Therapeutic compositions, methods, and uses for seizure control

Abstract

Described herein are compositions comprising bumetanide dibenzylamide for treating selected conditions of the central and peripheral nervous systems using non-synaptic mechanisms. More specifically, the present 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 treating seizure disorders, epilepsy, and related indications.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 63 / 398,480, filed August 16, 2022, which is incorporated herein by reference in its entirety.

[0002] Described herein are compositions comprising bumetanide dibenzylamide for treating selected conditions of the central and peripheral nervous systems using non-synaptic mechanisms. More specifically, the present 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 treating seizure disorders, epilepsy, and related indications. [Background technology]

[0003] Epilepsy is characterized by abnormal neuronal discharges and typically manifests as various types of seizures. Many anticonvulsants originally developed for the treatment of epilepsy and other seizure disorders have also been applied to the treatment of non-epileptic conditions, including neuropathic pain, mood disorders (such as bipolar affective disorder), and schizophrenia (for a review of the use of antiepileptic drugs in the treatment of non-epileptic conditions, see Rogawski and Loscher, Nat. Medicine, 10:685-692, 2004). Therefore, it has been suggested that epilepsy, neuropathic pain, and affective disorders share a common pathophysiological mechanism (Rogawski & Loscher, supra; Ruscheweyh & Sandkuhler, Pain 105:327-338, 2003), namely, a pathological increase in neuronal excitability, resulting in a correspondingly inappropriately high frequency of spontaneous neuronal firing. However, only some, but not all, antiepileptic drugs are effective in treating neuropathic pain, and furthermore, such antiepileptic drugs are effective only in a specific subset of patients with neuropathic pain (McCleane, Expert. Opin. Pharmacother. 5:1299-1312, 2004).

[0004] Epileptiform activity is identified by spontaneously occurring synchronous discharges of neuronal populations, which can be measured using electrophysiological techniques. This synchronous activity, which distinguishes epileptiform activity from non-epileptiform activity, is termed "hypersynchronization" to describe a state in which individual neurons become increasingly likely to discharge one another in a time-locked manner. Hypersynchronization is typically induced in experimental models of epilepsy by either increasing excitability or decreasing inhibitory synaptic currents. Therefore, hyperexcitability itself has been postulated to be a crucial feature involved in the generation and maintenance of epileptiform activity. Similarly, neuropathic pain has been thought to involve the conversion of neurons involved in pain transmission from a state of normal sensitivity to a state of hypersensitivity (Costigan & Woolf, Jnl. Pain 1:35-44, 2000). Therefore, the focus in developing treatments for both epilepsy and neuropathic pain has been on reducing neuronal hyperexcitability by either (a) suppressing action potential generation, (b) increasing inhibitory synaptic transmission, or (c) decreasing excitatory synaptic transmission.

[0005] Most currently used therapeutic drugs target synaptic activity in excitatory pathways, for example, by modulating the release or activity of excitatory neurotransmitters, enhancing inhibitory pathways, blocking ion channels involved in impulse generation, and / or acting as membrane stabilizers. Therefore, conventional drugs and therapeutic approaches for the treatment of epilepsy and neuropsychiatric disorders reduce neuronal excitability and inhibit synaptic firing. One serious drawback of these therapies is that they are nonselective and exert their effects on both normal and abnormal neuronal populations. This leads to negative, unintended side effects that can affect normal CNS functions, such as cognition, learning, and memory, and result in adverse physiological and psychological effects in treated patients. Common side effects include excessive sedation, dizziness, memory loss, and liver damage. However, it has been shown that hypersynchronous epileptiform activity can be dissociated from hyperexcitability, and that the cation-chloride cotransport inhibitor furosemide can reversibly block synchronous discharges without reducing hyperexcitable synaptic responses (Hochman et al. Science 270:99-102, 1995). Cation-chloride cotransporters (CCCs) are important regulators of neuronal chloride concentration that are thought to influence intercellular communication and various aspects of neuronal development, plasticity, and injury. The CCC gene family consists of three broad groups: Na + -Cl - cotransporter (NCC), K + -Cl - Cotransporter (KCC) and Na + -K + -2Cl -The Na-K-Cl cotransporter (NKCC) is a phosphodiesterase (PDS)-dependent pathway that inhibits Na-K-Cl cotransport in all cells and tissues. Loop diuretics, including furosemide, bumetanide, and benzmethanide, have previously suggested that furosemide may be useful in treating certain types of epilepsy (Medicina Espanola 61:280-281, 1969, and Brit. J. Clin. Pharmacol. 3:621-625, 1976). Bumetanide is a potentially more potent drug for treating epilepsy, but it also has a more pronounced diuretic effect. Therefore, there remains a need for methods and compositions for treating neuronal disorders that are not diuretic and that disrupt hypersynchronous neuronal activity without reducing neuronal excitability and spontaneous synchronization, which are necessary for normal peripheral and central nervous system function. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Rogawski and Loscher, Nat. Medicine, 10:685-692, 2004 [Non-patent document 2] Ruscheweyh&Sandkuhler,Pain 105:327-338,2003 [Non-patent document 3] McCleane,Expert.Opin.Pharmacother.5:1299-1312,2004 [Non-patent document 4] Costigan&Woolf,Jnl.Pain l:35-44,2000 [Non-patent document 5] Hochman et al.Science 270:99-102,1995 [Non-patent document 6] Medicina Espanola 61:280-281,1969 [Non-Patent Document 7] Brit.J.Clin.Pharmacol.3:621-625,1976 Summary of the Invention [Means for solving the problem]

[0007] In one embodiment of the present disclosure, bumetanide dibenzylamide demonstrated antiseizure activity in a non-human primate model, demonstrating a similar response to bumetanide in a rat anxiolytic bioassay as measured by NKCC suppression. In contrast, bumetanide exhibited potent diuretic effects, but treatment with bumetanide dibenzylamide did not increase urine output in the primate model. Comparison of the pharmacological activities of bumetanide and bumetanide dibenzylamide highlights the novel properties of bumetanide dibenzylamide as a potential antiseizure therapeutic agent that are not limited to its diuretic effect. Collectively, the data in this disclosure, along with published studies describing the antiseizure effects of bumetanide and furosemide, support the novel and unexpected properties of bumetanide dibenzylamide as an adjunctive antiseizure therapy. The observed antiseizure effects of bumetanide and furosemide are believed to be mediated through antagonism of NKCC1 on neurons and / or glial cells, while their diuretic effects are mediated through antagonism of renal NKCC2.

[0008] One embodiment of the present disclosure includes bumetanide derivatives and compositions containing such compounds that exhibit therapeutically effective seizure blockade substantially without a diuretic effect.

[0009] One embodiment of the present disclosure includes bumetanide derivatives and compositions containing such compounds that exhibit therapeutically effective seizure blockade substantially without a diuretic effect, where the ratio of seizure suppression to diuretic effect provides the therapeutic efficacy or therapeutic index. The therapeutic efficacy is the ratio of a measure of seizure suppression (e.g., seizure frequency, seizure intensity, etc.) to a measure of the diuretic effect of the therapeutic compound (e.g., urine volume, urine ion concentration, etc.).

[0010] One embodiment of the present disclosure includes a therapeutic effect based on a change in seizure frequency and plasma osmolality (as a measure of dehydration). One embodiment of the present disclosure includes a therapeutic effect based on an increase in interspike interval and a change in plasma osmolality (as a measure of dehydration). One embodiment of the present disclosure includes a therapeutic effect based on a change in seizure frequency and urine production over a given period of time. One embodiment of the present disclosure includes a therapeutic effect based on an increase in interspike interval and a change in urine production over a given period of time.

[0011] One embodiment of the present disclosure includes a therapeutic effect based on a reduction in seizure spike height or amplitude 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 seizure spike height or amplitude and a change in urine production over a given period of time. One embodiment of the present disclosure includes a therapeutic effect based on seizure frequency and a change in blood ions over time, the ions being selected from sodium, chloride, or magnesium. One embodiment of the present disclosure includes a therapeutic effect based on an increase in interspike interval and a change in blood ions over time, the ions being selected from sodium, chloride, magnesium, or pH. One embodiment of the present disclosure includes a therapeutic effect based on a reduction in seizure spike height or amplitude and a change in blood ions over time, the ions being selected from sodium, chloride, or magnesium.

[0012] 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 by EEG or other electrophysiological type 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, per week, or per month).

[0013] In one embodiment, the therapeutic effect is defined as the proportional change in seizure frequency or amplitude relative to urine output compared to baseline. In one embodiment, the therapeutic effect is the proportional change in seizure frequency or amplitude as determined objectively. In one embodiment, the therapeutic effect is the proportional change in seizure frequency or amplitude before and after treatment with a therapeutic compound.

[0014] In one embodiment, the change in frequency after treatment is at least a 50% decrease in seizure frequency. In one embodiment, the change in diuresis after treatment is less than about a 2-fold increase in urine production over a 24-hour period. In one embodiment, the change in frequency after treatment is more than a 50% decrease in seizure frequency. In one embodiment, the change in diuresis after treatment is no increase in urine production over a 24-hour period.

[0015] In one embodiment, the change in frequency after treatment is about a 50% to 100% decrease in seizure frequency, hi one embodiment, the change in diuresis after treatment is about a 0% to about a 100% increase in urine production over a 24 hour period.

[0016] In one embodiment, the therapeutic effect for a particular dose of a therapeutic compound is defined as follows: Treatment effect = [Seizure activity after treatment] / [Seizure activity before treatment]* [Diuresis after treatment] / [Diuresis before treatment]

[0017] The dose of the therapeutic compound may be less than that required to completely block seizure activity. If the dose is greater than that required to completely block seizure activity, a therapeutic effect different from zero is determined to ascertain the comparative effects of different therapeutic compounds.

[0018] One embodiment of the present disclosure includes bumetanide derivatives and compositions containing such compounds that provide an unexpectedly improved increase 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.

[0019] One embodiment of the present disclosure includes bumetanide derivatives and compositions containing such compounds that unexpectedly provide improved diuresis reduction when compared to other bumetanide derivatives.

[0020] One embodiment of the present disclosure includes bumetanide derivatives and compositions containing such compounds that exhibit a reduction in both seizure amplitude and frequency.

[0021] One embodiment of the present disclosure includes bumetanide derivatives and compositions containing such compounds in methods or uses for seizure control.

[0022] One embodiment of the present disclosure includes bumetanide derivatives and compositions containing such compounds that exhibit positive effects on synchronous activity without substantially affecting excitability. One aspect includes specific bumetanide derivatives and compositions containing such compounds that provide a therapeutic window of effect.

[0023] One embodiment of the present disclosure includes one or more bumetanide derivatives and compositions containing such compounds that provide seizure suppression, including a reduction in spike amplitude and interspike interval. 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.

[0024] 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 a seizure suppression / treatment effect. One aspect of the present disclosure includes determining a diuretic factor for a proposed anti-seizure treatment and then calculating a corresponding therapeutic factor. One aspect of the present disclosure includes the unique relationship between the dose of a bumetanide amide derivative and the lack of a diuretic effect. Bumetanide amide derivatives offer unexpected advantages over other bumetanide derivatives. Studies have shown significantly different effects for different derivatives and different formulations.

[0025] One embodiment of the present disclosure includes a method or use for treating patients refractory to conventional anticonvulsants, comprising administering bumetanide dibenzylamide. The present disclosure includes methods and uses that provide particular benefit to patients with epilepsy that is not adequately controlled by, or is otherwise refractory to, conventional therapies such as one or more of phenytoin, carbamazepine, valproate, lamotrigine, levetiracetam, ethosuximide, phenobarbital, and topiramate, by administering to the patient a pharmaceutical composition comprising bumetanide dibenzylamide.

[0026] One embodiment of the present disclosure includes a pharmaceutical composition comprising bumetanide dibenzylamide, bumetanide diethylamide, or bumetanide morpholinoamide, or a salt thereof, wherein the pharmaceutical composition has a therapeutic effect on seizure blockade in a patient.

[0027] In one embodiment, the therapeutic effect is a ratio of a measure of seizure suppression to a measure of diuretic effect for the patient. In one embodiment, the measure of seizure suppression is seizure frequency. In one embodiment, the measure of seizure suppression is seizure intensity. In one embodiment, the measure of seizure suppression is a change in the amplitude of pharmacologically or electrically induced seizure (epileptiform) activity as measured by EEG or other electrophysiological recording. In one embodiment, the measure of seizure suppression is a change in the frequency of pharmacologically or electrically induced seizure (epileptiform) activity as measured by EEG or other electrophysiological recording. In one embodiment, the measure of diuretic effect is urine volume. In one embodiment, the measure of diuretic effect is urinary ion concentration. In one embodiment, the therapeutic effect is based on a change in seizure frequency and plasma osmolality. In one embodiment, the therapeutic effect is based on an increase in interspike interval. One embodiment includes a reduction in interspike interval by about 50% to about 99%. One embodiment includes a therapeutic effect based on an increase in interspike interval and a change in plasma osmolality. One embodiment includes a therapeutic effect based on a change in seizure frequency and urine production over a given period of time. One embodiment includes a therapeutic effect based on an increase in interspike interval and a change in urine production over a given period of time. One embodiment includes a therapeutic effect based on a decrease in seizure spike height or amplitude and a change in plasma osmolality. One embodiment includes a therapeutic effect based on a decrease in seizure spike height or amplitude and a change in urine production over a given period of time. One embodiment includes a therapeutic effect based on seizure frequency and a change in blood ions over time, the ions being selected from sodium, magnesium chloride, or pH. One embodiment includes a therapeutic effect based on an increase in interspike interval and a change in blood ions over time, the ions being selected from sodium, magnesium chloride, or pH. One aspect includes the therapeutic effect being a reduction in seizure spike height or amplitude and an effect based on changes in blood ions over time, the ions being selected from sodium, chloride, or magnesium. One aspect includes the therapeutic effect being a proportional change in seizure frequency or amplitude relative to urine output compared to baseline.In one embodiment, the therapeutic effect is a proportional change in seizure frequency or amplitude in any objective determination. In one embodiment, the therapeutic effect is a proportional change in seizure frequency or amplitude before and after treatment with the composition. In one embodiment, the therapeutic effect is a proportional change in seizure frequency and amplitude before and after treatment with the composition. In one embodiment, the change in seizure frequency after treatment with the composition is at least a 50% reduction in seizure frequency. In one embodiment, the change in seizure frequency after treatment with the composition is a greater than 50% to 100% reduction in seizure frequency. In one embodiment, the measure of diuretic effect is less than about a 2-fold increase in urine production over 24 hours after treatment with the composition. In one embodiment, the measure of diuretic effect is no increase in urine production over 24 hours after treatment with the composition. In one embodiment, the measure of diuretic effect is about a 0% to about a 100% increase in urine production over 24 hours after treatment with the composition. In one embodiment, the therapeutic effect is determined based on an effective dose of the composition. In one embodiment, the therapeutic effect is: Treatment effect = [seizure activity after treatment] / [seizure activity before treatment]* [Diuresis after treatment] / [Diuresis before treatment] This includes being determined as follows.

