Formulations and methods for convenient and rapid diuresis for the treatment of edema

Abstract

A method and formulations are developed for enhancing solubility and improving the permeability of bumetanide across a mucosal membrane. The formulation includes bumetanide in various water-cosolvent mixtures, which include at least one of an alcohol, polyether, glyceride, pyrrolidone, or any combination thereof. In some embodiments, the formulation has a pH of about 3.5 to about 8.0. In some embodiments, the formulation enhances the solubility and permeability of the bumetanide. Also disclosed is a use of the formulation for rapidly achieving effective bumetanide plasma concentration leading to diuresis. In some embodiments, the formulation can be administered to a subject conveniently without injection in the treatment of edema associated with heart failure or other conditions.

Description

STRDS.003WO APPLICATIONFORMULATIONS AND METHODS FOR CONVENIENT AND RAPID DIURESIS FOR THE TREATMENT OF EDEMARELATED APPLICATIONS AND INCORPORATION BY REFERENCE

[0001] This application claims the benefit of U.S. Provisional Ser. No. 63 / 684578, filed August 19, 2024, which is hereby incorporated by reference in its entireties.BACKGROUND OF THE INVENTIONField of the Invention

[0002] Disclosed herein are formulations and methods for achieving rapid diuresis in subjects with edema. Some embodiments relate to compositions comprising a nasal spray or sublingual bumetanide administration.Description of the Related Art

[0003] There exists a need for improved formulations and methods for convenient rapid diuresis in certain patients such as those with decompensated heart failure and other conditions of edema.

[0004] Bumetanide is a loop diuretic indicated for the treatment of edema associated with congestive heart failure, and hepatic and renal diseases. The usual initial oral dose is 0.5-2mg and if not adequate, a second or third oral dose may be given at 4 to 5 hours intervals up to a daily maximum of lOmg. The intravenous dose is 0.5-lmg and can be given at intervals of 2-3 hours up to a daily maximum of lOmg.

[0005] Intravenous loop diuretics such as bumetanide (at a dose of 1 mg) have been shown to induce a much faster and greater water and sodium excretion compared to the same dose administered orally in human subjects. Thus, intravenous loop diuretics are recommended and constitute essential components for the treatment of acute heart failure. Recent studies have shown that patients with acute heart failure treated early with an intravenous loop diuretic after being admitted to the hospital (within a median time of 90 min) had significantly lower in-hospital mortality compared to those treated later. The benefit of early treatment with intravenous therapy in similar types of patients was also confirmed in another study. These data indicate that early loop diuretic treatment is desirable for acutedecompensated heart failure. If a product is available that would allow convenient administration and yet achieve rapid action, such therapy not only can offer a more user- friendly alternative to intravenous loop diuretics in the hospital setting, but also initiate such rapid effective therapy in the ambulatory setting. At present, loop diuretics including bumetanide, are primarily available as oral and intravenous formulations. There is a need to develop a formulation and method for intranasal and sublingual administration leading to rapid, safe, and effective action.SUMMARY OF THE INVENTION

[0006] Some disclosures herein relate to a composition. In some embodiments, the composition comprises a therapeutically effective dose of bumetanide. In some embodiments, the composition comprises a therapeutically effective dose of bumetanide, and a water cosolvent mixture comprising 1, 2, 3, or all 4 of: an alcohol, a polyether, a glyceride, and / or a pyrrolidone, wherein the composition is at a final pH that is between about 3.5 and about 8, as adjusted using NaOH and HC1. In some embodiments, the pH is any value between about 3.5 and about 8.0. In some embodiments, the composition is formulated for nasal or intranasal administration to a subject. In some embodiments, the composition is formulated for sublingual administration to a subject. In some embodiments, the composition is formulated such that bumetanide is capable of permeating a mucosal membrane. In some embodiments, the alcohol, polyether, glyceride, and / or pyrrolidone is at most about 80% of the composition In some embodiments, the pH is between about 4.0 and about 5.0. In some embodiments, the organic volume fraction of the composition is at least about 1%. In some embodiments, the organic volume fraction of the composition is at most about 80%. In some embodiments, the organic volume fraction of the composition is at most about 50%. In some embodiments, the solubility of bumetanide is at least about 2.5 mg / mL. In some embodiments, the permeability (Papp, as determined by PAMPA) of bumetanide in the composition is greater than that of bumetanide in water corresponding to a pH that provides targeted solubility. In some embodiments, the bumetanide is at a concentration that is at or between about 2.5 mg / mL and about 10 mg / mL. In some embodiments, the alcohol is an ethanol, a glycerol, or a glycofurol. In some embodiments, the composition comprises any one of a polyether, diethylene glycol monoethyl ether, a medium chain glyceride, one or more saturated polyglycolyzed C8-C10 glyceride, apyrrolidone, or any combination thereof. In some embodiments, the composition comprises any one of N-methyl pyrrolidone (NMP), dimethyl isosorbide ether (DMI), Tween 80, glycofurol, or any combination thereof.

[0007] Also disclosed herein is a composition comprising a therapeutically effective dose of a loop diuretic molecule; a water; and an organic fraction comprising at least 1, 2, 3, or 4 compound(s), wherein the compound(s) is / are selected from: an alcohol, a polyether, a glyceride, a pyrrolidone, or any combination thereof. In some embodiments, the composition is at a final pH that is about 3.5, about 4.0, about 4.5, about 5.0, about 5.5, about 6.0, about 7.0, about 7.5, about 8.0, or any value that is between about 3.5 and about 8.0. In some embodiments, the pH is adjusted using at least one of NaOH, KOH, HC1, or any combination thereof. In some embodiments, the alcohol is an ethanol, a glycerol, or a glycofurol. In some embodiments, the alcohol is glycofurol. In some embodiments, the polyether is dimethyl isosorbide (DMI). In some embodiments, the pyrrolidone is N-methyl- 2-pyrolidone (NMP). In some embodiments, the loop diuretic molecule is bumetanide.

[0008] In some embodiments, the composition is formulated for nasal, intranasal, oral, or sublingual administration to a subject. In some embodiments, the composition is formulated such that the loop diuretic molecule is capable of permeating a mucosal membrane. In some embodiments, the alcohol, polyether, glyceride, and / or pyrrolidone is independently present in the composition at an amount that is about 1%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, or any value that is between about 1% and about 80% of the total composition. In some embodiments, the pH is at about 4.0, about 4.5, about 5.0, or is any value that is between about 4.0 and about 5.0. In some embodiments, the organic fraction is at least about 1% of the total composition. In some embodiments, the organic fraction is at most about 80% of the total composition. In some embodiments, the organic fraction is at most about 50% of the total composition. In some embodiments, the solubility of the loop diuretic molecule is at least about 2.5 mg / mL. In some embodiments, the permeability (Papp, as determined by PAMPA) of the loop diuretic molecule in the composition is greater than that of loop diuretic molecule in water (permeability greater than that observed for aqueous formulation at about pH 7.0). In some embodiments, the loop diuretic molecule is at a concentration that is about 2.0, about 2.5, about 3.0, about 3.3, about3.5, about 4.0, about 4.5, about 5.0, about 5.5, about 6.0, about 6.5, about 7.0, about 7.5, about 8.0, about 8.5, about 9.0, about 9.5, about 10.0, or any value that is between about 2.0 and about 10.0 mg / mL. In some embodiments, the organic fraction comprises any 1, 2, 3, 4, or 5 of: a polyether, diethylene glycol monoethyl ether, a medium chain glyceride, one or more saturated polyglycolyzed C8-C10 glyceride, a pyrrolidone, or any combination thereof. In some embodiments, the organic fraction further comprises Tween 80 and / or a polyethylene glycol.

[0009] Some embodiments disclosed herein relate to a use of a composition of any one of the present embodiments. In some embodiments, the use of the composition is for treating a disease or disorder in a subject in need thereof. In some embodiments, the subject is mammalian and / or human. In some embodiments, the composition is optimized to achieve an effective dose and rapid plasma concentration upon administration to the subject. In some embodiments, the disease or disorder is an edema.

[0010] Some embodiments disclosed herein relate to a method for treating a disease or disorder in a subject in need thereof. In some embodiments, the method comprises administering the composition of any one of the embodiments of the present disclosure. In some embodiments, the subject is mammalian and / or human. In some embodiments, the composition is optimized to achieve an effective dose and rapid plasma concentration upon administration to the subject. In some embodiments, the disease or disorder is an edema. In some embodiments, the disease or disorder is an edema associated with heart failure.

[0011] Some embodiments disclosed herein relate to a method for treating a disease or disorder in a subject in need thereof. In some embodiments, the disease or disorder is an edema. In some embodiments, the method comprises administering a therapeutically effective dose of a composition, the composition comprising: a therapeutically effective dose of bumetanide; and a water co-solvent mixture comprising 1, 2, 3, or all 4 of: an alcohol, a polyether, a glyceride, and / or a pyrrolidone; wherein the composition is at a final pH that is between about 3.5 and about 8, as adjusted using NaOH and HC1. In some embodiments, the composition is formulated for nasal or intranasal administration to a subject. In some embodiments, the composition is administered intranasally. In some embodiments, the composition is formulated for sublingual administration to a subject. In some embodiments, the composition is administered sublingually. In some embodiments, the composition isformulated such that bumetanide is capable of permeating a mucosal membrane. In some embodiments, the method further comprises contacting a mucosal membrane of the subject with the composition. In some embodiments, the composition is optimized to achieve an effective dose and rapid plasma concentration upon administration to the subject. In some embodiments, the alcohol, polyether, glyceride, and / or pyrrolidone is at most about 80% of the composition. In some embodiments, the pH is between about 4.0 and about 5.0. In some embodiments, the organic volume fraction of the composition is at least about 1%. In some embodiments, the organic volume fraction of the composition is at most about 80%. In some embodiments, the organic volume fraction of the composition is at most about 50%. In some embodiments, the solubility of bumetanide is at least about 2.5 mg / mL. In some embodiments, the permeability (Papp, as determined by PAMPA) of bumetanide in the composition is greater than that of bumetanide in water. In some embodiments, the bumetanide is at a concentration that is between about 2.5 mg / mL and about 10.0 mg / mL. In some embodiments, the alcohol is an ethanol, a glycerol, or a glycofurol. In some embodiments, the composition comprises any one of a polyether, diethylene glycol monoethyl ether, a medium chain glyceride, one or more saturated poly glycolyzed C8-C10 glyceride, a pyrrolidone, or any combination thereof. In some embodiments, the composition comprises any one of N-methyl pyrrolidone (NMP), dimethyl isosorbide ether (DMI), Tween 80, glycofurol, or any combination thereof.BRIEF DESCRIPTION OF THE DRAWINGS

[0012] FIG. 1A depicts a non-limiting example graph of bumetanide solubility in aqueous solution plotted as a function of change in pH.

[0013] FIG. IB depicts a non-limiting example graph of bumetanide permeability in aqueous solution plotted as a function of change in pH.

[0014] FIG. 1C depicts a non-limiting example graph of bumetanide flux in aqueous solution plotted as a function of change in pH.

