Treatment of side effects caused by chemical denervation

Abstract

The invention relates to the treatment of side effects caused by chemical denervation. In particular, the present disclosure relates generally to methods of restoring neuromuscular delivery by topically administering to unreactive muscles an effective dose of a composition comprising an anti-cholinesterase agent. The present disclosure also relates to methods of reversing neurotoxin induced muscle paralysis or myasthenia comprising topically administering to a patient a composition comprising an anti-cholinesterase agent.

Description

[0001] This application is a divisional application of Chinese invention patent application 201780041576.4 (the invention name is "Treatment of side effects caused by chemical denervation", the application date is May 15, 2017, and the PCT application number is PCT / US2017 / 032636).

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 336,344, filed May 13, 2016, which is incorporated herein by reference in its entirety. Background of the Invention Field of the Invention

[0005] The present disclosure is generally directed to methods of restoring neuromuscular transmission by topically administering to unresponsive muscles an effective dose of a composition comprising an anticholinesterase agent. The present disclosure is also directed to methods of treating neurotoxin-induced muscle paralysis or weakness comprising topically administering to a patient a composition comprising an anticholinesterase agent. Background Art

[0006] Botulinum toxin has been used to treat more than 20 medical and cosmetic indications. Botulinum toxin is a nerve impulse "blocker". It attaches to nerve endings and blocks the release of chemical transmitters that activate muscles. These chemicals carry the "message" from the brain that tells the muscles to contract. If the message is blocked, the muscle will not contract.

[0007] The goal of topically applied botulinum toxin is to achieve muscle relaxation rather than muscle immobilization or complete blockade. However, in some cases, the effects of botulinum toxin have been observed to extend beyond the local injection site and are known as spread of toxin action. Side effects of botulinum toxin can be potentially life-threatening and in some cases temporarily disfiguring. Temporary but disfiguring side effects include: "drooping eyelids" (ptosis), which is caused by penetration of the neurotoxin into the muscles that lift the eyelids; or other unexpected fascio-muscular side effects known as "frozen face", "arched or drooping eyebrows", puffiness or swelling of the lips; and in some cases, pain or muscle weakness associated with medical procedures. There are currently no products approved for use in treating (e.g., rescuing) the effects of botulinum toxin. Summary of the invention

[0008] The present inventors have discovered that peripherally (ie, locally) acting anticholinesterase agents can be used as neurotoxin rescue agents.

[0009] Botulinum toxin acts presynaptically to block the release or exocytosis of acetylcholine (ACh) from the synapse. Figure 1). Once botulinum toxin is injected into the muscle, the neurotoxin is carried into the nerve endings. It prevents the binding of synaptic vesicles containing acetylcholine by cleaving SNAP-25, a protein that is important for the docking of vesicles to nerve endings. Therefore, neurotransmitter release into the synaptic cleft is inhibited, and muscle contraction cannot occur.

[0010] Studies have shown the ability of nerve endings at the neuromuscular junction to sprout after botulinum neurotoxin blocks neurotransmission. Sprouting that occurs after botulinum toxin poisons motor nerve endings has the ability to form functional synapses because it provides key proteins necessary for exocytosis. Sprouting forms to release ACh and direct electrical activity (de Paiva et al. (1999) Proc. Natl. Acad. Sci. 96 (6): 3200-3205). Although the factors involved in triggering this outcome are not fully understood, there is evidence that sprouting eventually decays once the original nerve activity is restored. Synaptic ACh concentrations can be increased by inhibiting the normal degradation with acetylcholinesterase (AChE). AChE is a powerful enzyme that triggers the rapid degradation of ACh. The acetylase is rapidly hydrolyzed, forming free enzyme and acetic acid. About 10,000 acetylcholine molecules are hydrolyzed per second in each active site ( al. (2013) Current Neuropharmacology 11(3):315-335).

[0011] Non-depolarizing neuromuscular blocking drugs (NMBDs) bind to the receptor as competitive antagonists, thus blocking the binding of ACh and preventing its ability to depolarize the receptor. Non-depolarizing NMBDs do not affect conformational changes in the receptor. Botulinum toxin is considered non-depolarizing, however, it has a different mechanism of action compared to NMBDs. There is no competitive blockade at the AChE receptor site. Nevertheless, the ACh receptor is trapped in a non-sensitized state rather than binding to ACh.

[0012] ACh diffuses across the membrane, binds to receptors at the motor end plate, and triggers myoelectric potential. AChE in the synaptic cleft destroys ACh and decomposes into acetate and choline. Choline is then reabsorbed to synthesize new ACh. Cholinesterase inhibitors (also known as anticholinesterase agents) act indirectly by passivating AChE. Specifically, cholinesterase inhibitors destroy AChE in the synaptic cleft so that it cannot attack the existing ACh molecules. Anticholinesterase agents / cholinesterase inhibitors are delivered to the synaptic cleft to extend the life of ACh, thereby increasing its concentration and enabling it to bind to ACh receptors. The timing of application is very important because when it is used in anesthesia, "antagonism of neuromuscular blockade should not be attempted with anticholinesterase agents until two twitches of four train-of-four twitch reactions can be detected, otherwise it will be invalid" (Srivastava and Hunter (2009) British Journal of Anesthesia, 103 (1): 1115-29). The guidelines suggest that evidence of recovery from induced neuromuscular blockade must be awaited. For recovery to occur, ACh must be present. In the case of terminal intoxication by botulinum toxin, this refers to the precondition of partial blockade or the need for increased concentrations of acetylcholine for spontaneous recovery of neuromuscular function after neuromuscular blocking agents.

