Methods of treating bile acid diarrhea

Abstract

The present disclosure relates to methods of treating bile acid diarrhea by administering a CFTR chloride channel inhibitor (CFTR-CCI). In some embodiments, the CFTR chloride channel inhibitor may be a benzopyrimidino-pyrrolo-oxazine-dione-CFTR-CCI (e.g., BPO-27), a PPQ-CFTR-CCI, a thiazolidinone-CFTR-CCI, or a glycine hydrazide-CFTR-CCI, or a pharmaceutically acceptable salt thereof.

Description

[0001] Government Rights

[0002] This invention was made with government support under Grant Nos. DK099803, DK072517, DK101373, EY013574, and DK089502 awarded by the National Institutes of Health. The government has certain rights in the invention. Technical Field

[0003] The present invention relates to methods of treating bile acid diarrhea by administering CFTR chloride channel inhibitors. Background Art

[0004] Bile acid diarrhea (BAD) is common after ileal resection and in Crohn's disease and is believed to occur in more than one-third of patients with diarrhea-predominant irritable bowel syndrome (IBS-D) or chronic functional diarrhea, with an estimated overall prevalence of up to 1% in Western countries. BAD can be caused by abnormalities in hepatic bile acid synthesis or the enterohepatic circulation, which results in the delivery of excessive bile acids to the colon, where they cause fluid secretion and increased motility.

[0005] Current therapies for primary BAD or BAD caused by ileal resection or Crohn's disease include bile acid binders such as cholestyramine, colestipol, and colesevelam, and farnesoid X receptor (FXR) agonists such as obeticholic acid are under development. For IBS-D, in which BAD may be involved in the pathogenesis of more than one-third of patients, FDA-approved therapies include the 5-HT3 antagonist alosetron, the mixed μ-opioid receptor agonist eluxadoline, and the broad-spectrum gut-specific antibiotic rifaximin. Other commonly used therapies for IBS-D include loperamide, bile acid sequestrants, antispasmodics, and tricyclic antidepressants. Some of these therapies are associated with serious side effects such as ischemic colitis associated with alosetron and pancreatitis associated with eluxadoline.

[0006] There remains an unmet need for improved and alternative therapies for BAD.

[0007] The cystic fibrosis transmembrane conductance regulator (CFTR) protein is a chloride channel expressed in certain mammalian epithelial cells, including intestinal epithelial cells. CFTR chloride channel function is associated with secretory diarrhea.

[0008] CFTR inhibitors have clinical applications in the therapy of secretory diarrhea. Secretory diarrhea caused by enterotoxins, such as cholera and traveler's diarrhea (enteropathogenic Escherichia coli), requires functional CFTR for the secretion of mainly chloride ions into the intestinal lumen, which in turn drives sodium and water secretion (see, e.g., Kunzelmann et al., Physiol. Rev. 82:245-89 (2002); Thiagarajah et al., Curr. Opin. Pharmacol. 3:594-9 (2003)). Cell culture and animal models have shown that intestinal chloride secretion in enterotoxin-mediated secretory diarrhea occurs mainly via CFTR (see, e.g., Clarke et al., Science 257:1125-28 (1992); Gabriel et al., Science 266:107-109 (1994); Kunzelmann and Mall, Physiol. Rev. 82:245-89 (2002); Field, 15 J. Clin. Invest. 111:931-43 (2003); and Thiagarajah et al., Gastroenterology 126:511-519 (2003)). Several classes of small molecule CFTR inhibitors have been previously described (see, e.g., the review by Verkman et al., Nat. Rev. Drug Discov. 8:153-71 (2009)).

[0009] U.S. Patent No. 9,062,073 describes a series of benzopyrimido-pyrrolo-oxazine-dione (BPO) compounds and certain pyrimido-pyrrolo-quinoxaline dione (PPQ) compounds, which can be used as CFTR chloride channel inhibitors. The BPO compounds have the general formula:

[0010]

[0011] wherein:

[0012] m is 1, 2, 3, or 4;

[0013] n is 1, 2, 3, 4, or 5;

[0014] p is an integer from 0 to 4;

[0015] q is an integer from 1 to 4;

[0016] R 1 is the same or different and independently at each occurrence H, halogen, haloalkyl, C1-C6 alkyl, —(CH2) p —C(O)—R 4a 、—S(O)2R 4a 、—NO2, or tetrazolyl;

[0017] R 1a Each occurrence is the same or different and independently is H, a halogen, a haloalkyl, a C1-C6 alkyl, —(CH2) p —C(O)—R 4a 、—S(O)2R 4a 、—NO2, or a tetrazolyl group;

[0018] R 2a and R 2b are each the same or different and independently are H, or a C1-C6 alkyl;

[0019] R 4a is —OR 7 、—NR 7 R 8 、—O(CH2) q —OC(O)R 7 、or an amino acid residue;

[0020] R 7 and R 8 are each the same or different and independently are H, C1-C 20 alkyl, a saccharide, or an amino acid residue; and

[0021] Z is an aryl or a heteroaryl,

[0022] wherein the amino acid residue is selected from the residues of: alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, phosphoserine, phosphothreonine, phosphotyrosine, 4-hydroxyproline, hydroxylysine, desmosine, isodesmosine, γ-carboxyglutamic acid, hippuric acid, octahydroindole-2-carboxylic acid, pepstatin, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, penicillamine, ornithine, 3-methylhistidine, norvaline, β-alanine, γ-aminobutyric acid, citrulline, homocysteine, homoserine, methylalanine, p-benzoyl-phenylalanine, phenylglycine, propargylglycine, sarcosine, methionine sulfone, tert-butylglycine, 3,5-dibromotyrosine, 3,5-diiodotyrosine, glycosylated threonine, glycosylated serine, and glycosylated asparagine.

[0023] The PPQ compound has the general formula:

[0024]

[0025] wherein:

[0026] m is 1, 2, 3, or 4;

[0027] n is 1, 2, 3, 4 or 5;

[0028] p is an integer from 0 to 4;

[0029] q is an integer from 1 to 4;

[0030] X is O or S;

[0031] R 1 is the same or different and independently H, halogen, haloalkyl, C 1-6 alkyl, —(CH2) p —C(O)—R 4a 、—S(O)2R 4a 、—NO2, or tetrazolyl;

[0032] R 1a is the same or different and independently H, halogen, haloalkyl, C 1-6 alkyl, —(CH2) p —C(O)—R 4a 、—S(O)2R 4a 、—NO2, or tetrazolyl;

[0033] R 2a and R 2b are each the same or different and independently H or C 1-6 alkyl;

[0034] R 4a is —OR 7 、—NR 7 R 8 、—O(CH2) q —OC(O)R 7 、an amino acid residue, or a peptide;

[0035] R 4 is H, —N(═O), C 1-6 alkyl, or haloalkyl;

[0036] R 5 is H, halogen, or C 1-6 alkyl;

[0037] R 6 is halogen, C 1-6 alkyl, or C 1-6 haloalkyl; and

[0038] R 7 and R 8 are each the same or different and independently H, C 1-20 alkyl, saccharide, amino acid residue, or peptide. SUMMARY OF THE INVENTION

[0039] Aspects of the present invention relate to methods of treating an individual suffering from bile acid diarrhea, comprising administering to the individual an amount of a CFTR chloride channel inhibitor effective to treat the bile acid diarrhea.

[0040] Other aspects of the present invention relate to methods of treating an individual suffering from bile acid diarrhea, comprising administering to the individual a pharmaceutical composition comprising an amount of a CFTR chloride channel inhibitor effective to treat the bile acid diarrhea.

[0041] In addition, aspects of the present invention also relate to methods of reducing intestinal fluid secretion in an individual in need thereof caused by activation of apical CFTR chloride channels in the intestinal epithelium induced by bile acids, comprising administering to the individual an amount of a CFTR chloride channel inhibitor effective to reduce the intestinal fluid secretion caused by activation of the apical CFTR chloride channels induced by bile acids.

[0042] Other aspects of the present invention relate to methods of reducing intestinal fluid secretion in an individual in need thereof caused by activation of apical CFTR chloride channels in the intestinal epithelium induced by bile acids, comprising administering to the individual a pharmaceutical composition comprising an amount of a CFTR chloride channel inhibitor effective to reduce the intestinal fluid secretion caused by activation of the apical CFTR chloride channels induced by bile acids.

[0043] Additional aspects of the present invention relate to methods of reducing apical CFTR chloride channel current in the intestinal epithelium of an individual in need thereof induced by bile acids, comprising administering to the individual an amount of a CFTR chloride channel inhibitor effective to reduce the apical CFTR chloride channel current induced by the bile acids.

[0044] Other aspects of the present invention relate to methods of reducing apical CFTR chloride channel current in the intestinal epithelium of an individual in need thereof induced by bile acids, comprising administering to the individual a pharmaceutical composition comprising an amount of a CFTR chloride channel inhibitor effective to reduce the apical CFTR chloride channel current induced by the bile acids.

[0045] In some aspects, the present invention relates to methods for treating diarrhea or alleviating symptoms associated with diarrhea in an individual who has undergone ileal resection, comprising administering to the individual an amount of a CFTR chloride channel inhibitor effective to treat the diarrhea or alleviate the symptoms of the diarrhea.

[0046] Other aspects of the present invention relate to methods for treating diarrhea or alleviating symptoms associated with diarrhea in an individual who has undergone ileal resection, comprising administering to the individual a pharmaceutical composition comprising an amount of a CFTR chloride channel effective to treat the diarrhea or alleviate the symptoms of the diarrhea. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 . CFTR inhibitors block bile acid-induced secretory currents in T84 cells. (A.) Short-circuit currents (Isc) in response to the indicated bile acids added to both apical and basolateral bathing solutions are shown. (R)-BPO-27 was added at 10 μM (representing 3 filters studied for cholic acid, ursodeoxycholic acid, and lithocholic acid; and 5 filters for deoxycholic acid, chenodeoxycholic acid, and their taurine conjugates). (B.) CDCA (0.75 mM) was added to both bathing solutions, and (R)-BPO-27 (10 μM) or CFTR inh -172 (10 μM) was added as indicated. (C.) Summary of peak CDCA-induced currents and current reduction after (R)-BPO-27 or CFTR inh -172 (ΔIsc, mean ± S.E.M., n = 7 - 8 filters per condition). (D.) Short-circuit currents in mouse colon showing the effect of CDCA (1 mM) added to the basolateral or apical bathing solution and (R)-BPO-27 (10 μM) added as indicated.

[0048] Figure 2 . CDCA acts from the apical side of the T84 cell monolayer to activate apical CFTR. (A.) CDCA was added to the basolateral or apical bathing solution. (Upper panel) Representative short-circuit current traces, (R)-BPO-27 added at 10 μM. (Lower panel) Summary of peak CDCA-induced currents (ΔIsc, mean ± S.E.M., n = 7 - 8, **p < 0.01, ns, not significant compared to zero effect). (B.) CDCA increasing from 0.75 mM to 2 mM was added to the basolateral bathing solution, followed by forskolin (10 μM added to both sides) and (R)-BPO-27 (10 μM). (C.) Short-circuit currents in T84 cells after basolateral membrane permeabilization with 250 μg / ml amphotericin B in the presence of a basolateral-to-apical solution Cl - gradient (basolateral [Cl - 120 mM, apical [Cl - 5 mM). CDCA (0.75 mM or 1 mM) was added to the apical bathing solution, followed by (R)-BPO-27 (10 μM).

[0049] Figure 3.The effect of CDCA on cAMP signaling in T84 cells is minimal. (A.) Short-circuit currents showing the effect of CDCA at the indicated concentrations then forskolin (10 μM) and subsequently (R)-BPO-27 (10 μM). (B.) Summary of peak forskolin-induced currents (ΔIsc, mean ± S.E.M., n = 4 - 6, **p < 0.01, ns, not significant compared to the forskolin response with 0 mM CDCA). (C.) cAMP in T84 cells measured 30 minutes after incubation with the indicated concentrations of CDCA in the presence or absence of 10 μM forskolin (mean ± S.E.M., n = 3 - 6, **p < 0.01, ns, not significant).