[0015] FIG. 2A depicts a non-limiting example graph of the change in mean saturated solubility of bumetanide in different fractions of aqueous glycofurol as a function of change in pH.

[0016] FIG. 2B depicts a non-limiting example graph of the change in mean saturated solubility of bumetanide in different fractions of aqueous N-methyl-2-pyrolidone (NMP) as a function of change in pH.

[0017] FIG. 2C depicts a non-limiting example graph of the change in mean saturated solubility of bumetanide in different fractions of aqueous dimethyl isosorbide (DMI) as a function of change in pH.

[0018] FIG. 3A depicts a non-limiting example graph of the change in mean permeability (determined by PAMPA) of bumetanide in different fractions of aqueous glycofurol as a function of change in pH.

[0019] FIG. 3B depicts a non-limiting example graph of the change in mean permeability (determined by PAMPA) of bumetanide in different fractions of aqueous NMP as a function of change in pH.

[0020] FIG. 3C depicts a non-limiting example graph of the change in mean permeability (determined by PAMPA) of bumetanide in different fractions of aqueous DMI as a function of change in pH.

[0021] FIG. 4 depicts a non-limiting example graph of the measured versus predicted bumetanide solubility of certain potential targeted formulations.

[0022] FIG. 5 depicts a non-limiting example graph for the Cmax of bumetanide after dosing with the formulations of D45 at pH 4, 4.5 and 6.5. Data was normalized to the dose of 1 mg / kg.DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

[0023] The present invention relates to formulations and methods of enhancing solubility and permeation of bumetanide across a mucosal membrane. The formulations and methods provided herein may be used for the treatment of edema associated with acute decompensated heart failure, for example. In some embodiments, the formulations and / or method can be implemented as part of administering a loop diuretic bumetanide to achieve rapid plasma concentration leading to rapid action following sublingual or intranasal administration.

[0024] Some embodiments disclosed herein relate to a water-cosolvent mixture that is relatively safe and / or well-tolerated by human subjects. In some embodiments, thewater-cosolvent mixture is capable of sufficiently solubilizing bumetanide so that an effective and safe dose can be administered intranasally and / or sublingually. In some embodiments, the administration is equivalent to about 1 mg oral administration of bumetanide. In some embodiments, the administration is much more rapidly absorbed by the subject compared to about 1 mg oral administration of bumetanide. In some embodiments, peak plasma concentration in a subject is achieved before an hour following administration. In some embodiments, peak plasma concentration in a subject is achieved at approximately at least about 2, about 5, about 10, about 15, about 20, about 25, about 30, or any integer that is between about 5 and about 30, minutes following administration. In some embodiments, the formulation is optimized to achieve a target of an effective dose but rapid plasma concentration upon intranasal or sublingual delivery of bumetanide. In some embodiments, the formulation comprises an organic component. In some embodiments, the organic component is at least about 1% of the total formulation. In some embodiments, the organic component is at most about 80% of the total formulation. In some embodiments, the organic component enhances bumetanide solubility. In some embodiments, the bumetamide solution is at a target concentration of about 2.5, about 3.0, about 3.5, about 4.0, about 5.0, about 5.5, about 6.0, about 6.5, about 7.0, about 7.5, about 8.0, about 8.5, about 9.0, about 9.5, about 10.0, or any value that is between about 2.5 and about 10.0 mg / ml. In some embodiments, the formulation is at a pH range of about 3.5 to about 8. In some embodiments, the formulation is at a pH range of about 4 to about 5.

[0025] In some embodiments, the method for identifying the specific formulation (composition) comprises: (1) initial screening of the bumetanide solubility vs pH in various water-cosolvent organic mixtures to achieve a bumetanide solubility of at least about 2.5 mg / ml and, (2) optimizing their permeability of selected suitable solutions (water-cosolvent mixtures) to achieve rapid absorption of bumetanide upon intranasal / sublingual administration.

[0026] Some embodiments disclosed herein relate to a method of treating the edema of a subject in need thereof. In some embodiments, the method comprises contacting a mucosal membrane of the subject with a formulation disclosed herein, thereby treating the edema of the subject. In some embodiments, contacting the mucosal membrane comprises intranasal administration. In some embodiments, contacting the mucosal membrane comprises sublingual administration.

[0027] Some embodiments disclosed herein relate to a method of preparing a formulation for treating the edema of a subject. In some embodiments, the method comprises: (a) adding bumetanide to an organic-aqueous solvent comprising an alcohol, a polyether, diethylene glycol monoethyl ether, a medium chain glyceride, one or more saturated polyglycolyzed C8-C10 glyceride, a pyrrolidone, or a combination thereof; (b) adjusting the pH of the organic-aqueous solvent comprising bumetanide from about 3.5 to about 8.0, or any value between about 3.5 and about 8.0, preferably from about 4.0 to about 5.0, wherein treating the edema comprises contacting a mucosal membrane of the subject with the formulation. In some embodiments, the alcohol, polyether, glyceride, and / or pyrrolidone is at most about 80% of the composition. In some embodiments, the alcohol, polyether, glyceride, and / or pyrrolidone is at most about 50% of the composition. In some embodiments, the alcohol, polyether, glyceride, and / or pyrrolidone is about 0%, about 1%, about 5%, about 10%, about 20%, about 25%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80% or any integer that is between about 0% and about 80%, of the total composition. In some embodiments, the formulations described herein comprise N-methyl pyrrolidone (NMP), Tween 80 or similar organic compounds. In some embodiments, the formulations described herein comprise N-methyl pyrrolidone (NMP), dimethyl isosorbide ether (DMI), Tween 80, glycofurol, or similar organic compounds in combination with each other and / or with one or more alcohol, a polyether, diethylene glycol monoethyl ether, a medium chain glyceride, one or more saturated polyglycolyzed C8-C10 glyceride, or a combination thereof. In some embodiments, the solubility of bumetanide is increased in the organic-aqueous solvent relative to the solubility of bumetanide in water. In some embodiments, the solubility of bumetanide is at least about 2.5 mg / mL. In some embodiments, the solubility of bumetanide is at least about 2.5, about 3, about 3.33, about 3.5, about 4, about 4.5, about 5, or about 10 mg / mL. In some embodiments, the solubility of bumetanide is any value between about 2.5 and about 10.0 mg / mL. In some embodiments, administration is performed through a spray. In some embodiments, the bumetanide is at an equivalent concentration of about 1 mg dose per 2 sprays. In some embodiments, a spray is about 100 uL, about 150 uL, about 200 uL, about 250 uL, or any integer between about 100 and about 250 uL. In some embodiments, the organic- aqueous solvent comprises an alcohol. In some embodiments, the alcohol is ethanol or glycerol or glycofurol In some embodiments, the organic component of the mixture has a concentrationof at least about 5%, and preferably at most about 80%. In some embodiments, the organic component of the mixture has a concentration of at most about 50%. In some embodiments, the formulation has a pH of about 3.0 to about 8.0, and preferably of about 4.0 to about 5.0.Terms

[0028] As used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, references to "the method" includes one or more methods, and / or steps of the type described herein which will become apparent to those persons skilled in the art upon reading this disclosure and so forth. "About" as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±30%, or ±20%, or ±10%, or ±5%, or even ±1% from the specified value, as such variations are appropriate for the disclosed compositions or to perform the disclosed methods.

[0029] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which this invention belongs.

[0030] Definition of standard chemistry terms may be found in reference works, including Carey and Sundberg "ADVANCED ORGANIC CHEMISTRY 4TH ED." Vols. A (2000) and B (2001), Plenum Press, New York. Unless otherwise indicated, conventional methods of mass spectroscopy, NMR, HPLC, protein chemistry, biochemistry, recombinant DNA techniques and pharmacology, within the skill of the art are employed. Unless specific definitions are provided, the nomenclature employed in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those known in the art. Standard techniques can be used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients. Reactions and purification techniques can be performed e.g., using kits of manufacturer's specifications or as commonly accomplished in the art or as described herein. The foregoing techniques and procedures can be generally performed of methods known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification.

[0031] As used herein, the terms “permeation” or “absorption,” unless specified otherwise, mean “penetration” of the active compound of a medicament through a mucosa. The terms “permeation” and “absorption” may be used interchangeably.

[0032] As used herein, the terms “mucosal” or “transmucosal” or “across a mucosal membrane,” unless specified otherwise, mean any route of administration via a mucosal membrane. Examples include, but are not limited to, sublingual, nasal, vaginal and rectal administration of a medicament or an active compound of a medicament.

[0033] As used herein, the term “subject” means animal and human.

[0034] The term “environment” or “environment of an administration” means an environment where an active compound of a medicament is absorbed by permeation across the mucosa. For example, when the administration is performed sublingually, the environment is saliva, which contains the drug and is “bathing” the sublingual mucosal membrane.

[0035] The term “loop diuretic” as used herein has its usual meaning, and refers to a therapeutic molecule whose administration to a mammalian and / or human subject causes diuresis. In some embodiments, the loop diuretic inhibits the reabsorption by the kidneys of an at least 1, 2, 3, 4 or 5 electrolyte(s). Non-limiting examples of electrolytes include sodium, potassium, chloride, calcium, and magnesium. In some embodiments, the loop diuretic is capable of inhibiting the sodium-potassium-chloride (Na+ / K+ / 2C1) co-transporter in the kidneys. In some embodiments, the loop diuretic has use in the treatment of edema such as due to heart failure, liver disease and kidney disease. In some embodiments, the loop diuretic may also be used to treat high blood pressure. In some embodiments, the loop diuretic has use against a disease or disorder in a subject having impaired kidney function. Non-limiting examples of loop diuretics include bumetanide, furosemide, ethacrynic acid, and torsemide.

[0036] The term “glycofurol” as used herein has its usual meaning, and refers to an ethoxylate of tetrahydrofurfuryl alcohol. In some embodiments, it comprises the structure of Formula (I):

[0037] In some embodiments, n is at least 1, 2, 3, or 4. Non-limiting example synonyms for glycofurol include: tetrahydrofurfuryl polyethylene glycol, 2,2,3,3-tetramethyl-l,3-dioxolane, tetrahydrofurfuryl alcohol polyethylene glycol ether, 2-(oxolan-2- ylmethoxy)ethanol, 2-((tetrahydrofurfuryl)oxy)ethanol, 2-((tetrahydrofuran-2- yl)methoxy)ethanol, tetraglycol, THFP, tetraglyco, glycofural, glycofurol75, glycofurolum 75, and glycofurol tetraglycol.

[0038] The method of embodiments which provides an environment with a certain pH includes providing the environment with a preferable pH during the administration of the medicament and making a suitable formulation of the medicament in such a way that the medicament itself can provide the environment with a desired pH. In some embodiments, the latter is preferred. In this case, buffering agents are preferably involved in the formulation.

[0039] The embodiment described herein can include calculating an estimated range of bumetanide quantity bumetanide that needs to be solubilized and then permeated or absorbed across the mucosal membrane to achieve a therapeutic effective concentration.

