Treatment of moderate to severe glabellar and lateral canthal wrinkles

Abstract

Disclosed herein are methods for treating glabellar and epicanthal wrinkles using liquid botulinum neurotoxin compositions.

Description

[Technical field]

[0001] Cross-reference statements This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 63 / 299,705, filed January 14, 2022, and U.S. Provisional Patent Application No. 63 / 399,127, filed August 18, 2022. The entire contents of these provisional applications are incorporated herein by reference.

[0002] Technical Field Described herein are methods and compositions for treating moderate to severe and severe glabellar lines (GL) and lateral canthal lines (LCL) using botulinum neurotoxin formulations. Also provided are liquid botulinum neurotoxin formulations and compositions. [Background technology]

[0003] The following description of the background of the present technology is provided merely to aid in the understanding of the present technology and is not admitted to describe or constitute prior art to the present technology.

[0004] Seven generally immunologically distinct botulinum neurotoxins (botulinum neurotoxin serotypes A, B, C, D, E, F, and G) have been characterized, each distinguished by neutralization by type-specific antibodies. As an example, BOTOX® is the trademark for the botulinum toxin type A (BoNT-A) purified neurotoxin complex commercially available from Allergan, Inc. (Irvine, Calif.). BOTOX® is a popular injectable cosmetic treatment that temporarily reduces the appearance of fine lines and wrinkles.

[0005] There are currently four BoNT-A products approved in the United States: BOTOX COSMETIC®, DYSPORT®, XEOMIN®, and JEUVEAU®. All of these products are stored in a lyophilized or freeze-dried state for stability reasons. Although each product contains BoNT-A as the active ingredient, the activity, stability, and safety of each product are not the same. On the contrary, many of these products have relatively variable activity, stability, and safety, all of which are further complicated by the need for physicians to reconstitute the aforementioned products before administering them.

[0006] Eyelid ptosis ("blepharoptosis") has been documented as an adverse event in many BoNT-A clinical trials, with incidence rates varying across treatment groups. Importantly, ptosis did not occur in any placebo group, indicating that it can be directly attributed to the injection of botulinum toxin. A review of trials totaling over 8000 patients found that the incidence of ptosis was approximately 2.5%. Specifically, incidences of ptosis were noted with Botox (3%) and Jeuveau (2%). Summary of the Invention [Problem to be solved by the invention]

[0007] Thus, there is currently a need for a safe botulinum neurotoxin preparation that does not cause ptosis or that causes ptosis less frequently and less severely, preferably in a liquid form suitable for storage and use in treatment. [Means for solving the problem]

[0008] The present disclosure relates generally to botulinum neurotoxin compositions and methods for treating lines and wrinkles, such as lateral canthal lines (LCL), forehead lines, and glabellar lines (GL).

[0009] In one aspect, the disclosure provides a method of treating upper facial wrinkles (e.g., glabellar lines (GL), lateral canthal lines (LCL), brow lines, or a combination thereof) in a human subject with a reduced risk of ptosis, comprising administering to the subject a therapeutically effective amount of a liquid composition comprising a botulinum neurotoxin, thereby reducing the appearance of the GL, LCL, brow lines, or a combination thereof, wherein the botulinum neurotoxin is less likely to cause ptosis than botulinum neurotoxin products with lower enzymatic activity or lower specific activity. In other words, a liquid composition comprising a botulinum neurotoxin for treating upper facial wrinkles (e.g., glabellar lines (GL), lateral canthal lines (LCL), forehead wrinkles, or combinations thereof) in a human subject with a low risk of ptosis is provided, said treatment comprising administering to the subject a therapeutically effective amount of a liquid composition comprising a botulinum neurotoxin, thereby reducing the appearance of the upper facial wrinkles (e.g., GL, LCL, forehead wrinkles, or combinations thereof), said botulinum neurotoxin being less likely to cause ptosis than a botulinum neurotoxin product having a lower enzymatic activity or a lower specific activity. The term "enzymatic activity" may alternatively be referred to herein as "L-chain proteolytic activity," indicating, for example, that the L-chain of the botulinum neurotoxin comprises a protease domain that cleaves a target (SNARE) protein.

[0010] This finding is surprising because when botulinum neurotoxins are utilized to treat upper facial wrinkles (as described above), a botulinum neurotoxin with a higher potency (e.g., higher enzymatic activity or higher specific activity) would be expected to be more likely to cause ptosis than a botulinum neurotoxin product with a lower potency (e.g., lower enzymatic activity or lower specific activity). Ptosis occurs when the botulinum neurotoxin spreads from the intended target muscle to the adjacent eyelid muscles and causes their flaccid paralysis (ptosis). Thus, while a botulinum neurotoxin with a higher potency may be desirable to achieve a high cosmetic effect, one anticipated weakness of such an approach is that the botulinum neurotoxin may be more likely to cause ptosis.

[0011] Another aspect provides a cosmetic method for treating upper facial wrinkles (e.g., glabellar lines (GL), lateral canthal lines (LCL), brow wrinkles, or combinations thereof) in a human subject with a low risk of ptosis, comprising administering to the subject a therapeutically effective amount of a liquid composition comprising a botulinum neurotoxin, thereby reducing the appearance of the upper facial wrinkles (e.g., GL, LCL, brow wrinkles, or combinations thereof), wherein the botulinum neurotoxin is less likely to cause ptosis than botulinum neurotoxin products with lower enzymatic activity or lower specific activity.

[0012] Another aspect provides a method of treating upper facial wrinkles (e.g., GL, LCL, brow wrinkles, or a combination thereof) in a subject, comprising administering to the subject a therapeutically effective amount of a botulinum neurotoxin (preferably a liquid composition comprising a botulinum neurotoxin), wherein the severity of the facial wrinkles is reduced and the subject experiences a reduced incidence of ptosis. Another aspect provides a method of treating upper facial wrinkles (e.g., GL, LCL, brow wrinkles, or a combination thereof) in a subject, comprising administering to the subject a therapeutically effective amount of a botulinum neurotoxin (preferably a liquid composition comprising a botulinum neurotoxin), wherein the severity of the facial wrinkles is reduced and the subject experiences a reduced incidence of ptosis compared to the incidence of ptosis in subjects treated with an alternative botulinum neurotoxin product having lower enzymatic activity or lower specific activity. In other words, a botulinum neurotoxin (preferably a liquid composition comprising a botulinum neurotoxin) is provided for treating upper facial wrinkles (e.g., GL, LCL, brow wrinkles, or a combination thereof) in a subject, wherein the severity of the facial wrinkles is reduced and the incidence of ptosis experienced by the subject is reduced. Another aspect provides a botulinum neurotoxin (preferably a liquid composition comprising a botulinum neurotoxin) for treating upper facial wrinkles (e.g., GL, LCL, brow wrinkles, or a combination thereof) in a subject, wherein the severity of the facial wrinkles is reduced and the incidence of ptosis experienced by the subject is reduced compared to the incidence of ptosis in subjects treated with an alternative botulinum neurotoxin product having lower enzymatic activity or lower specific activity.

[0013] When treating glabellar lines, botulinum neurotoxin is typically administered at up to two sites in the corrugator supercilii and one site in the procerus muscle.

[0014] When treating forehead wrinkles, botulinum neurotoxin is typically administered into up to five locations in the frontalis muscle.

[0015] When treating lateral canthal wrinkles, botulinum neurotoxin is typically administered at up to three sites in the lateral portion of the orbicularis oculi muscle.

[0016] The upper facial wrinkles may be glabellar wrinkles. The facial wrinkles may be moderate to severe facial wrinkles, such as moderate facial wrinkles or severe facial wrinkles. The incidence of ptosis may be less than 1%, preferably 0.9% or less. The total dose of botulinum neurotoxin administered (per consultation / visit / day) to treat glabellar wrinkles may be 40-60U (preferably 50U), where one unit is the amount of botulinum neurotoxin equivalent to the median lethal dose (LD50) calculated in mice. The total dose of botulinum neurotoxin administered (per consultation / visit / day) to treat lateral canthal wrinkles may be 50-70U (preferably 60U), where one unit is the amount of BoNT / A equivalent to the median lethal dose (LD50) calculated in mice.

[0017] In some embodiments, the subject does not experience serious adverse events. In some embodiments, the subject is less likely to experience ptosis compared to treatment with a botulinum neurotoxin product selected from Botox Cosmetic®, Xeomin®, and Jeuveau®. In some embodiments, the botulinum neurotoxin product with lower enzymatic activity is selected from Botox Cosmetic®, Xeomin®, and Jeuveau®.

[0018] The botulinum neurotoxin may have an enzymatic activity normalized at a botulinum neurotoxin concentration of about 1.16 BU / BoNT. Additionally or alternatively, the present disclosure provides a method of treating glabellar lines (GL), lateral canthal lines (LCL), forehead lines, or a combination thereof, with a reduced risk of ptosis in a human subject in need thereof, comprising administering to the subject a therapeutically effective amount of a liquid composition comprising a botulinum neurotoxin, thereby reducing the appearance of the GL, LCL, forehead lines, or a combination thereof, wherein the botulinum neurotoxin has an enzymatic activity normalized at a botulinum neurotoxin concentration of about 1.16 BU / BoNT. The human subject may administer a botulinum neurotoxin (e.g., an enzymatic activity normalized at a botulinum neurotoxin concentration of less than 1.16 BU / BoNT, or an enzymatic activity normalized at a botulinum neurotoxin concentration of less than about 2.0×10 8 The human subject may be a subject who is considered to be at risk of, or likely to develop, ptosis when administered a botulinum neurotoxin formulation having a specific activity of less than 1000 U / mg, such as Botox Cosmetic®, Xeomin®, and Jeuveau®. The human subject may also be a subject who suffers from ptosis and / or has a medical history of developing ptosis when administered a botulinum neurotoxin. In another embodiment, the human subject is not a subject who (i) is not at risk of developing ptosis, (ii) does not suffer from ptosis, and / or (iii) does not have a medical history of developing ptosis when administered any dose or enzymatic activity of botulinum neurotoxin.

[0019] The liquid composition can have an activity of about 97 BU / ml or greater, and optionally, the liquid composition can have an enzymatic activity normalized to a botulinum neurotoxin concentration of about 10. In another aspect, the disclosure provides a method of treating glabellar lines (GL), lateral canthal lines (LCL), forehead lines, or a combination thereof in a human subject, comprising administering to the subject a therapeutically effective amount of a liquid composition comprising a botulinum neurotoxin, thereby reducing the appearance of GL, LCL, forehead lines, or a combination thereof, wherein (i) the liquid composition has an activity of about 97 BU / ml or greater, and optionally, the liquid composition has an enzymatic activity normalized to a botulinum neurotoxin concentration of about 10.

[0020] In some embodiments of any of the aforementioned methods, the liquid formulation comprises about 2.0×10 8 BoNT specific activity is in U / mg of total protein, and specific activity (U / mg) is optionally measured using the mouse LD50 potency in U / ml divided by the total amount of protein determined by the μBCA method (mg / ml).

[0021] In some embodiments of any of the aforementioned methods, the liquid formulation has an enzymatic activity normalized to a botulinum neurotoxin concentration of about 1.16 BU / BoNT, and the enzymatic activity of the botulinum neurotoxin is measured by a BOTEST TM is arbitrarily determined using

[0022] In some embodiments of any of the aforementioned methods, the liquid formulation has a relative potency normalized to a botulinum neurotoxin concentration of about 1.20 units / BoNT, and the enzymatic activity of the botulinum neurotoxin is determined using a cell-based assay.

[0023] In some embodiments of any of the aforementioned methods, the liquid formulation does not include excipients of human or animal origin.

[0024] In some embodiments of any of the aforementioned methods, the incidence of ptosis may be less than 2%. The percentage of ptosis referred to herein preferably corresponds to the percentage of patients in a cohort (e.g., a cohort of at least 50 patients) that exhibit ptosis, and each patient in the cohort is administered with a BoNT formulation. This means that when the liquid composition described herein is administered to the target cohort, less than 2% of the cohort will experience ptosis. In some embodiments of any of the aforementioned methods, the incidence of ptosis is less than 2%, less than 1.9%, less than 1.8%, less than 1.7%, less than 1.5%, less than 1.4%, less than 1.3%, less than 1.2%, less than 1.1%, less than 1.0%, or as low as 0.9%. For example, the incidence of ptosis may be 1.0% or less. Preferably, the incidence of ptosis may be 0.9% or less. Thus, the incidence of ptosis following treatment with alternative botulinum neurotoxin products (eg, botulinum neurotoxin products with lower enzymatic activity or lower specific activity) may be greater than 2%.

[0025] In some embodiments of any of the aforementioned methods, the subject does not experience ptosis.

[0026] In some embodiments of any of the aforementioned methods, the subject is less likely to experience ptosis compared to treatment with a botulinum neurotoxin product selected from Botox Cosmetic®, Xeomin®, and Jeuveau®. In some embodiments of any of the aforementioned methods, the subject is less likely to experience ptosis compared to treatment with an alternative botulinum neurotoxin product that has been stored in a dry form (e.g., a lyophilized powder form) and reconstituted for administration to the subject.

[0027] In some embodiments of any of the aforementioned methods, the GL, LCL, or a combination thereof is moderate to severe.

[0028] In some embodiments of any of the aforementioned methods, the GL, LCL, or a combination thereof is severe or severe.

[0029] In some embodiments of any of the aforementioned methods, the liquid composition comprising a botulinum neurotoxin does not include an animal protein or a companion protein.

[0030] In some embodiments of any of the aforementioned methods, the subject is treated for GL only. In some embodiments of any of the aforementioned methods, the subject is treated for LCL only. In some embodiments of any of the aforementioned methods, the subject is treated for a combination of GL and LCL. In any of these embodiments, the GL and / or LCL may be on one or both sides of the subject's face.

[0031] In some embodiments of any of the aforementioned methods, the treatment provides a higher response rate than Botox Cosmetic®. In some embodiments of any of the aforementioned methods, the treatment provides a higher response rate than an alternative botulinum neurotoxin product that is stored in a dry form (e.g., a lyophilized powder form) and reconstituted for administration to a subject.

[0032] In some embodiments of any of the aforementioned methods, the liquid composition comprises about four buffers. For example, the liquid composition may comprise a first buffer present at a concentration of about 100 to about 300 mM or at a concentration of about 0.1 to 10 mg / mL. The liquid composition may comprise a second buffer present at a concentration of about 1 to about 25 mM or at a concentration of about 0.1 to 1.0 mg / mL. The liquid composition may comprise a third buffer present at a concentration of about 1 to about 25 mM or at a concentration of about 0.1 to 1.0 mg / mL. The liquid composition may comprise a fourth buffer present at a concentration of about 1 to about 25 mM or at a concentration of about 0.1 to 1.0 mg / mL. The liquid composition may comprise a fifth buffer present at a concentration of about 1 to about 25 mM or at a concentration of about 0.1 to 1.0 mg / mL. In some embodiments, the buffering agent is selected from the group consisting of sodium chloride, potassium chloride, sodium phosphate, potassium phosphate, disodium hydrogen phosphate dihydrate, and sodium dihydrogen phosphate dihydrate.

