Therapeutic Botulinum Neurotoxins

Abstract

A composition and method for cosmetic treatment is provided. [Solution] Disclosed herein are compositions and methods for therapeutic treatment. The use of "fast-acting" botulinum toxins in cosmetic treatments and / or to prevent or reduce scarring. "Fast-acting" refers to botulinum toxins that exert their effects in a patient more quickly than, for example, botulinum neurotoxin type A. "Fast-acting" botulinum toxins, such as type E, are also disclosed. [Selected Figure] Figure 1

Description

[Technical field]

[0001] The present specification relates to the use of neurotoxins in therapy. [Background technology]

[0002] Cosmetic or plastic surgery includes surgical and non-surgical procedures to enhance and reshape the structure of the body to enhance appearance and self-confidence. These procedures are becoming increasingly common. Summary of the Invention [Problem to be solved by the invention]

[0003] Disclosed herein are compositions and methods for cosmetic treatment. For example, disclosed embodiments include the use of a "fast-acting" botulinum toxin to treat, for example, glabellar lines. Disclosed herein are compositions and methods for use in minimizing scarring. For example, disclosed embodiments include the use of a "fast-acting" botulinum toxin to reduce muscle tension adjacent to a wound, thus preventing or reducing scarring. [Means for solving the problem]

[0004] In embodiments, activity of muscles adjacent to a skin incision or laceration is reduced, thus reducing or preventing scar formation. In embodiments, the disclosed methods include additional cosmetic procedures, for example, disclosed embodiments include administration of a fast-acting botulinum neurotoxin in combination with, for example, dermal filler injections, eye lifts, rhinoplasty, or the like. In an embodiment, the botulinum toxin is a "fast-recovery" toxin. In an embodiment, a "fast-acting" botulinum toxin is also a "fast-recovery" toxin. In embodiments, the cosmetic treatment may include additional doses of botulinum after an initial dose. In embodiments, the disclosed methods include administration of a fast-acting botulinum neurotoxin in combination with, for example, a slower-acting neurotoxin. In embodiments, the disclosed methods include administration of a fast-recovering botulinum neurotoxin, for example, in combination with a slow-recovering neurotoxin. In an embodiment, the dosage of the neurotoxin is expressed as the amount of protein. [Brief description of the drawings]

[0005] [Figure 1] FIG. 1 depicts injection sites used in cosmetic surgery. [Diagram 2] FIG. 1 is a graph showing the primary efficacy of the glabellar wrinkle treatment trial. [Diagram 3] 1 is a graph showing secondary efficacy of the glabellar wrinkle treatment trial. [Figure 4] 1 is a graph showing the effect of a single topical administration of the disclosed type E botulinum composition in a rat model of post-operative pain. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0006] The embodiments disclosed herein reduce local muscle activity, thereby reducing the appearance of cosmetic imperfections or irregularities, such as facial wrinkles. In embodiments, the cosmetic irregularities include frown lines, forehead wrinkles, "bunny" lines, laugh lines, jaw irregularities, platysmal bands, "marionette" lines, lip wrinkles, crow's feet, brow irregularities, combinations thereof, and the like. Embodiments include methods involving dermatological surgery, such as the treatment of actinic keratosis, seborrheic keratosis, basal cell carcinoma, squamous cell carcinoma, and other lesions or subcutaneous cysts. Administration sites useful in the practice of the disclosed embodiments may include the glabellar complex, including the corrugator supercilii and procerus muscles; the orbicularis oculi muscle; the superior lateral fibers of the orbicularis oculi muscle; the frontalis muscle; the nasalis muscle; the levator labii ala naris superioris muscle; the orbicularis oris muscle; the masseter muscle; the depressor anguli oris muscle; and the platysma muscle. Exemplary injection sites useful for treating glabellar wrinkles are shown in FIG. 1. Embodiments include the treatment of generalized wrinkles.

[0007] Disclosed embodiments may include, for example, treatment of skin disorders such as acne and the like. Disclosed embodiments may include treatment of inflammatory skin diseases. For example, disclosed embodiments may include treatment of psoriasis, eczema, and the like. The embodiments disclosed herein can reduce local muscle activity, thereby reducing the appearance of scars, such as scars caused by surgery. In embodiments, the surgery can include cosmetic surgery, such as rhinoplasty, eye lift, "tummy" tuck (abdominoplasty), or the like. In embodiments, the surgery can include other types of medical procedures, such as appendectomies, organ transplants, and the like. In embodiments, the method includes administration of the disclosed compositions adjacent to a wound. The embodiments disclosed herein can reduce local muscle activity, thereby reducing the appearance of scars, such as those caused by trauma. For example, following a traumatic injury, the disclosed embodiments can include administration of the disclosed compositions adjacent to the trauma, such as a laceration or cut. Useful administration sites for the practice of the disclosed embodiments may include any area where muscle activity is to be reduced. For example, when used in conjunction with facial cosmetic surgery, the disclosed embodiments may include administration to the glabellar complex, including the corrugator supercilii and procerus muscles; the orbicularis oculi muscle; the superior lateral fibers of the orbicularis oculi muscle; the frontalis muscle; the nasal muscle; the levator labii ala naris superioris muscle; the orbicularis oris muscle; the masseter muscle; the depressor anguli oris muscle; and the platysma muscle.

[0008] When used in conjunction with other surgical procedures, the disclosed embodiments may include administration to muscles of the arms, legs, torso, and the like, for example. The disclosed embodiments may include a method for preparing a surgical site prior to surgery to reduce muscle tension adjacent to the incision. The disclosed embodiments can, for example, promote the production of elastin, collagen, and the like. The disclosed embodiments can include methods of increasing skin elasticity. In embodiments, the compositions disclosed herein may include a fast-acting botulinum toxin, such as type E. In embodiments, the compositions disclosed herein may include a fast-recovering botulinum toxin, such as type E. In embodiments, the compositions disclosed herein may include a fast-acting, fast-recovering botulinum toxin, such as type E.

[0009] Definition: "Administration" or "administering" refers to the step of giving (i.e., administering) a pharmaceutical composition or active ingredient to a subject. The pharmaceutical compositions disclosed herein may be administered by any suitable route, but as described in the disclosed methods, the compositions are administered locally, for example, by intramuscular routes of administration, such as injection or by use of an implant. "Botulinum toxin" or "botulinum neurotoxin" refers to wild-type neurotoxins derived from Clostridium botulinum, as well as modified, recombinant, hybrid and chimeric botulinum toxins. Recombinant botulinum toxins have their light and / or heavy chains recombinantly produced by non-Clostridium species. As used herein, "botulinum toxin" encompasses botulinum toxin serotypes A, B, C, D, E, F, G and H. As used herein, "botulinum toxin" also includes both botulinum toxin complexes (i.e., 300, 600 and 900 kDa complexes) as well as pure botulinum toxins (i.e., approximately 150 kDa neurotoxin molecules), all of which are useful in the practice of the present invention. "Pure botulinum toxin" refers to a pure botulinum toxin or a botulinum toxin complex that has been isolated or substantially isolated from other proteins and impurities that may accompany a botulinum toxin when obtained by a culture or fermentation process. Thus, a pure botulinum toxin has at least 95%, and more preferably at least 99%, of the non-botulinum toxin proteins and impurities removed.

[0010] "Biocompatible" means that there is minimal inflammatory reaction at the implant site. "Clostridial neurotoxin" means a neurotoxin produced by or characteristic of a Clostridium bacterium, such as Clostridium botulinum, Clostridium butyricum, or Clostridium beratti, as well as a Clostridial neurotoxin recombinantly produced by a non-Clostridial species. "Completely free" (the term "consisting of") means that the substance cannot be detected or its presence identified within the detection range of the equipment or process being used. "Essentially free" means that only trace amounts of the substance can be detected, within the detection range of the equipment or process being used.

[0011] As used herein, "fast-acting" refers to a botulinum toxin that exerts an effect in a patient more rapidly than is exerted by, for example, botulinum neurotoxin type A. For example, the effect of a fast-acting botulinum toxin can be visually noticeable within 36 hours. As used herein, "fast-recovering" refers to a botulinum toxin whose effect in a patient decreases more quickly than that exerted by, for example, botulinum neurotoxin type A. For example, the effect of a fast-recovering botulinum toxin decreases within, for example, 120 hours, 150 hours, 300 hours, 350 hours, 400 hours, 500 hours, 600 hours, 700 hours, 800 hours, or the like. Botulinum toxin type A is known to have an effect for up to 12 months. However, the usual duration of intramuscular injection of botulinum neurotoxin type A is typically about 3 to 4 months. As used herein, "intermediate-acting" refers to a botulinum toxin that exerts its effect more slowly than a fast-acting toxin. "Neurotoxin" means a biologically active molecule with specific affinity for a neuronal cell surface receptor. Neurotoxins include both clostridial toxins as pure toxins and in complexes with one or more non-toxin, toxin-associated proteins.

[0012] "Patient" means a human or non-human subject receiving medical or veterinary care. "Pharmaceutical composition" refers to a formulation in which the active ingredient may be a botulinum toxin. The term "formulation" means that in addition to the botulinum neurotoxin active ingredient, at least one additional ingredient (e.g., but not limited to, albumin [such as human serum albumin or recombinant human albumin] and / or sodium chloride) is present in the pharmaceutical composition. Thus, a pharmaceutical composition is a formulation suitable for diagnostic, therapeutic or cosmetic administration to a subject, such as a human patient. The pharmaceutical composition may be in a lyophilized or vacuum-dried state, a solution formed after redissolving the lyophilized or vacuum-dried pharmaceutical composition with saline or water, for example, or as a solution that does not require redissolution. As already mentioned, the pharmaceutical composition may be liquid or solid. The pharmaceutical composition may be animal protein-free.

