Methods for Treating Botulinum Toxin Poisoning

Abstract

The present disclosure relates to a method of treating botulism comprising administering to a subject in need thereof an effective amount of 3,4-diaminopyridine or a pharma- ceutically acceptable salt thereof by continuous infusion, by single bolus injection, or orally. The present invention provides, for example, a method of treating botulism in a subject in need thereof comprising administering intravenously to the subject an effective amount of 3,4-diaminopyridine or an equivalent amount of a pharma- ceutically acceptable salt thereof in a pharma- ceutically acceptable vehicle by continuous infusion.

Description

[Background technology]

[0001] (background) Botulinum neurotoxins (BoNTs) are highly potent poisons produced by anaerobic bacteria of the genus Clostridium. The active neurotoxin is a heterodimer between a 100 kDa heavy chain (HC) and a 50 kDa light chain (LC). The HC mediates selective binding to endosomal receptors on the presynaptic membrane of peripheral neurons. After neuronal uptake by synaptic endocytosis, the LC migrates to the nerve terminal where it specifically cleaves neuronal soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins, which are essential for neurotransmitter release. Cleavage of SNARE proteins blocks the assembly of dense-core vesicle fusion complexes, preventing vesicle fusion and acetylcholine release. Increasing concentrations of cleaved SNARE proteins render the motor nerve terminal unable to reliably elicit muscle contractions, causing muscle weakness that progresses to flaccid paralysis.

[0002] Clinical symptoms of botulism typically appear 12 to 36 hours after exposure to BoNT and are caused by peripheral blockade of neurotransmission at the neuromuscular junction and autonomic nerve endings. At lethal doses, neuroparalytic symptoms appear as cranial nerve dysfunction that progresses rapidly to life-threatening respiratory depression. The only clinically approved treatment for botulism is postexposure prophylaxis with antitoxin, which blocks neuronal uptake of BoNT but has no effect on toxin molecules already bound to neurons or on those internalized within neurons. Due to the delay between neuronal uptake and the appearance of toxicity, substantial neuronal uptake may occur before symptoms appear. As a result, the majority of symptomatic patients with systemic botulism treated with antitoxin require several weeks of additional intensive care support to survive. BoNT is considered a Tier 1 select agent by the US government because even moderate outbreaks can have a devastating impact on local medical care resources. BoNT serotype A (BoNT / A) is the virulence agent responsible for approximately half of natural botulism cases in the US, and it is also the active ingredient in most neurotoxin-based medicines. Neurotoxin-based medicines, which are injected into muscles for medicinal or cosmetic purposes, can cause off-target effects if overdosed or misplaced. Off-target effects of localized BoNT injections can include unwanted localized muscle weakness or paralysis near the treatment site. As a result, treatment of natural and iatrogenic BoNT / A botulism is a high priority.

[0003] The clear limitations of antitoxin treatments have necessitated the search for antibotulinum therapies. Considerable efforts have been directed toward antidote treatments, primarily small molecule inhibitors (SMIs) that specifically block LC metalloprotease activity within nerve terminals. The development of SMIs is complicated by multiple factors, including the vast substrate-enzyme interface, topologically constrained active sites, high conformational flexibility, and the need for multiple SMIs to block structurally diverse toxin serotypes. As a result, to date, no SMIs have been approved. Alternatively, it has recently been reported that intraneuronal delivery of therapeutic antibodies has antidote efficacy in nonhuman primates, resulting in physiological reversal of botulism symptoms over a period of several days. This delay in therapeutic benefit is consistent with the need to regenerate intact SNARE proteins prior to symptomatic recovery from botulism, and highlights the possibility that LC inhibitors may not have an acute effect on toxic manifestations.

[0004] Given the limitations of antitoxins and the lack of approved intracellular SMIs, there is a critical need for rapidly acting symptomatic treatments for systemic and localized BoNT poisoning that preserve cholinergic neurotransmission until toxicity subsides. Summary of the Invention [Means for solving the problem]

[0005] (Brief Overview) The present disclosure relates to the use of 3,4-diaminopyridine and pharma- ceutically acceptable salts thereof in the treatment of botulism. In particular, the present disclosure relates to a method of treating botulism comprising administering an effective amount of 3,4-diaminopyridine or a pharma- ceutically acceptable salt thereof by continuous infusion, by injection in multiple doses, or by oral administration in multiple doses.

[0006] In one aspect, the disclosure provides a method of treating botulism in a subject in need thereof, comprising administering intravenously to the subject by continuous infusion an effective amount of 3,4-diaminopyridine, or an equivalent amount of a pharma- ceutically acceptable salt thereof, in a pharma- ceutically acceptable vehicle.

[0007] In some aspects, the effective amount of 3,4-diaminopyridine is infused at a rate of about 0.5 mg per kg of the subject's body weight per hour to about 3 mg per kg of the subject's body weight per hour.

[0008] In some aspects, the effective amount of 3,4-diaminopyridine is infused at a rate of about 1.4 mg per kg of the subject's body weight per hour.

[0009] In some aspects, the effective amount of 3,4-diaminopyridine is provided in a total daily dosage range of about 80 mg to about 160 mg of 3,4-diaminopyridine or an equivalent amount of a pharma- ceutically acceptable salt thereof.

[0010] In some aspects, the subject is a human subject.

[0011] In some aspects, administering the 3,4-diaminopyridine or an equivalent amount of a pharma- ceutically acceptable salt thereof by continuous infusion achieves a steady state plasma concentration of about 120 ng / mL of 3,4-diaminopyridine in the subject.

[0012] In some aspects, the method comprises administering the total daily dose of 3,4-diaminopyridine or an equivalent amount of a pharma- ceutically acceptable salt thereof by continuous infusion for multiple consecutive days.

[0013] In some aspects, the botulism is caused by botulinum neurotoxin serotype A.

[0014] In some aspects, the botulism involves localized botulinum neurotoxin intoxication.

[0015] In another aspect, the disclosure provides a method of treating botulism in a subject in need thereof, comprising administering to the subject an effective amount of 3,4-diaminopyridine, or an equivalent amount of a pharma- ceutically acceptable salt thereof, in a pharma- ceutically acceptable vehicle by multiple single bolus injections.

[0016] In some aspects, each of the multiple single bolus injections comprises from about 1 mg to about 3 mg of 3,4-diaminopyridine per kg of the subject's body weight, or an equivalent amount of a pharma- ceutically acceptable salt thereof.

[0017] In some aspects, each of the multiple single bolus injections comprises about 2 mg of 3,4-diaminopyridine per kg of the subject's body weight, or an equivalent amount of a pharma- ceutically acceptable salt thereof.

[0018] In some aspects, the effective amount of 3,4-diaminopyridine, or an equivalent amount of a pharma- ceutically acceptable salt thereof, is provided in a total daily dose range of about 80 mg to about 160 mg of 3,4-diaminopyridine.

[0019] In some aspects, the subject is a human subject.

[0020] In some aspects, administering the 3,4-diaminopyridine or an equivalent amount of a pharma- ceutically acceptable salt thereof by multiple single bolus injections achieves a steady state plasma concentration of about 120 ng / mL of 3,4-diaminopyridine in the subject.

[0021] In some aspects, the method comprises administering the total daily dose of 3,4-diaminopyridine or an equivalent amount of a pharma- ceutically acceptable salt thereof during each of multiple consecutive days.

[0022] In some aspects, the botulism is caused by botulinum neurotoxin serotype A.

[0023] In some aspects, the botulism involves localized botulinum neurotoxin intoxication.

[0024] In another aspect, the disclosure provides a method of treating botulism in a subject in need thereof, the method comprising orally administering to the subject an effective amount of 3,4-diaminopyridine phosphate.

[0025] In some aspects, the effective amount of 3,4-diaminopyridine phosphate is provided in a total daily dose equivalent to about 80 mg to about 160 mg of 3,4-diaminopyridine free base.

[0026] In some aspects, the total daily dose of 3,4-diaminopyridine phosphate is administered in multiple single doses per day.

[0027] In some aspects, oral administration of the 3,4-diaminopyridine phosphate achieves a steady state plasma concentration of about 120 ng / mL of 3,4-diaminopyridine in the subject.

[0028] In some aspects, the subject is a human subject.

[0029] In some aspects, the method includes administering the total daily dose of 3,4-diaminopyridine phosphate during each of multiple days.

[0030] In some aspects, the botulism is caused by botulinum neurotoxin serotype A.

[0031] In some aspects, the botulism involves localized botulinum neurotoxin intoxication.

[0032] Additional aspects and advantages of the disclosure will be set forth in part in the description which follows, and will arise from the description, or may be learned by the practice of the disclosure. The aspects and advantages of the disclosure will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims.

