PHARMACEUTICAL COMPOSITION

Abstract

The disclosure refers to methods for treating individuals exposed to neurotoxic agents with dantrolene or with a pharmaceutically acceptable salt thereof.

Description

1 / 51 “PHARMACEUTICAL COMPOSITION” Technical field

[0001] The disclosure relates to methods for treating individuals exposed to neurotoxic agents with dantrolene (or a dantrolene metabolite such as 5-hydroxy-dantrolene), or with a pharmaceutically acceptable salt thereof. Background

[0002] Neurotoxic agents, also referred to as nerve agents, are organophosphorus compounds that disrupt the transmission of nerve impulses in the nervous system by blocking acetylcholinesterase. These agents, whether in gaseous, aerosol, or liquid form, are extremely toxic and have a very rapid effect. Intoxication by neurotoxic agents produces a large number of symptoms, including unilateral or bilateral meiosis, headache, seizures, loss of consciousness, coma, and death. In particular, intoxication by neurotoxic agents produces seizures, which can rapidly progress to status epilepticus (SE), leading to seizure-related brain damage. Seizures induced by neurotoxic agents generally become refractory to standard antiepileptic therapy.

[0003] Medical care to treat neurotoxic agent poisoning will likely be delayed beyond the therapeutic window of opportunity to stop neurotoxic agent-induced seizures, and seizure-related brain damage will continue along a pathological cascade. Thus, drug therapy capable of interrupting the pathological cascade is needed to provide neuroprotection during the refractory phase of neurotoxic agent-induced seizures. Petition 870220113428, dated 06 / 12 / 2022, page 15 / 129 2 / 51 Summary of the invention

[0004] The disclosure relates to methods for treating individuals exposed to a neurotoxic agent with a pharmaceutical composition comprising an amount of dantrolene (or a dantrolene metabolite such as 5-hydroxy-dantrolene) or a pharmaceutically acceptable salt thereof. Brief description of the figures

[0005] Figure 1 shows the average body weight of the test group over time.

[0006] Figure 2A shows the results of the sucrose preference test on day 14 of the study.

[0007] Figure 2B shows the results of the sucrose preference test on day 15 of the study.

[0008] Figure 3 shows the results of the forced swimming test.

[0009] Figure 4 shows the mean necrosis scores of the frontoparietal cortex.

[0010] Figure 5A shows a photomicrograph of the frontoparietal cortex of an animal from Group E (positive control, water 50 minutes after the start of SE). Arrows mark faded and shrunken neurons. The necrosis score is 4.

[0011] Figure 5B shows a photomicrograph of the frontoparietal cortex of an animal from Group D (30 mg / kg of RYANODEX®, 20 minutes after the start of SE). Arrows mark normal neurons. The necrosis score is 0. Detailed description of illustrative incorporations

[0012] This disclosure can be more readily understood by reference to the following detailed description considered together with the accompanying figures and Petition 870220113428, dated 06 / 12 / 2022, p. 16 / 129 3 / 51 examples, which are part of this disclosure. It should be understood that this disclosure is not limited to the specific compositions or methods described and / or shown herein, and that the terminology used in this document is intended to describe particular embodiments by way of example only and is not intended to limit the claimed disclosure. Furthermore, as used in the descriptive report, including the appended claims, the singular forms a, an, oea include the plural and reference to a specific numerical value includes at least that specific value, unless the context clearly shows otherwise. All numerical ranges are inclusive and combinable.

[0013] The modifier about should be considered as disclosing the range defined by the absolute values ​​of the two extreme points. For example, the expression from about 2 to about 4 also discloses the range from 2 to 4. When used to modify a single number, the term about can refer to plus or minus 10% of the indicated number and includes the indicated number. For example, about 10% can indicate a range from 9% to 11%, and about 1 means from 0.9 to 1.1.

[0014] It should be understood that certain disclosure features which are, for clarity, described here in the context of separate embeddings, may also be provided in combination in a single embedding. Conversely, several disclosure features which are, for brevity, described in the context of a single embedding, may also be provided separately or in any subcombination.Furthermore, the reference to values ​​declared in the ranges includes any and all values ​​within that range. Petition 870220113428, dated 06 / 12 / 2022, page 17 / 129 4 / 51

[0015] When used herein, the term “pharmaceutical composition” shall mean a composition suitable for administration to humans and containing pharmaceutically acceptable excipients, for example, stabilizers, bulking agents, buffers, carriers, diluents, vehicles, solubilizers and binders. When used herein, pharmaceutical composition includes, but is not limited to, a liquid form ready for infusion or subcutaneous injection and intramuscular injection.

[0016] “Therapeutically effective amount refers to an amount of an active pharmaceutical agent described herein that is sufficient to inhibit, interrupt, or cause an improvement in a disorder or condition being treated in a particular individual or in a population of individuals. In certain embodiments, in a human or other mammal, a therapeutically effective amount may be determined experimentally in a laboratory or clinical setting, or may be the amount required by government guidelines for the particular disease and individual to be treated.

[0017] When used here, “improve” refers to a decrease in the severity of a disorder or condition being treated in a particular individual or population of individuals.

[0018] When used herein, “patient” or “individual” is intended to mean a mammal. Thus, the methods described herein are applicable to both human and non-human individuals.

[0019] When used herein, the term “pharmaceutically acceptable” means that which is pharmaceutically acceptable, for example, components, including containers, of a pharmaceutical composition, does not cause unacceptable loss of pharmacological activity or adverse side effects. Petition 870220113428, dated 06 / 12 / 2022, page 18 / 129 5 / 51 unacceptable. Examples of pharmaceutically acceptable components are provided in the United States Pharmacopeia (USP), the National Formulary (NF), adopted at the United States Pharmacopeia Convention held in Rockville, Maryland in 1990 and issued in 1996 by the US Food and Drug Administration (FDA) (both are incorporated herein by reference, including any drawings). Other grades of solutions or components that meet required limits and / or specifications that are outside of USP / NF may also be used.

[0020] When used herein, the term “pharmaceutically acceptable salt” means a salt of a disclosed compound that is pharmaceutically acceptable and possesses the desired pharmacological activity of the parent compound. In particular, such salts are non-toxic and may be acid addition salts and inorganic or organic base addition salts. Specifically, such salts include: salts formed when an acidic proton present in the parent compound is replaced by a metal ion, for example, an alkali metal ion, an alkaline earth metal ion, or an aluminum ion; or coordinates with an organic base such as ethanolamine, diethanolamine, triethanolamine, N-methylglucamine, and the like.Salts also include, by way of example only, sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium and the like; and when the compound contains a basic functionality, salts of non-toxic organic or inorganic acids, such as chlorides, bromides, tartrates, mesylate, acetates, maleates, oxalates and the like.

[0021] When used here, “neurotoxic agent” or “nerve agent” refers to compounds that affect the transmission of Petition 870220113428, dated 06 / 12 / 2022, page 19 / 129 6 / 51 nerve impulses in the nervous system. Neurotoxic agents are organophosphorus compounds, that is, they have the formula (R) 3P(O) , in which the R groups may be the same or different. Type G neurotoxic agents include O-pinacolyl methylphosphonofluoride (soman, GD), ethyl N,N-dimethylphosphoramidocyanide (tabun, GA), propan-2-yl methylphosphonofluoride (sarin, GB), cyclohexyl methylphosphonofluoride (cyclosarin, GF), and 2-(dimethylamino)ethyl (GV).Type V neurotoxic agents include O-cyclopentyl S-(2-diethylaminoethyl)methylphosphonothiolate (EA-3148), (S)-(ethyl {[2-(diethylamino)ethyl]sulfonyls}(ethyl)phosphonates) such as (S)-(ethyl {[2-(diethylamino)ethyl]sulfanyl}(ethyl)phosphinate) (VE), O,O-diethyl S-[2-(diethylamino)ethyl]phosphorothioate (VG), O-ethyl S-[2-(diethylamino)ethyl]methylphosphonothioate (VM), N,N-diethyl-2-(methyl-(2-methylpropoxy)phosphoryl)sulfanylethanamine (VR), and ethyl ({2-[bis(propan-2-yl)amino]ethyl}sulfanyl)(methyl)phosphinate (VX). The methods described here can be used to treat an individual exposed to one neurotoxic agent. The methods described here can also be used to treat an individual exposed to two or more neurotoxic agents.

[0022] When used here, the phrases resulting from exposure to a neurotoxic agent and due to exposure to a neurotoxic agent refer to effects that are a direct consequence of exposure to a neurotoxic agent, as well as effects that are a secondary consequence of exposure to a neurotoxic agent, as well as effects that are an indirect consequence of exposure to a neurotoxic agent.

[0023] Among other things, the disclosure refers to Petition 870220113428, dated 06 / 12 / 2022, p. 20 / 129 7 / 51 methods for treating an individual exposed to a neurotoxic agent with a pharmaceutical composition comprising an amount of dantrolene or a pharmaceutically acceptable salt thereof. The disclosure also refers to methods for treating an individual exposed to a neurotoxic agent with a pharmaceutical composition comprising an amount of a dantrolene metabolite, for example, 5-hydroxydantrolene, or a pharmaceutically acceptable salt thereof. For example, in some aspects, the methods described prevent neurological damage secondary to exposure to a neurotoxic agent. In other aspects, the methods described provide neuroprotective effects after exposure to a neurotoxic agent. In other aspects, the methods described improve brain tissue damage secondary to exposure to a neurotoxic agent. In other aspects, the methods described improve brain tissue damage secondary to status epilepticus due to exposure to a neurotoxic agent.In other aspects, the methods described prevent neuronal necrosis due to exposure to a neurotoxic agent. In other aspects, the methods described treat intracellular calcium overload due to exposure to a neurotoxic agent. In other aspects, the methods described improve intracellular calcium overload due to exposure to a neurotoxic agent. In other aspects, the methods described prevent intracellular calcium overload due to exposure to a neurotoxic agent.

