WO2019070753A1

WO2019070753A1 - Amino acid compositions and methods for treating diarrhea

Info

Publication number: WO2019070753A1
Application number: PCT/US2018/054018
Authority: WO
Inventors: Sadasivan Vidyasagar; Astrid GROSCHE; Reshu GUPTA
Original assignee: University Of Florida Research Foundation, Incorporated
Priority date: 2017-10-02
Filing date: 2018-10-02
Publication date: 2019-04-11

Abstract

Provided herein are amino acid compositions useful for increasing the translocation of the cystic fibrosis transmembrane conductance (CFTR) protein from the plasma membrane to the cytoplasm, particularly in epithelial cells of the GI tract (e.g., small intestine epithelial cells). Methods for reducing the amount of CFTR on the plasma membrane, decreasing anion secretion (e.g., decreasing the secretion of chloride and/or bicarbonate) in the GI tract, decreasing fluid loss in the GI tract (e.g., from the crypt), and increasing fluid absorption in the GI tract (e.g., in the villi) are also provided. These compositions and methods are useful in treating diarrhea (e.g., diarrhea caused by cholera or chronic diarrhea) in subjects in need thereof. Also provided in the present disclosure are methods, kits, and uses including or using amino acid compositions described herein.

Description

AMINO ACID COMPOSITIONS AND METHODS FOR TREATING DIARRHEA

RELATED APPLICATIONS

[0001] This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Application, U.S. S.N. 62/566,887, filed October 2, 2017, which is incorporated herein by reference.

BACKGROUND OF THE INVENTION

[0002] The CFTR channel normally allows chloride ions to flow out of cells. Cholera bacteria produce cholera toxin which binds to epithelial cells in the small intestine. This increases the concentration of cyclic adenosine monophosphate (cAMP) in the epithelial cells and activates the CFTR channels, so that the CFTR channels stay open and large amounts of chloride ions flow out. Osmotic gradients lead to the excretion of sodium ions and fluids, causing fluid loss, dehydration, and diarrhea. Severe acute diarrhea causes dehydration in subjects, from the loss of fluid and electrolytes. Diarrhea can result from active chloride ion secretion or decreased fluid absorption or a combination of both active secretion and decreased absorption. There is a need for therapies to treat diarrhea and the subsequent loss of fluids and electrolytes, including diarrhea caused by cholera.

BRIEF DESCRIPTION OF THE DRAWINGS

[0003] Figures 1A-1B show the experimental design for the rat perfusion experimental data shown in Figures 2-4. Figure 1A shows the effect of different perfusion rates of regular Ringer solution (RR) on intestinal absorption. Figure 1A shows the effect of perfusion time on intestinal absorption at 150 μΙ7ηιίη of regular Ringer solution (RR), wherein the fluid absorption was not stable initially, but stabilized as the temperature stabilized. Figure IB shows the effect of experimental conditions and surgery on rat body temperature, wherein it took approximately 90 minutes to reach a stable body temperature.

[0004] Figure 2 shows the intestinal absorption rate of Sprague-Dawley rats as described in Example 2 below, upon treatment with regular Ringer solution, Trioral (WHO-HO-ORS), 8AA-AD (an eight amino acid formulation of Isoleucine, Aspartic acid, Threonine, Lysine, Tyrosine, Serine, Valine, Glycine), 5AA (a five amino acid formulation of serine, threonine, tyrosine, valine, aspartic acid.), LS-5AA, and Speedlyte (commercial).

[0005] Figure 3 shows the intestinal absorption rate upon treatment of Sprague-Dawley rats as described in Example 2 below, upon treatment with CT-RR (control), CT-Ent-8AA- AD (an 8 amino acid formulation), CT-Trioral (WHO-formulation), CT-Ent-5AA (a 5 amino acid formulation), and CT-6AA-AD (new amino acid formulation provided in Table 1). All administered formulations had equal salt concentrations.

SUMMARY OF THE INVENTION

[0006] Certain amino acids decrease the chloride secretory channels from the membrane. On the other hand, one or more of selected amino acids could decrease proliferation, maturation and therefore decrease electrolyte and fluid absorptive machinery on the membrane, resulting in decreased fluid absorption. Net fluid absorption is as a result of active electrolyte and fluid secretion from the crypt and active fluid absorption from the villi.

Decreased electrolyte secretion from the crypt could enhance net fluid absorption. However, if certain amino acids inhibit absorptive processes from the villi, then the amplitude of the net fluid absorption will be diminished even if significant inhibition of the secretory processes occurs.

[0007] The amino acids that increase translocation of CFTR from the plasma membrane to the cytoplasm could be used to decrease electrolyte and fluid loss in acute severe diarrhea such as the diarrhea associated with cholera. Decreased expression of CFTR protein on the plasma membrane could also then decrease anion secretion (e.g. , secretion of chloride and/or bicarbonate (HCO3-)). There is a need for therapies to treat diarrhea and the subsequent loss of fluids and electrolytes, including diarrhea caused by cholera. Described herein are compositions including amino acids (e.g. free amino acids), methods, uses, and kits for treating diarrhea (e.g. , diarrhea caused by cholera or chronic diarrhea). In one aspect, the present disclosure provides compositions including free amino acids and methods for reducing the number of cystic fibrosis transmembrane conductance regulator (CFTR) proteins present on the plasma membrane of a cell. In another aspect, the present disclosure provides compositions including amino acids (e.g. , free amino acids) and methods for translocating CFTR from the plasma membrane to the cytoplasm of a cell. The cells may be, for example, from the GI tract (e.g. , from the small intestines), including, but not limited to, epithelial cells of the GI tract (e.g. , small intestine epithelial cells). The small intestine epithelial cells, in certain embodiments, are small intestine villus epithelial cells. In one aspect, provided are compositions including free amino acids and methods for decreasing the secretion and/or loss of anions (e.g. , chloride or bicarbonate) in the GI tract. In another aspect, the present disclosure provides compositions including free amino acids and methods for decreasing fluid loss in the GI tract (e.g. , from the crypt), and increasing fluid absorption in the GI tract (e.g. , in the villi). Without wishing to be bound by any particular theory, the method of decreasing fluid loss in the GI tract results from decreasing electrolyte loss in the GI tract.

[0008] In another aspect, the present disclosure provides compositions comprising, consisting essentially of, or consisting of two or more free amino acids selected from the group consisting of proline, glutamic acid, glutamine, leucine, alanine, aspartic acid, and phenylalanine for use in treating diarrhea. In a further aspect, the present invention provides use of the compositions including free amino acids to treat diarrhea in a subject (e.g. , a subject with cholera) in need thereof.

[0009] In certain embodiments, the composition useful in treating the conditions described herein comprises, consists essentially of, or consists of two or more free amino acids selected from the group consisting of proline, glutamic acid, glutamine, leucine, alanine, aspartic acid, and phenylalanine. In certain embodiments, the composition comprises, consists essentially of, or consists of three or more free amino acids selected from the group consisting proline, glutamic acid, glutamine, leucine, alanine, aspartic acid, and phenylalanine. In certain embodiments, the composition comprises, consists essentially of, or consists of four or more free amino acids selected from the group consisting of proline, glutamic acid, glutamine, leucine, alanine, aspartic acid, and phenylalanine. In certain embodiments, the composition comprises, consists essentially of, or consists of five or more free amino acids selected from the group consisting of proline, glutamic acid, glutamine, leucine, alanine, aspartic acid, and phenylalanine. In certain embodiments, the composition comprises, consists essentially of, or consists of six or more free amino acids selected from the group consisting of proline, glutamic acid, glutamine, leucine, alanine, aspartic acid, and phenylalanine. In certain embodiments, the composition comprises, consists essentially of, or consists of proline, glutamic acid, glutamine, leucine, alanine, aspartic acid, and phenylalanine.

[0010] In certain embodiments, in addition to the composition that comprises, consists essentially of, or consists of proline, glutamic acid, glutamine, leucine, alanine, aspartic acid, and phenylalanine, the composition further comprises, consists essentially of, or consists of one or more free amino acids selected from the group consisting of histidine, threonine, isoleucine, asparagine, and tryptophan. In certain embodiments, the composition further comprises, consists essentially of, or consists of two or more, three or more, or four or more, free amino acids selected from the group consisting of histidine, threonine, isoleucine, asparagine, and tryptophan. In certain embodiments, the composition further comprises, consists essentially of, or consists of histidine, threonine, isoleucine, asparagine, and tryptophan. In certain embodiments, the composition further comprises, consists essentially of or consists of histidine, threonine, isoleucine, asparagine, and tryptophan.

[0011] In certain embodiments, the composition comprises, consists essentially of, or consists of two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, or eleven or more free amino acids selected from the group consisting of proline, glutamic acid, glutamine, leucine, alanine, aspartic acid, phenylalanine, histidine, threonine, isoleucine, asparagine, and tryptophan. In certain embodiments, the composition comprises, consists essentially of, or consists of proline, glutamic acid, glutamine, leucine, alanine, aspartic acid, phenylalanine, histidine, threonine, isoleucine, asparagine, and tryptophan. In certain embodiments, the composition comprises, consists essentially of, or consists of proline, glutamic acid, glutamine, leucine, alanine, aspartic acid, and phenylalanine. In certain embodiments, the composition comprises, consists essentially of, or consists of threonine, phenylalanine, isoleucine, glutamic acid, aspartic acid, alanine, and asparagine. In certain embodiments, the composition consists essentially of, or consists of threonine, phenylalanine, isoleucine, glutamic acid, aspartic acid, alanine, and asparagine, and no other free amino acids. In certain embodiments, the solution comprises water. In certain embodiments, the composition further comprises a

pharmaceutically acceptable carrier, buffer, electrolyte, adjuvant, or excipient. In certain embodiments, the composition further comprises sugars, vitamins, electrolytes, minerals, proteins, or lipids. In certain embodiments, the composition further comprises sugars. In certain embodiments, the composition further comprises vitamins. In certain embodiments, the composition further comprises electrolytes. In certain embodiments, the composition further comprises minerals. In certain embodiments, the composition further comprises proteins. In certain embodiments, the composition further comprises lipids. In certain embodiments, the composition is sterile. In certain embodiments, the composition is formulated for enteral administration. In certain embodiments, the composition is formulated for oral administration.

[0012] Each of the free amino acids, if present in the composition, may be present in, for example, the following concentrations: proline at about 0.4 to about 1.5, about 0.7 to about 1.3, about 0.9 to about 1.1 grams/liter, or about 1.5 to about 1.7 grams/liter; glutamic acid at about 0.7 to about 1.7, about 0.9 to about 1.5, about 1.1 to about 1.3 grams/liter, or about 1.5 to about 1.7 grams/liter; glutamine at about 0.6 to about 1.6, about 0.8 to about 1.4, about 1.0 to about 1.2 grams/liter, or about 1.5 to about 1.7 grams/liter; leucine at about 0.05 to about 0.4, about 0.1 to about 0.3 grams/liter, or about 1.5 to about 1.7 grams/liter; alanine at about 0.4 to about 1.5, about 0.7 to about 1.3, at about 1.1 to about 2.1, about 1.3 to about 1.9, or about 1.5 to about 1.7 grams/liter; aspartic acid at about 0.4 to about 1.5, about 0.7 to about 1.3, at about 1.1 to about 2.1, about 1.3 to about 1.9, or about 1.5 to about 1.7 grams/liter; phenylalanine at 0.4 to about 1.5, about 0.7 to about 1.3, at about 1.1 to about 2.1, about 1.3 to about 1.9, or about 1.5 to about 1.7 grams/liter; histidine at 0.4 to about 1.5, about 0.7 to about 1.3, at about 1.1 to about 2.1, about 1.3 to about 1.9, or about 1.5 to about 1.7 grams/liter; threonine at about 0.4 to about 1.5, about 0.7 to about 1.3, about 0.9 to about 1.1 grams/liter, or about 1.5 to about 1.7 grams/liter; isoleucine at 0.4 to about 1.5, about 0.7 to about 1.3, at about 1.1 to about 2.1, about 1.3 to about 1.9, or about 1.5 to about 1.7 grams/liter; asparagine at 0.4 to about 1.5, about 0.7 to about 1.3, at about 1.1 to about 2.1, about 1.3 to about 1.9, or about 1.5 to about 1.7 grams/liter; tryptophan at 0.4 to about 1.5, about 0.7 to about 1.3, at about 1.1 to about 2.1, about 1.3 to about 1.9, or about 1.5 to about 1.7 grams/liter. In certain embodiments, the concentration is in grams of amino acid per liter of solution. In certain embodiments, the composition is formulated as a dry powder and reconstituted with water.

[0013] The details of certain embodiments of the invention are set forth in the Detailed Description of Certain Embodiments, as described below. Other features, objects, and advantages of the invention will be apparent from the Definitions, Examples, Figures, and Claims.

