Treatment of side-effects resulting from chemodenervation

Abstract

To provide methods of restoring neurotransmission in a botulinum toxin-induced non-responsive muscle.SOLUTION: The present invention relates generally to methods of restoring neurotransmission by locally administering an effective dose of a composition comprising an anticholinesterase to a non-responsive muscle. The present invention also relates to methods of reversing a neurotoxin-induced muscle paralysis or muscle weakness, the methods comprising administering a composition comprising an anticholinesterase to a patient.SELECTED DRAWING: None

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of U.S. Provisional Patent Application No. 62 / 336,344, filed on May 13, 2016, the entire contents of which are incorporated herein by reference.

Background Art

[0002] 1. Technical Field The present invention generally relates to a method for restoring neuromuscular transmission by locally administering a composition containing an effective dose of an anti - cholinesterase to non - responsive muscles. The present invention also relates to a method for treating neurotoxin - induced muscle paralysis or muscle weakness, which comprises locally administering a composition containing an anti - cholinesterase to a patient.

[0003] 2. Background Art Botulinum toxin is used to treat over 20 medical or cosmetic indications. Botulinum toxin is a nerve impulse "blocker". It binds to nerve endings and prevents the release of the chemical transmitters that activate muscles. These chemicals carry the "messages" from the brain that tell the muscles to contract. If the message is blocked, the muscle does not contract.

[0004] Local administration of botulinum toxin is targeted at achieving muscle relaxation and not at achieving muscle immobility or complete block. However, in some cases, the effects of botulinum toxin can be observed beyond the local injection site, which is referred to as diffusion of the toxin effect. The side effects of botulinum toxin can be life-threatening and in some cases may temporarily disfigure the appearance. The temporarily disfiguring side effects include "ptosis (blepharoptosis)" or "expressionless face" caused by the neurotoxin invading the muscle that raises the eyelid, other unintended facial muscle side effects known as "arched or drooping eyebrows", swelling or edema of the lips, and in some cases pain or muscle weakness associated with medical treatment. There are currently no approved products available for the treatment (e.g., rescue) of the effects of botulinum toxin.

Summary of the Invention

[0005] The inventors have found that peripherally (i.e., locally) acting anti-cholinesterases can be used as neurotoxin rescue agents.

[0006] Botulinum toxin acts to synaptically block the release or exocytosis of acetylcholine (ACh) from synapses (Figure 1). When botulinum toxin is injected into muscle, the neurotoxin is taken up by the nerve terminal. The neurotoxin prevents the binding of synaptic vesicles containing acetylcholine by cleaving SNAP-25, a protein important for the binding of vesicles to the nerve terminal. In this way, the release of neurotransmitter into the synaptic cleft is inhibited and muscle contraction may not occur.

[0007] Studies have shown the ability of nerve terminals at the neuromuscular junction to sprout after neurotransmission blockade by botulinum neurotoxin. Sprouting that occurs after intoxication of the motor nerve terminal by botulinum toxin has the ability to form functional synapses, as it is accompanied by the major proteins required for exocytosis. When sprouts are formed, ACh is released and electrical activity is induced (de Paiva et al. (1999) Proc. Natl. Acad. Sci. 96(6): 3200-3205). The factors involved in the induction of this outgrowth are not fully understood, but there is evidence indicating that the sprouts eventually regress when the original nerve activity is restored. Synaptic ACh concentration can be increased by inhibiting the degradation that normally occurs by acetylcholinesterase (AChE). AChE is a powerful enzyme that causes rapid breakdown of ACh. The acetylated enzyme is rapidly hydrolyzed, while free enzyme and acetic acid are formed. Approximately 10,000 molecules of acetylcholine are hydrolyzed per second at each active site (Colovic et al. (2013) Current Neuropharmacology 11(3):315-335).

[0008] Nondepolarizing neuromuscular blocking drugs (NMBDs) bind to the receptor as competitive antagonists, thereby blocking the binding of ACh and preventing its ability to depolarize the receptor. Nondepolarizing NMBDs do not affect the conformational changes at the receptor. Botulinum toxins are thought to be nondepolarizing, but they have a different mechanism of action compared to NMBDs. There is no competitive block at the AChE receptor site. Nevertheless, the ACh receptor is trapped in a desensitized state without binding ACh.