[0013] The inventors have discovered that targeted local parenteral administration of anticholinesterase agents to affected patients reverses / mitigates / treats muscle chemical denervation, such as that caused by administration of botulinum toxin. Targeted administration of anticholinesterase agents (e.g., by using small doses) to the afflicted 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 side effects of higher intravenous doses). In addition, the use of targeted drug delivery allows reversal of the damaged area without affecting the primary area originally targeted for treatment.

[0014] Thus, in one aspect, the present disclosure provides methods for restoring neuromuscular transmission by topically administering to unresponsive muscles an effective dose of a composition comprising an anticholinesterase agent.

[0015] In another aspect, the present disclosure provides a method of treating neurotoxin-induced (eg, botulinum toxin-induced) muscle paralysis or weakness comprising topically administering to a patient a composition comprising an anticholinesterase agent. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The accompanying drawings are included to provide a further understanding of the methods and apparatus of the present disclosure, and are incorporated in and constitute a part of this specification. The accompanying drawings illustrate one or more embodiments of the present disclosure, and together with the description serve to explain the principles and operations of the present disclosure.

[0017] Figure 1 The mechanism of action of botulinum toxin is explained.

[0018] Figure 2 Shown is food intake of rats treated with BOTOX alone (ie, without anticholinesterase treatment).

[0019] Figure 3 Shown are food intake on days 2, 7, 9, 11 and 14 after Botox injection for rats treated with anticholinesterase agents, and food intake for rats not treated with anticholinesterase agents.

[0020] Figure 4 is a line graph of food intake on days 2, 7, 9, 11 and 14 after Botox injection in rats treated with anticholinesterases and food intake in rats not treated with anticholinesterase agents.

[0021] Figure 5 Line graph of food intake following Botox injection in rats treated with the higher dosing regimen of the anticholinesterase agent and food intake in rats not treated with the anticholinesterase agent. DETAILED DESCRIPTION

[0022] Before describing the disclosed methods and materials, it is to be understood that the aspects described herein are not limited to specific embodiments, devices or configurations and as such may of course vary. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting unless explicitly defined herein.

[0023] Throughout this specification, unless the context clearly requires otherwise, the terms “comprise” and “include” and variations (for example, “comprises,” “comprising,” “includes,” “including”) will be understood to imply the inclusion of stated components, features, elements or steps, or combinations of components, features, elements or steps, but not the exclusion of any other integers or steps, or combinations of integers or steps.

[0024] 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.

[0025] Ranges may be expressed herein as from "about" one particular value and / or to "about" another particular value. When such ranges are expressed, another aspect includes from a particular value and / or to another particular value. Similarly, when the preposition "about" is used to express a numerical value as an approximate number, it is understood that the particular value forms another aspect. It is also understood that the endpoints of each range are important relative to the other endpoint, and independently of the other endpoint.

[0026] "Effective amount" means an amount of a compound that, when administered to a subject, is sufficient to effectively treat the conditions described herein. The amount of a compound that constitutes an "effective amount" will vary depending on the compound, the condition and its severity, and the age of the subject to be treated, but can be routinely determined by those skilled in the art.

[0027] As used herein, the term "muscle paralysis" means a complete loss of muscle function. Muscle paralysis may be accompanied by a loss of sensation (loss of perception) in the affected area.

[0028] The term "muscle weakness" as used herein means a partial loss of muscle function and / or a loss of muscle strength.

[0029] As used herein, the term "chemical denervation" (or "chemodenervation") refers to the loss of innervation (ie, blockade of neurotransmission) caused by a pharmacological agent (eg, a chemical compound).

[0030] In connection with the present disclosure, the methods and active materials described herein can be configured by one skilled in the art to meet desired requirements. Generally, the disclosed materials, methods and devices provide for targeted local parenteral administration of anticholinesterase agents to affected patients to restore neuromuscular transmission in drug-induced unresponsive muscles.

[0031] The neurotoxin botulinum toxin is widely used in cosmetic and medical procedures. Temporary but undesirable side effects result from the unintentional spread of the toxin to adjacent muscle structures, resulting in paralysis or weakness in unexpected areas. The action of the neurotoxin is to block the release of ACh at the neuromuscular junction. Anticholinesterase agents are used to indirectly increase ACh by degrading exogenous AChE. The binding of ACh to its receptor sites is necessary to maintain muscle transmission. The depth of blockade is an important factor in determining the efficacy of anticholinesterase agents in accelerating spontaneous recovery. For recovery or muscle reactivation to occur, ACh must be present. The term "depth of blockade" as used herein refers to the level occupied by postsynaptic receptors.

[0032] Due to the paralytic and diffuse properties of botulinum toxins used commercially, partial chemical denervation and / or spontaneous recovery will occur. Therefore, the extent or depth of the block will determine the speed of recovery. The inventors have discovered that peripherally acting anticholinesterase agents can be used as neurotoxin rescue agents that accelerate recovery time. The inventors have discovered that the timing of administration of the anticholinesterase agent, the concentration of the anticholinesterase agent, and the dosing period are important elements in the efficacy of the rescue treatment.

[0033] Thus, in one aspect, the present disclosure provides a method of restoring neuromuscular transmission by topically administering to an unresponsive muscle an effective dose of a composition comprising an anticholinesterase agent. In certain embodiments, the unresponsive muscle has been previously exposed to a neurotoxin. In certain embodiments, the unresponsive muscle has been previously exposed to a botulinum toxin.