[0024] The individuals described herein may be exposed to a neurotoxic agent via inhalation. In other aspects, individuals are exposed to a neurotoxic agent via consumption of a liquid or food that has been contaminated with a neurotoxic agent. In other aspects, individuals are exposed to a Petition 870220113428, dated 06 / 12 / 2022, page 21 / 129 8 / 51 neurotoxic agent via subcutaneous, intravenous or intramuscular administration of the agent to the individual.

[0025] In some respects, the methods refer to methods for protecting an individual from neural necrosis after the individual has been exposed to a neurotoxic agent. In these embodiments, a pharmaceutical composition comprising an amount of dantrolene (or a dantrolene metabolite such as 5-hydroxy-dantrolene) or a pharmaceutically acceptable salt thereof is administered to the individual after exposure to a neurotoxic agent. When used herein, the term protection from neural necrosis encompasses decreasing the severity of the effects of the neurotoxic agent or improving the effect of the neurotoxic agent or decreasing neural damage resulting from exposure to the neurotoxic agent. In some respects, the term protection from neural necrosis encompasses preventing neural necrosis in an individual who has been exposed to a neurotoxic agent.That is, subjects who are protected from neural necrosis by administration of the described dantrolene-containing compositions (or compositions comprising a dantrolene metabolite such as 5-hydroxy-dantrolene) perform better on neurobehavioral tests when compared with individuals exposed to a neurotoxic agent who were not administered the described compositions.

[0026] In some incorporations, the entire central nervous system of the individual is protected from neural necrosis. In some incorporations, the frontoparietal cortex, the hippocampus, and / or the thalamus are protected from neural necrosis. In other aspects, the frontoparietal cortex will be protected from neural necrosis. In other aspects, the hippocampus is protected from neural necrosis. In other incorporations, the thalamus is Petition 870220113428, dated 06 / 12 / 2022, page 22 / 129 9 / 51 protected from neural necrosis.

[0027] The presence and extent of neural necrosis can be determined using methods known in the art, including neurobehavioral tests, radiological tests, and pathology assessment.

[0028] The disclosure also refers to methods for protecting an individual from a decrease in central nervous system function resulting from exposure to a neurotoxic agent. These methods comprise administering to the individual a pharmaceutical composition comprising an amount of dantrolene (or a dantrolene metabolite, such as 5-hydroxy-dantrolene) or a pharmaceutically acceptable salt thereof after the individual has been exposed to a neurotoxic agent.

[0029] The disclosure also refers to methods for protecting an individual from central nervous system dysfunction resulting from exposure to a neurotoxic agent. These methods comprise administering to the individual a pharmaceutical composition comprising an amount of dantrolene (or a dantrolene metabolite, such as 5-hydroxy-dantrolene) or a pharmaceutically acceptable salt thereof after the individual has been exposed to a neurotoxic agent.

[0030] The disclosure also refers to methods for treating behavioral changes in an individual resulting from exposure to a neurotoxic agent. These methods comprise administering to the individual a pharmaceutical composition comprising an amount of dantrolene (or a dantrolene metabolite, such as 5-hydroxy-dantrolene) or a pharmaceutically acceptable salt thereof after the Petition 870220113428, dated 06 / 12 / 2022, page 23 / 129 10 / 51 individual having been exposed to a neurotoxic agent.

[0031] When used herein, protection from a decrease in central nervous system function includes decreasing the severity of the effects of the neurotoxic agent on the central nervous system or improving the effects of the neurotoxic agent on the central nervous system or decreasing the effects of the neurotoxic agent on the central nervous system. That is, individuals who are protected from a decrease in central nervous system function by administration of the described compositions perform better on neurobehavioral tests when compared with individuals exposed to the neurotoxic agent who were not administered the described compositions.

[0032] The disclosure also relates to methods for treating seizures induced by a neurotoxic agent in an individual who has been exposed to a neurotoxic agent. In some respects, the seizures treated are status epilepticus (SE). These methods comprise administering to the individual a pharmaceutical composition comprising an amount of dantrolene (or a dantrolene metabolite, such as 5-hydroxydantrolene) or a pharmaceutically acceptable salt thereof. When used herein, treatment of seizures induced by a neurotoxic agent results in a reduction in the severity or duration of the seizures. In other respects, treatment results in a reduction in both the severity and duration of the seizure.

[0033] The amount of dantrolene, or a pharmaceutically acceptable salt thereof, that is therapeutically effective in treating the individual according to any of the methods described, must be determined by a physician. Petition 870220113428, dated 06 / 12 / 2022, page 24 / 129 11 / 51 specialist in the technique. The therapeutically effective amount may be the amount needed to treat the individual with a single dose. Alternatively, the therapeutically effective amount may be the cumulative amount needed to treat the individual with more than one dose, for example, multiple doses, during chronic or prolonged treatment.

[0034] In those embodiments in which the individual is a human being, the therapeutically effective amount of dantrolene is 1 mg / kg to about 30 mg / kg, administered in one or two doses. In other aspects, the therapeutically effective amount of dantrolene is 1 mg / kg to about 20 mg / kg. In other aspects, the therapeutically effective amount of dantrolene is about 5 mg / kg to about 30 mg / kg. In other aspects, the therapeutically effective amount of dantrolene is about 10 mg / kg to about 30 mg / kg. In other aspects, the therapeutically effective amount of dantrolene is about 15 mg / kg to about 30 mg / kg. In other aspects, the therapeutically effective amount of dantrolene is about 20 mg / kg to about 30 mg / kg. In other respects, the therapeutically effective amount of dantrolene is approximately 5 mg / kg to approximately 20 mg / kg. In other respects, the therapeutically effective amount of dantrolene is approximately 5 mg / kg to approximately 15 mg / kg.In other respects, the therapeutically effective amount of dantrolene is about 5 mg / kg to about 10 mg / kg. In other respects, the therapeutically effective amount of dantrolene is about 10 mg / kg to about 20 mg / kg. In other respects, the therapeutically effective amount of dantrolene is about 2 mg / kg to about 10 mg / kg. Petition 870220113428, dated 06 / 12 / 2022, page 25 / 129 12 / 51 mg / kg, preferably from about 2 mg / kg to about 6 mg / kg. In other respects, the therapeutically effective amount of dantrolene is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or about 30 mg / kg. In some embodiments, the therapeutically effective amount of dantrolene to treat a human individual is greater than 30 mg / kg, for example, from 30 mg / kg to about 100 mg / kg, administered in one or two doses. In some formulations, the therapeutically effective amount of dantrolene to treat a human individual is approximately 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or approximately 100 mg / kg.

[0035] The amount of dantrolene metabolite (such as 5-hydroxy-dantrolene) or of a pharmaceutically acceptable salt thereof, which is therapeutically effective in treating the individual according to any of the methods described, should be determined by a physician skilled in the art. In those embodiments in which the individual is a human being, the therapeutically effective amount of dantrolene metabolite is from 1 mg / kg to about 30 mg / kg, administered in one or two doses. In other aspects, the therapeutically effective amount of dantrolene metabolite is from 1 mg / kg to about 20 mg / kg. In other aspects, the therapeutically effective amount of dantrolene metabolite is from about 5 mg / kg to about 30 mg / kg. In other aspects, the therapeutically effective amount of dantrolene metabolite is from about 10 mg / kg to about 30 mg / kg. In other respects, the therapeutically effective amount of the dantrolene metabolite is approximately 15 mg / kg to approximately 30 mg / kg.In other respects, the quantity is therapeutically important. Petition 870220113428, dated 06 / 12 / 2022, page 26 / 129 The therapeutically effective amount of the dantrolene metabolite is approximately 20 mg / kg to approximately 30 mg / kg. In other respects, the therapeutically effective amount of the dantrolene metabolite is approximately 5 mg / kg to approximately 20 mg / kg. In other respects, the therapeutically effective amount of the dantrolene metabolite is approximately 5 mg / kg to approximately 15 mg / kg. In other respects, the therapeutically effective amount of the dantrolene metabolite is approximately 5 mg / kg to approximately 10 mg / kg. In other respects, the therapeutically effective amount of the dantrolene metabolite is approximately 10 mg / kg to approximately 20 mg / kg. In other respects, the therapeutically effective amount of the dantrolene metabolite is from about 2 mg / kg to about 10 mg / kg, preferably from about 2 mg / kg to about 6 mg / kg. In other respects, the therapeutically effective amount of the dantrolene metabolite is from about 15 mg / kg to about 20 mg / kg.In other respects, the therapeutically effective amount of the dantrolene metabolite is approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or approximately 30 mg / kg. In some embodiments, the therapeutically effective amount of the dantrolene metabolite to treat a human individual is greater than 30 mg / kg, for example, from 30 mg / kg to approximately 100 mg / kg, administered in one or two doses. In some respects, the therapeutically effective amount of the dantrolene metabolite to treat a human individual is approximately 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or about 100 mg / kg.

[0036] In some aspects of disclosure, the timing of administration of the pharmaceutical composition comprising Petition 870220113428, dated 06 / 12 / 2022, page 27 / 129 14 / 51 dantrolene (or a dantrolene metabolite such as 5-hydroxy-dantrolene) or a pharmaceutically acceptable salt thereof, to the individual, after exposure to a neurotoxic agent, may affect the amount of protection from neural necrosis conferred to the individual.

[0037] In some aspects of disclosure, the timing of administration of the pharmaceutical composition comprising dantrolene (or a dantrolene metabolite such as 5-hydroxy-dantrolene) or a pharmaceutically acceptable salt thereof to the individual after exposure to a neurotoxic agent may affect the amount of decrease in central nervous system function conferred on the individual.

