 3 unit doses of modified BoNT / A are administered to 3 different sites of the orbicularis oculi muscle to treat lateral canthus lines (e.g., 8 unit doses are administered to 8 different sites).

[0058] The facial lines to be treated may be brow lines and lateral canthus lines, and the term "multiple sites" may mean up to 4, 5, 6, 7, 8, 9, 10 or 11 sites, where multiple sites include (or consist of) up to 2, 3, 4 or 5 sites on the frontalis muscle for treating brow lines and up to 2 or 3 sites on the orbicularis muscle for treating lateral canthus lines (e.g., up to 2 or 3 sites on the outer part of the orbicularis muscle). It is preferred that 5 unit doses are administered to 5 different sites on the frontalis muscle for treating brow lines, and 3 unit doses of modified BoNT / A are administered to 3 different sites on the orbicularis muscle for treating lateral canthus lines (e.g., 11 unit doses are administered to 11 different sites).

[0059] The facial lines to be treated may be glabellar lines, forehead lines and lateral canthal lines, and the term "multiple sites" may mean up to 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 sites, where the multiple sites include (or consist of): - up to two sites on the corrugator supercilii and one site on the procerus procerus to treat glabellar lines; - up to 2, 3, 4 or 5 sites on the frontalis muscle to treat forehead lines, and - Up to two or three sites on the orbicularis oculi muscle (eg, up to two or three sites on the outer portion of the orbicularis oculi muscle) to treat lateral canthal lines.

[0060] Preferred: - for treating glabellar lines, two unit doses of modified BoNT / A are administered to two different sites in the corrugator supercilii and one unit dose is administered to one site in the procerus muscle; - 5 unit doses are administered to 5 different sites of the frontalis muscle to treat forehead lines; and - for treating lateral canthal lines, three unit doses of modified BoNT / A are administered to three different sites of the orbicularis oculi muscle (e.g., its outer portion); - (eg, so that 11 unit doses are administered at 11 different sites).

[0061] The facial lines to be treated may be glabellar lines, forehead lines and lateral canthal lines, and the term "multiple sites" may mean up to 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 sites, where the multiple sites include (or consist of): - Up to two sites per corrugator supercilii and one site per procerus to treat glabellar lines; - up to 2, 3, 4 or 5 sites on the frontalis muscle to treat forehead lines, and - Up to 2 or 3 sites per orbicularis oculi muscle to treat lateral canthal lines (e.g., up to 2 or 3 sites per orbicularis oculi muscle in the outer portion) Preferred: - for treating glabellar lines, two unit doses of modified BoNT / A are administered at two different sites on the corrugator supercilii and one unit dose is administered at one site on the procerus muscle; - 5 unit doses are administered to 5 different sites of the frontalis muscle to treat forehead lines; and - To treat lateral canthal lines, three unit doses of modified BoNT / A are administered at three different sites per orbicularis oculi muscle (eg, its outer portion).

[0062] - (e.g., resulting in 16 unit doses being administered at 11 different sites).

[0063] Thus, the term "multiple sites" may mean up to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 sites. Preferably, the term "multiple sites" may refer to "up to 16 sites."

[0064] The term "multiple sites" may mean 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 sites. Preferably, the term "multiple sites" may refer to 16 sites.

[0065] Thus, the modified BoNT / A may be administered by intramuscular injection at up to 16 sites on the subject's face, e.g. - up to four sites in the corrugator supercilii (e.g., provided that the modified BoNT / A is administered to a maximum of two sites per corrugator supercilii) and one site in the procerus muscle to treat glabellar lines; - Up to five sites on the frontalis muscle to treat forehead lines, as well as - Up to six sites in the orbicularis oculi muscle to treat lateral canthal lines (provided, for example, that modified BoNT / A is administered to a maximum of three sites per orbicularis oculi muscle).

[0066] The multiple sites may be on the same muscle group (e.g., right and left corrugator supercilii, right and left orbicularis oculi). Similarly, the multiple sites may be on the same muscle (e.g., right or left corrugator supercilii or right or left orbicularis oculi). Similarly, the multiple sites may be on combinations of the muscles and muscle groups. Thus, a personalized treatment regimen may be provided.

[0067] The potency of modified BoNT / A for use according to the present invention is preferably determined using mouse LD 50 Assays in which one unit corresponds to a calculated median lethal dose (LD 50 ) preferably the calculated median lethal intraperitoneal dose in mice.

[0068] In particular, the amount of modified BoNT / A corresponding to 1 unit in the assay is preferably 24.04 pg.

[0069] The term "up to," when used in connection with a value (e.g., up to 3661 units), means up to and includes the recited value. Thus, by way of example, a reference to administering "up to 3661 units" of a modified BoNT / A includes administration of 3661 units of the modified BoNT / A as well as administration of less than 3661 units of the modified BoNT / A. As another example, a reference to administering "up to 80,000 pg" of a modified BoNT / A includes administration of 80,000 pg of the modified BoNT / A as well as administration of less than 80,000 pg of the modified BoNT / A.

[0070] The dose of modified BoNT / A is administered by intramuscular injection at multiple sites. A single unit dose is administered per site. The term "(single) unit dose is administered" means that substantially all of the (single) unit dose is administered. Preferably, only a residual amount (e.g., up to 1%, 0.1% or 0.01%) of the (single) unit dose may remain in the vial in which the modified BoNT / A was reconstituted. However, preferably, all of the (single) unit dose is administered. Depending on the muscle group, the (single) unit dose is usually administered to a muscle selected from the first group (described herein), 2x units are usually administered to a muscle selected from the second group (described herein), 3x units are usually administered to a muscle selected from the third group (described herein), and 5x units are usually administered to a muscle selected from the fourth group (described herein).

[0071] A unit dose can be expressed in terms of units of modified BoNT / A. For example, 24.04 pg can correspond to 1 unit of modified BoNT / A. For example, 2404 pg of BoN / A can be referred to as 100 units of modified BoNT / A.

[0072] The unit dose can be greater than 73 units of modified BoNT / A. The upper limit of the unit dose range can be 3,661, 3000, 2500, 2000, 1500, 1000, 500, or 100 units of modified BoNT / A. The lower limit of the unit dose range can be 75, 100, 150, 200, 300, or 500 units of modified BoNT / A. Preferably, the unit dose of modified BoNT / A is greater than 73 units to 3000 units, 100 units to 2500 units, more preferably 200 units to 2000 units.

[0073] As noted above, the unit dose of modified BoNT / A may be greater than 73 units. The upper limit of the unit dose range may be 300, 250, 200, 150, 100, or 50 units of modified BoNT / A, preferably the upper limit is 300 units. The lower limit of the unit dose range may be 75, 100, 150, 200, 300 units, preferably the lower limit is 100 units. Preferably, the unit dose of modified BoNT / A is 100-300 units of modified BoNT / A, for example 150-250 units.

[0074] The unit dose of the modified BoNT / A can be selected from 100 units to 300 units, 150 units to 250 units, and 175 units to 225 units.

[0075] The unit dose can be more than 1754pg to 8000pg of modified BoNT / A. In other words, the unit dose can be more than 1754pg and up to 8000pg of modified BoNT / A. The upper limit of the unit dose range can be 8,000, 7,000, 6,000, 5,000, 4,000, 3,000, 2,000 or 1,000pg of modified BoNT / A, preferably the upper limit is 5,000pg. The lower limit of the unit dose range can be 1,800, 2,000, 2,500, 3000 or 4,000pg of modified BoNT / A, preferably the lower limit is 1,800. The unit dose of modified BoNT / A may be selected from 1,800 to 8,000 pg, 2,000 to 6,000 pg, and most preferably 3,000 to 6,000 pg. The unit dose of modified BoNT / A may be 4,500 to 5,500 pg of modified BoNT / A, for example, 4,900 to 5,100 pg.

[0076] The unit dose can be greater than 1800 pg of modified BoNT / A. The unit dose can be greater than 1800 pg and up to 7000 pg of modified BoNT / A, for example, greater than 1800 pg and up to 6000 pg of modified BoNT / A. The unit dose can be greater than 2000 pg and up to 5000 pg of modified BoNT / A.

[0077] The unit dose can be greater than 2555 pg. For example, the unit dose can be greater than 2555 pg up to 8000 pg of modified BoNT / A, for example, greater than 2555 pg up to 6000 pg.

[0078] The unit dose can be 2000pg, 2500pg, 3000pg, 4000pg, 4500pg, 5000pg, 5500pg, 6000pg, 6500pg, 7000pg, 7500pg or 8000pg of the modified BoNT / A.

[0079] More preferably, the unit dose is 2000-3500 pg of modified BoNT / A. For example, the unit dose can be 2250 pg-3250 pg of modified BoNT / A. A particularly preferred unit dose can be about 2500 pg (e.g., 2500 pg ± 10%) of modified BoNT / A, for example, the unit dose can be preferably 2500 pg of modified BoNT / A. Another preferred unit dose can be about 3000 pg (e.g., 3000 pg ± 10%) of modified BoNT / A, for example, the unit dose can be 3000 pg of modified BoNT / A.

[0080] The unit dose may be 3500-5500 pg of modified BoNT / A. For example, the unit dose may be 3800 pg-4200 pg of modified BoNT / A. The unit dose may be 4800-5200 pg of modified BoNT / A. A particularly preferred unit dose may be about 4000 pg (e.g., 4000 pg ± 10%) of modified BoNT / A, for example, the unit dose may be 4000 pg of modified BoNT / A. Another particularly preferred unit dose may be about 5000 pg (e.g., 5000 pg ± 10%) of modified BoNT / A, for example, the unit dose may be 5000 pg of modified BoNT / A.

[0081] The unit dose can be expressed above in terms of "units" and in terms of the amount in pg. The unit dose of the modified BoNT / A can also be expressed simultaneously in both units and amount (pg).

[0082] When treating facial lines, the modified BoNT / A may be administered to multiple sites selected from up to two sites of the corrugator supercilii and one site of the procerus muscle to treat glabellar lines, up to five sites of the frontalis muscle to treat forehead lines, and up to three sites of the outer portion of the orbicularis oculi muscle to treat lateral canthal lines.

[0083] The total dose administered when carrying out the treatment regimen of the present invention can be up to 3661 units. In other words, the total amount of modified BoNT / A administered in a given treatment session can be up to 3661 units. The total dose can be up to 3500, 3000, 2500, 2000, 1500 or 1000 units of modified BoNT / A. Preferably, the total dose can be up to 3327.8 units of modified BoNT / A.

[0084] The total dose administered when carrying out the treatment regimen of the present invention can be up to 88000, 85000, 80000, 75000, 70000, 65000, 60000, 55000, 50000, 45000, 40000, 35000 or 30000 pg of modified BoNT / A, preferably up to 80000 pg of modified BoNT / A. Thus, the upper limit of the total dose can be 88000, 85000, 80000, 75000, 70000, 65000, 60000, 55000, 50000, 45000, 40000, 35000 or 30000 pg of modified BoNT / A, preferably 80000 pg of modified BoNT / A. The lower limit of the total dose can be 25000, 20000, 15000, 10000, 5000pg, 2000pg, 1000pg, 500pg or 50pg of modified BoNT / A. For example, the lower limit of the total dose can be 25000, 20000, 15000, 10000 or 5000pg of modified BoNT / A.

[0085] The total dose administered when carrying out the treatment regimen of the present invention can be 7,000pg to 8,000pg of modified BoNT / A. Thus, 7,000pg to 8,000pg of modified BoNT / A can be administered to a subject (e.g., by multiple unit doses as described herein). A particularly preferred total dose can be about 7,500pg (e.g., 7,500pg±10%) of modified BoNT / A, for example, the total dose can be 7,500pg of modified BoNT / A. Thus, about 7,500pg (e.g., 7,500pg±10%) of modified BoNT / A can be administered to a subject, for example, 7,500pg of modified BoNT / A can be administered to a subject (e.g., by multiple unit doses as described herein).

[0086] The total dose administered when carrying out the treatment regimen of the present invention can be 10,000pg to 20,000pg of modified BoNT / A. Thus, 10,000pg to 20,000pg of modified BoNT / A can be administered to a subject (e.g., by multiple unit doses as described herein). For example, the total dose can be 11,500pg to 12,500pg of modified BoNT / A. Thus, 11,500pg to 12,500pg of modified BoNT / A can be administered to a subject (e.g., by multiple unit doses as described herein). A particularly preferred total dose can be about 12,000pg (e.g., 12,000pg±10%) of modified BoNT / A, for example, the total dose can be 12,000pg of modified BoNT / A. Thus, about 12,000 pg (e.g., 12,000 pg ± 10%) of the modified BoNT / A can be administered to a subject, for example, 12,000 pg of the modified BoNT / A can be administered to a subject (e.g., by multiple unit doses as described herein).

[0087] For example, the total dose may be 12,000pg to 13,000pg of modified BoNT / A. Thus, 12,000pg to 13,000pg of modified BoNT / A may be administered to a subject (e.g., by multiple unit doses as described herein). A particularly preferred total dose may be about 12,500pg (e.g., 12,500pg±10%) of modified BoNT / A, e.g., the total dose may be 12,500pg of modified BoNT / A. Thus, about 12,500pg (e.g., 12,500pg±10%) of modified BoNT / A may be administered to a subject, e.g., 12,500pg of modified BoNT / A may be administered to a subject (e.g., by multiple unit doses as described herein).

[0088] The total dose administered to the subject may be 14,000pg to 16,000pg of modified BoNT / A. Thus, 14,000pg to 16,000pg of modified BoNT / A may be administered to the subject (e.g., by multiple unit doses as described herein). A particularly preferred total dose may be about 15,000pg (e.g., 15,000pg±10%) of modified BoNT / A, e.g., the total dose may be 15,000pg of modified BoNT / A. Thus, about 15,000pg (e.g., 15,000pg±10%) of modified BoNT / A may be administered to the subject, e.g., 15,000pg of modified BoNT / A may be administered to the subject (e.g., by multiple unit doses as described herein).

[0089] The total dose administered when carrying out the treatment regimen of the present invention can be 18,000pg to 26,000pg of modified BoNT / A. Thus, 18,000pg to 26,000pg of modified BoNT / A can be administered to a subject (e.g., by multiple unit doses as described herein). For example, the total dose can be 19,000pg to 21,000pg of modified BoNT / A. Thus, 19,000pg to 21,000pg of modified BoNT / A can be administered to a subject (e.g., by multiple unit doses as described herein). A particularly preferred total dose can be about 20,000pg (e.g., 20,000pg±10%) of modified BoNT / A, for example, the total dose can be 20,000pg of modified BoNT / A. Thus, about 20,000 pg (e.g., 20,000 pg ± 10%) of the modified BoNT / A can be administered to a subject, for example, 20,000 pg of the modified BoNT / A can be administered to a subject (e.g., by multiple unit doses as described herein).

[0090] The total dose administered when carrying out the treatment regimen of the present invention can be 22,000pg to 23,000pg of modified BoNT / A. Thus, 22,000pg to 23,000pg of modified BoNT / A can be administered to a subject (e.g., by multiple unit doses as described herein). A particularly preferred total dose can be about 22,500pg (e.g., 22,500pg±10%) of modified BoNT / A, e.g., the total dose can be 22,500pg of modified BoNT / A. Thus, about 22,500pg (e.g., 22,500pg±10%) of modified BoNT / A can be administered to a subject, e.g., 22,500pg of modified BoNT / A can be administered to a subject (e.g., by multiple unit doses as described herein).

[0091] The total dose administered to the subject may be 23,000pg to 25,000pg of modified BoNT / A. Thus, 23,000pg to 25,000pg of modified BoNT / A may be administered to the subject (e.g., by multiple unit doses as described herein). A particularly preferred total dose may be about 24,000pg (e.g., 24,000pg±10%) of modified BoNT / A, e.g., the total dose may be 24,000pg of modified BoNT / A. Thus, about 24,000pg (e.g., 24,000pg±10%) of modified BoNT / A may be administered to the subject, e.g., 24,000pg of modified BoNT / A may be administered to the subject (e.g., by multiple unit doses as described herein).

[0092] The total dose administered to the subject may be 24,000pg to 26,000pg of modified BoNT / A. Thus, 24,000pg to 26,000pg of modified BoNT / A may be administered to the subject (e.g., by multiple unit doses as described herein). A particularly preferred total dose may be about 25,000pg (e.g., 24,000pg±10%) of modified BoNT / A, e.g., the total dose may be 25,000pg of modified BoNT / A. Thus, about 25,000pg (e.g., 25,000pg±10%) of modified BoNT / A may be administered to the subject, e.g., 25,000pg of modified BoNT / A may be administered to the subject (e.g., by multiple unit doses as described herein).

[0093] The total dose administered to the subject may be 27,000pg to 28,000pg of modified BoNT / A. Thus, 27,000pg to 28,000pg of modified BoNT / A may be administered to the subject (e.g., by multiple unit doses as described herein). A particularly preferred total dose may be about 27,500pg (e.g., 27,500pg±10%) of modified BoNT / A, e.g., the total dose may be 27,500pg of modified BoNT / A. Thus, about 27,500pg (e.g., 27,500pg±10%) of modified BoNT / A may be administered to the subject, e.g., 27,500pg of modified BoNT / A may be administered to the subject (e.g., by multiple unit doses as described herein).

[0094] The total dose administered to the subject may be 31,000pg to 33,000pg of modified BoNT / A. Thus, 31,000pg to 33,000pg of modified BoNT / A may be administered to the subject (e.g., by multiple unit doses as described herein). A particularly preferred total dose may be about 32,000pg (e.g., 32,000pg±10%) of modified BoNT / A, e.g., the total dose may be 32,000pg of modified BoNT / A. Thus, about 32,000pg (e.g., 32,000pg±10%) of modified BoNT / A may be administered to the subject, e.g., 32,000pg of modified BoNT / A may be administered to the subject (e.g., by multiple unit doses as described herein).

[0095] The total dose administered to the subject may be 35,000pg to 37,000pg of modified BoNT / A. Thus, 35,000pg to 37,000pg of modified BoNT / A may be administered to the subject (e.g., by multiple unit doses as described herein). A particularly preferred total dose may be about 36,000pg (e.g., 36,000pg±10%) of modified BoNT / A, e.g., the total dose may be 36,000pg of modified BoNT / A. Thus, about 36,000pg (e.g., 36,000pg±10%) of modified BoNT / A may be administered to the subject, e.g., 36,000pg of modified BoNT / A may be administered to the subject (e.g., by multiple unit doses as described herein).

[0096] The total dose administered when carrying out the treatment regimen of the present invention may be 35,000-45,000 pg of modified BoNT / A. Thus, 35,000-45,000 pg of modified BoNT / A may be administered to a subject (e.g., by multiple unit doses as described herein). A particularly preferred total dose may be about 40,000 pg (e.g., 40,000 pg ± 10%) of modified BoNT / A, e.g., the total dose may be 40,000 pg of modified BoNT / A. Thus, about 40,000 pg (e.g., 40,000 pg ± 10%) of modified BoNT / A may be administered to a subject, e.g., 40,000 pg of modified BoNT / A may be administered to a subject (e.g., by multiple unit doses as described herein).

[0097] The total dose administered when carrying out the treatment regimen of the present invention may be 43,000-44,000 pg of modified BoNT / A. Thus, 43,000-44,000 pg of modified BoNT / A may be administered to a subject (e.g., by multiple unit doses as described herein). A particularly preferred total dose may be about 44,000 pg (e.g., 44,000 pg ± 10%) of modified BoNT / A, e.g., the total dose may be 44,000 pg of modified BoNT / A. Thus, about 44,000 pg (e.g., 44,000 pg ± 10%) of modified BoNT / A may be administered to a subject, e.g., 44,000 pg of modified BoNT / A may be administered to a subject (e.g., by multiple unit doses as described herein).

[0098] The total dose administered when implementing the treatment regimen of the present invention may be 60,000 to 70,000 pg of modified BoNT / A. Thus, 60,000 to 70,000 pg of modified BoNT / A may be administered to a subject (e.g., by multiple unit doses as described herein). A preferred total dose administered when implementing the treatment regimen of the present invention may be 63,000 to 65,000 pg of modified BoNT / A. Thus, 63,000 to 65,000 pg of modified BoNT / A may be administered to a subject (e.g., by multiple unit doses as described herein). A particularly preferred total dose may be about 64,000 pg (e.g., 64,000 pg ± 10%) of modified BoNT / A, e.g., the total dose may be 64,000 pg of modified BoNT / A. Thus, approximately 64,000 pg (e.g., 64,000 pg ± 10%) of the modified BoNT / A can be administered to a subject, for example, 64,000 pg of the modified BoNT / A can be administered to a subject (e.g., by multiple unit doses as described herein).

[0099] The total dose may be 12,000 pg to 13,000 pg of the modified BoNT / A, and the unit dose may be 2000 to 3500 pg of the modified BoNT / A. Thus, 12,000 pg to 13,000 pg of the modified BoNT / A may be administered to a subject, where the unit dose is 2000 to 3500 pg of the modified BoNT / A.

[0100] A particularly preferred total dose may be about 12,500 pg (e.g., 12,500 pg ± 10%) of modified BoNT / A, for example, the total dose may be 12,500 pg of modified BoNT / A, where the unit dose is 2000-3500 pg of modified BoNT / A. A preferred total dose is 12,500 pg of modified BoNT / A, where the unit dose is 2000-3500 pg of modified BoNT / A. A more preferred total dose is 12,500 pg of modified BoNT / A, where the unit dose is 2500 pg of modified BoNT / A. Thus, 12,500 pg of modified BoNT / A may be administered to a subject, where the unit dose is 2500 pg of modified BoNT / A.

[0101] The total dose may be 14,000 pg to 16,000 pg of the modified BoNT / A, and the unit dose may be 2000 to 3500 pg of the modified BoNT / A. Thus, 14,000 pg to 16,000 pg of the modified BoNT / A may be administered to a subject, where the unit dose is 2000 to 3500 pg of the modified BoNT / A.

[0102] A particularly preferred total dose may be about 15,000 pg (e.g., 15,000 pg ± 10%) of modified BoNT / A, for example, the total dose may be 15,000 pg of modified BoNT / A, where the unit dose is 2000-3500 pg of modified BoNT / A. A preferred total dose is 15,000 pg of modified BoNT / A, where the unit dose is 2000-3500 pg of modified BoNT / A. A more preferred total dose is 15,000 pg of modified BoNT / A, where the unit dose is 2500 pg of modified BoNT / A. Thus, 15,000 pg of modified BoNT / A may be administered to a subject, where the unit dose is 2500 pg of modified BoNT / A.

[0103] The total dose administered when practicing the treatment regimen of the present invention may be 18,000 pg to 26,000 pg of modified BoNT / A, where the unit dose is 3500 to 5500 pg of modified BoNT / A. Thus, 18,000 pg to 26,000 pg of modified BoNT / A may be administered to a subject, where the unit dose is 3500 to 5500 pg of modified BoNT / A.

