RU2817851C2 - Methods of using dantrolene for treating nerve agent exposure - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: group of inventions relates to methods of protecting a human subject from neuronal necrosis after exposure of a human subject to an organophosphate nerve agent, protecting a human subject from a decrease in central nervous system function resulting from exposure to an organophosphate nerve agent, to methods of preventing status epilepticus and preventing changes in behavior resulting from exposure to an organophosphate nerve substance, to a method of protecting a human subject from central nervous system dysfunction arising from exposure to an organophosphate nerve agent. Method of protecting a human subject from neuronal necrosis after exposing the human subject to an organophosphate nerve agent comprises administering to the human subject a pharmaceutical composition, containing from 1 mg/kg to 30 mg/kg of dantrolene or its pharmaceutically acceptable salt and 125 mg/kg of azoxime chloride, 2 mg/kg of atropine methylnitrate and 2 mg/kg of midazolam, where the pharmaceutical composition contains dantrolene or a pharmaceutically acceptable salt thereof, mannitol, polysorbate, povidone, an optional pH regulator and water, wherein the pharmaceutical composition is administered to human subject 24 hours or less after the subject is exposed to a nerve agent. Method of protecting a human subject exposed to an organophosphate nerve agent, comprises administering to a human subject a pharmaceutical composition containing from 1 mg/kg to 30 mg/kg of dantrolene or a pharmaceutically acceptable salt thereof and 125 mg/kg of azoxime chloride, 2 mg/kg of atropine methylnitrate and 2 mg/kg of midazolam, where the pharmaceutical composition contains dantrolene or a pharmaceutically acceptable salt thereof, mannitol, polysorbate, povidone, an optional pH regulator and water, wherein the pharmaceutical composition is administered to human subject 24 hours or less after the subject is exposed to a nerve agent. Method of protecting a human subject from a decrease in the function of the central nervous system arising from exposure to an organophosphate nerve substance, comprises administering to a human subject a pharmaceutical composition containing from 1 mg/kg to 30 mg/kg of dantrolene or a pharmaceutically acceptable salt thereof and 125 mg/kg of azoxime chloride, 2 mg/kg of atropine methylnitrate and 2 mg/kg of midazolam, where the pharmaceutical composition contains dantrolene or a pharmaceutically acceptable salt thereof, mannitol, polysorbate, povidone, an optional pH regulator and water, wherein the pharmaceutical composition is administered to human subject 24 hours or less after the subject is exposed to a nerve agent. Method of preventing status epilepticus involves administering to a human subject a pharmaceutical composition, containing from 1 mg/kg to 30 mg/kg of dantrolene or its pharmaceutically acceptable salt and 125 mg/kg of azoxime chloride, 2 mg/kg of atropine methylnitrate and 2 mg/kg of midazolam, where the pharmaceutical composition contains dantrolene or a pharmaceutically acceptable salt thereof, mannitol, polysorbate, povidone, an optional pH regulator and water, wherein the pharmaceutical composition is administered to human subject 24 hours or less after the subject is exposed to a nerve agent. Method of protecting a human subject from central nervous system dysfunction arising from exposure to an organophosphate nerve substance, comprises administering to a human subject a pharmaceutical composition containing from 1 mg/kg to 30 mg/kg of dantrolene or a pharmaceutically acceptable salt thereof and 125 mg/kg of azoxime chloride, 2 mg/kg of atropine methylnitrate and 2 mg/kg of midazolam, where the pharmaceutical composition contains dantrolene or a pharmaceutically acceptable salt thereof, mannitol, polysorbate, povidone, an optional pH regulator and water, wherein the pharmaceutical composition is administered to human subject 24 hours or less after the subject is exposed to a nerve agent. Method of preventing behavior changes arising from exposure to organophosphate nerve substance, comprises administering to a human subject a pharmaceutical composition containing from 1 mg/kg to 30 mg/kg of dantrolene or a pharmaceutically acceptable salt thereof and 125 mg/kg of azoxime chloride, 2 mg/kg of atropine methylnitrate and 2 mg/kg of midazolam, where the pharmaceutical composition contains dantrolene or a pharmaceutically acceptable salt thereof, mannitol, polysorbate, povidone, an optional pH regulator and water, wherein the pharmaceutical composition is administered to human subject 24 hours or less after the subject is exposed to a nerve agent.

EFFECT: group of inventions described above enables effective prevention and protection against the effect of nerve agents.

17 cl, 5 dwg, 5 tbl, 2 ex

Description

Cross references to related applications

This application claims the benefit of U.S. Provisional Application No. 62/554049, filed September 5, 2017, and U.S. Provisional Application No. 62/674406, filed May 21, 2018, which are incorporated herein by reference in their entirety.

Field of technology

The present invention relates to methods of treating subjects exposed to neurotoxic nerve agents with dantrolene (or a metabolite of dantrolene, such as 5-hydroxydantrolene) or a pharmaceutically acceptable salt thereof.

State of the art

Neurotoxic nerve agents, also called nerve agents, are organophosphorus compounds that interfere with the transmission of nerve impulses in the nervous system by blocking acetylcholinesterase. These substances, in gaseous, aerosol or liquid form, are extremely toxic and have a very rapid effect. Nerve agent poisoning causes a variety of symptoms, including unilateral or bilateral miosis, headache, seizures, loss of consciousness, coma, and death. Specifically, nerve agent poisoning causes seizures, which can rapidly progress to status epilepticus (SE), resulting in seizure-related brain damage. Nerve agent-induced seizures usually become refractory to standard antiepileptic therapy.

Medical care to treat nerve agent poisoning will likely be delayed beyond the therapeutic window of opportunity to terminate nerve agent-induced seizures, and seizure-related brain damage will continue throughout the pathological cascade. Thus, there is a need for drug treatments capable of interrupting the pathological cascade to provide neuroprotection during the refractory phase of nerve agent-induced seizures.

Essence

The present invention relates to methods of treating subjects exposed to a neurotoxic nerve agent with a pharmaceutical composition containing an amount of dantrolene (or a metabolite of dantrolene, such as 5-hydroxydantrolene), or a pharmaceutically acceptable salt thereof.

Brief description of the figures

In fig. Figure 1 shows the average body weight of the test group over time.

In fig. Figure 2A shows the results of the sucrose preference test on study day 14.

In fig. Figure 2B shows the results of the sucrose preference test on test day 15.

In fig. Figure 3 shows the results of the forced swim test.

In fig. Figure 4 shows the average rates of necrosis of the frontoparietal cortex.

In fig. Figure 5A shows a photomicrograph from the frontoparietal cortex of an animal in group E (positive control, water 50 minutes after the start of SE). Arrows indicate faded, wrinkled neurons. The necrosis score was 4.

In fig. Figure 5B shows a photomicrograph from the frontoparietal cortex of an animal in group D (30 mg/kg control, 20 minutes after the start of SE). Arrows mark normal neurons. The necrosis score was 0.

Detailed Description of Illustrative Embodiments

The present invention may be more readily understood by reference to the following detailed description taken in connection with the accompanying figures and examples, which form a part of this invention. It should be understood that the present invention is not limited to the specific compositions or methods described and/or shown herein, and that the terminology used herein is intended to describe specific embodiments by way of example only and is not intended to limit the claimed invention. Further, as used in the specification, including the appended claims, forms of the singular include the plural, and reference to a specific numerical value includes at least that specific value, unless the context clearly dictates otherwise. All ranges are included and combinable.

The "about" modifier should be thought of as revealing a range defined by the absolute values of the two endpoints. For example, the expression "from about 2 to about 4" also discloses the range "from 2 to 4." When used to change a single number, the term "about" can refer to plus or minus 10% of the specified number and includes the specified number. For example, "about 10%" could indicate a range from 9% to 11%, and "about 1" means from 0.9 to 1.1.

It should be appreciated that certain features of the invention, which for clarity are described herein in the context of individual embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which for brevity are described in the context of a single embodiment, may also be provided separately or in any subcombination. Additionally, reference to values specified in ranges includes each value within that range.

As used herein, the term “pharmaceutical composition” will mean a composition that is suitable for administration to humans and contains pharmaceutically acceptable excipients, for example, without limitation, stabilizers, bulking agents, buffers, carriers, diluents, solvents, solubilizers and binders. As used herein, the pharmaceutical composition includes, but is not limited to, a liquid form ready for subcutaneous injection or infusion and intramuscular injection.

"Therapeutically effective amount" refers to an amount of an active pharmaceutical agent described herein that is sufficient to inhibit, arrest, or improve a treatment-induced disorder or condition in a particular subject or population of subjects. In some embodiments, in a human or other mammal, a therapeutically effective amount may be determined experimentally in a laboratory or clinical setting, or may be an amount required by government guidelines for the particular disease and subject being treated.

As used herein, the term “alleviation” refers to reducing the severity of a disorder or condition being treated in a particular subject or population of subjects.