[0009] Acetylcholine (ACh) diffuses across the membrane, binds to receptors at the motor end plate, and induces a muscle action potential. Acetylcholinesterase (AChE) in the synaptic cleft breaks down ACh, hydrolyzing it into acetate and choline. Choline is then reabsorbed and used to synthesize new ACh. Cholinesterase inhibitors (also known as anti-cholinesterases) act indirectly by inactivating AChE. In particular, cholinesterase inhibitors prevent AChE from degrading ACh in the synaptic cleft, so that AChE cannot attack the molecules of ACh present. Delivery of an anti-cholinesterase / cholinesterase inhibitor to the synaptic cleft prolongs the lifespan of ACh, thereby increasing its concentration and enabling it to bind to ACh receptors. The timing of administration is as important in its use in anesthesia, where "antagonism of neuromuscular block by anti-cholinesterases should not be attempted until two of a series of four twitch responses become detectable, or it will be ineffective" (Srivastava and Hunter (2009) British Journal of Anesthesia, 103(1):1115-29). This guideline indicates the need to wait for evidence of recovery from the induced neuromuscular block. For recovery to occur, ACh must be present. In the case of botulinum toxin intoxication at the terminal, this indicates a precondition for partial block, or that natural recovery of neuromuscular function after the action of a neuromuscular blocker requires an increase in acetylcholine concentration.

[0010] The inventors have found that targeted, local parenteral administration of anti-cholinesterase to affected patients restores / reduces / treats muscle chemical denervation as caused by administration of botulinum toxin. Targeted administration of anti-cholinesterase (e.g., by using low doses) to the affected tissue / muscle reverses the unwanted effects. The methods described herein minimize the potential for systemic side effects, such as those resulting from first-pass metabolism in oral dosage forms or from higher intravenous drug doses. Furthermore, the use of targeted drug delivery enables recovery of the affected area without affecting the underlying area that is inherently targeted for treatment.

[0011] Thus, in one aspect, the present invention provides a method of restoring neuromuscular transmission by locally administering a composition comprising an effective dose of an anti-cholinesterase to non-responsive muscles.

[0012] In another aspect, the present invention provides a method of treating neurotoxin-induced (e.g., botulinum toxin-induced) muscle paralysis or muscle weakness, comprising locally administering a composition comprising an anti-cholinesterase to a patient. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to further understand the methods and devices of the present invention. The drawings illustrate one or more embodiments of the present invention and serve to explain the principles and operations of the present invention together with the description.

[0014]

Figure 1

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Figure 5

[0015] Detailed Description Before describing the disclosed methods and materials, it is to be understood that the aspects described herein are not limited to specific embodiments, apparatus, or configurations, and will, of course, vary. It is also understood that the terms used herein are for the purpose of describing only specific aspects and are not intended to be limiting, unless otherwise defined herein.

[0016] As used herein, unless the context requires otherwise, "comprise" and "include" and their variants (e.g., "comprises", "comprising", "includes", and "including") are taken to indicate the inclusion of the stated components, features, elements, or steps, or groups of components, features, elements, or steps, but not to preclude the inclusion of any other integer or step, or group of integers or steps.

[0017] As used in the specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise.

[0018] In this specification, ranges may be expressed as from "about" one particular value and / or to "about" another particular value. When such a range is expressed, another aspect includes from the one particular value and / or to the other particular value. Similarly, when values are expressed as approximations by use of the antecedent "about", that particular value is understood to form another aspect. Each endpoint of a range is significant both in relation to and independent of the other endpoint.

[0019] "Effective amount" refers to an amount of a compound that, when administered to a subject, is sufficient to achieve treatment of the condition described herein. The amount of the compound that constitutes an "effective amount" will vary depending on the compound, the disorder and its severity, and the age of the subject being treated, but can be determined routinely by one of ordinary skill in the art.

[0020] As used herein, the term "muscle paralysis" means complete loss of muscle function. Muscle paralysis may be accompanied by loss of sensation (sensory loss) in the affected area.

[0021] As used herein, the term "muscle weakness" means partial loss of muscle function and / or loss of muscle strength.

[0022] As used herein, the term "chemical denervation" or "chemical deinnervation" means loss of nerve supply (i.e., block of neurotransmission) caused by a substance (e.g., a chemical compound).

[0023] From the perspective of the present disclosure, the methods and active substances described herein can be set by those skilled in the art to meet the desired needs. Generally, the disclosed raw materials, methods, and devices provide targeted, topical parenteral administration of anti-cholinesterase to affected patients and restore neuromuscular transmission in drug-induced non-responsive muscles. For this purpose, it can be set by those skilled in the art. Generally, the disclosed raw materials, methods, and devices provide targeted, topical parenteral administration of anti-cholinesterase to affected patients and restore neuromuscular transmission in drug-induced non-responsive muscles.

[0024] Botulinum neurotoxins are used in a wide range of cosmetic and medical procedures. Temporary but undesirable side effects are due to the inadvertent spread of the toxin to adjacent muscle structures, causing paralysis or weakness in unintended areas. The neurotoxin acts to block the release of ACh at the neuromuscular junction. Anti-cholinesterase acts to indirectly increase ACh by breaking down endogenous AChE. Binding of ACh to the receptor site is necessary for maintaining muscle transmission. The depth of block is an important factor indicating the effectiveness of anti-cholinesterase in accelerating natural recovery. ACh needs to be present to cause muscle recovery or reactivation. As used herein, the term "depth of block" refers to the level of occupancy of the postsynaptic receptor.