[0034] In another aspect, the present disclosure provides a method of treating neurotoxin-induced muscle paralysis or weakness comprising topically administering to a patient a composition comprising an anticholinesterase agent. In certain embodiments, the neurotoxin is botulinum toxin.

[0035] Botulinum toxin is a neurotoxic protein produced by Clostridium botulinum and related species. Clostridium botulinum strains produce seven different neurotoxins, which are designated as AG types. All seven types have similar structures and molecular weights, consisting of heavy (H) chains and light (L) chains joined by disulfide bonds, and all of them interfere with neurotransmission by blocking the release of acetylcholine. Therefore, in one embodiment, the botulinum toxin of the present disclosure includes one or more of type A, type B, type C, type D, type E, type F and type G. In one embodiment, the botulinum toxin of the present disclosure includes one or more of type A, type B, type E and type F. In one embodiment, the botulinum toxin of the present disclosure includes one or more of type A and type B. In one embodiment, the botulinum toxin of the present disclosure is type A botulinum toxin.

[0036] Botulinum toxin type A is approved by the FDA for use in cosmetic procedures and is available under a variety of trade names, one of which is (Allergan, Irvine, CA, USA; referred to herein as "Botox"). Similarly, botulinum toxin is used to treat more than 20 medical conditions. The most common medical conditions include migraines, excessive sweating, muscle spasms, urinary incontinence, etc. The spread of toxin from the injection site or overuse of toxin can cause unwanted side effects in these procedures. For example, in the treatment of migraines, 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.

[0037] The method of the present disclosure requires a composition comprising an anticholinesterase agent. Anticholinesterase agents (i.e., cholinesterase inhibitors) are divided into two categories, organophosphorus compounds, which are irreversible; and carbamates, which are reversible. The former are generally more toxic, have a longer duration of action, and are generally associated with central nervous system (CNS) toxicity. Reversible anticholinesterase agents have been found to have medical uses for a variety of indications. For example, some reversible anticholinesterase agents are used to treat Alzheimer's disease because these can cross the blood-brain barrier to reach the CNS.

[0038] In some embodiments, the anticholinesterase agent of the present disclosure is a reversible anticholinesterase agent.In some embodiments, the anticholinesterase agent of the present disclosure is a reversible anticholinesterase agent having one or more groups selected from the group consisting of carbamate, tertiary ammonium, and quaternary ammonium.

[0039] In some embodiments, the anticholinesterase agent is selected from one or more of the following: physostigmine, neostigmine, ambenonium chloride, pyridostigmine, ambenonium chloride, demecarium bromide, rivastigmine, galantamine, donepezil, tacrine, 7-methoxytacrine, edrophonium, huperzine A, radotigid, and any derivatives and combinations thereof.

[0040] In some embodiments, the anticholinesterase agents of the present disclosure are selected from one or more of the following:

[0041] Neostigmine,

[0042] Tenxilong,

[0043] Pyridostigmine,

[0044] Physostigmine,

[0045] Lisdmin,

[0046] Donepezil,

[0047] Galantamine,

[0048] and combinations thereof.

[0049] In some embodiments of the present disclosure, the anticholinesterase agent is pyridostigmine, neostigmine, edrophonium, or a combination thereof.

[0050] In some embodiments of the present disclosure, the anticholinesterase agent is pyridostigmine. Pyridostigmine is not fat soluble and therefore acts peripherally. This property makes it ideal for use in muscle-related conditions. Pyridostigmine is also safer than neostigmine because it has a lower incidence of bradycardia and arrhythmias.

[0051] In some embodiments of the present disclosure, the anticholinesterase agent is a compound of formula (I):

[0052]

[0053] or a pharmaceutically acceptable salt thereof, wherein

[0054] Y is CR 3 or N + X - R 4 , wherein X is a halogen;

[0055] R1 is selected from hydrogen, C1-C6 alkyl, -CO(OH), -CO(C1-C6 alkoxy), -CO(NH2), -CONH(C1-C6 alkyl) and -CON(C1-C6 alkyl)2;

[0056] R2 is hydrogen, or R2 and R3 together with the atoms to which they are attached form an optionally substituted heterocyclic ring;

[0057] R3 is selected from C1-C6 alkyl, C1-C6 alkoxy, hydroxy C1-C6 alkyl, amino C1-C6 alkyl, (C1-C6 alkylamino) C1-C6 alkyl, (di-C1-C6 alkylamino)-C1-C6 alkyl, C1-C6 alkoxy-C1-C6 alkyl, -OH, -NH2, -NH(C1-C6 alkyl), -N(C1-C6 alkyl)2 and -N + (C1-C6 alkyl)3X - ;and

[0058] R4 is selected from C1-C6 alkyl, hydroxy C1-C6 alkyl or C1-C6 alkoxy-C1-C6 alkyl.

[0059] In some embodiments, the compound of formula (I) is wherein Y is C. In some embodiments, the compound of formula (I) is wherein Y is or N + X - , or Y is N + Br - or N + Cl - , or Y is N + Br - .

[0060] In some embodiments, the compound of formula (I) according to any of the preceding embodiments is wherein R1 is 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) is wherein R1 is selected from hydrogen, -CO(NH2), -CONH(C1-C6 alkyl) and -CON(C1-C6 alkyl)2. In some embodiments, the compound of formula (I) is wherein R1 is hydrogen. In some embodiments, the compound of formula (I) is wherein R1 is -CO(NH2), -CONH(C1-C6 alkyl) or -CON(C1-C6 alkyl)2. In some embodiments, the compound of formula (I) is wherein R1 is -CON(C1-C6 alkyl)2.