[0040] The embodiments described herein can include various formulations or compositions dependent on the dosage forms or routes of administration. For example, if a formulation or composition comprising a medicament is administered sublingually, it can be in the form of tablets, pills, pellets, powders, liquid, or sprays. Examples of other suitable formulations or compositions include but are not limited to, ointments, capsules, solutions, syrups, drops, granules, and suppositories. In any formulation or composition, the medicament can include a therapeutically effective amount of an active compound or a pharmaceutically acceptable form. As another example, if a formulation or composition comprising a medicament is administered intranasally, the formulation or composition can be in liquid form. Suitable liquid forms for intranasal administration are nasal sprays and nasal drops, for example.

[0041] In some embodiments, bumetanide is administered sublingually. Any method of making tablets, pills, pellets, powders, liquid, or sprays for sublingual administration can be used. To make tablets, granulated powder is pressed into a small tablet, for example. The tablet can disintegrate when mixed with saliva, resulting in solubilization and absorption of the drug. To obtain a desired pH range for permeation and / or absorption of the drug, a tablet formulation is made taking into account mixing with saliva, for example.

[0042] Alcohol powder can be used to make tablets for sublingual administration. As another example, polyethylene glycol (PEG) can be used to make tablets for sublingualadministration. Both alcohol powder and PEG are miscible with water. Exemplary liquid PEGs that can be used include but are not limited to, PEG200, PEG400, and PEG600. Exemplary waxy or solid PEGs that can be used include but are not limited to PEGs with an average molecular weight of greater than about 600 g / mol (PEG600), such as PEG3000, PEG3350, PEG4000, PEG6000, and PEG8000.

[0043] In some embodiments, bumetanide is administered intranasally. For intranasal administration, a formulation or composition can be in any of the forms described above, including a nasal spray or liquid drops, for example. A special device can be used for intranasal or sublingual administration of a set volume. Exemplary volumes for such devices can be in the range of about 10 pL to about 1.6 mL, which can be delivered to each of the two nostrils. Non-limiting exemplary volumes can be in the range of about 25 pL to about 1.0 mL, about 50 pL to about 800 pL, about 75 pL to about 600 pL, about 100 pl to about 500 pL, or about 200 pL to about 300 pL, per nostril for at least one nostril. Devices for intranasal administration are commercially available from Aptar, for example.

[0044] Intranasal (IN) drug administration, e.g. via nasal spray, is a convenient route of administration. This route of administration can achieve the following advantages relative to oral drug administration: (a) produce a faster effect, (b) the smaller amount of drug exposure to achieve the equal effect, and (c) administering without the need for water for swallowing. These advantages of IN administration is possible because of the leaky epithelium lining the nasal mucosa (as compared to intestinal epithelium), extensive vascular supply, relatively large surface area (about 9.6 m2including microvilli), and avoidance of first-pass metabolism. The relatively large surface area for drug absorption via IN route is also an advantage over the sublingual route. Although the sublingual route can also provide a rapid onset of effect, its much smaller surface area (about 26 cm2) is a limitation for drug absorption, especially if a large dose is needed.

[0045] To achieve good permeation and / or absorption at the sublingual or nasal sites, a low molecular weight (for example, of at most about 1 kD) is preferable, with a good membrane partition coefficient (a good log P), a good aqueous solubility, and a desirable pKa that could lead to ionization and favorable permeation at the physiologic pH of the nose. Since the physiological pH of the nose is about 6.4, a general recommendation is to keep the pH of the formulation between about pH 3.5 and about pH 8.0 to minimize nasal membrane irritation.In some embodiments, the recommended pH for bumetanide to achieve targeted solubility and optimal permeability is between about pH 4 and about pH 5.

[0046] Bumetanide has a molecular weight of 364.42, LogP 2.6, pKl of 3.6, and pK2 of 7.7 As an acidic compound, bumetanide’s aqueous solubility is pH dependent and reported to be less than 257 ug / ml. Thus bumetanide API solubility requires further improvement. Since a drug must be in the soluble form for rapid intranasal absorption to take place before exerting its therapeutic effect, achieving sufficient solubility and permeability are the fundamental steps for bumetanide IN formulation. To optimize and verify bumetanide IN formulation, determining its solubility and permeability experimentally at pH 3.5-8.0 will be preferred.Background on Bumetanide

[0047] When administered intranasally or sublingually, the thin nasal and sublingual membranes can provide more rapid absorption than oral administration. In addition, intranasal and sublingual routes of administration can bypass liver first metabolism and can yield greater bioavailability than oral administration. However, the aqueous solubility of bumetanide is pH dependent and is low at pH 4.0-5.0 (the lower end of pH suitable for nasal and sublingual delivery). This is a major obstacle for proper and convenient dosing in achieving rapid and sufficient absorption at these sites. To optimize mucosal permeation and / or absorption at these sites, a suitable solvent (such as an organic-aqueous mixture) that can improve solubility as well as achieve desired permeability at a pH range of about 3.5 to about 8.0, and preferably between about pH 4 and about pH 5, is to be targeted.

[0048] Bumetanide is a potent sulfamoylanthranilic acid derivative diuretic with a half-life of 1 to 1.5 hours, and 45% of its dose administered is excreted unchanged in the urine. Its molecular weight is 363.41, LogP2.6, pKl 3.6, pK2 7.7 but with its aqueous solubility of only about 257 ug / ml. Based upon these chemi cal / phy si cal properties, there exists a need to develop a new formulation and method to enhance solubilization and permeation in order to achieve rapid action in a convenient manner (such as via sublingual or intranasal administration) but also good tolerance to the sublingual and nasal membrane.

[0049] As bumetanide is an acidic compound, its solubility increases with increasing pH. Also, certain organic solvents can increase solubility relative to water.However, its permeability across mucosal membrane improves as pH decreases, as based on the well-known Henderson Hasselbatch equation. Since intravenous aqueous bumetanide solution (0.25 mg / ml at pH 7) has already been approved by FDA to be safe and effective for human use, suitable organic excipients will need to be added to enhance bumetanide solubility, but the pH must be adjusted to achieve effective plasma concentration rapidly to allow good permeation.

[0050] The effective concentration in the composition of the current invention is a bumetanide concentration range of about 2.5mg / ml to about 10 mg / mL

[0051] In some embodiments, the pH of the composition or formulation is between about 3.5 and about 8.0, preferably between about 4 and about 5.Formulation ReagentsAlcohols

[0052] In certain embodiments, the formulations and compositions for treating edema, increasing its solubility with optimal permeability of bumetanide at a given pH include at least one alcohol. An alcohol can have any number of carbon atoms in a chain. An alcohol can be a primary alcohol, a secondary alcohol, or a tertiary alcohol. Monohydric and polyhydric alcohols are known. Exemplary monohydric alcohols include methanol, ethanol, propanol, butanol, pentanol, hexanol, and others. Exemplary polyhydric alcohols include, for example, ethylene glycol, propylene glycol, glycerol (glycerin), glycofurol and others. In some embodiments, the alcohol is glycofurol. In some embodiments, glucofurol is present at a concentration that is at least about5% and at most about 80%. In some embodiments, glucofurol is present in the formulation at a concentration that is at most about 50%. In some embodiments, at least one of DMI, glycofurol, and / or NMP is present alone or in any combination in the formulation at a total percentage that is at least about 5% and at most about 80%, preferably wherein at least one of DMI, glycofurol, and / or NMP is present alone or in any combination in the formulation at a total percentage that is at most about 50%.

[0053] Some embodiments of the present disclosure relate to a formulation / composition comprising an ingredient list as “G# ” whereinis any number between about 1 and about 100. In this instance, “G” refers to glycofurol, and “#” refers to thepercentage of glycofurol present in the formulation. For example, a formulation comprising “G45” is a formulation comprising about 45% glycofurol.Polyethers

[0054] The herein-described formulations and compositions for treating edema, increasing its solubility with optimal permeability of bumetanide may, in certain embodiments, contain a polyether. Polyethers are polymers that contain more than one ether functional group. Polyethers can include, for example, dimethyl isosorbide (isosorbide dimethyl ether), polyethylene glycol (PEG), polyethylene oxide (PEG), polyoxyethylene (POE), polypropylene glycol (PPG), polytetramethylene glycol (PTMG), polytetramethylene ether glycol (PTMEG), and paraformaldehyde. Aromatic polyethers include, for example, polyphenyl ether (PPE) and poly(p-phenylene oxide) (PPO). In some embodiments, the polyether is polyethylene glycol (PEG). The molecular weight of polyethylene glycol (PEG) may range from about 300 g / mol to about 10,000,000 g / mol. In some embodiments, the polyether is isosorbide dimethyl ether or dimethyl isosorbide (DMI), In some embodiments, DMI is present in the formulation at a concentration that is at least about 5% and at most about 80%. In some embodiments, DMI is present in the formulation at a concentration that is at most about 50%. In some embodiments, at least one of DMI, glycofurol, and / or NMP is present alone or in any combination in the formulation at a total percentage that is at least about 5% and at most about 80%, preferably wherein at least one of DMI, glycofurol, and / or NMP is present alone or in any combination in the formulation at a total percentage that is at most about 50%.

[0055] Some embodiments of the present disclosure relate to a formulation / composition comprising an ingredient list as “D# ” whereinis any number between about 1 and about 100. In this instance, “D” refers to DMI, and “#” refers to the percentage of DMI present in the formulation. For example, a formulation comprising “D45” is a formulation comprising about 45% DMI.Glycerides

[0056] In certain embodiments, the formulations and compositions for treating edema, increasing the solubility with optimal bumetanide may include at least one or more glycerides. Glycerides are esters formed from glycerol and fatty acids. Exemplary glyceridesinclude mono-, di-, and triglycerides. In some embodiments, the formulations and compositions described herein contain medium-chain glycerides. In some embodiments, the formulations and compositions described herein contain polyglycolyzed C8-C10 glycerides. In some embodiments, the polyglycolyzed C8-C10 glyceride is a saturated polyglycolyzed C8- C10 glyceride. In some embodiments, the formulations and compositions described herein comprise a mixture of glycerides. Glycerides in a mixture can be unsaturated or saturated. In some embodiments, the mixture of glycerides comprises additional chemicals or compounds. In some embodiments, the glycerides comprise polyoxylglycerides. In some embodiments, the glycerides comprise capryl ocaproyl polyoxyl-8 glycerides or capryl ocaproyl macrogol-8 glycerides. In some embodiments, the glycerides comprise caprylic / capric glycerides. In some embodiments, caprylic / capric glycerides further comprise a polyethylene glycol, such as PEG- 8, for example. In some embodiments, the formulations and compositions described herein comprise LABRASOL.Pyrrolidone

[0057] In certain embodiments, the formulations and compositions for treating edema, increasing the solubility with optimal permeability of bumetanide may include at least one or more pyrrolidinone or pyrrolidone., a compound consisting of a 5-membered lactam. Exemplary pyrrolidones are N-methyl-2-pyrolidone (NMP) and 2-pyrrolidone. In some embodiments, NMP is present in the formulation at a concentration that is at least about 5% and at most about 80%. In some embodiments, NMP is present in the formulation at a concentration that is at most about 50%. In some other embodiments, the formulations described herein comprise of NMP in combination with one or more alcohol, a polyether, glyceride, or a combination thereof. In some embodiments, at least one of DMI, glycofurol, and / or NMP is present alone or in any combination in the formulation at a total percentage that is at least about 5% and at most about 80%, preferably wherein at least one of DMI, glycofurol, and / or NMP is present alone or in any combination in the formulation at a total percentage that is at most about 50%.