[0033] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed. [Brief description of the drawings]

[0034] [Figure 1] FIG. 1 shows BoNT / A potency determination and disease progression at 2.5 LD50 of BoNT / A in rats.

[0035] [Diagram 2] FIG. 2 shows the pharmacodynamic parameters of 3,4-DAP in naive rats.

[0036] [Figure 3A] FIG. 3A shows an overview of the experimental strategy for the experiment described in Example 1.

[0037] [Figure 3B] FIG. 3B shows the median±interquartile ratio (IQR) of toxicity signs over time for vehicle- and 3,4-DAP-treated rats for the experiment described in Example 1.

[0038] [Figure 3C] FIG. 3C shows the survival curves of vehicle- and 3,4-DAP-treated rats for the experiment described in Example 1.

[0039] [Figure 4] FIG. 4 shows that the infused dose of 3,4-DAP is linearly related to the 3,4-DAP steady-state plasma concentration in rats.

[0040] [Figure 5A] FIG. 5A shows an overview of the experimental strategy for the experiment described in Example 2.

[0041] [Figure 5B] FIG. 5B shows the median ±IQR of toxicity signs at the start of infusion and over time for vehicle- and 3,4-DAP-treated rats for the experiment described in Example 2.

[0042] [Figure 5C] FIG. 5C shows the survival curves of vehicle- and 3,4-DAP-treated rats for the experiment described in Example 2.

[0043] [Figure 5D] FIG. 5D shows the median toxicity signs over time for vehicle- and 3,4-DAP-treated rats for the experiment described in Example 2.

[0044] [Figure 5E] FIG. 5E shows the normalized body weight of surviving rats over time for the experiment described in Example 2.

[0045] [Figure 5F] Figure 5F shows the median ± IQR of toxicity signs in rats (n = 3) from the group infused with 1.44 mg / kg·h 3,4-DAP from days 1 to 5 for the experiment described in Example 2.

[0046] [Figure 6] 6A-6E show a comparison of diaphragm endplate success rate, endplate potential (EPP), miniEPP (mEPP), and quantal content (QC) between the following groups described in Example 2: BoNT naive rats; rats intoxicated with 110 U / kg BoNT / A and infused with 0.98 mg / kg h or 1.44 mg / kg h 3,4-DAP from days 1 to 14 and euthanized on day 21; and rats intoxicated with 110 U / kg BoNT / A and infused with 1.44 mg / kg h 3,4-DAP from days 1 to 5 and euthanized.

[0047] [Figure 7A]7A-7B are tables (Table 3) providing details regarding the statistical comparisons. [Figure 7B] 7A-7B are tables (Table 3) providing details regarding the statistical comparisons. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0048] (Detailed Description) The headings provided herein are not limitations of the various aspects of the disclosure, which can be defined by reference to the specification as a whole. It should also be understood that the terminology used herein is for the purpose of describing particular aspects only, and is not intended to be limiting, since the scope of the disclosure will be limited only by the appended claims.

[0049] 3,4-Diaminopyridine (also known as amifampridine or 3,4-DAP) may be a solution to the critical need for a fast-acting symptomatic treatment for systemic and localized BoNT poisoning that maintains cholinergic neurotransmission until toxicity subsides. 3,4-DAP has been used as a drug in the treatment of many rare muscle diseases (e.g., congenital myasthenic syndromes). 3,4-DAP is a potassium channel blocker that prolongs action potential duration by reversibly blocking voltage-gated potassium channels, facilitating presynaptic Ca2+ influx and increasing acetylcholine release. 3,4-DAP free base form is sold under the brand name Ruzurgi®, which is approved for treating LEMS in pediatric patients. The phosphate salt of 3,4-DAP is sold under the brand name Firdapse®, which is approved for treating LEMS in adults.

[0050] 3,4-DAP has been shown to reverse muscle paralysis in isolated mouse diaphragms intoxicated by multiple BoNT serotypes, and is particularly effective in treating serotype A. Short-term treatment with 3,4-DAP has been shown to improve respiratory function and prolong survival in mice with end-stage botulism, confirming symptomatic efficacy in vivo. However, 3,4-DAP has a short pharmacodynamic half-life. Prior to the experiments described below with respect to Examples 1 and 2, it remained unknown whether repeated or sustained administration of 3,4-DAP could maintain symptomatic benefits until neuromuscular function recovers from botulism paralysis (a process that can take several weeks) while avoiding symptoms of acute neurotoxicity (e.g., seizures) sometimes associated with repeated administration of 3,4-DAP.

[0051] (definition) For convenience, the meaning of some terms and phrases used in the specification, examples, and the appended claims are provided below. Unless otherwise indicated or implied from the context, the following terms and phrases have the meanings provided below. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this technology belongs. If there is an obvious discrepancy between the usage of a term in the art and the definition of the term provided herein, the definition provided herein shall prevail.

[0052] The articles "a," "an," and "the" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0053] As used herein, the term "about" means ±10% of the particular value, unless otherwise indicated.

[0054] The term "at least" before a plurality or series of numbers is understood to include the number adjacent to the term "at least," and all subsequent numbers or integers that may be logically included as is apparent from the context. When "at least" is before a series of numbers or a range, it is understood that "at least" may modify each of the numbers in the series or range.

[0055] As used herein, the terms "comprises," "comprising," "having," "including," "containing," and the like are open-ended terms meaning "including, but not limited to." When certain aspects disclosed herein "comprise" particular elements, it should be understood that the disclosure specifically contemplates and discloses aspects "consisting essentially of" and "consisting of" those elements.

[0056] As used herein, the terms "consists essentially of," "consisting essentially of," and the like, should be construed as semi-open-ended terms, meaning that no other components are included that materially affect the basic and novel characteristics of an aspect.

[0057] As used herein, the terms "consists of," "consisting of," and the like should be construed as limiting terms such that an aspect "consisting of" a particular set of elements excludes any element, step, or ingredient not specified in that aspect.

[0058] The terms "treat," "treating," and "treatment" refer to any indicator of success in treating or ameliorating an injury, disease, or condition, including any objective or subjective parameter, such as a reduction in symptoms; amelioration of symptoms; diminishing symptoms or making the injury, disease, or condition more tolerable to the patient; a slowing of the rate of degeneration or decline; or an improvement in the patient's physical or mental well-being. Treating or ameliorating symptoms may be based on objective or subjective parameters, including the results of a physical exam, a neuropsychiatric exam, or a psychiatric evaluation.

[0059] The phrase "effective amount" refers to a non-toxic, but sufficient amount of a drug or agent to provide the desired effect. The amount that is "effective" varies from subject to subject, depending on the age and general condition of the individual, the particular active agent or active agents, and the like. Thus, it is not always possible to specify an exact "effective amount." However, the appropriate "effective" amount in any particular case can be determined by one of ordinary skill in the art using routine experimentation. In general, the amount of 3,4-DAP that constitutes an "effective" amount for treating systemic BoNT poisoning depends on the dose of BoNT to which the subject was exposed. For example, without wishing to be bound by a particular theory, a larger total daily dose of 3,4-diaminopyridine may be required to achieve efficacy if the subject suffering from systemic BoNT poisoning was exposed to a larger amount of BoNT, whereas a smaller dose of 3,4-diaminopyridine may be effective if the subject was exposed to a smaller amount of BoNT. It is within the skill of a physician of ordinary skill in the art to determine the level of exposure to BoNT and select or titrate an "effective amount" of 3,4-diaminopyridine required to treat a subject in need thereof, regardless of whether the subject is suffering from systemic or localized BoNT poisoning.

[0060] The term "pharmaceutically acceptable salts" refers to salts of basic compounds (eg, 3,4-DAP) prepared from pharmaceutically acceptable inorganic acids and pharmaceutically acceptable organic acids.

[0061] The term "continuous infusion" refers to the administration of a fluid into the subcutaneous space or into a blood vessel over an extended period of time, which can be any suitable duration, such as from about 1 hour to about 24 hours (e.g., 1 hour, 2 hours, 3 hours, or 4 hours); or from about 1 day to about 21 days, or from about 1 week to about 12 weeks, or any suitable duration.

[0062] As used herein, the term "botulism" refers to a condition caused by systemic or localized intoxication with any serotype of BoNT (e.g., BoNT / A), including associated muscular and / or respiratory symptoms, regardless of the manner in which the condition was acquired.

[0063] As used herein, the term "toxic signs" refers to physiological signs associated with botulism, including respiratory signs (e.g., abdominal paradox and agonal respiratory pattern); and musculoskeletal signs (e.g., salivation, lethargy, and generalized paralysis).