[0038] In some aspects of the disclosure, the timing of administration of the pharmaceutical composition comprising dantrolene (or a dantrolene metabolite such as 5-hydroxy-dantrolene) or a pharmaceutically acceptable salt thereof, to the individual, after exposure to a neurotoxic agent, may affect the treatment of neurotoxic agent-induced seizures in the individual.

[0039] Compositions comprising dantrolene (or a dantrolene metabolite such as 5-hydroxy-dantrolene) or a pharmaceutically acceptable salt thereof may be administered chronically to the individual in two or more doses, i.e., over the course of two or more weeks, for example, 2, 3, 4, 5, 6, 7, 8 or more weeks after the individual has been exposed to the neurotoxic agent. Compositions comprising dantrolene (or a dantrolene metabolite such as 5-hydroxy-dantrolene) or a pharmaceutically acceptable salt thereof may be administered acutely to the individual in two or more doses, i.e., over the course of Petition 870220113428, dated 06 / 12 / 2022, page 28 / 129 15 / 51 less than two weeks, for example, during the course of hours or days, for example, during 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours or during 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 days after the individual has been exposed to the neurotoxic agent.

[0040] In some respects, with respect to the timing of the pharmaceutical composition comprising dantrolene (or a dantrolene metabolite such as 5-hydroxy-dantrolene) or a pharmaceutically acceptable salt thereof, the pharmaceutical composition comprising dantrolene (or a dantrolene metabolite such as 5-hydroxy-dantrolene) or a pharmaceutically acceptable salt thereof, at least one dose is administered to the individual 24 hours or less after the individual has been exposed to the neurotoxic agent. In some respects, the pharmaceutical composition comprising dantrolene (or a dantrolene metabolite such as 5-hydroxy-dantrolene) or a pharmaceutically acceptable salt thereof, at least one dose is administered to the individual 20 hours or less after the individual has been exposed to the neurotoxic agent.In some aspects, the pharmaceutical composition comprising dantrolene (or a dantrolene metabolite such as 5-hydroxydantrolene) or a pharmaceutically acceptable salt thereof, at least one dose is administered to the individual 16 hours or less after the individual has been exposed to the neurotoxic agent. In some aspects, the pharmaceutical composition comprising dantrolene (or a dantrolene metabolite such as 5-hydroxydantrolene) or a pharmaceutically acceptable salt thereof, at least one dose is administered to the individual 12 hours or less after the individual has been exposed. Petition 870220113428, dated 06 / 12 / 2022, page 29 / 129 16 / 51 to the neurotoxic agent. In some aspects, the pharmaceutical composition comprising dantrolene (or a dantrolene metabolite such as 5-hydroxy-dantrolene) or a pharmaceutically acceptable salt thereof, at least one dose is administered to the individual 8 hours or less after the individual has been exposed to the neurotoxic agent. In some aspects, the pharmaceutical composition comprising dantrolene (or a dantrolene metabolite such as 5-hydroxy-dantrolene) or a pharmaceutically acceptable salt thereof, at least one dose is administered to the individual 4 hours or less after the individual has been exposed to the neurotoxic agent. In some aspects, the pharmaceutical composition comprising dantrolene (or a dantrolene metabolite such as 5-hydroxy-dantrolene) or a pharmaceutically acceptable salt thereof, at least one dose is administered to the individual 2 hours or less after the individual has been exposed to the neurotoxic agent.In some aspects, the pharmaceutical composition comprising dantrolene (or a dantrolene metabolite such as 5-hydroxy-dantrolene) or a pharmaceutically acceptable salt thereof, at least one dose is administered to the individual 1 hour or less after the individual has been exposed to the neurotoxic agent. In some aspects, the pharmaceutical composition comprising dantrolene (or a dantrolene metabolite such as 5-hydroxy-dantrolene) or a pharmaceutically acceptable salt thereof, at least one dose is administered to the individual approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or approximately 24 hours after the individual has been exposed to a neurotoxic agent.

[0041] In some respects, the pharmaceutical composition comprising dantrolene (or a dantrolene metabolite such Petition 870220113428, dated 06 / 12 / 2022, page 30 / 129 17 / 51 (such as 5-hydroxy-dantrolene) or a pharmaceutically acceptable salt thereof, can deliver a therapeutically effective amount of dantrolene (or a dantrolene metabolite such as 5-hydroxy-dantrolene) to an individual exposed to a neurotoxic agent in one dose. In other respects, two or more doses of the pharmaceutical composition may be required to deliver a therapeutically effective amount of dantrolene (or a dantrolene metabolite such as 5-hydroxy-dantrolene) to an individual exposed to a neurotoxic agent. These additional dosages may be administered substantially simultaneously with the first dose. In those respects where 3 or more doses are administered, each dose may be separated in time from the administration of any other dose.

[0042] In some aspects of disclosure, the administration of dantrolene (or a dantrolene metabolite such as 5-hydroxy-dantrolene) to an individual exposed to a neurotoxic agent is an adjunctive therapy for neurotoxic agent exposure. Individuals exposed to a neurotoxic agent may also receive one or more antidotes for the neurotoxic agent. One class of antidotes for neurotoxic agent exposure is that of acetylcholinesterase reactivators, for example, asoxime chloride (HI-6). Another class of antidotes for neurotoxic agent exposure is that of reversible acetylcholine receptor antagonists, for example, atropine, for example, atropine methyl nitrate. Individuals exposed to neurotoxic agents may also receive anticonvulsant medication. Exemplary anticonvulsant medications include aldehydes (e.g., paraldehyde), aromatic allylic alcohols (e.g., stiripentol), benzodiazepines (e.g., Petition 870220113428, dated 06 / 12 / 2022, page 31 / 12918 / 51 example, clobazam, clonazepam, clorazepate, diazepam, midazolam, lorazepam, nitrazepam, temazepam, nimetazepam), barbiturates (e.g., phenobarbital, methylphenobarbital, barbexaclone), bromides (e.g., potassium bromide), carbamates (e.g., felbamate), carboxamides (e.g., carbamazepine, oxacarbazepine, eslicarbazepine acetate), fatty acids (e.g., valproic acid, sodium valproate, divalproex sodium, vigabatrin, progabide, tiagabine), topiramate, GABA analogs (e.g., gabapentin, pregabalin), hydantoins (e.g., ethotoin, phenytoin, mephenytoin, fosphenytoin), oxazolidinediones (e.g., paramethadione, trimetadione, etadione), propionates (e.g., beclamide), pyrimidinediones (e.g., primidone), pyrrolidines (e.g., brivaracetam, levitiracetam, seletracetam), succinimides (e.g., ethosuximide, phensuximide, messuximide), sulfonamides (e.g., acetazolamide, sultiame, methazolamide, zonisamide),Triazines (e.g., lamotrigine), ureas (e.g., feneturide, phenacemide), valproylamides (e.g., valpromide, valnoctamide), perampanel, and combinations thereof. In some respects, the anticonvulsant drug is a benzodiazepine, e.g., midazolam. In other respects, the anticonvulsant drug is a barbiturate. In still other respects, the anticonvulsant drug is a hydantoin. In some respects, the anticonvulsant drug is paraldehyde. In other respects, the anticonvulsant drug is a fatty acid. In other respects, the anticonvulsant drug is topiramate.

[0043] In those aspects where an antidote is administered to Petition 870220113428, dated 06 / 12 / 2022, p. 32 / 129 19 / 51 In an individual exposed to a neurotoxic agent, dantrolene (or a dantrolene metabolite such as 5-hydroxy-dantrolene) is administered after the antidote has been administered. For example, dantrolene may be administered after the administration of an acetylcholinesterase reactivator and / or after the administration of a reverse acetylcholine receptor antagonist.

[0044] In those aspects where an anticonvulsant drug is administered to an individual exposed to a neurotoxic agent, dantrolene (or a dantrolene metabolite such as 5-hydroxy-dantrolene) may be administered simultaneously with the administration of the anticonvulsant drug. Dantrolene (or a dantrolene metabolite such as 5-hydroxy-dantrolene) may also be administered substantially simultaneously with the administration of the anticonvulsant drug, for example, within about 5 minutes of the administration of the anticonvulsant drug. In other embodiments, dantrolene (or a dantrolene metabolite such as 5-hydroxy-dantrolene) is administered before the anticonvulsant drug is administered. In other embodiments, dantrolene (or a dantrolene metabolite such as 5-hydroxy-dantrolene) is administered after the administration of the anticonvulsant drug.

[0045] The pharmaceutical composition comprising dantrolene (or a dantrolene metabolite such as 5-hydroxydantrolene) or a pharmaceutically acceptable salt thereof may be administered intravenously. In other respects, the pharmaceutical composition comprising dantrolene (or a dantrolene metabolite such as 5-hydroxydantrolene) or a pharmaceutically acceptable salt thereof may be administered transdermally. In other respects, the Petition 870220113428, dated 06 / 12 / 2022, page 33 / 129 20 / 51 A pharmaceutical composition comprising dantrolene (or a dantrolene metabolite such as 5-hydroxy-dantrolene) or a pharmaceutically acceptable salt thereof may be administered intramuscularly. In other respects, the pharmaceutical composition comprising dantrolene (or a dantrolene metabolite such as 5-hydroxy-dantrolene) or a pharmaceutically acceptable salt thereof may be administered intraosseously. In other respects, the pharmaceutical composition comprising dantrolene (or a dantrolene metabolite such as 5-hydroxy-dantrolene) or a pharmaceutically acceptable salt thereof may be administered subcutaneously.

[0046] Preferred pharmaceutical compositions for use in the methods described include dantrolene (or a dantrolene metabolite such as 5-hydroxy-dantrolene) or a pharmaceutically acceptable salt thereof, mannitol, a polysorbate (e.g., polysorbate 80), a povidone (e.g., povidone K12), an optional pH adjuster (e.g., NaOH or HCl), and water.