As used herein, the term “patient” or “subject” is intended to refer to a mammal. Thus, the methods described herein are applicable to human and non-human subjects. The methods described herein are particularly applicable to humans.

The term “pharmaceutically acceptable” as used herein means that an item that is pharmaceutically acceptable, eg, components, including containers, of a pharmaceutical composition does not cause an unacceptable loss of pharmacological activity or unacceptable side effects. Examples of pharmaceutically acceptable components are provided in The United States Pharmacopeia (USP), The National Formulary (NF), adopted at the United States Pharmacopeial Convention, held in Rockville, Md. in 1990 and FDA Inactive Ingredient Guide 1990, 1996 issued by the U.S. Food and Drug Administration (both incorporated herein by reference, including any drawings). Other brands of solutions or components may also be used that meet necessary restrictions and/or specifications not included in the USP/NF.

The term "pharmaceutically acceptable salt" as used herein means a salt of a compound of the present invention that is pharmaceutically acceptable and that has the desired pharmacological activity of the parent compound. In particular, such salts are non-toxic and may be inorganic or organic acid addition salts and base addition salts. In particular, such salts include: salts formed when an acid proton present in the parent compound is either 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 further include, by way of example only, sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium and the like; and when the compound contains the basic functionality, salts of non-toxic organic or inorganic acids such as hydrochloride, hydrobromide, tartrate, mesylate, acetate, maleate, oxalate and the like.

As used herein, the term “neurotoxic nerve agent” or “nerve agent” refers to compounds that affect the transmission of nerve impulses in the nervous system. Nerve agents are organophosphorus compounds, meaning they have the formula (R) ₃ P(O), where each R group may be the same or different. Type "G" nerve agents include O-pinacolyl methylphosphonofluoridate (soman, GD), ethyl N,N-dimethylphosphoramidocyanidate (tabun, GA), propan-2-yl methylphosphonofluoridate (Sarin, GB), cyclohexyl methylphosphonofluoridate (cyclosarin, GF), and 2-(dimethylamino)ethyl (GV). Type "V" nerve agents include O-cyclopentyl S-(2-diethylaminoethyl) methylphosphonothiolate (EA-3148), (S)-(ethyl{[2-(diethylamino)ethyl]sulfonyl}(ethyl)phosphonates), such as (S)-(ethyl{[2-(diethylamino)ethyl]sulfanyl}(ethyl)phosphinate) (VE), O,O-diethyl S-[2-(diethylamino)ethyl]phosphorothioate (VG), S-[ 2-(diethylamino)ethyl]O-ethylmethylphosphonothioate (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 herein can be used to treat a subject exposed to a single nerve agent. The methods described herein can also be used to treat a subject exposed to two or more nerve agents.

As used herein, the expressions “as a result of exposure to a nerve agent” and “as a result of exposure to a nerve agent” refer to effects that are a direct consequence of exposure to a nerve agent, as well as effects that are a secondary consequence of exposure to a nerve agent. substance, as well as to effects that are an indirect consequence of exposure to a nerve agent.

Among other things, the invention relates to methods of treating a subject exposed to a nerve agent with a pharmaceutical composition containing an amount of dantrolene or a pharmaceutically acceptable salt thereof. The invention also relates to methods of treating a subject exposed to a nerve agent with a pharmaceutical composition containing an amount of a dantrolene metabolite, for example, 5-hydroxydantolerine or a pharmaceutically acceptable salt thereof. For example, in some aspects, the described methods prevent neurological damage secondary to exposure to a nerve agent. In other aspects, the described methods provide neuroprotective effects following exposure to a nerve agent. In other aspects, the described methods alleviate brain tissue damage secondary to exposure to a nerve agent. In other aspects, the described methods alleviate brain tissue damage secondary to status epilepticus secondary to nerve agent exposure. In other aspects, the described methods prevent neuronal necrosis due to exposure to a nerve agent. In other aspects, the described methods alleviate neuronal necrosis due to exposure to a nerve agent. In other aspects, the described methods treat intracellular calcium overload due to exposure to a nerve agent. In other aspects, the described methods alleviate intracellular calcium overload due to exposure to a nerve agent. In other aspects, the described methods prevent intracellular calcium overload due to exposure to a nerve agent.

Subjects described herein may be exposed to the nerve agent through inhalation. In other aspects, subjects are exposed to a nerve agent through transdermal penetration of the agent. In other aspects, subjects are exposed to the nerve agent through consumption of liquid or food that has been contaminated with the nerve agent. In other aspects, subjects are exposed to a nerve agent by subcutaneously, intravenously, or intramuscularly administering the substance to the subject.

In some aspects, the methods are directed to methods of protecting a subject from neuronal necrosis following exposure of the subject to a nerve agent. In these embodiments, a pharmaceutical composition containing an amount of dantrolene (or a metabolite of dantrolene, such as 5-hydroxydantrolene) or a pharmaceutically acceptable salt thereof is administered to a subject after the subject has been exposed to a nerve agent. As used herein, the term “protection” from neuronal necrosis includes reducing the severity of exposure to a nerve agent or attenuating the effects of a nerve agent or reducing neuronal damage caused by exposure to a nerve agent. In some aspects, “protecting” against neuronal necrosis includes preventing neuronal necrosis in a subject exposed to a nerve agent. That is, subjects who are “protected” from neuronal necrosis by administration of the disclosed dantrolene-containing compositions (or compositions containing a dantrolene metabolite such as 5-hydroxydantrolene) perform better on neurobehavioral tests compared with subjects exposed to the nerve agent who are not administered described compositions.

In some embodiments, the integrity of the subject's central nervous system is protected from neuronal necrosis. In some embodiments, the frontoparietal lobe, hippocampus, and/or thalamus are protected from neuronal necrosis. In other aspects, the frontoparietal lobe will be protected from neuronal necrosis. In other aspects, the hippocampus is protected from neuronal necrosis. In other embodiments, the thalamus is protected from neuronal necrosis.

The presence and extent of neuronal necrosis can be determined using methods known in the art, including neurobehavioral tests, radiological studies and pathological evaluation.

The invention also relates to methods of protecting a subject from decline in central nervous system function resulting from exposure to a nerve agent. These methods involve administering to a subject a pharmaceutical composition containing an amount of dantrolene (or a metabolite of dantrolene, such as 5-hydroxydantrolene) or a pharmaceutically acceptable salt thereof, following exposure of the subject to a nerve agent.

The invention also relates to methods of protecting a subject from central nervous system dysfunction resulting from exposure to a nerve agent. These methods involve administering to a subject a pharmaceutical composition containing an amount of dantrolene (or a metabolite of dantrolene, such as 5-hydroxydantrolene) or a pharmaceutically acceptable salt thereof, following exposure of the subject to a nerve agent.

The invention also relates to methods of treating behavioral changes in a subject resulting from exposure to a nerve agent. These methods involve administering to a subject a pharmaceutical composition containing an amount of dantrolene (or a metabolite of dantrolene, such as 5-hydroxydantrolene) or a pharmaceutically acceptable salt thereof, following exposure of the subject to a nerve agent.

As used herein, the term “protection” from reduction of central nervous system function includes reducing the severity of the effects of a nerve agent on the central nervous system or alleviating the effects of a nerve agent on the central nervous system or reducing the effects of a nerve agent on the central nervous system. . Thus, subjects who are "protected" from decline in central nervous system function by administration of the disclosed compositions perform better on neurobehavioral tests compared to nerve agent-exposed subjects who are not administered the disclosed compositions.

The invention also relates to methods of treating nerve agent-induced seizures in a subject exposed to the nerve agent. In some aspects, the treated seizures are status epilepticus (SE). These methods involve administering to a subject a pharmaceutical composition containing an amount of dantrolene (or a metabolite of dantrolene, such as 5-hydroxydantrolene) or a pharmaceutically acceptable salt thereof. As used herein, treatment of nerve agent-induced seizures results in a reduction in the severity or duration of the seizures. In other aspects, treatment results in a reduction in both the severity and duration of the seizure.

The amount of dantrolene or a pharmaceutically acceptable salt thereof that is therapeutically effective for treating a subject in accordance with any of the described methods will be determined by one skilled in the art. A therapeutically effective amount may be the amount required to treat a subject with a single dose. Alternatively, a therapeutically effective amount may be the total amount of dantrolene required to treat a subject with more than one dose, such as multiple doses, over a chronic or long-term course of treatment.

