Neurotoxin compositions with increased potency and duration of effect.

Abstract

Disclosed herein are neurotoxin compositions and methods that result in increased efficacy and extended duration of effect.

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 402,609, filed August 31, 2022, the entire contents of which are incorporated herein by reference.

[0002] (Technical field) The present disclosure relates to the formulation and use of neurotoxin compositions with increased duration and efficacy. [Background technology]

[0003] Clostridial neurotoxins are a family of closely related bacterial protein toxins, consisting of several antigenically distinct botulinum neurotoxins (BoNT) and tetanus neurotoxins (TeNT). BoNTs are secreted by C. botulinum in the form of multimeric complexes with a set of non-toxic proteins encoded by genes adjacent to the neurotoxin gene. These protein complexes range in size from 300 to 900 kDa. These large protein complexes consist of a 150 kDa neurotoxin moiety and a set of complexing proteins composed of non-toxic nonhemagglutinin proteins (or neurotoxin-binding proteins, NBPs) and several hemagglutinin proteins. These are known as neurotoxin-associated proteins (NAPs), also known as "complexing" or "accessory" proteins. Stabilized through noncovalent interactions, NAPs account for approximately 70% of the total mass.

[0004] In addition to type A BoNT, there are other serologically distinct types, serotypes B, C, D, E, F, and G, also produced by the Gram-positive bacterium Clostridium botulinum, each of which is distinguished by neutralizing type-specific antibodies.

[0005] BoNT induces muscle paralysis. The 150 kDa component has a light chain and a heavy chain linked by a disulfide bond. The toxin is internalized into neurons at the neuromuscular junction via the heavy chain. Once inside the cell, a reduction step occurs between the heavy and light chains, followed by cleavage of the light chain portion of the 150 kDa component by SNAP25, an anchor protein attached to the cell membrane. This cleavage prevents the effective docking of vesicles containing acetylcholine (Ach), a step required for the release of Ach at the neuromuscular junction to initiate muscle contraction. Without this release, the muscle cannot contract and remains flaccid. Botulinum toxins were initially identified as the source of food poisoning outbreaks.

[0006] The role of the accessory proteins is thought to be to protect the active toxin (the 150 kDa component) as it travels through the highly acidic environment of the stomach (botulinum infection is typically foodborne). Therefore, some theories posit that the accessory proteins are either useless or problematic upon administration, potentially eliciting an immune response, for example. It is also believed that once in solution, the accessory proteins rapidly dissociate into the 150 kDa molecule, rendering them therapeutically worthless. Neurotoxin suppliers are often required to state the "specific activity" of their BoNT products, a calculation that assumes the 150 kDa component is the only active component in BoNT.

[0007] BoNTs provide an effective means for treating both cosmetic and therapeutic conditions. However, limitations in efficacy and duration of effect can require higher doses and more frequent administration. Thus, compositions with increased efficacy and duration of effect are desirable. Summary of the Invention [Means for solving the problem]

[0008] The methods and compositions of the present disclosure provide increased efficacy and duration of effect compared to current botulinum neurotoxins, and also provide for their use in therapeutic and cosmetic applications.

[0009] The methods of the disclosure include administration of at least one clostridial neurotoxin, e.g., a BoNT, augmented with (or combined with) at least one additional NBP or NAP, e.g., an accessory protein such as Hn-17, Hn-33, or Hn-70, wherein the resulting amount of NBP or NAP is greater than the amount typically associated with the clostridial neurotoxin.

[0010] The methods of the disclosure include administration of at least one Clostridial neurotoxin, eg, a BoNT, in combination with at least one vasoconstrictor, eg, epinephrine.

[0011] The methods of the disclosure include administration of at least one clostridial neurotoxin, e.g., a BoNT, in combination with a vasoconstrictor and at least one NBP or NAP, e.g., an accessory protein such as Hn-17, Hn-33, or Hn-70, wherein the amount of NBP or NAP is greater than the amount typically associated with the clostridial neurotoxin.

[0012] The methods of the disclosure include combining a formulation comprising an accessory protein with a neurotoxin formulation prior to administration.

[0013] The methods of the present disclosure include combining a formulation comprising a vasoconstrictor with a neurotoxin formulation prior to administration.

[0014] The methods of the disclosure include combining a formulation comprising an accessory protein and a formulation comprising a vasoconstrictor with a neurotoxin formulation prior to administration.

[0015] The compositions of the present disclosure comprise at least one clostridial neurotoxin, e.g., a BoNT such as BoNT / A, augmented with (or combined with) at least one additional NBP or NAP, e.g., an accessory protein such as Hn-17, Hn-33, or Hn-70, wherein the resulting amount of NBP or NAP is greater than the amount typically associated with the clostridial neurotoxin.

[0016] The compositions of the present disclosure comprise at least one clostridial neurotoxin, e.g., a BoNT such as BoNT / A, combined with an additional active agent, such as a vasoconstrictor, and at least one NBP or NAP, e.g., an accessory protein, such as Hn-17, Hn-33, or Hn-70, wherein the amount of NBP or NAP is greater than that typically associated with the clostridial neurotoxin. [Brief explanation of the drawings]

[0017] [Figure 1] 1 shows exemplary injection sites (100, 120, 140) for the treatment of glabellar lines as disclosed herein. [Figure 2] Shown are the mean DAS scores for both paws for all animals in the control cohort plotted against each day. Error bars represent the standard deviation of all measurements. [Figure 3] Shown is the mean DAS score for both paws for all animals in the test cohort plotted against each day. Error bars represent the standard deviation of all measurements. All mice died on day 20. DETAILED DESCRIPTION OF THE INVENTION

[0018] The "potency" of BoNT refers to the amount of activity at a given time point. In clinical studies, the primary measurement is usually 30 days after administration and is measured by the number of responders at that time point. Potency can be measured using various assays, such as the amount of SNAP25 cleavage at a given time point after exposure to BoNT. There is a correlation between the degree of effect and the corresponding amount of BoNT used. The more toxin used, the greater the effect, until a saturation point is reached.

[0019] Duration of effect is a different concept and measures the duration of a given response or effect. Typically, it is measured from the onset of the effect to its disappearance. Duration of effect is thought to be a function of the time required for new SNAP25 complexes to form within the cell to facilitate attachment to ACh-containing vesicles and release of ACh into neural junctions.

[0020] Therefore, the difference between efficacy and durability can be summarized as follows: a. Efficacy - How many responders are there at any given time? i. For example, 1 point improvement - 85% at 1 hour b. Duration - how long does the reaction last? i. For example, a 1-point improvement lasts for 2 weeks

[0021] Duration of effect for multiple neurotoxin administrations is typically 3-4 months and may vary by indication, but for the glabellar area it is typically 3-4 months, and bladder treatment may provide longer duration of effect.

[0022] The degree of durability of response is also a function of how it is measured. Typically, a Kaplan-Meier analysis is performed, and durability is a function of when the response began and ended. End can be defined by the median, the time point at which less than 50% are responders. Some manufacturers define durability as when patients return to baseline, but this is not common practice in pharmaceuticals.

