Botulinum neurotoxin for therapy

Abstract

Disclosed herein are compositions and methods for therapeutic procedures. The use of "fast-acting" botulinum toxins for cosmetic treatments and / or to prevent or reduce scarring. "Fast-acting" refers to botulinum toxins that produce effects in patients more quickly than effects produced by, for example, botulinum neurotoxin type A. "Fast-acting" botulinum toxins, such as botulinum neurotoxin type E.

Description

[0001] This application is a divisional application of the international application filed on March 22, 2018, with application number PCT / US2018 / 023709 and the invention title "Botulinum neurotoxin for treatment". The international application entered the Chinese national phase on November 21, 2019, with application number 201880033758.1. Technical Field

[0002] This application relates to the use of neurotoxins in treatment. Background Technology

[0003] Cosmetic or plastic surgery includes both surgical and non-surgical procedures that can enhance and reshape the body, thereby improving appearance and boosting self-confidence. These procedures are becoming increasingly popular. Summary of the Invention

[0004] This document discloses compositions and methods for cosmetic treatments. For example, the disclosed embodiments include the use of "fast-acting" botulinum toxin for the treatment of, for example, glabellar lines.

[0005] This document discloses compositions and methods for minimizing scarring. For example, the disclosed embodiments include using a "fast-acting" botulinum toxin to reduce muscle tension near the wound, thereby preventing or reducing scarring.

[0006] In the implementation plan, muscle activity near skin incisions or tears is reduced, thereby reducing or preventing scar formation.

[0007] In the implementation scheme, the disclosed methods include other cosmetic procedures. For example, the disclosed implementation scheme includes combining the application of fast-acting botulinum toxin with, for example, dermal filler injections, eye lifts, rhinoplasty, etc.

[0008] In the implementation plan, botulinum toxin is a "rapid recovery" toxin.

[0009] In the implementation plan, "fast-acting" botulinum toxin is also called "rapid recovery" toxin.

[0010] In the implementation plan, cosmetic treatments may include a follow-up application of botulinum toxin after the initial application.

[0011] In the implementation plan, the disclosed method includes the combined application of a fast-acting botulinum neurotoxin with, for example, a slower-acting neurotoxin.

[0012] In the implementation scheme, the disclosed method includes the combined administration of a fast-recovering botulinum neurotoxin with, for example, a slower-recovering neurotoxin.

[0013] In the implementation plan, the neurotoxin dosage is expressed as the amount of protein. Attached Figure Description

[0014] Figure 1 The injection site is shown for use in cosmetic surgical procedures.

[0015] Figure 2 This demonstrates the main efficacy of research on the treatment of frown lines.

[0016] Figure 3 This demonstrates the secondary efficacy of research on the treatment of frown lines.

[0017] Figure 4 The efficacy of a single local application of the disclosed botulinum toxin type E composition is demonstrated in a rat model of postoperative pain. Detailed Implementation

[0018] The embodiments disclosed herein can reduce local muscle activity, thereby reducing cosmetic irregularities such as facial wrinkles. In these embodiments, cosmetic irregularities may include frown lines, forehead wrinkles, "rabbit" wrinkles, irregular smile lines, irregular chin lines, platysmal bands, "puppet" wrinkles, lip lines, crow's feet, irregular eyebrows, and combinations thereof.

[0019] The implementation plan includes methods that incorporate dermatological surgical procedures (such as treatments for actinic keratosis, seborrheic keratosis, basal cell carcinoma, squamous cell carcinoma, and other lesions or subcutaneous cysts).

[0020] For the disclosed embodiments, useful application sites may include the glabellar complex, including the corrugator supercilli and depressor supercilli muscles; the orbicularis oculi muscle; the superior lateral fibers of the orbicularis oculi muscle; the forehead; the nose; the levator labii superioris aleque nasi muscle; the orbicularis oris muscle; the masseter muscle; the depressor anguli oris muscle; and the platysma muscle. Exemplary injection sites useful in the treatment of glabellar lines include... Figure 1 As shown.

[0021] The treatment plan includes addressing severe wrinkles.

[0022] The disclosed implementation methods may include treatments for skin diseases such as acne. The disclosed implementation methods may include treatments for inflammatory skin diseases. For example, the disclosed implementation methods may include treatments for psoriasis, eczema, etc.

[0023] The embodiments disclosed herein can reduce local muscle activity, thereby reducing scar formation, such as surgical scars. In these embodiments, the surgical procedure may include cosmetic surgery, such as rhinoplasty, eyelid surgery, or belly-lifting surgery. In these embodiments, the surgical procedure may include other types of medical procedures, such as appendectomy or organ transplantation. In these embodiments, the method includes applying the disclosed composition near the wound.

[0024] The embodiments disclosed herein can reduce local muscle activity, thereby reducing scar formation, such as scars resulting from trauma. For example, following a traumatic injury, the disclosed embodiments may include applying the disclosed composition near the trauma (e.g., a laceration or amputation).

[0025] For the purposes of the disclosed embodiments, useful application sites may include any area where muscle activity is to be reduced. For example, when used in conjunction with facial cosmetic surgery procedures, the disclosed embodiments may include application to the following sites: the glabella complex, including the corrugator supercilii and depressor supercilii muscles; the orbicularis oculi muscle; the superior lateral fibers of the orbicularis oculi muscle; the forehead; the nose; the levator labii superioris muscle; the orbicularis oris muscle; the masseter muscle; the depressor anguli oris muscle; and the platysma muscle.

[0026] When used in combination with other surgical procedures, the disclosed implementation may include application to muscles such as those of the arm, leg, or torso.

[0027] The disclosed implementation may include methods for preparing the surgical site prior to the procedure in order to reduce muscle tension near the incision.

[0028] The disclosed implementation methods can promote the production of substances such as elastin and collagen. The disclosed implementation methods may include methods for increasing skin elasticity.

[0029] In embodiments, the compositions disclosed herein may comprise fast-acting botulinum toxin, such as type E.

[0030] In embodiments, the compositions disclosed herein may comprise rapid-recovery botulinum toxin, such as type E.

[0031] In embodiments, the compositions disclosed herein may comprise a fast-acting, rapidly recovering botulinum toxin, such as type E.

[0032] definition: "Administration" or "to administer" refers to the step of giving (i.e., administering) a pharmaceutical composition or active ingredient to a subject. The pharmaceutical compositions disclosed herein can be administered via many suitable routes, but as described in the disclosed methods, the compositions are administered locally via, for example, intramuscular administration (such as by injection or the use of an implant).

[0033] "Botulinum toxin" or "botulinum neurotoxin" refers to the substance produced by Clostridium botulinum (…). Clostridium botulinum Wild-type neurotoxins derived from clostridium botulinum, as well as modified, recombinant, hybrid, and chimeric botulinum toxins. Recombinant botulinum toxins can have properties derived from non-Clostridium botulinum (Clostridium difficile) Clostridial The light and / or heavy chains of the genus *Botulinum toxin* are prepared by recombinant synthesis. As used herein, "botulinum toxin" includes serotypes A, B, C, D, E, F, G, and H of botulinum toxin. "Botulinum toxin" as used herein also includes botulinum toxin complexes (i.e., complexes of 300, 600, and 900 kDa) and pure botulinum toxin (i.e., neurotoxic molecules of approximately 150 kDa), all of which can be used to carry out the present invention. "Purified botulinum toxin" refers to pure botulinum toxin or botulinum toxin complexes that are separated or substantially separated from other proteins and impurities, which may be accompanied by botulinum toxin obtained from culture or fermentation methods. Therefore, purified botulinum toxin may have at least 95%, more preferably at least 99%, of non-botulinum toxin proteins and impurities removed.

[0034] "Biocompatibility" refers to the absence of a significant inflammatory response at the implantation site.

[0035] "Clostridium neurotoxin" refers to neurotoxins produced by Clostridium bacteria such as Clostridium botulinum and Clostridium butyricum. Clostridium butyricum ) or Clostridium belladonnae ( Clostridium beratti Neurotoxins prepared or naturally derived from clostridium species, as well as clostridium neurotoxins recombined from non-clostridium species.

[0036] "Completely free of" (the term "composed of") means that the substance cannot be detected or its presence cannot be confirmed within the detection range of the instrument or method used.

[0037] "Very little or no" means that only trace amounts of the substance can be detected within the detection range of the instrument or method used.

[0038] As used in this article, "rapid-acting" means that botulinum toxin produces its effect in patients more quickly than that produced by, for example, botulinum toxin type A neurotoxin. For example, the effects of rapid-acting botulinum toxin can be seen within 36 hours.