In those embodiments where the subject is a human, the therapeutically effective amount of dantrolene is from 1 mg/kg to 30 mg/kg, administered in one or two doses. In other aspects, a therapeutically effective amount of dantrolene is from 1 mg/kg to about 20 mg/kg. In other aspects, a therapeutically effective amount of dantrolene is from about 5 mg/kg to about 30 mg/kg. In other aspects, a therapeutically effective amount of dantrolene is from about 10 mg/kg to about 30 mg/kg. In other aspects, a therapeutically effective amount of dantrolene is from about 15 mg/kg to about 30 mg/kg. In other aspects, a therapeutically effective amount of dantrolene is from about 20 mg/kg to about 30 mg/kg. In other aspects, a therapeutically effective amount of dantrolene is from about 5 mg/kg to about 20 mg/kg. In other aspects, a therapeutically effective amount of dantrolene is from about 5 mg/kg to about 15 mg/kg. In other aspects, a therapeutically effective amount of dantrolene is from about 5 mg/kg to about 10 mg/kg. In other aspects, a therapeutically effective amount of dantrolene is from about 10 mg/kg to about 20 mg/kg. In other aspects, a therapeutically effective amount of dantrolene is from about 2 mg/kg to about 10 mg/kg, preferably from about 2 mg/kg to about 6 mg/kg. In other aspects, a therapeutically effective amount of dantrolene is from about 15 mg/kg to about 20 mg/kg. In other aspects, a 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 approximately 30 mg/kg. In some embodiments, the therapeutically effective amount of dantrolene for treating a human is greater than 30 mg/kg, such as 30 mg/kg to about 100 mg/kg, administered in one or two doses. In some aspects, a therapeutically effective amount of dantrolene for human treatment is about 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or about 100 mg/kg.

The amount of dantrolene metabolite (such as 5-hydroxydantrolene) or a pharmaceutically acceptable salt thereof that is therapeutically effective for treating a subject in accordance with any of the described methods will be determined by one skilled in the art. In those embodiments where the subject is a human, 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 1 mg/kg to about 20 mg/kg. In other aspects, a therapeutically effective amount of dantrolene metabolite is from about 5 mg/kg to about 30 mg/kg. In other aspects, a therapeutically effective amount of dantrolene metabolite is from about 10 mg/kg to about 30 mg/kg. In other aspects, a therapeutically effective amount of dantrolene metabolite is from about 15 mg/kg to about 30 mg/kg. In other aspects, a therapeutically effective amount of dantrolene metabolite is from about 20 mg/kg to about 30 mg/kg. In other aspects, a therapeutically effective amount of dantrolene metabolite is from about 5 mg/kg to about 20 mg/kg. In other aspects, a therapeutically effective amount of dantrolene metabolite is from about 5 mg/kg to about 15 mg/kg. In other aspects, a therapeutically effective amount of dantrolene metabolite is from about 5 mg/kg to about 10 mg/kg. In other aspects, a therapeutically effective amount of dantrolene metabolite is from about 10 mg/kg to about 20 mg/kg. In other aspects, a therapeutically effective amount of 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 aspects, a therapeutically effective amount of dantrolene metabolite is from about 15 mg/kg to about 20 mg/kg. In other aspects, a therapeutically effective amount of dantrolene metabolite 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 approximately 30 mg/kg. In some embodiments, the therapeutically effective amount of dantrolene metabolite for treating a human is greater than 30 mg/kg, such as 30 mg/kg to about 100 mg/kg, administered in one or two doses. In some aspects, a therapeutically effective amount of dantrolene metabolite for human treatment is about 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or about 100 mg/kg.

In some aspects of the present invention, the timing of administration of a pharmaceutical composition containing dantrolene (or a metabolite of dantrolene, such as 5-hydroxydantrolene) or a pharmaceutically acceptable salt thereof following exposure to a nerve agent may influence the degree of protection against neuronal necrosis provided to the subject.

In some aspects of the present invention, the timing of administration of a pharmaceutical composition containing dantrolene (or a metabolite of dantrolene, such as 5-hydroxydantrolene) or a pharmaceutically acceptable salt thereof following exposure to a nerve agent may influence the amount of decline in central nervous system function experienced by the subject.

In some aspects of the present invention, the timing of administration of a pharmaceutical composition containing dantrolene (or a metabolite of dantrolene, such as 5-hydroxydantrolene) or a pharmaceutically acceptable salt thereof following exposure to a nerve agent may affect the treatment of nerve agent-induced seizures in a subject.

Compositions containing dantrolene (or a metabolite of dantrolene, such as 5-hydroxydantrolene) or a pharmaceutically acceptable salt thereof may be administered to a subject chronically, in two or more doses, i.e., over two or more weeks, for example, 2, 3, 4, 5 6, 7, 8 or more weeks after the subject was exposed to the nerve agent. Compositions containing dantrolene (or a metabolite of dantrolene, such as 5-hydroxydantrolene) or a pharmaceutically acceptable salt thereof may be administered to a patient acutely, in one or more doses, that is, over a period of less than two weeks, for example, over a period of several hours or days, for example, in 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 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 days after the subject was exposed to the nerve agent.

With respect to the dosage interval of a pharmaceutical composition containing dantrolene (or a metabolite of dantrolene, such as 5-hydroxydantrolene) or a pharmaceutically acceptable salt thereof, in some aspects, a pharmaceutical composition containing dantrolene (or a metabolite of dantrolene, such as 5-hydroxydantrolene) or a pharmaceutically acceptable salt thereof salt, at least one dose is administered to the subject 24 hours or less after the subject's exposure to the nerve agent. In some aspects, a pharmaceutical composition comprising dantrolene (or a metabolite of dantrolene, such as 5-hydroxydantrolene) or a pharmaceutically acceptable salt thereof, at least one dose is administered to a subject 20 hours or less after the subject's exposure to the nerve agent. In some aspects, a pharmaceutical composition comprising dantrolene (or a metabolite of dantrolene, such as 5-hydroxydantrolene) or a pharmaceutically acceptable salt thereof, at least one dose is administered to a subject 16 hours or less after the subject's exposure to the nerve agent. In some aspects, a pharmaceutical composition comprising dantrolene (or a metabolite of dantrolene, such as 5-hydroxydantrolene) or a pharmaceutically acceptable salt thereof, at least one dose is administered to a subject 12 hours or less after the subject's exposure to the nerve agent. In some aspects, a pharmaceutical composition comprising dantrolene (or a metabolite of dantrolene, such as 5-hydroxydantrolene) or a pharmaceutically acceptable salt thereof, at least one dose is administered to a subject 8 hours or less after the subject's exposure to the nerve agent. In some aspects, a pharmaceutical composition comprising dantrolene (or a metabolite of dantrolene, such as 5-hydroxydantrolene) or a pharmaceutically acceptable salt thereof, at least one dose is administered to a subject 4 hours or less after the subject's exposure to the nerve agent. In some aspects, a pharmaceutical composition comprising dantrolene (or a metabolite of dantrolene, such as 5-hydroxydantrolene) or a pharmaceutically acceptable salt thereof, at least one dose is administered to a subject 2 hours or less after the subject's exposure to the nerve agent. In some aspects, a pharmaceutical composition comprising dantrolene (or a metabolite of dantrolene, such as 5-hydroxydantrolene) or a pharmaceutically acceptable salt thereof, at least one dose is administered to a subject 1 hour or less after the subject's exposure to the nerve agent. In some aspects, a pharmaceutical composition comprising dantrolene (or a metabolite of dantrolene, such as 5-hydroxydantrolene) or a pharmaceutically acceptable salt thereof, at least one dose is administered to a subject over a period of about 1, 2, 3, 4, 5, 6, 7. 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or for approximately 24 hours after the subject is exposed to the nerve agent.

However, in some aspects, a pharmaceutical composition containing dantrolene (or a metabolite of dantrolene, such as 5-hydroxydantrolene) or a pharmaceutically acceptable salt thereof can deliver a therapeutically effective amount of dantrolene (or a metabolite of dantrolene, such as 5-hydroxydantrolene) to a subject exposed to a neurological -paralytic substance, in one dose. In other aspects, two or more doses of the pharmaceutical composition may be necessary to deliver a therapeutically effective amount of dantrolene (or a metabolite of dantrolene, such as 5-hydroxydantrolene) to a subject exposed to a nerve agent. For example, 2, 3, 4, 5, 6, 7, 8, 9, or 10 doses of the pharmaceutical composition may be needed to deliver a therapeutically effective amount of dantrolene (or a metabolite of dantrolene, such as 5-hydroxydantrolene) to a subject exposed to a nerve agent. . These additional doses can be administered substantially simultaneously with the first dose. In other aspects, additional doses are separated in time from the first dose. In those aspects where 3 or more doses are administered, each dose may be separated in time from the administration of any other dose.