[0023] The present disclosure is directed to methods and compositions for increasing the duration of efficacy of a Clostridial therapy, e.g., a BoNT therapy, compared to that achieved with current methods. For example, in embodiments, the duration of efficacy of a Clostridial neurotoxin therapy, e.g., a BoNT therapy, can be increased by, e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more compared to that achieved with current methods. The therapy can be applied, for example, to achieve a therapeutic or cosmetic effect.

[0024] The present disclosure is further directed to methods and compositions for increasing the efficacy of clostridial therapy, e.g., BoNT therapy, compared to that achieved with current methods. For example, in embodiments, the efficacy of clostridial neurotoxin therapy, e.g., BoNT therapy, can be increased by, e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more compared to that achieved with current methods. The therapy can be applied, for example, to achieve a therapeutic or cosmetic effect.

[0025] In embodiments, compositions of the present disclosure include a neurotoxin and an increased amount of at least one NBP or NAP, e.g., compared to the amount normally associated with BoNT. For example, each BOTOX® 900 kDa BoNT / A complex contains (or is associated with) three Hn-33 molecules. Embodiments of the present disclosure include enhancing the composition with additional NBPs or NAPs, e.g., Hn-33 molecules, thereby increasing the ratio of Hn-33 molecules relative to the number normally associated with the 900 kDa complex. The addition of an NBP or NAP, such as an accessory protein, can result in a formulation with increased efficacy, duration of effect, or both, e.g., by increasing BoNT uptake into cells. In embodiments, the amount of accessory protein normally associated with the 900 kDa complex is the amount present in the native form of the 900 kDa complex.

[0026] Further embodiments include compositions comprising at least one vasoconstrictor and a neurotoxin, such as BoNT. For example, this addition may aid in the cellular uptake of both the Hn-33 molecule and the 900 kDa complex by reducing vasoactivity in the area of ​​the neuromuscular junction, thereby allowing for greater uptake and increasing the duration of paralysis. The compositions of the present disclosure that include a vasoconstrictor may further include an increased amount of NBP or NAP compared to a standard or naturally occurring neurotoxin.

[0027] Further embodiments include methods of using the compositions of the present disclosure.

[0028] Further embodiments include methods of making the compositions of the present disclosure.

[0029] (definition) "Administering" means giving (i.e., administering) a pharmaceutical composition or active ingredient to a subject. The pharmaceutical compositions disclosed herein can be administered via a number of suitable routes, including intramuscular or subcutaneous routes, such as by injection (using a needle or needle-free system), topical administration, or by use of an implant.

[0030] "Botulinum toxin" or "botulinum neurotoxin" or "BoNT" refers to a neurotoxin derived from Clostridium botulinum, as well as modified, recombinant, hybrid, and chimeric BoNTs. Recombinant botulinum toxins may have light and / or heavy chains produced by recombination with non-clostridial species. As used herein, "botulinum toxin" encompasses BoNT serotypes A, B, C, D, E, F, G, and H. As used herein, "botulinum toxin" encompasses both BoNT complexes (i.e., 300, 600, and 900 kDa complexes) and pure BoNTs (i.e., approximately 150 kDa neurotoxin molecules), all of which are useful in practicing embodiments of the present disclosure. "Botulinum neurotoxin" also encompasses functional (i.e., biologically active) isoforms, homologs, orthologs, paralogs, and fragments of BoNT that exhibit at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, and no more than 60%, no more than 70%, no more than 80%, no more than 90%, or no more than 99% sequence identity with the amino acid sequence of wild-type BoNT, such as wild-type BoNT / A or the neurotoxic component of serotype A1 BoNT deposited in the GenBank database under accession number AAA23262.

[0031] "Clostridial neurotoxins" refers to neurotoxins produced by or native to Clostridial bacteria, such as Clostridium botulinum, Clostridium butyricum, or Clostridium beratti, as well as clostridial neurotoxins produced by recombinant means with non-clostridial species.

[0032] As used herein, "fast-acting" refers to a BoNT that provides an effect in a patient more quickly than, for example, one produced from BoNT / A. For example, the effect of a fast-acting BoNT (such as BoNT / E) can be provided within 36 hours.

[0033] As used herein, "rapid-recovery" refers to a BoNT whose effects in a patient decrease more quickly than those produced, for example, by BoNT / A. For example, the effects of a rapid-recovery BoNT (such as BoNT / E) may decrease within, for example, 120 hours, 150 hours, 300 hours, 350 hours, 400 hours, 500 hours, 600 hours, 700 hours, 800 hours, etc. However, the usual duration of intramuscular injection of BoNT / A is typically about 3 to 4 months.

[0034] "Neuromodulator" means a biologically active molecule that has specific affinity for a nerve cell surface receptor. "Neuromodulator" includes clostridial toxins both as pure toxins and those complexed with one or more non-toxic toxin-associated proteins.

[0035] "Neurotoxin" means a biologically active molecule that has specific affinity for a nerve cell surface receptor. "Neurotoxin" includes clostridial toxins both as pure toxins and those complexed with one or more non-toxic toxin-associated proteins.

[0036] "Patient" means a human or non-human subject receiving medical or veterinary care.

[0037] A "pharmaceutical composition" refers to a formulation in which the active ingredient may be a Clostridial toxin. The term "formulation" refers to the presence of at least one additional ingredient in a pharmaceutical composition in addition to the BoNT active ingredient, such as, but not limited to, albumin (human serum albumin or recombinant human albumin) and / or sodium chloride. Thus, a pharmaceutical composition is a preparation suitable for clinical, therapeutic, or cosmetic administration to a subject, such as a human or animal patient. A pharmaceutical composition may be in a lyophilized or vacuum-dried state, a solution formed after reconstitution of a lyophilized or vacuum-dried pharmaceutical composition with saline or water, or may exist as a solution that does not require reconstitution. As noted above, a pharmaceutical composition may be liquid, semisolid, or solid. A pharmaceutical composition may be free of animal proteins.

[0038] "Purified BoNT" refers to a pure BoNT or BoNT complex that has been isolated or substantially isolated from other proteins or impurities that may accompany the BoNT when obtained from a culture or fermentation process. Thus, purified BoNT can be at least 95%, and more preferably at least 99%, free of non-BoNT proteins and impurities.

[0039] "Therapeutic formulation" means a formulation that can be used to treat and alleviate a disorder or disease and / or symptoms associated therewith.

[0040] A "therapeutically effective amount" or "cosmetically effective amount" means the level, amount, or concentration of an agent (e.g., a Clostridial toxin or an agent as a pharmaceutical composition comprising a Clostridial toxin) needed to treat a disease, disorder, or condition without causing significant negative or adverse side effects.

[0041] "Treatment" means the alleviation or suppression (including partial suppression, significant suppression, near complete suppression, and complete suppression), amelioration, or prevention (either temporary or permanent), of a symptom, disease, disorder, or condition to achieve a desired therapeutic or cosmetic result, such as by healing an injury or damaged tissue, or by altering, altering, enhancing, ameliorating, mitigating, and / or beautifying an existing or recognized symptom, disease, disorder, or condition.