[0028] One embodiment includes an effective dose of the composition being the dose required to completely block seizure activity. One embodiment includes an effective dose of the composition exceeding the dose required to completely block seizures. One embodiment includes an effective dose of the composition being a dose that causes seizure suppression without causing a diuretic effect. One embodiment includes a composition having a positive effect on neuronal synchronous activity without substantially affecting neuronal excitability. One embodiment includes a composition providing a therapeutic window. One embodiment includes a composition comprising bumetanide dibenzylamide. One embodiment includes a composition comprising bumetanide morpholinoamide.

[0029] One embodiment of the present disclosure includes a method for treating seizures in a patient comprising administering a pharmaceutical composition of the present disclosure and reducing seizure activity in the patient without increasing the patient's urine output.

[0030] In one embodiment, the pharmaceutical composition is administered orally. In one embodiment, the pharmaceutical composition is administered once. In one embodiment, the pharmaceutical composition is administered once daily for a fixed number of consecutive days. In one embodiment, the anti-seizure effect of the pharmaceutical composition is mediated through antagonism of NKCC1 on neurons and / or glial cells. In one embodiment, the diuretic effect of the pharmaceutical composition is mediated through antagonism of renal NKCC2. In one embodiment, the pharmaceutical composition is administered once daily for a fixed number of consecutive days. In one embodiment, the pharmaceutical composition is administered to treat epilepsy. In one embodiment, the pharmaceutical composition is administered in combination with a conventional therapy for treating seizures. In one embodiment, urine output is measured by blood ion concentration imbalance. In one embodiment, urine output 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 embodiment, bumetanide dibenzylamide has a faster time to effect, as measured by a reduction in seizure frequency, than conventional antiepileptic agents. In one aspect, the 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 diary and recorded seizure activity, an increase in one or more of interictal (inter) and postictal (post) spikes, and a reduction in interictal activity measured by EEG.

[0031] One or more embodiments or aspects may be incorporated into different embodiments or aspects, even if not specifically described, i.e., all embodiments and aspects may be combined in any way to form additional embodiments. [Brief explanation of the drawings]

[0032] [Figure 1] 1 shows the results of using furosemide in a seizure model. [Figure 2]1 shows the effect of furosemide on afterdischarge (AD) thresholds. [Figure 3] 1 shows the results of using Keppra® in a stroke model. [Figure 4] Electrode placement in a primate seizure model is shown. [Figure 5] Representative EEG and AD activity are shown. [Figure 6] Measures of AD activity, duration, mean spike height, and envelope area are shown. [Figure 7] Shows the effect of Bikkuri Spike. [Figure 8] 1 shows the comparative effects of NPT2024 and bumetanide on interspike intervals. [Figure 9] The comparative effects of NPT2024 and bumetanide on spikes per minute are shown. [Figure 10] The comparative effects of NPT2024 and bumetanide on mean spike height are shown. [Figure 11] 1 shows the comparative effects of NPT2024 and bumetanide on urine production over time. [Figure 12] Figure 1 shows the stability of spikes from an acute bicuculline focus recorded with surface EEG electrodes. [Figure 13] 1 shows the comparative effects of Keppra® and bumetanide in the Bicuculline spike NHP seizure model (IV administration). [Figure 14] The computer-defined upper and lower portions of the bicuculline-generated spike are shown. [Figure 15] Representative images of spike height measurements in the bicuculline focus model are shown. [Figure 16] 1 shows EEG recordings from bicuculline foci following IV bumetanide and bumetanide dibenzylamide (NPT2042) administration: pre-treatment, post-treatment, and recovery. [Figure 17] Shown are oral bumetanide and NPT2042: surface EEG recordings from bicuculline foci. [Figure 18]1 shows the change in urine volume production over time in macaque monkeys after oral administration of bumetanide and NPT2042. [Figure 19] 1 shows the fear-potentiated startle model. [Figure 20] 1 shows rat fear-potentiated startle data in units of startle reflex amplitude per unit of potential. [Figure 21] 1 shows the percentage change in urine production over time showing the comparative effects of bumetanide, bumetanide morpholinoamide, bumetanide diethylamine, and bumetanide dibenzylamide on diuresis. [Figure 22] 1 shows urine production rates (urine rate mL / min) illustrating the comparative effects of bumetanide, bumetanide morpholinoamide, bumetanide diethylamine, and bumetanide dibenzylamide on diuresis. [Figure 23] 1 shows the mean urine production rates after treatment showing the comparative effects of bumetanide, bumetanide morpholinoamide, bumetanide diethylamine, and bumetanide dibenzylamide on diuresis. [Figure 24] 1 shows the mean pre-treatment urine rate (mL / min) compared to the maximum post-treatment urine rate (mL / min) after treatment with bumetanide methyl ester. [Figure 25] 1 shows the mean pre-treatment urine rate (mL / min) compared to the maximum post-treatment urine rate (mL / min) after treatment with bumetanide cyanomethyl ester. [Figure 26] 1 shows the mean pre-treatment urine rate (mL / min) compared to the maximum post-treatment urine rate (mL / min) after treatment with bumetanide N,N-diethylglycolamide ester. [Figure 27] 1 shows the mean pre-treatment urine rate (mL / min) compared to the maximum post-treatment urine rate (mL / min) after treatment with bumetanide benzyl ester. [Figure 28] 1 shows the effect of bumetanide dibenzylamide (NPT2042) administration on blood urea nitrogen in humans. [Figure 29] 1 shows the effect of bumetanide dibenzylamide (NPT2042) administration on creatinine in humans. [Figure 30]1 shows the effect of bumetanide dibenzylamide (NPT2042) administration on serum chloride in humans. [Figure 31] 1 shows the effect of bumetanide dibenzylamide (NPT2042) administration on urine specific gravity in humans. DETAILED DESCRIPTION OF THE INVENTION

[0033] definition As used herein, the terms "active ingredient," "active pharmaceutical ingredient," and "API" refer to a drug, active ingredient, compound, or substance, composition, or mixture thereof that provides a pharmacological, often beneficial, effect.

[0034] As used herein, the term "dose" refers to any form of formulation of the active ingredient that contains a sufficient amount to produce a therapeutic effect in a single administration.

[0035] As used herein, the term "dosage" refers to the administration of a particular amount, number, and frequency of doses over a specified period of time, typically daily.

[0036] As used herein, the term "active pharmaceutical ingredient load" or "drug load" refers to the amount (mass) of active pharmaceutical ingredient contained in a single soft capsule fill.

[0037] As used herein, the term "formulation" or "pharmaceutical composition" or "composition" refers to a drug in combination with a pharmaceutically acceptable excipient.

[0038] As used herein, the term "particle size distribution" (PSD) refers to the average particle size from a statistical distribution of particle size ranges described herein. The distribution can be Gaussian, normal, or non-normal.

[0039] 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 a distribution of particles are 100 μm or less.

[0040] As used herein, the term "patient" refers to any subject, including mammals and humans. A patient may have or be suspected of having a disease and therefore be treated with a drug. In some cases, a patient is a mammal such as a human, non-human primate, dog, cat, horse, cow, goat, pig, rabbit, rat, mouse, or a premature newborn, newborn, infant, juvenile, juvenile, or adult thereof. In some cases, as used herein, the term "patient" refers to a human (e.g., a male, female, or child). In some cases, as used herein, the term "patient" refers to an experimental animal in an animal model study. A patient or subject may be of any age, sex, or combination thereof.

[0041] As used herein, the terms "biological sample" or "sample" refer to a sample obtained from or derived from a patient. By way of example, a biological sample includes a material selected from the group consisting of bodily fluids, blood, whole blood, plasma, serum, mucous secretions, saliva, cerebrospinal fluid (CSF), bronchoalveolar lavage fluid (BALF), urine, ocular fluids (e.g., vitreous humor, aqueous humor), lymph, lymph node tissue, spleen tissue, bone marrow, and fluids from the ear cavity.

[0042] The term "treating" refers to administering therapy in an amount, manner, or mode effective to ameliorate a condition, symptom, disorder, or parameter associated with a disorder, or the probability thereof (e.g., a therapeutic effect).

[0043] In connection with the pharmacological descriptions, the following abbreviations may be used: [Table 1]

[0044] The term "prevention" refers to preventing or reducing the progression of a disorder to either a statistically significant degree or a degree detectable to one of ordinary skill in the art.

[0045] As used herein, the terms "essentially" or "substantially" mean to a great or significant extent, but not completely.

[0046] As used herein, the term "about" refers to any value, including both integer and fractional components, that is within a variance of up to ±10% of the value modified by the term "about."

[0047] Also described herein are pharmaceutical compositions and dosage forms that include one or more agents that reduce the rate at which the compositions described herein as an active ingredient decompose. Such agents, referred to herein as "stabilizers," include, but are not limited to, antioxidants such as ascorbic acid, pH buffers, salts, sugars, etc.

[0048] The phrase "solubilizer" is used to refer to an ingredient or group of ingredients that help solubilize a composition or part of a composition.

[0049] The terms "may be administered by injection," "injectable," or "injectability" refer to a combination of factors such as a certain force applied to the plunger of a syringe containing the formulations described herein and a certain temperature, a needle of a given bore connected to the outlet of such a syringe, and the time required to expel a certain volume of bumetanide dibenzylamide composition from the syringe through the needle.

[0050] The ranges for each ingredient in the formulations described represent the space in which it can be combined with other ingredients to arrive at a suitable alternative(s), with the ratio adjusted to a total of 100% w / w. The ranges provided are estimates based on available data.

[0051] Pharmaceutical Compositions of the Present Disclosure 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, decreased, combined, substituted, or omitted to provide a formulation that comprises about 100% by weight. Such compositions are disclosed herein as if they were expressly disclosed herein.

[0052] 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.

[0053] One embodiment described herein is a pharmaceutical composition formulated as an oral capsule. In one aspect, the composition comprises up to about 0.25% w / w to about 15% w / w of bumetanide dibenzylamide and one or more solubilizing agents. In one aspect, the solubilizing agent is a cosolvent. In one aspect, the solubilizing agent is a surfactant. In one aspect, the solubilizing agent comprises a triglyceride. In one aspect, the triglyceride comprises a medium-chain triglyceride. In one aspect, the triglyceride comprises a long-chain triglyceride. In one aspect, the triglyceride comprises a mixture of medium-chain triglycerides and long-chain triglycerides. In one aspect, the triglyceride comprises a polyoxylglyceride. In one embodiment, the polyoxylglyceride is selected from the group consisting of lauroyl polyoxylglyceride, linoleoyl polyoxylglyceride, oleoyl polyoxylglyceride, stearoyl polyoxylglyceride, caprylocaproyl polyoxylglyceride, and any combination thereof. In one embodiment, the triglyceride comprises a nonionic surfactant, a solubilizer, and an emulsifier. In one embodiment, the long-chain triglyceride is selected from the group consisting of polyoxyl 35 castor oil (Kolliphor EL), glyceryl monolinoleate (Maisine CC), and any combination thereof. In one aspect, the medium chain triglyceride is selected from the group consisting of caprylocaproyl polyoxylglyceride (Labrasol ALF), phosphatidylcholine (Phosal 75SA), caprylic / capric triglyceride (Captex 300), lauroylperoxyl-32 glyceride (Gelucire 44 / 14), sorbitan ester (Span 80), and any combination thereof.