[0058] Some embodiments of the present disclosure relate to a formulation / composition comprising an ingredient list as “N# ” whereinis any number between about 1 and about 100. In this instance, “N” refers to NMP, and “#” refers to thepercentage of NMP present in the formulation. For example, a formulation comprising “N45” is a formulation comprising about 45% NMP.Buffering Agents

[0059] Buffering Agents that can be used in the embodiments described herein will be known to those skilled in the art. Please see “Handbooks Pharmaceutical Excipients (Second Edition), edited by Ainley Wade and Paul J W Weller, The Pharmaceutical Press London, 1994,” which is incorporated herein by reference. Exemplified buffering agents include, but are not limited to, phosphates, such as sodium phosphate; phosphates monobasic, such as sodium dihydrogen phosphate and potassium dihydrogen phosphate; phosphates dibasic, such as disodium hydrogen phosphate and dipotassium hydrogen phosphate; citrates, such as sodium citrate (anhydrous or dehydrate); bicarbonates, such as sodium bicarbonate and potassium bicarbonate. The amount of buffering agents used in the formulations and methods described herein is readily determined by those skilled in the art, which depends on preferable pH values. Certain embodiments contemplated herein feature a formulation or composition having a pH of about 4.0, about 4.1, about 4.2, about 4.3, about 4.4, about 4.5, about 4.6, about4.7, about 4.8, about 4.9, about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, and any number or range in between.Carriers

[0060] The carrier suitably used in the embodiments described herein depends on the specific formulation or composition of the medicament. The carriers include, without limitation, fillers, binders, lubricants, diluents, sweetening and flavoring agents, preservatives, disintegrators, grilling agents, and permeation enhancers. Examples of the carriers include starch, gelatin, natural sugars, corn, natural and synthetic gums such as acacia, sodium alginate, methylcellulose, carboxymethylcellulose, polyethylene glycol, waxes, boric acid, sodium benzoate, sodium acetate, sodium chloride, agar, bentonite, agar gum, stearates such as sodium stearate, HPMC, palmitic acid, dimethyl sulfoxide, N,N-dimethyl acetamide, N,N- dimethylformamide, 2-pyrrolidone, l-methyl-2-pyrrolidone, l,5-dimethyl-2-pyrrolidone, 1- ethyl-2-pyrrolidone, 2-pyrrolidone-5-carboxylic acid, N,N-dimethyl-m-toluamide, urea, ethylacetate, 1 -dodecyl azacy cl oh eptan-2-one (Azone®), oleic acid, ethylene vinylacetate copolymer, polyvincyl chloride, polyethylene, polydiethyl phthalate.Exemplary Formulations for Enhancing Permeation of bumetanide Across a Mucosal Membrane

[0061] Described herein, in some embodiments, are formulations for enhancing the solubility of bumetanide across a mucosal membrane, comprising: (a) bumetanide; and (b) an organic-aqueous solvent comprising an alcohol, a glycol, diethylene glycol monoethyl ether, a medium chain glyceride, one or more saturated polyglycolyzed C8-C10 glyceride, glycofurol, NMP, DMI or a combination thereof; wherein the formulation has a pH of about 3.5 to about 8.0 or any value that is between about 3.5 and about 8.0, preferably about 4.0 to about 5.0, and wherein the organic-aqueous solvent enhances the solubility of bumetanide relative to the solubility of bumetanide in water. In some embodiments, the organic-aqueous solvent comprises an alcohol. In some embodiments, the alcohol is ethanol, glycerol, glycofurol, or any combination thereof. In some embodiments, the alcohol is present at a concentration that is at least about 5%, and preferably less than about 50% concentration. In some embodiments, the organic-aqueous solvent comprises a poly ether. In some embodiments, the poly ether is polyethylene glycol. In some embodiments, the polyethylene glycol is PEG 6000 or PEG 400. In some embodiments, the polyethylene glycol is present at a concentration that is at least about 5%, and preferably less than about 80%. In some embodiments, the polyethylene glycol is present at a concentration less than about 50%. In some embodiments, the polyether is DMI at a concentration that is at least about 5%, and preferably less than about 50% with a pH that is any value from about 3.5 to about 8.0, and preferably wherein the pH is at any value from about 4.0 to about 5.0. In some embodiments, the pyrrolidone is present at a concentration that is at least about 5%, and preferably less than about 80%. In some embodiments, the pyrrolidone is present at a concentration that is less than about 50%. In some embodiments, the pyrrolidone is NMP at a concentration that is at least about 5%, and preferably less than about 50% with a pH that is any value from about 3.5 to about 8.0, and preferably wherein the pH is at any value from about 4.0 to about 5.0.Exemplary Methods for Treating Edema

[0062] Described herein, in some embodiments, are methods of treating the edema of a subject in need thereof, comprising contacting a mucosal membrane of the subject with a formulation disclosed herein, thereby treating the edema of the subject. In some embodiments, contacting the mucosal membrane comprises intranasal administration. In some embodiments, contacting the mucosal membrane comprises sublingual administration.Exemplary Methods of Preparing Formulations for Treating Edema

[0063] Described herein, in some embodiments, are methods of preparing a formulation for treating the edema of a subject, comprising: (a) an appropriate amount (concentration) of bumetanide in an organic-aqueous solvent comprising an alcohol, a polyether, pyrrolidone, or a combination thereof; (b) adjusting the pH of the organic-aqueous solvent to any value that is between about 3.5 and about 8.0, preferably between about 4.0 and about 5.0; wherein treating the edema comprises contacting a mucosal membrane of the subject with the formulation. In some embodiments, the solubility of bumetanide is increased in the organic-aqueous solution relative to the solubility of bumetanide in water. In some embodiments, the permeability of bumetanide across the mucosal membrane is increased in the organic-aqueous solvent at low pH (e.g. about pH 4 to about pH 5) relative to the permeability of bumetanide in water at higher pH (e g. about pH 7 with good aqueous solubility). In some embodiments, the bioavailability of bumetanide is increased in the organic-aqueous solvent relative to that in water. In some embodiments, the organic-aqueous solvent comprises an alcohol. In some embodiments, the alcohol is glycerol or glycofurol. In some embodiments, the glycofurol is present at a concentration that is at least about 5%, and preferably less than about 50%. In some embodiments, the organic-aqueous solvent comprises a polyether. In some embodiments, the polyether is polyethylene glycol. In some embodiments, the polyethylene glycol is PEG 6000 or PEG 400. In some embodiments, the polyethylene glycol is present at a concentration that is at least about 5%, and preferably less than about 50% . In some embodiments, the polyether is DMI present at a concentration that is at least about 5%, and preferably less than about 50%. In some embodiments, the formulation has a pH of about any value that is between about 3.5 and about 8.0, preferably between about 4.0 and about 5.0. In some embodiments, the organic-aqueous solventcomprises pyrrolidone. In some embodiments, the pyrrolidone is NMP. In some embodiments, the NMP is present at a concentration that is at least about 5%, and preferably less than about 50%. In some embodiments, the formulation comprises any 2 or all 3 of DMI, NMP, and glycofurol. In some embodiments, the any two or three of DMI, NMP, and / or glycofurol are present in the formulation at a total concentration that is at least about 5%, and preferably less than about 80% of the total formulation. In some embodiments, the any two or three of DMI, NMP, and / or glycofurol are present in the formulation at a total concentration that is at most about 50% of the total formulation.

[0064] In some embodiments, the formulations described herein comprise an organic-aqueous solvent comprising more than one organic component. Exemplary organic solvent or component mixtures include, for example, glycofurol, NMP, and / or DMI in water. In some embodiments, at least 1, 2, or 3 of glycofurol, DMI and / or NMP is each independently present in the aqueous organic solvent mixture at a concentration that is at least about 5%, and preferably less than about 80%. In some embodiments, at least 1, 2, or 3 of: glycofurol, DMI and / or NMP is each independently present in the aqueous organic solvent mixture at a concentration that is less than about 50%. In some embodiments, the formulation comprises water. In some embodiments, water is present in the formulation at a concentration that is at least about 20%, and at most about 95% of the total formulation. In some embodiments, water is present in the formulation at a concentration that is at least about 50% of the total formulation. In some embodiments, the formulation further comprises at least one other solvent. In some embodiments, the at least one other solvent is present in the formulation at a concentration that is at least about 5%, and preferably less than about 80%. In some embodiments, the at least one other solvent is present in the formulation at a concentration that is less than about 50%. In some embodiments, the formulation comprising the at least one other solvent is at a pH that is any value between about 3.5 and about 8.0, preferably between about 4.0 and about 5.0. Formulations comprising more than one organic solvent or component in water can be used in any of the methods described herein.

[0065] In some embodiments, wherein the organic-aqueous solvent comprises more than one organic solvent or component, the second organic component is chosen for the purpose of enhancing at least one of property selected from the group solubility, stability,permeability, and safety. In some embodiments, the formulation has a pH of about 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, or any value that is between about 3.5 and about 8.0.

[0066] Although a patent application “Methods and compositions for treating edema refractory to oral diuretics” (Pub.NO.: US2021 / 0169833 Al; Appl. No.: 17 / 112, 899) by Radhakrishnan et al. has been filed on Dec 4, 2020, for intranasal and sublingual bumetanide, there are several important differences between the existing patent application and the current application. The existing application described the development of new formulations with a specific proposal to use KOH, arginine or Lysine, together with the buffering agent to increase the solubility of bumetanide by “maintaining a high solubility of the drug at a constant pH range, preferably pH 6-8”, and such formulation may include additional “permeation enhancers” composed of various organic compounds. The present patent described a method of new formulation development to use NaOH / HCl (same as that used in the injectable bumetanide formulation approved by FDA) to adjust and maintain pH range in combination with certain water-cosolvents to improve bumetanide solubility as well as to optimize permeability at pH 4-5 rather than at higher pH, such as pH 6-8. Thus the present application differs from the existing application in the composition and pH of the formulation solution. In addition, the so-called “permeation enhancers” proposed by Radhakrishnan et al in the existing patent have never been verified to enhance permeation at any given pH relative to an aqueous bumetanide solution. In fact, adding such / similar organic compounds to an aqueous bumetanide solution decreases bumetanide permeability at any given pH relative to aqueous bumetanide alone (FIGs. 3A-3C). Thus in comparison to the existing patented formulation at pH 6-8, the present formulation consisting of a sufficiently soluble bumetanide solution in well-established water-cosolvent mixtures maintained at about pH 4 to about pH 5 (corresponding to the lower tolerable pH of the nasal and sublingual membrane) using NaOH / HCl is expected to be safe as well as result in better permeability, based on the well- known Henderson-Hasselbalch equation and experimentally verified data in the present patent application.EXAMPLES

[0067] Additional embodiments are disclosed in further detail in the following examples, which are not in any way intended to limit the scope of the claims.EXAMPLE 1Intranasal Dosing and Formulation

[0068] Embodiments of an appropriate IN bumetanide dosing are required to achieve a therapeutic effect equivalent to an approved oral effective dose described herein. The estimation assumes that sufficient bumetanide solubility and permeability can lead to the desired bioavailability from equivalent IN and oral doses.

