Therapeutic composition, method and use for controlling epileptic seizure

Abstract

Described herein are compositions comprising bumetanib dibenzylamide for use in the treatment of selected conditions of the central and peripheral nervous system using non-synaptic mechanisms. More specifically, the present disclosure relates to methods and compositions for treating neurological disorders by administering an agent that disrupts hypersynchronous neuronal activity without reducing neuronal excitability. These compositions are useful in seizure conditions, epilepsy, and related indications.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 398,480, filed on August 16, 2022, which is incorporated herein by reference in its entirety. Technical Field

[0003] Described herein are compositions comprising Bumetanide Dibenzylamide for use in treating selected conditions of the central and peripheral nervous systems using non-synaptic mechanisms. More specifically, the disclosure relates to methods and compositions for treating neurological disorders by administering agents that disrupt hypersynchronous neuronal activity without reducing neuronal excitability. These compositions are useful for seizure disorders, epilepsy, and related indications. Background Art

[0004] Epilepsy is characterized by abnormal discharges of brain neurons and is usually manifested as various types of epileptic seizures. Many anticonvulsants originally developed for the treatment of epilepsy and other epileptic seizure disorders have also been applied to non-epileptic conditions, including neuropathic pain, mood disorders (such as bipolar disorder) and the treatment of schizophrenia (for a review of the use of anti-epileptic drugs in the treatment of non-epileptic conditions, see Rogawski and Loscher, "Natural Medicine (Nat. Medicine)", 10: 685-692, 2004). Therefore, it has been shown that epilepsy, neuropathic pain and mood disorders have a common pathophysiological mechanism (Rogawski and Loscher, supra; Ruscheweyh and Sandkuhler, "Pain (Pain)" 105: 327-338, 2003), i.e., a pathological increase in neuronal excitability, and an inappropriately high frequency of corresponding neuronal spontaneous discharges. However, only some and not all anti-epileptic drugs are effective in treating neuropathic pain, and further, such anti-epileptic drugs are only effective in certain subsets of patients with neuropathic pain (McCleane, Expert. Opin. Pharmacother. 5:1299-1312, 2004).

[0005] Epileptiform activity is identified as spontaneous synchronous discharges of neuronal populations that can be measured using electrophysiological techniques. This synchronous activity that distinguishes epileptiform activity from non-epileptic activity is called "hypersynchronization" because it describes a state in which individual neurons become increasingly likely to discharge each other in a time-locked manner. In experimental models of epilepsy, hypersynchronous activity is usually induced by increasing excitatory synaptic currents or by reducing inhibitory synaptic currents. Therefore, it is assumed that high excitability itself is a decisive feature involved in the generation and maintenance of epileptiform activity. Similarly, it is believed that neuropathic pain involves the conversion of neurons involved in pain transmission from a normal sensitive state to a supersensitive state (Costigan and Woolf, Journal of Pain (Jnl.Pain) 1: 35-44, 2000). Therefore, the development of treatments for both epilepsy and neuropathic pain has focused on inhibiting neuronal hyperexcitability by either: (a) inhibiting action potential generation; (b) increasing inhibitory synaptic transmission; or (c) reducing excitatory synaptic transmission.

[0006] Most of the drugs currently used for treatment target synaptic activity in excitatory pathways by, for example, regulating the release or activity of excitatory neurotransmitters, enhancing inhibitory pathways, blocking ion channels involved in pulse generation, and / or acting as membrane stabilizers. Therefore, conventional drugs and treatment methods for treating epilepsy and neuropsychiatric disorders reduce neuronal excitability and inhibit synaptic discharges. A serious disadvantage of these therapies is that they are non-selective and exert their effects on both normal and abnormal neuronal populations. This leads to negative and unplanned side effects, which may affect normal CNS functions such as cognition, learning and memory, and have adverse physiological and psychological effects on the treated patients. Common side effects include excessive sedation, dizziness, memory loss and liver damage. However, it has been shown that hypersynchronous epileptiform activity may not be associated with hyperexcitability, and furosemide, a cation chloride cotransport inhibitor, can reversibly block synchronous discharges without reducing hyperexcitatory synaptic responses (Hochman et al. Science 270: 99-102, 1995).

[0007] The cation-chloride cotransporters (CCCs) are important regulators of neuronal chloride concentrations and are believed to influence cell-to-cell communication and various aspects of neuronal development, plasticity, and trauma. The CCC gene family consists of three major classes: Na + -C1 - cotransporter (NCC), K + -C1 - cotransporter (KCC) and Na + -K + -2C1- Cotransporter (NKCC). The cotransport of Na-K-Cl in all cells and tissues is inhibited by loop diuretics, including furosemide, bumetanide and benzmetanide. Espinosa et al. and Ahmad et al. have previously proposed that furosemide may be used to treat certain types of epilepsy (Medicina Espanola 61:280-281, 1969; and Brit. J. Clin. Pharmacol. 3:621-625, 1976). Bumetanide is a more effective drug potentially used to treat epilepsy, but it also has a more obvious diuretic effect. Therefore, there is a continuing need for methods and compositions for treating neuronal disorders, which are not diuretics and destroy hypersynchronous neuronal activity without reducing the neuronal excitability and spontaneous synchronization required for the normal function of the peripheral and central nervous systems. Summary of the invention

[0008] In one embodiment of the present disclosure, bumetanide dibenzylamide exhibits anti-epileptic seizure activity in a non-human primate model and shows signs of NKCC inhibition in a rat anxiolytic bioassay by a similar response to bumetanide. In contrast, although bumetanide exhibits an effective diuretic effect, treatment with bumetanide dibenzylamide did not increase urine output in a primate model. Comparison of the pharmacological activity of bumetanide and bumetanide dibenzylamide highlights the novel properties of bumetanide dibenzylamide as a potential anti-epileptic seizure therapeutic agent that is not limited by diuretic effects. In summary, the data of the present disclosure, together with published studies describing the anti-epileptic seizure effects of bumetanide and furosemide, support the novel and unexpected properties of bumetanide dibenzylamide as an adjunctive anti-epileptic seizure treatment. The observed antiseizure effects of bumetanide and furosemide are believed to be mediated through their antagonism of neuronal and / or glial NKCC1, whereas their diuretic effects are mediated through antagonism of renal NKCC2.

[0009] One embodiment of the present disclosure includes bumetanide derivatives and compositions containing such compounds that exhibit therapeutically effective seizure blocking effects without substantial diuretic effects.

[0010] One embodiment of the present disclosure includes bumetanide derivatives and compositions containing such compounds that exhibit therapeutically effective seizure blockade without substantial diuresis, wherein the ratio of seizure control to diuresis provides the therapeutic effect or therapeutic coefficient. The therapeutic effect is the ratio of a measure of seizure inhibition (e.g., frequency of seizures, intensity of seizures, etc.) to a measure of diuresis of the therapeutic compound (e.g., urine volume, urine ion concentration, etc.).

[0011] One embodiment of the disclosure includes a therapeutic effect based on changes in seizure frequency and plasma osmolality as a measure of dehydration. One embodiment of the disclosure includes a therapeutic effect based on an increase in the interspike interval and changes in plasma osmolality as a measure of dehydration. One embodiment of the disclosure includes a therapeutic effect based on changes in seizure frequency and urine production over a given time period. One embodiment of the disclosure includes a therapeutic effect based on an increase in the interspike interval and changes in urine production over a given time period.

[0012] One embodiment of the present disclosure includes a therapeutic effect based on a reduction in the height or amplitude of seizure spikes and a change in plasma osmolality as a measure of dehydration. One embodiment of the present disclosure includes a therapeutic effect based on a reduction in the height or amplitude of seizure spikes and a change in urine production over a given time period. One embodiment of the present disclosure includes a therapeutic effect based on seizure frequency and changes in blood ions over time, wherein the ions are selected from sodium, chloride, or magnesium. One embodiment of the present disclosure includes a therapeutic effect based on an increase in the peak-to-peak interval and changes in blood ions over time, wherein the ions are selected from sodium, chloride, magnesium, or pH. One embodiment of the present disclosure includes a therapeutic effect based on a reduction in the height or amplitude of seizure spikes and changes in blood ions over time, wherein the ions are selected from sodium, chloride, or magnesium.

[0013] A change in seizure activity can be a change in the amplitude and / or frequency of pharmacologically or electrically induced seizure (epileptiform) activity as measured with an EEG or other type of electrophysiological recording. A change in seizure activity can be a change in the number of unprovoked seizures over a period of time (e.g., seizures per day, week, or month).

[0014] In one embodiment, the therapeutic effect is defined as the proportional change in seizure frequency or magnitude and urine output compared to baseline. In one embodiment, the therapeutic effect is the proportional change in seizure frequency or magnitude in any objective determination. In one embodiment, the therapeutic effect is the proportional change in seizure frequency or magnitude before and after treatment with a therapeutic compound.

[0015] In one embodiment, the change in frequency after treatment is a decrease in the frequency of seizures by at least 50%. In one embodiment, the change in diuresis after treatment is an increase in urine production of less than about two times in twenty-four hours. In one embodiment, the change in frequency after treatment is a decrease in the frequency of seizures by more than 50%. In one embodiment, the change in diuresis after treatment is no increase in urine production in twenty-four hours.

[0016] In one embodiment, the change in frequency after treatment is a decrease in the frequency of seizures by about 50% to 100%. In one embodiment, the change in diuresis after treatment is an increase in urine production within a twenty-four hour period in the range of about 0% to about 100%.

[0017] In one embodiment, the therapeutic effect of a particular dose of a therapeutic compound is defined as follows:

[0018]

[0019] The dose of the therapeutic compound may be lower than the dose required to completely block seizure activity. If the dose is higher than the amount required to completely block seizure activity, a therapeutic effect different from zero is determined, and the comparative effects of different therapeutic compounds are determined.

[0020] One embodiment of the present disclosure includes bumetanide derivatives and compositions containing such compounds that provide unexpectedly improved increases in seizure control when compared to other bumetanide derivatives. In other words, not all derivatives of bumetanide provide this effect. Rather, the amide derivatives appear to provide a unique effect.

[0021] One embodiment of the present disclosure includes bumetanide derivatives and compositions containing such compounds that provide unexpectedly improved reduction in diuresis when compared to other bumetanide derivatives.

[0022] One embodiment of the present disclosure includes bumetanide derivatives and compositions containing such compounds that demonstrate a reduction in both seizure amplitude and frequency.

[0023] One embodiment of the present disclosure includes bumetanide derivatives and compositions containing such compounds for use in methods or uses for epileptic seizure inhibition.

[0024] One embodiment of the present disclosure includes bumetanide derivatives and compositions containing such compounds that show a positive effect on synchronous activity without a substantial effect on excitability. One aspect includes specific bumetanide derivatives and compositions containing such compounds that provide a window of therapeutic effect.

[0025] One embodiment of the present disclosure includes one or more bumetanide derivatives and compositions containing such compounds that provide seizure inhibition comprising reducing spike amplitude and the interval between spikes. In one aspect, the amplitude is reduced by about 50% to about 99%, and the interval is reduced by about 50% to about 99%. In one aspect, the amplitude approaches zero (0) and the interval approaches infinity.

[0026] One embodiment of the present disclosure includes bumetanide derivatives and compositions containing such compounds administered at a therapeutically effective dose, which is one or more doses required to observe an epileptic seizure inhibitory / therapeutic effect. One aspect of the present disclosure includes determining the diuretic factor for the proposed anti-epileptic seizure treatment and then calculating the corresponding therapeutic factor. One aspect of the present disclosure includes the unique relationship between the dose of bumetanide amide derivatives and the lack of diuresis. The bumetanide amide derivatives provide unexpected benefits compared to other bumetanide derivatives. Testing has shown that the effects of different derivatives and different formulations are significantly different.

[0027] One embodiment of the present disclosure includes a method or use of treating a patient refractory to conventional anticonvulsant drugs, comprising administering bumetanide dibenzylamide. The present disclosure includes methods and uses of providing particular benefit to patients with epilepsy who are not well controlled by conventional therapy (e.g., one or more of phenytoin, carbamazepine, valproate, lamotrigine, levetiracetam, ethosuximide, phenobarbital, and topiramate) or who are otherwise refractory to conventional therapy by administering to the patient a pharmaceutical composition comprising bumetanide dibenzylamide.

[0028] One embodiment of the present disclosure includes a pharmaceutical composition comprising bumetanide dibenzylamide, bumetanide diethylamide, or bumetanide morpholineamide, or a salt thereof, wherein the pharmaceutical composition has a therapeutic effect on seizure blockade in a patient.

[0029] One aspect includes wherein the therapeutic effect is a ratio of a measure of seizure inhibition in a patient to a measure of diuresis. One aspect includes wherein the measure of seizure inhibition is the frequency of seizures. One aspect includes wherein the measure of seizure inhibition is the intensity of seizures. One aspect includes wherein the measure of seizure inhibition is a change in the amplitude of pharmacologically or electrically induced seizure (epileptic) activity, as measured by EEG or other electrophysiological type recordings. One aspect includes wherein the amplitude is reduced by about 50% to about 99% after treatment with the composition. One aspect includes wherein the measure of seizure inhibition is a change in the frequency of pharmacologically or electrically induced seizure (epileptic) activity, as measured by EEG or other electrophysiological type recordings. One aspect includes wherein the measure of diuresis is urine volume. One aspect includes wherein the measure of diuresis is urine ion concentration. One aspect includes wherein the therapeutic effect is based on changes in seizure frequency and plasma osmotic pressure. One aspect includes wherein the therapeutic effect is based on an increase in peak-to-peak intervals. One aspect includes wherein the peak-to-peak interval is reduced by about 50% to about 99%. On the one hand, it includes wherein the therapeutic effect is based on an increase in the peak-to-peak interval and a change in plasma osmotic pressure. On the one hand, it includes wherein the therapeutic effect is based on a change in the frequency of epileptic seizures and urine production in a given time period. On the one hand, it includes wherein the therapeutic effect is based on an increase in the peak-to-peak interval and a change in urine production in a given time period. On the one hand, it includes wherein the therapeutic effect is based on a decrease in the peak height or amplitude of epileptic seizures and a change in plasma osmotic pressure. On the one hand, it includes wherein the therapeutic effect is based on the effect of a decrease in the peak height or amplitude of epileptic seizures and a change in urine production in a given time period. On the one hand, it includes wherein the therapeutic effect is based on the effect of changes in the frequency of epileptic seizures and blood ions over time, wherein the ions are selected from sodium, magnesium chloride or pH. On the one hand, it includes wherein the therapeutic effect is based on an increase in the peak-to-peak interval and the effect of changes in blood ions over time, wherein the ions are selected from sodium, magnesium chloride or pH. On the one hand, it includes wherein the therapeutic effect is based on the effect of a decrease in the peak height or amplitude of epileptic seizures and the effect of changes in blood ions over time, wherein the ions are selected from sodium, magnesium chloride or pH. On the one hand, it includes wherein the therapeutic effect is a change in the ratio of the frequency or amplitude of epileptic seizures to the amount of urine compared to the baseline. On the one hand, it includes wherein the therapeutic effect is a change in the ratio of the frequency or amplitude of epileptic seizures in any objective determination. One aspect includes wherein the therapeutic effect is a change in the ratio of the frequency or amplitude of epileptic seizures before and after treatment with the composition. One aspect includes wherein the therapeutic effect is a change in the ratio of the frequency and amplitude of epileptic seizures before and after treatment with the composition. One aspect includes wherein the change in the frequency of epileptic seizures after treatment with the composition is a decrease in the frequency of epileptic seizures by at least 50%. One aspect includes wherein the change in the frequency of epileptic seizures after treatment with the composition is a decrease in the frequency of epileptic seizures by more than 50% to 100%. One aspect includes wherein the measure of diuresis is an increase in urine production of less than about two times within twenty-four hours after treatment with the composition.One aspect includes wherein the measure of diuresis is no increase in urine production within 24 hours after treatment with the composition. One aspect includes wherein the measure of diuresis is an increase in urine production within 24 hours after treatment with the composition by about 0% to about 100%. One aspect includes wherein the therapeutic effect is determined based on the effective dose of the composition. One aspect includes wherein the therapeutic effect is determined as:.