[0054] In one aspect, a pharmaceutical composition is described herein comprising from about 0.1% w / w to about 99.75% w / w of one or more solubilizing agents. In one aspect, the solubilizing agent comprises soybean oil. In one aspect, the solubilizing agent is in the oil phase. In one aspect, the solubilizing agent is selected from the group consisting of peanut oil, soybean oil, castor oil, corn oil, safflower oil, olive oil, apricot kernel oil, sesame oil, cottonseed oil, sunflower seed oil, palm oil, rapeseed oil, Maisine 35-1, Maisine CC (glyceryl monolinoleate), and any combination thereof. In one aspect, the solubilizing agent comprises a cosolvent. In one aspect, the solubilizing agent is selected from the group consisting of propylene glycol, Capryol™ 90 (propylene glycol monocaprylate), Lauroglycol™ 90 (propylene glycol monolaurate), glycerin, polyethylene glycol, and any combination thereof. In one embodiment, the solubilizer comprises an antioxidant. In one embodiment, the solubilizer is selected from the group consisting of alpha tocopherol, ascorbyl palmitate, ascorbic acid, butylated hydroxyanisole, butylated hydroxyl toluene, and any combination thereof. In one embodiment, the solubilizer comprises an antimicrobial preservative, a solvent, and a water-soluble co-solvent. In one embodiment, the solubilizer comprises a solvent and a water-soluble co-solvent. In one embodiment, the solubilizer 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. In one embodiment, the solubilizer is water. In one embodiment, the solubilizer is any diluent.

[0055] In one aspect, described herein are pharmaceutical compositions comprising about 0.5% w / w to about 1.8% w / w of bumetanide dibenzylamide. In one aspect, described herein are pharmaceutical compositions 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 bumetanide dibenzylamide, about 10% w / w to about 45% w / w polyoxyl 35 castor oil (Kolliphor EL), about 15% w / w to about 65% w / w glyceryl monolinoleate (Maisine CC), about 15% w / w to about 65% w / w soybean oil, about 0% w / w to about 15% w / w ethanol, and about 0% w / w to about 0.13% w / w 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.

[0056] One embodiment described herein is a pharmaceutical composition formulated as a nasal solution. In one aspect, the composition comprises bumetanide dibenzylamide in a solvent system. In one aspect, the composition comprises about 3 ml of solvent and about 28 mg to about 32 mg of bumetanide dibenzylamide. In one aspect, the solubilizing agent comprises a triglyceride. In one aspect, the triglyceride comprises a medium-chain triglyceride. In one aspect, the triglyceride comprises a long-chain triglyceride. In one aspect, the triglyceride comprises a mixture of medium-chain triglycerides and long-chain triglycerides. In one aspect, the triglyceride comprises a polyoxylglyceride. In one aspect, the polyoxylglyceride is selected from the group consisting of lauroyl polyoxylglyceride, linoleoyl polyoxylglyceride, oleoyl polyoxylglyceride, stearoyl polyoxylglyceride, caprylocaproyl polyoxylglyceride, and any combination thereof.

[0057] In one embodiment, the triglyceride comprises a nonionic surfactant, a solubilizer, and an emulsifier. In one embodiment, the solubilizer comprises caprylocaproyl polyoxylglyceride (Labrasol ALF). In one embodiment, the solvent system comprises one or more solubilizers. In one embodiment, the solubilizer comprises caprylocaproyl polyoxylglyceride (Labrasol ALF) and water. In one embodiment, the solubilizer comprises caprylocaproyl polyoxylglyceride (Labrasol ALF), propylene glycol, and water. In one embodiment, the solubilizer comprises caprylocaproyl polyoxylglyceride (Labrasol ALF), propylene glycol, PEG-400, and water. In one embodiment, the solubilizer comprises caprylocaproyl polyoxylglyceride (Labrasol ALF), propylene glycol, PEG-400, Vitamin E TPGS, and water. In one embodiment, the solubilizer comprises caprylocaproyl polyoxylglyceride (Labrasol ALF), propylene glycol, PEG-400, vitamin E TPGS, ethanol, and water. In one embodiment, the solubilizer comprises about 50 g of caprylocaproyl polyoxylglyceride (Labrasol ALF) per 50 g of solvent. In one embodiment, the solubilizer comprises about 25 g of caprylocaproyl polyoxylglyceride (Labrasol ALF) and about 25 g of water per 50 g of solvent. In one embodiment, the solubilizer comprises about 6 g of propylene glycol, about 40 g of PEG-400, and about 4 g of water per 50 g of solvent. In one embodiment, the solubilizer comprises about 5g of caprylocaproyl polyoxylglyceride (Labrasol ALF), about 6g of propylene glycol, about 35g of PEG-400, and about 4g of water per 50g of solvent. In one embodiment, the solubilizer comprises about 10g of propylene glycol, about 35g of PEG-400, about 0.5g of vitamin E TPGS, and about 4.5g of water per 50g of solvent. In one embodiment, the solubilizer comprises about 5g of caprylocaproyl polyoxylglyceride (Labrasol ALF), about 10g of propylene glycol, about 28.5g of PEG-400, about 0.5g of vitamin E TPGS, about 1g of ethanol, and about 4.5g of water per 50g of solubilizer.In one aspect, the solubilizing agent comprises glycofurol. In one aspect, the solubilizing agent comprises a penetrating agent and a solvent. In one aspect, the solubilizing agent comprises ethyl oleate. In one aspect, the solubilizing agent comprises an oily vehicle, a solvent, and a solvent.

[0058] In one embodiment, the pharmaceutical composition comprises about 3% w / v bumetanide dibenzylamide, about 11% w / v caprylocaproyl polyoxylglyceride (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 embodiment, 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 polyoxylglyceride (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 embodiment, the pharmaceutical composition comprises about 2.73% w / w bumetanide dibenzylamide, about 8% w / w caprylocaproyl polyoxylglyceride (Labrasol ALF), about 15% w / w propylene glycol, about 69.27% ​​w / w PEG-400, and about 5% w / w water. In one embodiment, the pharmaceutical composition comprises about 2.73% w / w bumetanide dibenzylamide, about 16% w / w caprylocaproyl polyoxylglyceride (Labrasol ALF), about 20% w / w propylene glycol, about 54.27% w / w PEG-400, and about 7% w / w water. In one embodiment, the pharmaceutical composition comprises about 2.73% w / w bumetanide dibenzylamide, about 5% w / w caprylocaproyl polyoxylglyceride (Labrasol ALF), about 4% w / w propylene glycol, about 78.27% w / w PEG-400, and about 10% w / w water.

[0059] 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 a different target dose of bumetanide dibenzylamide. In one aspect, if the drug substance shows some instability in water, the paste is determined to be a 100% non-aqueous formulation. In one embodiment, the rectal gel is formulated to contain about 0.6% w / w bumetanide dibenzylamide, about 10% w / w caprylocaproyl polyoxylglyceride (Labrasol ALF), about 9.15% w / w propylene glycol, about 53.84% w / w polyethylene glycol 600, about 3.6% w / w polyvinylpyrrolidone (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 embodiment, the rectal paste is formulated to contain about 0.6% w / w bumetanide dibenzylamide, about 10% w / w caprylocaproyl polyoxylglyceride (Labrasol ALF), about 9.15% w / w propylene glycol, about 66.75% w / w polyethylene glycol 600, about 3.6% w / w polyvinylpyrrolidone (K30), about 2.4% w / w poloxamer 407 (P407), and about 7.5% w / w polyethylene glycol 3350.

[0060] One embodiment described herein is a pharmaceutical composition formulated as a sublingual tablet. In one aspect, the formulated composition targets approximately 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, a sublingual tablet is formulated to contain about 15% w bumetanide dibenzylamide, about 20% w Ceolus KG (microcrystalline cellulose), about 51% w Mannogem EZ (spray-dried mannitol), about 7% w Polyplasdone XL (superdisintegrant), about 3% w Poloxamer 407 (wetting agent), about 1.5% w citric acid monohydrate, about 1% w Cabosil M5P (fumed silica), and about 1.5% w magnesium stearate. In one embodiment, a sublingual tablet is formulated to contain bumetanide dibenzylamide, one or more water-dispersible surfactants, one or more wetting agents, and one or more superdisintegrants. In one embodiment, the sublingual tablet is formulated to contain about 7.4% w bumetanide dibenzylamide, about 9.9% w lauroyl puroxyl-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 aluminometasilicate), about 0.5% w / w poloxamer 407 (a wetting agent), about 0.7% w citric acid monohydrate, about 2% w Cabosil M5P (fumed silica), about 54.3% w Polyplasdone XL (a superdisintegrant), and about 0.5% w magnesium stearate.

[0061] Common methods of treatment / uses / compounds for use One embodiment described herein, a preferred therapeutic agent and method of the present disclosure, is for use in treating other indications, such as seizures (e.g., partial seizure onset), epilepsy, and / or neuropathic pain, by modulating or disrupting synchronization of neuronal population activity in areas of enhanced synchronization by reducing NKCC cotransporter activity without a diuretic effect. One embodiment described herein is a preferred therapeutic agent and method of the present disclosure for treating seizures that cannot be suppressed by existing drug therapies, such as uncontrollable seizures, intractable seizures, refractory seizures, drug-resistant seizures, or medically resistant seizures. One embodiment described herein are preferred therapeutic agents and methods of the present disclosure for treating epilepsy syndromes such as Angelman syndrome, benign rolandic epilepsy, CDKL5 disorders, childhood absence epilepsy, Dravet syndrome, GLUT1 deficiency syndrome, hypothalamic hamartoma, infantile spasms (also known as West syndrome), Lennox-Gastaut, PCDH19, progressive myoclonic epilepsy, Rasmussen encephalitis, ring 20 syndrome, or reflex epilepsy.

[0062] In one embodiment described herein, preferred therapeutic agents and methods of the present disclosure are for use in treating epilepsy and / or neurological syndromes that are specific to children, including, but not limited to, Dravet syndrome, infantile spasms, Landau-Kleffner syndrome, Lennox-Gastaut syndrome, Rasmussen syndrome, benign rolandic epilepsy, benign occipital epilepsy, childhood absence epilepsy, juvenile myoclonus, Rett syndrome, Angelman syndrome, tuberous sclerosis, and / or Sturge-Weber syndrome. In one embodiment described herein, preferred therapeutic agents and methods of the present disclosure are for use in treating epilepsy and / or neurological syndromes that may be observed in adults or children.

[0063] Described herein are preferred therapeutic agents and methods of the present disclosure for treating one or more of the indications listed in the table below: [Table 2-1] [Table 2-2]

[0064] One embodiment described herein is a preferred therapeutic agent and method of the present disclosure for treating co-morbidities of epilepsy or seizures, such as depression, anxiety disorders, attention deficit hyperactivity disorder (ADHD), schizophreniform interictal psychosis, autism, and psychiatric disorders such as 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.

[0065] 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 agent and method of the present disclosure may be used to treat migraine with or without aura in adults. In another aspect, the preferred therapeutic agent and method of the present disclosure may be used for acute treatment of migraine with aura, acute treatment of migraine without aura, or chronic treatment for the prevention of migraine without aura.

[0066] One embodiment described herein is a preferred therapeutic agent and method of the present disclosure for treating mild, moderate, or severe anxiety. In one aspect, the preferred therapeutic agent and method of the present disclosure may be used for the acute and maintenance treatment of major depressive disorder (MDD) in adults and adolescents aged 12-17 years, or the acute treatment of generalized anxiety disorder (GAD) in adults. In one aspect, the preferred therapeutic agent and method of the present disclosure may be used for the acute and maintenance treatment of obsessive-compulsive disorder (OCD), the acute and maintenance treatment of bulimia nervosa, or the acute treatment of panic disorder (PD) with or without agoraphobia. In another aspect, the preferred therapeutic agent and method of the present disclosure may be used to treat acute depressive episodes associated with bipolar I disorder or to treat treatment-resistant depression.

[0067] In one embodiment described herein, preferred therapeutic agents and methods of the present disclosure 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). In one aspect, the obsessions or compulsions may cause significant distress, be time-consuming, or significantly interfere with social or occupational functioning to meet a DSM-III-R (circa 1989) diagnosis of OCD. Obsessions may be recurrent and persistent ideas, thoughts, images, or urges that are ego-dystopic. Compulsions may be repetitive, deliberate, and / or intentional behaviors performed in response to obsessions or performed in a stereotyped manner. Compulsions may be perceived by the individual as excessive or irrational.

[0068] One embodiment described herein is a preferred therapeutic agent and method of the present disclosure for use in treating patients with mild, moderate, or severe depression, anxiety associated with depression, anxiety associated with alcoholism, depression and / or anxiety associated with organic disease, psychotic depressive disorder with associated anxiety, including regressive depression and manic-depressive disorder. In one aspect, the preferred therapeutic agent and method of the present disclosure can be used to target neurotic symptoms such as anxiety, tension, depression, somatic symptoms and concerns, sleep disturbances, guilt, lack of energy, fear, worry, and distress.

[0069] One embodiment described herein is a preferred therapeutic agent and method of the disclosure for use in treating depression in patients with depressive neurosis (dysthymic disorder), manic depression, or major depressive disorder. In one aspect, the preferred therapeutic agent and method of the disclosure may be used for the short-term, long-term, and maintenance treatment of major depressive disorder (MDD), generalized anxiety disorder, diabetic peripheral neuropathic pain (DPNP), fibromyalgia (FM), or chronic musculoskeletal pain.

[0070] One embodiment described herein is a therapeutic agent and method of the present disclosure for treating progressive neurodegenerative disorders, including, for example, Alzheimer's disease. In one aspect, preferred therapeutic agents and methods of the present disclosure may be used to treat or halt the progression of one or more of Alzheimer's disease, amyotrophic lateral sclerosis, Friedreich's ataxia, Huntington's disease, Lewy body disease, Parkinson's disease, or spinal muscular atrophy.