[0050] Figure 4 .The action of CDCA in T84 cells involves Ca 2+ signaling. (A.) Short-circuit currents showing the effect of CDCA (1 mM) then (R)-BPO-27 (10 μM) after pretreatment with BAPTA-AM (30 μM) for 25 minutes. (B.) Summary of peak CDCA-induced currents with or without BAPTA-AM pretreatment (ΔIsc, mean ± S.E.M., n = 4 - 5, **p < 0.01). (C.) Cytoplasmic Ca 2+ concentration measured by Fluo-4 fluorescence. CDCA (0.75, 1 mM), ATP (100 μM), or carbachol (100 μM) was added to the apical bathing solution with or without pretreatment with BAPTA-AM (30 μM) for 30 minutes. (D.) Peak increase in Fluo-4 fluorescence (mean ± S.E.M., n = 3 - 6, **p < 0.01, ns, not significant).

[0051] Figure 5 .CDCA secretion response in primary cultures of human colonic epithelial cells consisting of planar monolayer cultures generated from colonoids. (A.) (Upper panel) Short-circuit currents showing the effect of the indicated CDCA concentrations added to the apical or basolateral bathing solution, then forskolin (10 μM) and (R)-BPO-27 (5 μM). (B.) Summary of peak CDCA-induced currents (ΔIsc, mean ± S.E.M., n = 9 - 13). (C.) Short-circuit currents showing the effect of CDCA (0.5 mM) then forskolin (10 μM) and (R)-BPO-27 (5 μM) after pretreatment with BAPTA-AM (30 μM) for 20 minutes. (D.) Fluorescence measurement of cytoplasmic Ca 2+ concentration upon addition of CDCA (0.75 mM) or ATP (100 μM).

[0052] Figure 6. CDCA-induced fluid secretion in closed intestinal loops in mice. (A.) Time course of CDCA-induced fluid secretion. 100 μl of CDCA or PBS in PBS (10 mM) was injected into the mid-jejunum closed loops, and the loops were excised at different times to measure loop weight and length (mean ± S.E.M., n = 5 - 9 loops per group, **p < 0.01). (B.) Concentration-dependence of the effect of CDCA in closed jejunal loops. 100 μl of CDCA or PBS was injected into the closed mid-jejunum loops (mean ± S.E.M., n = 3 - 14 loops per group, **p < 0.01, ns, not significant). (C.) Concentration-dependence of the effect of CDCA in closed colonic loops. 100 μl of CDCA or PBS was injected into the closed colonic loops (mean ± S.E.M., n = 3 - 5 loops per group, **p < 0.01, ns, not significant).

[0053] Figure 7 . (R)-BPO-27 inhibits CDCA-induced fluid secretion in closed intestinal loops in mice. (A.) Experimental protocol. (B.) (Left panel) Weight / length ratio of mid-jejunum loops injected with CDCA (10 mM) or PBS with or without BPO-27 (R or S enantiomer) (mean ± S.E.M., n = 6 - 7 S.E.M. per group), **p < 0.01, ns, not significant). (Right panel) Representative photographs of loops injected with CDCA and PBS with or without BPO-27. (C.) (Left panel) Weight / length ratio of colonic loops injected with CDCA (2.5, 10 mM) or PBS with or without (R)-BPO-27 (mean ± S.E.M., n = 4 - 7 loops per group, **p < 0.01, ns, not significant). (Right panel) Representative photographs of loops injected with CDCA and PBS with or without (R)-BPO-27. (D.) Weight / length ratio of mid-jejunum loops or colonic loops of cystic fibrosis (CFTR-deficient) mice injected with CDCA (2.5, 10 mM) (mean ± S.E.M., n = 4 loops per group, **p < 0.01, ns, not significant).

[0054] Figure 8. (R)-BPO-27 reduced the increase in fecal water content in a rat model of bile acid diarrhea. (A.) Experimental protocol (top panel) and photograph taken 10 minutes after infusion of 500 μL of PBS containing Evans blue dye into the mid-colon (bottom panel). (B.) Fecal output (wet weight) 4 hours after mid-colon infusion in rats with or without pre-treatment with (R)-BPO-27 (mean ± S.E.M., n = 4 rats in the PBS treatment group, n = 8 rats in the CDCA treatment group), *p < 0.05, ns, not significant). (C.) (Left panel) Fecal water content of feces collected in B (percentage of water from wet weight / dry weight measurements) (**p < 0.01, ns, not significant). (Right panel) Paired analysis showing the increase in fecal water content for each individual rat (fecal water at 0 - 4 hours in the same rat minus fecal water before infusion) (*p < 0.05, **p < 0.01, ns, not significant).

[0055] Detailed description of exemplary embodiments

[0056] The present invention can be understood by reference to the following detailed description which forms a part of this disclosure. The present invention is not limited to the specific methods, conditions or parameters described and / or illustrated herein, and the terms used herein are for the purpose of describing specific embodiments by way of example only and are not intended to limit the claimed invention.

[0057] Unless defined otherwise herein, scientific and technical terms associated with this application shall have the meaning commonly understood by one of ordinary skill in the art.

[0058] As used herein, the terms "composition", "compound", "drug", "pharmacological active agent", "active agent" or "medication" are used interchangeably herein to refer to one compound or a plurality of compounds or a composition of substances which, when administered to an individual (human or mammalian), induces a desired pharmacological and / or physiological effect by local and / or systemic action.

[0059] As used herein, the term "treatment" or "therapy" (and its various forms) includes prophylactic (e.g., preventive), curative or palliative treatment. As used herein, the term "treatment" includes alleviating or reducing at least one adverse or negative effect or symptom of a condition, disease or disorder.

[0060] The term "administer" means directly administering the compounds or compositions of the present invention.

[0061] The term "individual" is used herein to refer to a human or a domesticated mammal (e.g., dog, cat) to whom treatment with a compound according to the present invention, including prophylactic treatment, is provided.

[0062] In the present invention, the disclosed compounds can be prepared in the form of pharmaceutically acceptable salts. "Pharmaceutically acceptable salts" refers to derivatives of the disclosed compounds in which the parent compound is modified by formation of its acid or base salts. Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali metal or organic salts of acidic residues such as carboxylic acids; and the like. Pharmaceutically acceptable salts include conventional non-toxic salts or quaternary ammonium salts of the parent compound formed from, for example, non-toxic inorganic or organic acids. For example, such conventional non-toxic salts include those derived from inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, sulfamic acid, phosphoric acid, nitric acid, and the like; and salts prepared from organic acids such as acetic acid, propionic acid, succinic acid, glycolic acid, stearic acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, pamoic acid, maleic acid, hydroxy maleic acid, phenylacetic acid, glutamic acid, benzoic acid, salicylic acid, sulfanilic acid, 2-acetoxybenzoic acid, fumaric acid, toluenesulfonic acid, methanesulfonic acid, ethanedisulfonic acid, oxalic acid, hydroxyethanesulfonic acid, and the like. These pharmaceutically acceptable salts are prepared by methods known in the art, for example, by dissolving the free amine base in aqueous alcohol with an excess of acid, or by neutralizing the free carboxylic acid with an alkali metal base such as hydroxide or with an amine.

[0063] The present invention relates to a method of treating an individual suffering from bile acid diarrhea, comprising administering to the individual an effective amount of a CFTR chloride channel inhibitor for treating bile acid diarrhea.

[0064] In some aspects, the present invention relates to a method of treating an individual suffering from bile acid diarrhea. Bile acid diarrhea (BAD) is a clinically diagnosed condition in which diarrhea is thought to be caused by bile acid malabsorption or dysregulation. See, for example, M. Camilleri, Advances in understanding of bile acid diarrhea, Expert Rev Gastroenterol Hepatol. January 2014; 8(1):49–61. Methods of diagnosing BAD are known in the art. See, for example, id.

[0065] In some aspects of the methods disclosed herein, an amount of a CFTR chloride channel inhibitor (CFTR-CCI) is administered to an individual. As used herein, the CFTR chloride channel refers to the cystic fibrosis transmembrane conductance regulator (CFTR), which is a cAMP-activated chloride channel expressed in the epithelia of the lung, intestine, pancreas, testis, and other tissues. The term CFTR chloride channel inhibitor (or "CFTR-CCI") as used herein refers to any cystic fibrosis transmembrane conductance regulator chloride channel inhibitor described in the prior art that can be used for one or more medical purposes, including those described in: U.S. Patent Nos. 9,062,073, 7,235,573, 7,638,543, 7,414,037, and 7,888,332; U.S. Application Publication No. 2009 / 0253799; International Patent Application Publication Nos. WO 09 / 120803 and WO 09 / 146144; and Ma T, et al. Thiazolidinone CFTR inhibitor identified by high-throughput screening blocks cholera toxin-induced intestinal fluid secretion. J. Clin. Invest. 2002;110:1651–1658; Sonawane N, Verkman AS. Thiazolidinone CFTR inhibitors with improved water solubility identified by structure-activity analysis. Bioorg. Med. Chem. 2008;16:8187–8195, and Muanprasat C, et al. Discovery of glycine hydrazide pore-occluding CFTR inhibitors: mechanism, structure-activity analysis, and in vivo efficacy. J. Gen. Physiol. 2004;124:125–137. Each of these references is incorporated herein by reference in its entirety.

[0066] In some embodiments, the CFTR chloride channel inhibitor is a BPO CFTR chloride channel inhibitor (BPO-CFTR-CCI). The BPO-CFTR-CCI has the general structure

[0067]

[0068] The compounds contained

[0069] Wherein:

[0070] m is 1, 2, 3 or 4;

[0071] n is 1, 2, 3, 4 or 5;

[0072] p is an integer from 0 to 4;

[0073] q is an integer from 1 to 4;

[0074] R 1 is the same or different and independently H, halogen, haloalkyl, C1-C6 alkyl, —(CH2) p —C(O)—R 4a 、—S(O)2R 4a 、—NO2, or tetrazolyl;

[0075] R 1a is the same or different and independently H, halogen, haloalkyl, C1-C6 alkyl, —(CH2) p —C(O)—R 4a 、—S(O)2R 4a 、—NO2, or tetrazolyl;

[0076] R 2a and R 2b are each the same or different and independently H, or C1-C6 alkyl;

[0077] R 4a is —OR 7 、—NR 7 R 8 、—O(CH2) q —OC(O)R 7 、or an amino acid residue;

[0078] R 7 and R 8 are each the same or different and independently H, C1-C 20 alkyl, saccharide, or an amino acid residue; and

[0079] Z is aryl or heteroaryl,

[0080] Wherein the amino acid residue is selected from the following residues: alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, phosphoserine, phosphothreonine, phosphotyrosine, 4-hydroxyproline, hydroxylysine, desmosine, isodesmosine, γ-carboxyglutamic acid, hippuric acid, octahydroindole-2-carboxylic acid, pepstatinine, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, penicillamine, ornithine, 3-methylhistidine, norvaline, β-alanine, γ-aminobutyric acid, citrulline, homocysteine, homoserine, methylalanine, p-benzoyl-phenylalanine, phenylglycine, propargylglycine, sarcosine, methionine sulfone, tert-butylglycine, 3,5-dibromotyrosine, 3,5-diiodotyrosine, glycosylated threonine, glycosylated serine, and glycosylated asparagine.

[0081] In some embodiments, BPO-CFTR-CCI is a compound of the following formula

[0082]

[0083] Wherein:

[0084] R 1 is H, a halogen, or C 1-6 alkyl;

[0085] R 2 and R 3 are each independently the same or different and are H, a halogen, —NO2, C 1-6 alkyl, tetrazolyl, —S(O)2OR 7 , or —C(═O)OR 7 ;

[0086] R 5 is H, a halogen, or C 1-6 alkyl;

[0087] R 6 is a halogen, C 1-6 alkyl, or C 1-6 haloalkyl; and

[0088] R 7 is H, C 1-6 alkyl, a saccharide, an amino acid residue, or a peptide.