[0013] "Substantially free" means present at a level of less than 1 percent by weight of the culture medium, fermentation medium, pharmaceutical composition, or other substance in which the weight percent of the substance is calculated. As used herein, "booster administration" refers to an administration of botulinum following an initial administration of neurotoxin.

[0014] "Therapeutic formulation" means a formulation that can be used to treat and thereby alleviate a disorder or disease and / or symptoms associated therewith, such as a disorder or disease characterized by peripheral muscle activity. "Therapeutically effective amount" means the level, amount or concentration of an agent (e.g., a botulinum toxin or a pharmaceutical composition containing a botulinum toxin) required to treat a disease, disorder or condition without causing significant negative or adverse side effects. "Treate," "treating," or "treatment" means the alleviation or reduction (including slight reduction, significant reduction, near complete reduction, and complete reduction), resolution, or prevention (either temporary or permanent) of a disease, disorder, or condition to achieve a desired therapeutic or cosmetic result, for example, by healing damaged or impaired tissue, or by altering, altering, enhancing, ameliorating, reversing, and / or beautifying an existing or perceived disease, disorder, or condition. "Unit" or "U" refers to the amount of active BoNT standardized to have the equivalent neuromuscular blocking effect as one unit of commercially available botulinum neurotoxin type A.

[0015] Neurotoxin Compositions The embodiments disclosed herein include neurotoxin compositions, e.g., fast-acting neurotoxin compositions, e.g., botulinum type E compositions. Such neurotoxins may be formulated in any pharma- ceutically acceptable form and in any pharma-ceutically acceptable formulation. Neurotoxins may also be used in any pharma-ceutically acceptable form supplied by any manufacturer. The embodiments disclosed herein include neurotoxin compositions, such as fast-recovering neurotoxins. Such neurotoxins may be formulated in any pharma- ceutically acceptable form and in any pharma-ceutically acceptable formulation. Neurotoxins may also be used in any pharma-ceutically acceptable form supplied by any manufacturer. The embodiments disclosed herein may include multiple neurotoxins. For example, in embodiments, the disclosed compositions may include two neurotoxins, such as two botulinum neurotoxins, such as a fast-acting and a slower-acting neurotoxin, such as botulinum neurotoxin types E and A. In embodiments, the disclosed compositions may include a fragment of a botulinum neurotoxin, such as the 50 kDa light chain (LC). The neurotoxin may be produced by a Clostridium bacterium, such as Clostridium botulinum, Clostridium butyricum, or Clostridium veratti bacteria. Additionally, the neurotoxin may be a modified neurotoxin; a neurotoxin in which at least one amino acid has been removed, modified, or substituted, as compared to a native or wild-type neurotoxin. Additionally, the neurotoxin may be a recombinantly produced neurotoxin or a derivative or fragment thereof.

[0016] In embodiments, the disclosed type E compositions share 40% amino acid homology with type A, and both share the same basic domain structure consisting of two chains, a 100 kDa heavy chain (HC) and a 50 kDa light chain (LC), linked by disulfide bonds (Whelan 1992). The HC contains the receptor binding domain and the translocation domain, while the LC contains the synaptosomal associated protein (SNAP) enzymatic activity. The domain structure is the same structure shared by all botulinum neurotoxin serotypes. In disclosed embodiments, the neurotoxin is formulated in unit dosage form, for example, it may be provided as a sterile solution in a vial, or as a vial or sachet containing a lyophilized powder for reconstitution in a suitable solvent for injection, such as saline. In an embodiment, the botulinum toxin is formulated into a solution containing saline and pasteurized human serum albumin, which stabilizes the toxin and minimizes losses due to non-specific adsorption. The solution may be sterile filtered (0.2μ filter), filled into individual vials, and then vacuum dried to obtain a sterile lyophilized powder. At use, the powder may be reconstituted by the addition of sterile, preservative-free saline (sodium chloride 0.9% for injection).

[0017] In an embodiment, botulinum type E is supplied in a 5 mL vial as a sterile solution for injection at a nominal concentration of 20 ng / mL in 0.03 M sodium phosphate, 0.12 M sodium chloride and 1 mg / mL human serum albumin (HSA), pH 6.0. In one embodiment, botulinum type E is supplied in a 5 mL vial as a sterile solution for injection at a nominal concentration of 10 ng / mL in 0.03 M sodium phosphate, 0.12 M sodium chloride and 1 mg / mL HSA, pH 6.0. In one embodiment, botulinum type E is supplied in a 5 mL vial as a sterile solution for injection at a nominal concentration of 5 ng / mL in 0.03 M sodium phosphate, 0.12 M sodium chloride and 1 mg / mL HSA, pH 6.0. In one embodiment, botulinum type E is supplied in a 5 mL vial as a sterile solution for injection at a nominal concentration of 1 ng / mL in 0.03 M sodium phosphate, 0.12 M sodium chloride and 1 mg / mL HSA, pH 6.0.

[0018] Although the composition may contain only one type of neurotoxin, such as botulinum type E, the disclosed compositions may contain two or more types of neurotoxins, which may enhance the therapeutic effect against the disorder. For example, the composition administered to the patient may contain botulinum types A and E. Administering a single composition containing two different neurotoxins allows for a lower effective concentration of each neurotoxin than would be achieved if a single neurotoxin were administered to the patient, while still achieving the desired therapeutic effect. The composition administered to the patient may also contain other pharmacologic active ingredients, such as protein receptor or ion channel modulators, in combination with one or more neurotoxins. These modulators can contribute to the reduction of neurotransmission between various nerve cells. For example, the composition may contain GABA receptor modulators. A The drug may include a gamma aminobutyric acid (GABA) type A receptor modulator that enhances the inhibitory effects mediated by the GABA receptor. A The receptors inhibit neural activity by effectively short-circuiting the electrical current passing through the cell membrane. A Receptor modulators include GABA A It can increase the inhibitory effect of GABA receptors and reduce electrical or chemical signaling from nerve cells. AExamples of receptor modulators include benzodiazepines, such as diazepam, oxazepam, lorazepam, prazepam, alprazolam, halazepam, cordiazepoxide, and chlorazepate. The composition may also include a glutamate receptor modulator that reduces the excitatory effect mediated by glutamate receptors. Examples of glutamate receptor modulators include agents that inhibit current flux through AMPA, NMDA, and / or kainate-type glutamate receptors. The composition may also include agents that modulate dopamine receptors, such as antipsychotics, norepinephrine receptors, and / or serotonin receptors. The composition may also include agents that affect ion flux through voltage-dependent calcium, potassium, and / or sodium channels. Thus, the compositions used in the disclosed embodiments may include one or more neurotoxins, such as botulinum toxin, in addition to ion channel receptor modulators that may reduce neurotransmission.

[0019] How to use The methods disclosed herein may include administration of a fast-acting neurotoxin to a patient. In one preferred embodiment, the neurotoxin is botulinum type E. The methods disclosed herein may include administering to a patient a fast-recovering neurotoxin. In one preferred embodiment, the neurotoxin is botulinum type E. Before administering the compositions disclosed herein, the anatomy of the treatment site must be carefully considered. For example, in embodiments, the treatment goal is to inject into the area where the neuromuscular junction is most concentrated, if known. For example, for intramuscular administration, the position of the needle in the muscle can be confirmed by moving the muscle through its range of motion and observing the resulting movement of the needle end before injecting the muscle. General anesthesia, local anesthesia and sedation are used depending on the patient's age, the number of injection sites, and the patient's specific needs. More than one injection and / or injection site may be required to achieve the desired results. Also, some injections require the use of thin, hollow, TEFLON® coated needles guided by electromyography, depending on the muscle to be injected.

[0020] Administration of the disclosed compositions may be, for example, by injection into or around one or more of the following skeletal muscles: occipitalis, nasalis, orbicularis oris, depressor anguli oris, platysma, sternohyoid, serratus anterior, rectus abdominis, external oblique, tensor fasciae latae, brachioradialis, iliopsoas, psoas major, pubic, adductor longus, sartorius, gracilis, vastus lateralis, rectus femoris, vastus medialis, quadriceps tendon, patella, gastrocnemius, soleus, tibia, peroneus longus, tibialis anterior, patellar ligament, iliotibial band, and iliotibial band. Therapeutic injections may include injections into the thenar muscles, thenar muscles, flexor carpi ulnaris, flexor digitorum superficialis, palmaris longus, flexor carpi radialis, brachioradialis, pronator teres, brachialis, biceps brachii, triceps brachii, pectoralis major, deltoid, trapezius, sternocleidomastoid, masseter, orbicularis oculi, temporalis, galea aponeurosis, teres major, extensor digitorum, extensor carpi ulnaris, elbow, abductor pollicis longus, plantaris, calcaneus tendon, soleus, adductor magnus, gluteus maximus, gluteus medius, latissimus dorsi, infraspinatus, and combinations thereof, and the like.