[0030]

[0031] One aspect includes wherein the effective dose of the composition is the dose required to completely block epileptic seizure activity. One aspect includes wherein the effective dose of the composition is greater than the dose required to completely block epileptic seizures. One aspect includes wherein the effective dose of the composition is a dose that causes seizure inhibition without producing a diuretic effect. One aspect includes wherein the composition has a positive effect on neuronal synchronous activity without a substantial effect on neuronal excitability. One aspect includes wherein the composition provides a therapeutic effect window. One aspect is a composition comprising bumetanide dibenzylamide. One aspect is a composition comprising bumetanide morpholine amide.

[0032] One embodiment of the present disclosure includes a method for treating seizures in a patient, the method comprising: administering a pharmaceutical composition of the present disclosure; and reducing seizure activity in the patient without increasing urine output in the patient.

[0033] In one aspect, the pharmaceutical composition is administered orally. In one aspect, the pharmaceutical composition is administered once. In one aspect, the pharmaceutical composition is administered once a day for a fixed number of consecutive days. In one aspect, the anti-epileptic seizure effect of the pharmaceutical composition is mediated by its antagonism of NKCC1 of neurons and / or glial cells. In one aspect, the diuretic effect of the pharmaceutical composition is mediated by its antagonism of renal NKCC2. In one aspect, the pharmaceutical composition is administered once a day for a fixed number of consecutive days. In one aspect, the pharmaceutical composition is administered to treat epilepsy. In one aspect, the pharmaceutical composition is administered in combination with conventional therapy to treat epileptic seizures. In one aspect, urine volume is measured by an imbalance in blood ion concentrations. In one aspect, urine volume is measured by the magnitude of the diuretic effect calculated as the amount (concentration) of bumetanide in the blood compared to bumetanide dibenzylamide. In one aspect, the onset of action of bumetanide dibenzylamide, as measured by a reduction in the frequency of epileptic seizures, is faster than that of conventional anti-epileptic drugs. In one aspect, a reduction in seizure frequency is measured by one or more of: a time increment selected from one or more of hours, days, weeks, and months; a reduction in seizure activity from a seizure diary and records; an increase in one or more of interictal (between) and post-ictal (after) spikes; and a reduction in interictal activity as measured by EEG.

[0034] Although not specifically described, one or more embodiments or aspects may be incorporated into different embodiments or aspects. That is, all embodiments and aspects may be combined in any way to form another embodiment. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 Results using furosemide in a seizure model are presented.

[0036] Figure 2 The effects of furosemide on afterdischarge (AD) threshold are demonstrated.

[0037] Figure 3 Results using Keppra® in an epileptic seizure model are presented.

[0038] Figure 4 Demonstrated electrode placement in a primate epileptic seizure model.

[0039] Figure 5 Representative EEG and AD activities are shown.

[0040] Figure 6 AD activity, duration, mean spike height measurements, and envelope area are presented.

[0041] Figure 7 The effects of the bicuculline spike are demonstrated.

[0042] Figure 8 The comparative effects of NPT 2024 and bumetanide on the inter-peak interval are shown.

[0043] Fig. 9 The comparative effects of NPT 2024 and bumetanide on spikes per minute are shown.

[0044] Fig.10 The comparative effects of NPT 2024 and bumetanide on mean spike height are shown.

[0045] Fig.11 The comparative effects of NPT 2024 and bumetanide on urine production over time are presented.

[0046] Fig.12 The stability of spikes from acute bicuculline lesions recorded with surface EEG electrodes is demonstrated.

[0047] Fig.13 demonstrated that Keppra ® Comparative efficacy of bumetanide; IV administration.

[0048] Fig.14The top and bottom of the bicuculline-generated spike identified by computer are shown.

[0049] Fig.15 Representative graphs of spike height measurements in the bicuculline lesion model are shown.

[0050] Fig.16 Shown are EEG recordings from bicuculline lesions following IV bumetanide and bumetanide dibenzylamide (NPT 2042) administration: pre-treatment, post-treatment, and recovery.

[0051] Fig.17 Oral bumetanide and NPT 2042: Surface EEG recordings from bicuculline lesions are presented.

[0052] Fig.18 Shown are changes in urine volume production over time in cynomolgus monkeys following oral administration of bumetanide and NPT 2042.

[0053] Fig.19 The fear-potentiated startle model is presented.

[0054] Fig. 20 Data for fear-potentiated startle in rats are presented: unit startle reflex amplitude per unit voltage.

[0055] Fig.21 The percent change in urine production over time is presented to show the comparative effects of bumetanide, bumetanide morpholineamide, bumetanide diethylamide, and bumetanide dibenzylamide on diuresis.

[0056] Fig. 22 Urine production rates are presented to show the comparative effects of bumetanide, bumetanide morpholineamide, bumetanide diethylamide, and bumetanide dibenzylamide on diuresis (urine rate mL / min).

[0057] Fig.23 Mean urine production rates after treatment are presented to show the comparative effects of bumetanide, bumetanide morpholineamide, bumetanide diethylamide, and bumetanide dibenzylamide on diuresis.

[0058] Fig.24 Shown are mean pre-treatment urine rates (mL / min) compared to maximum post-treatment urine rates (mL / min) following treatment with bumetanide methyl ester.

[0059] Fig.25 Shown are mean pre-treatment urine rates (mL / min) compared to maximum post-treatment urine rates (mL / min) following treatment with bumetanide cyanomethyl ester.

[0060] Fig.26Shown are mean pre-treatment urine rates (mL / min) compared to maximum post-treatment urine rates (mL / min) following treatment with bumetanide NN-diethyl-2-hydroxyacetamide ester.

[0061] Fig. 27 Shown are mean pre-treatment urine rates (mL / min) compared to maximum post-treatment urine rates (mL / min) following treatment with bumetanide benzyl ester.

[0062] Fig.28 The effects of bumetanide dibenzylamide (NPT 2042) administration on human blood urea nitrogen are presented.

[0063] Fig.29 The effect of bumetanide dibenzylamide (NPT 2042) administration on human creatinine is presented.

[0064] Fig.30 The effects of bumetanide dibenzylamide (NPT 2042) administration on human serum chloride are presented.

[0065] Fig.31 The effect of bumetanide dibenzylamide (NPT 2042) administration on human urine specific gravity is presented. DETAILED DESCRIPTION

[0066] definition

[0067] As used herein, the terms "active ingredient," "active pharmaceutical ingredient" and "API" refer to an agent, active ingredient, compound or substance, composition or mixture thereof that provides a (usually beneficial) pharmacological effect.

[0068] As used herein, the term "dose" refers to any form of formulation containing an amount of active ingredient sufficient to produce a therapeutic effect by a single administration.

[0069] As used herein, the term "dose" refers to a specific amount, quantity, and frequency of administration of a dosage over a specified period of time, usually one (1) day.

[0070] As used herein, the term "active pharmaceutical ingredient loading" or "drug loading" refers to the amount (mass) of active pharmaceutical ingredient included in a single softgel capsule fill.

[0071] As used herein, the term "formulation" or "pharmaceutical composition" or "composition" refers to a drug in combination with a pharmaceutically acceptable excipient.

[0072] As used herein, the term average "particle size distribution" (PSD) refers to the average particle size of a statistical distribution of a range of particle sizes as described herein. The distribution may be Gaussian, normal or non-normal.

[0073] Terms such as "d90", "d50", and "d10" refer to the percentage of particle sizes that are less than a specified size, range, or distribution (e.g., 90%, 50%, or 10%, respectively). For example, "d90 ≤ 100 μm" means that 90% of the particle sizes within the particle distribution are less than or equal to 100 μm.

[0074] As used herein, the term "patient" refers to any subject including mammals and people. The patient may suffer from a disease or be suspected of having a disease and is therefore being treated with medication. In some cases, the patient is a mammal, such as humans, non-human primates, dogs, cats, horses, cattle, goats, pigs, rabbits, rats, mice, or its premature infants, newborns, infants, infancy, teenagers or adults. In some cases, as used herein, the term "patient" refers to people (e.g., men, women or children). In some cases, as used herein, the term "patient" refers to an experimental animal in an animal model study. The patient or subject can be any age, sex or a combination thereof.

[0075] As used herein, the term "biological sample" or "sample" refers to a sample obtained or derived from a patient. For example, a biological sample includes material selected from the group consisting of body fluids, blood, whole blood, plasma, serum, mucous secretions, saliva, cerebrospinal fluid (CSF), bronchoalveolar lavage fluid (BALF), urine, eye fluid (e.g., vitreous humor, aqueous humor), lymph fluid, lymph node tissue, spleen tissue, bone marrow, and fluid from the auditory cavity.

[0076] The term "treat" refers to administering treatment in an amount, manner or pattern effective (eg, therapeutically effective) to improve a condition, symptom, disorder or parameter associated with a disorder or the likelihood thereof.

[0077] The following abbreviations may be used in connection with pharmacological descriptions:

[0078]

[0079] The term "prevent" or "preventing" refers to preventing or reducing the progression of a disorder to a statistically significant extent or to an extent detectable by one skilled in the art.

[0080] As used herein, the terms "substantially" or "substantially" mean to a great or significant extent but not completely.

[0081] As used herein, the term "about" refers to any value, including integers and decimal places within a variation of up to ±10% of the value modified by the term "about".

[0082] Also described herein are pharmaceutical compositions and dosage forms comprising one or more agents that reduce the rate of decomposition of the compositions as described herein as active ingredients. Such agents, referred to herein as "stabilizers," include but are not limited to antioxidants such as ascorbic acid, pH buffers, salts, sugars, and the like.

[0083] The phrase 'solubilizer' is used to refer to an ingredient or group of ingredients that aids in solubilizing a composition or a part of a composition.

[0084] The phrase and term "can be administered by injection," "injectable," or "syringability" refers to a combination of factors such as a certain force applied to the plunger of a syringe containing a formulation described herein and a needle of a given inner diameter connected to the outlet of such syringe at a certain temperature, and the time required to extrude a certain volume of the bumetanide dibenzylamide composition from the syringe through the needle.

[0085] The ranges for each ingredient in the described formulations represent the space in which suitable substitutions can be obtained by combining with other ingredients in ratios adjusted to total 100% w / w. The ranges provided are estimates based on available data.

[0086] Pharmaceutical compositions of the present disclosure

[0087] One embodiment described herein is a pharmaceutical composition comprising bumetanide dibenzylamide. In one aspect, the composition comprises any of the formulations shown in the tables or examples described herein. Any of the components in the formulations described herein, shown in the tables, or illustrated in the examples may be increased, reduced, combined, substituted, or omitted to provide a formulation comprising about 100% by weight. Such compositions are hereby disclosed as if they were expressly disclosed herein.

[0088] One embodiment described herein is a pharmaceutical composition comprising bumetanide dibenzylamide and one or more solubilizing agents. Another embodiment described herein is a pharmaceutical composition comprising bumetanide dibenzylamide. Another embodiment described herein is a pharmaceutical composition further comprising one or more additional solvents. Another embodiment described herein is a pharmaceutical composition further comprising one or more surfactants, co-surfactants, emulsifiers, or wetting agents. Another embodiment described herein is a pharmaceutical composition consisting essentially of bumetanide dibenzylamide. Another embodiment described herein is a pharmaceutical composition consisting essentially of aqueous bumetanide dibenzylamide. Another embodiment described herein is a pharmaceutical composition comprising bumetanide dibenzylamide and one or more solubilizing agents. Another embodiment described herein is a pharmaceutical composition consisting essentially of bumetanide dibenzylamide and one or more solubilizing agents. In one aspect, the composition is a dry powder compressed into a tablet. In one aspect, the composition is a dry powder filled into a capsule. In one aspect, the composition is a dry powder extruded into a film. In one aspect, the composition is a dry powder extruded into a tablet. One embodiment described herein is a pharmaceutical composition comprising about 2.5 mg to about 42 mg of bumetanide dibenzylamide.

[0089] One embodiment described herein is a pharmaceutical composition formulated as an oral capsule. In one aspect, the composition includes up to about 0.25% w / w to about 15% w / w of bumetanide dibenzylamide and one or more solubilizers. In one aspect, the solubilizer is a cosolvent. In one aspect, the solubilizer is a surfactant. In one aspect, the solubilizer includes a triglyceride. In one aspect, the triglyceride includes a medium-chain triglyceride. In one aspect, the triglyceride includes a long-chain triglyceride. In one aspect, the triglyceride includes a mixture of medium-chain triglycerides and long-chain triglycerides. In one aspect, the triglyceride includes a polyoxyethylene glyceride. In one aspect, the polyoxyethylene glyceride is selected from the group consisting of lauroyl polyoxyethylene glyceride, linoleoyl polyoxyethylene glyceride, oleoyl polyoxyethylene glyceride, stearoyl polyoxyethylene glyceride, caprylocaproyl polyoxyethylene glyceride, and any combination thereof. In one aspect, the triglyceride includes a nonionic surfactant, a solubilizer, and an emulsifier. In one aspect, the long chain triglyceride is selected from the group consisting of: polyoxyethylene 35 castor oil (Kolliphor EL), monolinoleyl glyceride (Maisine CC), and any combination thereof. In one aspect, the medium chain triglyceride is selected from the group consisting of: caprylocaproyl polyoxyethylene glyceride (Labrasol ALF), phosphatidylcholine (Phosal 75SA), caprylic / capric triglyceride (Captex 300), lauroyl polyoxyethylene-32 glyceride (Gelucire 44 / 14), sorbitan esters (Span 80), and any combination thereof.

[0090] On the one hand, a pharmaceutical composition comprising one or more solubilizing agents of about 0.1% w / w to about 99.75% w / w is described herein. On the one hand, the solubilizing agent comprises soybean oil. On the one hand, the solubilizing agent is in an oil phase. On the one hand, the solubilizing agent is selected from the group consisting of peanut oil, soybean oil, castor oil, corn oil, safflower oil, olive oil, almond oil, sesame oil, cottonseed oil, sunflower seed oil, palm oil and rapeseed oil, Maisine 35-1, Maisine CC (monolinoleylglycerol) and any combination thereof. On the one hand, the solubilizing agent comprises a cosolvent. On the one hand, the solubilizing agent is selected from the group consisting of propylene glycol, Capryol™ 90 (propylene glycol monocaprylate), Lauroglycol™ 90 (propylene glycol monolaurate), glycerol, polyethylene glycol and any combination thereof. On the one hand, the solubilizing agent comprises an antioxidant. On the one hand, the solubilizing agent is selected from the group consisting of: alpha tocopherol, ascorbyl palmitate, ascorbic acid, butylated hydroxyanisole, butylated hydroxytoluene and any combination thereof. On the one hand, the solubilizing agent includes an antimicrobial preservative, a solvent and a water-soluble cosolvent. On the one hand, the solubilizing agent includes a solvent and a water-soluble cosolvent. On the one hand, the solubilizing agent is selected from the group consisting of: ethanol, propylene glycol, propylene glycol 300, propylene glycol 400, propylene glycol 600, oleyl alcohol and any combination thereof. On the one hand, the solubilizing agent is water. On the one hand, the solubilizing agent is any diluent.