[0033] In some embodiments of any of the foregoing methods, the liquid composition includes about one stabilizer, which may be an amino acid. For example, the amino acid may be selected from the group consisting of alanine, valine, leucine, isoleucine, methionine, phenylalanine, tyrosine, and tryptophan. In some embodiments, the amino acid is a D-isoform or an L-isoform. In some embodiments, the amino acid is present at a concentration of about 0.1 to about 3.0 mg / mL.

[0034] In some embodiments of any of the foregoing methods, the liquid composition comprises about one surfactant, which may be a non-ionic surfactant. In some embodiments, the non-ionic surfactant is present in a concentration of about 0.01% (v / v) to about 5.0% (v / v) or about 0.1 to about 3.0 mg / mL.

[0035] In some embodiments of any of the aforementioned methods, the botulinum neurotoxin is selected from the group consisting of botulinum neurotoxin types A, B, C, D, E, F, and G. In particular, the botulinum neurotoxin may be botulinum neurotoxin type A.

[0036] In some embodiments of any of the aforementioned methods, the pH of the liquid composition is between 6.6 and 6.9, hi some embodiments, the pH may be 6.75 or about 6.75.

[0037] In some embodiments of any of the aforementioned methods, the botulinum neurotoxin has a molecular weight of about 150 kDa.

[0038] In some embodiments of any of the aforementioned methods, the osmolality of the liquid composition is between 270 mosm / kg and 310 mosm / kg.

[0039] In some embodiments of any of the foregoing methods, between 1 and 100 units of a botulinum toxin are administered to the subject. In some embodiments, between 10 and 75 units of a botulinum toxin are administered to the subject. In some embodiments, between 25 and 75 units of a botulinum toxin are administered to the subject. In some embodiments, 10, 25, 30, 45, 50, 60, 75, or 90 units of a botulinum toxin are administered to the subject.

[0040] In some embodiments of any of the aforementioned methods, the liquid composition is administered by injection. The injection may be, for example, subcutaneous, transdermal, intradermal, or intramuscular. In some embodiments, the subject is injected multiple times in the glabellar region. In some embodiments, adjacent injection sites are about 0.5 to about 10 cm apart. In some embodiments, adjacent injection sites are about 1.5 to about 3 cm apart. In some embodiments, the injections are administered into the procerus muscle and the corrugator supercilii muscle on one or both sides of the face. In some embodiments, the injections are administered first into the procerus muscle, followed by the corrugator supercilii muscle on one or both sides of the face, moving from the midline to the outside. In some embodiments, all injections are administered about 1 cm above the supraorbital rim, medial to the pupil midline. In some embodiments, all injections are administered about 1 cm above the center of the eyebrow or about 1 cm above the bony supraorbital ridge. In some embodiments, the subject is injected multiple times below the lateral canthus, into the lateral portion of the orbicularis oculi muscle, and / or 1 to 2 cm from the orbital rim. In some embodiments, subjects receive multiple additional injections beneath the lateral canthus, into the lateral portion of the orbicularis oculi muscle, and / or 1-2 cm from the orbital rim.

[0041] In some embodiments of any of the foregoing methods, the method is repeated at intervals of about 1 month to about 6 months to inhibit recurrence of GL, LCL, or a combination thereof. In some embodiments, the method is repeated at intervals of about 3 months to about 6 months to inhibit said recurrence. In some embodiments, the method is repeated at intervals of about 4 months to inhibit said recurrence.

[0042] The present disclosure also provides a liquid composition comprising a botulinum neurotoxin for use in treating glabellar and / or epicanthal wrinkles in a subject as disclosed herein.

[0043] The present disclosure also provides the use of a liquid composition comprising a botulinum neurotoxin as disclosed herein for treating glabellar and / or epicanthal wrinkles.

[0044] The following detailed description is exemplary and explanatory but is not intended to be limiting. [Brief description of the drawings]

[0045] [Figure 1] FIG. 1 shows the MAS GL scale (resting) and the MAS GL scale (dynamic).

[0046] [Diagram 2] FIG. 2 shows the glabellar wrinkle severity scoring matrix.

[0047] [Diagram 3] FIG. 3 shows the results of ILA responder rates in glabellar QM1114-DP study subjects over time.

[0048] [Figure 4] FIG. 4 shows the Global Aesthetic Improvement Score (GAIS) analysis of glabellar QM1114-DP study subjects over time.

[0049] [Diagram 5] FIG. 5 shows the safety results (related adverse events) of the glabellar wrinkle QM1114-DP study.

[0050] [Figure 6] FIG. 6 shows the severity scoring matrix for lateral canthal wrinkles.

[0051] [Figure 7] FIG. 7 shows the results of ILA responder rate in subjects of the lateral canthal wrinkles QM1114-DP study over time.

[0052] [Figure 8] FIG. 8 shows the Global Aesthetic Improvement Score (GAIS) analysis of subjects in the Ectocanthal Wrinkle QM1114-DP study over time.

[0053] [Figure 9] FIG. 9 shows the safety results (related adverse events) of the lateral canthal wrinkles QM1114-DP study.

[0054] [Figure 10] FIG. 10 shows the results of ILA responder rates for glabellar lines over time.

[0055] [Figure 11] FIG. 11 shows the results of ILA responder rates for lateral canthal wrinkles over time.

[0056] [Figure 12] FIG. 12 shows the Global Aesthetic Improvement Score (GAIS) analysis.

[0057] [Figure 13] FIG. 13 shows a combined 2 grade improvement in glabellar lines.

[0058] [Figure 14] FIG. 14 shows the investigator scores for glabellar lines.

[0059] [Figure 15] FIG. 15 shows a combined 2 grade improvement in lateral canthal wrinkles.

[0060] [Figure 16] FIG. 16 shows the investigator scores for lateral canthal wrinkles.

[0061] [Figure 17] FIG. 17 shows the enzyme activity measured using BoTest™.

[0062] [Figure 18]Figure 18 shows the specific activity measured using BoTest™ normalized with a BoNT-A specific ELISA (BOLISA™). The top row shows the results from one repeat of the specific activity experiment in units of BoTest U / RBU. The bottom row shows U / pg.

[0063] [Figure 19] FIG. 19 shows the relative potency from the cell-based assay normalized to BoNT-A concentration.

[0064] [Figure 20] FIG. 20 shows a formulation (QM-1114) with a high level of purity (>98%) in the main peak of the UPLC-SEC.

[0065] [Figure 21] FIG. 21 shows the results from SDS-PAGE analysis in which no detectable impurities were observed, but only the bands expected from BoNT-A1.

[0066] [Figure 22] Figure 22 shows composite grade 2 responders for LCL at month 1 based on ILA and SLA assessments during maximum smile. 26 subjects treated with QM-1114 and 4 subjects treated with placebo had their assessments at month 1 performed remotely and were not included in this analysis. CMH, Cochran-Mantel-Haenszel; ILA, investigator live assessment; LCL, lateral canthal wrinkles; SLA, subject live assessment.

[0067] [Figure 23] Figure 23 shows the time to onset of treatment effect, specifically, time relative to initiation of LCL treatment based on subject diary.

[0068] [Figure 24] FIG. 24 shows the responder rates based on investigator assessment during maximum smile.

[0069] [Diagram 25]FIG. 25 shows the investigator-reported responder rate during maximum smile was ≥1 grade improvement in LCL severity.

[0070] [Figure 26] FIG. 26 shows the time for the lateral canthal crease (LCL) to return to baseline scores after treatment. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0071] The embodiments according to the present disclosure are described more fully below. However, aspects of the present disclosure may be embodied in different forms and should not be construed as being limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the present invention to those skilled in the art. The terms used in the description of this specification are merely for the purpose of describing specific embodiments, and are not intended to be limiting.

[0072] Unless the context indicates otherwise, it is specifically contemplated that the various features of the methods described herein may be used in any combination. The present disclosure also contemplates that in some embodiments, any feature or combination of features specified herein may be excluded or omitted. To illustrate, if a method is described herein as including steps A, B, and C, it is specifically contemplated that any or any combination of A, B, or C may be omitted and abandoned, either alone or in any combination.

[0073] Unless expressly stated otherwise, all specific embodiments, features, and terms are intended to include the described embodiment, feature, or term, and biological equivalents thereof.

[0074] I. Definition As used herein, the singular forms "a," "an," and "the" refer to both the singular and the plural, unless expressly stated to refer to the singular only.

[0075] It is to be understood, although not always explicitly stated, that all numerical designations are preceded by the term "about". The term "about" means that the numbers encompassed are not limited to the exact numbers set forth herein, but are intended to refer to numbers that are substantially in the vicinity of the numbers set forth, without departing from the scope of the present invention. As used herein, "about" is understood by those of ordinary skill in the art and will vary to some extent with the context in which it is used. If the use of this term is not clear to those of ordinary skill in the art given the context in which it is used, "about" will mean up to plus or minus 15%, 10%, 5%, 1%, or 0.1% of the particular term.

[0076] Also, as used herein, "and / or" refers to and includes any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative ("or").

[0077] As used herein, "administer", "administration", or "administering" refers to (1) providing, giving, dispensing, and / or prescribing, e.g., by or under the direction of a medical practitioner or his / her authorized agent, and (2) taking, taking, or consuming, e.g., by a medical practitioner or a subject. Administration includes, but is not limited to, oral, parenteral (e.g., intramuscular, intraperitoneal, intravenous, ICV, intracisternal injection or infusion, subcutaneous injection, or implantation), nasal inhalation spray, intravaginal, rectal, sublingual, urethral (e.g., urethral suppository), or topical routes of administration (e.g., gels, ointments, creams, aerosols, etc.), which may be formulated alone or together in suitable dosage unit formulations containing conventional non-toxic pharmacologic carriers, adjuvants, excipients, and vehicles appropriate for each route of administration. The present invention is not limited by route of administration, formulation, or dosing schedule. The pharmaceutical compositions disclosed herein are "administered locally" (topical administration), i.e., administered at or near the site where the therapeutic result is desired. Administration may be unilateral or bilateral, as needed for a given patient.

[0078] As used herein, "treat," "treating," or "treatment" includes reducing or improving the appearance of glabellar lines (GL) and / or lateral canthal lines (LCL) or one or more symptoms thereof, regardless of whether the GL or LCL is considered to be "cured" or "removed," and regardless of whether all symptoms are resolved. These terms also include inhibiting or preventing the progression of GL, LCL, and / or one or more symptoms thereof, as well as achieving a therapeutic and / or prophylactic benefit.

[0079] The term "botulinum toxin" means botulinum neurotoxin types A, B, C, D, E, F, or G either as a pure toxin (i.e., a neurotoxic component with a molecular weight of about 150 kDa) or as a botulinum toxin complex (molecular weight of about 300 to about 900 kDa), and includes recombinant, chimeric, hybrid, retargeted, and amino acid sequence-modified botulinum neurotoxins, but does not include botulinum toxins that are not neurotoxins, such as cytotoxic botulinum toxins C2 and C3.

[0080] "Local administration" means administration of a pharmaceutical agent to or near a target tissue, muscle, or subcutaneously by a non-systemic route (i.e., subcutaneous, intramuscular, subdermal, intradermal, subcutaneous, intraorgan (e.g., injection into the bladder wall or prostate), or transdermal routes). Local administration thus excludes systemic (i.e., into the blood circulation) routes of administration, such as intravenous or oral administration. Peripheral administration means administration to the periphery (i.e., on or within the face, limbs, trunk, or head of a patient), as opposed to administration to the viscera or enteric (i.e., into the internal organs).

[0081] By "pharmaceutical composition" is meant a formulation in which the active ingredient (active agent) can be a botulinum neurotoxin. By "formulation" is meant that at least one additional ingredient is present in the pharmaceutical composition in addition to the active agent. Thus, a pharmaceutical composition is a formulation suitable for cosmetic, diagnostic, or therapeutic administration (i.e., subcutaneous or intramuscular injection) to a subject, such as a human patient.

[0082] An "effective amount" is an amount sufficient to produce a beneficial or desired result, such as thinning, smoothing, or stretching of glabellar and / or lateral canthal wrinkles. As used herein, an effective amount would also include an amount sufficient to prevent the formation of glabellar or lateral canthal wrinkles from worsening or to reverse the formation of existing glabellar wrinkles. Thus, an exact "effective amount" cannot be specified. An effective amount can be administered in one or more administrations, applications, or dosages. Such delivery depends on many variables, including the duration for which the individual dosage units are used, the location of administration of the agent, the severity of glabellar wrinkles, the route of administration, and the like. However, it is understood that the specific dosage level of the therapeutic agent of the present disclosure for a particular subject will depend on a variety of factors, including the activity of the particular compound employed, the age, weight, overall health, sex, and diet of the subject, the time of administration, excretion rate, drug combination, and the severity and form of administration of the particular disorder being treated. Therapeutic dosages can generally be adjusted to optimize safety and efficacy. Dosages can be determined by a physician and adjusted, if necessary, to the observed effects of the treatment. Preferably, the effective amount does not cause side effects (eg, ptosis).

[0083] II. Liquid Compositions of BoNT Provided herein is a liquid composition comprising a botulinum neurotoxin (BoNT) and a buffering agent, which is suitable for storage as a liquid and is suitable for treating glabellar wrinkles without further reconstitution or mixing. In other words, the disclosed liquid composition is "ready to use" and does not require any kind of special preparation, unlike other BoNT products currently on the market. Thus, the liquid composition of the present invention can be contrasted with alternative botulinum neurotoxin products that are stored in a dry form (e.g., lyophilized powder form) and need to be reconstituted for administration to a subject.

[0084] Botulinum neurotoxin (BoNT) is a 150 kilodalton (kDa) protein dimer consisting of a 100 kDa heavy chain and a 50 kDa light chain. The two chains are linked by a disulfide bond between two cysteine ​​residues. The light chain is an enzyme that cleaves the 25 kDa synaptosomal associated protein (SNAP-25). The heavy chain mediates the binding and internalization of the toxin protein. Unlike other commercially available BoNTs, the botulinum neurotoxin in the present liquid composition (an exemplary embodiment of which is referred to herein as QM1114) is stable in liquid form and does not need to be reconstituted or mixed prior to use. In some embodiments, the BoNT formulated in the liquid composition is botulinum neurotoxin type A (BoNT-A1). As outlined in the Examples, an exemplary embodiment of the present liquid composition has been investigated in human clinical trials and is referred to as QM1114 (or QM1114-DP). QM1114 is therefore a representative embodiment of a liquid composition of the present invention, which is described in more detail below with reference to its excipients and properties.

[0085] Also, for purposes of this disclosure, the liquid compositions (e.g., QM1114) do not contain any human or animal-derived excipients, and such formulations can be manufactured using a totally animal-free process with a high level of purity using filtration and / or chromatography. The process may not include a precipitation step or lyophilization. The formulations may have a neutral or near-neutral pH (e.g., a pH of 6.5, 6.6, 6.7, 6.75, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5). In some embodiments, the pH may be about 6.75.