[0021] Administration of the disclosed compositions can include, for example, injection into or around one or more of the following nerves: axillary nerve, phrenic nerve, spinal ganglion, spinal cord, sympathetic ganglion chain, pudendal nerve, common palmar digital nerve, ulnar nerve, deep branch of the ulnar nerve, sciatic nerve, peroneal nerve, tibial nerve, saphenous nerve, interosseous nerve, superficial peroneal nerve, intermediate dorsalis cutaneous nerve, medial plantar nerve, medial dorsalis cutaneous nerve, deep peroneal nerve, muscular branch of the tibial nerve, infrapatellar branch of the saphenous nerve, common peroneal nerve, muscular branch of the femoral nerve, anterior cutaneous branch of the femoral nerve, muscular branch of the sciatic nerve, femoral nerve, ilioinguinal nerve, filum terminale, iliohypogastric This may include injections into the following nerves: obturator nerve, ulnar nerve, radial nerve, obturator nerve, radial nerve, subcostal nerve, intercostal nerve, dorsal branch of the intercostal nerve, medial cutaneous branch of the intercostal nerve, musculocutaneous nerve, deltoid nerve, vagus nerve, brachial plexus, supraclavicular nerve, facial nerve, auriculotemporal nerve, and combinations thereof, and the like. Smooth muscles suitable for administration of the disclosed compositions may include those in the walls of blood vessels, stomach walls, ureters, intestines, aorta (in the media layer), iris of the eye, prostate, digestive tract, airways, small arteries, arterioles, reproductive system (in both males and females), veins, kidney glomeruli (called mesangial cells), bladder, uterus, arrector pili muscles of the skin, ciliary muscles, sphincters, trachea, bile duct, and the like. The frequency and amount of injections under the disclosed method may be determined based on the nature and location of the particular area to be treated.However, in certain cases, repeated or additional injections may be desired to achieve optimal results.The frequency and amount of injections for each particular case may be determined by those skilled in the art.

[0022] In embodiments, administration of the fast-acting neurotoxin occurs prior to surgery, e.g., within 36 hours, 24 hours, 22 hours, 20 hours, 18 hours, 16 hours, 14 hours, 12 hours, 11 hours, 10 hours, 9 hours, 8 hours, 7 hours, 6 hours, 5 hours, 4 hours, 3 hours, 2 hours, 60 minutes, 50 minutes, 40 minutes, 30 minutes, 20 minutes, 10 minutes, 5 minutes, 2 minutes, etc. In embodiments, administration of the fast-acting neurotoxin occurs prior to surgery, for example, 36 hours or more prior to surgery, 24 hours or more prior to surgery, 22 hours or more prior to surgery, 20 hours or more prior to surgery, 18 hours or more prior to surgery, 16 hours or more prior to surgery, 14 hours or more prior to surgery, 12 hours or more prior to surgery, 11 hours or more prior to surgery, 10 hours or more prior to surgery, 9 hours or more prior to surgery, 8 hours or more prior to surgery, 7 hours or more prior to surgery, 6 hours or more prior to surgery, 5 hours or more prior to surgery, 4 hours or more prior to surgery, 3 hours or more prior to surgery, 2 hours or more prior to surgery, 60 minutes or more prior to surgery, 50 minutes or more prior to surgery, 40 minutes or more prior to surgery, 30 minutes or more prior to surgery, 20 minutes or more prior to surgery, 10 minutes or more prior to surgery, 5 minutes or more prior to surgery, 2 minutes or more prior to surgery, etc. In an embodiment, administration of the fast-acting neurotoxin is performed simultaneously with surgery. In embodiments, administration of the fast-acting neurotoxin occurs after surgery. For example, administration may occur within 1 minute after surgery, within 2 minutes after surgery, within 3 minutes after surgery, within 4 minutes after surgery, within 5 minutes after surgery, within 6 minutes after surgery, within 7 minutes after surgery, within 8 minutes after surgery, within 9 minutes after surgery, within 10 minutes after surgery, within 20 minutes after surgery, within 30 minutes after surgery, within 40 minutes after surgery, within 50 minutes after surgery, within 60 minutes after surgery, within 90 minutes after surgery, within 2 hours after surgery, within 3 hours after surgery, within 4 hours after surgery, within 5 hours after surgery, within 6 hours after surgery, within 7 hours after surgery, within 8 hours after surgery, within 9 hours after surgery, within 10 hours after surgery, within 11 hours after surgery, within 12 hours after surgery, etc.

[0023] The methods disclosed herein may include booster administrations of a fast-acting neurotoxin to a patient after an initial administration. Embodiments including booster administrations may further include evaluation by a physician or patient of the results of the previous neurotoxin administration. Such evaluation may include, for example, the use of photography, scanning, or the like.

[0024] In embodiments, the evaluation of the outcome of the first neurotoxin administration can be, for example, 6 hours after first administration, 8 hours after first administration, 10 hours after first administration, 12 hours after first administration, 14 hours after first administration, 16 hours after first administration, 18 hours after first administration, 24 hours after first administration, 30 hours after first administration, 36 hours after first administration, 42 hours after first administration, 48 hours after first administration, 54 hours after first administration, 60 hours after first administration, 66 hours after first administration, 72 hours after first administration, 78 hours after first administration, , 84 hours of first dose, 90 hours of first dose, 96 hours of first dose, 102 hours of first dose, 108 hours of first dose, 114 hours of first dose, 120 hours of first dose, 1 week of first dose, 2 weeks of first dose, 3 weeks of first dose, 4 weeks of first dose, 5 weeks of first dose, 6 weeks of first dose, 7 weeks of first dose, 8 weeks of first dose, 9 weeks of first dose, 10 weeks of first dose, 11 weeks of first dose, 12 weeks of first dose, etc.

[0025] In embodiments including a booster administration, the administration of the booster dose may occur, for example, within 6 hours of evaluation, 8 hours of evaluation, 10 hours of evaluation, 12 hours of evaluation, 14 hours of evaluation, 16 hours of evaluation, 18 hours of evaluation, 24 hours of evaluation, 30 hours of evaluation, 36 hours of evaluation, 42 hours of evaluation, 48 hours of evaluation, 54 hours of evaluation, 60 hours of evaluation, 66 hours of evaluation, 72 hours of evaluation, 78 hours of evaluation, 84 hours of evaluation, 90 hours of evaluation, 96 hours of evaluation, 102 hours of evaluation, 108 hours of evaluation, 114 hours of evaluation, 120 hours of evaluation, 1 week of evaluation, 2 weeks of evaluation, 3 weeks of evaluation, 4 weeks of evaluation, 5 weeks of evaluation, 6 weeks of evaluation, 7 weeks of evaluation, 8 weeks of evaluation, 9 weeks of evaluation, 10 weeks of evaluation, 11 weeks of evaluation, 12 weeks of evaluation, etc.

[0026] In embodiments, the booster dose may be administered, for example, 6 hours after the first dose, 8 hours after the first dose, 10 hours after the first dose, 12 hours after the first dose, 14 hours after the first dose, 16 hours after the first dose, 18 hours after the first dose, 24 hours after the first dose, 30 hours after the first dose, 36 hours after the first dose, 42 hours after the first dose, 48 hours after the first dose, 54 hours after the first dose, 60 hours after the first dose, 66 hours after the first dose, 72 hours after the first dose, 78 hours after the first dose, 80 hours after the first dose, 82 hours after the first dose, 84 hours after the first dose, 86 hours after the first dose, 88 hours after the first dose, 88 hours after the first dose, 89 hours after the first dose, 90 hours after the first dose, 92 hours after the first dose, 94 hours after the first dose, 96 hours after the first dose, 98 hours after the first dose, 98 hours after the first dose, 99 hours after the first dose, 100 hours after the first dose, 102 hours after the first dose, 104 hours after the first dose, 106 hours after the first dose, 108 hours after the first dose, 108 hours after the first dose, 109 hours after the first dose, 110 hours after the first dose, 120 hours after the first dose, 122 hours after the first dose, 124 hours after the first dose, 126 hours after the first dose, 128 hours after the first dose, 128 hours after the first dose, 130 hours after the first dose, 132 hours after the first dose, 136 hours after the first dose, 138 hours after the first dose, 140 hours after the first dose, 142 hours after the first dose, 144 hours after the first dose, 146 hours after the first dose, It may be performed within 84 hours, 90 hours of the first dose, 96 hours of the first dose, 102 hours of the first dose, 108 hours of the first dose, 114 hours of the first dose, 120 hours of the first dose, 1 week of the first dose, 2 weeks of the first dose, 3 weeks of the first dose, 4 weeks of the first dose, 5 weeks of the first dose, 6 weeks of the first dose, 7 weeks of the first dose, 8 weeks of the first dose, 9 weeks of the first dose, 10 weeks of the first dose, 11 weeks of the first dose, 12 weeks of the first dose, etc.

