Preparations and methods for treating diarrhea

Abstract

Described herein are amino acid formulations useful for treating diarrhea in subjects in need thereof. Such amino acid formulations and methods comprising administering the amino acid formulations to a subject can be used to treat diarrhea, particularly diarrhea caused by or associated with a bacterial infection, wherein secretagogue-stimulated anion secretion from the intestinal crypts results in at least one diarrheal symptom in the subject. Contemplated herein are the use of these amino acid formulations for treating diarrhea in general or diarrhea associated with secretagogue-stimulated anion secretion from the intestinal crypts, and their use in the preparation of a medicament for treating diarrhea in general or diarrhea associated with secretagogue-stimulated anion secretion from the intestinal crypts.

Description

[0001] Related applications

[0002] This application claims priority to U.S. Provisional Application No. 63 / 033,697, filed June 2, 2020, which is hereby incorporated by reference in its entirety for all purposes. Technical Field

[0003] Described herein are amino acid formulations useful for treating diarrhea in subjects in need thereof. The amino acid formulations and methods described herein are useful for treating diarrhea, particularly diarrhea caused by or associated with a bacterial infection, wherein secretagogue-stimulated anion secretion from the intestinal crypts results in at least one diarrheal symptom in such subjects. Also contemplated herein are uses of these amino acid formulations for treating diarrhea in general or diarrhea associated with secretagogue-stimulated anion secretion from the intestinal crypts, and for the preparation of medicaments for treating diarrhea in general or diarrhea associated with secretagogue-stimulated anion secretion from the intestinal crypts. Background Art

[0004] Secretory diarrhea remains a significant cause of mortality and morbidity worldwide, particularly in vulnerable populations such as children and the elderly. Indeed, infectious diarrhea is the leading cause of death in children, with fatal dehydration resulting from electrolyte and water losses and intestinal inflammation. Mortality from infectious diarrhea in children has decreased significantly since the introduction of the sugar-based World Health Organization Oral Rehydration Solution (WHO-ORS) in 1979. However, nearly 446,000 children still die from infectious diarrhea each year. Children <5 years of age and adults >70 years of age are at particularly high risk of death from diarrheal diseases. Summary of the Invention

[0005] The embodiments covered are defined by the claims, not by this Summary. This Summary is a high-level overview of various aspects and introduces some concepts that are further described in the Detailed Description below. This Summary is not intended to identify key or essential features of the claimed subject matter, nor is it intended to be used alone to determine the scope of the claimed subject matter. The subject matter should be understood by reference to the entire specification, any or all of the drawings, and appropriate portions of each claim.

[0006] In some embodiments, a formulation is provided for treating diarrhea in a subject in need thereof, wherein the formulation comprises a therapeutically effective combination of free amino acids consisting essentially of or consisting of a therapeutically effective amount of the free amino acids of proline and aspartic acid; and a therapeutically effective amount of at least one of the free amino acids of serine, threonine, glycine, alanine, arginine, or tyrosine, or any combination thereof; and optionally, the free amino acid of asparagine; or optionally, the monosaccharide glucose, at least one glucose-containing disaccharide, or any combination thereof, wherein the total concentration of the monosaccharide glucose, the at least one glucose-containing disaccharide, or any combination thereof is equal to or less than 90 mM; or optionally, at least one pharmaceutically acceptable carrier, buffer, electrolyte, adjuvant, excipient, or water, or any combination thereof; or any combination thereof; and wherein the therapeutically effective combination of free amino acids is sufficient to reduce at least one symptom associated with the diarrhea in the subject.

[0007] In some embodiments of the formulation, the concentration of proline is in the range of 0.5 mM to 13 mM; wherein the concentration of proline is in the range of 0.8 mM to 13 mM; wherein the concentration of proline is in the range of 2 mM to 10 mM; wherein the concentration of proline is in the range of 6 mM to 10 mM; wherein the concentration of proline is in the range of 8 mM to 10 mM; wherein the concentration of proline is in the range of 8.1 mM to 9.9 mM; or wherein the concentration of proline is 9 mM.

[0008] In some embodiments of the formulation, the concentration of aspartic acid is in the range of 1 mM to 18 mM; wherein the concentration of aspartic acid is in the range of 1 mM to 12 mM; wherein the concentration of aspartic acid is in the range of 8 mM to 12 mM; wherein the concentration of aspartic acid is in the range of 9 mM to 11 mM; or wherein the concentration of aspartic acid is 10 mM.

[0009] In some embodiments of the formulation, when serine is present, the concentration of serine is in the range of 1.5 mM to 12 mM; wherein the concentration of serine is in the range of 4 mM to 10 mM; wherein the concentration of serine is in the range of 8 mM to 12 mM; wherein the concentration of serine is in the range of 9 mM to 11 mM, or wherein the concentration of serine is 10 mM.

[0010] In some embodiments of the formulation, when threonine is present, the concentration of threonine is in the range of 0.5 mM to 12 mM; the concentration of threonine is in the range of 2 mM to 12 mM; wherein the concentration of threonine is in the range of 4 mM to 10 mM; wherein the concentration of threonine is in the range of 6 mM to 10 mM; wherein the concentration of threonine is in the range of 7.2 mM to 8.8 mM; or wherein the concentration of threonine is 8 mM.

[0011] In some embodiments of the formulation, when glycine is present, the concentration of glycine is in the range of 0.4 mM to 12 mM; wherein the concentration of glycine is in the range of 1 mM to 10 mM; wherein the concentration of glycine is in the range of 5 mM to 8 mM; wherein the concentration of glycine is in the range of 6 mM to 8 mM; wherein the concentration of glycine is in the range of 6.3 mM to 7.8 mM; or wherein the concentration of glycine is 7 mM.

[0012] In some embodiments of the formulation, when alanine is present, the concentration of alanine is in the range of 1.5 mM to 12 mM; wherein the concentration of alanine is in the range of 3 mM to 9 mM; wherein the concentration of alanine is in the range of 4 mM to 7 mM; wherein the concentration of alanine is in the range of 4 mM to 6 mM; wherein the concentration of alanine is in the range of 4.8 mM to 5.8 mM; or wherein the concentration of alanine is 5.3 mM.

[0013] In some embodiments of the formulation, when arginine is present, the concentration of arginine is in the range of 0.8 mM to 12 mM; wherein the concentration of arginine is in the range of 5 mM to 10 mM; wherein the concentration of arginine is in the range of 5 mM to 8 mM; wherein the concentration of arginine is in the range of 6 mM to 8 mM; wherein the concentration of arginine is in the range of 6 mM to 7.4 mM; or wherein the concentration of arginine is 6.7 mM.

[0014] In some embodiments of the formulation, when tyrosine is present, the concentration of tyrosine is in the range of 0.1 mM to 1.2 mM; the concentration of tyrosine is in the range of 0.5 mM to 1.2 mM; wherein the concentration of tyrosine is in the range of 0.8 mM to 1.2 mM; wherein the concentration of tyrosine is in the range of 0.9 mM to 1.2 mM; wherein the concentration of tyrosine is in the range of 1.0 mM to 1.3 mM; or wherein the concentration of tyrosine is 1.2 mM.

[0015] In some embodiments of the formulation, when asparagine is present, the concentration of asparagine is in the range of 2 mM to 12 mM; wherein the concentration of asparagine is in the range of 2 mM to 10 mM; or wherein the concentration of asparagine is in the range of 6 mM to 8 mM; or wherein the concentration of asparagine is in the range of 6.9 mM to 8.5 mM; wherein the concentration of asparagine is 7.7 mM.

[0016] In some embodiments of the formulation, the therapeutically effective combination of free amino acids consists essentially of or consists of therapeutically effective amounts of the free amino acids proline, aspartic acid, serine, threonine, glycine, alanine, arginine, and tyrosine.

[0017] In some embodiments of the formulation, the therapeutically effective combination of free amino acids consists essentially of or consists of therapeutically effective amounts of the free amino acids proline, aspartic acid, serine, threonine, glycine, alanine, arginine, and tyrosine, wherein the concentration of proline is 8-10 mM; wherein the concentration of aspartic acid is 9-11 mM; wherein the concentration of serine is 9-11 mM; wherein the concentration of threonine is 7-9 mM; wherein the concentration of glycine is 6-8 mM; wherein the concentration of alanine is 5-6 mM; wherein the concentration of arginine is 6-7 mM; and wherein the concentration of tyrosine is 0.8-1.2 mM.

[0018] In some embodiments of the formulation, the therapeutically effective combination of free amino acids consists essentially of or consists of therapeutically effective amounts of the free amino acids proline, aspartic acid, serine, threonine, glycine, alanine, arginine, and tyrosine, wherein the concentration of proline is 9 mM; wherein the concentration of aspartic acid is 10 mM; wherein the concentration of serine is 10 mM; wherein the concentration of threonine is 8 mM; wherein the concentration of glycine is 7 mM; wherein the concentration of alanine is 5.3 mM; wherein the concentration of arginine is 6.7 mM; and wherein the concentration of tyrosine is 1.2 mM.

