NEUROTOXIN COMPOSITIONS FOR USE IN IMPROVING LUNG FUNCTION

Disclosed herein are compositions and methods for use in reducing lung function inhibition.

FIELD

The present specification relates to the use of neurotoxins to reduce muscle and nerve activity in the vicinity of the lungs.

BACKGROUND

Surgical procedures can be very invasive, particularly those involving the torso. Recovery can be complicated by inflammation and pain, as well as reduced lung function caused by muscle and nerve activity.

SUMMARY

Disclosed herein are compositions and methods comprising neurotoxins and the use thereof to improve, maintain, or lessen the reduction of lung function, for example following a surgical procedure, for example a surgical procedure performed on the torso.

Disclosed embodiments can improve Slow Vital Capacity (SVC). Disclosed embodiments can reduce pain when performing this test.

Disclosed embodiments can improve Forced Vital Capacity (FCV). Disclosed embodiments can reduce pain when performing this test.

Disclosed embodiments can improve Forced Expiratory Volume 1 (FEV1). Disclosed embodiments can reduce pain when performing this test.

Disclosed embodiments can reduce breathing pain resulting from a reduction in lung function.

Disclosed embodiments comprise use of a “fast-acting” botulinum toxin.

Disclosed embodiments comprise use of a “fast-recovery” botulinum toxin.

In embodiments, the “fast-acting” botulinum toxin is also a “fast-recovery” toxin.

In embodiments, disclosed methods comprise additional surgical procedures. For example, disclosed embodiments comprise administration of a fast-acting botulinum neurotoxin in combination with, for example, a surgical procedure, treatment of an injury, or the like.

In embodiments, disclosed methods comprise administration of a fast-acting botulinum neurotoxin prior to a surgical procedure.

In embodiments, disclosed methods comprise administration of a fast-acting botulinum neurotoxin after a surgical procedure.

In embodiments, disclosed methods comprise administration of a fast-recovery botulinum neurotoxin prior to a surgical procedure.

In embodiments, disclosed methods comprise administration of a fast-recovery botulinum neurotoxin after a surgical procedure.

DETAILED DESCRIPTION

Embodiments disclosed herein can reduce local muscle activity, nerve activity, and pain sensation. This reduction can aid in treatment and recovery, for example recovery following an injury or a surgical procedure. In embodiments the surgical procedure can comprise any intentional disruption to the body, for example cosmetic surgery, dental surgery, and the like. Embodiments comprise administration to the trunk to reduce muscle activity in the vicinity of the lung and therefore improve, maintain, or lessen the reduction of lung function following a surgical procedure.

Administration sites useful for practicing disclosed embodiments can comprise any area where muscle and/or nerve activity is to be reduced. For example, in the case of a surgical procedure or injury to the trunk region of the body, disclosed embodiments can comprise administration to, for example, the external intercostals, the internal intercostals, the transverse abdominis, the infraspinatus, the rectus abdominis, the serratus anterior, the diaphragm, the external obliques, the internal obliques, the trapezius, the rhomboid major, the rhomboid minor, the levator scapulae, the levatores costarum, the longissimus, the multifidus, the splenius, the rectus abdominus, or combinations thereof.

In the case of surgery or injury involving the upper extremities, for example due to a surgical procedure or injury, disclosed embodiments can comprise administration to, for example, the pectoralis major, the pectoralis minor, the latissimus dorsi, the deltoid, the teres major, the biceps brachii, the triceps brachii, the brachialis, the brachioradialis, the palmaris longus, the flexor carpi radialis, the flexor digitorum superficialis, the extensor carpi radialis, the extensor digitorum, the extensor digiti minimi, the extensor carpi, the ulnaris, or combinations thereof.

In the case of surgery or an injury to the lower extremities, for example due to a surgical procedure or injury, disclosed embodiments can comprise, for example, administration to, for example, the iliopsoas, the sartorius, the gluteus maximus, the gluteus medius, the tensor fasciae latae, the adductor longus, the gracilis, the semimembranosus, the semitendinosus, the biceps femoris, the rectus femoris, the vastus lateralis, the vastus intermedium, the vastus medialis, the tibialis anterior, the gastrocnemius, the soleus, the peroneus longus, the peroneus brevis, or combinations thereof.

Disclosed embodiments can comprise methods for preparing a surgical site prior to the procedure.

In embodiments, compositions disclosed herein can comprise fast-acting botulinum toxins, for example, botulinum type E.

In embodiments, compositions disclosed herein can comprise fast-recovery botulinum toxins, for example, botulinum type E.

Definitions

“Administration,” or “to administer” means 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 a number of appropriate routs, including oral and intramuscular or subcutaneous routes of administration, such as by injection or use of an implant.

“Botulinum toxin” or “botulinum neurotoxin” means a neurotoxin derived fromClostridium botulinum, as well as modified, recombinant, hybrid and chimeric botulinum toxins. A recombinant botulinum toxin can have the light chain and/or the heavy chain thereof made recombinantly by a non-Clostridial species. “Botulinum toxin,” as used herein, encompasses the botulinum toxin serotypes A, B, C, D, E, F, G and H. “Botulinum toxin,” as used herein, also encompasses both a botulinum toxin complex (i.e. the 300, 600 and 900 kDa complexes) as well as pure botulinum toxin (i.e. the about 150 kDa neurotoxic molecule), all of which are useful in the practice of the present invention. “Purified botulinum toxin” means a pure botulinum toxin or a botulinum toxin complex that is isolated, or substantially isolated, from other proteins and impurities which can accompany the botulinum toxin as it is obtained from a culture or fermentation process. Thus, a purified botulinum toxin can have at least 95%, and more preferably at least 99% of the non-botulinum toxin proteins and impurities removed.

“Clostridial neurotoxin” means a neurotoxin produced from, or native to, a Clostridial bacterium, such asClostridium botulinum, Clostridium butyricumorClostridium beratti, as well as a Clostridial neurotoxin made recombinantly by a non-Clostridial species.

“Entirely free” (“consisting of” terminology) means that within the detection range of the instrument or process being used, a substance cannot be detected or its presence cannot be confirmed.

“Essentially free” means that only trace amounts of the substance can be detected.

“Fast-acting” as used herein refers to a botulinum toxin that produces effects in the patient more rapidly than those produced by, for example, a botulinum neurotoxin type A. For example, the effects of a fast-acting botulinum toxin can be produced within 36 hours.

“Fast-recovery” as used herein refers to a botulinum toxin that whose effects diminish in the patient more rapidly than those produced by, for example, a botulinum neurotoxin type A. For example, the effects of a fast-recovery botulinum toxin can diminish within, for example, 120 hours, 150 hours, 300 hours, 350 hours, 400 hours, 500 hours, 600 hours, 700 hours, 800 hours, or the like. It is known that botulinum toxin type A can have an efficacy for up to 12 months (European J. Neurology 6 (Supp 4): S111-S1150:1999), and in some circumstances for as long as 27 months, when used to treat glands, such as in the treatment of hyperhidrosis. See e.g. Bushara K., Botulinum toxin and rhinorrhea,Otolaryngol Head Neck Surg1996; 114(3):507, and The Laryngoscope 109:1344-1346:1999. However, the usual duration of an intramuscular injection of a botulinum neurotoxin type A is typically about 3 to 4 months.

“Forced Expiratory Volume 1” (FEV1) as used herein refers to how much air a person can exhale during a forced breath. The amount of air exhaled may be measured during the first (FEV1), second (FEV2), and/or third seconds (FEV3) of the forced breath. Forced vital capacity (FVC) is the total amount of air exhaled during the FEV test.

“Forced Vital Capacity” (FVC) as used herein refers to the amount of air which can be forcibly exhaled from the lungs after taking the deepest breath possible. FVC is used to help determine both the presence and severity of lung diseases.

“Intermediate-acting” as used herein refers to a botulinum toxin that produces effects more slowly than would a fast-acting toxin.