[0089] BPO-CFTR-CCI is described in U.S. Patent No. 9,062,073, which is incorporated herein by reference in its entirety.

[0090] In some embodiments, BPO-CFTR-CCI is (R)-BPO-27, which is a compound having the following structure:

[0091]

[0092] In other embodiments, the CFTR chloride channel inhibitor is a PPQ CFTR chloride channel inhibitor (PPQ-CFTR-CCI). PPQ-CFTR-CCI is of the general structure

[0093] comprising the compounds

[0094] wherein:

[0095] m is 1, 2, 3 or 4;

[0096] n is 1, 2, 3, 4 or 5;

[0097] p is an integer from 0 to 4;

[0098] q is an integer from 1 to 4;

[0099] X is O or S;

[0100] R 1 is the same or different and independently H, halogen, haloalkyl, C 1-6 alkyl, —(CH2) p —C(O)—R 4a 、—S(O)2R 4a 、—NO2, or tetrazolyl;

[0101] R 1a is the same or different and independently H, halogen, haloalkyl, C 1-6 alkyl, —(CH2) p —C(O)—R 4a 、—S(O)2R 4a 、—NO2, or tetrazolyl;

[0102] R 2a and R 2b are each the same or different and independently H or C 1-6 alkyl;

[0103] R 4a is —OR 7 、—NR 7 R 8 、—O(CH2) q —OC(O)R 7 、an amino acid residue, or a peptide;

[0104] R4 is H, —N(═O), C 1-6 alkyl or haloalkyl;

[0105] R 5 is H, halogen or C 1-6 alkyl;

[0106] R 6 is halogen, C 1-6 alkyl or C 1-6 haloalkyl; and

[0107] R 7 and R 8 are each independently the same or different and are H, C 1-20 alkyl, saccharide, amino acid residue, or peptide.

[0108] PPQ-CFTR-CCI is described in U.S. Patent No. 9,062,073, which is incorporated herein by reference in its entirety.

[0109] In some embodiments, the CFTR chloride channel inhibitor is a thiazolidinone CFTR chloride channel inhibitor (TD-CFTR-CCI). TD-CFTR-CCI has the general structure:

[0110] the compounds included

[0111] or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof,

[0112] wherein

[0113] Y is -NH- or absent;

[0114] W is ═CH-, -S-, -O-, -C(═S)-, or -C(═O)-;

[0115] Z1, Z2, Z3, Z4 and Z5 are each independently O or S;

[0116] J is C, S, O, or N;

[0117] Q is C or N;

[0118] R1, R2, R3 and R9 are each independently H, C 1-6 alkyl, alkoxy, halogen, -CF3, -CF2CF3, or -OCF3;

[0119] R5 is H, halogen, C 1-6 alkyl or absent;

[0120] X1, X2, X3, and X4 are each independently H, -OH, -SH, a halogen, tetrazolo, -P(=O)(OH)2, -C(=Z3)Z4H, -Z5-C(=Z3)Z4H, or -Z5-CH2-C(=Z3)Z4H; and

[0121] X5 is –O - , tetrazolo, -C(=O)OH, -O-C(=O)OH, or absent.

[0122] TD-CFTR-CCI also has the general structure:

[0123] The compounds included wherein X1 is trifluoromethyl;

[0124] X2 and X3 are independently selected from hydrogen and halogen groups;

[0125] Y1, Y2, and Y3 are independently selected from hydrogen, C1-C8 alkyl, C1-C7 alkoxy, carbonate, carbamate, carboxyl, halogen, nitro, azo, hydroxyl, and mercapto.

[0126] TD-CFTR-CCI is also a compound described in: Ma T, et al. Thiazolidinone CFTR inhibitor identified by high-throughput screening blocks cholera toxin-induced intestinal fluid secretion. J. Clin. Invest. 2002;110:1651–1658; and Sonawane N, Verkman AS. Thiazolidinone CFTR inhibitors with improved water solubility identified by structure-activity analysis. Bioorg. Med. Chem. 2008;16:8187–8195; U.S. Patent Nos. 7,235,573, 7,638,543, and International Patent Application Publication No. WO 09 / 120803. The entire contents of each of these references are incorporated herein by reference.

[0127] In some embodiments, TD-CFTR-CCI is CFTR inh -172, which has the structure

[0128] In some embodiments, the CFTR chloride channel inhibitor is a glycine hydrazide CFTR chloride channel inhibitor (GH-CFTR-CCI). The GH-CFTR-CCI is of the structure:

[0129] a compound contained therein, or a pharmaceutically acceptable salt or stereoisomer thereof,

[0130] wherein X1 is hydrogen or a substituted or unsubstituted, saturated linear or branched alkyl group;

[0131] Y is hydrogen or a substituted or unsubstituted, saturated linear or branched alkyl group;

[0132] R1 is an unsubstituted phenyl group,

[0133] a substituted phenyl group, wherein the phenyl group is substituted with one or more of hydroxyl, alkyl, and halogen,

[0134] a substituted or unsubstituted quinolinyl group,

[0135] a substituted or unsubstituted anthryl group, or

[0136] a substituted or unsubstituted naphthyl group;

[0137] R 2 is an unsubstituted phenyl group,

[0138] a substituted phenyl group, wherein the phenyl group is substituted with bromine or carboxyl,

[0139] bis(hydroxy)phenyl,

[0140] mono-(halogen)-mono(hydroxy)phenyl,

[0141] mono(halogen)-bis(hydroxy)phenyl,

[0142] mono(halogen)-tris(hydroxy)phenyl,

[0143] bis(halogen)-mono(hydroxy)phenyl,

[0144] bis(halogen)-bis(hydroxy)phenyl,

[0145] bis(halogen)-tris(hydroxy)phenyl,

[0146] mono(halogen)-mono(hydroxy)-mono(alkoxy)phenyl,

[0147] mono(halogen)-bis(hydroxy)-mono(alkoxy)phenyl,

[0148] mono(halogen)-mono(hydroxy)-bis(alkoxy)phenyl,

[0149] Mono(halogen)-di(hydroxy)-di(alkoxy)phenyl,

[0150] Di(halogen)-mono(hydroxy)-mono(alkoxy)phenyl,

[0151] Di(halogen)-di(hydroxy)-mono(alkoxy)phenyl,

[0152] Di(halogen)-mono(hydroxy)-di(alkoxy)phenyl; and

[0153] R3 is hydrogen or a substituted or unsubstituted alkyl group.

[0154] GH-CFTR-CCI is also of the general formula:

[0155] The compounds included

[0156] or a pharmaceutically acceptable salt or stereoisomer thereof,

[0157] wherein Y″ is a substituted or unsubstituted, saturated linear or branched alkyl group; or an amide or ether linker attached to a polar molecule, where the polar molecule is selected from a substituted or unsubstituted phenyl group, a polyoxyalkylene polyether, a polyethyleneimine, a disaccharide, a trisaccharide, a polyalkylimine, and a small aminoglucan;

[0158] R1 is an unsubstituted or substituted phenyl group, a substituted or unsubstituted quinolinyl group, a substituted or unsubstituted anthracenyl group, or a substituted or unsubstituted naphthyl group;

[0159] R 2 is an unsubstituted or substituted phenyl group; and

[0160] R3 is hydrogen or a substituted or unsubstituted alkyl group, thereby inhibiting CFTR

[0161] GH-CFTR-CCI is also of the general formula:

[0162]

[0163] The compounds included

[0164] or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof,

[0165] wherein:

[0166] R 1 and R 1 are the same or different and independently are an optionally substituted phenyl group, an optionally substituted heteroaryl group, an optionally substituted quinolinyl group, an optionally substituted anthracenyl group, or an optionally substituted naphthyl group;

[0167] R 2 、R 2 '、R3 and R 3 ', R 4 and R 4 ', R 5 and R 5 ', R 6 and R 6 ' are each the same or different and independently hydrogen, hydroxy, C₁₋₉ alkyl, C₁₋₉ alkoxy, carboxy, halogen, nitro, cyano, -SO₃H, -S(=O)₂NH₂, aryl, and heteroaryl;

[0168] R 13 and R 13 and R 14 and R 14 are each the same or different and independently hydrogen or C 1-8 alkyl;

[0169] X and X' are each the same or different linker moieties;

[0170] J and J' are each the same or different spacer moieties;

[0171] A is a polymeric subunit; and n is an integer between 0 and 2500.

[0172] GH-CFTR-CCI is also a compound described in: Muanprasat C, et al. Discovery of glycine hydrazide pore-occluding CFTR inhibitors: mechanism, structure-activity analysis, and in vivo efficacy. J. Gen. Physiol. 2004; 124:125–137; U.S. Patent Nos. 7,414,037 and 7,888,332; U.S. Application Publication No. 2009 / 0253799; International Patent Application Publication No. WO 09 / 146144. The entire contents of each of these references are incorporated herein by reference.

[0173] In some embodiments, GH-CFTR-CCI is GlyH-101, which has the structure

[0174] Other CFTR chloride channel inhibitors are glibenclamide, diphenylamine-2-carboxylate, 5-nitro-2-(3-phenylpropylamino)benzoate, and niflumic acid.

[0175] In some embodiments of the methods disclosed herein, a CFTR chloride channel inhibitor is administered to an individual, which is BPO-CFTR-CCI, PPQ-CFTR-CCI, TD-CFTR-CCI, or GH-CFTR-CCI.

[0176] In some embodiments of the methods disclosed herein, (R)-BPO-27, CFTR inh -172, GlyH-101, glibenclamide, diphenylamine-2-carboxylate, 5-nitro-2-(3-phenylpropylamino)benzoate, or niflumic acid; or a combination thereof is administered to an individual.

[0177] In some embodiments, the CFTR chloride channel inhibitor is BPO-CFTR-CCI.

[0178] In some embodiments, the CFTR chloride channel inhibitor is PPQ-CFTR-CCI.

[0179] In some embodiments, the CFTR chloride channel inhibitor is TD-CFTR-CCI. In some embodiments, TD-CFTR-CCI is CFTR inh -172.

[0180] In some embodiments, the CFTR chloride channel inhibitor is GH-CFTR-CCI. In some embodiments, GH-CFTR-CCI is GlyH-101.

[0181] In some embodiments, the CFTR chloride channel inhibitor is glibenclamide. In some embodiments, the CFTR chloride channel inhibitor is diphenylamine-2-carboxylate. In some embodiments, the CFTR chloride channel inhibitor is 5-nitro-2-(3-phenylpropylamino)benzoate. In some embodiments, the CFTR chloride channel inhibitor is niflumic acid.

[0182] In some embodiments of the methods of the present disclosure, a pharmaceutical composition comprising a pharmaceutical excipient and a certain amount of a CFTR chloride channel inhibitor is administered to an individual.

[0183] In some aspects, the present disclosure relates to methods of treating an individual suffering from bile acid diarrhea, comprising administering to the individual a pharmaceutical composition comprising a CFTR chloride channel inhibitor in an amount effective to treat bile acid diarrhea. In some embodiments, the CFTR chloride channel inhibitor is BPO-CFTR-CCI, PPQ-CFTR-CCI, TD-CFTR-CCI, or GH-CFTR-CCI. In some embodiments, the CFTR chloride channel inhibitor is BPO-CFTR-CCI. In some embodiments, BPO-CFTR-CCI is (R)-BPO-27. In some embodiments, the pharmaceutical composition further comprises (S)-BPO-27. In other embodiments, the CFTR chloride channel inhibitor is PPQ-CFTR-CCI. In other embodiments, the CFTR chloride channel inhibitor is TD-CFTR-CCI. In some embodiments, TD-CFTR-CCI is CFTR inh -172. In other embodiments, the CFTR chloride channel inhibitor is GH-CFTR-CCI. In some embodiments, GH-CFTR-CCI is GlyH-101. In still other embodiments, the CFTR chloride channel inhibitor is glibenclamide, diphenylamine-2-carboxylate, 5-nitro-2-(3-phenylpropylamino)benzoate or niflumic acid, or a combination thereof.