[0071] One embodiment described herein is a preferred therapeutic agent and method of the present disclosure, including use as monotherapy or adjunctive therapy. In one aspect, the preferred therapeutic agent and method of the present disclosure may be used as monotherapy for adults. In another aspect, the preferred therapeutic agent and method of the present disclosure may be used as adjunctive therapy with an additional therapeutic agent for adults. In another aspect, the preferred therapeutic agent and method of the present disclosure may be used as monotherapy for pediatric patients 2 years of age or older. In another aspect, the preferred therapeutic agent and method of the present disclosure may be used as adjunctive therapy with an additional therapeutic agent for pediatric patients 2 years of age or older. In another aspect, the preferred therapeutic agent and method of the present disclosure may be used as monotherapy for pediatric patients under 2 years of age. In another aspect, the preferred therapeutic agent and method of the present disclosure may be used as adjunctive therapy with an additional therapeutic agent for pediatric patients under 2 years of age.

[0072] The effective amount of the active pharmaceutical ingredient to be administered therapeutically will depend, for example, on the therapeutic situation and purpose. Those skilled in the art will understand that the appropriate dosage level for treatment will vary depending, in part, on the concentration of the bumetanide dibenzylamide composition, the dosing regimen in which the bumetanide dibenzylamide composition is being used, 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 can be titrated to obtain the optimal therapeutic effect.

[0073] As used herein, bumetanide dibenzylamide includes compositions and formulations containing bumetanide dibenzylamide, as the context requires.

[0074] The frequency of administration depends on the pharmacokinetic parameters of the therapeutic agent incorporated into the bumetanide dibenzylamide composition being used. The composition can be administered as a single dose, as two or more doses over an extended period of time (which may or may not contain the same amount of bumetanide dibenzylamide), or as a continuous infusion of an injectable formulation via an implanted device or catheter. Further refinement of the appropriate dosage is routinely performed by those skilled in the art and is within the scope of tasks routinely performed by them. Sublingual tablets can also be used for oral administration. The appropriate dosage can be ascertained through the use of appropriate dose-response data.

[0075] Opportunities for fine-tuning may include the use of sustained- or controlled-release oral capsules or tablets, or transdermal formulations. The intramuscular data presented below supports the development of a potential transdermal therapy. The intramuscular data demonstrate that bumetanide dibenzylamide is absorbed into the circulation via muscle microvasculature, thus avoiding first-pass metabolism. Therefore, bumetanide dibenzylamide should also be absorbed by skin microvasculature, making it suitable for a transdermal formulation.

[0076] The bumetanide dibenzylamide composition may be administered, for example, once, twice, three times, four times, five times, six times, or even more times per day. One or more doses may be administered, for example, for 1, 2, 3, 4, 5, 6, 7 days, or longer. One or more doses may be administered, for example, for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months, 1 year, 2 years, 3 years, 4 years, 5 years, more than 5 years, 10 years, decades, or longer. One or more doses may be administered at regular intervals until the subject or subject in need of epilepsy treatment or prevention no longer requires it. In one embodiment, the dose may be administered orally. In one embodiment, the dose may be administered sublingually. In one embodiment, the dose may be administered intravenously. In one embodiment, the dose may be administered rectally. In one aspect, the dose may be administered intramuscularly, hi one aspect, the dose may be administered intranasally, hi one aspect, the dose may be administered subcutaneously.

[0077] 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 the same time. In another embodiment, two or more different doses are administered at the same time. Such dual or different simultaneous doses can be used to provide an effective amount of the pharmaceutical composition to a subject in need thereof.

[0078] In one embodiment, the pharmaceutical compositions described herein may be used to treat, prevent, slow the progression of, delay the onset of, ameliorate, reduce the symptoms of, or prevent epilepsy.

[0079] 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 for 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, 1 month, 2 months, 3 months, 4 months, 6 months, 9 months, 1 year, 2 years, 3 years, 4 years, or even longer. In one aspect, some applications of the bumetanide dibenzylamide composition are administered more than once per day.

[0080] In another embodiment, the bumetanide dibenzylamide compositions described herein are provided as single-dose compositions, meaning that the container in which they are provided contains one pharmaceutical dose. In another embodiment, the compositions are provided as multi-dose compositions, meaning that they contain two or more therapeutic doses. Preferably, multi-dose compositions contain at least two doses. Such multi-dose bumetanide dibenzylamide compositions can be used for different subjects in need thereof, or are intended for use in one subject, with the remaining doses being stored after application of the first dose until needed. In another embodiment, the bumetanide dibenzylamide compositions are contained in one or more containers.

[0081] Critical to the efficacy of any pharmaceutical treatment is the overall systemic bioavailability of the pharmaceutical composition used in that treatment. Surprisingly, lipid formulations can be used to enhance the bioavailability and / or lymphatic absorption of pharmaceutical compositions. Log P is a measure of lipophilicity and is the octanol:water partition coefficient, expressed as the logarithmic ratio of molecules in octanol to water after mixing. The log P of bumetanide is approximately 2.61, the log P of bumetanide diethylamide is approximately 3.11, and the log P of bumetanide dibenzylamide is approximately 5.9. A log P of 3 indicates a 1000-fold higher concentration in octanol than in water, making bumetanide dibenzylamide approximately 1000 to 10,000 times more lipophilic than bumetanide.

[0082] Detailed method of treatment / use / compound for use As described herein, bumetanide dibenzylamide has been demonstrated to disrupt the synchronization of neuronal population activity in areas of enhanced synchrony. To avoid first-pass metabolism and increase overall systemic bioavailability, a bumetanide dibenzylamide administration composition was developed targeting four administration routes. The administration routes were selected based on the potential to maximize the bioavailability of bumetanide dibenzylamide and generate measurable systemic concentrations of bumetanide dibenzylamide. Because bumetanide dibenzylamide has been shown to be susceptible to high first-pass metabolism by the liver, the administration routes were selected to avoid hepatic metabolism.

[0083] Medically intractable epilepsy Seizures that cannot be controlled by existing drug therapies are referred to in several different ways, including "uncontrollable," "intractable," "refractory," "drug-resistant," or "medically resistant." It is estimated that 20% to 40% of patients with epilepsy (approximately 400,000 Americans) have intractable epilepsy. The combined indirect and direct costs of epilepsy in the United States are estimated to be $15.5 billion annually, with drug-resistant patients accounting for the majority of this cost. Despite the availability of numerous new drugs to treat epilepsy over the past decade, the effectiveness of these new drugs has not proven significantly superior to older drugs.

[0084] Most Common AEDs (and Their Use to Treat Other Neurological and Psychiatric Disorders in Addition to Epilepsy). The most commonly prescribed AEDs include valproate (VPA—the most commonly prescribed of all AEDs worldwide) and its derivatives divalproex sodium, carbamazepine (Tegretol), phenytoin (Dilantin), barbiturates (phenobarbitol and primidone), ethosuximide (Zarontin), clonazepam (Klonopin), lamotrigine (Lamictal), gabapentin (Neurontin), topiramate (Topamax), oxcarbazeipine (Trileptal), and zonisamide (Zonegran). In addition to their use to treat epilepsy, AEDs are also prescribed to treat many other neurological and psychiatric disorders.

[0085] Side Effects: All currently prescribed antiepileptic drugs (AEDs) are believed to mediate their antiepileptic effects by reducing neuronal or synaptic excitability. Because AEDs indiscriminately affect all neuronal or synaptic targets in the brain, regardless of whether they contribute to seizure activity, all AEDs also mediate a spectrum of cognitive, neurological, and psychiatric side effects. Approximately 25% of patients discontinue treatment due to intolerable side effects. Treatment failure and poor adherence are very common among patients experiencing AED-related side effects. The adverse effects of side effects can significantly impact the lives of patients' relatives and friends. Commonly occurring side effects of AEDs include memory impairment, fatigue, tremor, gastrointestinal symptoms, osteoporosis, depression, drowsiness, weight gain, and nausea. One study in the Netherlands estimated that the economic cost of epilepsy side effects (in addition to the direct and indirect costs of epilepsy itself) to patients in that country was US$26,675 per patient per year.

[0086] All commonly used AEDs have some effect on cognition, and these effects can have considerable implications for patients with epilepsy when important functions such as learning in children are involved. The most common adverse CNS effects of CNS drugs on cognition are sedation, somnolence, distractibility, insomnia, and dizziness.

[0087] Fatigue is a common side effect of most antiepileptic drugs. AED-induced fatigue is a chronic condition that can adversely affect patients' work, social interactions, and family. Stimulants such as amphetamine, dextroamphetamine, and methylphenidate are sometimes used to treat fatigue and daytime somnolence. However, these drugs can increase seizure intensity and lower the seizure threshold, so their use to treat fatigue in epilepsy patients is undesirable.

[0088] All antiepileptic drugs are believed to increase the risk of suicidal thoughts or behavior. This risk is of sufficient concern that the FDA issued safety warnings on December 15, 2008, and January 31, 2008, requiring all AED labels to include a warning about the increased risk of suicidal thoughts or behavior. This is particularly problematic because epilepsy and other medical and psychiatric conditions for which AEDs are prescribed (chronic pain, depression, bipolar disorder, and anxiety) all inherently pose a higher risk of suicidal behavior. For example, death by suicide is more common in people with epilepsy than in the general population (5% vs. 1.4%). Therefore, using AEDs to treat disorders already associated with a risk of suicidal behavior is expected to further increase that risk.

[0089] All antiepileptic drugs studied to date have been shown to have endocrine side effects in both men and women. These can adversely affect fertility, libido, thyroid function, and bone health. AEDs can alter sex hormone levels, potentially causing menstrual disorders, sexual problems, and decreased fertility. Other side effects that affect appearance include weight gain, alopecia (hair loss), acne, and masculine hair distribution in women.

[0090] Epilepsy treatment is affected by poor patient adherence to existing antiepileptic medications. As noted above, approximately 25% of patients discontinue treatment due to intolerable side effects. Up to 50% of all epilepsy patients experience adverse reactions to AEDs, negatively impacting tolerability and adherence. Even if side effects are not intolerable, unpleasant side effects may decrease patient adherence to taking AEDs as prescribed. Nonadherence in epilepsy is estimated to be in the 30% to 50% range [24,25]. Decreased AED adherence has been associated with a more than threefold increase in mortality

[26] . Periods of nonadherence in epilepsy patients have also been associated with significantly more emergency department visits, hospitalizations, injuries, and fractures.

[0091] Treatment of epilepsy is affected by comorbidities.

[0092] General: A recent study determined the prevalence of the most common comorbidities in men and women with epilepsy based on data from commercial health insurance plans. The top 10 comorbidities 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%). The top 10 comorbidities and their relative prevalence for men 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 among the top five comorbidities across all age groups. The presence of one comorbidity nearly tripled a member's health care costs compared with those of a member without a comorbidity.

[0093] Psychiatric Disorders - General: Epilepsy increases the likelihood of depression, anxiety disorders, attention deficit hyperactivity disorder (ADHD), schizophreniform interictal psychosis, autism, and suicidal behavior. Similarly, individuals with these psychiatric diagnoses and suicidal behavior are more likely to have epilepsy.

[0094] Sleep Disorders: Sleep deprivation is known to lower the seizure threshold in people with epilepsy. Sleep is vulnerable to its own set of disorders that can disrupt it. One example is obstructive sleep apnea (OSA). Both adults and children with intractable epilepsy are at much higher risk of developing OSA than the normal population.

[0095] Autism spectrum disorder (ASD): Epilepsy occurs much more frequently in individuals with autism. Between 11% and 39% of individuals with autism develop epilepsy.

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

[0096] Sleep disorders are common in children with autism, estimated to affect 40% to 80% of children.

[0097] The atypical antipsychotics risperidone and aripiprazole are approved by the Food and Drug Administration for the treatment of irritability and agitation in ASD. Both are associated with serious adverse events, including a lowered seizure threshold.

[0098] Migraine: The incidence of migraine is approximately 1% per year, with a 1-year prevalence of 11.7-13.2%. Patients with epilepsy have approximately a two-fold increased risk of developing migraine. Conversely, children with migraine have a three- to four-fold increased risk of developing epilepsy.

[0099] Comorbidities can worsen outcomes: patients with epilepsy who suffer from migraine are less likely to achieve epilepsy remission than patients with epilepsy alone. This is also evidence of the complex comorbidity of epilepsy, migraine, depression, and suicide.

[0100] Postictal Headache - 45% of people with epilepsy have headaches that follow a seizure, called postictal headache. These headaches last 6 to 24 hours or more and can be very disabling. Some medications used to treat these headaches may lower the seizure threshold, increasing the risk of further seizures.

[0101] Depression: The prevalence of depressive disorders in patients with epilepsy has been shown to range from 9% to 55%, depending on the sample population and assessment method. This contrasts with the prevalence in the general population, which is estimated to be 1% to 3% in men and 2% to 9% in women.

[0102] Medications commonly used to treat depression may lower the seizure threshold or increase the severity of seizures. Bupropion and tricyclic antidepressants decrease the seizure threshold. Selective serotonin reuptake inhibitors (SSRIs) may significantly prolong seizures.

[0103] Anxiety: The lifetime prevalence of anxiety is estimated to be 2.4 times higher in people with epilepsy than in people without epilepsy.

[0104] Psychosis: The risk of psychosis in patients with epilepsy may be 6-12 times higher than in the general population, with a prevalence of approximately 7-8%. All antipsychotics can lower the seizure threshold.

[0105] Attention Deficit Disorder (ADD) and Attention Deficit / Hyperactivity Disorder (ADHD): Approximately 20% of adults diagnosed with epilepsy also exhibit ADHD symptoms. Studies of pediatric epilepsy have found a 2.5- to 5.5-fold increased risk of ADHD compared with healthy controls. It is estimated that 2% to 7% of children with ADHD also have epilepsy.