DEFINITIONS

[0014] Descriptions and certain information relating to various terms used in the present disclosure are collected herein for convenience.

[0015] The term "agent" is used herein to refer to any substance, compound (e.g., molecule), supramolecular complex, material, or combination or mixture thereof. A compound may be any agent that can be represented by a chemical formula, chemical structure, or sequence. Example of agents, include, e.g., small molecules, polypeptides, nucleic acids (e.g., RNAi agents, antisense oligonucleotide, aptamers), lipids,

polysaccharides, etc. In general, agents may be obtained using any suitable method known in the art. The ordinary skilled artisan will select an appropriate method based, e.g., on the nature of the agent. An agent may be at least partly purified. In some embodiments an agent may be provided as part of a composition, which may contain, e.g., a counter-ion, aqueous or non-aqueous diluent or carrier, buffer, preservative, or other ingredient, in addition to the agent, in various embodiments. In some embodiments an agent may be provided as a salt, ester, hydrate, or solvate. In some embodiments an agent is cell-permeable, e.g., within the range of typical agents that are taken up by cells and acts intracellularly, e.g., within mammalian cells, to produce a biological effect. Certain compounds may exist in particular geometric or stereoisomeric forms. Such compounds, including cis- and irans-isomers, E- and Z-isomers, R- and S-enantiomers, diastereomers, (D)-isomers, (L)-isomers, (-)- and (+)- isomers, racemic mixtures thereof, and other mixtures thereof are encompassed by this disclosure in various embodiments unless otherwise indicated. Certain compounds may exist in a variety or protonation states, may have a variety of configurations, may exist as solvates [e.g., with water (i.e. hydrates) or common solvents] and/or may have different crystalline forms (e.g., polymorphs) or different tautomeric forms. Embodiments exhibiting such alternative protonation states, configurations, solvates, and forms are encompassed by the present disclosure where applicable. The term "agent" may also encompass a "therapeutic agent". The term "compound" and "agent" may be used interchangeably.

[0016] An "effective amount" or "effective dose" of an agent (or composition containing such agent) refers to the amount sufficient to achieve a desired biological and/or

pharmacological effect, e.g., when delivered to a cell or organism according to a selected administration form, route, and/or schedule. The phrases "effective amount" and

"therapeutically effective amount" are used interchangeably. As will be appreciated by those of ordinary skill in this art, the absolute amount of a particular agent or composition that is effective may vary depending on such factors as the desired biological or pharmacological endpoint, the agent to be delivered, the target tissue, etc. In certain embodiments, an effective amount is an amount sufficient to treat diarrhea {e.g. , diarrhea caused by cholera or chronic diarrhea). In certain embodiments, an effective amount is an amount sufficient to decrease the number of cystic fibrosis transmembrane conductance regulator (CFTR) proteins present on the plasma membrane of a cell. In certain embodiments, an effective amount is an amount sufficient to translocate CFTR from the plasma membrane to the cytoplasm. In certain embodiments, an effective amount is an amount sufficient to increase translocation of CFTR from the plasma membrane to the cytoplasm. In certain embodiments, an effective amount is an amount sufficient to decrease anion secretion {e.g. , chloride, bicarbonate) in the GI tract, decrease fluid loss in the GI tract {e.g. , from the crypt), or increase fluid absorption in the GI tract {e.g. , in the villi). Those of ordinary skill in the art will further understand that an "effective amount" may be contacted with cells or administered to a subject in a single dose, or through use of multiple doses, in various embodiments. [0001] By "negligible amount" it is meant that the amino acid present has no effect on the CFTR protein. Or, in certain embodiments, even if the amino acid is present in the composition, it is not present in an amount that would affect the translocation of CFTR to the plasma membrane, chloride ion transport, or the therapeutic effect of treating a subject in need thereof. In certain embodiments, a negligible amount is an amount wherein the total concentration of the amino acid is less than 100 mg/1, 50 mg/1, 10 mg/1, 5 mg/1, 1 mg/1, 0.5 mg/1, 0.1 mg/1, or 0.01 mg/1. In certain embodiments, a negligible amount is an amount wherein the total concentration of the amino acid is less than 100 mg/1. In certain

embodiments, a negligible amount is an amount wherein the total concentration of the amino acid is less than 50 mg/1. In certain embodiments, a negligible amount is an amount wherein the total concentration of the amino acid is less than 10 mg/1. In certain embodiments, a negligible amount is an amount wherein the total concentration of the amino acid is less than 5 mg/1. In certain embodiments, a negligible amount is an amount wherein the total concentration of the amino acid is less than 1 mg/1. In certain embodiments, a negligible amount is an amount wherein the total concentration of the amino acid is less than 0.5 mg/1. In certain embodiments, a negligible amount is an amount wherein the total concentration of the amino acid is less than 0.1 mg/1. In certain embodiments, a negligible amount is an amount wherein the total concentration of the amino acid is less than 0.01 mg/1.

[0017] 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 acids" encompasses the 21 amino acids encoded by the human genome (i.e., proteinogenic amino acids), amino acids encoded or produced by bacteria or single-celled organisms, and naturally derived amino acids. For the purposes of this disclosure, 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 essentially the same, unless otherwise noted. "Amino acids" also encompass derivatives thereof that retain substantially the same, or better, activity in terms of enhancing the survival, proliferation and/or development of stern cells and/or progenitor cells. The derivatives may be, for example, enantiomers, and include both the D- and L- forms of the amino acids. The derivatives may be derivatives of "natural" or "non- natural" amino acids (e.g., β-amino acids, homo-amino acids, proline derivatives, pyruvic acid derivatives, 3-substituted alanine derivatives, glycine derivatives, ring-substituted tyrosine derivatives, ring-substituted phenylalanine derivatives, linear core amino acids, and N-methyl amino acids), for example, selenocysteine, pyrrolysine, iodotyrosine, norleucine, or norvaline. Other amino acid derivatives include, but are not limited to, those that are synthesized by, for example, acylation, methylation, glycosylation, and/or halogenation of the amino acid. These include, for example, β-methyl amino acids, C-methyl amino acids, and N- methyl amino acids. The amino acids described herein may be present as free amino acids. The term "free amino acid" refers to an amino acid that is not part of a peptide or polypeptide (e.g., is not connected to another amino acid through a peptide bond). A free amino acid is free in solution, but may be associated with a salt or other component in solution.

[0018] The terms "protein," "peptide," and "polypeptide" are used interchangeably herein and refer to a polymer of amino acid residues linked together by peptide (amide) bonds. The terms refer to a protein, peptide, or polypeptide of any size, structure, or function. Typically, a protein, peptide, or polypeptide will be at least three amino acids long. A protein, peptide, or polypeptide may refer to an individual protein or a collection of proteins. One or more of the amino acids in a protein, peptide, or polypeptide may be modified, for example, by the addition of a chemical entity such as a carbohydrate group, a hydroxyl group, a phosphate group, a farnesyl group, an isofarnesyl group, a fatty acid group, a linker for conjugation, functionalization, or other modification, etc. A protein, peptide, or polypeptide may also be a single molecule or may be a multi-molecular complex. In some embodiments, a protein comprises a homodimer or a heterodimer. A protein, peptide, or polypeptide may be just a fragment of a naturally occurring protein or peptide. A protein, peptide, or polypeptide may be naturally occurring, recombinant, or synthetic, or any combination thereof. A protein may comprise different domains, for example, a nucleic acid binding domain (e.g., the gRNA binding domain of Cas9 that directs the binding of the protein to a target site) and a nucleic acid cleavage domain. In some embodiments, a protein comprises a proteinaceous part, e.g., an amino acid sequence constituting a nucleic acid binding domain, and an organic compound, e.g., a compound that can act as a nucleic acid cleavage agent. In some embodiments, a protein is in a complex with, or is in association with, a nucleic acid, e.g., RNA. In some embodiments, a protein comprises a ligand binding domain. In some embodiments, a protein comprises an active site (e.g., site of biological or enzymatic activity). In some embodiments, a protein comprises an allosteric site (e.g., site of a protein that can bind to a ligand that can be remote from an active site). Any of the proteins provided herein may be produced by any method known in the art. For example, the proteins provided herein may be produced via recombinant protein expression and purification, which is especially suited for fusion proteins comprising a peptide linker. Methods for recombinant protein expression and purification are well known, and include those described by Green and Sambrook, Molecular Cloning: A Laboratory Manual [4 ed., Cold Spring Harbor

Laboratory Press, Cold Spring Harbor, N.Y. (2012)], the entire contents of which are incorporated herein by reference.

[0019] The term "small molecule" as used herein, is an organic molecule that is less than about 2 kilodaltons (kDa) in mass. In some embodiments, the small molecule is less than about 1.5 kDa, or less than about 1 kDa. In some embodiments, the small molecule is less than about 800 daltons (Da), 600 Da, 500 Da, 400 Da, 300 Da, 200 Da, or 100 Da. Often, a small molecule has a mass of at least 50 Da. In some embodiments, a small molecule is non- polymeric. In some embodiments, a small molecule is not an amino acid. In some embodiments, a small molecule is not a nucleotide. In some embodiments, a small molecule is not a saccharide. In some embodiments, a small molecule contains multiple carbon-carbon bonds and can comprise one or more heteroatoms and/ or one or more functional groups important for structural interaction with proteins (e.g., hydrogen bonding), e.g., an amine, carbonyl, hydroxyl, or carboxyl group, and in some embodiments at least two functional groups. Small molecules often comprise one or more cyclic carbon or heterocyclic structures and/or aromatic or polyaromatic structures, optionally substituted with one or more of the above functional groups. In certain embodiments, the small molecule is a therapeutically active agent such as a drug (e.g., a molecule approved by the U.S. Food and Drug

Administration as provided in the Code of Federal Regulations (C.F.R.)). The small molecule may also be complexed with one or more metal atoms and/or metal ions. In this instance, the small molecule is also referred to as a "small organometallic molecule." Preferred small molecules are biologically active in that they produce a biological effect in animals, preferably mammals, more preferably humans. Small molecules include, but are not limited to, radionuclides and imaging agents. In certain embodiments, the small molecule is a drug. Preferably, though not necessarily, the drug is one that has already been deemed safe and effective for use in humans or animals by the appropriate governmental agency or regulatory body. For example, drugs approved for human use are listed by the FDA under 21 C.F.R. §§ 330.5, 331 through 361, and 440 through 460, incorporated herein by reference. All listed drugs are considered acceptable for use in accordance with the present invention.

[0020] The terms "composition" and "formulation" are used interchangeably.

[0021] The term "administer," "administering," or "administration" refers to implanting, absorbing, ingesting, injecting, inhaling, or otherwise introducing a compound described herein, or a composition thereof, in or on a subject. [0022] A "subject" may be any vertebrate organism in various embodiments. A subject may be individual to whom an agent is administered, e.g., for experimental, diagnostic, and/or therapeutic purposes or from whom a sample is obtained or on whom a procedure is performed. In some embodiments a subject is a mammal, e.g. a human, non-human primate, or rodent (e.g., mouse, rat, rabbit). In certain embodiments, the subject is a human. The human may be of either sex and may be at any stage of development. In certain embodiments, the subject has been diagnosed with diarrhea. In certain embodiments, the subject has been diagnosed with cholera.

[0023] "Treat," "treatment," "treating," and similar terms as used herein 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 age or composition (e.g., a pharmaceutical composition) to a subject. Treatment is typically undertaken in an effort to alter the course of a disease (which term is used to indicate any disease, disorder, syndrome or undesirable condition warranting or potentially warranting therapy) in a manner beneficial to the subject. The effect of treatment may include reversing, alleviating, reducing severity of, delaying the onset of, curing, inhibiting the progression of, and/or reducing the likelihood of occurrence or recurrence of the disease or one or more symptoms or manifestations of the disease. A therapeutic agent may be administered to a subject who has a disease or is at increased risk of developing a disease relative to a member of the general population. In some embodiments a therapeutic agent may be administered to a subject who has had a disease but no longer shows evidence of the disease. The agent may be administered, e.g., to reduce the likelihood of recurrence of evident disease. A therapeutic agent may be

administered prophylactically, e.g., before development of any symptom or manifestation of a disease. "Prophylactic treatment" refers to providing medical and/or surgical management to a subject who has not developed a disease or does not show evidence of a disease in order, e.g., to reduce the likelihood that the disease will occur or to reduce the severity of the disease should it occur. The subject may have been identified as being at risk of developing the disease (e.g., at increased risk relative to the general population or as having a risk factor that increases the likelihood of developing the disease.

[0024] The terms "condition," "disease," and "disorder" are used interchangeably.