[0104] A particularly preferred total dose may be about 20,000 pg (e.g., 20,000 pg ± 10%) of modified BoNT / A, for example, the total dose may be 20,000 pg of modified BoNT / A, where the unit dose is 3500-5500 pg of modified BoNT / A. A preferred total dose is 20,000 pg of modified BoNT / A, where the unit dose is 3500-5500 pg of modified BoNT / A. A more preferred total dose is 20,000 pg of modified BoNT / A, where the unit dose is 4000 pg of modified BoNT / A. Thus, 20,000 pg of modified BoNT / A may be administered to a subject, where the unit dose is 4000 pg of modified BoNT / A.

[0105] The total dose administered when practicing the treatment regimen of the present invention may be 23,000 pg to 25,000 pg of modified BoNT / A, where the unit dose is 3500 to 5500 pg of modified BoNT / A. Thus, 23,000 pg to 25,000 pg of modified BoNT / A may be administered to a subject, where the unit dose is 3500 to 5500 pg of modified BoNT / A.

[0106] A particularly preferred total dose may be about 24,000 pg (e.g., 24,000 pg ± 10%) of modified BoNT / A, for example, the total dose may be 24,000 pg of modified BoNT / A, where the unit dose is 3500-5500 pg of modified BoNT / A. A preferred total dose is 24,000 pg of modified BoNT / A, where the unit dose is 3500-5500 pg of modified BoNT / A. A more preferred total dose is 24,000 pg of modified BoNT / A, where the unit dose is 4000 pg of modified BoNT / A. Thus, 24,000 pg of modified BoNT / A may be administered to a subject, where the unit dose is 4000 pg of modified BoNT / A.

[0107] The total dose administered when carrying out the treatment regimen of the present invention may be 43,000-44,000 pg of modified BoNT / A, where the unit dose is 2000-3500 pg of modified BoNT / A. Thus, 43,000-44,000 pg of modified BoNT / A may be administered to a subject, where the unit dose is 2000-3500 pg of modified BoNT / A. A particularly preferred total dose may be about 44,000 pg (e.g., 44,000 pg ± 10%) of modified BoNT / A, where the unit dose is 2000-3500 pg of modified BoNT / A, for example, the total dose may be 44,000 pg of modified BoNT / A, where the unit dose is 2000-3500 pg of modified BoNT / A. Thus, about 44,000 pg (e.g., 44,000 pg ± 10%) of modified BoNT / A may be administered to a subject, where the unit dose is 2000-3500 pg of modified BoNT / A. 44,000 pg of modified BoNT / A may be administered to a subject, where the unit dose is preferably 2000-3500 pg of modified BoNT / A. For example, about 44,000 pg (e.g., 44,000 pg ± 10%) of modified BoNT / A may be administered to a subject, where the unit dose is about 2500 pg (e.g., 2500 pg ± 10%) of modified BoNT / A. 44,000 pg of modified BoNT / A may be administered to a subject, where the unit dose is more preferably 2500 pg of modified BoNT / A.

[0108] The total dose administered when carrying out the treatment regimen of the present invention may be 60,000-70,000 pg of modified BoNT / A, where the unit dose is 3500-5500 pg of modified BoNT / A. Thus, 60,000-70,000 pg of modified BoNT / A may be administered to a subject, where the unit dose is 3500-5500 pg of modified BoNT / A. A preferred total dose administered when carrying out the treatment regimen of the present invention may be 63,000-65,000 pg of modified BoNT / A, where the unit dose is 3500-5500 pg of modified BoNT / A. Thus, 63,000-65,000 pg of modified BoNT / A may be administered to a subject, where the unit dose is 3500-5500 pg of modified BoNT / A. A particularly preferred total dose may be about 64,000 pg (e.g., 64,000 pg ± 10%) of modified BoNT / A, where the unit dose is 3500-5500 pg of modified BoNT / A. For example, the total dose may be 64,000 pg of modified BoNT / A, where the unit dose is 3500-5500 pg of modified BoNT / A. Thus, about 64,000 pg (e.g., 64,000 pg ± 10%) of modified BoNT / A may be administered to a subject, where the unit dose is 3500-5500 pg of modified BoNT / A. About 64,000 pg (e.g., 64,000 pg ± 10%) of the modified BoNT / A may be administered to a subject, where the unit dose is preferably about 4000 pg (e.g., 4,000 pg ± 10%) of the modified BoNT / A. 64,000 pg of the modified BoNT / A may be administered to a subject, where the unit dose is more preferably 4000 pg of the modified BoNT / A.

[0109] The total dosage may vary depending on the facial line or combination of facial lines to be treated. Examples of preferred total dosages for any given facial line are discussed in more detail below.

[0110] The total dose administered when carrying out a treatment regimen for glabellar lines can be up to 30000, 25000, 20000, 15000, 10000 or 5000 pg of modified BoNT / A, preferably up to 25000 pg of modified BoNT / A. A particularly preferred total dose administered when carrying out a treatment regimen for glabellar lines can be about 12,500 pg (e.g., 12,500 pg ± 10%) of modified BoNT / A, for example, the unit dose can be 12,500 pg of modified BoNT / A. Another particularly preferred total dose administered when carrying out a treatment regimen for glabellar lines can be about 20,000 pg (e.g., 20,000 pg ± 10%) of modified BoNT / A, for example, the unit dose can be 20,000 pg of modified BoNT / A.

[0111] The total dose administered when carrying out a treatment regimen for glabeller wrinkles can be 7,000pg to 8,000pg of modified BoNT / A. Thus, to treat glabeller wrinkles, 7,000pg to 8,000pg of modified BoNT / A can be administered to a subject (e.g., by multiple unit doses as described herein). To treat glabeller wrinkles, a particularly preferred total dose can be about 7,500pg (e.g., m7,500pg±10%) of modified BoNT / A, e.g., to treat glabeller wrinkles, the total dose can be 7,500pg of modified BoNT / A. Thus, to treat glabeller lines, about 7,500 pg (e.g., 7,500 pg ± 10%) of the modified BoNT / A may be administered to a subject, for example, to treat glabeller lines, 7,500 pg of the modified BoNT / A may be administered to a subject (e.g., by multiple unit doses as described herein).

[0112] The total dose administered when carrying out a treatment regimen for glabellar lines can be 10,000pg to 20,000pg of modified BoNT / A. Thus, to treat glabellar lines, 10,000pg to 20,000pg of modified BoNT / A can be administered to a subject (e.g., by multiple unit doses as described herein). For example, to treat glabellar lines, the total dose can be 11,500pg to 12,500pg of modified BoNT / A. Thus, to treat glabellar lines, 11,500pg to 12,500pg of modified BoNT / A can be administered to a subject (e.g., by multiple unit doses as described herein). A particularly preferred total dose may be about 12,000 pg (e.g., 12,000 pg ± 10%) of modified BoNT / A, for example, to treat glabellar lines, the total dose may be 12,000 pg of modified BoNT / A. Thus, to treat glabellar lines, about 12,000 pg (e.g., 12,000 pg ± 10%) of modified BoNT / A may be administered to a subject, for example, to treat glabellar lines, 12,000 pg of modified BoNT / A may be administered to a subject (e.g., by multiple unit doses as described herein).

[0113] The total dose administered when carrying out a treatment regimen for glabellar lines can be 12,000pg to 13,000pg of modified BoNT / A. Thus, to treat glabellar lines, 12,000pg to 13,000pg of modified BoNT / A can be administered to a subject (e.g., by multiple unit doses as described herein). To treat glabellar lines, a particularly preferred total dose can be about 12,500pg (e.g., 12,500pg±10%) of modified BoNT / A, e.g., to treat glabellar lines, the total dose can be 12,500pg of modified BoNT / A. Thus, to treat glabellar lines, about 12,500 pg (e.g., 12,500 pg ± 10%) of the modified BoNT / A may be administered to a subject, for example, to treat glabellar lines, 12,500 pg of the modified BoNT / A may be administered to a subject (e.g., by multiple unit doses as described herein).

[0114] When carrying out a treatment regimen for glabellar lines, the total dose administered may be 18,000 pg to 26,000 pg of modified BoNT / A. Thus, to treat glabellar lines, 18,000 pg to 26,000 pg of modified BoNT / A may be administered to a subject (e.g., by multiple unit doses as described herein). For example, to treat glabellar lines, the total dose may be 19,000 pg to 21,000 pg of modified BoNT / A. Thus, to treat glabellar lines, 19,000 pg to 21,000 pg of modified BoNT / A may be administered to a subject (e.g., by multiple unit doses as described herein). For treating glabellar lines, a particularly preferred total dose may be about 20,000 pg (e.g., 20,000 pg ± 10%) of modified BoNT / A, for example, for treating glabellar lines, the total dose may be 20,000 pg of modified BoNT / A. Thus, for treating glabellar lines, about 20,000 pg (e.g., 20,000 pg ± 10%) of modified BoNT / A may be administered to a subject, for example, for treating glabellar lines, 20,000 pg of modified BoNT / A may be administered to a subject (e.g., by multiple unit doses as described herein).

[0115] The total dose administered when performing a treatment regimen for forehead lines can be up to 30,000, 25,000, 20,000, 15,000, 10,000 or 5,000 pg of modified BoNT / A, preferably up to 25,000 pg of modified BoNT / A.

[0116] The total dose administered when carrying out a treatment regimen for forehead wrinkles can be 12,000pg to 13,000pg of modified BoNT / A. Thus, to treat forehead wrinkles, 12,000pg to 13,000pg of modified BoNT / A can be administered to a subject (e.g., by multiple unit doses as described herein). To treat forehead wrinkles, a particularly preferred total dose can be about 12,500pg (e.g., 12,500pg±10%) of modified BoNT / A, e.g., to treat forehead wrinkles, the total dose can be 12,500pg of modified BoNT / A. Thus, to treat forehead lines, about 12,500 pg (e.g., 12,500 pg ± 10%) of the modified BoNT / A may be administered to a subject, for example, to treat forehead lines, 12,500 pg of the modified BoNT / A may be administered to a subject (e.g., by multiple unit doses as described herein).

[0117] The total dose administered when carrying out a treatment regimen to treat forehead wrinkles can be 18,000pg to 26,000pg of modified BoNT / A. Thus, to treat forehead wrinkles, 18,000pg to 26,000pg of modified BoNT / A can be administered to a subject (e.g., by multiple unit doses as described herein). For example, to treat forehead wrinkles, the total dose can be 19,000pg to 21,000pg of modified BoNT / A. Thus, to treat forehead wrinkles, 19,000pg to 21,000pg of modified BoNT / A can be administered to a subject (e.g., by multiple unit doses as described herein). For treating forehead wrinkles, a particularly preferred total dose may be about 20,000 pg (e.g., 20,000 pg ± 10%) of modified BoNT / A, for example, for treating forehead wrinkles, the total dose may be 20,000 pg of modified BoNT / A. Thus, for treating forehead wrinkles, about 20,000 pg (e.g., 20,000 pg ± 10%) of modified BoNT / A may be administered to a subject, for example, for treating forehead wrinkles, 20,000 pg of modified BoNT / A may be administered to a subject (e.g., by multiple unit doses as described herein).

[0118] The total dose administered when performing a treatment regimen for lateral canthal lines can be up to 30,000, 25,000, 20,000, 15,000, 10,000 or 5,000 pg of modified BoNT / A, preferably up to 25,000 pg of modified BoNT / A.

[0119] The total dose administered when carrying out a treatment regimen for lateral canthal wrinkles can be 7,000pg to 8,000pg of modified BoNT / A. Thus, to treat lateral canthal wrinkles, 7,000pg to 8,000pg of modified BoNT / A can be administered to a subject (e.g., by multiple unit doses as described herein). To treat lateral canthal wrinkles, a particularly preferred total dose can be about 7,500pg (e.g., 7,500pg±10%) of modified BoNT / A, e.g., to treat lateral canthal wrinkles, the total dose can be 7,500pg of modified BoNT / A. Thus, to treat lateral canthal lines, about 7,500 pg (e.g., 7,500 pg ± 10%) of the modified BoNT / A may be administered to a subject, for example, to treat lateral canthal lines, 7,500 pg of the modified BoNT / A may be administered to a subject (e.g., by multiple unit doses as described herein).

[0120] The total dose administered when carrying out a treatment regimen for lateral canthal wrinkles can be 10,000 pg to 20,000 pg of modified BoNT / A. Thus, to treat lateral canthal wrinkles, 10,000 pg to 20,000 pg of modified BoNT / A can be administered to a subject (e.g., by multiple unit doses as described herein). For example, to treat lateral canthal wrinkles, the total dose can be 11,500 pg to 12,500 pg of modified BoNT / A. Thus, to treat lateral canthal wrinkles, 11,500 pg to 12,500 pg of modified BoNT / A can be administered to a subject (e.g., by multiple unit doses as described herein). A particularly preferred total dose may be about 12,000 pg (e.g., 12,000 pg ± 10%) of modified BoNT / A, for example, to treat lateral canthal lines, the total dose may be 12,000 pg of modified BoNT / A. Thus, to treat lateral canthal lines, about 12,000 pg (e.g., 12,000 pg ± 10%) of modified BoNT / A may be administered to a subject, for example, to treat lateral canthal lines, 12,000 pg of modified BoNT / A may be administered to a subject (e.g., by multiple unit doses as described herein).

[0121] The total dose administered when carrying out a treatment regimen for lateral canthal wrinkles can be 14,000pg to 16,000pg of modified BoNT / A. Thus, to treat lateral canthal wrinkles, 14,000pg to 16,000pg of modified BoNT / A can be administered to a subject (e.g., by multiple unit doses as described herein). To treat lateral canthal wrinkles, a particularly preferred total dose can be about 15,000pg (e.g., 15,000pg±10%) of modified BoNT / A, e.g., to treat lateral canthal wrinkles, the total dose can be 15,000pg of modified BoNT / A. Thus, to treat lateral canthal lines, about 15,000 pg (e.g., 15,000 pg ± 10%) of the modified BoNT / A may be administered to a subject, for example, to treat lateral canthal lines, 15,000 pg of the modified BoNT / A may be administered to a subject (e.g., by multiple unit doses as described herein).

[0122] The total dose administered when carrying out a treatment regimen for lateral canthal wrinkles can be 23,000pg to 25,000pg of modified BoNT / A. Thus, to treat lateral canthal wrinkles, 23,000pg to 25,000pg of modified BoNT / A can be administered to a subject (e.g., by multiple unit doses as described herein). To treat lateral canthal wrinkles, a particularly preferred total dose can be about 24,000pg (e.g., 24,000pg±10%) of modified BoNT / A, for example, to treat lateral canthal wrinkles, the total dose can be 24,000pg of modified BoNT / A. Thus, to treat lateral canthal lines, about 24,000 pg (e.g., 24,000 pg ± 10%) of the modified BoNT / A may be administered to a subject, for example, to treat lateral canthal lines, 24,000 pg of the modified BoNT / A may be administered to a subject (e.g., by multiple unit doses as described herein).

[0123] When carrying out a treatment regimen for glabellar and forehead wrinkles, the total dose administered may be 18,000pg to 26,000pg of modified BoNT / A. Thus, to treat glabellar and forehead wrinkles, 18,000pg to 26,000pg of modified BoNT / A may be administered to a subject (e.g., by multiple unit doses as described herein). For example, to treat glabellar and forehead wrinkles, the total dose may be 19,000pg to 21,000pg of modified BoNT / A. Thus, to treat glabellar and forehead wrinkles, 19,000pg to 21,000pg of modified BoNT / A may be administered to a subject (e.g., by multiple unit doses as described herein). For treating glabellar and forehead lines, a particularly preferred total dose may be about 20,000 pg (e.g., 20,000 pg ± 10%) of modified BoNT / A, for example, for treating glabellar and forehead lines, a total dose may be 20,000 pg of modified BoNT / A. Thus, for treating glabellar and forehead lines, about 20,000 pg (e.g., 20,000 pg ± 10%) of modified BoNT / A may be administered to a subject, for example, for treating glabellar and forehead lines, 20,000 pg of modified BoNT / A may be administered to a subject (e.g., by multiple unit doses as described herein).

[0124] The total dose administered when carrying out a treatment regimen for glabellar and forehead wrinkles can be 24,000pg to 26,000pg of modified BoNT / A. Thus, to treat glabellar and forehead wrinkles, 24,000pg to 26,000pg of modified BoNT / A can be administered to a subject (e.g., by multiple unit doses as described herein). To treat glabellar and forehead wrinkles, a particularly preferred total dose can be about 25,000pg (e.g., 24,000pg±10%) of modified BoNT / A, for example, to treat glabellar and forehead wrinkles, the total dose can be 25,000pg of modified BoNT / A. Thus, to treat glabellar lines and forehead lines, about 25,000 pg (e.g., 25,000 pg ± 10%) of the modified BoNT / A may be administered to a subject, for example, to treat glabellar lines and forehead lines, 25,000 pg of the modified BoNT / A may be administered to a subject (e.g., by multiple unit doses as described herein).

[0125] The total dose administered when carrying out a treatment regimen for glabellar and forehead wrinkles can be 31,000pg to 33,000pg of modified BoNT / A. Thus, to treat glabellar and forehead wrinkles, 31,000pg to 33,000pg of modified BoNT / A can be administered to a subject (e.g., by multiple unit doses as described herein). To treat glabellar and forehead wrinkles, a particularly preferred total dose can be about 32,000pg (e.g., 32,000pg±10%) of modified BoNT / A, for example, to treat glabellar and forehead wrinkles, the total dose can be 32,000pg of modified BoNT / A. Thus, to treat glabellar lines and forehead lines, approximately 32,000 pg (e.g., 32,000 pg ± 10%) of the modified BoNT / A may be administered to a subject, for example, to treat glabellar lines and forehead lines, 32,000 pg of the modified BoNT / A may be administered to a subject (e.g., by multiple unit doses as described herein).

[0126] The total dose administered when carrying out a treatment regimen for glabellar and forehead wrinkles can be 35,000-45,000 pg of modified BoNT / A. Thus, to treat glabellar and forehead wrinkles, 35,000-45,000 pg of modified BoNT / A can be administered to a subject (e.g., by multiple unit doses as described herein). To treat glabellar and forehead wrinkles, a particularly preferred total dose can be about 40,000 pg (e.g., 40,000 pg±10%) of modified BoNT / A, e.g., to treat glabellar and forehead wrinkles, the total dose can be 40,000 pg of modified BoNT / A. Thus, to treat glabellar lines and forehead lines, about 40,000 pg (e.g., 40,000 pg ± 10%) of the modified BoNT / A may be administered to a subject, for example, to treat glabellar lines and forehead lines, 40,000 pg of the modified BoNT / A may be administered to a subject (e.g., by multiple unit doses as described herein).

[0127] The total dose administered when carrying out a treatment regimen for glabellar and lateral canthal lines can be 14,000pg to 16,000pg of modified BoNT / A. Thus, to treat glabellar and lateral canthal lines, 14,000pg to 16,000pg of modified BoNT / A can be administered to a subject (e.g., by multiple unit doses as described herein). To treat glabellar and lateral canthal lines, a particularly preferred total dose can be about 15,000pg (e.g., 15,000pg±10%) of modified BoNT / A, for example, to treat glabellar and lateral canthal lines, the total dose can be 15,000pg of modified BoNT / A. Thus, to treat glabellar lines and lateral canthal lines, about 15,000 pg (e.g., 15,000 pg ± 10%) of the modified BoNT / A may be administered to a subject, for example, to treat glabellar lines and lateral canthal lines, 15,000 pg of the modified BoNT / A may be administered to a subject (e.g., by multiple unit doses as described herein).

[0128] When carrying out a treatment regimen for glabellar and lateral canthal lines, the total dose administered may be 18,000 pg to 26,000 pg of modified BoNT / A. Thus, to treat glabellar and lateral canthal lines, 18,000 pg to 26,000 pg of modified BoNT / A may be administered to a subject (e.g., by multiple unit doses as described herein). For example, to treat glabellar and lateral canthal lines, the total dose may be 19,000 pg to 21,000 pg of modified BoNT / A. Thus, to treat glabellar and lateral canthal lines, 19,000 pg to 21,000 pg of modified BoNT / A may be administered to a subject (e.g., by multiple unit doses as described herein). For treating glabellar lines and lateral canthus lines, a particularly preferred total dose can be about 20,000 pg (e.g., 20,000 pg±10%) of modified BoNT / A, for example, for treating glabellar lines and lateral canthus lines, a total dose can be 20,000 pg of modified BoNT / A. Thus, for treating glabellar lines and lateral canthus lines, about 20,000 pg (e.g., 20,000 pg±10%) of modified BoNT / A can be administered to a subject, for example, for treating glabellar lines and lateral canthus lines, 20,000 pg of modified BoNT / A can be administered to a subject (e.g., by multiple unit doses as described herein).

[0129] The total dose administered when carrying out the treatment regimen for glabellar and lateral canthal lines can be 22,000pg to 23,000pg of modified BoNT / A. Thus, to treat glabellar and lateral canthal lines, 22,000pg to 23,000pg of modified BoNT / A can be administered to a subject (e.g., by multiple unit doses as described herein). To treat glabellar and lateral canthal lines, a particularly preferred total dose can be about 22,500pg (e.g., 22,500pg±10%) of modified BoNT / A, for example, to treat glabellar and lateral canthal lines, the total dose can be 22,500pg of modified BoNT / A. Thus, to treat glabellar lines and lateral canthal lines, about 22,500 pg (e.g., 22,500 pg ± 10%) of the modified BoNT / A may be administered to a subject, for example, to treat glabellar lines and lateral canthal lines, 22,500 pg of the modified BoNT / A may be administered to a subject (e.g., by multiple unit doses as described herein).

[0130] The total dose administered when carrying out a treatment regimen for glabellar and lateral canthal lines can be 23,000pg to 25,000pg of modified BoNT / A. Thus, to treat glabellar and lateral canthal lines, 23,000pg to 25,000pg of modified BoNT / A can be administered to a subject (e.g., by multiple unit doses as described herein). To treat glabellar and lateral canthal lines, a particularly preferred total dose can be about 24,000pg (e.g., 24,000pg±10%) of modified BoNT / A, for example, to treat glabellar and lateral canthal lines, the total dose can be 24,000pg of modified BoNT / A. Thus, to treat glabellar lines and lateral canthal lines, approximately 24,000 pg (e.g., 24,000 pg ± 10%) of the modified BoNT / A may be administered to a subject, for example, to treat lateral canthal lines, 24,000 pg of the modified BoNT / A may be administered to a subject (e.g., by multiple unit doses as described herein).

[0131] The total dose administered when carrying out a treatment regimen for glabellar and lateral canthal lines can be 27,000pg to 28,000pg of modified BoNT / A. Thus, to treat glabellar and lateral canthal lines, 27,000pg to 28,000pg of modified BoNT / A can be administered to a subject (e.g., by multiple unit doses as described herein). To treat glabellar and lateral canthal lines, a particularly preferred total dose can be about 27,500pg (e.g., 27,500pg±10%) of modified BoNT / A, for example, to treat glabellar and lateral canthal lines, the total dose can be 27,500pg of modified BoNT / A. Thus, to treat glabellar lines and lateral canthal lines, approximately 27,500 pg (e.g., 27,500 pg ± 10%) of the modified BoNT / A may be administered to a subject, for example, to treat glabellar lines and lateral canthal lines, 27,500 pg of the modified BoNT / A may be administered to a subject (e.g., by multiple unit doses as described herein).