“Neurotoxin” means a biologically active molecule with a specific affinity for a neuronal cell surface receptor. Neurotoxin includes Clostridial toxins both as pure toxin and as complexed with one to more non-toxin, toxin associated proteins.

“Opioid” as used herein refers to a substance that acts on opioid receptors.

“Patient” means a human or non-human subject receiving medical or veterinary care.

“Pharmaceutical composition” means a formulation in which an active ingredient can be a botulinum toxin. The word “formulation” means that there is at least one additional ingredient (such as, for example and not limited to, an albumin [such as a human serum albumin or a recombinant human albumin] and/or sodium chloride) in the pharmaceutical composition in addition to a botulinum neurotoxin active ingredient. A pharmaceutical composition is therefore a formulation which is suitable for diagnostic, therapeutic or cosmetic administration to a subject, such as a human patient. The pharmaceutical composition can be: in a lyophilized or vacuum dried condition, a solution formed after reconstitution of the lyophilized or vacuum dried pharmaceutical composition with saline or water, for example, or; as a solution that does not require reconstitution. As stated, a pharmaceutical composition can be liquid, semi-solid, or solid. A pharmaceutical composition can be animal-protein free.

“Slow Vital Capacity” (SVC) refers to a spirometry test that displays the volume of gas measured on a low complete expiration after a maximal inspiration without forced or rapid effort.

“Substantially free” means present at a level of less than one percent by weight of a culture medium, fermentation medium, pharmaceutical composition or other material in which the weight percent of a substance is assessed.

“Supplemental administration” as used herein refers to a botulinum administration that follows an initial neurotoxin administration.

“Therapeutic formulation” means a formulation that can be used to treat and thereby alleviate a disorder or a disease and/or symptom associated thereof, such as a disorder or a disease characterized by an activity of a peripheral muscle.

“Therapeutically effective amount” means the level, amount or concentration of an agent (e.g. such as a botulinum toxin or pharmaceutical composition comprising botulinum toxin) needed to treat a disease, disorder or condition without causing significant negative or adverse side effects.

“Treat,” “treating,” or “treatment” means an alleviation or a reduction (which includes some reduction, a significant reduction a near total reduction, and a total reduction), resolution or prevention (temporarily or permanently) of an disease, disorder or condition, so as to achieve a desired therapeutic or cosmetic result, such as by healing of injured or damaged tissue, or by altering, changing, enhancing, improving, ameliorating and/or beautifying an existing or perceived disease, disorder or condition.

“Unit” or “U” means an amount of active botulinum neurotoxin standardized to have equivalent neuromuscular blocking effect as a Unit of commercially available a botulinum neurotoxin type A.

“Wound” as used herein refers to a disruption to the skin, for example caused by injury or intentionally.

Embodiments disclosed herein comprise neurotoxin compositions, for example fast-acting, fast-recovery neurotoxins, for example botulinum type E. Such neurotoxins can be formulated in any pharmaceutically acceptable formulation in any pharmaceutically acceptable form. The neurotoxin can also be used in any pharmaceutically acceptable form supplied by any manufacturer.

The neurotoxin can be made by a Clostridial bacterium, such as by aClostridium botulinum, Clostridium butyricum, orClostridium berattibacterium. Additionally, the neurotoxin can be a modified neurotoxin; that is a neurotoxin that has at least one of its amino acids deleted, modified or replaced, as compared to the native or wild type neurotoxin. Furthermore, the neurotoxin can be a recombinantly produced neurotoxin or a derivative or fragment thereof.

In embodiments, a disclosed type E composition has 40% amino acid homology compared with type A, and they share the same basic domain structure consisting of 2 chains, a 100 kDa heavy chain (HC) and a 50 kDa light chain (LC), linked by a disulfide bond (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 can be provided as a sterile solution in a vial or as a vial or sachet containing a lyophilized powder for reconstituting in a suitable vehicle such as saline for injection.

In embodiments, the botulinum toxin is formulated in a solution containing saline and pasteurized human serum albumin, which stabilizes the toxin and minimizes loss through non-specific adsorption. The solution can be sterile filtered (0.2 μfilter), filled into individual vials, and then vacuum-dried to give a sterile lyophilized powder. In use, the powder can be reconstituted by the addition of sterile unpreserved normal saline (sodium chloride 0.9% for injection).

In an embodiment, botulinum type E is supplied in a sterile solution for injection with a 5-mL vial 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.

Although the composition may only contain a single type of neurotoxin, for example botulinum type E, disclosed compositions can include two or more types of neurotoxins, which can provide enhanced therapeutic effects of the disorders. For example, a composition administered to a patient can include botulinum types A and E. Administering a single composition containing two different neurotoxins can permit the effective concentration of each of the neurotoxins to be lower than if a single neurotoxin is administered to the patient while still achieving the desired therapeutic effects.

The composition administered to the patient can also contain other pharmaceutically active ingredients, such as, protein receptor or ion channel modulators, in combination with the neurotoxin or neurotoxins. These modulators may contribute to the reduction in neurotransmission between the various neurons. For example, a composition may contain gamma aminobutyric acid (GABA) type A receptor modulators that enhance the inhibitory effects mediated by the GABAAreceptor. The GABAAreceptor inhibits neuronal activity by effectively shunting current flow across the cell membrane. GABAAreceptor modulators may enhance the inhibitory effects of the GABAAreceptor and reduce electrical or chemical signal transmission from the neurons. Examples of GABAAreceptor modulators include benzodiazepines, such as diazepam, oxaxepam, lorazepam, prazepam, alprazolam, halazeapam, chordiazepoxide, and chlorazepate. Compositions may also contain glutamate receptor modulators that decrease the excitatory effects mediated by glutamate receptors. Examples of glutamate receptor modulators include agents that inhibit current flux through AMPA, NMDA, and/or kainate types of glutamate receptors.

Methods of Use

Methods disclosed herein can comprise administration of a neurotoxin, for example a fast-acting neurotoxin, to a patient. In embodiments, methods comprise administration of a neurotoxin in the vicinity of the lung, for example to or in the vicinity of the nerves and muscles whose activity can affect the lung.

In a preferred embodiment the neurotoxin is botulinum type E. Methods disclosed herein can comprise administration of an opioid to a patient. In embodiments, administration of the neurotoxin and the opioid can comprise different administration modes. For example, in embodiments, the neurotoxin can be administered via injection, while the opioid can be administered orally.

Methods disclosed herein can comprise supplemental administration of a fast-acting neurotoxin to a patient. Embodiments comprising supplemental administration can further comprise doctor or patient evaluation of the results of a prior neurotoxin administration.

Embodiments comprise administration of a fast-acting neurotoxin prior to a surgical procedure. In embodiments, the administration is performed, for example, within 48 hours before the procedure, within 47 hours before the procedure, within 46 hours before the procedure, within 45 hours before the procedure, within 44 hours before the procedure, within 43 hours before the procedure, within 42 hours before the procedure, within 41 hours before the procedure, within 40 hours before the procedure, within 39 hours before the procedure, within 38 hours before the procedure, within 37 hours before the procedure, within 36 hours before the procedure, within 35 hours before the procedure, within 34 hours before the procedure, within 33 hours before the procedure, within 32 hours before the procedure, within 31 hours before the procedure, within 30 hours before the procedure, within 29 hours before the procedure, within 28 hours before the procedure, within 27 hours before the procedure, within 26 hours before the procedure, within 25 hours before the procedure, within 24 hours before the procedure, within 23 hours before the procedure, within 22 hours before the procedure, within 21 hours before the procedure, within 20 hours before the procedure, within 19 hours before the procedure, within 18 hours before the procedure, within 17 hours before the procedure, within 16 hours before the procedure, within 15 hours before the procedure, within 14 hours before the procedure, within 13 hours before the procedure, within 12 hours before the procedure, within 11 hours before the procedure, within 10 hours before the procedure, within 9 hours before the procedure, within 8 hours before the procedure, within 7 hours before the procedure, within 6 hours before the procedure, within 5 hours before the procedure, within 4 hours before the procedure, within 3 hours before the procedure, within 2 hours before the procedure, within 60 minutes before the procedure, within 50 minutes before the procedure, within 40 minutes before the procedure, within 30 minutes before the procedure, within 20 minutes before the procedure, within 10 minutes before the procedure, within 5 minutes before the procedure, within 2 minutes before the procedure, or the like.