[0064] (Methods of Treating BoNT Poisoning) Systemic BoNT poisoning (e.g., botulism resulting from ingesting spoiled food or drink or from exposure to BoNT) can have serious consequences for affected subjects. Without proper treatment, subjects with systemic botulism can experience muscle paralysis, respiratory distress, and, if left untreated or treated too late, death from respiratory collapse.

[0065] Similarly, off-target effects resulting from the medicinal and / or cosmetic use of BoNT-based pharmaceuticals (e.g., to treat conditions including Meige syndrome, migraines, bruxism, facial wrinkles, limb spasticity, spasmodic dysphonia, etc.) are of significant concern, especially when the BoNT-based pharmaceutical is administered in amounts greater than prescribed or approved, when the BoNT-based pharmaceutical is administered in the wrong location, or when the BoNT-based pharmaceutical is administered properly but "leaks" into surrounding tissues. Off-target effects of BoNT-based pharmaceuticals include, but are not limited to, unwanted focal muscle weakness or paralysis, ptosis or other muscle ptosis, difficulty chewing, and weak or breathy voice resulting from vocal cord paralysis.

[0066] In one aspect, the disclosure provides a method of treating botulism in a subject in need thereof, comprising administering intravenously to the subject an effective amount of 3,4-diaminopyridine or an equivalent amount of a pharma- ceutically acceptable salt thereof in a pharma- ceutically acceptable vehicle by continuous infusion. The infusion of 3,4-DAP can continue for at least 1 hour (e.g., 1 hour to 24 hours) or for at least 1 day (e.g., 1 day to 21 days) or for at least 1 week (e.g., 1 week to 12 weeks).

[0067] In some aspects, the effective amount of 3,4-diaminopyridine can be infused at a rate of from about 0.1 mg per kg of the subject's body weight per hour to about 5.0 mg per kg of the subject's body weight per hour, e.g., from about 0.5 mg per kg per hour to about 4.5 mg per kg per hour, from about 1.0 mg per kg per hour to about 4.0 mg per kg per hour, from about 1.5 mg per kg per hour to about 3.5 mg per kg per hour, or from about 2.0 mg per kg per hour to about 3.0 mg per kg per hour.

[0068] In some aspects, the effective amount of 3,4-diaminopyridine is about 0.1 mg / kg of the subject's body weight per hour, about 0.2 mg / kg of the subject's body weight per hour, about 0.3 mg / kg of the subject's body weight per hour, about 0.4 mg / kg of the subject's body weight per hour, about 0.5 mg / kg of the subject's body weight per hour, about 0.6 mg / kg of the subject's body weight per hour, about 0.7 mg / kg of the subject's body weight per hour, about 0.8 mg / kg of the subject's body weight per hour, about 0.9 mg / kg of the subject's body weight per hour, ...0 mg / kg of the subject's body weight per hour, about 0.15 mg / kg of the subject's body weight per hour, about 0.16 mg / kg of the subject's body weight per hour, about 0.17 mg / kg of the subject's body weight per hour, about 0.18 mg / kg of the subject's body weight per hour, about 0.19 mg / kg of the subject's body weight per hour, about 0.20 mg / kg of the subject's body weight per hour, about 0.21 mg / kg of the subject's body weight per hour, about 0.22 mg / kg of the subject's body weight about 0.9 mg per g per hour, about 1.0 mg per kg of the subject's body weight per hour, about 1.1 mg per kg of the subject's body weight per hour, about 1.2 mg per kg of the subject's body weight per hour, about 1.3 mg per kg of the subject's body weight per hour, about 1.4 mg per kg of the subject's body weight per hour, about 1.5 mg per kg of the subject's body weight per hour, about 1.6 mg per kg of the subject's body weight per hour, about 1.7 mg per kg of the subject's body weight per hour, about 1. 8 mg per kg of the subject's body weight per hour, about 1.9 mg per kg of the subject's body weight per hour, about 2.0 mg per kg of the subject's body weight per hour, about 2.1 mg per kg of the subject's body weight per hour, about 2.2 mg per kg of the subject's body weight per hour, about 2.3 mg per kg of the subject's body weight per hour, about 2.4 mg per kg of the subject's body weight per hour, about 2.5 mg per kg of the subject's body weight per hour, about 2.6 mg per kg of the subject's body weight per hour, about 2.7 mg per kg of the subject's body weight per hour, about 2.8 mg per kg of the subject's body weight per hour, about 2.9 mg per kg of the subject's body weight per hour, about 3.1 mg per kg of the subject's body weight per hour, about 3.2 mg per kg of the subject's body weight per hour, about 3.3 mg per kg of the subject's body weight per hour, about 3.4 mg per kg of the subject's body weight per hour, about 3.5 mg per kg of the subject's body weight per hour, about 3.6 mg per kg of the subject's body weight per hour, about 3.7 mg per kg of the subject's body weight per hour, about 3.8 mg per kg of the subject's body weight per hour, about 3.9 mg per kg of the subject's body weight per hour, about 3.1 mg per kg of the subject's body weight per hour, about 3.2 mg per kg of the subject's body weight per hour, about 3.3 mg per kg of the subject's body weight per hour, about 3.4 mg per kg of the subject's body weight per hour, about 3.5 mg per kg of the subject's body weight per hour, about 3.6 mg per kg of the subject's body weight per hour, about 3.7 mg per kg of the subject's body weight per hour, about 3.8 mg per kg of the about 2.8 mg per g per hour, about 2.9 mg per kg of the subject's body weight per hour, about 3.0 mg per kg of the subject's body weight per hour, about 3.1 mg per kg of the subject's body weight per hour, about 3.2 mg per kg of the subject's body weight per hour, about 3.3 mg per kg of the subject's body weight per hour, about 3.4 mg per kg of the subject's body weight per hour, about 3.5 mg per kg of the subject's body weight per hour, about 3.6 mg per kg of the subject's body weight per hour, about 3.The subject's body weight may be infused at a rate of about 7 mg per kg of the subject's body weight per hour, about 3.8 mg per kg of the subject's body weight per hour, about 3.9 mg per kg of the subject's body weight per hour, about 4.0 mg per kg of the subject's body weight per hour, about 4.1 mg per kg of the subject's body weight per hour, about 4.2 mg per kg of the subject's body weight per hour, about 4.3 mg per kg of the subject's body weight per hour, about 4.4 mg per kg of the subject's body weight per hour, about 4.5 mg per kg of the subject's body weight per hour, about 4.6 mg per kg of the subject's body weight per hour, about 4.7 mg per kg of the subject's body weight per hour, about 4.8 mg per kg of the subject's body weight per hour, about 4.9 mg per kg of the subject's body weight per hour, or about 5.0 mg per kg of the subject's body weight per hour.

[0069] In some aspects, the infusion can occur over a period of about 1 hour to about 24 hours, e.g., about 2 hours to about 22 hours, about 4 hours to about 20 hours, about 6 hours to about 18 hours, about 8 hours to about 16 hours, or about 10 hours to about 14 hours.

[0070] In some aspects, the infusion may occur over about 1 hour, over about 2 hours, over about 3 hours, over about 4 hours, over about 5 hours, over about 6 hours, over about 7 hours, over about 8 hours, over about 9 hours, over about 10 hours, over about 11 hours, over about 12 hours, over about 13 hours, over about 14 hours, over about 15 hours, over about 16 hours, over about 17 hours, over about 18 hours, over about 19 hours, over about 20 hours, over about 21 hours, over about 22 hours, over about 23 hours, over about 24 hours.

[0071] In some aspects, infusion of 3,4-DAP (or a pharma- ceutically acceptable salt thereof) can be administered at any of the above rates for a period of time longer than one day, e.g., from about 3 to about 21 days, from about 5 to about 19 days, from about 7 to about 17 days, from about 9 to about 15 days, or from about 11 to about 13 days.

[0072] In some aspects, infusion of 3,4-DAP (or a pharma- ceutically acceptable salt thereof) can be administered at any of the above rates for about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 16 days, about 17 days, about 18 days, about 19 days, about 20 days, or about 21 days.

[0073] In some aspects, infusion of 3,4-DAP (or a pharma- ceutically acceptable salt thereof) can be administered at any of the above rates for a period of more than one week, e.g., from about 2 weeks to about 12 weeks, from about 3 weeks to about 11 weeks, from about 4 weeks to about 10 weeks, from about 5 weeks to about 9 weeks, or from about 6 weeks to about 8 weeks.

[0074] In some aspects, infusion of 3,4-DAP (or a pharma- ceutically acceptable salt thereof) can be administered at any of the above rates for about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 11 weeks, or about 12 weeks.