[0091] In one aspect, described herein is a pharmaceutical composition comprising about 0.5% w / w to about 1.8% w / w of bumetanide dibenzylamide. In one aspect, described herein is a pharmaceutical composition comprising about 9 mg of bumetanide dibenzylamide per capsule to about 12 mg of bumetanide dibenzylamide per capsule. In one aspect, described herein is a pharmaceutical composition comprising about 0% w / w to about 1.8% w / w of bumetanide dibenzylamide, about 10% w / w to about 45% w / w of polyoxyethylene 35 castor oil (Kolliphor EL), about 15% w / w to about 65% w / w of monolinoleylglycerol (Maisine CC), about 15% w / w to about 65% w / w of soybean oil, about 0% w / w to about 15% w / w of ethanol, and about 0% w / w to about 0.13% w / w of butylated hydroxytoluene. In one aspect, described herein is a pharmaceutical composition comprising about 1.75% w / w bumetanide dibenzylamide, about 32.37% w / w polyoxyl 35 castor oil (Kolliphor EL), about 31.30% w / w glyceryl monolinoleate (Maisine CC), about 31.30% w / w soybean oil, about 3.25% w / w ethanol, and about 0.3% w / w butylated hydroxytoluene.

[0092] One embodiment described herein is a pharmaceutical composition formulated as a nasal solution. In one aspect, the composition includes a solvent system containing bumetanide dibenzylamide. In one aspect, the composition includes about 3 ml of solvent and about 28 mg of bumetanide dibenzylamide to about 32 mg of bumetanide dibenzylamide. In one aspect, the solubilizer includes a triglyceride. In one aspect, the triglyceride includes a medium chain triglyceride. In one aspect, the triglyceride includes a long chain triglyceride. In one aspect, the triglyceride includes a mixture of medium chain triglycerides and long chain triglycerides. In one aspect, the triglyceride includes a polyoxyethylene glyceride. In one aspect, the polyoxyethylene glyceride is selected from the group consisting of lauroyl polyoxyethylene glyceride, linoleoyl polyoxyethylene glyceride, oleoyl polyoxyethylene glyceride, stearoyl polyoxyethylene glyceride, caprylocaproyl polyoxyethylene glyceride, and any combination thereof.

[0093] On the one hand, the triglyceride includes a nonionic surfactant, a solubilizing agent, and an emulsifier. On the one hand, the solubilizing agent includes Labrasol ALF. On the one hand, the solvent system includes one or more solubilizing agents. On the one hand, the solubilizing agent includes Labrasol ALF and water. On the one hand, the solubilizing agent includes Labrasol ALF, propylene glycol and water. On the one hand, the solubilizing agent includes Labrasol ALF, propylene glycol, PEG-400 and water. On the one hand, the solubilizing agent includes Labrasol ALF, propylene glycol, PEG-400, vitamin E TPGS and water. On the one hand, the solubilizing agent includes Labrasol ALF, propylene glycol, PEG-400, vitamin E TPGS and water. On the one hand, the solubilizing agent includes Labrasol ALF, propylene glycol, PEG-400, vitamin E TPGS, ethanol and water. On the one hand, the solubilizing agent includes about 50 g of caprylocaproyl polyoxyethylene glyceride (Labrasol ALF) per 50 g of solvent. On the one hand, the solubilizing agent includes about 25 g of caprylocaproyl polyoxyethylene glyceride (Labrasol ALF) per 50 g of solvent and about 25 g of water. On the one hand, the solubilizing agent includes about 6 g of propylene glycol, about 40 g of PEG-400 and about 4 g of water per 50 g of solvent. On the one hand, the solubilizing agent includes about 5 g of caprylocaproyl polyoxyethylene glyceride (Labrasol ALF) per 50 g of solvent, about 6 g of propylene glycol, about 35 g of PEG-400 and about 4 g of water per 50 g of solvent. On the one hand, the solubilizing agent includes about 10 g of propylene glycol, about 35 g or PEG-400, about 0.5 g of vitamin ETPGS and about 4.5 g of water per 50 g of solvent. On the one hand, the solubilizing agent includes about 5 g of caprylocaproyl polyoxyethylene glyceride (Labrasol ALF) per 50 g of solubilizing agent, about 10 g of propylene glycol, about 28.5 g of PEG-400, about 0.5 g of vitamin E TPGS, about 1 g of ethanol and about 4.5 g of water. On the one hand, the solubilizing agent includes glycogen. On the one hand, the solubilizing agent includes a permeation agent and a solvent. On the one hand, the solubilizing agent includes ethyl oleate. On the one hand, the solubilizing agent includes an oily vehicle, a solvent and a solvent.

[0094] In one aspect, the pharmaceutical composition comprises about 3% w / v bumetanide dibenzylamide, about 11% w / w caprylocaproyl polyoxyethylene glyceride (Labrasol ALF), about 13.26% w / v propylene glycol, about 73.94% w / v PEG-400, and about 8.8% w / w water. In one aspect, the pharmaceutical composition comprises about 0.01% w / w to about 40% w / w bumetanide dibenzylamide, about 5% w / w to about 100% w / w caprylocaproyl polyoxyethylene glyceride (Labrasol ALF), about 4% w / w to about 20% w / w propylene glycol, about 50% w / w to about 80% w / w PEG-400, and about 0% w / w to about 10% w / w water. In one aspect, the pharmaceutical composition comprises about 2.73% w / w bumetanide dibenzylamide, about 8% w / w caprylocaproyl polyoxyethylene glyceride (Labrasol ALF), about 15% w / w propylene glycol, about 69.27% ​​w / w PEG-400, and about 5% w / w water. In one aspect, the pharmaceutical composition comprises about 2.73% w / w bumetanide dibenzylamide, about 16% w / w caprylocaproyl polyoxyethylene glyceride (Labrasol ALF), about 20% w / w propylene glycol, about 54.27% w / w PEG-400, and about 7% w / w water. In one aspect, the pharmaceutical composition comprises about 2.73% w / w bumetanide dibenzylamide, about 5% w / w caprylocaproyl polyoxyethylene glyceride (Labrasol ALF), about 4% w / w propylene glycol, about 78.27% w / w PEG-400, and about 10% w / w water.

[0095] One embodiment described herein is a pharmaceutical composition formulated as a rectal paste. One embodiment described herein is a composition formulated as a rectal gel. In one aspect, the composition is formulated with a target of about 6 mg of bumetanide dibenzylamide per gram of composition based on a target dose of about 30 mg of bumetanide dibenzylamide in an amount of about 5 g of the composition. In one aspect, the composition is formulated with different target doses of bumetanide dibenzylamide. In one aspect, in cases where the drug substance exhibits some instability in water, the paste is determined to be a 100% non-aqueous formulation. In one aspect, the rectal gel is formulated to include about 0.6% w / w bumetanide dibenzylamide, about 10% w / w caprylocaproyl polyoxyethylene glyceride (Labrasol ALF), about 9.15% w / w propylene glycol, about 53.84% w / w polyethylene glycol 600, about 3.6% w / w polyvinyl pyrrolidone (K30), about 2.4% w / w poloxamer 407 (P407), about 0.41% w / w sodium carboxymethylcellulose (CMC), and about 20% w / w water. In one aspect, the rectal paste is formulated to include about 0.6% w / w bumetanide dibenzylamide, about 10% w / w caprylocaproyl polyoxyethylene glyceride (Labrasol ALF), about 9.15% w / w propylene glycol, about 66.75% w / w polyethylene glycol 600, about 3.6% w / w polyvinyl pyrrolidone (K30), about 2.4% w / w poloxamer 407 (P407), and about 7.5% w / w polyethylene glycol 3350.

[0096] One embodiment described herein is a pharmaceutical composition formulated as a sublingual tablet. In one aspect, the formulated composition targets about 30 mg of bumetanide dibenzylamide per tablet. In one aspect, a small tablet size is used. In one aspect, wetting and or dissolution of the composition occurs within 30 seconds. In one embodiment, the sublingual tablet is formulated to include bumetanide dibenzylamide, one or more wetting agents, and one or more superdisintegrants. In one aspect, the sublingual tablet is formulated to include about 15%w bumetanide dibenzylamide, about 20%w Ceolus KG (microcrystalline cellulose), about 51%w Mannogem EZ (spray-dried mannitol), about 7%w cross-linked polyvinylpyrrolidone XL (superdisintegrant), about 3%w poloxamer 407 (wetting agent), about 1.5%w citric acid monohydrate, about 1%w Cabosil M5P (fumed silicon dioxide), and about 1.5%w magnesium stearate. In one embodiment, the sublingual tablet is formulated to include bumetanide dibenzylamide, one or more water-dispersible surfactants, one or more wetting agents, and one or more superdisintegrants. In one aspect, the sublingual tablet is formulated to include about 7.4%w bumetanide dibenzylamide, about 9.9%w lauroyl polyoxyethylene-32 glyceride (Gelucire 44 / 14, a water-dispersible surfactant), about 9.9%w sorbitan ester (Span 80, a water-dispersible surfactant), about 14.8%w Neusilin US2 (magnesium aluminum silicate), about 0.5%w / w poloxamer 407 (wetting agent), about 0.7%w citric acid monohydrate, about 2%w Cabosil M5P (fumed silica), about 54.3%w crospovidone XL (superdisintegrant), and about 0.5%w magnesium stearate.

[0097] General treatment methods / applications / compounds used

[0098] In one embodiment described herein, preferred therapeutic agents and methods of the present disclosure are used to treat seizures (e.g., partial onset seizures), epilepsy, and / or other indications such as neuropathic pain by modulating or disrupting the synchrony of neuronal population activity in an area of ​​increased synchrony by reducing the activity of NKCC cotransporters without a diuretic effect. In one embodiment described herein, preferred therapeutic agents and methods of the present disclosure are used to treat seizures that cannot be controlled by existing drug therapeutics, such as uncontrolled seizures, intractable seizures, refractory seizures, drug-resistant seizures, or medically resistant seizures. One embodiment described herein is a preferred therapeutic agent and method of the present disclosure for treating an epilepsy syndrome, such as Angelman syndrome, benign rolandic epilepsy, CDKL5 disorder, childhood absence epilepsy, Dravet syndrome, GLUT1 deficiency syndrome, hypothalamic hamartoma, infantile spasms (also known as West syndrome), Lennox-Gastaut, PCDH19, progressive myoclonic epilepsy, Rasmussen's encephalitis, ring chromosome 20 syndrome, or reflex epilepsy.

[0099] In one embodiment described herein, preferred therapeutic agents and methods of the present disclosure are used to treat epilepsy and / or neurological syndromes that are specific to children, including but not limited to Dravett syndrome, infantile spasms, Landau-Kleffner syndrome, Lennox-Gasto syndrome, Rasmussen Syndrome, benign Rolando epilepsy, benign occipital epilepsy, childhood absence epilepsy, juvenile myoclonic epilepsy, Rett Syndrome, Angelman syndrome, tuberous sclerosis and / or SturgeWeber Syndrome. In one embodiment described herein, preferred therapeutic agents and methods of the present disclosure are used to treat epilepsy and / or neurological syndromes that may be observed in adults or children.

[0100] One embodiment described herein is a preferred therapeutic agent and method of the present disclosure for treating one or more of the indications listed in the following table:

[0101]

[0102] One embodiment described herein is a preferred therapeutic agent and method of the present disclosure for treating epilepsy or comorbidities of epileptic seizures, such as psychiatric disorders (e.g., depression, anxiety, attention deficit hyperactivity disorder (ADHD), schizophrenia-like interictal psychosis, autism, and suicidal behavior), sleep disorders, autism spectrum disorders, migraine, postictal headache, depression, anxiety, psychosis, attention deficit disorder (ADD) and attention-deficit / hyperactivity disorder (ADHD) or mental retardation.

[0103] One embodiment described herein is a preferred therapeutic agent and method of the present disclosure for treating migraine or tinnitus. In one aspect, the preferred therapeutic agents and methods of the present disclosure can be used to treat migraine with or without aura in adults. In another aspect, the preferred therapeutic agents and methods of the present disclosure can be used for the acute treatment of migraine with aura, the acute treatment of migraine without aura, or for the prevention of chronic treatment of migraine with or without aura.

[0104] One embodiment described herein is a preferred therapeutic agent and method of the present disclosure, which is used to treat mild, moderate or severe anxiety. On the one hand, the preferred therapeutic agent and method of the present disclosure can be used for the acute treatment and maintenance treatment of major depressive disorder (MDD) in adults and adolescents aged 12-17 years old, or for the acute treatment of generalized anxiety disorder (GAD) in adults. On the one hand, the preferred therapeutic agent and method of the present disclosure can be used for the acute treatment and maintenance treatment of obsessive-compulsive disorder (OCD), the acute treatment and maintenance treatment of bulimia nervosa (Bulimia Nervosa) or the acute treatment of panic disorder (PD) with or without agoraphobia. On the other hand, the preferred therapeutic agent and method of the present disclosure can be used to treat acute depressive episodes associated with bipolar disorder type I or for the treatment of refractory depression.

[0105] One embodiment described herein is a preferred therapeutic agent and method of the present disclosure, which can be used to treat obsessions and compulsions in patients with obsessive-compulsive disorder (OCD), major depressive disorder (MDD), panic disorder (PD), social anxiety disorder (SAD), premenstrual dysphoric disorder (PMDD) or post-traumatic stress disorder (PTSD). On the one hand, in order to meet the diagnosis of OCD in DSM-III-R (about 1989), obsessions or compulsions may cause significant distress, time-consuming or significantly interfere with social or occupational functioning. Obsessions can be recurring, persistent self-contradictory views, thoughts, impressions or impulses. Compulsions can be repeated, purposeful and / or intentional behaviors performed in response to obsessions or in a stereotyped manner. Compulsions may be considered excessive or unreasonable by people.

[0106] One embodiment described herein is a preferred therapeutic agent and method of the present disclosure for treating a patient suffering from the following psychoneurotic disorders: mild, moderate or severe depression; anxiety associated with depression; anxiety associated with alcoholism; depression and / or anxiety associated with organic disease; psychotic depression with associated anxiety (including menopausal depression and manic depression). In one aspect, the preferred therapeutic agent and method of the present disclosure can be used to target symptoms of psychoneurotic disorders, such as anxiety, tension, depression, somatic symptoms and worry, sleep disturbances, guilt, lack of energy, fear, anxiety and worry.

[0107] One embodiment described herein is a preferred therapeutic agent and method of the present disclosure for treating depression in a patient suffering from depressive neurosis (depressive disorder), manic depression, or major depressive disorder. In one aspect, the preferred therapeutic agent and method of the present disclosure can be used for short-term treatment, long-term treatment, and maintenance treatment of major depressive disorder (MDD), generalized anxiety disorder, diabetic peripheral neuropathic pain (DPNP), fibromyalgia (FM), or chronic musculoskeletal pain.

[0108] One embodiment described herein is a therapeutic agent and method of the present disclosure for treating a progressive neurodegenerative disorder including, for example, Alzheimer's disease. In one aspect, preferred therapeutic agents and methods of the present disclosure can be used to treat or halt disease progression in one or more of the following diseases: Alzheimer's disease, amyotrophic lateral sclerosis, Friedreich's ataxia, Huntington's disease, Lewy body disease, Parkinson's disease, or spinal muscular atrophy.

[0109] One embodiment described herein is a preferred therapeutic agent and method of the present disclosure, which includes use as a monotherapy or adjunct therapy. On the one hand, the preferred therapeutic agent and method of the present disclosure can be used as a monotherapy for adults. On the other hand, the preferred therapeutic agent and method of the present disclosure can be used as an adjunct therapy for adults together with other therapeutic agents. On the other hand, the preferred therapeutic agent and method of the present disclosure can be used as a monotherapy for children aged 2 years and above. On the other hand, the preferred therapeutic agent and method of the present disclosure can be used as an adjunct therapy for children aged 2 years and above together with other therapeutic agents. On the other hand, the preferred therapeutic agent and method of the present disclosure can be used as a monotherapy for children aged under 2 years old. On the other hand, the preferred therapeutic agent and method of the present disclosure can be used as an adjunct therapy for children aged under 2 years old together with other therapeutic agents.