[0086] The specific activity of the liquid compositions (e.g., QM1114) is as high as or higher than competing products. Specific activity can refer to how active a particular amount of toxin is, but is not necessarily a measure of activity (e.g., cell-based potency assay or BOTEST). TM ) by a measure of the amount of enzyme present during the activity measurement (e.g., BOLISA®). For example, the BoNT specific activity is approximately 1.5×10 8 , about 1.6×108 , about 1.7×10 8 , about 1.8×10 8 , about 1.9×10 8 , about 2.0×10 8 , about 2.1×10 8 , about 2.2×10 8 , about 2.3×10 8 , about 2.4×10 8 , or about 2.5 × 10 8 Specific activity (U / mg) is measured using the mouse LD potency in U / ml divided by the total protein (mg / ml) as determined by the μBCA method, although the specific activity may be greater than or equal to U / mg of total protein. In some embodiments, the specific activity is about 2.0×10 8 U / mg of total protein. In some embodiments, the disclosed liquid compositions (e.g., QM1114) have a higher specific activity than other clinical BoNT formulations, such as Botox Cosmetic®, Xeomin®, and Jeuveau®. In some embodiments, the disclosed liquid compositions (e.g., QM1114) have a higher specific activity than other clinical BoNT formulations that are stored in a dry form (e.g., a lyophilized powder form) and must be reconstituted for administration to a subject.

[0087] Liquid compositions (e.g., QM1114) may include buffers that include sodium, chloride, and / or phosphate ions. The addition of such ions is typically accomplished by the addition of buffer salts. In some embodiments, the formulation may include a saline phosphate buffer, and in some embodiments, no excipients of human or animal origin may be present.

[0088] For example, a liquid composition (e.g., QM1114) may include at least one chloride ion source, such as sodium chloride, potassium chloride, or another chloride ion source, at a concentration of about 100 to about 300 mM, e.g., 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300 mM, or at a concentration of about 1 to about 25 mM, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 mM. In some embodiments, a liquid composition (e.g., QM1114) may include multiple chloride ion sources at the same or different concentrations, for example, sodium chloride or another sodium or chloride ion source at a concentration of about 100 to about 300 mM, e.g., 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300 mM, and potassium chloride or another chloride ion source at a concentration of about 1 to about 25 mM, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 mM.In some embodiments, the one or more sodium / chloride ion sources are at the same or different concentrations in the range of about 0.1 to 10 mg / mL, e.g., 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7 , 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10 mg / mL, or any value therebetween.

[0089] Additionally or alternatively, the liquid composition (e.g., QM1114) may include at least one phosphate ion source, such as sodium phosphate, potassium phosphate, disodium hydrogen phosphate dihydrate, sodium dihydrogen phosphate dihydrate, or another phosphate ion source, at a concentration of about 1 to about 50 mM or about 5 to about 15 mM, e.g., 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 mM. In some embodiments, the liquid composition may include multiple phosphate ion sources at the same or different concentrations, for example, sodium phosphate, potassium phosphate, disodium hydrogen phosphate dihydrate, sodium dihydrogen phosphate dihydrate, or another phosphate ion source at a concentration of about 1 to about 50 mM or about 5 to about 15 mM, for example, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 mM, and a different phosphate ion source selected from sodium phosphate, potassium phosphate, disodium hydrogen phosphate dihydrate, sodium dihydrogen phosphate dihydrate, or another phosphate ion source at a concentration of about 1 to about 50 mM or about 5 to about 15 mM, for example, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 mM. In some embodiments, one or more phosphate ion sources may be present at the same or different concentrations ranging from about 0.1 to 1.0 mg / mL, for example, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 mg / mL, or any value therebetween.

[0090] In some embodiments, the liquid composition (e.g., QM1114) may include one to five or more buffering agents. Thus, the liquid composition (e.g., QM1114) may include one, two, three, four, or five or more buffering agents, including, but not limited to, sodium chloride, potassium chloride, sodium phosphate, potassium phosphate, disodium hydrogen phosphate dihydrate, or sodium dihydrogen phosphate dihydrate. The one, two, three, four, or five or more buffering agents may be present at the same or different concentrations. For example, in some embodiments, the first buffering agent (e.g., sodium chloride, potassium chloride, sodium phosphate, potassium phosphate, disodium hydrogen phosphate dihydrate, or sodium dihydrogen phosphate dihydrate) is present at a concentration of about 100 to about 300 mM, e.g., 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, , 230, 240, 250, 260, 270, 280, 290, or 300 mM, or at a concentration of about 0.1 to 10 mg / mL, e.g., 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5 ,2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, It may be present at a concentration of 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10 mg / mL, or any value therebetween.In some embodiments, the second buffering agent (e.g., sodium chloride, potassium chloride, sodium phosphate, potassium phosphate, disodium hydrogen phosphate dihydrate, or sodium dihydrogen phosphate dihydrate) may be present at a concentration of about 1 to about 25 mM, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 mM, or at a concentration of about 0.1 to 1.0 mg / mL, e.g., 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 mg / mL, or any value therebetween. In some embodiments, the third buffering agent (e.g., sodium chloride, potassium chloride, sodium phosphate, potassium phosphate, disodium hydrogen phosphate dihydrate, or sodium dihydrogen phosphate dihydrate) may be present at a concentration of about 1 to about 25 mM, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 mM, or at a concentration of about 0.1 to 1.0 mg / mL, e.g., 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 mg / mL, or any value therebetween. In some embodiments, the fourth buffering agent (e.g., sodium chloride, potassium chloride, sodium phosphate, potassium phosphate, disodium hydrogen phosphate dihydrate, or sodium dihydrogen phosphate dihydrate) may be present at a concentration of about 1 to about 25 mM, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 mM, or at a concentration of about 0.1 to 1.0 mg / mL, e.g., 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 mg / mL, or any value therebetween.In some embodiments, the fifth buffering agent (e.g., sodium chloride, potassium chloride, sodium phosphate, potassium phosphate, disodium hydrogen phosphate dihydrate, or sodium dihydrogen phosphate dihydrate) may be present at a concentration of about 1 to about 25 mM, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 mM, or at a concentration of about 0.1 to 1.0 mg / mL, e.g., 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 mg / mL, or any value therebetween.

[0091] Other ingredients may be included in the liquid composition to improve the stability or other properties of the composition. For example, applicable stabilizers include, but are not limited to, amino acids (e.g., alanine, valine, leucine, serine, threonine, lysine histidine, tryptophan, aspartic acid, or glutamic acid), sodium bisulfite, sodium citrate or other citrate salts, and the like. In some embodiments, the amino acid may be an amino acid with a hydrophobic side chain (e.g., alanine, valine, leucine, isoleucine, methionine, phenylalanine, tyrosine, and tryptophan). In some embodiments, the amino acid may be a D-isoform, and in some embodiments, the amino acid may be an L-isoform. Thus, in some embodiments, the liquid composition may contain at least one D- or L-amino acid (e.g., alanine, valine, leucine, serine, threonine, lysine histidine, tryptophan, aspartic acid, or glutamic acid) at a concentration of about 0.1 to about 3.0 mg / mL, about 0.5 to about 2.5 mg / mL, or about 0.75 to about 2.25 mg / mL. In some embodiments, the liquid composition may contain at least one D- or L-amino acid (e.g., alanine, valine, leucine, serine, threonine, lysine histidine, tryptophan, aspartic acid, or glutamic acid) at a concentration of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3.0 mg / mL, or any value therebetween.In some embodiments, the liquid composition may comprise at least one D- or L-amino acid (e.g., alanine, valine, leucine, serine, threonine, lysine histidine, tryptophan, aspartic acid, or glutamic acid) at a concentration of about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1.0, about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2.0, about 2.1, about 2.2, about 2.3, about 2.4, about 2.5, about 2.6, about 2.7, about 2.8, about 2.9, or about 3.0 mg / mL, or any value therebetween.

[0092] In some embodiments, the liquid composition (e.g., QM1114) may further comprise one or more surfactants (e.g., a non-ionic surfactant such as a polysorbate (e.g., polysorbate 80 or polysorbate 20) or nonoxynol; an anionic surfactant such as docusate; or a cationic surfactant such as a quaternary ammonium salt). Thus, in some embodiments, the liquid composition may comprise a non-ionic surfactant, including but not limited to, a polysorbate (e.g., polysorbate 80 or polysorbate 20) or nonoxynol. In some embodiments, the liquid composition may comprise an anionic surfactant, including but not limited to, docusate. In some embodiments, the liquid composition may comprise a cationic surfactant, including but not limited to, a quaternary ammonium salt. In some embodiments, the surfactant may be present at a concentration of about 0.01% (v / v) to about 5.0% (v / v), about 0.05% (v / v) to about 2.5% (v / v), or about 0.1% (v / v) to about 1.5% (v / v). In some embodiments, the at least one surfactant is present at a concentration of 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, It may be present at a concentration of 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9 or 5.0% (v / v), or any value therebetween.In some embodiments, the at least one surfactant is about 0.01, about 0.02, about 0.03, about 0.04, about 0.05, about 0.06, about 0.07, about 0.08, about 0.09, about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1.0, about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2.0, It may be present at a concentration of about 2.1, about 2.2, about 2.3, about 2.4, about 2.5, about 2.6, about 2.7, about 2.8, about 2.9, about 3.0, about 3.1, about 3.2, about 3.3, about 3.4, about 3.5, about 3.6, about 3.7, about 3.8, about 3.9, about 4.0, about 4.1, about 4.2, about 4.3, about 4.4, about 4.5, about 4.6, about 4.7, about 4.8, about 4.9, or about 5.0% (v / v), or any value therebetween. In some embodiments, the liquid composition may contain at least one surfactant at a concentration of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3.0 mg / mL, or any value therebetween. In some embodiments, the liquid composition may comprise at least one surfactant at a concentration of about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1.0, about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2.0, about 2.1, about 2.2, about 2.3, about 2.4, about 2.5, about 2.6, about 2.7, about 2.8, about 2.9, or about 3.0 mg / mL, or any value therebetween.

[0093] In some embodiments, the liquid composition may further comprise one or more emulsifiers (e.g., soy lecithin), wetting agents, excipients (such as lactose, mannitol, glucose, microcrystalline cellulose, colloidal silica, and starch), binders (such as hydroxypropylcellulose, polyvinylpyrrolidone, and magnesium aluminometasilicate), disintegrants (such as starch, L-hydroxypropylcellulose, carboxymethylcellulose, croscarmellose sodium, and cellulose calcium glycolate), lubricants (such as magnesium stearate), swelling agents (such as hydroxypropylcellulose, hydroxypropylmethylcellulose, carbopol, carboxymethylcellulose, polyvinyl alcohol, xanthan gum, and guar gum), swelling adjuvants (such as glucose, fructose, mannitol, xylitol, erythritol, maltose, trehalose, phosphates, citrates, silicates, glycine, glutamate, and arginine), and / or solubilizers (such as polyethylene glycol, propylene glycol, etc.).

[0094] In some embodiments, the liquid composition may include one to five or more buffering agents (e.g., sodium chloride, potassium chloride, sodium phosphate, potassium phosphate, disodium hydrogen phosphate dihydrate, or sodium dihydrogen phosphate dihydrate); one or more stabilizers (e.g., alanine, valine, leucine, serine, threonine, lysine histidine, tryptophan, aspartic acid, or glutamic acid); and one or more surfactants (e.g., a nonionic surfactant such as a polysorbate (e.g., polysorbate 80 or polysorbate 20) or nonoxynol; an anionic surfactant such as docusate; or a cationic surfactant such as a quaternary ammonium salt).In some embodiments, the liquid composition comprises: (i) a first buffering agent (e.g., sodium chloride, potassium chloride, sodium phosphate, potassium phosphate, disodium hydrogen phosphate dihydrate, or sodium dihydrogen phosphate dihydrate) at a concentration of about 100 to about 300 mM or at a concentration of about 0.1 to 10 mg / mL; (ii) a second buffering agent (e.g., sodium chloride, potassium chloride, sodium phosphate, potassium phosphate, disodium hydrogen phosphate dihydrate, or sodium dihydrogen phosphate dihydrate) at a concentration of about 1 to about 25 mM or at a concentration of about 0.1 to 1.0 mg / mL; (iii) a third buffering agent (e.g., sodium chloride, potassium chloride, sodium phosphate, potassium phosphate, disodium hydrogen phosphate dihydrate, or sodium dihydrogen phosphate dihydrate) at a concentration of about 1 to about 25 mM or at a concentration of about 0.1 to 1.0 mg / mL; (iv) a third buffering agent (e.g., sodium chloride, potassium chloride, sodium phosphate, potassium phosphate, disodium hydrogen phosphate dihydrate, or sodium dihydrogen phosphate dihydrate) at a concentration of about 1 to about 25 mM. (v) one or more stabilizers (e.g., alanine, valine, leucine, serine, threonine, lysine histidine, tryptophan, aspartic acid, or glutamic acid) at a concentration of about 0.1 to about 3.0 mg / mL; and (vi) one or more surfactants (e.g., a nonionic surfactant such as a polysorbate (e.g., polysorbate 80 or polysorbate 20) or nonoxynol; an anionic surfactant such as docusate; or a cationic surfactant such as a quaternary ammonium salt) at a concentration of about 0.05% (v / v) to about 2.5% (v / v) or about 0.1 to about 3.0 mg / mL. In some embodiments, the stabilizer may be an amino acid, and in some embodiments, the surfactant may be a non-ionic surfactant such as a polysorbate. For purposes of this disclosure, it should be understood that QM1114-drug (also referred to herein as "QM1114-DP" or simply "QM1114") may exemplify any of the preceding embodiments or any of the following embodiments.

[0095] The liquid composition (e.g., QM1114) may have a pH of 5.5 to 8. In some embodiments, the pH is 6.0 to 7.5, such as about 6.3, 6.35, 6.4, 6.45, 6.5, 6.55, 6.6, 6.65, 6.7, 6.75, 6.8, 6.85, 6.9, 6.95, 7.0, 7.05, 7.1, 7.15, 7.2, 7.25, 7.3, 7.35, 7.4, 7.45, or 7.5. In some embodiments, the pH is 6.6 to 6.9. Preferably, the liquid composition comprises an aqueous diluent, more preferably water, such as sterile water, water for injection, purified water, and sterile water for injection.

[0096] The liquid composition may contain the following components (e.g., in addition to BoNT) to provide a storage-stable liquid formulation of BoNT: a. a chloride ion source selected from NaCl at a concentration of 100-300 mM and KCl at a concentration of 1-25 mM; b. a plurality of phosphate ion sources selected from sodium phosphate, potassium phosphate, disodium hydrogen phosphate dihydrate, and sodium dihydrogen phosphate dihydrate, at concentrations between 1 and 50 mM; c. A non-ionic surfactant at a concentration of 0.01 to 5% (v / v); and d. an amino acid stabilizer selected from alanine, valine, leucine, isoleucine, methionine, phenylalanine, tyrosine, and tryptophan at a concentration of 0.1 to 3 mg / ml; may also include

[0097] The liquid composition of the present invention may include an amino acid that is tryptophan and / or tyrosine. Without wishing to be bound by theory, it is hypothesized that tryptophan and / or tyrosine can prevent oxidation of active proteins that may render them inoperative. Indeed, it is believed that amino acids added in molar excess over neurotoxins are oxidized first, preserving the neurotoxins. It is also hypothesized that tryptophan or tyrosine can neutralize the reaction decomposition products of surfactants such as polysorbates. Preferably, the amino acid is tryptophan. More preferably, the amino acid is L-tryptophan.