[0132] The total dose administered when carrying out a treatment regimen for glabellar and lateral canthal lines can be 31,000pg to 33,000pg of modified BoNT / A. Thus, to treat glabellar and lateral canthal lines, 31,000pg to 33,000pg of modified BoNT / A can be administered to a subject (e.g., by multiple unit doses as described herein). To treat glabellar and lateral canthal lines, a particularly preferred total dose can be about 32,000pg (e.g., 32,000pg±10%) of modified BoNT / A, for example, to treat glabellar and lateral canthal lines, the total dose can be 32,000pg of modified BoNT / A. Thus, to treat glabellar lines and lateral canthal lines, approximately 32,000 pg (e.g., 32,000 pg ± 10%) of the modified BoNT / A may be administered to a subject, for example, to treat glabellar lines and lateral canthal lines, 32,000 pg of the modified BoNT / A may be administered to a subject (e.g., by multiple unit doses as described herein).

[0133] The total dose administered when carrying out a treatment regimen for glabellar and lateral canthal lines can be 35,000pg to 37,000pg of modified BoNT / A. Thus, to treat glabellar and lateral canthal lines, 35,000pg to 37,000pg of modified BoNT / A can be administered to a subject (e.g., by multiple unit doses as described herein). To treat glabellar and lateral canthal lines, a particularly preferred total dose can be about 36,000pg (e.g., 36,000pg±10%) of modified BoNT / A, for example, to treat glabellar and lateral canthal lines, the total dose can be 36,000pg of modified BoNT / A. Thus, to treat glabellar lines and lateral canthal lines, approximately 36,000 pg (e.g., 36,000 pg ± 10%) of the modified BoNT / A may be administered to a subject, for example, to treat glabellar lines and lateral canthal lines, 36,000 pg of the modified BoNT / A may be administered to a subject (e.g., by multiple unit doses as described herein).

[0134] The total dose administered when carrying out a treatment regimen for glabellar and lateral canthal lines can be 43,000-44,000 pg of modified BoNT / A. Thus, to treat glabellar and lateral canthal lines, 43,000-44,000 pg of modified BoNT / A can be administered to a subject (e.g., by multiple unit doses as described herein). To treat glabellar and lateral canthal lines, a particularly preferred total dose can be about 44,000 pg (e.g., 44,000 pg ± 10%) of modified BoNT / A, e.g., to treat glabellar and lateral canthal lines, the total dose can be 44,000 pg of modified BoNT / A. Thus, to treat glabellar lines and lateral canthal lines, approximately 44,000 pg (e.g., 44,000 pg ± 10%) of the modified BoNT / A may be administered to a subject, for example, to treat glabellar lines and lateral canthal lines, 44,000 pg of the modified BoNT / A may be administered to a subject (e.g., by multiple unit doses as described herein).

[0135] The total dose administered when carrying out a treatment regimen for frown lines and lateral canthus lines can be 18,000 pg to 26,000 pg of modified BoNT / A. Thus, to treat frown lines and lateral canthus lines, 18,000 pg to 26,000 pg of modified BoNT / A can be administered to a subject (e.g., by multiple unit doses as described herein). For example, to treat frown lines and lateral canthus lines, the total dose can be 19,000 pg to 21,000 pg of modified BoNT / A. Thus, to treat frown lines and lateral canthus lines, 19,000 pg to 21,000 pg of modified BoNT / A can be administered to a subject (e.g., by multiple unit doses as described herein). For treating frown lines and lateral canthus lines, a particularly preferred total dose can be about 20,000pg (e.g., 20,000pg±10%) of modified BoNT / A, for example, for treating frown lines and lateral canthus lines, a total dose can be 20,000pg of modified BoNT / A. Thus, for treating frown lines and lateral canthus lines, about 20,000pg (e.g., 20,000pg±10%) of modified BoNT / A can be administered to a subject, for example, for treating frown lines and lateral canthus lines, 20,000pg of modified BoNT / A can be administered to a subject (e.g., by multiple unit doses as described herein).

[0136] The total dose administered when carrying out a treatment regimen for frown lines and lateral canthus lines can be 27,000pg to 28,000pg of modified BoNT / A. Thus, to treat frown lines and lateral canthus lines, 27,000pg to 28,000pg of modified BoNT / A can be administered to a subject (e.g., by multiple unit doses as described herein). To treat frown lines and lateral canthus lines, a particularly preferred total dose can be about 27,500pg (e.g., 27,500pg±10%) of modified BoNT / A, for example, to treat frown lines and lateral canthus lines, the total dose can be 27,500pg of modified BoNT / A. Thus, to treat forehead lines and lateral canthal lines, approximately 27,500 pg (e.g., 27,500 pg ± 10%) of the modified BoNT / A may be administered to a subject, for example, to treat forehead lines and lateral canthal lines, 27,500 pg of the modified BoNT / A may be administered to a subject (e.g., by multiple unit doses as described herein).

[0137] The total dose administered when carrying out a treatment regimen for frown lines and lateral canthus lines can be 31,000pg to 33,000pg of modified BoNT / A. Thus, to treat frown lines and lateral canthus lines, 31,000pg to 33,000pg of modified BoNT / A can be administered to a subject (e.g., by multiple unit doses as described herein). To treat frown lines and lateral canthus lines, a particularly preferred total dose can be about 32,000pg (e.g., 32,000pg±10%) of modified BoNT / A, for example, to treat frown lines and lateral canthus lines, the total dose can be 32,000pg of modified BoNT / A. Thus, to treat forehead lines and lateral canthal lines, approximately 32,000 pg (e.g., 32,000 pg ± 10%) of the modified BoNT / A may be administered to a subject, for example, to treat forehead lines and lateral canthal lines, 32,000 pg of the modified BoNT / A may be administered to a subject (e.g., by multiple unit doses as described herein).

[0138] The total dose administered when carrying out a treatment regimen for frown lines and lateral canthus lines can be 43,000-44,000 pg of modified BoNT / A. Thus, to treat frown lines and lateral canthus lines, 43,000-44,000 pg of modified BoNT / A can be administered to a subject (e.g., by multiple unit doses as described herein). To treat frown lines and lateral canthus lines, a particularly preferred total dose can be about 44,000 pg (e.g., 44,000 pg ± 10%) of modified BoNT / A, e.g., to treat frown lines and lateral canthus lines, the total dose can be 44,000 pg of modified BoNT / A. Thus, to treat forehead lines and lateral canthal lines, approximately 44,000 pg (e.g., 44,000 pg ± 10%) of the modified BoNT / A may be administered to a subject, for example, to treat forehead lines and lateral canthal lines, 44,000 pg of the modified BoNT / A may be administered to a subject (e.g., by multiple unit doses as described herein).

[0139] When carrying out a treatment regimen for glabellar lines, forehead lines and lateral canthus lines, the total dose administered may be 43,000-44,000 pg of modified BoNT / A. Thus, to treat glabellar lines, forehead lines and lateral canthus lines, 43,000-44,000 pg of modified BoNT / A may be administered to a subject (e.g., by multiple unit doses as described herein). To treat glabellar lines, forehead lines and lateral canthus lines, a particularly preferred total dose may be about 44,000 pg (e.g., 44,000 pg ± 10%) of modified BoNT / A, e.g., to treat glabellar lines, forehead lines and lateral canthus lines, the total dose may be 44,000 pg of modified BoNT / A. Thus, to treat glabellar lines, forehead lines and lateral canthal lines, approximately 44,000 pg (e.g., 44,000 pg ± 10%) of the modified BoNT / A may be administered to a subject, for example, to treat glabellar lines, forehead lines and lateral canthal lines, 44,000 pg of the modified BoNT / A may be administered to a subject (e.g., by multiple unit doses as described herein).

[0140] The total dose administered when implementing a treatment regimen for glabellar lines, forehead lines and lateral canthal lines can be 60,000-70,000 pg of modified BoNT / A. Thus, to treat glabellar lines, forehead lines and lateral canthal lines, 60,000-70,000 pg of modified BoNT / A can be administered to a subject (e.g., by multiple unit doses as described herein). The preferred total dose administered when implementing a treatment regimen for glabellar lines, forehead lines and lateral canthal lines can be 63,000-65,000 pg of modified BoNT / A. Thus, to treat glabellar lines, forehead lines and lateral canthal lines, 63,000-65,000 pg of modified BoNT / A may be administered to a subject (e.g., by multiple unit doses as described herein).To treat glabellar lines, forehead lines and lateral canthal lines, a particularly preferred total dose may be about 64,000 pg (e.g., 64,000 pg±10%) of modified BoNT / A, e.g., to treat glabellar lines, forehead lines and lateral canthal lines, the total dose may be 64,000 pg of modified BoNT / A. Thus, to treat glabellar lines, forehead lines and lateral canthal lines, approximately 64,000 pg (e.g., 64,000 pg ± 10%) of the modified BoNT / A may be administered to a subject, for example, to treat glabellar lines, forehead lines and lateral canthal lines, 64,000 pg of the modified BoNT / A may be administered to a subject (e.g., by multiple unit doses as described herein).

[0141] When carrying out a treatment regimen for glabellar lines, forehead lines and lateral canthus lines, the total dose administered can be 75,000pg to 88,000pg of modified BoNT / A. Thus, to treat glabellar lines, forehead lines and lateral canthus lines, 75,000pg to 88,000pg of modified BoNT / A can be administered to a subject (e.g., by multiple unit doses as described herein). To treat glabellar lines, forehead lines and lateral canthus lines, a particularly preferred total dose can be about 80,000pg (e.g., 80,000pg±10%) of modified BoNT / A, for example, to treat glabellar lines, forehead lines and lateral canthus lines, the total dose can be 80,000pg of modified BoNT / A. Thus, to treat glabellar lines, forehead lines and lateral canthal lines, approximately 80,000 pg (e.g., 80,000 pg ± 10%) of the modified BoNT / A may be administered to a subject, for example, to treat glabellar lines, forehead lines and lateral canthal lines, 80,000 pg of the modified BoNT / A may be administered to a subject (e.g., by multiple unit doses as described herein).

[0142] If each of glabellar lines, forehead lines and outer canthal lines are treated, the total dose can be up to 88,000 pg (eg, up to 80,000 pg).

[0143] One skilled in the art will take into account cases where the subject has recently undergone (or subsequently undergoes) additional treatment with a Clostridial neurotoxin (e.g., BoNT), for example as part of a cosmetic treatment or treatment for a different indication, and will adapt the treatment regimen appropriately, using routine techniques in the art.

[0144] Administration to multiple muscles according to the present invention preferably occurs in the same treatment session.

[0145] The modified BoNT / A of the present invention preferably has a longer duration of action compared to unmodified BoNT / A (e.g., Dysport®). The term "treating facial lines for a longer period than that treated by unmodified BoNT / A" may mean that facial lines are reduced for a longer period after administration of the modified BoNT / A of the present invention compared to administration of unmodified BoNT / A. The duration of action may be at least 1.25x, 1.5x, 1.75x, 2.0x, or 2.25x longer. The duration of action of the modified BoNT / A may be between 6 and 9 months. For example, the duration of action may be at least 4.5 months (from onset), 5.0 months, 5.5 months, 6 months, 6.5 months, 7.0 months, 7.5 months, 8.0 months, 8.5 months, or 9.0 months. In certain embodiments, the duration of action may be longer than 9.0 months. Preferably, the duration of action may be longer than 9.0 months. The reduction may be determined by comparison with a comparable control subject that is treated with unmodified BoNT / A and shows comparable symptoms. In a period in which the severity of one or more symptoms of the control subject is substantially the same (e.g., the same) as before unmodified BoNT / A treatment, the subject treated with modified BoNT / A according to the present invention may show at least 5%, 10%, 25% or 50% improvement in the comparable one or more symptoms when compared with the severity of one or more symptoms before treatment with modified BoNT / A. Unmodified BoNT / A is preferably SEQ ID NO:2, which exists in a two-chain form. In other words, unmodified BoNT / A described herein is preferably SEQ ID NO:2, which is a two-chain form.

[0146] Treatment may be repeated at an appropriate time period after administration of modified BoNT / A. Given that the duration of action is approximately twice that of unmodified BoNT / A (e.g., Dysport®), it is appropriate to allow a longer period between subsequent administrations than when the subject is treated with unmodified BoNT / A (e.g., Dysport®). The subject may be re-administered modified BoNT / A according to the present invention at least 18, 20, 25 or 30 weeks after the previous administration. For example, the subject may be re-administered modified BoNT / A according to the present invention at least 18 to 45 weeks, preferably 20 to 35 weeks, after the previous administration.

[0147] "Subject" as used herein may be a mammal, for example, a human or other mammal. Preferably, "subject" refers to a human subject. The subject is preferably a subject that does not have a pathology associated with the muscle to which the modified BoNT / A is administered. For example, it is preferred that the subject does not have blepharospasm or hemifacial spasm (e.g., typical hemifacial spasm). It is particularly preferred that the subject does not have blepharospasm.

[0148] The subject of the treatment according to the present invention may be a subject that is unsuitable for treatment with unmodified BoNT / A and / or with another Clostridial neurotoxin. The subject may be a subject that is resistant to treatment with unmodified BoNT / A and / or with another Clostridial neurotoxin. Resistance may occur due to the development of an immune response against the Clostridial neurotoxin, including the production of anti-Clostridial neurotoxin antibodies by the subject.

[0149] The term "treat" or "treating" as used herein refers to the cosmetic treatment of facial lines, and includes preventative treatments (e.g., to prevent the development of facial lines) as well as corrective treatments (treatment of subjects who already have facial lines). Thus, any method, modified BoNT / A for use, or use described herein may be appropriately described as a "cosmetic method for treating said facial lines with said modified BoNT / A." Additionally or alternatively, any method, modified BoNT / A for use, or use described herein may be appropriately described as a "non-therapeutic use of said modified BoNT / A for treating said facial lines."

[0150] Preferably, "treat" or "treating" as used herein means corrective treatment. The term "treat" or "treating" as used herein refers to facial lines and / or symptoms thereof. The treatments described herein may provide a reduction in the level of glabellar lines of at least 5%, 10%, 25% or 50% relative to the level of glabellar lines before treatment. The treatments described herein may provide a reduction in the level of forehead lines of at least 5%, 10%, 25% or 50% relative to the level of forehead lines before treatment. The treatments described herein may provide a reduction in the level of lateral canthal lines of at least 5%, 10%, 25% or 50% relative to the level of lateral canthal lines before treatment. The treatments described herein can provide a reduction in the level of glabellar lines and lateral canthus lines by at least 5%, 10%, 25% or 50% compared to the level of glabellar lines and lateral canthus lines before treatment.The treatments described herein can provide a reduction in the level of glabellar lines and lateral canthus lines by at least 5%, 10%, 25% or 50% compared to the level of glabellar lines and lateral canthus lines before treatment.The treatments described herein can provide a reduction in the level of glabellar lines and lateral canthus lines and lateral canthus lines by at least 5%, 10%, 25% or 50% compared to the level of glabellar lines and lateral canthus lines before treatment. The treatments described herein may provide a reduction in the level of glabellar lines, lateral canthal lines and forehead lines of at least 5%, 10%, 25% or 50% relative to the level of glabellar lines, lateral canthal lines and forehead lines pre-treatment.

[0151] BoNT / A is an example of a clostridial neurotoxin produced by bacteria of the genus Clostridium. Other examples of such clostridial neurotoxins include those produced by C. tetani and by C. botulinum (BoNT) serotypes B-G and X (see WO2018 / 009903 A2) as well as those produced by C. baratii and C. butyricum. The neurotoxins are highly potent and specific, and can poison neurons and other cells to which they are delivered. Clostridial toxins are some of the most potent toxins known. By way of example, botulinum neurotoxins have a median lethal dose (LD) in mice ranging from 0.5 to 5 ng / kg depending on the serotype. 50 ) value. Both tetanus and botulinum toxins act by inhibiting the function of affected neurons, specifically, the release of neurotransmitters. Botulinum toxins act at the neuromuscular junction and inhibit cholinergic transmission in the peripheral nervous system, whereas tetanus toxins act in the central nervous system.

[0152] In nature, clostridial neurotoxins (including BoNT / A) are synthesized as single-chain polypeptides that are post-translationally modified by a proteolytic cleavage event to form two polypeptide chains linked together by a disulfide bond. Cleavage occurs at a specific cleavage site, often referred to as the activation site (e.g., activation loop), located between cysteine ​​residues that provide the interchain disulfide bond. The active form of the toxin is this two-chain form. The two chains are referred to as the heavy chain (H chain), with a molecular weight of approximately 100 kDa, and the light chain (L chain), with a molecular weight of approximately 50 kDa. The H chain is linked to an N-terminal translocation component (H N domain) and the C-terminal targeting component (H C The cleavage site is located between the L chain and the translocation domain component. C After binding of the domain to its target neuron and internalization of the bound toxin into the cell by endosomes, H NThe domain translocates the L chain across the endosomal membrane into the cytosol, where the L chain provides the protease function (also known as a non-cytotoxic protease).

[0153] Non-cytotoxic proteases act by proteolytically cleaving intracellular transport proteins known as SNARE proteins (e.g., SNAP-25, VAMP or syntaxin) - see Gerald K (2002) "Cell and Molecular Biology" (4th edition) John Wiley & Sons, Inc. The acronym SNARE is derived from the term Soluble NSF Attachment Receptor, where NSF stands for N-ethylmaleimide-Sensitive Factor. SNARE proteins are essential for intracellular vesicle fusion and thus for secretion of molecules from cells via vesicle transport. The protease function is a zinc-dependent endopeptidase activity and shows high substrate specificity for SNARE proteins. Thus, once delivered to the desired target cell, non-cytotoxic proteases can inhibit cellular secretion from the target cell. The L-chain proteases of clostridial toxins are non-cytotoxic proteases that cleave SNARE proteins.

[0154] Given the ubiquity of SNARE proteins, clostridial neurotoxins, such as botulinum toxins, have been used successfully in a wide range of therapies.

[0155] For further details regarding the genetic basis of toxin production in C. botulinum and C. tetani, see Henderson et al (1997) in The Clostridia: Molecular Biology and Pathogenesis, Academic press.

[0156] As discussed above, clostridial neurotoxins are formed from two polypeptide chains, a heavy chain (H chain) having a molecular weight of approximately 100 kDa, and a light chain (L chain) having a molecular weight of approximately 50 kDa. The H chain contains a C-terminal targeting component (the receptor binding domain or H C domain) and N-terminal translocation component (H N domain).

[0157] Clostridial neurotoxin domains are described in more detail below.

[0158] Examples of light chain reference sequences include: Botulinum type A neurotoxin: amino acid residues 1-448 Botulinum type B neurotoxin: amino acid residues 1-440

[0159] The reference sequences identified above should be considered as a guide, as slight variations may occur according to subserotype. By way of example, US2007 / 0166332 (hereby incorporated by reference in its entirety) cites slightly different Clostridium sequences: Botulinum type A neurotoxin: Amino acid residues M1 to K448 Botulinum type B neurotoxin: Amino acid residues M1-K441

[0160] A translocation domain is a fragment of the H chain of a clostridial neurotoxin approximately equivalent to the amino-terminal half of the H chain, or the domain which corresponds to that fragment in an intact H chain.

[0161] Examples of reference translocation domains include: Botulinum type A neurotoxin - amino acid residues (449-871) Botulinum type B neurotoxin - amino acid residues (441-858)

[0162] The reference sequences identified above should be considered as a guide, as slight variations may occur according to subserotype. By way of example, US2007 / 0166332 (hereby incorporated by reference) cites slightly different Clostridium sequences: Botulinum type A neurotoxin - amino acid residues (A449~K871) Botulinum type B neurotoxin - amino acid residues (A442 to S858)

[0163] In view of the present invention, various BoNT / AHs containing a translocation domain N Regions that may be useful in embodiments of the present invention include those derived from the heavy chain of BoNT / A. N The region is approximately 410-430 amino acids long and contains the translocation domain. N Studies have shown that the total length of the region is not essential for the translocation activity of the translocation domain. Thus, aspects of this embodiment include, for example, BoNT / AH polypeptides that include a translocation domain having a length of at least 350 amino acids, at least 375 amino acids, at least 400 amino acids, or at least 425 amino acids. N Other aspects of this embodiment include, for example, a BoNT / AH gene that includes a translocation domain having a length of at most 350 amino acids, at most 375 amino acids, at most 400 amino acids, or at most 425 amino acids. N It may include regions.

[0164] H N The term refers to naturally occurring BoNT / AH N Modified BoNT / AHs having portions and non-naturally occurring amino acid sequences and / or synthetic amino acid residues N Preferably, the modified BoNT / AH N The moiety still demonstrates the translocation function described above.

[0165] Clostridial neurotoxin receptor binding domain (H C) Examples of reference sequences include: BoNT / A - N872~L1296 BoNT / B - E859~E1291

[0166] The approximately 50 kDa H of clostridial neurotoxins (e.g., BoNT) C The domain is H CC and H CN It contains two distinct structural features, called domains, each usually of about 25 kDa. The amino acid residues involved in receptor binding are mainly H CC It is believed to be located in the H domain of natural clostridial neurotoxins. C The domain may comprise approximately 400-440 amino acid residues. This fact is confirmed by the following publications, each of which is incorporated herein by reference in its entirety: Umland TC (1997) Nat. Struct. Biol. 4: 788-792; Herreros J (2000) Biochem. J. 347: 199-204; Halpern J (1993) J. Biol. Chem. 268: 15, pp. 11188-11192; Rummel A (2007) PNAS 104: 359-364; Lacey DB (1998) Nat. Struct. Biol. 5: 898-902; Knapp (1998) Am. Cryst. Assoc. Abstract Papers 25: 90; Swaminathan and Eswaramoorthy (2000) Nat. Struct. Biol. 7: 1751-1759; and Rummel A (2004) Mol. Microbiol. 51(3), 631-643.

[0167] (Reference)H CN Examples of domains include: Botulinum type A neurotoxin - amino acid residues (872-1110) Botulinum type B neurotoxin - amino acid residues (859-1097)

[0168] The above sequence positions may vary slightly according to serotype / subtype (see CN Further examples of domains include: Botulinum type A neurotoxin - amino acid residues (874-1110) Botulinum type B neurotoxin - amino acid residues (861-1097)

[0169] (Reference)H CC Examples of domains include: Botulinum type A neurotoxin - amino acid residues (Y1111 to L1296) Botulinum type B neurotoxin - amino acid residues (Y1098 to E1291)

[0170] WO2017 / 191315A1 (herein incorporated by reference) teaches modified BoNT / A (e.g., "chimeric clostridial neurotoxins") and methods for preparing and manufacturing same. Thus, botulinum neurotoxin A (BoNT / A) light chain and translocation domain (BoNT / AH) for use in the present invention are disclosed. N ) and the BoNT / B receptor binding domain (H C The modified BoNT / A including the IL-1 domain can be that taught in WO2017 / 191315A1.