[0042] "Unit" or "U" refers to the amount of active BoNT normalized to have the neuromuscular blocking effect equivalent to one unit of commercially available BoNT / A (e.g., onabotulinumtoxin A (BOTOX®)).

[0043] (Neurotoxin Composition) Embodiments disclosed herein include neurotoxin compositions. Such neurotoxins may be formulated in any pharmaceutically acceptable form and as any pharmaceutically acceptable formulation. Neurotoxins may be used in any pharmaceutically acceptable form supplied by any manufacturer. Embodiments of the present disclosure include the use of Clostridial neurotoxins.

[0044] The clostridial neurotoxin can be produced by a clostridial bacterium, such as Clostridium botulinum, Clostridium butyricum, or Clostridium beratti. Additionally, the neurotoxin can be a modified neurotoxin, which is a neurotoxin in which at least one amino acid has been deleted, modified, or substituted compared to a native or wild-type neurotoxin. Furthermore, the neurotoxin can be a recombinantly produced neurotoxin, or a derivative or fragment thereof.

[0045] The compositions of the present disclosure comprise at least one clostridial neurotoxin, e.g., a BoNT, augmented with at least one additional NBP or NAP, e.g., an accessory protein, wherein the amount of NBP or NAP is greater than that typically associated with the clostridial neurotoxin.

[0046] In embodiments, the accessory protein includes Hn-33. In embodiments, the amount of accessory protein can be increased by, for example, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 120%, 140%, 160%, 180%, 200%, 250%, 300%, or more compared to the normal amount naturally associated with the neurotoxin. In embodiments, the accessory protein can be combined with a different neurotoxin than that with which it is normally associated. For example, in embodiments including BoNT / E, BoNT / A Hn-33 can be added to the formulation.

[0047] In embodiments, the accessory protein comprises Hn-33. In embodiments, the amount of accessory protein can be increased, for example, by 0.5 molar amounts (0.5M), 1M, 1.5M, 2M, 2.5M, 3M, or more, relative to the normal amount naturally associated with the neurotoxin. In embodiments, the accessory protein can be combined with a different neurotoxin than that with which it is normally associated. For example, in embodiments including BoNT / E, BoNT / A Hn-33 can be added to the formulation.

[0048] Embodiments of the present disclosure include formulations comprising a neurotoxin, e.g., a Clostridial neurotoxin, such as BoNT, enhanced with at least one additional accessory protein. For example, each BoNT / A typically associates with three Hn-33 molecules. Thus, embodiments of the present disclosure comprising enhanced amounts of accessory protein can include formulations in which the ratio of Hn-33 to toxin molecules is greater than 3. In embodiments, the relative molar mixture ratio of neurotoxin to NAP is, for example, 1:1, 1:3, 1:5, 1:10, etc.

[0049] The compositions of the present disclosure include at least one Clostridial neurotoxin, e.g., BoNT, in combination with at least one vasoconstrictor, e.g., epinephrine. In further embodiments, the vasoconstrictor may include at least one of an alpha-adrenoceptor agonist, a vasopressin analog, norepinephrine, phenylephrine (Sudafed PE), dopamine, dobutamine, a migraine and headache medication (a serotonin (5-hydroxytryptamine) agonist or a triptan), and the like.

[0050] The compositions of the present disclosure comprise a vasoconstrictor and at least one clostridial neurotoxin, e.g., a BoNT, in combination with at least one additional NBP or NAP, e.g., an accessory protein, wherein the amount of NBP or NAP is greater than that typically associated with the clostridial neurotoxin.

[0051] In embodiments of the present disclosure, the neurotoxin is formulated in unit dosage form, for example, provided as a sterile liquid in a vial, or as a vial or sachet containing a lyophilized powder for reconstitution with a suitable solvent, such as saline, for injection. In embodiments including a vasoconstrictor, the vasoconstrictor can be added to the BoNT composition prior to use.

[0052] In one embodiment, BoNT / A is formulated in a solution containing pasteurized human serum albumin (HSA), which stabilizes the toxin and prevents loss through nonspecific absorption, and saline. This solution can be sterile filtered (0.2 μm filter), filled into individual vials, and then vacuum-dried to obtain a sterile, lyophilized powder. This powder can be reconstituted at the time of use by adding preservative-free sterile saline (0.9% sodium chloride for injection). In one embodiment, BoNT / A is supplied as a sterile solution for injection in a 5 mL vial at a nominal concentration of 20 ng / mL in a solution of 0.03 M sodium phosphate, 0.12 M sodium chloride, and 1 mg / mL HSA, pH 6.0.

[0053] Although a composition may contain only one neurotoxin, such as BoNT / A, the compositions of the present disclosure may contain two or more neurotoxins, which may provide an enhanced therapeutic effect for a disorder. For example, a composition administered to a patient may contain BoNT / A and BoNT / E. Administering a single composition containing two different neurotoxins allows for the effective concentration of each neurotoxin to be lower than when a single neurotoxin is administered to a patient, while still achieving the desired therapeutic effect. This type of "combination" composition may also provide the benefits of both neurotoxins, for example, a more rapid effect combined with a longer duration.

[0054] The compositions administered to a patient may also contain other pharmaceutically active ingredients, such as protein receptor or ion channel modulators, in combination with one or more neurotoxins. These modulators may contribute to the reduction of neurotransmission between various nerves. For example, the compositions may contain gamma-aminobutyric acid (GABA) type A receptor-mediated inhibitors, such as GABA receptors. A It may contain GABA receptor modulators. A The receptors inhibit neuronal activity by effectively blocking electrical current across the cell membrane. A Receptor modulators include GABA A It may enhance the inhibitory effect of GABA receptors, reducing electrical or chemical signal transmission from nerves. A Examples of receptor modulators include benzodiazepines such as diazepam, oxaxepam, lorazepam, prazepam, alprazolam, halazeapam, cordiazepoxide, and clorazepate. The composition may also include a glutamate receptor modulator that reduces the excitatory effects mediated by glutamate receptors. Examples of glutamate receptor modulators include agents that inhibit currents mediated by AMPA-type, NMDA-type, and / or kainate-type glutamate receptors.

[0055] (How to use) The neurotoxin compositions of the present disclosure can be injected into a patient using a needle or a needle-free device. In certain embodiments, the method includes injecting the composition into an individual via subdermal, subcutaneous, intramuscular, or superficial intramuscular injection. For example, administering includes injecting the composition through a 27-gauge needle, a 28-gauge needle, a 29-gauge needle, a 30-gauge needle, a 31-gauge needle, a 32-gauge needle, and / or a 33-gauge needle. In some embodiments, the method includes administering a composition comprising a botulinum toxin type A.

[0056] Administration of the compositions of the present disclosure can be carried out by any suitable means, for example, by syringe, catheter, needle, and other injection means. Injection can be into any area of ​​the mammalian body requiring treatment. Injection can be into any specific area, such as the epidermis, dermis, fat, muscle, nerve junction, or subcutaneous layer. Embodiments of the present disclosure can include avoiding injection into specific areas, for example, avoiding injection into at least one or both of the frontalis muscle region and the cervical paraspinal muscle region.

