Methods for Treating Bile Acid Diarrhea

Abstract

The present disclosure relates to methods of treating bile acid diarrhea by administering a CFTR chloride channel inhibitor.

Description

[Technical field]

[0001] Government Rights This invention was made with Government support under Grant Nos. DK099803, DK072517, DK101373, EY013574 and DK089502 from the National Institutes of Health. The Government has certain rights in this invention.

[0002] Technical Field The present invention relates to methods of treating bile acid diarrhea by administering a CFTR chloride channel inhibitor. [Background technology]

[0003] background Bile acid diarrhea (BAD) is common after ileal resection and in Crohn's disease, and is thought to occur in more than one-third of patients with diarrhea-predominant irritable bowel syndrome (IBS-D) or chronic functional diarrhea, with an estimated prevalence of up to 1% throughout the Western world. BAD may be caused by abnormalities in hepatic bile acid synthesis or enterohepatic circulation, which results in the delivery of excess bile acids to the colon, where they cause fluid secretion and increased motility.

[0004] Current treatments 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 in development.For IBS-D, where BAD is likely to be involved in the etiology of more than one-third of patients, FDA-approved treatments include the 5-HT3 antagonist alosetron, the mixed μ-opioid receptor agonist eluxadoline, and the broad-spectrum gut-specific antibiotic rifaximin.Other commonly used treatments for IBS-D include loperamide, bile acid sequestrants, antispasmodics, and tricyclic antidepressants.Some of these treatments are associated with significant side effects, such as ischemic colitis with alosetron and pancreatitis with eluxadoline.

[0005] There remains an unmet need for improved and alternative treatments for BAD.

[0006] The cystic fibrosis transmembrane conductance regulator (CFTR) protein is a chloride channel that is expressed in certain mammalian epithelial cells, including intestinal epithelial cells. CFTR chloride channel function is associated with secretory diarrhea.

[0007] CFTR inhibitors have clinical applications in the treatment of secretory diarrhea. Secretory diarrhea caused by enterotoxins, such as cholera and traveler's diarrhea (enteropathogenic E. coli), requires functional CFTR first for chloride secretion into the intestinal space, 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 demonstrated that intestinal chloride secretion in enterotoxin-mediated secretory diarrhea occurs primarily through 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 described previously (see, e.g., review by Verkman et al., Nat. Rev. Drug Discov. 8:153-71 (2009)).

[0008] No. 9,062,073 describes a series of benzopyrimido-pyrrolo-oxazine-dione (BPO) compounds and certain pyrimido-pyrrolo-quinoxalinedione (PPQ) compounds that are useful as CFTR chloride channel inhibitors. The BPO compounds have the following general formula:

[0009] [ka] where: m is 1, 2, 3, or 4; n is 1, 2, 3, 4 or 5; p is an integer from 0 to 4; q is an integer from 1 to 4; R 1 are the same or different and independently selected from H, halo, haloalkyl, C1-C6 alkyl, -(CH2) p -C(O)-R 4a , -S(O)2R 4a , -NO2, or tetrazolyl; R 1a are the same or different and independently selected from H, halo, haloalkyl, C1-C6 alkyl, -(CH2) p -C(O)-R 4a , -S(O)2R 4a , -NO2, or tetrazolyl; R 2a and R 2b are each the same or different and independently H, or C1-C6 alkyl; R 4a Yes, OR 7 , -NR 7 R 8 , -O(CH2) q -OC(O)R 7 or an amino acid residue; R 7 and R 8 are the same or different and independently represent H, C 20 an alkyl, saccharide, or amino acid residue; and Z is aryl or heteroaryl; Here, the amino acid residues are 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, demosine, isodemosine, gamma-carboxyglutamate, hippuric acid, octahydroindole-2-carboxylic acid, statin, 1,2,3,4- The glycosylated glycine residues are selected from the group consisting of tetrahydroisoquinoline-3-carboxylic acid, penicillamine, ornithine, 3-methylhistidine, norvaline, beta-alanine, gamma-aminobutyric acid, cirtulline, homocysteine, homoserine, methyl-alanine, para-benzoylphenylalanine, phenylglycine, propargylglycine, sarcosine, methionine sulfone, tert-butylglycine, 3,5-dibromotyrosine, 3,5-diiodotyrosine, glycosylated threonine, glycosylated serine and glycosylated asparagine.

[0010] PPQ compounds have the general formula:

[0011] [ka] where: m is 1, 2, 3, or 4; n is 1, 2, 3, 4 or 5; p is an integer from 0 to 4; q is an integer from 1 to 4; X is O or S; R 1 are the same or different and independently represent H, halo, haloalkyl, C 1-6 Alkyl, -(CH2) p -C(O)-R 4a , -S(O)2R 4a , -NO2, or tetrazolyl; R1a are the same or different and independently represent H, halo, haloalkyl, C 1-6 Alkyl, -(CH2) p -C(O)-R 4a , -S(O)2R 4a , -NO2, or tetrazolyl; R 2a and R 2b are each the same or different and independently H or C 1-6 is alkyl; R 4a Yes, OR 7 , -NR 7 R 8 , -O(CH2) q -OC(O)R 7 , an amino acid residue, or a peptide; R 4 is H, -N(=O), C 1-6 alkyl, or haloalkyl; R 5 is H, halo, or C 1-6 is alkyl; R 6 Halo, C 1-6 Alkyl or C 1-6 haloalkyl; and R 7 and R 8 are the same or different and independently represent H, C 1-20 It may be an alkyl, a saccharide, an amino acid residue, or a peptide. [Prior art documents] [Patent documents]

[0012] [Patent Document 1] U.S. Patent No. 9,062,073 [Non-patent literature]

[0013] [Non-Patent Document 1] Kunzelmann et al.,Physiol.Rev.82:245-89(2002) [Non-Patent Document 2] Thiagarajah et al.,Curr.Opin.Pharmacol.3:594-9(2003) [Non-Patent Document 3] Clarke et al.,Science 257:1125-28(1992) [Non-Patent Document 4] Gabriel et al.,Science 266:107-109(1994) [Non-Patent Document 5] KunzelmannandMall,Physiol.Rev.82:245-89(2002) [Non-Patent Document 6] Field,15 J.Clin.Invest.111:931-43(2003) [Non-Patent Document 7] Thiagarajah et al., Gastroenterology 126:511-519(2003) [Non-Patent Document 8] Verkman et al.,Nat.Rev.Drug Discov.8:153-71(2009) Summary of the Invention [Means for solving the problem]

[0014] An embodiment of the invention relates to a method of treating a subject having bile acid diarrhea, comprising administering to the subject an amount of a CFTR chloride channel inhibitor effective to treat bile acid diarrhea.

[0015] Another aspect of the invention relates to a method of treating a subject having bile acid diarrhea comprising administering to the subject a pharmaceutical composition comprising an amount of a CFTR chloride channel inhibitor effective to treat bile acid diarrhea.

[0016] Additionally, embodiments of the invention also relate to a method of reducing intestinal fluid secretion resulting from bile acid-induced activation of apical CFTR chloride channels in the intestinal epithelium in a subject in need thereof, comprising administering to the subject an amount of a CFTR chloride channel inhibitor effective to reduce intestinal fluid secretion resulting from bile acid-induced activation of the apical CFTR chloride channel.

[0017] Another aspect of the invention relates to a method of reducing intestinal fluid secretion resulting from bile acid-induced activation of apical CFTR chloride channels in the intestinal epithelium in a subject in need thereof, comprising administering to the subject a pharmaceutical composition comprising an amount of a CFTR chloride channel inhibitor effective to reduce intestinal fluid secretion resulting from bile acid-induced activation of apical CFTR chloride channels.

[0018] A further aspect of the invention relates to a method of reducing bile acid-induced apical CFTR chloride channel current in intestinal epithelium in a subject in need thereof, comprising administering to the subject an amount of a CFTR chloride channel inhibitor effective to reduce bile acid-induced apical CFTR chloride channel current.