[0047] A particularly preferred pharmaceutical composition comprising dantrolene (or a dantrolene metabolite such as 5-hydroxy-dantrolene) or a pharmaceutically acceptable salt thereof is RYANODEX® (dantrolene sodium, Eagle Pharmaceuticals, Woodcliff Lake, NJ). RYANODEX® is an injectable suspension provided as a sterile lyophilized powder. RYANODEX® is supplied in small 20 mL vials containing 250 mg of dantrolene sodium, 125 mg of mannitol, 25 mg of polysorbate 80, 4 mg of povidone K12 and sufficient sodium hydroxide or hydrochloric acid for pH adjustment. When reconstituted with 5 mL of sterile water for injection Petition 870220113428, dated 06 / 12 / 2022, page 34 / 129 21 / 51 USP, this produces a suspension.

[0048] The following examples are provided to illustrate some of the concepts described in this disclosure. While each example is considered to provide specific individual embodiments of the disclosure, none of the Examples should be considered as limiting the more general embodiments described herein. In the following examples, efforts have been made to ensure accuracy with respect to the numbers used (e.g., quantities, temperature, etc.), but some experimental errors and deviations should be accounted for. Examples Example 1 Study overview

[0049] The aim of the study was to determine whether RYANODEX® has neuroprotective effects in an established GD (soman) survival model in rats. Single doses of 10 or 30 mg / kg of RYANODEX® were administered by IV bolus injection 20 or 50 minutes after the onset of soman-induced seizures. Survival was facilitated by treatment with asoxime chloride (HI-6) thirty minutes before subcutaneous (SQ) injection of soman, atropine methylnitrate one minute after the onset of soman-induced seizures, and midazolam twenty minutes after the onset of soman-induced seizures that achieved a Racine score of at least 3. Controls included an untreated (naive) group of rats and another group that received sterile water 50 minutes after the onset of soman-induced seizures.

[0050] A series of neurobehavioral tests were performed over a period of approximately 28 days following single-dose GD exposure. On day 29, the rats were sacrificed. Petition 870220113428, dated 06 / 12 / 2022, page 35 / 129 22 / 51 under anesthesia via exsanguination and intracardiac perfusion. The brain of each rat was collected for microscopic neuropathological examination and the heart of each rat was collected for possible pathological examination.

[0051] The experimental subjects were male Sprague-Dawley rats cannulated with jugular veins, 8 weeks of age or older (Charles River Laboratories), weighing 300-500 g on the day of exposure. The sucrose preference test and the neurobehavioral forced swim test were used to assess possible deficits in tasks involving sensorimotor learning and / or integration. Materials

[0052] Soman (GD) - diluted with sodium chloride to 0.9%. Soman is an organic phosphorus neurotoxic agent that deactivates acetylcholine esterase (AChE) by forming an adduct with the enzyme. • Chemical name: pinacolyl methylphosphonofluoride • Formula: C7H16FO2P • Molecular weight: 182.17 • MRIGlobal Lot#: GD090415-DOC—1 • Primary standard ID: 13972-49-3 • Purity: 100% • Storage conditions: <4°C

[0053] HI-6 • Chemical name: [(E)-[1-[(4-carbamoylpyridin-1-io-1-yl)methoxymethyl]pyridin-2-ylidene]methyl]oxoazanium methanesulfonate (asoxime chloride) • Structure: Petition 870220113428, dated 06 / 12 / 2022, page 36 / 129 23 / 51 • Formula: C14H16Cl2N4O3 • Molecular weight: 359.207

[0054] Atropine methyl nitrate • Chemical name: (8,8-dimethyl-8-azoniabicyclo[3.2.1]octan-3-yl) 3-hydroxy-2-phenyl-propanoate nitrate • Structure: • Formula: C18H26N2O6 • Molecular weight: 366.414

[0055] Midazolam Chemical name: 8-chloro-6-(2-fluorophenyl)-1-methyl-4H Authorization 870220113428, dated 06 / 12 / 2022, page 37 / 129 24 / 51 imidazo[1,5-a][1,4]benzodiazepine • Structure: • Formula: C^H^ClFN • Molecular weight: 325.771

[0056] RYANODEX® is an FDA-approved drug for the treatment of malignant hyperthermia in conjunction with appropriate supportive measures and for the prevention of malignant hyperthermia in high-risk patients. Animals

[0057] For this study, 48 Sprague Dawley rats (plus 8 extras / replacements for a total of 56; Charles River Laboratories), aged 8 weeks or older and weighing 300-500 g, with jugular vascular catheters implanted by the supplier, were used. The rats were identified by tail tagging or ear tagging with a unique alphanumeric designation. (See, SOP MRI-1504, “Procedure for Identification of Animals and Cages”).

[0058] All rats included in the study were in good health, free from any signs of clinical disease, and possessed patented catheters suitable for dosing in Petition 870220113428, dated 06 / 12 / 2022, page 38 / 129 25 / 51 start of the study. After delivery, the rats were inspected for signs of health problems and quarantined in Spencer's conventional animal facilities for no less than 48 hours. A veterinarian examined the animals' health and cleared them for the study (see SOP MRI-1501, “Acclimation and / or Quarantine Procedures for Animals”). In accordance with SOP MRI-1500, the animals were weighed within three days of delivery. During the period from the day after the animal's arrival until the end of the study, the rats were handled by staff at least twice a day for acclimation and consistency throughout the study.

[0059] All rats were fed ad libitum Lab Diet certified rodent food, identified by source, batch number and certificate of analysis. The feed contained no contaminants that could affect the study results, as verified by the certificate of analysis (for each batch supplied by the supplier; see SOP MRI-1510, “Procedure for Feeding Animals”). Water bottles (one water bottle / cage) were provided with tap water throughout the study, except during the acclimation period and the sucrose preference test (see below), during which rats received either two water bottles containing tap water or one water bottle containing tap water and one water bottle containing a 1% sucrose solution.

[0060] The animals were housed in Tecniplast polycarbonate cages with metal hoods (Tecniplast, Petition 870220113428, dated 06 / 12 / 2022, page 39 / 129 26 / 51 (Phoenixville, PA) throughout the acclimation / quarantine period and shelf life. Animals were housed individually to prevent catheter damage. Rats were housed in environmentally controlled rooms with at least 10 air changes per hour. The rooms were maintained at a temperature of 20.0°C (68.0°F) to 26.1°C (79.0°F) and relative humidity of 50% ± 20% with a 12-hour light / dark cycle per day. Ambient temperature, humidity, and the 12 h:12 h reverse light / dark cycles were monitored by the Amega View monitoring system 24 h / day or by a hygrometer during the study (see SOP MRI-1170, “Temperature and Humidity Monitoring in Animal Rooms”). Doses

[0061] HI-6, soman, atropine methyl nitrate and midazolam: Single doses of HI-6 (IP, 125 mg / kg), soman (SC, 154 μg / kg, 1.4xLD50), atropine methyl nitrate (IM, 2 mg / kg), and midazolam (IM, 2 mg / kg) were selected based on previous experience with the model. This regimen was expected to induce seizures achieving a Racine score of at least 3 and an acceptable number of survivors for the follow-up study.

[0062] RYANODEX®: In a single-dose tolerability assessment in rats, IV bolus doses of 12, 40, and 60 mg / kg of RYANODEX® were administered to separate groups of rats cannulated via the jugular vein. Twelve mg / kg were well tolerated. A dose of 40 mg / kg resulted in slight body weight loss two days after administration, and 60 mg / kg resulted in moribund sacrifice approximately twenty-four hours later. Petition 870220113428, dated 06 / 12 / 2022, page 40 / 129 27 / 51 after administration for all five rats that received the dose. Single IV bolus doses of 10 and 30 mg / kg of RYANODEX® were selected for this study based on expected tolerability. Rat doses of 10 and 30 mg / kg of RYANODEX® are equivalent to human doses of approximately 2 and 6 mg / kg, respectively. Dosage schedule

[0063] A total of 48 plus up to 8 extra male Sprague-Dawley rats with jugular catheters implanted by the supplier were used in 6 groups, as shown in Table 1. Table 1. Plan for preliminary efficacy study with RYANODEX® Pretreatment Group with HI-6 (IP, 30 min before GD challenge) Soman GD (SQ) Atropine methyl nitrate (IM, 1 min post GD) Midazolam (IM, ~20 minutes after onset of seizure activity) RYANODEX® IV 20 or 50 min after seizure onset A (n=8) 125 mg / kg 154 μg / kg (1.4 x LD50) 2.0 mg / kg 2.0 mg / kg 50 min with 10 mg / kg B (n=8) 125 mg / kg 154 μg / kg (1.4 x LD50) 2.0 mg / kg 2.0 mg / kg 50 min with 30 mg / kg C (n=8) 125 mg / kg 154 μg / kg (1.4 x LD50) 2.0 mg / kg 2.0 mg / kg 20 min with 10 mg / kg D (n=8) 125 mg / kg 154 μg / kg (1.4 x LD50) 2.0 mg / kg 2.0 mg / kg 20 min with 30 mg / kg E(n=8) 125 mg / kg 154 μg / kg (1.4 x LD50) 2.0 mg / kg 2.0 mg / kg Sterile water control at 50 min F (N=8) 0 mg / kg 0 μg / kg 0 mg / kg 0 mg / kg NA Note: For groups C and D, RYANODEX® will be administered immediately after the midazolam injection.

[0064] Dose administration began with Group D, followed by Groups B, A, C, E, and F, respectively. Dose preparation

[0065] GD was prepared in cooled 0.9% sodium chloride Petition 870220113428, dated 06 / 12 / 2022, page 41 / 129 28 / 51 on ice according to SOP MRI-5821 “Preparation of Standards and Samples from Research Development and Testing Evaluation (RDTE) Dilute Solutions”.