[0184] In those embodiments in which a pharmaceutical composition comprising a CFTR chloride channel inhibitor that is (R)-BPO-27 is administered to an individual, the pharmaceutical composition may further comprise (S)-BPO-27. In these embodiments, the amount of (R)-BPO-27 in the pharmaceutical composition will be substantially equal to or greater than the amount of (S)-BPO-27 present in the pharmaceutical composition. For example, in some embodiments, the pharmaceutical composition may comprise a racemic mixture of (R / S)-BPO-27. In some aspects, (R)-BPO-27 will be present in an enantiomeric excess value (ee) compared to (S)-BPO-27. For example, (R)-BPO-27 may be present at about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% ee. In some embodiments, the pharmaceutical composition will comprise (R)-BPO-27, wherein the % ee is greater than 99%.

[0185] The amount of CFTR chloride channel inhibitor administered is an amount effective to treat bile acid diarrhea. The amount effective in this regard will vary depending on the characteristics and condition of the individual.

[0186] In some aspects of the disclosed methods, the amount of the CFTR chloride channel inhibitor is effective to reduce intestinal fluid secretion caused by bile acid diarrhea.

[0187] In other aspects of the disclosed methods, the amount of the CFTR chloride channel inhibitor is effective to reduce the activation of apical CFTR chloride channels induced by bile acids.

[0188] In some aspects of the disclosed methods, a second agent in an amount effective to treat bile acid diarrhea is also administered to the individual. In some embodiments, the second agent is a bile acid binder, a farnesoid X receptor (FXR) agonist, a 5-HT3 antagonist, an opioid receptor agonist, a mixed μ opioid receptor agonist, a broad-spectrum gut-specific antibiotic, an antispasmodic, or a tricyclic antidepressant. In some embodiments, the second agent is a bile acid binder, preferably cholestyramine, colestipol, or colesevelam. In other embodiments, the second agent is a farnesoid X receptor (FXR) agonist, preferably obeticholic acid. In other embodiments, the second agent is a 5-HT3 antagonist, preferably alosetron. In other embodiments, the second agent is an opioid receptor agonist, preferably loperamide. In other embodiments, the second agent is a mixed μ opioid receptor agonist, preferably eluxadoline. In other embodiments, the second agent is a broad-spectrum gut-specific antibiotic, preferably rifaximin.

[0189] In some aspects, the present invention relates to a method of reducing intestinal fluid secretion in an individual in need thereof caused by the activation of CFTR chloride channels in the intestinal epithelium induced by bile acids, comprising administering to the individual a CFTR chloride channel inhibitor in an amount effective to reduce intestinal fluid secretion caused by the activation of CFTR chloride channels induced by bile acids.

[0190] As used herein, intestinal fluid secretion refers to the secretion of fluid from the intestinal epithelium into the intestinal lumen.

[0191] In some embodiments, intestinal fluid secretion is caused by the activation of CFTR chloride channels in the intestinal epithelium induced by bile acids. In other embodiments, the CFTR chloride channel is the apical CFTR chloride channel in the intestinal epithelium. The apical CFTR chloride channel is the CFTR chloride channel located in the apical cell membrane (i.e., the cell membrane facing the intestinal lumen). Thus, in some embodiments, the present invention relates to a method of reducing intestinal fluid secretion in an individual in need thereof caused by the activation of the apical CFTR chloride channel in the intestinal epithelium induced by bile acids, comprising administering to the individual a CFTR chloride channel inhibitor in an amount effective to reduce intestinal fluid secretion caused by the activation of the apical CFTR chloride channel induced by bile acids.

[0192] In some aspects, intestinal fluid secretion is reduced by administering a CFTR chloride channel inhibitor that effectively reduces the amount of said intestinal fluid secretion. As used herein, a reduction in intestinal fluid secretion refers to a reduction in the amount of fluid secreted into the intestinal lumen relative to the amount of fluid secreted into the intestinal lumen in the absence of administration of a CFTR chloride channel inhibitor. Methods for measuring the reduction in fluid secretion are known to those skilled in the art and include measuring the water content of the intestinal lumen contents and the amount of diarrhea output.

[0193] In some embodiments, intestinal fluid secretion is reduced by administering a CFTR chloride channel inhibitor that is BPO-CFTR-CCI, PPQ-CFTR-CCI, TD-CFTR-CCI, or GH-CFTR-CCI. In some embodiments, the CFTR chloride channel inhibitor is BPO-CFTR-CCI. In some embodiments, the CFTR chloride channel inhibitor is PPQ-CFTR-CCI. In some embodiments, the CFTR chloride channel inhibitor is TD-CFTR-CCI. In some embodiments, the CFTR chloride channel inhibitor is GH-CFTR-CCI. In some embodiments, intestinal fluid secretion is reduced by administering a CFTR chloride channel inhibitor that is (R)-BPO-27, CFTR inh -172, GlyH-101, glibenclamide, diphenylamine-2-carboxylate, 5-nitro-2-(3-phenylpropylamino)benzoate, or niflumic acid or a combination thereof. In some embodiments, the CFTR chloride channel inhibitor is (R)-BPO-27. In some embodiments, the CFTR chloride channel inhibitor is CFTR inh -172. In some embodiments, the CFTR chloride channel inhibitor is GlyH-101. In some embodiments, the CFTR chloride channel inhibitor is glibenclamide. In some embodiments, the CFTR chloride channel inhibitor is diphenylamine-2-carboxylate. In some embodiments, the CFTR chloride channel inhibitor is 5-nitro-2-(3-phenylpropylamino)benzoate. In some embodiments, the CFTR chloride channel inhibitor is niflumic acid.

[0194] In this aspect of the invention, the amount of CFTR chloride channel inhibitor administered is an amount effective to reduce the amount of said intestinal fluid secretion caused by activation of the CFTR chloride channel (e.g., the apical CFTR chloride channel) induced by bile acids. The amount effective in this regard will vary depending on the characteristics and condition of the individual.

[0195] In some aspects, the present invention relates to methods of reducing intestinal fluid secretion in an individual in need thereof caused by activation of the CFTR chloride channel in the intestinal epithelium by bile acids, comprising administering to the individual a pharmaceutical composition comprising an amount of a CFTR chloride channel inhibitor effective to reduce intestinal fluid secretion caused by activation of the CFTR chloride channel by bile acids. In some embodiments, the CFTR chloride channel inhibitor is BPO-CFTR-CCI, PPQ-CFTR-CCI, TD-CFTR-CCI, or GH-CFTR-CCI. In some embodiments, the CFTR chloride channel inhibitor is BPO-CFTR-CCI. In some embodiments, BPO-CFTR-CCI is (R)-BPO-27. In some embodiments, the pharmaceutical composition further comprises (S)-BPO-27. In other embodiments, the CFTR chloride channel inhibitor is PPQ-CFTR-CCI. In other embodiments, the CFTR chloride channel inhibitor is TD-CFTR-CCI. In some embodiments, TD-CFTR-CCI is CFTR inh -172. In other embodiments, the CFTR chloride channel inhibitor is GH-CFTR-CCI. In some embodiments, GH-CFTR-CCI is GlyH-101. In still other embodiments, the CFTR chloride channel inhibitor is glibenclamide, diphenylamine-2-carboxylate, 5-nitro-2-(3-phenylpropylamino)benzoate or niflumic acid, or a combination thereof.

[0196] In other aspects, the present disclosure relates to methods of reducing bile acid-induced CFTR chloride channel current in the intestinal epithelium of an individual in need thereof, comprising administering to the individual an amount of a CFTR chloride channel inhibitor effective to reduce the bile acid-induced CFTR chloride channel current.

[0197] As used herein, "current" refers to the passage of ions through a channel. Thus, CFTR chloride channel current refers to the passage of chloride ions through the CFTR chloride channel.

[0198] In some embodiments, the CFTR chloride channel current is caused by activation of the apical CFTR chloride channel in the bile acid-induced intestinal epithelium. Thus, in some embodiments, the present invention relates to methods of reducing bile acid-induced apical CFTR chloride channel current in the intestinal epithelium of an individual in need thereof, comprising administering to the individual an amount of a CFTR chloride channel inhibitor effective to reduce the bile acid-induced apical CFTR chloride channel current.

[0199] In some embodiments, the CFTR chloride channel current in the intestinal epithelium induced by bile acids is reduced by administering a CFTR chloride channel inhibitor, which is BPO-CFTR-CCI, PPQ-CFTR-CCI, TD-CFTR-CCI, or GH-CFTR-CCI. In some embodiments, the CFTR chloride channel inhibitor is BPO-CFTR-CCI. In some embodiments, the CFTR chloride channel inhibitor is PPQ-CFTR-CCI. In some embodiments, the CFTR chloride channel inhibitor is TD-CFTR-CCI. In some embodiments, the CFTR chloride channel inhibitor is GH-CFTR-CCI. In some embodiments, the CFTR chloride channel current in the intestinal epithelium induced by bile acids is reduced by administering a CFTR chloride channel inhibitor, which is (R)-BPO-27, CFTR inh -172, GlyH-101, glibenclamide, diphenylamine-2-carboxylate, 5-nitro-2-(3-phenylpropylamino)benzoate, or niflumic acid or a combination thereof).

[0200] In some embodiments, the CFTR chloride channel inhibitor is (R)-BPO-27. In some embodiments, the CFTR chloride channel inhibitor is CFTR inh -172. In some embodiments, the CFTR chloride channel inhibitor is GlyH-101. In some embodiments, the CFTR chloride channel inhibitor is glibenclamide. In some embodiments, the CFTR chloride channel inhibitor is diphenylamine-2-carboxylate. In some embodiments, the CFTR chloride channel inhibitor is 5-nitro-2-(3-phenylpropylamino)benzoate. In some embodiments, the CFTR chloride channel inhibitor is niflumic acid.

[0201] In this aspect of the invention, the amount of the CFTR chloride channel inhibitor administered is an amount effective to reduce the bile acid-induced CFTR chloride channel (e.g., apical CFTR chloride channel) current. The amount effective in this regard will vary depending on the characteristics and condition of the individual.

[0202] In some aspects, the present invention relates to methods for reducing bile acid-induced CFTR chloride channel currents in the intestinal epithelium of an individual in need thereof, comprising administering to the individual a pharmaceutical composition comprising a CFTR chloride channel inhibitor in an amount effective to reduce said bile acid-induced CFTR chloride channel currents. In some embodiments, the CFTR chloride channel inhibitor is BPO-CFTR-CCI, PPQ-CFTR-CCI, TD-CFTR-CCI, or GH-CFTR-CCI. In some embodiments, the CFTR chloride channel inhibitor is BPO-CFTR-CCI. In some embodiments, BPO-CFTR-CCI is (R)-BPO-27. In some embodiments, the pharmaceutical composition further comprises (S)-BPO-27. In other embodiments, the CFTR chloride channel inhibitor is PPQ-CFTR-CCI. In other embodiments, the CFTR chloride channel inhibitor is TD-CFTR-CCI. In some embodiments, TD-CFTR-CCI is CFTR inh -172. In other embodiments, the CFTR chloride channel inhibitor is GH-CFTR-CCI. In some embodiments, GH-CFTR-CCI is GlyH-101. In still other embodiments, the CFTR chloride channel inhibitor is glibenclamide, diphenylamine-2-carboxylate, 5-nitro-2-(3-phenylpropylamino)benzoate or niflumic acid, or a combination thereof.

[0203] In some aspects, the present invention relates to methods for treating diarrhea or alleviating symptoms associated with diarrhea in an individual who has undergone ileal resection, comprising administering to the individual an amount of a CFTR chloride channel inhibitor effective to treat said diarrhea or alleviate the symptoms of said diarrhea. As used herein, the symptoms of diarrhea include one or more of the following: (1) increased water content of the feces, (2) increased frequency of defecation; (3) increased volume of fecal output; (4) abdominal pain; (5) abdominal distension; and (6) nausea.