[0098] The amino acid concentration may be about 0.1-5 mg / ml, e.g., 0.1-5 mg / ml, 0.25-3 mg / ml, e.g., about 0.25, 0.5, 1, 1.5, 2, or 3 mg / ml. Preferably, the amino acid (e.g., tryptophan and / or tyrosine) concentration is greater than 0.5 mg / ml. For example, the amino acid concentration may have a lower limit of greater than 0.5 mg / ml and an upper limit of 1, 1.5, 2, 3, 4, 5, 6, 7, or 8 mg / ml. Advantageously, concentrations greater than 5 mg / ml may represent insignificant concentrations of amino acids, but over that concentration the stabilizing effect may be particularly prevalent. See the examples in WO2017 / 203038A1, which is incorporated herein by reference.

[0099] The liquid composition may be stable over time. For example, the liquid composition may be stable at 2-8°C for 2 months. According to one embodiment, the liquid composition is stable at 2-8°C, for example at 5°C, for 3 months. According to a preferred embodiment, the liquid composition is stable at 2-8°C, for example at 5°C, for 6 months. According to one embodiment, the liquid composition is stable at 2-8°C, for example at 5°C, for 12 months. According to one embodiment, the liquid composition is stable at 2-8°C, for example at 5°C, for 18 months. According to one embodiment, the liquid composition is stable at 2-8°C, for example at 5°C, for 24 months. According to one embodiment, the liquid composition is stable at 2-8°C, for example at 5°C, for 36 months. According to one embodiment, the liquid composition is stable at room temperature, for example at 25°C, for 3 months. According to one embodiment, the liquid composition is stable at room temperature, for example at 25°C, for 6 months. According to one embodiment, the liquid composition is stable at 37°C for 2 months.

[0100] Stability can be assessed by comparing the activity of a botulinum neurotoxin over time. The activity of a botulinum neurotoxin can refer to the ability of the botulinum neurotoxin to bind to its target receptor on a cell, translocate its light chain into the cell, and / or cleave its target SNARE protein. Methods for measuring botulinum neurotoxin activity are well known in the art. Botulinum neurotoxin activity can be assessed, for example, by using a mouse lethality assay (LD50) as described above, a muscle tissue-based assay such as the mouse phrenic nerve hemidiaphragm assay (e.g., as described in Bigalke, H. and Rummel A., Toxins 7.12(201S):489S-490S), a cell-based assay (e.g., as described in WO201349508 or WO2012166943), or an extracellular proteolytic activity assay such as BoTest® (a botulinum neurotoxin detection kit available from BioSentinel).

[0101] Preferably, a composition according to the present invention is considered stable if it exhibits no more than a predetermined rate of activity loss at a predetermined temperature for a predetermined period of time.

[0102] According to one embodiment, the composition according to the invention is considered stable if there is no more than 30% loss of extracellular proteolytic activity at 2-8°C over 3, 6, 12, 18, 24 or 36 months, for example no more than 30% loss of extracellular proteolytic activity at 5°C over 6 months. Preferably, the composition according to the invention is considered stable if there is no more than 20% loss of extracellular proteolytic activity at 5°C over 3 months, more preferably no more than 6, 12, 18, 24 or 36 months. According to another embodiment, the composition according to the invention is considered stable if there is no more than 40% loss of extracellular proteolytic activity at room temperature, for example at 25°C, over 3 months. Preferably, the composition according to the invention is considered stable if there is no more than 30% loss of extracellular proteolytic activity at 25°C over 3 months, more preferably no more than 6 months at 25°C. According to another embodiment, the composition according to the invention is considered stable if there is no more than 50% loss of extracellular proteolytic activity at 37°C over 2 months. Extracellular proteolytic activity can be measured with the BoTest® assay.

[0103] According to one embodiment, the composition according to the invention is considered stable if there is no more than a 30% loss of MLD50 units at 2-8°C over 2, 3, 6, 12, 18, 24 or 36 months, for example no more than a 30% loss of MLD50 units at 5°C over 6 months. Preferably, the composition according to the invention is considered stable if there is no more than a 20% loss of MLD50 units at 5°C over 2 months, more preferably no more than a 3, 6, 12, 18, 24 or 36 months. According to another embodiment, the composition according to the invention is considered stable if there is no more than a 40% loss of MLD50 units at room temperature, for example at 25°C, over 2 or 3 months. Preferably, the composition according to the invention is considered stable if there is no more than a 30% loss of MLD50 units at 25°C over 3 months, more preferably no more than a 6 month loss of MLD50 units at 25°C. According to another embodiment, the composition according to the invention is considered stable if there is no more than a 50% loss of MLD50 units at 37°C over 2 months. The MLD50 units can be measured as set forth above.

[0104] The liquid composition may be suitable for injection into a patient, particularly a human patient. The amount of botulinum neurotoxin is generally determined by the mouse LD 2 , defined as the median intraperitoneal lethal dose in mice. 50 It is expressed in units of (lethal dose 50). The LD of botulinum toxin in mice 50 (MLD 50 LD units are not standardized units. In fact, the assays used by different manufacturers of commercial toxins vary, especially in the choice of diluent buffer. For example, the test used for BOTOX® uses saline as the diluent. Gelatin buffer is 50 It is believed to preserve the toxin at the high dilutions used in the assay, whereas some potency is thought to be lost when saline is used as a diluent.

[0105] In some embodiments, mouse LD 50 The dilution buffer used to determine the LD is gelatin phosphate buffer. For example, mouse LD 50can be determined as described in Hambleton, P. et al. Production, purification and toxoiding of Clostridium botulinum type A toxin. Eds. GE Jr Lewis, and PS Angel. Academic Press, Inc., New York, USA, 1981, p. 248. Briefly, a botulinum toxin sample is serially diluted in 0.2% (w / v) gelatin 0.07 M Na2HPO4 buffer (pH 6.5). Groups of mice (e.g., 4-8 mice per group) weighing approximately 20 g are injected intraperitoneally with a sample of the diluted toxin (e.g., 0.5 ml per mouse). Dilution groups, e.g., 5 dilution groups, are selected to span the 50% lethal dose. Mice are observed for up to 72 hours and the mouse lethal dose 50 (MLD 50) is determined. 50 ) is estimated.

[0106] The liquid composition according to the present invention preferably has a concentration of 4 to 10,000 LD per mL. 50 units of botulinum neurotoxin, more preferably 10 to 200 LD per mL 50 units of botulinum neurotoxin, e.g., 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200 LD per mL 50 The amount of botulinum neurotoxin can also be expressed in nanograms.

[0107] The liquid composition according to the present invention has an osmolality of 200 to 400 mosm / kg, preferably 270 to 310 mosm / kg, for example, 270, 275, 280, 285, 290, 295, 300, 305, or 310 mosm / kg, or any value therebetween.

[0108] According to one embodiment, the liquid composition according to the present invention comprises: - 4 to 10,000 LD50 units of botulinum neurotoxin per ml, - 0.001 to 15% v / v polysorbate, - 0.1-5 mg / ml tryptophan, - 10 to 500 mM NaCl, - 1 to 50 mM KCl, - 1 to 100 mM sodium phosphate It may comprise or consist essentially of It has a pH of 5.5 to 8 and is stable at 5°C for 2 months (e.g., 6 months). According to one embodiment, the liquid composition according to the present invention comprises: - 4 to 10,000 LD50 units of botulinum neurotoxin per ml, - 0.001 to 15% v / v polysorbate, - 0.1-5 mg / ml tryptophan, - 10 to 500 mM NaCl, - 1 to 50 mM KCl, - 1 to 100 mM disodium hydrogen phosphate dihydrate and sodium dihydrogen phosphate dihydrate It may comprise or consist essentially of It has a pH of 5.5 to 8 and is stable at 5°C for 2 months (e.g., 6 months). According to one embodiment, the liquid composition according to the present invention comprises: - botulinum neurotoxin at 10-2000 LD50 units per ml; - 0.005-2% v / v polysorbate, - 0.1-5 mg / ml tryptophan, - 25 to 300 mM NaCl, - 1 to 10 mM KCl, - 2 to 50 mM sodium phosphate It may comprise or consist essentially of It has a pH of 6.0 to 7.5 and is stable for 12 months at 5°C. According to one embodiment, the liquid composition according to the present invention comprises: - botulinum neurotoxin at 10-2000 LD50 units per ml; - 0.005-2% v / v polysorbate, - 0.1-5 mg / ml tryptophan, - 25 to 300 mM NaCl, - 1 to 10 mM KCl, - 2 to 50 mM disodium hydrogen phosphate dihydrate and sodium dihydrogen phosphate dihydrate It may comprise or consist essentially of It has a pH of 6.0 to 7.5 and is stable for 12 months at 5°C. According to one embodiment, the liquid composition according to the present invention comprises: - botulinum neurotoxin at 10-2000 LD50 units per ml; - 0.05-0.2% v / v polysorbate 80, - 0.1-5 mg / ml tryptophan, - 25 to 300 mM NaCl, - 1 to 10 mM KCl, - 2 to 50 mM sodium phosphate It may comprise or consist essentially of It has a pH of 6.0 to 7.5 and is stable for 12 months at 5°C. According to one embodiment, the liquid composition according to the present invention comprises: - botulinum neurotoxin at 10-2000 LD50 units per ml; - 0.05-0.2% v / v polysorbate 80, - 0.1-5 mg / ml tryptophan, - 25 to 300 mM NaCl, - 1 to 10 mM KCl, - 2 to 50 mM disodium hydrogen phosphate dihydrate and sodium dihydrogen phosphate dihydrate It may comprise or consist essentially of It has a pH of 6.0 to 7.5 and is stable for 12 months at 5°C. According to one embodiment, the liquid composition according to the present invention comprises: - Botulinum neurotoxin A, - 0.2% v / v polysorbate 80, - 1mg / ml tryptophan, - 140 mM NaCl, - 3 mM KCl, - 10mM Sodium Phosphate It may comprise or consist essentially of The pH of the above composition is about 6.6. According to one embodiment, the liquid composition according to the present invention comprises: - Botulinum neurotoxin A, - 0.2% v / v polysorbate 80, - 1mg / ml tryptophan, - 140 mM NaCl, - 3 mM KCl, - 10mM disodium hydrogen phosphate dihydrate and sodium dihydrogen phosphate dihydrate It may comprise or consist essentially of The pH of the above composition is about 6.6. According to one embodiment, the liquid composition according to the present invention comprises: - Botulinum neurotoxin A, - 0.04% v / v polysorbate 80, - 1mg / ml tryptophan, - 140 mM NaCl, - 3 mM KCl, - 10mM Sodium Phosphate It may comprise or consist essentially of The pH of the above composition is about 6.9. According to one embodiment, the liquid composition according to the present invention comprises: - Botulinum neurotoxin A, - 0.04% v / v polysorbate 80, - 1mg / ml tryptophan, - 140 mM NaCl, - 3 mM KCl, - 10mM disodium hydrogen phosphate dihydrate and sodium dihydrogen phosphate dihydrate It may comprise or consist essentially of The pH of the above composition is about 6.9. According to one embodiment, the liquid composition according to the present invention comprises: - Botulinum neurotoxin B, - 0.25% v / v polysorbate 20, - 4mg / ml tryptophan, - 140 mM NaCl, - 3 mM KCl, - 10mM Sodium Phosphate It may comprise or consist essentially of The pH of the above composition is about 7.4. According to one embodiment, the liquid composition according to the present invention comprises: - Botulinum neurotoxin B, - 0.25% v / v polysorbate 20, - 4mg / ml tryptophan, - 140 mM NaCl, - 3 mM KCl, - 10mM disodium hydrogen phosphate dihydrate and sodium dihydrogen phosphate dihydrate It may comprise or consist essentially of The pH of the above composition is about 7.4. According to one embodiment, the liquid composition according to the present invention comprises: - Botulinum neurotoxin A, - 0.01% v / v polysorbate 80, - 0.25mg / ml tryptophan, - 255 mM NaCl, - 2mM Sodium Phosphate It may comprise or consist essentially of The pH of the above composition is about 7.2. According to one embodiment, the liquid composition according to the present invention comprises: - Botulinum neurotoxin A, - 0.01% v / v polysorbate 80, - 0.25mg / ml tryptophan, - 255 mM NaCl, - 10 mM KCl, - 50mM Sodium Phosphate It may comprise or consist essentially of The pH of the above composition is about 6.3. According to one embodiment, the liquid composition according to the present invention comprises: - Botulinum neurotoxin A, - 1% v / v polysorbate 80, - 0.25mg / ml tryptophan, - 255 mM NaCl, - 50mM Sodium Phosphate It may comprise or consist essentially of The pH of the above composition is about 6.3. According to one embodiment, the liquid composition according to the present invention comprises: - Botulinum neurotoxin A, - 1% v / v polysorbate 80, - 3mg / ml tryptophan, - 255 mM NaCl, - 10 mM KCl, - 50mM Sodium Phosphate It may comprise or consist essentially of The pH of the above composition is about 7.2. According to one embodiment, the liquid composition according to the present invention comprises: - Botulinum neurotoxin A, - 1% v / v polysorbate 80, - 3mg / ml tryptophan, - 255 mM NaCl, - 10 mM KCl, - 50mM disodium hydrogen phosphate dihydrate and sodium dihydrogen phosphate dihydrate It may comprise or consist essentially of The pH of the above composition is about 7.2. According to one embodiment, the liquid composition according to the present invention comprises: - Botulinum neurotoxin A, - 0.1% v / v polysorbate 80, - 1.625mg / ml tryptophan, - 140 mM NaCl, - 3 mM KCl, - 10mM Sodium Phosphate It may comprise or consist essentially of The pH of the composition is about 6.75. According to one embodiment, the liquid composition according to the present invention comprises: - Botulinum neurotoxin A, - 0.1% v / v polysorbate 80, - 1.625mg / ml tryptophan, - 140 mM NaCl, - 3 mM KCl, - 10mM disodium hydrogen phosphate dihydrate and sodium dihydrogen phosphate dihydrate It may comprise or consist essentially of The pH of the composition is about 6.75. According to one embodiment, the liquid composition according to the present invention comprises: - Botulinum neurotoxin A, - 0.01% v / v polysorbate 80, - 1mg / ml tryptophan, - 140 mM NaCl, - 3 mM KCl, - 10mM Sodium Phosphate It may comprise or consist essentially of The pH of the above composition is about 6.75. According to one embodiment, the liquid composition according to the present invention comprises: - Botulinum neurotoxin A, - 0.01% v / v polysorbate 80, - 1mg / ml tryptophan, - 140 mM NaCl, - 3 mM KCl, - 10mM disodium hydrogen phosphate dihydrate and sodium dihydrogen phosphate dihydrate It may comprise or consist essentially of The pH of the composition is about 6.75. According to one embodiment, the liquid composition according to the present invention comprises: - Botulinum neurotoxin A, - 0.1% v / v polysorbate 80, - 1mg / ml tryptophan, - 140 mM NaCl, - 3 mM KCl, - 10mM Sodium Phosphate It may comprise or consist essentially of The pH of the composition is about 6.75. According to one embodiment, the liquid composition according to the present invention comprises: - Botulinum neurotoxin A, - 0.1% v / v polysorbate 80, - 1mg / ml tryptophan, - 140 mM NaCl, - 3 mM KCl, - 10mM disodium hydrogen phosphate dihydrate and sodium dihydrogen phosphate dihydrate It may comprise or consist essentially of The pH of the composition is about 6.75. According to one embodiment, the liquid composition according to the present invention comprises: - Botulinum neurotoxin A, - 1% v / v polysorbate 80, - 1mg / ml tryptophan, - 140 mM NaCl, - 3 mM KCl, - 10mM Sodium Phosphate It may comprise or consist essentially of The pH of the composition is about 6.75. According to one embodiment, the liquid composition according to the present invention comprises: - Botulinum neurotoxin A, - 1% v / v polysorbate 80, - 1mg / ml tryptophan, - 140 mM NaCl, - 3 mM KCl, - 10mM disodium hydrogen phosphate dihydrate and sodium dihydrogen phosphate dihydrate It may comprise or consist essentially of The pH of the composition is about 6.75. According to one embodiment, the liquid composition according to the present invention comprises: - Botulinum neurotoxin B, - 15% v / v polysorbate 20, - 1mg / ml tryptophan, - 140 mM NaCl, - 3 mM KCl, - 10mM Sodium Phosphate It may comprise or consist essentially of The pH of the above composition is about 7.4. According to one embodiment, the liquid composition according to the present invention comprises: - Botulinum neurotoxin B, - 15% v / v polysorbate 20, - 1mg / ml tryptophan, - 140 mM NaCl, - 3 mM KCl, - 10mM disodium hydrogen phosphate dihydrate and sodium dihydrogen phosphate dihydrate It may comprise or consist essentially of The pH of the above composition is about 7.4. According to one embodiment, the liquid composition according to the present invention comprises: - Botulinum neurotoxin B, - 15% v / v polysorbate 20, - 4mg / ml tryptophan, - 140 mM NaCl, - 3 mM KCl, - 10mM Sodium Phosphate It may comprise or consist essentially of The pH of the above composition is about 7.4. According to one embodiment, the liquid composition according to the present invention comprises: - Botulinum neurotoxin B, - 15% v / v polysorbate 20, - 4mg / ml tryptophan, - 140 mM NaCl, - 3 mM KCl, - 10mM disodium hydrogen phosphate dihydrate and sodium dihydrogen phosphate dihydrate It may comprise or consist essentially of The pH of the above composition is about 7.4. According to one embodiment, the liquid composition according to the present invention comprises: - Botulinum neurotoxin B, - 0.25% v / v polysorbate 20, - 4mg / ml tryptophan, - 140 mM NaCl, - 3 mM KCl, - 10mM Sodium Phosphate It may comprise or consist essentially of The pH of the above composition is about 7.4. According to one embodiment, the liquid composition according to the present invention comprises: - Botulinum neurotoxin B, - 0.25% v / v polysorbate 20, - 4mg / ml tryptophan, - 140 mM NaCl, - 3 mM KCl, - 10mM disodium hydrogen phosphate dihydrate and sodium dihydrogen phosphate dihydrate It may comprise or consist essentially of The pH of the above composition is about 7.4.