[0057] The methods of the present disclosure include administering at least one clostridial neurotoxin, e.g., BoNT, augmented with at least one additional NBP or NAP, e.g., an accessory protein, where the amount of NBP or NAP is greater than that typically associated with the clostridial neurotoxin. For example, each BoNT / A typically associates with three Hn-33 molecules. Thus, embodiments of the present disclosure may include formulations in which the ratio of Hn-33 to toxin molecules is greater than 3. In embodiments, the relative molar mixture ratio of neurotoxin to NAP is, for example, 1:1, 1:3, 1:5, 1:10, etc.

[0058] The embodiments disclosed herein may reduce the appearance of cosmetic imperfections or irregularities, such as facial lines, by reducing localized muscle activity. In embodiments, the cosmetic irregularities may include glabellar lines, forehead wrinkles, bunny lines, smile irregularities, jowl irregularities, platysma bands, marionette lines, lip wrinkles, crow's feet, brow irregularities, combinations thereof, and the like.

[0059] The methods of the disclosure include administration of at least one Clostridial neurotoxin, eg, a BoNT, in combination with at least one vasoconstrictor, eg, epinephrine.

[0060] The disclosed methods include administering at least one clostridial neurotoxin, e.g., BoNT, in combination with a vasoconstrictor and augmented with at least one NBP or NAP, e.g., an accessory protein, where the amount of NBP or NAP is greater than that typically associated with the clostridial neurotoxin. For example, each BoNT / A typically associates with three Hn-33 molecules. Thus, embodiments of the present disclosure may include formulations in which the ratio of Hn-33 to toxin molecules is greater than 3. In embodiments, the relative molar mixture ratio of neurotoxin to NAP is, for example, 1:1, 1:3, 1:5, 1:10, etc.

[0061] Administration sites useful for practicing embodiments of the present disclosure may include the glabellar complex (including the corrugator supercilii and procerus muscles), orbicularis oculi, the superior lateral fibers of the orbicularis oculi, the frontalis muscle, the nasalis muscle, the levator labii alae naris superioris, the orbicularis oculi muscle, the masseter muscle, the depressor anguli oris, and the platysma muscle. Exemplary injection sites useful for treating glabellar wrinkles are shown in Figure 1. Embodiments include the treatment of generalized wrinkles.

[0062] Embodiments of the present disclosure may include, for example, treatment of skin disorders such as acne, etc. Embodiments of the present disclosure may include treatment of inflammatory skin diseases, for example, psoriasis, eczema, etc.

[0063] Embodiments include methods involving dermatological surgical procedures, such as the treatment of actinic keratosis, seborrheic keratosis, basal cell carcinoma, squamous cell carcinoma, and other lesions or subcutaneous cysts.

[0064] In embodiments, the methods of the present disclosure may be used to treat symptoms, diseases, or conditions caused by or associated with hyperactive cholinergic innervation of muscles or exocrine glands in a patient, including, but not limited to, dystonia, spasticity, paratonia, dyskinesia, focal spasms, strabismus, tremors, tics, migraines, sialorrhea, and hyperhidrosis.

[0065] Embodiments disclosed herein may reduce local muscle activity to reduce the occurrence of scars, e.g., scars resulting from surgery. In embodiments, surgery may include cosmetic surgery, e.g., rhinoplasty, eye lift, tummy tuck, etc. In embodiments, surgery may include other types of medical procedures, e.g., appendectomy, organ transplant, etc. In embodiments, a method comprises administering a composition of the present disclosure near a wound.

[0066] Embodiments disclosed herein can reduce local muscle activity to reduce the occurrence of scarring, e.g., scarring resulting from trauma. For example, following trauma, embodiments of the present disclosure can include administering a composition of the present disclosure proximate to the trauma, e.g., tear or amputation.

[0067] Administration sites useful for practicing embodiments of the present disclosure can include any area in which muscle activity is to be reduced. For example, embodiments of the present disclosure can include administration to the glabellar complex (including the corrugator supercilii and procerus muscles), orbicularis oculi, superior lateral fibers of the orbicularis oculi, frontalis muscle, nasal muscles, levator labii ala naris superioris, orbicularis oculi, masseter muscle, depressor anguli oris, and platysma muscle.

[0068] When employed in combination with other surgical procedures, embodiments of the present disclosure may include administration to muscles of the face, arms, legs, torso, etc., for example.

[0069] Embodiments of the present disclosure include methods for preparing a surgical site prior to a procedure to reduce muscle tension near the incision.

[0070] Embodiments include administering neurotoxin at lower doses compared to methods known in the art, for example, the increased efficacy exhibited by the compositions and methods may allow lower doses to achieve the same duration of effect as higher doses.

[0071] Administration of the compositions of the present disclosure can be performed by syringe, catheter, needle, needleless system, and other injection means. Injection can be into any area of ​​the mammalian body in need of treatment, or into any specific area such as the epidermis, dermis, fat, muscle, nerve junction, or subcutaneous layer to affect treatment at the desired location.

[0072] The neurotoxin compositions of the present disclosure can be injected into a patient using a needle or a needle-free device. In certain embodiments, the method includes subdermal injection of the composition into an individual. For example, administration may include injecting the composition through a needle of about 30 gauge or smaller. In certain embodiments, the method includes administering a composition comprising a BoNT, e.g., BoNT / A, enhanced with additional accessory proteins relative to the amount typically associated with a neurotoxin. The composition may further comprise a vasoconstrictor.

[0073] More than one injection and / or injection site may be necessary to achieve the desired result, and depending on the intended location of the injection, some injections may require the use of a thin, hollow, Teflon-coated needle, which in certain embodiments is guided, for example, by electromyography.

[0074] The frequency and amount of injections in the methods of the present disclosure can be determined based on the nature and location of the particular area being treated. However, in certain cases, repeated injections may be required to achieve optimal results. The frequency and amount of injections for each particular case can be determined by those skilled in the art.

[0075] Although several examples of administration routes and dosages are provided, the appropriate administration route and dosage are typically determined individually by the attending physician. Such determinations are routine for those skilled in the art (see, for example, "Harrison's Principles of Internal Medicine," 14th ed., edited by Anthony Fauci et al., McGraw Hill, 1998). For example, the route and dosage for administration of the clostridial neurotoxin according to the present disclosure can be selected based on criteria such as the solubility characteristics of the selected neurotoxin, as well as the intensity and extent of the condition being treated.

[0076] The methods of the present disclosure can prevent or reduce pain, nausea, vomiting, photosensitivity, phonosensitivity, and combinations thereof.

[0077] In embodiments, the method comprises administering a therapeutically effective amount of at least one neurotoxin to a nerve associated with at least one of the frontalis, corrugator supercilii, procerus, masseter, occipitalis, temporalis, trapezius, and cervical paraspinal muscle regions.