[0061] In some embodiments, the compound of formula (I) according to any of the preceding embodiments is wherein R2 is hydrogen. In some embodiments, the compound of formula (I) according to any of the preceding embodiments is wherein R2 and R3 and the atoms to which they are attached together form an optionally substituted heterocycle. In some embodiments, the heterocycle is optionally substituted by one or more of the following: halogen, C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkoxy, -OH, -NH2, -NH(C1-C6 alkyl) or -(C1-C6 alkyl)2. In some embodiments, the heterocycle is octahydropyrrolo[2,3-b]pyrrole or pyrrolidine, each of which is optionally substituted by one or more of the following: 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 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.

[0062] In some embodiments, the compound of formula (I) according to any of the preceding embodiments is wherein R3 is selected from C1-C6 alkoxy, -OH, -NH2, -NH(C1-C6 alkyl), -N(C1-C6 alkyl)2 and -N + (C1-C6 alkyl)3X - In some embodiments, R3 is selected from C1-C6 alkoxy and -OH. In some embodiments, R3 is selected from -NH2, -NH(C1-C6 alkyl), -N(C1-C6 alkyl)2 and -N + (C1-C6 alkyl)3X - In some embodiments, R3 is -N + (C1-C6 alkyl)3X- In some embodiments, R3 is -N + (C1-C6 Alkyl)3Br - or -N + (C1-C6 alkyl)3Cl - .

[0063] In one embodiment, the method of the present disclosure requires targeted delivery of the composition. Targeted delivery of drugs to the site of action can enhance drug efficacy. Drug toxicity can be reduced by increasing the concentration of local active drug entering the afflicted tissue while minimizing exposure to other areas of the body. Local delivery avoids first-pass metabolism in the liver and gastrointestinal side effects. In addition, the total drug dose can be significantly reduced, thereby reducing the patient's exposure to systemic side effects that are off-target. This enables the use of anticholinesterase agents more widely and in beauty-related fields and other non-life-threatening conditions. In one embodiment of the present disclosure, the composition is applied directly to the affected muscle.

[0064] Targeted drug delivery can be achieved by parenteral injection using conventional techniques similar to those used in the administration of botulinum toxin, or by passive or active transdermal delivery. Passive transdermal delivery refers to a topical or other conventional skin patch or gel for delivering active drugs across the skin barrier without a driving force or perforation. In one embodiment of the present disclosure, the composition is administered parenterally (e.g., by intramuscular injection). Parenteral delivery (e.g., intramuscular injection) can be to unresponsive muscle areas (i.e., areas showing side effects such as muscle paralysis or muscle weakness) or in muscle structures recommended by medical professionals with a deep understanding of muscle characteristics.

[0065] In one embodiment of the present disclosure, the composition is transdermally applied. In one embodiment, the composition is applied as a transdermal patch or transdermal gel. Transdermal drug delivery allows the drug to enter the underlying tissue via the skin surface area. Considering that the highest incidence of the procedure will involve the facial area, any patch or topical preparation should remain in place within a limited time to allow the patient to resume normal daily activities. Once the drug has entered the subcutaneous space and the target tissue or muscle, it is desirable to remove the patch and remove any obstruction. Conventional patch technology is non-invasive, and is usually not required to be applied by a medical professional as required for injection. Skills and technicians will appreciate that performance (drug intake / delivery through the skin) depends primarily on drug properties, such as molecular size, lipophilicity, drug polarity and solubility. If it is necessary to improve drug delivery, those skilled in the art will be able to select suitable surfactants and / or penetration enhancers. If it is necessary to take effect faster, active transdermal technology (e.g., microneedles or other skin perforation techniques are to enhance delivery rate) can be used. Active methods include, for example, iontophoresis, electroporation, mechanical perturbation, and other energy-related techniques such as ultrasound and needle-free injection.

[0066] Other targeted delivery methods may include, but are not limited to, intramuscular, intradermal, subcutaneous or topical delivery.

[0067] Taking into account the localized characteristics of targeted delivery, an effective dose of anticholinesterase is delivered in a small dose, that is, lower than the clinical dose of the anticholinesterase when the anticholinesterase is administered orally or intravenously (usually for other therapeutic indications). The general guideline for converting oral administration to intravenous administration is to give patients 1 / 30 of the oral dose. Since targeted administration is directly injected into the tissue, its dose will be as low as 0.1 mg compared to intravenous administration, or at most 1 / 10 of the dose required for muscle relaxant reversal. The effective dose can be easily understood using the known physiological techniques used to evaluate muscle or nerve damage. Electromyography (EMG) measures the potential generated by muscle cells when they are electrically activated. Needle electrodes emit fast electrical pulses to nerves and measure the time it takes for muscles or nerves to contract. The speed of contraction is reported as conduction velocity. The conduction velocity can be measured before or after the injection of the reversal agent. The increase in the expected conduction velocity will be a measure of the efficiency of the dose response.

[0068] In certain embodiments, a small dose is about 4 / 5 to about 1 / 50 of the clinical dose of the anticholinesterase agent when the anticholinesterase agent is administered orally or intravenously for use (usually for other therapeutic indications). In some embodiments, a small dose is about 1 / 5 to about 1 / 50 of the oral or intravenous clinical dose, 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.