In some aspects of the disclosure, administration of dantrolene (or a metabolite of dantrolene, such as 5-hydroxydantrolene) to a subject exposed to a nerve agent is adjunctive therapy to the nerve agent exposure. Subjects exposed to the nerve agent may also be administered one or more nerve agent antidotes. One class of antidotes for nerve agent exposure are acetylcholinesterase reactivators, such as azoxime chloride (HI-6). Another class of antidotes for nerve agent effects are inverse acetylcholine receptor antagonists, such as atropine, such as atropine methyl nitrate. Subjects exposed to nerve agents may also be prescribed anticonvulsants. Examples of anticonvulsants are aldehydes (eg, paraldehyde), aromatic allylic alcohols (eg, stiripentol), benzodiazepines (eg, clobazam, clonazepam, clorazepate, diazepam, midazolam, lorazepam, nitrazepam, temazepam, nitrazepam, temazepam, nimetazepam), barbiturates (eg, phenobarbital, methylphenobarbital, barbexaclone), bromides (eg, potassium bromide), carbamates (eg, felbamate), carboxamides (eg, carbamazepine, oxcarbazepine, eslicarbazepine acetate), fatty acids (eg, valproic acid, sodium valproate, divalproex sodium, vigabatrin, progabide, tiagabine), topiramate, GABA analogs (eg, gabapentin, pregabalin), hydantoins (eg, ethotoin, phenytoin, mephenytoin, fosphenytoin), oxazolidinediones (eg, paramethadione, trimethadione, etadione), propionates (eg, beclamide), pyrimidinediones (eg e.g. primidone), pyrrolidines (e.g. brivaracetam, levitiracetam, seletracetam), succinimides (e.g. ethoxysimide, phensuximide, mesuximide), sulfonamides (e.g. acetazolamide, sulthiamide, methazolamide, zonisamide), triazines (e.g. lamotrigine), ureas (e.g. , feneturide, phenacemide), valproylamide (for example, valpromide, valnoctamide), perampanel and combinations thereof. In some aspects, the anticonvulsant is a benzodiazepine, such as midazolam. In other aspects, the anticonvulsant is a barbiturate. In still other aspects, the anticonvulsant is a hydantoin. In some aspects, the anticonvulsant is paraldehyde. In other aspects, the anticonvulsant is potassium bromide. In some aspects, the anticonvulsant is a fatty acid. In other aspects, the anticonvulsant is topiramate.

In those aspects where an antidote is administered to a subject exposed to a nerve agent, dantrolene (or a metabolite of dantrolene, such as 5-hydroxydantrolene) is administered after the antidote is administered. For example, dantrolene can be administered after administration of an acetylcholinesterase reactivator and/or after administration of an inverse acetylcholine receptor antagonist.

In those aspects where an anticonvulsant drug is administered to a subject exposed to a nerve agent, dantrolene (or a metabolite of dantrolene, such as 5-hydroxydantrolene) can be administered concomitantly with the administration of the anticonvulsant drug. Dantrolene (or a metabolite of dantrolene, such as 5-hydroxydantrolene) can be administered generally at the time of administration of the anticonvulsant, and also, for example, within about 5 minutes after administration of the anticonvulsant. In other embodiments, dantrolene (or a metabolite of dantrolene, such as 5-hydroxydantrolene) is administered prior to administration of the anticonvulsant. In other embodiments, dantrolene (or a metabolite of dantrolene, such as 5-hydroxydantrolene) is administered after administration of the anticonvulsant.

A pharmaceutical composition containing dantrolene (or a metabolite of dantrolene, such as 5-hydroxydantrolene) or a pharmaceutically acceptable salt thereof can be administered intravenously. In other aspects, a pharmaceutical composition containing dantrolene (or a metabolite of dantrolene, such as 5-hydroxydantrolene) or a pharmaceutically acceptable salt thereof can be administered transdermally. In other aspects, a pharmaceutical composition containing dantrolene (or a metabolite of dantrolene, such as 5-hydroxydantrolene) or a pharmaceutically acceptable salt thereof can be administered intramuscularly. In other aspects, a pharmaceutical composition containing dantrolene (or a metabolite of dantrolene, such as 5-hydroxydantrolene) or a pharmaceutically acceptable salt thereof can be administered intraosseously. In other aspects, a pharmaceutical composition containing dantrolene (or a metabolite of dantrolene, such as 5-hydroxydantrolene) or a pharmaceutically acceptable salt thereof can be administered subcutaneously.

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

A particularly preferred pharmaceutical composition containing dantrolene (or a metabolite of dantrolene, such as 5-hydroxydantrolene) or a pharmaceutically acceptable salt thereof is RYANODEX® (dantrolene sodium, Eagle Pharmaceuticals, Woodcliff Lake, NJ). RYANODEX® is an injectable suspension in the form of a sterile lyophilized powder. It comes in 20 ml vials containing 250 mg dantrolene sodium, 125 mg mannitol, 25 mg polysorbate 80, 4 mg povidone K12 and sufficient sodium hydroxide or hydrochloric acid to adjust the pH value. When reconstituted with 5 ml of Sterile Water for Injection USP, it gives a suspension.

The following examples are provided to illustrate some of the concepts described in the present invention. Although each example is believed to provide specific individual embodiments of the invention, none of the examples should be construed as limiting the more general embodiments described herein. In the following examples, efforts have been made to ensure accuracy with respect to the numerical values used (eg, quantity, temperature, etc.), but some experimental errors and deviations should be taken into account.

Examples

Example 1

Study Overview

The purpose of the study was to determine whether RYANODEX® has neuroprotective effects in a GD (soman) rat survival model. Single doses of 10 or 30 mg/kg RYANODEX® were administered by IV bolus injection 20 or 50 minutes after the onset of soman-induced seizures. Survival was enhanced by treatment with azoxime chloride (HI-6) thirty minutes before subcutaneous (SC) injection of Soman, atropine methyl nitrate one minute after SC injection of Soman, and midazolam twenty minutes after the onset of Soman-induced seizures, which reach a level of Racine scale of at least 3. Controls included one group of untreated (naïve) rats and another group that would receive sterile water 50 minutes after the onset of Soman-induced seizures.

A series of neurobehavioral tests were performed for approximately 28 days after a single exposure to GD. On day 29, all rats were sacrificed under anesthesia by exsanguination and intracardiac perfusion. The brain was collected from each rat for microscopic neuropathological examination, and the heart was collected from each rat for examination of possible pathology.

Experimental subjects were > 8-week-old male Sprague-Dawley rats (Charles River Laboratories) with jugular vein cannulation, weighing 300–500 g per exposure day. The sucrose preference test and neurobehavioral forced swim tests were used to assess potential impairment in tasks involving learning and/or sensorimotor integration.

Materials

Soman (GD) - diluted with 0.9% sodium chloride. Soman is an organophosphate nerve agent that deactivates acetylcholinesterase (AChE) by forming an adduct with the enzyme.

• Chemical name: pinacolyl methylphosphonofluoride

• Formula: C ₇ H ₁₆ FO ₂ P

• Molecular weight: 182.17

• MRIGlobal Lot#: GD090415-DOC-1

• Main standard ID: 13972-49-3

• Purity: 100%

• Storage conditions: <4°C

HI-6: Chemical name: [(E)-[1-[(4-carbamoylpyridin-1-ium-1-yl)methoxymethyl]pyridin-2-ylidene]methyl]oxoazanium;methanesulfonate (azoxime chloride)

Structure:

Formula: C ₁₄ H ₁₆ Cl ₂ N ₄ O ₃

Molecular weight: 359.207

Atropine methyl nitrate: Chemical name: (8,8-dimethyl-8-azoniumbicyclo[3.2.1]octan-3-yl)-3-hydroxy-2-phenylpropanoate;nitrate

Structure:

Formula: C ₁₈ H ₂₆ N ₂ O ₆

Molecular weight: 366.414

Midazolam : Chemical name: 8-chloro-6-(2-fluorophenyl)-1-methyl-4H-imidazo[1,5-a][1,4]benzodiazepine

Structure:

Formula: C ₁₈ H ₁₃ ClFN ₃

Molecular weight: 325.771

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

Animals

For this study, the study used 48 male Sprague-Dawley rats (plus 8 additional/replacements for a total of 56; Charles River Laboratories), > 8 weeks old and weighing 300–500 g, with jugular vein vascular catheters implanted by the supplier. Rats were identified by a tail tag or ear tag with a unique alphanumeric designation. (See SOP MRI-1504, “Procedure for Identification of Animals and Cages”).

All rats included in the study were in good condition, without any signs of clinical disease, and had patent catheters suitable for dosing at the start of the study. Upon delivery, animals were assessed for signs of deteriorating health and quarantined in Spencer's regular animal facility for a minimum of 48 hours. The veterinarian checked the health of the animals and released them for research. (See SOP MRI-1501, “Acclimation and/or Quarantine Procedures for Animals”). Animals were weighed within three days of delivery in accordance with SOP MRI-1500. From the day after animals arrived throughout the study, staff handled rats at least twice daily to ensure acclimatization and consistency throughout the study.

All rats were fed laboratory-certified rodent chow with source, lot number, and certificate of analysis identified, ad libitum. There were no contaminants in the feed that could interfere with the test results, as confirmed by the certificate of analysis (for each lot provided 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 were provided with two water bottles containing tap water or one one water bottle containing tap water and one water bottle containing 1% sucrose solution.