[0078] (neurotoxin dose) The neurotoxin is approximately 10 -3 In one embodiment, the neurotoxin may be administered in an amount of between about 10 U / kg and about 35 U / kg. -2 In another embodiment, the neurotoxin is administered in an amount of between about 10 U / kg and about 25 U / kg. -1 In one embodiment, the neurotoxin is administered in an amount between about 1 U / kg and about 15 U / kg. In another embodiment, the neurotoxin is administered in an amount between about 1 U / kg and about 10 U / kg. In many cases, administration of about 1 unit to about 300 units of a neurotoxin, such as botulinum type A, will provide effective therapeutic relief. In one embodiment, about 5 units to about 200 units of a neurotoxin, such as botulinum type A, may be used, while in another embodiment, about 10 units to about 100 units of a composition of the present disclosure may be administered locally to the target tissue.

[0079] In embodiments, administration is about 30 units, or about 35 units, or about 40 units, or about 45 units, or about 50 units, or about 55 units, or about 60 units, or about 65 units, or about 70 units, or about 75 units, or about 80 units, or about 85 units, or about 90 units, or about 95 units, or about 100 units, or about 105 units, or about 110 units, or about 115 units, or about 120 units, or about 125 units, or about 130 units, or about 135 units, or about 140 units, or about 145 units, or about 150 units, or about 155 units, or about 160 units, or about 165 units, or about The total dosage per treatment session may include about 170 units, or about 175 units, or about 180 units, or about 185 units, or about 190 units, or about 195 units, or about 200 units, or about 205 units, or about 210 units, or about 215 units, or about 220 units, or about 225 units, or about 230 units, or about 235 units, or about 240 units, or about 245 units, or about 250 units, or about 255 units, or about 260 units, or about 265 units, or about 270 units, or about 275 units, or about 280 units, or about 285 units, or about 290 units, or about 295 units, or about 300 units, etc.

[0080] In embodiments, administration is of 10 or more units, or 20 or more units, or 30 or more units, or 40 or more units, or 50 or more units, or 60 or more units, or 70 or more units, or 80 or more units, or 90 or more units, or 100 or more units, or 110 or more units, or 120 or more units, or 130 or more units, or 140 or more units, or 150 or more units of a neurotoxin, e.g., BoNT. It may include a total dosage per treatment session of 160 units or more, or 170 units or more, or 180 units or more, or 190 units or more, or 200 units or more, or 210 units or more, or 220 units or more, or 230 units or more, or 240 units or more, or 250 units or more, or 260 units or more, or 270 units or more, or 280 units or more, or 290 units or more, or 300 units or more, etc.

[0081] In embodiments, administration is of 10 units or less, or 20 units or less, or 30 units or less, or 40 units or less, or 50 units or less, or 60 units or less, or 70 units or less, or 80 units or less, or 90 units or less, or 100 units or less, or 110 units or less, or 120 units or less, or 130 units or less, or 140 units or less, or 150 units or less of a neurotoxin, e.g., botulinum type A neurotoxin. or a total dosage per treatment session of 160 units or less, or 170 units or less, or 180 units or less, or 190 units or less, or 200 units or less, or 210 units or less, or 220 units or less, or 230 units or less, or 240 units or less, or 250 units or less, or 260 units or less, or 270 units or less, or 280 units or less, or 290 units or less, or 300 units or less, etc.

[0082] In embodiments, administration may include a total annual dose of no more than 400 units, or no more than 500 units, or no more than 600 units, or no more than 700 units, or no more than 800 units, or no more than 900 units, or no more than 1000 units of neurotoxin, e.g., botulinum type A neurotoxin.

[0083] In embodiments, neurotoxin administration, e.g., botulinum administration, to nerves associated with at least one of the frontalis, corrugator supercilii, procerus, masseter, occipitalis, temporalis, trapezius, and cervical paraspinal muscle regions may include a total dosage per treatment session of about 40 units, or about 50 units, or about 60 units, or about 70 units, or about 80 units, or about 90 units, or about 100 units, or about 110 units, or about 120 units, or about 130 units, or about 140 units, or about 150 units, or about 160 units, or about 170 units, or about 180 units, or about 190 units, or about 200 units, or about 210 units, or the like, of BoNT.

[0084] In embodiments, the dosage per injection site t can be, for example, 2 units, 3 units, 4 units, 5 units, 10 units, 15 units, 20 units, between 2 and 10, between 4 and 8, between 4 and 6, between 5 and 20, between 5 and 15, between 5 and 10, between 10 and 15, etc.

[0085] In embodiments, the neurotoxin dosage is expressed in terms of protein amount or concentration. For example, in embodiments, the neurotoxin may be administered in an amount between about 2 ng and 20 ng. In one embodiment, the neurotoxin is administered to a target tissue, such as muscle, in an amount between about 0.3 ng and 19 ng, between about 0.4 ng and 18 ng, between about 0.5 ng and 17 ng, between about 0.6 ng and 16 ng, between about 0.7 ng and 15 ng, between about 0.8 ng and 14 ng, between about 0.9 ng and 13 ng, between about 1.0 ng and 12 ng, between about 1.5 ng and 11 ng, between about 2 ng and 10 ng, or between about 5 ng and 7 ng.

[0086] Ultimately, however, both the amount of toxin administered and the frequency of its administration will be at the discretion of the treating physician and will be commensurate with the efficacy and safety issues posed by the toxin.

[0087] Disclosed embodiments include treatments that can be repeated. For example, repeat administrations can be administered when a patient begins to experience symptoms. However, preferred embodiments include repeating treatments before symptoms return. Thus, disclosed embodiments include repeating treatments after 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, or more weeks. Repeat treatments can involve administration sites that are different from those used in previous treatments. For example, in embodiments, the frontalis muscle can be injected in an initial treatment and then not injected in subsequent treatments.

[0088] In the embodiments described herein, a controlled release system can be used to deliver the neurotoxin in vivo at a predetermined rate over a specific period of time. The controlled release system can be comprised of a neurotoxin incorporated into a carrier. The carrier can be a polymer or a bioceramic material. The controlled release system can be injected, inserted, or implanted into a selected location in the patient's body and remain there for an extended period of time during which the neurotoxin is released from the implant in a concentration and manner that provides the desired therapeutic effect.

[0089] Polymeric materials can release neurotoxins by diffusion, chemical reaction, or solvent activation, as well as by magnetic, ultrasonic, or temperature change factors. Diffusion can be from a reservoir or matrix. Chemical control can be by polymer degradation or drug cleavage from the polymer. Solvent activation can involve swelling of the polymer or osmotic effects.

[0090] (kit) Kits for carrying out embodiments of the present disclosure are also encompassed by the present disclosure. The kits may include a 30-gauge or smaller needle and a corresponding syringe. The kits may also include at least one Clostridial neurotoxin composition, such as a botulinum type A toxin composition. The neurotoxin composition may be provided in a syringe. The composition is injectable with a needle. The kits are designed in various forms based on the size of the syringe and needle and the volume of the injectable composition(s) contained therein; these requirements, in turn, are based on the specific deficiency for which the kit is designed to treat.