[0171] The modified BoNT / A may be referred to as a "chimeric clostridial neurotoxin." The term "chimeric clostridial neurotoxin" or "chimeric neurotoxin," as used herein, refers to a clostridial neurotoxin that is a combination of a clostridial neurotoxin light chain and a translocation domain (H) derived from a first clostridial neurotoxin serotype. N domain) and a receptor-binding domain (H) attributed to a second distinct Clostridial neurotoxin serotype. C Specifically, a modified BoNT / A (e.g., a chimeric Clostridial neurotoxin) for use in the present invention refers to a neurotoxin comprising (preferably consisting of) a botulinum neurotoxin A (BoNT / A) light chain and a translocation domain (H domain). N domain) and the BoNT / B receptor binding domain (HC The BoNT / A LH domain of a modified BoNT / A (e.g., a chimeric Clostridial neurotoxin) N The domain is BoNT / BH C The modified BoNT / A (e.g., chimeric Clostridial neurotoxin) of the present invention may be referred to as a chimeric botulinum neurotoxin. The chimeric Clostridial neurotoxin is also referred to herein as "BoNT / AB", "mrBoNT / AB" or "BoNT / AB chimera".

[0172] Light chains and heavy chains N The domains (optionally including a complete or partial activation loop, e.g., a complete activation loop if the modified BoNT / A is in a single-chain form and a truncated / partial activation loop if the modified BoNT / A is in a two-chain form) are collectively referred to as the LH N Sometimes called a domain. N The domain is therefore LH c Does not further include the domain.

[0173] The modified BoNT / A is essentially a botulinum neurotoxin A (BoNT / A) light chain and translocation domain (H N domain) and the BoNT / B receptor binding domain (H C domain).

[0174] The term "consists essentially of" as used in this context means that the modified BoNT / A does not further comprise one or more amino acid residues that impart additional functionality to the polypeptide when, for example, administered to a subject. In other words, the modified BoNT / A comprises a botulinum neurotoxin A (BoNT / A) light chain and a translocation domain (H N domain) and the BoNT / B receptor binding domain (H C A polypeptide "consisting essentially of" a botulinum neurotoxin A (BoNT / A) light chain and a translocation domain (H domain) is N domain) and the BoNT / B receptor binding domain (H CThe polypeptide may further comprise one or more amino acid residues (such as those of the nucleotide sequence of the polypeptide of the present invention) which do not confer an additional function to the polypeptide when, for example, administered to a subject. The additional function may include an enzymatic activity, a binding activity, and / or any physiological activity whatsoever.

[0175] The modified BoNT / A may include a non-clostridial neurotoxin sequence in addition to any clostridial neurotoxin sequence, so long as the non-clostridial neurotoxin sequence does not disrupt the ability of the modified BoNT / A (e.g., chimeric clostridial neurotoxin) to achieve its therapeutic effect. Preferably, the non-clostridial neurotoxin sequence does not have catalytic activity, e.g., enzymatic activity. In one embodiment, the modified BoNT / A of the present invention does not include a non-clostridial catalytic activity domain. In one embodiment, the modified BoNT / A does not include an additional catalytic activity domain. In one embodiment, the non-clostridial sequence does not bind to a cellular receptor. In other words, in one embodiment, the non-clostridial sequence is not a ligand of the cellular receptor. The cellular receptor may be a proteinaceous cellular receptor, e.g., an integral membrane protein. Examples of cellular receptors can be found in the IUPHAR Guide to Pharmacology Database, April 2019 edition, available at https: / / www.guidetopharmacology.org / download.jsp#db_reports. The non-clostridial neurotoxin sequence may include a tag, e.g., a His-tag, that aids in purification. In one embodiment, the modified BoNT / A of the invention does not include a label or a site for adding a label, e.g., a sortase acceptor or donor site.

[0176] Preferably, the modified BoNT / A comprises a botulinum neurotoxin A (BoNT / A) light chain and a translocation domain (H N domain) and the BoNT / B receptor binding domain (H C domain).

[0177] The modified BoNT / A comprises a light chain that can exhibit non-cytotoxic protease activity and can cleave SNARE proteins in the cytosol of target neurons. Cell-based and in vivo assays can be used to determine whether a Clostridial neurotoxin that comprises an L chain and a functional cell binding and translocation domain has non-cytotoxic protease activity. Assays such as Digit Abduction Score (DAS) assay, Dorsal Root Ganglion (DRG) assay, Spinal Cord Neuron (SCN) assay and Mouse Phrenic Nerve Hemidiaphragm (PNHD) assay are routine in the art. Suitable assays for determining non-cytotoxic protease activity can be those described in Aoki KR, Toxicon 39: 1815-1820; 2001 or Donald et al (2018), Pharmacol Res Perspect, e00446, 1-14, which are incorporated herein by reference.

[0178] When administered to a subject, the modified BoNT / A is preferably in its active two-chain form, in which the light and heavy chains are linked together by a disulfide bond. BoNT / A (e.g., modified BoNT / A) is defined herein by a polypeptide sequence (SEQ ID NO:), the L-chain portion of the sequence (SEQ ID NO:) may constitute the first chain of a two-chain Clostridial neurotoxin (e.g., a two-chain modified BoNT / A), and the H-chain portion of the sequence (SEQ ID NO:) may constitute the first chain of a two-chain Clostridial neurotoxin (e.g., a two-chain modified BoNT / A). N and H CThe domains together may constitute the second chain of a dichain clostridial neurotoxin (e.g., a dichain modified BoNT / A), where the first and second chains are linked together by a disulfide bond. Those skilled in the art will understand that the protease may cleave at one or more positions in the activation loop of a clostridial neurotoxin (e.g., a modified BoNT / A), preferably at two positions in the activation loop. If cleavage occurs at more than one position (preferably at two positions) in the activation loop, a small fragment of the C-terminal L chain portion of the sequence may not be present in the dichain clostridial neurotoxin sequence (e.g., a dichain modified BoNT / A). In view of this, the sequence of a dichain clostridial neurotoxin (e.g., a dichain modified BoNT / A) may differ slightly from that of the corresponding single-chain clostridial neurotoxin (e.g., a single-chain modified BoNT / A). The small fragment may be 1 to 15 amino acids. In particular, in one embodiment, when Lys-C is used to convert a single-chain modified BoNT / A to a two-chain modified BoNT / A, a small fragment of the C-terminal L-chain portion of the absent sequence can be SEQ ID NO: 9 or 10.

[0179] Most preferably, the modified BoNT / A for use in the present invention comprises a BoNT / A light chain and a translocation domain (BoNT / A LH N domain) and BoNT / BH C The BoNT / A LH domain may be included. N The domain is BoNT / BH C The modified BoNT / A is also referred to as a "BoNT / AB" or a "BoNT / AB chimera."

[0180] LH N The C-terminal amino acid residues of the domain are the same as those of the LH domain of BoNT / A. N and H C Separate domains 3 10 may correspond to the first amino acid residue of the helix, H C The N-terminal amino acid residues of the domain are the same as those of the LH domain in BoNT / B. N and H CSeparate domains 3 10 It may correspond to the second amino acid residue of the helix.

[0181] An example of a BoNT / A polypeptide sequence is provided as SEQ ID NO:2.

[0182] An example of a BoNT / B polypeptide sequence is provided as SEQ ID NO:8 (UniProt Accession No. B1INP5).

[0183] "BoNT / A LH N and H C Separate domains 3 10 References herein to "the first amino acid residue of the helix" include N and H C Separate domains 3 10 It refers to the N-terminal residue of the helix.

[0184] "BoNT / B LH N and H C Separate domains 3 10 References herein to "the second amino acid residue of the helix" include N and H C Separate domains 3 10 It refers to the amino acid residue following the N-terminal residue of the helix.

[0185] "3 10 A "helix" is a type of secondary structure found in proteins and polypeptides, along with α-helices, β-sheets, and reverse turns. 3 10The amino acids in the helix are arranged in a right-handed helical structure, with each full turn completed by three residues and 10 atoms separating the intramolecular hydrogen bonds between them. Each amino acid has 10 atoms in a ring formed by forming hydrogen bonds, corresponding to a 120° rotation in the helix (i.e., the helix has 3 residues per turn) and a translation of 2.0 Å (=0.2 nm) along the helix axis. Most importantly, the NH group of an amino acid forms a hydrogen bond with the C=O group of the amino acid three residues before it, and this repeated i+3→i hydrogen bond completes 3 cycles. 10 Define the helix. 3 10 A helix is ​​a standard concept in structural biology and is familiar to those of skill in the art.

[0186] These three 10 The helix corresponds to the four residues that form the actual helix plus two cap (or transition) residues, one at each end of these four residues. N and H C Separate domains 3 10 The term "helix" as used herein consists of those six residues.

[0187] By performing structural analysis and sequence alignment, LH N and H C Separate domains 3 10 The three helices were identified. 10 The helix is ​​located at its N-terminus (i.e., LH N at the C-terminal part of the domain) by an α-helix and at its C-terminus (i.e. C The N-terminal part of the domain is surrounded by β-strands. 10 The first (N-terminal) residue of a helix (the cap or transition residue) also corresponds to the C-terminal residue of this α-helix.

[0188] LH N and H C Separate domains 3 10The helices can be determined, for example, from the publicly available crystal structures of botulinum neurotoxins, such as 3BTA (http: / / www.rcsb.org / pdb / explore / explore.do?structureId=3BTA) and 1EPW (http: / / www.rcsb.org / pdb / explore / explore.do?structureId=1EPW) for botulinum neurotoxins A1 and B1, respectively.

[0189] We also used publicly available in silico modeling and alignment tools to characterize LH in other neurotoxins. N and H C Separate domains 3 10 The position of the helices can be determined, for example, by the homology modeling servers LOOPP (Learning, Observing and Outputting Protein Patterns, http: / / loopp.org), PHYRE (Protein Homology / analogY Recognition Engine, http: / / www.sbg.bio.ic.ac.uk / phyre2 / ) and Rosetta (https: / / www.rosettacommons.org / ), the protein superposition server SuperPose (http: / / wishart.biology.ualberta.ca / superpose / ), the alignment program Clustal Omega (http: / / www.clustal.org / omega / ) and several other tools / services listed in Internet Resources for Molecular and Cell Biologists (http: / / molbiol-tools.ca / ). N / H CN " The regions surrounding the junctions are highly structurally conserved, making them ideal regions for overlapping of different serotypes.

[0190] For example, the following methodology can be used to investigate the effect of these three 10 The sequence of the helix can be determined: 1. The structural homology modeling tool LOOP (http: / / loopp.org) may be used to obtain predicted structures of other BoNT serotypes based on the BoNT / A1 crystal structure (3BTA.pdb). 2. Copy the structure (pdb) file obtained in this way to H CN The N-terminus of the domain and the approximately 80 residues preceding it (H N This results in a highly structurally conserved "H N / H CN " area may be edited to preserve it; 3. Each serotype may be superimposed onto the 3BTA.pdb structure using the protein superposition server SuperPose (http: / / wishart.biology.ualberta.ca / superpose / ); 4. Investigate the overlapping pdb files and find 3 10 The helix is ​​the H C one may map it to the start of the domain and then identify the corresponding residues in other serotypes; 5. Other BoNT serotype sequences can be aligned using Clustal Omega to check that corresponding residues are correct.

[0191] LH determined by this method N , H C and 3 10 An example of a helical domain is shown below: [Table A]

[0192] Using structural analysis and sequence alignment, LH N and H C Separate domains 3 10 The posthelical β-strand is a conserved structure in all botulinum and tetanus neurotoxins and is involved in the formation of LH N and H C Separate domains 3 10It was found that if one starts at the first residue of the helix, it begins at the eighth residue (eg, at residue 879 in BoNT / A1).

[0193] BoNT / AB chimera is a BoNT / B derived H C LH from BoNT / A covalently linked to the domain N domain, where LH N The C-terminal amino acid residue of the domain is H C It corresponds to the 8th amino acid residue at the N-terminus of the β-strand located at the beginning (N-terminus) of the domain, and H C The N-terminal amino acid residue of the domain is H C It corresponds to the seventh amino acid residue at the N-terminus of the β-strand located at the beginning (N-terminus) of the domain.

[0194] BoNT / AB chimera is a BoNT / B-derived H C LH from BoNT / A covalently linked to the domain N domain, where LH N The C-terminal amino acid residues of the domain are the same as those of the LH domain of BoNT / A. N H corresponds to the C-terminal amino acid residue of the α-helix located at the end (C-terminus) of the domain. C The N-terminal amino acid residues of the domain are the LH N It corresponds to the amino acid residue immediately C-terminal to the C-terminal amino acid residue of the α-helix located at the end (C-terminus) of the domain.

[0195] The rationale for the design process of the BoNT / AB chimera was to ensure that the secondary structure was intact, thereby minimizing any changes to the tertiary structure and to the function of each domain. Without wishing to be bound by theory, 10It is hypothesized that not disrupting the four central amino acid residues of the helix ensures an optimal conformation of the modified BoNT / A (chimeric neurotoxin), thereby enabling the modified BoNT / A (chimeric neurotoxin) to exert its function to its full potential.

[0196] In fact, surprisingly, 3 10 The first amino acid residue of helix 1 and the third amino acid residue of BoNT / B 10 Retaining only the second amino acid residue after the helix not only allows for the production of a soluble, functional BoNT / AB chimera, but also leads to improved properties over other BoNT / AB chimeras, particularly increased potency, increased safety factor and / or longer duration of action (as well as increased safety factor and / or increased duration of action when compared to unmodified BoNT / A - e.g., SEQ ID NO:2, e.g., SEQ ID NO:2 in its dichain form).

[0197] BoNT / A light chain, BoNT / A translocation domain and / or BoNT / BH C The domain may be a modified BoNT / A light chain, a BoNT / A translocation domain, and / or a BoNT / BH C The BoNT / A light chain may be a modified BoNT / A light chain, a BoNT / A translocation domain, and / or a BoNT / BH domain or a derivative thereof, including, but not limited to, those described below. C The domain or derivative may be a BoNT / A light chain, a BoNT / A translocation domain, and / or a BoNT / BH C The BoNT / A light chain, the BoNT / A translocation domain and / or the BoNT / BH light chain may contain one or more amino acids that are modified compared to the native (unmodified) form of the domain, or may contain one or more amino acids that are modified compared to the native (unmodified) form of the domain. C It may contain one or more inserted amino acids not present in the native (unmodified) form of the domain. Examples include modified BoNT / A light chains, BoNT / A translocation domains and / or BoNT / BH domains. CThe domains may be any of the native (unmodified) BoNT / A light chain, the BoNT / A translocation domain and / or the BoNT / BH domain. C The domain sequence may have an altered amino acid sequence in one or more of the domains. Such alterations may alter its functional aspects, such as biological activity or persistence. Thus, in one embodiment, the BoNT / A light chain, the BoNT / A translocation domain and / or the BoNT / BH C The domains may include modified BoNT / A light chains, BoNT / A translocation domains and / or BoNT / BH C Domain or modified BoNT / A light chain, BoNT / A translocation domain and / or BoNT / BH C It is a domain derivative.

[0198] Modified BoNT / BH C The domain modifies binding to a target neuron, e.g., the native (unmodified) BoNT / BH C The BoNT / BH domain may have one or more modifications that provide higher or lower affinity binding when compared to the BoNT / BH domain. C Such modifications in the H domain alter binding to ganglioside and / or protein receptors on target neurons. C This may involve modifying residues in the ganglioside binding site or in the protein (e.g., synaptotagmin) binding site of the domain. Examples of such modified neurotoxins are described in WO2006 / 027207 and WO2006 / 114308, both of which are incorporated herein by reference in their entirety.

[0199] The modified light chain may have one or more modifications in its amino acid sequence, for example, modifications in the substrate binding or catalytic domains that may alter or modify the SNARE protein specificity of the modified light chain, preferably provided that said modifications do not catalytically inactivate said light chain. Examples of such modified neurotoxins are described in WO2010 / 120766 and US2011 / 0318385, both of which are incorporated herein by reference in their entirety.

[0200] BoNT / A-derived LH N The domain may correspond to amino acid residues 1-872 of SEQ ID NO:2, or a polypeptide sequence having at least 70% sequence identity thereto. N The domain may correspond to amino acid residues 1 to 872 of SEQ ID NO:2, or a polypeptide sequence having at least 80%, 90% or 95% sequence identity thereto. N The domain corresponds to amino acid residues 1-872 of SEQ ID NO:2.

[0201] BoNT / B-derived H C The domain may correspond to amino acid residues 860 to 1291 of SEQ ID NO:8, or a polypeptide sequence having at least 70% sequence identity thereto. C The domain may correspond to amino acid residues 860 to 1291 of SEQ ID NO:8, or a polypeptide sequence having at least 80%, 90% or 95% sequence identity thereto. C The domain corresponds to amino acid residues 860 to 1291 of SEQ ID NO:8.

[0202] Preferably, the BoNT / AB chimera is BoNT / A1 LH N Domain and BoNT / B1 H C More preferably, the LH domain is N The domain corresponds to amino acid residues 1 to 872 of BoNT / A1 (SEQ ID NO: 2), C The domain corresponds to amino acid residues 860 to 1291 of BoNT / B1 (SEQ ID NO:8).

[0203] Most preferably, BoNT / BH C The domain is H CCThe BoNT / BH further comprises at least one amino acid residue substitution, insertion, indel or deletion in the subdomain, which has the effect of increasing the binding affinity of the BoNT / B neurotoxin for human Syt II, as compared to the native BoNT / B sequence. CC Suitable amino acid residue substitutions, insertions, indels or deletions in the subdomains are disclosed in WO2013 / 180799, and in WO2016 / 154534 (both of which are incorporated herein by reference).

[0204] BoNT / BH CC Suitable amino acid residue substitutions, insertions, indels or deletions in the subdomains may include substitution mutations selected from the group consisting of V1118M, Y1183M, E1191M, E1191I, E1191Q, E1191T, S1199Y, S1199F, S1199L, S1201V, E1191C, E1191V, E1191L, E1191Y, S1199W, S1199E, S1199H, W1178Y, W1178Q, W1178A, W1178S, Y1183C, Y1183P and combinations thereof.

[0205] BoNT / BH CC Suitable amino acid residue substitutions, insertions, indels or deletions in the subdomain may further include a combination of two substitution mutations selected from the group consisting of E1191M and S1199L, E1191M and S1199Y, E1191M and S1199F, E1191Q and S1199L, E1191Q and S1199Y, E1191Q and S1199F, E1191M and S1199W, E1191M and W1178Q, E1191C and S1199W, E1191C and S1199Y, E1191C and W1178Q, E1191Q and S1199W, E1191V and S1199W, E1191V and S1199Y or E1191V and W1178Q.

[0206] BoNT / BH CCSuitable amino acid residue substitutions, insertions, indels or deletions in the subdomains may also include a combination of the three substitution mutations: E1191M, S1199W and W1178Q.

[0207] Preferably, BoNT / BH CC The amino acid residue substitution, insertion, indel or deletion in the subdomain includes a combination of two substitution mutations, E1191M and S1199Y. Such modifications are present, for example, in modified BoNT / A (e.g., BoNT / AB chimera) of SEQ ID NO:5 and SEQ ID NO:6. E1191M may correspond to position 1204 of SEQ ID NO:6, and S1199Y may correspond to position 1212. Thus, SEQ ID NO:6 may include 1204M and 1212Y.

[0208] The modification may be a modification compared to unmodified BoNT / B shown as SEQ ID NO:8, where the amino acid residue numbering is determined by alignment with SEQ ID NO:8. Since the presence of a methionine residue at position 1 of SEQ ID NO:8 (as well as the SEQ ID NOs corresponding to the modified BoNT / A polypeptides described herein) is optional, one of skill in the art will take into account the presence / absence of a methionine residue when determining the amino acid residue numbering. For example, if SEQ ID NO:8 contains a methionine, the position numbering will be as defined above (e.g., E1191 will be E1191 in SEQ ID NO:8). Alternatively, if a methionine is not present in SEQ ID NO:8, the amino acid residue numbering must be modified by -1 (e.g., E1191 will be E1190 in SEQ ID NO:8). Thus, the first methionine amino acid residue of the polypeptide sequence of the modified BoNT / A may be optional or absent. Similarly, similar considerations apply to the presence / absence of a methionine at position 1 of other polypeptide sequences described herein, and one of skill in the art would readily determine the correct amino acid residue numbering using routine techniques in the art.

[0209] A modified BoNT / A for use in the present invention may comprise a polypeptide sequence having at least 70% sequence identity to a polypeptide sequence selected from SEQ ID NOs: 3 to 7. For example, a polypeptide sequence having at least 80%, 90%, 95% or 99.9% sequence identity to a polypeptide sequence selected from SEQ ID NOs: 3 to 7. Preferably, a modified BoNT / A for use in the present invention may comprise (and more preferably consist of) a polypeptide sequence selected from SEQ ID NOs: 3 to 7.

[0210] It is preferred that the modified BoNT / A comprises a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 6. For example, a polypeptide sequence having at least 80%, 90%, 95% or 99.9% sequence identity to SEQ ID NO: 6. Most preferably, the modified BoNT / A for use in the present invention may comprise (and more preferably consist of) SEQ ID NO: 6.

[0211] The term "deletion," as used herein, refers to the removal of one or more amino acid residues of a polypeptide without replacement of one or more amino acid residues at the site of the deletion. Thus, if one amino acid residue is deleted from (for example) a polypeptide sequence having x number of amino acid residues, the resulting polypeptide will have x-1 amino acid residues.

[0212] The term "insertion / deletion" as used herein refers to the deletion of one or more amino acid residues of a polypeptide and the insertion at the deletion site of a different number of amino acid residues (more or fewer amino acid residues) as compared to the number of deleted amino acid residues. Thus, for an insertion / deletion in which two amino acid residues are deleted from (for example) a polypeptide sequence having a number x of amino acid residues, the resulting polypeptide has x-1 amino acid residues or x+≧1 amino acid residues. Insertions and deletions can be made in any order, sequentially or simultaneously.

[0213] The term "substitution" as used herein refers to the replacement of one or more amino acid residues with the same number of amino acid residues at the same sites. Thus, for a substitution in (for example) a polypeptide sequence having x number of amino acid residues, the resulting polypeptide also has x number of amino acid residues. Preferably, the substitution is at a single amino acid position.

[0214] The term "insertion," as used herein, refers to the addition of one or more amino acid residues to a polypeptide without the deletion of one or more amino acid residues of the polypeptide at the site of the insertion. Thus, if one amino acid residue is inserted into (for example) a polypeptide sequence having x number of amino acid residues, the resulting polypeptide will have x+1 amino acid residues.

[0215] Methods for modifying proteins by substitution, insertion or deletion of amino acid residues are known in the art. By way of example, amino acid modifications may be introduced by modification of the DNA sequence encoding BoNT / A (e.g., encoding unmodified BoNT / A). This can be accomplished using standard molecular cloning techniques, for example by site-directed mutagenesis, in which a short strand of DNA (oligonucleotide) encoding the desired amino acid(s) is used to replace the original coding sequence using a polymerase enzyme, or by inserting / deleting portions of the gene using various enzymes (e.g., ligases and restriction endonucleases). Alternatively, modified gene sequences can be chemically synthesized. Typically, modifications can be performed by modifying a nucleic acid encoding a native Clostridial neurotoxin (or a portion thereof) such that the modified BoNT / A (or a portion thereof) encoded by the nucleic acid contains the modification(s). Alternatively, a nucleic acid encoding a modified Clostridial neurotoxin (or a portion thereof) containing the modification(s) can be synthesized.