[0027] The methods disclosed herein can provide rapid onset effects (e.g., using fast-acting neurotoxins). For example, the disclosed embodiments can provide visually noticeable cosmetic effects, e.g., at 30 minutes, 45 minutes, 60 minutes, 75 minutes, 90 minutes, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 25 hours, 26 hours, 27 hours, 28 hours, 29 hours, 30 minutes, 35 minutes, 36 hours, 37 hours, 38 hours, 39 hours, 40 hours, 41 hours, 42 hours, 43 hours, 44 hours, 45 minutes, 46 minutes, 47 minutes, 48 ​​hours, 49 hours, 50 minutes, 51 minutes, 52 minutes, 53 minutes, 54 hours, 55 minutes, 56 hours, 57 hours, 58 hours, 59 hours, 60 minutes, 61 minutes, 62 minutes, 63 minutes, 64 hours, 65 minutes, 66 hours, 67 hours, 68 hours, 69 hours, 70 hours, 71 hours, 72 hours, 73 hours, 74 hours, 75 hours, 75 hours, 76 hours, 78 hours, 79 hours, 80 hours, 81 hours, 82 hours, 83 hours, 84 hours, 85 hours, 86 hours, 87 hours, 88 hours, 89 hours, 90 minutes, 91 hours, 92 hours, 93 hours, 94 hours, 95 In one embodiment, the method may be provided within 15 days after administration, 16 days after administration, 17 hours after administration, 18 hours after administration, 19 hours after administration, 20 hours after administration, 21 hours after administration, 22 hours after administration, 23 hours after administration, 24 hours after administration, 30 hours after administration, 36 hours after administration, 42 hours after administration, 48 hours after administration, 3 days after administration, 4 days after administration, 5 days after administration, 6 days after administration, 7 days after administration, 8 days after administration, 9 days after administration, 10 days after administration, 11 days after administration, 12 days after administration, etc. The methods disclosed herein can provide shorter term effects (e.g., with fast-acting neurotoxins). For example, the disclosed embodiments can provide a visually noticeable cosmetic effect that subsides within, for example, 3 days after administration, 4 days after administration, 5 days after administration, 6 days after administration, 7 days after administration, 8 days after administration, 9 days after administration, 10 days after administration, 11 days after administration, 12 days after administration, 13 days after administration, 14 days after administration, 15 days after administration, 16 days after administration, 17 days after administration, 18 days after administration, 19 days after administration, 20 days after administration, 21 days after administration, 22 days after administration, 23 days after administration, 24 days after administration, 25 days after administration, 26 days after administration, 27 days after administration, 28 days after administration, 29 days after administration, 30 days after administration, 45 days after administration, 60 days after administration, 75 days after administration, 90 days after administration, 105 days after administration, etc.

[0028] Side effects can be associated with botulinum injections. The disclosed embodiments can provide a neurotoxin treatment that results in fewer or shorter-lasting side effects than conventional neurotoxin treatments. For example, the disclosed embodiments can result in fewer (or shorter duration) outcomes such as double or blurred vision, eyelid paralysis (the subject is unable to fully open their eyelids), loss of facial muscle movement, hoarseness, bladder control dysfunction, shortness of breath, difficulty swallowing, difficulty speaking, death, and the like.

[0029] The method is particularly suitable for treating cosmetic imperfections that are usually the result of aging, environmental exposure, weight loss, childbirth, trauma, surgery, or a combination thereof. Aging and environmental exposure often cause wrinkles in various locations of the skin. On the other hand, weight loss and childbirth often cause stretch marks in various locations of the skin, especially the abdomen, lower body area, and legs. Trauma and surgery frequently result in scars in the trauma and surgical areas. Particular contour defects suitable for treatment with the disclosed method include, but are not limited to, frown lines, forehead lines, wrinkles, crow's feet, marionette lines, stretch marks, wounds, accidents, bites, surgery, or the like. Particularly suitable for treatment of the present invention are contour defects in areas such as the eyes, cheeks, nose, lips, forehead, and neck. Additionally, the disclosed embodiments can provide patients with more consistent, long-lasting cosmetic results. For example, a 20% variation in duration of effect with a longer-acting neurotoxin can result in a one-month difference in effective duration. With the disclosed fast-recovering neurotoxins, this 20% variation results in a less drastic difference in effective duration.

[0030] Additional administrations of a fast-acting neurotoxin can effectively correct or augment a previous administration of a cosmetic neurotoxin. For example, the methods disclosed herein may include additional administrations to correct uneven cosmetic results from a previous administration, or to increase the cosmetic results of a previous administration, or to accelerate the onset of results compared to results achieved with a non-fast-acting neurotoxin. The disclosed fast-acting neurotoxin composition may be administered using a needle or a needle-free device. In certain embodiments, the method includes injecting the composition subcutaneously into the individual. For example, the administration includes injecting the composition through a needle of about 30 gauge or less. In certain embodiments, the method includes administering a composition comprising botulinum toxin type E.

[0031] The injection of the composition may be performed by a syringe, catheter, needle, and other means for injection. The injection may be performed in any area of ​​the mammalian body requiring treatment, including but not limited to the face, neck, torso, arms, hands, legs, and feet. The injection may be performed in any location in a specific area, such as the epidermis, dermis, fat, muscle, or subcutaneous layer. The frequency and amount of injections under the disclosed method may be determined based on the nature and location of the particular cosmetic irregularity being treated. However, in certain cases, repeated injections may be desired to achieve optimal results. The frequency and amount of injections for each particular case may be determined by one skilled in the art. Although examples of routes of administration and dosages are provided, the appropriate routes of administration and dosages are generally determined by the attending physician on a case-by-case basis. Such determinations are routine for those skilled in the art. For example, the routes of administration and dosages of the disclosed clostridial neurotoxins of the present invention can be selected based on criteria such as the solubility characteristics of the selected neurotoxin and the intensity and extent of the cosmetic condition to be treated.

[0032] Fast-acting neurotoxins are around 10 -3 The neurotoxin may be administered in an amount of about 10 U / kg body weight to about 35 U / kg body weight. -2 In another embodiment, the neurotoxin is administered in an amount of about 10 U / kg to about 25 U / kg. -1 In one embodiment, the neurotoxin is administered in an amount of about 1 U / kg to about 15 U / kg. In another embodiment, the neurotoxin is administered in an amount of about 1 U / kg to about 10 U / kg. In many instances, administration of about 1 unit to about 500 units of a neurotoxin, such as a botulinum type E, provides effective therapeutic relief. In one embodiment, about 5 units to about 200 units of a neurotoxin, such as a botulinum type E, can be used, and in another embodiment, about 10 units to about 100 units of a neurotoxin, such as a botulinum type E, can be administered locally to a target tissue, such as muscle.

[0033] In embodiments, administration is about 4 units of neurotoxin, or about 5 units of neurotoxin, or about 6 units of neurotoxin, or about 7 units of neurotoxin, or about 8 units of neurotoxin, or about 10 units of neurotoxin, or about 15 units of neurotoxin, or about 20 units of neurotoxin, or about 30 units of neurotoxin, or about 40 units of neurotoxin, or about 50 units of neurotoxin, or about 60 units of neurotoxin, or about 70 units of neurotoxin, or about 80 units of neurotoxin, or about 90 units of neurotoxin. or about 100 units of neurotoxin, or about 110 units of neurotoxin, or about 120 units of neurotoxin, or about 130 units of neurotoxin, or about 140 units of neurotoxin, or about 150 units of neurotoxin, or about 160 units of neurotoxin, or about 170 units of neurotoxin, or about 180 units of neurotoxin, or about 190 units of neurotoxin, or about 200 units of neurotoxin, or about 210 units of neurotoxin, or about 220 units of neurotoxin, or about 230 units of neurotoxin, or About 240 units of neurotoxin, or about 250 units of neurotoxin, or about 260 units of neurotoxin, or about 270 units of neurotoxin, or about 280 units of neurotoxin, or about 290 units of neurotoxin, or about 290 units of neurotoxin, or about 300 units of neurotoxin, or about 310 units of neurotoxin, or about 320 units of neurotoxin, or about 330 units of neurotoxin, or about 340 units of neurotoxin, or about 350 units of neurotoxin, or about 360 units of neurotoxin, or about 370 units of neurotoxin units of neurotoxin, or about 380 units of neurotoxin, or about 390 units of neurotoxin, or about 400 units of neurotoxin, or about 410 units of neurotoxin, or about 420 units of neurotoxin, or about 430 units of neurotoxin, or about 440 units of neurotoxin, or about 450 units of neurotoxin, or about 460 units of neurotoxin, or about 470 units of neurotoxin, or about 480 units of neurotoxin, or about 490 units of neurotoxin, or about 500 units of neurotoxin, etc.

[0034] In an embodiment, administration is about 4 units of botulinum type E neurotoxin, or about 5 units of botulinum type E neurotoxin, or about 6 units of botulinum type E neurotoxin, or about 7 units of botulinum type E neurotoxin, or about 8 units of botulinum type E neurotoxin, or about 10 units of botulinum type E neurotoxin, or about 15 units of botulinum type E neurotoxin, or about 20 units of botulinum type E neurotoxin, or about 30 units of botulinum type E neurotoxin, or about 40 units of botulinum type E neurotoxin, or about 50 units of botulinum type E neurotoxin, or about 60 units of botulinum type E neurotoxin. or about 70 units of botulinum type E neurotoxin, or about 80 units of botulinum type E neurotoxin, or about 90 units of botulinum type E neurotoxin, or about 100 units of botulinum type E neurotoxin, or about 110 units of botulinum type E neurotoxin, or about 120 units of botulinum type E neurotoxin, or about 130 units of botulinum type E neurotoxin, or about 140 units of botulinum type E neurotoxin, or about 150 units of botulinum type E neurotoxin, or about 160 units of botulinum type E neurotoxin, or about 170 units of botulinum type E neurotoxin, or about 180 units of botulinum type E neurotoxin, or about 190 units of botulinum type E neurotoxin, or about 200 units of botulinum type E neurotoxin, or about 210 units of botulinum type E neurotoxin, or about 220 units of botulinum type E neurotoxin, or about 230 units of botulinum type E neurotoxin, or about 240 units of botulinum type E neurotoxin, or about 250 units of botulinum type E neurotoxin, or about 260 units of botulinum type E neurotoxin, or about 270 units of botulinum type E neurotoxin, or about 280 units of botulinum type E neurotoxin, or about 290 units of botulinum type E neurotoxin, about 290 units of botulinum type E neurotoxin, or about 300 units of botulinum type E neurotoxin, or about 310 units of botulinum type E neurotoxin, or about 320 units of botulinum type E neurotoxin, or about 330 units of botulinum type E neurotoxin, or about 340 units of botulinum type E neurotoxin, or about 350 units of neurotoxin, or about 360 units of botulinum type E neurotoxin, or about 370 units of botulinum type E neurotoxin, or about 380 units of botulinum type E neurotoxin, or about 390 units of botulinum type E neurotoxin,or about 400 units of botulinum type E neurotoxin, or about 410 units of botulinum type E neurotoxin, or about 420 units of botulinum type E neurotoxin, or about 430 units of botulinum type E neurotoxin, or about 440 units of botulinum type E neurotoxin, or about 450 units of botulinum type E neurotoxin, or about 460 units of botulinum type E neurotoxin, or about 470 units of botulinum type E neurotoxin, or about 480 units of botulinum type E neurotoxin, or about 490 units of botulinum type E neurotoxin, or about 500 units of botulinum type E neurotoxin, etc.