Animals were housed in a Tecniplast polycarbonate cage with a wire lid (Tecniplast, Phoenixville, PA) during the acclimation/quarantine and life periods. Animals were housed separately to prevent damage to the catheters. Rats were kept in climate-controlled rooms with at least 10 air changes per hour. The rooms were maintained at a temperature of 20°C to 26.11°C and a relative humidity of 50%±20% with a 12-hour light/dark cycle per day. Room temperature, humidity, and lighting cycles (12 hours:12 hours, alternating light/dark) were monitored using an AmegaView monitoring system 24 hours a day or using a hygrothermograph during the study (see SOP MRI-1170, “Temperature and Humidity Monitoring in Animal Rooms".

Doses

HI-6, soman, atropine methyl nitrate and midazolam: Single doses of HI-6 (i.p., 125 mg/kg); soman (s.c., 154 mcg/kg, 1.4 x LD₅₀), atropine methyl nitrate (i.m., 2 mg/kg); and midazolam (IM, 2 mg/kg) were selected based on previous experience with the model. This regimen was expected to produce seizures that would produce a Racine score of at least 3 and an acceptable number of survivors for subsequent study.

RYANODEX® : In an assessment of single dose tolerability in rats, intravenous bolus doses of 12, 40 and 60 mg/kg RYANODEX® were given to separate groups of jugular vein cannulated rats. Twelve mg/kg was well tolerated. The 40 mg/kg dose resulted in negligible body weight loss two days post-dose, and the 60 mg/kg dose resulted in experimental animal death approximately twenty-four hours post-dose for all five rats receiving this dose. Single intravenous bolus doses of 10 and 30 mg/kg RYANODEX® were selected for this study based on their expected tolerability. 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

A total of 48 plus up to 8 additional male Sprague-Dawley rats with vendor-implanted jugular venous catheters were used in 6 groups as shown in Table 1.

Table 1. Design for preliminary efficacy study with RYANODEX®

Group Pre-administration of HI-6 (i.p., 30 minutes before GD infection) soman “GD” (s/c) atropine methyl nitrate (IM, 1 min after GD) midazolam (IM, ~20 minutes after the onset of seizure activity) RYANODEX® IV 20 or 50 minutes after the onset of seizures A (n=8) 125 mg/kg 154 µg/kg (1.4 x LD ₅₀ ) 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 LD ₅₀ ) 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 LD ₅₀ ) 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 LD ₅₀ ) 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 LD ₅₀ ) 2.0 mg/kg 2.0 mg/kg Control of sterile water after 50 minutes F (N=8) 0 mg/kg 0 µg/kg 0 mg/kg 0 mg/kg N.A.

Note: For groups C and D, RYANODEX® was given immediately after the midazolam injection.

Dosing started with group D, followed by groups B, A, C, E and F, respectively.

Getting your dose

GD was prepared in ice-cold 0.9% sodium chloride according to SOP MRI-5821 “Preparation of Standards and Samples from Research Development and Testing Evaluation (RDTE) Dilute Solutions.”

RYANODEX® was prepared by adding 5 ml of sterile water for injection to one vial of lyophilized product.

Study plan

Animal preparation

Before or on study day 1 (GD exposure day), rats were transferred to the laboratory breeding facility.

Dosing

On study day 1, all animals in the AE groups received the cholinesterase reactivator, HI-6 (125 mg/kg) by the intraperitoneal (IP) route 30 min before GD challenge. All rats in the AE groups were exposed to GD (154 μg/kg or ~1.4 x _LD50 ) by a single subcutaneous injection. Approximately one minute after injection, GD for AE groups was administered by intramuscular injection of atropine methyl nitrate (AMN) (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 the AE groups received midazolam at a dose of 2 mg/kg intramuscularly. Group F did not receive HI-6, GD, AMN, midazolam, or RYANODEX®. RYANODEX® was administered through a Hamilton microsyringe, then the catheter was flushed with 0.2 ml of sterile water for injection to ensure complete delivery into the vein.

The initiation of seizure activity was determined by a score of “3” on the Racine scale, as described below:

1= immobilization and gaze fixation

2= shaking head, “shaking off wet dog”

3= forelimb clonus

4= bilateral forelimb clonus

5= bilateral forelimb clonus, rearing and loss of balance

If a rat did not reach this score of “3” approximately 20 min after GD exposure, it was replaced in the study.

As shown in Table 1, rats in groups A and C received a dose of 10 mg/kg RYANODEX® IV and rats in groups B and D received a dose of 30 mg/kg RYANODEX® IV 50 or 20 minutes after the onset of seizures (on the Racine scale “3”). Group E rats were administered control (sterile water for injection) intravenously in a volume comparable to the maximum dose of RYANODEX® 50 minutes after the onset of seizures.

Approximately 6 hours after GD challenge for AE groups, and then every morning and evening, rats were administered 5 ml of lactated Ringer's solution SC for the first 7 days after challenge to aid recovery. Once the animals have recovered from the challenge as indicated by observing them eating and drinking freely, moving around the cage freely, and showing no signs of dehydration (no dry skin on the back), SC rehydration therapy is discontinued. This was partly at the discretion of the veterinarian based on his/her assessment of the animals. In addition, after challenge for the same period of time (up to 7 days post-challenge), animals were offered hydrogels (ClearH ₂ O) and gruel (moistened food in a clay dish on the floor of the cage) to help them recover from the effects of GD administration.

Body mass

Body weights were recorded for each study animal within the first 72 hours of receipt from the supplier and then again on study days 1, 3, 5, 10, 17, 24, and 28. Body weight data were collected more frequently if the study director identified that animals continued to lose weight after challenge. If any animal lost more than 10% of its initial body weight, it was weighed daily until the animal gained rather than lost weight. The body weights of all groups are shown in Fig. 1. The body weight of all soman-exposed groups treated with RYANODEX® (groups A, B, C, D) recovered to the values of the negative control (group F) by study day 25.

Clinical observations

Observations of general health were carried out daily during the quarantine and acclimatization period. Animals were examined twice daily throughout the quarantine and acclimatization period, as well as throughout their lives. On study day 1, rats were examined before exposure. After exposure to GD, rats were frequently monitored to assess the initial appearance of seizures. After administration of midazolam, RYANODEX® and/or sterile water, animals were observed at least twice daily and euthanized at least once daily. Clinical observations including hunched posture, dehydration, rough hair coat, and lack of appetite were assessed and recorded. A modified Racine score was recorded at each observation point, if necessary. Any other abnormal clinical observations were also recorded at this time in the study log or on the appropriate data collection form according to SOP MRI-1528 “Procedure for Observation of Animals.” The time of death was documented; any animals found dead were recorded at the time of discovery.

Animal predisposition

Day 1 of the study was the day of soman exposure and RYANODEX® treatment. All rats that died were found dead (not euthanized in the agonal state). The protocol number of rats was 8 survivors/group, which was not achieved due to mortality associated with the high dose of soman. Overall survival was 27 of 45 rats exposed to soman (60%). See Table 2.

table 2

Group Mode Day of death in the study Mortality/Total (%) No. rats who completed the study A 10 mg/kg RYANODEX® after 50 min 1 (3 rats) ^(a) , 2, 15 5/10 (50%) 5 B 30 mg/kg RYANODEX® after 50 min 1, 2, 3, 8 4/9 (44.4%) 5 C 10 mg/kg RYANODEX® after 20 min 1, 15 2/8 (25%) 6 D 30 mg/kg RYANODEX® after 20 min 1, 6, 11 3/9 (33%) 6 E Water after 50 minutes 1 ^(b) , 2, 22, 23 4/9 (44.4%) 5 F No exposure/no treatment not provided 0/8 (0%) 8

(a) One rat from group A was found dead before receiving midazolam and RYANODEX®.

(b) One rat from group E was found dead before receiving water.

Model characteristics

Survival: 27 of 45 rats exposed to soman (60%) Time to onset of seizures 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 obtained on day 1: 4.2 ± 0.59
Range: 3-5 Clinical observations of seizures during the 28-day phase of life: Although the rats were not observed continuously, occasional Racine 5 seizures were observed in individual animals throughout the life phase. Body weight during the 28-day phase of life: A 15% loss of body weight occurred in all groups after exposure to soman; recovery to control levels by study day 25, except for positive control animals.

Neurobehavioral tests

Brain regions damaged by soman included the hippocampus, entorhinal, frontal, and parietal cortices. These areas contain structures and neural circuitry for learning, memory formation, information processing, and other cognitive processes. To evaluate the potential neuroprotective effects of RYANODEX®, rats were assessed using a series of established behavioral tests that require learning, memory, sensory-motor integration and adaptive responses.

The following tests were chosen: 1) sucrose preference test and 2) forced swim test.