[0019] In some embodiments of the formulation, the therapeutically effective combination of free amino acids consists essentially of or consists of therapeutically effective amounts of the free amino acids proline, aspartic acid, serine, threonine, glycine, alanine, arginine, and tyrosine, wherein the concentration of proline is 9-11 mM; wherein the concentration of aspartic acid is 15-17 mM; wherein the concentration of serine is 7-9 mM; wherein the concentration of threonine is 3-5 mM; wherein the concentration of glycine is 5-7 mM; wherein the concentration of alanine is 5-7 mM; wherein the concentration of arginine is 5-7 mM; and wherein the concentration of tyrosine is 0.8-1.2 mM.

[0020] In some embodiments of the formulation, the therapeutically effective combination of free amino acids consists essentially of or consists of therapeutically effective amounts of the free amino acids proline, aspartic acid, serine, threonine, glycine, alanine, arginine, and tyrosine, wherein the concentration of proline is 10 mM; wherein the concentration of aspartic acid is 15.9 mM; wherein the concentration of serine is 8 mM; wherein the concentration of threonine is 4.3 mM; wherein the concentration of glycine is 5.7 mM; wherein the concentration of alanine is 6.3 mM; wherein the concentration of arginine is 5.8 mM; and wherein the concentration of tyrosine is 1.2 mM.

[0021] In some embodiments of the formulation, the therapeutically effective combination of free amino acids consists essentially of or consists of therapeutically effective amounts of the free amino acids proline, aspartic acid, serine, threonine, alanine, arginine, and tyrosine.

[0022] In some embodiments of the formulation, the therapeutically effective combination of free amino acids consists essentially of or consists of therapeutically effective amounts of the free amino acids of proline, aspartic acid, serine, threonine, alanine, arginine, and tyrosine, wherein the concentration of proline is 11-13 mM; wherein the concentration of aspartic acid is 14-18 mM; wherein the concentration of serine is 4-6 mM; wherein the concentration of threonine is 4-6 mM; wherein the concentration of alanine is 3-5 mM; wherein the concentration of arginine is 4-7 mM; and wherein the concentration of tyrosine is 0.6-0.9 mM.

[0023] In some embodiments of the formulation, the therapeutically effective combination of free amino acids consists essentially of or consists of therapeutically effective amounts of the free amino acids proline, aspartic acid, serine, threonine, alanine, arginine, and tyrosine, wherein the concentration of proline is 12.3 mM; wherein the concentration of aspartic acid is 16.3 mM; wherein the concentration of serine is 4.8 mM; wherein the concentration of threonine is 5.3 mM; wherein the concentration of alanine is 4.3 mM; wherein the concentration of arginine is 5.8 mM; and wherein the concentration of tyrosine is 0.8 mM.

[0024] In some embodiments of the formulation, the formulation further comprises a pharmaceutically acceptable carrier, buffer, electrolyte, adjuvant, excipient, or water.

[0025] In some embodiments of the formulation, the formulation is sterile.

[0026] In some embodiments of the formulation, at least one free amino acid or each free amino acid comprises an L-amino acid.

[0027] In some embodiments of the formulations, the formulation does not comprise the monosaccharide glucose, at least one disaccharide containing glucose, or any combination thereof.

[0028] In some embodiments of the formulation, the formulation is a pharmaceutical formulation.

[0029] In some embodiments of the formulation, the formulation is formulated for administration by parenteral, pulmonary, inhalation, intranasal, enteral, intravenous, anal, or sublingual route.

[0030] In some embodiments of the formulation, the formulation is formulated for oral administration.

[0031] In some embodiments of the formulation, the subject is a mammal. In some embodiments of the formulation, the mammal is a human, cat, dog, pig, horse, cow, sheep, or goat. In some embodiments of the formulation, the mammal is a human. In some embodiments of the formulation, the human is 5 years old or younger; or the human is 70 years old or older.

[0032] In some embodiments of the formulation, the diarrhea is associated with a secretagogue-producing bacterial infection in the subject.

[0033] In some embodiments of the formulation, the bacteria comprise at least one of enterotoxigenic bacteria or enterotoxigenic bacteria, or any combination thereof.

[0034] In some embodiments of the formulation, the enterotoxigenic bacteria comprises Vibrio cholerae, Staphylococcus aureus, Bacillus cereus, Clostridium difficile, Clostridium perfringens, Staphylococcus aureus, Yersinia enterocolitica, Shigella dysenteriae, or enterotoxigenic Escherichia coli, or any combination thereof.

[0035] In some embodiments of the formulation, the at least one symptom associated with diarrhea comprises at least one of: loose, watery stools; abdominal cramps; abdominal pain; fever; blood in the stool; mucus in the stool; flatulence; or nausea; or any combination thereof.

[0036] In some embodiments of the formulation, reducing at least one symptom associated with diarrhea comprises reducing at least one of: the volume of loose, watery stools per day; or the frequency of loose, watery stools per day; or a combination thereof.

[0037] In some embodiments of the formulation, the formulation is formulated for use as a medicament for treating diarrhea.

[0038] In some embodiments of the formulations, there is provided a method for treating diarrhea in a subject in need thereof, the method comprising: administering to a subject in need thereof a formulation described herein, wherein the administration reduces at least one symptom associated with diarrhea in the subject.

[0039] In some embodiments, a formulation is provided wherein the formulation comprises a therapeutically effective combination of free amino acids consisting essentially of or consisting of a therapeutically effective amount of the free amino acids of proline and aspartate; and a therapeutically effective amount of at least one of the free amino acids of serine, threonine, glycine, alanine, arginine, or tyrosine, or any combination thereof; and optionally, a free amino acid of asparagine; or optionally, the monosaccharide glucose, at least one glucose-containing disaccharide, or any combination thereof, wherein the total concentration of the monosaccharide glucose, the at least one glucose-containing disaccharide, or any combination thereof is equal to or less than 90 mM; or optionally, at least one pharmaceutically acceptable carrier, buffer, electrolyte, adjuvant, excipient, or water, or any combination thereof; or any combination thereof; and wherein the therapeutically effective combination of free amino acids is sufficient to reduce at least one symptom associated with the diarrhea. In some embodiments thereof, the therapeutically effective combination of free amino acids consists essentially of or consists of a therapeutically effective amount of the free amino acids of proline, aspartate, serine, threonine, glycine, alanine, arginine, and tyrosine.

[0040] In some embodiments thereof, the therapeutically effective combination of free amino acids consists essentially of or consists of therapeutically effective amounts of the free amino acids proline, aspartic acid, serine, threonine, glycine, alanine, arginine, and tyrosine, wherein the concentration of proline is 8-10 mM; wherein the concentration of aspartic acid is 9-11 mM; wherein the concentration of serine is 9-11 mM; wherein the concentration of threonine is 7-9 mM; wherein the concentration of glycine is 6-8 mM;

[0041] wherein the concentration of alanine is 5-6 mM; wherein the concentration of arginine is 6-7 mM; and wherein the concentration of tyrosine is 0.8-1.2 mM.

[0042] In some embodiments thereof, the therapeutically effective combination of free amino acids consists essentially of or consists of therapeutically effective amounts of the free amino acids proline, aspartic acid, serine, threonine, glycine, alanine, arginine, and tyrosine,

[0043] wherein the concentration of proline is 9 mM; wherein the concentration of aspartic acid is 10 mM; wherein the concentration of serine is 10 mM; wherein the concentration of threonine is 8 mM; wherein the concentration of glycine is 7 mM; wherein the concentration of alanine is 5.3 mM; wherein the concentration of arginine is 6.7 mM; and wherein the concentration of tyrosine is 1.2 mM.

[0044] In some embodiments thereof, the therapeutically effective combination of free amino acids consists essentially of or consists of therapeutically effective amounts of the free amino acids proline, aspartic acid, serine, threonine, glycine, alanine, arginine, and tyrosine,

[0045] wherein the concentration of proline is 9-11 mM; wherein the concentration of aspartic acid is 15-17 mM; wherein the concentration of serine is 7-9 mM; wherein the concentration of threonine is 3-5 mM; wherein the concentration of glycine is 5-7 mM; wherein the concentration of alanine is 5-7 mM; wherein the concentration of arginine is 5-7 mM; and wherein the concentration of tyrosine is 0.8-1.2 mM.

[0046] In some embodiments thereof, the therapeutically effective combination of free amino acids consists essentially of or consists of therapeutically effective amounts of the free amino acids proline, aspartic acid, serine, threonine, glycine, alanine, arginine, and tyrosine, wherein the concentration of proline is 10 mM; wherein the concentration of aspartic acid is 15.9 mM; wherein the concentration of serine is 8 mM; wherein the concentration of threonine is 4.3 mM; wherein the concentration of glycine is 5.7 mM; wherein the concentration of alanine is 6.3 mM; wherein the concentration of arginine is 5.8 mM; and wherein the concentration of tyrosine is 1.2 mM.