[0025] Due to the paralysis and spread properties induced by botulinum toxin in commercial use, partial chemical denervation and / or natural recovery occur. Therefore, the state or depth of blockage determines the recovery rate. The inventors have found that peripherally acting anti-cholinesterase can be used as a nerve agent rescue agent that accelerates the recovery time. The inventors have found that the timing of anti-cholinesterase administration, the concentration of anti-cholinesterase, and the administration period are important factors in the effectiveness of the rescue treatment.

[0026] Accordingly, in one aspect, the present invention provides a method of restoring neuromuscular transmission by administering to a patient a composition comprising an anti-cholinesterase. In certain embodiments, the non-responsive muscle has been previously exposed to the nerve agent. In certain embodiments, the non-responsive muscle has been previously exposed to botulinum toxin.

[0027] In another aspect, the present invention provides a method of treating nerve agent-induced muscle paralysis or muscle weakness, comprising topically administering to a patient a composition comprising an anti-cholinesterase. In certain embodiments, the nerve agent is botulinum toxin.

[0028] Botulinum toxin is a neurotoxin protein produced by Clostridium botulinum and related species. Botulinum strains produce seven different neurotoxins named types A - G. All seven types have a similar structure and molecular weight and consist of a heavy (H) chain and a light (L) chain linked by a disulfide bond, and they all interfere with neurotransmission by blocking the release of acetylcholine. Accordingly, in certain embodiments, the botulinum toxin of the present invention comprises one or more of types A, B, C, D, E, F, and G. In certain embodiments, the botulinum toxin of the present invention comprises one or more of types A, B, E, and F. In certain embodiments, the botulinum toxin of the present invention comprises one or more of types A and B. In certain embodiments, the botulinum toxin of the present invention is type A botulinum toxin.

[0029] Botulinum toxin type A has been approved by the FDA for use in cosmetic procedures and is available under a variety of product names, one of which is BOTOX® (Allergan, Irvine, California, United States of America; "BOTOX" as used herein). Similarly, botulinum toxin is also used to treat more than 20 medical conditions. The most common medical conditions include migraine, hyperhidrosis, muscle spasm, urinary incontinence, etc. Diffusion of the toxin from the injection site or excessive use of the toxin can result in undesirable side effects in such procedures. For example, in the treatment of migraine, neck pain is the most common side effect experienced by about 9% of people in the botulinum toxin group compared to 3% in the placebo group.

[0030] The method of the present invention requires a composition containing an anti-cholinesterase. Anti-cholinesterases (i.e., cholinesterase inhibitors) are classified into two types: irreversible organophosphorus compounds and reversible carbamates. The former generally has higher toxicity, longer action time, and is often associated with central nervous system (CNS) toxicity. Reversible anti-cholinesterases have found applications in medicine for a wide range of indications. For example, some reversible anti-cholinesterases can cross the blood-brain barrier and reach the CNS, so they are used in the treatment of Alzheimer's disease.

[0031] In some embodiments, the anti-cholinesterase of the present invention is a reversible anti-cholinesterase. In some embodiments, the anti-cholinesterase of the present invention is a reversible anti-cholinesterase having one or more groups selected from carbamates, tertiary ammonium, and quaternary ammonium.

[0032] In some embodiments, the anti-cholinesterase is selected from physostigmine, neostigmine, ambenonium, pyridostigmine, ambenonium, demecarium, rivastigmine, galantamine, donepezil, tacrine, 7-methoxytacrine, edrophonium, huperzine A, ladostigil, and any derivatives thereof and combinations thereof.

[0033] In some embodiments, the anti-cholinesterase of the present invention is:

Chemical formula

[0034] In some embodiments of the present invention, the anti-cholinesterase is pyridostigmine, neostigmine, edrophonium or a combination thereof.

[0035] In some embodiments of the present invention, the anti-cholinesterase is pyridostigmine. Pyridostigmine is not lipid-soluble and is itself peripherally acting. This property makes it desirable for use in conditions related to muscles. Pyridostigmine is also more stable compared to neostigmine because it causes fewer bradycardia and arrhythmia.