[0066] RYANODEX® was prepared by adding 5 mL of sterile water for injection to a small vial of the lyophilized product. Experimental design Animal preparation

[0067] Before or on study day 1 (GD exposure day), rats were transferred to the diluted animal laboratory complex. Dosage

[0068] On study day 1, all animals in Groups AE received the cholinesterase reactivator, HI-6 (125 mg / kg) via intraperitoneal (IP) injection 30 minutes before the GD challenge. All rats in Groups AE were exposed to GD (154 μg / kg or ~1.4 x LD50) by a single subcutaneous injection. Approximately one minute after GD injection for Groups AE, atropine methyl nitrate (AMN) was administered via IM (2.0 mg / kg). Approximately 20 minutes after the onset of seizure activity following GD exposure as defined in Table 1 above, all animals in Groups AE received midazolam at an intramuscular dose of 2 mg / kg. Group E did not receive HI-6, GD, AMN, midazolam, or RYANODEX®. RYANODEX® was administered via a Hamilton microsyringe, and then the catheter was flushed with 0.2 mL of sterile water for injection to ensure complete delivery to the vein.

[0069] The onset of seizure activity was defined by a Petition 870220113428, dated 06 / 12 / 2022, p. 42 / 129 29 / 51 score of 3 on the Racine scale as described below: 1 = immobilization and fixed gaze 2 = head shaking 3 = forelimb clonus 4 = bilateral forelimb clonus 5 = bilateral clonus of the forelimb, elevation and loss of balance.

[0070] If a rat did not achieve this score of 3 after approximately 20 minutes after exposure to GD, it would be replaced in the study.

[0071] As shown in Table 1, rats in Groups A and C received a dose of 10 mg / kg of RYANODEX® IV and rats in Groups B and D received a dose of 10 mg / kg of RYANODEX® IV either 50 or 20 minutes after the onset of seizures (Racine scale of 3). Rats in Group E received a control dose (sterile water for injection) IV in a volume comparable to the maximum dose of RYANODEX® 50 minutes after the onset of seizures.

[0072] Approximately 6 hours after the GD challenge for Groups AE, and at each AM and PM, rats received 5 mL of Ringer's lactate solution SQ for the first 7 days after the challenge to aid recovery. Once the animals had recovered from the challenge, as indicated by observing them eating and drinking freely, moving freely in the cage, and showing no signs of dehydration (no skin drain on the back), SQ fluids ceased. This was partly at the veterinarian's discretion, based on their assessment of the animals. Additionally, after the challenge, during this same time period (up to 7 days after the challenge), the animals received hydrogels (ClearH2O) and gruel (moistened feed in a bowl on the floor of the cage). Petition 870220113428, dated 06 / 12 / 2022, page 43 / 129 30 / 51 cage) to help them recover from the effects of the GD challenge. Body weights

[0073] Body weights were recorded for each study animal within the first 72 hours after receipt from the supplier, and again on study days 1, 3, 5, 10, 17, 24, and 28. Body weights were collected more frequently if the study director discerned that the animals continued to lose weight after the challenge. If any animal lost more than 10% of its original body weight, it was weighed daily until it consistently gained, rather than lost, weight. Figure 1 shows the body weights of all groups. The body weights of all groups exposed to soman treated with RYANODEX® (Groups A, B, C, D) recovered to the negative control values ​​(Group F) on study day 25. Clinical observations

[0074] General health observations were conducted daily during the quarantine and acclimation period. Animals were treated twice daily during the quarantine and acclimation period and throughout their lives. On the first day of the study, rats were observed before exposure. After exposure to GD, rats were frequently monitored to assess the initial onset of seizures. After dosing with midazolam, RYANODEX®, and / or sterile water, animals were observed at least twice daily and at least once on the day of euthanasia. Clinical observations, including hunched posture, dehydration, rough coat, and inappetence, were assessed and recorded. The modified Racine score was recorded at each observation point, if applicable. Any other abnormal clinical observations Petition 870220113428, dated 06 / 12 / 2022, page 44 / 129 31 / 51 were also recorded at this time in the study notebook or appropriate data capture method as per SOP MRI-1528 “Procedure for Observations of Animals”. The time of death was documented; any animals found dead were documented at the time they were found. Animal disposal

[0075] The first day of the study was the day of exposure to soman and treatment with RYANODEX®. All rats that died were found dead (not sacrificed in a moribund condition). The number of rats specified in the protocol was 8 survivors / group, which was not achieved due to mortality associated with the high dose of soman. Overall survival was 27 of the 45 rats (60%) exposed to soman. See the Table 2. Table 2 Group Regimen Day and Mortality Study Mortality / Total (%) Number of rats that completed the study A 10 mg / kg of RYANODEX® over 50 min 1 (3 rats) (a), 2, 15 5 / 10 (50%) 5 B 30 mg / kg of RYANODEX® over 50 min 1, 2, 3, 8 4 / 9 (44.4%) 5 C 10 mg / kg of RYANODEX® over 20 min 1, 15 2 / 8 (25%) 6 D 30 mg / kg of RYANODEX® over 20 min 1, 6, 11 3 / 9 (335) 6 E Water over 50 min 1(b), 2, 22, 23 4 / 9 (44.4%) 5 F No exposure / no treatment Not applicable 0 / 8 (0%) 8 Petition 870220113428, dated 06 / 12 / 2022, page 45 / 129 32 / 51 (a) A rat from Group A was found dead before receiving midazolam and RYANODEX®. (b) A rat from Group E was found dead before receiving water. Model features Survival: 27 of 45 rats exposed to soman (60%) Time to seizure onset after soman injection: 6.8 minutes ± 1.93 Range: 4 - 11 minutes Time to maximum Racine score(a) after soman injection: 8.4 minutes ± 2.99 Range: 4 - 17 minutes Maximum Racine score reached on day 1: 4.2 minutes ± 0.59 Range: 3 - 5 minutes Clinical observations of seizures during day 28 of life Although rats were not observed continuously, isolated seizures of Racine scale 5 were observed in individual animals throughout the life stage Body weight during day 28 of life stage 15% body weight loss occurred for all groups after soman exposure; recovery to control levels by study day 25, EXCEPT for positive control animals Neurobehavioral tests

[0076] The brain areas damaged by soman exposure included the hippocampus and the entorhinal, frontal, and parietal cortices. These areas contain neural structures and circuits for learning, memory formation, information processing, and other cognitive processes. To assess the potential neuroprotective effects of RYANODEX®, rats were evaluated using a series of established behavioral tests that require learning, memory, and integration. Petition 870220113428, dated 06 / 12 / 2022, page 46 / 129 33 / 51 motor, sensory, and adaptive responses.

[0077] The tests selected were: (1) Sucrose preference test and (2) Forced swim test. Sucrose preference test

[0078] The sucrose preference test (SPT) utilizes the natural inclination of rats to prefer sugar water over plain water. It is an established test to measure pleasure-seeking behavior (hedonia) or lack thereof (anhedonia) and requires animals to adapt to changes in placement—left versus right—of bottles containing tap water and water with 1% sucrose. The SPT was conducted on study days 14 and 15.

[0079] Rats were housed individually with ad libitum access to food and water (a single water bottle in each cage) prior to the SPT. For the acclimation portion of the SPT, 2 water bottles were introduced into each rat's home cage for 5-6 days. The water bottles were fitted with clamp tubes to minimize leakage and were weighed approximately every 24 hours. After the acclimation phase, one water bottle was filled with approximately 200 mL of 1% aqueous sucrose solution and the other water bottle with approximately 200 mL of tap water. The left / right placement of the bottles was then switched and the amount of fluid remaining was again recorded twenty-four hours later. The amount (mL) of sucrose solution consumed was expressed as a percentage of the total volume of fluid consumed (sucrose water plus water) in each of the two 24-hour periods and compared between groups and days.

[0080] The SPT results are shown in Figures 2A Petition 870220113428, dated 06 / 12 / 2022, page 47 / 129 34 / 51 and 2B. On the second day of the test (Day 15), the positive control group (water in 50 minutes) did not maintain a preference for sucrose, unlike groups 3 and 4 which received RYANODEX®. Forced swimming test

[0081] The forced swim test (FST) was developed in the late 1970s by Porsolt as a quick way to track the effectiveness of antidepressant medications in rodents. The increased immobility that occurs at the end of the 5-minute FST in untreated (normal) rodents was interpreted to reflect behavioral despair, and its reversal with antidepressant drugs correlated with the antidepressant efficacy of these agents in humans. However, the validity of this test construct has been questioned for several reasons, including: (1) the acute effects of antidepressants are tested in the FST, whereas in clinically depressed patients, medications require 4–6 weeks for clinical improvement; (2) the dependent variable in the FST is the animal's acute response to the test and not a characteristic of the animal; and (3) the interpretation of fluctuating behavior as behavioral despair is anthropomorphic.It is now believed that the progressive immobility observed in untreated rats reflects an adaptive response to the acute stress of being placed in a container with no possibility of escape.

[0082] In the FST, swimming activity and immobility were measured in a cylindrical chamber (46 cm high x 30 cm in diameter) filled with water (30 cm high, 25°C). Thermometers were used to ensure that the water temperature was constant at 24-26°C for all animals. [The following sentence appears to be incomplete and possibly a fragment from another text: "They were carried out"] Petition 870220113428, dated 06 / 12 / 2022, page 48 / 129 35 / 51 Two swimming sessions were conducted, one as an initial 15-minute pre-test, followed 24 hours later by a second 5-minute test. The test sessions were video recorded. Time spent actively swimming and time spent stationary were scored for each minute of the FST.