[0025] A "therapeutically effective amount" of a compound described herein is an amount sufficient to provide a therapeutic benefit in the treatment of a condition or to delay or minimize one or more symptoms associated with the condition. A therapeutically effective amount of a compound means an amount of therapeutic agent, alone or in combination with other therapies, which provides a therapeutic benefit in the treatment of the condition. The term "therapeutically effective amount" can encompass an amount that improves overall therapy, reduces or avoids symptoms, signs, or causes of the condition, and/or enhances the therapeutic efficacy of another therapeutic agent. In certain embodiments, an effective amount is an amount sufficient to treat diarrhea (e.g., diarrhea caused by cholera or chronic diarrhea). In certain embodiments, an effective amount is an amount sufficient to decrease the number of cystic fibrosis transmembrane conductance regulator (CFTR) proteins present on the plasma membrane of a cell. In certain embodiments, an effective amount is an amount sufficient to translocate CFTR from the plasma membrane to the cytoplasm. In certain embodiments, an effective amount is an amount sufficient to increase translocation of CFTR from the plasma membrane to the cytoplasm. In certain embodiments, an effective amount is an amount sufficient to decrease anion secretion (e.g., chloride, bicarbonate) in the GI tract, decrease fluid loss in the GI tract (e.g., from the crypt), or increase fluid absorption in the GI tract (e.g., in the villi).

[0026] A "prophylactically effective amount" of a compound described herein is an amount sufficient to prevent a condition, or one or more symptoms associated with the condition or prevent its recurrence. A prophylactically effective amount of a compound means an amount of a therapeutic agent, alone or in combination with other agents, which provides a prophylactic benefit in the prevention of the condition. The term "prophylactically effective amount" can encompass an amount that improves overall prophylaxis or enhances the prophylactic efficacy of another prophylactic agent.

[0027] As used herein, the term "salt" refers to any and all salts, and encompasses pharmaceutically acceptable salts.

[0028] The term "carrier" may refer to any diluent, adjuvant, excipient, or vehicle with which a composition of the present disclosure is administered. Examples of suitable pharmaceutical carriers are described in Remington 's Essentials of Pharmaceuticals, 21^st ed., Ed. Felton, 2012, which is herein incorporated by reference.

[0029] Exemplary diluents include calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium phosphate lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch, cornstarch, powdered sugar, and mixtures thereof.

[0030] Pharmaceutically acceptable excipients used in the manufacture of provided pharmaceutical compositions include inert diluents, dispersing and/or granulating agents, surface active agents and/or emulsifiers, disintegrating agents, binding agents, preservatives, buffering agents, lubricating agents, and/or oils. Excipients such as cocoa butter and suppository waxes, coloring agents, coating agents, sweetening, flavoring, and perfuming agents may also be present in the composition. The exact amount of an amino acid composition required to achieve an effective amount will vary from subject to subject, depending, for example, on species, age, and general condition of a subject, severity of the side effects or disorder, identity of the particular compound, mode of administration, and the like. An effective amount may be included in a single dose (e.g. , single oral dose) or multiple doses (e.g. , multiple oral doses). In certain embodiments, when multiple doses are administered to a subject or applied to a tissue or cell, any two doses of the multiple doses include different or substantially the same amounts of an amino acid composition described herein. In certain embodiments, when multiple doses are administered to a subject or applied to a tissue or cell, the frequency of administering the multiple doses to the subject or applying the multiple doses to the tissue or cell is three doses a day, two doses a day, one dose a day, one dose every other day, one dose every third day, one dose every week, one dose every two weeks, one dose every three weeks, or one dose every four weeks. In certain embodiments, the frequency of administering the multiple doses to the subject or applying the multiple doses to the tissue or cell is one dose per day. In certain embodiments, the frequency of administering the multiple doses to the subject or applying the multiple doses to the tissue or cell is two doses per day. In certain embodiments, the frequency of administering the multiple doses to the subject or applying the multiple doses to the tissue or cell is three doses per day. In certain embodiments, when multiple doses are administered to a subject or applied to a tissue or cell, the duration between the first dose and 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 lifetime of the subject, tissue, or cell. In certain embodiments, the duration between the first dose and last dose of the multiple doses is three months, six months, or one year. In certain embodiments, the duration between the first dose and last dose of the multiple doses is the lifetime of the subject, tissue, or cell. In certain embodiments, a dose (e.g. , a single dose, or any dose of multiple doses) described herein includes independently 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, or between 1 g and 10 g, inclusive, of an amino acid composition described herein. In certain embodiments, a dose described herein includes independently between 1 mg and 3 mg, inclusive, of an amino acid composition described herein. In certain embodiments, a dose described herein includes independently between 3 mg and 10 mg, inclusive, of an amino acid composition described herein. In certain embodiments, a dose described herein includes independently between 10 mg and 30 mg, inclusive, of an amino acid composition described herein. In certain embodiments, a dose described herein includes independently between 30 mg and 100 mg, inclusive, of an amino acid composition described herein.

[0031] Dose ranges as described herein provide guidance for the administration of provided pharmaceutical compositions to an adult. The amount to be administered to, for example, a child or an adolescent can be determined by a medical practitioner or person skilled in the art and can be lower or the same as that administered to an adult.

[0032] The term "diarrhea" refers to a condition in which three or more unformed, loose, or watery stools occur within a 24-hour period. "Acute diarrhea" refers to diarrheal conditions that last no more than four weeks. "Chronic diarrhea" refers to diarrheal conditions that last more than four weeks. In certain embodiments, the diarrhea is caused by an infectious disease (e.g., a bacterial disease (e.g., cholera)). In certain embodiments, the diarrhea is caused by cholera.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

Compositions comprising amino acids for translocating CFTR to the cytoplasm

[0033] The CFTR protein is an ABC transporter protein that functions as an ATP-gated ion channel. When activated, CFTR allows chloride ions (CI^"), and other negatively charged ions such as thiocyanate ([SCN]^") and/or bicarbonate, to flow down their electrochemical gradient (e.g., passive diffusion or passive transport). Certain amino acids decrease the chloride secretory channels from the membrane. In comparison, one or more of certain amino acids could decrease proliferation, maturation and therefore decrease electrolyte and fluid absorptive machinery on the membrane, resulting in decreased fluid absorption. Net fluid absorption is as a result of active electrolyte and fluid secretion from the crypt and active fluid absorption from the villi. Decreased electrolyte secretion from the crypt could enhance net fluid absorption. When certain amino acids inhibit absorptive processes from the villi, then the net fluid absorption will be diminished even if significant inhibition of the secretory processes occurs. [0034] The selected amino acids that increase translocation of CFTR from the plasma membrane to the cytoplasm could be used to decrease electrolyte and fluid loss in acute severe diarrhea such as the diarrhea associated with cholera. Decreased expression of CFTR protein on the plasma membrane could also then decrease anion secretion (e.g. , secretion of chloride and/or bicarbonate (HCO3-)). In one aspect, the compositions provided herein translocate CFTR from the plasma membrane to the cytoplasm of a cell. In another aspect, the compositions provided herein increase the translocation of CFTR from the plasma membrane to the cytoplasm of a cell.

[0035] In one aspect, the composition comprises, consists essentially of, or consists of two or more free amino acids selected from the group consisting of proline, glutamic acid, glutamine, leucine, alanine, aspartic acid, and phenylalanine; and optionally,

pharmaceutically acceptable carriers, buffers, electrolytes, adjuvants, excipients, and an additional therapeutic agent. In certain embodiments, the composition consists essentially of, or consists of only the specified free amino acids and no other free amino acids, or a negligible amount of other free amino acids. The compositions include, in certain

embodiments, derivatives of the amino acids that are derivatives of "natural" or "non-natural" amino acids. The compositions include, in certain embodiments, salts and/or prodrugs of the amino acids. In certain embodiments, the composition further comprises sugars, vitamins, electrolytes, minerals, proteins, or lipids. In certain embodiments, the composition further comprises sugars. In certain embodiments, the composition further comprises vitamins. In certain embodiments, the composition further comprises electrolytes. In certain embodiments, the composition further comprises minerals. In certain embodiments, the composition further comprises proteins. In certain embodiments, the composition further comprises lipids. In certain embodiments, the composition further comprises an additional therapeutic agent. In certain embodiments, in addition to the composition that comprises, consists essentially of, or consists of proline, glutamic acid, glutamine, leucine, alanine, aspartic acid, and

phenylalanine, the composition further comprises one or more free amino acids selected from the group consisting of histidine, threonine, isoleucine, asparagine, and tryptophan.

[0036] In certain embodiments, the composition comprises, consists essentially of, or consists of three or more free amino acids selected from the group consisting of proline, glutamic acid, glutamine, leucine, alanine, aspartic acid, and phenylalanine. In certain embodiments, the composition comprises, consists essentially of, or consists of four or more free amino acids selected from the group consisting of proline, glutamic acid, glutamine, leucine, alanine, aspartic acid, and phenylalanine. In certain embodiments, the composition comprises, consists essentially of, or consists of five or more free amino acids selected from the group consisting of proline, glutamic acid, glutamine, leucine, alanine, aspartic acid, and phenylalanine. In certain embodiments, the composition comprises, consists essentially of, or consists of six or more free amino acids selected from the group consisting of proline, glutamic acid, glutamine, leucine, alanine, aspartic acid, and phenylalanine. In one aspect, the compositions provided herein translocate CFTR from the plasma membrane to the cytoplasm of a cell. In another aspect, the compositions provided herein increase the translocation of CFTR from the plasma membrane to the cytoplasm of a cell. In certain embodiments, the composition comprises, consists essentially of, or consists of proline. In certain embodiments, the composition comprises, consists essentially of, or consists of glutamic acid. In certain embodiments, the composition comprises, consists essentially of, or consists of glutamine. In certain embodiments, the composition comprises, consists essentially of, or consists of leucine. In certain embodiments, the composition comprises, consists essentially of, or consists of alanine. In certain embodiments, the composition comprises, consists essentially of, or consists of aspartic acid. In certain embodiments, the composition comprises, consists essentially of, or consists of phenylalanine. In certain embodiments, the composition comprises, consists essentially of, or consists of the free amino acids proline, glutamic acid, glutamine, leucine, alanine, aspartic acid, and phenylalanine. In certain embodiments, the composition comprises threonine, phenylalanine, isoleucine, glutamic acid, aspartic acid, alanine, and asparagine. In certain embodiments, the composition consists essentially of, or consists of threonine, phenylalanine, isoleucine, glutamic acid, aspartic acid, alanine, and asparagine.

[0037] In another embodiment, the composition comprises, consists essentially of, or consists of any two free amino acids selected from the group consisting of proline, glutamic acid, glutamine, leucine, alanine, aspartic acid, and phenylalanine. In certain embodiments, the composition comprises, consists essentially of, or consists of proline and glutamic acid. In certain embodiments, the composition comprises, consists essentially of, or consists of proline and glutamine. In certain embodiments, the composition comprises, consists essentially of, or consists of proline and leucine. In certain embodiments, the composition comprises, consists essentially of, or consists of proline and alanine. In certain embodiments, the composition comprises, consists essentially of, or consists of proline and aspartic acid. In certain embodiments, the composition comprises, consists essentially of, or consists of proline and phenylalanine. In certain embodiments, the composition comprises, consists essentially of, or consists of glutamic acid and glutamine. In certain embodiments, the composition comprises, consists essentially of, or consists of glutamic acid and leucine. In certain embodiments, the composition comprises, consists essentially of, or consists of glutamic acid and alanine. In certain embodiments, the composition comprises, consists essentially of, or consists of glutamic acid and aspartic acid. In certain embodiments, the composition comprises, consists essentially of, or consists of glutamic acid and

phenylalanine. In certain embodiments, the composition comprises, consists essentially of, or consists of glutamine and leucine. In certain embodiments, the composition comprises, consists essentially of, or consists of glutamine and alanine. In certain embodiments, the composition comprises, consists essentially of, or consists of glutamine and aspartic acid. In certain embodiments, the composition comprises, consists essentially of, or consists of glutamine and phenylalanine. In certain embodiments, the composition comprises, consists essentially of, or consists of leucine and alanine. In certain embodiments, the composition comprises, consists essentially of, or consists of leucine and aspartic acid. In certain embodiments, the composition comprises, consists essentially of, or consists of leucine and phenylalanine. In certain embodiments, the composition comprises, consists essentially of, or consists of alanine and aspartic acid. In certain embodiments, the composition comprises, consists essentially of, or consists of alanine and phenylalanine. In certain embodiments, the composition comprises, consists essentially of, or consists of aspartic acid and phenylalanine.