[0110] The effective amount of the active pharmaceutical ingredient to be administered therapeutically will depend, for example, on the therapeutic setting and the therapeutic goals. One of ordinary skill in the art will appreciate that the appropriate dosage level for treatment will vary depending, in part, on the concentration of the bumetanide dibenzylamide composition, the dosing regimen using the bumetanide dibenzylamide composition, the route of administration, and the size (weight or body surface area) of the subject and the condition (age and general health) of the patient. Thus, the dosage may be titrated to obtain the optimal therapeutic effect.

[0111] As used herein, bumetanide dibenzylamide includes compositions and formulations containing bumetanide dibenzylamide as will be relevant in the context.

[0112] The frequency of dosing will depend on the pharmacokinetic parameters of the therapeutic agent incorporated into the bumetanide dibenzylamide composition used. The composition may be administered as a single dose, as two or more doses (which may or may not contain the same amount of bumetanide dibenzylamide) over time, or by continuous infusion of the injection formulation via an implant device or catheter. Further refinement of the appropriate dose is routinely performed by one of ordinary skill in the art and is within the scope of his or her routinely performed tasks. Sublingual tablets may also be used for oral administration. Appropriate doses may be determined by use of appropriate dose-response data.

[0113] Opportunities for refinement may include sustained or controlled release oral capsules or tablets or the use of transdermal formulations. The intramuscular data presented below support the development of a potential transdermal therapy. The intramuscular data show that bumetanide dibenzylamide can be absorbed into the circulation through the microvasculature of the muscle and thereby avoid first-pass metabolism. Therefore, bumetanide dibenzylamide should also be absorbed by the dermal microvasculature, making it a transdermal formulation itself.

[0114] The bumetanide dibenzylamide composition can be administered, for example, once, twice, three times, four times, five times, six times or even more times per day. One or more doses can be administered, for example, for 1 day, 2 days, 3 days, four days, five days, six days, seven days or even longer. One or more doses can be administered, for example, for 1 week, 2 weeks, three weeks, four weeks or even longer. One or more doses can be administered, for example, for 1 month, 2 months, 3 months, four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, twelve months, one year, two years, three years, four years, five years, more than five years, ten years, decades or even longer. One or more doses can be administered at regular intervals until the subject or subject in need does not need treatment or prevention of epilepsy. In one aspect, the dose can be administered orally. In one aspect, the dose can be administered sublingually. In one aspect, the dose can be administered intravenously. In one aspect, the dose can be administered rectally. In one aspect, the dose can be administered intramuscularly. In one aspect, the dose can be administered intranasally. In one aspect, the dose can be administered subcutaneously.

[0115] In one embodiment, the pharmaceutical compositions described herein are administered simultaneously in one or more doses. For example, two or more identical doses are administered at one time. In another embodiment, two or more different doses are administered at one time. Such dual or different simultaneous doses can be used to provide an effective amount of the pharmaceutical composition to a subject in need.

[0116] In one embodiment, the pharmaceutical compositions described herein can be used to treat, arrest, slow down the progression of, delay the onset of, improve, alleviate the symptoms of, or prevent epilepsy.

[0117] In one embodiment, the bumetanide dibenzylamide compositions described herein are administered in a composition sufficient to provide a therapeutically effective amount in one application. In one aspect, one application of the bumetanide dibenzylamide composition is sufficient to last about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, one month, 2 months, 3 months, 4 months, 6 months, 9 months, one year, 2 years, 3 years, 4 years, or even longer. In one aspect, one application of the bumetanide dibenzylamide composition is administered more than once a day.

[0118] In another embodiment, the bumetanide dibenzylamide composition described herein is provided as a single dose, meaning that the container in which it is supplied contains one drug dose. In another embodiment, the composition is provided as a multi-dose composition, meaning that the container contains more than one therapeutic dose. Preferably, a multi-dose composition contains at least 2 doses. Such a multi-dose bumetanide dibenzylamide composition can be used for different subjects in need or intended for use with one subject, with the remaining doses stored after the first dose is applied until needed. In another embodiment, the bumetanide dibenzylamide composition is contained in one or more containers.

[0119] Crucial to the efficacy of any treatment delivered by a pharmaceutical composition is the overall systemic bioavailability of the pharmaceutical composition used for the treatment. Surprisingly, lipid formulations can be used to increase the bioavailability and / or lymphatic absorption of a pharmaceutical composition. LogP is a measure of lipophilicity and is an octal:water partition coefficient expressed as the logarithmic ratio of a molecule in octanol relative to a molecule in water after mixing. The LogP for bumetanide is about 2.61, the logP for bumetanide diethylamide is 3.11, and the logP for bumetanide dibenzylamide is about 5.9. A LogP of 3 indicates that the concentration in octanol is 1000 times that in water, and therefore the lipophilicity of bumetanide dibenzylamide is about 1000 to about 10,000 times that of bumetanide.

[0120] Detailed treatment methods / applications / compounds used

[0121] As described herein, bumetanide dibenzylamide was demonstrated to disrupt the synchronization of neuronal population activity in areas of synchronous enhancement. Dosage compositions of bumetanide dibenzylamide were developed for four (4) routes of administration in an attempt to bypass first-pass metabolism of the composition and increase overall systemic bioavailability. The route of administration was selected based on its potential to maximize the bioavailability of bumetanide dibenzylamide and produce measurable systemic concentrations of bumetanide dibenzylamide. Since bumetanide dibenzylamide has been shown to be susceptible to high first-pass metabolism by the liver, the route of administration was selected to avoid liver metabolism.

[0122] Drug-resistant epilepsy

[0123] Seizures that are not controlled by existing medications are referred to in a variety of ways, including “uncontrolled,” “refractory,” “intractable,” “resistant,” or “medically resistant.” An estimated 20%-40% of people with epilepsy (approximately 400,000 Americans) have refractory epilepsy. The indirect and direct costs of epilepsy in the United States are estimated to be $15.5 billion per year, with drug-resistant patients accounting for a large portion of these costs. Although many new drugs for epilepsy have become available in the past decade, these new drugs have not been shown to be significantly better than older drugs.

[0124] The most common AEDs (and their use to treat other neurological and psychiatric disorders besides epilepsy). The most commonly used AEDs include valproate (VPA – the most commonly prescribed of all AEDs worldwide) and its derivative divalproex sodium, carbamazepine (Tegretol), phenytoin (Dilantin), barbiturates (phenobarbital and primidone), ethosuximide (Zarontin), clonazepam (Klonopin), lamotrigine (Lamictal), gabapentin (Neurontin), topiramate (Topamax), oxcarbazepine (Trileptal), and zonisamide (Zonegran). In addition to their use to treat epilepsy, AEDs are used to treat many other neurological and psychiatric disorders.

[0125] Side effects: All currently prescribed anti-epileptic drugs (AEDs) are thought to mediate their anti-epileptic effects by reducing neuronal or synaptic excitability. Because AEDs indiscriminately affect all neuronal or synaptic targets in the brain, whether or not they cause seizure activity, all AEDs also mediate a range of cognitive, neurological, and psychiatric side effects. Approximately 25% of patients discontinue their treatment due to intolerable side effects. Treatment failure and poor compliance are common among patients who experience side effects from their AEDs. The negative consequences of side effects can seriously affect the lives of patients' relatives and friends. Common side effects of AEDs include memory problems, fatigue, tremors, gastrointestinal symptoms, osteoporosis, depression, drowsiness, weight gain, nausea, etc. A study in the Netherlands estimated that the economic cost of epilepsy side effects (in addition to the direct and indirect costs of epilepsy itself) per patient in the country is $26,675 per year.

[0126] All commonly used AEDs have some effects on cognition, and these effects can have considerable consequences for patients with epilepsy when critical functions are involved (e.g., learning in children). The most prevalent adverse CNS effects of CNS medications on cognition are sedation, somnolence, distractibility, insomnia, and dizziness.

[0127] Fatigue is a common side effect of most anti-epileptic drugs. Fatigue caused by AEDs is a chronic condition that may have a negative impact on the patient's work, social life and family. Stimulants such as amphetamine, dextroamphetamine and methylphenidate are sometimes used to treat fatigue and daytime sleepiness. However, these drugs may increase the intensity of epileptic seizures and lower the seizure threshold, and therefore these drugs should not be used to treat fatigue in patients with epilepsy.

[0128] All anti-epileptic drugs are thought to increase the risk of suicidal thoughts or behavior. This risk was of sufficient concern that the FDA issued safety alerts on December 15, 2008, and January 31, 2008, and currently requires that all AED labels include a warning about the increased risk of suicidal thoughts or behavior. This is particularly problematic because epilepsy and other disorders and psychiatric conditions (chronic pain, depression, bipolar disorder, and anxiety) that AEDs treat inherently carry an increased risk of suicidal behavior. For example, suicide deaths are more common in patients with epilepsy than in the overall population (5% vs. 1.4%). Therefore, using AEDs to treat conditions that are already associated with a risk of suicidal behavior would be expected to further increase that risk.

[0129] Every anti-epileptic drug studied to date has been shown to have endocrine side effects in both men and women. These may adversely affect fertility, sexual behavior, thyroid function, and bone health. AEDs can alter sex hormone levels, which can lead to menstrual irregularities, sexual problems, and decreased fertility. Other side effects that affect appearance include weight gain, baldness (hair loss), acne, and masculine hair distribution in women.

[0130] The treatment of epilepsy is impacted by poor patient adherence to currently available antiepileptic medications. As noted above, approximately 25% of patients discontinue their treatment due to intolerable side effects. Up to 50% of patients with epilepsy experience adverse reactions to AEDs, which in turn negatively impacts tolerability and adherence. Even if side effects are not intolerable, if they are unpleasant, they may reduce patient adherence to taking AEDs as prescribed. Estimates of nonadherence in epilepsy range from 30% to 50% [24,25]. Reduced AED adherence is associated with a greater than 3-fold increase in mortality

[26] . Periods of nonadherence in patients with epilepsy are also associated with significant increases in emergency department visits, hospitalizations, injuries, and fractures.

[0131] The treatment of epilepsy is affected by comorbidities.

[0132] Overview: A recent study determined the prevalence of the most common comorbidities in men and women with epilepsy based on data from commercial health plans. The top 10 comorbidities and their relative prevalence for women were psychiatric diagnoses (16%), hypertension (12%), asthma (11%), hyperlipidemia (11%), headache (7%), diabetes (6%), urinary tract infection (5%), hypothyroidism (5%), anemia (5%), and migraine (4%). For men, the top 10 comorbidities and their relative prevalence were psychiatric diagnoses (15%), hyperlipidemia (12%), hypertension (12%), asthma (8%), diabetes (5%), headache (4%), cancer (4%), coronary artery disease (3%), anemia (3%), and gastroesophageal reflux disease (3%). Seven of the top 10 comorbidities were common to both women and men. Psychiatric diagnoses were the only comorbidity in the top five for all age groups. The health care costs for a member with one comorbidity are approximately three times higher compared to the costs for a member without a comorbidity.

[0133] Psychiatric Disorders – Overview: Epilepsy increases the likelihood of depression, anxiety, attention deficit hyperactivity disorder (ADHD), schizophrenia-like interictal psychosis, autism, and suicidal behavior. Likewise, individuals with these psychiatric diagnoses and suicidal behavior are more likely to have epilepsy.

[0134] Sleep disturbances: It is well known that insufficient sleep can lower the seizure threshold in patients with epilepsy. Sleep is susceptible to its own set of disorders that can disrupt sleep. One example is obstructive sleep apnea [OSA]. Both adults and children with refractory epilepsy are at a much higher risk of developing OSA than the general population.

[0135] Autism Spectrum Disorder (ASD): Epilepsy occurs much more frequently in individuals with autism. Between 11% and 39% of individuals with autism will develop epilepsy.

[38] Epilepsy and autism co-exist in up to 20% of children with either disorder. Among children with autism, the highest prevalence of epilepsy is in those with intellectual disability.

[0136] Sleep disturbances are common among children with autism, with an estimated 40% to 80% of children affected.

[0137] The atypical antipsychotics risperidone and aripiprazole are approved by the U.S. Food and Drug Administration for the treatment of irritability and agitation in ASD. Both are associated with serious adverse events, including lowering of the seizure threshold.

[0138] Migraine: The incidence of migraine is about 1% per year, and the 1-year prevalence is 11.7%-13.2%. Patients with epilepsy also have an approximately two-fold increased risk of migraine. Conversely, children with migraine have a three- to four-fold increased risk of epilepsy.

[0139] Comorbidities may worsen the results. Patients with epilepsy who also have migraine are less likely to achieve epilepsy remission than those with epilepsy alone. This is also evidence of the complex comorbidity cluster of epilepsy, migraine, depression, and suicide.

[0140] Post-ictal headaches – 45% of people with epilepsy experience a headache after a seizure, called a post-ictal headache. These headaches last between 6 and 24 hours or longer and can be quite disabling. Many of the medications used to treat these headaches can lower the seizure threshold, increasing the risk of further seizures.

[0141] Depression: Depending on the sample population and assessment method, depression has been shown to occur in between 9% and 55% of patients with epilepsy. This contrasts with estimates of 1%-3% for men and 2%-9% for women in the general population.

[0142] Therapies commonly used to treat depression can lower the seizure threshold or increase the severity of seizures. Bupropion and tricyclic antidepressants lower the seizure threshold. Selective serotonin reuptake inhibitors (SSRIs) can significantly prolong seizures.

[0143] Anxiety: The lifetime prevalence of anxiety in people with epilepsy is estimated to be 2.4 times that of people without epilepsy.

[0144] Delirium: The risk of delirium in patients with epilepsy may be 6-12 times that of the general population (prevalence is approximately 7%-8%). All antipsychotics can lower the seizure threshold.

[0145] Attention Deficit Disorder (ADD) and Attention Deficit / Hyperactivity Disorder (ADHD): Nearly 20% of adults diagnosed with epilepsy also display symptoms of ADHD. Studies of pediatric epilepsy have found a 2.5- to 5.5-fold increased risk of ADHD compared to healthy controls. It is estimated that between 2% and 7% of children with ADHD also have epilepsy.

[0146] Stimulants such as amphetamine, dextroamphetamine, and methylphenidate are commonly used to treat attention deficit disorder (ADD) and attention-deficit / hyperactivity disorder (ADHD) in children and adults. These drugs can lower the seizure threshold and increase seizure severity.

[0147] Mental retardation: Epilepsy is one of the most common secondary disorders in individuals with mental retardation, and the prevalence increases with the severity of the mental retardation. Approximately 50% of individuals with severe learning disabilities will develop epilepsy. The lifetime prevalence of epilepsy in individuals with mental retardation (IQ < 70) is between 13% and 24%. Down syndrome is the most common genetic cause of mental retardation; estimates of the number of individuals with Down syndrome who experience epileptic seizures range from 5% to 10%. Currently available AEDs do not have adverse behavioral effects in individuals with mental retardation.

[0148] Others: Hyperlipidemia – The incidence in patients with epilepsy is 1.3 times that of controls. Population-based surveys show that patients with epilepsy have a higher incidence of hypertension, ischemic heart disease and diabetes.

[0149] Drug interactions may create problems in the treatment of seizure disorders.