[0106] Stimulants such as amphetamine, dextroamphetamine, and methylphenidate are commonly prescribed to treat attention deficit disorder (ADD) and attention deficit / hyperactivity disorder (ADHD) in children and adults. These medications may decrease the seizure threshold and increase the severity of seizures.

[0107] Mental Retardation: Epilepsy is one of the most common secondary disabilities among individuals with mental retardation, with prevalence increasing with the severity of intellectual disability. Approximately 50% of individuals with severe learning disabilities develop epilepsy. The lifetime prevalence of epilepsy in individuals with mental retardation (IQ<70) is 13%-24%. Down syndrome is the most common genetic cause of mental retardation, and the number of individuals with Down syndrome who have seizures is estimated to be 5%-10%. Currently available AEDs adversely affect the behavior of individuals with mental retardation.

[0108] Other: Hyperlipidemia - The incidence in epilepsy patients is 1.3 times higher than in controls. Population-based studies have reported higher rates of hypertension, ischemic heart disease, and diabetes in people with epilepsy.

[0109] Drug interactions can cause problems in treating seizure disorders.

[0110] The following medications may lower the seizure threshold and therefore increase the risk of seizures in people with epilepsy: acetylcholinesterase inhibitors - used to treat myasthenia gravis, glaucoma, postural orthostatic tachycardia syndrome, neuropsychiatric symptoms of Alzheimer's disease, dementia with Lewy bodies, Parkinson's disease, cognitive impairment in schizophrenia, and 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 - skeletal muscle relaxant for convulsions;

number

[0111] Rare epilepsy syndromes, including Angelman syndrome (occurring in 1 in 15,000 births), may be treated with the compounds and compositions of the present disclosure. Epilepsy is present in over 80% of affected individuals. Benign rolandic epilepsy accounts for approximately 15% of all childhood epilepsy. Seizures cease by age 15; CDKL5 disorders are so rare that they may not receive attention. There are 600 cases worldwide; childhood absence epilepsy accounts for 2-8% of people with epilepsy, usually resolving by adulthood; Dravet syndrome affects 1 in 30,000 people. Myoclonic seizures appear in 85% of children between the ages of 1 and 5 years. GLUT1 deficiency syndrome, likely occurring in 1 in 90,000 people, is thought to be underdiagnosed because many neurological disorders cause similar symptoms. Nearly all individuals experience frequent seizures during the first year of life. Hypothalamic hamartomas - 1 in 200,000 cases; infantile spasms (also known as West syndrome) - 2.5 to 6 in 10,000 births. They account for 30% of all epilepsy cases affecting infants. While they usually cease by age 4, most children are left with developmental disabilities, and one-fifth develop Lennox-Gastaut syndrome. Many clinicians believe the earlier seizure control, the better the outcome. Lennox-Gastaut syndrome accounts for 2 to 5% of childhood epilepsy cases. They usually persist through childhood and adolescence into adulthood. Seizures are very difficult to control with current treatments. PCDH19 - One in 10 girls who begin having seizures before age 5 may have PCDH19 epilepsy. It may be similar or mimic Dravet syndrome. In the United States, 15,000–30,000 people have PCEH19 epilepsy; progressive myoclonic epilepsy—not a single disorder but a group of syndromes with various names, including “severe myoclonic epilepsy of infancy (Dravet syndrome),” Unverricht-Lundborg disease (also called Baltic myoclonus), Lafora disease, and mitochondrial encephalopathy. It is difficult to control any of these patients with existing treatments; Rasmussen encephalitis—nothing is known about its incidence in different populations. Thought to be rare, clinicians worldwide are reporting patients with this syndrome. The outlook with current treatments is grim; the seizures are relentless.Ring 20 syndrome - rare; reflex epilepsy - a group of epilepsy syndromes in which certain stimuli (such as flickering lights) trigger seizures. 4-7% of people with epilepsy.

[0112] Side effects of monotherapy vs. polytherapy (combination therapy) In many cases, a single AED may provide partial but insufficient seizure control. For patients who are refractory to any single AED (monotherapy), better seizure control may be achieved by combining several different AEDs (polytherapy or combination therapy). Sometimes, up to four AEDs are administered to a single patient to attempt seizure control. However, administering multiple AEDs to a patient significantly increases the intensity and number of side effects. Some studies have shown that polytherapy has more severe and more cognitive side effects. The intensity and number of side effects increase with the number of AEDs a patient is taking.

[0113] The FDA label for bumetanide includes a statement that serum potassium should be monitored periodically and that potassium supplements or potassium-sparing diuretics should be added as needed. Periodic monitoring of other electrolytes is recommended in patients treated with high doses or for long periods, particularly those on a low-salt diet. Hyperuricemia may occur. Reported cases have been asymptomatic. Reversible elevations of BUN and creatinine may also occur, particularly in patients with renal failure, particularly in association with dehydration. Bumetanide may increase urinary calcium excretion with consequent hypocalcemia. Diuretics have been shown to increase urinary excretion of magnesium, which may lead to hypomagnesemia.

[0114] Ideally, it would be desirable to enhance the ability of loop diuretics to cross the blood-brain barrier. This would have the effect of achieving greater therapeutic efficacy in the brain and reducing diuretic effects. The present inventors have found that certain amide analogs of bumetanide have significant antiepileptic effects while dramatically reducing the diuretic effect compared to bumetanide. This is an unexpected finding. For example, Tollner et al. tested a bumetanide amide derivative (N,N-dimethylaminoethylamide) in a rat study and found that it did not result in elevated bumetanide levels. The authors of this study then chose to discontinue further testing of the amide derivative of bumetanide.

[0115] The method provides particular utility for patients with epilepsy that is not adequately controlled by conventional therapies such as phenytoin, carbamazepine, valproate, lamotrigine, levetiracetam, ethosuximide, phenobarbital, and topiramate, or whose epilepsy is otherwise refractory to conventional therapies.

[0116] Primate Model Here, we used a primate neocortical seizure model 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 to study intrinsic optical signals in the human and primate brains. These signals can 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 is generated either by electrically stimulating a small, localized cortical area with bipolar microelectrodes to generate afterdischarge activity (similar to what is done for intraoperative mapping of seizure foci in human patients; see below) or by applying various epileptiform agents (e.g., bicuculline, 4-AP) to a focal region over the hand motor cortex to generate an acute focus (similar to recordings from interictal foci in human patients; see below). We found that the data obtained from the primate seizure study were qualitatively similar to those found when conducting similar optical imaging studies in human patients (Haglund and Hochman, 2005), in that the morphology of electrophysiological activity recorded in EEG traces was similar between monkeys and humans. Notably, the spontaneous interictal spikes observed in humans, as shown below, resemble the neocortical spikes generated by bicuculline foci on the neocortex in the primate model. As shown in the second figure below, the afterdischarge protocol developed for the human intraoperative study was also used in the primate study. Similarly, the physical characteristics of optical signals from the brain generated by epileptiform activity (not shown here) (Haglund and Hochman, 2005, Haglund and Hochman, 2007) were similar.

[0117] Comparative Example-1: Furosemide (NKCC antagonist) blockade of spontaneous interictal spikes in human patients with medically refractory epilepsy (from Haglund and Hochman, 2005). Referring to Figure 1, the data in this figure were analyzed using the methods described herein for primate studies. The top trace shows data from an individual patient and indicates the change in spontaneous interictal spikes after furosemide administration. As performed in primates, electrophysiological activity was recorded from EEG electrodes placed on the cortical surface. The top two traces compare activity before and after IV administration of 20 mg of furosemide. The dark blue trace was recorded from the electrode at the interictal focus, and the overlaid light blue trace shows background activity from an electrode 1 cm away. Changes in spike frequency over time were determined using the same algorithm applied to the primate data and averaged across five patients and are shown in the bottom plot.

[0118] Comparative Example-2: Effect of furosemide (NKCC antagonist) on afterdischarge threshold in the cortex of human subjects (Haglund and Hochman, 2005). Referring to Figure 2A and B, a bipolar stimulating electrode (similar to those used in primate studies) was placed on the cortical surface as shown in the bottom center of the grayscale image. A recording electrode was placed within 1 cm of the stimulating electrode. Four-second stimuli (60 Hz; biphasic; 1 ms / phase) were delivered at various currents (similar protocol used in primate studies). Stimulation duration is represented by the blue box embedded at the beginning of the trace. Before furosemide treatment, the minimum current required to elicit after-discharge activity for at least 5 seconds over three consecutive trials was determined. This was defined as the "after-discharge threshold current" (A, top). Red horizontal bars on each trace indicate episodes of after-discharge activity. After furosemide administration, stimulation trials were performed every 2–5 minutes for the next 40 minutes. In this patient, after-discharge activity was suddenly blocked immediately after furosemide treatment (A, bottom trace). To determine whether the blockade of after-discharge activity was mediated by an increase in the after-discharge threshold, the stimulation current was incrementally increased (B). We found that afterdischarge episodes at least as long as those observed during the furosemide pre-test could be elicited by increasing the stimulation current, suggesting that furosemide increases the afterdischarge threshold.

[0119] Comparative Example-3: Quantification of the effect of NKCC2 antagonists on diuresis In rats, bumetanide is rapidly biotransformed to an inactive metabolite. Therefore, despite its potent diuretic properties in humans, bumetanide does not induce diuresis in rats (Schwartz, 1981. Metabolism of Bumetanide. J Clin Pharmacol. 12:555-563). Similarly, the diuretic effect of bumetanide in dogs is much lower than in humans due to the rapid renal excretion of bumetanide in dogs (Schwartz, 1981). Research in Hochman's laboratory at Duke has demonstrated that diuresis can be quantified using catheterized primates. This makes primates a more suitable species than other common laboratory species for comparing the diuretic effects of bumetanide analogs with bumetanide in a manner that is transferable to humans.

[0120] Comparative Example-4: Primate models are sensitive to the antiepileptic effects of drugs that have no antiepileptic effect in rats: Keppra (levetiracetam) is an excellent antiepileptic drug, but its development faced an uphill battle due to its well-known lack of antiepileptic efficacy in standard rat models of seizures. From the pharmacology overview provided to the FDA: "Two screening tests for antiepileptic drugs (AEDs), the maximal electroshock (MES) test and the maximal pentylenetetrazole (PTZ) test, did not show anticonvulsant activity. Levetiracetam lacked anticonvulsant activity against seizures induced by maximal stimulation with different chemical convulsants and showed only modest anticonvulsant activity in submaximal stimulation and threshold tests. One exception was the observed protection against seizures induced by pilocarpine and kainic acid."

[0121] As shown in Figure 3, the clear anticonvulsant effect of Keppra can be measured in our primate seizure model.

[0122] As Keppra did then, we believe that bumetanide mediates its antiepileptic effects through a mechanism unique to all currently approved AEDs. Primate models appear sufficiently sensitive to the anticonvulsant effects of bumenetanide and its analogs that they can be relied upon to study these classes of molecules in a manner that may be translatable to humans.

[0123] Furosemide and bumetanide block seizures while maintaining their rank in antagonizing NKCC. Furosemide at 10 mg / kg IV and bumetanide at 2 mg / kg IV. Furosemide's diuretic effect is approximately 10 times weaker than that of bumetanide. Bumetanide, fosphenatoin (Cerebyx), and pentothal (thiopental sodium) block the 4-AP burst. Keppra 2 x 40 mg / kg (levetiracetam) does not block the 4-AP burst. Thus, while primate models are sensitive to Keppra, it is well known that Keppra was ineffective in the standard AED rat model. Bumetanide and Keppra block bicuculline spikes, but fosphenatoin does not. Bumetanide and fosphenatoin block AD and hand tremor, but Keppra does not. [Example]

[0124] Example 1: Synthesis of bumetanide dibenzylamide (NPT2042) [ka]

[0125] Bumetanide dibenzamide can be made according to the procedures described in US Pat. No. 8,008,283, which is incorporated herein by reference in its entirety.

[0126] 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-dimethylaminopropyl)carbodiimide (EDC, 560 mg, 3.6 mmol) was added. After approximately 10 minutes, 1-hydroxybenzotriazole (HOBt, 392 mg, 2.9 mmol) was added and the solution was stirred for an additional 10 minutes. Dibenzylamine (1 mL, 5.2 mmol) was added and the reaction was stirred for 2 hours, at which point 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.

[0127] An initial set of amide analogs of bumetanide was initially screened by measuring their NKCC-mediated anxiolytic effects in a whole-animal rat bioassay and comparing their effects with bumetanide using the same method described by Krystal (2012). The rat anxiety model studies were conducted by NeuroInvestigations Inc. at the University of Lethbridge (Lethbridge, Canada). Candidates that induced anxiolytic effects were evaluated for diuretic and antiseizure effects in monkeys (cynomolgus monkeys, M. nemistrina) by Drs. Hochman and Haglund at Duke University Medical Center (Durham, NC). During these experiments, the monkeys were anesthetized and catheterized, and their urine was collected and measured in volumetric flasks, allowing quantification of the effects of various treatments on diuresis. Cynomolgus monkeys (M. nemistrina) were chosen as a relevant species to study the diuretic effects because they are thought to metabolize bumetanide (and therefore presumably its analogues) similarly to humans (Doyle 1982, Walmsley 1985).

[0128] Example 2: Experimental Setup and Test Method Bumetanide derivatives can be synthesized according to the methods described in U.S. Patent No. 8,008,283 (previously incorporated by reference). All tested derivatives were dissolved in PEG200 for IV formulation. For oral administration, they were administered as a CMC suspension with water, delivered by oral gavage at an equimolar dose equivalent to 10 mg / kg BUM.