[0036] In some embodiments of the present invention, the anti-cholinesterase is of formula (I):

Chemical formula

[0037] In some embodiments, the compound of formula (I) has Y as C. In some embodiments, the compound of formula (I) has Y as N + X - or Y is N + Br - or N + Cl - or Y is N + Br - or Y is N

[0038] In some embodiments, the compound of formula (I) according to any of the above embodiments has R1 selected from hydrogen, C1-C6 alkyl, -CO(NH2), -CONH(C1-C6 alkyl) and -CON(C1-C6 alkyl)2. In some embodiments, the compound of formula (I) has R1 selected from hydrogen, -CO(NH2), -CONH(C1-C6 alkyl), and -CON(C1-C6 alkyl)2. In some embodiments, the compound of formula (I) has R1 as hydrogen. In some embodiments, the compound of formula (I) has R1 as -CO(NH2), -CONH(C1-C6 alkyl), or -CON(C1-C6 alkyl)2. In some embodiments, the compound of formula (I) has R1 as -CON(C1-C6 alkyl)2.

[0039] In some embodiments, for the compound of formula (I) described in any of the above embodiments, R2 is hydrogen. In some embodiments, for the compound of formula (I) described in any of the above embodiments, R2 and R3 combine together to form an optionally substituted heterocycle. In some embodiments, the heterocycle may be substituted with one or more of halogen, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkoxy, -OH, -NH2, -NH(C1-C6 alkyl), or -N(C1-C6 alkyl)2. In some embodiments, the heterocycle is octahydropyrrolo[2,3-b]pyrrole or pyrrolidine, each of which may be optionally substituted with one or more of halogen, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkoxy, -OH, -NH2, -NH(C1-C6 alkyl) or -N(C1-C6 alkyl)2. In some embodiments, the heterocycle is octahydropyrrolo[2,3-b]pyrrole which may be optionally substituted with one or more of halogen, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkoxy, -OH, -NH2, -NH(C1-C6 alkyl) or -N(C1-C6 alkyl)2.

[0040] In some embodiments, for the compound of formula (I) described in any of the above embodiments, R3 is C1-C6 alkoxy, -OH, -NH2, -NH(C1-C6 alkyl), -N(C1-C6 alkyl)2 and -N + (C1-C6 alkyl)3X - selected from. In some embodiments, R3 is selected from C1-C6 alkoxy and -OH. In some embodiments, R3 is -NH2, -NH(C1-C6 alkyl), -N(C1-C6 alkyl)2 and -N + (C1-C6 alkyl)3X - selected from. In some embodiments, R3 is -N + (C1-C6 alkyl)3X - is. In some embodiments, R3 is -N + (C1-C6 alkyl)3Br - or -N+ (C1-C6 alkyl)3Cl - is.

[0041] In certain embodiments, the methods of the invention require targeted delivery of the composition. Targeted drug delivery to the site of action can enhance the effectiveness of the drug. By increasing the local active drug concentration in the affected tissue, drug toxicity can be reduced while minimizing exposure to other areas of the body. Local delivery avoids first-pass liver metabolism and gastrointestinal side effects. Additionally, the total drug amount is significantly lower, thereby reducing patient exposure from non-specific systemic side effects. This can enable the broad use of drugs of the anti-cholinesterase class and their use in the cosmetic and other non-life-threatening conditions. In certain embodiments, the composition is administered directly to the affected muscle.

[0042] Targeted drug delivery can be achieved by methods of parenteral injection using conventional techniques similar to those used in the administration of botulinum toxin, or by methods of passive or active transdermal delivery. Passive transdermal delivery refers to topical or other conventional skin patches or gels used to administer the active drug through the skin barrier without a driving force or perforation. In certain embodiments of the invention, the composition is administered parenterally (e.g., by intramuscular injection). Parenteral delivery (e.g., intramuscular injection) can be to non-responsive muscle regions (i.e., regions where side effects such as muscle paralysis or weakness are seen), or can be in muscle structures recommended by a medical professional with detailed knowledge of the muscle characteristics.

[0043] In certain embodiments of the present invention, the composition is administered transdermally. In certain embodiments, the composition is administered as a transdermal patch or a transdermal gel. Transdermal drug delivery enables the drug to enter the underlying tissue through the skin surface area. Considering that the highest frequency of the procedure relates to the facial area, any patch or topical formulation should stay in place for a limited time in order to allow the patient to resume normal daily activities. When the drug enters the subcutaneous space and the target tissue or muscle, it is preferred to remove the patch and any obstacles. Conventional patch technology is non-invasive and does not require administration by a medical professional as usually required for injections. Those skilled in the art recognize that performance (drug uptake / movement through the skin) depends mainly on drug properties such as molecular size, lipophilicity, drug polarity and solubility. Those skilled in the art can select appropriate surfactants and / or penetration enhancers if necessary to improve drug movement. If a more rapid onset of action is required, activated transdermal techniques can be applied (e.g., microneedles or other means of skin penetration to improve delivery rate). Activation methods include, for example, iontophoresis, electroporation, mechanical perturbation and other energy-related techniques such as ultrasound and needle-free injection.