[0150] The following medications may lower the seizure threshold and thus increase the risk of seizures in patients with epilepsy: Acetylcholinesterase inhibitors – used to treat: myasthenia gravis, glaucoma, postural tachycardia syndrome, neuropsychiatric symptoms of Alzheimer's disease, dementia with Lewy bodies, Parkinson's disease, cognitive impairment in schizophrenia, autism; Anticholinergics – used to treat gastrointestinal disorders, genitourinary disorders, respiratory disorders, sinus bradycardia, insomnia and dizziness; Antiemetics – used to treat nausea / vomiting; Antihistamines – suppress symptoms of allergic reactions; Baclofen – a skeletal muscle relaxant used to treat spasticity; Beta-blockers – Angina, atrial fibrillation, arrhythmias, congestive heart failure, essential tremor, glaucoma, hypertension, migraine prevention and treatment, mitral valve prolapse, myocardial infarction, pheochromocytoma; in combination with alpha-blockers, postural orthostatic tachycardia syndrome, anxiety symptom control (tachycardia, tremor) and hyperthyroidism, theophylline overdose; cephalosporins – antibiotics; cyclosporine – immunosuppressant, used for severe rheumatoid arthritis, severe psoriasis; dafopyridine – used to treat multiple sclerosis, spinal cord injury, Parkinson's disease; estrogen – oral contraceptives, hormone replacement therapy (administered after menopause to prevent osteoporosis, treat menopausal symptoms, prostate cancer; imipenem – antibiotics; iodinated contrast dye – radiocontrast agent; isoniazid – prevention and treatment of tuberculosis; lithium – bipolar disorder, severe depression and schizophrenia; local anesthetics – Seizures are a recognized side effect of administering local anesthetics; Methotrexate – chemotherapy for certain cancers, autoimmune conditions (including rheumatoid arthritis, juvenile dermatomyositis, psoriasis, psoriatic arthritis, lupus, sarcoidosis, Crohn's disease, eczema, and various forms of vasculitis); Metronidazole – antibiotic and antiprotozoal; Anesthetics – pain; Penicillin – antibiotic; Pyrimethamine – antimalarial, used for protozoal infections; Quinolones – antibacterial; Theophylline – chronic obstructive pulmonary disease (COPD), asthma, infantile apnea. Blocks the effects of adenosine, a neurotransmitter inhibitor that induces sleep, contracts smooth muscle, and relaxes heart muscle; Tramadol – pain.

[0151] More rare epilepsy syndromes that may be treated with the compounds and compositions of the present disclosure include Angelman syndrome – occurs in 1 / 15,000 newborns. More than 80% of affected individuals show epileptic symptoms; Benign Rolando epilepsy – accounts for approximately 15% of all epilepsy in children. Seizures cease by age 15 years; CDKL5 disorder – may be too rare to be of interest. 600 cases worldwide; Childhood absence epilepsy – occurs in 2%-8% of epilepsy patients. Usually disappears by adulthood; Dravet syndrome: affects 1 / 30,000. 85% of children develop myoclonic seizures between 1-5 years of age; GLUT1 deficiency syndrome – may occur in 1 / 90,000, but is considered underdiagnosed because many neurological conditions cause similar symptoms. Almost all individuals have frequent seizures beginning in the first year of life; Hypothalamic hamartoma – 1 / 200,000; Infantile spasms (also known as West syndrome) – 2.5-6 per 10,000 newborns. It accounts for 30% of all epilepsy cases affecting infants. It usually stops by age 4, but most children are developmentally impaired, and one in five will have Lennox-Gastaut syndrome. Many clinicians believe that the earlier seizures are controlled, the better they are; Lennox-Gastaut – It accounts for 2%-5% of childhood epilepsy. It usually lasts from childhood and adolescence into adulthood. Seizures are difficult to control with current therapies; PCDH19 – 1 in 10 girls who begin having seizures before age 5 may have PCDH19 epilepsy. It may overlap or look similar to Dravet syndrome. There are 15,000 – 30,000 people with PCEH19 epilepsy in the United States; Progressive Myoclonic Epilepsy – It is not a single condition, but includes a group of syndromes with different names, including severe myoclonic epilepsy of infancy (Dravet syndrome), Unverrict-Lundbord disease (also called Baltic myoclonus), Lafora disease, and mitochondrial encephalopathy. It is very difficult to control any of these patients with current treatments; Rasmussen's encephalitis – nothing is known about its incidence in different populations. This syndrome is thought to be rare, but clinicians around the world have described patients with it. The outlook is bleak with current treatments; Seizures are constant; Ring chromosome 20 syndrome – rare; Reflex epilepsy – a group of epilepsy syndromes in which a certain stimulus (such as a flashing light) triggers a seizure. 4%-7% of patients with epilepsy.

[0152] Monotherapy vs. polytherapy (combination therapy) side effects. Often, a single AED can provide partial but inadequate seizure control. For patients who are refractory to any single AED (monotherapy), better seizure control is sometimes obtained by combining several different AEDs (polytherapy or combination therapy). Sometimes, a single patient is given up to four AEDs to try to control seizures. However, the intensity and number of side effects increase significantly when patients are given more than one AED. Studies have shown that polytherapy has more severe and more cognitive side effects. The intensity and number of side effects increase as the number of AEDs a patient is taking increases.

[0153] The FDA labeling for bumetanide includes a statement that serum potassium should be measured periodically and potassium supplements or potassium-sparing diuretics added if necessary. Periodic determination of other electrolytes is recommended for patients receiving high-dose or long-term therapy, particularly those on a low-salt diet. Hyperuricemia may occur; none has been asymptomatic in cases reported to date. Reversible increases in BUN and creatinine may also occur, particularly associated with dehydration, and especially in patients with renal impairment. Bumex may increase urinary calcium excretion, leading to hypocalcemia. Diuretics have been shown to increase urinary magnesium excretion; this may lead to hypomagnesemia.

[0154] Ideally, it would be desirable to increase the ability of loop diuretics to cross the blood-brain barrier. This would have the effect of being able to achieve a greater therapeutic effect in the brain and reduce diuresis. We have found that certain amide analogs of bumetanide have profound antiepileptic effects with significantly reduced diuresis compared to bumetanide. This was an unexpected finding. For example, Tollner et al. tested a bumetanide amide derivative (N,N-dimethylaminoacetamide) in their rat study and found that it did not result in increased bumetanide levels. The authors of that study then chose to forgo further testing of the bumetanide amide derivative.

[0155] The methods of the present invention provide particular utility for patients with epilepsy that is not well controlled by conventional therapies (eg, phenytoin, carbamazepine, valproate, lamotrigine, levetiracetam, ethosuximide, phenobarbital, and topiramate) or that is otherwise refractory to conventional therapies.

[0156] Primate Model

[0157] A primate neocortical seizure model was used here to test the effects of NKCC antagonists on epileptiform activity. This model and the techniques used to analyze the data were originally developed by Haglund and Hochman for the purpose of studying endogenous light signals in the human and primate brains and how these signals could be used to map the propagation of seizure activity in the neocortex (Haglund et al. 1993; Haglund and Hochman, 2007). In primate studies, epileptiform activity was generated by electrically stimulating small focal cortical areas with bipolar microelectrodes to produce afterdischarge activity (similar to what is done for intraoperative localization of seizure foci in human patients; see below) or by applying various epileptiform-inducing agents (e.g., bicuculline, 4-AP) to focal areas of the hand motor cortex to produce acute lesions (similar to recordings from interictal lesions in human patients; see below). We found that the data obtained from the primate seizure studies were qualitatively similar to those we found when performing similar optical imaging studies in human patients (Haglund and Hochman, 2005), as the morphology of the electrophysiological activity recorded in the EEG traces was similar between monkeys and humans. In particular, the spontaneous interictal spikes observed in humans (shown below) were similar to the neocortical spikes generated by bicuculline lesions on the neocortex in the primate model. The afterdischarge protocol developed for the human intraoperative studies (shown in the second figure below) was also used in the primate studies. Likewise, the physical properties of the brain optical signals generated by epileptiform activity (not shown here) were similar (Haglund and Hochman, 2005; Haglund and Hochman, 2007).

[0158] Example Comparison Group-1: Furosemide (NKCC antagonist) blocks spontaneous interictal spikes in human patients with drug-refractory epilepsy (from Haglund and Hochman, 2005).

[0159] Reference Figure 1 , the data in this figure were analyzed using the methods described in this article for the primate studies. The top trace shows data from an individual patient to illustrate changes in spontaneous interictal spikes after furosemide administration. Electrophysiological activity was recorded from EEG electrodes placed on the cortical surface, as is done in primates. The top two traces compare activity before and after administration of 20 mg IV furosemide. The dark blue trace is recorded from electrodes at the interictal focus, and the superimposed light blue trace shows background activity from electrodes 1 cm away. Changes in spike frequency over time were determined using the same algorithm applied to the primate data, averaged across 5 patients, and are shown in the bottom graph.

[0160] Example Comparison Group-2: Effects of furosemide (NKCC antagonist) on afterdischarge thresholds in the cortex of human subjects (Haglund and Hochman, 2005).

[0161] Reference Figure 2 A and Figure 2 B , Bipolar stimulating electrodes (similar to those used in primate studies) were placed on the cortical surface, as shown in the grayscale image in the lower center of the figure. The recording electrode was placed within 1 cm of the stimulating electrode. Four-second stimulation (60 Hz; biphasic; 1 ms / phase) was delivered at different currents (similar to the protocol used in primate studies); the duration of stimulation is indicated by the blue box at the beginning of the inset trace. Before furosemide treatment, the minimum current required to elicit afterdischarge activity lasting at least 5 seconds in three consecutive trials was determined; this was defined as the “afterdischarge threshold current” (A . top). The red horizontal bar above each trace marks the onset of afterdischarge activity. After furosemide administration, stimulation trials were performed every 2–5 minutes for the next 40 minutes. In this patient, afterdischarge activity was abruptly blocked shortly after furosemide treatment (A . bottom trace). To determine whether the blockade of afterdischarge activity was mediated by an increase in the afterdischarge threshold, the stimulation current was gradually increased (B ). It was determined that afterdischarge bouts lasting at least as long as those observed during the pre-furosemide trial could be elicited by increasing the stimulation current. This result suggests that furosemide increases the afterdischarge threshold.

[0162] Example Comparison Group-3: Quantification of the Diuretic Effect of NKCC2 Antagonists

[0163] Rats rapidly bioconvert bumetanide to inactive metabolites, and thus bumetanide does not induce diuresis in rats, despite its potent diuretic effect in humans (Schwartz, 1981. Metabolism of bumetanide. J Clin Pharmacol 12:555-563). Similarly, the diuretic effect of bumetanide in dogs is much less than in humans because of the rapid renal excretion of bumetanide in dogs (Schwartz, 1981). Studies in the Hochman's lab at Duke University have shown that primates can be catheterized and diuretic effects quantified. This makes primates more suitable than other common laboratory species for comparing the diuretic effects of bumetanide analogs with bumetanide in a manner that is transferable to humans.

[0164] Example Comparative Group-4: Primate Model Sensitive to Antiepileptic Effects of Drugs that Have No Antiepileptic Effects in Rats:

[0165] Keppra (levetiracetam) is an excellent anti-epileptic drug, but faced an uphill battle during its development, as it is well known that it has no anti-epileptic activity in standard rat seizure models. According to the pharmacology review provided to the FDA: "There was no anticonvulsant activity in two screening tests for anti-epileptic drugs (AEDs), the maximal electroshock (MES) test and the maximal pentylenetetrazol (PTZ) test". Levetiracetam lacked anticonvulsant effects on seizures induced by maximal stimulation of different chemical convulsants, and submaximal stimulation showed a slight anticonvulsant effect, and the same was true in threshold tests, with an exception that protection against seizures induced by pilocarpine and kainic acid was observed.

[0166] like Figure 3 As shown, a clear anticonvulsant effect of Keppra could be measured in our primate seizure model.

[0167] As Keppra did at the time, we believe that bumetanide mediates its anticonvulsant effects through a mechanism that is unique to all currently approved AEDs. Primate models appear to exhibit sufficient sensitivity to the anticonvulsant effects of bumetanide and its analogs to allow for their use in studying such molecules in a manner that we believe is transferable to humans.

[0168] Furosemide and bumetanide prevented seizures; their rank order of antagonism to NKCC was preserved. Furosemide 10 mg / kg IV, bumetanide 2 mg / kg IV. The diuretic effect of furosemide is about 1 / 10 of that of bumetanide th . Bumetanide, fosphenytoin (Cerebyx), and pentothal (sodium thiopental) block 4-AP bursts. Keppra 2X 40 mg / kg (levetiracetam) does not block 4-AP bursts. Thus, primate models are sensitive to Keppra, but Keppra is not known to be effective in the standard AED rat model. Bumetanide and Keppra block bicuculline spikes, but fosphenytoin does not. Bumetanide and fosphenytoin block AD and hand twitches, but Keppra does not.

[0169] Examples

[0170] Example 1: Synthesis of Bumetanide Dibenzylamide (NPT 2042)

[0171]

[0172] Bumetanide dibenzylamide can be prepared according to the procedures set forth in U.S. Patent No. 8,008,283, which is incorporated herein by reference in its entirety.

[0173] Bumetanide dibenzylamide can be synthesized as described. Bumetanide (960 mg, 2.6 mmol) was dissolved in dimethylformamide (DMF, 10 mL) and 1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimide (EDC, 560 mg, 3.6 mmol) was added. After about 10 minutes, 1-hydroxybenzotriazole (HOBt, 392 mg, 2.9 mmol) was added and the solution was stirred for another 10 minutes. Dibenzylamine (1 mL, 5.2 mmol) was added and the reaction was stirred for 2 hours, at which time the reaction was complete by LC / MS. The reaction was poured into saturated ammonium chloride (20 mL) and extracted with ethyl acetate (2 x 100 mL). The ethyl acetate was washed with saturated sodium bicarbonate, water, and brine, and dried over anhydrous magnesium sulfate. The ethyl acetate was removed under reduced pressure to give 1.0 g (75%) of N,N-dibenzyl 3-aminosulfonyl-5-butylamino-4-phenoxybenzamide (bumetanide dibenzylamide) as a white solid.

[0174] An initial set of amide analogs of bumetanide were first screened by measuring their NKCC-mediated anxiolytic effects in a rat anxiety whole animal bioassay, and these effects were compared to bumetanide using the same methods as described in Krystal 2012. The rat anxiety model studies were performed by NeuroInvestigations, Inc. at the University of Lethbridge (Lethbridge, Canada). Drs. Hochman and Haglund of Duke University Medical Center (Durham, NC) evaluated the diuretic and antiseizure effects of the candidates that elicited anxiolytic effects in monkeys (Cynomolgus M. nemistrina). During these experiments, the monkeys were anesthetized and catheterized, and their urine was collected and measured in graduated vials so that the effects of various treatments on diuresis could be quantified. Cynomolgus M. nemistrina monkeys were chosen as a relevant species for studying diuretic effects because they are believed to metabolize bumetanide (and therefore likely its analogs) similarly to humans (Doyle 1982, Walmsley 1985).

[0175] Example 2: Experimental setup and testing methods

[0176] Bumetanide derivatives can be synthesized according to the methods described in U.S. Pat. No. 8,008,283, previously incorporated by reference, and all tested derivatives are dissolved in PEG 200 for IV formulation. For oral administration, suspension in CMC water was administered by oral gavage in equimolar amounts equivalent to 10 mg / kg BUM.

[0177] All data presented here used macaques (Macaca nemestrina, weighing 2-3 kg), whose care and treatment followed protocols approved by the Duke University Institutional Animal Care and Use Committee. Details regarding the treatment and surgical preparation for cortical AD stimulation and EEG recordings in macaques have been described ( Haglund et al., 1993 ; Haglund and Hochman, 2007 ; Tolner, E. A. et al., 2011).

[0178] Animals were artificially ventilated with 100% oxygen via cannula (Matrix VBS anesthesia machine and Hallowell EMC model 2002 ventilator) and maintained under pentobarbital anesthesia (1-2 mg / kg / hr). Oxygen saturation was measured from the tongue using a tongue sensor (Nellcor pulse oximeter) and was continuously monitored to maintain a constant saturation of 98%-100% throughout the experiment.

[0179] Fluid balance was controlled by monitoring intravenous fluid intake every 15 min and fluid output (via Foley catheter) every 30 min. Heart rate, blood pressure (Critikon Dinamap blood pressure monitor, model 8100), end-tidal pCO2, respiration, and O2 levels (Ohmeda 5250 RGM anesthesia monitor; Nellcor NBP-40 for SpO2) were monitored throughout the experiment based on EEG.