Sucrose preference test

The Sucrose Preference Test (SPT) takes advantage of the natural tendency of rats to prefer sugar water to regular water. This is a generally accepted test for measuring pleasure-seeking behavior (hedonia) or lack thereof (anhedonia) and requires animals to adapt to changes in the left and right placement of bottles containing tap water and a 1% aqueous sucrose solution. SPT was performed on study days 14 and 15.

Rats were housed individually with ad libitum access to food and water (one water bottle in each cage) before SPT. For the SPT acclimation phase, 2 water bottles were placed in each rat's home cage for 5–6 days. Water bottles were fitted with sippy cups to minimize leakage and were weighed approximately every 24 hours. After the acclimation phase, one water bottle was filled with approximately 200 ml of a 1% sucrose solution and the other water bottle with approximately 200 ml of tap water. Twenty-four hours later, the amount of liquid remaining in each bottle was recorded. The Left/Right placement of the bottles was then reversed, and the amount of liquid remaining in each bottle was recorded again twenty-four hours later. The amount (mL) of sucrose solution consumed was expressed as a percentage of total fluid intake (sucrose water plus water) for each of the two 24-hour periods and compared across groups and days.

The SPT results are shown in Fig. 2A and 2B. On the second day of the test (day 15), the positive control group (water after 50 minutes) did not maintain a preference for sucrose, unlike 3 of the 4 groups receiving RYANODEX®.

Forced swim test

The forced swim test (FST) was developed in the late 1970s by Porsolt as a rapid way to screen for the effectiveness of antidepressants in rodents. Increased immobility occurring toward the end of the 5-min FST in untreated (“normal”) rodents has been interpreted as reflecting “behavioral despair,” and its change with antidepressant drugs has been correlated with the antidepressant efficacy of those drugs in humans. However, the construct validity of this test has been questioned for many reasons, including: 1) the acute effects of antidepressants are tested in the FST, whereas in patients with clinical depression the drugs take 4–6 weeks to show clinical improvement; 2) the dependent variable in the FST is the animal's acute response to the test, not a characteristic of the animal; and 3) the interpretation of floating behavior as “behavioral despair” is anthropometric. It is now believed that the progressive immobility observed in naïve rats reflects an adaptive response to the acute stress of being confined to a container with no escape.

In the FST, swimming activity and immobility were measured in a glass cylindrical chamber (46 cm H x 30 cm D) filled with water (height 30 cm, 25°C). Thermometers were used to ensure that the water temperature was constant at 24–26°C for all animals. Two swimming sessions were conducted, one as an initial 15 min “pre-test”, followed 24 hours later by a second 5-min “test”. Testing sessions were recorded on video. Time spent actively swimming and time spent immobile were assessed for each minute of FST.

FST was performed on study days 25 and 26. On study day 25, each rat was placed in a water-filled Plexiglas cylinder for 15 min. On study day 26, each rat was placed back into the water-filled Plexiglas cylinder for a 5-min test. Time spent moving and immobile was assessed for each rat during each minute of testing. The FST results are depicted in Figure 3 . The RYANODEX® treated groups showed the same trend as the negative control (no treatment) group, i.e. a progressive increase in the time spent without moving during the 5-minute test duration.

Procedure for conducting neurobehavioral tests

The order of neurobehavioral tests was from least stressful to most stressful. The two tests were not performed on the same day. All tests were performed in the morning. Tests were performed as follows (after study day 1 as challenge day):

- On study day 8, sucrose preference test (lasts up to 8 days)

- On study day 25, forced swimming test (lasts 2 days)

The order of animals tested was randomized, and the group identity of rats undergoing both tests was masked so that the examiners performing these tests were blind to treatment group. Test results were assessed using video recordings.

Euthanasia and tissue collection and processing

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

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

The brains were left in paraformaldehyde fixative overnight at 4°C for at least 4 days, and then shipped in their individual vials containing fixative on ice packs overnight to HSRL, Inc. for rate. Each brain was evenly divided 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 nerve agents (eg, piriform cortex, entorhinal cortex, hippocampus, amygdala , thalamus, cerebellum). Sections were routinely processed, sectioned at 5 μm, embedded in paraffin, and stained with hematoxylin and eosin. Staff worked with anonymized data in relation to dose groups and treatments.

Neuropathology

7 brain sections from each rat were scored microscopically using a semi-quantitative 6-point scoring system. Microscopic lesions were assessed on a 6-point scale:

0=ok

1=1-5 affected cells per 40X microscope field of view

2=6-20 affected cells per 40X microscope field of view

3=21-50 affected cells per 40X microscope field of view

4=50% - 80% of affected cells in 40X microscope field of view

5 = >80% affected cells in 40X microscope field of view

Table 3 shows data on the pathology of the frontoparietal cortex, indicating the proportion of animals per group with necrosis.

Table 3

Necrosis assessment 0 1 2 3 4 5 Study group Fine 1-5% necrosis 6-20% necrosis 21-50% necrosis 51-80% necrosis >80% necrosis A: 10 mg/kg RYANODEX® 50 min 20 40 20 20 0 0 B: 30 mg/kg RYANODEX® 50 min 0 60 40 0 0 0 C: 10 mg/kg RYANODEX® 20 min 34 50 0 17 0 0 D: 30 mg/kg RYANODEX® 20 min 50 34 17 0 0 0 E: Water 50 min 20 0 0 60 20 0 F: no infection 100 0 0 0 0 0

The mean necrosis scores of the frontoparietal cortex are shown in Fig. 4.

Microphotographs are shown in Fig. 5A and 5B. Figure 5A shows a micrograph of an animal from group E, positive control, water, 50 minutes after the start of SE. Arrows indicate discolored, wrinkled neurons. This animal received a necrosis score of 4. In FIG. 5B is a photomicrograph of an animal from group D, 30 mg/kg RYANODEX® 20 minutes after the start of SE. Arrows indicate normal neurons. This animal received a necrosis score of 0.

Table 4 shows the microscopic pathology results. Animals in group F (no treatment) had no significant results. Group E (positive control) awaited the results. The decrease in the volume of necrosis of the frontoparietal cortex in rats treated with RYANODEX® compared to the positive control (group E) is consistent with the literature data.

Table 4

Brain area Mean (SD) Pathology Score for Necrosis A
10 mg/kg after 50 min B
30 mg/kg after 50 min C
10 mg/kg after 20 min D
30 mg/kg after 20 min E
Water after 50 minutes 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

Overall, animals treated with RYANODEX® performed better in neurobehavioral tests compared to untreated animals. Animals treated with RYANODEX® showed lower levels of cell necrosis in the cerebral cortex compared to untreated animals. Animals treated with RYANODEX® showed a dose- and time-dependent treatment effect on cell death. These neurobehavioral tests and neuropathology findings are compatible and appear to be directly related. RYANODEX® was well tolerated in all groups and no new safety signals were observed.

Example 2

Study Overview

The purpose of the study was to determine whether RYANODEX® has neuroprotective effects in a GD (soman) rat survival model. Single doses of 10 or 30 mg/kg RYANODEX® are administered by IV bolus injection 20 or 60 minutes after the onset of soman-induced seizures. Survival will be enhanced by treatment with azoxime chloride (HI-6) thirty minutes before subcutaneous (SC) injection of soman, atropine methyl nitrate one minute after SC injection of soman, and atropine sulfate, praldoxime chloride (2-PAM-Cl) and midazolam twenty minutes after the onset of Soman-induced seizures that reach a Racine scale level of at least 3. Controls include two separate groups: one group of untreated (naive) rats (negative control), and another group that receives or sterile water or sterile water plus mannitol vehicle, 60 minutes after the onset of Soman-induced seizures.

On day 2, all rats were sacrificed under anesthesia by exsanguination and intracardiac perfusion. The brain is collected from each rat for microscopic neuropathological examination, and the heart is collected from each rat for examination of possible pathology.

Experimental subjects were > 8-week-old male Sprague-Dawley rats (Charles River Laboratories) with jugular vein cannulation, weighing 300-500 g per day of exposure.

Materials

Soman (GD) - diluted with 0.9% sodium chloride. Soman is an organophosphate nerve agent that deactivates acetylcholinesterase (AChE) by forming an adduct with the enzyme.

• Chemical name: pinacolyl methylphosphonofluoride

• Formula: C ₇ H ₁₆ FO ₂ P

• Molecular weight: 182.17

• MRIGlobal Lot#: GD090415-DOC-1

• Main standard ID: 13972-49-3

• Purity: 100%

• Storage conditions: <4°C

Therapies promoting survival in this model are: 1) azoxime chloride (HI-6); 2) stropine methyl nitrate; 3) sling sulfate; 4) praldoxime chloride; 5) midazolam. HI-6 is an AChE reactivator that can displace organophosphates such as soman from acetylcholinesterase (AChE) and thereby reactivate the enzyme. Atropine sulfate is a centrally acting competitive and reversible antagonist of muscarinic acetylcholine receptors; atropine methylnitrate is a peripherally acting competitive and reversible antagonist of muscarinic acetylcholine receptors; both are used clinically to counteract the acute cholinergic syndrome caused by soman and other organophosphate nerve agents. Midazolam is a benzodiazepine for the treatment of seizures. These agents were chosen because oximes, atropine, and benzodiazepines are common clinical treatments for acute nerve agent/organophosphate toxicity. A three-drug regimen consisting of pralidoxime (an AChE reactivator), atropine, and diazepam (a benzodiazepine) is currently an FDA-approved countermeasure for military personnel.