[0129] All data presented herein were obtained using macaque monkeys (Macaca nemestrina, weighing 2-3 kg), whose care and treatment complied with protocols approved by the Duke Institutional Animal Care & Use Committee. Details regarding the treatment and surgical preparation of macaques for cortical AD stimulation and EEG recording have been described (Haglund et al., 1993; Haglund and Hochman, 2007; Tolner, E.A. et al., 2011).

[0130] Animals were intubated (Matrix VBS anesthesia machine and Hallowell EMC model 2002 ventilator) and ventilated with 100% oxygen and maintained under pentobarbital anesthesia (1–2 mg / kg / h). Oxygen saturation was measured from the tongue using a Nellcor Pulse Oximeter and continuously monitored to maintain a constant saturation of 98–100% throughout the experiment.

[0131] Fluid balance was controlled by monitoring intravenous fluid intake every 15 minutes and fluid output (via a Foley catheter) every 30 minutes. 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 in conjunction with EEG.

[0132] After partial craniectomy (25 mm diameter), the dura was peeled off and surface EEG recordings were performed using custom-built strip electrodes (2.5 mm electrode diameter, 1 cm interelectrode distance) placed over the sensorimotor cortex: one electrode over the hand motor cortex, one electrode over the sensory cortex, and a reference electrode attached to the skull at the mastoid process or a third surface electrode between the motor and sensory electrodes.

[0133] 0.5 mm of gelfoam soaked in 100 μM bicuculline (Sigma-Aldrich, St. Louis, Missouri, USA) 2 Acute bicuculline foci were generated in the motor cortex using pledgets (Codman & Shurtleff, Randolph, MA, USA). Pledgets were placed on the cortex for 5 minutes and replaced every hour for 5 minutes. Bicuculline-induced epileptiform spike activity stabilized after approximately 1 hour, after which data acquisition began. EEG signals from motor and sensory electrodes were continuously recorded (1000 Hz sampling, unfiltered; Axon Instrument Digidata 1440A system).

[0134] Stimulus-evoked afterdischarges (ADs) were elicited by a 4-second train of 60 Hz biphasic pulses at 4–20 mA using a bipolar stimulating electrode (5 mm interelectrode distance) driven by a constant current source (Ojemann Cortical Stimulator, Integra Life Sciences Corporation, NJ, USA) placed over the sensory cortex. The AD threshold was determined by stimulating at the lowest level that reliably elicited AD activity three consecutive times, and the threshold remained constant throughout the recording. AD activity was reliably elicited in the sensory cortex during the same experiment in which a bicuculline focus was created in the motor cortex. Animals were subjected to several AD trials, spaced 20–30 min apart. Each AD trial consisted of three to eight AD stimuli (1 min apart) during control conditions, during the onset of maximal CNS effects from treatment with bumetanide or a bumetanide derivative, and during the recovery period.

[0135] See Figure 4 for the experimental setup: Bicuculline or 4-AP was used to create an acute focus near the hand motor cortex. Three surface electrodes were positioned such that one electrode was over the hand motor cortex and the other was over the hand sensory cortex. The central electrode was used as a reference for differential recording. The recording electrode can also be used to stimulate the cortex by passing a current between the pair of electrodes.

[0136] Data were analyzed offline using custom-designed software, as previously described (Haglund and Hochman, 2005), using the R programming language. Bicuculline-evoked spike activity was quantified from motor cortex recordings. The effect of treatment on bicuculline-evoked spike activity was analyzed by comparing the peak response after treatment administration with the average value during a 5-minute control period before treatment administration. In bicuculline experiments, recovery from pharmacological treatment was tracked for 20–35 minutes, following a return to pretreatment spike activity. For technical reasons, some recordings had to be terminated before the recovery period.

[0137] Example 3: After-Discharge (AD) Activity Referring to Figure 5, the image on the bottom left shows the position of the stimulation electrodes on the surface of the monkey cortex (in this example, Ojemann stimulator electrodes are used, rather than the surface electrodes shown in Figure 1). The brain is electrically stimulated for 4 seconds at various current magnitudes until the "afterdischarge threshold" (i.e., the stimulation current magnitude just sufficient to induce AD) is determined. Stimulation artifacts are shown in the upper left trace (denoted by an asterisk). AD is indicated by the orange bar. In this case, 7 mA was not sufficient to induce AD, while 8 mA reliably produced AD. Therefore, the AD threshold in this case was 8 mA. In this way, the effect of treatment on epileptiform activity induced with this type of electrical stimulation can be quantified by measuring the change in AD threshold. (From Haglund and Hochman, 2007).

[0138] Example 4: Quantification of after-discharge activity Referring to Figure 6, this figure shows three aspects of AD activity that, in addition to the AD thresholds described in Figure 2, can be quantified using custom software developed by D. Hochman: 1) the duration of AD activity, 2) the mean spike height of AD activity, and 3) the area within the envelope of AD activity.

[0139] Example 5: Bicucullin spike activity See Figure 7, which shows consecutive EEG traces recorded from surface electrodes closest to the epileptic focus in the motor cortex of the hand (top trace) and the closest sensory cortex of the hand (bottom trace). In addition to visual comparison (i.e., left trace before treatment and right trace after treatment), spike activity can be quantified using software developed by D. Hochman, which measures 1) spike height, 2) spike frequency, and 3) interspike interval. Note that electrical stimulation-induced AD can be simultaneously generated and recorded at a location distant from the bicuculline focus without interference.

[0140] Example 6: Analysis of NPT2024 (bumetanide morpholinoamide; n=4) Referring to Figures 8-11, the results of a comparative study between the bumetanide morpholinoamide derivative and the parent bumetanide compound show a dramatic increase in interspike interval (Figure 8), a dramatic decrease in spikes per minute (Figure 9), a dramatic decrease in mean spike height (Figure 10), and the comparative effects of NPT2024 and bumetanide on urine production over time (Figure 11).

[0141] Example 7: Antiseizure Efficacy of Oral and Intravenous Bumetanide Dibenzylamide (NPT2042) in Non-Human Primates The purpose of this study was to evaluate the antiseizure efficacy of bumetanide dibenzylamide (NPT2042) in nonhuman primates (NHPs) compared with the parent compound bumetanide. As previously described, acute seizure foci were created on the neocortical surface in these animals using the GABAa antagonist bicuculline, generating epileptiform "bicuculline spikes." After seizure foci were created in these NHPs, a single dose of either NPT2042 or the reference control bumetanide was administered orally (PO) and intravenously (IV). The bicuculline focus primate seizure model was used in this study because previously obtained data from primate seizure studies were qualitatively similar to those observed when similar optical imaging studies were performed in patients with intractable epilepsy (Haglund and Hochman 2005). Specifically, the morphology and electrophysiological activity recorded by electroencephalography (EEG) measurements were similar between NHPs and humans. Bicuculline blocks the inhibitory effects of GABA receptors, inducing convulsions, which are thought to mimic epileptic seizures. Therefore, bicuculline has been used in laboratories for decades to study and test the anticonvulsant effects of putative antiseizure treatments (Schwartzkroin and Prince 1980). During the same experiments, the diuretic effects of NPT2042 and bumetanide were studied.

[0142] Results from the experiments show that both NPT2042 and bumetanide induced a near-complete blockade of bicuculline spikes in the NHP seizure model after administration of an intravenous dose of 2 mg / kg. Both NPT2042 and bumetanide at an oral dose of 10 mg / kg reduced the spike height and spike frequency of bicuculline spikes. These studies demonstrate the ability of NPT2042 and bumetanide to similarly suppress bicuculline spikes in the NHP seizure model.

[0143] The purpose of this study was to evaluate the antiseizure efficacy of bumetanide dibenzylamide (NPT2042) compared to bumetanide in non-human primates. Results from this study confirmed that NPT2042 is a candidate for further clinical development as an adjunctive antiseizure therapy in patients with medically refractory epilepsy. The study arms for the intravenous and oral administration routes are shown in Tables Ex7-1 and Ex7-2, respectively. [Table 3] [Table 4]

[0144] Bicuculline blocks the inhibitory effects of GABA receptors, inducing convulsions, which are thought to mimic epileptic seizures. Therefore, bicuculline has been used in laboratories for decades to study and test the anticonvulsant effects of putative antiseizure treatments (Schwartzkroin and Prince 1980). Data from primate seizure studies were qualitatively similar to those observed in similar optical imaging studies in human patients (Haglund and Hochman 2005), in that the morphology of electrophysiological activity recorded in EEG traces was similar between NHPs and humans.

[0145] Description of the acute bicuculline seizure focus model: An acute "seizure focus" was created on the cortical surface. Three surface electrodes were placed such that one motor electrode was over the motor cortex of the hand and one electrode was over the sensory cortex of the hand. The central electrode was used as a reference for differential recording. The recording electrode can also be used to stimulate the cortex by passing a current between the pair of electrodes. An image illustrating the placement of the surface electrodes, provided below, is shown in Figure 4.

[0146] An acute "seizure focus" was created in primates by placing a bicuculline-soaked pledget on the surface of the neocortex over the arm / hand motor cortex for approximately 20-40 minutes until stable spiking activity was observed (Figure 12, panel A below). The pledget was then removed from the cortex. Spiking persisted for at least 4-6 hours until the experiment was terminated. The effects of various treatments on bicuculline-induced spiking could then be studied by administering treatment immediately after a consistent spiking pattern was elicited.

[0147] Panel A of Figure 12 shows a continuous 70-minute segment of a trace recorded by surface EEG electrodes over the cortical region where the bicuculline focus was generated. Panels B and C show the first and last 60 seconds of the trace shown in A (the time indicated by the red arrow in A), plotted over a faster time course so that individual spikes can be seen. Importantly, no spontaneous significant changes in spike magnitude or frequency occur in this model.

[0148] A comparison of the effects of bumetanide and the standard of care treatment levetiracetam (Keppra®) on Bicuculline spikes is shown in Figure 13. The trace below shows that both bumetanide and Keppra inhibit Bicuculline spikes. Of note, Keppra has a clear (but short-lived) effect on Bicuculline spikes in this model, whereas it is notoriously ineffective in any of the standard animal models used to screen for anticonvulsant activity (Loscher and Honack 1993).

[0149] Experimental design: Anti-seizure experiment Macaque NHPs (Macaca nemestrina, weighing 2–3 kg) were used in this experiment. Details regarding the treatment and surgical preparation of macaques for cortical AD stimulation and EEG recording are briefly described below (Haglund et al. 1993; Haglund and Hochman 2007; Tolner et al. 2011).

[0150] Animals were intubated (Matrix VBS anesthesia machine and Hallowell EMC model 2002 ventilator) and ventilated with 100% oxygen and maintained under pentobarbital anesthesia (1–2 mg / kg / h). Oxygen saturation was measured from the tongue using a Nellcor Pulse Oximeter and continuously monitored to maintain a constant saturation of 98–100% throughout the experiment.

[0151] Fluid balance was controlled by monitoring intravenous fluid intake every 15 minutes and fluid output (via a Foley catheter) every 30 minutes. Heart rate, blood pressure (Critikon Dinamap blood pressure monitor, model 8100), end-tidal partial pressure of carbon dioxide (pCO2), respiration, and O2 levels (Ohmeda 5250 RGM anesthesia monitor; Nellcor NBP-40 for oxygen saturation [SpO2]) were monitored throughout the experiment in conjunction with EEG.

[0152] After partial craniectomy (25 mm diameter), the dura was peeled off and surface EEG recordings were performed using custom-built strip electrodes (2.5 mm electrode diameter, 1 cm interelectrode distance) placed over the sensorimotor cortex: one electrode over the hand motor cortex, one electrode over the sensory cortex, and a reference electrode attached to the skull at the mastoid process or a third surface electrode between the motor and sensory electrodes.

[0153] 0.5 mm of gelfoam soaked in 100 μM bicuculline (Sigma-Aldrich, St. Louis, MO, USA) 2 Acute bicuculline foci were generated in the motor cortex using pledgets (Codman & Shurtleff, Randolph, MA, USA). Pledgets were placed on the cortex for 5 minutes and replaced every hour for 5 minutes. Bicuculline-induced epileptiform spike activity stabilized after approximately 1 hour, after which data acquisition began. EEG signals from motor and sensory electrodes were continuously recorded (1000 Hz sampling, unfiltered; Axon Instrument Digidata 1440A system).

[0154] Test product was administered once stable bicuculline spikes were observed on the EEG tracing for each NHP.

[0155] Data were analyzed using custom-designed software in the R programming language as described in a published reference (Haglund and Hochman, 2005). Bicuculline-evoked spike activity was quantified from motor cortex recordings. The effect of the test product on bicuculline-evoked spike activity was analyzed by comparing the peak response after treatment administration with the average value during a 5-minute control period before test product administration (baseline). In bicuculline experiments, recovery after test product administration was tracked for 20–35 minutes, following a return to pretreatment spike activity. Due to technical reasons and practical limitations on staff time, some recordings had to be terminated before the recovery period was complete.

[0156] An example of an EEG trace recorded with a Bicucullin spike is shown in Figure 12.

[0157] Test Product: The investigational product is NPT2042 and bumetanide is the reference product or active control. Product descriptions for NPT2042 and bumetanide are listed in Table Ex7-3 and Table Ex7-4, respectively. [Table 5] [Table 6]

[0158] The investigational product, NPT2042, was synthesized by Synexis, Inc. (Research Triangle Park, NC) and supplied to Duke University. NPT2042 was stored at 2°C to 8°C. The lot number for NPT2042 was 025DAP012 (manufactured on February 22, 2007).

[0159] For PO experiments, a commercial source of the reference product bumetanide was procured by Duke University.