[0044] Additional methods of targeted delivery include, but are not limited to, intramuscular, intradermal, subcutaneous or topical delivery.

[0045] Considering the localized nature of target delivery, an effective dose of the anti-cholinesterase is administered at a low dose, i.e., an amount lower than the clinical dose of the anti-cholinesterase when administered for oral or intravenous use (when administered for other therapeutic indications). General guidelines for converting oral doses to intravenous doses are to give the patient 1 / 30 of the oral dose. Since targeted administration is by direct injection into the tissue, the dose can be as low as about 0.1 mg or up to 1 / 10 of the dose required for recovery of muscle relaxation compared to intravenous administration. The effective dose can be readily understood by using known physiological techniques that are always used to evaluate muscle or nerve dysfunction. Electromyography (EMG) measures the potential generated by muscle cells when the cells are electrically activated. A needle electrode emits a rapid electrical pulse into the nerve and measures the time it takes for the muscle or nerve to contract. The speed of contraction is reported as the conduction speed. The conduction speed can be measured before and after injection of the recovery agent. An increase in the expected conduction speed serves as a measure of the effectiveness of the dose response.

[0046] In certain embodiments, the low dose is about 4 / 5 to about 1 / 50 of the clinical dose of the anti-cholinesterase when administered for oral or intravenous use (normal dosing for other therapeutic indications). In some embodiments, the low dose is about 1 / 5 to about 1 / 50, or about 1 / 5 to about 1 / 20, or about 1 / 5 to about 1 / 10, or about 1 / 10 to about 1 / 50, or about 1 / 10 to about 1 / 50, or about 1 / 10 of the oral or intravenous clinical dose.

[0047] In some embodiments of the present invention, the anti-cholinesterase is administered at a dose of about 0.05 to 0.5 mg / kg, or at a dose of about 0.15 to 0.25 mg / kg, or at a dose of about 0.2 mg / kg. As is known in the art for the dosage guidelines provided for botulinum toxin injection, the dose of a particular anti-cholinesterase can be individually adjusted using methods such as needle electromyogram guidance or nerve stimulation as an indicator of response, based on the size, number and location of the muscles involved. For example, in the treatment of palmar hyperhidrosis, the dose is adjusted according to the size of the palm (e.g., cm 2 ). Those skilled in the art will recognize that the dose may be higher or lower depending on, among other factors, the activity of the anti-cholinesterase, its bioavailability, its metabolic rate and other pharmacokinetic properties, the method of administration and a variety of other factors. Those skilled in the art will also recognize that the anti-cholinesterase should be administered so as not to penetrate or diffuse into the systemic circulation so as to cause undesirable side effects.

[0048] In some embodiments of the present invention, the anti-cholinesterase can be administered immediately after a nerve toxin (e.g., botulinum toxin). For example, the anti-cholinesterase can be administered at least 1 minute, or at least 2 minutes, or at least 5 minutes, or at least 10 minutes, or at least 30 minutes after the nerve toxin. In some embodiments of the present invention, the anti-cholinesterase can be administered some time after the nerve toxin (e.g., botulinum toxin). For example, the anti-cholinesterase is administered at least 1 hour, or at least 6 hours, or at least 24 hours, or at least 2 days, or at least 3 days, or at least 4 days, or at least 5 days, or at least 6 days, or at least 7 days after the nerve toxin.

[0049] It is used to address the undesirable side effects caused by botulinum toxin, and it is important that the drug be available during an appropriate window for the application of anti-cholinesterase in the absence of specific monitoring. In certain embodiments, the drug is available when ACh is present. Thus, in the presence of complete block, the response is delayed. To determine when Ach is present, the patient may be monitored, for example, by electromyogram (EMG) or other tests. The use of a sustained release composition may make an appropriate treatment window available. The use of several administrations of the composition of the present invention may make an appropriate treatment window available. In certain embodiments, the composition of the present invention may be administered once (i.e., as a single dose). In certain embodiments, the composition of the present invention is administered 2, or 3, or 4 or more times.

[0050] Anti-cholinesterase is administered after the effects of the nerve toxin become apparent to the physician or patient. In some cases, it may take up to 7 days for the full effects of the nerve toxin to develop. Anti-cholinesterase may be used at any time after the appearance of undesirable side effects.