[0039] As used in this article, "rapid recovery" refers to a faster decline in the effectiveness of botulinum toxin in patients compared to the decline in effectiveness produced by, for example, botulinum toxin type A neurotoxin. For example, the effects of rapidly recovering botulinum toxin may decline within timeframes such as 120 hours, 150 hours, 300 hours, 350 hours, 400 hours, 500 hours, 600 hours, 700 hours, and 800 hours. The efficacy of botulinum toxin type A is known to last up to 12 months. However, the typical duration of action for intramuscularly injected botulinum toxin type A neurotoxin is usually around 3 to 4 months.

[0040] As used in this article, "intermediate effect" means that the effect of botulinum toxin is slower than that of fast-acting toxins.

[0041] "Neurotoxins" are bioactive molecules that have a specific affinity for receptors on the surface of neurons. Neurotoxins include clostridium toxins, which can be pure toxins or toxins complexed with one or more non-toxic or toxin-associated proteins.

[0042] "Patient" refers to a human or non-human subject receiving medical or veterinary care.

[0043] "Pharmaceutical composition" refers to a formulation in which the active ingredient may be botulinum toxin. The term "formulation" means that, in addition to the botulinum neurotoxin active ingredient, at least one other component (e.g., but not limited to albumin [e.g., human serum albumin or recombinant human albumin] and / or sodium chloride) is present in the pharmaceutical composition. Therefore, the 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: a solution formed under lyophilized or vacuum-dried conditions, for example, by reconstitution of the lyophilized or vacuum-dried pharmaceutical composition with saline or water; or a solution that does not require reconstitution. As mentioned above, the pharmaceutical composition may be liquid or solid. The pharmaceutical composition may not contain animal protein.

[0044] "Very little or no" means that the amount present is less than one percent by weight of the culture medium, fermentation medium, pharmaceutical composition, or other materials used to assess the weight percentage of the substances contained therein.

[0045] As used in this article, “supplementary administration” refers to botulinum toxin administration following the initial administration of neurotoxin.

[0046] "Therapeutic preparations" are preparations that can be used to treat and thereby alleviate symptoms of a condition or disease and / or associated with it, such as a condition or disease characterized by peripheral muscle activity.

[0047] "Therapeutic effective dose" refers to the level, amount, or concentration of an agent (such as botulinum toxin or a pharmaceutical composition containing botulinum toxin) required to treat a disease, condition, or symptom without causing significant negative or adverse side effects.

[0048] "Treatment" means the reduction or elimination (including some reduction, significant reduction, near-complete reduction, and complete reduction), elimination, or prevention (temporary or permanent) of a disease, symptom, or condition, thereby achieving a desired therapeutic or cosmetic outcome, such as by healing injured or damaged tissue, or by altering, changing, enhancing, improving, refining, and / or beautifying an existing or perceived disease, symptom, or condition.

[0049] "Unit" or "U" refers to the amount of active BoNT standardized to have neuromuscular blocking effects equivalent to units of commercially available botulinum toxin type A.

[0050] Neurotoxin Composition The embodiments disclosed herein include neurotoxin compositions, such as fast-acting neurotoxin compositions, like botulinum toxin type E compositions. Such neurotoxins can be formulated into any pharmaceutically acceptable dosage form. Neurotoxins can also be used in any pharmaceutically acceptable form available from any manufacturer.

[0051] The embodiments disclosed herein include neurotoxin compositions, such as rapidly recovering neurotoxin compositions. Such neurotoxins can be formulated into any pharmaceutically acceptable formulation in any pharmaceutically acceptable form. Neurotoxins can also be used in any pharmaceutically acceptable form available from any manufacturer.

[0052] The embodiments disclosed herein may include various neurotoxins. For example, in an embodiment, the disclosed composition may include two types of neurotoxins, such as two types of botulinum neurotoxins, such as fast-acting and slower-acting neurotoxins, such as type E and type A. In an embodiment, the disclosed composition may include a fragment of botulinum neurotoxin, such as a 50 kDa light chain (LC).

[0053] Neurotoxins can be produced from clostridium bacteria (e.g., Clostridium botulinum, Clostridium butyricum, or Clostridium betaine). Additionally, neurotoxins can be modified neurotoxins; that is, neurotoxins in which at least one amino acid has been removed, modified, or replaced compared to natural or wild-type neurotoxins. Furthermore, neurotoxins can be recombinant neurotoxins or their derivatives or fragments.

[0054] In the embodiments, the disclosed type E compositions have 40% amino acid homology compared to type A, and they share the same basic structural 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 a receptor-binding domain and a translocation domain, while the LC contains synaptosome-associated protein (SNAP) enzyme activity. This domain structure is a common structure shared by all botulinum neurotoxin serotypes.

[0055] In the disclosed embodiments, the neurotoxin is formulated into unit dosage forms; for example, it may be provided as a sterile solution in a vial, or as a vial or capsule containing lyophilized powder for reconstitution with a suitable medium (e.g., saline for injection).

[0056] In this implementation, botulinum toxin is formulated into a solution containing saline and pasteurized human serum albumin, which stabilizes the toxin and minimizes loss through nonspecific adsorption. The solution can be aseptically filtered (using a 0.2 μm filter), placed into individual vials, and then vacuum-dried to obtain a sterile lyophilized powder. In use, the powder can be reconstituted by adding sterile, preservative-free physiological saline (0.9% sodium chloride for injection).

[0057] In one embodiment, botulinum toxin type E is provided as a 5 mL vial of sterile injectable solution 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) at pH 6.0.

[0058] In one embodiment, botulinum toxin type E is provided as a 5 mL vial of sterile injectable solution at a nominal concentration of 10 ng / mL in 0.03 M sodium phosphate, 0.12 M sodium chloride, and 1 mg / mL HSA at pH 6.0.

[0059] In one embodiment, botulinum toxin type E is provided as a 5 mL vial of sterile injectable solution at a nominal concentration of 5 ng / mL in 0.03 M sodium phosphate, 0.12 M sodium chloride, and 1 mg / mL HSA at pH 6.0.

[0060] In one embodiment, botulinum toxin type E is provided as a 5 mL vial of sterile injectable solution at a nominal concentration of 1 ng / mL in 0.03 M sodium phosphate, 0.12 M sodium chloride, and 1 mg / mL HSA at pH 6.0.

[0061] While the compositions may contain only a single type of neurotoxin, such as botulinum toxin type E, the disclosed compositions may contain two or more types of neurotoxins, which can provide enhanced therapeutic effects on the condition. For example, a composition administered to a patient may include botulinum toxin types A and E. Administration of a single composition containing two different neurotoxins may result in an effective concentration of each neurotoxin lower than the effective concentration achieved when administering a single neurotoxin to a patient while still achieving the desired therapeutic effect. 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 help reduce neurotransmission between multiple neurons. For example, the composition may contain ingredients that enhance the neurotransmission of GABA-mediated neurotransmission. A GABA is a receptor-mediated inhibitory modulator of type A gamma-aminobutyric acid (GABA). A Receptors inhibit neural activity by effectively shunting electrical currents across the cell membrane. GABA A Receptor modulators can enhance GABA A GABA inhibits receptor activity and reduces electrical or chemical signal transmission in neurons. A Examples of receptor modulators include benzodiazepines, such as diazepam, oxaxepam, lorazepam, prazepam, alprazolam, halazepam, chordiazepoxide, and chlorazepate. The composition may also contain a glutamate receptor modulator that reduces the excitatory effects mediated by glutamate receptors. Examples of glutamate receptor modulators include agents that inhibit the currents across AMPA, NMDA, and / or fumarate-type glutamate receptors. The composition may also contain agents that modulate dopamine receptors (e.g., antipsychotics), norepinephrine receptors, and / or serotonin receptors. The composition may also contain agents that affect the ion flux through voltage-gated calcium, potassium, and / or sodium channels. Therefore, the compositions used in the disclosed embodiments may contain one or more neurotoxins, such as botulinum toxin, in addition to ion channel receptor modulators that can reduce neurotransmission.

[0062] How to use The methods disclosed herein may include administering a fast-acting neurotoxin to a patient. In a preferred embodiment, the neurotoxin is botulinum toxin type E.

[0063] The methods disclosed herein may include administering a rapidly recovering neurotoxin to a patient. In a preferred embodiment, the neurotoxin is botulinum toxin type E.

[0064] Before administering the compositions disclosed herein, the anatomy of the treatment site should be carefully considered. For example, in some embodiments, the treatment target is the area with the highest concentration of the neuromuscular junction (if known). For example, in the case of intramuscular administration, the needle's position in the muscle can be confirmed by passing the muscle through its range of motion and observing the resulting movement of the needle before injection. General anesthesia, local anesthesia, and sedation may be 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. Additionally, depending on the muscle to be injected, some injections may require the use of a fine, hollow TEFLON®-coated needle guided by electromyography.