[0038] In another embodiment, the composition comprises, consists essentially of, or consists of, any three free amino acids selected from the group consisting of proline, glutamic acid, glutamine, leucine, alanine, aspartic acid, and phenylalanine. In certain embodiments, the composition comprises, consists essentially of, or consists of proline, glutamic acid, and glutamine. In certain embodiments, the composition comprises, consists essentially of, or consists of proline, glutamic acid, and leucine. In certain embodiments, the composition comprises, consists essentially of, or consists of proline, glutamic acid, and alanine. In certain embodiments, the composition comprises, consists essentially of, or consists of proline, glutamic acid, and aspartic acid. In certain embodiments, the composition comprises, consists essentially of, or consists of proline, glutamic acid, and phenylalanine. In certain

embodiments, the composition comprises, consists essentially of, or consists of proline, glutamine, and leucine. In certain embodiments, the composition comprises, consists essentially of, or consists of proline, glutamine, and alanine. In certain embodiments, the composition comprises, consists essentially of, or consists of proline, glutamine, and aspartic acid. In certain embodiments, the composition comprises, consists essentially of, or consists of proline, glutamine, and phenylalanine. In certain embodiments, the composition comprises, consists essentially of, or consists of proline, leucine, and alanine. In certain embodiments, the composition comprises, consists essentially of, or consists of proline, leucine, and aspartic acid. In certain embodiments, the composition comprises, consists essentially of, or consists of proline, leucine, and phenylalanine. In certain embodiments, the composition comprises, consists essentially of, or consists of proline, aspartic acid, and phenylalanine. In certain embodiments, the composition comprises, consists essentially of, or consists of glutamic acid, glutamine, and leucine. In certain embodiments, the composition comprises, consists essentially of, or consists of glutamic acid, glutamine, and alanine. In certain embodiments, the composition comprises, consists essentially of, or consists of glutamic acid, glutamine, and aspartic acid. In certain embodiments, the composition comprises, consists essentially of, or consists of glutamic acid, glutamine, and phenylalanine. In certain embodiments, the composition comprises, consists essentially of, or consists of glutamic acid, leucine, and alanine. In certain embodiments, the composition comprises, consists essentially of, or consists of glutamic acid, leucine, and aspartic acid. In certain embodiments, the composition comprises, consists essentially of, or consists of glutamic acid, leucine, and phenylalanine. In certain embodiments, the composition comprises, consists essentially of, or consists of glutamic acid, alanine, and aspartic acid. In certain embodiments, the composition comprises, consists essentially of, or consists of glutamic acid, alanine, and phenylalanine. In certain embodiments, the composition comprises, consists essentially of, or consists of glutamic acid, aspartic acid, and phenylalanine.

[0039] In certain embodiments, the composition comprises, consists essentially of, or consists of glutamine, leucine, and alanine. In certain embodiments, the composition comprises, consists essentially of, or consists of glutamine, leucine, and aspartic acid. In certain embodiments, the composition comprises, consists essentially of, or consists of glutamine, leucine, and phenylalanine. In certain embodiments, the composition comprises, consists essentially of, or consists of glutamine, alanine, and aspartic acid. In certain embodiments, the composition comprises, consists essentially of, or consists of glutamine, alanine, and phenylalanine. In certain embodiments, the composition comprises, consists essentially of, or consists of glutamine, aspartic acid, and phenylalanine. In certain

embodiments, the composition comprises, consists essentially of, or consists of leucine, alanine, and aspartic acid. In certain embodiments, the composition comprises, consists essentially of, or consists of leucine, alanine, and phenylalanine. In certain embodiments, the composition comprises, consists essentially of, or consists of leucine, aspartic acid, and phenylalanine. In certain embodiments, the composition comprises, consists essentially of, or consists of alanine, aspartic acid, and phenylalanine.

[0040] In another embodiment, the composition comprises, consists essentially of, or consists of, any four free amino acids selected from the group consisting of proline, glutamic acid, glutamine, leucine, alanine, aspartic acid, and phenylalanine. In certain embodiments, the composition comprises, consists essentially of, or consists of proline, glutamic acid, glutamine, and leucine. In certain embodiments, the composition comprises, consists essentially of, or consists of proline, glutamic acid, glutamine, and alanine. In certain embodiments, the composition comprises, consists essentially of, or consists of proline, glutamic acid, glutamine, and aspartic acid. In certain embodiments, the composition comprises, consists essentially of, or consists of proline, glutamic acid, glutamine, and phenylalanine. In certain embodiments, the composition comprises, consists essentially of, or consists of proline, glutamic acid, leucine, and alanine. In certain embodiments, the composition comprises, consists essentially of, or consists of proline, glutamic acid, leucine, and aspartic acid. In certain embodiments, the composition comprises, consists essentially of, or consists of proline, glutamic acid, leucine, and phenylalanine. In certain embodiments, the composition comprises, consists essentially of, or consists of proline, glutamic acid, alanine, and aspartic acid. In certain embodiments, the composition comprises, consists essentially of, or consists of proline, glutamic acid, alanine, and phenylalanine. In certain embodiments, the composition comprises, consists essentially of, or consists of proline, glutamic acid, aspartic acid, and phenylalanine. In certain embodiments, the composition comprises, consists essentially of, or consists of proline, glutamine, leucine, and alanine. In certain embodiments, the composition comprises, consists essentially of, or consists of proline, glutamine, leucine, and aspartic acid. In certain embodiments, the composition comprises, consists essentially of, or consists of proline, glutamine, leucine, and phenylalanine. In certain embodiments, the composition comprises, consists essentially of, or consists of proline, glutamine, alanine, and aspartic acid. In certain embodiments, the composition comprises, consists essentially of, or consists of proline, glutamine, alanine, and phenylalanine. In certain embodiments, the composition comprises, consists essentially of, or consists of proline, glutamine, aspartic acid, and phenylalanine. In certain embodiments, the composition comprises, consists essentially of, or consists of proline, leucine, alanine, and aspartic acid. In certain

embodiments, the composition comprises, consists essentially of, or consists of proline, leucine, alanine, and phenylalanine. In certain embodiments, the composition comprises, consists essentially of, or consists of proline, leucine, aspartic acid, and phenylalanine. In certain embodiments, the composition comprises, consists essentially of, or consists of proline, alanine, aspartic acid, and phenylalanine.

[0041] In certain embodiments, the composition comprises, consists essentially of, or consists of glutamic acid, glutamine, leucine, and alanine. In certain embodiments, the composition comprises, consists essentially of, or consists of glutamic acid, glutamine, leucine, and aspartic acid. In certain embodiments, the composition comprises, consists essentially of, or consists of glutamic acid, glutamine, leucine, and phenylalanine. In certain embodiments, the composition comprises, consists essentially of, or consists of glutamic acid, glutamine, alanine, and aspartic acid. In certain embodiments, the composition comprises, consists essentially of, or consists of glutamic acid, glutamine, alanine, and phenylalanine. In certain embodiments, the composition comprises, consists essentially of, or consists of glutamic acid, glutamine, aspartic acid, and phenylalanine. In certain embodiments, the composition comprises, consists essentially of, or consists of glutamic acid, leucine, alanine, and aspartic acid. In certain embodiments, the composition comprises, consists essentially of, or consists of glutamic acid, leucine, alanine, and phenylalanine. In certain embodiments, the composition comprises, consists essentially of, or consists of glutamic acid, leucine, aspartic acid, and phenylalanine. In certain embodiments, the composition comprises, consists essentially of, or consists of glutamic acid, alanine, aspartic acid, and phenylalanine. In certain embodiments, the composition comprises, consists essentially of, or consists of glutamine, leucine, alanine, and aspartic acid. In certain embodiments, the composition comprises, consists essentially of, or consists of glutamine, leucine, alanine, and

phenylalanine. In certain embodiments, the composition comprises, consists essentially of, or consists of glutamine, leucine, aspartic acid, and phenylalanine. In certain embodiments, the composition comprises, consists essentially of, or consists of glutamine, alanine, aspartic acid, and phenylalanine. In certain embodiments, the composition comprises, consists essentially of, or consists of leucine, alanine, aspartic acid, and phenylalanine.

[0042] In another embodiment, the composition comprises, consists essentially of, or consists of any five free amino acids selected from the group consisting of proline, glutamic acid, glutamine, leucine, alanine, aspartic acid, and phenylalanine. In certain embodiments, the composition comprises, consists essentially of, or consists of proline, glutamic acid, glutamine, leucine, and alanine. In certain embodiments, the composition comprises, consists essentially of, or consists of proline, glutamic acid, glutamine, leucine, and aspartic acid. In certain embodiments, the composition comprises, consists essentially of, or consists of proline, glutamic acid, glutamine, leucine, and phenylalanine. In certain embodiments, the composition comprises, consists essentially of, or consists of proline, glutamic acid, glutamine, alanine, and aspartic acid. In certain embodiments, the composition comprises, consists essentially of, or consists of proline, glutamic acid, glutamine, alanine, and phenylalanine. In certain embodiments, the composition comprises, consists essentially of, or consists of proline, glutamic acid, glutamine, aspartic acid, and phenylalanine. In certain embodiments, the composition comprises, consists essentially of, or consists of proline, glutamic acid, leucine, alanine, and aspartic acid. In certain embodiments, the composition comprises, consists essentially of, or consists of proline, glutamic acid, leucine, alanine, and phenylalanine. In certain embodiments, the composition comprises, consists essentially of, or consists of proline, glutamic acid, leucine, aspartic acid, and phenylalanine. In certain embodiments, the composition comprises, consists essentially of, or consists of proline, glutamic acid, alanine, aspartic acid, and phenylalanine. In certain embodiments, the composition comprises, consists essentially of, or consists of proline, glutamine, leucine, alanine, and aspartic acid. In certain embodiments, the composition comprises, consists essentially of, or consists of proline, glutamine, leucine, alanine, and phenylalanine. In certain embodiments, the composition comprises, consists essentially of, or consists of proline, glutamine, leucine, aspartic acid, and phenylalanine. In certain embodiments, the composition comprises, consists essentially of, or consists of proline, glutamine, alanine, aspartic acid, and phenylalanine. In certain embodiments, the composition comprises, consists essentially of, or consists of proline, leucine, alanine, aspartic acid, and phenylalanine.

[0043] In certain embodiments, the composition comprises, consists essentially of, or consists of glutamic acid, glutamine, leucine, alanine, and aspartic acid. In certain

embodiments, the composition comprises, consists essentially of, or consists of glutamic acid, glutamine, leucine, alanine, and phenylalanine. In certain embodiments, the composition comprises, consists essentially of, or consists of glutamic acid, glutamine, leucine, aspartic acid, and phenylalanine. In certain embodiments, the composition comprises, consists essentially of, or consists of glutamic acid, glutamine, alanine, aspartic acid, and

phenylalanine. In certain embodiments, the composition comprises, consists essentially of, or consists of glutamic acid, leucine, alanine, aspartic acid, and phenylalanine. In certain embodiments, the composition comprises, consists essentially of, or consists of glutamine, leucine, alanine, aspartic acid, and phenylalanine.

[0044] In another embodiment, the composition comprises, consists essentially of, or consists of any six free amino acids selected from the group consisting of proline, glutamic acid, glutamine, leucine, alanine, aspartic acid, and phenylalanine. In certain embodiments, the composition comprises, consists essentially of, or consists of proline, glutamic acid, glutamine, leucine, alanine, and aspartic acid. In certain embodiments, the composition comprises, consists essentially of, or consists of proline, glutamic acid, glutamine, leucine, alanine, and phenylalanine. In certain embodiments, the composition comprises, consists essentially of, or consists of proline, glutamic acid, glutamine, leucine, aspartic acid, and phenylalanine. In certain embodiments, the composition comprises, consists essentially of, or consists of proline, glutamic acid, glutamine, alanine, aspartic acid, and phenylalanine. In certain embodiments, the composition comprises, consists essentially of, or consists of proline, glutamic acid, leucine, alanine, aspartic acid, and phenylalanine. In certain embodiments, the composition comprises, consists essentially of, or consists of proline, glutamine, leucine, alanine, aspartic acid, and phenylalanine. In certain embodiments, the composition comprises, consists essentially of, or consists of glutamic acid, glutamine, leucine, alanine, aspartic acid, and phenylalanine.

[0045] In certain embodiments, in addition to the composition that comprises, consists essentially of, or consists of proline, glutamic acid, glutamine, leucine, alanine, aspartic acid, and phenylalanine, the composition further comprises, consists essentially of, or consists of one or more free amino acids selected from the group consisting of histidine, threonine, isoleucine, asparagine, and tryptophan. In certain embodiments, the composition further comprises, consists essentially of, or consists of one or more free amino acids selected from the group consisting of histidine, threonine, isoleucine, asparagine, and tryptophan, and another amino acid. In certain embodiments, the composition further comprises histidine. In certain embodiments, the composition further comprises threonine. In certain embodiments, the composition further comprises isoleucine. In certain embodiments, the composition further comprises asparagine. In certain embodiments, the composition further comprises tryptophan. In certain embodiments, the composition further comprises, consists essentially of, or consists of two or more, three or more, or four or more, free amino acids selected from the group consisting of histidine, threonine, isoleucine, asparagine, and tryptophan. In certain embodiments, the composition further comprises, consists essentially of, or consists of histidine, threonine, isoleucine, asparagine, and tryptophan. In certain embodiments, the composition further comprises, consists essentially of or consists of histidine, threonine, isoleucine, asparagine, and tryptophan.