[0047] In some embodiments thereof, the therapeutically effective combination of free amino acids consists essentially of or consists of therapeutically effective amounts of the free amino acids proline, aspartic acid, serine, threonine, alanine, arginine, and tyrosine.

[0048] In some embodiments thereof, the therapeutically effective combination of free amino acids consists essentially of or consists of therapeutically effective amounts of the free amino acids proline, aspartic acid, serine, threonine, alanine, arginine, and tyrosine,

[0049] wherein the concentration of proline is 11-13 mM; wherein the concentration of aspartic acid is 14-18 mM; wherein the concentration of serine is 4-6 mM; wherein the concentration of threonine is 4-6 mM; wherein the concentration of alanine is 3-5 mM; wherein the concentration of arginine is 4-7 mM; and wherein the concentration of tyrosine is 0.6-0.9 mM.

[0050] In some embodiments thereof, the therapeutically effective combination of free amino acids consists essentially of or consists of therapeutically effective amounts of the free amino acids proline, aspartic acid, serine, threonine, alanine, arginine, and tyrosine, wherein the concentration of proline is 12.3 mM; wherein the concentration of aspartic acid is 16.3 mM; wherein the concentration of serine is 4.8 mM; wherein the concentration of threonine is 5.3 mM; wherein the concentration of alanine is 4.3 mM; wherein the concentration of arginine is 5.8 mM; and wherein the concentration of tyrosine is 0.8 mM. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Some embodiments of the present disclosure are described herein by way of example only with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is emphasized that the embodiments shown are by way of example and for purposes of illustrative discussion of the embodiments of the present disclosure. In this regard, the description using the drawings will provide those skilled in the art with a clear understanding of how the embodiments of the present disclosure may be practiced.

[0052] Figure 1 : Net fluid absorption in the presence of cholera toxin in the presence of the indicated formulations as determined by perfusion assay. Cholera toxin (CT); Ringer's solution (RR); Trioral (WHO oral rehydration solution).

[0053] Figure 2 : Effect of the presence or absence of secretagogues on net fluid absorption in the presence of the indicated agents. RR: Ringer's solution with 105 mM Na, 241 mOsm, pH 4.049 [n=6 (control) and n=4 (cholera toxin (CT))]; 8AA: aspartic acid, glycine, valine, isoleucine, threonine, tyrosine, serine, and lysine, 67 mM Na, 243 mOsm, pH 3.82 [n=6 (control) and n=8 (CT)]; WHO ORS (Trioral): 75 mM Na, 75 mM glucose, 221-225 mOsm, pH 7.5-7.8 [n=6 (control) and n=8 (CT)]; 6AA: aspartic acid, valine, isoleucine, threonine, tyrosine, and lysine, 67 mM Na, 245 mOsm, pH 3.7 [n=6 (control) and n=9 (CT)]; F3: aspartic acid, glycine, threonine, tyrosine, serine, proline, alanine and arginine, 67 mM Na, 221 mOsmo, pH 4.096 [n=0 (control) and n=4 (CT)].

[0054] Figure 3: Effect of secretagogues on net fluid absorption in the presence of the indicated agents. Saline: Ringer's solution with 105 mM Na, 241 mOsm, pH 4.049 [healthy (no secretagogue; diarrhea + secretagogue (in the presence of secretagogue)]; Trioral (WHO ORS): 75 mM Na, 75 mM glucose, 221-225 mOsm, pH 7.5-7.8 (in the presence of secretagogue); 8AA: aspartic acid, glycine, valine, isoleucine, threonine, tyrosine, serine, and lysine, 67 mM Na, 243 mOsm, pH 3.82 (in the presence of secretagogue)]; F3: aspartic acid, glycine, threonine, tyrosine, serine, proline, alanine, and arginine, 67 mM Na, 221 mOsm, pH 4.096 (in the presence of secretagogue). DETAILED DESCRIPTION

[0055] Among the benefits and improvements already disclosed, other objects and advantages of the present disclosure will become apparent from the following description taken in conjunction with the accompanying drawings. Detailed embodiments of the present disclosure are disclosed herein; however, it should be understood that the disclosed embodiments are merely illustrative of the present disclosure that can be embodied in various forms. Furthermore, each example provided with respect to the various embodiments of the present disclosure is intended to be illustrative and not restrictive.

[0056] The incidence of diarrheal disease is closely related to climate and economic development. Developing countries in sub-Saharan Africa and South Asia tend to have a high incidence of diarrheal disease. In developing countries, the main causes of diarrheal disease are infectious, including enterotoxin-producing bacteria such as Vibrio cholerae and enterotoxigenic Escherichia coli; viruses such as rotavirus; enteroinvasive bacteria such as Shigella and Salmonella; and parasites such as Entamoeba histolytica and Cryptosporidium parvum. For example, Vibrio cholerae causes at least 1-2% of severe diarrheal cases observed worldwide.

[0057] In the United States, enterocolitis induced by Salmonella is the most common cause of death due to foodborne illness associated with viruses, parasites or bacteria. In the United States, the serotype most often associated with this diarrheal disease syndrome is Salmonella serotype Typhimurium, which accounts for 26% of all Salmonella isolates reported to the Centers for Disease Control. Salmonella typhimurium invades the intestinal mucosa, but does not produce traditional enterotoxins. It triggers ileal secretion by causing changes in the active sodium and chloride transport mechanisms. Typhoid fever is caused by Salmonella enterica serotype Typhi (S. enterica serotype Typhi) adapted to humans, resulting in typhoid fever. Typhoid fever affects millions of people worldwide every year, with a morbidity rate of approximately 200,000 people per year.

[0058] The exemplary oral rehydration solutions (AA-ORS) described herein, comprising selected amino acids, exhibited surprising combined activity, as reflected by statistically significant and superior therapeutic properties, in terms of net chloride, sodium, and fluid absorption, when compared to WHO-ORS in an animal model system of human intestinal response, whether compared in the absence (normal, healthy) or presence (diseased) of secretagogues. The animal model system comprises normal, healthy (not toxin-exposed) and cholera toxin (CT)-exposed mouse and rat small intestine, thereby recapitulating human intestinal response in the absence or presence of secretagogues, respectively.

[0059] The exemplary AA-ORS described herein exhibit enhanced electrolyte absorption. The combination of amino acids in each exemplary AA-ORS exhibits synergistic effects on multiple criteria including net fluid absorption. See, e.g. Figure 1-3 As described herein, exemplary AA-ORS formulations (e.g., F1, F2, F3, and F4) were tested in the presence of CT in an anesthetized rat model for intestinal lumen perfusion. Lumen perfusion studies showed that F3 exhibited favorable properties in promoting net fluid absorption, as reflected in a statistically significant increase of approximately 2.3-fold in net fluid absorption in the presence of CT when compared to WHO-ORS in the presence of CT. F1 and F2 also exhibited favorable properties in promoting net fluid absorption, as reflected in a statistically significant increase of approximately 2.0-fold in net fluid absorption in the presence of CT when compared to WHO-ORS in the presence of CT. See, e.g. Figure 2 .

[0060] Lumen perfusion studies also demonstrated that FF3 exhibited favorable properties in promoting net fluid uptake, as reflected by a statistically significant increase of 56% in net fluid uptake in the presence of CT compared to 8AA in the presence of CT. F1 also exhibited favorable properties in promoting net fluid uptake, as reflected by a statistically significant increase of 42% in net fluid uptake in the presence of CT compared to 8AA in the presence of CT. F2 also exhibited favorable properties in promoting net fluid uptake, as reflected by a statistically significant increase of 31% in net fluid uptake in the presence of CT compared to 8AA in the presence of CT. In contrast, F4, which contains 8 free amino acids (many of which are the same as F1, F2, and F3), had a statistically significantly lower net fluid uptake relative to 8AA. See Figure 1 .

[0061] Exemplary formulations F1, F2, and F3 each contain the following free amino acids: proline, aspartic acid, serine, arginine, tyrosine, threonine, and alanine. F2 and F3 contain the free amino acids of proline, aspartic acid, serine, arginine, tyrosine, threonine, alanine, and glycine. F1 contains the free amino acids of proline, aspartic acid, serine, arginine, tyrosine, threonine, alanine, and asparagine.

[0062] Figure 1-3 The results presented in demonstrate that factors such as the supplementation of free amino acids present in the amino acid formulation and their concentration contribute synergistically to the activity of the amino acid formulation in terms of the net fluid absorption conferred thereby.