[0204] In some embodiments, the method for treating diarrhea or alleviating symptoms associated with diarrhea in an individual who has undergone ileal resection comprises administering to the individual a CFTR chloride channel inhibitor which is BPO-CFTR-CCI, PPQ-CFTR-CCI, TD-CFTR-CCI, or GH-CFTR-CCI. In some embodiments, the CFTR chloride channel inhibitor is BPO-CFTR-CCI. In some embodiments, the CFTR chloride channel inhibitor is PPQ-CFTR-CCI. In some embodiments, the CFTR chloride channel inhibitor is TD-CFTR-CCI. In some embodiments, the CFTR chloride channel inhibitor is GH-CFTR-CCI.

[0205] In some embodiments, a method for treating diarrhea or alleviating diarrhea-related symptoms in an individual who has undergone ileal resection, comprising administering to the individual a CFTR chloride channel inhibitor, which is (R)-BPO-27, CFTR inh -172, GlyH-101, glibenclamide, diphenylamine-2-carboxylate, 5-nitro-2-(3-phenylpropylamino)benzoate, or niflumic acid, or a combination thereof. In some embodiments, the CFTR chloride channel inhibitor is (R)-BPO-27. In some embodiments, the CFTR chloride channel inhibitor is CFTR inh -172. In some embodiments, the CFTR chloride channel inhibitor is GlyH-101. In some embodiments, the CFTR chloride channel inhibitor is glibenclamide. In some embodiments, the CFTR chloride channel inhibitor is diphenylamine-2-carboxylate. In some embodiments, the CFTR chloride channel inhibitor is 5-nitro-2-(3-phenylpropylamino)benzoate. In some embodiments, the CFTR chloride channel inhibitor is niflumic acid.

[0206] In this aspect of the invention, the amount of the CFTR chloride channel inhibitor administered is an amount effective to treat the diarrhea of the individual or alleviate the diarrhea-related symptoms of the individual. The amount effective in this regard will vary depending on the characteristics and condition of the individual.

[0207] In some aspects, the invention relates to a method for treating diarrhea or alleviating diarrhea-related symptoms in an individual who has undergone ileal resection, comprising administering to the individual a pharmaceutical composition comprising an amount of a CFTR chloride channel inhibitor effective to treat the diarrhea or alleviate the symptoms of the diarrhea. In some embodiments, the CFTR chloride channel inhibitor is BPO-CFTR-CCI, PPQ-CFTR-CCI, TD-CFTR-CCI, or GH-CFTR-CCI. In some embodiments, the CFTR chloride channel inhibitor is BPO-CFTR-CCI. In some embodiments, BPO-CFTR-CCI is (R)-BPO-27. In some embodiments, the pharmaceutical composition further comprises (S)-BPO-27. In other embodiments, the CFTR chloride channel inhibitor is PPQ-CFTR-CCI. In other embodiments, the CFTR chloride channel inhibitor is TD-CFTR-CCI. In some embodiments, TD-CFTR-CCI is CFTR inh-172. In other embodiments, the CFTR chloride channel inhibitor is GH-CFTR-CCI. In some embodiments, GH-CFTR-CCI is GlyH-101. In still other embodiments, the CFTR chloride channel inhibitor is glibenclamide, diphenylamine-2-carboxylate, 5-nitro-2-(3-phenylpropylamino)benzoate or niflumic acid, or a combination thereof.

[0208] In all methods of the present disclosure, the CFTR chloride channel inhibitor is administered to an individual. In some embodiments, the individual is a mammal. In other embodiments, the individual is a human.

[0209] In some embodiments, the individual has been diagnosed with Crohn's disease.

[0210] In other embodiments, the individual has been diagnosed with IBS-D.

[0211] In still other embodiments, the individual has been diagnosed with functional diarrhea.

[0212] In some aspects, the methods of the present invention result in a reduction in the fecal water content of an individual. The degree of water reduction can be determined by measuring the amount of water in the feces of the individual before administering the CFTR chloride channel inhibitor and comparing that amount to the amount of water in the feces of the individual after administering the CFTR chloride channel inhibitor.

[0213] In some embodiments, the reduction in the fecal water content of the individual is demonstrated by measurement using a clinical instrument such as the Bristol Stool Form Scale (BSFS). The BSFS, known in the art, assigns a consistency score to the feces of an individual ranging from 1 (hard) to 7 (watery). Thus, in some embodiments, the reduction in the fecal water content of an individual using the method of the present invention is demonstrated by a decrease in the score using the Bristol Stool Form Scale. In some embodiments, the reduction in the fecal water content of an individual using the method of the present invention is demonstrated by a 1-point decrease using the Bristol Stool Form Scale (e.g., 7 to 6, 6 to 5, 5 to 4, 4 to 3, 3 to 2, 2 to 1). In other embodiments, the reduction in the fecal water content of an individual using the method of the present invention is demonstrated by a 2-point decrease using the Bristol Stool Form Scale (e.g., 7 to 5, 6 to 4, 5 to 3, 4 to 2, 3 to 1). In other embodiments, the reduction in the fecal water content of an individual using the method of the present invention is demonstrated by a 3-point decrease using the Bristol Stool Form Scale (e.g., 7 to 4, 6 to 3, 5 to 2, 4 to 1). In other embodiments, the reduction in the fecal water content of an individual using the method of the present invention is demonstrated by a decrease of more than 3 points using the Bristol Stool Form Scale.

[0214] Those skilled in the art will understand that other clinical scales known in the art can be used to demonstrate a reduction in the fecal water content of an individual.

[0215] In some aspects, the methods of the invention result in a reduction in the bowel movement frequency of an individual. The degree of reduction in bowel movement frequency can be determined by comparing the bowel movement frequency of an individual before administration of a CFTR chloride channel inhibitor with the bowel movement frequency of the individual after administration of the CFTR chloride channel inhibitor. Here, the bowel movement frequency can be measured, for example, by interviewing the individual or administering an instrument designed to obtain this information to the individual. Methods for determining bowel movement frequency are known to those skilled in the art.

[0216] In other aspects, the methods of the invention result in a reduction in the fecal output of an individual. The degree of reduction in fecal output can be determined by measuring the fecal mass of an individual before administration of a CFTR chloride channel inhibitor and comparing that mass with the fecal mass of the individual after administration of the CFTR chloride channel inhibitor. Here, the mass can be measured by weight or by volume. Methods for measuring fecal output are known to those skilled in the art.

[0217] In other aspects, the methods of the invention result in a reduction in abdominal pain of an individual. The degree of reduction in abdominal pain can be determined by comparing the abdominal pain of an individual before administration of a CFTR chloride channel inhibitor with the abdominal pain of the individual after administration of the CFTR chloride channel inhibitor. Here, abdominal pain can be measured, for example, by interviewing the individual or administering an instrument designed to obtain this information to the individual. Methods for determining abdominal pain are known to those skilled in the art.

[0218] In other aspects, the methods of the invention result in a reduction in flatulence of an individual. The degree of reduction in flatulence can be determined by comparing the flatulence of an individual before administration of a CFTR chloride channel inhibitor with the flatulence of the individual after administration of the CFTR chloride channel inhibitor. Here, flatulence can be measured, for example, by interviewing the individual or administering an instrument designed to obtain this information to the individual. Methods for determining flatulence are known to those skilled in the art.

[0219] In other aspects, the methods of the invention result in a reduction in nausea of an individual. The degree of reduction in nausea can be determined by comparing the nausea of an individual before administration of a CFTR chloride channel inhibitor with the nausea of the individual after administration of the CFTR chloride channel inhibitor. Here, nausea can be measured, for example, by interviewing the individual or administering an instrument designed to obtain this information to the individual, such as a Nausea Questionnaire. Methods for measuring nausea are known to those skilled in the art.

[0220] This application also includes the following embodiments:

[0221] 1. A method for treating an individual suffering from bile acid diarrhea, comprising administering to the individual an amount of a CFTR chloride channel inhibitor effective to treat the bile acid diarrhea.

[0222] 2. The method according to embodiment 1, wherein the CFTR chloride channel inhibitor is BPO-CFTR-CCI, PPQ-CFTR-CCI, TD-CFTR-CCI, or GH-CFTR-CCI.

[0223] 3. The method according to embodiment 1 or embodiment 2, wherein the CFTR chloride channel inhibitor is BPO-CFTR-CCI.

[0224] 4. The method according to embodiment 3, wherein the BPO-CFTR-CCI is (R)-BPO-27.

[0225] 5. The method according to embodiment 1 or embodiment 2, wherein the CFTR chloride channel inhibitor is PPQ-CFTR-CCI.

[0226] 6. The method according to embodiment 1 or embodiment 2, wherein the CFTR chloride channel inhibitor is TD-CFTR-CCI.

[0227] 7. The method according to embodiment 6, wherein the TD-CFTR-CCI is CFTR inh -172.

[0228] 8. The method according to embodiment 1 or embodiment 2, wherein the CFTR chloride channel inhibitor is GH-CFTR-CCI.

[0229] 9. The method according to embodiment 8, wherein the GH-CFTR-CCI is GlyH-101.

[0230] 10. The method according to embodiment 1, wherein the CFTR chloride channel inhibitor is glibenclamide, diphenylamine-2-carboxylate, 5-nitro-2-(3-phenylpropylamino)benzoate or niflumic acid, or a combination thereof.

[0231] 11. A method for treating an individual suffering from bile acid diarrhea, comprising administering to the individual a pharmaceutical composition comprising an amount of a CFTR chloride channel inhibitor effective to treat the bile acid diarrhea.

[0232] 12. The method according to embodiment 11, wherein the CFTR chloride channel inhibitor is BPO-CFTR-CCI, PPQ-CFTR-CCI, TD-CFTR-CCI, or GH-CFTR-CCI.

[0233] 13. The method according to embodiment 11 or embodiment 12, wherein the CFTR chloride channel inhibitor is BPO-CFTR-CCI.

[0234] 14. The method according to embodiment 13, wherein the BPO-CFTR-CCI is (R)-BPO-27.

[0235] 15. The method according to embodiment 14, wherein the pharmaceutical composition further comprises (S)-BPO-27.

[0236] 16. The method according to embodiment 11 or embodiment 12, wherein the CFTR chloride channel inhibitor is PPQ-CFTR-CCI.

[0237] 17. The method according to embodiment 11 or embodiment 12, wherein the CFTR chloride channel inhibitor is TD-CFTR-CCI.

[0238] 18. The method according to embodiment 17, wherein the TD-CFTR-CCI is CFTR inh -172.

[0239] 19. The method according to embodiment 11 or embodiment 12, wherein the CFTR chloride channel inhibitor is GH-CFTR-CCI.

[0240] 20. The method according to embodiment 19, wherein the GH-CFTR-CCI is GlyH-101.

[0241] 21. The method according to embodiment 11, wherein the CFTR chloride channel inhibitor is glibenclamide, diphenylamine-2-carboxylate, 5-nitro-2-(3-phenylpropylamino)benzoate or niflumic acid, or a combination thereof.

[0242] 22. The method according to any one of the foregoing embodiments, wherein the amount of the CFTR chloride channel inhibitor effectively reduces intestinal fluid secretion caused by the bile acid diarrhea.

[0243] 23. The method according to any one of the foregoing embodiments, wherein the amount of the CFTR chloride channel inhibitor effectively reduces the activation of the apical CFTR chloride channel induced by bile acids.

[0244] 24. The method according to any one of the foregoing embodiments further comprises administering to the individual an amount of a second agent effective to treat the bile acid-induced diarrhea, wherein the second agent is a bile acid binder, a farnesoid X receptor (FXR) agonist, a 5-HT3 antagonist, an opioid receptor agonist, a mixed μ opioid receptor agonist, a broad-spectrum gut-specific antibiotic, an antispasmodic, or a tricyclic antidepressant.