[0065] Administration of the disclosed composition may include, for example, injection into or near one or more of the following skeletal muscles, such as the occipitofrontalis, nasalis, orbicularis oris, depressor anguli oris, platysma, sternohyoid, serratus anterior, rectus abdominis, external oblique, tensor fasciae latae, brachioradialis, iliacus, psoas major, pectineus, adductor longus, sartorius, gracilis, vastus lateralis, rectus femoris, vastus medialis, and the tendon of the quadriceps femoris. femoris, patella, gastrocnemius, soleus, tibia, fibularis longus, tibialis anterior, patellar ligament, iliotibial tract, hypothenar muscles, thenar muscles, flexor carpi ulnaris, flexor digitorum superficialis, palmaris longus, flexor carpi radialis, brachioradialis, pronator teres, brachialis, biceps brachii, triceps brachii, pectoralis major. Major, deltoid, trapezius, sternocleidomastoid, masseter, orbicularis oculiThe muscles include oculi, temporalis, epicranial aponeurosis, teres major, extensor digitorum, extensor carpi ulnaris, anconeus, abductor policis longus, plantaris, calcanel tendon, soleus, adductor magnus, gluteus maximus, gluteus medius, latissimus dorsi, intraspinatus, and combinations thereof.

[0066] Application of the disclosed composition may include, for example, injection into or near one or more of the following nerves, such as the axillary nerve, phrenic nerve, spinal ganglion, spinal cord, sympathetic ganglia 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 dorsal cutaneous nerve, medial plantar nerve, medial dorsolateral cutaneous nerve, deep peroneal nerve, and muscular branches of the tibial nerve. The following nerves are involved in the treatment of femoral nerves: the patellar branch of the saphenous nerve, the common peroneal nerve, the muscular branch of the femoral nerve, the anterior cutaneous branches of the femoral nerve, the muscular branches of the sciatic nerve, the femoral nerve, the iliolinguinal nerve, the filum terminal, the iliohypogastric region, the obturator nerve, the ulna, the radius, the subcostal region, the intercostal region, and the dorsal branches of the intercostal region.The intercostal nerve, medial cutaneous branches of the intercostal nerve, mucosa and skin, deltoid muscle, vagus nerve, brachial plexus, supraclavicular nerve, facial nerve, auriculotemporal nerve, and combinations thereof.

[0067] Smooth muscle suitable for application of the disclosed composition may include any of the following: blood vessel walls, stomach walls, ureters, intestines, aorta (membranous layer of blood vessels), iris of the eye, prostate, gastrointestinal tract, respiratory tract, small arteries, arterioles, reproductive tract (both sexual), veins, glomeruli of the kidneys (called glomerular mesangial cells), bladder, uterus, hair muscles of the skin, ciliary muscles, sphincter muscles, trachea, bile ducts, etc.

[0068] The injection frequency and volume under the disclosed method can be determined based on the nature and location of the specific area being treated. However, in some cases, repeated or supplementary injections may be necessary to achieve optimal results. The injection frequency and volume in each specific case can be determined by a person skilled in the art.

[0069] In the implementation plan, the rapid-acting neurotoxin is administered prior to the surgical procedure. Administered at the following times: 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, within 60 minutes, 50 minutes, 40 minutes, 30 minutes, 20 minutes, 10 minutes, 5 minutes, and 2 minutes before surgery.

[0070] In the implementation plan, the administration of the rapid-acting neurotoxin is performed prior to the surgical procedure. In the implementation plan, the administration is performed at the following times: at least 36 hours before surgery, at least 24 hours before surgery, at least 22 hours before surgery, at least 20 hours before surgery, at least 18 hours before surgery, at least 16 hours before surgery, at least 14 hours before surgery, at least 12 hours before surgery, at least 11 hours before surgery, at least 10 hours before surgery, at least 9 hours before surgery, at least 8 hours before surgery, at least 7 hours before surgery, at least 6 hours before surgery, at least 5 hours before surgery, at least 4 hours before surgery, at least 3 hours before surgery, at least 2 hours before surgery, at least 60 minutes before surgery, at least 50 minutes before surgery, at least 40 minutes before surgery, at least 30 minutes before surgery, at least 20 minutes before surgery, at least 10 minutes before surgery, at least 5 minutes before surgery, at least 2 minutes before surgery, etc.

[0071] In the implementation plan, the administration of fast-acting neurotoxins is carried out simultaneously with the surgical procedure.

[0072] In the implementation plan, the rapid-acting neurotoxin is administered after the surgical procedure. For example, administration may be performed within the following times: within 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, 90 minutes, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, and 12 hours after surgery.

[0073] The methods disclosed herein may include administering a follow-up dose of a rapid-acting neurotoxin to a patient after the initial administration. The implementation plan including follow-up administration may further include an assessment by a physician or patient of the outcome of the previous neurotoxin administration. Such an assessment may include the use of, for example, photographs, scans, etc.

[0074] In the implementation plan, the assessment of the results of the initial administration of the neurotoxin can be performed at the following times: for example, within 6 hours of the initial administration, within 8 hours of the initial administration, within 10 hours of the initial administration, within 12 hours of the initial administration, within 14 hours of the initial administration, within 16 hours of the initial administration, within 18 hours of the initial administration, within 24 hours of the initial administration, within 30 hours of the initial administration, within 36 hours of the initial administration, within 42 hours of the initial administration, within 48 hours of the initial administration, within 54 hours of the initial administration, within 60 hours of the initial administration, within 66 hours of the initial administration, and within 72 hours of the initial administration. Within the specified timeframes: within 78 hours of initial application, within 84 hours of initial application, within 90 hours of initial application, within 96 hours of initial application, within 102 hours of initial application, within 108 hours of initial application, within 114 hours of initial application, within 120 hours of initial application, within 1 week of initial application, within 2 weeks of initial application, within 3 weeks of initial application, within 4 weeks of initial application, within 5 weeks of initial application, within 6 weeks of initial application, within 7 weeks of initial application, within 8 weeks of initial application, within 9 weeks of initial application, within 10 weeks of initial application, within 11 weeks of initial application, and within 12 weeks of initial application.

[0075] In embodiments that include supplemental administration, the supplemental dose may be administered at the following times: for example, within 6 hours of assessment, within 8 hours of assessment, within 10 hours of assessment, within 12 hours of assessment, within 14 hours of assessment, within 16 hours of assessment, within 18 hours of assessment, within 24 hours of assessment, within 30 hours of assessment, within 36 hours of assessment, within 42 hours of assessment, within 48 hours of assessment, within 54 hours of assessment, within 60 hours of assessment, within 66 hours of assessment, within 72 hours of assessment, within 78 hours of assessment, within 84 hours of assessment, within 90 hours of assessment, within 96 hours of assessment, within 102 hours of assessment, within 108 hours of assessment, within 114 hours of assessment, within 120 hours of assessment, within 1 week of assessment, within 2 weeks of assessment, within 3 weeks of assessment, within 4 weeks of assessment, within 5 weeks of assessment, within 6 weeks of assessment, within 7 weeks of assessment, within 8 weeks of assessment, within 9 weeks of assessment, within 10 weeks of assessment, within 11 weeks of assessment, within 12 weeks of assessment, etc.

[0076] In the implementation plan, supplementary application may be performed, for example, within 6 hours of the initial application, within 8 hours of the initial application, within 10 hours of the initial application, within 12 hours of the initial application, within 14 hours of the initial application, within 16 hours of the initial application, within 18 hours of the initial application, within 24 hours of the initial application, within 30 hours of the initial application, within 36 hours of the initial application, within 42 hours of the initial application, within 48 hours of the initial application, within 54 hours of the initial application, within 60 hours of the initial application, within 66 hours of the initial application, within 72 hours of the initial application, and so on. Within 78 hours of application, within 84 hours of the first application, within 90 hours of the first application, within 96 hours of the first application, within 102 hours of the first application, within 108 hours of the first application, within 114 hours of the first application, within 120 hours of the first application, within 1 week of the first application, within 2 weeks of the first application, within 3 weeks of the first application, within 4 weeks of the first application, within 5 weeks of the first application, within 6 weeks of the first application, within 7 weeks of the first application, within 8 weeks of the first application, within 9 weeks of the first application, within 10 weeks of the first application, within 11 weeks of the first application, and within 12 weeks of the first application.

[0077] The methods disclosed herein can provide rapid-onset effects (e.g., using fast-acting neurotoxins). For example, the disclosed embodiments can provide visible cosmetic effects within the following timeframes: e.g., within 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, and 15 hours after application. Within 16 hours after application, within 17 hours after application, within 18 hours after application, within 19 hours after application, within 20 hours after application, within 21 hours after application, within 22 hours after application, within 23 hours after application, within 24 hours after application, within 30 hours after application, within 36 hours after application, within 42 hours after application, within 48 hours after application, within 3 days after application, within 4 days after application, within 5 days after application, within 6 days after application, within 7 days after application, within 8 days after application, within 9 days after application, within 10 days after application, within 11 days after application, within 12 days after application, etc.