[0063] In some embodiments, the concentration of each free amino acid present in the formulation is in the range of 0.1 mM to 20 mM or 0.5 mM to 20 mM. In some embodiments, the concentration of each free amino acid present in the formulation is in the range of 0.1 mM to 17 mM or 0.5 mM to 17 mM. In some embodiments, the concentration of each free amino acid present in the formulation is in the range of 0.1 mM to 15 mM or 0.5 mM to 15 mM. In some embodiments, the concentration of each free amino acid present in the formulation is in the range of 0.1 mM to 10 mM or 0.5 mM to 10 mM.

[0064] In some embodiments, the pH of the formulations described herein is in the range of 2.5 to 8.0, 3.0 to 8.0, 3.0 to 4.5, 3.0 to 4.2, 3.4 to 4.2, 3.5 to 4.2, 3.5 to 4.1, 3.5 to 8.0, 4.0 to 8.0, 4.5 to 8.0, 4.5 to 6.5, 5.5 to 6.5, 5.0 to 8.0, 5.5 to 8.0, 6.0 to 8.0, 6.5 to 8.0, 7.0 to 8.0, or 7.5 to 8.0. In some embodiments, the pH is about 3.7, 3.5, or 4.1.

[0065] As used herein, the phrase "reduction of at least one symptom associated with diarrhea" may be used to refer to at least one of: a decrease in the volume of loose, watery stools per day; or a decrease in the frequency of loose, watery stools per day; or a 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% decrease in any combination thereof.

[0066] As used herein, the term "bacterial secretagogue" may be used to refer to substances produced by bacteria that promote the secretion of anions from intestinal crypts.

[0067] As described herein, a reduction in at least one symptom associated with diarrhea can be determined by, for example, at least one of: measuring the volume and / or frequency of loose, watery stools per day using a standard volumetric measuring device (e.g., a sample collection container with tare measurement for quantitative purposes); or by counting the number of loose, watery stools per day and recording by some standard means; or any combination thereof.

[0068] The present inventors have established a model system in which net fluid absorption is assessed in the mammalian intestinal environment. More specifically, an anesthetized rat model for intestinal lumen perfusion has been developed that can be performed in the presence or absence of a secretagogue. The ability to perform the assay in the presence or absence of a secretagogue simulates the intestinal conditions of a subject simulating diarrheal disease caused by a bacterial infection, regardless of the ability of the bacteria to produce the secretagogue. Since dehydration is a serious and life-threatening consequence of diarrheal disease, the quantification of the increase in net fluid absorption conferred by the formulations described herein in the perfusion assay reflects the therapeutic benefit conferred by these formulations when used to treat subjects suffering from diarrheal disease.

[0069] Based on the results presented herein, F1, F2, and F3 were selected as exemplary formulations that demonstrated the ability to increase net fluid absorption relative to a negative control solution (Ringer's solution) and a statistically significant increase in net fluid absorption compared to WHO-ARS (Trioral), the standard of care for the treatment of diarrheal disease worldwide. The superiority of each of F1, F2, and F3 relative to Trioral in the ability to increase net fluid absorption was notable under all experimental conditions tested (regardless of secretagogue status), but was particularly pronounced in the presence of secretagogues. See, e.g., Figure 1 and 2 As described in detail herein, net fluid uptake assays performed in the presence of a secretagogue (e.g., forskolin or cholera toxin) serve as a model to mimic conditions in the intestine of a subject suffering from diarrheal disease caused by infection with secretagogue-producing bacteria.

[0070] As described herein, the increase in net fluid uptake can be determined, for example, by measuring the net fluid uptake in a perfusion assay using a mammalian lumen intestinal segment, and the quantitative measurement for a particular formulation can be compared to, for example, the net fluid uptake of a negative control solution in the perfusion assay to determine the percent increase relative to the negative control solution. In addition, the quantitative measurement for a particular formulation can be compared to, for example, the net fluid uptake of a positive control solution in the perfusion assay as a relative comparator for predicting efficacy.

[0071] Throughout the specification and claims, the following terms have the meanings explicitly associated herein, unless the context clearly indicates otherwise. As used herein, the expressions "in one embodiment," "in an embodiment," and "in some embodiments" do not necessarily refer to the same embodiment or embodiments, although they may. Furthermore, as used herein, the expressions "in another embodiment" and "in some other embodiments" do not necessarily refer to different embodiments, although they may. All embodiments of the present disclosure are intended to be combinable without departing from the scope or spirit of the present disclosure.

[0072] As used herein, the term "based on" is not exclusive and allows for being based on additional factors not described, unless the context clearly dictates otherwise. Additionally, throughout this specification, the meanings of "a," "an," and "the" include plural references. The meaning of "in..." includes "in" and "on."

[0073] An "effective amount" or "effective dose" of an agent (or a composition containing such an agent) refers to an amount sufficient to achieve a desired biological and / or pharmacological effect, for example, when delivered to a cell or organism according to a selected form of administration, route, and / or schedule. The phrases "effective amount" and "therapeutically effective amount" are used interchangeably. As will be understood by one of ordinary skill in the art, the absolute amount of a particular agent or composition that is effective may vary depending on factors such as the desired biological or pharmacological endpoint, the agent to be delivered, the target tissue, and the like. One of ordinary skill in the art will further understand that, in various embodiments, an "effective amount" may be contacted with a cell or administered to a subject in a single dose or by using multiple doses. In some embodiments, an effective amount is an amount that increases net fluid absorption in a cell. In some embodiments, an effective amount is an amount that increases net fluid absorption in a subject in need thereof. In some embodiments thereof, an effective amount is an amount that increases net fluid absorption in the intestinal tract of a subject in need thereof. In some embodiments, an effective amount is an amount that reduces at least one symptom of diarrhea associated with decreased fluid absorption. In some embodiments thereof, an effective amount is an amount that reduces at least one symptom of diarrhea associated with decreased fluid absorption in the intestinal tract of a subject. In some embodiments thereof, the effective amount is an amount that reduces at least one symptom of diarrhea associated with a bacterial infection in the intestinal tract of a subject. In some embodiments thereof, the effective amount is an amount that reduces at least one symptom of diarrhea associated with a bacterial infection in the intestinal tract of a subject, wherein the bacterial infection is associated with bacteria that produce a secretagogue. In some embodiments thereof, the effective amount is an amount that reduces at least one symptom of diarrhea associated with a bacterial infection in the intestinal tract of a subject, wherein the bacterial infection is associated with bacteria that do not produce a secretagogue.

[0074] As used herein, "treat," "treating," "treatment," and similar terms in the context of treating a subject refer to providing medical and / or surgical management of a subject. Treatment may include, but is not limited to, administering an agent or composition (e.g., a pharmaceutical composition) to a subject. As used herein, the term "treatment," or any grammatical variant thereof (e.g., treat, treat, treat, and treat, etc.), includes, but is not limited to, alleviating at least one symptom of a disease or condition; and / or reducing, suppressing, inhibiting, alleviating, or affecting the progression, severity, and / or extent of a disease or condition.

[0075] The therapeutic effect may also include reducing the likelihood of developing or recurring the disease or at least one symptom or manifestation of the disease. The therapeutic agent may be administered to a subject who has the disease or is at increased risk of developing the disease relative to members of the general population. In some embodiments, the therapeutic agent may be administered to a subject who has the disease but no longer shows evidence of the disease. The agent may be administered, for example, to reduce the likelihood of recurrence of the disease. The therapeutic agent may be administered prophylactically, i.e., before at least one symptom or manifestation of the disease appears.

[0076] "Prophylactic treatment" refers to providing medical and / or surgical management to a subject who does not yet have a disease or show evidence of a disease, e.g., to reduce the likelihood of developing the disease or to reduce the severity of the disease if it does occur. The subject may have been identified as being at risk for developing the disease (e.g., having an increased risk relative to the general population, or having risk factors that increase the likelihood of developing the disease).

[0077] As used herein, the term "amelioration" or any grammatical variations thereof (e.g., ameliorate, ameliorating, and amelioration, etc.) includes, but is not limited to, delaying the onset of or reducing the severity of a disease or condition (e.g., a disease or condition associated with decreased fluid absorption or complications thereof). As used herein, amelioration does not require the complete absence of symptoms.

[0078] The terms "condition," "disease," and "disorder" are used interchangeably.

[0079] In various embodiments, a "subject" can be any vertebrate organism. A subject can be an individual to whom a reagent is administered (e.g., for experimental, diagnostic, and / or therapeutic purposes) or from whom a sample is obtained or to whom a procedure is performed. In some embodiments, the subject is a mammal, such as a human; a non-human primate (e.g., an ape, chimpanzee, orangutan, monkey); or a domesticated animal, such as a dog, cat, rabbit, cow, bull, horse (including, for example, a foal), pig, sheep, goat, llama, mouse, and rat. In some embodiments, the subject is a human. The human or other mammal can be of either sex and can be at any stage of development. In some embodiments, the human or other mammal is a cub [e.g., a human cub; an infant (a human cub from birth to 1 year old), a juvenile (a toddler or young child under 5 years old), or an elderly subject (e.g., a person over 70 years old). In some embodiments, the subject has been diagnosed with diarrhea, which is associated with reduced fluid absorption.