[0019] Another aspect of the invention relates to a method of reducing bile acid-induced apical CFTR chloride channel current in intestinal epithelium in a subject in need thereof, comprising administering to the subject a pharmaceutical composition comprising an amount of a CFTR chloride channel inhibitor effective to reduce bile acid-induced apical CFTR chloride channel current.

[0020] In some embodiments, the present invention relates to a method for treating diarrhea or alleviating symptoms associated with diarrhea in a subject who has had an ileal resection, the method comprising administering to the subject an amount of a CFTR chloride channel inhibitor effective to treat or alleviate the symptoms of diarrhea.

[0021] Another aspect of the invention relates to a method of treating diarrhea or alleviating symptoms associated with diarrhea in a subject who has had an ileal resection, comprising administering to the subject a pharmaceutical composition comprising an amount of a CFTR chloride channel inhibitor effective to treat or alleviate the symptoms of diarrhea. [Brief description of the drawings]

[0022] [Figure 1-1] CFTR inhibitors block bile acid-induced secretory currents in T84 cells. (A.) Short circuit currents (Isc) showing responses to the indicated bile acids added to both the apical and basolateral bath solutions. (R)-BPO-27 is added at 10 μM (representing three filters tested for cholic acid, ursodeoxycholic acid, and lithocholic acid; and five filters tested for deoxycholic acid, chenodeoxycholic acid, and their tauroconjugates). [Figure 1-2] CFTR inhibitors block bile acid-induced secretory currents in T84 cells. (B.) CDCA (0.75 mM) was added to both bath solutions along with (R)-BPO-27 (10 μM) or CFTRinh-172 (10 μM) added as indicated. (C.) Summary of peak CDCA-induced currents and the decline in current after (R)-BPO-27 or CFTRinh-172 (ΔIsc, mean ± SEM, n = 7-8 filters per condition). (D.) Short-circuit currents in mouse colon showing the effect of adding CDCA (1 mM) to the basolateral or apical bath solutions along with (R)-BPO-27 (10 μM) added as indicated. [Figure 2-1] CDCA acts apically in T84 cell monolayers to activate apical CFTR. (A.) CDCA is added to the basolateral or apical bath solution. (Top) Representative short-circuit current curves with (R)-BPO-27 added at 10 μM. (Bottom) Summary of peak CDCA-evoked currents (ΔIsc, mean ± SEM, n=7-8, **p<0.01, ns, not significant compared to zero effect). [Figure 2-2]CDCA acts from the apical side in T84 cell monolayers to activate apical CFTR. (B.) Increasing amounts of CDCA from 0.75 to 2 mM are added to the basolateral bath solution, followed by forskolin (10 μM added on both sides) and (R)-BPO-27 (10 μM). (C.) Short-circuit current 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 or 1 mM) is added to the apical bath solution, followed by (R)-BPO-27 (10 μM). [Diagram 3] CDCA has minimal effect on cAMP signaling in T84 cells. (A.) Short circuit currents showing the effect of forskolin (10 μM) followed by (R)-BPO-27 (10 μM) after the indicated concentrations of CDCA. (B.) Summary of peak forskolin-evoked currents (ΔIsc, mean ± SEM, n=4-6, **p<0.01, ns, not significant compared to forskolin response with 0 mM CDCA). (C.) cAMP in T84 cells measured after 30 min of incubation with the indicated concentrations of CDCA with or without 10 μM forskolin (mean ± SEM, n=3-6, **p<0.01, ns, not significant). [Figure 4] CDCA action in T84 cells involves Ca2+ signaling. (A.) Short circuit currents showing the effect of CDCA (1 mM) after BAPTA-AM (30 μM) pretreatment for 25 min and subsequent (R)-BPO-27 (10 μM) treatment. (B.) Summary of peak CDCA-evoked currents with or without BAPTA-AM pretreatment (ΔIsc, mean ± SEM, n=4-5, **p<0.01). (C.) Cytoplasmic Ca2+ concentration measured by Fluo-4 fluorescence. CDCA (0.75, 1 mM), ATP (100 μM) or carbachol (100 μM) were added to the apical bath solution with or without BAPTA-AM (30 μM) pretreatment for 30 min. (D.) Peaks increased in Fluo-4 fluorescence (mean ± SEM, n=3-6, **p<0.01, ns, not significant). [Diagram 5]CDCA secretory responses in primary cultures of colony-forming human epithelial cells consisting of planar monolayer cultures generated from colonoids. (A.) (Top) Short-circuit currents showing the indicated concentrations of CDCA added to the apical or basolateral bath solution and the effect of subsequent addition of forskolin (10 μM) and (R)-BPO-27 (5 μM). (B.) Summary of peak CDCA-evoked currents (ΔIsc, mean ± SEM, n=9-13). (C.) Short-circuit currents showing the effect of CDCA (0.5 mM) after 20 min of BAPTA-AM (30 μM) pretreatment and subsequent treatment with forskolin (10 μM) and (R)-BPO-27 (5 μM). (D.) Fluorescence measurements of cytosolic Ca2+ concentration following addition of CDCA (0.75 mM) or ATP (100 μM). [Figure 6] CDCA-induced fluid secretion in obstructed intestinal loops in mice. (A.) Time course of CDCA-induced fluid secretion. 100 μl CDCA (10 mM) in PBS or PBS is injected into the obstructed mid-jejunum loops and the loops are excised at different times for measurement of loop weight and length (mean ± SEM, n = 5-9 loops per group, **p < 0.01). (B.) Concentration dependence of the CDCA effect in obstructed jejunum loops. 100 μl CDCA or PBS is injected into the obstructed mid-jejunum loops (mean ± SEM, n = 3-14 loops per group, **p < 0.01, ns, not significant). (C.) Concentration dependence of the CDCA effect in obstructed colonic loops. 100 μl CDCA or PBS is injected into the obstructed colonic loops (mean ± SEM, n = 3-5 loops per group, **p < 0.01, ns, not significant). [Figure 7](R)-BPO-27 inhibits CDCA-induced fluid secretion in obstructed intestinal loops in mice. (A.) Experimental protocol. (B.) (Left) Weight / length ratios of mid-jejunal loops injected with CDCA (10 mM) or PBS with or without BPO-27 (R or S enantiomer) (mean ± SEM, n = 6-7 loops per group, **p < 0.01, ns, not significant). (Right) Representative photographs of loops injected with CDCA and PBS with or without BPO-27. (C.) (Left) Weight / length ratios of colonic loops injected with CDCAA (2.5, 10 mM) with or without (R)-BPO-27 or PBS (mean ± SEM, n = 4-7 loops per group, **p < 0.01, ns, not significant). (Right) Representative photographs of loops injected with CDCA and PBS with or without (R)-BPO-27. (D.) Weight / length ratios of mid-jejunal or colonic loops from cystic fibrosis (CFTR-deficient) mice injected with CDCA (2.5, 10 mM) (mean ± SEM, n = 4 loops per group, **p < 0.01, ns, not significant). [Figure 8] (R)-BPO-27 reduces the increase in fecal water content in a rat model of bile acid diarrhea. (A.) Experimental protocol (top) and photograph taken 10 minutes after midcolonic infusion of 500 μL PBS containing Evans blue dye (bottom). (B.) Fecal output (wet weight) at 4 hours after midcolonic infusion in rats with or without (R)-BPO-27 pretreatment (mean ± SEM, n = 4 rats in PBS-treated group, n = 8 rats in CDCA-treated group, *p < 0.05, ns, not significant). (C.) (Left) Fecal water content (percentage of water from wet / dry weight measurements) from feces collected in B (**p < 0.01, ns, not significant). (Right) Paired analysis showing the increase in fecal water content for individual rats (division of pre-infusion fecal water from fecal water at 0–4 h in the same rat) (*p<0.05, **p<0.01, ns, not significant). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS 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 terminology used herein is for the purpose of describing particular embodiments by way of example only and is not intended to limit the invention as claimed.

[0024] Scientific and technical terms used in connection with this application have the meanings that are commonly understood by those of ordinary skill in the art, unless otherwise specified herein.