[0075] In some aspects, the effective amount of 3,4-diaminopyridine is in the range of about 1 mg to about 200 mg (e.g., about 5 mg to about 195 mg, about 10 mg to about 190 mg, about 15 mg to about 185 mg, about 20 mg to about 180 mg, about 25 mg to about 175 mg, about 30 mg to about 170 mg, about 35 mg to about 165 mg, about 45 mg to about 160 mg, about 50 mg to about 155 mg, about 50 mg to about 165 mg, about 50 mg to about 17 ... The present invention may be provided in a total daily dose of about 5 mg to about 145 mg, about 60 mg to about 140 mg, about 65 mg to about 135 mg, about 70 mg to about 130 mg, about 75 mg to about 125 mg, about 80 mg to about 120 mg, about 85 mg to about 115 mg, about 90 mg to about 110 mg, or about 95 mg to about 100 mg) of 3,4-diaminopyridine, or an equivalent amount of a pharma- ceutically acceptable salt thereof.

[0076] In some aspects, the effective amount of 3,4-diaminopyridine can be provided in a total daily dose of about 1 mg, about 5 mg, about 15 mg, about 20 mg, about 25 mg, about 30 mg, about 35 mg, about 40 mg, about 45 mg, about 50 mg, about 55 mg, about 60 mg, about 65 mg, about 70 mg, about 75 mg, about 80 mg, about 90 mg, about 95 mg, about 100 mg, about 105 mg, about 110 mg, about 115 mg, about 120 mg, about 125 mg, about 130 mg, about 135 mg, about 140 mg, about 145 mg, about 150 mg, about 155 mg, about 160 mg, about 165 mg, about 170 mg, about 175 mg, about 180 mg, about 185 mg, about 190 mg, about 195 mg, or about 200 mg.

[0077] In some aspects, the subject can be a human subject.

[0078] In some aspects, administering 3,4-diaminopyridine or a pharma- ceutically acceptable salt thereof as described herein may result in a 3,4-diaminopyridine steady-state plasma concentration in the subject of about 70 ng / mL to about 200 ng / mL (e.g., about 85 ng / mL to about 195 ng / mL, about 90 ng / mL to about 190 ng / mL, about 95 ng / mL to about 185 ng / mL, about 100 ng / mL to about 180 ng / mL, about 105 ng / mL to about 175 ng / mL, about 110 ng / mL to about 165 ng / mL, about 115 ng / mL to about 160 ng / mL, about 120 ng / mL to about 155 ng / mL, about 125 ng / mL to about 150 ng / mL, or about 130 ng / mL to about 145 ng / mL).

[0079] In some aspects, administering 3,4-diaminopyridine as described herein provides a 3,4-diaminopyridine steady state plasma concentration of about 70 ng / mL, about 75 ng / mL, about 80 ng / mL, about 85 ng / mL, about 90 ng / mL, about 95 ng / mL, about 100 ng / mL, about 105 ng / mL, about 110 ng / mL, about 115 ng / mL, about 120 ng / mL , about 125 ng / mL, about 130 ng / mL, about 135 ng / mL, about 140 ng / mL, about 145 ng / mL, about 150 ng / mL, about 155 ng / mL, about 160 ng / mL, about 165 ng / mL, about 170 ng / mL, about 175 ng / mL, about 180 ng / mL, about 185 ng / mL, about 190 ng / mL, about 195 ng / mL, or about 200 ng / mL.

[0080] In some aspects, the method may include administering the total daily dose of 3,4-diaminopyridine or an equivalent amount of a pharma- ceutically acceptable salt thereof by continuous infusion at any of the rates described herein for multiple consecutive days.

[0081] In some aspects, the botulism can be caused by botulinum neurotoxin serotype A.

[0082] In another aspect, the disclosure provides a method of treating botulism in a subject in need thereof, comprising administering to the subject an effective amount of 3,4-diaminopyridine, or an equivalent amount of a pharma- ceutically acceptable salt thereof, in a pharma- ceutically acceptable vehicle by multiple single bolus injections.

[0083] In some aspects, each of the multiple single bolus injections contains about 0.1 mg to about 5.0 mg per kg of the subject's body weight (e.g., about 0.5 mg / kg to about 4.5 mg / kg, about 1.0 mg / kg to about 4.0 mg / kg, about 1.5 mg / kg to about 3.5 mg / kg, or about 2.0 mg / kg to about 3.0 mg / kg) of 3,4-diaminopyridine or an equivalent amount of a pharma- ceutically acceptable salt thereof.

[0084] In some aspects, each of the multiple single bolus injections is about 0.1 mg / kg of the subject's body weight, or about 0.2 mg / kg of the subject's body weight, about 0.3 mg / kg of the subject's body weight, about 0.4 mg / kg of the subject's body weight, about 0.5 mg / kg of the subject's body weight, about 0.6 mg / kg of the subject's body weight, about 0.7 mg / kg of the subject's body weight, about 0.8 mg / kg of the subject's body weight, about 0.9 mg / kg of the subject's body weight, about 1.0 mg / kg of the subject's body weight, about 1.2 mg / kg of the subject's body weight, about 1.4 mg / kg of the subject's body weight, about 1.5 mg / kg of the subject's body weight, about 1.6 mg / kg of the subject's body weight, about 1.7 mg / kg of the subject's body weight, about 1.8 mg / kg of the subject's body weight, about 1.9 ...8 mg / kg of the subject's body weight, about 1.9 mg / kg of the subject's body weight, about 1.9 mg / kg of the subject's body weight, about 1.8 mg / kg of the subject's body weight, about 1.9 mg / kg of the subject's body weight, about 1.9 mg / kg of the subject's body weight, about 1.9 mg / kg of the subject's body weight .1 mg, about 1.2 mg per kg of the subject's body weight, about 1.3 mg per kg of the subject's body weight, about 1.4 mg per kg of the subject's body weight, about 1.5 mg per kg of the subject's body weight, about 1.6 mg per kg of the subject's body weight, about 1.7 mg per kg of the subject's body weight, about 1.8 mg per kg of the subject's body weight, about 1.9 mg per kg of the subject's body weight, about 2.0 mg per kg of the subject's body weight, about 2.1 mg per kg of the subject's body weight, about 2.2 mg per kg of the subject's body weight, about 2.3 mg per kg of the subject's body weight, about 2.4 mg / kg of the subject's body weight, about 2.5 mg / kg of the subject's body weight, about 2.6 mg / kg of the subject's body weight, about 2.7 mg / kg of the subject's body weight, about 2.8 mg / kg of the subject's body weight, about 2.9 mg / kg of the subject's body weight, about 3.0 mg / kg of the subject's body weight, about 3.1 mg / kg of the subject's body weight, about 3.2 mg / kg of the subject's body weight, about 3.3 mg / kg of the subject's body weight, about 3.4 mg / kg of the subject's body weight, about 3.5 mg / kg of the subject's body weight, about 3.6 mg / kg of the subject's body weight, about 3.7 mg / kg of the subject's body weight, about 3.8 mg / kg of the subject's body weight, about 3.9 ... about 3.6 mg per g, about 3.7 mg per kg of the subject's body weight, about 3.8 mg per kg of the subject's body weight, about 3.9 mg per kg of the subject's body weight, about 4.0 mg per kg of the subject's body weight, about 4.1 mg per kg of the subject's body weight, about 4.2 mg per kg of the subject's body weight, about 4.3 mg per kg of the subject's body weight, about 4.4 mg per kg of the subject's body weight, about 4.5 mg per kg of the subject's body weight, about 4.6 mg per kg of the subject's body weight, about 4.7 mg per kg of the subject's body weight, about 4.The composition may contain about 8 mg / kg of the subject's body weight, about 4.9 mg / kg of the subject's body weight, or about 5.0 mg / kg of the subject's body weight of 3,4-diaminopyridine, or an equivalent amount of a pharma- ceutically acceptable salt thereof.

[0085] In another aspect, the disclosure provides a method of treating botulism in a subject in need thereof, the method comprising orally administering to the subject an effective amount of 3,4-diaminopyridine phosphate.

[0086] In some aspects, the effective amount of 3,4-diaminopyridine phosphate is a total daily dose equivalent to about 1 mg to about 200 mg of 3,4-diaminopyridine free base (e.g., about 5 mg to about 195 mg, about 10 mg to about 190 mg, about 15 mg to about 185 mg, about 20 mg to about 180 mg, about 25 mg to about 175 mg, about 30 mg to about 170 mg, about 35 mg to about 165 mg, etc.). g, about 45 mg to about 160 mg, about 50 mg to about 155 mg, about 55 mg to about 145 mg, about 60 mg to about 140 mg, about 65 mg to about 135 mg, about 70 mg to about 130 mg, about 75 mg to about 125 mg, about 80 mg to about 120 mg, about 85 mg to about 115 mg, about 90 mg to about 110 mg, or about 95 mg to about 100 mg).