[0069] The desired dosing further requires an IN formulation of bumetanide API to provide an amount of bumetanide that will achieve a similar but significantly earlier effective concentration as that from the oral route. For example, if a Img oral dose is used, an IN dosing should lead to similar bioavailability but a much earlier peak time (Tmax) than that from the oral route. Since IN dose is administered either via a nasal spray or nose drop, the amount of IN dose can be estimated from the volume of bumetanide formulation solution administered intranasally times its solution concentration after adjustment for relative bioavailability. An example of the calculation is provided below.

[0070] As IN administration normally uses a volume of about 50 to about 200 uL per spray to each nostril (a spray volume larger than about 200 uL per nostril will most likely result in some volume dripping out of the nose). Thus, in some embodiments, the spray volume can be set at most about 200 uL per spray; for example at about 150uL per nostril or at 2 sprays of 150 uL for 2 nostrils. In some embodiments, the IN dose is equivalent to an about 1 mg oral dose.

[0071] Assuming absorption of bumetanide aqueous solution via IN administration can be estimated from the oral dose times bioavailability, which is about 80 to about 90%. Thus, its intranasal equivalent dose is expected to be about 0.9 times of the oral dose (similar to intravenous dose) but with a much earlier peak concentration. This is based on the known advantage of IN administration which can avoid first-pass liver metabolism and achieving rapid permeation via a nasal membrane. Thus, to administer an equivalent 1 mg oral dose will require about 0.5 mg per about 150 uL per nostril (or about l.Omg per about 300 uL for both nostrils delivery, corresponding to a soluble concentration of about 3.33 mg / mL bumetanide for the IN formulation. Using this concentration and dosing with 1 spray of about 150 uL per nostril, the total administered IN dose is about 1 mg, which corresponds to about 1.1 mg of an oral dose. A special device can be used for intranasal administration and can be delivered toeach of the two nostrils. Devices for intranasal administration are commercially available from Aptar, for example.EXAMPLE 2Screening for water-cosolvent mixtures to produce targeted formulations with desired solubility and optimal permeability

[0072] After an initial review of the physical and chemical properties of various alcohols, polyethers, glycerides, and pyrrolidones as well as after preliminary screening, 3 specific organic solvents (glycofurol, NMP, and DMI) were chosen to be cosolvents for the targeted formulations aimed to achieve bumetanide solubility of at least about 3.33 mg / ml and permeability greater than that observed for aqueous formulation at about pH 7.0. The following experimental approaches were carried out for confirming these suitable formulations: A. Achieving desired saturated solubility, B. Optimizing permeability, C. Designing targeted formulation, D. Confirming desired permeability using Calu-3 cell line study.Achieving desired saturated solubility - Materials1) Bumetanide Hydrochloride (bumetanide) (CAS No. 224785-91-5) was purchased from India Alembic Pharmaceutical Ltd, Gujarat-391450 India (Lot #1704002361).2) Furosemide (FUR) was purchased from Sigma-Aldrich (St. Louis, MO, USA).3) Acetonitrile (ACN) >99.5% ACS (CAS No. 75-05-8) was purchased from VWR Chemicals BDH®.4) Methanol (MeOH) was purchased from VWR Chemicals BDH®.5) Ethanol (EtOH) 190-Proof (CAS No. 64-17-5) was purchased from EMD Millipore (Burlington, MA, USA).6) Syringe Filter w / 0.2 um pore size Cellulose Acetate Membrane (Cat# 28145- 475) was purchased from VWR (Radnor, PA, USA).7) l-Methyl-2-Pyrrolidinone (NMP) (Lot 51K3683) was purchased from Sigma- Aldrich (St. Louis, MO, USA).8) Dimethyl isosorbide (DMI) (Cat# 906832; Batch# 0000173267) was purchased from Sigma-Aldrich (St. Louis, MO, USA).9) Tetrahydrofurfuryl alcohol polyethylene glycol ether (Glycofurol, Gly) (Cat# 8.21092.0250, Lot# S7906492040) was purchased from Sigma-Aldrich (St. Louis, MO, USA).10) Hydrochloric Acid (HC1) (Cat# 320331; Lot# SHBG2435V) was purchased from Sigma-Aldrich (St. Louis, MO, USA).11) Phosphate buffered saline (PBS, tablet) (Cat# P4417; Lot# SLCD5938) was purchased from Sigma-Aldrich (St. Louis, MO, USA).Equipment1) Accumet Basic pH meter was purchased from Fisher Scientific (Leicestershire, UK).2) Agilent 1260 Infinity HPLC system which consisted of a G1311B 1269 Quat Pump, a G7129 1260 vial sampler, and a G1315D 1260 DAD VAL detector was purchased from Agilent (Santa Clara, CA).3) Analytical Balance was purchased from Mettler-Toledo, LLC (Columbus, OH).Procedure

[0073] The solubility of bumetanide API in different cosolvent mixtures consisting of water and at least 1, 2, or 3 of: glycofurol, NMP, and DMI, was investigated and compared to solubility in pure water. Initially saturated bumetanide concentration in different percent aqueous-glycofurol or NMP or DMI solutions at various pH were determined (See Figs. 1 A- 3C). Based on these initial results, bumetanide concentrations in various pH and different fractions of any 1 or 2 of: glycofurol, NMP, and DMI were predicted (see Methods and Procedure of Predicting / Estimating Solubility below), Afterwards, certain selected solutions were confirmed and measured using an HPLC method (Table 1 below).Table 1: The measured and predicted solubility of potential targeted formulations compared to water reference

[0074] To prepare for the solubility measurement of these solutions, the pH of the measured solutions was adjusted using NaOH / HCl (use of NaOH is consistent with the intravenous bumetanide solutions approved by FDA). As disclosed herein, the measured solutions were prepared by the “shake-flask” method over 3 days to determine saturated solubility.

[0075] Briefly, increasing amounts of bumetanide API were added to different water-cosolvent mixtures until saturation. The saturated mixtures were adjusted with NaOH at different pH ( within the range of about pH 3.5 to about pH 8.0) with the use of a pH meter. The saturated solution was shaken slowly with a magnetic stirrer at room temperature or shaken rapidly several times a day for 24 hours or longer, up to 3 days. Afterward, the solutions were filtered using the VWR 0.2 um filter. The filtrate was then inspected for clarity and the solution concentration of bumetanide was determined by HPLC.HPLC method for bumetanide concentration determination

[0076] For concentration determination of the solubility as well as permeability studies, the samples were prepared for HPLC analysis by mixing 1 : 1 with about 5% to about 50% ACN similar to the published method. The standard curves were prepared in about 5% to about 50% ACN and mixed with PBS so that the analytical matrices were the same. The HPLC analysis was run through a Waters Spherisorb® 5pm ODS2, 4.6 x 250mm column (PN PSS831915, SN 017231083140 73) using Acetonitrile: 50 mM Potassium Phosphate Monobasic Buffer (46:54 v / v) isocratic flow at 1 mL / min for 10 minutes.Results and discussion

[0077] The validity of the assay was assessed according to FDA guidance with regards to linearity, sensitivity, repeatability, stability, precision, and accuracy. The calibration curve of bumetanide was linear over the concentration range of 0.2-200ug / ml. The correlation coefficient (R2) was greater than 0.99 for each of the 3 different runs. For quality control samples of 0.5, 10, and 200ug / ml, the relative standard deviation (RSD) values for precision were 1.8 to 6.1% (interday) and 0.07 to 4.1% (intraday). The accuracy (% bias) ranged from -4.2% to 2.2% (interday) and -0.9 to 3.4% (intraday). The lower limit of quantitation was 0.2ug / ml.

[0078] The active pharmaceutical ingredient, bumetanide, is an acidic drug, and its saturated solubility is dependent on pH. The saturated aqueous or water solubility of the active pharmaceutical ingredient (or the “API” of bumetanide) versus pH is shown in FIG. 1A. It should be noted that such saturated aqueous or water solubility of the active pharmaceutical ingredient versus pH if adjusted using KOH / buffering agent would show a lower bumetanide solubility at about < pH 6, but better solubility at about > pH 6 when compared to solutions using NaOH / HCl for pH adjustment (data on file not shown in FIG. 1A). Since bumetanide aqueous solubility at pH 7-8 (using NaOH / HCl adjustment) has already sufficient solubility of > 3.33mg / ml (see Table 1), the use of KOH to further enhance its solubility at pH 7-8 is no longer necessary. The use of NaOH / HCl to adjust bumetanide solution has already been approved by FDA and is safe.

[0079] The bumetanide solubility versus pH in different volume percentages of glycofurol, NMP, and / or DMI solutions relative to aqueous solubility is shown in FIGs. 2A- 2C. At any given pH, the solubility of bumetanide in these water-cosolvent mixtures is improved over that in aqueous solution.Optimizing permeability using PAMPA

[0080] The permeability of bumetanide was performed using a parallel artificial membrane permeability assay (PAMPA).

[0081] The permeability of bumetanide API in different solvents at room temperature and atmospheric pressure was screened using in vitro PAMPA. The PAMPA predicts the passive absorption of drugs and is suitable for studies with various solvents. Theunit of measurement is the apparent permeability (Papp) obtained at a steady state, expressed as cm / sec. Also, another associated measurement is the maximum flux (Jss) at a particular pH, expressed as the quantity of drug across the unit area per sec, which is calculated from the Papp and saturated solubility.Materials and Equipment1) Transport Receiver Plate (Cat# MATRNPS50) and Multi Screen-IP Filter Plate (Cat# MAIPN4550) were purchased from Millipore (Burlington, MA, USA).2) Bumetanide Hydrochloride (CAS No. 224785-91-5); India Alembic Pharmaceutical Ltd, Gujarat-391450 India (Lot #1704002361).3) Ethanol 190-Proof (CAS No. 64-17-5) was purchased from EMD Millipore (Burlington, MA, USA).4) Acetonitrile (Cat# BDH83639.400) was purchased from BDH Chemicals (Radnor, PA, USA).5) Dodecane (Cat# D221104), Sodium Phosphate monobasic (Cat# S0751), Sodium Phosphate dibasic (Cat# S0876) and Polyethylene Glycol 6000 (Cat# 8.07491) were purchased from Sigma-Aldrich (St. Louis, MO, USA).6) Lecithin, Refined Solid (Cat# 36486) was purchased from Alfa Aesar (Haverhill, MA, USA).7) Syringe Filter w / 0.2 um Cellulose Acetate Membrane (Cat# 28145-475) was purchased from VWR (Radnor, PA, USA).8) Polyethylene glycol (PEG) 400 (Lot 52081314) was purchased from EMD Millipore (Burlington, MA, USA).Procedure(a) Solution Preparation

[0082] Saturated solutions of bumetanide (3-5 mL) in different solvents were prepared by using an increasing amount of bumetanide and adjusted to the desired pH (range 3.5-8 with the use of a pH meter), as described in Example above. These saturated solutions were prepared and then filtered using a 0.2 um filter. The filtrates were used for permeation studies.(b) Permeation Studies using PAMPA

[0083] Bumetanide permeation studies were performed using the Parallel Artificial Membrane Permeation Assay (PAMPA) with the receiver plate and multi screen-IP filter plates. The PAMPA assay predicts passive absorption of drugs and is suitable for studies with a wide variety of solvents although its effect on DMI is not known. The permeation assay was carried out in triplicate or more and for a duration of 6 h. (For some solutions both 6 h and 24 h duration were carried out which showed that 6 h duration is sufficient to detect the concentration of the receiving chamber and can be sufficient for comparing different solutions) The donor chamber was initially coated with 5 ul of 3% (w / v) lecithin in dodecane before transferring 150 uL of desired sample into donor chamber. Then 300 pL of phosphate-buffered saline was transferred into the acceptor wells. After 6 hours of permeation, a sample was collected from the donor and receiver chamber for concentration analysis using HPLC as per the description above.