[0025] As used herein, the terms "composition," "compound," "drug," "pharmaceutical active agent," "active agent," or "medicine" are used interchangeably herein and refer to one or more chemical compounds or compositions that induce a desired pharmacological and / or physiological effect via local and / or systemic action when administered to a subject (human or mammal).

[0026] As used herein, the terms "treatment" or "therapy" (as well as their alternative forms) include preventative (e.g., prophylactic), curative, or palliative treatment. As used herein, the term "treating" includes alleviating or reducing at least one adverse or negative effect or symptom, symptom of a disease or disorder.

[0027] The term "administration" means either direct administration of a compound or composition of the present invention.

[0028] The term "subject" is used herein to refer to a human or domestic mammal (eg, dog, cat) to which treatment (including prophylactic treatment) with the compounds according to the invention is administered.

[0029] Within the scope of the present invention, the compounds of the present disclosure may be prepared in the form of pharmaceutically acceptable salts. "Pharmaceutically acceptable salts" refers to derivatives of the compounds of the present disclosure, in which the parent compound is modified by making its acid salt or base salt. Examples of pharmaceutically acceptable salts include, but are not limited to, mineral salts or organic acid salts of basic residues such as amines; alkali salts or organic salts of acidic residues such as carboxylic acids; and the like. Pharmaceutically acceptable salts include the conventional non-toxic salts or quaternary ammonium salts of the parent compound, for example, formed from 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, as well as 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, hydroxymaleic acid, phenylacetic acid, glutamic acid, benzoic acid, salicylic acid, sulfanilic acid, 2-acetoxybenzoic acid, fumaric acid, toluenesulfonic acid, methanesulfonic acid, ethanedisulfonic acid, oxalic acid, isethionic acid, and the like. These pharma- ceutically 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 the acid, or by neutralizing the free carboxylic acid with an alkali metal base or an amine, such as a hydroxide.

[0030] The present invention relates to a method of treating a subject having bile acid diarrhea comprising administering to the subject an amount of a CFTR chloride channel inhibitor effective to treat bile acid diarrhea.

[0031] In some embodiments, the present invention relates to a method for treating a subject with bile acid diarrhea. Bile acid diarrhea (BAD) is a clinically diagnosed condition in which diarrhea is believed to result from bile acid malabsorption or disregulation. See, for example, M. Camilleri, Advances in understanding of bile acid diarrhea, Expert Rev Gastroenterol Hepatol. 2014 January; 8(1): 49-61. Methods for diagnosing BAD are known in the art. See, for example, ibid.

[0032] In some embodiments of the methods disclosed herein, the subject is administered an amount of a CFTR chloride channel inhibitor (CFTR-CCI). As used herein, CFTR chloride channel refers to the cystic fibrosis transmembrane conductance regulator (CFTR), which is a cAMP-activated chloride channel expressed in epithelia in the lung, intestine, pancreas, testis, and other tissues. The term CFTR chloride channel inhibitor (or "CFTR-CCI"), as used herein, refers to any of the cystic fibrosis transmembrane conductance regulator chloride channel inhibitors described in the prior art as useful for one or more medical purposes, including those described in U.S. Pat. Nos. 9,062,073, 7,235,573, 7,638,543, 7,414,037, and 7,888,332; U.S. Patent Application No. 2009 / 0253799; International Patent Application Publication Nos. WO09 / 120803 and WO09 / 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.

[0033] In some embodiments, the CFTR chloride channel inhibitor is a BPOCFTR chloride channel inhibitor (BPO-CFTR-CCI). BPO-CFTR-CCI is a compound that falls within the following general formula:

[0034] [ka] where: m is 1, 2, 3, or 4; n is 1, 2, 3, 4 or 5; p is an integer from 0 to 4; q is an integer from 1 to 4; R 1 are the same or different and independently selected from H, halo, haloalkyl, C1-C6 alkyl, -(CH2) p -C(O)-R 4a , -S(O)2R 4a , -NO2, or tetrazolyl; R 1a are the same or different and independently selected from H, halo, haloalkyl, C1-C6 alkyl, -(CH2) p -C(O)-R 4a , -S(O)2R 4a , -NO2, or tetrazolyl; R 2a and R 2b are each the same or different and independently H, or C1-C6 alkyl; R 4a Yes, or 7 , -NR 7 R 8 , -O(CH2) q -OC(O)R 7 or an amino acid residue; R 7 and R 8 are the same or different and independently represent H, C 20an alkyl, saccharide, or amino acid residue; and Z is aryl or heteroaryl; Here, the amino acid residues are 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, demosine, isodemosine, gamma-carboxyglutamate, hippuric acid, octahydroindole-2-carboxylic acid, statin, 1,2,3,4- The glycosylated glycine residues are selected from the group consisting of tetrahydroisoquinoline-3-carboxylic acid, penicillamine, ornithine, 3-methylhistidine, norvaline, beta-alanine, gamma-aminobutyric acid, cirtulline, homocysteine, homoserine, methyl-alanine, para-benzoylphenylalanine, phenylglycine, propargylglycine, sarcosine, methionine sulfone, tert-butylglycine, 3,5-dibromotyrosine, 3,5-diiodotyrosine, glycosylated threonine, glycosylated serine and glycosylated asparagine.

[0035] In some embodiments, the BPO-CFTR-CCI is a compound of the following formula:

[0036] [ka] where: R 1 is H, halo, or C 1-6 is alkyl; R 2 and R 3 are the same or different and independently represent H, halo, -NO2, C 1-6 Alkyl, tetrazolyl, -S(O)2 or 7 , or -C(=O) or 7 and; R5 is H, halo, or C 1-6 is alkyl; R 6 Halo, C 1-6 Alkyl or C 1-6 haloalkyl; and R 7 , H, C 1-6 It may be an alkyl, a saccharide, an amino acid residue, or a peptide.

[0037] BPO-CFTR-CCI is described in US Pat. No. 9,062,073, which is incorporated herein by reference in its entirety.

[0038] In some embodiments, the BPO-CFTR-CCI is (R)-BPO-27, which is a compound having the following structure:

[0039] [ka]

[0040] In another embodiment, the CFTR chloride channel inhibitor is a PPQ CFTR chloride channel inhibitor (PPQ-CFTR-CCI). PPQ-CFTR-CCI is a compound that falls within the following general formula:

[0041] [ka] where: m is 1, 2, 3, or 4; n is 1, 2, 3, 4 or 5; p is an integer from 0 to 4; q is an integer from 1 to 4; X is O or S; R 1 are the same or different and independently represent H, halo, haloalkyl, C 1-6 Alkyl, -(CH2) p -C(O)-R 4a , -S(O)2R4a , -NO2, or tetrazolyl; R 1a are the same or different and independently represent H, halo, haloalkyl, C 1-6 Alkyl, -(CH2) p -C(O)-R 4a , -S(O)2R 4a , -NO2, or tetrazolyl; R 2a and R 2b are each the same or different and independently H or C 1-6 is alkyl; R 4a Yes, or 7 , -NR 7 R 8 , -O(CH2) q -OC(O)R 7 , an amino acid residue, or a peptide; R 4 is H, -N(=O), C 1-6 alkyl, or haloalkyl; R 5 is H, halo, or C 1-6 is alkyl; R 6 Halo, C 1-6 Alkyl or C 1-6 haloalkyl; and R 7 and R 8 are the same or different and independently represent H, C 1-20 It may be an alkyl, a saccharide, an amino acid residue, or a peptide.

[0042] PPQ-CFTR-CCI is described in US Pat. No. 9,062,073, which is incorporated herein by reference in its entirety.