[0091] The compositions of the present disclosure may be provided in pre-filled syringes.

[0092] (Composition preparation method) The compositions of the present disclosure can be prepared by adding at least one NBP or NAP, for example, an accessory protein such as Hn-17, Hn-33, or Hn-70, to a botulinum formulation. In embodiments, at least one NBP or NAP is added, and the composition is then lyophilized. In embodiments, at least one NBP or NAP is added immediately prior to administration.

[0093] In embodiments, at least one NBP or NAP may be naturally occurring or recombinant.

[0094] (Example) The following non-limiting examples are presented for illustrative purposes, with the sole intention of facilitating a more complete understanding of representative embodiments, and should not be construed as limiting any of the embodiments described herein.

[0095] Example 1 (Treatment of axillary hyperhidrosis) A 44-year-old man presented with a complaint of excessive underarm sweating. His physician recommended a Minor's starch-iodine test. For the Minor test, the skin was cleansed, shaved, and dried. A 3.5% alcoholic solution of iodine was applied to the affected area, and starch powder was sprinkled on top. When sweat came into contact with the iodine-starch mixture, it turned dark purple. This indicated a positive sweat test and provided a map of active eccrine glands.

[0096] The patient's physician diagnosed axillary hyperhidrosis and prescribed BoNT / A injections in the pattern shown by the minor test to induce remission. The injections were given subcutaneously (s / c) in the armpits in a grid pattern spaced approximately 1-2 cm apart. Each injection contained 10 units of BoNT / A potentiated with Hn-33, a dose 50% greater than that typically associated with the neurotoxin.

[0097] The patient experienced good results over six months after treatment.

[0098] Example 2 (Treatment for hyperhidrosis of the feet) A 66-year-old man presented with a complaint of excessive sweating from the soles of his feet. His physician diagnosed him with hyperhidrosis and prescribed BoNT / E injections to induce rapid relief. Injections were administered subcutaneously into the soles of his feet in a grid pattern approximately 1-2 cm apart. Each injection contained 5 ng of BoNT / E and 5% epinephrine in lidocaine.

[0099] The patient experienced good results over the four months following treatment.

[0100] Example 3 (Treatment for palmar hyperhidrosis) A 19-year-old toxin-naive male presented with symptoms of excessive sweating from the palms of his hands. His physician diagnosed hyperhidrosis and prescribed BoNT / A injections to induce rapid relief. Injections were administered to the palms in a grid pattern spaced approximately 1-2 cm apart. Each injection contained 10 units of BoNT / A potentiated with Hn-33, a 50% increase over the amount typically associated with the neurotoxin. The patient experienced good results over 6 months following treatment.

[0101] Example 4 (Treatment of crow's feet wrinkles) A 57-year-old man with crow's feet caused by years of sun exposure sought treatment from his physician. The physician recommended the composition disclosed herein, which was injected subdermally into either side of the patient's eye. Each injection contained 5 ng of BoNT / E and 1% epinephrine in lidocaine, with multiple injections given to either side of the eye. The crow's feet disappeared within approximately two days of treatment and remained reduced for five months.

[0102] Example 5 (Brow lift treatment) A 60-year-old neurotoxin-naive woman presented with symptoms of eyebrow extension below the brow bone. A physician recommended the composition disclosed herein, which was injected subdermally above each eye. Each injection contained 10 units of BoNT / A and 1% epinephrine in lidocaine, with multiple injections given on either side of the eye. The eye droopiness decreased within approximately two days and remained substantially alleviated for six months after administration.

[0103] Example 6 (Breast augmentation treatment) A 30-year-old woman elected to undergo breast augmentation surgery. Four hours before surgery, she received Hn-33-enhanced BoNT / B near the surgical incision, at a dose 50% greater than that typically associated with the neurotoxin. This administration reduces muscle tension in the incision area and minimizes scarring.

[0104] Example 7 (Treatment for breast reconstruction) A 30-year-old neurotoxin-naive woman elected to undergo breast reconstruction surgery. 14 hours before surgery, Hn-33-enhanced BoNT / F was administered near the surgical incision site at a dose 10% greater than that typically associated with neurotoxin. This administration reduces muscle tension in the incision area and minimizes scarring. An additional dose was administered two weeks after surgery.

[0105] Example 8 (episiotomy treatment) A 44-year-old woman underwent an episiotomy. Immediately after surgery, Hn-33-enhanced BoNT / F was administered to the tissues surrounding the surgical area at a dose 10% greater than that typically associated with the neurotoxin. Within 20 hours, there was a significant decrease in muscle and nerve activity around the wound.

[0106] Example 9 (Sports hernia treatment) A 28-year-old neurotoxin-naive man suffered from a sports hernia. His physician administered 4 ng of Hn-33-enhanced BoNT / F, a 100% dose higher than that typically associated with neurotoxin, into the tissues surrounding both hernias. Within 10 hours, there was a significant decrease in muscle and nerve activity around the wounds.

[0107] Example 10 (Treatment of acromioclavicular joint separation) A 48-year-old man suffered from acromioclavicular joint separation. His physician administered Hn-33-enhanced BoNT / A to the tissues surrounding both hernias at a dose 20% higher than that typically associated with the neurotoxin. Muscle and nerve activity in the injection areas decreased over a 6-month period.

[0108] Example 11 (Treatment for anterior cruciate ligament rupture) A 23-year-old woman suffered a torn anterior cruciate ligament. Six hours after the injury, her physician administered 10 units of BoNT / A and 1% epinephrine in lidocaine to the muscles surrounding the torn ligament. Within 30 hours, there was a significant decrease in muscle and nerve activity around the wound.

[0109] Example 12 (Treatment of neuropathic pain) A 42-year-old man experienced neuropathic pain in his foot. To relieve the patient's symptoms, approximately 10 units of BoNT / D and 0.7% epinephrine in lidocaine were injected near the nerves in his foot. The patient experienced a reduction in pain over a 7-month period.

[0110] Example 13 (episiotomy treatment) A 24-year-old woman underwent an episiotomy. Immediately after surgery, approximately 20 units of BoNT / A and 1% epinephrine in lidocaine were administered to the tissues surrounding the surgical area. The BoNT / A was potentiated with Hn-33, a dose 40% greater than that typically associated with the neurotoxin.

[0111] Example 14 (Treatment of frown lines) A 57-year-old man was treated for glabellar lines. Hn-33-enhanced BoNT / A, at a dose 40% greater than that typically associated with the neurotoxin, and 0.7% epinephrine in lidocaine were delivered in equal volumes to five injection sites (procerus, left and right corrugator supercilii, left and right corrugator supercilii lateralis, 0.1 mL, 5 units per site) in a standardized fashion (see Figure 1). The appearance of the glabellar lines was reduced over a 6-month period.