[0160] For IV experiments, a commercial source of the reference product bumetanide was procured by Synexis, Inc.

[0161] Preparation of test products Each NHP was weighed prior to the study procedure and administered equimolar doses of NPT2042 and bumetanide at 10 mg / kg PO and 2 mg / kg IV doses.

[0162] NPT2042 and bumetanide suspensions were prepared for oral delivery (by gavage) by sonicating NPT2042 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.

[0163] NPT2042 and bumetanide IV solutions were prepared by dissolving NPT2042 or bumetanide in 100% PEG-200. These solutions were supplied by Synexis, Inc.

[0164] Data analysis Data were analyzed using custom-designed software using the R programming language as previously described (Haglund and Hochman 2005).

[0165] To better quantify effect sizes from these recordings, an algorithm was applied to automatically detect the bases and tops of the bicuculline spikes described herein. The algorithm is "brute force" and works by moving a window just wide enough to retain a single spike across the data, ignoring all variations that are less than two standard deviations above background noise in the trace, and finding the maximum and minimum values ​​in the window (Haglund and Hochman 2005). An example visual representation of the output from this computer analysis is shown below in Figure 14. Here, the computer identified the tops of all spikes with the upper (red) dots and the bottoms of the corresponding spikes with the lower (blue) dots. The distance between the maximum and minimum values ​​was taken as the peak height and is shown in Figure 15.

[0166] From these data, calculations of the height of each spike and the time interval between each spike (interspike interval, ISI) were generated by computer. To reduce the possibility of short-lived (1 or 2 minute) random fluctuations biasing 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 NPT2042: 1) spike size during the 10-minute pretreatment interval and during the 10-minute interval surrounding the time of the spike identified as representing the largest change; 2) pretreatment and posttreatment interspike intervals during the same time interval in which spike size was obtained; and 3) time to 50% recovery—the time from the largest change in spike size or ISI during the pretreatment period to 50% of the mean value. Due to the smoothing, the standard deviation over the above 10-minute window was negligible and therefore not reported.

[0167] result Intravenous administration of bumetanide and NPT2042 In two experiments on two different animals, bumetanide (2 mg / kg) and NPT2042 (molar equivalent of 2 mg / kg bumetanide) were administered via IV injection into a vein in the arm via an IV line used during the experiment to maintain the animals' well-being.

[0168] EEG traces from this experiment are shown in Figure 16. The traces in row A of Figure 16 show 20 minutes of continuous recording beginning 3 minutes before administration of either bumetanide (A1) or NPT2042 (A2). The time of administration of the test products is indicated by the red vertical bar in these traces (at t = 3 minutes). The traces in row B of Figure 16 were selected from the time when a significant, nearly complete blockade of epileptiform activity was observed, beginning at t = 81 minutes post-injection for bumetanide (B1) and t = 72 minutes post-injection for NPT2042 (B2). The traces in row C of Figure 16 show the period of sudden recovery to the baseline pre-treatment spiking state after bumetanide treatment beginning at approximately t = 120 minutes post-injection (C1), and the period at the end of the experiment at t = 155 minutes post-injection of NPT2042 (C2), when recovery to baseline had not yet been observed. For practical reasons, the experiment in which NPT2042 was administered was terminated before determining the time to recovery after NPT2042 administration.

[0169] Oral administration of bumetanide and NPT2042 In two experiments in two different primates, once stable EEG traces were obtained for each NHP, bumetanide (10 mg / kg) and NPT2042 (molar equivalent of 10 mg / kg bumetanide) were administered orally by gavage. In contrast to the IV study, in which nearly complete blockade of epileptiform activity was evident and visually discernible from the raw data, the maximal changes induced by oral administration were more subtle. Figure 17 shows representative 3-minute pretreatment traces for bumetanide (A1) and NPT2042 (A2), as well as representative 3-minute traces during the maximal change for bumetanide (B1) and NPT2042 (B2). The traces in Figures A1 and A2 above represent 3 minutes of pretreatment spike activity (baseline). Treatment effects are depicted over 3-minute intervals in Figures B1 and B2. The results for PO bumetanide and NPT2042 show that both compounds transiently mediate decreases in spike size and frequency in Table Ex7-5. Due to small sample sizes and inter-animal variability, the experiment was not able to demonstrate quantitative differences between compounds. [Table 7]

[0170] conclusion The small sample sizes in these experiments limit what can be concluded. However, given the stability over time of bicuculline spikes from acute seizure foci created in monkey cortex and the near-complete blockade of those spikes immediately after intravenous administration of bumetanide or NPT2042, it seems likely that NPT2042, like bumetanide, reduces bicuculline-induced epileptiform activity in monkeys. When quantified during PO experiments, where the effects of treatment were less pronounced, both bumetanide and NPT2042 transiently reduced both spike size and frequency, with both appearing to recover to pretreatment values ​​immediately after treatment administration.

[0171] Example 8: Diuretic Effect of Orally Administered Bumetanide Dibenzylamide (NPT2042) in Non-Human Primates The purpose of this study was to evaluate the diuretic effects of bumetanide dibenzylamide (NPT2042) and bumetanide in nonhuman primates (NHPs). Urine output was measured from the same animals during the same experiments that investigated the effects of NPT2042 and bumetanide on epileptiform EEG activity (see Study No. NPT RD103). Urine from Foley-catheterized Macaca nemestrina monkeys was collected in volumetric flasks to allow for urine volume measurements at regular time intervals throughout the course of the experiment. A single dose of either NPT2042 or the reference control bumetanide was administered orally (PO) to anesthetized and catheterized primates.

[0172] These studies demonstrated that a 10 mg / kg oral dose of bumetanide induced a peak increase in urine production of at least 1500% above control values ​​in macaque monkeys, whereas an equimolar dose of the amide analog bumetanide dibenzylamide (NPT2042) did not induce a measurable diuretic response. It is important to note that the anti-seizure effects of NPT2042 were confirmed in the anti-seizure arm of these studies (Example 7, above). In these experiments, both NPT2042 and bumetanide at equimolar oral doses of 10 mg / kg reduced the spike height and spike frequency of bicuculline spikes.

[0173] Given the very large increase in diuresis induced by bumetanide and the lack of a measurable increase by NPT2042 in non-human primates, this study indicates that the diuretic effect of NPT2042 is much less than that of bumetanide in this limited sample set.

[0174] Research objectives and subjects The objective of the study was to evaluate the diuretic effect of bumetanide dibenzylamide (NPT2042) compared to bumetanide in non-human primates. Results from this study were used to confirm that NPT2042 is a drug candidate for further clinical development as an adjunctive anti-seizure therapy in patients with medically refractory epilepsy. A key attribute for drug candidate selection among NKCC antagonist analog candidates is reduced or decreased diuresis, so that the drug can be tolerated as a chronic treatment.

[0175] In this study, two primates were evaluated, one receiving bumetanide and the other receiving NPT2042 via oral gavage (Table Ex8-1). [Table 8]

[0176] Data obtained on assessment of urine output following oral administration of bumetanide and NPT2042 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 on faculty at Duke University Medical Center).

[0177] Details regarding the treatment and surgical preparation of macaques for EEG recording have been described ( Haglund and Hochman 2007 , Tolner et al. 2011 ).

[0178] Experimental design: Diuresis experiment For the urine output data cataloged in this technical report, animals were intubated (Matrix VBS anesthesia machine and Hallowell EMC Model 2002 ventilator) and ventilated with 100% oxygen 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 continuously monitored to maintain a constant 98-100% saturation throughout the experiment.

[0179] Test and control materials were administered after baseline urine volume measurements were obtained (up to 100 minutes prior to test article administration). Urine production was measured after administration of 10 mg / kg oral bumetanide (n=3) and 10 mg / kg molar equivalent (mol-Eq) oral bumetanide dibenzylamide (NPT2042, n=1).

[0180] Fluid balance was controlled by monitoring intravenous fluid intake every 15 minutes and fluid output (via a Foley catheter) every 10-15 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 in conjunction with EEG.

[0181] All urine samples were collected from the Foley catheter into volumetric flasks, and volumes were recorded at least two time points before administration of the test and reference materials (to obtain an average baseline / pre-treatment rate of urine production), for at least 1 hour before treatment administration, and every 15 minutes after treatment administration (for the rate of urine production after treatment) until peak urine output and recovery were observed.

[0182] Test Product The investigational product is NPT2042 and bumetanide is the reference product or active control. Product descriptions for NPT2042 and bumetanide are listed in Tables Ex8-2 and Ex8-3, respectively. [Table 9] [Table 10]

[0183] The investigational product, NPT2042, was synthesized by Synexis, Inc. (Research Triangle Park, NC) and supplied to Duke University. NPT2042 was stored at 2°C to 8°C. The lot number for NPT2042 was 025DAP012 (manufactured on February 22, 2007).

[0184] A commercial source of the reference product bumetanide was procured by Duke University.

[0185] Preparation of Test Products: Each NHP was weighed prior to the test procedure and administered NPT2042 and bumetanide at an equimolar (mol-Eq) dose of 10 mg / kg PO.

[0186] NPT2042 and bumetanide suspensions were prepared for oral delivery (by gavage) by sonicating NPT2042 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.

[0187] Data Analysis: Data were analyzed using custom-designed software using the R programming language.

[0188] result A molar equivalent of 10 mg / kg bumetanide was used, administered as a water-cmc suspension by oral delivery via oral gavage in anesthetized monkeys. Tables Ex8-4 and 8-5 show the mean changes in spike height and time to approximately 50% recovery after treatment. [Table 11] [Table 12]

[0189] Each panel in Figure 18 shows the percent change in urine volume (relative to baseline, pre-treatment levels) over time for an individual monkey after oral administration of bumetanide or NPT2042. The diuretic response to bumetanide is represented in Figure 18 by the graphs labeled bumetanide (1), (2), and (3), while the remaining graphs show the diuretic response to bumetanide dibenzylamide (NPT2042). The red line indicates the time at which treatment was administered orally at t=0. Time is given in minutes on the x-axis, and the percent change in urine volume produced (compared to pre-treatment urine production) is shown on the y-axis. Oral doses were 10 mg / kg bumetanide and the molar equivalent of NPT2042. Bumetanide induced increases in urine volume production of >1500% to 3000% of baseline values, while the bumetanide amide analog NPT2042 induced little to no increase in diuresis above baseline.

[0190] conclusion These studies showed that in macaque monkeys, bumetanide administered orally at 10 mg / kg induced a peak increase in urine production of at least 1500%, whereas the amide analog bumetanide dibenzylamide (NPT2042) did not induce a measurable diuretic response.

[0191] It is important to note that the seizure suppression effects of NPT2042 were confirmed in the anti-seizure arm of these studies (see Study No. NPT RD103). In these experiments, both NPT2042 and bumetanide at equimolar oral doses of 10 mg / kg reduced spike height and spike frequency of bicuculline spikes.

[0192] Given the very large increase in diuresis induced by bumetanide and the lack of a measurable increase by NPT2042 in non-human primates, it is likely that the diuretic effect of NPT2042 is much less than that of bumetanide, even in light of the small sample size.

[0193] Example 9: Anxiolytic effects of intravenous administration of bumetanide dibenzylamide (NPT2042) in rats The purpose of this study was to evaluate the central nervous system (CNS) effects (anxiolytic effects) of bumetanide dibenzylamide (NPT2042) in the rat fear-potentiated startle (FPS) model of conditioned anxiety (Lehmann et al. 2010, Krystal et al. 2012). The FPS model used in this study was the same model previously used to evaluate the anxiolytic effects of two NKCC antagonists, bumetanide and furosemide (Krystal et al. 2012).

[0194] The FPS test involves two training sessions in which an intrinsically aversive footshock stimulus is paired with a neutral cue stimulus, a light. During the test session, the presentation of this cue stimulus, the light, is then used to induce a potentiated startle. The FPS procedure consisted of five days of testing: baseline startle responses were collected on days 1 and 2, light / shock pairings were delivered on days 3 and 4, and fear-potentiated startle was administered on day 5.

[0195] On day 5, 30 minutes before the FPS test, animals were treated with either NPT2042 (35 mg / kg), bumetanide (35 mg / kg), or vehicle (dimethyl sulfoxide [DMSO] alone) via the cannulated jugular vein. The doses selected for this study were determined from a pilot study conducted by NeuroInvestigations, Inc. (Lethbridge, Canada) to determine the minimum dose of bumetanide that elicited a measurable response in the FPS model (data on file). Startle amplitude was measured and compared between NPT2042 vs. vehicle and bumetanide vs. vehicle.

[0196] The results show that NPT2042 induced anxiolytic effects in the FPS model similar in magnitude to those induced by an equivalent dose (mg / kg) of bumetanide, as determined by a reduction in startle amplitude to the shock-conditioned stimulus.

[0197] Research objectives and subjects The purpose of this study was to evaluate the anxiolytic effects of bumetanide dibenzylamide (NPT2042) in the rat FPS model. The results of this study confirm the suitability of NPT2042 as a drug candidate for adjunctive antiseizure therapy in patients with medically refractory epilepsy.