[0180] After craniectomy (25 mm in diameter), the dura was stripped away, and surface EEG recordings were performed using custom-made strip electrodes (electrode diameter, 2.5 mm; inter-electrode distance, 1 cm) placed over the sensorimotor cortex, with one electrode covering the hand motor cortex, one electrode covering the sensory cortex, and a reference electrode attached to the skull at the mastoid or to a third surface electrode between the motor and sensory electrodes.

[0181] The cells were then immersed in 0.5 mm 2Acute bicuculline lesions were created in the motor cortex with gelatin sponge pads (Codman & Shurtleff, Randolph, MA, USA). The pads were placed on the cortex for five minutes and fed for five minutes every hour. Bicuculline-induced epileptiform spiking activity stabilized after about 1 hour, and then data acquisition was started. EEG signals from motor and sensory cortical electrodes were recorded continuously (1000 Hz sampling, no filter; Digidata 1440A system from Axon Instrument).

[0182] Stimulus-evoked afterdischarges (ADs) were evoked by 4-second 60 Hz biphasic pulse trains of 4–20 mA using bipolar stimulating electrodes (5 mm inter-electrode distance) powered by a constant current source (Ojemann cortical stimulator, Integra Life Sciences, NJ, USA) placed over the sensory cortex, or by passing current between two of the three surface electrodes. The AD threshold was determined by three consecutive stimulations at the lowest level that reliably triggered AD activity, and the threshold was kept constant throughout the recording. AD activity was reliably elicited over the sensory cortex during the same experimental session in which bicuculline lesions were created over the motor cortex. Animals were subjected to several AD trials with 20–30 min intervals between trials. In each AD trial, 3–8 AD stimulations (1 min intervals) were performed during control conditions, during the period of maximal CNS effects from bumetanide or bumetanide derivative treatment, and during the recovery phase.

[0183] Reference Figure 4 : Experimental setup: Acute lesions were created near the hand motor cortex using bicuculline or 4-AP. Three surface electrodes were placed so that one electrode covered the hand motor cortex and the other covered the hand sensory cortex. The middle electrode was used as a reference for differential recording. The recording electrode could also be used to stimulate the cortex by passing current between a pair of electrodes.

[0184] As previously described ( Haglund and Hochman, 2005 ) Data were analyzed offline with custom-designed software using the R programming language. Bicuculline-induced spiking activity was quantified from motor cortex recordings. The effect of treatment on bicuculline-induced spiking was analyzed by comparing the peak response after treatment application to the average of a 5-min control period before treatment application. In the bicuculline experiments, recovery from drug treatment was followed for 20–35 min periods after recovery of pre-treatment spiking activity. For technical reasons, some recordings had to be terminated before the recovery phase.

[0185] Example 3: Afterdischarge (AD) activity

[0186] Reference Figure 5 The lower left image shows the placement of the stimulating electrodes on the surface of the monkey's cortex (in this example, electrodes from an Ojemann stimulator were used instead of Figure 1 ). The brain was electrically stimulated for 4 seconds at varying current intensities until the “afterdischarge threshold” was determined: the stimulation current intensity that was just enough to induce AD. The stimulation artifact is shown in the upper left trace, indicated by an asterisk (*). AD is shown by the orange bar. It can be seen that in this case, 7 mA was not sufficient to induce AD, but 8 mA reliably produced AD. Therefore, the AD threshold here was 8 mA. In this way, changes in the effect of treatment on epileptiform activity induced by this type of electrical stimulation can be quantified by measuring changes in the AD threshold. From Haglund and Hochman, 2007.

[0187] Example 4: Quantification of after-discharge activity

[0188] Reference Figure 6 ,Apart from Figure 2 In addition to the AD thresholds described in , this figure shows three aspects of AD activity that can be quantified using custom software developed by D. Hochman: 1) the duration of AD activity, 2) the average spike height of AD activity, and 3) the area within the envelope of AD activity.

[0189] Example 5: Bicuculline spike activity

[0190] Reference Figure 7 , which shows continuous EEG traces recorded from surface electrodes proximal to the epileptic focus in the hand motor cortex (upper trace) and proximal to the hand sensory cortex (lower trace). In addition to visual comparison (i.e., pre-treatment trace on the left vs. post-treatment trace on the right), spiking activity can be quantified using software developed by D. Hochman that measures 1) spike height, 2) spike frequency, and 3) inter-spike interval. It should be noted that electrical stimulation-induced ADs can be simultaneously generated and recorded at locations distant from the bicuculline focus without interference.

[0191] Example 6: Analysis of NPT 2024 (Bumetanide Morphamide; n = 4)

[0192] Reference Figures 8 to 11 , the comparative test results between the bumetanide morpholine amide derivative and the bumetanide parent compound showed a significant increase in the inter-peak interval ( Figure 8 ), the spikes per minute are significantly reduced ( Fig. 9 ), the average peak height is significantly reduced ( Fig.10 ), and the comparative effects of NPT 2024 and bumetanide on urine production over time ( Fig.11 ).

[0193] Example 7: Anti-seizure Effects of Orally and Intravenously Administered Bumetanide Dibenzylamide (NPT 2042) in Non-Human Primates

[0194] The aim of this study was to evaluate the antiseizure efficacy of bumetanide dibenzylamide (NPT 2042) in nonhuman primates (NHPs) and compare it with the parent bumetanide compound. In these animals, acute seizure lesions were created on the surface of the neocortex with the GABAa antagonist bicuculline to produce an epileptiform “bicuculline spike” as described above. After seizure lesions were created in these NHPs, single doses of NPT 2042 or the reference control bumetanide were administered orally (PO) and intravenously (IV). The bicuculline-lesion primate seizure model was used in this study because data previously obtained from primate seizure studies are qualitatively similar to those observed in similar optical imaging studies in patients with intractable epilepsy (Haglund and Hochman 2005). Specifically, the morphology and electrophysiological activity recorded in electroencephalographic (EEG) measurements were similar between NHPs and humans. Bicuculline blocks the inhibitory effects of GABA receptors and triggers convulsions that are thought to resemble epileptic seizures. Therefore, bicuculline has been used in the laboratory for decades to study and test the anticonvulsant effects of putative antiepileptic treatments (Schwartzkroin and Prince 1980). In the same experiments, the effects of NPT 2042 and bumetanide on diuresis were studied.

[0195] The experimental results indicate that both NPT 2042 and bumetanide elicited an almost complete blockade of bicuculline spikes following intravenous administration of a 2 mg / kg dose in the NHP seizure model. Oral doses of 10 mg / kg NPT 2042 and bumetanide both reduced the spike height and frequency of bicuculline spikes. These studies demonstrate that NPT 2042 and bumetanide have similar abilities to inhibit bicuculline spikes in the NHP seizure model.

[0196] The purpose of this study was to evaluate the anti-epileptic seizure efficacy of bumetanide dibenzylamide (NPT 2042) compared to bumetanide in non-human primates. The results of this study validate NPT 2042 as a candidate to advance clinical development as an adjunctive anti-epileptic seizure therapy for patients with drug-resistant epilepsy. The experimental groups for intravenous and oral administration routes are shown in Tables Ex 7-1 and Ex 7-2, respectively.

[0197] Table Ex 7-1 Test Groups and Sample Sizes: Intravenous Route

[0198]

[0199] IV = intravenous.

[0200] Table Ex 7-2 Trial Groups and Sample Size: Oral Route

[0201]

[0202] PO = oral.

[0203] Bicuculline blocks the inhibitory effects of GABA receptors and triggers convulsions that are thought to resemble epileptic seizures. Therefore, bicuculline has been used in the laboratory for decades to study and test the anticonvulsant effects of putative antiepileptic treatments (Schwartzkroin and Prince 1980). The data obtained from primate seizure studies are qualitatively similar to those observed from similar optical imaging studies in human patients, as the morphology of electrophysiological activity recorded in EEG traces is similar between NHPs and humans (Haglund and Hochman 2005).

[0204] Description of the Acute Bicuculline Seizure Focus Model: An acute "seizure focus" is created on the cortical surface. Three surface electrodes are placed so that the motor electrode covers the hand motor cortex and the other covers the hand sensory cortex. The middle electrode is used as a reference for differential recording. The recording electrode can also stimulate the cortex by passing current between a pair of electrodes. An image showing the placement of the surface electrodes is provided below, as shown in Figure 4 shown.

[0205] Acute “seizure lesions” were created in primates by placing bicuculline-soaked patches on the surface of the neocortex overlying the arm / hand motor cortex for approximately 20 to 40 minutes until stable spiking activity was observed ( Fig.12 , Figure A below). The patch is then removed from the cortex, and spiking will continue for at least 4 to 6 hours until the end of the experiment. The effects of various treatments on bicuculline-evoked spiking can then be studied by administering the treatment shortly after a consistent spiking pattern has been elicited.

[0206] Fig.12 Panel A in Figure 2 shows a continuous 70-minute segment of a trace recorded by a surface EEG electrode overlying a cortical area where a bicuculline lesion had been created. Panels B and C show the first and last 60 seconds of the trace shown in panel A (times indicated by red arrows in A), plotted over a faster time course so that individual spikes can be seen. Importantly, no spontaneous significant changes in the spike size or frequency of spiking occurred in this model.

[0207] Effect of bumetanide on bicuculline spike compared with standard of care treatment with levetiracetam (Keppra ® ) is shown in Fig.13 The lower trace shows that both bumetanide and Keppra inhibit the bicuculline spike. It is noteworthy that Keppra has a clear (albeit transient) effect on the bicuculline spike in this model, but it is known to have no effect in any of the standard animal models used to screen for anticonvulsant activity (Loscher and Honack 1993).

[0208] Experimental Design: Anticonvulsant Seizure Experiment

[0209] This experiment used macaque NHP (Macaca nemestrina, weighing 2 to 3 kg). Details regarding treatment and surgical preparation for cortical AD stimulation and EEG recording in macaques have been described previously (Haglund et al. 1993; Haglund and Hochman 2007; Tolner et al. 2011) and are briefly described below.

[0210] Animals were artificially ventilated with 100% oxygen via cannula (Matrix VBS anesthesia machine and Hallowell EMC model 2002 ventilator) and maintained under pentobarbital anesthesia (1 to 2 mg / kg / hr). Oxygen saturation was measured from the tongue using a tongue sensor (Nellcor pulse oximeter) and was continuously monitored to maintain a constant saturation of 98 to 100% throughout the experiment.

[0211] Fluid balance was controlled by monitoring intravenous fluid intake every 15 min and fluid output (via a Foley catheter) every 30 min. Heart rate, blood pressure (Critikon Dinamap blood pressure monitor, model 8100), end-tidal carbon dioxide partial pressure (pCO2), respiration, and O2 levels (Ohmeda 5250 RGM anesthesia monitor; Nellcor NBP-40 for oxygen saturation [SpO2]) were monitored throughout the experiment based on EEG.

[0212] After craniectomy (25 mm in diameter), the dura was stripped away, and surface EEG recordings were performed using custom-made strip electrodes (electrode diameter, 2.5 mm; inter-electrode distance, 1 cm) placed over the sensorimotor cortex, with one electrode covering the hand motor cortex, one electrode covering the sensory cortex, and a reference electrode attached to the skull at the mastoid or to a third surface electrode between the motor and sensory electrodes.

[0213] The 1:1 ratio of 1:1 was determined by soaking 0.5 mM of the 1:1 ratio in 100 μM bicuculline (Sigma-Aldrich, St. Louis, MO, USA). 2 Acute bicuculline lesions were created in the motor cortex with gelatin sponge pads (Codman & Shurtleff, Randolph, MA, USA). The pads were placed on the cortex for five minutes and fed for five minutes every hour. Bicuculline-induced epileptiform spiking activity stabilized after about 1 hour, and then data acquisition was started. EEG signals from motor and sensory cortical electrodes were recorded continuously (1000 Hz sampling, no filter; Digidata 1440A system from Axon Instrument).

[0214] Once stable bicuculline spikes were observed in the EEG trace of each NHP, the test product was administered.

[0215] Data were analyzed using the R programming language with custom-designed software as described in a published reference (Haglund and Hochman, 2005). Bicuculline-induced spiking activity was quantified from motor cortex recordings. The effect of the test product on bicuculline-induced spiking was analyzed by comparing the peak response after treatment administration to the average of a 5-min control period before test product administration (baseline). In the bicuculline experiments, recovery from test product administration was followed for a period of 20 to 35 min after recovery to pre-treatment spiking activity. Due to technical reasons and practical limitations on staff time, some recordings had to be terminated before the recovery phase was completed.

[0216] An example of an EEG trace recorded with bicuculline spikes is shown in Fig.12 middle.

[0217] Test product: The product under investigation was NPT 2042, and bumetanide was the reference product or active control. The product descriptions of NPT 2042 and bumetanide are listed in Table Ex 7-3 and Table Ex 7-4, respectively.

[0218] Table Ex 7-3 NPT 2042 Product Description

[0219]

[0220] Table Ex 7-4 Bumetanide Product Description

[0221]

[0222] The product under investigation (NPT 2042) was synthesized by Synexis, Inc. (Research Triangle Park, NC) and provided to Duke University. NPT 2042 was stored at 2°C to 8°C. NPT 2042 was lot number 025DAP012 and was manufactured on February 22, 2007.

[0223] A commercial source of the reference product bumetanide was provided by Duke University for use in PO experiments.

[0224] For the IV experiments, a commercial source of the reference product bumetanide was provided by Synexis.

[0225] Test product preparation

[0226] Each NHP was weighed prior to study procedures and administered NPT2042 and bumetanide at equimolar doses of 10 mg / kg PO and 2 mg / kg IV.

[0227] Each suspension of NPT 2042 and bumetanide for oral administration (by gavage) was prepared by sonicating NPT 2042 or bumetanide in a mixture of carboxymethylcellulose (CMC) and water. The suspensions were prepared immediately before use, stored at room temperature, and administered within 1 hour of preparation.

[0228] Each IV solution of NPT 2042 and bumetanide was prepared by dissolving NPT 2042 or bumetanide in 100% PEG-200. These solutions were provided by Scynexis.

[0229] Data analysis

[0230] Data were analyzed using the R programming language with custom-designed software as previously described (Haglund and Hochman 2005).

[0231] To better quantify the effect size of these records, an algorithm was applied to automatically detect the bases and tops of the bicuculline spikes, as described in the paper. The algorithm is a "brute force" and works by moving a window across the data that is wide enough to accommodate a single spike and finding the maximum and minimum through the window, ignoring all variations in the trace that are less than two standard deviations above the background noise (Haglund and Hochman 2005). An example of a visual representation of the output of this computer analysis is shown below. Fig.14 Here, the computer identifies all the tops of the peaks with the upper (red) points and the corresponding bottoms of the peaks with the lower (blue) points. The distance between the maximum and minimum values ​​is the peak height, as Fig.15 shown.

[0232] From these data, a computer generated calculation of the height of each spike and the time interval between each spike (interspike-to-spike interval, ISI). To reduce the possibility that brief (1 or 2 minutes) random fluctuations would bias the analysis, the data (spike size and ISI) were smoothed using a 3-minute moving average window. From these smoothed data, the following statistics were calculated for oral administration of bumetanide and NPT 2042: 1) spike size during the 10-minute interval before treatment and during the 10-minute interval before and after the determination of the spike time representing the maximum change; 2) the interspike-to-spike interval before and after treatment, during the same time interval in which the spike size was obtained; and 3) the time to 50% recovery – the time from the maximum change in spike size or ISI to 50% of the mean value during the pre-treatment period. Due to the smoothing, the standard deviation within the above 10-minute windows was negligible and is therefore not reported.

[0233] result

[0234] Intravenous administration of bumetanide and NPT 2042

[0235] In two experiments conducted on two different animals, bumetanide (2 mg / kg) and NPT 2042 (molar equivalent of 2 mg / kg bumetanide) were injected IV into an arm vein through an IV line used during the experiments to maintain the animals' health.