[0046] In certain embodiments, the composition comprises, consists essentially of, or consists of two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, or all twelve free amino acids selected from the group consisting of proline, glutamic acid, glutamine, leucine, alanine, aspartic acid, phenylalanine, histidine, threonine, isoleucine, asparagine, and tryptophan. In certain embodiments, the composition comprises, consists essentially of, or consists of two or more, amino acids selected from the group consisting of proline, glutamic acid, glutamine, leucine, alanine, aspartic acid, phenylalanine, histidine, threonine, isoleucine, asparagine, and tryptophan. In certain embodiments, the composition comprises, consists essentially of, or consists of three or more amino acids selected from the group consisting of proline, glutamic acid, glutamine, leucine, alanine, aspartic acid, phenylalanine, histidine, threonine, isoleucine, asparagine, and tryptophan. In certain embodiments, the composition comprises, consists essentially of, or consists of four or more amino acids selected from the group consisting of proline, glutamic acid, glutamine, leucine, alanine, aspartic acid, phenylalanine, histidine, threonine, isoleucine, asparagine, and tryptophan. In certain embodiments, the composition comprises, consists essentially of, or consists of five or more amino acids selected from the group consisting of proline, glutamic acid, glutamine, leucine, alanine, aspartic acid, phenylalanine, histidine, threonine, isoleucine, asparagine, and tryptophan. In certain embodiments, the composition comprises, consists essentially of, or consists of six or more amino acids selected from the group consisting of proline, glutamic acid, glutamine, leucine, alanine, aspartic acid, phenylalanine, histidine, threonine, isoleucine, asparagine, and tryptophan. In certain embodiments, the composition comprises, consists essentially of, or consists of seven or more amino acids selected from the group consisting of proline, glutamic acid, glutamine, leucine, alanine, aspartic acid, phenylalanine, histidine, threonine, isoleucine, asparagine, and tryptophan. In certain embodiments, the composition comprises, consists essentially of, or consists of eight or more amino acids selected from the group consisting of proline, glutamic acid, glutamine, leucine, alanine, aspartic acid, phenylalanine, histidine, threonine, isoleucine, asparagine, and tryptophan. In certain embodiments, the composition comprises, consists essentially of, or consists of nine or more amino acids selected from the group consisting of proline, glutamic acid, glutamine, leucine, alanine, aspartic acid, phenylalanine, histidine, threonine, isoleucine, asparagine, and tryptophan. In certain embodiments, the composition comprises, consists essentially of, or consists of ten or more amino acids selected from the group consisting of proline, glutamic acid, glutamine, leucine, alanine, aspartic acid, phenylalanine, histidine, threonine, isoleucine, asparagine, and tryptophan. In certain embodiments, the composition comprises, consists essentially of, or consists of eleven or more amino acids selected from the group consisting of proline, glutamic acid, glutamine, leucine, alanine, aspartic acid, phenylalanine, histidine, threonine, isoleucine, asparagine, and tryptophan.

[0047] In certain embodiments, the composition comprises, consists essentially of, or consists of, one or more free amino acids selected from the group consisting of lysine, threonine, valine, tyrosine, isoleucine, and aspartic acid. In certain embodiments, the composition comprises, consists essentially of, or consists of, two or more free amino acids selected from the group consisting of lysine, threonine, valine, tyrosine, isoleucine, and aspartic acid. In certain embodiments, the composition comprises, consists essentially of, or consists of, three or more free amino acids selected from the group consisting of lysine, threonine, valine, tyrosine, isoleucine, and aspartic acid. In certain embodiments, the composition comprises, consists essentially of, or consists of, four or more free amino acids selected from the group consisting of lysine, threonine, valine, tyrosine, isoleucine, and aspartic acid. In certain embodiments, the composition comprises, consists essentially of, or consists of, five or more free amino acids selected from the group consisting of lysine, threonine, valine, tyrosine, isoleucine, and aspartic acid.

[0048] In certain embodiments, the composition comprises, consists essentially of, or consists of, one amino acid selected from the group consisting of lysine, threonine, valine, tyrosine, isoleucine, and aspartic acid, and no other free amino acids. In certain

embodiments, the composition comprises, consists essentially of, or consists of, two amino acids selected from the group consisting of lysine, threonine, valine, tyrosine, isoleucine, and aspartic acid, and no other free amino acids. In certain embodiments, the composition comprises, consists essentially of, or consists of, three amino acids selected from the group consisting of lysine, threonine, valine, tyrosine, isoleucine, and aspartic acid, and no other free amino acids. In certain embodiments, the composition comprises, consists essentially of, or consists of, four amino acids selected from the group consisting of lysine, threonine, valine, tyrosine, isoleucine, and aspartic acid, and no other free amino acids. In certain embodiments, the composition comprises, consists essentially of, or consists of, five amino acids selected from the group consisting of lysine, threonine, valine, tyrosine, isoleucine, and aspartic acid, and no other free amino acids. In certain embodiments, the composition comprises, consists essentially of, or consists of, six amino acids selected from the group consisting of lysine, threonine, valine, tyrosine, isoleucine, and aspartic acid, and no other free amino acids. In certain embodiments, the composition comprises, consists essentially of, or consists of, lysine, threonine, valine, tyrosine, isoleucine, and aspartic acid, and no other free amino acids. [0049] In certain embodiments, the composition comprises, consists essentially of, or consists of, one amino acid selected from the group consisting of lysine, threonine, valine, tyrosine, isoleucine, and aspartic acid. In certain embodiments, the composition comprises, consists essentially of, or consists of, two amino acids selected from the group consisting of lysine, threonine, valine, tyrosine, isoleucine, and aspartic acid. In certain embodiments, the composition comprises, consists essentially of, or consists of, three amino acids selected from the group consisting of lysine, threonine, valine, tyrosine, isoleucine, and aspartic acid. In certain embodiments, the composition comprises, consists essentially of, or consists of, four amino acids selected from the group consisting of lysine, threonine, valine, tyrosine, isoleucine, and aspartic acid. In certain embodiments, the composition comprises, consists essentially of, or consists of, five amino acids selected from the group consisting of lysine, threonine, valine, tyrosine, isoleucine, and aspartic acid. In certain embodiments, the composition comprises, consists essentially of, or consists of, six amino acids selected from the group consisting of lysine, threonine, valine, tyrosine, isoleucine, and aspartic acid. In certain embodiments, the composition further comprises, one or more free amino acids selected from the group consisting of histidine, asparagine, tryptophan, proline, glutamic acid, glutamine, leucine, alanine, and phenylalanine.

[0050] In certain embodiments, the amino acids of the composition are free amino acids. In certain embodiments, the amino acids of the composition are L-amino acids. In certain embodiments, the amino acids of the composition are D-amino acids. In certain

embodiments, the amino acids of the composition are a combination of D- and L-amino acids.

[0051] In certain embodiments, the composition further comprises water.

[0052] In certain embodiments, the composition further comprises a buffer. Exemplary buffering agents include citrate buffer solutions, acetate buffer solutions, phosphate buffer solutions, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium glubionate, calcium gluceptate, calcium gluconate, D-gluconic acid, calcium

glycerophosphate, calcium lactate, propanoic acid, calcium levulinate, pentanoic acid, dibasic calcium phosphate, phosphoric acid, tribasic calcium phosphate, calcium hydroxide phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixtures, dibasic potassium phosphate, monobasic potassium phosphate, potassium phosphate mixtures, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, dibasic sodium phosphate, monobasic sodium phosphate, sodium phosphate mixtures, tromethamine, magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline, Ringer's solution, ethyl alcohol, and mixtures thereof.

[0053] In certain embodiments, phosphate ions, such as H₂P0₄ ^", HP0₄ ^2", and P0₄ ³ , are used to buffer the composition of the subject invention. In certain embodiments, the therapeutic composition uses HCO3^" or CO3^2" as a buffer. In other embodiments, the therapeutic composition does not use HCO3^" or CO3^2" as a buffer.

[0054] In certain embodiments, the composition comprises one or more electrolytes selected from, for example, Na⁺; K⁺; HCO3 ; CO3^2"; Ca²⁺; Mg²⁺; Fe²⁺; CI^"; phosphate ions, such as H₂P0₄ ^", HP0₄ ^2", and P0₄ ³ ; zinc; iodine; copper; iron; selenium; chromium; and molybdenum. In an alternative embodiment, the composition does not contain HCO3^" or CO3^2". In another alternative embodiment, the composition comprises HCO3^" and CO3^2" at a total concentration of less than 5 mg/1, or concentrations lower than 5 mg/1. In certain embodiments, the composition does not contain electrolytes. In certain embodiments, the composition does not contain glucose. In certain embodiments, the composition does not contain carbohydrates (e.g. , di-, oligo-, or polysaccharides). In certain embodiments, the composition does not contain any substrate of glucose transporters. For example, in certain embodiments the composition does not comprise one or more, or any, of Na⁺; K⁺; HCO3^"; CO3^2"; Ca²⁺; Mg²⁺; Fe₂; CI^"; phosphate ions, such as H₂P0₄ ^", HP0₄ ^2", and P0₄ ³ ; zinc; iodine; copper; iron; selenium; chromium; and molybdenum. In certain embodiments, the

composition is formulated as a dry powder and reconstituted with water. In certain

embodiments, the composition does not comprise, or only comprises negligible amounts of, the free amino acid cysteine. In certain embodiments, the composition does not comprise, or only comprises negligible amounts of, the free amino acid histidine.

[0055] Each of the free amino acids, if present in the composition, may be present in, for example, the following concentrations: Each of the free amino acids, if present in the composition, may be present in, for example, the following concentrations: proline at about 0.4 to about 1.5, about 0.7 to about 1.3, about 0.9 to about 1.1 grams/liter, or about 1.5 to about 1.7 grams/liter; glutamic acid at about 0.7 to about 1.7, about 0.9 to about 1.5, about 1.1 to about 1.3 grams/liter, or about 1.5 to about 1.7 grams/liter; glutamine at about 0.6 to about 1.6, about 0.8 to about 1.4, about 1.0 to about 1.2 grams/liter, or about 1.5 to about 1.7 grams/liter; leucine at about 0.05 to about 0.4, about 0.1 to about 0.3 grams/liter, or about 1.5 to about 1.7 grams/liter; alanine at about 0.4 to about 1.5, about 0.7 to about 1.3, at about 1.1 to about 2.1, about 1.3 to about 1.9, or about 1.5 to about 1.7 grams/liter; aspartic acid at about 0.4 to about 1.5, about 0.7 to about 1.3, at about 1.1 to about 2.1, about 1.3 to about 1.9, or about 1.5 to about 1.7 grams/liter; phenylalanine at 0.4 to about 1.5, about 0.7 to about 1.3, at about 1.1 to about 2.1, about 1.3 to about 1.9, or about 1.5 to about 1.7 grams/liter;

histidine at 0.4 to about 1.5, about 0.7 to about 1.3, at about 1.1 to about 2.1, about 1.3 to about 1.9, or about 1.5 to about 1.7 grams/liter; threonine at about 0.4 to about 1.5, about 0.7 to about 1.3, about 0.9 to about 1.1 grams/liter, or about 1.5 to about 1.7 grams/liter;

isoleucine at 0.4 to about 1.5, about 0.7 to about 1.3, at about 1.1 to about 2.1, about 1.3 to about 1.9, or about 1.5 to about 1.7 grams/liter; asparagine at 0.4 to about 1.5, about 0.7 to about 1.3, at about 1.1 to about 2.1, about 1.3 to about 1.9, or about 1.5 to about 1.7 grams/liter; tryptophan at 0.4 to about 1.5, about 0.7 to about 1.3, at about 1.1 to about 2.1, about 1.3 to about 1.9, or about 1.5 to about 1.7 grams/liter.

[0056] In certain embodiments, the free amino acid proline is present in the composition at the following concentrations: at about 0.4 to about 1.5, about 0.7 to about 1.3, about 0.9 to about 1.1 grams/liter, or about 1.5 to about 1.7 grams/liter. In certain embodiments, the free amino acid proline is present in the composition at about 0.4 to about 1.5 grams/liter. In certain embodiments, the free amino acid proline is present in the composition at about 0.7 to about 1.3 grams/liter. In certain embodiments, the free amino acid proline is present in the composition at about 0.9 to about 1.1 grams/liter. In certain embodiments, the free amino acid proline is present in the composition at about 1.5 to about 1.7 grams/liter. In certain embodiments, the free amino acid proline is present in the composition at about 1.0 or 1.5 grams/liter.