[0043] In some embodiments, the CFTR chloride channel inhibitor is a thiazolidinone CFTR chloride channel inhibitor (TD-CFTR-CCI). TD-CFTR-CCI is a compound that falls within the following general formula:

[0044] [ka] or a pharma- ceutically acceptable salt, prodrug or stereoisomer thereof; where Y is -NH- or absent; W is =CH-, -S-, -O-, -C(=S)-, or -C(=O)-; Z1, Z2, Z3, Z4, and Z5 are each independently O or S; J is C, S, O, or N; Q is C or N; R1, R2, R3, and R9 are each independently H, C 1-6 alkyl, alkoxy, halo, -CF3, -CF2CF3, or -OCF3; R5 is H, halo, C 1-6 alkyl or absent; X1, X2, X3, and X4 are each independently H, -OH, -SH, halo, tetrazolo, -P(=O)(OH)2, -C(=Z3)Z4H, -Z5-C(=Z3)Z4H, or -Z5-CH2-C(=Z3)Z4H; and X5 -O - , tetrazolo, -C(=O)OH, -OC(=O)OH or absent.

[0045] TD-CFTR-CCI is also a compound that falls within the general formula:

[0046] [ka] where X1 is trifluoromethyl; X2 and X3 are independently selected from hydrogen and a halo group; Y1, Y2 and Y3 are independently selected from hydrogen, C1-C8 alkyl, C1-C7 alkoxy, carbonate, carbamate, carboxyl, a halo group, a nitro group, an azo group, a hydroxyl group and a mercapto group.

[0047] TD-CFTR-CCI or 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. Each of these references is incorporated by reference herein in its entirety.

[0048] In some embodiments, TD-CFTR-CCI is a CFTR inh -172, which has the following structure:

[0049] [ka]

[0050] In some embodiments, the CFTR chloride channel inhibitor is a glycine hydrazine CFTR chloride channel inhibitor (GH-CFTR-CCI). GH-CFTR-CCI is a compound that includes the following structure:

[0051] [ka] or a pharma- ceutically acceptable salt or stereoisomer thereof; where X1 is hydrogen or substituted or unsubstituted, saturated linear or branched alkyl; Y is hydrogen or substituted or unsubstituted, saturated linear or branched alkyl; R1 is unsubstituted phenyl; substituted phenyl, wherein the phenyl is substituted with one or more of hydroxy, alkyl, and hydrogen; substituted or unsubstituted quinolinyl, substituted or unsubstituted anthracenyl, or substituted or unsubstituted naphthalenyl; R 2 is unsubstituted phenyl, substituted phenyl, wherein the phenyl is substituted with bromo or carboxy; di(hydroxy)phenyl, mono-(halo)-mono(hydroxy)phenyl, mono(halo)-di(hydroxy)phenyl, mono(halo)-tri(hydroxy)phenyl, di(halo)-mono(hydroxy)phenyl, di(halo)-di(hydroxy)phenyl, di(halo)-tri(hydroxy)phenyl, mono(halo)-mono(hydroxy)-mono(alkoxy)phenyl, mono(halo)-di(hydroxy)-mono(alkoxy)phenyl, mono(halo)-mono(hydroxy)-di(alkoxy)phenyl, mono(halo)-di(hydroxy)-di(alkoxy)phenyl, di(halo)-mono(hydroxy)-mono(alkoxy)phenyl, di(halo)-di(hydroxy)-mono(alkoxy)phenyl, di(halo)-mono(hydroxy)-di(alkoxy)phenyl; and R3 is hydrogen or substituted or unsubstituted alkyl.

[0052] GH-CFTR-CCI also includes compounds that fall within the general formula:

[0053] [ka] or a pharma- ceutically acceptable salt or stereoisomer thereof; where Y" is a substituted or unsubstituted, saturated linear or branched alkyl; 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 polyoxyalkyl polyether, a polyethyleneimine, a disaccharide, a trisaccharide, a polyalkylimine, and a small aminodextran; R1 is unsubstituted or substituted phenyl, substituted or unsubstituted quinolinyl, substituted or unsubstituted anthracenyl, or substituted or unsubstituted naphthalenyl; R 2 is unsubstituted or substituted phenyl; and R3 is hydrogen or substituted or unsubstituted alkyl, thereby inhibiting CFTR.

[0054] GH-CFTR-CCI also includes compounds that fall within the general formula:

[0055] [ka] or a pharma- ceutically acceptable salt, prodrug or stereoisomer thereof. where: R 1 and R 1 are the same or different and independently optionally substituted phenyl, optionally substituted heteroaryl, optionally substituted quinolinyl, optionally substituted anthracenyl, or optionally substituted naphthalenyl; R 2 , R 2’ , R 3 , R 3’ , R 4 , R 4’ , R 5 , R 5’ , R 6 , and R 6’are each the same or different and independently hydrogen, hydroxy, Ci g alkyl, Ci g alkoxy, carboxy, halo, nitro, cyano, -SO3H, -S(=O)2NH2, aryl, and heteroaryl; R 13 , R 13 , R 14 , and R 14 are each the same or different and independently hydrogen or C 1-8 is alkyl; X and X' are each linker moieties that are the same or different; J and J' are each spacer moieties that are the same or different; A is a polymer subunit; and n is an integer between 0 and 2,500.

[0056] 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. Patent Application Publication No. 2009 / 0253799; International Patent Application Publication No. WO 09 / 146144. The entirety of each of these references is incorporated herein by reference.

[0057] In some embodiments, the GH-CFTR-CCI is GlyH-101, which has the following structure:

[0058] [ka]

[0059] Other CFTR chloride channel inhibitors are glibenclamide, diphenylamine-2-carboxylate, 5-nitro-2-(3-phenylpropylamino)benzoate, and niflumic acid.

[0060] In some embodiments of the methods disclosed herein, the subject is administered a CFTR chloride channel inhibitor that is BPO-CFTR-CCI, PPQ-CFTR-CCI, TD-CFTR-CCI, or GH-CFTR-CCI.

[0061] In some embodiments of the methods disclosed herein, the subject is administered (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.

[0062] In some embodiments, the CFTR chloride channel inhibitor is BPO-CFTR-CCI.

[0063] In some embodiments, the CFTR chloride channel inhibitor is PPQ-CFTR-CCI.

[0064] In some embodiments, the CFTR chloride channel inhibitor is TD-CFTR-CCI. inh It is -172.

[0065] In some embodiments, the CFTR chloride channel inhibitor is GH-CFTR-CCI. In some embodiments, the GH-CFTR-CCI is GlyH-101.

[0066] 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.

[0067] In some embodiments of the disclosed methods, the subject is administered a pharmaceutical composition comprising a pharmaceutical excipient and an amount of a CFTR chloride channel inhibitor.

[0068] In some aspects, the disclosure relates to a method of treating a subject having bile acid diarrhea, the method comprising administering to the subject a pharmaceutical composition comprising an amount of a CFTR chloride channel inhibitor 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, the 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, the TD-CFTR-CCI is a CFTR chloride channel inhibitor. inh-172. In other embodiments, the CFTR chloride channel inhibitor is GH-CFTR-CCI. In some embodiments, the 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.

[0069] In those embodiments in which a subject is administered a pharmaceutical composition comprising a CFTR chloride channel inhibitor that is (R)-BPO-27, the pharmaceutical composition may also include (S)-BPO-27. In those embodiments, the amount of (R)-BPO-27 in the pharmaceutical composition is substantially the same as or greater than the amount of (S)-BPO-27 present in the pharmaceutical composition. For example, in some embodiments, the pharmaceutical composition may include a racemic mixture of (R / S)-BPO-27. In some aspects, (R)-BPO-27 is present in enantiomeric excess (ee) relative to (S)-BPO-27. For example, (R)-BPO-27 may be present in approximately 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 comprises greater than 99% % ee of (R)-BPO-27.

[0070] The amount of CFTR chloride channel inhibitor administered is an amount effective to treat bile acid diarrhea. In this regard, the amount that is effective will vary depending on the characteristics and condition of the subject.

[0071] In some embodiments of the disclosed methods, the amount of the CFTR chloride channel inhibitor is effective to reduce intestinal fluid secretion resulting from bile acid diarrhea.

[0072] In other embodiments of the disclosed methods, the amount of the CFTR chloride channel inhibitor is effective to attenuate bile acid-induced activation of the apical CFTR chloride channel.