[0083] The FST was performed on study days 25 and 26. On study day 25, each rat was placed in the water-filled plexiglass cylinder for 15 minutes. On study day 26, each rat was placed back in the water-filled plexiglass cylinder for the 5-minute test. Time spent moving and immobile was scored for each rat during each minute of the test. The FST results are shown in Figure 3. The groups treated with RYANODEX® showed a similar trend to the negative control group (no treatment), i.e., a progressive increase in time spent immobile during the 5-minute duration of the test. Order of neurobehavioral tests

[0084] The order of the neurobehavioral tests went from least stressful to most stressful. No two tests were performed on the same day. All tests took place in the morning. The tests were performed as follows (after study day 1 as challenge day): • On study day 8, sucrose preference test (lasting up to 8 days) • On study day 25, forced swim test (lasting 2 days).

[0085] The order of the animals tested was randomized and the identities of the groups of rats subjected to both tests were masked, so that the technicians who performed these tests were blind to the treatment group. The scoring of the test results was performed using the Petition 870220113428, dated 06 / 12 / 2022, page 49 / 129 36 / 51 video tapes. Euthanasia and tissue processing and collection

[0086] For each group on study day 29, all surviving rats were anesthetized via an IP injection of target doses < 200 mg / kg of ketamine and < 20 mg / kg of xylazine. The rats were transcardially perfused with heparinized saline followed by 0.4% paraformaldehyde (the perfusion protocol follows Rao et al. (2006)). The heart of each rat was collected and stored in fixative for possible evaluation.

[0087] After tissue collection, the animal carcasses were disposed of in accordance with SOP MRI-1526 “Disposal of Animal Carcasses and Medical Waste in Spencer Animal Care Facility”.

[0088] The brains were kept in paraformaldehyde fixative overnight at 4°C for at least 4 days and then shipped in their individual fixative-containing vials on ice packs overnight to HSRL, Inc., for evaluation. Each brain was uniformly sectioned into 7 sections, as described in Rao, et al. (2014) and Bolon, et al. (2013) to obtain maximum anatomical representation of the entire brain, including areas known to be affected by soman and other organophosphorus neurotoxic agents (e.g., piriform cortex, entorhinal cortex, hippocampus, amygdala, thalamus, cerebellum). The sections were routinely processed, sectioned to 5 microns, embedded in paraffin, and stained with hematoxylin and eosin. Personnel were blinded to dose groups and treatment. Neuropathology Petition 870220113428, dated 06 / 12 / 2022, page 50 / 129 37 / 51

[0089] The 7 brain sections of each rat were evaluated microscopically using a 6-point semi-quantitative scoring system. Microscopic lesions were classified on a 6-point scale: 0 = normal 1 = 1-5 cells affected per 40X microscopic field 2 = 6-20 cells affected per 40X microscopic field 3 = 21-50 cells affected per 40X microscopic field 4 = 50%-80% of cells affected per 40X microscopic field 5 = >80% of cells affected per 40X microscopic field.

[0090] Table 3 established the pathology data of the frontoparietal cortices, showing the proportion of animals by necrosis score per group. Table 3 Necrosis Score 0 1 2 3 4 5 Study Group Normal 1-5% necrosis 6-20% necrosis 21-50% necrosis 51-80% necrosis >80% necrosis A: 10 mg / kg of RYANODEX® 50 min 20 40 20 20 0 0 B: 30 mg / kg of RYANODEX® 50 min 0 60 40 0 ​​0 0 C: 10 mg / kg of RYANODEX® 20 min 34 50 0 17 0 0 D: 30 mg / kg of RYANODEX® 20 min 50 34 17 0 0 0 E: Water 50 min 20 0 0 60 20 0 F: No challenge 100 0 0 0 0 0

[0091] Figure 4 shows mean scores of necrosis of the frontoparietal cortex.

[0092] Figures 5A and 5B show photomicrographs. A Figure 5A shows a photomicrograph of an animal from Group E, the positive control, in water 50 minutes after the start of SE. Petition 870220113428, dated 06 / 12 / 2022, page 51 / 129 Arrows 38 / 51 mark faded and shrunken neurons. This animal received a necrosis score of 4. Figure 5B shows a photomicrograph of an animal from Group D, 30 mg / kg of RYANODEX® 20 minutes after the start of SE. Arrows mark normal neurons. This animal received a necrosis score of 0.

[0093] Table 4 shows the findings of microscopic pathology. Animals in Group F (untreated) did not show significant findings. Group E had expected findings. The reduced extent of necrosis to the frontoparietal cortex in rats treated with RYANODEX®, compared to the positive control (Group E), was consistent with reports in the literature. Table 4 Brain Location Average Pathology Score for Necrosis A 10 mg / kg in 50 min B 30 mg / kg in 50 min C 10 mg / kg in 20 min D 30 mg / kg in 20 min E Water in 50 min Hippocampus, CA3 1.4 (0.89) 1.0 (0.71) 0.8 (0.75) 1.8 (1.72) 0.8 (0.45) Hippocampus, CA2 4.8 (0.45) 4.2 (1.79) 3.5 (1.97) 4.7 (0.82) 5.0 (0.00) Hippocampus, CA1 4.4 (1.34) 4.8 (0.45) 3.7 (2.16) 4.7 (0.82) 5.0 (0.00) Entorhinal Cortex 4.0 (1.73) 3.6 (2.19) 4.0 (1.10) 3.2 (2.48) 3.6 (1.14) Frontoparietal cortex 1.4 (1.14) 1.4 (0.55) 1.0 (1.10) 0.7 (0.82) 2.6 (1.52) Piriform cortex 0.4 (0.89) 0.8 (1.30) 0.8 (0.98) 0.3 (0.82) 0.4 (0.89) Thalamus 3.4 (1.69) 3.0 (0.00) 2.3 (1.03) 3.7 (0.52) 4.2 (0.04) Conclusions

[0094] Overall, animals treated with RYANODEX® performed better on neurobehavioral tests compared to untreated animals. Animals treated with RYANODEX® showed a lower level of cellular necrosis in the cerebral cortex, Petition 870220113428, dated 06 / 12 / 2022, page 52 / 129 39 / 51 compared to untreated animals. Animals treated with RYANODEX® showed a dose- and time-dependent treatment effect impacting cell death. These neurobehavioral tests and pathological findings are consistent and appear to be directly correlated. RYANODEX® was well tolerated in all groups and no new safety signals were observed. Example 2 Study overview

[0095] The aim of the study was to determine whether RYANODEX® has neuroprotective effects in an established GD (soman) survival model in rats. Single doses of 10 or 30 mg / kg of RYANODEX® were administered by IV bolus injection 20 or 60 minutes after the onset of soman-induced seizures. Survival was facilitated by treatment with asoxime chloride (HI-6) thirty minutes after subcutaneous (SQ) injection of soman, atropine methyl nitrate one minute after SQ injection of soman, and atropine sulfate, praldoxime chloride (2-PAM-Cl), and midazolam twenty minutes after the onset of soman-induced seizures that reached a Racine score of at least 3. Controls included two separate groups: an untreated (naive) rat group (negative control) and another group that would receive sterile water or sterile water plus mannitol vehicle, 60 minutes after the onset of soman-induced seizures.

[0096] On day 2, all rats will be sacrificed under anesthesia via exsanguination and intracardiac perfusion. The brain will be collected from each rat for microscopic neuropathological examination, and the heart will be collected from each rat for possible pathological examination. Petition 870220113428, dated 06 / 12 / 2022, page 53 / 129 40 / 51

[0097] The experimental subjects will be male Sprague-Dawley rats cannulated with jugular veins, 8 weeks of age or older (Charles River Laboratories), weighing 300-500 g on the day of exposure. Materials

[0098] Soman (GD) - diluted with sodium chloride to 0.9%. Soman is an organic phosphorus neurotoxic agent that deactivates acetylcholine esterase (AChE) by forming an adduct with the enzyme. • Chemical name: pinacolyl methylphosphonofluoride • Formula: C7H16FO2P • Molecular weight: 182.17 • MRIGlobal Lot#: GD090415-DOC-1 • Primary standard ID: 13972-49-3 • Purity: 100% • Storage conditions: <4°C

[0099] The treatments to promote survival in this model will be: (1) asoxime chloride (HI-6); (2) stropine methyl nitrate; (3) stropine sulfate; ($) praldoxime chloride; (5) midazolam. HI-6 is an AChE reactivator that can displace organophosphates such as somam from acetylcholinesterase (AChE) and thus reactivate the enzyme. Atropine sulfate is a centrally acting, reversible, competitive antagonist of muscarinic acetylcholine receptors; atropine methyl nitrate is a peripherally acting, reversible, competitive antagonist of muscarinic acetylcholine receptors; both are used clinically to combat cholinergic syndrome induced by somam and other neurotoxic organophosphate agents. Midazolam is a benzodiazepine for the treatment of seizures. These agents Petition 870220113428, dated 06 / 12 / 2022, page 54 / 129 41 / 51 are selected because oximes, atropine, and benzodiazepines represent the accepted clinical treatment for acute neurotoxic agent / organophosphate toxicity. A three-drug regimen consisting of praldoxime (an AChE reactivator), atropine, and diazepam (a benzodiazepine) is a currently FDA-approved countermeasure for military personnel.

[0100] HI-6 • Chemical name: [(E)-[1-[(4-carbamoylpyridin-1-io-1-yl)methoxymethyl]pyridin-2-ylidene]methyl]oxoazanium methanesulfonate (asoxime chloride) • Structure: OH NHl • Formula: C14Hi6C12N4O3 • Molecular weight: 359.207

[0101] Atropine methyl nitrate • Chemical name: (8,8-dimethyl-8-azoniabicyclo[3.2.1]octan-3-yl) 3-hydroxy-2-phenyl-propanoate nitrate • Structure: Petition 870220113428, dated 06 / 12 / 2022, page 55 / 129 42 / 51 • Formula: C18H26N2O6 • Molecular weight: 366.414

[0102] Midazolam • Chemical name: 8-chloro-6-(2-fluorophenyl)-1-methyl-4H-imidazo[1,5-a][1,4]benzodiazepine • Structure: • Formula: C18H13ClFN3 • Molecular weight: 325.771

[0103] RYANODEX® (dantrolene sodium) is an approved drug. Petition 870220113428, dated 06 / 12 / 2022, page 56 / 129 FDA authorization 43 / 51 for the treatment of malignant hyperthermia in conjunction with appropriate supportive measures and for the prevention of malignant hyperthermia in high-risk patients.