[0084] Apparent permeability coefficient (Papp) determination, expressed as cm / sec, was calculated based on the following equation at steady state:VA = volume in the acceptor chamber t = duration of the permeability studyCA = drug concentration in the acceptor well at the end of the studyCE = equilibrium concentration in both wells, and

[0085] Steady-state flux (Jss). The Jss, expressed as ug / sec / cm2, was calculated based on the following equation:Jss Papp CD, whereCD = loading concentration in the donor chamber.Results and discussion

[0086] While the saturated solubility of all bumetanide solutions increases with pH as per adjustment using NaOH / HCl (FIGs. 1A and 2A-2C), their permeability decreases with increasing pH (FIGs. IB, and 3A-3C). These data further indicate that adding organiccosolvents to water generally decreases bumetanide Papp at a given pH when compared to that in an aqueous solution. However at lower pH, these organic cosolvents can in some embodiments improve bumetanide Papp.

[0087] Although the value of Papp for the bumetanide solutionversus pH can shift to a higher value when adjusting the solution using KOH / HC1, however, the Papp was better at a pH that was at least about pH 6 but lower at a pH that was higher than about pH 6 when compared to that from the solution adjusted by NaOH / HCl (data on fde, but not shown in FIG. IB). Despite the advantage of improved bumetanide Papp from KOH / HCl-adjusted solutions, its bumetanide solubility is too low at a pH less than about pH 6 to be used as an effective intranasal or sublingual formulation for the treatment of edema. On the other hand, at about pH 6.8 (solutions adjusted by KOH / HC1) , bumetanide’ s solubility is sufficient. However, bumetanide in KOH / HC1 has less permeability than in a solution adjusted by NaOH / HCl. Given that, in some embodiments, the solution is pH-adjusted using NaOH / HCl. Bumetanide flux in aqueous solution was also plotted as a function of change in pH; a poor trend was observed (FIG. 1C).

[0088] The solutions containing DMI at concentrations of at least about 15% (data not shown) resulted in many -fold higher Papp values compared to that in water. A higher DMI concentration (that is, at least about 30%) appeared to interfere with PAMPA and such results were considered not reliable and thus not included in the PAMPA study data. DMI concentrations of at least about 15% are not expected to interfere with the Calu-3 study (see below).Designing targeted formulation

[0089] Based on the aqueous bumetanide solubility versus pH (FIG. 1A) and a required solubility >3.33 mg / mL) for dosing (see Example 1, IN dosing and formulation section, 4th paragraph), this concentration can be achieved at about pH 7 (FIG. 1A and Table 1) At this pH, the mean Papp value for the bumetanide in aqueous solution is about 1.59E- 07cm / s (FIG. IB and Table 2). This Papp value and the corresponding flux ( 1.59E-07cm / s x 3.33 mg / mL) of 0.53 ug / s / cm2. are considered as minimum reference Papp and flux. Thus, for any water-cosolvent mixture that can achieve a solubility of at least about 3.33 mg / mL but with a PAMPA permeability of at least about 1.59E-07cm / s can be desirable and therefore canbe targeted. (PAMPA permeability is known to be useful for predicting the absorption and bioavailability of the majority of drugs with a passive diffusion mechanism).

[0090] Based on the Papp versus pH profile, a lower pH (i.e. < pH 7), can achieve improved permeability (FIG. IB). However, at < pH 7 the pure aqueous solution will not provide sufficient solubility (such as 3.33 mg / mL) required for effective IN dosing. Given that, any water-cosolvent-mixture (containing different percentages of organic compounds that are safe) capable of achieving solubility of at least about 3.33 mg / mL (e.g. at a lower pH, i.e. at about pH 7) as well as Papp that is at about 1.59E-07cm / s is, in some embodiments, targeted as the desired formulation.

[0091] To design the targeted formulation using various water-cosolvent mixtures (specific organic-aqueous mixtures with organic components, preferably at most comprising a less than about 80% organic fraction, more preferably at most comprising a less than about 50% organic fraction), the following approach of predicting / estimating their solubility (see below) in combination with determining their corresponding permeability were carried out (to achieve the desired solubility and Papp). Subsequently, the solubility and permeability of certain selected and potential targeted formulations were verified experimentally.Methods and Procedure of Predicting / Estimating Solubility

[0092] Since bumetanide is an acid, its Papp improves with decreasing pH. However, at a value of at most pH 3.5, the acidic solution is likely to be intolerable to the human nasal membrane. Thus in consideration of tolerance and optimixing Papp, the pH of the solution was adjusted to be at about pH 4-5. In some embodiments, the targeted formulation for bumetanide can be those water-cosolvent mixtures that can achieve a solubility at least about 3.33 mg / mL with a Papp that is at least about 1.59E-07cm / s at a preferable pH range of 4-5. To design and identify such formulation, the solubility was first predicted / estimated for various combinations (e.g. different volume fractions of organic components) at pH 4-5 using primarily the initial solubility data (see FIGs. 1 A-3C). Afterwards certain selected formulations and especially those predicted to achieve a concentration of > 3.33mg / mL would proceed to have their corresponding Papp determined, so as to identify those formulations with desired targeted values: solubility of at least about 3.33 mg / mL, and a Papp of at least about 1.59E-07 cm / s (i.e. above that of aqueous formulation at pH 7.0).a. Solubility Predictions

[0093] Equation 1 was used for the prediction / estimation of solubility of the watercosolvent mixture at any given pH, based on the equation:

[0094] logSm— logSw+ S(crC) Eq. 1

[0095] Where SM is the bumetanide solubility in the mixture, Sw is solubility in water, f is the cosolvent volume fraction, o is solubility power or the slope for each organic cosolvent, and £ is the summation of cosolvent compounds if cosolvent consists of more than one organic compound.

[0096] Plotting log (Sm / Sw) versus volume fraction should yield a straight line with a as the slope of the line when a single organic cosolvent compound with multiple volume fractions are used (for example, glycofurol at 15, 25, 35, or 45%, NMP at 10, 20, 30, 35, or 45%, or DMI at 10, 20, 30, or 45%, ). Based on preliminary data from plots at each pH (see FIGs. 1 A-3C), Eq. 1 was further used to predict / estimate Smconsisting of two or more organic mixtures (e.g. glycofurol at about 15% and NMP at about 20%, glycofural at about 20% and DMI at about 15%, NMP at about 20% and DMI at about 15%, etc.) at a given pH. The predicted solubility values of certain selected and potential targeted mixtures were then further measured and verified experimentally (See Determination of saturated solubility and Table 1).Results and discussion

[0097] The predicted versus measured Sm, as well as the bumetanide solubility of certain selected and potential targeted formulations are shown in Table 1 and FIG. 4. The corresponding permeability values of these solutions are as shown in Table 2:Table 2: Measured PAMPA Papp of certain potential targeted formulations compared to water reference.

[0098] These in vitro results indicate that while the prediction (see FIG. 4 ) under predicted the concentration of the measured bumetanide values, a number of specific mixtures(formulations) that had solubility >3.33 mg / ml and permeability > than that in aqueous solution can be potential suitable formulations, if their permeability can be further confirmed using Calu-3 fell line permeability study.Confirmation of the targeted formulations using Calu-3 cell line study

[0099] The Papp of soluble drugs determined by the Calu-3 cell line model has been shown to be relevant to in vivo nasal permeation results. As disclosed herein, the Calu-3 cell line model was utilized to confirm the validity of the targeted formulation in achievingequivalent or better Papp when compared to the reference water solution at pH 7. Several formulations initially estimated by PAMPA to be optimal formulations were tested using the Calu-3 method as described below.Materials1) Calu-3 cells were originally purchased from ATCC.2) Glacial acetic acid (>99% pure, CAS 64-19-7) was purchased from Alfa Aesar (Haverhill MA, USA).3) Acetonitrile (ACN) U99.5% ACS (CAS No. BDH83659400) was purchased from VWR Chemicals BDH®.4) Sodium Phosphate Monobasic Monohydrate (Phosphate Buffer ACS grade #BDH9268) was purchased from BDH Chemicals (Radnor, PA, USA).5) Sodium Hydroxide (NaOH) was purchased from Biobasic Canada Inc. (Markham, Ontario, Canada).6) Ethyl alcohol (EtOH), Pure 200-proof (Cat# 459844; Lot# SHBL6038).7) Bumetanide (Cat# B3023; Lot# BCBW8646).8) Tetrahydrofurfuryl alcohol polyethylene glycol ether (glycofurol, gly) (Cat# 8.21092.0250, Lot# S7906492 040)9) l-Methyl-2-Pyrrolidinone (NMP) (Cat# M-6762, Lot# 51K3683)10) Dimethyl isosorbide (DMI) Cat# 906832; Batch# 0000173267) was purchased from Sigma-Aldrich (St. Louis, MO, USA).11) Hydrochloric Acid (HC1) (Cat# 320331; Lot# SHBG2435V).12) Phosphate buffered saline (PBS, tablet) (Cat# P4417; Lot# SLCD5938) was purchased from Sigma-Aldrich (St. Louis, MO, USA).13) 8 mm Autosampler Vial Screw Thread Caps (HPLC Vial Caps) (Cat# C4013- 60 A).14) 8 mm Clear Glass Screw Thread Vials (HPLC Vials) (Cat# C4013-1).15) Corning® 12 mm Transwell® with 3.0 pm Pore Polycarbonate Membrane Insert: For Use With 12-Well Plates (Cat# 3402; Lot# 35724004).16) Furosemide, 97+%, ACROS Organics™ (Cat# 448970010; Lot# A0382402) was purchased from Thermo Fisher Scientific (Waltham, MA, USA)17) Standard Flat-Bottom Glass Insert for Standard Opening Vials (HPLC Vial Inserts) (Cat# 82028-448).18) Syringe Filter w / 0.2 um Cellulose Acetate Membrane (Cat# 28145-475) was purchased from VWR part of Avantor (Radnor, PA, USA).Equipment1) HPLC : An Agilent 1260 Infinity HPLC system was utilized for the assay, which consists of a G131 IB 1269 Quat Pump, a G7129 1260 vial sampler, and a G1315D 1260 DAD VAL detector (Agilent, Santa Clara, CA). Chemstation Version Rev C.01.07 SR2