[0109] The enzymatic activity of BoNT was also assayed using the BOTEST assay, which provides detection of BoNT proteolytic activity in an optimized reaction buffer. TM (For example, BOTEST TM This can be determined by the Matrix A Botulinum Neurotoxin Detection Kit (BioSentinel Catalog No. A1015). TM Intended uses of BOTEST include quantification and characterization of BoNT pharmaceutical formulations, drug discovery, high throughput screening applications, and detection applications. TM contains a reporter consisting of residues 141-206 of SNAP-25, a naturally occurring substrate for BoNT / A and E. The substrate is sandwiched between two fluorescent proteins, a cyan fluorescent protein (CFP) derivative and a yellow fluorescent protein (YFP) derivative. The CFP and YFP moieties form a donor / acceptor Förster resonance energy transfer (FRET) pair. The use of a FRET donor / acceptor pair separated by a substrate for BoNT allows for detection of BoNT proteolytic activity. In the absence of BoNT, the reporter is intact and the CFP and YFP moieties are in close proximity. When CFP is excited, energy transfer occurs to YFP by FRET. As a result, CFP emission is quenched and YFP fluoresces by FRET. In the presence of BoNT, the reporter is cleaved by the proteolytic activity of BoNT. The CFP and YFP moieties are physically separated and FRET no longer occurs. CFP emission is restored and YFP emission is reduced. Thus, BOTEST TMutilizes a fluorescent reporter that provides a ratiometric response when specifically cleaved by BoNT, providing an output as a relative fluorescent unit (RFU) or emission ratio. These outputs can be used to calculate Botulinum Units ("BU", which can be synonymous with Enzyme Units "U" or International Units "IU") / ml or BU / BoNT concentration (e.g. mg). In other words, the enzymatic activity of BoNT can also be determined by a first cell-based assay, such as one that provides detection of BoNT proteolytic activity in an optimized reaction buffer. Such a first cell-based assay includes a reporter consisting of residues 141-206 of SNAP-25, a naturally occurring substrate for BoNT / A and E. The substrate is sandwiched between two fluorescent proteins, a cyan fluorescent protein (CFP) derivative and a yellow fluorescent protein (YFP) derivative. The CFP and YFP moieties form a donor / acceptor Förster resonance energy transfer (FRET) pair. The use of a FRET donor / acceptor pair separated by a substrate for BoNT allows for detection of BoNT proteolytic activity. In the absence of BoNT, the reporter is intact and the CFP and YFP moieties are in close proximity. When CFP is excited, energy transfer occurs to YFP by FRET. As a result, CFP emission is quenched and YFP fluoresces by FRET. In the presence of BoNT, the reporter is cleaved by the proteolytic activity of BoNT. The CFP and YFP moieties are physically separated and FRET no longer occurs. CFP emission is restored and YFP emission is reduced. Thus, the first cell-based assay utilizes a fluorescent reporter that provides a ratiometric response when specifically cleaved by BoNT, which provides an output as a relative fluorescence unit (RFU) or emission ratio. These outputs can be used to calculate botulinum units ("BU", which can be synonymous with enzyme units "U" or international units "IU") / ml or BU / BoNT concentration (e.g. mg).

[0110] In some embodiments, the activity or specific activity of the formulation is determined by the BOTESTTM This can be confirmed using the Matrix A Botulinum Neurotoxin Detection Kit (BioSentinel, Catalog No. A1015). TM The Matrix A Botulinum Neurotoxin (BoNT) Detection Kit is an in vitro assay for the detection and quantification of BoNT serotype A (BoNT / A) in complex matrices such as serum, blood, food, water, bacterial supernatants, and pharmaceutical samples. TM The Matrix A kit contains two primary reagents: Matrix A beads and BOTEST TM Matrix A beads are composed of the A / E reporter and capture, concentrate, and isolate BoNT / A complexes or holotoxins in a complex matrix from samples ranging in size from 100 μl to 50 ml. And, BOTEST TM The A / E reporter detects the amount of captured BoNT / A, providing an assessment of BoNT / A activity and quantity. Depending on the matrix composition and sample size, picomolar sensitivity can be achieved in less than 3 hours and femtomolar sensitivity can be achieved in 24 hours. Matrix A beads consist of anti-BoNT / A antibodies covalently attached to proprietary and patented magnetic beads. These beads are added to a sample containing BoNT / A and incubated, allowing BoNT / A binding to occur. Interfering compounds that may interfere with the determination of BoNT / A activity are removed by washing the Matrix A beads. BOTEST TM The A / E reporter is modeled after naturally occurring substrates of proteolytic BoNT and detects the endopeptidase activity of BoNT / A. TMThe A / E reporter contains amino acids 141-206 of SNAP-25 and encompasses both the inactive binding and cleavage sites of BoNT / A. The reporter has a high affinity for BoNT / A and detects BoNT / A proteolytic activity rapidly and sensitively when incubated with BoNT / A bound to matrix A beads. One suitable protocol for this kit is described in the Examples below. In other words, the activity or specific activity of the formulation can be confirmed using a second cell-based assay, such as one for the detection and quantification of BoNT serotype A (BoNT / A) in complex matrices such as serum, blood, food, water, bacterial supernatants, and pharmaceutical samples. The second cell-based assay consists of two primary reagents, beads (consisting of anti-BoNT / A antibodies covalently bound to magnetic beads) and a reporter, a polypeptide modeled after the naturally occurring substrate of proteolytic BoNT, that detects the endopeptidase activity of BoNT / A. The beads capture, concentrate, and isolate BoNT / A complexes or holotoxins in a complex matrix from samples ranging in size from 100 μl to 50 ml. The reporter then detects the amount of captured BoNT / A, providing an assessment of BoNT / A activity and quantity. Depending on the matrix composition and sample size, picomolar sensitivity can be achieved in less than 3 hours, and femtomolar sensitivity can be achieved in 24 hours. As mentioned above, the beads consist of anti-BoNT / A antibodies covalently attached to magnetic beads. These beads are added to a sample containing BoNT / A and incubated, allowing BoNT / A binding to occur. Interfering compounds that may interfere with the determination of BoNT / A activity are removed by washing the beads. The reporter is modeled after a naturally occurring substrate for proteolytic BoNT and detects the endopeptidase activity of BoNT / A. The reporter includes amino acids 141-206 of SNAP-25, encompassing both the non-active binding site and the cleavage site of BoNT / A. The reporter has a high affinity for BoNT / A and upon incubation with BoNT / A bound to beads, rapidly and sensitively detects BoNT / A proteolytic activity. One suitable protocol for this kit is described in the Examples below.

[0111] For purposes of this disclosure, liquid compositions suitable for the disclosed treatments (e.g., QM1114) may be prepared using BOTEST TECHNOLOGY as described above and in the Examples. TM In some embodiments, the disclosed liquid compositions suitable for treatment (e.g., QM1114) may have an enzyme activity of at least 95, at least 96, at least 97, at least 98, at least 99, at least 100, at least 101, at least 102, at least 103, at least 104, at least 105, at least 106, at least 107, at least 108, at least 109, at least 110, at least 111, at least 112, at least 113, at least 114, or at least 115 BU / ml as determined by BOTEST. TM In some embodiments, the disclosed therapeutically suitable liquid compositions (e.g., QM1114) may have an enzyme activity of about 95, about 96, about 97, about 98, about 99, about 100, about 101, about 102, about 103, about 104, about 105, about 106, about 107, about 108, about 109, about 110, about 111, about 112, about 113, about 114, or about 115 BU / ml as determined by BOTEST. TM In some embodiments, the disclosed liquid compositions suitable for treatment (e.g., QM1114) may have an enzyme activity of 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, or 115 BU / ml as determined by BOTEST. TM In some embodiments, the disclosed therapeutically suitable liquid compositions (e.g., QM1114) may have an enzyme activity of about 95, about 96, about 97, about 98, about 99, about 100, about 101, about 102, about 103, about 104, about 105, about 106, about 107, about 108, about 109, about 110, about 111, about 112, about 113, about 114, or about 115 BU / ml as determined by BOTEST. TM(in other words, as determined in a first cell-based assay), may have an enzyme activity of 95-100, 96-100, 97-100, 98-100, 99-100, 96-99, 97-99, 98-99, or 97-98 BU / ml.

[0112] In some embodiments, the enzyme activity may be normalized to the BoNT concentration in a given formulation to take into account the relative activity levels of different BoNT enzymes (i.e., calculating the specific activity). For example, the specific activity or normalized activity may be calculated using the BOTEST TM (For example, BOTEST TMThe specific activity or normalized activity may be calculated by dividing the results of a Matrix A Botulinum Neurotoxin Detection Kit (BioSentinel, Catalog No. A1015) by the concentration or amount of BoNT in the formulation being tested. For example, the specific activity or normalized activity may be calculated by dividing the results of the first cell-based assay or the second cell-based assay by the concentration or amount of BoNT in the formulation being tested. In some embodiments, the disclosed liquid compositions suitable for treatment (e.g., QM1114) may have an enzymatic activity normalized to the BoNT concentration (i.e., specific activity) of at least 0.98, at least 0.99, at least 1.00, at least 1.01, at least 1.02, at least 1.03, at least 1.04, at least 1.05, at least 1.06, at least 1.07, at least 1.08, at least 1.09, at least 1.10, at least 1.11, at least 1.12, at least 1.13, at least 1.14, at least 1.15, at least 1.16, at least 1.17, at least 1.18, at least 1.19, or at least 1.20 BU / BoNT. In some embodiments, the disclosed liquid compositions suitable for treatment (e.g., QM1114) may have an enzyme activity normalized to a BoNT concentration of about 0.98, about 0.99, about 1.00, about 1.01, about 1.02, about 1.03, about 1.04, about 1.05, about 1.06, about 1.07, about 1.08, about 1.09, about 1.10, about 1.11, about 1.12, about 1.13, about 1.14, about 1.15, about 1.16, about 1.17, about 1.18, about 1.19, or about 1.20 BU / BoNT. In some embodiments, the disclosed liquid compositions suitable for treatment (e.g., QM1114) may have an enzyme activity normalized to a BoNT concentration in RBU of 0.98, 0.99, 1.00, 1.01, 1.02, 1.03, 1.04, 1.05, 1.06, 1.07, 1.08, 1.09, 1.10, 1.11, 1.12, 1.13, 1.14, 1.15, 1.16, 1.17, 1.18, 1.19, or 1.20 BU / BoNT.Additionally or alternatively, in some embodiments, the disclosed liquid compositions suitable for treatment (e.g., QM1114) may have an enzymatic activity normalized to the BoNT concentration (i.e., specific activity) of at least 0.12, at least 0.13, at least 0.14, at least 0.15, at least 0.16, at least 0.17, at least 0.18, at least 0.19, at least 0.20, at least 0.21, at least 0.22, at least 0.23, at least 0.24, or at least 0.25 BU / pg of BoNT. In some embodiments, the disclosed therapeutically suitable liquid compositions (e.g., QM1114) may have an enzymatic activity normalized to a BoNT concentration of about 0.12, about 0.13, about 0.14, about 0.15, about 0.16, about 0.17, about 0.18, about 0.19, about 0.20, about 0.21, about 0.22, about 0.23, about 0.24, or about 0.25 BU / pg of BoNT. In some embodiments, the disclosed therapeutically suitable liquid compositions (e.g., QM1114) may have an enzymatic activity normalized to a BoNT concentration of about 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, or 0.25 BU / pg of BoNT. For concentration-normalized activity, activity is BOTEST. TM and divided by the BoNT concentration, which can be determined using a BoNT-specific ELISA (e.g., BOLISA®), or the like.