[0035] Disclosed herein are methods for expressing and communicating neurotoxin dosages. In embodiments, the dosage is expressed as an amount of protein, e.g., in nanograms (ng). In embodiments, the neurotoxin may include a botulinum toxin. The methods disclosed herein may include administering to a patient a neurotoxin, e.g., a fast-acting neurotoxin, where the dose of the neurotoxin is expressed as an amount of protein, e.g., amount of protein per dose. In one embodiment, the fast-acting neurotoxin is a botulinum toxin, e.g., botulinum type E.

[0036] In embodiments, the dose of neurotoxin is expressed as the amount or concentration of protein. For example, in embodiments, the neurotoxin may be administered in an amount of about .2 ng to 20 ng. In one embodiment, the neurotoxin is administered to a target tissue, such as muscle, in an amount of about .3 ng to 19 ng, about .4 ng to 18 ng, about .5 ng to 17 ng, about .6 ng to 16 ng, about .7 ng to 15 ng, about .8 ng to 14 ng, about .9 ng to 13 ng, about 1.0 ng to 12 ng, about 1.5 ng to 11 ng, about 2 ng to 10 ng, about 5 ng to 7 ng, etc.

[0037] In embodiments, administration may comprise a total dose of 5 to 7 ng, 7 to 9 ng, 9 to 11 ng, 11 to 13 ng, 13 to 15 ng, 15 to 17 ng, 17 to 19 ng, and the like. In embodiments, administration may include a total dose of 5 ng or less, 6 ng or less, 7 ng or less, 8 ng or less, 9 ng or less, 10 ng or less, 11 ng or less, 12 ng or less, 13 ng or less, 14 ng or less, 15 ng or less, 16 ng or less, 17 ng or less, 18 ng or less, 19 ng or less, 20 ng or less, etc. In embodiments, administration may include a total dose of 5 ng or more, 6 ng or more, 7 ng or more, 8 ng or more, 9 ng or more, 10 ng or more, 11 ng or more, 12 ng or more, 13 ng or more, 14 ng or more, 15 ng or more, 16 ng or more, 17 ng or more, 18 ng or more, 19 ng or more, 20 ng or more, etc.

[0038] In embodiments, administration is of about 0.1 ng neurotoxin, 0.2 ng neurotoxin, 0.3 ng neurotoxin, 0.4 ng neurotoxin, 0.5 ng neurotoxin, 0.6 ng neurotoxin, 0.7 ng neurotoxin, 0.8 ng neurotoxin, 0.9 ng neurotoxin, 1.0 ng neurotoxin, 1.1 ng neurotoxin, 1.2 ng neurotoxin, 1.3 ng neurotoxin, 1.4 ng neurotoxin, 1.5 ng neurotoxin, 1.6 ng neurotoxin, 1.7 ng neurotoxin, 1.8 ng neurotoxin, 1.9 ng neurotoxin, 2.0 ng neurotoxin, 2.1 ng neurotoxin, 2.2 ng neurotoxin, 2.3 ng neurotoxin, 2.4 ng neurotoxin, 2.5 ng neurotoxin, 2.6 ng neurotoxin, 2.7 ng neurotoxin, 2.8 ng neurotoxin, 2.9 ng neurotoxin, 3.0 ng The total dose of neurotoxin may include 3.1 ng neurotoxin, 3.2 ng neurotoxin, 3.3 ng neurotoxin, 3.4 ng neurotoxin, 3.5 ng neurotoxin, 3.6 ng neurotoxin, 3.7 ng neurotoxin, 3.8 ng neurotoxin, 3.9 ng neurotoxin, 4.0 ng neurotoxin, 4.1 ng neurotoxin, 4.2 ng neurotoxin, 4.3 ng neurotoxin, 4.4 ng neurotoxin, 4.5 ng neurotoxin, 5 ng neurotoxin, 6 ng neurotoxin, 7 ng neurotoxin, 8 ng neurotoxin, 9 ng neurotoxin, 10 ng neurotoxin, 11 ng neurotoxin, 12 ng neurotoxin, 13 ng neurotoxin, 14 ng neurotoxin, 15 ng neurotoxin, 16 ng neurotoxin, 17 ng neurotoxin, 18 ng neurotoxin, 19 ng neurotoxin, 20 ng neurotoxin, and the like.

[0039] In embodiments, administration may be, for example, about 0.1 ng of botulinum type E neurotoxin, 0.2 ng of botulinum type E neurotoxin, 0.3 ng of botulinum type E neurotoxin, 0.4 ng of botulinum type E neurotoxin, 0.5 ng of botulinum type E neurotoxin, 0.6 ng of botulinum type E neurotoxin, 0.7 ng of botulinum type E neurotoxin, 0.8 ng of botulinum type E neurotoxin, 0.9 ng of botulinum type E neurotoxin, 1.0 ng of botulinum type E neurotoxin, 1.1 ng of botulinum type E neurotoxin, 1.2 ng of botulinum type E neurotoxin, or 1.4 ng of botulinum type E neurotoxin per injection. botulinum type E neurotoxin, 1.3ng botulinum type E neurotoxin, 1.4ng botulinum type E neurotoxin, 1.5ng botulinum type E neurotoxin, 1.6ng botulinum type E neurotoxin, 1.7ng botulinum type E neurotoxin, 1.8ng botulinum type E neurotoxin, 1.9ng botulinum type E neurotoxin, 2.0ng botulinum type E neurotoxin, 2.1ng botulinum type E neurotoxin, 2.2ng botulinum type E neurotoxin, 2.3ng botulinum type E neurotoxin, 2.4ng neurotoxin, 2.5ng neurotoxin, 2.6ng g of botulinum type E neurotoxin, 2.7 ng of botulinum type E neurotoxin, 2.8 ng of botulinum type E neurotoxin, 2.9 ng of botulinum type E neurotoxin, 3.0 ng of botulinum type E neurotoxin, 3.1 ng of botulinum type E neurotoxin, 3.2 ng of botulinum type E neurotoxin, 3.3 ng of botulinum type E neurotoxin, 3.4 ng of botulinum type E neurotoxin, 3.5 ng of botulinum type E neurotoxin, 3.6 ng of botulinum type E neurotoxin, 3.7 ng of botulinum type E neurotoxin, 3.8 ng of botulinum type E neurotoxin, 3. The dose may include 9 ng of botulinum type E neurotoxin, 4.0 ng of botulinum type E neurotoxin, 4.1 ng of botulinum type E neurotoxin, 4.2 ng of botulinum type E neurotoxin, 4.3 ng of botulinum type E neurotoxin, 4.4 ng of botulinum type E neurotoxin, 4.5 ng of botulinum type E neurotoxin, 5 ng of botulinum type E neurotoxin, 6 ng of botulinum type E neurotoxin, 7 ng of botulinum type E neurotoxin, 8 ng of botulinum type E neurotoxin, 9 ng of botulinum type E neurotoxin, 10 ng of botulinum type E neurotoxin, and the like. Ultimately, however, both the amount of toxin administered and the frequency of its administration will be at the discretion of the treating physician, commensurate with issues of safety and efficacy provided by the toxin.

[0040] A controlled release system may be used in the embodiments described herein to deliver neurotoxins in vivo at a predetermined rate for a specific time. In general, the release rate is determined by the design of the system and may be largely independent of environmental conditions such as pH. Controlled release systems are known that can deliver drugs over a period of years. In contrast, sustained release systems typically deliver drugs within 24 hours, and environmental factors may affect the release rate. Thus, the release rate of neurotoxins from implanted controlled release systems ("implants") is a function of the physiochemical properties of the implant carrier material and the drug itself. Typically, implants are made of inert materials that induce little or no host response. The controlled release system may include the neurotoxin incorporated into a carrier. The carrier may be a polymer or a bioceramic material. The controlled release system may be injected, inserted or implanted into a selected site in a patient's body and remain there for an extended period of time during which the neurotoxin is released by the implant in a manner and at a concentration that provides the desired therapeutic effect. The polymeric material can release the neurotoxin by diffusion, chemical reaction or solvent activation, as well as under the influence of magnetic, ultrasonic or temperature factors. Diffusion can be from a reservoir or matrix. Chemical control can result from degradation of the polymer or cleavage of the drug from the polymer. Solvent activation can include swelling of the polymer or osmotic effects.