[0069] In some embodiments of the present disclosure, the anticholinesterase agent is administered at a dose of about 0.05-0.5 mg / kg, or at a dose of about 0.15-0.25 mg / kg, or at a dose of about 0.2 mg / kg. As is known in the art regarding dosing guidelines provided for botulinum toxin injections, the specific dose of the anticholinesterase agent can be adjusted for the individual based on factors such as the size, number, and location of the muscles involved, as indicated by methods such as needle electromyography or nerve stimulation. For example, in the treatment of palmar hyperhidrosis, the size of the palm (e.g., cm) can be used to determine the amount of anticholinesterase agent administered. 2 ) to adjust the dosage. Those skilled in the art will appreciate that the dosage may be higher or lower depending on the activity of the anticholinesterase agent, the bioavailability of the anticholinesterase agent, its metabolic kinetics and other pharmacokinetic properties, the mode of administration, and a variety of other factors. Those skilled in the art will also appreciate that the anticholinesterase agent will be administered in such a way that it does not penetrate or diffuse into the systemic circulation to cause unwanted side effects.

[0070] In some embodiments of the present disclosure, the anticholinesterase agent may be administered immediately after a neurotoxin (e.g., botulinum toxin). For example, the anticholinesterase agent may 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 neurotoxin. In some embodiments of the present disclosure, the anticholinesterase agent may be administered at a certain time after a neurotoxin (e.g., botulinum toxin). For example, the anticholinesterase agent may be 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 neurotoxin.

[0071] For the application of anticholinesterase agents for solving the unwanted side effects caused by botulinum toxin and lacking specific monitoring, it is important that the drug is available during the appropriate window. In one embodiment, the drug is available when ACh is present. Therefore, the presence of complete blocking will delay the reaction. To determine whether ACh is present, the patient can be monitored by, for example, electromyography (EMG) or other tests. A suitable treatment window can be accessed by using a sustained release composition. A suitable treatment window can be accessed by using several applications of the disclosed composition. In one embodiment, the disclosed composition can be administered once (i.e., in a single dose). In one embodiment, the disclosed composition can be administered twice or three times or four times or more.

[0072] Anticholinesterase agents will be administered after the medical practitioner or patient has been clearly informed of the effects of the neurotoxin. In some cases, it may take 7 days to achieve the full effect of the neurotoxin. Anticholinesterase agents may be used at any time after evidence of unwanted side effects.

[0073] In one embodiment of the present disclosure, the composition is a sustained release composition. Sustained release formulations will ensure that the drug can function when acetylcholine becomes available. Sustained release compositions should avoid the need for multiple injections of the disclosed composition. It will also reduce the cost of care while increasing maximum efficacy and limited patient exposure and fewer side effects. In certain embodiments, the sustained release formulation releases the anticholinesterase over a period of time measured in days: for example, over a period of 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 over a period of time between 1 and 14 days or between 1 and 7 days or between 3 and 7 days or between 1 and 5 days or between 5 and 7 days, or over a period of time measured in hours: for example, up to about 3 hours or up to about 6 hours or up to about 12 hours or up to about 24 hours, or between about 3 hours to about 24 hours or between about 3 hours to about 18 hours or between about 3 hours to about 12 hours or between about 3 hours to about 6 hours or between about 6 hours to about 24 hours or between about 6 hours to about 18 hours or between about 6 hours to about 12 hours or between about 12 hours to about 24 hours or between about 18 hours and about 24 hours.

[0074] Suitable sustained release formulations are known to those skilled in the art, and the compositions of the present disclosure may be formulated in a variety of ways depending on the desired drug load, the desired drug delivery profile, and / or the desired release rate / pharmacokinetics.

[0075] The sustained-release composition of the present disclosure may be any formulation disclosed by 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 incorporated herein by reference in its entirety.

[0076] The sustained release composition of the present disclosure can be prepared by the method disclosed in U.S. Patent Publication No. 2012 / 0316108, which is incorporated herein by reference in its entirety. These preparations are single-phase gel preparations that require the use of phospholipids (20%-80%), preferably with a lower range of phospholipids to allow the use of syringes with thinner needles. The preparation may include pharmaceutical grade lecithin from eggs or soybeans actually used for parenteral products or oils determined to be used in injectable drug preparations. In the first step of formulating a phospholipid gel, a primary dispersion may include an emulsion or suspension, or may be referred to as an emulsion if oil is used. Therefore, in one embodiment, the composition of the present disclosure is a single-phase gel composition, which also includes 20-80wt% of phospholipids and 0.1-65wt% of water. In one embodiment, the composition of the present disclosure (e.g., a single-phase gel composition) can be extruded through a needle having a gauge of about 30G to about 33G. The sustained release single-phase gel formulation can be prepared by a method comprising: (a) mixing the components to form a primary dispersion comprising one or more phospholipids and excess water; (b) homogenizing the primary dispersion to form a nanodispersion having an average particle size of less than about 200 nm in diameter; (c) passing the nanodispersion through a 0.2 m or 0.45 micron filter; and (d) removing excess water to obtain a gel. In some embodiments, step c can be eliminated if the formulation is heat sterilized according to standard practices for injectable drugs.

[0077] Compositions comprising anticholinesterase agents may also include a suitable carrier, excipient or diluent. The precise nature of the carrier, excipient or diluent will depend on the desired properties of the composition, and may range from suitable or acceptable for veterinary use to suitable or acceptable for human use.

[0078] Compositions containing anticholinesterase agents can be prepared by conventional mixing, dissolving, granulating, sugar-coating, grinding, emulsifying, encapsulating, entrapping or lyophilizing processes. The compositions can be formulated in a conventional manner using one or more physiologically acceptable carriers, diluents, excipients or adjuvants that facilitate processing of the compound into pharmaceutically acceptable preparations.