[0078] The methods disclosed herein can provide effects over a shorter period of time (e.g., using rapidly recovering neurotoxins). For example, the embodiments disclosed herein can provide visible cosmetic effects that diminish over the following periods: e.g., within 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 45 days, 60 days, 75 days, 90 days, 105 days, etc.

[0079] Side effects may be associated with botulinum toxin injections. The disclosed implementation methods may provide neurotoxin treatment with fewer or shorter duration of side effects compared to conventional neurotoxin treatments.

[0080] For example, the disclosed implementation scheme may result in fewer occurrences (or shorter durations) of the following: double vision or blurred vision, eyelid paralysis (the subject is unable to fully open his / her eyelids), loss of facial muscle movement, hoarseness, loss of bladder control, shortness of breath, difficulty swallowing, difficulty speaking, death, etc.

[0081] The method of this invention is particularly suitable for treating cosmetic irregularities, which are typically the result of aging, environmental exposure, weight loss, childbirth, injury, surgery, or a combination thereof. Aging and environmental exposure typically produce wrinkles in different areas of the skin. On the other hand, weight loss and childbirth typically produce stretch marks in multiple locations of the skin, particularly in the stomach area, lower body region, and legs. Injuries and surgeries typically produce scars in the injured and surgical areas. Specific cosmetic defects suitable for treatment by the disclosed method include, but are not limited to, frown lines, worry lines, wrinkles, crow's feet, marionette lines, stretch marks, wounds, accidental injuries, bites, and surgical procedures. Cosmetic defects in areas such as the eyes, cheeks, nose, lips, forehead, and neck are particularly suitable for treatment by this invention.

[0082] Furthermore, the disclosed implementation methods can provide patients with more definite and lasting cosmetic results. For example, with longer-acting neurotoxins, a 20% difference in the duration of effect can result in a difference of one month in the effective duration. With the disclosed fast-recovery neurotoxin, this 20% difference does not result in a significant difference in the effective duration.

[0083] Supplemental application of fast-acting neurotoxins can effectively improve or enhance previous cosmetic neurotoxin applications. For example, the methods disclosed herein may include supplemental application that corrects uneven cosmetic results from previous applications, increases the cosmetic effect of previous applications, or accelerates the onset of results compared to results achieved using non-fast-acting neurotoxins.

[0084] The disclosed fast-acting neurotoxin compositions can be administered using needles or needle-free devices. In some embodiments, the method includes subcutaneous injection of the composition into an individual. For example, administration may include injecting the composition through a needle no larger than about 30 gauge. In some embodiments, the method includes administering a composition containing botulinum toxin type E.

[0085] The composition can be injected using syringes, catheters, needles, and other injection methods. Injection can be performed on any area of ​​the mammalian body requiring treatment, including but not limited to the face, neck, trunk, arms, hands, legs, and feet. Injection can be performed at any location within a specific area, such as the epidermis, dermis, fat, muscle, or subcutaneous layer.

[0086] The injection frequency and volume under the disclosed method can be determined based on the nature and location of the specific cosmetic irregularity being treated. However, in some cases, repeated injections may be necessary to achieve optimal results. The injection frequency and volume for each specific case can be determined by someone skilled in the art.

[0087] While examples of routes of administration and dosages are provided, the appropriate route of administration and dosage are typically determined by the attending physician based on the specific circumstances. This determination is routine for those skilled in the art. For example, the route of administration and dosage of the clostridial neurotoxin disclosed in this invention can be selected based on criteria such as the solubility characteristics of the chosen neurotoxin and the intensity and extent of the cosmetic condition being treated.

[0088] The dosage of fast-acting neurotoxin can be approximately 10. -3 The dosage of the neurotoxin is approximately 35 U / kg body weight to about 10 U / kg body weight. In one embodiment, the dosage of the neurotoxin is about 10 U / kg body weight. -2 The dosage of the neurotoxin is approximately 10 U / kg to about 25 U / kg. In another embodiment, the dosage of the neurotoxin is about 10 U / kg. -1The dosage of the neurotoxin is from about 1 U / kg to about 15 U / kg. In another embodiment, the dosage of the neurotoxin is from about 1 U / kg to about 10 U / kg. In many cases, the administration of about 1 unit to about 500 units of a neurotoxin, such as botulinum toxin type E, provides effective therapeutic relief. In one embodiment, about 5 units to about 200 units of a neurotoxin, such as botulinum toxin type E, can be used; in another embodiment, about 10 units to about 100 units of a neurotoxin, such as botulinum toxin type E, can be applied topically to target tissues such as muscle.

[0089] In the implementation scheme, the administration may include the following doses of neurotoxin: approximately 4 units of neurotoxin, or approximately 5 units of neurotoxin, or approximately 6 units of neurotoxin, or approximately 7 units of neurotoxin, or approximately 8 units of neurotoxin, or approximately 10 units of neurotoxin, or approximately 15 units of neurotoxin, or approximately 20 units of neurotoxin, or approximately 30 units of neurotoxin, or approximately 40 units of neurotoxin, or approximately 50 units of neurotoxin, or approximately 60 units of neurotoxin, or approximately 70 units of neurotoxin, or approximately 80 units of neurotoxin. Toxin, or approximately 90 units of neurotoxin, or approximately 100 units of neurotoxin, or approximately 110 units of neurotoxin, or approximately 120 units of neurotoxin, or approximately 130 units of neurotoxin, or approximately 140 units of neurotoxin, or approximately 150 units of neurotoxin, or approximately 160 units of neurotoxin, or approximately 170 units of neurotoxin, or approximately 180 units of neurotoxin, or approximately 190 units of neurotoxin, or approximately 200 units of neurotoxin, or approximately 210 units of neurotoxin, or approximately 220 units of neurotoxin, or approximately 2 30 units of neurotoxin, or approximately 240 units of neurotoxin, or approximately 250 units of neurotoxin, or approximately 260 units of neurotoxin, or approximately 270 units of neurotoxin, or approximately 280 units of neurotoxin, or approximately 290 units of neurotoxin, or approximately 290 units of neurotoxin, or approximately 300 units of neurotoxin, or approximately 310 units of neurotoxin, or approximately 320 units of neurotoxin, or approximately 330 units of neurotoxin, or approximately 340 units of neurotoxin, or approximately 350 units of neurotoxin, or approximately 360 units of neurotoxin. The neurotoxin is present in approximately 370 units, 380 units, 390 units, 400 units, 410 units, 420 units, 430 units, 440 units, 450 units, 460 units, 470 units, 480 units, 490 units, or 500 units.

[0090] In the implementation plan, the application may contain the following dosages of botulinum toxin type E neurotoxin: approximately 4 units of botulinum toxin type E neurotoxin, or approximately 5 units of botulinum toxin type E neurotoxin, or approximately 6 units of botulinum toxin type E neurotoxin, or approximately 7 units of botulinum toxin type E, or approximately 8 units of botulinum toxin type E neurotoxin, or approximately 10 units of botulinum toxin type E neurotoxin, or approximately 15 units of botulinum toxin type E neurotoxin, or approximately 20 units of botulinum toxin type E neurotoxin, or approximately 30 units of botulinum toxin type E neurotoxin, or approximately 40 units of botulinum toxin type E neurotoxin, or approximately 50 units of botulinum toxin type E neurotoxin, or approximately 60 units of botulinum toxin type E neurotoxin, or approximately 70 units of botulinum toxin type E neurotoxin, or approximately 80 units of botulinum toxin type E neurotoxin. Type E botulinum neurotoxin, or approximately 90 units of type E botulinum neurotoxin, or approximately 100 units of type E botulinum neurotoxin, or approximately 110 units of type E botulinum neurotoxin, or approximately 120 units of type E botulinum neurotoxin, or approximately 130 units of type E botulinum neurotoxin, or approximately 140 units of type E botulinum neurotoxin, or approximately 150 units of type E botulinum neurotoxin, or approximately 160 units of type E botulinum neurotoxin, or approximately 170 units of type E botulinum neurotoxin, or approximately 180 units of type E botulinum neurotoxin, or approximately 190 units of type E botulinum neurotoxin, or approximately 200 units of type E botulinum neurotoxin, or approximately 210 units of type E botulinum neurotoxin, or approximately 220 units of type E botulinum neurotoxin. Botulinum toxin type E neurotoxin, or approximately 230 units of botulinum toxin type E neurotoxin, or approximately 240 units of botulinum toxin type E neurotoxin, or approximately 250 units of botulinum toxin type E neurotoxin, or approximately 260 units of botulinum toxin type E neurotoxin, or approximately 270 units of botulinum toxin type E neurotoxin, or approximately 280 units of botulinum toxin type E neurotoxin, or approximately 290 units of botulinum toxin type E neurotoxin, or approximately 290 units of botulinum toxin type E neurotoxin, or approximately 300 units of botulinum toxin type E neurotoxin, or approximately 310 units of botulinum toxin type E neurotoxin, or approximately 320 units of botulinum toxin type E neurotoxin, or approximately 330 units of botulinum toxin type E neurotoxin, or approximately 340 units of botulinum toxin type E neurotoxin, or approximately 3 50 units of botulinum toxin type E, or approximately 360 units of botulinum toxin type E, or approximately 370 units of botulinum toxin type E, or approximately 380 units of botulinum toxin type E, or approximately 390 units of botulinum toxin type E, or approximately 400 units of botulinum toxin type E, or approximately 410 units of botulinum toxin type E, or approximately 420 units of botulinum toxin type E, or approximately 430 units of botulinum toxin type E, or approximately 440 units of botulinum toxin type E, or approximately 450 units of botulinum toxin type E, or approximately 460 units of botulinum toxin type E, or approximately 470 units of botulinum toxin type E, or approximately 480 units of botulinum toxin type E.This could be approximately 490 units of botulinum toxin type E, or approximately 500 units of botulinum neurotoxin type E, etc.