[0041] The implants may be prepared by mixing the desired amount of stabilized neurotoxin into a solution of the appropriate polymer in methylene chloride. The solution may be prepared at room temperature. The solution may then be transferred to a petri dish and the methylene chloride may be evaporated in a vacuum desiccator. Depending on the size of the implant desired and therefore the amount of neurotoxin formulated, an appropriate amount of the dry implant formulated with neurotoxin is compressed in a mold at about 8000 psi for 5 seconds or 3000 psi for 17 seconds to form an implant disk encapsulating the neurotoxin.

[0042] Preferably, the embedding material used is substantially non-toxic, non-carcinogenic, and non-immunogenic. Suitable embedding materials include poly(2-hydroxyethyl methacrylate) (p-HEMA), poly(N-vinylpyrrolidone) (p-NVP), + These include polymers such as poly(vinyl alcohol) (PVA), poly(acrylic acid) (PM), polydimethylsiloxane (PDMS), ethylene-vinyl acetate (EVAc) copolymers, polyvinylpyrrolidone / methyl acrylate copolymers, polymethyl methacrylate (PMMA), polylactic acid (PLA), polyglycolic acid (PGA), polyanhydrides, polyorthoesters, collagen and cellulose derivatives and bioceramics such as hydroxyapatite (HPA), tricalcium phosphate (TCP), and aluminocalcium phosphate (ALCAP). Lactic acid, glycolic acid and collagen may be used to make biodegradable implants. The implants may be biodegradable or bioerodible. The advantage of bioerodible implants is that they do not need to be removed from the patient. Bioerodible implants may be based on either membrane or matrix release of bioactive agents. Biodegradable microspheres prepared from PLA-PGA are known for subcutaneous or intramuscular administration.

[0043] Kits for carrying out the disclosed embodiments are also encompassed by the present disclosure. The kits may include a 30 gauge or smaller needle and a corresponding syringe. The kits also include a Clostridium neurotoxin composition, such as a botulinum type E toxin composition. The neurotoxin composition may be provided in a syringe. The composition is injectable through the needle. The kits are designed in various forms based on the dimensions of the syringe and needle and the volume of the injectable composition contained therein, which in turn is based on the particular cosmetic defect the kit is designed to treat. EXAMPLES

[0044] The following non-limiting examples are provided for illustrative purposes only to facilitate a more complete understanding of representative embodiments, and should not be construed as limiting any of the embodiments described herein.

[0045] (Example 1) Use of botulinum toxin type E to treat glabellar lines (GL) This first-in-human, randomized, double-blind, placebo-controlled, ascending dose cohort study enrolled 42 subjects who received EB-001 (a botulinum type E composition disclosed herein) (N=35) or placebo (N=7). The primary efficacy outcome was the proportion of subjects who demonstrated an investigator-assessed improvement of 2 grades (IR-2) in GL severity at maximum grimacing. Safety assessments included adverse events (AEs), laboratory tests, and physical examinations. IR-2 responses were observed beginning with the third cohort (EB-001) and were more frequent at higher doses. Onset of clinical benefit was within 24 hours, with duration ranging from 14 to 30 days for the highest dose. AE incidence was low, with mild to moderate headache being the most common. There were no serious AEs or ptosis, and no clinically significant changes in other safety assessments. In this clinical trial in GL, EB-001 demonstrated favorable safety and tolerability, as well as dose-dependent efficacy with an 80% response rate at the highest dose. Maximum clinical efficacy of EB-001 was observed within 24 hours and lasted for 14 to 30 days. This distinct profile of EB-001 supports its development into aesthetic and therapeutic applications where rapid onset and short duration of effect are desirable.

[0046] Botulinum neurotoxins inhibit the presynaptic release of acetylcholine and are among the most potent molecules in nature. When injected into muscle, botulinum neurotoxins inhibit neuromuscular transmission and produce dose-dependent local muscle relaxation. Purified botulinum neurotoxins, including serotypes A and B, have been developed as injectable drugs and are widely used to treat a variety of neuromuscular pathologies. Botulinum neurotoxin serotype E is a novel serotype that has not previously been developed for clinical use. Botulinum toxin type E has the most rapid onset of action and the shortest duration of action among all botulinum neurotoxins. Type E has a similar domain structure to type A, consisting of two protein chains, a 100 kDa heavy chain and a 50 kDa light chain linked by a disulfide bond. Type E inhibits neuromuscular transmission by cleaving the same presynaptic vesicle protein (synaptosome-associated protein 25) as type A, but at a different cleavage site than type A. Two binding sites on motor axons mediate high affinity recognition of nerve cells by botulinum neurotoxin. Binding is mediated first by cell surface gangliosides and then by specific protein receptors. These receptors are found on motor axon terminals at the neuromuscular junction. Both botulinum toxin types A and E have been shown to bind to a specific receptor, synaptic vesicle protein 2, which only these two serotypes have in common. This was the first clinical trial to evaluate the safety and efficacy of ascending doses of botulinum toxin type E in subjects with GL.

[0047] The study was a first-in-human evaluation of the safety and efficacy of EB-001, with a focus on the treatment of moderate to severe GL. EB-001 is a proprietary purified form of botulinum toxin type E formulated as an injectable solution (Bonti, Inc., Newport Beach, California, USA). The study was a randomized, double-blind, placebo-controlled, ascending dose cohort study conducted at two specialized clinical centers (Steve Yoelin, MD Medical Associates, Newport Beach, California, USA; Center for Dermatology Clinical Research, Fremont, California, USA). The study was approved by an Institutional Review Board (Aspire Institutional Review Board, Santee, California, USA) and conducted in accordance with the guidelines set by the Declaration of Helsinki. Written informed consent was received from all subjects prior to study participation.

[0048] A total of 42 healthy, toxin-naive, male and female subjects aged 18 to 60 years were enrolled in the study. Each subject's participation was to last approximately 6 weeks. Primary inclusion criteria were: presence of symmetric GL rated as moderate to severe at full frown, sufficient vision without glasses to accurately assess their own facial wrinkles (contact lens use was acceptable), and ability to comply with the requirements of the study. The main exclusion criteria were: any uncontrolled systemic disease or other medical condition, any medical condition that may have placed the subject at increased risk from exposure to botulinum neurotoxin (including diagnosed myasthenia gravis, Eaton-Lambert syndrome, amyotrophic lateral sclerosis, or any other medical condition that interferes with neuromuscular function), current or previous botulinum neurotoxin treatment, known immunological procedures or hypersensitivity to botulinum neurotoxin, prespecified dermatological procedures (e.g., noninvasive resurfacing, facial cosmetic procedures, topical / oral retinoid treatments) within 3 to 12 months prior to the study, and history of periorbital surgery or treatment. Women were not enrolled if they were pregnant, breastfeeding, or planning to become pregnant. Men with female partners of childbearing potential were enrolled only if they agreed to use dual contraception for 3 months after dosing.

[0049] At screening, subject demographics, medical history, and prior and concomitant medications were recorded, and alcohol / drug screens were performed.Standardized facial photographs were taken at baseline before treatment, at each follow-up visit, and at the end of the study, but photographs were not used to evaluate efficacy.

[0050] Seven cohorts (six subjects per cohort) were enrolled and received ascending doses of EB-001 or placebo in a 5:1 ratio. The highest recommended starting dose (with a 10-fold safety factor) in this first-in-human study was set based on the no-observed-toxicity level from preclinical safety and toxicity studies (data not published). From this, the base dose (cohort 1) was calculated and a sub-efficacious dose was determined, with cohorts 2 to 7 receiving 3, 9, 12, 16, 21, and 28 doses of the base dose, respectively, which corresponded to the sub- to maximally effective doses of EB-001. The total dose was administered in a standardized manner in equal volumes across five injection sites (0.1 mL per site into the procerus, left and right corrugator supercilii medialis, and left and right corrugator supercilii lateralis) (see Figure 1). Injections into the lateral corrugator supercilii were spaced approximately 1 cm above the supraorbital ridge. EB-001 was supplied in a 5 mL vial as a sterile solution for injection. Placebo was supplied in a matched vial without EB-001. Each subject completed visits at Screening (Days -30 to -1), Baseline / Injection (Day 0), Days 1, 2, 7, 14, and 30 (Study End), and Day 42 (Final Safety Follow-Up).

[0051] Safety was assessed by adverse events (AEs), laboratory tests, electrocardiogram (ECG), physical examination, vital signs (pulse rate, respiratory rate, and blood pressure), urine pregnancy test (for women of childbearing potential), and focused neurologic examination to assess potential spread of botulinum neurotoxin. Treatment-emergent adverse events (TEAEs) were defined as any AE that began or worsened in severity after exposure to study treatment. AEs and TEAEs were aggregated by system organ class and basic terms using the Medical Dictionary for Regulatory Activities (MedDRA, version 19.0). Serious AEs (SAEs, or AEs that met regulatory criteria for drug severity) and discontinuations due to AEs were also assessed. AE severity was recorded as mild, moderate, severe, or life-threatening events. Prior to enrollment in each dose cohort, a safety data review committee was convened to analyze all safety data from previous cohorts.