[0051] In certain embodiments of the present invention, the composition is a sustained release composition. The sustained release formulation ensures that the drug acts because acetylcholine becomes available. The sustained release composition should obviate the need for multiple injections of the composition of the present invention. It also reduces treatment costs while providing maximum effect with limited patient exposure and fewer undesirable side effects. In certain embodiments, the sustained release formulation releases the anti-cholinesterase over several days (e.g., 1 day, or 2 days, or 3 days, or 4 days, or 5 days, or 6 days, or 7 days, or 10 days, or 14 days, or 21 days, or 30 days, or a period of 1 day to 14 days, or a period of 1 day to 7 days, or a period of 3 days to 7 days, or a period of 1 day to 5 days, or a period of 5 days to 7 days), or over several hours (e.g., up to about 3 hours, or up to about 6 hours, or up to about 12 hours, or up to about 24 hours, or about 3 hours to about 24 hours, or about 3 hours to about 18 hours, or about 3 hours to about 12 hours, or about 3 hours to about 6 hours, or about 6 hours to about 24 hours, or about 6 hours to about 18 hours, or about 6 hours to about 12 hours, or about 12 hours to about 24 hours, or about 18 hours to about 24 hours).

[0052] Suitable sustained release formulations are known to those skilled in the art and the compositions of the present invention can be formulated in a variety of ways depending on the desired drug loading, desired drug delivery profile and / or desired release / pharmacokinetic rates.

[0053] The sustained release composition of the present invention can be any of the formulations disclosed in Rhee et al. ("Sustained-Release Injectable Drug Delivery," (2010) Pharmaceutical Technology, Vol. 2010 Supplement, Issue 6, pp. 1-7 source URL: http: / / www.pharmtech.com / sustained-release-injectable-drug-delivery), which is hereby incorporated by reference in its entirety.

[0054] The sustained-release composition of the present invention can be manufactured by the method disclosed in U.S. Patent Publication No. 2012 / 0316108, which is incorporated herein by reference in its entirety. These formulations are monolayer gel formulations that require the use of a low range of phospholipids (20% - 80%), preferably a syringe with finer needles. The formulation can include a pharmaceutical-grade lecithin derived from eggs or soybeans, such as those established for use in injectable pharmaceutical formulations or oils for parenteral formulations. The main dispersant can include an emulsion or suspension in the first step of formulating the phospholipid gel, or if an oil is used, the dispersant can exhibit an emulsion. Thus, in certain embodiments, the composition of the present invention is a monolayer gel composition further comprising 20 - 80% by weight of phospholipid and 0.1% - 65% by weight of water. In certain embodiments, the composition of the present invention (e.g., monolayer gel composition) can be extruded through a needle having a gauge of about 30 gauge to about 33 gauge. The sustained-release monolayer gel formulation can be produced by a method comprising: (a) forming a first dispersant comprising one or more phospholipids and excess water; (b) homogenizing the first dispersant to form a nano-dispersant having an average particle diameter of less than about 200 nm; (c) passing the nano-dispersant through a 0.2 μm or 0.45 μm filter; and (d) removing the excess water to obtain a gel. In some embodiments, step c may be excluded if the formulation is heat sterilized by standard practice for injectable drugs.

[0055] The composition containing an anti-cholinesterase further comprises a suitable carrier, excipient, or diluent. The exact nature of the carrier, excipient, or diluent depends on the performance required for the composition and can range from suitable or acceptable for animal use to suitable or acceptable for human use.

[0056] A composition containing an anti-cholinesterase can be produced by conventional methods of mixing, dissolving, granulating, sugar coating, powdering, emulsifying, encapsulating, enclosing, or lyophilizing processes. The composition can be formulated by conventional methods using one or more physiologically acceptable carriers, diluents, excipients, or adjuvants that facilitate processing the compound into a pharmaceutically useful formulation.

[0057] An anti-cholinesterase can be formulated in a pharmaceutical composition, by itself or in the form of a hydrate, solvate, N-oxide, or pharmaceutically acceptable salt, as described above. Generally, such salts are more soluble in aqueous solution than the corresponding free acids and bases, although salts with lower solubility than the free acids and bases can also be formed.

[0058] If desired, the compositions of the present invention can be presented in ready-to-use, single-use delivery formulations (including formulations designed for administration by injection). This avoids problems of cross-contamination and waste. Depending on the activity of the treatment center, the frequency of use of rescue products may not require a multi-use sterile container system such as a vial. Useful injectable formulations include sterile suspensions, solutions, or emulsions of the compound in aqueous or oily vehicles. The composition also includes formulating substances such as suspending agents, stabilizers, and / or dispersing agents. Formulations for injection can be in unit dosage form, for example, in the form of ampoules or multi-dose containers, and may contain added preservatives. Alternatively, the injectable formulation can be provided in powder form for reconstitution with a suitable vehicle before use, including but not limited to sterile non-pyrogenic water, buffer solutions, dextrose solutions, etc. For this purpose, the anti-cholinesterase can be dried and reconstituted by any technique known in the art such as lyophilization before use.