Embodiments comprise administration of a fast-acting neurotoxin prior to a surgical procedure. In embodiments, the administration is performed, for example, within 48 hours or less before the procedure, within 47 hours or less before the procedure, within 46 hours or less before the procedure, within 45 hours or less before the procedure, within 44 hours or less before the procedure, within 43 hours or less before the procedure, within 42 hours or less before the procedure, within 41 hours or less before the procedure, within 40 hours or less before the procedure, within 39 hours or less before the procedure, within 38 hours or less before the procedure, within 37 hours or less before the procedure, within 36 hours or less before the procedure, within 35 hours or less before the procedure, within 34 hours or less before the procedure, within 33 hours or less before the procedure, within 32 hours or less before the procedure, within 31 hours or less before the procedure, within 30 hours or less before the procedure, within 29 hours or less before the procedure, within 28 hours or less before the procedure, within 27 hours or less before the procedure, within 26 hours or less before the procedure, within 25 hours or less before the procedure, within 24 hours or less before the procedure, within 23 hours or less before the procedure, within 22 hours or less before the procedure, within 21 hours or less before the procedure, within 20 hours or less before the procedure, within 19 hours or less before the procedure, within 18 hours or less before the procedure, within 17 hours or less before the procedure, within 16 hours or less before the procedure, within 15 hours or less before the procedure, within 14 hours or less before the procedure, within 13 hours or less before the procedure, within 12 hours or less before the procedure, within 11 hours or less before the procedure, within 10 hours or less before the procedure, within 9 hours or less before the procedure, within 8 hours or less before the procedure, within 7 hours or less before the procedure, within 6 hours or less before the procedure, within 5 hours or less before the procedure, within 4 hours or less before the procedure, within 3 hours or less before the procedure, within 2 hours or less before the procedure, within 60 minutes or less before the procedure, within 50 minutes or less before the procedure, within 40 minutes or less before the procedure, within 30 minutes or less before the procedure, within 20 minutes or less before the procedure, within 10 minutes or less before the procedure, within 5 minutes or less before the procedure, within 2 minutes or less before the procedure, or the like.

Embodiments comprise administration of a fast-acting neurotoxin following a surgical procedure. In embodiments, the administration is performed, for example, within 48 hours or less after the procedure, within 47 hours or less after the procedure, within 46 hours or less after the procedure, within 45 hours or less after the procedure, within 44 hours or less after the procedure, within 43 hours or less after the procedure, within 42 hours or less after the procedure, within 41 hours or less after the procedure, within 40 hours or less after the procedure, within 39 hours or less after the procedure, within 38 hours or less after the procedure, within 37 hours or less after the procedure, within 36 hours or less after the procedure, within 35 hours or less after the procedure, within 34 hours or less after the procedure, within 33 hours or less after the procedure, within 32 hours or less after the procedure, within 31 hours or less after the procedure, within 30 hours or less after the procedure, within 29 hours or less after the procedure, within 28 hours or less after the procedure, within 27 hours or less after the procedure, within 26 hours or less after the procedure, within 25 hours or less after the procedure, within 24 hours or less after the procedure, within 23 hours or less after the procedure, within 22 hours or less after the procedure, within 21 hours or less after the procedure, within 20 hours or less after the procedure, within 19 hours or less after the procedure, within 18 hours or less after the procedure, within 17 hours or less after the procedure, within 16 hours or less after the procedure, within 15 hours or less after the procedure, within 14 hours or less after the procedure, within 13 hours or less after the procedure, within 12 hours or less after the procedure, within 11 hours or less after the procedure, within 10 hours or less after the procedure, within 9 hours or less after the procedure, within 8 hours or less after the procedure, within 7 hours or less after the procedure, within 6 hours or less after the procedure, within 5 hours or less after the procedure, within 4 hours or less after the procedure, within 3 hours or less after the procedure, within 2 hours or less after the procedure, within 60 minutes or less after the procedure, within 50 minutes or less after the procedure, within 40 minutes or less after the procedure, within 30 minutes or less after the procedure, within 20 minutes or less after the procedure, within 10 minutes or less after the procedure, within 5 minutes or less after the procedure, within 2 minutes or less after the procedure, or the like.

Embodiments comprise administration of a fast-acting neurotoxin following an injury. For example, in embodiments, the fast-acting neurotoxin can be administered within 5 minutes of an injury occurring, within 10 minutes an injury, within 15 minutes of an injury, within 20 minutes of an injury, within 25 minutes of an injury, within 30 minutes of an injury, within 35 minutes of an injury, within 40 minutes of an injury, within 45 minutes of an injury, within 50 minutes of an injury, within 55 minutes of an injury, within 60 minutes of an injury, within 65 minutes of an injury, within 70 minutes of an injury, within 75 minutes of an injury, within 80 minutes of an injury, within 85 minutes of an injury, within 90 minutes of an injury, within 95 minutes of an injury, within 100 minutes of an injury, within 110 minutes of an injury, within 2 hours of an injury, within 2 hours of an injury, within 3 hours of an injury, within 4 hours of an injury, within 5 hours of an injury, within 6 hours of an injury, within 7 hours of an injury, within 8 hours of an injury, within 9 hours of an injury, within 10 hours of an injury, within 11 hours of an injury, within 12 hours of an injury, within 13 hours of an injury, within 14 hours of an injury, within 15 hours of an injury, within 16 hours of an injury, within 17 hours of an injury, within 18 hours of an injury, within 19 hours of an injury, within 20 hours of an injury, within 21 hours of an injury, within 22 hours of an injury, within 23 hours of an injury, within 1 day of an injury, within 2 days of an injury, within 3 days of an injury, within 4 days of an injury, within 5 days of an injury, within 6 days of an injury, within 7 days of an injury, within 8 days of an injury, within 9 days of an injury, within 10 days of an injury, or the like.

In embodiments, administration of the fast-acting neurotoxin is performed concurrently with a surgical procedure. In embodiments, administration of an opioid is performed concurrently with a surgical procedure.

In embodiments, administration of the fast-acting neurotoxin is performed after administration of an opioid. For example, administration can be performed, within 1 minute after administration of an opioid, within 2 minutes after administration of an opioid, within 3 minutes after administration of an opioid, within 4 minutes after administration of an opioid, within 5 minutes after administration of an opioid, within 6 minutes after administration of an opioid, within 7 minutes after administration of an opioid, within 8 minutes after administration of an opioid, within 9 minutes after administration of an opioid, within 10 minutes after administration of an opioid, within 20 minutes after administration of an opioid, within 30 minutes after administration of an opioid, within 40 minutes after administration of an opioid, within 50 minutes after administration of an opioid, within 60 minutes after administration of an opioid, within 90 minutes after administration of an opioid, within 120 minutes after administration of an opioid, within 180 minutes after administration of an opioid, within 240 minutes after administration of an opioid, within 300 minutes after administration of an opioid, or the like.