[0073] In some aspects of the method of the present disclosure, the subject is also administered an amount of a second agent effective for treating bile acid diarrhea. 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 another embodiment, the second agent is a mixed μ-opioid receptor agonist, preferably eluxadoline.In another embodiment, the second agent is a broad-spectrum gut-specific antibiotic, preferably rifaximin.

[0074] In some embodiments, the invention relates to a method of reducing intestinal fluid secretion resulting from bile acid-induced activation of CFTR chloride channels in the intestinal epithelium in a subject in need thereof, comprising administering to the subject an amount of a CFTR chloride channel inhibitor effective to reduce intestinal fluid secretion resulting from bile acid-induced activation of CFTR chloride channels.

[0075] As used herein, intestinal secretion refers to the secretion of fluid from the intestinal epithelium into the mesenteric space.

[0076] In some embodiments, the intestinal fluid secretion is due to bile acid-induced activation of the CFTR chloride channel in the intestinal epithelium. In other embodiments, the CFTR chloride channel is an apical CFTR chloride channel in the intestinal epithelium. The apical CFTR chloride channel is a CFTR chloride channel located in the apical cell membrane (i.e., the cell membrane facing the mesentery). Thus, in some embodiments, the present invention relates to a method of reducing intestinal fluid secretion due to bile acid-induced activation of the apical CFTR chloride channel in the intestinal epithelium in a subject in need thereof, comprising administering to the subject an amount of a CFTR chloride channel inhibitor effective to reduce intestinal fluid secretion due to bile acid-induced activation of the apical CFTR chloride channel.

[0077] In some embodiments, intestinal fluid secretion is reduced by administering an amount of a CFTR chloride channel inhibitor effective to reduce intestinal fluid secretion.As used herein, reducing 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 without 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 contents of the intestinal lumen and diarrheal discharge.

[0078] 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 (R)-BPO-27, CFTRinh In some embodiments, the CFTR chloride channel inhibitor is (R)-BPO-27. In some embodiments, the CFTR chloride channel inhibitor is CFTR-172, GlyH-101, glibenclamide, diphenylamine-2-carboxylate, 5-nitro-2-(3-phenylpropylamino)benzoate, or niflumic acid, or a combination thereof. ... 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.

[0079] In this aspect of the invention, the amount of CFTR chloride channel inhibitor administered is effective to reduce intestinal fluid secretion resulting from bile acid-induced activation of a CFTR chloride channel (e.g., an apical CFTR chloride channel). In this regard, the amount that is effective will vary depending on the characteristics and condition of the subject.

[0080] In some aspects, the invention relates to a method of reducing intestinal fluid secretion resulting from bile acid-induced activation of CFTR chloride channels in the intestinal epithelium in a subject in need thereof, comprising administering to the subject a pharmaceutical composition comprising an amount of a CFTR chloride channel inhibitor effective to reduce intestinal fluid secretion resulting from bile acid-induced activation of CFTR chloride channels. 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, the 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 inh -172. In other embodiments, the CFTR chloride channel inhibitor is GH-CFTR-CCI. In some embodiments, the 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.

[0081] In another aspect, the disclosure relates to a method of reducing bile acid-induced CFTR chloride channel current in an intestinal epithelium in a subject in need thereof, the method comprising administering to the subject an amount of a CFTR chloride channel inhibitor effective to reduce bile acid-induced CFTR chloride channel current.

[0082] As used herein, "current" refers to the passage of ions through a channel. Thus, a CFTR chloride channel current refers to the passage of chloride ions through a CFTR chloride channel.

[0083] In some embodiments, the CFTR chloride channel current results from bile acid-induced activation of the apical CFTR chloride channel in intestinal epithelium. Thus, in some embodiments, the invention relates to a method of reducing bile acid-induced apical CFTR chloride channel current in intestinal epithelium in a subject in need thereof, comprising administering to the subject an amount of a CFTR chloride channel inhibitor effective to reduce the bile acid-induced apical CFTR chloride channel current.

[0084] In some embodiments, bile acid-induced CFTR chloride channel currents in the intestinal epithelium are 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, bile acid-induced CFTR chloride channel currents in the intestinal epithelium are reduced by administering (R)-BPO-27, CFTR inh It is reduced by administering a CFTR chloride channel inhibitor such as -172, GlyH-101, glibenclamide, diphenylamine-2-carboxylate, 5-nitro-2-(3-phenylpropylamino)benzoate or niflumic acid, or a combination thereof.

[0085] 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.

[0086] In this embodiment of the invention, the amount of CFTR chloride channel inhibitor administered is an amount effective to reduce bile acid-induced CFTR chloride channel (e.g., apical CFTR chloride channel) current. In this regard, the amount that is effective will vary depending on the characteristics and condition of the subject.

[0087] In some aspects, the invention provides a method of reducing bile acid-induced CFTR chloride channel current in an intestinal epithelium in a subject in need thereof, comprising administering to the subject a pharmaceutical composition comprising an amount of a CFTR chloride channel inhibitor effective to reduce bile acid-induced CFTR chloride channel current. 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, the 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 inh -172. In other embodiments, the CFTR chloride channel inhibitor is GH-CFTR-CCI. In some embodiments, the 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.

[0088] In some embodiments, the present invention relates to a method for treating diarrhea or alleviating symptoms associated with diarrhea in a subject who has undergone ileal resection, comprising administering to the subject an amount of a CFTR chloride channel inhibitor effective to treat or alleviate the symptoms of diarrhea.As used herein, symptoms of diarrhea include one or more of the following: (1) increased fecal water content, (2) increased stool frequency; (3) increased fecal output; (4) abdominal pain; (5) abdominal distension; and (6) nausea.

[0089] In some embodiments, a method for treating diarrhea or alleviating symptoms associated with diarrhea in a subject who has had an ileal resection comprises administering to the subject 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.

[0090] In some embodiments, the method for treating diarrhea or alleviating symptoms associated with diarrhea in a subject who has undergone ileal resection comprises administering to the subject (R)-BPO-27, CFTR inh The method includes administering to the subject a CFTR chloride channel inhibitor that is CFTR-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.

[0091] In this embodiment of the invention, the amount of CFTR chloride channel inhibitor administered is an amount effective to treat diarrhea or alleviate symptoms associated with diarrhea in a subject. In this regard, the amount that is effective will vary depending on the characteristics and condition of the subject.

[0092] In some aspects, the invention relates to a method of treating diarrhea or alleviating symptoms associated with diarrhea in a subject who has undergone ileal resection, the method comprising administering to the subject a pharmaceutical composition comprising an amount of a CFTR chloride channel inhibitor effective to treat diarrhea or alleviate symptoms of 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, the 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, the TD-CFTR-CCI is a CFTR chloride channel inhibitor. inh-172. In other embodiments, the CFTR chloride channel inhibitor is GH-CFTR-CCI. In some embodiments, the 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.

[0093] In all of the methods of the present disclosure, a CFTR chloride channel inhibitor is administered to a subject. In some embodiments, the subject is a mammal. In other embodiments, the subject is a human.

[0094] In some embodiments, the subject has been diagnosed with Crohn's disease.

[0095] In other embodiments, the subject has been diagnosed with IBS-D.

[0096] In still other embodiments, the subject has been diagnosed with functional diarrhea.

[0097] In some embodiments, the methods of the present invention result in a reduction in the water content of the subject's feces. The degree of water reduction can be measured by measuring the amount of water in the subject's feces before administering a FTR chloride channel inhibitor and comparing this amount to the amount of water in the subject's feces after administering a CFTR chloride channel inhibitor.