[0057] In certain embodiments, the free amino acid glutamic acid is present in the composition at the following concentrations: at about 0.7 to about 1.7, about 0.9 to about 1.5, about 1.1 to about 1.3 grams/liter, or about 1.5 to about 1.7 grams/liter. In certain

embodiments, the free amino acid glutamic acid is present in the composition at about 0.7 to about 1.7 grams/liter. In certain embodiments, the free amino acid glutamic acid is present in the composition at about 0.9 to about 1.5 grams/liter. In certain embodiments, the free amino acid glutamic acid is present in the composition at about 1.1 to about 1.3 grams/liter. In certain embodiments, the free amino acid glutamic acid is present in the composition at about 1.5 to about 1.7 grams/liter. In certain embodiments, the free amino acid glutamic acid is present in the composition at about 1.0 or 1.5 grams/liter.

[0058] In certain embodiments, the free amino acid glutamine is present in the composition at the following concentrations: at about 0.6 to about 1.6, about 0.8 to about 1.4, about 1.0 to about 1.2 grams/liter, or about 1.5 to about 1.7 grams/liter. In certain embodiments, the free amino acid glutamine is present in the composition at about 0.7, 1.0, 1.2, or 1.5 grams/liter. In certain embodiments, the free amino acid leucine is present in the composition at the following concentrations: at about 0.05 to about 0.4, about 0.1 to about 0.3 grams/liter, or about 1.5 to about 1.7 grams/liter. In certain embodiments, the free amino acid leucine is present in the composition at about 0.1, 0.2, 0.5, 0.7, 1.0, 1.2, or 1.5 grams/liter. In certain embodiments, the free amino acid alanine is present in the composition at the following concentrations: at about 0.4 to about 1.5, about 0.7 to about 1.3, at about 1.1 to about 2.1, about 1.3 to about 1.9, or about 1.5 to about 1.7 grams/liter. In certain embodiments, the free amino acid alanine is present in the composition at about 0.5, 0.7, 1.0, 1.2, or 1.5 grams/liter. In certain embodiments, the free amino acid aspartic acid is present in the composition at the following concentrations: at about 0.4 to about 1.5, about 0.7 to about 1.3, at about 1.1 to about 2.1, about 1.3 to about 1.9, or about 1.5 to about 1.7 grams/liter. In certain

embodiments, the free amino acid aspartic acid is present in the composition at about 0.5, 0.7, 1.0, 1.2, or 1.5 grams/liter.

[0059] In certain embodiments, the free amino acid phenylalanine is present in the composition at the following concentrations: at 0.4 to about 1.5, about 0.7 to about 1.3, at about 1.1 to about 2.1, about 1.3 to about 1.9, or about 1.5 to about 1.7 grams/liter. In certain embodiments, the free amino acid phenylalanine is present in the composition at about 0.5, 0.7, 1.0, 1.2, or 1.5 grams/liter. In certain embodiments, the free amino acid histidine is present in the composition at the following concentrations: at 0.4 to about 1.5, about 0.7 to about 1.3, at about 1.1 to about 2.1, about 1.3 to about 1.9, or about 1.5 to about 1.7 grams/liter. In certain embodiments, the free amino acid histidine is present in the

composition at about 0.5, 0.7, 1.0, 1.2, 1.5, or 1.7 grams/liter. In certain embodiments, the free amino acid threonine is present in the composition at the following concentrations: at about 0.4 to about 1.5, about 0.7 to about 1.3, about 0.9 to about 1.1 grams/liter, or about 1.5 to about 1.7 grams/liter. In certain embodiments, the free amino acid threonine is present in the composition at about 0.5, 0.7, 1.0, 1.2, 1.5, or 1.7 grams/liter. In certain embodiments, the free amino acid isoleucine is present in the composition at the following concentrations: isoleucine at 0.4 to about 1.5, about 0.7 to about 1.3, at about 1.1 to about 2.1, about 1.3 to about 1.9, or about 1.5 to about 1.7 grams/liter; asparagine at 0.4 to about 1.5, about 0.7 to about 1.3, at about 1.1 to about 2.1, about 1.3 to about 1.9, or about 1.5 to about 1.7 grams/liter. In certain embodiments, the free amino acid isoleucine is present in the composition at about 0.5, 0.7, 1.0, 1.2, 1.5, or 1.7 grams/liter. In certain embodiments, the free amino acid tryptophan is present in the composition at the following concentrations: at 0.4 to about 1.5, about 0.7 to about 1.3, at about 1.1 to about 2.1, about 1.3 to about 1.9, or about 1.5 to about 1.7 grams/liter. In certain embodiments, the free amino acid tryptophan is present in the composition at about 0.5, 0.7, 1.0, 1.2, 1.5, or 1.7 grams/liter.

[0060] In one embodiment, the total osmolarity of the composition is from about 100 mosm to about 280 mosm, or any value therebetween. Preferably, the total osmolarity is from about 150 to about 260 mosm. In another embodiment, the composition has a total osmolarity that is any value lower than about 280 mosm. In one embodiment, the total osmolarity of the composition is from about 100 mosm to about 280 mosm. In certain embodiments, the total osmolarity is from about 150 to about 260 mosm.

[0061] The composition may have a pH ranging from about 2.5 to about 8.5. In certain embodiments, the pH of the composition ranges from about 2.5 to about 6.5, about 3.0 to about 6.0, about 3.5 to about 5.5, about 3.9 to about 5.0, or about 4.2 to about 4.6. In other embodiments, the pH of the composition ranges from about 6.5 to about 8.5, about 7.0 to about 8.0, or about 7.2 to about 7.8. In certain embodiments, the composition has a pH from, for example, about 2.5 to about 8.5. In certain embodiments, the composition has a pH from about 2.5 to about 6.5, about 2.5 to about 6.0, about 3.0 to about 6.0, about 3.5 to about 6.0, about 3.9 to about 6.0, about 4.2 to about 6.0, about 3.5 to about 5.5, about 3.9 to about 5.0, or about 4.2 to about 4.6. In other embodiments, the pH is about 6.0 to about 8.0, about 6.0 to about 7.5, about 6.5 to about 8.5, about 7.0 to about 8.5, about 7.0 to about 8.0, about 7.0 to about 7.5, about 7.2 to about 8.0, or about 7.2 to about 7.8.

[0062] Compositions described herein can be prepared by any method known in the art of pharmacology. In general, such preparatory methods include bringing the amino acids described herein (e.g., the "free amino acid composition") into association with a carrier or excipient, and/or one or more other accessory ingredients, and then, if necessary and/or desirable, shaping, and/or packaging the product into a desired single- or multi-dose unit.

[0063] Relative amounts of the active ingredient, the pharmaceutically acceptable excipient, and/or any additional ingredients in a pharmaceutical composition described herein will vary, depending upon the identity, size, and/or condition of the subject treated and further depending upon the route by which the composition is to be administered. The composition may comprise between 0.1% and 100% (w/w) active ingredient.

[0064] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active ingredient is mixed with at least one inert, pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and/or (a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol, and silicic acid, (b) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia, (c) humectants such as glycerol, (d) disintegrating agents such as agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, (e) solution retarding agents such as paraffin, (f) absorption accelerators such as quaternary ammonium compounds, (g) wetting agents such as, for example, cetyl alcohol and glycerol monostearate, (h) absorbents such as kaolin and bentonite clay, and (i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets, and pills, the dosage form may include a buffering agent.

[0065] Liquid dosage forms for oral and parenteral administration include

pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active ingredients, the liquid dosage forms may comprise inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (e.g., cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. Besides inert diluents, the oral compositions can include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents. In certain embodiments for parenteral administration, the conjugates described herein are mixed with solubilizing agents such as Cremophor^®, alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins, polymers, and mixtures thereof.

Methods for increasing translocation of CFTR to the cytoplasm, decreasing secretion and/or loss of anions, decreasing fluid loss, and increasing fluid absorption in the GI tract

[0066] The present disclosure provides compositions for use in reducing the number of cystic fibrosis transmembrane conductance regulator (CFTR) proteins present on the plasma membrane of a cell. In one aspect, the present disclosure provides a method for translocating CFTR from the plasma membrane to the cytoplasm of a cell of a subject in need thereof, the method comprising administering to the subject a composition of free amino acids as described herein an effective amount of a composition comprising free amino acids of the present disclosure. Thus, in one aspect, the present disclosure provides a method for increasing the translocation of CFTR from the plasma membrane to the cytoplasm of a cell of a subject in need thereof, the method comprising administering to the subject an effective amount of a composition comprising free amino acids of the present disclosure. In certain embodiments, the cell is a GI tract cell. In certain embodiments, the cell is an epithelial GI tract cell. In certain embodiments, the cell is a small intestine epithelial cell. In certain embodiments, the cell is an intestine epithelial cell. In certain embodiments, the cell is a small intestine villus epithelial cell.

[0067] In certain embodiments, the composition comprises, consists essentially of, or consists of two or more free amino acids selected from the group consisting of proline, glutamic acid, glutamine, leucine, alanine, aspartic acid, and phenylalanine. In certain embodiments, the composition comprises, consists essentially of, or consists of three or more; four or more; five or more; or six or more; free amino acids selected from the group consisting of proline, glutamic acid, glutamine, leucine, alanine, aspartic acid, and

phenylalanine. The composition further comprises, in certain embodiments, one or more free amino acids selected from the group consisting of histidine, threonine, isoleucine, asparagine, and tryptophan. In certain embodiments, the composition comprises, consists essentially of, or consists of threonine, phenylalanine, isoleucine, glutamic acid, aspartic acid, alanine, and asparagine.

[0068] In one aspect, the present disclosure provides a method for increasing the translocation of CFTR from the plasma membrane to the cytoplasm of a cell of a subject in need thereof, the method comprising administering to the subject an effective amount of a composition comprising, consisting essentially of, or consisting of one or more free amino acids selected from the group consisting of lysine, threonine, valine, tyrosine, isoleucine, and aspartic acid. In certain embodiments, the composition comprises free amino acids of the present disclosure.

[0069] In certain embodiments, the composition comprises, consists essentially of, or consists of one or more free amino acids selected from the group consisting of lysine, threonine, valine, tyrosine, isoleucine, and aspartic acid. In certain embodiments, the composition comprises, consists essentially of, or consists of two or more; three or more; four or more; five or more; or six or more; free amino acids selected from the group consisting of lysine, threonine, valine, tyrosine, isoleucine, and aspartic acid. The composition further comprises, in certain embodiments, one or more free amino acids selected from the group consisting of histidine, asparagine, tryptophan, proline, glutamic acid, glutamine, leucine, alanine, and phenylalanine.

[0070] In certain embodiments, compositions of the present disclosure comprise one or more free amino acids that increase the translocation of CFTR to the cytoplasm of a cell of a subject in need thereof. In certain embodiments, compositions of the present disclosure may include one or more free amino acids that are not for increasing the translocation of CFTR to the cytoplasm, but may instead provide alternative beneficial properties to the composition (e.g., maintain a specific pH or osmolality). In certain embodiments, compositions of the present disclosure may include ingredients that are not for increasing the translocation of CFTR to the cytoplasm, but may instead provide alternative beneficial properties to the composition (e.g., maintain a specific pH or osmolarity).

[0071] The CFTR protein is an ABC transporter protein that functions as an ATP-gated ion channel. When activated, CFTR allows chloride ions (CI^"), and other negatively charged ions such as thiocyanate ([SCN]^") and/or bicarbonate (HCO-3), to flow down their

electrochemical gradient (e.g., passive diffusion or passive transport).

[0072] Thus, in one aspect, the present disclosure provides a method for decreasing anion secretion in the GI tract, the method comprising administering to a subject in need thereof a composition comprising, consisting essentially of, or consisting of two or more free amino acids selected from the group consisting of proline, glutamic acid, glutamine, leucine, alanine, aspartic acid, and phenylalanine. In one aspect, the present disclosure provides a method for decreasing anion loss in the GI tract, the method comprising administering to a subject in need thereof a composition comprising, consisting essentially of, or consisting of two or more free amino acids selected from the group consisting of proline, glutamic acid, glutamine, leucine, alanine, aspartic acid, and phenylalanine. The composition further comprises, in certain embodiments, one or more free amino acids selected from the group consisting of histidine, threonine, isoleucine, asparagine, and tryptophan. In certain embodiments, the composition comprises, consists essentially of, or consists of two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, or eleven or more free amino acids selected from the group consisting of proline, glutamic acid, glutamine, leucine, alanine, aspartic acid, phenylalanine, histidine, threonine, isoleucine, asparagine, and tryptophan. In certain embodiments, the anion is chloride. In certain embodiments, the anion is bicarbonate.