[0216] If the polypeptide sequence of the modified BoNT / A described herein includes, for example, a tag for purification, such as a His-tag, the tag is optional. Preferably, the tag is removed prior to use of the modified BoNT / A according to the present invention.

[0217] As discussed above, the modified BoNT / A described herein has increased tissue retention properties, which also provide increased potency and / or duration of action, and can allow for increased dosage without any additional negative effects. One way these advantageous properties may be defined is in terms of the safety factor of the modified BoNT / A. In this regard, the undesired effects of clostridial toxins (caused by the diffusion of the toxin away from the site of administration) can be experimentally evaluated by measuring the percentage of body weight loss in relevant animal models (e.g., mice, where weight loss is detected within 7 days of administration). Conversely, the desired on-target effects of clostridial toxins can be experimentally evaluated by the digit abduction score (DAS) assay, which is a measure of muscle paralysis. The DAS assay can be performed by injection of 20 μl of clostridial neurotoxin formulated in gelatin phosphate buffer into the mouse gastrocnemius / soleus muscle complex, followed by assessment of digit abduction scores using the method of Aoki (Aoki KR, Toxicon 39: 1815-1820; 2001). In the DAS assay, mice are briefly suspended by their tails to elicit a characteristic startle response in which the mouse extends its hind limbs and abducts its hind digits. After clostridial neurotoxin injection, various degrees of digit abduction are scored on a 5-point scale (0=normal to 4=maximal reduction in digit abduction and leg extension).

[0218] The safety factor of a neurotoxin (e.g., a modified BoNT / A of the present invention (or unmodified BoNT / A for comparison)) may then be expressed as the ratio between the amount of toxin required for a 10% reduction in body weight (measured at peak effect within the first 7 days after dosing in mice) and the amount of toxin required for a DAS score of 2. Thus, a high safety factor score is desirable and indicates a neurotoxin that can effectively paralyze a target muscle with few unwanted off-target effects. The modified BoNT / A of the present invention has a higher safety factor than the safety factor of a comparable unmodified (native) BoNT / A.

[0219] A high safety margin is particularly advantageous in therapy since it represents an increased therapeutic index. In other words, this means that a reduced dosage can be used compared to an alternative Clostridial neurotoxin therapeutic agent, and / or an increased dosage can be used without any additional (e.g., adverse) effects. The possibility of using a higher dose of the neurotoxin without additional effects is particularly advantageous, since a higher dose usually leads to a longer duration of action of the neurotoxin.

[0220] The efficacy of the modified BoNT / A is determined by the expression of a given DAS score, e.g., a DAS score of 2 (ED 50 The potency of modified BoNT / A may also be expressed as the EC 50 Dose, e.g., EC in a cellular assay measuring SNAP25 cleavage by modified BoNT / A 50 It may be expressed as a dose.

[0221] The duration of action of the modified BoNT / A may be expressed as the time required to recover a DAS score of 0 following administration of a given dose of neurotoxin to the mouse gastrocnemius / soleus muscle complex, for example, the minimum dose of neurotoxin that leads to a DAS score of 4.

[0222] Thus, in one embodiment, a modified BoNT / A of the invention has a safety factor of greater than 7 (e.g., at least 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50), and the safety factor is greater than the DAS ED 50 Dose of toxin required for -10% body weight change (pg / mouse) divided by (pg / mouse) [ED 50 Dose required to produce a DAS score of 0.05 to 0.25, for example, ...

[0223] In one embodiment, the modified BoNT / A of the present invention has a safety factor of at least 10. In one embodiment, the modified BoNT / A of the present invention has a safety factor of at least 15.

[0224] Preferably, the modified BoNT / A has a safety factor of at least 10 (eg, a safety factor of 10), more preferably at least 12 or 13 (eg, 14-15).

[0225] The modified BoNT / A may have a safety factor of greater than 7, up to 50, for example, 8-45, 10-20, or 12-15.

[0226] In use, the modified BoNT / A of the invention is in a dichain form.

[0227] The modified BoNT / A for use in the present invention may comprise a polypeptide sequence having at least 70% sequence identity to a polypeptide sequence selected from SEQ ID NOs: 3 to 7. For example, a polypeptide sequence having at least 80%, 90%, 95% or 99.9% sequence identity to a polypeptide sequence selected from SEQ ID NOs: 3 to 7. Preferably, the modified BoNT / A for use in the present invention may comprise (and more preferably consist of) a polypeptide sequence selected from SEQ ID NOs: 3 to 7. Of the modified BoNT / A neurotoxins (e.g., chimeric Clostridium neurotoxins), SEQ ID NO: 6 is preferred.

[0228] Thus, it is preferred that a modified BoNT / A (e.g., a chimeric Clostridial neurotoxin) comprises a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 6. More preferably, a polypeptide sequence having at least 80%, 90%, 95% or 99.9% sequence identity to SEQ ID NO: 6. Most preferably, a modified BoNT / A (e.g., a chimeric Clostridial neurotoxin) for use in the present invention may comprise (and more preferably consist of) SEQ ID NO: 6.

[0229] The two-chain modified BoNT / A of the present invention may comprise a light chain portion of a polypeptide sequence having at least 70%, 80%, 90%, 95%, 99.9% or 100% sequence identity to any one of SEQ ID NOs: 3-7 which constitutes the first chain of the two-chain modified BoNT / A, and a heavy chain portion of a polypeptide sequence having at least 70%, 80%, 90%, 95%, 99.9% or 100% sequence identity to any one of SEQ ID NOs: 3-7 which together constitute the second chain of the two-chain modified BoNT / A. N and H C domain, where the first and second chains are linked by a disulfide bond.

[0230] When cleavage occurs at more than one position (preferably at two positions) within the activation loop of a modified BoNT / A that contains a polypeptide sequence having at least 70%, 80%, 90%, 95%, 99.9% or 100% sequence identity to any one of SEQ ID NOs: 3-7, a small fragment of the C-terminal L-chain portion of the sequence having at least 70%, 80%, 90%, 95%, 99.9% or 100% sequence identity to any one of SEQ ID NOs: 3-7 may not be present in the two-chain modified BoNT / A. In view of this, the sequence of a two-chain modified BoNT / A (e.g., comprising a polypeptide sequence having at least 70%, 80%, 90%, 95%, 99.9% or 100% sequence identity to any one of SEQ ID NOs: 3-7) may differ slightly from the sequence of a corresponding single-chain modified BoNT / A comprising a polypeptide sequence having at least 70%, 80%, 90%, 95%, 99.9% or 100% sequence identity to any one of SEQ ID NOs: 3-7. The subfragment may be 1 to 15 amino acids. In particular, in one embodiment, when Lys-C is used to convert a single-chain modified BoNT / A to a two-chain Clostridial neurotoxin, the subfragment of the C-terminal L-chain portion of the absent sequence may be SEQ ID NO: 9 or 10.

[0231] Preferably, the two-chain modified BoNT / A of the invention may comprise a light chain portion of a polypeptide sequence having at least 70%, 80%, 90%, 95%, 99.9% or 100% sequence identity to SEQ ID NO:6 constituting the first chain of the two-chain modified BoNT / A, and a heavy chain portion of a polypeptide sequence having at least 70%, 80%, 90%, 95%, 99.9% or 100% sequence identity to SEQ ID NO:6 constituting together the second chain of the two-chain modified BoNT / A. N and H C domain, and the first and second chains are linked together by disulfide bonds.

[0232] In the case where cleavage occurs at more than one position (preferably at two positions) within the activation loop of a modified BoNT / A that includes a polypeptide sequence having at least 70%, 80%, 90%, 95%, 99.9% or 100% sequence identity to SEQ ID NO: 6, a small fragment of the C-terminal L chain portion of the sequence having at least 70%, 80%, 90%, 95%, 99.9% or 100% sequence identity to SEQ ID NO: 6 may not be present in the two-chain modified BoNT / A. In light of this, the sequence of a two-chain modified BoNT / A (e.g., including a polypeptide sequence having at least 70%, 80%, 90%, 95%, 99.9% or 100% sequence identity to SEQ ID NO: 6) may differ slightly from the sequence of a corresponding single-chain modified BoNT / A that includes a polypeptide sequence having at least 70%, 80%, 90%, 95%, 99.9% or 100% sequence identity to SEQ ID NO: 6. The subfragment can be 1 to 15 amino acids. In particular, in one embodiment, when Lys-C is used to convert a single-chain modified BoNT / A to a modified BoNT / A, the subfragment of the C-terminal L-chain portion of the absent sequence can be SEQ ID NO: 9 or 10.

[0233] In a particularly preferred embodiment, the two-chain modified BoNT / A comprises (or consists of) a light chain comprising a polypeptide sequence having at least 70%, 80%, 90%, 95% or 99.9% sequence identity to SEQ ID NO: 11 or 12 (preferably SEQ ID NO: 11) and a heavy chain comprising a polypeptide sequence having at least 70%, 80%, 90%, 95% or 99.9% sequence identity to SEQ ID NO: 13, where the light chain and the heavy chain are linked together by a disulfide bond. More preferably, the two-chain modified BoNT / A comprises (or consists of) a light chain comprising SEQ ID NO: 11 or 12 (preferably SEQ ID NO: 11) and a heavy chain comprising SEQ ID NO: 13, where the light chain and the heavy chain are linked together by a disulfide bond. Even more preferably, the two-chain modified BoNT / A comprises (or consists of) a light chain having SEQ ID NO: 11 and a heavy chain having SEQ ID NO: 13, where the light chain and the heavy chain are linked together by a disulfide bond. The disulfide bond is preferably formed by and / or between cysteine ​​residue 429 of SEQ ID NO:11 or 12 and cysteine ​​residue 6 of SEQ ID NO:13.

[0234] In a preferred embodiment, the modified BoNT / A (e.g., chimeric Clostridial neurotoxin) of the present invention does not include a therapeutic or diagnostic agent (e.g., a nucleic acid, protein, peptide, or small molecule therapeutic or diagnostic agent) in addition to the light and heavy chains. For example, in one embodiment, the modified BoNT / A (e.g., chimeric Clostridial neurotoxin) may not include a therapeutic or diagnostic agent that is covalently or non-covalently associated therewith. Thus, the modified BoNT / A (e.g., chimeric Clostridial neurotoxin) of the present invention preferably does not function as a delivery vehicle for additional therapeutic or diagnostic agents.

[0235] In embodiments in which the modified BoNT / A described herein (e.g., a chimeric Clostridial neurotoxin) has a tag (e.g., a His-tag) and / or linker for purification, the tag and / or linker are optional.

[0236] The modified BoNT / A is preferably in an uncomplexed form (i.e., it may not contain the complexing proteins present in a naturally occurring Clostridial neurotoxin complex, e.g., a BoNT / A complex). Examples of such complexing proteins include neurotoxin-associated proteins (NAPs) and non-toxic non-hemagglutinin components (NTNHs). However, it is preferred that the modified BoNT / A is a recombinant modified BoNT / A. 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 (e.g., a chimeric Clostridial neurotoxin).

[0237] In one embodiment, a nucleic acid (e.g., DNA) is provided that comprises a nucleic acid sequence that encodes a modified BoNT / A. In one embodiment, the nucleic acid sequence is prepared as part of a DNA vector that includes a promoter and a terminator. The nucleic acid sequence can be selected from any of the nucleic acid sequences described herein.

[0238] In a preferred embodiment, the vector has a promoter selected from the following: Promoter / Inducer / Normal induction conditions Tac (hybrid) / IPTG / 0.2mM (0.05~2.0mM) AraBAD / L-arabinose / 0.2% (0.002~0.4%) T7-lac operator / IPTG / 0.2mM (0.05~2.0mM)

[0239] In another preferred embodiment, the vector has a promoter selected from: Promoter / Inducer / Normal induction conditions Tac (hybrid) / IPTG / 0.2mM (0.05~2.0mM) AraBAD / L-arabinose / 0.2% (0.002~0.4%) T7-lac operator / IPTG / 0.2mM (0.05~2.0mM) T5-lac operator / IPTG / 0.2mM (0.05~2.0mM)

[0240] The nucleic acid molecule may be produced using any suitable process known in the art. Thus, the nucleic acid molecule may be produced using chemical synthesis techniques. Alternatively, the nucleic acid molecule of the present invention may be produced using molecular biology techniques.

[0241] The DNA constructs of the present invention are preferably designed on a computer and then synthesized by conventional DNA synthesis techniques.

[0242] The above nucleic acid sequence information is optionally modified for codon bias according to the final host cell (eg, E. coli) expression system to be utilized.

[0243] The terms "nucleotide sequence" and "nucleic acid" are used synonymously herein. Preferably, the nucleotide sequence is a DNA sequence.

[0244] The modified BoNT / A of the present invention can exist as a single chain or as two chains. However, it is also possible that the modified BoNT / A is a single chain in which the L chain is linked to the H chain (or a component thereof, e.g., H) via a disulfide bond. N It is preferred that the polypeptide exists as two chains linked together in a single chain (domain).

[0245] The production of a single-chain modified BoNT / A having a light chain and a heavy chain may be achieved using a method that includes expressing a nucleic acid encoding the modified BoNT / A in an expression host, lysing the host cell to provide a host cell homogenate containing the single-chain modified BoNT / A, and isolating the single-chain modified BoNT / A. The single-chain modified BoNT / A described herein can be proteolytically processed using a method that includes contacting the single-chain modified BoNT / A with a protease (e.g., Lys-C) that hydrolyzes a peptide bond in the activation loop of the modified BoNT / A, thereby converting the single-chain modified BoNT / A into the corresponding two-chain modified BoNT / A (e.g., the light chain and the heavy chain are linked together by a disulfide bond). The two-chain modified BoNT / A can preferably be obtained by such a method.

[0246] The term "obtainable" as used herein also encompasses the term "obtained." In one embodiment, the term "obtainable" means obtained.

[0247] Therefore, the modified BoNT / A used in the present invention is preferably a two-chain modified BoNT / A that is generated from a single-chain BoNT / A, the single-chain BoNT / A comprising or consisting of the polypeptide sequence described herein. For example, it is preferred that the modified BoNT / A used in the present invention is a two-chain modified BoNT / A that is generated from a polypeptide that comprises a polypeptide sequence having at least 70% (e.g., at least 80%, 90%, 95% or 99.9%) sequence identity to SEQ ID NO: 6. Most preferably, the modified BoNT / A used in the present invention is a two-chain modified BoNT / A that is generated from a polypeptide that comprises (and even more preferably consists of) SEQ ID NO: 6. Thus, in some embodiments, the modified BoNT / A is a two-chain modified BoNT / A that can be obtained by a method that includes a light chain (L chain) linked to a heavy chain (H chain) via a disulfide bond and contacting the single-chain modified BoNT / A with a protease that hydrolyzes the peptide bond in its activation loop, thereby converting the single-chain modified BoNT / A into the corresponding two-chain modified BoNT / A. Thus, in some embodiments, the modified BoNT / A is a two-chain modified BoNT / A, in which the light chain (L chain) is linked to the heavy chain (H chain) via a disulfide bond, and can be obtained by a method comprising contacting a single-chain modified BoNT / A comprising SEQ ID NO: 6 with a protease that hydrolyzes the peptide bond in its activation loop, thereby converting the single-chain modified BoNT / A into the corresponding two-chain modified BoNT / A. In some embodiments, the modified BoNT / A is a two-chain modified BoNT / A, in which the L chain is linked to the H chain via a disulfide bond, and can be obtained by a method comprising contacting a single-chain modified BoNT / A consisting of SEQ ID NO: 6 with a protease that hydrolyzes the peptide bond in its activation loop, thereby converting the single-chain modified BoNT / A into the corresponding two-chain modified BoNT / A.

[0248] The protease used to cleave activation loop is preferably Lys-C. Suitable proteases and methods for cleaving activation loop to generate two-chain clostridial neurotoxins are taught in WO2014 / 080206, WO2014 / 079495 and EP2677029A2, which are incorporated herein by reference. Lys-C can cleave the activation loop C-terminal of one or more lysine residues present therein. If Lys-C cleaves activation loop more than once, those skilled in the art will understand that the small peptide of the activation loop of two-chain modified BoNT / A may not exist when compared with the sequence number shown herein.

[0249] The term "one or more," as used herein, may mean at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20. In one embodiment, when "one or more" precedes a list, "one or more" may mean all of the members of the list. Similarly, the term "at least one," as used herein, may mean at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20. In one embodiment, when "at least one" precedes a list, "at least one" may mean all of the members of the list.

[0250] The term "disorder" as used herein also encompasses "disease." In one embodiment, a disorder is a disease.

[0251] The modified BoNT / A of the invention may be formulated in any suitable manner for administration to a subject, for example, as part of a pharmaceutical composition, which includes the modified BoNT / A of the invention and a pharma- ceutically acceptable carrier, excipient, adjuvant, propellant, and / or salt.

[0252] Liquid dosage forms are usually prepared using modified BoNT / A and a pyrogen-free sterile medium. Depending on the vehicle and concentration used, modified BoNT / A can be dissolved or suspended in the vehicle. In preparing a solution, modified BoNT / A can be dissolved in the vehicle, the solution is made isotonic by adding sodium chloride as necessary, sterilized by filtration through a sterile filter using aseptic techniques, and then filled into suitable sterile vials or ampoules and sealed. Alternatively, if the solution stability is sufficient, the solution in the sealed container may be sterilized by autoclaving. Advantageously, additives such as buffers, solubilizers, stabilizers, preservatives or bactericides, suspending or emulsifying agents, and or local anesthetics can be dissolved in the vehicle.

[0253] By filling pre-sterilized ingredients into sterile containers using aseptic technique in a sterile area, dry powders can be prepared that are dissolved or suspended in a suitable medium prior to use. Alternatively, the ingredients can be dissolved in a suitable container using aseptic technique in a sterile area. The product is then lyophilized and the container is aseptically sealed.

[0254] Parenteral suspensions suitable for the routes of administration described herein are prepared in substantially the same manner, except that the sterile components are suspended in the sterile vehicle instead of dissolved, and sterilization cannot be accomplished by filtration. The components may be isolated in a sterile state or may be sterilized after isolation, for example, by gamma irradiation.

[0255] Advantageously, a suspending agent, such as polyvinylpyrrolidone, can be included in the composition(s) to facilitate uniform distribution of the components.

[0256] In one embodiment, the present invention provides a unit dosage form of modified botulinum neurotoxin A (BoNT / A) for treating upper facial lines, comprising: a. More than 73 units of modified BoNT / A; or b. modified BoNT / A greater than 1754 pg (e.g., greater than 1800 pg); and c. optionally, pharma- ceutically acceptable carriers, excipients, adjuvants and / or salts; Here, the modified BoNT / A comprises a BoNT / A light chain and translocation domain and a BoNT / B receptor binding domain (H C domain).

[0257] In one aspect, the present invention provides a unit dosage form of a modified botulinum neurotoxin A (BoNT / A) for treating upper facial lines, the unit dosage form comprising: a. More than 73 units to 332.8 units of modified BoNT / A (where 1 unit is the calculated median lethal dose (LD) in mice) 50 ), or b. More than 1754 pg (preferably more than 1800 pg) to 8000 pg of modified BoNT / A, and c. optionally, pharma- ceutically acceptable carriers, excipients, adjuvants and / or salts; d. wherein the modified BoNT / A comprises a BoNT / A light chain and a translocation domain (H N domain) and the BoNT / B receptor binding domain (H C domain).

[0258] The unit dosage form may include: a. 100 units to 200 units of modified BoNT / A, or b. 4000pg to 6000pg of modified BoNT / A.

[0259] A unit dosage form for treating facial wrinkles may contain 73 units to 332.8 units of modified BoNT / A. The upper limit of said range may be 300, 250, 200, 150 or 100 units of modified BoNT / A, preferably the upper limit is 200 units. The lower limit of said range may be 80, 90, 100, 150, 200 or 250 units of modified BoNT / A, preferably the lower limit is 100 units. Preferably, the unit dosage form contains 90 to 250 units of modified BoNT / A, for example 100 units to 200 units.

[0260] A unit dosage form for treating facial wrinkles may contain 1755pg to 8000pg of modified BoNT / A. The upper limit of the range may be 7500, 7000, 6500, 6000, 5500, 5000, 4500, 4000, 3500 or 3000pg of modified BoNT / A, preferably the upper limit is 7000pg. The lower limit of the range may be 1800, 2000, 2,500, 3,000, 3,500, 4,000, 4,500 or 5,000pg of modified BoNT / A, preferably the lower limit is 2000pg. Preferably, the unit dosage form contains 2000pg to 7000pg of modified BoNT / A, for example 4,000pg to 6,000pg.

[0261] The unit dosage form may contain 1800-7000 pg, 2000-6500 pg, 2500-6000 pg of the modified BoNT / A, preferably 4000-5500 pg of the modified BoNT / A.

[0262] The unit dosage form may contain more than 1754 pg to 8000 pg of modified BoNT / A. The upper limit of the unit dosage form may be 8,000, 7,000, 6,000, 5,000, 4,000, 3,000, 2,000 or 1,000 pg of modified BoNT / A, preferably the upper limit is 5,000 pg. The lower limit of the unit dosage form may be 1,800, 2,000, 2,500, 3000 or 4,000 pg of modified BoNT / A, preferably the lower limit is 1,800 pg of modified BoNT / A. The unit dosage form may contain 1,800 to 8,000 pg or 2,000 to 6,000 pg of modified BoNT / A, more preferably 3,000 to 6,000 pg of modified BoNT / A. The unit dosage form can be 4,500 to 5,500 pg of modified BoNT / A, for example, 4,900 to 5,100 pg of modified BoNT / A.

[0263] The unit dosage form may contain more than 1800 pg of modified BoNT / A. The unit dosage form may contain more than 1800 pg and up to 7000 pg of modified BoNT / A, for example, more than 1800 pg and up to 6000 pg of modified BoNT / A. The unit dosage form may contain more than 2000 pg and up to 5000 pg of modified BoNT / A.

[0264] A unit dosage form can contain more than 2555 pg. For example, a unit dosage form can contain more than 2555 pg up to 8000 pg of modified BoNT / A, for example, more than 2555 pg up to 6000 pg of modified BoNT / A.

[0265] The unit dosage form may contain 2000pg, 2500pg, 3000pg, 4000pg, 4500pg, 5000pg, 5500pg, 6000pg, 6500pg, 7000pg, 7500pg or 8000pg of the modified BoNT / A.

[0266] More preferably, the unit dosage form may contain 2000-3500 pg of modified BoNT / A. For example, the unit dosage form may contain 2250 pg-3250 pg of modified BoNT / A. A particularly preferred unit dosage form may contain about 2500 pg (e.g., 2500 pg ± 10%) of modified BoNT / A, for example, the unit dosage form may preferably contain 2500 pg of modified BoNT / A. Another suitable unit dosage form may contain about 3000 pg (e.g., 3000 pg ± 10%) of modified BoNT / A, for example, the unit dosage form may contain 3000 pg of modified BoNT / A.