[0236] The EEG traces from this experiment are Fig.16 Indicated in. Fig.16 The A-row traces in FIG. 5 show a continuous 20-minute recording starting 3 minutes before the administration of bumetanide (A1) or NPT 2042 (A2). The time of test product administration is shown by the red vertical bar in these traces (at t = 3 minutes). Fig.16 The B traces in Figure 1 were selected from the time when a deep, nearly complete blockade of epileptiform activity was observed, starting at t = 81 min after injection of bumetanide (B1) and t = 72 min after injection of NPT 2042 (B2). Fig.16 The C trace in Figure 2 shows a period of abrupt recovery to baseline pre-treatment spike conditions after bumetanide treatment started at approximately t = 120 minutes after injection (C1) and at the end of the experiment at t = 155 minutes after NPT 2042 injection (C2), where recovery to baseline had not yet been observed. For practical reasons, the experiment with NPT 2042 administration ended before the recovery time after NPT 2042 administration could be determined.

[0237] Oral administration of bumetanide and NPT 2042

[0238] In two experiments in two different primates, bumetanide (10 mg / kg) and NPT 2042 (molar equivalent of 10 mg / kg bumetanide) were administered PO by oral gavage once a stable EEG trace was obtained for each NHP. Compared with the IV studies, where a near-complete blockade of epileptiform activity was evident and could be discerned visually from the raw data, the maximal changes induced by oral administration were more subtle. Fig.17 Representative 3-minute traces before treatment for bumetanide (A1) and NPT 2042 (A2), and representative 3-minute traces during the period of maximum change for bumetanide (B1) and NPT 2042 (B2) are shown. The traces in panels A1 and A2 above represent 3-minute spike activity before treatment (baseline). Treatment effects are presented in panels B1 and B2 as 3-minute intervals. The results for PO bumetanide and NPT 2042 show that both compounds transiently mediate a decrease in spike size and frequency (Table Ex 7-5). Due to the small sample size and variability between animals, this experiment was not able to demonstrate quantitative differences between compounds.

[0239] Table Ex 7-5 Effects of PO Bumetanide and NPT 2042 on the Bicuculline-Produced Spike

[0240]

[0241] in conclusion

[0242] Conclusions drawn from small sample sizes in these experiments are limited. However, given the stability of bicuculline spikes over time in acute seizure foci created on monkey cortex and the nearly complete blockade of this spike shortly after intravenous administration of bumetanide or NPT2042, NPT 2042 appears to reduce bicuculline-induced epileptiform activity in monkeys, similar to bumetanide. When quantified during the PO experiments (in which the treatment effects were smaller), both bumetanide and NPT 2042 appeared to transiently reduce both spike size and frequency shortly after treatment administration, and both returned to pretreatment values.

[0243] Example 8: Diuretic Effects of Orally Administered Bumetanide Dibenzylamide (NPT 2042) in Nonhuman Primates

[0244] The purpose of this study was to evaluate the diuretic effects of bumetanide dibenzylamide (NPT 2042) and bumetanide in nonhuman primates (NHPs). Urine output was measured in the same animals during the same experiments investigating the effects of NPT 2042 and bumetanide on epileptiform EEG activity (see study number NPT RD 103). Urine was collected into graduated bottles from Foley-catheterized Macacanemestrina monkeys so that urine volume could be measured at fixed intervals throughout the experiment. A single dose of NPT 2042 or the reference control bumetanide was administered orally (PO) to anesthetized, intubated primates.

[0245] These studies demonstrated that oral administration of a 10 mg / kg dose of bumetanide increased peak urine production in cynomolgus monkeys by at least 1500% of control values, whereas equimolar doses of the amide analog bumetanide dibenzylamide (NPT 2042) did not induce a measurable diuretic response. Of note, the seizure suppressive effects of NPT 2042 were demonstrated in the anti-seizure arm of these studies (Example 7, supra). In these experiments, equimolar oral doses of 10 mg / kg NPT2042 and bumetanide both reduced the spike height and frequency of bicuculline spikes.

[0246] Considering the large increase in diuresis induced by bumetanide in nonhuman primates and the lack of any measurable increase with NPT 2042, this study suggests that NPT 2042 has a much lower diuretic effect than bumetanide in this limited sample set.

[0247] Study Aims and Objectives

[0248] The purpose of this study was to evaluate the diuretic effect of bumetanide dibenzylamide (NPT 2042) compared to bumetanide in nonhuman primates. The results of this study were used to validate NPT 2042 as a drug candidate to advance into clinical development as an adjunctive antiseizure therapy for patients with drug-refractory epilepsy. A key attribute in selecting a drug candidate among NKCC antagonist analog candidates is reduced or decreased diuretic effect so the drug can be tolerated as a long-term treatment.

[0249] In this experiment, two primates were evaluated, one receiving bumetanide and the other receiving NPT 2042 by oral gavage, Table Ex 8-1.

[0250] Table Ex 8-1 Test groups and sample size

[0251]

[0252] PO = oral.

[0253] The data used to assess urine volume obtained after oral administration of bumetanide and NPT 2042 were obtained during studies evaluating the effects of these molecules on bicuculline-mediated spikes and cortical EEG recordings. These primate studies were conducted in Dr. Hochman's laboratory while he was a faculty member at Duke University Medical Center.

[0254] Details regarding the treatment and surgical preparation of macaques for EEG recordings have been described (Haglund and Hochman 2007; Tolner et al. 2011).

[0255] Experimental design: diuresis experiment

[0256] For the urine output data presented in this technical report, in brief, animals were artificially ventilated with 100% oxygen via cannula (Matrix VBS anesthesia machine and Hallowell EMC model 2002 ventilator) and maintained under pentobarbital anesthesia (1-2 mg / kg / h). Oxygen saturation was measured from the tongue using a tongue sensor (Nellcor pulse oximeter) and was continuously monitored to maintain a constant saturation of 98%-100% throughout the experiment.

[0257] Test and reference materials were administered after baseline urine volume measurements were obtained (up to 100 minutes prior to test article administration). Urine production was measured following administration of 10 mg / kg oral bumetanide (n=3) and oral administration of 10 mg / kg of oral bumetanide dibenzylamide (NPT 2042; n=1) at a molar equivalent (mol-Eq).

[0258] Fluid balance was controlled by monitoring intravenous fluid intake every 15 minutes and fluid output (via Foley catheter) every 10 to 15 minutes until the animals returned to pretreatment urine production levels. Heart rate, blood pressure (Critikon Dinamap blood pressure monitor, model 8100), end-tidal pCO2, respiration, and O2 levels (Ohmeda 5250 RGM anesthesia monitor; Nellcor NBP-40 for SpO2) were monitored throughout the experiment based on EEG.

[0259] All urine samples were collected from Foley catheters into graduated bottles, and the volume was recorded for at least two time points before test article and reference material administration at least one hour before treatment administration (to obtain the average baseline / pre-treatment rate of urine production) and every 15 minutes after treatment administration until peak urine volume and recovery (post-treatment urine production rate) were observed.

[0260] Test products

[0261] The product under investigation was NPT 2042, and bumetanide was the reference product or active control. The product descriptions of NPT 2042 and bumetanide are listed in Tables Ex 8-2 and Ex 8-3, respectively.

[0262] Table Ex 8-2 Test product information

[0263]

[0264] Table Ex 8-3 Bumetanide Product Description

[0265]

[0266] The product under investigation (NPT 2042) was synthesized by Synexis, Inc. (Research Triangle Park, NC) and provided to Duke University. NPT 2042 was stored at 2°C to 8°C. NPT 2042 was lot number 025DAP012 and was manufactured on February 22, 2007.

[0267] The commercial source of the reference product bumetanide was provided by Duke University.

[0268] Test Product Preparation: Each NHP was weighed prior to study procedures and administered NPT 2042 and bumetanide at equimolar (mol-Eq) doses of 10 mg / kg PO.

[0269] Each suspension of NPT 2042 and bumetanide for oral administration (by gavage) was prepared by sonicating NPT 2042 or bumetanide in a mixture of carboxymethylcellulose (CMC) and water. The suspensions were prepared immediately before use, stored at room temperature, and administered within 1 hour of preparation.

[0270] Data Analysis: Data were analyzed using custom-designed software using the R programming language.

[0271] result

[0272] A molar equivalent of 10 mg / kg bumetanide was used. It was administered orally via oral gavage to anesthetized monkeys in the form of a water-cmc suspension. Tables Ex 8-4 and 8-5 show the mean change in spike height after treatment and the time to approximately 50% recovery.

[0273]

[0274] Table Ex 8-4 Changes in seizure activity after bumetanide and NPT 2042.

[0275]

[0276] Table Ex 8-5 Changes in seizure activity after morpholine amide and diethylamide.

[0277] Fig.18 Each graph in the Figures shows the percent change in urine volume produced over time for each monkey following oral administration of bumetanide or NPT 2042 (relative to baseline, pre-treatment levels). Fig.18 The graphs labeled bumetanide (1), (2), and (3) in the figure represent the diuretic response to bumetanide, and the remaining graphs show the diuretic response to bumetanide dibenzylamide (NPT 2042). The red line indicates the time of oral administration of treatment at t = 0. The time in minutes is given on the x-axis, and the percent change in urine volume produced (compared to urine production before treatment) is shown on the y-axis. The oral dose was 10 mg / kg bumetanide and a molar equivalent of NPT 2042. Bumetanide elicited an increase in urine volume production of more than 1500% to 3000% from baseline values, while the bumetanide amide analog (NPT 2042) elicited little or no increase in diuresis relative to baseline.

[0278] in conclusion

[0279] These studies demonstrated that oral administration of 10 mg / kg bumetanide elicited at least a 1500% increase in peak urine production in cynomolgus monkeys, whereas the amide analog bumetanide dibenzylamide (NPT 2042) did not elicit any measurable diuretic response.

[0280] It is worth mentioning that the seizure suppressive effects of NPT 2042 were confirmed in the anti-seizure arm of these studies (see study number NPT RD 103). In these experiments, equimolar oral doses of 10 mg / kg NPT 2042 and bumetanide both reduced the spike height and frequency of bicuculline spikes.

[0281] Given that bumetanide induced a large increase in diuresis in nonhuman primates, whereas NPT 2042 did not have any measurable increase, it appears that NPT 2042 has a much lower diuretic effect than bumetanide, even with a small sample size.

[0282] Example 9: Anxiolytic Effects of Bumetanide Dibenzylamide (NPT 2042) Administered Intravenously in Rats

[0283] The aim of this study was to evaluate the effects of bumetanide dibenzylamide (NPT 2042) on the central nervous system (anxiolytic effects) in the fear-potentiated startle (FPS) model of conditioned anxiety in rats (Lehmann et al. 2010; Krystal et al. 2012). The FPS model used in this study is the same model used previously to evaluate the anxiolytic effects of two NKCC antagonists, bumetanide and furosemide (Krystal et al. 2012).

[0284] The FPS test consists of two training sessions in which an intrinsically aversive foot shock is paired with a neutral cue light. In the test session, presentation of this cue light is subsequently used to elicit startle enhancement. The FPS procedure consists of five days of testing; baseline startle responses are collected on days 1 and 2, light / shock pairings are presented on days 3 and 4, and fear-potentiated startle is administered on day 5.

[0285] On day 5, animals were treated with NPT 2042 (35 mg / kg), bumetanide (35 mg / kg), or vehicle (dimethyl sulfoxide [DMSO] alone) via jugular vein cannulation 30 minutes before FPS testing. The doses selected for this study were based on a pilot study conducted by NeuroInvestigations (Lethbridge, Canada) that determined the minimum dose of bumetanide that elicited a measurable response in the FPS model (data on file). Startle amplitudes were measured and compared between NPT2042 and vehicle and between bumetanide and vehicle.

[0286] The results indicate that NPT 2042 elicited anxiolytic effects in the FPS model that were similar in magnitude to those elicited by an equivalent (mg / kg) dose of bumetanide, as determined by a reduction in the startle amplitude of the footshock conditioned stimulus.

[0287] Study Aims and Objectives

[0288] The purpose of this study was to evaluate the anxiolytic effects of bumetanide dibenzylamide (NPT 2042) in the rat FPS model. The results of this study validate NPT 2042 as a drug candidate suitable for advancement to primate studies for further evaluation as an adjunctive anti-epileptic treatment in patients with drug-refractory epilepsy.

[0289] In this study, the anxiolytic effects of NPT 2042 were tested in the FPS anxiety model (vehicle control / DMSO, n=51; bumetanide, n=14; NPT 2042, n=15) using Long-Evans male adult (3 to 4 months old) rats. See Table Ex 9-1.

[0290] Table Ex 9-1 Test Group and Sample Size

[0291]

[0292] For the purpose of screening bumetanide analogs, the rat model is the most reasonable model to measure CNS responses and can also be used to test and compare a range of bumetanide analog candidates. Several standard rat epilepsy models were also considered, but these models are highly labor intensive and / or require large numbers of animals to produce statistically significant comparisons of the CNS effects of bumetanide versus bumetanide analogs and between each other. However, rat associative anxiety models, particularly the fear-potentiated startle (FPS) model, demonstrate extremely robust bumetanide-mediated NKCC-sensitized responses that vary depending on the potency and concentration of the NKCC antagonist, produce large and reproducible responses (thus, requiring the use of far fewer animals), and are much less labor intensive than rat seizure models (Krystal et al. 2012). Of note, many anti-seizure drugs also have anxiolytic effects (Mula et al. 2007), and rat anxiety models reliably predict therapeutic CNS responses in humans (Calabrese 2008) (see also Table 3 (Krystal et al. 2012).

[0293] These rat studies were designed to test CNS effects only (in this case, reduced anxiety), and not to anticipate a diuretic effect of bumetanide, as rats rapidly metabolize and bioconvert bumetanide to inactive metabolites via oxidation of its N-butyl side chain prior to its initiation of diuresis via renal NKCC2 antagonism (Schwartz 1981). Since bumetanide analog candidates, including NPT 2042, have the same N-butyl side chain, they are expected to be similarly susceptible to this metabolism. Therefore, a diuretic effect has been evaluated in primate studies and this is documented in report NPT RD102 (NeuroPro Therapeutics, Inc. 2022).

[0294] Experimental Design: Fear-potentiated startle

[0295] The published FPS protocol was followed (Lehmann et al. 2010) and described below.

[0296] These studies were conducted using male adult (3 to 4 months old) Long-Evans rats housed at the University of Lethbridge Zoo under the direction of Dr. Janice Sutherland. Rat housing consisted of plexiglass cages with wood chip bedding, shared with two or three rats. The temperature of the colony room (20°C to 21°C) was controlled by a 12-hour light / 12-hour dark cycle starting at 7:00 am each day. Food and water were provided ad libitum. 72 hours prior to the experiment, the rats were anesthetized with isoflurane, and a cannula was implanted in the right external jugular vein of each rat for the purpose of administering the test article. Rats were housed in individual cages thereafter, and the cannulas were flushed daily to ensure patency.

[0297] All behavioral tests were performed during the light cycle (7:00 AM to 7:00 PM). Testing occurred between 9:00 AM and 3:00 PM. A different randomly selected rat was used for each group (i.e., no rat was retested in more than one group). All testing was performed in room with ambient light.