[0091] This document discloses methods for expressing and delivering neurotoxin doses. In embodiments, the dose is expressed in the amount of protein, such as nanograms (ng). In embodiments, the neurotoxin may comprise botulinum toxin.

[0092] The methods disclosed herein may include administering a neurotoxin, such as a rapid-acting neurotoxin, to a patient, wherein the dose of the neurotoxin is expressed in terms of the amount of protein, such as the amount of protein administered per dose. In one embodiment, the rapid-acting neurotoxin is a botulinum toxin, such as botulinum toxin type E.

[0093] In the implementation scheme, the dosage of the neurotoxin is expressed as the amount or concentration of protein. For example, in the implementation scheme, the amount of neurotoxin administered may be from about 0.2 ng to 20 ng. In one implementation scheme, the neurotoxin is administered to a target tissue, such as muscle, in amounts of about 0.3 ng to 19 ng, about 0.4 ng to 18 ng, about 0.5 ng to 17 ng, about 0.6 ng to 16 ng, about 0.7 ng to about 15 ng, about 0.8 ng to 14 ng, about 0.9 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.

[0094] In the implementation plan, the total dose that may be administered is 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, etc.

[0095] In the implementation plan, the total dose that may be administered is up to 5 ng, up to 6 ng, up to 7 ng, up to 8 ng, up to 9 ng, up to 10 ng, up to 11 ng, up to 12 ng, up to 13 ng, up to 14 ng, up to 15 ng, up to 16 ng, up to 17 ng, up to 18 ng, up to 19 ng, up to 20 ng, etc.

[0096] In the implementation plan, the total dose that may be administered is at least 5 ng, at least 6 ng, at least 7 ng, at least 8 ng, at least 9 ng, at least 10 ng, at least 11 ng, at least 12 ng, at least 13 ng, at least 14 ng, not less than 15 ng, at least 16 ng, at least 17 ng, at least 18 ng, at least 19 ng, at least 20 ng, etc.

[0097] In the implementation plan, the total dose of the neurotoxins that may be included is as follows: 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. ng neurotoxin, 3.0 ng neurotoxin, 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, etc.

[0098] In the implementation scheme, administration may include the following dosage per injection: for example, approximately 0.1 ng of botulinum toxin type E, 0.2 ng of botulinum toxin type E, 0.3 ng of botulinum toxin type E, 0.4 ng of botulinum toxin type E, 0.5 ng of botulinum toxin type E, 0.6 ng of botulinum toxin type E, 0.7 ng of botulinum toxin type E, 0.8 ng of botulinum toxin type E, 0.9 ng of botulinum toxin type E, 1.0 ng of botulinum toxin type E, 1.1 ng of botulinum toxin type E, 1.2 ng of botulinum toxin type E, 1.3 ng of botulinum toxin type E, 1.4 ng of botulinum toxin type E, 1.5 ng of botulinum toxin type E, 1.6 ng of botulinum toxin type E, 1.7 ng of botulinum toxin type E, 1.8 ng of botulinum toxin type E, etc. Botulinum toxin type E neurotoxin, 1.9 ng Botulinum toxin type E neurotoxin, 2.0 ng Botulinum toxin type E neurotoxin, 2.1 ng Botulinum toxin type E neurotoxin, 2.2 ng Botulinum toxin type E neurotoxin, 2.3 ng Botulinum toxin type E neurotoxin, 2.4 ng neurotoxin, 2.5 ng neurotoxin, 2.6 ng Botulinum toxin type E neurotoxin, 2.7 ng Botulinum toxin type E neurotoxin, 2.8 ng Botulinum toxin type E neurotoxin, 2.9 ng Botulinum toxin type E neurotoxin, 3.0 ng Botulinum toxin type E neurotoxin, 3.1 ng Botulinum toxin type E neurotoxin, 3.2 ng Botulinum toxin type E neurotoxin, 3.3 ng Botulinum toxin type E neurotoxin, 3.4 ng Botulinum toxin type E neurotoxin, 3.5 ng Botulinum toxin type E neurotoxin, 3.6 ng Botulinum toxin type E neurotoxin, 3.7 ng Botulinum toxin type E neurotoxin, 3.8 ng Botulinum toxin type E neurotoxin, 3.9 ng Botulinum toxin type E neurotoxin, 4.0 ng Botulinum toxin type E neurotoxin, 4.1 ng Botulinum toxin type E neurotoxin, 4.2 ng Botulinum toxin type E neurotoxin, 4.3 ng Botulinum toxin type E neurotoxin, 4.4 ng Botulinum toxin type E neurotoxin, 4.5 ng Botulinum toxin type E neurotoxin, 5 ng Botulinum toxin type E neurotoxin, 6 ng Botulinum toxin type E neurotoxin, 7 ng Botulinum toxin type E neurotoxin, 8 ng Botulinum toxin type E neurotoxin, 9 ng Botulinum toxin type E neurotoxin, 10 ng Botulinum toxin type E neurotoxin, etc.

[0099] Ultimately, however, the dosage and frequency of toxin administration will be determined at the discretion of the treating physician, based on safety and the effects of the toxin. Controlled-release systems can be used in the embodiments described herein to deliver neurotoxins in vivo at a predetermined rate over a specific time period. Typically, the release rate is determined by the system design and can be largely independent of environmental conditions such as pH. Controlled-release systems that can deliver drugs over several years are known. In contrast, sustained-release systems typically deliver drugs over 24 hours or less, and environmental factors can affect the release rate. Therefore, the release rate of a neurotoxin from an implanted controlled-release system (“implant”) varies depending on the physicochemical properties of the carrier implant material and the drug itself. Typically, implants are made of inert materials that elicit little or no host response.

[0100] Controlled-release systems may include neurotoxins incorporated into a carrier. The carrier may be a polymer or a bioceramic material. Controlled-release systems can be injected, inserted, or implanted at selected sites in a patient's body and remain there for an extended period, during which time the neurotoxin is released by the implant in a manner and concentration that provides the desired therapeutic efficacy.

[0101] Polymer materials can release neurotoxins due to diffusion, chemical reactions, solvent activation, and exposure to magnetic, ultrasonic, or temperature changes. Diffusion can originate from the container or matrix. Chemical control can be attributed to polymer degradation or drug cleavage from the polymer. Solvent activation can include polymer swelling or permeation.

[0102] The implant can be prepared by incorporating the desired amount of stabilized neurotoxin into a solution of a suitable polymer dissolved in dichloromethane. This solution can be prepared at room temperature. The solution can then be transferred to a petri dish, and the dichloromethane evaporated in a vacuum desiccator. Depending on the desired implant size and therefore the amount of neurotoxin incorporated, a suitable amount of the dried, neurotoxin-infused implant is compressed in a mold at approximately 8000 psi for 5 seconds or at 3000 psi for 17 seconds to form an implant disc encapsulating the neurotoxin.

[0103] Preferably, the implant material used is substantially non-toxic, non-carcinogenic, and non-immunogenic. Suitable implant materials include polymers such as poly(2-hydroxyethyl methacrylate) (p-HEMA), poly(N-vinylpyrrolidone) (p-NVP)+, poly(vinyl alcohol) (PVA), poly(acrylic acid) (PM), polydimethylsiloxane (PDMS), ethylene-vinyl acetate (EVAc) copolymer, polyvinylpyrrolidone / methyl acrylate copolymer, polymethyl methacrylate (PMMA), poly(lactic acid) (PLA), poly(glycolic acid) (PGA), polyanhydride, poly(orthoester), collagen and cellulose derivatives, and bioceramics such as hydroxyapatite (HPA), tricalcium phosphate (TCP), and calcium aluminum phosphate (ALCAP). Lactic acid, glycolic acid, and collagen can be used to manufacture biodegradable implants.