In embodiments comprising a supplemental administration, evaluation of the results of the initial neurotoxin administration can be performed within, for example, 6 hours of the initial administration of neurotoxin, 8 hours of the initial administration, 10 hours of the initial administration, 12 hours of the initial administration, 14 hours of the initial administration, 16 hours of the initial administration, 18 hours of the initial administration, 24 hours of the initial administration, 30 hours of the initial administration, 36 hours of the initial administration, 42 hours of the initial administration, 48 hours of the initial administration, 54 hours of the initial administration, 60 hours of the initial administration, 66 hours of the initial administration, 72 hours of the initial administration, 78 hours of the initial administration, 84 hours of the initial administration, 90 hours of the initial administration, 96 hours of the initial administration, 102 hours of the initial administration, 108 hours of the initial administration, 114 hours of the initial administration, 120 hours of the initial administration, 1 week of the initial administration, 2 weeks of the initial administration, 3 weeks of the initial administration, 4 weeks of the initial administration, 5 weeks of the initial administration, 6 weeks of the initial administration, 7 weeks of the initial administration, 8 weeks of the initial administration, 9 weeks of the initial administration, 10 weeks of the initial administration, 11 weeks of the initial administration, 12 weeks of the initial administration, or the like.

In embodiments comprising a supplemental administration, the supplemental neurotoxin administration can be performed within, for example, 6 hours of the initial administration of neurotoxin, 8 hours of the initial administration, 10 hours of the initial administration, 12 hours of the initial administration, 14 hours of the initial administration, 16 hours of the initial administration, 18 hours of the initial administration, 24 hours of the initial administration, 30 hours of the initial administration, 36 hours of the initial administration, 42 hours of the initial administration, 48 hours of the initial administration, 54 hours of the initial administration, 60 hours of the initial administration, 66 hours of the initial administration, 72 hours of the initial administration, 78 hours of the initial administration, 84 hours of the initial administration, 90 hours of the initial administration, 96 hours of the initial administration, 102 hours of the initial administration, 108 hours of the initial administration, 114 hours of the initial administration, 120 hours of the initial administration, 1 week of the initial administration, 2 weeks of the initial administration, 3 weeks of the initial administration, 4 weeks of the initial administration, 5 weeks of the initial administration, 6 weeks of the initial administration, 7 weeks of the initial administration, 8 weeks of the initial administration, 9 weeks of the initial administration, 10 weeks of the initial administration, 11 weeks of the initial administration, 12 weeks of the initial administration, or the like.

Methods disclosed herein can provide rapid-onset effects (for example, using a fast-acting neurotoxin). For example, disclosed embodiments can reduce muscle activity, nerve activity, and pain sensation within, for example, 30 minutes or less after administration, 45 minutes or less after administration, 60 minutes or less after administration, 75 minutes or less after administration, 90 minutes or less after administration, 2 hours or less after administration, 3 hours or less after administration, 4 hours or less after administration, 5 hours or less after administration, 6 hours or less after administration, 7 hours or less after administration, 8 hours or less after administration, 9 hours or less after administration, 10 hours or less after administration, 11 hours or less after administration, 12 hours or less after administration, 13 hours or less after administration, 14 hours or less after administration, 15 hours or less after administration, 16 hours or less after administration, 17 hours or less after administration, 18 hours or less after administration, 19 hours or less after administration, 20 hours or less after administration, 21 hours or less after administration, 22 hours or less after administration, 23 hours or less after administration, 24 hours or less after administration, 30 hours or less after administration, 36 hours or less after administration, 42 hours or less after administration, 48 hours or less after administration, 3 days or less after administration, 4 days or less after administration, 5 days or less after administration, 6 days or less after administration, 7 days or less after administration, or the like.

Methods disclosed herein can provide reduction in muscle activity, nerve activity, and pain sensation for a shorter duration (for example, using a fast-recovery neurotoxin). For example, disclosed embodiments can provide a reduction in muscle activity and pain sensation that subsides within, for example, 3 days or less after administration, 4 days or less after administration, 5 days or less after administration, 6 days or less after administration, 7 days or less after administration, 8 days or less after administration, 9 days or less after administration, 10 days or less after administration, 11 days or less after administration, 12 days or less after administration, 13 days or less after administration, 14 days or less after administration, 15 days or less after administration, 16 days or less after administration, 17 days or less after administration, 18 days or less after administration, 19 days or less after administration, 20 days or less after administration, 21 days or less after administration, 22 days or less after administration, 23 days or less after administration, 24 days or less after administration, 25 days or less after administration, 26 days or less after administration, 27 days or less after administration, 28 days or less after administration, 29 days or less after administration, 30 days or less after administration, 45 days or less after administration, 60 days or less after administration, 75 days or less after administration, 90 days or less after administration, 105 days or less after administration, or the like.

Side-effects can be associated with botulinum injections. Disclosed embodiments can provide neurotoxin treatments that result in fewer side effects, or side effects of a shorted duration, than conventional neurotoxin treatments. For example, disclosed embodiments can result in fewer (or shorter duration) instances of double vision or blurred vision, eyelid paralysis (subject cannot lift eyelid all the way open), loss of facial muscle movement, hoarseness, loss of bladder control, shortness of breath, difficulty in swallowing, difficulty speaking, death, and the like.

Further, disclosed embodiments can provide reduced muscle and nerve activity and reduced pain sensation of a more-certain duration. For example, with a longer acting neurotoxin, a 20% variance in duration of effects can result in a month's difference in effective duration. With the disclosed fast-recovery neurotoxins, this 20% variance produces a much less drastic difference in effective duration.

Disclosed fast-acting neurotoxin compositions can be injected into the patient using a needle or a needleless device. In certain embodiments, the method comprises sub-dermally injecting the composition in the individual. For example, administering may comprise injecting the composition through a needle no greater than about 30 gauge. In certain embodiments, the method comprises administering a composition comprising a botulinum toxin type E.

Administration of the disclosed compositions can be carried out by syringe, catheters, needles and other means for injecting. The injection can be performed on any area of the mammal's body that is in need of treatment, including, but not limited to, face, neck, torso, arms, hands, legs, and feet. The injection can be into any position in the specific area such as epidermis, dermis, fat, muscle, or subcutaneous layer.

Before injecting any muscle group, careful consideration is given to the anatomy of the muscle group, the aim being to inject the area with the highest concentration of neuromuscular junctions, if known. Before injecting the muscle, the position of the needle in the muscle can be confirmed by putting the muscle through its range of motion and observing the resultant motion of the needle end. General anesthesia, local anesthesia and sedation are used according to the age of the patient, the number of sites to be injected, and the particular needs of the patient. More than one injection and/or sites of injection may be necessary to achieve the desired result. Also, some injections, depending on the muscle to be injected, may require the use of fine, hollow, Teflon®-coated needles, guided by electromyography.

Smooth muscles suitable for administration of disclosed compositions can comprise any of walls of blood vessels, walls of stomach, ureters, intestines, in the aorta (tunica media layer), iris of the eye, prostate, gastrointestinal tract, respiratory tract, small arteries, arterioles, reproductive tracts (both genders), veins, glomeruli of the kidneys (called mesangial cells), bladder, uterus, arrector pili of the skin, ciliary muscle, sphincter, trachea, bile ducts, and the like.

The frequency and the amount of injection under the disclosed methods can be determined based on the nature and location of the particular area being treated. In certain cases, however, repeated injection may be desired to achieve optimal results. The frequency and the amount of the injection for each particular case can be determined by the person of ordinary skill in the art.

Although examples of routes of administration and dosages are provided, the appropriate route of administration and dosage are generally determined on a case by case basis by the attending physician. Such determinations are routine to one of ordinary skill in the art (see for example, Harrison's Principles of Internal Medicine (1998), edited by Anthony Fauci et al., 14th edition, published by McGraw Hill). For example, the route and dosage for administration of a Clostridial neurotoxin according to the present disclosed invention can be selected based upon criteria such as the solubility characteristics of the neurotoxin chosen as well as the intensity and scope of the condition being treated.