[0080] "Negligible amount" means that the free amino acid present does not increase fluid absorption in, for example, an enteral perfusion assay. Alternatively, in some embodiments, even if the free amino acid is present in the formulation, it is not present in an amount that would affect fluid absorption in, for example, an enteral perfusion assay or the therapeutic effect of treating a subject in need thereof. In some embodiments, a negligible amount is an amount wherein the total concentration of the free amino acid is less than 100 mg / l, 50 mg / l, 10 mg / l, 5 mg / l, 1 mg / l, 0.5 mg / l, 0.1 mg / l, or 0.01 mg / l. In some embodiments, a negligible amount is an amount wherein the total concentration of the free amino acid is less than 100 mg / l. In some embodiments, a negligible amount is an amount wherein the total concentration of the free amino acid is less than 50 mg / l. In some embodiments, a negligible amount is an amount wherein the total concentration of the free amino acid is less than 10 mg / l. In some embodiments, a negligible amount is an amount wherein the total concentration of the free amino acid is less than 5 mg / l. In some embodiments, a negligible amount is an amount wherein the total concentration of the free amino acid is less than 1 mg / l. In some embodiments, a negligible amount is an amount wherein the total concentration of free amino acids is less than 0.5 mg / l. In some embodiments, a negligible amount is an amount wherein the total concentration of free amino acids is less than 0.1 mg / l. In some embodiments, a negligible amount is an amount wherein the total concentration of free amino acids is less than 0.01 mg / l.

[0081] The term "amino acid" encompasses all known amino acids comprising an amine (-NH2) functional group, a carboxyl (-COOH) functional group, and a side chain ("R") group specific to each amino acid. "Amino acid" encompasses the 21 amino acids encoded by the human genome (i.e., proteinogenic amino acids), amino acids encoded or produced by bacteria or unicellular organisms, and naturally derived amino acids. For the purposes of this disclosure, unless otherwise indicated, the conjugate acid form of amino acids with basic side chains (arginine, lysine, and histidine) or the conjugate base form of amino acids with acidic side chains (aspartic acid and glutamic acid) are substantially identical. "Amino acid" also encompasses derivatives thereof that maintain substantially the same activity in, for example, an intestinal perfusion assay in increasing fluid absorption. The derivatives can be, for example, enantiomers, and include the D and L forms of amino acids. The derivatives can be derivatives of "natural" or "non-natural" amino acids (e.g., β-amino acids, homoamino acids, proline derivatives, pyruvate derivatives, 3-substituted alanine derivatives, glycine derivatives, ring-substituted tyrosine derivatives, ring-substituted phenylalanine derivatives, linear core amino acids and N-methyl amino acids), such as selenocysteine, pyrrolysine, iodinated tyrosine, norleucine or norvaline. Other amino acid derivatives include, but are not limited to, those synthesized by, for example, acylation, methylation, glycosylation and / or halogenation of amino acids. These include, for example, β-methyl amino acids, C-methyl amino acids and N-methyl amino acids. The amino acids described herein can exist in the form of free amino acids. The term "free amino acid" refers to an amino acid that is not part of a peptide or polypeptide (e.g., not linked to another amino acid by a peptide bond). A free amino acid is free in solution (as opposed to being linked to at least one other amino acid by, for example, a dipeptide bond), but can associate with salts or other components in solution.

[0082] As used herein, the term "salt" refers to any and all salts and encompasses pharmaceutically acceptable salts.

[0083] The term "carrier" may refer to a diluent, adjuvant, excipient, or vehicle with which the formulations described herein are administered. Examples of suitable pharmaceutical carriers are described in Remington's Essentials of Pharmaceuticals, 21st edition, ed. Felton, 2012, which is incorporated herein by reference.

[0084] Exemplary salts for inclusion in the formulations described herein include sodium chloride, potassium chloride, calcium chloride, magnesium chloride or trisodium citrate, sodium bicarbonate, sodium gluconate, phosphate buffer using monosodium phosphate, disodium phosphate or trisodium phosphate, or any combination thereof.

[0085] Exemplary diluents include calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, dibasic calcium phosphate, cellulose, microcrystalline cellulose, kaolin, sodium chloride, and mixtures thereof.

[0086] Pharmaceutically acceptable excipients for use in the manufacture of the pharmaceutical formulations described herein include inert diluents, dispersants and / or granulating agents, surfactants and / or emulsifiers, disintegrants, binders, preservatives, buffers, lubricants and / or oils. Excipients such as cocoa butter and suppository waxes, colorants, coating agents, and flavoring agents may also be present in the composition.

[0087] The exact amount of the amino acid preparation or composition required for realizing effective dose will vary between subjects, depending on, for example, the species, age and general condition of the subject, the severity of side effect or disease, mode of administration, etc. Effective dose can be included in a single dose (e.g., a single oral dose) or multiple doses (e.g., a plurality of oral doses). In certain embodiments, when multiple doses are applied to a subject or multiple doses are applied to tissue or cell, any two doses in multiple doses include different amounts or substantially the same amount of amino acid composition as herein described. In certain embodiments, when multiple doses are applied to a subject or multiple doses are applied to tissue or cell, the frequency of multiple doses applied to a subject or multiple doses is three doses per day, two doses per day, one dose per day, one dose every other day, one dose every three days, one dose per week, one dose every two weeks, one dose every three weeks, or one dose every four weeks. In certain embodiments, the frequency of multiple doses applied to a subject or multiple doses is one dose per day. In certain embodiments, the frequency of multiple doses applied to a subject or multiple doses is two doses per day. In certain embodiments, the frequency of multiple doses applied to a subject or multiple doses is three doses per day. In some embodiments, when multiple doses are administered to a subject or applied to a tissue or cell, the duration between the first dose and the last dose of the multiple doses is one day, two days, four days, one week, two weeks, three weeks, one month, two months, three months, four months, six months, nine months, one year, two years, three years, four years, five years, seven years, ten years, fifteen years, twenty years, or the life of the subject, tissue, or cell. In some embodiments, the duration between the first dose and the last dose of the multiple doses is three months, six months, or one year. In some embodiments, the duration between the first dose and the last dose of the multiple doses is the life of the subject, tissue, or cell.

[0088] In some embodiments, the doses described herein (e.g., a single dose, or any of a plurality of doses) independently include between 0.1 μg and 1 μg, between 0.001 mg and 0.01 mg, between 0.01 mg and 0.1 mg, between 0.1 mg and 1 mg, between 1 mg and 3 mg, between 3 mg and 10 mg, between 10 mg and 30 mg, between 30 mg and 100 mg, between 100 mg and 300 mg, between 300 mg and 1,000 mg, between 1 g and 10 g, between 1 g and 15 g, or between 1 g and 20 g (inclusive) of an amino acid composition described herein. In some embodiments, the doses described herein independently include between 1 mg and 3 mg (inclusive) of an amino acid composition described herein. In some embodiments, the doses described herein independently include between 3 mg and 10 mg (inclusive) of an amino acid composition described herein. In some embodiments, the doses described herein independently include between 10 mg and 30 mg (inclusive) of an amino acid composition described herein. In some embodiments, the dosages described herein independently comprise between 30 mg and 100 mg, inclusive, of an amino acid composition described herein.

[0089] The dosage ranges described herein provide guidance for administering the pharmaceutical compositions described herein to adults. The amount administered to, for example, children or adolescents can be determined by a physician or person skilled in the art and can be lower than or equal to the amount administered to adults.

[0090] All prior patents, publications, and test methods cited herein are incorporated by reference in their entirety.