[0104] Implant materials can be biodegradable or bioresorbable. The advantage of bioresorbable implants is that they do not require removal from the patient. Bioresorbable implants can be released based on membranes or matrices containing bioactive substances. Biodegradable microspheres prepared from PLA-PGA are known for subcutaneous or intramuscular administration.

[0105] This disclosure also includes a kit for implementing the disclosed embodiments. The kit may include a needle of gauge 30 or smaller and a corresponding syringe. The kit also includes a clostridium neurotoxin composition, such as a botulinum toxin type E composition. The neurotoxin composition may be provided in the syringe. The composition can be injected through the needle. The kit is designed in various forms depending on the size of the syringe and needle and the volume of the injectable composition contained therein, which in turn is based on designing the kit for treating specific cosmetic defects.

[0106] Example The following non-limiting embodiments are provided for illustrative purposes only to provide a more complete understanding of representative implementations. These embodiments should not be construed as limiting any implementation described in this specification.

[0107] Example 1 Uses of botulinum toxin type E for treating frown lines (GL) This first-in-human, randomized, double-blind, placebo-controlled, escalating-dose cohort study enrolled 42 participants who received either EB-001 (the botulinum toxin type E composition disclosed herein) (N = 35) or placebo (N = 7). The primary outcome of efficacy was the proportion of participants who achieved an investigator-rated grade 2 (IR-2) improvement in GL severity at maximum frowning. Safety assessments included adverse events (AEs), laboratory tests, and physical examinations. IR-2 responses were observed starting with the third cohort (EB-001), with increased response rates observed at higher doses. At the highest dose, clinical effects were observed within 24 hours and lasted 14 to 30 days. AEs were infrequent, with mild to moderate headache being the most common. There were no serious AEs or ptosis, and no significant clinical changes were observed in other safety assessments.

[0108] In this GL clinical study, EB-001 demonstrated good safety and tolerability, as well as dose-dependent efficacy with a response rate of 80% at the highest dose. The maximum clinical effect of EB-001 was visible within 24 hours and lasted for 14 to 30 days. This distinctive profile of EB-001 supports its development for cosmetic and therapeutic applications where rapid onset and short duration of action are desirable.

[0109] Botulinum neurotoxin, which inhibits the presynaptic release of acetylcholine, is one of the most promising molecules found in nature. When injected into muscle, it inhibits neuromuscular transmission and produces 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 diseases. Botulinum neurotoxin serotype E is a novel serotype that has not yet been developed for clinical use. Botulinum neurotoxin type E has the fastest 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 disulfide bonds). 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. Two binding sites on the motor axon mediate the high affinity recognition of botulinum neurotoxin for nerve cells. Binding is initially mediated by cell surface gangliosides, and then by specific protein receptors. These receptors are located on the motor axon terminals at the neuromuscular junction. It has been shown that both botulinum toxin type A and type E bind to the specific receptor synaptic vesicle protein 2, and that only these two serotypes share this receptor. This is the first clinical study evaluating the safety and efficacy of escalating doses of botulinum toxin type E in GL subjects.

[0110] This study is the first human in vivo evaluation of the safety and efficacy of EB-001 for 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). This was a randomized, double-blind, placebo-controlled, dose-escalation cohort study conducted at two specialist clinical centers (Steve Yoelin, MD Medical Associates, Newport Beach, California, USA; Center for Dermatology Clinical Research, Fremont, California, USA). This study was approved by the Institutional Review Board (Aspire Institutional Review Board, Santee, California, USA) and conducted in accordance with the guidelines established in the Declaration of Helsinki. Written informed consent was obtained from all participants prior to participation.

[0111] This study recruited 42 healthy, toxin-naïve male and female participants aged 18 to 60 years. Participation lasted approximately 6 weeks for each participant. The primary inclusion criteria were: moderate to severe bilateral symmetrical GL (glove fold) at maximum frowning; adequate visual acuity without glasses (contact lenses were permitted) to accurately assess facial wrinkles; and the ability to meet the study requirements. The primary exclusion criteria were: any uncontrolled systemic disease or other medical condition; any medical condition that might increase the risk of exposure to botulinum neurotoxin (including confirmed myasthenia gravis, Eaton-Lambert syndrome, amyotrophic lateral sclerosis, or any other condition interfering with neuromuscular function); current or previous botulinum neurotoxin treatment; known immune or hypersensitivity reactions to botulinum neurotoxin; pre-specified dermatological procedures (non-ablative surface resurfacing, facial cosmetic procedures, topical / oral retinoid therapy, etc.) within 3 to 12 months of the study period; and previous periorbital surgery or treatment. Women who are pregnant, breastfeeding, or planning to become pregnant will not be recruited. Men with fertile female partners will only be recruited if they agree to use dual contraception for three months after starting the medication.

[0112] During screening, participants' demographic information, medical history, and prior and concomitant medications were recorded, and alcohol / drug screening was performed. Standardized facial photography was conducted at baseline before treatment and at each follow-up visit throughout the study, but these photos were not used for efficacy assessment.

[0113] Seven cohorts (six subjects per cohort) were recruited and assigned to receive escalating doses of EB-001 or placebo in a 5:1 ratio. The maximum starting dose recommended in this first-in-human study (with a 10-fold safety margin) was based on the level of no observed adverse events from preclinical safety and toxicity studies (unpublished data). Accordingly, a baseline dose was calculated (cohort 1) and designated as sub-effective, with cohorts 2 through 7 receiving 3, 9, 12, 16, 21, and 28 times the baseline dose, respectively. This represents the sub-effective to maximum effective dose of EB-001. All doses were delivered in a standard manner at five injection sites with equal volumes (0.1 mL per site into the depressor supercilii, left medial corrugator supercilii, right medial corrugator supercilii, left lateral corrugator supercilii, and right lateral corrugator supercilii) (see [link to relevant documentation]). Figure 1 The injection site is approximately 1 cm above the brow bone in the corrugator supercilii muscle. EB-001 is provided as a sterile injectable solution in 5 mL vials. A placebo is provided in the same vials but without EB-001.

[0114] Each subject completed visits at screening (day -30 to day -1), at baseline / injection (day 0), at days 1, 2, 7, 14 and 30 (end of study), and at day 42 (final safety follow-up).

[0115] Safety assessments were conducted using adverse events (AEs), laboratory tests, electrocardiograms (ECGs), physical examinations, vital signs (pulse rate, respiratory rate, and blood pressure), urine pregnancy tests (for women of childbearing potential), and focused neurological examinations to evaluate the potential spread of botulinum neurotoxin. Treatment-induced adverse events (TEAEs) were defined as any adverse event that began or worsened in severity after exposure to the study treatment. AEs and TEAEs were summarized by systemic organ class and preferred terminology using the Medical Dictionary for Regulatory Activities (MedDRA, version 19.0). Serious AEs (SAEs or AEs meeting the medical severity regulatory criteria) and discontinuation due to AEs were also assessed. The severity of AEs was recorded as mild, moderate, severe, or life-threatening. Prior to inclusion in each dose cohort, the Safety Data Review Board met to analyze all safety data from previous cohorts.

[0116] Participants' glossitis severity (GL) at maximum frowning and at rest was assessed using the Facial Wrinkles Scale (FWS) at screening, baseline, and days 1, 2, 7, 14, and 30. Assessments were performed by both investigators and participants. The FWS is a widely accepted measure for assessing facial line severity. In this study, a 4-point scale indicating GL severity was used: 0 = none, 1 = mild, 2 = moderate, 3 = severe. Participants were considered treatment responders if they achieved at least a 2-grade improvement (reduction) (IR-2) according to the investigator's FWS assessment. The primary efficacy variable was the proportion of IR-2 responders at maximum frowning at any time from the baseline visit to day 30. Another efficacy endpoint of interest was the proportion of responders achieving an investigator-assessed FWS grade of zero or mild at days 1, 2, 7, 14, or 30 (through visit analysis).

[0117] Two analytical populations were pre-specified: the safety population and the efficacy population. Subjects receiving placebo were combined for all analyses. The safety population included all subjects who received the study treatment and subsequently underwent at least one safety assessment. All TEAEs and SAEs were aggregated by treatment group. Researchers reviewed all safety parameters, including laboratory tests, ECG, physical examination, vital signs, urine pregnancy tests, and focused neurological examinations, and assessed their clinical significance. The efficacy population was the modified intention-to-treat (mITT) population, defined as all randomized subjects who received at least one dose of the study treatment and underwent at least one post-baseline efficacy assessment. Demographic and baseline characteristics analyses were performed on the mITT population. Medical history was based on the safety population, coded using MedDRA, and aggregated by system organ class and preferred terminology. Previous and concomitant medications were based on the safety population, coded using the World Health Organization Anatomical Therapeutic Chemical (WHO) classification index, and aggregated by drug class and treatment group. Efficacy analyses were performed using the mITT population. FWS grades were summarized using frequency counts and response rates (%) over treatment and study days. Analysis was performed using Fisher's exact test at a significance level of 0.05 to compare the proportion of IR-2 responders in each EB-001 cohort with placebo (pooled).