[0245] 25. The method according to embodiment 24, wherein the second agent is a bile acid binder, preferably cholestyramine, colestipol, or colesevelam.

[0246] 26. The method according to embodiment 24, wherein the second agent is a farnesoid X receptor (FXR) agonist, preferably obeticholic acid.

[0247] 27. The method according to embodiment 24, wherein the second agent is a 5-HT3 antagonist, preferably alosetron.

[0248] 28. The method according to embodiment 24, wherein the second agent is an opioid receptor agonist, preferably loperamide.

[0249] 29. The method according to embodiment 24, wherein the second agent is a mixed μ opioid receptor agonist, preferably eluxadoline.

[0250] 30. The method according to embodiment 24, wherein the second agent is a broad-spectrum gut-specific antibiotic, preferably rifaximin.

[0251] 31. A method of reducing intestinal fluid secretion in an individual in need thereof caused by activation of the apical CFTR chloride channel in intestinal epithelium induced by bile acids, comprising administering to the individual an amount of a CFTR chloride channel inhibitor effective to reduce the intestinal fluid secretion caused by activation of the apical CFTR chloride channel induced by bile acids.

[0252] 32. The method according to embodiment 31, wherein the CFTR chloride channel inhibitor is BPO-CFTR-CCI, PPQ-CFTR-CCI, TD-CFTR-CCI, or GH-CFTR-CCI.

[0253] 33. The method according to embodiment 31 or embodiment 32, wherein the CFTR chloride channel inhibitor is BPO-CFTR-CCI.

[0254] 34. The method according to embodiment 33, wherein the BPO-CFTR-CCI is (R)-BPO-27.

[0255] 35. The method according to embodiment 31 or embodiment 32, wherein the CFTR chloride channel inhibitor is PPQ-CFTR-CCI.

[0256] 36. The method according to embodiment 31 or embodiment 32, wherein the CFTR chloride channel inhibitor is TD-CFTR-CCI.

[0257] 37. The method according to embodiment 36, wherein the TD-CFTR-CCI is CFTR inh -172.

[0258] 38. The method according to embodiment 31 or embodiment 32, wherein the CFTR chloride channel inhibitor is GH-CFTR-CCI.

[0259] 39. The method according to embodiment 38, wherein the GH-CFTR-CCI is GlyH-101.

[0260] 40. The method according to embodiment 31, wherein the CFTR chloride channel inhibitor is glibenclamide, diphenylamine-2-carboxylate, 5-nitro-2-(3-phenylpropylamino)benzoate or niflumic acid, or a combination thereof.

[0261] 41. A method for reducing intestinal fluid secretion caused by activation of the apical CFTR chloride channel in intestinal epithelium induced by bile acids in an individual in need thereof, comprising administering to the individual a pharmaceutical composition comprising a CFTR chloride channel inhibitor in an amount effective to reduce the intestinal fluid secretion caused by activation of the apical CFTR chloride channel induced by bile acids.

[0262] 42. The method according to embodiment 41, wherein the CFTR chloride channel inhibitor is BPO-CFTR-CCI, PPQ-CFTR-CCI, TD-CFTR-CCI, or GH-CFTR-CCI.

[0263] 43. The method according to embodiment 41 or embodiment 42, wherein the CFTR chloride channel inhibitor is BPO-CFTR-CCI.

[0264] 44. The method according to embodiment 43, wherein the BPO-CFTR-CCI is (R)-BPO-27.

[0265] 45. The method according to embodiment 44, wherein the pharmaceutical composition further comprises (S)-BPO-27.

[0266] 46. The method according to embodiment 41 or embodiment 42, wherein the CFTR chloride channel inhibitor is PPQ-CFTR-CCI.

[0267] 47. The method according to embodiment 41 or embodiment 42, wherein the CFTR chloride channel inhibitor is TD-CFTR-CCI.

[0268] 48. The method according to embodiment 47, wherein the TD-CFTR-CCI is CFTR inh -172.

[0269] 49. The method according to embodiment 41 or embodiment 42, wherein the CFTR chloride channel inhibitor is GH-CFTR-CCI.

[0270] 50. The method according to embodiment 49, wherein the GH-CFTR-CCI is GlyH-101.

[0271] 51. The method according to embodiment 41, wherein the CFTR chloride channel inhibitor is glibenclamide, diphenylamine-2-carboxylate, 5-nitro-2-(3-phenylpropylamino)benzoate or niflumic acid, or a combination thereof.

[0272] 52. A method of reducing bile acid-induced apical CFTR chloride channel current in the intestinal epithelium of an individual in need thereof, comprising administering to the individual an amount of a CFTR chloride channel inhibitor effective to reduce the bile acid-induced apical CFTR chloride channel current.

[0273] 53. The method according to embodiment 52, wherein the CFTR chloride channel inhibitor is BPO-CFTR-CCI, PPQ-CFTR-CCI, TD-CFTR-CCI, or GH-CFTR-CCI.

[0274] 54. The method according to embodiment 52 or embodiment 53, wherein the CFTR chloride channel inhibitor is BPO-CFTR-CCI.

[0275] 55. The method according to embodiment 54, wherein the BPO-CFTR-CCI is (R)-BPO-27.

[0276] 56. The method according to embodiment 52 or embodiment 53, wherein the CFTR chloride channel inhibitor is PPQ-CFTR-CCI.

[0277] 57. The method according to embodiment 52 or embodiment 53, wherein the CFTR chloride channel inhibitor is TD-CFTR-CCI.

[0278] 58. The method according to embodiment 57, wherein the TD-CFTR-CCI is CFTR inh -172.

[0279] 59. The method according to embodiment 52 or embodiment 53, wherein the CFTR chloride channel inhibitor is GH-CFTR-CCI.

[0280] 60. The method according to embodiment 59, wherein the GH-CFTR-CCI is GlyH-101.

[0281] 61. The method according to embodiment 52, wherein the CFTR chloride channel inhibitor is glibenclamide, diphenylamine-2-carboxylate, 5-nitro-2-(3-phenylpropylamino)benzoate or niflumic acid, or a combination thereof.

[0282] 62. A method of reducing apical CFTR chloride channel current in intestinal epithelium induced by bile acids in an individual in need thereof, comprising administering to the individual a pharmaceutical composition comprising a CFTR chloride channel inhibitor in an amount effective to reduce the bile acid-induced apical CFTR chloride channel current.

[0283] 63. The method according to embodiment 62, wherein the CFTR chloride channel inhibitor is BPO-CFTR-CCI, PPQ-CFTR-CCI, TD-CFTR-CCI, or GH-CFTR-CCI.

[0284] 64. The method according to embodiment 62 or embodiment 63, wherein the CFTR chloride channel inhibitor is BPO-CFTR-CCI.

[0285] 65. The method according to embodiment 64, wherein the BPO-CFTR-CCI is (R)-BPO-27.

[0286] 66. The method according to embodiment 65, wherein the pharmaceutical composition further comprises (S)-BPO-27.

[0287] 67. The method according to embodiment 62 or embodiment 63, wherein the CFTR chloride channel inhibitor is PPQ-CFTR-CCI.

[0288] 68. The method according to embodiment 62 or embodiment 63, wherein the CFTR chloride channel inhibitor is TD-CFTR-CCI.

[0289] 69. The method according to embodiment 68, wherein the TD-CFTR-CCI is CFTRinh -172。

[0290] 70. The method according to embodiment 62 or embodiment 63, wherein the CFTR chloride channel inhibitor is GH-CFTR-CCI.

[0291] 71. The method according to embodiment 70, wherein the GH-CFTR-CCI is GlyH-101.

[0292] 72. The method according to embodiment 62, wherein the CFTR chloride channel inhibitor is glibenclamide, diphenylamine-2-carboxylate, 5-nitro-2-(3-phenylpropylamino)benzoate or niflumic acid, or a combination thereof.

[0293] 73. A method for treating diarrhea or alleviating diarrhea-related symptoms in an individual who has undergone ileectomy, comprising administering to the individual an amount of a CFTR chloride channel inhibitor effective to treat the diarrhea or alleviate the symptoms of the diarrhea.

[0294] 74. The method according to embodiment 73, wherein the CFTR chloride channel inhibitor is BPO-CFTR-CCI, PPQ-CFTR-CCI, TD-CFTR-CCI, or GH-CFTR-CCI.

[0295] 75. The method according to embodiment 73 or embodiment 74, wherein the CFTR chloride channel inhibitor is BPO-CFTR-CCI.

[0296] 76. The method according to embodiment 75, wherein the BPO-CFTR-CCI is (R)-BPO-27.

[0297] 77. The method according to embodiment 73 or embodiment 74, wherein the CFTR chloride channel inhibitor is PPQ-CFTR-CCI.

[0298] 78. The method according to embodiment 73 or embodiment 74, wherein the CFTR chloride channel inhibitor is TD-CFTR-CCI.

[0299] 79. The method according to embodiment 78, wherein the TD-CFTR-CCI is CFTR inh -172。

[0300] 80. The method according to embodiment 73 or embodiment 74, wherein the CFTR chloride channel inhibitor is GH-CFTR-CCI.

[0301] 81. The method according to embodiment 80, wherein the GH-CFTR-CCI is GlyH-101.

[0302] 82. The method according to embodiment 73, wherein the CFTR chloride channel inhibitor is glibenclamide, diphenylamine-2-carboxylate, 5-nitro-2-(3-phenylpropylamino)benzoate or niflumic acid, or a combination thereof.

[0303] 83. A method for treating diarrhea or alleviating symptoms associated with diarrhea in an individual who has undergone ileectomy, comprising administering to the individual a pharmaceutical composition comprising a CFTR chloride channel inhibitor in an amount effective to treat the diarrhea or alleviate the symptoms of the diarrhea.

[0304] 84. The method according to embodiment 83, wherein the CFTR chloride channel inhibitor is BPO-CFTR-CCI, PPQ-CFTR-CCI, TD-CFTR-CCI, or GH-CFTR-CCI.

[0305] 85. The method according to embodiment 83 or embodiment 84, wherein the CFTR chloride channel inhibitor is BPO-CFTR-CCI.

[0306] 86. The method according to embodiment 85, wherein the BPO-CFTR-CCI is (R)-BPO-27.

[0307] 87. The method according to embodiment 86, wherein the pharmaceutical composition further comprises (S)-BPO-27.

[0308] 88. The method according to embodiment 83 or embodiment 84, wherein the CFTR chloride channel inhibitor is PPQ-CFTR-CCI.

[0309] 89. The method according to embodiment 83 or embodiment 84, wherein the CFTR chloride channel inhibitor is TD-CFTR-CCI.

[0310] 90. The method according to embodiment 89, wherein the TD-CFTR-CCI is CFTR inh -172.

[0311] 91. The method according to embodiment 83 or embodiment 84, wherein the CFTR chloride channel inhibitor is GH-CFTR-CCI.

[0312] 92. The method according to embodiment 91, wherein the GH-CFTR-CCI is GlyH-101.

[0313] 93. The method according to embodiment 83, wherein the CFTR chloride channel inhibitor is glibenclamide, diphenylamine-2-carboxylate, 5-nitro-2-(3-phenylpropylamino)benzoate or niflumic acid, or a combination thereof.

[0314] 94. The method according to any one of the preceding embodiments, wherein the individual is a human.

[0315] 95. The method according to any one of the preceding embodiments, wherein the individual has been diagnosed with Crohn's disease.

[0316] 96. The method according to any one of embodiments 1 to 94, wherein the individual has been diagnosed with IBS-D.

[0317] 97. The method according to any one of embodiments 1 to 94, wherein the individual has functional diarrhea.

[0318] 98. The method according to any one of the preceding embodiments, wherein the administration results in a reduction in the fecal water content of the individual.

[0319] 99. The method according to embodiment 98, wherein the reduction in the fecal water content of the individual is demonstrated by a decrease in the score of the individual on the Bristol Stool Form Scale.