[0113] BOLISA® or Botulinum Neurotoxin Sandwich ELISA can be used to detect and quantify the total mass of BoNT from a liquid matrix using a traditional sandwich ELISA approach. Several different BOLISA® kits are available from BioSentinel for various serotypes (e.g., BoNT / A Catalog No. A1029; BoNT / B Catalog No. A1045; BoNT / B4 Catalog No. A1048; BoNT / C Catalog No. A1042; BoNT / E Catalog No. A1034). In the BOLISA® kit (in other words, BoNT-specific ELISA), an anti-BoNT antibody is bound to a solid support and a sample containing BoNT is contacted with the support. BoNT present in the sample is allowed to bind to the antibody and all other materials are removed or washed away. A second detectably labeled antibody that binds to the same serotype of BoNT can be added and the amount of BoNT in the sample can be determined by detecting or quantifying the output of the detectable label (e.g., fluorescence, enzyme activity, colorimetric change, etc.). The BOLISA® assay (or in other words, a BoNT-specific ELISA) is particularly useful for calculating specific activity (i.e., the level of activity in a formulation divided by the concentration of BoNT in the formulation). The output of the BOLISA® assay (or in other words, a BoNT-specific ELISA) can be weight / ml (e.g., mg, μg, or pg / ml) or relative BOLISA® units (RBU) / ml.

[0114] Relative potency or enzymatic activity can also be determined using cell-based potency assays (CBPAs). CBPAs can assess key steps of BoNT action: receptor binding, internalization-translocation, and catalytic activity, and can therefore be used in place of the standard mouse bioassays described above. For example, one particularly useful CBPA is described in PCT / IB2021 / 056210 (WO2022 / 009182) and includes: (a) distributing approximately two different BoNT samples into approximately two containers containing cells expressing SNAP25 protein, where the first BoNT sample is a reference sample of known potency and the second BoNT sample is a test sample of unknown potency; (b) incubating the cells with BoNT for a period of time; (c) determining the ratio of cleaved SNAP25 protein to uncleaved SNAP25 protein corresponding to the reference sample and the test sample; and (d) determining the potency of the test sample relative to the reference sample. Optionally, a third BoNT sample, a quality control sample of known potency, can be dispensed into a third container to serve as a positive control. In some embodiments, (c) can include subjecting the cleaved and uncleaved SNAP25 proteins to quantification by Western blot and densitometry. In some embodiments, the period is about 6, 12, 16, 20, 24, 32, 40, 48, or 56 hours. In some embodiments, the approximately two containers each include multiple wells. In some embodiments, the approximately two different BoNT samples are serially diluted into the multiple wells. In some aspects, the approximately two containers are tissue culture plates. In some embodiments, the approximately two containers are 48-well plates, 96-well plates, 384-well plates, or 1536-well plates. In some embodiments, the cells are attached or adhered to the approximately two containers. In some embodiments, the cells naturally express SNAP25. In some embodiments, the cells express heterologous SNAP25. In some embodiments, the cells are non-neuronal cells. In some embodiments, the cell is genetically engineered. In some embodiments, the cell is a neuronal cell.In some embodiments, the neural cells are motor neurons. In some embodiments, the cells are treated with a non-proliferative agent. In some embodiments, the non-proliferative agent inhibits γ-secretase. In some aspects, the non-proliferative agent is DAPT. In some embodiments, at the end of (b), a protease inhibitor is added to about two of the vessels. In some embodiments, the cells are lysed after incubating the cells with BoNT. In some embodiments, the cells are lysed by sonication. In some embodiments, the cells are lysed by adding a lysis agent. In some embodiments, the lysis agent comprises a detergent. This assay demonstrates LD in mice. 50 It provides the same efficacy data as current animal-based efficacy assays, without the need for reliance on experimentation.

[0115] Another possible CBPA can utilize human induced pluripotent stem cells (hiPSCs) derived from motor neurons and SNAP25 cleavage detected by Western blot. Another example is a CBPA utilizing differentiated human neuroblastoma SiMa cells, where potency can be determined using a sandwich ELISA that measures BoNT / A-dependent intracellular increase in cleaved SNAP25. Exemplary such assays are disclosed in, for example, Salas et al., Botulinum neurotoxin serotype A specific cell-based potency assay to replace the mouse bioassay, PLOS ONE, 2012, 7(11):e49516.

[0116] In some embodiments, the relative potency of the disclosed therapeutic liquid compositions (e.g., QM1114) as determined in a cell-based assay (e.g., a hiPSC-based assay or a SiMa cell-based assay as described above) can be about 115, about 116, about 117, about 118, about 119, about 120, about 121, about 122, about 123, about 124, about 125, about 126, about 127, about 128, about 129, about 130, about 131, about 132, about 133, about 134, about 135, about 136, about 137, about 138, about 139, or about 140 CBpA units. In some embodiments, the relative potency of the disclosed therapeutic liquid compositions (e.g., QM1114) can be at least 115, at least 116, at least 117, at least 118, at least 119, at least 120, at least 121, at least 122, at least 123, at least 124, at least 125, at least 126, at least 127, at least 128, at least 129, at least 130, at least 131, at least 132, at least 133, at least 134, at least 135, at least 136, at least 137, at least 138, at least 139, or at least 140 CBpA units as determined by cell-based ELISA. In some embodiments, the relative potency of the disclosed therapeutic liquid compositions (e.g., QM1114) as determined in a cell-based assay (e.g., a hiPSC-based assay or a SiMa cell-based assay as described above) can be 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, or 140 Units.

[0117] In some aspects, potency or activity can be normalized to the BoNT concentration in a given formulation to take into account the relative activity levels of different BoNT enzymes (i.e., calculating specific activity). For example, in some embodiments, a disclosed therapeutically suitable liquid composition (e.g., QM1114) can have a potency normalized to BoNT concentration of at least 1.20, at least 1.21, at least 1.22, at least 1.23, at least 1.24, at least 1.25, at least 1.26, at least 1.27, at least 1.28, at least 1.29, at least 1.30, at least 1.31, at least 1.32, at least 1.33, at least 1.34, at least 1.35, at least 1.36, at least 1.37, at least 1.38, at least 1.39, or at least 1.40 CBpA units / BoNT concentration (e.g., mg). In some embodiments, the disclosed liquid compositions suitable for treatment (e.g., QM1114) can have a potency normalized to BoNT concentration of about 1.20, about 1.21, about 1.22, about 1.23, about 1.24, about 1.25, about 1.26, about 1.27, about 1.28, about 1.29, about 1.30, about 1.31, about 1.32, about 1.33, about 1.34, about 1.35, about 1.36, about 1.37, about 1.38, about 1.39, or about 1.40 CBpA units / BoNT concentration (e.g., mg). In some embodiments, the disclosed therapeutic liquid compositions (e.g., QM1114) may have a BoNT concentration-normalized potency of 1.20, 1.21, 1.22, 1.23, 1.24, 1.25, 1.26, 1.27, 1.28, 1.29, 1.30, 1.31, 1.32, 1.33, 1.34, 1.35, 1.36, 1.37, 1.38, 1.39, or 1.40 CBpA units / mg of BoNT concentration. For concentration-normalized potency / activity, the potency or activity can be determined by a cell-based assay (e.g., the cell-based assays disclosed in PCT / IB2021 / 056210 as described above), the concentration of the formulation can be determined by an ELISA assay (e.g., BOLISA®), or the like.

[0118] III. Treatment Provided herein is a method of treating moderate to severe glabellar and / or lateral canthal wrinkles in a human subject, comprising administering to the subject a therapeutically effective amount of a liquid composition comprising a botulinum neurotoxin (e.g., QM1114 or a "ready to use" liquid composition), thereby reducing the appearance of moderate to severe glabellar wrinkles. The liquid composition may correspond to any of the preceding embodiments disclosed in II. above.

[0119] In addition to utilizing a "ready-to-use" liquid composition (e.g., QM1114) that does not need to be reconstituted or mixed, the disclosed treatments can be further distinguished from conventional BoNT-based treatments in that the liquid compositions contain a BoNT (e.g., BoNT / A) that is more enzymatically active than competing products (e.g., XEOMIN®, BOTOX®) when normalized with the BoNT concentration in the formulation. The "competitive products" described herein can also be referred to as BoNT formulations that are stored in a dry form (e.g., lyophilized powder form) and must be reconstituted for administration to a subject. As a result, the liquid compositions (e.g., QM1114) can contain less BoNT than competing products, and therefore are generally safer and less likely to cause adverse events or side effects, such as ptosis. Thus, the methods and uses disclosed herein may include selecting a subject who is at risk of developing ptosis, who has previously suffered from ptosis, or who has previously been treated with a BoNT formulation (e.g., GL, LCL, or a combination thereof) and experienced ptosis. In some embodiments, the methods and uses disclosed herein may include selecting a subject who has not previously been treated with a BoNT formulation (e.g., GL, LCL, or a combination thereof).

[0120] The higher the enzyme activity level in the liquid composition (e.g., QM1114), the higher the relative efficacy (when normalized to BoNT concentration) compared to competing products (e.g., XEOMIN®, BOTOX®). With higher activity and higher efficacy, the disclosed methods provide more rapid onset of results in terms of reduction in GL and LCL, longer lasting results, and less incidence of side effects (e.g., ptosis) compared to competing products (e.g., XEOMIN®, BOTOX®).

[0121] Thus, the present disclosure provides methods of treating GL and LCL with a BoNT, such as QM1114, that are safer and / or have fewer or less severe side effects than competing products (e.g., BOTOX COSMETIC®, XEOMIN®, and JEUVEAU®). The present disclosure also provides methods of treating GL and LCL with a BoNT, such as QM1114, that are less likely to cause ptosis than competing products (e.g., BOTOX COSMETIC®, XEOMIN®, and JEUVEAU®). The present disclosure also provides methods of treating GL and LCL with a BoNT, such as QM1114, that have a more rapid onset of therapeutic effect than competing products (e.g., BOTOX COSMETIC®, XEOMIN®, and JEUVEAU®). The present disclosure also provides methods of treating GL and LCL with a BoNT, such as QM1114, that provide therapeutic benefit for a longer period of time than competing products (e.g., BOTOX COSMETIC®, XEOMIN®, and JEUVEAU®).

[0122] Additionally or alternatively, the present disclosure provides a method of treating glabellar lines (GL), lateral canthal lines (LCL), or a combination thereof, with a low risk of ptosis in a human subject in need thereof, comprising administering to the subject a therapeutically effective amount of a liquid composition comprising a botulinum neurotoxin, thereby reducing the appearance of the GL, LCL, or a combination thereof, wherein the botulinum neurotoxin has an enzymatic activity normalized to a botulinum neurotoxin concentration of at least 1.16 BU / BoNT. The human subject is administered a liquid composition containing a botulinum neurotoxin (e.g., an enzymatic activity normalized to a botulinum neurotoxin concentration of less than 1.16 BU / BoNT, or about 2.0×10 8 The human subject may be a subject who is considered to be at risk of, or likely to develop, ptosis when administered a botulinum neurotoxin formulation having a specific activity of less than 1000 U / mg, e.g., Botox Cosmetic®, Xeomin®, and Jeuveau®. The human subject may also be a subject who suffers from ptosis and / or has a medical history of developing ptosis when administered a botulinum neurotoxin. In another embodiment, the human subject is not a subject who (i) is not at risk of developing ptosis, (ii) does not suffer from ptosis, and / or (iii) does not have a medical history of developing ptosis when administered any dose or enzymatic activity of botulinum neurotoxin.

[0123] Specifically with regard to ptosis, the present disclosure provides evidence of a ptosis rate with QM1114 that is significantly lower than that observed with other competing products. For example, a review of studies with a total of over 8000 patients found a ptosis rate of about 2.5%. Specifically, incidences of ptosis were observed for Botox (3%) and Jeuveau (2%). As shown in Example 1, ptosis rates observed when treating LCL / GL with QM1114 were as low as 0.9% in some studies. Thus, in some embodiments of the disclosed treatments (treatments for LCL, GL, or combinations thereof), ptosis rates may be less than 2%, less than 1.9%, less than 1.8%, less than 1.7%, less than 1.5%, less than 1.4%, less than 1.3%, less than 1.2%, less than 1.1%, less than 1.0%, or as low as 0.9%. In some embodiments of the disclosed treatments (treatments for LCL, GL, or combinations thereof), the ptosis rate may be about 2%, about 1.9%, about 1.8%, about 1.7%, about 1.5%, about 1.4%, about 1.3%, about 1.2%, about 1.1%, about 1.0%, or about 0.9%. In some embodiments of the disclosed treatments (treatments for LCL, GL, or combinations thereof), the ptosis rate may be 1.9%, 1.8%, 1.7%, 1.5%, 1.4%, 1.3%, 1.2%, 1.1%, 1.0%, or 0.9% or less.

[0124] Further, in some embodiments, the present disclosure provides a method of treating lateral canthal creases (LCL) in a subject, comprising administering a liquid composition of BoNT (e.g., QM1114), which treatment has a higher success rate than treating LCL with BOTOX COSMETIC (registered trademark) (in other words, a BoNT formulation that is stored in a dry form (e.g., a lyophilized powder form) and must be reconstituted for administration to a subject).

[0125] In some embodiments, 1-100 units of a botulinum toxin are administered to the subject. In some embodiments, 10-75 units of a botulinum toxin are administered to the subject. In some embodiments, 25-75 units of a botulinum toxin are administered to the subject. In some embodiments, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 units of a botulinum toxin are administered to the subject. In some embodiments, 50-250 units of a botulinum toxin are administered to the subject. In some embodiments, 75-200 units of a botulinum toxin are administered to the subject. In some embodiments, the concentration of the liquid composition administered to a subject is between 1 and 300 units / mL of botulinum toxin, e.g., 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, or 300 units / mL.

[0126] In some embodiments, the composition is administered by injection. In some embodiments, the injection is subcutaneous, transdermal, intradermal, or intramuscular. In some embodiments, the method includes administering multiple injections to the glabellar region. In some embodiments, adjacent injection sites are about 0.5-10 cm apart. In some embodiments, adjacent injection sites are about 1.5-3 cm apart. In some embodiments, the injection may be unilateral. In some embodiments, the injection may be bilateral.

[0127] In some embodiments, injections are made into the procerus and corrugator supercilii muscles on each side of the face, and in some embodiments, injections are made in a specific order, for example, first the procerus, then the corrugator supercilii muscles on each side of the face, moving from the midline outwards, in some embodiments, all injections are made about 1 cm above the superior orbital rim, medial to the midpupil line.

[0128] In some embodiments, all injections are given in the center of the eyebrow or at least 1 cm above the bony supraorbital ridge.