[0118] Of the 59 participants selected for the study, 43 were enrolled in one of seven cohorts. One participant did not receive treatment, and the remaining 42 were enrolled in the mITT and safety populations (35 received EB-001 and 7 received placebo). 41 participants completed the study, with one lost to follow-up. Table 1 shows the demographic and baseline characteristics of the mITT population. The mean age (age range) of participants in the EB-001 (pooled) group and the placebo (pooled) group were 47.9 years (22–60 years) and 50.4 years (32–57 years), respectively. The majority of participants were female (EB-001 = 91.4%; placebo = 85.7%) and white (71.4% in both groups). The baseline mean (standard deviation [SD]) of GL at maximum frown, as assessed by the researchers, was 2.6 (0.50) for the EB-001 group and 2.9 (0.38) for the placebo group. The EB-001 group and the placebo group were well balanced, with no significant differences between the groups.

[0119] exist Figure 2 In this study, the proportion of subjects achieving an IR-2 response to GL severity at maximal frowning was represented by the dose cohorts at any time from the baseline visit to day 30 in the mITT population. In cohort 3, 40% of subjects were IR-2 responders. This response rate was the same or higher in all higher-dose cohorts, with IR-2 response rates of 80% in cohorts 6 and 7. Cohorts 6 and 7 showed a significantly higher percentage of IR-2 responders than placebo (P = 0.046). Figure 3 The proportion of subjects with a grade of no or mild GL at maximum frowning at any time from baseline visit to day 30 was summarized in each cohort. The percentage of responders was higher in cohorts 2 through 7 (inclusive) compared to the placebo group, reaching 60% to 100% in cohorts 3 and higher doses. In cohorts 3 through 7, most were observed to be non-responsive or mildly responsive on days 1, 2, and / or 7. In cohorts 3, 5, 6, and 7, one responder (20%) was observed on day 14; and in cohorts 3 and 5, one responder (20%) was observed on day 30. Safety results support the safety of all assessed doses of EB-001 administered via IM injection in this population. No clinically significant changes from baseline were observed in any subject's neurological examination, ECG, physical examination, or laboratory tests.

[0120] Five subjects treated with EB-001 reported TEAEs, while no subjects in the placebo group reported TEAEs. No SAEs were reported, and no TEAEs led to study interruption. All TEAEs were mild or moderate in severity. Events of sore throat and flu-like symptoms were considered treatment-independent. Three subjects reported TEAEs for headache; one of these was considered treatment-related. There was no dose-related increase in the incidence of headache. No ptosis or other TEAE events that could be related to toxin spread were reported.

[0121] To the best of our knowledge, this is the first controlled clinical trial of a botulinum toxin type E product for any aesthetic or therapeutic purpose. This first-in-human study of EB-001—a novel purified form of botulinum toxin type E administered via intramimetic injection—achieved its goal of evaluating the safety, tolerability, and effective dose range of EB-001. Dose responses were observed, with a higher proportion of treatment responders in the higher-dose EB-001 cohorts. IR-2 responses were observed starting in cohort 3 and increased in the higher-dose cohorts, suggesting that the effective dose range of EB-001 is likely the doses used in cohorts 4 through 7. Cohorts 6 and 7 had an IR-2 responder rate of 80%, similar to approved botulinum toxin type A products. Subjects with a FWS grade of zero or mild were observed starting in cohort 2. In terms of onset of action, treatment responses were observed as early as 24 hours after administration, supporting previous reports that botulinum toxin type E has a faster onset of action than type A.

[0122] Regarding the duration of effect, defined as the proportion of respondents with no or mild response, one subject in most of the five higher-dose cohorts was observed to have the effect for 14 days, and one subject in two of the five higher-dose cohorts was observed to have the effect for 30 days. All doses of EB-001 showed good tolerability with no local injection site reactions. No SAEs or serious TEAEs were reported, and no discontinuation of treatment was due to TEAEs. The most common TEAE, headache, was mild to moderate in severity, and there were no other treatment-related AEs. There were no events of ptosis at any dose level, nor any events possibly related to toxin spread. Therefore, the clinical safety and tolerability profile of this study appears favorable. The efficacy and safety of EB-001 are promising and support its potential as a unique treatment option for treating GL and other facial cosmetic uses. Its rapid onset of action can meet the unmet need of those seeking rapid treatment of facial wrinkles before unforeseen social or professional events. The limited duration of effect may be beneficial for individuals who may be considering their first Botox neurotoxin treatment and are unwilling to undergo it long-term. Compared to the 12-week duration of effect for Botox type A products, EB-001 treatment allows them to assess cosmetic results within a shorter duration. In this first-in-class clinical study in GL subjects, EB-001 demonstrated good safety and tolerability in all cohorts. Five of the seven cohorts showed higher response rates compared to placebo, supporting the efficacy of EB-001 in reducing GL severity. Similar to approved Botox type A products, the two highest doses provided an 80% response rate. Compared to the known time course of action for type A products, the clinical effects of EB-001 were visible within 24 hours (onset) and lasted for 14–30 days (duration). This distinguishable clinical characteristic supports the future development of EB-001 in facial aesthetics and key therapeutic uses requiring rapid onset and short duration of action.

[0123] Table S-1: Dosage escalation regimen Example 2 Uses of botulinum toxin type E in treating crow's feet A 57-year-old man developed crow's feet due to years of sun exposure and sought treatment from a doctor. The doctor recommended the composition disclosed in this article, which was injected subcutaneously into one eye. Approximately 3 units of botulinum toxin type E were administered at each injection site, with several injections performed on each eye. The crow's feet disappeared within about 2 days after treatment, and the reduction in crow's feet remained for two months.

[0124] Example 3 Uses of botulinum toxin type E in eyebrow lift surgery A 60-year-old woman's eyebrows extended below her brow bone. The physician recommended a composition as disclosed in this article, injected subcutaneously above each eye. Approximately 10 units of botulinum toxin type E were administered at each injection site, with several injections made on either side of the eye. The drooping of the eyebrows decreased within approximately 2 days and was largely resolved within 3 months of application.

[0125] Example 4 Uses of botulinum toxin type E for breast augmentation A 30-year-old woman opted for breast augmentation surgery. Four hours before the surgery, botulinum toxin type E was applied near the surgical incision site. This reduced muscle tension in the incision area, minimizing scarring. A follow-up dose was administered two weeks post-surgery.

[0126] Example 5 Uses of botulinum toxin type E in breast reconstruction A 30-year-old woman opted for breast reconstruction surgery. Fourteen hours prior to the surgery, botulinum toxin type E was applied near the surgical incision site. This reduced muscle tension in the incision area, minimizing scarring. A follow-up dose was administered two weeks post-surgery.

[0127] Example 6 Uses of botulinum toxin type E in the treatment of episiotomy A 44-year-old woman underwent an episiotomy. Immediately after the surgery, 4 ng of botulinum toxin type E was administered to the tissue surrounding the surgical area. Within 20 hours, muscle and nerve activity around the wound was significantly reduced.

[0128] Example 7 Uses of botulinum toxin type E in the treatment of sports hernia A 28-year-old man suffered from a sports-type hernia. His doctor administered 4 ng of botulinum toxin type E to the tissue surrounding both hernias. Within 10 hours, muscle and nerve activity around the injury was significantly reduced.

[0129] Example 8 Uses of botulinum toxin type E in the treatment of shoulder dislocation A 48-year-old man suffered from a hernia in his shoulder. His doctor administered 4 ng of botulinum toxin type E to the tissue surrounding the two hernias. Within 16 hours, muscle and nerve activity around the injury was significantly reduced.

[0130] Example 9 Uses of botulinum toxin type E in the treatment of torn ACL A 23-year-old woman suffered a torn ACL. Six hours after the injury, her doctor administered 7 ng of botulinum toxin type E to the muscles surrounding the torn ligament. Within 30 hours, muscle and nerve activity around the wound was significantly reduced.

[0131] Finally, it should be understood that although various aspects of this specification are emphasized by reference to specific embodiments, those skilled in the art will readily appreciate that these disclosed embodiments are merely illustrative of parts of the subject matter disclosed herein. Therefore, it should be understood that the disclosed subject matter is by no means limited to the specific methods, schemes, and / or reagents described herein. Similarly, various modifications, alterations, or alternative combinations of the disclosed subject matter can be made based on the teachings herein without departing from the spirit of this specification. Finally, the terminology used herein is for the purpose of describing specific embodiments only and is not intended to limit the scope of this disclosure, which is defined solely by the claims. Therefore, embodiments of this disclosure are not limited to those precisely shown and described.