HI-6: Chemical name: [(E)-[1-[(4-carbamoylpyridin-1-ium-1-yl)methoxymethyl]pyridin-2-ylidene]methyl]oxoazanium;methanesulfonate (azoxime chloride)

Structure:

Formula: C ₁₄ H ₁₆ Cl ₂ N ₄ O ₃

Molecular weight: 359.207

Atropine methyl nitrate : Chemical name: (8,8-dimethyl-8-azoniumbicyclo[3.2.1]octan-3-yl)-3-hydroxy-2-phenylpropanoate;nitrate

Structure:

Formula: C ₁₈ H ₂₆ N ₂ O ₆

Molecular weight: 366.414

Midazolam : Chemical name: 8-chloro-6-(2-fluorophenyl)-1-methyl-4H-imidazo[1,5-a][1,4]benzodiazepine

Structure:

Formula: C ₁₈ H ₁₃ ClFN ₃

Molecular weight: 325.771

RYANODEX® (dantrolene sodium) is an FDA-approved drug for the treatment of malignant hyperthermia in combination with appropriate supportive measures and for the prevention of malignant hyperthermia in high-risk patients.

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

Structure:

Molecular weight: 399

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: C ₆ H ₁₄ O ₆

Animals

For this study, the study is using 120 male Sprague-Dawley rats (plus 20 additional/replacements for a total of 140; Charles River Laboratories), > 8 weeks old and weighing 300–500 g, with jugular vein vascular catheters implanted by the supplier. Rats are identified by a tail tag or ear tag with a unique alphanumeric designation. (See, for example, SOP MRI-1504, “Procedure for Identification of Animals and Cages”).

All rats included in the study were in good condition, without any signs of clinical disease, and had patent catheters suitable for dosing at the start of the study. Upon delivery, animals are assessed for signs of deteriorating health and quarantined in a regular animal facility for at least 48 hours. The veterinarian checks the health of the animals and releases them for research (see, for example, SOP MRI-1501, “Acclimation and/or Quarantine Procedures for Animals”). Animals are weighed within three days of delivery. From the day following animals' arrival throughout the study, staff handled rats at least twice daily for acclimatization and consistency throughout the study.

All rats are provided with certified rodent laboratory chow, indicating the source, batch number and certificate of analysis, ad libitum. There are no contaminants present in the feed that could interfere with the test results, as evidenced by the certificate of analysis (for each batch provided by the supplier; see, for example, SOP MRI-1510, “Procedure for Feeding Animals”). Water bottles (one water bottle/cage) are provided with tap water throughout the study.

Animals are housed in a Tecniplast polycarbonate cage with a wire lid (Tecniplast, Phoenixville, PA) during the acclimation/quarantine period and life. Animals are housed separately to prevent damage to catheters. Rats are kept in climate-controlled rooms with at least 10 air changes per hour. The rooms are maintained at a temperature of 20°C to 26.11°C and a relative humidity of 50%±20% with a 12-hour light/dark cycle per day. Room temperature, humidity, and lighting cycles (12 hours:12 hours, alternating light/dark) are monitored using the AmegaView monitoring system 24 hours a day or using a hygrothermograph during the study (see, for example, SOP MRI-1170, “Temperature and Humidity Monitoring in Animal Rooms".

Doses

HI-6, soman, atropine sulfate, atropine methyl nitrate, 2-PAM Cl and midazolam : Single doses of HI-6 (IP, 125 mg/kg); soman (s.c., 154 mcg/kg, 1.4 x LD ₅₀ ), atropine methyl nitrate (i.m., 2 mg/kg); atropine sulfate (0.45 mg/kg), 2-PAM Cl (25 mg/kg), and midazolam (IM, 1.8 mg/kg) were selected based on previous experience with the model. Inclusion of HI-6, atropine methyl nitrate, and midazolam is necessary to ensure adequate survival. This regimen is expected to produce seizures with a Racine score of at least 3 and an acceptable number of survivors for subsequent study.

RYANODEX® : In assessing single dose tolerability in rats, intravenous bolus doses of 12, 40 and 60 mg/kg RYANODEX® are given to separate groups of jugular vein cannulated rats. 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

A total of 120 plus up to 20 additional male Sprague-Dawley rats with vendor-implanted jugular venous catheters were used in 6 groups, as shown in the table below. The number of animals is 20 in groups. All group assignments will be randomized with the requirement that each dose group will be represented by at least one animal in each animal group.

Table. Assignment of treatment type to groups

Group Pre-administration of HI-6 (i.p., 30 minutes before GD infection) soman “GD” (s/c) atropine methyl nitrate (IM, 1 min after GD) midazolam (IM, ~20 minutes after the onset of seizure activity) RYANODEX® IV 20 or 60 minutes after the onset of seizures A (n=20) 125 mg/kg 154 µg/kg (1.4 x LD ₅₀ ) 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 LD ₅₀ ) 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 LD ₅₀ ) 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 LD ₅₀ ) 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 LD ₅₀ ) 2.0 mg/kg 2.0 mg/kg Sterile water (control) or sterile water + mannitol (control) after 50 minutes F (N=20) 0 mg/kg 0 µg/kg 0 mg/kg 0 mg/kg N.A.

Note: For groups C and D, RYANODEX® was given immediately after the midazolam injection.

Getting your dose

GD was prepared in ice-cold 0.9% sodium chloride according to, for example, SOP MRI-5821 “Preparation of Standards and Samples from Research Development and Testing Evaluation (RDTE) Dilute Solutions.”

RYANODEX® is prepared by adding 5 ml of sterile water for injection (without bacteriostatic agent) to one vial of lyophilized product.

HI-6 is prepared to a concentration of 250 mg/ml by adding sterile 0.9% saline solution to a syringe ampoule.

Atropine methyl nitrate is prepared to a concentration of 4 mg/ml by adding sterile water for injection (WFI) to the syringe ampoule.

Atropine sulfate is prepared to a concentration of 0.8 mg/ml with sterile saline.

2-PAM Cl is prepared to a concentration of 100 mg/ml with sterile saline.

Atropine sulfate and 2-PAM Cl are then mixed 1:1 and administered intramuscularly at a dose of 0.5 ml/kg.

Midazolam is purchased from the supplier as a 10 mg/mL solution and administered as is.

Study plan

Animal preparation

Before or on study day 1 (GD exposure day), rats were transferred to the study site.

Dosing

On study day 1, all animals were weighed to the nearest gram, and all treatment doses were calculated based on these weights. AE groups received the cholinesterase reactivator, HI-6 (125 mg/kg) by intraperitoneal (IP) route 30 min before GD challenge. All rats in the AE groups were exposed to GD (154 μg/kg (308 μg/ml) or ~1.4 x _LD50 ) by a single subcutaneous injection. Approximately one minute after injection, GD for AE groups was administered by intramuscular injection of atropine methyl nitrate (AMN) (2.0 mg/kg). Approximately 20 minutes after the onset of seizure activity following GD exposure, as defined in the table above, all animals in the AE groups received atropine sulfate (0.45 mg/kg) mixed with 2-PAM-Cl (25 mg/kg) intramuscularly, and received midazolam at a dose of 1.8 mg/kg intramuscularly. Group F did not receive HI-6, GD, AMN, midazolam, or RYANODEX®. RYANODEX® was administered using a 100 µl Hamilton microsyringe or a ^1cc luer lock syringe depending on the volume of dose to be administered, then the catheter was flushed with at least 0.2 ml of sterile water for injection to ensure complete delivery into a vein.

The initiation of seizure activity was determined by a score of “3” on the Racine scale, as described below:

1= immobilization and gaze fixation

2= shaking head, “shaking off wet dog”

3= forelimb clonus, rhythmic ear pounding accompanied by eye blinking and facial clonus

4= bilateral forelimb clonus

5= bilateral forelimb clonus, rearing and loss of balance

If a rat did not reach this “3” score approximately 20 min after GD exposure, it was replaced in the study.

As shown in the table above, rats in groups A and C received a dose of 10 mg/kg RYANODEX® IV and rats in groups B and D received a dose of 30 mg/kg RYANODEX® IV 60 or 20 minutes after the onset of seizures. seizures (according to the Racine scale “3”). Group E rats are administered a control (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 .

Approximately 6 hours after the GD challenge for groups A-E, rats were administered 5 ml of lactated Ringer's solution to aid recovery.