[0079] The anticholinesterase agent can be formulated as a pharmaceutical composition by itself or as a hydrate, solvate, N-oxide or pharmaceutically acceptable salt (as previously described). Generally, such salts are more soluble in aqueous solution than the corresponding free acids and bases, but salts with lower solubility than the corresponding free acids and bases may also be formed.

[0080] If necessary, the compositions of the present disclosure may exist as ready-to-use single-use delivery formulations (including those designed for injection). This will avoid cross-contamination and waste problems. Depending on the activity of the treatment center, the frequency of use of the rescue product will likely not require the use of a sterile container system that can be used multiple times, such as vials. Available injection preparations include sterile suspensions, solutions or emulsions of active compounds in aqueous or oily carriers. The composition may also include a formulation, such as a suspending agent, a stabilizer and / or a dispersant. Injectable preparations may exist in unit dose form, for example, in an ampoule or in a multi-dose container, and may include an added preservative. Alternatively, injectable preparations may be provided in powder form to be reconstituted with a suitable carrier before use, including but not limited to sterile pyrogen-free water, buffer, glucose solution, etc. For this reason, the anticholinesterase agent may be dried (such as lyophilized) by any technique known in the art and reconstituted before use.

[0081] Injectable preparations can be launched (in commercial use or in the development process) using a variety of methods. These may include, but are not limited to, oil-based injections, injectable drug suspensions, injectable microspheres or in-situ systems, excipients, and polymers for drug reservoirs that dissolve over time. The selection of technology will depend on the following major factors: drug loading in the matrix, required drug delivery curve, release rate / pharmacokinetics (therapeutic window). Oil-based injectable solutions and injectable drug suspensions will release control in a few days to a few weeks, while polymer-based microspheres and in-situ gels or drug reservoirs are reported to be sustainable for several months. The ideal final formulation will be limited to the total volume that needs to be injected or otherwise administered to the patient.

[0082] If desired, the compositions of the present disclosure may be present in a kit. The kit may be accompanied by instructions for administration.

[0083] If desired, the compositions of the present disclosure may be present in a packaging or dispensing device which may contain one or more unit dosage forms containing the compound. The packaging may, for example, comprise metal or plastic foil, such as a blister pack. The packaging or dispensing device may be provided with instructions for administration.

[0084] Example

[0085] Animal models and methods

[0086] To demonstrate the efficacy of anticholinesterase agents in promoting spontaneous recovery after botulinum toxin injection, a rat model outlined by Moon et al. (Maxillofacial Plastic and Reconstructive Surgery (2015) 37:46) was used. In this case, botulinum toxin was injected into the masseter muscle of the rats and the impending changes in food intake were monitored. The purpose of the experiment was to induce paralysis of the jaw muscles in rats using botulinum neurotoxin. It was expected that this would have an adverse effect on food intake and the ability of anticholinesterase agents to promote the recovery process compared to injections was subsequently tested.

[0087] The test material is a product approved for injection. (Allergan, Irvine, CA, USA) was reconstituted with saline for injection to the required dose. The dose of Botox used in the experiment was 5 100 μL / unit. Pyridostigmine (Sandoz Inc., Princeton, NJ, USA) was used as a ready-to-use injection solution (5 mg / ml).

[0088] SD rats, which usually weigh between 375 and 400 g, were subjected to a Botox control or treated with the anticholinesterase agent pyridostigmine. The weight and food intake of each group of rats (usually 3-5) were monitored for about 1 week as a baseline measurement. Subsequently, each group received a Botox injection into the masseter muscle. Rat weight and food intake were tracked for a total of 2-4 weeks.

[0089] It is expected that inducing jaw muscle paralysis will lead to reduced food intake and possible weight loss, followed by recovery as the effects of Botox fade over time. After Botox administration, some groups of rats received reversal agent injections into the masseter muscle. Treatment groups differed in dose, dosing regimen, and frequency of reversal agent administration. Each animal was briefly anesthetized with isoflurane before injection and weighed daily. Rats were fed granular rat chow. This differs from the Moon experiment in that a powdered diet was used. It was considered that granular diet would be a more representative measure of muscle atrophy and chewing ability.

[0090] Botox (5 100 μl / unit) was injected into the masseter muscle of rats. A comparable volume of saline was injected at the beginning of the experiment to standardize the injection procedure and dose delivery. Although Botox below 5 units / side was tested (0.1-2.5 units), these lower doses did not affect food intake compared to baseline, thus suggesting that the experimental model does not show a dose response. It is suspected that limitations of the animal model have a weakening effect on the recovery results.

[0091] To limit the potential for dehydration of the animals during a period of muscle paralysis and severe impact on food intake, rats were injected intravenously with sterile lactated Ringer's solution.

[0092] Botulinum toxin control

[0093] like Figure 2 As shown in , rats were not able to achieve 100% recovery even after 37 days using only Botox (i.e., without anticholinesterase treatment). This is consistent with the duration of action reported by the manufacturer in the product labeling, i.e., the effects of the neurotoxin can last for 90+ days in patients. However, this is inconsistent with the results shown by Moon et al., which showed that near-complete recovery occurred spontaneously after only 10 days. The reason for the difference in results may be due to the different biopotencies of the neurotoxins used. Botox was not specifically stated in the Materials and Methods. The potencies between different manufacturers of botulinum toxin type A are not comparable, and this is clearly stated in the literature and in the product labels of the different manufacturers.