[0132] Certain embodiments described herein include the best modes known to the inventors for carrying out the methods and apparatus described herein. Of course, variations of these described embodiments will be apparent to those skilled in the art after reading the foregoing description. Therefore, this disclosure includes all modifications and equivalents of the subject matter set forth in the appended claims, as permitted by applicable law. Moreover, unless otherwise indicated herein or clearly contradicted by the context, any combination of the above embodiments in all possible variations is included in this disclosure.

[0133] The grouping of alternative embodiments, elements, or steps in this disclosure should not be construed as limiting. Each member of a group may be mentioned and claimed individually or in any combination with other members of the groups disclosed herein. For convenience and / or patentability, one or more members of a group may be included in or removed from that group. When any such inclusion or removal occurs, the specification is deemed to contain the modified group and thus satisfy the written description of the entire Markush group as used in the appended claims.

[0134] Unless otherwise stated, all figures used in this specification and claims to represent features, items, quantities, parameters, characteristics, terms, etc., should be understood to be modified by the term "about" in all cases. As used herein, "about" means that the feature, item, quantity, parameter, characteristic, or term covers a range of plus or minus ten percent above and below the value of the stated feature, item, quantity, parameter, or term. Therefore, unless stated to the contrary, the numerical parameters presented in the specification and appended claims are approximate values ​​that can vary. At least, no attempt should be made to apply the doctrine of equivalents to limit the scope of the claims; each number shown should be interpreted at least as obtained based on the reported significant figures and by applying ordinary rounding techniques. Although the ranges and values ​​of numbers that set forth a broad scope of this disclosure are approximate, the ranges and values ​​set forth in the specific embodiments are reported as precisely as possible. However, any range or value inherently contains some errors, which must be caused by the standard deviation found in their respective test measurements. The description of numerical ranges herein is intended only as a shorthand method for independently referring to each individual value falling within that range. Unless otherwise stated herein, each individual value in the range is incorporated into this specification as if it were individually referenced herein.

[0135] Unless otherwise stated herein or clearly contradicted by the context, the terms “a,” “the,” and similar references (particularly in the context of the following claims) used in the context of describing this disclosure should be construed as covering both the singular and plural. Unless otherwise stated herein or clearly contradicted by the context, all methods described herein may be performed in any suitable order. The use of any and all embodiments or exemplary language (e.g., “for example”) provided herein is intended only to better illustrate the disclosure and does not constitute a limitation on the scope originally claimed. No language in this specification should be construed as representing any unclaimed element necessary for the practice of the embodiments disclosed herein.

[0136] The specific embodiments disclosed herein may be further limited in the claims using the language "consisting of..." or "substantially consisting of...". When used in the claims, whether submitted or added by amendment, the transitional term "consisting of..." excludes any element, step, or ingredient not specified in the claim. The transitional term "substantially consisting of..." limits the scope of the claim to the specified materials or steps and materials or steps that do not substantially affect the essential and novel features. The embodiments of the invention claimed thereby are inherently or clearly described herein and are capable of being practiced.

Claims

1. A cosmetic treatment method for human patients with frown lines, comprising administering, by injection, a total dose of botulinum neurotoxin serum type E in the amount of about 0.2 ng to about 20 ng. The total dose was divided into five approximately equal doses of botulinum neurotoxin serum type E and administered via intramuscular injection to the following muscles: depressor supercilii, left medial corrugator supercilii, right medial corrugator supercilii, left lateral corrugator supercilii, and right lateral corrugator supercilii. The clinical effects of the aforementioned cosmetic treatment are achieved within 24 hours of the administration of botulinum neurotoxin serum type E.

2. The method of claim 1, wherein the botulinum neurotoxin serotype E is pure botulinum neurotoxin serotype E of about 150 kDa.

3. The method of claim 1, wherein the total dose comprises an amount of about 2 ng to about 10 ng.

4. The method of claim 2, wherein the total dose is about 0.2 ng to about 20 ng of pure botulinum neurotoxin serum type E of about 150 kDa.

5. The method of claim 1, wherein the clinical effect comprises a change in the Facial Wrinkles Scale (FWS) grade at maximum frowning, as assessed by the investigator, from moderate to severe to no or mild after application of botulinum neurotoxin serotype E.

6. The method of claim 5, wherein the total dose is about 0.2 ng to about 20 ng of pure botulinum neurotoxin serum type E of about 150 kDa.

7. The method of claim 1, wherein about 150 U of pure about 150 kDa botulinum neurotoxin serum type E is injected into at least one muscle.

8. A method for cosmetic treatment of human patients with moderate to severe glabellar lines, comprising administering to the patient a therapeutically effective amount of pure botulinum neurotoxin serum type E, approximately 150 kDa. The therapeutically effective dose is approximately 0.2 ng to approximately 20 ng of pure botulinum neurotoxin serum type E, approximately 150 kDa. The therapeutically effective dose is administered via five intramuscular injections, each containing approximately an equal amount of pure botulinum neurotoxin serum type E (approximately 150 kDa), with one injection into each of the following muscles: depressor supercilii, left medial corrugator supercilii, right medial corrugator supercilii, left lateral corrugator supercilii, and right lateral corrugator supercilii. The clinical efficacy of the aforementioned cosmetic treatment was achieved within 48 hours following the application of pure botulinum neurotoxin serum type E (approximately 150 kDa); and The clinical effects described include changes in the Facial Wrinkles Scale (FWS) grade at maximum frowning, as assessed by the investigator, from moderate or severe before the application of pure botulinum neurotoxin serotype E at approximately 150 kDa to none or mild after the application of pure botulinum neurotoxin serotype E at approximately 150 kDa.

9. A method for cosmetic treatment of a human patient with glabellar lines, comprising injecting a total dose of botulinum neurotoxin serum type E of about 0.2 ng to about 20 ng into the area of ​​the patient to be treated. The total dose was divided into five approximately equal doses of botulinum neurotoxin serum type E and administered via intramuscular injection to the following muscles: depressor supercilii, left medial corrugator supercilii, right medial corrugator supercilii, left lateral corrugator supercilii, and right lateral corrugator supercilii. The duration of clinical effect of the cosmetic treatment following the application of botulinum neurotoxin serum type E is approximately 14 to approximately 30 days, and the clinical effect includes a change in the Facial Wrinkles Scale (FWS) grade at maximum frowning, as assessed by the investigator before the application of botulinum neurotoxin serum type E, from moderate or severe to none or mild after the application of botulinum neurotoxin serum type E.

10. The method of claim 9, wherein the botulinum neurotoxin serotype E is pure botulinum neurotoxin serotype E of about 150 kDa.

11. The method of claim 9, wherein the total dose comprises about 2 ng to about 10 ng of botulinum neurotoxin serotype E.

12. The method of claim 10, wherein the total dose is about 0.2 ng to about 20 ng of pure botulinum neurotoxin serotype E of about 150 kDa.

13. The method of claim 9, wherein the method is statistically significantly superior to comparable methods in which a placebo is administered to achieve a grade 2 or better improvement (reduction) on the Facial Wrinkles Scale (FWS) assessment (IR-2) at the time of maximum frowning at one (1), two (2), seven (7), 14 or 30 days after the administration.

14. The method of claim 13, wherein the total dose is about 0.2 ng to about 20 ng of pure botulinum neurotoxin serotype E of about 150 kDa.

15. The method of claim 9, wherein about 150 U of pure about 150 kDa botulinum neurotoxin serum type E is injected into at least one muscle.

16. A method for cosmetic treatment of human patients with moderate to severe glabellar lines, comprising administering to the patient a therapeutically effective amount of pure botulinum neurotoxin serum type E, approximately 150 kDa. The therapeutically effective dose comprises a total dose of approximately 150 kDa pure botulinum neurotoxin serum type E in amounts of approximately 2 ng to approximately 10 ng, and The therapeutically effective dose is administered via five intramuscular injections, each containing approximately equal amounts of pure botulinum neurotoxin serum type E of about 150 kDa, with one injection into each of the following muscles: depressor supercilii, left medial corrugator supercilii, right medial corrugator supercilii, left lateral corrugator supercilii, and right lateral corrugator supercilii.

17. The method of claim 16, wherein the method is statistically significantly superior to comparable methods in which a placebo is administered to achieve a grade 2 or better improvement (reduction) on the Facial Wrinkles Scale (FWS) assessment (IR-2) at the time of maximum frowning at one (1), two (2), seven (7), 14 or 30 days after the administration.

18. The method of claim 16, wherein about 150 U of pure about 150 kDa botulinum neurotoxin serum type E is injected into at least one muscle.

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