[0052] At screening, baseline, and days 1, 2, 7, 14, and 30, subjects' GL was assessed using the Facial Wrinkle Scale (FWS) at maximum frown and at rest. Assessments were completed by the investigator and the subject. The FWS is a widely accepted method used for the assessment of facial wrinkle severity. In this study, the 4-point scale of GL severity was as follows: 0=none, 1=mild, 2=moderate, and 3=severe. Subjects were considered treatment responders if they achieved at least a 2-grade improvement (reduction) (IR-2) based on the investigator's FWS assessment. The primary efficacy variable was the proportion of IR-2 responders at maximum frown at any post-baseline visit up to day 30. An additional efficacy outcome of interest was the proportion of responders who achieved an investigator-assessed FWS grade of none or mild (analyzed by visit) at days 1, 2, 7, 14, or 30.

[0053] Two analysis populations were prespecified: safety and efficacy populations. Subjects who received placebo were pooled for all analyses. The safety population included all subjects who received study treatment and subsequently had at least one safety assessment. All TEAEs and SAEs were aggregated by treatment group. All safety parameters, including laboratory tests, ECG, physical examination, vital signs, urine pregnancy test, and focused neurological examination, were reviewed by the investigator and assessed for clinical significance. The efficacy population was the modified intent-to-treat (mITT) population, defined as all randomized subjects who received at least one dose of study treatment and had at least one post-baseline efficacy assessment. Analyses of demographic and baseline characteristics were performed on the mITT population. Medical histories were based on the safety population and were coded using MedDRA and aggregated by system organ class and preferred terminology. Prior and concomitant medications were based on the safety population and were coded using the World Health Organization Anatomical Therapeutic Chemical classification index and aggregated by drug class and treatment group. Efficacy analyses were performed using the mITT population. FWS grades were aggregated by treatment and study date using frequency counts and percentage response rates. Analyses comparing the proportion of IR-2 responders in each EB-001 cohort versus (pooled) placebo were performed using Fisher's exact test at a significance level of 0.05.

[0054] Of the 59 subjects selected for the study, 43 were enrolled in one of the seven cohorts. One subject did not receive treatment, resulting in 42 subjects being included in the mITT and safety populations (35 EB-001-treated and 7 placebo-treated). One subject was lost to follow-up, and 41 subjects completed the study. Demographic and baseline characteristics of the mITT population are shown in Table 1. The mean (range) age of subjects was 47.9 (22-60) years and 50.4 (32-57) years for EB-001-treated (pooled) versus placebo-treated (pooled) subjects, respectively. The majority of subjects were female (EB-001=91.4%; placebo=85.7%) and white (71.4% in both groups). Baseline mean (standard deviation [SD]) investigator-assessed GL at maximal frown was 2.6 (0.50) and 2.9 (0.38) in the EB-001 and placebo groups, respectively. The EB-001 and placebo groups were well balanced with no substantial between-group differences.

[0055] The percentage of subjects in the mITT population who achieved an IR-2 response for GL severity at maximal frown at any post-baseline visit through day 30 is shown by dose cohort in Figure 2. In cohort 3, 40% of subjects were IR-2 responders. This response rate was similar or higher in all higher dose cohorts, with cohorts 6 and 7 having 80% IR-2 responders. Cohorts 6 and 7 showed significantly higher percentages of IR-2 responders versus placebo (P=0.046). Figure 3 summarizes the percentage of subjects in each cohort with investigator-assessed FWS grade of no GL or mild GL at maximal frown at any post-baseline visit through day 30. Cohorts 2 to 7 (inclusive) had a higher percentage of responders versus placebo, with cohorts 3 and above achieving rates of 60% to 100%. No or mild responses were most commonly observed on days 1, 2, and / or 7 in cohorts 3 through 7. One responder (20%) was observed on day 14 in cohorts 3, 5, 6, and 7, and on day 30 in cohorts 3 and 5. Safety results support the safety of EB-001 at all evaluated doses administered as an IM injection in this population. No clinically significant changes from baseline in neurological examination, ECG, physical examination, or laboratory tests were observed in any subject.

[0056] Five subjects treated with EB-001 reported TEAEs, none in the placebo group. No SAEs were reported, and no TEAEs led to discontinuation of the study. All TEAEs were mild or moderate in severity. Sore throat and flu-like symptom events were considered unrelated to treatment. Three subjects reported a TEAE of headache, one of which was considered related to treatment. There was no dose-related increase in the occurrence of headache. There were no developments of ptosis or other TEAEs that appeared to be related to the spread of toxins.

[0057] To our knowledge, this is the first controlled clinical trial of a botulinum toxin type E product in any aesthetic or therapeutic use. This first-in-human study of EB-001, a novel purified form of botulinum toxin type E administered IM, served its objectives of evaluating the safety, tolerability, and effective dose range of EB-001. A dose response was observed, with a greater proportion of treatment responders in cohorts receiving higher doses of EB-001. IR-2 responses were observed beginning in cohort 3 and increased in the higher dose cohorts, suggesting that an effective dose range for EB-001 may be the dose used in cohorts 4 through 7. Cohorts 6 and 7 had 80% IR-2 responders, a response rate similar to that of approved botulinum toxin type A products. Subjects achieving none or mild FWS grades were observed beginning in cohort 2. In terms of onset of effect, therapeutic responses were observed as early as 24 hours after administration, supporting previous reports suggesting that botulinum toxin type E has a more rapid onset than type A.

[0058] Duration of response, defined as the proportion of responders with none or mild responses, was observed in the majority of the five higher dose cohorts by day 14, and in one subject in two of the five higher dose cohorts by day 30. All doses of EB-001 were well tolerated without local injection site reactions. No SAEs or serious TEAEs were reported, and there were no discontinuations due to TEAEs. The most common TEAE, headache, was mild or moderate in severity, and there were no other treatment-related AEs. There were no incidents of ptosis at any dose level, and no events likely related to toxin diffusion. Thus, the clinical safety and tolerability profile appears favorable in this study. The efficacy and safety profile of EB-001 is promising, supporting its potential as a unique treatment option in the treatment of GL and other facial aesthetic applications. Rapid onset can satisfy an unmet need for people seeking rapid treatment of facial wrinkles before an unexpected social or professional event. Limited duration of effect can be beneficial for people considering using botulinum neurotoxin treatment for the first time and are unwilling to make long-term commitments. EB-001 treatment will allow people to evaluate the aesthetic effect over a shorter duration compared to the 12-week duration of effect of botulinum toxin type A products. In this first-of-its-kind clinical trial in subjects with GL, EB-001 demonstrated favorable safety and tolerability in all cohorts. Five of the seven cohorts demonstrated numerically higher response rates compared to placebo. This supports the efficacy of EB-001 in reducing the severity of GL. The two highest doses resulted in an 80% response rate, similar to approved botulinum toxin type A products. In contrast to the known time course of type A products, the clinical effect of EB-001 was noted within 24 hours (onset) and continued for 14 to 30 days (duration). This distinct clinical profile supports the future advancement of EB-001 for aesthetic and critical therapeutic uses on the face where rapid onset and short duration of effect are desirable.

[0059] [Table 1] (Example 2) Use of botulinum toxin type E to treat crow's feet A 57-year-old man with crow's feet caused by years of sun exposure seeks treatment from his doctor. The doctor recommends the composition disclosed herein, which is injected subcutaneously into each of the patient's eyes. Each injection site receives approximately 3 units of botulinum toxin type E, with multiple injections on either side of the eye. The crow's feet disappear approximately 2 days after treatment and remain reduced for 2 months.

[0060] (Example 3) Use of Botulinum Toxin Type E for Brow Lift A 60 year old woman has eyebrows that extend below the brow bone. A physician recommends the compositions disclosed herein, which are injected subcutaneously above each eye. Each injection site receives approximately 10 units of botulinum toxin type E, with multiple injections on either side of the eye. Eyebrow droop is reduced within approximately 2 days and is substantially relieved for 3 months after administration.

[0061] (Example 4) Use of botulinum toxin type E for breast augmentation A 30 year old woman elects to have breast augmentation surgery. Four hours prior to surgery, botulinum toxin type E is administered proximal to where the surgical incision will be made. The administration reduces muscle tension in the incision area, resulting in minimal scarring. An additional dose is administered two weeks after surgery.

[0062] (Example 5) Use of botulinum toxin type E in breast reconstruction A 30 year old woman elects to have breast reconstruction surgery. 14 hours prior to surgery, botulinum toxin type E is administered proximal to where the surgical incision will be made. Administration reduces muscle tension in the incision area, resulting in minimal scarring. An additional dose is administered two weeks after surgery.

[0063] (Example 6) Use of botulinum toxin type E to treat episiotomy A 44 year old woman undergoes an episiotomy. Immediately after surgery, 4 ng of botulinum toxin type E is administered to the tissue surrounding the surgical area. Within 20 hours, there is a significant decrease in muscle and nerve activity around the wound.

[0064] (Example 7) Use of botulinum toxin type E to treat sports hernias A 28-year-old man suffers from a sports hernia. His physician administers 4 ng of botulinum toxin type E to the tissues surrounding both hernias. Within 10 hours, there is a significant decrease in muscle and nerve activity around the injury.

[0065] (Example 8) Use of botulinum toxin type E to treat shoulder spondylolisthesis A 48-year-old man suffers from a spondylolisthesis of the shoulder. His physician administers 4 ng of botulinum toxin type E to the tissues surrounding both herniated discs. Within 16 hours, there is a significant decrease in muscle and nerve activity around the injury.