[0094] Results: Timing of administration

[0095] As explained above, the acceleration of spontaneous recovery / reversal depends on the presence of acetylcholine at the neuromuscular junction. Anticholinesterases are used to inhibit the action of cholinesterase in breaking down acetylcholine into its building blocks choline and acetate. Acetylcholine is normally taken up into the synapse and regenerated by choline reprocessing.

[0096] The experiments conducted proved that early administration of anticholinesterase agents was ineffective. The appropriate timing, that is, the timing of causing an enhanced recovery response compared to the Botox control, occurred in our animal model after about the 6th day (i.e., 6 days after the botulinum toxin injection). Due to the lack of AChE response, and without being limited by any theory, it is possible that acetylcholine is not available until this time. It is expected that if the dose of neurotoxin is low enough to cause continuous muscle relaxation and not completely fixed, a more immediate response will be seen. For example, in the case of botulinum toxin side effects, a faster time response will be expected. Side effects are usually the result of poor injection techniques that diffuse the toxin effect or enter the target area. It has been reported that side effects can take weeks to months to resolve in human use. At the same time, the product insert of Botox cosmetics states that the dosage is designed to achieve partial chemical denervation. In the case where the side effects originate from the use of botulinum toxin treatment in humans, the reaction time can be immediate.

[0097] Results: Enhancement of spontaneous recovery

[0098] like Figure 3 As shown in Figure 2, rats were given (Pyridostigmine Bromide) Injection. The dose of 0.5 mg / side was 100 μl injected into the masseter muscle of rats on the indicated days. The mean relative increase in food intake was 26.3%, based on data collected after 6 days post-injection.

[0099] like Figure 4 As shown in Treated animals showed full recovery of food intake by approximately day 26, whereas control animals had not recovered by day 37. Also note that recovery was still ongoing 14 days after the last treatment day.

[0100] Result: Endplate poisoning

[0101] Nausea, vomiting and diarrhea are among the muscarinic side effects that can be observed from the administration of anticholinesterase agents. In the toxicity studies of pyridostigmine carried out in SD rats, the toxic signs caused by pyridostigmine include eye mucous secretions, nasal discharge, hypoactivity, collapse, ataxia, diarrhea, hunchback posture, emaciation and death (Battell, Preclinical Toxicology of New Drugs, Report 8740-86-2, April 4, 1986, JG Gage Principle Investigator). Therefore, the adverse effects signs of rats in the present embodiment are monitored. Treatment of rats by the disclosed method shows no side effects.

[0102] It is important to achieve an effective dose of anticholinesterase without affecting the poisoning of the endplate terminals. No doses have been reported for intramuscular injection into muscle tissue. As previously discussed, the depth of blockade during administration will affect recovery efficiency. As a parallel approach to intravenous use of cholinesterase inhibition to reverse neuromuscular blockade at the end of anesthesia, there is no advantage in administering before the onset of spontaneous recovery. In addition, cholinesterase inhibitors reach an upper limit at higher doses. In this animal model, it was demonstrated that giving more anticholinesterases would not induce an improved response. Without being limited to a specific theory, it can be assumed that higher doses can lead to higher concentrations of acetylcholine at the endplate because exogenous enzymes cannot be used to eliminate excess acetylcholine. Accumulation can lead to endplate poisoning, leading to a constant state of depolarization. This serves as its own form of depolarization blockade.

[0103] Since the preferred anticholinesterase agents are of limited duration of action, recovery will occur over time as exogenous cholinesterase levels are reestablished.

[0104] Figure 5 It was shown that animals treated with the higher dose regimen experienced a lag in recovery of food intake. It is assumed that this lag in response is due to temporary intoxication of the terminal plate extremities. Recovery did not occur until 10+ days after Botox fixation, compared to the control group using only Botox (which began to recover spontaneously after 6 days). The higher dose in this experiment was defined as a dose of 0.25 mg / side injected twice daily starting from day 3 until day 14.

[0105] in conclusion

[0106] In human subjects, it is expected that the rescue or reversal of the partial chemical denervation state will show a faster response. As previously explained, the presence of natural acetylcholine is a prerequisite for establishing nerve conduction. The advent of side effects is considered to be the result of toxin diffusion, which leads to off-target effects. This is most likely because of off-target muscle relaxation (partial denervation) compared with complete muscle fixation or paralysis. According to the mouse food intake rodent model (which shows a lower sensitivity to the dose-response curve of the paralysis caused by neurotoxins), those skilled in the art expect that the results in the human model will be more significant. This is because the actual situation of the beauty and medical uses of neurotoxins in humans is not expected to reach a completely fixed state, especially in the manifestation of side effects. The target dose of the expected neurotoxin triggers a state of muscle relaxation. Compared with complete denervation, this will lead to the existence of partial denervation. At the same time, the human model does not need to chew. In the mouse food intake model, chewing is a complex process and can be affected by multiple factors in driving motor function. Therefore, the state of partial chemical denervation will not be reached. This limitation leads to a delayed onset of reaction because acetylcholine will be available until nerve sprouting begins.

[0107] In summary, this application involves the following aspects:

[0108] 1. A method of restoring neuromuscular transmission by topically administering to an unresponsive muscle an effective amount of a composition comprising an anticholinesterase agent, wherein the unresponsive muscle has been previously exposed to a neurotoxin.

[0109] 2. The method of claim 1, wherein the neurotoxin comprises botulinum toxin.

[0110] 3. The method of claim 2, wherein the botulinum toxin is botulinum toxin type A.