[0073] Thus, in one aspect, the present disclosure provides a method for decreasing anion secretion in the GI tract, the method comprising administering to a subject in need thereof a composition comprising, consisting essentially of, or consisting of one or more free amino acids selected from the group consisting of lysine, threonine, valine, tyrosine, isoleucine, and aspartic acid. In one aspect, the present disclosure provides a method for decreasing anion loss in the GI tract, the method comprising administering to a subject in need thereof a composition comprising, consisting essentially of, or consisting of one or more free amino acids selected from the group consisting of lysine, threonine, valine, tyrosine, isoleucine, and aspartic acid. The composition further comprises, in certain embodiments, one or more free amino acids selected from the group consisting of histidine, asparagine, tryptophan, proline, glutamic acid, glutamine, leucine, alanine, and phenylalanine. In certain embodiments, the composition comprises, consists essentially of, or consists of one or more, two or more, three or more, four or more, five or more free amino acids selected from the group consisting of lysine, threonine, valine, tyrosine, isoleucine, and aspartic acid. In certain embodiments, the anion is chloride. In certain embodiments, the anion is bicarbonate.

[0074] In one aspect, increased translocation of CFTR from the plasma membrane to the cytoplasm of a cell of a subject decreases fluid loss and/or electrolyte loss in the GI tract. In another aspect, the present disclosure provides a method for decreasing fluid loss in the GI tract, the method comprising administering to a subject in need thereof a composition described herein. In certain embodiments, the present disclosure provides a method for decreasing fluid loss in the crypt of the small intestine, the method comprising administering to a subject in need thereof a composition described herein. In certain embodiments, the decreasing fluid loss in the GI tract comprises decreasing fluid loss from the crypt. In certain embodiments, the decreasing fluid loss in the GI tract comprises decreasing fluid loss from the crypt of the small intestine. In certain embodiments, the method further comprises decreasing electrolyte loss in the GI tract. In certain embodiments, the method further comprises decreasing electrolyte loss in the crypt of the small intestine. Without wishing to be bound by any particular theory, the method of decreasing fluid loss in the GI tract results from decreasing electrolyte loss in the GI tract. The electrolyte or electrolytes, in certain embodiments, include, but are not limited to Na⁺; K⁺; HCO3^"; Ca²⁺; Mg²⁺; CI^"; and phosphate ions, such as HP0₄ ^2".

[0075] In one aspect, increased translocation of CFTR from the plasma membrane to the cytoplasm of a cell of a subject increases fluid absorption in the GI tract. In another aspect, the present disclosure provides a method for increasing fluid absorption in the GI tract, the method comprising administering to a subject in need thereof a composition described herein. In certain embodiments, the present disclosure provides a method for increasing fluid absorption in the villi of the small intestine, the method comprising administering to a subject in need thereof a composition described herein. In certain embodiments, the increasing fluid absorption in the GI tract comprises increasing fluid absorption in the villi of the small intestine. In certain embodiments, the increasing fluid absorption in the GI tract comprises increasing fluid absorption in the villi.

Methods for treatment of diarrhea

[0076] As discussed above, diarrhea causes dehydration in subjects, from the loss of fluid and electrolytes. The CFTR channel normally allows chloride ions to flow out of cells.

Cholera bacteria produce cholera toxin which binds to epithelial cells in the small intestine, which increases the number of cAMP molecules in the epithelial cells and activates the CFTR channels, so that the CFTR channels stay open and large amounts of the chloride ions flow out. Osmotic gradients lead to the excretion of sodium ions and fluids, causing fluid loss, dehydration and diarrhea. In one aspect, the present disclosure provides a method for treating a gastrointestinal disease in a subject in need thereof, the method comprising administering to the subject a composition described herein. In one aspect, the present disclosure provides a method for treating diarrhea in a subject in need thereof, the method comprising

administering to the subject a composition described herein. In certain embodiments, the diarrhea comprises severe acute diarrhea. In certain embodiments, the diarrhea is caused by an infectious disease. In certain embodiments, the diarrhea is caused by a bacterial infectious disease. In certain embodiments, the diarrhea is caused by cholera. In certain embodiments, the severe acute diarrhea is caused by cholera. In certain embodiments, the severe acute diarrhea is caused by an infectious organism. In certain embodiments, the diarrhea is caused by bacteria. The bacteria, in certain embodiments, is gram-negative. The bacteria, in certain embodiments, is gram-positive. In certain embodiments, the diarrhea is caused by

Escherichia coli. In certain embodiments, the diarrhea is caused by shigella. In certain embodiments, the diarrhea is caused by a parasite. In certain embodiments, the diarrhea is caused by Cryptosporidium. In certain embodiments, the diarrhea is caused by a virus. In certain embodiments, the diarrhea is caused by rotavirus. In certain embodiments, the diarrhea comprises acute bloody diarrhea. In certain embodiments, the diarrhea comprises chronic diarrhea. The composition, in some embodiments, comprises, consists essentially of, or consists of two or more free amino acids selected from the group consisting of proline, glutamic acid, glutamine, leucine, alanine, aspartic acid, and phenylalanine. In certain embodiments, the composition further comprises, one or more free amino acids selected from the group consisting of histidine, threonine, isoleucine, asparagine, and tryptophan. [0077] In another aspect, provided is a composition comprising, consisting essentially of, or consisting of two or more free amino acids selected from the group consisting of proline, glutamic acid, glutamine, leucine, alanine, aspartic acid, and phenylalanine for use in treating diarrhea (e.g. , diarrhea caused by cholera or chronic diarrhea). In certain embodiments, the composition further comprises, one or more free amino acids selected from the group consisting of histidine, threonine, isoleucine, asparagine, and tryptophan. In certain embodiments, the composition comprises, consists essentially of, or consists of two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, or eleven or more free amino acids selected from the group consisting of proline, glutamic acid, glutamine, leucine, alanine, aspartic acid, phenylalanine, histidine, threonine, isoleucine, asparagine, and tryptophan. In a further aspect, provided is a composition described herein for reducing the number of CFTR proteins present on the plasma membrane of a cell, translocating CFTR from the plasma membrane to the cytoplasm of a cell, increasing translocation of CFTR from the plasma membrane to the cytoplasm of a cell, decreasing anion secretion in the GI tract, decreasing anion loss in the GI tract, decreasing fluid loss in the GI tract, or increasing fluid absorption in the GI tract.

[0078] In certain embodiments, provided is a method for treating diarrhea in a subject in need thereof, the method comprising administering to the subject a composition comprising, consisting essentially of, or consisting of one or more free amino acids selected from the group consisting of lysine, threonine, valine, tyrosine, isoleucine, and aspartic acid, and no other free amino acids. In another aspect, provided is a composition comprising, consisting essentially of, or consisting of one or more free amino acids selected from the group consisting of lysine, threonine, valine, tyrosine, isoleucine, and aspartic acid for use in treating diarrhea (e.g. , diarrhea caused by cholera or chronic diarrhea). In certain

embodiments, the composition further comprises, one or more free amino acids selected from the group consisting of histidine, asparagine, tryptophan, proline, glutamic acid, glutamine, leucine, alanine, and phenylalanine. In certain embodiments, the composition comprises, consists essentially of, or consists of one or more, two or more, three or more, four or more, five or more, or six free amino acids selected from the group consisting of lysine, threonine, valine, tyrosine, isoleucine, and aspartic acid. In a further aspect, provided is a composition described herein for reducing the number of CFTR proteins present on the plasma membrane of a cell, translocating CFTR from the plasma membrane to the cytoplasm of a cell, increasing translocation of CFTR from the plasma membrane to the cytoplasm of a cell, decreasing anion secretion in the GI tract, decreasing anion loss in the GI tract, decreasing fluid loss in the GI tract, or increasing fluid absorption in the GI tract. In a further aspect, provided are uses of the compositions described herein, in treating diarrhea (e.g. , diarrhea caused by cholera or chronic diarrhea). In certain embodiments, provided are uses of the compositions described herein, for reducing the number of CFTR proteins present on the plasma membrane of a cell, translocating CFTR from the plasma membrane to the cytoplasm of a cell, increasing translocation of CFTR from the plasma membrane to the cytoplasm of a cell, decreasing anion secretion (e.g. , decreasing the secretion of chloride and/or bicarbonate) in the GI tract, decreasing the loss of anions (e.g. , decreasing the loss of chloride and/or bicarbonate) in the GI tract, decreasing fluid loss in the GI tract, or increasing fluid absorption in the GI tract.

Kits

[0079] Another aspect of the present disclosure relates to kits for use in administering the compositions described herein. The kit may be useful in a method of the disclosure. The kit may also include instructions on how to use the materials in the kit. Kits are also provided for using or administering the inventive particle, or pharmaceutical compositions and/or nutraceutical compositions thereof. The particle may be provided in convenient dosage units for administration to a subject. The kit may include multiple dosage units. For example, the kit may include 1-100 dosage units. In certain embodiments, the kit includes a week supply of dosage units, or a month supply of dosage units. In certain embodiments, the kit includes an even longer supply of dosage units. The kits may also include devices for administering the particles or a pharmaceutical composition thereof. Exemplary devices include syringes, spoons, measuring devices, amongst others. The kit may optionally include instructions for administering the inventive particles (e.g. , prescribing information). In one aspect, the kit comprises a composition comprising two or more free amino acids selected from the group consisting of proline, glutamic acid, glutamine, leucine, alanine, aspartic acid, and phenylalanine; and instructions for administering to a subject or contacting a biological sample with the composition. In certain embodiments, the composition further comprises, one or more free amino acids selected from the group consisting of histidine, threonine, isoleucine, asparagine, and tryptophan. In one aspect, the kit comprises a composition comprising one or more free amino acids selected from the group consisting of lysine, threonine, valine, tyrosine, isoleucine, and aspartic acid; and instructions for administering to a subject or contacting a biological sample with the composition. In certain embodiments, the composition further comprises, one or more free amino acids selected from the group consisting of histidine, asparagine, tryptophan, proline, glutamic acid, glutamine, leucine, alanine, and phenylalanine.

EXAMPLES

[0080] In order that the invention described herein may be more fully understood, the following examples are set forth. The examples described in this application are offered to illustrate the methods, compositions, and systems provided herein and are not to be construed in any way as limiting their scope.

Example 1. Effects of Selected Amino Acids on CFTR translocation from the plasma membrane to the cytoplasm of a cell

[0081] To study the effect of amino acids on CFTR translocation from the plasma membrane to the cytoplasm, studies were first done using isolated small intestinal loops that were filled with Ringer's solution or Ringer's solution containing each of the amino acids or ringer solution containing a combination of amino acids in the presence of cholera toxin. Ringer's solution contained l(^g/lml cholera toxin. Ringer's solution containing each of the individual amino acids (alanine, arginine, asparagine, aspartic acid, cysteine, glutamate, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine) were used in the isolated intestinal loops and incubated for a period of 45 minutes with cholera toxin. The tissues were then isolated, the volume of fluid in the intestinal loops was measured and the tissues were homogenized for protein characterization using western blot analysis. Western blot analysis showed that certain amino acids (threonine, phenylalanine, isoleucine, glutamate, aspartic acid, alanine, asparagine) decreased the protein levels of CFTR in the brush border membrane vesicles isolated from small intestinal epithelial cells incubated with cholera toxin. When these amino acids were used in combination in isolated intestinal loops, there was significant decrease in CFTR protein on brush border membrane vesicles isolated from the small intestinal villus epithelial cells and net fluid secretion in the presence of cholera toxin.

Example 2: Rat Perfusion Experiments

[0082] The small intestine was used for the studies herein as 80% of electrolyte and fluid absorption occurs in the small intestine. Rats (Sprague-Dawley, male, 350-500 g, 9-12 weeks) underwent fasting for 12 hours before being administered isoflurane as anesthesia and undergoing abdominal surgery lasting between 20-25 minutes. During surgery, rats experienced a body temperature decrease to 92 °F. The distal jejunum and proximal Ileum (5- 9 cm) were perfused at a rate of 150 μΙ7ηιίη. Perfusion equilibration lasted 90 minutes to allow for stabilization (Figure 1A), which coincided with body temperature stabilization back to 97 °F (Figure IB).

[0083] Perfusion sampling took place via administration of 3 x 15 minutes of Ringer solution followed by switching to test solution and allowing 15 minutes for equilibration. Next, 2 x 15 minutes of test solution was administered, before switching to Ringer and allowing 15 minutes of equilibration. Finally, 3 x 15 minutes of Ringer was administered. Tissue and blood sampling was carried, before the subject was euthanized.