[0104] Dantrolene sodium Chemical name: 1-[[[5-(4-nitrophenyl)-2-furanyl]methylene]amino]-2,4-imidazolidinedione sodium salt hydrate Structure: Molecular weight: 399

[0105] Excipients: Mannitol (25 mg / mL) polysorbate 80 (5 mg / mL); povidone K12 (0.8 mg / mL) 5% mannitol solution: 5% mannitol solution (in sterile water) Formula: C6H14O6 Animals

[0106] For this study, 120 male Sprague Dawley rats (plus 20 extras / replacements for a total of 140; Charles River Laboratories), aged 8 weeks or older and weighing 300-500 g, with jugular vascular catheters implanted by the supplier, were used. The rats were identified by tail tagging or ear tagging with a unique alphanumeric designation. (See, SOP MRI-1504, “Procedure for Identification of Animals and Cages”).

[0107] All rats included in the study were in good condition. Petition 870220113428, dated 06 / 12 / 2022, p. 57 / 129 44 / 51 healthy rats, free from any signs of clinical disease and possessing appropriate patented dosing catheters at the start of the study. After delivery, the rats were inspected for signs of health problems and quarantined in conventional animal facilities for no less than 48 hours. A veterinarian examined the animals' health and cleared them for the study (see SOP MRI1501, “Acclimation and / or Quarantine Procedures for Animals”). The animals were weighed within three days of delivery. During the period from the day after the animal's arrival until the end of the study, the rats were handled by staff at least twice daily for acclimation and consistency throughout the study.

[0108] All rats were provided with Lab Diet certified rodent food, identified by source, batch number and certificate of analysis, ad libitum. No contaminants were present in the feed that could affect the study results, as verified by the certificate of analysis (for each batch supplied by the supplier; see SOP MRI-1510, “Procedure for Feeding Animals”). Water bottles (one water bottle / cage) were provided with tap water throughout the study.

[0109] The animals were housed in Tecniplast polycarbonate cages with metal hoods (Tecniplast, Phoenixville, PA) throughout the acclimation / quarantine period and shelf life. The animals were housed individually to avoid damage to the catheters. The rats were housed in environmentally controlled rooms, with fur Petition 870220113428, dated 06 / 12 / 2022, pp. 58 / 129 45 / 51 minus 10 air changes per hour. The rooms were maintained at a temperature of 20.0°C (68.0°F) to 26.1°C (79.0°F) and relative humidity of 50% ± 20% with a 12-hour light / dark cycle per day. Ambient temperature, humidity, and the 12 h:12 h reverse light / dark cycles were monitored by the Amega View monitoring system 24 h / day or by a hygrometer during the study (see SOP MRI-1170, “Temperature and Humidity Monitoring in Animal Rooms”). Doses

[0110] HI-6, soman, atropine sulfate, atropine methyl nitrate, 2-PAM Cl, and midazolam: Single doses of HI-6 (IP, 125 mg / kg), soman (SC, 154 μg / kg, 1.4xLD50), atropine methyl nitrate (IM, 2 mg / kg), atropine sulfate (0.45 mg / kg), 2PAM Cl (25 mg / kg), and midazolam (IM, 2 mg / kg) were selected based on prior experience with the model. The inclusion of HI-6, atropine methyl nitrate, and midazolam is necessary to ensure adequate survival. This regimen was expected to induce seizures achieving a Racine score of at least 3 and an acceptable number of survivors for the follow-up study.

[0111] RYANODEX®: In a single-dose tolerability assessment in rats, IV bolus doses of 12, 40, and 60 mg / kg of RYANODEX® were administered to separate groups of rats cannulated into the jugular vein. RYANODEX® doses of 10 and 30 mg / kg in rats are equivalent to human doses of approximately 2 and 6 mg / kg, respectively. Dosage schedule

[0112] A total of 120 plus up to 20 Sprague-Dawley rats Petition 870220113428, dated 06 / 12 / 2022, page 59 / 129 46 / 51 extra males with jugular catheters implanted by the supplier were used in 6 groups, as shown in the Table below. The animals were distributed into platoons of 20. All group tasks were randomly assigned with the stipulation that each dose group be represented by at least one animal in each platoon. Table. Treatment assignment by group Pretreatment Group with HI-6 (IP, 30 min before GD challenge) Soman GD (SQ) Atropine methyl nitrate (IM, 1 min after GD) Midazolam (IM, ~20 minutes after seizure onset) RYANODEX® IV 20 or 60 min after seizure onset A (n=20) 125 mg / kg 154 μg / kg (1.4 x LD50) 2.0 mg / kg 2.0 mg / kg 60 min with 10 mg / kg B (n=20) 125 mg / kg 154 μg / kg (1.4 x LD50) 2.0 mg / kg 2.0 mg / kg 60 min with 30 mg / kg C (n=20) 125 mg / kg 154 μg / kg (1.4 x LD50) 2.0 mg / kg 2.0 mg / kg 20 min with 10 mg / kg D (n=20) 125 mg / kg 154 μg / kg (1.4 x LD50) 2.0 mg / kg 2.0 mg / kg 20 min with 30 mg / kg E (n=20) 125 mg / kg 154 μg / kg (1.4 x LD50) 2.0 mg / kg 2.0 mg / kg Sterile water (control) or sterile water + mannitol (control) in 50 min F (N=20) 0 mg / kg 0 μg / kg 0 mg / kg 0 mg / kg NA Note: For groups C and D, RYANODEX® was administered immediately. Petition 870220113428, dated 06 / 12 / 2022, pp. 60 / 129 47 / 51 after midazolam injection. Dose preparation

[0113] GD was prepared in 0.9% sodium chloride cooled on ice in accordance with SOP MRI-5821 “Preparation of Standards and Samples from Research Development and Testing Evaluation (RDTE) Dilute Solutions”.

[0114] RYANODEX® was prepared by adding 5 mL of sterile water for injection to a small vial of the lyophilized product.

[0115] HI-6 was prepared at a concentration of 250 mg / mL by adding sterile 0.9% saline solution to an injection vial.

[0116] Atropine methyl nitrate was prepared at a concentration of 4 mg / mL by adding sterile water for injection (WFI) to an injection vial.

[0117] Atropine sulfate was prepared at a concentration of 8 mg / mL with sterile brine.

[0118] 2-PAM Cl was prepared at a concentration of 100 mg / mL with sterile brine.

[0119] Atropine sulfate and 2-PAM Cl were mixed in a 1:1 ratio and administered IM at 0.5 mL / kg.

[0120] Midazolam was purchased from a supplier as a 10 mg / mL solution and was administered as is. Experimental design Animal preparation

[0121] Before or on study day 1 (GD exposure day), rats were transferred to the test site. Dosage Petition 870220113428, dated 06 / 12 / 2022, pp. 61 / 129 48 / 51

[0122] On study day 1, all animals were weighed to the nearest gram and all treatment doses were calculated based on those weights. Groups AE received the cholinesterase reactivator, HI-6 (125 mg / kg) via intraperitoneal (IP) injection 30 minutes before the GD challenge. All rats in Groups AE were exposed to GD (154 μg / kg (308 μg / mL) ~1.4 x LD50) by a single subcutaneous injection. Approximately one minute after GD injection for Groups AE, atropine methyl nitrate (AMN) was administered IM (2.0 mg / kg). Approximately 20 minutes after the onset of seizure activity following exposure to GD as defined in the table above, all animals in Groups AE received atropine sulfate (0.45 mg / kg) mixed with 2-PAM Cl (25 mg / kg) intramuscularly and midazolam at an intramuscular dose of 1.8 mg / kg. Group F did not receive HI-6, GD, AMN, midazolam, or RYANODEX®.RYANODEX® was administered via a 100 μL Hamilton microsyringe or Luer locking syringe (1 cm3), depending on the dose volume to be administered, and then the catheter was flushed with 0.2 mL of sterile water for injection to ensure complete delivery to the vein.

[0123] The onset of seizure activity was defined by a score of 3 on the Racine scale as described below: 1 = immobilization and fixed gaze 2 = head bobbing 3 = Forelimb clonus, rhythmic ear twitching accompanied by eye blinking and facial clonus. 4 = Bilateral forelimb clonus. 5 = Bilateral forelimb clonus, elevation and loss of balance Petition 870220113428, dated 06 / 12 / 2022, p. 62 / 129 49 / 51

[0124] If a rat did not achieve this score of 3 after approximately 20 minutes after exposure to GD, it would be replaced in the study.

[0125] As shown in the Table above, rats in Groups A and C received a dose of 10 mg / kg of RYANODEX® IV and rats in Groups B and D received a dose of 30 mg / kg of RYANODEX® IV either 60 or 20 minutes after the onset of seizures (Racine scale of 3). Rats in Group E received a control dose (sterile water or 5% mannitol solution (the amount of mannitol delivered by a 30 mg / kg dose of RYANODEX®) in sterile water for injection) IV in a volume comparable to the maximum dose of RYANODEX® 60 minutes after the onset of seizures.