[0129] In some embodiments, the composition may be administered to treat, prevent, or ameliorate lateral canthal wrinkles (LCL). In some embodiments, the treatment may include about three injections (one injection per injection site). For example, the treatment may include 1, 2, 3, 4, or 5 injections. The location of the injection may be adjusted depending on the individual subject's LCL wrinkle pattern. Depending on the individual subject's wrinkle pattern, if the wrinkles in the LCL area are above and below the lateral canthus, injections were administered, for example, as described in FIG. 12A. Alternatively, if the individual subject's LCL wrinkles are primarily below the lateral canthus, injections were administered, for example, as described in FIG. 12B. In some embodiments, the injection point may be the outer portion of the orbicularis oculi muscle, about 1-2 cm from the orbital rim, if applicable. Some embodiments may include three injections of equal volume (100 μl) administered to each side of the face (i.e., six injections total).

[0130] In some embodiments, the composition may be administered to treat, prevent, or improve both GL and LCL (i.e., simultaneously). In some embodiments, the treatment may include, for example, 11 injections (one injection per injection site) of equal volume (100 μl) administered to the GL area (five injections) and the LCL area (three injections on each side of the face). In some embodiments, the simultaneous treatment of GL and LCL may include 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 or more injections. For example, for GL, five injection sites may include two injections in each corrugator muscle and one injection in the procerus muscle, and for LCL, the location of the injection may be adjusted depending on the individual subject's LCL wrinkle pattern. Depending on the individual subject's wrinkle pattern, if the wrinkles in the LCL area were above and below the lateral canthus, the injections may be performed as described in FIG. 12A. Alternatively, if the wrinkles in the LCL of an individual subject were primarily below the lateral canthus, the injections may be performed as described in FIG. 12B. In some embodiments, the injection point for treating LCL may be the lateral portion of the orbicularis oculi muscle, approximately 1-2 cm from the orbital rim, if applicable.

[0131] In some embodiments, the method is repeated at intervals of about 3 months to about 6 months to inhibit said recurrence. In some embodiments, the method is repeated at intervals of about 4 months to inhibit said recurrence.

[0132] The methods provided herein provide a temporary reduction in the appearance of glabellar and / or canthal wrinkles in a subject. The effectiveness of treatment can be evaluated by methods known to those skilled in the art. Exemplary evaluation methods are provided below and in the Examples.

[0133] a. 4-point photo scale for glabellar severity: Investigator Live Assessment (GL-ILA) The validated 4-point glabellar severity photo scale includes two grading systems: one for investigator live assessment at maximum frown and one for investigator live assessment at rest. The scale represents severity of glabellar wrinkles from none (grade 0) to mild (grade 1), moderate (grade 2) to severe (grade 3). Each grade is also represented by a separate photograph and description. Investigators are trained in the use of the 4-point photo scale. Investigators use the 4-point photo scale for direct, real-life comparison with the subject's face at screening, baseline (pre-treatment), and all post-treatment visits. Subjects perform their own assessment of glabellar wrinkle severity independent of the investigator's assessment. Subjects are asked to assess their own glabellar wrinkles at maximum frown using a static 4-point category scale at screening, baseline (pre-treatment), and all post-treatment visits. [Table 1]

[0134] b. Global Aesthetic Improvement Scale (GAIS) Subjects will rate the global aesthetic improvement of their maximum frown glabellar lines compared to their pre-treatment appearance at all post-treatment visits using the following categorical scale: [Table 2]

[0135] Subjects are asked, "How would you rate the change in appearance of your glabellar lines (the lines between your eyebrows) at maximum frown compared to just before the injection?"

[0136] Subjects are instructed to select the one rating that best represents the degree to which their maximal frown glabellar appearance has changed compared to baseline. Subjects are allowed to review the baseline photograph to assist them in rating.

[0137] c.Diary card Subjects are asked to record their assessment of their study treatment response on a diary card from the day after treatment (Day 1) through Day 7 (Study Visit 3). Subjects are asked to answer the following question with a "yes" or "no": "Since receiving the injections, have you noticed an improvement in the appearance of your glabellar lines (the lines between the eyebrows)?". Subjects will complete the diary card each day and return the diary to the study center at the Day 7 visit.

[0138] d.FACE-Q The FACE-Q is a patient-reported outcome tool to assess the experience and outcome of cosmetic facial procedures from the subject's perspective. The FACE-Q consists of more than 40 scales, covering four domains: satisfaction with facial appearance, health-related quality of life, side effects, and care process. Each domain has one or more scales that function independently. For the purpose of this study, and taking into account the condition being treated, psychological functioning scales were selected for subjects to complete at the time points stated in the assessment schedule.

[0139] e. Facial wrinkle treatment satisfaction questionnaire These questions ask the subject to think about the area of ​​their face that was treated in their most recent procedure. The subject selects the answer that best matches how much they agree with the given statement. An exemplary questionnaire is shown below. There is only one answer to each question. [Table 3]

[0140] f. Satisfaction with Wrinkles (SWL) Questionnaire At baseline (pre-treatment) and all post-treatment visits, subjects are asked to complete a validated SWL questionnaire developed by Galderma.

[0141] g. Independent Photo Jury (IPR) Three IPRs, blinded to the subject's randomized treatment, will perform a photographic assessment of each subject's GL at maximum frown using a validated 4-point glabellar severity photo scale. The IPR assessment will be performed on all subjects at the end of the study. The IPR will use the scale to compare photographs of each subject's GL at maximum frown at baseline and each post-treatment visit. The IPR score will be determined as the median of the scores of the three judges. The IPR will not be involved in any other aspect of the study. EXAMPLES

[0142] Example 1 Efficacy and Safety of QM1114-DP for the Treatment of Moderate to Severe Glabellar Folds (Study 43QM1602) A multi-center, randomized, double-blind, placebo-controlled study evaluating the efficacy and safety of QM1114 Drug Product (DP) for the treatment of moderate to severe glabellar wrinkles was conducted in 300 subjects at 10 sites in the United States and Canada. QM1114-DP is an example of a liquid composition of botulinum neurotoxin (e.g., BoNT / A) of the present invention that includes at least four buffering agents as outlined in the description above. Thus, references to QM1114-DP throughout these examples refer to an exemplary "liquid composition containing botulinum neurotoxin" of the present invention. Group 1 (treatment group) consisted of 225 subjects who received a single treatment of QM1114-DP (5 units) at baseline. Group 2 (placebo group) consisted of 75 subjects who received a single placebo treatment at baseline. Efficacy and safety of the treatment were measured at follow-up visits on days 7, 14, and 1, 2, 3, 4, 5, and 6 after administration.

[0143] The primary efficacy endpoint was measured using the "composite responder rate" for glabellar lines. The composite responder rate for glabellar lines is assessed using two rating scales at maximum frown at month 1: the Investigator Live Assessment (ILA) 4-point photographic scale and the Subject Self-Assessment (SSA) static 4-point categorical scale. A "composite responder" refers to a subject who achieves a score of 0 or 1 on glabellar line severity and at the same time an improvement of at least 2 grades from baseline on both the ULA and SSA scales. Figure 2 shows the glabellar line severity scoring matrix.

[0144] The primary efficacy outcome of the study showed that 82.9% of subjects in the treatment group (165 / 199) were combined responders compared with none in the placebo group (0%: 0 / 67). The primary efficacy outcome (combined rate) measured at 1 month was higher than placebo and was statistically significant (p<0.001).

[0145] To analyze secondary efficacy, the ILA responder rate over time was measured. Figure 3 shows the results of the ILA responder rate over time. At every time point, the treatment group showed a significantly higher ILA response rate (p<0.001) compared to the placebo group. See Figure 3.

[0146] Next, an exploratory efficacy analysis was performed using subject Global Aesthetic Improvement Score (GAIS) analysis. In this analysis, responders are subjects who respond as "improved," "much improved," or "very improved" on the subject GAIS. In this example, subject response rates on the GAIS were high (the majority rated improved or better through 6 months). See Figure 4. Subjects in the treatment group reported onset of response as early as day 0. The median onset of response for the treatment group was 2 days.

[0147] Safety: The overall incidence of adverse events (AEs) was comparable to placebo. All serious adverse events (SAEs) were assessed unrelated to study drug or injection treatment. All treatment-emergent adverse events (TEAEs - also referred to as "related adverse events") were mild or moderate in intensity, with the most frequent TEAEs being headache, ptosis, and bruising. See Figure 5. No subjects died or experienced a TEAE leading to early study discontinuation.

[0148] conclusion Efficacy: QM-1114-DP demonstrated statistically significant differences versus placebo in the primary and secondary endpoints in reducing glabellar lines in subjects.

[0149] Onset: Subjects in the treatment group reported noticing a beneficial effect as early as day 0, with a median onset of 2 days.

[0150] Safety: The overall safety of QM-1114-DP was tolerable with a favorable risk / benefit profile. Treatment groups showed very low rates of ptosis (0.9%).

[0151] Example 2 Efficacy and Safety of QM1114-DP for the Treatment of Moderate to Severe Ectocanthal Wrinkles (Study 43QM1901) A multicenter, randomized, double-blind, placebo-controlled study evaluating the efficacy and safety of QM1114-DP for the treatment of moderate to severe epicanthal wrinkles was conducted in 300 subjects at 10 sites in the United States and Canada. Group 1 (treatment group) consisted of 225 subjects who received a single treatment of QM1114-DP (5 units) at baseline. Group 2 (placebo group) consisted of 75 subjects who received a single placebo treatment at baseline. Efficacy and safety of the treatment were measured at follow-up visits at days 7 and 14 and at months 1, 2, 3, 4, 5, and 6 after dosing.

[0152] The primary efficacy endpoint was measured using the "composite responder rate" for lateral canthal wrinkles. The composite responder rate for lateral canthal wrinkles is assessed using two rating scales at maximum smile at month 1: the Investigator Live Assessment (ILA) 4-point photographic scale and the Subject Live Assessment (SLA) static 4-point categorical scale. A "composite responder" refers to a subject who achieves a score of 0 or 1 on the severity of lateral canthal wrinkles and at the same time an improvement of at least 2 grades from baseline on both the ULA and SSA scales. Figure 6 shows the severity scoring matrix for lateral canthal wrinkles.

[0153] The primary efficacy outcome of the study showed that 51.8% of subjects (106 / 204) in the treatment group were combined responders compared with only 1.4% of subjects (1 / 69) in the placebo group. The primary efficacy outcome (combined rate) measured at 1 month was higher than placebo and was statistically significant (p<0.001).

[0154] To analyze secondary efficacy, the ILA responder rate over time was measured. Figure 7 shows the results of the ILA responder rate over time. At every time point, the treatment group showed a significantly higher ILA response rate (p<0.001) compared to the placebo group. See Figure 7.

[0155] Next, an exploratory efficacy analysis was performed using a subject Global Aesthetic Improvement Score (GAIS) analysis. In this analysis, responders are subjects who respond as "improved," "much improved," or "very improved" on the subject GAIS. In this example, the subject responder rate on the GAIS was high (the majority rated improved or better through 6 months). See Figure 8. Subjects in the treatment group reported onset of response as early as day 0. The median onset of response for the treatment group was 2 days.

[0156] Safety: The overall incidence of adverse events (AEs) was comparable to placebo. All serious adverse events (SAEs) were assessed unrelated to study drug or injection treatment. All treatment-emergent adverse events (TEAEs - also referred to as "related adverse events") were mild or moderate in intensity, with injection site bruising being the most frequent TEAE. See Figure 9. No subjects died or experienced a TEAE leading to early study discontinuation.

[0157] conclusion Efficacy: QM-1114-DP demonstrated statistically significant differences versus placebo in the primary and secondary endpoints in reducing subjects' lateral canthal wrinkles.

[0158] Onset: Subjects in the treatment group reported noticing a beneficial effect as early as day 0, with a median onset of 2 days.

[0159] Safety: The overall safety of QM-1114-DP was tolerable with a favorable risk / benefit profile. The most common related AE was injection site bruising (4.8% in the treatment group vs. 4.1% in the placebo group).

[0160] Example 3 Efficacy and Safety of QM1114-DP for the Treatment of Moderate to Severe Canthal and Glabellar Folds (Alone or in Combination) (Study 43QM1902) A multicenter, randomized, double-blind, placebo-controlled study evaluating the efficacy and safety of QM1114-DP for the treatment of moderate to severe lateral canthal wrinkles (LCL) and glabellar wrinkles (GL) (alone or in combination) was conducted in 413 subjects at 12 sites in the United States and Canada. Group A (LCL monotherapy group) consisted of 118 subjects who received a single treatment of QM1114-DP (5 units) in LCL and a single placebo treatment in GL at baseline. Group B (GL monotherapy group) consisted of 118 subjects who received a single treatment of QM1114-DP (5 units) in GL and a single placebo treatment in LCL at baseline. Group C (LCL / GL combination therapy group) consisted of 118 subjects who received a single treatment of QM1114-DP (5 units) in LCL and a single treatment of QM1114-DP (5 units) in GL at baseline. Finally, group D (placebo group) consisted of 59 subjects who received a single treatment of placebo for LCL and a single treatment of placebo for GL at baseline. Efficacy and safety of treatment were measured at follow-up visits on days 7 and 14 and at months 1, 2, 3, 4, 5, and 6 after administration.

[0161] The primary efficacy endpoint for glabellar lines was measured using the "composite responder rate" for glabellar lines. The composite responder rate for glabellar lines is assessed using two rating scales at maximum frown at month 1: the Investigator Live Assessment (ILA) 4-point photographic scale and the Subject Self-Assessment (SSA) static 4-point categorical scale. A "composite responder" refers to a subject who achieves a score of 0 or 1 on glabellar line severity and at the same time an improvement of at least 2 grades from baseline on both the ULA and SSA scales. Figure 2 shows the glabellar line severity scoring matrix.

[0162] The primary efficacy outcome measure of glabellar lines showed that 71.1% of subjects (75 / 106) in the GL monotherapy group were combined responders, 72% of subjects (78 / 108) in the LCL / GL combination therapy group were combined responders, and none of the placebo group subjects (0%: 0 / 55) were combined responders. The primary efficacy outcome measure at 1 month (combined rate) was higher than placebo, with statistically significant results (p<0.001).

[0163] The primary efficacy endpoint for lateral canthal wrinkles was measured using the "composite responder rate" for lateral canthal wrinkles. The composite responder rate for lateral canthal wrinkles is assessed using two rating scales at maximum smile at month 1: the Investigator Live Assessment (ILA) 4-point photographic scale and the Subject Live Assessment (SLA) static 4-point categorical scale. A "composite responder" refers to a subject who achieves a score of 0 or 1 on the severity of lateral canthal wrinkles and at the same time an improvement of at least 2 grades from baseline on both the ULA and SSA scales. Figure 6 shows the severity scoring matrix for lateral canthal wrinkles.

[0164] The primary efficacy outcome measure of lateral canthal wrinkles showed that 45.1% of subjects (53 / 117) in the LCL monotherapy group were combined responders, 55% of subjects (59 / 108) in the LCL / GL combination therapy group were combined responders, and none of the placebo subjects (0%: 0 / 55) were combined responders. The primary efficacy outcome measure at 1 month (combined rate) was higher than placebo, with statistically significant results (p<0.001).