[0091] Detailed Description of Some Embodiments

[0092] In some embodiments, the composition comprises, consists essentially of, or consists of the free amino acids of proline (P) and aspartic acid (D), and the free amino acids of at least one of serine (S), threonine (T), glycine (G), alanine (A), arginine (R), or tyrosine (Y), and optionally asparagine (N). Different combinations of this embodiment are presented in List 1 as follows: Eight AA set: P, D, S, T, G, A, R, and Y. In some embodiments thereof, the composition comprises, consists essentially of, or consists of the free amino acids of P, D, S, T, G, A, R, and Y (F3 and F2). In some embodiments thereof, the composition comprises, consists essentially of, or consists of the free amino acids of P, D, S, T, N, A, R, and Y (8AA-OP). Seven AA subsets: P, D, S, T, G, A, and R; P, D, S, T, G, A, and Y; P, D, S, T, G, R, and Y; P, D, S, T, A, R, and Y; P, D, S, G, A, R, and Y; and P, D, T, G, A, R, and Y. In some embodiments thereof, the composition comprises, consists essentially of, or consists of: P, D, S, T, G, A, and R; P, D, S, T, G, A, and Y; P, D, S, T, G, R, and Y; P, D, S, T, A, R, and Y; P, D, S, G, A, R, and Y; or the free amino acids of P, D, T, G, A, R, and Y. In some embodiments thereof, N may replace G. Six AA subsets: P, D, S, T, G, and A; P, D, S, T, G, and R; P, D, S, T, G, and Y; P, D, S, T, A, and R; P, D, S, T, A, and Y; P, D, S, T, R, and Y; P, D, S, G, A, and R; P, D, S, G, A, and Y; P, D, S, G, R, and Y; P, D, S, A, R, and Y; P, D, T, G, A, and R; P, D, T, G, A, and Y; P, D, T, G, R, and Y; P, D, T, A, R, and Y; and P, D, G, A, R, and Y. In embodiments thereof, the composition comprises, consists essentially of, or consists of P, D, S, T, G, and A; P, D, S, T, G, and R; P, D, S, T, G, and Y; P, D, S, T, A, and R; P, D, S, T, A, and Y; P, D, S, T, R, and Y; P, D, S, G, A, and R; P, D, S, G, A, and Y; P, D, S, G, R, and Y; P, D, S, A, R, and Y; P, D, T, G, A, and R; P, D, T, G, A, and Y; P, D, T, G, R, and Y; P, D, T, A, R, and Y; or the free amino acids of P, D, G, A, R, and Y. In some embodiments thereof, N may replace G.Five AA subsets: P, D, S, T and G; P, D, S, T and A; P, D, S, T and R; P, D, S, T and Y; P, D, S, G and A; P, D, S, G and R; P, D, S, G and Y; P, D, S, A and R; P, D, S, A and Y; P, D, S, R and Y; P, D, T, G and A; P, D, T, G and R; P, D, T, G and Y; P, D, T, A and R; P, D, T, A and Y; P, D, T, R and Y; P, D, G, A and R; P, D, G, A and Y; P, D, G, R and Y; and P, D, A, R and Y. In embodiments thereof, the composition comprises, consists essentially of, or consists of: D, S, T, and Y; P, D, S, G, and A; P, D, S, G, and R; P, D, S, G, and Y; P, D, S, A, and R; P, D, S, A, and Y; P, D, S, R, and Y; P, D, T, G, and A; P, D, T, G, and R; P, D, T, G, and Y; P, D, T, A, and R; P, D, T, A, and Y; P, D, T, R, and Y; P, D, G, A, and R; P, D, G, A, and Y; P, D, G, R, and Y; or the free amino acids of P, D, A, R, and Y. In some embodiments thereof, N may replace G. Four AA subsets: P, D, S, and T; P, D, S, and G; P, D, S, and A; P, D, S, and R; P, D, S, and Y; P, D, T, and G; P, D, T, and A; P, D, T, and R; P, D, T, and Y; P, D, G, and A; P, D, G, and R; P, D, G, and Y; P, D, A, and R; P, D, A, and Y; and P, D, R, and Y. In embodiments thereof, the composition comprises, consists essentially of, or consists of: P, D, S, and T; P, D, S, and G; P, D, S, and A; P, D, S, and R; P, D, S, and Y; P, D, T, and G; P, D, T, and A; P, D, T, and R; P, D, T, and Y; P, D, G, and A; P, D, G, and R; P, D, G, and Y; P, D, A, and R; P, D, A, and Y; or the free amino acids of P, D, R, and Y. In some embodiments thereof, N may replace G. Triple AA subset: P, D, and S; P, D, and T; P, D, and G; P, D, and A; and P, D, and R. In embodiments thereof, the composition comprises, consists essentially of, or consists of: P, D, and S; P, D, and T; P, D, and G; P, D, and A; or the free amino acids of P, D, and R. In some embodiments thereof, N may replace G.

[0093] Thus, encompassed herein are formulations (e.g., pharmaceutical formulations) comprising the selected eight amino acids (P, D, S, T, G, A, R, and Y) of F3 and F2, and subsets thereof comprising three, four, five, six, or seven amino acid subsets of the selected eight amino acids, and their use for increasing net fluid absorption in the intestine of a subject in need thereof and / or treating diarrhea associated with decreased fluid absorption and for the preparation of a medicament for treating diarrhea associated with decreased fluid absorption. In some embodiments, the diarrhea is caused by secretagogue-producing bacteria. In some embodiments, N can replace G in the formulations described herein. The above reasoning also applies to any combination of two (P, D), three, four, five, six, or seven amino acid subsets of the selected eight amino acids (P, D, S, T, G, A, R, and Y) described herein.

[0094] Among other things, encompassed herein are formulations (e.g., pharmaceutical formulations) comprising the selected eight amino acids (P, D, S, T, N, A, R, and Y) of F1 and subsets thereof comprising three, four, five, six, or seven amino acid subsets of the selected eight amino acids, and their use for increasing net fluid absorption in the intestine of a subject in need thereof and / or treating diarrhea associated with decreased fluid absorption and for the preparation of a medicament for treating diarrhea associated with decreased fluid absorption. In some embodiments, the diarrhea is caused by secretagogue-producing bacteria. The above reasoning also applies to any combination of two (P, D), three, four, five, six, or seven amino acid subsets of the selected eight amino acids (P, D, S, T, N, A, R, and Y) described herein.

[0095] Exemplary formulations described herein are presented in Tables 1-4 below (mmol refers to mmol / liter in each formulation presented below):

[0096]

[0097] Several control solutions were used in the experimental results presented in this article, including:

[0098] Negative control - Ringer's solution containing 105 mM Na, 241 mOsm, pH 4.049

[0099] Comparative solution - 8AA: Aspartic acid, glycine, valine, isoleucine, threonine, tyrosine, serine and lysine, 67 mM Na, 243 mOsm, pH 3.82. Perfusion (mean) = 3.83

[0100] Comparative solution - WHO ORS (Trioral): 75 mM Na, 75 mM glucose, 221-225 mOsm, pH 7.5-7.8. Perfusion (mean) = 2.56

[0101] Comparative solution - 6AA: aspartic acid, valine, isoleucine, threonine, tyrosine and lysine, 67 mM Na, 245 mOsm, pH 3.7

[0102] F3: Aspartic acid, glycine, threonine, tyrosine, serine, proline, alanine, and arginine (57.2 mM). Na (67 mM), 221 mOsmo, pH 4.096 (-&n=4)

[0103] In some embodiments, the formulations described herein may optionally contain the monosaccharide glucose, at least one disaccharide containing glucose, or any combination thereof, wherein the total concentration of the monosaccharide glucose, at least one disaccharide containing glucose, or any combination thereof is equal to or less than 90 mM. In embodiments thereof, the monosaccharide glucose, at least one disaccharide containing glucose, or any combination thereof is equal to or less than 85 mM; the monosaccharide glucose, at least one disaccharide containing glucose, or any combination thereof is equal to or less than 80 mM; the monosaccharide glucose, at least one disaccharide containing glucose, or any combination thereof is equal to or less than 75 mM; the monosaccharide glucose, at least one disaccharide containing glucose, or any combination thereof is equal to or less than 70 mM; the monosaccharide glucose, at least one disaccharide containing glucose, or any combination thereof is equal to or less than 65 mM; the monosaccharide glucose, at least one disaccharide containing glucose, or any combination thereof is equal to or less than 60 mM; the monosaccharide glucose, at least one disaccharide containing glucose, or any combination thereof is equal to or less than 55 mM; the monosaccharide glucose, at least one disaccharide containing glucose, or any combination thereof is equal to or less than 50 mM; the monosaccharide glucose, at least one disaccharide containing glucose, or any combination thereof is equal to or less than 10 mM; or any combination thereof.

[0104] In embodiments thereof, the monosaccharide glucose, at least one disaccharide containing glucose, or any combination thereof is in the range of 10-90 mM; in the range of 10-85 mM; in the range of 10-80 mM; in the range of 10-75 mM; in the range of 10-70 mM; in the range of 10-65 mM; in the range of 10-60 mM; in the range of 10-55 mM; in the range of 10-50 mM; in the range of 10-45 mM; in the range of 10-40 mM; in the range of 10-35 mM; in the range of 10-30 mM; in the range of 10-25 mM; in the range of 10-20 mM; in the range of 5-90 mM; in the range of 5-85 mM; in the range of 5-80 mM; in the range of 5-75 mM; in the range of 5-70 mM; in the range of 5-65 mM. in the range of 5-60 mM; in the range of 5-55 mM; in the range of 5-50 mM; in the range of 5-45 mM; in the range of 5-40 mM; in the range of 5-35 mM; in the range of 5-30 mM; in the range of 5-25 mM; in the range of 5-20 mM; in the range of 1-90 mM; in the range of 1-85 mM; in the range of 1-80 mM; in the range of 1-75 mM; in the range of 1-70 mM; in the range of 1-65 mM; in the range of 1-60 mM; in the range of 1-55 mM; in the range of 1-50 mM; in the range of 1-45 mM; in the range of 1-40 mM; in the range of 1-35 mM; in the range of 1-30 mM; in the range of 1-25 mM; or in the range of 1-20 mM.