[0320] 100. The method according to any one of the preceding embodiments, wherein the administration results in a reduction in the defecation frequency of the individual.

[0321] 101. The method according to any one of the preceding embodiments, wherein the administration results in a reduction in the fecal output of the individual.

[0322] 102. The method according to any one of the preceding embodiments, wherein the administration results in a reduction in abdominal pain of the individual.

[0323] 103. The method according to any one of the preceding embodiments, wherein the administration results in a reduction in flatulence of the individual.

[0324] 104. The method according to any one of the preceding embodiments, wherein the administration results in a reduction in nausea of the individual. Examples

[0325] The following examples are provided to provide a better understanding of the subject matter described herein. These examples should not be construed as limiting the described subject matter. It should be understood that the examples and embodiments described herein are for illustrative purposes only, and various modifications or changes made in accordance therewith will be apparent to those skilled in the art and will be included within the scope of the present invention and may be made without departing from the scope of the present invention.

[0326] Abbreviation

[0327] BAD: Bile acid diarrhea

[0328] BAPTA-AM: 1,2-Bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetra(acetoxymethyl ester)

[0329] BPO-27: 6-(5-Bromofuran-2-yl)-7,9-dimethyl-8,10-dioxo-11-phenyl-7,8,9,10-tetrahydro-6H-benzo[b]pyrimido[4',5':3,4]pyrrolo[1,2-d][1,4]oxazine-2-carboxylic acid

[0330] CaCC: Calcium-activated chloride channel

[0331] CDCA: Chenodeoxycholic acid

[0332] CFTR: Cystic fibrosis transmembrane conductance regulator

[0333] CFTR inh -172: 4-[[4-Oxo-2-thioxo-3-[3-(trifluoromethyl)phenyl]-5-thiazolidinylidene]methyl]benzoic acid

[0334] DCA: Deoxycholic acid

[0335] DRA: Down-regulated in adenoma

[0336] ENaC: Epithelial sodium channel

[0337] FGF-19: Fibroblast growth factor 19

[0338] FXR: Farnesoid X receptor

[0339] GlyH-101: N-2-Naphthyl-[(3,5-dibromo-2,4-dihydroxyphenyl)methylene]glycine hydrazide

[0340] IBS-D: Irritable bowel syndrome with diarrhea

[0341] IC 50 : Half maximal inhibitory concentration

[0342] Chemical

[0343] Unless otherwise noted, all chemicals were purchased from Sigma-Aldrich (St. Louis, MO). The sodium salts of taurochenodeoxycholic acid and taurodeoxycholic acid were purchased from Spectrum Chemicals (Gardena, CA). All bile acid solutions were prepared in PBS, except for lithocholic acid, which was dissolved in DMSO. BAPTA-AM was purchased from EMD Millipore (Billerica, MA). Forskolin was purchased from Lc Laboratories (Woburn, MA). CFTR inh -172 and (R)-BPO-27 were synthesized and purified as described. See, e.g., Snyder, D.S., Tradtrantip, L., Yao, C., Kurth, M.J., and Verkman, A.S. (2011) Potent, metabolically stable benzopyrimido-pyrrolo-oxazine-dione (BPO) CFTR inhibitors for polycystic kidney disease. J Med Chem 54, 5468-5477; Snyder, D.S., Tradtrantip, L., Battula, S., Yao, C., Phuan, P.W., Fettinger, J.C., Kurth, M.J., and Verkman, A.S. (2013) Absolute configuration and biological properties of enantiomers of CFTR inhibitor BPO-27. ACS Med Chem Lett 4, 456-459; Ma, T., Thiagarajah, J.R., Yang, H., Sonawane, N.D., Folli, C., Galietta, L.J., and Verkman, A.S. (2002) Thiazolidinone CFTR inhibitor identified by high-throughput screening blocks cholera toxin-induced intestinal fluid secretion. J Clin Invest 110, 1651-1658.

[0344] Example 1. T84 Cell Culture

[0345] T84 cells (ATCC CCL-248) were cultured in a 1:1 mixture of DMEM / Ham's F-12 medium supplemented with 10% FBS, 100 U / mL penicillin, and 100 μg / mL streptomycin. Cells were grown at 37 °C in 5% CO2 / 95% air on Snapwell inserts (Costar Corning, Horseheads, NY) and used 7 to 14 days after seeding.

[0346] Example 2. Human colonoid cultures

[0347] Human colonoid cultures were generated from de-identified tissue samples (except for age and gender) obtained from endoscopic or surgical procedures from three different individuals. Human cultures were generated from isolated intestinal crypts embedded in Matrigel (Corning, Tewksbury, MA) in 24-well plates and cultured in the presence of Wnt3A, R-spondin-1, and Noggin in undifferentiated medium (UDM) as described in: Zachos, N.C. et al. (2016) Human enteroids / colonoids and intestinal organoids functionally recapitulate normal intestinal physiology and pathophysiology. J Biol Chem 291, 3759-3766; In, J., et al. (2016) Enterohemorrhagic escherichia coli reduce mucus and intermicrovillar bridges in human stem cell-derived colonoids. Cell Mol Gastroenterol Hepatol 2, 48-62. Matrigel cultures in UDM medium were used for the proliferation of colonoids. To generate monolayer cultures, colonoids were minced in Cultrex organoid harvesting solution (Trevigen, Gaithersburg, MD), debris was collected by centrifugation and resuspended in UDM. Colonoid fragments (100 μL) were seeded onto 0.4 μm pore polyester membrane 24-well cell culture inserts (Transwell; Corning, Tewksbury, MA) pre-coated with human collagen IV (34 μg / mL; MilliporeSigma). Monolayer cells were cultured in UDM at 37 °C, 5% CO2. Under these conditions, cultures reached confluence within 7 to 14 days as monitored by transepithelial electrical resistance.

[0348] Example 3. Short-circuit current measurement

[0349] T84 cells were mounted in an Ussing chamber and bathed in a symmetrical HCO3 buffer solution containing (in mM): 120 NaCl, 5 KCl, 1 MgCl2, 1 CaCl2, 10 D-glucose, 5 HEPES, and 25 NaHCO3 (pH 7.4). The solution was gassed with 95% O2 / 5% CO2 and maintained at 37 °C. In one study, to measure apical Cl conductance, a basolateral-to-apical Cl gradient was applied, where the basolateral half-chamber contained (in mM): 120 NaCl, 1 MgCl2, 1 CaCl2, 10 D-glucose, 5 HEPES, and 25 NaHCO3 (pH 7.4); in the apical solution, 120 mM NaCl was replaced with 5 mM NaCl and 115 mM Na-gluconate, and the basolateral membrane was permeabilized with 250 μg / ml amphotericin B. The short-circuit current was measured using an EVC4000 multi-channel voltage clamp (World Precision Instruments, Sarasota, FL). - In the buffer solution. The solution was gassed with 95% O2 / 5% CO2 and maintained at 37 °C. In one study, to measure apical Cl - conductance, a basolateral-to-apical Cl - gradient was applied, where the basolateral half-chamber contained (in mM): 120 NaCl, 1 MgCl2, 1 CaCl2, 10 D-glucose, 5 HEPES, and 25 NaHCO3 (pH 7.4); in the apical solution, 120 mM NaCl was replaced with 5 mM NaCl and 115 mM Na-gluconate, and the basolateral membrane was permeabilized with 250 μg / ml amphotericin B. The short-circuit current was measured using an EVC4000 multi-channel voltage clamp (World Precision Instruments, Sarasota, FL).

[0350] For intestinal short-circuit current measurement, CD1 mice were anesthetized with isoflurane. The colon was removed, rinsed with ice-cold Krebs buffer, opened along the mesenteric border, and full-thickness fragments were mounted in a miniature Ussing chamber (area 0.7 cm 2 , World Precision Instruments). The half-chambers were filled with oxygenated Krebs-bicarbonate solution.

[0351] Measurements in human colonoid cultures were performed as described in (18). The apical and basolateral half-chambers were filled with Krebs-Ringer bicarbonate (KBR) buffer gassed with 95% O2 / 5% CO2 at 37 °C. The basolateral half-chamber was supplemented with 10 mM glucose, and the apical half-chamber was supplemented with 10 mM mannitol to maintain osmotic balance.

[0352] Example 4. Intracellular Ca 2+ and cAMP measurement

[0353] T84 cells were seeded in 96-well black-walled microplates. Seventy-two hours after seeding, the confluent cells were loaded with Fluo-4 NW (Invitrogen, Carlsbad, CA). For Ca 2+Measurement, Fluo-4 fluorescence was measured using a FluoStar Optima microplate reader (BMG LabTechnologies, Durham, North Carolina) at an excitation / emission wavelength of 485 / 538 nm. In some studies, cells were pretreated with BAPTA-AM for 30 minutes. Ca in human colonoid cultures 2+ Measurements were performed in monolayers transduced with the fluorescent Ca 2+ sensor Adeno-GCsMP6s. For cAMP assays, T84 cells were grown in 24-well plates, treated with CDCA and / or forskolin for 30 minutes, lysed by repeated freeze / thaw, centrifuged to remove cell debris, and cAMP in the supernatant was assayed using a cAMP immunoassay kit according to the manufacturer's instructions (R&D Systems, Minneapolis, MN).

[0354] Example 5. Intestinal fluid secretion in mouse closed intestinal loops

[0355] CD1 mice (8 to 10 weeks old) were given 5% glucose water but no solid food 24 hours before the experiment. (R)-BPO-27 (5 mg / kg) or vehicle (5% DMSO, 10% Kolliphor HS in saline) was administered intraperitoneally to the mice 60 minutes before creating a closed intestinal (mid-jejunum or distal colon) loop and injecting CDCA or PBS vehicle. This dose of (R)-BPO-27 has previously been shown to provide therapeutic serum levels in mice for several hours. See Cil, O., et al. (2017) Benzopyrimido-pyrrolo-oxazine-dione CFTR inhibitor (R)-BPO-27 for antisecretory therapy of diarrheas caused by bacterial enterotoxins. FASEB J 31, 751-760. To create the loop, the mice were anesthetized with isoflurane and their body temperature was maintained at 36°C to 38°C using a heating pad during the surgery. A small abdominal incision was made to expose the intestine. For the mid-jejunum closed-loop model, a 2-cm to 3-cm loop was created by sutures. The loop was injected with 100 μl of PBS containing CDCA or PBS vehicle. The abdominal incision was closed with sutures and the mice were allowed to recover from anesthesia. In the colon closed-loop model, the mice received an enema (500 μl of mineral oil) 12 hours before the surgery to clear the colon of solids, as previously reported (Haggie, P.M., et al. (2018) SLC26A3 inhibitor identified in small molecule screen blocks colonic fluid absorption and reduces constipation. JCI Insight 3, e121370) and a 1-cm to 2-cm closed loop was formed by sutures. The intestinal loop was surgically removed at the designated time and the loop length and weight were measured to quantify fluid secretion. In some experiments, CF mice (homozygous for ΔF508, 8 to 10 weeks old) were used.

[0356] Example 6. Rat Model of Bile Acid Diarrhea

[0357] Female Sprague-Dawley rats (8 to 10 weeks old) were administered 500 μl of CDCA (10 mM in PBS) or PBS vehicle by mid-colon infusion using the mid-section of a flexible plastic tube (size 15, 78 mm long, Instech Laboratories, Plymouth Meeting, PA). (R)-BPO-27 (10 mg / kg) (or vehicle control) was administered intraperitoneally to the rats 30 minutes before the infusion. (R)-BPO-27 was dissolved in saline containing 5% DMSO and 10% Kolliphor HS. After the infusion, the rats were placed individually in metabolic cages and had free access to water and food. Fecal samples were collected before the infusion (for baseline fecal water content) and within 4 hours after the infusion. Feces were weighed immediately upon visualization to determine the wet weight. To determine fecal water content, fecal samples were dried at 70 °C for 24 hours, and the water content was calculated as (wet weight - dry weight) / wet weight.