[0059] Injectable formulations can be dispersed using a range of methods (commercial use or in development). These include, but are not limited to, oil-based injectables, injectable drug suspensions, injectable microspheres or in-situ systems, excipients, and polymers for long-lasting drug depots. The technology selection is based on the following main factors: drug loading within the matrix, the required drug delivery profile, and the rate of release / pharmacokinetics (the therapeutic window). Oil-based injectable solutions and injectable drug suspensions control release for periods of days to weeks, while polymer-based microspheres and in-situ gels or drug depots are reported to last for months. The desired final formulation limits the bulk volume that needs to be injected into the patient or administered by another method.

[0060] The compositions of the present invention may be present in a kit, if desired. Instructions for administration may be attached to the kit.

[0061] The compositions of the present invention may be present in a sachet or dispensing device that, if desired, contains one or more dosage forms comprising the compound. The sachet contains a metal or plastic foil, for example, in the form of a blister pack. Instructions for administration may be attached to the pack or dispensing device.

Examples

[0062] Animal Models and Methods To demonstrate the effectiveness of an anti-cholinesterase in accelerating the natural recovery after botulinum toxin injection, the rat model described by Moon et al. (Maxillofacial Plastic and Reconstructive Surgery (2015) 37:46) was employed. In this case, botulinum toxin was injected into the masseter muscle of rats, and subsequent changes in food intake were monitored. The aim of the study was to induce paralysis in the jaw muscles of rats using botulinum neurotoxin. This was thought to have a negative impact on food intake, and the ability of an equivalent injection of anti-cholinesterase to accelerate the subsequent recovery process was tested.

[0063] The test substance was a product approved for injection. Botulinum toxin, BOTOX® (Allergan, Irvine, California, United States of America), was reconstituted to the desired amount using injectable saline. The dose of BOTOX used in the test was 5 units in 100 μl. For pyridostigmine, Regonol® (Sandoz Inc., Princeton, New Jersey, United States of America) was used in the ready-to-inject solution (5 mg / ml).

[0064] Sprague Dawley rats with a typical body weight of 375 - 400 g were subjected to treatment with BOTOX control or pyridostigmine, an anti-cholinesterase. The body weight and food intake of each group of rats (generally 3 - 5) were monitored for about one week as baseline measurements. Thereafter, BOTOX was injected into each masseter muscle. The body weight and food intake of the rats were followed for a total of 2 - 4 weeks.

[0065] The induction of masseter muscle paralysis results in a decrease in food intake and potential weight loss, and as the effect of BOTOX decreases over time, it is thought to result in subsequent recovery. After BOTOX administration, some rat groups are injected with a recovery agent into the masseter muscle. The treatment groups differ in dose, dosing regimen, and frequency of recovery agent administration. Each animal is briefly anesthetized with isoflurane before injection and once-daily dosing. The rats were fed pelleted rodent diet. This differed from Moon's test in that ground diet was used. Pelleted diet was considered a more representative measure of muscle atrophy and chewing ability.

[0066] BOTOX (5 units in 100 μl) was injected into the masseter muscle of the rats. To standardize the injection method and dose delivery, the same volume of saline was injected at the start of the experiment. In any case, low doses (0.1 - 2.5 units), less than 5 units per side of BOTOX, were tested. These low doses did not affect food intake compared to the baseline, thus indicating that this test model does not show a dose response. The limitations in the animal model are thought to contribute to the inhibitory effect on the recovery results.

[0067] To limit dehydration in animals during periods of muscle paralysis and severely affected food intake, rats were injected with sterile lactated Ringer's solution via the tail vein.

[0068] Botulinum Toxin Control As shown in Figure 2, rats treated with BOTOX only (i.e., untreated with anti-cholinesterase) did not reach 100% recovery even after 37 days. This is consistent with the reporting period in the manufacturer's package insert in terms of the possibility that the nerve toxin can persist in patients for more than 90 days. However, it is not consistent with the results shown by Moon et al. who had almost complete spontaneous recovery in just 10 days. The reason for the difference in results is thought to be due to the different biological potencies of the nerve toxins used. BOTOX is not specifically mentioned in the Materials and Methods. The potencies among manufacturers of various botulinum toxin type A are not equivalent, which is clearly identified under the literature and the package inserts of various manufacturers.

[0069] Results: Timing of Administration As previously explained, the acceleration of spontaneous recovery / reversal depends on the presence of acetylcholine at the neuromuscular junction. Anti-cholinesterase acts to inhibit the activity of the enzyme cholinesterase by breaking down acetylcholine into its basic units, choline and acetic acid. Acetylcholine is normally taken up into the synapse and regenerated by the reprocessing of choline.