[0298] Animals were trained and tested in four identical stabilometer apparatus (Med-Associates). Each rat was placed in a small plexiglass cylinder. The base of each stabilometer consisted of four 6-mm-diameter stainless steel rods spaced 18 mm apart, through which electric shocks could be delivered. Movement of the cylinder resulted in displacement of the accelerometer, where a voltage was generated that was proportional to the velocity of the cage displacement. Startle amplitude was defined as the maximum accelerometer voltage occurring within 0.25 s after delivery of the startle stimulus. The analog output of the accelerometer was amplified, digitized on a scale of 0 to 4096 units, and stored on a microcomputer. Each stabilometer was enclosed in a ventilated, light- and sound-attenuated box. All sound level measurements were made using a precision sound level meter. The noise of a ventilation fan attached to the side wall of each wooden box produced an overall background noise level of 64 dB. The startle stimulus was a 50-ms burst of white noise (5-ms rise to decay time) generated by a white noise generator. The visual conditioned stimulus was the illumination of a light bulb near the white noise source. The unconditioned stimulus was a 0.6 mA foot shock of 0.5 s duration, produced by four constant current shockers located in the chamber. The presentation and order of all stimuli were controlled by a computer. The fear-enhanced startle procedure consisted of 5 days of testing. Baseline startle responses were collected on days 1 and 2, light / shock pairings were provided on days 3 and 4, and fear-enhanced startle testing was performed on day 5. Animals received treatment with test compounds or vehicle on day 5.

[0299] See also Fig.19 Overview of the FPS Model. The details describing the daily procedures are described in the following sections.

[0300] Day 1 and 2: Matching

[0301] On days 1 and 2, rats were placed individually in a Plexiglas cylinder and presented 30 startle stimuli 3 min later with an inter-stimulus interval of 30 sec. An intensity of 105 dB was used. On the second day, the mean startle amplitude of the 30 startle stimuli was used to assign rats into treatment groups with similar means.

[0302] Day 3 and 4: Training

[0303] On days 3 and 4, rats were placed individually in a Plexiglas cylinder. During the first 3 min in the chamber, the rats were acclimated and then given 10 sets of conditioned stimulus (CS)-shock pairings. The shock was delivered during the last 0.5 s of a 3.7-s CS, with an average inter-trial interval of 4 min (range, 3 to 5 min).

[0304] Day 5: Testing

[0305] Animals received study drug (NPT 2042 [35 mg / kg] or bumetanide [35 mg / kg]) or vehicle (DMSO alone) via jugular vein cannulation 30 min prior to testing on day 5.

[0306] For testing, rats were placed in the same startle box in which they were trained and, after 3 min of habituation, presented 18 startle-eliciting stimuli (all 105 dB). These initial startle stimuli were used to habituate the rats to the acoustic startle stimuli again. Thirty seconds after the last stimulus, each animal received 60 startle stimuli, half of which were presented alone (startle-alone trials) and the other half were presented 3.2 seconds after the onset of the 3.7-second CS (CS startle trials). All startle stimuli were presented with an average inter-stimulus interval of 30 seconds, randomly varying between 20 and 40 seconds.

[0307] Test products

[0308] The drug under investigation was NPT 2042, and the control products were bumetanide (active control) and DMSO (vehicle control). The product descriptions of NPT 2042 and bumetanide are listed in Table Ex 9-2 and Table Ex 9-3, respectively.

[0309] Table Ex 9-2 NPT 2042 Product Description

[0310]

[0311] Table Ex 9-3 Bumetanide Product Description

[0312]

[0313] The study drug (NPT 2042) was synthesized by Synexis (Research Triangle Park, NC) and supplied to the University of Lethbridge Zoo. NPT 2042 was stored at 2°C to 8°C. NPT 2042 (lot number 009MPS023) was manufactured on October 10, 2005.

[0314] Commercial sources of the active control product bumetanide and vehicle control DMSO were purchased from the University of Lethbridge Zoo.

[0315] Test product preparation

[0316] The dosing solution is prepared immediately prior to use, stored at room temperature prior to use, and administered within 3 hours of preparation.

[0317] Each rat was weighed prior to study procedures and administered NPT 2042 and bumetanide at a dose of 35 mg / kg IV.

[0318] Each IV solution of NPT 2042 and bumetanide was prepared by dissolving NPT 2042 or bumetanide in 100% DMSO.

[0319] Data Analysis: Data were entered into an Excel spreadsheet and SPSS for data analysis. Independent sample t-tests were used to compare each treatment group. The statistical programming language R was used to generate plots and perform statistical analyses. Welch two-sample t-tests (one-sided) were used to compare NPT 2042 to vehicle and bumetanide to vehicle.

[0320] result

[0321] Bumetanide and NPT 2042 significantly reduced the increase in startle amplitude during footshock conditioning compared with rats treated with vehicle alone (vehicle mean = 165.8 [standard error (SE) = 21.6], p = 0.02652; NPT 2042 mean = 105 [SE = 24.4], p = 0.03512), and bumetanide mean = 97.6 (SE = 26.3; p = 0.02652). Fig. 20 .

[0322] The 95% t-confidence intervals for the means were NPT 2042: [52.6, 157.4], bumetanide: [41.0, 154.2], and vehicle: [122.3, 209.3].

[0323] These data suggest that NPT 2042 at a dose of 35 mg / kg has similar CNS effects to bumetanide at a dose of 35 mg / kg in the FPS model of conditioned anxiety in rats.

[0324] No rats died in any of the test groups.

[0325] Example 10

[0326] All three derivatives: bumetanide diethylamide, bumetanide N-morpholinamide, and bumetanide dibenzylamide resulted in lower percent changes in urine production over time compared to bumetanide. Fig.21 .

[0327] Example 11

[0328] All three derivatives: bumetanide diethylamide, bumetanide N-morpholinamide, and bumetanide dibenzylamide resulted in lower rates of urine production over time compared to bumetanide. Fig. 22 .

[0329] Example 12

[0330] All three derivatives: bumetanide diethylamide, bumetanide N-morpholinamide, and bumetanide dibenzylamide resulted in lower mean rates of urine production compared to bumetanide. Fig.23 .

[0331] Example 13

[0332] In direct contrast to the demonstrated results with amide compounds, the ester prodrug of bumetanide continued to exhibit diuretic effects. Fig.23 , Figure 24 to Figure 27 Make a comparison.

[0333] The mean pre-treatment urine rates (mL / min) were very low compared to the maximum post-treatment urine rates (mL / min) following treatment with esters including bumetanide methyl ester, bumetanide cyanomethyl ester, bumetanide NN-diethyl-2-hydroxyacetamide ester, and bumetanide benzyl ester. Figure 24 to Figure 27 All animals were administered the corresponding ester at a molar dose equivalent to 2 mg / kg bumetanide. Administration was performed intravenously. As shown, the diuretic effect of the bumetanide derivatives was unpredictable.

[0334] Those skilled in the art to which the present disclosure belongs may make modifications without departing from the spirit or characteristics of the present disclosure, particularly in view of the foregoing teachings, thereby causing other embodiments that employ the principles of the present disclosure. Therefore, the described embodiments should be regarded in all respects as merely illustrative and not restrictive, and the scope of the present disclosure is therefore indicated by the appended claims rather than by the foregoing description or drawings. Therefore, although the present disclosure has been described with reference to specific embodiments, modifications of structure, sequence, materials, etc. that are obvious to those skilled in the art still fall within the scope of the protection claimed.

[0335] Example 14

[0336] The purpose of this study was to evaluate the effects of bumetanide dibenzylamide (NPT 2042) on the human central nervous system (anxiolytic effects) to assess the safety and pharmacokinetics (PK) of single and repeated ascending doses of NPT 2042 in healthy adult subjects. Subjects were given eight capsules, each containing 16 mg of bumetanide dibenzylamide, every 24 hours. Pharmacokinetic (PK) blood samples were collected and analyzed before dosing to determine baseline levels. Analyses were also performed on Days 1, 3, 4, 5, 6, 7, and 8 after dosing. Samples were analyzed for BUN (blood urea nitrogen), creatinine, serum chloride, and urine specific gravity. Diuresis is indicated by a disproportionate increase in serum BUN compared to creatinine (BUN increase is not proportional to creatinine), a decrease in urine specific gravity, and an increase in serum chloride. As Figure 28 to Figure 29 As shown, there was no evidence of diuresis. The data indicate that bumetanide dibenzylamide may act similarly to a renal NKCC inhibitor. Figure 28 to Figure 29 The gray horizontal lines in the graph indicate the upper and lower ranges of acceptable normal values. Control subjects (placebo) are represented by alternating black and white dashed lines and gray dots.

[0337] Those skilled in the art to which the present disclosure pertains may make modifications without departing from the spirit or characteristics of the present disclosure, particularly in light of the foregoing teachings, thereby resulting in other embodiments employing the principles of the present disclosure.

[0338] Therefore, the described embodiments should be considered in all respects as illustrative only and not restrictive, and the scope of the present disclosure is therefore indicated by the appended claims rather than by the foregoing description or drawings. Therefore, although the present disclosure has been described with reference to specific embodiments, modifications of structure, sequence, material, etc. that are obvious to those skilled in the art still fall within the scope of the claimed protection.

Claims

1. A pharmaceutical composition comprising bumetanide dibenzylamide, bumetanide diethylamide or bumetanide morpholineamide, wherein the pharmaceutical composition has a therapeutic effect on blocking epileptic seizures in a patient.

2. The composition of claim 1, wherein the therapeutic effect is a ratio of a measure of seizure suppression to a measure of diuresis in the patient.

3. The composition of any one of claims 1 to 2, wherein the measure of seizure inhibition is the frequency of seizures.

4. A composition according to any one of claims 1 to 3, wherein the measure of seizure inhibition is the intensity of the seizure.

5. The composition according to any one of claims 1 to 4, wherein the measure of seizure inhibition is a change in the amplitude of pharmacologically or electrically induced seizure (epileptic) activity as measured with EEG or other electrophysiological type recordings.

6. The composition of claim 5, wherein the amplitude is reduced by about 50% to about 99% after treatment with the composition.

7. A composition according to any one of claims 1 to 6, wherein the measure of seizure inhibition is a change in the frequency of pharmacologically or electrically induced seizure (epileptic) activity as measured with EEG or other electrophysiological type recordings.

8. A composition according to any one of claims 1 to 7, wherein the measure of the diuretic effect is urine volume.

9. A composition according to any one of claims 1 to 8, wherein the measure of the diuretic effect is urine ion concentration.

10. The composition according to any one of claims 1 to 9, wherein the therapeutic effect is based on changes in seizure frequency and plasma osmolality.

11. The composition according to any one of claims 1 to 10, wherein the therapeutic effect is based on an increase in the peak-to-peak interval.

12. The composition of claim 11, wherein the peak-to-peak interval is reduced by about 50% to about 99%.

13. The composition according to any one of claims 1 to 12, wherein the therapeutic effect is based on an increase in the inter-peak interval and a change in plasma osmolality.

14. The composition of any one of claims 1 to 13, wherein the therapeutic effect is based on changes in seizure frequency and urine production over a given period of time.

15. The composition of any one of claims 1 to 14, wherein the therapeutic effect is based on an increase in inter-peak intervals and changes in urine production over a given period of time.

16. The composition of any one of claims 1 to 15, wherein the therapeutic effect is based on a reduction in seizure spike height or amplitude and a change in plasma osmolality.

17. The composition of any one of claims 1 to 16, wherein the therapeutic effect is based on a reduction in seizure spike height or amplitude and changes in urine production over a given period of time.

18. The composition according to any one of claims 1 to 17, wherein the therapeutic effect is an effect based on changes in seizure frequency and blood ions over time, wherein the ions are selected from sodium, chloride, magnesium or pH.

19. The composition according to any one of claims 1 to 18, wherein the therapeutic effect is an effect based on an increase in the peak-to-peak interval and changes in blood ions over time, wherein the ions are selected from sodium, chloride and magnesium.

20. The composition according to any one of claims 1 to 19, wherein the therapeutic effect is based on the effect of reduction in seizure spike height or amplitude and changes in blood ions over time, wherein the ions are selected from sodium, chloride and magnesium.

21. The composition of any one of claims 1 to 20, wherein the therapeutic effect is a change in the ratio of seizure frequency or amplitude to urine output compared to baseline.

22. The composition of any one of claims 1 to 21, wherein the therapeutic effect is a proportional change in seizure frequency or amplitude in any objective determination.

23. The composition of any one of claims 1 to 22, wherein the therapeutic effect is the proportional change in epileptic seizure frequency or amplitude before and after treatment with the composition.

24. The composition of any one of claims 1 to 23, wherein the therapeutic effect is the proportional change in frequency and amplitude of epileptic seizures before and after treatment with the composition.

25. The composition of any one of claims 1 to 24, wherein the change in seizure frequency following treatment with the composition is a decrease in the frequency of seizures by at least 50%.

26. The composition of any one of claims 1 to 25, wherein the change in seizure frequency following treatment with the composition is a decrease in the frequency of seizures occurring by more than 50% to 100%.

27. A composition according to any one of claims 1 to 26, wherein The diuretic effect is measured as less than about a two-fold increase in urine production within twenty-four hours following treatment with the composition.

28. A composition according to any one of claims 1 to 27, wherein The diuretic effect is measured as the absence of an increase in urine production within twenty-four hours following treatment with the composition.

29. A composition according to any one of claims 1 to 28, wherein The diuretic effect is measured as an increase in urine production of about 0% to about 100% within twenty-four hours following treatment with the composition.

30. The composition of any one of claims 1 to 29, wherein the therapeutic effect is determined based on an effective dose of the composition.

31. The composition of claim 30, wherein the therapeutic effect is determined as:

32. A composition according to any one of claims 30 to 31, wherein An effective dose of the composition is that dose required to completely block seizure activity.

33. A composition according to any one of claims 30 to 32, wherein The effective dose of the composition is higher than the dose required to completely block epileptic seizures.

34. A composition according to any one of claims 30 to 33, wherein An effective dose of the composition is a dose that causes seizure suppression without producing a diuretic effect.

35. The composition of any one of claims 1 to 34, wherein the composition has a positive effect on neuronal synchronous activity without having a substantial effect on neuronal excitability.

36. The composition of any one of claims 1 to 35, wherein the composition provides a therapeutic effect window.

37. The composition of any one of claims 1 to 36, wherein the composition comprises bumetanide dibenzylamide.

38. The composition of any one of claims 1 to 37, wherein the composition comprises bumetanide morpholine amide.

39. A method for treating epileptic seizures in a patient, the method comprising: Administering a pharmaceutical composition according to any one of claims 1 to 38; Reducing the patient's seizure activity without increasing the patient's urine output.

40. The method of claim 39, wherein the pharmaceutical composition is administered orally.

41. The method of any one of claims 39 to 40, wherein the pharmaceutical composition is administered once.

42. The method of any one of claims 39 to 41, wherein the pharmaceutical composition is administered once daily for a fixed number of consecutive days.

43. The method according to any one of claims 39 to 42, wherein the anti-epileptic seizure effect of the pharmaceutical composition is mediated through its antagonism of NKCC1 on neurons and / or glial cells.

44. The method of any one of claims 39 to 43, wherein the diuretic effect of the pharmaceutical composition is mediated through its antagonistic effect on renal NKCC2.

45. The method of any one of claims 39 to 44, wherein the pharmaceutical composition is administered once daily for a fixed number of consecutive days.

46. ​​The method of any one of claims 39 to 45, wherein the pharmaceutical composition is administered to treat epilepsy.

47. The method of any one of claims 39 to 46, wherein the pharmaceutical composition is administered in combination with conventional therapy to treat epileptic seizures.

48. The method of any one of claims 39 to 47, wherein urine volume is measured by blood ion concentration imbalance.

49. The method of any one of claims 39 to 48, wherein urine volume is measured by the magnitude of the diuretic effect calculated as the amount (concentration) of bumetanide in the blood compared to bumetanide dibenzylamide.

50. The method of any one of claims 39 to 49, wherein bumetanide dibenzylamide has a faster onset of action as measured by a reduction in seizure frequency than traditional anti-epileptic drugs.

51. The method of claim 50, wherein the reduction in seizure frequency is measured by one or more of: a) a time increment selected from one or more of hours, days, weeks and months; b) reduction in seizure activity recorded in seizure diaries and records; c) an increase in one or more of the interictal (between) and postictal (after) spikes; and d) Decreased interictal activity as measured by EEG.

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