[0267] The unit dosage form may contain 3500-5500 pg of modified BoNT / A. For example, the unit dosage form may contain 3800 pg-4200 pg of modified BoNT / A. The unit dosage form may contain 4800-5200 pg of modified BoNT / A. A particularly suitable unit dosage form may contain about 4000 pg (e.g., 4000 pg ± 10%) of modified BoNT / A, for example, the unit dosage form may contain 4000 pg of modified BoNT / A. Another particularly preferred unit dosage form may contain about 5000 pg (e.g., 5000 pg ± 10%) of modified BoNT / A, for example, the unit dosage form may preferably contain 5000 pg of modified BoNT / A.

[0268] Also provided is a kit comprising: a. a unit dosage form as previously defined herein, and b. instructions for use thereof in treating facial lines; and c. Optionally, a diluent.

[0269] Also provided is a kit comprising: a. a unit dosage form as previously defined herein, and b. instructions for use thereof in treating glabellar lines; and c. Optionally, a diluent.

[0270] Also provided is a kit comprising: a. a unit dosage form as previously defined herein, and b. instructions for use thereof in treating forehead lines; and c. Optionally, a diluent.

[0271] Also provided is a kit comprising: a. a unit dosage form as previously defined herein, and b. instructions for use thereof in the treatment of lateral canthal lines; and c. Optionally, a diluent.

[0272] Also provided is a kit comprising: a. a unit dosage form as previously defined herein, and b. instructions for use thereof in the treatment of glabellar lines, forehead lines, and epicanthal lines; and c. Optionally, a diluent.

[0273] The modified BoNT / A in unit dosage form can include a polypeptide sequence having at least 70% sequence identity to any one of SEQ ID NOs: 3-7. For example, a polypeptide sequence having at least 80%, 90%, 95% or 99.9% sequence identity to any one of SEQ ID NOs: 3-7.

[0274] It is preferred that the modified BoNT / A in unit dosage form comprises a polypeptide sequence having at least 70% sequence identity to SEQ ID NO: 6. For example, a polypeptide sequence having at least 80%, 90%, 95% or 99.9% sequence identity to SEQ ID NO: 6. Most preferably, the modified BoNT / A will comprise (and more preferably consist of) SEQ ID NO: 6.

[0275] The embodiments relating to the various therapeutic uses of the invention are applicable to the methods, compositions (eg, unit dosage forms) and kits of the invention, and vice versa.

[0276] sequence homology

[0277] Any of a variety of sequence alignment methods can be used to determine percent identity, including, but not limited to, global, local and hybrid methods, such as segment approach methods. Protocols for determining percent identity are routine within the skill of the art. Global methods align sequences from the beginning to the end of the molecule and determine the best alignment by summing the scores of individual residue pairs and by imposing gap penalties. Non-limiting methods include, for example, CLUSTAL W, see, e.g., Julie D. Thompson et al., CLUSTAL W: Improving the Sensitivity of Progressive Multiple Sequence Alignment Through Sequence Weighting, Position- Specific Gap Penalties and Weight Matrix Choice, 22(22) Nucleic Acids Research 4673-4680 (1994), and iterative refinement, see, e.g., Osamu Gotoh, Significant Improvement in Accuracy of Multiple Protein. Sequence Alignments by Iterative Refinement as Assessed by Reference to Structural Alignments, 264(4) J. MoI. Biol. 823-838 (1996). Local methods align sequences by identifying one or more conserved motifs shared by all of the input sequences.Non-limiting methods include, for example, Match-box, see e.g., Eric Depiereux and Ernest Feytmans, Match-Box: A Fundamentally New Algorithm for the Simultaneous Alignment of Several Protein Sequences, 8(5) CABIOS 501 -509 (1992), Gibbs sampling, see e.g., CE Lawrence et al., Detecting Subtle Sequence Signals: A Gibbs Sampling Strategy for Multiple Alignment, 262(5131 ) Science 208-214 (1993), Align-M, see e.g., Ivo Van WaIIe et al., Align-MA New Algorithm for Multiple Alignment of Highly Divergent Sequences, 20(9) Bioinformatics:1428-1435 (2004).

[0278] Thus, percent sequence identity is determined by conventional methods. See, e.g., Altschul et al., Bull. Math. Bio. 48: 603-16, 1986 and Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915-19, 1992. Briefly, two amino acid sequences are aligned to optimize the alignment score using a gap opening penalty of 10, a gap extension penalty of 1, and the "blosum 62" scoring matrix of Henikoff and Henikoff (ibid.) as shown below (amino acids are designated by standard single letter codes).

[0279] The "percent sequence identity" between two or more nucleic acid or amino acid sequences is a function of the number of identical positions shared by the sequences.Accordingly, percent identity can be calculated as the number of identical nucleotides / amino acids divided by the total number of nucleotides / amino acids and multiplied by 100.The calculation of percent sequence identity can also take into account the number of gaps and the length of each gap that need to be introduced to optimize the alignment of two or more sequences.The sequence comparison and percent identity determination between two or more sequences can be carried out using a certain mathematical algorithm, such as BLAST, that will be familiar to those skilled in the art.

[0280] Alignment scores for determining sequence identity

number

[0281] The percent identity is then calculated as: [total number of matches identified × 100] / [length of the longer sequence and number of gaps introduced in the longer sequence to align the two sequences]

[0282] Substantially homologous polypeptides are characterized as having one or more amino acid substitutions, deletions, or additions. These changes are preferably of a minor nature, such as conservative amino acid substitutions (see below) and other substitutions that do not significantly affect folding or activity of the polypeptide, usually small deletions of one to about 30 amino acids, and small amino- or carboxyl-terminal extensions, such as an amino-terminal methionine residue, small linker peptides of up to about 20-25 residues, or affinity tags.

[0283] Conservative Amino Acid Substitutions

[0284] Basicity: Arginine Lysine Histidine Acidic: Glutamic acid Aspartic acid polarity: glutamine Asparagine Hydrophobicity: Leucine Isoleucine Ballin Aromatic: Phenylalanine Tryptophan Tyrosine small: glycine Alanine Serine Threonine Methionine

[0285] In addition to the 20 standard amino acids, non-standard amino acids (e.g., 4-hydroxyproline, 6-N-methyllysine, 2-aminoisobutyric acid, isovaline and α-methylserine) can be substituted for the amino acid residues of the polypeptides of the present invention. A limited number of non-conservative amino acids, amino acids that are not encoded by the genetic code and unnatural amino acids can be substituted for the polypeptide amino acid residues. The polypeptides of the present invention can also include non-naturally occurring amino acid residues.

[0286] Non-naturally occurring amino acids include, but are not limited to, trans-3-methylproline, 2,4-methano-proline, cis-4-hydroxyproline, trans-4-hydroxy-proline, N-methylglycine, allo-threonine, methyl-threonine, hydroxy-ethylcysteine, hydroxyethylhomo-cysteine, nitro-glutamine, homoglutamine, pipecolic acid, t-leucine, norvaline, 2-azaphenylalanine, 3-azaphenyl-alanine, 4-azaphenyl-alanine, and 4-fluorophenylalanine. Several methods are known in the art for incorporating non-naturally occurring amino acid residues into proteins. For example, an in vitro system can be used in which nonsense mutations are suppressed using chemically aminoacylated suppressor tRNAs. Methods for synthesizing amino acids and aminoacylating tRNAs are known in the art. Transcription and translation of plasmids containing nonsense mutations are carried out in a cell-free system that includes E. coli S30 extracts and commercially available enzymes and other reagents. Proteins are purified by chromatography. See, 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 the second method, translation is carried out in Xenopus oocytes by microinjection of mutated mRNA and chemically aminoacylated suppressor tRNA (Turcatti et al., J. Biol. Chem. 271:19991-8, 1996). Within the third method, E. coli cells are cultured in the absence of the natural amino acid to be replaced (e.g., phenylalanine) and in the presence of the desired non-naturally occurring amino acid(s) (e.g., 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, or 4-fluorophenylalanine).Non-naturally occurring amino acids are incorporated into polypeptides in place of their natural counterparts. See Koide et al., Biochem. 33:7470-6, 1994. Naturally occurring amino acid residues can be converted to non-naturally occurring species by in vitro chemical modification. To further expand the scope of substitutions, chemical modification can be combined with site-directed mutagenesis (Wynn and Richards, Protein Sci. 2:395-403, 1993).

[0287] A limited number of non-conservative amino acids, amino acids that are not encoded by the genetic code, non-naturally occurring amino acids, and unnatural amino acids may be substituted for amino acid residues in the polypeptides of the invention.

[0288] Essential amino acids in the polypeptides of the invention can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine scanning mutagenesis (Cunningham and Wells, Science 244: 1081-5, 1989). Along with mutations of putative contact site amino acids, the sites of biological interaction can also be determined by physical analysis of the structure, such as those determined by techniques such as nuclear magnetic resonance, crystallography, electron diffraction or photoaffinity labeling. See, for example, de Vos et al., Science 255:306-12, 1992; Smith et al., J. Mol. Biol. 224:899-904, 1992; Wlodaver et al., FEBS Lett. 309:59-64, 1992. The identity of essential amino acids can also be inferred from analysis of homology with related components (e.g., translocation or protease components) of the polypeptides of the invention.

[0289] Multiple amino acid substitutions can be made and tested using known methods of mutagenesis and screening, such as those disclosed by Reidhaar-Olson and Sauer (Science 241:53-7, 1988) or Bowie and Sauer (Proc. Natl. Acad. Sci. USA 86:2152-6, 1989). Briefly, these authors disclose methods in which two or more positions in a polypeptide are simultaneously randomized, functional polypeptides are selected, and the mutagenized polypeptides are then sequenced to determine the range of permissible substitutions at each position. Other methods that can be used include phage display (e.g., Lowman et al., Biochem. 30:10832-7, 1991; Ladner et al., U.S. Pat. No. 5,223,409; Huse, WIPO Publication WO 92 / 06204) and region-directed mutagenesis (Derbyshire et al., Gene 46:145, 1986; Ner et al., DNA 7:127, 1988).

[0290] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs. Singleton, et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 20 ED., John Wiley and Sons, New York (1994) and Hale & Marham, THE HARPER COLLINS DICTIONARY OF BIOLOGY, Harper Perennial, NY (1991) provide those skilled in the art with a general dictionary of many of the terms used in this disclosure.

[0291] The present disclosure is not limited by the exemplary methods and materials disclosed herein, and any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the present disclosure. Numeric ranges include the numbers that define the range. Unless otherwise specified, each nucleic acid sequence is written from left to right in 5' to 3' orientation, and amino acid sequences are written from left to right in amino to carboxy orientation.

[0292] The headings provided herein are not limitations of the various aspects or embodiments of the disclosure.

[0293] Amino acids are referred to herein using the name, three-letter abbreviation or single-letter abbreviation of the amino acid. The term "protein" as used herein includes proteins, polypeptides and peptides. As used herein, the term "amino acid sequence" is synonymous with the term "polypeptide" and / or the term "protein". In some instances, the term "amino acid sequence" is synonymous with the term "peptide". In some instances, the term "amino acid sequence" is synonymous with the term "enzyme". The terms "protein" and "polypeptide" are used synonymously herein. In the present disclosure and claims, conventional single-letter and three-letter codes for amino acid residues may be used. The three-letter codes for amino acids defined in accordance with the IUPACIUB Joint Commission on Biochemical Nomenclature (JCBN). It is also understood that a polypeptide may be encoded by more than one nucleotide sequence due to the degeneracy of the genetic code.

[0294] Other definitions of terms may appear throughout this specification. Before exemplary embodiments are described in more detail, it should be understood that the present disclosure is not limited to the specific embodiments described, and may vary as such. Since the scope of the present disclosure is defined only by the appended claims, it should also be understood that the technical terms used in this specification are merely for the purpose of describing specific embodiments, and are not intended to be limiting.

[0295] Where a range of values ​​is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit, between the upper and lower limits of that range is also specifically disclosed, unless the context clearly dictates otherwise. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the disclosure. The upper and lower limits of these smaller ranges may be independently included or excluded in the range, and each range where either, neither, or both limits are included in the smaller range is also encompassed within the disclosure, subject to any specifically excluded limits in the stated range. Where a stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0296] It must be noted that, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to "botulinum neurotoxin A" includes a plurality of such candidate agents, 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.

[0297] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application, and nothing herein should be construed as an admission that such publications constitute prior art to the claims appended hereto.

[0298] Embodiments of the present invention will now be described, by way of example only, with reference to the following figures and examples. [Brief description of the drawings]

[0299] [Figure 1] FIG. 1 shows the FDA-approved dosages of Dysport® for treating facial lines in adults. [Diagram 2] 2 shows SDS-PAGE of purified recombinant BoNT / AB chimeras 1, 2 and 3A (SEQ ID NOs: 3-4, respectively). Lanes are labeled "marker" (molecular weight marker), "-DTT" (oxidized BoNT / AB chimera sample) and "+DTT" (reduced BoNT / AB chimera sample). [Diagram 3] FIG. 3 shows cleavage of SNAP-25 in rat spinal cord neurons by recombinant BoNT / AB chimeras 1, 2 and 3A (SEQ ID NO: 3, 4 and 5, respectively, converted to a two-chain form). Cultured rat primary spinal cord neurons (SCN) were exposed to various concentrations of recombinant BoNT / AB chimeras 1, 2 or 3A for 24 hours at 37° C. in a humidified atmosphere containing 10% CO2. Cells were then lysed using 1× NuPAGE buffer supplemented with DTT and benzonase. Samples were transferred to microcentrifuge tubes, heated at 90° C. for 5 minutes on a heat block, stored at −20° C., and then analyzed for SNAP-25 cleavage by Western blot. SNAP-25 was detected using a polyclonal antibody (Sigma #S9684) that detects both full-length and cleaved forms of SNAP-25. Anti-rabbit HRP (Sigma #A6154) was used as the secondary antibody. [Figure 4]Figure 4 shows the mouse digit abduction scoring assay. Mice were injected into the gastrocnemius-soleus complex muscle of one hind limb under brief general anesthesia, and muscle weakness was measured using the digit abduction score (DAS) on a 0-4 scale. The highest DAS value for each dose was determined and plotted against dose, and the data was fitted to a 4-parameter logistic equation to determine the ED50 and the dose leading to DAS 4 (DAS 4 dose) values. [Diagram 5] 5 shows SDS-PAGE of purified recombinant BoNT / AB chimeras 3B and 3C (SEQ ID NOs: 6 and 7, respectively). Lanes are labeled "marker" (molecular weight marker), "-DTT" (oxidized BoNT / AB chimera sample) and "+DTT" (reduced BoNT / AB chimera sample). [Figure 6] FIG. 6 shows cleavage of SNAP-25 by unmodified BoNT / A and BoNT / AB chimeras 3B and 3C (SEQ ID NOs: 2, 6 and 7, respectively) in human induced pluripotent stem cell-derived peripheral neurons (PERI.4U-Axiogenesis, Germany). PERI.4U cells were exposed to various concentrations of recombinant BoNT / A or BoNT / AB chimeras 3B or 3C for 24 hours at 37° C. in a humidified CO2 atmosphere containing 5% CO2. Cells were then lysed using 1× NuPAGE buffer supplemented with DTT and benzonase. Samples were transferred to microcentrifuge tubes, heated at 90° C. for 5 minutes on a heat block, stored at −20° C., and then analyzed for SNAP-25 cleavage by Western blot. SNAP-25 was detected using a polyclonal antibody (Sigma #S9684) that detects both full-length and cleaved forms of SNAP-25. Anti-rabbit HRP (Sigma #A6154) was used as the secondary antibody. [Figure 7]Figure 7 shows the duration of muscle weakness over time in a mouse digit abduction scoring assay. Mice were injected into the gastrocnemius-soleus complex muscle of one hind limb under brief general anesthesia, and muscle weakness was measured using the digit abduction score (DAS) on a 0-4 scale. Animals from the group injected with the lowest dose that induced a DAS of 4 during the first 4 days of injection were monitored until complete recovery of muscle weakness to a DAS of 0 (no observed muscle weakness). [Figure 8] FIG. 8 shows injection sites for intramuscular administration of modified BoNT / A for treating glabellar lines. [Figure 9] FIG. 9 shows injection sites for intramuscular administration of modified BoNT / A for treating glabellar and forehead lines. [Figure 10] FIG. 10 shows the injection sites for intramuscular administration of modified BoNT / A for treating lateral canthal lines only. [Figure 11] FIG. 11 shows injection sites for intramuscular administration of modified BoNT / A for treating glabellar lines, forehead lines, and lateral canthal lines.

[0300] Sequence Listing

[0301] In any of the following SEQ ID NOs, where the first Met amino acid residue or the corresponding first codon is shown, said residue / codon is optional.

[0302] SEQ ID NO:1 (Nucleotide sequence of unmodified BoNT / A)

[0303] SEQ ID NO:2 (polypeptide sequence of unmodified BoNT / A)

[0304] SEQ ID NO:3 (polypeptide sequence of modified BoNT / "chimera 1") MPFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLN PPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGG STIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNGY GSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPN RVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKA KSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIYTEDNFVKFFKV LNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTKLKNFT GLFEFYKLLCVRGIITSKTKSLDKGYNKALNDLCIKVNNWDLFFSPSEDNFTNDLNKGEE ITSDTNIEAAEENISLDLIQQYYLTFNFDNEPENISIENLSSDIIGQLELMPNIERFPNG KKYELDKYTMFHYLRAQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSDYVKKVNKATEA AMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYKDDFVGALIFSG AVILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALSKRNEKWDEVYKYIVTNWLAK VNTQIDLIRKKMKEALENQAEATKAIINYQYNQYTEEEKNNINFNIDDLSSKLNESINKA MININKFLNQCSVSYLMNSMIPYGVKRLEDFDASLKDALLKYIYDNRGTLIGQVDRLKDK VNNTLSTDIPFQLSKYVDNQRLLSTFTEYIKSEILNNIILNLRYKDNNLIDLSGYGAKVE VYDGVELNDKNQFKLTSSANSKIRVTQNQNIIFNSVFLDFSVSFFWIRIPKYKNDGIQNYI HNEYTIINCMKNNSGWKISIRGNRIIWTLIDINGKTKSVFFEYNIREDISEYINRWFFVT ITNNLNNAKIYINGKLESNTDIKDIREVIANGEIIFKLDGDIDRTQFIWMKYFSIFNTEL SQSNIEERYKIQSYSEYLKDFWGNPLMYNKEYYMFNAGNKNSYIKLKKDSPVGEILTRSK YNQNSKYINYRDLYIGEKFIIRRKSNSQSINDDIVRKEDYIYLDFFNLNQEWRVYTYKYF KKEEMKLFLAPIYDSDEFYNTIQIKEYDEQPTYSCQLLFKKDEESTDEIGLIGIHRFYES GIVFEEYKDYFCISKWYLKEVKRKPYNLKLGCNWQFIPKDEGWTEHHHHHHHHHH

[0305] SEQ ID NO:4 (polypeptide sequence of modified BoNT / "chimera 2") MPFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLN PPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGG STIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNGY GSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPN RVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKA KSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIYTEDNFVKFFKV LNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTKLKNFT GLFEFYKLLCVRGIITSKTKSLDKGYNKALNDLCIKVNNWDLFFSPSEDNFTNDLNKGEE ITSDTNIEAAEENISLDLIQQYYLTFNFDNEPENISIENLSSDIIGQLELMPNIERFPNG KKYELDKYTMFHYLRAQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSDYVKKVNKATEA AMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYKDDFVGALIFSG AVILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALSKRNEKWDEVYKYIVTNWLAK VNTQIDLIRKKMKEALENQAEATKAIINYQYNQYTEEEKNNINFNIDDLSSKLNESINKA MININKFLNQCSVSYLMNSMIPYGVKRLEDFDASLKDALLKYIYDNRGTLIGQVDRLKDK VNNTLSTDIPFQLSKYVDNQRLLSTFTEYIKNIIELGGGGSELSEILNNIILNLRYKDNN LIDLSGYGAKVEVYDGVELNDKNQFKLTSSANSKIRVTQNQNIIFNSVFLDFSVSFWIRI PKYKNDGIQNYIHNEYTIINCMKNNSGWKISIRGNRIIWTLIDINGKTKSVFFEYNIRED ISEYINRWFFVTITNNNLNNAKIYINGKLESNTDIKDIREVIANGEIIFKLDGDIDRTQFI WMKYFSIFNTELSQSNIEERYKIQSYSEYLKDFWGNPLMYNKEYYMFNAGNKNSYIKLKK DSPVGEILTRSKYNQNSKYINYRDLYIGEKFIIRRKSNSQSINDDIVRKEDYIYLDFFNL NQEWRVYTYKYFKKEEMKLFLAPIYDSDEFYNTIQIKEYDEQPTYSCQLLFKKDEESTDE IGLIGIHRFYESGIVFEEYKDYFCISKWYLKEVKRKPYNLKLGCNWQFIPKDEGWTEHHH HHHHHHH

[0306] SEQ ID NO:5 (polypeptide sequence of modified BoNT / A "chimera 3A") MPFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLN PPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGG STIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNGY GSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPN RVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKA KSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIYTEDNFVKFFKV LNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTKLKNFT GLFEFYKLLCVRGIITSKTKSLDKGYNKALNDLCIKVNNWDLFFSPSEDNFTNDLNKGEE ITSDTNIEAAEENISLDLIQQYYLTFNFDNEPENISIENLSSDIIGQLELMPNIERFPNG KKYELDKYTMFHYLRAQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSDYVKKVNKATEA AMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYKDDFVGALIFSG AVILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALSKRNEKWDEVYKYIVTNWLAK VNTQIDLIRKKMKEALENQAEATKAIINYQYNQYTEEEKNNINFNIDDLSSKLNESINKA MININKFLNQCSVSYLMNSMIPYGVKRLEDFDASLKDALLKYIYDNRGTLIGQVDRLKDK VNNTLSTDIPFQLSKYVDNQRLLSTFTEYIKNILNNIILNLRYKDNNLIDLSGYGAKVEV YDGVELNDKNQFKLTSSANSKIRVTQNQNIIFNSVFLDFSVSFWIRIPKYKNDGIQNYIH NEYTIINCMKNNSGWKISIRGNRIIWTLIDINGKTKSVFFEYNIREDISEYINRWFFVTI TNNLNNAKIYINGKLESNTDIKDIREVIANGEIIFKLDGDIDRTQFIWMKYFSIFNTELS QSNIEERYKIQSYSEYLKDFWGNPLMYNKEYYMFNAGNKNSYIKLKKDSPVGEILTRSKY NQNSKYINYRDLYIGEKFIIRRKSNSQSINDDIVRKEDYIYLDFFNLNQEWRVYTYKYFK KEEMKLFLAPIYDSDEFYNTIQIKEYDEQPTYSCQLLFKKDEESTDEIGLIGIHRFYESG IVFEEYKDYFCISKWYLKEVKRKPYNLKLGCNWQFIPKDEGWTEHHHHHHHHHH