[0098] In some embodiments, the reduction in water content in the subject's stool is performed, for example, by measurement using a clinical instrument such as the Bristol Fecal Foam Scale (BSFS). The BSFS, known in the art, assigns a consistency score to the subject's stool ranging from 1 (firm) to 7 (watery). Thus, in some embodiments, the reduction in water content of the subject's stool using the methods of the invention is demonstrated by a lowering score using the Bristol Fecal Foam Scale. In some embodiments, the reduction in water content of the subject's stool using the methods of the invention is demonstrated by a 1-point reduction (e.g., from 7 to 6, from 6 to 5, from 5 to 4, from 4 to 3, from 3 to 2, from 2 to 1) using the Bristol Fecal Foam Scale. In other embodiments, the reduction in water content of the subject's stool using the methods of the invention is demonstrated by a 2-point reduction (e.g., from 7 to 5, from 6 to 4, from 5 to 3, from 4 to 2, from 3 to 1) using the Bristol Fecal Foam Scale. In other embodiments, a reduction in the water content of a subject's feces using the methods of the invention is demonstrated by a reduction of 3 points using the Bristol Fecal Foam Scale (e.g., from 7 to 4, from 6 to 3, from 5 to 2, from 4 to 1). In other embodiments, a reduction in the water content of a subject's feces using the methods of the invention is demonstrated by a reduction of more than 3 points using the Bristol Fecal Foam Scale.

[0099] It will be understood by those of skill in the art that the reduction in the water content of a subject's feces may also be demonstrated using other clinical scales known in the art.

[0100] In some embodiments, the method of the present invention results in a reduction in the frequency of defecation in a subject. The degree of reduction in defecation frequency can be measured by comparing the defecation frequency of a subject before administration of a CFTR chloride channel inhibitor with the defection frequency of a subject after administration of a CFTR chloride channel inhibitor. Here, the defecation frequency can be measured, for example, by interviewing the subject or by using a device designed to elicit this information on the subject. Methods for measuring defecation frequency are known in the art.

[0101] In another embodiment, the method of the present invention results in a reduction in the fecal output of the subject. The degree of reduction in fecal output can be measured by measuring the amount of feces of the subject before administration of a CFTR chloride channel inhibitor and comparing this amount to the amount of feces of the subject after administration of a CFTR chloride channel inhibitor, where this amount can be measured by weight or volume. Methods for measuring fecal output are known in the art.

[0102] In another embodiment, the method of the present invention provides a reduction in abdominal pain in a subject. The degree of abdominal pain reduction can be measured by comparing the abdominal pain of the subject before administration of a CFTR chloride channel inhibitor with the abdominal pain of the subject after administration of a CFTR chloride channel inhibitor. Here, abdominal pain can be measured by interviewing the subject or by administering a device to the subject that is designed to elicit this information. Methods for measuring abdominal pain are known in the art.

[0103] In another embodiment, the method of the present invention results in a reduction in abdominal distension in a subject. The degree of reduction in abdominal distension can be measured by comparing the abdominal distension of the subject before administration of a CFTR chloride channel inhibitor with the abdominal distension of the subject after administration of a CFTR chloride channel inhibitor. Here, abdominal distension can be measured by interviewing the subject or by administering to the subject a device designed to elicit this information. Methods for measuring abdominal distension are known in the art.

[0104] In another embodiment, the method of the present invention results in a reduction in nausea in a subject. The degree of reduction in nausea can be measured by comparing the nausea of ​​the subject before administration of a CFTR chloride channel inhibitor with the nausea of ​​the subject after administration of a CFTR chloride channel inhibitor. Here, nausea can be measured by interviewing the subject or by administering to the subject a device designed to elicit this information. Methods for measuring nausea are known in the art. EXAMPLES

[0105] The following examples are provided to provide a better understanding of the subject matter described herein. It is understood that the examples and embodiments described herein are for illustrative purposes only, and that various modifications or changes therein will be apparent to those skilled in the art and are within the scope of the present invention and can be made without departing from this scope.

[0106] Abbreviation BAD: Bile acid diarrhea BAPTA-AM: 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester) 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 CaCC: Calcium-activated chloride channel CDCA: chenodeoxycholic acid CFTR: Cystic fibrosis transmembrane conductance regulator CFTR inh -172: 4-[[4-oxo-2-thioxo-3-[3-(trifluoromethyl)phenyl]-5-thiazolidinylidene]methyl]benzoic acid DCA: Deoxycholic acid DRA: Downregulated in adenomas ENaC: epithelial sodium channel FGF-19: Fibroblast growth factor 19 FXR: Farnesoid X receptor GlyH-101: N-2-naphthalenyl-[(3,5-dibromo-2,4-dihydroxyphenyl)methylene]glycine hydrazine IBS-D: Diarrhea-predominant irritable bowel syndrome I C 50 : Half inhibitory concentration

[0107] chemicals Unless otherwise specified, all chemicals were purchased from Sigma-Aldrich (St. Louis, MO). Tauro-chenodeoxycholic acid and sodium salt of tauro-deoxycholic 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, DS, Tradtrantip, L., Yao, C., Kurth, MJ, and Verkman, AS (2011) Potent, metabolically stable benzopyrimido-pyrrolo-oxazine-dione (BPO) CFTR inhibitors for polycystic kidney disease. J Med Chem 54, 5468-5477; Snyder, DS, Tradtrantip, L., Battula, S., Yao, C., Phuan, PW, Fettinger, JC, Kurth, MJ, and Verkman, AS (2013) Absolute composition and biological properties of enantiomers of CFTR inhibitor BPO-27. ACS Med Chem Lett 4,456-459;Ma,T.,Thiagarajah,JR,Yang,H.,Sonawane,ND,Folli,C.,Galietta,LJ,and Verkman,AS(2002)Thiazolidinone CFTR inhibitor identified by high-throughput screening blocks cholera toxin-induced intestinal fluid secretion.J Clin Invest 110,1651-1658.

[0108] Example 1. T84 cell culture T84 cells (ATCC CCL - 248) are 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 are grown on Snapwell inserts (Costar Corning, Horseheads, NY) at 37°C in 5% CO2 / 95% air and used 7-14 days after plating.

[0109] Example 2. Human coronoid cultures Human colonoid cultures are generated from anonymized (except for age and sex) tissue samples obtained by endoscopic or surgical procedures from three different subjects. Human cultures are made from isolated intestinal crypts embedded in Matrigel (Corning, Tewksbury, MA) in 24-well plates and cultured in the presence of undifferentiated medium (UDM) containing Wnt3A, R-spondin-1 and Noggin as described below: 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 are used for propagation of colonoids. To generate monolayer cultures, colonoids are ground in Cultrex Organoid Harvesting solution (Trevigen, Gaithersburg, MD) and fragments are collected by centrifugation and resuspended in UDM. Colonoid fragments (100 μL) are seeded onto 0.4 μm pore polyester membrane 24-well cell culture inserts (Transwell; Corning, Tewksbury, MA) precoated with human collagen IV (34 μg / mL; Millipore Sigma). Monolayers are cultured in UDM at 37°C and 5% CO2. Under these conditions, cultures reach confluency between 7 and 14 days, as monitored by transepithelial resistance.

[0110] Example 3. Short circuit current measurement T84 cells were mounted in an Ussing chamber and incubated with equal volumes of HCO3- The cells are bathed in a buffered solution (in mM): 120 NaCl, 5 KCl, 1 MgCl2, 1 CaCl2, 10 D-glucose, 5 HEPES, and 25 NaHCO3 (pH 7.4). The solution is aerated with 95% O2 / 5% CO2 and maintained at 37°C. In one study, apical Cl - For conductance measurements, Cl was transferred from the basolateral to the apical - A gradient is applied where the basolateral hemi-chamber contains (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 is replaced by 5 mM NaCl and 115 mM Na-gluconate, and the basolateral membrane is permeabilized with 250 μg / ml amphotericin B. Short-circuit currents are measured using an EVC4000 multichannel voltage clamp (World Precision Instruments, Sarasota, Fla.).

[0111] For intestinal short circuit current measurements, CD1 mice are anesthetized with isoflurane. The colon is excised, washed with ice-cold Krebs buffer, dissected along the mesenteric edge, and full thickness sections are inserted into micro-Ussing chambers (area 0.7 cm). 2 The hemi-chamber is filled with oxygenated Krebs-bicarbonate solution.