[0087] In some aspects, the effective amount of 3,4-diaminopyridine phosphate may be provided in a total daily dose equivalent to about 1 mg, about 5 mg, about 15 mg, about 20 mg, about 25 mg, about 30 mg, about 35 mg, about 40 mg, about 45 mg, about 50 mg, about 55 mg, about 60 mg, about 65 mg, about 70 mg, about 75 mg, about 80 mg, about 90 mg, about 95 mg, about 100 mg, about 105 mg, about 110 mg, about 115 mg, about 120 mg, about 125 mg, about 130 mg, about 135 mg, about 140 mg, about 145 mg, about 150 mg, about 155 mg, about 160 mg, about 165 mg, about 170 mg, about 175 mg, about 180 mg, about 185 mg, about 190 mg, about 195 mg, or about 200 mg.

[0088] In some aspects, the total daily dose of 3,4-diaminopyridine phosphate may be administered in multiple single doses per day.

[0089] In some aspects, administering 3,4-diaminopyridine phosphate as described herein may result in a steady state 3,4-diaminopyridine plasma concentration of about 70 ng / mL to about 200 ng / mL (e.g., about 85 ng / mL to about 195 ng / mL, about 90 ng / mL to about 190 ng / mL, about 95 ng / mL to about 185 ng / mL, about 100 ng / mL to about 180 ng / mL, about 105 ng / mL to about 175 ng / mL, about 110 ng / mL to about 165 ng / mL, about 115 ng / mL to about 160 ng / mL, about 120 ng / mL to about 155 ng / mL, about 125 ng / mL to about 150 ng / mL, or about 130 ng / mL to about 145 ng / mL) in the subject.

[0090] In some aspects, administering 3,4-diaminopyridine phosphate as described herein provides a 3,4-diaminopyridine steady state plasma concentration of about 70 ng / mL, about 75 ng / mL, about 80 ng / mL, about 85 ng / mL, about 90 ng / mL, about 95 ng / mL, about 100 ng / mL, about 105 ng / mL, about 110 ng / mL, about 115 ng / mL, about 120 ng / mL, or about 130 ng / mL. mL, about 125 ng / mL, about 130 ng / mL, about 135 ng / mL, about 140 ng / mL, about 145 ng / mL, about 150 ng / mL, about 155 ng / mL, about 160 ng / mL, about 165 ng / mL, about 170 ng / mL, about 175 ng / mL, about 180 ng / mL, about 185 ng / mL, about 190 ng / mL, about 195 ng / mL, or about 200 ng / mL.

[0091] In some aspects, the method can include administering the total daily dose of 3,4-diaminopyridine phosphate during each of multiple days.

[0092] It has been surprisingly discovered that continuous infusion of 3,4-DAP or repeated administration of 3,4-diaminopyridine can reduce toxic symptoms and maintain survival in subjects suffering from botulism during the acute phase of the disease, which can persist for up to several weeks without causing symptoms of neurotoxicity. For example, in the experiment described below with reference to Example 2, toxic symptoms in rat subjects lethally challenged with BoNT resolved after a 14-day course of continuous infusion of 3,4-diaminopyridine. Effective daily doses are discussed elsewhere herein.

[0093] The therapeutic efficacy of 3,4-diaminopyridine and its salts in the present method can be determined by the clinician, for example, by using any suitable evaluation, which may include, but is not limited to, analysis of recorded diaphragm endplate potentials, visual observation and analysis of toxicity signs, questionnaire surveys of responsive human subjects, and the like.

[0094] (Pharmaceutical preparations) In some aspects, the present disclosure provides compositions suitable for parenteral administration.For example, 3,4-diaminopyridine can be formulated for parenteral administration by injection (for example, by bolus injection or continuous infusion).The preparation for injection can be provided in unit dosage form (for example, in ampoules or in multi-dose containers) with additional preservatives.The composition can take the form of suspension, solution, or emulsion in oily or aqueous vehicle, and can include formulary agents such as suspending agents, stabilizing agents, and / or dispersing agents.

[0095] When an effective amount of 3,4-diaminopyridine is administered by intravenous, cutaneous, or subcutaneous injection, the composition may be in the form of a pyrogen-free parenterally acceptable aqueous solution. The preparation of such parenterally acceptable solutions, with due attention to pH, isotonicity, stability, and the like, is within the skill of the art. In some aspects, compositions for intravenous, cutaneous, or subcutaneous injection typically include an isotonic vehicle.

[0096] Pharmaceutical compositions for parenteral administration may include aqueous solutions of 3,4-diaminopyridine. In addition, suspensions of 3,4-diaminopyridine may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils or synthetic fatty acid esters. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension. If necessary, the suspension may also contain suitable stabilizers or agents that increase the solubility of 3,4-diaminopyridine to allow for the preparation of highly concentrated solutions. Alternatively, the composition may be in powder form for constitution with a suitable vehicle (e.g., sterile pyrogen-free water) before use.

[0097] 3,4-Diaminopyridine can also be injected parenterally (e.g., intravenously, intramuscularly, subcutaneously, or intracoronarily). For parenteral administration, the 3,4-diaminopyridine composition can be used in the form of a sterile aqueous solution which may contain other substances that make the solution isotonic with blood (e.g., salts, or simple sugars such as mannitol or glucose).

[0098] As a further aspect, the present disclosure may include a kit that includes one or more compounds or compositions packaged in a manner that facilitates their use to carry out the method of treating botulism described herein. In one aspect, the kit may include a compound or composition described herein as useful for carrying out the method (e.g., a composition that includes 3,4-diaminopyridine) packaged in a container (e.g., a sealed bottle or container) with a label attached to the container or included in the kit that describes the use of the compound or composition to carry out the method of the present disclosure. In a particular aspect, the compound or composition may be packaged in a unit dosage form. The kit may further include a device (e.g., a syringe or drip bag) suitable for administering the composition according to the intended route of administration. In another aspect, the 3,4-diaminopyridine or a pharma- ceutically acceptable salt thereof may be a lyophilizate. In this case, the kit may further include an additional container that includes a solution useful for reconstituting the lyophilizate.

[0099] In some aspects, the effective amount of 3,4-diaminopyridine or a pharma- ceutically acceptable salt thereof may be provided and / or administered in a pharmaceutical formulation suitable for oral administration (e.g., a tablet formulation of its phosphate salt sold by Catalyst Pharmaceuticals, Inc. under the trade name Firdapse®).

[0100] In some aspects, the oral formulation can be selected from the group consisting of solid oral formulation, semi-solid oral formulation, and liquid oral formulation.In some aspects, the solid oral formulation can be a tablet, a pill, a sugar-coated tablet, a powder, a granule, or a capsule.Suitable liquid formulations include, for example, aqueous suspensions, solutions, elixirs, and syrups.Suitable semi-solid formulations include, for example, oral gels.

[0101] Orally administered pharmaceutical preparations may contain conventional excipients known in the art and may be prepared by conventional methods. Orally administered pharmaceutical preparations of the present disclosure may contain one or more pharma-ceutically acceptable excipients. Suitable excipients include fillers (e.g., sugars, e.g., lactose or sucrose), mannitol, sodium saccharin or sorbitol, magnesium carbonate, cellulose preparations and / or calcium phosphate (e.g., tricalcium phosphate or calcium hydrogen phosphate) and binders (starch paste (e.g., using corn starch, wheat starch, rice starch, potato starch), gelatin, tragacanth, methylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, and / or polyvinylpyrrolidone, etc.). If desired, disintegrants (e.g., the above-mentioned starches and carboxymethyl starches, cross-linked polyvinylpyrrolidone, agar, or alginic acid or its salts (e.g., sodium alginate) and sodium starch glycolate) may be added. Suitable excipients also include flow-regulating agents and lubricants (e.g., silica, talc, stearic acid or salts thereof (e.g., magnesium stearate or calcium stearate), and / or polyethylene glycol) to provide a pharma- ceutically palatable preparation; sweeteners (e.g., fructose, aspartame, or saccharin); flavoring agents (e.g., peppermint, oil of wintergreen, or cherry); coloring agents; and preservatives. In addition, dyes or pigments may be added to the tablets or dragee coatings, for example, for identification or to characterize active compound dose combinations. Other examples of suitable pharmaceutical excipients are described in Remington's Pharmaceutical Sciences, pp. 1447-1676 (ed. Alfonso R. Gennaro, 19th ed., 1995), which is incorporated herein by reference. In one aspect, the excipients are pharmaceutical grade excipients.