[0112] Example 15 (Treatment of frown lines) A 27-year-old man was treated for glabellar lines. Hn-33-enhanced BoNT / E, at a dose 70% greater than that typically associated with the neurotoxin, and 0.5% epinephrine in lidocaine were delivered in equal volumes to five injection sites (procerus, left and right corrugator supercilii medialis, and left and right corrugator supercilii lateralis, 0.1 mL, 5 units per site) in a standardized fashion (see Figure 1). The appearance of the glabellar lines was reduced over a 7-month period.

[0113] Example 16 (Treatment of frown lines) A 47-year-old woman was treated for glabellar lines. Hn-33-enhanced BoNT / B, at a dose 20% greater than that typically associated with the neurotoxin, was delivered in equal volumes to five injection sites (procerus, left and right corrugator supercilii medialis, and left and right corrugator supercilii lateralis, 0.1 mL, 5 units per site) in a standardized fashion (see Figure 1). The appearance of the glabellar lines was reduced over a 5-month period.

[0114] Example 17 (Treatment of chronic migraines) Patients with chronic migraines were treated via injection of 15 U of BoNT / A boosted with Hn-33, a dose 20% greater than that typically associated with the neurotoxin, at multiple sites along the trigeminal nerve, including the frontalis, corrugator supercilii, proximal, and occipital muscles.

[0115] Patients reported fewer migraines over the 24 weeks following treatment.

[0116] Example 18 (Treatment of chronic migraines) Patients with chronic migraines were treated via injection of 20 U of BoNT / E boosted with Hn-33, an 80% increase over the amount typically associated with the neurotoxin, at multiple sites along the trigeminal nerve, including the frontalis, corrugator supercilii, proboscis, masseter, and occipital muscles.

[0117] Patients reported fewer migraines over the 24 weeks following treatment.

[0118] Example 19 (Treatment of recurrent migraines) Patients with chronic migraine were treated with 15 U of BoNT / A injections at multiple sites along the trigeminal nerve, including the frontalis, corrugator supercilii, proximal, and occipital muscles. No injections were made into the cervical paraspinal muscle region. Patients also received 30 U of BoNT / A (15 U bilaterally) orally into the pterygopalatine fossa.

[0119] Patients reported fewer migraines over the 16 weeks following treatment.

[0120] Example 20 (Composition Testing) Recovery studies in mice after intramuscular administration of different formulations of Ebolus samples with Hn-33.

[0121] Study Objective - Evaluation of muscle weakness by DAS scoring and rotarod assay system for sponsor's test article in Nu / NU or Balb / c mice.

[0122] [Table 1]

[0123] experiment Dose: 50μl. Injection volume: 100 μl. Injection sites: Samples were injected into both the left and right paws. All dilutions were prepared in 1x PBS. Negative control: 1x PBS. Positive control I: In-house produced BoNT / A complex drug substance (10 units): without Hn-33 added. Positive control II (FI): Ebolus sample (10 units): No Hn-33 added. Sample 1 (FII): Ebolus sample (10 units) mixed with native Hn-33 (1:1 molar ratio). Sample 2 (FIII): Evolus sample (10 units) mixed with natural Hn-33 (at a molar ratio of 1:3). Sample 3 (FIV): Evolus sample (10 units) mixed with native Hn-33 (at a molar ratio of 1:5). Sample 4 (FV): Evolus sample (10 units) mixed with recombinant Hn-33 (at a molar ratio of 1:5). Sample 5 (FVI): Evolus sample (10 units) mixed with native Hn-33 (at a molar ratio of 1:10). Sample 6 (FVII): In-house produced BoNT / A complex drug substance sample (10 units) mixed with recombinant Hn-33 (at a molar ratio of 1:5).

[0124] DAS Scoring Procedure: Scores were recorded independently by all observers and collected at the end of the final reading. Neither the person who injected the mice nor the person who assessed the DAS scores knew the samples (all preparations were coded). Results: A) In controls, 1x PBS was injected intramuscularly into both hind limbs of each mouse under anesthesia.

[0125] As shown in Figure 2, for the positive control II-FI (Ebolus formulated BoNT / A complex, without Hn-33), 100 μl of 10 units of formulated BoNT / A complex (FI) was injected intramuscularly into both hind limbs of each mouse under anesthesia.

[0126] The mean DAS scores for both hind paws of all animals in this cohort were plotted against their respective dates, with error bars indicating the standard deviation of all measurements.

[0127] For Formulated Complex I-FII (Ebolus formulated BoNT / A complex with Hn-33 added at a 1:1 ratio), the Ebolus sample was mixed with native Hn-33 in 1x PBS at a 1:1 molar ratio, as seen in Figure 3. 100 μl of 10 units of formulated BoNT / A complex (FII) was injected intramuscularly into both hind limbs of each mouse under anesthesia.

[0128] The mean DAS scores for both hind paws of all animals in this cohort are plotted against their respective dates. Error bars indicate the standard deviation of all measurements. All mice died on day 20.

[0129] Thus, Figure 3 shows the effect of boosting the Type A BoNT composition with 1 M Hn-33 compared to the control (Figure 2), with a DAS score of approximately 4 maintained for most of the day up to day 10, whereas the DAS score of the control group fell below 4 by day 5.

[0130] In closing, while aspects of the present disclosure have been emphasized with reference to particular embodiments, it should be readily understood by those skilled in the art that these disclosed embodiments are merely illustrative of the principles of the subject matter disclosed herein. Therefore, it should be understood that the disclosed subject matter is in no way limited to the particular methods, protocols, and / or reagents, etc., disclosed herein. Thus, various modifications or variations of the disclosed subject matter, or various alternative configurations of the disclosed subject matter, are possible in accordance with the teachings herein without departing from the spirit of the present disclosure. Finally, the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present disclosure, which is defined solely by the claims. Therefore, embodiments of the present disclosure are not limited to the exact same as shown and described.

[0131] Certain embodiments are described herein, including the best mode known to the inventors for carrying out the methods and apparatus described herein. Of course, variations on these described embodiments will be apparent to those skilled in the art upon reading the foregoing description. Accordingly, this disclosure includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Furthermore, any combination of the above-described embodiments in all possible variations thereof is encompassed by the present disclosure unless otherwise specified herein or otherwise clearly contradicted by context.

[0132] The collections of alternative embodiments, elements, and steps in the present disclosure are not to be construed as limiting. Each collection element may be referenced and claimed individually or in combination with other collection elements disclosed herein. It is anticipated that one or more elements of a collection may be added to or deleted from a group for reasons of convenience and / or patentability. When such additions or deletions occur, the specification is deemed to include the modified group, which therefore satisfies all the recitation requirements of the Markush group used in the appended claims.

[0133] Unless otherwise specified, features, items, amounts, parameters, properties, periods, and the like used in the specification and claims are understood to be modified in all instances by the term "about." As used herein, the term "about" encompasses the feature, item, amount, parameter, property, or period so qualified, plus or minus a range of plus or minus 10 percent above or below the relevant feature, item, amount, parameter, property, or period. Accordingly, unless expressly stated to the contrary, the numerical parameters set forth in this specification and the appended claims are approximations that may vary. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical designation should be construed in light of, at the very least, the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and values ​​setting forth the broad scope of the present disclosure are approximations, the numerical ranges and values ​​set forth in the specific examples are reported as precisely as possible. However, any numerical ranges and values ​​inherently contain certain errors necessarily resulting from the standard deviation found in the corresponding testing measurements. The recitation of numerical ranges of values ​​herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within that range, and unless otherwise stated herein, each separate value of the numerical range is incorporated herein as if each separate value were individually indicated herein.