[0198] In this study, adult male Long-Evans rats (3-4 months old) were used to test the anxiolytic effects of NPT2042 in the FPS anxiety model (vehicle control / DMSO, n=51; bumetanide, n=14; NPT2042, n=15). See Table Ex9-1. [Table 13]

[0199] For the purpose of screening bumetanide analogs, the rat model was the most reasonable model for measuring CNS responses and was also feasible for testing and comparing a series of bumetanide analog candidate compounds. Several standard rat epilepsy models were also considered, but these models were very labor-intensive and / or required large numbers of animals to statistically significantly compare the CNS effects of bumetanide to bumetanide analogs and each other. However, rat associative anxiety models, particularly the fear-potentiated startle (FPS) model, demonstrated very robust bumetanide-mediated NKCC-sensitive responses that varied depending on the potency and concentration of the NKCC antagonist, produced large and reproducible responses (thus requiring the use of far fewer animals), and were far less labor-intensive than rat seizure models (Krystal et al. 2012). Of note, some anti-seizure medications 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 in Krystal et al. 2012).

[0200] These rat studies were intended solely to test CNS effects (in this case, anxiety reduction); diuretic effects were not anticipated because rats rapidly metabolize bumetanide and biotransform it to an inactive metabolite via oxidation of its N-butyl side chain before bumetanide can induce diuresis via antagonism of renal NKCC2 (Schwartz 1981). Potential bumetanide analogs (including NPT2042) share this same N-butyl side chain, so they are expected to be similarly vulnerable to such metabolism. Therefore, diuretic effects have been evaluated in primate studies, which are described in report NPT RD102 (NeuroPro Therapeutics Inc. 2022).

[0201] Experimental design: fear-potentiated startle The published FPS protocol (Lehmann et al. 2010) was followed and is described below.

[0202] Adult male (3-4 months old) Long-Evans rats were housed at the University of Lethbridge vivarium and used in these studies under the direction of Janice Sutherland, PhD. Rat housing consisted of Plexiglas cages with sawdust bedding shared by two or three rats. The colony room was temperature-controlled (20-21°C) with a 12-hour light / 12-hour dark cycle beginning at 7:00 AM daily. Food and water were provided ad libitum. 72 hours before the experiment, 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 compound. Rats were then placed in individual cages, and the cannula was flushed daily to ensure patency.

[0203] All behavioral testing was performed during the light cycle (7 AM to 7 PM). Testing took place between 9 AM and 3 PM. Randomly selected different rats were used for each group (i.e., rats were not retested in multiple groups). All testing was performed under ambient room light.

[0204] Animals were trained and tested with four identical stabilimeter devices (Med-Associates). Each rat was placed in a small Plexiglas cylinder. The floor of each stabilimeter consisted of four 6-mm-diameter stainless steel rods spaced 18 mm apart, through which shocks could be delivered. Cylinder movement resulted in accelerometer displacement, and the resulting potential was proportional to the rate of cage displacement. Startle amplitude was defined as the maximum accelerometer potential occurring during the first 0.25 s after the startle stimulus was delivered. The analog output of the accelerometer was amplified, digitized on a scale of 0–4096 units, and stored on a microcomputer. Each stabilimeter was placed in a ventilated, light- and sound-attenuating 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 resulted in an overall background noise level of 64 dB. The startle stimulus was a 50-ms burst of white noise (with a 5-ms onset-decay time) generated by a white noise generator. The visual conditioned stimulus was the illumination of a light bulb adjacent to a white noise source. The unconditioned stimulus was a 0.5-second, 0.6 mA foot shock generated by four constant-current shockers positioned outside the chamber. All stimulus presentation and sequencing was computer-controlled. The fear-potentiated startle procedure consisted of five days of testing. On days 1 and 2, baseline startle responses were collected; on days 3 and 4, light / shock pairings were delivered; and on day 5, fear-potentiated startle testing was performed. Animals were treated with test compound or vehicle on day 5.

[0205] For an overview of the FPS model, see Figure 19. Details describing the daily procedures are provided in the following sections.

[0206] Day 1 and Day 2: Matching On days 1 and 2, rats were individually placed in Plexiglas cylinders and presented with 30 startle stimuli at 30-second interstimulus intervals after 3 minutes. An intensity of 105 dB was used. The mean startle amplitude across the 30 startle stimuli on day 2 was used to assign rats to treatment groups with similar mean values.

[0207] Days 3 and 4: Training On days 3 and 4, rats were placed individually in Plexiglas cylinders. After an initial 3-minute acclimatization period, rats were delivered 10 conditioned stimulus (CS)-shock pairings. The shock was delivered during the last 0.5 seconds of the 3.7-second CS, with a mean intertrial interval of 4 minutes (range 3-5 minutes).

[0208] Day 5: Exam On day 5, 30 minutes before testing, animals were administered study drug (NPT2042 [35 mg / kg] or bumetanide [35 mg / kg]) or vehicle (DMSO alone) via a cannulated jugular vein.

[0209] For testing, rats were placed in the same startle box used for training and, after a 3-minute acclimatization period, presented with 18 startle-inducing stimuli (all 105 dB). These initial startle stimuli were used to re-habituate the rats to the acoustic startle stimuli. Thirty seconds after the final of these stimuli, each animal received 60 startle stimuli. Half of the stimuli were presented alone (startle-alone trials), and the other half were presented 3.2 seconds after the onset of a 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.

[0210] Test Product The investigational drug was NPT2042 and the control products were bumetanide (active control) and DMSO (vehicle control). Product descriptions for NPT2042 and bumetanide are listed in Tables Ex9-2 and Ex9-3, respectively. [Table 14] [Table 15]

[0211] The investigational drug, NPT2042, was synthesized by Synexis (Research Triangle Park, NC) and supplied to the University of Lethbridge vivarium. NPT2042 was stored at 2°C to 8°C. NPT2042 lot number 009MPS023 was manufactured on October 10, 2005.

[0212] Commercial sources of the active control product bumetanide and vehicle control DMSO were procured by the University of Lethbridge vivarium.

[0213] Preparation of test products Dosing solutions were prepared immediately before use, stored at room temperature before use, and administered within 3 hours of preparation.

[0214] Each rat was weighed prior to the test procedure and administered NPT2042 and bumetanide at a dose of 35 mg / kg IV.

[0215] NPT2042 and bumetanide IV solutions were prepared by dissolving NPT2042 or bumetanide in 100% DMSO.

[0216] Data Analysis: For data analysis, data were entered into an Excel spreadsheet and SPSS. An independent samples t-test was used to compare treatment groups. The statistical programming language R was used to generate plots and perform statistical analysis. A Welch two-sample t-test (one-tailed) was used to compare NPT2042 vs. vehicle and bumetanide vs. vehicle.

[0217] result Both bumetanide and NPT2042 induced a significantly smaller increase in shock-conditioned stimulus startle amplitude than rats treated with vehicle alone (Vehicle mean = 165.8 [standard error (SE) = 21.6], p = 0.02652; NPT2042 mean = 105 [SE = 24.4], p = 0.03512; bumetanide mean = 97.6 [SE = 26.3], p = 0.02652). See Figure 20.

[0218] The 95% t-confidence intervals for the means were NPT2042: [52.6, 157.4], bumetanide: [41.0, 154.2], and vehicle: [122.3, 209.3].

[0219] These data indicate that a 35 mg / kg dose of NPT2042 has similar CNS effects as a 35 mg / kg dose of bumetanide in the rat FPS model of conditioned anxiety.

[0220] No rats died in any of the test groups.

[0221] Example 10 All three derivatives, bumetanide diethylamide, bumetanide N-morpholinoamide, and bumetanide dibenzylamide, resulted in a lower rate of change in urine production over time compared to bumetanide. See Figure 21.

[0222] Example 11 All three derivatives, bumetanide diethylamide, bumetanide N-morpholinoamide, and bumetanide dibenzylamide, resulted in a lower rate of urine production over time compared to bumetanide. See Figure 22.

[0223] Example 12 All three derivatives, bumetanide diethylamide, bumetanide N-morpholinoamide, and bumetanide dibenzylamide, resulted in a lower mean rate of urine production over time compared to bumetanide. See Figure 23.

[0224] Example 13 In stark contrast to the demonstrated results associated with the amide compounds, the ester prodrug of bumetanide continued to exhibit diuretic effects. Compare Figure 23 with Figures 24-27.

[0225] The mean pre-treatment urine rates (mL / min) were significantly lower compared to the maximum post-treatment urine rates (mL / min) following treatment with the esters (including bumetanide methyl ester, bumetanide cyanomethyl ester, bumetanide N,N-diethylglycolamido ester, and bumetanide benzyl ester). See Figures 24-27. All animals received the respective ester at a dose that was the molar equivalent of 2 mg / kg bumetanide. Administration was intravenous. As shown, the diuretic effect of bumetanide derivatives is unpredictable.

[0226] Those skilled in the art to which this disclosure pertains, particularly in light of the foregoing teachings, may make modifications resulting in other embodiments employing the principles of the present disclosure without departing from its spirit or character. The described embodiments are therefore to be considered in all respects merely illustrative and not restrictive, the scope of the present disclosure being accordingly indicated by the appended claims rather than by the foregoing description or drawings. Consequently, while the present disclosure has been described with reference to particular embodiments, modifications in structure, arrangement, materials, and the like that are obvious to those skilled in the art will still be within the scope of the claims.

[0227] Example 14 The objectives of this study were to evaluate the central nervous system (CNS) effects (anxiolytic effects) of bumetanide dibenzylamide (NPT2042) in humans and to assess the safety and pharmacokinetics (PK) of single and multiple ascending doses of NPT2042 in healthy adult subjects. Subjects received eight capsules, each containing 16 mg of bumetanide dibenzylamide, every 24 hours. Pharmacokinetic (PK) blood samples were collected and analyzed pre-dose to establish baseline levels. Analysis was also performed on days 1, 3, 4, 5, 6, 7, and 8 post-dose. 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 (an increase in BUN disproportionate to creatinine), a decrease in urine specific gravity, and an increase in serum chloride. As shown in Figures 28-29, there was no evidence of diuresis. The data indicate that bumetanide dibenzylamide likely acts as a renal NKCC inhibitor. The gray horizontal lines in Figures 28-29 indicate the upper and lower limits of accepted normal values. Control subjects (placebo) are indicated by the white and black dashed lines and gray dots.

[0228] Those skilled in the art to which this disclosure pertains, particularly in light of the foregoing teachings, may make modifications resulting in other embodiments employing the principles of the present disclosure without departing from its spirit or character.

[0229] The described embodiments are, therefore, to be considered in all respects merely illustrative and not restrictive, the scope of the present disclosure being, accordingly, indicated by the appended claims rather than by the foregoing description or drawings. Consequently, although the present disclosure has been described with reference to particular embodiments, modifications in structure, arrangement, materials, and the like that are obvious to those skilled in the art will still be within the scope of the claims.

Claims

1. 1. A pharmaceutical composition comprising bumetanide dibenzylamide, bumetanide diethylamide, or bumetanide morpholinoamide, wherein the pharmaceutical composition has a therapeutic effect on seizure blockade 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 diuretic effect on the patient.

3. 3. The composition of claim 1 or 2, wherein the measure of seizure suppression is seizure frequency.

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

5. 5. The composition of any one of claims 1 to 4, wherein the measure of seizure suppression is a change in the amplitude of pharmacologically or electrically induced seizure (epileptiform) activity as measured by EEG or other electrophysiological type of recording.

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

7. 7. The composition of any one of claims 1 to 6, wherein the measure of seizure suppression is a change in the frequency of pharmacologically or electrically induced seizure (epileptiform) activity as measured by EEG or other electrophysiological type of recording.

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

9. The composition according to any one of claims 1 to 8, wherein the measure of the diuretic effect is a urinary 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 interspike interval.

12. 12. The composition of claim 11, wherein the interspike 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 interspike 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 attack frequency and urine production over a given period of time.

15. The composition according to any one of claims 1 to 14, wherein the therapeutic effect is based on an increase in interspike interval and a change in urine production over a given period of time.

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

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

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

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

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

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

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

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

25. 25. The composition of any one of claims 1 to 24, wherein the change in seizure frequency after treatment with the composition is at least a 50% reduction in the frequency of seizure occurrence.

26. 26. The composition of any one of claims 1 to 25, wherein the change in seizure frequency after treatment with the composition is between greater than 50% and 100% reduction in the frequency of occurrence of the seizures.

27. 27. The composition of any one of claims 1 to 26, wherein the diuretic effect is measured by less than about a two-fold increase in urine production over a 24-hour period following treatment with the composition.

28. 28. The composition of any one of claims 1 to 27, wherein the measure of the diuretic effect is the lack of an increase in urine production over a 24 hour period following treatment with the composition.

29. 29. The composition of any one of claims 1 to 28, wherein the measure of the diuretic effect is an increase in urine production of about 0% to about 100% over a 24 hour period 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 therapeutic effect is Treatment effect = [seizure activity after treatment] / [seizure activity before treatment] * [Diuresis after treatment] / [Diuresis before treatment] 31. The composition of claim 30, wherein the

32. 32. The composition of claim 30 or 31, wherein the effective dose of the composition is the dosage required to completely block seizure activity.

33. 33. The composition of any one of claims 30 to 32, wherein the effective dose of the composition exceeds the dosage required to completely block seizures.

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

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

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

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

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

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

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

41. 41. The method of claim 39 or 40, wherein the pharmaceutical composition is administered once.

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

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

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

47. 47. The method of any one of claims 39 to 46, wherein the pharmaceutical composition is administered in combination with a conventional therapy for treating stroke.

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

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

50. 50. The method of any one of claims 39 to 49, wherein bumetanide dibenzylamide has a faster time to effect as measured by reduction in seizure frequency than conventional antiepileptic drugs.

51. the reduction in seizure frequency is a) a time increment selected from one or more of hours, days, weeks, and months; b) seizure diary and recorded reduction in seizure activity; c) an increase in one or more of interictal (inter) and postictal (post) spikes; and d) a decrease in interictal activity as measured by EEG.

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