[0105] In certain embodiments, the therapeutic composition does not contain any sugar, including any monosaccharide, disaccharide, oligosaccharide, polysaccharide and carbohydrate. In certain embodiments, the therapeutic composition does not contain glucose and / or any disaccharide, oligosaccharide, polysaccharide and carbohydrate that can be hydrolyzed into glucose. In certain embodiments, the composition does not contain lactose. In certain embodiments, the therapeutic composition does not contain fructose and / or galactose, and / or any disaccharide, oligosaccharide, polysaccharide and carbohydrate that can be hydrolyzed into fructose and / or galactose.

[0106] As used herein, the term "consisting essentially of" limits the scope of ingredients and steps to the specified materials or steps, and those materials or steps that do not materially affect the basic and novel features of the invention, such as the compositions for treating diarrhea associated with impaired fluid absorption, their uses, and methods for treating diarrhea associated with impaired fluid absorption. For example, by using "consisting essentially of," the therapeutic composition is free of any ingredients not explicitly recited in the claims, including, but not limited to, free amino acids, dipeptides, oligopeptides, or polypeptides, or proteins; and monosaccharides, disaccharides, oligosaccharides, polysaccharides, and carbohydrates that have a direct therapeutic effect on treating diarrhea associated with impaired fluid absorption. In the context of "consisting essentially of," the change in therapeutic effect conferred by the additional ingredients can be determined based on the change in fluid absorption in a rat intestinal perfusion assay, where an increase or decrease of up to 1%, 2%, 3%, 4%, or 5% can fall within the scope of the term "consisting essentially of." Additionally, by using the term "consisting essentially of," the composition can include substances that do not have a therapeutic effect on treating diarrhea associated with impaired fluid absorption.

[0107] Variations, modifications, and changes to the embodiments of the present disclosure described above will be apparent to those skilled in the art. All such variations, modifications, changes, etc. are intended to fall within the spirit and scope of the present disclosure, which is limited only by the appended claims.

[0108] Although several embodiments of the present disclosure have been described, it should be understood that these embodiments are illustrative only and not restrictive, and that many modifications may be apparent to those skilled in the art. For example, all dimensions discussed herein are provided as examples only and are intended to be illustrative rather than restrictive.

[0109] Any feature or element explicitly identified in this specification may also be specifically excluded as a feature or element of an embodiment of the disclosure as defined in the claims.

[0110] The disclosure described herein may be practiced in the absence of any element or elements, limitation or limitations not specifically disclosed herein. The terms and expressions employed are used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions to exclude any equivalents of the features shown and described, or portions thereof, but it is recognized that various modifications are possible within the scope of the disclosure.

[0111] Exemplary methods include: electrophysiological techniques: a) measuring benzamiloride-sensitive current (electrogenic sodium current mediated by ENaC), bumetanide-sensitive current, and transepithelial resistance in an Ussing chamber; b) using 22Na to determine net Na absorption and use 36 Cl flux studies using Ussing chambers for chloride secretion; and c) permeability assays using fluorescein isothiocyanate (FITC)-dextran (4KD) added directly to the chamber.

[0112] Ussing Chamber - Sodium Flux (Universal)

[0113] Small intestinal mucosal tissue (ileum and jejunum) from 8-week-old male Swiss mice was mounted in an Ussing chamber containing isotonic Ringer's solution bubbled with 95% O2 and 5% CO2 and maintained at 37°C throughout the experiment. After allowing the tissue to stabilize, conductance (G; expressed as mS / cm 2 ) and paired intestinal tissues based on similar conductance. Sodium radioisotopes ( 22 Na) was added to the basolateral or apical (hot) side of each tissue pair. Ringer samples were collected from the contralateral (cold) side every 15 minutes. Samples were analyzed using a gamma counter. 22 Na activity, and the unidirectional net sodium flux (Jnet; μeq·cm 2 ·h -1 ).

[0114]

[0115] [CPM = counts per minute, CPM1 = previous sample, CPM2 = next sample; blank = not added 22 =Na; 9 / 10 = dilution factor per sample (0.5 mL to 5 mL); 5 = chamber volume (5 mL); 4 = time factor (15 min to 60 min); 140 = sodium concentration; hot CPM = activity of "hot" sample; cold CPM = activity of "cold" sample; 10 = volume factor of hot sample (0.1 mL to 1 mL); 0.3 = intestinal surface area (cm 2 )]

[0116] Molecular biology techniques: mRNA expression of ENaC (α, β, and γ), claudin 1, 2, 5, 7, and 8, occludin, and E-cadherin), acid-sensing ion channel (ASIC1a), and aquaporins 1 and 5 were performed by qRT-PCR.

[0117] Western blot analysis and immunohistochemistry: Western blot analysis and / or immunohistochemistry to determine the protein levels and expression of ENaC (α, β, and γ), tight junction proteins (claudins 1, 2, 5, 7, and 8, occludin, and E-cadherin), acid-sensing ion channel (ASIC1a), and aquaporins 1 and 5.

[0118] Examples

[0119] Example 1

[0120] Methods: The small intestine of mice was used to study electrolyte absorption and intestinal barrier function (short-circuit current Isc and conductance G), and chloride ( 36 Cl) and sodium ( 22 The net movement of sodium (Na) was studied in Ussing chamber flux studies to quantify anion and cation absorption capacity. The absorption and secretion properties of AA-ORS were screened in the presence and absence of the cAMP secretagogue: forskolin (Fsk), which serves as a cholera toxin-like secretagogue. Individual mouse small intestinal loops were incubated in Ringer's solution (RR) or each of the designated exemplary AA-ORS to determine the absorption and secretion properties of these formulations, and brush border membrane vesicles (BBMVs) were isolated from the small intestinal loops to determine the molecular properties of the transporters that mediate these absorption / secretion properties. The exemplary AA-ORS described herein were evaluated using a number of criteria, including but not limited to: sodium absorption capacity, chloride secretory activity, effects on barrier function, and protein expression levels of transporters or channels that mediate sodium and chloride absorption.

[0121] The exemplary AA-ORS described herein exhibit enhanced electrolyte absorption. The combination of free amino acids in each exemplary AA-ORS exhibits synergistic effects on a variety of criteria including net fluid absorption. See, e.g. Figure 1-3 . Exemplary AA-ORS formulations (e.g., F1, F4, F2, and F3) were tested in the presence of CT in an anesthetized rat model for intestinal lumen perfusion. Lumen perfusion studies showed that F3 exhibited favorable properties in promoting net fluid absorption, as reflected in a statistically significant increase of approximately 2.3-fold in net fluid absorption in the presence of CT when compared to WHO-ORS in the presence of CT. F1 and F2 also exhibited favorable properties in promoting net fluid absorption, as reflected in a statistically significant increase of approximately 2.0-fold in net fluid absorption in the presence of CT when compared to WHO-ORS in the presence of CT. See, e.g. Figure 2 .

[0122] Lumen perfusion studies also demonstrated that FF3 exhibited favorable properties in promoting net fluid uptake, as reflected by a statistically significant increase of 56% in net fluid uptake in the presence of CT compared to 8AA in the presence of CT. F1 also exhibited favorable properties in promoting net fluid uptake, as reflected by a statistically significant increase of 42% in net fluid uptake in the presence of CT compared to 8AA in the presence of CT. F2 also exhibited favorable properties in promoting net fluid uptake, as reflected by a statistically significant increase of 31% in net fluid uptake in the presence of CT compared to 8AA in the presence of CT. In contrast, F4, which contains 8 free amino acids (many of which are the same as F1, F2, and F3), had a statistically significantly lower net fluid uptake relative to 8AA. See Figure 1 .

[0123] Exemplary formulations F1, F2, and F3 each contain the following free amino acids: proline, aspartic acid, serine, arginine, tyrosine, threonine, and alanine. F2 and F3 contain the free amino acids of proline, aspartic acid, serine, arginine, tyrosine, threonine, alanine, and glycine. F1 contains the free amino acids of proline, aspartic acid, serine, arginine, tyrosine, threonine, alanine, and asparagine.

[0124] Figure 1-3 The results presented in demonstrate that factors such as the supplementation of free amino acids present in the amino acid formulation and their concentration contribute synergistically to the activity of the amino acid formulation in terms of the net fluid absorption conferred thereby.