[0255] was used to control the operation and data analysis.2) Nanopure Water Filtration System: Bamstead Nanopure Diamond Life Science UV / UF System (Cat# DI 1931) purchased from APS Water Services Corporation (Lake Balboa, CA, USA).3) Analytical Balance (Cat# AB54-5) was purchased from Mettler-Toledo, LLC (Columbus, OH, USA).4) pH Meter: Fisherbrand™ accumet™ AB 15 Basic pH / mV / °C Meters was purchased from Thermo Fisher Scientific (Waltham, MA, USA).5) Epithelial Volt / Ohm (TEER) Meter: EVOM™ (Serial# 63309 G02D) was purchased from World Precision Instruments (Sarasota, FL, USA).Procedure(a) Solution preparation

[0100] Bumetanide solutions (ranging from about 0.5 to about 1.5 mg / mL) in different solvents were prepared and pH adjusted to be similar to that in each targeted formulation using a pH meter. The bumetanide solutions were prepared by fully dissolving each powder by vortexing, followed by overnight mixing on a rotating platform. Afterward, the solutions were filtered using a 0.2 um filter. The filtrates were then used for the Calu-3 permeation study.(b) Culture of Calu-3 and preparation of monolayers

[0101] The study was carried out similarly as that described previously. Calu-3, a human bronchial submucosal gland carcinoma cell line, grown in DMEM:Ham’s F-12 (1 : 1) mixture was supplemented with 10% FBS and 1% penicillin / streptomycin solution. The cells were harvested with 0.25% trypsin-EDTA and seeded on polycarbonate filters (pore size: 3.0 pm, growth area: 1.2 cm2, 12 wells / plate, Corning) at a density of 5 x 103cells / well. The culture medium was changed every 2 days over the course of the experiment. The monolayer was used for in vitro transport studies, 10 days after seeding.(c) In-vitro permeation / permeability study using Calu-3 cell line model

[0102] The formulations selected for this study were found to be not toxic to the Calu-3 cells as inspected after a 2 hour incubation with these formulations in a pilot evaluation study. At the beginning of the present permeability study, TEER of the monolayer was measured and found to be around 200 to around 500 Them2(an indication of cell integrity). Afterward, the growth medium was aspirated and the upper and lower chambers washed with a transport medium (TM: Hank’s balanced salts solution supplemented with 15 mM glucose and 10 mM HEPES buffer, pH 7.4).

[0103] Following 10 min of incubation, the solution in the apical chamber was replaced with the studied bumetanide solutions. Aliquots of the sample (100 pL) were taken from the basal side at 15, 30, 45, 60, 90, and 120 mins under a BSL2 hood. A 100 pL aliquot of transport medium was added to replenish the volume each time, and TEER measurements were determined to verify cell viability.(d) HPLC assay preparation and measurement

[0104] 100 pL samples from the receiving and apical chambers were either mixed with 100 pL of 50% MeOH and 10 pL internal standard or diluted with 100 pL medium and internal standard. The supernatant was taken after centrifugation for HPLC analysis. HPLC Analysis was performed, as described above (see Determination of saturated solubility).(e) Calculation of Papp

[0105] Papp, expressed in cm / sec, was calculated from the samples analyzed by HPLC using the equation: whereindQ / dt is the appearance of the drug in the receiving chamber (nmol / sec), A is the surface area of the monolayer (1.12 cm2), and Co is the initial concentration of the drug in the apical chamber.Results and discussion

[0106] The mean values of bumetanide Papp from several targeted formulations are shown in Table 3 :Table 3: Papp of certain targeted formulations in comparison to water-Calu-3

[0107] The Papp values of 18 selected formulations were all confirmed to be higher than that from the reference water solution. The formulation DMI 45% at pH 4.0 and 4,5 both achieved Papp 4-fold better than the reference water solution at pH 7. Since these 2 solutions can also achieve bumetanide solubility of >3.33 mg / ml at pH 4-5, they can be potentially optimal formulations, although there are also many other suitable candidates that meet the targeted value initially proposed.

[0108] The present approach / method as described is likely to be reliable in developing many useful bumetanide formulations at pH 4-5 for intranasal and sublingual administration resulting in effective and rapid plasma concentrations to achieve rapid diuresis in the treatment of edema.Conclusion

[0109] The results of the Calu-3 model study further confirmed the validity of the targeted formulation initially identified by PAMPA.

[0110] The present approach / method as described is likely to be reliable in developing many useful bumetanide formulations at about pH 4 to about pH 5 for intranasal and sublingual administration resulting in effective and rapid plasma concentrations to achieve rapid diuresis in the treatment of edema.EXAMPLE 3Comparison of IN, sublingual, intravenous and oral administration of selected formulations in rats[OHl] This example describes the determination of bioavailability / pharmacokinetics of bumetanide in animals following IN, sublingual (SL), intravenous and oral administration of selected formulations. The formulations were selectedbased on results from previous in vitro studies and also in order to understand how variations in the formulation and route of administration can affect the pharmacokinetics of bumetanide.

[0112] Compared to oral administration, IN and SL administration allows quicker absorption as well as for compounds to bypass liver metabolism. Thus, IN / SL administration can lead to faster peak concentration time (shorter Tmax) and greater bioavailability compared to oral administration as well as potentially achieve similar rapid effect as that from intravenous administration. Prior to this in vivo study, whether the effect of certain bumetanide formulation and route of administration (e.g. IN administration) can optimize bumetanide bioavailability and achieve rapid Tmax were unknown and could not be accurately predicted.Materials1) Acetonitrile (Cat# BDH83639.400)2) Methanol (MeOH) (Cat# BDH20864; Lot# 0000269772)3) Potassium Phosphate Monobasic (ACS grade) (Cat# BDH9268; Lot# 1906C155) was purchased from BDH Chemicals (Radnor, PA, USA).4) NaOH (Sodium Hydroxide) (Cat# SB0617, Lot# C26S617R0S) was purchased from Biobasic Canada Inc. (Markham, Ontario, Canada)5) Ethyl alcohol, Pure 200-proof (Cat# 459844; Lot# SHBL6038) was purchased from EMD Millipore (Burlington, MA, USA).6) Bumetanide (Cat# B3023; Lot# BCBW8646)7) Dimethyl isosorbide (Cat# 906832; Batch# 0000173267)8) Glycofurol (Tetrahydrofurfuryl alcohol polyethylene glycol ether) (Cat# 8.21092.0250, Lot# S7906492 040)9) HC1 (Hydrochloric Acid) (Cat# 320331 ; Lot# SHBG2435V)10) NMP (1 -Methyl -2-Pyrrolidinone) (Cat# M-6762, Lot# 51K3683)11) Phosphate buffered saline (PBS, tablet) (Cat# P4417; Lot# SLCD5938) was purchased from Sigma-Aldrich (St. Louis, MO, USA).12) 8mm Autosampler Vial Screw Thread Caps (HPLC Vial Caps) (Cat# C4013- 60A)13) 8mm Clear Glass Screw Thread Vials (HPLC Vials) (Cat# C4013-1)14) Falcon™ Cell Culture Inserts: For Use With 12-Well Plates (Cat# 353181 ; Lot# 1069005)15) Furosemide, 97+%, ACROS Organics™ (Cat# 448970010; Lot# A0382402) was purchased from Thermo Fisher Scientific (Waltham, MA, USA)16) Standard Flat-Bottom Glass Insert for Standard Opening Vials (HPLC Vial Inserts) (Cat# 82028-448)17) Syringe Filter w / 0.2 um Cellulose Acetate Membrane (Cat# 28145-475) was purchased from VWR part of Avantor (Radnor, PA, USA).Instruments1) Nanopure Water Filtration System-Barnstead Nanopure Diamond Life Science UV / UF System (Cat# DI 1931) purchased from APS Water Services Corporation (Lake Balboa, CA, USA)2) Analytical Balance (Cat# AB54-5) was purchased from Mettler-Toledo, LLC (Columbus, OH, USA)3) pH Meter -Fisherbrand™ accumet™ AB 15 Basic pH / mV / °C Meters was purchased from Thermo Fisher Scientific (Waltham, MA, USA)4) LCMS- The liquid chromatographic system consisted of an API3200 LC / MS / MS system (Sciex, Framingham, MA, USA) and two Shimadzu LC-20AD Prominence Liquid Chromatography pumps equipped with an SIL-20A Prominence autosampler (Shimadzu, Colombia, MD, USA). Analyst 1.5 was used to control the operation and data analysis.In vivo study procedure

[0113] Prior to the in vivo study, the nasal spray solution was prepared using bumetanide API (~ 5 mg / mL) solubilized in different water-cosolvent mixtures consisting of glycofurol, NMP, and DMI at pH 4.0-4.5 and at pH 6.5. (See Tables 4 and 6). The nasal spray solutions were filtered (0.22 pm filter)

[0114] The study was carried out in 3 parts: A, B and C. In all studies, bumetanide aqueous solution and organic-aqueous solutions were prepared using the same protocol as described in Example 2. Sprague Dawley rats with jugular vein cannula inserted were ordered from Envigo RMS, Inc. (Indianapolis, IN). After arrival, the rats were adjusted to the animalvivarium environment of the Western University of Health Sciences for one week before the pharmacokinetic study. The anesthesia and study procedure were carried out similar to a previously published study (28). PO dosing was achieved via gavage.

[0115] A subset of rats was given formulations per os (PO, or orally) containing bumetanide (~ 0.5-0.7 mg / kg). The other subset of rats was given formulations by IN or SL route containing bumetanide (~ 0.5 mg / kg). The IN rat group was given formulations through a micropipette 12.5 uL per nostril.

[0116] For Part A of study, 12 formulations were selected for administration. After administration of the formulations, a 200 uL sample of blood was obtained from rats at 0, 2, 5, 10, 15, 20, 30, 45, 60, 120 and 180 min. For Part B of study, 8 formulations were selected. After administration of each IN formulation, the same volume (200uL) blood samples were obtained at each time point, except the last sample was obtained at 120 min. ( The Tl / 2 of bumetanide in the rat was quite short based on Part A study). For both Part A and B of study, the rats were placed in the cage with normal activity before further drug administration. At one week after initial formulation administration, the hematocrit levels of the rats would have returned to normal, as verified previously by the blood plasma of randomly selected rats in our laboratory. Thus based on such hematocrit response, general physical activity, and patency of the cannula, the rats were crossed over to a different formulation treatment one week later with same blood sampling schedule. For Part C of study, only formulation D45 (pH 6.5) was administered. Blood samples were collected at 2, 5, 10, 20 and 60 min (3 rats for each time point) before sacrificing the animals.