[0102] In some aspects, 3,4-diaminopyridine or its salt can be micronized before preparing its oral formulation. Methods known in the art can be used for micronization of 3,4-diaminopyridine or its salt. For example, traditional micronization techniques based on friction (e.g., milling, bashing and grinding) can be used to reduce particle size. A typical industrial mill is composed of a cylindrical metal drum that usually contains steel balls. As the drum rotates, the inner balls collide with solid particles, thereby crushing the solid particles into smaller diameters. In the case of grinding, solid particles are formed when the grinding units of the device rub against each other while the solid particles are trapped between them. Methods such as crushing and cutting can also be used to reduce particle diameter. Crushing uses a hammer-like tool to break solids into smaller particles by impact. Cutting uses a sharp blade to cut coarse solid pieces into smaller solid pieces. In addition, modern micronization methods that use supercritical fluids in the micronization process can be used. These methods use supercritical fluids to induce a state of supersaturation, which results in the precipitation of individual particles. Suitable techniques include the RESS (Rapid Expansion of Supercritical Solution) process, the SAS (Supercritical Anti-Solvent) method, and the PGSS (Particle from Gas Saturated Solution) method. These modern techniques allow for greater tunability of the process. Parameters such as relative pressure and temperature, solute concentration, and antisolvent to solvent ratio can be altered to adjust for the desired particle size. The supercritical fluid method provides for more precise control over particle diameter, particle size distribution, and morphology consistency.

[0103] In some aspects, the micronized 3,4-diaminopyridine or salt thereof suitable for use in the oral formulation of the present disclosure can be a composition in which 90% or more of the particles have a particle size of 20 microns or less (i.e., ≦20 μm).In some aspects, the oral pharmaceutical formulation of the present disclosure comprises micronized 3,4-diaminopyridine phosphate.In some aspects, 90% or more of the particles in the micronized 3,4-diaminopyridine phosphate have a particle size of 20 microns or less. EXAMPLES

[0104] (Example) The methods of treatment described herein are further detailed herein with reference to the following examples. These examples are provided for illustrative purposes only, and the aspects described herein should in no way be construed as being limited to these examples. Rather, the aspects should be construed to include any and all variations that become evident as a result of the teachings provided herein.

[0105] Example 1 Continuous administration of 3,4-DAP extends survival after lethal challenge with BoNT / A. To confirm that 3,4-DAP reverses botulinum toxicity in rats, the effects of repeated administration of 3,4-DAP were first evaluated in rats lethally challenged with 110 U / kg BoNT / A intravenously (2.5 LD50; see Figure 1A).

[0106] Figure 1 generally illustrates BoNT / A potency determination and disease progression at 2.5LD50 in rats. Figure 1A illustrates the determination of rat intravenous LD50. Rats were administered 22U / kg to 69U / kg of BoNT / A by tail vein injection and monitored for survival at 24-h intervals until day 7. Surviving rats were lively, alert, and responsive on day 7, and showed reduced toxic signs of botulism. The LD50 was calculated from the survival outcomes using simple linear regression. Figure 1B illustrates the progression of toxic signs in rats (n=12) challenged with 110U / kg (2.5LD50) of BoNT / A. Figure 1C illustrates the survival curves of rats derived from Figure 1B. Details regarding statistical comparisons are shown in Figures 7A-7B (Table 3).

[0107] The treatment initiation times were based on the onset of toxic signs (FIG. 1B) and time to death (FIG. 1C) in the toxicity efficacy study, while the treatment intervals were estimated from the duration of respiratory effects observed in naive rats treated with 2 mg / kg 3,4-DAP (determined as shown in FIG. 2). In naive rats, a single dose of ≦8 mg / kg 3,4-DAP or 15 successive injections of 2 mg / kg 3,4-DAP did not induce physiological indicators of acute neurotoxicity (see "Bolus Infusion" column in Table 1 below), suggesting that the treatment regimen was well tolerated. [Table 1]

[0108] Figure 2 generally illustrates 3,4-DAP pharmacodynamic parameters in naive rats. Rats implanted with diaphragmatic electrodes and maintained in metabolic cages were administered vehicle saline or 2 mg / kg 3,4-DAP by subcutaneous injection (n=4 per group). Figure 2A outlines the experimental strategy. Figures 2B-D show that 3,4-DAP treatment (trend line "a") induces a transient increase in VO2 (Figure 2B), respiratory rate (Figure 2C) and tidal volume (Figure 2D). Mean values ​​were compared to vehicle (trend line "b") at each time point using a two-way repeated measures ANOVA followed by Sidak multiple comparison test. For Figure 2B, samples were collected in 5-minute bins, whereas for Figures 2C and 2D, values ​​were collected at 30-minute intervals. **** =p<0.0001; *** =p<0.001; ** = p < 0.01; and * = p<0.05. Details regarding statistical comparisons are in Figures 7A-B (Table 3).

[0109] Figure 3 generally shows that repeated administration of 3,4-DAP reverses clinical signs of botulism and prolongs survival. Rats were treated with 2 mg / kg 3,4-DAP or saline vehicle by 15 consecutive injections at 90 min intervals starting 32 h after intoxication (n=6 each; Figure 3A). In a previous study, 2 mg / kg 3,4-DAP reversed respiratory and skeletal muscle depression in mice intoxicated with BoNT / A. 3,4-DAP pharmacokinetic properties were similar between rats and mice, suggesting that 2 mg / kg 3,4-DAP is also effective in reversing acute botulism in rats.

[0110] Figure 3B shows the median ± interquartile ratio (IQR) of toxic signs over time for vehicle-treated rats (trend line "a") and 3,4-DAP-treated rats (trend line "b") (two-way repeated measures ANOVA and Sidak's multiple comparison test at each time point). At the start of treatment, intoxicated rats showed signs of systemic botulism, including paradoxical breathing, dysphagia, and limb weakness. Compared to vehicle, 3,4-DAP reduced toxic signs within 30 minutes and maintained symptomatic benefit throughout the treatment period.

[0111] FIG. 3C shows survival curves (Mantel-Cox log-rank test) for vehicle-treated rats (trend line "a") and 3,4-DAP-treated rats (trend line "b"). Grey boxes in panels B and C indicate the treatment period. Details regarding statistical comparisons are in FIG. 7A-7B (Table 3). 3,4-DAP significantly increased survival from 0% (vehicle) to 100% (3,4-DAP; p=0.002) at the time of the last injection. Toxicity signs worsened approximately 1.5 hours after cessation of 3,4-DAP treatment, and rats died 2-4 hours after the last injection. Taken together, the data shown in FIG. 3B and FIG. 3C show a transient but robust effect of repeated bolus injections of 3,4-DAP on toxicity signs and survival in rats with end-stage botulism.

[0112] Example 2 (Continuous infusion of 3,4-DAP reversed the toxic symptoms and enabled survival.) Abstract: 3,4-DAP was infused continuously for 13 days into rats lethally challenged with BoNT serotype A. Clinically relevant doses of 3,4-DAP stabilized toxic signs and permitted survival without adverse effects or re-emergence of toxicity after cessation of treatment. Diaphragm endplate recordings from infused rats revealed significant neuromuscular depression at day 5 that was partially reversed in surviving rats at day 21, providing a functional mechanism for antidotal efficacy. These data indicate a strong translational potential of 3,4-DAP in the treatment of clinical botulism.

[0113] Figure 4 generally shows that infusion dose is linearly related to 3,4-DAP steady-state concentration. To determine whether the therapeutic benefit of 3,4-DAP could be maintained over longer periods, lethally intoxicated rats were continuously infused with 3,4-DAP through a subcutaneous catheter. First, the steady-state relationship between 3,4-DAP infusion dose and serum concentration was established by measuring 3,4-DAP serum levels after 24-h infusion of 0 mg / kg·h, 0.36 mg / kg·h, 0.72 mg / kg·h, or 1.44 mg / kg·h of 3,4-DAP (Figure 4A; n=7 rats per dose). The relationship between infusion dose rate (IDR) and CSS was determined by linear regression. CSS was linearly related to infusion dose, which showed that there was no saturation effect up to 1.44 mg / kg·h of 3,4-DAP (Figure 4B; best-fit equation is shown above the graph). No behavioral signs of neurotoxicity were observed during the infusion at any dose (see "Infusion" column in Table 1 above). Details regarding statistical comparisons are provided in Figures 7A-7B (Table 3).