[0070] The experiments conducted showed that early administration of anti-cholinesterase was not effective. The appropriate timing, i.e., the timing at which an enhanced recovery response is induced as compared to the BOTOX control, occurs approximately 6 days later (i.e., 6 days after injection of the botulinum toxin) in our animal model. Due to the lack of response by AChE and without being bound by any theory, acetylcholine is thought not to be available until this point. If the dose of the nerve poison is low enough to maintain muscle relaxation rather than complete immobility, a more immediate response is thought to be seen. For example, a faster response time is thought to be the case with botulinum toxin side effects. The side effects are the result of diffusion of the toxin effect or lack of injection technique into the target area. It has been reported that in human use, the side effects take several weeks to several months to recover. Also, the package insert for BOTOX Cosmetic states that the dose is designed to achieve partial chemical denervation. In the case of side effects resulting from treatment with botulinum toxin in humans, the response time can be immediate.

[0071] Results: Synergistic Effect of Spontaneous Recovery As shown in Figure 3, rats were injected with Regonol® (pyridostigmine bromide) on days 2, 7, 9, 11, and 14. The dose of Regonol® was 0.5 mg, 100 μl injected per side of the masseter muscle of the rats on a specific day. The average relative increase in food intake was 26.3% based on data collected after injection over a 6-day period.

[0072] As shown in Figure 4, animals treated with Regonol® showed complete recovery of food intake by approximately 26 days, while control animals had not yet recovered even at 37 days. Also notable is that recovery continued beyond the final treatment at 14 days.

[0073] Results: Endplate Poisoning Among the muscarinic side effects that can be observed from the administration of anti-cholinesterase, there are nausea, vomiting and diarrhea. In the toxicity test for pyridostigmine in Sprague Dawley rats, the signs of pyridostigmine-induced toxicity included lacrimation, rhinorrhea, decreased activity, weakness, ataxia, diarrhea, kyphosis, emaciated appearance and death (Battelle, Preclinical Toxicology of New Drugs, Report 8740-86-2, April 4, 1986, J. G Gage Principle Investigator). As a result, the rats in this example were monitored for signs of pathological effects. The rats treated by the disclosed method showed no side effects.

[0074] It is important to reach an effective dose of anti-cholinesterase without affecting the poisoning of the endplate terminal. Doses for intramuscular injection into muscle tissue have not been reported. As described above, the depth of block at the time of administration affects the effectiveness of recovery. There is no advantage in administering prior to the start of spontaneous recovery in parallel with the intravenous use of cholinesterase inhibitors that reverse neuromuscular block at the end of anesthesia. Furthermore, cholinesterase inhibitors reach an upper limit at high doses. In the animal model of the present application, it has been shown that giving more anti-cholinesterase induces an improved response. Without being bound by a particular theory, it is thought that at high doses the acetylcholine concentration at the motor endplate can be higher because the endogenous enzyme is not available to remove the excess acetylcholine. The accumulation leads to endplate poisoning and a continuous state of depolarization. This acts as a unique form of depolarization block.

[0075] Since the preferred anti-cholinesterase has a limited duration of action, recovery occurs gradually as the endogenous cholinesterase enzyme levels are re-established.

[0076] Figure 5 shows that animals treated with a high-dose regimen experienced a delay in the recovery of food intake. This delay in response is thought to be due to temporary poisoning of the endplate terminals. Recovery does not occur until more than 10 days after BOTOX immobilization, compared to a BOTOX-only control that begins to recover spontaneously after 6 days. The elevated dosing in this experiment was defined as a dose of 0.25 mg / day injected twice daily from day 3 to day 14.

[0077] Conclusion In human subjects, rescue or recovery from a state of partial chemical denervation is expected to show a more rapid response. As previously explained, naturally occurring acetylcholine is a precursor for establishing neurotransmission. The occurrence of side effects is known to result from the spread of toxins that cause off-target effects. This is most likely due to off-target muscle relaxation (partial chemical denervation) as compared to complete muscle immobilization or paralysis. Based on rodent food intake models that show low sensitivity to the dose-response profile for neurotoxin-induced paralysis, one of ordinary skill in the art would expect the results in human models to be more pronounced. This is because the actual situation in both the cosmetic and medical use of neurotoxins in humans is not expected to reach a state of complete immobility, especially in the signs of side effects. This gives rise to the presence of partial denervation as opposed to complete denervation. Also, the human model does not require chewing. In the rodent food intake model, chewing is a complex process and can be affected by many factors in the drive of motor function. As a result, a state of partial chemical denervation cannot be achieved. This limitation results in a delay in response because acetylcholine is not available until the onset of nerve sprouting.

[0078] The examples and embodiments described herein are for illustrative purposes only, and various modifications or changes will be presented to those of ordinary skill in the art and are understood to be included within the concept and scope of this application and the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference for all purposes.

Claims

【Claim 1】 The invention described in the specification or drawings of this application.

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