The fast-acting neurotoxin can be administered in an amount of between about 10−3U/kg and about 35 U/kg. In an embodiment, the neurotoxin is administered in an amount of between about 10−2U/kg and about 25 U/kg. In another embodiment, the neurotoxin is administered in an amount of between about 10−1U/kg and about 15 U/kg. In another embodiment, the neurotoxin is administered in an amount of between about 1 U/kg and about 10 U/kg. In many instances, an administration of from about 1 unit to about 500 units of a neurotoxin, such as a botulinum type E, provides effective therapeutic relief. In an embodiment, from about 5 units to about 200 units of a neurotoxin, such as a botulinum type E, can be used and in another embodiment, from about 10 units to about 100 units of a neurotoxin, such as a botulinum type E, can be locally administered into a target tissue such as a muscle.

In embodiments, administration can comprise a dose of about 10 units of a neurotoxin, or about 20 units of a neurotoxin, or about 30 units of a neurotoxin, or about 40 units of a neurotoxin, or about 50 units of a neurotoxin, or about 60 units of a neurotoxin, or about 70 units of a neurotoxin, or about 80 units of a neurotoxin, or about 90 units of a neurotoxin, or about 100 units of a neurotoxin, or about 110 units of a neurotoxin, or about 120 units of a neurotoxin, or about 130 units of a neurotoxin, or about 140 units of a neurotoxin, or about 150 units of a neurotoxin, or about 160 units of a neurotoxin, or about 170 units of a neurotoxin, or about 180 units of a neurotoxin, or about 190 units of a neurotoxin, or about 200 units of a neurotoxin, or about 210 units of a neurotoxin, or about 220 units of a neurotoxin, or about 230 units of a neurotoxin, or about 240 units of a neurotoxin, or about 250 units of a neurotoxin, or about 260 units of a neurotoxin, or about 270 units of a neurotoxin, or about 280 units of a neurotoxin, or about 290 units of a neurotoxin, or about 290 units of a neurotoxin, or about 300 units of a neurotoxin, or about 310 units of a neurotoxin, or about 320 units of a neurotoxin, or about 330 units of a neurotoxin, or about 340 units of a neurotoxin, or about 350 units of a neurotoxin, or about 360 units of a neurotoxin, or about 370 units of a neurotoxin, or about 380 units of a neurotoxin, or about 390 units of a neurotoxin, or about 400 units of a neurotoxin, or about 410 units of a neurotoxin, or about 420 units of a neurotoxin, or about 430 units of a neurotoxin, or about 440 units of a neurotoxin, or about 450 units of a neurotoxin, or about 460 units of a neurotoxin, or about 470 units of a neurotoxin, or about 480 units of a neurotoxin, or about 490 units of a neurotoxin, or about 500 units of a neurotoxin, or the like.

In embodiments, the dose of the neurotoxin is expressed in protein amount or concentration. For example, in embodiments the neurotoxin can be administered in an amount of between about 0.2 ng and 20 ng. In an embodiment, the neurotoxin is administered in an amount of between about 0.3 ng and 19 ng, about 0.4 ng and 18 ng, about 0.5 ng and 17 ng, about 0.6 ng and 16 ng, about 0.7 ng and 15 ng, about 0.8 ng and 14 ng, about 0.9 ng and 13 ng, about 1.0 ng and 12 ng, about 1.5 ng and 11 ng, about 2 ng and 10 ng, about 5 ng and 7 ng, and the like, into a target tissue such as a muscle.

In embodiments, administration can comprise a total dose of between 5 and 7 ng, between 7 and 9 ng, between 9 and 11 ng, between 11 and 13 ng, between 13 and 15 ng, between 15 and 17 ng, between 17 and 19 ng, or the like.

In embodiments, administration can comprise a total dose of not more than 5 ng, not more than 6 ng, not more than 7 ng, not more than 8 ng, not more than 9 ng, not more than 10 ng, not more than 11 ng, not more than 12 ng, not more than 13 ng, not more than 14 ng, not more than 15 ng, not more than 16 ng, not more than 17 ng, not more than 18 ng, not more than 19 ng, not more than 20 ng, or the like.

In embodiments, administration can comprise a total dose of not less than 5 ng, not less than 6 ng, not less than 7 ng, not less than 8 ng, not less than 9 ng, not less than 10 ng, not less than 11 ng, not less than 12 ng, not less than 13 ng, not less than 14 ng, not less than 15 ng, not less than 16 ng, not less than 17 ng, not less than 18 ng, not less than 19 ng, not less than 20 ng, or the like.

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

In embodiments, administration can comprise a dose per administration of about 0.1 ng of a neurotoxin, 0.2 ng of a neurotoxin, 0.3 ng of a neurotoxin, 0.4 ng of a neurotoxin, 0.5 ng of a neurotoxin, 0.6 n of a neurotoxin, 0.7 ng of a neurotoxin, 0.8 ng of a neurotoxin, 0.9 ng of a neurotoxin, 1.0 ng of a neurotoxin, 1.1 ng of a neurotoxin, 1.2 ng of a neurotoxin, 1.3 ng of a neurotoxin, 1.4 ng of a neurotoxin, 1.5 ng of a neurotoxin, 1.6 ng of a neurotoxin, 1.7 ng of a neurotoxin, 1.8 ng of a neurotoxin, 1.9 ng of a neurotoxin, 2.0 ng of a neurotoxin, 2.1 ng of a neurotoxin, 2.2 ng of a neurotoxin, 2.3 ng of a neurotoxin, 2.4 ng of a neurotoxin, 2.5 ng of a neurotoxin, 2.6 ng of a neurotoxin, 2.7 ng of a neurotoxin, 2.8 ng of a neurotoxin, 2.9 ng of a neurotoxin, 3.0 ng of a neurotoxin, 3.1 ng of a neurotoxin, 3.2 ng of a neurotoxin, 3.3 ng of a neurotoxin, 3.4 ng of a neurotoxin, 3.5 ng of a neurotoxin, 3.6 n of a neurotoxin, 3.7 n of a neurotoxin, 3.8 n of a neurotoxin, 3.9 ng of a neurotoxin, 4.0 ng of a neurotoxin, 4.1 ng of a neurotoxin, 4.2 ng of a neurotoxin, 4.3 ng of a neurotoxin, 4.4 ng of a neurotoxin, 4.5 ng of a neurotoxin, 5 ng of a neurotoxin, 6 ng of a neurotoxin, 7 ng of a neurotoxin, 8 ng of a neurotoxin, 9 ng of a neurotoxin, 10 ng of a neurotoxin, or the like.

In embodiments, the patient's total bi-monthly protein dose can be limited to, for example, 5 ng of a neurotoxin, 6 ng of a neurotoxin, 7 ng of a neurotoxin, 8 ng of a neurotoxin, 9 ng of a neurotoxin, 10 ng of a neurotoxin, 11 ng of a neurotoxin, 12 ng of a neurotoxin, 13 ng of a neurotoxin, 14 ng of a neurotoxin, 15 ng of a neurotoxin, 16 ng of a neurotoxin, 17 ng of a neurotoxin, 18 ng of a neurotoxin, 19 ng of a neurotoxin, 20 ng of a neurotoxin, or the like.

In embodiments, the patient's total monthly protein dose can be limited to, for example, 5 ng of a neurotoxin, 6 ng of a neurotoxin, 7 ng of a neurotoxin, 8 ng of a neurotoxin, 9 ng of a neurotoxin, 10 ng of a neurotoxin, 11 ng of a neurotoxin, 12 ng of a neurotoxin, 13 ng of a neurotoxin, 14 ng of a neurotoxin, 15 ng of a neurotoxin, 16 ng of a neurotoxin, 17 ng of a neurotoxin, 18 ng of a neurotoxin, 19 ng of a neurotoxin, 20 ng of a neurotoxin, or the like.

In embodiments, the patient's total yearly protein dose can be limited to, for example, 25 ng of a neurotoxin, 35 ng of a neurotoxin, 45 ng of a neurotoxin, 55 ng of a neurotoxin, 65 ng of a neurotoxin, 75 ng of a neurotoxin, 85 ng of a neurotoxin, 95 ng of a neurotoxin, 105 ng of a neurotoxin, 125 ng of a neurotoxin, 145 ng of a neurotoxin, 165 ng of a neurotoxin, 185 ng of a neurotoxin, 200 ng of a neurotoxin, or the like.