[0307] SEQ ID NO:6 (Polypeptide sequence of modified BoNT / A "chimera 3B")

[0308] SEQ ID NO: 7 (Polypeptide sequence of modified BoNT / A "chimera 3C") MPFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLN PPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGG STIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNGY GSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPN RVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKA KSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIYTEDNFVKFFKV LNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTKLKNFT GLFEFYKLLCVRGIITSKTKSLDKGYNKALNDLCIKVNNWDLFFSPSEDNFTNDLNKGEE ITSDTNIEAAEENISLDLIQQYYLTFNFDNEPENISIENLSSDIIGQLELMPNIERFPNG KKYELDKYTMFHYLRAQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSDYVKKVNKATEA AMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYKDDFVGALIFSG AVILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALSKRNEKWDEVYKYIVTNWLAK VNTQIDLIRKKMKEALENQAEATKAIINYQYNQYTEEEKNNINFNIDDLSSKLNESINKA MININKFLNQCSVSYLMNSMIPYGVKRLEDFDASLKDALLKYIYDNRGTLIGQVDRLKDK VNNTLSTDIPFQLSKYVDNQRLLSTFTEYIKNILNNIILNLRYKDNNLIDLSGYGAKVEV YDGVELNDKNQFKLTSSANSKIRVTQNQNIIFNSVFLDFSVSFFWIRIPKYKNDGIQNYIH NEYTIINCMKNNSGWKISIRGNRIIWTLIDINGKTKSVFFEYNIREDISEYINRWFFVTI TNNLNNNAKIYINGKLESNTDIKDIREVIANGEIIFKLDGDIDRTQFIWMKYFSIFTELS QSNIEERYKIQSYSEYLKDFWGNPLMYNKEYYMFNAGNKNSYIKLKKDSPVGEILTRSKY NQNSKYINYRDLYIGEKFIIRRKSNSQSINDDIVRKEDYIYLDFFNLNQEWRVYTYKYFK KEEEKLFLAPISDSDEFYNTIQIKEYDEQPTYSCQLLFKKDEESTDEIGLIGIHRFYESG IVFEEYKDYFCISKWYLKEVKRKPYNLKLGCNWQFIPKDEGWTE

[0309] SEQ ID NO:8 (Polypeptide sequence of BoNT / B) MPVTINNFNYNDPIDNNNIIMMEPPFARGTGRYYKAFKITDRIWIIPERYTFGYKPEDFN KSSGIFNRDVCEYYDPDYLNTNDKKNIFLQTMIKLFNRIKSKPLGEKLLEMIINGIPYLG DRRVPLEEFNTNIASVTVNKLISNPGEVERKKGIFANLIIFGPGPVLNENETIDIGIQNH FASREGFGGIMQMKFCPEYVSVFNNVQENKGASIFNRRGYFSDPALILMHELIHVLHGLY GIKVDDLPIVPNEKKFFMQSTDAIQAEELYTFGGQDPSIITPSTDKSIYDKVLQNFRGIV DRNLKVLVCISDPNININIYKNKFKDKYKFVEDSEGKYSIDVESFDKLYKSLMFGFTETN IAENYKIKTRASYFSDSLPPVKIKNLLDNEIYTIEEGFNISDKDMEKEYRGQNKAINKQA YEEISKEHLAVYKIQMCKSVKAPGICIDVDNEDLFFIADKNSFSDDLSKNERIEYNTQSN YIENDFPINELILDTDLISKIELPSENTESLTDFNVDVPVYEKQPAIKKIFTDENTIFQY LYSQTFPLDIRDISLTSSFDDALLFSNKVYSFFSMDYIKTANKVVEAGLFAGWVKQIVND FVIEANKSNTMDKIADISLIVPYIGLALNVGNETAKGNFENAFEIAGASILLEFIPELLI PVVGAFLLESYIDNKNKIIKTIDNALTKRNEKWSDMYGLIVAQWLSTVNTQFYTIKEGMY KALNYQAQALEEIIKYRYNIYSEKEKSNINIDFNDINSKLNEGINQAIDNINNFINGCSV SYLMKKMIPLAVEKLLDFDNTLKKNLLNYIDENKLYLIGSAEYEKSKVNKYLKTIMPFDL SIYTNTILIEMFNKYNSEILNNIILNLRYKDNNLIDLSGYGAKVYDGVELNDKNQFK LTSSANSKIRVTQNQNIIFNSVFLDFSVSFWIRIPKYKNDGIQNYIHNEYTIINCMKNNS GWKISIRGNRIIWTLIDINGKTKSVFFEYNIREDISEYINRWFFVTITNNNLNNAKIYING KLESNTDIKDIREVIANGEIIFKLDGDIDRTQFIWMKYFSIFTELSQSNIEERYKIQSY SEYLKDFWGNPLMYNKEYYMFNAGNKNSYIKLKKDSPVGEILTRSKYNQNSKYINYRDLY IGEKFIIRRKSNSQSINDDIVRKEDYIYLDFFNLNQEWRVYTYKYFKKEEEKLFLAPISD SDEFYNTIQIKEYDEQPTYSCQLLFKKDEESTDEIGLIGIHRFYESGIVFEEYKDYFCIS KWYLKEVKRKPYNLKLGCNWQFIPKDEGWTE

[0310] SEQ ID NO:9 - C-terminal light chain fragment TKSLDKGYNK

[0311] SEQ ID NO:10 - C-terminal light chain fragment 2 SLDKGYNK

[0312] SEQ ID NO:11 - Two-chain light chain 1 PFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGGSTIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNGYGSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPNRVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKAKSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIYTEDNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTKLKNFTGLFEFYKLLCVRGIITSK

[0313] Sequence number 12 - Double-stranded L chain 2 PFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGGSTIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNGYGSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPNRVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKAKSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIYTEDNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTKLKNFTGLFEFYKLLCVRGIITSKTK

[0314] Sequence number 13 - Double-stranded H chain ALNDLCIKVNNWDLFFSPSEDNFTNDLNKGEEITSDTNIEAAENISLDLIQQYYLTFNFDNEPENISIENLSSDIIGQLELMPNIERFPNGKKYELDKYTMFHYLRAQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSDYVKKVNKATEAAMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPANLIGNMLYKDDFVGALIFSGAV ILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALSKRNEKWDEVYKYIVTNWLAKVNTQIDLIRKKMKEALENQAEEATKAIINYQYNQYTEEEKNNINFNIDDLSSKLNESINKAMININKFLNQCSVSYLMNSMIPYGVKRLEDFDASLKDALLKYIYDNRGTLIGQVDRLKDKVNNTLSTDIPFQLSKYVDNQRLLSTFTEYIKNILNN IILNLRYKDNNLIDLSGYGAKVEVYDGVELNDKNQFKLTSSANSKIRVTQNQNIIFNSVFLDFSVSFWIRIPKYKNDGIQNYIHNEYTIINCMKNNSGWKISIRGNRIIWTLIDINGKTKSVFFEYNIREDISEYINRWFFVTITNNLNNAKIYINGKLESNTDIKDIREVIANGEIIFKLDGDIDRTQFIWMKYFSIFNTELSQSNIEERYKI QSYSEYLKDFWGNPLMYNKEYYMFNAGNKNSYIKLKDSPVGEILTRSKYNQNSKYINYRDLYIGEKFIIRRKSNSQSINDDIVRKEDYIYLDFFNNLNQEWRWYTYKYFKKEEMKLFLAPIYDSDEFYNTIQIKEYDEQPTYSCQLLFKKDEESTDEIGLIGIHRFYESGIVFEEYKDYFCISKWYLKEVKRKPYNLKGCNWQFIPKDEGWTE

example

[0315] [example1]

[0316] Cloning, expression and purification of BoNT / AB chimeras

[0317] BoNT / AB chimeric constructs 1, 2, 3A, 3B and 3C (SEQ ID NOs: 3-7, respectively) were constructed using standard molecular biology techniques from DNA encoding the parent serotype molecules and appropriate oligonucleotides. These were then modified to include a C-terminal His 10 The A-tags were cloned into the pJ401 expression vector and transformed into BLR(DE3) E. coli cells for overexpression. The cells were grown in 2L baffled Erlenmeyer flasks containing 1L of modified Terrific Broth (mTB) supplemented with the appropriate antibiotics at 37°C and 225 RPM shaking. 600 When the β reached >0.5, the incubator temperature was reduced to 16° C. and then induced 1 hour later with 1 mM IPTG for 20 hours at 225 RPM shaking to allow expression of the recombinant BoNT / AB constructs.

[0318] Harvested cells were lysed by sonication and clarified by centrifugation at 4500 RPM for 1 hour at 4°C. The recombinant BoNT / AB chimera molecule was then extracted in ammonium sulfate and purified by standard fast protein liquid chromatography (FPLC) techniques, which involved the use of a hydrophobic interaction resin for capture and an anion exchange resin for intermediate purification steps. The partially purified molecule was then proteolytically cleaved with endoprotease Lys-C to obtain the active dichain, which was further purified using a second hydrophobic interaction resin to obtain the final BoNT / AB chimera.

[0319] Decahistadine Tag (H 10 For the BoNT / AB chimera molecules (chimeras 1, 2, 3A) having the .sup.1-hydroxypropyl ether (H.sub.2O) and .sup.2-hydroxypropyl ether (H.sub.2O), the capture step involved the use of immobilized nickel resin instead of hydrophobic interaction resin.

[0320] The sequences of each chimera are shown in Table 1. [Table 1]

[0321] [Example 2]

[0322] Comparison of BoNT / AB chimeras 1, 2, and 3A

[0323] C-terminal His 10 BoNT / AB chimeras 1, 2 and 3A carrying the tag and the E1191M / S1199Y double mutation were purified and tested for functional activity as described in Example 1 (FIG. 2).

[0324] Rat spinal cord neuron SNAP-25 cleavage assay

[0325] Primary cultures of rat spinal cord neurons (SCN) were prepared and grown for 3 weeks in 96-well tissue culture plates (as described in Masuyer et al., 2011, J. Struct. Biol. Structure and activity of a functional derivative of Clostridium botulinum neurotoxin B, and Chaddock et al., 2002, Protein Expr. Purif. Expression and purification of catalytically active, non-toxic endopeptidase derivatives of Clostridium botulinum toxin type A). Serial dilutions of BoNT / AB were prepared in SCN feeding medium. Growth medium from the wells to be treated was collected and filtered (0.2 μm filter). 125 μL of filtered medium was added back to each test well. Then 125 μL of diluted toxin was added to the plate (triplicate wells). Treated cells were incubated at 37° C., 10% CO2 for 24±1 h.

[0326] Analysis of BoNT activity using a SNAP-25 cleavage assay

[0327] After treatment, BoNT was removed and cells were washed once with PBS (Gibco, UK). Cells were lysed in 1x NuPAGE lysis buffer (Life Technologies) supplemented with 0.1 M dithiothreitol (DTT) and 250 units / mL benzonase (Sigma). Lysate proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes. Membranes were probed with a primary antibody specific for SNAP-25 (Sigma #S9684), which recognizes uncleaved SNAP-25 as well as SNAP-25 cleaved by BoNT / A endopeptidase. The secondary antibody used was anti-rabbit IgG conjugated with HRP (Sigma #A6154). Bands were detected by enhanced chemiluminescence and imaged using pXi6 Access (Synoptics, UK). Band intensities were determined using GeneTools software (Syngene, Cambridge, UK) and the percentage of cleaved SNAP-25 for each concentration of BoNT was calculated. Data were fitted to a four-parameter logistic equation to determine pEC using GraphPad Prism version 6 (GraphPad). 50 was calculated.

[0328] Table 2 below shows the pECs determined for chimeras 1, 2 and 3A in the rat SCN SNAP-25 cleavage assay. 50 These results indicate that the three BoNT / AB chimeras retained the ability to enter rat spinal cord neurons and cleave their target substrates. However, chimera 3A was more potent in this assay than chimeras 1 and 2 (see also FIG. 3). [Table 2]

[0329] Digital Abduction Scoring (DAS) Assay

[0330] The method for measuring the activity of BoNT / AB chimeras 1, 2 and 3A in the DAS assay is based on the startle response of mice when briefly suspended by their tails, the toes-spreading reflex. This reflex is scored as the digit abduction score (DAS) and is inhibited after administration of BoNT into the gastrocnemius-soleus muscle of the hind limb. Mice are briefly suspended by their tails to elicit the characteristic startle response in which the animals extend their hind limbs and then abduct their toes. (Aoki et al. 1999, Eur. J. Neurol.; 6 (suppl. 4) S3-S10).

[0331] On the day of injection, mice were anesthetized with 3% isoflurane in oxygen in an induction chamber. Each mouse received an intramuscular injection of the BoNT / AB chimera or vehicle (phosphate buffer containing 0.2% gelatin) in the gastrocnemius-soleus muscle of the right hind leg.

[0332] After neurotoxin injection, the variable degree of finger abduction was scored on a scale of 0 to 4, where 0 = normal and 4 = maximal reduction in finger abduction and leg extension. The ED50 was determined by nonlinear adjustment analysis using the mean of the maximal effect at each dose. The mathematical model used was a four-parameter logistic model.

[0333] The DAS was performed every 2 hours during the first day after dosing, then three times per day for four days.

[0334] Figure 4 shows the fitted curves of chimeras 1, 2 and 3A (SEQ ID NOs: 3, 4 and 5, respectively, converted to two-chain form). The chimera 3A curve is shifted to the left, which means that a lower dose of chimera 3A achieved a similar DAS response compared to chimeras 1 and 2, thus indicating that chimera 3A is more potent than the others in the mouse DAS assay, see also the table below (Table 3) which provides the calculated ED50 of each chimera and the value of the dose leading to DAS 4 (highest score).

[0335] Table 3 below shows the EDs determined for unmodified recombinant BoNT / A1 (rBoNT / A1 converted to a two-chain form - SEQ ID NO: 2) and chimeras 1, 2 and 3A in the mouse DAS assay. 50 and DAS 4 doses. These results indicate that of the three chimeras, chimera 3A has the highest in vivo efficacy in inducing muscle weakness. The studies shown in Figure 4 and Table 3 were performed in mice obtained from Charles River Laboratories. [Table 3]

[0336] [Example 3]

[0337] Comparison of BoNT / AB chimeras 3B, 3C and unmodified BoNT / A1

[0338] The untagged BoNT / AB chimeras 3B and 3C (sequence numbers 6 and 7), with and without the E1191M / S1199Y double mutation, respectively, were purified as described in Example 1 (Figure 5) and tested for functional activity using unmodified BoNT / A (sequence number 2 converted to a two-chain form) as a reference.

[0339] Human pluripotent stem cell SNAP-25 cleavage assay

[0340] Cryopreserved PERI.4U-cells were purchased from Axiogenesis (Cologne, Germany). Thawing and plating of cells was performed as recommended by the manufacturer. Briefly, cryovials containing cells were thawed for 2 minutes in a water bath at 37°C. After gentle suspension, cells were transferred to a 50 mL tube. The cryovial was washed with 1 mL of Peri.4U® Thawing Medium supplied by the manufacturer, and the medium was transferred dropwise to the cell suspension into the 50 mL tube, after which another 2 mL of Peri.4U® Thawing Medium was added dropwise to the 50 mL tube. Cells were then counted using a hemocytometer. After this, another 6 mL of Peri.4U® Thawing Medium was added to the cell suspension. Cell pellets were obtained by centrifugation at 260 x g (e.g., 1,100 RPM) at room temperature for 6 minutes. Cells were then resuspended in the complete Peri.4U® culture medium supplied by the manufacturer. Cells were plated on cell culture plates coated with poly-L-ornithine and laminin at 1 cm 2 Cells were plated at a density of 50,000–150,000 cells per well. Cells were cultured at 37°C in a humidified CO2 atmosphere, with complete medium changes every 2–3 days during culture.

[0341] For toxin treatment, serial dilutions of BoNT were prepared in Peri.4U® culture medium. The medium from the wells to be treated was collected and filtered (0.2 μm filter). 125 μL of filtered medium was added back to each test well. Then, 125 μL of diluted toxin was added to the plate (triplicate wells). Treated cells were incubated at 37° C., 10% CO2 for 48±1 hours.

[0342] Analysis of BoNT activity using a SNAP-25 cleavage assay

[0343] After treatment, BoNT was removed and cells were washed once with PBS (Gibco, UK). Cells were lysed in 1x NuPAGE lysis buffer (Life Technologies) supplemented with 0.1 M dithiothreitol (DTT) and 250 units / mL benzonase (Sigma). Lysate proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes. Membranes were probed with a primary antibody specific for SNAP-25 (Sigma #S9684), which recognizes uncleaved SNAP-25 as well as SNAP-25 cleaved by BoNT / A endopeptidase. The secondary antibody used was anti-rabbit IgG conjugated with HRP (Sigma #A6154). Bands were detected by enhanced chemiluminescence and imaged using pXi6 Access (Synoptics, UK). Band intensities were determined using GeneTools software (Syngene, Cambridge, UK) and the percentage of cleaved SNAP-25 for each concentration of BoNT was calculated. Data were fitted to a four-parameter logistic equation to determine pEC using GraphPad Prism version 6 (GraphPad). 50 was calculated.

[0344] Figure 6 shows that chimeras 3B and 3C were more potent than rBoNT / A1 in cleaving SNAP-25 in induced human pluripotent stem cells, with the former being significantly more potent, which can be explained by a double mutation that increases the affinity of chimera 3B for the human synaptotagmin II protein receptor present in these cells (Figure 6, Table 4). [Table 4]

[0345] Digital Abduction Scoring (DAS) Assay – Safety Factor

[0346] The method for measuring the activity of BoNT in the DAS assay is based on the startle response of mice when briefly suspended by their tails, the toe-spreading reflex. This reflex is scored as the digit abduction score (DAS) and is inhibited after administration of BoNT into the gastrocnemius-soleus muscle of the hind limb. Mice are briefly suspended by their tails to elicit a characteristic startle response in which the animals extend their hind limbs and then abduct their toes. (Aoki et al. 1999, Eur. J. Neurol.; 6 (suppl. 4) S3-S10).

[0347] On the day of injection, mice were anesthetized with 3% isoflurane in oxygen in an induction chamber. Each mouse received an intramuscular injection of BoNT or vehicle (phosphate buffer containing 0.2% gelatin) in the gastrocnemius-soleus muscle of the right hind leg.

[0348] After neurotoxin injection, the variable degree of finger abduction was scored on a scale of 0 to 4, where 0 = normal and 4 = maximal reduction in finger abduction and leg extension. The ED50 was determined by nonlinear adjustment analysis using the mean of the maximal effect at each dose. The mathematical model used was a four-parameter logistic model.

[0349] DAS were performed every 2 hours for all doses during the first day post-dosing, then 3 times per day for 4 days. Animals in the vehicle and lowest dose injected groups that induced a DAS of 4 during the first 4 days of injection were then monitored until complete recovery of muscle weakness to a DAS of 0 (no observed muscle weakness).

[0350] To calculate the safety rate, all animals were weighed the day before toxin injection (D0) and then once a day throughout the study period. The mean body weight, its standard deviation and standard error of the mean were calculated daily for each dose group. The safety rate of BoNT (-10%ΔBW / ED 50 To obtain the ED of the BoNT being studied, doses were administered at any time during the study where the mean body weight of a dose group was 10% lower than the mean body weight of that same dose group at D0. 50A lethal dose was defined as the dose at which at least one animal in that dose group died.

[0351] Figure 7 shows the duration of muscle weakness over time in a mouse digit abduction scoring assay for unmodified BoNT / A, chimera 3B and chimera 3C (sequence numbers 2, 6 and 7 converted to two-chain form), indicating that the chimeras have a longer duration of action.

[0352] Table 5 below shows the EDs determined for rBoNT / A1 and chimeras 3B and 3C in the mouse DAS assay. 50 and DAS 4 doses. The table also provides the total duration of action of the DAS 4 doses until full recovery of muscle weakness to a DAS of 0 (no muscle weakness observed). Additionally, the table provides the mouse lethal dose and safety factor (-10%ΔBW / ED 50 ) are shown. In comparison to rBoNT / A1, chimeras 3B and 3C have a longer duration of action, a better safety rate and a higher lethal dose. The studies shown in FIG. 7 and Table 5 were performed in mice obtained from Janvier Laboratories. [Table 5]

[0353] [Example 4]

[0354] Dosage regimen for treating glabellar lines

[0355] The modified BoNT / A is provided as a lyophilized powder in 2 mL clear glass vials containing 15 ng of modified BoNT / A per vial. The lyophilized powder is reconstituted with a mixture of sterile sodium chloride 0.9% v / w preservative-free solution and diluent (formulation buffer containing only the modified BoNT / A excipients). After reconstitution, the solution is further diluted as needed.

[0356] Follow the injection regimen shown in Figure 8 to treat moderate to severe glabellar lines.

[0357] The unit dose is 20-300 pg (2-35 units).

[0358] Intramuscular injections are given at up to five sites according to unit dose. The maximum total dose administered is 1500 pg (177 units).

[0359] [Example 5]

[0360] Dosage regimen for treating glabellar and forehead lines

[0361] The modified BoNT / A is provided as a lyophilized powder in 2 mL clear glass vials containing 15 ng of modified BoNT / A per vial. The lyophilized powder is reconstituted with a mixture of sterile sodium chloride 0.9% v / w preservative-free solution and diluent (formulation buffer containing only the modified BoNT / A excipients). After reconstitution, the solution is further diluted as needed.

[0362] Follow the injection regimen shown in FIG. 9 to treat moderate to severe glabellar and forehead lines.

[0363] The unit dose is 20-300 pg (2-35 units).

[0364] Intramuscular injections are administered at up to 10 sites according to unit dose. The maximum total dose administered is 3000 pg (355 units).

[0365] [Example 6]

[0366] Dosage regimen for treating lateral canthal lines

[0367] The modified BoNT / A is provided as a lyophilized powder in 2 mL clear glass vials containing 15 ng of modified BoNT / A per vial. The lyophilized powder is reconstituted with a mixture of sterile sodium chloride 0.9% v / w preservative-free solution and diluent (formulation buffer containing only the modified BoNT / A excipients). After reconstitution, the solution is further diluted as needed.

[0368] Follow the injection regimen shown in Figure 10 to treat moderate to severe lateral canthal lines.

[0369] The unit dose is 20-300 pg (2-35 units).

[0370] Intramuscular injections are given at up to six sites according to unit dose. The maximum total dose administered is 1800 pg (213 units).

[0371] [Example 7]

[0372] Dosage regimen for treating glabellar, forehead and lateral canthal lines

[0373] The modified BoNT / A is provided as a lyophilized powder in 2 mL clear glass vials containing 15 ng of modified BoNT / A per vial. The lyophilized powder is reconstituted with a mixture of sterile sodium chloride 0.9% v / w preservative-free solution and diluent (formulation buffer containing only the modified BoNT / A excipients). After reconstitution, the solution is further diluted as needed.

[0374] Follow the injection regimen shown in FIG. 11 to treat moderate to severe glabellar, forehead and lateral canthal lines.

[0375] The unit dose is 20-300 pg (2-35 units).