[0165] To analyze the secondary efficacy of glabellar lines, the ILA responder rate over time was measured. Figure 10 shows the results of the ILA responder rate of glabellar lines over time. At every time point, the treatment groups (both the GL monotherapy group and the GL / LCL combination therapy group) showed significantly higher ILA response rates (p<0.001) compared to the placebo group. See Figure 10.

[0166] To analyze the secondary efficacy of lateral canthal wrinkles, the ILA responder rate over time was measured. Figure 11 shows the results of the ILA responder rate over time for lateral canthal wrinkles. At every time point, the treatment groups (both the LCL monotherapy group and the GL / LCL combination therapy group) showed significantly higher ILA response rates (p<0.001) compared to the placebo group. See Figure 11.

[0167] Next, an exploratory efficacy analysis was performed using subject Global Aesthetic Improvement Score (GAIS) analysis. In this analysis, responders were subjects who responded as "improved," "much improved," or "very improved" on the subject GAIS. In this example, both GL and LCL subject responder rates on the GAIS were higher compared to placebo controls (most rated improved or better through 5 months). See Figure 12. Subjects in the treatment group reported onset of response as early as day 0. Median onset of response for the treatment group was 2-4 days.

[0168] Safety: The overall incidence of adverse events (AEs) was comparable to placebo. All serious adverse events (SAEs) were assessed independent of study drug or injection procedure. All treatment-emergent adverse events (TEAEs - also referred to as "related adverse events") were mild or moderate in intensity, with the most frequent TEAEs being headache (4.3%), injection site bruising (2.6%), and ptosis (1.7%, only seen in the GL monotherapy group). No subjects died or experienced a TEAE leading to early study discontinuation.

[0169] Example 4 Comparison of the Effects of QM-114-DP and Other Botulinum Toxins on Glabellar Lines In a comparative study, QM-1114-DP was compared to other available botulinum toxin products.

[0170] Composite 2-grade improvement in glabellar lines: QM-1114-DP demonstrated superior composite 2-grade improvement in glabellar lines at week 4 (day 30) compared to Daxi40U (daxibotulinumtoxinA) and Jeuveau EV-002 (prabotulinumtoxinA). Briefly, QM-1114-DP demonstrated approximately 82.9% composite 2-grade improvement in glabellar lines compared to 73.8% for Daxi40U and 70.4% for Jeuveau EV-002. See FIG. 13.

[0171] Glabellar Investigator Scores: QM-1114-DP demonstrated comparable sustained investigator scores of none to mild glabellar lines up to 24 weeks (150 days) when compared to Daxi40U and Botox. See Figure 14.

[0172] Composite 2-grade improvement in lateral canthal wrinkles: QM-1114-DP demonstrated superior composite 2-grade improvement in lateral canthal wrinkles at Week 4 (Day 30) compared to Botox. See Figure 15.

[0173] Glabellar Investigator Score: QM-1114-DP showed comparable sustained investigator scores of none to mild for glabellar lines up to 24 weeks (150 days) when compared to Daxi40U and Botox (onabotulinumtoxinA).Interestingly, it was noted that no subjects with Daxi40U and Botox had investigator scores of none or mild after 4 weeks.See Figure 16.

[0174] Comparison of enzyme activity: Enzyme activity was measured using BoTest TM (See Figures 17 and 18.) TM Assay (BOTEST TMFor the Matrix A Botulinum Neurotoxin Detection Kit (catalog number A1015 from BioSentinel), a common buffer was used for all toxins to eliminate any effects from the excipients and look only at the toxins. Briefly, samples were dispensed into microtiter wells and matrix binding buffer was added to each sample. Resuspended Matrix A beads were dispensed into each well containing sample and the plate was incubated. Matrix beads were washed with Matrix Wash Buffer. The wash buffer was removed and the wells were incubated with BoTest containing DTT. TM The reaction buffer was replaced with BoTest TM After addition of the master stock, the plate was covered and incubated, followed by a fluorescent read. The enzyme activity patterns observed appeared very similar in both assays.

[0175] In Figure 18, normalized values ​​(i.e., specific activity) for each product were calculated by dividing the activity (U / ml) by the amount of BoNT in the formulation as determined using a BoNT-A specific ELISA (BOLISA®).

[0176] The enzyme activity experiments were repeated twice and the results are shown in the table below. [Table 4] [Table 5]

[0177] Comparison of relative potency: We also analyzed how much toxin protein is present in QM1114-DP and competitors using a cell-based assay in which hiPSCs are evaluated for BoNT / A-dependent intracellular increase of cleaved SNAP25 detected via Western blot. A common buffer was used in the CBA test. The amount of Botulinum neurotoxin type A was determined using a BoNT-A specific ELISA (also using a common buffer). When the activity and potency are normalized (by dividing the activity and potency by the amount of toxin), it can be seen that QM1114-DP has the best potency per toxin molecule. Therefore, QM1114-DP is a more potent toxin than the other toxins (e.g., 90% better than Botox). See Figure 19.

[0178] In Figure 19, relative potency was determined using a cell-based assay performed in common buffer and normalized for the protein amount of each product. U / pg was calculated by dividing the relative potency by the amount of BoNT in the formulation as determined using a BoNT-A specific ELISA (BOLISA®).

[0179] conclusion QM1114-DP is superior to other botulinum toxins (e.g., Daxi40U, Jeuveau EV-002, and Botox). In particular, QM1114-DP outperformed both Xeomin and Botox in terms of efficacy and activity. For example, QM1114-DP has a better responder rate for treating LCL and GL than other botulinum toxins. QM1114-DP is also less likely to cause adverse side effects such as ptosis or causes less severe ptosis than other botulinum toxins. Because the toxin activity per unit (IU) is higher for QM1114-DP, the probability or severity of any potential adverse events may be minimized since less QM1114-DP can be used to obtain the same degree of treatment. Finally, QM1114-DP has a faster onset and longer duration of effect than other tested toxins.

[0180] Example 5 QM1114-DP is pure and complex-free QM-1114-DP was analyzed for protein purity using ultra-performance liquid chromatography size-exclusion chromatography (UPLC-SEC) and SDS-PAGE. UPLC-SEC analysis showed that QM-1114-DP was purified using Water BEH TM It was shown that the proteins were separated on a SEC column and analyzed using absorbance (A280). SDS-PAGE was performed on both reduced and non-reduced material and stained with colloidal Coomassie, allowing for quantification of impurities.

[0181] As shown in Figures 20 and 21, the QM-1114-DP preparations were pure and complex-free, with BoNT-A exhibiting only the expected bands observed in SDS-PAGE analysis.

[0182] Example 6 Treatment of Lateral Canthal Lines (LCL) QM-1114-DP was shown to have a high composite grade ≥2 responder rate (83%) for the treatment of glabellar lines at Month 1, with a duration of response of up to 6 months and onset in more than one-third of subjects at Day 1. Therefore, further studies were performed to evaluate a single dose of 60 U of QM-1114-DP compared to placebo for the treatment of moderate to severe LCL.

[0183] Subjects (N=303) with moderate to severe LCL were randomized 3:1 to QM-1114-DP60U or placebo. LCL severity was assessed by the LCL Investigator Live Assessment (LCL-ILA) 4-point photo scale, the LCL Subject Live Assessment (LCL-SLA) 4-point photo scale, and subject-reported response onset, subject satisfaction. Adverse events were also tracked. The primary endpoint of the study was the rate of composite grade 2 responders assessed using the LCL-ILA and LCL-SLA at maximum smile at month 1. Subjects who achieved a score of none (0) or mild (1) on LCL severity and had at least a 2-grade improvement from baseline on both the LCL-ILA and LCL-SLA scales at maximum smile were defined as composite grade 2 responders.

[0184] The LCL composite grade 2 responder rate was higher with relaBoNT-A versus placebo (51.8% vs. 1.4%, P<0.001) (Figure 22). Median time to onset of response was 2 days, with 34% of subjects reporting onset on day 1 (Figure 23). Investigators reported none or mild severity in 88% of subjects at 1 month, with response maintained in 23% at 6 months (Figure 24). Investigators reported ≥1 grade improvement in 93% of subjects at 1 month, with response maintained in 36% at 6 months (Figure 25).

[0185] In addition, one exploratory endpoint was the time to return to baseline. This endpoint was evaluated in subjects who achieved a score of 0 or 1 on both the LCL-ILA 4-point photo scale and the LCL-SLA 4-point photo scale during maximum smile. As shown in Figure 26, the median time to return to baseline simultaneously on ILA and SLA was 173 days (24.7 weeks). Thus, QM-1114-DP provides a long-lasting treatment for LCL.

Claims

1. 1. A liquid composition comprising a botulinum neurotoxin for use in a method for treating glabellar lines (GL), lateral canthal lines (LCL), or a combination thereof in a human subject, comprising: (i) the method comprises administering to the subject a therapeutically effective amount of the liquid composition, thereby reducing the appearance of GL, LCL, or a combination thereof, wherein the liquid composition has an activity of at least about 97 BU / ml or greater; or (ii) the human subject has a low risk of ptosis, and the method comprises administering to the subject a therapeutically effective amount of the liquid composition, thereby reducing the appearance of GL, LCL, or a combination thereof, and the botulinum neurotoxin is less likely to cause ptosis than botulinum neurotoxin products with lower enzymatic activity or lower specific activity. liquid composition.

2. The liquid composition of claim 1, wherein the subject experiences no serious adverse events.

3. 3. The liquid composition of claim 1 or 2, wherein the subject is less likely to experience ptosis compared to treatment with a botulinum neurotoxin product selected from BOTOX COSMETIC®, XEOMIN®, and JEUVEAU®.

4. 3. The liquid composition of claim 1, wherein the botulinum neurotoxin product with lower enzymatic activity is selected from BOTOX COSMETIC®, XEOMIN®, and JEUVEAU®.

5. The liquid composition contains about 2.0 x 10 ８ having a BoNT specific activity in U / mg of total protein, said specific activity (U / mg) being optionally measured using the mouse LD50 potency in U / ml divided by the total amount of protein (mg / ml) as determined by the μBCA method; The liquid composition has an enzymatic activity normalized to a botulinum neurotoxin concentration of at least about 1.10 BU / RBU or at least about 0.17 BU / pg, and the enzymatic activity of the botulinum neurotoxin is measured by a BOTEST ＴＭ and / or the liquid composition has a relative potency, normalized to a botulinum neurotoxin concentration, of at least about 1.35 CBpA units / BoNT, wherein the enzymatic activity of the botulinum neurotoxin is determined using a cell-based assay. The liquid composition according to claim 1 or 2.

6. the liquid composition does not contain excipients of human or animal origin, and / or Contains no animal or companion proteins The liquid composition according to claim 1 or 2.

7. 3. The liquid composition of claim 1 or 2, wherein the incidence of ptosis is less than 2%, less than 1.9%, less than 1.8%, less than 1.7%, less than 1.5%, less than 1.4%, less than 1.3%, less than 1.2%, less than 1.1%, less than 1.0%, or as low as 0.9%.

8. The liquid composition of claim 1 or 2, wherein the subject does not experience ptosis.

9. 3. The liquid composition of claim 1 or 2, wherein GL, LCL, or a combination thereof is moderate to severe, or severe or very severe.

10. The subject has a combination of GL and LCL; The treatment provides a higher response rate than BOTOX COSMETIC®; and / or The liquid composition comprises at least four buffering agents; The liquid composition according to claim 1 or 2.

11. 3. The liquid composition of claim 1, wherein the liquid composition comprises a first buffering agent present at a concentration of about 100 to about 300 mM or about 0.1 to 10 mg / mL.

12. 12. The liquid composition of claim 11, wherein the liquid composition comprises a second buffer, a third buffer, a fourth buffer, and / or a fifth buffer present at a concentration of about 1 to about 25 mM or about 0.1 to 1.0 mg / mL.

13. the buffering agent is selected from the group consisting of sodium chloride, potassium chloride, sodium phosphate, potassium phosphate, disodium hydrogen phosphate dihydrate, and sodium dihydrogen phosphate dihydrate; and / or The liquid composition comprises at least one stabilizer, which may be an amino acid. The liquid composition of claim 10.

14. The amino acid is selected from the group consisting of alanine, valine, leucine, isoleucine, methionine, phenylalanine, tyrosine, and tryptophan; the amino acid is a D-isoform or an L-isoform; and / or 14. The liquid composition of claim 13, wherein the amino acid is present at a concentration of about 0.1 to about 3.0 mg / mL.

15. 3. The liquid composition of claim 1, wherein the liquid composition comprises at least one surfactant, which may be a non-ionic surfactant.

16. 16. The liquid composition of claim 15, wherein the non-ionic surfactant is present at a concentration of about 0.01% (v / v) to about 5.0% (v / v) or about 0.1 to about 3.0 mg / mL.

17. 3. The liquid composition according to claim 1, wherein the botulinum neurotoxin is selected from the group consisting of botulinum neurotoxin types A, B, C, D, E, F, and G.

18. The botulinum neurotoxin is botulinum neurotoxin type A; The pH of the liquid composition is 6.6 to 6.9; The botulinum neurotoxin has a molecular weight of about 150 kDa; The osmotic pressure of the liquid composition is 270 mosm / kg to 310 mosm / kg; 1 to 100 units of botulinum toxin are administered to the subject; and / or 10, 25, 30, 45, 50, 60, 75, or 90 units of botulinum toxin are administered to the subject; The liquid composition according to claim 1 or 2.

19. 3. The liquid composition of claim 1, wherein the liquid composition is administered by injection.

20. The injection may be subcutaneous, transdermal, intradermal, or intramuscular; The subject receives multiple injections in the glabellar area; adjacent injection sites are about 0.5 to about 10 cm apart; and / or adjacent injection sites are about 1.5 to about 3 cm apart; 20. The liquid composition of claim 19.

21. The injections are made into the procerus and corrugator supercilii muscles on one or both sides of the face; and / or 20. The liquid composition of claim 19, wherein the injections are made first into the procerus muscle, followed by the corrugator supercilii muscles on one or both sides of the face, moving from the midline outwards.

22. All of the above injections are given approximately 1 cm above the supraorbital rim, medial to the mid-pupillary line; and / or 20. The liquid composition of claim 19, wherein all of said injections are made in the center of the eyebrow or at least 1 cm above the bony supraorbital ridge.

23. The subject receives multiple injections below the lateral canthus, into the lateral portion of the orbicularis oculi muscle, and / or 1-2 cm from the orbital rim; The subject receives multiple additional injections below the lateral canthus, into the lateral portion of the orbicularis oculi muscle, and / or 1-2 cm from the orbital rim; repeating the method at intervals of about 1 month to about 6 months to inhibit recurrence of GL, LCL, or a combination thereof; repeating the above method at intervals of about 3 months to about 6 months; and / or Repeat the above procedure at intervals of about 4 months.

20. The liquid composition of claim 19.

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