[0134] As used in the context of the foregoing disclosure (particularly in the context of the claims that follow), the singular terms "a," "the," "the," and "the" should be construed to cover both the singular and the plural, unless otherwise stated herein or otherwise clearly contradicted by the context. All of the methods described herein can be performed in any suitable order, unless otherwise stated herein or otherwise clearly contradicted by the context. Any and all examples or illustrative language (e.g., "etc.") presented herein are intended merely to further clarify the disclosure and are not intended to impose limitations on the scope of the claims without their use. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the embodiments disclosed herein.

[0135] Certain embodiments disclosed herein may be further limited in the claims using the phrases "consisting of" or "consisting essentially of." When used in a claim, whether as originally filed or added by amendment, the transitional phrase "consisting of" excludes any element, step, or ingredient not specified in the claim. The transitional phrase "consisting essentially of" limits the scope of the claim to the materials or steps specified in the claim and to materials or steps that do not materially affect the basic and novel characteristic(s). The claimed embodiments of the present disclosure are inherently or explicitly described and enabled by this specification.

[0136] [Note] [Appendix 1] A composition comprising a clostridial neurotoxin and a clostridial neurotoxin-associated protein (NAP), wherein said NAP is present in an amount at least 10% greater than the amount normally associated with said toxin.

[0137] [Appendix 2] 2. The composition of claim 1, wherein the NAP is present in an amount 20% greater than the amount normally associated with the toxin.

[0138] [Appendix 3] 3. The composition of claim 2, wherein the NAP is present in an amount that is 40% greater than the amount normally associated with the toxin.

[0139] [Appendix 4] 4. The composition of claim 3, wherein the NAP is present in an amount that is 60% greater than the amount normally associated with the toxin.

[0140] [Appendix 5] 2. The composition of claim 1, wherein the NAP comprises a clostridial accessory protein.

[0141] [Appendix 6] 6. The composition of claim 5, wherein the accessory protein comprises Hn-33.

[0142] [Appendix 7] 2. The composition of claim 1, further comprising a vasoconstrictor.

[0143] [Appendix 8] 8. The composition of claim 7, wherein the vasoconstrictor comprises epinephrine.

[0144] [Appendix 9] 9. The composition of claim 8, wherein the epinephrine is present in an amount of at least 0.5%.

[0145] [Appendix 10] 10. A method of treating a cosmetic imperfection, comprising administering to an individual the composition of claim 1.

[0146] [Appendix 11] 11. The method of claim 10, wherein the cosmetic imperfection is a wrinkle.

[0147] [Appendix 12] 12. The method of claim 11, wherein the cosmetic imperfection is a condition selected from the group consisting of marionette lines, glabellar lines, crow's feet, brow furrows, and combinations thereof.

[0148] [Appendix 13] 11. The method of claim 10, wherein the composition further comprises a vasoconstrictor.

[0149] [Appendix 14] 14. The method of claim 13, wherein the vasoconstrictor comprises epinephrine.

[0150] [Appendix 15] 10. A method of treating muscle comprising administering to an individual the composition of claim 1.

[0151] [Appendix 16] 16. The method of claim 15, wherein the total dose of the Clostridial neurotoxin is 50-200 units.

[0152] [Appendix 17] 17. The method of claim 16, wherein the composition is administered by injection or topically.

[0153] [Appendix 18] 18. The method of claim 17, wherein the composition is administered by injection.

[0154] [Appendix 19] 18. The method of claim 17, wherein the clostridial neurotoxin comprises BoNT / A.

[0155] [Appendix 20] 18. The method of claim 17, wherein the clostridial neurotoxin comprises BoNT / B.

[0156] [Appendix 21] 18. The method of claim 17, wherein the clostridial neurotoxin comprises BoNT / C.

[0157] [Appendix 22] 18. The method of claim 17, wherein the clostridial neurotoxin comprises BoNT / D.

[0158] [Appendix 23] 18. The method of claim 17, wherein the clostridial neurotoxin comprises BoNT / E.

[0159] [Appendix 24] 18. The method of claim 17, wherein the clostridial neurotoxin comprises BoNT / F.

[0160] [Appendix 25] 18. The method of claim 17, wherein the clostridial neurotoxin comprises BoNT / G.

Claims

1. A composition comprising a clostridial neurotoxin and a clostridial neurotoxin-associated protein (NAP), wherein said NAP is present in an amount at least 10% greater than the amount normally associated with said toxin.

2. 10. The composition of claim 1, wherein said NAP is present in an amount 20% greater than the amount normally associated with said toxin.

3. 3. The composition of claim 2, wherein said NAP is present in an amount 40% greater than the amount normally associated with said toxin.

4. 4. The composition of claim 3, wherein said NAP is present in an amount 60% greater than the amount normally associated with said toxin.

5. The composition of claim 1 , wherein the NAP comprises a clostridial accessory protein.

6. The composition of claim 5, wherein the accessory protein comprises Hn-33.

7. The composition of claim 1 further comprising a vasoconstrictor.

8. The composition of claim 7 , wherein the vasoconstrictor comprises epinephrine.

9. 9. The composition of claim 8, wherein the epinephrine is present in an amount of at least 0.5%.

10. 10. A method of treating a cosmetic imperfection comprising administering to an individual the composition of claim 1.

11. The method of claim 10, wherein the cosmetic imperfection is a wrinkle.

12. 12. The method of claim 11, wherein the cosmetic imperfection is a condition selected from the group consisting of marionette lines, glabellar lines, crow's feet, brow furrows, and combinations thereof.

13. The method of claim 10 , wherein the composition further comprises a vasoconstrictor.

14. 14. The method of claim 13, wherein the vasoconstrictor comprises epinephrine.

15. A method of treating muscle comprising administering to an individual the composition of claim 1.

16. 16. The method of claim 15, wherein the total dose of the Clostridial neurotoxin is 50-200 units.

17. 17. The method of claim 16, wherein the composition is administered by injection or topically.

18. 18. The method of claim 17, wherein the composition is administered by injection.

19. 18. The method of claim 17, wherein the clostridial neurotoxin comprises BoNT / A.

20. 18. The method of claim 17, wherein the clostridial neurotoxin comprises BoNT / B.

21. 18. The method of claim 17, wherein the clostridial neurotoxin comprises BoNT / C.

22. 18. The method of claim 17, wherein the clostridial neurotoxin comprises BoNT / D.

23. 18. The method of claim 17, wherein the clostridial neurotoxin comprises BoNT / E.

24. 18. The method of claim 17, wherein the clostridial neurotoxin comprises BoNT / F.

25. 18. The method of claim 17, wherein the clostridial neurotoxin comprises BoNT / G.

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