[0117] After completion of all blood sampling, the samples were centrifuged and plasma collected and stored for analysis using Liquid Chromatography Tandem Mass Spectrometry (LC / MS / MS, Sciex API4000 and Agilent HPLC 1200 system) The assay used rat plasma to construct the standard curve and tolbutamide was used as the internal standard (IS).Determination of the plasma concentration of bumetanide

[0118] Analysis of the bumetanide concentration in blood plasma was conducted using Liquid Chromatography Tandem Mass Spectrometry (LC / MS / MS, Sciex API4000 and Agilent HPLC 1200 system). During the validation experiments, each calibration standard andQC sample was prepared by spiking of a specified amount of bumetanide HC1 (from USP) and tolbutamide was served as the internal standard after mixing with the rat plasma. Standard stock solutions were made by diluting the stock solution (lOOOug / ml) with 50% ACN to make 10, 20, 100, 200, 1000, 200, 10000 and 20000 ng / mL solutions. Tolbutamide standard stock solution (5 pg / mL) was prepared by weighing approximately 1 mg tolbutamide and transferring it to a 100 mL volumetric flask. It was dissolved with about 50mL of ACN and water was added to the mark. Samples are extracted with 300 pL of acetonitrile, centrifuged and 10 ul supernatant was injected to LCMS instrument for analysis.

[0119] Bumetanide and tolbutamide were detected using multiple-reactionmonitoring for each of the respective analyte. The average assay intra-day accuracy ranged from 89.9-102.9% with inter-day accuracy ranged from 91.8-128.0% The R2of the calibration curves was 0.9981 for the concentration of 0.1 -lOOOng / mk Precision, defined as the coefficient of variation (CV) = (standard deviation / mean of replicate measurements XI 00%) ranged 1.5- 4.7%. for intra-day samples and 4.4-15.7% for inter-day samples. The lower limit of quantitation was 1 ng / mLResults: Part A of study

[0120] Twelve different bumetanide formulations were administered via IN, PO, IV and SL routes. The specific formulations and routes of administration are shown in Table 4. Bumetanide pharmacokinetics corresponding to Study Part A study are shown in Table 5 and FIG. 5.Table 4: Formulations for Part A of studyTable 5: Pharmacokinetic parameters from different formulationsNormalized to 1 mg / kgNormalized to 1 mg / kg*AUC up to 120 min instead of 180 minute presented due to lack of confidence of last time point at 180 minResults: Part B of study

[0121] A total of 8 formulations (6 organic aqueous mixture administered by IN route and 2 aqueous formulations administered each administered as either IN or PO route. The IN and PO dosages are shown in Table 6. The pharmacokinetic parameters of all the formulations at different are shown in Table 7.Table 6: Formulations for Parts B and C of studyTable 7: Pharmacokinetic parameters from different formulations (Part B of Study)Part C of StudyThe formulation D45 (pH6.5) was administered by IN route and after administration, only one sample was collected at each time point (3 rats / time point). The dosage was labeled corresponding to formulation 9* in Table 6. The Pharmacokinetics of IN administered formulation D45 (pH6.5) are shown in Table 8 and Cmax shown in Figure 5.Table 8; Pharmacokinetic parameters of D45 at pH of 4, 4,5, 6,5 (Part C in comparison toPart B result)Discussion and Conclusion

[0122] All IN cosolvent formulations when normalized to 1 mg / kg dose, had higher Cmax when compared to that from reference IN water formulation. Also, all IN formulations had substantially shorter Tmax as well as higher Cmax and AUC when compared to oral administration. Among organic-aqueous cosolvent mixtures, D45 at about pH 4 and about pH 4.5 achieved highest Cmax as well as AUC with a short Tmax. Thus, these in vivo data confirm the results from in vitro screening using PAMPA and Calu 3 cell line studies.

[0123] In conclusion, the results from Examples 2 and 3 studies found bumetanide formulations in D, N and G (up to about 45% alone or in combination at about pH 4 to about pH 4.5) can produce short Tmax, higher Cmax and higher bioavailability than a solution at a pH that is greater than about pH 5. Such formulations also have sufficient bumetanide solubility at any integer that is between pH 4.0 and about pH 5.0 and can yield rapid therapeutic effect when administered intranasally, but is unlikely to achieve same result with these IN formulations at physiologic pH.EXAMPLE 4Methods of Administering Bumetanide

[0124] This example describes methods of intranasal and sublingual administration of bumetanide.

[0125] Bumetanide is added to its desired water-cosolvent mixture targeted to achieve sufficient solubility (e.g. > 3.33mg / ml) with pH adjusted for optimal permeation. Any organic-aqueous mixture or solvent can be used, preferably D, N, and G, and can include including any other organic-aqueous solvent that is relatively safe or well tolerated by a human subject and that the water-cosolvent mixture is capable of sufficiently solubilizing and also achieving desired permeation by adjusting pH corresponding to about 4 to about 5.

[0126] Improved solubility of the bumetanide with sufficient permeability can lead to sufficient and rapid permeation across a mucosal membrane. Permeation of bumetanide across the mucosal membrane can occur at pH range of about 3.5 to about 8.0, preferably at about pH 4 to about pH 5. As a result of improved permeation at lower pH, the bioavailability of bumetanide can be potentially increased to achieve a rapid and effective Tmax, when administered intranasally or sublingually with an appropriate formulation. Moreover, greater bioavailability can be achieved upon intranasal and / or sublingual administration (if properly administered for optimal absorption) as compared to oral administration at the same dosage.

[0127] Bumetanide solution can be delivered mucosally (e.g. intranasally) in different forms including a spray. As an example, an amount of bumetanide can be added to a water-cosolvent mixture to deliver a desired amount of the bumetanide in a 150 uL volume per spray, either intranasally or sublingually. Using a specific formulation targeted to achieve sufficient solubility (e.g. > 3.33 mg / ml) with permeability equivalent to or better than the reference water solution (at pH 7) can lead to sufficient permeation across the mucosal membrane.

[0128] The bumetanide solution with improved solubility and permeation as described above is administered for rapid treatment of edema, for example.

Claims

WHAT IS CLAIMED IS:

1. A composition comprising: a therapeutically effective dose of a loop diuretic molecule; a water; and an organic fraction comprising at leastl, 2, 3, or 4 compound(s), wherein the compound(s) is / are selected from: an alcohol, a polyether, a glyceride, a pyrrolidone, or any combination thereof.

2. The composition of claim 1, wherein the composition is at a final pH that is about 3.5, about 4.0, about 4.5, about 5.0, about 5.5, about 6.0, about 7.0, about 7.5, about 8.0, or any value that is between about 3.5 and about 8.0.

3. The composition of claim 1 or 2, wherein the pH is adjusted using at least one of NaOH, KOH, HC1, or any combination thereof.

4. The composition of any one of claims 1-3, wherein the alcohol is an ethanol, a glycerol, or a glycofurol.

5. The composition of claim 4, wherein the alcohol is glycofurol.

6. The composition of any one of claims 1-5, wherein the polyether is dimethyl isosorbide (DMI).

7. The composition of any one of claims 1-6, wherein the pyrrolidone is N-methyl- 2-pyrolidone (NMP).

8. The composition of any one of claims 1-7, wherein the loop diuretic molecule is bumetanide.

9. The composition of any one of claims 1-8, wherein the composition is formulated for nasal, intranasal, oral, or sublingual administration to a subject.

10. The composition of any one of claims 1-9, wherein the composition is formulated such that the loop diuretic molecule is capable of permeating a mucosal membrane.

11. The composition of any one of claims 1-10, wherein the alcohol, poly ether, glyceride, and / or pyrrolidone is independently present in the composition at a percentage that is about 1, about 5, about 10, about 15, about 20, about 25, about 30, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, or any value that is between about 1% and about 80% of the total composition.

12. The composition of any one of claims 1 -1 1, wherein the pH is at about 4.0, about 4.5, about 5.0, or is any value that is between about 4.0 and about 5.0.

13. The composition of any one of claims 1-12, wherein the organic fraction is at least about 1% of the total composition.

14. The composition of any one of claims 1-13, wherein the organic fraction is at most about 80% of the total composition.

15. The composition of any one of claims 1-14, wherein the organic fraction is at most about 50% of the total composition.

16. The composition of any one of claims 1-15, wherein the solubility of the loop diuretic molecule is at least about 2.5 mg / mL.

17. The composition of any one of claims 1-16, wherein the permeability (Papp, as determined by PAMPA) of the loop diuretic molecule in the composition is greater than that of loop diuretic moleculein water, adjusted to the same pH.

18. The composition of any one of claims 1-11, wherein the loop diuretic molecule is at a concentration that is about 2.0, about 2.5, about 3.0, about 3.3, about 3.5, about 4.0, about 4.5, about 5.0, about 5.5, about 6.0, about 6.5, about 7.0, about 7.5, about 8.0, about 8.5, about 9.0, about 9.5, about 10.0, or any value that is between about 2.0 and about 10.0 mg / mL.

19. The composition of any one of claims 1-18, wherein the organic fraction comprises any 1, 2, 3, 4, or 5 of: a polyether, diethylene glycol monoethyl ether, a medium chain glyceride, one or more saturated polyglycolyzed C8-C 10 glyceride, a pyrrolidone, or any combination thereof.

20. The composition of any one of claims 1-19, wherein the organic fraction further comprises Tween 80 and / or a polyethylene glycol.

21. Use of a composition of any one of claims 1-20 for treating a disease or disorder in a subject in need thereof.

22. The use of claim 21, wherein the subject is mammalian and / or human.

23. The use of claim 21 or 22, wherein the composition is optimized to achieve an effective dose and rapid plasma concentration upon administration to the subject.

24. The use of any one of claims 21-23, wherein the disease or disorder is an edema.

25. A method for treating a disease or disorder in a subject in need thereof, the method comprising administering the composition of any one of claims 1-20.

26. The method of claim 25, wherein the subject is mammalian and / or human.

27. The method of claim 25 or 26, wherein the composition is optimized to achieve an effective dose and rapid plasma concentration upon administration to the subject.

28. The method of any one of claims 25-27, wherein the disease or disorder is an edema.

29. A method of treating disease or disorder in a subj ect in need thereof, the method comprising administering a therapeutically effective dose of a composition, the composition comprising: a therapeutically effective dose of bumetanide; and a water co-solvent mixture comprising 1, 2, 3, or all 4 of: an alcohol, a polyether, a glyceride, and / or a pyrrolidone; wherein the composition is at a final pH that is between about 3.5 and about 8, as adjusted using NaOH and HC1.

30. The method of claim 29, wherein the composition is formulated for nasal, oral, sublingual, or intranasal administration to a subject.

31. The method of any one of claims29-30, wherein the composition is formulated such that bumetanide is capable of permeating a mucosal membrane.

32. The method of any one of claims 29-31, wherein the method further comprises contacting a mucosal membrane of the subject with the composition.

33. The method of any one of claims 29-32, wherein the disease or disorder is an edema.

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