[0066] (Example 9) Use of botulinum toxin type E to treat anterior cruciate ligament rupture A 23-year-old woman suffers from a torn anterior cruciate ligament. Six hours after the injury, the attending physician administers 7 ng of botulinum toxin type E into the muscle surrounding the torn ligament. Thirty hours later, there is a significant decrease in muscle and nerve activity around the wound. In closing, although aspects of the present specification are highlighted by reference to specific embodiments, it should be understood that those skilled in the art will readily recognize that these disclosed embodiments are merely illustrative of the principles of the subject matter disclosed herein. Thus, it should be understood that the disclosed subject matter is in no way limited to the specific methods, protocols, and / or reagents, etc., described herein. Thus, various modifications or alterations to the disclosed subject matter, or alternative configurations, may be made in accordance with the teachings herein without departing from the spirit of the present specification. Finally, the terms used herein are intended only to describe particular embodiments, and are not intended to limit the scope of the present disclosure, which is defined solely by the claims. Thus, the embodiments of the present disclosure are not limited to the exact embodiments shown and described. 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 of these described embodiments will become 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. Moreover, any combination of all possible variations of the foregoing embodiments is encompassed by the disclosure unless otherwise indicated herein or otherwise clearly contradicted by context. Groups of alternative embodiments, elements, or steps of the present disclosure should not be construed as limiting. Each group member may be referenced or claimed alone or in any combination with other group members disclosed herein. It is anticipated that one or more members of a group may be included in or deleted from a group for reasons of convenience and / or patentability. When such inclusion or deletion occurs, the specification is deemed to include the group as modified so as to satisfy the written description of all Markush groups used in the appended claims.

[0067] Unless otherwise indicated, all numbers expressing features, items, amounts, parameters, properties, periods, etc. used in the specification and claims should be understood to be modified in all instances by the term "about". As used herein, the term "about" means that the feature, item, amount, parameter, property, or period so modified includes a range of plus or minus 10 percent above and below the value of the stated feature, item, amount, parameter, property, or period. Thus, unless specifically indicated 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 indication should be construed at least in light of the number of reported significant digits and by applying ordinary roughening techniques. Notwithstanding that the numerical ranges and values ​​setting forth the broad scope of the 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 their respective testing measurements. The recitation of numerical ranges of values ​​herein is merely intended to serve as a shorthand method of individually expressing that each separate value falls within the range. Unless otherwise indicated herein, each individual value of the numerical range is incorporated herein as if each individual value were individually set forth herein.

[0068] The terms "a," "an," "the," and similar referents as used in the context of describing this disclosure (particularly in the context of the claims below) should be construed to include both the singular and the plural, unless otherwise indicated herein or otherwise clearly contradicted by the context. All methods described herein may be performed in any suitable order, unless otherwise indicated herein or otherwise clearly contradicted by the context. The use of any and all examples or exemplary language presented herein (e.g., "such as") is intended merely to better clarify the disclosure and does not present a limitation on the scope otherwise claimed. No language herein should be construed as indicating any non-claimed element essential to the practice of the embodiments disclosed herein.

[0069] Specific embodiments disclosed herein may be further limited in the claims using the words consisting of or consisting essentially of. When used in a claim, whether as filed or added by amendment, the transitional term "consisting of" excludes any element, step, or ingredient not specified in the claim. The transitional term "consisting essentially of" limits the scope of the claim to those materials or steps specified and which do not materially affect the basic and novel characteristics. The embodiments of the present disclosure so claimed are essentially or expressly described and enabled herein.

Claims

1. A pharmaceutical composition comprising botulinum neurotoxin serotype E for use in a method of performing a cosmetic treatment on a human patient having glabellar lines, said method comprising administering to the patient by injection the pharmaceutical composition such that a total dose of botulinum neurotoxin serotype E is between about 0.2 ng and about 20 ng; The total dose is divided into five approximately equal doses of botulinum neurotoxin serotype E, and the approximately equal doses are administered by intramuscular injection into the following muscles: procerus, left medial corrugator supercilii, right medial corrugator supercilii, left lateral corrugator supercilii, and right lateral corrugator supercilii; and The pharmaceutical composition, wherein the onset of clinical efficacy of the cosmetic treatment is achieved within 24 hours after administration of botulinum neurotoxin serotype E.

2. The pharmaceutical composition described in claim 1, wherein the botulinum neurotoxin serotype E is a pure botulinum neurotoxin serotype E of approximately 150 kDa.

3. The pharmaceutical composition of claim 1, wherein the total dose of botulinum neurotoxin serotype E is between about 2 ng and about 10 ng.

4. The pharmaceutical composition of claim 1, wherein the clinical effect comprises a change in investigator-assessed Facial Wrinkle Scale (FWS) grade during maximal frown from moderate to severe prior to administration of botulinum neurotoxin serotype E to none or mild following administration of botulinum neurotoxin serotype E.

5. The pharmaceutical composition described in claim 4, wherein the botulinum neurotoxin serotype E is a pure botulinum neurotoxin serotype E of approximately 150 kDa.

6. The pharmaceutical composition of claim 2, wherein about 150 units of pure botulinum neurotoxin serotype E of about 150 kDa is injected into at least one muscle.

7. A pharmaceutical composition comprising about 150 kDa pure botulinum neurotoxin serotype E for use in a method of performing a cosmetic treatment on a human patient having moderate to severe glabellar wrinkles, said method comprising administering the pharmaceutical composition such that a therapeutically effective amount of about 150 kDa pure botulinum neurotoxin serotype E is administered to the patient; said therapeutically effective amount being between about 0.2 ng and about 20 ng; The therapeutically effective amount is administered in five intramuscular injections, each injection containing an approximately equal amount of pure botulinum neurotoxin serotype E of about 150 kDa, with one injection administered to the following muscles: procerus, left corrugator supercilii, right corrugator supercilii, left corrugator supercilii lateralis, and corrugator supercilii lateralis. Each of the following is carried out: The onset of clinical efficacy of the cosmetic treatment is achieved within 48 hours after administration of the pure botulinum neurotoxin serotype E of about 150 kDa; and The clinical efficacy comprises a change in Facial Wrinkle Scale (FWS) grade at maximum frown as assessed by the investigator from moderate to severe prior to administration of about 150 kDa pure botulinum neurotoxin serotype E to none or mild following administration of about 150 kDa pure botulinum neurotoxin serotype E.

8. A pharmaceutical composition comprising botulinum neurotoxin serotype E for use in a method of performing a cosmetic treatment on a human patient having glabellar lines, the method comprising administering to the patient by injection the pharmaceutical composition such that a total dose of the botulinum neurotoxin serotype E is between about 0.2 ng and about 20 ng; the total dose is divided into five approximately equal doses of botulinum neurotoxin serotype E, the approximately equal doses being administered by intramuscular injection into the following muscles: procerus, left corrugator supercilii, right corrugator supercilii, left corrugator supercilii lateralis, and right corrugator supercilii lateralis; The pharmaceutical composition, wherein the duration of the clinical effect of the cosmetic treatment ranges from about 14 days to about 30 days after administration of botulinum neurotoxin serotype E, and the clinical effect comprises a change in Facial Wrinkle Scale (FWS) grade at maximum frown as assessed by an investigator from moderate to severe before administration of botulinum neurotoxin serotype E to none or mild after administration of botulinum neurotoxin serotype E.

9. The pharmaceutical composition of claim 8, wherein the botulinum neurotoxin serotype E is a pure botulinum neurotoxin serotype E of approximately 150 kDa.

10. The pharmaceutical composition described in claim 8, wherein the total dose of botulinum neurotoxin serotype E is between about 2 ng and about 10 ng.

11. The pharmaceutical composition of claim 8, wherein the method is statistically significantly superior to an equivalent method administered a placebo in achieving a 2 or greater grade improvement (reduction) (IR-2) in maximal frown Facial Wrinkle Scale (FWS) assessment at 1, 2, 7, 14, or 30 days after administration.

12. The pharmaceutical composition of claim 11, wherein the botulinum neurotoxin serotype E is a pure botulinum neurotoxin serotype E of approximately 150 kDa.

13. The pharmaceutical composition of claim 9, wherein about 150 units of pure botulinum neurotoxin serotype E of about 150 kDa is injected into at least one muscle.

14. A pharmaceutical composition comprising about 150 kDa pure botulinum neurotoxin serotype E for use in a method of performing a cosmetic treatment on a human patient having moderate to severe glabellar wrinkles, said method comprising administering the pharmaceutical composition such that a therapeutically effective amount of about 150 kDa pure botulinum neurotoxin serotype E is administered to the patient; said therapeutically effective amount being between about 2 ng and about 10 ng; The therapeutically effective amount is administered in five intramuscular injections, each injection containing approximately equal amounts of about 150 kDa pure botulinum neurotoxin serotype E, with one injection administered to each of the following muscles: procerus, left corrugator supercilii, right corrugator supercilii, left corrugator supercilii lateralis, and right corrugator supercilii lateralis. The pharmaceutical composition, wherein each of the above steps is carried out.

15. The pharmaceutical composition of claim 14, wherein the method is statistically significantly superior to an equivalent method administered a placebo in achieving a 2 or greater grade improvement (reduction) (IR-2) in maximal frown Facial Wrinkle Scale (FWS) assessment at 1, 2, 7, 14, or 30 days after administration.

16. The pharmaceutical composition of claim 14, wherein about 150 units of pure botulinum neurotoxin serotype E of about 150 kDa is injected into at least one muscle.

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