[0084] The formulation of 6AA solution is provided in Table 1 below.

Table 1: Formulation of 6AA

[0085] Different solutions were tested to determine the absorption rate of each. Ringer (RR), Trioral (WHO formulation), 8AA-AD (8 amino acid formulation), 5AA (5 amino acid oncology formulation), LS-5AA (Enterade 5 amino acid, low sodium formulation), and Speedlyte® were compared to one another (Figure 2). Finally, CT-RR (control experiment wherein the intestine was perfused with cholera toxin and then with cholera toxin containing ringer solution), CT-Ent 8 AA-AD (an 8 amino acid formulation in cholera treated exposed tissues), CT-Trioral (WHO formulation in the presence of cholera toxin), CT-Ent 5 AA (a 5 amino acid formulation in the presence of cholera toxin), and CT-6 AA-AD (new

antidiarrheal amino acid formulation in the presence of cholera toxin) were compared to one another (Figure 3). The new exemplary antidiarrheal amino acid formulation (CT-6 AA-AD had the highest absorption rate. For CT-6 AA-AD all amino acids helped move CFTR from the plasma membrane into the cytoplasm, while the 5 and 8 amino acids formulations included amino acids that were not stimulating secretion, increasing amino acid coupled sodium absorption, tightening mucosal barrier, and increasing proliferation. [0086] The total osmolality or osmotic concentration (where osmoles is "Osm") of the solutions tested appear below in Table 2. Osmotic concentration is defined as the number of osmoles (Osm) of solute per liter (L) of solution.

Table 2: Solutions and Total Osmolarity (in Osm)

EQUIVALENTS AND SCOPE

[0087] While several embodiments of the present invention have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and/or structures for performing the functions and/or obtaining the results and/or one or more of the advantages described herein, and each of such variations and/or modifications is deemed to be within the scope of the present invention. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and/or configurations will depend upon the specific application or applications for which the teachings of the present invention is/are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto; the invention may be practiced otherwise than as specifically described and claimed. The present invention is directed to each individual feature, system, article, material, kit, and/or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and/or methods, if such features, systems, articles, materials, kits, and/or methods are not mutually inconsistent, is included within the scope of the present invention.

[0088] The indefinite articles "a" and "an," as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean "at least one." [0089] The phrase "and/or," as used herein in the specification and in the claims, should be understood to mean "either or both" of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Other elements may optionally be present other than the elements specifically identified by the "and/or" clause, whether related or unrelated to those elements specifically identified unless clearly indicated to the contrary. Thus, as a non-limiting example, a reference to "A and/or B," when used in conjunction with open-ended language such as "comprising" can refer, in one embodiment, to A without B (optionally including elements other than B); in another embodiment, to B without A (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

[0090] As used herein in the specification and in the claims, "or" should be understood to have the same meaning as "and/or" as defined above. For example, when separating items in a list, "or" or "and/or" shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as "only one of or "exactly one of," or, when used in the claims, "consisting of," will refer to the inclusion of exactly one element of a number or list of elements. In general, the term "or" as used herein shall only be interpreted as indicating exclusive alternatives (i.e. "one or the other but not both") when preceded by terms of exclusivity, such as "either," "one of," "only one of," or "exactly one of." "Consisting essentially of," when used in the claims, shall have its ordinary meaning as used in the field of patent law.

[0091] As used herein in the specification and in the claims, the phrase "at least one," in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase "at least one" refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, "at least one of A and B" (or, equivalently, "at least one of A or B," or, equivalently "at least one of A and/or B") can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another

embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

[0092] In the claims, as well as in the specification above, all transitional phrases such as "comprising," "including," "carrying," "having," "containing," "involving," "holding," and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases "consisting of and "consisting essentially of shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03.

Claims

CLAIMS What is claimed is:

1. A method for treating diarrhea in a subject in need thereof, the method comprising administering to the subject a composition comprising, consisting essentially of, or consisting of one or more free amino acids selected from the group consisting of lysine, threonine, valine, tyrosine, isoleucine, and aspartic acid, and no other free amino acids.

2. The method of claim 1, wherein the diarrhea is caused by cholera.

3. The method of any one of claims 1-2, wherein the composition comprises, consisting essentially of, or consisting of two or more free amino acids selected from the group consisting of lysine, threonine, valine, tyrosine, isoleucine, and aspartic acid, and no other free amino acids.

4. The method of any one of claims 1-3, wherein the composition comprises, consisting essentially of, or consisting of three or more free amino acids selected from the group consisting of lysine, threonine, valine, tyrosine, isoleucine, and aspartic acid, and no other free amino acids.

5. The method of any one of claims 1-4, wherein the composition comprises, consisting essentially of, or consisting of four or more free amino acids selected from the group consisting of lysine, threonine, valine, tyrosine, isoleucine, and aspartic acid, and no other free amino acids.

6. The method of any one of claims 1-5, wherein the composition comprises, consisting essentially of, or consisting of five or more free amino acids selected from the group consisting of lysine, threonine, valine, tyrosine, isoleucine, and aspartic acid, and no other free amino acids.

7. The method of any one of claims 1-6, wherein the composition comprises, consisting essentially of, or consisting of all six free amino acids selected from the group consisting of lysine, threonine, valine, tyrosine, isoleucine, and aspartic acid, and no other free amino acids.

8. The method of any one of claims 1-7, wherein the composition further comprises water.

9. The method of any one of claims 1-8, wherein the composition further comprises a pharmaceutically acceptable carrier, buffer, electrolyte, adjuvant, or excipient.

10. The method of any one of claims 1-9, wherein the composition is sterile.

11. The method of any one of claims 1-10, wherein the composition is formulated for oral administration.

12. A method for reducing the number of cystic fibrosis transmembrane conductance regulator (CFTR) proteins present on the plasma membrane of a cell, the method comprising administering to a subject in need thereof a composition comprising, consisting essentially of, or consisting of, two or more free amino acids selected from the group consisting of proline, glutamic acid, glutamine, leucine, alanine, aspartic acid, and phenylalanine.

13. The method of claim 12, wherein the composition further comprises one or more amino acids selected from the group consisting of histidine, threonine, isoleucine, asparagine, and tryptophan.

14. The method of claim 12, wherein the CFTR protein is translocated from the plasma membrane to the cytoplasm.

15. The method of claim 12, wherein the cell is a GI tract cell.

16. The method of claim 15, wherein the cell is an epithelial GI tract cell.

17. The method of claim 16, wherein the cell is a small intestine epithelial cell.

18. The method of claim 17, wherein the cell is a small intestine villus epithelial cell.

19. A method for reducing the number of cystic fibrosis transmembrane conductance regulator (CFTR) proteins present on the plasma membrane of a cell, the method comprising administering to a subject in need thereof a composition comprising, consisting essentially of, or consisting of, one or more free amino acids selected from the group consisting of lysine, threonine, valine, tyrosine, isoleucine, and aspartic acid.

20. The method of claiml9, wherein the composition further comprises one or more amino acids selected from the group consisting of histidine, asparagine, tryptophan, proline, glutamic acid, glutamine, leucine, alanine, and phenylalanine.

21. The method of claim 19, wherein the CFTR protein is translocated from the plasma membrane to the cytoplasm.

22. The method of claim 19, wherein the cell is a GI tract cell.

23. The method of claim 22, wherein the cell is an epithelial GI tract cell.

24. The method of claim 23, wherein the cell is a small intestine epithelial cell.

25. The method of claim 24, wherein the cell is a small intestine villus epithelial cell.

26. A method of translocating CFTR from the plasma membrane to the cytoplasm of a cell.

27. A method of decreasing anion secretion in the GI tract, the method comprising administering to a subject in need thereof a composition comprising, consisting essentially of, or consisting of, two or more free amino acids selected from the group consisting of proline, glutamic acid, glutamine, leucine, alanine, aspartic acid, and phenylalanine.

28. The method of claim 27, wherein the anion is chloride.

29. The method of claim 27, wherein the anion is bicarbonate.

30. A method of decreasing anion secretion in the GI tract, the method comprising administering to a subject in need thereof a composition comprising, consisting essentially of, or consisting of, one or more free amino acids selected from the group consisting of lysine, threonine, valine, tyrosine, isoleucine, and aspartic acid.

31. The method of claim 30, wherein the anion is chloride.

32. The method of claim 30, wherein the anion is bicarbonate.

33. A method of decreasing fluid loss in the GI tract, the method comprising

administering to a subject in need thereof a composition comprising, consisting essentially of, or consisting of, two or more free amino acids selected from the group consisting of proline, glutamic acid, glutamine, leucine, alanine, aspartic acid, and phenylalanine.

34. The method of claim 33, comprising decreasing electrolyte loss in the GI tract.

35. The method of claim 33, wherein the decreasing fluid loss in the GI tract comprises decreasing fluid loss from the crypt.

36. A method of decreasing fluid loss in the GI tract, the method comprising

administering to a subject in need thereof a composition comprising, consisting essentially of, or consisting of, one or more free amino acids selected from the group consisting of lysine, threonine, valine, tyrosine, isoleucine, and aspartic acid.

37. The method of claim 36, comprising decreasing electrolyte loss in the GI tract.

38. The method of claim 36, wherein the decreasing fluid loss in the GI tract comprises decreasing fluid loss from the crypt.

39. A method of increasing fluid absorption in the GI tract, the method comprising administering to a subject in need thereof a composition comprising two or more free amino acids selected from the group consisting of proline, glutamic acid, glutamine, leucine, alanine, aspartic acid, and phenylalanine.

40. The method of claim 39, wherein the increasing fluid absorption in the GI tract comprises increasing fluid absorption in the villi.

41. A method of increasing fluid absorption in the GI tract, the method comprising administering to a subject in need thereof a composition comprising one or more free amino acids selected from the group consisting of lysine, threonine, valine, tyrosine, isoleucine, and aspartic acid.

42. The method of claim 41, wherein the increasing fluid absorption in the GI tract comprises increasing fluid absorption in the villi.

43. A composition comprising, consisting essentially of, or consisting of, two or more free amino acids selected from the group consisting of proline, glutamic acid, glutamine, leucine, alanine, aspartic acid, and phenylalanine, and no other free amino acids for use in treating diarrhea.

44. A composition consisting essentially of, or consisting of, two or more free amino acids selected from the group consisting of proline, glutamic acid, glutamine, leucine, alanine, aspartic acid, and phenylalanine, and no other free amino acids for use in treating diarrhea.

45. A composition comprising, consisting essentially of, or consisting of, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, or eleven or more free amino acids selected from the group consisting of proline, glutamic acid, glutamine, leucine, alanine, aspartic acid, phenylalanine, histidine, threonine, isoleucine, asparagine, and tryptophan, and no other free amino acids.

46. A composition consisting essentially of, or consisting of, threonine, phenylalanine, isoleucine, glutamic acid, aspartic acid, alanine, and asparagine, and no other free amino acids.

47. A composition comprising, consisting essentially of, or consisting of, one or more free amino acids selected from the group consisting of lysine, threonine, valine, tyrosine, isoleucine, and aspartic acid, and no other free amino acids for use in treating diarrhea.

48. A composition consisting essentially of, or consisting of, one or more free amino acids selected from the group consisting of lysine, threonine, valine, tyrosine, isoleucine, and aspartic acid, and no other free amino acids for use in treating diarrhea.

49. A composition comprising, consisting essentially of, or consisting of, one or more, two or more, three or more, four or more, five or more, or six free amino acids selected from the group consisting of lysine, threonine, valine, tyrosine, isoleucine, and aspartic acid, and no other free amino acids.

50. A composition consisting essentially of, or consisting of, lysine, threonine, valine, tyrosine, isoleucine, and aspartic acid, and no other free amino acids.

51. Use of a composition to treat diarrhea in a subject in need thereof, wherein the composition comprises two or more free amino acids selected from the group consisting of proline, glutamic acid, glutamine, leucine, alanine, aspartic acid, and phenylalanine.

52. Use of a composition to treat diarrhea in a subject in need thereof, wherein the composition comprises one or more free amino acids selected from the group consisting of lysine, threonine, valine, tyrosine, isoleucine, and aspartic acid.

53. A kit comprising:

a composition comprising, consisting essentially of, or consisting of, two or more free amino acids selected from the group consisting of proline, glutamic acid, glutamine, leucine, alanine, aspartic acid, and phenylalanine; and

instructions for administering to a subject or contacting a biological sample with the composition.

54. A kit comprising:

a composition comprising, consisting essentially of, or consisting of, one or more free amino acids selected from the group consisting of lysine, threonine, valine, tyrosine, isoleucine, and aspartic acid; and