[0126] Approximately 6 hours after the GD challenge for the AE Groups, the rats received 5 mL of Ringer's lactate solution SQ to aid in recovery. Body weights

[0127] Body weights were recorded for each study animal within the first 72 hours after receipt from the supplier, and again on study days 1 and 2. Any animal that did not gain weight between the first weighing upon receipt and the weighing on study day 1 would be replaced. Clinical observations

[0128] General health observations were conducted daily during the quarantine and acclimation period. The animals were treated twice daily during the quarantine and acclimation period and throughout their lives. On the first day of the study, the rats were observed and weighed before exposure. After exposure to GD, the rats were continuously monitored to document the onset of seizures. After Petition 870220113428, dated 06 / 12 / 2022, page 63 / 129 50 / 51 dosage of midazolam, RYANODEX® and / or control solution, animals were observed at 1, 2, 4 and 6 hours after seizure onset, and at least once on the day of euthanasia. Clinical observations, including arched posture, dehydration, rough coat and inappetence were assessed and recorded. The modified Racine score was recorded at each observation point, if applicable. Any other abnormal clinical observations were also recorded at this time in the study notebook or appropriate data capture form as per SOP MRI-1528 “Procedure for Observations of Animals”. The time of death was documented; any animals found dead were documented at the time they were found. Euthanasia and tissue processing and collection

[0129] For each group on study day 2, all surviving rats were deeply anesthetized by an IP injection of >75 mg / kg of pentobarbital solution. The rats were transcardially perfused with heparinized saline followed by 0.4% paraformaldehyde or 10% neutral buffered formalin (the perfusion protocol follows Rao et al. (2006)). The heart of each rat was collected and stored in fixative for possible evaluation.

[0130] After tissue collection, animal carcasses were disposed of in accordance with, for example, SOP MRI-1526 “Disposal of Animal Carcasses and Medical Waste in Spencer Animal Care Facility”.

[0131] Brains were stored in paraformaldehyde at 4°C, drained the following day and Petition 870220113428, dated 06 / 12 / 2022, pp. 64 / 129 51 / 51 brains were replaced with cold PBS and kept at 4°C overnight. The following day, the PBS was drained and replaced with 10% sucrose solution and stored overnight at 4°C, then transferred to 10% neutral buffered formalin overnight. Alternatively, the brain remained in 10% formalin fixative for at least 4 days at room temperature and then shipped. Each brain was uniformly sectioned into 7 sections, as described in Rao et al. (2014) and Bolon et al. (2013) to obtain maximum anatomical representation of the entire brain, including areas known to be affected by soman and other organophosphorus neurotoxic agents (e.g., piriform cortex, entorhinal cortex, hippocampus, amygdala, thalamus, cerebellum). The sections were routinely processed, sectioned to 5 microns, embedded in paraffin, and stained with hematoxylin and eosin. Personnel were blindfolded to dose groups and treatment. Neuropathology

[0132] The 7 brain sections of each rat were evaluated microscopically using a 6-point semi-quantitative scoring system. Microscopic lesions were classified on a 6-point scale. Neurobehavioral tests

[0133] Optionally, prior to euthanasia, but after exposure to GD on day 1, the rats were subjected to neurobehavioral tests such as those described in Example 1. Petition 870220113428, dated 06 / 12 / 2022, pp. 65 / 129

Claims

1 / 5 CLAIMS 1. Pharmaceutical composition, characterized in that it comprises from 1 mg / kg to 30 mg / kg of dantrolene or a pharmaceutically acceptable salt thereof, an anticonvulsant drug, mannitol, a polysorbate, a povidone, an optional pH adjuster, and water, for use in a method for protecting an individual from neural necrosis after the individual has been exposed to a nerve agent, the method comprising administering said pharmaceutical composition to the human individual 24 hours or less after exposure, the anticonvulsant drug being midazolam.

2. Composition, according to claim 1, characterized in that the frontoparietal cortex, hippocampus and / or thalamus are protected from neural necrosis.

3. Pharmaceutical composition, according to claim 1, characterized in that it comprises from 1 mg / kg to 30 mg / kg of dantrolene or a pharmaceutically acceptable salt thereof, an anticonvulsant drug, mannitol, a polysorbate, a povidone, an optional pH adjuster, and water, for use in a method for treating a human subject exposed to an organophosphate nerve agent, wherein the method comprises administering said pharmaceutical composition to the human subject 24 hours or less after exposure, wherein the anticonvulsant drug is midazolam.

4. Pharmaceutical composition, characterized by comprising from 1 mg / kg to 30 mg / kg of dantrolene or a pharmaceutically acceptable salt thereof, an anticonvulsant drug, mannitol, a polysorbate, a povidone, an optional pH adjuster, and water, for use in a method for protecting an individual from a decrease in the function of the central nervous system resulting from exposure to the nerve agent, the method comprising administering said pharmaceutical composition to the human individual 24 hours or less after exposure, the anticonvulsant drug being midazolam.

5. Pharmaceutical composition, characterized by comprising from 1 mg / kg to 30 mg / kg of dantrolene or a pharmaceutically acceptable salt thereof, an anticonvulsant drug, mannitol, a polysorbate, a povidone, an optional pH adjuster, and water, for use in a method for treating seizures induced by a nerve agent, in particular epileptic conditions, in a human individual, the method comprising administering said pharmaceutical composition to the human individual 24 hours or less after exposure to the nerve agent, the anticonvulsant drug being midazolam.

6. Pharmaceutical composition, characterized by comprising from 1 mg / kg to 30 mg / kg of dantrolene or a pharmaceutically acceptable salt thereof, an anticonvulsant drug, mannitol, a polysorbate, a povidone, an optional pH adjuster, and water, for use in a method for protecting a human subject from central nervous system dysfunction resulting from exposure to an organophosphate nerve agent, the method comprising administering said pharmaceutical composition to the human subject 24 hours or less after exposure, the anticonvulsant drug being midazolam.

7. Pharmaceutical composition, characterized by comprising from 1 mg / kg to 30 mg / kg of dantrolene or a pharmaceutically acceptable salt thereof, an anticonvulsant drug, mannitol, a polysorbate, a povidone, an optional pH adjuster, and water, for use in a method for treating behavioral changes in a human subject resulting from exposure to the organophosphate nerve agent, the method comprising administering said pharmaceutical composition to the human subject 24 hours or less after exposure, the anticonvulsant drug being midazolam.

8. Pharmaceutical composition, for use in accordance with any one of claims 1 to 7, characterized in that the nerve agent is an acetylcholinesterase inhibitor.

9. Pharmaceutical composition, for use in accordance with any one of claims 1 to 8, characterized in that the nerve agent is an organophosphate.

10. Pharmaceutical composition, for use according to any of claims 1 to 9, characterized in that the nerve agent is O-pinacolyl methylphosphonofluoridate (soman), ethyl N,N-dimethylphosphoramidocyanidate (tabun), propano-2-yl methylphosphonofluoridate (sarin), cyclohexyl methylphosphonofluoridate (cyclosarin), or 2-(dimethylamino)ethyl (GV).

11. Pharmaceutical composition, for use according to any of claims 1 to 9, characterized in that the nerve agent is O-cyclopentyl S-(2-diethylamino-ethyl)-methylphosphonothiolate (EA-3148), (S)-(ethyl {[2-(diethylamino)ethyl]sulfanyl}(ethyl) phosphinate) (VE), O,O-diethyl S-[2-(diethylamino)ethyl] phosphorothioate (VG), S-[2-(diethylamino)ethyl] O-ethylmethyl-phosphonothioate (VM), N,N-diethyl-2-(methyl-(2-methyl-propoxy)phosphoryl)sulfanyl-ethanamine (VR), or ({2-[bis(propan-2-yl)amino]ethyl}sulfanyl) Petition 870220113428, dated 06 / 12 / 2022, page. 68 / 129 4 / 5 ethyl (methyl)phosphinate (VX).

12. Pharmaceutical composition, for use in accordance with any of claims 1 to 11, characterized in that the pharmaceutical composition is administered to a human being in one or more doses.

13. Pharmaceutical composition, for use in accordance with any of claims 1 to 12, characterized in that it further comprises providing the human individual with an acetylcholinesterase reactivator, a reverse acetylcholine receptor antagonist, or a combination thereof.

14. Pharmaceutical composition, for use according to claim 13, characterized in that the acetylcholinesterase reactivator is asoxime chloride (HI-6).

15. Pharmaceutical composition, for use according to claim 13, characterized in that the reverse antagonist of acetylcholine receptors is methyl atropine nitrate.

16. Pharmaceutical composition, for use according to claim 13, characterized in that the pharmaceutical composition is administered after the administration of the acetylcholinesterase reactivator.

17. Pharmaceutical composition, for use according to claim 13, characterized in that the pharmaceutical composition is administered after the administration of the acetylcholinesterase reactivator and after the administration of the reverse antagonist of acetylcholine receptors.

18. Pharmaceutical composition, for use according to claim 17, characterized in that the pharmaceutical composition is administered concomitantly or substantially concomitantly with the administration of the anticonvulsant medication.

19. Pharmaceutical composition, for use according to claim 17, characterized in that the pharmaceutical composition is administered after the administration of the anticonvulsant drug.

20. Pharmaceutical composition, for use in accordance with any of claims 1 to 19, characterized in that the pharmaceutical composition is administered intravenously, subcutaneously, intramuscularly, intraosseously or transdermally.

21. Pharmaceutical composition, for use in accordance with any of claims 1 to 20, characterized in that the pharmaceutical composition comprises dantrolene or a pharmaceutically acceptable salt thereof, mannitol, a polysorbate, povidone, an optional pH adjuster, and water.

22. Pharmaceutical composition, for use in accordance with any of claims 1 to 21, characterized in that administration of the pharmaceutical composition comprising dantrolene or a pharmaceutically acceptable salt thereof results in improved behavioral performance when compared with a human subject exposed to the nerve agent who was not administered the pharmaceutical composition comprising dantrolene or a pharmaceutically acceptable salt thereof. Petition 870220113428, dated 06 / 12 / 2022, pp. 70 / 129

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