[0111] 4. A method of treating botulinum toxin-induced muscle paralysis or weakness, the method comprising topically administering to a patient a composition comprising an anticholinesterase agent.

[0112] 5. The method of claim 4, wherein the botulinum toxin is botulinum toxin type A.

[0113] 6. The method of any one of items 1-4, wherein the anticholinesterase agent is a reversible anticholinesterase agent having one or more groups selected from the group consisting of carbamate, tertiary ammonium, and quaternary ammonium.

[0114] 7. A method as described in any one of items 1-4, wherein the anticholinesterase agent is selected from one or more of the following: neostigmine, edrophonium, pyridostigmine, physostigmine, rivastigmine, donepezil, galantamine and combinations thereof.

[0115] 8. A method as described in any one of items 1-4, wherein the anticholinesterase agent is pyridostigmine.

[0116] 9. The method of any one of items 1-6, wherein the anticholinesterase agent is of the formula:

[0117]

[0118] or a pharmaceutically acceptable salt thereof, wherein

[0119] Y is CR 3 or N + X - R 4 , wherein X is a halogen;

[0120] R1 is selected from hydrogen, C1-C6 alkyl, -CO(OH), -CO(C1-C6 alkoxy), -CO(NH2), -CONH(C1-C6 alkyl) and -CON(C1-C6 alkyl)2;

[0121] R2 is hydrogen, or R2 and R3 together with the atoms to which they are attached form an optionally substituted heterocyclic ring;

[0122] R3 is selected from C1-C6 alkyl, C1-C6 alkoxy, hydroxy C1-C6 alkyl, amino C1-C6 alkyl, (C1-C6 alkylamino) C1-C6 alkyl, (di C1-C6 alkylamino) C1-C6 alkyl, C1-C6 alkoxy C1-C6 alkyl, -OH, -NH2, -NH(C1-C6 alkyl), -N(C1-C6 alkyl)2 and -N + (C1-C6 alkyl)3X - ;and

[0123] R4 is selected from C1-C6 alkyl, hydroxy C1-C6 alkyl or C1-C6 alkoxy C1-C6 alkyl.

[0124] 10. The method of any one of items 1-9, wherein the composition is administered directly to the affected muscle.

[0125] 11. The method of any one of items 1-10, wherein the composition is administered parenterally (eg, by intramuscular injection).

[0126] 12. The method of any one of items 1-10, wherein the composition is administered transdermally (eg, topically or as a patch).

[0127] 13. The method of claim 12, wherein the transdermal administration is a transdermal patch or a transdermal gel.

[0128] 14. The method of any one of items 1-13, wherein the composition is a sustained release composition.

[0129] 15. A method as described in claim 14, wherein the release is carried out over a period of 1 day, 7 days or 30 days or any combination thereof.

[0130] 16. The method of any one of items 1-15, wherein the composition is a single-phase gel composition further comprising 20-80 wt% of phospholipids and 0.1-65 wt% of water.

[0131] 17. The method of claim 16, wherein the gel composition is extrudable through a needle of about 30G to about 33G.

[0132] 18. A method as described in any one of items 1-17, wherein the anticholinesterase agent is administered at a dose of about 0.05-0.5 mg / kg, or about 0.15-0.25 mg / kg, or about 0.2 mg / kg.

[0133] 19. The method of any one of items 1-18, wherein the anticholinesterase agent is administered at a low dose.

[0134] 20. The method of claim 19, wherein the low dose is about 4 / 5 to 1 / 50 of the clinical dose of the anticholinesterase agent when the anticholinesterase agent is administered orally or intravenously.

[0135] It should be understood that the embodiments and implementations described herein are for illustrative purposes only, and that those skilled in the art may suggest various modifications or variations thereto, and that these various modifications or variations are included within the spirit and scope of the present application and within the scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference herein for all purposes.

Claims

1. Use of a composition comprising an anticholinesterase agent for the preparation of a medicament for restoring neuromuscular transmission in unresponsive muscles that have been previously exposed to botulinum toxin as a result of cosmetic and medical procedures, wherein the composition is formulated for intramuscular or intradermal injection or for transdermal administration.

2. The method of claim 1, wherein the unresponsive muscles include muscle weakness.

3. The method of claim 1, wherein the botulinum toxin is type A, type B, type C, type D, type E, type F or type G botulinum toxin.

4. The use according to any one of claims 1 to 3, wherein the anticholinesterase agent is a reversible anticholinesterase agent having one or more groups selected from the group consisting of carbamate, tertiary ammonium and quaternary ammonium.

5. The use according to any one of claims 1 to 3, wherein the anticholinesterase agent is selected from one or more of the following: neostigmine, edrophonium, pyridostigmine, physostigmine, rivastigmine, donepezil, galantamine, and combinations thereof.

6. The use according to any one of claims 1 to 3, wherein the anticholinesterase agent is pyridostigmine.

7. The use according to any one of claims 1 to 2, wherein the transdermal administration is a transdermal patch or a transdermal gel.

8. The use according to any one of claims 1 to 2, wherein the composition is a sustained release composition.

9. The use of claim 7, wherein the release is over a period of 1 day, 7 days or 30 days or any combination thereof.

10. The use of any one of claims 1-2, wherein the anticholinesterase agent is administered at a dose of about 0.05-0.5 mg / kg, or at a dose of about 0.15-0.25 mg / kg, or at a dose of about 0.2 mg / kg.

11. The use of any one of claims 1-2, wherein the anticholinesterase agent is administered at a low dose.

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