Body mass

Body weights were recorded for each study animal within the first 72 hours of receipt from the supplier and then again on study days 1 and 2. Any animal that did not gain weight between the first receipt weighing and the study day 1 weighing was replaced.

Clinical observations

Observations of general health are carried out daily during the quarantine and acclimatization period. Animals are examined twice a day during the entire period of quarantine and acclimatization, as well as throughout their lives. On study day 1, rats were examined and weighed prior to exposure. After GD exposure, rats were continuously monitored to document the initial onset of seizures. After administration of midazolam, RYANODEX® and/or control solution, animals are examined at 1, 2, 4 and 6 hours after the onset of seizures and at least once on the day of euthanasia. Clinical observations including hunched posture, dehydration, rough coat, and lack of appetite are assessed and recorded. The Racine scale score is recorded at each observation point, if necessary. Any other abnormal clinical observations were also recorded at this time in the study log or on an appropriate data collection form according to, for example, SOP MRI-1528 “Procedure for Observation of Animals.” The time of death was documented; any animals found dead were recorded at the time of discovery.

Euthanasia and tissue collection and processing

For each group, on study day 2, all surviving rats were placed under general anesthesia with an intraperitoneal injection of >75 mg/kg pentobarbital solution. Rats are perfused transcardially with heparinized saline followed by 0.4% paraformaldehyde or 10% neutral buffered formalin (perfusion protocol follows Rao, et al (2006). The heart of each rat is collected and stored in fixative for possible evaluation.

After tissue collection, animal remains are disposed of in accordance with, for example, SOP MRI-1526 “Disposal of Animal Carcasses and Medical Waste in the Spencer Animal Care Facility.”

The brain is stored overnight at 4°C in paraformaldehyde, the next day it is evacuated and replaced with cold PBS and kept at 4°C overnight. The next day, the PBS was discarded and replaced with 10% sucrose solution and stored at 4°C overnight and then transferred to 10% neutral buffered formaldehyde overnight. Alternatively, the brain is left in 10% formalin fixative for at least 4 days at room temperature and then removed. Each brain is evenly divided into 7 slices as described in Rao, et al (2014) and Bolon, et al (2013), to obtain maximum anatomical representation of the entire brain, including regions known to be affected by soman and other organophosphorus nerve agents (eg, piriform cortex, entorhinal cortex, hippocampus, amygdala , thalamus, cerebellum). Sections are typically processed, sectioned at 5 μm, embedded in paraffin, and stained with hematoxylin and eosin. Staff work with anonymized data regarding dose groups and treatments.

Neuropathology

7 brain sections from each rat are scored microscopically using a semi-quantitative 6-point system. Microscopic lesions are assessed on a 6-point scale.

Neurobehavioral tests

Optionally before euthanasia, but after day 1 of GD exposure, rats are subjected to neurobehavioral tests such as those described in Example 1.

Claims (29)

1. A method of protecting a human subject from neuronal necrosis after exposure of the human subject to an organophosphate nerve agent, comprising administering to the human subject a pharmaceutical composition containing from 1 mg/kg to 30 mg/kg of dantrolene or a pharmaceutically acceptable salt thereof and 125 mg /kg azoxime chloride, 2 mg/kg atropine methyl nitrate and 2 mg/kg midazolam; wherein the pharmaceutical composition contains dantrolene or a pharmaceutically acceptable salt thereof, mannitol, polysorbate, povidone, an optional pH adjuster and water; wherein the pharmaceutical composition is administered to a human subject 24 hours or less after the subject's exposure to the nerve agent. 2. The method according to claim 1, where the frontoparietal cortex, hippocampus and/or thalamus are protected from neuronal necrosis. 3. A method of protecting a human subject exposed to an organophosphate nerve agent, comprising administering to the human subject a pharmaceutical composition containing from 1 mg/kg to 30 mg/kg dantrolene or a pharmaceutically acceptable salt thereof and 125 mg/kg azoxime chloride, 2 mg/kg atropine methyl nitrate and 2 mg/kg midazolam; wherein the pharmaceutical composition contains dantrolene or a pharmaceutically acceptable salt thereof, mannitol, polysorbate, povidone, an optional pH adjuster and water; wherein the pharmaceutical composition is administered to a human subject 24 hours or less after the subject's exposure to the nerve agent. 4. A method of protecting a human subject from a decrease in the function of the central nervous system resulting from exposure to an organophosphate nerve agent, comprising administering to the human subject a pharmaceutical composition containing from 1 mg/kg to 30 mg/kg of dantrolene or a pharmaceutically acceptable salt thereof and 125 mg/kg azoxime chloride, 2 mg/kg atropine methyl nitrate and 2 mg/kg midazolam; wherein the pharmaceutical composition contains dantrolene or a pharmaceutically acceptable salt thereof, mannitol, polysorbate, povidone, an optional pH adjuster and water; wherein the pharmaceutical composition is administered to a human subject 24 hours or less after the subject's exposure to the nerve agent. 5. A method for preventing status epilepticus, comprising administering to a human subject a pharmaceutical composition containing from 1 mg/kg to 30 mg/kg dantrolene or a pharmaceutically acceptable salt thereof and 125 mg/kg azoxime chloride, 2 mg/kg atropine methyl nitrate and 2 mg/kg kg midazolam; wherein the pharmaceutical composition contains dantrolene or a pharmaceutically acceptable salt thereof, mannitol, polysorbate, povidone, an optional pH adjuster and water; wherein the pharmaceutical composition is administered to a human subject 24 hours or less after the subject's exposure to the nerve agent. 6. A method of protecting a human subject from central nervous system dysfunction resulting from exposure to an organophosphate nerve agent, comprising administering to the human subject a pharmaceutical composition containing from 1 mg/kg to 30 mg/kg of dantrolene or a pharmaceutically acceptable salt thereof and 125 mg /kg azoxime chloride, 2 mg/kg atropine methyl nitrate and 2 mg/kg midazolam; wherein the pharmaceutical composition contains dantrolene or a pharmaceutically acceptable salt thereof, mannitol, polysorbate, povidone, an optional pH adjuster and water; wherein the pharmaceutical composition is administered to a human subject 24 hours or less after the subject's exposure to the nerve agent. 7. A method of preventing behavioral changes resulting from exposure to an organophosphate nerve agent, comprising administering to a human subject a pharmaceutical composition containing from 1 mg/kg to 30 mg/kg dantrolene or a pharmaceutically acceptable salt thereof and 125 mg/kg azoxime chloride, 2 mg/kg atropine methyl nitrate and 2 mg/kg midazolam; wherein the pharmaceutical composition contains dantrolene or a pharmaceutically acceptable salt thereof, mannitol, polysorbate, povidone, an optional pH adjuster and water; wherein the pharmaceutical composition is administered to a human subject 24 hours or less after the subject's exposure to the nerve agent. 8. The method of any one of the preceding claims, wherein the nerve agent is O-pinacolyl methylphosphonofluoridate (soman), ethyl N,N-dimethylphosphoramidocyanidate (tabun), propan-2-yl methylphosphonofluoridate (sarin), cyclohexyl methylphosphonofluoridate (cyclosarin) or 2 -(dimethylamino)ethyl (GV). 9. Method according to any one of paragraphs. 1-8, where the nerve agent is O-cyclopentyl S-(2-diethylaminoethyl) 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-ethylmethylphosphonothioate (VM), N,N-diethyl-2-( methyl (2-methylpropoxy)phosphoryl)sulfanylethanamine (VR) or ethyl ({2-[bis(propan-2-yl)amino]ethyl}sulfanyl)(methyl)phosphinate (VX). 10. The method of any one of the preceding claims, wherein the pharmaceutical composition is administered to a human subject in one or more doses. 11. The method according to any of the preceding claims, wherein the pharmaceutical composition is administered after administration of the acetylcholinesterase reactivator. 12. The method according to any of the preceding claims, wherein the pharmaceutical composition is administered after administration of an acetylcholinesterase reactivator and after administration of an inverse acetylcholine receptor antagonist. 13. The method of claim 12, wherein the pharmaceutical composition is administered simultaneously or substantially simultaneously with the administration of the anticonvulsant. 14. The method according to claim 12, where the pharmaceutical composition is administered after administration of the anticonvulsant. 15. The method according to any of the preceding claims, wherein the pharmaceutical composition is administered intravenously, subcutaneously, intramuscularly, intraosseously or transdermally. 16. The method according to any of the preceding claims, wherein the pharmaceutical composition contains dantrolene or a pharmaceutically acceptable salt thereof, mannitol, polysorbate, povidone, an optional pH adjuster and water. 17. The method of any one of the preceding claims, wherein administration of a pharmaceutical composition containing dantrolene or a pharmaceutically acceptable salt thereof results in improved neurobehavioral performance compared to a human subject exposed to a nerve agent who has not been administered a pharmaceutical composition containing dantrolene or a pharmaceutically acceptable salt thereof.

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