[0114] Figure 5 generally shows that continuous infusion of 3,4-DAP has both symptomatic and antidotal effects in lethally poisoned rats. For efficacy studies, rats were lethally poisoned with 2.5 LD50 of BoNT / A and randomly assigned to treatment or vehicle groups. Signs of toxicity, body weight, and survival were monitored at 24-hour intervals. Grey boxes indicate the infusion period.

[0115] Figure 5A shows an overview of the experimental strategy. Catheterized rats were intoxicated by intravenous injection of 110 U / kg BoNT / A. Beginning 24-27 h after intoxication, continuous infusions were initiated with saline vehicle (n=14) or 0.54 mg / kg·h 3,4-DAP (target CSS=70 ng / mL; n=8), 0.98 mg / kg·h 3,4-DAP (target CSS=126 ng / mL; n=10), or 1.44 mg / kg·h 3,4-DAP (target CSS=186 ng / mL; n=8) 27 h after intoxication. These infusion doses were intended to produce 3,4-DAP blood levels that were within the clinical range produced by oral administration.

[0116] Figure 5B shows toxicity signs at the start of the infusion (p = 0.36; Kruskal-Wallis test), data are presented as median ± IQR. At the start of the infusion, 92.3% (36 / 39) of rats showed a cumulative toxicity sign score > 1, and there were no differences in toxicity signs between groups.

[0117] Figure 5C shows survival curves for each treatment group (Mantel-Cox log-rank test; overall p<0.0001). Trend line "a" corresponds to the saline vehicle group. Trend line "b" corresponds to the group receiving 0.54 mg / kg h of 3,4-DAP. Trend line "c" corresponds to the group receiving 0.98 mg / kg h of 3,4-DAP. Trend line "d" corresponds to the group receiving 1.44 mg / kg h of 3,4-DAP. These designations are consistent throughout panels C-E. Significance indices indicate pairwise comparisons made relative to the saline vehicle group. Median survival time for vehicle-injected rats was 2.5 days (range: 2.0-4.5 days). Of all treatment conditions, infusion of 3,4-DAP improved survival proportion (p<0.0001) and median survival time (p<0.0001) compared with vehicle. Pairwise analysis revealed a clear dose-dependent effect of 3,4-DAP infusion rate on survival. Treatment with 0.54 mg / kg·h 3,4-DAP nearly doubled median survival time compared with vehicle (4.7 days; p=0.015), but only 12.5% ​​of rats survived to day 21 (1 / 8; p=0.36 compared with vehicle). Infusion of 0.98 mg / kg·h or 1.44 mg / kg·h 3,4-DAP resulted in 90% survival (p<0.0001 compared with vehicle) and 100% survival (p<0.0001 compared with vehicle), respectively. For a target CSS of at least about 120 ng / mL, the human equivalent total daily dose can be from about 1 mg / day to about 200 mg / day. A summary of outcomes in rats receiving continuous infusion is shown in Table 2 below. [Table 2]

[0118] FIG. 5D shows the median toxicity signs over time for each group (2-way ANOVA and Tukey; overall p<0.0001). Significance indices indicate pairwise comparisons made against vehicle. Toxicity signs stabilized in surviving rats by day 2 as moderate paradoxical breathing, limb weakness, and salivation, and resolved by day 14, at which point infusion was stopped. Toxicity signs were ameliorated by infusion of 0.98 mg / kg h or 1.44 mg / kg h 3,4-DAP compared to vehicle (p<0.0001), indicating that continuous infusion maintained symptomatic benefit.

[0119] Figure 5E shows the mean ± SD of normalized body weight for surviving rats in the 0.98 mg / kg h and 1.44 mg / kg h treatment groups (two-way ANOVA; p = 0.54). After cessation of infusion, surviving rats remained active, alert, and responsive, with no rebound of symptoms until day 21 (Figure 5D), and gradually regained body weight (Figure 5E). Video recordings showed that 3,4-DAP-injected rats were active 21 days after challenge with 2.5 LD50 of BoNT / A. Video recordings were collected on day 21 after BoNT / A intoxication, and they depicted the rats' activities. Surviving rats showed high levels of activity, including grooming, exploration, and food consumption.

[0120] Figure 5F shows the median ± IQR of toxic signs in rats (n = 3) infused with 1.44 mg / kg·h 3,4-DAP from days 1 to 5. Treatment was discontinued on day 5, and toxic signs were monitored at 6-h intervals. **** =p<0.0001, *=p<0.05. Details regarding statistical comparisons are in Figure 7A-7B (Table 3). To determine whether the detoxification effect required continuous infusion of 3,4-DAP, BoNT / A-intoxicated rats (n=3) were infused with 1.44 mg / kg h of 3,4-DAP from days 1 to 5 and then switched to saline vehicle. This time point of day 5 was chosen because (1) 100% of vehicle-injected rats died by day 5 (indicating a decline in respiratory function below a level sufficient for survival) and (2) toxicity signs reached a maximum between days 3 and 5 in 3,4-DAP-injected surviving rats (suggesting that paralysis had reached peak severity). After cessation of 3,4-DAP infusion, toxicity signs worsened within 3 to 6 hours, and all rats died within 12 hours. The need for continued treatment with 3,4-DAP beyond day 5 suggested that the effectiveness of detoxification resulted from a sustained symptomatic benefit.

[0121] Rats surviving a lethal BoNT / A challenge showed significant recovery of neurophysiological function from days 5 to 21. Figure 6 generally shows the time-dependent recovery of diaphragm endplate potentials in intoxicated rats infused with 3,4-DAP. To identify mechanisms contributing to the recovery of respiratory function in 3,4-DAP-treated rats, evoked endplate potentials (EPPs) and spontaneous miniature endplate potentials (mEPPs) were recorded from diaphragm muscle fibers and compared between naive rats, surviving rats euthanized 21 days after BoNT / A challenge (7 days after cessation of 3,4-DAP infusion), and rats euthanized 5 days after BoNT / A challenge (4 days after the start of infusion of 1.44 mg / kg h 3,4-DAP; Figure 3A). Phrenic nerve stimulation with trains of 10 impulses at 0.2 Hz produced EPPs with 100% success rate in naive rats (Figure 3A).

[0122] Figures 6B-E show scatter plots of the mean ± SEM for (B) EPP amplitude, (C) mEPP frequency, (D) QC, and (E) mEPP amplitude. For B-E, means were compared using two-way ANOVA followed by Tukey's multiple comparison test.**** =p<0.0001. Details on statistical comparisons are in Figure 7A-B (Table 3). After BoNT / A treatment, the mean EPP success rate was reduced to 60.5 ± 5.3% at day 5 (p<0.0001 compared to naïve), and recovered to 75.4 ± 3.5% (0.98 mg / kg h; p<0.0001 compared to day 5) and 79.7 ± 2.5% (1.44 mg / kg h; p<0.0001 compared to day 5) at day 21. Similarly, the mean EPP amplitude (Figure 3B), miniature EPP (mEPP) frequency (Figure 3C) and quantal content (QC; Figure 3D), which estimates the number of vesicles that fuse during each nerve stimulation, were significantly suppressed at day 5 and partially recovered at day 21. In contrast, mEPP amplitude was unchanged after intoxication, consistent with the finding that BoNT reduced mEPP frequency but not quantal size (Fig. 6E). There were no differences in EPP amplitude (p>0.99), mEPP frequency (p>0.99), or QC (p>0.99) between the 0.98 mg / kg·h and 1.44 mg / kg·h treatment groups, indicating that infusion of 3,4-DAP from days 1 to 14 had no dose-dependent effect on the recovery of neurotransmission at day 21. That is, 3,4-DAP infusion maintained survival during a period when phrenic neurotransmission was markedly suppressed.

[0123] (Consideration) The examples described herein show that continuous infusion of 3,4-DAP from day 1 to day 14 has dose-dependent symptomatic and antidote efficacy in rats challenged with 2.5 LD50 of BoNT / A. Rats remained asymptomatic after treatment was discontinued, despite neurophysiological evidence of residual damage in the diaphragm motor endplate. Collectively, these data show that infusion of a human-equivalent dose of 3,4-DAP maintained a symptomatic benefit that may allow survival from lethal exposure to botulinum.

[0124] During the development of a continuous infusion system to prolong the pharmacodynamic effects of 3,4-DAP in the treatment of botulism toxemia, it was determined that the 3,4-DAP infusion rate could be adjusted to maximize symptomatic benefit while avoiding the neurological effects of high doses and breakthrough of symptoms between doses. There was no evidence of functional toxicity or loss of efficacy throughout the infusion period, suggesting that a 13-day continuous infusion could be well tolerated. By extension, the sustained effectiveness of 3,4-DAP in treating botulism suggests that continuous infusion may also be effective in treating patients with LEMS.