[0358] Example 7. Statistics

[0359] Data are expressed as mean ± S.E.M. Statistical analysis was performed using the Prism 5 GraphPad software package (San Diego, CA). Student's t-test was used for statistical comparison when there were two groups, or one-way ANOVA was used for statistical comparison when there were three or more groups. A p-value < 0.05 was considered statistically significant.

[0360] CFTR inhibition blocks bile acid-induced secretory responses in T84 cells

[0361] In preliminary studies, multiple bile acids were screened by short-circuit current measurements to determine their ability to induce a secretory response in the T84 colonic epithelial cell line. See Example 3. When added to both the apical (mucosal) and basolateral (serosal) bathing solutions, the bile acids CDCA and DCA and their taurine conjugates produced robust secretory currents that were blocked by (R)-BPO-27 ( Figure 1 A). No increase in short-circuit current was observed for cholic acid and ursodeoxycholic acid, and only a small transient response was observed for lithocholic acid.

[0362] The increase in short-circuit current after CDCA application was completely blocked by the selective and chemically unrelated inhibitors (R)-BPO-27 and CFTR inh -172( Figure 1 B, Figure 1 C). Concentration-dependent studies showed an IC for (R)-BPO-27 inhibition in this model of 50 < 1 μM (data not shown). Short-circuit current measurements in the distal colon of mice showed that upon addition of CDCA to the apical bathing solution, the current increased by 5.2 ± 1.4 μA / cm2 (Mean ± S.E.M., n = 3), which was completely reversed by (R)-BPO-27 (-5.2 ± 1.7 μA / cm 2 )( Figure 1 D). No increase in short-circuit current was observed upon addition of CDCA to the basolateral bathing solution.

[0363] To study the directionality of the action of CDCA in T84 cells, CDCA was added to the apical or basolateral bathing solution. Figure 2 A shows a robust current response upon addition of 0.75 mM or 1 mM CDCA to the apical solution, while addition of 1 mM CDCA to the basolateral solution had no effect. Addition of up to 2 mM CDCA to the basolateral solution was found to have little effect ( Figure 2 B). Forskolin was added at the end of the study as a positive control to demonstrate the integrity of the T84 cell monolayer. The action of CDCA on apical CFTR and its inhibition by (R)-BPO-27 were confirmed in short-circuit current measurements of basolaterally permeabilized T84 cell monolayers in the presence of an apical-to-basolateral Cl - concentration gradient, where the short-circuit current provides a direct measure of apical CFTR Cl - currents ( Figure 2 C).

[0364] The possible role of CDCA on the cAMP and Ca 2+ signaling pathways in T84 cells was investigated. See Example 4. Up to 0.75 mM CDCA did not significantly affect the short-circuit response to addition of the cAMP agonist forskolin, but a small decrease in forskolin-induced current was observed at 1 mM CDCA ( Figure 3 A, Figure 3 B). In each case, the increased current was completely blocked by (R)-BPO-27. Intracellular cAMP measurements showed that up to 1 mM CDCA by itself did not increase cAMP. However, 0.75 mM and 1 mM CDCA increased cAMP in response to maximal (10 μM) forskolin ( Figure 3 C).

[0365] In T84 cells, the CDCA-induced increase in short-circuit current was largely blocked by pretreatment with the Ca 2+ chelator BAPTA-AM ( Figure 4 A, Figure 4 B). Measurement of intracellular Ca 2+ concentration using the fluorescent sensor Fluo-4 showed an increase in Ca 2+Continued to increase, and the magnitude was comparable to the peak Ca observed with the cholinergic agonist carbachol or the purinergic agonist ATP 2+ elevation ( Figure 4 C, Figure 4 D). The Ca 2+ elevation was largely blocked by pretreatment with BAPTA-AM. These experiments support the involvement of Ca 2+ signaling in the secretory response to CDCA in T84 cells, which was unexpected because the secretory response appears to be mediated entirely by CFTR (cAMP-activated Cl - channels) rather than by Ca 2+ -activated Cl - channels.

[0366] Study of primary human colonoid cultures

[0367] The major findings obtained using T84 cells were investigated in primary human colonoid cultures, including the CFTR Cl - secretory response to apical CDCA and the involvement of Ca 2+ signaling. See Example 2. As observed in T84 cells, CDCA produced a concentration-dependent increase in short-circuit current when added to the apical but not the basolateral bathing solution, which was reversed by (R)-BPO-27 ( Figure 5 A, Figure 5 B). In addition, pretreatment with BAPTA-AM greatly reduced the increase in short-circuit current ( Figure 5 C), and CDCA elevated cytoplasmic Ca 2+ concentration, with a peak magnitude comparable to that produced by ATP ( Figure 5 D), although this was more transient than that observed in T84 cells.

[0368] BPO-27 inhibits fluid secretion in closed mouse intestinal loops

[0369] A closed intestinal loop model was established in mice to study the secretory effect of CDCA in vivo. See Example 5. Initial studies were performed on mid-jejunal loops because it was technically easy to obtain multiple loops in a single animal and they had extensive previous use in studying intestinal fluid secretion in cholera and traveler's diarrhea models. Figure 6 A shows that injection of 10 mM CDCA into mid-jejunal loops produced a large accumulation of fluid within 1 hour. A CDCA concentration-dependent study using loop fluid measured at 2 hours showed a significant increase in loop fluid accumulation with 5 mM and 10 mM CDCA ( Figure 6 B). Similar experiments in closed distal colon loops showed significant loop fluid accumulation with 2.5 mM CDCA ( Figure 6 C).

[0370] To study the effects of CFTR inhibition, (R)-BPO-27 was administered 60 minutes before creating the closed intestinal loops and injecting CDCA (or control PBS vehicle). Figure 7 A). In closed mid-jejunum loops, the active BPO-27 enantiomer, (R)-BPO-27, caused a significant 55% reduction in loop fluid accumulation, while the inactive enantiomer, (S)-BPO-27, had no significant effect. Figure 7 B). A greater ∼70% reduction in loop fluid accumulation was found in closed colon loops with (R)-BPO-27. Figure 7 C).

[0371] Since CFTR inhibition only partially blocks loop fluid accumulation, it was investigated whether CFTR-dependent mechanisms might be partly caused by the effects of CDCA. For these experiments, closed loop studies were performed in cystic fibrosis mice lacking functional CFTR. Compared to control loops injected with PBS vehicle, closed mid-jejunum loops and distal colon closed loops injected with 10 mM CDCA showed a small but significantly greater loop fluid content, indicating that mechanisms other than CFTR also play a role in CDCA-induced fluid accumulation, which based on previous studies may include inhibition of Na + / H + and Cl - / HCO3 - exchangers (absorptive processes), increased paracellular permeability, or stimulation of mucus secretion. See Alrefai, W.A., et al. (2007) Taurodeoxycholate modulates apical Cl - / OH - exchange activity in Caco2 cells. Dig Dis Sci 52, 1270 - 1278; Pallagi-Kunstar, E., et al. (2015) Bile acids inhibit Na + / H + exchanger and Cl - / HCO3 - exchanger activities via cellular energy breakdown and Ca 2+Overload in human colonic crypts. Pflugers Arch 467, 1277 - 1290; Sarathy, J., et al. (2017) The yin and yang of bile acid action on tight junctions in a model colonic epithelium. Physiol Rep 5, e13294; Barcelo, A., et al. (2001) Effect of bile salts on colonic mucus secretion in isolated vascularly perfused rat colon. Dig Dis Sci 46, 1223 - 1231.

[0372] BPO - 27 reduces fecal water content in a rat model of bile acid diarrhea

[0373] A model was established to test the effect of (R)-BPO-27, in which CDCA (or vehicle control) was administered to rats by mid-colonic infusion, and feces were collected within 4 hours to measure fecal weight and water content (as wet / dry weight ratio) ( Figure 8A , upper panel). See Example 6. As seen with Evans blue dye, the infused fluid was rapidly distributed through the colonic lumen in this model ( Figure 8A , lower panel). A greater amount of feces was collected from rats infused with CDCA within 4 hours, and the fecal water content increased significantly by ~25% ( Figure 8B 、 Figure 8C ). Although (R)-BPO-27 pretreatment did not affect fecal volume or water content in rats receiving mid-colonic infusion of PBS, the increase in fecal water content in rats receiving CDCA infusion was inhibited by ~55% ( Figure 8C ).

[0374] Without being bound by theory, the data herein support the CFTR inhibitory utility of CFTR inhibitors, particularly (R)-BPO-27, in the treatment of bile acid-related diarrhea. Short-circuit current measurements in T84 cells and human colonic crypt cultures showed a secretory current in response to apical surface exposure of cells to CDCA, which was completely reversed by CFTR inhibition. Complete inhibition of the secretory current by (R)-BPO-27 was also seen in ex vivo mouse colonic tissues exposed to CDCA. Systemic administration of (R)-BPO-27 largely prevented fluid accumulation in closed colonic loops in mice and prevented an increase in fecal water after intracolonic infusion of a solution containing CDCA. These results suggest that CFTR is the major secretory ion channel in CDCA-induced diarrhea in human colonic cell cultures and rodents.

[0375] This application claims the benefit of priority of U.S. Provisional Application No. 62 / 860,539, filed on June 12, 2019, the entire content of which is incorporated herein by reference.

Claims

1. Use of BPO-27 in the preparation of a medicament for treating an individual suffering from bile acid diarrhea.

2. The use according to claim 1, wherein the BPO-27 is (R)-BPO-27.

3. The use according to claim 1 or 2, wherein the medicament comprises an amount of BPO-27 effective to reduce intestinal fluid secretion caused by the bile acid diarrhea.

4. The use according to any one of the preceding claims, wherein the medicament comprises an amount of BPO-27 effective to reduce the activation of bile acid-induced apical CFTR chloride channels.

5. The use according to any one of the preceding claims, wherein the medicament further comprises an amount of a second agent for treating the bile acid diarrhea, wherein the second agent is a bile acid binder, a farnesoid X receptor (FXR) agonist, a 5-HT3 antagonist, an opioid receptor agonist, a mixed μ opioid receptor agonist, a broad-spectrum gut-specific antibiotic, an antispasmodic, or a tricyclic antidepressant.

6. The use according to claim 5, wherein the second agent is a bile acid binder, preferably cholestyramine, colestipol, or colesevelam.

7. The use according to claim 5, wherein the second agent is a farnesoid X receptor (FXR) agonist, preferably obeticholic acid.

8. The use according to claim 5, wherein the second agent is a 5-HT3 antagonist, preferably alosetron.

9. The use according to claim 5, wherein the second agent is an opioid receptor agonist, preferably loperamide.

10. The use according to claim 5, wherein the second agent is a mixed μ opioid receptor agonist, preferably eluxadoline.

11. The use according to claim 5, wherein the second agent is a broad-spectrum gut-specific antibiotic, preferably rifaximin.

12. The use according to any one of the preceding claims, wherein the bile acid diarrhea results from Crohn's disease.

13. The use according to any one of claims 1 to 11, wherein the bile acid diarrhea results from IBS-D.

14. The use according to any one of claims 1 to 11, wherein the bile acid diarrhea results from functional diarrhea.

15. The use according to any one of the preceding claims, wherein the medicament causes a reduction in fecal water content.

16. The use according to claim 15, wherein the reduction in fecal water content is demonstrated by a decrease in the score on the Bristol Stool Form Scale.

17. The use according to any one of the preceding claims, wherein the medicament causes a reduction in bowel movement frequency.

18. The use according to any one of the preceding claims, wherein the medicament causes a reduction in fecal output.

19. The use according to any one of the preceding claims, wherein the medicament causes a reduction in abdominal pain.

20. The use according to any one of the preceding claims, wherein the medicament causes a reduction in abdominal distension.

21. The use according to any one of the preceding claims, wherein the medicament causes a reduction in nausea.

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