[0125] Example 2

[0126] Studies can be conducted to evaluate the efficacy of the amino acid formulations described herein. Such studies may include formulations that are considered standard of care for treating subjects with acute gastroenteritis. Acute gastroenteritis is a disease state that occurs when edible food or water contaminated by pathogenic microorganisms (such as Clostridium perfringens, Vibrio cholerae, Escherichia coli) or their toxins. The symptoms of acute gastroenteritis include at least one of nausea, vomiting, diarrhea or abdominal pain, or any combination thereof. WHO-ORS is a standard low osmotic pressure glucose ORS, an exemplary formulation for treating diarrhea, which is routinely used in studies for treating diarrhea. Therefore, WHO-ORS can serve as a positive control in the studies described herein.

[0127] Research Design and Methods

[0128] Design: Randomized, double-blind, two-unit study

[0129] Study population: The study subjects were male children (to facilitate separate collection of urine and feces).

[0130] Inclusion criteria: Age: 6 months to 36 months; diarrhea duration ≤ 48 hours; some degree of dehydration (clinically judged according to hospital accepted practice); written informed consent from parents / guardians.

[0131] Exclusion criteria: severe malnutrition; patients with diarrhea caused by cholera; systemic diseases; bloody diarrhea; any congenital anomaly or condition; and records of taking antibiotics and / or antidiarrheal drugs within 48 hours before hospitalization.

[0132] Study Procedure

[0133] Screening: All male children aged 6-36 months who present with diarrhea (onset ≤ 48 hours) and some degree of dehydration will be screened by a research nurse for possible study inclusion. All screened children will remain in the hospital for a thorough clinical history and physical examination. Weight and height will be measured, and dehydration will be corrected according to standard rehydration protocols.

[0134] Enrollment: Participants enrolled in the study will be assigned to one of the ORS groups according to a predetermined randomization schedule. Enrolled children will then be randomized into two groups, one of which will be treated with either the exemplary amino acid ORS described herein or the WHO ORS.

[0135] Blinding Techniques: All participants in the study will be blinded to the identity of the specific product administered. Product category will be unblinded only after statistical analysis has been performed.

[0136] Case Management: Study patients will be placed on a bed with diarrhea. Initial treatment with, for example, WHO-ORS or the exemplary amino acid ORS described herein will be administered using body weight at an estimated dose of 5-10 ml / kg after each loose stool, according to hospital guidelines, to maintain ongoing losses. Adjustments may be necessary, using smaller or larger volumes to maintain hydration depending on measured losses. Children will be fed with a spoon. Assessment and treatment will continue until the diarrhea resolves. Each participating child will be closely monitored for any adverse events, such as worsening diarrhea or increased vomiting, and their severity and duration will be noted and recorded for analysis.

[0137] Laboratory research

[0138] Routine: Blood will be obtained by venipuncture upon admission and 24 hours after enrollment to determine serum electrolytes, and blood glucose will be determined. A complete blood count, serum creatinine, routine stool examination, bacterial culture, and rotavirus antigen will also be performed on all patients only upon admission. Other clinical laboratory tests may be performed if clinically indicated.

[0139] definition

[0140] Duration of in-hospital diarrhea: time in hours from randomization to resolution of diarrhea.

[0141] Resolution of diarrhea: passage of the last liquid or semi-liquid stool or absence of a stool for 12 hours before a soft / formed stool.

[0142] Stool output: Stool weight expressed in g / kg admission body weight for each time period (ie, every 24 hours and for the entire duration of diarrhea).

[0143] ORS and fresh water intake: The total volume of ORS or fresh water consumed per kg of admission body weight (in ml) for each time period.

[0144] Sample size calculation and outcome (primary and exploratory) variables

[0145] Sample size: Based on similar studies, using the pooled standard deviation reported within each outcome, a 20% clinically significant improvement yields effect sizes of 0.4 to 0.7 (duration) and 0.3 (stool output).

[0146] Outcome measures / variables

[0147] Primary outcome: Duration of diarrhea in hospital (hours)

[0148] Exploratory outcomes: Fecal output (g / kg body weight) during the first 24 hours of hospitalization, further divided into two 12-hour periods; total fecal output during hospitalization (g / kg body weight); ORS intake (g / kg body weight) during the first 24 hours of hospitalization; total ORS intake (ml / kg body weight); weight change (between pre-randomization and post-treatment); urine output (g / kg body weight) during the first 24 hours of hospitalization; total urine output (g / kg body weight) during hospitalization.

[0149] Before statistical analysis of efficacy endpoints is performed, differences between treatment groups with respect to baseline characteristics will be examined. If any differences are considered clinically important, subgroup analyses of relevant endpoints will be provided.

[0150] Depending on the normality of the distribution of quantitative variables, t-tests or Mann-Whitney U tests will be used to compare stool output, vomitus output, ORS intake, and / or duration of diarrhea. Primary outcomes will be compared with and without adjustment for design effects (age strata) and patient characteristics (etiology, clinical characteristics at admission, nutritional status). All analyses will be performed individually for children according to the protocol and on an intention-to-treat basis for all children.

Claims

1. A formulation for treating diarrhea in a subject in need thereof, wherein the formulation comprises: A therapeutically effective combination of free amino acids, wherein the therapeutically effective combination of free amino acids consists of: Alanine, arginine, asparagine, aspartic acid, proline, serine, threonine, tyrosine; or Alanine, arginine, aspartic acid, glycine, proline, serine, threonine, tyrosine, Optionally, The monosaccharide glucose, at least one disaccharide containing glucose, or Any combination thereof, wherein the total concentration of the monosaccharide glucose, the at least one glucose-containing disaccharide, or any combination thereof is equal to or less than 90 mM; or Optionally, at least one pharmaceutically acceptable carrier, buffer, electrolyte, adjuvant, excipient or water, or any combination thereof; or any combination thereof; and wherein the therapeutically effective combination of free amino acids is sufficient to reduce at least one symptom associated with the diarrhea in the subject.

2. The formulation of claim 1 , wherein the concentration of proline is in the range of 0.5 mM to 13 mM; wherein the concentration of aspartic acid is in the range of 1 mM to 18 mM; wherein the concentration of serine is in the range of 1.5 mM to 12 mM; wherein the concentration of threonine is in the range of 0.5 mM to 12 mM; wherein when glycine is present, the concentration of glycine is in the range of 0.4 mM to 12 mM; wherein the concentration of alanine is in the range of 1.5 mM to 12 mM; wherein the concentration of arginine is in the range of 0.8 mM to 12 mM; wherein the concentration of tyrosine is in the range of 0.1 mM to 1.2 mM; wherein when asparagine is present, the concentration of asparagine is in the range of 2 mM to 12 mM.

3. The formulation according to claim 1 or 2, wherein the therapeutically effective combination of free amino acids consists of: A therapeutically effective amount of the free amino acids proline, aspartic acid, serine, threonine, glycine, alanine, arginine and tyrosine.

4. The preparation according to claim 1 or 2, The preparation further comprises a pharmaceutically acceptable carrier, buffer, electrolyte, adjuvant, excipient or water, and / or wherein the preparation is sterile, and / or wherein the formulation does not comprise the monosaccharide glucose, at least one glucose-containing disaccharide, or any combination thereof, and / or wherein the preparation is a pharmaceutical preparation, and / or wherein the formulation is formulated for administration by parenteral, pulmonary, inhalation, intranasal, enteral, intravenous, anal or sublingual route, and / or wherein the formulation is formulated for oral administration, and / or The preparation is formulated for use as a medicament for treating diarrhea.

5. The formulation of claim 1 or 2, wherein at least one of the free amino acids or each of the free amino acids comprises an L-amino acid.

6. The preparation of claim 1 or 2, wherein the subject is a mammal.

7. The formulation of claim 6, wherein the mammal is a human, cat, dog, pig, horse, cow, sheep, or goat.

8. The formulation of claim 7, wherein the human is 5 years old or younger; or wherein the human is 70 years old or older.

9. The formulation of claim 1 or 2, wherein the diarrhea is associated with a secretagogue-producing bacterial infection in the subject.

10. The preparation according to claim 9, wherein the bacteria comprises at least one of enterotoxin-producing bacteria or enterotoxigenic bacteria, or any combination thereof.

11. The formulation of claim 10, wherein the enterotoxigenic bacteria comprises Vibrio cholerae, Staphylococcus aureus, Bacillus cereus, Clostridium difficile, Clostridium perfringens, Staphylococcus aureus, Yersinia enterocolitica, Shigella dysenteriae, or enterotoxigenic Escherichia coli, or any combination thereof.

12. The formulation of claim 1 or 2, wherein the at least one symptom associated with diarrhea comprises at least one of: loose stools, watery stools; abdominal cramps; abdominal pain; fever; blood in the stool; mucus in the stool; flatulence; or nausea; or any combination thereof, or Wherein reducing the at least one symptom associated with diarrhea comprises reducing at least one of: the volume of loose, watery stools per day; or the frequency of loose, watery stools per day; or any combination thereof.

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