Ultimately, however, both the quantity of toxin administered and the frequency of its administration will be at the discretion of the physician responsible for the treatment and will be commensurate with questions of safety and the effects produced by the toxin.

In embodiments, the dose of the opioid can be, for example, between 0.1 and 100 mg, between 1 and 100 mg, between 4 and 95 mg, between 6 and 90 mg, between 8 and 85 mg, between 10 and 80 mg, between 20 and 60 mg, and the like.

In embodiments, the dose of the opioid can be, for example, at least 1 mg, at least 2 mg, at least 4 mg, at least 6 mg, at least 8 mg, at least 10 mg, at least 12 mg, at least 14 mg, at least 16 mg, at least 18 mg, at least 20 mg, at least 22 mg, at least 24 mg, at least 26 mg, at least 28 mg, at least 30 mg, at least 32 mg, at least 34 mg, at least 36 mg, at least 38 mg, at least 40 mg, at least 42 mg, at least 44 mg, at least 46 mg, at least 48 mg, at least 50 mg, at least 55 mg, at least 60 mg, at least 65 mg, at least 70 mg, at least 75 mg, at least 80 mg, at least 85 mg, at least 95 mg, at least 100 mg, or the like.

In embodiments, the dose of the opioid can be, for example, not more than 1 mg, not more than 2 mg, not more than 4 mg, not more than 6 mg, not more than 8 mg, not more than 10 mg, not more than 12 mg, not more than 14 mg, not more than 16 mg, not more than 18 mg, not more than 20 mg, not more than 22 mg, not more than 24 mg, not more than 26 mg, not more than 28 mg, not more than 30 mg, not more than 32 mg, not more than 34 mg, not more than 36 mg, not more than 38 mg, not more than 40 mg, not more than 42 mg, not more than 44 mg, not more than 46 mg, not more than 48 mg, not more than 50 mg, not more than 55 mg, not more than 60 mg, not more than 65 mg, not more than 70 mg, not more than 75 mg, not more than 80 mg, not more than 85 mg, not more than 95 mg, not more than 100 mg, or the like.

A controlled release system can be used in the embodiments described herein to deliver a neurotoxin in vivo at a predetermined rate over a specific time period. Generally, release rates are determined by the design of the system, and can be largely independent of environmental conditions such as pH. Controlled release systems which can deliver a drug over a period of several years are known. Contrarily, sustained release systems typically deliver drug in 24 hours or less and environmental factors can influence the release rate. Thus, the release rate of a neurotoxin from an implanted controlled release system (an “implant”) is a function of the physiochemical properties of the carrier implant material and of the drug itself. Typically, the implant is made of an inert material which elicits little or no host response.

A controlled release system can be comprised of a neurotoxin incorporated into a carrier. The carrier can be a polymer or a bio-ceramic material. The controlled release system can be injected, inserted or implanted into a selected location of a patient's body and reside therein for a prolonged period during which the neurotoxin is released by the implant in a manner and at a concentration which provides a desired therapeutic efficacy.

Polymeric materials can release neurotoxins due to diffusion, chemical reaction or solvent activation, as well as upon influence by magnetic, ultrasound or temperature change factors. Diffusion can be from a reservoir or matrix. Chemical control can be due to polymer degradation or cleavage of the drug from the polymer. Solvent activation can involve swelling of the polymer or an osmotic effect.

Implants may be prepared by mixing a desired amount of a stabilized neurotoxin into a solution of a suitable polymer dissolved in methylene chloride. The solution may be prepared at room temperature. The solution can then be transferred to a Petri dish and the methylene chloride evaporated in a vacuum desiccator. Depending upon the implant size desired and hence the amount of incorporated neurotoxin, a suitable amount of the dried neurotoxin incorporating implant is compressed at about 8000 p.s.i. for 5 seconds or at 3000 p.s.i. for 17 seconds in a mold to form implant discs encapsulating the neurotoxin.

An implant material can be biodegradable or bioerodible. An advantage of a bioerodible implant is that it does not need to be removed from the patient. A bioerodible implant can be based upon either a membrane or matrix release of the bioactive substance. Biodegradable microspheres prepared from PLA-PGA are known for subcutaneous or intramuscular administration.

A kit for practicing disclosed embodiments is also encompassed by the present disclosure. The kit can comprise a 30 gauge or smaller needle and a corresponding syringe. The kit can also comprise a Clostridial neurotoxin composition, such as a botulinum type E toxin composition. The neurotoxin composition may be provided in the syringe. The composition is injectable through the needle. The kits are designed in various forms based the sizes of the syringe and the needles and the volume of the injectable composition contained therein, which in turn are based on the specific deficiencies the kits are designed to treat.

EXAMPLES

The following non-limiting Examples are provided for illustrative purposes only in order to facilitate a more complete understanding of representative embodiments. This example should not be construed to limit any of the embodiments described in the present specification.

Use of Botulinum Toxin Type E to Treat Glabellar Lines

This first-in-human, randomized, double-blinded, 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 efficacy primary outcome was the proportion of subjects with a 2-grade investigator-rated (IR-2) improvement in GL severity at maximum frown. Safety evaluations included adverse events (AEs), laboratory tests, and physical examinations. An IR-2 response was observed starting in the third cohort (EB-001), with increased rates observed at higher doses. Onset of clinical effect was within 24 hours, with a duration ranging between 14 and 30 days for the highest doses. AE incidence was low, with the most common being mild to moderate headache. There were no serious AEs or ptosis, and no clinically significant changes in other safety assessments.

In this clinical study in GL, EB-001 showed favorable safety and tolerability, and dose dependent efficacy with an 80% response rate at the highest dose. EB-001 maximum clinical effect was seen within 24 hours and lasted between 14 and 30 days. This differentiated EB-001 profile supports its development for aesthetic and therapeutic applications where fast onset and short duration of effect are desirable.

Botulinum neurotoxins, which inhibit the pre-synaptic release of acetylcholine, are among the most potent molecules in nature. When injected into muscles, 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 conditions. Botulinum neurotoxin serotype E is a novel serotype that has not been developed for clinical use to date. Botulinum toxin type E has the fastest onset and the shortest duration of action of all the Botulinum neurotoxins. Type E has similar domain structure to type A, consisting of 2 protein chains, a 100 kDa heavy chain and a 50 kDa light chain linked by a disulfide bond. 2 Type E inhibits neuromuscular transmission by cleaving the same presynaptic vesicular protein (synaptosomal associated protein 25) as type A, but at a different cleavage site. Two binding sites on motor axons mediate the high affinity recognition of nerve cells by Botulinum neurotoxins. 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. Botulinum toxin types A and E have both been shown to bind the specific receptor synaptic vesicle protein 2, and only these two serotypes share this receptor. This was the first clinical study to evaluate the safety and efficacy of ascending doses of Botulinum toxin type E in subjects with GL.

This study was a first-in-human evaluation of the safety and efficacy of EB-001 and focused on the treatment of moderate to severe GL. EB-001 is a proprietary purified form of Botulinum toxin type E, formulated as a liquid for injection (Bonti, Inc., Newport Beach, Calif., USA). This was a randomized, double-blinded, placebo-controlled, ascending-dose cohort study conducted at 2 expert clinical centers (Steve Yoelin, MD Medical Associates, Newport Beach, Calif., USA; Center for Dermatology Clinical Research, Fremont, Calif., USA). This study was approved by an Institutional Review Board (Aspire Institutional Review Board, Santee, Calif., USA) and was conducted in accordance with the guidelines set by the Declaration of Helsinki. Written informed consent was received from all subjects prior to their participation.