[0112] Measurements in human colonoid cultures are performed as described (18). The apical and basolateral hemichambers are filled with Krebs-Ringer bicarbonate (KBR) buffer aerated with 95% O2 / 5% CO2 at 37°C. The basolateral hemichamber is supplemented with 10 mM glucose and the apical hemichamber with 10 mM mannitol to maintain osmotic balance.

[0113] Example 4. Intracellular Ca 2+ and cAMP measurement T84 cells are plated in 96-well black-walled microplates. Confluent cells are loaded with Fluo-4 NW (Invitrogen, Carlsbad, Calif.) 72 hours after plating. 2+ For measurements, Fluo-4 fluorescence is measured at excitation / emission wavelengths of 485 / 538 nm on a FluoStar fluorescent plate reader (BMG Lab Technologies, Durham, North Carolina). In some studies, cells are pretreated with BAPTA-AM for 30 min. Ca in human colonoid cultures 2+ Measurements were performed on monolayers and were analyzed using fluorescent Ca 2+ For cAMP assays, T84 cells are grown in 24-well plates, treated with CDCA and / or forskolin for 30 min, lysed by repeated freeze / thawing, centrifuged to remove cell debris, and supernatants are assayed for cAMP using the Parameter cAMP Immunoassay Kit according to the manufacturer's instructions (R&D Systems, Minneapolis, NM).

[0114] Example 5. Intestinal fluid secretion in obstructed intestinal loops in mice CD1 mice (8-10 weeks old) are allowed access to 5% dextrose water but are deprived of chow for 24 hours prior to the experiment. Mice are administered (R)-BPO-27 (5mg / kg) or vehicle (5% DMSO, 10% Kolliphor HS in saline) intraperitoneally 60 minutes prior to incision of an obstructed intestinal (mid-jejunum or distal colon) loop and injection of CDCA or PBS vehicle. This dose of (R)-BPO-27 was pre-indicated to provide therapeutic serum levels in mice over 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. For the dissection of the loop, mice are anesthetized with isoflurane and body temperature is maintained at 36-38°C with a heating pad during surgery. A small abdominal incision is made to expose the intestine. For the mid-jejunum obstructed loop model, a 2-3 cm loop is sutured. The loop is injected with 100 μl PBS containing CDCA or PBS vehicle. The abdominal incision is sutured closed and the mouse is allowed to recover from anesthesia. As previously reported (Haggie, PM, et al. (2018) SLC26A3 inhibitor identified in small molecule screen blocks colonic fluid absorption and reduces constipation. JCI Insight 3, e121370), in the colonic obstructed loop model, mice are given an enema (500 μl mineral oil) 12 hours before surgery to wash out the colonic solids, and a 1-2 cm obstructed loop is sutured. Intestinal loops are surgically removed at specified times and loop length and weight are measured to determine the amount of fluid secretion. In some experiments, CF mice (ΔF508 homozygous, 8-10 weeks old) are used.

[0115] Example 6. Rat model of bile acid diarrhea Female Sprague-Dawley rats (8-10 weeks old) are administered 500 μl CDCA (in 10 mM PBS) or PBS vehicle by midcolonic infusion using flexible plastic tubing (15 gauge, 78 mm long, Instech Laboratories, Plymouth Meeting, PA). Rats are administered (R)-BPO-27 (10 mg / kg) (or vehicle control) intraperitoneally 30 minutes prior to infusion. (R)-BPO-27 is dissolved in saline containing 5% DMSO and 10% Kolliphor HS. After infusion, rats are individually housed in metabolic cages with free access to water and food. Fecal samples are collected prior to infusion (for baseline fecal water content) and for 4 hours after infusion. Feces are weighed immediately when visualized to determine wet weight. To measure fecal water content, fecal samples are dried at 70° C. for 24 hours and the water content is calculated as (wet weight−dry weight) / wet weight.

[0116] Example 7. Statistics Data are presented as mean±SEM. Statistical analysis is performed using the Prism 5 GraphPad Software package (San Diego, CA). Statistical comparisons are performed using Student's t-test when there are two groups or one-way analysis of variance when there are three or more groups. A p-value of less than 0.05 is considered statistically significant.

[0117] CFTR inhibition blocks bile acid-induced secretory responses in T84 cells In the first study, several bile acids are screened for their ability to induce a pro-secretory response in T84 colonic epithelial cell line by short-circuit current measurement. See Example 3. When added to both apical (mucosal) and basolateral (serosa) bath solutions, the bile acids CDCA and DCA and their taurine conjugates generate a robust secretory current that is blocked by (R)-BPO-27 (Figure 1A). No increase in short-circuit current is seen for cholic acid and ursodeoxycholic acid, and only a small transient response is seen with lithocholic acid.

[0118] The increase in short-circuit current after CDCA application was observed in the presence of selective and chemically unrelated inhibitors, (R)-BPO-27 and CFTR. inh This inhibition is completely blocked by -172 (Figure 1B, C). Concentration-dependence studies show an IC of less than 1 μM for (R)-BPO-27 inhibition in this model. 50 (Data not shown). Short-circuit current measurements in mouse distal colon showed that addition of CDCA to the apical bath solution increased current by 5.2 ± 1.4 μA / cm 2 (mean ± SEM, n = 3), which was completely reversed by (R)-BPO-27 (-5.2 ± 1.7 μA / cm 2 ) (FIG. 1D). No increase in short-circuit current is seen following addition of CDCA to the basolateral bath solution.

[0119] To examine the profile of CDCA action in T84 cells, CDCA is added to either the apical or basolateral bath solution. Figure 2A shows a robust current response upon addition of 0.75 or 1 mM CDCA to the apical solution, with no effect of 1 mM CDCA added to the basolateral solution. Addition of up to 2 mM CDCA to the basolateral solution had only a minor effect (Figure 2B), and as a positive control, forskolin was added at the end of the study to demonstrate the integrity of the T84 cell monolayer. CDCA action on apical CFTR and its inhibition by (R)-BPO-27 was examined by measuring apical to basolateral Cl - This was confirmed in short-circuit current measurements in basolateral membrane permeabilized T84 cell monolayers in the presence of a concentration gradient, where the short-circuit current was determined by the apical CFTRCl- It provides a direct measurement of the current (Figure 2C).

[0120] cAMP and Ca in T84 cells 2+ Potential CDCA effects on signal transduction pathways are examined. See Example 4. CDCA up to 0.75 mM does not significantly affect the short-circuit current response to addition of the cAMP agonist forskolin, although a small reduction in forskolin-induced current is seen with 1 mM CDCA (FIG. 3A,B). In each case, the increased current was completely blocked by (R)-BPO-27. Intracellular cAMP measurements show that CDCA up to 1 mM does not increase cAMP by itself. However, 0.75 and 1 mM CDCA increase cAMP in response to maximal (10 μM) forskolin (FIG. 3C).

[0121] CDCA-induced increase in short-circuit current in T84 cells was associated with Ca 2+ Pretreatment with the chelator BAPTA-AM (Figure 4A, B) largely blocks intracellular Ca2+ signaling using the fluorescent sensor Fluo-4. 2+ Measurements of the peak Ca concentration observed after 0.75 or 1 mM CDCA with the cholinergic agonist carbachol or the purinergic agonist ATP (Figure 4C,D) 2+ Sustained Ca with comparable strength to the increase 2+ It shows an increase. 2+ These experiments unexpectedly demonstrated that CDCA inhibits the Ca2+ receptor signaling pathway in T84 cells. 2+ Supporting the involvement of the secretory response in signal transduction, because the secretory response is mediated by CFTR and cAMP-activated Cl - is mediated entirely by Ca channels, 2+ activated Cl - This is because it is thought not to be mediated by a channel.

[0122] Studies in primary human coronoid cultures CFTR Cl to apical CDCA -Secretory responses and Ca 2+ The main findings obtained with T84 cells, including the involvement of signal transduction, are examined in primary human colonoid cultures. See Example 2. As seen with T84 cells, CDCA produces a concentration-dependent increase in short-circuit current when added to the apical bath solution, which is reversed by (R)-BPO-27, but not when added to the basolateral bath solution (Fig. 5A, B). The increase in short-circuit current is also greatly reduced by BAPTA-AM pretreatment (Fig. 5C), and CDCA produces a cytoplasmic Ca2+ signaling pathway that is more transient than that seen in T84 cells, but with a peak intensity comparable to that produced by ATP. 2+ This results in an increase in concentration (Figure 5D).