[0376] Intramuscular injections are administered at up to 16 sites according to unit dose. The maximum total dose administered is 4800 pg (569 units).

[0377] [Example 8]

[0378] Further characterization of modified BoNT / A (SEQ ID NO:6 converted to a dichain form)

[0379] SEQ ID NO:6, converted to a modified BoNT / A (e.g., BoNT / AB chimera) two-chain form, was used in mouse LD 50 The assay yielded a result of 1.202 ng / kg. Thus, 1 unit of SEQ ID NO:6 corresponds to 24.04 pg in this assay.

[0380] Additionally, the BoNT / AB chimera was tested in a rat DAS assay to determine duration of action (as in Example 6) when compared to Dysport®. The results are shown in Table 6 below: [Table 6]

[0381] In conclusion, the duration of action of BoNT / AB was significantly higher than that of Dysport® and similar to that of SEQ ID NO: 6. It is therefore expected that the unit dose and administration regimen for SEQ ID NO: 6 may be similarly applied to BoNT / AB to provide improved treatment of facial wrinkles.

[0382] [Example 9]

[0383] Calculation of Unit Dose of Modified BoNT / A (SEQ ID NO: 6 Converted to Dichain Form) for Upper Facial Lines

[0384] Taking into account the preclinical pharmacology data, a suitable unit dose range (UD) for administration of modified BoNT / A in humans was calculated.

[0385] For SEQ ID NO: 6, DAS ED of 13 pg / kg 50 was calculated. ED 50is considered to be the minimum pharmacologically active dose, which is approximately 300-fold lower than the no observed adverse effect level (NOAEL) of 4 ng / kg in the same animal species. The ED of 13 pg / kg of SEQ ID NO:6 in rats 50 corresponds to a 0.8 ng dose for a human weighing 60 kg.

[0386] Therefore, a lower unit dose limit of 20 pg was selected. An upper unit dose limit of 1500 pg was selected, which is lower than the NOAEL of 4 ng / kg obtained from both non-clinical safety species (rat and monkey) translated to a human dose of 60 kg body weight.

[0387] Given the improved safety profile, one maximum total dose for the treatment of upper facial lines was established at 24,000 pg, derived from the NOAEL of 4 ng / kg from both non-clinical safety species (rat and monkey) translated into a human dose of 60 kg body weight.

[0388] [Example 10]

[0389] Dosage regimen for treating glabellar lines using modified BoNT / A (SEQ ID NO: 6 converted to a dichain form)

[0390] The modified BoNT / A is provided as a lyophilized powder in vials containing 36 ng of modified BoNT / A per vial. The lyophilized powder is reconstituted.

[0391] Follow the injection regimen shown in Figure 8 to treat moderate to severe glabellar lines.

[0392] The unit dose is 20 to 1500 pg (0.8 to 62 units).

[0393] Intramuscular injections are given at up to five sites according to unit dose. The maximum total dose administered is 7500 pg (312 units).

[0394] [Example 11]

[0395] Dosage regimen for treating glabellar and forehead lines using modified BoNT / A (SEQ ID NO: 6 converted to a dichain form)

[0396] The modified BoNT / A is provided as a lyophilized powder in vials containing 36 ng of modified BoNT / A per vial. The lyophilized powder is reconstituted.

[0397] Follow the injection regimen shown in FIG. 9 to treat moderate to severe glabellar and forehead lines.

[0398] The unit dose is 20 to 1500 pg (0.8 to 62 units).

[0399] Intramuscular injections are administered in unit doses up to 10. The maximum total dose administered is 15,000 pg (624 units).

[0400] [Example 12]

[0401] Dosage regimen for treating lateral canthal lines using modified BoNT / A (SEQ ID NO: 6 converted to a dichain form)

[0402] The modified BoNT / A is provided as a lyophilized powder in vials containing 36 ng of modified BoNT / A per vial. The lyophilized powder is reconstituted.

[0403] Follow the injection regimen shown in Figure 10 to treat moderate to severe lateral canthal lines.

[0404] The unit dose is 20 to 1500 pg (0.8 to 62 units).

[0405] Intramuscular injections are administered at up to 6 sites according to unit dose. The maximum total dose administered is 9000 pg (374 units).

[0406] [Example 13]

[0407] Dosage regimen for treating glabellar, forehead, and lateral canthal lines using modified BoNT / A (SEQ ID NO: 6 converted to a dichain form)

[0408] The modified BoNT / A is provided as a lyophilized powder in vials containing 36 ng of modified BoNT / A per vial. The lyophilized powder is reconstituted.

[0409] Follow the injection regimen shown in FIG. 11 to treat moderate to severe glabellar, forehead and lateral canthal lines.

[0410] The unit dose is 20 to 1500 pg (0.8 to 62 units).

[0411] Intramuscular injections are administered at up to 16 sites according to unit dose. The maximum total dose administered is 24,000 pg (998 units).

[0412] [Example 14]

[0413] Safety & Efficacy of Modified BoNT / A (SEQ ID NO:6 Converted to Dichain Form) in Humans

[0414] An integrated Phase I / II multicenter, double-blind, randomized, Dysport and placebo-controlled, dose escalation and dose-finding study was conducted to evaluate the safety and efficacy of modified BoNT / A (SEQ ID NO: 6 converted to bichain form) in the treatment of moderate to severe upper facial lines for adults. The upper facial lines include glabellar lines, forehead lines and lateral canthal lines. The study involved human sequential dose escalation in a unique cohort of patients with adult upper facial lines. The upper facial lines include glabellar lines, forehead lines and lateral canthal lines. Each muscle was injected with a unit dose of modified BoNT / A, Dysport or placebo. Six cohorts received different (escalating) amounts of modified BoNT / A (SEQ ID NO: 6 converted to bichain form). The total dose range was 0.1-6.0 ng. Modified BoNT / A was injected into up to 16 sites across the upper facial region (5 sites in the glabellar lines, 5 sites in the forehead lines, and 3 sites on each side in the lateral canthal lines - see Figure 11).

[0415] The results showed that, despite the exceptionally high doses per muscle, all unit and total doses of modified BoNT / A tested (i.e., up to 6,000 pg) were effective and safely tolerated, with no adverse effects observed. This indicates that modified BoNT / A does not diffuse away from the injection site, highlighting the exceptional safety profile of modified BoNT / A (SEQ ID NO: 6). Based on these findings, it can be trusted that significantly higher unit doses can be administered per muscle without adverse effects resulting. Furthermore, given the lack of systemic diffusion of the toxin, it can be trusted that higher unit doses, up to 13-14x, can be administered without safety concerns.

[0416] Thus, unit doses of up to 5,000 pg + / - 10% each were selected for treatment of upper facial lines to be administered up to 16x across the upper facial area (e.g., 5 sites in the glabellar lines, 5 sites in the forehead lines and 3 sites on each side in the lateral canthal lines). The total dose administered during a treatment session would therefore be up to 80,000 (+ / - 10%), respectively.

[0417] For example, a unit dose of up to 5,000 pg + / - 10% was selected for treatment of glabellar lines (2 doses per corrugator supercilii and 1 dose in the procerus) for a total dose of up to 25,000 pg in the glabellar region, approximately 10-fold lower than the NOAEL of 4 ng / kg from both non-clinical safety species (rat and monkey) translated to a human dose for a body weight of 60 kg.

[0418] Indeed, as described in Example 21, five injections (two per corrugator supercilii and one in the procerus), each at a 4,000 pg unit dose (to provide a total dose of 20,000 pg), were well tolerated.

[0419] [Example 15]

[0420] Dosage regimen for treating glabellar lines using modified BoNT / A (SEQ ID NO: 6 converted to a dichain form)

[0421] The modified BoNT / A is provided as a lyophilized powder in vials containing 36 ng of modified BoNT / A per vial. The lyophilized powder is reconstituted.

[0422] Treat moderate to severe glabellar lines according to the injection regimen shown in Figure 11, i.e. up to two unit doses per corrugator supercilii and one in the procerus muscle.

[0423] The unit dose is >1754pg to 5,000ng. The total dose of modified BoNT / A administered during treatment is up to 88,000pg. This is more than 14x the maximum total dose of Dysport® that can be administered during treatment of facial wrinkles without approaching toxicity limits (a concern with conventional treatment regimens). Thus, clinicians can tailor treatment to patients with the knowledge that a total dose of 88,000pg can be administered without any concerns of toxicity, thereby allowing treatment of additional muscles of interest and / or ensuring that each muscle and / or site receives an effective dose.

[0424] [Example 16]

[0425] Dosage regimen for treating glabellar and forehead lines using modified BoNT / A (SEQ ID NO: 6 converted to a dichain form)

[0426] The modified BoNT / A is provided as a lyophilized powder in vials containing 36 ng of modified BoNT / A per vial. The lyophilized powder is reconstituted.

[0427] Treat moderate to severe glabellar and forehead lines according to the injection regimen shown in Figure 9, i.e. up to 2 unit doses per corrugator supercilii and 1 in the procerus muscle, and up to 5 unit doses in the frontalis muscle.

[0428] The unit dose is >1754pg to 5,000ng. The total dose of modified BoNT / A administered during treatment is up to 88,000pg. This is more than 14x the maximum total dose of Dysport® that can be administered during treatment of facial wrinkles without approaching toxicity limits (a concern with conventional treatment regimens). Thus, clinicians can tailor treatment to patients with the knowledge that a total dose of 88,000pg can be administered without any concerns of toxicity, thereby allowing treatment of additional muscles of interest and / or ensuring that each muscle and / or site receives an effective dose.

[0429] [Example 17]

[0430] Dosage regimen for treating lateral canthal lines using modified BoNT / A (SEQ ID NO: 6 converted to a dichain form)

[0431] The modified BoNT / A is provided as a lyophilized powder in vials containing 36 ng of modified BoNT / A per vial. The lyophilized powder is reconstituted.

[0432] Treat moderate to severe lateral canthal lines according to the injection regimen shown in Figure 10, i.e. up to 3 unit doses per orbicularis oculi muscle.

[0433] The unit dose is >1754pg to 5,000ng. The total dose of modified BoNT / A administered during treatment is up to 88,000pg. This is more than 14x the maximum total dose of Dysport® that can be administered during treatment of facial wrinkles without approaching toxicity limits (a concern with conventional treatment regimens). Thus, clinicians can tailor treatment to patients with the knowledge that a total dose of 88,000pg can be administered without any concerns of toxicity, thereby allowing treatment of additional muscles of interest and / or ensuring that each muscle and / or site receives an effective dose.

[0434] [Example 18]

[0435] Dosage regimen for treating glabellar, forehead, and lateral canthal lines using modified BoNT / A (SEQ ID NO: 6 converted to a dichain form)

[0436] The modified BoNT / A is provided as a lyophilized powder in vials containing 36 ng of modified BoNT / A per vial. The lyophilized powder is reconstituted.

[0437] Treat moderate to severe glabellar, forehead and lateral canthal lines according to the injection regimen shown in Figure 11, i.e. up to 2 unit doses per corrugator supercilii and 1 in the procerus muscle, as well as up to 5 unit doses in the frontalis muscle, and up to 3 unit doses per orbicularis oculi muscle.

[0438] The unit dose is >1754pg to 5,000ng. The total dose of modified BoNT / A administered during treatment is up to 88,000pg. This is more than 14x the maximum total dose of Dysport® that can be administered during treatment of facial wrinkles without approaching toxicity limits (a concern with conventional treatment regimens). Thus, clinicians can tailor treatment to patients with the knowledge that a total dose of 88,000pg can be administered without any concerns of toxicity, thereby allowing treatment of additional muscles of interest and / or ensuring that each muscle and / or site receives an effective dose.

[0439] [Example 19]

[0440] Treating Patients with Upper Facial Lines

[0441] John, age 55, presents with severe glabellar, forehead and canthal lines. Modified BoNT / A (SEQ ID NO: 6) is administered by one or more unit doses (1 x unit dose = 5000 pg) into the following muscles as shown below: - 2 unit doses to each corrugator supercilii (so 4 unit doses across the corrugator supercilii) and 1 site on the procerus to treat glabellar lines; - 5 unit doses into the frontalis muscle to treat forehead lines, and - 3 unit doses in the outer part of each orbicularis oculi muscle (hence 6 unit doses in the entire orbicularis oculi muscle) to treat the lateral canthal lines.

[0442] The total dose administered is 80,000 ng (+ / - 10%) of modified BoNT / A (SEQ ID NO:6 converted to the two-chain form).

[0443] All wrinkles (wrinkles) are reduced, leaving skin with a clear, youthful appearance, and due to the long-term modified BoNT / A, John does not require further treatment beyond 9 months. Therefore, John receives less frequent injections (e.g., once a year) when compared to a comparable subject receiving unmodified BoNT / A. Furthermore, Bill does not show any side effects due to the improved safety profile of modified BoNT / A.

[0444] [Example 20]

[0445] Safety & Efficacy of Modified BoNT / A (SEQ ID NO:6 Converted to Dichain Form) in Humans

[0446] SEQ ID NO:6 (converted to a two-chain form) was administered to human subjects by intramuscular injection. Subjects were administered 2x 15,000pg unit doses (i.e., 30,000pg total), 2x 25,000pg unit doses (i.e., 50,000pg total), or 2x 36,000pg unit doses (i.e., 72,000pg total) of modified BoNT / A (SEQ ID NO:6 converted to a two-chain form).

[0447] The results showed that, despite exceptionally high doses per muscle (e.g., for unit doses of 25,000 pg and 36,000 pg), all unit doses of modified BoNT / A tested were effective in muscle paralysis, were safely tolerated, and no adverse effects were observed, indicating that modified BoNT / A does not diffuse away from the injection site and highlighting the exceptional safety profile of modified BoNT / A (SEQ ID NO: 6 converted to a two-chain form).

[0448] The unit doses and total doses described herein in the context of or for treating facial lines are well within the dose ranges shown to be safe and effective for the advantageous modified BoNT / A molecules described herein, providing flexibility to the clinician in terms of treatment options, including taking advantage of the advantageous properties and exceptional safety profile of the modified BoNT / A (e.g., SEQ ID NO: 6 converted to a two-chain form).

[0449] [Example 21]

[0450] Safety & Efficacy of Modified BoNT / A (SEQ ID NO:6 Converted to Dichain Form) in the Treatment of Glabellar Lines

[0451] SEQ ID NO:6 (converted to the two-chain form) was administered to human subjects by intramuscular injection.

[0452] One cohort of subjects received five injections of a 2,500 pg unit dose (two in each corrugator supercilii and one in the procerus) to provide a total dose of 12,500 pg. Another cohort of subjects received five injections of a 4,000 pg unit dose (two in each corrugator supercilii and one in the procerus) to provide a total dose of 20,000 pg.

[0453] Glabellar lines were reduced. Results showed that, despite the exceptionally high doses per muscle, all unit doses of modified BoNT / A tested were safely tolerated and no adverse effects were observed. This indicates that modified BoNT / A does not diffuse away from the injection site, highlighting the exceptional safety profile of modified BoNT / A (SEQ ID NO: 6 converted to a two-chain form).

[0454] All publications mentioned in the above specification are incorporated herein by reference. Various modifications and variations of the described methods and systems of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with certain preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in the art of biochemistry and biotechnology or related fields are intended to be within the scope of the following claims. [Explanation of symbols]

[0455] [Figure 1] Indication: Recommended Concentration: Recommended DYSPORT Dose: Recommended Dysport Dose Glabellar Lines, Adults: Glabellar Lines, Adults 12 Units / 0.1 mL or 20 Units / 0.1 mL: 12 Units / 0.1 mL or 20 Units / 0.1 mL 50 Units, divided in five equal aliquots of 10 Units (0.08 mL) each or 50 Units, divided in five equal aliquots of 10 Units (0.05 mL) each: 50 Units, divided in five equal aliquots of 10 Units (0.08 mL) each or 50 Units, divided in five equal aliquots of 10 Units (0.05 mL) each [Figures 2 and 5] Marker: Marker [Figures 2-7] SEQ ID NO: Sequence number [Figures 3 and 6] % SNAP-25 cleavage: SNAP-25 cleavage percentage in rat spinal cord neurones: Un-Tr: Untreated [Figure 4] Mean DAS: Average DAS Dose (pg / mouse): Dose (pg / mouse) [Figure 6] in human pluripotent stem cells [Figure 7] Time (day): Time (day) [Figures 8, 9, and 11] Glabellar line injection point: Glabellar line injection point [Figures 9 and 11] Forehead lines injection point: Forehead lines injection point [Figure 11] Lateral canthal line injection point: Injection point for the lateral canthal line

Claims

1. A pharmaceutical product comprising the modified double-chain chimeric botulinum neurotoxin BoNT / AB for use in the treatment of facial wrinkles. Here, BoNT / AB is administered by intramuscular injection at multiple sites on the target face. Multiple body parts can be selected from the following: To treat frown lines, up to two areas of the corrugator supercilii muscle and one area of ​​the procerus muscle, Up to five areas of the frontalis muscle for treating forehead wrinkles, as well as To treat wrinkles at the outer corner of the eye, up to three areas of the orbicularis oculi muscle, Modified BoNT / AB includes the light chain and translocation domain (HN domain) of BoNT / A and the receptor-binding domain (HC domain) of BoNT / B.

2. The pharmaceutical product according to claim 1, wherein the unit dose of modified BoNT / AB is greater than 1754 pg.

3. The pharmaceutical product according to claim 1 or 2, wherein the unit dose of modified BoNT / AB is greater than 1754 pg and up to 8,000 pg.

4. The pharmaceutical product according to claim 2, wherein the unit dose of modified BoNT / AB is greater than 1754 pg and up to 5,000 pg.

5. The pharmaceutical product according to claim 4, wherein the unit dose is a modified BoNT / AB of 2500 pg.

6. The pharmaceutical product according to claim 1, wherein the total dose of modified BoNT / AB administered during treatment is a maximum of 80,000 pg.

7. The pharmacopoeia according to claim 1, wherein the modified BoNT / AB has a safety factor greater than 7, where the safety factor is calculated as the dose of toxin required for a -10% change in body weight as measured as pg / mouse, divided by DAS ED 50 as measured as pg / mouse, where ED 50 is the dose required to produce a DAS score of 2.

8. The pharmaceutical product according to claim 1 for use in the treatment of frown lines, wherein modified BoNT / AB is administered at two sites on the corrugator supercilii muscle and one site on the procerus muscle.

9. The pharmaceutical product according to claim 1, wherein the modified BoNT / AB is administered at a total of five sites.

10. The pharmaceutical product according to claim 8 or 9, wherein the total dose administered for frown lines or forehead wrinkles is 12,000 pg to 13,000 pg of modified BoNT / AB, and the unit dose is 2,000 pg to 3,000 pg of modified BoNT / AB.

11. The pharmacopoeia according to claim 10, wherein the total dose administered for frown lines or forehead wrinkles is 12,500 pg of modified BoNT / AB.

12. The pharmaceutical product according to claim 1, wherein the total dose administered for frown lines or forehead wrinkles is 19,000 pg to 21,000 pg of modified BoNT / AB, and the unit dose is 3,500 to 4,500 pg of modified BoNT / AB.

13. The pharmacopoeia according to claim 12, wherein the total dose administered for frown lines or forehead wrinkles is 20,000 pg of modified BoNT / AB.

14. The pharmacopoeia according to claim 1 for the treatment of forehead wrinkles, wherein modified BoNT / AB is administered at five sites of the frontalis muscle.

15. The pharmacopoeia according to claim 1, wherein the modified BoNT / AB is for use in the treatment of wrinkles of the outer corner of the eye, and the modified BoNT / AB is administered at a total of six sites.

16. The pharmacopoeia according to claim 15, wherein the modified BoNT / AB is for use in the treatment of wrinkles of the outer corner of the eye, and the modified BoNT / AB is administered at three sites per orbicularis oculi muscle.

17. The pharmaceutical product according to claim 1, wherein the total dose administered for wrinkles of the outer corner of the eye is 14,000 pg to 16,000 pg of modified BoNT / AB, and the unit dose is 2,000 to 3,000 pg of modified BoNT / AB.

18. The pharmacopoeia according to claim 17, wherein the total dose administered for wrinkles of the outer corner of the eye is 15,000 pg of modified BoNT / AB.

19. The pharmaceutical product according to claim 15, wherein the total dose administered for wrinkles of the outer corner of the eye is 23,000 pg to 25,000 pg of modified BoNT / AB, and the unit dose is 3,500 pg to 4,500 pg of modified BoNT / AB.

20. The pharmacopoeia according to claim 19, wherein the total dose administered for wrinkles of the outer corner of the eye is 24,000 pg of modified BoNT / AB.

21. Modified BoNT / AB is for use in treating frown lines, forehead wrinkles, and outer canthal wrinkles, - Modified BoNT / AB is administered to two sites on the corrugator supercilii muscle and one site on the procerus muscle to treat frown lines between the eyebrows. - Modified BoNT / AB is administered to five sites in the frontalis muscle, and - The pharmacopoeia according to claim 1, wherein modified BoNT / AB is administered at three sites of the orbicularis oculi muscle to treat wrinkles of the outer corner of the eye.

22. The pharmaceutical product according to claim 21, wherein the total dose for treating frown lines, forehead wrinkles, and outer canthal wrinkles is 43,000 to 44,000 pg of modified BoNT / AB, and the unit dose is 2,000 to 3,000 pg of modified BoNT / AB.

23. The pharmaceutical product according to claim 21, wherein the total dose for treating frown lines, forehead wrinkles, and outer canthal wrinkles is 63,000 to 65,000 pg of modified BoNT / AB, and the unit dose is 3,500 to 4,500 pg of modified BoNT / A.

24. The pharmaceutical product according to claim 1, wherein the modified BoNT / AB is administered to site 8, 10, 12, 14, or 16, or the modified BoNT / AB is administered to site 7, 9, 11, 13, or 15.

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

6.

26. The pharmaceutical product according to claim 1, wherein modified BoNT / AB comprises the light chain and transposition domain of BoNT / A and the HC domain of BoNT / B, where 24.04 pg of modified BoNT / AB corresponds to the calculated median lethal dose (LD50) in mice.

27. ​​A unit dosage form of the modified double-chain chimeric botulinum neurotoxin BoNT / AB, wherein the unit dosage form includes: a. More than 73 units of modified BoNT / AB, where 1 unit is the amount of modified BoNT / AB corresponding to the calculated median lethal dose (LD50) in mice, or b. More modifications than 1754pg BoNT / AB, Here, modified BoNT / AB includes the light chain and transposition domain (HN domain) of BoNT / A and the receptor-binding domain (HC domain) of BoNT / B.

28. a. More than 73 units and up to 332.8 units of modified BoNT / AB, or b. More than 1754pg, up to a maximum of 8000pg of modified BoNT / AB The unit dosage form according to claim 27, including the above.

29. The unit dosage form according to claim 27 or 28, further comprising a pharmaceutically acceptable carrier, excipient, adjuvant, and / or salt.

30. A kit including the following: a. The unit dosage form described in claim 27, and b. Instructions for use of the unit dosage form in the treatment of frown lines, forehead wrinkles, and / or wrinkles around the outer corners of the eyes.

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