A total of 42 healthy toxin-naïve male and female subjects, ages 18 to 60 years, were enrolled in the study. Each subject's participation was to last approximately 6 weeks. The main inclusion criteria were: the presence of bilaterally symmetrical GL of moderate to severe rating at maximum frown, sufficient visual acuity without the use of eyeglasses (contact lens use acceptable) to accurately assess their facial wrinkles, and the ability to conform with study requirements. The main criteria for exclusion were: any uncontrolled systemic disease or other medical condition, any medical condition that may have put the subject at increased risk with exposure to Botulinum neurotoxin (including diagnosed myasthenia gravis, Eaton-Lambert syndrome, amyotrophic lateral sclerosis, or any other condition that interfered with neuromuscular function), current or prior Botulinum neurotoxin treatment, known immunization or hypersensitivity to Botulinum neurotoxin, pre-specified dermatological procedures within 3 to 12 months of the study (non-ablative resurfacing, facial cosmetic procedures, topical/oral retinoid therapy, etc.), and prior periorbital surgery or treatment. Women were not enrolled if they were pregnant, lactating, or planning to become pregnant. Men with female partner(s) of childbearing potential were enrolled only if they agreed to use dual methods of contraception for 3 months following dosing.

At Screening, subject demographics, medical history, and prior and concomitant medications were recorded and an alcohol/drug screen was performed. Standardized facial photography was performed at Baseline prior to treatment, and at every follow-up visit through the end of the study, but the photographs were not used for efficacy evaluations.

Seven cohorts (6 subjects per cohort) were enrolled and received ascending doses of EB-001 or placebo in a 5:1 ratio. The maximum recommended starting dose (with a 10-fold safety factor) in this first-in-human study was developed based on the no observed adverse effect levels from a preclinical safety and toxicity study (unpublished data). From this, a base dose (Cohort 1) was calculated and determined to be sub-efficacious, and Cohorts 2 to 7 received 3, 9, 12, 16, 21, and 28 times the base dose, respectively. This represented sub-efficacious to maximum-efficacious doses of EB-001. The total dose was delivered at 5 injection sites in equal volumes (0.1 mL per site into the procerus, left and right medial corrugators, and left and right lateral corrugators) in a standardized fashion (seeFIG. 1). The spacing of injections into the lateral corrugators was approximately 1 cm above the supraorbital ridge. EB-001 was supplied in a sterile solution for injection in a 5-mL vial. The placebo was supplied in identical vials without EB-001.

Safety was evaluated by adverse events (AEs), laboratory testing, electrocardiograms (ECGs), physical examinations, vital signs (pulse rate, respiratory rate, and blood pressure), urine pregnancy tests (for women of childbearing potential), and focused neurologic examinations to evaluate for the potential spread of Botulinum neurotoxin. Treatment-emergent AEs (TEAEs) were defined as any AE that started or worsened in severity after exposure to study treatment. AEs and TEAEs were summarized by system organ class and preferred term using the Medical Dictionary for Regulatory Activities (MedDRA, version 19.0). Serious AEs (SAEs, or AEs that fulfilled regulatory criteria for medical seriousness), and discontinuation due to AEs were also evaluated. Severity of AEs was recorded as mild, moderate, severe, or life threatening. Before enrollment of each dosing cohort, a safety data review committee met to analyze all safety data from the previous cohort(s).

At Screening, Baseline, and Days 1, 2, 7, 14, and 30, the subject's GL were assessed at maximum frown and at rest using the Facial Wrinkle Scale (FWS). Evaluations were completed by the investigator and the subject. The FWS is a widely accepted measure used for the evaluation of facial line severity. In the present study, the 4-point scale indicating severity of GL was as follows: 0=none, 1=mild, 2=moderate, 3=severe. Subjects were considered as treatment responders if they achieved at least a 2-grade improvement (reduction) based on the investigator's FWS assessment (IR-2). The primary efficacy variable was the proportion of IR-2 responders at maximum frown at any post baseline visit through Day 30. An additional efficacy endpoint of interest was the proportion of responders achieving an investigator-assessed FWS grade of none or mild at Days 1, 2, 7, 14, or 30 (analyzed by visit).

Two analysis populations were pre-specified, a safety and an efficacy population. Subjects receiving placebo were pooled for all analyses. The safety population included all subjects who received study treatment and had at least 1 safety assessment thereafter. All TEAEs and SAEs were summarized by treatment group. All safety parameters, including laboratory testing, ECGs, physical exams, vital signs, urine pregnancy tests, and focused neurologic examinations, were reviewed and evaluated for clinical significance by the investigators. The efficacy population was the modified intent-to-treat (mITT) population, defined as all randomized subjects who received at least 1 dose of study treatment and had at least 1 post baseline efficacy assessment. Analyses of demographics and baseline characteristics were performed on the mITT population. Medical history was based on the safety population and coded using MedDRA and summarized by system organ class and preferred term. Prior and concomitant medications were based on the safety population and coded using the World Health Organization Anatomical Therapeutic Chemical classification index and summarized by drug class and treatment group. Efficacy analyses were performed using the mITT population. FWS grades were summarized by treatment and study day using frequency counts and rates of response (%). An analysis comparing the proportion of IR-2 responders in each EB-001 cohort versus placebo (pooled) was performed using Fisher's exact test with a 0.05 level of significance.

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

The proportions of subjects in the mITT population achieving an IR-2 response for GL severity at maximum frown at any postbaseline visit through Day 30 are presented by dose cohort inFIG. 2. In Cohort 3, 40% of subjects were IR-2 responders. This responder rate was the same or greater in all higher dose cohorts, with Cohorts 6 and 7 having 80% IR-2 responders. Cohorts 6 and 7 demonstrated significantly greater percentages of IR-2 responders versus placebo (P=0.046).FIG. 3summarizes the proportions of subjects in each cohort with investigator-assessed FWS grades of none or mild GL at maximum frown, at any post baseline visit through Day 30. Cohorts 2 to 7 (inclusive) had greater percentages of responders versus placebo, with rates of 60% to 100% achieved for Cohorts 3 and higher. In Cohorts 3 to 7, most none or mild responses were observed at Days 1, 2, and/or 7. One responder (20%) was observed at Day 14 in Cohorts 3, 5, 6 and 7 and at Day 30 in Cohorts 3 and 5. The safety results support the safety of all evaluated doses of EB-001, administered as IM injections, in this population. No clinically significant changes from baseline in neurologic examinations, ECGs, physical examinations, or laboratory tests were observed for any subject.

Five subjects treated with EB-001 reported TEAEs, and none in placebo group. No SAEs were reported and no TEAE led to discontinuation of the study. All TEAEs were mild or moderate in severity. The events of sore throat and flu like symptoms were considered unrelated to treatment. Three subjects reported TEAEs of headache, 1 of which was considered related to treatment. There was no dose-related increase in the incidence of headaches. There were no events of ptosis or other TEAE possibly related to spread of toxin.

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, fulfilled its objectives of evaluating the safety, tolerability, and efficacious dose-range of EB-001. A dose response was observed, with greater proportions of treatment responders in the higher dosing cohorts of EB-001. An IR-2 response was observed starting with Cohort 3 and increased in higher dose cohorts, suggesting that the efficacious dose range of EB-001 may be at doses used in Cohorts 4 to 7. Cohorts 6 and 7 had 80% IR-2 responders, a response rate similar to approved Botulinum toxin type A products. Subjects achieving none or mild FWS grades were observed starting at Cohort 2. In terms of onset of effect, treatment response was observed as early as 24 hours following dosing, which supports prior reports suggesting that Botulinum toxin type E has a faster onset than type A.