[0123] BPO-27 inhibits fluid secretion in obstructed mouse intestinal loops An obstructed intestinal loop model is established in mice to investigate the secretagogue effect of CDCA in vivo. See Example 5. Initial studies are done in mid-jejunal loops because of the technical ease of obtaining multiple loops in one animal and its extensive prior use in studying intestinal fluid secretion in models of cholera and traveler's diarrhea. Figure 6A shows that injection of 10 mM CDCA in mid-jejunal loops produces intense fluid accumulation at 1 hour. CDCA concentration-dependence studies with loop fluid measured at 2 hours show a significant increase in loop fluid accumulation with 5 and 10 mM CDCA (Figure 6B). Similar experiments in obstructed distal colon loops show significant loop fluid accumulation with 2.5 mM CDCA (Figure 6C).

[0124] To study the effects of CFTR inhibition, (R)-BPO-27 is administered 60 min prior to the creation of an obstructed intestinal loop and injection of CDCA (or control PBS vehicle) (Figure 7A). In obstructed mid-jejunal loops, the active BPO-27 enantiomer, (R)-BPO-27, caused a significant 55% reduction in loop fluid accumulation, whereas the inactive enantiomer, (S)-BPO-27, had no significant effect (Figure 7B). A larger reduction of loop fluid accumulation by approximately 70% with (R)-BPO-27 was found in obstructed colonic loops (Figure 7C).

[0125] Because CFTR inhibition largely but not completely blocks loop fluid accumulation, we investigate whether CFTR-dependent mechanisms may be partially responsible for the CDCA effect. For these experiments, closed loop studies are performed in cystic fibrosis mice lacking functional CFTR. Closed loops of mid-jejunum and distal colon injected with 10 mM CDCA show slightly but significantly greater loop fluid content compared to control loops injected with PBS vehicle, suggesting that mechanisms other than CFTR also play a role in CDCA-induced fluid accumulation. This suggests that Na + / H + and Cl - / HCO3 - Based on previous studies, this may include inhibition of exchangers (pro-absorptive processes), increased paracellular permeability, or stimulation of mucus secretion. Alrefai, WA, 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. See Dig Dis Sci 46, 1223-1231.

[0126] BPO-27 reduces fecal water content in a rat model of bile acid diarrhea A model is established to test (R)-BPO-27 effects in which rats are administered CDCA (or vehicle control) by midcolonic infusion and feces are collected over 4 hours for measurement of fecal weight and water content (by wet-to-dry weight ratio) (Figure 8A, top). See Example 6. In this model, the infused liquid is rapidly distributed throughout the colonic lumen, as shown with Evans Blue dye (Figure 8A, bottom). Rats infused with CDCA have a higher amount of feces collected over 4 hours and fecal water content is significantly increased by about 25% (Figure 8B, C). In rats receiving midcolonic infusion of PBS, (R)-BPO-27 pretreatment does not affect fecal volume or water content, but the increase in fecal water content in rats receiving CDCA infusion is abolished by about 55% (Figure 8C).

[0127] Without being bound by theory, the data herein support the utility of CFTR inhibition by CFTR inhibitors, particularly (R)-BPO-27, in the treatment of bile acid-associated diarrhea. Short-circuit current measurements in T84 cells and human colonoid cultures show secretory currents in response to exposure of the apical cell surface to CDCA, which completely reverses CFTR inhibition. Complete inhibition of secretory currents by (R)-BPO-27 is also seen in ex vivo CDCA-exposed mouse colon tissue. Systemically administered (R)-BPO-27 largely prevents fluid accumulation in obstructed colonic loops in mice in vivo and prevents the increase in fecal water after intracolonic infusion of CDCA-containing solutions. These results implicate CFTR as the major secretory ion channel in CDCA-induced diarrhea in human colon cell cultures and in rodents.

[0128] This application claims priority to U.S. Patent No. 62 / 860,539, filed June 12, 2019, the entirety of which is incorporated by reference herein.

Claims

1. (a) a method for treating a subject having bile acid diarrhea, comprising administering to said subject a CFTR chloride channel inhibitor, said CFTR chloride channel inhibitor being BPO-27, in an amount effective to treat bile acid diarrhea; (b) a CFTR chloride channel inhibitor, BPO-27, in an amount effective to reduce intestinal fluid secretion due to bile acid-induced activation of apical CFTR chloride channels in the intestinal epithelium for reducing intestinal fluid secretion due to bile acid-induced activation of apical CFTR chloride channels in a subject in need of such reduction; (c) comprising a CFTR chloride channel inhibitor that is BPO-27 in an amount effective to reduce bile acid-induced apical CFTR chloride channel current for reducing bile acid-induced apical CFTR chloride channel current in intestinal epithelium in a subject in need thereof; or (d) treating diarrhea or alleviating symptoms associated with diarrhea in a subject who has undergone ileal resection, comprising a CFTR chloride channel inhibitor that is BPO-27 in an amount effective to treat said diarrhea or alleviate the symptoms of said diarrhea; Pharmaceutical compositions.

2. The pharmaceutical composition of claim 1, further comprising (S)-BPO-27.

3. 10. The pharmaceutical composition of claim 1, wherein the amount of the CFTR chloride channel inhibitor is effective to reduce intestinal fluid secretion resulting from bile acid diarrhea.

4. 2. The pharmaceutical composition of claim 1, wherein the amount of the CFTR chloride channel inhibitor is effective to attenuate bile acid-induced activation of the apical CFTR chloride channel.

5. 3. The pharmaceutical composition of claim 1 or 2, further comprising a second drug in an amount effective to treat bile acid diarrhea, wherein the second drug 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. (a) the bile acid binder is cholestyramine, colestipol, or colesevelam; (b) the farnesoid X receptor (FXR) agonist is obeticholic acid; (c) the 5-HT3 antagonist is alosetron; (d) the opioid receptor agonist is loperamide; (e) the mixed μ-opioid receptor agonist is eluxadoline, or (f) the broad-spectrum gut-specific antibiotic is rifaximin; The pharmaceutical composition according to claim 5.

7. The pharmaceutical composition according to any one of claims 1 to 6, wherein the subject is a human.

8. The method of claim 1 , wherein the subject has been diagnosed with Crohn's disease.

9. The method of claim 1, wherein the subject has been diagnosed with IBS-D.

10. The pharmaceutical composition of claim 1 , wherein the subject has functional diarrhea.

11. The pharmaceutical composition, when administered to the subject, (a) resulting in a decrease in the water content of the feces of said subject; (b) resulting in a decrease in the frequency of bowel movements in said subject; (c) resulting in a decrease in fecal output in the subject; (d) resulting in relief of abdominal pain in said subject. (e) causing relief of abdominal bloating in said subject; or (f) resulting in relief of nausea in the subject. The pharmaceutical composition of claim 1.

12. 12. The pharmaceutical composition of claim 11, wherein the reduction in water content of the subject's stool is demonstrated by a reduction in the subject's score on the Bristol Fecal Foam Scale.

13. A pharmaceutical composition according to any one of claims 1 to 3 and 5 to 12 for treating a subject with bile acid diarrhea.

14. A pharmaceutical composition according to any one of claims 1, 2 and 4 to 12 for reducing intestinal fluid secretion due to bile acid-induced activation of apical CFTR chloride channels in the intestinal epithelium.

15. The pharmaceutical composition according to any one of claims 1, 2 and 5 to 12, for decreasing bile acid-induced apical CFTR chloride channel current in intestinal epithelium.

16. 13. The pharmaceutical composition of any one of claims 1, 2 and 5 to 12 for treating diarrhea or alleviating symptoms associated with diarrhea in a subject who has undergone ileal resection.

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