[0125] Compared with the marked decrease in neurotransmission observed in endplate recordings at day 5, neurotransmission was markedly improved in isolated diaphragms from surviving rats at day 21, although it was still suppressed compared to naive rats. These data indicate that (1) the symptomatic benefit of ≥0.98 mg / kg h 3,4-DAP on cholinergic neurotransmission is sufficient to maintain survival at an otherwise lethal time point, and (2) synaptic function is sufficiently restored by day 14 to support survival after cessation of 3,4-DAP. Nonetheless, these data clearly demonstrate a marked acute suppression of neurotransmission at day 5 and a marked recovery at day 21 in surviving rats after lethal challenge with BoNT / A.

[0126] Symptomatic food-borne botulism cases typically require mechanical ventilation for a median duration of 1.5 to 2 weeks. The primary clinical benefit of administering antitoxin after the onset of symptoms is a reduction in the duration of disease, suggesting that the antitoxin reduces the intracellular toxin load and therefore the severity of intoxication. Because the efficacy of 3,4-DAP is inversely related to the severity of intoxication, symptomatic treatment with 3,4-DAP may be expected to be more effective in reversing the symptoms of botulism in patients treated with the antitoxin. 3,4-DAP acts orthogonally to the antitoxin and has a low risk of adverse drug interactions. Multimodality treatment with antitoxin and 3,4-DAP may offer several advantages over monotherapy with either drug by accelerating recovery from botulism, reducing the risk of life-threatening hospital-acquired disease, reducing treatment costs, and freeing up limited resources for other critically ill patients. Combined with an intracellular antidote that removes or inactivates the BoNT light chain, this multimodality therapy may be a comprehensive treatment strategy for botulism. Furthermore, the majority of botulism patients continue to exhibit neuropathy and muscle weakness after discharge. Although the pathophysiology responsible for the persistent symptoms remains unknown, they may be related to suppressed neurotransmission (as suggested by Figure 3) and may be similarly susceptible to 3,4-DAP treatment.

[0127] Previous efforts to understand the mechanism of BoNT toxicity on neurotransmission have relied on either supraphysiological intoxication of isolated diaphragm muscle preparations or local administration of paralytic doses to skeletal muscle, neither of which recapitulate the toxicokinetics of a lethal systemic challenge.This was the first animal model in which neurotransmission changes in response to a lethal systemic botulinum challenge were functionally correlated with physiological metrics during recovery.

[0128] Cases of botulism usually involve less than 5LD50, but in rare cases, exposure may exceed 100LD50. Without wishing to be bound by a particular theory, it is believed that 3,4-DAP may be less effective in more severely paralyzed muscles, suggesting that 3,4-DAP may have reduced efficacy in cases involving higher BoNT doses. However, such reduced efficacy may be at least partially reversed as neuromuscular junction repair progresses, thereby reducing overall recovery time.

[0129] In summary, continuous infusion of 3,4-DAP had both symptomatic and antidote effects in rats challenged with a lethal dose of BoNT / A. Therapeutic benefit emerged at 3,4-DAP exposure levels produced by standard clinical dosing. Survival required >5 days of 3,4-DAP infusion, suggesting that the antidote outcome emerged from a sustained symptomatic effect. These data indicate the strong translational potential of 3,4-DAP as a treatment for clinical botulism caused by the serotypes most commonly associated with human disease.

[0130] The present disclosure now being fully described, it will be appreciated by those skilled in the art that the present disclosure can be practiced within a wide and equivalent range of conditions, formulations, and parameters without affecting the scope of the present invention or any aspect thereof.

[0131] Other aspects of the present disclosure will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. It is intended that the specification be considered as exemplary only, with a true scope and spirit of the invention being indicated by the appended claims.

[0132] All patents, patent applications and publications cited herein are hereby incorporated by reference in their entirety.

Claims

1. 1. A composition for treating botulism in a subject in need thereof, the composition comprising 3,4-diaminopyridine or a pharmaceutically acceptable salt thereof in a pharmaceutically acceptable vehicle, wherein an effective amount of 3,4-diaminopyridine or an equivalent amount of the pharmaceutically acceptable salt thereof in the pharmaceutically acceptable vehicle is administered intravenously to the subject by continuous infusion.

2. 10. The composition of claim 1, wherein the effective amount of 3,4-diaminopyridine is infused at a rate of from about 0.5 mg per kg of the subject's body weight per hour to about 3 mg per kg of the subject's body weight per hour.

3. 3. The composition of claim 2, wherein the effective amount of 3,4-diaminopyridine is infused at a rate of about 1.4 mg per kg of body weight of the subject per hour.

4. 10. The composition of claim 1, wherein the effective amount of 3,4-diaminopyridine is provided in a total daily dose ranging from about 80 mg to about 160 mg of 3,4-diaminopyridine or an equivalent amount of a pharmaceutically acceptable salt thereof.

5. The composition of claim 4 , wherein the subject is a human subject.

6. 10. The composition of claim 1, wherein administration of the 3,4-diaminopyridine or an equivalent amount of a pharmaceutically acceptable salt thereof achieves a steady state plasma concentration of about 120 ng / mL of 3,4-diaminopyridine in the subject.

7. 10. The composition of claim 1, wherein the total daily dose of 3,4-diaminopyridine or an equivalent amount of a pharmaceutically acceptable salt thereof is administered by continuous infusion for multiple consecutive days.

8. The composition of any one of claims 1 to 7, wherein the botulism is caused by botulinum neurotoxin serotype A.

9. The composition of any one of claims 1 to 7, wherein the botulism comprises localized botulinum neurotoxin intoxication.

10. 1. A composition for treating botulism in a subject in need thereof, the composition comprising 3,4-diaminopyridine or a pharmaceutically acceptable salt thereof in a pharmaceutically acceptable vehicle, wherein an effective amount of 3,4-diaminopyridine or an equivalent amount of the pharmaceutically acceptable salt thereof in the pharmaceutically acceptable vehicle is administered to the subject by multiple single bolus injections.

11. 11. The composition of claim 10, wherein each of the multiple single bolus injections comprises from about 1 mg to about 3 mg of 3,4-diaminopyridine per kg of the subject's body weight, or an equivalent amount of a pharmaceutically acceptable salt thereof.

12. 12. The composition of claim 11, wherein each of the multiple single bolus injections comprises about 2 mg of 3,4-diaminopyridine per kg of the subject's body weight, or an equivalent amount of a pharmaceutically acceptable salt thereof.

13. 11. The composition of claim 10, wherein the effective amount of 3,4-diaminopyridine or an equivalent amount of a pharmaceutically acceptable salt thereof is provided in a total daily dose range of about 80 mg to about 160 mg of 3,4-diaminopyridine.

14. The composition of claim 13 , wherein the subject is a human subject.

15. 14. The composition of claim 13, wherein administration of the 3,4-diaminopyridine or an equivalent amount of a pharmaceutically acceptable salt thereof achieves a steady state plasma concentration of about 120 ng / mL of 3,4-diaminopyridine in the subject.

16. 11. The composition of claim 10, wherein the total daily dose of 3,4-diaminopyridine or an equivalent amount of a pharmaceutically acceptable salt thereof is administered during each of multiple consecutive days.

17. The composition of any one of claims 10 to 16, wherein the botulism is caused by botulinum neurotoxin serotype A.

18. The composition of any one of claims 10 to 16, wherein the botulism comprises localized botulinum neurotoxin intoxication.

19. 1. A composition for treating botulism in a subject in need thereof, the composition comprising 3,4-diaminopyridine phosphate, wherein an effective amount of 3,4-diaminopyridine phosphate is orally administered to the subject.

20. 20. The composition of claim 19, wherein the effective amount of 3,4-diaminopyridine phosphate is provided in a total daily dose equivalent to about 80 mg to about 160 mg of 3,4-diaminopyridine free base.

21. 20. The composition of claim 19, wherein the total daily dose of 3,4-diaminopyridine phosphate is administered in multiple single doses per day.

22. 20. The composition of claim 19, wherein administration of the 3,4-diaminopyridine phosphate achieves a steady-state plasma concentration of about 120 ng / mL of 3,4-diaminopyridine in the subject.

23. 20. The composition of claim 19, wherein the subject is a human subject.

24. 20. The composition of claim 19, wherein the total daily dose of 3,4-diaminopyridine phosphate is administered during each of multiple days.

25. The composition of any one of claims 19 to 24, wherein the botulism is caused by botulinum neurotoxin serotype A.

26. 25. The composition of any one of claims 19 to 24, wherein the botulism comprises localized botulinum neurotoxin intoxication.

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