Regarding the duration of effect defined as the proportion of responders with a none or mild rating, an effect was observed through Day 14 in 1 subject in most of the 5 higher dose cohorts, and through Day 30 in 1 subject in 2 of the 5 higher dose cohorts. All doses of EB-001 showed good tolerability with no local injection site reactions. There were no SAEs or severe TEAEs reported, and no discontinuations due to a TEAE. The most common TEAE of headache was mild or moderate in severity, and there were no other treatment related AEs. There were no events of ptosis at any dose levels, and no events potentially related to spread of toxin. Therefore, the clinical safety and tolerability profile seems favorable in this study. The efficacy and safety profiles of EB-001 are promising and support the potential of EB-001 as a unique treatment option in the treatment of GL and other facial aesthetic uses. The fast onset can fulfill an unmet need for individuals seeking a rapid treatment for facial wrinkles before unexpected social or professional events. The limited duration of effect can be beneficial for individuals who may be considering first time use of a Botulinum neurotoxin treatment, and are unwilling to make a longer-term commitment. An EB-001 treatment would allow them to assess the aesthetic effect over a shorter duration of effect compared with the 12-week duration of effect of Botulinum toxin type A products. In this first clinical study in subjects with GL, EB-001 showed favorable safety and tolerability in all cohorts. Five out of the 7 cohorts showed numerically higher response rates compared to placebo, supporting the efficacy of EB-001 in the reduction of GL severity. The 2 highest doses provided 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 seen within 24 hours (onset) and lasted between 14-30 days (duration). This differentiated clinical profile supports the future development of EB-001 for facial aesthetic and key therapeutic uses, where fast onset and short duration of effect are desirable.

TABLE S-1Dose Escalation SchemeTotal EB-Dose atDoses at MedialDose at Lateral001 DoseProcerusCorrugatorsCorrugatorsCohort1(ng)2(ng)2(ng)(ng)10.1EB-001EB-001 into right and leftEB-001 into right and left(0.02)corrugators (0.02 each)corrugators (0.02 each)20.3EB-001EB-001 into right and leftEB-001 into right and left(0.06)corrugators (0.06 each)corrugators (0.06 each)30.9EB-001EB-001 into right and leftEB-001 into right and left(0.18)corrugators (0.18 each)corrugators (0.18 each)41.2EB-001EB-001 into right and leftEB-001 into right and left(0.24)corrugators (0.24 each)corrugators (0.24 each)51.6EB-001EB-001 into right and leftEB-001 into right and left(0.32)corrugators (0.32 each)corrugators (0.32 each)62.1EB-001EB-001 into right and leftEB-001 into right and left(0.42)corrugators (0.42 each)corrugators (0.42 each)72.8EB-001EB-001 into right and leftEB-001 into right and left(0.56)corrugators (0.56 each)corrugators (0.56 each)

Use of Botulinum Toxin Type E in Connection with Surgery to the Trunk

A 57 year old man requires bypass surgery. 24 hours prior to the surgery, the patient's doctor administers 10 ng of type E botulinum toxin to the muscles and nerves in the vicinity of the lungs. Within 24 hours, muscle activity in the area surrounding the lungs is greatly reduced, and the reduction in lung function that often accompanies such surgery is reduced.

Use of Botulinum Toxin Type E in Connection with Surgery to the Trunk

A 19 year old man requires gall bladder surgery. 12 hours prior to the surgery, the patient's doctor administers 10 ng of type E botulinum toxin to the muscles and nerves in the vicinity of the lungs. Within 36 hours, muscle activity in the area surrounding the lungs is greatly reduced, and the reduction in lung function that often accompanies such surgery is reduced.

Use of Botulinum Toxin Type E in Connection with Surgery to the Trunk

A 19 year old man requires surgery following a gunshot wound. 1 hour prior to the surgery, the patient's doctor administers 10 ng of type E botulinum toxin to the muscles and nerves in the vicinity of the lungs. The doctor also administers an opioid to the patient. Within 20 hours, muscle activity in the area surrounding the lungs is greatly reduced, and the reduction in lung function that often accompanies such surgery is prevented.

Pulmonary Function Testing

Pulmonary Function Test (SVC, FVC, and FEV1)

Performance of FEV1/FVC in pain models is challenging for 2 reasons:a. the surgery by itself can impact PFT performance,b. spirometry testing may lead to complications at surgical site

The performance of PFT testing in the immediate post-surgical period is challenging especially in surgeries that involve the chest or upper abdomen. The maximal effort exerted during performance of FEV1/FVC increases the risk of complications at the surgical site such as wound dehiscence or bleeding (Koc et al. 2015). Abdominoplasty is reported to negatively affect PFT in the first few post-operative days, but they seem to recover around 2-4 weeks (Tercan et al. 2002, Rodrigues et al. 2013). This is thought to be related, at least partly, to hyperactivity of rectus abdominus muscles and associated pain. Thus, PFT assessment for potential spread of toxin is likely to be confounded by impact of muscle spasm surrounding the surgical site.

After expert consultations regarding the potential issues with PFT in the early post-operative period, the consensus was that such testing may be limited by pain induced by deep inhalation after mammoplasty and therefore may impact the utility of the PFT results.

The International guidelines on spirometry recommended that testing be avoided for 6 weeks post-operatively. A more recent recommendation (Cooper 2010) suggests that PFT testing can be reasonably performed 3 weeks post-surgery.

Evaluation of SVC will be performed at Screening, 24 hours (Day 2), 48 hours (Day 3), 72 hours (Day 4), 96 hours (Days 5), and Days 8 and 29. In addition to SVC, FEV1/FVC testing will be performed at screening and on Day 29 (EOS/ET).

When appropriate, PFTs will be performed by a trained respiratory unit technician after all pain assessments have been made. When possible, the same trained respiratory unit technician will be used. Prior to performing the PFT examination, the subject should rest for 15 minutes. The procedure using spirometer will be explained to subjects before the start of each test.

All PFTs will utilize standard, open circuit technique (Miller et al. 2005). Subjects should be seated for all PFTs performed, with both feet on the floor. Nose clips are required.

Slow Vital Capacity (SVC)a. Instruct the subject to breathe normally through the pneumotach for at least four consecutive breaths are stable before beginning SVC.b. Instruct the subject to inspire slowly and maximally and then exhale slowly and maximally.c. Instruct the subject to return to normal breathing.

An acceptable test is one with no hesitation as cough could significantly affect the accuracy. SVCs should agree within 5% or 150 mL. If one of the 3 assessments is not within 5% or 150 mL of the other 2, it should be repeated once. From the three acceptable tests, the largest value should be recorded in the e-CRF.

In the event when the SVC change is more than 40% reduction and the absolute SVC is less than 2 L beginning at 48 hours post-dose, a repeat SVC test will be conducted approximately 4 hours later. Use of incentive spirometry, if not on schedule, will be performed approximately 1 hour prior.

Forced Vital Capacity and Forced Expiratory Volume 1 (FEV1/FVC)a. Instruct the subject to breathe normally through the pneumotach for at least four consecutive breaths are stable before beginning testing.b. Instruct the subject to take a deep breath in, as large as possible, and blow out as hard and as fast as possible, and keep going until there is no air left.c. Instruct the subject to return to normal breathing.d. Repeat at least two additional times to obtain three successful tests.

In closing, it is to be understood that although aspects of the present specification are highlighted by referring to specific embodiments, one skilled in the art will readily appreciate that these disclosed embodiments are only illustrative of the principles of the subject matter disclosed herein. Therefore, it should be understood that the disclosed subject matter is in no way limited to a particular methodology, protocol, and/or reagent, etc., described herein. As such, various modifications or changes to or alternative configurations of the disclosed subject matter can be made in accordance with the teachings herein without departing from the spirit of the present specification. Lastly, the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present disclosure, which is defined solely by the claims. Accordingly, embodiments of the present disclosure are not limited to those precisely as shown and described.

Certain embodiments are described herein, comprising the best mode known to the inventor for carrying out the methods and devices described herein. Of course, variations on these described embodiments will become apparent to those of ordinary skill in the art upon reading the foregoing description. Accordingly, this disclosure comprises all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described embodiments in all possible variations thereof is encompassed by the disclosure unless otherwise indicated herein or otherwise clearly contradicted by context.